Methods for synthesizing targeting ligand-conjugated nucleotide phosphoramidites

Abstract

Disclosed are methods for making targeting ligand-conjugated nucleotide phosphoramidites known as adem-A-GalNAc phosphoramidites, which can be used in the synthesis of oligonucleotides, such as therapeutic oligonucleotides.

Description

[Technical Field]

[0001] The present disclosure relates generally to improved methods for making nucleotide phosphoramidites, i.e., the targeted ligand-conjugated nucleotide phosphoramidite known as (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-(((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2-cyanoethoxy)(diisopropylamino)phosphanyl)oxy)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (adem-A-GalNAc phosphoramidite), which can be used in the production of therapeutic oligonucleotides. [Background technology]

[0002] Oligonucleotides are short polymeric sequences of nucleotides that have a wide range of uses, including as primers, probes, and therapeutic agents. Oligonucleotides, such as therapeutic oligonucleotides, can be chemically synthesized using various known methods. Various chemical modifications can be made to one or more of the nucleotides in a therapeutic oligonucleotide to introduce improved properties for in vivo administration (e.g., to stabilize the oligonucleotide against nucleases, to increase the cellular uptake of the oligonucleotide, and / or to enhance other pharmacodynamic and / or pharmacokinetic properties of the oligonucleotide).

[0003] WO 2016 / 100401 describes a method for making amidites known as adem-A-GalNAc phosphoramidites to improve therapeutic oligonucleotides for in vivo administration. The method described therein uses lengthy process steps that increase production costs and result in a less robust and efficient manufacturing route, especially when applied to large-scale commercial production.

[0004] In view of the above, there is a need for improved methods for making adem-A-GalNAc phosphoramidites. Summary of the Invention

[0005] The present disclosure describes methods for making adem-A-GalNAc phosphoramidites. In one example, (1) silylation of N-benzoyl adenosine to obtain N-(9-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide; (2) protecting N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide as the methylthiomethyl ether to obtain N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide; (3) forming a pentaacetate on (2R,3R,4R,5R,6R)-2-amino-3,4,5,6-tetrahydroxyhexanal hydrochloride to obtain (2R,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)-tetrahydro-2H-pyran-2,4,5-triyl triacetate; (4) forming a glycosidic bond on (2R,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)-tetrahydro-2H-pyran-2,4,5-triyl triacetate to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(hex-5-en-1-yloxy)tetrahydro-2H-pyran-3,4-diyl diacetate; (5) oxidizing (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(hex-5-en-1-yloxy)tetrahydro-2H-pyran-3,4-diyl diacetate to obtain 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid; (6) amidating 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-hydroxyethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate; (7) (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((6aR,8R,9R,9aR)-8-(6-benzamido-9H-purin-9-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-9-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate, (2R,3R,4R ,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-hydroxyethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate to replace the sulfoxide on N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide; (8) (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate, )-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((6aR,8R,9R,9aR)-8-(6-benzamido-9H-purin-9-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-9-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate; (9) (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyldiacetate tritylation of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate to obtain tetrahydro-2H-pyran-3,4-diyl diacetate; (10) (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-(((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2-cyanoethoxy)(diisopropylamino)phosphanyl)oxy)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (adem-A-GalNAc phosphoramidite) and phosphorylating (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate to give

[0006] Another example is: (1') providing a compound comprising N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide; providing a compound comprising (2')(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-hydroxyethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate; (3') (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((6aR,8R,9R,9aR)-8-(6-benzamido-9H-purin-9-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-9-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (2R,3R,4 displacing the sulfoxide on N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide with R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-hydroxyethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate; (4') (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (2R,3R,4R,5R,6 deprotecting R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((6aR,8R,9R,9aR)-8-(6-benzamido-9H-purin-9-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-9-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate; (5')(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyldiacetate tritylation of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate to obtain tetrahydro-2H-pyran-3,4-diyl diacetate; (6')(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-(((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2-cyanoethoxy)(diisopropylamino)phosphanyl)oxy)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (adem-A-GalNAc phosphoramidite) and phosphorylating (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate to give

[0007] In some examples, N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide is (1) silylation of N-benzoyl adenosine to obtain N-(9-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide; (2) protecting N-(9-((6aR,8R,9R,9aR)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide as the methylthiomethyl ether to obtain N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide.

[0008] In some examples, (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-hydroxyethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate is (1) forming a pentaacetate on (2R,3R,4R,5R,6R)-2-amino-3,4,5,6-tetrahydroxyhexanal hydrochloride to obtain (2S,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)-tetrahydro-2H-pyran-2,4,5-triyl triacetate; (2) forming a glycosidic bond on (2R,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)-tetrahydro-2H-pyran-2,4,5-triyl triacetate to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(hex-5-en-1-yloxy)tetrahydro-2H-pyran-3,4-diyl diacetate; (3) oxidizing (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(hex-5-en-1-yloxy)tetrahydro-2H-pyran-3,4-diyl diacetate to obtain 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid; (4) amidating 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-hydroxyethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate.

[0009] An advantage of the methods herein is that the materials used are available on a commercial scale.

[0010] An advantage of the methods herein is that they facilitate less resource intensive, cheaper and / or more efficient production of adem-A-GalNAc phosphoramidites.

[0011] An advantage of the methods herein is that they are based on a convergent approach to making adem-A-GalNAc phosphoramidites, which reduces the number of steps required to generate adem-A-GalNAc phosphoramidites and improves efficiency compared to linear approaches. [Brief explanation of the drawings]

[0012] Further advantages, benefits, features and objects will become more readily apparent from a consideration of the following detailed description, which refers to the following drawings. [Figure 1A] 1 shows an exemplary scheme for making adem-A-GalNAc phosphoramidite. [Figure 1B] 1 shows an exemplary scheme for making adem-A-GalNAc phosphoramidite. DETAILED DESCRIPTION OF THE INVENTION

[0013] overview Chemical modifications can be introduced into therapeutic oligonucleotides to confer desirable properties under certain conditions, such as those experienced after their in vivo administration. These modifications can be introduced into the base, sugar, and / or phosphate groups of one or more nucleotides of the oligonucleotide. Such modifications include, for example, those designed to (i) stabilize the oligonucleotide against nucleases or other enzymes that degrade or interfere with the structure or activity of the oligonucleotide, (ii) increase cellular uptake of the oligonucleotide, and / or (iii) improve the pharmacokinetic properties of the oligonucleotide.

[0014] For example, therapeutic oligonucleotides can include targeting ligands at the 5' or 3' end or on internal nucleotides to selectively target the oligonucleotides to desired cells, tissues, and / or organs. One such targeting ligand is N-acetylgalactosamine (GalNAc), which can be incorporated into therapeutic oligonucleotides, including in the form of adem-A-GalNAc phosphoramidite. See WO 2016 / 100401.

[0015] Abbreviations and Definitions 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 belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods, exemplary methods and materials are described herein.

[0016] In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that there is one and only one element. Thus, the indefinite article "a" or "an" normally means "at least one."

[0017] Furthermore, the use of "including" and other forms such as "include," "includes," and "included" is not limiting.

[0018] Certain abbreviations used herein are as follows:

[0019] "AcOH" refers to acetic acid (C2H4O2), "Ac2O" refers to acetic anhydride (C4H6O3), "CDI" refers to 1,1'-carbonyldiimidazole, "DCM" refers to dichloromethane (CHCl2), "DIPDS" refers to 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, and "DMAP" refers to 4-dimethylaminopyridine (C7H 10 N2), "DMSO" refers to dimethyl sulfoxide (C2H6OS), "DMSO-d6" refers to deuterated dimethyl sulfoxide (C2D6OS), and "DMTr-Cl" refers to 4,4'-dimethoxytriphenylmethyl chloride (C 21 H 19 "DNA" refers to deoxyribonucleic acid, "EA" refers to ethyl acetate (C4H8O2), "ES / MS" refers to electrospray mass spectrometry, "eq" refers to equivalents, "GalNAc" refers to N-acetylgalactosamine, "HPLC" refers to high performance liquid chromatography, "hr" refers to hours, "Me" refers to methyl (-CH3), "MeOH" refers to methanol (C4O), "min" refers to minutes, and "MTBE" refers to methyl tert-butyl ether (C5H 12 "NIS" refers to N-iodosuccinimide (C4H4INO2), "NMI" refers to 1-methylimidazole, "NMM" refers to N-methylmorpholine, "RNA" refers to ribonucleic acid, and "TEA" refers to triethylamine (C6H 15N), "TfOH" refers to trifluoromethanesulfonic acid (CF3SO3H), "THF" refers to tetrahydrofuran (C4H8O), and "V" refers to volume.

[0020] Certain definitions used herein are as follows:

[0021] As used herein, "about" means within a statistically significant range of values, e.g., a specified concentration, length, molecular weight, pH, sequence similarity, time frame, temperature, volume, etc. Such values ​​or ranges can be within 20%, within 15%, within 10%, or more typically within 5% of a given value or range. Alternatively, with respect to biological systems or processes, "about" can mean within an order of magnitude, e.g., within 5-fold, or more typically within 2-fold, of a given value. The acceptable variation encompassed by "about" will depend on the system under study and can be readily appreciated by one of ordinary skill in the art.

[0022] As used herein, "modified nucleobase" means a nucleobase comprising a modified purine or pyrimidine base (e.g., adenine A, guanine G, cytosine C), thymine (T), and uracil U). Examples of modified nucleobases include, but are not limited to, diaminopurine and derivatives thereof, alkylated purines or pyrimidines, acylated purines or pyrimidines, thiolated purines or pyrimidines, etc. Other modified nucleobases include 1-methyladenine, 2-methyladenine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentyladenine, N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethycytosine, 4-acetylcytosine, 1-methylguanine, 2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, 5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil, 5- Purine and pyrimidine analogs include, but are not limited to, (carboxyhydroxymethyl)uracil, 5-(methylaminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil, 2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, pseudouracil, 1-methylpseudouracil, queosine, hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyarninopurine, nitropyrrolyl, nitroindolyl, and difluorotolyl, 6-thiopurine, and 2,6-diaminopurine. Alternatively, the modified nucleobase may not contain a nitrogen atom (i.e., a universal base. See also WO 2003 / 040395. Alternatively, the modified nucleobase is abasic (i.e., does not include a nucleobase).

[0023] As used herein, "modified nucleoside" refers to a nucleoside comprising a modified or universal nucleobase and / or a modified sugar. A modified or universal nucleobase (also referred to herein as a base analog) can be located at the 1'-position of the sugar moiety and refers to a nucleobase other than adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U) at the 1'-position. In some instances, a modified nucleotide does not contain a nucleobase (abasic). A modified sugar (also referred to herein as a sugar analog) comprises a modified deoxyribose or ribose moiety (e.g., the modification occurs at the 2'-, 3'-, 4'-, or 5'-carbon position of the sugar). Modified sugars can also contain non-natural alternative carbon structures, such as those present in bridged nucleic acids ("BNA"), locked nucleic acids ("LNA"), and / or unlocked nucleic acids ("UNA").

[0024] As used herein, "modified nucleotide" refers to a nucleotide comprising a modified or universal nucleobase, as described above, a modified sugar, as described above, and / or a modified phosphate or phosphate group. The modified phosphate can be a modification of a phosphate or phosphate group that does not occur in naturally occurring nucleotides, including non-naturally occurring phosphate mimetics as known in the art. Modified phosphate or phosphate groups also include non-naturally occurring internucleotide linkage groups, including both phosphorus-containing and non-phosphorus-containing linkage groups as known in the art. Suitable modified or universal nucleobases, modified sugars, and modified phosphates or phosphate groups are described herein.

[0025] As used herein, "nucleobase" refers to a heterocyclic nitrogenous base that can pair with a complementary nucleobase or nucleobase analog (i.e., a nucleobase derivative) to form Watson-Crick hydrogen bonding and stacking interactions when the nucleobase is incorporated into a polymer structure. Naturally occurring heterocyclic nitrogenous bases include purines and pyrimidines, such as adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U).

[0026] As used herein, "nucleoside" means a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar moiety (e.g., deoxyribose, ribose, or an analog thereof). Naturally occurring heterocyclic nitrogenous bases include adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U).

[0027] As used herein, "nucleoside phosphoramidite" means a derivative of a natural or synthetic nucleoside in which the reactive hydroxy and exocyclic amino groups present in the natural or synthetic nucleoside are suitably protected to prevent undesired side reactions during nucleic acid synthesis.

[0028] As used herein, "nucleotide" refers to an organic compound having a nucleoside (e.g., a nucleic acid base such as adenine, cytosine, guanine, thymine, or uracil, and a pentose sugar such as ribose or 2'-deoxyribose) and a phosphate group. "Nucleotides" can function as monomer units of nucleic acid polymers such as DNA and ribonucleic acid (RNA).

[0029] As used herein, "nucleotide phosphoramidite" means a derivative of a natural or synthetic nucleotide in which the reactive hydroxy and exocyclic amino groups present in the natural or synthetic nucleotide are appropriately protected to prevent undesired side reactions during nucleic acid synthesis.

[0030] As used herein, "oligonucleotide" refers to a short nucleic acid (e.g., less than about 100 nucleotides in length) of ribonucleotides, deoxyribonucleotides, or a combination thereof. An oligonucleotide may be single-stranded (ss) or double-stranded (ds). An oligonucleotide may or may not have a duplex region. As a non-limiting set of examples, an oligonucleotide may be, but is not limited to, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a dicer substrate interfering RNA (dsiRNA), an antisense oligonucleotide (ASO), a short siRNA, or a ss siRNA.

[0031] As used herein, "phosphoramidite" means a nitrogen-containing trivalent phosphorus derivative that may have the formula: (RO)2PNR2.

[0032] As used herein, a "protecting group" refers to a group that reversibly renders a functional group unreactive under certain conditions of a desired reaction. After the desired reaction, the protecting group can be removed to deprotect the protected functional group. The protecting group should be removable under conditions that do not substantially reduce the amount of the molecule (i.e., oligonucleotide) being synthesized.

[0033] As used herein, "ribonucleotide" means a natural or modified nucleotide that has a hydroxyl group at the 2' position of the sugar moiety.

[0034] As used herein, a "targeting ligand" refers to a chemical moiety that facilitates the entry of an oligonucleotide, such as an RNAi agent, into a cell. It can be a compound (e.g., an amino sugar, carbohydrate, cholesterol, lipid, or polypeptide) that selectively binds to a cognate compound (e.g., a receptor) in a tissue or cell of interest and can be conjugated to another substance to target the other substance to the tissue or cell of interest. For example, a targeting ligand can be conjugated to an oligonucleotide for the purpose of targeting the oligonucleotide to a specific tissue or cell of interest. The targeting ligand can selectively bind to a cell surface receptor. Thus, when conjugated to an oligonucleotide, the targeting ligand facilitates delivery of the oligonucleotide to a specific cell through selective binding to a receptor expressed on the surface of the cell and endosomal internalization by the cell of a complex comprising the oligonucleotide, targeting ligand, and receptor. Furthermore, the targeting ligand can be conjugated to the oligonucleotide via a linker that is cleaved after or during cellular internalization, thereby releasing the oligonucleotide from the targeting ligand within the cell.

[0035] composition adem-A-GalNAc phosphoramidite: The structure of adem-A-GalNAc phosphoramidite is as follows:

[0036] [ka] See, for example, WO 2016 / 100401.

[0037] adem-A-GalNAc modified oligonucleotides: The adem-A-GalNAc phosphoramidite can be incorporated into an oligonucleotide, such as a therapeutic oligonucleotide. In some cases, the adem-A-GalNAc phosphoramidite can be incorporated at the 3' end of the oligonucleotide. In other cases, the adem-A-GalNAc phosphoramidite can be incorporated at the 5' end of the oligonucleotide. In still other cases, the adem-A-GalNAc phosphoramidite can be incorporated at both the 5' and 3' ends of the oligonucleotide. In still other cases, the adem-A-GalNAc phosphoramidite can be incorporated at one or more internal positions of the oligonucleotide. See, e.g., WO 2016 / 100401, WO 2021 / 188795, WO 2022 / 032288, and WO 2022 / 221430.

[0038] Oligonucleotides (e.g., ds oligonucleotides, such as targeting ligand-conjugated oligonucleotides) can be prepared using methods and / or techniques known to those skilled in the art, such as, for example, conventional nucleic acid solid-phase synthesis. The nucleotides of the oligonucleotide can be assembled on a suitable nucleic acid synthesizer using standard nucleotide or nucleoside precursors (e.g., phosphoramidites). Automated nucleic acid synthesizers, including DNA / RNA synthesizers, are commercially available from, for example, Applied Biosystems (Foster City, CA), BioAutomation (Irving, TX), and GE Healthcare Life Sciences (Pittsburgh, PA).

[0039] In some cases, oligonucleotide synthesis steps may be performed in alternating order to obtain the desired compound. Other synthetic chemical transformations, protecting groups (e.g., present on bases for hydroxyl, amino, etc.), and protecting group methodologies (protection and deprotection) useful in synthesizing oligonucleotides are known in the art, see, for example, Larock, "Comprehensive Organic Transformations," VCH Publishers (1989); Greene & Wuts, "Protective Groups in Organic Synthesis," 2000; nd Ed., John Wiley & Sons (1991), Fieser & Fieser, "Fieser and Fieser's Reagents for Organic Synthesis", John Wiley & Sons (1994), and Paquette, ed., "Encyclopedia of Reagents for Organic Synthesis", John Wiley & Sons (1995).

[0040] Pharmaceutical Composition: The adem-A-GalNAc modified oligonucleotide (or a pharmaceutically acceptable salt thereof, e.g., trifluoroacetate, acetate, or hydrochloride) can be incorporated into a pharmaceutical composition comprising an effective amount of an adem-A-GalNAc-containing oligonucleotide and a pharmaceutically acceptable carrier, delivery agent, or excipient. See, e.g., WO 2016 / 100401, WO 2021 / 188795, WO 2022 / 032288, and WO 2022 / 221430.

[0041] Various formulations have been developed to facilitate the use of oligonucleotides. In some instances, oligonucleotides can be delivered to an individual or cellular environment using formulations that minimize degradation, facilitate delivery and / or uptake, or provide other beneficial properties to the oligonucleotides in the formulation. In some instances, oligonucleotides can be formulated in buffers such as phosphate-buffered saline, liposomes, micellar structures, and capsids.

[0042] In some instances, oligonucleotides can react with inorganic and organic acids / bases to form pharmaceutically acceptable acid / base addition salts. In some instances, forming pharmaceutically acceptable acid / base addition salts improves the in vivo compatibility and / or efficacy of the oligonucleotide. Pharmaceutically acceptable salts and general methodologies for preparing them are well known in the art (e.g., Stahl et al., "Handbook of Pharmaceutical Salts: Properties, Selection and Use," 2004). nd Revised Edition (Wiley-VCH, 2011). Pharmaceutically acceptable salts for use herein include sodium, trifluoroacetate, hydrochloride, and acetate salts.

[0043] In some instances, a pharmaceutical composition can be formulated to be compatible with its intended route of administration, including, but not limited to, parenteral (e.g., intravenous, intramuscular, intraperitoneal, intradermal, and subcutaneous), oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration.

[0044] Additionally, factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations can be contemplated by those skilled in the art of preparing such pharmaceutical formulations, and therefore, various dosages and treatment regimens may be desirable.

[0045] In some examples, the pharmaceutical composition may include one or more additional therapeutic agents.

[0046] method How to make adem-A-GalNAc phosphoramidite: The method for making adem-A-GalNAc phosphoramidite or a salt thereof can include the steps described herein, which may, but need not, be performed in the order described. However, other orders are contemplated. Furthermore, individual or multiple steps may be performed in parallel and / or overlapping time, and / or individually or in multiple repeated steps. The product of each of the following steps can be recovered by conventional methods, including chromatography, crystallization, evaporation, extraction, filtration, precipitation, and trituration.

[0047] In one example, adem-A-GalNAc phosphoramidite can be prepared according to the following method, which is as follows: (1) to obtain N-(9-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide,

[0048] [ka] silylation of N6-benzoyl adenosine;

[0049] [ka] (2) protecting N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide as the methylthiomethyl ether to obtain N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide;

[0050] [ka] (3) forming a pentaacetate on (2R,3R,4R,5R,6R)-2-amino-3,4,5,6-tetrahydroxyhexanal hydrochloride to obtain (2R,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)-tetrahydro-2H-pyran-2,4,5-triyl triacetate;

[0051] [ka] (4) forming a glycosidic bond on (2R,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)-tetrahydro-2H-pyran-2,4,5-triyl triacetate to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(hex-5-en-1-yloxy)tetrahydro-2H-pyran-3,4-diyl diacetate;

[0052] [ka] (5) oxidizing (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(hex-5-en-1-yloxy)tetrahydro-2H-pyran-3,4-diyl diacetate to obtain 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid;

[0053] [ka] (6) amidating 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-hydroxyethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate;

[0054] [ka] (7) (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((6aR,8R,9R,9aR)-8-(6-benzamido-9H-purin-9-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-9-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate, (2R,3R,4R ,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-hydroxyethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate to replace the sulfoxide on N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide;

[0055] [ka] (8) (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate, )-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((6aR,8R,9R,9aR)-8-(6-benzamido-9H-purin-9-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-9-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate;

[0056] [ka] (9) (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyldiacetate tritylation of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate to obtain tetrahydro-2H-pyran-3,4-diyl diacetate;

[0057] [ka] (10) (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2-cyanoethoxy)(diisopropylamino)phosphanyl)oxy)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (a phosphorylating (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate to obtain (dem-A-GalNAc phosphoramidite);

[0058] [ka] may include:

[0059] In another example, adem-A-GalNAc phosphoramidite can be prepared according to the following method, which is as follows: (1') providing a compound comprising N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide; providing a compound comprising (2')(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-hydroxyethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate; (3') (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((6aR,8R,9R,9aR)-8-(6-benzamido-9H-purin-9-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-9-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (2R,3R,4 displacing the sulfoxide on N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide with R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-hydroxyethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate; (4') (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (2R,3R,4R,5R,6 deprotecting R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((6aR,8R,9R,9aR)-8-(6-benzamido-9H-purin-9-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-9-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate; (5')(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyldiacetate tritylation of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate to obtain tetrahydro-2H-pyran-3,4-diyl diacetate; (6')(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2-cyanoethoxy)(diisopropylamino)phosphanyl)oxy)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (adem-A) and phosphorylating (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate.

[0060] In yet another example, adem-A-GalNAc phosphoramidite can be prepared according to the following method, which is as follows: to obtain (1''')(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((6aR,8R,9R,9aR)-8-(6-benzamido-9H-purin-9-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-9-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate. displacing the sulfoxide on N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide with 4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-hydroxyethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate;

[0061] [ka] to obtain (2''')(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate. deprotecting 6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((6aR,8R,9R,9aR)-8-(6-benzamido-9H-purin-9-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-9-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate;

[0062] [ka] (3''')(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyldiazolidinyl tritylation of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate to obtain acetate;

[0063] [ka] (4''')(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-(((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2-cyanoethoxy)(diisopropylamino)phosphanyl)oxy)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate( phosphorylating (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate to obtain adem-A-GalNAc phosphoramidite;

[0064] [ka] may include:

[0065] In some examples, N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide can be prepared according to the following method, which is as follows: (1) to obtain N-(9-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide,

[0066] [ka] silylation of N6-benzoyl adenosine;

[0067] [ka] (2) protecting N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide as the methylthiomethyl ether to obtain N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide;

[0068] [ka] may include:

[0069] In some examples, (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-hydroxyethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate can be prepared according to the following method, which is as follows: (1) forming a pentaacetate on (2R,3R,4R,5R,6R)-2-amino-3,4,5,6-tetrahydroxyhexanal hydrochloride to obtain (2R,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)-tetrahydro-2H-pyran-2,4,5-triyl triacetate;

[0070] [ka] (2) forming a glycosidic bond on (2R,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)-tetrahydro-2H-pyran-2,4,5-triyl triacetate to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(hex-5-en-1-yloxy)tetrahydro-2H-pyran-3,4-diyl diacetate;

[0071] [ka] (3) oxidizing (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(hex-5-en-1-yloxy)tetrahydro-2H-pyran-3,4-diyl diacetate to obtain 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid;

[0072] [ka] (4) amidating 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-hydroxyethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate;

[0073] [ka] may include: [Example]

[0074] The following non-limiting examples are provided for illustrative purposes and are not intended to limit the scope of the present disclosure.

[0075] Example 1: N-(9-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide,

[0076] [ka] Synthesis of.

[0077] Method: N6-benzoyladenosine (1 kg, 2.693 mol) was dissolved in dry pyridine. TiPDS-Cl (1.02 kg, 3.23 mol) was added dropwise to the solution while maintaining the temperature at 5°C. The solution was warmed to 25°C, and the mixture was then stirred at 25°C for 2 hours and then quenched with MeOH (800 mL). The solution was concentrated in vacuo to remove pyridine, and DCM (10 L) was added. The solution was washed with 30% aqueous citric acid (10 L x 2) and 20% aqueous NaCl (10 L) to give a DCM solution (13.3 kg) of the title compound.

[0078] Results: Weight: 1.357 kg (determined by HPLC in solution); Purity: 92.4%; Yield: 82.1%; ES-MS m / z 614.5 (M+H).

[0079] Example 2: N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide,

[0080] [ka] Synthesis of.

[0081] Procedure: A solution of N-(9-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide (13.3 kg) from Example 1 in DCM was diluted with DMSO (4.0 L). The solution was concentrated under vacuum below 45° C. to remove DCM. Toluene (6.8 L) was then added to the solution. Acetic acid (4.27 kg) and acetic anhydride (3.1 kg) were added sequentially at 10° C. The reaction was heated to 50° C. and stirred for 42 hours. The reaction was quenched at 20° C. by the dropwise addition of MeOH (4 L). The solution was then diluted with EA (14 L) and washed with 25% aqueous K2CO3 (14 L × 2) and saturated aqueous NaCl (14 L × 1). The organic phase was concentrated to 2 V under vacuum and then diluted with n-heptane / EA (2:1; 6.5 L). The solution was filtered through silica gel (7 kg), and the silica gel was eluted with n-heptane / EA (100%, approximately 1:1). The filtrate was concentrated to 1 V to 2 V and then diluted with EA (2.7 L). The resulting solution was added to n-heptane / HO (5 V / 2 V). The mixture was stirred for 1 h and then filtered to give the title compound (933 g, purity 59%) as an off-white solid.

[0082] result: 1H NMR(400MHz,DMSO-d6)δ11.24(s,1H),8.67(s,1H),8.53(s,1H),8.06(m,2H),7.66(m,1H),7.56( ES-MS m / z 674.5(M+H).

[0083] Example 3: (2R,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)-tetrahydro-2H-pyran-2,4,5-triyl triacetate,

[0084] [ka] Synthesis of.

[0085] Method: Under a N2 atmosphere at 20°C, (2R,3R,4R,5R)-2-amino-3,4,5,6-tetrahydroxyhexanal hydrochloride (387 g, 1.79 mol) was dissolved in pyridine (8 V, 3096 mL). A catalytic amount of DMAP (153 g, 1.26 mol) was added, and the mixture was stirred at the same temperature. The mixture was cooled to 0°C-10°C, and AcO (6 V, 2322 mL) was added dropwise, and the mixture was stirred at the same temperature. The solution was warmed to 20°C and stirred for 18 h. Toluene (12 V) was added to the reaction solution at 20°C, and the mixture was stirred at 20°C for 30 min. The solution was then concentrated, and MeOH (5 V) was added and stirred at 20°C for 2 h. The solid was filtered, and the resulting cake was washed with MeOH. The solid was then dried under vacuum to give the title compound (600 g, 86%) as a white solid.

[0086] Result: Weight: 1H NMR(400MHz,DMSO-d6)δ7.89(d,J=9.3Hz,1H),5.64(d,J=8.8Hz,1H),5.29-5.24(m,1H),5.06(dd,J=11.4,3.4Hz, ES-MS m / z 406.8(M+NH4).

[0087] Example 4: (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(hex-5-en-1-yloxy)tetrahydro-2H-pyran-3,4-diyl diacetate,

[0088] [ka] Synthesis of.

[0089] Procedure: (2R,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)-tetrahydro-2H-pyran-2,4,5-triyl triacetate (150 g, 0.39 mol) from Example 3 was dissolved in DCM (10 V, 1.5 L). 5-Hexenol (44.34 g, 1.15 equiv., 44.9 mol) was added to the mixture, and the mixture was stirred at 20 °C for 10-15 min. FeCl (37.4 g, 0.6 equiv., 37.95 mol) was added to the mixture at 20 °C under a N atmosphere. The mixture was then stirred at 20 °C until complete conversion (i.e., approximately 16 h). After the reaction was judged complete, HO (5 V) was added, and the mixture was stirred for 30 min. The mixture was extracted with DCM (2 × 10 V). The organic layers were combined, dried over Na2SO4, filtered, and concentrated to 1 V. n-Heptane (10 V-15 V) was added at 20 °C, and the mixture was stirred for 4 h. The solid was filtered and dried to give the title compound (149 g, 90%) as a white solid.

[0090] result: 1H NMR(400MHz,DMSO-d6)δ7.80(d,J=9.1Hz,1H),5.75(s,2H),5.24-5.16(m,1H),5.03-4.91(m,3H),4.49(d,J=8.5Hz,1H),4.02(s,3H),3.92-3.81(m ,1H),3.79-3.63(m,1H),3.49-3.36(m,1H),2.10(s,3H),2.05-1.95(m,6 ES-MS m / z 430(M+H).

[0091] Example 5: 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid,

[0092] [ka] Synthesis of.

[0093] Procedure: (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(hex-5-en-1-yloxy)tetrahydro-2H-pyran-3,4-diyl diacetate (143 g, 0.33 mol) from Example 4 was dissolved in DCM:CHCN:HO (3.5 V:3.5 V:4.75 V) at 20 °C, and the mixture was stirred for 20 min. The mixture was cooled to 0 °C-5 °C, and NaIO (284 g, 4 equiv., 1.32 mol) was added, followed by RuCl·HO (1.723 g, 0.027 equiv., 0.00891 mol). The reaction mixture was stirred at 5°C to 20°C, and an additional amount of NaIO4 (2 equiv., 0.66 mol) was added, and the mixture was stirred for an additional 2 h. The periodate was filtered off with DCM (2 V). The organic layer was separated, and the aqueous layer was back-extracted with DCM (2 x 10 V) by saturating the aqueous layer with NaCl. The organic layers were combined and concentrated to 1 V to 2 V. MTBE was added dropwise (10 V to 15 V) with constant stirring over 2 to 4 h. The solid was filtered, and the cake was washed with MTBE (2 V). The solid was dried to give the title compound (140 g, 79%) as a white solid.

[0094] result: 1 H NMR(400MHz,DMSO-d6)δ=11.98(br s,1H),7.80(d,J=9.3Hz,1H),5.21(d,J=3.4Hz,1H),4.96(dd,J=11.3,3.4Hz,1H),4.49(d,J=8.4Hz,1H),4.09-3.96(m,3H),3.87(td,J=11.1 ,8.9Hz,1H),3.81-3.61(m,1H),3.47-3.38(m,1H),2.20(s,2H),2.10( s,3H),2.00(s,4H),1.89(s,3H),1.77(s,3H),1.55-1.43(m,5H);ES-MS m / z 448(M+H).

[0095] Example 6: (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-hydroxyethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate,

[0096] [ka] Synthesis of.

[0097] Procedure: 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (470 g, 1.05 mol) from Example 5 was dissolved in DCM (4.7 L). CDI (205 g, 1.26 mol) was added at 5° C., and the mixture was stirred at 20° C. for 2 h. The resulting mixture was added to a solution of 2-(2-aminoethoxy)ethan-1-ol (132.5 g, 1.26 mol) in DCM (940 mL) at 5° C. The reaction mixture was warmed to 20° C. and stirred for 2 h. Tartaric acid (379 g, 2.4 eq, 2.52 mol) in THF (3.76 L) was added to the reaction solution at 5° C. The mixture was stirred at 5° C. for 1 h and then filtered. The filtrate was concentrated, diluted with DCM (4 V), and then filtered through silica gel (2.35 kg). The silica gel was eluted with a gradient from 100% DCM to DCM / MeOH (20 / 1), and the fractions containing the compound were collected and combined. The solution was concentrated and diluted to 10 V with THF to give 4.0 kg of a THF solution of the title compound.

[0098] Results: Weight: 468 g (determined by HPLC of THF solution); Purity: 98.3%; Yield: 83.3%; ES-MS m / z 535.4 (M+H).

[0099] Example 7: (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((6aR,8R,9R,9aR)-8-(6-benzamido-9H-purin-9-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-9-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate,

[0100] [ka] Synthesis of.

[0101] Method: N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide (508.8 g, assay: 93.6%, 0.71 mol) was dissolved in (((2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-hydroxyethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (3.874 kg, assay: 11.7%, 0.85 mol) from Example 6 in THF. The solution was concentrated to remove HO and then diluted with THF (9.5 L). 4 Å molecular sieves (476 g) were added, and the mixture was stirred at 20°C for 30 minutes. NIS (191 g, 1.2 equiv., 0.852 mol) and TfOH (213 g, 2.0 equiv., 1.42 mol) were added sequentially at −35°C to −20°C, and the mixture was stirred at that temperature for 2 hours and then at 5°C for an additional 3 hours. The solution was cooled to −35°C to −20°C, and the reaction was quenched with TEA (210 g) at −35°C to −20°C. The reaction mixture was filtered, and the filtrate was washed with 10% aqueous NaSO (4.8 L) and saturated aqueous NaCl (4.8 L) to give 10.05 kg of a THF solution of the title compound.

[0102] Results: Weight: 763.8 g (determined by HPLC); Purity: 86.7%; Yield: 93.2%; ES-MS m / z 1160.9 l (M+H).

[0103] Example 8: (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate,

[0104] [ka] Synthesis of.

[0105] Method: A THF solution of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((6aR,8R,9R,9aR)-8-(6-benzamido-9H-purin-9-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-9-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate from Example 7 (9.9 kg, assay: 7.6%; 0.65 mol) was concentrated to remove HO and then diluted to 7.5 L with THF. TEA (197 g, 3.0 equiv., 1.95 mol) and TEA·3HF (314 g, 3.0 equiv., 1.95 mol) were added at 5°C, and the mixture was stirred at that temperature for 12 h. DCM (7.5 L) was added, and the mixture was concentrated under vacuum to 5 V–6 V. DCM (7.5 L × 2) was added again. The solution was washed successively with 7% aqueous NaHCO3 (7.5 L) and saturated aqueous NaCl (7.5 L) to give 12.5 kg of a DCM solution of the title compound.

[0106] Results: Weight: 550 g (determined by quantitative HPLC); Purity: 86.5%; Yield: 92.4%; ES-MS m / z 918.6 (M+H).

[0107] Example 9: (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate,

[0108] [ka] Synthesis of.

[0109] Method: A solution of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (1.35 kg, HPLC assay: 4.4%, 0.54 mol) from Example 8 in DCM was concentrated to 5V to 6V. DCM (5 L) was then added to dilute the solution. 4 Å molecular sieves (500 g) were added, and the mixture was stirred at 5° C. for 30 minutes. NMM (221 g, 4.0 equiv., 4.16 mol) and DMTr-Cl (203 g, 1.1 equiv., 0.594 mol; dissolved in 1.5 L of DCM) were added sequentially, and the mixture was stirred at 5 °C for 2 h. The mixture was filtered, and the solution was washed with 7% aqueous NaHCO (2.5 L) and water (2.5 L × 2). The DCM solution was concentrated to 2 V to 3 V, and EA (5 L) was added. The solution was concentrated to 3 V to 4 V, and then n-heptane / MTBE (12.5 L / 12.5 L) was added. The resulting mixture was purified by chromatography on silica gel (4 kg) using a gradient from 100% EA to 1 / 1 EA / acetone. The appropriate fractions were concentrated to an oil. A solution of EA + 0.5% TEA (5 L) was added, and the mixture was concentrated to 1.5 L. The EA solution was added to n-heptane / MTBE (5.45 L / 1.09 L + 0.5% TEA) at 5°C. The mixture was stirred at 5°C ± 5°C for 1 hour, then filtered, and the wet cake was washed with n-heptane (1 L). The wet cake was again purified by chromatography on silica gel (4.35 kg) using a gradient from 100% EA to 1 / 1 EA / acetone. The appropriate fractions were concentrated to an oil. EA + 0.5% TEA (1.08 L) was added, and the EA solution was added to n-heptane / MTBE (4.5 L / 0.9 L + 0.5% TEA) at 5°C. The mixture was stirred at 5°C for 1 hour, then filtered, and the wet cake was washed with n-heptane (1 L) and dried under vacuum at 45°C to give the title compound (381 g, 55%).

[0110] result: 1 H NMR(400MHz,CDCl3)δ9.01(s,1H),8.63(s,1H),δ8.18(s,1H),δ7.98(d,2H),δ7.57-7.53(m,1H),7.48-7.44(m,2H),7.3 8-7.31(m,2H),7.21-7.10(m,8H),6.79-6.71(m,5H),6.39(d,1H),6.2(d,1H),5.3-5.24(m,1H),5.15(dd,1H),4.94-4.8 8(m,1H),4.76(2,2H),4.58(d,1H),4.49-4.44(m,1H),4.29-4.22(m,1H),4.11-3.92(m,4H),3.87-3.75(m,3H),3.71(s, ES-MS m / z 1220.2(M+H).

[0111] Example 10: (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-(((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2-cyanoethoxy)(diisopropylamino)phosphanyl)oxy)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (adem-A-GalNAc phosphoramidite),

[0112] [ka] Synthesis of.

[0113] Procedure: (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (246 g, HPLC assay: 94.9%; 0.19 mol) from Example 9 was added to a solution of NMI (16.0 g, 1.0 equiv., 0.19 mol) and DCM (2.46 L). The solution was concentrated to 5 V and DCM (1.23 L) was added. The solution was then cooled to 5°C, and 4 Å molecular sieves (234 g) and tetrazole (6.7 g, 0.5 equiv., 0.95 mol) were added sequentially. The solution was purged with N2, and bis(diisopropylamino)-phosphanyloxypropanenitrile (92.3 g, 1.6 equiv., 0.304 mol) was added at 5°C. The mixture was warmed to 20°C and then stirred at that temperature for 4 hours. The mixture was filtered, and the solution was washed with 7% aqueous NaHCO3 (1.17 L x 1), water (1.87 L x 1), and saturated aqueous NaCl (1.17 L x 2). The DCM solution was concentrated to an oil, and EA (3.5 L) / MTBE (7.0 L) was added. The solution was washed with DMF / HO (1 / 1, 3.5 L × 2), saturated aqueous NaCl (3.5 L), DMF / HO (1 / 1, 3.5 L × 2), and saturated aqueous NaCl (3.5 L × 2). TEA (55 mL, 0.5% in MTBE, 0.39 mol) was added, and the resulting solution was dried over NaSO (750 g) for 1 hour. The mixture was filtered and concentrated to 5 V. The solution was added to n-heptane / MTBE (3 / 2, 0.96 L / 0.64 L + 0.5% TEA) at 5 °C, and the mixture was stirred at that temperature for 1 hour. The mixture was filtered, and the filter cake was washed with n-heptane / MTBE (3 / 2, 0.3 L / 0.2 L + 0.5% TEA). The solid was dissolved in EA+0.5% TEA (0.2 L) and the solution was added to n-heptane / MTBE (3 / 2, 0.96 L / 0.64 L+0.5% TEA) at 5°C.The mixture was filtered and the solid was washed with n-heptane / MTBE (3 / 2, 50 mL / 33 mL + 0.5% TEA). The solid was dried under vacuum at 50° C. for 61 hours to give the title compound (289.55 g, yield: 100%) as a white solid.

[0114] result: 1 H NMR(400MHz,DMSO-d6)δ11.26(s,1H),8.71-8.60(m,2H),7.82(d,1H),7.79 -7.72(m,1H),7.69-7.61(m,1H),7.59-7.53(m,2H),7.43-7.35(m,2H),7.32 -7.16(m,7H),6.90-6.76(m,4H),6.29-6.20(m,1H),5.28-5.16(m,2H),5.0 1(dd,1H),4.83-4.70(m,3H),4.52(d,1H),4.36-4.24(m,1H),4.11-3.99(m, 3H),3.95-3.80(m,2H),3.78-3.66(m,8H),3.65-3.53(m,2H),3.5-3.37(m, 3H),3.34-3.19(m,5H),3.13-3.05(m,3H),2.82-2.66(m,2H),2.18(s,3H),2 .06-2.03(m,2H),2.00(s,3H),1.91(s,3H),1.78(s,3H),1.54-1.39(m,4H), 1.29-1.20(m,2H),1.19-1.12(m,9H),1.11-1.09(m,4H),1.07-1.03(m,2H). 31 P-NMR(400MHz,DMSO-d6)δ149.36.HRMS(ESI)C 71 H 90 N9O 20 P(M+H) + The calculated value is 1420.6112, and the measured value is 1420.6083 / 1420.6093.

Claims

1. 1. A method of making a compound represented by the structure: 【Chemistry 1】 silylation of N6-benzoyladenosine to obtain N-(9-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilosin-8-yl)-9H-purin-6-yl)benzamide; protecting N-(9-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide as the methylthiomethyl ether to obtain N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide; forming a pentaacetate on (2R,3R,4R,5R)-2-amino-3,4,5,6-tetrahydroxyhexanal hydrochloride to obtain (2R,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)-tetrahydro-2H-pyran-2,4,5-triyl triacetate; forming a glycosidic bond on (2R,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)-tetrahydro-2H-pyran-2,4,5-triyl triacetate to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(hex-5-en-1-yloxy)tetrahydro-2H-pyran-3,4-diyl diacetate; oxidizing (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(hex-5-en-1-yloxy)tetrahydro-2H-pyran-3,4-diyl diacetate to obtain 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid; amidating 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-hydroxyethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate; (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-hydroxyethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate, sulfo-N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxin-8-yl)-9H-purin-6-yl)benzamide displacing the oxide to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((6aR,8R,9R,9aR)-8-(6-benzamido-9H-purin-9-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-9-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate; (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((6aR,8R,9R,9aR)-8-(6-benzamido-9H-purin-9-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilosin-9-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-di deprotecting (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate; (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate was tritylated to give (2R,3R,4R ,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate; (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate was phosphorylated to give (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate. and obtaining)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2-cyanoethoxy)(diisopropylamino)phosphanyl)oxy)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (adem-A-GalNAc phosphoramidite).

2. 1. A method of making a compound represented by the structure: 【Chemistry 2】 providing a compound comprising N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide; providing a compound comprising (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-hydroxyethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate; (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-hydroxyethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate, sulfo-N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisiloxin-8-yl)-9H-purin-6-yl)benzamide displacing the oxide to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((6aR,8R,9R,9aR)-8-(6-benzamido-9H-purin-9-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-9-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate; (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((6aR,8R,9R,9aR)-8-(6-benzamido-9H-purin-9-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilosin-9-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-di deprotecting (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate; (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate was tritylated to give (2R,3R,4R ,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate; (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate was phosphorylated to give (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxytetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate. and obtaining)-6-((5-((2-(2-((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(((2-cyanoethoxy)(diisopropylamino)phosphanyl)oxy)tetrahydrofuran-3-yl)oxy)methoxy)ethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (adem-A-GalNAc phosphoramidite).

3. N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide silylation of N6-benzoyladenosine to obtain N-(9-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilosin-8-yl)-9H-purin-6-yl)benzamide; and protecting N-(9-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide as the methylthiomethyl ether to provide N-(9-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-((methylthio)methoxy)tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisirosin-8-yl)-9H-purin-6-yl)benzamide.

4. (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-hydroxyethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate forming a pentaacetate on (2R,3R,4R,5R)-2-amino-3,4,5,6-tetrahydroxyhexanal hydrochloride to obtain (2S,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)-tetrahydro-2H-pyran-2,4,5-triyl triacetate; forming a glycosidic bond on (2S,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)-tetrahydro-2H-pyran-2,4,5-triyl triacetate to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(hex-5-en-1-yloxy)tetrahydro-2H-pyran-3,4-diyl diacetate; oxidizing (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-(hex-5-en-1-yloxy)tetrahydro-2H-pyran-3,4-diyl diacetate to obtain 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)-tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid; amidating 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid to obtain (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((5-((2-(2-hydroxyethoxy)ethyl)amino)-5-oxopentyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate.

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