5'-MODIFIED CARBOCYCLIC RIBONUCLEOTIDE DERIVATIVES AND METHODS OF USE - Patent application

Abstract

The present disclosure relates to phosphomimetic derivatives of formula (I) or (II): JPEG2025508785000193.jpg74170 and pharma- ceutically acceptable salts thereof, as well as related nucleic acid agents and conjugates. The present disclosure also relates to the use of the phosphomimetic derivatives (e.g., in nucleic acid agents and conjugates) in the treatment or prevention of disease.

Description

[Technical field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Application No. 63 / 312,554, filed February 22, 2022, the entire contents of which are incorporated herein by reference.

[0002] Incorporating sequence tables The contents of the electronic sequence listing (SANB_005_001WO_SeqList_ST26.xml, size: 105,372 bytes, created on: February 17, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] Argonaute 2 (Ago2) belongs to the AGO protein family and plays a key role in the biogenesis of small RNA (smRNA). Ago2 is a key component of the RISC loading complex (RLC), which binds and loads double-stranded RNA (dsRNA) into the RISC complex for processing and degradation of target mRNA. The composition of nucleotides in an RNA molecule has been shown to affect the binding of Ago2 to RNA. Various modifications to the structure of nucleotides in an RNA molecule have been tested. Thus, there is a continuing need for RNAs with improved potency and / or stability for degrading mRNAs by chemical modifications to the RNA, such as modifications to the RNA backbone. The present application addresses this need. Summary of the Invention

[0004] The present invention relates to modification of the 5'-terminal nucleotide of an oligonucleotide, such as RNA. More specifically, the present disclosure relates to cyclopentyl-based nucleotides, such as cyclopentyl-based nucleotides comprising 4'-ethylphosphonate or 4'-vinylphosphonate as described herein. Oligonucleotides (e.g., RNA) comprising the cyclopentyl-based nucleotides of the present disclosure exhibit advantageous properties, such as improved metabolic stability against nucleases and phosphatases, while maintaining or even improving RISC binding affinity and intrinsic potency.

[0005] In some embodiments, the present disclosure provides a compound of formula (I) or (II): [ka] or a pharma- ceutically acceptable salt thereof, wherein: B is H or a nucleobase moiety; X is H, halogen, or -OR X and R X is H, C1-C6 alkyl, or -(C1-C6 alkyl)-(C6-C 10 aryl), C1-C6 alkyl or -(C1-C6 alkyl)-(C6-C 10 aryl) is one or more R Xa optionally replaced by Each R Xa are independently halogen, C1-C6 alkyl, or -O-(C1-C6 alkyl), where C1-C6 alkyl or -O-(C1-C6 alkyl) is optionally substituted with one or more halogens; Y is H, C1-C6 alkyl optionally substituted with one or more halogens, -P(R Y )2, -P(OR Y )(N(R Y )2), -P(=O)(OR Y )R Y , -P(=S)(OR Y )R Y , -P(=O)(SR Y )R Y , -P(=S)(SR Y)R Y , -P(=O)(OR Y )2, -P(=S)(OR Y )2, -P(=O)(SR Y )2, -P(=S)(SR Y ) 2, or a hydroxy protecting group; Each R Y is independently H or C1-C6 alkyl optionally substituted with one or more halogen or cyano; Z is P(R Z )2, -P(OR Z )(N(R Z )2), -P(=O)(OR Z )R Z , -P(=S)(OR Z )R Z , -P(=O)(SR Z )R Z , -P(=S)(SR Z )R Z , -P(=O)(OR Z )2, -P(=S)(OR Z )2, -P(=O)(SR Z )2, or -P(=S)(SR Z )2, Each R Z is independently H or C1-C6 alkyl optionally substituted with one or more halogen or cyano; R 1 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; R 2 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; R 3 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; R 4 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; [ka] represents a single bond or a double bond, Each R 6are independently H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; A compound or a pharma- ceutically acceptable salt thereof is provided.

[0006] In some embodiments, the disclosure provides a nucleic acid agent, or a pharma- ceutically acceptable salt thereof, wherein the nucleic acid agent comprises an oligonucleotide comprising one or two 5' terminal units, each 5' terminal unit independently comprising: [ka] where the variables B, R 1 , R 2 , R 3 , R 4 , R 6 wherein, X, and Z are described herein and ## indicates the bond to the remainder of the oligonucleotide, or a pharma- ceutically acceptable salt thereof.

[0007] In some embodiments, the present disclosure provides a conjugate or a pharma- ceutically acceptable salt thereof, the conjugate comprising: (i) a nucleic acid agent comprising one or two 5' terminal units covalently attached to an oligonucleotide, wherein each 5' terminal unit is independently [ka] where the variables B, R 1 , R 2 , R 3 , R 4 , R 6 , X, and Z are as described herein, and ## indicates the attachment to the remainder of the oligonucleotide; and (ii) one or more ligands covalently attached to the nucleic acid agent or a pharma- ceutically acceptable salt thereof.

[0008] In some embodiments, the present disclosure provides compounds that are isotopic derivatives of the compounds disclosed herein.

[0009] In some embodiments, the disclosure provides a pharmaceutical composition comprising a compound, nucleic acid agent, or conjugate described herein.

[0010] In some embodiments, the present disclosure provides a method of modulating expression of a target gene in a subject, the method comprising administering to the subject a conjugate described herein.

[0011] In some embodiments, the disclosure provides a method of delivering a nucleic acid agent to a subject, the method comprising administering to the subject a conjugate described herein.

[0012] In some embodiments, the present disclosure provides a method of treating or preventing a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate described herein.

[0013] In some embodiments, the present disclosure provides a conjugate as described herein for modulating expression of a target gene in a subject.

[0014] In some embodiments, the disclosure provides a conjugate as described herein for delivery of a nucleic acid agent to a subject.

[0015] In some embodiments, the present disclosure provides a conjugate as described herein for treating or preventing a disease in a subject in need thereof.

[0016] In some embodiments, the present disclosure provides the use of a conjugate described herein in the manufacture of a medicament for modulating expression of a target gene in a subject.

[0017] In some embodiments, the disclosure provides for the use of a conjugate described herein in the manufacture of a medicament for delivering a nucleic acid agent to a subject.

[0018] In some embodiments, the disclosure provides the use of a conjugate described herein in the manufacture of a medicament for treating or preventing a disease in a subject in need thereof.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In this specification, the singular includes the plural unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. All practices, patent applications, patents, and other references described herein are incorporated by reference. References cited herein are not admitted to be prior art to the invention described in the claims. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are merely illustrative and are not intended to be limiting. In the event of a conflict between the chemical structure and the name of a compound disclosed herein, the chemical structure will control.

[0020] Other features and advantages of the invention will become apparent from the following detailed description and claims. [Brief description of the drawings]

[0021] [Figure 1] 1A and 1B are a series of graphs showing gene silencing activity of siRNA compounds in the liver on day 5 after a single subcutaneous injection of 0.5 mg / kg in CD-I female mice, followed by administration of HDI (human gene plasmid, 20 μg) on ​​day 4.

[0022] [Diagram 2] 2A-2E are a series of graphs showing gene silencing activity of siRNA compounds in multiple extrahepatic tissues at day 10 following a single subcutaneous injection of 3 mg / kg of siRNA compounds in CD-I female mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Chemical modifications have been introduced into oligonucleotides to introduce properties that may be desirable under certain conditions, for example, to stabilize them against nucleases or other enzymes that degrade them or interfere with their structure or activity. For example, synthetic oligonucleotides generally terminate with a 5'-hydroxyl or 3'-hydroxyl group. The terminal hydroxyl group can be replaced with a phosphate group, which can be used, for example, to attach a linker, adapter, or label, or to directly attach the oligonucleotide to another nucleic acid. In addition, it has been reported that a phosphate group at the 5' end enhances the interaction of certain nucleic acid inhibitor molecules with Ago2. However, oligonucleotides with a 5'-phosphate group are generally susceptible to degradation by phosphatases or other enzymes, which can limit their bioavailability in vivo. It is therefore desirable to develop modifications to the 5'-terminal nucleotide of oligonucleotides, such as nucleic acid inhibitor molecules, that provide the functional effect of a phosphate group but are more stable in the environmental conditions to which the oligonucleotide is exposed when administered to a subject. Such phosphate analogs would be more resistant to phosphatases and other enzymes, with minimal adverse effects on the function of the oligonucleotide (e.g., with minimal reduction in gene target knockdown when used as an RNAi inhibitor molecule).

[0024] Limited approaches have been reported for modifying the 5'-terminal nucleotide of oligonucleotides. However, there continues to be a need for novel phosphomimetic derivatives of the 5'-terminal nucleotide and conjugates thereof that are metabolically stable to nucleases, phosphatases, and the like, while retaining maintained or improved RISC binding affinity and intrinsic potency of the oligonucleotide.

[0025] The present disclosure provides phosphomimetic derivatives of the 5'-terminal nucleotide. Without wishing to be bound by theory, it is understood that the phosphomimetic derivatives, when incorporated into oligonucleotides (e.g., at the 5'-end of the antisense strand), can improve Ago2 binding / loading, improve the metabolic stability of the oligonucleotide, and therefore improve the potency and duration of the oligonucleotide (e.g., siRNA molecule).

[0026] The present disclosure further provides nucleic acid agents and conjugates comprising the phosphomimetic derivatives for nucleic acid delivery. The present disclosure also relates to the use of the phosphomimetic derivatives, nucleic acid agents, and conjugates, for example, in the delivery of nucleic acids and / or in the treatment or prevention of diseases.

[0027] 5'-end unit compounds of the present disclosure In some embodiments, the present disclosure provides a compound of formula (I) or (II): [ka] or a pharma- ceutically acceptable salt thereof, wherein: B is H or a nucleobase moiety; X is H, halogen, or -OR X and R X is H, C1-C6 alkyl, or -(C1-C6 alkyl)-(C6-C 10 aryl), C1-C6 alkyl or -(C1-C6 alkyl)-(C6-C 10 aryl) is one or more R Xa optionally replaced by Each R Xa are independently halogen, C1-C6 alkyl, or -O-(C1-C6 alkyl), where C1-C6 alkyl or -O-(C1-C6 alkyl) is optionally substituted with one or more halogens; Y is H, C1-C6 alkyl optionally substituted with one or more halogens, -P(R Y )2, -P(OR Y )(N(R Y )2), -P(=O)(ORY )R Y , -P(=S)(OR Y )R Y , -P(=O)(SR Y )R Y , -P(=S)(SR Y )R Y , -P(=O)(OR Y )2, -P(=S)(OR Y )2, -P(=O)(SR Y )2, -P(=S)(SR Y )2, or a hydroxy protecting group; Each R Y is independently H or C1-C6 alkyl optionally substituted with one or more halogen or cyano; Z is P(R Z )2, -P(OR Z )(N(R Z )2), -P(=O)(OR Z )R Z , -P(=S)(OR Z )R Z , -P(=O)(SR Z )R Z , -P(=S)(SR Z )R Z , -P(=O)(OR Z )2, -P(=S)(OR Z )2, -P(=O)(SR Z )2, or -P(=S)(SR Z )2, Each R Z is independently H or C1-C6 alkyl optionally substituted with one or more halogen or cyano; R 1 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; R 2 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; R 3 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; R 4is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; [ka] represents a single bond or a double bond, Each R 6 are independently H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; A compound or a pharma- ceutically acceptable salt thereof is provided.

[0028] For the compounds of the present disclosure, the variables B, X, R X , R Xa , Y, R Y , Z, R Z , R 1 , R 2 , R 3 , R 4 , and R 6 may each be selected from the groups described herein, where applicable, and the variables B, X, R X , R Xa , Y, R Y , Z, R Z , R 1 , R 2 , R 3 , R 4 , and R 6 Any group described herein with respect to any of the variables B, X, R X , R Xa , Y, R Y , Z, R Z , R 1 , R 2 , R 3 , R 4 , and R 6 It is to be understood that one or more of the remaining groups may be combined with any group described herein.

[0029] Variables B, X, R X , R Xa , Y, R Y , Z, and R Z In some embodiments, B is H.

[0030] In some embodiments, B is a nucleobase moiety.

[0031] The term "nucleobase moiety" as used herein refers to a nucleobase that is attached to the remainder of a compound, e.g., via an atom of the nucleobase or a functional group thereof.

[0032] In some embodiments, the nucleobase moiety is adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U).

[0033] In some embodiments, the nucleobase moiety is a modified nucleobase.

[0034] In some embodiments, the modified nucleobase is 5-methylcytosine.

[0035] In some embodiments, the modified nucleobase is hypoxanthine, xanthine, or 7-methylguanine.

[0036] In some embodiments, the modified nucleobase is 5,6-dihydrouracil, 5-methylcytosine, or 5-hydroxymethylcytosine.

[0037] In some embodiments, the nucleobase moiety is an artificial nucleobase.

[0038] In some embodiments, the artificial nucleobase is isoguanine, isocytosine, 2-amino-6-(2-thienyl)purine, or pyrrole-2-carbaldehyde.

[0039] In some embodiments, X is H.

[0040] In some embodiments, X is not H.

[0041] In some embodiments, X is halogen (eg, F, Cl, Br, or I).

[0042] In some embodiments, X is F or Cl.

[0043] In some embodiments, X is F.

[0044] In some embodiments, X is -OR X It is.

[0045] In some embodiments, X is -OH.

[0046] In some embodiments, X is not --OH.

[0047] In some embodiments, X is -O-(C1-C6 alkyl) (e.g., C1-C6 alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0048] In some embodiments, X is -OCH3.

[0049] In some embodiments, X is -O-(C1-C6 alkyl)-O-(C1-C6 alkyl) (e.g., C1-C6 alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0050] In some embodiments, X is -OCH2CH2OCH3.

[0051] In some embodiments, X is one or more R Xa -O-(C1-C6 alkyl)-(C6-C 10 aryl).

[0052] In some embodiments, X is -O-(C1-C6 alkyl)-(C6-C 10 aryl).

[0053] In some embodiments, X is [ka] It is.

[0054] In some embodiments, X is one or more R Xa Optionally replaced with [ka] It is.

[0055] In some embodiments, X is optionally substituted with one or more halogens. [ka] It is.

[0056] In some embodiments, X is optionally substituted with one or more C1-C6 alkyl or -O-(C1-C6 alkyl). [ka] wherein C1-C6 alkyl or -O-(C1-C6 alkyl) is optionally substituted with one or more halogens.

[0057] In some embodiments, R X is H.

[0058] In some embodiments, R X is not H.

[0059] In some embodiments, R X is one or more R Xa and C1-C6 alkyl optionally substituted with (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0060] In some embodiments, R Xis C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br, or I), or -O-(C1-C6 alkyl) (e.g., C1-C6 alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogens.

[0061] In some embodiments, R X is C1 to C6 alkyl (for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0062] In some embodiments, R X is methyl, ethyl, or propyl.

[0063] In some embodiments, R X is methyl.

[0064] In some embodiments, R X is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0065] In some embodiments, R X is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) substituted with one or more -O-(C1-C6 alkyl) (e.g., C1-C6 alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl), where -O-(C1-C6 alkyl) is optionally substituted with one or more halogens.

[0066] In some embodiments, R X is one or more R Xa -(C1-C6 alkyl)-(C6-C 10 aryl).

[0067] In some embodiments, R X is -(C1-C6 alkyl)-(C6-C6 alkyl) optionally substituted with one or more halogens (e.g., F, Cl, Br, or I); 10 aryl), C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl), or -O-(C1-C6 alkyl) (e.g., C1-C6 alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl), where C1-C6 alkyl or -O-(C1-C6 alkyl) is optionally substituted with one or more halogens.

[0068] In some embodiments, R X is -(C1-C6 alkyl)-(C6-C 10 aryl).

[0069] In some embodiments, at least one R Xa is a halogen (e.g., F, Cl, Br, or I).

[0070] In some embodiments, at least one R Xa is F or Cl.

[0071] In some embodiments, at least one R Xa is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0072] In some embodiments, at least one R Xa is C1 to C6 alkyl (for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0073] In some embodiments, at least one R Xa is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0074] In some embodiments, at least one R Xa is -O-(C1-C6 alkyl) optionally substituted with one or more halogen (e.g., F, Cl, Br, or I).

[0075] In some embodiments, at least one R Xa is -O-(C1-C6 alkyl).

[0076] In some embodiments, at least one R Xa is -O-(C1-C6 alkyl) substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0077] In some embodiments, Y is H.

[0078] In some embodiments, Y is not H.

[0079] In some embodiments, Y is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0080] In some embodiments, Y is C1-C6 alkyl (eg, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0081] In some embodiments, Y is methyl, ethyl, or propyl.

[0082] In some embodiments, Y is -P(R Y )2, -P(OR Y )(N(R Y )2), -P(=O)(OR Y )R Y , -P(=S)(OR Y )R Y , -P(=O)(SR Y )R Y , -P(=S)(SR Y )R Y , -P(=O)(OR Y )2, -P(=S)(OR Y )2, -P(=O)(SR Y )2, -P(=S)(SR Y )2.

[0083] In some embodiments, Y is -P(R Y )2.

[0084] In some embodiments, Y is -PH2.

[0085] In some embodiments, Y is -P(OR Y )(N(R Y )2).

[0086] In some embodiments, Y is -P(OH)(NH2).

[0087] In some embodiments, Y is -P(O(C1-C6 alkyl))(N(C1-C6 alkyl)2), where C1-C6 alkyl is optionally substituted with one or more halogen or cyano.

[0088] In some embodiments, Y is -P(=O)(OR Y )R Y It is.

[0089] In some embodiments, Y is -P(=O)(OH)(C1-C6 alkyl), where C1-C6 alkyl is optionally substituted with one or more halogen or cyano.

[0090] In some embodiments, Y is -P(=S)(OR Y )R Y It is.

[0091] In some embodiments, Y is -P(=S)(OH)(C1-C6 alkyl), where C1-C6 alkyl is optionally substituted with one or more halogen or cyano.

[0092] In some embodiments, Y is -P(=O)(SR Y )R Y It is.

[0093] In some embodiments, Y is -P(=O)(SH)(C1-C6 alkyl), where C1-C6 alkyl is optionally substituted with one or more halogen or cyano.

[0094] In some embodiments, Y is -P(=S)(SR Y )R Y It is.

[0095] In some embodiments, Y is -P(=S)(SH)(C1-C6 alkyl), where C1-C6 alkyl is optionally substituted with one or more halogen or cyano.

[0096] In some embodiments, Y is -P(=O)(OR Y )2.

[0097] In some embodiments, Y is -P(=O)(OH).

[0098] In some embodiments, Y is -P(=S)(OR Y )2.

[0099] In some embodiments, Y is -P(=S)(OH)2.

[0100] In some embodiments, Y is -P(=O)(SR Y )2.

[0101] In some embodiments, Y is -P(=O)(SH)2.

[0102] In some embodiments, Y is -P(=S)(SR Y )2.

[0103] In some embodiments, Y is -P(=S)(SH)2.

[0104] In some embodiments, Y is a hydroxy protecting group (eg, silyl, Tr, DMTr, acyl, or benzyl).

[0105] In some embodiments, Y is silyl (eg, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or triisopropylsilyl).

[0106] In some embodiments, Y is triphenylmethyl (Tr) or 4,4'-dimethoxytrityl (DMTr).

[0107] In some embodiments, Y is optionally substituted acyl (eg, optionally substituted acetyl) or benzyl.

[0108] In some embodiments, Y is not a hydroxy protecting group (eg, silyl, Tr, DMTr, acyl, or benzyl).

[0109] In some embodiments, at least one R Y is H.

[0110] In some embodiments, each R Y is H.

[0111] In some embodiments, at least one R Y is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogen (e.g., F, Cl, Br, or I) or cyano.

[0112] In some embodiments, each R Y is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogen (e.g., F, Cl, Br, or I) or cyano.

[0113] In some embodiments, at least one R Y is H and at least one R Y is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogen or cyano.

[0114] In some embodiments, when X is -OH, Y is not H or a hydroxy protecting group (eg, silyl, Tr, DMTr, acyl, or benzyl).

[0115] In some embodiments, when X is -OH, Y is C1-C6 alkyl optionally substituted with one or more halogens, -P(R Y )2, -P(OR Y )(N(R Y )2), -P(=O)(OR Y )R Y , -P(=S)(ORY )R Y , -P(=O)(SR Y )R Y , -P(=S)(SR Y )R Y , -P(=O)(OR Y )2, -P(=S)(OR Y )2, -P(=O)(SR Y )2, or -P(=S)(SR Y )2.

[0116] In some embodiments, when Y is H or a hydroxy protecting group (eg, silyl, Tr, DMTr, acyl, or benzyl), X is not -OH.

[0117] In some embodiments, when Y is H or a hydroxy protecting group (e.g., silyl, Tr, DMTr, acyl, or benzyl), X is H, halogen, or -OR X where R X is C1-C6 alkyl or -(C1-C6 alkyl)-(C6-C 10 aryl), C1-C6 alkyl or -(C1-C6 alkyl)-(C6-C 10 aryl) is one or more R Xa is optionally replaced by

[0118] In some embodiments, Z is -P(R Z )2

[0119] In some embodiments, Z is -PH2.

[0120] In some embodiments, Z is -P(OR Z )(N(R Z )2).

[0121] In some embodiments, Z is -P(OH)(NH2).

[0122] In some embodiments, Z is -P(O(C1-C6 alkyl))(N(C1-C6 alkyl)2), where C1-C6 alkyl is optionally substituted with one or more halogen or cyano.

[0123] In some embodiments, Z is -P(=O)(OR Z )R Z It is.

[0124] In some embodiments, Z is -P(=O)(OH)(C1-C6 alkyl), wherein C1-C6 alkyl is optionally substituted with one or more halogen or cyano.

[0125] In some embodiments, Z is -P(=S)(OR Z )R Z It is.

[0126] In some embodiments, Z is -P(=S)(OH)(C1-C6 alkyl), wherein C1-C6 alkyl is optionally substituted with one or more halogen or cyano.

[0127] In some embodiments, Z is -P(=O)(SR Z )R Z It is.

[0128] In some embodiments, Z is -P(=O)(SH)(C1-C6 alkyl), wherein C1-C6 alkyl is optionally substituted with one or more halogen or cyano.

[0129] In some embodiments, Z is -P(=S)(SR Z )R Z is

[0130] In some embodiments, Z is -P(=S)(SH)(C1-C6 alkyl), wherein C1-C6 alkyl is optionally substituted with one or more halogen or cyano.

[0131] In some embodiments, Z is -P(=O)(OR Z )2.

[0132] In some embodiments, Z is -P(=O)(OH).

[0133] In some embodiments, Z is -P(=O)(O(C1-C6 alkyl))(OH).

[0134] In some embodiments, Z is -P(=O)(OCH3)(OH).

[0135] In some embodiments, Z is -P(=O)(O(C1-C6 alkyl))2.

[0136] In some embodiments, Z is -P(=O)(OCH3)2.

[0137] In some embodiments, Z is -P(=S)(OR Z )2.

[0138] In some embodiments, Z is -P(=S)(OH)2.

[0139] In some embodiments, Z is -P(=O)(SR Z )2.

[0140] In some embodiments, Z is -P(=O)(SH)2.

[0141] In some embodiments, Z is -P(=S)(SR Z )2.

[0142] In some embodiments, Z is -P(=S)(SH)2.

[0143] In some embodiments, at least one R Z is H.

[0144] In some embodiments, each R Z is H.

[0145] In some embodiments, at least one R Z is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogen (e.g., F, Cl, Br, or I) or cyano.

[0146] In some embodiments, at least one R Z is C1 to C6 alkyl (for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0147] In some embodiments, each R Z is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogen (e.g., F, Cl, Br, or I) or cyano.

[0148] In some embodiments, each R Z is C1 to C6 alkyl (for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0149] In some embodiments, at least one R Z is H and at least one R Z is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogen (e.g., F, Cl, Br, or I) or cyano.

[0150] In some embodiments, at least one R Zis H and at least one R Z is C1 to C6 alkyl (for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0151] Variable R 1 , R 2 , R 3 , R 4 , and R 6 In some embodiments, R 1 is H.

[0152] In some embodiments, R 1 is a halogen (e.g., F, Cl, Br, or I).

[0153] In some embodiments, R 1 is F or Cl.

[0154] In some embodiments, R 1 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0155] In some embodiments, R 1 is C1 to C6 alkyl (for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0156] In some embodiments, R 1 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0157] In some embodiments, R 2is H.

[0158] In some embodiments, R 2 is a halogen (e.g., F, Cl, Br, or I).

[0159] In some embodiments, R 2 is F or Cl.

[0160] In some embodiments, R 2 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0161] In some embodiments, R 2 is C1 to C6 alkyl (for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0162] In some embodiments, R 2 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0163] In some embodiments, R 3 is H.

[0164] In some embodiments, R 3 is a halogen (e.g., F, Cl, Br, or I).

[0165] In some embodiments, R 3 is F or Cl.

[0166] In some embodiments, R 3is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0167] In some embodiments, R 3 is C1 to C6 alkyl (for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0168] In some embodiments, R 3 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0169] In some embodiments, R 4 is H.

[0170] In some embodiments, R 4 is a halogen (e.g., F, Cl, Br, or I).

[0171] In some embodiments, R 4 is F or Cl.

[0172] In some embodiments, R 4 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0173] In some embodiments, R 4 is C1 to C6 alkyl (for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0174] In some embodiments, R 4 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0175] In some embodiments, each R 6 is H.

[0176] In some embodiments, at least one R 6 is halogen (e.g., F, Cl, Br, or I) or C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0177] In some embodiments, at least one R 6 is a halogen (e.g., F, Cl, Br, or I).

[0178] In some embodiments, at least one R 6 is F or Cl.

[0179] In some embodiments, at least one R 6 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) optionally substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0180] In some embodiments, at least one R 6 is C1 to C6 alkyl (for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl).

[0181] In some embodiments, at least one R 6 is C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl, or hexyl) substituted with one or more halogens (e.g., F, Cl, Br, or I).

[0182] In some embodiments, R 1 , R 2 , R 3 , R 4 , and R 6 Each of is H.

[0183] Exemplary embodiments of the compounds In some embodiments, the compound has formula (I'-1), (I'-2), (II'-1), or (II'-2): [ka] or a pharma- ceutically acceptable salt thereof.

[0184] In some embodiments, the compound has formula (IA) or (II-A): [ka] or a pharma- ceutically acceptable salt thereof.

[0185] In some embodiments, the compound has formula (I-A'-1), (I-A'-2), (II-A'-1), or (II-A'-2): [ka] or a pharma- ceutically acceptable salt thereof.

[0186] In some embodiments, the compound has formula (IB-1), (IB-2), (II-B-1), or (II-B-2): [ka] or a pharma- ceutically acceptable salt thereof.

[0187] In some embodiments, the compound has formula (I-B'-1), (I-B'-2), (I-B'-3), (I-B'-4), (II-B'-1), (II-B'-2), (II-B'-3), or (II-B'-4): [ka] [ka] or a pharma- ceutically acceptable salt thereof.

[0188] In some embodiments, the compound is [ka] or a pharma- ceutically acceptable salt thereof, wherein: Y is -P(R Y )2, -P(OR Y )(N(R Y )2), -P(=O)(OR Y )R Y , -P(=S)(OR Y )R Y , -P(=O)(SR Y )R Y , -P(=S)(SR Y )R Y , -P(=O)(OR Y )2, -P(=S)(OR Y )2, -P(=O)(SR Y )2, -P(=S)(SR Y )2, or a hydroxy protecting group (e.g., silyl) (e.g., trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or triisopropylsilyl), triphenylmethyl (Tr), 4,4′-dimethoxytrityl (DMTr), substituted acyl (e.g., optionally substituted acetyl), or benzyl); Each R Yis independently H or C1-C6 alkyl optionally substituted with one or more halogen or cyano; Z is -P(R Z )2, -P(OR Z )(N(R Z )2), -P(=O)(OR Z )R Z , -P(=S)(OR Z )R Z , -P(=O)(SR Z )R Z , -P(=S)(SR Z )R Z , -P(=O)(OR Z )2, -P(=S)(OR Z )2, -P(=O)(SR Z )2, or -P(=S)(SR Z )2, Each R Z is independently H or C1-C6 alkyl optionally substituted with one or more halogen or cyano.

[0189] In some embodiments, the compound is [ka] [ka] [ka] [ka] [ka] or a pharma- ceutically acceptable salt thereof, wherein B is adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U).

[0190] In some embodiments, the compound is selected from the compounds set forth in Table E and pharma- ceutically acceptable salts thereof. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0191] In some embodiments, the disclosure provides compounds that are isotopic derivatives of any one of the compounds of the formulas disclosed herein (eg, isotopically labeled compounds).

[0192] It will be appreciated that isotopic derivatives can be prepared using any of a variety of art-recognized techniques. For example, isotopic derivatives can generally be prepared by following the procedures disclosed in the schemes and / or examples herein, substituting a non-isotopically labeled reagent for an isotopically labeled reagent.

[0193] In some embodiments, the isotopic derivatives are deuterium-labeled compounds.

[0194] In some embodiments, an isotopic derivative is a deuterium-labeled compound of any one of the compounds of the formulas disclosed herein.

[0195] As used herein, the term "isotopic derivative" refers to a derivative of a compound in which one or more atoms are isotopically enriched or labeled. For example, an isotopic derivative of a compound of formula (I) or (II) is isotopically enriched with or labeled with one or more isotopes compared to the corresponding compound of formula (I) or (II). In some embodiments, an isotopic derivative is 2 H, 13 C. 14 C. 15 N, 18 O.29 Si, 32 P, and 34 S. In some embodiments, the isotopic derivatives are deuterium-labeled compounds (i.e., enriched or labeled with one or more atoms selected from S and / or S). 2 In some embodiments, the compound is 2 H-labeled compound. In some embodiments, the compound is 13 C-labeled compounds or 14 C labeled compound. In some embodiments, the compound is 18 F labeled compound. In some embodiments, the compound is 123 I-labeled compound, 124 I-labeled compound, 125 I-labeled compound, 129 I-labeled compound, 131 I-labeled compound, 135 I-labeled compounds, or any combination thereof. In some embodiments, the compounds are 32 P-labeled compounds or 32 In some embodiments, the compound is 33 S-labeled compound, 34 S-labeled compound, 35 S-labeled compound, 36 S-labeled compounds, or any combination thereof.

[0196] It will be appreciated that isotopic derivatives can be prepared using any of a variety of art-recognized techniques. For example, isotopic derivatives can generally be prepared by following the procedures disclosed in the schemes and / or examples described herein, substituting an isotopic labeled reagent for a non-isotopically labeled reagent.

[0197] It will also be appreciated that isotopic substitution may confer certain therapeutic advantages, such as greater metabolic stability, for example increased in vivo half-life or reduced required dosage.

[0198] For the avoidance of doubt, when a group is defined herein as being "as described herein," it is to be understood that the group encompasses the broadest definition as well as each and every specific definition appearing first for that group.

[0199] It will be understood that the compounds disclosed herein may be presented in one particular configuration. Such a particular configuration should not be interpreted as limiting the disclosure to one or another isomer, tautomer, positional isomer, or stereoisomer, nor does it exclude mixtures of isomers, tautomers, positional isomers, or stereoisomers. In some embodiments, the presentation of a compound in a particular configuration herein is intended to encompass and refer to each of the available isomers, tautomers, positional isomers, and stereoisomers of the compound, or any mixtures thereof, and the presentation is further intended to refer to the particular configuration of the compound.

[0200] It will be understood that the compounds disclosed herein may be presented without a specified configuration (e.g., without a specified stereochemistry). Such presentation is intended to encompass all available isomers, tautomers, positional isomers, and stereoisomers of the compound. In some embodiments, presentation of a compound herein without a specified configuration is intended to refer to each of the available isomers, tautomers, positional isomers, and stereoisomers of the compound, or any mixture thereof.

[0201] As used herein, the term "isomerism" refers to compounds that have the same molecular formula but differ in the sequence of bonding of the atoms or the arrangement of the atoms in space. Compounds that have the same molecular formula but differ in the nature or sequence of bonding of the atoms or the arrangement of the atoms in space are termed "isomers". Isomers that have different arrangements of the atoms in space are termed "stereoisomers". Stereoisomers that are not mirror images of each other are termed "diastereomers" and those that are non-superimposable mirror images of each other are termed "enantiomers". When a compound has an asymmetric center, for example, when a compound is bonded to four different groups, a pair of enantiomers can be adopted. Enantiomers can be characterized by the absolute configuration of their asymmetric center and described by the R and S ordering rules of Cahn and Prelog, or by the way in which a molecule rotates the plane of polarized light to designate it as dextrorotatory or levorotatory ((+) or (-) isomers, respectively). Chiral compounds can exist as individual enantiomers or as mixtures thereof. A mixture containing equal proportions of enantiomers is called a "racemic mixture".

[0202] The compounds of the present disclosure may have one or more asymmetric centers, and therefore such compounds can be produced as individual (R) or (S) stereoisomers, or as mixtures thereof. Unless otherwise specified, the description or naming of a particular compound in the present specification and claims is intended to include both individual enantiomers and mixtures thereof, or racemates, etc. Methods for determining stereochemistry and separating stereoisomers are well known in the art (see discussion in Chapter 4 of "Advanced Organic Chemistry", 4th edition J. March, John Wiley and Sons, New York, 2001), for example, by synthesis from optically active starting materials or by resolution of racemates. Some of the compounds of the present disclosure may have geometric isomeric centers (E and Z isomers). It is to be understood that the present disclosure encompasses all optical, diastereomeric, and geometric isomers, and mixtures thereof, that have inflammasome inhibitory activity.

[0203] As used herein, the term "chiral center" refers to a carbon atom bonded to four nonidentical substituents.

[0204] As used herein, the term "chiral isomer" refers to a compound having at least one chiral center. Compounds with two or more chiral centers may exist as individual diastereomers or as a mixture of diastereomers, termed a "diastereomeric mixture." When one chiral center is present, a stereoisomer may be characterized by the absolute configuration (R or S) of that chiral center. Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are ranked according to the Cahn-Ingold-Prelog ranking rules (Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem. Educ. 1964, 41, 116).

[0205] As used herein, the term "geometric isomers" refers to diastereomers resulting from hindered rotation about a double bond or a cycloalkyl linker (e.g., 1,3-cyclobutyl). These configurations are distinguished in names by the prefixes cis and trans or Z and E, which indicate that the groups are on the same or opposite sides of a double bond in a molecule according to the Cahn-Ingold-Prelog rules.

[0206] It is to be understood that the compounds of the present disclosure may be represented as different chiral or geometric isomers. It is also to be understood that when a compound has chiral or geometric isomers, all isomers are intended to be included within the scope of the present disclosure, and the naming of the compound does not exclude any isomers (it is understood that not all isomers have the same level of activity).

[0207] It is understood that the structures and other compounds discussed in this disclosure include all atropic isomers thereof. It is also understood that not all atropic isomers have the same level of activity.

[0208] As used herein, the term "atropic isomer" refers to a type of stereoisomer in which the atoms of the two isomers are arranged differently in space. Atropic isomers arise from restricted rotation caused by the prevention of rotation of large groups around a central bond. Such atropic isomers typically exist as mixtures, but recent advances in chromatographic techniques have made it possible to separate mixtures of two atropic isomers in selective cases.

[0209] As used herein, the term "tautomer" refers to one of two or more structural isomers that exist in equilibrium and are easily converted from one isomer to another. This conversion involves the formal migration of a hydrogen atom by switching adjacent conjugated double bonds. Tautomers exist as a mixture of a set of tautomers in solution. In solutions where tautomerization is possible, a chemical equilibrium of the tautomers is achieved. The exact ratio of tautomers depends on several factors, including temperature, solvent, and pH. The concept of tautomers that are interconvertible by tautomerization is called tautomerism. Of the various types of tautomerism that are possible, two are commonly observed. In keto-enol tautomerism, a simultaneous shift of electrons and hydrogen atoms occurs. Ring-chain tautomerism occurs as a result of an aldehyde group (-CHO) in a sugar molecule reacting with one of the hydroxyl groups (-OH) in the same molecule, giving the cyclic (ring-shaped) form exhibited by glucose.

[0210] It should be understood that the compounds of the present disclosure may be represented as different tautomers. When a compound has tautomers, it should also be understood that all tautomers are intended to be included within the scope of the present disclosure, and that the naming of the compound does not exclude any tautomers. It will be understood that certain tautomers may have a higher level of activity than others.

[0211] It should be understood that the compounds of any formula described herein include the compounds themselves as well as their salts and solvates (where applicable). Salts can be formed, for example, between an anion and a positively charged group (e.g., amino) on the substituted compounds disclosed herein. Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and acetate (e.g., trifluoroacetate).

[0212] As used herein, the term "pharmaceutically acceptable anion" refers to an anion suitable for forming a pharmaceutically acceptable salt. Similarly, salts can also be formed between a cation and a negatively charged group (e.g., carboxylate) on the substituted compounds disclosed herein. Suitable cations include sodium, potassium, magnesium, calcium, and ammonium cations, such as tetramethylammonium and diethylamine. The substituted compounds disclosed herein also include those salts that contain a quaternary nitrogen atom.

[0213] It is understood that the compounds of the present disclosure, for example, salts of the compounds, can exist in hydrated or non-hydrated (anhydrous) form, or as solvates with other solvent molecules. Non-limiting examples of hydrates include monohydrates, dihydrates, etc. Non-limiting examples of solvates include ethanol solvates, acetone solvates, etc.

[0214] As used herein, the term "solvate" refers to a solvent addition form that contains a stoichiometric or non-stoichiometric amount of solvent. Some compounds have a tendency to trap a certain molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. When the solvent is water, the solvate formed is a hydrate, and when the solvent is alcohol, the solvate formed is an alcoholate. A hydrate is formed by the combination of one or more water molecules with one molecule of a substance in which the water retains its molecular state as H2O.

[0215] As used herein, the term "analog" refers to compounds that are structurally similar to each other but have slightly different compositions (such as when one atom is replaced by an atom of a different element, or when a particular functional group is present, or when one functional group is replaced by another). Thus, an analog is a compound that is similar or comparable to a reference compound in function and appearance, but not in terms of structural origin.

[0216] As used herein, the term "derivative" refers to compounds that have a common core structure and are substituted with various groups as described herein.

[0217] As used herein, the term "bioisomer" refers to a compound obtained by exchanging an atom or group of atoms with another broadly similar atom or group of atoms. The purpose of bioisosteric replacement is to create a new compound with similar biological properties as the parent compound. Bioisosteric replacement may be physicochemically or topologically based. Examples of bioisosteres of carboxylic acids include, but are not limited to, acylsulfonamides, tetrazoles, sulfonates, and phosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96, 3147-3176, 1996.

[0218] It is also understood that any one of the specific compounds of the formulas disclosed herein may exist in solvated and unsolvated forms, such as, for example, hydrated forms. Suitable pharma-ceutically acceptable solvates are, for example, hydrates, such as hemihydrates, monohydrates, dihydrates, or trihydrates. It is understood that the present disclosure encompasses all such solvated forms that have inflammasome inhibitory activity.

[0219] It should also be understood that any one particular compound of the formula disclosed herein may exhibit polymorphism, and the present disclosure encompasses all such forms or mixtures thereof that have inflammasome inhibitory activity.In general, it is known that crystalline materials can be analyzed using conventional techniques such as X-ray powder diffraction analysis, differential scanning calorimetry, thermogravimetry, diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, near infrared (NIR) spectroscopy, solution and / or solid-state nuclear magnetic resonance spectroscopy.The moisture content of such crystalline materials can be measured by Karl Fischer analysis.

[0220] Compounds of any one of the formulas disclosed herein may exist in a number of different tautomeric forms, and reference herein to any one of the compounds of the formulas includes all such forms. For the avoidance of doubt, compounds may exist in one of several tautomeric forms, and even if only one of them is specifically described or shown, all others are included in the formulas disclosed herein. Examples of tautomers include keto, enol, and enolate forms, such as in the tautomeric pairs: keto / enol (illustrated below), imine / enamine, amide / iminoalcohol, amidine / amidine, nitroso / oxime, thioketone / enethiol, nitro / acinitoro. [ka]

[0221] Compounds of any one of the formulas disclosed herein that contain an amine function may also form N-oxides. Reference herein to compounds of any one of the formulas disclosed herein that contain an amine function also includes N-oxides. When a compound contains several amine functions, one or more nitrogen atoms may be oxidized to form N-oxides. Specific examples of N-oxides include N-oxides of tertiary amines or nitrogen atoms of nitrogen-containing heterocycles. N-oxides can be formed by treating the corresponding amine with an oxidizing agent such as hydrogen peroxide or a peracid (e.g., a percarboxylic acid). See, for example, Advanced Organic Chemistry, by Jerry March, 4th Edition, Wiley Interscience, page). More specifically, N-oxides can be made by the procedure of LW Deady (Syn. Comm. 1977, 7, 509-514), in which an amine compound is reacted with metachloroperbenzoic acid (mCPBA) in an inert solvent such as dichloromethane.

[0222] The compound of any one of the formulas disclosed herein may be administered in the form of a prodrug that is broken down in the human or animal body to release the compound of the present disclosure. Prodrugs may be used to change the physical properties and / or pharmacokinetic properties of the compound of the present disclosure. Prodrugs can be formed when the compound of the present disclosure contains a suitable group or substituent to which a property-modifying group can be attached.

[0223] Thus, the present disclosure includes any one of the compounds of the formulae disclosed herein as defined above when they are made available by organic synthesis and when they are made available in the human or animal body by cleavage of their prodrugs. Thus, the present disclosure includes any one of the compounds of the formulae disclosed herein produced by organic synthesis means and compounds produced in the human or animal body by metabolism of precursor compounds, and any one of the compounds of the formulae disclosed herein may be synthetically produced compounds or metabolically produced compounds.

[0224] Suitable pharma- ceutically acceptable prodrugs of a compound of any one of the formulae disclosed herein are those that are based on sound medical judgment that are free of undesirable pharmacological activity and are suitable for administration to the human or animal body without undue toxicity. Various forms of prodrugs are described, for example, in the following documents: a) Methods in Enzymology, Vol. 42, p. 309-396, edited by K. Widder, et al. (Academic Press, 1985); b) Design of Pro-drugs, edited by H. Bundgaard, (Elsevier, 1985); c) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H. Bundgaard, Chapter 5 "Design and Application of Pro-drugs", by H. Bundgaard p. 113-191 (1991); d) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992); e) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77, 285 (1988); f) N. Kakeya, et al., Chem. Pharm. Bull., 32, 692 (1984); g) T. Higuchi and V. Stella, “Pro-Drugs as Novel Delivery Systems”, ACS Symposium Series, Volume 14; and h) E. Roche (editor), “Bioreversible Carriers in Drug Design”, Pergamon Press, 1987.

[0225] The in vivo effects of a compound of any one of the formulae disclosed herein may be exerted in part by one or more metabolites formed in the human or animal body following administration of a compound of any one of the formulae disclosed herein. As mentioned above, the in vivo effects of a compound of any one of the formulae disclosed herein may also be exerted by metabolism of a precursor compound (prodrug).

[0226] Suitably, the present disclosure excludes any individual compound that does not have biological activity as defined herein.

[0227] Nucleic acid agents containing 5' terminal units In some embodiments, the disclosure provides a nucleic acid agent, or a pharma- ceutically acceptable salt thereof, wherein the nucleic acid agent comprises an oligonucleotide comprising one or two 5' terminal units covalently attached to the oligonucleotide, each 5' terminal unit independently comprising: [ka] where the variables B, R 1 , R 2 , R 3 , R 4 , R 6 wherein, X, and Z are described herein and ## indicates the bond to the remainder of the oligonucleotide, or a pharma- ceutically acceptable salt thereof.

[0228] In some embodiments, the nucleic acid agent comprises a single-stranded RNA (eg, a single-stranded siRNA).

[0229] In some embodiments, the nucleic acid agent comprises a single-stranded RNA (e.g., a single-stranded siRNA) in which one 5' terminal unit is covalently linked at the 5' terminal position of the single-stranded RNA (e.g., the single-stranded siRNA).

[0230] In some embodiments, the nucleic acid agent comprises double-stranded RNA (eg, double-stranded siRNA).

[0231] In some embodiments, a nucleic acid agent comprises a double-stranded RNA (eg, a double-stranded siRNA) and at least one 5' end unit.

[0232] In some embodiments, a nucleic acid agent comprises a double-stranded RNA (eg, a double-stranded siRNA) and one or two 5' end units.

[0233] In some embodiments, the nucleic acid agent comprises a double-stranded RNA (e.g., a double-stranded siRNA) and one or two 5' end units, the 5'-terminal unit is attached (e.g., at the 5'-terminal position) to the sense strand of a double-stranded RNA (e.g., a double-stranded siRNA); and / or The 5' terminal unit is attached (eg, at the 5' terminal position) to the antisense strand of a double-stranded RNA (eg, a double-stranded siRNA).

[0234] In some embodiments, the nucleic acid agent comprises a double-stranded RNA (e.g., a double-stranded RNA (e.g., a double-stranded siRNA) and a single 5' terminal unit, where the 5' terminal unit is attached to (e.g., at the 5' terminal position) the sense strand of the double-stranded RNA (e.g., the double-stranded siRNA).

[0235] In some embodiments, the nucleic acid agent comprises a double-stranded RNA (e.g., a double-stranded siRNA) and one 5' terminal unit, where the 5' terminal unit is attached to the antisense strand of the double-stranded RNA (e.g., the double-stranded siRNA) (e.g., at the 5' terminal position).

[0236] In some embodiments, the nucleic acid agent comprises a double-stranded RNA (e.g., a double-stranded siRNA) and two 5' end units, one 5' terminal unit is attached (e.g., at the 5' terminal position) to the sense strand of a double-stranded RNA (e.g., a double-stranded siRNA); The other 5' terminal unit is attached (eg, at the 5' terminal position) to the antisense strand of a double-stranded RNA (eg, a double-stranded siRNA).

[0237] In some embodiments, the 5' end unit in a nucleic acid agent is [ka] or a pharma- ceutically acceptable salt thereof, wherein: Z is -P(R Z )2, -P(OR Z )(N(R Z )2), -P(=O)(OR Z )R Z , -P(=S)(OR Z )R Z , -P(=O)(SR Z )R Z , -P(=S)(SR Z )R Z , -P(=O)(OR Z )2, -P(=S)(OR Z )2, -P(=O)(SR Z )2, or -P(=S)(SR Z )2, Each R Z is independently H or C1-C6 alkyl optionally substituted with one or more halogen or cyano.

[0238] In some embodiments, the 5' end unit in a nucleic acid agent is [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or a pharma- ceutically acceptable salt thereof, wherein B is adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U).

[0239] In some embodiments, the 5' end unit in a nucleic acid agent is selected from the 5' end units set forth in Table N. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9]

[0240] Conjugates containing 5' end units As used herein, the term "conjugate" refers to a compound or complex that includes one or more ligands and a nucleic acid agent covalently attached to one or two 5'-end units of the disclosure.

[0241] In some embodiments, the present disclosure provides a conjugate or a pharma- ceutically acceptable salt thereof, the conjugate comprising: (i) a nucleic acid agent comprising an oligonucleotide comprising one or two 5' terminal units covalently attached to the oligonucleotide, wherein each 5' terminal unit is independently [ka] where the variables B, R 1 , R 2 , R 3 , R 4 , R 6 , X, and Z are as described herein, and ## indicates the attachment to the remainder of the oligonucleotide; and (ii) one or more ligands covalently attached to the nucleic acid agent or a pharma- ceutically acceptable salt thereof.

[0242] In some embodiments, the conjugate comprises a single stranded RNA (eg, a single stranded siRNA), one or more ligands, and a 5' end unit.

[0243] In some embodiments, the conjugate comprises a single-stranded RNA (e.g., a single-stranded siRNA), one or more ligands, and one 5'-terminal unit, where the 5'-terminal unit is covalently linked at the 5'-terminal position of the single-stranded RNA (e.g., a single-stranded siRNA).

[0244] In some embodiments, the conjugate comprises a double-stranded RNA (eg, a double-stranded siRNA), one or more ligands, and at least one 5' end unit.

[0245] In some embodiments, the conjugate comprises a double-stranded RNA (eg, a double-stranded siRNA), one or more ligands, and one or two 5'-end units.

[0246] In some embodiments, the conjugate comprises a double-stranded RNA (e.g., a double-stranded siRNA), one or more ligands, and one or two 5' end units; the 5'-terminal unit is attached to the sense strand of a double-stranded RNA (e.g., a double-stranded siRNA) (e.g., at the 5'-terminal position); and / or The 5' terminal unit is attached (eg, at the 5' terminal position) to the antisense strand of a double-stranded RNA (eg, a double-stranded siRNA).

[0247] In some embodiments, the conjugate comprises a double-stranded RNA (e.g., a double-stranded siRNA), one or more ligands, and a 5'-end unit, wherein the 5'-end unit is attached to (e.g., at the 5'-end position) the sense strand of the double-stranded RNA (e.g., the double-stranded siRNA).

[0248] In some embodiments, the conjugate comprises a double-stranded RNA (e.g., a double-stranded siRNA), one or more ligands, and a 5'-end unit, where the 5'-end unit is attached to (e.g., at the 5'-end position) the antisense strand of the double-stranded RNA (e.g., the double-stranded siRNA).

[0249] In some embodiments, the conjugate comprises a double-stranded RNA (e.g., a double-stranded siRNA), one or more ligands, and two 5' end units; one 5' terminal unit is attached (e.g., at the 5' terminal position) to the sense strand of a double-stranded RNA (e.g., a double-stranded siRNA); The other 5' terminal unit is attached (eg, at the 5' terminal position) to the antisense strand of a double-stranded RNA (eg, a double-stranded siRNA).

[0250] In some embodiments, the 5' end unit in the conjugate is [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or a pharma- ceutically acceptable salt thereof, wherein B is adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U).

[0251] In some embodiments, the 5' end unit in the conjugate is selected from the 5' end units listed in Table C. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9]

[0252] 5' end unit As used herein, "5'-End Unit" or "5'-end unit" refers to the portion corresponding to a phosphomimetic derivative in which Y is replaced with a bond to a nucleic acid agent.

[0253] In some embodiments, the 5' end unit is of formula (I) where Y is replaced with a linkage to a nucleic acid agent.

[0254] In some embodiments, the 5' end unit is of formula (I'-1), (I'-2), (II'-1), or (II'-2), where Y is replaced with a linkage to a nucleic acid agent.

[0255] In some embodiments, the 5' end unit is of formula (IA) or (II-A), where Y is replaced with a linkage to a nucleic acid agent.

[0256] In some embodiments, the 5' end unit is of formula (I-A'-1), (I-A'-2), (II-A'-1), or (II-A'-2), where Y is replaced with a linkage to a nucleic acid agent.

[0257] In some embodiments, the 5' end unit is of formula (IB-1), (IB-2), (II-B-1), or (II-B-2), where Y is replaced with a linkage to a nucleic acid agent.

[0258] In some embodiments, the 5' end unit is of formula (I-B'-1), (I-B'-2), (I-B'-3), (I-B'-4), (II-B'-1), (II-B'-2), (II-B'-3), or (II-B'-4), where Y is replaced with a linkage to the nucleic acid agent.

[0259] In some embodiments, the 5' terminal unit prior to attachment is a phosphomimetic derivative as described herein.

[0260] In some embodiments, the 5' terminal unit prior to attachment is a compound of formula (I) or a pharma- ceutically acceptable salt thereof.

[0261] In some embodiments, the 5' terminal unit prior to attachment is a compound of formula (I'-1), (I'-2), (II'-1), or (II'-2), or a pharma- ceutically acceptable salt thereof.

[0262] In some embodiments, the 5' terminal unit prior to conjugation is a compound of formula (IA) or (II-A), or a pharma- ceutically acceptable salt thereof.

[0263] In some embodiments, the 5' terminal unit prior to conjugation is a compound of formula (I-A'-1), (I-A'-2), (II-A'-1), or (II-A'-2), or a pharma- ceutically acceptable salt thereof.

[0264] In some embodiments, the 5' terminal unit prior to attachment is a compound of formula (IB-1), (IB-2), (II-B-1), or (II-B-2), or a pharma- ceutically acceptable salt thereof.

[0265] In some embodiments, the 5' terminal unit prior to conjugation is a compound of formula (I-B'-1), (I-B'-2), (I-B'-3), (I-B'-4), (II-B'-1), (II-B'-2), (II-B'-3), or (II-B'-4), or a pharma- ceutically acceptable salt thereof.

[0266] In some embodiments, the 5' terminal unit prior to conjugation is a compound selected from the compounds set forth in Table E and pharma- ceutically acceptable salts thereof.

[0267] Ligand As used herein, the term "ligand" refers to a moiety that, when covalently attached to a nucleic acid agent (e.g., an oligonucleotide), is capable of mediating entry into or facilitating delivery to a target site (e.g., a target cell or tissue).

[0268] In some embodiments, the ligand comprises a sugar ligand moiety (eg, N-acetylgalactosamine (GalNAc)) that can direct uptake of the oligonucleotide to the liver.

[0269] In some embodiments, the ligand binds to the asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand binds to the liver (e.g., via the ASGPR), such as to liver parenchymal cells.

[0270] Suitable ligands include, but are not limited to, those described in Winkler (Ther. Deliv., 2013, 4(7):791-809), PCT Patent Application Publication Nos. WO / 2016 / 100401, WO / 2012 / 089352, and WO / 2009 / 082607, and U.S. Patent Application Publication Nos. 2009 / 0239814, 2012 / 0136042, 2013 / 0158824, and 2009 / 0247608, each of which is incorporated by reference.

[0271] In some embodiments, the ligand comprises a carbohydrate moiety.

[0272] As used herein, a "carbohydrate moiety" refers to a moiety that includes one or more monosaccharide units (which may be linear, branched, or cyclic), each having at least six carbon atoms and having an oxygen, nitrogen, or sulfur atom attached to each carbon atom. In some embodiments, the carbohydrate moiety includes a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide. In some embodiments, the carbohydrate moiety includes an oligosaccharide including about 4-9 monosaccharide units. In some embodiments, the carbohydrate moiety includes a polysaccharide (e.g., starch, glycogen, cellulose, or a polysaccharide gum).

[0273] In some embodiments, the carbohydrate moiety comprises a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide.

[0274] In some embodiments, the carbohydrate moiety comprises an oligosaccharide (eg, containing from about 4 to about 9 monosaccharide units).

[0275] In some embodiments, the carbohydrate moiety comprises a polysaccharide (eg, starch, glycogen, cellulose, or a polysaccharide gum).

[0276] In some embodiments, the ligand is capable of binding to a human asialoglycoprotein receptor (ASGPR), for example, human asialoglycoprotein receptor 2 (ASGPR2).

[0277] In some embodiments, the carbohydrate moiety comprises a sugar (eg, one, two, or three sugars).

[0278] In some embodiments, the carbohydrate moiety comprises galactose or a derivative thereof (eg, one, two, or three galactose or a derivative thereof).

[0279] In some embodiments, the carbohydrate moiety comprises N-acetylgalactosamine or a derivative thereof (eg, one, two, or three N-acetylgalactosamines or derivatives thereof).

[0280] In some embodiments, the carbohydrate moiety comprises an N-acetyl-D-galactosylamine or derivative thereof (eg, one, two, or three N-acetyl-D-galactosylamines or derivatives thereof).

[0281] In some embodiments, the carbohydrate moiety comprises an N-acetylgalactosamine (eg, one, two, or three N-acetylgalactosamines).

[0282] In some embodiments, the carbohydrate moiety comprises an N-acetyl-D-galactosylamine (eg, one, two, or three N-acetyl-D-galactosylamines).

[0283] In some embodiments, the carbohydrate moiety comprises mannose or a derivative thereof (eg, mannose-6-phosphate).

[0284] In some embodiments, the carbohydrate moiety further comprises a linking moiety that attaches one or more sugars (eg, N-acetyl-D-galactosylamine) to the 5' terminal unit.

[0285] In some embodiments, the linking moiety comprises a thioether (eg, thiosuccinimide or its hydrolyzed analogues), a disulfide, a triazole, a phosphorothioate, a phosphodiester, an ester, an amide, or any combination thereof.

[0286] In some embodiments, the linking moiety is a three-arm linking moiety.

[0287] Suitable ligands include, but are not limited to, those disclosed in PCT Publication Nos. WO / 2015 / 006740, WO / 2016 / 100401, WO / 2017 / 214112, WO / 2018 / 039364, and WO / 2018 / 045317, each of which is incorporated herein by reference.

[0288] In some embodiments, the ligand is [ka] (e.g., one, two, or three [ka] ).

[0289] In some embodiments, the ligand is [ka] (e.g., one, two, or three [ka] ).

[0290] In some embodiments, the ligand is [ka] (e.g., one, two, or three [ka] ).

[0291] In some embodiments, the ligand is [ka] (e.g., one, two, or three [ka] ).

[0292] In some embodiments, the ligand is [ka] (e.g., one, two, or three [ka] ).

[0293] In some embodiments, the ligand is [ka] (e.g., one, two, or three [ka] ).

[0294] In some embodiments, the ligand is [ka] (e.g., one, two, or three [ka] ).

[0295] In some embodiments, the ligand is [ka] (e.g., one, two, or three [ka] ).

[0296] In some embodiments, the ligand is [ka] Includes.

[0297] In some embodiments, the ligand comprises: [ka] Includes.

[0298] In some embodiments, the ligand is [ka] Includes.

[0299] In some embodiments, the ligand is [ka] Includes.

[0300] In some embodiments, the ligand is [ka] Includes.

[0301] In some embodiments, the ligand is [ka] Includes.

[0302] In some embodiments, the ligand is [ka] Includes.

[0303] In some embodiments, the ligand is [ka] Includes.

[0304] In some embodiments, the ligand comprises a lipid moiety (eg, one, two, or three lipid moieties).

[0305] In some embodiments, the lipid moiety is C-C 24 The fatty acids may include fatty acids, cholesterol, vitamins, sterols, phospholipids (eg, one, two, or three of them), or any combination thereof.

[0306] In some embodiments, the ligand comprises a peptide moiety (eg, one, two, or three peptide moieties).

[0307] In some embodiments, the peptide moiety comprises an integrin, an insulin, a glucagon-like peptide (eg, one, two, or three of them), or any combination thereof.

[0308] In some embodiments, the ligand comprises an antibody moiety (eg, transferrin).

[0309] In some embodiments, the ligand comprises one, two, or three antibody moieties (eg, transferrin).

[0310] In some embodiments, the ligand comprises an oligonucleotide (eg, an aptamer or CpG).

[0311] In some embodiments, the ligand comprises one, two, or three oligonucleotides (eg, an aptamer or CpG).

[0312] In some embodiments, the ligand is one, two, or three sugars (e.g., N-acetyl-D-galactosylamine); 1, 2 or 3 lipid moieties, 1, 2 or 3 peptide moieties, 1, 2, or 3 antibody moieties, 1, 2, or 3 oligonucleotides, or any combination thereof Includes.

[0313] Other aspects of nucleic acid agents In some embodiments, the nucleic acid agent is linked to a ligand (eg, GalNAc).

[0314] In some embodiments, the nucleic acid agent is linked to the ligand via an internal or terminal nucleotide of the nucleic acid agent.

[0315] In some embodiments, the nucleic acid agent comprises an oligonucleotide.

[0316] In some embodiments, a nucleic acid agent (eg, an oligonucleotide) includes one or more phosphate groups or one or more phosphate group analogs.

[0317] In some embodiments, the 5' terminal unit is attached to the oligonucleotide via a phosphate group or a phosphate group analog in the nucleic acid agent.

[0318] In some embodiments, the oligonucleotide has a length of from 1 to 100 nucleotides, from 1 to 80 nucleotides, from 1 to 60 nucleotides, or from 1 to 50 nucleotides.

[0319] In some embodiments, the oligonucleotides are 1-40 nucleotides, 10-40 nucleotides, 12-35 nucleotides, 15-30 nucleotides, 18-25 nucleotides, or 20-23 nucleotides in length. In some embodiments, the oligonucleotides are 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the oligonucleotides are 19, 20, 21, 22, or 23 nucleotides in length.

[0320] In some embodiments, the nucleic acid agent comprises RNA, DNA, or a mixture thereof.

[0321] In some embodiments, the oligonucleotide comprises RNA, DNA, or a mixture thereof.

[0322] In some embodiments, the nucleic acid agent comprises RNA.

[0323] In some embodiments, the oligonucleotide is RNA.

[0324] In some embodiments, the oligonucleotide is an siRNA (e.g., a single stranded siRNA (e.g., a hairpin single stranded siRNA) or a double stranded siRNA), a microRNA, an anti-microRNA, a microRNA mimic, an anti-miR, an antagomir, a dsRNA, a ssRNA, an aptamer, an immunostimulatory oligonucleotide, a decoy oligonucleotide, a splice altering oligonucleotide, a triplex forming oligonucleotide, a G-quadruplex, or an antisense oligonucleotide.

[0325] In some embodiments, the nucleic acid agent comprises double-stranded RNA (dsRNA), the double-stranded RNA comprising a sense strand and an antisense strand as described herein.

[0326] In some embodiments, the oligonucleotide is a double-stranded RNA (ds-RNA), where the double-stranded RNA comprises a sense strand and an antisense strand as described herein.

[0327] In some embodiments, a nucleic acid agent comprises a double-stranded siRNA (ds-siRNA), the double-stranded siRNA comprising a sense strand and an antisense strand as described herein.

[0328] In some embodiments, the oligonucleotide is a double-stranded siRNA (ds-siRNA), wherein the double-stranded siRNA comprises a sense strand and an antisense strand as described herein.

[0329] The sense strand is also known as the passenger strand, and it is understood that the terms "sense strand" and "passenger strand" are used interchangeably herein.

[0330] The antisense strand is also known as the guide strand, and it is understood that the terms "antisense strand" and "guide strand" are used interchangeably herein.

[0331] In some embodiments, the oligonucleotide is an iRNA.

[0332] The term "iRNA" refers to an RNA agent that can downregulate the expression of a target gene (e.g., siRNA), e.g., an endogenous or pathogen target RNA. Without wishing to be bound by theory, iRNAs can act by one or more of a number of mechanisms, including post-transcriptional cleavage of the target mRNA (referred to in the art as RNAi) or pre-transcriptional or pre-translational mechanisms. iRNAs can include a single strand or can include two or more strands, e.g., double-stranded iRNAs. When an iRNA is single-stranded, the iRNA can include a 5' modification, including one or more phosphate groups or one or more phosphate group analogs. In some embodiments, an iRNA is double-stranded. In some embodiments, one or both strands of a double-stranded iRNA can be modified, e.g., 5' modified.

[0333] An iRNA typically comprises a region of sufficient homology with a target gene and is of sufficient length, in terms of nucleotides, so that the iRNA or a fragment thereof can mediate downregulation of the target gene. An iRNA is or comprises a region that is at least partially, and in some embodiments completely, complementary to a target RNA. There need not be perfect complementarity between the iRNA and the target, but the correspondence can be sufficient so that the iRNA or its cleavage product can induce sequence-specific silencing, for example, by RNAi cleavage of the target RNA (e.g., mRNA).

[0334] Nucleotides in an iRNA may be modified (e.g., one or more nucleotides include a 2'-F or 2'-OCH3 group or may be a nucleotide surrogate). Single-stranded or double-stranded regions of an iRNA may be modified or include nucleotide surrogates, e.g., an unpaired region or a hairpin structure region (e.g., a region connecting two complementary regions) may have a modification or nucleotide surrogate. Modifications to stabilize one or more of the 3' or 5' ends of an iRNA, e.g., against exonucleases. Modifications include C3 (or C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C210, C22, C23, C24, C25, C26, C27, C28, C29, C30, C31, C32, C33, C34, C35, C36, C37, C38, C39, C40, C41, C42, C43, C44, C45, C46, ​​C47, C48, C49, C41, C42, C43, C44, C45, C46, ​​C47, C48, C49, C50, C51, C52, C53, C54, C55, C56, C57, C58, C59, C60, C61, C62, C63, C64, C65, C66, C77, C78, ​​C79, C81, C82, C83, C84, C85, C86, C87, C88, C89, C91, C92, C93, C94, C95, C96 12) Amino linkers, thiol linkers, carboxyl linkers, non-nucleotide spacers (C3, C6, C9, C 12 , abasic, triethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents that are provided as phosphoramidites and have an additional DMT-protected hydroxyl group, which allows for multiple couplings during RNA synthesis. Modifications can also include, for example, the use of modifications at the 2'OH group of the ribose sugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine, instead of ribonucleotides, and modifications at the phosphate group, e.g., phosphothioate modifications. In some embodiments, different strands contain different modifications.

[0335] In some embodiments, the strands are selected such that the iRNA comprises a single stranded or unpaired region at one or both ends of the molecule. A double stranded iRNA may have an overhang, e.g., one or two 5' or 3' overhangs (e.g., a 3' overhang of at least 2-3 nucleotides). In some embodiments, the iRNA has an overhang, e.g., a 3' overhang, at least 2 (e.g., 2 or 3) nucleotides in length at each end. The overhangs may be the result of one strand being longer than the other, or the result of two strands of the same length being staggered.

[0336] In some embodiments, the length of the duplex region between the strands of the iRNA is between 6 and 30 nucleotides in length. In some embodiments, the duplex region is between 15 and 30, most preferably 18, 19, 20, 21, 22, and 23 nucleotides in length. In some embodiments, the duplex region is between 6 and 20 nucleotides in length, most preferably 6, 7, 8, 9, 10, 11, and 12 nucleotides in length.

[0337] The oligonucleotides may be those described in U.S. Patent Publication Nos. 2009 / 0239814, 2012 / 0136042, 2013 / 0158824, or 2009 / 0247608, which are incorporated herein by reference.

[0338] In some embodiments, the oligonucleotide is a siRNA.

[0339] In some embodiments, the oligonucleotide is a single stranded siRNA.

[0340] In some embodiments, the oligonucleotide is a double-stranded siRNA, eg, a double-stranded siRNA described herein.

[0341] As used herein, a "single-stranded siRNA" refers to an siRNA that is composed of a single strand and contains a duplex region formed by intrastrand pairing, and may be, for example, a hairpin structure or a panhandle structure. A single-stranded siRNA may be antisense to a target molecule.

[0342] Single-stranded siRNA may be long enough to enter RISC and participate in the cleavage of target mRNA via RISC.Single-stranded siRNA is at least 14 nucleotides long, and in some embodiments, at least 15, 20, 25, 29, 35, 40, or 50 nucleotides long.In some embodiments, it is less than 200, 100, 80, 60, 50, 40, or 30 nucleotides long.

[0343] In some embodiments, the single stranded siRNA has a length of 10-40 nucleotides, 12-35 nucleotides, 15-30 nucleotides, 18-25 nucleotides, or 20-23 nucleotides. In some embodiments, the single stranded siRNA has a length of 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the single stranded siRNA has a length of 20, 21, 22, or 23 nucleotides.

[0344] The hairpin siRNA may have a duplex region equal to or at least 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region may be less than 200, 100, or 50 nucleotide pairs in length. In some embodiments, the duplex region ranges from 15-30, 17-23, 19-23, and 19-21 nucleotide pairs in length. The hairpin may have a single-stranded overhang or terminal unpaired region. In some embodiments, the overhang is at least 2 (e.g., 2 or 3) nucleotides in length. In some embodiments, the overhang is on the sense side of the hairpin, and in some embodiments, on the antisense side of the hairpin.

[0345] In some embodiments, the oligonucleotide is a double-stranded siRNA.

[0346] As used herein, a "double-stranded siRNA" is an siRNA that contains two or more strands, and in some cases contains two strands, that are capable of forming a duplex structural region by interstrand hybridization.

[0347] In some embodiments, the sense strand of the double stranded siRNA may be equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotides in length. It may be equal to or less than 200, 100, or 50 nucleotides in length. The range may be 13-36, 17-25, 19-23, 19-21, 21-23, or 20-22 nucleotides in length.

[0348] In some embodiments, the sense strand is 10-40 nucleotides, 12-35 nucleotides, 13-36 nucleotides, 15-30 nucleotides, 18-25 nucleotides, or 20-23 nucleotides in length. In some embodiments, the sense strand is 13-36 nucleotides in length. In some embodiments, the sense strand is 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the sense strand is 20, 21, 22, or 23 nucleotides in length.

[0349] In some embodiments, the sense strand has a length of 18, 19, 20, 21, or 22 nucleotides.In some embodiments, the sense strand has a length of 20, 21, or 22 nucleotides.

[0350] In some embodiments, the antisense strand of the double stranded siRNA may be equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotides in length. It may be equal to or less than 200, 100, or 50 nucleotides in length. The range may be 17-25, 18-31, 19-23, 19-21, 21-23, or 20-22 nucleotides in length.

[0351] In some embodiments, the antisense strand is 10-40 nucleotides, 12-35 nucleotides, 15-30 nucleotides, 18-31 nucleotides, 18-25 nucleotides, or 20-23 nucleotides in length. In some embodiments, the antisense strand is 18-31 nucleotides in length. In some embodiments, the antisense strand is 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the antisense strand is 20, 21, 22, or 23 nucleotides in length.

[0352] In some embodiments, the antisense strand has a length of 20, 21, 22, 23, or 24 nucleotides.In some embodiments, the antisense strand has a length of 21, 22, or 23 nucleotides.

[0353] In some embodiments, the sense strand has a length of 13-36 nucleotides and the antisense strand has a length of 18-31 nucleotides. In some embodiments, the sense strand has a length of 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides and the antisense strand has a length of 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the sense strand has a length of 18, 19, 20, 21, or 22 nucleotides and the antisense strand has a length of 20, 21, 22, 23, or 24 nucleotides. In some embodiments, the sense strand has a length of 20, 21, or 22 nucleotides and the antisense strand has a length of 21, 22, or 23 nucleotides.

[0354] In some embodiments, the sense strand has a length of 18 nucleotides and the antisense strand has a length of 20 nucleotides.

[0355] In some embodiments, the sense strand has a length of 19 nucleotides and the antisense strand has a length of 21 nucleotides.

[0356] In some embodiments, the sense strand has a length of 20 nucleotides and the antisense strand has a length of 22 nucleotides.

[0357] In some embodiments, the sense strand has a length of 21 nucleotides and the antisense strand has a length of 23 nucleotides.

[0358] In some embodiments, the sense strand has a length of 22 nucleotides and the antisense strand has a length of 24 nucleotides.

[0359] The double-stranded portion of the double-stranded siRNA may be equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotides in length. It may be equal to or less than 200, 100, or 50 nucleotides in length. Ranges may be 15-30, 17-23, 19-23, and 19-21 nucleotides in length.

[0360] In some embodiments, the siRNA is sufficiently large such that it can be cleaved by an endogenous molecule, eg, Dicer, to generate smaller siRNAs, eg, siRNA agents.

[0361] The sense and antisense strands may be selected such that the double-stranded siRNA comprises a single strand or unpaired region at one or both ends of the molecule. Thus, the double-stranded siRNA may comprise a sense and antisense strand paired to comprise an overhang, for example, one or two 5' or 3' overhangs, or a 3' overhang of 1-3 nucleotides. The overhang may be the result of one strand being longer than the other, or may be the result of two strands of the same length being staggered. Some embodiments have at least one 3' overhang. In some embodiments, both ends of the siRNA molecule have a 3' overhang. In some embodiments, the overhang is 2 nucleotides.

[0362] In some embodiments, the length of the duplex region is 15-30, or 18, 19, 20, 21, 22, and 23 nucleotides long, for example, within the range of ssiRNAs discussed above. ssiRNAs can be similar in length and structure to the natural Dicer-processed products derived from long dsiRNAs. Also included are embodiments in which the two strands of the ssiRNA are linked, for example, covalently linked. Hairpins or other single-stranded structures that provide the necessary duplex region, and 3' overhangs are also contemplated.

[0363] In some embodiments, the siRNA is a dsRNA.

[0364] In some embodiments, the dsRNA comprises a sense strand having a length of 13-36 nucleotides, 15-30 nucleotides, 18-25 nucleotides, or 20-22 nucleotides.

[0365] In some embodiments, the dsRNA comprises an antisense strand having a length of 18-31 nucleotides, 19-27 nucleotides, 20-24 nucleotides, or 21-23 nucleotides.

[0366] In some embodiments, the dsRNA comprises a sense strand having a length of 13-36 nucleotides, 15-30 nucleotides, 18-25 nucleotides, or 20-22 nucleotides, and an antisense strand having a length of 18-31 nucleotides, 19-27 nucleotides, 20-24 nucleotides, or 21-23 nucleotides.

[0367] In some embodiments, the dsRNA comprises a sense strand having a length of 20-22 nucleotides and an antisense strand having a length of 21-23 nucleotides.

[0368] In some embodiments, in the dsRNA, the antisense strand has a 3' overhang, eg, a 3' overhang of at least 2 nucleotides.

[0369] In some embodiments, the dsRNA comprises at least one modified phosphate backbone, eg, phosphorothioate, methylphosphonate, or phosphorodithioate.

[0370] In some embodiments, the dsRNA is linked to a ligand (eg, GalNAc) via an internal or terminal nucleotide of the dsRNA.

[0371] The siRNA described herein (including double-stranded siRNA and single-stranded siRNA) can mediate the silencing of target RNA, for example, mRNA, for example, the transcript of a gene coding for a protein. For convenience, such mRNA is also referred to herein as the mRNA to be silenced. Such gene is also referred to as a target gene. Generally, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNA other than mRNA, for example, tRNA and viral RNA, can also be targeted.

[0372] As used herein, the phrase "mediate RNAi" refers to the ability to silence target RNA in a sequence-specific manner. Without wishing to be bound by theory, it is believed that silencing uses the RNAi mechanism or process and guide RNA, for example, 21-23 nucleotide ssiRNA.

[0373] In some embodiments, siRNA is "sufficiently complementary" to target RNA, e.g., target mRNA, so that the siRNA silences the production of the protein encoded by the target mRNA. In another embodiment, siRNA is "fully complementary" to target RNA, e.g., target RNA, and siRNA anneals to form a hybrid consisting of Watson-Crick base pairs exclusively in the region of perfect complementarity. "Sufficiently complementary" target RNA can include an internal region (e.g., at least 10 nucleotides) that is completely complementary to the target RNA. Furthermore, in some embodiments, siRNA specifically discriminates between single nucleotide differences. In this case, siRNA mediates RNAi only when perfect complementarity is found in the region of single nucleotide difference (e.g., within 7 nucleotides).

[0374] MicroRNA: MicroRNAs (miRNAs) are a highly conserved class of small RNA molecules that are transcribed from DNA in the genomes of plants and animals but are not translated into proteins. Processed miRNAs are single-stranded RNA molecules of approximately 17-25 nucleotides (nt) that are incorporated into the RNA-induced silencing complex (RISC) and have been identified as important regulators of development, cell proliferation, apoptosis, and differentiation. They are believed to play a role in regulating gene expression by binding to the 3' untranslated region of specific mRNAs. RISC mediates downregulation of gene expression by translational inhibition, transcriptional cleavage, or both. RISC has also been implicated in transcriptional silencing in the nuclei of various eukaryotic organisms.

[0375] The number of miRNA sequences identified to date is large and growing, examples of which can be found, for example, in "miRBase: microRNA sequences, targets and gene nomenclature" Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright A J. NAR, 2006, 34, Database Issue, D140-D144; "The microRNA Registry" Griffiths-Jones S. NAR, 2004, 32, Database Issue, D109-D111.

[0376] Antisense oligonucleotides: In some embodiments, the nucleic acid is an antisense oligonucleotide to a target polynucleotide. The term "antisense oligonucleotide" or simply "antisense" is meant to include oligonucleotides that are complementary to a target polynucleotide sequence. An antisense oligonucleotide is a single strand of DNA or RNA that is complementary to a selected sequence, for example, a target gene mRNA. Antisense oligonucleotides are believed to inhibit gene expression by binding to complementary mRNA. Binding to a target mRNA can cause inhibition of gene expression by binding to it and thereby preventing translation of the complementary mRNA strand or by causing degradation of the target mRNA. Antisense DNA can be used to target a specific complementary (coding or non-coding) RNA. If binding occurs, this DNA / RNA hybrid can be degraded by the enzyme RNase H. In some embodiments, an antisense oligonucleotide comprises about 10 to about 50 nucleotides, more preferably about 15 to about 30 nucleotides. The term also includes antisense oligonucleotides that may not be completely complementary to a desired target gene. Thus, it is contemplated that non-target specific activity may be found using antisense, or that an antisense sequence that contains one or more mismatches with the target sequence may be most preferred for a particular application.

[0377] Antisense oligonucleotides have been shown to be effective and targeted inhibitors of protein synthesis, and can therefore be used to specifically inhibit protein synthesis by target genes.The effectiveness of antisense oligonucleotides for protein synthesis inhibition has been well established.For example, the synthesis of polygalacturonase and muscarinic type 2 acetylcholine receptor is inhibited by antisense oligonucleotides against their respective mRNA sequences (U.S. Patent Nos. 5,739,119 and 5,759,829, each of which is incorporated by reference). Additionally, examples of antisense inhibition have been shown for the nuclear protein cyclin, the multidrug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABAA receptors, and human EGF (Jaskulski et al., Science. 1988 Jun. 10; 240(4858): 1544-6; Vasanthakumar and Ahmed, Cancer Commun. 1989; 1(4): 225-32; Peris et al., Brain Res Mol Brain Res. 1998 Jun. 15; 57(2): 310-20; U.S. Patent Nos. 5,801,154, 5,789,573, 5,718,709, and 5,610,288, each of which is incorporated by reference). Additionally, antisense constructs have been described that can be used to inhibit and treat various abnormal cell proliferations, such as cancer (U.S. Pat. Nos. 5,747,470, 5,591,317, and 5,783,683, each of which is incorporated by reference).

[0378] Methods for producing antisense oligonucleotides are known in the art and can be easily adapted to produce antisense oligonucleotides targeting any polynucleotide sequence. Selection of antisense oligonucleotide sequences specific for a given target sequence is based on analysis of the selected target sequence and determination of secondary structure, Tm, binding energy, and relative stability. Antisense oligonucleotides may be selected based on their relative inability to form dimers, hairpins, or other secondary structures that reduce or inhibit specific binding to the target mRNA in a host cell. Highly preferred target regions of mRNA include regions at or near the AUG translation initiation codon and sequences substantially complementary to the 5' region of the mRNA. These secondary structure analyses and target site selection studies can be performed, for example, using OLIGO primer analysis software version 4 (Molecular Biology Insights) and / or BLASTN2.0.5 algorithm software (Altschul et al., Nucleic Acids Res. 1997, 25(17):3389-402).

[0379] Antagomir: Antagomir is an RNA-like oligonucleotide with various modifications for pharmacological properties such as RNAse protection and improved uptake into tissues and cells. It differs from normal RNA, for example, by complete 2'-O-methylation of sugars, phosphorothioate backbone, and cholesterol moiety, for example, at the 3' end. Antagomir may be used to efficiently silence endogenous miRNA by forming a duplex containing antagomir and endogenous miRNA, thereby preventing miRNA-induced gene silencing. An example of antagomir-mediated miRNA silencing is the silencing of miR-122, described in Krutzfeldt et al, Nature, 2005, 438:685-689, which is expressly incorporated herein by reference in its entirety. AntagomirRNA may be synthesized using standard solid-phase oligonucleotide synthesis protocols. See U.S. Patent Application Publication Nos. 2007 / 0123482 and 2007 / 0213292, each of which is incorporated herein by reference.

[0380] Antagomirs include ligand-binding monomer subunits and monomers for oligonucleotide synthesis. Exemplary monomers are described in US Patent Application Publication No. 2005 / 0107325, which is incorporated by reference in its entirety. Antagomirs can have a ZXY structure, for example, as described in WO2004 / 080406, which is incorporated by reference in its entirety. Antagomirs can form complexes with amphiphilic moieties. Exemplary amphiphilic moieties for use with oligonucleotide agents are described in WO2004 / 080406, which is incorporated by reference in its entirety.

[0381] Aptamers: Aptamers are nucleic acid or peptide molecules that bind to a particular molecule of interest with high affinity and specificity (Tuerk and Gold, Science 249:505 (1990); Ellington and Szostak, Nature 346:818 (1990), which are incorporated by reference in their entireties). DNA or RNA aptamers have been successfully generated that bind many different entities, from large proteins to small organic molecules. See Eaton, Curr. Opin. Chem. Biol. 1:10-16 (1997), Famulok, Curr. Opin. Struct. Biol. 9:324-9 (1999), and Hermann and Patel, Science 287:820-5 (2000), which are incorporated by reference in their entireties. Aptamers may be RNA or DNA based and may include riboswitches. Riboswitches are parts of mRNA molecules that can directly bind to small target molecules and whose binding to the target affects the activity of genes. Thus, mRNAs that contain riboswitches are directly involved in regulating their own activity depending on the presence or absence of the target molecule. In general, aptamers are engineered by repeated rounds of in vitro selection or equivalently SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to bind to various molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. Aptamers may be prepared by any known method, including synthetic, recombinant, and purification methods, and may be used alone or in combination with other aptamers specific for the same target. Additionally, as described more fully herein, the term "aptamer" specifically includes "secondary aptamers" that include consensus sequences obtained by comparing two or more known aptamers to a given target.

[0382] Ribozymes: In another embodiment, the nucleic acid-lipid particle is associated with a ribozyme, which is an RNA molecular complex that contains a specific catalytic domain with endonuclease activity (Kim and Cech, Proc Natl Acad Sci USA. 1987 December; 84(24):8788-92; Forster and Symons, Cell. 1987 April 24; 49(2):211-20). For example, many ribozymes catalyze phosphoester transfer reactions with high specificity, often cleaving only one of several phosphates in an oligonucleotide substrate (Cech et al., Cell. 1981 December;27(3 Pt 2):487-96; Michel and Westhof, J Mol Biol. 1990 Dec.5;216(3):585-610; Reinhold-Hurek and Shub, Nature. 1992 May 14;357(6374):173-6). This specificity results from the requirement that the substrate be bound by specific base-pairing interactions to the ribozyme's internal guide sequence ("IGS") prior to chemical reaction.

[0383] Currently, at least six basic types of naturally occurring enzymatic RNAs are known. Each is capable of catalyzing the hydrolysis of RNA phosphodiester bonds in trans under physiological conditions (and thus capable of cleaving other RNA molecules). In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through a target binding portion of the enzymatic nucleic acid that is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, an enzymatic nucleic acid first recognizes a target RNA, then binds to said target RNA by complementary base pairing, and when bound to the correct site, acts enzymatically to cleave the target RNA. Such strategic cleavage of the target RNA destroys its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to seek another target and can repeatedly bind and cleave new targets.

[0384] The enzymatic nucleic acid molecule may be formed, for example, of a hammerhead, hairpin, Hepatitis delta virus, group I intron, or RNaseP RNA (in conjunction with an RNA guide sequence), or Neurospora VS RNA motif. Specific examples of hammerhead motifs are described in Rossi et al. Nucleic Acids Res. 1992 Sep. 11; 20(17): 4559-65. Examples of hairpin motifs are described in Hampel et al. (European Patent Application No. EP 0360257), Hampel and Tritz, Biochemistry 1989 Jun. 13; 28(12): 4929-33; Hampel et al., Nucleic Acids Res. 1990 Jan. 25; 18(2): 299-304, and U.S. Patent No. 5,631,359. An example of a Hepatitis delta virus motif is described in Perrotta and Been, Biochemistry. 1992 Dec. 1;31(47):11843-52, an example of an RNase P motif is described in Guerrier-Takada et al., Cell. 1983 December;35(3 Pt 2):849-57, a Neurospora VS RNA ribozyme motif is described in Collins (Saville and Collins, Cell. 1990 May 18;61(4):685-96; Saville and Collins, Proc Natl Acad Sci USA. 1991 Oct. 1;88(19):8826-30; Collins and Olive, Biochemistry. 1993 Mar. 23;32(11):2795-9), and an example of a group I intron is described in U.S. Pat. No. 4,987,071. The important characteristics of the enzymatic nucleic acid molecule used are that it has a specific substrate binding site that is complementary to one or more regions of the DNA or RNA of the target gene, and that it has nucleotide sequences within or surrounding that substrate binding site that confer RNA cleavage activity to the molecule. Thus, ribozyme constructs need not be limited to the specific motifs mentioned herein.

[0385] Methods for making ribozymes that target any polynucleotide sequence are known in the art. Ribozymes can be designed as described in International Patent Application Publication Nos. WO93 / 23569 and WO94 / 02595 (each of which is specifically incorporated herein by reference) and synthesized for in vitro and in vivo testing as described therein.

[0386] Ribozyme activity can be optimized by varying the length of the ribozyme binding arms or chemically synthesizing ribozymes with modifications that prevent degradation by serum ribonucleases (see, e.g., International Patent Applications WO 92 / 07065, WO 93 / 15187, and WO 91 / 03162, European Patent Application Publication No. 92110298.4, U.S. Pat. No. 5,334,711, and International Patent Application Publication No. WO 94 / 13688, which describe various chemical modifications that can be made to the sugar moiety of enzymatic RNA molecules), modifications that enhance efficacy in cells, and removal of stem II bases to shorten RNA synthesis times and reduce chemical requirements.

[0387] Immunostimulatory oligonucleotides: The nucleic acids associated with lipid particles include immunostimulatory oligonucleotides (ISS, single-stranded or double-stranded) that are immunostimulatory and can induce an immune response when administered to a subject, which may be a mammal or other patient. ISS include, for example, certain palindromic structures that result in hairpin secondary structures (Yamamoto S., et al. (1992) J. Immunol. 148: 4072-4076, which is incorporated by reference in its entirety) or CpG motifs, and other known ISS features, such as multi-G domains (see WO96 / 11266, which is incorporated by reference in its entirety).

[0388] The immune response may be an innate or adaptive immune response. The immune system is divided into a more innate immune system and an adaptive immune system in vertebrates, the latter being further divided into humoral cellular components. In some embodiments, the immune response may be mucosal.

[0389] In some embodiments, the immunostimulatory nucleic acid is only immunostimulatory when administered in combination with a lipid particle, and is not immunostimulatory when administered in "free form." Such oligonucleotides are considered to be immunostimulatory.

[0390] An immunostimulatory nucleic acid is considered to be non-sequence specific if it does not need to specifically bind to and reduce the expression of a target polynucleotide in order to elicit an immune response. Thus, a particular immunostimulatory nucleic acid may contain a sequence that corresponds to a region of a naturally occurring gene or mRNA and still be considered a non-sequence specific immunostimulatory nucleic acid.

[0391] In some embodiments, the immunostimulatory nucleic acid or oligonucleotide comprises at least one CpG dinucleotide. The oligonucleotide or CpG dinucleotide may be unmethylated or methylated. In another embodiment, the immunostimulatory nucleic acid comprises at least one CpG dinucleotide with a methylated cytosine. In some embodiments, the nucleic acid comprises a single CpG dinucleotide, and the cytosine in the CpG dinucleotide is methylated. In an alternative embodiment, the nucleic acid comprises at least two CpG dinucleotides, and at least one cytosine in the CpG dinucleotide is methylated. In a further embodiment, each cytosine in the CpG dinucleotide present in the sequence is methylated. In another embodiment, the nucleic acid comprises a plurality of CpG dinucleotides, and at least one of the CpG dinucleotides comprises a methylated cytosine.

[0392] Attachment between the 5' end unit, the nucleic acid agent, and the ligand In some embodiments, the linkage between the 5' end unit and the nucleic acid agent is a bond.

[0393] In some embodiments, the linkage between the 5' terminal unit and the nucleic acid agent is a moiety (eg, a moiety that includes a cleavable group).

[0394] Groups can be cleavable or non-cleavable. Suitable groups include, for example, -NR-, -C(=O)-, -C(=O)NH-, -S(=O)-, -S(=O)2-, -S(=O)2NH-, or chains of atoms such as, but not limited to, alkylene, alkenylene, alkynylene, aryl alkylene, aryl alkenylene, aryl alkynylene, heteroaryl alkylene, heteroaryl alkenylene, heteroaryl alkynylene, heterocyclyl alkylene, heterocyclyl alkenylene, heterocyclyl alkynylene, arylene, heteroarylene, heterocyclylene, cycloalkylene, hetero ... alkylene, cycloalkenylene, alkylarylalkylene, alkylarylalkenylene, alkylarylalkynylene, alkenylarylalkylene, alkenylarylalkenylene, alkenylarylalkynylene, alkynylarylalkylene, alkynylarylalkenylene, alkynylarylalkynylene, alkylheteroarylalkylene, alkylheteroarylalkenylene, alkylheteroarylalkynylene, alkenylheteroarylalkylene, alkenylheteroarylalkenylene, alkynylarylalkynylene, arylheteroarylalkynylene, alkynylheteroarylalkylene, alkynylheteroarylalkenylene, alkynylheteroarylalkynylene, alkylheterocyclylalkylene, alkylheterocyclylalkenylene, alkylheterocyclylalkynylene, alkenylheterocyclylalkylene, alkenylheterocyclylalkenylene, alkenylheterocyclylalkynylene, alkynylheterocyclylalkylene, alkynylheterocyclylalkenylene, alkynylheterocyclylalkynylene, alkylaryl and methylene, -O-, -S-, -S(=O)-, -S(=O)2-, -NR-, -C(=O)-, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycle, and wherein R is hydrogen, acyl, aliphatic, or substituted aliphatic.

[0395] A cleavable group is one that is sufficiently stable outside a cell, but which is cleaved once inside a target cell to release the two moieties it carries. In a preferred embodiment, the cleavable group is cleaved at least 10 times faster, preferably at least 100 times faster, in the target cell or under a first reference condition (which can, for example, be selected to mimic or represent intracellular conditions) than in the subject's blood or under a second reference condition (which can, for example, be selected to mimic or represent conditions found in blood or serum).

[0396] Cleavable groups are sensitive to cleaving agents, such as pH, redox potential, or the presence of degrading molecules. In general, cleaving agents are found to be more abundant or at higher levels or activity within cells than in serum or blood. Such degrading agents include redox agents that are selective for a particular substrate or have no substrate specificity (e.g., oxidizing or reducing enzymes, or reducing agents such as mercaptans present in cells that can degrade redox cleavable groups by reduction), esterases, endosomes or agents that can create an acidic environment (e.g., those that result in a pH of 5 or less), enzymes that can hydrolyze or degrade acid cleavable groups by acting as a general acid, peptidases (which may be substrate specific), and phosphatases.

[0397] Cleavable groups such as disulfide bonds can be sensitive to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers have a cleavable group that is cleaved at a preferred pH (thereby releasing the cationic lipid from the ligand intracellularly or into a desired compartment of the cell).

[0398] The conjugate may contain a cleavable group that is cleavable by a specific enzyme. The type of cleavable group incorporated into the conjugate depends on the cell to be targeted. For example, a liver targeting ligand can be linked to a cationic lipid via a chemical moiety that contains an ester group. Hepatocytes are rich in esterases, and therefore this group is cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types that are rich in esterases include lung, renal cortex, and testicular cells.

[0399] Coupling groups containing peptide bonds can be used when targeting cell types that are rich in peptidases, such as hepatocytes and synovial cells.

[0400] In general, the suitability of a candidate cleavable group can be evaluated by testing the ability of a degrading agent (or condition) to cleave the candidate group. It is also desirable to test the candidate cleavable group for its ability to withstand cleavage in blood or when in contact with other non-target tissues. Thus, the relative susceptibility to cleavage between a first condition and a second condition can be determined, where the first condition is selected to indicate cleavage in target cells and the second condition is selected to indicate cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, cells, cell cultures, organ or tissue cultures, or whole animals. It may be useful to perform initial evaluations in cell-free or culture conditions and confirm by further evaluations in whole animals. In a preferred embodiment, a useful candidate compound is cleaved at least 2, 4, 10, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0401] Redox-cleavable groups. One class of cleavable groups is redox-cleavable groups that are cleaved upon reduction or oxidation. An example of a reductively cleavable group is a disulfide bond group (-SS-). To determine whether a candidate cleavable group is a suitable "reductively cleavable linking group" or is suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one can refer to the methods described herein. Candidates can be evaluated by incubating with dithiothreitol (DTT) or other reducing agents using, for example, reagents known in the art that mimic the cleavage rate observed in cells (e.g., target cells). Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In a preferred embodiment, the candidate compound is cleaved up to 10% in blood. In a preferred embodiment, a useful candidate compound is degraded at least 2, 4, 10, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of a candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic the intracellular medium and compared to conditions selected to mimic the extracellular medium.

[0402] Phosphate-based cleavable groups. Phosphate-based cleavable groups are cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that cleaves a phosphate group in a cell is an enzyme such as a phosphatase in the cell. In some embodiments, the phosphate-based linking group is -OP(=O)(OR k )-O-, -OP(=S)(OR k )-O-, -OP(=S)(SR k )-O-, -SP(=O)(OR k )-O-, -OP(=O)(OR k )-S-, -SP(=O)(OR k )-S-, -OP(=S)(OR k )-s-, -SP(=S)(OR k )-O-, -OP(=O)(R k)-O-, -OP(=S)(R k )-O-, -SP(=O)(R k )-O-, -SP(=S)(R k )-O-, -SP(=O)(R k )-S- or -OP(=S)(R k )-S-. In some embodiments, the phosphate based linking group is -OP(=O)(OH)-O-, -OP(=S)(OH)-O-, -OP(=S)(SH)-O-, -SP(=O)(OH)-O-, -OP(=O)(OH)-S-, -SP(=O)(OH)-S-, -OP(=S)(OH)-S-, -SP(=S)(OH)-O-, -OP(=O)(H)-O-, -OP(=S)(H)-O-, -SP(=O)(H)-O-, -SP(=S)(H)-O-, -SP(=O)(H)-S-, or -OP(=S)(H)-S-. In some embodiments, the phosphate based linking group is -OP(=O)(OH)-O-.

[0403] Acid-cleavable groups. Acid-cleavable groups are linking groups that are cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable group is cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0, or less) or by an agent such as an enzyme that can act as a general acid. In cells, certain low pH organelles such as endosomes and lysosomes can provide a cleavage environment for acid-cleavable linking groups. Examples of acid-cleavable groups include, but are not limited to, hydrazones, esters, and amino acid esters. Acid-cleavable groups can have the general formula: -C=NN-, C(O)O, or -OC(O). A preferred embodiment is where the carbon attached to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0404] Ester-based cleavable groups. Ester-based cleavable groups are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linking groups have the general formula: -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0405] Peptide-based cleavable groups. Peptide-based cleavable groups are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable groups are peptide bonds formed between amino acids to generate oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to generate peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to generate peptides and proteins, and do not include amide functional groups altogether. Peptide-based cleavable linking groups have the general formula: -NHCHR A C(O)NHCHR B C(O)—, where R A and R Bare the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above. As used herein, "carbohydrate" refers to a carbohydrate itself, which may be one or more monosaccharide units having at least six carbon atoms, each of which has an oxygen, nitrogen, or sulfur atom attached to each carbon atom, or a compound having as part of it a carbohydrate moiety, which may be one or more monosaccharide units, each of which has at least six carbon atoms, each of which has an oxygen, nitrogen, or sulfur atom attached to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4-9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include sugars of C5 or more (preferably C5-C8), and disaccharides and trisaccharides include sugars having two or three monosaccharide units (preferably C5-C8).

[0406] It has previously been reported that certain 1'-amino 2'-OTBS carbocyclic phosphoramidites have been prepared and incorporated into natural oligonucleotides as a means for attaching fluorophores for labeling of oligonucleotides (Org. Lett. 2021, 23, 6735-6739, incorporated herein by reference). Without wishing to be bound by theory, the compounds, nucleic acid agents, and conjugates of the present disclosure may differ from the previously reported 1'-amino 2'-OTBS carbocyclic phosphoramidites in various aspects, including chemical structure, oligonucleotides attached, use of the conjugates, and / or synthetic approaches.

[0407] Synthesis method In some aspects, the disclosure provides methods of preparing the disclosed compounds.

[0408] In some aspects, the disclosure provides compounds obtainable or obtainable by the methods for preparing the compounds described herein.

[0409] In some aspects, the disclosure provides intermediates described herein that are suitable for use in the processes for preparing the compounds described herein.

[0410] The compounds of the present disclosure can be prepared by any suitable technique known in the art. Specific processes for preparing these compounds are further described in the accompanying examples.

[0411] In the description of synthetic methods provided herein, and in any reference synthetic methods used to prepare starting materials, it is understood that all reaction conditions proposed, including choice of solvent, reaction atmosphere, reaction temperature, experimental time, and work-up procedures, can be selected by one of ordinary skill in the art.

[0412] It is understood by one skilled in the art of organic synthesis that the functionality present on various portions of the molecule must be compatible with the reagents and reaction conditions used.

[0413] It will be understood that during the synthesis of the compounds of the present disclosure or during the synthesis of certain starting materials in the processes defined herein, it may be desirable to protect certain substituents to prevent undesired reactions. A skilled chemist will understand when such protection is necessary and how to install and subsequently remove such protecting groups. For examples of protecting groups, see one of the many general texts on the subject, for example 'Protective Groups in Organic Synthesis' by Theodora Green (publisher: John Wiley & Sons). Protecting groups may be removed by any convenient method described in the literature or known to a skilled chemist as suitable for removing the protecting group in question, such a method being selected to effect removal of the protecting group with minimal effect on other groups in the molecule. Thus, when a reactant contains a group such as, for example, amino, carboxy, or hydroxy, it may be desirable to protect that group in some of the reactions mentioned herein.

[0414] For example, a suitable protecting group for an amino or alkylamino group is, for example, an acyl group, for example an alkanoyl group, such as acetyl, an alkoxycarbonyl group, for example a methoxycarbonyl, ethoxycarbonyl or t-butoxycarbonyl group, an arylmethoxycarbonyl group, for example benzyloxycarbonyl, or an aroyl group, for example benzoyl. Suitable protecting groups for hydroxy or alkylhydroxy groups may be, for example, acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl ether (MEM), dimethoxytrityl (DMT), methoxymethyl ether (MOM), methoxytrityl (MMT), p-methoxybenzyl ether (PMB), p-methoxyphenyl ether (PMP), pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (triphenylmethyl, Tr), silyl ethers (e.g. trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), triisopropylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS) ethers), methyl ethers, or ethoxyethyl ethers (EE). Suitable protecting groups for 1,2-diols may be, for example, acetals. A suitable protecting group for 1,3-diols can be, for example, tetraisopropyldisiloxanylidene (TIPDS).

[0415] The deprotection conditions for the above protecting groups necessarily vary with the choice of protecting group. Thus, for example, acyl groups such as alkanoyl or alkoxycarbonyl groups, or aroyl groups, may be removed by hydrolysis with a suitable base, such as an alkali metal hydroxide, for example lithium hydroxide or sodium hydroxide. Alternatively, acyl groups such as tert-butoxycarbonyl groups may be removed by treatment with a suitable acid, for example hydrochloric acid, sulfuric acid, phosphoric acid, or trifluoroacetic acid, and arylmethoxycarbonyl groups such as benzyloxycarbonyl groups may be removed by hydrogenation with a catalyst, for example palladium on carbon, or by treatment with a Lewis acid, such as boron tris (trifluoroacetic acid). A suitable alternative protecting group for primary amino groups is the phthaloyl group, which may be removed by treatment with an alkylamine, for example dimethylaminopropylamine, or hydrazine.

[0416] Suitable protecting groups for hydroxyl groups are, for example, acyl groups, e.g., alkanoyl groups such as acetyl, aroyl groups, e.g., benzoyl, or arylmethyl groups, e.g., benzyl. The deprotection conditions for the above protecting groups necessarily vary with the choice of protecting group. Thus, for example, acyl groups such as alkanoyl groups or aroyl groups can be removed by hydrolysis with a suitable base, e.g., an alkali metal hydroxide, e.g., lithium, sodium hydroxide, or ammonia. Alternatively, arylmethyl groups such as benzyl groups can be removed by hydrogenation, e.g., over a catalyst, e.g., palladium on carbon.

[0417] A suitable protecting group for a carboxy group is, for example, an esterifying group, such as a methyl or ethyl group (which may be removed by hydrolysis with a base such as sodium hydroxide), or, for example, a tert-butyl group (which may be removed by treatment with an acid, for example an organic acid such as trifluoroacetic acid), or, for example, a benzyl group (which may be removed by hydrogenation over a catalyst such as palladium on carbon).

[0418] Advantageously, the reaction of the compounds is carried out in the presence of a suitable solvent, which is preferably inert under the respective reaction conditions. Examples of suitable solvents include hydrocarbons such as hexane, petroleum ether, benzene, toluene or xylene; chlorinated hydrocarbons such as trichloroethylene, 1,2-dichloroethane, tetrachloromethane, chloroform or dichloromethane; alcohols such as methanol, ethanol, isopropanol, n-propanol, n-butanol or tert-butanol; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran (THF), 2-methyltetrahydrofuran, cyclopentyl methyl ether (CPME), methyl tert-butyl ether (MTBE) or dioxane; ethylene glycol, propylene glycol, propylene glycol ether ... These include, but are not limited to, glycol ethers such as glycol monomethyl ether, monoethyl ether, or ethylene glycol dimethyl ether (diglyme); ketones such as acetone, methyl isobutyl ketone (MIBK), or butanone; amides such as acetamide, dimethylacetamide, dimethylformamide (DMF), or N-methylpyrrolidone (NMP); nitriles such as acetonitrile; sulfoxides such as dimethylsulfoxide (DMSO); nitro compounds such as nitromethane or nitrobenzene; esters such as ethyl acetate or methyl acetate, or mixtures of the above solvents or with water.

[0419] The reaction temperature is preferably about -100°C to 300°C depending on the reaction step and conditions used.

[0420] The reaction time generally ranges from a fraction of a minute to several days, depending on the reactivity of each compound and the respective reaction conditions. Suitable reaction times are easily determined by methods known in the art, such as reaction monitoring. Based on the above reaction temperatures, suitable reaction times generally range from 10 minutes to 48 hours.

[0421] Moreover, by utilizing the procedures described herein in combination with ordinary skills in the art, additional compounds of the present disclosure can be readily prepared. Those skilled in the art will readily understand that known variations of the conditions and processes of the following preparative procedures can be used to prepare these compounds.

[0422] As will be appreciated by those skilled in the art of organic synthesis, the compounds of the present disclosure are readily accessible by various synthetic routes, some of which are illustrated in the accompanying examples. Those skilled in the art will easily recognize what kind of reagents and reaction conditions are used to obtain the compounds of the present disclosure, and how to apply and adapt them in any particular case, whenever necessary or useful. In addition, some of the compounds of the present disclosure can be easily synthesized by converting certain functional groups present in the compounds of the present disclosure or in suitable precursor molecules thereof to other functional groups, for example by applying standard synthetic methods such as reduction, oxidation, addition, or substitution reactions, which are well known to those skilled in the art. Similarly, those skilled in the art will apply synthetic protecting groups (or protective groups), whenever necessary or useful. Suitable protecting groups and methods for their introduction and removal are well known to those skilled in the art of chemical synthesis, and are described in detail, for example, in PGM Wuts, T.W. Greene, "Greene's Protective Groups in Organic Synthesis", 4th edition (2006) (John Wiley & Sons).

[0423] A general route for preparing the compounds of the present application is depicted in Scheme 1 herein: [ka]

[0424] Biological assays The compounds, nucleic acid agents, or conjugates designed, selected, prepared, and / or optimized by the above methods can be characterized, when produced, using various assays known to those skilled in the art to determine whether the compounds, nucleic acid agents, or conjugates have biological activity. For example, the compounds, nucleic acid agents, or conjugates can be characterized by conventional assays, including but not limited to the assays described below, to determine whether they have the desired activity, such as target binding activity and / or specificity and / or stability.

[0425] Furthermore, high-throughput screening can be used to speed up the analysis using such assays. As a result, it may be possible to rapidly screen the activity of the molecules described herein using techniques known in the art. General methodologies for performing high-throughput screening are described, for example, in Devlin (1998) High Throughput Screening, Marcel Dekker and U.S. Patent No. 5,763,263. High-throughput assays can use one or more different assay techniques, including but not limited to those described below.

[0426] A variety of in vitro or in vivo biological assays may be suitable for detecting the effects of the disclosed compounds, nucleic acid agents, or conjugates. These in vitro or in vivo biological assays may include, but are not limited to, enzyme activity assays, electrophoretic mobility shift assays, reporter gene assays, in vitro cell viability assays, and assays described herein.

[0427] In some embodiments, the biological assays are described in the Examples herein.

[0428] Pharmaceutical Compositions In some aspects, the disclosure provides pharmaceutical compositions comprising a compound, nucleic acid agent, or conjugate of the disclosure as an active ingredient.

[0429] As used herein, the term "composition" is intended to encompass a product containing the specified ingredients in the specified amounts, and any product resulting directly or indirectly from a combination of the specified ingredients in the specified amounts.

[0430] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium, including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal. In many cases, it will be preferable to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol and sorbitol, sodium chloride, etc. Prolonged absorption of the injectable compositions can be achieved by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0431] Sterile solution for injection can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or combination of the above-listed components as required, followed by sterilization by filtration.Generally, dispersion is prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other components required from the above-listed ones.For the preparation of sterile powder for preparation of sterile solution for injection, the preparation method is vacuum drying and freeze-drying, whereby the powder of active ingredient and any additional ingredient is obtained from the solution that has been previously sterilized and filtered.

[0432] The formulation of the present disclosure may be in the form of an aqueous solution comprising an aqueous vehicle. The aqueous vehicle component may comprise water and at least one pharma- ceutically acceptable excipient. Suitable acceptable excipients include those selected from the group consisting of solubility enhancers, chelating agents, preservatives, isotonicity agents, viscosity formulation / suspension agents, buffers, and pH adjusters, and mixtures thereof.

[0433] Any suitable solubility enhancer may be used. Examples of solubility enhancers include cyclodextrins such as those selected from the group consisting of hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, randomly methylated β-cyclodextrin, ethylated β-cyclodextrin, triacetyl-β-cyclodextrin, peracetylated-β-cyclodextrin, carboxymethyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, 2-hydroxy-3-(trimethylammonio)propyl-β-cyclodextrin, glucosyl-β-cyclodextrin, sulfated β-cyclodextrin (S-β-CD), maltosyl-β-cyclodextrin, β-cyclodextrin sulfobutyl ether, branched β-cyclodextrins, hydroxypropyl-β-cyclodextrin, randomly methylated β-cyclodextrin, and trimethyl-β-cyclodextrin, and mixtures thereof.

[0434] Any suitable chelating agent may be used. Examples of suitable chelating agents include those selected from the group consisting of ethylenediaminetetraacetic acid and its metal salts, disodium edetate, trisodium edetate, and tetrasodium edetate, and mixtures thereof.

[0435] Any suitable preservative can be used. Examples of preservatives include quaternary ammonium, such as benzalkonium halide (preferably benzalkonium chloride), chlorhexidine gluconate, benzethonium chloride, cetylpyridinium chloride, benzyl bromide, phenylmercuric nitrate, phenylmercuric acetate, phenylmercuric neodecanoate, merthiolate, methylparaben, propylparaben, sorbic acid, potassium sorbate, sodium benzoate, sodium propionate, ethyl p-parahydroxybenzoate, propylaminopropyl biguanide, butyl p-hydroxybenzoate, and sorbic acid, and mixtures thereof.

[0436] The aqueous vehicle may also include an isotonicity agent to adjust tonicity (osmotic pressure), which may be selected from the group consisting of glycols (such as propylene glycol, diethylene glycol, triethylene glycol), glycerol, dextrose, glycerin, mannitol, potassium chloride, and sodium chloride, and mixtures thereof.

[0437] The formulation may contain a pH adjuster to adjust the formulation to an acceptable pH (typically in the pH range of about 5.0 to about 9.0, more preferably about 5.5 to about 8.5, particularly about 6.0 to about 8.5, about 7.0 to about 8.5, about 7.2 to about 7.7, about 7.1 to about 7.9, or about 7.5 to about 8.0). The pH adjuster is typically a mineral acid or metal hydroxide base selected from the group of potassium hydroxide, sodium hydroxide, and hydrochloric acid, and mixtures thereof, preferably sodium hydroxide and / or hydrochloric acid. These acidic and / or basic pH adjusters are added to adjust the formulation to a target acceptable pH range. Thus, depending on the formulation, it may not be necessary to use both an acid and a base, and the addition of either an acid or a base may be sufficient to bring the mixture to the desired pH range.

[0438] The aqueous vehicle may also contain a buffer to stabilize the pH, which, if used, is selected from the group consisting of phosphate buffers (such as sodium dihydrogen phosphate and disodium hydrogen phosphate), borate buffers (such as boric acid or its salts, including disodium tetraborate), citrate buffers (such as citric acid or its salts, including sodium citrate), and ε-aminocaproic acid, and mixtures thereof.

[0439] According to a further aspect of the present disclosure there is provided a pharmaceutical composition comprising a compound of the present disclosure as defined above or a pharma- ceutically acceptable salt, hydrate or solvate thereof, in association with a pharma- ceutically acceptable diluent or carrier.

[0440] The compositions of the present disclosure may be in a suitable form for oral use (e.g., as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), topical use (e.g., as creams, ointments, gels, or aqueous or oily solutions or suspensions), administration by inhalation (e.g., as a finely divided or liquid aerosol), administration by insufflation (e.g., as a finely divided powder), or parenteral administration (e.g., as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular, intraperitoneal, or intramuscular administration, or as a suppository for rectal administration).

[0441] The compositions of the present disclosure may be obtained by conventional procedures using conventional pharmaceutical excipients well known in the art. Thus, compositions intended for oral use may contain, for example, one or more coloring agents, sweeteners, flavoring agents, and / or preservatives.

[0442] An effective amount of a compound of the present disclosure for use in therapy is an amount sufficient to treat or prevent, slow the progression of, and / or alleviate the symptoms associated with an inflammasome-associated condition referred to herein.

[0443] An effective amount of a compound of the present disclosure for use in therapy is an amount sufficient to treat, slow the progression of, and / or alleviate the symptoms associated with an inflammasome-associated condition referred to herein.

[0444] The amount of a compound of formula (I) or (II) administered for therapeutic or prophylactic purposes will necessarily vary according to the nature and severity of the condition, the age and sex of the animal or patient, and the route of administration, in accordance with well-known medical guidelines.

[0445] How to use In some aspects, the present disclosure provides a method of modulating (e.g., reducing or eliminating) expression of a target gene in a subject, the method comprising administering to the subject a conjugate of the present disclosure.

[0446] In some aspects, the present disclosure provides a method of modulating (e.g., reducing or eliminating) expression of a target gene in a cell or tissue of a subject, the method comprising administering to the subject a conjugate of the present disclosure.

[0447] In some aspects, the present disclosure provides a method of delivering a nucleic acid agent to a subject, the method comprising administering to the subject a conjugate of the present disclosure.

[0448] In some aspects, the present disclosure provides a method of treating or preventing a disease in a subject in need of such treatment or prevention, comprising administering to the subject a therapeutically effective amount of a conjugate of the present disclosure.

[0449] In some aspects, the present disclosure provides a conjugate of the present disclosure for modulating (e.g., reducing or eliminating) expression of a target gene in a subject.

[0450] In some aspects, the present disclosure provides a conjugate of the present disclosure for modulating (e.g., reducing or eliminating) expression of a target gene in a cell or tissue of a subject.

[0451] In some aspects, the present disclosure provides a conjugate of the present disclosure for delivery of a nucleic acid agent to a subject.

[0452] In some aspects, the present disclosure provides a conjugate of the present disclosure for treating or preventing a disease in a subject in need of such treatment or prevention.

[0453] In some aspects, the disclosure provides use of a conjugate of the disclosure in the manufacture of a medicament for modulating (e.g., reducing or eliminating) expression of a target gene in a subject.

[0454] In some aspects, the disclosure provides for the use of a conjugate of the disclosure in the manufacture of a medicament for modulating (e.g., reducing or eliminating) expression of a target gene in a cell or tissue of a subject.

[0455] In some aspects, the disclosure provides for the use of a conjugate of the disclosure in the manufacture of a medicament for delivering a nucleic acid agent to a subject.

[0456] In some aspects, the present disclosure provides the use of a conjugate of the present disclosure in the manufacture of a medicament for treating or preventing a disease in a subject in need of such treatment or prevention.

[0457] In some embodiments, the subject is a cell.

[0458] In some embodiments, the subject is a tissue.

[0459] In some embodiments, the subject is a human.

[0460] In some embodiments, the target gene is Factor VII, Eg5, PCSK9, TPX2, ApoB, SAA, TTR, HBV, HCV, RSV, PDGF beta 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, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2 / Neu gene, topoisomerase I gene, topoisomerase II alpha gene, p73 gene, p21(WAF1 / CIP1) gene, p27(KIP1) gene, PPM1D gene, RAS gene, caveolin I gene, MIB I gene, MTAI gene, M68 gene, mutation of a tumor suppressor gene, p53 tumor suppressor gene, LDHA, or a combination thereof.

[0461] In some embodiments, the disease is characterized by unwanted expression of the target gene.

[0462] In some embodiments, administration reduces or eliminates expression of the target gene in the subject.

[0463] In some embodiments, the disease is a viral infection, such as an HCV, HBV, HPV, HSV, or HIV infection.

[0464] In some embodiments, the disease is cancer.

[0465] In some embodiments, the cancer is selected from the group consisting of 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, Wilms' tumor, leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), and / or chronic myelogenous leukemia (CLM). ), chronic myelomonocytic leukemia (CMML), liver cancer, hepatoma, hepatoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, B cell lymphoma, non-Hodgkin's lymphoma, diffuse large cell B cell lymphoma, mantle cell lymphoma, T cell lymphoma, non-Hodgkin's lymphoma, precursor T lymphoblastic lymphoma / leukemia, peripheral T cell lymphoma, multiple myeloma, nasopharyngeal carcinoma (NPC), neuroblastoma, oropharyngeal cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, pseudopapillary tumor, acinar cell carcinoma, prostate cancer, prostate adenocarcinoma, skin cancer, melanoma, malignant melanoma, cutaneous melanoma, small intestine cancer, gastric cancer (stomach gastric cancer, gastric carcinoma, gastrointestinal stromal tumor (GIST), uterine cancer, or uterine sarcoma.

[0466] In some embodiments, the cancer is liver cancer, liver cancer, hepatoma, hepatocellular carcinoma, cholangiocarcinoma, or hepatoblastoma.

[0467] In some embodiments, the disease is a proliferative disease, an inflammatory disease, an autoimmune disease, a neurological disease, an eye disease, a respiratory disease, a metabolic disease, a skin disease, an auditory disease, a liver disease, a kidney disease, or an infectious disease, hi some embodiments, the disease is a liver disease.

[0468] definition Unless otherwise stated, the following terms used in the specification and claims have the meanings given below.

[0469] While not wishing to be limited to this description, it is understood that while various options for the variables are described herein, the present disclosure is intended to encompass workable embodiments having combinations of options, and the present disclosure may be interpreted as excluding non-workable embodiments resulting from a particular combination of options.

[0470] As used herein, "alkyl", "C1, C2, C3, C4, C5, or C6 alkyl" or "C1-C6 alkyl" is intended to include C1, C2, C3, C4, C5, or C6 straight chain (linear) saturated aliphatic hydrocarbon groups and C3, C4, C5, or C6 branched saturated aliphatic hydrocarbon groups. For example, C1-C6 alkyl is intended to include C1, C2, C3, C4, C5, and C6 alkyl groups. Examples of alkyl include, but are not limited to, moieties having 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, or n-hexyl. In some embodiments, a straight chain or branched alkyl has 6 or fewer carbon atoms (e.g., C1-C6 for straight chain, C3-C6 for branched chain), and in other embodiments, a straight chain or branched alkyl has 4 or fewer carbon atoms.

[0471] As used herein, the term "optionally substituted alkyl" refers to an unsubstituted alkyl or an alkyl having specified substituents replacing one or more hydrogen atoms on one or more carbons of the hydrocarbon backbone. Such substituents may include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonate, phosphinate, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or aromatic or heteroaromatic moieties.

[0472] As used herein, the term "alkenyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one double bond. For example, the term "alkenyl" includes straight chain alkenyl groups (such as ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.) and branched alkenyl groups. In some embodiments, a straight chain or branched alkenyl group has six or fewer carbon atoms in its backbone (e.g., C2-C6 for straight chain, C3-C6 for branched chain). The term "C2-C6" includes alkenyl groups containing 2-6 carbon atoms. The term "C3-C6" includes alkenyl groups containing 3-6 carbon atoms.

[0473] As used herein, the term "optionally substituted alkenyl" refers to an unsubstituted alkenyl or an alkenyl having specified substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms. Such substituents may include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonate, phosphinate, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, heterocyclyl, alkylaryl, or aromatic or heteroaromatic moieties.

[0474] As used herein, the term "alkynyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond. For example, "alkynyl" includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl) and branched alkynyl groups. In some embodiments, a straight-chain or branched alkynyl group has six or fewer carbon atoms in its backbone (e.g., C2-C6 for straight chain, C3-C6 for branched chain). The term "C2-C6" includes alkynyl groups containing 2-6 carbon atoms. The term "C3-C6" includes alkynyl groups containing 3-6 carbon atoms. As used herein, a "C2-C6 alkenylene linker" or "C2-C6 alkynylene linker" is intended to include a C2, C3, C4, C5, or C6 chain (linear or branched) divalent unsaturated aliphatic hydrocarbon group. For example, a C2-C6 alkenylene linker includes C2, C3, C4, C5, and C6 alkenylene linker groups.

[0475] As used herein, the term "optionally substituted alkynyl" refers to unsubstituted alkynyl or alkynyl having specified substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms. Such substituents may include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonate, phosphinate, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or aromatic or heteroaromatic moieties.

[0476] Other optionally substituted moieties (such as optionally substituted cycloalkyl, heterocycloalkyl, aryl, or heteroaryl) include both unsubstituted moieties and moieties with one or more of the specified substituents. For example, substituted heterocycloalkyl includes those substituted with one or more alkyl groups, such as 2,2,6,6-tetramethyl-piperidinyl and 2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridinyl.

[0477] As used herein, the term "cycloalkyl" refers to an alkyl group having 3 to 30 carbon atoms (e.g., C 12 , C3~C 10, or C3-C8), a saturated or partially unsaturated hydrocarbon monocyclic or polycyclic (e.g., fused, bridged, or spirocyclic) system. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, 1,2,3,4-tetrahydronaphthalenyl, adamantyl, and the like. In the case of a polycyclic cycloalkyl, only one of the rings in the cycloalkyl need be non-aromatic.

[0478] As used herein, unless otherwise specified, the term "heterocycloalkyl" refers to a saturated or partially unsaturated 3-8 membered monocyclic, 7-12 membered bicyclic (fused, bridged, or spiro) or 11-14 membered tricyclic ring system (fused, bridged, or spiro) having one or more heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur (e.g., O, N, S, P, Se), e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or e.g., 1, 2, 3, 4, 5, or 6 heteroatoms.Examples of heterocycloalkyl groups include piperidinyl, piperazinyl, pyrrolidinyl, dioxanyl, tetrahydrofuranyl, isoindolinyl, indolinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl, oxiranyl, azetidinyl, oxetanyl, thietanyl, 1,2,3,6-tetrahydropyridinyl, tetrahydropyranyl, dihydropyranyl, pyranyl, morpholinyl, tetrahydrothiopyranyl, 1,4-diazepanyl, 1,4-oxazepam ... 2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, 2-oxa-6-azaspiro[3.3]heptanyl, 2,6-diazaspiro[3.3]heptanyl, 1,4-dioxa-8-azaspiro[4.5]decanyl, 1,4-dioxaspiro[4.5]decanyl, 1-oxaspiro[4.5]decanyl, 1-azaspiro[4.5]decanyl, 3'H-spiro[cyclohexane-1,1'-isobenzofuran]-yl, 7'H-spiro[cyclohexa 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[3.1.0]hexan-3-yl, 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazolyl, 3,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidinyl, 4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridinyl, 5,6,7,8-tetrahydropyrido[4 ,3-d]pyrimidinyl, 2-azaspiro[3.3]heptanyl, 2-methyl-2-azaspiro[3.3]heptanyl, 2-azaspiro[3.5]nonanyl, 2-methyl-2-azaspiro[3.5]nonanyl, 2-azaspiro[4.5]decanyl, 2-methyl-2-azaspiro[4.5]decanyl, 2-oxa-azaspiro[3.4]octanyl, 2-oxa-azaspiro[3.4]octan-6-yl, 5,6-dihydro-4H-cyclopenta[b]thiophenyl, and the like.In the case of polycyclic heterocycloalkyls, only one of the rings in the heterocycloalkyl need be non-aromatic (eg, 4,5,6,7-tetrahydrobenzo[c]isoxazolyl).

[0479] As used herein, the term "aryl" includes groups that have aromatic character, including "conjugated" or polycyclic systems with one or more aromatic rings, and do not contain heteroatoms in the ring structure. The term aryl includes both monovalent and divalent species. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl, and the like. Conveniently, the aryl is phenyl.

[0480] As used herein, the term "heteroaryl" is intended to include a stable 5-, 6-, or 7-membered monocyclic aromatic heterocyclic ring or a 7-, 8-, 9-, 10-, 11-, or 12-membered bicyclic aromatic heterocyclic ring consisting of carbon atoms and one or more heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or, e.g., 1, 2, 3, 4, 5, or 6 heteroatoms. The nitrogen atom may be substituted or unsubstituted (i.e., N or NR, where R is H or other defined substituents). The nitrogen and sulfur heteroatoms may be optionally oxidized (i.e., N→O and S(O)). p(wherein p=1 or 2). Note that the total number of S and O atoms in the aromatic heterocycle is not more than 1. Examples of heteroaryl groups include pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, isothiazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like. Heteroaryl groups can also be fused or bridged with alicyclic or non-aromatic heterocyclic rings to form polycyclic systems (e.g., 4,5,6,7-tetrahydrobenzo[c]isoxazolyl). In some embodiments, heteroaryl is thiophenyl or benzothiophenyl. In some embodiments, heteroaryl is thiophenyl. In some embodiments, heteroaryl is benzothiophenyl.

[0481] Furthermore, the terms "aryl" and "heteroaryl" include polycyclic aryl and heteroaryl groups, e.g., tricyclic, bicyclic, such as naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzimidazole, benzothiophene, quinoline, isoquinoline, naphthyridine, indole, benzofuran, purine, benzofuran, deazapurine, indolizine.

[0482] Cycloalkyl, heterocycloalkyl, aryl, or heteroaryl rings may contain at one or more ring positions (e.g., a ring-forming carbon or a heteroatom such as N) a substituent as described above, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, amino The aryl and heteroaryl groups can be substituted with carbonyl, alkylthiocarbonyl, phosphate, phosphonate, phosphinate, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or aromatic or heteroaromatic moieties. The aryl and heteroaryl groups can also be fused or bridged with alicyclic or non-aromatic heterocyclic rings to form polycyclic systems (e.g., methylenedioxyphenyl, such as tetralin, benzo[d][1,3]dioxol-5-yl, etc.).

[0483] As used herein, the term "substituted" means that any one or more hydrogen atoms on the designated atom are replaced with one selected from the designated group, provided that the normal valence of the designated atom is not exceeded and the substitution results in a stable compound. When the substituent is oxo or keto (i.e., =O), two hydrogen atoms on the atom are replaced. Keto substituents do not occur in aromatic moieties. As used herein, a ring double bond is a double bond formed between two adjacent ring atoms (e.g., C=C, C=N, or N=N). "Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation from a reaction mixture to a useful purity and formulation into an effective therapeutic agent.

[0484] When a bond to a substituent is shown to cross a bond connecting two atoms in a ring, such substituent may be bonded to any atom in the ring. When substituents are listed without indicating the atom to which such substituent is bonded to the remainder of the compound of a given formula, such substituent may be bonded through any atom in such formula. Combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0485] When any variable (e.g., R) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-2 R groups, then that group may be optionally substituted with up to 2 R groups, and R at each occurrence is selected independently of the definition of R. Additionally, combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0486] As used herein, the term "hydroxy" or "hydroxyl" refers to -OH or -O - The formula includes groups having the formula:

[0487] As used herein, the term "halo" or "halogen" refers to fluoro, chloro, bromo, and iodo.

[0488] The terms "haloalkyl" or "haloalkoxy" refer to an alkyl or alkoxy substituted with one or more halogen atoms.

[0489] As used herein, the term "optionally substituted haloalkyl" refers to an unsubstituted haloalkyl having specified substituents replacing one or more hydrogen atoms on one or more hydrocarbon backbone carbon atoms. Such substituents may include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonate, phosphinate, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or aromatic or heteroaromatic moieties.

[0490] As used herein, the term "alkoxy" or "alkoxyl" includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently bonded to an oxygen atom. Examples of alkoxy or alkoxyl groups include, but are not limited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups. Examples of substituted alkoxy groups include halogenated alkoxy groups. Alkoxy groups can be substituted with groups such as alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonate, phosphinate, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Examples of halogen substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, and trichloromethoxy.

[0491] As used herein, phrases such as "one or more of A, B, or C," "one or more of A, B, or C," "one or more of A, B, and C," "one or more of A, B, and C," "selected from the group consisting of A, B, and C," "selected from A, B, and C," and the like are used interchangeably and, unless otherwise specified, they all refer to a selection from the group consisting of A, B, and / or C, i.e., one or more A, one or more B, one or more C, or any combination thereof.

[0492] It should be understood that the present disclosure provides methods for synthesizing the compounds, nucleic acid agents, and conjugates described herein.The present disclosure also provides detailed methods for synthesizing the various compounds, nucleic acid agents, and conjugates of the present disclosure according to the schemes described herein and the schemes shown in the examples.

[0493] It should be understood that throughout the description, when a composition is described as having, including, or comprising a particular component, the composition is also intended to consist essentially of or consist of the recited components. Similarly, when a method or process is described as having, including, or comprising particular process steps, the process also consists essentially of or consists of the recited processing steps. Furthermore, it should be understood that the order of steps for performing certain acts is not critical so long as the invention remains operable. Moreover, two or more steps or acts may be performed simultaneously.

[0494] It is to be understood that the synthetic processes of the present disclosure can tolerate a wide variety of functional groups and therefore can employ a variety of substituted starting materials. Although the processes generally provide the desired final compound at or near the end of the overall process, in some cases it may be desirable to further convert the compound to a pharma-ceutically acceptable salt thereof.

[0495] It should be understood that the compounds, nucleic acid agents, and conjugates of the present disclosure can be prepared in a variety of ways using commercially available starting materials, using compounds known in the literature, or from intermediates that can be readily prepared, by using standard synthetic methods and procedures that are known to those skilled in the art or that will become apparent to those skilled in the art in light of the teachings herein. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be obtained from the relevant scientific literature or standard textbooks in the field. Examples of such methods and procedures include, but are not limited to, any one or several sources, such as Smith, MB, March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5 th edition,John Wiley&Sons:New York,2001;Greene,TW,Wuts,PGM,Protective Groups in Organic Synthesis,3 rd edition, John Wiley & Sons: New York, 1999; R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents forganic Synthesis, John Wiley and Sons (1995), which are incorporated herein by reference, are useful and recognized reference texts in organic synthesis known to those of skill in the art.

[0496] Those skilled in the art will note that the order of certain steps, such as the introduction and removal of protecting groups, may be altered during the reaction sequences and synthetic schemes described herein. Those skilled in the art will recognize that certain groups may need to be protected from reaction conditions by the use of protecting groups. Protecting groups can also be used to distinguish similar functional groups in a molecule. A list of protecting groups and methods for introducing and removing these groups can be found in Greene, TW, Wuts, PGM, Protective Groups in Organic Synthesis, 3 rd edition, John Wiley & Sons: New York, 1999.

[0497] Unless otherwise specified, any description of a method of treatment or prevention should be understood to include the use of the compounds, nucleic acid agents, and conjugates to provide the treatment or prevention described herein. Furthermore, unless otherwise specified, any description of a method of treatment or prevention should be understood to include the use of the compounds, nucleic acid agents, and conjugates to prepare a medicament for treating or preventing such a condition. Treatment or prevention includes treatment or prevention of humans or non-human animals, including rodents and other disease models.

[0498] Unless otherwise specified, any description of a method of treatment should be understood to include the use of the compounds, nucleic acid agents, and conjugates to perform the treatments described herein.Furthermore, unless otherwise specified, any description of a method of treatment should be understood to include the use of the compounds, nucleic acid agents, and conjugates to prepare medicaments for treating such conditions.Treatment includes the treatment of humans or non-human animals, including rodents and other disease models.

[0499] As used herein, the term "subject" is interchangeable with the term "subject in need thereof," both of which refer to a subject having a disease or at high risk of developing a disease. "Subject" includes mammals. The mammal may be, for example, a human or a suitable non-human mammal, such as a primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep, or pig. The subject may also be a bird or poultry. In some embodiments, the mammal is a human. The subject in need thereof may be a subject previously diagnosed or identified as having a disease or disorder disclosed herein. The subject in need thereof may be a subject suffering from a disease or disorder disclosed herein. Alternatively, the subject in need thereof may be a subject at high risk of developing such a disease or disorder relative to the general population (i.e., a subject predisposed to developing such a disorder relative to the general population). The subject in need thereof may have a refractory or resistant disease or disorder disclosed herein (i.e., a disease or disorder disclosed herein that is not or has not yet responded to treatment). The subject may be resistant at the start of treatment or may become resistant during treatment. In some embodiments, the subject in need thereof has undergone and failed all known effective treatments for the disease or disorder disclosed herein. In some embodiments, the subject in need thereof has undergone at least one prior treatment.

[0500] As used herein, the term "treatment" or "treating" refers to the management and care of a patient for the purpose of combating a disease, condition, or disorder, and includes administering a compound of the present disclosure, or a pharma- ceutically acceptable salt, polymorph, or solvate thereof, to alleviate symptoms or complications of the disease, condition, or disorder, or to eliminate the disease, condition, or disorder. The term "treating" may also include the treatment of an in vitro cell or animal model. Reference to "treating" or "treatment" includes the alleviation of established symptoms of a pathology. Thus, "treating" or "treatment" of a disease state, disorder, or condition includes (1) preventing or delaying the appearance of clinical symptoms of the disease state, disorder, or condition that occurs in humans who may be afflicted with or predisposed to the disease state, disorder, or condition, but who have not yet experienced or exhibited clinical or subclinical symptoms of the disease state, disorder, or condition; (2) inhibiting the disease state, disorder, or condition, i.e., preventing, alleviating, or delaying the onset or recurrence of the disease (in the case of maintenance therapy) or at least one clinical or asymptomatic symptom thereof; or (3) ameliorating or attenuating the disease, i.e., causing regression of the disease state, disorder, or symptom, or at least one clinical or asymptomatic symptom thereof.

[0501] It is to be understood that the compounds, nucleic acid agents, and conjugates of the present disclosure, or pharma- ceutically acceptable salts, polymorphs, or solvates thereof, can be or may be used to prevent the associated disease, condition, or disorder, or to identify suitable candidates for such purposes.

[0502] As used herein, the terms "preventing," "prevent" or "protecting against" refer to reducing or eliminating the onset of symptoms or complications of such a disease, condition, or disorder.

[0503] It should be understood that the present disclosure also provides pharmaceutical compositions comprising any compound, nucleic acid agent, or conjugate described herein in combination with at least one pharma- ceutically acceptable excipient or carrier.

[0504] As used herein, the term "pharmaceutical composition" refers to a formulation that contains the disclosed compound, nucleic acid agent, or conjugate in a form suitable for administration to a subject. In some embodiments, the pharmaceutical composition is in bulk or unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump in an aerosol inhaler, or a vial. The amount of active ingredient (e.g., the disclosed compound or its salt, hydrate, solvate, or isomer) in a unit dose of the composition is an effective amount and varies according to the specific treatment involved. Those skilled in the art understand that it may be necessary to make routine variations in the amount depending on the age and condition of the patient. The dosage also varies according to the route of administration. Various routes are contemplated, such as oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalation, buccal, sublingual, intrapleural, intrathecal, intranasal, etc. Dosage forms for topical or transdermal administration of a compound of the present disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. In some embodiments, the active compound is mixed under sterile conditions with a pharma- ceutically acceptable carrier, and with any required preservatives, buffers, or propellants.

[0505] As used herein, the term "pharmacologically acceptable" refers to compounds, nucleic acid agents, conjugates, anions, cations, materials, compositions, carriers, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment.

[0506] As used herein, "a pharma- ceutically acceptable excipient" means an excipient that is generally safe, non-toxic, and not biologically or otherwise undesirable and is useful in preparing pharmaceutical compositions, and includes excipients that are acceptable for veterinary and human pharmaceutical use. As used in the specification and claims, "a pharma- ceutically acceptable excipient" includes both one excipient and more than one excipient.

[0507] It is understood that the pharmaceutical compositions of the present disclosure are formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral (intravenous, intradermal, subcutaneous, oral (e.g., ingestion), inhalation, transdermal (topical), and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may contain the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be placed into glass or plastic ampoules, disposable syringes, or multiple dose vials.

[0508] It should be understood that the compounds or pharmaceutical compositions of the present disclosure can be administered to a subject in many well-known ways currently used for chemotherapy treatment. For example, in the case of cancer treatment, the compounds of the present application may be directly injected into a tumor, injected into the bloodstream or body cavity, or taken orally, or applied through the skin using a patch. The dose selected should be sufficient to constitute an effective treatment, but not so high as to cause unacceptable side effects. It is advisable to closely monitor the patient's condition (e.g., disease or disorder disclosed herein) and health status during and for a reasonable period after treatment.

[0509] As used herein, the term "therapeutically effective amount" refers to an amount of an agent to treat, ameliorate, or prevent an identified disease or condition, or to exert a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The exact effective amount for a subject will depend on the subject's weight, size, health, the nature and extent of symptoms, and the therapeutic agent or combination of therapeutic agents selected for administration. The therapeutically effective amount for a given situation can be determined by routine experimentation within the skill and judgment of the clinician.

[0510] As used herein, the term "therapeutically effective amount" refers to an amount of an agent to treat or ameliorate an identified disease or condition, or to exert a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The exact effective amount for a subject will depend on the subject's weight, size, health, the nature and extent of symptoms, and the therapeutic agent or combination of therapeutic agents selected for administration. The therapeutically effective amount for a given situation can be determined by routine experimentation within the skill and judgment of the clinician.

[0511] It is understood that for any compound, the therapeutically effective amount can be estimated initially in cell culture assays, e.g., tumor cell cell culture assays, or in animal models, usually rats, mice, rabbits, dogs, or pigs. Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine effective doses and routes of administration in humans. Therapeutic / prophylactic efficacy and toxicity can be assessed by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED 50 (the dose that is therapeutic in 50% of the population) and LD 50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the LD 50 / ED 50Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The dosage may vary within this range depending on the dosage form used, sensitivity of the patient, and the route of administration.

[0512] Dosage and administration methods are adjusted to provide sufficient levels of the active agent or to maintain the desired effect. Factors that may be considered include the severity of the condition, the subject's general health, the subject's age, weight, and sex, diet, timing and frequency of administration, drug combinations, reaction sensitivities, and tolerability / response to treatment. Long-acting pharmaceutical compositions may be administered every 3-4 days, every week, or once every two weeks, depending on the half-life and clearance rate of the particular formulation.

[0513] Pharmaceutical compositions containing the active compounds of the present disclosure may be manufactured in a generally known manner, such as by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more pharma- ceutically acceptable carriers, including excipients and / or auxiliaries, that facilitate the processing of the active compounds into preparations that can be used as pharmaceuticals. Of course, the appropriate formulation depends on the route of administration selected.

[0514] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippan, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium, including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, etc. Prolonged absorption of the injectable compositions can be achieved by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0515] Sterile injection solution can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or combination of the above-listed components as required, followed by sterilization by filtration.Generally, dispersion is prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other components required from the above-listed ones.For the preparation of sterile powder for preparation of sterile injection solution, the preparation method is vacuum drying and freeze-drying, whereby the powder of active ingredient and any additional ingredient is obtained from its solution that has been previously sterilized and filtered.

[0516] Oral compositions generally include an inert diluent or an edible pharma- ceutically acceptable carrier. They can be placed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, in which case the compound in the fluid carrier is applied orally, swished in the mouth, and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavor.

[0517] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, such as a gas such as carbon dioxide, or a nebulizer.

[0518] For intranasal administration, the compound is delivered in a solution or in a solid formulation. In some embodiments, the compound is delivered in a solution as a mist, drops, or wipe. In some embodiments, the compound is delivered as a powder. In some embodiments, the compound is included in a kit, which further includes an intranasal applicator.

[0519] Systemic administration can also be carried out by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for permeating the barrier is used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be carried out by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams generally known in the art.

[0520] The active compounds can be prepared with pharma- ceutically acceptable carriers that prevent the compound from being rapidly eliminated from the body, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable and biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for preparing such formulations will be clear to those skilled in the art. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions, including liposomes targeted to infected cells with monoclonal antibodies against viral antigens, can also be used as pharma-ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0521] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate oral or parenteral compositions in dosage unit form.Dosage unit form as used herein refers to a physically separate unit suitable as a unitary dosage for treatment subject, each unit containing a predetermined amount of active compound calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.The specification of dosage unit form of the present disclosure is determined and directly depends on the inherent characteristics of active compound and the specific therapeutic effect to be achieved.

[0522] In therapeutic applications, the dosage of pharmaceutical compositions used according to the present disclosure will vary depending on the agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or professional administering the treatment, among other factors influencing the dosage selected. In general, the dosage should be sufficient to slow and preferably cause regression of tumor growth, preferably even to cause complete regression of the cancer. Dosages may range from about 0.01 mg / kg per day to about 5000 mg / kg per day. An effective amount of an agent is an amount that produces an objectively identifiable improvement by a clinician or other qualified observer. Improved survival and growth indicate regression. As used herein, the term "dosage effective manner" refers to an amount of an active compound that produces a desired biological effect in a subject or cell.

[0523] It will be appreciated that the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0524] It is to be understood that the compounds, nucleic acid agents, or conjugates of the present disclosure can further form salts, and all of these forms are also contemplated as being within the scope of the disclosure as set forth in the claims.

[0525] As used herein, the term "pharmaceutically acceptable salts" refers to derivatives of the compounds of the present disclosure, where the parent compound is modified by making an acid or base salt thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral organic acid salts of basic residues such as amines, alkali organic salts of acidic residues such as carboxylic acids, and the like. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic organic acids. For example, such conventional non-toxic salts include 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, bicarbonate, carbonic acid, citric acid, edetic acid, ethanedisulfonic acid, 1,2-ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, glycolarsanilic acid, hexylresorcylic acid, hydrabamic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxymaleic acid, hydroxynaphthoic acid, isethionic acid, lactic acid, lactobionic acid, laurylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, naphsylic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, subacetic acid, These include those derived from inorganic and organic acids selected from the following commonly occurring amino acids: succinic acid, sulfamic acid, sulfanilic acid, sulfuric acid, tannic acid, tartaric acid, toluenesulfonic acid, and commonly occurring amino acids such as glycine, alanine, phenylalanine, arginine, and the like.

[0526] In some embodiments, the pharma- ceutically acceptable salt is a sodium salt, a potassium salt, a calcium salt, a magnesium salt, a diethylamine salt, a choline salt, a meglumine salt, a benzathine salt, a tromethamine salt, an ammonia salt, an arginine salt, or a lysine salt.

[0527] Other examples of pharma- ceutically acceptable salts include hexanoic acid, cyclopentanepropionic acid, pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, muconic acid, etc. The present disclosure also encompasses salts formed by replacing acidic protons present in the parent compound with metal ions, e.g., alkali metal ions, alkaline earth ions, or aluminum ions, or by coordinating with organic bases, such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. It is understood that in the salt form, the ratio of the compound to the cation or anion of the salt can be 1:1 or any ratio other than 1:1, for example, 3:1, 2:1, 1:2, or 1:3.

[0528] It should be understood that all references to pharma- ceutically acceptable salts include the solvent addition forms (solvates) or crystal forms (polymorphs) of the same salt as defined herein.

[0529] The compound or a pharma- ceutically acceptable salt thereof may be administered orally, nasally, transdermally, pulmonary, inhalation, buccal, sublingually, intraperitoneally, subcutaneously, intramuscularly, intravenously, rectally, intrathoracically, intrathecally, and parenterally. In some embodiments, the compound is administered orally. Those skilled in the art will recognize the advantages of certain routes of administration.

[0530] The dosing regimen using the compound is selected depending on a variety of factors, including the type, species, age, weight, sex, and condition of the patient, the severity of the condition being treated, the route of administration, the renal and hepatic function of the patient, and the particular compound or salt thereof being used. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progression of the condition. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to counter or arrest the progression of the condition.

[0531] Techniques for formulation and administration of the compounds of this disclosure are described in Remington: the Science and Practice of Pharmacy, 1999. th edition, Mack Publishing Co., Easton, PA (1995). In one embodiment, the compounds described herein and their pharma- ceutically acceptable salts are used in pharmaceutical preparations in combination with a pharma- ceutically acceptable carrier or diluent. Suitable pharma- ceutically acceptable carriers include inert solid fillers or diluents and sterile aqueous organic solutions. The compounds are present in such pharmaceutical compositions in an amount sufficient to provide the desired dosage within the range described herein.

[0532] All percentages and ratios used herein are by weight unless otherwise specified. Other features and advantages of the present disclosure are apparent from the various examples. The examples provided illustrate different components and methodologies useful in implementing the present disclosure. These examples do not limit the disclosure described in the claims. Based on the present disclosure, one skilled in the art can identify and use other components and methodologies useful for implementing the present disclosure.

[0533] In the synthesis schemes described herein, for the sake of simplicity, compounds may be depicted in one specific configuration.Such specific configurations should not be interpreted as limiting the present disclosure to one or another isomer, tautomer, positional isomer, or stereoisomer, nor do they exclude mixtures of isomers, tautomers, positional isomers, or stereoisomers.However, it will be understood that a given isomer, tautomer, positional isomer, or stereoisomer may have a higher level of activity than another isomer, tautomer, positional isomer, or stereoisomer.

[0534] All publications and patent documents cited in this specification are incorporated by reference as if each such publication or document was specifically and individually indicated to be incorporated by reference. The citation of publications and patent documents is not intended as an admission that any is pertinent prior art, nor is it an admission that it constitutes the content or date of such pertinent prior art. Although the invention has been described above by written description, those skilled in the art will recognize that the invention can be practiced in various embodiments, and that the foregoing description and the following examples are illustrative and not limiting of the scope of the claims that follow.

[0535] Exemplary embodiment No. Exemplary embodiment 1. A compound of formula (I) or (II): [ka] or a pharma- ceutically acceptable salt thereof, wherein: B is H or a nucleobase moiety; X is H, halogen, or -OR X and R X is H, C1-C6 alkyl, or -(C1-C6 alkyl)-(C6-C 10 aryl), C1-C6 alkyl or -(C1-C6 alkyl)-(C6-C 10 aryl) is one or more RXa optionally replaced by Each R Xa are independently halogen, C1-C6 alkyl, or -O-(C1-C6 alkyl), where C1-C6 alkyl or -O-(C1-C6 alkyl) is optionally substituted with one or more halogens; Y is H, C1-C6 alkyl optionally substituted with one or more halogens, -P(R Y )2, -P(OR Y )(N(R Y )2), -P(=O)(OR Y )R Y , -P(=S)(OR Y )R Y , -P(=O)(SR Y )R Y , -P(=S)(SR Y )R Y , -P(=O)(OR Y )2, -P(=S)(OR Y )2, -P(=O)(SR Y )2, -P(=S)(SR Y ) 2, or a hydroxy protecting group; Each R Y is independently H or C1-C6 alkyl optionally substituted with one or more halogen or cyano; Z is P(R Z )2, -P(OR Z )(N(R Z )2), -P(=O)(OR Z )R Z , -P(=S)(OR Z )R Z , -P(=O)(SR Z )R Z , -P(=S)(SR Z )R Z , -P(=O)(OR Z )2, -P(=S)(OR Z )2, -P(=O)(SR Z )2, or -P(=S)(SR Z )2, Each R Z is independently H or C1-C6 alkyl optionally substituted with one or more halogen or cyano; R 1 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; R 2 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; R 3 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; R 4 is H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; [ka] represents a single bond or a double bond, Each R 6 are independently H, halogen, or C1-C6 alkyl optionally substituted with one or more halogens; A compound or a pharma- ceutically acceptable salt thereof.

[0536] Exemplary embodiment 2. A nucleic acid agent, or a pharma- ceutically acceptable salt thereof, wherein the nucleic acid agent comprises an oligonucleotide comprising one or two 5' terminal units, each 5' terminal unit independently being: [ka] where the variables B, R 1 , R 2 , R 3 , R 4 , R 6 , X, and Z are as described herein, and ## indicates the bond to the remainder of the oligonucleotide, or a nucleic acid agent or a pharma- ceutically acceptable salt thereof.

[0537] Exemplary embodiment 3. A conjugate or a pharma- ceutically acceptable salt thereof, comprising: (i) a nucleic acid agent comprising one or two 5' terminal units covalently attached to an oligonucleotide, wherein each 5' terminal unit is independently [ka] where the variables B, R 1 , R 2 , R 3 , R 4 , R 6 , X, and Z are as described herein, ## indicates the attachment to the remainder of the oligonucleotide, and (ii) one or more ligands covalently attached to the nucleic acid agent or a pharma- ceutically acceptable salt thereof.

[0538] Exemplary embodiment No. 4. The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein B is H.

[0539] The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein exemplary embodiment 5.B is a nucleobase moiety.

[0540] Exemplary embodiment 6. The compound, nucleic acid agent, or conjugate of any one of the preceding exemplary embodiments, wherein the nucleobase moiety is adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U).

[0541] Exemplary embodiment No. 7. The compound, nucleic acid agent, or conjugate of any one of the preceding exemplary embodiments, wherein the nucleobase moiety is a modified nucleobase.

[0542] Exemplary embodiment 8. A compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein the nucleobase moiety is an artificial nucleobase.

[0543] Exemplary embodiment No. 9. The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein X is H.

[0544] Exemplary embodiment 10. The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein X is a halogen.

[0545] Exemplary embodiment No. 11. X is -OR X The compound, nucleic acid agent, or conjugate of any one of the preceding exemplary embodiments, wherein

[0546] Exemplary embodiment No. 12. The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein X is -OH.

[0547] Exemplary embodiment No. 13. The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein X is -O-(C1-C6 alkyl).

[0548] Exemplary embodiment No. 14. A compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein X is -O-(C1-C6 alkyl)-O-(C1-C6 alkyl).

[0549] Exemplary embodiment 15. X is one or more R Xa -O-(C1-C6 alkyl)-(C6-C 10 The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein R is H, R is aryl.

[0550] Exemplary embodiment No. 16. X is -O-(C1-C6 alkyl)-(C6-C 10 The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein R is H, R is aryl.

[0551] Exemplary embodiment No. 17.R X The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein

[0552] Exemplary embodiment No. 18.R Xis C1-C6 alkyl optionally substituted with one or more halogens, or -O-(C1-C6 alkyl) optionally substituted with one or more halogens.

[0553] Exemplary embodiment No. 19.R X is optionally substituted with one or more halogens, C1-C6 alkyl, or -O-(C1-C6 alkyl), 10 The compound, nucleic acid agent, or conjugate of any one of the preceding exemplary embodiments, wherein -O-(C1-C6 alkyl) is optionally substituted with one or more halogens.

[0554] Exemplary embodiment No. 20.R X -(C1-C6 alkyl)-(C6-C 10 The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein R is H, R is aryl.

[0555] Exemplary embodiment No. 21. The compound, nucleic acid agent, or conjugate of any one of the preceding exemplary embodiments, wherein Y is H.

[0556] Exemplary embodiment No. 22. A compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein Y is C1-C6 alkyl optionally substituted with one or more halogens.

[0557] Exemplary embodiment No. 23. Y is -P(R Y )2, -P(OR Y )(N(R Y )2), -P(=O)(OR Y )R Y , -P(=S)(OR Y )R Y , -P(=O)(SR Y )R Y , -P(=S)(SR Y )R Y , -P(=O)(ORY )2, -P(=S)(OR Y )2, -P(=O)(SR Y )2, -P(=S)(SR Y The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein:

[0558] Exemplary embodiment No. 24. The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein Y is a hydroxy protecting group.

[0559] Exemplary embodiment No. 25. The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein Y is silyl.

[0560] Exemplary embodiment No. 26. The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein Y is triphenylmethyl (Tr) or 4,4-dimethoxytrityl (DMTr).

[0561] Exemplary embodiment No. 27. The compound, nucleic acid agent, or conjugate of any one of the preceding exemplary embodiments, wherein Y is optionally substituted acyl or benzyl.

[0562] Exemplary embodiment 28. At least one R Y The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein

[0563] Exemplary embodiment 29. At least one R Y The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein is C1-C6 alkyl optionally substituted with one or more halogen or cyano.

[0564] Exemplary embodiment 30. At least one R Y is H and at least one R YThe compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein is C1-C6 alkyl optionally substituted with one or more halogen or cyano.

[0565] Exemplary embodiment No. 31. The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein when X is -OH, then Y is not H or a hydroxy protecting group.

[0566] Exemplary embodiment No. 32. When X is -OH, Y is C1-C6 alkyl optionally substituted with one or more halogens, -P(R Y )2, -P(OR Y )(N(R Y )2), -P(=O)(OR Y )R Y , -P(=S)(OR Y )R Y , -P(=O)(SR Y )R Y , -P(=S)(SR Y )R Y , -P(=O)(OR Y )2, -P(=S)(OR Y )2, -P(=O)(SR Y )2, or -P(=S)(SR Y The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein:

[0567] Exemplary embodiment No. 33. The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein when Y is H or a hydroxy protecting group, then X is not -OH.

[0568] Exemplary embodiment No. 34. When Y is H or a hydroxy protecting group, X is H, a halogen, or -OR X and -OR X is C1-C6 alkyl or -(C1-C6 alkyl)-(C6-C 10 aryl), C1-C6 alkyl or -(C1-C6 alkyl)-(C6-C 10 aryl) is one or more RXa The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, optionally substituted with:

[0569] Exemplary embodiment No. 35. Z is -P(R Z The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein:

[0570] Exemplary embodiment No. 36. The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein Z is -PH2.

[0571] Exemplary embodiment No. 37. Z is -P(OR Z )(N(R Z The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein:

[0572] Exemplary embodiment No. 38. Z is -P(=O)(OR Z )R Z The compound, nucleic acid agent, or conjugate of any one of the preceding exemplary embodiments, wherein

[0573] Exemplary embodiment No. 39. Z is -P(=S)(OR Z )R Z The compound, nucleic acid agent, or conjugate of any one of the preceding exemplary embodiments, wherein

[0574] Exemplary embodiment No. 40. Z is -P(=O)(SR Z )R Z The compound, nucleic acid agent, or conjugate of any one of the preceding exemplary embodiments, wherein

[0575] Exemplary embodiment No. 41. Z is -P(=S)(SR Z )R Z The compound, nucleic acid agent, or conjugate of any one of the preceding exemplary embodiments, wherein

[0576] Exemplary embodiment No. 42. Z is -P(=O)(OR Z The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein:

[0577] Exemplary embodiment No. 43. Z is -P(=S)(OR Z The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein:

[0578] Exemplary embodiment No. 44. Z is -P(=O)(SR Z The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein:

[0579] Exemplary embodiment No. 45. Z is -P(=S)(SR Z The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein:

[0580] Exemplary embodiment 46. At least one R Z The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein

[0581] Exemplary embodiment 47. At least one R Z The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein is C1-C6 alkyl optionally substituted with one or more halogen or cyano.

[0582] Exemplary embodiment 48. At least one R Z is H and at least one R Z The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein is C1-C6 alkyl optionally substituted with one or more halogen or cyano.

[0583] Exemplary embodiment No. 49.R 1The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein

[0584] Exemplary embodiment No. 50.R 1 The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein is a halogen.

[0585] Exemplary embodiment No. 51.R 1 The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein is C1-C6 alkyl optionally substituted with one or more halogens.

[0586] Exemplary embodiment No. 52.R 2 The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein

[0587] Exemplary embodiment No. 53.R 2 The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein is a halogen.

[0588] Exemplary embodiment No. 54.R 2 The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein is C1-C6 alkyl optionally substituted with one or more halogens. .

[0589] Exemplary embodiment No. 55.R 3 The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein

[0590] Exemplary embodiment No. 56.R 3 The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein is a halogen.

[0591] Exemplary embodiment No. 57.R 3The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein is C1-C6 alkyl optionally substituted with one or more halogens.

[0592] Exemplary embodiment No. 58.R 4 The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein

[0593] Exemplary embodiment No. 59.R 4 The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein is a halogen.

[0594] Exemplary embodiment No. 60.R 4 The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein is C1-C6 alkyl optionally substituted with one or more halogens.

[0595] Exemplary embodiment No. 61.R 6 The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein

[0596] Exemplary embodiment No. 62.R 6 The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein is a halogen.

[0597] Exemplary embodiment No. 63.R 6 The compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments, wherein is C1-C6 alkyl optionally substituted with one or more halogens.

[0598] Exemplary embodiment No. 64.R 1 , R 2 , R 3 , R 4 , and R 6 The compound, nucleic acid agent, or conjugate of any one of the preceding exemplary embodiments, wherein each of is H.

[0599] Exemplary embodiment 65. A compound represented by formula (I'-1), (I'-2), (II'-1), or (II'-2): [ka] or a pharma- ceutically acceptable salt thereof.

[0600] Exemplary embodiment 66. Compounds represented by formula (IA) or (II-A): [ka] or a pharma- ceutically acceptable salt thereof.

[0601] Exemplary embodiment 67. A compound represented by formula (I-A'-1), (I-A'-2), (II-A'-1), or (II-A'-2): [ka] or a pharma- ceutically acceptable salt thereof.

[0602] Exemplary embodiment No. 68. A compound of formula (IB-1), (IB-2), (II-B-1), or (II-B-2): [ka] or a pharma- ceutically acceptable salt thereof.

[0603] Exemplary embodiment No. 69. A compound represented by formula (I-B'-1), (I-B'-2), (I-B'-3), (I-B'-4), (II-B'-1), (II-B'-2), (II-B'-3), or (II-B'-4): [ka] [ka] or a pharma- ceutically acceptable salt thereof.

[0604] Exemplary embodiment 70. The compound is [ka] or a pharma- ceutically acceptable salt thereof, wherein: Y is -P(R Y )2, -P(OR Y )(N(R Y )2), -P(=O)(OR Y )R Y , -P(=S)(OR Y )R Y , -P(=O)(SR Y )R Y , -P(=S)(SR Y )R Y , -P(=O)(OR Y )2, -P(=S)(OR Y )2, -P(=O)(SR Y )2, -P(=S)(SR Y )2, or a hydroxy protecting group (e.g., silyl (e.g., trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, or triisopropylsilyl), triphenylmethyl (Tr), 4,4′-dimethoxytrityl (DMTr), substituted acyl (e.g., optionally substituted acetyl), or benzyl); Each R Y is independently H or C1-C6 alkyl optionally substituted with one or more halogen or cyano; Z is -P(R Z )2, -P(OR Z )(N(R Z )2), -P(=O)(OR Z )R Z , -P(=S)(OR Z )R Z , -P(=O)(SR Z )R Z, -P(=S)(SR Z )R Z , -P(=O)(OR Z )2, -P(=S)(OR Z )2, -P(=O)(SR Z )2, or -P(=S)(SR Z )2, Each R Z is independently H or C1-C6 alkyl optionally substituted with one or more halogen or cyano; A compound according to any one of the preceding exemplary embodiments.

[0605] Exemplary embodiment 71. The compound is [ka] [ka] [ka] [ka] [ka] or a pharma- ceutically acceptable salt thereof, wherein B is adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U).

[0606] Exemplary embodiment No. 72. The compound of any one of the preceding exemplary embodiments, wherein the compound is selected from the compounds set forth in Table E and pharma- ceutically acceptable salts thereof.

[0607] Exemplary embodiment No. 73. A compound which is an isotopic derivative of a compound according to any one of the preceding exemplary embodiments.

[0608] Exemplary embodiment No. 74. The nucleic acid agent of any one of the preceding exemplary embodiments, comprising single-stranded RNA.

[0609] Exemplary embodiment No. 75. The nucleic acid agent of any one of the preceding exemplary embodiments, comprising double-stranded RNA.

[0610] Exemplary embodiment 76. A double-stranded RNA (e.g., a double-stranded siRNA) and one or two 5'-end units, the 5'-terminal unit is attached (e.g., at the 5'-terminal position) to the sense strand of a double-stranded RNA (e.g., a double-stranded siRNA); and / or The 5' terminal unit is attached (e.g., at the 5' terminal position) to the antisense strand of a double-stranded RNA (e.g., a double-stranded siRNA); The nucleic acid agent according to any one of the preceding exemplary embodiments.

[0611] Exemplary embodiment 77. The 5' terminal unit in the nucleic acid agent is [ka] or a pharma- ceutically acceptable salt thereof, wherein: Z is -P(R Z )2, -P(OR Z )(N(R Z )2), -P(=O)(OR Z )R Z , -P(=S)(OR Z )R Z , -P(=O)(SR Z )R Z , -P(=S)(SR Z )R Z , -P(=O)(OR Z )2, -P(=S)(OR Z )2, -P(=O)(SR Z )2, or -P(=S)(SR Z )2, Each R Z is independently H or C1-C6 alkyl optionally substituted with one or more halogen or cyano; The nucleic acid agent according to any one of the preceding exemplary embodiments.

[0612] Exemplary embodiment 78. The 5' terminal unit in the nucleic acid agent is [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or a pharma- ceutically acceptable salt thereof, wherein B is adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U).

[0613] Exemplary embodiment No. 79. The nucleic acid agent of any one of the preceding exemplary embodiments, wherein the 5' terminal unit in the nucleic acid agent is selected from the 5' terminal units set forth in Table N.

[0614] Exemplary embodiment No. 80. A conjugate according to any one of the preceding exemplary embodiments, comprising a single-stranded RNA (e.g., a single-stranded siRNA), one or more ligands, and one 5'-terminal unit.

[0615] Exemplary embodiment No. 81. A conjugate according to any one of the preceding exemplary embodiments, comprising a double-stranded RNA (e.g., a double-stranded siRNA), one or more ligands, and one or two 5'-terminal units.

[0616] Exemplary embodiment 82. A nucleic acid sequence comprising a double-stranded RNA (e.g., a double-stranded siRNA), one or more ligands, and one or two 5'-terminal units, the 5'-terminal unit is attached (e.g., at the 5'-terminal position) to the sense strand of a double-stranded RNA (e.g., a double-stranded siRNA); and / or The 5' terminal unit is attached (e.g., at the 5' terminal position) to the antisense strand of a double-stranded RNA (e.g., a double-stranded siRNA); The conjugate according to any one of the preceding exemplary embodiments.

[0617] Exemplary embodiment 83. The 5' terminal unit in the conjugate is [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or a pharma- ceutically acceptable salt thereof, wherein B is adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U).

[0618] Exemplary embodiment No. 84. A conjugate according to any one of the preceding exemplary embodiments, wherein the 5' end unit is selected from the conjugates set forth in Table C.

[0619] Exemplary embodiment No. 85. A nucleic acid agent or conjugate according to any one of the preceding exemplary embodiments, wherein the ligand comprises a carbohydrate moiety.

[0620] Exemplary embodiment No. 86. The nucleic acid agent or conjugate of any one of the preceding exemplary embodiments, wherein the carbohydrate moiety comprises a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide.

[0621] Exemplary embodiment No. 87. The nucleic acid agent or conjugate of any one of the preceding exemplary embodiments, wherein the carbohydrate moiety comprises galactose or a derivative thereof.

[0622] Exemplary embodiment 88. The ligand is [ka] The nucleic acid agent or conjugate of any one of the preceding exemplary embodiments, comprising:

[0623] Exemplary embodiment 89. The ligand is [ka] The nucleic acid agent or conjugate of any one of the preceding exemplary embodiments, comprising:

[0624] Exemplary embodiment 90. The ligand is: [ka] The nucleic acid agent or conjugate of any one of the preceding exemplary embodiments, comprising:

[0625] Exemplary embodiment 91. The ligand is: [ka] The nucleic acid agent or conjugate of any one of the preceding exemplary embodiments, comprising:

[0626] Exemplary embodiment 92. The ligand is [ka] The nucleic acid agent or conjugate of any one of the preceding exemplary embodiments, comprising:

[0627] Exemplary embodiment 93. The ligand is [ka] The nucleic acid agent or conjugate of any one of the preceding exemplary embodiments, comprising:

[0628] Exemplary embodiment 94. The ligand is [ka] The nucleic acid agent or conjugate of any one of the preceding exemplary embodiments, comprising:

[0629] Exemplary embodiment 95. The ligand is: [ka] The nucleic acid agent or conjugate of any one of the preceding exemplary embodiments, comprising:

[0630] Exemplary embodiment 96. The ligand is [ka] The nucleic acid agent or conjugate of any one of the preceding exemplary embodiments, comprising:

[0631] Exemplary embodiment 97. The ligand is [ka] The nucleic acid agent or conjugate of any one of the preceding exemplary embodiments, comprising:

[0632] Exemplary embodiment 98. The ligand is: [ka] The nucleic acid agent or conjugate of any one of the preceding exemplary embodiments, comprising:

[0633] Exemplary embodiment 99. The ligand is: [ka] The nucleic acid agent or conjugate of any one of the preceding exemplary embodiments, comprising:

[0634] Exemplary embodiment 100. The ligand comprises: [ka] The nucleic acid agent or conjugate of any one of the preceding exemplary embodiments, comprising:

[0635] Exemplary embodiment 101. The ligand is [ka] The nucleic acid agent or conjugate of any one of the preceding exemplary embodiments, comprising:

[0636] Exemplary embodiment 102. The ligand is [ka] The nucleic acid agent or conjugate of any one of the preceding exemplary embodiments, comprising:

[0637] Exemplary embodiment 103. The ligand is [ka] The nucleic acid agent or conjugate of any one of the preceding exemplary embodiments, comprising:

[0638] Exemplary embodiment No. 104. A nucleic acid agent or conjugate according to any one of the preceding exemplary embodiments, wherein the ligand comprises a lipid.

[0639] Exemplary embodiment No. 105. A nucleic acid agent or conjugate according to any one of the preceding exemplary embodiments, wherein the ligand comprises a peptide moiety.

[0640] Exemplary embodiment No. 106. A nucleic acid agent or conjugate according to any one of the preceding exemplary embodiments, wherein the ligand comprises an antibody moiety.

[0641] Exemplary embodiment No. 107. The nucleic acid agent or conjugate of any one of the preceding exemplary embodiments, wherein the nucleic acid agent comprises an oligonucleotide.

[0642] Exemplary embodiment No. 108. A nucleic acid agent or conjugate according to any one of the preceding exemplary embodiments, wherein the nucleic acid agent comprises one or more phosphate groups or one or more phosphate group analogs.

[0643] Exemplary embodiment No. 109. A nucleic acid agent or conjugate according to any one of the preceding exemplary embodiments, wherein the linker unit is attached to the nucleic acid agent via a phosphate group or a phosphate group analog in the nucleic acid agent.

[0644] Exemplary embodiment No. 110. The nucleic acid agent or conjugate of any one of the preceding exemplary embodiments, wherein the nucleic acid agent comprises RNA.

[0645] Exemplary embodiment 111. The nucleic acid agent or conjugate of any one of the preceding exemplary embodiments, wherein the oligonucleotide is an siRNA, microRNA, anti-microRNA, microRNA mimic, anti-miR, antagomir, dsRNA, ssRNA, aptamer, immunostimulatory oligonucleotide, decoy oligonucleotide, splice variant oligonucleotide, triplex forming oligonucleotide, G-quadruplex, or antisense oligonucleotide.

[0646] Exemplary embodiment No. 112. A pharmaceutical composition comprising a compound, nucleic acid agent, or conjugate according to any one of the preceding exemplary embodiments.

[0647] Exemplary embodiment No. 113. A method for regulating expression of a target gene in a subject, comprising administering to the subject a conjugate described in any one of the preceding exemplary embodiments.

[0648] Exemplary embodiment No. 114. A method for delivering a nucleic acid agent to a subject, comprising administering to the subject a conjugate described in any one of the preceding exemplary embodiments.

[0649] Exemplary embodiment No. 115. A method for treating or preventing a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a conjugate according to any one of the preceding exemplary embodiments.

[0650] Exemplary embodiment No. 116. A conjugate according to any one of the preceding exemplary embodiments for modulating expression of a target gene in a subject.

[0651] Exemplary embodiment No. 117. A conjugate according to any one of the preceding exemplary embodiments for delivering a nucleic acid agent to a subject.

[0652] Exemplary embodiment No. 118. A conjugate according to any one of the preceding exemplary embodiments for treating or preventing a disease in a subject in need thereof.

[0653] Exemplary embodiment No. 119. Use of a conjugate according to any one of the preceding exemplary embodiments in the manufacture of a medicament for modulating expression of a target gene in a subject.

[0654] Exemplary embodiment No. 120. Use of a conjugate according to any one of the preceding exemplary embodiments in the manufacture of a medicament for delivering a nucleic acid agent to a subject.

[0655] Exemplary embodiment No. 121. Use of a conjugate according to any one of the preceding exemplary embodiments in the manufacture of a medicament for treating or preventing a disease in a subject in need thereof.

[0656] Exemplary embodiment No. 122. The method, conjugate, or use according to any one of the preceding exemplary embodiments, wherein the subject is a human. EXAMPLES

[0657] Example 1. Synthesis of 5'-end unit compounds. [ka]

[0658] ((3aR,4R,6R,6aS)-6-Amino-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)methanol (1-2). To a solution of compound 1-1 (50.0 g, 272.28 mmol, 1 equiv, HCl salt) in MeOH (300 mL) was added TsOH (59.0 g, 310.40 mmol, 1.14 equiv) and dimethoxypropane (198.5 g, 1.91 mol, 233.53 mL, 7 equiv) at 25 °C. The reaction mixture was stirred at 25 °C under N for 5 h. The reaction mixture was quenched with 7 Mn H / MeOH and concentrated under reduced pressure to give a crude residue. The residue was redissolved in 2M KCO (500 mL), extracted with EA / DCM (1 / 1, 1000 mL×3), dried over NaSO, filtered and concentrated under reduced pressure to give crude 1-2 (43.2 g), which was used in the next step without further purification. 1 H NMR: 400MHz, DMSO-d 6, δ4.47(d,J=6.0Hz,1H),4.14(d,J=6.0Hz,1H),3.45-3.40(m,3H),3.38-3. 23(m,1H),2.16-2.08(m,2H),1.25(s,3H),1.24-1.21(m,2H),1.08(s,3H).

[0659] (E)-3-Ethoxy-N-(((3aS,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxol-4-yl)carbamoyl)acrylamide (1-3). To a solution of compound 1-2 (38.7 g, 206.69 mmol, 1 equiv.) in DMF (230 mL) was added isocyanate (58.3 g, 413.38 mmol, 2 equiv.) at -10°C. The mixture was stirred at 25°C for 16 h and then concentrated under vacuum to give crude compound 1-3 as a yellow oil, which was used in the next step without further purification.

[0660] 1-((3aS,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]-dioxol-4-yl)pyrimidine-2,4(1H,3H)-dione (1-4). A solution of compound 1-3 (8.48 g, 25.83 mmol, 1 equiv.) in NH3·H2O (61.4 g, 578.14 mmol, 67.47 mL, 33%, 22.39 equiv.) was stirred at 90 °C for 16 h. The mixture was then cooled to 25 °C and concentrated under vacuum. The residue was purified by column chromatography (SiO2, DCM:MeOH=50:1 to 5:1) to give compound 1-4 (37.0 g, 131.07 mmol, 63.4% yield) as a yellow solid.

[0661] 1-((1R,2S,3R,4R)-2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl)pyrimidine-2,4(1H,3H)-dione (1-5). A solution of compound 1-4 (37.0 g, 131.07 mmol, 1 equiv.) in AcOH (233.10 g, 3.11 mol, 222 mL, 80% purity, 23.69 equiv.) was stirred at 80 °C for 3 h. The mixture was then concentrated under vacuum and coevaporated with toluene (100 mL x 3) and pyridine (100 mL x 3). The residue was purified by column chromatography (SiO2, DCM:MeOH = 50:1 to 5:1) to give compound 1-5 (24.0 g, 99.08 mmol, 75.6% yield) as a yellow solid. 1 H NMR:400MHz,DMSO-d6,δ7.67(d,J=8.0Hz,1H),5.58(d,J=8.0Hz,1H),4.85(d,J=6.4Hz,1H),4.67-4.64(m,3H), 4.56(t,J=1.4Hz,1H),4.36-3.98(m,1H),3.71(s,1H),3.45-3.38(m,3H),2.01-1.92(m,2H),1.27-1.23(m,1H).

[0662] (5aR,7R,8R,8aR)-8-Hydroxy-7-(hydroxymethyl)-5a,7,8,8a-tetrahydro-2H,6H-cyclopenta-[4,5]oxazolo[3,2-a]pyrimidin-2-one (1-6). To a solution of compound 1-5 (30.0 g, 123.85 mmol, 1 equiv.) in DMF (300 mL) was added DPC (39.80 g, 185.78 mmol, 1.5 equiv.) and NaHCO3 (5.20 g, 61.93 mmol, 2.41 mL, 0.5 equiv.). The mixture was stirred at 120° C. for 16 h. The reaction was then cooled to 25° C., poured into MTBE (3000 mL), and filtered. The residue was further concentrated in vacuo to give crude 1-6 (27.8 g) as a brown solid, which was used directly in the next step without further purification.

[0663] 1-((1R,2S,3R,4R)-3-hydroxy-4-(hydroxymethyl)-2-methoxycyclopentyl)pyrimidine-2,4(1H,3H)-dione (1-7). To a solution of compound 1-6 (5.55 g, 24.75 mmol, 1 equiv) in MeOH (55 mL) was added trimethyl borate (5.14 g, 49.51 mmol, 5.59 mL, 2 equiv), trimethoxymethane (2.63 g, 24.75 mmol, 2.71 mL, 1 equiv), and NaHCO3 (20.8 mg, 247.53 μmol, 9.63 μL, 0.01 equiv). The mixture was stirred at 140 °C for 16 h in a 100 mL autoclave. The mixture was then cooled to 25 °C and concentrated under vacuum. The residue was purified by column chromatography (SiO2, DCM:MeOH=50:1 to 5:1) to give compound 1-7 (11.0 g, 42.93 mmol, yield 34.7%) as a yellow solid. 1 H NMR: 400MHz, DMSO-d 6,δ11.24(s,1H),7.70(d,J=8.0Hz,1H),5.59(d,J=8.0Hz,1H),4.75-4.71(m,2H),4.62(d,J=4.8Hz,1H),3.94-3.73( m,1H),3.41-3.40(m,1H),3.39-3.38(m,2H),3.25(s,3H),3.17-3.16(m,1H),2.06-1.95(m,2H),1.27-1.25(m,1H).

[0664] 1-((1R,2S,3R,4R)-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-hydroxy-2-methoxycyclopentyl)pyrimidine-2,4(1H,3H)-dione (1-8). To a solution of compound 1-7 (11.0 g, 42.93 mmol, 1 equiv) in pyridine (110 mL) was added DMTrCl (17.5 g, 51.51 mmol, 1.2 equiv) at 25° C. The mixture was stirred at 25° C. for 1 h, quenched with MeOH (5 mL), and concentrated in vacuo. The residue was dissolved in EtOAc (200 mL) and washed with aqueous citric acid (100 mL) and brine (100 mL). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo to give crude compound 1-8 (24.0 g, crude) as a yellow solid, which was used in the next step without further purification.

[0665] 1-((1R,2S,3R,4R)-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)-2-methoxycyclopentyl)pyrimidine-2,4(1H,3H)-dione (1-9). To a solution of compound 1-8 (24.0 g, 42.93 mmol, 1 equiv) in DCM (168 mL) was added imidazole (7.31 g, 107.32 mmol, 2.5 equiv) and TBSCl (7.76 g, 51.51 mmol, 6.31 mL, 1.2 equiv). The mixture was stirred at 25 °C for 16 h. The reaction was then extracted with DCM (200 mL) and washed with aqueous NaHCO3 (200 mL) and brine (200 mL). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo to give compound 1-9 (28.9 g, crude) as a yellow oil, which was used in the next step without further purification.

[0666] 1-((1R,2S,3R,4R)-3-((tert-butyldimethylsilyl)oxy)-4-(hydroxymethyl)-2-methoxycyclopentyl)pyrimidine-2,4(1H,3H)-dione (1-10). To a solution of compound 1-9 (28.9 g, 42.92 mmol, 1 equiv) in DCM (180 mL) was added dodecane-1-thiol (17.4 g, 85.84 mmol, 20.56 mL, 2 equiv) and DCA (22.1 g, 171.68 mmol, 14.10 mL, 4 equiv) at 0 °C. The reaction was stirred at 25 °C for 2 h. The mixture was then washed with aqueous NaHCO3 (200 mL), extracted with DCM (200 mL), and washed with brine (200 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under vacuum. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 2 / 1 to 0 / 1) to give compound 1-10 (9.70 g, 26.18 mmol, 61.00% yield) as a yellow solid. 1 H NMR: 400MHz, DMSO-d 6,δ11.25(1H),7.71(d,J=8.0Hz,1H),5.62-5.59(m,1H),4.79-4.73(m,2H),4.14-4.13(m,1H),3.78-3.75(m,1H),3.41- 3.38(m,2H),3.23(s,3H),2.11-2.05(m,1H),1.96-1.94(m,1H),1.23-1.22(m,1H),0.87(s,9H),0.07(d,J=5.6Hz,6H).

[0667] Diethyl ((E)-2-((1R,2R,3S,4R)-2-((tert-butyldimethylsilyl)oxy)-3-methoxy-4-(2-oxo-3,4-dihydropyrimidin-1(2H)-yl)cyclopentyl)vinyl)phosphonate (1-11). To a solution of compound 1-10 (8.70 g, 23.48 mmol, 1 equiv) in ACN (174 mL) was added IBX (8.55 g, 30.53 mmol, 1.3 equiv). The mixture was stirred at 80° C. for 1 h and then cooled to 25° C. The reaction was filtered and the filtrate was concentrated under vacuum to give the crude aldehyde (7.50 g) as a white solid. The aldehyde was redissolved in THF (30 mL). To a solution of phosphonate (9.39 g, 32.56 mmol, 1.6 equiv.) in THF (45 mL), t-BuOK (1 M, 30.53 mL, 1.5 equiv. in t-BuOH) was added dropwise at 0 °C. The mixture was stirred at 0 °C for 0.5 h. Then, at 0 °C, the mixture was slowly added to the solution of aldehyde prepared above. The resulting mixture was stirred at 0 °C for 1 h and further stirred at 25 °C for 1 h. The reaction was then poured into aqueous NH4Cl (200 mL), extracted with ethyl acetate (200 mL × 2), and washed with brine (200 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under vacuum. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 5 / 1 to 0 / 1) to give compound 1-11 (10.2 g, containing about 30% phosphonate) as a yellow oil. 1H NMR:400MHz,CDCl3,δ8.96(s,1H),7.15(d,J=8.0Hz,1H),6.81-6.69(m,1H),5.80-5.71(m,2H),4.44-4.43(m,1H),4.18-4.07(m,6H),3. 85(t,J=5.6Hz,1H),3.38(s,3H),2.83-2.75(m,1H),2.36-2.29(m,1H),1.86-1.78(m,4H),1.36-1.24(m,7H),0.91(s,9H),0.10(s,6H).

[0668] Diethyl ((E)-2-((1R,2R,3S,4R)-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-hydroxy-3-methoxycyclopentyl)vinyl)phosphonate (1-12). To a solution of compound 1-11 (3.00 g, 5.97 mmol, 1 equiv.) in MeOH (30 mL) was added NHF (2.21 g, 59.69 mmol, 10 equiv.). The reaction was stirred at 65 °C for 16 h. The mixture was then cooled to 25 °C and concentrated under vacuum. The residue was purified by column chromatography (SiO2, DCM:MeOH = 50:1 to 5:1) to give compound 1-12 (1.40 g, 3.60 mmol, 60.40% yield) as a yellow solid. 1 H NMR:400MHz,CDCl3,δ8.84(s,1H),7.13(d,J=8.0Hz,1H),6.89-6.77(m,1H),5.81-5.75(m,1H),4.54-4.50(m,1H),4.14- 4.07(m,5H),3.93-3.91(m,1H),3.45(s,3H),2.77-2.73(m,2H),2.36-2.28(m,1H),1.85-1.76(m,1H),1.37-1.32(m,6H).

[0669] 2-Cyanoethyl ((1R,2S,3R,5R)-5-((E)-2-(diethoxyphosphoryl)vinyl)-3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-methoxycyclopentyl)diisopropylphosphoramidite (1-13). To a solution of compound 1-12 (1.40 g, 3.60 mmol, 1 equiv) in DCM (14 mL) was added DCI (510.9 mg, 4.33 mmol, 1.2 equiv) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (1.63 g, 5.41 mmol, 1.72 mL, 1.5 equiv) at 25° C. The reaction was stirred at 25° C. for 1 h. The mixture was then diluted with DCM (100 mL) and washed with aqueous NaHCO3 (100 mL) and brine (100 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 3 / 1 to 0 / 1, 0.1% TEA) to give compound 1-13 (C-VP) (1.40 g, 2.38 mmol, 66% yield) as a white solid. 1 H NMR:400MHz,CD3CN,δ9.11(s,1H),7.38-7.34(m,1H),6.80-6.66(m,1H),5. 87-5.73(m,1H),5.60-5.58(m,1H),4.62-4.58(m,1H),4.26-4.03(m,1H),4. 02-3.59(m,9H),3.37-3.33(m,3H),2.97-2.85(m,1H),2.74-2.62(m,2H),2 .33-2.24(m,1H),1.73-1.65(m,1H),1.29-1.24(m,6H),1.20-1.13(m,12H).

[0670] Diethyl (2-((1R,2R,3S,4R)-2-((tert-butyldimethylsilyl)oxy)-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-methoxycyclopentyl)ethyl)phosphonate (1-14). To a solution of compound 1-11 (7.20 g, 14.33 mmol, 1 equiv) in MeOH (72 mL) was added Pd / C (3.6 g, 10% purity) and AcOH (2.27 g, 37.77 mmol, 2.16 mL, 2.64 equiv) at 25 °C. The reaction was stirred under H2 (15 psi) at 25 °C for 2 h. The mixture was then filtered and concentrated in vacuo to give crude compound 1-14 (7.23 g) as a yellow oil, which was used in the next step without further purification.

[0671] Diethyl (2-((1R,2R,3S,4R)-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-hydroxy-3-methoxycyclopentyl)ethyl)phosphonate (1-15). To a solution of compound 1-14 (7.23 g, 14.33 mmol, 1 equiv.) in MeOH (72 mL) was added NHF (5.31 g, 143.27 mmol, 10 equiv.). The reaction was stirred at 65 °C for 16 h. The mixture was then cooled to 25 °C and concentrated under vacuum. The residue was purified by column chromatography (SiO2, DCM:MeOH=50:1 to 5:1) to give compound 1-15 (3.70 g, 9.48 mmol, 66.2% yield) as a yellow oil. 1 H NMR:400MHz,DMSO-d6,δ11.2(s,1H),7.74(d,J=8.0Hz,1H),5.58(d,J=7.6Hz,1H),4.67-4.63(m,2H),4.00-3.96(m, 4H),3.73-3.71(m,2H),3.26(s,3H),2.08-2.01(m,1H),1.81-1.70(m,4H),1.51-1.44(m,1H),1.23(t,J=7.0Hz,6H).

[0672] 2-Cyanoethyl ((1R,2S,3R,5R)-5-(2-(diethoxyphosphoryl)ethyl)-3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-methoxycyclopentyl)diisopropylphosphoramidite (1-16). To a solution of compound 1-15 (2.70 g, 6.92 mmol, 1 equiv) in DCM (27 mL) was added DCI (980.2 mg, 8.30 mmol, 1.2 equiv) and 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (3.13 g, 10.37 mmol, 3.30 mL, 1.5 equiv) at 25° C. The reaction was stirred at 25° C. for 1 h. The mixture was then diluted with DCM (100 mL) and washed with aqueous NaHCO3 (100 mL) and brine (100 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 3 / 1 to 0 / 1, 0.1% TEA) to give compound 1-16 (C-EP) (3.9 g, 70% yield) as a white oil. 1 H NMR:400MHz,CD3CN,δ9.09(s,1H),7.38-7.35(m,1H),5.60-5.57(m,1H),4. 67-4.62(m,1H),4.05-4.00(m,5H),3.99-3.62(m,5H),3.34-3.31(m,3H),2. 69-2.66(m,2H),2.29-2.21(m,1H),2.12-2.06(m,1H),1.89-1.68(m,3H),1 .60-1.50(m,1H),1.38-1.32(m,1H),1.30-1.24(m,6H),1.22-1.14(m,12H).

[0673] 2-Cyanoethyl((1R,2S,3R,5R)-5-(2-(dimethoxyphosphoryl)ethyl)-3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-methoxycyclopentyl)diisopropylphosphoramidite (2-16) [ka] Using a procedure similar to that described above to prepare 1-16, the dimethyl ester of the carbocyclic uridine phosphoramidite 2-16 was prepared.

[0674] Example 2. General procedure for oligonucleotide synthesis Oligonucleotides were synthesized according to standard oligonucleotide synthesis procedures using 2' modified phosphoramidites (Hongene Biotech), the carbocyclic nucleotide phosphoramidites described above for the antisense strand, and G1b GalNAc phosphoramidites for the sense strand (PCT / US2022 / 039517).

[0675] To prepare the antisense strand bearing the monomethyl-protected carbocyclic nucleotide [5'-C-MeEPmU], the synthesized oligonucleotide was deprotected under standard cleavage and deprotection conditions using concentrated ammonia (28-30 wt%) at 45 °C for 1 day, simultaneously removing one methyl group.

[0676] To prepare the antisense strand containing the fully deprotected carbocyclic nucleotide [5'-C-EPmU], the solid support column was first treated with TMSI / pyridine / CH2Cl2 at room temperature for 1 h and quenched with 2-mercaptoethanol in TEA / CH3CN solution, and then the standard cleavage and deprotection conditions described above were used to obtain the fully cleaved and deprotected antisense strand.

[0677] The crude oligonucleotides were analyzed by SAX-HPLC and HR-LC-MS and purified by SAX-HPLC. The pure fractions were combined, concentrated, desalted, and lyophilized to obtain purified sense and antisense strands. The sense and antisense strands were then redissolved in water and annealed to obtain duplexes based on a 1:1 molar ratio.

[0678] Example 3. mRNA knockdown activity of siRNA molecules incorporating 5'-carbocyclic nucleotides against target genes in mouse liver The siRNA compounds listed in Table 1 were used to examine gene silencing activity in mouse liver. These siRNA molecules incorporating 5'-C-MeEP or 5'-C-EP were compared to siRNA molecules containing 5'-(E)-vinylphosphonate (5'-VP) or 5'-hydroxyl group (5'-OH). As shown in Figures 1A and 1B, both 5'-C-MeEP and 5'-C-EP showed improved knockdown potency in mouse liver compared to 5'-VP and 5'-OH.

[0679] CD-1 female mice were subcutaneously administered 0.5 mg / kg of siRNA molecules. The control group received phosphate-buffered saline (PBS). Four days after treatment, animals were hydrodynamically injected (HDI) with 20 μg of target gene 2 in pcDNA3.1(+) via the tail vein. One day after treatment, mice were sacrificed. Liver tissue was collected and stored in RNAlater® at 4°C overnight and transferred to -80°C after removal from RNAlater for mRNA analysis. Target mRNA reduction was measured by qPCR using a CFX384TOUCH™ Real-Time PCR Detection System (BioRad Laboratories, Inc., Hercules, CA). All samples were normalized to PBS-treated control animals and plotted using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA).

[0680] Example 4. mRNA knockdown activity of siRNA molecules incorporating carbocyclic nucleotides against target genes in mouse extrahepatic tissues Gene silencing activity in mouse extrahepatic tissues was studied using fully chemically modified siRNA molecules conjugated with specific targeting ligands for extrahepatic tissues. These siRNA molecules incorporating 5'-C-MeEP or 5'-C-EP were compared to 5'-(E)-vinylphosphonate (5'-VP) or 5'-hydroxyl group (5'-OH). As shown in Figures 2A-2E, both 5'-C-MeEP and 5'-C-EP showed improved knockdown potency compared to 5'-OH and at least equivalent or improved activity to 5'-VP in multiple extrahepatic tissues.

[0681] CD-1 female mice were subcutaneously administered 3 mg / kg of siRNA molecules. The control group received phosphate-buffered saline (PBS). Ten days after treatment, the mice were sacrificed. Multiple extrahepatic tissues were collected and stored in RNAlater® at 4°C overnight and transferred to -80°C after removal from RNAlater for mRNA analysis. Target mRNA reduction was measured by qPCR using the CFX384TOUCH™ Real-Time PCR Detection System (BioRad Laboratories, Inc., Hercules, CA). All samples were normalized to PBS-treated control animals and plotted using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA). [Table 4] The lowercase f / m indicate 2'-deoxy-2'-fluoro (2'-F) and 2'-O-methyl (2'-OMe) sugar modifications to adenosine, cytidine, guanosine, and uridine, respectively; s indicates a phosphorothioate (PS) bond; and VP, C-EPmU, C-MeEPmU, and G1b indicate the chemical structures shown below. [ka]

[0682] Equivalent The details of one or more embodiments of the present disclosure are described in the above description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described herein. Other features, objects, and advantages of the present disclosure will be apparent from the above description and the claims. In this specification and the appended claims, the singular forms include plural referents unless otherwise clearly indicated by the context. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications cited herein are incorporated by reference.

[0683] The foregoing description is presented for purposes of illustration only and is not intended to limit the disclosure to the precise form disclosed, but rather is intended to be limited by the claims appended hereto.

Claims

1. Compounds of formula (I) or (II): 【Chemistry 1】 or a pharmaceutically acceptable salt thereof, wherein: B is H or a nucleobase moiety; X is H, halogen, or -OR Ｘ and R Ｘ is H, C １ ~C ６ alkyl, or -(C １ ~C ６ alkyl)-(C ６ ~C １０ aryl), and １ ~C ６ Alkyl or -(C １ ~C ６ alkyl)-(C ６ ~C １０ aryl) may be one or more R Ｘａ optionally replaced by Each R Ｘａ are independently halogen, C １ ~C ６ Alkyl, or —O—(C １ ~C ６ alkyl), and １ ~C ６ Alkyl or —O—(C １ ~C ６ alkyl) is optionally substituted with one or more halogens; Y is H, C optionally substituted with one or more halogens １ ~C ６ Alkyl, -P(R Ｙ ) ２ , -P(OR Ｙ ) (N (R Ｙ ) ２ ), -P(=O)(OR Ｙ ) R Ｙ , -P(=S)(OR)R Ｙ , -P(=O)(SR Ｙ ) R Ｙ , -P(=S)(SR)R Ｙ , -P(=O)(OR Ｙ ) ２ , -P(=S)(OR Ｙ ) ２ , -P(=O)(SR Ｙ ) ２ , -P(=S)(SR Ｙ ) ２ or a hydroxy protecting group, Each R Ｙ are independently H or C optionally substituted with one or more halogens or cyanos. １ ~C ６ is alkyl, Z is P(R Ｚ ), -P(OR ２ ), -P(=O)(OR Ｚ )(N(R Ｚ ), -P(=O)(OR ２ ), -P(=O)(OR Ｚ ), -P(=S)(OR Ｚ ), -P(=S)(OR Ｚ ), -P(=O)(SR Ｚ ), -P(=S)(SR Ｚ ), -P(=O)(OR Ｚ ), -P(=S)(OR Ｚ ), -P(=O)(SR Ｚ ), -P(=S)(SR Ｚ ), or -P(=S)(SR ２ ), -P(=S)(OR Ｚ ), -P(=O)(SR ２ ), or -P(=S)(SR Ｚ ), and is​​​​​​ Each R Ｚ are independently H or C optionally substituted with one or more halogens or cyanos. １ ~C ６ is alkyl, R １ is H, halogen, or C optionally substituted with one or more halogens １ ~C ６ is alkyl, R ２ is H, halogen, or C optionally substituted with one or more halogens １ ~C ６ is alkyl, R ３ is H, halogen, or C optionally substituted with one or more halogens １ ~C ６ is alkyl, R ４ is H, halogen, or C optionally substituted with one or more halogens １ ~C ６ is alkyl, 【Chemistry 2】 indicates a single bond, Each R ６ are independently H, halogen, or C optionally substituted with one or more halogens. １ ~C ６ is alkyl, The compound or a pharmaceutically acceptable salt thereof.

2. The compound, 【Chemistry 3-1】 【Chemistry 3-2】 and pharmaceutically acceptable salts thereof.

3. The compound 【Chemistry 4】 or a pharmaceutically acceptable salt thereof.

4. A nucleic acid agent or a pharmaceutically acceptable salt thereof, wherein the nucleic acid agent comprises an oligonucleotide comprising one or two 5' terminal units, each 5' terminal unit independently comprising: 【Transformation 5】 wherein: B is H or a nucleobase moiety; X is H, halogen, or —OR X ; R X is H, C 1 -C 6 alkyl, or —(C 1 -C 6 alkyl)-(C 6 -C 10 aryl), wherein said C 1 -C 6 alkyl or —(C 1 -C 6 alkyl)-(C 6 -C 10 aryl) is optionally substituted with one or more R Xa ; each R Xa is independently halogen, C 1 -C 6 alkyl, or —O—(C 1 -C 6 alkyl), wherein said C 1 -C 6 alkyl or —O—(C 1 -C 6 alkyl) is optionally substituted with one or more halogens; Z is -P(RZ) 2, -P(ORZ)(N(RZ)2), -P(=O)(ORZ)RZ, -P(=S)(ORZ)RZ, -P(=O)(SRZ)RZ , -P(=S)(SRZ)RZ, -P(=O)(ORZ)2, -P(=S)(ORZ)2, -P(=O)(SRZ)2, or P(=S)(SRZ)2; each R Z is independently H or C 1 -C 6 alkyl optionally substituted with one or more halogen or cyano; R 1 is H, halogen, or C 1 -C 6 alkyl optionally substituted with one or more halogens; R 2 is H, halogen, or C 1 -C 6 alkyl optionally substituted with one or more halogens; R 3 is H, halogen, or C 1 -C 6 alkyl optionally substituted with one or more halogens; R 4 is H, halogen, or C 1 -C 6 alkyl optionally substituted with one or more halogens; R 6 is H, halogen, or C 1 -C 6 alkyl optionally substituted with one or more halogens; 【Transformation 6】 represents a single bond; ## indicates the bond to the remainder of the oligonucleotide, A nucleic acid agent or a pharmaceutically acceptable salt thereof.

5. A nucleic acid agent comprising: 【Chemistry 7-1】 【Chemistry 7-2】 【Transformation 7-3】 【Chemistry 7-4】 The nucleic acid agent of claim 4, comprising one or two of the following:

6. The method of claim 1, wherein the 5' terminal unit is: 【Transformation 8】 The nucleic acid agent according to claim 4, wherein

7. A conjugate or a pharmaceutically acceptable salt thereof, comprising: (i) one or more nucleic acid agents, each of which is selected from the following: an oligonucleotide comprising one or two 5' terminal units covalently attached to said oligonucleotide, wherein each 5' terminal unit is independently 【Chemistry 9】 an oligonucleotide which is (ii) one or more ligands covalently attached to the one or more nucleic acid agents, During the ceremony: B is H or a nucleobase moiety; X is H, halogen, or —OR X ; R X is H, C 1 -C 6 alkyl, or —(C 1 -C 6 alkyl)-(C 6 -C 10 aryl), wherein said C 1 -C 6 alkyl or —(C 1 -C 6 alkyl)-(C 6 -C 10 aryl) is optionally substituted with one or more R Xa ; each R Xa is independently halogen, C 1 -C 6 alkyl, or —O—(C 1 -C 6 alkyl), wherein said C 1 -C 6 alkyl or —O—(C 1 -C 6 alkyl) is optionally substituted with one or more halogens; Z is -P(RZ) 2, -P(ORZ)(N(RZ)2), -P(=O)(ORZ)RZ, -P(=S)(ORZ)RZ, -P(=O)(SRZ)RZ , -P(=S)(SR Z )R Z , -P(=O)(OR Z ) 2 , -P(=S)(OR Z ) 2 , -P(=O)(SR Z ) 2 , or -P(=S)(SR Z ) 2 ; each R Z is independently H or C 1 -C 6 alkyl optionally substituted with one or more halogen or cyano; R 1 is H, halogen, or C 1 -C 6 alkyl substituted with one or more halogens; R 2 is H, halogen, or C 1 -C 6 alkyl optionally substituted with one or more halogens; R 3 is H, halogen, or C 1 -C 6 alkyl optionally substituted with one or more halogens; R 4 is H, halogen, or C 1 -C 6 alkyl optionally substituted with one or more halogens; 【Chemistry 10】 represents a single bond; each R 6 is independently H, halogen, or C 1 -C 6 alkyl optionally substituted with one or more halogens; and ## indicates the bond to the remainder of the oligonucleotide, or a pharmaceutically acceptable salt thereof.

8. 8. The conjugate of claim 7, wherein B is H.

9. 8. The conjugate of claim 7, wherein B is a nucleobase moiety.

10. 8. The conjugate of claim 7, wherein the nucleobase moiety is adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U).

11. X is -OR Ｘ The conjugate of claim 7, wherein

12. 8. The conjugate of claim 7, wherein X is --OH.

13. X is —O—(C １ ~C ６ 8. The conjugate of claim 7, wherein the aryl group is aryl, ...

14. X is —O—(C １ ~C ６ alkyl)-O-(C １ ~C ６ 8. The conjugate of claim 7, wherein the aryl group is aryl, ...

15. X is one or more R Ｘａ -O-(C １ ~C ６ alkyl)-(C ６ ~C １０ 8. The conjugate of claim 7, wherein the aryl is aryl.

16. X is —O—(C １ ~C ６ alkyl)-(C ６ ~C １０ 8. The conjugate of claim 7, wherein the aryl is aryl.

17. Z is -P(R Ｚ ) ２ 8. The conjugate of claim 7, wherein the conjugate is -PH 2 , -PH 2 , or -P(OR Z )(N(R Z ) 2 ).

18. Z is -P(=O)(OR Ｚ ) R Ｚ 8. The conjugate of claim 7, which is -P(=S)(OR Z )R Z , -P(=O)(SR Z )R Z , -P(=S)(SR Z )R Z , -P(=O)(OR Z ) 2 , -P(=S)(OR Z ) 2 , -P(=O)(SR Z ) 2 , or -P(=S)(SR Z ) 2.

19. A conjugate comprising: 【Chemistry 11-1】 【Chemistry 11-2】 【Chemistry 11-3】 【Chemistry 11-4】 The conjugate of claim 7, comprising one or two of:

20. The 5' terminal unit is 【Chemistry 12】 The conjugate of claim 7, wherein 21. The 5' terminal unit of claim 1, 【Chemistry 13】 The conjugate of claim 7, wherein 22. The 5' terminal unit of claim 1, 【Chemistry 14】 The conjugate of claim 7, wherein

23. the one or more ligands are 【Chemistry 15】 The conjugate of claim 7, comprising:

24. The conjugate of claim 7 , wherein the one or more ligands comprise a lipid, a peptide moiety, or an antibody moiety.

25. A pharmaceutical composition comprising the conjugate of any one of claims 7 to 24 and at least one pharmaceutically acceptable excipient or carrier.

26. 26. The pharmaceutical composition of claim 25 for modulating expression of a target gene in a subject or for delivering a nucleic acid agent to a subject.

Images