Small Molecule Inhibitors of STING Signaling Compositions and Methods of Use

Abstract

The compound of the present application or its pharma- ceutically acceptable salt can interact with stimulator of interferon genes (STING) protein and attenuate its activity.In one embodiment of the present invention, the inhibitor compound binds to STING and attenuates the downstream signal transduction of STING.The pharmaceutical compositions and methods involving such compounds as STING modulators are further provided herein.

Description

[Technical field]

[0001] The present invention relates to compositions of inhibitors of stimulator of interferon genes (STING) and methods of using said inhibitors of STING. [Background technology]

[0002] Human STING (TMEM173:NM_198282) is a 379 amino acid transmembrane-spanning integral protein that controls innate immune signaling triggered by cytosolic DNA species produced by invading microbes. STING is potently activated through interaction with cyclic dinucleotides (CDNs), such as cyclic-di-AMP, which can be secreted by bacteria, including Listeria monocytogenes. Conversely, cytosolic double-stranded deoxyribonucleic acid (dsDNA) species, which can include microbial DNA or self-DNA leaked from the nucleus, can trigger STING signaling after binding to cGAS (cyclic GMP-AMP synthase), a 522 amino acid protein that, in the presence of ATP and GTP, catalyzes the production of a type of CDN called cGAMP (cyclic [G(2',5')pA(3',5')p]), which contains one 2'-5' phosphodiester bond and a canonical 3-5' bond. CDN binding results in the relocation of STING, complexed with the IRF3 kinase TANK-binding kinase 1 (TBK1), to the perinuclear region of the cell. Association with CDNs enables STING to activate the transcription factors IRF3 and NF-κB, which stimulate the production of type I interferons (IFNs) and proinflammatory cytokines, promoting adaptive immunity. Summary of the Invention

[0003] In one aspect, the present application provides a compound of formula I:

[0004] [ka]

[0005] (wherein R1, R2, R3, R4 and R5 are independently selected from the group consisting of -H, -halogen, -(C1-C6)alkyl, -(C3-C6)cycloalkyl, -(C1-C6)haloalkyl, -(C1-C6)alkoxy, -(C1-C6)haloalkoxy, -(C2-C6)alkenyl, -(C2-C6)alkynyl, -(C1-C6)dialkylether, -(C1-C6)alkyl(C3-C6)cycloalkylether, -(C1-C6)alkylarylether, -nitro, -CN, -OH, -COOH, -SH, -NH2, -NH(C1-C4)alkyl, and -N((C1-C4)alkyl)2; R6, R7 and R8 are independently selected from the group consisting of -H, -halogen, R is an atom selected from the group consisting of carbon and nitrogen; R is an atom selected from the group consisting of carbon and nitrogen; 10 is an atom selected from the group consisting of carbon, sulfur, nitrogen and oxygen; R 11 is an atom selected from the group consisting of sulfur, nitrogen and oxygen, or a pharma- ceutically acceptable salt, hydrate, ester, solvate, prodrug, stereoisomer, or tautomer thereof.

[0006] In another aspect, the present application relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present application and a pharma- ceutically acceptable carrier.

[0007] In one embodiment of the present application, the method of regulating (e.g., inhibiting or stimulating) STING protein comprises applying a STING inhibitor. The method comprises administering to a subject in need thereof an effective amount of a compound of the present application or a pharma- ceutically acceptable salt or ester thereof, or a pharmaceutical composition of the present application. In one embodiment, the STING protein is a human STING protein.

[0008] In one embodiment of the present application, a method of treating or preventing a disease caused by or associated with STING expression, activity, and / or function (e.g., deregulation of STING expression, activity, and / or function) comprises application of a STING inhibitor. The method further comprises administering to a subject in need thereof an effective amount of a STING inhibitor compound of the present application or a pharma- ceutically acceptable salt or ester thereof, or a pharmaceutical composition of the present application.

[0009] Another aspect of the present application relates to a method of treating or preventing a disease associated with deregulation of one or more intracellular pathways in which the STING protein is involved (e.g., deregulation of intracellular dsDNA-mediated type I interferon activation), comprising administering to a subject in need thereof an effective amount of a STING compound of the present application or a pharma- ceutically acceptable salt or ester thereof, or a pharmaceutical composition of the present application.

[0010] Another aspect of the present application pertains to kits comprising a compound of the present application or a pharma- ceutically acceptable salt or ester thereof, or a pharmaceutical composition of the present application.

[0011] Another aspect of the present application relates to a compound of the present application or a pharma- ceutically acceptable salt or ester thereof, or a pharmaceutical composition of the present application, for use in the manufacture of a medicament for modulating (e.g., inhibiting or stimulating) STING protein, for treating or preventing a disease caused by or associated with STING expression, activity, and / or function (e.g., deregulation of STING expression, activity, and / or function), or for treating or preventing a disease associated with deregulation of one or more intracellular pathways in which STING protein plays a role (e.g., deregulation of intracellular dsDNA-mediated type I interferon activation).

[0012] Another aspect of the present application relates to the use of a compound of the present application or a pharma- ceutically acceptable salt or ester thereof, or a pharmaceutical composition of the present application, in the manufacture of a medicament for modulating (e.g., inhibiting or stimulating) STING protein, for treating or preventing a disease caused by or associated with STING expression, activity, and / or function (e.g., deregulation of STING expression, activity, and / or function), or for treating or preventing a disease associated with deregulation of one or more intracellular pathways in which STING protein plays a role (e.g., deregulation of intracellular dsDNA-mediated type I interferon activation).

[0013] Another aspect of the present application relates to a compound of the present application or a pharma- ceutically acceptable salt or ester thereof, or a pharmaceutical composition of the present application, for use in modulating (e.g., inhibiting or stimulating) STING protein, in treating or preventing a disease caused by or associated with STING expression, activity, and / or function (e.g., deregulation of STING expression, activity, and / or function), or in treating or preventing a disease associated with deregulation of one or more intracellular pathways in which STING protein plays a role (e.g., deregulation of intracellular dsDNA-mediated type I interferon activation).

[0014] Another aspect of the present application relates to the use of a compound of the present application or a pharma- ceutically acceptable salt or ester thereof, or a pharmaceutical composition of the present application, in modulating (e.g., inhibiting or stimulating) STING protein, in treating or preventing a disease caused by or associated with STING expression, activity, and / or function (e.g., deregulation of STING expression, activity, and / or function), or in treating or preventing a disease associated with deregulation of one or more intracellular pathways in which the STING protein is involved (e.g., deregulation of intracellular dsDNA-mediated type 1 interferon activation).

[0015] The present application provides inhibitors of the STING protein that are therapeutic agents in the treatment or prevention of diseases such as cancer inflammation, and other immune disorders.

[0016] Details of the present disclosure are described in the accompanying description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, illustrative methods and materials are described herein. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. Other features, objects, and advantages of the present disclosure will become apparent from the specification and claims. In this specification and the appended claims, the singular form also includes the plural form unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0017] Various embodiments of the invention will be explained in more detail with reference to the following drawings: [Brief description of the drawings]

[0018] [Figure 1A]Figure 1A shows the inhibitory effect of compound W1 on cells transfected with the ISD of STING (W1) at concentrations of 10 μM (493) and 50 μM (490), where W1 was placed on hTERT-pIFNβ-Glu cells (hTERT) cells that had been stably transfected for 15 hours with a type I IFNβ promoter driving luciferase and a CMV promoter driving SEAP, and transfected with double-stranded DNA at 3 μg / ml. According to one embodiment of the present invention, inhibition of IFNβ expression was measured by luciferase induction 24 hours after transfection of the ISD, DMSO is shown (230) and ISD treatment alone (220) was used as control; [Figure 1B] FIG. 1B shows quantitative real-time PCR (qPCR) of Cxcl10 in normal hTERT cells 6 hours after ISD transfection with / without W1 treatment as in FIG. 1A according to one embodiment of the present invention, where Mock is shown (210) and ISD treatment alone (220) was used as control; [Figure 1C] FIG. 1C is a photograph showing Western blot analysis of STING, phospho-TBK1 (pTBK1) and phospho-IRF3 (pIRF3) performed 6 hours after ISD transfection with / without W1 treatment as in FIG. 1A according to one embodiment of the present invention, where Mock is shown (210) and ISD treatment alone (220) was used as a control; [Figure 1D] FIG. 1D is a photograph showing confocal analysis of STING trafficking performed 6 hours after ISD transfection with / without W1 treatment similar to FIG. 1A according to one embodiment of the present invention; [Figure 1E] FIG. 1E is a photograph showing a Western blot analysis in mouse fibroblasts (MEFs) treated similarly to FIG. 1A, according to one embodiment of the present invention; [Figure 1F] FIG. 1F is a photograph showing confocal analysis in MEFs treated similarly to FIG. 1A, according to one embodiment of the present invention; [Figure 1G]FIG. 1G shows qPCR in MEFs treated similarly to FIG. 1A according to one embodiment of the present invention; [Figure 1H] FIG. 1H shows the survival rate of knockout (TKO) mice treated with W1 according to one embodiment of the present invention (4-week-old TKO mice were administered W1 (25 μg / mouse) intraperitoneally twice a week for 5 months), with a control shown (210); [Figure 2A] Figure 2A shows hTERT-pIFNβ-Glu cells treated with W2 at 50 μM (490), 25 μM (492) or 10 μM (493) for 24 hours and transfected with double-stranded DNA (ISD) at 3 μg / ml. According to one embodiment of the present invention, inhibition of IFNβ expression was measured by luciferase induction 24 hours after transfection of ISD, Mock is shown (210) and ISD treatment alone (220) was used as control; [Figure 2B] FIG. 2B shows qPCR of IFNβ in normal hTERT cells 6 hours after ISD transfection with and without W2 treatment similar to FIG. 2A , according to one embodiment of the present invention, with DMSO shown (230) and ISD treatment alone (220) used as control; [Figure 2C] FIG. 2C is a photograph showing Western blot analysis of STING, phospho-STING (pSTING), pTBK1, and pIRF3 performed 6 hours after ISD transfection with and without W2 treatment as in FIG. 2A according to one embodiment of the present invention, where DMSO is shown (230) and ISD treatment alone (220) was used as a control; [Figure 2D] FIG. 2D shows an IFNβ luciferase assay performed in hTERT cells 6 hours after ISD transfection with / without W3 treatment at 50 μM (490), 25 μM (492) or 10 μM (493) as in FIG. 2A according to one embodiment of the present invention, Mock is shown (210) and ISD treatment only (220) was used as control; [Figure 2E]FIG. 2E shows qPCR of Cxcl10 performed in hTERT cells 6 hours after ISD transfection with / without W3 treatment at 50 μM (490) or 10 μM (493) as in FIG. 2A according to one embodiment of the present invention, Mock is shown (210) and ISD treatment only (220) was used as control; [Figure 2F] FIG. 2F is a photograph showing Western blot analysis of STING, pSTING, β-actin and pIRF3 performed in hTERT cells 6 hours after ISD transfection with / without W3 treatment at 50 μM (490), 25 μM (492) or 10 μM (493) as in FIG. 2A according to one embodiment of the present invention, where Mock is shown (210) and ISD treatment alone (220) was used as a control; [Figure 3A] Figure 3A shows that W11 and W15 at 25 μM (491), 12.5 μM (495) or 6.2 μM (496) were placed on hTERT-pIFNβ-Glu cells for 24 hours and the cells were transfected with double-stranded DNA (ISD) at 3 μg / ml. According to one embodiment of the present invention, inhibition of IFNβ expression was measured by luciferase induction 24 hours after transfection of ISD, Mock is shown (210) and ISD treatment alone (220) was used as control; [Figure 3B] FIG. 3B shows photographs of Western blot analysis of pTBK1, pIRF3, and pSTING (controls: STING, and β-actin) performed 6 hours after ISD transfection with and without treatment with W11 and W15 according to one embodiment of the present invention, DMSO is shown (230) and ISD treatment alone (220) was used as a control; [Figure 3C] FIG. 3C shows photographs of confocal analysis for IRF3 translocation performed 6 hours after ISD transfection treatment with W11 and W15 according to one embodiment of the present invention, where Mock is shown (210) and ISD treatment alone (220) was used as control; [Figure 3D]Figure 3D shows IFNβ1 qPCR performed in normal hTERT cells 6 hours after ISD transfection with / without W11, W15 and W-151 (240) according to one embodiment of the present invention, Mock is shown (210) and ISD treatment only (220) was used as control; [Figure 3E] Figure 3E shows qPCR of Cxcl10 performed in normal hTERT cells 6 hours after ISD transfection with / without W11, W15 and W-151 (240) according to one embodiment of the present invention, Mock is shown (210) and ISD treatment only (220) was used as control; [Figure 3F] Figure 3F shows qPCR of CCL5 performed in normal hTERT cells 6 hours after ISD transfection with / without W11, W15 and W-151 (240) according to one embodiment of the present invention, Mock is shown (210) and ISD treatment only (220) was used as control; [Figure 3G] Figure 3G shows photographs of Western blot analysis of pTBK1, pIRF3 and pSTING (controls: STING, and β-actin) performed 6 hours after ISD transfection with / without W11, W15 and W-151 (240) according to one embodiment of the present invention, DMSO is shown (230) and ISD treatment alone (220) was used as a control; [Figure 3H] FIG. 3H shows IFNβ1 qPCR performed on MEFs treated with W11, W15 and W-151 (240) according to one embodiment of the present invention, Mock is shown (210) and treated as in FIG. 3A, ISD treatment only (220) was used as a control; [Figure 3I] FIG. 3I shows qPCR of CCL5 performed on MEFs treated with W11, W15 and W-151 (240) according to one embodiment of the present invention, Mock is shown (210) and treated as in FIG. 3A, ISD treatment only (220) was used as a control; [Figure 4A]Figure 4A shows the inhibitory effect of compounds W1-W5 on cells transfected with ISD of STING at a concentration of 50 μM, where compounds W1-W5 were placed on hTERT cells that had been stably transfected for 15 hours with type I IFNβ promoter driving luciferase and CMV promoter driving SEAP, and transfected with double-stranded DNA at 3 μg / ml. According to one embodiment of the present invention, inhibition of IFNβ expression was measured by luciferase induction 24 hours after transfection of ISD, Mock is shown (210) and ISD treatment alone (220) was used as control; [Figure 4B] Figure 4B shows the inhibitory effect of compounds W1-W26 on cells transfected with ISD of STING at a concentration of 5 μM, where compounds W1-W26 were placed on hTERT cells that had been transfected with type I IFNβ promoter driving luciferase and CMV promoter driving SEAP for 15 hours and transfected with double-stranded DNA at 3 μg / ml. According to one embodiment of the present invention, inhibition of IFNβ expression was measured by luciferase induction 24 hours after transfection of ISD, Mock is shown (210) and ISD treatment alone (220) was used as control; [Figure 5A] Figure 5A shows photographs of heart tissue from wild-type mice treated with PBS three times a week for two months and stained with hematoxylin and eosin (H&E) for heart tissue at the end of the experiment; [Figure 5B] Figure 5B shows photographs of cardiac tissue from wild-type mice treated with inhibitor W15 (200 μg / mouse) three times a week for two months and stained with hematoxylin and eosin (H&E) for cardiac tissue at the end of the experiment; [Figure 5C] Figure 5C shows photographs of cardiac tissue from Trex1 KO mice treated with PBS three times a week for two months and stained with hematoxylin and eosin (H&E) for cardiac tissue at the end of the experiment; [Figure 5D]Figure 5D shows photographs of cardiac tissue from Trex1 KO mice treated with inhibitor W15 (200 μg / mouse) three times a week for two months and stained with hematoxylin and eosin (H&E) for cardiac tissue at the end of the experiment; [Figure 5E] Figure 5E shows plots of fold changes in TNF measured by qPCR in cardiac tissue from wild-type and Trex1 KO mice treated with PBS or inhibitor W15 (200 μg / mouse) three times a week for 2 months; [Figure 5F] Figure 5F shows plots of fold changes in IL-6 measured by qPCR in cardiac tissue from wild-type and Trex1 KO mice treated with PBS or inhibitor W15 (200 μg / mouse) three times a week for 2 months; [Figure 5G] FIG. 5G shows a plot of the fold change in IL-1β measured by qPCR in cardiac tissue from wild-type and Trex1 KO mice treated with PBS or inhibitor W15 (200 μg / mouse) three times a week for two months; and [Figure 6] Figure 6 shows qPCR analysis of Cxcl10 performed on hTERT stably transfected with the type I IFNβ promoter driving luciferase and the CMV promoter driving SEAP for 15 h and stimulated with ISD for 24 h, treated with compounds W15, W28-W35 compared to H-151(240) at 5 μM for 24 h. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] STING is a cellular innate immune receptor essential for controlling the transcription of many host defense genes, including type I IFNs and proinflammatory cytokines, and subsequently recognizing CDNs or aberrant DNA species within the cytosol of cells. Sources of DNA can include the genomes of invading pathogens, such as Herpes Simplex 1 (HSV1), while CDNs are known to be secreted by bacteria, such as Listeria monocytogenes. Potent activators of the STING pathway can assemble self-DNA that is thought to have leaked from the host cell's own nucleus, following cell division, or even as a result of DNA damage. Such self-DNA may be responsible for causing a wide variety of autoinflammatory diseases, such as systemic lupus erythematosus (SLE) or Aicardi-Goutières syndrome (AGS), and may even be associated with inflammation-related cancers. Recent insights into the regulation of STING signaling have generated much-needed information related to the pathogenesis of inflammatory diseases and provide novel opportunities to develop novel anti-inflammatory compounds targeting this pathway.

[0020] Therefore, there is a need for novel small molecule compounds that bind to and inhibit the activity of STING, i.e., STING inhibitors. This application addresses this need. The identified molecules disrupted STING-regulated inflammatory cytokine production in mouse and human cells. The compounds further demonstrated in vivo efficacy, reducing autoinflammatory disease in mice. In one embodiment of the present invention, the identified molecules prevent STING-dependent disease and other forms of chronic innate immune disease. Thus, the compounds of the present invention can regulate the activity of STING, and thus may provide beneficial therapeutic effects in the treatment of diseases, disorders and / or conditions in which STING regulation is beneficial, such as inflammation, graft-versus-host disease, allergy and autoimmune diseases, infectious diseases, cancer, and pre-cancerous syndromes.

[0021] The present application relates to compounds of formula I, which have been shown to potently and selectively antagonize STING proteins (e.g., human STING proteins). In one embodiment, the compounds of the present application have the formula I:

[0022] [ka]

[0023] (wherein R1, R2, R3, R4 and R5 are independently selected from the group consisting of -H, -halogen, -(C1-C6)alkyl, -(C3-C6)cycloalkyl, -(C1-C6)haloalkyl, -(C1-C6)alkoxy, -(C1-C6)haloalkoxy, -(C2-C6)alkenyl, -(C2-C6)alkynyl, -(C1-C6)dialkylether, -(C1-C6)alkyl(C3-C6)cycloalkylether, -(C1-C6)alkylarylether, -nitro, -CN, -OH, -COOH, -SH, -NH2, -NH(C1-C4)alkyl, and -N((C1-C4)alkyl)2; R6, R7 and R8 are independently selected from the group consisting of -H, -halogen, R is an atom selected from the group consisting of carbon and nitrogen; R is an atom selected from the group consisting of carbon and nitrogen; 10 is an atom selected from the group consisting of carbon, sulfur, nitrogen and oxygen; R 11 is an atom selected from the group consisting of sulfur, nitrogen and oxygen), or a pharma- ceutically acceptable salt, hydrate, ester, solvate, prodrug, stereoisomer, or tautomer thereof.

[0024] A compound of formula I, wherein R9 is carbon.

[0025] R 10 is sulfur.

[0026] R 11 is nitrogen.

[0027] A compound of formula I, wherein R2, R4, R5 and R6 are -H.

[0028] A compound of formula I, wherein R7 is -CH3.

[0029] A compound of formula I, wherein R8 is -N(CH3)2.

[0030] A compound of formula I, wherein R3 is -OCH3.

[0031] A compound of formula I, wherein R1 is selected from the group consisting of -OCH3, -CF3, and -CH2CH3.

[0032] R9 is carbon and R 10 is sulfur and R 11 is nitrogen.

[0033] R2, R4, R5 and R6 are -H, R7 is -CH3, R8 is -N(CH3)2, R9 is carbon, and R 10 is sulfur and R 11 is nitrogen.

[0034] R2, R4, R5 and R6 are -H, R3 is -OCH3, R7 is -CH3, R8 is -N(CH3)2, R9 is carbon, and R 10 is sulfur and R 11 is nitrogen.

[0035] R2, R4, R5 and R6 are -H, R7 is -CH3, R8 is -N(CH3)2, R9 is carbon, and R 10is sulfur and R 11 is nitrogen and R1 is selected from the group consisting of -OCH3, -CF3, and -CH2CH3.

[0036] R2, R3, R4, R5 and R6 are -H, R7 is -CH3, R8 is -N(CH3)2, R9 is carbon, and R 10 is sulfur and R 11 is nitrogen.

[0037] R2, R3, R4, R5 and R6 are -H, R7 is -CH3, R8 is -N(CH3)2, R9 is carbon, and R 10 is sulfur and R 11 is nitrogen and R1 is selected from the group consisting of -OCH3, -CF3, and -CH2CH3.

[0038] Further embodiments Embodiments contemplated herein include the following embodiments P1 to P34.

[0039] Embodiment P1. Formula I:

[0040] [ka]

[0041] (wherein R1, R2, R3, R4 and R5 are independently selected from the group consisting of -H, -halogen, -(C1-C6)alkyl, -(C3-C6)cycloalkyl, -(C1-C6)haloalkyl, -(C1-C6)alkoxy, -(C1-C6)haloalkoxy, -(C2-C6)alkenyl, -(C2-C6)alkynyl, -(C1-C6)dialkylether, -(C1-C6)alkyl(C3-C6)cycloalkylether, -(C1-C6)alkylarylether, -nitro, -CN, -OH, -COOH, -SH, -NH2, -NH(C1-C4)alkyl, and -N((C1-C4)alkyl)2; R6, R7 and R8 are independently selected from the group consisting of -H, -halogen, R is an atom selected from the group consisting of carbon and nitrogen; R is an atom selected from the group consisting of carbon and nitrogen; 10 is an atom selected from the group consisting of carbon, sulfur, nitrogen and oxygen; R 11 is an atom selected from the group consisting of sulfur, nitrogen and oxygen, or a pharma- ceutically acceptable salt, hydrate, ester, solvate, prodrug, stereoisomer, or tautomer thereof.

[0042] Embodiment P2. The compound of embodiment P1 wherein R9 is carbon.

[0043] Embodiment P3. A compound of Embodiment P1 or Embodiment P2 wherein R9 is sulfur.

[0044] Embodiment P4. The compound of any one of Embodiments P1 through P3 wherein R9 is nitrogen.

[0045] Embodiment P5. The compound of any one of Embodiments P1 through P4 wherein R2, R4, R5 and R6 are -H.

[0046] Embodiment P6. The compound of any one of Embodiments P1 through P5 wherein R7 is -CH3.

[0047] Embodiment P7. The compound of any one of Embodiments P1 through P6 wherein R8 is -N(CH3)2.

[0048] Embodiment P8. The compound of any one of Embodiments P1 through P7 wherein R3 is -OCH3.

[0049] Embodiment P9. The compound of any one of Embodiments P1 through P8 wherein R1 is selected from the group consisting of -OCH3, -CF3, and -CH2CH3.

[0050] Embodiment P10. R6 is -H, R7 is -CH3, R8 is -N(CH3)2, R9 is carbon and R 10 is sulfur, R 11 A compound of embodiment P1, wherein is nitrogen.

[0051] Embodiment P11. The compound of embodiment P10 wherein R2, R4 and R5 are -H.

[0052] Embodiment P12. A compound of Embodiment P10 or Embodiment P11 wherein R3 is -OCH3.

[0053] Embodiment P13. The compound of any one of Embodiments P10-P12 wherein R1 is selected from the group consisting of -OCH3, -CF3, and -CH2CH3.

[0054] Embodiment P14. R2, R3, R4, R5 and R6 are -H, R7 is -CH3, R8 is -N(CH3)2, R9 is carbon and R 10 is sulfur and R 11 A compound of embodiment P1, wherein is nitrogen.

[0055] Embodiment P15. A compound of Embodiment P14 wherein R1 is selected from the group consisting of -OCH3, -CF3, and -CH2CH3.

[0056] Embodiment P16. The compound of embodiment P1 which is 4-(4-{[2-(4-ethylphenyl)-1,3-thiazol-4-yl]methyl}piperazin-1-yl)-N,N,6-trimethylpyrimidin-2-amine and for use in treating or preventing disease.

[0057] Embodiment P17. The compound of embodiment P1 which is an inhibitor that modulates the signaling of a STING protein.

[0058] Embodiment P18. A compound of embodiment P16, wherein the disease is selected from the group consisting of graft-versus-host disease, inflammation, autoinflammation, cancer-associated inflammation, systemic lupus erythematosus and Aicardi-Goutières syndrome.

[0059] Embodiment P19. The compound of embodiment P1 which is N,N,4-trimethyl-6-[4-({2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-4-yl}methyl)piperazin-1-yl]pyrimidin-2-amine and for use in treating or preventing disease.

[0060] Embodiment P20. The compound of embodiment P19 which is an inhibitor that modulates the signaling of a STING protein.

[0061] Embodiment P21. The compound of embodiment P19, wherein the disease is selected from the group consisting of graft-versus-host disease, inflammation, autoinflammation, cancer-associated inflammation, systemic lupus erythematosus and Aicardi-Goutières syndrome.

[0062] Embodiment P22. A pharmaceutical composition for use in inhibiting a STING protein comprising a therapeutically effective amount of Compound I or a pharma- ceutically acceptable salt, hydrate, ester, solvate, prodrug, stereoisomer, or tautomer thereof and a pharma- ceutically acceptable carrier, wherein Compound I is:

[0063] [ka]

[0064] (wherein R1, R2, R3, R4 and R5 are independently selected from the group consisting of -H, -halogen, -(C1-C6)alkyl, -(C3-C6)cycloalkyl, -(C1-C6)haloalkyl, -(C1-C6)alkoxy, -(C1-C6)haloalkoxy, -(C2-C6)alkenyl, -(C2-C6)alkynyl, -(C1-C6)dialkylether, -(C1-C6)alkyl(C3-C6)cycloalkylether, -(C1-C6)alkylarylether, -nitro, -CN, -OH, -COOH, -SH, -NH2, -NH(C1-C4)alkyl, and -N((C1-C4)alkyl)2; R6, R7 and R8 are independently selected from the group consisting of -H, -halogen, R is an atom selected from the group consisting of carbon and nitrogen; R is an atom selected from the group consisting of carbon and nitrogen; 10 is an atom selected from the group consisting of carbon, sulfur, nitrogen and oxygen; R 11 is an atom selected from the group consisting of sulfur, nitrogen and oxygen.

[0065] Embodiment P23. A method for treating a human subject suffering from a disease with an inhibitor of STING protein, comprising: determining whether the human subject has dysfunctional activity of STING protein by: i) isolating a sample from the human subject having the disease; ii) performing a PCR assay on the sample to determine the functional activity of STING protein in a cell population; and iii) if the human subject has upregulated dysfunctional activity of STING, identifying a selected inhibitor treatment; and orally treating the human subject with the selected inhibitor treatment.

[0066] Embodiment P24. The inhibitor treatment comprises a therapeutically effective amount of Compound I, wherein Compound I is:

[0067] [ka]

[0068] (wherein R1, R2, R3, R4 and R5 are independently selected from the group consisting of -H, -halogen, -(C1-C6)alkyl, -(C3-C6)cycloalkyl, -(C1-C6)haloalkyl, -(C1-C6)alkoxy, -(C1-C6)haloalkoxy, -(C2-C6)alkenyl, -(C2-C6)alkynyl, -(C1-C6)dialkylether, -(C1-C6)alkyl(C3-C6)cycloalkylether, -(C1-C6)alkylarylether, -nitro, -CN, -OH, -COOH, -SH, -NH2, -NH(C1-C4)alkyl, and -N((C1-C4)alkyl)2; R6, R7 and R8 are independently selected from the group consisting of -H, -halogen, R is an atom selected from the group consisting of carbon and nitrogen; R is an atom selected from the group consisting of carbon and nitrogen; 10 is an atom selected from the group consisting of carbon, sulfur, nitrogen and oxygen; R 11 is an atom selected from the group consisting of sulfur, nitrogen, and oxygen.

[0069] Embodiment P25. R6 is -H, R7 is -CH3, R8 is -N(CH3)2, R9 is carbon and R 10 is sulfur, R 11 The method of embodiment P24, wherein is nitrogen.

[0070] Embodiment P26. R2, R3, R4, R5 and R6 are -H, R7 is -CH3, R8 is -N(CH3)2, R9 is carbon and R 10 is sulfur and R 11The method of embodiment P24, wherein is nitrogen.

[0071] Embodiment P27. The method of any one of embodiments P24 to P26, for treating a disease caused by or associated with STING protein expression, activity and / or function, or associated with upregulation of one or more intracellular pathways in which STING protein is involved.

[0072] Embodiment P28. 2-Methyl-4-nitro-N-[5-(trifluoromethyl)-1H-1,3-benzodiazol-2-yl]benzene-1-sulfonamide; 2-(3,4-dimethoxyphenyl)-4-[[4-(6-methyl-2-propan-2-ylpyrimidin-4-yl)piperazin-1-yl]methyl]-1,3-thiazole; 4-(4-{[2-(3,4-dimethoxyphenyl)-1,3-thiazol-4-yl]methyl}piperazin-1-yl)-N,N ,6-Trimethyl-1,2-dihydropyrimidin-2-amine;N,N,4-Trimethyl-6-[4-({2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-4-yl}methyl)piperazin-1-yl]pyrimidin-2-amine;4-(4-{[2-(4-ethylphenyl)-1,3-thiazol-4-yl]methyl}piperazin-1-yl)-N,N,6-trimethylpyrimidin-2-amine;4-(4-{[2-(4-ethylphenyl)-1,3-thiazol-4-yl]methyl}piperazin-1-yl)-N,N,6-trimethylpyrimidin-2-amine 4-(4-(4-({2-(4-cyclopropylphenyl)-1,3-thiazol-4-yl]methyl}piperazin-1-yl)-NN6-trimethylpyrimidin-2-amine;NN4-trimethyl-6-[4-({2-[4-(propan-2-yl)phenyl]-1,3-thiazol-4-yl}methyl)piperazin-1-yl]pyrimidin-2-amine;4-(4-(4-{[2-(4-cyclopropylphenyl)-1,3-thiazol-4-yl]methyl}piperazin-1-yl)-NN6-trimethylpyrimidin-2-amine 4-[4-({2-[4-(1.1-difluoroethyl)phenyl]-1.3-thiazol-4-yl}methyl)piperazin-1-yl]-N-N-trimethylpyrimidin-2-amine and N-N-trimethyl-6-[4-({2-[4-(2.2.2-trifluoroethyl)phenyl]-1.3-thiazol-4-yl}methyl)piperazin-1-yl]pyrimidin-2-amine.

[0073] Embodiment P29. A pharmaceutical composition comprising a compound of any one of embodiments P1-P15 or P28, and further comprising a pharma- ceutically acceptable and physiologically compatible excipient.

[0074] Embodiment P30. The compound of any one of embodiments P1-P15 or P28-P29 which is an inhibitor that modulates STING protein signaling.

[0075] Embodiment P31. A compound of any one of embodiments P1-P15 or P28-P29 for use in treating or preventing a disease.

[0076] Embodiment P32. The compound of embodiment 31, wherein the disease is selected from the group consisting of graft-versus-host disease, inflammation, autoinflammation, cancer-associated inflammation, systemic lupus erythematosus and Aicardi-Goutières syndrome.

[0077] Embodiment P33. A composition comprising a compound of any one of embodiments P1-P15 or P28-P29 in the form of a liposomal particle, nanoparticle, PEGylated compound or lipid nanoparticle.

[0078] Embodiment P34. A composition comprising a compound of any one of embodiments P1-P15 or P28-P29 and a lipid nanoparticle (LNP), wherein the LNP comprises a polymer-conjugated lipid; a sterol; a phospholipid; and an ionizable lipid.

[0079] Non-limiting illustrative compounds of the present application include those in Table I.

[0080] [Table 1] TIFF2024521048000008.tif226159TIFF2024521048000009.tif230159TIFF2024521048000010.tif225159TIFF20245210480 00011.tif242159TIFF2024521048000012.tif242159TIFF2024521048000013.tif242159TIFF2024521048000014.tif217159

[0081] [Table 2] TIFF2024521048000016.tif232159TIFF2024521048000017.tif142159*% means blockage of IRF3 translocation induced by dsDNA (ISD); ND-not determined.

[0082] Some of the aforementioned compounds may contain one or more asymmetric centers and therefore may exist in various isomeric forms, such as stereoisomers and / or diastereomers. Thus, the compounds of the present application may be in the form of individual enantiomers, diastereomers or geometric isomers, or may be in the form of a mixture of stereoisomers. In one embodiment, the compounds of the present application are enantiopure compounds. In another embodiment, a mixture of stereoisomers or diastereomers is provided.

[0083] Another aspect is an isotopically labeled compound of any of the formulae defined herein. Such compounds may be isotopically labeled with a radioactive material (e.g., 3 H, 2 H, 14 C. 13 C. 18 F, 35 S, 32 P, 125 I, and 131 I) .Such compounds are useful for drug metabolism studies and diagnostic and therapeutic applications.

[0084] Efficacy is also IC 50 The IC value can also be determined by a low IC determined under substantially similar conditions. 50 Compounds with high IC 50 In some embodiments, the substantially similar conditions include determining the level of binding of a known STING ligand to a STING protein in the presence of a compound of the present application, in vivo or in vitro.

[0085] In one embodiment, the compounds of the present application are useful as therapeutic agents and thus may be useful in treating diseases caused by or associated with STING expression, activity, and / or function (e.g., deregulation of STING expression, activity, and / or function) or diseases associated with one or more intracellular pathways in which STING is involved (e.g., regulation of intracellular DNA-mediated type I interferon activation), such as those described herein.

[0086] A "selective STING modulator" can be identified, for example, by comparing the ability of a compound to modulate STING expression / activity / function with its ability to modulate other proteins or STING proteins from other species. In some embodiments, selectivity is measured by the EC 50 or IC 50 can be identified by measuring

[0087] The compounds of the present application are defined herein by their chemical structures and / or chemical names. When a compound is referred to by both a chemical structure and a chemical name, and the chemical structure and chemical name do not coincide, the chemical structure shall be determinative of the compound's identity.

[0088] The recitation of a list of chemical groups in any definition of a variable herein includes a definition of that variable as any one group or combination of listed groups. The recitation of an embodiment for a variable herein includes that embodiment as any one embodiment or in combination with any other embodiment or portion thereof.

[0089] In another aspect, the present application provides a method for synthesizing the compounds disclosed herein.The synthesis of the compounds of the present application can be found in the present specification and in the following examples.Another embodiment is a method for producing a compound of any formula herein using any one of the reactions defined herein, or a combination thereof.The method can include the use of one or more intermediates or chemical reagents defined herein.

[0090] The present application also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present application, or a pharma- ceutically acceptable salt or ester thereof, and a pharma- ceutically acceptable carrier.

[0091] Another aspect of the present application relates to a kit comprising a compound of the present application or a pharma- ceutically acceptable salt or ester thereof, or a pharmaceutical composition of the present application.In another aspect, the present application provides a kit comprising a compound capable of modulating STING activity selected from one or more compounds disclosed herein, or a pharma- ceutically acceptable salt or ester thereof, optionally in combination with a second agent, and instructions for use.

[0092] Another aspect of the present application relates to a compound of the present application or a pharma- ceutically acceptable salt or ester thereof, or a pharmaceutical composition of the present application, for use in the manufacture of a medicament for modulating (e.g., inhibiting or stimulating) a STING protein for treating or preventing a disease caused by or associated with STING expression, activity, and / or function (e.g., deregulation of STING expression, activity, and / or function) or for treating or preventing a disease associated with deregulation of one or more intracellular pathways in which the STING protein is involved (e.g., deregulation of intracellular dsDNA-mediated type I interferon activation).

[0093] Another aspect of the present application relates to the use of a compound of the present application or a pharma- ceutically acceptable salt or ester thereof, or a pharmaceutical composition of the present application, in the manufacture of a medicament for modulating (e.g., inhibiting or stimulating) a STING protein, for treating or preventing a disease caused by or associated with STING expression, activity, and / or function (e.g., deregulation of STING expression, activity, and / or function), or for treating or preventing a disease associated with deregulation of one or more intracellular pathways in which the STING protein is involved (e.g., deregulation of intracellular dsDNA-mediated type I interferon activation).

[0094] Another aspect of the present application relates to a compound of the present application or a pharma- ceutically acceptable salt or ester thereof, or a pharmaceutical composition of the present application, for use in modulating (e.g., inhibiting or stimulating) STING protein in treating or preventing a disease caused by or associated with STING expression, activity, and / or function (e.g., deregulation of STING expression, activity, and / or function), or in treating or preventing a disease associated with deregulation of one or more intracellular pathways in which STING protein is involved (e.g., deregulation of intracellular dsDNA-mediated type I interferon activation).

[0095] Another aspect of the present application relates to the use of a compound of the present application or a pharma- ceutically acceptable salt or ester thereof, or a pharmaceutical composition of the present application, in antagonizing a STING protein in treating or preventing a disease caused by or associated with STING expression, activity, and / or function (e.g., deregulation of STING expression, activity, and / or function), or in treating or preventing a disease associated with deregulation of one or more intracellular pathways in which the STING protein is involved (e.g., deregulation of intracellular dsDNA-mediated type I interferon activation).

[0096] Methods for the synthesis of compounds The compounds of the present application can be prepared from commercially available starting materials, a wide variety of methods using compounds known in the literature, or from previously prepared intermediates by using standard synthetic methods and procedures known to those skilled in the art or that will become apparent to those skilled in the art in light of the teachings herein. Standard synthetic methods and procedures for the preparation of organic molecules and the transformation and manipulation of functional groups can be obtained from the relevant scientific literature or standard textbooks in the art. Classical texts such as, but not limited to one or several sources, Smith, MB, March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition, John Wiley & Sons: New York, 2001; and Greene, TW, Wuts, PGM, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons: New York, 1999 are useful and recognized reference textbooks of organic synthesis known to those skilled in the art. The following description of synthetic methods is presented to illustrate, but not limit, the general procedures for the preparation of the compounds of the present application. Although the processes generally provide the desired final compound at or near the end of the overall process, in certain cases it may be desirable to further convert the compound to a pharma-ceutically acceptable salt, ester, or prodrug thereof. Suitable synthetic routes are depicted in the following schemes:

[0097] Example 22: Inhibitor identification Purified STING protein was used as bait to identify binding compounds using thermal shift assays (TSA). A library containing 250,000 compounds (Enamine, Monmouth Jct., NJ) was surveyed. Compounds that bound to STING were then investigated for their ability to inhibit STING signaling (see Examples 1-4 below). In the presence of cytosolic DNA species, STING becomes activated to promote transcription of cytokines such as type I IFN. Compounds that bind to STING via TSA were then investigated for their ability to prevent cytosolic DNA species from activating type I interferon (IFN) promoters using a live cell assay. The assay consisted of immortalized human fibroblasts (hTERT) stably transfected with a type I IFN promoter driving luciferase and a CMV promoter driving SEAP (hTERT-pIFNβ-Glu). The ability of small molecules to prevent activation of type I IFN promoters and transcription of luciferase, but not SEAP in response to cytosolic DNA species, can be evaluated. Activation of STING by CDNs typically also activates transcription factors NF-κB and IRF3, both of which are required to induce transcriptional stimulation of type I IFN promoters, but not CMV promoters (IRF3 and NF-κB transcription factor binding sites are not contained in the CMV promoter). Inhibition of STING activation by small molecules identified in the first screen (HTS) and reduction of the level of luciferase in response to transfection of cytosolic DNA can be investigated.

[0098] As used herein, the phrase "STING intracellular pathway" includes the IRF3 and NF-κB pathways.

[0099] The compounds of the present application can be prepared in many ways that are well known to those skilled in the art of organic synthesis.As an example, the compounds of the present application can be synthesized using the methods described below, together with synthetic methods known in the art of organic synthetic chemistry, or variations thereof as would be understood by those skilled in the art.Preferred methods include, but are not limited to, the methods described below.

[0100] The compounds of the present application can be synthesized by the following steps outlined in the following schemes, which consist of different sequences of assembling intermediates: Starting materials are either commercially available, or prepared by known procedures reported in the literature, or as illustrated.

[0101] The compounds of the present application can be prepared as pharma- ceutically acceptable addition salts by reacting the free base form of the compound with a pharma- ceutically acceptable inorganic or organic acid. Alternatively, pharma- ceutically acceptable base addition salts of the compounds of the present application can be prepared by reacting the free acid form of the compound with a pharma- ceutically acceptable inorganic or organic base. Pharmaceutically acceptable salts can include various counterions, such as counterions of inorganic or organic acids, inorganic or organic bases, or counterions obtained by counterion exchange.

[0102] Acids and bases useful in the methods herein are known in the art. Acid catalysts can be any acidic chemical, inorganic in nature (e.g., hydrochloric acid, sulfuric acid, nitric acid, aluminum trichloride) or organic in nature (e.g., camphorsulfonic acid, p-toluenesulfonic acid, acetic acid, ytterbium triflate). Acids are useful in either catalytic or stoichiometric amounts to facilitate chemical reactions. Bases can be any basic chemical, inorganic in nature (e.g., sodium bicarbonate, potassium hydroxide) or organic in nature (e.g., triethylamine, pyridine). Bases are useful in either catalytic or stoichiometric amounts to facilitate chemical reactions.

[0103] Alternatively, salt forms of the compounds of the present application can be prepared using salts of starting materials or intermediates. The free acid or free base forms of the compounds of the present application can be prepared from the corresponding base addition salt or acid addition salt form, respectively. For example, the compounds of the present application in the form of an acid addition salt can be converted to the corresponding free base by treating with a suitable base (e.g., ammonium hydroxide solution, sodium hydroxide, etc.). The compounds of the present application in the form of a base addition salt can be converted to the corresponding free acid by treating with a suitable acid (e.g., hydrochloric acid, etc.).

[0104] Those skilled in the art will recognize whether a stereocenter exists in the compounds disclosed herein. Thus, the present application includes both possible stereoisomers (unless specified in the synthesis), including racemates as well as individual enantiomers and / or diastereomers. When a compound is desired as a single enantiomer or diastereomer, it may be obtained by stereospecific synthesis or by resolution of the final product or any convenient intermediate. Resolution of the final product, intermediate, or starting material may be affected by any suitable method known in the art. See, for example, "Stereochemistry of Organic Compounds" by EL Eliel, SH Wilen, and LN Mander (Wiley-lnterscience, 1994).

[0105] Compounds of the present application containing non-pyrroloquinoxaline nitrogens can be converted to N-oxides by treatment with an oxidizing agent (e.g., 3-chloroperoxybenzoic acid (m-CPBA) and / or hydrogen peroxide) to provide other compounds of the present application. Thus, all of the depicted and claimed non-pyrroloquinoxaline nitrogen-containing compounds may be converted to N-oxides as shown and their N-oxide derivatives (N→O or N), when permitted by valence and structure. + -O -(which may be designated as N-hydroxy or N-alkoxy). Additionally, in other cases, the nitrogens in the non-pyrroloquinoxaline compounds of the present application can be converted to N-hydroxy or N-alkoxy compounds. For example, N-hydroxy compounds can be prepared by oxidation of the parent amine with an oxidizing agent such as m-CPBA. All of the depicted and claimed non-pyrroloquinoxaline nitrogen-containing compounds are also considered to be converted, when permitted by valence and structure, to both the compounds as depicted and their N-hydroxy (i.e., N-OH) and N-alkoxy (i.e., N-OR, where R is a substituted or unsubstituted C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, 3-14 membered carbocyclic or 3-14 membered heterocyclic) derivatives.

[0106] Prodrugs of the compounds of the present application can be prepared by methods known to those skilled in the art (e.g., for further details, see Saulnier et al., (1994), Bioorganic and Medicinal Chemistry Letters, Vol. 4, p. 1985, which is expressly incorporated herein by reference in its entirety for all purposes). For example, suitable prodrugs can be prepared by reacting underivatized compounds of the present application with a suitable carbamylating agent (e.g., 1,1-acyloxyalkylcarbonochloridate, para-nitrophenyl carbonate, etc.). Specifically, the central N-acetic acid moiety of the compounds of the present invention, as well as other related carboxylic acid groups, can be modified through techniques known in the art to produce effective prodrugs of the present invention.

[0107] Derivatives of the protected compounds of the present application can be prepared by means known to those skilled in the art. A detailed description of the techniques applicable to the creation of protecting groups and their removal can be found in TW Greene, "Protecting Groups in Organic Chemistry", 3rd Edition, John Wiley and Sons, Inc., 1999, which is expressly incorporated herein by reference in its entirety for all purposes.

[0108] The compounds of the present application can be conveniently prepared, or formed during the process of the present application, as solvates (e.g., hydrates). Hydrates of the compounds of the present application can be conveniently prepared by recrystallization from an aqueous / organic solvent mixture using organic solvents such as dioxin, tetrahydrofuran or methanol.

[0109] Optical isomers may be prepared from their respective optically active precursors by the procedures described herein or by resolution of racemic mixtures. Resolution can be carried out in the presence of a resolving agent, by chromatography, or by repeated crystallization, or by some combination of these techniques known to those skilled in the art. Further details regarding resolution can be found in Jacques, et al., Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981), the entirety of which is expressly incorporated herein by reference for all purposes.

[0110] The synthesized compounds may be separated from the reaction mixture and further purified by methods such as column chromatography, high pressure liquid chromatography, or recrystallization. As one of ordinary skill in the art can appreciate, additional methods of synthesizing the compounds of the formulae herein will be apparent to one of ordinary skill in the art. Furthermore, the various synthetic steps may be carried out in a different order or sequence to provide the desired compounds. In addition, the solvents, temperatures, reaction periods, etc. defined herein are for illustrative purposes only, and one of ordinary skill in the art will recognize that variations in the reaction conditions can produce the desired bridged macrocyclic products of the present application. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. Muts, Protective Groups in Organic Synthesis, 2nd Edition, John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof, which are expressly incorporated by reference herein in their entireties for all purposes.

[0111] The compounds of the present application may be modified by appending various functionalities via any synthetic means defined herein to enhance selected biological properties. Such modifications are known in the art and include those that enhance biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), enhance oral availability, enhance solubility to allow administration by injection, alter metabolism, and alter the rate of excretion.

[0112] Biological assays The biological activity of the compounds of the present application can be measured by various biochemical or cellular assays known to those skilled in the art. Non-limiting examples of biochemical and cellular assays are listed in the Examples below.

[0113] Pharmaceutical Compositions In another aspect, a pharmaceutical composition is provided, comprising a therapeutically effective amount of a compound of the present application, or a pharma- ceutically acceptable salt or ester thereof, and a pharma- ceutically acceptable carrier.

[0114] The compounds of the present application may be administered as pharmaceutical compositions by any conventional route, in particular internally, for example orally, e.g. in the form of tablets or capsules, or parenterally, for example in the form of injectable solutions or suspensions, or topically, for example in the form of lotions, gels, ointments or creams, or nasally or in the form of suppositories.

[0115] Pharmaceutical compositions containing the compounds of the present application in free form or in the form of pharma- ceutically acceptable salts in association with at least one pharma- ceutically acceptable carrier or diluent may be prepared in a conventional manner by mixing, granulating or coating.For example, oral compositions may be tablets or gelatin capsules containing the active ingredient together with a) diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine; b) lubricants such as silica, talcum, stearic acid, its magnesium or calcium salts and / or polyethylene glycol; for tablets, also c) binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidone; optionally d) disintegrants such as starch, agar, alginic acid or its sodium salts, or effervescent mixtures; and / or e) absorbents, colorants, flavors and sweeteners.Injectable compositions may be aqueous isotonic solutions or suspensions, and suppositories may be prepared from fatty emulsions or suspensions. The compositions may be sterilized and / or may contain adjuvants such as preservatives, stabilizers, wetting or emulsifying agents, solution promoters, salts and / or buffers to adjust osmotic pressure. In addition, they may also contain other therapeutically valuable substances. Formulations suitable for transdermal application include an effective amount of the compound of the present application with a carrier. The carrier may include an absorbable pharmacologically acceptable solvent to assist in passage through the host's skin. For example, the transdermal device may be in the form of a dressing, including a backing member, a reservoir containing the compound and optionally a carrier, optionally a rate-controlling barrier for delivering the compound to the host's skin at a controlled, predetermined rate over an extended period of time, and a means for securing the device to the skin. Matrix transdermal formulations may also be used. Formulations suitable for topical application, e.g., to the skin and eyes, are preferably aqueous solutions, ointments, creams or gels well known in the art. Solubilizers, stabilizers, tonicity enhancers, buffers and preservatives may be included.

[0116] The pharmaceutical compositions of the present application comprise a therapeutically effective amount of the compounds of the present application formulated together with one or more pharma- ceutically acceptable carriers. As used herein, the term "pharma-ceutically acceptable carrier" refers to any type of non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary. Some examples of substances which may act as pharma- ceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, saturated vegetable fatty acids such as glycine, sorbic acid, or potassium sorbate, protamine sulfate, partial glyceride mixtures of water, salts or electrolytes, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene polyoxypropylene block polymers, wool fat, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and sodium carboxymethylcellulose, ethylcellulose and cellulose acetate. and its derivatives; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax, oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water, isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants, which can also be present in the composition at the discretion of the formulator.

[0117] The pharmaceutical compositions of the present application may be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (such as by powders, ointments, or eye drops), bucally, or as an oral spray or nasal drops.

[0118] Liquid dosage forms for oral administration may include pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.In addition to active compounds, liquid dosage forms may include inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.Besides inert diluents, oral compositions may also include adjuvants, such as wetting agents, emulsifying suspending agents, sweeteners, flavorings and aromatics.

[0119] Injectable preparations, for example, sterile injectable aqueous suspensions or oily suspensions, may be formulated by known techniques using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions, suspensions or emulsions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, USP and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any brand of fixed oil may be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0120] In order to prolong the effect of a drug, it is often desirable to delay the absorption of the drug from subcutaneous or intramuscular injection.This can be achieved by using a liquid suspension of crystalline or amorphous material with poor water solubility.The rate of absorption of the drug then depends on the dissolution rate, which can also depend on crystal size and crystalline form.Alternatively, delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drug in an oil vehicle.

[0121] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compound of the present application with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol or a suppository wax, which is solid at ambient temperature but liquid at body temperature and therefore will melt in the rectum or vaginal cavity and release the active compound.

[0122] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols.

[0123] The active compound may also be in microencapsulated form with one or more excipients as described above. The solid dosage forms of tablets, dragees, capsules, pills, and granules may be prepared with coatings and shells, such as enteric coatings, release control coatings, and other coatings well known in pharmaceutical formulation technology. In such solid dosage forms, the active compound may be mixed with at least one inert diluent, such as sucrose, lactose, or starch. Such dosage forms may also contain, as is common practice, additional substances other than inert diluents, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, the dosage forms may also contain buffering agents.

[0124] The dosage form for topical or transdermal administration of the compound of the present application includes ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches.The active compound is mixed under sterile conditions with a pharma- ceutically acceptable carrier and any necessary preservatives or buffers, if required.Ophthalmic preparations, ear drops, eye ointments, powders and solutions are also considered to be within the scope of the present application.

[0125] The ointments, pastes, creams and gels may contain, in addition to the active compounds of the present application, excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0126] Powders and sprays can contain, in addition to the compounds of this application, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons.

[0127] Transdermal patch has the additional advantage of providing controlled delivery of compound to the body.Such dosage forms can be prepared by dissolving or dispensing the compound in a suitable medium.Absorption enhancers can also be used to increase the flux of the compound across the skin.The rate can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

[0128] For any compound, the therapeutically effective amount can be estimated initially, for example, in cell culture assays of neoplastic cells, or in animal models, usually rats, mice, rabbits, dogs, or pigs. Animal models may also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine useful dosages and routes of administration in humans. Therapeutic / prophylactic efficacy and toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, ED 50 (the dose that is therapeutically effective in 50% of the population) and LD 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, and this ratio, LD 50 / ED 50 Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The dosage may vary within this range depending on the dosage form used, sensitivity of the patient, and the route of administration.

[0129] Dosage and administration are adjusted to provide sufficient levels of the active agent or to maintain the desired effect. Factors that may be considered include the severity of the disease state, the subject's overall health, the subject's age, weight, and sex, diet, time and frequency of administration, drug combinations, reaction sensitivities, and tolerance / response to treatment. Long-acting pharmaceutical compositions may be administered every 3-4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.

[0130] The amount of active ingredient (e.g., a compound of the present disclosure or a formulation of its salt, hydrate, solvate or isomer) in a unit dose of the composition is an effective amount and varies depending on the specific treatment involved. Those skilled in the art will understand that it may be necessary to periodically vary the dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. A wide variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalation, buccal, sublingual, intrapleural, intrathecal, intranasal, and the like. Dosage forms for topical or transdermal administration of the compounds of the present application include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. In one embodiment, the active compound is mixed under sterile conditions with a pharma- ceutically acceptable carrier and any preservatives, buffers, or propellants required.

[0131] Pharmaceutical compositions containing the active compounds of the present application may be generally prepared by means of known methods, such as conventional mixing, dissolving, granulating, dragee-making, pulverizing, emulsifying, encapsulating, encapsulating, or lyophilizing processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more pharma- ceutically acceptable carriers, including excipients and / or auxiliaries that facilitate the processing of the active compounds into preparations that can be used pharma- ceutically. Of course, the appropriate formulation depends on the route of administration selected.

[0132] Techniques for formulating and administering the disclosed compounds of this application can be found in Remington: the Science and Practice of Pharmacy, 19th Edition, Mack Publishing Co., Easton, PA (1995), which is expressly incorporated herein by reference in its entirety for all purposes. In one embodiment, the compounds described herein, and their pharmaceutically acceptable salts, are used in pharmaceutical preparations in combination with a pharmaceutically acceptable carrier or diluent. Suitable pharmaceutically acceptable carriers include inert solid fillers or diluents and sterile aqueous or organic solutions. The compounds are present in such pharmaceutical compositions in an amount sufficient to provide the desired dosage within the range described herein.

[0133] How to use In one aspect, the present application provides a method for inhibiting a STING protein, the method comprising administering to a subject in need thereof an effective amount of a compound of the present application or a pharma- ceutically acceptable salt or ester thereof, or a pharmaceutical composition of the present application.

[0134] In some embodiments, modulation of STING protein activity is achieved by IC 50 In some embodiments, modulation of STING protein activity is measured by EC 50 It is measured by:

[0135] The compounds of the application (e.g., compounds of any of the formulas described herein, compounds selected from any of the compounds described herein) can treat or prevent a disease, which is caused by or associated with STING expression, activity, and / or function (e.g., deregulation of STING expression, activity, and / or function), or is associated with deregulation of one or more intracellular pathways in which the STING protein is involved (e.g., deregulation of intracellular dsDNA-mediated type I interferon activation).

[0136] In one aspect, the present application provides a method for treating or preventing a disease, the disease being caused by or associated with STING expression, activity, and / or function (e.g., deregulation of STING expression, activity, and / or function). The method comprises administering to a subject in need thereof an effective amount of a STING inhibitor compound of the present application or a pharma- ceutically acceptable salt or ester thereof, or a pharmaceutical composition of the present application. In one aspect, the disease is a STING-mediated disorder.

[0137] In one aspect, the present application provides a method for treating or preventing a disease associated with deregulation of one or more intracellular pathways in which the STING protein is involved (e.g., deregulation of intracellular dsDNA-mediated type I interferon activation), comprising administering to a subject in need thereof an effective amount of a STING inhibitor compound of the present application or a pharma- ceutically acceptable salt or ester thereof, or a pharmaceutical composition of the present application.

[0138] In one embodiment, the present application provides a method of treating or preventing any of the diseases, disorders, and conditions described herein, and the subject is a human. In one embodiment, the present application provides a method of treatment. In one embodiment, the present application provides a method of prevention.

[0139] As inhibitors of STING protein, the compounds and compositions of the present application are particularly useful for treating or reducing the severity of a disease, condition, or disorder in which STING protein or one or more intracellular pathways in which STING is involved are implicated in the disease, condition, or disorder. In one embodiment, the present application provides a method for treating or reducing the severity of a disease, condition, or disorder with STING inhibitor compounds that modulate the binding of cyclic dinucleotides (CDNs), including non-canonical cyclic dinucleotides such as 2'3'cGAMP, to STING protein. In one embodiment, the present application provides a method for treating or reducing the severity of a disease, condition, or disorder with compounds that modulate the synthesis of type I interferons and / or type I IFN responses and other cytokines, chemokines (STING-inducible proteins).

[0140] In one aspect, the present application also provides a method for treating or preventing cell proliferation disorders such as hyperplasia, dysplasia, or precancerous lesions. Dysplasia is an early form of precancerous lesion that can be identified in a biopsy by a pathologist. The compounds of the present application may be administered to prevent hyperplasia, dysplasia, or precancerous lesions from continuing to expand or becoming cancerous. Examples of precancerous lesions may occur in skin, esophageal tissue, breast, and cervical intraepithelial tissue.

[0141] In one embodiment, the disease or disorder includes, but is not limited to, an immune disorder, an autoimmune, a cell proliferative disease or disorder, cancer, inflammation, graft versus host, transplantation, gastrointestinal disorders, rheumatoid arthritis, systemic erythroderma, cachexia, a neurodegenerative disease or disorder, a neurological disease or disorder, cardiac dysfunction, or a microbial infection (e.g., an infection caused by a virus, a bacterial, and / or a fungal infection, a parasite, or other microorganism).

[0142] In one embodiment, the disease or disorder is a cell proliferative disease or disorder.

[0143] As used herein, the term "cell proliferative disorder" refers to a condition in which unregulated or abnormal proliferation of cells, or both, can lead to the development of an undesirable condition or disease, which may or may not be cancer. Exemplary cell proliferative diseases or disorders encompass a wide variety of conditions in which cell division is deregulated. Exemplary cell proliferative disorders include, but are not limited to, neoplasms, benign tumors, malignant tumors, precancerous conditions, intraepithelial neoplasia, encapsulated tumors, metastatic tumors, liquid tumors, solid tumors, immunological tumors, hematological tumors, cancers, carcinomas, leukemias, lymphomas, sarcomas, and rapidly dividing cells. The term "rapidly dividing cells" as used herein is defined as any cell that divides at a rate that exceeds or is faster than that expected or observed between adjacent or juxtaposed cells in the same tissue. Cell proliferative diseases or disorders include precancerous conditions or conditions. Cell proliferative diseases or disorders include cancer.

[0144] In one embodiment, the proliferative disease or disorder is non-cancerous. In one embodiment, the non-cancerous disease or disorder includes, but is not limited to, rheumatoid arthritis; inflammation; autoimmune diseases; lymphoproliferative conditions; acromegaly; rheumatoid spondylitis; osteoarthritis; gout; other arthritic conditions; sepsis; septic shock; endotoxin shock; gram-negative sepsis; toxic shock syndrome; asthma; adult respiratory distress syndrome; chronic obstructive pulmonary disease; chronic pulmonary inflammation; inflammatory bowel disease; Crohn's disease; skin-related hyperproliferative disorders; psoriasis; eczema; atopic dermatitis; Dermatitis;Hyperpigmentation disorders;Ocular-related hyperproliferative disorders;Age-related macular degeneration;Ulcerative colitis;Pancreatic fibrosis;Hepatic fibrosis;Acute and chronic kidney disease;Irritable bowel syndrome;Pyresis;Restenosis;Cerebral malaria;Stroke and ischemic injury;Neurologic trauma;Alzheimer's disease;Huntington's disease;Parkinson's disease;Acute and chronic pain;Allergic rhinitis;Allergic conjunctivitis;Chronic heart failure;Acute coronary syndromes;Cachexia;Malaria;Leprosy ; leishmaniasis; Lyme disease; Reiter's syndrome; acute synovitis; muscle degeneration, bursitis; tendonitis; tenosynovitis; herniated, ruptured, or prolapsed disc syndrome; osteopetrosis; thrombosis; restenosis; silicosis; pulmonary sarcosis; bone resorption diseases such as osteoporosis; graft versus host reaction; fibroadipose hyperplasia; spinocerebellar ataxia type 1; CLOVES syndrome; harlequin ichthyosis; macrodactyly syndrome; Proteus syndrome (Wiedemann syndrome); LEOPARD syndrome; systemic sclerosis; multiple sclerosis; lupus; fibromyalgia; AIDS and other viral diseases such as shingles, herpes simplex type I or II, influenza virus, and cytomegalovirus; diabetes mellitus; hemihyperplasia multiple lipomatosis syndrome; megalencephaly; rare hypoglycemia, Klippel-Trenaunay syndrome; hamartoma; Cowden syndrome; or hyperproliferative hyperglycemia.

[0145] In one embodiment, the proliferative disease or disorder is cancer. In one embodiment, the cancer is lung cancer, colon cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, brain cancer, kidney cancer, ovarian cancer, stomach cancer, skin cancer, bone cancer, gastric cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, hepatocellular carcinoma, papillary renal carcinoma, squamous cell carcinoma of the head and neck, leukemia, lymphoma, myeloma, or a solid tumor.

[0146] The term "cancer" includes, but is not limited to, the following cancers: breast; ovary; cervix; prostate; testis, genitourinary tract; esophagus; larynx, glioblastoma; neuroblastoma; stomach; skin, keratoacanthoma; lung, epidermoid carcinoma, large cell carcinoma, small cell carcinoma, lung adenocarcinoma; bone; colon; colorectal; adenoma; pancreas, adenocarcinoma; thyroid, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma; seminoma; melanoma; sarcoma; bladder cancer; liver cancer and biliary tract; kidney carcinoma; spinal cord disorders; lymphatic disorders, Hodgkin, hairy cell; buccal cavity and pharynx (oral cavity), lips, tongue, mouth, pharynx; small intestine; colon, rectum, large intestine, rectum, brain and central nervous system; chronic myeloid leukemia (CML), and leukemia. The term "cancer" includes, but is not limited to, the following cancers: myeloma, lymphoma, or a cancer selected from gastric, renal, or and the following cancers: head and neck cancer, oropharangeal cancer, non-small cell lung cancer (NSCLC), endometrial cancer, hepatocellular carcinoma, non-Hodgkin's lymphoma, and lung cancer.

[0147] The term "cancer" also refers to any cancer caused by the proliferation of malignant neoplastic cells, such as tumors, neoplasms, carcinomas, sarcomas, leukemias, lymphomas, and the like. For example, cancers include, but are not limited to, mesothelioma, leukemias and lymphomas, such as cutaneous T-cell lymphoma (CTCL), non-cutaneous peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATLL), B-cell lymphoma, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, lymphoma, and lymphomas associated with human T-cell lymphoproliferative virus (HTLV) such as multiple myeloma, non-Hodgkin's lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hodgkin's lymphoma, Burkitt's lymphoma, adult T-cell leukemia lymphoma, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), or hepatocellular carcinoma. Further examples include solid tumors common in adults, such as myelodysplastic syndromes, pediatric solid tumors such as brain tumors, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors, and soft tissue sarcomas, head and neck cancer (e.g., oral cavity, larynx, nasopharynx, and esophagus), genitourinary cancer (e.g., prostate, bladder, kidney, uterus, ovary, testes), lung cancer (e.g., small cell and non-small cell), breast cancer, pancreatic cancer, melanoma and other skin cancers, stomach cancer, brain tumors, tumors associated with Gorlin syndrome (e.g., medulloblastoma, meningioma, etc.), and liver cancer. Additional representative forms of cancer that may be treated by the subject compounds include, but are not limited to, skeletal or smooth muscle cancer, stomach cancer, small intestine cancer, rectal cancer, salivary gland cancer, endometrial cancer, adrenal cancer, anal cancer, rectal cancer, parathyroid cancer, and pituitary cancer.

[0148] The cancer may also include colon cancer, familial adenomatous polyposis cancer and hereditary nonpolyposis colorectal cancer, or melanoma. Further, cancers include, but are not limited to, carcinoma of the labia, larynx, hypopharynx, tongue, salivary gland, gastric carcinoma, adenocarcinoma, thyroid cancer (medullary and papillary thyroid cancer), renal carcinoma, renal parenchymal carcinoma, cervical cancer, uterine cancer, endometrial cancer, choriocarcinoma, testicular cancer, urinary tract cancer, melanoma, glioblastoma, astrocytoma, meningioma, medulloblastoma and brain tumors such as peripheral neuroectodermal tumors, gallbladder cancer, bronchial carcinoma, multiple myeloma, basal cell tumor, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing's sarcoma, and plasmacytoma.

[0149] Cancer may also include colorectal cancer, thyroid cancer, breast cancer, and lung cancer; and myeloproliferative disorders such as polycythemia vera, thrombocythemia, myeloid metaplasia with myelofibrosis, chronic myelogenous leukemia, chronic myelomonocytic leukemia, hypereosinophilic syndrome, juvenile myelomonocytic leukemia, and systemic mastocytosis. In one embodiment, the compounds of the present application are useful for treating hematopoietic disorders, particularly acute myelogenous leukemia (AML), chronic-myelogenous leukemia (CML), acute promyelocytic leukemia, and acute lymphocytic leukemia (ALL).

[0150] Representative cancers also include adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, anorectal cancer, cancer of the anal canal, appendix cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, basal cell carcinoma, skin cancer (non-melanoma), biliary tract cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, urinary bladder cancer, bone and joint cancer, osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor, brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodeimal tumor, tumor), visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma / carcinoma, carcinoid tumor, gastrointestinal tract, nervous system cancer, nervous system lymphoma, central nervous system cancer, central nervous system lymphoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, lymphoid neoplasms, mycosis fungoides, Seziary syndrome, endometrial cancer, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric (stomach cancer), gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor glioma, head and neck cancer, stem cell (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, eye cancer, islet cell tumor (endocrine pancreas), Kaposi's sarcoma, renal cancer, kidney cancer, renal cancer, laryngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkin's lymphoma, primary central nervous system lymphoma, Waldenström's macroglobulinemia, medulloblastoma, melanoma, intraocular (eye) melanoma, Merkel cell carcinoma, malignant mesothelioma, mesothelioma, metastatic squamous cell carcinoma of the neck, oral cancer, tongue cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorder, chronic myelogenous leukemialeukemia, acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorder, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer, ovarian cancer, ovarian epithelial cancer, ovarian low malignant potential tumor, pancreatic cancer, islet cell carcinoma, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Ewing family of sarcoma tumors, Kaposi's sarcoma, soft tissue sarcoma, uterine cancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), Merkel cell skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer These may include, but are not limited to, supratentorial primitive neuroectodermal tumor, testicular cancer, pharyngeal cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis ureter and other urinary organs, gestational trophoblastic tumor, urethral cancer, endometrial carcinoma of the uterus, sarcoma, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0151] A "cell proliferative disorder of the blood system" is a cell proliferative disease or disorder involving cells of the blood system. Cell proliferative disorders of the blood system may include lymphoma, leukemia, myeloid neoplasm, mast cell neoplasm, myelodysplasia, benign monoclonal gammopathy, lymphomatoid granulomatosis, lymphomatoid papulosis, polycythemia vera, chronic myelocytic leukemia, idiopathic myeloid metaplasia, and essential thrombocythemia. Cell proliferative disorders of the blood system may include hyperplasia, dysplasia, and metaplasia of cells of the blood system. The compounds and compositions of the present application may be used to treat a cancer selected from the group consisting of blood cancer or blood cell proliferative disorders. Cancers of the blood can include multiple myeloma, lymphomas (including Hodgkin's lymphoma, non-Hodgkin's lymphoma, childhood lymphoma, and lymphomas of lymphocytic and cutaneous origin), leukemias (including childhood leukemia, hairy cell leukemia, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, chronic myelogenous leukemia, and mast cell leukemia), myeloid neoplasms, and mast cell neoplasms.

[0152] A "cell proliferative disorder of the lung" is a cell proliferative disease or disorder involving cells of the lung. A cell proliferative disorder of the lung may include all forms of cell proliferative disorders affecting lung cells. A cell proliferative disorder of the lung may include lung cancer, precancerous or precancerous conditions of the lung, benign growths or lesions of the lung, and malignant growths or lesions of the lung, as well as metastatic lesions in tissues and organs of the body other than the lung. The compounds and compositions of the present application may be used to treat lung cancer or cell proliferative disorders of the lung. Lung cancer may include all forms of cancer of the lung. Lung cancer may include malignant lung neoplasms, carcinoma in situ, typical carcinoid tumors, and atypical carcinoid tumors. Lung cancer may include small cell lung cancer ("SCLC"), non-small cell lung cancer ("NSCLC"), squamous cell carcinoma, adenocarcinoma, small cell carcinoma, large cell carcinoma, adenosquamous carcinoma, and mesothelioma. Lung cancer may include "scar carcinoma," bronchoalveolar carcinoma, giant cell carcinoma, spindle cell carcinoma, and large cell neuroendocrine carcinoma. Lung cancer may include lung neoplasms with histologic and ultrastructural heterogeneity (e.g., mixed cell types).

[0153] Pulmonary cell proliferative disorders may also include pulmonary hyperplasia, metaplasia, and dysplasia. Pulmonary cell proliferative disorders may include asbestos-induced hyperplasia, squamous metaplasia, and benign reactive mesothelial metaplasia. Pulmonary cell proliferative disorders may include replacement of columnar epithelium with stratified squamous epithelium, and mucosal dysplasia. Individuals exposed to inhaled harmful environmental substances, such as tobacco smoke and asbestos, may be at higher risk of developing pulmonary cell proliferative disorders. Previous pulmonary diseases that may predispose individuals to developing pulmonary cell proliferative disorders may include chronic interstitial lung disease, necrotizing lung disease, scleroderma, rheumatoid disease, sarcoidosis, interstitial pneumonia, tuberculosis, recurrent pneumonia, idiopathic pulmonary fibrosis, granuloma, asbestosis, fibrosing alveolitis, and Hodgkin's disease.

[0154] A "cell proliferative disorder of the colon" is a cell proliferative disorder involving cells of the colon. Cell proliferative disorders of the colon may include colon cancer. The compounds and compositions of the present application may be used to treat colon cancer or cell proliferative disorders of the colon. Colon cancer may include all forms of cancer of the colon. Colon cancer may include sporadic and hereditary colon cancer. Colon cancer may include malignant colon neoplasms, carcinoma in situ, typical carcinoid tumors, and atypical carcinoid tumors. Colon cancer may include adenocarcinoma, squamous cell carcinoma, and adenosquamous cell carcinoma. Colon cancer may be associated with a genetic syndrome selected from the group consisting of hereditary nonpolyposis colorectal cancer, familial adenomatous polyposis, Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's syndrome, and juvenile polyposis. Colon cancer may be caused by a genetic syndrome selected from the group consisting of hereditary nonpolyposis colorectal cancer, familial adenomatous polyposis, Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's syndrome, and juvenile polyposis.

[0155] Cell proliferative disorders of the colon may also include colon cancer, colon precancerous conditions, colon adenomatous polyps, and colon metachronous lesions. Cell proliferative disorders of the colon may include adenomas. Cell proliferative disorders of the colon may be characterized by colon hyperplasia, metaplasia, and dysplasia. Previous colon diseases that may predispose an individual to develop cell proliferative disorders of the colon may include previous colon cancer. Current colon diseases that may predispose an individual to develop cell proliferative disorders of the colon may include Crohn's disease and ulcerative colitis. Cell proliferative disorders of the colon may be associated with mutations in genes selected from the group consisting of p53, ras, FAP, and DCC. An individual may be at increased risk of developing cell proliferative disorders of the colon due to the presence of mutations in genes selected from the group consisting of p53, ras, FAP, and DCC.

[0156] "Pancreatic cell proliferative disorder" refers to a cell proliferative disorder involving pancreatic cells. The compounds and compositions of the present application may be used to treat pancreatic cancer or pancreatic cell proliferative disorder. Pancreatic cell proliferative disorder may include all forms of cell proliferative disorder affecting pancreatic cells. Pancreatic cell proliferative disorder may include pancreatic cancer, precancerous or precancerous conditions of the pancreas, pancreatic hyperplasia, and pancreatic dysplasia, benign growths or lesions of the pancreas, and malignant growths or lesions of the pancreas, as well as metastatic lesions in tissues and organs of the body other than the pancreas. Pancreatic cancer includes all forms of cancer of the pancreas. Pancreatic cancer may include tubular adenocarcinoma, adenosquamous carcinoma, pleomorphic giant cell carcinoma, mucinous carcinoma, osteoclast-like giant cell carcinoma, mucinous cystadenocarcinoma, acinar carcinoma, unclassified large cell carcinoma, small cell carcinoma, pancreatoblastoma, papillary tumor, mucinous cystadenoma, papillary cystic tumor, and serous cystadenoma. Pancreatic cancer may also include pancreatic tumors with histological and ultrastructural heterogeneity (e.g., mixed cell types).

[0157] "Cell proliferative disorder of the prostate" refers to a cell proliferative disorder involving cells of the prostate. The compounds and compositions of the present application may be used to treat prostate cancer or cell proliferative disorder of the prostate. Cell proliferative disorder of the prostate may include all forms of cell proliferative disorder that affect prostate cells. Cell proliferative disorder of the prostate may include prostate cancer, precancerous or precancerous conditions of the prostate, benign growths or lesions of the prostate, and malignant growths or lesions of the prostate, as well as metastatic lesions in tissues and organs of the body other than the prostate. Cell proliferative disorder of the prostate may include hyperplasia, metaplasia, and dysplasia of the prostate.

[0158] A "cell proliferative disorder of the skin" is a cell proliferative disorder involving cells of the skin. The compounds and compositions of the present application may be used to treat skin cancer or cell proliferative disorders of the skin. Cell proliferative disorders of the skin may include all forms of cell proliferative disorders affecting skin cells. Cell proliferative disorders of the skin may include precancerous or precancerous conditions of the skin, benign growths or lesions of the skin, melanoma, malignant melanoma and other malignant growths or lesions of the skin, and metastatic lesions in tissues and organs of the body other than the skin. Cell proliferative disorders of the skin may include hyperplasia, metaplasia, and dysplasia of the skin.

[0159] "Cell proliferative disorder of the ovary" is a cell proliferative disorder involving cells of the ovary. The compounds and compositions of the present application may be used to treat ovarian cancer or cell proliferative disorder of the ovary. Cell proliferative disorder of the ovary may include all forms of cell proliferative disorder affecting cells of the ovary. Cell proliferative disorder of the ovary may include precancerous or precancerous conditions of the ovary, benign growths or lesions of the ovary, ovarian cancer, malignant growths or lesions of the ovary, and metastatic lesions in tissues and organs of the body other than the ovary. Cell proliferative disorder of the skin may include ovarian cell hyperplasia, metaplasia, and dysplasia.

[0160] A "cell proliferative disorder of the breast" is a cell proliferative disorder involving cells of the breast. The compounds and compositions of the present application may be used to treat breast cancer or cell proliferative disorders of the breast. Cell proliferative disorders of the breast may include all forms of cell proliferative disorders affecting breast cells. Cell proliferative disorders of the breast may include breast cancer, precancerous or precancerous conditions of the breast, benign growths or lesions of the breast, and malignant growths or lesions of the breast, as well as metastatic lesions in body tissues and organs other than the breast. Cell proliferative disorders of the breast may include hyperplasia, metaplasia, and dysplasia of the breast.

[0161] In one embodiment, the disease or disorder includes, but is not limited to, a disease or disorder caused by or associated with Entamoeba histolytica, Pneumocystis carinii, Trypanosoma cruzi, Trypanosoma brucei, Leishmania mexicana, Clostridium histolyticum, Staphylococcus aureus, foot and mouth disease virus, or Crithidia fasciculata, as well as a disease or disorder associated with osteoporosis, autoimmunity, schistosomiasis, malaria, tumor metastasis, metachromatic leukodystrophy, muscular dystrophy, or muscle atrophy.

[0162] Additional examples of diseases or disorders include, but are not limited to, diseases or disorders caused by or associated with veterinary and human pathogenic protozoa, intracellular parasites of the phyla Apicomplexa or Sarcoflagellates, Trypanosoma, Plasmodium, Leishmania, Babesia and Theileria, Cryptosporidia, Sacrocystida, Amoebae, Coccidia, and Trichomonadia. For example, diseases or disorders include, for example, tropical malaria caused by Plasmodium falciparum; vivax caused by Plasmodium vivax or Plasmodium ovale; malaria caused by Plasmodium malariae; toxoplasmosis caused by Toxoplasma gondii; coccidiosis caused by Isospora belli; intestinal sarcocystis caused by Sarcocystis suihominis; dysentery caused by Entamoeba histolytica; cryptosporidiosis caused by Cryptosporidium parvum; Trypanosoma cruzi; Chagas disease caused by Trypanosoma cruzi; sleeping sickness, cutaneous, visceral and other forms of leishmaniasis caused by Trypanosoma brucei rhodesiense or Trypanosoma gambiense;Theileria parva, the agent that causes East Coast fever in bovine, Trypanosoma congolense congolense or Trypanosoma vivax vivax, Trypanosoma brucei brucei, the agent that causes cow-breeding fever in Africa, Trypanosoma brucei evansi, the agent that causes surra disease, Babesia bigemina, the agent that causes Texas fever in cattle and buffaloes, Babesia bovis, the agent that causes European bovine babesiosis and babesiosis in dogs, cats and sheep, Sarcocystis ovicanis, and ovifelis pathogens causing Sarcocystiosis in sheep, cattle and pigs, Cryptosporidia, pathogens causing Cryptosporidioses in cattle and birds, Eimeria and Isospora species, pathogens causing coccidiosis in rabbits, cattle, sheep, goats, pigs and birds, particularly chickens and turkeys, and other veterinary pathogens. Rickettsia includes species such as Rickettsia felis, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi, Rickettsia conorii, and Rickettsia africae, which cause diseases such as typhus, rickettsialpox, boutonneill fever, African tick fever, Rocky Mountain spotted fever, Australian tick typhus, Flinders Island spotted fever, and Queensland tick typhus;

[0163] In one embodiment, the disease or disorder is caused by or associated with one or more bacteria. Examples of bacteria include, but are not limited to, gram-positive microorganisms (e.g., Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and E. faecium, Streptococcus pneumoniae), and gram-negative microorganisms (e.g., Pseudomonas aeruginosa, Burkholderia cepacia, Xanthomonas maltophila, Escherichia coli, Enterobacter species, Klebsiella pneumoniae, and Salmonella species).

[0164] In one embodiment, the disease or disorder is caused by or associated with one or more fungi, including, but not limited to, Candida albicans, Histoplasma neoformans, Coccidioides immitis, and Penicillium marneffei.

[0165] In one embodiment, the disease or disorder is a disease or disorder of the nervous system. In one embodiment, the disease or disorder of the nervous system involves the central nervous system (e.g., the brain, brainstem and cerebellum), the peripheral nervous system (e.g., the cranial nerves), and / or the autonomic nervous system (e.g., parts located in both the central nervous system and the peripheral nervous system).

[0166] Examples of disorders of the nervous system include acquired epileptic aphasia; acute disseminated encephalomyelitis; adrenoleukodystrophy; age-related macular degeneration; agenesis of the corpus callosum; cognitive impairment; Aicardi syndrome; Alexander disease; Alpers disease; alternating hemiplegia; Alzheimer's disease; vascular dementia; amyotrophic lateral sclerosis; anencephaly; Angelman syndrome; hemangiomatosis; anoxia; aphasia; apraxia; arachnoid cysts; arachnoiditis; Arnold-Chiari malformation; arteriovenous malformations; Asperger's syndrome; ataxia-telangiectasia. telegiectasia);attention deficit hyperactivity disorder;autism;autonomic dysfunction;back pain;Batten disease;Behçet's disease;Bell's palsy;benign essential blepharospasm;benign focal;muscular atrophy;benign intracranial hypertension;Binswanger's disease;blepharospasm;Bloch-Sulzberger syndrome;brachial plexus injury;brain abscess;brain trauma;brain tumors (including glioblastoma multiforme);spinal tumors;Brown-Séquard syndrome;Cavan disease;carpal tunnel syndrome;burning pain;central pain syndromes;central pontine myelinolysis;head injury;cerebral aneurysm;cerebral arteriosclerosis;cerebral atrophy;cerebral gigantism;cerebral palsy;Charcot-Marie-Tooth disease;chemotherapy-induced Neuropathic neuropathy and neuropathic pain;Chiari malformation;Choreae;Chronic inflammatory demyelinating polyneuropathy;Chronic pain;Chronic regional pain syndrome;Coffin-Lowry syndrome;Coma including persistent vegetative state;Congenital bilateral facial palsy;Corticobasal degeneration;Cranial arteritis;Craniosynostosis;Creutzfeldt-Jakob disease;Cumulative trauma disorder;Cushing's syndrome;Giant cytomegalic inclusion disease;Cytomegalic virus infection;Dancing eyes-dancing feet syndrome;Dandie-Walker syndrome;Dawson's disease;Domorsia syndrome;Dejerine-Klumpke palsy palsy);dementia;dermatomyositis;diabetic neuropathy;diffuse sclerosis;autonomic dysregulation;dysgraphia;dyslexia;dystonia;early infantile epileptic encephalopathy;empty sella syndrome;encephalitis;encephalocele;cerebral trigeminal region angiomatosis;epilepsy;Erb palsy;essential tremor;Fabry disease;Fahr syndrome;syncope;familial spastic paralysis;febrile convulsions;Fisher syndrome;Friedreich ataxia;frontotemporal dementia and other "tauopathies";Gaucher disease;Gerstmann syndrome;giant cell arteritis;giant cell inclusion disease;globoid cell leukodystrophy;Guillain-Barré syndrome;HTLV-1-associated myelopathy;Hallervorden-Spatz syndrome;Head trauma;Hemiface paralysis;Hereditary spastic paraplegia;Polyneuropathy-type hereditary ataxia;Herpes zoster oticus;Hirayama syndrome;HIV-associated dementia and neuropathy (also neurological signs of AIDS);Holoprosencephaly;Huntington's disease and other polyglutamine repeat diseases;Hydranencephaly;Hydrocephalus;Hyperadrenal insufficiency;Hypoxia;Immune-mediated infarcted encephalomyelitis;inclusion body myositis;incontinentia pigmenti;infantile phytanic acid storage disease;infantile Refsum disease;infantile spasms;inflammatory myopathy;intracranial cyst;intracranial hypertension;Joubert syndrome;Kerns-Sayre syndrome;Kennedy disease, Kinsbone syndrome;Klippel-Feil syndrome;Krabbe disease;Kugelberg-Welander disease;kuru;Lafora disease;Lambert-Eaton myasthenic syndrome;Landau-Kleffner syndrome;lateral myelopathy Wallenberg syndrome; learning disabilities; Leigh's disease; Lennox-Gustaut syndrome; Lesch-Nyhan syndrome; leukodystrophy; dementia with Lewy bodies; alimentary defects; locked-in syndrome; Lou Gehrig's disease (i.e. motor neuron disease or amyotrophic lateral sclerosis); lumbar disc disease; Lyme disease - neurological sequelae; Machado-Joseph disease; megaencephalopathy; megalencephaly; Merkelson-Rosenthal syndrome; Meniere's disease; meningitis; Menkes disease; metachromatic leukodystrophy; microcephaly; migraine; Miller Fisher syndrome; ministroke; mitochondrial myopathy; Moebius syndrome; unilateral upper limb muscular atrophy; motor neuron disease; Moyamoya disease; mucopolysaccharidosis; multi-infarct dementia dementia);multifocal motor neuropathy;multiple sclerosis and other demyelinating disorders;multiple system atrophy with orthostatic hypotension;progressive muscular dystrophy;myasthenia gravis;myelinoclastic diffuse sclerosis;infantile myoclonic encephalopathy;myoclonus;myopathy;myotonia congenita;narcolepsy;neurofibromatosis;neuroleptic malignant syndrome;neurological manifestations of AIDS;neurological sequelae of lupus;neuromyotonia;neuronal ceroid lipofuscinosis;neuronal migration disorder;Niemann-Pick disease;O'Sullivan-McLeod syndrome;occipital neuralgia;Occult spinal malunion sequence;Ohtahara syndrome;Olivopontocerebellar atrophy;Opsoclonus-myoclonus;Optic neuritis;Orthostatic hypotension;Overuse syndrome;Paresthesia;Parkinson's disease;Congenital paramyotonia;Paraneoplastic syndrome;Seizures;Parry-Romberg syndrome;Pelizaeus-Merzbacher disease;Periodic paralysis;Peripheral neuropathy;Painful neuropathy and neuropathic pain;Persistent vegetative state;Pervasive developmental disorder;Phosphotic sneeze reflex;Phytanic acid storage disease;Pick's disease;Compressed nerve;Lower Pituitary tumors;Polymyositis;Porencephaly;Post-polio syndrome;Postherpetic neuralgia;Postinfectious encephalomyelitis;Orthostatic hypotension;Prader-Willi syndrome;Primary lateral sclerosis;Prion disease;Progressive facial hemiatrophy;Progressive multifocal leukoencephalopathy;Progressive sclerosing gray matter dystrophy;Progressive supranuclear palsy;Pseudotumor cerebri;Ramsay Hunt syndrome (types I and II);Rasmussen encephalitis;Reflex sympathetic dystrophy syndrome;Refsum's disease;Repetitive movement disorder;Repetitive stress injury;Restless legs syndrome;Retrovirus Luss-associated myelopathy;Rett syndrome;Reye's syndrome;Choreae;Sandhoff disease;Schilder's disease;Schisencephaly;Septo-optic dysplasia;Shaken baby syndrome;Herpes zoster;Shy-Drager syndrome;Sjogren's syndrome;Sleep apnea;Sotos syndrome;Spasticity;Spina bifida;Spinal cord injury;Spinal tumor;Spinal muscular atrophy;Stiff-person syndrome;Stroke;Sturge-Weber syndrome;Subacute sclerosing panencephalitis;Subcortical arteriosclerotic encephalopathy;Sydenham's chorea;Collapse;Syringomyelia;Tardive dyskinesia;Tay-Sachs These conditions include, but are not limited to, temporal arteritis, tethered spinal cord syndrome, Thomsen's disease, thoracic outlet syndrome, painful tics, Todd's palsy, Tourette's syndrome, transient ischemic attacks, transmissible spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremors, trigeminal neuralgia, tropical spastic paraparesis, tuberous sclerosis, vascular dementia (multiple infarct dementia), vasculitis including temporal arteritis, von Hippel-Lindau disease, Wallenberg syndrome, Werdnig-Hoffmann disease, West syndrome, whiplash injury, Williams syndrome, Wilson's disease, and Zellweger syndrome.

[0167] Examples of neurodegenerative diseases also include adrenoleukodystrophy (ALD), Alexander disease, Alper's disease, Alzheimer's disease, amyotrophic lateral sclerosis (Lou Gehrig's disease), ataxia-telangiectasia, Batten disease (also known as Speelmeyer-Voght-Sjögren-Batten disease), bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, familial fatal insomnia, frontotemporal lobar degeneration, Huntington's disease, HIV-associated dementia, Kennedy disease, Krabbe disease, dementia with Lewy bodies, neuroborreliosis, Machado-Joseph disease (spinal These conditions may include, but are not limited to, cerebellar degeneration type 3), multiple system atrophy, multiple sclerosis, narcolepsy, Niemann-Pick disease, Parkinson's disease, Pelizaeus-Merzbacher disease, Pick's disease, primary lateral sclerosis, prion diseases, progressive supranuclear palsy, Refsum disease, Sandhoff disease, Schilder's disease, subacute combined spinal degeneration secondary to pernicious anemia, Speelmeyer-Voght-Sjogren-Batten disease (also known as Batten disease), spinocerebellar ataxias (multiple types with varying features), spinal muscular atrophy, Steele-Richardson-Olszewski disease, tabes dorsalis, and toxic encephalopathy.

[0168] In one embodiment, the disease or disorder is an autoimmune disease. Examples of autoimmune diseases include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, chronic inflammatory diseases with polygenic susceptibility, Crohn's disease (CD), and inflammatory bowel disease (IBD), including ulcerative colitis (UC).

[0169] In one embodiment, the disease or disorder is inflammation, arthritis, rheumatoid arthritis, spondyiarthropathies, gouty arthritis, osteoarthritis, juvenile arthritis, and other arthritic conditions, systemic lupus erthematosus (SLE), skin-related disorders, psoriasis, eczema, disorders, dermatitis, neuroinflammation, allergies, pain, neuropathic pain, fever, lung disease, lung inflammation, adult respiratory distress syndrome, pulmonary sarcoidosis, pulmonary inflammatory disease, pulmonary inflammatory disorders, pulmonary sarcoidosis, pulmonary inflammatory disease ... sarcoisosis), asthma, silicosis, chronic pulmonary inflammatory disease, and chronic obstructive pulmonary disease (COPD), cardiovascular disease, arteriosclerosis, myocardial infarction (including post-myocardial infarction indications), thrombosis, congestive heart failure, cardiac reperfusion injury, and complications associated with hypertension and / or heart failure, such as vascular organ damage, restenosis, cardiomyopathies, stroke including ischemic and hemorrhagic stroke, reperfusion injury, renal reperfusion injury, ischemia including stroke and cerebral ischemia, and ischemia from cardiac / coronary artery bypass, neurodegenerative disorders, liver disease and nephritis, gastrointestinal disorders, inflammatory bowel disease, Crohn's disease, gastritis, irritable bowel syndrome, ulcerative colitis, ulcer disease, peptic ulcer, viral and bacterial infections, sepsis, septic shock, gram-negative sepsis, malaria, meningitis, HIV infection, opportunistic infections, cachexia secondary to infection or malignancy, cachexia secondary to acquired immune deficiency syndrome (AIDS), AIDS, ARC (AIDS-related phobia), pneumonia, herpes viruses, myalgia due to infection, influenza, autoimmune diseases, graft-versus-host reaction and allograft rejection, treatment of bone resorption diseases, osteoporosis, multiple sclerosis, cancer, leukemia, lymphoma, colorectal cancer, brain cancer, bone cancer, epithelial cell-derived neoplasia (epithelial carcinoma), basal cell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer, oral cavity cancer, esophageal cancer, small intestine cancer, stomach cancer cancer, colon cancer, liver cancer, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, skin cancer, squamous and / or basal cell carcinoma, prostate cancer, renal cell carcinoma, and other known cancers that affect epithelial cells throughout the body, chronic myelogenous leukemia,The primary disease is a chronic myeloid leukemia (CML), acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL), neoplasia, angiogenesis including metastasis, central nervous system disorders, central nervous system disorders with an inflammatory or apoptotic component, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinal cord injury, peripheral neuropathy, or B-cell lymphoma.

[0170] In one embodiment, the disease or disorder is selected from autoimmune diseases, inflammatory diseases, proliferative and hyperproliferative diseases, immune-mediated diseases, bone diseases, metabolic diseases, neurological and neurodegenerative diseases, cardiovascular diseases, hormone-related diseases, allergies, asthma, and Alzheimer's disease. In one embodiment, the disease or disorder is selected from proliferative disorders and immune disorders.

[0171] As modulators of STING protein, the compounds and compositions of the present application are also useful in evaluating, studying, or testing biological samples. One aspect of the present application relates to modulating the activity of STING protein in a biological sample, comprising contacting the biological sample with a compound or composition of the present application.

[0172] As used herein, the term "biological sample" refers to in vitro or ex vivo samples, including but not limited to cell cultures or extracts thereof; biopsies or extracts thereof obtained from mammals; and blood, saliva, urine, feces, semen, tears, or other bodily fluids or extracts thereof. Modulation (e.g., inhibition or stimulation) of protein kinase activity in biological samples is useful for a wide variety of purposes known to those skilled in the art. Examples of such purposes include, but are not limited to, blood transfusions, organ transplants, and biological specimen storage.

[0173] Another aspect of the present application relates to the study of STING protein in biological and pathological phenomena; the study of intracellular signaling pathways mediated by STING protein. Examples of such uses include, but are not limited to, biological assays, such as enzyme assays and cell-based assays.

[0174] The activity of the compounds and compositions of the present application as STING modulators may be assayed in vitro, in vivo, or in cell lines. In vitro assays include assays that determine the modulation (e.g., inhibition or stimulation) of STING ligand binding to STING protein through competitive binding assays. Alternative in vitro assays may quantify the ability of agonists to bind to protein kinases, measured by either radiolabeling or fluorescent labeling of the agonist prior to binding, isolation of the ligand / protein complex, and determination of the amount of radiolabel / fluorescent label bound. Detailed conditions for assaying the compounds utilized in the present application as inhibitors of STING protein are described in the examples below.

[0175] In accordance with the foregoing, the present application provides a method for preventing or treating any of the above-mentioned diseases or disorders in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of a compound of the present application or an enantiomer, diastereomer, stereoisomer, or pharma- ceutically acceptable salt thereof, or a pharmaceutical composition of the present application. For any of the above uses, the required dosage will vary depending on the mode of administration, the particular condition to be treated, and the desired effect.

[0176] The compounds and compositions of the present application may be administered in therapeutically effective amounts in combination therapy with one or more therapeutic agents (pharmaceutical combinations) or modalities, such as anti-proliferative, anti-cancer, immunomodulatory, or anti-inflammatory agents, and / or non-pharmaceutical therapies. For example, synergistic effects may occur with anti-proliferative, anti-cancer, immunomodulatory, or anti-inflammatory substances. When the compounds of the present application are administered in combination with other therapies, the dosage of the co-administered compound will of course vary depending on the type of co-administered drug, the particular drug used, the condition being treated, and the like.

[0177] Combination therapy may include administration to a subject of a subject compound further in combination with one or more other biologically active components, such as, but not limited to, a second STING modulator (inhibitor or stimulator), a modulator (inhibitor or stimulator) of the cGAS-CDN-STING axis, or a modulator (inhibitor or stimulator) involved in intracellular dsDNA-mediated type I interferon activation. US Patent Application Publication No. 16 / 717,325, filed December 17, 2019, to N. Barber, is incorporated herein by reference in its entirety for all purposes. Other biologically active components may also include antiproliferative agents, anticancer agents (e.g., chemotherapeutic agents), immunomodulators, antibodies, and the like. For example, the compounds of the present application may be used in combination with other pharmacologic active compounds, preferably compounds that can enhance the agonist effect of the compounds of the present application. The compounds of the present application may be administered simultaneously (in a single formulation or in separate formulations) or subsequent to other drug therapies or treatment modalities. In general, combination therapy contemplates the administration of two or more drugs during a single cycle or course of treatment.

[0178] In one embodiment, the chemotherapeutic agent is an alkylating agent; an antibiotic; an antimetabolite; an antidote; an interferon; a polyclonal or monoclonal antibody; an EGFR inhibitor; a HER2 inhibitor; a histone deacetylase inhibitor; a hormone; a mitosis inhibitor; an MTOR inhibitor; a multikinase inhibitor; a serine / threonine kinase inhibitor; a tyrosine kinase inhibitor; a VEGF / VEGFR inhibitor; a taxane or taxane derivative, an aromatase inhibitor, an anthracycline, a microtubule targeting agent, a topoisomerase poison, an inhibitor of a molecular target or enzyme (e.g., a kinase inhibitor), a cytidine analog, or any chemotherapeutic, antitumor or antiproliferative agent listed at www.cancer.org / docroot / cdg / cdg_0.asp, last visited on April 27, 2020.

[0179] Representative alkylating agents include, but are not limited to, cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan (Alkeran); carmustine (BiCNU); busulfan (Busulfex); lomustine (CeeNU); dacarbazine (DTIC-Dome); oxaliplatin (Eloxatin); carmustine (Gliadel); Ifosfamide (Ifex); mechlorethamine (Mastergen); busulfan (Myleran); carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide (Temodar); thiotepa (Thioplex); bendamustine (Trenda); or streptozotocin (Zanosar).

[0180] Representative antibiotics include, but are not limited to, doxorubicin (Adriamycin); doxorubicin liposomal (Doxil); mitoxantrone (Novantrone); bleomycin (Blenoxane); daunorubicin (Cerubicin); daunorubicin liposomal (Daunoxome); dactinomycin (Cosmegen); epirubicin (Ellence); idarubicin (Idamycin); plicamycin (Mithracin); mitomycin (Mutamycin); pentostatin (Nipent); or valrubicin (Valstar).

[0181] Representative antimetabolites include, but are not limited to, fluorouracil (Adrucil); capecitabine (Xeloda); hydroxyurea (Hydrea); mercaptopurine (Purinethol); pemetrexed (Alimta); fludarabine (Fludara); nelarabine (Alanon); cladribine (Cladribine Nova Plus); clofarabine (Clolar); cytarabine (Cytosar-U); decitabine (Dacogen); cytarabine liposomal (DepoCyt); hydroxyurea (Droxia); pralatrexate (Folotin); floxuridine (FUDR); gemcitabine (Gemzar); cladribine (Leustatin); fludarabine (Oforta); methotrexate (MTX; Rheumatrex); methotrexate (Trexall); thioguanine (Tabloid); TS-1 or cytarabine (Tarabine PFS).

[0182] Representative antidotes include, but are not limited to, amifostine (Ethyl) or mesna (Mesnex).

[0183] Representative interferons include, but are not limited to, interferon alpha-2b (Intron A) or interferon alpha 2a (Roferon-A).

[0184] Representative polyclonal or monoclonal antibodies include trastuzumab (Herceptin); ofatumumab (Arzera); bevacizumab (Avastin); rituximab (Rituxan); cetuximab (Erbitux); panitumumab (Vectibix); tositumomab / iodine 131 These include, but are not limited to, tositumomab (Bexar); alemtuzumab (Campath); ibritumomab (Zevalin; In-111; Y-90 Zevalin); gemtuzumab (Mylotarg); eculizumab (Soliris) or denosumab.

[0185] Representative EGFR inhibitors include, but are not limited to, gefitinib (Iressa); lapatinib (Tykerb); cetuximab (Erbitux); erlotinib (Tarceva); panitumumab (Vectibix); PKI-166; canertinib (CI-1033); matuzumab (Emd7200) or EKB-569.

[0186] Representative HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin); lapatinib (Tykerb) or AC-480.

[0187] Representative histone deacetylase inhibitors include, but are not limited to, vorinostat (Zolinza).

[0188] Representative hormones include, but are not limited to, tamoxifen (Soltamox; Nolvadex); raloxifene (Evista); megestrol (Megace); leuprolide (Lupron; Lupron Depo; Eligard; Viadur); fulvestrant (Faslodex); letrozole (Femara); triptorelin (Trelstar LA; Trelstar Depo); exemestane (Aromasin); goserelin (Zoladex); bicalutamide (Casodex); anastrozole (Arimidex); fluoxymesterone (Androxy; Halotestin); medroxyprogesterone (Provera; Depo-Provera); estramustine (Emcyt); flutamide (Eurexin); toremifene (Fareston); degarelix (Filmagon); nilutamide (Nilandrone); abarelix (Prenaxis); or testolactone (Teslac).

[0189] Representative mitotic inhibitors include, but are not limited to, paclitaxel (Taxol; Onxol; Abraxane); docetaxel (Taxotere); vincristine (Oncovin; Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopofos; Bepcid); teniposide (Bumone); ixabepilone (Ixempra); nocodazole; epothilones; vinorelbine (Navelbine); camptothecin (CPT); irinotecan (Camptosar); topotecan (Hycamtin); amsacrine or lamellarin D (LAM-D).

[0190] Representative MTOR inhibitors include, but are not limited to, everolimus (Afinitor) or temsirolimus (Torisel); rapamune, ridaforolimus; or AP23573.

[0191] Representative multikinase inhibitors include, but are not limited to, sorafenib (Nexavar); sunitinib (Sutent); BIBW 2992; E7080; Zd6474; PKC-412; motesanib; or AP24534.

[0192] Representative serine / threonine kinase inhibitors include, but are not limited to, ruboxistaurin; eril / easudil hydrochloride; flavopiridol; seliciclib (CYC202; roscovitine); SNS-032 (BMS-387032); Pkc412; bryostatin; KAI-9803; SF1126; VX-680; Azd1152; Arry-142886 (AZD-6244); SCIO-469; GW681323; CC-401; CEP-1347 or PD 332991.

[0193] Representative tyrosine kinase inhibitors include erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec); sorafenib (Nexavar); sunitinib (Sutent); trastuzumab (Herceptin); bevacizumab (Avastin); rituximab (Rituxan); lapatinib (Tykerb); cetuximab (Erbitux); panitumumab (Vectibix); and everolim. These include, but are not limited to, sirolimus (Afinitor); alemtuzumab (Campath); gemtuzumab (Mylotarg); temsirolimus (Torisel); pazopanib (Votrient); dasatinib (Sprycel); nilotinib (Tasigna); vatalanib (Ptk787; ZK222584); CEP-701; SU5614; MLN518; XL999; VX-322; Azd0530; BMS-354825; SKI-606 CP-690; AG-490; WHI-P154; WHI-P131; AC-220; or AMG888.

[0194] Representative VEGF / VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin); sorafenib (Nexavar); sunitinib (Sutent); ranibizumab; pegaptanib; or vandetinib.

[0195] Representative microtubule targeting drugs include, but are not limited to, paclitaxel, docetaxel, vincristine, vinblastine, nocodazole, epothilones, and navelbine.

[0196] Representative topoisomerase poisons include, but are not limited to, teniposide, etoposide, adriamycin, camptothecin, daunorubicin, dactinomycin, mitoxantrone, amsacrine, epirubicin, and idarubicin.

[0197] Representative taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxol.

[0198] Representative and common chemotherapy, antineoplastic, and antiproliferative agents include altretamine (Hexalen); isotretinoin (Accutane; Amnesty; Claravis; Sotret); tretinoin (Vesanoid); azacitidine (Vidaza); bortezomib (Velcade); asparaginase (Elspar); levamisole (Ergamisole); mitotane (Lysodren); procarbazine (Matulene); pegaspargase (Oncaspar); denileukin diftitox (Ontac); porfimer (Photofrin); aldesleukin (Proleukin); lenalidomide (Revlimid); bexarotene (Targretin); thalidomide (Salomid); temsirolimus (Torisel); arsenic trioxide (Trisenox); verteporfin (Visudyne); mimosine (Leusenor); (1M tegafur-0.4M These include, but are not limited to, 5-chloro-2,4-dihydroxypyrimidine-1M potassium oxonate) or lovastatin.

[0199] Representative kinase inhibitors include bevacizumab (targets VEGF), BIBW 2992 (targets EGFR and Erb2), cetuximab / Erbitux (targets Erb1), imatinib / Gleevec (targets Bcr-Abl), trastuzumab (targets Erb2), gefitinib / Iressa (targets EGFR), ranibizumab (targets VEGF), pegaptanib (targets VEGF), erlotinib / Tarceva (targets Erb1), nilotinib (targets Bcr-Abl), and lapatinib ( Erb1 and Erb2 / Her2 targets), GW-572016 / lapatinib ditosylate (HER2 / Erb2 targets), panitumumab / vectibix (EGFR targets), vandetanib (RET / VEGFR targets), E7080 (multiple targets including RET and VEGFR), Herceptin (HER2 / Erb2 targets), PKI-166 (EGFR targets), canertinib / CI-1033 (EGFR targets), s Nitinib / SU-11464 / Sutent (targets EGFR and FLT3), Matuzumab / Emd7200 (targets EGFR), EKB-569 (targets EGFR), Zd6474 (targets EGFR and VEGFR), PKC-412 (targets VEGR and FLT3), Vatalanib / Ptk787 / ZK222584 (targets VEGR), CEP-701 (targets FLT3), SU5614 (targets FLT3), MLN5 18 (targets FLT3), XL999 (targets FLT3), VX-322 (targets FLT3), Azd0530 (targets SRC), BMS-354825 (targets SRC), SKI-606 (targets SRC), CP-690 (targets JAK), AG-490 (targets JAK), WHI-P154 (targets JAK), WHI-P131 (targets JAK), sorafenib / nexavar (RAF kinase,These include, but are not limited to, VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-β, KIT, FLT-3, and RET), dasatinib / Sprycel (BCR / ABL and Src), AC-220 (targets Flt3), AC-480 (targets all HER proteins, "panHER"), motesanib diphosphate (targets VEGF1-3, PDGFR, and c-kit), denosumab (targets RANKL, inhibits SRC), AMG888 (targets HER3), and AP24534 (multiple targets including Flt3).

[0200] In one embodiment, the compound may be administered in combination with one or more separate pharmaceutical agents, such as chemotherapeutic, immunotherapeutic, or adjunctive therapeutic agents.

[0201] As used herein, "combination therapy" or "co-therapy" includes the administration of a compound of the present application, or a pharmaceutically acceptable salt or ester thereof, and at least one second agent as part of a specific treatment plan intended to provide a beneficial effect from the synergy of these therapeutic agents. The beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic synergy resulting from the combination of therapeutic agents. The co-administration of these therapeutic agents is typically carried out over a defined period of time (usually minutes, hours, days or weeks, depending on the combination selected). "Combination therapy" may, but generally does not, intend to encompass the administration of two or more of these therapeutic agents as part of separate monotherapy plans that incidentally and arbitrarily result in the combination of the present application.

[0202] "Combination therapy" is intended to encompass administration of these therapeutic agents in a sequential manner, where each therapeutic agent is administered at a different time, as well as where administration of these therapeutic agents, or at least two of the therapeutic agents, is substantially simultaneous. Substantially simultaneous administration can be achieved, for example, by administering to the subject a single capsule with a fixed ratio of each therapeutic agent, or a plurality of single capsules of each therapeutic agent. Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any suitable route, including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The therapeutic agents can be administered by the same route or by different routes. For example, the first therapeutic agent of the selected combination may be administered by intravenous injection, while the other therapeutic agents of the combination may be administered orally. Alternatively, for example, all therapeutic agents may be administered orally, or all therapeutic agents may be administered by intravenous injection. The order in which the therapeutic agents are administered is not narrowly important.

[0203] "Combination therapy" also encompasses the administration of the above-mentioned therapeutic agents further combined with other biologically active ingredients and non-drug therapeutic agents (e.g., surgery or radiation therapy). Combination therapy further includes non-drug treatments, which may be performed at any appropriate time, so long as the beneficial effects from the cooperation of the combination of therapeutic agent and non-drug treatment are achieved. For example, where appropriate, the beneficial effects are still achieved when the non-drug treatment is temporarily removed from the administration of the therapeutic agent, perhaps for several days or even weeks.

[0204] definition Listed below are definitions of various terms used in this application. These definitions apply to the terms as they are used throughout the specification and claims, either individually or as part of a larger group, unless otherwise limited in specific instances.

[0205] The transitional term "comprising" is synonymous with "including," "containing," or "characterized by" and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0206] The transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim, but does not exclude additional components or steps not related to the invention, such as impurities normally associated with compositions.

[0207] The transitional phrase "consisting essentially of" limits the scope of a claim to the particular materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention.

[0208] As used herein, the term "alkyl" refers, in certain embodiments, to a saturated straight or branched chain hydrocarbon group containing 1 to 6 carbon atoms. Examples of C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, and n-hexyl groups.

[0209] As used herein, the term "perhaloalkyl" refers to a saturated, straight or branched chain, in certain embodiments, hydrocarbon group of 1 to 6 carbon atoms that does not contain hydrogen atoms bonded to carbons. Examples of C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, and n-hexyl groups. As used herein, halo refers to a halogen atom.

[0210] As used herein, the term "alkenyl" refers, in certain embodiments, to a monovalent group derived from a hydrocarbon moiety containing 2 to 6 carbon atoms having at least one carbon-carbon double bond. The double bond may or may not be the point of attachment to another group. Alkenyl groups include, but are not limited to, for example, ethynyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, and the like.

[0211] As used herein, the term "alkynyl" refers, in certain embodiments, to a monovalent group derived from a hydrocarbon moiety containing 2 to 6 carbon atoms with at least one carbon-carbon triple bond. The triple bond may or may not be the point of attachment to another group. Alkyl groups include, but are not limited to, for example, ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, and the like.

[0212] The term "alkoxy" refers to an --O-alkyl group.

[0213] As used herein, the terms "hal," "halo," and "halogen" refer to an atom selected from fluorine, chlorine, bromine, and iodine.

[0214] As used herein, the term "cycloalkyl" refers to a monovalent group derived from a monocyclic or polycyclic saturated or partially unsaturated carbocyclic compound. Examples of C3-C8 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl, and cyclooctyl; 12 Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptyl, and bicyclo[2.2.2]octyl.

[0215] As used herein, the term "cycloalkenyl" refers to a monovalent group derived from a monocyclic or polycyclic saturated or partially unsaturated carbocyclic compound containing at least one carbon-carbon double bond. Examples of C4-C8 cycloalkenyl include, but are not limited to, cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclopentenyl, and cyclooctenyl.

[0216] As used herein, the term "aryl" refers to a mono- or polycyclic carbocyclic ring system having one or more aromatic rings, fused or non-fused, including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like.

[0217] As used herein, the term "aralkyl" refers to an alkyl residue, such as those described herein, attached to an aryl ring, such as those described herein. Examples include, but are not limited to, benzyl, phenyl, and the like.

[0218] As used herein, the term "heteroaryl" refers to a mono- or polycyclic (e.g., bicyclic, or tricyclic or higher) fused or non-fused group or ring system having at least one aromatic ring having from 5 to 10 ring atoms, where one ring atom is selected from S, O, and N; 0, 1, or 2 ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon. Heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, quinoxalinyl, indazolyl, cinnolinyl, phthalazinyl, pyridazinyl, indolyl, acridinyl, benzoquinolinyl, pyrimidinyl, purinyl, pyrrolopyrimidinyl, quinoxalinyl, quinazolinyl, indazolinyl, and phthalazinyl.

[0219] As used herein, the term "heteroaralkyl" refers to an alkyl residue, such as those described herein, attached to a heteroaryl ring, such as those described herein. Examples include, but are not limited to, pyridinylmethyl, pyrimidinylethyl, and the like.

[0220] According to the present application, any aryl, substituted aryl, heteroaryl and substituted heteroaryl described herein can be any aromatic group. The aromatic group can be substituted or unsubstituted.

[0221] As used herein, the term "heterocyclyl" refers to a non-aromatic monocyclic or polycyclic (e.g., bicyclic, or tricyclic or higher) fused or non-fused group or ring system having 3 to 10 ring atoms, where one ring atom is selected from S, O, and N; 0, 1, or 2 ring atoms are additional heteroatoms independently selected from S, O, and N; and the remaining ring atoms are carbon. Representative heterocycloalkyl groups include, but are not limited to, [1,3]dioxolanyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuryl, and the like.

[0222] The term "alkylamino" refers to a group having the structure -NH(C 12 It refers to groups having the formula -NH(C1-C6 alkyl), for example -NH(C1-C6 alkyl), where C1-C6 alkyl is as previously defined.

[0223] The term "dialkylamino" refers to a group having the structure -NH(C 12 It refers to a group having the formula -NH(C1-C6 alkyl)2, for example -NH(C1-C6 alkyl)2, where C1-C6 alkyl is as previously defined.

[0224] The term "acyl" includes residues derived from acids, including, but not limited to, carboxylic acids, carbamic acids, carbonic acids, sulfonic acids, and phosphorous acids. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, and aliphatic phosphates. Examples of aliphatic carbonyls include, but are not limited to, acetyl, propionyl, 2-fluoroacetyl, butyryl, 2-hydroxyacetyl, and the like.

[0225] The term "ester" includes compounds or moieties which contain a carbon or a heteroatom bound to an oxygen atom which is bonded to the carbon of a carbonyl group. The term "ester" includes alkoxycarboxy groups such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, and the like.

[0226] As described herein, the compounds of the present application and the moieties present in the compounds may optionally be substituted with one or more substituents, as generally exemplified above or as exemplified by specific classes, subclasses, and species of the present application. It will be understood that the phrase "optionally substituted" is used interchangeably with the phrase "substituted or unsubstituted". In general, the term substituted, whether preceded by the term optionally or not, refers to the replacement of a hydrogen group in a given structure with a group of a specified substituent. Unless otherwise specified, an optionally substituted group may have a substituent at each substitutable position of the group, and when one or more positions in any given structure may be substituted with one or more substituents selected from a specified group, the substituents may be the same or different at each position. As used herein, the terms "optionally substituted," "optionally substituted alkyl," "optionally substituted alkenyl," "optionally substituted alkynyl," "optionally substituted cycloalkyl," "optionally substituted cycloalkenyl," "optionally substituted aryl," "optionally substituted heteroaryl," "optionally substituted aralkyl," "optionally substituted heteroaralkyl," "optionally substituted heterocyclyl," and any other optionally substituted groups include, but are not limited to, -F, -CI, -Br, -I, -OH, protected hydroxy, -NO, -CN, -NH, protected amino, -NH-C1-C2 12 -Alkyl, -NH-C2~C 12 -Alkenyl, -NH-C2~C 12 -Alkenyl, -NH-C3~C 12 -Cycloalkyl, -NH-aryl, -NH-heteroaryl, -NH-heterocycloalkyl, -dialkylamino, -diarylamino, -diheteroarylamino, -O-C 12 -Alkyl, -O-C2~C 12 -Alkenyl, -O-C2~C 12 -Alkenyl, -O-C3~C 12 -Cycloalkyl, -O-aryl, -O-heteroaryl, -O-heterocycloalkyl, -C(O)-C1-C12 -Alkyl, -C(O)-C2~C 12 -Alkenyl, -C(O)-C2-C 12 -Alkenyl, -C(O)-C 12 -Cycloalkyl, -C(O)-aryl, -C(O)-heteroaryl, -C(O)-heterocycloalkyl, -CONH2, -CONH-C1-C 12 -Alkyl, -CONH-C2~C 12 -Alkenyl, -CONH-C2~C 12 -Alkenyl, -CONH-C3~C 12 -Cycloalkyl, -CONH-aryl, -CONH-heteroaryl, -CONH-heterocycloalkyl, -OCO2-C1~C 12 -Alkyl, -OCO2-C2~C 12 -Alkenyl, -OCO2-C2~C 12 -Alkenyl, -OCO2-C3~C 12 -Cycloalkyl, -OCO2-aryl, -OCO2-heteroaryl, -OCO2-heterocycloalkyl, -OCONH2, -OCONH-C1~C 12 -Alkyl, -OCONH-C2~C 12 -Alkenyl, -OCONH-C2~C 12 -Alkenyl, -OCONH-C3~C 12 -Cycloalkyl, -OCONH-aryl, -OCONH-heteroaryl, -OCONH-heterocycloalkyl, -NHC(O)-C1-C 12 -Alkyl, -NHC(O)-C2~C 12 -Alkenyl, -NHC(O)-C 12 -Alkenyl, -NHC(O)-C 12 -Cycloalkyl, -NHC(O)-aryl, -NHC(O)-heteroaryl, -NHC(O)-heterocycloalkyl, -NHCO2-C1~C 12 -Alkyl, -NHCO2-C2~C 12 -Alkenyl, -NHCO2-C2~C 12 -Alkenyl, -NHCO2-C3~C 12-Cycloalkyl, -NHCO2-aryl, -NHCO2-heteroaryl, -NHCO2-heterocycloalkyl, NHC(O)NH2, -NHC(O)NH-C1~C 12 -Alkyl, -NHC(O)NH-C2~C 12 -Alkenyl, -NHC(O)NH-C 12 -Alkenyl, -NHC(O)NH-C 12 -Cycloalkyl, -NHC(O)NH-aryl, -NHC(O)NH-heteroaryl, NHC(O)NH-heterocycloalkyl, -NHC(S)NH2, -NHC(S)NH-C1~C 12 -Alkyl, -NHC(S)NH-C2~C 12 -Alkenyl, -NHC(S)NH-C2~C 12 -Alkenyl, -NHC(S)NH-C3~C 12 -Cycloalkyl, -NHC(S)NH-aryl, -NHC(S)NH-heteroaryl, -NHC(S)NH-heterocycloalkyl, -NHC(NH)NH2, -NHC(NH)NH-C1~C 12 -Alkyl, -NHC(NH)NH-C2~C 12 -Alkenyl, -NHC(NH)NH-C2~C 12 -Alkenyl, -NHC(NH)NH-C3~C 12 -Cycloalkyl, -NHC(NH)NH-aryl, -NHC(NH)NH-heteroaryl, -NHC(NH)NHheterocycloalkyl, -NHC(NH)-C1-C 12 -Alkyl, -NHC(NH)-C2~C 12 -Alkenyl, -NHC(NH)-C2~C 12 -Alkenyl, -NHC(NH)-C3~C 12 -Cycloalkyl, -NHC(NH)-aryl, -NHC(NH)-heteroaryl, -NHC(NH)-heterocycloalkyl, -C(NH)NH-C1-C 12 -Alkyl, -C(NH)NH-C2~C 12 -Alkenyl, -C(NH)NH-C2~C 12 -Alkenyl, C(NH)NH-C3~C 12-Cycloalkyl, -C(NH)NH-aryl, -C(NH)NH-heteroaryl, -C(NH)NHheterocycloalkyl, -S(O)-C1-C 12 -Alkyl, -S(O)-C2~C 12 -Alkenyl, -S(O)-C2~C 12 -Alkenyl, -S(O)-C3~C 12 -Cycloalkyl, -S(O)-aryl, -S(O)-heteroaryl, -S(O)-heterocycloalkyl-SO2NH2, -SO2NH-C1~C 12 -Alkyl, -SO2NH-C2~C 12 -Alkenyl, -SO2NH-C2~C 12 -Alkenyl, -SO2NH-C3~C 12 -Cycloalkyl, -SO2NH-aryl, -SO2NH-heteroaryl, -SO2NH-heterocycloalkyl, -NHSO2-C1~C 12 -Alkyl, -NHSO2-C2~C 12 -Alkenyl, -NHSO2-C2~C 12 -Alkenyl, -NHSO2-C3~C 12 -Cycloalkyl, -NHSO2-aryl, -NHSO2-heteroaryl, -NHSO2-heterocycloalkyl, -CH2NH2, -CH2SO2CH3, -aryl, -arylalkyl, -heteroaryl, -heteroarylalkyl, -heterocycloalkyl, -C3~C 12 -Cycloalkyl, polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, -methoxyethoxy, -SH, -S-C 12 -Alkyl, -S-C2~C 12 -Alkenyl, -S-C2~C 12 -Alkenyl, -S-C3~C 12 Refers to groups that are substituted or unsubstituted by the independent replacement of one, two, or three or more hydrogen atoms thereon, with substituents including, but not limited to, -cycloalkyl, -S-aryl, -S-heteroaryl, -S-heterocycloalkyl, or methylthiomethyl.

[0227] The term "subject" as used herein refers to a mammal. Thus, a subject may refer to, for example, a dog, cat, horse, cow, pig, guinea pig, etc. Preferably, the subject is a human. When the subject is a human, the subject may be referred to herein as a patient.

[0228] "Treat", "treating" and "treatment" refer to a method of alleviating or reducing a disease and / or its associated symptoms.

[0229] As used herein, "preventing" or "prevent" describes the reduction or elimination of the onset of symptoms or complications of a disease, condition or disorder.

[0230] The terms "disease," "disorder," and "condition" are used interchangeably unless the context clearly indicates otherwise.

[0231] As used herein, the term "therapeutically effective amount" of a compound or pharmaceutical composition of the present application means a sufficient amount of the compound or pharmaceutical composition to alleviate the symptoms of a disorder in a subject. As is well understood in the medical arts, a therapeutically effective amount of a compound or pharmaceutical composition of the present application will be at a reasonable benefit / risk ratio applicable to any medical treatment. However, it will be understood that the total daily usage of the compounds and compositions of the present application will be determined by the attending physician within the scope of sound medical judgment. The specific modulatory (e.g., inhibitory or stimulatory) dose for any particular patient will depend on a wide variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used, the age, weight, general health, sex and diet of the patient; the time of administration, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination or simultaneously with the specific compound used; and factors well known in the medical arts.

[0232] As used herein, the phrase "pharmacologically acceptable" refers to those compounds, substances, compositions, carriers, and / or dosage forms that 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.

[0233] As used herein, the term "pharmaceutically acceptable salts" refers to those salts of compounds formed by the process of the present application that are suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., within the scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19 (1977), which is expressly incorporated herein by reference in its entirety for all purposes. Salts can be prepared in situ during the final isolation and purification of the compounds of the present application, or separately by reacting a free base or acid functional group with a suitable acid or base.

[0234] Examples of pharma- ceutically acceptable salts include, but are not limited to, non-toxic acid addition salts: salts formed with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, and perchloric acids, or organic acids such as acetic, maleic, tartaric, citric, succinic, or malonic acids. Other pharma- ceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobiolate, and the like. Representative salts include, but are not limited to, phosphate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, 7-toluenesulfonate, undecanoate, valerate, and the like. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharma- ceutically acceptable salts include sulfonates and arylsulfonates when suitable non-toxic ammonium, quaternary ammonium, and amine cations are formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1-6 carbon atoms, and the like.

[0235] As used herein, the term "pharmaceutical acceptable ester" refers to an ester of a compound formed by the process of the present application that is hydrolyzed in vivo, including those that are readily decomposed in the human body to leave the parent compound or a salt. Suitable ester groups include, for example, those derived from pharmaceutical acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, each alkyl or alkenyl moiety advantageously having 6 or fewer carbon atoms. Examples of specific esters include, but are not limited to, formates, acetates, propionates, butyrates, acrylates and ethylsuccinates.

[0236] The term "pharmaceutically acceptable prodrugs" as used herein refers to those prodrugs of the compounds formed by the processes of the present application, and where possible, zwitterionic forms of the compounds of the present application, that are, within the scope of sound medical judgment, effective for their intended use without undue toxicity, irritation, allergic response, etc., commensurate with a reasonable benefit / risk ratio, and suitable for use in contact with the tissues of humans and lower animals.

[0237] As used herein, "prodrug" means a compound that is convertible in vivo by metabolic means (e.g., hydrolysis) to yield any compound defined by the formulations of the present application. Various forms of prodrugs are described, for example, in Bundgaard, (eds.), Design of Prodrugs, Elsevier (1985); Widder, et al. (eds.), Methods in Enzymology, vol. 4, Academic Press (1985); Krogsgaard-Larsen, et al., (eds.), "Design and Application of Prodrugs, Textbook of Drug Design and Development, Chapter 5, 113-191 (1991); Bundgaard, et al., Journal of Drug Deliver Reviews, 8:1-38 (1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285 et seq. (1988); Higuchi and Stella (eds.), Prodrugs as Novel Drug Delivery Systems, American Chemical Society (1975); and Bernard Testa & Joachim Mayer, "Hydrolysis In Drug And Prodrug Development, vol. 1, pp. 113-191 (1992)," all of which are expressly incorporated by reference in their entireties for all purposes. These methods are known in the art, as discussed in "Metabolism: Chemistry, Biochemistry And Enzymology", John Wiley and Sons, Ltd. (2002).

[0238] "Pharmaceutically acceptable excipient" means an excipient that is generally safe, non-toxic, and not biologically or otherwise undesirable and is useful in preparing pharmaceutical compositions, and includes excipients that are acceptable for veterinary as well as human pharmaceutical use. As used in the specification and claims, "pharmaceutically acceptable excipient" includes both one or more of such excipients.

[0239] The present application also encompasses pharmaceutical compositions comprising pharma- ceutically acceptable prodrugs of the compounds of the present application, and methods of administering the same to treat disorders. For example, compounds of the present application having free amino, amide, hydroxy, or carboxyl groups can be converted to prodrugs. Prodrugs include compounds in which an amino acid residue, or a polypeptide chain of two or more (e.g., 2, 3, or 4) amino acid residues is covalently linked via an amide or ester bonded to a free amino, hydroxy, or carboxylic acid group of the compounds of the present application. Amino acid residues include, but are not limited to, the 20 naturally occurring amino acids, commonly designated by their three-letter code, and include 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, 3-methylhistidine, norvaline, beta-alanine, gamma-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine, and methionine sulfone. Additional types of prodrugs are also encompassed. For example, free carboxyl groups can be derivatized as amides or alkyl esters. Free hydroxyl groups may be derivatized using groups including but not limited to hemisuccinic acid, phosphate ester, dimethylaminoacetate, and phosphoryloxymethyloxycarbonyl, as reviewed in Advanced Drug Delivery Reviews, 1996, 19, 1-15, which is expressly incorporated herein by reference in its entirety for all purposes.Also included are carbamate prodrugs of hydroxyl and amino groups, such as carbonate prodrugs of hydroxyl groups, sulfonate esters, and sulfate esters.Also included are derivatizations of hydroxyl groups, such as (acyloxy)methyl and (acyloxy)ethyl ethers, where the acyl group may be an alkyl ester, optionally substituted with groups including but not limited to ether, amine, and carboxylic acid functional groups, or the acyl group is an amino acid ester, as described above.This type of prodrug is described in J.Med.Chem.1996, 39, 10, which is expressly incorporated herein by reference in its entirety for all purposes.Free amines may also be derivatized as amides, sulfonamides or phosphonamides.All of these prodrug moieties may incorporate groups including, but not limited to, ether, amine and carboxylic acid functionalities.

[0240] Combinations of substituents and variables envisioned by this application are only those that result in the formation of stable compounds. As used herein, the term "stable" refers to compounds that retain sufficient stability to permit their manufacture and maintain compound integrity for a period of time sufficient to be useful for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject).

[0241] When any variable (e.g., R1) occurs more than one time in any constituent or formula of a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with one or more R moieties, R at each occurrence is selected independently from the definitions of R. Also, combinations of substituents and / or variables are permissible only if such combinations result in stable compounds within the normal valences of the designated atoms.

[0242] In addition, some of the compounds of the present application have one or more double bonds or one or more asymmetric centers. Such compounds may occur as racemates, racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures, and other stereoisomeric forms that may be defined in terms of absolute stereochemistry as (R)- or (S)-, or (D)- or (L)- of amino acids. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless otherwise specified, it is intended that the compounds include both E and Z geometric isomers. The configuration of any carbon-carbon double bonds appearing herein has been selected merely for convenience and is not intended to designate a particular configuration unless so stated in the text; thus, any carbon-carbon double bond depicted herein as trans may be cis, trans, or a mixture of both in any proportion. All such isomeric forms of such compounds are expressly included in the present application.

[0243] "Isomerism" means compounds that have identical molecular formulae but differ in the sequence of bonding of their atoms or the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are called "stereoisomers". Stereoisomers that are not mirror images of one another are called "diastereoisomers" and stereoisomers that are non-superimposable mirror images of each other are called "enantiomers" or sometimes optical isomers. A mixture containing equal amounts of individual enantiomeric forms of opposite chirality is called a "racemic mixture".

[0244] A carbon atom bonded to four non-identical substituents is called a "chiral center." "Chiral isomer" means a compound having at least one chiral center. Compounds with one or more chiral centers can exist as either individual diastereomers or mixtures of diastereomers called "diastereomeric mixtures." When one chiral center is present, a stereoisomer can be characterized by the absolute configuration (R or S) of its chiral center, e.g., carbon. Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are ranked according to the Cahn-Ingold-Prelog ranking rules (Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem. Educ. 1964, 41, 116).

[0245] "Geometric isomer" means diastereomers whose existence is due to hindered rotation about a double bond. These configurations are also distinguished in their names by the prefixes cis and trans, or Z and E, indicating that the groups are on the same or opposite sides of a double bond in the molecule according to the Cahn-Ingold-Prelog precedence rules.

[0246] Additionally, the structures and other compounds discussed in this application include all atropic isomers thereof. An "atropic isomer" is a type of stereoisomer in which the atoms of two isomers are arranged differently in space. Atropic isomers owe their existence to restricted rotation caused by hindrance of rotation of large groups about a central bond. Such atropic isomers typically exist as mixtures, but as a result of recent advances in chromatographic techniques; it has become possible to separate mixtures of two atropic isomers in selected cases.

[0247] A "tautomer" is one of two or more structural isomers that exist in equilibrium and are easily converted from one isomeric form to another. This conversion results in the formal migration of a hydrogen atom accompanied by the interchange of adjacent conjugated double bonds. Tautomers exist as a mixture of a set of tautomers in solution. In solids, one tautomer usually predominates. In solutions where tautomerization is possible, a chemical equilibrium of the tautomers will be reached. The exact ratio of the tautomers depends on several factors, including temperature, solvent, and pH. The concept of tautomers that are interconvertible by tautomerization is called tautomerism.

[0248] Of the various types of possible tautomerism, two are commonly observed. In keto-enol tautomerism, a simultaneous shift of electrons and hydrogen atoms occurs. Ring-chain tautomerism occurs as a result of an aldehyde group (-CHO) of a sugar molecule reacting with one of the hydroxyl groups (-OH) of the same molecule to give a cyclic (ring-shaped) form, as shown by glucose. Common tautomer pairs are: ketone-enol, amide-nitrile, lactam-lactim, amide-imidic acid tautomerism in heterocycles (e.g., nucleobases such as guanine, thymine, and cytosine), amine-enamine, and enamine-enamine. Compounds of the present application may also be represented in multiple tautomeric forms, and in such cases, the present application expressly includes all tautomeric forms of the compounds described herein (e.g., alkylation of a ring system may result in alkylation at multiple sites, and the present application expressly includes all such reaction products).

[0249] In this application, the structural formula of a compound may in some cases be convenient to represent a particular isomer, but this application includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, etc. In this application, the structural formula of a compound may in some cases be convenient to represent a particular isomer, but this application includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, etc.

[0250] Additionally, the compounds of the present application, for example, salts of the compounds, can exist in either hydrated or non-hydrated (anhydrous) form or as solvates with other solvent molecules. Non-limiting examples of hydrates include monohydrates, dihydrates, etc. Non-limiting examples of solvates include ethanol solvates, acetone solvates, etc.

[0251] "Solvate" refers to a solvent addition form that contains either stoichiometric or non-stoichiometric amounts of solvent. Some compounds have a tendency to trap solvent molecules in a fixed molar ratio in the crystalline solid state, forming a solvate. When the solvent is water, the solvate formed is a hydrate; and when the solvent is alcohol, the solvate formed is an alcoholate. Hydrates are formed by the combination of one or more molecules of water with a molecule of a substance, in which the water retains its molecular state as H2O.

[0252] In this specification, the structural formulas of compounds may in some cases conveniently represent certain isomers, but this application includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, and the like.

[0253] Working Example The purity of all compounds was greater than 95% and was analyzed on a Waters LC / MS system. 1 H NMR was obtained at 400 MHz. Chemical shifts were: 1 H NMR is reported relative to dimethylsulfoxide (δ=2.50). Data are reported as (br=broad, s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet).

[0254] Abbreviations used in the following examples and elsewhere herein are as follows: AcOH acetic acid; atm air; BMDM bone marrow derived macrophage; BOC2O di-tert-butyl dicarbonate; cGAMP cyclic [G(2',5')pA(3',5')p]; cGAS cyclic guanosine monophosphate-adenosine monophosphate synthase; CuSO4 copper sulfate; CDCl3 deuterated chloroform; CDN cyclic dinucleotide; DCM dichloromethane; DIEA N,N-diisopropylethylamine; DMA N,N-dimethylacetamide; DMAP 4-dimethylaminopyridine; DMF N,N-dimethylformamide; DMSO dimethylsulfoxide; DMSO-d6 deuterated dimethylsulfoxide; dsDNA double-stranded deoxyribonucleic acid; EDCI 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; ESI electrospray ionization; EtOAc ethyl acetate; H&E Hematoxylin and eosin;HCl hydrochloric acid;h hours;HPLC high performance liquid chromatography;immortalized human fibroblasts stably transfected with a type I IFN promoter driving hTERT luciferase and a CMV promoter driving SEAP or pIFNβ-Glu;IFN interferon;IP intraperitoneal;IRF3 interferon regulatory factor 3;ISD interferon-stimulated DNA;LCMS liquid chromatography-mass spectrometry;mL milliliter;MeCN acetonitrile;MEF mouse embryonic fibroblast;MeOH methanol;mg milligram;mmol millimole;MgSO4 magnesium sulfate;MHz megahertz;min minutes;MS mass spectrometry;Na2CO3 sodium carbonate;NaHCO3 sodium bicarbonate;NF-kB nuclear factor kappa-light-chain-enhancer of activated B cells;NMR nuclear magnetic resonance;PCR polymerase chain reaction;PO oral;STING stimulator of interferon genes;Tf triflate; TKO TREX1 knockout; Pd2(dba)3 tris(dibenzylideneacetone)dipalladium(0); Pd(PPh3)2Cl2 bis(triphenylphosphine)palladium(II) dichloride; ppm parts per million; PCR polymerase chain reaction; PTSA paratoluenesulfonic acid; qPCR quantitative real-time PCR;rt room temperature; t-BuOH tert-butanol; TBAF tetra-n-butylammonium fluoride; TBK1 TANK-binding kinase 1; THF tetrahydrofuran; TFA trifluoroacetic acid; TMS trimethylsilane; TLC thin layer chromatography; TNF tumor necrosis factor; TSA thermal shift assay; μL microliter; WT wild type. ;

[0255] Example 1: Identification of small molecule inhibitors of STING. In the presence of cytosolic DNA species, STING becomes activated to promote transcription of cytokines such as type I IFN. Compounds that bind to STING via TSA were then investigated for their ability to prevent cytosolic DNA species from activating type I interferon (IFN) promoters using a live cell assay. The assay consisted of immortalized human fibroblasts (hTERT) stably transfected with a type I IFN promoter driving luciferase and a CMV promoter driving SEAP (hTERT-pIFNβ-Glu). The live cell assay was previously disclosed in PCT / US2019 / 025380, entitled IFN-Beta Reporter System for Immortalized Primary Cells, inventor Glen N. Barber, filed April 2, 2018, and published as International Application No. 2019195285, which is incorporated herein by reference in its entirety for all purposes. The ability of small molecules to prevent activation of the type I IFN promoter and transcription of luciferase, but not SEAP, in response to cytosolic DNA species was evaluated. Activation of STING by CDNs typically also activates transcription factors NF-κB and IRF3, both of which are required to induce transcriptional stimulation of the type I IFN promoter, but not the CMV promoter, because IRF3 and NF-κB transcription factor binding sites are not contained in the CMV promoter. The assay identified small molecule compound W1 in the first screen (HTS), where the level of luciferase was not able to activate the type I IFN promoter or induce expression of luciferase (see Tables I and II).

[0256] Compound W1 (50 μM) was placed on hTERT-pIFNβ-Glu cells (hTERT cells stably transfected with the type I IFNβ promoter driving luciferase and the CMV promoter driving SEAP) for 24 h and transfected with double-stranded DNA at 3 μg / ml (also called ISD treatment) to inhibit activation of the type I IFN promoter in response to the presence of cytosolic DNA (Figure 1A). Inhibition of IFNβ expression was measured by luciferase induction 24 h after transfection of the ISD. ISD-only treatment was used as a control.

[0257] FIG 1B shows qPCR of Cxcl10 in normal hTERT cells 6 hours after ISD transfection with / without W1 treatment as in FIG 1A. FIG 1C shows Western blot analysis of STING, phospho-TBK1 (pTBK1) and phospho-IRF3 (pIRF3) performed 6 hours after ISD transfection with / without W1 treatment as in FIG 1A. FIG 1D shows confocal analysis of STING trafficking performed 6 hours after ISD transfection with / without W1 treatment as in FIG 1A.

[0258] The ability of compound W1 administered to normal hTERT cells to prevent cytosolic DNA from activating STING was evaluated (Figure 1B-H). This was accomplished by examining whether analogue W1 compound inhibited type I IFN production using ELISA assays and quantitative real-time PCR (qPCR). The data showed that compound W1 dramatically reduced IFNβ expression and production in hTERT cells (Figure 1B). In addition, the levels of STING phosphorylation (STING is phosphorylated after activation) and subsequent phosphorylation of TBK1, a kinase of IRF3, were evaluated. Phosphorylation of TBK1 and IRF3 is required for activation of the type I IFN promoter. The results suggested that compound W1 strongly prevented the phosphorylation of STING and the subsequent phosphorylation of TBK1 and IRF3 (Figure 1C). Compound W1 reproducibly blocked STING activity and effectively prevented STING function, STING phosphorylation, and STING trafficking required for TBK1 and IRF3 phosphorylation (Figure 1D). Thus, W1 binds to STING and disrupts STING function in human cells.

[0259] MEFs were treated with W1 and then stimulated with cytoplasmic DNA (ISD) to examine Cxcl10 expression and STING-dependent phosphorylation of TBK1 and IRF3 by qPCR. Figure 1E shows Western blot analysis of STING, phospho-TBK1 (pTBK1) and phospho-IRF3 (pIRF3) in MEFs treated as in Figure 1A. Figure 1F shows confocal analysis in MEFs treated as in Figure 1A. Figure 1G shows qPCR in MEFs treated as in Figure 1A. Figures 1E-G show that W1 was able to inhibit STING activation. The production of STING-induced cytokine Cxcl10 in response to ISD was dramatically reduced in the presence of compound W1.

[0260] Figure 1H shows the survival rate of knockout (TKO) mice treated with W1 (4-week-old TKO mice were intraperitoneally administered W1 (25 μg / mouse) twice a week for 5 months). Saline with 5% DMSO was administered as a control (210).

[0261] FIG. 2A shows hTERT-pIFNβ-Glu cells treated with analog W2 compound (at concentrations of 50 μM, 25 μM, and 10 μM) for 24 hours and transfected with double-stranded DNA (ISD) at 3 μg / ml. Inhibition of IFNβ expression was measured by luciferase induction 24 hours after transfection of ISD. ISD-only treatment was used as a control. FIG. 2B shows qPCR of IFNβ in normal hTERT cells 6 hours after ISD transfection with / without W2 treatment as in FIG. 2A. FIG. 2C shows Western blot analysis of STING, phospho-STING (pSTING), phospho-TBK1 (pTBK1), and phospho-IRF3 (pIRF3) performed 6 hours after ISD transfection with / without W2 (as in FIG. 2A), with ISD-only treatment used as a control. Figure 2D shows IFNβ luciferase assays performed in hTERT cells 6 h after ISD transfection with / without W2 (as in Figure 2A ), with ISD-only treatment used as control. Figure 2E shows qPCR of Cxcl10 performed in hTERT cells 6 h after ISD transfection with / without W2 (as in Figure 2A ), with ISD-only treatment used as control;

[0262] Figure 2F shows Western blot analysis of STING, pSTING, β-actin, and pIRF3 performed in hTERT cells 6 h after ISD transfection with / without W3 (as in Figure 2A ), with ISD-only treatment used as control.

[0263] FIG. 3A shows that W11 and W15 at 50 μM were placed on hTERT-pIFNβ-Glu cells for 24 hours and cells were transfected with double-stranded DNA (ISD) at 3 μg / ml. Inhibition of IFNβ expression was measured by luciferase induction 24 hours after transfection of ISD. FIG. 3B shows Western blot analysis of pTBK1, pIRF3 and pSTING (control: STING, and β-actin) performed 6 hours after ISD transfection with / without treatment. FIG. 3C shows confocal analysis for IRF3 translocation performed 6 hours after ISD transfection with / without inhibitors, with Mock and ISD only treatments used as controls. FIG. 3D shows qPCR of IFNβ1 performed in normal hTERT cells 6 hours after ISD transfection with / without inhibitors. H-151 (N-(4-ethylphenyl)-N'-1H-indol-3-yl-urea, CAS 941987-60-6, IC50 0.47 ± 0.23 μM) is a synthetic STING inhibitor (InvivoGen, San Diego, CA) and was used as a control (240). Figure 3E shows qPCR of Cxcl10 performed in normal hTERT cells 6 hours after ISD transfection with / without inhibitors. Figure 3F shows qPCR of CCL5 performed in normal hTERT cells 6 hours after ISD transfection with / without inhibitors. Figure 3G shows Western blot analysis of pTBK1, pIRF3 and pSTING (control: STING, and β-actin) performed 6 hours after ISD transfection with / without treatment. Figure 3H qPCR of IFNβ1 performed in MEFs treated as in Figure 3A. FIG. 3I qPCR of CCL5 performed on MEFs treated as in FIG. 3A .

[0264] Mouse embryonic fibroblasts (MEFs) were obtained from E13.5 embryos by standard procedures. Bone marrow-derived macrophages (BMDMs) were isolated from the hind femurs of 8- to 10-week-old WT and Trex1 KO (TKO) mice. Bone marrow-derived hematopoietic cells were cultured for 10 to 14 days in complete DMEM (Invitrogen) containing 10 ng / ml mouse recombinant colony-stimulating factor (M-CSF, R&D Systems).

[0265] Figure 4A shows the inhibitory effect of compounds W1-W5 on cells transfected with the ISD of STING at a concentration of 50 μM, where compounds W1-W5 were placed on hTERT cells stably transfected for 15 h with a type I IFNβ promoter driving luciferase and a CMV promoter driving SEAP, and transfected with double-stranded DNA at 3 μg / ml. Inhibition of IFNβ expression was measured by luciferase induction 24 h after transfection of the ISD, where Mock is shown (210) and ISD treatment alone (220) were used as controls.

[0266] Figure 4B shows the inhibitory effect of compounds W1-W26 on cells transfected with the ISD of STING at a concentration of 5 μM, where compounds W1-W26 were placed on hTERT cells stably transfected for 15 h with a type I IFNβ promoter driving luciferase and a CMV promoter driving SEAP, and transfected with double-stranded DNA at 3 μg / ml. Inhibition of IFNβ expression was measured by luciferase induction 24 h after transfection of the ISD, where Mock is shown (210) and ISD treatment alone (220) were used as controls.

[0267] FIG. 5A shows a photograph of heart tissue from a wild-type mouse treated with PBS three times a week for two months and stained with hematoxylin and eosin (H&E) for heart tissue at the end of the experiment. FIG. 5B shows a photograph of heart tissue from a wild-type mouse treated with inhibitor W15 (200 μg / mouse) three times a week for two months and stained with hematoxylin and eosin (H&E) for heart tissue at the end of the experiment. FIG. 5C shows a photograph of heart tissue from a Trex1 KO mouse treated with PBS three times a week for two months and stained with hematoxylin and eosin (H&E) for heart tissue at the end of the experiment. FIG. 5D shows a photograph of heart tissue from a Trex1 KO mouse treated with inhibitor W15 (200 μg / mouse) three times a week for two months and stained with hematoxylin and eosin (H&E) for heart tissue at the end of the experiment.

[0268] Figure 5E shows plots of fold change in TNF measured by qPCR in cardiac tissue from wild-type mice (225) and Trex1 KO mice (235) treated with PBS (210) or inhibitor W15 (200 μg / mouse) three times a week for two months. Figure 5F shows plots of fold change in IL-6 measured by qPCR in cardiac tissue from wild-type mice (225) and Trex1 KO mice (235) treated with PBS (210) or inhibitor W15 (200 μg / mouse) three times a week for two months. Figure 5G shows plots of fold change in IL-1β measured by qPCR in cardiac tissue from wild-type mice (225) and Trex1 KO mice (235) treated with PBS (210) or inhibitor W15 (200 μg / mouse) three times a week for two months.

[0269] Figure 6 shows qPCR analysis performed on stably transfected hTERT with a type I IFNβ promoter driving luciferase and a CMV promoter driving SEAP treated with compounds W15, W28-W35 compared to H-151 (240) (InvivoGen, San Diego), a selective STING inhibitor, at 5 μM in 0.5% DMSO. Inhibition of Cxcl10 expression was measured by luciferase induction 24 hours after stimulation with ISD for 24 hours; Mock is shown (210) and ISD treatment alone (220) were used as controls.

[0270] Example 2: Purification of STING protein for binding assay: A DNA sequence encoding human STING CBD-CTT(152-379) was inserted into the pET 26b-6XHis-pelB(-) vector between the NdeI / XhoI sites (STING152-379H). Protein was expressed in E. coli BL21 DE3 RIPL Codon Plus cells. E. coli cells were induced with 0.2 mM IPTG and grown overnight at 10°C when the cell density reached 0.5-0.6. Cells were spun down and lysed in cell lysis buffer (20 mM Tris pH 7.5, 300 mM NaCl, 5 mM DTT and protease inhibitor cocktail tablets (completely EDTA-free, Roche 11873580001). Protein lysates were French pressed (10-15 times) using an EmulsiFlex-C3 French Press (Avestin, Inc.) and imidazole was added to a final concentration of 50 mM. Cell debris was removed by centrifugation at 20,000 rpm at 4°C. The supernatant was applied to a 5 ml HisTrap HP column (GE Healthcare 17-5247-01) at very slow speed. Before applying the cell lysate, the column was equilibrated with cell lysis buffer containing 50 mM imidazole. The column containing STING protein was washed extensively using 3 column volumes of cell lysis / binding buffer. Elution buffer (20 mM Tris pH 7.5, 300 mM NaCl, 5 mM The protein was eluted with 100 mM DTT, 300 mM imidazole. The eluted fraction was applied to an S200 chromatography column to isolate pure STING dimers. Fractions containing STING protein dimers were pooled, centrifuged, and flash frozen for future use.

[0271] Example 3: Thermal high-throughput screening: A fluorometer (qPCR device with 320-well capacity) with temperature control was used to calibrate the denaturation profile of purified STING (STING152-379H). Once this was established, purified STING (5 μl; 1 μM) was mixed with 2.5 ul of DMSO and compound (final 1% DMSO with 2.5% SYPRO Orange. Total reaction mixture was 10 μL in 50 μM HEPES and 100 mM NaCl. SYPRO Orange binds non-specifically to hydrophobic surfaces. Upon protein unfolding, exposed hydrophobic surfaces will bind the dye and displace water, resulting in an increase in fluorescence. Stability curves and their midpoints (melting temperature, Tm, also known as hydrophobic exposure temperature, Th) were obtained by gradually increasing the temperature to unfold the protein and measuring the fluorescence at each point. Curves were measured for STING only and STING + small molecule and ΔTM was calculated. The temperature ramp was 0.1 degrees Celsius / sec. Approximately 250,000 compounds were screened.

[0272] Example 4: Luciferase assay: hTERT-BJ1 telomerase fibroblasts (hTERT-pIFNβ-Glu) stably expressing the luciferase gene under the control of the interferon-beta promoter and the SEAP gene under the control of the CMV promoter were generated in the Barber laboratory. Luciferase assays were performed using the Secrete-Pair Dual Luminescence Assay Kit from GeneCopoeia according to the manufacturer's protocol.

[0273] Example 5: Immunoblot analysis: Equal amounts of protein were resolved on sodium dodecyl sulfate (SDS)-polyacrylamide gels and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking with 5% blocking reagent, the membranes were incubated with various primary antibodies (and appropriate secondary antibodies). Images were resolved using the enhanced chemiluminescence system ECL (Thermo Scientific) and detected by autoradiography (Kodak). Antibodies: A rabbit polyclonal antibody against STING was developed in our laboratory as previously described in Ishikawa et al., 2008; other antibodies were obtained from the following sources: β-actin (Sigma-Aldrich), p-IRF3 (Cell Signaling), IRF3 (Santa Cruz Biotechnology), p-p65 (Cell Signaling), p65 (Cell Signaling), p-TBK1 (Cell Signaling), TBK1 (Abcam), cGAS (Cell Signaling).

[0274] Example 6: Interferon-β Elisa Analysis: Interferon-β Elisa was performed using either the IFNβ human ELISA kit from Invitrogen or the human IFNβ ELISA kit from PBL Interferon Source according to the manufacturer's protocol.

[0275] Example 7: Immunofluorescence microscopy: Cells were cultured and treated in their appropriate media on Lab-Tek II chamber slides. Cells were fixed with 4% paraformaldehyde for 15 min at 37°C and permeabilized with 0.05% Triton X-100 for 5 min at room temperature. Immunostaining was performed with rabbit anti-STING polyclonal, rabbit anti-IRF3 (Santa Cruz Biotechnology) or rabbit anti-p65 (Cell Signaling) followed by fluorescently conjugated secondary antibodies (FITC goat anti-rabbit) (Invitrogen).

[0276] Example 8: Quantitative real-time PCR (qPCR) Total RNA was reverse transcribed using M-MLV reverse transcriptase (Promega). Real-time PCR was performed for innate immune genes and inflammatory cytokines using Taqman gene expression assays (Applied Biosystems).

[0277] Example 9: Mouse treatment: Heterozygous mice were crossed to generate Trex1 heterozygous knockout mice (Trex1 + / - ) were generated. Mouse genotypes from tail biopsies were determined using real-time PCR with specific probes designed by a commercial vendor (Transnetyx) for each gene. Trex1 KO (TKO) mice (4-5 weeks old) were injected intraperitoneally with 25 μg of Z811 per mouse in 200 μL of 1% DMSO in PBS. Mice were injected three times a week for 5 months. Survival rates were assessed.

[0278] Example 10: Histopathology. Mice were euthanized and cardiac tissues were fixed in 10% formalin for 48 h.

[0279] Example 11: Stimulation of the immune system to promote potent antibody and cytotoxic T cell responses is essential to protect us against microbial infections and cancer (1). These responses are controlled by cellular innate immune sensors, e.g., STING, RIG-I / MDA5, or Toll-like receptors, and have evolved to detect microbial infection of cells primarily through the recognition of pathogen-derived nucleic acids, an event that triggers the transcription of numerous host defense-related proteins, including proinflammatory cytokines. However, in recent years, it has become clear that deregulation (chronic activation) of these same pathways can lead to a wide variety of autoimmune diseases.

[0280] We designed an assay and performed a high-throughput screen to detect small molecule compounds that bind to STING. After screening, we found 250,000 compounds, a family of STING-binding and inhibitory molecules. Unexpectedly, the compounds may be useful in preventing STING-dependent diseases and other forms of chronic innate immune-mediated diseases.

[0281] Cytokine overproduction is known to be involved in the pathogenesis of inflammatory diseases. Many patients show high titers of anti-nucleic acid antibodies (ANA), circulating DNA or even nucleosomes. Self-DNA is therefore implicated in potentially manifesting these types of diseases with abnormal innate immune signaling, possibly due to an inability to distinguish between self and foreign nucleotides. The discovery of the STING-regulated cytosolic DNA-enhanced innate immune pathway subsequently led to research into the plausible involvement of this pathway in autoimmune / autoinflammatory diseases. Several animal models have been recognized to exhibit inflammatory diseases. For example, deoxyribonuclease I (DNase I)-deficient mice exhibit inflammatory autoimmune symptoms such as immune complex nephritis. More seriously, macrophage lysosomal DNA endonuclease DNase II-deficient mice die prenatally due to the toxic effects of type I IFN overproduction. DNase III (also called TREX1)-deficient mice exhibit inflammation-associated diseases and have an average lifespan of 2 months. Human patients with DNase deficiencies, such as TREX1, suffer from a severe form of systemic lupus erythematosus (SLE) called Aicardi-Goutières syndrome (AGS). These disorders are now known to be caused by the host's inability to degrade self-DNA, which accumulates and activates the STING sensing pathway, generating chronic, harmful cytokine production. Indeed, mice deficient in DNase II or DNase III are fully viable when bred onto a STING-deficient background. STING has also been shown to affect inflammation-induced cancer and inflammatory bowel disease (IBD) (U.S. Patent Application Serial No. 16 / 284,975, filed February 25, 2019, by inventor Glen N. Barber, entitled CANCER TREATMENT AND DIAGNOSIS, incorporated herein by reference in its entirety for all purposes). Finally, mutations in the STING gene itself that result in constitutive activation of STING have been described in patients with infancy-onset STING-associated vasculopathy (SAVI), characterized by systemic inflammation and severe scaling lesions of the fingers, toes, nose, cheeks, and ears ( 26 ).Collectively, the STING pathway may provide novel targets that may be therapeutically regulated to help prevent a wide variety of inflammatory diseases in humans. The unexpected discovery of small molecules and novel analogs that bind and inhibit STING function may be useful in treating chronic STING-associated autoinflammatory diseases.

[0282] Two novel STING-based assays are used in high-throughput screening. Using these assays, compounds that bind to STING and inhibit its specific signaling can be identified. Based on the structure, chemistry is developed to generate a novel series of STING inhibitors. Lead compounds are selected for further evaluation, prevention of inflammatory diseases. Lead compounds bind to and stabilize STING and can be administered systemically. The compounds are human and mouse specific and can prevent AGS in vivo (survival rates were significantly improved over several months). Unexpectedly, novel STING inhibitors that exhibit anti-inflammatory activity may be useful for the treatment of a wide variety of autoinflammatory disorders.

[0283] Development of novel STING-activating compounds: Purified STING protein was used as bait to identify binding compounds using a thermal shift assay (TSA). Of the 250,000 screened compounds, achieved using an enamine library, several STING-binding small molecules were examined for their ability to inhibit STING signaling. In the presence of cytosolic DNA species, STING becomes activated and promotes the transcription of cytokines such as type I IFN. Compounds that bind to STING via TSA were then examined using a live cell assay for their ability to prevent cytosolic DNA species from activating type I interferon (IFN) promoters. The assay involved immortalized human fibroblasts (hTERT) stably transfected with a type I IFN promoter driving luciferase and a CMV promoter driving SEAP (hTERT-pIFNβ-Glu). The live cell assay was developed and patented in the Barber lab. The ability of small molecules to prevent activation of type I IFN promoters and transcription of luciferase, but not SEAP, in response to cytosolic DNA species was evaluated. Activation of STING by CDNs typically also activates transcription factors NF-κB and IRF3, both of which are required to induce transcriptional stimulation of type I IFN promoters, but not CMV promoters (IRF3 and NF-κB transcription factor binding sites are not contained in the CMV promoter). Thus, if STING activation is inhibited by small molecules identified in the first screen (HTS), the level of luciferase should be reduced in response to transfection of cytosolic DNA.

[0284] STING signaling plays a key role in promoting such inflammatory diseases. Therefore, novel STING inhibitors may be useful in preventing STING-dependent diseases and other forms of chronic innate immune-driven diseases. Other techniques are disclosed in U.S. Utility Application No. 17 / 246,480, entitled SMALL MOLECULAR INHIBITORS OF STING SIGNALING COMPOSITIONS AND METHODS OF USE, filed April 30, 2021, inventor Glen N. Barber, which is incorporated herein by reference in its entirety for all purposes.

[0285] Example 12: Synthesis of compound W1.

[0286] 5-(Trifluoromethyl)-1H-benzo[d]imidazol-2-amine (0.1 g, 1 equiv, 0.497 mmol) and 2-methyl-4-nitrobenzenesulfonyl chloride (0.14 g, 1.2 equiv, 0.596 mmol) were mixed in pyridine (20 mL) and the mixture was heated at 100 °C for 16 h. After cooling to room temperature, pyridine was evaporated under reduced pressure and the residue was purified by reverse phase HPLC (water / methanol, 0–50% in 2–8 min, 30 mL / min, column: SunFire 100 × 19 mm) to give 2-methyl-4-nitro-N-(5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)benzenesulfonamide (W1, 0.09 g) as a light brown solid. 1 H NMR (400MHz, DMSO-d6), δ, ppm: 12.31 (s, 2H), 8.37~7.97 (m, 3H), 7.56 (s, 1H), 7.46 (d, J = 6.1Hz, 2H), 2.76 (s, 3H). LCMS:m / z401.2[M+H] + .

[0287] [ka]

[0288] Example 13: Synthesis of compound W2.

[0289] [ka]

[0290] 2-Isopropyl-4-methyl-6-(piperazin-1-yl)pyrimidine (0.1 g, 1 eq, 0.454 mmol), 4-(chloromethyl)-2-(3,4-dimethoxyphenyl)thiazole hydrochloride (0.139 g, 1 eq, 0.454 mmol) and N-isopropyl-N-methylpropan-2-amine (0.129 g, 2.2 eq, 0.999 mmol) were mixed in ethanol (20 mL) and the mixture was heated at 78° C. for 16 h. After cooling to room temperature, the solvent was evaporated under reduced pressure and the residue was purified by reverse phase HPLC (water / acetonitrile, column: SunFire 100×19 mm) to give 2-(3,4-dimethoxyphenyl)-4-((4-(2-isopropyl-6-methylpyrimidin-4-yl)piperazin-1-yl)methyl)thiazole (W2, 0.105 g) as a yellow solid. 1 H NMR(400MHz,DMSO-d6),δ,ppm:8.07(s,1H),7.61(d,J=1.7Hz,1H),7.46(d,J=2.1Hz,2H),7.3 3(s,1H),7.16(s,1H),4.55(s,2H),2.46(s,3H),2.09(m,1H),0.97~0.82(m,2H),0.62(m,2H). LCMS:m / z454.2[M+H] + .

[0291] Example 14: Synthesis of intermediate 660.

[0292] Triethylamine (0.g, 1.1 equiv., mmol) was added to a solution of 2,4-dichloro-6-methylpyrimidine (3 g, 1 equiv., mmol) in ethanol (100 mL) and the mixture was stirred at room temperature for 30 min. tert-Butyl piperazine-1-carboxylate (g, 1 equiv., mmol) was added to the resulting solution and the resulting mixture was stirred at room temperature for 16 h. The solvent was evaporated under reduced pressure and water (100 mL) was added to the residue. The mixture was extracted with dichloromethane (3×30 mL). The combined organic phases were washed with brine (2×30 mL), dried over sodium sulfate and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography (chloroform / MTBE) to give tert-butyl 4-(2-chloro-6-methylpyrimidin-4-yl)piperazine-1-carboxylate (660) (3.5 g). LCMS: m / z 313.2 [M+H] + .

[0293] [ka]

[0294] Example 15: Synthesis of intermediate 610.

[0295] To a solution of 4-(2-chloro-6-methylpyrimidin-4-yl)piperazine-1-carboxylate (0.5 g, 1 eq, 1.6 mmol) in dioxane (10 mL) was added an aqueous solution of dimethylamine (40%, 2.5 mL) and the mixture was heated at 100° C. for 16 h. After cooling to room temperature, the solvent was evaporated under reduced pressure and water (30 mL) was added to the residue. The mixture was extracted with dichloromethane (3×20 mL). The combined organic phases were washed with brine (2×20 mL), dried over sodium sulfate and concentrated to dryness under reduced pressure. The resulting residue was dissolved in trifluoroacetic acid (10 mL) and the mixture was stirred at room temperature for 48 h and then concentrated to dryness to give N,N,4-trimethyl-6-(piperazin-1-yl)pyrimidin-2-amine ditrifluoroacetate (610, 0.58 g), which was used in the next step without further purification. LCMS: 222.2m / z [M+H]+ .

[0296] [ka]

[0297] Example 16: Synthesis of intermediates 620-650 following the procedure described for intermediate 610 (see above) using the amines shown in Table III.

[0298] [Table 3] TIFF2024521048000023.tif78159

[0299] Example 17: Synthesis of compound W3.

[0300] N,N,4-Trimethyl-6-(piperazin-1-yl)pyrimidin-2-amine ditrifluoroacetate (610, 0.1 g, 1 eq, 0.222 mmol), 4-(chloromethyl)-2-(3,4-dimethoxyphenyl)thiazole hydrochloride (0.068 g, 1 eq, 0.222 mmol) and potassium carbonate (0.153 g, 5 eq, 1.11 mmol) were suspended in acetonitrile (20 mL) and the mixture was stirred at room temperature for 24 h. The solvent was evaporated under reduced pressure and water (30 mL) was added to the residue. The mixture was extracted with dichloromethane (3 x 20 mL). The combined organic phases were washed with brine (2 x 20 mL), dried over sodium sulfate and concentrated to dryness under reduced pressure. The residue was purified by reverse phase HPLC (water / acetonitrile, column: SunFire 100×19 mm) to give 4-(4-((2-(3,4-dimethoxyphenyl)thiazol-4-yl)methyl)piperazin-1-yl)-N,N,6-trimethylpyrimidin-2-amine (W3, 0.062 g) as a yellow solid. 1H NMR(400MHz,DMSO-d6)δ7.45(t,J=7.4Hz,3H),7.05(d,J=8.4Hz,1H),5.91(s,1H),3 .83(d,J=10.5Hz,6H),3.67(s,2H),3.54(s,4H),3.32(d,J=9.6Hz,4H),2.10(s,3H). LCMS:m / z455.2[M+H] + .

[0301] [ka]

[0302] Example 18: Synthesis of compounds W6-W24 following the procedure described for compound W3. Compounds, structures, and property data are shown in Table IV.

[0303] [Table 4] TIFF2024521048000026.tif229159TIFF2024521048000027.tif240159TIFF202 4521048000028.tif240159TIFF2024521048000029.tif237159TIFF20245210480 00030.tif209159TIFF2024521048000031.tif203159TIFF2024521048000032.t if212159TIFF2024521048000033.tif229159TIFF2024521048000034.tif122159

[0304] Example 19: Synthesis of intermediate 670.

[0305] [ka]

[0306] tert-Butyl piperazine-1-carboxylate (1 g, 1 eq, 5.37 mmol), 4-(chloromethyl)-2-(3,4-dimethoxyphenyl)thiazole hydrochloride (1.64 g, 1 eq, 5.37 mmol) and calcium carbonate (3.7 g, 5 eq, 26.85 mmol) were suspended in acetonitrile (150 mL) and the mixture was stirred at room temperature for 24 h. The solvent was evaporated under reduced pressure and water (300 mL) was added to the residue. The mixture was extracted with dichloromethane (3×50 mL). The combined organic phases were washed with brine (2×50 mL), dried over sodium sulfate and concentrated to dryness under reduced pressure. The resulting residue was dissolved in trifluoroacetic acid (30 mL) and the mixture was stirred at room temperature for 48 h, then concentrated to dryness to give 2-(3,4-dimethoxyphenyl)-4-(piperazin-1-ylmethyl)thiazole trifluoroacetate (670) (2.4 g), which was used in the next step without further purification. LCMS: 320.2 m / z [M+H] + .

[0307] Example 20: Synthesis of compound W4.

[0308] To a solution of 670 (0.1 g, 1 eq, 0.182 mmol) in dioxane (10 mL) was added 4-chloro-N,N,6-trimethyl-1,3,5-triazin-2-amine (0.032 g, 1 eq, 0.182 mmol) and N-isopropyl-N-methylpropan-2-amine (0.118 g, 5 eq, 0.91 mmol). The mixture was heated at 100° C. for 16 h. After cooling to room temperature, the solvent was evaporated under reduced pressure and water (30 mL) was added to the residue. The mixture was extracted with dichloromethane (3×20 mL). The combined organic phases were washed with brine (2×20 mL), dried over sodium sulfate and concentrated to dryness under reduced pressure. The resulting residue was purified by reverse-phase HPLC (water / acetonitrile, column: SunFire 100×19 mm) to give 4-(4-{[2-(3,4-dimethoxyphenyl)-1,3-thiazol-4-yl]methyl}piperazin-1-yl)-N,N,6-trimethyl-1,3,5-triazin-2-amine (W4, 0.033 g) as a white solid.1 H NMR(400MHz,DMSO-d6)δ7.44(d,J=3.9Hz,3H),7.05(d,J=8.4Hz,1H),3.83(d,J=10. 7Hz,6H), 3.70(d,J=28.3Hz,6H),3.08(d,J=31.7Hz,8H),2.41(s,2H),2.14(s,3H). LCMS:m / z456.0[M+H]+.

[0309] [ka]

[0310] Example 21: Synthesis of compounds W5 and W25-W28 following the procedure described for compound W4. Compounds and characterization data are shown in Table V.

[0311] [Table 5] TIFF2024521048000038.tif190159TIFF2024521048000039.tif102159

[0312] Example 23: The term "functionality" as used herein means that the nano-inhibitor has biological activity to activate STING. The STA compositions of the present invention are useful for the treatment of cancer, inflammation and other disorders. The term "therapeutic level" refers to a level of the nano-inhibitor above normal physiological levels or in a subject prior to administration of the nano-inhibitor composition. As provided herein, the composition includes a delivery vehicle. As used herein, the term "delivery vehicle" includes any standard pharmaceutical carrier, diluent, excipient, etc. generally intended for use in connection with the administration of biologically active agents, including nucleic acids. The compositions and particularly the delivery vehicles described herein can deliver the nano-inhibitor to target cells. In an embodiment, the delivery vehicle is a lipid nanoparticle.

[0313] The term "polymer-conjugated lipid" refers to a polymer (e.g., polyethylene glycol (PEG), polypropylene glycol, polyvinylpyrrolidone, poly(N-(2-hydroxypropyl)methacrylamide) and PEGylated liposomes with different functional groups including methoxy (OCH3), amino (NH2), carboxyl (COOH), and hydroxyl (OH) moieties) conjugated with lipids. For example, PEG can be conjugated with myristoyl diglyceride to produce DMG-PEG2000. Alternatively, PEG can be conjugated with DSPE, a water-soluble derivative of phosphatidylethanolamine with (18:0) stearic acid acyl chains, to produce DSPE-PEG2000. PEG-conjugated lipids can incorporate a variety of functionalized PEG end groups including amine, carboxylic acid, azide, aldehyde, thiol, and hydroxyl moieties. PEG-conjugated lipids improve circulation time, drug stability, compatibility with different routes of administration, and assist in achieving targeted drug delivery. In an alternative embodiment of the invention, a branched polymer (e.g., poly(oligo(ethylene glycol) methyl ether methacrylate, i.e., poly(tri(ethylene glycol) methyl ether methacrylate, poly(tetra(ethylene glycol) methyl ether methacrylate, poly(penta(ethylene glycol) methyl ether methacrylate, poly(hexa(ethylene glycol) methyl ether methacrylate, poly(hepta(ethylene glycol) methyl ether methacrylate, poly(octa(ethylene glycol) methyl ether methacrylate, poly(noan(ethylene glycol) methyl ether methacrylate)) can be conjugated to a lipid.

[0314] In addition to PEG-conjugated lipids, "sterols" or unsaturated steroid alcohols can be used to enhance the stability of the LNPs. Sterols can include natural sterols and sterols with unnatural ring bonds. Sterols can be used to aid in the efficiency of introducing nano-inhibitors into cells. Varying the nature of the sterol component can also alter the efficiency of introducing nano-inhibitors into cells. Natural sterols include cholesterol, cholesterol sulfate, desmosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, dihydrolanosterol, zymosterol, lathosterol, 14-demethyl-lanosterol, 8(9)-dehydrocholesterol, 8(14)-dehydrocholesterol, FF-MAS, diosgenin, dehydroepiandrosterone (DHEA) sulfate, DHEA, sitosterol, lanosterol-95, zymostenol, sitostanol, campestanol, campesterol, 7-dehydrodesmosterol, pregnenolone, dihydro-T-MAS, dehydro-T-MAS, These include 5-avenasterol, brassicasterol, dihydro FF-MAS, 24-methylenecholesterol, 3β-hydroxy-7-oxo-5-cholestenoic acid, 7α-hydroxy-3-oxo-4-cholestenoic acid, 3β,7α-dihydroxy-5-cholestenoic acid, 3β,7β-dihydroxy-5-cholestenoic acid, 3β-hydroxy-5-cholestenoic acid, 3-oxo-4-cholestenoic acid, 3β,7α,24S-trihydroxy-5-cholestenoic acid, 3β,24S-dihydroxy-5-cholestenoic acid, 3β,7α,25-trihydroxy-5-cholestenoic acid, and 3β,25-OH-7-oxo-5-cholestenoic acid.

[0315] "Phospholipid" refers to a molecule having a hydrophilic head group and an aliphatic chain attached to an alcohol moiety. The nature of the head group, aliphatic chain, and alcohol can be used to generate a wide variety of phospholipids. The aliphatic chain includes saturated acyl chains, saturated alkyl chains, unsaturated acyl chains, unsaturated alkyl chains, saturated acyl chains with ether bonds, saturated alkyl chains with ester bonds, unsaturated acyl chains with ether bonds, and unsaturated alkyl chains with ester bonds. Glycerophospholipids and sphingomyelin are phospholipids that differ based on the alcohol moiety. Phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, cardiolipin, dipalmitoyl, dimyristoyl, distearoylphosphatidylcholine, dioleoyl, distearoyl PC, and L-α-phosphatidylcholine. In one embodiment of the present invention, the alcohol of the phospholipid can be a C3 alcohol. In an alternative embodiment of the invention, the phospholipid may include a C4-C8 alcohol.

[0316] "Ionizable lipids" are a class of lipid molecules that remain neutral at physiological pH but become protonated under acidic conditions. Ionizable lipids promote endosomal escape and reduce toxicity of LNPs. Ionizable lipids include 7-[(2-hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]heptyl 2-octyldecanoate, DODMA (MBN305A), DLin-KC2-DMA, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (D-Lin-MC3-DMA, or MC3), 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}heptadecan-9-yl octanoate (SM-102), and bis(2-hexyldecanoate)[(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) (ALC-0315).

[0317] The term "LNP" refers to lipid nanoparticles. LNPs refer to particles made of lipids (e.g., cationic lipids, non-cationic lipids, conjugated lipids and / or sterols that prevent nanoparticle aggregation) and a nanoinhibitor, which is encapsulated within the lipid (e.g., the nanoinhibitor).

[0318] In one embodiment of the present invention, the LNP formulation can have four major components other than the nucleic acid. In one embodiment of the present invention, the LNP includes a phospholipid, a sterol, an ionizable lipid, and a polymer-conjugated lipid. In one embodiment of the present invention, to generate a nano-inhibitor, the phospholipid can be distearoylphosphatidylcholine, the sterol can be cholesterol, the ionizable lipid can be MC3, the PEG-conjugated lipid can be DMG-PEG2000, and the diameter of the spherical liponanoparticle can be approximately 88 nm, approximately meaning +-10 nm. In one embodiment of the present invention, the cholesterol can be 35-45% of the LNP composition. In one embodiment of the present invention, the LNP includes distearoylphosphatidylcholine (DSPC), cholesterol, an MC3-like lipid, and a PEG-conjugated lipid. The phospholipid and cholesterol promote the stability and structural integrity of the LNP. The ionizable lipid promotes electrostatic interactions with the negatively charged nucleic acid, aiding in intracellular delivery. The polymer-conjugated lipid improves the solubility of the LNP in serum and circulation by preventing the particles from agglomerating, but retains good biocompatibility and has good tolerability properties. In one embodiment of the invention, the nano-inhibitor particles may be approximately 88 nm in size, where approximately means plus or minus 10 percent. In one embodiment of the invention, the inhibitor is approximately 50% encapsulated in the nano-inhibitor. In this range, approximately means plus or minus 20 percent. In an alternative embodiment of the invention, the inhibitor is approximately 75% encapsulated in the nano-inhibitor. In this range, approximately means plus or minus 10 percent. In another embodiment of the invention, the inhibitor is at least approximately 90% encapsulated in the nano-inhibitor. In this range, approximately means plus or minus 5 percent. In another alternative embodiment of the invention, the inhibitor is approximately 98% encapsulated in the nano-inhibitor. In this range, approximately means plus or minus 1 percent. In one embodiment of the invention, the inhibitor is approximately 98% encapsulated in the nano-inhibitor at a concentration of 0.2 mg / mL of dsDNA in the LNP in PBS.LNPs are extremely useful for systemic applications because they can exhibit extended circulatory lifetime following intravenous (iv) injection, can accumulate at distal sites (e.g., sites physically separated from the site of administration), and can deliver inhibitors to sites distal to the site of administration.

[0319] In one embodiment of the present invention, LNPs containing distearoylphosphatidylcholine, cholesterol, MC3, and DMG-PEG2000 are dissolved in ethanol and rapidly mixed with inhibitor in an aqueous buffer at a pH where the ionizable lipid is positively charged (pH approximately 4, meaning approximately +-pH 1). The resulting dispersion is then dialyzed against normal saline buffer to remove residual ethanol and raise the pH above the pKa of the cationic lipid (pH approximately 7.4, meaning approximately +-pH 0.5) to produce the final nano-inhibitor.

[0320] Example 24: All statistical analyses were performed by Student's t-test, and data were considered significantly different when P<0.05.

[0321] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed within the scope of this application. All patents, patent applications, and references cited herein are hereby expressly incorporated by reference.

[0322] Exemplary embodiments of the methods, systems, and components of the present invention are described herein. As noted elsewhere, these exemplary embodiments are set forth for illustrative purposes only and are not limiting. Other embodiments are possible and are included in the present invention. Such embodiments will be apparent to those skilled in the relevant art based on the teachings contained herein. For example, it is envisioned that the outer diameter outlet of the inlet tube can be tapered or non-tapered, and the outer diameter inlet of the outlet tube can be tapered or non-tapered, regardless of the actual shapes depicted in the various figures and embodiments described above.

[0323] Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

Claims

1. Formula I: 【Chemistry 1】 (In the formula, R １ is selected from the group consisting of -H, -halogen, -(C1-C6)alkyl, -(C3-C6)cycloalkyl, -(C1-C6)haloalkyl, -(C1-C6)alkoxy, -(C2-C6)haloalkoxy, -(C2-C6)alkenyl, -(C2-C6)alkynyl, -(C1-C6)dialkylether, -(C1-C6)alkyl(C3-C6)cycloalkylether, -(C1-C6)alkylarylether, -nitro, -CN, -OH, -COOH, -SH, -NH2, -NH(C1-C4)alkyl, and -N((C1-C4)alkyl)2; R ２ , R ３ , R ４ and R ５ are independently -H, -halogen, -(C １ ~C ６ ) alkyl, -(C ３ ~C ６ ) cycloalkyl, -(C １ ~C ６ ) haloalkyl, -(C １ ~C ６ ) alkoxy, -(C １ ~C ６ ) haloalkoxy, -(C ２ ~C ６ ) alkenyl, -(C ２ ~C ６ ) alkynyl, -(C １ ~C ６ ) dialkyl ether, -(C １ ~C ６ ) alkyl(C ３ ~C ６ ) cycloalkyl ether, -(C １ ~C ６ ) Alkyl aryl ether, -nitro, -CN, -OH, -COOH, -SH, -NH ２ , -NH(C １ ~C ４ ) alkyl, and —N((C １ ~C ４ ) alkyl) ２ R is selected from the group consisting of ６ , R ７ and R 8 are independently -H, -halogen, -(C １ ~C ６ ) alkyl, -(C ３ ~C ６ ) cycloalkyl, -(C １ ~C ６ ) haloalkyl, -(C １ ~C ６ ) alkoxy, -(C １ ~C ６ ) haloalkoxy, -(C ２ ~C ６ ) alkenyl, -(C ２ ~C ６ ) alkynyl, -(C １ ~C ６ ) dialkyl ether, -(C １ ~C ６ ) alkyl(C ３ ~C ６ ) cycloalkyl ether, -(C １ ~C ６ ) Alkyl aryl ether, -nitro, -CN, -OH, -COOH, -SH, -SO ２ Alkyl, -CF ３ , -(C １ ~C ６ ) Alkyl CF ３ , -NH ２ , -NH(C １ ~C ４ ) alkyl, and —N((C １ ~C ４ ) alkyl) ２ R is selected from the group consisting of ９ is an atom selected from the group consisting of carbon and nitrogen, R １０ is an atom selected from the group consisting of carbon, sulfur, nitrogen and oxygen; R １１ is an atom selected from the group consisting of sulfur, nitrogen and oxygen, or a pharma- ceutically acceptable salt, hydrate, ester, solvate, prodrug, stereoisomer, or tautomer thereof.

2. R ９ The compound of claim 1 , wherein is carbon.

3. R １０ The compound of claim 1 , wherein is sulfur.

4. R １１ The compound according to claim 2 or 3, wherein is nitrogen.

5. R ９ is carbon, R １０ is sulfur, R １１ The compound of claim 1 , wherein is nitrogen.

6. R １ -H, -CF ３ , and -CH ２ CH ３ 6. The compound of claim 5 selected from the group consisting of:

7. 2-Methyl-4-nitro-N-[5-(trifluoromethyl)-1H-1,3-benzodiazol-2-yl]benzene-1-sulfonamide; 2-(3,4-dimethoxyphenyl)-4-[[4-(6-methyl-2-propan-2-ylpyrimidin-4-yl)piperazin-1-yl]methyl]-1,3-thiazole ； N,N,4-trimethyl-6-[4-({2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-4-yl}methyl)piperazin-1-yl]pyrimidin-2-amine; 4-(4-{[2-(4-ethylphenyl)-1,3-thiazol-4-yl]methyl}piperazin-1-yl)-N,N,6-trimethylpyrimidin-2-amine; 4-(4-{[2-(4-ethylphenyl)-1,3-thiazol-4-yl]methyl}piperazin-1-yl)-N,N,6-trimethylpyrimidin-2-amine; N.N. 4-trimethyl-6-[4-({2-[4-(propan-2-yl)phenyl]-1.3-thiazol-4-yl}methyl)piperazin-1-yl]pyrimidin-2-amine; 4-(4-{[2-(4-cyclopropylphenyl)-1.3-thiazol-4-yl]methyl}piperazin-1-yl)-N.N. 6-trimethylpyrimidin-2-amine; 4-[4-({2-[4-(1.1-difluoroethyl)phenyl]-1.3-thiazol-4-yl}methyl)piperazin-1-yl]-N.N. 6-trimethylpyrimidin-2-amine and N.N. A compound selected from the group consisting of 4-trimethyl-6-[4-({2-[4-(2.2.2-trifluoroethyl)phenyl]-1.3-thiazol-4-yl}methyl)piperazin-1-yl]pyrimidin-2-amine.

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