WO2019241787A1 - Nouveaux inhibiteurs cycliques de la gmp-amp synthase (cgaz) et leur procédé d'utilisation - Google Patents

Nouveaux inhibiteurs cycliques de la gmp-amp synthase (cgaz) et leur procédé d'utilisation Download PDF

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WO2019241787A1
WO2019241787A1 PCT/US2019/037502 US2019037502W WO2019241787A1 WO 2019241787 A1 WO2019241787 A1 WO 2019241787A1 US 2019037502 W US2019037502 W US 2019037502W WO 2019241787 A1 WO2019241787 A1 WO 2019241787A1
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compound
pharmaceutically acceptable
formula
acceptable salt
cgas
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WO2019241787A9 (fr
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Hang Hubert YIN
Rosaura PADILLA-SALINAS
Zhijian Chen
Lijun Sun
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The Regents Of The University Of Colorado A Body Corporate
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
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    • C07D251/14—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hydrogen or carbon atoms directly attached to at least one ring carbon atom
    • C07D251/16—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hydrogen or carbon atoms directly attached to at least one ring carbon atom to only one ring carbon atom
    • C07D251/18—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hydrogen or carbon atoms directly attached to at least one ring carbon atom to only one ring carbon atom with nitrogen atoms directly attached to the two other ring carbon atoms, e.g. guanamines
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    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
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    • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

Definitions

  • This invention relates generally to the fields of biology, chemistry and medicine. More particularly, it concerns methods and compositions relating to autoimmunity and inflammation.
  • Cytosolic DNA including cytoplasmic chromatin fragments, endogenous nuclear, or mitochondrial DNA, and DNA arising from intracellular pathogens, triggers a powerful innate immune response. It is sensed by cyclic GMP-AMP synthase (cGAS), which elicits the production of type I interferons by generating the second messenger 2'3'-cyclic-GMP-AMP (cGAMP), activating the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP- AMP synthase linked to stimulator of interferon genes) pathway, leading to short-term inflammation, but also to chronic inflammation that has been linked to the onset and progression of autoimmunity.
  • cGAS cyclic GMP-AMP synthase linked to stimulator of interferon genes
  • cGAS systemic lupus erythematosus
  • RA rheumatoid arthritis
  • IBD inflammatory bowel disease
  • Aicardi-Gouties syndrome Aicardi-Gouties syndrome
  • microbial RNA with specific features can be recognized by Retinoic acid-inducible Gene I (RIG-I) or Melanoma Differentiation- Associated protein 5 (Mda5), which activate Mitochondrial Anti-Viral Signaling protein (MAVS).
  • RIG-I Retinoic acid-inducible Gene I
  • Mda5 Melanoma Differentiation- Associated protein 5
  • MAVS Mitochondrial Anti-Viral Signaling protein
  • TK1 Tank-Binding Kinase 1
  • IKK Inhibitor of KB Kinase
  • cytosolic DNA is detected by cGAS, a primary DNA sensor that belongs to the nucleotidyltransferase enzyme family.
  • cGAS catalyzes the formation of cyclic dinucleotide, cyclic GMP-AMP (cGAMP or c[G(2’,5’)pA(3’,5’)p]).
  • cGAMP binds to the adaptor protein stimulator of interferon genes (STING) and triggers its cellular trafficking and activation of TBK1 and IKK complexes.
  • TBK1 and IKK activate the transcription factors Interferon regulatory factor 3 (IRF3) and Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-KB), which are essential for induction of type I interferons and other inflammatory cytokines.
  • IRF3 Interferon regulatory factor 3
  • NF-KB Nuclear Factor kappa-light-chain-enhancer of activated B cells
  • cGAS does not distinguish self- from non-self-DNA, therefore aberrant accumulation of self-DNA in the cytoplasm can induce unwanted immune response.
  • Normal cells deploy multiple DNases including Trexl to keep cytoplasm clear of DNA.
  • endogenous DNA can activate cGAS-STING pathway.
  • Aicardi-Gouties syndrome Aicardi-Gouties syndrome
  • Gain-of-function mutations within STING in human patients are linked to early onset STING-associated vasculopathy, an autoinflammatory disease.
  • cGAS-STING signaling has also been shown to promote cancer growth and metastasis through modulation of the tumor microenvironment.
  • Crystal structures of the cGAS dimer bound to dsDNA have provided valuable insight into the activation mechanism of cGAS.
  • the central role of cGAS-STING pathway in inflammation, autoimmunity, cancer, and tumor progression has spurred intensive investigations toward the identification and characterization of small molecule inhibitors for cGAS, including RU.521, PF-06928215, suramin, and X6.
  • these modulators all have demonstrated antagonistic effects on cGAS, the inhibitors are also associated with drawbacks that may limit their utility as cellular chemical tools.
  • RU.521 is a potent small molecule inhibitor of murine cGAS (mcGAS), and PF-06928215 only inhibits human cGAS (hcGAS) but lacks cellular activity.
  • Suramin, an approved drug, and amino acridine, X6 were also identified as viable cGAS inhibitors.
  • suramin and X6 inhibit hcGAS and mcGAS, respectively, through the displacement of DNA from cGAS.
  • suramin is active in human cells, the inhibitor suffers from off-target effects through inhibition of the Toll-like receptor (TLR) 3 dsRNA sensing pathway. Indeed, only few small molecule cGAS inhibitors exist, highlighting the urgent need to discover new chemical scaffolds that can selectively inhibit hcGAS in cells.
  • TLR Toll-like receptor
  • the inventors have developed small molecule inhibitors that directly target cytosolic cyclic-GMP-AMP (cGAMP) Synthase (cGAS), thereby inhibiting the cGAS/Stimulator of Interferon Genes (STING) pathway and providing new therapeutic paths for down regulating the cytosolic DNA sensing pathway, regulating inflammation and ultimately preventing or treating autoimmune diseases and disorders.
  • cGAMP cytosolic cyclic-GMP-AMP
  • STING Interferon Genes
  • cGAS novel small molecule human cGAS
  • CU-32 and CU-76 selectively inhibit the DNA pathway in human cells but had no effect on RIG-I-MAVS or Toll-like Receptor pathways.
  • CU-32 and CU-76 represent a new class of hcGAS inhibitors with activity in cells and provide a new chemical scaffold for designing probes to study cGAS function and development of autoimmune therapeutics.
  • novel small molecule inhibitors CU-32, CU-76, and analogs selectively inhibit the DNA pathway representing a new class of hcGAS inhibitors with cellular activity in human THP-l cells. More specifically, the inhibitory activity of CU-32 is specific for hcGAS versus mcGAS in cells. Moreover, the novel small molecule inhibitors CU-32 and CU- 76 are selective for the cytosolic DNA pathway over other NA-sensing pathways such as RIG-I- MAVS and endosomal TLRs. Such small molecule inhibitors provide a new chemical scaffold for developing hcGAS inhibitors with potential therapeutic applications and a much-needed small molecule chemical probe for studying cGAS biology and cGAS related disorders in human cells.
  • This disclosure therefore provides therapeutic strategies for the treatment of inflammation and autoimmune diseases associated with chronic inflammation and autoimmunity due to cGAS activation.
  • a compound of Formula I, II, III, IV or claim 4 or a pharmaceutically acceptable salt, solvate, stereoisomer, tautomer, or prodrug thereof, for use in medical therapy.
  • a pharmaceutical composition comprising (a) a compound of Formula I, II, III, IV or claim 4, or a pharmaceutically acceptable salt, solvate, stereoisomer, tautomer, or prodrug thereof, and (b) a pharmaceutically acceptable carrier, for use in medical therapy.
  • a method for treating a disease or condition for which modulation of cGAS activity is beneficial comprising: administering to a patient in need thereof, a therapeutically effective amount of a compound of I, II, III, IV, or claim 4, or a pharmaceutically acceptable salt thereof.
  • a method for treating a disease or condition for which modulation of cGAS is beneficial comprising: administering to a patient in need thereof, a therapeutically effective amount of a combination comprising a compound of Formula I, II, III, IV or claim 4, or a pharmaceutically acceptable salt thereof, and at least one further therapeutic agent.
  • a pharmaceutical composition comprising a compound of Formula I, II, III, IV or claim 4, or a pharmaceutically acceptable salt thereof, for use in the treatment of a disease or condition for which modulation of cGAS is beneficial.
  • a pharmaceutical composition comprising: a compound of Formula I, II, III, IV or claim 4, or a pharmaceutically acceptable salt thereof, and at least one further therapeutic agent, for use in the treatment of a disease or condition for which modulation of cGAS is beneficial.
  • a pharmaceutical composition comprising: a compound of Formula I, II, III, IV or claim 4, or a pharmaceutically acceptable salt thereof, at least one further therapeutic agent, and one or more of pharmaceutically acceptable excipients, for use in the treatment of a disease or condition for which modulation of cGAS is beneficial.
  • a method of treating an inflammatory, allergic or autoimmune disease comprising: administering to a patient in need thereof a therapeutically effective amount of a compound of Formula I, II, III, IV or claim 4, or a pharmaceutically acceptable salt thereof.
  • the inflammatory, allergic or autoimmune diseases is systemic lupus erythematosus, psoriasis, insulin-dependent diabetes mellitus (IDDM), scleroderma, Aicardi Gourtiers syndrome, dermatomyositis, inflammatory bowel diseases, multiple sclerosis, rheumatoid arthritis or Sjogren's syndrome (SS).
  • IDDM insulin-dependent diabetes mellitus
  • SS Sjogren's syndrome
  • a method for treating an inflammatory, allergic or autoimmune disease comprising: administering to a patient in need thereof, a therapeutically effective amount of a combination comprising a compound of Formula I, II, III, IV or claim 4, or a pharmaceutically acceptable salt thereof, and at least one further therapeutic agent.
  • a pharmaceutical composition comprising a compound of Formula I, II, III, IV or claim 4, or a pharmaceutically acceptable salt thereof, for the treatment of an inflammatory, allergic or autoimmune disease.
  • a pharmaceutical composition comprising a compound of Formula I, II, III, IV or claim 4, or a pharmaceutically acceptable salt thereof, and at least one further therapeutic agent, for the treatment of an inflammatory, allergic or autoimmune disease.
  • a method of treating an infectious disease comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula I, II, III, IV or claim 4, or a pharmaceutically acceptable salt thereof.
  • infectious disease is a viral, bacterial or parasite infection.
  • a method for treating an infectious disease comprising: administering to a patient in need thereof, a therapeutically effective amount of a combination comprising a compound of Formula I, II, III, IV or claim 4, or a pharmaceutically acceptable salt thereof, and at least one further therapeutic agent.
  • a pharmaceutical composition comprising a compound of Formula I, II, III, IV or claim 4, or a pharmaceutically acceptable salt thereof, for use in the treatment of an infectious disease.
  • a pharmaceutical composition comprising a compound of Formula I, II, III, IV or claim 4, or a pharmaceutically acceptable salt thereof, and at least one further therapeutic agent, for use in the treatment of an infectious disease.
  • a compound of Formula I, II, III, IV or claim 4, or a pharmaceutically acceptable salt thereof, for use in the treatment of a senescence-related disease comprising: administering to a patient in need thereof a therapeutically effective amount of a compound of Formula I, II, III, IV or claim 4, or a pharmaceutically acceptable salt thereof.
  • the senescence-related disease is atherosclerosis, myocardial infarction, Alzheimer's disease, Parkinson's diseases, Huntington's disease, amyotrophic lateral sclerosis, hepatitis, renal disease, diabetes, cancer and aging.
  • a method for treating a senescence-related disease comprising: administering to a patient in need thereof, a therapeutically effective amount of a combination comprising a compound of Formula I, II, III, IV or claim 4, or a pharmaceutically acceptable salt thereof, and at least one further therapeutic agent.
  • a pharmaceutical composition comprising a compound of Formula I, II, III, IV or claim 4, or a pharmaceutically acceptable salt thereof, for the treatment of a senescence-related disease.
  • a pharmaceutical composition comprising a compound of Formula I, II, III, IV or claim 4, or a pharmaceutically acceptable salt thereof, and at least one further therapeutic agent, for the treatment of a senescence-related disease.
  • FIG. 1 Residues mediating important cGAS-cGAS and cGAS-DNA interactions and the design of an unprecedented strategy for inhibiting hcGAS.
  • FIG. 9 A Schematic of mcGAS and dsDNA interaction surfaces. The residues highlighted in red are involved in the dimer interface of cGAS and mediate cGAS-DNA interactions.
  • FIG. 9B Close-up view of the grid generated on the PPI of mcGAS. For the virtual high throughput screen of the Maybridge and Enamine drug databases, the grid box was generated to target the PPI of mcGAS with incorporation of important residues mediating the PPI interactions.
  • FIG. 2 Preliminary SAR studies for hcGAS inhibitors.
  • A Targets synthesized to identify key protein binding structural motifs.
  • B Structures of additional derivatives designed to probe inhibitor interactions.
  • FIG. 3 SAR in vitro results for analogs bearing an NH-heterocycles at the six position.
  • Table 4 (A) Key targets synthesized to identify protein binding structural motifs.
  • B Structure of additional derivatives designed to probe inhibitor interactions. (Table 4 for chemical structure of compounds 31-3).
  • FIG. 4 CU-76 and CU-32 selectively inhibit the cytosolic DNA sensing, but not the RNA sensing pathway.
  • IRF3 interferon regulatory factor 3
  • the (IRF)2 dimer was not detected at 30 and 100 mM for CU-76 and at 100 mM for CU-32 confirming the compounds effectiveness for inhibiting the cGAS-STING pathway.
  • At 10, 30, and 100 mM CU-76 and CU-32 did not reduce dimerization of IRF3 induced by the RIG-I-MAVs pathways confirming the specificity of these compounds for the DNA pathway.
  • (B) Effects of CU-76 and CU-32 treatment on the production of interferon beta- 1 alpha (IFN-b) (ELISA measurements) in THP-l cells stimulated with interferon-stimulatory DNA (ISD) (upper panel and Sendai virus (Sev).
  • CU-32 and CU-76 (10, 30, and 100 mM) suppressed levels of IFN-b in media for ISD stimulated THP-l cells in a dose dependent manner further supporting the compounds specificity for the DNA pathway.
  • the levels of IFN-b in media were not reduced in THP-l cells stimulated with Sev showing CU-32 and CU-76 (10, 30, and 100 mM) do not display off-target effects for the RNA pathway.
  • FIG. 5 Effect of CU-32 and CU-76 on mouse cGAS.
  • A In vitro concentration-dependent inhibition of mcGAS enzymatic activity in an ATP consumption assay by CU-32 and CU-76. The IC50 value reported represents the mean value.
  • B RAW-ISG-luc cells were transfected with interferon-stimulatory DNA (ISD) or infected with Sendai virus (SEV) for 16 h in the presence of serial concentrations of compounds or DMSO, followed by measurement of ISRE reporter expression using luminescence. CU-32 and CU-76 were ineffective for suppressing the enzymatic activity of mcGAS in RAW 264.7 cells.
  • ISD interferon-stimulatory DNA
  • SEV Sendai virus
  • FIG. 6 Molecular docking studies for CU-45 and CU-76 cGAS inhibitors.
  • A Close-up view of the binding site of CU-76 on the cGAS dimer interface. CU-76 may potentially disrupt the interface of the mcGAS dimer by inserting aside the Zn loop.
  • B Schematic representation of residues around the molecules (left, using MOE v.2014.0901) and close-up view of the binding site on the protein interface (right, using PYMOL v2.0.7) are shown.
  • methylation of -NH2 abrogates H-bond interaction with GLU386.
  • contacts within 3 A are shown. See FIGS. 24A and 24B.
  • FIG. 7 Synthesis of 1, 2a and related compounds.
  • A Schematic representation of the one-step condensation cyclisation reaction for the synthesis of 4-amino-6-(arylamino)-l,3,5- triazine-2-carboxylate derivatives.
  • B Schematic representation of select 6-subsituted 4-amino- 6-(arylamino)-l,3,5-triazine derivatives.
  • C Synthetic routes for targets are summarized in the supplemental information (Fig. 17)
  • FIG. 8 In vitro validation studies of the ten lead hits, Related to Figure 2A.
  • the inhibitory activity of the indicated compounds was evaluated by the measurement of ATP consumption from hcGAS-mediated 2’,3’-cGAMP synthesis.
  • the compounds in DMSO were added at 0.1 mM and 1 mM concentrations to a reaction mixture containing 20mM Tris-Cl, 5mM MgCl2, 0.2mg/ml bovine serum albumin (BSA), O.Olmg/ml Herring testis DNA (HT-DNA), O. lmM GTP, 0.006mM ATP, and 30nM human cGAS protein, incubated at 37 DC for 20min.
  • BSA bovine serum albumin
  • HT-DNA O.Olmg/ml Herring testis DNA
  • O. lmM GTP O. lmM GTP
  • 0.006mM ATP 0.006mM ATP
  • Remaining ATP levels was measured by adding 40 Dl of KinaseGlo (Promega) and reading luminescence. Reactions omitting cGAS and reactions without compounds but DMSO were considered 100% and 0% inhibition, respectively. Inhibition of cGAS enzymatic activity at 100 mM was observed for one compound, Z918, (-20% inhibition) of the ten hits identified from the screen. The IC50 for hit Z918, was estimated. A full dose-response curve was not conducted for the Z918 and the IC50 was estimated to be 100 mM.
  • FIG. 9 depicts a schematic representation of the pathway from cGAS activation to STING and type I interferons and pro-inflammatory cytokines.
  • the interaction point of inhibitory molecules of this disclosure in this pathway is indicated at the‘X’.
  • FIG. 10A illustrates the functional portions of a core chemical structure of cGAS inhibitory compounds of this disclosure.
  • (B) shows modifications made to the core chemical structure and the resulting cGAS inhibitory activity.
  • FIG. 11 illustrates further strategies for structure activity relationship (SAR) studies of functional portions of a core chemical structure of cGAS inhibitors of this disclosure.
  • FIGS. 12 (A)-(C) each depict chemical modifications made to the hydrophilic portion of the core chemical structure, and the resulting cGAS inhibitory activity.
  • FIG. 13 depicts chemical modifications made to the hydrophobic portion of the core chemical structure, and the resulting cGAS inhibitory activity.
  • FIG. 14 depicts chemical modifications made to the 4-amino position on the 1,3,5- triazine motif within the core chemical structure, and the resulting cGAS inhibitory activity.
  • FIG. 15 depicts the effect of CoCl 2 on the in vitro measured cGAS inhibitory activity of compounds of this disclosure.
  • FIG. 16 shows the results of specificity testing of compound CU-32 on endosomal TLR signaling pathways in the presence of natural ligands.
  • (B) shows the results of specificity testing of compound 32 on endosomal TLR8 signaling in the presence of ssRNA ligands.
  • FIG. 8C shows a dose-response curve for compound CU-32 demonstrating that compound CU-32 does not inhibit TLR8.
  • D shows the results of a fluorescence polarization assay evaluating the intercalation of compounds CU-32 and CU-40 with dsDNA.
  • FIG. 17A-N Supplementary synthesis schemes.
  • FIG. 18 Cellular toxicity of hcGAS inhibitors in THEM cells. The toxicity was examined by treating THP-l cells with sample compound in DMSO (1% final concentration) for 16 hours. Cell survival rate was determined by measuring the intracellular ATP levels and comparing to DMSO treated cells. Cells of top analogs were non-toxic at low concentrations (0.3 and 3 mM) with only partial toxicity at 300 mM.
  • FIG. 19 cGAS inhibitors do not intercalate DNA.
  • CU-32 and selective active and inactive targets were examined for their ability to intercalate DNA.
  • the assay was performed in a final volume of 30 pL in a 384-solid bottom opaque plates.
  • Each well contained 10 pL microliters of HEN buffer (10 mM HEPES pH 7.5, 1 mM EDTA pH 7.5, 100 mM NaCl), 10 pL of a solution of 150 nM acridine orange in HEN buffer, 10 pL of a solution of 45-bp dsDNA at pgmU 1 , and 10 pL of compound.
  • Serial dilutions of compounds in DMSO were prepared using HEN buffer.
  • mP millipolarization
  • Mitoxantrone a known DNA intercalator was used at 50 mM as a positive control, while DMSO alone was used as negative control.
  • CU-32 and analogs were determined to not intercalate with DNA.
  • FIG. 20 Cellular activity of CU-l and CU-lb in THP1 cells.
  • THP1 cells were transfected with 2pg/ml of ISD in the presence of indicated concentrations of CU-l, CU-lb (structures shown in right panel), or DMSO for 3 hours.
  • Cell lysates were subjected to native PAGE and western blot using an anti-IRF3 antibody (left panel).
  • Activation of the pathway was indicated by dimerization of IRF3, shown as (IRF3) 2 .
  • the (IRF) 2 dimer was not detected at 100 mM for CU-l suggesting the compound is effective for inhibiting the cGAS-STING pathway in THP1 cells while CU-lb was inactive at 10, 30 and 100 mM in cells.
  • FIG. 21 Selective inhibition of CU-32 does not affect the cGAS/STING pathways in murine cells.
  • RAW-Dual mouse macrophage cells transfected with IRF-Luc/KI-[MIP-2]SEAP reporter genes were treated with G3YSD.
  • At 3, 10, and 30 mM CU-32 does not modulate type-l IFN transcription mediated by cGAS in RAW-Dual cells stimulated with G3-YSD.
  • the data was normalized as [(raw data - untreated cells)/(ligand + solvent control - untreated cells)].
  • Ligand + solvent is 100% activation, and untreated cells are 0% activation.
  • the result of one representative biological replicate for two independent days is plotted with the error bars representing the standard deviation of three technical replicates for one independent biological replicate.
  • FIG. 22 Effect of CU-32 on DNA and RNA nucleic acid sensing pathways.
  • HEK Human embryonic kidney
  • hTLR human toll-like receptor
  • SEAP embryonic alkaline phosphatase
  • Ligand + solvent is 100% activation, and untreated cells are 0% activation.
  • the result of one representative biological replicate for three independent days is plotted with the error bars representing the standard deviation of three technical replicates for one independent biological replicate.
  • B At 10, 25 and 50 mM CU-32 does not modulate the NF-KB inhibition induced by ssRNA-Lyo-40 in HEK 293 TLR8 cells. See above for data normalization.
  • C RAW 264.7 macrophage cells were incubated with CU-32 for 16 h. Activation of TLR7 results in the activation of NO synthase and the production of NO in RAW 264.7 cells. The NO level was monitored as an indicator of R848-induced TLR7 activation to evaluate the compound inhibitory activity.
  • FIG. 23 Dose-response curves for compound 17-26, Related to Table 1.
  • IC 50 derived from dose-response curves for the measurement of ATP consumption from hcGAS- mediated 2’,3’-cGAMPsynthesis.
  • Serial dilutions of compounds in DMSO were added to a reaction mixture containing 20mM Tris-Cl, 5mM MgCl 2 , 0.2mg/ml bovine serum albumin (BSA), O.Olmg/ml Herring testis DNA (HT-DNA), O. lmM GTP, 0.006mM ATP, and 30nM human cGAS protein, incubated at 37°C for 20 min.
  • BSA bovine serum albumin
  • HT-DNA O.Olmg/ml Herring testis DNA
  • O. lmM GTP O. lmM GTP
  • 0.006mM ATP 0.006mM ATP
  • Remaining ATP levels was measured by adding 40m1 of KinaseGlo (Promega) and reading luminescence. Reactions omitting cGAS and reactions without compounds but DMSO were considered 100% and 0% inhibition, respectively. IC50 values were deduced from non-linear fitting of [inhibitor] vs response in Prism 8. Unless otherwise noted all IC50 values represent mean.
  • FIG. 24 Molecular docking studies for CU-76 and CU-45, Related to Figure 5A and 5B.
  • A The grid box set for docking CU-76 and CU-45.
  • B Schematic representation showing that methylation of -NH 2 abrogates the H-bond interaction of CU-45 with GLU386 (left: CU-76, right CU-45).
  • FIG. 25 EMSA of recombinant human cGAS and ISD.
  • EMSA Electrophoretic mobility shift assay
  • ISD Electrophoretic mobility shift assay
  • This disclosure provides potent and selective inhibitors of cGAS and the cGAS-STING pathway and therapies for treating inflammation and autoimmune diseases associated with chronic inflammation and autoimmunity due to cGAS activation. These therapies provide therapeutic strategies for treatment of severe debilitating diseases associated with IFN-I.
  • structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention.
  • structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13C- or l4C-enriched carbon are within the scope of this invention.
  • Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention.
  • the singular forms“a”,“and”, and“the” include plural referents unless the context clearly dictates otherwise.
  • reference to“a compound” includes a plurality of such compounds
  • reference to“the method” includes reference to one or more methods, method steps, and equivalents thereof known to those skilled in the art, and so forth.
  • compositions ot“a compound of the invention includes all solvates, complexes, polymorphs, radiolabeled derivatives, tautomers, stereoisomers, and optical isomers of the compounds of the cGAS inhibitors generally described herein, and salts thereof, unless otherwise specified.
  • the term“docking” refers to orienting, rotating, translating a chemical entity in the binding pocket, domain, molecule or molecular complex or portion thereof based on distance geometry or energy. Docking may be performed by distance geometry methods that find sets of atoms of a chemical entity that match sets of sphere centers of the binding pocket, domain, molecule or molecular complex or portion thereof. See Meng et al. J. Comp. Chem. 4: 505-524 (1992). Sphere centers are generated by providing an extra radius of given length from the atoms (excluding hydrogen atoms) in the binding pocket, domain, molecule or molecular complex or portion thereof.
  • Real-time interaction energy calculations, energy minimizations or rigid-body minimizations can be performed while orienting the chemical entity to facilitate docking.
  • interactive docking experiments can be designed to follow the path of least resistance. If the user in an interactive docking experiment makes a move to increase the energy, the system will resist that move. However, if that user makes a move to decrease energy, the system will favor that move by increased responsiveness. (Cohen et al., J. Med. Chem. 33 :889-894 (1990)). Docking can also be performed by combining a Monte Carlo search technique with rapid energy evaluation using molecular affinity potentials.
  • the term“designed” “rational design” refers to an agent (i) whose structure is or was selected by the hand of man; (ii) that is produced by a process requiring the hand of man; and/or (iii) that is distinct from natural substances and other known agents.
  • the term“autoimmune disease,” refers to a disease wherein a patient's immune system is producing an unwanted immune response to one or more of their own proteins.
  • Non-limiting examples may be selected from the group consisting of: systemic lupus erythematosus (SLE), lupus nephritis (LN), rheumatoid arthritis, juvenile rheumatoid arthritis, Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus, Reynaud's syndrome, Sjorgen's disease, scleroderma, polymyositis and gl omerul onephriti s .
  • SLE systemic lupus erythematosus
  • LN lupus nephritis
  • monogenic disorder refers to a disease that is the result of a single defective gene on the autosomes.
  • Representative monogenic disorders may include rare monogenic disorders, such as Aicardi-Goutiere's Syndrome (AGS).
  • Aicardi-Goutiere's Syndrome Aicardi-Goutiere's Syndrome
  • Representative examples of autoimmune diseases include STING-Associated Vasculopathy with onset in Infancy (SAVI), and spondyloenchondrodysplasia (SPENCD).
  • a “biological sample” encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay.
  • the definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived there from and the progeny thereof.
  • the definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as polynucleotides.
  • the term“biological sample” encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, and particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it: (b) inhibiting the disease, i.e., arresting its development; (c) relieving the disease, i.e., causing regression of the disease; (d) protection from or relief of a symptom or pathology caused by cGAS activity or activation; (e) reduction, decrease, inhibition, amelioration, or prevention of onset, severity, duration, progression, frequency or probability of one or more symptoms or pathologies associated with cGAS activity or activation; and (f) prevention or inhibition of a worsening or progression of symptoms or pathologies associated with cGAS activity or activation.
  • the terms“individual,”“subject,” and“patient,” used interchangeably herein, refer to a mammal, including, but not limited to, murines, simians, humans, mammalian farm animals, mammalian sport animals, and mammalian pets.
  • the subject herein is human.
  • R-group or“substituent” refers to a single atom (for example, a halogen atom) or a group of two or more atoms that are covalently bonded to each other, which are covalently bonded to an atom or atoms in a molecule to satisfy the valency requirements of the atom or atoms of the molecule, typically in place of a hydrogen atom.
  • R-group s/substituents include alkyl groups, hydroxyl groups, alkoxy groups, acyloxy groups, mercapto groups, and aryl groups.
  • “Substituted” or“substitution” refer to replacement of a hydrogen atom of a molecule or an R-group with one or more additional R-groups such as halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-l-yl, piperazin-l-yl, nitro, sulfate, or other R-groups.
  • additional R-groups such as halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino
  • “Acyl” refers to a group having the structure RCO-, where R may be alkyl, or substituted alkyl.“Lower acyl” groups are those that contain one to six carbon atoms.
  • acyloxy refers to a group having the structure RCOO-, where R may be alkyl or substituted alkyl.“Lower acyloxy” groups contain one to six carbon atoms.
  • Alkenyl refers to a cyclic, branched or straight chain group containing only carbon and hydrogen, and unless otherwise mentioned typically contains one to twelve carbon atoms, and contains one or more double bonds that may or may not be conjugated. Alkenyl groups may be unsubstituted or substituted.“Lower alkenyl” groups contain one to six carbon atoms.
  • alkoxy refers to a straight, branched or cyclic hydrocarbon configuration and combinations thereof, including from 1 to 20 carbon atoms, preferably from 1 to 8 carbon atoms (referred to as a“lower alkoxy”), more preferably from 1 to 4 carbon atoms, that include an oxygen atom at the point of attachment.
  • An example of an“alkoxy group” is represented by the formula -OR, where R can be an alkyl group, optionally substituted with an alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, alkoxy or heterocycloalkyl group.
  • Suitable alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, sec-butoxy, tert- butoxy cyclopropoxy, cyclohexyloxy, and the like.
  • alkyl refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like.
  • A“lower alkyl” group is a saturated branched or unbranched hydrocarbon having from 1 to 6 carbon atoms. Preferred alkyl groups have 1 to 4 carbon atoms.
  • Alkyl groups may be“substituted alkyls” wherein one or more hydrogen atoms are substituted with a substituent such as halogen, cycloalkyl, alkoxy, amino, hydroxyl, aryl, alkenyl, or carboxyl.
  • a lower alkyl or (Ci-C 6 )alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl;
  • (C3-C 6 )cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl;
  • (C 3 - C 6 )cycloalkyl(C l -C 6 )alkyl can be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or 2- cyclohexylethyl;
  • (Ci-C 6 )alkoxy can be methoxy, ethoxy, propoxy
  • Alkynyl refers to a cyclic, branched or straight chain group containing only carbon and hydrogen, and unless otherwise mentioned typically contains one to twelve carbon atoms, and contains one or more triple bonds. Alkynyl groups may be unsubstituted or substituted.“Lower alkynyl” groups are those that contain one to six carbon atoms.
  • halogen refers to fluoro, bromo, chloro, and iodo substituents.
  • Aryl refers to a monovalent unsaturated aromatic carbocyclic group having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl), which can optionally be unsubstituted or substituted.
  • amino refers to an R-group having the structure -NH 2 , which can be optionally substituted with, for example, lower alkyl groups, to yield an amino group having the general structure -NHR or -NR 2 .
  • Niro refers to an R-group having the structure -N0 2 .
  • aliphatic as applied to cyclic groups refers to ring structures in which any double bonds that are present in the ring are not conjugated around the entire ring structure.
  • aromatic refers to ring structures which contain double bonds that are conjugated around the entire ring structure, possibly through a heteroatom such as an oxygen atom or a nitrogen atom.
  • Aryl groups, pyridyl groups and furan groups are examples of aromatic groups.
  • the conjugated system of an aromatic group contains a characteristic number of electrons, for example, 6 or 10 electrons that occupy the electronic orbitals making up the conjugated system, which are typically un-hybridized p-orbitals.
  • “Pharmaceutical compositions” are compositions that include an amount (for example, a unit dosage) of one or more of the disclosed compounds together with one or more non-toxic pharmaceutically acceptable additives, including carriers, diluents, and/or adjuvants, and optionally other biologically active ingredients.
  • Such pharmaceutical compositions can be prepared by standard pharmaceutical formulation techniques such as those disclosed in Remington's Pharmaceutical Sciences , Mack Publishing Co., Easton, Pa. (l9th Edition).
  • salts or esters refers to salts or esters prepared by conventional means that include salts, e.g., of inorganic and organic acids, including but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid, tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid, maleic acid, salicylic acid, benzoic acid, phenylacetic acid, mandelic acid, and the like.
  • inorganic and organic acids including but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid, tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid, maleic acid, salicylic acid, benzoic acid,
  • salts of the compounds are those wherein the counter-ion is pharmaceutically acceptable.
  • salts of acids and bases which are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.
  • the pharmaceutically acceptable acid and base addition salts as mentioned above are meant to comprise the therapeutically active non-toxic acid and base addition salt forms which the compounds can form.
  • the pharmaceutically acceptable acid addition salts can conveniently be obtained by treating the base form with such appropriate acid.
  • Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric and the like acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e.
  • salt forms can be converted into the free base form by treatment with an appropriate base.
  • the compounds containing an acidic proton may also be converted into their non-toxic metal or amine addition salt forms by treatment with appropriate organic and inorganic bases.
  • Appropriate base salt forms comprise, for example, the ammonium salts, the alkali and earth alkaline metal salts, e.g. the lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, e.g. the benzathine, N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine, and the like.
  • A“therapeutically effective amount” of the disclosed compounds is a dosage of the compound that is sufficient to achieve a desired therapeutic effect, such as promotion of cell cycle, mitotic catastrophe, promotion of apoptosis, inhibition of angiogenesis or an anti-tumor or anti-metastatic effect, inhibition of TNF-alpha activity, inhibition of immune cytokines, or treatment of a neurodegenerative disease.
  • a therapeutically effective amount is an amount sufficient to achieve tissue concentrations at the site of action that are similar to those that are shown to modulate angiogenesis, TNF-alpha activity, or immune cytokines, in tissue culture, in vitro, or in vivo.
  • a therapeutically effective amount of a compound may be such that the subject receives a dosage of about 0.1 pg/kg body weight/day to about 1000 mg/kg body weight/day, for example, a dosage of about 1 pg/kg body weight/day to about 1000 pg/kg body weight/day, such as a dosage of about 5 pg/kg body weight/day to about 500 pg/kg body weight/day.
  • stereoisomer refers to a molecule that is an enantiomer, diastereomer or geometric isomer of a molecule.
  • Stereoisomers unlike structural isomers, do not differ with respect to the number and types of atoms in the molecule's structure but with respect to the spatial arrangement of the molecule's atoms. Examples of stereoisomers include the (+) and (-) forms of optically active molecules.
  • the present invention relates to compounds of Formula (I) or a prodrug, therapeutically active metabolite, hydrate, solvate, or pharmaceutically acceptable salt thereof (hereinafter compounds of this disclosure):
  • Ri is wherein Xi and X are independently N, NH, O, or S;
  • R 2 is -NH , -NR 4 R 5 , -OR 4 , -CF 3 , -N0 2 ;
  • SR4 or R 2 is phenyl optionally substituted with halogen, -OH, -OR4, -CN, -S02R4, -C02R4, -CF3, -C(0)H, -Ci- 6 alkyl, or -NHC(0)CH 3 ;
  • X 7 is S, O, NH, or NR, and n is 0-12; each R is independently H, -OH, or -NH 2 ; or R is Ci- 6 alkyl optionally substituted with one or more of halogen, -OH, -NR 4 R 5 , or Ci -6 cycloalkyl, or R 4 is Ci- 6 cycloalkyl, or R is Ci- 6 aryl optionally substituted with one or more of halogen, -OH, -CN or -NHR 5 ; and, each R 5 is independently H, halogen, -NH 2 , -OH, -CN, -S0 2 Me, -C0 2 Me, -CF 3 , -CHO, -
  • R 5 is -Ci- 6 alkyl optionally substituted with one or more of halogen, -NH 2 , -OH, -CN; or R 5 is -Ci- 6 aryl optionally substituted with one or more of halogen, -OH, -CN, -NH 2 , -Ci -6 alkyl.
  • R 2 is phenyl optionally substituted with one or more of halogen, -OH, -CN, Ci -6 alkyl;
  • the present invention relates to compounds of Formula (II) or a prodrug, therapeutically active metabolite, hydrate, solvate, or pharmaceutically acceptable salt thereof (hereinafter compounds of this disclosure):
  • each X 8 is independently O, NH, S, or CH;
  • R 6 is -NH 2 , -OH, -NRxRc , or -NS0 2 R 8 ;
  • R 7 is halogen, -OH, -NH 2 , NR 8 Rg, -OR 8 , -CN, -CF 3 , or -N0 2 ;
  • R 8 is H, -OH, or -NH 2 ; or R 8 is Ci- 6 alkyl optionally substituted with halogen, -OH, -NR4R5, or Ci -6 cycloalkyl, or R 8 is Ci- 6 cycloalkyl, or R 8 is Ci- 6 aryl optionally substituted with halogen, -OH, -CN or -NHR 5 ; and,
  • R 9 is H, halogen, -NH 2 , -OH, -CN, -S0 2 Me, -C0 2 Me, -CF 3 , -CHO, -OMe, -SiR 3 , - C0 2 R 4 ,
  • R 9 is -Ci- 6 alkyl optionally substituted with one or more of halogen, -NH 2 , -OH, or -CN; or R 9 is -Ci- 6 aryl optionally substituted with one or more of halogen, -OH, -CN, -NH 2 , or -Ci- 6 alkyl.
  • Additional embodiments may include a compound having the chemical structure of Formula (III) or a prodrug, therapeutically active metabolite, hydrate, solvate, or pharmaceutically acceptable salt thereof:
  • compositions may include a compound having the chemical structure of Formula (IV) or a prodrug, therapeutically active metabolite, hydrate, solvate, or pharmaceutically acceptable salt thereof:
  • the invention may include the compounds 0-78 listed below, also identified as CUO-78, or cGAS 0-78.
  • cGAS 56 and cGAS 44 include hetero cyclic substituents at the 2-position which are tolerated and exhibit cGAS inhibition or modulation activity.
  • the invention may include the compound of Formula I, or a pharmaceutically acceptable salt thereof, comprising the structure selected from the group:
  • This disclosure also provides treatments of autoimmune diseases by the administration of a compound of this disclosure.
  • Compounds of this disclosure are useful for the treatment of autoimmune diseases including systemic lupus erythematosus (SLE), lupus nephritis (LN), rheumatoid arthritis, juvenile rheumatoid arthritis, Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP).
  • SLE systemic lupus erythematosus
  • LN lupus nephritis
  • ITP idiopathic thrombocytopenic purpura
  • TTP thrombotic thrombocytopenic purpura
  • autoimmune thrombocytopenia multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus, Reynaud's syndrome, Sjorgen's disease, scleroderma, polymyositis, and glomerulonephritis.
  • This disclosure also provides treatments of monogenic disorders by the administration of a compound of this disclosure.
  • Compounds of this disclosure are useful for the treatment of monogenic disorders including AGS, SAVI, or SPENCD.
  • the monogenic disorder is AGS.
  • the treatment of the autoimmune disease and/or monogenic disorder involves inhibition of cGAS activity.
  • this disclosure also provides the use of a cGAS inhibitor compound of this disclosure, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of an autoimmune disease or monogenic disorder.
  • this disclosure provides a cGAS inhibitor compound of this disclosure, or a pharmaceutically acceptable salt thereof, for use in the treatment of an autoimmune disease or monogenic disorder.
  • These methods of treatment may include the administration of a pharmaceutical composition described herein.
  • this disclosure also provides pharmaceutical compositions comprising one or more CGAS inhibitor compounds of this disclosure useful in the methods of treatment of this disclosure, these pharmaceutical compositions or formulations may include a compound of this disclosure and a pharmaceutically acceptable carrier, diluent, or excipient.
  • compositions/formulations are useful for administration to a subject, in vivo or ex vivo.
  • Pharmaceutical compositions and formulations include carriers or excipients for administration to a subject.
  • pharmaceutically acceptable and “physiologically acceptable” mean a biologically compatible formulation, gaseous, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact.
  • Such formulations include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery.
  • Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents.
  • Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules and crystals.
  • Supplementary active compounds can also be incorporated into the compositions.
  • the formulations may, for convenience, be prepared or provided as a unit dosage form. In general, formulations are prepared by uniformly and intimately associating the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • a tablet may be made by compression or molding. Compressed tablets may be prepared by compressing, in a suitable machine, an active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent.
  • Molded tablets may be produced by molding, in a suitable apparatus, a mixture of powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide a slow or controlled release of the active ingredient therein.
  • Cosolvents and adjuvants may be added to the formulation.
  • cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters.
  • Adjuvants include, for example, surfactants such as, soya lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone.
  • Supplementary active compounds e.g., preservatives, antioxidants, antimicrobial agents including biocides and biostats such as antibacterial, antiviral and antifungal agents
  • Preservatives and other additives include, for example, antimicrobials, anti-oxidants, chelating agents and inert gases (e.g., nitrogen).
  • Pharmaceutical compositions may therefore include preservatives, antimicrobial agents, anti-oxidants, chelating agents and inert gases.
  • Preservatives can be used to inhibit microbial growth or increase stability of the active ingredient thereby prolonging the shelf life of the pharmaceutical formulation.
  • Suitable preservatives include, for example, EDTA, EGTA, benzalkonium chloride or benzoic acid or benzoates, such as sodium benzoate.
  • Antioxidants include, for example, ascorbic acid, vitamin A, vitamin E, tocopherols, and similar vitamins or provitamins.
  • compositions can optionally be formulated to be compatible with a particular route of administration.
  • routes of administration include administration to a biological fluid, an immune cell (e.g., T or B cell) or tissue, mucosal cell or tissue (e.g., mouth, buccal cavity, labia, nasopharynx, esophagus, trachea, lung, stomach, small intestine, vagina, rectum, or colon), neural cell or tissue (e.g., ganglia, motor or sensory neurons) or epithelial cell or tissue (e.g., nose, fingers, ears, cornea, conjunctiva, skin or dermis).
  • an immune cell e.g., T or B cell
  • mucosal cell or tissue e.g., mouth, buccal cavity, labia, nasopharynx, esophagus, trachea, lung, stomach, small intestine, vagina, rectum, or colon
  • neural cell or tissue e.g.
  • compositions include carriers (excipients, diluents, vehicles or filling agents) suitable for administration to any cell, tissue or organ, in vivo, ex vivo (e.g., tissue or organ transplant) or in vitro, by various routes and delivery, locally, regionally or systemically.
  • Exemplary routes of administration for contact or in vivo delivery which a CGAS inhibitor can optionally be formulated include inhalation, respiration, intubation, intrapulmonary instillation, oral (buccal, sublingual, mucosal), intrapulmonary, rectal, vaginal, intrauterine, intradermal, topical, dermal, parenteral (e.g., subcutaneous, intramuscular, intravenous, intradermal, intraocular, intratracheal and epidural), intranasal, intrathecal, intraarticular, intracavity, transdermal, iontophoretic, ophthalmic, optical (e.g., corneal), intraglandular, intraorgan, and intralymphatic.
  • parenteral e.g., subcutaneous, intramuscular, intravenous, intradermal, intraocular, intratracheal and epidural
  • parenteral e.g., subcutaneous, intramuscular, intravenous, intradermal, intraocular, intratracheal and epi
  • Formulations suitable for parenteral administration include aqueous and non-aqueous solutions, suspensions or emulsions of the compound, which may include suspending agents and thickening agents, which preparations are typically sterile and can be isotonic with the blood of the intended recipient.
  • aqueous carriers include water, saline (sodium chloride solution), dextrose (e.g., Ringer's dextrose), lactated Ringer's, fructose, ethanol, animal, vegetable or synthetic oils.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose).
  • the formulations may be presented in unit-dose or multi- dose kits, for example, ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring addition of a sterile liquid carrier, for example, water for injections, prior to use.
  • penetrants can be included in the pharmaceutical composition.
  • Penetrants are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • the active ingredient can be formulated into aerosols, sprays, ointments, salves, gels, pastes, lotions, oils or creams as generally known in the art.
  • compositions typically include ointments, creams, lotions, pastes, gels, sprays, aerosols or oils.
  • Carriers which may be used include Vaseline, lanolin, polyethylene glycols, alcohols, transdermal enhancers, and combinations thereof.
  • An exemplary topical delivery system is a transdermal patch containing an active ingredient.
  • compositions include capsules, cachets, lozenges, tablets or troches, as powder or granules.
  • Oral administration formulations also include a solution or a suspension (e.g., aqueous liquid or a non-aqueous liquid; or as an oil-in- water liquid emulsion or a water-in-oil emulsion).
  • compositions can be formulated in a dry powder for delivery, such as a fine or a coarse powder having a particle size, for example, in the range of 20 to 500 microns which is administered in the manner by inhalation through the airways or nasal passage.
  • effective dry powder dosage levels typically fall in the range of about 10 to about 100 mg.
  • Appropriate formulations, wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient.
  • aerosol and spray delivery systems and devices also referred to as“aerosol generators” and“spray generators,” such as metered dose inhalers (MDI), nebulizers (ultrasonic, electronic and other nebulizers), nasal sprayers and dry powder inhalers can be used.
  • MDIs typically include an actuator, a metering valve, and a container that holds a suspension or solution, propellant, and surfactant (e.g., oleic acid, sorbitan trioleate, lecithin).
  • surfactant e.g., oleic acid, sorbitan trioleate, lecithin
  • MDIs typically use liquid propellant and typically, MDIs create droplets that are 15 to 30 microns in diameter, optimized to deliver doses of 1 microgram to 10 mg of a therapeutic.
  • Nebulizers are devices that turn medication into a fine mist inhalable by a subject through a face mask that covers the mouth and nose. Nebulizers provide small droplets and high mass output for delivery to upper and lower respiratory airways. Typically, nebulizers create droplets down to about 1 micron in diameter.
  • DPI Dry-powder inhalers
  • DPIs can be used to deliver the compounds of the invention, either alone or in combination with a pharmaceutically acceptable carrier.
  • DPIs deliver active ingredient to airways and lungs while the subject inhales through the device.
  • DPIs typically do not contain propellants or other ingredients, only medication, but may optionally include other components.
  • DPIs are typically breath-activated, but may involve air or gas pressure to assist delivery.
  • compositions can be included as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
  • a suitable base comprising, for example, cocoa butter or a salicylate.
  • pharmaceutical compositions can be included as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient a carrier, examples of appropriate carriers which are known in the art.
  • compositions and methods of the invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20.sup.th ed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) l8.sup.th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) l2.sup.th ed., Merck Publishing Group, Whitehouse, N.J.; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) l l .sup.th ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford, N.Y., pp. 253-315).
  • the CGAS inhibitors may be packaged in unit dosage forms for ease of administration and uniformity of dosage.
  • A“unit dosage form” as used herein refers to a physically discrete unit suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of compound optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filling agent) which, when administered in one or more doses, is calculated to produce a desired effect (e.g., prophylactic or therapeutic effect or benefit).
  • Unit dosage forms can contain a daily dose or unit, daily sub-dose, or an appropriate fraction thereof, of an administered compound.
  • Unit dosage forms also include, for example, capsules, troches, cachets, lozenges, tablets, ampules and vials, which may include a composition in a freeze-dried or lyophilized state; a sterile liquid carrier, for example, can be added prior to administration or delivery in vivo.
  • Unit dosage forms additionally include, for example, ampules and vials with liquid compositions disposed therein.
  • Unit dosage forms further include compounds for transdermal administration, such as“patches” that contact with the epidermis of the subject for an extended or brief period of time.
  • the individual unit dosage forms can be included in multi-dose kits or containers. Pharmaceutical formulations can be packaged in single or multiple unit dosage forms for ease of administration and uniformity of dosage.
  • the CGAS inhibitor(s) may be administered in accordance with the methods at any frequency as a single bolus or multiple dose e.g., one, two, three, four, five, or more times hourly, daily, weekly, monthly or annually or between about 1 to 10 days, weeks, months, or for as long as appropriate. Exemplary frequencies are typically from 1-7 times, 1-5 times, 1-3 times, 2-times or once, daily, weekly or monthly. Timing of contact, administration ex vivo or in vivo delivery can be dictated by the infection, reactivation, pathogenesis, symptom, pathology or adverse side effect to be treated. For example, an amount can be administered to the subject substantially contemporaneously with, or within about 1-60 minutes or hours of the onset of a symptom or adverse side effect of autoimmune diseases or inflammation, or treatment.
  • Doses may vary depending upon whether the treatment is therapeutic or prophylactic, the onset, progression, severity, frequency, duration, probability of or susceptibility of the symptom, the type of virus infection, reactivation or pathogenesis to which treatment is directed, clinical endpoint desired, previous, simultaneous or subsequent treatments, general health, age, gender or race of the subject, bioavailability, potential adverse systemic, regional or local side effects, the presence of other disorders or diseases in the subject, and other factors that will be appreciated by the skilled artisan (e.g., medical or familial history). Dose amount, frequency or duration may be increased or reduced, as indicated by the clinical outcome desired, status of the infection, reactivation, pathology or symptom, or any adverse side effects of the treatment or therapy. The skilled artisan will appreciate the factors that may influence the dosage, frequency and timing required to provide an amount sufficient or effective for providing a prophylactic or therapeutic effect or benefit.
  • the CGAS inhibitor(s) will be administered as soon as practical.
  • a CGAS inhibitor can be administered prior to, concurrently with or following administration of the subject.
  • Doses can be based upon current existing treatment protocols, empirically determined, determined using animal disease models or optionally in human clinical studies.
  • a subject may be administered in single bolus or in divided/metered doses, which can be adjusted to be more or less according to the various consideration set forth herein and known in the art.
  • Dose amount, frequency or duration may be increased or reduced, as indicated by the status of autoimmune or inflammation disease condition, reactivation or pathogenesis, associated symptom or pathology, or any adverse side effect(s).
  • control or a particular endpoint for example, reducing, decreasing, inhibiting, ameliorating or preventing onset, severity, duration, progression, frequency or probability of one or more symptoms associated with an autoimmune or inflammation disease condition, reactivation or pathogenesis of one or more symptoms or pathologies associated with or caused by an autoimmune or inflammation disease condition.
  • kits containing a pharmaceutical composition of this disclosure, prescribing information for the composition, and a container.
  • Example 1 Strategy for discovering a small molecule hcGAS inhibitor.
  • FIG. 1 A Crystallographic studies of the 2:2 mcGAS complex with DNA revealed several key residues involved in the PPI and cGAS-DNA interaction for mcGAS and are shown in FIG. 1 A. Notably, point mutations studies demonstrated that Lys335 (Lys347 in human cGAS) is involved in mediating the formation of the cGAS dimer. cGAS activity was also abolished in cGAS mutants with point mutations of both Lys335 and Lys382 (Lys 394 in humans), demonstrating their critical role for cGAS function. Furthermore, it was demonstrated hcGAS can be inhibited by aspirin mediated acetylation of either Lys384, Lys394, or Lys494 in patient cells.
  • Example 2 Identification of Novel cGAS Inhibitor Molecules by High Throughput In Silico Screening.
  • the inventors performed a high throughput virtual screen (HTVS) of drug-like libraries against the cGAS/dsDNA complex to identify novel small molecule hcGAS inhibitors.
  • HTVS high throughput virtual screen
  • An in- silico screen of the Maybridge (53,000 compounds) and Enamine (1.7 million compounds) Hit finder libraries using the Glide 5.6 program was conducted using reported co-crystal structures of recombinant mcGAS, since hcGAS-DNA complex was unknown until recently.
  • FIG. 1B based on the findings described above, the inventors generated the grid on the PPI with incorporation of the residues involved in the dsDNA binding site (crystal structure PDB ID: 406 A).
  • This in silico screen identified ten small molecules hits (see FIG. 8 and Table 2).
  • the selection of the candidate molecules was based on four criteria: (1) predicted binding energy and spatial complementarity; (2) reasonable chemical structures found in the dsDNA-binding site of cGAS; (3) existence of at least one hydrogen bond between the ligand and one of the dsDNA- recognizing residues on the cGAS surface; (4) drug-like properties analysis.
  • Drug-like properties considered by the inventors follow Lipinski’s rule of five and include properties such as molecular weight, hydrogen bond or, hydrogen bond acceptor, Lipophilicity (log P), and human oral absorption.
  • cGAS consumes 100% ATP without the inhibitors in this assay, and a titration with half-log increments is conducted with the sample compound. The concentration of the sample at 50% of ATP consumption is utilized to determine the IC50 .
  • the cytosolic DNA sensor cGAS forms an oligomeric complex with DNA and undergoes switch-like conformational changes in the activation loop.
  • IRF3 dimerization assay with human monocyte THP-l cells was used for cell-based studies. For this assay, stimulation of cGAS/STING pathway is induced with dsDNA, which causes STING to activate IKK, and TBK1. TBK1 phosphorylates STING and recruits IRF3 for phosphorylation by TBK1. The phosphorylated IRF3 dimerizes and activates the expression of type I inteferons (FIG. 9). Readout of cellular dimerization was measured by Western Blotting to detect the IRF2 dimer.
  • Example 4 Synthesis overview of the designed hcGAS inhibitor 1 and related molecules.
  • Methyl 4-amino-6-[(4-fluorophenyl)amino]- l,3,5-triazine-2-carboxylate (1) was synthesized using a reported one-pot cyclisation condensation reaction of the N'-(azaniumylmethanimidoyl)-N-(4-fluorophenyl)guanidine chloride with dimethyl oxalate (See FIG. 7A).
  • the present inventors next focused on systematically altering the 4-amino group and the heterocyclic core (see Figure 2A and 2B).
  • pyrimidine 10 lacking the 4-NH 2 group was active at 100 mM, implying a critical role for the 4-NH 2 group.
  • Replacement of the 1,3,5- triazine core with a benzene ring (11) abrogated the bioactivity, indicating an electron deficient heterocyclic core is necessary.
  • the inventors ruled out 4-aminopyridine 12 (>1000 mM) and pyrimidine 10 (>100 mM) scaffolds because inhibition was only observed at very high concentrations.
  • the methylated derivatives of 8 (CU-45) and 9 were prepared to further investigate the impact of the amine substitution.
  • Example 6 Improving CU-l potency through SAR.
  • the ethynyl group is a nonclassical bioisostere that has a polarized -CH moiety, and it is a weak hydrogen bond donor.
  • replacement of -I with the ethynyl moiety (24) did not improve the potency, which explained that halogen bonding may not be the dominating factor.
  • the inventors modified the 6-position of the l,3,5-triazine core and the 3- and 5- positions of the NH-phenylamino motif.
  • Replacement of the ester group with heterocycles, such as l,3,4-oxaziazole (27), N- pyrazole (28), and aryl- 1,2, 3 -triazoles (29 and 30) only showed modest inhibitory activity while benzimidazole (31) and indole (32) were inactive (see Table 4 for chemical structures).
  • 3,4,5-trisubstituted and 3, 5 -di substituted phenylamino rings were examined to thoroughly explore additional substitutions on the NH-phenylamino motif.
  • the inventors introduced two F- atoms at the 3- and 5-positions of the NH-phenylamino motif and a ⁇ 3-fold increase of inhibitory potency with compound CU-76 (25) was achieved showing a low micromolar IC50 (0.24 ⁇ 0.0l mM) value.
  • Selected target compounds from the SAR studies were tested in a high throughput fluorescence polarization (FP) assay for their capacity to intercalate DNA following the protocol developed for RU.521.
  • the five compounds tested showed 0% DNA intercalation compared to mitoxantrone, a known DNA intercalator. (see Figure 19).
  • the biological investigation for CU- 32 and CU-76 was prioritized based on stability, potency, and lack of DNA interaction for further testing in cellular assays.
  • Example 7 CU-32 analogs selectively inhibit cGAS pathway in human cells.
  • the inventors used ELISA to measure IFN-b production from these cells following ISD transfection or Sendai virus infection.
  • CU-32 and CU-76 suppressed levels of IFN-b in the media dose-dependently; however, IFN-b levels I response to Sendai virus were not affected ( Figure 4B), confirming the effectiveness and specificity of these compounds.
  • the inventors also confirmed the inhibitory activity of CU-32 and analogs was not the result of toxicity, as the top inhibitors had no effect on cell viability up to 30 mM, with only partial toxicity at 300 mM ( Figure 18).
  • the carboxylic acid derivatives of CU-1 and CU-32 were also prepared and determined to have an in vitro IC 50 value of 3.8F1.9 mM (lb) and 0.59 ⁇ 0.3 mM (19b), see Figure 4A. for chemical structures.
  • Utilizing a prodrug strategy to optimize effectiveness and“drug like” properties, such as permeability and target selectivity, is a possibility based on the in vitro activities for the carboxylic acid derivatives of lb and 19b.
  • lb did not display antagonistic activity toward the cGAS-STING pathway IRF dimerization assay (See Figure 20).
  • the present inventors cannot effectively conclude the active drug is the carboxylic acid of the corresponding methyl ester inhibitor (1) since two amide derivatives also inhibited hcGAS in vitro and lacked cellular activity.
  • the inventors speculate the carboxylic acid (lb) and amides (15 and 16) lack cellular activity due to poor permeability caused by the 4-NH 2 and 2-COOH functional groups.
  • Example 8 CU-32 does not inhibit Toll-like receptor pathways.
  • TLR pathways which are membrane localized pathogen recognition receptors of the innate immune system.
  • Various TLRs recognize different viral or bacterial membrane components or nucleic acids.
  • the present inventors used human embryonic kidney (HEK) cell lines each ectopically expressing a TLR together with NF-KB-inducible SEAP (secreted embryonic alkaline phosphatase).
  • HEK human embryonic kidney
  • TLR ligands including poly(TC) for TLR3, LPS for TLR4, R848 and ssRNA for TLR7/8, and CpG-ODN for TLR9, in the presence of CU-32 or DMSO, and activation of TLR signaling was evaluated by measurement of SEAP activity in the media.
  • TLR ligands including poly(TC) for TLR3, LPS for TLR4, R848 and ssRNA for TLR7/8, and CpG-ODN for TLR9
  • the inventors speculate the insertion of the inhibitor molecules aside the Zn loop disturbs the interface of the dimer, thus inhibiting the dimerization through an allosteric effect (conformational change).
  • the present inventors also hypothesize the 4-NH 2 (donor) may have an H-bond interaction with GLET386, Figure 6A.
  • CU-45 cannot interact with GLET-386 mainly because there is not a H (donor) on the N-atom and due to steric clash with the methyl groups, see Figure 6B.
  • the in vitro results for CU-45 and CU-9 (0% cGAS inhibition) are consistent with our hypothesis.
  • the molecular docking studies also indicate CU-76 and analogs may bind to different pocket compared to other cGAS inhibitors.
  • cGAS protein caused mobility shift of ISD (lane2) and reduction of the unbound DNA. This effect was reversed by adding Quinacrine (lane 2-4), a compound known to disrupt cGAS:DNA binding (cite PMID: 25821216), see Figure 25.
  • Quinacrine lane 2-4
  • CU-76 lane 9-11
  • CU-32 and CU-76 do not disrupt cGAS:DNA intercalation.
  • Example 10 Synthesis and Structure Activity Relationship (SAR) of Inhibitors Targeting cGAS.
  • ester functionality was identified as a metabolically unstable group, as it can be hydrolyzed by esterases. This presents the possibility of utilizing a prodrug strategy to optimize effectiveness and“drug like” properties such as permeability and target selectivity.
  • a prodrug strategy to optimize effectiveness and“drug like” properties such as permeability and target selectivity.
  • a >20-fold increase in potency was observed with CU-2 in vitro.
  • the lower in vitro activity of CU-0 could be due to poor permeability caused by the additional free amino group on the hydrophilic anthranilic acid motif.
  • ester and amide functionalities were examined and the ester group was identified as the most active functional group (FIG. 10B).
  • Cellular assays revealed that compounds CU-l and CU-6 prevented IRF3 dimerization, suggesting cGAS inhibition occurs.
  • the inventors rationalized that the methyl ester of compounds CU-l and CU-6 could be metabolized to the pharmacologically active carboxylic acid derivatives, and that CU-9 was inactive because the amide cannot be easily hydrolyzed.
  • the carboxylic acid in compound CU-l 7, and the alcohol in compound CU-2 did not inhibit cGAS in the cell-based assay further supporting a prodrug hypothesis and the importance of the ester functionality.
  • the core chemical scaffold including the 1,2, 3-triazole motif provided promising SAR results and therefore the inventors undertook additional SAR studies to identify substituents that further enhance the in vitro cGAS inhibitory activity of these compounds (FIGS. 12B and 12C).
  • Example 12 Optimization of the Hydrophobic Motif.
  • the inventors undertook additional SAR testing of N-aryl derivatives and heterocyclic motifs to identify substituents that further enhance the in vitro cGAS inhibitory activity of these compounds (FIGS. 11 and 13).
  • the inventors undertook additional SAR testing of primary, secondary, and tertiary amine derivatives of the 4-amino position in the l,3,5-triazine core (FIGS. 11 and 14). The results indicate that the amine group is necessary for inhibitory activity.
  • Example 13 Effects of CoCl2 on Bioactivitv of cGAS Inhibitor Analogs.
  • cGAS activity increases in the presence of CoCl 2 (or ZnCl 2 ) because the Zn-binding domain facilitates dsDNA recognition.
  • the inventors rationalized that differences in the in vitro and cellular results could be attributed to the metal ion.
  • Co2+ and Zn2+ complexes with triazine- based ligands are known, and the analogs have potential to bind to M2+.
  • the inventors examined compound toxicity by treating THP-l cells with sample compounds in DMSO (1% final concentration) for 16 hours. Measuring intracellular ATP levels and comparing to DMSO treated cells determined the cell survival rate. The inventors’ top analogs were nontoxic at low concentrations (0.3 and 3mM) with partial toxicity at 300 mM.
  • the inventors used compound CU-32 to evaluate whether the inhibitors of cGAS-STING affect other innate immune signaling pathways beyond dsDNA. Endosomal Toll-like signaling (TLR3, 7, 8, and 9) pathways are also major nucleic acid sensing pathways for dsRNA, ssRNA, and CpG methylated DNA.
  • TLR3, 7, 8, and 9 Endosomal Toll-like signaling pathways are also major nucleic acid sensing pathways for dsRNA, ssRNA, and CpG methylated DNA.
  • the inventors therefore used a human embryonic kidney cell (Hek)-Blue TLR cell-based assay to evaluate the specificity of the signaling inhibition of the inhibitors of this disclosure. Briefly, Hek 293 cells were transfected with the appropriate hTLR gene and an inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene was used to evaluate compound potency and TLR specificity.
  • SEAP inducible secreted embryonic alkaline
  • the SEAP reporter gene was fused to five NF-kB and AP-l sites. Stimulation of the hTLR was induced with a natural ligand or small molecule chemical ligand (i.e., Poly(TC), R848, ssRNA-Lyo40, ORN-06, or ODN-2006) (ssRNA-Lyo40 and ORN-06 are GU-rich oligonucleotide complexed with LyoVec (Invivogen)). This activates NF-kb and AP-l, which induces the production of SEAP protein.
  • a natural ligand or small ligand i.e., Poly(TC), R848, ssRNA-Lyo40, ORN-06, or ODN-2006
  • ssRNA-Lyo40 and ORN-06 are GU-rich oligonucleotide complexed with LyoVec (Invivogen)
  • TLR ligand i.e., 1 pg/mL R848, 5 pg/mL ssRNA/LyoVec, 5 pg/mL ORN-06, or 1 pg/mL ODN- 2006.
  • the cells were incubated for 18-20 hours and assayed for NF-kB signaling using a SEAP assay.
  • Quanit-Blue (Invivogen) medium for quantification of alkaline phosphatase was used to monitor expression of SEAP via detection of SEAP reporter protein secreted by cells. The compounds were considered active if they decreased SEAP levels by a decrease in absorbance at 620 nm.
  • FIG. 16A the present inventors demonstrate that compound CU-32 was unable to suppress activation of endosomal TLR signaling pathways in the presence of natural ligands.
  • FIG. 16B demonstrates that CU-32 was unable to suppress activation of endosomal TLR8 signaling in the presence of ssRNA ligands.
  • FIG. 16C shows a dose-response curve for compound CU-32 demonstrating that compound CU-32 does not inhibit TLR8 stimulated with ssRNA-Lyo-40 (compound ZH-9a is a control).
  • Fluorescence polarization is a powerful approach by which alterations in the apparent molecular weight of a fluorescent probe (or tracer) in solution are indicated by changes in the polarization of the sample’s emitted light.
  • the inventors used the FP assay to interrogate the molecular interaction between compounds of this disclosure and dsDNA using acridine orange, an organic compound used as a nucleic acid- selective fluorescent cationic dye that is cell permeable and interacts with DNA and RNA by intercalation or electrostatic interactions.
  • FIG. 16D shows the results of FP assay evaluating the intercalation of compounds CU-32 and CU-40 with dsDNA. Mitoxantrone, a known DNA-intercalating agent, was included as a positive control. Compounds CU-32 and CU-40 do not intercalate with dsDNA.
  • High throughput virtual screening was performed against the cGAS/dsDNA complex structure.
  • the Enamine drug database (1.3 million small molecules) and Maybridge library (50,000 small molecules) was docked into the dsDNA-binding domain of cGAS (PDB: 406A).
  • Glide maestro protocol was used for the virtual screening using Schrodinger software.
  • the grid was generated on the protein-protein interface with incorporation of important residues involved in dsDNA binding, See Figure 1.
  • the protocol includes addition of hydrogens, restrained energy-minimizations of the protein structure with the Optimized Potentials for Liquid Simulations-All Atom (OPLS-AA) force field, and finally setting up the Glide grids using the Protein and Ligand Preparation Module. All compounds were first docked and ranked using High Throughput Virtual Screening Glide, continued with standard precision docking (SP) Glide for the top 10,000 compounds. To reduce the number of compounds in the library, after performing HTVS screening, the remaining 10% was docked using the more accurate and computationally intensive SP docking, after which the remaining 10% was docked using Extra-precision. The top ranked compounds were re- ranked by predicted binding energy. The compounds were filtered by Lipkinski’s rule of five and reactive functionality. It performed docking of the drug compounds in the different phases like HTVS, SP, XP (Extra-precision).
  • Selection of the candidate molecules was based on four criteria: (1) predicted binding energy and spatial complementarity; (2) reasonable chemical structures found in the dsDNA- binding site of cGAS; (3) existence of at least one hydrogen bond between the ligand and one of the dsDNA-recognizing residues on the cGAS surface; (4) drug-like properties analysis.
  • Drug- like properties follow Lipinski’s rule of five and include properties such as molecular weight, hydrogen bond or, hydrogen bond acceptor, Lipophilicity (log P), and human oral absorption. Ten of these molecules were selected by chemical and geometrical properties for experimental evaluation (Table 1).
  • HEK human embryonic kidney
  • SEAP embryonic alkaline phosphatase reporter gene
  • the SEAP reporter gene is fused to five NF-kB and AP-l sites. Stimulation of the hTLR is induced with natural ligand or small molecule chemical ligand (R848, Invivogen). This activates NF-kb and AP-l, which induces the production of SEAP protein.
  • Growth media for cell maintenance was prepared using DMEM media with 10% FBS, 1% L-glutamine, 1% Penicillin/Streptomycin and supplemental antibiotics (10 pg/mL blasticidin and 100 pg/mL zeocin) per manufacture’s recommendations.
  • ETn-supplemented test media was prepared using DMEM media with 10% FBS (deactivated), 1% L-glutamine, and 1% Penicillin/Streptomycin (Note: supplemental antibiotics were not added). 100,000 cells/well or 70,000 cells/well were plated in a tissue culture treated 96-well (Costar 3596) in un-supplemented DMEM test media. Cells were then treated with appropriate concentration of compound, natural TLR ligand (5 pg/mL Poly(LC), 20 ng LPS, 1 pg/mL R848, 1 pg/mL CpG-ODN, or ssRNA/LyoVec, (Invivogen).
  • natural TLR ligand 5 pg/mL Poly(LC), 20 ng LPS, 1 pg/mL R848, 1 pg/mL CpG-ODN, or ssRNA/LyoVec, (Invivogen).
  • Quanti-Blue (Invivogen) medium for quantification of alkaline phosphatase was used to monitor expression of SEAP via detection of SEAP reporter protein secreted by cells.
  • the compounds were considered active if they decreased SEAP levels as indicated by a decrease in absorbance at 620 nm.
  • the data was normalized as [(raw data - untreated cells)/(ligand + solvent control - untreated cells)].
  • Ligand + solvent is 100% activation, and untreated cells are 0% activation.
  • the result of one representative biological replicate for three independent days is plotted with the error bars representing the standard deviation of three technical replicates for one independent biological replicate.
  • the result of one representative biological replicate for three independent days is plotted with the error bars representing the standard deviation of three technical replicates for one independent biological replicate.
  • Raw 264.7 cells were plated on day one at 375,000 cells/mL in a tissue culture treated 96- well plate.
  • the cells were plated in supplemented RPMI medium (10% fetal bovine serum, 1% L-glutamine, 1% Penicillin/Streptomycin) and incubated at 37 °C.
  • supplemented media was removed from the cells, and the unsupplemented RPMI was added (100 pL).
  • the cells were treated with 1 pg/mL R848 (90 pL) (Invivogen) and varying concentrations of the appropriate organic compound (10 pL). The final volume in each well was 200 pL.
  • the 96-well plate was incubated with the organic compound for 18-24 hours at 37 °C.
  • the NO assay uses an aryldiazonium intermediate to convert 2,3-diaminonapthalene to fluorescent l(H)-naphthotriazole in the presence of NO. As NO is produced in the TLR inflammatory response, this readout provides information on the extent of TLR signaling.
  • [macrophage inflammatory protein-2 (MIP-2)]- secreted embryonic alkaline phosphatase (SEAP) reporter genes and an inducible Lucia luciferase gene (Luc) were used to evaluate compound potency for murine macrophages.
  • the Lucia luciferase gene is under the control of an ISG54 minimal promoter with IFN-stimulated response elements. Stimulation of cGAS was induced with G3YSD, a cGAS agonist (Invivogen). This activates the IRF pathway, which induces the production of the Luciferase protein.
  • Growth media for cell maintenance was prepared using DMEM media with 10 % FBS 1% L-glutamine, 1% Penicillin/Streptomycin and supplemental antibiotics (100 pg/mL normocin and 200 pg/mL zeocin) per manufacture’s recommendations to select for cGAS and IRF-Lucia/KI-[MIP-2]SEAP reporter expression.
  • ETn-supplemented test media was prepared using DMEM media with 10% FBS (heat deactivated), 1% L-glutamine, and 1% Penicillin/Streptomycin (Note: supplemental antibiotics were not added). 100,000 cells/well were plated in a tissue culture treated 96-well (Costar 3596) in un-supplemented DMEM test media. Cells were then treated with appropriate concentration of compound, and 1 pg/mL G3YSD ligand. The cells were incubated for 18-20 hours and assayed for IRF signaling using a Lucia luciferase assay.
  • Quanit-Luc (Invivogen) medium for quantification of luciferase was used to monitor the expression of luciferase via detection of Lucia luciferase reporter protein secreted by cells.
  • the compounds were considered active if they decreased luciferase levels as indicated by a decrease in luminescence relative light units (RLU).
  • RLU luminescence relative light units
  • the data was normalized with 100% untreated cells as the negative control and 100% cells treated with cGAS ligand (1 pg/mL G3YSD) as the positive control. All data for cell-based assays is represented as the average and standard deviation of three biological replicates, unless otherwise noted.
  • the crystal structure of murine cGAS (mcGAS) -DNA(2:2) complex (PDB: 406A) was used. Similar to the approach described above for the HTVS, we processed 406 A prior to docking and only the monomer mcGAS was retained. The DNA and water molecules were removed. The compounds were first optimized using the GaussView v.5.0.9 and Gaussion v.9.5(Method: b3lyp, Basic set: 6-3 l+g(d,p), pseudo potential for I: sdd). We prepared the ligands and protein receptor with AutoDockTools-l .5.6 (added hydrogens and gaslessnesser charges, set rotatable bonds for ligands etc.).
  • the GridBox was generated at the cGAS-cGAS interface, Zn loop(K382 E386) and a7 helix(K335) involved (Figure S8A.).
  • the docking parameters were all set to default (Number of Genetic Algorithm Runs: 50).
  • the protein structure was set to be rigid.
  • redocking was conducted with select residues (Lys335, Lys382, Glu386) in the GridBox being flexible.
  • CU-76 was overlapped with mcGAS- DNA(2:2) complex together (Figure S8B.).
  • Mass spectrometry was performed at the mass- spectrometry facility of the Biofrontiers Institute at the University of Colorado Boulder. High resolution mass spectra were obtained using a Waters Synapt G2 QToF HR-MS using an ESI ionization mode. Infrared spectra are reported in cm-l and recorded using a Agilent Cary 630 FT/IR instrument and opitcal rotations were measured on JASCO P-1030 and are reported as an average of data points.
  • the arylbiguanide salt (1.0 mmol) in anhydrous ethanol or methanol (4.4 M) was added to a mixture of sodium ethoxide (1.2 mmol) in anhydrous ethanol (0.34 M). After stirring the solution for 3 h at rt, the mixture was filtered through a pad of celite. The filtrate was concentrated by rotary evaporation. The residue was dissolved in hot ethanol and filtered through a pad of celite. The filtrate was concentrated by rotary evaporation to afford the desired arylbiguanide base and was used without further purification.
  • Methyl 4-amino-6-((4-fluorophenyl)amino)-l,3,5-triazine-2-carboxylate Following the general free base procedure C, a mixture of l-carbamimidamido-N-(4- fluorophenyl)methanimidamide hydrochloride (400 mg, 1.73 mmol) and NaOEt (118 mg, 1.73 mmol) was stirred in EtOH (0.34 M) at rt for 3 h.
  • N2-(4-Fluorophenyl)-6-(methoxymethyl)-l, 3, 5-triazine-2, 4-diamine The general procedure was followed using l-carbamimidamido-N-(4-fluorophenyl)methanimidamide hydrochloride (1.83 g, 7.9 mmol), NaOMe (513 mg, 9.5 mmol), and in anhydrous MeOH (23 mL). The corresponding arylbiguanide base, ethylmethoxyacetate (0.95 mL, 8.06 mmol), and 15 mL MeOH were added and then heated at reflux for 24 h. The reaction mixture was cooled to rt and concentrated to afford a white solid.
  • 6-(Aminomethyl)-N2-(4-fluorophenyl)-l, 3, 5-triazine-2, 4-diamine Following the general procedure A using 4-amino-6-((4-fluorophenyl)amino)-l,3,5-triazine-2-carboxamide (240 mg, 0.967 mmol) and L1AIH 4 (110 mg, 2.90 mmol) in anhydrous THF (0.1 M). the reaction was stirred at rt and monitored by TLC. After 6.5 h, the reaction was quenched using the Feiser protocol to afford a yellow residue.
  • Fluorophenyl)amino]-6-(methylamino)-l,3,5-triazine-2-carbonitrile (0.614 mmol) was weighed into a flame dried around bottom flask and dissolved with anhydrous MeOH (0.25 M). Then freshly distilled BF 3 OEt 2 (4.91 mmol) was added and refluxed. After 12 h, the mixture was cooled to rt and diluted with H 2 0 (2 mL). The mixture was extracted with CH 2 Cl 2 (3 x 5 mL). The organic mixture was dried with Na 2 S0 4 , filtered, and concentrated by rotary evaporation to afford a solid.
  • 6-((4-Fluorophenyl)amino)pyrimidine-4-carboxylic acid General pyrimidine synthesis procedure was followed using pyrimidine-4-carboxylate (209 mg, 1.21 mmol) and 4- fluoroaniline (0.116 mL, 1.21 mmol) in 2-propanol (2.0 mL, 0.58 M) and 37% HC1 (2.18 mmol, 0.214 mL). The reaction was stirred at 100 °C for 19 h. The product hydrolyzed quantitatively to the corresponding carboxylic acid. Purification by column chromatography (eluent 10% MeOH:CH 2 Cl2) provided 5 (157 mg, 92% pure) in 52% yield as a yellow solid: m.p.
  • Methyl 3-amino-5-((4-fluorophenyl)amino)benzoate A mixture of methyl 3-((4- fluorophenyl)amino)-5-nitrobenzoate (0.517 mmol) and 10 mol% Pd/C in 3.7 mL anhydrous methanol was stirred at rt under 1.1 atm of 3 ⁇ 4 for 12 h. The reaction progress was monitored by TLC using 5% MeOH:CH 2 Cl2. After 12 h, the catalyst was removed by filtration using Celite® and methanol. The filtrate was concentrated by rotary evaporation to afford a brown solid.
  • Methyl 4-amino-6-((4-fluorophenyl)amino)picolinate A mixture of methyl 6-((4- fluorophenyl)amino)-4-nitropicolinate (0.0549 mmol) and 20 mol% Pd/C (0.01098 mmol) in anhydrous methanol (0.8 mL) was stirred at rt under 1.1 atm 3 ⁇ 4 pressure. TLC was ued to monitor rhe reaction progress. (5% MeOH:CH 2 Cl 2 ). After 2 h, the catalyst was removed by filtration using Celite ® . The solid residue was washed with methanol and the filtrate was concentrate by rotary evaporation to afford a purple residue.
  • Methyl 4-amino-6-((4-fluorophenyl)(methyl)amino)-l,3,5-triazine-2-carboxylate Following the general free base procedure C, a mixture of aryl biguanide salt (2.5 g, 10.0 mmol) and NaOEt (885 mg, 13.0 mmol) was stirred in EtOH (0.3 M) at rt for 3 h. Following the general procedure D, a mixture of dimethyloxalate (3.5 g, 30.0 mmol) and the arylbiguanide base in anhydrous MeOH (0.27 M) was stirred at 25 °C for lh and then refluxed overnight.
  • aryl biguanide salt 2.5 g, 10.0 mmol
  • NaOEt 885 mg, 13.0 mmol
  • Methyl 4-amino-6-(phenylamino)-l,3,5-triazine-2-carboxylate Following the general free base procedure C, a mixture of l-carbamimidamido-N-phenylmethanimidamide hydrochloride (1.0 g, 4.681 mmol) and NaOEt (318 mg, 4.68 mmol) was stirred in EtOH (0.34 M) at rt for 3 h. Following the general procedure D, a mixture of dimethyloxalate (1.6 g, 14.0 mmol) and the arylbiguanide base in anhydrous MeOH (0.27 M) was stirred at 25 °C for 3 h and then refluxed overnight.
  • Methyl 4-amino-6-((4-iodophenyl)amino)-l,3,5-triazine-2-carboxylate The general procedure C was followed using l-carbamimidamido-N-(4-iodophenyl)methanimidamide hydrochloride (4.0 g, 11.8 mmol), sodium methoxide (829 mg, 15.34 mmol), and dimethyloxalate (4.2 g, 35.4 mmol) in anhydrous MeOH (35 mL and 20 mL, respectively) at rt to reflux for 24 h.
  • Methyl 4-amino-6-((3-iodophenyl)amino)-l,3,5-triazine-2-carboxylate The general procedure C was followed using l-carbamimidamido-N-(3-iodophenyl)methanimidamide hydrochloride (300 mg, 0.883 mmol), NaOMe (62.2 mg, 1.15 mmol), and 2.6 mL in anhydrous MeOH. Following the general procedure D, the corresponding arylbiguanide base, dimethyl oxalate (313 mg, 2.65 mmol) 5.3 mL MeOH at reflux for 12 h. The reaction mixture was cooled to rt and concentrated to afford a grey-purple solid.
  • Methyl 4-amino-6-((2-iodophenyl)amino)-l,3,5-triazine-2-carboxylate The general procedure C was followed using l-carbamimidamido-N-(2-iodophenyl)methanimidamide hydrochloride (400 mg, 1.08 mmol), NaOMe (75.7 mg, 1.40 mmol), and 2.7 mL in anhydrous MeOH. The corresponding arylbiguanide base, dimethyl oxalate (383 mg, 3.24 mmol) 1.8 mL MeOH at reflux 18 h. The reaction mixture was cooled to rt.
  • Methyl 4-amino-6-((4-hydroxyphenyl)amino)-l,3,5-triazine-2-carboxylate The general procedure C was followed using l-carbamimidamido-N-(4-hydroxyphenyl)methanimidamide hydrochloride (700 mg, 3.05 mmol), NaOMe (200 mg, 3.7 mmol), and 7.6 mL in anhydrous MeOH. The corresponding arylbiguanide base, dimethyl oxalate (1.1 g, 9.2 mmol) 10 mL MeOH at reflux 12 h.
  • Methyl 4-amino-6-((4-methoxyphenyl)amino)-l,3,5-triazine-2-carboxylate Following the general free base procedure C a l-carbamimidamido-N-(4-methoxyphenyl)methanimidamide hy drochl on de Error! Bookmark not defme(1 ⁇ (200 mg, 0.821 mmol) and NaOEt (67 mg, 0.985 mmol) was stirred in EtOH (0.34 M) at rt for 3 h.
  • Methyl 4-amino-6-((4-ethynylphenyl)amino)-l,3,5-triazine-2-carboxylate To a dry THF solution of Methyl 4-amino-6-((4-((trimethylsilyl)ethynyl)phenyl)amino)-l,3,5-triazine-2- carboxylate (45 mg, 0.132 mmol) was added a solution of tetrabutylammonium fluoride (45 mg, 0.172 mmol) dropwise at 25 °C under N 2 . The mixture was stirred at rt and monitored by TLC.
  • Methyl 4-amino-6-((3,4,5-trifluorophenyl)amino)-l,3,5-triazine-2-carboxylate The general procedure B was followed using l-carbamimidamido-N-(3,4,5-trifluorophenyl)methanimidamide hydrochloride (400 mg, 1.49 mmol), NaOMe (97 mg, 1.79 mmol), and 3.7 mL in anhydrous MeOH. The corresponding arylbiguanide base, dimethyl oxalate (531 mg, 4.5 mmol) 5.0 mL MeOH at reflux 12 h.
  • Methyl 4-amino-6-[(3,5-difluoro-4-iodophenyl)amino]-l,3,5-triazine-2-carboxylate The general procedure C was followed using l-carbamimidamido-N-(3,5-difluoro-4- iodophenyl)methanimidamide hydrochloride (65 mg, 0.173 mmol), NaOMe (28 mg, 0.519 mmol), and 0.340 mL in anhydrous MeOH. The corresponding arylbiguanide base, dimethyl oxalate (20.4 mg, 0.173 mmol) 0.340 mL MeOH at reflux for 12 h.
  • N-2-(4-Iodophenyl)-6-(l, 3, 4-oxadiazol-2-yl)-l, 3, 5-triazine-2, 4-diamine Triethyl orthofomate (0.216 mL) was added to a mixture of FeCl 3 (1.8 mg, 0.011 mmol), L-proline (1.2 mg, 0.011 mmol) and Et 3 N (3 pL, 0.022 mmol), and the resulting solution was stirred for 1 h at rt. The hydrazide substrate was added, and the mixture was stirred at 80 °C for 12 h. After cooling, the reaction mixture was washed with Et 2 0 (3 x 0.5 mL).
  • N 2 -(4-Iodophenyl)-6-(lH-pyrazol-l-yl)-l, 3, 5-triazine-2, 4-diamine Pyrazole (22 mg, 0.317 mmol), 6-chloro-N 2 -(4-iodophenyl)-l, 3, 5-triazine-2, 4-diamine (100 mg, 0.288 mmol), and K 2 C0 3 (44 mg, 0.318 mmol) were weighed into a microwave vial with a stir bar.
  • 6-Azido-N 2 -(4-iodophenyl)-l, 3, 5-triazine-2, 4-diamine (50 mg, 0.141 mmol) and copper iodide (8.1 mg, 0.042 mmol) were in anhydrous DMSO (0.214 mL) under N 2 . After 15 min, 2-ethynyl aniline (48 pL, 0.423 mmol) was added to the mixture and stirred at rt for 2 h. The reaction progress was monitored using TLC until completion. The reaction mixture was diluted with 10% NH 3 aq (1 mL), and the mixture was extracted with EtOAc (3 x 2 mL).
  • 6-(lH-Indol-l-yl)-N 2 -(4-iodophenyl)-l, 3, 5-triazine-2, 4-diamine Indole (33.7 mg, 0.288 mmol), 6-chloro-N 2 -(4-iodophenyl)-l, 3, 5-triazine-2, 4-diamine (100 mg, 0.288 mmol), and K 2 C0 3 (51.7 mg, 0.374 mmol) were weighed into a microwave vial with a stir bar. Anhydrous DMSO (1.0 mL) was added to the sealed vial. The reaction mixture was stirred at 120 °C overnight.
  • the reaction mixture was diluted with EtOAc (5 mL) and 5% aq LiCl (5 x 5 mL). The organic mixture was separated, and the aqueous mixture was extracted with EtOAc (3 x 5 mL). The combined organic mixtures were washed with 5% aq LiCl (5 x 5 mL). The organic mixture was dried with Na 2 S0 4 , filtered, and concentrated by rotary evaporation to afford a light brown residue. The residue was purified by flash column chromatography (1% MeOH:CH 2 Cl2) to afford a white solid (35.3 mg, 99% pure) in 29% yield: m.p.
  • Methyl 4-amino-6-((4-chlorophenyl)amino)-l,3,5-triazine-2-carboxylate Following the general free base procedure C, a mixture of l-carbamimidamido-N-(4- chlorophenyl)methanimidamide hydrochloride (8.9 g, 35.8 mmol) and NaOEt ( 2.4 mg, 35.8 mmol) was stirred in MeOH (70 mL) at rt for 22 h.
  • Methyl 4-amino-6-((4-bromophenyl)amino)-l,3,5-triazine-2-carboxylate Following the general free base procedure C, a mixture of N-(4-bromophenyl)-l- carbamimidamidomethanimidamide hydrochloride Eriw! Bookmark not defmed ⁇ (12.4 g, 42.5 mmol) and NaOEt ( 2.9 g, 42.5 mmol) was stirred in EtOH (70 mL) at rt for 22 h.
  • Methyl 4-amino-6-((4-nitrophenyl)amino)-l,3,5-triazine-2-carboxylate Following the general free base procedure C a mixture of l-carbamimidamido-N-(4- nitrophenyl)methanimidamide hydrochloride (15.8 g, 61.1 mmol) and NaOEt ( 4.2 g, 61.1 mmol) was stirred in EtOH (100 mL) at rt for 22 h.
  • Methyl 4-amino-6-((4-cyanophenyl)amino)-l,3,5-triazine-2-carboxylate The general procedure C was followed using l-carbamimidamido-N-(4-cyanophenyl)methanimidamide (496 mg, 2.08 mmol), NaOMe (145.9 mg, 2.70 mmol), and 6.9 mL in anhydrous MeOH. Following the general procedure D, a mixture of the corresponding arylbiguanide base, dimethyl oxalate (737 mg, 6.24 mmol) 4.0 mL MeOH was stirred at 35 °C for 0.5 h and then refluxed for 22 h.
  • Methyl 4-amino-6-((4-iodophenyl)amino)-l,3,5-triazine-2-carboxylate 75 mg, 0.202 mmol
  • PdCl2(PPh 3 )2 76 mg, 0.108 mmol
  • Cul 10 mg, 0.054 mmol
  • the flask was sealed and placed under vacuum then backfilled with N2.
  • trimethyl silyl acetylene (0.157 mL, 0.592 mmol), Et 3 N (0.524 mL, 3.77 mmol), and THF (1.8 mL) were added, and the mixture was stirred at rt overnight.
  • Methyl 4-amino-6-((4-iodophenyl)amino)-l,3,5-triazine-2-carboxylate 50 mg, 0.135 mmol
  • PdCl2(PPh 3 ) 2 (19 mg, 0.027 mmol)
  • Cul 2.6 mg, 0.014 mmol
  • the flask was sealed and placed under vacuum then backfilled with N2.
  • ethynylcyclopropane (12.6 pL, 0.149 mmol), Et 3 N (0.131 mL, 0.945 mmol), and THF (0.43 mL) were added, and the mixture was stirred at rt overnight.
  • Methyl 4-amino-6-[(6-iodopyridin-3-yl)amino]-l,3,5-triazine-2-carboxylate The general procedure C was followed using l-Carbamimidamido-N-(6-iodopyridin-3-yl)methanimidamide hydrochloride (54) (787 mg, 2.31 mmol), NaOMe (374 mg, 6.93 mmol), and 4.6 mL in anhydrous MeOH.
  • Methyl 4-amino-6-(quinolin-6-ylamino)-l,3,5-triazine-2-carboxylate The general procedure C was followed using l-carbamimidamido-N-(quinolin-6-yl)methanimidamide hydrochloride (250 mg, 0.944 mmol), NaOMe (153 mg, 2.83 mmol), and 2.7 mL in anhydrous MeOH. The corresponding arylbiguanide base and dimethyl oxalate (3.34 mg, 2.83 mmol) in anhydrous 3.5 mL MeOH at reflux for 22 h. The reaction mixture was cooled to rt.
  • N-[2-(l- ⁇ 4-amino-6-[(4-iodophenyl)amino]-l,3,5-triazin-2-yl ⁇ -lH-l,2,3-triazol-4- yl)phenyl]methanesulfonamide 6-Azido-N 2 -(4-iodophenyl)-l, 3, 5-triazine-2, 4-diamine (37 mg, 0.104 mmol), Et 3 NH (14.5 pL, 0.104 mmol) and copper iodide (5.9 mg, 0.031 mmol) were in anhydrous DMSO (0.416 mL) under N 2.
  • N-(2- N-(2- ethynylphenyl)methanesulfonamide (20.3 mg, 0.104 mmol) was added to the mixture and stirred at rt for 2 h.
  • the reaction progress was monitored using TLC until completion.
  • the reaction mixture was diluted with 10% NH 3 aq (1 mL), and the mixture was extracted with EtOAc (3 x 2 mL). The combined organic mixture was washed several times with H 2 0 and then dried with Na 2 S0 4 , filtered, and concentrated by rotary evaporation to afford a brown residue.
  • 6-Azido-N 2 -(4-iodophenyl)-l, 3, 5-triazine-2, 4-diamine (50 mg, 0.141 mmol) and copper iodide (8.1 mg, 0.0423 mmol) were in anhydrous DMSO (0.214 mL) under N 2. After 15 min, 1- ethylnyl-4-fluorobenzene (49 pL, 0.423 mmol) was added to the mixture and stirred at rt for 2 h. The reaction progress was monitored using TLC until completion. The reaction mixture was diluted with 10% NH 3 aq (1 mL), and the mixture was extracted with EtOAc (3 x 2 mL).
  • Methyl 4-amino-6-((3,5-difluorophenyl)amino)-l,3,5-triazine-2-carboxylate Following the general free base procedure C, a mixture of l-carbamimidamido-N-(3,5- difluorophenyl)methanimidamide hydrochloride 1 (152 mg, 0.607 mmol) and NaOEt (41 mg, 0.607 mmol) was stirred in EtOH (5 mL) at rt for 24 h.
  • Methyl 4-amino-6-((3,5-dimethylphenyl)amino)-l,3,5-triazine-2-carboxylate Following the general free base procedure C, a mixture of l-carbamimidamido-N-(3,5- dimethylphenyl)methanimidamide hydrochloride (226 mg, 0.933 mmol) and NaOEt ( 64 mg, 0.933 mmol) was stirred in EtOH (5 mL) at rt for 22 h.
  • Methyl 4-amino-6-((3-fluoro-4-iodophenyl)amino)-l,3,5-triazine-2-carboxylate The general procedure C was followed using l-carbamimidamido-N-(3-fluoro-4- iodophenyl)methanimidamide hydrochloride (200 mg, 0.559 mmol), NaOMe (118 mg, 2.18 mmol), and 1.6 mL in anhydrous MeOH. The corresponding arylbiguanide base, dimethyl oxalate (198 mg, 1.68 mmol) 1.8 mL MeOH at reflux 18 h. The reaction mixture was cooled to rt.
  • l-Carbamimidamido-N-(2-iodophenyl)methanimidamide hydrochloride The general procedure B was followed using 2-iodoaniline (1.0 g, 4.57 mmol) and dicyandiamide (384 mg, 4.57 mmol) in 1.5 mL 3 M HC1 at 100 °C for 48 h.
  • l-Carbamimidamido-N-(4-hydroxyphenyl)methanimidamide hydrochloride Into a microwave vessel was added 4-aminophenol (649 mg, 5.95 mmol), dicyandiamide (500 mg, 5.95 mmol), and (0.83 mL, 6.55 mmol). Then acetonitrile (7.9 mL) was added and the mixture was stirred at 150 °C. After 4.5 hours, a purple precipitate formed. The precipitate was dissolved in MeOH and stirred for 15 min, then concetrated by rotary evaporation to afford a purple solid.
  • N'-(Azaniumylmethanimidoyl)-N-(6-iodopyridin-3-yl)guanidine chloride The general procedure B was followed using 5-amino-2-iodopyridine (500 mg, 2.27 mmol) and dicyandiamide (191 mg, 2.27 mmol) in 0.91 mL 3 M HC1 and 0.91 mL H 2 0 at 100 °C for 24 h.
  • N'-(zaniumylmethanimidoyl)-N-(4-fluorophenyl)-N-methylguanidine chloride The general procedure B was followed using 4-fluoromethyl aniline (0.5 mL, 4.4 mmol) and dicyandiamide (370 mg, 4.4 mmol) in 1.5 mL 3 M HC1 at 90 °C for 24 h. The reacttion mixture was cooled for 1 h at rt.
  • 6-Chloro-N 2 -(4-fluorophenyl)-N 4 ,N 4 -dimethyl-1, 3, 5-triazine-2, 4-diamine To a solution of 4,6-dichloro-N-(4-fluorophenyl)-l,3,5-triazin-2-amine (above) (300 mg, 1.16 mmol) in acetone was added K 2 C0 3 (160 mg, 1.16 mmol) and dimethylamine (0.58 mL, 1.16 mmol). The mixture was stirred at 40 °C for 6 h. The solvent was removed by rotary evaporation and ice water (11 mL) was added.
  • Methyl 3-[(4-fluorophenyl)amino]-5-nitrobenzoate Methyl 3-bromo-5-nitrobenzoate (2.58 mmol), cesium carbonate (3.87 mmol), and 2, 2'-bis(diphenylphosphino)- 1,1 '-binaphthyl (BINAP) (0.194 mmol) were weighed into a Schlenck flask, and the flask was purged with N 2. Anhydrous toluene (25 mL) was then added to the flask. Pd 2 (dba) 2 was weighed into a separate microwave vial and dissolved in 1.0 mL of toluene.
  • 6-Bromo-4-nitropicolinic acid To a solution of 2-bromo-6-methyl-4-nitro-pyridine (2.07 mmol) in concentrated H2SO4, Cr0 3 (8.28 mmol) was added at 0 °C. The resulting solution was stirred at rt for 4 h. The mixture was then heated to 70 °C for 30 min and then cooled to rt. Ice cold H 2 0 (13 mL) was added slowly to afford a dark green heterogeneous solution. The mixture was allowed to stand at -20 °C overnight.
  • Methyl 6-((4-fluorophenyl)amino)-4-nitropicolinate Methyl 6-bromo-4-nitropicolinate (65) (0.766 mmol), 4-fluoro aniline (0.919 mmol), t-BuOK (1.07 mmol), were weighed into a
  • 6-Azido-N 2 -(4-iodophenyl)-l, 3, 5-triazine-2, 4-diamine Ammonium hydroxide (0.375mL, 3.21 mmol) was added to 4-azido-6-chloro-N-(4-iodophenyl)-l,3,5-triazin-2-amine (400 mg, 1.07 mmol) in THF (2.9 mL). The reaction was refluxed overnight. The solution turned into a yellow color and a white solid precipitated. The mixture was concentrated by rotary evaporation to afford a white solid.
  • 6-(Azidomethyl)-N 2 -(4-iodophenyl)-l, 3, 5-triazine-2, 4-diamine A mixture of 6- (chloromethyl)-N 2 -(4-iodophenyl)-l, 3, 5-triazine-2, 4-diamine (100 mg, 0.277 mmol) and sodium azide (54 mg, 0.830) were weighed into a microwave vial with a stir bar. Acetonitrile (2.8 mL) was added and the reaction vessel was sealed. The reaction was heated at reflux overnight. The mixture was cooled to rt and concentrated by rotary evaporation to afford a white residue, which was dissolved with 3 mL CH2CI2.
  • Ar defined as aryl group.
  • IC50 defined as half maximal inhibitory concentration.
  • Ar defined as aryl group.
  • IC50 defined as half maximal inhibitory concentration.
  • Ar defined as aryl group.
  • IC50 defined as half maximal inhibitory concentration. a In vitro IC50 derived from dose-response curve for the measurement of ATP consumption from cGAS- mediated 2’,3’-cGAMP synthesis.
  • Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway Science 339 , 786-791.

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Abstract

L'invention concerne des procédés de traitement de maladies liées à l'activation de cGAS. L'invention concerne des inhibiteurs à petites molécules de cGAS et des compositions pharmaceutiques, ainsi que leurs utilisations dans le traitement de maladies auto-immunes ou de l'inflammation.
PCT/US2019/037502 2018-06-15 2019-06-17 Nouveaux inhibiteurs cycliques de la gmp-amp synthase (cgaz) et leur procédé d'utilisation WO2019241787A1 (fr)

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WO2021209484A1 (fr) * 2020-04-16 2021-10-21 F. Hoffmann-La Roche Ag Dérivés de benzimidazole
WO2022051634A1 (fr) * 2020-09-03 2022-03-10 Immunesensor Therapeutics, Inc. Composés de quinoléine antagonistes de cgas
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WO2022238335A1 (fr) 2021-05-12 2022-11-17 Boehringer Ingelheim International Gmbh Dérivés de pyridine ayant des substituants cycliques liés à c en tant qu'inhibiteurs de cgas
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US11603351B2 (en) 2017-07-11 2023-03-14 Vertex Pharmaceuticals Incorporated Carboxamides as modulators of sodium channels
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