Multivalent cargo carrying composites and uses thereof

Abstract

Compounds comprising multivalent sugars that bind to nucleic acids are provided. These compounds contain shikimic acid-derived cores and are useful in delivering nucleic acids to cells or tissues, e.g., for use in therapeutic treatment. Also provided are pharmaceutical compositions comprising the above compounds and medical uses thereof, including their use in the treatment or prevention of conditions such as liver disease.

Description

[0001] Multivalent sugar compounds that bind to nucleic acids are provided. These compounds can be used to deliver nucleic acids to cells or tissues, for example, for use in therapeutic treatments. In particular, these compounds contain specific monosaccharides capable of binding to the desialylate glycoprotein receptor (ASGPR). Such compounds can be used, for example, to target the delivery of therapeutic oligonucleotides to cells, such as hepatocytes. Pharmaceutical compositions comprising the above-described compounds and their medical uses are also provided, including their use in the treatment or prevention of conditions such as liver diseases.

[0002] Cross-references to related applications

[0003] This specification claims priority to GB Patent Application No. 2311334.3 (filed July 24, 2023). The entire text of the aforementioned patent application is incorporated herein by reference. Summary of the Invention

[0004] There are numerous methods for targeted delivery of cargo portions, including therapeutic nucleic acids, to cells. One approach is to tether the cargo to one or more sugar ligands to utilize sugar-binding proteins associated with the target cell. Exemplary sugar-binding proteins include cell membrane surface receptors, such as glycoprotein receptors, including the desialylate glycoprotein receptor (ASGPR). ASGPRs have been shown to be highly expressed on the surface of mammalian hepatocytes and cancer cell lines; they are also expressed in other cell types, although generally at lower levels. Thus, ASGPRs represent promising targets for liver delivery of cargo, including therapeutic nucleic acid agents (see, for example, D'Souza et al., J. Control Release, (2015) 203:126-139). Ligands that can be used to target ASGPRs include monosaccharides, such as N-acetylgalactosamine (GalNAc).

[0005] Target-binding complexes typically contain more than one ligand and / or more than one cargo moiety. This functionality can be achieved by using a branched core structure with connectors for coupling the ligand and / or cargo moiety to the core. In this way, complexes with an appropriate number and arrangement of functional moieties for the desired application can be prepared. Examples of multivalent (e.g., branched) complexes are described in the following international patent publications: WO 2014 / 179620 (Isis Pharmaceuticals, Inc.); WO 2015 / 177668 A1 (Pfizer Inc.); WO 2009 / 073809 A2 (Alnylam Pharmaceuticals, Inc.); WO 2012 / 083046 A2 (Arrowhead Research Corporation); WO2017 / 156012 A1 (Arrowhead Pharmaceuticals, Inc.); WO 2016 / 100401 A1 (Dicerna Pharmaceuticals, Inc.); WO 2017 / 174657 A1 (Silence Therapeutics GmbH); and WO2019 / 092280 A1 (Silence Therapeutics GmbH). Complexes of the aforementioned types utilize different coupling chemistry to link the ligand and cargo portion to the branched core structure, and also utilize different core structures. However, alternative chemical functional groups are needed to couple the cargo moiety and ligand to prepare complexes that can be used to deliver the cargo moiety into cells. This disclosure seeks to address this need by providing, for example, novel compounds and complexes for use in targeting therapeutic nucleic acids into cells such as hepatocytes.

[0006] In short, the compounds and complexes disclosed herein contain one or more monosaccharide ligands and one or more nucleic acids. These are linked together using a multidentate "splitter" derived from or chemically related to shikimic acid. The use of shikimic acid and its analogues in the preparation of branched units provides a novel way of coupling the targeting and cargo portions, which can have advantages over known methods, including, for example, the use of simple natural precursors. The compounds of the present invention exhibit useful properties such as improved targeting, increased activity, and / or improved in vivo pharmacokinetic properties. Summary of the Invention

[0007] This disclosure includes the following aspects and implementation methods, which are presented as clauses 1 to 50:

[0008] 1. A compound having the structure of formula (I):

[0009]

[0010] Or its pharmaceutically acceptable salt, wherein:

[0011] X is selected from covalent bonds, -CH2-, and -C(O)-;

[0012] Y is either -O or -NR-.

[0013] Where R is selected from -H and -(C1-C6) alkyl;

[0014] It represents a carbon-carbon single or double bond, provided that X is a covalent bond. It represents a carbon-carbon single bond; and

[0015] G 1 To G 4 Each independently represents either the -[spacer]-[ligand] part or the -[connector]-[cargo] part, where each ligand is independently a monosaccharide and each cargo is independently a nucleic acid, provided that G... 1 To G 4 At least one of them represents the -[spacer]-[ligand] part and G 1 To G 4 At least one of them represents the [chain]-[joint]-[cargo] part.

[0016] 2. The compound or a pharmaceutically acceptable salt thereof as described in Clause 1, wherein X is -C(O)-.

[0017] 3. A compound or a pharmaceutically acceptable salt thereof as described in Clause 1 or Clause 2, wherein Y is -NR-, for example, wherein Y is -NH- or -N(CH3)-.

[0018] 4. The compound or a pharmaceutically acceptable salt thereof according to any one of clauses 1 to 3, wherein Indicates a single key.

[0019] 5. The compound or a pharmaceutically acceptable salt thereof according to any one of Clauses 1 to 4, wherein each ligand is N-acetylgalactosamine (GalNAc).

[0020] 6. The compound or a pharmaceutically acceptable salt thereof according to any one of Clauses 1 to 5, wherein each of the goods is independently selected from antisense oligonucleotides (ASO), immunostimulatory oligonucleotides, decoy oligonucleotides, splice-altering oligonucleotides, splice-converting oligonucleotides, triplet-forming oligonucleotides, siRNA, saRNA, microRNA, microRNA mimics, antimiR, double-stranded RNA, single-stranded RNA, ribozymes, aptamers, mirror aptamers, CRISPR oligonucleotides, and G-quadruplexes.

[0021] 7. A compound or a pharmaceutically acceptable salt thereof according to any one of Clauses 1 to 6, wherein each spacer group independently comprises a chain of 2 to 20 atoms selected from C, N, O, S and P, for example wherein each spacer group is independently selected from linear alkylene groups (which may optionally be interrupted by one or more amide and / or phosphate groups) and polyethylene glycol.

[0022] 8. A compound or a pharmaceutically acceptable salt thereof according to any one of clauses 1 to 7, wherein [joint-chain] in each case independently represents a straight chain portion comprising 8 to 30 atoms.

[0023] 9. A compound or a pharmaceutically acceptable salt thereof according to any one of clauses 1 to 8, wherein said compound has a structure of formula (Ia) or formula (Ib):

[0024]

[0025] ,

[0026] Where X, Y, G 1 G 2 G 3 and G 4 As defined in any of the foregoing clauses, in formula (Ib) It represents a carbon-carbon single bond.

[0027] 10. A compound or a pharmaceutically acceptable salt thereof according to any one of clauses 1 to 3 and 5 to 8, wherein said compound has the structure of formula (Id):

[0028] ,

[0029] Where X, Y, G 1 G 2 G 3 and G 4 As defined in any of the foregoing clauses, and It represents a double bond.

[0030] 11. A compound having the structure of formula (II):

[0031]

[0032] Or its pharmaceutically acceptable salt, wherein:

[0033] X is selected from covalent bonds, -CH2-, and -C(O)-;

[0034] Y is either -O or -NR-.

[0035] Where R is selected from -H and -(C1-C6) alkyl;

[0036] It represents a carbon-carbon single or double bond, provided that X is a covalent bond. Indicates a carbon-carbon single bond;

[0037] In each case, the spacer group independently represents a portion of a straight chain containing 6 to 20 atoms (e.g., selected from C, N, O, S, and P) that attaches the ligand to the rest of the molecule;

[0038] The ligand is a monosaccharide independently in each case;

[0039] The term "joint" and "chain" together refer to the part that attaches cargo to the rest of the molecule, such as a straight chain containing 8 to 30 atoms; and

[0040] The goods are nucleic acid.

[0041] 12. A compound or a pharmaceutically acceptable salt thereof as described in Clause 11, wherein X is -C(O)- and Y is -NR- (e.g., wherein R is -H or -CH3).

[0042] 13. A compound or a pharmaceutically acceptable salt thereof as described in Clause 11 or Clause 12, wherein Indicates a single key.

[0043] 14. A compound or a pharmaceutically acceptable salt thereof according to any one of clauses 11 to 13, wherein said compound has a structure of formula (IIa) or formula (IIb):

[0044] ,

[0045] Wherein X, Y, ligand, spacer, chain, connector, and cargo are defined according to any of the foregoing clauses, wherein in formula (IIb) It represents a carbon-carbon single bond.

[0046] 15. A compound or a pharmaceutically acceptable salt thereof as described in Clause 11 or Clause 12, wherein said compound has the structure of formula (IId):

[0047]

[0048] Wherein X, Y, ligand, spacer, chain, connector, and cargo are defined in accordance with any of the foregoing clauses, and It represents a double bond.

[0049] 16. A compound having the structure of formula (III):

[0050]

[0051] Or a pharmaceutically acceptable salt thereof, wherein X, Y, spacer, chain, connector and goods are as defined in any of the foregoing clauses, and It represents a carbon-carbon single or double bond, provided that X is a covalent bond. It represents a carbon-carbon single bond.

[0052] 17. The compound or a pharmaceutically acceptable salt thereof as described in Clause 16, wherein:

[0053] X is -C(O)-;

[0054] Y is -O or -NR-, where R is selected from -H and -(C 1-6 )alkyl;

[0055] Indicates a single key;

[0056] The spacer group, in each case, independently represents a straight chain containing 6 to 20 atoms selected from C, N, O, S, and P;

[0057] The term "joint" and "chain" together represent a straight chain containing 8 to 30 atoms; and

[0058] The goods are nucleic acid.

[0059] 18. A compound or a pharmaceutically acceptable salt thereof as described in Clause 16 or Clause 17, wherein said compound has a structure of formula (IIIa) or formula (IIIb):

[0060]

[0061] ,

[0062] Wherein X, Y, spacer base, tether, joint, and cargo are defined according to any of the foregoing clauses, wherein in formula (IIIb) It represents a carbon-carbon single bond.

[0063] 19. A compound or a pharmaceutically acceptable salt thereof as described in Clause 16, wherein said compound has the structure of formula (IIId):

[0064] ,

[0065] Wherein X, Y, spacer base, tether, joint, and cargo are defined according to any of the foregoing clauses, and It represents a double bond.

[0066] 20. A compound having the structure of formula (IV):

[0067]

[0068] Or its pharmaceutically acceptable salt, wherein:

[0069] Z independently represents, in each case, a portion of a straight chain containing 1 to 16 atoms (e.g., selected from C, N, O, S, and P);

[0070] m is independently selected from an integer from 1 to 6 in each case;

[0071] R is selected from -H and -(C1-C6) alkyl; and

[0072] Chains, joints, and goods are defined as in any of the foregoing clauses.

[0073] 21. The compound or a pharmaceutically acceptable salt thereof as described in Clause 20, wherein Z is independently selected in each case from... -(C1-C 16 )alkylene-, -(C2-C 16 ) imidene-,

[0074] -(C1-C 12 )alkylene-C(O)-, -(C2-C 12 ) imidene-C(O)-,

[0075] -(C1-C8)alkylene-C(O)NR'-(C1-C6)alkylene-、

[0076] -(C2-C8)-alkenyl-C(O)NR'-(C1-C6)alkyl-,

[0077] -(C1-C8)alkylene-C(O)NR'-(C2-C6)alkenyl-and

[0078] -(C2-C8)imenyl-C(O)NR'-(C2-C6)imenyl-(wherein) (in each case, R' represents the attachment point with the oxygen atom in GalNac), where R' is independently selected from -H and -(C1-C6) alkyl groups in each case.

[0079] 22. A compound or a pharmaceutically acceptable salt thereof as described in Clause 20 or Clause 21, wherein each Z is independently selected from...

[0080] -(C1-C6)alkylene-C(O)NR'-(C1-C4)alkylene- (where R' is independently selected from -H and -(C1-C6)alkyl in each case), for example, where Z is -(C4)alkylene-C(O)NH-(C3)alkylene-.

[0081] 23. A compound or a pharmaceutically acceptable salt thereof according to any one of clauses 20 to 22, wherein m is independently selected from 1, 2, 3 and 4 in each case.

[0082] 24. A compound or a pharmaceutically acceptable salt thereof according to any one of clauses 20 to 23, wherein said compound has a structure of formula (IVa) or formula (IVb):

[0083]

[0084] ,

[0085] Z, m, R, chain, joint, and cargo are defined as in any of the foregoing clauses.

[0086] 25. A compound having the structure of formula (V):

[0087] ,

[0088] Or its pharmaceutically acceptable salt, wherein:

[0089] A independently represents, in each case, a portion of a straight chain containing 1 to 16 atoms (e.g., selected from C, N, O, S, and P);

[0090] n is an integer independently selected from 1 to 6 in each case;

[0091] R is selected from -H and -(C1-C6) alkyl; and

[0092] Chains, joints, and goods are defined as in any of the foregoing clauses.

[0093] 26. The compound described in Clause 25 or a pharmaceutically acceptable salt thereof, wherein A is independently selected in each case from... -(C1-C 16 )alkylene- and

[0094] -(C2-C 16 ) imidene- (wherein) In each case, it represents the attachment point with the GalNac oxygen atom.

[0095] 27. A compound or a pharmaceutically acceptable salt thereof as described in Clause 25 or Clause 26, wherein n is independently selected from 1, 2, 3 and 4 in each case.

[0096] 28. A compound or a pharmaceutically acceptable salt thereof according to any one of clauses 25 to 27, wherein said compound has a structure of formula (Va) or formula (Vb):

[0097]

[0098] ,

[0099] Wherein A, n, R, chain, joint and goods are as defined in any of the foregoing clauses.

[0100] 29. A compound having the structure of formula (IX):

[0101] ,

[0102] Or its pharmaceutically acceptable salt, wherein:

[0103] Z independently represents, in each case, a portion of a straight chain containing 1 to 16 atoms (e.g., selected from C, N, O, S, and P) that attaches the GalNAc ligand to the rest of the molecule;

[0104] m is independently selected from an integer from 1 to 6 in each case;

[0105] R is selected from -H and -(C1-C6) alkyl; and

[0106] Chains, joints, and goods are defined as in any of the foregoing clauses.

[0107] 30. A compound having the structure of formula (XI):

[0108]

[0109] in:

[0110] Z is selected independently in each case. -(C1-C 12 )alkylene- and

[0111] -(C1-C6)alkylene-C(O)NR'-(C1-C4)alkylene-, wherein R' is independently selected from -H and -(C1-C6)alkyl in each case (where R' is selected from -H and -(C1-C6)alkyl). (In each case, it represents the attachment point with the GalNac oxygen atom).

[0112] m is independently selected from an integer from 1 to 6 in each case;

[0113] R is selected from -H and -(C1-C6) alkyl;

[0114] q is an integer from 1 to 12;

[0115] D is selected from (i) -C(O)NH- and (ii) -NHC(O)-(CH2). t -C(O)NH- (where t is an integer from 1 to 6);

[0116] s is an integer from 0 to 12; and

[0117] Goods as defined herein (e.g., ASO or siRNA).

[0118] 31. A compound, said compound being selected from:

[0119]

[0120] And its pharmaceutically acceptable salts, wherein nucleic acids represent goods as defined in any of the foregoing clauses.

[0121] 32. The compound or a pharmaceutically acceptable salt thereof as described in Clause 31, wherein the nucleic acid is represented as ASO or siRNA.

[0122] 33. A compound or a pharmaceutically acceptable salt thereof as defined in any of clauses 1 to 32, having an IC50 concentration of less than about 20 nM against ASGPR. 50 Values ​​(e.g., less than approximately 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, or 2 nM IC) 50 The binding affinity is represented by the value.

[0123] 34. A compound or a pharmaceutically acceptable salt thereof as defined in any one of Clauses 1 to 32, having binding kinetics to ASGPR characterized by a Kd value of less than about 8 nM (e.g., a Kd value of less than about 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM or 0.5 nM).

[0124] 35. A compound or a pharmaceutically acceptable salt thereof as defined in any one of clauses 1 to 32, having an IC50 concentration of less than about 50 nM. 50 Values ​​(e.g., less than about 20 nM, 10 nM, or 5 nM IC) 50 The knockdown of gene expression in HEK293 cells was characterized by a value (value).

[0125] 36. A compound or a pharmaceutically acceptable salt thereof as defined in any one of Clauses 1 to 32, having the activity of knocking down gene expression in tissues such as the liver in vivo, wherein the compound is capable of knocking down the level of a target mRNA in the tissue by at least about 40% (e.g., at least about 45%, 50%, 55%, 60%, 65% or 70%).

[0126] 37. A pharmaceutical composition comprising a compound as defined in any one of clauses 1 to 36 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient or carrier.

[0127] 38. A compound or a pharmaceutically acceptable salt thereof according to any one of Clauses 1 to 36, or a pharmaceutical composition according to Clause 37, for use in a therapeutic manner.

[0128] 39. A compound or a pharmaceutically acceptable salt thereof according to any one of Clauses 1 to 36, or a pharmaceutical composition according to Clause 37, for use in the treatment of conditions selected from: liver disease (e.g., liver cancer), hereditary diseases, hemophilia and bleeding disorders, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), viral hepatitis, rare diseases (e.g., acromegaly), metabolic diseases, cardiovascular diseases, obesity, thalassemia, liver injury (e.g., drug-induced liver injury), hemochromatosis, alcoholic liver disease, alcohol dependence, anemia, and anemia of chronic disease.

[0129] 40. The compound, pharmaceutically acceptable salt, or pharmaceutical composition used as described in Clause 39, wherein the condition is selected from NASH, NAFLD, metabolic diseases, and cardiovascular diseases.

[0130] 41. The compound, pharmaceutically acceptable salt, or pharmaceutical composition used as described in Clause 39, wherein the condition is NASH.

[0131] 42. The compound, pharmaceutically acceptable salt, or pharmaceutical composition used according to Clause 39, wherein the condition is a metabolic disorder selected from hypercholesterolemia, dyslipidemia, and hypertriglyceridemia.

[0132] 43. A compound having the structure of formula (VI):

[0133]

[0134] Or a pharmaceutically acceptable salt thereof, wherein X, Y, spacer group and chain are defined as in any of the preceding clauses; It represents a carbon-carbon single or double bond, provided that X is a covalent bond. R represents a carbon-carbon single bond; R" is an acyl group, -C(O)aryl group, or -H; and J is -CO2H, -OH, -C(O)O-(pentafluorophenyl), -N3, or phosphoramide.

[0135] 44. A method for preparing a compound of formula (III) as defined in any one of clauses 16 to 19, characterized in that a compound of formula (VI) as defined in clause 43 is reacted with a compound having the following structure.

[0136] Q - [Goods]

[0137] The goods are defined according to any of the preceding clauses, and Q represents a group that is reactive to group J as defined in clause 43.

[0138] 45. The method according to Clause 44, wherein Q represents a group containing BCN and J is -N3.

[0139] 46. ​​A compound having the structure of formula (X):

[0140]

[0141] Or a pharmaceutically acceptable salt thereof, wherein Z, R, m and the chain are defined as in any of the preceding clauses; R" is an acyl, -C(O)aryl or -H; and J is -CO2H, -OH, -C(O)O-(pentafluorophenyl), -N3 or phosphoramide.

[0142] 47. A method for preparing a compound of formula (IX) as defined in Clause 29, characterized in that the compound of formula (X) as defined in Clause 46 is reacted with a compound having the following structure.

[0143] Q - [Goods]

[0144] The goods are defined according to any of the preceding clauses, and Q represents a group that is reactive to group J as defined in clause 46.

[0145] 48. A compound having the structure of formula (XII):

[0146]

[0147] Or a pharmaceutically acceptable salt thereof, wherein Z, R, m and q are as defined in any of the preceding clauses; D' is selected from (i) the bond, and (ii) -NHC(O)-(CH2). t - (where t is as described herein), R" is an acyl, -C(O)aryl, or -H; and J is -CO2H, -OH, -C(O)O-(pentafluorophenyl), -N3, or phosphoramidite.

[0148] 49. The compound according to clause 48, wherein J is -CO2H or -C(O)O-(pentafluorophenyl).

[0149] 50. A method for preparing a compound of formula (XI) as defined in Clause 30, characterized in that a compound of formula (XII) as defined in Clause 48 or Clause 49 is reacted with a compound having the following structure.

[0150] Q - [Goods]

[0151] The goods are defined according to any of the preceding clauses, and Q represents a group that is reactive to group J as defined in clause 48 or clause 49. Attached Figure Description

[0152] Figure 1 A schematic example of a compound of this disclosure is shown. The cargo nucleic acid is attached to the remainder of the molecule via a linker and a tie strand. The separator portion binds to the tie strand on one side and to multiple ligands on the other side, with a spacer base between the ligand and the separator in each case. The complex shown carries three ligands, but other numbers of ligands are also possible (e.g., by employing different separators). In operation, the ligand (which may be, for example, the GalNAc portion) typically binds to a cell or tissue-associated receptor or other biomolecule; this facilitates association with and / or uptake into a cell or tissue where the cargo (e.g., siRNA) can exert a biological effect.

[0153] Figure 2 The results of an in vivo assay evaluating MALAT-1 mRNA knockdown in hepatocytes following a single 50 nmol / kg dose are shown (Example 3). The relative levels of MALAT-1 mRNA in the liver are shown compared to a PBS control (100%, left column). The middle column shows mRNA levels after treatment with naked MALAT-1 ASO. The right column shows mRNA levels after treatment with compound 1 of Example 1.

[0154] Figure 3 The pharmacokinetics of ASO exposure following a single dose of 50 nmol / kg are shown (Example 3). The left column shows the exposure in the liver (nmol / kg) after treatment with naked MALAT-1 ASO. The right column shows the exposure in the liver after treatment with compound 1 of Example 1.

[0155] Figure 4Results of an in vivo assay evaluating MALAT-1 mRNA knockdown in renal cells following a single 50 nmol / kg dose are shown (Example 3). The relative levels of MALAT-1 mRNA in the kidneys are shown compared to a PBS control (100%, left column). The middle column shows mRNA levels after treatment with naked MALAT-1 ASO. The right column shows mRNA levels after treatment with compound 1 from Example 1.

[0156] Figure 5 The pharmacokinetics of ASO exposure following a single dose of 50 nmol / kg are shown (Example 3). The left column shows the renal exposure (nmol / kg) after treatment with naked MALAT-1 ASO. The right column shows the renal exposure after treatment with compound 1 of Example 1.

[0157] Figure 6 The ANGPTL3 protein concentration measured prior to subcutaneous injection in the in vivo assay of Example 3 is shown. ANGPTL3 levels (ng / ml) are shown for the PBS control (column 1, left), the control siRNA cluster of Example 1 (column 2, middle), and compound 2 (column 3, right). No statistical significance was observed between groups.

[0158] Figure 7 The plasma ANGPTL3 protein concentration on day 28 is shown (Example 3). Column 1 (left-handed side) shows the results after administration of PBS as a control. Columns 2 and 3 show the exposure results after treatment with the control siRNA cluster of Example 1 and compound 2, respectively. Statistical analysis was performed using two-way ANOVA.

[0159] Figure 8 The ANGPTL3 protein concentration in plasma, corrected for PBS control, is shown over time (Example 3). The black line and circle (top) indicate the PBS control values. The light gray line and circle (middle) indicate the results for the control siRNA cluster in Example 1, and the dark gray line and circle (bottom) indicate the results for compound 2.

[0160] Figure 9 The liver Angptl3 mRNA levels from the in vivo study of Example 3 are shown. Values ​​are normalized relative to the PBS control (column 1, left). Columns 2 and 3 show the relative exposure after treatment with the control siRNA cluster of Example 1 and after treatment with compound 2, respectively.

[0161] Figure 10The relative levels of MALAT1 mRNA in the liver (compared to RPS16 mRNA) from the in vivo study of Example 3 are shown. Values ​​were normalized relative to the PBS control (column 1, 100%). Column 2 shows the knockdown of MALAT1 using the control ASO cluster (reference) of Example 1, while columns 3-8 show the knockdown using compounds 1, 2c, 3, 5, 6, and 7, respectively.

[0162] Figure 11 The relative levels of PPIB mRNA in the liver (compared to RPS16 mRNA) from the in vivo study of Example 3 are shown. Values ​​were normalized relative to the PBS control (column 1, 100%). Column 2 shows the knockdown of PPIB using the control ASO cluster (reference) of Example 1, while columns 3 and 4 show the knockdown using compounds 2b and 12, respectively.

[0163] Figure 12 The relative levels of MALAT1 mRNA in the liver (compared to RPS16 mRNA) from the in vivo study of Example 3 are shown. Values ​​were normalized relative to the PBS control (column 1, 100%). Column 2 shows the knockdown of MALAT1 using the control ASO cluster (reference) of Example 1, while columns 3-6 show the knockdown using compounds 8-11, respectively.

[0164] Figure 13 The liver from the in vivo study in Example 3 is shown. Figure 13 A) and kidneys ( Figure 13 B) Relative levels of MALAT1 mRNA. Values ​​were normalized relative to the PBS control (column 1, 1.0 in each case). Columns 2-4 show the knockdown of MALAT1 using compounds 2c, 16a, and 15a, respectively. Detailed Implementation

[0165] Although specific embodiments of this disclosure will now be described with reference to description and examples, it should be understood that such embodiments are merely examples and illustrate only a small fraction of the many possible specific embodiments in which the principles of this disclosure may be applied. In view of the benefits of this disclosure, various changes and modifications will be apparent to those skilled in the art and are considered to be within the spirit and scope of this disclosure as further defined in the appended claims.

[0166] definition

[0167] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, exemplary methods, apparatuses, and materials are described hereafter. All technical and patent publications referenced herein are incorporated herein by reference in their entirety.

[0168] Unless otherwise stated, the practice of this disclosure will employ conventional techniques such as chemical synthesis, tissue culture, immunology, molecular biology, microbiology, cell biology, recombinant DNA, etc., which are within the scope of the art. See, for example, Michael R. Green and Joseph Sambrook, *Molecular Cloning* (4th ed., Cold Spring Harbor Laboratory Press, 2012); Ausubel et al. (2007), *Current Protocols in Molecular Biology*; *Methods in Enzymology* (Academic Press, Inc., NY); MacPherson et al. (1991), *PCR 1: A Practical Approach* (IRL Press at Oxford University Press); MacPherson et al. (1995), *PCR 2: A Practical Approach*; Harlow and Lane (1999), *Antibodies, A Laboratory Manual*; Freshney (2005), *Culture of Animal Cells: A Manual of Basic Technique*, 5th ed.; Gait (1984), *Oligonucleotide Synthesis*; US Patent No. 4,683,195; Hames and Higgins (1984), *Nucleic Acid*. Hybridization; Anderson (1999) Nucleic Acid Hybridization; edited by Hames and Higgins (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; edited by Miller and Calos (1987) Gene Transfer Vectors for Mammalian Cells (ColdSpring Harbor Laboratory);Edited by Makrides (2003) Gene Transfer and Expression in Mammalian Cells; edited by Mayer and Walker (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); edited by Herzenberg et al. (1996) Weir's Handbook of Experimental Immunology; Manipulating the Mouse Embryo: ALaboratory Manual, 3rd Edition (Cold Spring Harbor Laboratory Press (2002)); Sohail (editor) (2004) Gene Silencing by RNA Interference: Technology and Application (CRC Press). ;

[0169] All numerical names (e.g., pH, temperature, time, concentration, molecular weight, etc., including ranges) are approximate values ​​that vary (+) or (-) in increments of, for example, 0.1 or 1.0, where appropriate. It should be understood that, although not always explicitly stated, all numerical names are preceded by the term "about," which is used to indicate a typical level of variability. For example, a numerical name "about" for a given value may vary by ±10% of said value; alternatively, the variation may be ±5%, ±2%, or ±1% of the value. It should also be understood that, although not always explicitly stated, the reagents described herein are merely exemplary, and their equivalents are known in the art.

[0170] Unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” as used in the specification and claims include plural references. For example, the term “cell” includes a plurality of cells, including mixtures thereof. Unless specifically stated or obvious from the context, the term “or” as used herein should be understood as inclusive. The term “comprising” is used herein to mean the phrase “including but not limited to,” and may be used interchangeably with it.

[0171] As used herein, the terms “comprising” or “including” are intended to mean that a composition and method includes the listed elements without excluding other elements. When used to define compositions and methods, “consisting substantially of” should mean excluding other elements that are of any essential significance to the stated purpose. Thus, a composition consisting substantially of the elements defined herein does not exclude trace contaminants from separation and purification methods, as well as pharmaceutically acceptable carriers such as phosphate-buffered saline, preservatives, etc. “Containing” should mean excluding other components beyond trace elements and substantial method steps for administering the compositions of this disclosure or process steps for producing the compositions or achieving the intended results. Embodiments defined by each of these transitional terms are within the scope of this disclosure. The term “comprising” is used herein to cover and disclose corresponding statements in which the term “comprising” is replaced by “consisting substantially of” or “consisting of”.

[0172] "GalNAc" refers to 2-(acetamido)-2-deoxy-D-galactopyranose, commonly referred to as N-acetylgalactosamine in the literature. In this article, "GalNAc" or "N-acetylgalactosamine" refers to the β-form, namely 2-(acetamido)-2-deoxy-β-D-galactopyranose.

[0173] As used herein, the term "cargo" refers to a chemical or biological entity suitable for targeting and / or delivery to cells or tissues, such as as part of the compounds disclosed herein. The cargo according to this disclosure is a nucleic acid.

[0174] As used herein, the term "nucleic acid" includes nucleic acids selected from the group consisting of DNA, RNA, peptide nucleic acids (PNA), and locked nucleic acids (LNA). Nucleic acids can be functional nucleic acids, for example, those selected from the group consisting of mRNA, microRNA, shRNA, combinations of RNA and DNA, siRNA, siNA, antisense nucleic acids (e.g., antisense oligonucleotides (ASO)), ribozymes, aptamers, and mirror aptamers. "Peptide nucleic acids" are polymers similar to DNA or RNA, wherein the backbone consists of repeating amino acid units (typically N-(2-aminoethyl)-glycine) linked by peptide bonds. Various purine and pyrimidine bases are linked to the backbone via methylene bridges and carbonyl groups. "Lock nucleic acids" are nucleic acids in which a 2' hydroxyl group is linked, for example, to the 4' carbon of the same ribose via a methylene bridge.

[0175] A “double-stranded region” refers to a region in two complementary or substantially complementary oligonucleotides that form a double helix with each other by Watson-Crick base pairing or any other means that allows complementary or substantially complementary oligonucleotide chains to form a double helix. For example, an oligonucleotide chain having 21 nucleotide units can pair with another oligonucleotide chain having 21 nucleotide units, but only 19 nucleotides on each chain are complementary or substantially complementary, such that the “double-stranded region” consists of 19 base pairs. The remaining base pairs can exist as 5' and 3' overhangs or as a single-stranded region. Furthermore, 100% complementarity is not required within a double-stranded region; substantially complementary complementarity is allowed within a double-stranded region. Substantial complementarity refers to the complementarity between the chains, allowing them to anneal under biological conditions. Techniques for empirically determining whether two chains can anneal under biological conditions are well known in the art. Alternatively, two chains can be synthesized and added together under biological conditions to determine whether they anneal to each other. The first and second chains forming at least one portion of a double-stranded region can be completely complementary and at least partially complementary to each other.

[0176] As used in this article in connection with nucleotides, the term "debasement" refers to a portion lacking a base at the 1' position or having other chemical groups replacing the base, such as 3',3'-linked or 5',5'-linked deoxy-debasement ribose derivatives.

[0177] As used in this article, the nucleic acid-related term "alternation" refers to something happening one after another in a regular manner. In other words, alternation means that something happens in a sequential, repeated manner. For example, if one nucleotide is modified, the next consecutive nucleotide is not modified, and the subsequent consecutive nucleotides are modified, and so on.

[0178] As used herein, the terms “inhibition,” “downregulation,” or “reduction” in relation to gene expression mean the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits (e.g., mRNA), or the activity of one or more proteins or protein subunits reduced to levels observed in the absence of the nucleic acids of this disclosure; for example, expression may be reduced to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less of the levels observed in the absence of inhibitors.

[0179] As used herein, “protruding end” has its normal and conventional meaning in the art as a single-stranded portion of a nucleic acid that extends beyond the terminal nucleotides of the complementary strand in a double-stranded nucleic acid. The term “flat end” includes double-stranded nucleic acids, where both strands terminate at the same position, regardless of whether the terminal nucleotides are base-paired.

[0180] "Pharmaceutically acceptable carriers" are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-delaying agents, etc., compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or reagent is incompatible with the active compound, its use in the compositions of this disclosure should be considered.

[0181] The terms “subject,” “individual,” or “patient” are used interchangeably herein and generally refer to a vertebrate, such as a mammal. Mammals include, but are not limited to, rodents, farm animals, sport animals, pets, and primates; such as rats, rats, rabbits, apes, cattle, sheep, pigs, dogs, cats, horses, and humans. In one specific implementation, the mammal is a human.

[0182] "Administration" is defined herein as a means of delivering a drug or a composition containing such drug to a subject in a manner that causes the drug to come into contact with the subject's body (e.g., within the subject's body). Such administration can be performed via any route, including but not limited to oral, transdermal (e.g., through the vagina, rectum, or oral mucosa), injection (e.g., subcutaneous, intravenous, parenteral, intraperitoneal, or central nervous system access), or inhalation (e.g., oral or nasal). Administration can also include delivering a substance or composition to a portion of the surface of the subject's body, such as by topical application to the skin. Of course, pharmaceutical preparations are given in forms suitable for each route of administration.

[0183] The “treatment” of a disease includes: (1) disease prevention, i.e., preventing the development of clinical symptoms of the disease in patients who may be susceptible to the disease but have not yet experienced or shown symptoms of the disease; (2) disease suppression, i.e., stopping or reducing the development of the disease or its clinical symptoms; and / or (3) disease mitigation, i.e. causing the disappearance of the disease or its clinical symptoms. Patients or individuals may be susceptible to the disease due to the presence of gene mutations associated with it.

[0184] "Effective amount" or "therapeutic effective amount" is an amount sufficient to achieve a beneficial or desired outcome. An effective amount can be administered, applied, or dosed, either once or multiple times. This delivery depends on many variables, including the time period for which a single dose unit is used, the bioavailability of the therapeutic agent, the route of administration, etc. However, it should be understood that the specific dose level of the therapeutic agent of this disclosure for any particular subject depends on a variety of factors, including, for example, the activity of the specific compound used, the subject's age, weight, general health condition, sex, and diet, the time of administration, the rate of excretion, the combination of drugs, the severity of the specific condition being treated, and the form of administration. Therapeutic doses can generally be titrated to optimize safety and efficacy. Typically, dose-response relationships from in vitro and / or in vivo tests can initially provide useful guidance regarding the appropriate dose to be administered to a patient. Generally, one would expect to administer an amount of compound effective to achieve serum levels equivalent to concentrations found to be effective in vitro. The determination of these parameters is entirely within the scope of the art. These considerations, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks. Consistent with this definition, as used herein, the term "therapeutic effective dose" is an amount sufficient to treat (e.g., improve) one or more symptoms associated with the condition. The total daily dose may be administered as a single dose or in divided doses, and may fall outside the typical range given herein at the physician's discretion.

[0185] When used in conjunction with the nucleic acid-containing compounds and compositions of this disclosure, the term "delivery" generally refers to some active targeting of the nucleic acid to a target cell or tissue. Therefore, delivery of nucleic acids using the compounds and compositions of this disclosure generally results in target cells or tissues being exposed to a higher level of nucleic acid than would occur if the same nucleic acid were administered without the remainder of the complex (e.g., when administered as "naked" nucleic acid).

[0186] As used herein, the terms "pharmaceuticalally acceptable excipient" or "pharmaceuticalally acceptable carrier" encompass any standard pharmaceutical ingredient, such as that described in Remington's Pharmaceutical Sciences (20th edition, Mack Publishing Co. 2000). Such excipients include carriers (such as phosphate-buffered saline solutions, water, and emulsions, such as oil / water or water / oil emulsions), and various types of wetting agents. Pharmaceutical compositions may also include stabilizers, preservatives, adjuvants, fillers, binders, lubricants, etc.

[0187] As used herein, the term "alkyl" refers to a saturated straight-chain or branched radical consisting essentially of carbon atoms and a corresponding number of hydrogen atoms. The term "alkylene" has a corresponding meaning associated with divalent radicals. Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, etc. Other alkyl groups will be apparent to those skilled in the art in view of the benefits of this disclosure. The terms "(C1-C3)alkyl," "(C1-C6)alkyl," etc., have equivalent meanings of a saturated straight-chain or branched radical consisting essentially of 1 to 3 (or 1 to 6) carbon atoms and a corresponding number of hydrogen atoms. The definition of "alkyl" also applies to the context of other groups containing alkyl groups, such as "-O(C1-C3)alkyl." The term "haloalkyl" refers to an alkyl group substituted with one or more halogens. Exemplary haloalkyl groups include trifluoromethyl, trifluoroethyl, difluoroethyl, pentafluoroethyl, chloromethyl, etc. One or more carbon atoms in the skeleton of an alkyl group may be replaced (or bonded to) a heteroatom via a multiple bond (e.g., a double bond); for example, a carbon atom of an alkyl group may be bonded to oxygen via a double bond (i.e., replaced by an oxo group to provide a carbonyl functional group). The presence of such a substituent does not prevent the carbon skeleton of the radical from being considered an alkyl group. In embodiments, the alkyl group is linear. The alkyl groups of this disclosure may be replaced by one or more optional substituents as defined herein.

[0188] As used herein, the term "alkenyl" refers to an unsaturated straight-chain or branched radical consisting essentially of carbon atoms and a corresponding number of hydrogen atoms, containing at least one carbon-carbon double bond. The term "alkenyl" has a corresponding meaning associated with divalent radicals. Exemplary alkenyl groups include vinyl, propenyl, propenyl-2-alkenyl, isopropenyl, butenyl, 2-methyl-propenyl, and 2-methyl-propenyl-2-alkenyl. The terms "(C2-C6)alkenyl," etc., have equivalent meanings, namely, unsaturated straight-chain or branched radicals consisting essentially of 2 to 6 carbon atoms and a corresponding number of hydrogen atoms. The alkenyl groups of this disclosure may be substituted with one or more optional substituents as defined herein.

[0189] As used herein, the term "aryl" refers to an aromatic radical having at least six carbon atoms forming a ring (i.e., ring atoms). It should be understood that aryl groups can be monocyclic or polycyclic (e.g., fused). In the case of polycyclic aryl groups, additional rings are present, such as one or more additional rings, all of which contain at least three carbon atoms (i.e., ring atoms). Examples of aryl groups include phenyl and naphthyl. An aryl group may contain six to ten carbon atoms in the ring portion of the group, and it may be monocyclic or polycyclic (e.g., fused). In an embodiment, the aryl group is phenyl. The aryl groups of this disclosure may be substituted with one or more optional substituents as defined herein.

[0190] As used herein, the term "cycloalkyl" means a saturated radical having at least 3 to 9 carbon atoms forming a ring (i.e., ring atoms). Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. It should be understood that cycloalkyl groups can be monocyclic or polycyclic (e.g., fused, bridged, or spirocyclic). In the case of polycyclic cycloalkyl groups, additional rings are present, such as one or more additional rings, all of which contain 3 to 7 carbon atoms (i.e., ring atoms). Exemplary cycloalkyl groups having such additional rings include bicyclic [1.1.1]pentyl. One or more ring atoms of a cycloalkyl group may be substituted by a heteroatom through a double bond (i.e., bonded to a heteroatom) (e.g., a cycloalkyl group substituted with an oxo group). The presence of such a substituent does not prevent the carbon skeleton of the radical from being considered a cycloalkyl group. The cycloalkyl groups of this disclosure may be substituted by one or more optional substituents as defined herein.

[0191] As used herein, the term "heterocyclic alkyl" refers to a saturated radical having at least 3 to 10 ring-forming atoms (i.e., ring atoms), wherein at least 1 to 9 of the ring atoms are carbon, and the remaining at least 1 to 9 ring atoms (i.e., heterocyclic atoms) are independently selected from the group consisting of nitrogen, sulfur, and oxygen. The heterocyclic alkyl ring may have an oxosubstituent, typically adjacent to the heteroatom (e.g., 2-oxopyrrolyl), but the oxygen atom is not part of the ring and is not included in the number of ring atoms. The presence of such a substituent does not prevent the radical's ring (or rings) from being considered a heterocyclic alkyl group. Exemplary heterocyclic alkyl groups include tetrahydrofuranyl, piperidinyl, morpholinyl, and piperazineyl. In the case of polycyclic heterocyclic groups, additional rings are present, such as one or more additional rings, all of which contain 3 to 7 ring atoms selected from carbon, nitrogen, sulfur, and oxygen. The additional rings may be saturated, or partially or completely unsaturated (e.g., having aromatic properties). Polycyclic heterocyclic groups include fused, bridged, and spirocyclic systems. When a polycyclic heterocyclic alkyl group contains an unsaturated fused ring, the group is typically not bonded to the remainder of the molecule via that fused ring. The heterocyclic alkyl groups disclosed herein may be substituted with one or more optional substituents as defined herein.

[0192] The term "phosphate" is generally used herein to refer to a radical (or diradical) group comprising a central phosphorus atom bonded to four oxygen atoms (one via a double bond). The oxygen atom bonded to phosphorus via a single bond may represent an attachment point to the rest of the molecule, or may carry a hydrogen atom, or may be considered to carry a negative charge (e.g., in the case of a salt). The term "thiophosphate" is generally used herein to refer to phosphate ester analogs in which at least one oxygen atom is replaced by sulfur; such groups are also referred to herein as "thiophosphates".

[0193] "Optional substituents" are groups covalently attached to a portion of the carbon atom (generally via a portion of the carbon atom and typically replacing a hydrogen atom on said carbon atom). Optional substituents can be selected as groups that do not significantly alter the steric and / or electronic properties of the molecule. In embodiments, each optional substituent is independently selected from the group consisting of: halogens (e.g., -F, -Cl, -Br, or -I); -OH; -SH; -NH2; -NHMe; -NMe2; -(C1-C3)alkyl (e.g., -Me or -Et); and 3- or 4-membered cycloalkyl or heterocycloalkyl groups (e.g., cyclopropyl or epoxides), which may optionally be substituted by one or more halogens. A group defined as "optionally substituted" can be unsubstituted or substituted with one or more substituents (e.g., 1, 2, 3, 4, 5, 6, or more substituents). In embodiments, the substituted group has 1 to 4 substituents, for example, 1, 2, or 3 substituents. In embodiments, the substituted group has 1 or 2 substituents. In the implementation scheme, the substituted group has 3 substituents.

[0194] As used herein, the terms “halogenated” and “halogen” refer to fluorine, chlorine, bromine, or iodine. These terms are used interchangeably and can refer to a halogen radical group or the halogen atom itself. Given the context in which these terms are used in this disclosure, those skilled in the art will be able to readily identify them. In an embodiment, the halogen is fluorine.

[0195] The compounds disclosed herein are described in particular by structural formulas. It should be understood that these formulas typically show only one form of the compound (e.g., resonance form, tautomer form, etc.), and some compounds may exist in more than one such form. This will be apparent to those skilled in the art. This disclosure includes all possible tautomers of the compounds characterized by the structural formulas herein, including as a single tautomer or as any mixture of tautomers in any proportion. It should also be understood that some compounds of the invention may exist in one or more isomers (e.g., stereoisomers). This disclosure includes all possible stereoisomers, enantiomers, diastereomers, etc., of the compounds described above and below, as well as their cis and trans forms and conformational isomers. Purification and separation of isomers can be accomplished by the methods described below and by techniques known in the art. For example, optical isomers of a compound can be obtained by resolving racemic mixtures of its diastereomeric salts (e.g., using an optically active acid or base, or by forming covalent diastereomers). Different methods for separating optical isomers include the use of chiral chromatography (e.g., HPLC columns using chiral phases), with or without conventional derivatization. Enzymatic separation with or without derivatization can also be useful, and the optically active compounds of this disclosure can also be obtained by chiral synthesis utilizing optically active starting materials. This disclosure includes all possible stereoisomers of the compounds described herein, as a single stereoisomer, or as any mixture of said stereoisomers (e.g., (R)- or (S)- isomers) in any proportion.

[0196] The compounds disclosed herein may exist in the form of free acids or bases, or as addition salts with suitable acids or bases. Methods for forming salts are described below and are also known in the art (see, for example, Berge et al., JPharm Sci. (1977) 66:1-19). As used herein, when used in conjunction with salts, the term "pharmaceutically acceptable" refers to a salt of the currently disclosed compounds that can be administered without any resulting significant undesirable biological effects or any resulting harmful interactions with any other component of a pharmaceutical composition that may contain it.

[0197] The description of a list of chemical groups in any definition of a variable herein includes the definition of that variable as any single group or combination of the listed groups. The description of embodiments of a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or part thereof.

[0198] The compounds, compositions, and methods provided herein may be combined with one or more of any other compounds, compositions, and methods provided herein.

[0199] This article uses the following abbreviations and empirical formulas:

[0200]

[0201]

[0202]

[0203] compound

[0204] This disclosure provides compounds containing monosaccharides, which are particularly useful for targeting nucleic acid cargo portions to specific locations in the body (e.g., cell and / or tissue types). Figure 1 As schematically illustrated, the compound has several "arms" carrying a targeting ligand and a cargo portion. These are then typically linked together by a "separator" portion (also called a "branching unit") having the appropriate functionality to connect the arms of the compound together. This disclosure particularly relates to compounds in which the separator is derived from shikimic acid or chemically related to it, wherein the ligand-containing arms can be attached to a hydroxyl group on a cyclohexyl ring. The use of shikimic acid and its analogues in the preparation of the branching unit provides a novel way of coupling the targeting and cargo portions, which can have advantages over known methods, including, for example, the use of simple natural precursors. The compounds of the present invention exhibit useful properties such as improved targeting, increased activity, and / or improved in vivo pharmacokinetic properties.

[0205] Therefore, from a first aspect, this disclosure provides a compound or a pharmaceutically acceptable salt thereof having the structure of formula (I):

[0206]

[0207] in:

[0208] X is selected from covalent bonds, -CH2-, and -C(O)-;

[0209] Y is either -O or -NR-.

[0210] Where R is selected from -H and -(C1-C6) alkyl;

[0211] It represents a carbon-carbon single or double bond, provided that X is a covalent bond. It represents a carbon-carbon single bond; and

[0212] G 1 To G 4 Each independently represents either the -[spacer]-[ligand] part or the -[connector]-[cargo] part, where each ligand is independently a monosaccharide and each cargo is independently a nucleic acid, provided that G... 1 To G 4At least one of them represents the -[spacer]-[ligand] part and G 1 To G 4 At least one of them represents the [chain]-[joint]-[cargo] part.

[0213] In one implementation, X is -C(O)-. In other implementations, X is a covalent bond.

[0214] In the embodiments, Y is -NR-, for example, where R is selected from -H and -(C1-C3)alkyl. In the embodiments, R is -H. In the embodiments, R is -CH3. In the embodiments, Y is -NH-. In the embodiments, Y is -N(CH3)-.

[0215] In one embodiment, X is -C(O)- and Y is -NR-, for example, where R is selected from -H and -(C1-C3)alkyl. In another embodiment, X is -C(O)- and Y is -NH-. In yet another embodiment, X is -C(O)- and Y is -N(CH3)-. In still another embodiment, X is covalently bonded and Y is -NR-, for example, where R is selected from -H and -(C1-C3)alkyl.

[0216] In the implementation plan, This indicates a single bond. In other implementations, It represents a double bond.

[0217] In the implementation scheme, the compound is a compound having the structure of formula (Ia):

[0218]

[0219] Or a pharmaceutically acceptable salt thereof, wherein X, Y, G 1 G 2 G 3 and G 4 As defined in this article.

[0220] In the implementation scheme, the compound is a compound having the structure of formula (Ib):

[0221]

[0222] Or a pharmaceutically acceptable salt thereof, wherein X, Y, G 1 G 2 G 3 and G 4 As defined in this article, and Indicates a single key.

[0223] In other embodiments, the compound is a compound having the structure of formula (Ic):

[0224]

[0225] Or a pharmaceutically acceptable salt thereof, wherein X, Y, G 1 G 2 G 3 and G 4 As defined in this article, and Indicates a single key.

[0226] In other embodiments, the compound is a compound having the structure of formula (Id):

[0227]

[0228] Or a pharmaceutically acceptable salt thereof, wherein X, Y, G 1 G 2 G 3 and G 4 As defined in this article, and It represents a double bond.

[0229] Each "ligand" present in the compounds of this disclosure is a monosaccharide. The monosaccharide may be selected to have affinity for the hepatic desialylate glycoprotein receptor (ASGPR). In embodiments, the target ASGPR is located on the surface of mammalian hepatocytes. In embodiments, the ligand is GalNAc. In embodiments, the compounds of this disclosure have 2, 3, or 4 (e.g., 2 or 3) ligands, all of which are GalNAc.

[0230] The “linking” and “connector” groups work together to link the cargo portion (or portions) to the remainder of the molecule, i.e., to the separator. Suitable linking and connecting groups will be apparent to those skilled in the art based on the present description and its embodiments. In embodiments, the [linking]-[connector] portion is obtained by reacting a linking precursor (which may be bonded to the remainder of the molecule, i.e., to the spacer / ligand construct) with a connecting precursor (which may be bonded to the cargo) to link the two together. Suitable methods for reacting the linking and connecting precursors are described in the following examples and are known to those skilled in the art. Examples of such methods include “click” chemistry reactions, such as copper-catalyzed cycloaddition between azides and alkynes (see, for example, Fantoni et al., Chem. Rev. (2021) 121(12):7122–7154). In other embodiments, a [tether]-[connector] moiety is introduced into the complex without attaching the cargo, and the cargo is subsequently coupled to the connector. The complex can be conveniently coupled to the cargo in a final step of solid-phase synthesis, for example, where the [tether]-[connector] moiety initially introduces a phosphate group at its distal end, which can react with the cargo nucleic acid on the solid phase to give the final compound (see, for example, Beaucage, Curr Opin Drug Discov Dev (2008) 11(2):203-216). Other examples of tethers and / or connectors that may be used according to this disclosure are available in, for example, International Patent Publications WO 2014 / 179620, WO 2015 / 177668, WO 2009 / 073809, WO The contents of each of these patents, found in WO 2012 / 083046, WO 2017 / 156012, WO 2016 / 100401, WO 2017 / 174657 and WO 2019 / 092280, are incorporated herein by reference in their entirety.

[0231] In embodiments, the linker and connector together represent a straight chain portion comprising 8 to 30 atoms that connects the cargo to the remainder of the molecule. In embodiments, the linker and connector together represent a straight chain with optional substitutions of 8 to 30 atoms selected from C, N, O, S, and P, such as chains of 9 to 24 atoms, 10 to 20 atoms, or 12 to 18 atoms. In embodiments, the linker and connector together comprise one or more groups selected from amides (e.g., obtained by reacting carboxylic acids or acid derivatives with amines), esters (e.g., obtained by reacting carboxylic acids or acid derivatives with alcohols (such as aliphatic or aromatic alcohols)), or 1,2,3-triazoles (e.g., obtained by reacting azides with alkynes). In embodiments, the linker and connector together comprise groups represented by (e.g., composed of):

[0232] -(CH2) q-D-(CH2) s -

[0233] Where q is an integer from 1 to 12 (e.g., 2 to 8, such as 6); D is selected from (i) a direct bond, (ii) -C(O)NH-, (iii) a group containing 1,2,3-triazole, and (iv) -NHC(O)-(CH2). t -C(O)NH-, where t is an integer from 0 to 6 (e.g., 1 to 4, such as 2); and s is an integer from 0 to 12 (e.g., 2 to 8, such as 6). This group can optionally be substituted, and This indicates the attachment point to the cargo. It should be understood that the [tether]-[connector] groups shown above are typically attached to the cargo via phosphate groups. Therefore, in embodiments, the tether and connector together comprise groups as shown above, which are used in... The marked position also contains a phosphate group (e.g., -OP(=O)(OH)O-). In the embodiments, q is an integer from 1 to 12 (e.g., 2 to 8, such as 6); D is selected from (i) a direct bond, (ii) -C(O)NH- and (iii) a group containing 1,2,3-triazole; and s is an integer from 0 to 12 (e.g., 2 to 8, such as 6).

[0234] In the implementation scheme, D is a direct bond. Therefore, the tandem and connector together may contain -(CH2). q (CH2) s - The group represents (e.g., composed of), where q is an integer from 1 to 12 (e.g., 6), and s is an integer from 0 to 12 (e.g., 6). In an embodiment, s is 0, in which case the chain and connector together comprise -(CH2). q - indicates a group (e.g., composed of), where q is an integer from 1 to 12 (e.g., 6).

[0235] In other embodiments, D is -C(O)NH-. Therefore, the chain and connector together can contain -(CH2). q -C(O)NH-(CH2) s - The group (e.g., composed of) represents a group, where q is an integer from 1 to 12 (e.g., 6), and s is an integer from 0 to 12 (e.g., 6). In the embodiment, q is 6 and s is 6.

[0236] In another embodiment, D is a group containing 1,2,3-triazole, such as a group obtained by reacting BCN with an azide. Therefore, in this embodiment, D contains a group... or (For example, composed of), where † indicates the relationship with -(CH2). s - The attachment point. In the implementation, the tether and connector together comprise a... The group to be represented (e.g., composed of), where q and s are as defined herein (e.g., where q is 6 and s is 6).

[0237] In another implementation, D is -NHC(O)-(CH2). t -C(O)NH-, and t is an integer from 0 to 6 (e.g., 1 to 4, such as 2). In the embodiment, the tether and connector together comprise -(CH2) q -NHC(O)-(CH2) t -C(O)NH-(CH2) s - The group to be represented (e.g., composed of), where q and s are as defined herein (e.g., where q is 6 and s is 6).

[0238] In other embodiments, the chain and connector together comprise (e.g., constitute) groups represented by the following:

[0239] -(CH2) u -C(O)NH-(CH2) v -E-

[0240] Where u is an integer from 0 to 11 (e.g., 1 to 7, such as 5); v is an integer from 1 to 10 (e.g., 2 to 5, such as 3); and E is a pyrrolidine-containing group. This group may optionally be substituted, and This indicates the attachment point to the cargo. It should be understood that the [tether]-[connector] groups shown above are typically attached to the cargo via phosphate groups. Therefore, in embodiments, the tether and connector together comprise groups as shown above, which are used in... The marked position also contains a phosphate group (e.g., -OP(=O)(OH)O-).

[0241] In the implementation scheme, E includes... The indicated group (e.g., composed of), where † indicates the attachment point with the cargo. In an embodiment, the tether and connector together comprise a group composed of... The group to be represented (e.g., composed of), where u and v are as defined herein (e.g., where u is 5 and v is 3).

[0242] As will be understood, the [chain-connector] portion can conceptually be "separated" in several ways to obtain separate chain and connector components. In an embodiment, the [chain-connector] portion is generated by reacting a cargo-containing portion (which includes a connector or a portion thereof) with a ligand-containing precursor (which includes a chain or a portion thereof). Thus, in an embodiment, the chain is formed by the group -(CH2). q -or-(CH2) q -C(O)- is used, where q is as defined herein. Similarly, in the embodiment, the connector is formed by the group -D-(CH2). s - Or -NH-(CH2) s - This indicates that D and s are as defined herein. In the implementation, the kinetic chain is -(CH2). q - where q is 6, and the connector is -D-(CH2). s - Where D is a direct bond and s is 0. In other embodiments, the chain is -(CH2). q -C(O)-, where q is 6, and the connector is -NH-(CH2). s - , where s is 6. In other embodiments, the ligand is -(CH2). q -NH-, where q is 6, and the connector is -C(O)-(CH2). t -C(O)NH-(CH2) s - Where t is 2 and s is 6. In other embodiments, the chain is -(CH2). q -NHC(O)-(CH2) t -C(O)-, where q is 6 and t is 2, and the connector is -NH-(CH2). s - Where s is 6. In another implementation, the kinetic chain is -(CH2). q - where q is 6, and the connector is -D-(CH2). s - Where D is a functional group And s is 6. In a further implementation, the tandem is -(CH2). u -C(O)- (e.g., where u is 5), and the connector is -NH-(CH2). v -E (For example, where v is 3 and E is a pyrrolidine-containing group as defined herein).

[0243] The "cargo" group of the compounds disclosed herein is a nucleic acid. These can be single-stranded or double-stranded. In one embodiment, the cargo is an oligonucleotide. In another embodiment, the cargo is selected from antisense oligonucleotides (ASO), immunostimulatory oligonucleotides, decoy oligonucleotides, splice-modifying oligonucleotides, splice-converting oligonucleotides, triplet-forming oligonucleotides, siRNA, saRNA, microRNA, microRNA mimics, antimiR, double-stranded RNA, single-stranded RNA, ribozymes, aptamers, mirror aptamers, CRISPR oligonucleotides, and G-quadruplexes. In one embodiment, the cargo is an antisense oligonucleotide (ASO). In another embodiment, the cargo is siRNA.

[0244] A "spacer group" comprises a chain of atoms (typically carbon atoms) with one or more heteroatoms (e.g., independently selected from N, O, S, and P) optionally inserted to connect the ligand to the separator portion (i.e., the shikimic acid-derived core). In embodiments, the spacer group comprises a chain of 2-20 atoms selected from C, N, O, S, and P (e.g., a chain of 7-14 atoms). Exemplary spacer groups include linear alkylene groups (which may optionally be interrupted by one or more amide and / or phosphate groups) and polyethylene glycol. Based on the present description and its embodiments, suitable spacer groups will be apparent to those skilled in the art. Other examples of spacer bases used in this disclosure can be found, for example, in International Patent Publications WO 2014 / 179620, WO 2015 / 177668, WO2009 / 073809, WO 2012 / 083046, WO 2017 / 156012, WO 2016 / 100401, WO 2017 / 174657 and WO2019 / 092280, the contents of each of which are incorporated herein by reference in their entirety.

[0245] In the implementation, the spacer bases (e.g., each spacer base) have the formula... -Z-NH-C(O)-(CH2) m -†, where: Z independently represents a portion of a straight chain containing 1 to 16 atoms (e.g., selected from C, N, O, S, and P); and m independently selects an integer from 1 to 6 in each case, where † indicates the attachment site between the spacer group and the ligand, and † indicates the attachment site with the separator portion (i.e., the shikimic acid-derived core). In the embodiments, This indicates the attachment point to the oxygen atom of the ligand (e.g., the GalNac oxygen atom).

[0246] In the implementation scheme, Z is selected independently in each case. -(C1-C 16 )alkylene-,

[0247] -(C2-C 16 ) imidene-, -(C1-C 12 )alkylene-C(O)-, -(C2-C 12 ) imidene-C(O)-,

[0248] -(C1-C8)alkylene-C(O)NR'-(C1-C6)alkylene-、

[0249] -(C2-C8)-alkenyl-C(O)NR'-(C1-C6)alkyl-,

[0250] -(C1-C8)alkylene-C(O)NR'-(C2-C6)alkenyl-and

[0251] -(C2-C8)imenyl-C(O)NR'-(C2-C6)imenyl-(wherein) In each case, R' represents the attachment point with the GalNac oxygen atom, where R' is independently selected from -H and -(C1-C6) alkyl groups in each case. In the embodiments, Z is independently selected from...

[0252] -(C1-C 12 )alkylene- and -(C1-C6)alkylene-C(O)NR'-(C1-C4)alkylene-, wherein R' is independently selected from -H and -(C1-C6)alkyl in each case. In the embodiments, each Z is independently selected from... -(C1-C 12 )alkylene-, such as -(C1-C 10 )alkylene-,

[0253] -(C2-C8)alkylene- or -(C4-C7)alkylene-, for example, where Z is n-hexene. In other embodiments, each Z is independently selected from...

[0254] -(C1-C6)alkylene-C(O)NR'-(C1-C4)alkylene- (where R' is independently selected from -H and -(C1-C6)alkyl in each case), such as -(C2-C5)alkylene-C(O)NR'-(C2-C4)alkylene-, for example, where Z is -(C4)alkylene-C(O)NH-(C3)alkylene-. In the embodiments, each Z is the same.

[0255] In each implementation, m is independently selected from an integer from 1 to 5. In each implementation, m is independently selected from 1, 2, 3, and 4. In each implementation, m is independently selected from 1, 2, and 3. In each implementation, each m is the same. In each implementation, m is 1. In other implementations, m is 2. In other implementations, m is 3.

[0256] In the implementation, m is independently selected from integers from 1 to 4, for example, 1 to 3. In the implementation, m is independently 1 or 2. In the implementation, m is 2. In the implementation, Z is positive hexadecimal and m is 2.

[0257] In the implementation plan, G 1 To G 3 Independently representing the -[spacer]-[ligand] part (e.g., as defined herein), and G 4 The term represents the [chain]-[joint]-[cargo] portion (e.g., as described herein). From this perspective, this disclosure provides a compound or a pharmaceutically acceptable salt thereof having the structure of formula (II):

[0258]

[0259] in:

[0260] X is selected from covalent bonds, -CH2-, and -C(O)-;

[0261] Y is either -O or -NR-.

[0262] Where R is selected from -H and -(C1-C6) alkyl;

[0263] It represents a carbon-carbon single or double bond, provided that X is a covalent bond. Indicates a carbon-carbon single bond;

[0264] In each case, the spacer group independently represents a straight-chain portion containing 6 to 20 atoms (e.g., selected from C, N, O, S, and P) that attaches the ligand to the rest of the molecule;

[0265] The ligands are monosaccharides independently in each case (e.g., GalNAc).

[0266] The term "joint" and "chain" together refer to the part that attaches cargo to the rest of the molecule, such as a straight chain containing 8 to 30 atoms; and

[0267] The goods are nucleic acid.

[0268] In one implementation, X is -C(O)-. In other implementations, X is a covalent bond.

[0269] In the embodiments, Y is -NR-, for example, where R is selected from -H and -(C1-C3)alkyl. In the embodiments, R is -H. In the embodiments, R is -CH3. In the embodiments, Y is -NH-. In the embodiments, Y is -N(CH3)-.

[0270] In one embodiment, X is -C(O)- and Y is -NR-, for example, where R is selected from -H and -(C1-C3)alkyl. In another embodiment, X is -C(O)- and Y is -NH-. In yet another embodiment, X is -C(O)- and Y is -N(CH3)-. In still another embodiment, X is covalently bonded and Y is -NR-, for example, where R is selected from -H and -(C1-C3)alkyl.

[0271] In the implementation plan, This indicates a single bond. In other implementations, It represents a double bond.

[0272] In the implementation scheme, the compound is a compound having the structure of formula (IIa):

[0273]

[0274] Or a pharmaceutically acceptable salt thereof, wherein X, Y, ligand, spacer, chain linker and cargo are as defined herein.

[0275] In the implementation scheme, the compound is a compound having the structure of formula (IIb):

[0276]

[0277] Or a pharmaceutically acceptable salt thereof, wherein X, Y, ligand, spacer, linker, and cargo are as defined herein, and Indicates a single key.

[0278] In other embodiments, the compound is a compound having the structure of formula (IIc):

[0279]

[0280] Or a pharmaceutically acceptable salt thereof, wherein X, Y, ligand, spacer, linker, and cargo are as defined herein, and Indicates a single key.

[0281] In other embodiments, the compound is a compound having the structure of formula (IId):

[0282]

[0283] Or a pharmaceutically acceptable salt thereof, wherein X, Y, ligand, spacer, linker, and cargo are as defined herein, and It represents a double bond.

[0284] In specific embodiments, the ligand in each case is N-acetylglucosamine (“GalNAc”), which can facilitate or promote the binding of the compound to ASGPR. This can target the delivery of cargo molecules to cells and tissues expressing such receptors. From this perspective, this disclosure provides a compound or a pharmaceutically acceptable salt thereof having the structure of formula (III):

[0285]

[0286] Wherein X, Y, spacer base, chain, joint, and cargo are as defined herein, and It represents a carbon-carbon single or double bond, provided that X is a covalent bond. It represents a carbon-carbon single bond.

[0287] In the implementation scheme: X is -C(O)-; Y is -O or -NR-, where R is selected from -H and -(C 1-6 )alkyl; The term "single bond" indicates a single bond; the spacer group, in each case independently, represents a portion of a straight chain containing 6 to 20 atoms (e.g., selected from C, N, O, S, and P) that attaches the ligand to the rest of the molecule; the linker and tie chain together represent a portion that attaches the cargo to the rest of the molecule, such as a straight chain containing 8 to 30 atoms; and the cargo is a nucleic acid.

[0288] In the implementation scheme, the compound is a compound having the structure of formula (IIIa):

[0289]

[0290] X, Y, spacer base, tether, connector, and cargo are as defined herein. In the implementation scheme, Indicates a single key.

[0291] In the implementation scheme, the compound is a compound having the structure of formula (IIIb):

[0292]

[0293] Wherein X, Y, spacer base, chain, joint, and cargo are as defined herein, and Indicates a single key.

[0294] In other embodiments, the compound is a compound having the structure of formula (IIIc):

[0295]

[0296] Wherein X, Y, spacer base, chain, joint, and cargo are as defined herein, and Indicates a single key.

[0297] In other embodiments, the compound is a compound having the structure of formula (IIId):

[0298]

[0299] Wherein X, Y, spacer base, chain, joint, and cargo are as defined herein, and It represents a double bond.

[0300] In a specific embodiment, the cargo-containing arm is attached to the separator via an amide, and the spacer group comprises an amide. From this perspective, the present disclosure provides a compound or a pharmaceutically acceptable salt thereof having the structure of formula (IV):

[0301]

[0302] in:

[0303] Z independently represents, in each case, a portion of a straight chain containing 1 to 16 atoms (e.g., selected from C, N, O, S, and P) that attaches the GalNAc ligand to the rest of the molecule;

[0304] m is independently selected from an integer from 1 to 6 in each case;

[0305] R is selected from -H and -(C1-C6) alkyl; and

[0306] Chains, joints, and cargo are as defined herein.

[0307] In the implementation scheme, Z is selected independently in each case. -(C1-C 16 )alkylene-,

[0308] -(C2-C 16 ) imidene-, -(C1-C 12 )alkylene-C(O)-, -(C2-C 12 ) imidene-C(O)-,

[0309] -(C1-C8)alkylene-C(O)NR'-(C1-C6)alkylene-、

[0310] -(C2-C8)-alkenyl-C(O)NR'-(C1-C6)alkyl-,

[0311] -(C1-C8)alkylene-C(O)NR'-(C2-C6)alkylene- and -(C2-C8)alkylene-C(O)NR'-(C2-C6)alkylene- (wherein) In each case, R' represents the attachment point with the GalNac oxygen atom, where R' is independently selected from -H and -(C1-C6) alkyl groups in each case. In the embodiments, Z is independently selected from... -(C1-C 16 )alkylene-,

[0312] -(C2-C 16 ) imidene-, -(C1-C 12 )alkylene-C(O)-, -(C2-C 12 ) imidene-C(O)-,

[0313] -(C1-C8)alkylene-C(O)NR'-(C1-C6)alkylene-、

[0314] -(C2-C8)-alkenyl-C(O)NR'-(C1-C6)alkyl-,

[0315] -(C1-C8)alkylene-C(O)NR'-(C2-C6)alkenyl-and

[0316] -(C2-C8)imenyl-C(O)NR'-(C2-C6)imenyl-(wherein) In each case, R' represents the attachment point with the GalNac oxygen atom, where R' is independently selected from -H and -(C1-C6) alkyl groups in each case. In the embodiments, Z is independently selected from...

[0317] -(C1-C 12 )alkylene- and -(C1-C6)alkylene-C(O)NR'-(C1-C4)alkylene-, wherein R' is independently selected from -H and -(C1-C6)alkyl in each case. In the embodiments, each Z is independently selected from... -(C1-C 12 )alkylene-, such as -(C1-C 10 )alkylene-,

[0318] -(C2-C8)alkylene- or -(C4-C7)alkylene-, for example, where Z is n-hexene. In other embodiments, each Z is independently selected from...

[0319] -(C1-C6)alkylene-C(O)NR'-(C1-C4)alkylene- (where R' is independently selected from -H and -(C1-C6)alkyl in each case), such as -(C2-C5)alkylene-C(O)NR'-(C2-C4)alkylene-, for example, where Z is -(C4)alkylene-C(O)NH-(C3)alkylene-. In the embodiments, each Z is the same.

[0320] In each implementation, m is independently selected from an integer from 1 to 5. In each implementation, m is independently selected from 1, 2, 3, and 4. In each implementation, m is independently selected from 1, 2, and 3. In each implementation, each m is the same. In each implementation, m is 1. In other implementations, m is 2. In other implementations, m is 3.

[0321] In the implementation scheme, R is -H. In the implementation scheme, R is -CH3.

[0322] In the implementation scheme, the compound is a compound having the structure of formula (IVa):

[0323]

[0324] Z, m, R, chain, connector, and cargo are as defined herein.

[0325] In the implementation scheme, the compound is a compound having the structure of formula (IVb):

[0326]

[0327] Z, m, R, chain, connector, and cargo are as defined herein.

[0328] In other embodiments, the compound is a compound having the structure of formula (IVc):

[0329]

[0330] Z, m, R, chain, connector, and cargo are as defined herein. On the other hand, the cargo-containing arm is attached to the separator via an amide, and the spacer group comprises a reverse amide. From this perspective, this disclosure provides a compound or a pharmaceutically acceptable salt thereof having the structure of formula (V):

[0331]

[0332] in:

[0333] A independently represents, in each case, a portion of a straight chain containing 1 to 16 atoms (e.g., selected from C, N, O, S, and P) that attaches the GalNAc ligand to the rest of the molecule;

[0334] n is an integer independently selected from 1 to 6 in each case;

[0335] R is selected from -H and -(C1-C6) alkyl; and

[0336] Chains, joints, and cargo are as defined herein.

[0337] In the implementation scheme, A is selected independently in each case. -(C1-C 16 )alkylene- and

[0338] -(C2-C 16 ) imidene- (wherein) In each case, A represents the attachment point with the GalNac oxygen atom. In the implementation, A is independently selected in each case.

[0339] -(C1-C 12 )alkylene-, such as -(C2-C 10 )alkylene- or -(C4-C8)alkylene-, for example, where A is n-pentane. In the embodiments, each A is the same.

[0340] In each implementation, n is independently selected from an integer from 1 to 5. In each implementation, n is independently selected from 1, 2, 3, and 4. In each implementation, n is independently selected from 2, 3, and 4. In each implementation, each n is the same. In each implementation, n is 3.

[0341] In the implementation scheme, R is -H. In the implementation scheme, R is -CH3.

[0342] In the implementation scheme, the compound is a compound having the structure of formula (Va):

[0343]

[0344] A, n, R, chain, connector, and cargo are as defined herein.

[0345] In the implementation scheme, the compound is a compound having the structure of formula (Vb):

[0346]

[0347] A, n, R, chain, connector, and cargo are as defined herein.

[0348] In other embodiments, the compound is a compound having the structure of formula (Vc):

[0349]

[0350] A, n, R, chain, connector, and cargo are as defined herein.

[0351] In a specific embodiment, the shikimic acid core is not fully saturated. From this perspective, this disclosure provides a compound or a pharmaceutically acceptable salt thereof having the structure of formula (IX):

[0352]

[0353] in:

[0354] Z independently represents, in each case, a portion of a straight chain containing 1 to 16 atoms (e.g., selected from C, N, O, S, and P) that attaches the GalNAc ligand to the rest of the molecule;

[0355] m is independently selected from an integer from 1 to 6 in each case;

[0356] R is selected from -H and -(C1-C6) alkyl; and

[0357] Chains, joints, and cargo are as defined herein.

[0358] In the implementation scheme, each Z is independently selected. -(C1-C 12 )alkylene-, such as

[0359] -(C2-C8)alkylene- or -(C4-C7)alkylene-, for example, where Z is n-hexene (where...) (In each case, it represents the attachment point with the GalNAc oxygen atom). In the implementation, each Z is the same.

[0360] In each implementation, m is independently selected from integers 1, 2, and 3. In each implementation, each m is the same. In one implementation, m is 1. In other implementations, m is 2. In other implementations, m is 3.

[0361] In the implementation scheme, R is -H. In the implementation scheme, R is -CH3.

[0362] In the implementation scheme, the compound is a compound having the structure of formula (IXa):

[0363]

[0364] Z, m, R, chain, connector, and cargo are as defined herein.

[0365] In specific embodiments, the spacer group and the linker-chain component comprise alkylene chains and amide groups, such as those defined herein. From this perspective, the present disclosure provides a compound or a pharmaceutically acceptable salt thereof having the structure of formula (XI):

[0366]

[0367] Z, m, R, q, D, s, and [goods] are as defined herein.

[0368] In the implementation plan:

[0369] Z is selected independently in each case. -(C1-C 12 )alkylene- and

[0370] -(C1-C6)alkylene-C(O)NR'-(C1-C4)alkylene-, wherein R' is independently selected from -H and -(C1-C6)alkyl in each case (where R' is selected from -H and -(C1-C6)alkyl). (In each case, it represents the attachment point with the GalNac oxygen atom).

[0371] m is independently selected from an integer from 1 to 6 in each case;

[0372] R is selected from -H and -(C1-C6) alkyl;

[0373] q is an integer from 1 to 12;

[0374] D is selected from (i) -C(O)NH- and (ii) -NHC(O)-(CH2). t-C(O)NH- (where t is an integer from 1 to 6);

[0375] s is an integer from 0 to 12; and

[0376] Goods as defined herein (e.g., ASO or siRNA).

[0377] In the implementation scheme, each Z is independently selected. -(C1-C 12 Alkylene-. In an embodiment, each Z is n-hexylene. In an embodiment, each Z is n-hexylene and each m is 2. In other embodiments, each Z is independently selected from...

[0378] -(C1-C6)alkylene-C(O)NR'-(C1-C4)alkylene- (wherein R' is independently selected from -H and -(C1-C6)alkyl in each case). In the embodiments, each Z is -(CH2)4-C(O)NH-(CH2)3-. In the implementation scheme, each Z is -(CH2)4-C(O)NH-(CH2)3- and each m is 2.

[0379] In the implementation scheme, R is hydrogen.

[0380] In one implementation, D is -C(O)NH-, q is an integer from 1 to 8, and s is an integer from 1 to 8. In another implementation, D is -C(O)NH-, q is 5, and s is 6. In other implementations, D

[0381] It is -NHC(O)-(CH2) t -C(O)NH- (where t is an integer from 1 to 6), q is an integer from 1 to 8, and s is an integer from 1 to 8. In the implementation, D is -NHC(O)-(CH2). t -C(O)NH- (where t is 4), q is 4, and s is 6.

[0382] In the implementation scheme, the compound is a compound having the structure of formula (XIa):

[0383]

[0384] Z, m, R, q, D, s, and [goods] are as defined herein.

[0385] In specific embodiments, the spacer group and the linker-chain component comprise specific alkylene chains and amide groups. From this perspective, the present disclosure provides a compound or a pharmaceutically acceptable salt thereof having the structure of formula (XIII):

[0386]

[0387] Wherein: each p is independently selected from 0 and 1; It represents a carbon-carbon single or double bond; and goods are as defined herein (e.g., ASO or siRNA).

[0388] In the implementation, each p is 1. In the implementation, each p is 0. In the implementation, This represents a carbon-carbon single bond. In the implementation scheme, This represents a carbon-carbon double bond. In the implementation, each p is 1, and This represents a carbon-carbon double bond. In other embodiments, each p is 1, and This represents a carbon-carbon single bond. In other embodiments, each p is 0, and This represents a carbon-carbon double bond. In other embodiments, each p is 0, and This indicates a carbon-carbon single bond. In the embodiment, the carbon atom attached to the carbonyl group on the shikimic acid core has an (S) configuration.

[0389] In the embodiments, the compound is a compound shown in any of the examples described below, such as a compound that can be obtained (or obtained by) the method described in Example 1. From this perspective, this disclosure provides a compound selected from:

[0390]

[0391] And its pharmaceutically acceptable salts, wherein “nucleic acid” refers to cargo nucleic acid as defined herein.

[0392] In the implementation scheme, the compound is selected from:

[0393]

[0394] And its pharmaceutically acceptable salts, wherein “nucleic acid” refers to cargo nucleic acid as defined herein.

[0395] In the implementation scheme, the compound is selected from:

[0396]

[0397] And its pharmaceutically acceptable salts, wherein “nucleic acid” refers to cargo nucleic acid as defined herein.

[0398] In one embodiment, the nucleic acid is an ASO (e.g., a MALAT1 ASO having a sequence containing SEQ ID NO: 1). In other embodiments, the nucleic acid is siRNA (e.g., an ANGPTL3 siRNA having a sequence containing SEQ ID NO: 2 and / or SEQ ID NO: 3). In another embodiment, the siRNA is a PPIB siRNA (e.g., having a sequence containing SEQ ID NO: 4, 5 and / or 6 (e.g., containing SEQ ID NO: 4 and 6, or containing SEQ ID NO: 5 and 6)).

[0399] In the implementation scheme, the compound is selected from:

[0400]

[0401] And its pharmaceutically acceptable salts, wherein “nucleic acid” means cargo nucleic acid as defined herein. In embodiments, the nucleic acid is an ASO (e.g., a MALAT1 ASO having a sequence comprising SEQ ID NO: 1). In other embodiments, the nucleic acid is siRNA. In embodiments, the siRNA is ANGPTL3 siRNA (e.g., having a sequence comprising SEQ ID NO: 2 and / or SEQ ID NO: 3). In other embodiments, the siRNA is PPIB siRNA (e.g., having a sequence comprising SEQ ID NO: 4, 5 and / or 6 (e.g., comprising SEQ ID NO: 4 and 6, or comprising SEQ ID NO: 5 and 6)).

[0402] In one embodiment, the compound is compound 1 as defined below or a pharmaceutically acceptable salt thereof. In another embodiment, the compound is compound 2a as defined below or a pharmaceutically acceptable salt thereof.

[0403] In the embodiments, the compound is selected from compounds 1, 2a, 2b, 2c, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15a, 15b, 16a, 16b, 17, 18, 19, 20, and 21 as defined below, and their pharmaceutically acceptable salts.

[0404] In embodiments, the compounds of this disclosure are characterized based on their binding affinity to ASGPR (e.g., human ASGPR) (e.g., as measured by the FRET assay as described in Example 2 below). In embodiments, the compounds have an IC50 of less than about 10 nM. 50 Values, such as ICs less than approximately 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, or 2nM. 50 Value. In embodiments, the compound has an IC50 value of less than about 2.0 nM (e.g., less than about 1.5 nM, 1.0 nM, or 0.5 nM). 50 value.

[0405] In embodiments, the compounds of this disclosure are characterized according to their binding kinetics to ASGPR (e.g., human ASGPR) (e.g., as measured by an SPR assay as described in Example 2 below). In embodiments, the compounds have a Kd value of less than about 8 nM, for example, less than about 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In embodiments, the compounds have a Kd value of less than about 1.5 nM (e.g., less than about 1.0 nM or 0.5 nM). In embodiments, the compounds have a Kd value of less than about 20 nM, for example, less than about 16 nM, 12 nM, or 10 nM.

[0406] In embodiments, the compounds of this disclosure are characterized based on their binding affinity to natural ASGPR (e.g., mouse ASGPR) (e.g., as measured by fluorescence polarization assays as described in Example 2 below). In embodiments, the compounds have an IC50 of less than about 10 nM. 50 Values, such as ICs less than approximately 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, or 2nM. 50 Value. In embodiments, the compound has an IC50 value of less than about 2.0 nM (e.g., less than about 1.5 nM or 1.0 nM). 50 Value. In the embodiments, the compound has an IC50 value of less than about 0.5 nM. 50 value.

[0407] In the embodiments, the compounds of this disclosure are characterized based on their activity in gene expression in knockdown HEK293 cells (e.g., in HEK293 cells overexpressing ASGPR) (e.g., as measured by qPCR as described in Example 2 below). In the embodiments, the compounds have an IC50 of less than about 1000 nM. 50 Values, such as ICs less than approximately 800 nm, 700 nm, 600 nm, or 500 nm. 50 Value. In other embodiments, the compound has an IC50 value of less than about 50 nM. 50Values, such as ICs less than approximately 20 nm or 10 nm. 50 Value. In the embodiments, the compound has an IC50 value of less than about 5 nM. 50 value.

[0408] In other embodiments, the compounds of this disclosure are characterized based on their activity in gene expression knockdown in PHH cells (e.g., PHH cells overexpressing ASGPR) (e.g., as measured by qPCR as described in Example 2 below). In embodiments, the compounds have an IC50 of less than about 100 nM. 50 Values, such as ICs less than approximately 80 nM, 70 nM, 60 nM, or 50 nM. 50 Value. In other embodiments, the compound has an IC50 value of less than about 50 nM. 50 Values, such as ICs less than approximately 20 nm or 10 nm. 50 Value. In the embodiments, the compound has an IC50 value of less than about 5 nM. 50 value.

[0409] In embodiments, the compounds of this disclosure are characterized based on their activity in knocking down gene expression in tissues (such as the liver) in vivo (e.g., as measured according to the assay described in Example 3 below). In embodiments, the compounds can knock down the level of target mRNA by at least about 40%, for example, at least about 45%, 50%, or 55%. In embodiments, the compounds can knock down the level of target mRNA by at least about 60%, for example, at least about 65% or 70%. In embodiments, at least about 40% knockdown is achieved in the liver but not in one or more other tissues (e.g., not in the kidney).

[0410] Nucleic acid

[0411] As defined herein, the compounds disclosed herein are particularly suitable for delivering nucleic acids to cells and tissues. Therefore, the “cargo” group of the compounds disclosed herein is a nucleic acid. As used herein, the term “nucleic acid” includes nucleic acids selected from the group consisting of DNA, RNA, PNA, and LNA. Nucleic acids can be functional nucleic acids, for example, wherein the functional nucleic acid is selected from the group consisting of mRNA, microRNA, shRNA, combinations of RNA and DNA, siRNA, siNA, antisense nucleic acids (e.g., antisense oligonucleotides (ASO)), ribozymes, aptamers, and mirror aptamers. In embodiments, the nucleic acid is selected from siRNA and ASO. In embodiments, the nucleic acid is siRNA. In other embodiments, the nucleic acid is ASO.

[0412] Nucleic acids can be of any length and can have any number of nucleotides, making them effective for the intended purpose (e.g., RNAi). In embodiments, siRNA ranges from 15 to 30 nucleotides. Using Watson-Crick base pairing, the double-stranded region of double-stranded RNA can range from 15 to 30 nucleotide base pairs. The double-stranded region can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs. In embodiments, nucleic acids have 19 to 23 base pairs. For example, the length of nucleic acid can be 19, 20, 21, 22, or 23 base pairs. Double-stranded RNAi can be blunt-ended at one or both ends. Double-stranded RNAi can have one or more nucleotide overhangs at one or both ends on one or both strands. The length of the overhang can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides.

[0413] For any of the foregoing aspects, the nucleic acid can be a modified nucleic acid. Modification can be selected from substitution or insertion using analogs of nucleic acids or bases, and chemical modification of the base, sugar, or phosphate moiety. For example, the nucleic acid can: a) be blunt at both ends; b) have a protruding end at one end and a blunt end at the other end; c) have protruding ends at both ends. One or more nucleotides on the first and / or second strand can be modified to form a modified nucleotide. One or more nucleotides of the odd number of nucleotides in the first strand can be modified. One or more nucleotides of the even number of nucleotides in the first strand can be modified by at least a second modification, wherein the at least second modification is different from the modification on one or more added nucleotides. At least one nucleotide of the one or more modified even number of nucleotides can be adjacent to at least one nucleotide of the one or more modified odd number of nucleotides. The nucleic acid of this disclosure can be a modified nucleotide of the first strand having a displacement of at least one nucleotide relative to an unmodified or differently modified nucleotide of the second strand.

[0414] The nucleic acid disclosed herein may comprise two strands containing nucleotides capable of interfering with gene expression. Repression may be complete or partial and may result in targeted downregulation of gene expression. The nucleic acid may comprise two separate polynucleotide chains; a first chain, which may also be a guide chain; and a second chain, which may also be a guest chain. The first and second chains may be part of the same complementary polynucleotide molecule, which "folds" to form a double-stranded molecule. The nucleic acid may be an siRNA molecule. The first chain may also be referred to as the antisense chain. The second chain may also be referred to as the sense chain.

[0415] Nucleic acids may comprise ribonucleotides, modified ribonucleotides, deoxynucleotides, deoxyribonucleotides, or nucleotide analogs. Nucleic acids may also comprise a double-stranded nucleic acid portion or double-stranded region formed by all or a portion of a first strand (also referred to in the art as the leader strand) and all or a portion of a second strand (also referred to in the art as the guest strand). A double-stranded region is defined as beginning with a first base pair formed between the first and second strands and ending with a last base pair formed between the first and second strands, including the endpoints.

[0416] Depending on the length of the nucleic acid, a perfect base complementarity match between the first and second strands is not necessarily required. However, the first and second strands must be able to hybridize under physiological conditions. The complementarity between the first and second strands in at least one duplex region can be perfect because there are no nucleotide mismatches or added / deleted nucleotides in either strand. Alternatively, the complementarity may be imperfect. The complementarity can be at least 70%, 75%, 80%, 85%, 90%, or 95%. The first and second strands may each contain a complementary region consisting of at least 15 consecutive nucleotides differing by no more than 3 nucleotides.

[0417] Unmodified polynucleotides (especially ribonucleotides) may be readily degraded by cellular nucleases, and therefore, modified and / or altered nucleotides may be included in the nucleic acids of this disclosure. One or more nucleotides on the second and / or first strand of the nucleic acids of this disclosure may be modified. Modification of the nucleic acids of this disclosure generally provides a powerful tool for overcoming potential limitations, including but not limited to the inherent in vitro and in vivo stability and bioavailability of natural RNA molecules. Nucleic acids according to this disclosure can be modified by chemical modification. Modified nucleic acids can also minimize the likelihood of inducing interferon activity in humans. Modification can also enhance the functional delivery of nucleic acids to target cells. The modified nucleic acids of this disclosure may comprise one or more chemically modified ribonucleotides of either or both of the first or second strand. Ribonucleotides may comprise chemical modifications of base, sugar, or phosphate moieties. Ribonucleic acids can be modified by substitution or insertion with analogs of nucleic acids or bases.

[0418] One or more nucleotides of the nucleic acid disclosed herein may be modified. The nucleic acid may contain at least one modified nucleotide. The modified nucleotide may be on the first strand. The modified nucleotide may be on the second strand. The modified nucleotide may be in a double-stranded region. The modified nucleotide may be outside the double-stranded region, i.e., in a single-stranded region. The modified nucleotide may be on the first strand and may be outside the double-stranded region. The modified nucleotide may be on the second strand and may be outside the double-stranded region. The 3'-terminal nucleotide of the first strand may be a modified nucleotide. The 3'-terminal nucleotide of the second strand may be a modified nucleotide. The 5'-terminal nucleotide of the first strand may be a modified nucleotide. The 5'-terminal nucleotide of the second strand may be a modified nucleotide.

[0419] The nucleic acid disclosed herein may have one modified nucleotide, or the nucleic acid disclosed herein may have about 2-4 modified nucleotides, or the nucleic acid may have about 4-6 modified nucleotides, about 6-8 modified nucleotides, about 8-10 modified nucleotides, about 10-12 modified nucleotides, about 12-14 modified nucleotides, about 14-16 modified nucleotides, about 16-18 modified nucleotides, about 18-20 modified nucleotides, about 20-22 modified nucleotides, about 22-24 modified nucleotides, 24-26 modified nucleotides, or about 26-28 modified nucleotides. In each case, compared with the same nucleic acid but without the modified nucleotide, the nucleic acid containing the modified nucleotide retains at least 50% of its activity. Compared with the same nucleic acid but without the modified nucleotide, the nucleic acid may retain 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or higher of its activity.

[0420] The modified nucleotide can be purine or pyrimidine. At least half of the purines can be modified. At least half of the pyrimidines can be modified. All purines can be modified. All pyrimidines can be modified. The modified nucleotide can be selected from the group consisting of: 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, debased nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholinonucleotides, aminophosphates, nucleotides containing non-natural bases, nucleotides containing 5'-thiophosphate groups, nucleotides containing 5'-phosphate groups or 5'-phosphate analogs, and terminal nucleotides linked to cholesterol derivatives or dodecanoic acid bis(decanoic acid) groups.

[0421] Nucleic acids may contain modified nucleotides, wherein the bases are selected from 2-aminoadenosine, 2,6-diaminopurine, inosine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine), 6-azpyrimidine, 6-alkylpyrimidine (e.g., 6-methyluridine), propyne, etc. q-nucleotide (quesosine), 2-thiouridine, 4-thiouridine, huaitin, huaitoxy, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5'-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N 6 -Methyladenosine (m 6 A) 7-Methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methoxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N 6 -Isopentenyl adenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid, and 2-thiocytidine.

[0422] The nucleic acids discussed in this article include unmodified RNA as well as polymers of RNA and nucleoside substitutes that have been modified, for example, to improve efficacy. Unmodified RNA refers to molecules in which the components of the nucleic acid (i.e., the sugar, base, and phosphate moieties) are the same or substantially the same as those naturally occurring (e.g., those naturally occurring in the human body). Modified nucleotides, as used herein, refer to nucleotides in which one or more components of the nucleic acid (i.e., the sugar, base, and phosphate moieties) differ from those naturally occurring. Although they are referred to as modified nucleotides, due to the modification, they will of course include molecules that are not nucleotides, such as polynucleotide molecules, in which the ribose phosphate backbone is replaced by a non-ribose phosphate construct that allows inter-chain hybridization; that is, the modified nucleotide mimics the ribose phosphate backbone.

[0423] Many modifications that occur within nucleic acids as described herein will be repeated within polynucleotide molecules, such as modifications of bases or phosphate moieties or non-linked oxygens of phosphate moieties. In some cases, modifications will occur at all possible positions / nucleotides in the polynucleotide, but in many cases, they will not occur. Modifications may occur only at the 3' or 5' ends, or only in terminal regions, such as positions on terminal nucleotides or in the last 2, 3, 4, 5, or 10 nucleotides of the strand. Modifications may occur in double-stranded regions, single-stranded regions, or both. Modifications may occur only in double-stranded regions of the nucleic acids of this disclosure, or only in single-stranded regions of the nucleic acids of this disclosure. Phosphothiophosphate modifications at non-linked oxygen positions may occur only at one or both ends, or only in terminal regions, such as positions on terminal nucleotides or in the last 2, 3, 4, or 5 nucleotides of the strand, or may occur in double-stranded and / or single-stranded regions, particularly at the ends. The 5' or 3' ends may be phosphorylated.

[0424] The stability of the nucleic acid disclosed herein can be increased by including specific bases at the overhang, or by including modified nucleotides at the single-stranded overhang (e.g., at the 5' or 3' overhang, or both). Purine nucleotides may be included at the overhang. All or some bases at the 3' or 5' overhang may be modified. Modifications may include modifications at the 2' OH group of the ribose, the use of deoxyribonucleotides instead of ribonucleotides, and modifications at the phosphate group, such as thiophosphate modifications. The overhang does not need to be homologous to the target sequence.

[0425] Nucleases can hydrolyze phosphodiester bonds in nucleic acids. However, chemical modifications to nucleic acids can impart improved properties and make oligonucleotides more stable to nucleases. As used herein, modified nucleic acids may include one or more of the following:

[0426] (i) Change (e.g., replace) one or two non-linked phosphate oxygens and / or one or more linked phosphate oxygens (even at the 5' and 3' ends of the nucleic acids disclosed herein, also referred to as linking).

[0427] (ii) Change (e.g., replace) the components of ribose (e.g., the 2' hydroxyl group on the ribose).

[0428] (iii) Replace the phosphoric acid portion with a "dephosphoric acid" connector;

[0429] (iv) Modifying or replacing naturally occurring bases;

[0430] (v) Replace or modify the ribose-phosphate backbone;

[0431] (vi) Modify the 3' or 5' end of RNA, for example by removing, modifying or replacing the terminal phosphate group, or by concatenating a portion (e.g., a fluorescently labeled portion) to the 3' or 5' end of RNA.

[0432] The substitution, modification, or alteration of terms indicates differences from naturally occurring molecules.

[0433] Examples of modified phosphate groups include thiophosphates, selenophosphates, borane phosphates, borane phosphates, hydrophosphonates, aminophosphates, alkyl or aryl phosphonates, and phosphate triesters. In dithiophosphates, both unlinked oxygen atoms are substituted with sulfur. One, each, or both unlinked oxygen atoms in the phosphate group can independently be any of S, Se, B, C, H, N, or OR (where R is alkyl or aryl). Phosphate ester links can also be modified by replacing the linking oxygen with nitrogen (bridged aminophosphates), sulfur (bridged thiophosphates), and carbon (bridged methylene phosphonates). Substitution can occur at the terminal oxygen. Substitution of unlinked oxygen with nitrogen is possible.

[0434] Modified nucleotides can include modifications to sugar groups. The 2' hydroxyl group (OH) can be modified or replaced by many different "oxygen" or "deoxy" substituents. Examples of "oxygen"-2' hydroxyl group modifications include alkoxy or aryloxy groups (OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), O(CH2CH2O). n CH2CH2OR; "locked" nucleic acid (LNA), in which the 2' hydroxyl group is connected to the 4' carbon of the same ribose, for example, via a methylene bridge; O-amine (amine = NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino) and aminoalkoxy, O(CH2). n Amines (e.g., amine = NH₂; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino). "Deoxygenation" modifications include hydrogen halogenation; amino groups (e.g., NH₂, alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acids); NH(CH₂CH₂NH). n CH2CH2-amine (amine = NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, or diheteroarylamino), -NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl, and alkynyl groups, which may optionally be substituted with, for example, an amino functional group. Other substituents in some embodiments include 2'-methoxyethyl, 2'-OCH3, 2'-O-allyl, 2'-C-allyl, and 2'-fluorine.

[0435] The sugar group may also contain one or more carbon atoms with a stereochemical configuration opposite to that of the corresponding carbon in ribose. Therefore, modified nucleotides may contain sugars, such as arabinose. Modified nucleotides may also include "debased" sugars, which lack a nucleotide at the C-I' position. These debased sugars may also contain modifications at one or more constituent sugar atoms.

[0436] The 2' modification can be used in combination with one or more phosphate linker modifications (e.g., thiophosphates). The phosphate group can be replaced by a phosphorus-free linker. Examples of replaceable phosphate groups include siloxanes, carbonates, carboxymethyl groups, urethanes, amides, thioethers, ethylene oxide linkers, sulfonates, sulfonamides, thiomethyl acetals, methyl acetals, oximes, methyleneimino, methylenemethylimino, methylenehydrazine, methylenedimethylhydrazine, and methyleneoxymethylimino. In some embodiments, the replacement may include methylenecarbonylamino and methylenemethylimino groups.

[0437] The phosphate linker and ribose can be replaced with nuclease-resistant nucleotides. Examples include morpholino, cyclobutyl, pyrrolidine, and peptide nucleic acid (PNA) nucleoside substitutes. In some embodiments, PNA substitutes may be used.

[0438] The 3' and 5' ends of oligonucleotides can be modified. Such modifications can occur at the 3' or 5' end of the molecule, or at both ends. They can include modifying or replacing one or more atoms of the terminal phosphate group or phosphate molecule. For example, the 3' and 5' ends of the oligonucleotide can be conjugated to other functional molecular entities, such as labeled moieties, fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3, or Cy5 dyes), or protecting groups (based on, for example, sulfur, silicon, boron, or esters). Functional molecular entities can be attached to sugars via phosphate groups and / or linkers. The terminal atom of the linker can be attached to or replace the linking atom of the phosphate group or the C-3' or C-5' O, N, S, or C group of the sugar. Alternatively, the linker can be attached to or replace the terminal atom of a nucleotide substitute (e.g., PNA). These spacer groups or linkers can include, for example, -(CH2). n -、-(CH2)-NH-、-(CH2) n O-, -(CH2) n S-, O(CH2CH2O) n CH2CH2OH (e.g., where n=3 or 6), debased sugar, amide, carboxyl group, amine, hydroxyamine, hydroxyimine, thioether, disulfide, thiourea, sulfonamide or morpholino, or biotin and fluorescein reagents. The 3' end may be a -OH group.

[0439] Other examples of end-modification include dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, desafeline, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), and lipophilic carriers (e.g., cholesterol, bile acids, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geraniol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl) Lithocholic acid, O3-(oleoyl)cholic acid, dimethoxytriphenylmethyl or phenoxazine) and peptide conjugates (e.g., antennal foot peptide, Tat peptide), alkylating agents, phosphate esters, amino groups, thiol groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption promoters (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, diimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, tetraza macrocyclic Eu3+ complexes).

[0440] Terminal modifications can be added for a variety of reasons, including modulating activity or resistance to degradation. Terminal modifications that can be used to modulate activity include modifying the 5' end with phosphate or a phosphate analog. The nucleic acids disclosed herein on the first or second strand may be 5' phosphorylated or contain a phosphoryl analog at the 5' end. 5'-phosphate modifications include those compatible with RISC-mediated gene silencing. Suitable modifications include: 5' monophosphate ((HO)2(O)PO-5'); 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-monothiophosphate (thiophosphate; (HO)2(S)PO-5'); 5'-monodithiophosphate (dithiophosphate) Phosphate esters; (HO)(HS)(S)PO-5'), 5'-thiophosphate esters ((HO)2(O)PS-5'); any additional combination of oxygen / sulfur-substituted monophosphates, diphosphates and triphosphates (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-aminophosphate esters ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-, (OH)2(O)P-5'-CH2-), 5'-vinylphosphonates, 5'-alkyl ether phosphonates (R=alkyl ether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-).

[0441] The nucleic acids disclosed herein may contain one or more phosphate-thioester modifications at one or more ends of the first and / or second strand. Optionally, each or any end of the first strand may contain one, two, or three phosphate-thioester modified nucleotides. Optionally, each or any end of the second strand may contain one, two, or three phosphate-thioester modified nucleotides. Optionally, both ends of the first strand and the 5' end of the second strand may contain two phosphate-thioester modified nucleotides. A phosphate-thioester modified nucleotide refers to a nucleotide whose bond with an adjacent nucleotide contains a phosphate-thioester group instead of a standard phosphate group.

[0442] Terminal modifications can also be used to monitor distribution, and in this case, the group to be added can include a fluorophore, such as fluorescein or Alexa dye. Terminal modifications can also be used to enhance uptake; cholesterol is a useful modification for this. Terminal modifications can also be used to crosslink RNA agents to another part.

[0443] Adenine, guanine, cytosine, and uracil are the most common bases in RNA. These bases can be modified or substituted to provide RNA with improved properties. For example, nuclease-resistant oligonucleotides can be prepared using these bases or synthetic and natural nucleobases (e.g., inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanine, or tuberculin) and any of the modifications described above. Alternatively, analogs of substitutions or modifications of any of the bases described above and "universal bases" can be used. Examples include 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudoruracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-aminoallyluracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and others. It contains 8-substituted adenine and guanine, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine, 5-substituted pyrrolidine, 6-azapyrimidine, and N-2, N-6, and O-6 substituted purines (including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine), dihydrouracil, 3-deazo-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkylcytosine, 7-deazoadenine, N... 6 N 6 -Dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N 3 -Methyluracil, substituted 1,2,4-triazoles, 2-pyridones, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N <4> -Acetylcytosine, 2-Thiocytosine, N 6 -Methyladenine, N 6 -Isopentyladenine, 2-Methylthio-N 6 -Isoprene adenine, N-methylguanine, or O-alkylated base.

[0444] Certain portions may be attached to the 5' end of the first or second strand and include debased ribose moieties, debased deoxyribose moieties, modified debased ribose and debased deoxyribose moieties, including 2'O-alkyl modifications; reverse debased ribose and debased deoxyribose moieties and their modifications, C6-imino-Pi; mirror nucleotides, including L-DNA and L-RNA; 5'OMe nucleotides; and nucleotide analogs, including 4',5'-methylene nucleotides; 1-(β-D-erythrofuranosyl) nucleotides; 4'-thionucleotides, carbocyclic nucleotides; 5'-Amino-alkyl phosphates; 1,3-diamino-2-propyl phosphates, 3-aminopropyl phosphates; 6-aminohexyl phosphates; 12-aminododecyl phosphates; hydroxypropyl phosphates; 1,5-dehydrated hexitol nucleotides; α-nucleotides; threo-pentafuranosyl nucleotides; acyclic 3',4'-open-ring nucleotides; 3,4-dihydroxybutyl nucleotides; 3,5-dihydroxypentyl nucleotides, 5'-5'-reverse debasement moiety; 1,4-butanediol phosphates; 5'-amino; and bridged or unbridged methylphosphonates and 5'-mercaptoyl moietyes. Nucleic acids of this disclosure may include one or more reverse nucleotides, such as reverse thymidine or reverse adenine (see, for example, Takei et al., J Biol Chem (2002) 277(26):23800-23806).

[0445] The nucleic acid disclosed herein may comprise debased nucleotides. The nucleic acid may comprise one or more nucleotides modified on the second strand and / or the first strand. Alternating nucleotides may be modified to form modified nucleotides. In alternating nucleotides, one nucleotide may be modified with a first modification, the next consecutive nucleotide may be modified with a second modification, and subsequent consecutive nucleotides may be modified with a first modification, and so on, wherein the first and second modifications are distinct.

[0446] RNA modification

[0447] Modification of the siRNA molecules disclosed herein generally provides a powerful tool for overcoming potential limitations, including but not limited to the inherent in vitro and in vivo stability and bioavailability of natural RNA molecules. The siRNAs according to this disclosure can be chemically modified. Modified siRNAs can also minimize the likelihood of activating interferon activity in humans. Modifications can also enhance the functional delivery of siRNAs to target cells. The modified siRNAs of this disclosure may contain one or more chemically modified ribonucleotides, either antisense or sense strands. Ribonucleotides may contain chemical modifications of bases, sugars, or phosphate moieties. Ribonucleic acid can be modified by substitution or insertion with analogs of nucleic acids or bases.

[0448] One or more nucleotides of the siRNA disclosed herein may contain modified bases. In one aspect, the siRNA comprises at least one nucleotide containing a modified base. In one embodiment, the modified base is on the antisense strand. In another embodiment, the modified base is on the sense strand. In another embodiment, the modified base is in the double-stranded region. In another embodiment, the modified base is outside the double-stranded region, i.e., in the single-stranded region. In another embodiment, the modified base is on the antisense strand and outside the double-stranded region. In another embodiment, the modified base is on the sense strand and outside the double-stranded region. In another embodiment, the 3'-terminal nucleotide of the antisense strand is a nucleotide having a modified base. In another embodiment, the 3'-terminal nucleotide of the sense strand is a nucleotide having a modified base. In another embodiment, the 5'-terminal nucleotide of the antisense strand is a nucleotide having a modified base. In another embodiment, the 5'-terminal nucleotide of the sense strand is a nucleotide having a modified base.

[0449] The modified bases can be purines or pyrimidines. In another embodiment, at least half of the purines are modified. In another embodiment, at least half of the pyrimidines are modified. In another embodiment, all purines are modified. In another embodiment, all pyrimidines are modified. In another embodiment, the siRNA may comprise nucleotides containing the modified bases, wherein the bases are selected from 2-aminoadenosine, 2,6-diaminopurine, inosine, pyridine-4-one, pyridine-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine), 6-azpyrimidine, 6-alkylpyrimidine (e.g., 6-methyluridine), propyne, q-nucleoside, 2-thiouridine, 4-thiouridine, huaitin, huaitoxy, 4-acetyl Cytidine, 5-(carboxyhydroxymethyl)uridine, 5'-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, β-D-galactosyl guanosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyl adenosine, β-D-mannosyl guanosine, uridine-5-oxyacetic acid, and 2-thiocytidine.

[0450] On the other hand, the siRNA disclosed herein contains debased nucleotides. As used herein, nucleotides with modified bases do not include debased nucleotides.

[0451] Sugar modification

[0452] On the other hand, modifications to the sugar moiety are involved. One or more nucleotides of the siRNA disclosed herein may comprise a modified ribose moiety. Modifications at the 2'-position, where the 2'-OH is substituted, include non-limiting examples selected from: alkyl, substituted alkyl, alkylaryl-, arylalkyl-, -F, -Cl, -Br, -CN, -CF3, -OCF3, -OCN, -O-alkyl, -S-alkyl, HS-alkyl-O, -O-alkenyl, -S-alkenyl, -N-alkenyl, -SO-alkyl, -alkyl-OSH, -alkyl-OH, -O-alkyl-OH, -O-alkyl-SH, -S-alkyl The substituents include -OH, -S-alkyl-SH, -alkyl-S-alkyl, -alkyl-O-alkyl, -ONO2, -NO2, -N3, -Ή2, alkylamino, dialkylamino-, aminoalkyl-, aminoalkoxy, aminoacyl-, aminoacyl-, -ONH2, -O-aminoalkyl, -O-amino, -O-aminoacyl, heterocyclic alkyl-, heterocyclic aryl-, aminoalkylamino-, polyalkylamino-, substituted silyl-, methoxyethyl- (MOE), alkenyl, and alkynyl. It also includes "locked" nucleic acids (LNAs) in which the 2' hydroxyl group is, for example, linked to the 4' carbon of the same ribose via a methylene bridge as a 2' modification of this disclosure. In embodiments, the substituents are 2'-methoxyethyl, 2'-O-CH3, 2'-O-allyl, 2'-C-allyl, and 2'-fluoro(2'-F).

[0453] Modifying groups

[0454] In one aspect, the antisense double-stranded region comprises multiple sets of modified nucleotides, referred to herein as "modifying groups," wherein each modifying group consists of one or more identically modified nucleotides, wherein each modifying group is side-attached to a second set of nucleotides on one or both sides, referred herein as "flank groups," wherein each of the flanking groups consists of one or more unmodified nucleotides or nucleotides modified in a manner different from the nucleotides of the modifying group. In one embodiment, each modifying group in the antisense double-stranded region is identical, i.e., each modifying group consists of an equal number of identically modified nucleotides. In another embodiment, each flanking group has an equal number of nucleotides. In another embodiment, each flanking group is identical. In another embodiment, the nucleotides of the modifying groups in the antisense double-stranded region contain modified bases. In another embodiment, the nucleotides of the modifying groups contain a modified phosphate backbone. In another embodiment, the nucleotides of the modifying groups contain a modified 2' position.

[0455] On the other hand, the sense double-stranded region includes multiple sets of modifying groups, each modifying group consisting of one or more identically modified nucleotides, wherein each modifying group is flanked by flanking groups on one or both sides, and each flanking group consists of one or more unmodified nucleotides or nucleotides modified in a manner different from the nucleotides of the modifying group. In one embodiment, each modifying group in the sense double-stranded region is identical. In another embodiment, each flanking group has an equal number of nucleotides. In another embodiment, each flanking group is identical. In another embodiment, the nucleotides of the modifying groups in the sense double-stranded region contain modified bases. In another embodiment, the nucleotides of the modifying groups contain a modified phosphate backbone. In another embodiment, the nucleotides of the modifying groups contain a modified 2' position.

[0456] In another embodiment, both the antisense double-stranded region and the sense double-stranded region contain multiple modifying groups, each of which consists of one or more identically modified nucleotides, wherein each modifying group is flanked by flanking groups on one or both sides, and each flanking group consists of one or more unmodified nucleotides or nucleotides modified in a manner different from the nucleotides of the modifying group. In one embodiment, each modifying group in the antisense double-stranded region and the sense double-stranded region is identical. In another embodiment, each flanking group in the antisense double-stranded region and the sense double-stranded region has an equal number of nucleotides. In another embodiment, each flanking group in the antisense double-stranded region and the sense double-stranded region is identical. In another embodiment, the nucleotides of the modifying groups in the antisense double-stranded region and the sense double-stranded region each contain the same modifying group and the same flanking groups. In another embodiment, the nucleotides of the modifying groups in the antisense double-stranded region and the sense double-stranded region each contain a modified base. In another embodiment, the nucleotides of the modifying groups in the antisense double-stranded region and the sense double-stranded region each contain a modified phosphate backbone. In another embodiment, the nucleotides of the modified groups in the antisense double-stranded region and the sense double-stranded region each contain a modified 2' position.

[0457] Modification of the phosphate backbone

[0458] On the other hand, modifications to the phosphate backbone are involved. All or part of the nucleotides of the siRNA disclosed herein can be linked by phosphodiester bonds, as found in unmodified nucleic acids. However, the siRNA disclosed herein may contain modified phosphodiester bonds. The phosphodiester bonds of the antisense or sense strand can be modified to independently contain at least one heteroatom selected from nitrogen and sulfur. In one embodiment, the phosphate group of the ribonucleotide linked to the adjacent ribonucleotide is replaced by a modifying group. In one embodiment, the modifying group replacing the phosphate group is selected from thiophosphate, methylphosphonate, dithiophosphate, or aminophosphate.

[0459] 5' and 3' end modifications

[0460] The siRNA disclosed herein may comprise a nucleic acid molecule having one or more modified nucleotides, abase nucleotides, acyclic nucleotides, or deoxyribonucleotides at the 5'- or 3'-terminus of either or both of the sense strand and antisense strand. The 5'-terminal nucleotide of the antisense strand and / or the sense strand may be phosphorylated. In another embodiment, the 5'-terminal nucleotide of the antisense strand is phosphorylated, and the 5'-terminal nucleotide of the sense strand has a free hydroxyl group (5'-OH). In another embodiment, the 5'-terminal nucleotide of the antisense strand is phosphorylated, and the 5'-terminal nucleotide of the sense strand is modified. In another embodiment, the 5'-terminal nucleotide of the antisense strand carries a 5'E vinylphosphonate ester.

[0461] Modifications to the 5'- and 3'-terminal nucleotides are not limited to the 5' and 3' positions on these terminal nucleotides. Examples of terminal nucleotide modifications include, but are not limited to, biotin, reverse (deoxy)base removal, amino, fluorine, chlorine, bromine, CN, CF, methoxy, imidazole, carboxylic esters, thioesters, C1-C... 10 Lower alkyl, substituted lower alkyl, alkylaryl or arylalkyl, OCF3, OCN, O-, S- or N-alkyl; O-, S- or N-alkenyl; SO-CH3; SO2CH3; ONO2; NO2, N3; heterocyclic alkyl; heterocyclic alkylaryl; aminoalkylamino; polyalkylamino or substituted silyl, especially as described, for example, in PCT patent publication WO 99 / 54459 or European patent EP0 586 520 B1 or EP 0 618 925 B1, each of which is incorporated herein by reference in its entirety.

[0462] In another embodiment, the terminal 3' nucleotide or both terminal 3'-nucleotides on either or both of the antisense or sense strands are 2'-deoxynucleotides. In another embodiment, the 2'-deoxynucleotide is 2'-deoxy-pyrimidine. In yet another embodiment, the 2'-deoxynucleotide is 2'-deoxy-thymidine.

[0463] shRNA (short hairpin loop RNA) and linked siRNA

[0464] On the other hand, shRNA and linked siRNA are involved. The antisense and sense strands can be covalently linked to each other. This linkage can occur between any nucleotides that form the antisense and sense strands, respectively, and can be formed by covalent or non-covalent linkage. Covalent linkage can be formed by one or more times using compounds selected from the group containing methylene blue and bifunctional groups, linking the two strands at one or more sites respectively. In embodiments, the bifunctional group is selected from the group containing bis(2-chloroethyl)amine, N-acetyl-N'-(p-acetaldehydebenzoyl)cystamine, 4-thiouracil, and psoralen.

[0465] Furthermore, the antisense and sense strands can be linked by a ring structure. The ring structure can be composed of non-nucleic acid polymers (such as polyethylene glycol). The 5' end of the antisense strand can be attached to the 3' end of the sense strand, or vice versa. The ring can be composed of nucleic acids, locked nucleic acids (LNAs), peptide nucleic acids (PNAs), or can be formed from polymers. The ring length can be sufficient to covalently link the two strands through a foldback mechanism using the ring structure or similar structures.

[0466] The ribonucleic acid construct can be incorporated into a suitable vector system. In an embodiment, the vector contains a promoter for expressing RNAi. The promoter can be selected from any promoter known in the art, such as pol III, U6, H1, or 7SK.

[0467] The nucleic acid according to this disclosure may contain one or more phosphate-thionucleotide internucleotide bonds. Phospho-thionucleotide internucleotide bonds may be distributed throughout the nucleotide sequence and may appear in any number at any position. The nucleic acid may contain one to ten phosphate-thionucleotide internucleotide bonds. The antisense strand may have at least one phosphate-thionucleotide modification at each end. The antisense strand may have 1-3 phosphate-thionucleotide modifications at each end. For example, the antisense strand may have 2 phosphate-thionucleotide modifications at each end. The sense strand may have at least one phosphate-thionucleotide modification at the 3' end. The sense strand may have 1-3 phosphate-thionucleotide modifications at the 3' end. For example, the sense strand may have 2 phosphate-thionucleotide modifications at the 3' end.

[0468] siRNA with overhangs

[0469] The overhangs at the 3' or 5' end of the sense or antisense strand can be selected from the group consisting of 1, 2, 3, 4, and 5 nucleotides in length. Alternatively, the siRNA molecule can be blunt-ended at both ends and can have a length of 16 to 29 consecutive nucleotides. In one embodiment, the siRNA molecule is blunt-ended at one end, and the double-stranded or duplex portion of the siRNA molecule has a length selected from 16 to 29 consecutive nucleotides. In one embodiment, the siRNA molecule has overhangs at both ends of either strand, and the double-stranded or duplex portion of the siRNA molecule has a length of 16 to 29 consecutive nucleotides. The overhangs may contain at least one deoxyribonucleotide and / or TT dinucleotide.

[0470] Those skilled in the art will understand that the modifications described above, including modifications to the sugar moiety, patterns, 5' and 3' end modifications, protrusions, formulations, delivery, dosage, and delivery routes, can be applied equivalently to any type of RNAi molecule, and are not limited to siRNA.

[0471] The nucleic acids disclosed herein can be produced using conventional methods in the art, including in vitro (e.g., uncontrolled transcription) or in vivo chemical synthesis or expression of nucleic acids. For example, solid-phase chemical synthesis or expression vectors can be used. In one embodiment, the expression vector can generate the nucleic acids of this disclosure in target cells. Methods for synthesizing and purifying the nucleic acid molecules described herein are known to those skilled in the art.

[0472] Pharmaceutical Composition

[0473] In one aspect, this disclosure provides a pharmaceutical composition comprising a compound described herein (e.g., a compound of formula (I)) or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient or carrier.

[0474] In some embodiments, the pharmaceutical composition comprises a compound of formula (I) (e.g., a compound of formula (Ia) or (Ib)) or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition comprises a compound of formula (II) (e.g., a compound of formula (IIa) or (IIb)) or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition comprises a compound of formula (III) (e.g., a compound of formula (IIIa) or (IIIb)) or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition comprises a compound of formula (IV) (e.g., a compound of formula (IVa) or (IVb)) or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition comprises a compound of formula (V) (e.g., a compound of formula (Va) or (Vb)) or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition comprises a compound of formula (IX) (e.g., a compound of formula (IXa)) or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition comprises a compound of formula (XI) (e.g., a compound of formula (XIa)) or a pharmaceutically acceptable salt thereof. In the embodiments, the pharmaceutical composition comprises a compound of formula (XIII).

[0475] Nucleic acids and conjugated compounds can be delivered to cells in vitro and in vivo using a variety of methods known to those skilled in the art, including direct contact with cells or in combination with one or more agents that promote targeting and / or delivery to cells. Such agents and methods include liposome complexes, liposomes, iontophoresis, hydrogels, cyclodextrins, nanocapsules, microspheres and nanospheres, and protein carriers. Nucleic acid / carrier combinations can be delivered locally in vivo via direct injection or by using an infusion pump.

[0476] The compositions disclosed herein may comprise surface-modified liposomes (PEG-modified, long-circulating, or stealth liposomes) containing poly(ethylene glycol) lipids. These formulations provide a method for increasing their stability by preventing the aggregation and fusion of liposomes or liposome complex solutions. The formulations also offer the additional in vivo benefits of opsonization and elimination of the mononuclear phagocytic system (MPS or RES), thereby enabling the encapsulated drug to have longer blood circulation time and enhanced tissue exposure. Such liposomes may selectively accumulate in tumors by extravasation and capture in angiogenic target tissues. Long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes known to accumulate in MPS tissues (see, for example, Liu et al., J. Biol. Chem. (1995) 42:24864-24780; and PCT publications WO 96 / 10391; WO 96 / 10390; and WO 96 / 10392, the contents of each of which are incorporated herein by reference in their entirety). Long-circulating liposomes can also protect siRNA from nuclease degradation.

[0477] The pharmaceutical compositions disclosed herein can be used as pharmaceuticals or diagnostic agents. For example, one or more compounds of this disclosure (e.g., siRNA conjugates) can be combined with a delivery medium (e.g., liposomes) and excipients (such as carriers or diluents). Other agents, such as preservatives and stabilizers, may also be added. Methods for delivering nucleic acid-containing molecules are known in the art and are within the knowledge of those skilled in the art. The compounds of this disclosure (e.g., siRNA conjugates) can also be administered in combination with other therapeutic compounds, alone or simultaneously, for example as a unit dose of a combination.

[0478] The pharmaceutical composition may be a sterile, injectable aqueous suspension or solution, or in lyophilized form. In one embodiment, the pharmaceutical composition comprises a lyophilized liposome complex or an aqueous suspension of a liposome complex. In another embodiment, the liposome complex comprises a compound of the present disclosure. Such liposome complexes can be used to deliver the compounds of the present disclosure to target cells in vitro or in vivo.

[0479] The pharmaceutical compositions and drugs disclosed herein can be administered to a subject (e.g., a mammal) in a pharmaceutically effective dose. The mammal may be selected from humans, dogs, cats, horses, cattle, pigs, goats, sheep, mice, rats, hamsters, and guinea pigs. In the embodiments, the subject is a human.

[0480] The compounds or compositions disclosed herein (e.g., compositions comprising double-stranded siRNA) can be delivered to a subject via a variety of routes. Exemplary routes include subcutaneous, intramuscular, intradermal, intravenous, local, rectal, anal, vaginal, nasal, lung, and ocular administration. Administration can be local (including ocular, vaginal, rectal, intranasal, and transdermal), oral, or parenteral. Parenteral administration includes intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection, or intrathecal or intraventricular administration. In embodiments, the compounds of this disclosure are delivered in vivo by a method selected from intravenous, subcutaneous, intramuscular, or intradermal injection or by inhalation. In one embodiment, the compounds of this disclosure are delivered by intravenous injection or infusion.

[0481] The route and / or site of administration can be chosen to enhance targeting. For example, intramuscular injection into the muscle of interest would be a logical choice to target muscle cells. Lung cells can be targeted by administering the composition in aerosol form. Vascular endothelial cells can be targeted by coating a balloon catheter with a compound or composition and mechanically introducing nucleic acids.

[0482] The pharmaceutical compositions disclosed herein can be formulated, for example, using conventional carriers or excipients for administration in solid or liquid form. Using techniques known in the art, the compositions may be suitable for, for example, oral administration (e.g., as a solution, suspension, tablet, or capsule), parenteral administration (e.g., as a solution, dispersion, suspension, or emulsion, or as a dry powder for reconstitution), or topical administration (e.g., as a cream, ointment, patch, or spray to be applied to the skin).

[0483] Medical use

[0484] The compounds disclosed herein act as regulators of nucleic acids in vivo, which makes them available for the treatment of a variety of diseases and conditions.

[0485] From this perspective, this disclosure provides the use of the disclosed compound (or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or salt thereof) in a therapeutic context. In a related aspect, the use of the disclosed compound (or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or salt thereof) as a medicament is provided. In another related aspect, a method of treating a subject in need is provided, the method comprising administering to the subject an effective amount of the disclosed compound (or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or salt thereof). In yet another related aspect, the use of the disclosed compound (or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or salt thereof) in the manufacture of a medicament is provided.

[0486] In particular, the compounds of this disclosure can target cells and / or tissues carrying ASGP receptors and can be used to treat such cells and tissues by delivering therapeutic nucleic acids. In this respect, this disclosure provides the compounds of this disclosure (or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising the compound or salt) for use in treating a symptom in a subject, which can be treated by delivering a therapeutic nucleic acid to cells and / or tissues of the subject expressing ASGPR. In a related aspect, this disclosure provides a method for improving the therapeutic activity of a therapeutic nucleic acid in treating a symptom in a subject, which can be treated by delivering the therapeutic nucleic acid to cells and / or tissues of the subject expressing ASGPR, the method comprising conjugating the therapeutic nucleic acid to one or more molecules to form the compounds of this disclosure (or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising the compound or salt). In another related aspect, this disclosure provides a method for treating a symptom in a subject, the symptom being treatable by delivering a therapeutic nucleic acid to the subject’s ASGPR-expressing cells and / or tissues, wherein the method comprises delivering the therapeutic nucleic acid as part of a compound of this disclosure (or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound or salt thereof).

[0487] Examples of conditions that can be treated by delivery of the disclosed compounds (or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising the compound or salt) include liver diseases (e.g., liver cancer, such as hepatocellular carcinoma), hereditary diseases, hemophilia and bleeding disorders, liver fibrosis, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), viral hepatitis, rare diseases (e.g., acromegaly), metabolic diseases (e.g., hypercholesterolemia, dyslipidemia, hypertriglyceridemia), cardiovascular diseases, obesity, thalassemia, liver injury (e.g., drug-induced liver injury), hemochromatosis, alcoholic liver disease, alcohol dependence, anemia, and anemia of chronic disease. In embodiments, the condition is selected from NASH, NAFLD, metabolic diseases, and cardiovascular diseases. In an embodiment, the condition is NASH.

[0488] In another aspect, this disclosure provides the use of the disclosed compounds (or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising the compound or salt) in the treatment of a condition as defined herein. In a related aspect, use of the disclosed compounds (or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising the compound or salt) in the treatment of a condition as defined herein is provided. In another related aspect, a method of treating a condition in a subject in need is provided, the method comprising administering to the subject an effective amount of the disclosed compound (or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising the compound or salt), wherein the condition is as defined herein. In a further related aspect, use of the disclosed compounds (or pharmaceutically acceptable salts thereof, or pharmaceutical compositions comprising the compound or salt) in the manufacture of a medicament for the treatment of a condition as defined herein is provided. In embodiments of the foregoing aspects, the condition is selected from NASH, NAFLD, metabolic diseases (e.g., selected from hypercholesterolemia, dyslipidemia, and hypertriglyceridemia), and cardiovascular diseases. In an embodiment, the condition is NASH.

[0489] Delivery of nucleic acids to cells

[0490] On the other hand, a method is provided for delivering nucleic acids to cells using a compound (or a pharmaceutically acceptable salt thereof) according to this disclosure, wherein the cells carry (e.g., express) a binding partner of a ligand of the compound. The method includes the step of contacting the cells with the compound. The method can be used in vitro (e.g., for diagnostic or research purposes) or in vivo (e.g., for diagnostic or therapeutic purposes). In an embodiment, the method is an in vitro method.

[0491] In one embodiment, the binding partner is an ASGPR (e.g., human ASGPR), and the compound carries at least one monosaccharide ligand (e.g., the compound carries at least one GalNAc moiety). In another embodiment, the cell is a hepatocyte, such as a mammalian hepatocyte, like a human hepatocyte. In yet another embodiment, the cell is a malignant hepatocyte (e.g., hepatocellular carcinoma cell).

[0492] In one embodiment, the method includes contacting cells carrying (e.g., expressing) ASGPR (e.g., hepatocytes) with a compound carrying at least one GalNAc moiety, such as a nucleic acid delivery agent comprising formula (II), or a compound of formula (V), (VI), (VII), or (VIII), or a pharmaceutically acceptable salt thereof. In another embodiment, the method includes contacting cells carrying (e.g., expressing) ASGPR (e.g., hepatocytes) with a compound carrying at least one GalNAc moiety, such as a compound of formula (I), (II), (III), (IV), (V), (IX), (XI), or (XIII), or a pharmaceutically acceptable salt thereof.

[0493] Methods and intermediates

[0494] This disclosure also provides methods for preparing compounds as described herein, as well as intermediates that can be used to prepare said compounds.

[0495] From this perspective, the present disclosure provides a compound having the structure of formula (VI).

[0496]

[0497] Or a pharmaceutically acceptable salt thereof, wherein X, Y, spacer group and chain linker are as defined herein; It represents a carbon-carbon single or double bond, provided that X is a covalent bond. R represents a carbon-carbon single bond; R" is

[0498] Acyl, -C(O)aryl, or -H; and J is -CO2H, -OH, -C(O)O-(pentafluorophenyl), -N3, or phosphoramidite.

[0499] In the implementation scheme, the compound is a compound having the structure of formula (VIa):

[0500]

[0501] Or a pharmaceutically acceptable salt thereof, wherein X, Y, R", spacer group, chain linker and J are as defined herein, and It represents a carbon-carbon single or double bond, provided that X is a covalent bond. It represents a carbon-carbon single bond.

[0502] In the implementation scheme, the compound is a compound having the structure of formula (VIb):

[0503]

[0504] Or a pharmaceutically acceptable salt thereof, wherein X, Y, R", spacer group, chain linker and J are as defined herein, and It represents a carbon-carbon single bond.

[0505] In the implementation scheme, the compound is a compound having the structure of formula (VIc):

[0506]

[0507] Or a pharmaceutically acceptable salt thereof, wherein X, Y, R", spacer group, chain linker and J are as defined herein, and It represents a carbon-carbon single bond.

[0508] In the implementation scheme, the compound is a compound having the structure of formula (VId):

[0509]

[0510] Or a pharmaceutically acceptable salt thereof, wherein X, Y, R", spacer group, chain linker and J are as defined herein, and It represents a carbon-carbon double bond.

[0511] On the other hand, this disclosure provides a compound having the structure of formula (VII):

[0512]

[0513] Or a pharmaceutically acceptable salt thereof, wherein Z, R, R", m, chain and J are as defined herein.

[0514] In the implementation scheme, the compound is a compound having the structure of formula (VIIa):

[0515]

[0516] Or a pharmaceutically acceptable salt thereof, wherein Z, R, R", m, chain and J are as defined herein.

[0517] In the implementation scheme, the compound is a compound having the structure of formula (VIIb):

[0518]

[0519] Or a pharmaceutically acceptable salt thereof, wherein Z, R, R", m, chain and J are as defined herein.

[0520] In the implementation scheme, the compound is a compound having the structure of formula (VIIc):

[0521]

[0522] Or a pharmaceutically acceptable salt thereof, wherein Z, R, R", m, chain and J are as defined herein.

[0523] On the other hand, this disclosure provides a compound having the structure of formula (VIII):

[0524]

[0525] Or a pharmaceutically acceptable salt thereof, wherein A, R, R'', n, chain and J are as defined herein.

[0526] In the implementation scheme, the compound is a compound having the structure of formula (VIIIa):

[0527]

[0528] Or a pharmaceutically acceptable salt thereof, wherein A, R, R'', n, chain and J are as defined herein.

[0529] In the implementation scheme, the compound is a compound having the structure of formula (VIIIb):

[0530]

[0531] Or a pharmaceutically acceptable salt thereof, wherein A, R, R'', n, chain and J are as defined herein.

[0532] In the implementation scheme, the compound is a compound having the structure of formula (VIIIc):

[0533]

[0534] Or a pharmaceutically acceptable salt thereof, wherein A, R, R'', n, chain and J are as defined herein.

[0535] On the other hand, this disclosure provides a compound having the structure of formula (X):

[0536]

[0537] Or a pharmaceutically acceptable salt thereof, wherein Z, R, R", m, chain and J are as defined herein.

[0538] In the implementation scheme, the compound is a compound having the structure of formula (Xa):

[0539]

[0540] Or a pharmaceutically acceptable salt thereof, wherein Z, R, R", m, chain and J are as defined herein.

[0541] On the other hand, this disclosure provides a compound having the structure of formula (XII):

[0542]

[0543] Or a pharmaceutically acceptable salt thereof, wherein Z, R, R'', m, q and J are as defined herein, and wherein D is selected from (i) the bond and (ii) -NHC(O)-(CH2). t - (where t is as described herein). In embodiments, D' is a bond and J is -CO2H or -C(O)O-(pentafluorophenyl). In other embodiments, D' is -NHC(O)-(CH2). t - (for example, where t is 6), and J is -CO2H or -C(O)O-(pentafluorophenyl).

[0544] In the implementation scheme, the compound is a compound having the structure of formula (XIIa):

[0545]

[0546] Or a pharmaceutically acceptable salt thereof, wherein Z, R, R", m, q, D' and J are as defined herein.

[0547] On the other hand, this disclosure provides a compound having the structure of formula (XIV):

[0548]

[0549] Or a pharmaceutically acceptable salt thereof, wherein R", p and J are as defined herein, and Represents a carbon-carbon single or double bond. In an embodiment, R" is an acyl group. In an embodiment, J is -CO2H, -OH, or -C(O)O-(pentafluorophenyl). In an embodiment, R" is an acyl group, and J is -C(O)O-(pentafluorophenyl).

[0550] In a related aspect, a method is provided for preparing a compound of formula (III) or a pharmaceutically acceptable salt thereof, characterized in that a compound of formula (VI) is reacted with a compound having the following structure.

[0551] Q - [Goods]

[0552] Wherein the goods are as defined herein, and Q represents a group that is reactive to group J as defined herein (e.g., where Q represents a group containing BCN). In an embodiment, the compound of formula (III) is a compound of formula (IIIa), and the compound of formula (VI) is a compound of formula (VIa). In an embodiment, the compound of formula (III) is a compound of formula (IIIb), and the compound of formula (VI) is a compound of formula (VIb). In an embodiment, the compound of formula (III) is a compound of formula (IIIc), and the compound of formula (VI) is a compound of formula (VIc). In an embodiment, the compound of formula (III) is a compound of formula (IIId), and the compound of formula (VI) is a compound of formula (VId).

[0553] In another related aspect, a method is provided for preparing a compound of formula (IV) or a pharmaceutically acceptable salt thereof, characterized in that a compound of formula (VII) is reacted with a compound having the following structure.

[0554] Q - [Goods]

[0555] Wherein the goods and Q are as defined herein. In an embodiment, the compound of formula (IV) is a compound of formula (IVa), and the compound of formula (VII) is a compound of formula (VIIa). In an embodiment, the compound of formula (IV) is a compound of formula (IVb), and the compound of formula (VII) is a compound of formula (VIIb). In an embodiment, the compound of formula (IV) is a compound of formula (IVc), and the compound of formula (VII) is a compound of formula (VIIc).

[0556] In another related aspect, a method is provided for preparing a compound of formula (V) or a pharmaceutically acceptable salt thereof, characterized in that a compound of formula (VIII) is reacted with a compound having the following structure.

[0557] Q - [Goods]

[0558] Wherein the goods and Q are as defined herein. In an embodiment, the compound of formula (V) is a compound of formula (Va), and the compound of formula (VIII) is a compound of formula (VIIIa). In an embodiment, the compound of formula (V) is a compound of formula (Vb), and the compound of formula (VIII) is a compound of formula (VIIIb). In an embodiment, the compound of formula (V) is a compound of formula (Vc), and the compound of formula (VIII) is a compound of formula (VIIIc).

[0559] In another related aspect, a method is provided for preparing a compound of formula (IX) or a pharmaceutically acceptable salt thereof, characterized in that the compound of formula (X) is reacted with a compound having the following structure.

[0560] Q - [Goods]

[0561] Wherein the goods and Q are as defined herein. In the embodiments, the compound of formula (IX) is a compound of formula (IXa), and the compound of formula (X) is a compound of formula (Xa).

[0562] In another related aspect, a method is provided for preparing a compound of formula (XI) or a pharmaceutically acceptable salt thereof, characterized in that the compound of formula (XII) is reacted with a compound having the following structure.

[0563] Q - [Goods]

[0564] Wherein the goods and Q are as defined herein. In the embodiments, the compound of formula (XI) is a compound of formula (XIa), and the compound of formula (XII) is a compound of formula (XIIa).

[0565] In another related aspect, a method is provided for preparing a compound of formula (XIII) or a pharmaceutically acceptable salt thereof, characterized in that a compound of formula (XIV) is reacted with a compound having the following structure.

[0566] Q - [Goods]

[0567] Goods and Q are as defined herein.

[0568] In embodiments of the above method, Q represents a BCN-containing group, and J is -N3. In other embodiments of the above method, Q represents an amide-containing group (e.g., a hexylamine-containing group), and J is -CO2H or -C(O)O-(pentafluorophenyl) (e.g., -C(O)O-(pentafluorophenyl)).

[0569] Having been generally described herein, the following non-limiting embodiments are provided to further illustrate this disclosure.

[0570] Example

[0571] General synthesis scheme

[0572] The following scheme, scheme 1, illustrates exemplary methods and examples for preparing compounds according to this disclosure:

[0573]

[0574] Option 1

[0575] According to Scheme 1, in step 1, shikimic acid is reacted with an exemplary chain (where n can be, for example, 4, 5, or 6, and R...). a This indicates a nitrogen-protecting group or an N-alkyl group, such as methyl, reacting to form compound 1. In step 2, compound 1 is reacted with... t The acrylate is reacted to form compound 2. In the third step, the shikimic acid core of compound 2 is reduced, for example, using Pd-C / MeOH, followed by treatment with HCl to produce tricarboxylic acid compound 3. In step 4, compound 3 is reacted with compound 4 (containing an acetylated GalNac group attached to an alkylamine chain, where m can be, for example, 4, 5, or 6) to produce compound 5. In step 5, the free alcohol of the chain group is oxidized to a carboxylic acid, which is then reacted with pentafluorophenyl trifluoroacetate in step 6 to give compound 7. In step 7, compound 7 is treated to remove the O-acetyl group on the GalNAc ligand, and then reacted with a cargo-containing compound (shown in Scheme 1 with an exemplary hexylamino linker), followed by treatment with, for example, ammonia, to form compound 8.

[0576] The following scheme, scheme 2, illustrates another exemplary manner and embodiment for preparing the compound according to this disclosure:

[0577]

[0578] Option 2

[0579] According to scheme 2, in step 1, compound 1 (where n can be, for example, 4, 5 or 6, and R) is made... a The nitrogen-protecting group or N-alkyl group (e.g., methyl) reacts with iodoacetic acid to give compound 9. In step 2, the shikimic acid core of compound 9 is reduced, for example, using Pd-C / MeOH, followed by treatment with HCl to produce tricarboxylic acid compound 10. In step 3, compound 10 is reacted with compound 4 (containing an acetylated GalNac group attached to an alkylamine chain, where m can be, for example, 4, 5, or 6) to produce compound 11. In step 4, the free alcohol of the chain group is oxidized to a carboxylic acid, which is then reacted with pentafluorophenyl trifluoroacetate in step 5 to give compound 13. In step 6, compound 13 is treated to remove the O-acetyl group on the GalNAc ligand, and then reacted with a cargo-containing compound (shown in scheme 2 with an exemplary hexylamino linker), followed by treatment with, for example, ammonia, to form compound 14.

[0580] The following scheme, scheme 3, illustrates another exemplary manner and embodiment for preparing the compound according to this disclosure:

[0581]

[0582] Option 3

[0583] According to scheme 3, in step 1, compound 1 (where n can be, for example, 4, 5 or 6, and R) is made... a The reaction of a nitrogen-protecting group or N-alkyl group (e.g., methyl) with acrylonitrile yields compound 15. In step 2, the shikimic acid core of compound 9 (illustrated with Pd-C in scheme 3) is reduced, and the compound is treated with Boc2O to yield compound 16. In step 3, the protecting group is removed with HCl to yield triamine compound 17. In step 4, compound 17 is reacted with compound 18 (containing an acetylated GalNac group attached to a functionalized alkyl ester, where p can be, for example, 3, 4, or 5) to produce compound 19. In step 5, the free alcohol is oxidized to a carboxylic acid (compound 20), which is then reacted with pentafluorophenyl trifluoroacetate in step 6 to yield compound 21. In step 7, compound 21 is treated to remove the O-acetyl group on the GalNAc ligand, and then reacted with a cargo-containing compound (shown with an exemplary hexylamino linker in scheme 3), followed by treatment with, for example, ammonia, to form compound 22.

[0584] Alternative joining methods for attaching the cargo portion to the remainder of the molecule are shown in Scheme 4 below:

[0585]

[0586] Option 4

[0587] Scheme 4 illustrates in a general manner how a ligand-containing precursor (e.g., compound 5, compound 11, or compound 19) can react with a reagent (e.g., 2-cyanoethyl N,N-diisopropylchlorophosphine (CEP-Cl)) to produce a phosphoramidite derivative, which can be coupled to a nucleotide or oligonucleotide to form the compounds disclosed herein. The coupling step can be carried out, for example, on a solid phase.

[0588] In the general schemes described above, for example after the catalytic hydrogenation step, no specific stereochemistry is specified for the reduced shikimic acid ring at the α-position of the amide. In the following synthetic schemes, this position is shown as having an amide configuration on the same side of the cyclohexane ring as the oxygen atom at the para position of the group (i.e., those substituents are in a cis-1,4 relationship). The catalytic hydrogenation step in the schemes described herein typically yields a single stereoisomer (as evaluated by, for example, HPLC), which, based on spatial considerations, is expected to be a cis isomer. Other synthetic methods can be used to prepare the trans isomer.

[0589] Experimental techniques

[0590] At 400 MHz and 500 MHz 1 1H NMR spectra were recorded on a Bruker Avance DRX-400 and a Bruker Avance DPX-500 spectrometer, respectively, with the chemical shift (δ in ppm) in the solvent dimethyl sulfoxide-d6 (DMSO-d6) being 2.5 ppm at the cited temperature as reference. Coupling constants (J) are given in Hertz.

[0591] Liquid chromatography / mass spectrometry (LC / MS) was obtained on a UPLC Acquity Waters instrument, a Sedere light scattering detector, and a SQ Waters mass spectrometer, using UV detection DAD 210 < λ < 400 nm and a column Acquity UPLC CSH C18 1.7 µm, size 2.1 x 30 mm, with the mobile phase H2O + 0.1% HCO2H / CH3CN + 0.1% HCO2H.

[0592] Unless otherwise stated, all synthetic reactions were carried out under an inert atmosphere. In the following examples, when the source of the starting product is not specified, it should be understood that the product is a known compound (e.g., a compound commercially available from a supplier such as Sigma-Aldrich).

[0593] Synthesis of precursor and cargo molecules

[0594] Oligonucleotide synthesis

[0595] Oligonucleotides were synthesized on a CUTAG CPG support (Sigma-Aldrich, 25 - 35 μmol / g) on a K&A system at a 10 μmol scale. Oligonucleotides were also synthesized on an ÄKTA OligoPilot Plus 10 synthesizer (Cytiva) at a 32 μmol scale using standard synthesis protocols. Nucleotide phosphoramidites were purchased from Sigma-Aldrich or WuXi. Linker phosphoramidites were purchased from Glen Research or WuXi. All cEt phosphoramidites were obtained from Pharmaron. 5'-Amino-modifier C6 was obtained from Glen Research. Polystyrene solid support (Primer support 5G) was purchased from Cytiva. UV purity was determined using ion-pairing LCMS and stated at 260 nm. Yields are given based on the initial resin loading and the oligonucleotide content of the final product, as calculated from the UV absorption.

[0596] The sequences of the synthetic oligonucleotides representing the ASO (MALAT1) cargo or the siRNA (ANGPTL3) cargo are shown in the table below:

[0597]

[0598] mC represents 5'-methylcytidine.

[0599] HA represents hexylamine

[0600] InvAb indicates reverse debasing.

[0601] Indicates thiophosphate bond

[0602] The uppercase letters represent 2'-F in SEQ ID NO: 2 and 3, and DNA in SEQ ID NO: 1.

[0603] Bold text indicates 2'-O,4'-C-methylene locked nucleic acid

[0604] Lowercase letters indicate 2'-Ome

[0605] The sequences of the synthetic oligonucleotides representing siRNA (PPIB) cargoes are shown in the table below:

[0606]

[0607] P represents phosphate.

[0608] f represents 2'-fluorine

[0609] m represents 2'-OMe (mC represents 2'-O-methylcytidine in SEQ ID NO: 4-6)

[0610] Indicates thiophosphate bond

[0611] General Synthesis Program

[0612] Use this procedure unless otherwise specified.

[0613] Before use, phosphorus amide was dissolved in DNA-grade acetonitrile (ACN) to a final concentration of 0.1 M (3 equivalents). Detrimethylation was performed using a 3% (v / v) DCM solution of dichloroacetic acid (contact time 5 × 35 s). Bi- and tri-antennae linkers were deprotected using bi-detrimethylation. Activator 42 was used as the coupling activator (0.25 M in ACN). The phosphorus amide recycling time was 4 min for DNA structural units and 10 min for all 2'-modified structural units. Linker phosphorus amides were coupled using triple coupling for 10 min, and GalNAc phosphorus amides were coupled using quadruple coupling. Hydroxyxanol was dissolved in pyridine (0.2 M) and used as a thiolation agent with a contact time of 5 min. The oxidizing agent solution, purchased from Sigma-Aldrich, was used unchanged with a contact time of 9 s. End-capping was performed by in-situ mixing of equal volumes of capping solution A (9.1 vol% acetic anhydride in tetrahydrofuran (THF) solution) and capping solution B (THF / N-methylimidazolium / pyridine 80:10:10 vol%) (contact time 50 s). If necessary, after final 5'-detriphenylmethylation, the cyanoethyl backbone was removed with 20 vol% diethylamine in ACN solution (contact time 7 × 1 min). The oligonucleotides were cleaved from the solid support and further deprotected by treatment with methanol-ammonia (3M) at 55 °C for 15–20 h, followed by purification on a reversed-phase column using ion-paired HPLC. For siRNA compounds, single strands were prepared separately as equal concentrations of mQ aqueous solution, mixed in equal volumes, heated to 95 °C for 5 min, and then cooled to room temperature for 1 h.

[0614] Oligonucleotides were synthesized according to a standard procedure, except that a 5'-amino-modifier C6 (0.2 M, 10 min contact time, double coupling) was introduced as the final coupling step. The oligonucleotides were purified while retaining the 5'-monomethoxytriphenylmethyl (MMT) protecting group. After removing the MMT group in aqueous acetic acid (pH 4.5), the free 5'NH₂ was reacted with the pentafluorophenyl ester of the GalNAc moiety. Three equivalents of the ester were dissolved in ACN and added to a solution of the oligonucleotides in borate buffer (pH 9). The final product was then purified by normal-phase HPLC.

[0615] For siRNA synthesis, the guest strand (PO to HA) was synthesized using a standard oligonucleotide synthesis procedure. The guide strand was synthesized according to a general synthesis protocol. For annealing, the guest and guide strands were dissolved separately in PBS buffer to prepare equimolar solutions of each strand. The two solutions were then mixed. Next, annealing was performed under standard conditions (95°C for 5 min, followed by slow cooling to 20°C with gentle shaking for 1 hour). QC gel analysis was performed. The product solution was adjusted to 1 mM in PBS buffer.

[0616] To prepare the siRNA-containing complex, a 5'-hexylamine-containing transit chain, deprotected from MMT, was reacted with a pentafluorophenyl ester (PFP-ester) of the GalNAc moiety. The PFP-ester was dissolved in ACN / THF / DMF and added to a solution of hexylamine-containing oligonucleotides in borate buffer (pH 9). The conjugate product was then purified by HPLC. Once the transit and guide chains containing the GalNAc conjugate were synthesized, siRNA synthesis was performed following a standard annealing procedure. The chain solution in PBS buffer was mixed and heated at 95°C for 5 minutes, then slowly cooled to 20°C (1 h) with gentle shaking. QC gelation was performed to adjust the concentration, and re-annealing was performed if necessary. The compound was stored as a 1 mm PBS buffer solution.

[0617] Synthesis of BCN-modified hexylamine MALAT1 ASO :

[0618]

[0619] First, MALAT1-hexylamine-modified ASO (50 mg) (all PS, PO to hexylamine) was dissolved in 500 µL of BBS solvent. NHS-activated BCN (1.5 equivalents, 4 mg) was dissolved in 200 µL of THF and added to the MALAT1 solution. The reaction mixture became turbid. The reaction mixture was sonicated for several minutes with stirring. LCMS showed conversion after 2 hours. Stirring was continued overnight. The oligonucleotides were precipitated by adding 2 mL of anhydrous EtOH and 0.2 mL of 3M sodium acetate (9:1 v / v). The white precipitate was centrifuged at 4 °C for 15 min. The supernatant was removed, and the mixture was washed once more with 2 mL of ethanol. The precipitate, dissolved in 5 mL of water, was lyophilized overnight to provide a white solid. The resulting product was dissolved in 1 mL of deionized water and used directly for the next step. LCMS m / z ES - Expected value: 5681.704; Measured values: 1894.9 (z = 3), 1420.7 (z = 4), 1136.34 (z = 5).

[0620] Synthesis of precursor (1)

[0621]

[0622] Synthesis scheme :

[0623]

[0624] In part (A), SOCl2 (10.48 ml, 143.55 mmol) was added dropwise to a solution of (3R,4S,5R)-3,4,5-trihydroxycyclohexane-1-en-1-carboxylic acid (1-1) (50 g, 287.11 mmol) in MeOH (1000 ml) at 0 °C under argon atmosphere. The resulting mixture was stirred at 60 °C for 3 hours. The solvent was removed under reduced pressure to obtain a crude product. The crude solid was ground with EtOAc (500 ml) / hexane (500 ml) and stirred for 2 hours to obtain a solid. The solid was collected by filtration and dried under vacuum to give methyl (3R,4S,5R)-3,4,5-trihydroxycyclohexane-1-en-1-carboxylic acid (1-2) (48.0 g, 89%) as a white solid.

[0625] ¹H-NMR (300MHz, DMSO, 20℃) δ 1.99–2.13 (m, 1H), 2.39 (dt, J = 4.7, 2.5Hz, 1H), 2.42–2.49 (m, 1H), 2.51 (q, J = 1.9Hz, 1H), 3.5–3.62 (m, 2H), 3.67 (s, 3H), 3.86 (dt, J = 5.8, 4.1Hz, 1H), 4.18–4.26 (m, 1H), 6.62 (d, J = 2.7Hz, 1H). LCMS m / z ESI expected value 188.0, measured value 187.1 ([MH]). - ).

[0626] Sodium hydride (7.01 g, 175.36 mmol) was added to a DMF (10 g, 53.14 mmol) solution (1-2) at 0 °C. After stirring for 45 min, tert-butyl 2-bromoacetate (124 g, 635.71 mmol) was added to the reaction mixture. The reaction mixture was warmed to room temperature and stirred for 16 h. The reaction mixture was quenched with ice water (100 ml), extracted with EtOAc (200 ml), and washed successively with saturated brine (2 × 100 ml). The organic layer was dried over Na2SO4, filtered, and evaporated to give the crude product. The crude product was purified by rapid silica gel chromatography with an elution gradient of 0% to 40% EtOAc in petroleum ether. The pure fraction was evaporated to dryness to give 2,2',2''-(((1R,2S,3R)-5-(methoxycarbonyl)cyclohexyl-4-ene-1,2,3-triyl)tri(oxy))triacetic acid tritert-butyl ester (1-3) (8.00 g, 28.4%), which was a colorless oil.

[0627] ¹H-NMR (400MHz, DMSO, 27℃) δ 1.43 (d, J = 5.8Hz, 27H), 2.16–2.33 (m, 1H), 2.52–2.61 (m, 1H), 3.70 (s, 3H), 3.82–3.94 (m, 2H), 4–4.12 (m, 3H), 4.13–4.28 (m, 4H), 6.77 (d, J = 2.8Hz, 1H). LCMS m / z ESI expected value 530.2, measured value 548.2 ([M+NH₄]). + ).

[0628] A solution of (1-3) (8 g, 15.08 mmol) and Pd-C (10%) (1.604 g, 1.51 mmol) in MeOH (90 mL) was stirred for 3 hours at 1 atm under a hydrogen atmosphere and at room temperature. The mixture was filtered through a diatomaceous earth mat. The solvent was removed under reduced pressure to give 2,2',2''-(((1R,3R)-5-(methoxycarbonyl)cyclohexane-1,2,3-triyl)tri(oxy))triacetic acid tritert-butyl ester (1-4) (6.80 g, 85%) as a yellow oil. The product was used directly in the next step without further purification. Compounds (1-4) were isolated as single diastereomers. The stereocenter α of the amide was described as (S), and hydrogenation was expected to occur on the least sterically hindered surface of the alkene.

[0629] ¹H-NMR (400MHz, DMSO, 27℃) δ 1.42 (d, J = 2.0Hz, 27H), 1.56–1.99 (m, 4H), 3.17 (d, J = 5.2Hz, 1H), 3.55–3.63 (m, 3H), 3.75–3.89 (m, 3H), 4–4.12 (m, 4H), 4.18 (d, J = 5.5Hz, 2H). LCMS m / z ESI expected value 532.2, measured value 550.2 ([M+NH₄]). + ).

[0630] Trimethyltin hydroxide (11.54 g, 63.83 mmol) was added to a 200 mL solution of DCE (1-4) (6.8 g, 12.77 mmol) at room temperature under nitrogen atmosphere. The resulting mixture was stirred at 85 °C for 16 hours. The reaction mixture was filtered through diatomaceous earth. The solvent was removed under reduced pressure to give (3R,5R)-3,4,5-tris(2-(tert-butoxy)-2-oxoethoxy)cyclohexane-1-carboxylic acid (crude). The crude product was purified by rapid silica gel chromatography with an elution gradient of 10% to 80% EtOAc in petroleum ether. The purified fraction was evaporated to dryness to give (1-5) (1.700 g, 25.7%) as a colorless oil.

[0631] 1H-NMR (300MHz, DMSO, 22℃) δ 1.42 (d, J = 1.5Hz, 27H), 1.61 (td, J =14.6, 13.6, 9.3Hz, 2H), 1.86 (dd, J = 24.3, 13.2Hz, 2H), 3.17 (d, J = 4.7Hz,2H), 3.82 (tt, J = 6.6, 3.2Hz, 2H), 4.03 (t, J = 3.1Hz, 3H), 4.10 (dd, J =4.9, 2.1Hz, 1H), 4.18 (s, 2H), 12.21 (brs, 1H). LCMS m / z ESI expected value 518.2, measured value 536.3 ([M+H2O]) + ).

[0632] DIEA (2.53 mL, 14.46 mmol) was added to a solution of (1–5) (1.5 g, 2.89 mmol), 6-azidohexyl-1-amine hydrochloride (1.034 g, 5.78 mmol), HOBt (0.886 g, 5.78 mmol), and EDC (1.109 g, 5.78 mmol) in 25 mL of DMF at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature for 16 hours. The reaction was quenched with water (10 mL), and the solvent was removed under reduced pressure. The crude product was purified by rapid silica gel chromatography with an elution gradient of 0% to 30% EtOAc in petroleum ether. The pure fraction was evaporated to dryness to give 2,2',2''-(((1R,3R)-5-((6-azidohexyl)carbamoyl)cyclohexane-1,2,3-triyl)tri(oxy))tri-tert-butyl triacetate (1-6) (0.600 g, 32.3%), which is a yellow oil.

[0633] ¹H-NMR (300MHz, DMSO, 22℃) δ 1.23–1.34 (m, 6H), 1.43 (d, J = 1.2Hz, 27H), 1.48–1.74 (m, 7H), 3.02 (dd, J = 12.2, 5.9Hz, 2H), 3.31 (s, 4H), 3.76–3.87 (m, 2H), 4.02 (dd, J = 9.0, 5.0Hz, 3H), 4.18 (s, 2H), 7.7–7.82 (m, 1H). LCMS m / z ESI expected value 642.3, measured value 643.3 ([M+H]). + ).

[0634] TFA (6.59 mL, 85.56 mmol) was added to a 10 mL solution of DCM (1-6) (550 mg, 0.86 mmol) at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature for 2.5 hours. The solvent was removed under reduced pressure to give a yellow oily product: 2,2',2''-(((1R,3R)-5-((6-azidohexyl)carbamoyl)cyclohexane-1,2,3-triyl)tri(oxy))triacetic acid (1-7) (400 mg, 99%). The product was ready for use in the next step without further purification.

[0635] ¹H-NMR (300MHz, DMSO, 25℃) δ 1.16–1.44 (m, 10H), 1.52 (p, J = 6.9Hz, 3H), 1.59–1.71 (m, 3H), 2.79 (d, J = 5.0Hz, 1H), 3.00 (q, J = 6.4Hz, 2H), 3.51–3.67 (m, 2H), 3.84 (d, J = 9.4Hz, 2H), 4.07 (dd, J = 5.3, 2.6Hz, 3H), 7.76 (t, J = 5.7Hz, 1H). Three protons have been exchanged. LCMS m / z ESI expected value 474.4, observed value 475.2 ([M+H]). + ).

[0636] In part (B), DIEA (5.86 mL, 33.52 mmol) was added to a solution of 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)valerate (1-9) (3 g, 6.70 mmol), EDC (2.57 g, 13.41 mmol), HOBt (1.848 g, 12.07 mmol), and (3-aminopropyl)carbamate tert-butyl ester (1.752 g, 10.06 mmol) in DMF (60 mL) under nitrogen atmosphere at room temperature. The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into water (250 mL), extracted with EtOAc (300 mL), and washed with saturated brine (3 × 250 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to give the crude product. The crude product was purified by rapid silica gel chromatography with an elution gradient of 0% to 40% EtOAc in petroleum ether. The purified fraction was evaporated to dryness to give (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((3-((tert-butoxycarbonyl)amino)propyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (1-10) (2.500 g, 61.8%) as a yellow oil.

[0637] 1H-NMR (300MHz, DMSO, 26℃) δ 1.38 (s, 14H), 1.78 (s, 3H), 1.90 (s, 3H), 2.01 (s, 4H), 2.11 (s, 3H), 2.91 (q, J = 6.7Hz, 2H), 3.02 (q, J = 6.7Hz,2H), 3.36–3.49 (m, 1H), 3.56–3.65 (m, 1H), 3.66–3.79 (m, 1H), 3.88 (q, J =9.3Hz, 1H), 4.03 (s, 3H), 4.49 (d, J = 8.4Hz, 1H), 4.98 (dd, J = 11.2, 3.4Hz, 1H), 5.22 (d, J = 3.4Hz, 1H), 6.75 (s, 1H), 7.57–8.1 (m, 2H). LCMS m / z ESI expected value 603.3, measured value 604.1 ([M+H]). + ).

[0638] At room temperature under nitrogen atmosphere, a 4M dioxane solution of hydrochloric acid (20.71 mL, 82.83 mmol) was added to a 20 mL solution of DCM (1-10) (2.5 g, 4.14 mmol). The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure to give (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-(((3-aminopropyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (1-11) (1.890 g, 91%) as a yellow oil. The product was used directly in the next step without further purification.

[0639] 1H-NMR (300MHz, DMSO, 26℃) δ 1.50 (s, 6H), 1.78 (d, J = 2.9Hz, 3H), 1.90 (s, 3H), 2.00 (d, J = 3.5Hz, 4H), 2.11 (s, 3H), 2.76 (d, J = 7.4Hz, 3H), 3.01–3.15 (m, 3H), 3.40 (dt, J = 12.8, 5.9Hz, 2H), 3.83–3.93 (m, 1H), 3.96–4.06 (m, 3H), 4.52 (d, J = 8.5Hz, 1H), 4.98 (dd, J = 11.2, 3.3Hz, 1H), 5.22 (d, J = 3.4Hz, 1H), 7.64 (s, 3H). LCMS m / z ESI expected value 503.2, measured value 502.2 (ES-, [MH]). + ).

[0640] In part (C), under nitrogen atmosphere, DIEA (1.988 ml, 11.38 mmol) was added to a solution of (1-7) (450 mg, 0.95 mmol), (1-11) (1672 mg, 3.32 mmol), EDC (909 mg, 4.74 mmol), and HOBt (654 mg, 4.27 mmol) in 10 ml of DCM. The resulting mixture was stirred at room temperature for 15 hours. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by rapid silica gel chromatography with an elution gradient of 0% to 25% MeOH in DCM. The purified fraction was evaporated to dryness and then purified again by rapid silica gel chromatography with an elution gradient of 0% to 20% MeOH in DCM. The pure fraction was evaporated to dryness to give a brown oily substance (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-(((((2,2'-(((1R,2S,3R,5S)-3-(2-((3-(5-(((2S,3S,4S,5S,6S)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentamido)propyl)amino) )-2-oxoethoxy)-5-((6-azidohexyl)carbamoyl)cyclohexane-1,2-diyl)bis(oxy))bis(acetyl))bis(azinaldiyl))bis(propane-3,1-diyl))bis(azinaldiyl))bis(5-oxopentane-5,1-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (1-8) (360 mg, 19.66%).

[0641] 1H-NMR (400MHz, DMSO, 24℃) δ 1.19 (t, J = 7.3Hz, 46H), 1.38–1.62 (m,7H), 1.78 (s, 3H), 1.89 (s, 3H), 2.00 (s, 3H), 2.11 (s, 5H), 3.05 (q, J =7.3Hz, 36H), 3.25–3.52 (m, 8H), 3.62–4.12 (m, 11H), 4.50 (d, J = 8.5Hz, 1H), 4.97 (dd, J = 11.3, 3.5Hz, 1H), 5.22 (d, J = 3.4Hz, 1H), 7.31 (dddd, J =27.1, 8.1, 6.8, 1.1Hz, 4H), 7.55 (dt, J = 8.3, 1.1Hz, 2H), 7.8–7.91 (m, 4H). LCMS m / z ESI +Expected value: 1929.9, measured value: 966.3 (z = 2).

[0642] Sodium methoxide (210 mg, 1.17 mmol) was added to a MeOH (10 mL) solution of (1-8) (250 mg, 0.13 mmol) at 0 °C under nitrogen atmosphere. The resulting mixture was stirred at room temperature for 2 hours. AcOH (0.067 mL, 1.17 mmol) was added to the reaction mixture. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 25% MeCN in water (0.05% TFA). The pure fraction was evaporated to dryness to give a white solid N,N'-(((2,2'-(((1R,2S,3R,5S)-3-(2-((3-(5-(((2S,3S,4S,5S,6S)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentamido)propyl)amino)-2-oxoethoxy)-5-(( 6-Azide-hexyl)carbamoyl)cyclohexane-1,2-diyl)bis(oxy))bis(acetyl))bis(azanediyl))bis(propane-3,1-diyl))bis(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamide)(1) (74.0 mg, 36.8%).

[0643] 1H-NMR (400MHz, DMSO, 25℃) δ 1.21–1.59 (m, 26H), 1.78 (d, J =17.0Hz, 14H), 1.97–2.18 (m, 6H), 2.26–2.51 (m, 1H), 3.08 (dtd, J = 26.7,13.2, 12.7, 6.7Hz, 13H), 3.30 (q, J = 6.9Hz, 6H), 3.4–3.45 (m,3H), 3.53 (ddd,J = 13.9, 11.0, 5.6Hz, 5H), 3.64–3.77 (m, 8H), 3.86 (d, J = 14.5Hz, 2H), 3.94–4.12 (m, 4H), 4.22 (d, J = 8.4Hz, 2H), 4.48–4.77 (m, 8H), 6.68 (s, 1H), 6.98 (dddd, J = 17.3, 7.7, 6.6, 1.2Hz, 4H), 7.41 (d, J = 8.1Hz, 3H), 7.51 (d,J = 8.2Hz, 2H), 7.69 (dd, J = 9.3, 3.5Hz, 2H), 7.76–7.97 (m, 7H). LCMS m / z ESI + Expected value: 1551.8, measured value: 777.0 (z = 2).

[0644] Synthesis of precursor (2)

[0645]

[0646] Synthesis scheme :

[0647]

[0648]

[0649] In part (A), a solution of (((6-bromohexyl)oxy)methyl)benzene (2-1) (100 g, 184.37 mmol) in ethanol (3000 mL) in 2 M MeNH₂ was stirred at room temperature for 70 hours. The solvent was removed under reduced pressure to give 6-(benzyloxy)-N-methylhexyl-1-amine (2-2) (70.0 g, 85%) as a white solid. The product could be used directly in the next step without further purification.

[0650] ¹H-NMR (500MHz, DMSO, 24℃) δ 1.32 (dp, J = 11.1, 7.0, 5.6Hz, 4H), 1.55 (dq, J = 13.3, 7.0, 6.5Hz, 4H), 2.54 (s, 3H), 2.77–2.92 (m, 2H), 3.42 (t, J = 6.5Hz, 2H), 4.45 (s, 2H), 7.18–7.43 (m, 5H), 8.38 (s, 1H). LCMS m / z ESI expected value 221.3, measured value 222.2 ([M+H]). + ).

[0651] (2-2) (35 g, 158.12 mmol) was added to a solution of N-ethyl-N-isopropylpropyl-2-amine (83 mL, 474.37 mmol), shikimic acid (30.3 g, 173.94 mmol), BOP (105 g, 237.19 mmol), and DMAP (19.32 g, 158.12 mmol) in 700 mL of DMF. The resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was poured into water (1 L), extracted with EtOAc (2 × 1 L), the organic layer was washed with saturated brine (2 × 1 L), dried over Na2SO4, filtered, and evaporated to give a brown gel. The crude product was purified by rapid silica gel chromatography with an elution gradient of 0% to 20% MeOH in DCM. The pure fraction was evaporated to dryness to give (3R,4S,5R)-N-(6-(benzyloxy)hexyl)-3,4,5-trihydroxy-N-methylcyclohex-1-ene-1-carboxamide (2-3) (50.0 g, 84%), which is a yellow oil.

[0652] 1H-NMR (500MHz, DMSO, 26℃) δ 1.21–1.25 (m, 2H), 1.33 (s, 2H), 1.41–1.56 (m, 4H), 1.88–1.97 (m, 1H), 2.33–2.42 (m, 1H), 2.81 (s, 2H), 2.92 (s,1H), 3.25 (d, J = 26.1Hz, 3H), 3.41 (t, J = 6.5Hz, 2H), 3.50 (dd, J = 7.1,4.0Hz, 1H), 3.83 (dt, J = 7.1, 5.1Hz, 1H), 4.13 (s, 1H), 4.45 (s, 2H), 5.48 (t, J = 2.2Hz, 1H), 6.73–6.74 (m, 1H), 7.25–7.38 (m, 5H), 8.15 (d, J = 5.2Hz, 1H). LCMS m / z ESI expected value 377.2, measured value 378.2 ([M+H]). + ).

[0653] Cs₂CO₃ (85 g, 262.26 mmol) was added to a solution of (2-3) (30 g, 79.47 mmol) and tert-butyl acrylate (698 mL, 4768.45 mmol) in t-BuOH (1500 mL). The resulting mixture was stirred mechanically for 3 days at room temperature. The reaction mixture was diluted with EtOAc (3000 mL). The solution was filtered through diatomaceous earth. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by rapid silica gel chromatography with an elution gradient of 0% to 45% EtOAc in petroleum ether. The purified fraction was evaporated to dryness to give tritert-butyl 3,3',3''-(((1R,2S,3R)-5-((6-(benzyloxy)hexyl)(methyl)carbamoyl)cyclohex-4-ene-1,2,3-triyl)tri(oxy))tripropionate (45 g, crude product 2, containing dimer). The crude product 2 was purified by rapid silica gel (330 g silica gel column) chromatography with an elution gradient of 0% to 35.5% EtOAc in petroleum ether. The pure fraction was evaporated to dryness to give 3,3',3''-(((1R,2S,3R)-5-((6-(benzyloxy)hexyl)(methyl)carbamoyl)cyclohex-4-ene-1,2,3-triyl)tri(oxy))tripropionate tritert-butyl ester (2-4) (31.5 g, 52.0%) as a colorless oil.

[0654] ¹H-NMR (300MHz, DMSO, 22℃) δ 1.39 (d, J = 1.8Hz, 34H), 1.99 (s, 2H), 2.40 (dq, J = 7.4, 4.5, 3.9Hz, 7H), 2.83 (d, J = 24.1Hz, 3H), 3.25 (s, 2H), 3.41 (t, J = 6.4Hz, 2H), 3.58–3.83 (m, 8H), 3.97–4.06 (m, 1H), 4.44 (s, 2H), 5.51 (s, 1H), 7.21–7.41 (m, 5H). LCMS m / z ESI expected value 761.4, measured value 762.6 ([M+H]). + ).

[0655] Pd / C (10%, 50 w / w% water) (2.79 g, 2.62 mmol) was added to a MeOH (200 mL) solution of (2-4), and the mixture was stirred at 50 °C for 18 hours under a hydrogen atmosphere (20 atm). The reaction mixture was filtered through a Buchner funnel. The solvent was removed under reduced pressure, and the resulting oil was dissolved in acetonitrile and concentrated to dryness to give 3,3',3''-(((1R,3R)-5-((6-hydroxyhexyl)(methyl)carbamoyl)cyclohexane-1,2,3-triyl)tri(oxy))tripropionate tritert-butyl ester (2-5) (8.50 g, 96%) as a colorless oil. The product was used directly in the next step without further purification. Compound (2-5) was isolated as a single diastereomer. The stereocenter α of the amide was described as (S), and hydrogenation was expected to occur on the least sterically hindered surface of the alkene.

[0656] 1H-NMR (300MHz, DMSO, 23℃) δ 1.09–1.29 (m, 4H), 1.34–1.45 (m, 27H), 1.56 (t, J = 11.5Hz, 7H), 2.21–2.47 (m, 6H), 2.75 (d, J = 9.4Hz, 2H), 2.95(s, 2H), 3.25 (q, J = 8.3, 7.2Hz, 2H), 3.33–3.51 (m, 4H), 3.65 (dt, J = 16.2,9.7Hz, 6H), 3.79 (dt, J = 10.9, 5.6Hz, 2H), 4.33 (td, J = 5.2, 2.2Hz, 1H). LCMS m / z ESI expected value 673.4, measured value 674.4 ([M+H]). + ).

[0657] At room temperature, TFA (22.87 mL, 296.79 mmol) was added to a 20 mL solution of DCM (2-5) (2.0 g, 2.97 mmol). The resulting mixture was stirred at room temperature for 3 hours. The solvent was removed under reduced pressure to give 3,3',3''-(((1R,2S,3R,5S)-5-(N-methyl(6-(2,2,2-trifluoroacetoxy)hexyl)carbamoyl)cyclohexane-1,2,3-triyl)tri(oxy))tripropionic acid (2-6) (1.500 g, 84%) as a colorless oil. The product could be used directly in the next step without further purification.

[0658] ¹H-NMR (300MHz, DMSO, 25℃) δ 1.29–1.39 (m, 4H), 1.54 (d, J = 8.8Hz, 4H), 1.68 (p, J = 6.6Hz, 4H), 2.43 (d, J = 6.3Hz, 6H), 2.95 (s, 3H), 3.23 (t, J = 7.4Hz, 3H), 3.63 (dqt, J = 15.5, 9.6, 4.9Hz, 11H). LCMS m / z ESI expected value 601.2, measured value 602.3 ([M+H]). + ).

[0659] In part (B), Pd-C (10%, 50 w / w% water) (0.641 g, 0.60 mmol) was added to a solution of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((6-((((benzyloxy)carbonyl)amino)hexyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (2-10) (5 g, 8.61 mmol) (12) in EtOH (100 mL) at room temperature. The resulting solution was stirred at room temperature under a hydrogen atmosphere for 16 hours. The reaction mixture was filtered through diatomaceous earth to give a crude product, which was washed with MeOH (3 × 150 mL). The solvent was removed under reduced pressure to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((6-aminohexyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (2-9) (3.60 g, 94%), which was a colorless oil and was used without further purification.

[0660] 1H-NMR (300MHz, DMSO, 24℃) δ 1.29 (d, J = 16.4Hz, 6H), 1.46 (s, 2H), 1.78 (d, J = 1.2Hz, 3H), 1.90 (s, 3H), 2.00 (s, 3H), 2.09 (d, J = 9.1Hz, 8H), 3.34–3.49 (m, 1H), 3.70 (dt, J = 9.7, 6.1Hz, 1H), 3.78–3.93 (m, 1H), 4.03 (s, 2H), 4.49 (d, J = 8.5Hz, 1H), 4.97 (dd, J = 11.2, 3.4Hz, 1H), 5.22 (d, J =3.4Hz, 1H), 7.83 (d, J = 9.2Hz, 1H). LCMS m / z ESI expected value 446.2, measured value 447.2 ([M+H]). + ).

[0661] (2-6) (0.5 g, 0.83 mmol) was added to a solution of N-ethyl-N-isopropylpropyl-2-amine (1.452 mL, 8.31 mmol), PyBOP (1.838 g, 4.16 mmol), (2-9) (1.299 g, 2.91 mmol), and DMAP (0.305 g, 2.49 mmol) in 25 mL of DMF. The resulting mixture was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 38% MeCN in water (0.1% FA). The purified fraction was evaporated to dryness to give a yellow oil. The oily substance was dissolved in acetonitrile and water, and then lyophilized to obtain methyl acetate [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[3-[(1R,3R)-2,3-bis[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxohexylamino]-3-oxo-propoxy]-5-[6-hydroxyhexyl(methyl)carbamoyl]cyclohexyloxy]propionylamino]hexyloxy]tetrahydropyran-2-yl]acetate (2-7) (1.000 g, 67.2%), which was a yellow solid.

[0662] 1H-NMR (300MHz, DMSO, 25℃) δ 1.11–1.31 (m, 17H), 1.32–1.58 (m, 21H), 1.77 (s, 9H), 1.90 (s, 8H), 2.00 (s, 9H), 2.11 (s, 8H), 2.17–2.38 (m, 6H), 2.75 (d, J = 10.8Hz, 3H), 2.97 (d, J = 9.9Hz, 2H), 3–3.13 (m, 5H), 3.22 (d, J= 10.4Hz, 3H), 3.35–3.42 (m, 5H), 3.5–3.78 (m, 12H), 3.87 (dt, J = 11.2, 8.8Hz, 3H), 4.03 (s, 8H), 4.35 (dt, J = 15.4, 5.3Hz, 1H), 4.49 (d, J = 8.4Hz, 3H), 4.97 (dd, J = 11.2, 3.4Hz, 3H), 5.22 (d, J = 3.4Hz, 3H), 7.45–8.24 (m, 6H). LCMS m / z ESI expected value 1789.9, measured value 1790.7 ([M+H]). + ).

[0663] At 0 °C, chromium-sulfuric acid (1.228 mL, 2.46 mmol) was added to a solution of (2-7) (2.2 g, 1.23 mmol) in acetone (25 mL). The resulting mixture was stirred at room temperature for 16 hours. The reaction was quenched with i-PrOH (50 mL), and the solvent was removed under reduced pressure. The crude product was diluted with DCM (100 mL) and washed successively with water (100 mL), saturated NaHCO3 (2 × 100 mL), and saturated brine (100 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to obtain the crude product. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 40% MeCN in water (0.1% FA). The pure fraction was evaporated to dryness to obtain a colorless oily substance. The product was then lyophilized to obtain a white solid 6-((3R,5R)-3,4,5-tris(3-((6-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)hexyl)amino)-3-oxopropoxy)-N-methylcyclohexane-1-carbamoyl)hexanoic acid (2-8) (2.300 g, 82%).

[0664] 1H-NMR (500MHz, DMSO, 25℃) δ 1.17–1.31 (m, 15H), 1.32–1.35 (m, 7H), 1.37–1.41 (m, 7H), 1.49–1.67 (m, 7H), 1.77 (s, 9H), 1.90 (s, 8H), 2.00 (s, 8H), 2.11 (s, 8H), 2.20 (dt, J = 15.0, 7.5Hz, 2H), 2.25–2.34 (m, 5H), 2.66–2.84 (m, 3H), 2.95 (s, 2H), 2.98–3.14 (m, 6H), 3.17–3.33 (m, 3H), 3.33–3.46(m, 4H), 3.5–3.59 (m, 5H), 3.6–3.79 (m, 7H), 3.87 (dt, J = 11.5, 8.9Hz, 3H), 4.03 (q, J = 4.0Hz, 9H), 4.49 (dd, J = 8.4, 1.4Hz, 3H), 4.97 (dd, J = 11.2, 3.4Hz, 3H), 5.22 (d, J = 3.4Hz, 3H), 7.6–8.04 (m, 6H). LCMS m / z ESI + Expected value: 1803.8, measured value: 903.4 (z = 2).

[0665] At room temperature, perfluorophenyl 2,2,2-trifluoroacetic acid (0.286 mL, 1.66 mmol) was added to a solution of (2-8) (1 g, 0.55 mmol) and DIEA (0.290 mL, 1.66 mmol) in DMF (20 mL). The resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with EtOAc (50 mL) and washed successively with 1 M NaHSO4 (4 × 50 mL), saturated NaHCO3 (2 × 50 mL), and saturated brine (50 mL). The organic layer was dried with Na2SO4, filtered, and evaporated to dryness. Then, it was lyophilized to obtain a yellow solid 6-[methyl-[(3R,5R)-3,4,5-tris[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxohexylamino]-3-oxo-propoxy]cyclohexanecarbonyl]amino]hexanoic acid (2,3,4,5,6-pentafluorophenyl) ester (2) (1.02 g, 93%).

[0666] 1H-NMR (300MHz, DMSO, 22℃) δ 0.95–1.3 (m, 15H), 1.40 (d, J = 27.5Hz,19H), 1.62–1.72 (m, 2H), 1.77 (s, 8H), 1.89 (s, 8H), 1.99 (s,9H), 2.10 (s,8H), 2.17–2.38 (m, 6H), 2.78 (dt, J = 10.2, 3.6Hz, 4H), 2.88–3.11 (m, 8H), 3.17–3.36 (m, 3H), 3.37–3.46 (m, 5H), 3.49–3.79 (m, 11H), 3.87 (q, J = 9.2Hz, 3H), 4.01 (d, J = 3.4Hz, 8H), 4.49 (d, J = 8.4Hz, 3H), 4.97 (dd, J = 11.2, 3.4Hz, 3H), 5.21 (d, J = 3.4Hz, 3H), 7.65–7.92 (m, 5H), 11.63 (s, 1H). LCMS m / z ESI expected value 1969.8, measured value 1971.2 ([M+H]). + ).

[0667] Synthesis of precursor (3)

[0668]

[0669] Synthesis scheme :

[0670]

[0671] Sodium hydride (2.96 g, 73.91 mmol) was added to a THF (100 mL) solution of (3R,4S,5R)-N-(6-(benzyloxy)hexyl)-3,4,5-trihydroxy-N-methylcyclohexyl-1-ene-1-carboxamide (2-3) (3 g, 7.95 mmol) in nitrogen at 0 °C. The mixture was stirred at 0 °C for 2 h, and then 15-crown-5 (0.875 g, 3.97 mmol) and 2-iodoacetic acid (8.87 g, 47.68 mmol) were added to the mixture. The resulting mixture was stirred at room temperature for 20 h. The reaction was quenched with saturated NH4Cl (15 mL), and the solvent was removed under reduced pressure. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 50% MeCN in water (0.05% TFA). The pure fraction was evaporated to dryness to give 2,2',2''-(((1R,2S,3R)-5-((6-(benzyloxy)hexyl)(methyl)carbamoyl)cyclohex-4-ene-1,2,3-triyl)tri(oxy))triacetic acid (3-1) (2.000 g, 45.6%), which is a yellow gel.

[0672] ¹H-NMR (400MHz, DMSO, 23℃) δ 1.12–1.39 (m, 4H), 1.54 (d, J = 12.8Hz, 4H), 2.21 (ddd, J = 40.1, 16.3, 8.7Hz, 1H), 2.86 (d, J = 43.7Hz, 3H), 3.42 (s, 5H), 3.82 (d, J = 19.2Hz, 3H), 4.03–4.39 (m, 6H), 4.44 (s, 2H), 5.80 (d, J = 71.3Hz, 1H), 7.31 (dp, J = 19.2, 7.2, 6.8Hz, 5H). Three protons have been exchanged. LCMS m / z ESI expected value 551.2, measured value 552.2 ([M+H]) + ).

[0673] Pd / C (10%) (1.929 g, 1.81 mmol) was added to a solution of (3-1) (1 g, 1.81 mmol) in 25 mL of MeOH. The resulting mixture was stirred at room temperature under hydrogen for 16 hours. The reaction mixture was filtered through diatomaceous earth and washed with methanol (200 mL). The solvent was removed under reduced pressure, and the mixture was dried under vacuum to give 2,2',2''-(((1R,2S,3R)-5-((6-hydroxyhexyl)(methyl)carbamoyl)cyclohexane-1,2,3-triyl)tri(oxy))triacetic acid (3-2) (0.500 g, 59.5%) as a colorless oil, which was used without further purification. Compound (3-2) was isolated as a single diastereomer. The stereocenter α of the amide was described as (S), and hydrogenation was expected to occur on the least sterically hindered surface of the alkene.

[0674] ¹H-NMR (400MHz, DMSO, 23℃) δ 1.16–1.32 (m, 4H), 1.34–1.47 (m, 4H), 1.51 (s, 2H), 1.71 (d, J = 39.1Hz, 2H), 2.08 (s, 1H), 2.94–3.03 (m, 2H), 3.37 (q, J = 6.1Hz, 5H), 3.68–4.1 (m, 9H). LCMS m / z ESI expected value 463.2, measured value 464.2 ([M+H]). + ).

[0675] DMAP (211 mg, 1.73 mmol) was added to a solution of (3-2) (400 mg, 0.86 mmol), (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((6-aminohexyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (2-9) (1349 mg, 3.02 mmol), BOP (763 mg, 1.73 mmol), and DIEA (0.452 mL, 2.59 mmol) in 10 mL of DMF. The resulting mixture was stirred at room temperature for 2 hours. The reaction was quenched with water (5 mL), and the solvent was removed under reduced pressure. The crude product was purified by rapid silica gel chromatography with an elution gradient of 7% to 10% MeOH in DCM. The pure fraction was evaporated to dryness to obtain a yellow gel-like substance (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((((2,2'-(((1R,2S,3R,5S)-3-(2-((6-(((2S,3S,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy) (3-3) (370 mg, 24.51%). ((6-hydroxyhexyl)(methyl)carbamoyl)cyclohexane-1,2-diyl)bis(oxy)bis(acetyl)bis(azanediyl)bis(hexane-6,1-diyl)bis(oxy)bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (3-3).

[0676] 1H-NMR (400MHz, DMSO, 25℃) δ 1.22–1.26 (m, 14H), 1.45 (s, 14H), 1.71 (d, J = 11.1Hz, 2H), 1.77–1.78 (m, 11H), 1.89 (s, 9H), 1.99 (s, 9H), 2.10 (s,9H), 2.78 (s, 1H), 2.93–3.03 (m, 7H), 3.42 (s, 10H), 3.66–3.69 (m, 4H), 3.89(dd, J = 11.7, 2.9Hz, 3H), 3.92–3.99 (m, 6H), 4.02 (d, J = 2.0Hz, 8H), 4.97 (dd, J = 11.2, 3.4Hz, 3H), 5.03–5.12 (m, 3H), 5.21 (d, J = 3.4Hz, 3H), 6.95–7.01 (m, 1H), 7.70 (dd, J = 9.3, 3.4Hz, 6H), 7.75–7.87 (m, 5H), 8.19–8.24 (m, 1H). LCMS m / z expected value 1747.8, measured value 1749.6 ([M+H]). + ).

[0677] At 0 °C, chromium-sulfuric acid (0.172 mL, 0.34 mmol) was added to a solution of (3-3) (300 mg, 0.17 mmol) in acetone (10 mL). The resulting mixture was stirred at room temperature for 3 hours. The reaction was quenched with isopropanol (15 mL), and the mixture was poured into ice water (20 mL). The solvent was removed under reduced pressure. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 60% MeCN in water (0.1% TFA). The pure fraction was evaporated to dryness to give 6-((1S,3R,4S,5R)-3,4-bis(2-((6-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)hexyl)amino)-2-oxoethoxy)-5-(2-((6-(((2S,3S,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)hexyl)amino)-2-oxoethoxy)-N-methylcyclohexane-1-carbamoyl)hexanoic acid (3-4) (110 mg, 36.4%), which was a green solid.

[0678] 1H-NMR (400MHz, DMSO, 23℃) δ 1.24 (s, 15H), 1.37–1.5 (m, 15H), 1.62–1.74 (m, 4H), 1.77 (s, 9H), 1.89 (s, 9H), 1.99 (s, 9H), 2.10 (s, 9H), 2.19(t, J = 6.1Hz, 2H), 2.78 (s, 1H), 2.97 (s, 3H), 3.08 (s, 6H), 3.24 (s, 2H), 3.39 (dd, J = 9.9, 6.6Hz, 6H), 3.88 (ddd, J = 19.4, 9.8, 6.8Hz, 9H), 3.94–3.98 (m, 3H), 3.99–4.06 (m, 10H), 4.48 (d, J = 8.5Hz, 3H), 4.96 (dd, J =11.3, 3.4Hz, 3H), 5.21 (d, J = 3.4Hz, 3H), 7.64–7.86 (m, 6H). LCMS m / z ESI + Expected value: 1761.8, measured value: 882.1 (z = 2).

[0679] At room temperature, perfluorophenyl 2,2,2-trifluoroacetic acid (0.054 mL, 0.31 mmol) was added to a solution of (3-4) (110 mg, 0.06 mmol) and DIEA (0.065 mL, 0.37 mmol) in DMF (8 mL). The resulting mixture was stirred at room temperature for 45 minutes. The reaction mixture was diluted with EtOAc (50 mL) and washed successively with 1 M NaHSO4 (3 × 50 mL), saturated NaHCO3 (2 × 50 mL), and saturated brine (2 × 50 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to dryness. The residue was lyophilized to give a white solid (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((((2,2'-(((1R,2S,3R,5S)-3-(2-((6-(((2S,3S,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy) Hexyl)amino)-2-oxoethoxy)-5-(methyl(6-oxo-6-(perfluorophenoxy)hexyl)carbamoyl)cyclohexane-1,2-diyl)bis(oxy))bis(acetyl))bis(azanediyl))bis(hexane-6,1-diyl))bis(oxy))bis(5-acetamido-2-(acetoxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (3) (90 mg, 74.8%).

[0680] 1H-NMR (400MHz, DMSO, 23℃) δ 1.14–1.31 (m, 22H), 1.34–1.55 (m, 16H), 1.68 (dt, J = 14.0, 7.5Hz, 6H), 1.77 (s, 8H), 1.89 (s, 7H), 1.99 (s, 8H), 2.10 (s, 8H), 2.78 (dt, J = 7.4, 4.4Hz, 3H), 2.98 (d, J = 5.4Hz, 2H), 3.02–3.17 (m, 5H), 3.24–3.31 (m, 2H), 3.41 (dd, J = 10.1, 6.5Hz, 3H), 3.53–3.78 (m, 5H), 3.79–3.98 (m, 8H), 3.99–4.27 (m, 8H), 4.3–4.61 (m, 3H), 4.79–5.08 (m, 3H), 5.21 (d, J = 3.4Hz, 3H), 7.59–8.01 (m, 5H), 11.45 (s, 1H). LCMS m / z ESI expected value 1927.8, measured value 1929.1 ([M+H]). + ).

[0681] Synthesis of precursor (4)

[0682]

[0683] Synthesis scheme :

[0684]

[0685]

[0686] In fraction (A), at room temperature, (bromomethyl)benzene (10.78 mL, 90.80 mmol) was added to a solution of 6-hydroxyhexanoic acid (4-1) (10 g, 75.67 mmol) and TEA (31.6 mL, 227.00 mmol) in DCM (200 mL). The resulting mixture was stirred at room temperature for 24 hours. The reaction mixture was poured into water (250 mL), extracted with DCM (2 × 250 mL), and the organic layer was dried over Na2SO4, filtered, and evaporated to dryness. The crude product was purified by rapid silica gel chromatography with an elution gradient of 10% to 70% EtOAc in petroleum ether. The purified fraction was evaporated to dryness to give benzyl 6-hydroxyhexanoate (4-2) (7.00 g, 41.6%) as a colorless oil.

[0687] ¹H-NMR (300MHz, DMSO, 24℃) δ 1.22–1.34 (m, 2H), 1.34–1.47 (m, 2H), 1.55 (p, J = 7.4Hz, 2H), 2.35 (t, J = 7.4Hz, 2H), 3.37 (t, J = 6.4Hz, 2H), 4.35 (brs, 1H), 5.09 (s, 2H), 7.23–7.52 (m, 5H). LCMS m / z ESI expected value 222.1, measured value 223.2 ([M+H]). + ).

[0688] In part (B), TMs-OTf (5.10 mL, 28.25 mmol) was added to a solution of (2S,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-trimethyltriacetate (4-3) (10 g, 25.68 mmol) in 100 mL of DCE at room temperature. The resulting mixture was stirred at 60 °C for 1 hour. After cooling to room temperature, TEA (5.37 mL, 38.53 mmol) was added dropwise to the mixture and stirred for 10 minutes. The reaction mixture was diluted with DCM (50 mL) and washed successively with saturated NaHCO3 (100 mL) and saturated brine (100 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to dryness to obtain (5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazol-6,7-dimethyldiacetate (4-4) (7.50 g, 89%), a light yellow oil. The product can be used directly in the next step without further purification.

[0689] ¹H-NMR (300MHz, DMSO, 25℃) δ 1.96 (d, J = 1.5Hz, 3H), 2.02 (d, J = 2.2Hz, 6H), 2.07 (s, 3H), 3.92–4 (m, 1H), 4–4.17 (m, 2H), 4.27 (ddd, J = 7.5, 5.1, 2.8Hz, 1H), 4.89 (dd, J = 6.8, 3.9Hz, 1H), 5.25 (dd, J = 3.9, 2.9Hz, 1H), 6.06 (d, J = 7.0Hz, 1H). LCMS m / z ESI expected value 329.1, measured value 330.2 ([M+H]). + ).

[0690] At room temperature, (4-2) (5.57 g, 25.05 mmol) was added to a DCE (75 mL) solution of (4-4) (7.5 g, 22.78 mmol) and 4A molecular sieve (3.6 g, 0.00 mmol). After stirring the reaction at 60 °C for 30 min, TMs-OTf (4.12 mL, 22.78 mmol) was added. The resulting mixture was then stirred at 60 °C for another 2 h. The reaction mixture was poured into saturated NaHCO3 (200 mL) and extracted with DCM (2 × 200 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to dryness to give (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((6-(benzyloxy)-6-oxohexyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (4-5) (7.50 g, 59.7%), a yellow oil. The product can be used directly in the next step without further purification.

[0691] 1H-NMR (300MHz, DMSO, 24℃) δ 1.29 (q, J = 8.6Hz, 2H), 1.49 (dq, J =20.3, 7.0, 6.4Hz, 4H), 1.90 (s, 3H), 2.00 (d, J = 1.1Hz, 6H), 2.10 (s, 3H), 2.34 (t, J = 7.3Hz, 2H), 3.41 (dt, J = 9.9, 6.5Hz, 1H), 3.69 (dt, J = 11.6, 6.1Hz, 1H), 3.8–3.9 (m, 1H), 4.03 (d, J = 7.1Hz, 4H), 4.19–4.31 (m, 1H), 4.49 (d, J = 8.5Hz, 1H), 4.97 (dd, J = 11.3, 3.4Hz, 1H), 5.23 (dd, J = 10.7, 3.4Hz, 1H), 7.36 (d, J = 3.8Hz, 5H), 7.81 (d, J = 9.2Hz, 1H). LCMS m / z ESI expected value 551.2, measured value 552.4 ([M+H]). + ).

[0692] A solution of (4-5) (7.0 g, 12.69 mmol) and Pd / C (10%, 50 w / w% water) (1.351 g, 1.27 mmol) in MeOH (80 mL) was stirred for 2 hours at 1 atm under a hydrogen atmosphere and at room temperature. The reaction mixture was filtered through diatomaceous earth. The solvent was removed under reduced pressure to give 6-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)hexanoic acid (4-6) (5.00 g, 85%) as a yellow oil. The product was used directly in the next step without further purification.

[0693] 1H-NMR (300MHz, DMSO, 22℃) δ 1.23–1.32 (m, 2H), 1.48 (t, J = 7.7Hz,4H), 1.89 (s, 3H), 2.00 (s, 3H), 2.07 (s, 3H), 2.10 (s, 3H), 2.18 (t, J =7.4Hz, 2H), 3.3–3.47 (m, 2H), 3.69 (dt, J = 9.8, 6.1Hz, 1H), 3.87 (dt, J =11.3, 8.8Hz, 1H), 4.14–4.29 (m, 1H), 4.49 (d, J = 8.5Hz, 1H), 4.88–5.12 (m, 2H), 5.21 (d, J = 3.4Hz, 1H), 7.81 (d, J = 9.2Hz, 1H), 11.96 (brs, 1H). LCMS m / z ESI expected value 461.1, measured value 484.2 ([M+Na]). + ).

[0694] At room temperature, perfluorophenyl 2,2,2-trifluoroacetic acid (1.821 g, 6.50 mmol) was added to a solution of (4-6) (2.5 g, 5.42 mmol) and DIEA (1.183 mL, 6.77 mmol) in DMF (50 mL). The resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with DCM (200 mL) and washed successively with 1 M NaHSO4 (3 × 100 mL), saturated NaHCO3 (3 × 100 mL), and saturated brine (2 × 100 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to dryness to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((6-oxo-6-(perfluorophenoxy)hexyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (4-7) (2.400 g, 70.6%), a light yellow oil. This product solidified upon standing. No further purification was required before use in the next step.

[0695] 1H-NMR (500MHz, DMSO, 25℃) δ 1.32–1.45 (m, 2H), 1.53 (p, J = 6.7Hz, 2H), 1.6–1.71 (m, 2H), 1.77 (d, J = 3.4Hz, 3H), 1.90 (s, 3H), 2.00 (s, 3H),2.11 (s, 3H), 2.72–2.85 (m, 2H), 3.41–3.77 (m, 2H), 3.88 (dq, J = 10.8,8.4Hz, 1H), 4.04 (s, 2H), 4.09–4.28 (m, 1H), 4.50 (t, J = 7.2Hz, 1H), 4.87–5.01 (m, 1H), 5.22 (t, J = 2.9Hz, 1H), 7.82 (dd, J = 9.2, 4.8Hz, 1H). LCMS m / z ESI expected value 627.1, measured value 650.2 ([M+Na]). + ).

[0696] In part (C), under argon atmosphere, acrylonitrile (126 mL, 1907.38 mmol) was added to a solution of (3R,4S,5R)-N-(6-(benzyloxy)hexyl)-3,4,5-trihydroxy-N-methylcyclohexyl-1-ene-1-carboxamide (2-3) (12 g, 31.79 mmol) and Cs₂CO₃ (31.1 g, 95.37 mmol) in tert-butanol (200 mL). The resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with EtOAc, the solids were filtered off, and the filtrate was evaporated to dryness. The crude product was purified by rapid silica gel chromatography with an elution gradient of 0% to 100% EtOAc in petroleum ether, followed by 0% to 20% MeOH in EtOAc. The pure fraction was evaporated to dryness to give (3R,4S,5R)-N-(6-(benzyloxy)hexyl)-3,4,5-tris(2-cyanoethoxy)-N-methylcyclohex-1-ene-1-carboxamide (4-8) (10.00 g, 58.6%), which is a yellow oil.

[0697] 1H-NMR (400MHz, DMSO, 17℃) δ 1.22 (h, J = 6.1Hz, 2H), 1.33 (t, J =7.6Hz, 2H), 1.53 (td, J = 13.7, 6.8Hz, 4H), 2.08 (s, 3H), 2.52–2.61 (m, 1H), 2.76 (q, J = 9.6, 6.0Hz, 7H), 2.93 (s, 2H), 3.2–3.36 (m, 2H), 3.41 (t, J =6.5Hz, 2H), 3.67–3.89 (m, 6H), 4.18 (t, J = 3.7Hz, 1H), 4.45 (s, 2H), 5.62–5.67 (m, 1H), 7.25–7.4 (m, 5H). LCMS m / z ESI expected value 536.3, measured value 537.4 ([M+H]). + ).

[0698] A solution of (4-8) (2 g, 3.73 mmol), 10% Pd / C (7.93 g, 7.45 mmol), and Boc-anhydride (4.33 mL, 18.63 mmol) in MeOH (130 mL) was stirred at 50 °C for 2 days under a hydrogen atmosphere at 20 atm. The reaction mixture was filtered and the filtrate was concentrated to dryness. The residue was dissolved in MeOH (200 mL), and equal volumes of Pd / C (10%) and Boc-anhydride were added. The mixture was stirred again at 50 °C for 2 days under a hydrogen atmosphere at 20 atm. The reaction mixture was filtered through diatomaceous earth. The filtrate was concentrated to dryness. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 60% MeCN in water (0.1% NH4HCO3). The pure fraction was evaporated to dryness to give di-tert-butyl dicarboxylate (4-9) (1R,3R)-2-(3-((tert-butoxycarbonyl)amino)propoxy)-5-((6-hydroxyhexyl)(methyl)carbamoyl)cyclohexane-1,3-diyl)bis(oxy))bis(propane-3,1-diyl))dicarboxylate (4-9) (0.200 g, 7.05%), which was a colorless liquid.

[0699] ¹H-NMR (300MHz, DMSO, 24℃) δ 1.20 (d, J = 24.3Hz, 6H), 1.37 (s, 29H), 1.58 (s, 10H), 2.52 (s, 1H), 2.77 (s, 3H), 2.98 (d, J = 7.8Hz, 6H), 3.23 (t, J = 7.3Hz, 2H), 3.35–3.54 (m, 8H), 3.54–3.68 (m, 3H), 4.33 (t, J = 5.2Hz, 1H), 6.61–6.82 (m, 2H). One proton has been exchanged. LCMS m / z ESI expected value 760.5, observed value 761.3 ([M+H]). + ).

[0700] The solution of (4-9) (181 mg, 0.24 mmol) in DCM / TFA = 1:1 (12 mL) was stirred at room temperature for 2.5 hours. The solvent was removed under reduced pressure to give (1S,3R,4S,5R)-3,4,5-tris(3-aminopropoxy)-N-(6-hydroxyhexyl)-N-methylcyclohexane-1-carboxamide (4-10) (110 mg, 100%) as a yellow oil. The product could be used directly in the next step without further purification.

[0701] 1H-NMR (300MHz, DMSO, 24℃) δ 1.24 (d, J = 2.7Hz, 3H), 1.31–1.44 (m,3H), 1.57 (q, J = 11.1, 9.8Hz, 4H), 1.75 (dq, J = 17.2, 7.8, 7.1Hz, 9H),2.53–2.63 (m, 1H), 2.78 (s, 2H), 2.87 (p, J = 7.6Hz, 5H), 2.96 (s, 2H), 3.24(t, J = 7.2Hz, 2H), 3.50 (q, J = 6.1Hz, 5H), 3.58–3.77 (m, 4H), 4.38 (td, J =6.6, 2.9Hz, 2H), 7.80 (brs, 6H). LCMS m / z ESI expected value 460.3, measured value 461.3 ([M+H]). + ).

[0702] At room temperature, DIEA (0.279 mL, 1.60 mmol) was added to solutions of (4-10) and (4-7) (627 mg, 0.80 mmol) in 25 mL of DMF. The resulting mixture was stirred at room temperature for 4 hours. The solvent was removed under reduced pressure. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 50% MeCN in water (0.1% FA). The pure fraction was evaporated to dryness to give methyl acetate [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[3-[(1R,3R)-2,3-bis[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxohexanoylamino]propoxy]-5-[6-hydroxyhexyl(methyl)carbamoyl]cyclohexyloxy]propanylamino]-6-oxo-hexyloxy]tetrahydropyran-2-yl]acetate (4-11) (200 mg, 55.9%), which was a yellow solid.

[0703] 1H-NMR (300MHz, DMSO, 24℃) δ 1.17–1.32 (m, 14H), 1.32–1.61 (m, 1), 1.77 (s, 9H), 1.90 (s, 9H), 2.00 (s, 9H), 2.11 (s, 8H), 2.16–2.33 (m, 8H), 2.71 (s, 1H), 2.86 (d, J = 94.2Hz, 3H), 3.03 (s, 6H), 3.24 (s, 3H), 3.36–3.46(m, 4H), 3.55–3.76 (m, 14H), 3.87 (q, J = 9.4Hz, 3H), 4.03 (q, J = 4.0Hz, 8H), 4.49 (d, J = 8.5Hz, 3H), 4.97 (dd, J = 11.2, 3.4Hz, 3H), 5.22 (d, J = 3.5Hz, 3H), 7.79 (dd, J = 30.8, 7.3Hz, 6H), 11.99 (brs, 1H). LCMS m / z ESI expected value 1789.9, measured value 1790.8 ([M+H]). + ).

[0704] At 0 °C, a solution of chromium sulfuric acid in H₂SO₄ (0.098 mL, 0.20 mmol) was added to a solution of (4-11) (175 mg, 0.10 mmol) in acetone (20 mL). The resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was quenched with i-PrOH (10 mL) and the solvent was removed under reduced pressure. The crude product was diluted with DCM (50 mL) and washed successively with water (50 mL), saturated NaHCO₃ (2 × 50 mL), and saturated brine (50 mL). The organic layer was dried over Na₂SO₄, filtered, and evaporated to dryness. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 40% MeCN in water (0.1% FA). The pure fraction was evaporated to dryness to give 6-((3R,5R)-3,4,5-tris(3-(6-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)hexamido)propoxy)-N-methylcyclohexane-1-carbamoyl)hexanoic acid (4-12) (124 mg, 70.3%), which was a yellow solid.

[0705] 1H-NMR (500MHz, DMSO, 25℃) δ 1.17–1.32 (m, 14H), 1.32–1.61 (m, 1), 1.77 (s, 9H), 1.90 (s, 9H), 2.00 (s, 9H), 2.11 (s, 8H), 2.16–2.33 (m, 8H),2.71 (s, 1H), 2.86 (d, J = 94.2Hz, 3H), 3.03 (s, 6H), 3.24 (s, 3H), 3.36–3.46(m, 4H), 3.55–3.76 (m, 11H), 3.87 (q, J = 9.4Hz, 3H), 4.03 (q, J = 4.0Hz, 8H), 4.49 (d, J = 8.5Hz, 3H), 4.97 (dd, J = 11.2, 3.4Hz, 3H), 5.22 (d, J = 3.5Hz, 3H), 7.79 (dd, J = 30.8, 7.3Hz, 6H), 11.99 (brs, 1H). LCMS m / z ESI expected value 1803.8, measured value 1804.6 ([M+H]). + ).

[0706] At room temperature, perfluorophenyl 2,2,2-trifluoroacetic acid (0.023 mL, 0.13 mmol) was added to a DMF (50 mL) solution of (4-12) (80 mg, 0.04 mmol) and DIEA (0.023 mL, 0.13 mmol). The resulting mixture was stirred at room temperature for 20 minutes. The reaction mixture was diluted with EtOAc (50 mL) and washed successively with 1 M NaHSO4 (4 × 50 mL), saturated NaHCO3 (2 × 50 mL), and saturated brine (50 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to dryness. The residue was lyophilized to give 6-[methyl-[(3R,5R)-3,4,5-tris[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyhexanoylamino]propoxy]cyclohexanecarbonyl]amino]hexanoic acid (2,3,4,5,6-pentafluorophenyl) ester (4) (67.0 mg, 77%) as a gray solid.

[0707] 1H-NMR (400MHz, DMSO, 21℃) δ 1.19–1.31 (m, 14H), 1.39–1.5 (m, 13H), 1.52–1.67 (m, 13H), 1.77 (s, 8H), 1.89 (s, 8H), 1.97–2.06 (m, 13H), 2.10 (s,8H), 2.69–2.85 (m, 5H), 2.88–3 (m, 7H), 3.03–3.16 (m, 6H), 3.53–3.76 (m,10H), 3.79–3.94 (m, 3H), 3.96–4.07 (m, 9H), 4.48 (d, J = 8.5Hz, 3H), 4.96 (dd, J = 11.2, 3.4Hz, 3H), 5.21 (d, J = 3.4Hz, 3H), 7.69–7.87 (m, 5H). One proton has been exchanged. LCMS m / z ESI + Expected value: 1969.8, measured value: 986.2 (z = 2).

[0708] Alternatively, perfluorophenyl 2,2,2-trifluoroacetic acid (0.029 mL, 0.18 mmol) was added to a solution of (4-12) (100 mg, 0.06 mmol) and DIEA (0.048 mL, 0.28 mmol) in DCM (2 mL) at room temperature. The resulting mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with EtOAc (50 mL) and washed successively with 1 M NaHSO4 (4 × 20 mL), saturated NaHCO3 (2 × 20 mL), and saturated brine (20 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to dryness. The residue was diluted with 5 mL of MeCN / water (1:1) and lyophilized to give (4) (108.0 mg, 88%) as a beige solid. The product was ready for use in the next reaction without any further purification. LCMS m / z expected value 1969.863, found value 986.8 [M+H] + (z = 2).

[0709] Synthesis of precursor (5)

[0710]

[0711] Synthesis scheme :

[0712]

[0713] 6-((tert-butyldimethylsilyl)oxy)hexyl-1-amine (5 g, 21.60 mmol) was added to a solution of (3R,4S,5R)-3,4,5-trihydroxycyclohexyl-1-en-1-carboxylic acid (4.14 g, 23.76 mmol), EDC (8.70 g, 45.37 mmol), HOBt (6.62 g, 43.20 mmol), and DIEA (11.32 mL, 64.81 mmol) in DMF (50 mL). The resulting mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by rapid silica gel chromatography with an elution gradient of 0% to 5% MeOH in DCM. The pure fraction was evaporated to dryness to give (3R,4S,5R)-N-(6-((tert-butyldimethylsilyl)oxy)hexyl)-3,4,5-trihydroxycyclohex-1-en-1-carboxamide (5-2) (4.40 g, 72%), which was a yellow liquid.

[0714] 1H-NMR (400MHz, DMSO, 24℃) δ 0.10 (d, J = 1.7Hz, 6H), 0.85 (d, J =6.3Hz, 9H), 1.24 (s, 2H), 1.33–1.49 (m, 6H), 1.94–2.02 (m, 2H), 3.07–3.09 (m,2H), 3.56 (m, 4H), 4.03 (q, J = 7.1Hz, 1H), 4.17 (d, J = 3.8Hz, 1H), 6.27(dd, J = 3.4, 1.7Hz, 1H), 7.23–7.32 (m, 2H), 7.80 (q, J = 1.8, 1.4Hz, 1H). The expected value of LCMS m / z is 387.2, and the measured value is 388.4.

[0715] Cs₂CO₃ (24.13 g, 74.07 mmol) was added to a solution of tert-butyl acrylate (197 mL, 1346.78 mmol) and (3R,4S,5R)-N-(6-((tert-butyldimethylsilyl)oxy)hexyl)-3,4,5-trihydroxycyclohex-1-en-1-carboxamide (5-2) (8.7 g, 22.45 mmol) in t-BuOH (800 mL). The resulting mixture was stirred at room temperature for 3 days. The reaction mixture was diluted with EtOAc (1 L) and filtered through a diatomaceous earth filter. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by rapid silica gel chromatography with an elution gradient of 0% to 50% EtOAc in petroleum ether. The pure fraction was evaporated to dryness to give tri-tert-butyl 3,3',3''-(((1R,2S,3R)-5-((6-((tert-butyldimethylsilyl)oxy)hexyl)carbamoyl)cyclohex-4-en-1,2,3-triyl)tri(oxy))tripropionate (5-3) (8.30 g, 47.9%), which was a yellow oil. The crude product could be used directly in the next step without further purification.

[0716] ¹H-NMR (400MHz, DMSO, 24℃) δ 0.86 (s, 9H), 1.12 (s, 2H), 1.40 (dd, J = 4.3, 1.3Hz, 31H), 1.47–1.58 (m, 2H), 2.36–2.43 (m, 8H), 3.06 (d, J = 6.6Hz, 2H), 3.49 (t, J = 6.2Hz, 1H), 3.65–3.77 (m, 9H), 3.92 (dt, J = 21.1, 4.6Hz, 1H), 6.26–6.31 (m, 1H), 7.87 (tdd, J = 8.2, 5.6, 2.8Hz, 1H). Three protons were exchanged. The expected value of LCMS m / z is 771.5, and the measured value is 772.4.

[0717] TBAF (1M THF solution) (9.45 mL, 9.45 mmol) was added to a THF solution of 3,3',3''-(((1R,2S,3R)-5-((6-((tert-butyldimethylsilyl)oxy)hexyl)carbamoyl)cyclohex-4-en-1,2,3-triyl)tri(oxy))tripropionate (5-3) (7.3 g, 9.45 mmol). The resulting mixture was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by rapid silica gel chromatography with an elution gradient of 0% to 80% EtOAc in petroleum ether. The pure fraction was evaporated to dryness to give 3,3',3''-(((1R,2S,3R)-5-((6-hydroxyhexyl)carbamoyl)cyclohex-4-ene-1,2,3-triyl)tri(oxy))tri-tert-butyl tripropionate (5-4) (1.500 g, 24.12%), which was a colorless oil.

[0718] 1H-NMR (400MHz, DMSO, 23℃) δ 1.18–1.31 (m, 4H), 1.37–1.45 (m, 29H), 2.16 (dd, J = 18.2, 3.6Hz, 1H), 2.32–2.47 (m, 7H), 3.06 (q, J = 6.6Hz, 2H), 3.17 (d, J = 5.2Hz, 3H), 3.37 (m, 2H), 3.73 (m, 6H), 4.10 (q, J = 5.2Hz, 1H), 4.33 (t, J = 5.1Hz, 1H), 6.29 (d, J = 2.5Hz, 1H), 7.87 (q, J = 5.8, 4.5Hz, 1H). One proton is exchanged. LCMS m / z expected value 657.4, measured value 658.5.

[0719] A solution of 3,3',3''-(((1R,2S,3R)-5-((6-hydroxyhexyl)carbamoyl)cyclohex-4-en-1,2,3-triyl)tri(oxy))tripropionate tritert-butyl ester (5-4) (1.5 g, 2.28 mmol) and Pd-C (10%) (0.485 g, 0.46 mmol) in MeOH (30 mL) was stirred for 16 hours at room temperature under a hydrogen atmosphere at 1 atm. The reaction mixture was filtered through diatomaceous earth. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by rapid silica gel chromatography with an elution gradient of 10% to 70% EtOAc in petroleum ether. The pure fraction was evaporated to dryness to give 3,3',3''-(((1R,2S,3R,5S)-5-((6-hydroxyhexyl)carbamoyl)cyclohexane-1,2,3-triyl)tri(oxy))tri-tert-butyl tripropionate (5-5) (1.000 g, 66.5%), which is a colorless oil.

[0720] ¹H-NMR (400MHz, DMSO, 21℃) δ 1.05–1.3 (m, 5H), 1.35–1.45 (m, 30H), 1.47–1.67 (m, 4H), 2.18–2.47 (m, 7H), 2.99 (q, J = 6.5Hz, 2H), 3.35–3.45 (m, 3H), 3.47–3.72 (m, 7H), 3.78 (m, 1H), 4.33 (t, J = 5.2Hz, 1H), 7.69 (q, J = 6.5, 5.6Hz, 1H). LCMS m / z expected value 659.4, measured value 660.2.

[0721] At room temperature, TFA (3.15 mL, 40.92 mmol) was added to a solution of 0.9 g (1.36 mmol) of tritert-butyl tripropionate (5-5) in DCM (10 mL). The resulting mixture was stirred at room temperature for 3 hours. The solvent was removed under reduced pressure to give 3,3',3''-(((1R,2S,3R,5S)-5-((6-(2,2,2-trifluoroacetoxy)hexyl)carbamoyl)cyclohexane-1,2,3-triyl)tri(oxy))tripropionate (5-6) (0.790 g, 99%) as a yellow oil. The product was used directly in the next step without further purification.

[0722] 1H-NMR (300MHz, DMSO, 23℃) δ 1.26–1.4 (m, 6H), 1.46–1.63 (m, 4H), 1.64–1.72 (m, 2H), 2.29 (p, J = 6.1Hz, 1H), 2.35–2.49 (m, 7H), 2.99 (q, J =6.4Hz, 2H), 3.41 (d, J = 8.3Hz, 1H), 3.59–3.67 (m, 5H), 3.72–3.83 (m, 2H), 4.37 (t, J = 6.6Hz, 2H), 7.70 (t, J = 5.5Hz, 1H), 12.61 (s, 3H). LCMS m / z expected 587.2, observed 588.2.

[0723] At room temperature, perfluorophenyl 2,2,2-trifluoroacetic acid (0.921 mL, 5.36 mmol) was added to a solution of 3,3',3''-(((1R,2S,3R,5S)-5-((6-(2,2,2-trifluoroacetoxy)hexyl)carbamoyl)cyclohexane-1,2,3-triyl)tri(oxy))tripropionic acid (5-6) (0.7 g, 1.19 mmol) and DIEA (1.353 mL, 7.74 mmol) in DCM (20 mL). The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with DCM (20 mL) and washed successively with 1 M NaHSO4 (2 × 25 mL), saturated NaHCO3 (2 × 15 mL), and saturated brine (30 mL). The organic layer was dried over Na₂SO₄, filtered, and evaporated to obtain a yellow oily product: tris(perfluorophenyl)3,3',3''-(((1R,2S,3R,5S)-5-((6-(2,2,2-trifluoroacetoxy)hexyl)carbamoyl)cyclohexane-1,2,3-triyl)tri(oxy))tripropionate (5-7) (1.200 g, 93%). The product can be used directly in the next step without further purification.

[0724] ¹H-NMR (300MHz, DMSO, 23℃) δ 1.27–1.43 (m, 6H), 1.53–1.72 (m, 6H), 2.32 (d, J = 10.1Hz, 1H), 2.87–3.11 (m, 11H), 3.74–3.92 (m, 5H), 3.98 (m, 1H), 4.36 (t, J = 6.6Hz, 2H), 7.71 (t, J = 5.6Hz, 1H). LCMS m / z expected value 1085.1, measured value 1086.0.

[0725] N-Ethyl-N-isopropylpropyl-2-amine (1.126 mL, 6.45 mmol) was added to a solution of tris(perfluorophenyl)3,3',3''-(((1R,2S,3R,5S)-5-((6-(2,2,2-trifluoroacetoxy)hexyl)carbamoyl)cyclohexane-1,2,3-triyl)tri(oxy))tripropionate (5-7) (700 mg, 0.64 mmol) and (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((6-aminohexyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (1152 mg, 2.58 mmol) in DMF (10 mL). The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with water (15 mL). The solvent was removed under reduced pressure to give the crude product. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 50% MeCN in water (0.1% FA). The purified fraction was evaporated to dryness to give methyl acetate [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[3-[(1R,3R)-2,3-bis[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxohexylamino]-3-oxo-propoxy]-5-(6-hydroxyhexylcarbamoyl)cyclohexyloxy]propionylamino]hexyloxy]tetrahydropyran-2-yl]acetate (5-8) as a yellow solid (450 mg, 39.3%).

[0726] 1H NMR (300MHz, DMSO, 23℃) δ 1.25 (s, 16H), 1.47 (t, J = 26.6Hz,17H), 1.78 (s, 9H), 1.90 (s, 9H), 2.00 (s, 10H), 2.10 (d, J = 8.4Hz, 12H), 2.29 (d, J = 7.7Hz, 5H), 3.02 (d, J = 6.4Hz, 5H), 3.17 (q, J = 6.5Hz, 3H), 3.41 (d, J = 9.6Hz, 10H), 3.53–3.79 (m, 9H), 3.87 (q, J = 9.3Hz, 3H), 4.03(s, 9H), 4.49 (d, J = 8.4Hz, 3H), 4.97 (dd, J = 11.2, 3.4Hz, 3H), 5.22 (d, J = 3.4Hz, 3H), 7.48–8.16 (m, 6H), 9.41 (s, 1H). LCMS m / z expected value 1775.8, measured value 1776.8 ([M+H]). + ).

[0727] At 0 °C, chromium sulfuric acid (0.225 mL, 0.45 mmol) was added to a solution of methyl acetate [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[3-[(1R,3R)-2,3-bis[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxohexylamino]-3-oxo-propoxy]-5-(6-hydroxyhexylcarbamoyl)cyclohexylamino]propionylamino]hexyloxy]tetrahydropyran-2-yl]acetate (5-8) (400 mg, 0.23 mmol) in acetone (10 mL). The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with i-PrOH (20 ml), diluted with DCM (100 ml), and washed successively with saturated NaHCO3 (2 × 120 ml) and saturated brine (2 × 120 ml). The organic layer was dried over Na2SO4, filtered, and evaporated to obtain the crude product. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 40% MeCN in water (0.1% FA). The pure fraction was evaporated to dryness to give 6-((3R,5R)-3,4,5-tris(3-((6-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)hexyl)amino)-3-oxopropoxy)cyclohexane-1-carbamoyl)hexanoic acid (5-9) (200 mg, 49.6%) as a yellow solid.

[0728] 1H NMR (500MHz, DMSO, 23℃) δ 1.25 (d, J = 5.6Hz, 15H), 1.37 (q, J =7.0Hz, 8H), 1.46 (p, J = 6.9, 6.5Hz, 8H), 1.53 (d, J = 24.7Hz, 4H), 1.78 (s,9H), 1.90 (s, 9H), 2.00 (s, 9H), 2.11 (s, 10H), 2.27 (ddt, J = 19.8, 14.7,6.9Hz, 7H), 3.01 (dp, J = 18.6, 6.4, 5.9Hz, 8H), 3.41 (dd, J = 10.1, 6.3Hz, 4H), 3.51–3.75 (m, 11H), 3.87 (q, J = 9.5Hz, 3H), 4.02 (q, J = 3.9Hz, 9H), 4.50 (dd, J = 8.5, 3.4Hz, 3H), 4.98 (dt, J = 11.4, 2.9Hz, 3H), 5.22 (d, J = 3.4Hz, 3H), 7.6–8.04 (m, 7H). LCMS m / z expected value 1789.8, measured value 1791.8 ([M+H]). + ).

[0729] At room temperature, perfluorophenyl 2,2,2-trifluoroacetic acid (0.032 mL, 0.18 mmol) was added to a solution of (5-9) (110 mg, 0.06 mmol) and DIEA (0.054 mL, 0.31 mmol) in 2 mL of DCM. The resulting mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with EtOAc (50 mL) and washed successively with 1 M NaHSO4 (4 × 20 mL), saturated NaHCO3 (2 × 20 mL), and saturated brine (20 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to dryness. The residue was diluted with 5 mL of MeCN / water (1:1) and lyophilized to give (5) (107.0 mg, 89%) as a beige solid. The product was ready for use in the next reaction without any further purification. LCMS m / z expected value 1955.8, found value 979.4 [M+H] + (z = 2).

[0730] Synthesis of precursor (6)

[0731]

[0732] Synthesis scheme

[0733]

[0734] At room temperature, 1 g (1.31 mmol) of 3,3',3''-(((1R,2S,3R)-5-((6-(benzyloxy)hexyl)(methyl)carbamoyl)cyclohex-4-ene-1,2,3-triyl)tri(oxy))tripropionate tritert-butyl ester (2a-4) was added to HBr (48% aqueous solution, 25 mL). The resulting mixture was stirred at room temperature for 1 hour. The resulting mixture was then stirred at 60 °C for 16 hours. The solvent was removed under reduced pressure. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 25% MeCN in water (0.1% TFA). The pure fraction was evaporated to dryness to obtain 3,3',3''-(((1R,2S,3R)-5-((6-hydroxyhexyl)(methyl)carbamoyl)cyclohex-4-ene-1,2,3-triyl)tri(oxy))tripropionic acid (6-1) (0.160 g, 24.21%), which is a light yellow gel.

[0735] ¹H-NMR (400MHz, DMSO, 21℃) δ 1.08–1.74 (m, 9H), 2.01 (d, 1H), 2.35–2.48 (m, 6H), 2.83 (d, J = 40.9Hz, 3H), 3.25 (s, 2H), 3.38 (t, J = 6.5Hz, 1H), 3.63–3.83 (m, 8H), 4.04 (s, 2H), 4.37 (t, J = 6.6Hz, 1H), 5.50 (s, 1H), 12.19 (brs, 2H). LCMS m / z expected value 503.2, measured value 504.3 ([M+H]). +Add 3,3',3''-(((1R,2S,3R)-5-((6-hydroxyhexyl)(methyl)carbamoyl)cyclohex-4-en-1,2,3-triyl)tri(oxy))tripropionic acid (6-1) (160 mg, 0.32 mmol) to N-ethyl-N-isopropylpropyl-2-amine (1.110 mL, 6.35 mmol) and ((1H-benzo[d][1,2,3]triazol-1-yl)oxy)tri (Dimethylamino)phosphonium hexafluorophosphate (V) (703 mg, 1.59 mmol), (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((6-aminohexyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (2-9) (709 mg, 1.59 mmol), and DMAP (116 mg, 0.95 mmol) in DMF (8 mL). The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 40% MeCN in water (0.1% FA). The pure fraction was evaporated to dryness to give methyl acetate [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[3-[(1R,5R,6S)-5,6-bis[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxohexylamino]-3-oxo-propoxy]-3-[6-hydroxyhexyl(methyl)carbamoyl]cyclohex-3-en-1-yl]oxopropionylamino]hexyloxy]tetrahydropyran-2-yl]acetate (6-2) (100 mg, 17.59%), which was a light yellow solid.

[0736] 1H NMR (300MHz, DMSO, 28℃) δ 1.2–1.46 (m, 32H), 1.77 (s, 9H), 1.90 (s, 8H), 2.00 (s, 11H), 2.11 (s, 9H), 2.22–2.39 (m, 7H), 2.65–2.93 (m, 6H), 2.94–3.08 (m, 6H), 3.6–3.79 (m, 13H), 3.8–3.93 (m, 4H), 4.03 (s, 11H), 4.49 (d, J = 8.4Hz, 3H), 4.97 (dd, J = 11.2, 3.4Hz, 3H), 5.22 (d, J = 3.3Hz, 3H), 5.49 (s, 1H), 7.76–7.94 (m, 5H). LCMS m / z expected value 1787.8, measured value 895.5 ([M+H]).+ (z = 2).

[0737] At 0 °C, chromium sulfuric acid (0.056 mL, 0.11 mmol) was added to a solution of methyl acetate [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[3-[(1R,5R,6S)-5,6-bis[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxohexylamino]-3-oxo-propoxy]-3-[6-hydroxyhexyl(methyl)carbamoyl]cyclohex-3-en-1-yl]oxopropionylamino]hexyloxy]tetrahydropyran-2-yl]acetate (6-2) (100 mg, 0.06 mmol) in acetone (10 mL). The resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was quenched with isopropanol (5 mL) and diluted with DCM (20 mL). The reaction mixture was poured into water (20 mL) and extracted with DCM (2 × 20 mL). The organic layer was washed with NaHCO3 (2 × 20 mL) and saturated brine (20 mL), dried over Na2SO4, filtered, and evaporated to give a white solid. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 40% MeCN in water (0.1% FA). The pure fraction was evaporated to dryness to give 6-((3R,4S,5R)-3,4,5-tris(3-((6-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)hexyl)amino)-3-oxopropoxy)-N-methylcyclohex-1-en-1-carbamoyl)hexanoic acid (6-3) (53.0 mg, 52.6%) as a white solid.

[0738] 1H NMR (400MHz, DMSO, 20℃) δ 1.24 (s, 17H), 1.32–1.53 (m, 16H), 1.77(s, 9H), 1.90 (s, 9H), 2.00 (s, 10H), 2.11 (s, 10H), 2.23–2.41 (m, 7H), 2.74–2.9 (m, 3H), 2.96–3.08 (m, 6H), 3.24 (s, 3H), 3.59–3.81 (m, 11H), 3.82–3.92(m, 3H), 3.98–4.11 (m, 10H), 4.49 (d, J = 8.4Hz, 3H), 4.97 (dd, J = 11.2, 3.2Hz, 3H), 5.22 (d, J = 3.3Hz, 3H), 5.49 (s, 1H), 7.7–8.03 (m, 6H). LCMS m / z expected value 1081.8, measured value 902.3 ([M+H]). + (z = 2).

[0739] At room temperature, perfluorophenyl 2,2,2-trifluoroacetic acid (0.025 mL, 0.15 mmol) was added to a solution of (6-3) (53 mg, 0.03 mmol) and DIEA (0.051 mL, 0.29 mmol) in 2 mL of DCM. The resulting mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with EtOAc (50 mL) and washed successively with 1 M NaHSO4 (4 × 20 mL), saturated NaHCO3 (2 × 20 mL), and saturated brine (50 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to dryness. The residue was diluted with 5 mL of MeCN / water (1:1) and lyophilized to give (6) (40.0 mg, 69%) as a light brown solid. The product was ready for use in the next reaction without any further purification.

[0740] Synthesis of precursor (7)

[0741]

[0742] Synthesis scheme :

[0743]

[0744] DIEA (2.93 mL, 16.77 mmol) was added to a solution of 3,3',3''-(((1R,2S,3R,5S)-5-(methyl(6-(2,2,2-trifluoroacetoxy)hexyl)carbamoyl)cyclohexane-1,2,3-triyl)tri(oxy))tripropionic acid, trifluoroacetic acid (1 g, 1.40 mmol), tert-butyl (2-6) carbamate (1.461 g, 8.38 mmol), EDC (2.68 g, 13.97 mmol), and HOBt (2.140 g, 13.97 mmol) in DMF (10 mL). The resulting mixture was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 100% MeCN in water (0.1% NaHCO3). The pure fraction was evaporated to dryness to give a yellow oily substance, (((3,3'-(((1R,2S,3R,5S)-2-(3-((3-((tert-butoxycarbonyl)amino)propyl)amino)-3-oxopropoxy)-5-((6-hydroxyhexyl)(methyl)carbamoyl)cyclohexane-1,3-diyl)bis(oxy))bis(propionyl))bis(azanediyl))bis(propane-3,1-diyl))dicarbamate (7-1) (0.300 g, 22.04%).

[0745] ¹H NMR (300MHz, DMSO, 23℃) δ 1.37 (s, 38H), 1.48 (dd, J = 12.7, 6.0Hz, 11H), 2.08–2.33 (m, 8H), 2.77 (s, 2H), 2.85–2.96 (m, 10H), 2.97–3.11 (m, 8H), 3.5–3.8 (m, 9H). LCMS m / z expected value 973.6, measured value 974.6 ([M+H]). + ).

[0746] At 0 °C, TFA (3.08 mL, 40.03 mmol) was added to a solution of tert-butyl carbamate (7-1) (300 mg, 0.31 mmol) in 3 mL of DCM. The resulting mixture was stirred at 0 °C for 2 hours. The solvent was removed under reduced pressure to obtain 6-((1S,3R,4S,5R)-3,4,5-tris(3-((3-aminopropyl)amino)-3-oxopropoxy)-N-methylcyclohexane-1-carbamoyl)hexyl 2,2,2-trifluoroacetate (7-2) (100 mg, 42.2%), a yellow oil. The product can be used directly in the next step without further purification.

[0747] 1H NMR (400MHz, DMSO, 23℃) δ 1.23 (d, J = 7.5Hz, 2H), 1.41 (d, J =7.2Hz, 2H), 1.52 (d, J = 9.5Hz, 4H), 1.62–1.74 (m, 10H), 2.31 (dq, J = 12.0,6.4Hz, 7H), 2.75–2.79 (m, 6H), 2.84–2.9 (m, 3H), 3.09–3.15 (m, 6H), 3.21–3.29(m, 2H), 3.39 (ddd, J = 17.9, 9.4, 4.6Hz, 2H), 3.55–3.68 (m, 7H), 4.38 (td, J = 6.6, 3.6Hz, 2H), 8.08 (t, J = 5.4Hz, 3H). LCMS m / z expected value 769.4, measured value 770.4 ([M+H]). + ).

[0748] DIEA (0.681 mL, 3.90 mmol) was added to a solution of 6-((1S,3R,4S,5R)-3,4,5-tris(3-((3-aminopropyl)amino)-3-oxopropoxy)-N-methylcyclohexane-1-carbamoyl)hexyl 2,2,2-trifluoroacetate (7-2) (300 mg, 0.39 mmol) and (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-oxo-5-(perfluorophenoxy)pentyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (6-12) (1434 mg, 2.34 mmol) in DMF (5 mL). The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 100% MeCN in water (0.1% FA). The pure fraction was evaporated to dryness to give a colorless oily product, methyl [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[5-[3-[3-[(1R,3R)-2,3-bis[3-[3-[5-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxopentanoylamino]propylamino]-3-oxo-propoxy]-5-[6-hydroxyhexyl(methyl)carbamoyl]cyclohexyloxy]propionylamino]propylamino]-5-oxo-pentanoyl]tetrahydropyran-2-yl]acetate (7-3) (300 mg, 39.2%).

[0749] 1H NMR (400MHz, DMSO, 20℃) δ 1.24 (s, 2H), 1.78 (s, 18H), 1.95 (d, J= 42.6Hz, 46H), 2.28 (p, J = 5.6, 5.0Hz, 13H), 2.95 (s, 3H), 3.02 (s, 2H),3.54–3.7 (m, 20H), 3.7–3.78 (m, 9H), 4.48 (d, J = 8.4Hz, 9H), 4.97 (dd, J =11.3, 3.4Hz, 6H), 7.83 (q, J = 8.5, 7.0Hz, 12H), 8.14 (s, 3H). LCMS m / z expected value 1960.9, measured value 1962.3 ([M+H]) + ).

[0750] At 0 °C, chromium sulfuric acid (0.071 mL, 0.14 mmol) was added to a solution of methyl acetate [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[5-[3-[3-[(1R,3R)-2,3-bis[3-[3-[5-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxopentanoylamino]propylamino]-3-oxo-propoxy]-5-[6-hydroxyhexyl(methyl)carbamoyl]cyclohexylamino]propanoylamino]propylamino]-5-oxo-pentanoyl]tetrahydropyran-2-yl]acetate (7-3) (140 mg, 0.07 mmol) in acetone (5 mL). The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 100% MeCN in water (0.05% FA). The purified fraction was evaporated to dryness to give 6-((3R,5R)-3,4,5-tris(3-((3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentamido)propyl)amino)-3-oxopropoxy)-N-methylcyclohexane-1-carbamate)hexanoic acid (7-4) (64.0 mg, 45.4%) as a white solid.

[0751] 1H NMR (500MHz, DMSO, 24℃) δ 1.20 (d, J = 7.2Hz, 1H), 1.24 (s, 1H), 1.4–1.59 (m, 27H), 1.78 (s, 9H), 1.90 (s, 9H), 2.00 (s, 9H), 2.05 (t, J =7.0Hz, 7H), 2.11 (s, 10H), 2.28 (dq, J = 13.2, 6.8Hz, 6H), 2.71 (s, 1H), 2.95(s, 3H), 2.99–3.08 (m, 12H), 3.40 (t, J = 4.4Hz, 3H), 3.42 (t, J = 4.8Hz,3H), 3.60 (dt, J = 16.0, 5.2Hz, 5H), 3.71 (tq, J = 15.5, 9.7, 7.4Hz, 6H), 3.84–3.92 (m, 3H), 4.03 (h, J = 4.1Hz, 9H), 4.46–4.55 (m, 3H), 4.98 (dd, J = 11.2, 3.5Hz, 3H), 5.22 (d, J = 3.4Hz, 3H), 7.68–8.05 (m, 8H). LCMS m / z expected value 1974.9, measured value 1976.70 ([M+H]). + ).

[0752] At room temperature, perfluorophenyl 2,2,2-trifluoroacetic acid (0.026 mL, 0.15 mmol) was added to a solution of (7-4) (60 mg, 0.03 mmol) and DIEA (0.053 mL, 0.3 mmol) in 2 mL of DCM. The resulting mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with EtOAc (50 mL) and washed successively with 1 M NaHSO4 (4 × 20 mL), saturated NaHCO3 (2 × 20 mL), and saturated brine (50 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to dryness. The residue was diluted with 5 mL of MeCN / water (1:1) and lyophilized to give (7) (24.0 mg, 37%) as a light brown solid. The product was ready for use in the next reaction without any further purification. LCMS m / z expected value 2140.9, found value 1072.5 [M+H] + (z = 2).

[0753] Synthesis of precursor (8)

[0754]

[0755] Synthesis scheme :

[0756]

[0757] Under nitrogen atmosphere at 0 °C, TBDPS-Cl (153 mL, 597.11 mmol) was slowly added to a solution of 11-bromoundecane-1-ol (100 g, 398.08 mmol) (8-1) and imidazole (81 g, 1194.23 mmol) in DCM (1500 mL). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into water (1000 mL), and the organic layer was washed successively with saturated NaHCO3 (2 × 1000 mL) and saturated NaCl (2 × 1000 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to give ((11-bromoundecyl)oxy)(tert-butyl)diphenylsilane (8-2) (180 g, 92%) as a yellow oil. The product could be used directly in the next step without further purification.

[0758] ¹H NMR (300MHz, DMSO, 22℃) δ 0.99 (s, 9H), 1.23 (d, J = 4.6Hz, 10H), 1.35 (q, J = 6.9, 6.5Hz, 4H), 1.51 (p, J = 6.4Hz, 2H), 1.77 (p, J = 6.8Hz, 2H), 3.50 (d, J = 13.4Hz, 2H), 3.63 (d, J = 12.6Hz, 2H), 7.38–7.54 (m, 6H), 7.58–7.64 (m, 4H). LCMS m / z expected value 488.2, no quality signal observed.

[0759] A solution of ((11-bromoundecyl)oxy)(tert-butyl)diphenylsilane (8-2) (170 g, 347.21 mmol) in NH3·H2O / dioxane = 1:1 (2500 mL) was stirred in a high-pressure reactor at 100 °C for 16 hours. The solvent was removed under reduced pressure to obtain a yellow oily product of 11-((tert-butyldiphenylsilyl)oxy)undecane-1-amine (8-3) (120 g, 81%). The product could be used directly in the next step without further purification.

[0760] ¹H NMR (300MHz, DMSO, 23℃) δ 0.99 (s, 9H), 1.17–1.37 (m, 14H), 1.49 (dp, J = 14.5, 6.8Hz, 4H), 2.66 (t, J = 7.3Hz, 2H), 3.64 (d, J = 12.6Hz, 2H), 7.4–7.51 (m, 6H), 7.56–7.65 (m, 4H). Two protons exchanged. LCMS m / z expected 425.3, observed 426.2. ([M+H]) + ).

[0761] 11-((tert-butyldiphenylsilyl)oxy)undecane-1-amine (8-3) (55.0 g, 129.20 mmol) was added to a solution of (3R,4S,5R)-3,4,5-trihydroxycyclohexyl-1-en-1-carboxylic acid (22.5 g, 129.20 mmol), EDC (52.0 g, 271.31 mmol), HOBt (39.6 g, 258.39 mmol), and DIEA (67.7 mL, 387.59 mmol) in DMF (750 mL). The resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with H2O (1 L) and evaporated to give the crude product. The crude product was purified by rapid silica gel chromatography with an elution gradient of 0% to 10% MeOH in DCM. The pure fraction was evaporated to dryness to give (3R,4S,5R)-N-(11-((tert-butyldiphenylsilyl)oxy)undecyl)-3,4,5-trihydroxycyclohex-1-ene-1-carboxamide (8-4) (53 g, 70%), which was a yellow oil.

[0762] 1H NMR (300MHz, DMSO, 20℃) δ 0.99 (s, 9H), 1.32 (s, 8H), 1.37–1.64(m, 6H), 1.98 (dd, J = 17.7, 4.7Hz, 1H), 2.46 (s, 1H), 2.78–2.87 (m, 1H),2.97–3.14 (m, 2H), 3.50 (d, J = 5.9Hz, 2H), 3.64 (t, J = 6.3Hz, 3H), 3.82 (d,J = 5.9Hz, 1H), 4.17 (s, 1H), 4.51 (s, 1H), 4.70 (d, J = 27.6Hz, 2H), 6.56 (s, 1H), 7.43–7.46 (m, 5H), 7.59–7.63 (m, 5H), 7.74–7.84 (m, 1H). LCMS m / z expected value 581.3, measured value 582.4 ([M+H]). + ).

[0763] Cs₂CO₃ (98 g, 300.75 mmol) was added to a solution of (3R,4S,5R)-N-(11-((tert-butyldiphenylsilyl)oxy)undecyl)-3,4,5-trihydroxycyclohexyl-1-en-1-carboxamide (50 g, 85.93 mmol) and tert-butyl acrylate (8-4) (755 mL, 5155.80 mmol) in t-BuOH (1000 mL). The resulting mixture was stirred at room temperature for 7 days. The reaction mixture was diluted with EtOAc (2500 mL). The solution was filtered through diatomaceous earth. Solvent was removed under reduced pressure to yield a yellow oily product, tri-tert-butyl 3,3',3''-(((1R,2S,3R)-5-((11-((tert-butyldiphenylsilyl)oxy)undecyl)carbamoyl)cyclohex-4-en-1,2,3-triyl)tri(oxy))tripropionate (8-5) (75 g, 90%). The product can be used directly in the next step without further purification.

[0764] 1H NMR (400MHz, DMSO, 21℃) δ 0.95 (s, 4H), 0.98 (d, J = 3.4Hz, 9H), 1.22 (s, 12H), 1.28–1.56 (m, 34H), 2.13–2.46 (m, 6H), 3.06 (d, J = 6.4Hz,1H), 3.20 (s, 1H), 3.57–3.81 (m, 7H), 4.03 (q, J = 7.1Hz, 1H), 5.84 (dd, J =10.2, 1.8Hz, 1H), 7.38–7.48 (m, 6H), 7.58–7.63 (m, 4H), 7.85 (q, J = 7.2, 6.4 Hz, 1H). LCMS m / z expected value 965.6, measured value 988.8 ([M+Na]). + ).

[0765] A THF solution of TBAF (76 mL, 76.06 mmol) was added to a THF solution of 3,3',3''-(((1R,2S,3R)-5-((11-((tert-butyldiphenylsilyl)oxy)undecyl)carbamoyl)cyclohexyl-4-ene-1,2,3-triyl)tri(oxy))tripropionate (8-5) (70 g, 72.44 mmol). The resulting mixture was stirred at room temperature for 6 hours. The solvent was removed under reduced pressure to give a yellow oily product of 3,3',3''-(((1R,2S,3R)-5-((11-hydroxyundecyl)carbamoyl)cyclohexyl-4-ene-1,2,3-triyl)tri(oxy))tripropionate (8-6) (53 g, 99%). The product was ready for use in the next step without further purification.

[0766] ¹H NMR (400MHz, DMSO, 19℃) δ 0.95 (d, J = 6.3Hz, 18H), 1.18–1.35 (m, 18H), 1.36–1.42 (m, 14H), 1.56 (ddt, J = 13.4, 9.4, 4.7Hz, 5H), 2.36–2.46 (m, 2H), 3.11–3.23 (m, 5H), 3.56–3.63 (m, 2H), 3.63–3.81 (m, 3H), 6.16–6.35 (m, 1H), 7.71 (d, J = 2.1Hz, 1H). LCMS m / z expected value 729.5, measured value 728.5 ([MH]). - ).

[0767] At room temperature, Pd-C (10%, wet) (5.85 g, 5.49 mmol) was added to a solution of 3,3',3''-(((1R,2S,3R)-5-((11-hydroxyundecyl)carbamoyl)cyclohexane-4-ene-1,2,3-triyl)tri(oxy))tripropionate tritert-butyl ester (8-6) (50 g, 68.68 mmol) in 1500 mL of EtOH. The resulting mixture was stirred under hydrogen at room temperature for 16 hours. The mixture was filtered through a diatomaceous earth filter. The solvent was removed under reduced pressure to give 100 g, crude 3,3',3''-(((1R,2S,3R,5S)-5-((11-hydroxyundecyl)carbamoyl)cyclohexane-1,2,3-triyl)tri(oxy))tripropionate as a yellow oil. The crude product was purified by rapid silica gel chromatography with an elution gradient of 0% to 30% THF in petroleum ether. The pure fraction was evaporated to dryness to give 3,3',3''-(((1R,2S,3R,5S)-5-((11-hydroxyundecyl)carbamoyl)cyclohexane-1,2,3-triyl)tri(oxy))tri-tert-butyl tripropionate (8-7) (60.0 g, 60%) as a colorless oil.

[0768] 1H NMR (400MHz, DMSO, 18℃) δ 1.1–1.3 (m, 17H), 1.3–1.47 (m, 29H), 1.49–1.58 (m, 2H), 1.59–1.69 (m, 1H), 1.76 (m, 1H), 2.2–2.33 (m, 1H), 2.34–2.48 (m, 5H), 2.85–3.06 (m, 2H), 3.37 (d, J = 5.6Hz, 4H), 3.64 (m, 6H), 3.78(dt, J = 9.6, 5.6Hz, 1H), 4.33 (m, 1H), 7.64–7.73 (m, 1H). LCMS m / z expected value 729.5, measured value 730.7 ([M+H]) + ).

[0769] At room temperature, TFA (10.55 mL, 136.99 mmol) was added to a 20 mL solution of tritert-butyl tripropionate (8-7) (2 g, 2.74 mmol) in DCM. The resulting mixture was stirred at room temperature for 3 hours. The solvent was removed under reduced pressure to give 3,3',3''-(((1R,2S,3R,5S)-5-((11-(2,2,2-trifluoroacetoxy)undecyl)carbamoyl)cyclohexane-1,2,3-triyl)tri(oxy))tripropionate (8-8) (1.780 g, 99%) as a yellow oil. The product was used directly in the next step without further purification.

[0770] 1H NMR (300MHz, DMSO, 24℃) δ 1.02–1.46 (m, 18H), 1.54 (q, J = 7.8Hz,3H), 1.63–1.8 (m, 2H), 1.99 (s, 2H), 2.2–2.35 (m, 1H), 2.36–2.47 (m, 4H), 2.98 (q, J = 6.6Hz, 2H), 3.39 (ddd, J = 12.6, 9.2, 5.2Hz, 1H), 3.49–3.85 (m,6H), 4.03 (q, J = 7.1Hz, 1H), 4.37 (t, J = 6.6Hz, 2H), 7.63–7.73 (m, 1H). Three protons were exchanged. The expected LCMS m / z value was 657.3, and the observed value was 658.1 ([M+H]). + ).

[0771] At room temperature, perfluorophenyl 2,2,2-trifluoroacetic acid (1.999 mL, 11.63 mmol) was added to a solution of 3,3',3''-(((1R,2S,3R,5S)-5-((11-(2,2,2-trifluoroacetoxy)undecyl)carbamoyl)cyclohexane-1,2,3-triyl)tri(oxy))tripropionic acid (8-8) (1.7 g, 2.58 mmol) and DIEA (2.93 mL, 16.80 mmol) in DMF (25 mL). The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with EtOAc (20 mL) and washed successively with 1 M NaHSO4 (2 × 25 mL), saturated NaHCO3 (2 × 15 mL), and saturated brine (10 mL). The organic layer was dried over Na₂SO₄, filtered, and evaporated to obtain a yellow oily product, tris(perfluorophenyl)3,3',3''-(((1R,2S,3R,5S)-5-((11-(2,2,2-trifluoroacetoxy)undecyl)carbamoyl)cyclohexane-1,2,3-triyl)tri(oxy))tripropionate (8-9) (2.500 g, 84%). The product can be used directly in the next step without further purification.

[0772] 1H NMR (300MHz, DMSO, 24℃) δ 0.81–1 (m, 10H), 1.04 (d, J = 1.1Hz, 2H), 1.09–1.43 (m, 15H), 1.60 (td, J = 18.0, 16.2, 10.2Hz, 5H), 2.92–3.07 (m,2H), 3.1–3.29 (m, 3H), 3.34–3.91 (m, 3H), 3.92–4.18 (m, 1H), 4.37 (td, J =6.7, 2.1Hz, 1H), 7.64–7.69 (m, 1H). LCMS m / z expected value 1155.2, measured value 1156.4 ([M+H]) + ).

[0773] At room temperature, a solution of tris(perfluorophenyl)3,3',3''-(((1R,2S,3R,5S)-5-((11-(2,2,2-trifluoroacetoxy)undecyl)carbamoyl)cyclohexane-1,2,3-triyl)tri(oxy))tripropionate (8-9) (1.5 g, 1.30 mmol) in DMF (20.0 mL) was added to a solution of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((6-aminohexyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (2.086 g, 4.67 mmol) (2-9) and DIEA (2.267 mL, 12.98 mmol) in DMF (50 mL). The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with water (200 mL) and extracted with DCM (3 × 200 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to obtain the crude product. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 50% MeCN in water (0.1% FA). The pure fraction was evaporated to dryness to give methyl acetate [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[3-[(1R,3R)-2,3-bis[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxohexylamino]-3-oxo-propoxy]-5-(11-hydroxyundecylcarbamoyl)cyclohexyloxy]propionylamino]hexyloxy]tetrahydropyran-2-yl]acetate (8-10) (1.500 g, 62.6%), which was a white solid.

[0774] 1H NMR (300MHz, DMSO, 23℃) δ 1.24 (s, 27H), 1.42 (d, J = 24.4Hz,15H), 1.55 (s, 3H), 1.77 (s, 9H), 1.90 (s, 9H), 2.00 (s, 9H), 2.11 (s, 9H), 2.29 (q, J = 7.1Hz, 7H), 3.02 (q, J = 7.0Hz, 8H), 3.28–3.49 (m, 7H), 3.49–3.78 (m, 11H), 3.87 (q, J = 9.7, 9.3Hz, 3H), 3.94–4.1 (m, 9H), 4.32 (t, J =5.1Hz, 1H), 4.49 (d, J = 8.4Hz, 3H), 4.97 (dd, J = 11.3, 3.4Hz, 3H), 5.22 (d, J = 3.4Hz, 3H), 7.55–7.93 (m, 7H). LCMS m / z expected value 1845.9, measured value 1847.7.

[0775] At 0 °C, chromium sulfuric acid (0.650 mL, 1.30 mmol) was added to a solution of methyl acetate [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[3-[(1R,3R)-2,3-bis[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxohexylamino]-3-oxo-propoxy]-5-(11-hydroxyundecylcarbamoyl)cyclohexylamino]propionylamino]hexyloxy]tetrahydropyran-2-yl]acetate (8-10) in acetone (30 mL). The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with i-PrOH (25 mL), diluted with DCM (100 mL), and washed successively with saturated NaHCO3 (100 mL) and saturated brine (100 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to give the crude product. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 30% MeCN in water (0.1% FA). The purified fraction was evaporated to dryness to give 11-((3R,5R)-3,4,5-tris(3-((6-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)hexyl)amino)-3-oxopropoxy)cyclohexane-1-carbamate)undecanoic acid (8-11) (0.400 g, 33.1%) as a white solid.

[0776] 1H NMR (500MHz, DMSO, 25℃) δ 1.24 (d, J = 5.1Hz, 25H), 1.32–1.4 (m,8H), 1.41–1.62 (m, 12H), 1.77 (s, 9H), 1.90 (s, 9H), 2.00 (s, 9H), 2.11 (s,9H), 2.19 (t, J = 7.4Hz, 2H), 2.22–2.33 (m, 6H), 2.86–3.1 (m, 8H), 3.41 (dt,J = 9.7, 6.5Hz, 4H), 3.5–3.79 (m, 11H), 3.87 (m, 3H), 3.94–4.09 (m, 9H), 4.49 (dd, J = 8.4, 1.8Hz, 3H), 4.97 (dd, J = 11.2, 3.4Hz, 3H), 5.22 (d, J = 3.4Hz, 3H), 7.69 (dt, J = 57.9, 5.6Hz, 2H), 7.77–7.93 (m, 5H), 11.95 (s, 1H). The expected LCMS m / z value is 1859.9, and the measured value is 1861.2.

[0777] At room temperature, perfluorophenyl 2,2,2-trifluoroacetic acid (0.028 mL, 0.16 mmol) was added to a solution of (8-11) (100 mg, 0.05 mmol) and DIEA (0.047 mL, 0.27 mmol) in 2 mL of DCM. The resulting mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with EtOAc (50 mL) and washed successively with 1 M NaHSO4 (4 × 20 mL), saturated NaHCO3 (2 × 20 mL), and saturated brine (50 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to dryness. The residue was diluted with 5 mL of MeCN / water (1:1) and lyophilized to give (7) (95.0 mg, 87%) as a light brown solid. The product was ready for use in the next reaction without any further purification. LCMS m / z expected value 2025.9, found value 1014.6 [M+H] + (z = 2).

[0778] Synthesis of precursor (9)

[0779]

[0780] Synthesis scheme :

[0781]

[0782] At room temperature, tert-butyl (9-hydroxynonyl)carbamate (6.93 g, 26.72 mmol) was added to a solution of (5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazol-6,7-dimethyldiacetate (8 g, 24.29 mmol) (9-1) and 4A MS (8 g, 0.00 mmol) in DCE (80 mL). After stirring the reaction at 60 °C for 30 min, TMS-OTf (4.39 mL, 24.29 mmol) was added. The resulting mixture was stirred at 60 °C for 2 h. The reaction mixture was poured into saturated NaHCO3 (200 mL) and extracted with DCM (2 × 250 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((9-((tert-butoxycarbonyl)amino)nonyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (9-2) (8.50 g, 59.4%), a brown oil. The product could be used directly in the next step without further purification.

[0783] 1H NMR (300MHz, DMSO, 23℃) δ 1.21–1.27 (m, 14H), 1.37 (s, 9H), 1.43–1.48 (m, 1H), 1.77 (s, 3H), 1.90 (s, 3H), 2.00 (s, 3H), 2.11 (s, 3H), 2.65(t, J = 7.2Hz, 1H), 2.88 (q, J = 6.5Hz, 2H), 3.36–3.46 (m, 2H), 3.65–3.77 (m,1H), 3.81–3.91 (m, 1H), 4.49 (d, J = 8.5Hz, 1H), 4.97 (dd, J = 11.2, 3.4Hz, 1H), 5.22 (d, J = 3.4Hz, 1H), 6.76 (t, J = 5.7Hz, 1H), 7.79–7.88 (m, 1H). LCMS m / z expected value 588.3, ​​measured value 589.3.

[0784] At room temperature, TFA (26 mL, 337.48 mmol) was added to a solution of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((9-((tert-butoxycarbonyl)amino)nonyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (9-3) (6.5 g, 11.04 mmol) in 130 mL of DCM. The resulting mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, DCM (3 × 50 mL) was added and evaporated to give a brown oily product of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((9-aminononyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (9-3) (5.30 g, 98%). The product was ready for use in the next step without further purification.

[0785] 1H NMR (300MHz, DMSO, 23℃) δ 1.18–1.37 (m, 16H), 1.77 (s, 3H), 1.89(s, 3H), 2.00 (s, 3H), 2.11 (s, 3H), 3.34–3.47 (m, 1H), 3.66–3.75 (m, 1H), 3.82–3.93 (m, 1H), 4.38 (t, J = 6.6Hz, 1H), 4.49 (d, J = 8.5Hz, 1H), 4.97 (dd, J = 11.2, 3.4Hz, 1H), 5.22 (d, J = 3.4Hz, 1H), 7.73 (s, 4H), 13.78–14.01 (m, 1H). LCMS m / z expected value 488.2, measured value 489.2.

[0786] At 0 °C, a DMF (30 mL) solution of tris(perfluorophenyl)3,3',3''-(((1R,2S,3R,5S)-5-((6-(2,2,2-trifluoroacetoxy)hexyl)carbamoyl)cyclohexane-1,2,3-triyl)tri(oxy))tripropionate (2.3 g, 2.12 mmol) (5-7) was added to a DMF (80 mL) solution of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((9-aminononyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (9-3) (4.14 g, 8.47 mmol) and DIEA (2.405 mL, 13.77 mmol). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with water (200 mL) and extracted with DCM (3 × 200 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to obtain the crude product. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 50% MeCN in water (0.1% FA). The pure fraction was evaporated to dryness to give methyl acetate [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[9-[3-[(1R,3R)-2,3-bis[3-[9-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxonylamino]-3-oxo-propoxy]-5-(6-hydroxyhexylcarbamoyl)cyclohexyloxy]propionylamino]nonoxy]tetrahydropyran-2-yl]acetate (9-4) (0.950 g, 23.56%), which was a white solid.

[0787] 1H NMR (300MHz, DMSO, 23℃) δ 1.24 (s, 48H), 1.34–1.42 (m, 12H), 1.46 (s, 2H), 1.55 (s, 1H), 1.77 (s, 9H), 1.90 (s, 9H), 2.00 (s, 9H), 2.11 (s,9H), 2.23–2.32 (m, 7H), 2.94–3.1 (m, 9H), 3.35–3.44 (m, 6H), 3.55–3.63 (m,1H), 3.67–3.74 (m, 4H), 3.82–3.92 (m, 3H), 4.48 (d, J = 8.5Hz, 3H), 4.93–5.02 (m, 3H), 5.22 (d, J = 3.4Hz, 3H), 7.75 (t, J = 5.6Hz, 1H), 7.78–7.88 (m, 5H). LCMS m / z expected value 1902.0, measured value 1903.3.

[0788] At 0 °C, chromium sulfuric acid (0.499 mL, 1.00 mmol) was added to a solution of methyl acetate [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[9-[3-[(1R,3R)-2,3-bis[3-[9-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxonylamino]-3-oxo-propoxy]-5-(6-hydroxyhexylcarbamoyl)cyclohexylamino]propionylamino]nonoxy]tetrahydropyran-2-yl]acetate (9-4) (950 mg, 0.50 mmol) in acetone (15 mL). The resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was quenched with i-PrOH (15 mL), diluted with DCM (60 mL), and washed successively with saturated NaHCO3 (60 mL) and saturated brine (60 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to give the crude product. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 50% MeCN in water (0.1% FA). The purified fraction was evaporated to dryness to give 6-((3R,5R)-3,4,5-tris(3-((9-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)nonyl)amino)-3-oxopropoxy)cyclohexane-1-carbamate)hexanoic acid (9-5) (294 mg, 30.8%) as a white solid.

[0789] 1H NMR (400MHz, DMSO, 20℃) δ 1.24 (s, 31H), 1.33–1.62 (m, 22H), 1.77 (s, 9H), 1.90 (s, 9H), 2.00 (s, 9H), 2.11 (s, 9H), 2.16–2.33 (m, 9H), 2.95–3.09 (m, 8H), 3.37–3.44 (m, 3H), 3.54–3.78 (m, 11H), 3.87 (q, J = 11.1,8.8Hz, 3H), 4.02 (s, 9H), 4.48 (d, J = 8.5Hz, 3H), 4.93–5.01 (m, 3H), 5.22 (d, J = 3.4 Hz, 3H), 7.6–7.91 (m, 7H), 12.00 (s, 1H). LCMS m / z expected value 1916.0, measured value 1917.9.

[0790] At room temperature, perfluorophenyl 2,2,2-trifluoroacetic acid (0.027 mL, 0.16 mmol) was added to a solution of (9-5) (100 mg, 0.05 mmol) and DIEA (0.045 mL, 0.26 mmol) in 2 mL of DCM. The resulting mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with EtOAc (50 mL) and washed successively with 1 M NaHSO4 (4 × 20 mL), saturated NaHCO3 (2 × 20 mL), and saturated brine (50 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to dryness. The residue was diluted with 6 mL of MeCN / water (1:1) and lyophilized to give (9) (89.0 mg, 82%) as a light brown solid. The product was ready for use in the next reaction without any further purification. LCMS m / z expected value 2081.9, found value 1042.4 [M+H] + (z = 2).

[0791] Synthesis of precursor (10)

[0792]

[0793] Synthesis scheme :

[0794]

[0795] (3R,4S,5R)-3,4,5-trihydroxycyclohexyl-1-en-1-carboxylic acid (10.02 g, 57.53 mmol) was added to a solution of N-1-Z-1,6-diaminohexane·HCl (15 g, 52.30 mmol), EDC (21.06 g, 109.83 mmol), HOBt (16.02 g, 104.60 mmol), and DIEA (27.4 mL, 156.90 mmol) in 100 mL of DMF. The resulting mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by rapid silica gel chromatography with an elution gradient of 0% to 30% MeOH in DCM. The pure fraction was evaporated to dryness to give (6-((3R,4S,5R)-3,4,5-trihydroxycyclohex-1-en-1-carbamate)hexyl)carbamate (10-2) (20.00 g, 94%), which was a white gel.

[0796] 1H-NMR (500MHz, DMSO, 25℃) δ 1.25 (s, 4H), 1.39 (p, J = 7.1Hz, 4H), 1.91–2.04 (m, 1H), 2.98 (q, J = 6.6Hz, 2H), 3.07 (m, 3H), 3.49 (t, J = 5.4Hz,2H), 3.81 (q, J = 5.4Hz, 1H), 4.17 (s, 1H), 4.51 (s, 1H), 4.66 (s, 1H), 4.75(s, 1H), 5.01 (s, 2H), 6.27 (dt, J = 3.5, 1.8Hz, 1H), 7.28–7.46 (m, 5H), 7.79 (t, J = 5.7 Hz, 1H). LCMS m / z expected value 406.2, measured value 407.2.

[0797] At room temperature, Cs₂CO₃ (26.5 g, 81.19 mmol) was added to a solution of (6-((3R,4S,5R)-3,4,5-trihydroxycyclohexyl-1-en-1-carbamate)hexyl)carbamate (10⁻²) (10 g, 24.60 mmol) and tert-butyl acrylate (216 mL, 1476.09 mmol) in t-BuOH (750 mL). The resulting mixture was stirred at 40 °C for 5 days. The reaction mixture was filtered through diatomaceous earth. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by rapid silica gel chromatography with an elution gradient of 0% to 60% EtOAc in petroleum ether. The pure fraction was evaporated to dryness to give 3,3',3''-(((1R,2S,3R)-5-((6-((((benzyloxy)carbonyl)amino)hexyl)carbamoyl)cyclohex-4-ene-1,2,3-triyl)tri(oxy))tripropionate tritert-butyl ester (10-3) (10.20 g, 52.4%), which was a colorless oil.

[0798] 1H-NMR (300MHz, CDCl3, 22 ° C) δ 1.35 (d, J = 3.9Hz, 4H), 1.42–1.58 (m, 31H), 2.33–2.62 (m, 8H), 3.25 (dt, J = 21.5, 6.5Hz, 4H), 3.68–3.97 (m, 8H), 5.11 (s, 2H), 5.91 (s, 1H), 6.30 (s, 1H), 7.31–7.48 (m, 5H). Two protons are exchanged. LCMS m / z expected value 790.4, measured value 791.4.

[0799] A solution of 3,3',3''-(((1R,2S,3R)-5-((6-((((benzyloxy)carbonyl)amino)hexyl)carbamoyl)cyclohex-4-ene-1,2,3-triyl)tri(oxy))tripropionic acid (10⁻³) (5 g, 6.32 mmol) in formic acid (75 mL) was stirred at room temperature for 2.5 hours. The solvent was removed under reduced pressure to give a colorless oily product of 3,3',3''-(((1R,2S,3R)-5-((6-((((benzyloxy)carbonyl)amino)hexyl)carbamoyl)cyclohex-4-ene-1,2,3-triyl)tri(oxy))tripropionic acid (10⁻⁴) (3.80 g, 97%). The product was ready for use in the next step without further purification.

[0800] ¹H-NMR (300MHz, CDCl₃, 22℃) δ 1.33 (dd, J = 20.0, 11.4Hz, 8H), 2.37 (d, J = 17.9Hz, 1H), 2.5–2.92 (m, 6H), 3.24 (d, J = 29.1Hz, 3H), 3.57–4.31 (m, 8H), 5.13 (d, J = 7.6Hz, 2H), 6.50 (d, J = 70.0Hz, 2H), 7.36 (s, 4H), 8.07 (s, 5H). Three protons were exchanged. LCMS m / z expected value 622.2, observed value 623.2.

[0801] At room temperature, a DMF (40 mL) solution of (2R,3R,4R,5R)-5-acetamido-2-(acetoxymethyl)-6-((6-aminohexyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (2-9) (10.90 g, 24.41 mmol) was added to N-ethyl-N-isopropylpropyl-2-amine (14.92 mL, 85.44 mmol) and ((1H-benzo[d][1,2,3]triazol-1-yl)oxy The mixture was prepared in 60 mL of DMF in the form of tris(dimethylamino)phosphonium hexafluorophosphate (V) (11.88 g, 26.85 mmol), 3,3',3''-(((1R,2S,3R)-5-((6-((((benzyloxy)carbonyl)amino)hexyl)carbamoyl)cyclohex-4-ene-1,2,3-triyl)tri(oxy))tripropionic acid (10⁻⁴) (3.8 g, 6.10 mmol), and DMAP (2.237 g, 18.31 mmol). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into ice water (150 mL) and extracted with DCM / MeOH = 10:1 (5 × 125 mL). The organic layer was dried over Na₂SO₄, filtered, and evaporated to give the crude product. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 58% MeCN in water (0.1% FA). The purified fraction was evaporated to dryness to give methyl acetate [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[3-[(1R,5R,6S)-5,6-bis[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxohexylamino]-3-oxo-propoxy]-3-[6-(benzyloxycarbonylamino)hexylcarbamoyl]cyclohex-3-en-1-yl]oxopropionylamino]hexyloxy]tetrahydropyran-2-yl]acetate (10-5) (9.00 g, 77%) as a white solid.

[0802] 1H-NMR (300MHz, CDCl3, 22℃) δ 1.3–1.61 (m, 32H), 1.93–2.21 (m, 40H), 2.38–2.69 (m, 7H), 3.16–3.25 (m, 7H), 3.58 (d, J = 47.8Hz, 5H), 3.77–3.97 (m,13H), 3.98–4.3 (m, 11H), 4.68 (d, J = 8.4Hz, 3H), 5.12 (d, J = 10.9Hz, 3H), 5.37 (d, J = 3.4Hz, 3H), 6.38 (s, 1H), 6.76–7.05 (m, 5H), 7.36 (d, J = 3.9Hz, 6H), 7.46–7.56 (m, 1H), 7.77–7.9 (m, 1H). LCMS m / z expected value 1906.9, measured value 1908.1.

[0803] TMS-I (2.140 mL, 15.72 mmol) was added to a solution of [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[3-[(1R,5R,6S)-5,6-bis[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxohexylamino]-3-oxo-propoxy]-3-[6-(benzyloxycarbonylamino)hexylcarbamoyl]cyclohex-3-en-1-yl]oxopropionylamino]hexyloxy]tetrahydropyran-2-yl]acetate (10⁻⁵) (3 g, 1.57 mmol) in 30 mL of MeCN. The resulting mixture was stirred at room temperature for 15 minutes. At 0 °C, TEA (2.191 mL, 15.72 mmol) was added to the reaction mixture. The mixture was stirred at room temperature for 15 minutes. The solvent was removed under reduced pressure to give methyl acetate [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[3-[(1R,5R,6S)-5,6-bis[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxohexylamino]-3-oxo-propoxy]-3-(6-aminohexylcarbamoyl)cyclohexyl-3-en-1-yl]oxopropionylamino]hexyloxy]tetrahydropyran-2-yl]acetate (10⁻⁶) (2.60 g, 93%) as a yellow solid. The product could be used directly in the next step without further purification.

[0804] 1H NMR (400MHz, CDCl3, 21℃) δ 1.33 (d, J = 13.4Hz, 10H), 1.49 (d, J= 7.3Hz, 57H), 1.91–2.08 (m, 23H), 2.54 (d, J = 31.4Hz, 3H), 3.35–3.61 (m,4H), 3.61–4 (m, 7H), 4.0–4.3 (m, 5H), 4.83 (dd, J = 12.1, 9.1Hz, 1H), 5.32(s, 14H), 6.47–6.68 (m, 1H), 6.93 (dd, J = 27.6, 6.9Hz, 1H), 7.22–7.29 (m, 1H), 7.33–7.48 (m, 2H), 7.48–7.68 (m, 1H), 7.86–8.31 (m, 2H). LCMS m / z expected value 1772.8, measured value 1773.9.

[0805] Dihydrofuran-2,5-dione (0.259 g, 2.59 mmol) was added to [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[3-[(1R,5R,6S)-5,6-bis[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran- [2-yl]oxohexylamino]-3-oxo-propoxy]-3-(6-aminohexylcarbamoyl)cyclohex-3-en-1-yl]oxopropionylamino]hexyloxy]tetrahydropyran-2-yl]acetate (10⁻⁶) (2.3 g, 1.30 mmol), TEA (0.904 mL, 6.48 mmol), and DMAP (0.016 g, 0.13 mmol) in EA (40 mL). The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 30% MeCN in water (0.1% FA). The pure fraction was evaporated to dryness to give 4-oxo-4-((6-((3R,4S,5R)-3,4,5-tris(3-((6-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)hexyl)amino)-3-oxopropoxy)cyclohex-1-en-1-carbamate)hexyl)amino)butyric acid (10-7) (1.799 g, 74.1%), which was a white solid.

[0806] 1H-NMR (500MHz, DMSO, 24℃) δ 1.03–1.3 (m, 19H), 1.37 (h, J = 6.8Hz, 10H), 1.45 (q, J = 6.7Hz, 6H), 1.78 (s, 9H), 1.90 (s, 9H), 2.00 (s, 10H), 2.11 (s, 10H), 2.25–2.37 (m, 7H), 2.41 (t, J = 7.0Hz, 2H), 3.03 (tt, J =12.2, 6.9Hz, 9H), 3.41 (dt, J = 10.0, 6.6Hz, 3H), 3.59–3.8 (m, 10H), 3.87(dt, J = 11.1, 8.8Hz, 3H), 4.03 (h, J = 4.2Hz, 10H), 4.49 (d, J = 8.4Hz, 3H), 4.97 (dd, J = 11.2, 3.4Hz, 3H), 5.22 (d, J = 3.4Hz, 3H), 6.28 (s, 1H), 7.66–7.94 (m, 8H), 11.98 (brs, 1H). LCMS m / z expected 1872.9, observed 1874.1.

[0807] At room temperature, perfluorophenyl 2,2,2-trifluoroacetic acid (0.028 mL, 0.16 mmol) was added to a solution of (10⁻⁷) (100 mg, 0.05 mmol) and DIEA (0.046 mL, 0.27 mmol) in 2 mL of DCM. The resulting mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with EtOAc (50 mL) and washed successively with 1 M NaHSO₄ (4 × 20 mL), saturated NaHCO₃ (2 × 20 mL), and saturated brine (50 mL). The organic layer was dried over Na₂SO₄, filtered, and evaporated to dryness. The residue was diluted with 6 mL of MeCN / water (1:1) and lyophilized to give (10) (78.0 mg, 72%) as a light brown solid. The product was ready for use in the next reaction without any further purification. LCMS m / z expected value 2038.9, found value 1020.8 [M+H] + (z = 2).

[0808] Synthesis of precursor (11)

[0809]

[0810] Synthesis scheme :

[0811]

[0812] Methyl acetate [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[3-[(1R,5R,6S)-5,6-bis[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxohexylamino]-3-oxo-propoxy]-3-[6-(benzyloxycarbonylamino)hexylcarbamoyl]cyclohex-3-en-1-yl]oxopropionylamino]hexyloxy]tetrahydropyran-2-yl]acetate (10⁻⁵) (1.0 g, 0.52 mmol) was dissolved in MeOH (20 mL), and Pd-C (10% wet) (0.056 g, 0.05 mmol) was added, followed by purging with hydrogen three times. The reaction mixture was stirred for 20 hours at 1 atm under a hydrogen atmosphere and at room temperature. The mixture was filtered through a diatomaceous earth pad. The solvent was removed under reduced pressure to give methyl [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[3-[(1R,3R)-2,3-bis[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxohexylamino]-3-oxo-propoxy]-5-(6-aminohexylcarbamoyl)cyclohexyloxy]propionylamino]hexyloxy]tetrahydropyran-2-yl]acetate (11-1) (0.800 g, 86%) as a white solid. The product could be used directly in the next step without further purification.

[0813] 1H NMR (300MHz, DMSO, 23℃) δ 1.22 (d, J = 20.4Hz, 16H), 1.41 (d, J =28.1Hz, 19H), 1.78 (s, 9H), 1.90 (s, 9H), 2.00 (s, 10H), 2.11 (s, 9H), 2.29(d, J = 8.8Hz, 7H), 2.69–2.83 (m, 2H), 3.02 (d, J = 6.0Hz, 7H), 3.22–3.5 (m,6H), 3.55–3.78 (m, 10H), 3.8–3.95 (m, 3H), 4.03 (s, 9H), 4.49 (d, J = 8.4Hz, 3H), 4.97 (dd, J = 11.2, 3.4Hz, 3H), 5.22 (d, J = 3.4Hz, 3H), 7.69–7.97 (m, 7H). LCMS m / z expected value 1775.9, measured value 1776.7.

[0814] DMAP (0.144 g, 1.18 mmol) was added to a solution of [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[3-[(1R,3R)-2,3-bis[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxohexylamino]-3-oxo-propoxy]-5-(6-aminohexylcarbamoyl)cyclohexyloxy]propionylamino]hexyloxy]tetrahydropyran-2-yl]acetate (11-1) (0.7 g, 0.39 mmol) and dihydrofuran-2,5-dione (0.079 g, 0.79 mmol) in DCM (20 mL). The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 30% MeCN in water (0.1% FA). The purified fraction was evaporated to dryness to give 4-oxo-4-((6-((3R,5R)-3,4,5-tris(3-((6-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)hexyl)amino)-3-oxopropoxy)cyclohexane-1-carbamoyl)hexyl)amino)butyric acid (11-2) (0.306 g, 41.4%) as a white solid.

[0815] 1H-NMR (300MHz, DMSO, 23℃) δ 1.25 (s, 18H), 1.41 (d, J = 28.5Hz,20H), 1.78 (s,9H), 1.90 (s, 9H), 2.00 (s, 9H), 2.11 (s, 8H), 2.27 (q, J =5.5, 3.9Hz, 10H), 2.88–3.14 (m, 10H), 3.42 (dd, J = 10.2, 6.2Hz, 5H), 3.66(m, 11H), 3.88 (dt, J = 11.1, 8.8Hz, 3H), 4.03 (d, J = 3.7Hz, 8H), 4.51 (dd,J = 8.4 (2.3 Hz, 3H), 4.98 (dt, J = 11.2, 2.5 Hz, 3H), 5.22 (d, J = 3.4 Hz, 3H), 7.49–8.04 (m, 7H), 8.19 (s, 1H). One proton is exchanged. LCMS m / z expected 1874.9, observed 1876.2.

[0816] At room temperature, perfluorophenyl 2,2,2-trifluoroacetic acid (0.037 mL, 0.21 mmol) was added to a solution of (11-2) (100 mg, 0.05 mmol) and DIEA (0.056 mL, 0.32 mmol) in 2 mL of DCM. The resulting mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with EtOAc (50 mL) and washed successively with 1 M NaHSO4 (4 × 20 mL), saturated NaHCO3 (2 × 20 mL), and saturated brine (50 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to dryness. The residue was diluted with 6 mL of MeCN / water (1:1) and lyophilized to give (11) (83.0 mg, 76%) as a light brown solid. The product was ready for use in the next reaction without any further purification. LCMS m / z expected value 2040.9, found value 1022.0 [M+H] + (z = 2).

[0817] Synthesis of precursor (12)

[0818]

[0819] Synthesis scheme :

[0820]

[0821] DIEA (9.37 mL, 53.63 mmol) was added to a solution of (3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidine-3-ol (5 g, 11.92 mmol), 4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)butyric acid (4.65 g, 14.30 mmol), and O-(benzotriazol-1-yl)-NN-N',N'-tetramethylurea hexafluorophosphate (5.42 g, 14.30 mmol) in DCM (100 mL) at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into water (200 mL) and extracted with DCM (3 × 150 mL). The organic layer was dried over Na₂SO₄, filtered, and evaporated to give a yellow solid of (4-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxypyrrolidine-1-yl)-4-oxobutyl)carbamate (9H-fluorene-9-yl)methyl ester (12-2) (7.00 g, 81%). The product could be used directly in the next step without further purification.

[0822] 1H NMR (400MHz, DMSO, 22℃) δ 1.63 (s, 2H), 1.79–2.11 (m, 2H), 2.25 (hept, J = 7.1Hz, 2H), 3.1–3.38 (m, 3H), 3.72 (t, J = 2.1Hz, 6H), 4.07–4.24(m, 2H), 4.25–4.43 (m, 3H), 6.79–6.92 (m, 4H), 7.13–7.26 (m, 7H), 7.26–7.35(m, 7H), 7.40 (t, J = 7.5Hz, 2H), 7.44–7.52 (m, 1H), 7.68 (d, J = 7.3Hz, 2H), 7.76 (dt, J = 7.8, 1.0Hz, 1H), 7.88 (dd, J = 7.6, 4.6Hz, 2H). Two protons are exchanged. LCMS m / z expected value 726.3, observed value 727.5 ([M+H]). + ).

[0823] Piperidine (49.6 mL, 500.78 mmol) was added to a solution of (4-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxypyrrolidine-1-yl)-4-oxobutyl)carbamate (9H-fluorene-9-yl)methyl ester (12-2) (7 g, 9.63 mmol) in DCM (50 mL). The resulting mixture was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by rapid silica gel chromatography with an elution gradient of 0% to 30% MeOH in DCM (0.1% TEA). The product was then further purified by rapid C18 chromatography with an elution gradient of 0% to 56% MeCN in water (0.1% NH4HCO3). The pure fraction was evaporated to dryness to give 4-amino-1-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxypyrrolidine-1-yl)but-1-one (12-3) as a white solid (3.00 g, 61.7%).

[0824] 1H NMR (300MHz, DMSO, 23℃) δ 1.63 (dp, J = 14.7, 7.8Hz, 2H), 1.85 (ddd, J = 12.9, 8.3, 4.7Hz, 1H), 1.95–2.12 (m, 1H), 2.19–2.39 (m, 2H), 2.55(d, J = 6.4Hz, 1H), 2.66 (t, J = 7.0Hz, 1H), 2.99 (dd, J = 8.9, 3.2Hz, 1H), 3.11–3.29 (m, 1H), 3.42 (ddd, J = 39.4, 11.3, 4.3Hz, 1H), 3.59 (dd, J = 10.7, 5.1 Hz, 1H), 3.74 (s, 6H), 4.08–4.23 (m, 1H), 4.24–4.48 (m, 2H), 6.89 (ddd, J = 9.0, 3.6, 1.1 Hz, 4H), 7.21 (ddd, J = 8.8, 6.3, 2.1 Hz, 5H), 7.27–7.39 (m, 4H). Two protons are exchanged. LCMS m / z expected value 504.2, measured value 505.4 ([M+H]). + ).

[0825] At room temperature, 4-amino-1-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxypyrrolidone-1-yl)but-1-one (12-3) (4.61 g, 9.13 mmol) was added to a solution of 6-[methyl-[(3R,5R)-3,4,5-tris[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxohexamido]propoxy]cyclohexanecarbonyl]amino]hexanoic acid (2,3,4,5,6-pentafluorophenyl) ester (2) (12 g, 6.09 mmol) and DIEA (5.32 mL, 30.44 mmol) in DMF (240 mL). The resulting mixture was stirred at room temperature for 20 hours. The reaction mixture was diluted with H2O (300 mL). The mixture was extracted with IPA / CHCl3 = 3:1 (5 × 500 mL). The organic layer was washed with saturated brine (750 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to obtain the crude product. The crude product was purified by rapid silica gel chromatography with an elution gradient of 0% to 20% MeOH in DCM (0.1% TEA), followed by rapid C18 chromatography with an elution gradient of 0% to 60% MeCN in water (0.1% NH4HCO3). The pure fraction was evaporated to dryness to give a yellow gel-like substance, [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[3-[(1R,3R)-2,3-bis[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxohexylamino]-3- Methyl acetate (12-4) (10.00 g, 71.7%), methyl oxo-propoxy]-5-[[6-[[4-[(2S,4R)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hydroxy-pyrrolidone-1-yl]-4-oxo-butyl]amino]-6-oxo-hexyl]-methyl-carbamoyl]cyclohexyloxy]propionylamino]hexyloxy]tetrahydropyran-2-yl]acetate.

[0826] 1H NMR (300MHz, DMSO, 24℃) δ 1.25 (s, 15H), 1.37 (s, 7H), 1.46 (s,10H), 1.77 (s, 9H), 1.90 (s, 8H), 2.00 (s, 10H), 2.09 (d, J = 7.6Hz, 14H),2.27 (s, 8H), 2.98 (dd, J = 23.1, 6.5Hz, 13H), 3.59 (s, 8H), 3.74 (s, 18H),3.88 (q, J = 9.5Hz, 4H), 4.03 (s, 8H), 4.15 (s, 3H), 4.25–4.56 (m, 6H), 4.97 (dd, J = 11.3, 3.4Hz, 4H), 5.22 (d, J = 3.4Hz, 3H), 6.88–6.91 (m, 4H), 7.20 (t, J = 2.1Hz, 5H), 7.3–7.32 (m, 4H), 7.83 (d, J = 9.0Hz, 6H). LCMS m / z expected value 2290.1, measured value 1145.1 ([MH]). - (z = 2).

[0827] At 0 °C, DMAP (0.376 g, 3.08 mmol) was added to succinic anhydride (0.205 g, 2.05 mmol) and [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[3-[(1R,3R)-2,3-bis[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]]. Methyl acetate (12-4) (2.35 g, 1.03 mmol) was added to a solution of DCM (30 mL). The resulting mixture was stirred at room temperature for 20 hours. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by rapid silica gel chromatography with an elution gradient of 0% to 25% MeOH in DCM (0.1% TEA), followed by rapid C18 chromatography with an elution gradient of 0% to 45% MeCN in water (0.2% TEA) (0.1% TEA). The pure fraction was evaporated to dryness to obtain the product. The product was dissolved in MeCN / water (v / v = 1:10, 20 mL) and lyophilized to obtain 4-(((3R,5S)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-1-(4-(6-(((3R,5R)-3,4,5-tris(3-((6-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)hexyl)amino)-3-oxopropoxy)-N-methylcyclohexane-1-carbamoyl)hexamido)butyryl)pyrrolidine-3-yl)oxy)-4-oxobutyric acid (12-5) (0.598 g, 24.40%) as a white solid.

[0828] 1H NMR (300MHz, DMSO, 23℃) δ 0.87–1.29 (m, 15H), 1.31–1.68 (m, 22H), 1.77 (s, 9H), 1.90 (s, 9H), 2.00 (s, 10H), 2.11 (s, 10H), 2.19–2.35 (m, 8H), 2.48 (d, J = 6.3Hz, 4H), 2.69–2.81 (m, 2H), 2.93 (d, J = 3.8Hz, 2H), 3.02 (p,J = 6.4, 6.0Hz, 9H), 3.22 (d, J = 5.5Hz, 3H), 3.40 (dt, J = 9.5, 6.1Hz, 5H), 3.58 (q, J = 12.1, 9.2Hz, 5H), 3.63–3.72 (m, 6H), 3.74 (s, 8H), 3.87 (dt, J =11.3, 8.9Hz, 3H), 4.03 (s, 9H), 4.20 (s, 1H), 4.49 (d, J = 8.4Hz, 3H), 4.97 (dd, J = 11.2, 3.4Hz, 3H), 5.22 (d, J = 3.4Hz, 3H), 5.38 (s, 1H), 6.88 (dt, J= 8.9, 2.3Hz, 4H), 7.21 (ddt, J = 8.9, 4.6, 2.3Hz, 5H), 7.25–7.39 (m, 4H), 7.66–7.97 (m, 7H), 12.25 (brs, 1H). LCMS m / z expected value 2390.1, measured value 1194.8 ([MH]). - (z = 2).

[0829] Synthesis of 12 on polystyrene solid support :

[0830] Add 200 mg of amino-derived polystyrene solid support to a 4 mL glass vial. Add 1.2 mL of acetonitrile. Shake the vial to prepare a homogeneous suspension. Add HBTU (112 mg, 4 equivalents) and succinic acid substrate 12-5 (177 mg, 1 equivalent), and shake the vial for 10 minutes. Add N-ethyldiisopropylamine (0.08 mL, 6 equivalents) to the reaction mixture and continue shaking overnight. Filter the support and wash with 2 × 5 mL of acetonitrile. Dry the support under vacuum and transfer it to another 4 mL glass vial. Further treat the solid support with a capping reagent (2.4 mL) to cap unreacted amines and shake for 3 hours (the capping reagent was prepared by mixing 1.2 mL of capping reagent A with 0.6 mL of capping reagent B1 and 0.6 mL of capping reagent B2 - all from Sigma-Aldrich, Novabiochem). ® The support was filtered and washed with 5 mL of acetonitrile, 5 mL of ethanol, and then with 5 mL of acetonitrile. The support was then vacuum-dried overnight at room temperature (290 mg, 109.5 μmol / g loading).

[0831] Synthesis of precursor (13)

[0832]

[0833] Synthesis scheme :

[0834]

[0835] At room temperature, TMS-OTf (25.5 mL, 141.26 mmol) was added to a solution of (2S,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-trimethyltriacetate (50 g, 128.42 mmol) in DCE (500 mL). The resulting mixture was stirred at 60 °C for 1 hour. After cooling to room temperature, TEA (26.8 mL, 192.63 mmol) was added dropwise to the mixture and stirred for 10 minutes. The reaction mixture was diluted with DCM (250 mL) and washed successively with saturated NaHCO3 (500 mL) and saturated brine (500 mL). The organic layer was dried over Na₂SO₄, filtered, and evaporated to obtain (5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazol-6,7-dimethyldiacetate (13-2) (50.0 g, 118%), a brown oil. The product was ready for use in the next step without further purification.

[0836] 1H-NMR (500MHz, DMSO, 25℃) δ 1.95 (d, J = 1.4Hz, 3H), 2.01 (d, J = 3.4Hz, 6H), 2.07 (s, 3H), 3.95 (m, 1H), 4.02–4.17 (m, 2H), 4.26 (m, 1H), 4.89 (m, 1H), 5.24 (m, 1H), 6.05 (d, J = 7.0Hz, 1H). LCMS m / z expected value 329.1, measured value 330.1.

[0837] HCl (4N in dioxane) (90 mL, 360.00 mmol) was added to a solution of tert-butyl (9-hydroxynonyl)carbamate (9 g, 34.70 mmol) in DCM (90 mL). The resulting solution was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure to give 9-aminononyl-1-ol hydrochloride (13-8) (7.70 g, 96%) as a white solid. The product was ready for use in the next step without further purification.

[0838] 1H-NMR (300MHz, CDCl3, 22 ° C) δ 1.26 (s, 10H), 1.35–1.45 (m, 2H), 1.55 (m, 2H), 2.67–2.79 (m, 2H), 3.37 (t, J = 6.5Hz, 2H), 4.07–4.54 (m, 1H). LCMS m / z expected value 159.2, measured value 160.1.

[0839] DIEA (24.82 mL, 142.13 mmol) was added to a solution of 9-aminononyl-1-ol hydrochloride (11 g, 47.38 mmol) in DCM (200 mL) over 10 minutes. (2,5-dioxopyrrolidone-1-yl)benzyl carbonate (14.76 g, 59.22 mmol) was added to the mixture. The resulting solution was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by rapid silica gel chromatography with an elution gradient of 0% to 5% MeOH in DCM. The purified fraction was evaporated to dryness to give (9-hydroxynonyl)carbamate (13-9) (13.75 g, 99%) as a white solid.

[0840] 1H-NMR (300MHz, CDCl3, 22 °C) δ 1.23 (s, 10H), 1.38 (m, 4H), 2.96 (q, J = 6.6Hz, 2H), 3.33–3.4 (m, 2H), 4.32 m (J = 5.2, 1.7Hz, 1H), 4.99 (s, 2H), 7.18–7.25 (m, 1H), 7.28–7.36 (m, 5H). LCMS m / z expected value 293.2, measured value 294.1.

[0841] At room temperature, (9-hydroxynonyl)carbamate (13-9, 14.34 g, 48.89 mmol) was added to a solution of (5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazol-6,7-dimethyldiacetate (13-2, 23 g, 48.89 mmol) and molecular sieve (2.3 g) in DCE (250 mL). After stirring the reaction at room temperature for 30 min, TMS-OTf (8.83 mL, 48.89 mmol) was added. The resulting mixture was stirred at 60 °C for 2 h. The reaction mixture was quenched with saturated NaHCO3 (300 mL), extracted with DCM (3 × 250 mL), and washed with saturated brine (2 × 300 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to give a dark brown oil. The crude product was purified by rapid silica gel chromatography with an elution gradient of 0% to 80% EtOAc in petroleum ether. The purified fraction was evaporated to dryness to give (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((9-((((benzyloxy)carbonyl)amino)nonyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (13-3) (27.0 g, 89%), which was a yellow liquid.

[0842] 1H-NMR (300MHz, CDCl3, 22℃) δ 1.24 (s, 10H), 1.34–1.5 (m, 4H), 1.77(s, 3H), 1.90 (s, 3H), 2.00 (d, J = 1.5Hz, 3H), 2.11 (s, 3H), 2.97 (q, J =6.6Hz, 2H), 3.36–3.46 (m, 1H), 3.70 (m, J = 9.8, 6.1Hz, 1H), 3.8–4.02 (m,2H), 4.06–4.16 (m, 1H), 4.48 (d, J = 8.5Hz, 1H), 4.98 (d, J = 14.4Hz, 3H),5.23 (m, J = 9.9, 3.6Hz, 2H), 7.19–7.29 (m, 1H), 7.31–7.37 (m, 5H), 7.83 (d,J = 9.2Hz, 1H). LCMS m / z expected value 622.3, measured value 623.3.

[0843] At room temperature, trimethyliodosilane (17.14 mL, 120.44 mmol) was added to a solution of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((9-((((benzyloxy)carbonyl)amino)nonyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetic acid (13-3, 15 g, 24.09 mmol) in MeCN (150 mL). The resulting mixture was stirred at room temperature for 15 minutes. The solvent was removed under reduced pressure. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 35% MeCN in water (0.1% TFA). The pure fraction was evaporated to dryness to give (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((9-aminononyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (13-4) (14.00 g, 119%), a yellow gel-like substance.

[0844] 1H-NMR (300MHz, CDCl3, 22℃) δ 1.26 (d, J = 6.6Hz, 10H), 1.50 (m, J =16.1, 8.2Hz, 4H), 1.77 (s, 3H), 1.89 (s, 3H), 2.00 (s, 3H), 2.10 (s, 3H),2.69–2.84 (m, 2H), 3.41 (m, J = 9.8, 6.4Hz, 1H), 3.70 (m, J = 9.7, 6.1Hz,1H), 3.87 (m, J = 11.2, 8.8Hz, 1H), 3.97–4.06 (m, 3H), 4.48 (d, J = 8.4Hz,1H), 4.96 (m, J = 11.2, 3.4Hz, 1H), 5.22 (d, J = 3.4Hz, 1H), 7.84 (d, J = 9.3Hz, 1H). LCMS m / z expected value 488.3, ​​measured value 489.3.

[0845] Under nitrogen atmosphere, DIEA (3.65 mL, 20.88 mmol) was added to a solution of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((9-aminononyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (13-4, 6.12 g, 12.53 mmol), 3,3',3''-(((1R,2S,3R)-5-((6-(((benzyloxy)carbonyl)amino)hexyl)carbamoyl)cyclohex-4-ene-1,2,3-triyl)tri(oxy))tripropionic acid (13-5, 1.3 g, 2.09 mmol), BOP (3.69 g, 8.35 mmol), and DMAP (0.765 g, 6.26 mmol) in DMF (70 mL). The resulting mixture was stirred at room temperature for 16 hours. Solvent was removed under reduced pressure. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 63% MeCN in water (0.1% TFA). The pure fraction was evaporated to dryness to give methyl acetate [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[9-[3-[(1R,5R,6S)-5,6-bis[3-[9-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxonylamino]-3-oxo-propoxy]-3-[6-(benzyloxycarbonylamino)hexylcarbamoyl]cyclohex-3-en-1-yl]oxopropionylamino]nonoxy]tetrahydropyran-2-yl]acetate (13-6) (2.400 g, 56%), which was a yellow solid.

[0846] 1H-NMR (300MHz, CDCl3, 22℃) δ 1.23 (s, 34H), 1.41 (d, J = 24.1Hz,16H), 1.76 (s, 9H), 1.89 (s, 9H), 1.99 (s, 9H), 2.10 (s, 9H), 2.12–2.17 (m,1H), 2.2–2.35 (m, 7H), 2.99 (d, J = 14.4Hz, 10H), 3.40 (m, J = 9.4, 6.3Hz,4H), 3.63–3.76 (m, 11H), 4.08 (d, J = 19.3Hz, 12H), 4.48 (d, J = 8.5Hz, 3H), 4.92–5.02 (m, 5H), 5.21 (d, J = 3.4Hz, 3H), 6.27 (s, 1H), 7.23 (t, J = 5.6Hz, 1H), 7.28–7.37 (m, 5H), 7.76 (t, J = 5.4Hz, 1H), 7.82 (m, J = 7.9, 4.9Hz, 6H). LCMS m / z expected value 2033.1, measured value 1018.1 (M+1, z=2).

[0847] At room temperature, TMS-I (1.004 mL, 7.37 mmol) was added to a solution of [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[9-[3-[(1R,5R,6S)-5,6-bis[3-[9-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxonylamino]-3-oxo-propoxy]-3-[6-(benzyloxycarbonylamino)hexylcarbamoyl]cyclohex-3-en-1-yl]oxopropionylamino]nonoxy]tetrahydropyran-2-yl]acetate (13-6, 1.5 g, 0.74 mmol) in MeCN (20 mL). The reaction mixture was stirred at room temperature for 15 minutes. The solvent was removed under reduced pressure. DCM (50 ml × 4) was added to the residue and concentrated to dryness to give a brown solid of methyl [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[9-[3-[(1R,5R,6S)-5,6-bis[3-[9-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxynonylamino]-3-oxo-propoxy]-3-(6-aminohexylcarbamoyl)cyclohex-3-en-1-yl]oxypropionylamino]nonoxy]tetrahydropyran-2-yl]acetate (13-7) (1.3 g, 0.684 mmol, 93%). The product was ready for the next step without further purification.

[0848] 1H-NMR (500MHz, DMSO, 25℃) δ 1.21–1.28 (m, 30H), 1.29–1.41 (m, 15H), 1.48–1.57 (m, 7H), 1.76 (s, 3H), 1.80 (s, 6H), 1.89 (d, J = 2.0Hz, 9H), 1.99(d, J = 1.9Hz, 10H), 2.10 (s, 10H), 2.25 (q, J = 6.8Hz, 4H), 2.32 (t, J =6.6Hz, 2H), 2.71–2.81 (m, 2H), 2.98–3.03 (m, 6H), 3.07 (t, J = 6.8Hz, 2H), 3.36–3.42 (m, 3H), 3.54–3.62 (m, 3H), 3.66 (d, J = 3.5Hz, 1H), 3.68–3.76 (m,6H), 3.99–4.04 (m, 6H), 4.13 (t, J = 6.5Hz, 2H), 4.16–4.22 (m, 2H), 4.48 (d,J = 8.1Hz, 1H), 4.64 (d, J = 16.3Hz, 1H), 4.71–4.85 (m, 3H), 4.94–5.03 (m,3H), 5.2–5.32 (m, 3H), 6.27 (s, 1H), 7.28–7.33 (m, 1H), 7.4–7.57 (m, 3H), 7.76–7.83 (m, 3H), 7.98 (d, J = 8.1Hz, 2H). LCMS m / z expected value 1899.0, measured value 951.1 (M+1, z=2).

[0849] At room temperature under nitrogen atmosphere, DIEA (0.956 mL, 5.47 mmol) was added to [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[9-[3-[(1R,5R,6S)-5,6-bis[3-[9-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxonylamino]- Methyl acetate (13-7, 1.3 g, 0.68 mmol), O-(benzotriazol-1-yl)-NN-N',N'-tetramethylurea hexafluorophosphate (1.557 g, 4.10 mmol), and adipic acid (1.000 g, 6.84 mmol) in a 20 mL solution of DMF were mixed. The resulting mixture was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 50% MeCN in water (0.1% TFA). The product was not pure enough and was purified by rapid C18 chromatography with an elution gradient of 0% to 43% MeCN in water (0.1% TFA). The pure fraction was evaporated to dryness to give 6-oxo-6-((6-((3R,4S,5R)-3,4,5-tris(3-((9-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)nonyl)amino)-3-oxopropoxy)cyclohex-1-en-1-carbamate)hexyl)amino)hexanoic acid (13-8) (0.303 g, 22%), which was a white solid.

[0850] 1H-NMR (500MHz, DMSO, 24℃) δ 1.24 (t, J = 5.6Hz, 29H), 1.34 (d, J =21.3Hz, 15H), 1.43–1.49 (m, 5H), 1.53 (p, J = 6.6Hz, 5H), 1.78 (d, J =19.9Hz, 9H), 1.89 (d, J = 1.7Hz, 9H), 1.99 (s, 9H), 2.04 (t, J = 6.8Hz, 2H), 2.10 (s, 10H), 2.19 (t, J = 6.9Hz, 2H), 2.22–2.29 (m, 4H), 2.31 (t, J =6.6Hz, 2H), 2.34–2.41 (m, 1H), 2.97–3.07 (m, 10H), 3.35–3.43 (m, 3H), 3.56–3.6 (m, 2H), 3.63–3.76 (m, 8H), 3.98–4.05 (m, 7H), 4.13 (t, J = 6.4Hz, 3H), 4.15–4.24 (m, 3H), 4.48 (d, J = 8.5Hz, 1H), 4.83 (d, J = 3.5Hz, 3H), 4.93–5.03 (m, 3H), 5.21–5.32 (m, 3H), 6.27 (s, 1H), 7.71–7.84 (m, 5H), 7.98 (d, J= 8.1Hz, 3H). LCMS m / z expected value 2027.1, measured value 1014.8 (M+1, z=2).

[0851] At room temperature, perfluorophenyl 2,2,2-trifluoroacetic acid (0.065 mL, 0.38 mmol) was added to a solution of (13-8) (110 mg, 0.05 mmol) and DIEA (0.095 mL, 0.54 mmol) in 2 mL of DCM. The resulting mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with EtOAc (50 mL) and washed successively with 1 M NaHSO4 (4 × 20 mL), saturated NaHCO3 (2 × 20 mL), and saturated brine (20 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to dryness. The residue was diluted with 5 mL of MeCN / water (1:1) and lyophilized to give (13) (108 mg, 91%) as a grayish-white solid. The product was ready for use in the next reaction without any further purification. LCMS m / z expected value 2193.05, found value 1098.1 [M+H] + (z = 2).

[0852] Synthesis of precursor (14)

[0853]

[0854] Synthesis scheme :

[0855]

[0856] At 0 °C, perfluorophenyl 2,2,2-trifluoroacetic acid (1.738 mL, 10.12 mmol) was added to a solution of 3,3',3''-(((1R,2S,3R)-5-((6-((((benzyloxy)carbonyl)amino)hexyl)carbamoyl)cyclohex-4-ene-1,2,3-triyl)tri(oxy))tripropionic acid (14-1) (1.4 g, 2.25 mmol) and DIEA (2.55 mL, 14.61 mmol) in DCM (30 mL). The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with DCM (150 mL) and washed successively with 1 M NaHSO4 (2 × 100 mL), saturated NaHCO3 (2 × 100 mL), and saturated brine (100 mL). The organic layer was dried over Na₂SO₄, filtered, and evaporated to obtain a yellow oily product: tris(perfluorophenyl)3,3',3''-(((1R,2S,3R)-5-((6-((((benzyloxy)carbonyl)amino)hexyl)carbamoyl)cyclohex-4-en-1,2,3-triyl)tri(oxy))tripropionate (14-2) (2.400 g, 95%). The product can be used directly in the next step without further purification.

[0857] 1H-NMR (500MHz, DMSO, 25℃) δ 1.25 (d, J = 6.6Hz, 8H), 2.15–2.25 (m, 1H), 2.37–2.48 (m, 2H), 2.96–3.06 (m, 8H), 3.71–3.99 (m, 9H), 4.12–4.2 (m, 1H), 4.99 (s, 2H), 6.3–6.37 (m, 1H), 7.21 (t, J = 5.7Hz, 1H), 7.3–7.36 (m, 5H), 7.83–7.91 (m, 1H). LCMS m / z expected value 1120.2, measured value 1143.3.

[0858] At room temperature, DIEA (2.69 mL, 15.39 mmol) was added to a solution of tris(perfluorophenyl)3,3',3''-(((1R,2S,3R)-5-((6-((((benzyloxy)carbonyl)amino)hexyl)carbamoyl)cyclohex-4-ene-1,2,3-triyl)tri(oxy))tripropionate (2.3 g, 2.05 mmol) and tert-butyl 5-aminovalerate (1.600 g, 9.23 mmol) in DMF (30 mL). The resulting mixture was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 90% MeCN in water (0.1% NH4HCO3). The pure fraction was evaporated to dryness to give a yellow oily substance, 5,5',5''-((3,3',3''-(((1R,2S,3R)-5-((6-((((benzyloxy)carbonyl)amino)hexyl)carbamoyl)cyclohex-4-ene-1,2,3-triyl)tri(oxy))tri(propionyl))tri(azanediyl))tritert-tert-butyl trivalerate (14-3) (1.500 g, 67%).

[0859] 1H-NMR (300MHz, DMSO, 23℃) δ 1.3–1.47 (m, 48H), 2.15–2.28 (m, 10H), 2.87–3.09 (m, 12H), 3.56–3.8 (m, 9H), 4.00 (s, 1H), 4.99 (s, 2H), 6.26 (s, 1H), 7.27–7.35 (m, 5H), 7.69–7.9 (m, 5H). LCMS m / z expected value 1087.7, measured value 1110.7.

[0860] At room temperature, 10 mL of TFA was added to a solution of 1.4 g (1.29 mmol) of tritert-tert-butyl trivalerate (14-3) in DCM (30 mL). The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure to obtain a yellow oily substance, 5,5',5''-((3,3',3''-(((1R,2S,3R)-5-((6-(((benzyloxy)carbonyl)amino)hexyl)carbamoyl)cyclohex-4-en-1,2,3-triyl)tri(oxy))tri(propionyl))tri(azanediyl))trivaleric acid (14-4) (1.100 g, 83%). The product can be used directly in the next step without further purification.

[0861] 1H-NMR (500MHz, DMSO, 25℃) δ 1.2–1.27 (m, 5H), 1.35–1.42 (m, 10H), 1.44–1.51 (m, 6H), 2.17–2.22 (m, 6H), 2.23–2.29 (m, 4H), 2.32 (t, J = 6.5Hz,2H), 2.94–3.09 (m, 11H), 3.63–3.78 (m, 8H), 3.99–4.04 (m, 1H), 5.00 (s, 2H),6.27 (d, J = 2.9Hz, 1H), 7.21 (t, J = 5.7Hz, 1H), 7.27–7.38 (m, 5H), 7.74–7.87 (m, 4H), 10.59 (brs, 3H). LCMS m / z expected value 919.5, measured value 920.2.

[0862] At 0 °C, perfluorophenyl 2,2,2-trifluoroacetic acid (0.748 mL, 4.35 mmol) was added to a solution of 5,5',5''-((3,3',3''-(((1R,2S,3R)-5-((6-(((benzyloxy)carbonyl)amino)hexyl)carbamoyl)cyclohex-4-en-1,2,3-triyl)tri(oxy))tri(propionyl))tri(azanediyl))trivalve acid (14-4) (1 g, 0.97 mmol) and DIEA (1.098 mL, 6.29 mmol) in DCM (30 mL). The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with DCM (80 mL) and washed successively with 1 M NaHSO4 (2 × 70 mL), saturated NaHCO3 (2 × 70 mL), and saturated brine (100 mL). The organic layer was dried over Na₂SO₄, filtered, and evaporated to obtain a yellow oily product: tris(perfluorophenyl)5,5',5''-((3,3',3''-(((1R,2S,3R)-5-((6-((((benzyloxy)carbonyl)amino)hexyl)carbamoyl)cyclohex-4-en-1,2,3-triyl)tri(oxy))tri(propionyl))tri(azanediyl))trivalve ester (14-5) (1.200 g, 87%). The product can be used directly in the next step without further purification.

[0863] 1H-NMR (500MHz, DMSO, 25℃) δ 1.17–1.28 (m, 6H), 1.38 (q, J = 7.3,6.8Hz, 5H), 1.43–1.52 (m, 6H), 1.6–1.69 (m, 5H), 2.1–2.41 (m, 9H), 2.71–2.82(m, 5H), 2.96 (q, J = 6.6Hz, 2H), 3.02–3.11 (m, 7H), 3.62–3.81 (m, 8H), 4.99(d, J = 1.8Hz, 2H), 6.28 (s, 1H), 7.21 (t, J = 5.7Hz, 1H), 7.26–7.39 (m, (5H), 7.75–7.92 (m, 4H). LCMS m / z expected value 1417.4, measured value 1418.4.

[0864] At room temperature, DIEA (1.108 mL, 6.35 mmol) was added to a solution of tris(perfluorophenyl)5,5',5''-((3,3',3''-(((1R,2S,3R)-5-((6-(((benzyloxy)carbonyl)amino)hexyl)carbamoyl)cyclohex-4-ene-1,2,3-triyl)tri(oxy))tri(propionyl))tri(azanediyl))trivalve ester (14-5) (1.2 g, 0.85 mmol) and (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((6-aminohexyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (2.187 g, 3.81 mmol) in DMF (20 mL). The resulting mixture was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 60% MeCN in water (0.1% TFA). The pure fraction was evaporated to dryness to give methyl acetate [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[5-[3-[(1R,5R,6S)-5,6-bis[3-[[5-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxohexylamino]-5-oxo-pentyl]amino]-3-oxo-propoxy]-3-[6-(benzyloxycarbonylamino)hexylcarbamoyl]cyclohex-3-en-1-yl]oxopropionylamino]pentamido]hexyloxy]tetrahydropyran-2-yl]acetate (14-6) (1.400 g, 75%), which was a gray solid.

[0865] 1H-NMR (300MHz, DMSO, 22℃) δ 1.06–1.28 (m, 18H), 1.29–1.47 (m, 27H), 1.76 (s, 9H), 1.88 (s, 9H), 1.98 (s, 9H), 2.09 (s, 9H), 2.18–2.36 (m, 8H), 2.99 (d, J = 7.8Hz, 17H), 3.32–3.46 (m, 4H), 3.59–3.75 (m, 11H), 3.8–3.91 (m,4H), 4.01 (s, 11H), 4.47 (d, J = 8.5Hz, 3H), 4.93 (d, J = 3.4Hz, 2H), 4.95–5.01 (m, 4H), 5.20 (d, J = 3.4Hz, 3H), 6.26 (s, 1H), 7.21 (d, J = 5.6Hz, 1H), 7.28–7.36 (m, 5H), 7.73 (t, J = 5.6Hz, 3H), 7.83 (t, J = 8.6Hz, 7H). LCMS m / z expected value 2204.1, measured value 1103.3.

[0866] At room temperature, TMS-I (0.432 mL, 3.17 mmol) was added to [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[5-[3-[(1R,5R,6S)-5,6-bis[3-[[5-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-( [Acetoxymethyl]tetrahydropyran-2-yl]oxohexylamino]-5-oxo-pentyl]amino]-3-oxo-propoxy]-3-[6-(benzyloxycarbonylamino)hexylcarbamoyl]cyclohex-3-en-1-yl]oxopropionylamino]pentamido]hexyloxy]tetrahydropyran-2-yl]methyl acetate (14-6) (700 mg, 0.32 mmol) in a 20 mL solution of MeCN. The resulting mixture was stirred at room temperature for 15 minutes. The solvent was removed under reduced pressure. DCM (50 ml × 4) was added to the residue and concentrated to dryness to give a yellow solid of methyl [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[5-[3-[(1R,5R,6S)-5,6-bis[3-[5-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxohexylamino]-5-oxo-pentyl]amino]-3-oxo-propoxy]-3-(6-aminohexylcarbamoyl)cyclohex-3-en-1-yl]oxopropionylamino]pentamido]hexyloxy]tetrahydropyran-2-yl]acetate (14-7) (650 mg, 99%). The product can be used directly in the next step without further purification.

[0867] 1H-NMR (500MHz, DMSO, 25℃) δ 1.11–1.56 (m, 51H), 1.78 (d, J =19.1Hz, 9H), 1.88 (d, J = 1.9Hz, 9H), 1.98 (s, 9H), 2.01–2.05 (m, 8H), 2.09(s, 9H), 2.25 (q, J = 6.5Hz, 4H), 2.76 (q, J = 6.8Hz, 2H), 2.91–3.11 (m,15H), 3.3–3.46 (m, 3H), 3.65–3.74 (m, 9H), 3.82–3.89 (m, 2H), 3.98–4.04 (m,10H), 4.43–4.49 (m, 2H), 4.95 (dd, J = 11.2, 3.5Hz, 2H), 5.20 (d, J = 3.5Hz, 2H), 6.26 (s, 1H), 7.72–7.87 (m, 9H), 7.98 (d, J = 8.0Hz, 1H). Two protons have been exchanged. LCMS m / z expected value 2070.1, observed value 1036.3 (M+1, z=2).

[0868] At room temperature under nitrogen atmosphere, DIEA (0.548 mL, 3.14 mmol) was added to [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[5-[3-[(1R,5R,6S)-5,6-bis[3-[[5-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxohexylamino]-5-oxo- Methyl acetate (14-7) (650 mg, 0.31 mmol), adipic acid (229 mg, 1.57 mmol), and O-(benzotriazol-1-yl)-NN-N',N'-tetramethylurea hexafluorophosphate (357 mg, 0.94 mmol) were added to a DMF solution (15 mL). The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure to give the crude product. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 50% MeCN in water (0.1% TFA). The pure fraction was evaporated to dryness to give 6-oxo-6-((6-((3R,4S,5R)-3,4,5-tris(3-((5-((6-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)hexyl)amino)-5-oxopentyl)amino)-3-oxopropoxy)cyclohex-1-en-1-carbamoyl)hexyl)amino)hexanoic acid (14-8) (344 mg, 50%), which is a yellow solid.

[0869] 1H-NMR (500MHz, DMSO, 25℃) δ 1.13–1.28 (m, 15H), 1.3–1.55 (m, 32H), 1.79 (d, J = 19.4Hz, 9H), 1.89 (s, 9H), 1.99 (s, 9H), 2.01–2.05 (m, 8H), 2.10(s, 9H), 2.12–2.14 (m, 1H), 2.19 (t, J = 6.9Hz, 2H), 2.22–2.28 (m, 4H), 2.3–2.41 (m, 3H), 2.91–3.09 (m, 17H), 3.3–3.44 (m, 3H), 3.54–3.64 (m, 1H), 3.66–3.8 (m, 9H), 3.84–3.89 (m, 2H), 3.92–4.07 (m, 10H), 4.08–4.24 (m, 2H), 4.48 (d, J = 8.4Hz, 2H), 4.83 (d, J = 3.5Hz, 1H), 4.92–5.03 (m, 3H), 5.21 (d, J = 3.4Hz, 2H), 5.32 (d, J = 3.2Hz, 1H), 6.27 (s, 1H), 7.58–7.91 (m, 10H), 7.99 (d, J = 8.1Hz, 1H). One proton has been exchanged. The expected value of LCMS m / z is 2198.1, and the measured value is 1100.6 (M+1, z=2).

[0870] At room temperature, perfluorophenyl 2,2,2-trifluoroacetic acid (0.109 mL, 0.64 mmol) was added to a solution of (14-8) (200 mg, 0.09 mmol) and DIEA (0.159 mL, 0.91 mmol) in 2 mL of DCM. The resulting mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with EtOAc (50 mL) and washed successively with 1 M NaHSO4 (4 × 20 mL), saturated NaHCO3 (2 × 20 mL), and saturated brine (20 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to dryness. The residue was diluted with 5 mL of MeCN / water (1:1) and lyophilized to give (14) (202.0 mg, 89%) as a white solid. The product was ready for use in the next reaction without any further purification. LCMS m / z expected value 2364.1, found value 1183.6 [M+H] + (z = 2).

[0871] Synthesis of precursor (15)

[0872]

[0873] Synthesis scheme :

[0874]

[0875] At room temperature, 20 mL of TFA was added to a 20 mL solution of 2 g (2.62 mmol) of tritert-butyl tripropionate (15-1) of 15-3-(((1R,2S,3R)-5-((4-((((benzyloxy)carbonyl)amino)butyl)carbamoyl)cyclohex-4-ene-1,2,3-triyl)tri(oxy))tripropionate. The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure to give 2 g (3.36 mmol, 99%) of 3,3',3''-(((1R,2S,3R)-5-((4-((((benzyloxy)carbonyl)amino)butyl)carbamoyl)cyclohex-4-ene-1,2,3-triyl)tri(oxy))tripropionate (15-2) as a yellow oil. The product was ready for use in the next step without further purification.

[0876] 1H-NMR (300MHz, DMSO, 23℃) δ 1.37–1.44 (m, 4H), 2.08–2.33 (m, 2H), 2.43 (m, J = 6.7, 5.2, 2.6Hz, 6H), 3.03 (m, J = 24.2, 5.7Hz, 4H), 3.71–3.79(m, 8H), 4.08 (s, 1H), 5.01 (s, 2H), 6.21–6.32 (m, 1H), 7.23 (d, J = 5.8Hz,1H), 7.32–7.36 (m, 5H), 7.91 (t, J = 5.6Hz, 1H), 12.10 (s, 3H). The expected value of LCMS m / z is 594.2, and the measured value is 595.3.

[0877] DIEA (5.87 mL, 33.64 mmol) was added to a solution of 3,3',3''-(((1R,2S,3R)-5-((4-((((benzyloxy)carbonyl)amino)butyl)carbamoyl)cyclohex-4-ene-1,2,3-triyl)tri(oxy))tripropionic acid (15-2) (2 g, 3.36 mmol), (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((6-aminohexyl)oxy)tetrahydro-2H-pyran-3,4-dimethyldiacetate (6.61 g, 14.80 mmol), BOP (5.95 g, 13.45 mmol), and DMAP (1.233 g, 10.09 mmol) in DMF (30 mL) under nitrogen atmosphere at room temperature. The resulting mixture was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by rapid C18 chromatography with an elution gradient of 0% to 60% MeCN in water (0.1% TFA). The pure fraction was evaporated to dryness to give methyl acetate [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-[6-[3-[(1R,5R,6S)-5,6-bis[3-[6-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxohexylamino]-3-oxo-propoxy]-3-[4-(benzyloxycarbonylamino)butylcarbamoyl]cyclohex-3-en-1-yl]oxopropionylamino]hexyloxy]tetrahydropyran-2-yl]acetate (15-3) (2.500 g, 40%), which was a yellow solid.

[0878] 1H-NMR (500MHz, DMSO, 25℃) δ 1.24 (m, J = 14.4, 9.8, 7.4Hz, 12H), 1.38 (m, J = 22.0, 14.0, 6.9Hz, 13H), 1.46 (d, J = 6.7Hz, 4H), 1.77 (s, 9H),1.89 (s, 9H), 1.99 (s, 9H), 2.07 (s, 2H), 2.10 (s, 9H), 2.23–2.35 (m, 6H), 3.03 (m,...

Claims

1. A compound having the structure of formula (I): Or its pharmaceutically acceptable salt, wherein: X is selected from covalent bonds, -CH2-, and -C(O)-; Y is either -O or -NR-. Where R is selected from -H and -(C1-C6) alkyl; It represents a carbon-carbon single or double bond, provided that X is a covalent bond. It represents a carbon-carbon single bond; and G 1 To G 4 Each independently represents either the -[spacer]-[ligand] part or the -[connector]-[cargo] part, where each ligand is independently a monosaccharide and each cargo is independently a nucleic acid, provided that G... 1 To G 4 At least one of them represents the -[spacer]-[ligand] part and G 1 To G 4 At least one of them represents the [chain]-[joint]-[cargo] part.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein X is -C(O)-.

3. The compound of claim 1 or claim 2 or a pharmaceutically acceptable salt thereof, wherein Y is -NR-, for example, wherein Y is -NH- or -N(CH3)-.

4. The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein Indicates a single key.

5. The compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, wherein each ligand is N-acetylgalactosamine (GalNAc).

6. The compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, wherein each product is independently selected from antisense oligonucleotides (ASO), immunostimulatory oligonucleotides, decoy oligonucleotides, splice-altering oligonucleotides, splice-conversion oligonucleotides, triplet-forming oligonucleotides, siRNA, saRNA, microRNA, microRNA mimics, antimiR, double-stranded RNA, single-stranded RNA, ribozymes, aptamers, mirror aptamers, CRISPR oligonucleotides, and G-quadruplexes.

7. The compound of any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof, wherein each spacer group independently comprises a chain of 2 to 20 atoms selected from C, N, O, S and P, for example wherein each spacer group is independently selected from linear alkylene groups (which can optionally be interrupted by one or more amide and / or phosphate groups) and polyethylene glycol.

8. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein [joint-chain] in each case independently represents a straight chain portion comprising 8 to 30 atoms.

9. The compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure of formula (Ia) or formula (Ib): ， Where X, Y, G 1 G 2 G 3 and G 4 As defined in any of the preceding claims, wherein in formula (Ib) It represents a carbon-carbon single bond.

10. The compound according to any one of claims 1 to 3 and 5 to 8, or a pharmaceutically acceptable salt thereof, wherein the compound has the structure of formula (Id): ， Where X, Y, G 1 G 2 G 3 and G 4 As defined in any of the preceding claims, and It represents a double bond.

11. A compound having the structure of formula (II): Or its pharmaceutically acceptable salt, wherein: X is selected from covalent bonds, -CH2-, and -C(O)-; Y is either -O or -NR-. Where R is selected from -H and -(C1-C6) alkyl; It represents a carbon-carbon single or double bond, provided that X is a covalent bond. Indicates a carbon-carbon single bond; In each case, the spacer group independently represents a portion of a straight chain containing 6 to 20 atoms (e.g., selected from C, N, O, S, and P) that attaches the ligand to the rest of the molecule; The ligand is a monosaccharide independently in each case; The term "joint" and "chain" together refer to the part that attaches cargo to the rest of the molecule, such as a straight chain containing 8 to 30 atoms; and The goods are nucleic acid.

12. The compound of claim 11 or a pharmaceutically acceptable salt thereof, wherein X is -C(O)- and Y is -NR- (e.g., wherein R is -H or -CH3).

13. The compound of claim 11 or claim 12, or a pharmaceutically acceptable salt thereof, wherein... Indicates a single key.

14. The compound according to any one of claims 11 to 13, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure of formula (IIa) or formula (IIb): ， Wherein X, Y, ligand, spacer, chain, connector, and cargo are defined according to any one of the preceding claims, wherein in formula (IIb) It represents a carbon-carbon single bond.

15. The compound of claim 11 or claim 12, or a pharmaceutically acceptable salt thereof, wherein the compound has the structure of formula (IId): ， Wherein X, Y, ligand, spacer, chain, connector, and cargo are defined according to any one of the preceding claims, and It represents a double bond.

16. A compound having the structure of formula (III): Or a pharmaceutically acceptable salt thereof, wherein X, Y, spacer, chain, connector, and cargo are as defined in any one of the preceding claims, and It represents a carbon-carbon single or double bond, provided that X is a covalent bond. It represents a carbon-carbon single bond.

17. The compound of claim 16 or a pharmaceutically acceptable salt thereof, wherein: X is -C(O)-; Y is either -O- or -NR-. Where R is selected from -H and -(C 1-6 )alkyl; Indicates a single key; The spacer group, in each case, independently represents a straight chain containing 6 to 20 atoms selected from C, N, O, S, and P; The term "joint" and "chain" together represent a straight chain containing 8 to 30 atoms; and The goods are nucleic acid.

18. The compound of claim 16 or claim 17, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure of formula (IIIa) or (IIIb): ， Wherein X, Y, spacer base, chain, joint, and cargo are defined according to any one of the preceding claims, wherein in formula (IIIb) It represents a carbon-carbon single bond.

19. The compound of claim 16 or a pharmaceutically acceptable salt thereof, wherein the compound has the structure of formula (IIId): ， Wherein X, Y, spacer base, chain, joint, and cargo are defined according to any one of the preceding claims, and It represents a double bond.

20. A compound having the structure of formula (IV): Or its pharmaceutically acceptable salt, wherein: Z independently represents, in each case, a portion of a straight chain containing 1 to 16 atoms (e.g., selected from C, N, O, S, and P); m is independently selected from an integer from 1 to 6 in each case; R is selected from -H and -(C1-C6) alkyl; and Chains, connectors, and cargo are as defined in any of the preceding claims.

21. The compound of claim 20 or a pharmaceutically acceptable salt thereof, wherein Z is independently selected in each case from... -(C1-C 16 )alkylene-, -(C2-C 16 ) imidene-, -(C1-C 12 )alkylene-C(O)-, -(C2-C 12 ) imidene-C(O)-, -(C1-C8)alkylene-C(O)NR'-(C1-C6)alkylene-、 -(C2-C8)-alkenyl-C(O)NR'-(C1-C6)alkyl-, -(C1-C8)alkylene-C(O)NR'-(C2-C6)alkenyl-and -(C2-C8)imenyl-C(O)NR'-(C2-C6)imenyl-(wherein) (in each case, R' represents the attachment point with the oxygen atom in GalNac), where R' is independently selected from -H and -(C1-C6) alkyl groups in each case.

22. The compound of claim 20 or claim 21, or a pharmaceutically acceptable salt thereof, wherein each Z is independently selected from... -(C1-C6)alkylene-C(O)NR'-(C1-C4)alkylene- (where R' is independently selected from -H and -(C1-C6)alkyl in each case), for example, where Z is -(C4)alkylene-C(O)NH-(C3)alkylene-.

23. The compound according to any one of claims 20 to 22 or a pharmaceutically acceptable salt thereof, wherein m is independently selected from 1, 2, 3 and 4 in each case.

24. The compound according to any one of claims 20 to 23, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure of formula (IVa) or formula (IVb): ， Z, m, R, chain, connector, and cargo are defined according to any one of the preceding claims.

25. A compound having the structure of formula (V): ， Or its pharmaceutically acceptable salt, wherein: A independently represents, in each case, a portion of a straight chain containing 1 to 16 atoms (e.g., selected from C, N, O, S, and P); n is an integer independently selected from 1 to 6 in each case; R is selected from -H and -(C1-C6) alkyl; and Chains, connectors, and cargo are as defined in any of the preceding claims.

26. The compound of claim 25 or a pharmaceutically acceptable salt thereof, wherein A is independently selected in each case from... -(C1-C 16 )alkylene- and -(C2-C 16 ) imidene- (wherein) In each case, it represents the attachment point with the GalNac oxygen atom.

27. The compound of claim 25 or claim 26 or a pharmaceutically acceptable salt thereof, wherein n is independently selected from 1, 2, 3 and 4 in each case.

28. The compound according to any one of claims 25 to 27, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure of formula (Va) or formula (Vb): ， Wherein A, n, R, chain, connector, and cargo are as defined in any of the preceding claims.

29. A compound having the structure of formula (IX): ， Or its pharmaceutically acceptable salt, wherein: Z independently represents, in each case, a portion of a straight chain containing 1 to 16 atoms (e.g., selected from C, N, O, S, and P) that attaches the GalNAc ligand to the rest of the molecule; m is independently selected from an integer from 1 to 6 in each case; R is selected from -H and -(C1-C6) alkyl; and Chains, connectors, and cargo are as defined in any of the preceding claims.

30. A compound having the structure of formula (XI): in: Z is selected independently in each case. -(C1-C 12 )alkylene- and -(C1-C6)alkylene-C(O)NR'-(C1-C4)alkylene-, wherein R' is independently selected from -H and -(C1-C6)alkyl in each case (where R' is selected from -H and -(C1-C6)alkyl). (In each case, it represents the attachment point with the GalNac oxygen atom). m is independently selected from an integer from 1 to 6 in each case; R is selected from -H and -(C1-C6) alkyl; q is an integer from 1 to 12; D is selected from (i) -C(O)NH- and (ii) -NHC(O)-(CH2). t -C(O)NH- (where t is an integer from 1 to 6); s is an integer from 0 to 12; and Goods as defined herein (e.g., ASO or siRNA).

31. A compound, said compound being selected from: And its pharmaceutically acceptable salts, wherein the nucleic acid represents the goods as defined in any of the preceding claims.

32. The compound of claim 31 or a pharmaceutically acceptable salt thereof, wherein the nucleic acid represents ASO or siRNA.

33. The compound or its pharmaceutically acceptable salt claimed or defined according to any one of claims 1 to 32, having an IC50 concentration of less than about 20 nM against ASGPR. 50 Values ​​(e.g., less than approximately 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, or 2 nM IC) 50 The binding affinity is represented by the value.

34. The compound or its pharmaceutically acceptable salt claimed or defined in any one of claims 1 to 32, having binding kinetics to ASGPR characterized by a Kd value of less than about 8 nM (e.g., a Kd value of less than about 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM or 0.5 nM).

35. The compound or a pharmaceutically acceptable salt thereof claimed or defined according to any one of claims 1 to 32, having an IC50 concentration of less than about 50 nM. 50 Values ​​(e.g., less than about 20 nM, 10 nM, or 5 nM IC) 50 The knockdown of gene expression in HEK293 cells was characterized by a value (value).

36. The compound or a pharmaceutically acceptable salt thereof claimed or defined in any one of claims 1 to 32, having the activity of knocking down gene expression in tissues such as the liver in vivo, wherein the compound is capable of knocking down the level of target mRNA in the tissue by at least about 40% (e.g., at least about 45%, 50%, 55%, 60%, 65%, or 70%).

37. A pharmaceutical composition comprising a compound claimed in any one of claims 1 to 36 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient or carrier.

38. The compound of any one of claims 1 to 36 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 37, for use in a therapeutic manner.

39. The compound of any one of claims 1 to 36 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 37, for use in the treatment of conditions selected from: liver disease (e.g., liver cancer), hereditary diseases, hemophilia and bleeding disorders, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), viral hepatitis, rare diseases (e.g., acromegaly), metabolic diseases, cardiovascular diseases, obesity, thalassemia, liver injury (e.g., drug-induced liver injury), hemochromatosis, alcoholic liver disease, alcohol dependence, anemia, and anemia of chronic disease.

40. The compound, pharmaceutically acceptable salt, or pharmaceutical composition used according to claim 39, wherein the condition is selected from NASH, NAFLD, metabolic diseases, and cardiovascular diseases.

41. The compound, pharmaceutically acceptable salt, or pharmaceutical composition used according to claim 39, wherein the condition is NASH.

42. The compound, pharmaceutically acceptable salt, or pharmaceutical composition used according to claim 39, wherein the condition is a metabolic disorder selected from hypercholesterolemia, dyslipidemia, and hypertriglyceridemia.

43. A compound having the structure of formula (VI): #imgpt72# Or a pharmaceutically acceptable salt thereof, wherein X, Y, spacer group and chain are defined according to any one of the preceding claims; #imgpt73# represents a carbon-carbon single or double bond, provided that X is a covalent bond and #imgpt74# represents a carbon-carbon single bond; R" is an acyl, -C(O)aryl or -H; and J is -CO2H, -OH, -C(O)O-(pentafluorophenyl), -N3 or phosphoramide.

44. A method for preparing a compound of formula (III) claimed according to any one of claims 16 to 19, characterized in that, React the compound of formula (VI) claimed in claim 43 with a compound having the following structure. Q - [Goods] The goods are defined according to any one of the preceding claims, and Q represents a group that is reactive to group J as defined in claim 43.

45. The method of claim 44, wherein Q represents a group containing BCN, and J is -N3.

46. ​​A compound having the structure of formula (X): #imgpt75# Or a pharmaceutically acceptable salt thereof, wherein Z, R, m and the chain are as defined in any of the preceding claims; R" is an acyl, -C(O) aryl or -H; and J is -CO2H, -OH, -C(O)O-(pentafluorophenyl), -N3 or phosphoramide.

47. A method for preparing a compound of formula (IX) claimed in claim 29, characterized in that, React the compound of formula (X) claimed according to claim 46 with a compound having the following structure. Q - [Goods] The goods are defined according to any one of the preceding claims, and Q represents a group that is reactive to group J as defined in claim 46.

48. A compound having the structure of formula (XII): #imgpt76# Or a pharmaceutically acceptable salt thereof, wherein Z, R, m and q are as defined in any one of the preceding claims; D' is selected from (i) the bond and (ii) -NHC(O)-(CH2). t - (where t is as described in this article), R" is an acyl group, -C(O) aryl group, or -H; and J is -CO2H, -OH, -C(O)O-(pentafluorophenyl), -N3 or phosphoramide.

49. The compound according to claim 48, wherein J is -CO2H or -C(O)O-(pentafluorophenyl).

50. A method for preparing a compound of formula (XI) as defined in claim 30, characterized in that, React the compound of formula (XII) as defined in claim 48 or claim 49 with a compound having the following structure. Q - [Goods] The goods are defined according to any one of the preceding claims, and Q represents a group that is reactive to group J as defined in claim 48 or claim 49.

Images