WO2018009652A1

WO2018009652A1 - Compounds, compositions, and methods for the treatment of disease

Info

Publication number: WO2018009652A1
Application number: PCT/US2017/040886
Authority: WO
Inventors: Radhakrishnan P. Iyer; Anjaneyulu Sheri; Seetharamaiyer Padmanabhan; Geeta MEHER; Shenghua Zhou; Sreerupa CHALLA
Original assignee: Sperovie Biosciences, Inc.
Priority date: 2016-07-06
Filing date: 2017-07-06
Publication date: 2018-01-11
Also published as: TW201803886A

Abstract

Disclosed are compounds and compositions for the induction of expression of a pattern recognition receptor (e.g., STING) and methods of use thereof.

Description

COMPOUNDS, COMPOSITIONS, AND METHODS FOR THE

TREA TMENT OF DISEASE

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. provisional application Nos. 62/359,092, filed July 6, 2016; 62/363,115, filed July 15, 2016, and 62/508,854, filed May 19, 2017, the contents of each of which are hereby incorporated by reference in their entireties.

FIELD OF THE DISCLOSURE

This disclosure relates to compounds and compositions that induce expression of pattern recognition receptors and methods of use for the treatment of microbial infections.

BACKGROUND OF DISCLOSURE

A key feature of the innate immune system is the recognition and elimination of microbial pathogens. Identification of these pathogenic invaders occurs through host recognition of evolutionarily conserved microbial structures known as pathogen-associated molecular patterns (PAMPs) (Jensen, S. and Thomsen, A.R. J Virol (2012) 86:2900-2910). These PAMPs include a wide array of molecular structures, such as nucleic acids, lipopolysaccharides, and glycoproteins that may be broadly shared by multiple microbial species and are critical to their survival and/or pathogenicity. Host recognition may occur by multiple pathways, such as activation of pattern recognition receptors (PRRs), which ultimately lead to downstream signaling events and culminate in the mounting of an immune response.

To date, several PRRs have been identified that serve as sensors of pathogenic infection. For example, the retinoic acid-inducible gene-I (RIG-I) protein is a DNA helicase that also functions as a sensor of microbial-derived RNA. RIG-I is important factor in host recognition of RNA viruses from a variety of different viral families, including Flaviviridae (e.g., West Nile virus, Hepatitis C virus, Japanese encephalitis virus, Dengue virus), Paramyxoviridae (e.g., Sendai virus, Newcastle disease virus, Respiratory syncytial virus (RSV), Measles virus), Rhabdoviridae (e.g., Rabies virus), Orthomyxoviridae (e.g., influenza A virus, influenza B virus), and Arenaviridae (e.g., Lassa virus). The stimulator of interferon genes (STING) is a cytoplasmic adaptor protein that activates the TBK1-IRF3 signaling complex, resulting in induction of interferons (IFN-β) and other immune pathway proteins. Other PRRs also play a role in sensing microbial-derived nucleic acids, including NOD2, LGP2, MDA5, and a number of Toll-like receptors (TLRs) that are expressed on the cell surface and within endosomal compartments. A major obstacle of many currently available antiviral therapies relates to the emergence of drug resistant variants that occurs upon extended use. In addition, many available treatments require persistent and long-term therapy, which often results in unwanted side effects and the risk of relapse upon treatment discontinuation. Further, many viruses can be subdivided into different genotypes, and certain drugs developed against one genotype may not be active against other genotypes. In contrast, the use of small molecule mimics of viral- derived RNA capable of PRR induction provides an alternate approach to the treatment of viral infection, as these compounds may be agnostic to genotype, may possess both direct antiviral activity as well as the ability to activate the host immune response, and potentially limit the development of drug resistance and toxicity. As such, there exists a need for a new generation of therapies that induce expression of PRRs for use in the treatment of disease and as diagnostic tools.

SUMMARY OF DISCLOSURE

The present disclosure features compounds, compositions, and methods that induce expression of pattern recognition receptors (PRRs). The present disclosure further includes methods of use for the treatment of microbial infections.

In one aspect, the present isclosure describes a compound of Formula (I):

Formula (I)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

Z is either S or O

each of B¹ and B² is independently a purinyl nucleobase or pyrimidinyl nucleobase; each of X¹ and X² is independently O or S;

each of Y¹ and Y² is independently O, S, or R⁵;

each of L¹ and L² is independently absent, C₁-C₆ alkyl or C₁-C₆ heteroalkyl, wherein each C₁-C₆ alkyl and C₁-C₆ heteroalkyl is optionally substituted with R⁶; each of R¹ and R² is independently hydrogen, halo, -CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), or OR⁷;

each of R³ and R⁴ is independently hydrogen, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), C₁-C₂₀ heteroalkyl (e.g., C₁-C₆ heteroalkyl), cycloalkyl, heterocyclyl, OC(O)OC₁-C2o alkyl (e.g., C₁- C₆ alkyl), aryl, or heteroaryl, wherein each C₁-C₂₀ alkyl, C₁-C₂₀ heteroalkyl, cycloalkyl, heterocyclyl, aryl, OC(O)0 C₁-C₂₀ alkyl (e.g., C₁-6 alkyl), and heteroaryl is optionally substituted with 1-5 R⁸;

each R⁵ is independently hydrogen or C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl);

R⁶ is halo, -CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OR⁷, oxo, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each C₁-C₂₀ alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-5 R⁹;

R⁷ is hydrogen, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each C₁-C₂₀ alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-5 R⁹;

each R⁸ is independently C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), O-aiyl, OC(O)NR₅-C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), S(O)₂NR₅-aryl, NR₅C(O)-aryl, NR₅R₅C(O)-aryl, C(O)-aryl, C(O)- heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl, OC(O)-C₁-C₂₀ alkyl (e.g., C₁-C₆), OC(O)0-C₁- C₂₀ alkyl (e.g., C₁-C₆), wherein each C₁-C₂₀ alkyl, O-aiyl, OC(O)NR₅-C₁-C₂₀ alkyl,

S(O)₂NR₅-aryl, NR₅C(O)-aryl, CH₂NR₅C(O)-aryl, C(O)-aryl, C(O)-heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl, OC(O)-C₁-C₂₀ alkyl (e.g., C₁-C₆), OC(O)0-C₁-C₂₀ alkyl (e.g., C₁-C₆), is optionally substituted by 1-5 R⁹; and

each R⁹ is independently C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), halo, -CN, OH, O-C₁-C₂₀ alkyl, O-C₁-C₂₀ heteroalkyl, O-aiyl, O-heteroaiyl.

In some embodiments, the com ound is a compound of Formula (I-a):

Formula (I-a)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: each of B¹ and B² is independently a purinyl nucleobase or pyrimidinyl nucleobase; each of X¹ and X² is independently O or S;

each of Y¹ and Y² is independently O, S, or R⁵;

each of L¹ and L² is independently absent, C₁-C₆ alkyl or C₁-C₆ heteroalkyl, wherein each C₁-C₆ alkyl and C₁-C₆ heteroalkyl is optionally substituted with R⁶;

each of R¹ and R² is independently hydrogen, halo, -CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), or OR⁷;

each of R³ and R⁴ is independently hydrogen, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), C₁-C₂₀ heteroalkyl (e.g., C₁-C₆ heteroalkyl), cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each C₁-C₂₀ alkyl, C₁-C₂₀ heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substituted with 1-5 R⁸;

R⁵ is hydrogen or C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl);

each R⁸ is independently C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), C(O)-aryl, C(O)-heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl, wherein each C₁-C₂₀ alkyl, C(O)-aryl, C(O)-heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl is optionally substituted by 1-5 R⁹; and

each R⁹ is independently C₁-C₂₀ alkyl, halo, -CN, OH, O-C₁-C₂₀ alkyl, O-C₁-C₂₀ heteroalkyl, O-aiyl, or O-heteroaiyl.

In some embodiments the compound is a compound of Formulas I-b (I-c), (I-d), or (I-e):

Formula (I-b) Formula (I-c)

Formula (I-d) Formula (I-e) or a pharmaceutically acceptable salt thereof, wherein each of B¹, B², X¹, X², Y¹, Y², L¹, L², R¹, R², R³, R⁴, and subvariables thereof are defined as for Formula (I).

In some embodiments, B¹ is a purinyl nucleobase. In some embodiments, B² is a pyrimidinyl nucleobase. In some embodiments, B¹ is a purinyl nucleobase and B² is a pyrimidinyl nucleobase.

In some embodiments, B l is adenosinyl or guanosinyl. In some embodiments, B2 is cytosinyl, thyminyl, or uracilyl. In some embodiments, B¹ is adenosinyl or guanosinyl and B² is cytosinyl, thyminyl, or uracilyl. In some embodiments, each of B¹ and B² is independently uracilyl.

In some embodiments, each of R¹ and R² is independently hydrogen, halo, or OR⁶. In some embodiments, each of R¹ and R² is independently halo (e.g., fluoro). In some embodiments, each of R¹ and R² is not hydrogen or OR⁷.

In some embodiments, X¹ is O. In some embodiments, X² is O. In some

embodiments, each of X¹ and X² is O.

In some embodiments, Y¹ is O or S. In some embodiments, Y² is O or S. In some embodiments, each of Y¹ and Y² is independently O or S. In some embodiments, one of Y¹ or Y² is O and the other of Y¹ or Y² is S. In some embodiments, each of Y¹ or Y² is

independently S. In some embodiments, each of Y¹ or Y² is independently O.

In some embodiments, L¹ is C₁-C₆ alkyl (e.g., CFh). In some embodiments, L² is C₁- C₆ alkyl (e.g., CFh). In some embodiments, each of L¹ and L² is independently C₁-C₆ alkyl

(e.g., CH₂). In some embodiments, R is hydrogen, aryl, or heteroaryl, wherein aryl and heteroaryl is optionally substituted with 1-5 R⁸. In some embodiments, R³ is aryl or heteroaryl, each of which is optionally substituted with 1-5 R⁸. In some embodiments, R³ is phenyl substituted with 1 R⁸.

In some embodiments, R⁴ is independently hydrogen, aryl, or heteroaryl, wherein aryl and heteroaryl is optionally substituted with 1-5 R⁸. In some embodiments, R⁴ is aryl or heteroaryl, each of which is optionally substituted with 1-5 R⁸. In some embodiments, R⁴ is phenyl substituted with 1 R⁸.

In some embodiments, each of R³ and R⁴ is independently hydrogen, aryl, or heteroaryl, wherein aryl and heteroaryl is optionally substituted with 1-5 R⁸.

In some embodiments, R³ is aryl or heteroaryl, each of which is optionally substituted with 1-5 R⁸, and R⁴ is hydrogen.

In some embodiments, R³ is phenyl substituted with 1 R⁸ and R⁴ is hydrogen. In some embodiments, each of R³ and R⁴ is independently phenyl substituted with 1 R⁸.

In some embodiments, each of Y¹ and Y² is O and each of R³ and R⁴ is independently hydrogen. In some embodiments, Y² is O and R⁴ is hydrogen. In some embodiments, each of Y¹ and Y² is independently S and each of R³ and R⁴ is independently substituted with 1 R⁸. In some embodiments, Y¹ is S and R³ is substituted with 1 R⁸.

In some embodiments, R⁸ is OC(O)-aryl optionally substituted by 1-5 R⁹ (e.g., 1 R⁹). In some embodiments, R⁹ is O-C₁-C₁2 alkyl (e.g., 0-CH2(CH2)8CH3). In some embodiments, R⁹ is O-C₁-C₁o alkyl (e.g., 0-CH₂(CH₂)8CH3). In some embodiments, R⁹ is O-C₁-Cs alkyl (e.g., 0-CH₂(CH₂)₆CH₃). In some embodiments, R⁹ is O-C₁-C₆ alkyl (e.g., O- CH₂(CH₂)₄CH₃).

In some embodiments, the com ound is represented by Formula (I-f):

Formula (I-f)

armaceutically acceptable salt or stereoisomer thereof, wherein: each of B¹ and B² is independently a purinyl nucleobase or pyrimidinyl nucleobase; each of X¹ and X² is independently O or S;

each of Y¹ and Y² is independently O, S, or R⁵;

each of R¹ and R² is independently halo;

each of R³ and R⁴ is independently hydrogen, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each C₁-C₆ alkyl, C₁-C₆ heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substituted with 1-5 R⁸;

R⁵ is hydrogen or C₁-C₆ alkyl;

R⁶ is halo, -CN, C₁-C₆ alkyl, OR⁷, oxo, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each C₁-C₆ alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-5 R⁹;

R⁷ is hydrogen, C₁-C₆ alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each C₁-C₆ alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1- 5 R⁹;

each R⁸ is independently C₁-C₆ alkyl, C(O)-aryl, C(O)-heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl, wherein each C₁-C₆ alkyl, C(O)-aryl, C(O)-heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl is optionally substituted by 1-5 R⁹; and

In some embodiments, the com ound is represented by Formula (I-g):

Formula (I-g)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

each of B¹ and B² is independently a purinyl nucleobase or pyrimidinyl nucleobase; each of X¹ and X² is independently O; each of Y¹ and Y² is independently O or S;

each of L¹ and L² is independently absent or C₁-C₆ alkyl;

each of R¹ and R² is independently halo or OH;

each of R³ and R⁴ is independently hydrogen or aryl optionally substituted with 1-5

R

each R⁸ is independently OC(O)-aryl optionally substituted by 1-5 R⁹;

each R⁹ is independently O-C₁-C₂₀ alkyl.

In some embodiments, the compound is selected from Table 1 :

10

or a pharmaceutically acceptable salt thereof.

In one aspect, the present disclosure describes a method of treating a microbial infection in a subject, the method comprising administering to the subject a compound of Formula (I),

Formula (I)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

Z is either S or O

each of Y¹ and Y² is independently O, S, or NR⁵;

each of R¹ and R² is independently hydrogen, halo, -CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), or OR⁷; each of R³ and R⁴ is independently hydrogen, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), C₁-C₂₀ heteroalkyl (e.g., C₁-C₆ heteroalkyl), cycloalkyl, heterocyclyl, OC(O)OC₁-C2o alkyl (e.g., C₁- C₆ alkyl), aryl, or heteroaryl, wherein each C₁-C₂₀ alkyl, C₁-C₂₀ heteroalkyl, cycloalkyl, heterocyclyl, aryl, OC(O)0 C₁-C₂₀ alkyl (e.g., C₁-6 alkyl), and heteroaryl is optionally substituted with 1-5 R⁸;

In some embodiments, the present disclosure describes a method of treating a microbial infection in a subject, the method comprising administering to the subject a compound of Formula (I-a),

Formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

each of Y¹ and Y² is independently O, S, or R⁵;

R⁵ is hydrogen or C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl);

In one aspect, the present disclosure describes a method of inducing the expression of a pattern recognition receptor in a subject suffering from a microbial infection, the method comprising administering to the subject a compound of Formula (I),

Formula (I)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

Z is either S or O

each of B ¹ and B² is independently a purinyl nucleobase or pyrimidinyl nucleobase; each of X¹ and X² is independently O or S;

each of Y¹ and Y² is independently O, S, or R⁵;

each R⁸ is independently C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), O-aiyl, OC(O)NR₅-C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), S(O)₂NR₅-aryl, NR₅C(O)-aryl, NR₅R₅C(O)-aryl, C(O)-aryl, C(O)- heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl, OC(O)-C₁-C₂₀ alkyl (e.g., C₁-C₆), OC(O)0-C₁- C₂₀ alkyl (e.g., C₁-C₆), wherein each C₁-C₂₀ alkyl, O-aiyl, OC(O)NR₅-C₁-C₂₀ alkyl, S(O)2 R₅-aryl, R₅C(O)-aryl, CH2 R₅C(O)-aryl, C(O)-aryl, C(O)-heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl, OC(O)-C₁-C₂₀ alkyl (e.g., C₁-C₆), OC(O)0-C₁-C₂₀ alkyl (e.g., C₁-C₆), is optionally substituted by 1-5 R⁹; and

In some embodiments, the present disclosure describes a method of inducing the expression of a pattern recognition receptor in a subject suffering from a microbial infection, the method comprising administerin to the sub ect a compound of Formula (I-a),

Formula (I-a)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

each of Y¹ and Y² is independently O, S, or R⁵;

R⁵ is hydrogen or C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl);

R⁶ is halo, -CN, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), OR⁷, oxo, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each C₁-C₂₀ alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-5 R⁹; R is hydrogen, C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each C₁-C₂₀ alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-5 R⁹;

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are graphs showing the evaluation of percent (%) IRF induction by compound 1 and compound 1 1 administration compared with 2',3 '-cGAMP in wild type THPl cells (FIG. 1 A) and THPl cells in which STING has been knocked out (FIG. IB).

FIGS. 2A-2B are graphs showing the evaluation of percent (%) NF-κβ induction by compound 1 and compound 1 1 compared with 2',3 '-cGAMP in wild type THPl cells (FIG. 2A) and THPl cells in which STING has been knocked out (FIG. 2B).

FIGS. 3A-3B are graphs showing the percent (%) cell death caused by compound 1 and compound 1 1 compared with 2',3 '-cGAMP in wild type THPl cells (FIG. 3 A) and THPl cells in which STING has been knocked out (FIG. 3B).

FIG. 4 is a graph depicting the percent (%) cell death caused by compound 4 in wild type THPl cells.

FIG. 5 is a graph depicting the percent (%) IRF induction by compound 4 in wild type THPl cells.

FIGS. 6A-6B are graphs depicting the percent (%) IRF induction (FIG. 6A) and percent (%) NF-κβ induction in THPl cells caused by compound 1 administration.

FIGS. 7A-7B are graphs indicating the level of ISG54 ISRE-luc activity (FIG. 7A) and NF-KP-1UC activity (FIG. 7B) of compound 1, compound 2, compound 3, and 2',3 '- cGAMP at varying concentrations as fold increase over DMSO in HEK293 cells.

FIG. 8 is a graph indicating the level of ISG54 ISRE-luc activity of compound 1, compound 2, compound 3, and 2',3 '-cGAMP at varying concentrations as fold increase over DMSO in HEK293 cells. FIG. 9 is a chart showing the IRF -type I interferon activity in THP1 cells upon administration of exemplary compounds of the present disclosure.

FIG. 10A, FIG. 10B and FIG. IOC. show IRF induction by exemplary compounds.

FIG. 11 A, FIG. 11B and FIG. 11C. show IRF induction by exemplary compounds.

FIGS. 12A-12B are bar graphs showing RSV infection and RSV percentage infection when RSVA2 infected A459 cells were treated with vehicle (DMSO) or Compound 1. FIG. 12A shows RSV infection (RSV titer) calculated by performing a viral plaque assay when RSV infected cells were treated with vehicle (DMSO), 50 μΜ, 100 μΜ or 200 μΜ of Compound 1. FIG. 12B shows RSV percentage infection calculated based on the viral titer shown in FIG. 12A when RSV infected cells were treated with vehicle (DMSO), 50 μΜ, 100 μΜ or 200 μΜ of Compound 1 (FIG. 12B). 100% infection represents RSV infection in vehicle treated cells.

FIGS. 13A-13G are graphs showing the effect on HCV RNA replication in THP-1 calls when treated with increasing concentrations of Compound 1.

FIGS. 14A-14B are graphs showing the cell viability of Vero and A549 cells measured by MTT method when treated with increasing concentrations of DMSO (FIG. 14 A) or Compound 1 (FIG. 14B).

FIG. 15 is a bar graph showing the virus yield of Junin virus at 24 and 48 hours post infection in A549 cells when treated with Compound 1. Virus yield diminished 1 log in A549 infected cells treated with Compound 1 compared to untreated A549 infected cells, both at 24 and 48 hours post infection (h p.i.).

FIG. 16 is a bar graph showing the virus yield of Dengue virus serotype 2 (DSV2) at 24 and 48 hours post infection in A549 cells when treated with Compound 1. Virus yield diminished 1 log in A549 cells infected with DSV2 compared to untreated A549 infected cells at 24 hours post infection (h p.i.). At 48 hours post infection, no significant difference was found. FIGS. 17A-17B are bar graphs showing the percent (%) IRF induction (FIG. 17A) and percent (%) NF-κΒ (FIG. 17B) when THP1 dual cell are treated with different concentrations of Compound (Cmd) 1, Cmd 1A, and Cmd IB. The THP1 dual cells carry both the secreted embryonic alkaline phosphatase (SEAP) reporter gene which is under the control of an IFN-β minimal promoter fused to five copies of the NF-κΒ consensus transcription response element and the Lucia reporter gene which is under the control of an ISG54 minimal promoter. FIGS. 18A-18D are graphs showing the induction of IRF (FIGS. 18A-18B) and F- KB (FIGS. 18C-18D) by Cmd 1. The results in FIGS. 18A-18D indicate that Cmd 1 is taken up by cells without the use of transfection agents.

FIGS.19A-19B are graphs showing the induction of IRF by Cmd 3, and indicate that Cmd 3 is taken up by cells without the use of transfection agents.

FIGS. 20A-20D are graphs showing the induction of IRF (FIGS. 20A-20B) and F- KB (FIGS. 20C-20D) by Cmd 12, and indicate that Cmd 12 is taken up by cells without the use of transfection agents.

FIGS. 21A-21D are graphs showing the induction of IRF (FIGS. 21A-21B) and F- KB (FIGS. 21C-21D) by Cmd 13, and indicate that Cmd 13 is taken up by cells without the use of transfection agents.

FIGS. 22A-22D are graphs showing the induction of IRF (FIGS. 22A-22B) and F- KB (FIGS. 22C-22D) by Cmd 14, and indicate that Cmd 14 is taken up by cells without the use of transfection agents.

FIGS. 23A-23D are graphs showing the induction of IRF (FIGS. 23A-23B) and F- KB (FIGS. 23C-23D) by Cmd 15, and indicate that Cmd 15 is taken up by cells without the use of transfection agents.

FIGS.24A-24B are bar graphs comparing the relative induction of IRF (FIG. 24A) and F-κΒ (FIG. 24B) by Cmd 1, Cmd 3, Cmd 12, Cmd 13, Cmd 14, and Cmd 15 at various concentrations.

FIGS. 25A-25B are graphs showing the stability of Cmd 1 in serum (FIG. 25A) and in microsomes (FIG. 25B). In FIGS. 25A-25B, Peak 1 and Peak 2 represent Cmds 1-A and 1- B, respectively.

FIGS. 26A-26B are graphs showing the stability of Cmd 15 in serum (FIG. 26 A) and in microsomes (FIG. 26B). In FIGS. 26A-26B, Peak 1 and Peak 2 represent Cmds 15-A and 15-B, respectively.

FIGS. 27A-27B are bar graphs comparing the induction of IRF (FIG. 27A) and F- KB (FIG. 27B) by Cmd 15 and its isomers, Cmd 15-A and Cmd 15-B.

FIG. 28 is a bar graph showing the induction of apoptosis through % cytoxicity of THPl cells when treated with various concentrations of Cmd 15 and its isomers, Cmd 15-A and Cmd 15-B. FIGS. 29A-29B are graphs showing the effect on type 1 IFN signaling in cells when treated with various concentrations of Cmd 1 and 2',3'-cGAMP. As shown in FIGs. 29A- 29B, the binding of Cmd 1 to STING activates type 1 IFN signaling, similar to the activation of type 1 IFN signaling observed with 2',3'-cGAMP.

FIG. 30 is a bar graph showing the effect on type 1 IFN signaling in mouse macrophages when treated with various concentrations of Cmd 1 and 2',3'-cGAMP. As shown in FIG 30, Cmd 1 is highly active in mouse macrophages in activating type 1 IFN signaling, similar to the activation of type 1 IFN signaling observed with 2',3'-cGAMP.

FIGS. 31A-31B are graphs showing the effect on type 1 IFN signaling when Human monocytes (THP1 cells) and Mouse macrophages (RAW) are treated with various concentrations. As shown in FIGs. 31A-31B, Cmd 1, Cmd 5, Cmd 12, Cmd 13, Cmd 14, and Cmd 15 are more active in human monocytes (FIG. 31 A) and mouse macrophages (FIG. 3 IB) than the natural STING ligand 3',3'-cGAMP.

FIGS. 32A-32B are graphs showing the induction of type I IFN signaling in HEK293 (FIG. 32A) and THP1 (FIG. 32B) cells treated with Cmd 1 and its isomers Cmd 1A (Cmd 1- PK1) and Cmd IB (Cmd 1-PK2).

FIGS. 33A-33B are bar graph showing the effects on type III interferon (IL-29) production in THP1 cells treated with various concentrations of Cmd 1 and Cmd 15. As shown in FIGS. 33A-33B, Cmd 1 and Cmd 15 induce type III interferon (IL-29) production in THP1 cells (FIG. 33 A), and indicating that both Cmd 1 and Cmd 15 are taken up by cells without use of a transfection reagent (FIG. 33B).

FIGS. 34A-34B are graphs comparing the induction of type I IFN signaling in THP1 cells treated with Cmd 1, Cmd 13, Cmd 15.

FIGS. 35A-35B are bar graphs comparing the induction of IRF (FIG. 35 A) and NF- KB (FIG. 35B) when THP dual cells were treated with various concentrations of DMSO, Cmd 15, or Cmd 16.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure relates to methods of inducing the expression of a PRR (e.g., STING) in a subject. In some embodiments, the method comprises administration of a compound of Formula (I) or pharmaceutically acceptable salt thereof.

Definitions As used herein, the articles "a" and "an" refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.

"About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values.

As used herein, the term "acquire" or "acquiring" as the terms are used herein, refer to obtaining possession of a physical entity (e.g., a sample, e.g., blood sample or liver biopsy specimen), or a value, e.g., a numerical value, by "directly acquiring" or "indirectly acquiring" the physical entity or value. "Directly acquiring" means performing a process (e.g., an analytical method) to obtain the physical entity or value. "Indirectly acquiring" refers to receiving the physical entity or value from another party or source (e.g., a third party laboratory that directly acquired the physical entity or value). Directly acquiring a value includes performing a process that includes a physical change in a sample or another substance, e.g., performing an analytical process which includes a physical change in a substance, e.g., a sample, performing an analytical method, e.g., a method as described herein, e.g., by sample analysis of bodily fluid, such as blood by, e.g., mass spectroscopy, e.g. LC-MS.

As used herein, the terms "induce" or "induction of refer to the increase or enhancement of a function, e.g., the increase or enhancement of the expression of a pattern recognition receptor (e.g, STING). In some embodiments, "induction of PRR expression" refers to induction of transcription of PRR RNA, e.g., STING RNA (e.g., mRNA, e.g., an increase or enhancement of), or the translation of a PRR protein, e.g., the STING protein (e.g., an increase or enhancement of). In some embodiments, induction of PRR expression (e.g., STING expression) refers to the increase or enhancement of the concentration of a PRR RNA, e.g., or STING RNA (e.g., mRNA) or the STING protein, e.g., in a cell. In some embodiments, induction of PRR expression (e.g., STING expression) refers to the increase of the number of copies of PRR RNA, e.g., STING RNA (e.g., mRNA) or PRR protein, e.g., the STING protein, e.g., in a cell. In some embodiments, to induce expression of a PRR (e.g., STING) may refer to the initiation of PRR RNA (e.g., STING RNA (e.g., mRNA)) or transcription or PRR protein (e.g., STING protein) translation. In some embodiments, to induce expression of a PRR (e.g., STING) may refer to an increase in the rate of PRR RNA (e.g., STING RNA (e.g., mRNA)) transcription or an increase in the rate of PRR protein (e.g., STING protein) expression. As used herein, the terms "activate" or "activation" refer to the stimulation or triggering of a function, e.g., of a downstream pathway, e.g., a downstream signaling pathway. In some embodiments, activation of a pattern recognition receptor (PRR) (e.g., STING) refers to the stimulation of a specific protein or pathway, e.g., through interaction with a downstream signaling partner (e.g., IFN-β promoter stimulator 1 (IPS-1), IRF3, IRF7, NF-KB, interferons (e.g., IFN-a or IFN-β) and/or cytokines). In some embodiments, activation is distinct from the induction of expression of a PRR. In some embodiments, a PRR may be activated without resulting in an induction of PRR expression (e.g., expression of STING). In some embodiments, activation may include induction of expression of a PRR (e.g., STING). In some embodiments, activation of a PRR may trigger the induction of expression of a PRR (e.g., STING) by about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%), about 80%, about 90%, about 95%, or more compared to a reference standard (e.g., basal expression levels of a PRR (e.g., STING)).

As used herein, an amount of a compound, conjugate, or substance effective to treat a disorder (e.g., a disorder described herein), "therapeutically effective amount," "effective amount" or "effective course" refers to an amount of the compound, substance, or composition which is effective, upon single or multiple dose administration(s) to a subject, in treating a subject, or in curing, alleviating, relieving or improving a subject with a disorder (e.g., a microbial infection) beyond that expected in the absence of such treatment.

As used herein, the term "latent" refers to a microbial infection (e.g., a viral infection) in which the microbe has entered a dormant stage (e.g., in a cell) and is no longer

substantially replicating. In some embodiments, viral latency refers to the lysogenic portion of the viral replication cycle. In some embodiments, latency may refer to microbial infection of a host (e.g., a subject described herein). In these cases, the infected subject may experience symptoms related to the infection, or alternatively may be substantially asymptomatic.

As used herein, the terms "prevent" or "preventing" as used in the context of a disorder or disease, refer to administration of an agent to a subject, e.g., the administration of a compound of the present disclosure (e.g., compound of Formula (I)) to a subject, such that the onset of at least one symptom of the disorder or disease is delayed as compared to what would be seen in the absence of administration of said agent.

As used herein, the terms "reference treatment" or "reference standard" refer to a standardized level or standardized treatment that is used as basis for comparison. In some embodiments, the reference standard or reference treatment is an accepted, well known, or well characterized standard or treatment in the art. In some embodiments, the reference standard describes an outcome of a method described herein. In some embodiments, the reference standard describes a level of a marker (e.g., a level of induction of a PRR, e.g., STING) in a subject or a sample, e.g., prior to initiation of treatment, e.g., with a compound or composition described herein. In some embodiments, the reference standard describes a measure of the presence of, progression of, or severity of a disease or the symptoms thereof, e.g., prior to initiation of treatment, e.g., with a compound or composition described herein.

As used herein, the term "subject" is intended to include human and non-human animals. Exemplary human subjects include a human patient having a disorder, e.g., a disorder described herein, or a normal subject. The term "non-human animals" includes all vertebrates, e.g., non-mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates, domesticated and/or agriculturally useful animals, e.g., sheep, dogs, cats, cows, pigs, etc. In exemplary embodiments of the present disclosure, the subject is a woodchuck (e.g., an Eastern woodchuck (Marmota monax)).

As used herein, the terms "treat" or "treating" a subject having a disorder or disease refer to subjecting the subject to a regimen, e.g., the administration of a compound of Formula (I) or pharmaceutically acceptable salt thereof, or a composition comprising Formula (I) or pharmaceutically acceptable salt thereof, such that at least one symptom of the disorder or disease is cured, healed, alleviated, relieved, altered, remedied, ameliorated, or improved. Treating includes administering an amount effective to alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder or disease, or the symptoms of the disorder or disease. The treatment may inhibit deterioration or worsening of a symptom of a disorder or disease.

As used herein, the term "Cmd" refers to the word "compound" or "Compound", and all of them are used interchangeably.

Numerous ranges, e.g., ranges for the amount of a drug administered per day, are provided herein. In some embodiments, the range includes both endpoints. In other embodiments, the range excludes one or both endpoints. By way of example, the range can exclude the lower endpoint. Thus, in such an embodiment, a range of 250 to 400 mg/day, excluding the lower endpoint, would cover an amount greater than 250 that is less than or equal to 400 mg/day.

Definitions The term "alkyl," as used herein, refers to a monovalent saturated, straight- or branched-chain hydrocarbon such as a straight or branched group of 1-12, 1-10, or 1-6 carbon atoms, referred to herein as C₁-C₁2 alkyl, C₁-C₁0 alkyl, and C₁-C₆ alkyl, respectively.

Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl, iso-pentyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl, and the like.

The terms "alkenyl" and "alkynyl" are art-recognized and refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond, respectively. Exemplary alkenyl groups include, but are not limited to, -CH=CH₂ and -CH₂CH=CH₂.

The term "alkylene" refers to the diradical of an alkyl group.

The terms "alkenylene" and "alkynylene" refer to the diradicals of an alkenyl and an alkynyl group, respectively.

The term "methylene unit" refers to a divalent -CH2- group present in an alkyl, alkenyl, alkynyl, alkylene, alkenylene, or alkynylene moiety.

The term "carbocyclic ring system", as used herein, means a monocyclic, or fused, spiro-fused, and/or bridged bicyclic or polycyclic hydrocarbon ring system, wherein each ring is either completely saturated or contains one or more units of unsaturation, but where no ring is aromatic.

The term "carbocyclyl" refers to a radical of a carbocyclic ring system.

Representative carbocyclyl groups include cycloalkyl groups (e.g., cyclopentyl, cyclobutyl, cyclopentyl, cyclohexyl and the like), and cycloalkenyl groups (e.g., cyclopentenyl, cyclohexenyl, cyclopentadienyl, and the like).

The term "aromatic ring system" is art-recognized and refers to a monocyclic, bicyclic or polycyclic hydrocarbon ring system, wherein at least one ring is aromatic.

The term "aryl" refers to a radical of an aromatic ring system. Representative aryl groups include fully aromatic ring systems, such as phenyl, naphthyl, and anthracenyl, and ring systems where an aromatic carbon ring is fused to one or more non-aromatic carbon rings, such as indanyl, phthalimidyl, naphthimidyl, or tetrahydronaphthyl, and the like.

The term "heteroalkyl" refers to an "alkyl" moiety wherein at least one of the carbone molecules has been replaced with a heteroatom such as O, S, or N.

The term "heteroaromatic ring system" is art-recognized and refers to monocyclic, bicyclic or polycyclic ring system wherein at least one ring is both aromatic and comprises a heteroatom; and wherein no other rings are heterocyclyl (as defined below). In certain instances, a ring which is aromatic and comprises a heteroatom contains 1, 2, 3, or 4 independently selected ring heteroatoms in such ring.

The term "heteroaryl" refers to a radical of a heteroaromatic ring system.

Representative heteroaryl groups include ring systems where (i) each ring comprises a heteroatom and is aromatic, e.g., imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrrolyl, furanyl, thiophenyl pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl; (ii) each ring is aromatic or carbocyclyl, at least one aromatic ring comprises a heteroatom and at least one other ring is a hydrocarbon ring or e.g., indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl,

pyrido[2,3-b]-l,4-oxazin-3(4H)-one, 5,6,7,8-tetrahydroquinolinyl and

5,6,7,8-tetrahydroisoquinolinyl; and (iii) each ring is aromatic or carbocyclyl, and at least one aromatic ring shares a bridgehead heteroatom with another aromatic ring, e.g.,

4H-quinolizinyl. In certain embodiments, the heteroaryl is a monocyclic or bicyclic ring, wherein each of said rings contains 5 or 6 ring atoms where 1, 2, 3, or 4 of said ring atoms are a heteroatom independently selected from N, O, and S.

The term "heterocyclic ring system" refers to monocyclic, or fused, spiro-fused, and/or bridged bicyclic and polycyclic ring systems where at least one ring is saturated or partially unsaturated (but not aromatic) and comprises a heteroatom. A heterocyclic ring system can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted.

The term "heterocyclyl" refers to a radical of a heterocyclic ring system.

Representative heterocyclyls include ring systems in which (i) every ring is non-aromatic and at least one ring comprises a heteroatom, e.g., tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl; (ii) at least one ring is non-aromatic and comprises a heteroatom and at least one other ring is an aromatic carbon ring, e.g., 1,2,3,4-tetrahydroquinolinyl,

1,2,3,4-tetrahydroisoquinolinyl; and (iii) at least one ring is non-aromatic and comprises a heteroatom and at least one other ring is aromatic and comprises a heteroatom, e.g.,

3,4-dihydro-lH-pyrano[4,3-c]pyridine, and l,2,3,4-tetrahydro-2,6-naphthyridine. In certain embodiments, the heterocyclyl is a monocyclic or bicyclic ring, wherein each of said rings contains 3-7 ring atoms where 1, 2, 3, or 4 of said ring atoms are a heteroatom independently selected from N, O, and S.

The term "saturated heterocyclyl" refers to a radical of heterocyclic ring system wherein every ring is saturated, e.g., tetrahydrofuran, tetrahydro-2H-pyran, pyrrolidine, piperidine and piperazine.

"Partially unsaturated" refers to a group that includes at least one double or triple bond. A "partially unsaturated" ring system is further intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic groups (e.g., aryl or heteroaryl groups) as herein defined. Likewise, "saturated" refers to a group that does not contain a double or triple bond, i.e., contains all single bonds.

The term "nucleobase" as used herein, is a nitrogen-containing biological compounds found linked to a sugar within a nucleoside— the basic building blocks of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The primary, or naturally occurring, nucleobases are cytosine (DNA and RNA), guanine (DNA and RNA), adenine (DNA and RNA), thymine (DNA) and uracil (RNA), abbreviated as C, G, A, T, and U, respectively. Because A, G, C, and T appear in the DNA, these molecules are called DNA-bases; A, G, C, and U are called RNA-bases. Adenine and guanine belong to the double-ringed class of molecules called purines (abbreviated as R). Cytosine, thymine, and uracil are all pyrimidines. Other nucleobases that do not function as normal parts of the genetic code, are termed non-naturally occurring.

As used herein, the definition of each expression, e.g., alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.

As described herein, compounds of the present disclosure may contain "optionally substituted" moieties. In general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at each position. Combinations of substituents envisioned under this disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term "stable", as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

Pattern Recognition Receptors

The disclosure presented herein features methods for the activation and induction of PRR expression (e.g., STING expression) in a subject, e.g., a subject with a microbial infection (e.g., a viral infection, bacterial infection, fungal infection, or parasitic infection). Pattern recognition receptors (PRRs) are a broad class of proteins which recognize pathogen- associated molecular patterns (PAMPs) conserved within pathogenic invaders. PAMPs are typically products of biosynthetic pathways that are essential to the survival and/or infectivity of the pathogen, e.g., lipopolysaccharides, glycoproteins, and nucleic acids. Recognition of PAMPs by their cognate PRRs activates signaling pathways that result in the production of immune defense factors such as pro-inflammatory and anti-inflammatory cytokines, type I interferons (IFN-a, IFN-β), and/or interferon stimulated genes (ISGs). It is well known that induction of innate immune signaling also results in the activation of T cell responses as well as the induction of adaptive immunity. These downstream immune effects are essential for clearance of the virus through apoptosis and killing of infected cells through cytotoxic T lymphocytes and other defense mechanisms. It is also well known that interferons act on ISRE (interferon response elements) that can trigger the production of ISGs, which play an important role in antiviral cellular defense.

The stimulator of interferon genes (STING) is a cytosolic microbial-derived DNA sensor that has been shown to be particularly sensitive to double-stranded DNA and cyclic dinucleotides (e.g., cyclic di-GMP) (Burdette, D. L. and Vance, R. E. (2013) Nat Immunol 14: 19-26). Two molecules of STING form a homodimer mediated by an a-helix present in the C-terminal dimerization domain, and molecular binding studies have revealed that each STING dimer binds one molecule of microbial nucleic acids, e.g., DNA or a cyclic dinucleotide. Upon ligand binding, STING activates the innate immune response through interaction with RIG-I and IPS-1, resulting in interferon production (e.g., IFN-a and IFN-β) and other downstream signaling events. Since its discovery, STING has been shown to function as a critical sensor of viruses (e.g., adenovirus, herpes simplex virus, hepatitis B virus, vesicular stomatitis virus, hepatitis C virus), bacteria (e.g., Listeria monocytogenes, Legionella pneumopholia, Mycobacterium tuberculosis) and protozoa {Plasmodium falciparum, Plasmodium berghei). In addition, STING has been shown to play a major role in the innate immune response against tumor antigens, driving dendritic cell activation and subsequent T cell priming in several cancers (Woo, S.R. et al. Trends in Immunol (2015) 36:250-256).

Another class of PRRs includes RIG-I, which is the founding member of a family of PRRs termed RIG-I-like receptors (RLRs) that primarily detect RNA derived from foreign sources. It is a critical sensor of microbial infection (e.g., viral infection) in most cells and is constitutively expressed at low levels in the cytosol. After ligand binding, the expression of RIG-I is rapidly enhanced, leading to increased RIG-I concentrations in the cell (Jensen, S. and Thomsen, A.R. J Virol (2012) 86:2900-2910; Yoneyama M. et al. Nat Immunol (2004) 5 :730-737). RIG-I is an ATP-dependent helicase containing a central DExD/H box ATPase domain and tandem N-terminal caspase-recruiting domains (CARDs) that mediate downstream signaling. The C-terminus of RIG-I comprises an ssRNA/dsRNA-binding domain that when unbound acts to silence CARD function at the N-terminus. Without wishing to be bound by theory, it is believed that upon recognition of target RNA structures, two N-terminal CARDs are exposed, allowing for interaction with the CARD of a downstream binding partner, IFN-β promoter stimulator 1 (IPS- 1), also known as

mitochondrial antiviral signaling molecule (MAVS) and CARDIF. This interaction in turn triggers further downstream signaling, such as induction of IRF3, IRF7, NF-κΒ, IFNS, and cytokine production that results in the initiation of the host immune response.

Other RLRs are homologous to RIG-I and function in a similar manner, including MDA5, LGP2, and RNase L. MDA5 is highly homologous to RIG-I, and has been shown to be crucial for triggering a cytokine response upon infection with picornaviruses (e.g., encephalomyocarditis virus (EMCV), Theiler's virus, and Mengo virus), Sendai virus, rabies virus, West Nile virus, rabies virus, rotavirus, murine hepatitis virus, and murine norovirus. LPG2 lacks a CARD domain found in RIG-I and MDA5, which is responsible for direct interaction with IPS- 1 to initiate downstream signaling. As such, LPG2 is believed to behave as a modulator of the innate immune response in conjunction with other CARD-bearing RLRs such as RIG-I and MDA5.

Another class of PRRs encompasses the nucleotide-binding and oligomerization domain (NOD)-like receptors, or NLR family (Caruso, R. et al, Immunity (2014) 41 :898- 908), which includes the microbial sensor NOD2. NOD2 is composed of an N-terminal CARD, a centrally-located nucleotide-binding oligomerization domain, and a C-terminal leucine rich repeat domain that is responsible for binding microbial PAMPs, such as bacterial peptidoglycan fragments and microbial nucleic acids. Ligand binding activates NOD2 and is believed to drive interaction with the CARD-containing kinase RIPK2, which in turn activates a number of downstream proteins including NF-κΒ, MAPK, IRF7, and IRF3, the latter of which results in the induction of type 1 interferons. NOD2 is expressed in a diverse set of cell types, including macrophages, dendritic cells, paneth cells, epithelial cells (e.g., lung epithelial cells, intestinal epithelia), and osteoblasts. NOD2 has been established as a sensor of infection by variety of pathogenic invaders, such as protozoa (e.g., Toxoplasma gondii and Plasmodium berghei), bacteria (e.g., Bacillus anthracis, Borrelia burgdorferi, Burkholderia pseudomallei, Helicobacter hepaticus, Legionella pneumophilia,

Mycobacterium tuberculosis, Propionibacterium acne, Porphyromonas gingivalis,

Salmonella enterica, and Streptococcus pneumonia), and viruses (e.g., respiratory syncytial virus and murine norovirus-1) (Moreira, L. O. and Zamboni, D. S. Front Immunol (2012) 3 : 1- 12). Recent work has shown that mutation of NOD2 may contribute to inflammatory diseases such as Crohn's disease, resulting in an aberrant inflammatory response upon stimulation.

Compounds

The present disclosure features compounds and methods for the induction of PRR expression (e.g., STING expression) in a subject (e.g., a subject with a microbial infection, e.g., a viral infection, a bacterial infection, a fungal infection, or a parasitic infection), comprising administration of a compound of Formula (I) or a prodrug or pharmaceutically acceptable salt thereof.

In some embodiments, the present disclosure features a compound of Formula (I) in which the 3'-OH end of one nucleoside is joined to the 5'-OH of the second nucleoside through a linkage as shown. In some other embodiments, the 2' -OH end of one nucleoside may be joined to the 5' -OH of the second nucleoside through a linkage.

In some embodiments, the com ound is a compound of Formula (I):

Z is either S or O

each of Y¹ and Y² is independently O, S, or NR⁵;

In some embodiments, the compound is a compound of Formula (I-a):

Formula (I-a)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each of B¹ and B² is independently a purinyl nucleobase or pyrimidinyl nucleobase; each of X¹ and X² is independently O or S; each of Y¹ and Y² is independently O, S, or R⁵; each of L¹ and L² is independently absent, C₁-C₂₀ alkyl or C₁-C₂₀ heteroalkyl, wherein each C₁-C₂₀ alkyl and C₁- C₂₀ heteroalkyl is optionally substituted with R⁶; each of R¹ and R² is independently hydrogen, halo, -CN, C₁-C₂₀ alkyl, or OR⁷; each of R³ and R⁴ is independently hydrogen, C₁- C₂₀ alkyl, C₁-C₂₀ heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each C₁- C₂₀ alkyl, C₁-C₂₀ heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substituted with 1-5 R⁸; R⁵ is hydrogen or C₁-C₂₀ alkyl; R⁶ is halo, -CN, C₁-C₂₀ alkyl, OR⁷, oxo, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each C₁-C₂₀ alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-5 R⁹; R⁷ is hydrogen, C₁-C₂₀ alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each C₁-C₂₀ alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-5 R⁹; each R⁸ is

independently C₁-C₂₀ alkyl, C(O)-aryl, C(O)-heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl, wherein each C₁-C₂₀ alkyl, C(O)-aryl, C(O)-heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl is optionally substituted by 1-5 R⁹; and each R⁹ is independently C₁-C₂₀ alkyl, halo, -CN, OH, O-C₁-C₂₀ alkyl, O-C₁-C₂₀ heteroalkyl, O-aiyl, or O-heteroaiyl.

In some embodiments, the compound is a compound of Formulas (I-b), (I-c), (I-d), or

(I-e):

Formula (I-b) Formula (I-c)

Formula (I-d) Formula (I-e) or a pharmaceutically acceptable salt thereof, wherein each of B¹, B², X¹, X², Y¹, Y², L¹, L², R¹, R², R³, R⁴, and subvariables thereof as previously described.

In an embodiment, the nucleobase of B¹ or B² is a naturally occurring nucleobase, e.g., a naturally occurring purinyl nucleobase or a naturally occurring pyrimidinyl nucleobase. In an embodiment, the nucleobase of B¹ or B² is a modified nucleobase, e.g., a chemically modified purinyl nucleobase or pyrimidinyl nucleobase.In some embodiments, B¹ is a purinyl nucleobase and B² is a pyrimidinyl nucleobase. In some embodiments, B¹ is adenosinyl or guanosinyl and B² is cytosinyl, thyminyl, or uracilyl. In some embodiments, each of B¹ and B² is uracilyl.

In some embodiments, B¹ is adenosinyl or guanosinyl. In some embodiments, B² is cytosinyl, thyminyl, or uracilyl. In some embodiments each of B¹ or B² is selected from:

wherein "^™™" indicates the linkage of the nucleobase to the ribonse ring.

In some embodiments, one of B¹ or B² is selected from a naturally occurring nucleobase and the other of B¹ or B² is a modified nucleobase. In some embodiments, one of B¹ or B² is adenosinyl, guanosinyl, thyminyl, cytosinyl, or uracilyl, and the other of B¹ or B² is 5'-methylcytosinyl, 5'-fluorouracilyl, 5'-propynyluracilyl, or 7-deazaadenosinyl.

In some embodiments, B¹ is adenosinyl or guanosinyl. In some embodiments, B² is cytosinyl, thyminyl, or uracilyl. In some embodiments, B¹ is adenosinyl or guanosinyl and B² is cytosinyl, thyminyl, or uracilyl. In some embodiments, each of B¹ and B² is

independently uracilyl. In some embodiments, each of B¹ and B² is independently adenosinyl.

In some embodiments, X¹ is O. In some embodiments, X² is O. In some

embodiments, each of X¹ and X² is independently O.

In some embodiments, Y¹ is O or S. In some embodiments, Y² is O or S. In some embodiments, each of Y¹ and Y² is independently O or S. In some embodiments, one of Y¹ or Y² is O and the other of Y¹ or Y² is S. In some embodiments, each of Y¹ or Y² is independently S. In some embodiments, each of Y¹ or Y² is independently O.

In some embodiments, each of L¹ and L² is independently C₁-C₆ alkyl (e.g., CH2). In some embodiments, L² is C₁-C₆ alkyl (e.g., CH2). In some embodiments, each of L¹ and L² is independently C₁-C₆ alkyl (e.g., CH2).

In some embodiments, R³ is hydrogen, aryl, or heteroaryl, wherein aryl and heteroaryl is optionally substituted with 1-5 R⁸. In some embodiments, R³ is aryl or heteroaryl, each of which is optionally substituted with 1-5 R⁸. In some embodiments, R³ is phenyl substituted with 1 R⁸. In some embodiments, R⁴ is independently hydrogen, aryl, or heteroaryl, wherein aryl and heteroaryl is optionally substituted with 1-5 R⁸. In some embodiments, R⁴ is aryl or heteroaryl, each of which is optionally substituted with 1-5 R⁸. In some embodiments, R⁴ is phenyl substituted with 1 R⁸.

In some embodiments, each of R³ and R⁴ is independently hydrogen, aryl, or heteroaryl, wherein aryl and heteroaryl is optionally substituted with 1-5 R⁸. In some embodiments, R³ is aryl or heteroaryl, each of which is optionally substituted with 1-5 R⁸, and R⁴ is hydrogen. In some embodiments, R³ is phenyl substituted with 1 R⁸ and R⁴ is hydrogen. In some embodiments, each of R³ and R⁴ is independently phenyl substituted with 1 R⁸.

In some embodiments, R⁸ is OC(O)-aryl optionally substituted by 1-5 R⁹ (e.g., 1 R⁹). In some embodiments, R⁹ is O-C₁-C₂₀ alkyl (e.g., 0-CH2(CH2)8CH3). In some embodiments, R⁹ is O-C₁-C₁2 alkyl (e.g., 0-CH₂(CH₂)8CH3). In some embodiments, R⁹ is O-C₁-C₁o alkyl (e.g., 0-CH2(CH2 CH3). In some embodiments, R⁹ is O-C₁-Cs alkyl (e.g., O- CH₂(CH₂)6CH3). In some embodiments, R⁹ is O-C₁-C₆ alkyl (e.g., 0-CH₂(CH₂)4CH3).

In some embodiments, the com ound is represented by Formula (I-f):

Formula (I-f)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each of B¹ and B² is independently a purinyl nucleobase or pyrimidinyl nucleobase; each of X¹ and X² is independently O or S; each of Y¹ and Y² is independently O, S, or R⁵; each of L¹ and L² is independently absent, C₁-C₆ alkyl or C₁-C₆ heteroalkyl, wherein each C₁-C₆ alkyl and C₁-C₆ heteroalkyl is optionally substituted with R⁶; each of R¹ and R² is independently halo; each of R³ and R⁴ is independently hydrogen, C₁-C₂₀ alkyl, C₁-C₆ heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each C₁-C₂₀ alkyl, C₁-C₆ heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substituted with 1-5 R⁸; R⁵ is hydrogen or C₁- C₂₀ alkyl; R⁶ is halo, -CN, C₁-C₂₀ alkyl, OR⁷, oxo, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each C₁-C₂₀ alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-5 R⁹; R⁷ is hydrogen, C₁-C₂₀ alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each C₁-C₂₀ alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-5 R⁹; each R⁸ is independently C₁-C₂₀ alkyl, C(O)-aryl, C(O)- heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl, wherein each C₁-C₂₀ alkyl, C(O)-aryl, C(O)- heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl is optionally substituted by 1-5 R⁹; and each R⁹ is independently C₁-C₂₀ alkyl, halo, -CN, OH, O-C₁-C₂₀ alkyl, O-C₁-C₂₀ heteroalkyl, O-aiyl, or O-heteroaiyl.

In some embodiments, th m ound is represented by Formula (I-g):

Formula (I-g)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each of B¹ and B² is independently a purinyl nucleobase or pyrimidinyl nucleobase; each of X¹ and X² is independently O; each of Y¹ and Y² is independently O or S; each of L¹ and L² is independently absent or C₁-C₆ alkyl; each of R¹ and R² is independently halo or OH; each of R³ and R⁴ is independently hydrogen or aryl optionally substituted with 1-5 R⁸; each R⁸ is independently OC(O)-aryl optionally substituted by 1-5 R⁹; and each R⁹ is independently O- C₁-C₂₀ alkyl.

In some embodiments, the compound is selected from a compound depicted in Table

1 :



60

61

Structure Compound Number

13

14

15

In an embodiment, a compound described herein a in the form of a pharmaceutically acceptable salt. Exemplary salts are described herein, such as ammonium salts. In some embodiments, the compound is a mono-salt. In some embodiments, the compound is a di- salt. A compound of Formula (I) is a small molecule nucleic acid hybrid (cyclic dinucleotide) compound that combines both antiviral and immune modulating activities. The latter activity mediates, for example, controlled apoptosis of virus-infected hepatocytes via stimulation of the innate immune response, similar to what is also achieved by IFN-a therapy in patients suffering from a viral infection.

Without wishing to be bound by theory, the mechanism of action of a compound of Formula (I) may be dissected into two components. The first component entails the host immune stimulating activity of a compound of Formula (I), which may induce endogenous IFNs via the activation of a PRR, e.g., RIG-I, NOD2, and STING. Activation may occur by binding of a compound of Formula (I) to the nucleotide binding domain of a PRR (e.g., STING), as described previously, and may further result in the induction of PRR expression (e.g., STING expression).

The second component of the mechanism of action of a compound of Formula (I) involves its direct antiviral activity, which inhibits the synthesis of viral nucleic acids by steric blockage of the viral polymerase. The block may be achieved by interaction of a compound of Formula (I) with a PRR (e.g., STING) as described earlier that then in turn may prevent the polymerase enzyme from engaging with the nucleic acid template for replication (e.g., viral-derived RNA). In some embodiments, the compound of Formula (I) directly engages with a PRR (e.g., STING). In some embodiments, the compound of Formula (I) directly engages with a PRR (e.g., STING) and induces a downstream pathway (e.g., IFN signaling).

The compounds provided herein may contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers, and diastereomeric mixtures. All such isomeric forms of these compounds are expressly included within the scope. Unless otherwise indicated when a compound is named or depicted by a structure without specifying the stereochemistry and has one or more chiral centers, it is understood to represent all possible stereoisomers of the compound. The compounds provided herewith may also contain linkages (e.g., carbon-carbon bonds, phosphorus-oxygen bonds, or phosphorus-sulfur bonds) or substituents that can restrict bond rotation, e.g. restriction resulting from the presence of a ring or double bond.

In some embodiments, the method described herein comprises administration of a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some

embodiments, the compound of Formula (I) comprises an isomer (e.g., an Rp-isomer or Sp isomer) or a mixture of isomers (e.g., Rp-isomers or Sp isomers) of a compound of Formula (I).

Methods of Use

The present disclosure relates to methods for inducing the expression of a PRR (e.g., STING) in a subject through administration of a compound of Formula (I) or a

pharmaceutically acceptable salt thereof. In some embodiments, the subject may be suffering from a condition described below, e.g., a viral infection (e.g., viral latency), a bacterial infection, or a cancer.

Treatment of Viral Infections

Pattern recognition receptors such as STING, RIG-I, and NOD2, have been shown to be an important factor in host recognition of a large number of RNA viruses from a variety of different viral families. In some embodiments, the methods of inducing expression of PRRs (e.g., STING) disclosed herein comprise administration of a compound of Formula (I) or a pharmaceutically acceptable salt thereof to a subject infected with a microbial infection. In some embodiments, the microbial infection is a virus. In some embodiments, the virus is a RNA virus (e.g., a double-stranded RNA (dsRNA) virus, a single-stranded RNA (ssRNA) virus (e.g., a positive-strand (sense) ssRNA virus or a negative- strand (antisense) ssRNA virus), or a ssRNA retrovirus) or a DNA virus (e.g., a dsDNA virus, ssDNA virus, or a dsDNA retrovirus). In some embodiments, the virus may be a Group I, Group II, Group III, Group IV, Group V, Group VI, or Group VII class of virus, e.g., according to the Baltimore classification system.

In some embodiments, the virus is dsRNA virus, e.g., a Group III virus. In some embodiments, expression of a PRR (e.g., STING) is induced through host-produced or viral- derived RNA. In some embodiments, the virus is a dsRNA virus, and is a member of the Birnaviridae, Chrysoviridae, Cystoviridae, Endornaviridae, Hypoviridae,

Megabirnaviridae, Partitiviridae, Picobirnaviridae, Reoviridae, or Totiviridae families, or other family of dsRNA virus. Exemplary dsRNA viruses and virus genera include, but are not limited to, Picobirnavirus, Rotavirus, Seadornavirus, Coltivirus, Orbivirus, and

Orthoreovirus, or a subtype, species, or variant thereof.

In some embodiments, the virus is ssRNA virus, e.g., a positive-strand (sense) ssRNA virus, e.g., a Group IV virus. In some embodiments, expression of a PRR (e.g., STING) is induced through host-produced or viral-derived RNA. In some embodiments, the virus is a positive-strand (sense) ssRNA virus, and is a member of the Arteriviridae, Coronaviridae, Mesoniviridae, Roniviridae, Dicistroviridae, Iflaviridae, Marnaviridae, Piconaviridae, Secoviridae, Alphaflexiviridae, Betaflexiviridae, Gammaflexiviridae, Tymoviridae, Alphatetraviridae, Alvernaviridae, Astroviridae, Barnaviridae, Bromoviridae, Caliciviridae, Carmotetraviridae, Closteroviridae, Flaviviridae, Leviviridae, Luteoviridae, Narnaviridae, Nodaviridae, Permutotetraviridae, Potyviridae, Togaviridae, or Virgaviridae families, or other family of positive-strand (sense) ssRNA virus. Exemplary positive-strand (sense) ssRNA viruses and virus genera include, but are not limited to, Yellow fever virus, West Nile virus, Hepatitis C virus, Dengue fever virus, Rubella virus, Ross River virus, Sindbis virus, Chikungya virus, Norwalk virus, Japanese encephalitis virus, Tick-borne encephalitis virus, St. Louis encephalitis virus, Murray Valley encephalitis virus, Kyasanur Forest disease virus (e.g., Monkey disease virus), Western Equine encephalitis virus, Eastern Equine encephalitis virus, Venezuelan Equine encephalitis virus, Sapporo virus, Norovirus, Sapovirus, Calicivirus, Parechovirus, Hepatitis A virus, Rhinovirus (e.g., Rhinovirus A, Rhinovirus B, and Rhinovirus C), Enterovirus (e.g., Enterovirus A,

Enterovirus B, Enterovirus C (e.g., poliovirus), Enterovirus D, Enterovirus E, Enterovirus F, Enterovirus G, or Enterovirus H), Apthovirus (e.g., Foot and mouth disease virus),

Nidovirales (e.g., Cavally virus, Nam Dinh virus, Middle East respiratory syndrome coronavirus (MERS-CoV), Coronavirus HKU1, Coronavirus NL63, SARS-CoV,

Coronavirus OC43, and Coronavirus 229E), Benyvirus, Blunevirus, Cilevirus, Hepevirus (e.g., Hepatitis E virus), Higrevirus, Idaeovirus, Negevirus, Ourmiavirus, Polemovirus, Sobemovirus, or Umbravirus, or a subtype, species, or variant thereof.

In some embodiments, the virus is a member of the genus Norovirus, or a subtype, species, or variant thereof. In some embodiments, the virus is the Norwalk virus, Hawaii virus, Snow Mountain virus, Mexico virus, Desert Shield virus, Southampton virus, Lordsdale virus, or Wilkinson virus, or a subtype or variant thereof. In some embodiments, the virus is a member of the genus Norovirus and can be classified as genogroup GI, genogroup Gil, genogroup GUI, genogroup GIV, or genogroup GV.

In some embodiments, the virus is ssRNA virus, e.g., a negative- strand (antisense) ssRNA virus, e.g., a Group V virus. In some embodiments, expression of a PRR (e.g., STING) is induced through host-produced or viral-derived RNA. In some embodiments, the virus is a negative- strand (antisense) ssRNA virus, and is a member of the Bornaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, Nyamiviridae, Arenaviridae, Bunyaviridae, Ophioviridae, or Orthomyxoviridae families, or other family of negative- strand (antisense) ssRNA virus. Exemplary negative- strand (antisense) ssRNA viruses and virus genera include, but are not limited to, Brona disease virus, Ebola virus, Marburg virus, Measles virus, Mumps virus, Nipah virus, Hendra virus, Respiratory syncytial virus, Influenza and Parainfluenza viruses, Metapneumovirus, Newcastle disease virus, Deltavirus (e.g., Hepatitis D virus), Dichohavirus, Emaravirus, Nyavirus, Tenuivirus, Varicosavirus, or a subtype, species, or variant thereof.

In some embodiments, the virus is an ssRNA retrovirus (ssRNA RT virus), e.g., a Group VI virus. In some embodiments, expression of a PRR (e.g., STING) is induced through host-produced or viral-derived RNA. In some embodiments, the virus is an ssRNA RT virus and is a member of the Metaviridae, Pseudoviridae, or Retrovir idae families, or other family of ssRNA RT virus. Exemplary ssRNA RT viruses and virus genera include, but are not limited to, Metavirus, Err antivirus, Alpharetrovirus (e.g., Avian leukosis virus, Rous sarcoma virus), Betaretrovirus (e.g., Mouse mammary tumor virus), Gammaretrovirus (e.g., Murine leukemia virus, Feline leukemia virus), Deltaretrovirus (e.g., human T- lymphotropic virus), Epsilonretrovirus (e.g., Walleye dermal sarcoma virus), Lentivirus (e.g., Human immunodeficiency virus 1 (HIV)), or a subtype, species, or variant thereof.

In some embodiments, the virus is a DNA virus, e.g., a dsDNA virus or an ssDNA virus. In some embodiments, the virus is a dsDNA virus, e.g., a Group I virus, and expression of a PRR (e.g., STING) is induced through host-produced or viral-derived RNA. In some embodiments, the virus is a dsDNA virus and is a member of the Myovir idae, Podoviridae, Siphoviridae, Alloherpesviridae, Herpesviridae, Malacoherpesviridae, Lipothrixviridae, Rudiviridae, Adenoviridae, Ampullaviridae, Ascoviridae, Asfarviridae, Baculoviridae, Bicaudaviridae , Clavaviridae , Corticoviridae, Fuselloviridae ,

Globuloviridae, Guttaviridae, Hytrosaviridae, Iridoviridae, Marseilleviridae, Nimaviridae, Pandoraviridae, Papillomaviridae, Phycodnaviridae, Polydnaviruses, Polymaviridae, Poxviridae, Sphaerolipoviridae, Tectiviridae, or Turriviridae families, or other family of dsDNA virus. Exemplary dsDNA viruses and virus genera include, but are not limited to, Dinodnavirus, Nudivirus, smallpox, human herpes virus, Varicella Zoster virus,

polyomavirus 6, polyomavirus 7, polyomavirus 9, polyomavirus 10, JC virus, BK virus, KI virus, WU virus, Merkel cell polyomavirus, Trichodysplasia spinulosa-associated polyomavirus, MX polyomavirus, Simian virus 40, or a subtype, species, or variant thereof.

In some embodiments, the virus is an ssDNA virus, e.g., a Group II virus, and expression of a PRR (e.g., STING) is induced through host-produced or viral-derived RNA. In some embodiments, the virus is an ssDNA virus and is a member of the Anelloviridae, Bacillariodnaviridiae, Bidnaviridae, Circoviridae, Geminiviridae, Inoviridae, Microviridae, Nanoviridae, Parvoviridae, or Spiraviridae families, or other family of ssDNA virus.

Exemplary ssDNA viruses and virus genera include, but are not limited to, Torque teno virus, Torque teno midi virus, Torque teno mini virus, Gyrovirus, Circovirus, Parvovirus B19, Bocaparvovirus, Dependoparvovirus, Erythroparvovirus, Protoparvovirus,

Tetraparvovirus, Bombyx mori densovirus type 2, lymphoidal parvo-like virus,

Hepatopancreatic parvo-like virus, or a subtype, species, or variant thereof.

In some embodiments, the virus is a dsDNA reverse transcriptase (RT) virus, e.g., a Group VII virus, and expression of a PRR (e.g., STING) is induced through host-produced or viral-derived RNA. In some embodiments, the virus is a dsDNA RT virus and is a member of the Hepadnaviridae, or Caulimoviridae families, or other family of dsDNA RT virus. Exemplary dsDNA RT viruses and virus genera include, but are not limited to, Hepatitis B virus, or a subtype, species, or variant thereof.

In some embodiments, the virus (e.g., a virus described herein) is latent, e.g., within a cell. In some embodiments, the virus is an RNA virus (e.g., a double-stranded RNA (dsRNA) virus, a single-stranded RNA (ssRNA) virus (e.g., a positive-strand (sense) ssRNA virus or a negative-strand (antisense) ssRNA virus), or a ssRNA retrovirus) or a DNA virus (e.g., a dsDNA virus, ssDNA virus, or a dsDNA retrovirus) and is latent, e.g., within a cell. In some embodiments, the virus is a Group I, Group II, Group III, Group IV, Group V, Group VI, or Group VII class of virus, e.g., according to the Baltimore classification system, and is latent, e.g., within a cell.

In some embodiments, the virus is an RNA virus (e.g., an RNA virus described herein) and is latent, e.g., within a cell. In some embodiments, the virus is an ssRNA retrovirus (ssRNA RT virus), e.g., a Group VI virus, and is latent, e.g., within a cell. In some embodiments, the virus is the human immunodeficiency virus 1 (HIV)), or a subtype, species, or variant thereof, and is latent, e.g., within a cell.

In some embodiments, the methods of inducing expression of a PRR (e.g., STING) in a subject suffering from a viral infection disclosed herein result in an increase in PRR expression (e.g., STING expression). In some embodiments, expression of a PRR (e.g., STING) is induced by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about 4, about 5, about 7.5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 150, about 200, about 250, about 500, about 1000, about 1500, about 2500, about 5000, about 10,000, or more. In some embodiments, induction of expression of a PRR (e.g., STING) occurs within about 5 minutes of administration of a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, induction of expression of a PRR (e.g., STING) occurs within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours or more following administration of a compound of Formula (I) or a pharmaceutically acceptable salt thereof to a subject.

Treatment of Bacterial Infections

Recent studies have shown that PRRs (e.g., STING) play a critical role in host recognition of bacterial infections stemming from a variety of species (Dixit, E. and Kagan, J.C. Adv Immunol (2013) 117:99-125). In some cases, bacteria may secrete nucleic acids during the exponential growth phase (e.g., Listeria monocytogenes; Abdullah, Z. et al, EMBO J (2012) 31 :4153-4164), which in turn are detected by PRRs such as RIG-I and thus promote the induction of further PRR expression. In other cases, such as for Legionella pneumophila, bacterial DNA enters into the cytosol over the course of infection and is transcribed into an RNA ligand for RIG-I (Chiu, Y. H. et al, Cell (2009) 138:576-591), thus triggering downstream PRR-mediated signaling events. PRR expression (e.g., STING expression) may further be induced upon recognition of RNA released during phagocytotic uptake of bacteria. Additionally, bacterial cell wall components such as peptidoglycans (e.g., muramyl dipeptide, i.e., MDP) may serve as ligands for activation and induction of PRRs, namely NOD2, and bacterial-derived nucleic acids such as cyclic dinucleotides (e.g., cyclic di-GMP) may bind to and activate PRRs, in particular STING. In some embodiments, the expression of one or more PRRs may be induced through other means not explicitly recited herein

In some embodiments, the methods of inducing expression of a PRR (e.g., STING) disclosed herein comprise administration of a compound of Formula (I) or a

pharmaceutically acceptable salt thereof to a subject infected with a microbial infection, e.g., a bacterial infection.

In some embodiments, the methods of inducing expression of a PRR (e.g., STING) disclosed herein comprise administration of a compound of Formula (I-a) or a

pharmaceutically acceptable salt thereof to a subject infected with a microbial infection, e.g., a bacterial infection. In some embodiments, the bacterium is a Gram-negative bacterium or a Gram-positive bacterium. Exemplary bacteria include, but are not limited to, Listeria (e.g., Listeria monocytogenes), Francisella (e.g., Francisella tularensis), Mycobacteria (e.g., Mycobacteria tuberculosis), Brucella (e.g., Brucella abortis),

Streptococcus (e.g., group B Streptococcus), Legionella (e.g., Legionella pneumophila), Escherichia (e.g., Escherichia coli), Pseudomonas (e.g., Psuedomonas aeruginosa), Salmonella (e.g., Salmonella typhi), Shigella (e.g., Shigella flexneri), Campylobacter (e.g., Campylobacter jejuni), Clostridium (e.g., Clostrodium botulinum), Enterococcus (e.g., Enterococcus faecalis), Vibrio (e.g., Vibrio cholera), Yersinia (e.g., Yersinia pestis), Staphylococcus (e.g., Staphylococcus aureus), or other genera, species, subtypes, or variants thereof.

In some embodiments, the methods of inducing expression of a PRR (e.g., STING) in a subject suffering from a bacterial infection disclosed herein result in an increase in PRR expression (e.g., STING expression). In some embodiments, expression of a PRR (e.g., STING) is induced by a factor of about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about 4, about 5, about 7.5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 75, about 100, about 150, about 200, about 250, about 500, about 1000, about 1500, about 2500, about 5000, about 10,000, or more. In some embodiments, induction of expression of a PRR (e.g., STING) occurs within about 5 minutes of administration of a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, induction of expression of a PRR (e.g., STING) occurs within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 10 hours, about 12 hours or more following administration of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

Pharmaceutical Compositions

The present disclosure features methods for inducing the expression of a PRR (e.g., STING) in a subject, the methods comprising administering a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

While it is possible for the compound of the present disclosure (e.g., a compound of Formula (I)) to be administered alone, it is preferable to administer said compound as a pharmaceutical composition or formulation, where the compounds are combined with one or more pharmaceutically acceptable diluents, excipients or carriers. The compounds according to the present disclosure may be formulated for administration in any convenient way for use in human or veterinary medicine. In certain embodiments, the compounds included in the pharmaceutical preparation may be active itself, or may be a prodrug, e.g., capable of being converted to an active compound in a physiological setting. Regardless of the route of administration selected, the compounds of the present disclosure, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present disclosure, are formulated into a pharmaceutically acceptable dosage form such as described below or by other conventional methods known to those of skill in the art.

The amount and concentration of compounds of the present disclosure (e.g., a compound of Formula (I)) in the pharmaceutical compositions, as well as the quantity of the pharmaceutical composition administered to a subject, can be selected based on clinically relevant factors, such as medically relevant characteristics of the subject (e.g., age, weight, gender, other medical conditions, and the like), the solubility of compounds in the

pharmaceutical compositions, the potency and activity of the compounds, and the manner of administration of the pharmaceutical compositions. For further information on Routes of Administration and Dosage Regimes the reader is referred to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

Thus, another aspect of the present disclosure provides pharmaceutically acceptable compositions comprising a therapeutically effective amount or prophylactically effective amount of a compound described herein (e.g., a compound of Formula (I)), formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present disclosure may be specially formulated for administration in solid or liquid form, including those adapted for oral or parenteral administration, for example, by oral dosage, or by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension.

However, in certain embodiments the subject compounds may be simply dissolved or suspended in sterile water. In certain embodiments, the pharmaceutical preparation is non- pyrogenic, i.e., does not elevate the body temperature of a patient.

The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of the compound other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example,

subcutaneous administration.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable carrier" as used herein means a

pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, stabilizing agent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject antagonists from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) ascorbic acid; (17) pyrogen-free water; (18) isotonic saline; (19) Ringer's solution; (20) ethyl alcohol; (21) phosphate buffer solutions; (22) cyclodextrins such as Captisol®; and (23) other non-toxic compatible substances such as antioxidants and antimicrobial agents employed in

pharmaceutical formulations.

As set out above, certain embodiments of the compounds described herein may contain a basic functional group, such as an amine, and are thus capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term

"pharmaceutically acceptable salts" in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present disclosure. These salts can be prepared in situ during the final isolation and purification of the compounds of the present disclosure, or by separately reacting a purified compound of the present disclosure in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like (see, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66: 1-19).

In other cases, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of the compound of the present disclosure (e.g., a compound of Formula (I). These salts can likewise be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like (see, for example, Berge et al., supra).

Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

The pharmaceutically acceptable carriers, as well as wetting agents, emulsifiers, lubricants, coloring agents, release agents, coating agents, sweetening, flavoring agents, perfuming agents, preservatives, antioxidants, and other additional components may be present in an amount between about 0.001% and 99% of the composition described herein. For example, said pharmaceutically acceptable carriers, as well as wetting agents, emulsifiers, lubricants, coloring agents, release agents, coating agents, sweetening, flavoring agents, perfuming agents, preservatives, antioxidants, and other additional components may be present from about 0.005%, about 0.01%, about 0.05%, about 0.1%, about 0.25%, about 0.5%, about 0.75%, about 1%, about 1.5%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%), about 75%, about 85%>, about 90%, about 95%, or about 99% of the composition described herein.

Pharmaceutical compositions of the present disclosure may be in a form suitable for oral administration, e.g., a liquid or solid oral dosage form. In some embodiments, the liquid dosage form comprises a suspension, a solution, a linctus, an emulsion, a drink, an elixir, or a syrup. In some embodiments, the solid dosage form comprises a capsule, tablet, powder, dragee, or powder. The pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages. Pharmaceutical compositions may comprise, in addition to the compound described herein (e.g., a compound of Formula (I)) or a

pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, and may optionally further comprise one or more pharmaceutically acceptable excipients, such as, for example, stabilizers (e.g., a binder, e.g., polymer, e.g., a precipitation inhibitor, diluents, binders, and lubricants.

In some embodiments, the composition described herein comprises a liquid dosage form for oral administration, e.g., a solution or suspension. In other embodiments, the composition described herein comprises a solid dosage form for oral administration capable of being directly compressed into a tablet. In addition, said tablet may include other medicinal or pharmaceutical agents, carriers, and or adjuvants. Exemplary pharmaceutical compositions include compressed tablets (e.g., directly compressed tablets), e.g., comprising a compound of the present disclosure (e.g., a compound of Formula (I)) or a pharmaceutically acceptable salt thereof.

Formulations of the present disclosure include those suitable for parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about 99 percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent. Pharmaceutical compositions of this disclosure suitable for parenteral administration comprise compounds of the present disclosure in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a compound of the present disclosure (e.g., a compound of Formula (I)), it may be desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered form of the compound of the present disclosure is accomplished by dissolving or suspending compound in an oil vehicle.

In some embodiments, it may be advantageous to administer the compound of the present disclosure (e.g., a compound of Formula (I)) in a sustained fashion. It will be appreciated that any formulation that provides a sustained absorption profile may be used. In certain embodiments, sustained absorption may be achieved by combining a compound of the present disclosure with other pharmaceutically acceptable ingredients, diluents, or carriers that slow its release properties into systemic circulation. Routes of Administration

The compounds and compositions used in the methods described herein may be administered to a subject in a variety of forms depending on the selected route of

administration, as will be understood by those skilled in the art. Exemplary routes of administration of the compositions used in the methods described herein include topical, enteral, or parenteral applications. Topical applications include but are not limited to epicutaneous, inhalation, enema, eye drops, ear drops, and applications through mucous membranes in the body. Enteral applications include oral administration, rectal

administration, vaginal administration, and gastric feeding tubes. Parenteral administration includes intravenous, intraarterial, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrastemal, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal,

intrapulmonary, intrathecal, rectal, and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time. In certain embodiments of the present disclosure, a composition described herein comprising a compound of Formula (I) is administered orally. In other embodiments of the present disclosure, a composition described herein comprising a compound of Formula (I) is administered parenterally (e.g., intraperitoneally). In certain embodiments of the present disclosure, a composition described herein comprising a compound of Formula (I-a) is administered orally. In other embodiments of the present disclosure, a composition described herein comprising a compound of Formula (I-a) is administered parenterally (e.g., intraperitoneally).

For intravenous, intraperitoneal, or intrathecal delivery or direct injection, the composition must be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier can be an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin. The choice of the route of administration will depend on whether a local or systemic effect is to be achieved. For example, for local effects, the composition can be formulated for topical administration and applied directly where its action is desired. For systemic, long term effects, the composition can be formulated for enteral administration and given via the digestive tract. For systemic, immediate and/or short term effects, the composition can be formulated for parenteral administration and given by routes other than through the digestive tract.

Dosages

The compositions of the present disclosure are formulated into acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients in the compositions of the present disclosure (e.g., a compound of Formula (I)) may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular

compositions of the present disclosure employed, the route of administration, the time of administration, the rate of absorption of the particular agent being employed, the duration of the treatment, other drugs, substances, and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the composition required. For example, the physician or veterinarian can start doses of the substances of the present disclosure employed in the composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a composition of the present disclosure will be that amount of the substance which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Preferably, the effective daily dose of a therapeutic composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

Preferred therapeutic dosage levels are between about 0.1 mg/kg to about 1000 mg/kg (e.g., about 0.2 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1000 mg/kg) of the composition per day administered (e.g., orally or intraperitoneally) to a subject afflicted with the disorders described herein (e.g., HBV infection). Preferred prophylactic dosage levels are between about 0.1 mg/kg to about 1000 mg/kg (e.g., about 0.2 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1000 mg/kg) of the composition per day administered (e.g., orally or intraperitoneally) to a subject. The dose may also be titrated (e.g., the dose may be escalated gradually until signs of toxicity appear, such as headache, diarrhea, or nausea).

The frequency of treatment may also vary. The subject can be treated one or more times per day (e.g., once, twice, three, four or more times) or every so-many hours (e.g., about every 2, 4, 6, 8, 12, or 24 hours). The composition can be administered 1 or 2 times per 24 hours. The time course of treatment may be of varying duration, e.g., for two, three, four, five, six, seven, eight, nine, ten, or more days, two weeks, 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, or more than one year. For example, the treatment can be twice a day for three days, twice a day for seven days, twice a day for ten days. Treatment cycles can be repeated at intervals, for example weekly, bimonthly or monthly, which are separated by periods in which no treatment is given. The treatment can be a single treatment or can last as long as the life span of the subject (e.g., many years).

Patient Selection and Monitoring

The methods of the present disclosure described herein entail administration of a compound of Formula (I) or a pharmaceutically acceptable salt thereof to a subject to induce expression of a PRR (e.g., STING). In some embodiments, the subject is suffering from or is diagnosed with a condition, e.g., a microbial infection. Accordingly, a patient and/or subject can be selected for treatment using a compound of Formula (I) or a pharmaceutically acceptable salt thereof by first evaluating the patient and/or subject to determine whether the subject is infected with a microbial infection (e.g., a viral infection or bacterial infection). A subject can be evaluated as infected with a microbial infection (e.g., a viral infection or bacterial infection) using methods known in the art. The subject can also be monitored, for example, subsequent to administration of a compound described herein (e.g., a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is an adult. In some embodiments, the subject is suffering from a microbial infection (e.g., a viral infection, a bacterial infection, a fungal infection, or a parasitic infection). In some embodiments, the subject is suffering from a viral infection (e.g., an infection caused by an RNA virus or a DNA virus). In some embodiments, the subject is suffering from a bacterial infection.

In some embodiments, the subject is infected with a virus. In some embodiments, the subject is infected with a virus, and the virus is in a latent stage. In some embodiments, the subject is infected with an RNA virus (e.g., a double-stranded RNA (dsRNA) virus, a single-stranded RNA (ssRNA) virus (e.g., a positive-strand (sense) ssRNA virus or a negative- strand (antisense) ssRNA virus), or a ssRNA retrovirus) or a DNA virus (e.g., a dsDNA virus, ssDNA virus, or a dsDNA retrovirus) and the virus is in a latent stage. In some embodiments, the subject is infected with a Group I, Group II, Group III, Group IV, Group V, Group VI, or Group VII class of virus, e.g., according to the Baltimore

classification system, and the virus is in a latent stage. In some embodiments, the subject is infected with an RNA virus (e.g., an RNA virus described herein), and the virus is in a latent stage. In some embodiments, the virus is an ssRNA retrovirus (ssRNA RT virus), e.g., a Group VI virus, and is latent, e.g., within a cell. In some embodiments, the virus is the human immunodeficiency virus 1 (HIV)), or a subtype, species, or variant thereof, and is latent, e.g., within a cell.

In some embodiments, the subject is infected with a ssRNA virus, e.g., a positive- strand (sense) ssRNA virus, e.g., a Group IV virus. In some embodiments, the subject is infected with a Norovirus, or a subtype, species, or variant thereof. In some embodiments, the subject is infected with the Norwalk virus, Hawaii virus, Snow Mountain virus, Mexico virus, Desert Shield virus, Southampton virus, Lordsdale virus, or Wilkinson virus, or a subtype or variant thereof. In some embodiments, the subject is infected with a member of the genus Norovirus, e.g., Norovirus genogroup GI, genogroup Gil, genogroup GUI, genogroup GIV, or genogroup GV.

In some embodiments, the subject is infected with a virus and is symptomatic. In some embodiments, the subject is infected with a virus and is asymptomatic. In some embodiments, the subject is infected with an ssRNA retrovirus (ssRNA RT virus), e.g., a Group VI virus, and is asymptomatic.

Combination Therapies

In some embodiments, additional therapeutic agents may be administered with compositions of the present disclosure for the treatment of a microbial infection (e.g., a viral infection, a bacterial infection, a fungal infection, or a parasitic infection) or any symptom or associated condition thereof. When combination therapy is employed, the additional therapeutic agent(s) can be administered as a separate formulation or may be combined with any of the compositions described herein.

For example, any of the methods described herein may further comprise the administration of a therapeutically effective amount of an additional agent. In some embodiments, the additional agent is an antiviral agent, an antibacterial agent, or an anticancer agent. In some embodiments, the antiviral agent comprises an interferon, a nucleoside analog, a non-nucleoside antiviral, or an immune enhancer (e.g., a non-interferon immune enhancer or a small molecule immune enhancer). In some embodiments, the antiviral agent is a capsid inhibitor, an entry inhibitor, a secretion inhibitor, a microRNA, an anti sense RNA agent, an RNAi agent, or other agent designed to inhibit viral RNA or DNA. In some embodiments, the antiviral agent is selected from entecavir, lamuvidine, adefovir, darunavir, sofosbuvir, telaprevir, tenofovir, zidovudine, and ribavirin. In some embodiments, the antibacterial agent is selected from gentamicin, kanamycin, streptomycin,

chloramphenicol, ceftobiprole, amoxicillin, penicillin, bacitracin, tetracycline, rifabutin, tigecycline, and vancomycin.

EXAMPLESThe disclosure is further illustrated by the following examples and synthesis schemes, which are not to be construed as limiting this disclosure in scope or spirit to the specific procedures herein described. It is to be understood that the examples are provided to illustrate certain embodiments and that no limitation to the scope of the disclosure is intended thereby. It is to be further understood that resort may be had to various other embodiments, modifications, and equivalents thereof which may suggest themselves to those skilled in the art without departing from the spirit of the present disclosure and/or scope of the appended claims.

Abbreviations used in the following examples and elsewhere herein are:

DCA dichloroacetic acid

DCC N,N'-dicyclohexylcarbodiimide DCM dichloromethane

DMAP 4-dimethylaminopyridine

ETT 5-(ethylthio)- IH-tetrazole

h hours

IPA isopropyl alcohol

LCMS liquid chromatography-mass spectrometry

MeOH methanol

PTSA p-Toluenesulfonic acid

r.t. room temperature

THF tetrahydrofuran

TLC thin-layer chromatography

Example 1. Synthesis of exemplary compounds of the disclosure

Cmd 9 Cmd 3

Step 1: Synthesis of 5'-OH-3'-Levulinyl-2'F-dA (2):

Levulinic acid (2.148 g, 18.5 mmol) was dissolved in dry-dioxane (50 mL) and the solution was cooled to 5-10 °C on an ice-water bath. DCC (1.939 g, 9.4 mmol) was added portion wise over 1 h. The ice-water bath was removed and the mixture was allowed to warm to room temperature and stirred for 2 hours. The dicyclohexyl urea precipitate was filtered off, and the precipitate washed with dry-dioxane (10 mL). The filtrate was then added to a solution of 5'DMT-2'F-3'OH-dA ((1), 5.0 g, 7.4 mmol) in dry pyridine (50 mL) and catalytic amount of DMAP was added under argon. After stirring for 2 hours at room temperature the mixture was evaporated to dryness. The residue was dissolved in DCM (150 mL) and washed with 5% NaHCCb (100 mL) and brine (100 mL). The organic phase was separated, dried over Na₂S04 and concentrated under reduced pressure to give the desired product (2) as a white solid. The product was carried onto the next step without further purification.

Step 2 (Tritylation): Synthesis of (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro- 2-(hydroxymethyl) tetrahydrofuran-3-yl 4-oxopentanoate (3):

5'-OH-3'-Levulinyl-2'F-dA (1) was dissolved in DCM (100 mL) and water (1.33 mL, 74 mmol) was then added. 6% DC A in DCM (100 mL) was added, reaction mixture was stirred at room temperature for 10-15 min. The reaction mixture was quenched by addition of methanol (25 mL). The resulting mixture was washed with 5% NaHCCb solution (150 mL) and brine (150 mL). The organic layers were separated, dried over Na₂S0₄ and concentrated under reduced pressure to give a crude residue. The crude residue was purified on combi- flash silica gel column chromatography eluting with 0-5% MeOH in DCM to give 3.45 g (62% yield) of the desired product (3) as a white solid.

Step 3: (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((2- cyanoethoxy)(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-4-fluoro-2- (hydroxymethyl)tetrahydrofuran-3-yl)oxy)phosphorothioyl)oxy)methyl)-4- fluorotetrahydrofuran-3-yl 4-oxopentanoate (5)

(1) (i) (Coupling):

5'OH-3'-Levulinylated-2'F-deoxy-Adinosine ((3), 700 mg, 1.48 mmol) and 5'DMT-2'F- 3 'CED-Phosphoamidite-deoxy -Uridine ((4), 1.66 g, 2.22 mmol) mixture was dried under high vacuum for 1-2 hours. Argon was flushed over R.B. flask containing reaction mixture. Anhydrous acetonitrile (40 mL) was added to reaction mixture followed by ETT (279 mg, 2.146 mmol) in acetonitrile solution (5.0 mL) under an atmosphere of argon. The resulting mixture was stirred at room temperature under argon for 2 h. Once TLC analysis showed completion of the reaction, water was added (80 μΕ, 2 equivalents to amidite). The resulting mixture was carried onto the next step.

(ii) (Sulfurization) :

In a silanized flask, Beaucage reagent (3H-BD) (592 mg, 2.96 mmol) was dissolved in acetonitrile (5.0 mL). The above coupling reaction mixture containing (5) was added to the solution of sulfurizing reagent (3H-BD) in acetonitrile under an atmosphere of argon and the resulting mixture was stirred at room temperature for 45 minutes to allow the sulfurization reaction to go to completion. Methanol (10 mL) was then added and the resulting mixture was stirred for 30 min. The reaction mixture was concentrated under reduced pressure. The crude residue was dissolved in DCM (100 mL) and washed with water (75 mL). The organic layer was separated, dried over Na₂SC"4, and used in the next step (detritylation).

(2) Detritylation: The DCM solution containing the product from the previous step was cooled to ice-water bath in a R.B. flask. 5% PTSA solution in DCM:MeOH (7:3, 100 mL) was added and the resulting mixture stirred for 15 minutes to allow the detritylation reaction to go to completion. Water (50 mL) was then added and the resulting mixture was stirred for another 15 minutes. The reaction mixture was transferred to separatory funnel water and the phases were separated. The organic layer was washed 5% NaHCCb solution (100 mL) until the pH of the aqueous layer was adjusted to above 7.0. The organic layer was then dried over Na₂S0₄ and concentrated under reduced pressure. The crude product was purified using Combiflash silica gel column chromatography eluting with 0-5% MeOH in DCM to give 960 mg of the desired product (5) as white solid.

Step 4 (Levulinyl group deprotection): 0-(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9- yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl) 0-(2-cyanoethyl) O- ((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-4-fluoro-2- (hydroxymethyl)tetrahydrofuran-3-yl) phosphorothioate (6)

3'-Leculinyl protected dinucleotide thiophosphate was treated with 0.5M hydrazine monohydrate in a mixture of pyridine:acetic acid (3 :2) and the resulting mixture was stirred at room temperature for 15 minutes. Once TLC analysis showed reaction completion, 2,4- pentanedione (2.0 mL) was then added to the reaction mixture in order to quench unreacted hydrazine hydrate. The volatiles were removed under reduced pressure and the resulting mixture was partitioned between 25% IPA in DCM (50 mL) and water (50 mL). The organic layers were separated and concentrated to dryness under reduced pressure to give a thick liquid, which was co-evaporated with toluene (2 x 15 mL) to give a crude residue. The crude product was purified using Combiflash silica gel column chromatography eluting with 0-10% MeOH in DCM to give 725 mg of the desired product (6) as white solid.

Step 5a: N-(9-((2R,3R,3aR,7aR,9R,10R,10aR,14aR)-5,12-bis(2-cyanoethoxy)-9-(2,4- dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3,10-difluoro-5,12-disulfidooctahydro-2H,7H- difuro[3,2-d:3',2'-j] [l,3,7,9]tetraoxa[2,8]diphosphacyclododecin-2-yl)-9H-purin-6- yl)benzamide (7)

(i) Cyclization: Dinucleotide phosphorothioate trimester (6) (1 equivalent) and 2- cyanoethyl tetra isopropyl phosphorodiamidite (bisamidite) (1 equivalent) were dissolved in a mixture of dry acetonitrile and dry DCM (2: 1, 30 mL). Disopropylaminotetrazolide (1 equivalent) was then added to the reaction mixture in 4 portions over a period of 1 hour under an inert atmosphere. The solution was stirred for an additional 2 h at r.t. ETT (2.0 equivalent) was then added and the reaction mixture stirred overnight. Deoxygenated water (29 [iL) was then added to reaction mixture. The crude product was carried on to the next step without further purification.

(ii) Sulfurization (Synthesis of protected cyclic phosphorothiodiphosphate): Beaucage reagent (3H-BD) (2.0 equivalent) was dissolved in acetonitrile in a silanized flask. One portion of above cyclization product (two thirds) was added to sulfurizing reagent under an atmosphere of argon. The resulting mixture was stirred at room temperature for 45 minutes. Methanol (10 mL) was then added and the reaction mixture was stirred for 30 minutes. The solvents were removed under reduced pressure and the crude residue was dissolved in DCM (50 mL) and washed with water (50 mL). The combined organic layers were dried over

Na₂S04 and concentrated under reduced pressure. The crude product was purified using Combiflash silica gel column chromatography eluting with 0-10 MeOH in DCM to give 150 mg of desired product (7).

Step 5b (Oxidation): N-(9-((2R,3R,3aR,7aR,9R,10R,10aR,14aR)-5,12-bis(2-cyanoethoxy)- 9-(2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3,10-difluoro-5-oxido-12-sulfidooctahydro- 2H,7H-difuro[3,2-d:3S2'-j] [l^,7,9]tetraoxa[2,8]diphosphacyclododecin-2-yl)-9H-purin- 6-yl)benzamide (8)

TBHP (4.0 equivalent) was added to a stirred solution of second portion of cyclization product from Step 5(a) (i) (one third) at 0 °C and the reaction mixture was warmed to r.t. over 15 minutes. Excess TBHP was then quenched by the addition of saturated sodium bisulfite solution. The resulting mixture was evaporated under reduced pressure. The resulting residue was dissolved in DCM (25 mL) and washed with water (20 mL). The organic layers were separated, dried over Na₂S04, and concentrated under reduced pressure to give crude product. The crude product was purified using Combiflash silica gel column chromatography eluting with 0-10% MeOH in DCM to give 60 mg of desired product (8).

Synthesis of Compound 2: Ammonium (2R,3R,3aR,7aR,9R,10R,10aR,14aR)-2-(6- amino-9H-purin-9-yl)-9-(2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3,10- difluorooctahydro-2H,7H-difuro[3,2-d:3',2'- j][l,3,7,9]tetraoxa[2,8]diphosphacyclododecine-5,12-bis(thiolate) 5,12-dioxide

Cmd 2

Protected cyclic phosphorothiodiphosphate (8) (60 mg) was dissolved in cone.

H4OH (2.0 mL) and stirred at r.t. overnight. Once LCMS showed reaction completion, reaction mixture was evaporated under reduced pressure to remove the ammonia. The water layer was washed with ethyl acetate (5 x 5 mL), separated and lyophilized to provide 100 mg of Compound 2 as white fluffy solid. Synthesis of Compound 4: ((((2R,3R,3aR,7aR,9R,10R,10aR,14aR)-2-(6-amino-9H- purin-9-yl)-9-(2,6-dioxo-2H-l,3-oxazin-3(6H)-yl)-3,10-difluoro-5,12-dioxidooctahydro- 2H,7H-difuro[3,2-d:3',2'-j] [l,3,7,9]tetraoxa[2,8]diphosphacyclododecine-5,12- diyl)bis(sulfanediyl))bis(methylene))bis(4,l-phenylene) bis(4-(decyloxy)benzoate)

Compound 2 (25 mg) was dissolved in water (250 μΐ.) and a solution of 4- (iodomethyl)phenyl 4-(decyloxy)benzoate (42 mg) in a mixture of THF:Acetone (1 : 1, 2.0 mL) was then added. The pH of the reaction mixture was approximately 3.5-4.0. The reaction mixture was then stirred at r.t. for 40 hours. The crude product was purified using Combiflash silica gel column chromatography eluting with 0-10% IP A in DCM to give 25 mg of

Compound 4 as yellowish brown solid.

Synthesis of Compound 3: Ammonium (2R,3R,3aR,7aR,9R,10R,10aR,14aR)-2-(6- amino-9H-purin-9-yl)-9-(2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)-3,10-difluoro-12- sulfidooctahydro-2H,7H-difuro[3,2-d:3',2'- j][l,3,7,9]tetraoxa[2,8]diphosphacyclododecin-5-olate 5,12-dioxide

Cmd 3

Protected cyclic phosphoro monothiodiphosphate (8) (60 mg) was dissolved in cone. NH4OH (5.0 mL) and stirred at r.t. overnight. Once LCMS showed reaction completion, the reaction mixture was concentrated under reduced pressure to remove the ammonia. The water layer was washed with ethyl acetate (5 x 5 mL), separated, and lyophilized to provide 50 mg of Compound 3 as a white fluffy solid.

Synthesis of Compound 9: Ammonium (2R,3R,3aR,7aR,9R,10R,10aR,14aR)-2-(6- amino-9H-purin-9-yl)-12-((4-((4-(decyloxy)benzoyl)oxy)benzyl)thio)-9-(2,4-dioxo-3,4- dihydropyrimidin-l(2H)-yl)-3,10-difluorooctahydro-2H,7H-difuro[3,2-d:3',2'- j] [l,3,7,9]tetraoxa[2,8]diphosphacyclododecin-5-olate 5,12-dioxide

Cmd 9

Compound 3 (20 mg) was dissolved in water (200 μΐ.) and a solution of 4- (iodomethyl) phenyl 4-(decyloxy)benzoate (18 mg), in a mixture of THF:Acetone (1 : 1, 1.4 mL) was added. The pH of the reaction mixture was approximately 4.0. The reaction mixture then stirred at r.t. overnight. The solvent was removed under reduced pressure and the crude product was redissolved in water: acetonitrile (1 : 1, 2.0 mL). The resulting precipitate

(unreacted alkylating reagent) was then removed by centrifugation. The mother liquor was lyophilized to provide 12 mg of crude product which was purified by using C₁s Sep pack column (Waters, 4.0 g) using 0.2 M ammonium acetate buffer. The compound was eluted with acetonitrile: water (1 : 1). The pure fractions were collected and lyophilized to provide 5-6 mg of Compound 9 as a white fluffy solid.

Example 2. In vitro induction of IRF and NF-κβ in THP1 cells

Table 2: ECso values for exemplary compounds of the disclosure

Compound No. IRF ECso (nM) NF-κβ ECso (nM)

13 26.7 144

14 350 860

15 29.5 107

17 12.7 >50

19 >10 >10

20 2.1 5.1

21 0.034 0.42

22 0.024 0.028

23 0.007 0.073

24 0.029 0.107

25 0.004 0.01

26 1.18 2.33

27 1.63 3

28 0.005 0.072

29 0.016 0.33

31 >20 >20

32 1 >10

33 0.004 0.055

34 0.011 0.192

Example 3. In vitro activation of ISG54 and NF-κβ in HEK293 cells

In this experiment, HEK293 cells (SZ14) stably expressing either the ISG54 ISRE- luc reporter or the ΝΡ-κβ-luc reporter gene were treated in duplicate with an exemplary compound of the disclosure (e.g., compound 1, compound 2, and compound 3) or 2',3'- cGAMP as a control, each in digitonin buffer for 5 hours, in order to screen for potential STING agonists. ISG54 or NF-κβ activity was determined using the Steady-glo buffer system (Promega), and are summarized in FIGS. 7A-7B and FIG. 8. Data are shown as fold induction over cells that received DMSO (compound carrier) alone as the mean, +/- standard deviation of duplicate wells per stimulant.

Example 4. Evaluation of IRF-type I IFN activity in THP cells and Raw-Lucia cells

THP 1 -dual cells were treated in triplicate with exemplary compounds of the disclosure in lipofectamine (e.g., compound 2 or compound 3) or 2',3'-cGAMP in lipofectamine as a control at varying concentrations for 22 hours. Levels of IRF-inducible luciferase reporter activity in the cell culture supernatants were assayed using the Quanti- luc reagent, and are summarized in FIG. 9. Data are shown as fold induction over cells that received DMSO (compound carrier) alone as the mean, +/- standard deviation of duplicate wells per stimulant. Alternatively, TUP 1 -Dual cells (Human monocytes)) and Raw-Lucia cells (Mouse macrophages (RAW)) in 96-well plate were stimulated in triplicate with a compound disclosed herein alone for 24 hrs. Activity of secreted luciferase in cell culture supernatant was measured using Invivogen's Quanti-luc. Data are shown as fold induction over DMSO treated cells (mean ± standard deviation of triplicate wells per stimulant).

As shown in FIGs. 31A-31B, Cmd 1, Cmd 5, Cmd 12, Cmd 13, Cmd 14, and Cmd 15 are more active in human monocytes (FIG. 31 A) and mouse macrophages (FIG. 3 IB) than the natural STING ligand 3',3'-cGAMP.

Example 5. Efficacy of exemplary compounds against norovirus, RSV, Junin Virus, Dengue Virus and HCV.

A Replicon of Norovirus strain GI NoV in HG23 (hepatoma) cell line was used and activity assessed by RNA hybridization and quantitative PCR. Cytotoxicity was measured through neutral red method. Infected cells were treated with a compound disclosed herein or 2'-C-methylcytidine (positive control). The results are shown in Table 3 below.

Table 3: Antiviral activity of the compounds against Norovirus

Compound 1 showed a high selectivity index, almost of 300, against norovirus strain GI NoV. CCso was 100 μΜ while ECso resulted of 0.342 μΜ for the HG23 cell line. Efficacy of Compound 1 against RSV.

RSVA2-infected (0.5 MOI) A549 cells were used and viral titer was estimated by viral plaque assays. The RSV infected cells were treated with DMSO or 50 μΜ, 100 μΜ, or 200 μΜ of a compound disclosed herein. RSV percentage infection was calculated based on the viral titer values. 100% infection represents RSV infection in vehicle treated cells. For vehicle vs. a compound of the disclosure treated cells p < 0.05 using a Student's t test. Treatment of RSVA-2 A549 cells with 50 μΜ, 100 μΜ, and 200 μΜ of Compound 1 all decreased RSV titer and RSV percentage infection compared to vehicle (FIGs. 12A and 12B)

Efficacy of Compound 1 against Junin Virus and Dengue Virus.

Activity against Junin (JUNV) and Dengue virus serotype 2 (DSV-2) was conducted using strain JV 4454 and DENV-2 (strain NGC) respectively in Vero cells and extracellular DENV/JUNV yields were determined by plaque assays. Cytotoxicity assays were done in parallel by neutral red, MTT or MTS methods. Infected cells were treated with vehicle or a compound disclosed herein.

As shown in FIG. 15 A, virus yield diminished 1 log in A549 infected cells treated with Compound 1 compared to untreated A549 infected cells, both at 24 and 48 hours post infection (h p.i.). The virus yield of Dengue virus serotype 2 (DSV2) diminished 1 log in A549 cells infected with DSV2 compared to untreated A549 infected cells at 24 hours post infection (h p.i.). At 48 hours post infection, no significant difference was found. (FIG. 15B)

Efficacy of Compound 1 against HCV

Activity against HCV genotypes la andlb was tested using a capture fusion assay. THP-1 cells were briefly exposed to donor serum fused with Huh7 derivative cells and qPCR was used to assess HCV replication. C₆lls were treated with various concentrations of a compound disclosed herein.

As shown in FIGs. 13A-13G, HCV RNA replication was decreased upon treatment with increasing concentrations of Compound 1.

Compound 1 elicited potent antiviral activity against all tested RNA viruses with ECso ranging from 0.34 to 5.5 μΜ, and with high selectivity index. Consistent with its mechanism of action, the STING agonist Compound 1 showed potent antiviral activity against several RNA viruses including hemorrhagic fever viruses.

Example 6: Evaluation of Induction of IRF and NF-KB

THP1 dual cells grown in complete media were treated with various concentrations of a compound of the present disclosure or DMSO control. Dual cells carry both secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of an IFN-β minimal promoter fused to five copies of the NF-kJ3 consensus transcriptional response element to measure NF-kJ3 activity and Lucia reporter gene under the control of an ISG54 minimal promoter to measure IRF activity. After 20 h incubation, IRF activity was assessed using QUANTI-luc to measure levels of Lucia and NF-kB activity was determined by measure SEAP levels at 620-655 nm. % induction was calculated from fold change in luminescence/absorbance compared to DMSO treated sample. Any negative values were given base value 1 for plotting data in log scale for accurate demonstration of dose response. ECso values were generated by curve fit in Xlfit. C₆lls grown in complete media were treated with various concentrations of a compound of the disclosure or DMSO control. Dual cells carry both secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of an IFN-β minimal promoter fused to five copies of the NF-kB consensus transcriptional response element to measure NF-kB activity and Lucia reporter gene under the control of an ISG54 minimal promoter to measure IRF activity. After 20 h incubation, IRF activity was assessed using QUANTI-luc to measure levels of Lucia and NF-kB activity was determined by measure SEAP levels at 620-655 nm. % induction was calculated from fold change in

luminescence/absorbance compared to DMSO treated sample. ECso values are generated by curve fit in Xlfit.

Cmd 1, Cmd 1A, Cmd IB, Cmd 12, Cmd 13, Cmd 14, Cmd 15 all show induction of IRF and NF-kB. (See FIGS. 17A, 17B, 18A-18D, 19A-19B, 20A-20D, 21A-21D, 22A- 22D, 23A-23D, and 24A-24B) The results indicate that Cmd 1, Cmd 1A, Cmd IB, Cmd 12, Cmd 13, Cmd 14, Cmd 15 are taken up by cells without the use of transfection agents. Cmd 3 showed no NF-kB activity (FIGS. 19A-19B).

FIGS. 27A-27B and 35A-35B compare the induction of IRF (FIGS. 27A and 35A) and NF-KB (FIGS. 27B and 35B) by Cmd 15, Cmd 15-A and Cmd 15-B (isomers of Cmd 15), and Cmd 16.

Example 7. Determination of Stability of exemplary compounds

Serum Stability Study: 0.5 mM of a compound disclosed herein is incubated with Rabbit Serum for various time points at 37 °C. The reactions are quenched with addition of 1 mL Acetonitrile. The supernatant with compound was collected after snap freezing and centrifuging @ 4 °C for 5 min. The supernatant with compound was later analyzed in HPLC.

Microsome Stability Study: 0.5 mM of a compound disclosed herein is incubated with Human microsomes for various time points at 37 °C. The reactions are initiated with 20 mM NADPH, incubated, then quenched with addition of 1 mL Acetonitrile. The supernatant with compound was collected after snap freezing and centrifuging at 4 °C for 5 min. The supernatant with compound was later analyzed in HPLC.

As can be seen in FIGS. 25A-25B, the isomers of Cmd 1, Cmpl-A and Cmd 1-B, are stable in Rabbit serum and Human microsomes. Peak 1 and peak 2 represent Cmds 1-A and 1-B. Cmd 15 is also stable in Rabbit serum and Human microsomes. FIGS. 26A-26B show the stability of the isomers of Cmd 1, Cmd 15 1-A and Cmd 15B in Rabbit serum and Human microsomes.

Example 8. Determination of cytotoxicity of exemplary compounds

The cytotoxicity of exemplary compounds in THP1 cells was assessed using C₆ll titer Glo Assay (Promega). THPl dual cells grown in complete media were treated with various concentrations of compounds or DMSO control. The C₆llTiter-Glo® Luminescent C₆ll Viability/cytotoxicity was a determined by assessing number of viable cells in culture based on quantitation of the ATP present through a "glow-type" luminescent signal, produced by the luciferase reaction. % apoptosis was calculated from fold change in luminescence compared to DMSO treated sample.

FIG. 28 shows the induction of apoptosis through % cytoxicity of THPl cells when treated with various concentrations (5 μΜ, 14 μΜ, 41 μΜ, 123 μΜ, 370 μΜ, 1111 μΜ, 3333 μΜ, and 1000 μΜ) of Cmd 15 and its isomers, Cmd 15- A and Cmd 15-B.

Example 9. Quantification of STING binding

SZ14 HEK293 cells stably expressing the ISG54 ISRE-luc reporter gene were treated with a compounds disclosed herein, 2 3 -cGAMP (natural STING ligand), or DMSO in the presence of digitonin for 5-6 hrs. ISRE-luciferase activity was determined and normalized to DMSO treated cells (mean ± standard deviation of triplicate wells per stimulant).

As shown in FIGS. 29A-29B, the binding of Cmd 1 to STING activates type 1 IFN signaling, similar to the activation of type 1 IFN signaling observed with 2',3'-cGAMP.

Alternatively, raw-ISG-Dual cells in 96-well plates were stimulated in triplicate with compound/lipo, cGAMP/lipo complex or compound alone for 22-24 hours at 37 °C, 5% CO2. Activity of secreted luciferase in cell culture supernatant was measured using Invivogen Quanti-luc. Data are shown as fold induction over DMSO treated cells (mean ± standard deviation of triplicate wells per stimulant). As shown in FIG 30, Cmd 1 is highly active in mouse macrophages in activating type 1 IFN signaling, similar to the activation of type 1 IFN signaling observed with 2',3'- cGAMP.

Example 10. Induction of Type III IFN (IL-29) production in THP cells by exemplary compounds

TUP 1 -Dual (WT) cells were treated in triplicate with an exemplary compound alone or cGAMP/lipo for 21 hrs. Level of IL-29 in culture supernatant was determined using ELISA. Results shown are the average ± standard deviation of duplicate wells.

Treatment of cells with Cmd 1 and Cmd 15 induced type III interferon (IL-29) production in THP1 cells (FIG. 33A). This indicates that both Cmd 1 and Cmd 15 are taken up by cells without use of a transfection reagent (FIG. 33B).

Example 11: Induction of Type I IFN production in THP cells by exemplary compounds

SZ14 cells (HEK293 stably expressing ISG54 ISRE-luc reporter gene) were treated in triplicate with compound/digitonin buffer for 5 hrs. ISG54 ISRE-luc activity was determined using Promega Steady-Glo luciferase assay buffer and normalized to DMSO treated cells (mean ± standard deviation of triplicate wells).

Alternatively, TUP 1 -Dual (WT) cells were treated in triplicate with compound alone for 3-22h. IRF-type I IFN activity was determined using Quanti-luc buffer and normalized to DMSO treated cells (mean ± standard deviation of triplicate wells). FIG. 32A-B and FIG. 34

FIGS. 32A-32B and 34A-34B show the induction of type I IFN signaling in HEK293 (FIG. 32A) and THP1 (FIG. 32B) cells treated with Cmd 1 and its isomers Cmd 1A (Cmd 1- PK1) and Cmd IB (Cmd 1-PK2). Cmd 1, Cmd 1-A, Cmd 1-B, Cmd 13 and Cmd 15 all induce type I IFN signaling compared to control.

EQUIVALENTS

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety. While this disclosure has been described with reference to specific aspects, it is apparent that other aspects and variations may be devised by others skilled in the art without departing from the true spirit and scope of the disclosure. The appended claims are intended to be construed to include all such aspects and equivalent variations. Any patent, publication, or other disclosure material, in whole or in part, that is said to be incorporated by reference herein is incorporated herein only to the extent that the incorporated material does not conflict with existing definitions, statements, or other disclosure material set forth in this disclosure. As such, and to the extent necessary, the disclosure as explicitly set forth herein supersedes any conflicting material incorporated herein by reference.

While this disclosure has been particularly shown and described with reference to embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the disclosure encompassed by the appended claims.

Claims

What is claimed is:

1. A compound of Formula I):

Formula (I)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

Z is either S or O;

each of Y¹ and Y² is independently O, S, or NR⁵;

each R⁸ is independently C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), O-aiyl, OC(O)NR₅-C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), S(O)₂NR₅-aryl, NR₅C(O)-aryl, N(R₅)₂C(O)-aryl, C(O)-aryl, C(O)- heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl, OC(O)-C₁-C₂₀ alkyl (e.g., C₁-C₆), OC(O)0-C₁- C₂₀ alkyl (e.g., C₁-C₆), wherein each C₁-C₂₀ alkyl, O-aiyl, OC(O)NR₅-C₁-C₂₀ alkyl,

2. A compound of Formula I-a):

Formula (I-a)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

each of Y¹ and Y² is independently O, S, or NR⁵;

R⁵ is hydrogen or C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl);

3. The compound of claim 2, wherein the compound is a compound of Formulas (I-b), (I-c), (I-d or (I-e):

Formula (I-d) Formula (I-e) or a pharmaceutically acceptable salt thereof, wherein each of B¹, B², X¹, X², Y¹, Y², L¹, L², R¹, R², R³, R⁴, and subvariables thereof are defined as in claim 1.

4. The compound of any one of the preceding claims, wherein B¹ is a purinyl nucleobase and B² is a pyrimidinyl nucleobase.

5. The compound of any one of the preceding claims, wherein B¹ is adenosinyl or guanosinyl and B² is cytosinyl, thyminyl, or uracilyl.

6. The compound of any one of the preceding claims, wherein B¹ is adenosinyl, and B² is uracilyl.

7. The compound of any one of the preceding claims, wherein each of R¹ and R² is independently hydrogen, halo, or OR⁶.

8. The compound of any one of the preceding claims, wherein each of R¹ and R² is independently halo (e.g., fluoro).

9. The compound of any one of the preceding claims, wherein each of R¹ and R² is not hydrogen or OR⁷.

10. The compound of any one of the preceding claims, wherein each of X¹ and X² is independently O.

11. The compound of any one of the preceding claims, wherein each of Y¹ and Y² is independently O or S.

12. The compound of any one of the preceding claims, wherein one of Y¹ or Y² is O and the other of Y¹ or Y² is S.

13. The compound of any one of the preceding claims, wherein each of Y¹ or Y² is independently S.

14. The compound of any one of the preceding claims, wherein each of Y¹ or Y² is independently O.

15. The compound of any one of the preceding claims, wherein each of L¹ and L² is independently C₁-C₆ alkyl (e.g., CH2).

16. The compound of any one of the preceding claims, wherein each of R³ and R⁴ is independently hydrogen, aryl, or heteroaiyl, wherein aryl and heteroaiyl is optionally substituted with 1-5 R⁸.

17. The compound of any one of the preceding claims, wherein R³ is aryl or heteroaiyl, each of which is optionally substituted with 1-5 R⁸, and R⁴ is hydrogen.

18. The compound of any one of the preceding claims, wherein R³ is phenyl substituted with 1 R⁸ and R⁴ is hydrogen.

19. The compound of any one of the preceding claims, wherein each of R³ and R⁴ is independently phenyl substituted with 1 R⁸.

20. The compound of any one of the preceding claims, wherein each of Y¹ and Y² is O and each of R³ and R⁴ is independently hydrogen.

21. The compound of any one of the preceding claims, wherein Y² is O and R⁴ is hydrogen.

22. The compound of any one of the preceding claims, wherein each of Y¹ and Y² is independently S and each of R³ and R⁴ is independently substituted with 1 R⁸.

23. The compound of any one of the preceding claims, wherein Y¹ is S and R³ is substituted with 1 R⁸.

24. The compound of any one of the preceding claims, wherein R⁸ is OC(O)-aryl optionally substituted by 1-5 R⁹ (e.g., 1 R⁹).

25. The compound of claim 24, wherein R⁹ is O-C₁-C₁2 alkyl (e.g., 0-CH₂(CH2>CH3).

26. The compound of claim 1, wherein the compound is represented by Formula (I-f):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

each of Y¹ and Y² is independently O, S, or NR⁵;

each of R¹ and R² is independently halo;

R⁵ is hydrogen or C₁-C₆ alkyl;

27. The compound of claim 1, wherein the compound is represented by Formula (I-g):

Formula (I-g)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

each of B¹ and B² is independently a purinyl nucleobase or pyrimidinyl nucleobase; each of X¹ and X² is independently O;

each of Y¹ and Y² is independently O or S;

each of L¹ and L² is independently absent or C₁-C₆ alkyl;

each of R¹ and R² is independently halo or OH;

R⁸;

each R⁸ is independently OC(O)-aryl optionally substituted by 1-5 R⁹; and each R⁹ is independently O-C₁-C₂₀ alkyl.

28. The compound of any one of the preceding claims, wherein the compound is selected from Table 1 :

Structure Compound Number

13

14

15

or a pharmaceutically acceptable salt thereof.

29. A method of treating a microbial infection in a subject, the method comprising administering to the subject a compound of Formula (I),

Formula (I)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

Z is either S or O

each of Y¹ and Y² is independently O, S, or NR⁵;

each R⁸ is independently C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), O-aiyl, OC(O)NR₅-C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl), S(O)₂NR₅-aryl, NR₅C(O)-aryl, NR₅R₅C(O)-aryl, C(O)-aryl, C(O)- heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl, OC(O)-C₁-C₂₀ alkyl (e.g., C₁-C₆), OC(O)0-C₁- C₂₀ alkyl (e.g., C₁-C₆), wherein each C₁-C₂₀ alkyl, O-aiyl, OC(O)NR₅-C₁-C₂₀ alkyl, S(O)2NR₅-aryl, NR₅C(O)-aryl, CH2NR₅C(O)-aryl, C(O)-aryl, C(O)-heteroaryl, OC(O)-aryl, or OC(O)-heteroaryl, OC(O)-C₁-C₂₀ alkyl (e.g., C₁-C₆), OC(O)0-C₁-C₂₀ alkyl (e.g., C₁-C₆), is optionally substituted by 1-5 R⁹; and

30. A method of treating a microbial infection in a subject, the method comprising administering to the subject a com ound of Formula (I-a),

Formula (I-a)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

each of Y¹ and Y² is independently O, S, or NR⁵;

R⁵ is hydrogen or C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl);

31. A method of inducing the expression of a pattern recognition receptor in a subject suffering from a microbial infection, the method comprising administering to the subject a compound of Formula (I),

Formula (I)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

Z is either S or O

each of Y¹ and Y² is independently O, S, or NR⁵;

32. A method of inducing the expression of a pattern recognition receptor in a subject suffering from a microbial infection, the method comprising administering to the subject a compound of Formula (I-a),

Formula (I-a)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

each of B¹ and B² is independently a purinyl nucleobase or pyrimidinyl nucleobase; each of X¹ and X² is independently O or S; each of Y¹ and Y² is independently O, S, or NR⁵;

R⁵ is hydrogen or C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl);

33. A method of treating a viral infection in a subject, the method comprising

administering to the subject a com ound of Formula (I),

Formula (I)

harmaceutically acceptable salt or stereoisomer thereof, wherein: Z is either S or O

each of Y¹ and Y² is independently O, S, or NR⁵;

34. A method of treating a viral infection in a subject, the method comprising administering to the subject a compound of Formula (I-a),

Formula (I-a)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

each of Y¹ and Y² is independently O, S, or NR⁵;

R⁵ is hydrogen or C₁-C₂₀ alkyl (e.g., C₁-C₆ alkyl);

35. The method of claim 33 or 34, wherein the viral infection is Hepatitis C virus, Norovirus, Junin virus, Respiratory syncytial virus, or Dengue virus.