4′-phosphate analogs and oligonucleotides containing them

Abstract

This document discloses oligonucleotides, such as nucleic acid inhibitor molecules, having a 4′-phosphate analogue, and methods for using them, for example, to regulate the expression of target genes in cells. The phosphate analogue is bonded to the 4′-carbon of the sugar moiety (e.g., ribose or deoxyribose or an analogue thereof) of the 5′-terminal nucleotide of the oligonucleotide. Typically, the phosphate analogue is an oxymethylphosphonate, wherein the oxygen atom of the oxymethyl group is bonded to the 4′-carbon of the sugar moiety or an analogue thereof.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 383,207, filed September 2, 2016, and U.S. Provisional Patent Application No. 62 / 393,401, filed September 12, 2016, and relies on the filing dates of those U.S. Provisional Patent Applications, the entire disclosure of which is incorporated herein by reference. Background of the Invention

[0004] Oligonucleotides are polymeric sequences of nucleotides (RNA, DNA, and their analogues). Nucleic acid inhibitor molecules are oligonucleotides that regulate intracellular RNA levels and have shown early promise in the treatment of cancer, viral infections, and genetic disorders. Nucleic acid inhibitor molecules can regulate RNA expression through a different set of mechanisms, including RNA interference (RNAi).

[0005] RNAi is a conserved pathway found in most eukaryotes, in which a double-stranded RNA molecule (dsRNA) inhibits the expression of a target gene having a sequence complementary to that dsRNA. In a typical RNAi pathway, the longer dsRNA is cleaved by the cleavage enzyme Dicer into a shorter RNA duplex called small interfering RNA (“siRNA”). siRNA has been shown to associate with cleavage enzyme, trans-activating response RNA-binding protein (TRBP), and Argonaute 2 (“Ago2”) to form a complex, sometimes referred to as the RNA-induced silencing complex (“RISC”). Ago2 is a nuclease that uses the antisense strand (also known as the guide strand) of the siRNA to guide the sequence-specific cleavage of the target mRNA.

[0006] Various double-stranded RNAi inhibitor molecular structures have been developed over the years. For example, early work on RNAi inhibitor molecules focused on double-stranded nucleic acid molecules mimicking natural siRNA, where each strand has a size of 19-25 nucleotides and at least one 3′ overhang with 1 to 5 nucleotides (see, for example, U.S. Patent No. 8,372,968). Subsequently, longer double-stranded RNAi inhibitor molecules were developed, which were processed in vivo by cleatase into active RNAi inhibitor molecules (see, for example, U.S. Patent No. 8,883,996). Subsequent work developed extended double-stranded nucleic acid inhibitor molecules, where at least one end of at least one strand extends beyond the double-stranded target region of the molecule, including structures where one of the strands comprises a thermodynamically stable tetracyclic structure (see, for example, U.S. Patent Nos. 8,513,207, 8,927,705, WO 2010 / 033225, and WO 2016 / 100401). Those structures include single-strand extensions (on one or both sides of the molecule) and double-strand extensions.

[0007] Single-stranded nucleic acid inhibitor molecules are also known in the art. For example, recent work has demonstrated the activity of ssRNAi inhibitor molecules (see, for example, Matsui et al., 2016, 24(5): 946-55). Furthermore, antisense molecules have been used for decades to reduce the expression of specific target genes. Pelechano and Steinmetz, Nature Review Genetics, 2013, 14: 880-93. Numerous variations on common themes related to these structures have been developed for a range of targets. Other single-stranded nucleic acid inhibitor molecules include, for example, microRNAs, riboproteinases, microRNA antagomir, and aptamers, all of which are known in the art.

[0008] In some cases, chemical modifications have been introduced into nucleic acid inhibitor molecules to introduce properties that may be desired under specific conditions, such as those experienced after in vivo administration. These modifications include those designed, for example, to stabilize against nucleases or other enzymes that degrade or interfere with the structure or activity of oligonucleotides, to increase cellular uptake of oligonucleotides, or to improve the pharmacokinetic properties of oligonucleotides.

[0009] For example, synthetic oligonucleotides are typically terminated with a 5′-hydroxy or 3′-hydroxy group. It is possible to replace the terminal hydroxyl group with a phosphate ester group, which can be used, for example, to connect adapters, integrators, or as a label, or to directly link the oligonucleotide to another nucleic acid. Furthermore, it has been reported that the 5′-terminal phosphate ester group enhances the interaction between certain nucleic acid inhibitor molecules and Ago2. However, oligonucleotides with a 5′-phosphate ester group are generally readily degraded by phosphatases or other enzymes, which may limit their bioavailability in vivo.

[0010] Therefore, it is desirable to develop modifications to the 5′-terminal nucleotide of oligonucleotides, such as nucleic acid inhibitor molecules, that provide the functional role of the phosphate ester group, but are more stable to the environmental conditions to which the oligonucleotide will be exposed when administered to a subject. These phosphate ester analogs will be more resistant to phosphatases and other enzymes, while minimizing any negative impacts on the function of the oligonucleotide (e.g., minimizing any reduction in gene target knockdown when used in RNAi inhibitor molecules). Invention Overview

[0012] This application discloses oligonucleotides containing 4′-phosphate analogs. Suitable oligonucleotides include nucleic acid inhibitor molecules, such as dsRNAi inhibitor molecules, antisense oligonucleotides, microRNAs, ribonucleases, microRNA antagonists, aptamers, and ssRNAi inhibitor molecules.

[0013] The phosphate ester analogs disclosed herein are bonded to the 4′-carbon of the sugar moiety (e.g., ribose or deoxyribose or an analogue thereof) of the 5′-terminal nucleotide (“N1 nucleotide”) of the oligonucleotide as described herein. Typically, the phosphate ester analog is an oxymethylphosphonate, wherein the oxygen atom of the oxymethyl group is bonded to the 4′-carbon of the sugar moiety or an analogue thereof. In other embodiments, the phosphate ester analog is a thiomethylphosphonate or an aminomethylphosphonate, wherein the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is bonded to the 4′-carbon of the sugar moiety or an analogue thereof.

[0014] In some embodiments, the 4′-oxymethylphosphonate is represented by -O-CH2-PO(OH)2 or -O-CH2-PO(OR)2, wherein R is independently selected from H, CH3, alkyl, or protecting group. In some embodiments, the alkyl group is CH2CH3.

[0015] In one respect, the phosphate ester analog-modified nucleic acid inhibitor molecules described herein can be used to regulate the expression of target genes in cells. These phosphate ester analog-modified nucleic acid inhibitor molecules can be formulated into pharmaceutical compositions with pharmaceutically acceptable excipients and used to regulate the expression of target genes and treat patients in need.

[0016] In some aspects, this disclosure relates to an oligonucleotide comprising a 5′-terminal nucleotide containing a 4′-oxymethylphosphonate, wherein the 4′-oxymethylphosphonate is -O-CH2-PO(OH)2 or -O-CH2-PO(OR)2, and wherein R is independently selected from H, CH3, alkyl, or a protecting group. In some embodiments, the alkyl group is CH2CH3.

[0017] In some aspects, this disclosure relates to an oligonucleotide comprising a 5′-terminal nucleotide represented by Formula I or Formula II as described herein. In some embodiments, the 5′-terminal nucleotide is represented by Formula I as described herein. In some embodiments, the oligonucleotide is represented by Formula I and X2 is OH, F, OCH2CH2OCH3, or OCH3 and R8 is absent or wherein X2 is O and R8 is a glutathione-sensitive moiety.

[0018] In some aspects, this disclosure relates to an oligonucleotide comprising a 5′-terminal nucleotide represented by Formula III as described herein. In some embodiments of the oligonucleotide, X2 is OH, F, or OCH3 and R8 is absent.

[0019] In some embodiments of the oligonucleotides described herein, R a and R b It is hydrogen; R a It is CH3 or CH2CH3 and R b It is hydrogen; or R a and R b Each is either CH3 or CH2CH3.

[0020] In some respects, this disclosure relates to an oligonucleotide comprising a 5′-terminal nucleotide represented by Formula IV as described herein.

[0021] In some respects, this disclosure relates to an oligonucleotide comprising a 5′-terminal nucleotide represented by formula V as described herein.

[0022] In some aspects, this disclosure relates to an oligonucleotide comprising a 5′-terminal nucleotide represented by Formula VI as described herein. In some embodiments, the sugar moiety is a furanose.

[0023] In some embodiments, the oligonucleotide is a double-stranded RNAi inhibitor molecule comprising a first strand and a second strand, wherein the first strand is a sense strand and the second strand is an antisense strand. In some embodiments, the double-stranded RNAi inhibitor molecule comprises a complementary region of 15 to 45 nucleotides between the sense and antisense strands. In some embodiments, the complementary region between the sense and antisense strands is 20 to 30 nucleotides. In some embodiments, the complementary region between the sense and antisense strands is 21 to 26 nucleotides. In some embodiments, the complementary region between the sense and antisense strands is 19 to 24 nucleotides. In some embodiments, the complementary region between the sense and antisense strands is 19 to 21 nucleotides.

[0024] In some embodiments, the 5′-terminal nucleotide is located on the antisense strand. In some embodiments, the 5′-terminal nucleotide is located on the sense strand.

[0025] In some embodiments, the double-stranded RNAi inhibitor molecule contains a tetracycle.

[0026] In some embodiments, the oligonucleotide is a single-stranded oligonucleotide. In some embodiments, the single-stranded oligonucleotide is a conventional antisense oligonucleotide, ribonucleotide, or aptamer.

[0027] In some embodiments, the single-stranded oligonucleotide is a single-stranded RNAi inhibitor molecule. In some embodiments, the single-stranded RNAi inhibitor molecule has a length of 14-50 nucleotides. In some embodiments, the single-stranded RNAi inhibitor molecule has a length of about 16-30, 18-22, or 20-22 nucleotides.

[0028] In some embodiments, the oligonucleotide further comprises at least one delivery agent, wherein the at least one delivery agent is conjugated to the oligonucleotide to facilitate the transmembrane transport of the oligonucleotide. In some embodiments, the delivery agent is selected from the group consisting of carbohydrates, peptides, lipids, vitamins, and antibodies. In some embodiments, the delivery agent is selected from N-acetylgalactosamine (GalNAc), mannose-6-phosphate, galactose, oligosaccharides, polysaccharides, cholesterol, polyethylene glycol, folate, vitamin A, vitamin E, lithocholic acid, and cationic lipids.

[0029] In some embodiments, the oligonucleotide is contained within lipid nanoparticles. In some embodiments, the oligonucleotide is a naked oligonucleotide.

[0030] In some aspects, this disclosure relates to a pharmaceutical composition comprising an oligonucleotide (e.g., a nucleic acid inhibitor molecule) containing a 4′-phosphate analog as described herein and a pharmaceutically acceptable excipient; and a method of using the pharmaceutical composition to reduce the expression of a target gene in a subject, the method comprising administering the pharmaceutical composition to a subject in need in an amount sufficient to reduce the expression of the target gene. In some embodiments, the administration comprises systemic administration.

[0031] In some aspects, this disclosure relates to a nucleoside phosphoramide, wherein the nucleoside phosphoramide is represented by formula X or formula XI as described herein. In some embodiments of the nucleoside phosphoramide, M1 is O and X is... 10 It is O. In some embodiments of the nucleoside phosphorus amide, X2 is O and R8 is the glutathione-sensitive moiety. In some embodiments of the nucleoside phosphorus amide, X2 is F, OCH2CH2OCH3, or OCH3 and R8 is absent. c and R d Each is either CH3 or CH2CH3.

[0032] In some aspects, this disclosure relates to a nucleoside phosphoramide, wherein the nucleoside phosphoramide is represented by formula XII as described herein. In some embodiments of the nucleoside phosphoramide, R c and R d Each is independently selected from CH3, CH2CH3, or a protecting group. In some embodiments of the nucleoside phosphoridamide, X2 is F or OCH3 and R8 is absent. In some embodiments of the nucleoside phosphoridamide, X2 is O and R8 is a glutathione-sensitive moiety.

[0033] In some respects, this disclosure relates to a nucleoside phosphoramide, wherein the nucleoside phosphoramide is represented by formula XIII as described herein.

[0034] In some respects, this disclosure relates to a nucleoside phosphoramide, wherein the nucleoside phosphoramide is represented by formula XIV as described herein.

[0035] In some aspects, this disclosure relates to a nucleoside phosphoramide, wherein the nucleoside phosphoramide is represented by formula XV as described herein. In some embodiments, the sugar moiety is a furanose. In some embodiments, R c and R d Each is either CH3 or CH2CH3. Brief description of the attached diagram

[0037] Figure 1ATwo representative control double-stranded RNAi inhibitor molecules as described in the examples are depicted: control compound (5′-OH, 2′-F) and control compound (5′-PO4, 2′-F). Except for the 5′-OH or 5′-PO4 of the N1 nucleotide of the leader strand, control compound (5′-OH, 2′-F) and control compound (5′-PO4, 2′-F) are identical.

[0038] Figure 1B Two representative double-stranded RNAi inhibitor molecules as described in the examples are depicted: test compound (fully deprotected, 2′-F) and test compound (monomethyl protected, 2′-F). Except for the 4′-oxymethylphosphonate group on the N1 nucleotide of the leader strand, test compound (fully deprotected, 2′-F) and test compound (monomethyl protected, 2′-F) are identical, wherein the former test compound has a fully deprotected phosphonate group and the latter test compound has a monomethyl protecting group on the phosphonate moiety. Except for the N1 nucleotide of the leader strand, test compound (fully deprotected, 2′-F) and test compound (monomethyl protected, 2′-F) are identical to control compound (5′-OH, 2′-F) and control compound (5′-PO4, 2′-F). Figure 1A The control compound is the same as the test compound, wherein the control compound has 5′-OH or 5′-PO4 and the test compound has 4′-oxymethylphosphonate.

[0039] Figure 1C Two representative control double-stranded RNAi inhibitor molecules as described in the examples are depicted: control compound (5′-OH, 2′-OMe) and control compound (5′-PO4, 2′-OMe). The control compound (5′-OH, 2′-OMe) and control compound (5′-PO4, 2′-OMe) are identical except for the 5′-OH or 5′-PO4 of the N1 nucleotide of the leader strand.

[0040] Figure 1DTwo representative double-stranded RNAi inhibitor molecules as described in the examples are depicted: test compound (fully deprotected, 2′-OMe) and test compound (monomethyl protected, 2′-OMe). Except for the 4′-oxymethylphosphonate group on the N1 nucleotide of the leader strand, test compound (fully deprotected, 2′-OMe) and test compound (monomethyl protected, 2′-OMe) are identical, wherein the former test compound has a fully deprotected phosphonate group and the latter test compound has a monomethyl protecting group on the phosphonate moiety. Except for the N1 nucleotide of the leader strand, test compound (fully deprotected, 2′-OMe) and test compound (monomethyl protected, 2′-OMe) are identical to control compound (5′-OH, 2′-OMe) and control compound (5′-PO4, 2′-OMe). Figure 1C The control compound is the same as the test compound, wherein the control compound has 5′-OH or 5′-PO4 and the test compound has 4′-oxymethylphosphonate.

[0041] Figure 2A -D describes the configuration as shown in Example 8, such as in the use of RNAiMax (Thermo Fisher Scientific Inc., RockVille, MD) measured the difference between the target gene A mRNA and the control compound (5′-OH, 2′-F) 48 hours after transfecting the compound into HEK293 cells. Figure 2A ) and control compound (5′-PO4, 2′-F)( Figure 2B Compared to the tested compound (completely deprotected, 2′-F), Figure 2C ) and test compound (monomethyl protected, 2′-F) ( Figure 2D The effectiveness of IC 50 ).

[0042] Figure 3A -B describes the test compound in monkey hepatocytes as measured by knockdown of target gene A mRNA 24 hours after transfection (completely deprotected, 2′-F), as described in Example 9, in the absence of a cationic lipid transfection agent. Figure 3A ) and test compound (monomethyl protected, 2′-F) Figure 3B The effectiveness of IC 50 ).

[0043] Figure 4A -B describes the test compound in human hepatocytes as measured by knockdown of target gene A mRNA 48 hours after transfection (completely deprotected, 2′-F), as described in Example 10, in the absence of a cationic lipid transfection agent. Figure 4A ) and test compound (monomethyl protected, 2′-F) Figure 4B The effectiveness of IC 50 ).

[0044] Figure 5A The relative abundance of the guide chain of the control compound (5′-OH, 2′-OMe); the control compound (5′-PO4, 2′-OMe); the test compound (completely deprotected, 2′-OMe); and the metabolite (“M1”) of the control compound (5′-PO4, 2′-OMe) having 5′-OH instead of 5′-PO4 is depicted after incubation in rat liver tritosomes as described in Example 11.

[0045] Figure 5B The relative abundance of the guide chain of a mixture of the test compound (monomethyl protected, 2′-F) and its metabolites after incubation in decontaminated lysosomes of rat liver, as described in Example 11, is depicted. The metabolite mixture includes a major metabolite having the same structure as the guide chain of the test compound (fully deprotected, 2′-F).

[0046] Figure 5C The relative abundance of the guide chain of the test compound (monomethyl protected, 2′-OMe) and its metabolite (“M2”) in mouse liver samples after in vivo administration of 3 mg (“mpk”) of the test compound (monomethyl protected, 2′-OMe) per kilogram of body weight was depicted, as described in Example 11. M2 has the same structure as the guide chain of the test compound (fully deprotected, 2′-OMe).

[0047] Figure 6A The efficacy of the control compound (5′-OH, 2′-F) compared to control PBS injection, such as administration of 1 mg per kilogram body weight (“mpk”) of the control compound (5′-PO4, 2′-F) in vivo; or the test compound (completely deprotected, 2′-F) in mice 3 days after administration, as described in Example 12, was measured by knockdown of the target gene A mRNA.

[0048] Figure 6B The efficacy in mice, as described in Example 12, compared to control PBS injection, was measured by knockdown of the target gene B mRNA 4 days after administration of either the control compound (5′-OH, 2′-OMe) at 1 mg / kg body weight (“mpk”) or the test compound (completely deprotected, 2′-OMe).

[0049] Figure 7The in vivo potency in mice, as described in Example 12, was depicted by knockdown of the target gene A mRNA 10 days after administration of the test compound (monomethyl protected, 2′-F) at doses of 0.3 mg (“mpk”), 1 mpk, and 3 mpk per kilogram of body weight.

[0050] Figure 8 The in vivo potency in mice, measured by knockdown of the target gene B mRNA, is shown as described in Example 12, at 3 and 10 days after administration of the test compound (fully deprotected, 2′-OMe) and the test compound (monomethyl protected, 2′-OMe) at a dose of 0.3 mg (“mpk”) or 1 mpk per kilogram of body weight.

[0051] Figure 9A The results of a time-course study in cynomolgus monkeys, as described in Example 13, are shown, measured by knockdown of the target gene B mRNA, at 14, 28, and 56 days after administration of the control compound (5′-OH, 2′-OMe) and the test compound (completely deprotected, 2′-OMe) per kilogram in vivo.

[0052] Figure 9B Results of a time-course study in cynomolgus monkeys, as described in Example 13, were obtained by knockdown of target gene B mRNA at 14, 28, and 56 days after administration of 3 mg / kg of the test compound (fully deprotected, 2′-OMe) and the test compound (monomethyl protected, 2′-OMe). Invention Details

[0054] definition

[0055] To make this disclosure easier to understand, certain terms are defined below. Further definitions for the following and other terms may be set forth in the specification. If a term's definition described below is inconsistent with the definition in an application or patent incorporated herein by reference, the definition set forth in this application shall be used to interpret the meaning of that term.

[0056] Unless the context clearly specifies otherwise, the singular forms “a / an” and “described” as used in this specification and the appended claims include plural references. Thus, for example, reference to “a method” includes one or more methods and / or steps of the type described herein and / or that will become apparent to a person skilled in the art upon reading this disclosure.

[0057] 5′-Terminal nucleotide: As used herein, the term “5′-terminal nucleotide” refers to a nucleotide located at the 5′ end of an oligonucleotide. 5′-terminal nucleotides may also be referred to as “N1 nucleotides” in this application.

[0058] Acyl group: As used herein, the term "acyl group" refers to alkyl carbonyl, cycloalkyl carbonyl, and aryl carbonyl moieties.

[0059] Aliphatic group: As used herein, the term "aliphatic group" refers to a saturated and unsaturated straight-chain (i.e., unbranched) or branched hydrocarbon, which is optionally substituted with one or more functional groups. The term "substituted aliphatic group" refers to an aliphatic moiety with substituents.

[0060] Alkoxy group: As used herein, the term "alkoxy group" refers to an alkyl group that is connected to a molecular part via an oxygen atom.

[0061] Alkenyl: As used herein, the term "alkenyl" refers to a straight-chain or branched hydrocarbon group having at least one carbon-carbon double bond and having carbon atoms in the range of about 2 to about 20. "Substituted alkenyl" refers to an alkenyl group further having one or more substituents. "Lower alkenyl" as used herein refers to an alkenyl moiety having 2 to about 6 carbon atoms.

[0062] Alkyl: As used herein, the term "alkyl" refers to a straight-chain or branched hydrocarbon group having 1 to about 20 carbon atoms. When it appears herein, a range such as "C1-C6 alkyl" means that an alkyl group can contain only 1, 2, 3, etc., up to and including 6 carbon atoms, although the term "alkyl" also includes cases where no range of carbon atoms is specified. For example, the term "alkyl" can refer to C1-C6 alkyl groups. 10 The sub-range (e.g., C1-C6). “Substituted alkyl” refers to an alkyl moiety with a substituent. As used herein, “lower alkyl” refers to an alkyl moiety having one to about six carbon atoms.

[0063] Alkylamino: As used herein, the term "alkanoylamino" refers to an alkyl group containing an amine functional group. Alkanoylamino groups can be substituted or unsubstituted.

[0064] Alkynyl: As used herein, “alkynyl” refers to a straight-chain or branched hydrocarbon group having at least one carbon-carbon triple bond and having carbon atoms in the range of about 2 to about 20. “Substituted alkynyl” refers to an alkynyl group further having one or more substituents. “Lower alkynyl” as used herein refers to an alkynyl moiety having about 2 to about 6 carbon atoms.

[0065] About: As used herein, the term “about” or “approximately” when applied to one or more values ​​of interest means a value similar to the reference value stated herein. In some embodiments, unless otherwise stated or otherwise apparent from the context, the term “about” or “approximately” means a range of values ​​falling within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in either direction (unless such a value would exceed 100% of the possible value).

[0066] Aptamer: As used herein, the term "aptamer" refers to an oligonucleotide that has a binding affinity for a specific target, including nucleic acids, proteins, specific whole cells, or specific tissues. Aptamers can be obtained using methods known in the art, such as in vitro selection from a large pool of random sequences of nucleic acids. Lee et al., Nucleic Acid Res., 2004, 32: D95-D100.

[0067] MicroRNA antagonists: As used herein, “microRNA antagonists” refers to oligonucleotides that have binding affinity to specific targets of the guide strand, including exogenous RNAi inhibitor molecules or natural microRNAs (Krutzfeldt et al., Nature 2005, 438(7068): 685-689).

[0068] Antisense strand: A double-stranded RNAi inhibitor molecule comprises two oligonucleotide strands: an antisense strand and a sense strand. The antisense strand or a region thereof is partially, substantially, or completely complementary to a corresponding region of the target nucleic acid. Furthermore, the antisense strand or a region thereof of the double-stranded RNAi inhibitor molecule is partially, substantially, or completely complementary to the sense strand or a region thereof of the double-stranded RNAi inhibitor molecule. In some embodiments, the antisense strand may also contain non-complementary nucleotides of the target nucleic acid sequence. Non-complementary nucleotides may be on either side of the complementary sequence or on both sides of the complementary sequence. In some embodiments, where the antisense strand or a region thereof is partially or substantially complementary to the sense strand or a region thereof, the non-complementary nucleotides may be located between one or more complementary regions (e.g., one or more mismatches). The antisense strand of the double-stranded RNAi inhibitor molecule is also referred to as the guide strand.

[0069] Aromatic group: As used herein, the term "aromatic group" refers to a planar ring having a delocalized π-electron system containing 4n+2π electrons, where n is an integer. An aromatic ring can be formed of 5, 6, 7, 8, 9, or more than 9 atoms. The term "aromatic" is intended to include carbocyclic aryl (e.g., phenyl) and heterocyclic aryl (or "heteroaryl" or "heteroaromatic") groups (e.g., pyridine). The term includes monocyclic or fused-ring polycyclic rings, i.e., rings sharing adjacent carbon atom pairs. "Substituted aromatic group" refers to an aromatic group further having one or more substituents.

[0070] Aryl: As used herein, the term "aryl" refers to an aromatic monocyclic or polycyclic group having 5 to 19 carbon atoms. "Substituted aryl" refers to an aryl group further comprising one or more substituents.

[0071] Canonical RNA inhibitor molecules: As used in this article, "canonical RNA inhibitor molecules" refers to two strands of nucleic acid, each 21 nucleotides long, with a central complementary region of 19 base pairs for forming a double-stranded nucleic acid and two nucleotide overhangs at each 3′ end.

[0072] Complementarity: As used herein, the term "complementarity" refers to a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid chain) that allows the two nucleotides to form base pairs with each other. For example, a purine nucleotide complementary to a pyrimidine nucleotide of an opposing nucleic acid can be base-paired together by forming hydrogen bonds. In some embodiments, complementary nucleotides can be base-paired in a Watson-Crick manner or by any other manner that allows the formation of a stable duplex. "Complete complementarity" or 100% complementarity means that each nucleotide monomer of the first oligonucleotide chain or a segment of the first oligonucleotide chain can form a base pair with each nucleotide monomer of the second oligonucleotide chain or a segment of the second oligonucleotide chain. Less than 100% complementarity means that some, but not all, nucleotide monomers of two oligonucleotide chains (or two segments of two oligonucleotide chains) can form base pairs with each other. "Basic complementarity" means that two oligonucleotide chains (or segments of two oligonucleotide chains) exhibit 90% or greater complementarity with each other. "Complementary" refers to the complementarity between the target mRNA and the nucleic acid inhibitor molecule, which reduces the amount of protein encoded by the target mRNA.

[0073] Complementary strand: As used in this article, the term “complementary strand” refers to a strand of a double-stranded nucleic acid inhibitor molecule that is partially, substantially, or completely complementary to the other strand.

[0074] Conventional antisense oligonucleotides: As used herein, “conventional antisense oligonucleotides” refers to single-stranded oligonucleotides that inhibit the expression of a targeted gene through one of the following mechanisms: (1) steric hindrance, for example, antisense oligonucleotides interfere with steps in the sequence of events involved in gene expression and / or the production of proteins by directly interfering with, for example, gene transcription, splicing of precursor mRNA, and translation of mRNA; (2) inducing RNase H to enzymatically digest the RNA transcript of the target gene; (3) inducing RNase L to enzymatically digest the RNA transcript of the target gene; (4) inducing RNase P to enzymatically digest the RNA transcript of the target gene; (5) inducing double-stranded RNase to enzymatically digest the RNA transcript of the target gene; and (6) a combination of steric hindrance and induction of enzymatic digestive activity within the same antisense oligonucleotide. Conventional antisense oligonucleotides do not have the same RNAi mechanism of action as RNAi inhibitor molecules. RNAi inhibitor molecules can be distinguished from conventional antisense oligonucleotides in several ways, including requiring Ago2 to be combined with the RNAi antisense strand so that the antisense strand guides the Ago2 protein to one or more intended targets, and where Ago2 is required to silence the target.

[0075] CRISPR RNA: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a microbial nuclease system involved in the defense of invading bacteriophages and plasmids. Wright et al., Cell, 2016, 164: 29-44. This prokaryotic system has been adapted to edit target nucleic acid sequences of interest in the genome of eukaryotic cells. Cong et al., Science, 2013, 339: 819-23; Mali et al., Science, 2013, 339: 823-26; Woo Cho et al., Nat. Biotechnology, 2013, 31(3): 230-232. As used herein, the term "CRISPR RNA" refers to a nucleic acid comprising a "CRISPR" RNA (crRNA) portion and / or a trans-activating crRNA (tracrRNA) portion, wherein the CRISPR portion has a first sequence and a second sequence (also referred to as a tracer partner sequence) substantially or completely complementary to the target nucleic acid portion, the second sequence being sufficiently complementary to the tracrRNA portion such that the tracer partner sequence and the tracrRNA portion hybridize to form a guide RNA. The guide RNA forms a complex with a nuclease, such as a Cas nuclease (e.g., Cas9), and guides the nuclease to mediate the cleavage of the target nucleic acid. In some embodiments, the crRNA portion is fused with the tracrRNA portion to form a chimeric guide RNA. Jinek et al., Science, 2012, 337: 816-21. In some embodiments, the first sequence of the crRNA portion comprises about 16 to about 24 nucleotides, preferably about 20 nucleotides, which hybridizes with the target nucleic acid. In some embodiments, the guide RNA is about 10 to 500 nucleotides. In other embodiments, the guide RNA is about 20 to 100 nucleotides.

[0076] Cycloalkyl: As used herein, the term “cycloalkyl” refers to a cyclic (i.e., cyclic) hydrocarbon group containing 3 to 12 carbons, such as 3 to 8 carbons and 3 to 6 carbons. “Substituted cycloalkyl” refers to a cycloalkyl group further having one or more substituents.

[0077] Delivery agents: As used herein, the term "delivery agent" refers to a transfection agent or ligand that complexes or binds to an oligonucleotide and mediates its entry into the cell. The term includes cationic liposomes, for example, which have a net positive charge that binds to the negative charge of the oligonucleotide. The term also includes conjugates such as GalNAc and cholesterol, which can be covalently linked to the oligonucleotide to guide delivery to certain tissues. Other specific suitable delivery agents are also described herein.

[0078] Deoxyribonucleic acid: As used herein, the term "deoxyribonucleic acid" refers to a nucleotide having a hydrogen group at the 2′ position of the sugar moiety.

[0079] Disulfides: As used herein, the term "disulfide" refers to compounds containing the following groups: Typically, each sulfur atom is covalently bonded to a hydrocarbon group. In some embodiments, at least one sulfur atom is covalently bonded to a group other than a hydrocarbon group. This connection is also referred to as an SS bond or a disulfide bridge.

[0080] Double helix: As used in this article with respect to nucleic acids (e.g., oligonucleotides), the term "double helix" refers to a double helix structure formed by the pairing of complementary bases in two antiparallel sequences of nucleotides.

[0081] Excipients: As used herein, the term “excipient” refers to a non-therapeutic agent that may be included in a composition, for example, to provide or promote a desired consistency or stabilizing effect.

[0082] Furanose: As used herein, the term "furanose" refers to a carbohydrate having a five-membered ring structure, wherein the ring structure has four carbon atoms and one oxygen atom and is represented by formula XVII:

[0083]

[0084] In formula XVII, the numbers represent the positions of the four carbon atoms in the five-membered ring structure.

[0085] Glutathione: As used herein, the term "glutathione" (GSH) refers to a tripeptide having the structure of formula XVIII. GSH is present in cells at concentrations of approximately 1 mM–10 mM. GSH reduces glutathione-sensitive bonds, including disulfide bonds. In this process, glutathione is converted to its oxidized form, glutathione disulfide (GSSG). Once oxidized, glutathione can be reduced back by glutathione reductase using NADPH as an electron donor.

[0086]

[0087] Glutathione-sensitive compounds or glutathione-sensitive moieties: As used herein, the terms “glutathione-sensitive compounds” or “glutathione-sensitive moieties” are used interchangeably and refer to any compound (e.g., oligonucleotide, nucleotide, or nucleoside) or moieties containing at least one glutathione-sensitive bond, such as a disulfide bridge or sulfonyl group. As used herein, “glutathione-sensitive oligonucleotide” is an oligonucleotide containing at least one nucleotide containing a glutathione-sensitive bond.

[0088] Halogen: As used herein, the terms “halogen” and “halogen” are interchangeable and refer to an atom selected from fluorine, chlorine, bromine and iodine.

[0089] Halogenated alkyl: As used herein, the term “halogenated alkyl” refers to an alkyl group having one or more halogen atoms attached thereto, and examples include groups such as chloromethyl, bromoethyl, and trifluoromethyl.

[0090] Heteroaryl: As used herein, the term "heteroaryl" refers to an aromatic ring system containing at least one heteroatom selected from nitrogen, oxygen, and sulfur. A heteroaryl ring may be fused with or otherwise connected to one or more heteroaryl rings, aromatic or non-aromatic hydrocarbon rings, or heterocyclic alkyl rings.

[0091] Heterocycle: As used herein, the term "heterocycle" or "heterocyclic group" refers to a non-aromatic cyclic (i.e., cyclic) group containing one or more heteroatoms (e.g., N, O, S, etc.) as part of a ring structure and having a range of 3 to 14 carbon atoms. "Substituted heterocyclic group" or "substituted heterocycle" refers to a heterocyclic group further having one or more substituents.

[0092] Internucleotide linker: As used herein, the term "internucleotide linker" or "internucleotide bond" refers to a chemical group capable of covalently linking two nucleoside moieties. Typically, such chemical group is a phosphorus-containing linker group containing a phosphate or phosphite group. Phosphate linkers are intended to include phosphodiester bonds, dithiophosphate bonds, thiophosphate bonds, phosphotriester bonds, thiocarbonyl phosphonate bonds, thiocarbonyl phosphotriester bonds, phosphoramide bonds, phosphonate bonds, and / or borane phosphate bonds. Many phosphorus bonds are well known in the art, such as those disclosed in the following U.S. patents: U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050. In other embodiments, the oligonucleotide contains one or more phosphorus-free internucleotide linking groups, such as short-chain alkyl or cycloalkyl internucleotide bonds, mixed heteroatom and alkyl or cycloalkyl internucleotide bonds, or one or more short-chain heteroatom or heterocyclic internucleotide bonds, including but not limited to those having a siloxane backbone; sulfide, sulfoxide and sulfone backbones; formylacetyl and thioformylacetyl backbones; methyleneformylacetyl and thioformylacetyl backbones; nucleoacetyl backbones; olefin-containing backbones; aminosulfonate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; and those with amide backbones.Phosphorus-free bonds are well known in the art, as disclosed, for example, in the following U.S. patents: U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439.

[0093] Loop: As used herein, the term "loop" refers to a structure formed by a single strand of nucleic acid, in which complementary regions of specific single-stranded nucleotides hybridize in a manner that excludes double-stranded formation or Watson-Crick base pairing between the complementary regions. A loop is a single-stranded nucleotide region of any length. Examples of loops include unpaired nucleotides present in structures such as hairpins and tetracycles.

[0094] MicroRNAs: As used herein, the terms “microRNA,” “mature microRNA,” “miRNA,” and “miR” are interchangeable and refer to non-coding RNA molecules encoded in the genomes of plants and animals. Typically, mature microRNAs are about 18–25 nucleotides in length. In some cases, highly conserved endogenously expressed microRNAs regulate gene expression by binding to the 3′-untranslated region (3′-UTR) of a specific mRNA. Some mature microRNAs appear to originate from long endogenous primary microRNA transcripts (also known as precursor microRNAs, primary microRNAs, pri-mir, pri-miR, or primary precursor microRNAs), which often have a length of several hundred nucleotides (Lee et al., EMBO J., 2002, 21(17), 4663–4670).

[0095] Modified nucleosides: As used herein, the term "modified nucleoside" refers to a nucleoside containing one or more of a modified or generic nucleobase or a modified sugar. A modified or generic nucleobase (also referred to herein as a base analog) is generally located at the 1′ position of the nucleoside sugar moiety and refers to a nucleobase at the 1′ position other than adenine, guanine, cytosine, thymine, and uracil. In some embodiments, the modified or generic nucleobase is a nitrogenous base. In some embodiments, the modified nucleobase does not contain a nitrogen atom. See, for example, U.S. Patent Application Publication No. 20080274462. In some embodiments, the modified nucleotide does not contain a nucleobase (base-free). Modified sugars (also referred to herein as sugar analogs) include modified deoxyribose or ribose moieties, for example, wherein modification occurs at the 2′, 3′, 4′, or 5′ carbon position of the sugar. Modified sugars may also include non-natural alternative carbon structures, such as those present in: locked nucleic acids (“LNA”) (see, for example, Koshkin et al. (1998), Tetrahedron, 54, 3607-3630); bridged nucleic acids (“BNA”) (see, for example, U.S. Patent No. 7,427,672 and Mitsuoka et al. (2009), Nucleic Acids Res., 37(4): 1225-38); and unlocked nucleic acids (“UNA”) (see, for example, Snead et al. (2013), Molecular Therapy-Nucleic Acids, 2, e103 (doi: 10.1038 / mtna.2013.36)). Suitable modifications or generic nucleobases or modified sugars are described herein within the context of this disclosure.

[0096] Modified nucleotide: As used herein, the term "modified nucleotide" refers to a nucleotide containing one or more of a modified or generic nucleotide base, a modified sugar, or a modified phosphate ester. A modified or generic nucleotide base (also referred to herein as a base analog) is generally located at the 1′ position of the nucleotide sugar moiety and refers to a nucleotide base at the 1′ position other than adenine, guanine, cytosine, thymine, and uracil. In some embodiments, the modified or generic nucleotide base is a nitrogenous base. In some embodiments, the modified nucleotide base does not contain a nitrogen atom. See, for example, U.S. Patent Application Publication No. 20080274462. In some embodiments, the modified nucleotide does not contain a nucleotide base (base-free). Modified sugars (also referred to herein as sugar analogs) include modified deoxyribose or ribose moieties, for example, wherein modifications occur at the 2′, 3′, 4′, or 5′ carbon positions of the sugar. Modified sugars may also include non-naturally substituted carbon structures, such as those present in: locked nucleic acids (“LNA”) (see, for example, Koshkin et al. (1998), Tetrahedron, 54, 3607-3630); bridged nucleic acids (“BNA”) (see, for example, U.S. Patent No. 7,427,672 and Mitsuoka et al. (2009), Nucleic Acids Res., 37(4): 1225-38); and unlocked nucleic acids (“UNA”) (see, for example, Snead et al. (2013), Molecular Therapy-Nucleic Acids, 2, e103 (doi: 10.1038 / mtna.2013.36)). Modified phosphate groups refer to modifications of phosphate groups that are not present in natural nucleotides and include non-naturally present phosphate ester mimics as described herein. Modified phosphate groups also include non-naturally occurring internucleotide linking groups, including both phosphorus-containing and phosphorus-free linking groups, as described herein. Suitable or generic modifications of nucleobases, modified sugars, or modified phosphates are described herein within the context of this disclosure.

[0097] Naked oligonucleotides: As used herein, the term “naked oligonucleotide” refers to oligonucleotides that are not formulated in protective lipid nanoparticles or other protective formulations and are therefore exposed to the blood and endosome / lysosome compartments when administered in vivo.

[0098] Natural nucleosides: As used herein, the term "natural nucleosides" refers to a heterocyclic nitrogenous base in an N-glycosidic bond with a sugar (such as deoxyribose or ribose or its analogues). Natural heterocyclic nitrogenous bases include adenine, guanine, cytosine, uracil, and thymine.

[0099] Natural nucleotides: As used herein, the term "natural nucleotide" refers to a heterocyclic nitrogenous base in an N-glycosidic bond to a sugar (e.g., ribose or deoxyribose or similar), which is linked to a phosphate ester group. Natural heterocyclic nitrogenous bases include adenine, guanine, cytosine, uracil, and thymine.

[0100] Nucleic acid inhibitor molecules: As used herein, the term "nucleic acid inhibitor molecule" refers to an oligonucleotide molecule that reduces or eliminates the expression of a target gene, wherein the oligonucleotide molecule contains a region that specifically targets a sequence in the mRNA of the target gene. Typically, the target region of a nucleic acid inhibitor molecule contains a sequence that is sufficiently complementary to a sequence on the target gene mRNA to guide the action of the nucleic acid inhibitor molecule on the designated target gene. Nucleic acid inhibitor molecules may include ribonucleotides, deoxyribonucleotides, and / or modified nucleotides.

[0101] Nucleoside: As used in this article, the term “nucleoside” refers to a natural nucleotide or a modified nucleoside.

[0102] Nucleotides: As used in this article, the term “nucleotide” refers to natural nucleotides or modified nucleotides.

[0103] Nucleotide position: As used herein, the term "nucleotide position" refers to the position of a nucleotide in an oligonucleotide, starting from the 5' end. For example, nucleotide position 1 refers to the 5'-terminal nucleotide of an oligonucleotide.

[0104] Oligonucleotide: As used herein, the term "oligonucleotide" refers to a polymeric form of nucleotides ranging from 2 to 2500 nucleotides. Oligonucleotides can be single-stranded or double-stranded. In some embodiments, an oligonucleotide has 500-1500 nucleotides, and typically, for example, the oligonucleotide described herein is used in gene therapy. In some embodiments, an oligonucleotide is single-stranded or double-stranded and has 7-100 nucleotides. In some embodiments, an oligonucleotide is single-stranded or double-stranded and has 15-100 nucleotides. In another embodiment, an oligonucleotide is single-stranded or double-stranded and has 15-50 nucleotides, and typically, for example, the oligonucleotide described herein is a nucleic acid inhibitor molecule. In another embodiment, an oligonucleotide is single-stranded or double-stranded and has 25-40 nucleotides, and typically, for example, the oligonucleotide described herein is a nucleic acid inhibitor molecule. In yet another embodiment, the oligonucleotide is single-stranded or double-stranded and has 19-40 or 19-25 nucleotides, typically, for example, described herein as a double-stranded nucleic acid inhibitor molecule and forming a double helix of at least 18-25 base pairs. In other embodiments, the oligonucleotide is single-stranded and has 15-25 nucleotides, typically, for example, described herein as a single-stranded RNAi inhibitor molecule. Typically, the oligonucleotide contains one or more phosphorus-containing internucleotide linking groups, as described herein. In other embodiments, the internucleotide linking groups are phosphorus-free bonds, as described herein.

[0105] Overhang: As used herein, the term "overhang" refers to one or more terminal non-base-paired nucleotides at any end of either strand of a double-stranded nucleic acid inhibitor molecule. In some embodiments, the overhang is generated by a strand or region extending beyond the end of the complementary strand that forms a duplex with the first strand or region. One or both of the two oligonucleotide regions capable of forming a duplex via hydrogen bonding of base pairs may have 5′ and / or 3′ ends that extend beyond the complementary 3′ and / or 5′ ends shared by the two polynucleotides or regions. The single-stranded region extending beyond the 3′ and / or 5′ ends of the duplex is referred to as an overhang.

[0106] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" comprises a pharmacologically effective amount of a phosphate ester analog-modified oligonucleotide and a pharmaceutically acceptable excipient. As used herein, "pharmacologically effective amount," "therapeuticly effective amount," or "effective amount" refers to the amount of the phosphate ester analog-modified oligonucleotide of this disclosure that effectively produces the intended pharmacological, therapeutic, or preventative outcome.

[0107] Pharmaceutically acceptable excipients: As used herein, “pharmaceuticalally acceptable excipients” means that the excipient is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions) in proportion to a reasonable benefit / risk ratio.

[0108] Phosphorous amides: As used herein, the term "phosphorous amide" refers to a nitrogen-containing trivalent phosphorus derivative. Examples of suitable phosphorous amides are described herein.

[0109] Potency: As used herein, “potency” refers to the amount of oligonucleotide or other drug that must be administered in vivo or in vitro to achieve a specific level of activity against a desired target in cells. For example, an oligonucleotide that inhibits the expression of its target by 90% at a dose of 1 mg / kg in a subject has greater potency than an oligonucleotide that inhibits the expression of its target by 90% at a dose of 100 mg / kg in a subject.

[0110] Protecting group: As used herein, the term "protecting group" is used in a conventional chemical sense as a group that reversibly renders a functional group unreactive under certain conditions of the desired reaction. After the desired reaction, the protecting group can be removed to deprotect the protected functional group. All protecting groups should be removable under conditions that do not degrade a significant proportion of the orthosynthesized molecule.

[0111] Ribonucleotides: As used herein, the term "ribonucleotide" refers to a natural or modified nucleotide that has a hydroxyl group at the 2′ position of its sugar moiety.

[0112] Ribozyme: As used herein, the term "ribozyme" refers to a catalytic nucleic acid molecule that specifically recognizes and cleaves different target nucleic acid sequences, which may be DNA or RNA. Each ribozyme has a catalytic component (also referred to as a "catalytic domain") and a target sequence binding component, the target sequence binding component consisting of two binding domains, one on each side of the catalytic domain.

[0113] RNAi inhibitor molecules: As used herein, the term “RNAi inhibitor molecule” refers to (a) a double-stranded nucleic acid inhibitor molecule (“dsRNAi inhibitor molecule”) having a sense strand (passenger) and an antisense strand (guide), wherein the antisense strand or a portion thereof is used by the Argonaute 2 (Ago2) endonuclease to cleave target mRNA; or (b) a single-stranded nucleic acid inhibitor molecule (“ssRNAi inhibitor molecule”) having a single antisense strand, wherein the antisense strand (or a portion thereof) is used by the Ago2 endonuclease to cleave target mRNA.

[0114] Sense strand: A double-stranded RNAi inhibitor molecule comprises two oligonucleotide chains: an antisense strand and a sense strand. The sense strand or a region thereof is partially, substantially, or completely complementary to the antisense strand or a region thereof of the double-stranded RNAi inhibitor molecule. In some embodiments, the sense strand may also contain nucleotides that are non-complementary to the antisense strand. Non-complementary nucleotides may be on either side of the complementary sequence or on both sides of the complementary sequence. In some embodiments, where the sense strand or a region thereof is partially or substantially complementary to the antisense strand or a region thereof, the non-complementary nucleotides may be located between one or more complementary regions (e.g., one or more mismatches). The sense strand is also referred to as a guest strand.

[0115] Substituent or substituted: As used herein, the term "substituent" or "substituted" means that a hydrogen group in a given structure is replaced by a substituent group. When more than one position in any given structure can be substituted by more than one substituent, the substituents at each position can be the same or different, unless otherwise specified. As used herein, the term "substituted" is considered to include all permissible substituents compatible with organic compounds. Permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. This disclosure is not intended to be limited in any way by the permissible substituents of organic compounds.

[0116] Sulfonyl group: As used herein, the term "sulfonyl group" refers to a compound containing a divalent group -SO2-. In some embodiments, the sulfur atom is covalently bonded to two carbon atoms and two oxygen atoms. In other embodiments, the sulfur atom is covalently bonded to a carbon atom, a nitrogen atom, and two oxygen atoms.

[0117] Systemic administration: As used in this article, "systemic administration" refers to the absorption or accumulation of a drug in the bloodstream throughout the body, and its subsequent distribution throughout the body.

[0118] Target site: As used herein, the terms “target site,” “target sequence,” “target nucleic acid,” “target region,” and “target gene” are used interchangeably and refer to an RNA or DNA sequence that is “targeted,” for example, for cleavage mediated by an RNAi inhibitor molecule that contains a sequence in its lead / antisense region that is partially, substantially, completely, or sufficiently complementary to the target sequence.

[0119] Tetracyclic: As used herein, the term "tetracyclic" refers to a ring (single-stranded region) that forms a stable secondary structure that contributes to the stability of adjacent Watson-Crick hybridization nucleotides. Unrestricted by theory, tetracyclics can stabilize adjacent Watson-Crick base pairs through stacking interactions. Furthermore, interactions between nucleotides in tetracyclics include, but are not limited to, non-Watson-Crick base pairing, stacking interactions, hydrogen bonding, and contact interactions (Cheong et al., Nature 1990; 346(6285): 680-2; Heus and Pardi, Science 1991; 253(5016): 191-4). Tetracyclics impart an increase in the melting temperature (Tm) of adjacent duplexes, which is higher than the melting temperature expected from simple model ring sequences composed of random bases. For example, the tetracycle can impart a hairpin comprising a double strand having a length of at least two base pairs with a melting temperature of at least 50°C, at least 55°C, at least 56°C, at least 58°C, at least 60°C, at least 65°C, or at least 75°C in 10 mM NaHPO4. The tetracycle can contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. In some embodiments, the tetracycle consists of four nucleotides. In some embodiments, the tetracycle consists of five nucleotides.

[0120] Examples of tetracyclic RNAs include the tetracyclic UNCG family (e.g., UUCG), the tetracyclic GNRA family (e.g., GAAA), and the CUUG tetracyclic RNA. (Woese et al., PNAS, 1990, 87(21): 8467-71; Antao et al., Nucleic Acids Res., 1991, 19(21): 5901-5). Examples of tetracyclic DNAs include the tetracyclic d(GNNA) family (e.g., d(GTTA)), the tetracyclic d(GNRA) family, the tetracyclic d(GNAB) family, the tetracyclic d(CNNG) family, and the tetracyclic d(TNCG) family (e.g., d(TTCG)). (Nakano et al., Biochemistry, 2002, 41(48): 14281-14292; Shinji et al., Nippon Kagakkai Koen Yokoshu, 2000, 78(2): 731).

[0121] I. Introduction

[0122] This application provides phosphate ester analog-modified oligonucleotides, such as nucleic acid inhibitor molecules. The 5′-terminal nucleotide of the oligonucleotide of interest is modified with a phosphate ester-containing portion as described herein. The modifications of this invention are particularly suitable for in vivo use because they can help protect oligonucleotides against phosphatases and / or nucleases, such as exonucleases, present in the blood and / or cells, such as in the endosome / lysosomal compartments of cells. Typically, phosphate ester analog-modified oligonucleotides are nucleic acid inhibitor molecules, such as dsRNAi inhibitor molecules, antisense oligonucleotides, ribonucleotides, aptamers, microRNAs, and ssRNAi inhibitor molecules.

[0123] Nucleosides modified with phosphate ester analogs, including a phosphoramidite moiety, are also provided, which can be used to synthesize oligonucleotides having a 5′-terminal nucleotide containing a phosphate ester analog according to the present disclosure.

[0124] II. Phosphate ester analog-modified oligonucleotides

[0125] One aspect relates to an oligonucleotide, such as a nucleic acid inhibitor molecule, wherein said oligonucleotide comprises a 4′-phosphate analog, typically at the 5′-terminal nucleotide. Typically, the 4′-phosphate analog is an oxymethylphosphonate, wherein the oxygen atom of the oxymethyl group is bonded to the 4′-carbon of the sugar moiety or its analogue. In other embodiments, the phosphate analog is a thiomethylphosphonate or an aminomethylphosphonate, wherein the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is bonded to the 4′-carbon of the sugar moiety or its analogue.

[0126] In some embodiments, the 4′-phosphate analog is an oxymethylphosphonate. Typically, the oxymethylphosphonate is represented by -O-CH2-PO(OH)2 or -O-CH2-PO(OR)2, wherein R is independently selected from H, CH3, an alkyl group, CH2CH2CN, CH2OCOC(CH3)3, CH2OCH2CH2Si(CH3)3, or a protecting group. In some embodiments, the alkyl group is CH2CH3. More typically, R is independently selected from H, CH3, or CH2CH3.

[0127] 1. Equations I and II

[0128] In some embodiments, the oligonucleotide comprises a 5′-terminal nucleotide represented by Formula I or Formula II:

[0129]

[0130] Where R a and R bEach is independently selected from hydrogen, CH3, CH2CH3, CH2CH2CN, CH2OCOC(CH3)3, CH2OCH2CH2Si(CH3)3 or a protecting group;

[0131] Where B is a natural nucleobase, a modified nucleobase, a universal base, or does not exist;

[0132] Where M1 represents O, S, NR′, CR′R″;

[0133] R4, R5, R6, or R7 are each independently selected from hydrogen, halogen, OH, C1-C6 alkyl, C1-C6 haloalkyl, or two of R4, R5, R6, and R7 together form a 5- to 8-membered ring, wherein the ring optionally contains heteroatoms;

[0134] Where X1 is absent or is selected from O, S, NR′ or CR′R″;

[0135] Where Y is an internucleotide linker group that connects the 5′-terminal nucleotide to the oligonucleotide;

[0136] R8 is either a glutathione-sensitive component or absent;

[0137] Wherein, if R8 is the glutathione-sensitive moiety, then X2 is O, S, Se, or NR′; or if R8 is absent, then X2 is H, OH, SH, NH2, halogen, optionally substituted alkoxy, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkylthio, optionally substituted alkylamino, or dialkylamino, wherein one or more methylene groups of the alkyl, alkenyl, and alkynyl groups may be interrupted by one or more of O, S, S(O), SO2, N(R′), C(O), N(R′)C(O)O, OC(O)N(R′), optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclic, or optionally substituted cycloalkyl, O, S, Se, or NHR′; and

[0138] R′ and R″ are each independently hydrogen, halogen, substituted or unsubstituted aliphatic group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, substituted or unsubstituted heterocyclic group, or substituted or unsubstituted cycloalkyl group.

[0139] In some implementations, the 5′-terminal nucleotide is represented by Formula I.

[0140] In some implementations, the 5′-terminal nucleotide is represented by Formula II.

[0141] In some implementations, B is a natural nucleobase.

[0142] In some implementations, M1 is O.

[0143] In some implementations, the halogen is fluorine.

[0144] In some embodiments, R4, R5, R6, and R7 are independently selected from hydrogen, fluorine, CH3, or C1-C6 alkyl. Typically, R4, R5, R6, and R7 are hydrogen.

[0145] In some implementations, X1 is 0.

[0146] In some implementations, R a and R b It is hydrogen. In some implementations, R a It is CH3 and R b It is hydrogen. In some implementations, R a and R b It is CH3. In some implementations, R a It is CH2CH3 and R b It is hydrogen. In some implementations, R a and R b It is CH2CH3.

[0147] In some implementations, M1 is O, X2 is O, and R4, R5, R6, and R7 are hydrogen.

[0148] In some embodiments, X2 is O, S, Se, or NHR′, wherein R′ is selected from hydrogen, halogen, substituted or unsubstituted aliphatic group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, substituted or unsubstituted heterocyclic group, or substituted or unsubstituted cycloalkyl group, and R8 is a glutathione-sensitive moiety. Typically, X2 is O and R8 is a glutathione-sensitive moiety, and the 5′-terminal nucleotide is represented by Formula I.

[0149] In some embodiments, X2 is a halogen or optionally substituted alkoxy group and R8 is absent. Typically, X2 is F, OCH2CH2OCH3, or OCH3 and R8 is absent and the 5′-terminal nucleotide is represented by Formula I.

[0150] In some implementations, M1 is O, X2 is O, R4, R5, R6 and R7 are hydrogen, B is a natural nucleobase; X1 is absent or O, and the 5′-terminal nucleotide is represented by formula I.

[0151] 2. Formula III

[0152] In some embodiments, the oligonucleotide comprises a 5′-terminal nucleotide represented by Formula III:

[0153]

[0154] Where Ra and R b Each is independently selected from hydrogen, CH3, CH2CH3, CH2CH2CN, CH2OCOC(CH3)3, CH2OCH2CH2Si(CH3)3 or a protecting group;

[0155] Where B is a natural nucleobase, a modified nucleobase, a universal base, or does not exist;

[0156] Where Y is an internucleotide linker group that connects the 5′-terminal nucleotide to the oligonucleotide; and

[0157] Where X2 is OH, F, OCH3 or OCH2CH2OCH3 and R8 is absent, or where X2 is O and R8 is the glutathione-sensitive moiety.

[0158] In some implementations, B is a natural nucleobase.

[0159] In some implementations, R a and R b Each is independently selected from hydrogen, CH3, and CH2CH3.

[0160] In some implementations, X2 is F or OCH3 and R8 is absent.

[0161] In some implementations, X2 is O and R8 is the glutathione-sensitive moiety.

[0162] In some implementations, R a and R b It is hydrogen, R8 does not exist, and X2 is either F or OCH3.

[0163] In some implementations, R a It is CH3, R b It is hydrogen, R8 does not exist, and X2 is either F or OCH3.

[0164] In some implementations, R a and R b It is CH3, R8 does not exist, and X2 is F or OCH3.

[0165] In some implementations, R a It is CH2CH3, R b It is hydrogen, R8 does not exist, and X2 is either F or OCH3.

[0166] In some implementations, R a and R b It is CH2CH3, R8 does not exist, and X2 is F or OCH3.

[0167] Formula IV

[0168] In some embodiments, the oligonucleotide comprises a 5′-terminal nucleotide represented by formula IV:

[0169]

[0170] Where B is a natural nucleobase, a modified nucleobase, a universal base, or does not exist;

[0171] Where Y is an internucleotide linker group that connects the 5′-terminal nucleotide to the oligonucleotide; and

[0172] Where X2 is OH, F, OCH3 or OCH2CH2OCH3.

[0173] In some implementations, B is a natural nucleobase.

[0174] In some implementations, X2 is F or OCH3.

[0175] 4. Equation V

[0176] In some embodiments, the oligonucleotide comprises a 5′-terminal nucleotide represented by formula V:

[0177]

[0178] Where B is a natural nucleobase, a modified nucleobase, a universal base, or does not exist;

[0179] Where Y is an internucleotide linker group that connects the 5′-terminal nucleotide to the oligonucleotide; and

[0180] Where X2 is OH, F, OCH3 or OCH2CH2OCH3.

[0181] In some implementations, B is a natural nucleobase.

[0182] In some implementations, X2 is F or OCH3.

[0183] 5. Formula VI

[0184] In one embodiment, the oligonucleotide comprises a 5′-terminal nucleotide, wherein the 5′-terminal nucleotide is represented by formula VI:

[0185]

[0186] Where R a and R b Each is independently selected from hydrogen, CH3, CH2CH3, CH2CH2CN, CH2OCOC(CH3)3, CH2OCH2CH2Si(CH3)3 or a protecting group;

[0187] Where V is O;

[0188] Where Z is a nucleoside containing the sugar moiety;

[0189] Where Y is an internucleotide linker group that connects the 5′-terminal nucleotide to the oligonucleotide; and

[0190] V is bonded to the 4′-carbon of the sugar moiety.

[0191] Typically, the sugar moiety is a furanose and V is bonded to the 4′-carbon of the furanose.

[0192] In some implementations, R a and R b It is hydrogen. In some implementations, R a It is CH3 and R b It is hydrogen. In some implementations, R a and R b It is CH3. In some implementations, R a It is CH2CH3 and R b It is hydrogen. In some implementations, R a and R b It is CH2CH3.

[0193] 6. Equation VII

[0194] In one embodiment, the oligonucleotide comprises a 5′-terminal nucleotide, wherein the 5′-terminal nucleotide is represented by formula VII:

[0195]

[0196] Where R1 is O or S;

[0197] R2 and R3 are each independently selected from OH, SH, NH2, OCH3, OR9, OCH2CH2CN, OCH2OCOC(CH3)3 and OCH2OCH2CH2Si(CH3)3, wherein R9 is an alkyl group and wherein OH, SH and NH2 are optionally protected by a protecting group;

[0198] Where V is O, S, NR′, CR′R″, and R′ and R″ are each independently hydrogen, halogen, substituted or unsubstituted aliphatic group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, substituted or unsubstituted heterocyclic group, or substituted or unsubstituted cycloalkyl group.

[0199] Where Z is a nucleoside containing the sugar moiety;

[0200] Where Y is an internucleotide linker group that connects the 5′-terminal nucleotide to the oligonucleotide; and

[0201] V is bonded to the 4′-carbon of the sugar moiety.

[0202] Typically, the sugar moiety is a furanose and V is bonded to the 4′-carbon of the furanose.

[0203] In some embodiments, R2 or R3 is independently selected from OH, OCH3, or OR9, wherein R9 is a C1-C6 alkyl group. In some embodiments, R9 is CH2CH3.

[0204] Normally, R1 is 0.

[0205] In some embodiments, R1 is O; R2 is OH, OCH3, or OCH2CH3; and R3 is OH, OCH3, or OCH2CH3. In some embodiments, R1 is O; R2 is OH; and R3 is OH. In some embodiments, R1 is O; R2 is OCH3 or OCH2CH3; and R3 is OH. In some embodiments, R1 is O; R2 is OCH3; and R3 is OH. In some embodiments, R1 is O and R2 and R3 are OCH2CH3. In some embodiments, R1 is O; R2 is OCH2CH3; and R3 is OH. In some embodiments, R1 is O and R2 and R3 are OCH2CH3.

[0206] 7. Formula VIII or Formula IX

[0207] In some embodiments, this disclosure provides an oligonucleotide comprising a 5′-terminal nucleotide represented by formula VIII or formula IX:

[0208]

[0209] Where R1 is O or S;

[0210] R2 and R3 are each independently selected from OH, SH, NH2, OCH3, OR9, OCH2CH2CN, OCH2OCOC(CH3)3 and OCH2OCH2CH2Si(CH3)3, wherein R9 is an alkyl group and wherein OH, SH and NH2 are optionally protected;

[0211] Where W is N or S; and

[0212] B, M1, R4, R5, R6, R7, R8, X1, X2 and Y are as described in Formula I or Formula II.

[0213] In some implementations, W is N.

[0214] In some implementations, W is S.

[0215] In some implementations, R1 is 0.

[0216] In some embodiments, R2 or R3 is independently selected from OH, OCH3, or OR9, wherein R9 is a C1-C6 alkyl group. In some embodiments, R9 is CH2CH3.

[0217] Normally, R1 is 0.

[0218] Oligonucleotides comprising 4′-phosphate analogs as described herein may comprise any nucleotide sequence of interest. In some embodiments, the oligonucleotides of formulas I-IX have 7 to 100 nucleotides. In another embodiment, the oligonucleotides of formulas I-IX have 15 to 50 nucleotides. In yet another embodiment, the oligonucleotides of formulas I-IX have 25 to 40 nucleotides. In yet another embodiment, the oligonucleotides of formulas I-IX have 19 to 25 nucleotides.

[0219] A. Nucleic acid inhibitor molecules

[0220] In some embodiments, the oligonucleotide containing a 4′-phosphate analog is a nucleic acid inhibitor molecule. Various oligonucleotide structures have been used as nucleic acid inhibitor molecules, including single-stranded and double-stranded oligonucleotides, and any of these various oligonucleotides can be modified to include nucleotides modified with 4′-phosphate analogs as described herein, including the 5′-terminal nucleotide of any of Formulas I-IX.

[0221] Double-stranded nucleic acid inhibitor molecules

[0222] In some embodiments, the nucleic acid inhibitor molecule described herein is a double-stranded RNAi inhibitor molecule having a sense (or guest) strand and an antisense (or guide) strand and containing at least one nucleotide having a 4′-phosphate analog as described herein. As described above, various double-stranded RNAi inhibitor molecular structures are known in the art, including, for example: (a) double-stranded nucleic acid molecules, wherein each strand has a size of 19-25 nucleotides and has at least one 3′-protrusion with 1 to 5 nucleotides (see, for example, U.S. Patent No. 8,372,968); (b) longer double-stranded RNAi inhibitor molecules processed in vivo by cleatase into active RNAi inhibitor molecules (see, for example, U.S. Patent No. 8,883,996); and (c) double-stranded nucleic acid inhibitor molecules, wherein at least one end of at least one strand extends beyond the double-stranded target region of the molecule, including structures in which one of the strands comprises a thermodynamically stable tetracyclic structure (see, for example, U.S. Patent Nos. 8,513,207, 8,927,705, WO 2010 / 033225, and WO 2016 / 100401, the disclosures of which relating to these double-stranded nucleic acid inhibitor molecules are incorporated herein by reference).

[0223] In some embodiments of the dsRNAi inhibitor molecule, the sense strand and antisense strand range from 15 to 66, 25 to 40, or 19 to 25 nucleotides. In some embodiments, the sense strand is 18 to 66 nucleotides long. In some embodiments, the sense strand is 18 to 25 nucleotides long. In some embodiments, the sense strand is 18, 19, 20, 21, 22, 23, or 24 nucleotides long. In some of those embodiments, the sense strand is 25 to 45 nucleotides long. In some embodiments, the sense strand is 30 to 40 nucleotides long. In some embodiments, the sense strand is 36, 37, 38, 39, or 40 nucleotides long. In some embodiments, the sense strand is 25 to 30 nucleotides long. In some of those embodiments, the sense strand is 25, 26, or 27 nucleotides long.

[0224] In some embodiments of the dsRNAi inhibitor molecule, the antisense strand has a length of 18 to 66 nucleotides. Typically, the antisense strand contains a sequence that is fully complementary to a sequence in the target gene mRNA to guide the action of the nucleic acid inhibitor molecule on the target gene. In some embodiments, the antisense strand contains a sequence that is completely complementary to a sequence contained in the target gene mRNA, wherein the completely complementary sequence has a length of 18 to 40 nucleotides. In some of those embodiments, the antisense strand has a length of 20 to 50 nucleotides. In some embodiments, the antisense strand has a length of 20 to 30 nucleotides. In some embodiments, the antisense strand has a length of 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides. In some embodiments, the antisense strand has a length of 35 to 40 nucleotides. In some of those embodiments, the antisense strand has a length of 36, 37, 38, or 39 nucleotides.

[0225] In some embodiments of the dsRNAi inhibitor molecule, the sense and antisense strands form a double-stranded structure having 15 to 50 base pairs. In some embodiments, the double-stranded region has a length of 15 to 45 base pairs, more typically 15 to 30 base pairs, such as 18 to 30, more typically 18 to 26 or 21 to 26, such as 19 to 23, and in some cases, 19 to 21 base pairs. In some embodiments, the double-stranded region has a length of 19, 20, 21, 22, 23, 24, 25, or 26 base pairs.

[0226] In some embodiments, the dsRNAi inhibitor molecule may further comprise one or more single-stranded nucleotide overhangs. Typically, the dsRNAi inhibitor molecule has single-stranded overhangs of 1-10, 1-4, or 1-2 nucleotides. The single-stranded overhangs are typically located at the 3' end of the sense strand and / or the 3' end of the antisense strand. In some embodiments, the single-stranded overhang of 1-10, 1-4, or 1-2 nucleotides is located at the 5' end of the antisense strand. In some embodiments, the single-stranded overhang of 1-10, 1-4, or 1-2 nucleotides is located at the 5' end of the sense strand. In some embodiments, the single-stranded overhang of 1-2 nucleotides is located at the 3' end of the antisense strand. In some embodiments, the single-stranded overhang of 10 nucleotides is located at the 5' end of the antisense strand. In some embodiments, the dsRNAi inhibitor molecule has blunt ends, typically located at the 5' end of the antisense strand.

[0227] In some embodiments, the dsRNAi inhibitor molecule comprises a sense strand and an antisense strand, and a double-stranded region having 19-21 nucleotides in length, wherein the sense strand has a length of 19-21 nucleotides and the antisense strand has a length of 21-23 nucleotides and includes a single-stranded overhang of 1-2 nucleotides at its 3′ end.

[0228] In some embodiments, the dsRNAi inhibitor molecule has an antisense strand of 21 nucleotides in length and a sense strand of 21 nucleotides in length, wherein a 3′-sense strand overhang of two nucleotides is present on the right side of the molecule (the 3′ end of the sense strand / the 5′ end of the antisense strand), and a single-stranded overhang of two nucleotides is present at the 3′ end of the antisense strand. In such a molecule, a double-stranded region of 19 base pairs is present.

[0229] In some embodiments, the dsRNAi inhibitor molecule has an antisense strand of 23 nucleotides in length and a sense strand of 21 nucleotides in length, wherein a blunt end is present on the right side of the molecule (the 3′ end of the sense strand / the 5′ end of the antisense strand) and a 3′-sense strand overhang of two nucleotides is present on the left side of the molecule (the 5′ end of the sense strand / the 3′ end of the antisense strand). In such a molecule, a double-stranded region of 21 base pairs is present.

[0230] In some embodiments, the dsRNAi inhibitor molecule comprises a sense strand and an antisense strand, and a double-stranded region having 18-34 nucleotides, wherein the sense strand has a length of 25-34 nucleotides and the antisense strand has a length of 26-38 nucleotides and includes 1-5 single-stranded nucleotides at its 3′ end. In some embodiments, the sense strand is 26 nucleotides, the antisense strand is 38 nucleotides and has a single-stranded overhang of 2 nucleotides at its 3′ end and a single-stranded overhang of 10 nucleotides at its 5′ end, and the sense strand and the antisense strand form a double-stranded region of 26 nucleotides. In some embodiments, the sense strand is 25 nucleotides, the antisense strand is 27 nucleotides and has a single-stranded overhang of 2 nucleotides at its 3′ end, and the sense strand and the antisense strand form a double-stranded region of 25 nucleotides.

[0231] In some embodiments, the dsRNAi inhibitor molecule includes a stem and a loop. Typically, the 3′ or 5′ terminal region of the transient strand of the dsRNAi inhibitor molecule forms the stem and loop structure.

[0232] In some embodiments, the dsRNAi inhibitor molecule contains a stem and a tetracyclic ring. In embodiments where the dsRNAi inhibitor molecule contains a stem and a tetracyclic ring, the guest strand contains both a stem and a tetracyclic ring and is in the range of 20-66 nucleotides in length. Typically, the leader strand and the guest strand are separate strands, each with a 5′ end and a 3′ end, and do not form a continuous oligonucleotide (sometimes referred to as a “nicked” structure).

[0233] In some of those embodiments, the guide strand has a length of 15 to 40 nucleotides. In some embodiments, the extension of the guest strand containing the stem and four rings is at the 3' end of the strand. In some other embodiments, the extension of the guest strand containing the stem and four rings is at the 5' end of the strand.

[0234] In some embodiments, the transit chain of the dsRNAi inhibitor molecule containing a stem and a tetracycle has a length of 34 to 40 nucleotides and the guide chain of the dsRNAi inhibitor molecule contains 20 to 24 nucleotides, wherein the transit chain and the guide chain form a double-stranded region having 18 to 24 nucleotides.

[0235] In some embodiments, the dsRNAi inhibitor molecule comprises: (a) a transient chain containing a stem and a tetracycle and having a length of 36 nucleotides, wherein the first 20 nucleotides from the 5′ end are complementary to the guide chain and the subsequent 16 nucleotides form the stem and tetracycle; and (b) a guide chain having a length of 22 nucleotides and having a single-stranded overhang of two nucleotides at its 3′ end, wherein the guide chain and the transient chain are separate chains and do not form a continuous oligonucleotide (see, for example...). Figures 1A-1D ).

[0236] In some embodiments, the dsRNAi inhibitor molecule comprises one or more deoxyribonucleotides. Typically, the dsRNAi inhibitor molecule contains fewer than five deoxyribonucleotides. In some embodiments, the dsRNAi inhibitor molecule comprises one or more ribonucleotides. In some embodiments, all nucleotides in the dsRNAi inhibitor molecule are ribonucleotides.

[0237] In some embodiments, the 5′-terminal nucleotide of any one of Formulas I-IX is located on the transit strand of a double-stranded nucleic acid inhibitor molecule, such as a dsRNAi inhibitor molecule. In another embodiment, the 5′-terminal nucleotide of any one of Formulas I-IX is located on the leader strand. In yet another embodiment, the 5′-terminal nucleotide of any one of Formulas I-IX is located on both the leader strand and the transit strand. In one embodiment, the 5′-terminal nucleotide of any one of Formulas I-IX is located in a double-stranded region. In some embodiments, the 5′-terminal nucleotide of any one of Formulas I-IX is located in a salient region.

[0238] Single-stranded nucleic acid inhibitor molecules

[0239] In some embodiments, the nucleic acid inhibitor molecule is a single-stranded nucleic acid inhibitor molecule containing a 5′-terminal nucleotide according to any one of Formulas I-IX. Single-stranded nucleic acid inhibitor molecules are known in the art. For example, recent results have demonstrated the activity of ssRNAi inhibitor molecules (see, for example, Matsui et al., Molecular Therapy, 2016, 24(5): 946-55). Furthermore, antisense molecules have been used for decades to reduce the expression of specific target genes. Pelechano and Steinmetz, Nature Review Genetics, 2013, 14: 880-93. Numerous variations on the common theme of these structures have been developed for a range of targets. Single-stranded nucleic acid inhibitor molecules include, for example, conventional antisense oligonucleotides, microRNAs, riboside enzymes, aptamers, microRNA antagonists, and ssRNAi inhibitor molecules, all of which are known in the art.

[0240] In some embodiments, the nucleic acid inhibitor molecule is an ssRNAi inhibitor molecule having 14-50, 16-30, or 15-25 nucleotides. In other embodiments, the ssRNAi inhibitor molecule has 18-22 or 20-22 nucleotides. In some embodiments, the ssRNAi inhibitor molecule has 20 nucleotides. In other embodiments, the ssRNAi inhibitor molecule has 22 nucleotides.

[0241] In some embodiments, the nucleic acid inhibitor molecule is a single-stranded oligonucleotide that inhibits exogenous RNAi inhibitor molecules or natural microRNAs. In some embodiments, the nucleic acid inhibitor molecule is a single-stranded antisense oligonucleotide having 8-80, 14-50, 16-30, 12-25, 12-22, 14-20, 18-22, or 20-22 nucleotides. In some embodiments, the single-stranded antisense oligonucleotide has 18-22 nucleotides, such as 18-20 nucleotides.

[0242] In some embodiments, the antisense oligonucleotide or a portion thereof is completely complementary to the target nucleic acid or a specific portion thereof. In some embodiments, the antisense oligonucleotide or a portion thereof is complementary to at least 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive nucleotides of the target nucleic acid. In some embodiments, the antisense oligonucleotide contains no more than 5, 4, 3, 2 or 1 non-complementary nucleotides relative to the target nucleic acid or a portion thereof. It is possible to reduce the length of the antisense oligonucleotide and / or introduce mismatched bases without eliminating activity.

[0243] B. Nucleotide modification

[0244] In addition to the 4′-phosphate analogs described herein, the modified oligonucleotides of this disclosure may also include modifications. Typically, multiple nucleotide subunits of a nucleic acid inhibitor molecule are modified to improve various characteristics of the molecule, such as resistance to nucleases or reduced immunogenicity. See, for example, Bramsen et al. (2009), Nucleic Acids Res., 37, 2867-2881. Many nucleotide modifications have been used in the field of oligonucleotides, particularly for nucleic acid inhibitor molecules. These modifications can be made at any part of the nucleotide, including the sugar moiety, phosphate ester bond, and nucleobase. In some embodiments of the nucleic acid inhibitor molecule, one to each nucleotide is modified at the 2′-carbon of the sugar moiety using, for example, 2′-carbon modifications known in the art and described herein. Typical examples of 2′-carbon modifications include, but are not limited to, 2′-F, 2′-O-methyl (“2′-OMe” or “2′-OCH3”), and 2′-O-methoxyethyl (“2′-MOE” or “2′-OCH2CH2OCH3”). As described in this article, modifications can also occur at other parts of the sugar portion of a nucleotide, such as the 5′-carbon.

[0245] In some embodiments, as previously described, the ring structure modifying the sugar moiety includes, but is not limited to, locked nucleic acid (“LNA”) structures, bridging nucleic acid (“BNA”) structures, and unlocking nucleic acid (“UNA”) structures.

[0246] As is known in the art and as described herein, modified nucleobases include nucleobases at the 1′ position other than adenine, guanine, cytosine, thymine, and uracil. A typical example of a modified nucleobase is 5′-methylcytosine.

[0247] The naturally occurring nucleotide bonds in RNA and DNA are 3′ to 5′ phosphodiester bonds. As known in the art and as described herein, modified phosphodiester bonds include non-naturally occurring internucleotide linking groups, including nucleotide bonds containing phosphorus atoms and nucleotide bonds without phosphorus atoms. Typically, as described herein, the nucleic acid inhibitor molecule contains one or more phosphorus-containing internucleotide linking groups. In other embodiments, as described herein, one or more of the nucleotide linking groups of the nucleic acid inhibitor molecule are phosphorus-free bonds. In some embodiments, the nucleic acid inhibitor molecule contains one or more phosphorus-containing internucleotide linking groups and one or more phosphorus-free internucleotide linking groups.

[0248] In some embodiments, one or two nucleotides of the nucleic acid inhibitor molecule are reversibly modified with a glutathione-sensitive moiety. Typically, the glutathione-sensitive moiety is located at the 2′-carbon of the sugar moiety and contains a sulfonyl group or a disulfide bridge. In some embodiments, the glutathione-sensitive moiety is compatible with methods for synthesizing phosphoramide oligonucleotides, such as those described, for example, in International Patent Application No. PCT / US2017 / 048239, which is incorporated herein by reference in its entirety. In some embodiments, more than two nucleotides of the nucleic acid inhibitor molecule are reversibly modified with a glutathione-sensitive moiety. In some embodiments, most nucleotides are reversibly modified with a glutathione-sensitive moiety. In some embodiments, all or substantially all nucleotides of the nucleic acid inhibitor molecule are reversibly modified with a glutathione-sensitive moiety.

[0249] At least one glutathione-sensitive moiety is typically located at the 5′ or 3′-terminal nucleotide of a single-stranded nucleic acid inhibitor molecule or at the 5′ or 3′-terminal nucleotide of the guest strand or guide strand of a double-stranded nucleic acid inhibitor molecule. However, at least one glutathione-sensitive moiety can be located at any nucleotide of interest in the nucleic acid inhibitor molecule.

[0250] In some embodiments, the nucleic acid inhibitor molecule is fully modified, wherein every nucleotide of the fully modified nucleic acid inhibitor molecule is modified. In some embodiments, the fully modified nucleic acid inhibitor molecule does not contain reversible modifications. In some embodiments, at least one of the leader strand or transit strand of a single-stranded or double-stranded nucleic acid inhibitor molecule, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, is modified.

[0251] In some embodiments, the fully modified nucleic acid inhibitor molecule is modified with one or more reversible glutathione-sensitive moieties. In some embodiments, substantially all nucleotides of the nucleic acid inhibitor molecule are modified. In some embodiments, more than half of the nucleotides of the nucleic acid inhibitor molecule are modified with chemical modifications other than reversible modifications. In some embodiments, less than half of the nucleotides of the nucleic acid inhibitor molecule are modified with chemical modifications other than reversible modifications. Modifications may occur in groups on the nucleic acid inhibitor molecule, or the modified nucleotides may be distributed.

[0252] In some embodiments of the nucleic acid inhibitor molecule, one to each nucleotide is modified at the 2′-carbon. In some embodiments, at the 2′-carbon, the nucleic acid inhibitor molecule (or its sense strand and / or antisense strand) is partially or completely modified using, for example, a 2′-carbon modification known in the art and described herein. In some embodiments of the nucleic acid inhibitor molecule, one to each phosphorus atom is modified and one to each nucleotide is modified at the 2′-carbon. In some embodiments, the modification at the 2′-carbon is one or more of 2′-F, 2′-OMe, and / or 2′-MOE. In some embodiments, the modification at the 2′-carbon is 2′-F and / or 2′-OMe (i.e., the sense strand and / or antisense strand of a single-stranded or double-stranded oligonucleotide) partially or completely modified by 2′-F and / or 2′-OMe. In some embodiments, the single-stranded oligonucleotide contains one or more nucleotides reversibly modified with a glutathione-sensitive moiety.

[0253] C. Other 4′-phosphate analog-modified oligonucleotides

[0254] Although the 4′-phosphate analogs disclosed herein are typically incorporated into nucleic acid inhibitor molecules, other nucleic acids can be modified to include nucleotides modified with 4′-phosphate analogs as described herein (e.g., the 5′-terminal nucleotide of any of Formulas I-IX). Modified oligonucleotides of this disclosure may include any oligonucleotide of interest, wherein the presence of a phosphate analog at the 5′-terminal nucleotide is desired. For example, other nucleic acids that may be modified according to the teachings of this application include other therapeutic nucleic acids, such as oligonucleotides for gene therapy or for gene editing, such as CRISPR nucleic acid molecules. See, for example, Cong et al., Science, 2013, 339: 819-23; Mali et al., Science, 2013, 339: 823-26; Woo Cho et al., Nat. Biotechnology, 2013, 31(3): 230-232. Furthermore, oligonucleotides containing phosphate analogs of this disclosure may also be used in vitro. Such oligonucleotides include, for example, probes, primers, adapters, adaptors, or gene fragments.

[0255] III. Nucleoside phosphoramids containing phosphate ester analogs

[0256] Another aspect of this disclosure relates to nucleoside phosphoramids comprising a 4′-phosphate analog as described herein, which can be used in standard oligonucleotide synthesis methods. Typically, the phosphate analog is an oxymethylphosphonate, wherein the oxygen atom of the oxymethyl group is bonded to the 4′-carbon of the sugar moiety or its analogue. In other embodiments, the phosphate analog is a thiomethylphosphonate or an aminomethylphosphonate, wherein the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is bonded to the 4′-carbon of the sugar moiety or its analogue.

[0257] In some embodiments, the oxymethylphosphonate is represented by -O-CH2-PO(OR)2, wherein R is independently selected from CH3, an alkyl group, CH2CH2CN, CH2OCOC(CH3)3, CH2OCH2CH2Si(CH3)3, or a protecting group. In some embodiments, the alkyl group is CH2CH3. More generally, R is independently selected from CH3, CH2CH3, or a protecting group.

[0258] 1. Equations X and XI

[0259] In some embodiments, the nucleoside phosphoramidide of this disclosure is represented by formula X or formula XI:

[0260]

[0261] Wherein B, M1, R4, R5, R6, R7, R8 and X2 are as described in Formula I or Formula II;

[0262] Where R c and R d Each is independently selected from CH3, CH2CH3, CH2CH2CN, CH2OCOC(CH3)3, CH2OCH2CH2Si(CH3)3 or a protecting group;

[0263] Where X 10 It does not exist or is selected from O, S, NR′ or CR′R″; and

[0264] Where R 10 It is phosphorus amide.

[0265] In some embodiments, the phosphate ester analog-modified nucleoside phosphoramidide is represented by formula X.

[0266] In some embodiments, the phosphate ester analog-modified nucleoside phosphoramidide is represented by formula XI.

[0267] In some implementations, B is a natural nucleobase.

[0268] In some implementations, M1 is O.

[0269] In some embodiments, R4, R5, R6, and R7 are independently selected from hydrogen, fluorine, CH3, or C1-C6 alkyl. Typically, R4, R5, R6, and R7 are hydrogen.

[0270] In some embodiments, X2 is O, a halogen, or an optionally substituted alkoxy group.

[0271] In some implementations, R c and R d It is CH3. In some implementations, R c and R d It is CH2CH3.

[0272] In some implementations, M1 is O, X2 is O, and R4, R5, R6, and R7 are hydrogen.

[0273] In some embodiments, X2 is O, S, Se, or NHR′, wherein R′ is selected from hydrogen, halogen, substituted or unsubstituted aliphatic group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, substituted or unsubstituted heterocyclic group, or substituted or unsubstituted cycloalkyl group, and R8 is a glutathione-sensitive moiety. Typically, X2 is O and R8 is a glutathione-sensitive moiety.

[0274] In some embodiments, X2 is a halogen or optionally substituted alkoxy group and R8 is absent. Typically, X2 is F, OCH2CH2OCH3, or OCH3 and R8 is absent.

[0275] In some implementations, R c and R d It is CH3, R8 does not exist, and X2 is F or OCH3.

[0276] In some implementations, R c and R d It is CH2CH3, R8 does not exist, and X2 is F or OCH3.

[0277] In some embodiments, the phosphoramidite has the formula -P(OR) x )-N(R y )2, where R x Choose from the group consisting of arbitrarily substituted methyl, 2-cyanoethyl and benzyl groups, wherein each R y Choose from the group consisting of ethyl and isopropyl groups with optional substitutions.

[0278] In some embodiments, the phosphate ester analog-modified nucleoside phosphoramid is identical to formula X or XI, except that the oxygen atom bonded to the sugar moiety of the nucleoside is replaced by a sulfur or nitrogen atom.

[0279] 2.Formula XII

[0280] In some embodiments, the nucleoside phosphoramidide of this disclosure is represented by formula XII:

[0281]

[0282] Where R c and R d Each is independently selected from CH3, CH2CH3, CH2CH2CN, CH2OCOC(CH3)3, CH2OCH2CH2Si(CH3)3 or a protecting group;

[0283] Where B is a natural nucleobase, a modified nucleobase, a universal base, or does not exist;

[0284] Where R 10 It is phosphorus amide; and

[0285] Where X2 is OH, F, OCH3 or OCH2CH2OCH3 and R8 is absent, or where X2 is O and R8 is the glutathione-sensitive moiety.

[0286] In some implementations, B is a natural nucleobase.

[0287] In some implementations, R c and R d Each is independently selected from CH3 and CH2CH3.

[0288] In some implementations, X2 is F or OCH3 and R8 is absent.

[0289] In some implementations, X2 is O and R8 is the glutathione-sensitive moiety.

[0290] In some implementations, R c and R d It is CH3, R8 does not exist, and X2 is F or OCH3.

[0291] In some implementations, R c and R d It is CH2CH3, R8 does not exist and X2 is F or OCH3.

[0292] 3.Formula XIII

[0293] In some embodiments, nucleoside phosphoramide is represented by formula XIII:

[0294]

[0295] Where B is a natural nucleobase, a modified nucleobase, a universal base, or does not exist;

[0296] Where R 10 It is phosphorus amide; and

[0297] Where X2 is OH, F, OCH3 or OCH2CH2OCH3.

[0298] In some implementations, B is a natural nucleobase.

[0299] In some implementations, X2 is F or OCH3.

[0300] 4. Formula XIV

[0301] In some embodiments, nucleoside phosphoramide is represented by formula XIV:

[0302]

[0303] Where B is a natural nucleobase, a modified nucleobase, a universal base, or does not exist;

[0304] Where R 10 It is phosphorus amide; and

[0305] Where X2 is OH, F, OCH3 or OCH2CH2OCH3.

[0306] In some implementations, B is a natural nucleobase.

[0307] In some implementations, X2 is F or OCH3.

[0308] 5. Formula XV

[0309] In some embodiments, the phosphate ester analog-modified nucleoside phosphoramids of this disclosure are represented by formula XV:

[0310]

[0311] Where R c and R d Each is independently selected from CH3, CH2CH3, CH2CH2CN, CH2OCOC(CH3)3, CH2OCH2CH2Si(CH3)3 or a protecting group;

[0312] Where V is O;

[0313] Z1 is a nucleoside containing both phosphoramidite and a sugar moiety; and

[0314] V is bonded to the 4′-carbon of the sugar moiety.

[0315] Typically, the sugar moiety is a furanose and V is bonded to the 4′-carbon of the furanose.

[0316] In some implementations, R c and R d It is CH3. In some implementations, R c and R d It is CH2CH3.

[0317] 6. Formula XVI

[0318] In some embodiments, the phosphate ester analog-modified nucleoside phosphoramid of this disclosure is represented by formula XVI:

[0319]

[0320] Where R1 is O or S;

[0321] R2 and R3 are each independently selected from protected OH, protected SH or protected NH2, OCH3, OR9, OCH2CH2CN, OCH2OCOC(CH3)3 and OCH2OCH2CH2Si(CH3)3, wherein R9 is an alkyl group;

[0322] Where V is O, S, NR′, CR′R″, and R′ and R″ are each independently hydrogen, halogen, substituted or unsubstituted aliphatic group, substituted or unsubstituted aryl group, substituted or unsubstituted heteroaryl group, substituted or unsubstituted heterocyclic group, or substituted or unsubstituted cycloalkyl group.

[0323] Z1 is a nucleoside containing both phosphoramidite and a sugar moiety; and

[0324] V is bonded to the 4′-carbon of the sugar moiety.

[0325] Typically, the sugar moiety is a furanose and V is bonded to the 4′-carbon of the furanose.

[0326] Normally, V is O.

[0327] In some embodiments, R2 or R3 is independently selected from protected OH, OCH3, or OR9, wherein R9 is a C1-C6 alkyl group. In some embodiments, R9 is CH2CH3.

[0328] Normally, R1 is 0.

[0329] In some embodiments, R1 is O; R2 is protected OH, OCH3, or OCH2CH3; and R3 is OCH3 or OCH2CH3. In some embodiments, R1 is O; R2 is protected OH; and R3 is protected OH. In some embodiments, R1 is O; R2 is OCH3 or OCH2CH3; and R3 is protected OH. In some embodiments, R1 is O; R2 is OCH3; and R3 is protected OH. In some embodiments, R1 is O and R2 and R3 are OCH2CH3. In some embodiments, R1 is O; R2 is OCH2CH3; and R3 is protected OH. In some embodiments, R1 is O and R2 and R3 are OCH2CH3.

[0330] Protective base

[0331] In some embodiments of 4′-phosphate analog-modified nucleoside phosphoramids, the protecting group is linked to B (i.e., a natural, modified, or generic nucleobase). Suitable protecting groups for B include acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl, benzoyl, 9-fluorenylmethoxycarbonyl, phenoxyacetyl, dimethylformamidinium, dibutylformamidinium, and N,N-diphenylcarbamate.

[0332] In some embodiments, the protecting group is attached to the hydroxyl group in the aforementioned nucleoside phosphoramidite. Suitable protecting groups for the hydroxyl group in the aforementioned nucleoside phosphoramidite include any protecting group compatible with solid-phase oligonucleotide synthesis, including but not limited to dimethoxytriphenylmethyl, monomethoxytriphenylmethyl, and / or triphenylmethyl. A typical example is 4,4′-dimethoxytriphenylmethyl (DMTr), which can be readily cleaved under acidic conditions (e.g., in the presence of dichloroacetic acid (DCA), trichloroacetic acid (TCA), trifluoroacetic acid (TFA), or acetic acid).

[0333] Other typical hydroxyl protecting groups include trialkylsilyl groups, such as tert-butyldimethylsilyl (TBDMS). The TBDMS group is stable under acidic conditions used to remove the DMT group during the synthesis cycle, but can be removed after cleavage and deprotection of the RNA oligomer by various methods, such as using a solution of tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (THF) or using triethylamine hydrofluoric acid. Other typical hydroxyl protecting groups include tert-butyldiphenylsilyl ethers (TBDPS), which can be removed with, for example, ammonium fluoride.

[0334] IV. Nucleotides

[0335] In the aforementioned oligonucleotides and nucleosides containing 4′-phosphate analogs, B represents a native nucleobase, a modified nucleobase, or a universal nucleobase. Suitable native nucleobases include purine and pyrimidine bases, such as adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U). Suitable modified nucleobases include diaminopurines and their derivatives, alkylated purines or pyrimidines, acylated purines or pyrimidines, thiolated purines or pyrimidines, etc.

[0336] Other suitable modified nucleobases include analogs of purines and pyrimidines. Suitable analogs include, but are not limited to, 1-methyladenine, 2-methyladenine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentyladenine, N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine, 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, and 5-chlorouracil. 5-Iodouracil, 5-Ethyluracil, 5-Propyluracil, 5-Methoxyuracil, 5-Hydroxymethyluracil, 5-(Carboxyhydroxymethyl)uracil, 5-(Methylaminomethyl)uracil, 5-(Carboxymethylaminomethyl)uracil, 2-Thiouracil, 5-Methyl-2-Thiouracil, 5-(2-Bromovinyl)uracil, uracil-5-hydroxyacetic acid, uracil-5-hydroxyacetic acid methyl ester, pseudouracil, 1-Methylpseudouracil, queosine, hypoxanthine, xanthine, 2-Aminopurine, 6-Hydroxyaminopurine, nitropyrrole, nitroindolyl and difluorotolyl, 6-Thiopurine and 2,6-diaminopurine nitropyrrole, nitroindolyl and difluorotolyl.

[0337] Typically, nucleobases contain nitrogenous bases. In some embodiments, nucleobases do not contain nitrogen atoms. See, for example, U.S. Patent Application Publication No. 20080274462. A universal nucleobase is a base that can pair with more than one of the bases commonly found in naturally occurring nucleic acids and thus can substitute for such naturally occurring bases in a double helix. The base does not need to be able to pair with every naturally occurring base. For example, some bases pair only with purines or selectively with purines, or only with pyrimidines or selectively with pyrimidines. Universal nucleobases can pair via hydrogen bonds formed through Watson-Crick or non-Watson-Crick interactions (e.g., Hoogsteen interactions). Representative universal nucleobases include inosine and its derivatives.

[0338] In some embodiments, one or more nucleotides of the oligonucleotide of the present invention have no nucleobase attached to the 1′ position of the sugar ring. Such nucleotides are referred to as abase-free.

[0339] V. Other substituents in Formula I-XVI

[0340] In Formulas I to XVI, where appropriate, suitable aliphatic groups typically contain about 2 to about 10 carbon atoms, more typically about 2 to about 6 carbon atoms, such as about 2 to about 5 carbon atoms.

[0341] In Formula I to Formula XVI, where appropriate, suitable alkyl groups typically contain about 1 to about 10 carbon atoms, more typically about 2 to about 6 carbon atoms, such as about 2 to about 5 carbon atoms.

[0342] In formulas I-XVI, suitable alkoxy groups include, where appropriate, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy, and n-hexyloxy.

[0343] In formulas I-XVI, suitable cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc., when appropriate.

[0344] In Formulas I-XVI, suitable heteroatoms include oxygen, sulfur, and nitrogen, where appropriate. Representative heterocycles include pyrrolidinyl, pyrazolinyl, pyrazolinyl, imidazolinyl, imidazolinyl, piperidinyl, piperazineyl, and others. azolealkyl, isopropyl Alkyl, morpholino, thiazolyl, isothiazolyl, and tetrahydrofuranyl. Representative heteroaryl groups include furanyl, thiophene, pyridyl, pyrrolithyl, N-lower alkylpyrrolo, pyrimidinyl, pyrazinyl, and imidazolyl.

[0345] In formulas I to XVI, suitable alkenyl groups include vinyl, allyl, and 2-methyl-3-heptene, and suitable alkynyl groups include propyne and 3-hexyne.

[0346] In formulas I-XVI, suitable aryl groups include phenyl, naphthyl, etc., and suitable heteroaryl groups include pyridyl, furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, phenylthio or thiophene, quinolinyl, indolyl, thiazolyl, etc.

[0347] In formulas I to XVI, where appropriate, suitable alkylamino groups include -CH2CH2CH2NH- or CH2CH2NH-.

[0348] VI. Methods for synthesizing oligonucleotides

[0349] The 4′-phosphate analog-modified oligonucleotides described in this application can be prepared using a variety of synthetic methods known in the art, including standard phosphorusamide methods. Any phosphorusamide synthetic method can be used to synthesize the 4′-phosphate analog-modified oligonucleotides of the present invention. In some embodiments, phosphorusamide is used in a solid-phase synthetic method to generate a reactive intermediate phosphite compound, which is then oxidized using known methods to produce phosphonate-modified oligonucleotides, typically having phosphodiester or thiophosphate nucleotide internucleotide bonds. The oligonucleotide synthesis of this disclosure can be carried out in either direction using methods known in the art: from 5′ to 3′ or from 3′ to 5′.

[0350] Therefore, in another aspect, this disclosure relates to methods for synthesizing oligonucleotides using 4′-phosphate analog-modified nucleosides, as described above and represented, for example, by formulas X-XVI. Typically, the 4′-phosphate analog-modified nucleoside is incorporated as a terminal nucleotide of the synthesized oligonucleotide. More typically, the phosphate analog-modified nucleoside is incorporated as a 5′-terminal nucleotide of the synthesized oligonucleotide.

[0351] In some embodiments, a method for synthesizing oligonucleotides includes (a) covalently linking a nucleoside to a solid support; (b) coupling a nucleoside phosphorous acid to a reactive hydroxyl group on the nucleoside of step (a) to form an internucleotide bond therebetween, wherein any uncoupled nucleoside on the solid support is capped with a capping agent; (c) oxidizing the internucleotide bond using an oxidizing agent; and (d) repeatedly performing steps (b) through (c) using a subsequent nucleoside phosphorous acid to form an oligonucleotide, wherein at least one of the nucleoside of step (a), the nucleoside phosphorous acid of step (b), or the subsequent nucleoside phosphorous acid of step (d) comprises a phosphonate-containing moiety as described herein. Typically, the coupling, capping / oxidation, and optional deprotection steps are repeated until the oligonucleotide reaches the desired length and / or sequence, after which it is cleaved from the solid support.

[0352] VII. Pharmaceutical Composition

[0353] This disclosure provides pharmaceutical compositions comprising a 4′-phosphate analog modified nucleic acid inhibitor molecule and a pharmaceutically acceptable excipient.

[0354] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient and a therapeutically effective amount of a nucleic acid inhibitor molecule, wherein the nucleic acid inhibitor molecule comprises at least one nucleotide comprising a phosphate ester analog as described herein.

[0355] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient and a therapeutically effective amount of a nucleic acid inhibitor molecule, wherein the nucleic acid inhibitor molecule comprises at least one nucleotide containing a 4′-phosphate analog, represented by any one of Formulas I-IX as previously described.

[0356] Although the pharmaceutical compositions typically contain nucleic acid inhibitor molecules, they can also be prepared using other therapeutic nucleic acids (e.g., gene therapy oligonucleotides or CRISPR oligonucleotides) that have been modified with 4′-phosphate analogs as described herein.

[0357] VIII. Pharmaceutically acceptable excipients

[0358] Pharmaceutically acceptable excipients that can be used in this disclosure are conventional. Remington's Pharmaceutical Sciences, EW Martin, Mack Publishing Co., Easton, PA, 15th edition (1975) describes compositions and formulations suitable for the delivery of one or more therapeutic compositions. Some examples of materials that can be used as pharmaceutically acceptable excipients include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; malt; gelatin; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; buffers, such as magnesium hydroxide and aluminum hydroxide; (isotonic saline; Ringer's solution); ethanol; pH buffer solutions; polyols, such as glycerol, propylene glycol, polyethylene glycol, etc.; and other non-toxic and compatible substances used in pharmaceutical formulations.

[0359] IX. Dosage Form

[0360] Pharmaceutical compositions containing oligonucleotides (e.g., nucleic acid inhibitor molecules) containing 4′-phosphate analogs can be formulated with conventional excipients for any intended route of administration.

[0361] Typically, the pharmaceutical compositions disclosed herein comprise nucleic acid inhibitor molecules containing 4′-phosphate analogs as described herein, and are formulated in liquid form for parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection. Dosage forms suitable for parenteral administration typically contain one or more suitable media for parenteral administration, including, for example, sterile aqueous solutions, saline, low molecular weight alcohols such as propylene glycol, polyethylene glycol, vegetable oils, gelatin, fatty acid esters such as ethyl oleate, etc. Parenteral formulations may contain sugars, alcohols, antioxidants, buffers, antibacterial agents, solutes or suspending agents or thickeners that make the formulation isotonic with the blood of the intended recipient. Appropriate fluidity can be maintained, for example, by using surfactants. Liquid formulations may be lyophilized and stored for subsequent use after reconstitution with a sterile injectable solution.

[0362] The pharmaceutical composition may also be formulated for other routes of administration, including topical or transdermal, rectal or vaginal, ocular, nasal, buccal or sublingual administration.

[0363] X. Delivery agent

[0364] Oligonucleotides containing 4′-phosphate ester analogs (e.g., nucleic acid inhibitor molecules) can be mixed, encapsulated, conjugated, or otherwise associated with: other molecules, molecular structures, or mixtures of compounds, including, for example, liposomes and lipids, such as those disclosed in U.S. Patent Nos. 6,815,432, 6,586,410, 6,858,225, 7,811,602, 7,244,448, and 8,158,601; and polymeric materials, such as those disclosed in U.S. Patent Nos. 6,835,393, 7,374,778, 7,737,108, 7,718,193, 8,137,695, and U.S. Patent Application Publications Nos. 2011 / 0143434, 2011 / 0129921, 2011 / 0123636, 2011 / 0143435, and 2011 / 0142951. Those disclosed in 2012 / 0021514, 2011 / 0281934, 2011 / 0286957 and 2008 / 0152661; capsids, pseudocapsules or receptor-targeting molecules used to assist in uptake, distribution or absorption.

[0365] In some embodiments, oligonucleotides containing 4′-phosphate analogs (e.g., nucleic acid inhibitor molecules) are formulated in lipid nanoparticles (LNPs). Lipid-nucleic acid nanoparticles typically form spontaneously when lipids are mixed with nucleic acids to form a complex. Depending on the desired particle size distribution, a hot barrel extruder, such as... An extruder (Northern Lipids, Inc.) extrudes the resulting nanoparticle mixture through a polycarbonate membrane (e.g., with a 100 nm cutoff). To prepare lipid nanoparticles for therapeutic use, it may be desirable to remove the solvent (e.g., ethanol) used to form the nanoparticles and / or the exchange buffer; this can be accomplished, for example, by dialysis or tangential flow filtration. Methods for preparing lipid nanoparticles containing nucleic acid inhibitor molecules are known in the art, such as those disclosed, for example, in U.S. Patent Application Publications Nos. 2015 / 0374842 and 2014 / 0107178.

[0366] In some embodiments, the LNP comprises a lipid core containing cationic liposomes and polyethylene glycolated lipids. The LNP may also comprise one or more encapsulated lipids, such as cationic lipids, structural or neutral lipids, sterols, polyethylene glycolated lipids, or mixtures thereof.

[0367] In some embodiments, the oligonucleotides of the present invention are covalently conjugated with a ligand that guides the delivery of the oligonucleotide to the tissue of interest. Many such ligands have been explored. See, for example, Winkler, Ther. Deliv., 2013, 4(7): 791-809. For example, the oligonucleotides of the present invention can be conjugated with multiple sugar ligand moieties (e.g., N-acetylgalactosamine (GalNAc)) to guide the oligonucleotide uptake into the liver. See, for example, WO 2016 / 100401. Other ligands that can be used include, but are not limited to, mannose-6-phosphate, cholesterol, folate, transferrin, and galactose (see, for example, WO 2012 / 089352 for other specific exemplary ligands). Typically, when the oligonucleotide is conjugated with a ligand, the oligonucleotide is administered as a naked oligonucleotide, wherein the oligonucleotide is not formulated in an LNP or other protective coating. In some embodiments, each nucleotide within the naked oligonucleotide is modified at the 2′ position of the sugar moieties, typically by 2′-F or 2′-OMe modification.

[0368] These pharmaceutical compositions can be sterilized using conventional sterilization techniques or can be sterile filtered. The resulting aqueous solution can be packaged for use as is, or lyophilized, with the lyophilized formulation combined with sterile aqueous excipients prior to application. The pH of the formulation will typically be 3 to 11, more preferably 5 to 9 or 6 to 8, and most preferably 7 to 8, such as 7 to 7.5. Pharmaceutical compositions in solid form can be packaged into multiple single-dose units, each single-dose unit containing a fixed amount of one or more of the above-mentioned agents, such as in a sealed package of tablets or capsules. Pharmaceutical compositions in solid form can also be packaged in containers to obtain flexible dosages, such as in squeeze tubes designed for topical application of creams or ointments.

[0369] The pharmaceutical compositions disclosed herein are suitable for therapeutic use. Therefore, one aspect of this disclosure provides a pharmaceutical composition that can be used to treat a subject, including but not limited to a person suffering from a disease or condition, by administering an effective amount of the pharmaceutical composition of this disclosure to the subject.

[0370] In some embodiments, this disclosure is characterized by the use of a therapeutically effective amount of the pharmaceutical composition as described herein for the manufacture of a medicament for the treatment of patients in need.

[0371] XI. Instructions for Use

[0372] The nucleic acid inhibitor molecules containing 4′-phosphate analogs described herein can be used in methods for regulating the expression of target genes in cells. Typically, such methods involve introducing a nucleic acid inhibitor molecule containing a 4′-phosphate analog into a cell in an amount sufficient to regulate the expression of the target gene. In some embodiments, the method is performed in vivo. The method can also be performed in vitro or ex vivo. In some embodiments, the cells are mammalian cells, including but not limited to human cells.

[0373] In some embodiments, the oligonucleotides (e.g., nucleic acid inhibitor molecules) containing 4′-phosphate analogs described herein can be used in methods for treating patients in need. Typically, such methods involve administering a therapeutically effective amount of a pharmaceutical composition comprising a nucleic acid inhibitor molecule containing a 4′-phosphate analog as described herein to a patient in need.

[0374] In some embodiments, the pharmaceutical compositions disclosed herein can be used to treat or prevent symptoms associated with viral infection in patients in need. One embodiment relates to a method of treating a viral infection, the method comprising administering a pharmaceutical composition to a subject, the pharmaceutical composition comprising a therapeutically effective amount of an oligonucleotide (e.g., a nucleic acid inhibitor molecule) containing a 4′-phosphate analog as described herein. Non-limiting examples of such viral infections include HCV, HBV, HPV, HSV, or HIV infection.

[0375] In some embodiments, the pharmaceutical compositions disclosed herein can be used to treat or prevent cancer-related symptoms in patients in need. One embodiment relates to a method of treating cancer, the method comprising administering a pharmaceutical composition to a subject, the pharmaceutical composition comprising a therapeutically effective amount of a 4′-phosphate analog-modified nucleic acid inhibitor molecule as described herein. Non-limiting examples of such cancers include biliary tract cancer, bladder cancer, transitional cell carcinoma, urothelial carcinoma, brain cancer, glioma, astrocytoma, breast cancer, metaplastic carcinoma, cervical cancer, cervical squamous cell carcinoma, rectal cancer, colorectal cancer, colon cancer, hereditary nonpolyposis colorectal cancer, colorectal adenocarcinoma, gastrointestinal stromal tumor (GIST), endometrial cancer, endometrial stromal sarcoma, esophageal cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, ocular melanoma, uveal melanoma, gallbladder cancer, gallbladder adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, transitional cell carcinoma, urothelial carcinoma, etc. Skin cancer, nephroblastoma, leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloid monocytic leukemia (CMML), liver cancer, liver carcinoma, hepatocellular carcinoma, hepatocellular carcinoma, bile duct epithelial carcinoma, hepatoblastoma, lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, B-cell lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, T-cell lymphoma, non-Hodgkin lymphoma, precursor T-cell lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma, multiple myeloma, nasopharyngeal carcinoma (NPC), neuroblastoma, oropharyngeal carcinoma, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic duct adenocarcinoma, pseudopapillary tumor, acinar cell carcinoma. Prostate cancer, prostate adenocarcinoma, skin cancer, melanoma, malignant melanoma, cutaneous melanoma, small intestine cancer, stomach cancer, gastric cancer, gastrointestinal stromal tumor (GIST), uterine cancer, or uterine sarcoma. Generally, this disclosure is characterized by a method of treating liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, cholangiocarcinoma, and hepatoblastoma by administering a therapeutically effective amount of a pharmaceutical composition as described herein.

[0376] In some embodiments, the pharmaceutical compositions disclosed herein can be used to treat or prevent symptoms associated with proliferative, inflammatory, autoimmune, neurological, ocular, respiratory, metabolic, dermatological, auditory, liver, kidney, or infectious diseases. One embodiment relates to a method of treating a proliferative, inflammatory, autoimmune, neurological, ocular, respiratory, metabolic, dermatological, auditory, liver, kidney, or infectious disease, said method comprising administering a pharmaceutical composition to a subject, said pharmaceutical composition comprising a therapeutically effective amount of a 4′-phosphate analog-modified nucleic acid inhibitor molecule as described herein. Typically, the disease or symptom is a liver disease.

[0377] In some embodiments, this disclosure provides a method for reducing the expression of a target gene in a subject, the method comprising administering a pharmaceutical composition to a subject in need in an amount sufficient to reduce the expression of the target gene, wherein the pharmaceutical composition comprises a 4′-phosphate analog modified nucleic acid inhibitor molecule as described herein and a pharmaceutically acceptable excipient, also as described herein.

[0378] In some implementations, the 4′-phosphate analog modified nucleic acid inhibitor molecule is an RNAi inhibitor molecule as described herein, including dsRNAi inhibitor molecules or ssRNAi inhibitor molecules.

[0379] The target gene can be a target gene from any mammal, such as a human target gene. Any gene can be silenced using the method of the present invention. Exemplary target genes include, but are not limited to, factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, HBV, HCV, RSV, PDGFβ gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1 / 2 gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, cyclin D gene, VEGF gene, EGFR gene, cyclin A gene, cyclin E gene, WNT-1 gene, β-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, susceptin gene, Her2 / Neu gene, topoisomerase I gene, topoisomerase IIα gene, p73 gene, and p21(WAF1 / CIP1). Genes, p27(KIP1) gene, PPM1D gene, RAS gene, caveolin I gene, MIB I gene, MTAI gene, M68 gene, mutations in tumor suppressor genes, p53 tumor suppressor gene, LDHA and its combinations.

[0380] In some embodiments, the 4′-phosphate analog-modified nucleic acid inhibitor molecule silences the target gene and can therefore be used to treat subjects suffering from conditions characterized by unwanted expression of the target gene or at risk of such conditions. For example, in some embodiments, the 4′-phosphate analog-modified nucleic acid inhibitor molecule of the present invention silences the β-catenin gene and can therefore be used to treat subjects suffering from conditions characterized by unwanted β-catenin expression, such as adenocarcinoma or hepatocellular carcinoma, or at risk of such conditions.

[0381] Typically, the oligonucleotides (e.g., nucleic acid inhibitor molecules) containing 4′-phosphate analogs of the present invention are administered intravenously or subcutaneously. However, the pharmaceutical compositions disclosed herein can also be administered by any method known in the art, including, for example, oral, buccal, sublingual, rectal, vaginal, urethral, ​​topical, intraocular, intranasal, and / or intracardiac administration, which may include tablets, capsules, granules, aqueous suspensions, gels, sprays, suppositories, ointments, creams, etc.

[0382] In some embodiments, the pharmaceutical composition is delivered to the relevant tissues or cells of a subject or organism, such as the liver, via systemic administration (e.g., intravenous or subcutaneous administration). In other embodiments, the pharmaceutical composition is delivered via local or systemic administration. In some embodiments, the pharmaceutical composition is delivered to the relevant tissues or cells, such as lung cells and tissues, such as via lung delivery, via local administration.

[0383] The therapeutically effective amount of the compounds disclosed herein can depend on the route of administration and the patient’s physical characteristics, such as the subject’s body size and weight, the extent of disease progression or penetration, the subject’s age, health, and sex.

[0384] In some implementations, the 4′-phosphate analog-modified oligonucleotides as described herein are administered at doses of 20 micrograms to 10 milligrams per kilogram of recipient body weight per day, 100 micrograms to 5 milligrams per kilogram of recipient body weight per day, or 0.5 milligrams to 2.0 milligrams per kilogram of recipient body weight per day.

[0385] The pharmaceutical compositions disclosed herein can be administered daily or intermittently. For example, intermittent administration of the compounds disclosed herein may be administered 1 to 6 days a week, 1 to 6 days a month, once a week, once every other week, once a month, once every other month, or once or twice a year, or divided into multiple doses per year, month, week, or day. In some embodiments, intermittent administration may mean cyclical administration (e.g., daily administration for one day, one week, or for two to eight consecutive weeks, followed by a rest period with no administration for one week, one month, two months, three months, or six months or longer) or it may mean administration every other day, every other week, every other month, or every other year.

[0386] In any of the treatment methods of the present invention, the compound may be administered to the subject alone as a monotherapy or in combination with other therapies known in the art. Example

[0387] Example 1: Synthesis of phosphoramide 1

[0388] Scheme 1 below describes the synthesis of the following nucleoside phosphorimide containing a diethyl-protected oxymethylphosphonate: (2R,3S,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((diethoxyphosphoryl)methoxy)-4-methoxytetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphorimide (phosphorimide 1).

[0389] .

[0390] Option 1

[0391] Synthesized benzoic acid (2R,3R,4R,5R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl ester (1B).

[0392] A solution of 2′-O-methyluridine (150 g, 580.9 mmol) in pyridine (1.5 L) was cooled in an ice bath. Tert-butylchlorodimethylsilane (96.3 g, 639.0 mmol) was added to the solution in several portions over 15 minutes. The reaction mixture was stirred at room temperature for 5 hours. The reaction mixture was then cooled in an ice bath. Benzoyl chloride (165.5 g, 1.2 mol) was added dropwise to the reaction mixture over 15 minutes. The reaction mixture was continuously stirred at room temperature for 12 hours, then diluted with ethyl acetate (2 L). The solution was washed with water (3 L × 3), saturated NaHCO3 solution (1 L × 2), and brine (1 L). The organic layer was dried over Na2SO4, filtered, and concentrated under vacuum to give a pale yellow residue (500 g, crude product) of 1B, which was used directly for the next step.

[0393] Synthesize (2R,3R,4R,5R)-5-(3-((benzoxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methoxytetrahydrofuran-3-yl ester (1C).

[0394] The product (500 g, crude) (1B) from the previous step was dissolved in DMF (5 L). The solution was cooled in an ice bath. Benzyl chloromethyl ether (74.2 g, 1.16 mol) and DBU (239.8 g, 1.58 mol) were added, and the reaction mixture was heated to room temperature and stirred for 16 hours. The reaction mixture was quenched with 0.1 N HCl (2 L) and diluted with ethyl acetate (2 L). The organic layer was separated. It was then washed with water and brine, dried over Na2SO4, filtered, and concentrated under vacuum. The crude substance was purified by silica gel chromatography, eluting with CH2Cl2∶MeOH (20∶1) to give the title product 1C (500 g, 837.9 mmol) as a yellow oil.

[0395] 1H NMR: (CD3OD, 400MHz): δ7.93-7.95 (d, J=7.2Hz, 2H), 7.85-7.87 (d, J=8.0Hz, 1H), 7.50-7.52 (d, J=7.2Hz, 1H), 7.36- 7.40 (t, J=8.0Hz, 2H), 7.12-7.19 (m, 5H), 5.90-5.91 (d, J=3.2Hz, 1 H), 5.54-5.56 (d, J=8.4Hz, 1H), 5.35 (s, 2H), 5.24-5.27 (t, J=5.6 Hz, 1H), 4.55 (d, J = 1.6Hz, 2H), 4.28-4.29 (d, J = 5.6Hz, 1H), 3.93-4.01 (m, 1H), 3.81-3.93 (t, J = 6.8Hz, 1H), 3.32 (s, 3H), 0.81-0.84 (d, J = 7.6Hz, 10H), 0.00 (s, 6H); Measured m / z values: [M+H] + =597.2.

[0396] Synthesized benzoic acid (2R,3R,4R,5R)-5-(3-((benzoxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-(hydroxymethyl)-4-methoxytetrahydrofuran-3-yl ester (1D).

[0397] A solution of 1C (250 g, 419 mmol) in MeOH (1.5 L) was placed in an ice bath, and acetyl chloride (24.9 g, 502.7 mmol) was added dropwise over 15 minutes. The reaction mixture was heated to room temperature and stirred for 2 hours. Ag₂CO₃ (138.6 g, 502.7 mmol) was added to the reaction mixture and stirred for 1 hour. The reaction mixture was filtered and concentrated under vacuum to give the title compound 1D (400 g, crude) as a yellow oil.

[0398] 1H NMR: (CD3OD, 400MHz): δ8.02-8.05 (m, 3H), 7.57-7.59 (d, J=7.6Hz, 1H), 7.44-7.48 (t, J=7.6Hz, 2H), 7.19-7.27 (q, J=7.2Hz, 5H), 6.03-6.05 (d, J=9.2Hz, 1H), 5.72-5.74 (d, J=8.0Hz, 1H), 5.41-5.44 (t, J=8.8Hz, 3H), 4.63 (s, 2H), 4.29-4.31 (t, J=2.4Hz, 1 4.17-4.19 (t, J = 5.2 Hz, 1H), 3.79-3.88 (m, 2H), 3.37 (s, 3H); Measured m / z values: [M+H] + =482.2.

[0399] Synthesis of (2S,3S,4R,5R)-3-(benzoyloxy)-5-(3-((benzooxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-carboxylic acid (1E).

[0400] [Acetoxy(phenyl)-iodoalkyl]acetate (293.7 g, 912 mmol) was added to a suspension of 1D (200 g, 414.5 mmol) and TEMPO (15.64 g, 99.48 mmol) in water (1 L) and CH3CN (1 L). The reaction mixture was stirred at room temperature for 12 hours and then diluted with ethyl acetate. The organic layer was separated and washed with water and brine, dried over Na2SO4, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography, eluting with CH2Cl2:MeOH (20:1) to give the title product 1E (150 g, 837.9 mmol) as a yellow oil.

[0401] 1H NMR: (CD3OD, 400MHz): δ8.21-8.23 (d, J=8.0Hz, 1H), 7.93-7.97 (t, J=7.6Hz, 2H), 7.52 (s, 1H), 7.37-7.41 (t, J=4.6Hz, 2H), 7.10-7.20(m, 4H), 7.02-7.06(m, 2H), 6.95(s, 1H), 6.08-6.09 (d, J = 5.6 Hz, 1H), 5.69-5.71 (d, J = 8.0 Hz, 1H), 5.60-5.62 (t, J = 4.0 Hz, 1H), 5.31-5.36 (t, J = 9.6 Hz, 2H), 4.54 (s, 2H), 4.11-4.13 (t, J = 4.8 Hz, 1H), 3.29 (s, 3H); Measured m / z values ​​[M+H] + =497.2.

[0402] Synthesize (2R,3S,4R,5R)-2-acetoxy-5-(3-((benzomethoxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl ester (1F)

[0403] 1E (20 g, 40.3 mmol) and Pb(OAc)4 (53.6 g, 120.8 mmol) were added to a dry flask. The reaction mixture was purged with argon, followed by the addition of DMF (150 mL). The reaction mixture was protected from light and stirred at room temperature for 16 hours. It was quenched with water (600 mL) and diluted with ethyl acetate (400 mL). The resulting suspension was filtered through a diatomaceous earth filter. The solid was washed with ethyl acetate. The organic layer was separated and concentrated under vacuum. The crude material was purified by silica gel chromatography, eluting with petroleum ether:ethyl acetate (3:1) to give the title product 1F (7 g, 13.7 mmol) as an α / β mixture.

[0404] 1H NMR: (CD3OD, 400MHz): δ8.10-8.13 (t, J=7.6Hz, 3H), 7.65-7.69 (t, J=5.6Hz, 3H), 7.54-7.58 (t, J=8.0Hz, 3H), 7.26-7.35 (m, 9H), 6.35-6.37 (t, J=6.8Hz, 2H), 5.89-5.91 (d, J=8.0Hz, 1H), 5.68-5.69 (d, J = 4.0 Hz, 1H), 5.48-5.50 (t, J = 1.6 Hz, 3H), 4.68-4.71 (t, J = 7.2 Hz, 3H), 4.54-4.57 (q, J = 4.8 Hz, 1H), 3.44 (s, 4H), 2.21 (s, 3H); Measured m / z values ​​[M+H] + =511.2.

[0405] Synthesize (2R,3S,4R,5R)-5-(3-((benzoxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((diethoxyphosphoryl)methoxy)-4-methoxytetrahydrofuran-3-yl ester (1G)

[0406] The reaction was carried out under argon atmosphere. Diethyl (hydroxymethyl)phosphonate (26.4 g, 156.7 mmol) and the boron trifluoride diethyl ether complex (27.8 g, 196.0 mmol) were added to a solution of 1F (20 g, 39.2 mmol) in anhydrous CH2Cl2 (130 mL). The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with water and extracted with ethyl acetate. The organic layer was separated, washed with brine, dried over Na2SO4, and concentrated under vacuum. The crude material was purified by silica gel chromatography, eluted with petroleum ether:ethyl acetate (3:1 to 1:1), to give the title compound 1G (7 g, 13.7 mmol) as a white foam.

[0407] 1H NMR: (CD3OD, 400MHz): δ8.10-8.13 (t, J=7.6Hz, 3H), 7.65-7.69 (t, J=5.6Hz, 3H), 7.54-7.58 (t, J=8.0Hz, 3H), 7.26-7.35 (m, 9H), 6.35-6.37 (t, J=6.8Hz, 2H), 5.89-5.91 (d, J=8.0Hz, 1H), 5.68-5.69 (d, J = 4.0 Hz, 1H), 5.48-5.50 (t, J = 1.6 Hz, 3H), 4.68-4.71 (t, J = 7.2 Hz, 3H), 4.54-4.57 (q, J = 4.8 Hz, 1H), 3.44 (s, 4H), 2.21 (s, 3H); Measured m / z values ​​[M+H] + =619.2.

[0408] Synthesize benzoic acid (2R,3S,4R,5R)-2-((diethoxyphosphoryl)methoxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl ester (1H).

[0409] The solution of 1 G (9 g, 14.6 mmol) in TFA (90 mL) was stirred at 80 °C for 30 min and then concentrated under vacuum. The crude substance was purified by silica gel chromatography, eluting with CH2Cl2∶MeOH (70∶1) to give the title compound 1H (6.8 g, 13.7 mmol) as a white foam.

[0410] 1 ¹H NMR (CD₃OD, 400MHz): δ 11.54 (s, 1H), 8.03–8.04 (d, J = 7.6 Hz, 2H), 7.61 (s, 5H), 6.26–6.28 (d, J = 6.8 Hz, 1H), 5.73–5.76 (m, 1H), 5.55–5.56 (d, J = 4.4 Hz, 1H), 5.39 (s, 1H), 4.49–4.50 (t, J = 4.4 Hz, 1H), 4.02–4.14 (m, 11H), 3.18 (s, 3H), 1.24–1.30 (m, 6H); m / z measured values ​​[M+H] + =499.2.

[0411] Synthesis of diethyl phosphonate ((((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxy-4-methoxytetrahydrofuran-2-yl)oxy)methyl)phosphonate (1I)

[0412] The solution of 1H (5 g, 10 mmol) in ammonia-methanol solution (7 N, 50 mL) was stirred at room temperature for 16 hours. The reaction mixture was concentrated under vacuum. The crude substance was purified by silica gel chromatography, eluting with CH2Cl2∶MeOH (70∶1) to give the title compound 1I (3.3 g, 25.4 mmol) as a white foam.

[0413] 1 ¹H NMR (CD₃OD, 400MHz): δ 11.53 (s, 1H), 8.02–8.04 (t, J = 7.2Hz, 2H), 7.59–7.74 (m, 4H), 6.27–6.28 (d, J = 7.8Hz, 1H), 5.74–5.76 (d, J = 8.0Hz, 1H), 5.55–5.56 (d, J = 4.4Hz, 1H), 5.39 (s, 1H), 4.49–4.50 (t, J = 4.8Hz, 1H), 4.02–4.13 (m, 7H), 3.32 (s, 3H), 1.25–1.30 (m, 7H); m / z measured [M+H] + =395.1.

[0414] Synthesize 2-cyanoethyl((2R,3S,4R,5R)-2-((diethoxyphosphoryl)methoxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-3-yl)diisopropylphosphonamide (phosphonamide 1).

[0415] DIPEA (2.4 g, 18.3 mmol) was added to a solution of 1I (4 g, 10.1 mmol) in anhydrous CH2Cl2 (40 mL), followed by the addition of 3-[chloro-(diisopropylamino)phosphonyl]oxypropionitrile (3.4 g, 14.2 mmol). The reaction mixture was stirred at room temperature for 2 hours, then quenched with MeOH. The reaction mixture was diluted with ethyl acetate and washed with saturated NaHCO3, water, and brine. The organic layer was concentrated under vacuum. The crude material was purified by silica gel chromatography, eluting with CH2Cl2:MeOH (70:1) to give the title compound, phosphoramide 1 (2.9 g, 10.1 mmol), as a white solid.

[0416] 1H NMR (CD3OD, 400MHz): δ9.13 (s, 1H), 7.54-7.59 (q, J=8.4 Hz, 1H), 6.17-6.19 (d, J=7.2Hz, 1H), 5.68-5.70 (d, J=8.0Hz, 1H), 5.08-5.16 (d, J=28.8Hz, 1H), 4.38-4.40 (d, J=9.2Hz, 1H), 4.07- 4.12 (m, 5H), 3.83-3.86 (d, J=8.8Hz, 1H), 3.63 (s, 5H), 3.33-3.37 (d, J=14.4Hz, 3H), 2.66-2.70 (q, J=5.6Hz, 2H), 1.27-1.30 (m, 6H), 1.17-1.21 (q, J=6.0Hz, 2H). 31 P NMR (CD3CN, 162MHz): δ 151.54, 150.57, 19.84; measured m / z [M+H] + =595.2.

[0417] Example 2: Synthesis of phosphoramide 2

[0418] Scheme 2 below describes the synthesis of the following nucleoside phosphorimide comprising a diethyl-protected oxymethylphosphonate: 2-cyanoethyl((2R,3R,4R,5R)-2-((diethoxyphosphoryl)methoxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluorotetrahydrofuran-3-yl)diisopropylphosphorimide (phosphorimide 2). Phosphorimide 2 was prepared following the procedure described in Example 1.

[0419]

[0420] Option 2

[0421] phosphoramide 2 1 The 1H NMR spectrum (CD3CN, 400MHz) is as follows: δ 7.57-7.59 (d, J = 8.2Hz, 1H) 6.26-6.35 (m, 1H) 5.70-5.73 (q, J = 4.8Hz, 1H) 5.21-5.34 (m, 2H) 4.45 (m, 1H) 4.13-4.17 (m, 5H) 4.13 (m, 3H) 3.70-3.72 (m, 2H) 2.69-2.74 (m, 2H) 1.31-1.35 (m, 6H) 1.21-1.24 (q, J = 2.0Hz, 13H). (The last part, "phosphoramide 2," appears to be an unrelated fragment and is left untranslated.) 19The F NMR (CD3CN, 376MHz) spectra are as follows: δ -212.04, -212.04 (m, 0.6F); -215.00, -215.02 (m, 0.4F). (The text abruptly ends here, likely due to an incomplete sentence or missing information.) 31 The P NMR (162MHz, CDCl3) spectrum is as follows: δ 19.39, 19.26, 151.9, 151.3; measured m / z values ​​[M+H] + =583.2.

[0422] Example 3: Synthesis of phosphoramide 3

[0423] Scheme 3 below describes the synthesis of the following nucleoside phosphorimide comprising a dimethyl-protected oxymethylphosphonate: (2R,3S,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-((dimethoxyphosphoryl)methoxy)-4-methoxytetrahydrofuran-3-yl(2-cyanoethyl)diisopropylphosphonimide (phosphonimide 3). Phosphonimide 3 was prepared following the procedure described in Example 1.

[0424]

[0425] Option 3

[0426] Example 4: Synthesis of phosphoramide 4

[0427] Scheme 4 below describes the synthesis of the following nucleoside phosphorimide comprising a dimethyl-protected oxymethylphosphonate: 2-cyanoethyl((2R,3R,4R,5R)-2-((dimethoxyphosphoryl)methoxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluorotetrahydrofuran-3-yl)diisopropylphosphorimide. Phosphorimide 4 was prepared following the procedure described in Example 1.

[0428]

[0429] Option 4

[0430] Example 5: Synthesis of phosphoramide 5 and phosphoramide 5′

[0431] Synthesize carbocyclic nucleoside phosphorimides having a 4′-oxymethylphosphonate ester. Carbocyclic nucleosides represent a class of nucleoside analogs having a cyclopentane ring replacing the tetrahydrofuran ring of the nucleoside; this is a modification that can confer antiviral properties to nucleoside analogs. See, for example, U.S. Patent No. 6,001,840. In other words, a carbocyclic nucleoside is a nucleoside analog in which the oxygen atom of the furanose ring of the sugar moiety is replaced by a carbon atom.

[0432] Scheme 5 below describes the synthesis of the following carbocyclic nucleoside phosphorimides enantiomers containing diethyl-protected oxymethylphosphonates: 1) 2-cyanoethyl((1S,2S,4R)-2-((diethoxyphosphoryl)methoxy)-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclopentyl)diisopropylphosphorimide (phosphorimide 5) and 2) 2-cyanoethyl((1R,2R,4S)-2-((diethoxyphosphoryl)methoxy)-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)cyclopentyl)diisopropylphosphorimide (phosphorimide 5′).

[0433]

[0434] Option 5

[0435] The reagents and conditions used for the synthesis of 5I and 5I′ (step ai of scheme 5) are disclosed in Drake et al., J. Chem. Soc., Perkin Trans. 1, 1996, 2739 and are as follows: a) m-CPBA, DCM; b) K2CO3, Ac2O, H2O, DMSO; c) K2CO3, MeOH; d) t-BuSi(OTf)2, dimethylpyridine, DMF; e) (EtO)2POCH2OTf, n-BuLi, THF; f) NH4F, MeOH; g) 3-benzoyl-2H-112-pyrimidin-2,4(3H)-dione; PPh3, DIAD; h) NH4OH, MeOH; and i) SFC separation. The final steps involve reacting 5I and 5I′ with 3-((chloro(diisopropylamino)phosphoalkyl)oxy)propionitrile, DIPEA, and DCM to form phosphoramide 5 and phosphoramide 5I′.

[0436] 1 H NMR (400MHz, CDCl3): δ9.02 (br s, 1H), 7.62 (dd, J=1.6, 8.2 Hz, 1H), 5.62 (d, J=8.0Hz, 1H), 5.32-5.14 (m, 1H), 4.46 (br d, J=9.4Hz, 1H), 4.20-4.08(m, 4H), 4.06-3.96(m, 1H), 3.94-3.73(m, 4H), 3.72- 3.57(m, 2H), 2.76-2.65(m, 2H), 2.59-2.48(m, 1H), 2.33-2.19(m, 2H), 1.75(br d, J=13.9Hz, 1H), 1.32 (br t, J=7.0Hz, 6H), 1.25-1.15 (m, 12H); 31P NMR (162MHz, CD3CN) δ 147.53, 20.45, 20.36; m / z measured [M+H] + =563.5.

[0437] Example 6: Synthesis of an oligonucleotide containing 4′-oxymethylphosphonate at the 5′ end using dimethyl phosphonate phosphoramide

[0438] The following control compounds were synthesized using 2′-modified nucleoside phosphoramids, specifically 2′-F and 2′-OMe-modified nucleoside phosphoramids: (5′-OH, 2′-F); (5′-PO4, 2′-F); (5′-OH, 2′-OMe); and (5′-PO4, 2′-OMe). Figure 1A and Figure 1C The test compound (completely deprotected, 2′-F); the test compound (monomethyl protected, 2′-F); the test compound (completely deprotected, 2′-OMe); and the test compound (monomethyl protected, 2′-OMe) were also synthesized using 2′-F and 2′-OMe modified nucleoside phosphoramidides. Figure 1B (and Figure 1D). Each compound contains a leader chain of 22 nucleotides and a guest chain of 36 nucleotides, wherein the guest chain contains four nucleotides in a tetracyclic ring, each of which is conjugated to a polyethylene glycol-GalNAc ligand. See also Figure 1A -D. Except for nucleotide position 1, the control and test compounds share the same primary sequence of the target gene A mRNA, the same transit strand, and the same chemical modification pattern on the guide strand. Some compounds contain 2′-F and others contain 2′-OMe, and each test compound contains a phosphate ester analog (4′-oxymethylphosphonate) not present in the control compound. See also Figure 1A -D. In every compound, all nucleotides are modified at the 2′-carbon of the sugar ring.

[0439] Oligonucleotides were synthesized on a solid support in the 3′ to 5′ direction using a commercially available oligonucleotide synthesizer. A standard oligonucleotide synthesis protocol was used. The coupling time was 300 seconds, and 5-ethylthio-1H-tetrazole (ETT) was used as the activator. Triphosphite oxidation was performed using iodine solution.

[0440] To synthesize the guide chain of a test compound having a phosphate ester analog at the N1 nucleotide of the guide chain, a 2′-modified nucleoside phosphoramidite containing a 4′-oxymethylphosphonate is coupled to the 5′ end of each guide chain. More specifically, phosphoramidite 3 (Example 3) or phosphoramidite 4 (Example 4), as shown below, is coupled to the 5′ end of the guide chain of each test compound.

[0441]

[0442] Phosphite 3 and phosphite 4 each contain two methyl-protected oxygen atoms in their phosphonate groups. However, depending on the deprotection step used, one or both of these methyl groups are removed, resulting in a 5′-terminal nucleotide having one methyl-protected oxygen atom in its phosphonate group, as shown in test compounds (monomethyl protected, 2′-F) and test compounds (monomethyl protected, 2′-OMe) (see [link]). Figure 1B and Figure 1D ); or having a fully deprotected phosphonate group (without methyl-protected oxygen atom), as shown in both the test compound (fully deprotected, 2′-F) and the test compound (fully deprotected, 2′-OMe) (see Figure 1B and Figure 1D ).

[0443] Monomethyl-protected 4′-oxymethylphosphonate oligonucleotides can be prepared using ammonia. To prepare the leading chains of the test compounds (monomethyl-protected, 2′-F) and (monomethyl-protected, 2′-OMe), oligonucleotides already coupled to a solid support with phosphoramidite 3 or phosphoramidite 4 were suspended in a mixture of concentrated ammonia (28 wt%–30 wt%) and heated at 55 °C for 17 hours to complete the cleavage from the solid support and remove the protecting groups, including one methyl group of the phosphonate group, from the oligonucleotides. Test compounds (monomethyl-protected, 2′-F) and (monomethyl-protected, 2′-OMe) (see...) Figure 1B and Figure 1D The 5′-terminal nucleotide of the leading strand of ) is shown below, where R is F and OMe respectively.

[0444]

[0445] Fully deprotected 4′-oxymethylphosphonate oligonucleotides can be prepared using trimethylsilyl iodide reagent (“TMSI”). To prepare the leading chains of the test compounds (fully deprotected, 2′-F) and (fully deprotected, 2′-OMe), oligonucleotides already coupled to a solid support with phosphoramidite 3 or phosphoramidite 4 were treated with TMSI / pyridine solution in CH2Cl2 at room temperature. After 30–45 minutes, the reaction was quenched with a solution of 1 M 2-mercaptoethanol in TEA / CH3CN (1:1). Following the TMSI step, a standard oligonucleotide procedure for deprotection and cleavage from the solid support was applied to obtain the fully deprotected 4′-oxymethylphosphonate oligonucleotide leading chain. Test compounds (fully deprotected, 2′-F) and (fully deprotected, 2′-OMe) (see [link to test compound description]). Figure 1B and Figure 1D The 5′-terminal nucleotide of the leading strand of ) is shown below, where R is F and OMe respectively.

[0446]

[0447] After deprotection and cleavage, the crude oligonucleotides were analyzed and purified by high-performance liquid chromatography (HPLC) (Integrated DNA Technologies, Coralville, Iowa). The obtained oligonucleotide solutions were collected, concentrated, and desalted with water. Finally, the oligonucleotides were lyophilized into powder.

[0448] The above process is then repeated to prepare complementary oligonucleotide transit chains containing monovalent GalNAc-conjugated nucleotides at each of positions 27-30. GalNAc-conjugated phosphoramidine synthon is prepared using click chemistry or acetal linkers to link the GalNAc ligand to the 2′-carbon using methods known in the art (see, for example, WO 2016 / 100401). The GalNAc-conjugated phosphoramidine synthon is incorporated into four consecutive positions (27-30) of the transit chain. The transit chain does not contain 4′-oxymethylphosphonate.

[0449] Four dsRNAi inhibitor molecules were obtained by forming duplexes by mixing each of the two complementary strands (guide and passer) in a 1:1 molar ratio: test compound (completely deprotected, 2′-F); test compound (monomethyl protected, 2′-F); test compound (completely deprotected, 2′-OMe); and test compound (monomethyl protected, 2′-OMe). See also Figure 1B and Figure 1D .

[0450] As described above, four control dsRNAi inhibitor molecules were also prepared (control compound (5′-OH, 2′-F); control compound (5′-PO4, 2′-F); control compound (5′-OH, 2′-OMe); and control compound (5′-PO4, 2′-OMe)), except that none of the control compounds contained a 4′-oxymethylphosphonate nucleotide. See also Figure 1A and Figure 1C The synthesis utilizes a native phosphate ester (5′-pO4) at the 5′-carbon of the 5′-terminal nucleotide of the guide chain. 2- The control compounds (5′-PO4, 2′-F) and (5′-PO4, 2′-OMe) contain a free hydroxyl group (5′-OH) at the 5′-carbon of the 5′-terminal nucleotide of the leading chain.

[0451] Example 7: Synthesis of an oligonucleotide containing 4′-oxymethylphosphonate at the 5′ end using diethyl phosphonate phosphoramide

[0452] The oligonucleotide synthesis procedure described in Example 6 was also repeated using diethyl phosphonate phosphoramide to synthesize additional dsRNA inhibitor molecules. More specifically, phosphoramide 1 (Example 1) or phosphoramide 2 (Example 2) shown below was coupled to the 5′ end of the oligonucleotide guide strand.

[0453]

[0454] Phosphite 1 and phosphite 2 each contain two ethyl-protected oxygen atoms in their phosphonate groups. However, depending on the deprotection step used, one or both of the ethyl groups are removed to produce a 5′-terminal nucleotide having one ethyl-protected oxygen atom in its phosphonate group or having a completely deprotected phosphonate group (without ethyl-protected oxygen atoms).

[0455] Monoethyl-protected 4′-oxymethylphosphonate oligonucleotides can be prepared using ammonia. To prepare the oligonucleotide leader chain with a monoethyl-protected 5′-terminal nucleotide, the oligonucleotide, already coupled to a solid support with phosphoramide 1 or phosphoramide 2, is suspended in a mixture of concentrated ammonia (28 wt%–30 wt%) and heated at 55°C for 17 hours to complete cleavage from the solid support and removal of the protecting group, including one ethyl group of the phosphonate, from the oligonucleotide. The 5′-terminal nucleotide of the leader chain with a monoethyl-protected phosphonate group is shown below.

[0456]

[0457] Fully deprotected 4′-oxymethylphosphonate oligonucleotides can be prepared using trimethylsilyl iodide reagent (“TMSI”). To prepare an oligonucleotide leader chain with a fully deprotected 5′-terminal nucleotide, the oligonucleotide already bound to a solid support coupled with phosphoramidite 1 or phosphoramidite 2 was treated with a TMSI / pyridine solution in CH2Cl2 at room temperature. After 30–45 minutes, the reaction was quenched with a solution of 1 M 2-mercaptoethanol in TEA / CH3CN (1:1). Following the TMSI step, a standard oligonucleotide procedure for deprotection and cleavage from the solid support was applied to obtain the fully deprotected 4′-oxymethylphosphonate oligonucleotide leader chain. The 5′-terminal nucleotide of the fully deprotected leader chain is shown below.

[0458]

[0459] After deprotection and cleavage, the crude oligonucleotides were analyzed and purified by high-performance liquid chromatography (HPLC) (Integrated DNA Technologies, Coralville, Iowa). The obtained oligonucleotide solutions were collected, concentrated, and desalted with water. Finally, the oligonucleotides were lyophilized into powder.

[0460] The above process is then repeated to prepare complementary oligonucleotide transit chains containing monovalent GalNAc-conjugated nucleotides at each of positions 27-30. GalNAc-conjugated phosphoramidine synthon is prepared using click chemistry or acetal linkers to link the GalNAc ligand to the 2′-carbon using methods known in the art (see, for example, WO 2016 / 100401). The GalNAc-conjugated phosphoramidine synthon is incorporated into four consecutive positions (27-30) of the transit chain. The transit chain does not contain 4′-oxymethylphosphonate.

[0461] A dsRNAi inhibitor molecule is obtained by forming a double strand by mixing each of two complementary strands (guide and guest) in a 1:1 molar ratio. Each dsRNAi inhibitor molecule contains a 22-base-pair guide strand with a 4′-oxymethylphosphonate at nucleotide position 1 and a 36-base-pair guest strand without any 4′-oxymethylphosphonate, wherein the guest strand contains four nucleotides in a tetracyclic ring, each of which is conjugated to a polyethylene glycol-GalNAc ligand.

[0462] Example 8: In vitro potency (IC50) of test compounds transfected into cells using cationic lipid transfection agents 50 )

[0463] Used in 96-well plates according to the manufacturer's specifications. RNAiMax (ThermoFisher Scientific Inc., Rockville, MD) reverse-stained HEK293 cells with the dsRNAi inhibitor molecule prepared in Example 6. RNAiMax (Thermo Fisher Scientific Inc., Rockville, MD) is a cationic lipid formulation designed to enhance the transfection efficiency of RNAi inhibitor molecules in multiple cell types. HEK293 cells were also transfected with the gene A plasmid. The final concentration of the dsRNAi inhibitor molecule ranged from 1000 pM to 0.0128 pM. HEK293 cells were added to 96-well plates at a density of 12,000 cells / well, and the plates were incubated at 37°C for 48 hours. After 48 hours, 30 μl of ISCRIPT was added to each well. TM Cells were lysed using lysis buffer (Bio-Rad Laboratories, Hercules, CA). Subsequently, 22 μl of the lysate was transferred to a fresh plate and cDNA was prepared using a large-volume cDNA reverse transcription kit (Applied Biosystems Corporation, Carlsbad, CA). Quantitative PCR was performed at 55 °C using a target sequence normalized relative to the human SFRS9-F569 (HEX) gene. Graphs were plotted using GraphPadPrism (GraphPad Software Inc., La Jolla, CA), and IC50 was calculated. 50 value.

[0464] Figure 2A -D describes the use of After RNAiMax (Thermo Fisher Scientific Inc., Rockville, MD) was transfected into HEK293 cells, the in vitro activities of the control compound (5′-OH, 2′-F), the control compound (5′-PO4, 2′-F), the test compound (completely deprotected, 2′-F), and the test compound (monomethyl protected, 2′-F) were compared. The control compound (5′-OH, 2′-F) with 5′-OH had an IC50 of approximately 10.3 pM. 50 This is in contrast to the activity (IC50 ≥ 7 pM) of the control compound (5′-PO4, 2′-F) which has 5′-PO4 but not 5′-OH. 50 )quite. Figure 2A-B. Since 5′-PO4 is considered important for Ago2 loading, these results indicate that the 5′-OH of the control compound (5′-OH, 2′-F) is converted to 5′-PO4 by a kinase in the cytoplasm of transfected cells. The test compound (fully deprotected, 2′-F) had similar activity to the control compound (IC50 7.8 pM). 50 This indicates that the fully deprotected 4′-oxymethylphosphonate is an effective phosphate ester analog. Figure 2C The test compound (monomethyl protected, 2′-F) showed better performance in this assay than the test compound (completely deprotected, 2′-F) (IC50 7.8 pM). 50 Low activity (24.8 pM IC50) 50 This may be attributed to the inefficient removal of the methyl protecting group from the compound (monomethyl protected, 2′-F) tested under these assay conditions. Figure 2D Not intending to be bound by any theory, it is believed that removing the methyl protecting group from the 4′-oxomethylphosphonate of the test compound (monomethyl protected, 2′-F) (resulting in a fully deprotected 4′-oxomethylphosphonate) allows for more efficient Ago2 loading.

[0465] Example 9: In vitro potency of test compounds transfected into monkey hepatocytes without the use of cationic lipid transfection agents

[0466] Primary monkey hepatocytes were obtained from Life Technologies (Carlsbad, CA), thawed, and plated according to the manufacturer's protocol. BIOCOAT TM In 96-well plates. After 4-6 hours of plating, replace the medium with 90 μl of Williams E incubation medium per well. Serially dilute the test compound (completely deprotected, 2′-F) and the test compound (monomethyl protected, 2′-F) from 1 μM to 12.8 pM (5-fold reduction). In the absence of cationic lipid transfection agents, such as... In the case of Thermo Fisher Scientific, Inc., 10 μl of the test compound was added to the corresponding well. The plate was incubated at 37°C for 24 hours to test the knockdown of the RNA target. Target RNA was extracted and purified using the SV96 Total RNA Isolation System (Promega, Madison, WI) according to the manufacturer's protocol. cDNA was prepared using a large-capacity cDNA reverse transcription kit (Applied Biosystems Corporation, Carlsbad, CA). Quantitative PCR was performed at 60°C using RNA targets normalized relative to Homo sapiens peptidyl prolyl isomerase B (PPIB). Graphs were plotted and IC50 was calculated using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA). 50 value.

[0467] Figure 3A -B describes the activity of the test compound (completely deprotected, 2′-F) and the test compound (monomethyl protected, 2′-F) in primary monkey hepatocytes after transfection (“self-delivery”) without the use of a cationic lipid transfection agent. These conditions are considered to represent the in vivo conditions encountered by dsRNAi inhibitor molecules more closely than the transfection protocol used in Example 8. More specifically, it is believed that without the use of transfection agents that can be used to isolate and protect oligonucleotides, such as… In the case of RNAiMax (Thermo Fisher Scientific Inc., Rockville, MD), dsRNAi inhibitor molecules may be more directly exposed to the enzymes and conditions of the cellular endosome compartment. This may result in more efficient removal of the methyl protecting group of, for example, the test compound (monomethyl protected, 2′-F) compared to lipid transfection described in Example 8. Consistent with this, the test compound (fully deprotected, 2′-F) and the test compound (monomethyl protected, 2′-F) showed comparable activity (IC50) after self-delivery transfection. 50 1.2nM and IC 50 3.4 nM). Figure 3A -B.

[0468] Example 10: In vitro potency of test compounds transfected into human hepatocytes without the use of cationic lipid transfection agents

[0469] Frozen human hepatocytes (Triangle Research Laboratories, Durham, NC; Lot No.: HUM4111B) were thawed and plated into 96-well collagen I-coated plates (BD Biosciences) in hepatocyte plating medium (Triangle Research Laboratories) according to the manufacturer's instructions. After 4 hours, the medium was replaced with serum-free maintenance medium (Triangle Research Laboratories, Durham, NC). Test compounds (fully deprotected, 2′-F) and test compounds (monomethyl protected, 2′-F) were serially diluted from 1 μM to 0.13 nM and added to the medium in the absence of cationic lipid transfection agents, such as... Incubate for 24 hours using the medium (Thermo Fisher Scientific, Inc.). The next day, change the medium and culture the cells for another 24 hours.

[0470] After the incubation period, cells were lysed and RNA was prepared using the SV96 total RNA isolation system (Promega, Madison, WI) according to the manufacturer's protocol. cDNA was prepared using a high-capacity cDNA reverse transcription kit (Applied Biosystems Corporation, Carlsbad, CA). Quantitative PCR was then performed using gene A-specific primers and probes normalized relative to the housekeeping genes HPRT1 and IPO8. Gene A mRNA expression levels were normalized relative to sham-treated cells, and dose-response curves were plotted using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA). IC50 was estimated using a three-parameter model. 50 value.

[0471] Figure 4A -B depicts the activities of the test compounds (completely deprotected, 2′-F) and (monomethyl protected, 2′-F) in primary human hepatocytes after transfection without the use of cationic lipid transfection agents. Similar to the monkey hepatocyte self-delivery experiment described above, these conditions are considered to be closer to the in vivo conditions encountered by dsRNAi inhibitor molecules than the transfection protocol in Example 8. Consistent with the results in Example 9, both test compounds showed comparable activity (IC50). 50 0.7nM and IC 50(0.9 nM), indicating that under these conditions the methyl protecting group in the test compound (monomethyl protected, 2′-F) can be removed more effectively to produce a fully deprotected phosphonate group, as in the test compound (fully deprotected, 2′-F). Figure 4A -B.

[0472] Example 11: Testing the stability of the compound

[0473] To assess the in vitro stability of the 4′-oxymethylphosphonate compounds, 3 μM of the control compound (5′-OH, 2′-OMe), the control compound (5′-PO4, 2′-OMe), and the test compound (completely deprotected, 2′-OMe) were incubated in 1 mg / mL rat liver decontaminated lysosomes (Sekisui Xenotech, Kansas City, KS). The rat liver decontaminated lysosomes were lysosomes derived from rat hepatocytes that had been treated with Triton WR 1339 (also known as Tyloxapol). The stability was then assessed using 96 wells / 100 mg lysosomes according to the manufacturer's instructions. OTX TM Two control compounds and one test compound were extracted from the lysosomal matrix using column SPE plates (Phenomenex, Torrance, CA) and 96-well vacuum manifolds. The (Biotage, Charlotte, NC) solvent evaporation unit evaporated the eluent and restored it in water, and the eluent was analyzed by LC-MS.

[0474] Using ACQUITY The instrument (Waters Corporation, Milford, MA) delivers a mobile phase containing a buffer additive at a rate of 0.4 mL / min using ACQUITY. Chromatographic separation of oligonucleotides was performed using a BEH C18 column with a 1.7 μm particle size, reversed-phase ultra-high performance liquid chromatography (2.1 mm × 50 mm) column (Waters Corporation, Milford, MA). The column temperature was maintained at 70 °C, and the sample injection volume was 10 μL or 15 μL. The chromatography was performed under negative ion mode and electrospray ionization (ESI) conditions. A G2S high-resolution time-of-flight mass spectrometer (Waters Corporation, Milford, MA) was used to detect control and test compounds and their metabolites. Promises deconvolution was employed. TMThe software (Novatia, Newtown, PA) obtains the mass of the zero-charge molecular ion via charge-state deconvolution. The control and test compounds, as well as their metabolites, are identified by comparing the experimentally determined mass with the expected theoretical molecular weight.

[0475] Figure 5A The stability of the guide chains of the control and test compounds after incubation in decontaminated lysosomes of rat liver was depicted. Phosphatases in the decontaminated lysosomes removed the 5′-PO4 group of the control compound (5′-PO4, 2′-OMe). Within 2 hours of incubation with decontaminated lysosomes, the guide chain of the control compound (5′-PO4, 2′-OMe) was undetectable and replaced by a metabolite (“M1”) of the guide chain of the control compound, which possesses 5′-OH instead of 5′-PO4. Figure 5A The chemical structure of the 5′-terminal nucleotide of the metabolite is identical to that of the 5′-terminal nucleotide of the leading strand of the control compound (5′-OH, 2′-OMe). No cleavage of the phosphonate from the test compound (fully deprotected, 2′-OMe) was observed during the 24-hour incubation period. The test compound (fully deprotected, 2′-OMe) also exhibited improved metabolic stability compared to the control compound (5′-OH, 2′-OMe). Figure 5A These data indicate that the fully deprotected 4′-oxymethylphosphonate at the 5′-terminal nucleotide of the guide strand is resistant to phosphatase-mediated cleavage. A side-by-side comparison of the 5′-terminal nucleotides of the control compound (5′-PO4, 2′-OMe) and the test compound (fully deprotected, 2′-OMe) is shown below.

[0476]

[0477] In the relevant experiments, 1.7 μM of the test compound (monomethyl protected, 2′-F) was incubated in decontaminated rat liver lysosomes (Sekisui Xenotech, Kansas City, KS) at 1.2 U / mL (acid phosphatase activity). Samples were extracted from the lysosomal matrix by UPLC as described above, and the presence of the test compound and related metabolites was analyzed. Over time, the level of the leading chain of the test compound (monomethyl protected, 2′-F) steadily decreased and was replaced in the sample by a mixture of metabolites (including the main component with the same structure as the test compound (fully deprotected, 2′-F)). This indicates that under these conditions, the leading chain of the test compound (monomethyl protected, 2′-F) was converted to that of the test compound (fully deprotected, 2′-F). Figure 5BAfter 48 hours, the mixture of metabolites was present at approximately 80% of the original amount of the test compound (fully monomethyl protected, 2′-F), indicating that the fully deprotected 4′-oxymethylphosphonate located at the 5′-terminal nucleotide of the guide strand is resistant to phosphatase-mediated cleavage. Figure 5B .

[0478] To assess the in vivo stability of the 4′-oxymethylphosphonate compound, two male CD1 mice were administered the test compound (monomethyl protected, 2′-OMe) at 3 mpk, and at each time point, the liver was treated and analyzed by reversed-phase ion-pair ultra-high performance liquid chromatography (RP-IP-UPLC) and high-resolution mass spectrometry (HRMS). Frozen tissue was transferred to a Covaris TissueTube ultra-thick pulverizer bag (Covaris, Woburn, MA), rapidly frozen in liquid nitrogen, and pulverized using a Cryoprep pulverizer (Covaris, Woburn, MA). The sample was then returned to a Safe-Lock tube (Eppendorf, Hauppauge, NY) and 1 mL was added. OTX TM Lysis loading buffer (Phenomenex, Torrance, CA). Homogenize tissues for 3 minutes at 30 Hz using TissueLyser II (Qiagen, Frederick, MD). Then centrifuge samples at 20,000 rpm for 15 minutes at 4°C. Use 100 mg of the solution in 96 wells according to the manufacturer's instructions. OTX TM (Phenomenex, Torrance, CA) solid-phase extraction plates were used to extract the test compound (monomethyl protected, 2′-F) and its metabolites from the supernatant. The final eluent was frozen, lyophilized, and resuspended in 80 μL of water for analysis by RP-IP-UPLC-HRMS.

[0479] Using ACQUITY The instrument (Waters Corporation, Milford, MA) delivers a mobile phase containing a buffer additive at a rate of 0.4 mL / min using ACQUITY. Oligonucleotide BEH C18 column with 1.7 μm particle size, reversed-phase ultra-high performance liquid chromatography (2.1 mm × 50 mm) (Waters Corporation, Milford, MA) was used for chromatographic separation. The column temperature was maintained at 70 °C and the sample injection volume was 40 μL. The chromatography was performed using negative ion mode and electrospray ionization (ESI) conditions. A G2S high-resolution time-of-flight mass spectrometer (Waters Corporation, Milford, MA) was used to detect the guide chain of the test compound (monomethyl protected, 2′-F) and its metabolites. Promises deconvolution was used. TM The software (Novatia, Newtown, PA) obtains the mass of the zero-charge-state molecular ion via charge-state deconvolution. The guide chains of the test compounds (monomethyl protected, 2′-OMe) and their metabolites are identified by comparing the experimentally determined masses with the expected theoretical molecular weights. This is achieved through PROMASS DECONVOLUTION. TM The software calculates the signal intensity of the guide chain of the test compound (monomethyl protected, 2′-OMe) and related metabolites and derives it from the deconvolution signal intensity of the charge state.

[0480] By 48 hours, the amount of the leading chain of the test compound (monomethyl protected, 2′-OMe) had steadily decreased to about 30%. Figure 5C As the amount of the leading chain of the test compound (monomethyl protected, 2′-OMe) decreased, the metabolite (M2) with the same structure as the test compound (fully deprotected, 2′-OMe) steadily increased, reaching about 20% at 50 hours and exceeding 30% at 175 hours, indicating that the methyl group of 4′-oxymethylphosphonate is converted to a hydroxyl group in vivo. Figure 5C .

[0481] Example 12: Testing the in vivo activity of the compound in mice

[0482] Subcutaneous administration of the double-stranded nucleic acid inhibitor molecule at the dosage levels described below to female CD-1 mice at a volume of 10 μL / g. The control group was given phosphate-buffered saline (PBS). Animals were sacrificed at 72 or 240 hours post-treatment. The left medial lobe of the liver was removed, and a 1-4 mm extract was placed in a 96-well plate on dry ice. The extract was then processed using a CFX384 TOUCH. TM qPCR was performed using a real-time PCR detection system (BioRad Laboratories, Inc., Hercules, CA) to measure the reduction in target mRNA. All samples were normalized relative to PBS-treated control animals and plotted using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA).

[0483] In the first experiment, mice were subcutaneously administered the control compound (5′-OH, 2′-F), the control compound (5′-PO4, 2′-F), and the test compound (completely deprotected, 2′-F) at 1 mpk. Figure 1A and Figure 1B As shown, the three compounds are identical except for the nucleotide at position 1 of the guide strand, with the control compound having a 5′-OH or 5′-PO4 group and the test compound having a fully deprotected 4′-oxymethylphosphonate. Inhibition of target gene AmRNA expression was measured on day 3 post-dose. Compared to the two control compounds at the same dose, the test compound (fully deprotected, 2′-F) showed significantly enhanced gene silencing activity. Figure 6A These data suggest that metabolically stable 4′-oxymethylphosphonates enhance the in vivo activity of RNAi inhibitor molecules.

[0484] In the second experiment, control compound (5′-OH, 2′-OMe) and test compound (completely deprotected, 2′-OMe) were administered subcutaneously to female CD-1 mice at 1 mpk. Figure 1C As shown in Figure 1D, these compounds are identical except for the nucleotide at position 1 of the guide strand, with the control compound having a 5′-OH and the test compound having a fully deprotected 4′-oxymethylphosphonate. Inhibition of target gene B mRNA expression was measured on day 4 post-dose. The same trend was observed, with the test compound showing significantly enhanced gene silencing activity compared to the control compound at the same dose, indicating that 4′-oxymethylphosphonate enhances the in vivo activity of the dsRNAi inhibitor molecule. Figure 6B .

[0485] In the third experiment, the test compound (monomethyl protected, 2′-F) was administered subcutaneously to female CD-1 mice at body weights of 0.3 mpk, 1 mpk, and 3 mpk. Inhibition of target gene A mRNA expression was measured on day 10 post-administration. The test compound (monomethyl protected, 2′-F) showed a dose-dependent knockdown of target gene mRNA expression. Figure 7 .

[0486] In the fourth experiment, the test compound (completely deprotected, 2′-OMe) and the test compound (monomethyl protected, 2′-OMe) were administered subcutaneously to female CD-1 mice at 0.3 mpk or 1 mpk. Figure 1DAs shown, the two test compounds are identical except for the 4′-oxymethylphosphonate nucleotide at position 1 of the guide strand; one of the 4′-oxymethylphosphonates is completely deprotected while the other is protected by a single methyl group (i.e., monomethyl protection). Inhibition of target gene B mRNA expression was measured on days 3 and 10 post-dose. Both compounds showed dose-dependent knockdown and similar potency at both doses and time points. Figure 8 Not intending to be bound by any theory, it is believed that the monomethyl ester of 4′-oxomethylphosphonate can be converted in vivo into the completely deprotected 4′-oxomethylphosphonate.

[0487] Example 13: Testing the in vivo activity of compounds in non-human primates

[0488] In the first experiment, male and female cynomolgus monkeys were administered the control compound (5′-OH, 2′-OMe) and the test compound (completely deprotected, 2′-OMe) at a dose of 3 mg / kg body weight. (See Figure 1C and...) Figure 1D As shown, the two compounds are identical except for the nucleotide at position 1 of the guide strand, with the control compound having a 5′-OH group and the test compound having a fully deprotected 4′-oxymethylphosphonate. In a second experiment, male and female cynomolgus monkeys were administered the test compound (fully deprotected, 2′-OMe) and the test compound (monomethyl protected, 2′-OMe) at a dose of 3 mg / kg body weight. Figure 1D As shown, the two test compounds are identical except for the 4′-oxomethylphosphonate on the nucleotide at position 1 of the guide strand; one of the 4′-oxomethylphosphonates is completely deprotected and the other is protected by a single methyl group (i.e., monomethyl protection). The double-stranded nucleic acid inhibitor molecule was administered subcutaneously at a volume of 10 ml / kg. The control group was given phosphate-buffered saline (PBS).

[0489] Animals were fasted overnight prior to all sample collection. On study days – 7, 14, 28, and 56 – animals were sedated and approximately 20 mg of percutaneous liver biopsy samples were collected. Tissue samples were weighed and divided in half for use in… Store or keep at -70°C. (Use CFX384 TOUCH) TM qPCR was performed using a real-time PCR detection system (BioRad Laboratories, Inc., Hercules, CA) to measure the reduction of target mRNA. All animal samples were first normalized relative to their own pre-drug control samples, then normalized relative to PBS-treated control animals, and plotted using GraphPadPrism software (GraphPad Software Inc., La Jolla, CA).

[0490] In the first experiment, at days 14 and 28, the test compound (completely deprotected, 2′-OMe) showed better mRNA reduction activity compared to the control compound (5′-OH, 2′-OMe), indicating that the presence of 4′-oxymethylphosphonate enhances the in vivo activity of the RNAi inhibitor molecule in cynomolgus monkeys. Figure 9A In the second experiment, both test compounds (fully deprotected and monomethyl protected) showed similar activity at all time points. Figure 9B .

Claims

1. An oligonucleotide comprising a 5'-terminal nucleotide, wherein the 5'-terminal nucleotide is represented by Formula III: Where R a and R b Both are hydrogen, or R a and R b They are hydrogen and CH3, respectively. Where B is uracil; Wherein Y is an internucleotide linker group that connects the 5'-terminal nucleotide to the oligonucleotide; and Where X2 is either F or OCH3 and R8 does not exist.

2. An oligonucleotide comprising a 5'-terminal nucleotide, wherein the 5'-terminal nucleotide is represented by formula IV: Where B is uracil; Wherein Y is an internucleotide linker group that connects the 5'-terminal nucleotide to the oligonucleotide; and Where X2 is either F or OCH3.

3. An oligonucleotide comprising a 5'-terminal nucleotide, wherein the 5'-terminal nucleotide is represented by formula V: Where B is uracil; Wherein Y is an internucleotide linker group that connects the 5'-terminal nucleotide to the oligonucleotide; and Where X2 is either F or OCH3.

4. The oligonucleotide according to any one of claims 1-3, wherein the oligonucleotide is a double-stranded RNAi inhibitor molecule comprising a first strand and a second strand, wherein the first strand is a sense strand and the second strand is an antisense strand.

5. The oligonucleotide of claim 4, wherein the double-stranded RNAi inhibitor molecule comprises a complementary region between the sense strand and the antisense strand having 15 to 45 nucleotides.

6. The oligonucleotide of claim 5, wherein the complementary region between the sense strand and the antisense strand is 20 to 30 nucleotides.

7. The oligonucleotide of claim 6, wherein the complementary region between the sense strand and the antisense strand is 21 to 26 nucleotides.

8. The oligonucleotide of claim 5, wherein the complementary region between the sense strand and the antisense strand is 19 to 24 nucleotides.

9. The oligonucleotide of claim 8, wherein the complementary region between the sense strand and the antisense strand is 19 to 21 nucleotides.

10. The oligonucleotide of claim 4, wherein the 5'-terminal nucleotide is located on the antisense strand.

11. The oligonucleotide of claim 4, wherein the 5'-terminal nucleotide is located on the sense strand.

12. The oligonucleotide of claim 4, wherein the double-stranded RNAi inhibitor molecule contains a tetracycle.

13. The oligonucleotide according to any one of claims 1-3, wherein the oligonucleotide is a single-stranded oligonucleotide.

14. The oligonucleotide of claim 13, wherein the single-stranded oligonucleotide is a single-stranded RNAi inhibitor molecule.

15. The oligonucleotide of claim 13, wherein the single-stranded oligonucleotide is a conventional antisense oligonucleotide, ribonucleotide, or aptamer.

16. The oligonucleotide of claim 14, wherein the single-stranded RNAi inhibitor molecule has a length of 14 to 50 nucleotides.

17. The oligonucleotide of claim 16, wherein the single-stranded RNAi inhibitor molecule has a length of 16-30, 18-22, or 20-22 nucleotides.

18. The oligonucleotide according to any one of claims 1-3, 5-12 and 14-17, wherein the oligonucleotide is a naked oligonucleotide.

19. An oligonucleotide conjugated with at least one delivery agent to facilitate the transmembrane transport of the oligonucleotide across a cell, wherein the oligonucleotide is an oligonucleotide according to any one of claims 1-3, 5-12, and 14-17.

20. The oligonucleotide of claim 19, wherein the at least one delivery agent is selected from the group consisting of carbohydrates, peptides, lipids and vitamins.

21. The oligonucleotide of claim 20, wherein the at least one delivery agent is selected from N-acetylgalactosamine (GalNAc), mannose-6-phosphate, galactose, oligosaccharides, polysaccharides, cholesterol, polyethylene glycol, folate, vitamin A, vitamin E, lithocholic acid, and cationic lipids.

22. The oligonucleotide of claim 19, wherein the at least one delivery agent is an antibody.

23. A pharmaceutical composition comprising the oligonucleotide according to claim 4 and a pharmaceutically acceptable excipient.

24. A pharmaceutical composition comprising an oligonucleotide according to any one of claims 5-12.

25. A pharmaceutical composition comprising the oligonucleotide according to claim 13 and a pharmaceutically acceptable excipient.

26. A pharmaceutical composition comprising an oligonucleotide according to any one of claims 14-16.

27. Use of the pharmaceutical composition according to claim 23 or 25 in the preparation of a medicament for reducing the expression of a target gene in a subject in need.

28. The use according to claim 27, wherein the drug is formulated for systemic administration.

29. The oligonucleotide according to any one of claims 1-3, wherein the oligonucleotide is a double-stranded oligonucleotide comprising a first strand and a second strand.

30. The oligonucleotide of claim 29, wherein each strand of the double-stranded oligonucleotide has a length of 15-100 or 15-50 nucleotides.

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