cGAS INHIBITORS AND METHODS OF USE THEREOF

Abstract

The present disclosure relates generally to novel inhibitors of human cGAS and methods of treating a cGAS-related disease or disorder in a patient.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 309,894, filed February 14, 2022, which is incorporated by reference in its entirety.

[0002] Statement of government support This invention was made with Government support under Contract No. 5250037401 (RO1AG051390) awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Technical Field The present teachings relate generally to novel chemical compounds and methods useful for treating cGAS-related diseases or disorders. [Background technology]

[0004] background Innate immunity is considered a frontline cellular stress response that defends host cells against invading pathogens and initiates signaling to the adaptive immune system. These processes are triggered by conserved pathogen-associated molecular patterns (PAMPs) through sensing by diverse pattern recognition receptors (PRRs) and subsequent activation of cytokine and type I interferon gene expression. Primary antigen-presenting cells, such as monocytes, macrophages, and dendritic cells, produce interferons and are crucial in eliciting T-cell and B-cell adaptive immune system responses. Primary PRRs detect abnormal, i.e., mislocalized, immature, or unmodified nucleic acids, either on the cell surface, inside the lysosomal membrane, or in the cytosol.

[0005] Cyclic GMP-AMP synthase (cGAS / MB2lDl) is a major sensor of cytosolic dsDNA of pathogen origin or mislocalization of self-dsDNA in the nucleus or mitochondria. Binding of dsDNA to cGAS activates the synthesis of a diffusible cyclic dinucleotide, c[G(2',5')pA(3',5')p], termed cGAMP, which translocates to and activates the endoplasmic reticulum membrane-bound adaptor protein, stimulator of interferon genes (STING / TMEM173).

[0006] Activated STING recruits and activates TANK-binding kinase 1 (TBK1), which in turn phosphorylates the interferon regulatory factor (IRF) family of transcription factors that induce mRNA expression of cytokines and type I interferons. Type I interferons are expressed from more than 10 IFNA genes and one IFNB1 gene.

[0007] The essential role of cGAS in sensing dsDNA has been demonstrated in various pathogenic bacteria, viruses, and retroviruses. In addition, cGAS is essential for various other biological processes, such as the recognition of ruptured micronuclei in the surveillance of cellular senescence and potential cancer cells.

[0008] While the cGAS pathway is important for host defense against invading pathogens, cellular stress and genetic factors can also lead to the accumulation of self-dsDNA in the cytosol, for example, through nuclear or mitochondrial leakage. This can trigger an autoinflammatory response. Aicardi-Goutières syndrome (AGS), a severe autoinflammatory immune-mediated disorder that resembles lupus, is caused by loss-of-function mutations in TREX1, the major DNA exonuclease responsible for the degradation of abnormal DNA in the cytosol. Knockout of cGAS in TREX1-deficient mice prevents otherwise lethal autoimmune responses, supporting cGAS as a drug target and driver of interferonopathies. Similarly, embryonic lethality caused by deficiency of DNase II, an endonuclease responsible for the degradation of excess DNA in lysosomes during endocytosis, is fully rescued by additional knockout of STING or cGAS. Therefore, inhibition of cGAS represents an important therapeutic strategy to prevent autoinflammatory diseases that involve anti-dsDNA antibodies in their pathogenesis. Systemic lupus erythematosus (SLE) may be one such disease [Pisetsky, Nat Rev Rheumatol 12, 102-110 (2016)].

[0009] In a model of Parkinson's disease, loss of dopaminergic neurons from the substantia nigra pars compacta, motor defects, and inflammation observed in aged parkin knockout mutant mice that accumulate mutations in mitochondrial DNA are rescued by loss of STING, suggesting that inflammation promotes this phenotype (Sliter D, 2018). cGAS and STING have attracted the interest of structural biologists and medicinal chemists for the identification of inhibitors and / or activators. In silico screening efforts using murine cGAS-DNA crystal structures led to the identification of quinacrine, a well-characterized small molecule antimalarial drug, as a promising cGAS inhibitor [An et ah, Immunol. 194, 4089-4093 (2015)]. However, instead of directly binding to and inhibiting cGAS, quinacrine, a known DNA intercalator, was found to indirectly affect cGAS activity by preventing the activation of the enzyme through disruption of dsDNA conformation, and significant off-target effects were observed through interference with the RIG-I pathway.

[0010] We investigated cGAS activation in microglia from tauopathy mice and human AD brains and identified tau-induced cytosolic mtDNA leakage as a potential mechanism driving a cGAS-dependent type I interferon response in microglia. Using a combination of behavioral, electrophysiological and single-nuclear (sn)RNA-seq, we demonstrated the strong protective effect of cGAS inhibition against synaptic and cognitive deficits without affecting pathogenic tau burden and discovered a surprising mechanistic basis for the resilience induced by cGAS inhibition. Our findings link the maladaptive immune response to the transcriptional network of Mef2c, an AD risk gene involved in cognitive resilience to amyloid and tau pathology in AD patients, and support cGAS inhibitors as a novel therapeutic approach to improve cognitive resilience in AD.

[0011] Prodrugs are inactive derivatives of drugs that are designed to accumulate in target organs and then undergo activation under physiological conditions. Some prodrugs can also undergo enzyme-mediated activation and can be designed to undergo both passive and receptor-mediated active cellular uptake.

[0012] Small molecule inhibitors specific for cGAS or their prodrugs would be of great value in treating diseases caused by inappropriate cGAS activity and the resulting undesirable type I interferon activity. Examples of such autoimmune diseases include Aicardi-Goutières syndrome (AGS) and systemic lupus erythematosus (SLE), a complex chronic systemic autoimmune disease that affects over 1.5 million Americans. Current treatments for SLE require immunosuppressive regimens with debilitating adverse side effects. Other possible applications associated with the suppression of undesirable type I interferon activity may include the treatment of inflammatory bowel disease (IBD) and neurodegenerative diseases.

[0013] There is a need for cGAS inhibitors that can circulate for long periods in tissues and plasma or that can increase brain concentrations through receptor-mediated permeation across the blood-brain barrier (BBB). Summary of the Invention

[0014] overview The present disclosure relates to compounds of formula (I), (II), or (III): TIFF2025507382000001.tif33133 or a pharma- ceutically acceptable salt thereof, During the ceremony, R 1 is heteroaryl, halogen, aryl, cyclic amine, hydroxy, -OC(O)alkyl, -NH 2 , -N(H)CO-alkyl, or alkoxy; R 2 is H, alkyl, -CHF 2 , -CF 3 , -CN, -OR c, halogen, or heterocyclyl; R 3 and R 4 are independently H, halogen, -CHF 2 , -CF 3 , -CN, -OR c , or -OCF 3 and; R 5a and R 5b is independently H, alkyl, aryl, or cycloalkyl; R 6 is N(H)R a , O-alkyl, OH, -CO 2 R d Is it; Or, R 5a and R 6 taken together form a 5- to 6-membered heterocyclyl; R 7a and R 7b are independently H or alkyl, or together with the carbon to which they are attached form a 3-membered aliphatic carbocyclic ring; R a is H, alkyl, -COR b , -CON(H)R b , or -CO 2 R e and; R b is alkyl, or aryl; R c is H, alkyl, or -C(O)-alkyl; R d is H or alkyl; R e is alkyl, or aryl; X is NH, NMe, NEt, O, or S; and TIFF2025507382000002.tif2128 is a single or double bond.

[0015] The present disclosure also relates to pharmaceutical compositions comprising compounds of Formula (I), (II) or (III).

[0016] The present disclosure further provides methods of treating a cGAS-associated autoimmune disease or disorder in a subject using the compounds of the present disclosure.

[0017] The present disclosure further provides methods of inhibiting an inflammatory response in a subject using the compounds of the present disclosure.

[0018] The present disclosure further provides methods of inhibiting dsDNA-induced interferon expression in a subject using the compounds of the present disclosure.

[0019] The present disclosure further provides methods of treating a neurodegenerative disease or disorder in a subject using the compounds of the present disclosure.

[0020] The present disclosure further provides methods of treating epilepsy in a subject using the compounds of the present disclosure.

[0021] The present disclosure further provides methods of treating viral infection-associated dementia in a subject using the compounds of the present disclosure.

[0022] The present disclosure further provides a method of treating a COVID-associated neurological disorder in a subject using a compound of the present disclosure.

[0023] The present disclosure further relates to a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a compound of the present disclosure.

[0024] These and other features, aspects, and advantages of the present disclosure will become better understood with reference to the following drawings, associated description, and claims. [Brief description of the drawings]

[0025] [Figure 1A]Figures 1A-1M show that the cGAS-STING pathway is activated in the hippocampus of tauopathy mice and human AD brains. Figure 1A is a volcano plot of RNA-seq data from bulk hippocampal tissue from 8-9 month old P301S and non-transgenic mice. Red dots represent genes with |log2 fold change|>0.5. Wald test was used. All other genes are shown in blue. Selected upregulated interferon genes are labeled. n=7 non-transgenic, n=6 P301S; Figure 1B is a gene set enrichment analysis showing hallmark pathways associated with the top 500 DEGs upregulated in P301S compared to non-transgenic samples; Figure 1C is a gene set enrichment analysis showing the top transcription factors associated with the top 500 DEGs upregulated in P301S compared to non-transgenic samples; Figure 1D is an Ingenuity Pathway Analysis (IPA) prediction of cGAS as an upstream regulator of upregulated DEGs identified using an activation z-score > 1 and a p-value of overlap < 0.05; Figure 1E is a western blot for phosphorylated Tank-binding kinase (pTBK1), total Tank-binding kinase 1 (TBK1) and GAPDH using hippocampal tissue lysates. Lanes 1-7: non-transgenic (ntg); Lanes 8-14: P301S; Figure 1F is the ratio of pTBK1 / TBK1 from (E) showing significantly higher phospho-TBK1 in P301S compared to non-transgenic hippocampus. **p=0.0015 Student's two-tailed t-test; Figure 1G is a representative immunofluorescence image of non-transgenic and P301S hippocampi labeled with anti-Iba1 (green) and anti-STING antibodies (scale bar = 50 µm); Figure 1H is a quantification of Iba1 and Sting immunofluorescence intensity, showing increased Iba1 coverage and Iba1-Sting overlap in the P301S hippocampus. Results are presented as average intensity measurements from 3-4 cross sections per animal. p-value <0.05.Student's two-tailed t-test (n=5 Ntg, n=5 P301S); Figure 1I is a diagram showing the number of patients and brain regions used for single-nucleus 10x genomics sequencing (n=7, 4 males and 3 females); Figure 1J is a UMAP plot showing expression of microglial marker genes INPP5D and CSF1R as well as STAT1 and cGAS (MB21D1) in snRNA-Seq of human microglial populations; Figure 1K is a gene set enrichment analysis showing relevant hallmark pathways enriched in cGAS-expressing microglia; Figure 1L is a representative western blot for pTBK1 and GAPDH using human frontal cortex brain lysates. Lanes 1-3: non-AD (Braak stage 0); Lanes 4-6: AD (Braak stage 6); Figure 1M is the ratio of pTBK1 / GAPDH from (L) showing significantly higher phospho-TBK1 in human AD compared to non-AD brains. ** p < 0.01, two-tailed Student's t test (n = 10 non-AD, n = 8 AD). [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Figure 1E] See legend to Figure 1A. [Figure 1F] See legend to Figure 1A. [Figure 1G] See legend to Figure 1A. [Figure 1H] See legend to Figure 1A. [Figure 1I] See legend to Figure 1A. [Figure 1J] See legend to Figure 1A. [Figure 1K] See legend to Figure 1A. [Figure 1L] See legend to Figure 1A. [Figure 1M] See legend to Figure 1A. [Figure 2A]Figures 2A-2J show that interferon activation in tau-stimulated microglia is mediated by cGAS and mitochondrial DNA leakage. Figure 2A is quantification of IFNB by ELISA and CXCL10 and CCL5 proteins by MagPix multiplex ELISA in culture medium supernatants from untreated (control) and tau-treated (tau) primary mouse microglia. IFNB: n=7, ** p=0.0016, paired t-test. CXCL10 and CCL5, n=5, *** p=0.0004, ** p=0.0031, unpaired t-test. [Figure 2B] Figures 2A-2J show that interferon activation in tau-stimulated microglia is mediated by cGAS and mitochondrial DNA leakage. Figure 2B is a representative Western blot for phosphorylated Tank-binding kinase (pTBK1), total Tank-binding kinase 1 (TBK1) and GAPDH using mouse primary microglial cell lysates (lane 1: untreated; lane 2: treated with tau fibrils). [Figure 2C] Figures 2A-2J show that interferon activation in tau-stimulated microglia is mediated by cGAS and mitochondrial DNA leakage. Figure 2C is the ratio of pTBK1 / TBK1 from Figure 2A showing significantly higher phospho-TBK1 in primary microglia treated with tau fibrils (tau) compared to untreated (control). *, p<0.05, Student's paired t-test (n=3). [Figure 2D] Figures 2A-2J show that interferon activation in tau-stimulated microglia is mediated by cGAS and mitochondrial DNA leakage. Figure 2D is an electron micrograph of primary mouse microglia treated with tau fibrils and immunogold labeled for antibodies against tau (L=lysosomes, M=mitochondria). [Figure 2E]Figures 2A-2J show that interferon activation in tau-stimulated microglia is mediated by cGAS and mitochondrial DNA leakage. Figure 2E shows the ratio of mitochondrial DNA (Nd2) to genomic DNA (Tert) measured by RT-qPCR on DNA extracts of BV2 IfnB luciferase reporter cells treated for 7 days with ddC (40 or 80 μg / ml) or EtBr (50 or 100 ng / ml) to generate mtDNA-depleted (ρ°) cells. Values ​​are normalized to untreated samples (n=2), ****p<0.0001, one-way ANOVA. [Figure 2F] Figures 2A-2J show that interferon activation in tau-stimulated microglia is mediated by cGAS and mitochondrial DNA leakage. In Figure 2F, control and mtDNA-depleted (ρ°) IfnB luciferase-reporter BV2 cells were stimulated or not with tau fibrils. IfnB signal and viability were measured 16 hours later. IfnB-luciferase signal is shown normalized to Cell TiterGlo signal to correct for viability / cell number (n=3), ****p<0.0001, two-way ANOVA. [Figure 2G] Figures 2A-2J show that interferon activation in tau-stimulated microglia is mediated by cGAS and mitochondrial DNA leakage. Figure 2G shows bulk RNA-seq analysis of Cgas+ / + and Cgas- / - primary cultured microglia treated or not with tau fibrils or HT-stranded DNA (n=3 per condition). Venn diagram showing overlap of genes upregulated by dsDNA and tau treatment in Cgas+ / + microglia. Log fold change >1 and FDR<0.05. [Figure 2H]Figures 2A-2J show that interferon activation in tau-stimulated microglia is mediated by cGAS and mitochondrial DNA leakage. Figure 2H shows the top five reactome pathways represented by upregulated DEGs that are common to dsDNA and tau-treated Cgas+ / + microglia. FDR<0.05. [Figure 2I] Figures 2A-2J show that interferon activation in tau-stimulated microglia is mediated by cGAS and mitochondrial DNA leakage. Figure 2I is a heatmap summary of interferon-stimulated genes that are lower in Cgas- / - compared to Cgas+ / + microglia stimulated with HT-DNA or tau. [Figure 2J] Figures 2A-2J show that interferon activation in tau-stimulated microglia is mediated by cGAS and mitochondrial DNA leakage. Figure 2J is a string interaction plot of genes from (I) including interferon genes including Stat1, Sp100 and Ddx60. [Figure 3A]Figures 3A-3J show that partial or complete loss of Cgas attenuates tauopathy-associated microglial interferon signature. Figure 3A is a dot plot showing normalized cell type expression of Cgas (Mb21d1) and Sting (Tmem173) in single nucleus sequencing (snRNA-Seq) samples; Figure 3B is a UMAP plot showing strong expression of marker genes P2ry12, Siglech, Sall1 and Csf1r in snRNA-Seq microglial populations (n=6 per genotype); Figure 3C is a UMAP plot colored according to microglial subclusters and divided by genotype; Figure 3D is a violin plot showing expression levels of homeostasis genes (P2ry12, Siglech), disease-related genes (Apoe, Itgax) and interferon genes (Parp14, Stat1, Trim30a, Rnf213) in microglial clusters; Figure 3E is a violin plot showing expression levels of homeostasis genes (P2ry12, Siglech), disease-related genes (Apoe, Itgax) and interferon genes (Parp14, Stat1, Trim30a, Rnf213) in microglial clusters compared to P301S Cgas+ / - and P301S Cgas+ / + microglia. FIG. 3F is a representative 63× confocal image of immunostaining for phosphor-STAT1 in the CA1 stratum radiatum of mouse hippocampus (scale bar=10 μm); FIG. 3G is the average intensity of phosphor-STAT1 measured in the CA1 striatum radiatum. Each circle represents the average intensity measurement of three images per animal. Statistical comparisons were performed using two-way ANOVA.Data are reported as mean ± SEM (n=6 Cgas+ / +, n=8 Cgas+ / -, n=7 Cgas- / -, n=6 P301S Cgas+ / +, n=9 P301S Cgas+ / -, n=5 P301S Cgas- / -) (Cgas+ / + vs. P301S Cgas+ / +: p=0.0002, P301S Cgas+ / + vs. P301S Cgas+ / -: p=0.0002, P301S Cgas+ / + vs. P301S Cgas- / -: p=0.0415); Figure 3H is a heatmap showing associations between gene modules and genotypes; Figure 3G is an analysis of disease module 1 and 2 markers compared to disease-associated, early response and late response microglial signatures. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 3F] See legend to Figure 3A. [Figure 3G] See legend to Figure 3A. [Figure 3H] See legend to Figure 3A. [Figure 3I] See legend to Figure 3A. [Figure 3J] See legend to Figure 3A. [Figure 4A]Figures 4A-4I show that loss of Cgas rescues tauopathy-induced hippocampal synaptic toxicity and memory impairment. Figure 4A: Cumulative exploration distance during the hidden trials (sessions 1-12) of the Morris Water Maze (MVM) assessment of spatial learning and memory in 7-8 month old P301S cGAS+ / +, P301S cGAS+ / - and P301S cGas- / - and their non-transgenic littermates. Males and females were tested on separate days. Data presented here represent both sexes combined. n=12 cGAS+ / +, n=11 cGAS+ / -, n=11 cGAS- / -, n=8 P301S cGAS+ / +, n=17 P301S cGAS+ / -, n=6 P301S cGas- / -. Two-way ANOVA. ****, p<0.0001; Figure 4B: Percentage of mean time spent in the target quadrant or in the non-target (other) quadrant over the 24-h probe course of the MVM assessment. Paired two-tailed Student's t-test; Figure 4C: Percentage of mean time spent in the target quadrant or in the non-target (other) quadrant over the 72-h probe course of the MVM assessment. Paired two-tailed Student's t-test; Figure 4D: Field excitatory postsynaptic potentials (fEPSPs) were recorded in the dentate gyrus molecular layer and LTP was induced by applying a TBS protocol (arrow) to the perforant path. Representative traces show fEPSPs before and after LTP induction (top). Scale bars, 0.4 mV and 5 ms.fEPSP slope measurements made up to 60 min after TBS were normalized to the average baseline fEPSP slope before LTP induction (bottom, n = 8–11 cross sections from 3–4 mice per group); in Figure 4E, LTP magnitude was calculated from the normalized average fEPSP slope 55–60 min after TBS application (n = 8–11 cross sections from 3–4 mice per group; *, p < 0.05, **, p < 0.01; one-way ANOVA, Bonferroni post hoc analysis); in Figure 4F, granule, C Figure 4G is a pie chart summarizing the percentage of DEGs from the clusters relating to the dentate gyrus (DG), CA1 and CA2 / 3 clusters; Figure 4H is a representative confocal image of the CA1 radial striatum of the hippocampus labeled with PSD95 antibody (scale bar = 10 µm); Figure 4I is the average intensity of PSD-95 puncta measured in the CA1 radial striatum. Each circle represents the average intensity measurement of 3-5 images per animal. Statistical comparisons were performed using one- or two-way ANOVA (Cgas+ / + vs. P301S Cgas+ / +: p=0.0275, P301S Cgas+ / + vs. P301S Cgas+ / -: p=0.0002, P301S Cgas+ / + vs. P301S Cgas- / -: p=0.0415); data are reported as mean ± SEM. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 4E] See legend to Figure 4A. [Figure 4F] See legend to Figure 4A. [Figure 4G] See legend to Figure 4A. [Figure 4H] See legend to Figure 4A. [Figure 4I] See legend to Figure 4A. [Figure 5A]Figures 5A-5L show that loss of cGAS rescues the expression of Mef2c and its target genes in tauopathy neurons. Figure 5A is a volcano plot showing representative differentially expressed genes upregulated in P301S Cgas- / - compared to P301S Cgas+ / + excitatory neurons (log2FC>0.1, FDR<0.05). [Figure 5B] Figures 5A-5L show that loss of cGAS rescues the expression of Mef2c and its target genes in tauopathy neurons. Figure 5B is a representative 63x confocal image of immunostaining for NRG1 in the CA1 stratum radiatum of the mouse hippocampus (scale bar = 10 µm). [Figure 5C] Figures 5A-5L show that loss of cGAS rescues the expression of Mef2c and its target genes in tauopathy neurons. Figure 5C shows the mean intensity of NRG1 measured in the CA1 radial striatum. Each circle represents the mean intensity measurement of three images per animal. Statistical comparisons were performed using two-way ANOVA. Data are reported as mean ± SEM (n=11 Cgas+ / +, n=6 P301S Cgas+ / +, n=8 P301S Cgas+ / -, n=6 P301S Cgas- / -. Cgas+ / + vs. P301S Cgas+ / +: p=0.0016, P301S Cgas+ / + vs. P301S Cgas- / -: p=0.0234). [Figure 5D] Figures 5A-5L show that loss of cGAS rescues the expression of Mef2c and its target genes in tauopathy neurons. Figure 5D is a volcano plot showing representative differentially expressed genes upregulated in P301S Cgas- / - compared to P301S Cgas+ / + inhibitory neurons (log2FC>0.1, FDR<0.05). [Figure 5E]Figures 5A-5L show that loss of cGAS rescues the expression of Mef2c and its target genes in tauopathy neurons. Figure 5E is a representative 25x confocal image of immunostaining for Mef2c and NeuN in the CA1 pyramidal layer of the mouse hippocampus (scale bar = 50um). [Figure 5F] Figures 5A-5L show that loss of cGAS rescues the expression of Mef2c and its target genes in tauopathy neurons. Figure 5F shows the average intensity of Mef2c in Mef2c+, NeuN+ neurons in the CA1 pyramidal layer. Each circle represents the average intensity measurement of three images per animal. *, p<0.05, unpaired t-test. Data are reported as mean ± SEM (n=4 P301S Cgas+ / +, n=5 P301S Cgas- / -). [Figure 5G] Figures 5A-5L show that loss of cGAS rescues the expression of Mef2c and its target genes in tauopathy neurons. Figure 5G is a Venn diagram of the overlap between excitatory neuron DEGs, inhibitory neuron DEGs and MEF2C target genes. [Figure 5H] Figure 5A-5L show that loss of cGAS rescues the expression of Mef2c and its target genes in tauopathy neurons. Figure 5H is a heatmap showing the overlap between excitatory / inhibitory neuronal DEGs and lists of transcription factor target genes (MEF2A, MEF2C, FOSL2, JUNB) and activity-induced upregulated genes (ARGs and scARGs). The number in each box represents the odds ratio of overlap. [Figure 5I] Figures 5A-5L show that loss of cGAS rescues the expression of Mef2c and its target genes in tauopathy neurons. Figure 5I is a heatmap of expression of significant DEGs that are MEF2C targets in WT, P301S, and P301S cgas- / - excitatory neuron clusters (p.adj<0.5, logFC>=0.1 or <=-0.1). [Figure 5J]Figures 5A-5L show that loss of cGAS rescues the expression of Mef2c and its target genes in tauopathy neurons. Figure 5J is a heatmap of expression of significant DEGs that are MEF2C targets in WT, P301S, and P301S cgas- / - inhibitory neuron clusters (p.adj<0.5, logFC>=0.1 or <=-0.1). [Figure 5K] Figures 5A-5L show that loss of cGAS rescues the expression of Mef2c and its target genes in tauopathy neurons. Figure 5K is a dot plot showing the expression of significantly upregulated DEGs by cgas deletion that are positively correlated with human cognitive resilience in excitatory neuron clusters (p.adj<0.5, logFC>=0.1). [Figure 5L] Figures 5A-5L show that loss of cGAS rescues the expression of Mef2c and its target genes in tauopathy neurons. Figure 5L shows the expression of significantly upregulated DEGs by cgas deletion that positively correlate with human cognitive resilience in inhibitory neuron clusters (p.adj<0.5, logFC>=0.1). [Figure 6A]Figures 6A-6I show that brain-permeable cGAS inhibitors upregulate MEF2C target genes and prevent synapse loss and impairment of spatial learning and memory. Figure 6A is a Venn diagram of the overlap between P301S TDI DEGs versus P301S Veh DEGs in excitatory neurons, inhibitory neurons and MEF2C target genes; Figure 6B is a heatmap showing the overlap between excitatory / inhibitory neuron DEGs and lists of transcription factor target genes (MEF2A, MEF2C, FOSL2, JUNB) and activity-induced upregulated genes (ARGs and scARGs). Numbers in each box represent overlapping odds ratios; Figure 6C is a dot plot showing the expression of significantly upregulated DEGs that are MEF2C targets in Ntg control, Ntg TDI, P301S control, and P301S TDI excitatory neuron clusters (p.adj<0.5, logFC>=0.1); Figure 6D is a dot plot showing the expression of significantly upregulated DEGs that are MEF2C targets in Ntg control, Ntg TDI, P301S control, and P301S TDI inhibitory neuron clusters (p.adj<0.5, logFC>=0.1); Figure 6E is a novel object recognition test for Ntg and P301S mice fed 150 mg / kg TDI-6570 or control diet for 3 months. F: everyday objects, N: novel objects (n=9 Ntg control, n=6 Ntg TDI-6570, n=5 P301S control, n=12 P301S TDI6570). Statistical comparisons were performed using two-way ANOVA. *, p<0.05, **, p<0.01; data are reported as mean ± SEM; Figure 6F is a representative confocal image of the CA1 radial striatum of the hippocampus labeled with PSD95 antibody (scale bar = 10 µm); Figure 6G is the mean intensity of PSD95 puncta measured in the CA1 radial striatum. Each circle represents the mean intensity measurement of one image. Three to five images were acquired per animal. Statistical comparisons were performed using mixed models (n=13 Ntg control, n=12 Ntg TDI-6570, n=9 P301S control, n=12 P301S TDI-6570.Ntg control vs P301S control: p=0.0302, P301S control vs P301S TDI-6570: p=0.0427); Figure 6H is a representative confocal image of the CA1 radial striatum of the hippocampus labeled with vGAT antibody (scale bar = 10 µm); Figure 6I is the average intensity of vGAT puncta measured in the CA1 radial striatum. Each circle represents the average intensity measurement of one image. Three to five images were acquired per animal. Statistical comparisons were performed using mixed models (n=12 Ntg control, n=11 Ntg TDI-6570, n=8 P301S control, n=13 P301S TDI-6570. Ntg control vs P301S control: p=0.0453, P301S control vs P301S TDI-6570: p=0.0318). [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 6E] See legend to Figure 6A. [Figure 6F] See legend to Figure 6A. [Figure 6G] See legend to Figure 6A. [Figure 6H] See legend to Figure 6A. [Figure 6I] See legend to Figure 6A. [Figure 7]We show that Cgas deletion also altered the transcriptome of inhibitory neurons in tauopathy. Subclustering of pan-interneuron markers GAD1 and GAD2 positive neuronal populations identified nine inhibitory neuronal subpopulations (Figure 6A) (Arneson et al., 2018; Cembrowski et al., 2016). We found that Cgas deletion rescued tauopathy-induced downregulation of a subset of interneuron markers, such as Pvalb, Vip, Reln, and Lhx6, but not Sst or Cck. In addition, analysis of DEGs from interneurons revealed that Cgas deletion led to upregulation of genes involved in GABA signaling, including the GABA receptor Gabbr2 and the GABA transporter Slc6a, supporting the restoration of interneuron function by Cgas deletion (Figure 6B). Interestingly, among the genes upregulated by Cgas deletion, we found that many are involved in regulating neuronal excitability and seizure activity, including potassium channels and interacting subunits Kcnc1, Kcnc2, Kacnip1, and sodium channel Scn1a (Figure 6C). Indeed, Cgas deletion led to a marked upregulation of anti-seizure genes, including Scn1a, that were decreased in interneurons of P301S mice (Figure 6D). Deficiency of Scn1a leads to Dravet syndrome, an intractable childhood epilepsy with generalized tonic-clonic seizures. [Figure 8A] Figures 8A-8D show a working model illustrating the cGAS-IFN-MEF2c axis in tauopathy. Under disease / vulnerability conditions, pathogenic tau activates a cGAS-dependent interferon response via leakage of mtDNA in microglia and reduction of the MEF2c transcriptional network in excitatory and inhibitory neurons, leading to cognitive impairment. Loss of cGAS reduces the interferon response and enhances the Mef2c transcriptional network in microglia, resulting in cognitive resilience. [Figure 8B]Figures 8A-8D show a working model illustrating the cGAS-IFN-MEF2c axis in tauopathy. Under disease / vulnerability conditions, pathogenic tau activates a cGAS-dependent interferon response via leakage of mtDNA in microglia and reduction of the MEF2c transcriptional network in excitatory and inhibitory neurons, leading to cognitive impairment. Loss of cGAS reduces the interferon response and enhances the Mef2c transcriptional network in microglia, resulting in cognitive resilience. [Figure 8C] Figures 8A-8D show a working model illustrating the cGAS-IFN-MEF2c axis in tauopathy. Under disease / vulnerability conditions, pathogenic tau activates a cGAS-dependent interferon response via leakage of mtDNA in microglia and reduction of the MEF2c transcriptional network in excitatory and inhibitory neurons, leading to cognitive impairment. Loss of cGAS reduces the interferon response and enhances the Mef2c transcriptional network in microglia, resulting in cognitive resilience. [Figure 8D] Figures 8A-8D show a working model illustrating the cGAS-IFN-MEF2c axis in tauopathy. Under disease / vulnerability conditions, pathogenic tau activates a cGAS-dependent interferon response via leakage of mtDNA in microglia and reduction of the MEF2c transcriptional network in excitatory and inhibitory neurons, leading to cognitive impairment. Loss of cGAS reduces the interferon response and enhances the Mef2c transcriptional network in microglia, resulting in cognitive resilience. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Detailed Description While the concepts of the present disclosure will be illustrated and described in detail in the drawings and description herein, the results in the drawings and their description are to be considered as exemplary and not limiting in nature; it will be understood that only illustrative embodiments have been shown and described, and that all changes and modifications that come within the spirit of the disclosure are desired to be protected.

[0027] Unless otherwise defined, scientific and technical terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] The entire contents of any and all patent, non-patent and bibliographic documents cited herein are incorporated by reference herein, except that in the event of any disclosure or definition that conflicts with this specification, the disclosure or definition in this specification shall control.

[0029] In each of the above and each of the following embodiments, the formulae should be understood to include and represent not only all pharma- ceutically acceptable salts of the conjugate formulae. It is recognized that certain functional groups, such as hydroxy, amino and the like, form complexes and / or coordination complexes with water and / or various solvents in various physical forms of the compounds of formula (I) or (II). It is understood that the formulae depicted throughout this disclosure include and represent hydrates and / or solvates of the compounds of formula (I), (II) or (III). It should also be understood that the non-hydrates and / or non-solvates of the compounds of formula (I), (II) or (III) are described by such formulae, as well as the hydrates and / or solvates of the compounds of formula (I), (II) or (III).

[0030] definition For convenience, before further description of the present disclosure, some terms used in the specification, examples and appended claims are summarized here.These definitions should be read in light of the remainder of the present disclosure and should be understood in the same manner as understood by those skilled in the art.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0031] In order that this disclosure may be more readily understood, certain terms and phrases are defined below and throughout the specification.

[0032] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0033] The term "and / or" as used herein and in the claims should be understood to mean "either or both" of the elements connected thereby, i.e., elements that are conjunctive in some cases and disjunctive in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., as "one or more" of the elements connected thereby. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to the elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B", when used in conjunction with open-ended language such as "comprising", can refer to A only (optionally including elements other than B); or B only (optionally including elements other than A); or even both A and B (optionally including other elements); and so forth.

[0034] When used in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., including at least one of the elements of the number or list, but also including more than one of them, and optionally including additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or "consisting of," when used in the claims, refer to the inclusion of exactly one element of the elements of the number or list. In general, the term "or," when used in the specification, shall be interpreted to refer to exclusive alternatives (i.e., "one or the other but not both") only when preceded by an exclusive term, such as "either," "one of," "only one of," or "exactly one of." When used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0035] As used herein and in the claims, the phrase "at least one" should be understood to refer to a list of one or more elements and to mean at least one element selected from any one or more of the elements in that list, but not necessarily including at least one of each and every element specifically listed within the list of elements, and not excluding any combination of elements in that list. This definition also allows for the optional presence of elements other than those specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer to at least one A, optionally including more than one, and no B (and optionally including elements other than B); or at least one B, optionally including more than one, and no A (and optionally including elements other than A); or at least one further A, optionally including more than one, and at least one B, optionally including more than one (and optionally including other elements), etc.

[0036] Also, unless expressly indicated to the contrary, in any method claimed herein that includes more than one step or act, it should be understood that the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are described.

[0037] As in the specification above, in the claims, all transitional phrases, such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are to be understood to mean open ended, i.e., including but not limited to.

[0038] Various compounds contained in the compositions of the present disclosure may exist in specific geometric or stereoisomeric forms. In addition, the polymers of the present disclosure may also be optically active. The present disclosure contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (d)-isomers, (l)-isomers, racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the present disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are intended to be included in the present disclosure.

[0039] By way of example, if a particular enantiomer of a compound of the present disclosure is desired, it may be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary, in which case the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, if the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, a diastereomeric salt is formed with a suitable optically active acid or base, and the diastereomers so formed are subsequently resolved by fractional crystallization or chromatographic means well known in the art, after which the pure enantiomer is recovered.

[0040] Structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds produced by the replacement of hydrogen with deuterium or tritium, or carbon with 13C- or 14C-enriched carbon are within the scope of the disclosure.

[0041] The term "prodrug" as used herein includes compounds that are converted to therapeutically active agents under physiological conditions. A common method for making a prodrug is to include a moiety selected to be hydrolyzed under physiological conditions to reveal the desired molecule. The prodrug may be converted by the enzymatic activity of the host animal.

[0042] The phrase "pharmacologically acceptable excipient" or "pharmacologically acceptable carrier" as used herein means a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulant, involved in carrying or transporting a chemical of interest from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, not harmful to the patient, and substantially non-pyrogenic. Some examples of materials which can serve as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes. wax);(9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil;(10) glycols, such as propylene glycol;(11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol;(12) esters, such as ethyl oleate and ethyl laurate;(13) agar;(14) buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid;(16) pyrogen-free water;(17) isotonic saline;(18) Ringer's solution;(19) ethyl alcohol;(20) phosphate buffer; and(21) other non-toxic compatible substances used in pharmaceutical formulations. The pharmaceutical compositions of the present disclosure are non-pyrogenic, i.e., do not induce a significant temperature elevation when administered to a patient.

[0043] The term "pharmaceutically acceptable salts" refers to relatively non-toxic inorganic and organic acid addition salts of a compound. These salts can be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and laurylsulfonate (see, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19.).

[0044] In other cases, the compounds useful in the disclosed methods may contain one or more acidic functional groups and therefore can form pharma-ceutically acceptable salts with pharma-ceutically acceptable bases. In these cases, the term "pharma-ceutically acceptable salts" refers to the relatively non-toxic inorganic and organic base addition salts of the compounds. These salts can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compounds in their free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharma-ceutically acceptable metal cation, with ammonia, or with a pharma-ceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium and aluminum salts, and the like. Representative organic amines useful for forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like (see, for example, Berge et al., supra).

[0045] A "therapeutically effective amount" (or "effective amount") of a compound for use in treatment refers to the amount of compound in a preparation that, when administered (to a mammal, e.g., a human) as part of a desired dosing regimen, alleviates symptoms, improves the condition, or delays the onset of a disease state, e.g., in accordance with clinically accepted standards, such that a disease or condition is treated, e.g., at a reasonable benefit / risk ratio compatible with any medical treatment, or for cosmetic purposes.

[0046] The term "prophylactic or therapeutic" treatment is art-recognized and includes administering to a patient one or more compounds of the present disclosure. If the compound is administered prior to the appearance of clinical symptoms of an undesired condition (e.g., a disease or other undesired condition of a host animal), the treatment is prophylactic (i.e., protects the host from the occurrence of the undesired condition), whereas if the compound is administered after the appearance of the undesired condition, the treatment is therapeutic (i.e., is intended to reduce, ameliorate, or stabilize an existing undesired condition or its side effects).

[0047] The term "patient" or "subject" refers to a mammal suffering from a disease, disorder, or condition. The patient or subject can be a primate, canine, feline, or equine. The patient can be an avian subject. The bird can be a domestic bird, such as a chicken. The bird can be a poultry. The patient or subject can be a human.

[0048] Aliphatic chains include the classes of alkyl, alkenyl and alkynyl as defined below. Linear aliphatic chains are limited to unbranched carbon chain moieties. As used herein, the term "aliphatic group" refers to straight-chain, branched-chain or cyclic aliphatic hydrocarbon groups, including saturated and unsaturated aliphatic groups such as alkyl, alkenyl or alkynyl groups.

[0049] "Alkyl" refers to a fully saturated, cyclic or acyclic, branched or unbranched carbon chain moiety having the specified number of carbon atoms, or up to 30 carbon atoms if not specified. For example, alkyl of 1 to 8 carbon atoms refers to moieties such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl, as well as moieties that are positional isomers of these moieties. Alkyl of 10 to 30 carbon atoms includes decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, trocontanyl, Pentacosenyl and hexacosenyl A straight chain or branched chain alkyl can have 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C3-C30 for branched chain), or 20 or fewer carbon atoms. An alkyl group can be substituted or unsubstituted.

[0050] As used herein, the term "alkylene" refers to an alkyl group having a specified number of carbon atoms, e.g., 2-12 carbon atoms, containing two points of attachment to the remainder of the compound on its longest carbon chain. Non-limiting examples of alkylene groups include methylene-(CH2)-, ethylene-(CH2CH2)-, n-propylene-(CH2CH2CH2)-, isopropylene-(CH2CH(CH3))-, and the like. Alkylene groups can be cyclic or acyclic, branched or unbranched carbon chain moieties, and may be optionally substituted with one or more substituents.

[0051] "Cycloalkyl" means monocyclic or bicyclic or bridged or spirocyclic or polycyclic saturated carbocyclic rings, each having from 3 to 12 carbon atoms. In various aspects, cycloalkyls have from 3-10 carbon atoms in their ring structure, or from 3-6 carbons in the ring structure. Cycloalkyl groups can be substituted or unsubstituted.

[0052] Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group as defined above but having 1-10 carbons or 1-6 carbon atoms in its backbone structure, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. Similarly, "lower alkenyl" and "lower alkynyl" have similar chain lengths. Substituents designated herein as alkyl can be lower alkyl.

[0053] "Alkenyl" refers to any cyclic or acyclic, branched or unbranched, unsaturated carbon chain moiety having the specified number of carbon atoms or up to 26 carbon atoms if there is no limit to the specified number of carbon atoms; and having one or more double bonds within the moiety. Alkenyls of 6 to 26 carbon atoms are exemplified by the various isomeric forms of hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, eicosenyl, heneicosoenyl, docosenyl, tricosenyl, and tetracosenyl, where the unsaturated bond may be located anywhere within the moiety and may have either the (Z) or (E) configuration around the double bond.

[0054] "Alkynyl" refers to a hydrocarbyl moiety within the scope of alkenyl, but which has one or more triple bonds within the moiety.

[0055] The term "alkylthio" refers to an alkyl group, as defined above, having a sulfur moiety attached thereto. The "alkylthio" moiety can be represented by one of -(S)-alkyl, -(S)-alkenyl, -(S)-alkynyl, and -(S)-(CH2)m-R1, where m and R1 are defined below. Representative alkylthio groups include methylthio, ethylthio, and the like. The term "alkoxyl" or "alkoxy", as used herein, refers to an alkyl group, as defined below, having an oxygen moiety attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. Thus, the substituent of an alkyl that makes the alkyl an ether is or resembles an alkoxyl, and can be represented by, for example, one of -O-alkyl, -O-alkenyl, -O-alkynyl, -O-(CH2)m-R10, where m and R10 are described below.

[0056] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, for example, amines of the formula: TIFF2025507382000003.tif11128, where R11 and R12 each independently represent hydrogen, an alkyl, an alkenyl, -(CH2)m-R10, or R11 and R12 together with the N atom to which they are attached complete a heterocycle having 4-8 atoms in its structure; R10 represents an alkenyl, an aryl, a cycloalkyl, a cycloalkenyl, a heterocyclyl, or a polycyclyl; and m is zero or an integer ranging from 1 to 8. In some examples, only one of R11 or R12 can be a carbonyl, e.g., R11, R12, and the nitrogen do not together form an imide. R11 and R12 each independently represent hydrogen, an alkyl, an alkenyl, or -(CH2)m-R10. Thus, the term "alkylamine" as used herein means an amine group as defined above having a substituted or unsubstituted alkyl attached thereto, i.e., at least one of R11 and R12 is an alkyl group. An amino group or an alkylamine is basic, which means that it has a conjugate acid with a pKa>7.00, i.e., the protonated form of these functional groups has a relative pKa to water of greater than about 7.00.

[0057] The term "amide" as used herein refers to the group: TIFF2025507382000004.tif17128, where each R13 independently represents a hydrogen or a hydrocarbyl group, or two R13 together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in its structure.

[0058] The term "aryl" as used herein includes 3-12 membered substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon (i.e., carbocyclic aryl), or 3-12 membered substituted or unsubstituted monocyclic aromatic groups in which one or more atoms are heteroatoms (i.e., heteroaryl). In various aspects, the aryl group includes a 5-12 membered ring, or a 6-10 membered ring. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, where at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Carbocyclic aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like. Heteroaryl groups include substituted or unsubstituted aromatic 3-12 membered ring structures, 5-12 membered rings, or 5-10 membered rings, whose ring structures contain 1-4 heteroatoms. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine. Aryl and heteroaryl can be monocyclic, bicyclic, or polycyclic. Each instance of an aryl group can be independently optionally substituted, i.e., unsubstituted ("unsubstituted aryl"), or substituted with one or more substituents; for example, 1-5 substituents, 1-4 substituents, 1-3 substituents, 1-2 substituents, or just one substituent ("substituted aryl").The aromatic ring may be substituted at one or more ring positions with one or more substituents, such as halogen, azide, alkyl, aryl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amide, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moiety, fluoroalkyl (such as trifluoromethyl), cyano, etc. For example, the aryl group can be an unsubstituted C5-C12 aryl or the aryl group can be a substituted C5-C10 aryl.

[0059] The terms "halo", "halide", or "halogen" as used herein means halogen, including, for example, but not limited to, fluoro, chloro, bromo, iodo, and the like, in both radioactive and non-radioactive forms. Halo can be selected from the group consisting of fluoro, chloro, and bromo.

[0060] "Heterocyclyl" or "heterocyclic group" refers to a 3- to 12-membered ring structure, a 5- to 12-membered ring, or a 5- to 10-membered ring whose ring structure contains one to four heteroatoms. The heterocycle can be monocyclic, bicyclic, spirocyclic, or polycyclic. The heterocycle can be saturated or unsaturated. Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolene, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The heterocyclic ring may be substituted at one or more positions with substituents such as those described above, such as, for example, halogen, alkyl, aryl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, sulfamoyl, sulfinyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, -CN, etc.

[0061] Heteroaryl rings are an embodiment of heterocyclyl groups. The phrase "heterocyclyl group" includes fused ring species, including those containing fused aromatic and non-aromatic groups. Representative heterocyclyl groups include, but are not limited to, piperidynyl, piperazinyl, morpholinyl, furanyl, pyrrolidinyl, pyridinyl, pyrazinyl, pyrimidinyl, triazinyl, thiophenyl, tetrahydrofuranyl, pyrrolyl, oxazolyl, imidazolyl, triazyolyl, tetrazolyl, benzoxazolinyl, and benzimidazolinyl groups. For example, heterocyclyl groups include, but are not limited to, TIFF2025507382000005.tif52158, where X 5 is H, (C 1 ~C 20 ) alkyl, (C 6 ~C 20 ) represents an aryl or amine protecting group (e.g., t-butyloxycarbonyl group), and the heterocyclyl group may be substituted or unsubstituted. Nitrogen-containing heterocyclyl groups are heterocyclyl groups that contain a nitrogen atom as an atom in the ring. In some embodiments, the heterocyclyl is other than thiophene or substituted thiophene. In some embodiments, the heterocyclyl is other than furan or substituted furan.

[0062] The term "carbonyl" is art recognized and refers to a group having the formula: TIFF2025507382000006.tif12128, wherein X' is a bond or represents oxygen, nitrogen or sulfur, R14 represents hydrogen, alkyl, alkenyl, -(CH2)m-R10 or a pharma- ceutically acceptable salt, and R15 represents hydrogen, alkyl, alkenyl, or -(CH2)m-R10, where m and R10 are as defined above. When X' is oxygen and R14 or R15 is not hydrogen, the formula represents an "ester". When X' is oxygen and R14 is as defined above, the moiety is referred to herein as a carboxyl group, and in particular, when R14 is hydrogen, the formula represents a "carboxylic acid". When X' is oxygen and R15 is hydrogen, the formula represents a "formate". In general, when the oxygen atom of the above formula is replaced by sulfur, the formula represents a "thiocarbonyl" group. When X' is a sulfur and R14 or R15 is not hydrogen, the formula represents a "thioester" group. When X' is a sulfur and R14 is hydrogen, the formula represents a "thiocarboxylic acid" group. When X' is a sulfur and R15 is hydrogen, the formula represents a "thioformate" group. On the other hand, when X' is a bond and R14 is not hydrogen, the formula represents a "ketone" group. When X' is a bond and R14 is hydrogen, the formula represents an "aldehyde" group.

[0063] The term "amide," as used herein, refers to a group having the formula C(O)NRR, where R is defined herein and can each independently be, for example, hydrogen, alkyl, aryl, or each R, together with the nitrogen atom to which they are attached, can form a heterocyclyl group.

[0064] As used herein, the term "nitro" refers to -NO 2 the term "halogen" refers to -F, -Cl, -Br, or -I; the term "sulfhydryl" refers to -SH; the term "hydroxyl" refers to -OH; the term "sulfonyl" refers to -SO 2the term "azido" means -N3; ​​the term "cyano" means -CN; the term "isocyanato" means -NCO; the term "thiocyanato" means -SCN; the term "isothiocyanato" means -NCS; and the term "cyanato" means -OCN.

[0065] As used herein, the definition of each expression, e.g., alkyl, m, n, etc., when it occurs more than one time in any structure, is intended to be independent of its definition elsewhere in the same structure.

[0066] The term "substituted" refers to a moiety having a substituent replacing a hydrogen on one or more carbons of the backbone. It is understood that "substituted" or "substituted with" includes the implicit proviso that such substitution is subject to the permissible valences of the substituted atom and substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, and the like. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituent of organic compounds described herein that satisfies the valence of the heteroatom. The substituents may include any of the substituents described herein, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aryl, or aromatic or heteroaromatic moieties. Substituents on the substituted alkyl may be selected from C1-6 alkyl, C3-6 cycloalkyl, halogen, carbonyl, cyano, or hydroxyl. Substituents on the substituted alkyl may be selected from fluoro, carbonyl, cyano, or hydroxyl. It will be appreciated by those skilled in the art that the substituents themselves may be substituted, if appropriate. Unless specifically stated as "unsubstituted," reference to a chemical moiety herein is understood to include substituted variants.For example, reference to an "aryl" group or moiety implicitly includes both substituted and unsubstituted variants.

[0067] The term "substituted" as used herein also refers to the following groups: halogen (e.g., F, Cl, Br, and I), R, OR, ROH (e.g., CH 2 OH), OC(O)N(R) 2 , CN, NO, NO 2 , O.N.O. 2 , azide, CF 3 , OCF 3 , methylenedioxy, ethylenedioxy, (C 3 ~C 20 ) Heteroaryl, N(R) 2 , Si(R) 3 , SR, SOR, SO 2 R, SO 2 N(R) 2 , S.O. 3 R, P(O)(OR) 2 , OP(O)(OR) 2 , C(O)R, C(O)C(O)R, C(O)CH 2 C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R) 2 , C(O)N(R)OH, OC(O)N(R) 2 , C(S)N(R) 2 , (CH 2 ) 0~2 N(R)C(O)R, (CH 2 ) 0~2 N(R)N(R) 2 , N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R) 2 , N(R)SO 2 R, N(R)SO 2 N(R) 2 , N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R) 2 , N(R)C(S)N(R) 2 , N(COR)COR, N(OR)R, C(=NH)N(R) 2, C(O)N(OR)R, or C(=NOR)R, where R is hydrogen, (C 1 ~C 20 ) alkyl, (C 6 ~C 20 ) an aryl, heterocyclyl or polyalkylene oxide group, for example of the formula -(CH 2 CH 2 O) f -R-OR, -(CH 2 CH 2 CH 2 O) g -R-OR, -(CH 2 CH 2 O) f (CH 2 CH 2 CH 2 O) g-R-OR polyalkylene oxide groups, each of which in turn may be substituted or unsubstituted, and f and g are each independently an integer from 1 to 50 (e.g., 1 to 10, 1 to 5, 1 to 3, or 2 to 5). Substituted also includes groups substituted with one or more groups, including, but not limited to, the following groups: fluoro, chloro, bromo, iodo, amino, amido, alkyl, hydroxy, alkoxy, alkylamido, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxy, haloalkyl, hydroxy, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, thio, alkylthio, arylthiol, alkylsulfonyl, alkylsulfinyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino, and dialkylamido. When there are two or more adjacent substituents, the substituents can be linked to form a carbocyclic or heterocyclic ring. Such adjacent groups can be in a vicinal or germinal relationship, or they can be adjacent on the ring, for example, in an ortho configuration. Each instance of substituted is understood to be independent. For example, a substituted aryl can be substituted with bromo, and a substituted heterocycle on the same compound can be substituted with alkyl. It is contemplated that the substituted group can be substituted with one or more non-fluoro groups. As another example, the substituted group can be substituted with one or more non-cyano groups. As another example, the substituted group can be substituted with one or more groups other than haloalkyl. As yet another example, the substituted group can be substituted with one or more groups other than tert-butyl. As yet a further example, the substituted group can be substituted with one or more groups other than trifluoromethyl.As yet a further example, a substituted group may be substituted with one or more groups other than nitro, other than methyl, other than methoxymethyl, other than dialkylaminosulfonyl, other than bromo, other than chloro, other than amido, other than halo, other than benzodioxepinyl, other than polycyclic heterocyclyl, other than polycyclic substituted aryl, other than methoxycarbonyl, other than alkoxycarbonyl, other than thiophenyl, or other than nitrophenyl, or groups corresponding to combinations of such descriptions. Additionally, substituted is also understood to include fluoro, cyano, haloalkyl, tert-butyl, trifluoromethyl, nitro, methyl, methoxymethyl, dialkylaminosulfonyl, bromo, chloro, amido, halo, benzodioxepinyl, polycyclic heterocyclyl, polycyclic substituted aryl, methoxycarbonyl, alkoxycarbonyl, thiophenyl, and nitrophenyl groups.

[0068] For purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87 (inside cover).

[0069] Compounds of the Disclosure The present disclosure relates to a compound represented by formula (I) or (II): The compound of TIFF2025507382000007.tif33128 or a pharma- ceutically acceptable salt thereof, During the ceremony, R 1 is heteroaryl, halogen, aryl, cyclic amine, hydroxy, -OC(O)alkyl, -NH 2 , -N(H)CO-alkyl, or alkoxy; R 2 is H, alkyl, -CHF 2 , -CF 3 , -CN, -OR c , halogen, or heterocyclyl; R 3 and R 4are independently H, halogen, -CHF 2 , -CF 3 , -CN, -OR c , or -OCF 3 and; R 5a and R 5b is independently H, alkyl, aryl, or cycloalkyl; R 6 is N(H)R a , O-alkyl, OH, -CO 2 R d Is it; Or, R 5a and R 6 taken together form a 5- to 6-membered heterocyclyl; R 7a and R 7b are independently H or alkyl, or together with the carbon to which they are attached form a 3-membered aliphatic carbocyclic ring; R a is H, alkyl, -COR b , -CON(H)R b , or -CO 2 R e and; R b is alkyl, or aryl; R c is H, alkyl, or -C(O)-alkyl; R d is H or alkyl; R e is alkyl, or aryl; X is NH, NMe, NEt, O, or S; and TIFF2025507382000008.tif2128 is a single or double bond.

[0070] The present invention relates to a compound of formula (I). The present invention relates to a compound of formula (II). The present invention also relates to a prodrug of a compound of formula (I) or (II) or a pharma- ceutically acceptable salt thereof.

[0071] R1 R can be a substituted or unsubstituted heteroaryl. 1 R can be an N-containing heteroaryl. 1 R can be a substituted or unsubstituted C-C or C-N linked monocyclic or bicyclic heteroaryl. 1 R can be substituted or unsubstituted aryl. 1 R can be ortho, meta or para aminophenyl, methoxyphenyl, or fluorophenyl. 1 R can be substituted or unsubstituted aryl. 1 R can be a halogen. 1 R can be a substituted or unsubstituted cyclic amine. 1 R can be functionalized and non-functionalized azacyclobutane, azacyclobutanone, azacyclopentane, azacyclopentanone. 1 R can be hydroxy. 1 R can be a substituted or unsubstituted -OC(O)alkyl. 1 is -NH 2 It can be. R 1 R can be a substituted or unsubstituted -N(H)COalkyl. 1 R can be alkoxy. 1 is C 1 ~C 4 It can be an alkoxy.

[0072] R 1 teeth, It could be TIFF2025507382000009.tif140163.

[0073] R 2 can be H. 2 R can be substituted or unsubstituted alkyl. 2 is C 1~4R can be alkyl. 2 -CHF 2 It can be. R 2 -CF 3 It can be. R 2 R can be -CN. 2 -OR c It can be. R 2 R can be -OH. 2 can be OMe. R 2 R can be a halogen. 2 can be Cl or F. R 2 R can be a substituted or unsubstituted heterocyclyl. 2 R can be a substituted or unsubstituted 5-membered heterocyclyl. 2 can be a substituted or unsubstituted 6-membered heterocyclyl.

[0074] R 3 can be H, R 3 R can be a halogen. 3 can be Cl or F. R 3 -CHF 2 It can be. R 3 -CF 3 It can be. R 3 R can be -CN. 3 -OR c It can be. R 3 -OCF 3 It can be.

[0075] R 4 can be H, R 4 R can be a halogen. 4 can be Cl or F. R 4 -CHF 2 It can be. R 4 -CF 3 It can be. R 4R can be -CN. 4 -OR c It can be. R 4 -OCF 3 It can be.

[0076] R 3 and R 4 can be the same, for example, R 3 and R 4 can both be halogen, for example Cl.

[0077] R 3 and R 4 can be different, e.g., R 3 and R 4 can both be halogens, but R 3 is Cl, and R 4 is F or R 3 is H and R 4 is a halogen.

[0078] R 5a can be H. 5a R can be substituted or unsubstituted alkyl. 5a R can be methyl. 5a R can be substituted and unsubstituted aryl. 5a can be a substituted or unsubstituted cycloalkyl.

[0079] R 5b can be H. 5b R can be substituted or unsubstituted alkyl. 5b R can be methyl. 5b R can be substituted or unsubstituted aryl. 5b can be a substituted or unsubstituted cycloalkyl.

[0080] R 5a and R5b can be the same, for example, R 5a and R 5b R can both be methyl. 5a and R 5b can be different, e.g., R 5a can be H, R 5b can be methyl.

[0081] R 6 is N(H)R a It can be. R 6 NH 2 It can be. R 6 can be N(H)C(O)OtBu. 6 R can be O-alkyl. 6 R can be OH. 6 -CO 2 R d It can be. R 6 -OCH 3 , -OC 2 H 5 , -OC 3 H 7 , -OC 4 H 9 , -OC 5 H 11 , -OC 6 H 13 , -OC 7 H 15 , -OC 8 H 17 , -OC 10 H 21 , -OC 12 H 25 , -OC 14 H 29 or -OC 16 H 33 It can be. R 6 -NHCH 3 , -NHC 2 H 5 , -NHC 3 H 7 , -NHC 4 H 9 , -NHC 6 H 13, -NHC 7 H 15 , -NHC 8 H 17 , -NHC 10 H 21 , -NHC 14 H 29 or -NHC 16 H 33 It can be.

[0082] R 5a and R 6 can be taken together to form a substituted or unsubstituted 5-membered heterocyclyl. 5a and R 6 can be taken together to form a substituted or unsubstituted 6-membered heterocyclyl.

[0083] R 7a can be H. 7a can be alkyl.

[0084] R 7b can be H. 7b can be alkyl.

[0085] R 7a and R 7b can be the same, for example, R 7a and R 7b R can both be methyl. 7a and R 7b can be different, e.g., R 7a can be H, R 7b can be methyl.

[0086] R 7a and R 7b can be taken together with the carbon to which they are attached to form a three-membered aliphatic carbocyclic ring.

[0087] R a can be H. aR can be substituted or unsubstituted alkyl. a -COR b It can be. R a -CON(H)R b It can be. R a -CO 2 R e It can be. R a -CO 2 It can be tBu.

[0088] R b R can be substituted or unsubstituted alkyl. b can be a substituted or unsubstituted aryl.

[0089] R c can be H. c R can be substituted or unsubstituted alkyl. c can be a substituted or unsubstituted --C(O)-alkyl.

[0090] R d can be H. d can be substituted or unsubstituted alkyl.

[0091] R e R can be substituted or unsubstituted alkyl. e can be a substituted or unsubstituted aryl.

[0092] X can be NH. X can be NMe. X can be NEt. X can be O. X can be S.

[0093] TIFF2025507382000010.tif2128 can be a single bond. TIFF2025507382000011.tif2128 can be a double bond.

[0094] The compound of formula (I) The compound may be selected from TIFF2025507382000012.tif105140.

[0095] The compound of formula (I) The compound may be selected from TIFF2025507382000013.tif50155.

[0096] The compound of formula (II) TIFF2025507382000014.tif46166.

[0097] The compound of formula (I) or (II) can be a compound from Table 1.

[0098] All diastereomers of the compounds of Formulas (I)-(II) are contemplated herein.

[0099] Also contemplated herein are isotopic isomers, which are compounds in which one or more atoms in a compound are replaced with an isotope of that atom. Thus, for example, the present disclosure provides compounds in which one or more hydrogen atoms are replaced with deuterium or fluorine atoms are replaced with 19 This relates to compounds which are substituted with F atoms.

[0100] The compounds of the present disclosure can be synthesized by any method known in the art.

[0101] Treatment Method The present disclosure relates to a method of treating a cGAS-associated disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of any one of the aforementioned compounds.

[0102] The present disclosure relates to a method of inhibiting an inflammatory response comprising administering to a subject in need thereof a therapeutically effective amount of any one of the aforementioned compounds.

[0103] The present disclosure relates to a method of inhibiting dsDNA-induced interferon expression comprising administering to a subject in need thereof a therapeutically effective amount of any one of the aforementioned compounds.

[0104] The present disclosure further provides a method of treating a neurodegenerative disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of any one of the aforementioned compounds.

[0105] Neurodegenerative diseases or disorders include AIDS dementia complex, Alzheimer's disease, amyotrophic lateral sclerosis, adrenoleukodystrophy, Alexander disease, Alpers disease, ataxia-telangiectasia, Batten disease, bovine spongiform encephalopathy (BSE), Canavan disease, corticobasal degeneration, Creutzfeldt-Jakob disease, dementia with Lewy bodies, fatal familial insomnia, frontotemporal lobar degeneration, Huntington's disease, Kennedy disease, Krabbe disease, Lyme disease, Machado-Joseph disease, multiple sclerosis, multiple system atrophy, neuroacanthocytosis, Niemann-Pick disease, Parkinson's disease, Pick's disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum disease, Sandhoff disease, diffuse myelinoclastic sclerosis, and diffuse myelinoclastic palsy. sclerosis, spinocerebellar ataxia, subacute combined spinal degeneration, tabes dorsalis, Tay-Sachs disease, toxic encephalopathy, transmissible spongiform encephalopathy, and wobbly hedgehog syndrome. In one embodiment, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, and Parkinson's disease.

[0106] The neurodegenerative disease or disorder may be selected from the group consisting of Alzheimer's disease, Parkinson's disease, a tauopathy, or frontotemporal dementia.

[0107] The present disclosure further provides methods of treating epilepsy, including Dravet syndrome and other drug-resistant seizures.

[0108] The present disclosure further provides methods of treating viral infection-associated dementia, such as HIV dementia.

[0109] The present disclosure further provides methods of treating neurological disorders associated with COVID.

[0110] Pharmaceutical Compositions, Routes of Administration, and Dosages In certain aspects, the present disclosure is directed to a pharmaceutical composition comprising a compound of the present disclosure and a pharma- ceutically acceptable carrier. In certain aspects, the pharmaceutical composition comprises a plurality of compounds of the present disclosure and a pharma- ceutically acceptable carrier.

[0111] In certain embodiments, the pharmaceutical composition of the present disclosure further comprises at least one additional pharma- ceutical active agent other than the compound of the present disclosure.The at least one additional pharma-ceutical active agent can be an agent useful in the treatment of ischemia-reperfusion injury.

[0112] Pharmaceutical compositions of the present disclosure can be prepared by combining one or more compounds of the present disclosure with a pharma- ceutically acceptable carrier and, optionally, one or more additional pharma- ceutically active agents.

[0113] As stated above, "effective amount" refers to any amount sufficient to achieve a desired biological effect. By selecting from a variety of active compounds and considering factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and mode of administration, in combination with the teachings provided herein, one can design an effective prophylactic or therapeutic treatment regimen that does not cause substantial undesired toxicity and is effective in treating a particular subject. The effective amount for any particular application may vary depending on factors such as the disease or condition being treated, the particular compound of the present disclosure being administered, the size of the subject, or the severity of the disease or condition. Those of ordinary skill in the art can empirically determine the effective amount of a particular compound of the present disclosure and / or other therapeutic agent without necessitating undue experimentation. The maximum dose, i.e., the highest safe dose, may be used according to some medical judgment. Multiple doses per day may be envisioned to achieve an appropriate systemic level of the compound. The appropriate systemic level may be determined, for example, by measuring the patient's peak or sustained drug plasma levels. "Dose" and "administration" are used interchangeably herein.

[0114] In general, the daily oral dose of the compound is about 0.01 milligrams / kg per day to 1000 milligrams / kg per day for human subjects. Oral doses ranging from 0.5 to 50 milligrams / kg per day, administered once or multiple times, can produce therapeutic results. The dosage can be appropriately adjusted to achieve the desired drug levels, either locally or systemically, depending on the mode of administration. For example, intravenous administration can vary from one to several orders of magnitude lower per day. If the subject responds inadequately at such doses, even higher doses (or higher doses effective via a different, more localized delivery route) can be used, as long as patient tolerance allows. Multiple doses per day are envisioned to achieve adequate systemic levels of the compound.

[0115] For any compound described herein, therapeutically effective amount can be determined from animal models first.Therapeutically effective dose can also be determined from human data for compounds that have been tested in humans and for compounds known to exhibit similar pharmacological activity, such as other related active agents.Higher doses may be required for parenteral administration.Applied doses can be adjusted based on the relative bioavailability and efficacy of the administered compound.Adjusting doses to achieve maximum efficacy based on the above and other methods is well within the skill of the skilled artisan, as is well known in the art.

[0116] In clinical use, any compound of the disclosure may be administered in an amount equal to or equivalent to 0.2 to 2000 milligrams (mg) of compound per kilogram (kg) of subject body weight per day. Compounds of the disclosure may be administered in a dose equal to or equivalent to 2 to 2000 mg of compound per kg of subject body weight per day. Compounds of the disclosure may be administered in a dose equal to or equivalent to 20 to 2000 mg of compound per kg of subject body weight per day. Compounds of the disclosure may be administered in a dose equal to or equivalent to 50 to 2000 mg of compound per kg of subject body weight per day. Compounds of the disclosure may be administered in a dose equal to or equivalent to 100 to 2000 mg of compound per kg of subject body weight per day. Compounds of the disclosure may be administered in a dose equal to or equivalent to 200 to 2000 mg of compound per kg of subject body weight per day. When a precursor or prodrug of a compound of the disclosure is to be administered rather than the compound itself, it is administered in an amount equivalent to, i.e., sufficient to deliver, the above-mentioned amounts of the compound of the invention.

[0117] Formulations of the compounds of the present disclosure can be administered to human subjects in therapeutically effective amounts. Typical dose ranges are about 0.01 micrograms / kg to about 2 mg / kg of body weight per day. The dosage of the drug to be administered is likely to depend on variables such as the type and extent of the disorder, the overall health of the particular subject, the specific compound being administered, the excipients used to formulate the compound, and its route of administration. Routine experimentation may be used to optimize the dosage and dosing frequency for any particular compound.

[0118] The compounds of the present disclosure can be administered at concentrations ranging from about 0.001 micrograms / kg to greater than about 500 mg / kg. For example, concentrations can be 0.001 micrograms / kg, 0.01 micrograms / kg, 0.05 micrograms / kg, 0.1 micrograms / kg, 0.5 micrograms / kg, 1.0 micrograms / kg, 10.0 micrograms / kg, 50.0 micrograms / kg, 100.0 micrograms / kg, 500 micrograms / kg, 1.0 mg / kg, 5.0 mg / kg, 10.0 mg / kg, 15.0 mg / kg, 20.0 mg / kg, 25.0 mg / kg, 30.0 mg / kg, 35.0 mg / kg, 40.0 mg / kg, 45.0 mg / kg, 50.0 mg / kg, 50.0 mg / kg, 60.0 mg / kg, 70.0 mg / kg, 80.0 mg / kg, 85.0 mg / kg, 90.0 mg / kg, 100.0 mg / kg, 120.0 mg / kg, 130.0 mg / kg, 140.0 mg / kg, 150.0 mg / kg, 160.0 mg / kg, 170.0 mg / kg, 180.0 mg / kg, 190.0 mg / kg, 200.0 mg / kg, 220.0 mg / kg, 230.0 mg / kg, 240.0 mg / kg, 250.0 mg / kg, 260.0 mg / kg, 270.0 mg / kg, 280.0 mg / kg, 290.0 mg / kg, 300.0 mg / kg, 310.0 mg / kg, 320.0 mg / kg 0 mg / kg, 30.0 mg / kg, 35.0 mg / kg, 40.0 mg / kg, 45.0 mg / kg, 50.0 mg / kg, 60.0 mg / kg, 70.0 mg / kg, 80.0 mg / kg, 90.0 mg / kg, 100.0 mg / kg, 150.0 mg / kg, 200.0 mg / kg, 250.0 mg / kg, 300.0 mg / kg, 350.0 mg / kg, 400.0 mg / kg, 450.0 mg / kg, to greater than about 500.0 mg / kg or any increment therebetween. All values ​​and ranges between these values ​​and ranges should be understood to be encompassed by the present invention.

[0119] The compounds of the present disclosure can be administered at dosages ranging from about 0.2 milligrams / kg / day to greater than about 100 mg / kg / day. For example, dosages can range from 0.2 mg / kg / day to 100 mg / kg / day, 0.2 mg / kg / day to 50 mg / kg / day, 0.2 mg / kg / day to 25 mg / kg / day, 0.2 mg / kg / day to 10 mg / kg / day, 0.2 mg / kg / day to 7.5 mg / kg / day, 0.2 mg / kg / day to 5 mg / kg / day, 0.25 mg / kg / day to 100 mg / kg / day, 0.25 mg / kg / day to 50 mg / kg / day, 0.25 mg / kg / day to 25 ... ~10mg / kg / day, 0.25mg / kg / day ~7.5mg / kg / day, 0.25mg / kg / day ~5mg / kg / day, 0.5mg / kg / day ~50mg / kg / day, 0.5mg / kg / day ~25mg / kg / day, 0.5mg / kg / day ~20mg / kg / day, 0.5mg / kg / day ~15mg / kg / day, 0.5mg / kg / day ~10mg / kg / day, 0.5mg / kg / day ~7.5mg / kg / day, 0.5mg / kg / day ~5mg / kg / day, 0.75mg / kg / day ~5 0mg / kg / day, 0.75mg / kg / day ~ 25mg / kg / day, 0.75mg / kg / day ~ 20mg / kg / day, 0.75mg / kg / day ~ 15mg / kg / day, 0.75mg / kg / day ~ 10mg / kg / day, 0.75mg / kg / day day~7.5mg / kg / day, 0.75mg / kg / day~5mg / kg / day, 1.0mg / kg / day~50mg / kg / day, 1.0mg / kg / day~25mg / kg / day, 1.0mg / kg / day~20mg / kg / day, 1.0mg / kg / day It can be 2mg / kg / day to 50mg / kg / day, 2mg / kg / day to 25mg / kg / day, 2mg / kg / day to 20mg / kg / day, 2mg / kg / day to 15mg / kg / day, 2mg / kg / day to 10mg / kg / day, 2mg / kg / day to 7.5mg / kg / day, or 2mg / kg / day to 5mg / kg / day.

[0120] The compounds of the present disclosure can be administered at dosages ranging from about 0.25 milligrams / kg / day to about 25 mg / kg / day. For example, dosages can range from 0.25 mg / kg / day, 0.5 mg / kg / day, 0.75 mg / kg / day, 1.0 mg / kg / day, 1.25 mg / kg / day, 1.5 mg / kg / day, 1.75 mg / kg / day, 2.0 mg / kg / day, 2.25 mg / kg / day, 2.5 mg / kg / day, 2.75 mg / kg / day, 3.0 mg / kg / day, 3.25 mg / kg / day, 3.5 mg / kg / day, 3.75 mg / kg / day, 4.0 ... kg / day, 4.25mg / kg / day, 4.5mg / kg / day, 4.75mg / kg / day, 5mg / kg / day, 5.5mg / kg / day, 6.0mg / kg / day, 6.5mg / kg / day, 7.0mg / kg / day, 7.5mg / kg / day, 8.0mg / kg / day, 8.5mg / kg / day, 9.0mg / kg / day, 9.5mg / kg / day, 10mg / kg / day, 11mg / kg / day, 12mg / kg / day, 13mg / kg / day, 1 4mg / kg / day, 15mg / kg / day, 16mg / kg / day, 17mg / kg / day, 18mg / kg / day, 19mg / kg / day, 20mg / kg / day, 21mg / kg / day, 22mg / kg / day, 23mg / day kg / day, 24mg / kg / day, 25mg / kg / day, 26mg / kg / day, 27mg / kg / day, 28mg / kg / day, 29mg / kg / day, 30mg / kg / day, 31mg / kg / day, 32mg / kg / day , 33 mg / kg / day, 34 mg / kg / day, 35 mg / kg / day, 36 mg / kg / day, 37 mg / kg / day, 38 mg / kg / day, 39 mg / kg / day, 40 mg / kg / day, 41 mg / kg / day, 42 mg / kg / day, 43 mg / kg / day, 44 mg / kg / day, 45 mg / kg / day, 46 mg / kg / day, 47 mg / kg / day, 48 mg / kg / day, 49 mg / kg / day, or 50 mg / kg / day.

[0121] The compound or precursor thereof can be administered at a concentration ranging from 0.01 micromolar to greater than or equal to 500 micromolar. For example, doses can be 0.01 micromolar, 0.02 micromolar, 0.05 micromolar, 0.1 micromolar, 0.15 micromolar, 0.2 micromolar, 0.5 micromolar, 0.7 micromolar, 1.0 micromolar, 3.0 micromolar, 5.0 micromolar, 7.0 micromolar, 10.0 micromolar, 15.0 micromolar, 20.0 micromolar, 25.0 micromolar, 30.0 micromolar, 35.0 micromolar, 40. 0 micromolar, 45.0 micromolar, 50.0 micromolar, 60.0 micromolar, 70.0 micromolar, 80.0 micromolar, 90.0 micromolar, 100.0 micromolar, 150.0 micromolar, 200.0 micromolar, 250.0 micromolar, 300.0 micromolar, 350.0 micromolar, 400.0 micromolar, 450.0 micromolar, to greater than about 500.0 micromolar or any increment therebetween, it being understood that all values ​​and ranges between these values ​​and ranges are encompassed by the present invention.

[0122] The compound or its precursor can be administered at a concentration ranging from 0.10 micrograms / mL to 500.0 micrograms / mL. For example, concentrations can be 0.10 micrograms / mL, 0.50 micrograms / mL, 1 micrograms / mL, 2.0 micrograms / mL, 5.0 micrograms / mL, 10.0 micrograms / mL, 20 micrograms / mL, 25 micrograms / mL, 30 micrograms / mL, 35 micrograms / mL, 40 micrograms / mL, 45 micrograms / mL, 50 micrograms / mL, 60.0 micrograms / mL, 70.0 micrograms / mL, 80.0 micrograms / mL, 90.0 micrograms / mL, 100.0 micrograms / mL, 110.0 micrograms / mL, 120.0 micrograms / mL, 130.0 micrograms / mL, 140.0 micrograms / mL, 150.0 micrograms / mL, 160.0 micrograms / mL, 170.0 micrograms / mL, 180.0 micrograms / mL, 190.0 micrograms / mL, 200.0 micrograms / mL, 210.0 micrograms / mL, 220.0 micrograms / mL, 230.0 micrograms / mL, 240.0 micrograms / mL, 250.0 micrograms / mL, 260.0 micrograms / mL, 270.0 micrograms / mL, 280.0 micrograms / mL, 290.0 micrograms / mL, 300.0 micrograms / mL, 350.0 micrograms / mL, 360.0 micrograms / mL, 370.0 micrograms / mL, 380.0 micrograms / mL, The concentration may be from about 500.0 micrograms / mL or any increment thereof, up to about 500.0 micrograms / mL, 80.0 micrograms / mL, 90.0 micrograms / mL, 100.0 micrograms / mL, 150.0 micrograms / mL, 200.0 micrograms / mL, 250.0 micrograms / mL, 250.0 micrograms / mL, 300.0 micrograms / mL, 350.0 micrograms / mL, 400.0 micrograms / mL, 450.0 micrograms / mL, to about 500.0 micrograms / mL or any increment thereof. All values ​​and ranges between these values ​​and ranges should be understood to be encompassed by the present invention.

[0123] The formulations of the present disclosure can be administered in pharma- ceutically acceptable solutions that may routinely contain pharma- ceutically acceptable concentrations of salts, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.For use in treatment, an effective amount of the compound can be administered to a subject by any mode that delivers the compound to the desired surface.Administering pharmaceutical compositions can be accomplished by any means known to those skilled in the art.Routes of administration include, but are not limited to, intravenous, intramuscular, intraperitoneal, intravesical (bladder), oral, subcutaneous, direct injection (e.g., into tumor or abscess), mucosa (e.g., external application to eye), inhalation, and topical.

[0124] For intravenous and other parenteral routes of administration, the compounds of the present disclosure can be formulated as lyophilized preparations, as lyophilized preparations of liposome-entrapped or encapsulated active compounds, as lipid complexes in aqueous suspension, or as salt complexes.Lyophilized preparations are generally reconstituted in a suitable aqueous solution, such as sterile water or saline, immediately prior to administration.

[0125] For oral administration, the compound can be easily formulated by combining the active compound with pharma- ceutically acceptable carriers well known in the art. Such carriers allow the compound of the present disclosure to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., to be orally ingested by the subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipients, and optionally the mixture obtained is ground, and the mixture of granules is processed to obtain tablets or dragee cores, after adding suitable auxiliary agents as required. Suitable excipients are, in particular, sugars, including fillers such as lactose, sucrose, mannitol or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP), etc. If desired, disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, e.g., sodium alginate. Optionally, the oral preparations may also be formulated in saline or buffers, e.g., EDTA for neutralizing internal acidic conditions, and may be administered without any carrier.

[0126] Oral dosage forms of the compounds of the present disclosure are also contemplated. The compounds of the present disclosure may be chemically modified to effect oral delivery of the derivatives. In general, the contemplated chemical modifications are to attach at least one moiety to the compound itself, where the moiety (a) inhibits acid hydrolysis; and (b) allows uptake from the stomach or intestine into the bloodstream. Also desirable is an increase in the overall stability of the compound and an increase in circulation time in the body. Examples of such moieties include polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, and polyproline. Abuchowski and Davis, "Soluble Polymer-Enzyme Adducts", In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY, pp. 367-383 (1981);Newmark et al., J Appl Biochem 4:185-9 (1982). Other polymers that can be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane.For pharmaceutical applications, as indicated above, polyethylene glycol moieties are preferred.

[0127] The location of release of the compound of the present disclosure can be the stomach, the small intestine (duodenum, jejunum or ileum), or the large intestine.Those skilled in the art can utilize formulations that do not dissolve in the stomach but release the substance in the duodenum or other places in the intestine.Release can avoid the harmful effects of the stomach environment by either protecting the compound of the present disclosure or releasing the compound beyond the stomach environment, such as the intestine.

[0128] To ensure adequate gastric resistance, a coating that is impermeable to at least pH 5.0 is essential. Examples of the more common inactive ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and shellac. These coatings may be used as mixed membranes.

[0129] A coating or mixture of coatings may also be used on tablets, which are not intended for gastric protection. This may include sugar coatings, or coatings that make the tablet easier to swallow. Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutics (e.g., powder); for liquid forms, a soft gelatin shell may be used. The shell material for cachets can be thick starch or other edible paper. For pills, lozenges, moist tablets, or powder tablets, moist mashing techniques may be used.

[0130] The therapeutic agent may be included in the formulation as fine multiparticulates in the form of granules or pellets about 1 mm in size. Formulation of material for capsule administration may be as a powder, low compression plugs or even tablets. The therapeutic agent may also be prepared by compression.

[0131] Colorants and flavoring agents may also be included. For example, the compounds of the present disclosure may be formulated (e.g., by liposome or microsphere encapsulation) and then further incorporated into edible products, such as refrigerated beverages, that contain colorants and flavoring agents.

[0132] Inert substances may be used to dilute or bulk up the therapeutic agent. These diluents may also include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts, including calcium triphosphate, magnesium carbonate and sodium chloride, may be used as bulking agents. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

[0133] Disintegrants may be included in the formulation of therapeutic agents into solid dosage forms. Materials used as disintegrants include, but are not limited to, starch, including the starch-based commercial disintegrant Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethylcellulose, natural sponge and bentonite may all be used. Another form of disintegrant is an insoluble cation exchange resin. Powdered gums may be used as disintegrants and binders, and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

[0134] Binders may be used to hold the therapeutic substances together to form hard tablets and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methylcellulose (MC), ethylcellulose (EC) and carboxymethylcellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC) can also be used together in an alcoholic solution to granulate the therapeutic agent.

[0135] Antifriction agents may be included in the formulation of therapeutic agents to prevent sticking during the formulation process.Lubricants may be used as a layer between therapeutic agents and the mold wall, and may include, but are not limited to, stearic acid, including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes.Soluble lubricants may also be used, such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycols of various molecular weights, Carbowax 4000 and 6000.

[0136] Glidants may be added which will improve the flow of the drug during formulation and aid in reconstitution during compression. Glidants can include starch, talc, pyrogenic silica and hydrated silicoaluminate.

[0137] Surfactants may be added as wetting agents to aid in the dissolution of therapeutic agents into the aqueous environment. Surfactants may include anionic detergents such as sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate. Cationic detergents that can be used include benzalkonium chloride and benzethonium chloride. Nonionic detergents that can be included in the formulation as surfactants include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid esters, methylcellulose and carboxymethylcellulose. These surfactants can also be present in the formulation of the compounds or derivatives of the present disclosure either alone or as a mixture in different ratios.

[0138] Orally usable pharmaceutical preparations include push-fit capsules made of gelatin, as well as soft sealed capsules made of gelatin and plasticizers such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with fillers such as lactose, binders such as starch, and / or lubricants such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, active compounds can be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers can be added. Microspheres formulated for oral administration can also be used. Such microspheres are well defined in the art. All formulations for oral administration should be in a dosage suitable for such administration.

[0139] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0140] For local administration, the compounds may be formulated as solutions, gels, ointments, creams, suspensions, etc., as is well known in the art. Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal buccal or pulmonary administration.

[0141] For administration by inhalation, the compound for use according to the present disclosure can be conveniently delivered in the form of aerosol spray presentation from pressurized pack or nebulizer, using suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.In the case of pressurized aerosol, dosage unit can be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, for example, gelatin, for use in inhaler or insufflator can be formulated, containing a powder mix of the compound and suitable powder base, for example, lactose or starch.

[0142] Also contemplated herein is pulmonary delivery of the compounds disclosed herein (or salts thereof). The compounds are delivered to the lungs of a mammal during inhalation and cross the lung epithelial lining into the bloodstream. Other reports of inhaled molecules include Adjei et al., Pharm Res 7:565-569 (1990); Adjei et al., Int J Pharmaceutics 63:135-144 (1990) (leuprolide acetate); Braquet et al., J Cardiovasc Pharmacol 13(suppl. 5):143-146 (1989) (endothelin-1); Hubbard et al., Annal Int Med 3:206-212 (1989) (a1-antitrypsin); Smith et al., 1989, J Clin Invest 84:1145-1146 (a-1-proteinase); Oswein et al., 1990, "Aerosolization of Proteins", Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March (recombinant human growth hormone); Debs et al., 1988, J Immunol 140:3482-3488 (interferon-γ and tumor necrosis factor α) and Platz et al., U.S. Patent No. 5,284,656 (granulocyte colony-stimulating factor; incorporated by reference). Methods and compositions for pulmonary delivery of drugs for systemic effect are described in Wong et al., U.S. Patent No. 5,451,569, issued September 19, 1995 (incorporated by reference).

[0143] A wide range of mechanical devices designed for pulmonary delivery of therapeutic products are contemplated for use in the practice of the present disclosure, including, but not limited to, nebulizers, metered dose inhalers and dry powder inhalers, all of which are well known to those skilled in the art.

[0144] Some specific examples of commercially available devices suitable for practicing the present disclosure are the Ultravent nebulizer manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer manufactured by Marquest Medical Products, Englewood, Colo.; the Ventolin metered dose inhaler manufactured by Glaxo Inc., Research Triangle Park, North Carolina; and the Spinhaler powder inhaler manufactured by Fisons Corp., Bedford, Mass.

[0145] All such devices require the use of suitable formulations for dispensing the compounds of the present disclosure. Typically, each formulation is specific to the type of device used, and may use appropriate propellant substances in addition to the usual diluents, adjuvants and / or carriers useful in therapy. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is envisioned. Also, the chemically modified compounds of the present disclosure may be prepared in different formulations depending on the type of chemical modification or the type of device used.

[0146] Formulations suitable for use in either jet or ultrasonic nebulizers typically contain the disclosed biologically active compound dissolved in water at a concentration of about 0.1-25 mg per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for inhibitor stabilization and osmolality adjustment). Nebulizer formulations may also contain a surfactant to reduce or prevent surface-induced aggregation of the disclosed compound caused by atomization of the solution upon aerosol formation.

[0147] The formulation for use in metered dose inhalation device generally comprises a finely divided powder containing the compound of the present disclosure suspended in a propellant with a surfactant.The propellant can be any conventional material used for this purpose, such as chlorofluorocarbon, hydrochlorofluorocarbon, hydrofluorocarbon, or hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, or combinations thereof.Suitable surfactants include sorbitan trioleate and soybean lecithin.Oleic acid can also be useful as a surfactant.

[0148] Formulations for dispensing from a powder inhalation device include finely divided dry powders containing the compounds of the present disclosure and may also include bulking agents such as lactose, sorbitol, sucrose, or mannitol in amounts to facilitate dispersion of the powder from the device, e.g., 50-90% by weight of the formulation. Compounds of the present disclosure may be prepared in particulate form with an average particle size of less than 10 micrometers (mm), or 0.5-5 mm, for delivery to the deep lung.

[0149] Nasal delivery of the pharmaceutical compositions of the present disclosure is also contemplated. Nasal delivery allows the pharmaceutical compositions of the present disclosure to pass directly to the bloodstream without the need to administer the therapeutic product to the nose and then deposit the product in the lungs. Formulations for nasal delivery include those using dextran or cyclodextran.

[0150] For nasal administration, a useful device is a small hard bottle with a metered dose sprayer attached.In one embodiment, the metered dose is delivered by drawing the solution of the pharmaceutical composition of the present disclosure into a chamber of a specified volume, which has an opening sized to aerosolize the aerosol formulation by forming a spray when the liquid in the chamber is compressed.The chamber is compressed to administer the pharmaceutical composition of the present disclosure.In a particular embodiment, the chamber is a piston arrangement.Such devices are commercially available.

[0151] Alternatively, a plastic squeeze bottle is used that has an opening or hole sized to aerosolize the aerosol formulation by forming a spray when squeezed. The hole is usually found at the top of the bottle, which is generally tapered to partially fit into the nasal cavity so that the aerosol formulation is administered efficiently. Nasal inhalers can provide a metered dose of the aerosol formulation so that a measured dose of the drug is administered.

[0152] When it is desired to deliver the compound systemically, the compound can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion.The preparation for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with the addition of preservatives.The composition can take the form of a suspension, solution or emulsion in oily or aqueous medium, and can contain formulating agents such as suspending agents, stabilizing agents and / or dispersing agents.

[0153] Pharmaceutical preparations for parenteral administration include aqueous solutions of active compounds in water-soluble form.In addition, suspensions of active compounds may be prepared as oily injection suspensions if necessary.Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes.Aqueous injection suspensions may contain substances that increase the viscosity of suspensions, such as sodium carboxymethylcellulose, sorbitol or dextran.Optionally, suspensions may also contain suitable stabilizers or agents that increase the solubility of compounds, allowing the preparation of highly concentrated solutions.

[0154] Alternatively, the active compound may be in powder form for reconstitution with a suitable vehicle, eg, sterile pyrogen-free water, before use.

[0155] The compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

[0156] In addition to the above formulations, the compounds can be formulated as depot preparations. Such long-acting preparations can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as sparingly insoluble salts.

[0157] The pharmaceutical compositions may also include suitable solid or gel phase carriers or excipients, examples of which include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

[0158] Suitable liquid or solid pharmaceutical preparation forms include, for example, aqueous solutions or saline solutions for inhalation, microencapsulated, encochleated, coated on fine gold particles, contained in liposomes, nebulized, aerosolized, pellets for intradermal implantation, or dried on a sharp object for rubbing into the skin. Pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops, or preparations for extended release of active compounds, in which excipients and additives and / or auxiliary agents, such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners, or solubilizers, are routinely used as described above. Pharmaceutical compositions are suitable for use in various drug delivery systems. For a brief review of drug delivery methods, see Langer R, Science 249:1527-33 (1990).

[0159] The compounds of the present disclosure and optionally other therapeutic agents may be administered per se (pure form) or in the form of a pharmaceutically acceptable salt. When used in medicine, the salt should be pharmaceutically acceptable, but it may be convenient to prepare its pharmaceutically acceptable salt using a non-pharmaceutically acceptable salt. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Such salts can also be prepared as alkali metal or alkaline earth metal, for example, sodium, potassium, or calcium salts of the carboxylic acid group.

[0160] Suitable buffering agents include acetic acid and salts (1-2% w / v), citric acid and salts (1-3% w / v), boric acid and salts (0.5-2.5% w / v), and phosphoric acid and salts (0.8-2% w / v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w / v), chlorobutanol (0.3-0.9% w / v), parabens (0.01-0.25% w / v), and thimerosal (0.004-0.02% w / v).

[0161] The pharmaceutical composition of the present disclosure contains an effective amount of the compound as described herein and optionally a therapeutic substance in a pharma- ceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to humans or other vertebrates. The term "carrier" refers to a natural or synthetic organic or inorganic component with which an active ingredient is combined to facilitate application. The components of the pharmaceutical composition are also capable of being mixed with the compounds of the present disclosure and with each other in such a way that there is no interaction that would substantially impair the desired pharmaceutical effectiveness.

[0162] The therapeutic agent, specifically including but not limited to the compounds of the present disclosure, may be provided in particles. Particles, as used herein, refer to nanoparticles or microparticles (or in some examples larger particles) that may be composed in whole or in part of the compounds of the present disclosure or other therapeutic agents as described herein. The particles may contain the therapeutic agent in a core surrounded by a coating, including but not limited to an enteric coating. The therapeutic agent may also be dispersed throughout the particle. The therapeutic agent may also be adsorbed within the particle. The particles may be of any order of release kinetics, including zero-order release, first-order release, second-order release, delayed release, sustained release, immediate release, and any combination thereof. The particles may contain, in addition to the therapeutic agent, any of the materials routinely used in the pharmaceutical and medical fields, including but not limited to erodible, non-erodible, biodegradable or non-biodegradable materials or combinations thereof. The particles may be microcapsules containing the compounds of the present disclosure in solution or in a semi-solid state. The particles may be of virtually any form.

[0163] Both non-biodegradable and biodegradable polymeric materials can be used in the manufacture of particles for delivering therapeutic substances. Such polymers can be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired. Bioadhesive polymers of particular interest include the bioerodible hydrogels described in Sawhney HS et al. (1993) Macromolecules 26:581-7, the teachings of which are incorporated herein. These include polyhyaluronic acid, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).

[0164] The therapeutic substance may be contained in a controlled release system. The term "controlled release" is intended to refer to any drug-containing formulation in which the manner and profile of drug release from the formulation is controlled. It refers to immediate release formulations as well as non-immediate release formulations, including but not limited to sustained release formulations and delayed release formulations. The term "sustained release" (also referred to as "extended release") is used in its conventional sense to refer to a drug formulation that provides sustained release of drug over an extended period of time, and can result in a substantially constant blood drug concentration over an extended period of time. The term "delayed release" is used in its conventional sense to refer to a drug formulation in which there is a time delay between administration of the formulation and the release of drug therefrom. "Delayed release" may or may not involve sustained release of drug over an extended period of time, and therefore may or may not be a "sustained release".

[0165] For the treatment of chronic conditions, the use of long-term sustained release implants may be particularly suitable. "Long-term" release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days and up to 30-60 days. Long-term sustained release implants are well known to those of ordinary skill in the art and include some of the release systems described above.

[0166] Other suitable modifications and adaptations to the compositions and methods described herein will be readily apparent from the description of the disclosure contained herein in light of the information known to those skilled in the art, and may be made without departing from the scope of the disclosure or any of its aspects, as will be appreciated by those of ordinary skill in the relevant art. Although the disclosure has now been described in detail, the same will be more clearly understood by reference to the following examples, which are included herein for illustrative purposes only and are not intended to be limiting of the disclosure. EXAMPLES

[0167] Having now generally described the invention, the same will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to be limiting of the invention.

[0168] material and method mouse P301S transgenic mice (https: / / www.jax.org / strain / 008169) were crossed with cGas knockout mice (https: / / www.jax.org / strain / 026554) to generate littermate P301S cGas+ / + mice, P301S cGas+ / - and littermate P301S cGas- / - mice, as well as non-transgenic littermates. Mice of both sexes were used for behavioral, histological and biochemical analyses. At 7–8 months of age, mice were subjected to behavioral testing and were not used for other experiments. At 9–10 months of age, the same mice were used for pathology and RNA-seq studies. For TDI-6570 in vivo treatment, 6–7 month old P301S and non-transgenic littermate mice were used for dietary experiments and assayed for behavior and histology at 9–10 months. Mice were housed under specific pathogen-free conditions under a 12-h light / dark cycle, and all mouse protocols were approved by the Institutional Animal Care and Use Committee at the University of California, San Francisco.

[0169] RNA isolation Brains harvested from freshly perfused mice were dissected to isolate hippocampi and cortex. Hippocampi were divided into two halves and frozen at -80°C until experimentation. To isolate RNA, hippocampi were thawed on ice for 30 min and then homogenized. Briefly, hippocampi were passed through a solution of RLT buffer containing 1% β-mercaptoethanol using a 21G needle. Following homogenization, samples were spun down briefly and then frozen at -80°C overnight. The next day, samples were thawed on ice and then spun down at 14000 rpm for 6 min at 4°C. RNA isolation was performed on hippocampal lysates according to the manufacturer's protocol (RNeasy mini-kit, Qiagen). Isolated RNA was sent to Weill Cornell Medicine Genomics Core for analysis of RNA quality and integrity. All samples passed QC and RNA sequencing libraries were prepared for sequencing using NovaSeq. For validation real-time qPCR experiments, cDNA was prepared using iScript Reverse Transcription supermix (BioRad). Rt-qPCR was performed in triplicate using SYBR green PCR master mix (Applied Biosystems) on an ABI7900HT sequence detector (Applied biosystems). The change in Ct value between the transcript of interest and mouse GAPDH was calculated. Then, 2 ΔCt Relative gene expression was determined using and then expressed as relative fold change.

[0170] Western Blot: For mouse brain samples, half of the hippocampus was mechanically homogenized in RIPA buffer containing protease and phosphatase inhibitors (Millipore Sigma) on ice. 50ug of hippocampal lysate was used to analyze protein expression in the brain. Samples were loaded onto a NuPage Bis-Tris gel (ThermoFisher) and run in SDS running buffer at 150V for approximately 2.5 hours. Gels were transferred to methanol-activated nitrocellulose membranes (BioRad) overnight in a cold room. Membranes were washed 3×10 minutes in TBS containing 0.01% Triton X-100 (TBST) and blocked for 1 hour in 5% milk TBST. Appropriate primary antibodies were diluted in 1% milk TBST and incubated overnight at 4°C. The next day, membranes were washed 3×10 minutes in TBST and then incubated with appropriate secondary antibodies in 1% milk TBST at room temperature for 1 hour. Membranes were washed again to minimize non-specific binding and then treated with ECL (BioRad) for 60 seconds and developed in the dark. Blots were scanned at 300 DPI and quantified using ImageJ.

[0171] For human brain samples, frontal cortex lysates were prepared as previously described (Min et al., 2010). Briefly, human or mouse brain tissues were lysed in RIPA buffer containing protease inhibitor cocktail (Sigma), 1 mM phenylmethylsulfonyl fluoride (Sigma), phosphatase inhibitor cocktail (Roche) and HDAC inhibitors including 5 mM nicotinamide (Sigma) and 1 mM trichostatin A (Sigma). After sonication, lysates were centrifuged at 170,000 g for 15 min at 4°C. Supernatants were collected and analyzed by Western blot. Protein concentrations were measured by BCA assay (Thermo Scientific).

[0172] For cultured primary microglia, 1-2 million cells were lysed in M-PER™ Mammalian Protein Extraction Reagent (Thermo Scientific) supplemented with HALT protease and phosphatase inhibitor cocktail (Thermo Scientific) and 150 mM NaCl. Samples were rotated for 10 min at 4°C. Lysates were clarified by centrifugation at 16000×g for 15 min at 4°C. Protein concentrations were measured by BCA assay.

[0173] Nuclei isolation from frozen mouse hippocampus Hippocampal isolation from frozen mouse hippocampi was adapted with modifications from previous studies (Grubman et al., 2019; Habib et al., 2017). All procedures were performed on ice or at 4°C. Briefly, postmortem brain tissue was placed in 1500 μl of Sigma nuclei PURE lysis buffer (Sigma, NUC201-1KT) and homogenized using a Dounce tissue grinder (Sigma, D8938-1SET) with 20 strokes of pestle A and 15 strokes of pestle B. The homogenized tissue was filtered through a 35 μm cell strainer and then centrifuged at 600 g for 5 min at 4°C and lysed with 1% BSA, 20 mM DTT and 0.2 U μl of 1% BSA. -1 The cells were washed three times with 1 ml of PBS containing recombinant RNase inhibitor. The nuclei were then centrifuged at 600 g for 5 min at 4° C. and resuspended in 800 μl of PBS containing 0.04% BSA and 1×DAPI, followed by removal of cell debris by FACS sorting. The suspension of FACS-sorted DAPI-stained nuclei was counted and diluted to a concentration of 1000 nuclei per microliter in PBS containing 0.04% BSA.

[0174] Droplet-based single-nucleus RNA sequencing For droplet-based snRNA-seq, libraries were prepared using Chromium Single Cell 3' Reagent Kits v3 (10x Genomics, PN-1000075) according to the manufacturer's protocol. snRNA-seq libraries were sequenced on a NovaSeq 6000 sequencer (Illumina) for 100 cycles.

[0175] Analysis of droplet-based single-nucleus RNA-seq data from human brain tissue Gene counts were obtained by aligning reads to the mm10 genome using Cell Ranger software (v.3.1.0) (10x Genomics). To take into account unspliced ​​nuclear transcripts, reads mapped to pre-mRNA were counted. Cell barcodes were called using Cell Ranger 3.1.0 default parameters. We further excluded genes expressed in only two types of cells, i.e., cells with unique gene counts >4,000 or <200, and cells with a high fraction of mitochondrial reads (>5%). DoubletFinder (McGinnis et al., 2019) was used to predict potential doublet cells separately for each sample, and high-confidence doublets were removed. Normalization and clustering were performed using the Seurat package v3.2.2 (Stuart et al., 2019). Briefly, counts for all nuclei were scaled by multiplying the total library size by a scale factor (10,000) and transformed into log space. A set of 2000 highly variable genes was identified using SCTransform from the sctransform R package in variable stabilization mode. This was converted back to the corrected unique molecular identifier (UMI) count matrix, the log-transformed data matrix, and the Pearson residuals from a regularized negative binomial regression model. Principal component analysis (PCA) was performed on all genes and t-SNE was run on the top 20 PCs. Cell clusters were identified using Seurat functions FindNeighbors (using the top 20 PCs) and FindClusters (resolution = 0.02). In this analysis, the neighborhood size parameter pK was estimated using the mean variance normalized bimodality coefficient (BCmvn) approach using 20 PCs and automatically setting pN to 0.25. For each cluster, we assigned a cell type label using statistical enrichment on a set of marker genes (Lake et al., 2018; Wang et al., 2018) and manual assessment of gene expression on a small set of known marker genes.Differentially expressed gene analysis was performed using FindMarkers function and MAST (Finak et al., 2015). To identify gene ontology and pathways enriched in differentially expressed genes (DEGs), the (MSigDB) gene annotation database (Liberzon et al., 2011; Subramanian et al., 2005) was used to analyze the DEGs. To control for multiple testing, we used the Benjamini-Hochberg (BH) approach to limit the false discovery rate (FDR). For trajectory analysis, Seurat objects were converted to cds objects and analyzed using Monocle 3 (Cao et al., 2019; Qiu et al., 2017; Trapnell et al., 2014). Moran's I spatial autocorrelation analysis was performed to identify gene modules significantly associated with microglial trajectories. Target gene enrichment analysis was performed using the GeneOverlap package in R. Briefly, MEF2C, MEF2A, JunB and FOSL2 target gene lists, as well as the human cognitive resilience gene list (kindly shared by Dr. Li Huei Tsai) and P301S Cgas- / - vs. P301S EN / IN DEG list were used as input for comparison. Results include overlap p-values ​​and odds ratios to examine associations between the two databases.

[0176] antibody Antibodies used for immunofluorescence analysis were as follows: secondary antibodies used were Alexa fluor donkey anti-rabbit / goat 488 and anti-mouse 555, and Jackson donkey anti-goat 555 at 1:500 (Invitrogen).

[0177] Antibodies against STING (D2P2F, Cell Signaling Technology, 1:300), anti-IBA1 (ab5076, Abcam, 1:500), anti-PSD-95 (1:500, Millipore MAB1596), anti-vGAT (Ab5062, Millipore, 1:500), anti-pSTAT1 (mAb9167, Cell Signaling, 1:500), anti-NRG1 (MA5-12896, Invitrogen, 1:100), MC1 (a generous gift from Dr. Peter Davis), and anti-MEF2C (MAB6786, R&D systems 1:200).

[0178] Antibodies used for Western blots were: STING (as above), TBK1 (D1B4, Cell Signaling Technology, 1:1000), pTBK1 (D52C2, Cell Signaling Technology, 1:500), Caspase-3 (9661, Cell Signaling Technology, 1:1000), GAPDH (MAB374, Millipore, 1:10000 and GTX100118, GeneTex, 1:10000). Secondaries used were anti-rabbit HRP (401393, Calbiochem, 1:2000) or anti-mouse HRP (401253, Calbiochem, 1:2000), anti-NeuN (ABN78, Millipore, 1:500). For immunogold-labeled electron microscopy, an antibody against tau (A0024, Agilent Technologies, 1:1000) was used.

[0179] Immunofluorescence: Hemibrains from transcardially perfused mice were placed in 4% paraformaldehyde for 48 h at 4 °C, followed by 48 h in 30% sucrose in PBS. Sections were cut coronally at 40 μm using a freezing microtome (Leica) and placed in cryoprotective medium at -20 °C until use. 8–10 free-floating sections per mouse. All washing steps were 3 × 5 min. Sections were washed in TBST (0.01% Triton X-100), permeabilized with TBST (0.5% Triton X-100) for 15 min, and then washed again. Sections were then placed in antigen unmasking solution (citrate buffer, pH 6.0, h-3300) and, if necessary, placed in a 90 °C incubator for 30 min. Sections were washed and then blocked in 10% normal donkey serum (NDS, Vector BMK-2202) in PBST for 2 hours at room temperature. Primary antibodies were diluted in 5% NDS PBST and incubated overnight at 4°C. The next day, sections were washed extensively and incubated in appropriate secondary antibodies (1:500, Invitrogen) for 1 hour. Sections were washed, mounted on slides, and imaged using a Keyence BZ-X700 microscope. For pSTAT1 imaging, the CA1 region of mouse brain sections was imaged with a Zeiss Apotome 20x objective (Carl Zeiss). Images were acquired with a Z-stack spaced at 7um intervals with a step size of 1um. For MEF2C, images of mouse brain CA1 pyramidal region were acquired using a 25x objective on a Zeiss LSM880 confocal microscope (Carl Zeiss). Maximum projection images were generated using 4x1 tile scans and a Z-stack of 12um with a step size of 3μm, and images were stitched for each section. Slides were imaged using a Zeiss LSM 880 confocal microscope for PSD-95, vGAT and NRG1. Quantification was performed using area percentages based on thresholding determined using negative and positive controls using ImageJ software (NIH). For higher resolution, images were acquired by LSM 880 confocal microscopy using a 40x objective and Zen Black image acquisition software.The CA1 region of the hippocampus was imaged in 2 × 2 tile scans over a total distance of 15 μm per slice, with Z-stacks spaced 1 μm apart. Final images were processed with maximum intensity projection.

[0180] behavioral research cGas+ / + mice, P301S cGas+ / - and littermate P301S cGas- / - mice were compared with their respective non-transgenic or P301S littermates. Experimenters were blinded to mouse genotypes throughout the experiment. Male and female mice were tested on separate days.

[0181] Cgas+ / +, Cgas+ / - and Cgas- / - mice were compared to their respective P301S transgenic littermates. Male and female mice were tested on separate days and the experimenters were blinded to the mouse genotypes throughout the experiment.

[0182] For experiments involving TDI-6570 treatment, male P301S and non-transgenic littermates fed TDI-6570 or control diet were used.

[0183] Morris Water Maze The water maze consisted of a pool (122 cm diameter) containing opaque water (20 ± 1 °C) and a platform (10 cm diameter) 1.5 cm below the water surface. Three different images were attached to the walls of the room as spatial cues. Hidden platform training (days 1-7) consisted of 14 sessions (2 per day, 2 h apart), each with 2 trials. Mice were placed in the pool in alternating quadrants for each trial. A trial ended when the mouse settled on the platform or after 60 s had elapsed. 24 and 72 h after training, mice were tested in a probe trial in which the hidden platform was removed and the mouse was allowed to swim for 60 s. Before the 24 and 72 h probe trials, mice underwent 7 days of hidden platform training. The visible platform test was performed 24 h after the last probe trial. Performance was measured with an EthoVision video tracking system (Noldus Information Technology).

[0184] elevated cross maze The maze consisted of two open arms measuring 15 x 2 inches with no walls and two closed arms with walls 6.5 inches high, 30.5 inches above the ground. One hour prior to testing, mice were moved to the testing room and allowed to acclimate to dim lighting. Mice were placed individually at the intersection of the open and closed arms of the maze and allowed to explore the maze for 10 minutes.

[0185] Open Field Mice were placed individually into a brightly lit automated activity chamber (San Diego Instruments) equipped with an array of infrared photocells connected to a computer. Open field activity was recorded for 5 min. Recorded beam breaks were used to calculate total time of activity.

[0186] Novel object recognition test Mice were habituated to an opaque open-field arena (40×40 cm) in two 10-min trials spaced 2 days apart before being challenged with object recognition. 24 h after the second arena habituation trial, two identical objects (glass bottles) were placed in the center of each arena. Mice were allowed to explore these objects in one 15-min trial. The day after object habituation, one of the identical objects was replaced with a novel object (DUPLO® block structure) during a 15-min test period. Video recording and tracking (Ethovision v15, Noldus, Wageningen, the Netherlands) were used to determine the total distance traveled. An experimenter blinded to the groups manually scored the time that mice spent exploring each object. Preference was calculated based on the total time that each mouse spent exploring both objects.

[0187] Electrophysiology Brains were quickly dissected from anesthetized mice and placed in ice-cold stripping solution (gassed with 95% O2-5% CO2, pH ~7.4) containing (in mM) 210 sucrose, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 7 glucose, 2 MgSO4, and 0.5 CaCl2. Horizontal slices (400 μm thick) were made on a vibratome, and the slices were then incubated for 30 min in artificial cerebrospinal fluid (ACSF) (gassed with 95% O2-5% CO2, pH ~7.4) warmed to 35 °C containing (in mM) 119 NaCl, 2.5 KCl, 26.2 NaHCO3, 1 NaH2PO4, 11 glucose, 1.3 MgSO4, and 2.5 CaCl2. The slices were then kept at room temperature in oxygenated ACSF until recordings were performed.

[0188] Field recordings were performed in the dentate gyrus molecular layer of acute horizontal brain slices placed in a recording chamber. Slices were submerged in oxygenated ACSF continuously perfused at 30 °C. A glass recording electrode (pipette resistance of approx. 3 MΩ) was filled with ACSF and lowered approx. 50 μm into the molecular layer of the dorsal wing of the dentate gyrus. A bipolar tungsten electrode (FHC, Bowdoin, ME) located approx. 150 μm from the recording electrode was used to stimulate the perforant path input to the dentate gyrus. Stimulation pulses were generated by a Model 2100 Isolated Pulse Stimulator (AM Systems). Responses were elicited every 30 s with stimulus intensities ranging from 5 to 40 μA for a stimulus duration of 0.5 ms. After recording fEPSPs in response to stimuli of 5–40 μA, the stimulus intensity was adjusted to elicit 30% of the maximum fEPSP slope to set a baseline for LTP recordings. LTP recordings were performed in the presence of picrotoxin (100 μM, Sigma). After recording baseline fEPSPs for 20 min, theta burst stimulation (TBS) was administered, consisting of 10 bursts (4 pulses, 100 Hz) each 200 ms apart, with 10 theta bursts given every 15 s. Stimulation intensity was increased to a level of 60% of the maximum fEPSP slope only during TBS, and then returned to the stimulation intensity used during baseline recording after TBS. fEPSP slopes were normalized to the baseline response before LTP induction. Recordings were performed using a Multiclamp 700B amplifier (Molecular Devices), digitized at 10 kHz, acquired and analyzed using WinLTP software (version 1.11b, University of Bristol). Recordings and analysis were performed blinded to mouse genotype.

[0189] Microglia culture and isolation BV2 microglia culture. The BV2 microglia cell line was maintained in growth medium-DMEM (Thermo Fisher) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone) and 1% penicillin / streptomycin (Life Technologies) in a HERAcell 150i incubator (Caisson Labs) at 37 °C with 5% CO2. BV2 microglia were serially passaged when plates reached 80–90% confluency.

[0190] Isolation and culture of postnatal primary microglia. Primary microglial cells were harvested from 1-3 day old mouse pups (P1-P3). Briefly, brain cortex was isolated and finely minced. Tissue was dissociated in 0.25% trypsin-EDTA at 37°C for 10 min, vortexed every 5 min. Trypsin was neutralized with complete medium (DMEM (Thermo Fisher) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone)), filtered through a 70 μm cell strainer (BD Falcon), and pelleted by centrifugation at 1500 rpm. Mixed glial cultures were maintained in vitro for 7-10 days at 37°C and 5% CO2 in growth medium. Once bright round cells began to appear in the mixed glial cultures, recombinant mouse granulocyte-macrophage colony-stimulating factor (1 ng / ml, Life Technologies) was added to encourage microglial proliferation. After 48–72 h, primary microglial cells were harvested by mechanical agitation and plated onto poly-L-lysine-coated T-75 flasks (Corning) in Growth Medium. Assays were performed 24–48 h after microglial plating.

[0191] Interferon-β reporter system Molecular cloning. BV2 microglia were transiently transfected with the interferon-β reporter plasmid pNiFty3-I-Lucia (Invivogen) using Lipofectamine 2000 (ThermoFisher). Transfection medium was removed 6 hours post-transfection and replaced with complete growth medium. Selection with Zeocin was performed for 3 weeks. Resistant cells were plated as single clones in 96-well plates (Corning). Genomic DNA was isolated from the clones to confirm integration of the luciferase reporter. Clones were then verified for induction of interferon in response to cGas agonist.

[0192] Luciferase reporter assay. BV2 IfnB reporter microglia were stimulated with 0–10ug of HT-DNA (Sigma) or cyclic GAMP (Invivogen). DNA was delivered to cells using Lipofectamine 2000 (ThermoFisher). Luciferase levels secreted into the medium were measured using Quanti-Luc (Invivogen). Luminescence was measured on a BioTek Synergy hybrid reader.

[0193] cGas agonist assay Cell viability. BV2 microglia were treated with 0–100 uM TDI 6570 solubilized in DMSO. 24 h after treatment, cell viability was measured using the Cell Titer Glo assay (Promega). Briefly, cells in 96-well plates were equilibrated at room temperature for 30 min and then lysed. Luminescence was measured on a BioTek Synergy hybrid reader.

[0194] Functional assays. Primary postnatal microglia or BV2 IfnB reporter microglia were treated with 0–50 uM TDI6570 with or without HT-DNA transfection. RNA was isolated, cDNA was prepared, and the levels of cGas-related genes were assayed using the following primers: TIFF2025507382000015.tif36146. Alternatively, luciferase levels in the medium were measured as above.

[0195] Mitochondrial DNA depletion assay BV2 IfnB reporter microglia were treated with 50–100 ng / ml ethidium bromide (EtBr, Sigma Aldrich) or 40–80 μg / ml dideoxycytidine (ddC, Sigma Aldrich) for 7 days in DMEM supplemented with 10% FBS (GIBCO), 100 units / mL penicillin, and 100 μg / mL streptomycin. On day 7, cells were detached from the plate by scraping. A portion of the cells was saved for mtDNA quantification PCR assay. The remaining cells were plated in DMEM F12 supplemented with 100 units / mL penicillin, 100 μg / mL streptomycin, and left undisturbed for 48 h. Cells were then treated with 0N4R tau fibrils or ABT-737+Q-VD-Oph (ABT+QVD, 10 μM each, SelleckChem) for 24 h before assaying luciferase reporter activity and cell viability.

[0196] For mtDNA depletion qPCR, DNA was extracted from cell pellets using the DNeasy Blood and Tissue Kit (QIAGEN). The ratio of mtDNA (Nd2) to genomic DNA (Tert) was determined using the following primer pairs: Measured by SybrGreen real-time PCR (Bio-rad Laboratories) using TIFF2025507382000016.tif36128.

[0197] Enzyme-Linked Immunosorbent Assay (ELISA) and Multiplex Bead-Based Immunoassays Cell culture medium from cultured primary mouse microglia was collected 24 hours after stimulation and clarified by centrifugation at 2000 rpm for 5 minutes. Supernatants were diluted 1:10 and assayed using the VeriKine-HS™ Mouse IFN Beta Serum ELISA Kit (PBL Assay Science) according to the manufacturer's instructions. CXCL10 and CCL5 were measured using the MagPix System with the MILLIPLEX MAP Mouse Cytokine / Chemokine Magnetic Bead kit (Millipore).

[0198] Electron microscopy Electron microscopy experiments were performed by the electron microscopy core facility at Weill Cornell Medicine. Cells were washed with serum-free medium or appropriate buffers and then fixed with modified Karmovsky fixative of 2.5% glutaraldehyde, 4% paraformaldehyde, and 0.02% picric acid in 0.1 M sodium cacodylate buffer at pH 7.2. Following a second fixation in 1% osmium tetroxide, 1.5% potassium ferricyanide, samples were dehydrated through a graded ethanol series and embedded in situ in LX-112 resin (Ladd Research Industries). Ultrathin sections were cut using a Diatome diamond knife (Diatome, USA, Hatfield, PA) on a Leica Ultracut S ultramicrotome (Leica, Vienna, Austria). Sections were collected on copper grids and further counterstained with lead citrate. For immunolabeling, sections were collected on 200-mesh nickel grids. Briefly, sections were rehydrated in PBS. Unreacted aldehydes were quenched with 50 mM glycine in PBS, followed by blocking with a number of secondary ABs (Aurion, EMS) for 15 min at RT. Incubation with primary ABs was performed overnight at 4 °C in PBS-c (PBS + 0.2% BSA-c [Aurion, EMS]). The next day, sections were washed six times in PBS-c and incubated with secondary antibodies (Aurion gold conjugate, 1:100 in BB) for 1–2 h at RT. Washes were performed in PBS-c, followed by water. Sections were then fixed with 2.5% glutaraldehyde in 0.1 M buffer, washed in PBS, counterstained with uranyl acetate, and air-dried after a final wash in water. Samples were viewed under a JEM 1400 electron microscope (JEOL, USA, Inc., Peabody, MA) operated at 120 kV. Digital images were taken with a Veleta 2K × 2K CCD camera (Olympus-SIS, Germany).

[0199] Pharmacokinetic evaluation of TDI-6570 To study the pharmacokinetics, TDI-6570 (50 mg / kg, as a suspension in 0.5% methylcellulose solution in water containing 0.2% Tween 80) was administered intraperitoneally (IP) to 8-week-old CD-1 male mice (n=3 per time point) and both plasma (EDTA-K2) and brain tissue were collected at various time points (0.5, 2, 4, 8 and 24 hours after drug administration). Transcardial perfusion with saline was performed prior to brain collection. Bioanalysis of brain tissue and plasma extracts was performed by LC-MS / MS.

[0200] statistical analysis Statistical analysis was performed using GraphPad Prism 8. All data are expressed as mean ± SEM.

[0201] chemistry All commercially available chemicals and solvents were reagent grade and used without further purification. All air-sensitive reactions were carried out under argon protection. Column chromatography was performed using a silica gel SNAP column. Analytical thin-layer chromatography was performed using Merck 250 μM silica gel F 254 Preparative thin-layer chromatography was performed on Merck 1000 μM silica gel F plates. 254 The identity of each product was confirmed by mass spectrometry and CDCl as the solvent unless otherwise stated. 3 Chemical shifts were determined using NMR using a 350 nm NMR spectroscopy with a 350 nm NMR spectroscopy. Chemical shifts are reported in δ values ​​(ppm) downfield from TMS as internal standard. 1 H data are reported as follows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, br=broad, m=multiplet), coupling constant (Hz), integration.

[0202] result The cGas-STING pathway is activated in the hippocampus of tauopathy mice and human AD brains (Figure 1). Figure 1A is a volcano plot of RNA-seq data from bulk hippocampal tissue from 8-9 month old P301S and non-transgenic mice. Red dots represent genes with |log2 fold change|>0.5. Wald test was used. All other genes are shown in blue. Selected upregulated interferon genes are labeled. n=7 non-transgenic, n=6 P301S; Figure 1B is a gene set enrichment analysis showing hallmark pathways associated with the top 500 DEGs upregulated in P301S compared to non-transgenic samples; Figure 1C is a gene set enrichment analysis showing the top transcription factors associated with the top 500 DEGs upregulated in P301S compared to non-transgenic samples; Figure 1D is an IPA prediction of cGAS as an upstream regulator of upregulated DEGs identified using activation z-score >1 and overlap p-value <0.05; Figure 1E is a western blot for phosphorylated Tank-binding kinase (pTBK1), total Tank-binding kinase 1 (TBK1) and GAPDH using hippocampal tissue lysates. Lanes 1-7: non-transgenic (ntg); Lanes 8-14: P301S; Figure 1F is the ratio of pTBK1 / TBK1 from (E) showing significantly higher phospho-TBK1 in P301S compared to non-transgenic hippocampus. **p=0.0015 Student's two-tailed t-test; Figure 1G is a representative immunofluorescence image of non-transgenic and P301S hippocampi labeled with anti-Iba1 (green) and anti-STING antibodies (scale bar = 50 µm); Figure 1H is a quantification of Iba1 and Sting immunofluorescence intensity, showing increased Iba1 coverage and Iba1-Sting overlap in the P301S hippocampus. Results are presented as average intensity measurements from 3-4 cross sections per animal. p-value <0.05.Student's two-tailed t-test (n=5 Ntg, n=5 P301S); Figure 1I is a diagram showing the number of patients and brain regions used for single-nucleus 10x genomics sequencing (n=7, 4 males and 3 females); Figure 1J is a UMAP plot showing expression of microglial marker genes INPP5D and CSF1R as well as STAT1 and cGAS (MB21D1) in snRNA-Seq of human microglial populations; Figure 1K is a gene set enrichment analysis showing relevant hallmark pathways enriched in cGAS-expressing microglia; Figure 1L is a representative western blot for pTBK1 and GAPDH using human frontal cortex brain lysates. Lanes 1-3: non-AD (Braak stage 0); Lanes 4-6: AD (Braak stage 6); Figure 1M is the ratio of pTBK1 / GAPDH from (L) showing significantly higher phospho-TBK1 in human AD compared to non-AD brains. ** p < 0.01, two-tailed Student's t test (n = 10 non-AD, n = 8 AD).

[0203] Interferon activation in tau-stimulated microglia is mediated by cGAS and mitochondrial DNA leakage (Figure 2) Figure 2A is a quantification of IFNB by ELISA and CXCL10 and CCL5 proteins by MagPix multiplex ELISA in culture medium supernatants from untreated (control) and tau-treated (tau) primary mouse microglia. IFNB: n=7, ** p=0.0016, paired t-test; CXCL10 and CCL5, n=5, *** p=0.0004, ** p=0.0031, unpaired t-test; Figure 2B is a representative Western blot for phosphorylated Tank-binding kinase (pTBK1), total Tank-binding kinase 1 (TBK1) and GAPDH using mouse primary microglial cell lysates (lane 1: untreated; lane 2: treated with tau fibrils); Figure 2C is the ratio of pTBK1 / TBK1 from Figure 2A showing significantly higher phospho-TBK1 in primary microglia treated with tau fibrils (tau) compared to untreated (control). *, p<0.05, Student's paired t-test (n=3); Figure 2D is an electron micrograph of primary mouse microglia treated with tau fibrils and immunogold-labeled for antibodies against tau (L=lysosomes, M=mitochondria); Figure 2E is the ratio of mitochondrial DNA (Nd2) to genomic DNA (Tert) measured by RT-qPCR on DNA extracts of BV2 IfnB luciferase reporter cells treated for 7 days with ddC (40 or 80 μg / ml) or EtBr (50 or 100 ng / ml) to generate mtDNA-depleted (ρ°) cells. Values ​​are normalized to untreated samples (n=2), **** p<0.0001, one-way ANOVA; In Figure 2F, control and mtDNA-depleted (ρ°) IfnB luciferase-reporter BV2 cells were stimulated or not with tau fibrils. IfnB signal and viability were measured 16 h later. IfnB-luciferase signal is shown normalized to Cell TiterGlo signal to correct for viability / cell number (n=3), ****p<0.0001, 2-way ANOVA; Figure 2G is bulk RNA-seq analysis of Cgas+ / + and Cgas- / - primary microglia treated or not with tau fibrils or HT-strand DNA (n=3 per condition).Venn diagram showing overlap of genes upregulated by dsDNA and tau treatment in Cgas+ / + microglia. Log fold change >1 and FDR <0.05; Figure 2H is the top 5 reactome pathways represented by upregulated DEGs common to dsDNA and tau treated Cgas+ / + microglia. FDR <0.05; Figure 2I is a heatmap summary of interferon stimulated genes lower in Cgas- / - compared to HT-DNA or tau stimulated Cgas+ / + microglia; Figure 2J is a string interaction plot of genes from (I) including interferon genes including Stat1, Sp100 and Ddx60.

[0204] Partial or complete loss of cGas attenuates tauopathy-associated microglial interferon signature (Figure 3) Figure 3A is a dot plot showing normalized cell type expression of Cgas (Mb21d1) and Sting (Tmem173) in single nucleus sequencing (snRNA-Seq) samples; Figure 3B is a UMAP plot showing strong expression of marker genes P2ry12, Siglech, Sall1 and Csf1r in snRNA-Seq microglial populations (n=6 per genotype); Figure 3C is a UMAP plot colored according to microglial subclusters and divided by genotype; Figure 3D is a violin plot showing expression levels of homeostasis genes (P2ry12, Siglech), disease-related genes (Apoe, Itgax) and interferon genes (Parp14, Stat1, Trim30a, Rnf213) in microglial clusters; Figure 3E is a violin plot showing expression levels of homeostasis genes (P2ry12, Siglech), disease-related genes (Apoe, Itgax) and interferon genes (Parp14, Stat1, Trim30a, Rnf213) in microglial clusters compared to P301S Cgas+ / - and P301S Cgas+ / + microglia. FIG. 3F is a representative 63× confocal image of immunostaining for phosphor-STAT1 in the CA1 stratum radiatum of the mouse hippocampus (scale bar = 10 μm); FIG. 3G is the average intensity of phosphor-STAT1 measured in the CA1 stratum radiatum. Each circle represents the average intensity measurement of three images per animal. Statistical comparisons were performed using two-way ANOVA. Data are reported as mean ± SEM (n=6 Cgas+ / +, n=8 Cgas+ / -, n=7 Cgas- / -, n=6 P301S Cgas+ / +, n=9 P301S Cgas+ / -, n=5 P301S Cgas- / -) (Cgas+ / + vs. P301S Cgas+ / +: p=0.0002, P301S Cgas+ / + vs. P301S Cgas+ / -: p=0.0002, P301S Cgas+ / + vs. P301S Cgas- / -: p=0.0415); Figure 3H is a heatmap showing associations between gene modules and genotypes; Figure 3G is an analysis of disease module 1 and 2 markers compared to disease-associated, early response and late response microglial signatures.

[0205] Partial or complete loss of cGAS rescues tauopathy-induced hippocampal synaptic toxicity and memory impairment (Figure 4) Figure 4A: Cumulative exploration distance during the hidden trials (sessions 1-12) of the Morris Water Maze (MVM) assessment of spatial learning and memory in 7-8 month old P301S cGAS+ / +, P301S cGAS+ / - and P301S cGas- / - and their non-transgenic littermates. Males and females were tested on separate days. Data presented here represent both sexes combined. n=12 cGAS+ / +, n=11 cGAS+ / -, n=11 cGas- / -, n=8 P301S cGAS+ / +, n=17 P301S cGAS+ / -, n=6 P301S cGas- / -. Two-way ANOVA. ****, p<0.0001; Figure 4B: Percentage of mean time spent in the target quadrant or in the non-target (other) quadrant over the 24-h probe course of the MVM assessment. Paired two-tailed Student's t-test; Figure 4C: Percentage of mean time spent in the target quadrant or in the non-target (other) quadrant over the 72-h probe course of the MVM assessment. Paired two-tailed Student's t-test; Figure 4D: Field excitatory postsynaptic potentials (fEPSPs) were recorded in the dentate gyrus molecular layer and LTP was induced by applying a TBS protocol (arrow) to the perforant path. Representative traces show fEPSPs before and after LTP induction (top). Scale bars, 0.4 mV and 5 ms.fEPSP slope measurements made up to 60 min after TBS were normalized to the average baseline fEPSP slope before LTP induction (bottom, n = 8–11 cross sections from 3–4 mice per group); in Figure 4E, LTP magnitude was calculated from the normalized average fEPSP slope 55–60 min after TBS application (n = 8–11 cross sections from 3–4 mice per group; *, p < 0.05, **, p < 0.01; one-way ANOVA, Bonferroni post hoc analysis); in Figure 4F, granule, C Figure 4G is a pie chart summarizing the percentage of DEGs from the clusters relating to the dentate gyrus (DG), CA1 and CA2 / 3 clusters; Figure 4H is a representative confocal image of the CA1 radial striatum of the hippocampus labeled with PSD95 antibody (scale bar = 10 µm); Figure 4I is the average intensity of PSD-95 puncta measured in the CA1 radial striatum. Each circle represents the average intensity measurement of 3-5 images per animal. Statistical comparisons were performed using one- or two-way ANOVA (Cgas+ / + vs. P301S Cgas+ / +: p=0.0275, P301S Cgas+ / + vs. P301S Cgas+ / -: p=0.0002, P301S Cgas+ / + vs. P301S Cgas- / -: p=0.0415); data are reported as mean ± SEM.

[0206] Loss of cGAS rescues the expression of Mef2c and its target genes in tauopathy neurons (Figure 5) Figure 5A is a volcano plot showing representative differentially expressed genes upregulated in P301S Cgas- / - compared to P301S Cgas+ / + excitatory neurons (log2FC>0.1, FDR<0.05); Figure 5B is a representative 63x confocal image of immunostaining for NRG1 in the CA1 stratum radiatum of the mouse hippocampus (scale bar = 10 μm); Figure 5C is the average intensity of NRG1 measured in the CA1 stratum radiatum. Each circle represents the average intensity measurement of three images per animal. Statistical comparisons were performed using two-way ANOVA. Data are reported as mean ± SEM (n = 11 Cgas+ / +, n = 6 P301S Cgas + / + , n=8 P301S Cgas + / - , n=6 P301S Cgas - / - Cgas+ / + vs. P301S Cgas+ / +: p=0.0016, P301S Cgas+ / + vs. P301S Cgas- / -: p=0.0234); Figure 5D shows the P301S Cgas + / + P301S Cgas compared with inhibitory neurons - / -Figure 5A is a volcano plot showing representative differentially expressed genes upregulated in the CA1 pyramidal layer of mouse hippocampus (log2FC>0.1, FDR<0.05); Figure 5E is a representative 25x confocal image of Mef2c and NeuN immunostaining in the CA1 pyramidal layer of mouse hippocampus (scale bar=50um); Figure 5F is the average intensity of Mef2c in Mef2c+, NeuN+ neurons in the CA1 pyramidal layer. Each circle represents the average intensity measurement of three images per animal. *, p<0.05, unpaired t-test. Data are reported as mean ± SEM (n=4 P301S Cgas+ / +, n=5 P301S Cgas- / -); Figure 5G is a Venn diagram of overlap between excitatory neuron DEGs, inhibitory neuron DEGs and MEF2C target genes; Figure 5H is a heatmap showing overlap between excitatory / inhibitory neuron DEGs and lists of transcription factor target genes (MEF2A, MEF2C, FOSL2, JUNB) and activity-induced expression-change genes (ARGs and scARGs). Numbers in each box represent overlapping odds ratios; Figure 5I is a heatmap of expression of significant DEGs that are MEF2C targets in WT, P301S, and P301S Cgas- / - excitatory neuron clusters (p.adj<0.5, logFC>=0.1 or <=-0.1); Figure 5J is a heatmap of expression of significant DEGs that are MEF2C targets in WT, P301S, and P301S Cgas- / - excitatory neuron clusters (p.adj<0.5, logFC>=0.1 or <=-0.1); Heatmap of expression of significant DEGs that are MEF2C targets in Cgas- / - inhibitory neuron clusters (p.adj<0.5, logFC>=0.1 or <=-0.1); Fig. 5K is a dot plot showing expression of significantly upregulated DEGs by Cgas deletion that are positively correlated with human cognitive resilience in excitatory neuron clusters (p.adj<0.5, logFC>=0.1); Fig. 5L is a dot plot showing expression of significantly upregulated DEGs by Cgas deletion that are positively correlated with human cognitive resilience in inhibitory neuron clusters (p.adj<0.5, logFC>=0.1).

[0207] Brain-permeable cGAS inhibitors upregulate MEF2C target genes and prevent synapse loss and impairment of spatial learning and memory (Figure 6) Figure 6A is a Venn diagram of the overlap between P301S TDI DEGs versus P301S Veh DEGs in excitatory neurons, inhibitory neurons and MEF2C target genes; Figure 6B is a heatmap showing the overlap between excitatory / inhibitory neuron DEGs and lists of transcription factor target genes (MEF2A, MEF2C, FOSL2, JUNB) and activity-induced upregulated genes (ARGs and scARGs). Numbers in each box represent overlapping odds ratios; Figure 6C is a dot plot showing the expression of significantly upregulated DEGs that are MEF2C targets in Ntg control, Ntg TDI, P301S control, and P301S TDI excitatory neuron clusters (p.adj<0.5, logFC>=0.1); Figure 6D is a dot plot showing the expression of significantly upregulated DEGs that are MEF2C targets in Ntg control, Ntg TDI, P301S control, and P301S TDI inhibitory neuron clusters (p.adj<0.5, logFC>=0.1); Figure 6E is a novel object recognition test for Ntg and P301S mice fed 150 mg / kg TDI-6570 or control diet for 3 months. F: everyday objects, N: novel objects (n=9 Ntg control, n=6 Ntg TDI-6570, n=5 P301S control, n=12 P301S TDI6570). Statistical comparisons were performed using two-way ANOVA. *, p<0.05, **, p<0.01; data are reported as mean ± SEM; Figure 6F is a representative confocal image of the CA1 radial striatum of the hippocampus labeled with PSD95 antibody (scale bar = 10 µm); Figure 6G is the mean intensity of PSD95 puncta measured in the CA1 radial striatum. Each circle represents the mean intensity measurement of one image. Three to five images were acquired per animal. Statistical comparisons were performed using mixed models (n=13 Ntg control, n=12 Ntg TDI-6570, n=9 P301S control, n=12 P301S TDI-6570.Ntg control vs P301S control: p=0.0302, P301S control vs P301S TDI-6570: p=0.0427); Figure 6H is a representative confocal image of the CA1 radial striatum of the hippocampus labeled with vGAT antibody (scale bar = 10 µm); Figure 6I is the average intensity of vGAT puncta measured in the CA1 radial striatum. Each circle represents the average intensity measurement of one image. Three to five images were acquired per animal. Statistical comparisons were performed using mixed models (n=12 Ntg control, n=11 Ntg TDI-6570, n=8 P301S control, n=13 P301S TDI-6570. Ntg control vs P301S control: p=0.0453, P301S control vs P301S TDI-6570: p=0.0318). Following the remarkable protective effect of cGAS depletion against P301S behavioral impairment, small molecule cGAS inhibitors were developed, starting with the known cGAS inhibitor TDI-6570. 1 TDI-6570 inhibits both mouse and human cGAS with submicromolar activity (IC 50 TDI-6570 inhibits the β-lactamase inhibitor β-lactamase (β-lactamase inhibitor ... 1 It was prepared on a multigram scale using a four-step process previously described by.

[0208] Working model illustrating the cGAS-IFN-MEF2c axis in tauopathy (Figure 7). Under disease / vulnerability conditions, pathogenic tau activates a cGAS-dependent interferon response via leakage of mtDNA in microglia and reduction of the MEF2c transcriptional network in excitatory and inhibitory neurons, leading to cognitive impairment. Loss of cGAS reduces the interferon response and enhances the Mef2c transcriptional network in microglia, resulting in cognitive resilience.

[0209] Cgas deletion altered the transcriptome of inhibitory neurons in tauopathy (Figure 8). Subclustering of pan-interneuron markers GAD1 and GAD2 positive neuronal populations identified nine inhibitory neuronal subpopulations (Figure 8A) (Arneson et al., 2018; Cembrowski et al., 2016). We found that Cgas deletion rescued tauopathy-induced downregulation of a subset of interneuron markers, such as Pvalb, Vip, Reln, and Lhx6, but not Sst or Cck. In addition, analysis of DEGs from interneurons revealed that Cgas deletion led to upregulation of genes involved in GABA signaling, including the GABA receptor Gabbr2 and the GABA transporter Slc6a, helping to restore interneuron function by Cgas deletion (Figure 8B). Interestingly, among the genes upregulated by Cgas deletion, we found that many are involved in regulating neuronal excitability and seizure activity, including potassium channels and interacting subunits Kcnc1, Kcnc2, Kacnip1, and sodium channel Scn1a (Figure 8C). Indeed, Cgas deletion led to a marked upregulation of anti-seizure genes, including Scn1a, that were decreased in interneurons of P301S mice (Figure 8D). Deficiency of Scn1a leads to Dravet syndrome, an intractable childhood epilepsy with generalized tonic-clonic seizures.

[0210] The cGAS-STING pathway is activated in tau transgenic mice and human AD brains To characterize gene expression changes associated with tauopathy, bulk RNA sequencing of P301S tau transgenic and non-transgenic hippocampi (8–9 months old) was performed. Differentially expressed gene (DEG) analysis revealed a marked upregulation of interferon genes, as well as enrichment of interferon response factor (IRF) and ISRE (interferon sensitive response element) transcription factor motifs in P301S compared to non-transgenic hippocampi (Figure 1A–C). IPA of predicted upstream regulators of upregulated DEGs substantiated numerous components of interferon signaling, including Ifnar1, Stat1 and Irf3. Notably, components of the cGAS-STING pathway, including cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING) and tank-binding kinase 1 (TBK1), were also predicted activators of upregulated DEGs (Figure 1D).

[0211] We tested cGAS-STING pathway activation by immunoblotting for TBK1 phosphorylation and observed increased TBK1 phosphorylation in P301S hippocampal lysates (Figure 1E-F). In addition, immunofluorescence labeling was performed and significantly increased microglial STING expression was observed in P301S compared to non-transgenic hippocampi (Figure 1G-H). Taken together, these studies indicate that the cGAS-STING pathway is activated in P301S tauopathy mice.

[0212] To investigate the involvement of cGAS in the microglial interferon response in human AD, we assessed the levels of TBK1 phosphorylation in a cohort of postmortem brain samples from healthy vs. AD (Braak stage 0 vs. VI) and showed that pTBK1 levels were significantly elevated in human AD brains (Figure 1L-M). In addition, we examined an independent cohort of AD single nucleus RNA-seq (snRNA-seq) of the midfrontal cortex of 11 AD patients (6 males and 5 females, Figure 1I) with the ApoE3 / 4 genotype. snRNA-seq experiments were performed and analyzed as previously described (Sayed et al., 2021). Sequencing reads obtained from multiplets were filtered out using DoubletFinder (McGinnis et al., 2019) and reads from low-quality nuclei were removed by thresholding gene counts, UMI counts and percent mitochondrial genes per nucleus (Figure S2). Analysis of INPP5D- and CSF1R-positive microglia identified a subpopulation of STAT1-positive microglia that overlapped with cells expressing CGAS (Figure 1J). + Hallmark pathway analysis of microglia revealed enrichment for complement, interferon gamma and alpha responses (Figure 1K). Taken together, the results indicate that cGAS-STING signaling contributes to interferon activation in human AD.

[0213] Tau fibrils activate microglial interferon signaling through cGAS activation and mitochondrial DNA leakage To determine whether pathogenic tau directly activates interferon signaling in microglia, we used multiplex ELISA to measure the levels of IFNB, CXCL10 and CCL5 proteins in culture medium from primary microglia treated with tau fibrils and confirmed their induction in response to tau (Figure 2A). Tau treatment also led to robust TBK1 phosphorylation, indicating that tau directly induces activation of cGAS-STING signaling in microglia (Figure 2B-C).

[0214] Next, we investigated the mechanism of interferon activation after tau treatment. To determine the subcellular localization of tau fibrils in microglia, we performed electron microscopy of tau-treated primary microglia with immunogold labeling for tau 24 hours after treatment. Surprisingly, tau was found to localize to mitochondria in addition to lysosomes, their expected location after phagocytosis (Figure 2D). We reasoned that tau in mitochondria might trigger the release of mtDNA that can activate cGAS and interferon signaling. To facilitate quantification of interferon response, we generated an IfnB luciferase reporter BV2 cell line. These cells upregulate IfnB in response to tau fibrils as well as known cGAS agonists such as cGAMP and herring testis DNA (HT-DNA). We then generated mtDNA-depleted cells (ρ° cells) by treating IfnB BV2 cells with low doses of ethidium bromide (EtBr) or dideoxycytidine (ddC) (Hashiguchi and Zhang-Akiyama, 2009; Kaguni, 2004). qPCR for mitochondrial and nuclear genes confirmed dose-dependent depletion of mtDNA after ddC and EtBr treatment (Figure 2E). To examine whether our system can reliably measure the interferon response induced by mtDNA leakage, we co-treated IfnB luciferase BV2 cells with the Bcl2 inhibitor ABT-737 and the caspase inhibitor Q-VD-OPH (QVD) to cause mtDNA leakage without apoptosis induction (Rongvaux et al., 2014; White et al., 2014). ABT-737+QVD treatment activated IfnB-dependent luciferase expression, which was significantly attenuated with ddC- or EtBr-induced mtDNA depletion. Notably, both ddC and EtBr treatment significantly attenuated the microglial IfnB response to tau fibrils in a dose-dependent manner, strongly supporting the involvement of mtDNA as part of the tau-dependent IfnB response in microglia (Figure 2F).

[0215] To investigate whether cGAS mediates the tau-induced interferon response, we - / - and Cgas + / + Primary microglia were treated with tau fibrils or HT-DNA and subjected to RNA sequencing. Microglial responses to HT-DNA were monitored by Cgas - / - The transcriptomic response in microglia is dependent on cGAS, as revealed by the complete elimination of the transcriptomic response. In comparison, cgas deletion only reduced some tau-stimulated inflammation and cytokine expression. Nevertheless, further analysis revealed a striking overlap of the microglial responses induced by HT-DNA and tau, with nearly 80% of the genes upregulated by tau also being upregulated by HT-DNA (Figure 2G). Pathway enrichment terms such as interferon-α and β signaling and interferon-γ signaling were overrepresented pathways in these shared DEGs, suggesting that HT-DNA and tau engage similar interferon-responsive genes to promote microglial inflammation (Figure 2H). Indeed, cgas deletion reduced expression of a subset of interferon-stimulated genes, including Stat1, Ddx60, Isg20, Rnf213, Parp12, Ifi35 and Sp100, in microglia treated with HT-DNA or tau fibrils (Figure 2I-J). Thus, microglial cGAS promotes tau-induced interferon and inflammatory responses, overlapping with those induced by cytosolic DNA and mediated at least in part by mitochondrial DNA leakage.

[0216] Loss of cGAS ameliorates tauopathy-associated microglial interferon signature in vivo To directly assess the role of cGAS in tauopathy, we transformed P301S mice into Cgas - / -We crossed transgenic and non-transgenic mice with 10- to 15-month-old mice to generate cohorts of transgenic and non-transgenic littermates expressing two copies, one copy, or no functional Cgas. + / + , P301S Cgas + / + , P301S Cgas + / - and P301S Cgas - / - Single-nuclear RNA sequencing (snRNA-Seq) of hippocampus from mice was performed. Stringent quality control measures were implemented to remove sequencing reads derived from multiplets using DoubletFinder (McGinnis et al., 2019) and remove reads from low-quality nuclei by thresholding gene counts, UMI counts and percent mitochondrial genes per nucleus. Unsupervised clustering was then performed to group the resulting 234,474 high-quality nuclei into transcriptionally distinct clusters that represented all of the major cell types in the brain.

[0217] In our snRNA-seq data, Cgas (Mb21d1) and Sting (Tmem173) were detected only in microglial clusters characterized by strong expression of microglial markers such as Csf1r, P2ry12 and Siglech (Figure 3A-B). To analyze how reduction of Cgas affects the microglial response to tauopathy, we further subclustered microglia into four microglial subpopulations, revealing that Cgas + / +While the samples consisted primarily of cluster 1 microglia, all four clusters were found in tauopathy samples, demonstrating a robust conversion of microglial state in tauopathy as previously reported (Sayed et al., 2021) (Figure 3C). Cluster 1 expressed high levels of homeostatic genes P2ry12 and Siglech, while clusters 2, 3, and 4 showed reduced expression of these homeostatic genes, as well as concomitant upregulation of disease-associated genes (Apoe, Itgax) and interferon-stimulated genes (Stat1, Parp14, Trim30a, and Rnf213) (Figure 3D). Cluster 3 was specifically enriched for interferon genes, distinct from cluster 4, which was enriched for well-established disease-associated microglial phenotypes (DAMs) reported in mouse amyloid models (Keren-Shaul et al., 2017) (Figure 3D). Cgas + / + Compared with microglia, the expression of genes related to interferon response (Trim 30a, Trim 30b, Stat1, Ddx60, Rnf213, Parp14, and others) was significantly increased in P301S Cgas + / + The suppression of interferon response in microglia was much stronger in mouse microglia (Figure 3E). To verify the suppression of interferon response in microglia by reduction of cGAS, we performed immunofluorescence labeling of phospho-STAT1. + / + Increased pSTAT1 signaling in microglia in the hippocampal CA1 region of P301S Cgas mice + / - and P301S Cgas - / - It was significantly decreased in the brain (Figure 3F-G).

[0218] Using trajectory analysis to model microglial transformation from a homeostatic to a disease signature (Trapnell et al., 2014), we identified three gene modules (Figure 3H) that were significantly associated with microglial disease transformation. Gene ontology analysis was performed to identify overrepresented gene signatures associated with the two microglial disease modules. Disease module 1 (D1) markers were associated with enrichment terms such as cell activation, defense response and immune response system, while disease module 2 (D2) markers were enriched for genes involved in response to viruses and response to type I interferon. D1 genes included DAM genes such as Apoe, Lyz2 and Itgax, while D2 was characterized by expression of interferon genes. P301S Cgas + / + Compared to P301S Cgas + / - and P301S Cgas - / - Microglia showed no changes in the D1 module, but decreased D2 (Figure 3I). Moreover, D2 correlated most strongly with the delayed response microglia (LRM) signature, which was associated with synaptic and neuronal loss and cognitive impairment in a p25-induced model of neurodegeneration (Figure 3J) (Mathys et al., 2017).

[0219] Loss of cGAS rescues tauopathy-induced memory impairment and hippocampal synaptic loss and plasticity Our data showed that loss of cGAS fine-tuned the microglial response to suppress a subset of disease response modules. Tauopathy-induced spatial learning and memory affected by Cgas deletion were assessed using the Morris Water Maze (MWM) behavioral test. To assess spatial learning, hidden platform trials were conducted over six consecutive days, and the cumulative distance traveled to reach the platform in each trial was measured (Figure 4A). Cgas + / - and Cgas - / - The mouse is Cgas + / + While phenocopying the P301S Cgas mice, + / +Mice showed impaired learning compared to non-transgenic mice, exhibiting significantly longer exploration distances in the final hidden platform trial (session 12). + / - and P301S Cgas - / - Mice scored Cgas in the hidden platform trial. + / + P301S mice performed similarly to cGAS loss mice, indicating the strong protection induced by cGAS loss (Figure 4A). - / - Mice took significantly more time to explore the target platform quadrant compared to the other quadrants, whereas P301S Cgas + / + were unable to discriminate the target quadrant from the others (Figure 4B). The beneficial effects of cGAS loss on spatial memory persisted in the 72-h probe trial, demonstrating a robust rescue of spatial memory impairment (Figure 4C). Swimming speed, vision, overall activity and anxiety levels were unchanged across all genotypes, supporting the specific effects of cGAS loss on spatial learning and memory in tauopathy mice.

[0220] Tau-induced deficits in hippocampal synaptic plasticity have been implicated in tauopathy-associated memory loss (Tracy et al., 2016). + / + and P301S Cgas - / - In slices, long-term potentiation (LTP) was induced to similar levels at the early stage of LTP. However, the magnitude of LTP was significantly greater than that of P301S Cgas by 60 min after induction. - / - P301S Cgas compared to slice + / + This is because the P301S Cgas + / + We show that the late LTP impairment in the hippocampus was rescued by cGAS deletion ( Figure 4D-E ), thus preventing tau-mediated plasticity deficits in hippocampal circuits.

[0221] To assess how loss of cGAS affects neuronal changes in hippocampal circuits, we performed subclustering analysis of excitatory neuron populations and identified 10 transcriptionally distinct subpopulations of excitatory neurons (Figure 4F), the majority of which showed non-overlapping expression of dentate granule, CA1, and CA2 / 3 neuron-specific subtype markers (Arneson et al., 2018; Cembrowski et al., 2016; Dong et al., 2009; Sarkar et al., 2018) (Figure 4F). - / - and P301S Cgas + / + Comparing excitatory neurons in the CA1 and CA2 subpopulations, we observed enrichment of DEGs in clusters expressing CA1 markers and to a lesser extent CA3, but not in those expressing granule cell markers (Figure 4G). The effect of cGAS on CA1 hippocampal synapses was further examined by immunofluorescent labeling and quantification of density of PSD-95, an excitatory postsynaptic terminal marker, in the radial striatum. cGAS loss resulted in a dose-dependent rescue of tauopathy-induced PSD-95 loss in CA1 pyramidal neurons (Figure 4H-I). Using the conformation-specific tau antibody MC1, we observed no difference in the accumulation of insoluble tau aggregates in the hippocampus and entorhinal cortex of transgenic mice. These findings support the idea that cGAS loss confers synaptic and cognitive resilience in the presence of tau pathology.

[0222] Deletion of Cgas increases the expression of Mef2c and its target genes in excitatory and inhibitory neurons To further analyze how cGAS loss confers protection from tau toxicity, we examined DEGs in hippocampal excitatory and inhibitory neurons. Top DEGs in excitatory neurons included genes involved in transcriptional regulation and chromatin remodeling (Mef2c, Satb1, Satb2), genes regulating excitability (potassium channel regulator, Dpp10), and genes involved in synapse maintenance (Nrg1 Pcdh7, Pcdh5) (Jaitner et al., 2016; Li et al., 2008; Li et al., 2017; Wang et al., 2020) (Figure 5A). Using immunofluorescence labeling to NRG1 in CA1, we verified that NRG1 protein levels were indeed downregulated in tauopathy and rescued by deletion of Cgas (Figure 5B-C). Cgas deletion also altered the transcriptome of inhibitory neurons in tauopathy. Subclustering of the pan-interneuron marker GAD1 and GAD2 positive neuronal populations identified a subpopulation of nine inhibitory neurons (Arneson et al., 2018; Cembrowski et al., 2016). Genes upregulated by Cgas deletion in interneurons included genes involved in GABAergic signaling, GABA transporters (Slc6a1), GABA receptors (Gabbr2), and ion channels that regulate neuronal excitability and firing, such as Shaw-type potassium channels (Kcnc1, Kcnc2). Genes downregulated by Cgas deletion also included calcium channels (Cacnb2, Cacna1e) and ryanodine receptor calcium release channels (Ryr3) (Figure 5D).

[0223] Among them, P301S Cgas - / -The top DEG upregulated in both excitatory and inhibitory neurons in mice was Mef2c, a transcription factor implicated in late-onset AD, which has recently been linked to cognitive resilience in the AD brain ( Barker et al., 2021 ). Using immunohistochemistry, we identified the P301S Cgas - / - We confirmed elevated MEF2C expression in the hippocampus of mice. To investigate whether MEF2C is a key regulator of neuronal responses to Cgas deletion, we investigated the expression of DEGs (P301S Cgas) in both excitatory and inhibitory neurons that harbor MEF2C target genes. - / - Compared to P301S, P301S Cgas - / - We observed a striking overrepresentation of MEF2C target genes in excitatory and inhibitory neurons (Figure 5G). The transcriptomic changes in these neurons were specific to the MEF2C network, but not to other MEF2 family members or transcription factors regulating neuronal activity, as there was no strong overlap between DEGs and MEF2A, FOSL2 and JUNB target genes using two published datasets of transcriptional changes following neuronal activity (Barker et al., 2021) (Figure 5G).

[0224] Cgas loss induced a specific enhancement of the MEF2C transcriptional network as both a transcriptional activator and repressor in excitatory and inhibitory neurons in P301S mice (Figure 5I, J). In excitatory neurons (ENs), MEF2C target genes rescued by Cgas deletion in P301S mice included genes involved in axon guidance, dendritic growth and synapse maintenance (Tenm3, Unc5d, Nrxn1, Lzts1 Ptprd, Fhod3, Hs6st2), as well as genes in the regulation of calcium signaling / homeostasis (Cacng3, Ncald, Slc24a3) (Figure 5I). Similarly, Cgas deletion in inhibitory neurons (INs) also rescued MEF2C target genes involved in axon guidance, growth and synapse maintenance (Tenm3, Unc5d, Lzts1, Ctnnd2, Cdh8, Sipa1l1), as well as genes regulating calcium signaling / homeostasis (Ncald, Camk4) (Figure 5J). Consistent with the finding that MEF2C overexpression improved hyperexcitability in P301S mice (Barker et al., 2021), we found that Cgas deletion also rescued genes involved in regulating neuronal excitability, such as potassium channel and regulatory subunits Kcnab2, Dpp10 in ENs (Figure 5I) and Kcnj9, Kcnip2 in INs (Figure 5J). Among them, MEF2C target genes downregulated by Cgas deletion in both EN and IN were genes involved in Eph / ephrin signaling (Eph6 and Eph7) (Figure 5I-J), whose blockade was found to promote regeneration during injury (Teng et al., 2019). - / - Further examination of MeF2C target genes upregulated in ENs and INs revealed multiple overlaps with genes involved in cognitive resilience in AD brains, including Ncald, Rasgef1b, Igsf3 and R3hdm2 (Figure 5K-L). Thus, an enhanced MEF2C transcriptional network is involved in the upregulation of Cgas in the presence of tau pathology. - / - It may trigger underlying protective mechanisms in neurons.

[0225] Cgas deletion resulted in a marked upregulation of antiseizure genes, including Scn1a, which was reduced in interneurons of P301S mice (Figure 6A-D). Scn1a deficiency leads to Dravet syndrome, an intractable childhood epilepsy with generalized tonic-clonic seizures.

[0226] Pharmaceutical inhibition of cGAS prevents synapse loss and improves cognition in tauopathy mice To determine the efficacy of TDI-6570 in preventing tau-mediated neurotoxicity, cohorts of 6-month-old Ntg and P301S mice were fed 150 mg / kg TDI-6570 or control diet for 3 months. The effects of TDI-6570 on excitatory and inhibitory neuronal populations were first examined by single-nucleus RNA-seq. Consistent with the genetic deletion of Cgas, Mef2c was the top upregulated gene in inhibitory neuronal clusters. To assess whether the MEF2C transcriptional network was significantly enriched in neurons of TDI-6570-treated P301S mice, we again performed overlap analysis between DEGs and MEF2C target genes in both excitatory and inhibitory neurons (Figure 7A). Consistent with the results from gene deletion of Cgas, we observed a strong enrichment of MEF2C target genes in P301S TDI-6570 DEGs of both excitatory and inhibitory neurons, but no strong enrichment of MEF2A target genes, neuronal activity-regulated transcription factors FOSL and JUNB target genes, nor activity-induced genes (ARGs and scARGs) (Figure 7B). We also identified many MEF2C target genes among the top upregulated genes in excitatory and inhibitory neuron clusters in P301S TDI-6570 versus P301S (Figure 7C-D).

[0227] A novel object recognition test paradigm was performed to evaluate the effect of cGAS inhibition on memory. P301S mice fed a control diet showed a deficit in recognizing novel (N) objects among familiar (F) objects (Figure 7E). Notably, this deficit was rescued in mice fed TDI-6750, indicating that cGAS inhibition can ameliorate memory impairments observed in tauopathy (Figure 7E). Given the protective effect of Cgas deletion on synapses in tauopathy, we pursued whether cGAS inhibition could also alter synaptic density in the hippocampus of P301S mice. Immunofluorescence staining of excitatory synapses (PSD-95, Figure 7F-G) and inhibitory synapses (vGAT, Figure 7H-I) showed that chronic treatment with TDI-6570 rescued P301S-dependent synapse loss. Thus, pharmacological inhibition of cGAS prevented synapse loss and cognitive impairment in tauopathy, likely via enhancing the MEF2C transcriptional network and associated cognitive resilience.

[0228] Our current study linked an overactive cGAS-interferon antiviral response with reduced MEF2C-related cognitive resilience. In disease states, tau-induced cGAS hyperactivation promotes microglial type I interferon responses and reduces neuronal MEF2C transactivation, rendering neurons susceptible to tau toxicity (Figure 8). Meanwhile, ablation of cGAS reduces microglial type I interferon responses and enhances neuronal MEF2C transcriptional networks, leading to cognitive resilience against tau pathology (Figure 8). Also, pharmacological inhibition of cGAS with TDI-6570 enhances MEF2C target genes and restores synaptic integrity and memory, supporting the therapeutic potential of targeting the cGAS-MEF2c axis to improve resilience and treat AD.

[0229] Our analysis of two independent datasets revealed that tau induces strong cGAS activation and type I interferon response in tauopathy mice, consistent with a recent study performed in cultured microglia (Jin et al., 2021). Activation of interferon response in AD brains and mouse models has been reported in previous studies and found to be involved in complement-related synapse loss (Roy et al., 2020). Using snRNA-seq of human AD microglia, we identified a subpopulation of microglia with overlapping expression of cGAS and STAT1, indicating enrichment for complement and interferon response. Our finding that levels of pTBK1 were elevated in AD brains versus normal brains further supports that cGAS-STING activation may underlie the interferon response in AD. Activation of STING signaling has been linked to the pathogenesis of other neurodegenerative diseases, and inhibiting STING activation has been shown to prevent the deleterious effects of the interferon response in Parkinson's disease, Huntington's disease and ALS (Sharma et al., 2020; Sliter et al., 2018; Yu et al., 2020), supporting a centralized and detrimental role of cGAS-STING activation in neurodegeneration.

[0230] The expression of Cgas in the brain is most enriched in microglia, and our in vitro study provides direct evidence that tau induces cGAS-dependent interferon signaling in microglia. As a sensor of cytosolic DNA, cGAS can be activated by DNA leakage from mitochondria or nuclei (Rongvaux et al., 2014;White et al., 2014). We showed that after phagocytosis, tau is found not only in lysosomes but also in mitochondria. It is possible that tau entry into mitochondria causes mitochondrial DNA leakage into the cytosol, which leads to cGAS-STING activation. Indeed, by depleting mtDNA using two independent methods, we showed that tau-induced interferon response was reduced in a dose-dependent manner in mtDNA-depleted microglia. These findings are consistent with the observation that TDP-43 overexpression leads to mtDNA release and cGAS-STING activation, resulting in neuroinflammation. Studies have shown that TDP-43 may enter the mitochondrial matrix via the mitochondrial transport inner membrane translocase TIM22, which leads to mitochondrial destabilization and mtDNA leakage (Yu et al., 2020). It is not known whether TIM22 or other mitochondrial translocases are involved in tau entry into mitochondria. It is believed that mitochondrial stress induced by tau entry into mitochondria directly or indirectly leads to mtDNA leakage. Despite clear evidence of mtDNA in the tau-induced microglial cGAS-interferon response, our study does not exclude the possibility of a contribution of cytosolic DNA of nuclear origin, such as increased cytosolic chromatin fragments as a result of genomic instability, which may stimulate cGAS-STING activation.

[0231] Cgas deletion in tau-stimulated microglia attenuated interferon and inflammatory signaling in vitro and in vivo. Our snRNA-seq data showed that rather than abolishing tau-induced microglial responses to tauopathies, Cgas deletion fine-tuned the microglial response, with only interferon-related genes selectively reduced, while DAM responses were largely unaffected. Studies have shown that in tauopathies, glial interferon signaling may drive synapse elimination by microglia via excessive complement activity (Dejanovic et al., 2018; Litvinchuk et al., 2018).

[0232] MEF2C is dramatically downregulated in neurons of patients with HIV-associated dementia, suggesting that an interplay between antiviral signaling and MEF2C transcriptional networks may exist in the brain (Yelamanchili et al., 2010). Whether microglial interferon can downregulate neuronal Mef2c remains to be elucidated. Mouse models with cell type-specific deletion of cGAS are needed to further analyze the association between cGAS and regulators of cognitive resilience and neuronal excitability.

[0233] In AD brains, the accumulation of amyloid plaques and neurofibrillary tangles occurs decades before clinical symptoms appear, and is an indicator of long-term cognitive resilience in healthy aging. Harnessing and promoting the brain's intrinsic cognitive resilience mechanisms may lead to effective treatments. Our finding that cGAS inactivation induces significant protection in the presence of tau pathology supports an exciting new class of therapeutic strategies that may be effective even after the development of plaque and tangle pathology. This suggests that cgas - / - Together with the fact that the mice were healthy and fertile, this supports further evaluation of pharmacological inhibition of cGAS as a promising treatment strategy for AD.

[0234] Synthesis. All new compounds were confirmed using 1H NMR (Table 1) and mass spectrometry and / or LC-MS analysis.

[0235] 1. Indole Derivatives. We focused on new indole derivatives that have not been previously described and are known to have no cGAS activity. These compounds were prepared using readily available starting materials that can be obtained in 3-4 steps from commercially available compounds as described in Scheme 1.

[0236] 1.1. Compounds 1 and 2 (Scheme 1A). Commercially available 2-fluoro-3-chloronitrobenzene was converted to indole A-2 in four steps (Lama, et. al, WO2019153002A1). Intermediate A-2 was reacted with 2-acetoxyacetyl chloride to give amide A-3, which underwent Pd-catalyzed bromide-hydroxide conversion to give compound 1 or Suzuki coupling with boronic acid to give compound 1 (Scheme 1A).

[0237] TIFF2025507382000017.tif58166Scheme 1A. Synthesis of compounds 1 and 2

[0238] Compound A-2. Commercially available compound A-1 underwent a four-step transformation including bromination, nitro reduction to amine, hydrazine formation and indole synthesis as previously described (Lama, et. al, WO2019153002A1) to give A-2 HCl salt (6.2 g, crude) as a white solid. ESI [M+H] = 304.9 / 302.9

[0239] Compound A-3. Dioxane (20 mL) and saturated Na 2 CO 3 To a mixture of A-2 (1.5 g, 4.9 mmol, 1 equiv.) in aqueous solution (10 mL) was added (2-chloro-2-oxo-ethyl)acetate (4.7 g, 34.4 mmol, 7.0 equiv.), and the mixture was then stirred at 25° C. for 1 h. 2Quench with O (10 mL), extract with EtOAc (30 mL × 3), wash with brine (15 mL × 2), and add Na 2 SO 4 Drying, filtering, concentrating, and column chromatography (SiO 2 , petroleum ether:THF, 1:0 to 1:1) to give A-3 (1.2 g, 2.9 mmol, 57.7% yield) as a yellow solid. ESI [M+H] = 404.9 / 402.9

[0240] Compound A-4. A mixture of A-3 (1.1 g, 2.7 mmol, 1 equiv.) in DMF (30 mL) was treated with NaH (218 mg, 5.5 mmol, 60% purity, 2 equiv.) and CH 3 I (773.6 mg, 5.5 mmol, 2 equiv.) was added at 0° C., and then the mixture was stirred at 25° C. for 1 h. The mixture was quenched with 1N HCl (15 mL) and extracted with EtOAc (50 mL×3). The organic layer was washed with brine (10 mL×5) and diluted with Na 2 SO 4 The mixture was dried at 40° C., filtered, concentrated, and purified by preparative HPLC (TFA condition; column: Phenomenex luna C18 250×50 mm×10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 50%-70%, 10 min) to give A-4 (370 mg, 885.9 umol, 32.5% yield) as a white solid. ESI [M+H] = 419.0 / 417.0

[0241] Compound 1. Dioxane (2.4 mL) and H 2 A-4 (100 mg, 239.4 umol, 1 equiv.) in O (0.9 mL), Pd 2 (dba) 3 A mixture of t-BuXphos (22 mg, 23.9 umol, 0.1 equiv.), t-BuXphos (10 mg, 23.9 umol, 0.1 equiv.) and KOH (27 mg, 478.9 umol, 2 equiv.) was degassed with N 2 Purge the mixture three times with N 2 The mixture was stirred at 100° C. for 1 hour under atmospheric pressure. 2The mixture was quenched with O (15 mL) and extracted with EtOAc (50 mL x 3). The organic layer was washed with brine (10 mL x 5) and diluted with Na 2 SO 4 The mixture was dried at 40° C., filtered, concentrated and purified by preparative HPLC (TFA condition; column: Phenomenex Luna 80×30 mm×3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 20%-45%, 8 min) to give 1-(7-chloro-6-fluoro-9-hydroxy-5-methyl-3,4-dihydro-1H-pyrido[4,3-b]indol-2-yl)-2-hydroxy-ethanone 1 (69 mg, 212.4 umol, 88.7% yield, 96.3% purity) as a yellow solid.

[0242] Compound 2 (General Method A). H 2 A-4 (50 mg, 120 umol, 1.0 equiv), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-ol (106 mg, 479 umol, 4.0 equiv), Pd(dppf)Cl in 0 (0.5 mL) and dioxane (1.5 mL). 2 (9mg, 12umol, 0.1eq), K 3 PO 4 (102 mg, 479 umol, 4.0 equiv.) was mixed with N 2 The mixture was stirred at 80° C. for 12 hours under atmospheric pressure, and then H 2 The organic layer was diluted with 2 mL of O and extracted with EtOAc (3 mL x 3). 2 SO 4 The mixture was dried at 40° C., filtered, concentrated and purified by preparative HPLC (column: Phenomenex Luna 80×30 mm×3 um; mobile phase: [water (TFA)-ACN]; B%: 20%-50%, 8 min) to give compound 2 (6 mg, 13 umol, 11.3% yield, 92.6% purity) as a yellow gum.

[0243] 1.2. Compound 3 (Scheme 1B). Prepared similarly as described for compound 1, except for the Pd-catalyzed conversion of the bromo functionality in compound A-7 to the phenol in A-8, and using starting material A-5.

[0244] TIFF2025507382000018.tif51165Scheme 1B. Synthesis of compound 3

[0245] Compound A-6. Commercially available compound A-5 was converted to A-6 in four steps as described in Scheme 1A, A-1 to A-2. The title product was obtained as a pale pink solid (5.5 g, crude, HCl). TIFF2025507382000019.tif19156

[0246] Compound A-7. A solution of A-6 (700 mg, 2.2 mmol, 1.0 equiv) and TEA (664 mg, 6.6 mmol, 3.0 equiv) in DCM (10 mL) was treated with Boc 2 O (2.9 g, 13.1 mmol, 6.0 equiv.) and DMAP (27 mg, 218.7 umol, 0.1 equiv.) were added and stirred at 40° C. for 12 h. Water (10 mL) was added and extracted with DCM (10 mL×3). The combined organic layer was diluted with Na 2 SO 4 Drying, filtering, concentrating, and column chromatography (SiO 2 , petroleum ether:ethyl acetate=20:1 to 15:1) to produce compound A-7 (550 mg, 1.1 mmol, yield 48.3%) as a white solid. TIFF2025507382000020.tif12157

[0247] Compound A-8. Dioxane (4 mL) and H 2 Compound A-7 (480 mg, 922.7 umol, 1.0 equiv.) in O (1.5 mL), Pd 2 (dba) 3 A mixture of (85 mg, 92.3 umol, 0.1 equiv.), t-BuXphos (39 mg, 92.3 umol, 0.1 equiv.) and KOH (104 mg, 1.9 mmol, 2.0 equiv.) was added to N 2 The mixture was stirred at 100° C. for 1 hour under atmospheric pressure. 2 Add 200 mL of EtOAc (5 mL), extract with EtOAc (20 mL x 3), and add Na 2 SO4 The mixture was dried at 40° C., filtered, concentrated and purified by preparative HPLC (TFA condition; column: Phenomenex luna C18 100×40 mm×5 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 40%-80%, 8 min) to produce A-8 (110 mg, 240.5 umol, 26.1% yield) as a white solid. TIFF2025507382000021.tif19164

[0248] Compound A-9 (General Method B). A mixture of compound A-8 (80 mg, 174.9 umol, 1.0 equiv) in HCl / MeOH (4 mL) (4 M) was stirred at 30° C. for 18 h. The reaction mixture was concentrated and dried under vacuum to give A-9 (50 mg, crude) as a light brown solid. ESI [M+H] = 257.0 / 259.0

[0249] Compound A-10. DCM (1 mL) and saturated NaHCO 3 To a mixture of A-9 (35 mg, 136.1 umol, 1.0 equiv) in aqueous solution (0.5 mL) was added 2-acetoxyacetyl chloride (149 mg, 1.1 mmol, 8.0 equiv) and the resulting mixture was stirred at 15° C. for 1 h. Water (2 mL) was added and extracted with DCM (5 mL×3). The combined organic layers were washed with Na 2 SO 4 The mixture was dried at 40° C., filtered, concentrated and dried under vacuum to give A-10 (50 mg, crude) as a yellow solid. ESI [M+H] = 357.0 / 359.0

[0250] Compound 3 (General Method C). A solution of compound A-10 (50 mg, 140.0 umol, 1.0 equiv.) in DCM (2 mL) was added with LiOH H 2 O (1M, 1.4 mL, 10.0 equiv.) was added and the mixture was stirred at 15° C. for 1 h. The reaction was quenched with HCl (2 mL, 1M), extracted with DCM (5 mL×3), and diluted with Na 2 SO 4It was dried at 40° C., filtered, concentrated and purified by preparative HPLC (TFA condition; column: Phenomenex Luna C18 150×30 mm×5 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 1%-35%, 8 min) to give compound 3 (5 mg, 14.0 umol, yield 10.0%, purity 100.0%) as a yellow gum.

[0251] 1.3. Compounds 4 and 5 (Scheme 1C). The title compounds were prepared in five steps using A-6. First, compound A-6 was Boc-protected to give A-11, then N-methylated to give A-12. The latter underwent Suzuki coupling with N-methylpyrazoleboronic acid derivatives to give A-13. Subsequent deprotection to A-14 followed by reaction with 2-hydroxypropanoic acid or 2-methyl-2-hydroxypropanoic acid gave compounds 4 and 5.

[0252] TIFF2025507382000022.tif56150 Scheme 1C. Synthesis of compounds 4 and 5

[0253] Compound A-11. A solution of compound A-6 (700 mg, 2.0 mmol, 1.0 equiv., HCl) in THF (8 mL) was diluted with saturated Na 2 CO 3 Aqueous solution (2 mL) and Boc 2 O (429 mg, 2.0 mmol, 1.0 equiv) was added. The mixture was stirred at 15° C. for 1 h. Water (2 mL) was added, extracted with EtOAc (10 mL×3), and Na 2 SO 4 Drying, filtering, concentrating, and column chromatography (SiO 2 , petroleum ether:THF, 10:1 to 2:1) to afford A-11 (670 mg, 1.6 mmol, 81.2% yield) as a yellow solid. TIFF2025507382000023.tif12152

[0254] Compound A-12. A solution of A-11 (580 mg, 1.4 mmol, 1.0 equiv) in DMF (5 mL) was treated with NaH (110 mg, 2.8 mmol, 60.0% purity, 2.0 equiv) and CH 3 I (235 mg, 1.7 mmol, 1.2 equiv) was added at 0° C. The resulting mixture was stirred at 20° C. for 1 h. The reaction mixture was diluted with saturated NH 4 Quenched with aqueous Cl (10 mL), extracted with EtOAc (10 mL x 3), and 2 SO 4 The residue was dried at rt, filtered, concentrated and triturated with EtOAc (5 mL) to afford A-12 (520 mg, 1.2 mmol, 84.4% yield, 97.3% purity) as a white solid. TIFF2025507382000024.tif19151

[0255] Compound A-13. Dioxane (4.5 mL) and H 2 A-12 (250 mg, 575.8 umol, 1.0 equiv), 1-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole (144 mg, 691.0 umol, 1.2 equiv), Pd(PPh 3 ) 4 (67mg, 57.6umol, 0.1eq) and Na 2 CO 3 A mixture of (183 mg, 1.7 mmol, 3.0 equiv.) was degassed with N 2 Purge three times with N 2 The mixture was stirred at 80° C. for 4 hours under atmospheric pressure. 2 Dilute with 20 mL of O (10 mL), extract with EtOAc (10 mL x 3), and 2 SO 4 The mixture was dried at 40° C., filtered, concentrated and purified by preparative HPLC (TFA condition; column: Phenomenex Luna 80×30 mm×3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 50%-85%, 8 min) to give tert-butyl A-13 (310 mg, crude) as a green solid. TIFF2025507382000025.tif26160

[0256] Compound A-14. A solution of A-13 (260 mg, 597.2 umol, 1.0 equiv) in HCl / MeOH (4M, 5 mL) was stirred at 15° C. for 1 h. The reaction mixture was concentrated to yield A-14 (192 mg, crude HCl salt) as a brown solid. ESI [M+H] = 335.1 / 337.1

[0257] Compound 4 (General Method D). To a solution of A-14 (50 mg, 134.5 umol, 1.0 equiv, HCl) and DIEA (70 mg, 538.1 umol, 4.0 equiv) in DMF (1 mL), 2-hydroxypropanoic acid (24 mg, 269.0 umol, 2.0 equiv), HATU (77 mg, 201.8 umol, 1.5 equiv) were added and the mixture was stirred at 30°C for 1 h. The mixture was filtered to obtain the filtrate, which was purified by preparative HPLC (TFA condition; column: Phenomenex Luna 80 x 30 mm x 3 um; mobile phase: [water (TFA)-ACN]; B%: 23%-53%, 8 min) to produce 4 (7 mg, 15.5 umol, 11.5% yield, 93.2% purity) as a yellow solid.

[0258] Compound 5. Obtained by method D as described for 4 using intermediate A-14 and 2-hydroxy-2-methylpropanoic acid.

[0259] 1.4. Compounds 6-8 (Scheme 1D). The title compounds were prepared using intermediate A-12. First, intermediate A-12 was deprotected under acidic conditions and the resulting amine A-15 was reacted with NBoc-glycine to give A-16. Subsequent Boc-deprotection of A-16 gave compound 6. Separately, Suzuki coupling of A-16 with a pyrimidine boronic acid derivative gave compound 7, which was followed by Boc-deprotection to give 8.

[0260] TIFF2025507382000026.tif57166Scheme 1D. Synthesis of compounds 6-8

[0261] Compound A-15. Boc deprotection in A-12 using methanolic HCl gave A-15 HCl salt as a yellow solid. ESI [M+H] = 334.9 / 332.9

[0262] Compound A-16. To a solution of A-15 (10 mg, 30.0 umol, 1.0 equiv.) and NMM (12 mg, 119.8 umol, 4.0 equiv.) in DMF (1 mL), EDCI (9 mg, 44.9 umol, 1.5 equiv.), HOBt (6 mg, 44.9 umol, 1.5 equiv.) and N-Boc glycine (11 mg, 59.9 umol, 2.0 equiv.) were added at 0°C, and the mixture was stirred at 20°C for 1 h. The reaction mixture was filtered and purified by preparative HPLC (column: Phenomenex Luna 80×30 mm×3 um; mobile phase: [water (TFA)-ACN]; B%: 45%-85%, 8 min) to produce A-16 (6 mg, 11.9 umol, 39.9% yield, 100.0% purity) as a white solid. TIFF2025507382000027.tif19165

[0263] Compound 6. Prepared by Boc deprotection in A-16 using method B. The title product (6 mg, 13.3 umol, 43.7% yield, 97.0% purity) was obtained as a yellow solid.

[0264] Compound 7. Dioxane (0.9 mL) and H 2 A-16 (12 mg, 24.4 umol, 1.0 equiv), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine (6 mg, 29.3 umol, 1.2 equiv), Pd(PPh) in 2H2O (0.3 mL) 3 ) 4 (3mg, 2.4umol, 0.1eq) and Na 2 CO 3 (8 mg, 73.3 umol, 3.0 equiv.) was degassed with N 2 Purge the mixture three times with N 2The mixture was stirred under atmosphere at 80° C. for 2 hours. The reaction mixture was concentrated and purified by preparative HPLC (column: Phenomenex Luna 80×30 mm×3 um; mobile phase: [water (TFA)-ACN]; B%: 35%-65%, 8 min) to produce compound 7 (11 mg, 23.0 umol, yield 94.0%, purity 99.4%) as a yellow gum.

[0265] Compound 8). Prepared by Boc deprotection in compound 7 using method B to give 8 (12 mg, 30.4 umol, 74.4% yield, 98.7% purity) as a white solid.

[0266] 1.5. Compound 9 (Scheme 1E). Compound 9 can be prepared by the reaction of compound 7 with NaBH 3 It was prepared by reaction with CN followed by Boc deprotection under acidic conditions.

[0267] TIFF2025507382000028.tif27135Scheme 1E. Synthesis of compound 9 (ADI035-3)

[0268] Compound A-17. A mixture of compound 7 (70 mg, 142.8 umol, 1.0 equiv.) in AcOH (1 mL) was treated with NaBH 3 CN (11 mg, 171.3 umol, 1.2 equiv) was added at -5°C and stirred at 25°C for 1 h. The mixture was diluted with saturated Na 2 CO 3 The mixture was diluted with aqueous solution (5 mL) and extracted with EtOAc (10 mL×3). The organic layer was washed with brine (3 mL×5) and diluted with Na 2 SO 4 The extract was dried at 40° C., filtered, concentrated and purified by preparative HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 um; mobile phase: [water (NH 4 HCO 3 )-ACN]; B%: 30%-60%, 10 min) to give A-17 (6 mg, 12.2 umol, 8.5% yield) as a white solid. ESI [M+H] = 492.2 / 494.2

[0269] Compound 9. Obtained as a yellow gum using Method B (2 mg, 6.3 umol, 62.0% yield, 100.0% purity).

[0270] 1.6. Compounds 10-12 (Scheme 1F). Key intermediate A-12 was subjected to Suzuki coupling with N-methylpyrazoleboron-pinacol (Pin) to give compound A-18, which was subsequently deprotected by Boc to give the free amine A-19, followed by amide formation with N-Boc glycine to give compound 10, which was then deprotected by Boc to give the free amine 11. Compound 12 was prepared by the reaction of 11 with NaBH as described above for compounds 7-8. 3 Obtained by CN reduction.

[0271] TIFF2025507382000029.tif58166Scheme 1F. Synthesis of compounds 10-12 (Scheme 1F)

[0272] Compound A-18. Dioxane (9 mL) and H 2 Compound A-12 (580 mg, 1.3 mmol, 1.0 equiv.), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole (334 mg, 1.6 mmol, 1.2 equiv.), Pd(PPh 3 ) 4 (154 mg, 133.4 umol, 0.1 equivalent), Na 2 CO 3 A mixture of (425 mg, 4.0 mmol, 3.0 equiv.) was degassed with N 2 Purge the mixture three times with N 2 The mixture was stirred at 80° C. for 2 hours under atmospheric pressure. 2 Dilute with 20 mL of 2H2O (10 mL), extract with EtOAc (10 mL x 5), and 2 SO 4 The mixture was dried at 40° C., filtered, concentrated and purified by preparative HPLC (column: Phenomenex Luna 80×30 mm×3 um; mobile phase: [water (TFA)-ACN]; B%: 50%-80%, 8 min) to produce compound A-18 (210 mg, 482.4 umol, yield 36.1%, purity 100.0%) as a yellow solid. TIFF2025507382000030.tif19166

[0273] Compound A-19. Obtained as a yellow solid by carrying out N-Boc deprotection in A-18 using Method B (3.44 mg, 9.43 umol, 41.04% yield, 91.861% purity). TIFF2025507382000031.tif19164

[0274] Compound 10. Prepared by reacting A-24 with N-Boc glycine using method C. The title product 10 (7 mg, 14.1 umol, 47.3% yield, 95.9% purity) was obtained as a yellow solid.

[0275] Compound 11. N-Boc deprotection of compound 10 using methanolic HCl (Method B) gave 11 (11 mg, 26.5 umol, 43.4% yield, 97.4% purity) as a yellow solid.

[0276] Compound 12. A solution of 11 (15 mg, 30.5 umol, 1.0 equiv) in TFA (0.5 mL) was added with NaBH 3 CN (10 mg, 152.3 umol, 5.0 equiv) was added at 0° C. and the mixture was stirred at 15° C. for 0.5 h. The mixture was then concentrated and saturated Na 2 CO 3 water (10 mL), extracted with EtOAc (10 mL x 3), and 2 SO 4 The extract was dried at 40° C., filtered, concentrated and purified by preparative HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 um; mobile phase: [water (NH 4 HCO 3 )-ACN]; B%: 25% to 55%, 10 min) to afford 12 (1.0 mg, 2.5 umol, 8.2% yield, 97.0% purity) as a yellow gum.

[0277] 1.7. Compounds 13-18 (Scheme 1G). The title compounds were prepared in 2-3 steps using indole derivatives A-6 and A-15. First, A-6 and A-15 were reacted with Boc-protected proline to give amides A-20 and A-21, which were then subjected to appropriate boronic acid derivatives to give compounds 13-15. These products were then subjected to Boc deprotection to give the free amine compounds 16-18.

[0278] TIFF2025507382000032.tif45166Scheme 1G. Synthesis of compounds 13-18

[0279] 1.8. Compounds 19 and 20 (Scheme 1H). The title compounds were prepared in three steps using compound A-6, where A-6 underwent amide formation with N-Boc protected aminocyclopropyl carboxylic acid and amino-iso-propyl carboxylic acid to give A-22 and A-24, respectively. Suzuki coupling of A-22 with aminopyridine boronic acid and A-24 with pyrazole boronic acid derivatives gave compounds A-23 and A-25. Finally, the latter products were N-Boc deprotected to give 19 and 20, respectively.

[0280] TIFF2025507382000033.tif67166 Scheme 1H. Synthesis of compounds 19 and 20

[0281] Compound A-22. Compound A-6 was reacted with Boc-protected 2-aminocyclopropyl carboxylic acid using method C to give A-22 as a white solid (140 mg, 278.21 umol, 99.17% yield).

[0282] Compound A-23. Compound A-22 was coupled with 2-aminopyridine-5-boronic acid derivative using Method A to give A-23 (8.58 mg, 12.88 umol, 9.30% yield, 94.97% purity, TFA) as a white solid. TIFF2025507382000034.tif27165

[0283] Compound 19 (ADI-51-9). NBoc deprotection in A-23 using method B gave compound 19 (1.48 mg, 2.95 μmol, 30.58% yield, 90.62% purity) as the HCl salt.

[0284] Compound A-24. Compound A-6 was reacted with Boc-protected 2-amino-iso-propyl carboxylic acid using method C to give A-24 (50 mg, crude). ESI [M+H] = 506.0 / 504.0

[0285] Compound A-25. Compound A-24 was coupled with a pyrazole-3-boronic acid derivative using Method A to give A-25 (2.78 mg, 5.65 umol, 28.52% yield) as a yellow gum. TIFF2025507382000035.tif19162

[0286] Compound 20 (ADI-47-13). NBoc deprotection in A-25 using method B gave compound 20 (3.39 mg, 8.41 umol, 8.28% yield, 97.31% purity) as a yellow gum.

[0287] 2. Benzofuran Derivatives. We designed, prepared, and evaluated benzofuran derivatives to mimic indole compounds. The synthesis of various benzofuran derivatives was accomplished starting from commercially available materials as described in Sections 2.1-2.x.

[0288] 2.1. Compound 21 (Scheme 2A). The synthesis of compound 21 began with the reaction of boronic acid B-1 with N-hydroxyphthalimide to give compound B-2. The latter was reacted with MeNH 2 Removal of the phthalic acid by reaction with gave B-3, which was reacted with 4-tetrahydropyridone to give the key intermediate B-4. Subsequent reaction with 2-acetoxyacetyl chloride and deprotection of the acetate group gave compounds B-5 and then 21.

[0289] TIFF2025507382000036.tif51129Scheme 2A. Synthesis of compound 21

[0290] Compound B-2. A mixture of (2,3-dichlorophenyl)boronic acid B-1 (11.7 g, 61.3 mmol, 2.0 equiv.), 2-hydroxyisoindoline-1,3-dione (5.0 g, 30.7 mmol, 1.0 equiv.), Py (2.7 g, 33.7 mmol, 1.1 equiv.), 4A MS (2.0 g) and CuCl (3.0 g, 30.7 mmol, 1.0 equiv.) in DCE (200 mL) was degassed and purified with O 2 The mixture was then purged with O three times. 2 The mixture was stirred at 40° C. for 48 hours under atmospheric pressure. The mixture was diluted with water (50 mL), extracted with DCM (100 mL×3), and diluted with Na 2 SO 4 The mixture was dried at 40° C., filtered, concentrated and purified by preparative HPLC (TFA condition; column: Phenomenex luna C18 250 mm×100 mm×10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 40%-70%, 20 min) to produce compound B-2 (550 mg, 1.8 mmol, yield 5.8%) as a brown solid. TIFF2025507382000037.tif12137

[0291] Compound B-3. Compound B-2 (550 mg, 1.8 mmol, 1 equiv.) and MeNH in EtOH 2 (30.0% MeNH in EtOH 2 , 8 mL) was stirred for 1 h at 15° C. The reaction mixture was concentrated to yield compound B-3 (840 mg, crude) as a brown solid. TIFF2025507382000038.tif19159

[0292] Compound B-4. A mixture of compound B-3 (840 mg, 4.7 mmol, 1.0 equiv.) in AcOH (10 mL) was added with H 2 SO 4(1 mL) was added, followed by tert-butyl 4-oxopiperidine-1-carboxylate (1.1 g, 5.7 mmol, 1.2 equiv). The mixture was stirred at 15° C. for 5 min and then heated to 100° C. for 12 h. The reaction mixture was concentrated and purified by preparative HPLC (TFA conditions; column: Phenomenex luna C18 250×50 mm×10 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 15%-45%, 10 min) to produce B-4 (350 mg, 982.8 umol, 20.8% yield, TFA salt) as a brown solid. TIFF2025507382000039.tif12170

[0293] Compound B-5. To a mixture of compound B-4 (100 mg, 280.8 umol, 1.0 equiv, TFA) and TEA (57 mg, 561.6 umol, 2.0 equiv) in DCM (1 mL), 2-acetoxyacetyl chloride (58 mg, 421.2 umol, 1.5 equiv) was added at 0° C. and stirred at 15° C. for 1 h. The mixture was quenched with MeOH (0.5 mL) and concentrated to yield B-5 (100 mg, crude) as a brown solid. ESI [M+H] = 342.0 / 344.0

[0294] Compound 21. Compound B-5 (100 mg, 292.3 umol, 1.0 equiv.) and LiOH H in MeOH (2 mL). 2 A mixture of 25 mg, 584.5 umol, 2.0 equiv. and 1.0 sulphate was stirred at 15° C. for 1 h. The mixture was concentrated and purified by rep-HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 um; mobile phase: [water (10 mM NH 4 HCO 3 )-ACN]; B%: 35% to 65%, 10 min) to produce compound 21 (8 mg, 27.6 umol, 9.4% yield, 100.0% purity) as a white solid.

[0295] 2.2. Compounds 22-26 and 28-29 (Scheme 2B). To prepare the title compounds, the key benzofuran intermediates B-20 and B-21 were synthesized starting from commercially available compounds B-6 and B-7 via boronic acids B-12 and B-13, respectively (Scheme 2B). Here, compounds B-6 and B-7 were reacted with sodium methoxide to give B-8 and B-9, which were then reacted with sodium methoxide to give B-10 and B-11, respectively (Scheme 2B). 2 Pin 2 The resulting products B-10 and B-11 were treated with sodium periodate to give B-12 and B-13. The latter product was reacted with N-hydroxyphthalimide to give the resulting products B-14 and B-15, which were then reacted with MeNH in ethanol to give 2 The benzofurans B-18 and B-19 were then hydrolyzed using 1,2-dichlorophenyl ether to give compounds B-16 and B-17. B-16 and B-17 were then coupled with N-Boc-4-tetrahydropyridone to afford the resulting benzofurans B-18 and B-19 using BBr 3 The latter compound was reacted with Na to give the key intermediates B-20 and B-21. 2 CO 3 The products B-22 and B-23 were converted to triflates B-24 and B-25 by reaction with trifluoromethanesulfonic anhydride. B-24 and B-25 then underwent Suzuki coupling with the appropriate heterocyclic boronic acid derivatives to give compounds B-(26-32), which were deprotected with acetic acid to give the title products 22-26 and 28-29. The method described here is for compound B-7, and compound B-6 is treated similarly.

[0296] TIFF2025507382000040.tif121166 Scheme 2B. Synthesis of benzofuran compounds 22–26 and 28–29

[0297] Compound B-9. A mixture of compound B-7 (10.0 g, 41.0 mmol, 1.0 equiv.) and NaOMe (8.9 g, 164.0 mmol, 4.0 equiv.) in THF (100 mL) was degassed and purified with N 2 Purge the mixture three times with N 2 The mixture was stirred at 75° C. under atmospheric pressure for 4.5 hours. 2 Dilute with 20 mL of O (50 mL), extract with EtOAc (80 mL x 3), and 2 SO 4 The mixture was dried over 1000 ml of ethyl acetate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate = 95:5 to 9:1) to produce B-9 (4.6 g, 18.0 mmol, 43.8% yield) as a white solid. TIFF2025507382000041.tif12128

[0298] Compound B-11. B-9 (4.2 g, 16.4 mmol, 1.0 equiv.) in dioxane (60 mL), Pin 2 B 2 (12.5g, 49.2mmol, 3.0eq), Pd(dppf)Cl 2 ·CH 2 Cl 2 A mixture of (1.3 g, 1.6 mmol, 0.1 equiv) and KOAc (4.8 g, 49.2 mmol, 3.0 equiv) was degassed with N 2 Purge the mixture three times with N 2 The mixture was stirred at 90° C. for 12 hours under atmospheric pressure. 2 The mixture was diluted with 20 mL of EtOAc (50 mL x 3) and extracted with EtOAc (50 mL x 3). The organic layer was then washed with Na 2 SO 4 Drying, filtering, concentrating, and column chromatography (SiO 2 , petroleum ether:ethyl acetate=1:0 to 1:1) to afford B-11 (2.2 g, 7.3 mmol, 44.2% yield) as a green oil. TIFF2025507382000042.tif19161

[0299] Compound B-13. H 2 B-11 (2.2 g, 7.1 mmol, 1.0 equiv.), NaIO in 2HO (10 mL) and ACN (10 mL). 4 (6.1 g, 28.4 mmol, 4.0 equiv.) and NH 4 A mixture of OAc (2.2 g, 28.4 mmol, 4.0 equiv.) was stirred for 1 h at 25° C. The reaction mixture was filtered and purified by preparative HPLC (column: Phenomenex luna C18 (250×70 mm, 15 um); mobile phase: [water (TFA)-ACN]; B%: 15%-45%, 30 min) to produce B-13 (1.2 g, 5.2 mmol, 73.4% yield) as a white solid. TIFF2025507382000043.tif19155

[0300] Compound B-15. A mixture of B-13 (740 mg, 3.4 mmol, 1.0 equiv.), 2-hydroxyisoindoline-1,3-dione (1.1 g, 6.7 mmol, 2.0 equiv.), Py (292 mg, 3.7 mmol, 1.1 equiv.), 4A MS (1 g) and CuCl (332 mg, 3.4 mmol, 1.0 equiv.) in DCE (30 mL) was degassed and cooled to 50° C. for 1 h. 2 The mixture was then purged with O three times. 2 The mixture was stirred at 25° C. for 12 hours under atmospheric pressure. The reaction mixture was filtered and then diluted with H 2 Dilute with 200 mL of HO (10 mL), extract with DCM (30 mL x 3), and 2 SO 4 Drying, filtering, concentrating, and column chromatography (SiO 2 , petroleum ether:ethyl acetate=1:0-5:1) to produce B-15 (510 mg, 1.5 mmol, 45.0% yield) as a white solid. TIFF2025507382000044.tif12145

[0301] Compound B-17. MeNH 2A mixture of B-15 (500 mg, 1.5 mmol, 1.0 equiv) in (30.0% purity, 6 mL, in EtOH) was stirred for 1 h at 25° C. The reaction mixture was concentrated to yield B-17 (550 mg, crude) as a yellow oil. TIFF2025507382000045.tif5164

[0302] Compound B-19. A mixture of B-17 (500 mg, 2.4 mmol, 1.0 equiv) in AcOH (3 mL) was added with H 2 SO 4 (0.3 mL) was added, followed by tert-butyl 4-oxopiperidine-1-carboxylate (575 mg, 2.9 mmol, 1.2 equiv). The mixture was stirred at 15° C. for 5 min and then heated to 100° C. for 12 h. The mixture was diluted with saturated Na 2 CO 3 The mixture was quenched with aqueous solution (30 mL) until pH>8, extracted with EtOAc (10 mL×3), and then diluted with Na 2 SO 4 The mixture was dried at 40° C., filtered, concentrated and purified by preparative HPLC (column: Phenomenex C18 80×40 mm×3 um; mobile phase: [water (NH 4 HCO 3 )-ACN]; B%: 15% to 65%, 8 min) to produce B-19 (80 mg, 294.0 umol, 12.2% yield) as a brown solid. TIFF2025507382000046.tif19160

[0303] Compound B-21. CHCl 3 To a mixture of B-19 (10 mg, 36.8 umol, 1.0 equiv) in 1 mL of BBr 3 (558mg, 220.5umol, 215uL, 6.0eq) was added at -60°C. The mixture was stirred at 25°C for 3 hours. The mixture was diluted with saturated Na 2 CO 3 The mixture was quenched with aqueous solution (30 mL) until pH>8, extracted with EtOAc (10 mL×3), and then diluted with Na 2 SO 4Drying at 40° C., filtration and concentration afforded B-21 (15 mg, crude) as a brown oil. ESI [M+H] = 258.0 / 260.0

[0304] Compound B-23. Compound B-21 was reacted with 2-acetoxyacetyl chloride as described in the conversion of compound A-2 to A-3 to give B-23.

[0305] Compound B-25. A mixture of B-23 (280 mg, 781.75 umol, 1 equiv.) in DCM (4 mL) and TEA (395 mg, 3.91 mmol, 5 equiv.) in DCM (1 mL) was treated with Tf 2 O (551.40 mg, 1.95 mmol, 2.5 equiv.) was added at 0 °C, and N 2 The mixture was stirred at 25°C for 1 hour under atmospheric pressure, concentrated, and then cooled to 37°C. 2 The mixture was diluted with 200 mL of 2H2O (5 mL) and extracted with EtOAc (10 mL x 3). The organic layer was washed with brine (5 mL x 3). The organic layer was washed with Na 2 SO 4 Drying at 40° C., filtration and concentration afforded B-25 (110 mg, crude) as a brown oil. ESI [M+Na] = 511.8 / 510.0

[0306] Compound B-(28-32). Compound B-25 was coupled with a substituted boronic acid ester derivative using method A to give B-(28-32).

[0307] Compounds 24-26 and 28-29. Ester hydrolysis in B-(26-32) using method C gave compounds 24-26 and 28-29.

[0308] 2.3. Compounds 27 and 30-31 (Scheme 2C). Triflate B-25 was converted to the boronic acid derivative B-33 and reacted with bromo-heterocycles to give compounds B-(34-36), which were then deprotected to give compounds 27 and 30-31.

[0309] TIFF2025507382000047.tif45166Scheme 2C. Synthesis of benzofuran derivatives 27 and 30-31

[0310] Compound B-33. B-25 (130 mg, 265.18 μmol, 1 equiv.), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (336.69 mg, 1.33 mmol, 5 equiv.), Pd(dppf)Cl in dioxane (6 mL). 2 A mixture of (19.40 mg, 26.52 μmol, 0.1 equiv.), KOAc (78.07 mg, 795.54 μmol, 3 equiv.) was degassed with N 2 Purge the mixture three times with N 2 The mixture was stirred at 110° C. for 12 hours under atmospheric pressure. 2 The mixture was diluted with 200 mL of EtOAc (10 mL) and extracted with EtOAc (15 mL x 3). The combined organic layers were washed with Na 2 SO 4 The mixture was dried over 1000 ml of ethyl acetate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate=1:1 to 1:2) to produce B-33 (120 mg, 256.34 μmol, yield 96.67%) as a white solid.

[0311] Compounds B-34-36. Compounds B-33 were coupled with substituted bromide derivatives using Method A to produce B-34-36 (1.56 mg, 1.95 μmol, 3.04% yield, 94% purity) as pale yellow solids.

[0312] Compounds 27, 30 and 31. Ester hydrolysis of compounds B-34-36 using Method C gave compounds 27, 30 and 31.

[0313] 2.4. Compounds 32-35 (Scheme 2D). The synthesis of compounds 32-35 was accomplished using intermediate B-21 described above, which underwent peptide coupling with Boc-protected glycine and 2-methylalanine to give compounds B-37 and B-38. The latter product was converted to ethyl acetate by the addition of Et 3 Reaction with trifluoromethanesulfonic anhydride in the presence of N gave triflates B-39 and B-40, which underwent Suzuki coupling with the appropriate bromo-heterocycles to give B-(41–44), followed by Boc deprotection to give compounds 32–35.

[0314] TIFF2025507382000048.tif60166Scheme 2D. Synthesis of compounds 32-35

[0315] Compound B-37. To a mixture of B-21 (45 mg, 152.8 umol, 1.0 equiv, HCl), 2-(tert-butoxycarbonylamino)acetic acid (54 mg, 305.5 umol, 2.0 equiv), and NMM (62 mg, 611.1 umol, 4.0 equiv) in DMF (1 mL) was added EDCI (44 mg, 229.2 umol, 1.5 equiv), HOBt (31 mg, 229.2 umol, 1.5 equiv), and stirred at 25° C. for 1 h. The mixture was then washed with saturated Na 2 CO 3 The mixture was quenched with aqueous solution (1 mL) and extracted with EtOAc (5 mL×3). The organic layer was washed with brine (3 mL×5) and diluted with Na 2 SO 4 Drying, filtering, concentrating, and column chromatography (SiO 2 , petroleum ether:ethyl acetate=5:1 to 3:1) to produce B-37 (45 mg, 108.4 umol, 70.9% yield) as a white solid. TIFF2025507382000049.tif19158

[0316] Compound B-39. A mixture of B-37 (40 mg, 96.3 umol, 1.0 equiv) and TEA (97 mg, 963.2 umol, 10.0 equiv) in DCM (1 mL) was degassed with N2 Purge three times with N 2 Tf in DCM (0.2 mL) under atmospheric pressure 2 O (136 mg, 481.6 umol, 5.0 equiv) was added slowly at 0° C. The mixture was then diluted with N 2 The mixture was stirred at 25° C. for 1 h under atmospheric pressure. 2 The mixture was quenched with O (2 mL) and extracted with EtOAc (5 mL x 2). 2 SO 4 Dry, filter, concentrate, and run on preparative TLC (SiO 2 , petroleum ether:ethyl acetate=2:1) ​​to give B-39 (48 mg, 87.7 umol, 91.1% yield) as a yellow oil. ESI [MH] = 544.9 / 546.9

[0317] Compounds B-41-42. Compounds B-39 were coupled with substituted boronic ester derivatives using Method A to produce B-41-42 (1.56 mg, 1.95 μmol, 3.04% yield, 94% purity) as pale yellow solids.

[0318] Compounds 32-35. NBoc deprotection in B-41-44 using method B gave compounds 32-35.

[0319] 2.5. Compounds 36-38 (Scheme 2E). The synthesis of compounds 36-38 was achieved using compounds B-16 and B-17, which were reacted with 1-Boc-3-piperidone to give the key benzofuran intermediates B-45 and B-46. 2 N-Boc protection of these products using O gave compounds B-47 and B-48, which were then treated with BBr3 to give the amino-phenols B-49 and B-50. The latter products were converted to the next key intermediates B-53 and B-54 by addition of an acetoxyacetyl linker to give the amides B-51 and B-52, followed by treatment with trifluoromethanesulfonic anhydride / TEA in dichloromethane.

[0320] TIFF2025507382000050.tif81143Scheme 2E. Synthesis of compounds 36-38

[0321] Compound B-46. A solution of B-17 (1.9 g, 9.13 mmol, 1 equiv.), tert-butyl 3-oxopiperidine-1-carboxylate (2.18 g, 10.96 mmol, 1.2 equiv.) in AcOH (20 mL) was added with H 2 SO 4 (4 mL) was added. The mixture was stirred at 90° C. for 1 h. The reaction mixture was diluted with saturated aqueous NaOH (30 mL) at 0° C. to adjust pH=9. The mixture was extracted with 2-Me-THF (20 mL×3). The combined organic layers were washed with Na 2 SO 4 The mixture was dried at rt, filtered and concentrated under reduced pressure to yield B-46 (2.88 g, crude) as a white solid.

[0322] Compound B-48. A solution of B-46 (2.88 g, 10.58 mmol, 1 equiv.) in saturated aqueous NaOH (20 mL), THF (30 mL) was treated with Boc 2 O (4.62 g, 21.17 mmol, 4.86 mL, 2 equiv) was added at 0° C. The mixture was stirred at 25° C. for 1 h. The mixture was concentrated and extracted with 2-Me-THF (20 mL×3). The combined organic layers were washed with Na 2 SO 4 The mixture was dried over 1000 ml of ethyl acetate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate = 10:1 to 1:1) to give B-48 (780 mg, 2.10 mmol, 19.80% yield) as a yellow solid. ESI [M-Boc+H] = 272.0 / 274.0

[0323] Compound B-50. CHCl 3 To a solution of B-48 (630 mg, 1.69 mmol, 1 equiv) in (10 mL) was added BBr 3 (4.24 g, 16.92 mmol, 1.63 mL, 10 equiv) was added at -60°C. The mixture was stirred at 25°C for 1 h. The reaction mixture was cooled to 0°C and Na2 CO 3 (100 mL). The solution was concentrated and quenched with CHCl 3 was removed and used directly in the next step without further purification.

[0324] Compound B-52. Saturated NaHCO 3 To a solution of B-50 (50 mg, 135 umol, 1.0 equiv, HCl) in water (1 mL) and dioxane (1 mL), (2-chloro-2-oxo-ethyl)acetate (74 mg, 540 umol, 4.0 equiv) was added at 0° C. and stirred at 25° C. for 16 h, then H 2 The organic layer was diluted with 2 mL of O (3 mL) and extracted with EtOAc (5 mL x 3). 2 SO 4 The mixture was dried at 40° C., filtered and concentrated to give B-52 (60 mg, crude) as a yellow solid.

[0325] Compound B-54. A mixture of B-52 (70 mg, 162 umol, 1.0 equiv) and TEA (328 mg, 3 mmol, 20.0 equiv) in DCM (1 mL) was diluted with Tf 2 O (229 mg, 811 umol, 5.0 equiv.) was added at 0°C, and N 2 The mixture was stirred at 25° C. for 1 hour under atmospheric pressure, diluted with H2O (2 mL), and extracted with EtOAc (3 mL×3). 2 SO 4 Dry, filter, concentrate, and run on preparative TLC (SiO 2 , petroleum ether:EtOAc=1:1) to afford B-54 (66 mg, 117 umol, 72.2% yield) as a yellow solid.

[0326] Compounds B-55-57. Compounds B-54 were coupled with substituted boronic ester derivatives using Method A to give B-55-57 (1.56 mg, 1.95 μmol, 3.04% yield, 94% purity) as pale yellow solids.

[0327] Compounds 36-38. Ester hydrolysis of compounds B-55-57 using method C gave compounds 32-35.

[0328] 2.6. Compounds 39-41 (Scheme 2F). The synthesis of compounds 39-41 is achieved by using intermediate B-50 and reacting it with N-Boc glycine to give B-58. The latter is reacted with Tf 2 Reaction with O gave triflate B-59, which underwent Suzuki reaction with heterocycle-boronic acid derivatives to give compounds B-(60-62), followed by Boc deprotection to give the title products 39-41.

[0329] TIFF2025507382000051.tif55150 Scheme 2F. Synthesis of compounds 39-41

[0330] Compound B-58. To a solution of B-50 (476 mg, 1.62 mmol, 1 equiv., HCl) in DMF (5 mL) was added 2-(tert-butoxycarbonylamino)acetic acid (849.26 mg, 4.85 mmol, 3 equiv.) and DIEA (626.56 mg, 4.85 mmol, 3 equiv.), HATU (921.66 mg, 2.42 mmol, 1.5 equiv.). The mixture was stirred at 0° C. for 1 h. The reaction mixture was diluted with H 2 The mixture was diluted with 20 mL of 2H2O (10 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (10 mL x 3) and 2 SO 4 The mixture was dried over 1000 ml of ethyl acetate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate = 1:1 to 0:1) to give B-58 (380 mg, 915.07 μmol, 56.63% yield) as a yellow solid. ESI [M+H] = 414.9 / 416.9

[0331] Compound B-59. A mixture of B-58 (370 mg, 890.99 μmol, 1 equiv.), TEA (450.80 mg, 4.45 mmol, 5 equiv.) and Tf in DCM (5 mL) was added. 2A mixture of 2H2O (628.46 mg, 2.23 mmol, 2.5 equiv) was degassed and cooled to 0 °C with N 2 Purge the mixture three times with N 2 The mixture was stirred at 25° C. for 1 hour under atmospheric pressure. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was 2 The mixture was diluted with 20 mL of EtOAc (10 mL) and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with Na 2 SO 4 The mixture was dried over 1000 ml of ethyl acetate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate=0:1-1:1) to give B-59 (220 mg, 401.95 μmol, 45.11% yield) as a yellow oil. + ] = 568.9 / 570.9

[0332] Compound B-(60-62). Compound B-59 was coupled with a substituted boronic acid ester derivative using method A to produce B-(60-62).

[0333] Compounds 39–41. NBoc deprotection in B-(60–62) using method B gave compounds 39–41 as pale yellow solids.

[0334] 3. Benzothiofuran derivatives 3.1. Compounds 42-44 (Scheme 3A). The synthesis of compounds 42-44 began with the methylation of phenol C-1 followed by bromination to give bromide C-2. Pd-catalyzed reaction with ethyl 3-mercaptopropionate afforded compound C-3, which was reacted with N-Boc-3,3-epoxy-piperidine followed by oxidation of the resulting alcohol to give compound C-4. The latter underwent phosphoric acid-mediated cyclization to give key intermediate C-5, which was then reacted with 2-acetoxyacetyl chloride to give intermediate C-6, which served as a precursor for compounds 42-44 as well as other analogues.

[0335] TIFF2025507382000052.tif80165Scheme 3A. Synthesis of compounds 42-44

[0336] 3.2. Compounds 45-52 (Scheme 3B). The synthesis of compounds 45-52 started from compounds C-7 and C-8, which underwent a series of reactions as described above for the synthesis of compound C-5, to give intermediates C-13 and C-14 via alcohols C9 / C10 and ketones C11 / C12. The methoxy groups in compounds C-13 and C-14 were replaced by BBr. 3 Deprotection with ethyl acetate afforded the aminophenols C-15 and C16, which were reacted with 2-acetoxyacetyl chloride to give compounds C-17 and C-18. The phenols were converted to triflates C-19 and C-20, and Pd-catalyzed reaction with boronic acid derivatives followed by acetic acid deprotection under basic conditions afforded the target compounds 45-52 via the acetate precursors C-21 and C-(22-27) (Scheme 3B). Detailed methods are provided below for the conversion of compounds C-8 to 46-48, and compound C-7 was similarly converted to 45.

[0337] TIFF2025507382000053.tif123166Scheme 3B. Synthesis of compounds 45-52

[0338] Compound C-10. To a solution of compound C-8 (2.0 g, 7 mmol, 1.0 equiv.), tert-butyl 7-oxa-3-azabicyclo[4.1.0]heptane-3-carboxylate (1.4 g, 7 mmol, 1.1 equiv.) in MeOH (30 mL), NaOH (247 mg, 6 mmol, 0.9 equiv.) was added and stirred at 80° C. for 2 h. H 2 The mixture was quenched with O (20 mL) and extracted with DCM (20 mL x 5). 2 SO 4 Drying, filtering, concentrating, and column chromatography (SiO 2 , petroleum ether:EtOAc = 3:2 to 3:2) to afford C-10 (2.4 g, 6 mmol, yield 86.0%) as a colorless oil. TIFF2025507382000054.tif27164

[0339] Compound C-12. To a solution of C-10 (2.4 g, 5.8 mmol, 1.0 equiv.) in DCM (20 mL), DMP (3.0 g, 7 mmol, 1.2 equiv.) was added and stirred at 15 °C for 2 h. The mixture was then cooled to 10 °C with saturated NaHCO 3 aqueous solution (30 mL) and saturated Na 2 S 2 O 3 The mixture was quenched with aqueous solution (30 mL) and extracted with DCM (50 mL×5). 2 SO 4 The mixture was dried at 40° C., filtered, concentrated and purified by preparative HPLC (column: Phenomenex Luna 80×30 mm×3 um; mobile phase: [water (TFA)-ACN]; B%: 45%-80%, 8 min) to give C-12 (3.3 g, crude) as a yellow oil. TIFF2025507382000055.tif19164

[0340] Compound C-14. H 3 PO 4 A solution of C-12 (3.0 g, 7 mmol, 1.0 equiv) in 1M NaOH (30 mL, 85.0% purity) was stirred at 130 °C for 12 h, quenched with 5M NaOH (150 mL) to pH = 14, and extracted with DCM (150 mL x 4). The organic layer was washed with Na 2 SO 4 The mixture was dried at 40° C., filtered, concentrated and purified by preparative HPLC (column: Phenomenex luna C18 (250×70 mm, 15 um); mobile phase: [water (TFA)-ACN]; B%: 25%-55%, 20 min) to give C-14 (600 mg, 2 mmol, 28.2% yield) as a light brown solid. TIFF2025507382000056.tif19158

[0341] Compound C-16. CHCl 3 A solution of compound C-14 (530 mg, 2 mmol, 1.0 equiv.) in 10 mL of BBr at -60 °C was added. 3(10.4 g, 42 mmol, 23 eq.) was added and stirred at 30° C. for 48 h. Then, the mixture was cooled to 0° C. and saturated with Na 2 CO 3 Quench with aqueous solution (20 mL) to pH=9 and CHCl 3 and H 2 C-16 (504 mg, crude) in O was obtained as a pale yellow liquid. ESI [M+H] = 274.0 / 276.0

[0342] Compound C-18. DCM (40mL), Na 2 CO 3 (30 mL) and CHCl 3 To a solution of compound C-16 (500 mg, 2 mmol, 1.0 equiv.) in 10 mL of H 2 O, add 2-acetoxyacetyl chloride (996 mg, 7 mmol, 4.0 equiv.), stir at 15° C. for 1 h, and then add H 2 The organic layer was diluted with 20 mL of O and extracted with DCM (40 mL x 3). 2 SO 4 Drying, filtering, concentrating, and column chromatography (SiO 2 , petroleum ether:EtOAc = 3:7 to 3:7) to give the O-acetyl derivative of C-18 (470 mg, crude) as a pale yellow solid. ESI [M+H] = 473.9 / 475.9

[0343] The above-mentioned O-acetyl derivative of C-18 (50 mg, 105 umol, 1.0 equiv.) and Cs in ACN (1 mL) 2 CO 3 The mixture of (137 mg, 422 umol, 4.0 equiv) was stirred at 30° C. for 1 h, then filtered, quenched with 1N HCl (0.1 mL) to pH=4, and extracted with EtOAc (30 mL×3). The organic layer was washed with Na 2 SO 4 Drying at 40° C., filtration and concentration gave compound C-18 (60 mg, crude) as a brown oil. ESI [M+H] = 373.9 / 375.9

[0344] Compound C-20. A solution of C-18 (50 mg, 134 umol, 1.0 equiv.) and TEA (54 mg, 534 umol, 4.0 equiv.) in DCM (1 mL) was added with Tf 2 Add 2.0O (75 mg, 267 umol, 2.0 equiv.) at 0°C, stir at 15°C for 1 h, and add saturated Na 2 CO 3 The mixture was quenched with aqueous solution (1 mL) and extracted with DCM (30 mL x 3). 2 SO 4 Drying at 40° C., filtration and concentration afforded C-20 (80 mg, crude) as a brown oil. ESI [M+H] = 505.9 / 507.9

[0345] Compound C-(21-27). Compound C-14 was coupled with a substituted boronic acid ester derivative using method A to produce C-(22-27) as a brown solid. Compound C-21 was prepared similarly.

[0346] Compounds 45-52. Ester hydrolysis of compounds C-21-27 using method C gave compounds 45-52.

[0347] 3.3. Compounds 53-56 (Scheme 3C). The synthesis of compounds 53-56 began with the reaction of intermediate C-16 with N-Boc-protected glycine and 2-methylalanine to give compounds C-28 and C-29. The latter compound was reacted with trifluoromethanesulfonic anhydride to give compounds C-30 and C-31, which underwent Pd-catalyzed Suzuki coupling with heterocycle-boronic acid derivatives to give compounds C-(32-35), followed by Boc deprotection to give compounds 53-56.

[0348] TIFF2025507382000057.tif75166Scheme 3C. Synthesis of compounds 53-56

[0349] Compound C-28. To a solution of C-16 (484.16 mg, 2.38 mmol, 2 equiv.) in DMF (50 mL) was added DIEA (461.83 mg, 3.57 mmol, 622.41 uL, 3 equiv.), 6,7-dichloro-1,2,3,4-tetrahydrobenzothiopheno[3,2-c]pyridin-9-ol (370 mg, 1.19 mmol, 1 equiv., HCl) and HATU (905.81 mg, 2.38 mmol, 2 equiv.). The mixture was stirred at 25° C. for 1 h and then washed with saturated NaCl. 2 CO 3 Add aqueous solution (10 mL) and stir at 15°C for 12 hours. 2 The mixture was diluted with 20 mL of 2H2O and extracted with EtOAc (15 mL x 3). The organic layer was washed with brine (5 mL x 3). The organic layer was washed with Na 2 SO 4 Drying, filtering, concentrating, and column chromatography (SiO 2 , petroleum ether:ethyl acetate=1:2) to give C-28 (500 mg, 1.09 mmol, 91.38% yield) as a yellow oil. ESI [M-Boc+H] = 359.1 / 361.0

[0350] Compound C-30. C-28 (500 mg, 1.09 mmol, 1 equiv.) in DCM (60 mL) and TEA (551 mg, 5 mmol, 5 equiv.) in DCM (5 mL) and Tf in DCM (5 mL). 2 A mixture of N2O (768 mg, 2.72 mmol, 2.5 equiv.) at 0 °C was 2 Stir at 25°C for 1 hour under atmospheric pressure and add H 2 The organic layer was diluted with 200 mL of HO (10 mL) and extracted with DCM (30 mL x 3). The organic layer was washed with brine (10 mL x 3). The organic layer was washed with Na 2 SO 4 Drying, filtering, concentrating, and column chromatography (SiO 2 , petroleum ether:ethyl acetate=1:1) to give C-30 (200 mg, 338.15 umol, 31.07% yield) as a white solid. ESI [M+Na] = 612.9 / 614.7

[0351] Compound C-(32-35). Compound C-30 was coupled with a substituted boronic ester derivative using Method A to give C-(32-35) as a white solid.

[0352] Compounds 53-56. NBoc deprotection of C-(32-35) using method B gave compounds 53-56 as white solids.

[0353] 3.4. Compound 57 (Scheme 3D). The synthesis of 57 began with the peptide coupling of amine C-16 with N-Boc protected (R)-proline to give compound C-36. The phenol was reacted with Tf 2 Reaction with O gave triflate C-37, the latter of which underwent Suzuki coupling with 2-amino-pyridine boronic acid derivatives to give compound C-38, followed by Boc deprotection to give the title product 57 (Scheme 3D).

[0354] TIFF2025507382000058.tif66130Scheme 3D. Synthesis of compound 57

[0355] Compound C-36. To a solution of C-16 (150 mg, 482.89 umol, 1 equiv., HCl) in DMF (15 mL), DIEA (187 mg, 1 mmol, 3 equiv.), (2R)-1-tert-butoxycarbonylpyrrolidine-2-carboxylic acid (208 mg, 966 umol, 2 equiv.) and HATU (367 mg, 965 umol, 2 equiv.) were added and stirred at 25° C. for 1 h. The mixture was then cooled to 30° C. with a 10% NaCl solution. 2 CO 3 Add aqueous solution (10 mL) and stir at 15°C for 12 hours. 2 The mixture was diluted with 20 mL of 2H2O and extracted with EtOAc (15 mL x 3). The organic layer was washed with brine (5 mL x 3). The organic layer was washed with Na 2 SO 4 Drying, filtering, concentrating, and column chromatography (SiO 2, petroleum ether:ethyl acetate=1:2) to give C-36 (200 mg, 424.27 umol, 87.86% yield) as a brown solid. ESI [M-Boc+H] = 371.0 / 372.9

[0356] Compound C-37. C-36 (120 mg, 254.56 umol, 1 equiv.) in DCM (4 mL) and TEA (129 mg, 1 mmol, 5 equiv.) in DCM (1 mL) and Tf in DCM (1 mL). 2 A mixture of N2O (180 mg, 636 umol, 2.5 equiv.) at 0 °C was 2 The mixture was stirred at 25°C for 1 hour under atmospheric pressure, concentrated, and then cooled to 37°C. 2 The mixture was diluted with 200 mL of HO (5 mL) and extracted with EtOAc (5 mL x 3). The organic layer was washed with brine (5 mL x 3). The organic layer was washed with Na 2 SO 4 Drying, filtering, concentrating, and column chromatography (SiO 2 , petroleum ether:ethyl acetate=1:1) to give C-37 (100 mg, 165.71 umol, 65.10% yield) as a white solid. ESI [M-Boc+H] = 502.9 / 504.7

[0357] Compound C-38. Compound C-37 was coupled with a substituted boronic ester derivative using Method A to produce C-38 (9.74 mg, 17.79 umol, 21.47% yield) as a white solid. TIFF2025507382000059.tif27161

[0358] Compound 57. Ester hydrolysis of compound C-38 using method C gave compound 57 (15 mg, 32 umol, 25.01% yield, HCl) as a white solid.

[0359] 3.5. Compound 58 (Scheme 3E). The synthesis of compound 58 began with nucleophilic displacement of the bromide in N-Boc-3-bromopyridone with the thiol in 2,3-dichlorobenzenethiol to give the thioether C-39, which underwent PPA-mediated cyclization to give the benzothiofuran intermediate C-40. The latter was reacted with 2-acetoxyacetyl chloride and the resulting product C-41 underwent basic hydrolysis to give the target compound 58.

[0360] TIFF2025507382000060.tif36133Scheme 3E. Synthesis of compound 51

[0361] Compound C-39. 2,3-Dichloro-thiophenol (5.0 g, 27.9 mmol, 1.0 equiv.), tert-butyl 3-bromo-4-oxo-piperidine-1-carboxylate (7.8 g, 27.9 mmol, 1.0 equiv.) and Na in DMF (50 mL). 2 CO 3 (5.9 g, 55.9 mmol, 2.0 equiv.) was stirred at 30° C. for 1.5 h. The mixture was 2 Quench with O (40 mL), extract with EtOAc (50 mL × 3), wash with brine (20 mL × 5), and add Na 2 SO 4 The extract was dried at 4°C, filtered, concentrated, and purified by preparative HPLC (neutral conditions; column: Phenomenex Titank C18 Bulk 250×100 mm 10 u; mobile phase: [water (10 mM NH 4 HCO 3 )-ACN]; B%: 50%~80%, 20 min) to afford C-39 (7.7 g, 20.5 mmol, 73.3% yield) as a yellow oil. TIFF2025507382000061.tif19165

[0362] Compound C-40. A solution of PPA (2 mL) was stirred at 100° C. for 30 min. Compound C-39 (200 mg, 531.5 umol, 1.0 equiv.) was then added and stirred at 140° C. for 2 h. The mixture was diluted with H 2The mixture was added with 10 mL of HO, adjusted to pH > 8 with 10 mL of aqueous NaOH (aqt. aq.), extracted with 3×50 mL of EtOAc, washed with 5×20 mL of brine, and diluted with 10 mL of Na 2 SO 4 The extract was dried at 40° C., filtered, concentrated, and purified by preparative HPLC (neutral conditions; column: Phenomenex Gemini-NX C18 75×30 mm×3 um; mobile phase: [water (10 mM NH 4 HCO 3 )-ACN]; B%: 35%-65%, 8 min) to produce C-40 (27 mg, 103.2 umol, 19.4% yield, 100.0% purity) as a yellow solid. TIFF2025507382000062.tif19162

[0363] Compound C-41. To a solution of C-40 (170 mg, 658.5 umol, 1.0 equiv) and TEA (200 mg, 2.0 mmol, 3.0 equiv) in DCM (3 mL) was added 2-acetoxyacetyl chloride (539 mg, 4.0 mmol, 6.0 equiv). The mixture was stirred at 15° C. for 1 h. The mixture was then cooled to 10° C. for 1 h. 2 The mixture was quenched with O (5 mL), extracted with DCM (10 mL x 3), and then cooled to 30° C. 2 SO 4 The mixture was dried at 40° C., filtered, concentrated and purified by preparative HPLC (TFA condition; column: Phenomenex Luna C18 150×30 mm×5 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 28%-58%, 8 min) to give C-41 (130 mg, 361.1 umol, 54.8% yield, 99.5% purity) as a brown solid. TIFF2025507382000063.tif19163

[0364] Compound 58. A solution of C-41 (100 mg, 279.1 umol, 1.0 equiv) in MeOH (2 mL) was added with LiOH H 2 2H2O (35 mg, 837.4 umol, 3.0 equiv.) was added. The mixture was stirred at 25° C. for 12 h. The mixture was concentrated and H 2 Dilute with 200 mL of HO (5 mL), extract with DCM (10 mL × 3), and2 SO 4 The mixture was dried at 40° C., filtered, concentrated and purified by preparative HPLC (TFA condition; column: Phenomenex Luna 80×30 mm×3 um; mobile phase: [water (0.1% TFA)-ACN]; B%: 23%-53%, 8 min) to give compound 58 (32 mg, 101.2 umol, yield 36.3%, purity 100.0%) as a pale yellow solid.

[0365] 3.6. Compounds 59-66 (Scheme 3F). To prepare compounds 59-66, we started with intermediates C-7 and C-8 above, which were reacted with 1-Boc-3-bromo-4-pyrimidinone to give the thioethers C-42 and C-43. Oxidation of the alcohols afforded ketones at C-44 and C-45, and phosphoric acid mediated cyclization afforded the key benzothiofuran derivatives C-46 and C-47. The thioethers C-42 and C-43 were reacted with 1-Boc-3-bromo-4-pyrimidinone to give the thioethers C-42 and C-43. Oxidation of the alcohols afforded ketones at C-44 and C-45, and phosphoric acid mediated cyclization afforded the key benzothiofuran derivatives C-46 and C-47. 3 The methoxy groups were deprotected using , and the resulting products C-48 and C-49 were converted to triflates C-50 and C-51. Suzuki coupling of the triflates with appropriate heterocycle-boronic acid derivatives gave compounds C-52 and C-(53–59), and elimination of acetic acid in the latter compounds afforded the target products 59–66 (Scheme 3F).

[0366] TIFF2025507382000064.tif107166Scheme 3F. Synthesis of compounds 59-66

[0367] Compound C-43. To a mixture of C-8 (100 mg, 338.8 umol, 1.0 equiv) in EtOH (2 mL) was added EtONa (69 mg, 1.0 mmol, 3.0 equiv). The mixture was stirred at 25° C. for 1 h. To the mixture was added tert-butyl 3-bromo-4-oxo-piperidine-1-carboxylate (283 mg, 1.0 mmol, 3.0 equiv) and stirred at 25° C. for 1 h. The mixture was diluted with H 2 Quench with O (10 mL), extract with EtOAc (10 mL x 3), and 2 SO 4Dry, filter, concentrate, and run on preparative TLC (SiO 2 , petroleum ether:ethyl acetate=5:1) to give compound C-43 (100 mg, 246.1 umol, 72.7% yield) as a colorless oil. TIFF2025507382000065.tif19158

[0368] Compound C-45. A solution of PPA (0.5 mL) was stirred at 100 °C for 30 min. Then C-43 (20 mg, 49.2 umol, 1.0 equiv.) was added and stirred at 140 °C for 2 h. The reaction mixture was diluted with MeOH (5 mL) and purified by preparative HPLC (column: Phenomenex Luna 80 × 30 mm × 3 um; mobile phase: [water (TFA)-ACN]; B%: 15%-45%, 8 min) to produce C-45 (1 mg, 4.2 umol, 8.4% yield, 98.0% purity) as a pale yellow solid. TIFF2025507382000066.tif19163

[0369] Compound C-47. CHCl 3 To a mixture of C-45 (820 mg, 2.9 mmol, 1.0 equiv) in 10 mL of BBr 3 (2.1 g, 8.5 mmol, 3.0 equiv) was added at -60°C. The mixture was stirred at 25°C for 12 h and then saturated Na 2 CO 3 The mixture was quenched with aqueous solution (20 mL) until pH>8 and extracted with DCM (15 mL×3). 2 SO 4 The extract was dried at 40° C., filtered, concentrated and purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150×40 mm×10 um; mobile phase: [water (NH 3 H 2 O+NH 4 HCO 3 )-ACN]; B%: 20%-50%, 8 min) to produce C-47 (110 mg, 367.6 umol, 12.9% yield, 91.6% purity) as a brown solid. TIFF2025507382000067.tif12140

[0370] Compound C-49. DCM (4 mL) and saturated Na 2 CO 3 To a solution of C-47 (390 mg, 1.26 mmol, 1 equiv, HCl) in water (4 mL) was added (2-chloro-2-oxo-ethyl)acetate (514.26 mg, 3.77 mmol, 404.93 uL, 3 equiv). The mixture was stirred at 15° C. for 3 h. The mixture was diluted with H 2 The organic layer was diluted with 20 mL of O and extracted with EtOAc (30 mL x 3). 2 SO 4 The mixture was dried over 1000 ml of ethyl acetate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO 2 , DCM:THF=1:0-5:1) to afford C-49 (280 mg, 748.19 umol, 59.59% yield) as a brown solid. TIFF2025507382000068.tif19161

[0371] Compound C-51. A mixture of C-49 (40 mg, 106.88 umol, 1 equiv.) and TEA (216.31 mg, 2.14 mmol, 20 equiv.) in DCM (1 mL) was degassed with N 2 The solution was purged three times and then dissolved in DCM (0.5 mL) of Tf 2 O (90.47 mg, 320.65 umol, 3 equiv.) was added at 0° C., and the mixture was then diluted with N 2 The mixture was stirred at 25° C. for 12 hours under atmospheric pressure. 2 The organic layer was diluted with O (10 mL) and extracted with DCM (15 mL x 3). 2 SO 4 The mixture was dried at 40° C., filtered and concentrated to give C-51 (130 mg, crude) as a brown solid. ESI [M+H] = 506.0 / 508.0

[0372] Compounds C-(52-59). Compounds C-50-51 were coupled with substituted boronic acid ester derivatives using Method A to produce C-52-59 as brown solids.

[0373] Compounds 59-66. Ester hydrolysis of compounds C (52-59) using method C gave compounds 59-66.

[0374] 3.7. Compounds 67–72 (Scheme 3G). The synthesis of 67–72 was carried out by reacting triflate C-51 with dipinacolato-diborane (B 2 Pin 2 ) together, followed by Suzuki coupling of the resulting boronic acid derivative C-60 with a bromoheterocycle to give compounds C-(61-66), which was then hydrolyzed to afford the title products.

[0375] TIFF2025507382000069.tif58166Scheme 3G. Synthesis of compounds 67-72

[0376] Compound C-60. C-51 (660 mg, 1.30 mmol, 1 equiv.), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (993.08 mg, 3.91 mmol, 3 equiv.), Pd(dppf)Cl in dioxane (20 mL). 2 A mixture of (95.38 mg, 130.36 μmol, 0.1 equiv.), KOAc (383.81 mg, 3.91 mmol, 3 equiv.) was degassed with N 2 Purge the mixture three times with N 2 The mixture was stirred at 80° C. under atmospheric pressure for 12 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate = 1:0 to 0:1) to give C-60 (430 mg, 888.06 μmol, 68.13% yield) as a white solid. ESI [M+H] = 483.9 / 485.7

[0377] Compound C-(61-66). Compound C-60 was coupled with a substituted boronic ester derivative using Method A to produce C-(61-66) as a white solid.

[0378] Compounds 67-72. Ester hydrolysis of compounds C-54-56 using method C gave compounds 67-72.

[0379] 3.8. Compounds 73-84 (Scheme 3H). The synthesis of compounds 73-84 was accomplished starting from compound C-47, reacting the latter with N-Boc-protected glycine and 2-methylalanine. The resulting amide-alcohols C-67 and C-68 were converted to triflates C-69 and C-70 by reaction with trifluoromethanesulfonic anhydride, followed by the synthesis of the heterocycle-boronic acid derivatives (R 1 Treatment with BPin afforded compounds C-(71–83). Finally, C-(71–83) was stirred in methanolic HCl to give 73–85 as HCl salts.

[0380] TIFF2025507382000070.tif80166 Scheme 3H. Synthesis of compounds 73-84

[0381] Compound C-67. To a mixture of C-49 (10 mg, 36.5 umol, 1.0 equiv), N-boc glycine (19 mg, 109.4 umol, 3.0 equiv) and NMM (15 mg, 145.9 umol, 4.0 equiv) in DMF (1 mL), EDCI (10 mg, 54.7 umol, 1.5 equiv) and HOBt (7 mg, 54.7 umol, 1.5 equiv). The mixture was stirred at 25 °C for 1 h. The reaction mixture was filtered and purified by preparative HPLC (TFA condition; column: Phenomenex Luna 80 × 30 mm × 3 um; mobile phase: [water (TFA)-ACN]; B%: 40%-75%, 8 min) to produce C-67 (8 mg, 17.8 umol, 48.7% yield, 100.0% purity) as a white solid. TIFF2025507382000071.tif19165

[0382] Compound C-69. A mixture of C-67 (75 mg, 173.9 umol, 1.0 equiv) and TEA (352 mg, 3.5 mmol, 20.0 equiv) in DCM (1.0 mL) was degassed with N2 The solution was purged three times and dissolved in DCM (0.5 mL). 2 O (245 mg, 869.4 umol, 5.0 equiv.) was added at 0° C., and the mixture was then diluted with N 2 The mixture was stirred under atmospheric pressure at 25° C. for 1 hour. The reaction mixture was concentrated and purified by column chromatography (SiO 2 The mixture was purified by hexane distillation (petroleum ether:ethyl acetate = 3:2 to 3:2) to give C-69 (93 mg, 165.1 umol, 95% yield) as a brown solid. ESI [MH] = 561.0 / 563.0

[0383] Compound C-(71-83). Compound C-69 was coupled with substituted boronic acid ester derivatives using method A to produce C-71-82 as white solids. Compound C-83 was prepared similarly.

[0384] Compounds 73-85. NBoc deprotection of C-(71-83) using method B gave compounds 73-85 as white solids.

[0385] 3.9. Compounds 86-93 (Scheme 3I). To prepare compounds 86-93, triflate C-69 was converted to the boronic acid derivative C-84 and reacted with bromoheterocycles as required to give compounds C-(85-92). The latter underwent N-Boc deprotection using methanolic HCl to give the title products.

[0386] TIFF2025507382000072.tif44166Scheme 3I. Synthesis of compounds 86-93

[0387] Compound C-84. C-69 (1.6 g, 2.84 mmol, 1 equiv.), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (1.44 g, 5.68 mmol, 2 equiv.), Pd(dppf)Cl in dioxane (50 mL). 2A mixture of (207.80 mg, 283.99 umol, 0.1 equiv.) and KOAc (836.15 mg, 8.52 mmol, 3 equiv.) was degassed with N 2 Purge the mixture three times with N 2 The mixture was stirred at 80° C. under atmospheric pressure for 12 hours. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate = 1:1 to 1:2) to afford C-84 (1.5 g, 2.77 mmol, yield 97.58%) as a white solid. TIFF2025507382000073.tif19160

[0388] Compound C-(85-92). Compound C-84 was coupled with a substituted boronic ester derivative using Method A to produce C-(85-92) as a white solid.

[0389] Compounds 86-93. NBoc deprotection of C-(85-92) using Method B gave compounds 86-93 as white solids. Compounds C-(85-92). Compounds C-84 was coupled with a substituted boronic ester derivative using Method A to give C-(85-92) as white solids.

[0390] Compounds 86-93. NBoc deprotection of C(85-92) using method B gave compounds 86-93 as white solids.

[0391] 3.10. Compounds 94-97 (Scheme 3J) TIFF2025507382000074.tif45165Scheme 3J. Synthesis of compounds 94-97

[0392] Compound C-93. MeCN (15 mL) and H 2 To a solution of C-84 (1 g, 1.85 mmol, 1 equiv.) in 20O (6 mL), NaIO 4 (1.58g, 7.39mmol, 409.48uL, 4eq) and NH 4OAc (569.62 mg, 7.39 mmol, 4 equiv.) was added. The mixture was stirred at 70° C. for 12 h. The reaction mixture was filtered and purified by preparative HPLC (column: Phenomenex luna C18 250×50 mm×10 um; mobile phase: [water (TFA)-ACN]; B%: 30%-70%, 10 min) to produce C-93 (350 mg, 762.28 umol, 41.26% yield) as a pale yellow solid. LCMS: m / z 458.9 [M+H] +

[0393] Compound C-(94-97). C-93 (30 mg, 65.34 umol, 1 equiv.), substituted cyclic secondary amine (98.01 umol, 1.5 equiv.), Cu(OAc) in DMF (2 mL). 2 A mixture of Py (35.60 mg, 196.02 umol, 3 equiv.) and Py (25.84 mg, 326.70 umol, 5 equiv.) was degassed and diluted with O 2 The mixture was then purged with O three times. 2 The reaction mixture was stirred at 25° C. for 1 hour under atmospheric pressure. 2 The mixture was diluted with 2 mL of O and extracted with EtOAc (5 mL x 3). The combined organic layers were washed with Na 2 SO 4 The residue was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150×40 mm×10 um; mobile phase: [water (NH 4 HCO 3 )-ACN]; B%: 50%-85%, 8 min) to afford C-(94-97) as a white solid.

[0394] Compounds 94-97. NBoc deprotection of intermediate C-(94-97) using method B gave compounds 94-97 as white solids.

[0395] 4. Pyridoimidazole derivatives 4.1. Compounds 98-101 (Scheme 4A) TIFF2025507382000075.tif87164 Scheme J. Synthesis of compounds 97-101

[0396] Compound D-2. A mixture of D-1 (980 mg, 6.0 mmol, 1.0 equiv.), tert-butyl 3-bromo-4-oxo-piperidine-1-carboxylate (5.0 g, 18.0 mmol, 3.0 equiv.) and HCl (1 M, 601 uL, 0.1 equiv.) in toluene (10 mL) was stirred at 110° C. for 24 h. The mixture was stirred at 10° C. for 24 h. 2 Quench with O (10 mL), extract with EtOAc (50 mL x 3), and 2 SO 4 The mixture was dried over 1000 ml of ethyl acetate, filtered and concentrated under reduced pressure to give a residue which was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate = 1:0 to 78:22) to produce D-2 (1.1 g, 3.2 mmol, yield 53.5%) as a white solid. TIFF2025507382000076.tif12163

[0397] Compound D-3. A mixture of D-2 (330 mg, 964.3 umol, 1.0 equiv) in THF (4 mL) was degassed and purified with N 2 The mixture was purged three times, and n-BuLi (2.5M, 578uL, 1.5eq) was added at -78°C and stirred for 0.5h. Then the mixture was diluted with I 2 (367 mg, 1.4 mmol, 1.5 eq.) was added and N 2 The mixture was stirred at 0° C. for 1 hour under atmospheric pressure. 4 Quenched with aqueous Cl (5 mL), extracted with EtOAc (20 mL x 3), and 2 SO 4 The mixture was dried over 400° C., filtered and concentrated under reduced pressure to give a residue which was purified by column chromatography (SiO 2 , petroleum ether:ethyl acetate = 6:1 to 4:1) to produce D-3 (350 mg, 748 umol, yield 77.5%) as a yellow solid. TIFF2025507382000077.tif19160

[0398] Compounds D-4 and D-5. Compound D-3 was coupled with a substituted boronic acid ester derivative using Method A to produce D-4 and D-5 as white solids.

[0399] tert-Butyl 10,11-dichloro-13-pyrimidin-5-yl-1,4,8-triazatricyclo[7.4.0.02,7]trideca-2(7),8,10,12-tetraene-4-carboxylate, D-4 (70 mg, 166.6 umol, 91.7% yield). TIFF2025507382000078.tif19155

[0400] tert-Butyl 10,11-dichloro-13-(1-methylpyrazol-4-yl)-1,4,8-triazatricyclo[7.4.0.02,7]trideca-2(7),8,10,12-tetraene-4-carboxylate, D-5 (70 mg, 165.8 umol, 91.3% yield) as a brown oil. TIFF2025507382000079.tif19161

[0401] Compounds D6 and D7. To a mixture of D-4 and D-5 (65 mg, 154.6 umol, 1.0 equiv) in DCM (1.5 mL) was added TFA (0.5 mL) at 0° C. The mixture was stirred at 25° C. for 1 h. The reaction was concentrated to give D6 and D7 (70 mg, crude, TFA) as a brown oil, which was used in the next step without further purification.

[0402] Compounds D8 and D-10. To a mixture of D6 and D7 (60 mg, 138.2 umol, 1.0 equiv, TFA) and TEA (70 mg, 690.9 umol, 5 equiv) in DCM (1 mL) was added (2-chloro-2-oxo-ethyl)acetate (28 mg, 207.3 umol, 1.5 equiv) at 0° C. The mixture was stirred at 25° C. for 1 h. The mixture was diluted with Na 2 CO 3 (2 mL), extracted with EtOAc (3 mL x 5), and the organic layer was washed with Na 2 SO 4Drying at rt, filtration and concentration under reduced pressure afforded D8 and D-10 (50 mg, crude) as brown solids which were used in the next step without further purification.

[0403] Compounds 98 and 99. Ester hydrolysis of compounds D8 and D-10 using Method C gave compounds 98 and 99.

[0404] Compounds D11 and D-12. To a mixture of D6 / D7 (25 mg, 69.7 umol, 1.0 equiv, HCl) and 2-(tert-butoxycarbonylamino)acetic acid (24 mg, 139.4 umol, 2.0 equiv) in DMF (1 mL) was added NMM (28 mg, 278.8 umol, 4.0 equiv), EDCI (20 mg, 104.6 umol, 1.5 equiv) and HOBT (14 mg, 104.6 umol, 1.5 equiv). The mixture was stirred at 25° C. for 1 h. The mixture was then cooled to 5° C. for 1 h. 2 The mixture was quenched with O (3 mL), extracted with EtOAc (3 mL × 5), and the organic layer was washed with brine (3 mL × 3) and Na 2 SO 4 Drying at rt, filtration and concentration under reduced pressure gave D11 and D-1 (25 mg, crude) as a brown solid, which was used in the next step without further purification.

[0405] Compounds 100 and 101. To a mixture of D11 / D-12 (20 mg, 41.9 umol, 1.0 equiv) in DCM (0.9 mL) was added TFA (0.3 mL) at 0° C. and then stirred at 25° C. for 1 h. The reaction was concentrated and washed with saturated Na 2 CO 3 water (5 mL), extracted with EtOAc (5 mL x 5), and 2 SO 4 The extract was dried at 40° C., filtered, concentrated and purified by preparative HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 um; mobile phase: [water (NH 4 HCO 3)-ACN]; B%: 15% to 45%, 10 min) to afford 100 and 101 (2 mg, 4.3 umol, 10.3% yield, 100.0% purity) as pale yellow solids.

[0406] (Table 1) Spectral data TIFF2025507382000080.tif195170TIFF2025507382000081.tif213170TIFF2025507382000082.tif184170TIFF2025507382000083.t if211170TIFF2025507382000084.tif191170TIFF2025507382000085.tif207170TIFF2025507382000086.tif208170TIFF20255073820 00087.tif202170TIFF2025507382000088.tif199170TIFF2025507382000089.tif200170TIFF2025507382000090.tif205170TIFF202 5507382000091.tif193170TIFF2025507382000092.tif189170TIFF2025507382000093.tif196170TIFF2025507382000094.tif201170

[0407] Cell-free mouse and human cGAS enzyme inhibition assays (Table 2). The activity of all compounds (in Table 1) against m-cGAS (30 nM) and / or h-cGAS (100 nM) was measured in Tris-HCl (20 mM, pH 7.4), NaCl (150 mM), MnCl 2 (0.2 mM, h-cGAS) or MgCl 2Conversion of ATP and GTP (100 μM each) to cGAMP in the presence of dsDNA (5 μg / mL) in a reaction buffer composed of m-cGAS (5 mM) and Tween-20 (0.01%), as well as residual ATP concentration, was determined by measuring using the Kinase-Glo® Max Luminescent Kinase Assay (Promega, Madison, WI) as described (Lama, et. al., 2019). Briefly, 10 μl of a master mix of 0.4 mM ATP, 0.4 mM GTP, 0.02 mg / mL dsDNA in reaction buffer supplemented with 2 mM DTT was added to reaction wells containing TDI-6570 or TDI-8246 (2× the desired concentration) in the same buffer (20 μL) using a Thermo 8-channel Multidrop Combi dispenser. Then, 10 μl of 4× m-cGAS (0.120 μM) or h-cGAS (0.4 μM) solution in reaction buffer supplemented with 2 mM DTT was added to the appropriate wells. A similar set of reactions without cGAS or inhibitors was set up by adding buffer only. Plates were sealed and reactions were incubated at 37° C. (1 h for m-cGAS, 3 h for h-cGAS) and stopped by adding 40 μl of Kinase-Glo® Max. Luminescence was recorded in relative light units (RLU) using a Biotek Synergy H1 Hybrid plate reader (BioTek, Winooski, VT). ATP depletion was normalized to positive (no cGAS) and negative (with cGAS) controls as follows: % inhibition=100×(RLU sample−RLU average negative control) / (RLU average positive control−RLU average negative control). All reactions were performed in triplicate and calculated using GraphPad Prism 9. Values ​​are the mean IC 50 The values ​​are ±SD.

[0408] Table 2. Activity of cGAS inhibitors in cell-free enzyme assays TIFF2025507382000095.tif47128TIFF2025507382000096.tif21588TIFF2025507382000097.tif21588TIFF2025507382000098.tif21588TIFF2025507382000099.tif150128Note: 0-24% cell-free activity is represented as "-"; 25-49% as +; 50-74% as ++; 75-100% as +++.

[0409] Evaluation of active cGAS inhibitors in THP1 cells (Table 3). Cellular activity of all compounds (in Table 3) was determined using THP1-Dual™ cells as described (Lama et. al., 2019). THP1-Dual™ cells (Invivogen) (50000 cells in 100ul of medium) were seeded into each well of a 96-well plate and treated with various dilutions of compounds for 4 hours. Cells were then transfected with DNA (2ug / ml) and lipofectamine and cells were incubated overnight. Ifnb expression was measured in cell supernatants using Quantiluc, nfkb expression using Quantiblue and cell viability was determined using Quantiblue. Promega® CellTiter-Glo® It was judged using.

[0410] Table 3: Examples of biological evaluation in the THP1 assay TIFF2025507382000100.tif205148TIFF2025507382000101.tif215148TIFF2025507382000102.tif220148Note: Cell viability <24% in THP1 cells is represented as "-", similarly, +, ++ and +++ mean that the compound exhibits 25-49%; 50-74%; or 75-100% cell viability, respectively.

[0411] Data from pharmacokinetic, off-target effects, protein binding and metabolic studies for selected compounds are provided in Tables 4-7.

[0412] Table 4. Summary of plasma (top) and brain (bottom) PK parameters TIFF2025507382000103.tif112152TIFF2025507382000104.tif133152

[0413] Table 5. Off-target effects of compounds 48 and 63 TIFF2025507382000105.tif78170

[0414] Table 6. Protein binding results of cGAS inhibitor fraction in mouse brain homogenate TIFF2025507382000106.tif1011701 The test compounds were not detectable in the buffer side (below limit of detection, BLOD), and the LOD was used as the peak area to calculate the cutoff value. Two compounds were unstable with % remaining values ​​below 50%. 3 As a control, TDI-6570 was used as a mouse-selective cGAS inhibitor.

[0415] Table 7. Metabolic stability of test compounds TIFF2025507382000107.tif111166TIFF2025507382000108.tif138170Note: NCF: no cofactor. No NADPH is added to the NCF sample (exchanged to buffer) during the 60 min incubation. If less than 60% NCF remains, non-NADPH dependent metabolism is likely occurring. R 2 : Correlation coefficient of linear regression to determine rate constants (see raw data worksheet) T 1 / 2 :Half-life CL int(ミクロソーム) : Inherent clearance CL int(ミクロソーム) =0.693 / T 1 / 2 / mg microsomal protein / mL CL int(肝臓) =CL int(ミクロソーム) × microsomal protein mg / liver weight g × liver weight g / body weight kg

[0416] INCORPORATION BY REFERENCE All U.S. patents and published U.S. patent applications cited herein are hereby incorporated by reference.

[0417] Equivalent Those skilled in the art will recognize, or be able to ascertain without undue routine experimentation, many equivalents to the various embodiments of the disclosure described herein, such equivalents being encompassed by the scope of the appended claims.

Claims

[Claim 1] Formula (I) or (II): or a pharmaceutically acceptable salt thereof, During the ceremony, R 1 is heteroaryl, halogen, aryl, cyclic amine, hydroxy, -OC(O) alkyl, -NH 2 , —N(H)CO-alkyl, or alkoxy; R 2 is H, alkyl, -CHF 2 , -CF 3 , -CN, -OR c , halogen, or heterocyclyl; R 3 and R 4 are independently H, halogen, -CHF 2 , -CF 3 , -CN, -OR c , or -OCF 3 and R 5a and R 5b is independently H, alkyl, aryl, or cycloalkyl; R 6 is N(H)R a , O-alkyl, OH, -CO 2 R d Is it; Or, R 5a and R 6 taken together to form a 5- or 6-membered heterocyclyl; R 7a and R 7b are independently H or alkyl, or together with the carbon to which they are attached form a 3-membered aliphatic carbocyclic ring; R a is H, alkyl, -COR b , -CON(H)R b , or -CO 2 R e and R b is alkyl or aryl; R c is H, alkyl, or —C(O)-alkyl; R d is H or alkyl; R e is alkyl or aryl; X is NH, NMe, NEt, O, or S; and is a single or double bond, The compound or a pharmaceutically acceptable salt thereof.

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