Small molecule SLC15A4 inhibitors with anti-inflammatory activity

Abstract

Small molecule inhibitors of SLC15A4 and methods of their use to treat pDC-mediated diseases and conditions are disclosed.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 981,907, filed February 26, 2020, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to novel compounds and methods useful for inhibiting SLC15A4. [Background technology]

[0003] Recognition of infectious pathogens relies on a set of germline-encoded immune sensors known as pattern receptors (TLRs) and NOD-like receptors (NLRs). TLRs are membrane sensors that scan the extracellular environment for microbial PAMPs, while NLRs monitor the cytoplasmic environment. Viral and bacterial nucleic acids are prominent PAMPs recognized by several TLRs, including TLR3, TLR7, TLR8, and TLR9. Ligand binding to these sensors triggers signaling events that lead to the expression of several immune response genes, including proinflammatory cytokines, stimulatory immune cytokines, chemokines, and costimulatory molecules that enhance pathogen killing. 1-2 However, inappropriate recognition of host nucleic acids can lead to autoimmune and autoinflammatory conditions. 3-6 Autoimmunity emerges through several concurrent mechanisms related to the presence of autoreactive immune cell subsets and the loss of immune tolerance. The loss of tolerance during central and peripheral differentiation of the adaptive immune response can lead to the unrestrained activation of autoreactive B and T cells, which, with the help of innate immune cells, can induce autoimmunity. TLR signaling plays an essential role in the activation of the adaptive immune system by inducing the production of proinflammatory cytokines, and continuous activation or dysregulation of TLR signaling directly contributes to the pathogenesis of autoimmunity. 7In particular, a very important finding was that activation of endolysosomal nucleic acid-sensing TLRs and production of type I interferon (IFN-I) by APC class plasmacytoid dendritic cells (pDCs) are central driving events in pathogenesis. 8 .

[0004] pDCs are a specialized dendritic cell subset of recirculating cells that act as early sentinels in pathogen surveillance. pDCs recognize microbial and endogenous nucleic acids via TLR7 and TLR9 (TLR7 / 9). 9、10 In response to TLR7 / 9, pDCs produce approximately 1000 times more type 1 IFN (IFN-I) than other cell types. Activation of TLR7 / 9 in pDCs can also induce other cytokines (IL-12, IL-6, TNFα) and inflammatory chemokines. 11 Evidence also suggests that pDCs activate B cells, act as APCs, and promote immune regulation and tolerance. 12-15 Given their central role in inflammation, it is perhaps not surprising that pDCs are causative effectors in the pathogenesis of several autoimmune disorders, such as lupus and psoriasis. One of the strongest links between pDCs and autoimmune disease is in systemic lupus erythematosus (SLE). 8 In most mouse models, lupus is dependent on IFN-I, and genetic deletion or neutralization of IFN-I signaling can prevent or ameliorate the disease. 7 Furthermore, approximately 70% of SLE patients show elevated IFN-I signatures. 16 Clinical trials with IFN-I receptor-neutralizing antibodies are currently underway, with promising results in lupus patients in the clinic. pDCs have also been detected in the cerebrospinal fluid of patients with multiple sclerosis (MS). 17 , which accumulates in demyelinated lesions in inflamed MS brains 18 . Summary of the Invention [Problem to be solved by the invention]

[0005] Small molecule immunomodulatory drugs have been developed to control harmful immune responses during inflammation, transplantation, and autoimmune conditions. Corticosteroids, calcineurin inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDs), mTOR inhibitors, and kinase inhibitors have been used to treat autoimmune conditions and improve transplantation efficiency. More recently, therapeutic agents such as Gilenya and Tecfidera have shown efficacy in ameliorating disease progression and symptoms in patients with multiple sclerosis. However, most of these treatments are general inflammation modulators or broad-spectrum immunosuppressants that can engage signaling pathways common to nearly all cell types, resulting in undesirable side effects that limit their use. 19 Monoclonal antibody (mAb) therapies directed against specific immune targets have been developed (e.g., checkpoint inhibitors (anti-CTLA-4), anti-TNF) and have shown some success, but they often only work in a subset of patients and are often associated with severe immune-related adverse events (irAEs). 19、20 There are currently no available therapies that target pDC-mediated production of IFN-I, a central driver of many autoimmune conditions. 21 Thus, there is an unmet need for novel small molecule therapeutics for many pDC-mediated conditions.

[0006] SLC15A4 plays a central role in pDC-mediated inflammation and autoimmunity. Solute transporter gene family 15 member 4 (SLC15A4), also known as proton / histidine transporter 1 (PHT1), is a 12-transmembrane protein whose gene expression is primarily restricted to APCs, specifically pDCs and B cells. 22-24 SLC15A4 is a member of the SLC15 family, which also includes the proton / histidine transporter SLC15A3 (PHT2) and the di- / tripeptide transporters SLC15A1 (PEPT1) and SLC15A2 (PEPT2). Both SLC15A3 and SLC15A4 contain an acidic dileucine motif that mediates their localization to endosomes / lysosomes and are annotated as di- or tripeptide cotransporters. 25Lysosomes and endosomes are acidic, suggesting that SLC15A3 and SLC15A4, which share 60% sequence identity, exploit the outward proton gradient to transport short peptides into the cytoplasm. 25、26 However, the substrates of endolysosomal SLC15A3 and SLC15A4 have not been established. Several studies have demonstrated that SLC15A4 transports bacterial peptidoglycans, such as MDP and Tri-DAP, which are ligands for the immune sensors NOD1 and NOD2, leading to their activation. 27-30 SLC15A4 has also been closely linked to TLR7 / 9-mediated signaling and IFN-I production. Specifically, studies have shown that both SLC15a4 loss-of-function mutants (termed "weak") and knockout mice give rise to pDCs with defective IFN-I, TNF-α, IL-6, and IL-12 production upon TLR stimulation, but otherwise display normal development. 31、29、30、32 This defect is not due to impaired TLR ligand uptake or IFN-I secretion, but affects both TLR7 and TLR9 signaling pathways. Importantly, Slc15a4-deficient mice exhibit significantly reduced lupus symptoms and an extended lifespan. 32 Although SLC15A3 and SLC15A4 are thought to have similar functions, the disease-modifying effects of SLC15A4 mutations and deletions suggest that there are significant functional differences between these two transporters or that expression of both is required to ensure optimal function. Furthermore, genome-wide association studies (GWAS) have revealed that SLC15A4 (but not SLC15A3) is closely associated with inflammatory diseases such as systemic lupus erythematosus (SLE) and inflammatory bowel disease (IBD). 33、34However, the precise mechanisms by which SLC15A4 contributes to these processes remain to be elucidated. Nevertheless, the essential pathogenic role of the pDC / TLR / IFN-I axis and the ameliorative effect of SLC15A4 loss of function in mouse models on autoimmune diseases establish SLC15A4 as a key regulator of inflammation and provide a strong basis for the characterization and development of SLC15A4 inhibitors.

[0007] SLC Biology and Chemical Probe Discovery. The solute-like transporter (SLC) family of proteins is the largest group of membrane transporters with 456 members distributed across 52 subfamilies. SLCs have not only been implicated in many disorders resulting from genetic polymorphisms, but also have established roles in tumorigenesis, autoimmune diseases, and metabolic disorders. 35-37 Despite their importance, a significant portion (>30%) of SLCs remains insufficiently or completely characterized, and the majority (>80%) lack chemical probes. 38 One of the biggest obstacles is their complex integral membrane morphology, which requires an intact membrane to maintain their native functional properties. The difficulty in expressing and purifying SLCs in their native state limits the use of traditional high-throughput screening (HTS) approaches and general in vitro biochemical investigations to annotate their substrate range, measure transport rates, and examine the effects of various perturbations (e.g., mutations, inhibitors) on transport. 38 Due to inherent technical challenges, only 10 human SLCs have known structures, and few exist in multiple conformations or with substrate or drug bound states, limiting the potential for structure-based drug design. 39 Cell- and animal-based models for studying SLCs are similarly challenging, as genetic perturbations are complicated by overlapping specificities, compensatory mechanisms, and toxicity, thereby limiting studies to only a subset of SLCs and sometimes obscuring the relative contribution of transporters to the studied function or phenotype. 35、36、40、41Given these challenges, new approaches to study SLC biology and develop useful SLC-targeting chemical probes are desperately needed.

[0008] Previous studies have revealed that SLC15A4 plays a unique and important role in the production of IFN-I and other inflammatory cytokines in pDCs and the development of autoimmune conditions, raising the possibility of SLC15A4 as a therapeutic target for such diseases. However, SLC15A4 has not yet been drugged, and no inhibitors have been disclosed. This application not only describes a possible chemical proteomic strategy to elucidate the mechanism by which SLC15A4 regulates TLR signaling, but also evaluates the therapeutic potential of SLC15A4 for the treatment of pDC-mediated conditions.

[0009] There are no clinically approved drugs that specifically target pDCs and their IFN-I production and nucleotide-binding TLR signaling, central factors in the pathogenesis of many autoimmune conditions such as lupus, Crohn's disease, irritable bowel syndrome (IBS), type 1 diabetes, psoriasis, and even MS. Importantly, SLC15A4 is primarily expressed in antigen-presenting cells that directly contribute to the pathogenesis of autoimmune conditions, specifically pDCs, B cells, and macrophages, making it a highly plausible therapeutic target for the development of compounds that selectively suppress inflammation. [Means for solving the problem]

[0010] The applicants have discovered novel SLC154A inhibitor compounds and evaluated the biochemical potency (e.g., % IFNα±SD inhibition and % transport inhibition assessment in human pDCs), potency, and utility of both representative examples of such compounds.

[0011] In various embodiments, the present disclosure relates to compounds of formula (I) or (II) or a pharmaceutically acceptable salt thereof: [Formula 1] During the ceremony, X is NR 4 or S; R 1 is H or -C(O)-C 1-10 is alkyl; R 2 is a heterocycle or aryl; R 3 is a halogen, -CHF2, or -CF3; R 4 -H, -C 1-10 Alkyl, -C(O)-C 1-10 Alkyl, -C(O)-C 3-10 Cycloalkyl, -S(O)2-C 1-10 alkyl, or [Case 2] and; n is 0, 1, 2, 3, or 4.

[0012] In various embodiments, the disclosure relates to the compounds of FIG.

[0013] In various embodiments, the present disclosure relates to methods of treating conditions mediated by pDCs, B cells, macrophages, or monocytes. [Brief explanation of the drawings]

[0014] [Figure 1]Diagram of fragment-based ligand mapping in cells (FbLMiC). Overview of the method and dedicated chemical library. (A) Fully functionalized fragment (FFF) probes, consisting of drug-like fragments and search tags, enable covalent capture of fragment-binding protein targets directly within cells upon UV irradiation. The fragment-target and fragment-interaction sites can be identified and quantified by mass spectrometry and gel-based methods. (B) General structure of the FFF library, showing the constant affinity tag region (red), including photoreactive (diazirine) and latent affinity (alkyne) groups, and the variable region (blue), which contains the fragment recognition elements for binding to proteins. Example fragments are shown. (C) A subset of proteins for which FFF coordination provided the first evidence of druggability (non-Drugbank) and functional classification for established druggable proteins (Drugbank). (46,47) [Figure 2A] This figure depicts the chemoproteomic development of SLC chemical probes. (A) Summary of confirmed fragment-SLC interactions using FbLMiC in HEK293T, K562, and human PBMC cells. An SLC is considered an FFF target if it is reproducibly enriched >5-fold (at least in biological replicates) relative to a control methyl-FFF probe and is chemically selective (e.g., not enriched by all FFFs). The x-axis represents the SLC subfamily, and the y-axis represents the proportion of the SLC subfamily considered an FFF target. (B) Structures of FFF3, a fragment-based inhibitor (CP22), and a control compound (CP26) for a previously published (46) functional study of SLC25A20. (C) FFF3 probe-labeled sites (brown) mapped to the homologous SLC25A20 structure (brown). Example MS1 chromatograms are shown for probe-labeled tryptic peptides shown in blue. (D) CP22 increases long-chain acylcarnitine content in HSC5 cells. Data = mean ± SD; **p<0.01, ***p<0.001, and ****p<0.0001 for treatment groups; n=3-5. [Figure 2B]This figure depicts the chemoproteomic development of SLC chemical probes. (A) Summary of confirmed fragment-SLC interactions using FbLMiC in HEK293T, K562, and human PBMC cells. An SLC is considered an FFF target if it is reproducibly enriched >5-fold (at least in biological replicates) relative to a control methyl-FFF probe and is chemically selective (e.g., not enriched by all FFFs). The x-axis represents the SLC subfamily, and the y-axis represents the proportion of the SLC subfamily considered an FFF target. (B) Structures of FFF3, a fragment-based inhibitor (CP22), and a control compound (CP26) for a previously published (46) functional study of SLC25A20. (C) FFF3 probe-labeled sites (brown) mapped to the homologous SLC25A20 structure (brown). Example MS1 chromatograms are shown for probe-labeled tryptic peptides shown in blue. (D) CP22 increases long-chain acylcarnitine content in HSC5 cells. Data = mean ± SD; **p<0.01, ***p<0.001, and ****p<0.0001 for treatment groups; n=3-5. [Figure 2C]This figure depicts the chemoproteomic development of SLC chemical probes. (A) Summary of confirmed fragment-SLC interactions using FbLMiC in HEK293T, K562, and human PBMC cells. An SLC is considered an FFF target if it is reproducibly enriched >5-fold (at least in biological replicates) relative to a control methyl-FFF probe and is chemically selective (e.g., not enriched by all FFFs). The x-axis represents the SLC subfamily, and the y-axis represents the proportion of the SLC subfamily considered an FFF target. (B) Structures of FFF3, a fragment-based inhibitor (CP22), and a control compound (CP26) for a previously published (46) functional study of SLC25A20. (C) FFF3 probe-labeled sites (brown) mapped to the homologous SLC25A20 structure (brown). Example MS1 chromatograms are shown for probe-labeled tryptic peptides shown in blue. (D) CP22 increases long-chain acylcarnitine content in HSC5 cells. Data = mean ± SD; **p<0.01, ***p<0.001, and ****p<0.0001 for treatment groups; n=3-5. [Figure 2D]This figure depicts the chemoproteomic development of SLC chemical probes. (A) Summary of confirmed fragment-SLC interactions using FbLMiC in HEK293T, K562, and human PBMC cells. An SLC is considered an FFF target if it is reproducibly enriched >5-fold (at least in biological replicates) relative to a control methyl-FFF probe and is chemically selective (e.g., not enriched by all FFFs). The x-axis represents the SLC subfamily, and the y-axis represents the proportion of the SLC subfamily considered an FFF target. (B) Structures of FFF3, a fragment-based inhibitor (CP22), and a control compound (CP26) for a previously published (46) functional study of SLC25A20. (C) FFF3 probe-labeled sites (brown) mapped to the homologous SLC25A20 structure (brown). Example MS1 chromatograms are shown for probe-labeled tryptic peptides shown in blue. (D) CP22 increases long-chain acylcarnitine content in HSC5 cells. Data = mean ± SD; **p<0.01, ***p<0.001, and ****p<0.0001 for treatment groups; n=3-5. [Figure 3A] Figure 1. Chemical proteomic development of SLC15A4 chemical probes. (A) Structure of the FFF probe identified in a proteomic experiment as engaging SLC15A4 (see text for experimental description). All probes were tested for their ability to inhibit IFN-I production in human pDCs, with 5 showing the highest activity. The structurally similar 6 was not observed to bind to SLC15A4 or inhibit IFN-I production. (B) Gel-based competition readout showing the interaction of 5 competing with excess 5-comp in human PBMCs. (C) Isotopic reductive demethylation heat map showing 5-enriched proteins (20 mM) competed by excess 5-comp or 6-comp (80 mM) in human PBMCs. The inset shows the identification of the top 15 competing targets. (D) Example MS1 of SLC15A4 tryptic peptides from a competition experiment. [Figure 3B]Figure 1. Chemical proteomic development of SLC15A4 chemical probes. (A) Structure of the FFF probe identified in a proteomic experiment as engaging SLC15A4 (see text for experimental description). All probes were tested for their ability to inhibit IFN-I production in human pDCs, with 5 showing the highest activity. The structurally similar 6 was not observed to bind to SLC15A4 or inhibit IFN-I production. (B) Gel-based competition readout showing the interaction of 5 competing with excess 5-comp in human PBMCs. (C) Isotopic reductive demethylation heat map showing 5-enriched proteins (20 mM) competed by excess 5-comp or 6-comp (80 mM) in human PBMCs. The inset shows the identification of the top 15 competing targets. (D) Example MS1 of SLC15A4 tryptic peptides from a competition experiment. [Figure 3C] Figure 1. Chemical proteomic development of SLC15A4 chemical probes. (A) Structure of the FFF probe identified in a proteomic experiment as engaging SLC15A4 (see text for experimental description). All probes were tested for their ability to inhibit IFN-I production in human pDCs, with 5 showing the highest activity. The structurally similar 6 was not observed to bind to SLC15A4 or inhibit IFN-I production. (B) Gel-based competition readout showing the interaction of 5 competing with excess 5-comp in human PBMCs. (C) Isotopic reductive demethylation heat map showing 5-enriched proteins (20 mM) competed by excess 5-comp or 6-comp (80 mM) in human PBMCs. The inset shows the identification of the top 15 competing targets. (D) Example MS1 of SLC15A4 tryptic peptides from a competition experiment. [Figure 3D]Figure 1. Chemical proteomic development of SLC15A4 chemical probes. (A) Structure of the FFF probe identified in a proteomic experiment as engaging SLC15A4 (see text for experimental description). All probes were tested for their ability to inhibit IFN-I production in human pDCs, with 5 showing the highest activity. The structurally similar 6 was not observed to bind to SLC15A4 or inhibit IFN-I production. (B) Gel-based competition readout showing the interaction of 5 competing with excess 5-comp in human PBMCs. (C) Isotopic reductive demethylation heat map showing 5-enriched proteins (20 mM) competed by excess 5-comp or 6-comp (80 mM) in human PBMCs. The inset shows the identification of the top 15 competing targets. (D) Example MS1 of SLC15A4 tryptic peptides from a competition experiment. [Figure 4A] Figure 1 shows that the SLC15A4 chemical probe suppresses proinflammatory cytokines (IFN-I and IL-6) in primary mouse and human pDCs. Suppression of IFN-I production in isolated human (A, B) and mouse (C) pDCs. (D) Suppression of IL-6 in primary mouse pDCs. Mean ± SD (n=3). [Figure 4B] Figure 1 shows that the SLC15A4 chemical probe suppresses proinflammatory cytokines (IFN-I and IL-6) in primary mouse and human pDCs. Suppression of IFN-I production in isolated human (A, B) and mouse (C) pDCs. (D) Suppression of IL-6 in primary mouse pDCs. Mean ± SD (n=3). [Figure 4C] Figure 1 shows that the SLC15A4 chemical probe suppresses proinflammatory cytokines (IFN-I and IL-6) in primary mouse and human pDCs. Suppression of IFN-I production in isolated human (A, B) and mouse (C) pDCs. (D) Suppression of IL-6 in primary mouse pDCs. Mean ± SD (n=3). [Figure 4D]Figure 1 shows that the SLC15A4 chemical probe suppresses proinflammatory cytokines (IFN-I and IL-6) in primary mouse and human pDCs. Suppression of IFN-I production in isolated human (A, B) and mouse (C) pDCs. (D) Suppression of IL-6 in primary mouse pDCs. Mean ± SD (n=3). [Figure 5A] Development of the SLC15A4 transport reporter assay. (A) Fluorescence micrographs of stable A549 cells transfected with SLC15A4-mCherry WT (top) and mutants (L14A, L15A, L318A, and V319A, bottom). SLC15A4 mutant expression is localized to the plasma membrane. (B) Schematic of the SLC15A4 NFkB transporter assay performed in a 96-well format. (C) SLC15A4 mutants, but not WT, produce luciferase signals upon treatment with MDP or Tri-DAP. Luciferase expression is suppressed in the presence of triptolide (an NFkB inhibitor) and 5, but not 6. Mean ± SD (n=3). [Figure 5B] Development of the SLC15A4 transport reporter assay. (A) Fluorescence micrographs of stable A549 cells transfected with SLC15A4-mCherry WT (top) and mutants (L14A, L15A, L318A, and V319A, bottom). SLC15A4 mutant expression is localized to the plasma membrane. (B) Schematic of the SLC15A4 NFkB transporter assay performed in a 96-well format. (C) SLC15A4 mutants, but not WT, produce luciferase signals upon treatment with MDP or Tri-DAP. Luciferase expression is suppressed in the presence of triptolide (an NFkB inhibitor) and 5, but not 6. Mean ± SD (n=3). [Figure 5C]Development of the SLC15A4 transport reporter assay. (A) Fluorescence micrographs of stable A549 cells transfected with SLC15A4-mCherry WT (top) and mutants (L14A, L15A, L318A, and V319A, bottom). SLC15A4 mutant expression is localized to the plasma membrane. (B) Schematic of the SLC15A4 NFkB transporter assay performed in a 96-well format. (C) SLC15A4 mutants, but not WT, produce luciferase signals upon treatment with MDP or Tri-DAP. Luciferase expression is suppressed in the presence of triptolide (an NFkB inhibitor) and 5, but not 6. Mean ± SD (n=3). [Figure 6A] Figure 1 shows SAR studies and functional evaluation of SLC15A4 inhibitors. (A) Structure of 5-comp and two general synthetic routes for SAR studies. (B) Structures of 5-comp analogs. (C) Representative cytotoxicity profiles of isolated human pDCs treated with 10 mM of each compound after 24 hours. (C) Representative cytotoxicity profiles of isolated human pDCs treated with 10 mM of each compound after 24 hours. Read by Cell Titer Glo. (D) Correlation plot of IFN-I suppression (x-axis) versus transport inhibition (y-axis) for 10 mM of each compound. (E) Dose-dependent suppression in primary human pDCs with lead analog 8. Mean ± SD (n=3). [Figure 6B] Figure 1 shows SAR studies and functional evaluation of SLC15A4 inhibitors. (A) Structure of 5-comp and two general synthetic routes for SAR studies. (B) Structures of 5-comp analogs. (C) Representative cytotoxicity profiles of isolated human pDCs treated with 10 mM of each compound after 24 hours. (C) Representative cytotoxicity profiles of isolated human pDCs treated with 10 mM of each compound after 24 hours. Read by Cell Titer Glo. (D) Correlation plot of IFN-I suppression (x-axis) versus transport inhibition (y-axis) for 10 mM of each compound. (E) Dose-dependent suppression in primary human pDCs with lead analog 8. Mean ± SD (n=3). [Figure 6C]Figure 1 shows SAR studies and functional evaluation of SLC15A4 inhibitors. (A) Structure of 5-comp and two general synthetic routes for SAR studies. (B) Structures of 5-comp analogs. (C) Representative cytotoxicity profiles of isolated human pDCs treated with 10 mM of each compound after 24 hours. (C) Representative cytotoxicity profiles of isolated human pDCs treated with 10 mM of each compound after 24 hours. Read by Cell Titer Glo. (D) Correlation plot of IFN-I suppression (x-axis) versus transport inhibition (y-axis) for 10 mM of each compound. (E) Dose-dependent suppression in primary human pDCs with lead analog 8. Mean ± SD (n=3). [Figure 6D] Figure 1 shows SAR studies and functional evaluation of SLC15A4 inhibitors. (A) Structure of 5-comp and two general synthetic routes for SAR studies. (B) Structures of 5-comp analogs. (C) Representative cytotoxicity profiles of isolated human pDCs treated with 10 mM of each compound after 24 hours. (C) Representative cytotoxicity profiles of isolated human pDCs treated with 10 mM of each compound after 24 hours. Read by Cell Titer Glo. (D) Correlation plot of IFN-I suppression (x-axis) versus transport inhibition (y-axis) for 10 mM of each compound. (E) Dose-dependent suppression in primary human pDCs with lead analog 8. Mean ± SD (n=3). [Figure 6E] Figure 1 shows SAR studies and functional evaluation of SLC15A4 inhibitors. (A) Structure of 5-comp and two general synthetic routes for SAR studies. (B) Structures of 5-comp analogs. (C) Representative cytotoxicity profiles of isolated human pDCs treated with 10 mM of each compound after 24 hours. (C) Representative cytotoxicity profiles of isolated human pDCs treated with 10 mM of each compound after 24 hours. Read by Cell Titer Glo. (D) Correlation plot of IFN-I suppression (x-axis) versus transport inhibition (y-axis) for 10 mM of each compound. (E) Dose-dependent suppression in primary human pDCs with lead analog 8. Mean ± SD (n=3). [Figure 7-1] 1 is a table summarizing IFNα suppression and transport inhibition in human pDCs. [Figure 7-2]1 is a table summarizing IFNα suppression and transport inhibition in human pDCs. [Figure 8-1] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-2] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-3] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-4] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-5] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-6] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-7] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-8] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-9] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-10] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-11] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-12] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-13] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-14] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-15] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-16] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-17] IC of SLC15A4 inhibitors50 This is a table. [Figure 8-18] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-19] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-20] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-21] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-22] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-23] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-24] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-25] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-26] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-27] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-28] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-29] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-30] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-31] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-32] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-33] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-34] IC of SLC15A4 inhibitors 50This is a table. [Figure 8-35] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-36] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-37] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-38] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-39] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-40] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-41] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-42] IC of SLC15A4 inhibitors 50 This is a table. [Figure 8-43] IC of SLC15A4 inhibitors 50 This is a table. [Figure 9-1] 1 is a series of graphs showing that SLC15A4 inhibitors block MDP transport in a dose-dependent manner, where AJ2-3A and AJ2-30 are active controls and AJ2-18 and AJ2-22 are inactive controls. [Figure 9-2] 1 is a series of graphs showing that SLC15A4 inhibitors block MDP transport in a dose-dependent manner, where AJ2-3A and AJ2-30 are active controls and AJ2-18 and AJ2-22 are inactive controls. [Figure 10] FIG. 1 shows that compounds that bind to SLC15A4 block endogenous NOD signaling in THP cells. [Figure 11-1] FIG. 1 shows that compounds that bind to SLC15A4 block endogenous NOD signaling in human and mouse macrophages. AJ2-30 is an active control, and AJ2-18 is an inactive control. [Figure 11-2]FIG. 1 shows that compounds that bind to SLC15A4 block endogenous NOD signaling in human and mouse macrophages. AJ2-30 is an active control, and AJ2-18 is an inactive control. [Figure 12-1] FIG. 1 shows that SLC15A4 inhibitors suppress TLR9-mediated B cell activation. [Figure 12-2] FIG. 1 shows that SLC15A4 inhibitors suppress TLR9-mediated B cell activation. [Figure 13] FIG. 1 shows that SLC15A4 inhibitors are inactive in immune cells from SLC15A4 feeble mice; AJ2-3A and AJ2-30 are active controls, and AJ2-18 and AJ2-22 are inactive controls. [Figure 14-1] Figure 1 shows the in vivo efficacy of SLC15A4 inhibitors in a simple inflammation model. Mice were co-injected with compound (or vehicle) and CpG (TLR9), and serum was collected and cytokines were measured 6 hours later (single dose). AJ2-3 and AJ2-30 are active controls, and AJ2-22 is an inactive control. [Figure 14-2] Figure 1 shows the in vivo efficacy of SLC15A4 inhibitors in a simple inflammation model. Mice were co-injected with compound (or vehicle) and CpG (TLR9), and serum was collected and cytokines were measured 6 hours later (single dose). AJ2-3 and AJ2-30 are active controls, and AJ2-22 is an inactive control. [Figure 14-3] Figure 1 shows the in vivo efficacy of SLC15A4 inhibitors in a simple inflammation model. Mice were co-injected with compound (or vehicle) and CpG (TLR9), and serum was collected and cytokines were measured 6 hours later (single dose). AJ2-3 and AJ2-30 are active controls, and AJ2-22 is an inactive control. [Figure 15-1] 1 shows the structures of SLC15A4 inhibitors AJ2-1 to AJ2-92 and AJ2-CP53. [Figure 15-2] 1 shows the structures of SLC15A4 inhibitors AJ2-1 to AJ2-92 and AJ2-CP53. [Figure 15-3]1 shows the structures of SLC15A4 inhibitors AJ2-1 to AJ2-92 and AJ2-CP53. [Figure 15-4] 1 shows the structures of SLC15A4 inhibitors AJ2-1 to AJ2-92 and AJ2-CP53. [Figure 15-5] 1 shows the structures of SLC15A4 inhibitors AJ2-1 to AJ2-92 and AJ2-CP53. [Figure 15-6] 1 shows the structures of SLC15A4 inhibitors AJ2-1 to AJ2-92 and AJ2-CP53. [Figure 15-7] 1 shows the structures of SLC15A4 inhibitors AJ2-1 to AJ2-92 and AJ2-CP53. [Figure 15-8] 1 shows the structures of SLC15A4 inhibitors AJ2-1 to AJ2-92 and AJ2-CP53. [Figure 15-9] 1 shows the structures of SLC15A4 inhibitors AJ2-1 to AJ2-92 and AJ2-CP53. DETAILED DESCRIPTION OF THE INVENTION

[0015] In various embodiments, the disclosure relates to compounds that inhibit SLC15A4, hi various embodiments, the compounds are selective for SLC15A4.

[0016] The compounds are useful in the treatment of conditions mediated by pDCs, B cells, macrophages or monocytes.

[0017] definition For convenience, before further description of the present disclosure, certain specific terms employed in the specification, examples, and appended claims are collected here. These definitions should be understood as understood by one of ordinary skill in the art when read in light of the remainder of the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0018] In order that this disclosure may be more readily understood, certain terms and phrases are defined below and throughout the specification.

[0019] 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.

[0020] The phrase "and / or," as used in the specification and claims of this application, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are sometimes present in conjunction and other times present without conjunction. Multiple elements listed with "and / or" should be construed as the same type, i.e., "one or more" of the elements so conjoined. Other elements may be present other than the elements specifically identified by the "and / or" clause, whether related or not to the elements specifically identified. Thus, as a non-limiting example, "A and / or B," when used in conjunction with open-ended language such as "comprising," in one embodiment may refer to A only (optionally including elements other than B); in another embodiment, may refer to B only (optionally including elements other than A); in yet another embodiment, may refer to both A and B (optionally including other elements), etc.

[0021] As used herein and in the claims of this application, "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 inclusive, i.e., the inclusion of at least one of, and a plurality of, several elements or a list of elements, and optionally, additional unlisted items. Only terms expressly specified to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," shall refer to the inclusion of exactly one element of several elements or a list of elements. In general, the term "or," as used herein, shall be interpreted to indicate exclusive alternatives (i.e., "either / or, but not both") only when preceded by terms of exclusivity, e.g., "any of," "one of," "only one of," "exactly one of," "consisting essentially of," and, when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0022] As used herein in the specification and claims, the phrase "at least one" in connection with a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, nor excluding any combination of elements in the list of elements. This definition also allows for elements other than the specifically identified elements in the list of elements to which the phrase "at least one" refers, whether related to those specifically identified elements or not. 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, in one embodiment, refer to at least one A, optionally including a plurality, and no B (and optionally including elements other than B); in another embodiment, refer to B, optionally including a plurality, and no A (and optionally including elements other than A); in yet another embodiment, refer to at least one A, optionally including a plurality, and at least one B, optionally including a plurality (and optionally including other elements), etc.

[0023] It is to be understood that, unless expressly specified to the contrary, in any method claimed herein that includes multiple steps or actions, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are recited.

[0024] In the claims, as well as in the above specification, all transitional phrases, such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are to be understood to be open-ended, i.e., to mean inclusive, but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" are closed or semi-closed transitional phrases, respectively, as set forth in Section 2111.03 of the United States Patent Office Manual of Patent Examining Procedures.

[0025] The various compounds contained in the compositions of the present disclosure may exist in particular 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.

[0026] For 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 cleaved to yield the pure desired enantiomer. Alternatively, if the molecule contains a basic functional group such as amino, or an acidic functional group such as carboxyl, diastereomeric salts may be formed with an appropriate optically active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, followed by recovery of the pure enantiomers.

[0027] Structures depicted herein are also intended to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, the replacement of a hydrogen by deuterium or tritium, or the replacement of a carbon by 13 C or 14 Compounds produced by substituting C-enriched carbon are within the scope of this disclosure.

[0028] The term "prodrug," as used herein, encompasses compounds that are converted under physiological conditions into therapeutically active agents. A common method for making a prodrug is to include certain moieties that are hydrolyzed under physiological conditions to yield the desired molecule. In other embodiments, the prodrug is converted by enzymatic activity in the host animal.

[0029] As used herein, the phrase "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" means a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating substance, that is involved in the delivery or transport of a chemical substance 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 injurious to the patient, and substantially non-pyrogenic. Examples of substances that can function as pharmaceutically 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 wax; and (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and dairy. (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) buffers, 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. In various embodiments, the pharmaceutical compositions of the present disclosure are non-pyrogenic, i.e., they induce little or no increase in body temperature when administered to a patient.

[0030] The term "pharmaceutically acceptable salts" refers to relatively non-toxic, inorganic and organic acid addition salts of compounds. These salts can be prepared in situ during the final isolation and purification of the compounds, 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, e.g., Berge et al. (1977) "Pharmaceutical Salts," J. Pharm. Sci. 66:1-19).

[0031] In other cases, compounds useful in the methods of the present disclosure may contain one or more acidic functional groups, thereby capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. In these instances, the term "pharmaceutically acceptable salts" refers to the relatively non-toxic, inorganic and organic base addition salts of the compounds. These salts can also be prepared in situ during the final isolation and purification of the compounds, or can be prepared by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, ammonia, or a pharmaceutically acceptable organic primary, secondary, or tertiary amine. Representative alkali or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts. Representative organic amines useful for forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like (see, e.g., Berge et al., supra).

[0032] With respect to use in treatment, a "therapeutically effective amount" (or "effective amount") of a compound refers to the amount of compound in a preparation that, when administered (to a mammal, e.g., a human) as part of a desired regimen, alleviates symptoms, reverses symptoms, or delays the onset of disease symptoms according to clinically accepted criteria for the disorder or condition being treated, or for cosmetic purposes, e.g., at a reasonable benefit / risk ratio applicable to any medical treatment.

[0033] The term "prophylactic or therapeutic" treatment is art-recognized and includes administration to a host of one or more of the subject compositions. If administered prior to the clinical manifestation of an undesired condition (e.g., a disease or other undesired condition in the host animal), the treatment is prophylactic (i.e., it protects the host from developing the undesired condition), whereas if administered after the manifestation of the undesired condition, the treatment is therapeutic (i.e., it is intended to reduce, ameliorate, or stabilize an existing undesired condition or its side effects).

[0034] The term "patient" or "subject" refers to a mammal in need of a particular treatment. In various embodiments, the patient or subject is a primate, dog, cat, or horse. In various embodiments, the patient or subject is human.

[0035] Aliphatic chains include alkyl, alkenyl, and alkynyl groups as defined below. A linear aliphatic chain is limited to an unbranched carbon chain portion. As used herein, the term "aliphatic group" refers to a linear, branched, or cyclic aliphatic hydrocarbon group, including saturated and unsaturated aliphatic groups, such as alkyl, alkenyl, or alkynyl groups.

[0036] "Alkyl," unless otherwise specified, 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. 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, and tetracosyl. In various embodiments, straight or branched chain alkyls have 30 or fewer carbon atoms (e.g., C1-C5 for straight chain). 30 , C3 to C for branched chains 30 ), or 20 or fewer carbon atoms in its backbone. An alkyl group can be substituted or unsubstituted.

[0037] As used herein, the term "alkylene" refers to an alkyl group having a specified number of carbons, e.g., 2 to 12 carbon atoms, including two points of attachment to the remainder of the compound on its longest carbon chain. Non-limiting examples of alkylene groups include methylene-(CH)-, ethylene-(CHCH)-, n-propylene-(CHCHCH)-, and isopropylene-(CHCH(CH))-. Alkylene groups can be cyclic or acyclic, branched or unbranched carbon chain moieties, and may be substituted with one or more substituents.

[0038] "Cycloalkyl" means monocyclic, bicyclic, bridged, spirocyclic, or polycyclic saturated carbocyclic rings, each having from 3 to 12 carbon atoms. Likewise, some cycloalkyls have from 3 to 10 carbon atoms in their ring structure, and some have from 3 to 6 carbons in the ring structure. Cycloalkyl groups can be substituted or unsubstituted.

[0039] Unless the number of carbons is otherwise specified, "lower alkyl," as used herein, refers to an alkyl group as defined above but having 1 to 10 carbons, 1 to 6 carbon atoms, in its backbone structure, e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. Similarly, "lower alkenyl" and "lower alkynyl" have similar chain lengths. Throughout this application, alkyl groups can be lower alkyls. In various embodiments, a substituent designated herein as alkyl is a lower alkyl.

[0040] "Alkenyl" refers to any cyclic or acyclic, branched or unbranched unsaturated carbon chain moiety having a specified number of carbon atoms, or up to 26 carbon atoms if no limit on the number of carbon atoms is specified, and having one or more double bonds within the moiety. Examples of alkenyls of 6 to 26 carbon atoms include hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, eicosenyl, heneicosoenyl, docosenyl, tricosenyl, and tetracosenyl in various isomeric forms, where the unsaturated bond can be located anywhere within the moiety and can have either the (Z) or (E) configuration around the double bond.

[0041] "Alkynyl" refers to a hydrocarbyl moiety within the scope of alkenyl, but containing one or more triple bonds within the moiety.

[0042] The term "alkylthio" refers to an alkyl group, as defined above, having a sulfur moiety attached thereto. In various embodiments, the "alkylthio" moiety includes -(S)-alkyl, -(S)-alkenyl, -(S)-alkynyl, and -(S)-(CH) m -R 1 and m and R 1is defined below. Representative alkylthio groups include methylthio and ethylthio. 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, and tert-butoxy. An "ether" is two hydrocarbons covalently linked by an oxygen. Thus, an alkyl substituent that makes an alkyl an ether is an alkoxyl or an alkoxyl-like group, such as -O-alkyl, -O-alkenyl, -O-alkynyl, -O-(CH2) m -R 10 can be expressed by one of m and R 10 are described below.

[0043] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, for example, a moiety that can be represented by the formula: [C3] In the formula, R 11 , R 12 and R 13 are each independently hydrogen, alkyl, alkenyl, -(CH2) m -R 10 or R 11 and R 12 together with the N atom to which they are attached form a heterocycle having 4 to 8 atoms in the ring structure, and R 10 represents alkenyl, aryl, cycloalkyl, cycloalkenyl, heterocycle, or polycyclyl, and m is zero or an integer ranging from 1 to 8. In various embodiments, R 11 or R 12 Only one of the groups can be a carbonyl, e.g., R 11 , R 12 and the nitrogen together do not form an imide. In various further embodiments, R 11 and R 12 (and optionally R 13) are each independently hydrogen, alkyl, alkenyl, or -(CH) m -R 10 Thus, the term "alkylamine" as used herein refers to an amine having a substituted or unsubstituted alkyl attached thereto, i.e., R 11 and R 12 and n is an alkyl group. In certain embodiments, the amino group or alkylamine is basic, which means that the group has a pK a >7.00, i.e., the protonated form of these functional groups has a pK relative to water above about 7.00. a It means having.

[0044] The term "amide," as used herein, refers to the group: [C4] In the formula, each R 14 independently represent hydrogen or a hydrocarbyl group, or two R 14 together with the N atom to which they are attached form a heterocyclic ring with 4 to 8 atoms in the ring structure.

[0045] As used herein, the term "aryl" includes 3- to 12-membered substituted or unsubstituted monocyclic aryl groups in which each atom of the ring is carbon (i.e., carbocyclic aryl) or one or more atoms are heteroatoms (i.e., heteroaryl). Aryl groups include 5- to 12-membered rings and 6- to 10-membered rings. 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, at least one of which is aryl, and the other cyclic rings may be, for example, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclic. Carbocyclic aryl groups include benzene, naphthalene, phenanthrene, phenol, and aniline. Heteroaryl groups include substituted or unsubstituted aromatic 3- to 12-membered ring structures, 5- to 12-membered rings, and 5- to 10-membered rings, whose ring structures contain 1 to 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 aryl group can be independently substituted, i.e., unsubstituted ("unsubstituted aryl") or substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 4 substituents, 1 to 3 substituents, 1 to 2 substituents or exactly 1 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, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocycle, aryl or heteroaryl moiety, fluoroalkyl (e.g., trifluoromethyl), cyano, etc. For example, in various embodiments, the aryl group may be an unsubstituted C5-C6 12In various embodiments, the aryl group can be a substituted C5-C 10 It can be aryl.

[0046] The terms "halo," "halide," or "halogen," as used herein, mean halogen, including, but not limited to, fluoro, chloro, bromo, iodo, and the like, in both radioactive and non-radioactive forms. In various embodiments, halo is selected from the group consisting of fluoro, chloro, and bromo.

[0047] The terms "heterocyclic" or "heterocyclic group" refer to 3- to 12-membered ring structures, 5- to 12-membered rings, or 5- to 10-membered rings, whose ring structures include one to four heteroatoms. Heterocycles can be monocyclic, bicyclic, spirocyclic, or polycyclic. Heterocyclic 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, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The heterocycle may be optionally substituted at one or more positions with substituents such as those described above, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, sulfamoyl, sulfinyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclic, aryl, or heteroaryl moiety, -CF, -CN, and the like.

[0048] The term "carbonyl" is art-recognized and includes a moiety represented by the formula: [C5] wherein X′ is a bond, or represents oxygen or sulfur; R 15 is hydrogen, alkyl, alkenyl, -(CH2) m -R 10 or a pharmaceutically acceptable salt thereof, R 16 is hydrogen, alkyl, alkenyl or -(CH2) m -R 10 where m and R 10 is as defined above. X' is oxygen and R 15 or R 16 Where X' is oxygen and R 15 is as defined above, the moiety is referred to herein as a carboxyl group, and in particular R 15 Where X' is hydrogen, the formula represents a "carboxylic acid". 16 is hydrogen, the formula represents a "formate." In general, where the oxygen atom of the above formula is replaced by a sulfur, the formula represents a "thiocarbonyl" group. 15 or R 16 Where X' is not hydrogen, the formula represents a "thioester" group. 15 Where X' is a sulfur and R 16 is hydrogen, the formula represents a "thioformate" group. On the other hand, if X' is a bond, and R 15 Where X' is a bond and R is not hydrogen, the above formula represents a "ketone" group. 15 Where is hydrogen, the above formula represents an "aldehyde" group.

[0049] As used herein, the term "nitro" means -NO2, the term "halogen" means -F, -Cl, -Br, or -I, the term "sulfhydryl" means -SH, the term "hydroxyl" means -OH, the term "sulfonyl" means -SO2-, the 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.

[0050] As used herein, the definition of each expression, e.g., alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.

[0051] The term "substituted" refers to moieties having substituents replacing a hydrogen on one or more carbon atoms of the backbone. It should be clear that "substituted" or "substituted with" includes the implicit proviso that such substitution is consistent with the allowed valencies 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, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aryl and non-aryl substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For the 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 valencies of the heteroatoms. Substituents can include any substituent described herein, for example, halogen, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocycle, araalkyl, or aryl or heteroaryl moiety. In various embodiments, substituents on a substituted alkyl are C 1-6 Alkyl, C 3-6In various embodiments, the substituents on substituted alkyl are selected from fluoro, carbonyl, cyano, or hydroxyl. It will be apparent to those skilled in the art that the substituents themselves can be substituted, where appropriate. Reference to a chemical moiety herein is understood to include substituted versions, unless specifically stated as "unsubstituted." For example, reference to an "aryl" group or moiety implicitly includes both substituted and unsubstituted versions.

[0052] For purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, inside cover of the Handbook of Chemistry and Physics, 67th Ed., 1986-87.

[0053] Exemplary Compounds of the Present Disclosure In various embodiments, the present disclosure relates to a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof: [C6] During the ceremony, X is NR 4 or S; R 1 is H or -C(O)-C 1-10 is alkyl; R 2 is a heterocycle or aryl; R 3 is a halogen, -CHF2, or -CF3; R 4 -H, -C 1-10 Alkyl, -C(O)-C 1-10 Alkyl, -C(O)-C 3-10 Cycloalkyl, -S(O) 2- C 1-10 alkyl, or [C7] and; n is 0, 1, 2, 3, or 4.

[0054] In some embodiments, the compound is a compound of formula (I): In some embodiments, the compound is a compound of formula (II):

[0055] In some embodiments, R 1 is H. In some embodiments, R 1 is -C(O)-C 1-10 In some embodiments, R 1 is —C(O)—C1H3. In some embodiments, R 1 is —C(O)—C2H5. In some embodiments, R 1 is —C(O)—C3H7. In some embodiments, R 1 is —C(O)—C4H9.

[0056] In some embodiments, R 2 is an unsubstituted heterocycle. In some embodiments, R 2 is a substituted heterocycle. In some embodiments, the heterocycle is monocyclic. In some embodiments, the heterocycle is bicyclic. In some embodiments, the heterocycle is tricyclic. In some embodiments, the heterocycle is aryl. In some embodiments, the heterocycle is non-aryl. In some embodiments, R 2 is unsubstituted aryl. In some embodiments, R 2 is a substituted aryl.

[0057] In some embodiments, R 2 is substituted with at least one substituent selected from halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, sulfamoyl, sulfinyl, alkylthio, sulfonyl, ketone, heterocyclic, aryl or heteroaryl moiety, -CHF2-CF3, -CN. 2 When is substituted with two or more substituents, the substituents may be the same or different.

[0058] In some embodiments, R 2 teeth, [C8] is selected from the group consisting of:

[0059] In some embodiments, R 2 teeth, [C9] is.

[0060] In some embodiments, R 2 teeth [C10] is.

[0061] In some embodiments, R 2 teeth, [C11] is.

[0062] In some embodiments, R 2 teeth, [C12] is.

[0063] In some embodiments, R 2 teeth, [C13] is selected from the group consisting of:

[0064] In some embodiments, R 3 is F. In some embodiments, R 3 is Cl. In some embodiments, R 3 is Br. In some embodiments, R 3 is -CHF2. In some embodiments, R 3 is -CF3.

[0065] In some embodiments, R 4 is H. In some embodiments, R4 Ha-C 1-10 In some embodiments, R 4 is methyl, ethyl, i-propyl, n-propyl, t-butyl, i-butyl, or n-butyl. 4 is methyl.

[0066] In some embodiments, R 4 is -C(O)-C 1-10 In some embodiments, R 4 is —C(O)-methyl, —C(O)-ethyl, —C(O)-i-propyl, —C(O)-n-propyl, —C(O)-t-butyl, —C(O)-i-butyl, or —C(O)-n-butyl. 4 is -C(O)-C 5-10 It is alkyl.

[0067] In some embodiments, R 4 teeth, [C14] is.

[0068] In some embodiments, R 4 is -C(O)-C3H7.

[0069] In some embodiments, R 4 is -C(O)-C 3-10 In some embodiments, R 4 is —C(O)-cyclopropyl. In some embodiments, R 4 is -C(O)-cyclohexyl.

[0070] In some embodiments, R 4 is -S(O)2-C 1-10 In some embodiments, R 4is —S(O)-methyl, —S(O)-ethyl, —S(O)-i-propyl, —S(O)-n-propyl, —S(O)-t-butyl, —S(O)-i-butyl, or —S(O)-n-butyl. 4 is -S(O)2-C 5-10 It is alkyl.

[0071] In some embodiments, R 4 is -S(O)2-C3H7.

[0072] In some embodiments, R 4 teeth [C15] is.

[0073] In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4.

[0074] In some embodiments, the compound of formula (I) is [C16] is selected from the group consisting of:

[0075] In some embodiments, the compound of formula (I) [C17] is.

[0076] In some embodiments, the compound of formula (I) is [C18] is selected from the group consisting of:

[0077] In some embodiments, the compound of formula (I) is [C19] is.

[0078] In some embodiments, the compound of formula (II) is [C20] is selected from the group consisting of:

[0079] In some embodiments, the compound of formula (I) is [C21] is selected from.

[0080] In some embodiments, the compound of formula (I) is [C22] is selected from the group consisting of:

[0081] In some embodiments, the compound of formula (I) is [C23] is.

[0082] In some embodiments, the compound of formula (I) is [C24] is.

[0083] In some embodiments, the compound of formula (I) is [C25] is.

[0084] Exemplary Pharmaceutical Compositions In various embodiments, the present disclosure relates to a pharmaceutical composition comprising any one of the compounds disclosed herein and a pharmaceutically acceptable carrier.

[0085] Patients, including but not limited to humans, can be treated by administering to the patient an effective amount of an active compound or a pharmaceutically acceptable prodrug or salt thereof in the presence of a pharmaceutically acceptable carrier or diluent. The active agent can be administered by any suitable route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid or solid form.

[0086] The concentration of active compound in the drug composition will depend on the absorption, inactivation and excretion rate of the drug, as well as other factors known to those skilled in the art.It should be noted that dosage values ​​also vary depending on the severity of the symptoms to be improved.It should be further understood that for a particular subject, specific dosage regimens should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the composition, and the concentration ranges described herein are merely exemplary and do not limit the scope or implementation of the claimed compositions.The active ingredient can be administered at a single time, or can be divided into several small doses administered at varying time intervals.

[0087] In various embodiments, the mode of administration of the active compound is oral. Oral compositions generally include an inert diluent or an edible carrier. These can be enclosed in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition.

[0088] Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients, or compounds of similar nature: binders, such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, Primogel, or corn starch; lubricants, such as magnesium stearate or steroteles; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavorings, such as peppermint, methyl salicylate, or orange flavoring. If the dosage unit is a capsule, it may contain, in addition to the above-mentioned materials, a liquid carrier, such as fatty oil. In addition, dosage unit may contain various other materials that modify the physical form of the dosage unit, such as sugar coating, shellac, or other enteric agents.

[0089] The compounds can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum, etc. A syrup may contain, in addition to the active compounds, sucrose or other sweetening agents as a sweetening agent and various preservatives, dyes and colorings and flavors.

[0090] The compound or its pharmaceutically acceptable prodrug or salt can also be mixed with other active substances that do not interfere with the desired action, or with materials that supplement the desired action, such as antibiotics, antifungals, anti-inflammatory agents, or other antivirals, for example, but not limited to, nucleoside compounds. Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can contain the following components: a sterile diluent, such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate, or phosphate, and an agent for adjusting tonicity, such as sodium chloride or glucose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0091] If administered intravenously, carriers include saline and phosphate buffered saline (PBS).

[0092] In various embodiments, the active compound is formulated with a carrier that protects the compound from rapid elimination from the body, such as sustained-release formulations, including, but not limited to, implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. For example, enteric coating compounds can be used to protect against cleavage by stomach acid. Methods for preparing such formulations are apparent to those skilled in the art. Suitable materials can also be obtained commercially.

[0093] Liposomal suspensions (including, but not limited to, liposomes targeted to infected cells bearing monoclonal antibodies against viral antigens) are also pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811 (incorporated by reference). For example, liposomal formulations can be prepared by dissolving a suitable lipid (e.g., stearoylphosphatidylethanolamine, stearoylphosphatidylcholine, arachadoylphosphatidylcholine, and cholesterol) in an inorganic solvent, followed by evaporation of the solvent, leaving a thin film of dried lipid on the surface of the container. An aqueous solution of the active compound is then introduced into the container. The container is then manually swirled to remove lipid material from the sides of the container and disperse lipid aggregates, thereby forming a liposomal suspension.

[0094] Exemplary Methods of the Present Disclosure In various embodiments, the disclosure relates to a method of treating a disease or condition mediated by pDCs, B cells, macrophages, or monocytes, comprising administering to a subject in need thereof a therapeutically effective amount of any one of the above-described compounds. In some embodiments, the pDC-mediated disease is lupus, Crohn's disease, irritable bowel syndrome (IBS), type I diabetes, dermatomyositis, Sjogren's syndrome, psoriasis, or any type I interferon-driven interferonopathy. In some embodiments, the pDC-mediated disease is multiple sclerosis (MS). [Example]

[0095] The present disclosure summarized herein will be more readily understood by reference to the following examples, which are provided solely for the purpose of illustrating aspects and embodiments of the present disclosure and are not intended to limit the disclosure.

[0096] Example 1. General schemes and procedures for the preparation of compounds of the present disclosure. The synthesis of various compounds is illustrated in Figure 6A.

[0097] Example 2. Representative Preparation of Compounds of the Disclosure (A) Chemical materials Chemicals and reagents were purchased from commercial vendors, including Sigma-Aldrich, Fisher Scientific, Cmbi-Blocks, MedChemExpress, Alfa Aesar, and AstaTerch, and were used as received without further purification unless otherwise noted. Anhydrous solvents were purchased from Sigma-Aldrich in Sure / Seal™ formulations. All reactions were monitored by thin-layer chromatography (TLC, Merck silica gel 60F-254 plates). Plates were stained with p-anisaldehyde (2.5% p-anisaldehyde, 1% AcOH, 3.5% H2SO4 (concentrated) in 95% EtOH), ninhydrin (0.3% ninhydrin (wt / vol), 97:3 EtOH-AcOH), KMnO4 (1.5 g KMnO4, 10 g K2CO3, and 1.25 mL 10% NaOH in 200 mL water), iodine, or directly visualized under UV light. Reaction purification was performed using flash chromatography (230-400 mesh silica gel), Biotage®, or preparative thin-layer chromatography (pTLC, Analtech, 500-2000 μm thickness). NMR spectra were recorded on a Bruker DPX-400 or Bruker AV-600 spectrometer in the indicated solvents. Multiplicities are reported using the following abbreviations: s singlet; d doublet; t triplet; q quartet; p quintet; m multiplet; br broad; dd ​​double doublet; dt triple doublet; td double triplet. Chemical shifts are reported in ppm relative to the residual solvent peak, and J values ​​are reported in Hz. Mass spectrometer data were collected on an Agilent 6120 single quadrupole LC / MS instrument (ESI, low resolution).

[0098] (B) Compound synthesis and characterization data: a) General synthetic scheme 1: [C26]

[0099] General Procedure 1: Coupling Procedure for the Synthesis of Benzo[d]imidazole Amine Intermediate (S1) To a dry round-bottom flask containing a solution of commercially available 2-aminobenzimidazole derivative (1.0 equiv.) and the corresponding aldehyde (1.0 equiv.) in anhydrous methanol was added K2CO3 (3.0 equiv.), and the reaction mixture was heated at 50 °C for 16–30 h. The solvent was filtered to remove excess potassium carbonate, and sodium triacetoxyhydride (1.5 equiv.) was added to the solution at 0 °C. The resulting mixture was stirred at room temperature for 3–5 h. After completion of the reaction (monitored by TLC), the solvent was removed by rotary evaporation, and the crude mixture was diluted with water, saturated aqueous NaHCO3, and extracted with ethyl acetate. The combined extracts were dried over Na2SO4, filtered, concentrated under reduced pressure, and purified by biotage column chromatography to give the corresponding amine (S1).

[0100] General Procedure 2: Coupling of the amine intermediate (S1) with an acid To a vial containing the corresponding amine intermediate (S1, 1 equiv.) in DCM (60 mM relative to S1) was added commercially available butyric acid or 3-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)propanoic acid (1.1 equiv.), DIPEA (3.0 equiv.), EDC-HCl (1.5 equiv.), and HOBt (1.5 equiv.). The reaction mixture was stirred at room temperature for 4 hours to overnight, until TLC indicated completion of the reaction. The crude mixture was diluted with DCM, first with saturated aqueous NH4Cl and saturated aqueous NaHCO3, then dried over anhydrous Na2SO4, and the volatiles were removed by rotary evaporation. The crude product was purified by PTLC or flash chromatography to give the corresponding product.

[0101] General Procedure 3: Coupling of the amine intermediate (S1) with an acid To a solution of the corresponding butyric acid or 3-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)propanoic acid in DMF (60 mM relative to S1), HATU (1.1 equiv.) and DIPEA (3.0 equiv.) were added at 0°C. The resulting mixture was stirred for 5 min, the corresponding amine intermediate (S1) was added, and the resulting mixture was stirred at room temperature until the amine was completely consumed, as indicated by TLC. The crude mixture was diluted with cold water, extracted with ethyl acetate, dried over anhydrous Na2SO4, and the volatiles were removed by rotary evaporation. The crude product was purified by PTLC or flash column chromatography to give the corresponding product.

[0102] General Procedure 4: Coupling Procedure with Acid Chlorides for Amide Synthesis To a solution of the corresponding amine (S1, 1.0 equiv.) in DCM (0.1 M) was added triethylamine (1.1 equiv.), followed by the slow addition of the corresponding acid chloride (1.0 equiv.) at 0°C. The resulting mixture was stirred at room temperature until the amine was completely consumed, as indicated by TLC. The crude mixture was diluted with DCM and washed first with saturated aqueous NH4Cl and saturated aqueous NaHCO3, then dried over anhydrous Na2SO4, and the volatiles were removed by rotary evaporation. The crude product was purified by PTLC or Biotage® to give the corresponding product.

[0103] General Procedure 5: Coupling Procedure for the Synthesis of N-Alkyl-Containing Molecules To a solution (0.1 M) of the corresponding amine (S1, 1.0 equiv.) in DMF, dry K2CO3 (2.0 equiv.) was added, followed by the corresponding alkyl iodide or 3-(but-3-yn-1-yl)-3-(2-iodoethyl)-3H-diazirine (2.0 equiv.) at room temperature. The resulting mixture was stirred at 50 °C until the amine was completely consumed, typically over 18–24 h, as indicated by TLC. The crude mixture was diluted with cold water and extracted with ethyl acetate. The combined extracts were dried over anhydrous Na2SO4, and the volatiles were removed by rotary evaporation. The crude product was purified by PTLC or flash column chromatography to give the corresponding product.

[0104] b) General synthesis scheme 2: [C27]

[0105] 6-Bromo-9-ethylcarbazole-3-carbaldehyde (S2): 9-Ethylcarbazole-3-carbaldehyde (2 g, 8.95 mmol) was dissolved in DMF (15 mL) and the solution was cooled in an ice bath. A solution of N-bromosuccinimide (1.91 g, 10.74 mmol) in DMF (10 mL) was added dropwise over 10 minutes. The reaction mixture was stirred at room temperature for 2 hours. The mixture was then poured into ice water and extracted with ethyl acetate. The combined extracts were dried over anhydrous Na2SO4, and the volatiles were removed by rotary evaporation. The crude product was purified by flash column chromatography to give the corresponding 6-bromo-9-ethylcarbazole-3-carbaldehyde product (S2).

[0106] General Procedure 6: Suzuki Coupling Procedure for the Synthesis of (S3) A solution of 6-bromo-9-ethylcarbazole-3-carbaldehyde (0.822 mmol), boronic acid (0.986 mmol), and potassium carbonate (0.246 mmol) in dimethoxyethane (9 mL) and water (3 mL) was degassed by bubbling argon for 5 min, followed by the addition of tetrakis(triphenylphosphino)palladium (47 mg, 0.041 mmol). The resulting mixture was stirred at 80 °C for 6–8 h. After cooling, the reaction mixture was filtered through Celite, diluted with water, and extracted with ethyl acetate. The combined extracts were dried over anhydrous NaSO, and the volatiles were removed by rotary evaporation. The crude product was purified by flash column chromatography using ethyl acetate / hexane to give the corresponding product (S3).

[0107] c) General synthetic scheme 3: [C28]

[0108] Step 1: Synthesis of intermediate (S4): To a stirred solution of 4-bromophenylhydrazine (1.1 equiv.) in AcOH, substituted cyclohexanone (1.1 equiv.) was added, and the reaction mixture was stirred at 118 °C for 3 h. After cooling, the acetic acid was removed by rotary evaporation, and the reaction mixture was diluted with water and saturated aqueous NaHCO and extracted with ethyl acetate. The combined extracts were dried over anhydrous NaSO, and the volatiles were removed by rotary evaporation. The crude product was purified by flash column chromatography using ethyl acetate / hexane to give the corresponding product (S4).

[0109] Step 2: Synthesis of intermediate (S5): To a stirred solution of (S4) (1 equivalent) in DMF, a suspension of sodium hydride (1.1 equivalents) (60% in mineral oil) was slowly added over 10 minutes at 0°C. The resulting mixture was stirred in a cold ice bath for 15 minutes. A solution of ethyl iodide (1.5 equivalents) was added dropwise over 5 minutes. The resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined extracts were dried over anhydrous Na2SO4, and the volatiles were removed by rotary evaporation. The crude product was purified by flash column chromatography using ethyl acetate / hexane to give the corresponding product (S5).

[0110] Step 3: Synthesis of intermediate (S6): To a stirred solution of (S5) (1 equivalent) in THF was added n-butyllithium (1.1 equivalents) at −78° C. under an argon atmosphere. The resulting mixture was stirred for 20 minutes, and DMF (3 equivalents) was added. The reaction mixture was stirred at room temperature for 2 hours. The mixture was then poured into ice-cold ammonium chloride solution and extracted with ethyl acetate. The combined extracts were dried over anhydrous NaSO, and the volatiles were removed by rotary evaporation. The crude product was purified by flash column chromatography to give the corresponding aldehyde (S6).

[0111] d) General synthesis scheme 4: [C29]

[0112] Step 1: Synthesis of intermediate (S7): n-Butyllithium (1.1 equiv.) was added to a stirred solution of the corresponding Wittig salt (1.0 equiv.) in THF at −78°C under an argon atmosphere. The resulting mixture was stirred for 20 minutes, and a solution of 9-ethyl-3-carbazolecarboxaldehyde (1.0 equiv.) in THF was added. The reaction mixture was stirred at room temperature for 2 hours. The mixture was poured into an ice-cold solution of ammonium chloride and extracted with ethyl acetate. The combined extracts were dried over anhydrous NaSO, and the volatiles were removed by rotary evaporation. The crude product was purified by flash column chromatography to give the corresponding aldehyde (S7).

[0113] Step 2: Synthesis of Intermediate (S8): To a solution of Intermediate (S7) in methanol, 5 wt% Pd / C was added, and the resulting mixture was stirred under a hydrogen atmosphere at room temperature for 8 hours. After completion of the reaction, the reaction mixture was filtered through a short pad of Celite, washed with methanol, dried by rotary evaporation, dissolved in DMF, and a solution of N-bromosuccinimide (1.2 equiv.) in DMF was added dropwise at 0°C over 10 minutes. The reaction mixture was stirred at room temperature for 2 hours. The mixture was poured into ice water and extracted with ethyl acetate. The combined extracts were dried over anhydrous Na2SO4, and the volatiles were removed by rotary evaporation. The crude product was purified by flash column chromatography to give the corresponding bromo product (S8).

[0114] Step 3: Synthesis of intermediate (S9): To a stirred solution of (S8) (1.0 equiv.) in THF at −78° C. under an argon atmosphere, n-butyllithium (1.1 equiv.) was added. The resulting mixture was stirred for 20 minutes, and DMF (3.0 equiv.) was added. The reaction mixture was stirred at room temperature for 2 hours. The mixture was then poured into an ice-cold solution of ammonium chloride and extracted with ethyl acetate. The combined extracts were dried over anhydrous NaSO, and the volatiles were removed by rotary evaporation. The crude product was purified by flash column chromatography to give the corresponding aldehyde (S9).

[0115] [C30]

[0116] 1-(2-(((1H-indol-5-yl)methyl)amino)-1H-benzo[d]imidazol-1-yl)butan-1-one (AJ2-1): Synthesized according to general procedure 4 according to Scheme 1 and purified by biotage (hexane / EtOAc, 6:4) to afford AJ2-1 as an off-white solid (17 mg, 62%). 1 H NMR (400 MHz, CDCl3) δ 8.21 (s, 1H), 8.16 (s, 1H), 7.68 (dt, J = 1.6, 0.8 Hz, 1H), 7.47 (dd, J = 7.8, 1.2 Hz, 1H), 7.38 (ddd, J = 8.4, 2.5, 1.7 Hz, 2H), 7.26 - 7.20 (m, 4H), 7.06 (ddd, J = 8.6, 7.5, 1.3 Hz, 1H), 6.53 - 6.55 (m, 1H), 4.85 (d, J = 5.2 Hz, 2H), 2.99 (t, J = 7.2 Hz, 2H), 1.84 (p, J = 7.3 Hz, 2H), 1.08 (t, J = 7.4 Hz, 3H).LCMS C 20 H 21 Calculated for NO: 333.2 (M+H + ), Actual value: 333.2.

[0117] [C31] N-((1H-indol-5-yl)methyl)-N-(benzo[d]thiazol-2-yl)butyramide (AJ2-2): Synthesized according to general procedure 2 according to Scheme 1 and purified by PTLC (hexane / EtOAc, 4:2) to afford AJ2-2 as a brown solid (8 mg, 62%). 1H NMR (400 MHz, CDCl3) δ 8.17 (s, 1H), 7.84 (dt, J = 7.7, 1.1 Hz, 1H), 7.79 (dt, J = 8.2, 0.9 Hz, 1H), 7.48 - 7.45 (m, 1H), 7.40 (ddd, J = 8.3, 7.2, 1.3 Hz, 1H), 7.36 - 7.27 (m, 2H), 7.20 (dd, J = 3.2, 2.4 Hz, 1H), 7.09 (dd, J = 8.5, 1.8 Hz, 1H), 6.48 (ddd, J = 3.1, 2.0, 1.0 Hz, 1H), 5.74 (s, 2H), 2.62 (t, J = 7.3 Hz, 2H), 1.72 (q, J = 7.4 Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H). 20 H 20 Calculated value of N3OS: 350.1 (M+H + ), Actual value: 350.0.

[0118] [C32] N-((5-Bromo-1H-indol-3-yl)methyl)-1H-benzo[d]imidazol-2-amine (AJ2-3A): Synthesized according to Scheme 1 and purified by biotage (hexane / EtOAc, 3:7) to afford AJ2-3A as a light brown solid (160 mg, 64%). 1 H NMR (400 MHz, CD3OD) δ 7.74 (s, 1H), 7.28 (s, 1H), 7.23 (dd, J = 8.6, 0.6 Hz, 1H), 7.20 - 7.12 (m, 3H), 6.94 (dd, J = 5.8, 3.2 Hz, 2H), 4.64 (d, J = 0.8 Hz, 2H).LCMS C 16 H 14 Calculated for BrN4: 341.0 (M+H + ), Actual value: 340.9.

[0119] [C33] 1-(2-(((5-Bromo-1H-indol-3-yl)methyl)amino)-1H-benzo[d]imidazol-1-yl)butan-1-one (AJ2-3): Synthesized according to Scheme 1 and general procedure 4 and purified by biotage (hexane / EtOAc, 3:2) to afford AJ2-3 as a brown solid (32 mg, 64%). 1 H NMR (400 MHz, CDCl3) δ 8.19 (s, 1H), 8.03 (s, 1H), 7.84 (dd, J = 1.7, 0.9 Hz, 1H), 7.49 (dd, J = 7.9, 1.2 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.32 - 7.27 (m, 3H), 7.25 (dd, J = 1.8, 0.9 Hz, 1H), 7.08 (ddd, J = 8.1, 7.5, 1.3 Hz, 1H), 4.88 (d, J = 4.3 Hz, 2H), 2.99 (t, J = 7.2 Hz, 2H), 1.84 (h, J = 7.4 Hz, 2H), 1.08 (t, J = 7.4 Hz, 3H).LCMS C 20 H 20 Calculated for BrNO: 411.1 (M+H + ), Actual value: 411.1.

[0120] [C34] 1-(2-((isoquinolin-5-ylmethyl)amino)-1H-benzo[d]imidazol-1-yl)butan-1-one (AJ2-4): Synthesized according to Scheme 1 and general procedure 4 and purified by biotage (hexane / EtOAc, 3:2) to afford AJ2-4 as a light brown solid (7 mg, 54%). 1H NMR (400 MHz, CD3OD) δ 9.17 (s, 1H), 8.40 (d, J = 6.1 Hz, 1H), 7.96 (dd, J = 7.2, 4.6 Hz, 2H), 7.76 (dd, J = 7.2, 1.2 Hz, 1H), 7.62 - 7.54 (m, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.24 - 7.20 (m, 1H), 7.12 (td, J = 7.7, 1.0 Hz, 1H), 7.07 - 6.99 (m, 1H), 5.09 (s, 2H), 3.00 (t, J = 7.1 Hz, 2H), 1.73 (q, J = 7.3 Hz, 2H), 0.98 (t, J = 7.4 Hz, 3H). LCMS C 21 H 21 Calculated for NO: 345.2 (M+H + ), Actual value: 345.2.

[0121] [C35] 1-(2-(((1H-Pyrrolo[2,3-b]pyridin-3-yl)methyl)amino)-1H-benzo[d]imidazol-1-yl)butan-1-one (AJ2-5): Synthesized according to Scheme 1 and general procedure 4 and purified by biotage (hexane / EtOAc, 4:2) to afford AJ2-5 as a brown solid (12 mg, 62%). 1H NMR (400 MHz, CDCl3) δ 10.42 (s, 1H), 8.33 (dd, J = 4.8, 1.5 Hz, 1H), 8.10 (t, J = 5.2 Hz, 1H), 8.05 (dd, J = 7.9, 1.5 Hz, 1H), 7.50 (dd, J = 8.0, 1.3 Hz, 1H), 7.44 - 7.38 (m, 2H), 7.30 - 7.24 (m, 2H), 7.13 - 7.05 (m, 2H), 4.93 (dd, J = 5.1, 0.8 Hz, 2H), 2.98 (t, J = 7.2 Hz, 2H), 1.83 (q, J = 7.3 Hz, 2H), 1.07 (t, J = 7.4 Hz, 3H).LCMS C 19 H 20 Calculated for NO: 334.1 (M+H + ), Actual value: 334.1.

[0122] [C36] 1-(2-((pyrazolo[1,5-a]pyridin-5-ylmethyl)amino)-1H-benzo[d]imidazol-1-yl)butan-1-one (AJ2-6): Synthesized according to Scheme 1 and general procedure 4 and purified by biotage (hexane / EtOAc, 4:2) to afford AJ2-6 as a brown solid (6 mg, 52%). 1H NMR (400 MHz, CDCl3) δ 8.45 (d, J = 7.2 Hz, 1H), 8.34 (s, 1H), 7.95 (d, J = 2.3 Hz, 1H), 7.55 (s, 1H), 7.45 (t, J = 8.3 Hz, 2H), 7.29 (d, J = 0.9 Hz, 2H), 7.16 - 7.06 (m, 1H), 6.82 (dd, J = 7.2, 2.0 Hz, 1H), 6.49 (d, J = 2.3 Hz, 1H), 4.83 (d, J = 5.9 Hz, 2H), 3.06 (t, J = 7.2 Hz, 2H), 1.92 (q, J = 7.3 Hz, 2H), 1.14 (t, J = 7.4 Hz, 3H).LCMS C 19 H 20 Calculated for NO: 334.16 (M+H + ), Actual value: 334.16.

[0123] [C37] 1-(2-((3,4-Dimethoxybenzyl)amino)-1H-benzo[d]imidazol-1-yl)butan-1-one (AJ2-7): Synthesized according to Scheme 1 and general procedure 4 and purified by biotage (hexane / EtOAc, 3:2) to afford AJ2-7 as a brown solid (12 mg, 72%). 1H NMR (400 MHz, CDCl3) δ 8.14 (t, J = 5.5 Hz, 1H), 7.45 (ddd, J = 7.9, 1.3, 0.5 Hz, 1H), 7.38 (dt, J = 8.2, 0.8 Hz, 1H), 7.24 (dd, J = 7.7, 1.0 Hz, 1H), 7.06 (ddd, J = 8.2, 7.5, 1.3 Hz, 1H), 6.98 - 6.93 (m, 2H), 6.87 - 6.81 (m, 1H), 4.70 (d, J = 5.4 Hz, 2H), 3.88 (s, 3H), 3.87 (s, 3H), 2.99 (t, J = 7.2 Hz, 2H), 1.86 (q, J = 7.3 Hz, 2H), 1.09 (t, J = 7.4 Hz, 3H). 20 H 24 N3O3; 354.2 (M+H + ) Calculated value: Measured value: 354.2.

[0124] [C38] 1-(2-(((1-Benzyl-1H-indol-5-yl)methyl)amino)-1H-benzo[d]imidazol-1-yl)butan-1-one (AJ2-8): Synthesized according to Scheme 1 and general procedure 4 and purified by biotage (hexane / EtOAc, 3:2) to afford AJ2-8 as an off-white solid (14 mg, 74%). 1H NMR (400 MHz, CDCl3) δ 8.13 (s, 1H), 7.71 - 7.65 (m, 1H), 7.46 (dd, J = 7.9, 1.2 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.33 - 7.27 (m, 3H), 7.25 - 7.23 (m, 2H), 7.15 (d, J = 3.2 Hz, 1H), 7.10 (dd, J = 4.5, 2.1 Hz, 1H), 7.09 - 7.02 (m, 2H), 6.53 (dd, J = 3.1, 0.8 Hz, 1H), 5.32 (s, 2H), 4.84 (d, J = LCMS C 27 H 27 Calculated for NO: 423.2 (M+H + ), Actual value: 423.2.

[0125] [C39] 1-(2-(((1H-Benzo[d]imidazol-5-yl)methyl)amino)-1H-benzo[d]imidazol-1-yl)butan-1-one (AJ2-9): Synthesized according to Scheme 1 and general procedure 4 and purified by PTLC (DCM / MeOH, 9:1) to afford AJ2-9 as an off-white solid (6 mg, 48%). 1 H NMR (400 MHz, MeOD) δ 8.05 (s, 1H), 7.57 (d, J = 1.5 Hz, 1H), 7.50 (d, J = 8.3 Hz, 1H), 7.24 (dd, J = 8.3, 1.7 Hz, 1H), 7.15 ~ 7.12 (m, LCMS C 19 H20 Calculated for NO: 334.2 (M+H + ), Actual value: 334.16.

[0126] [C40]

[0127] (2-(((1H-indol-5-yl)methyl)amino)-1H-benzo[d]imidazol-1-yl)(cyclopropyl)methanone (AJ2-10): Synthesized according to Scheme 1 and general procedure 4 and purified by PTLC (hexane / ethyl acetate 3:2) to afford AJ2-10 as an off-white solid (11 mg, 54%). 1 H NMR (400 MHz, CDCl3) δ 8.30 (s, 1H), 7.68 (t, J = 5.2 Hz, 1H), 7.58 (s, 1H), 7.51 (d, J = 8.1 Hz, 1H), 7.40 (d, J = 7.9 Hz, 1H), 7.26 (d, J = 8.4 Hz, 1H), 7.15 (dd, J = 14.9, 2.5 Hz, 4H), 6.98 (t, J = 7.8 Hz, 1H), 6.44 (d, J = 3.2 Hz, 1H), 4.75 (d, J = 4.6 Hz, 2H), 2.41 (tt, J = 8.3, 4.6 Hz, 1H), 1.32 - 1.24 (m, 2H), 1.12 (dd, J = 7.8, 3.4 Hz, 2H). 13 C NMR (151 MHz, CDCl3) δ 174.96, 154.41, 143.92, 135.36, 130.69, 129.28, 129.26, 128.07, 124.78, 124.73, 124.61, 122.32, 120.14, 117.07, 112.78, 111.36, 111.31, 102.62, 102.56, 47.56, 16.72, 10.26.LCMS C 20 H 19 Calculated for NO: 331.1 (M+H + ), Actual value: 331.1.

[0128] [C41] N-((1H-indol-5-yl)methyl)-1-(propylsulfonyl)-1H-benzo[d]imidazol-2-amine (AJ2-11): Synthesized according to Scheme 1 and general procedure 4 and purified by PTLC (hexane / ethyl acetate 3:2) to afford AJ2-11 as an off-white solid (5 mg, 43%). 1 H NMR (400 MHz, DMSO) δ 11.05 (s, 1H), 7.56 (d, J = 1.6 Hz, 1H), 7.54 - 7.48 (m, 1H), 7.36 (d, J = 8.3 Hz, 1H), 7.34 - 7.28 (m, 2H), 7.22 - 7.14 (m, 2H), 7.09 (t, J = 5.9 Hz, 1H), 7.05 (td, J = 7.7, 1.2 Hz, 1H), 6.39 (dd, J = 2.0, 0.9 Hz, 1H), 4.69 (d, J = 5.8 Hz, 2H), 3.65 - 3.56 (m, 2H), 1.60 - 1.48 (m, 2H), 0.82 (t, J = 7.4 Hz, 3H). 13 C NMR (151 MHz, DMSO) δ 152.60, 142.79, 135.63, 131.68, 129.76, 128.01, 126.12, 124.89, 121.49, 121.13, 119.41, 116.76, 112.22, 111.76, 101.42, 54.76, 47.20, 16.88, 12.49.LCMS C 19 H 21 Calculated value for N4O2S: 369.1 (M+H + ), Actual value: 469.13.

[0129] [C42] 1-(2-(((1H-indol-5-yl)methyl)amino)-1H-benzo[d]imidazol-1-yl)-3,5,5-trimethylhexan-1-one (AJ2-12): Synthesized according to Scheme 1 and general procedure 4 and purified by PTLC (hexane / ethyl acetate 3:2) to afford AJ2-12 as an off-white solid (6 mg, 45%). 1 H NMR (400 MHz, CDCl3) δ 8.38 (s, 1H), 8.19 (t, J = 5.2 Hz, 1H), 7.68 - 7.64 (m, 1H), 7.46 (dd, J = 7.9, 1.3 Hz, 1H), 7.40 - 7.33 (m, 2H), 7.25 - 7.19 (m, 3H), 7.11 - 7.00 (m, 2H), 6.52 (dd, J = 2.0, 0.9 Hz, 1H), 4.84 (d, J = 5.1 Hz, 2H), 3.00 - 2.94 (m, 1H), 2.89 - 2.84 (m, 1H), 2.38 - 2.26 (m, 2H), 1.38 (d, J = 3.9 Hz, 1H), 1.10 (s, 3H), 1.01 (d, J = 6.3 Hz, 1H), 0.93 (s, 9H). ). LCMS C 25 H 31 Calculated for NO: 403.2 (M+H + ), Actual value: 403.2.

[0130] [C43] 1-(2-(((1H-indol-5-yl)methyl)amino)-1H-benzo[d]imidazol-1-yl)-3-cyclopentylpropan-1-one (AJ2-13): Synthesized according to Scheme 1 and general procedure 4 and purified by PTLC (hexane / ethyl acetate 3:2) to afford AJ2-13 as an off-white solid (8 mg, 47%). 1H NMR (400 MHz, CDCl3) δ 8.20 (s, 1H), 8.18 (t, J = 4.4 Hz, 1H), 7.69 - 7.66 (m, 1H), 7.47 (dd, J = 8.0, 1.2 Hz, 1H), 7.42 - 7.35 (m, 2H), 7.25 - 7.17 (m, 4H), 7.10 - 7.04 (m, 2H), 6.54 (dd, J = 2.0, 1.0 Hz, 1H), 4.84 (d, J = 5.1 Hz, 2H), 3.08 - 2.97 (m, 2H), 2.37 (s, 2H), 1.85 - 1.77 (m, 4H), 1.69 - 1.63 (m, 5H).LCMS C 24 H 27 Calculated for NO: 387.2 (M+H + ), Actual value: 387.2.

[0131] [C44]

[0132] (2-(((1H-indol-5-yl)methyl)amino)-1H-benzo[d]imidazol-1-yl)(cyclohexyl)methanone (AJ2-14): Synthesized according to general procedure 4 according to Scheme 1 and purified by PTLC (hexane / ethyl acetate 3:2) to afford AJ2-14 as an off-white solid (6 mg, 47%). 1H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 8.15 (t, J = 5.2 Hz, 1H), 7.58 (d, J = 1.6 Hz, 1H), 7.38 (dd, J = 7.9, 1.2 Hz, 1H), 7.27 (d, J = 8.3 Hz, 1H), 7.22 - 7.13 (m, 4H), 7.04 - 6.96 (m, 2H), 6.49 - 6.42 (m, 1H), 4.74 (d, J = 5.1 Hz, 2H), 3.12 - 3.07 (m, 1H), 2.02 - 1.93 (m, 2H), 1.87 - 1.81 (m, 2H), 1.75 - 1.65 (m, 2H), 1.57 - 1.50 (m, 2H), 1.37 (dt, J = 12.7, 3.3 Hz, 2H). 13 C NMR (151 MHz, CDCl3) δ 178.08, 155.28, 143.83, 135.37, 129.83, 129.18, 128.08, 124.87, 124.78, 122.35, 120.39, 120.17, 117.12, 112.82, 111.36, 102.62, 47.67, 44.79, 29.04, 28.73, 25.63, 25.47, 25.42.LCMS C 23 H 25 Calculated for NO: 373.2 (M+H + ), Actual value: 373.2.

[0133] [C45] 1-(2-(((1H-indol-5-yl)methyl)amino)-1H-benzo[d]imidazol-1-yl)-3-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)propan-1-one (AJ2-15): Synthesized according to Scheme 1 and general procedure 1 and purified by biotage (hexane / ethyl acetate 3:2) to afford AJ2-15 as a colorless liquid (14 mg, 54%). 1H NMR (400 MHz, CDCl3) δ 8.36 (s, 1H), 8.06 (t, J = 5.2 Hz, 1H), 7.67 (s, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.30 - 7.20 (m, 4H), 7.06 (t, J = 7.8 Hz, 1H), 6.52 (s, 1H), 4.84 (d, J = 5.1 Hz, 2H), 2.76 (t, J = 7.4 Hz, 2H), 2.10 - 1.98 (m, 5H), 1.73 (d, J = 7.3 Hz, 2H).LCMS C 24 H 23 Calculated for NO: 411.2 (M+H + ), Actual value: 411.0.

[0134] [C46] 1-(2-(((6-Methoxypyridin-3-yl)methyl)amino)-1H-benzo[d]imidazol-1-yl)butan-1-one (AJ2-16): Synthesized according to Scheme 1 and general procedure 4 and purified by biotage (hexane / ethyl acetate 3:2) to afford AJ2-16 as an off-white solid (12 mg, 47%). 1H NMR (400 MHz, DMSO) δ 8.41 (t, J = 6.1 Hz, 1H), 8.22 (d, J = 2.4 Hz, 1H), 7.78 (dd, J = 8.5, 2.5 Hz, 1H), 7.57 (d, J = 8.1 Hz, 1H), 7.27 (dd, J = 7.9, 1.4 Hz, 1H), 7.17 (td, J = 7.6, 1.1 Hz, 1H), 7.03 (ddd, J = 8.3, 7.5, 1.3 Hz, 1H), 6.78 (d, J = 8.5 Hz, 1H), 4.59 (d, J = 6.1 Hz, 2H), 3.82 (s, 3H), 3.10 (t, J = 7.0 Hz, 2H), 1.73 (q, J = 7.2 Hz, 3H), 1.02 (t, J = 7.4 Hz, 3H). LCMS C 18 H 21 Calculated value for N4O2: 325.1 (M+H + ), Actual value: 325.0.

[0135] [C47] 1-(2-(((5-Fluoro-1H-indol-3-yl)methyl)amino)-1H-benzo[d]imidazol-1-yl)butan-1-one (AJ2-17A): Synthesized according to Scheme 1 and general procedure 1 and purified by biotage (hexane / ethyl acetate 4:6) to afford AJ2-17A as a brown solid (74 mg, 54%). 1 H NMR (400 MHz, CD3OD_SPE) δ 7.32 - 7.24 (m, 2H), 7.16 (d, J = 7.6, Hz, 2H), 6.92 (d, J = 7.1 Hz, 2H), 6.82 (td, J = 9.2, 2.6 Hz, 1H), 4.63 (s, 2H).LCMS C 16 H 14 Calculated value of FN4: 281.1 (M+H + ), Actual value: 281.1.

[0136] [C48] 1-(2-(((5-Fluoro-1H-indol-3-yl)methyl)amino)-1H-benzo[d]imidazol-1-yl)butan-1-one (AJ2-17): Synthesized according to Scheme 1 and general procedure 4 and purified by biotage (hexane / ethyl acetate 3:2) to afford AJ2-17 as an off-white solid (5 mg, 42%). 1 H NMR (400 MHz, CDCl3) δ 8.47 (s, 1H), 8.07 (t, J = 5.0 Hz, 1H), 7.49 (dd, J =7.9, 1.2 Hz, 1H), 7.42 - 7.36 (m, 1H), 7.33 (dd, J = 9.5, 2.5 Hz, 1H), 7.29 - 7.26 (m, 1H), 7.26 - 7.22 (m, 2H), 7.07 (ddd, J = 8.6, 7.5, 1.3 Hz, 1H), 6.94 (td, J = 9.1, 2.5 Hz, 1H), 4.88 (dd, J = 5.0, 0.8 Hz, 2H), 2.97 (t, J = 7.2 Hz, 2H), 1.84 (p, J = 7.3 Hz, 2H), 1.07 (t, J = 7.4 Hz, 3H). LCMS C 20 H 19 Calculated for FNO: 351.2 (M+H + ), Actual value: 351.0.

[0137] [C49] N-((1H-indol-5-yl)methyl)-N-(2-oxo-1,2,3,4-tetrahydroquinolin-3-yl)butyramide (AJ2-18): Synthesized according to Scheme 1 and general procedure 4 and purified by biotage (hexane / ethyl acetate 6:4) to afford AJ2-18 as an off-white solid (8 mg, 47%). 1H NMR (400 MHz, CDCl3) δ 8.46 (s, 1H), 8.31 (s, 1H), 7.48 (d, J = 1.6 Hz, 1H), 7.32 (d, J = 8.3 Hz, 1H), 7.19 - 7.13 (m, 1H), 7.02 (dd, J = 8.3, 1.7 Hz, 2H), 6.87 (d, J = 7.6 Hz, 1H), 6.79 (td, J = 7.4, 1.1 Hz, 1H), 6.62 (dd, J = 7.9, 1.1 Hz, 1H), 6.49 - 6.42 (m, 1H), 5.00 - 4.85 (m, 1H), 4.77 (d, J = 17.1 Hz, 1H), 4.63 (d, J = 17.0 Hz, 1H), 3.34 (t, J = 14.8 Hz, 1H), 2.69 (dd, J = 15.3, 6.7 Hz, 1H), 2.51 - 2.32 (m, 2H), 1.68 (q, J = 7.4 Hz, 2H), 0.88 (t, J = 7.4 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 174.69, 169.14, 136.32, 135.33, 128.58, 128.32, 128.13, 127.62, 125.12, 122.95, 122.50, 120.65, 118.54, 115.14, 111.61, 102.50, 55.30, 51.88, 35.64, 30.32, 18.75, 13.90.LCMS C 22 H 24 Calculated value for N3O2: 362.1 (M+H + ), Actual value: 362.1.

[0138]

[50] 1-(2-((Dibenzo[b,d]furan-4-ylmethyl)amino)-1H-benzo[d]imidazol-1-yl)butan-1-one (AJ2-19): Synthesized according to Scheme 1 and general procedure 4 and purified by biotage (hexane / ethyl acetate 3:2) to afford AJ2-19 as a white solid (14 mg, 62%). 1 H NMR (400 MHz, CDCl3) δ 7.95 (dd, J = 7.7, 1.4 Hz, 1H), 7.89 (dd, J = 7.8, 1.3 Hz, 1H), 7.60 (dt, J = 8.2, 0.9 Hz, 1H), 7.54 (dd, J = 7.5, 1.2 Hz, 1H), 7.46 (dd, J = 8.4, 1.4 Hz, 2H), 7.41 - 7.36 (m, 1H), 7.36 - 7.33 (m, 1H), 7.31 (d, J = 7.6 Hz, 1H), 7.28 - 7.23 (m, 2H), 7.06 (dd, J = 8.5, 1.3 LCMS C 24 H 22 Calculated value for N3O2: 384.2 (M+H + ), Actual value: 384.2.

[0139] [C51] 1-(2-(((5-(4-Fluorophenyl)-1H-pyrazol-4-yl)methyl)amino)-1H-benzo[d]imidazol-1-yl)butan-1-one (AJ2-20): Synthesized according to Scheme 1 and general procedure 4 and purified by PTLC (DCM / MeOH 9:1) to afford AJ2-20 as a brown viscous liquid (6 mg, 42%). 1H NMR (400 MHz, CDCl3) δ 8.01 (t, J = 5.5 Hz, 1H), 7.70 (s, 1H), 7.62 - 7.51 (m, 2H), 7.41 (dd, J = 16.1, 8.0 Hz, 2H), 7.24 (d, J = 7.3 Hz, 3H), 7.20 - 7.07 (m, 3H), 6.98 (s, 1H), 4.71 (d, J = 4.8 Hz, 2H), 2.97 (t, J = 7.2 Hz, 2H), 1.93 - 1.78 (m, 2H), 1.08 (t, J = 7.4 Hz, 3H).LCMS C 21 H 21 Calculated for FNO: 378.1 (M+H + ), Actual value: 378.0.

[0140] [C52] 1-(4-(((1-Butyryl-1H-benzo[d]imidazol-2-yl)amino)methyl)-5-(4-fluorophenyl)-1H-pyrazol-1-yl)butan-1-one (AJ2-21): Synthesized according to Scheme 1 and general procedure 4 and purified by PTLC (DCM / MeOH 9.5:0.5) to afford AJ2-21 as a brown viscous liquid (4 mg, 48%). 1 H NMR (400 MHz, CDCl3) δ 8.34 (s, 1H), 8.10 (t, J = 5.3 Hz, 1H), 7.76 - 7.66 (m, 2H), 7.49 - 7.35 (m, 2H), 7.31 - 7.24 (m, 1H), 7.18 - 7.05 (m, 3H), 4.77 (d, J = 5.4, 2H), 3.14 (t, J = 7.4 Hz, 2H), 2.99 (t, J = 7.2 Hz, 2H), 1.88 - 1.80 (m, 4H), 1.09 (t, J = 7.4 Hz, 3H), 1.04 (t, J = 7.4 Hz, 3H). LCMS C 25 H 27 Calculated value for FN5O2: 448.2 (M+H + ), Actual value: 448.1.

[0141] [C53] N-((1H-indol-5-yl)methyl)-N-(2,3-dihydro-1H-inden-2-yl)butyramide (AJ2-22): Synthesized according to Scheme 1 and general procedure 4 and purified by biotage (hexane / ethyl acetate 7:3) to afford AJ2-22 as a brown viscous liquid (17 mg, 68%). 1 H NMR (400 MHz, CDCl3) δ 8.79 (s, 1H), 8.55 (s, 0.39 H), 7.41 (d, J = 1.8 Hz, 1H), 7.39 - 7.32 (m, 1.61 H), 7.26 (d, J = 8.4 Hz, 0.5 H), 7.19 (t, J = 2.8 Hz, 1.12 H), 7.17 - 7.12 (m, 1.93 H), 7.00 (d, J = 8.4 Hz, 0.45 H), 6.95 - 6.93 (dd, J = 8.4, 1.9 Hz, 1.19 H), 6.50 (t, J = 2.7Hz, 1.10H), 6.45 (s, 0.42 H), 5.58 - 5.50 (m, 1.14 H), 4.90 (t, J = 8.2 Hz, 0.43 H), 4.74 (s, 0.89 H), 4.64 (s, 2.20 H), 3.17 - 2.93 (m, 6.63 H), 2.57 (t, J = 7.6 Hz, 0.93 H), 2.32 (t, J = 7.5 Hz, 2.28 H), 1.83 (q, J = 7.5 Hz, 1.01H), 1.74 - 1.65 (m, 2.49 H), 1.04 (t, J = 7.4 Hz, 1.43 H), 0.89 (t, J = 7.4 Hz, 3.53H). Note: Rotamers observed. LCMS C 22 H 24 Calculated for NO: 333.1 (M+H + ), Actual value: 333.1.

[0142] [C54] N-((1H-Benzo[d]imidazol-2-yl)methyl)-N-((1H-indol-5-yl)methyl)butyramide (AJ2-23): Synthesized according to Scheme 1 and general procedure 4 and purified by PTLC to afford AJ2-23 as a viscous liquid (13 mg, 57%). 1 H NMR (400 MHz, CDCl3) δ 10.50 (s, 1H), 9.17 (s, 1H), 7.79 - 7.69 (m, 1H), 7.47 - 7.39 (m, 2H), 7.28 - 7.23 (m, 4H), 6.89 (dd, J = 8.3, 1.7 Hz, 1H), 6.53 - 6.47 (m, 1H), 4.70 (s, 2H), 4.69 (s, 2H), 2.48 (t, J = 7.5 Hz, 2H), 1.74 (h, J = 7.4 Hz, 2H), 0.96 (t, J = 7.4 Hz, 3H).LCMS C 21 H 23 Calculated for NO: 347.1 (M+H + ), Actual value: 347.1.

[0143] [C55] 1-(2-(((1H-indol-5-yl)methyl)amino)-5-fluoro-1H-benzo[d]imidazol-1-yl)butan-1-one (AJ2-24): Synthesized according to Scheme 1 and general procedure 4 and purified by biotage (hexane / ethyl acetate 6:4) to afford AJ2-24 as a viscous liquid (6 mg, 43%). 1H NMR (400 MHz, CDCl3) δ 8.24 (s, 1H), 8.22 (s, 1H), 7.68 (d, J = 1.6 Hz, 1H), 7.39 (dd, J = 8.3, 0.9 Hz, 1H), 7.31 - 7.26 (m, 1H), 7.23 - 7.24 (m, 2H), 7.14 (dd, J = 9.2, 2.6 Hz, 1H), 6.75 (td, J = 9.0, 2.6 Hz, 1H), 6.55 - 6.53 (m, 1H), 4.83 (d, J = 5.2 Hz, 2H), 2.94 (t, J = 7.2 Hz, 2H), 1.84 (h, J = 7.3 Hz, 2H), 1.08 (t, J = 7.4 Hz, 3H). LCMS C 20 H 20 Calculated for FNO: 351.1 (M+H + ), Actual value: 351.1.

[0144] [C56] N-((9-Ethyl-9H-carbazol-3-yl)methyl)-1H-benzo[d]imidazol-2-amine (AJ2-25A): Synthesized according to Scheme 1 and general procedure 1 and purified by biotage (hexane / ethyl acetate 4:6) to afford AJ2-25A as a yellow solid (254 mg, 78%). 1 H NMR (400 MHz, DMSO) δ 8.15 (d, J = 1.6 Hz, 1H), 8.10 (dt, J = 7.8, 1.0 Hz, 1H), 7.61 - 7.54 (m, 2H), 7.51 (dd, J = 8.5, 1.7 Hz, 1H), 7.43 (ddd, J = 8.3, 7.1, 1.2 Hz, 1H), 7.27 (s, 1H), 7.21 - 7.11 (m, 3H), 6.88 (dd, J = 5.8, 3.2 Hz, 2H), 4.67 (d, J = 5.4 Hz, 2H), 4.42 (q, J = 7.1 Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H). LCMS C 20 H21 Calculated value of FN4: 341.1 (M+H + ), Actual value: 341.0.

[0145] [C57] 1-(2-(((9-Ethyl-9H-carbazol-3-yl)methyl)amino)-1H-benzo[d]imidazol-1-yl)butan-1-one (AJ2-25): Synthesized according to Scheme 1 and general procedure 4 and purified by biotage (hexane / ethyl acetate 6:4) to afford AJ2-25 as a yellow solid (16 mg, 68%). 1 H NMR (400 MHz, CDCl3) δ 8.21 (s, 1H), 8.14 (s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.54 (dd, J = 8.4, 1.5 Hz, 1H), 7.51 - 7.44 (m, 2H), 7.44 - 7.37 (m, 3H), 7.26 - 7.19 (m, 2H), 7.11 - 7.03 (m, 1H), 4.94 (d, J = 5.2 Hz, 2H), 4.38 (q, J = 7.2 Hz, 2H), 3.00 (t, J = 7.2 Hz, 2H), 1.85 (h, J = 7.4 Hz, 2H), 1.49 - 1.37 (m, 3H), 1.14 - 1.04 (m, 3H). LCMS C 26 H 27 Calculated for NO: 411.2 (M+H + ), Actual value: 411.0.

[0146] [C58] N-((1H-indol-5-yl)methyl)-N-(2-(azepan-1-yl)-2-phenylethyl)butyramide (AJ2-26): Synthesized according to Scheme 1 and general procedure 4 and purified by PTLC (hexane / ethyl acetate 7:3) to afford AJ2-26 as a colorless liquid (22 mg, 74%). 1H NMR (400 MHz, CD2Cl2) δ 8.97 (s, 1H), 7.67 - 7.48 (m, 2H), 7.42 - 7.31 (m, 2H), 7.25 (d, J = 8.3 Hz, 2H), 7.13 (t, J = 2.6 Hz, 2H), 6.69 (dd, J = 8.4, 1.7 Hz, 1H), 6.36 (t, J = 2.4 Hz, 1H), 4.41 - 4.29 (m, 2H), 3.62 - 3.51 (m, 2H), 3.05 (s, 2H), 2.29 - 2.09 (m, 3H), 1.79 - 1.69 (m, 3H), 1.50 (dt, J = 14.8, 9.3 Hz, 9H), 0.80 (d, J = 7.4 Hz, 3H). Note: Rotamers were observed. LCMS C 27 H 36 Calculated for NO: 418.2 (M+H + ), Actual value: 418.1.

[0147] [C59] 1-(2-(((1-Phenyl-1H-pyrazol-4-yl)methyl)amino)-1H-benzo[d]imidazol-1-yl)butan-1-one (AJ2-27): Synthesized according to Scheme 1 and general procedure 4 and purified by PTLC (hexane / ethyl acetate 6:4) to afford AJ2-27 as a colorless liquid (16 mg, 62%). 1 H NMR (400 MHz, CDCl3) δ 8.11 (t, J = 5.6 Hz, 1H), 8.01 (d, J = 0.8 Hz, 1H), 7.77 (d, J = 0.7 Hz, 1H), 7.69 - 7.63 (m, 2H), 7.49 - 7.37 (m, 4H), 7.31 - 7.24 (m, 3H), 7.08 (ddd, J = 8.5, 7.5, 1.3 Hz, 1H), 4.71 (d, J = 5.5 Hz, 2H), 3.00 (t, J = 7.2 Hz, 2H), 1.87 (h, J = 7.3 Hz, 2H), 1.09 (t, J = 7.4 Hz, 3H). LCMS C21 H 22 Calculated for NO: 360.1 (M+H + ), Actual value: 360.1.

[0148]

[60] N-((5-Bromo-1H-indol-3-yl)methyl)-1-methyl-1H-benzo[d]imidazol-2-amine A (AJ2-28): Synthesized according to Scheme 1 and purified by biotage (DCM / MeOH; 9:1) to afford AJ2-28 as a brown solid (178 mg, 65%). 1 H NMR (400 MHz, DMSO) δ 11.15 (s, 1H), 7.94 (d, J = 1.9 Hz, 1H), 7.42 (d, J = 2.4 Hz, 1H), 7.33 (d, J = 8.5 Hz, 1H), 7.26 - 7.22 (m, 1H), 7.18 (dd, J = 8.6, 2.0 Hz, 1H), 7.14 - 7.10 (m, 1H), 7.01 - 6.97 (m, 1H), 6.97 - 6.87 (m, 2H), 4.69 (d, J = 5.6 Hz, 2H), 3.48 (s, 3H). 13 C NMR (101 MHz, DMSO) δ 155.65, 143.07, 135.83, 135.44, 129.10, 126.24, 123.93, 121.90, 120.64, 118.66, 115.35, 113.86, 113.50, 111.63, 107.63, 38.12, 28.69.LCMS(ESI) C 17 H 16 Calculated for BrN4: 355.0 (M+H + ), Actual value: 354.9.

[0149]

[61] N-((1H-indol-5-yl)methyl)-1-methyl-1H-benzo[d]imidazol-2-amine (AJ2-29): Synthesized according to Scheme 1 and purified by biotage (DCM / MeOH; 9:1) to afford AJ2-29 as a brown solid (165 mg, 72%). 1 H NMR (400 MHz, CDCl3) δ 8.40 (s, 1H), 7.70 (d, J = 1.6 Hz, 1H), 7.53 (dt, J = 7.7, 1.0 Hz, 1H), 7.42 - 7.36 (m, 1H), 7.31 - 7.24 (m, 3H), 7.13 (ddd, J = 7.7, 5.0, 3.7 Hz, 1H), 7.10 - 7.05 (m, 2H), 6.55 (d, J = 1.1 Hz, 1H), 4.81 (d, J = 5.1 Hz, 2H), 4.24 (d, J = 5.5 Hz, 1H), 3.46 (s, 3H).LCMS C 17 H 17 Calculated value for N4: 277.1 (M+H + ), Actual value: 277.1.

[0150]

[62] N-((9-Ethyl-9H-carbazol-3-yl)methyl)-1-methyl-1H-benzo[d]imidazol-2-amine (AJ2-30): Synthesized according to Scheme 1 and purified by biotage (hexane / ethyl acetate; 3:7) to afford AJ2-30 as a yellow solid (248 mg, 76%). 1H NMR (400 MHz, DMSO) δ 8.17 (t, J = 1.1 Hz, 1H), 8.12 (dt, J = 7.8, 1.0 Hz, 1H), 7.61 - 7.53 (m, 3H), 7.43 (ddd, J = 8.3, 7.1, 1.2 Hz, 1H), 7.23 (t, J = 5.9 Hz, 1H), 7.23 - 7.13 (m, 3H), 7.00 - 6.84 (m, 2H), 4.75 (d, J = 5.8 Hz, 2H), 4.42 (q, J = 7.1 Hz, 2H), 3.55 (s, 3H), 1.28 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 154.38, 142.32, 140.31, 139.55, 135.03, 128.90, 126.27, 125.89, 123.13, 122.67, 121.24, 120.52, 120.38, 119.61, 118.96, 116.53, 108.68, 108.59, 107.05, 48.26, 37.63, 28.24, 13.82.LCMS C 23 H 23 Calculated value for N4: 355.1 (M+H + ), Actual value: 355.1.

[0151]

[63] N-((9-Ethyl-9H-carbazol-3-yl)methyl)-N-(1-methyl-1H-benzo[d]imidazol-2-yl)butyramide (AJ2-31): Synthesized according to Scheme 1 and general procedure 3 and purified by biotage (DCM / MeOH; 9.5:0.5) to afford AJ2-31 as a white solid (64 mg, 52%). 1H NMR (400 MHz, DMSO) δ 8.15 - 7.86 (m, 2H), 7.64 (d, J = 7.6 Hz, 1H), 7.57 (d, J = 8.2 Hz, 1H), 7.50 (dd, J = 11.8, 8.1 Hz, 2H), 7.45 - 7.43 (m, 1H), 7.35 (d, J = 8.5 Hz, 1H), 7.25 (p, J = 7.4 Hz, 2H), 7.19 - 7.12 (m, 1H), 5.07 (s, 2H), 4.40 (q, J = 7.1 Hz, 2H), 3.39 (s, 3H), 2.00 (s, 2H), 1.62 - 1.48 (m, 2H), 1.28 (t, J = 7.1 Hz, 3H), 0.81 (d, J = 7.7 Hz, 3H). 13 C NMR (151 MHz, DMSO) δ 171.61, 147.34, 139.80, 139.16, 138.30, 133.94, 126.53, 125.82, 125.15, 122.23, 121.58, 121.29, 119.82, 119.61, 118.74, 118.12, 110.28, 108.53, 108.33, 50.42, 36.34, 34.55, 28.66, 17.24, 13.05, 12.90. Note: Rotamers observed. LCMS C 27 H 28 Calculated for NO: 425.2 (M+H + ), Actual value: 425.1.

[0152]

[64] 3-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)-N-((9-ethyl-9H-carbazol-3-yl)methyl)-N-(1-methyl-1H-benzo[d]imidazol-2-yl)propanamide (AJ2-32): Synthesized according to Scheme 1 and general procedure 3 and purified by biotage (DCM / MeOH; 9.5:0.5) to afford AJ2-32 as a light brown viscous liquid (12 mg, 46%). 1H NMR (400 MHz, CDCl3) δ 8.00 (d, J = 7.8 Hz, 1H), 7.97 (d, J = 1.6 Hz, 1H), 7.83 (dd, J = 6.9, 2.1 Hz, 1H), 7.50 - 7.46 (m, 1H), 7.41 (dt, J = 8.3, 1.0 Hz, 1H), 7.39 - 7.26 (m, 4H), 7.25 - 7.18 (m, 2H), 5.19 (s, 2H), 4.34 (q, J = 7.2 Hz, 2H), 3.05 (s, 3H), 1.99 (td, J = 7.4, 2.6Hz, 3H), 1.95 - 1.81 (m, 4H), 1.62 (t, J = 7.4 Hz, 2H), 1.42 (t, J = 7.2 Hz, 3H). Note: Rotamers observed. LCMS C 31 H 31 Calculated for NO: 503.2 (M+H + ), Actual value: 503.0.

[0153]

[65] N-((5-Bromo-1H-indol-3-yl)methyl)-3-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)-N-(1-methyl-1H-benzo[d]imidazol-2-yl)propanamide (AJ2-32): Synthesized according to Scheme 1 and general procedure 3 and purified by PTLC (DCM / MeOH; 9.5:0.5) to afford AJ2-33 as a light brown viscous liquid (4 mg, 27%). 1H NMR (400 MHz, CDCl3) δ 8.33 (d, J = 8.8 Hz, 1H), 7.81 (d, J = 2.0 Hz, 1H), 7.59 - 7.52 (m, 2H), 7.48 (dd, J = 8.8, 2.0 Hz, 1H), 7.16 (dd, J = 7.8, 4.2 Hz, 1H), 7.15 - 7.11 (m, 2H), 5.12 (s, 1H), 4.85 (s, 2H), 4.30 (s, 1H), 3.72 - 3.59 (m, 3H), 3.52 (s, 3H), 3.35 (s, 2H), 2.68 (t, J = 7.4 Hz, 2H), 2.36 (t, J = 7.6 Hz, 1H), 2.11 - 1.97 (m, 7H), 1.73 (s, 2H). Note: Rotamers observed. LCMS C 25 H 24 Calculated for BrNO: 503.1 (M+H + ), Actual value: 503.0.

[0154]

[66] 1-(2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethyl)-N-((9-ethyl-9H-carbazol-3-yl)methyl)-1H-benzo[d]imidazol-2-amine (AJ2-34): Synthesized according to Scheme 1 and general procedure 5 and purified by biotage (hexane / ethyl acetate; 4:6) to afford AJ2-34 as a yellow viscous liquid (43 mg, 64%). 1H NMR (400 MHz, CDCl3) δ 8.17 (dd, J = 1.7, 0.7 Hz, 1H), 8.10 (dt, J = 7.9, 1.0 Hz, 1H), 7.60 - 7.54 (m, 2H), 7.52 - 7.38 (m, 3H), 7.25 - 7.21 (m, 1H), 7.18 - 7.14 (m, 1H), 7.13 - 7.05 (m, 2H), 4.92 (d, J = 5.1 Hz, 2H), 4.59 (t, J = 5.2 Hz, 1H), 4.38 (q, J = 7.2 Hz, 2H), 3.79 - 3.69 (m, 2H), 1.93 - 1.84 (m, 4H), 1.80 (t, J = 2.7 Hz, 1H), 1.46 - 1.42 (m 5H). 13 C NMR (101 MHz, CDCl3) δ 153.72, 142.47, 140.30, 139.53, 133.94, 128.81, 126.23, 125.89, 123.11, 122.67, 121.61, 120.55, 120.37, 119.84, 118.96, 116.78, 108.70, 108.60, 107.20, 82.52, 69.62, 48.33, 37.62, 36.80, 32.20, 26.63, 13.84, 13.16.LCMS C 29 H 29 Calculated value for N6: 461.2 (M+H + ), Actual value: 461.0.

[0155]

[67] 3-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)-1-(2-(((6-methoxypyridin-3-yl)methyl)amino)-1H-benzo[d]imidazol-1-yl)propan-1-one (AJ2-35): Synthesized according to Scheme 1 and general procedure 3 and purified by biotage (hexane / ethyl acetate; 4:6) to afford AJ2-35 as a light brown viscous liquid (12 mg, 62%). 1H NMR (400 MHz, CDCl3) δ 8.26 - 8.16 (m, 1H), 8.02 (t, J = 5.6 Hz, 1H), 7.67 (dd, J = 8.5, 2.5 Hz, 1H), 7.44 (ddd, J = 7.9, 1.2, 0.6 Hz, 1H), 7.32 - 7.24 (m, 2H), 7.08 (dd, J = 8.1, 1.3 Hz, 1H), 6.73 (dd, J = 8.5, 0.7 Hz, 1H), 4.69 (d, J = 5.6 Hz, 2H), 3.93 (s, 3H), 2.85 - 2.75 (m, 2H), 2.15 - 1.95 (m, 5H), 1.74 (t, J = 7.3 Hz, 2H).LCMS C 22 H 23 Calculated value for N6O2: 403.1 (M+H + ), Actual value: 403.0.

[0156]

[68] N-((1H-indol-5-yl)methyl)-3-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)-N-(2,3-dihydro-1H-inden-2-yl)propanamide (AJ2-36): Synthesized according to Scheme 1 and general procedure 2 and purified by biotage (hexanes / ethyl acetate; 4:6) to afford AJ2-36 as a light brown viscous liquid (12 mg, 46%). 1H NMR (400 MHz, CDCl3) δ 8.33 (s, 1H), 7.44 - 7.35 (m, 3H), 7.34 - 7.22 (m, 2H), 7.21 - 7.12 (m, 2H), 7.03 - 6.91 (m, 2H), 6.53 (t, J = 2.7 Hz, 1H), 6.47 (s, 1H), 5.52 (q, J = 8.6 Hz, 1H), 4.84 - 4.66 (m, 1H), 4.60 (s, 2H), 3.18 - 3.11 (m, 2H), 3.01 - 2.97 (m, 4H), 2.31 (t, J = 7.6 Hz, 1H), 2.08 (t, J = 7.5 Hz, 2H), 1.98 - 1.88 (m 3H), 1.83 (t, J = 7.4 Hz, 2H), 1.71 (t, J = 7.5 Hz, 1H), 1.56 (t, J = 7.5 Hz, 2H). Note: Rotamers were observed. LCMS C 26 H 27 Calculated for NO: 411.2 (M+H + ), Actual value: 411.2.

[0157]

[69] 1-(2-(((5-Bromo-1H-indol-3-yl)methyl)amino)-1H-benzo[d]imidazol-1-yl)-4,4,4-trifluorobutan-1-one (AJ2-37): Synthesized according to Scheme 1 and general procedure 4 and purified by biotage (DCM / MeOH; 9.5:0.5) to afford AJ2-37 as a light brown viscous liquid (12 mg, 46%). 1H NMR (400 MHz, CDCl3) δ 8.18 (s, 1H), 7.89 (s, 1H), 7.84 (dd, J = 1.7, 0.8 Hz, 1H), 7.51 (dd, J = 8.0, 1.3 Hz, 1H), 7.37 - 7.31 (m, 2H), 7.31 - 7.27 (m, 3H), 7.11 (ddd, J = 8.1, 7.5, 1.2 Hz, 1H), 4.89 (dd, J = 5.1, 0.8 Hz, 2H), 3.35 - 3.24 (m, 2H), 2.74 - 2.55 (m, 2H).LCMS C 20 H 17 Calculated for BrFNO: 465.0 (M+H + ), Actual value: 466.8.

[0158] [C70] N-((1,6,7,8-Tetrahydrocyclopenta[g]indol-3-yl)methyl)-1H-benzo[d]imidazol-2-amine (AJ2-38): Synthesized according to Scheme 1 and purified by biotage (DCM / MeOH; 9.5:0.5) to afford AJ2-38 as an off-white solid (64 mg, 72%). 1 H NMR (400 MHz, CD3OD) δ 7.38 (d, J = 8.0 Hz, 1H), 7.22 (dd, J = 5.8, 3.2 Hz, 2H), 7.17 (s, 1H), 6.99 (dd, J = 5.8, 3.2 Hz, 2H), 6.91 (d, J = 8.0 Hz, 1H), 4.70 (s, 2H), 2.97 (dt, J = 23.9, 7.3 Hz, 4H), 2.11 (p, J = 7.4 Hz, 2H). 13C NMR (101 MHz, CD3OD) δ 154.74, 137.63, 136.51, 133.87, 125.45, 125.30, 122.36, 120.45, 116.22, 115.69, 112.07, 111.32, 47.52, 47.31, 47.09, 38.70, 32.64, 29.49, 25.02.LCMS C 19 H 19 Calculated value for N4: 303.1 (M+H + ), Actual value: 303.1.

[0159] [C71] 1-(2-(((1,6,7,8-Tetrahydrocyclopenta[g]indol-3-yl)methyl)amino)-1H-benzo[d]imidazol-1-yl)butan-1-one (AJ2-39): Synthesized according to Scheme 1 and general procedure 4 and purified by biotage (hexane / ethyl acetate; 5:5) to afford AJ2-39 as a white solid (64 mg, 72%). 1 H NMR (400 MHz, CDCl3) δ 8.06 (s, 1H), 7.92 (s, 1H), 7.54 - 7.45 (m, 2H), 7.38 (d, J = 8.1 Hz, 1H), 7.28 - 7.21 (m, 2H), 7.06 (td, J = 7.8, 1.5 Hz, 2H), 4.93 (d, J = 4.9 Hz, 2H), 3.04 (t, J = 7.3 Hz, 4H), 2.96 (t, J = 7.2 Hz, 2H), 2.21 (p, J = 7.4 Hz, 2H), 1.82 (h, J = 7.4 Hz, 2H), 1.06 (t, J = 7.4 Hz, 3H). LCMS C 23 H 25 Calculated for NO: 373.2 (M+H + ), Actual value: 373.1.

[0160] [C72]

[0161] (S)—N-(1H-Benzo[d]imidazol-2-yl)-2-(6-methoxynaphthalen-2-yl)propanamide (AJ2-40): Synthesized according to Scheme 1 and general procedure 2 and purified by biotage (hexane / ethyl acetate; 3:7) to afford AJ2-40 as a white solid (67 mg, 68%). 1 H NMR (400 MHz, DMSO) δ 12.07 (s, 1H), 11.74 (s, 1H), 7.87 - 7.73 (m, 3H), 7.54 (dd, J = 8.6, 1.8 Hz, 1H), 7.42 - 7.38 (m, 2H), 7.29 (d, J = 2.6 Hz, 1H), 7.15 (dd, J = 9.0, 2.6 Hz, 1H), 7.06 (t, J = 4.4 Hz, 2H), 4.13 (q, J = 7.0 Hz, 1H), 3.86 (s, 3H), 1.55 (d, J = 6.9 Hz, 3H).LCMS C 21 H 20 Calculated value for N3O2: 346.1 (M+H + ), Actual value: 346.0.

[0162] [C73]

[0163] (S)—N-(2-(6-Methoxynaphthalen-2-yl)propyl)-1H-benzo[d]imidazol-2-amine (AJ2-41): Synthesized according to Scheme 1 and purified by biotage (hexane / ethyl acetate; 3:7) to afford AJ2-41 as a light brown solid (22 mg, 57%). 1H NMR (400 MHz, CDCl3) δ 7.49 (d, J = 8.5 Hz, 1H), 7.42 (d, J = 8.9 Hz, 1H), 7.28 (d, J = 1.9 Hz, 1H), 7.17 (dd, J = 5.8, 3.2 Hz, 2H), 7.05 - 7.02 (m, 2H), 7.01 - 6.92 (m, 3H), 3.82 (s, 3H), 3.57 (dd, J = 12.8, 6.1 Hz, 1H), 3.36 (dd, J = 12.8, 8.5 Hz, 1H), 2.95 (q, J = 7.1 Hz, 1H), 1.15 (d, J = 6.9 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 157.42, 155.10, 138.80, 137.18, 133.47, 129.08, 128.92, 127.26, 125.71, 125.59, 120.81, 118.85, 112.06, 105.58, 55.29, 49.77, 39.63, 19.33.LCMS C 21 H 22 Calculated for NO: 332.1 (M+H + ), Actual value: 332.1.

[0164] [C74]

[0165] (S)-1-(2-((2-(6-Methoxynaphthalen-2-yl)propyl)amino)-1H-benzo[d]imidazol-1-yl)butan-1-one (AJ2-42): Synthesized according to Scheme 1 and general procedure 4 and purified by biotage (hexane / ethyl acetate; 6:4) to afford AJ2-42 as a light brown solid (22 mg, 57%). 1H NMR (400 MHz, CDCl3) δ 7.89 (t, J = 5.4 Hz, 1H), 7.70 (dd, J = 8.5, 5.7 Hz, 2H), 7.64 (d, J = 1.8 Hz, 1H), 7.44 - 7.38 (m, 2H), 7.33 (d, J = 8.1 Hz, 1H), 7.22 (td, J = 7.7, 1.1 Hz, 1H), 7.16 - 7.09 (m, 2H), 7.05 - 7.00 (m, 1H), 3.91 (s, 3H), 3.88 (dd, J = 13.5, 7.0 Hz, 1H), 3.81 - 3.74 (m, 1H), 3.39 - 3.23 (m, 1H), 2.90 (td, J = 7.1, 1.0 Hz, 2H), 1.78 (h, J = 7.4 Hz, 3H), 1.45 (d, J = 7.0 Hz, 3H), 1.02 (t, J = 7.4 Hz, 3H).LCMS C 25 H 28 Calculated value for N3O2: 402.2 (M+H + ), Actual value: 402.1.

[0166] [C75] N-(1H-Benzo[d]imidazol-2-yl)-2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamide (AJ2-43): Synthesized according to Scheme 1 and purified by biotage (hexanes / ethyl acetate; 4:6) to afford AJ2-43 as a light brown solid (42 mg, 64%). 1 H NMR (400 MHz, CDCl3) δ 7.74 - 7.64 (m, 2H), 7.58 - 7.42 (m, 4H), 7.22 (dd, J = 6.0, 3.2 Hz, 2H), 6.92 (d, J = 2.5 Hz, 1H), 6.82 (d, J = LCMS C 26H 22 Calculated for ClN4O3: 473.1 (M+H + ), Actual value: 472.9.

[0167] [C76] N-(1H-Benzo[d]imidazol-2-yl)-2-(6-chloro-9H-carbazol-3-yl)propanamide (AJ2-44): Synthesized according to general procedure 2 and purified by biotage (hexane / ethyl acetate; 4:6) to afford AJ2-44 as an off-white solid (24 mg, 54%). 1 H NMR (400 MHz, DMSO) δ 11.36 (s, 1H), 8.23 ​​(s, 2H), 8.17 (d, J = 2.1 Hz, 1H), 8.09 (d, J = 8.1 Hz, 1H), 7.49 (d, J = 8.5 Hz, 1H), 7.43 - 7.39 (m, 1H), 7.39 - 7.30 (m, 3H), 7.19 (dd, J = 5.9, 3.2 Hz, 2H), 7.11 (dd, J = 8.2, 1.5 Hz, 1H), 3.84 (t, J = 7.1 Hz, 1H), 1.44 (d, J = 7.1 Hz, 3H).LCMS C 22 H 18 Calculated for ClNO: 389.1 (M+H + ), Actual value: 389.0.

[0168] [C77] N-(2-(6-chloro-9H-carbazol-3-yl)propyl)-1H-benzo[d]imidazol-2-amine (AJ2-45): Synthesized according to Scheme 1 and general procedure 1 and purified by biotage (hexane / ethyl acetate; 3:7) to afford AJ2-45 as a brown solid (8 mg, 42%). 1H NMR (400 MHz, CDCl3) δ 8.19 (s, 1H), 7.93 (d, J = 1.8 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.33 (dd, J = 4.1, 1.3 Hz, 1H), 7.20 (dd, J = 5.8, 3.2 Hz, 2H), 7.04 (dd, J = 5.8, 3.2 Hz, 2H), 6.96 - 6.87 (m, 2H), 3.62 (dd, J = 12.7, 5.6 Hz, 1H), 3.36 (dd, J = 12.8, 9.0 Hz, 1H), 3.04 (t, J = 7.4 Hz, 1H), 1.24 (d, J = 7.0 Hz, 3H).LCMS C 22 H 20 Calculated for ClN4: 375.1 (M+H + ), Actual value: 375.1.

[0169] [C78] 3-(((1H-Benzo[d]imidazol-2-yl)amino)methyl)-1H-indole-6-carbonitrile (AJ2-46): Synthesized according to Scheme 1 and general procedure 1 and purified by biotage (hexane / ethyl acetate; 3:7) to afford AJ2-46 as a brown solid (35 mg, 58%). 1 H NMR (400 MHz, CD3OD) δ 7.83 - 7.75 (m, 2H), 7.58 (d, J = 0.9 Hz, 1H), 7.30 (dd, J = 8.2, 1.5 Hz, 1H), 7.23 (dd, J = 5.8, 3.2 Hz, 2H), 7.00 (dd, J = 5.8, 3.2 Hz, 2H), 4.77 (d, J = 0.8 Hz, 2H). LCMS C 17 H 14 Calculated value of N5: 288.1 (M+H + ), Actual value: 288.1.

[0170] [C79] N-((5-chloro-1H-indol-3-yl)methyl)-1H-benzo[d]imidazol-2-amine (AJ2-47): Synthesized according to Scheme 1 and general procedure 1 and purified by biotage (hexane / ethyl acetate; 3:7) to afford AJ2-47 as a brown solid (43 mg, 64%). 1 H NMR (400 MHz, CDCl3) δ 8.38 (s, 1H), 7.49 (d, J = 2.0 Hz, 1H), 7.29 - 7.23 (m, 2H), 7.20 (d, J = 8.6 Hz, 1H), 7.11 (dd, J = 8.6, 2.0 Hz, 1H), 7.05 (dd, J = 5.8, 3.2 Hz, 2H), 6.99 (s, 1H), 5.12 (s, 1H), 4.61 (s, 2H). LCMS C 16 H 14 Calculated for ClN4: 297.0 (M+H + ), Actual value: 297.0.

[0171] [C80] 9-Ethyl-6-(((1-methyl-1H-benzo[d]imidazol-2-yl)amino)methyl)-9H-carbazole-3-carbonitrile (AJ2-48): Synthesized according to Scheme 1 and general procedure 1 and purified by biotage (hexanes / ethyl acetate; 3:7) to afford AJ2-48 as a brown solid (32 mg, 65%). 1 H NMR (400 MHz, CDCl3) 1H NMR (400 MHz, CDCl3) δ 7.89 (dd, J = 3.4, 1.6 Hz, 2H), 7.48 (dd, J = 8.4, 1.7 Hz, 1H), 7.42 (dt, J = 8.5, 1.6 Hz, 2H), 7.15 (dd, J = 16.5, 8.5 Hz, 2H), 7.19 - 7.12 (m, 2H), 6.89 (dd, J = 7.7, 1.3 Hz, 1H), 6.00 (s, 1H), 4.82 (s, 2H), 4.08 (q, J = 7.1 Hz, 2H), 3.34 (s, 3H), 1.26 (t, J = 7.2 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 154.84, 142.15, 141.58, 139.69, 135.13, 131.07, 128.58, 127.33, 125.01, 122.61, 121.84, 121.13, 120.74, 119.90, 119.52, 115.89, 109.09, 108.95, 107.11, 100.85, 47.42, 37.77, 28.35, 13.78.LCMS C 24 H 22 Calculated value of N5: 380.1 (M+H + ), Actual value: 380.1.

[0172] [chemical 81] N-((9-Ethyl-3,3-dimethyl-2,3,4,9-tetrahydro-1H-carbazol-6-yl)methyl)-1-methyl-1H-benzo[d]imidazol-2-amine (AJ2-49): Synthesized according to Scheme 3 and general procedure 1 and purified by biotage (hexanes / ethyl acetate; 3:7) to afford AJ2-49 as a brown solid (32 mg, 65%). 1H NMR (400 MHz, CDCl3) δ 7.59 - 7.46 (m, 2H), 7.31 - 7.18 (m, 2H), 7.12 (ddd, J = 7.9, 6.3, 2.3 Hz, 1H), 7.09 - 7.03 (m, 2H), 4.78 (d, J = 4.8 Hz, 2H), 4.22 (d, J = 5.5 Hz, 1H), 4.08 (q, J = 7.2 Hz, 2H), 3.40 (d, J = 2.1 Hz, 3H), 2.69 (t, J = 6.3 Hz, 2H), 2.51 (d, J = 1.6 Hz, 2H), 1.69 (t, J = 6.4 Hz, 2H), 1.31 (t, J = 7.2 Hz, 3H), 1.04 (s, 6H). 13 C NMR (101 MHz, CDCl3) δ 154.41, 142.41, 135.69, 135.01, 134.67, 128.36, 127.87, 121.16, 119.49, 117.84, 116.51, 108.88, 108.84, 106.96, 48.68, 37.66, 36.09, 35.06, 30.10, 28.20, 28.06, 19.61, 15.52.LCMS C 25 H 31 Calculated value for N4: 387.2 (M+H + ), Actual value: 387.1.

[0173] [chemical 82] N-((9-Ethyl-3,3-dimethyl-2,3,4,9-tetrahydro-1H-carbazol-6-yl)methyl)-1H-benzo[d]imidazol-2-amine (AJ2-50): Synthesized according to Scheme 3 and general procedure 1 and purified by biotage (hexane / ethyl acetate; 6:4) to afford AJ2-50 as a brown solid (45 mg, 62%). 1H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 1.6 Hz, 1H), 7.23 (dd, J = 5.8, 3.2 Hz, 2H), 7.19 (d, J = 8.3 Hz, 1H), 7.10 (dd, J = 8.4, 1.7 Hz, 1H), 7.02 (dd, J = 5.8, 3.2 Hz, 2H), 4.61 (s, 2H), 4.05 (q, J = 7.2 Hz, 2H), 2.69 (t, J = 6.3 Hz, 2H), 2.46 (d, J = 1.6 Hz, 2H), 1.69 (t, J = 6.4 Hz, 2H), 1.30 (t, J = 7.2 Hz, 3H), 1.04 (s, 6H).LCMS C 24 H 29 Calculated value for N4: 373.2 (M+H + ), Actual value: 373.1.

[0174] [chemical 83] 1-Butyl-N-((9-ethyl-3,3-dimethyl-2,3,4,9-tetrahydro-1H-carbazol-6-yl)methyl)-1H-benzo[d]imidazol-2-amine (AJ2-51): Synthesized according to Scheme 3 and General Procedure 1 according to General Procedure 4 and purified by biotage (hexane / ethyl acetate; 6:4) to afford AJ2-51 as a brown solid (22 mg, 56%). 1H NMR (400 MHz, CDCl3) δ 7.45 (dd, J = 7.2, 1.3 Hz, 2H), 7.19 - 7.12 (m, 2H), 7.06 - 6.99 (m, 1H), 7.00 - 6.94 (m, 2H), 4.76 (d, J = 3.8 Hz, 2H), 3.96 (q, J = 7.2 Hz, 2H), 3.80 (t, J = 7.3 Hz, 2H), 2.59 (t, J = 6.4 Hz, 2H), 2.40 (d, J = 1.5 Hz, 2H), 1.60 (td, J = 6.9, 6.4, 2.5 Hz, 4H), 1.32 - 1.25 (m, 2H), 1.22 (q, J = 6.9 Hz, 4H), 0.81 (t, J = 7.3 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 153.04, 139.89, 135.64, 134.52, 133.84, 128.07, 127.77, 121.51, 121.07, 120.12, 117.67, 115.74, 108.83, 107.55, 48.43, 42.29, 37.62, 36.09, 35.00, 30.96, 30.06, 28.04, 20.18, 19.59, 15.51, 13.79. 28 H 37 Calculated value for N4: 428.2 (M+H + ), Actual value: 429.2.

[0175] [chemical 84] 1-Butyl-N-((9-ethyl-9H-carbazol-3-yl)methyl)-1H-benzo[d]imidazol-2-amine (AJ2-52): Synthesized according to general procedure 1 according to general procedure 5 and purified by biotage (hexanes / ethyl acetate; 5:5) to afford AJ2-52 as a yellow solid (18 mg, 62%). 1H NMR (400 MHz, CDCl3) δ 8.12 (d, J = 1.7 Hz, 1H), 8.07 (dt, J = 7.8, 1.0 Hz, 1H), 7.58 - 7.51 (m, 2H), 7.47 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.43 - 7.34 (m, 2H), 7.27 - 7.21 (m, 1H), 7.15 - 7.11 (m, 1H), 7.07 (dd, J = 3.8, 1.0 Hz, 2H), 4.90 (d, J = 4.9 Hz, 2H), 4.35 (q, J = 7.3 Hz, 3H), 3.82 (t, J = 7.2 Hz, 2H), 1.77 - 1.61 (m, 2H), 1.41 (t, J = 7.2 Hz, 3H), 1.39 - 1.29 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 153.90, 142.19, 140.32, 139.55, 134.61, 128.99, 126.16, 125.90, 123.13, 122.67, 121.17, 120.50, 120.26, 119.60, 118.97, 116.51, 108.71, 108.59, 107.37, 48.28, 42.10, 37.63, 31.12, 20.24, 13.83, 13.79. 26 H 29 Calculated value for N4: 397.2 (M+H + ), Actual value: 397.2.

[0176] [C85] N-((1H-indol-5-yl)methyl)-1-butyl-1H-benzo[d]imidazol-2-amine (AJ2-53): Synthesized according to general procedure 1 according to general procedure 5 and purified by biotage (hexane / ethyl acetate; 6:4) to afford AJ2-53 as a light brown liquid (16 mg, 56%). 1H NMR (400 MHz, CDCl3) δ 9.13 (s, 1H), 7.51 (d, J = 1.7 Hz, 1H), 7.45 - 7.38 (m, 1H), 7.21 (d, J = 8.3 Hz, 1H), 7.11 - 6.95 (m, 5H), 6.38 (ddd, J = 3.0, 2.0, 0.9 Hz, 1H), 4.67 (s, 2H), 4.57 (s, 1H), 3.70 (t, J = 7.2 Hz, 2H), 1.57 (dq, J = 9.6, 7.4 Hz, 2H), 1.33 - 1.12 (m, 3H), 0.79 (t, J = 7.3 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 153.76, 141.46, 135.55, 134.37, 129.41, 128.06, 125.21, 122.22, 121.31, 120.13, 119.80, 116.06, 111.61, 107.51, 102.20, 48.37, 42.12, 31.02, 20.21, 13.77.LCMS C 20 H 23 Calculated value for N4: 319.1 (M+H + ), Actual value: 319.1.

[0177] [chemical 86] 1-Butyl-N-(dibenzo[b,d]furan-4-ylmethyl)-1H-benzo[d]imidazol-2-amine (AJ2-54): Synthesized according to general procedure 1 according to general procedure 4 and purified by biotage (hexane / ethyl acetate; 6:4) to afford AJ2-54 as a light brown liquid (24 mg, 68%). 1H NMR (400 MHz, CDCl3) δ 7.95 (ddd, J = 7.7, 1.4, 0.7 Hz, 1H), 7.87 (dd, J = 7.7, 1.3 Hz, 1H), 7.59 - 7.50 (m, 3H), 7.48 - 7.44 (m, 1H), 7.39 - 7.27 (m, 2H), 7.14 - 7.10 (m, 1H), 7.08 - 7.04 (m, 2H), 5.12 (d, J = 5.4 Hz, 2H), 4.84 (t, J = 5.6 Hz, 1H), 3.84 (t, J = 7.2 Hz, 2H), 1.76-1.63 (m, 2H), 1.41 - 1.28 (m, 2H), 0.86 (t, J = 7.3 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 156.04, 154.37, 153.90, 142.23, 134.66, 127.33, 127.09, 124.40, 124.21, 123.10, 123.00, 122.58, 121.15, 120.85, 120.07, 119.61, 116.61, 111.66, 107.36, 43.02, 42.16, 31.13, 20.25, 13.74.LCMS C 24 H 24 Calculated for NO: 370.1 (M+H + ), Actual value: 370.1.

[0178] [chemical 87] 1-Butyl-N-((6-methoxypyridin-3-yl)methyl)-1H-benzo[d]imidazol-2-amine (AJ2-55): Synthesized according to general procedure 1 according to general procedure 4 and purified by biotage (hexanes / ethyl acetate; 4:6) to afford AJ2-55 as a light brown liquid (18 mg, 62%). 1H NMR (400 MHz, CDCl3) δ 8.11 (t, J = 1.8 Hz, 1H), 7.60 (ddd, J = 8.6, 2.6, 1.4 Hz, 1H), 7.41 (dd, J = 7.5, 1.3 Hz, 1H), 7.09 - 6.98 (m, 3H), 6.64 (dd, J = 8.5, 0.9 Hz, 1H), 4.60 (s, 3H), 3.84 (s, 3H), 3.81 (t, J = 7.3 Hz, 2H), 1.72 - 1.59 (m, 2H), 1.36 - 1.22 (m, 2H), 0.85 (t, J = 7.3 Hz, 3H). LCMS C 18 H 23 Calculated for NO: 311.2 (M+H + ), Actual value: 311.2.

[0179] [C88] N-(benzo[d][1,3]dioxol-5-ylmethyl)-1-butyl-1H-benzo[d]imidazol-2-amine (AJ2-56): Synthesized by general procedure 1 according to general procedure 4 and purified by biotage (hexane / ethyl acetate; 4:6) to afford AJ2-56 as a light brown liquid (8 mg, 56%). 1 H NMR (400 MHz, CDCl3) δ 7.39 (dt, J = 7.7, 1.0 Hz, 1H), 7.03 - 6.97 (m, 1H), 7.05 - 7.00 (m, 2H), 6.81 (d, J = 1.7 Hz, 1H), 6.75 (dd, J = 7.9, 1.7 Hz, 1H), 6.67 (d, J = 7.9 Hz, 1H), 5.84 (s, 2H), 4.55 (d, J = 4.5 Hz, 2H), 4.48 (s, 1H), 3.77 (t, J = 7.2 Hz, 2H), 1.68 - 1.55 (m, 2H), 1.34 - 1.21 (m, 2H), 0.84 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 153.61, 147.89, 147.06, 141.60, 134.44, 132.52, 121.26, 121.16, 119.77, 116.29, 108.48, 108.30, 107.46, 101.08, 47.37, 42.17, 31.09, 20.23, 13.79.LCMS C 19 H 22 Calculated value for N3O2: 324.1 (M+H + ), Actual value: 324.1.

[0180] [C89] 1-(2-(diethylamino)ethyl)-N-((9-ethyl-9H-carbazol-3-yl)methyl)-1H-benzo[d]imidazol-2-amine (AJ2-57): Synthesized according to general procedure 1 and purified by biotage (DCM / methanol; 9.5:0.5) to afford AJ2-57 as a yellow solid (35 mg, 74%). 1 H NMR (400 MHz, CDCl3) δ 8.06 (d, J = 1.7 Hz, 1H), 8.00 (dt, J = 7.8, 1.0 Hz, 1H), 7.94 (s, 1H), 7.50 - 7.45 (m, 2H), 7.39 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 7.35 - 7.25 (m, 2H), 7.14 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 7.04 (td, J = 7.5, 1.5 Hz, 1H), 7.00 - 6.89 (m, 2H), 4.74 (d, J = 3.9 Hz, 2H), 4.28 (q, J = 7.2 Hz, 2H), 3.91 - 3.81 (m, 2H), 2.67 - 2.58 (m, 2H), 2.28 (q, J = 7.2 Hz, 4H), 1.33 (t, J = 7.2 Hz, 3H), 0.61 (t, J = 7.1 Hz, 6H).LCMS C 28 H 34 Calculated value for N5: 440.2 (M+H +), Actual value: 440.1.

[0181] [C90] N-((5-Bromo-1H-indol-3-yl)methyl)-1-butyl-1H-benzo[d]imidazol-2-amine (AJ2-58): Synthesized according to Scheme 1 and general procedure 5 and purified by biotage (hexane / ethyl acetate; 4:6) to afford AJ2-58 as a light brown liquid (6 mg, 43%). 1 H NMR (400 MHz, CDCl3) δ 9.17 (s, 1H), 7.51 - 7.46 (m, 1H), 7.43 - 7.37 (m, 1H), 7.24 (s, 1H), 7.06 - 7.00 (m, 3H), 6.99 - 6.89 (m, LCMS C 20 H 22 Calculated for BrN4: 397.0 (M+H + ), Actual value: 397.0.

[0182] [C91] N-((9-Ethyl-9H-carbazol-3-yl)methyl)-1-phenethyl-1H-benzo[d]imidazol-2-amine (AJ2-59): Synthesized according to Scheme 1 and general procedure 5 and purified by biotage (hexane / ethyl acetate; 6:4) to afford AJ2-59 as a pale yellow liquid (12 mg, 47%). 1H NMR (400 MHz, CDCl3) δ 7.96 (d, J = 7.7 Hz, 1H), 7.91 (s, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.40 - 7.27 (m, 3H), 7.20 (d, J = 8.4 Hz, 1H), 7.14 - 7.09 (m, 1H), 7.09 - 7.03 (m, 4H), 6.99 (td, J = 7.6, 1.2 Hz, 1H), 6.95 - 6.87 (m, 3H), 4.61 (s, 2H), 4.22 (q, J = 7.2 Hz, 2H), 4.08 (t, J = 6.6 Hz, 2H), 2.92 (t, J = 6.6 Hz, 2H), 1.30 (t, J = 7.2 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 152.73, 140.20, 139.47, 137.86, 128.95, 128.87, 127.96, 127.12, 126.26, 125.76, 122.91, 122.67, 122.04, 120.75, 120.51, 120.44, 118.88, 115.54, 108.50, 107.67, 48.07, 44.63, 37.55, 35.01, 13.79.LCMS C 30 H 29 Calculated value for N4: 445.2 (M+H + ), Actual value: 445.1.

[0183] [C92] 1-Benzyl-N-((9-ethyl-9H-carbazol-3-yl)methyl)-1H-benzo[d]imidazol-2-amine (AJ2-60): Synthesized according to general procedure 1 and purified by biotage (hexane / ethyl acetate; 6:4) to afford AJ2-60 as a pale yellow solid (43 mg, 67%). 1H NMR (400 MHz, CDCl3) δ 7.93 (dd, J = 7.9, 1.0 Hz, 1H), 7.86 (d, J = 1.7 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.38 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 7.33 - 7.27 (m, 2H), 7.23 - 7.16 (m, 4H), 7.13 (td, J = 7.4, 6.9, 1.0 Hz, 1H), 7.10 - 7.01 (m, 3H), 6.98 (d, J = 4.1 Hz, 2H), 5.01 (d, J = 2.3 Hz, 2H), 4.76 (d, J = 4.3 Hz, 2H), 4.51 (s, 1H), 4.24 (q, J = 7.2 Hz, 2H), 1.31 (t, J = 7.2 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 154.05, 140.26, 139.45, 135.36, 134.72, 129.16, 128.70, 128.11, 126.55, 125.87, 125.82, 123.05, LCMS C 29 H 27 Calculated value for N4: 431.2 (M+H + ), Actual value: 431.1.

[0184] [C93] N-((9-Ethyl-3-(trifluoromethyl)-2,3,4,9-tetrahydro-1H-carbazol-6-yl)methyl)-1-methyl-1H-benzo[d]imidazol-2-amine (AJ2-61): Synthesized according to Scheme 3 and general procedure 1 and purified by biotage (hexane / ethyl acetate; 5:5) to afford AJ2-61 as a brown solid (24 mg, 72%). 1H NMR (400 MHz, CDCl3) δ 7.37 ‐ 7.31 (m, 2H), 7.14 ‐ 7.07 (m, 2H), 6.95 (dtd, J = 16.4, 7.3, 1.4 Hz, 2H), 6.90 ‐ 6.85 (m, 1H), 4.98 (s, 1H), 4.66 (s, 2H), 3.90 (qd, J = 7.3, 3.0 Hz, 2H), 3.25 (s, 3H), 2.85 (dd, J = 15.1, 5.2 Hz, 1H), 2.77 ‐ 2.66 (m, 1H), 2.66 ‐ 2.51 (m, 2H), 2.40 ‐ 2.25 (m, 1H), 2.24 - 2.14 (m, 1H), 1.71 (qd, J = 12.2, 5.9 Hz, 1H), 1.18 (t, J = 7.2 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 154.27, 141.58, 135.59, 134.83, 134.34, 129.48, 129.01, 126.99, 126.71, 121.59, 121.24, 119.66, 117.48, 116.02, 109.05, 107.08, 106.44, 48.23, 39.50 (q, J = 27.0 Hz), 37.74, 28.27, 22.29 (d, J = 3.0 Hz),, 20.96, 20.71 (d, J = 2.9 Hz), 15.48. 19 F NMR (376 MHz, CDCl3) δ -72.78 (d, J = 8.4 Hz). LCMS C 24 H 26 Calculated value of F3N4: 427.2 (M+H + ), actual measured value: 427.2.

[0185]

Chemical 94

[0186] [C95] N-(1H-Benzo[d]imidazol-2-yl)-9-ethyl-N-methyl-9H-carbazole-3-carboxamide (AJ2-63): Synthesized according to general procedure 3 and purified by biotage (hexane / ethyl acetate; 6:4) to afford AJ2-63 as a brown solid (34 mg, 63%). 1 H NMR (400 MHz, CDCl3) δ 11.62 (s, 1H), 8.40 (d, J = 1.6 Hz, 1H), 8.14 (dt, J = 7.9, 0.9 Hz, 1H), 7.73 (dd, J = 8.5, 1.7 Hz, 2H), 7.59 - 7.44 (m, 4H), 7.33 - 7.29 (m, 1H), 4.43 (q, J = 7.3 Hz, 2H), 3.82 (s, 3H), 1.48 (t, J = 7.2 Hz, 3H). LCMS C 23 H 21 Calculated for NO: 369.1 (M+H + ), Actual value: 369.1.

[0187] [C96] 1-Butyl-N-((9-ethyl-3-(trifluoromethyl)-2,3,4,9-tetrahydro-1H-carbazol-6-yl)methyl)-1H-benzo[d]imidazol-2-amine (AJ2-64): Synthesized according to general procedure 5 according to Scheme 3 and purified by biotage (hexane / ethyl acetate; 6:4) to afford AJ2-64 as a brown solid (42 mg, 64%). 1H NMR (400 MHz, CDCl3) δ 7.46 - 7.37 (m, 2H), 7.24 - 7.13 (m, 2H), 7.08 - 6.94 (m, 3H), 4.97 - 4.80 (m, 1H), 4.74 (d, J = 3.1 Hz, 2H), 4.01 - 3.94 (m, 2H), 3.79 (t, J = 7.2 Hz, 2H), 2.99 - 2.87 (m, 1H), 2.86 - 2.79 (m, 1H), 2.75 - 2.56 (m, 2H), 2.47 - 2.33 (m, 1H), 2.30 - 2.21 (m, 1H), 1.84 - 1.73 (m, 1H), 1.65 - 1.58 (m, 2H), 1.31 - 1.18 (m, 5H), 0.82 (t, J = 7.3 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 153.36, 135.59, 134.37, 134.13, 129.40, 128.91, 126.94, 126.63, 121.66, 121.36, 119.92, 117.51, 116.00, 109.13, 107.47, 106.53, 48.30, 42.23, 39.54 (q, J = 27.0 Hz) 37.77, 31.03, 22.32, 22.29, 20.99, 20.70, 20.21, 15.48, 13.76. 19 F NMR (376 MHz, CDCl3) δ -72.89 (d, J = 8.4 Hz). LCMS C 27 H 32 Calculated value for F3N4: 369.2 (M+H + ), Actual value: 369.2.

[0188] [C97] N-((5-Bromo-1H-indol-3-yl)methyl)-1-(2-(diethylamino)ethyl)-1H-benzo[d]imidazol-2-amine (AJ2-65): Synthesized according to general scheme 3 and purified by biotage (hexane / ethyl acetate; 4:6) to afford AJ2-65 as a yellow solid (24 mg, 66%). 1 H NMR (400 MHz, DMSO) δ 11.15 (s, 1H), 7.87 (d, J = 2.0 Hz, 1H), 7.43 (d, J = 2.5 Hz, 1H), 7.41 - 7.30 (m, 2H), 7.24 (d, J = 7.5 Hz, 1H), 7.23 - 7.10 (m, 2H), 6.93 (dt, J = 20.8, 7.1 Hz, 2H), 4.66 (d, J = 3.7 Hz, 2H), 4.00 (t, J = 5.8 Hz, 2H), 2.57 (t, J = 5.7 Hz, 2H), 2.38 (q, J = 7.1 Hz, 4H), 0.72 (t, J = 7.1 Hz, 6H).LCMS C 22 H 27 Calculated for BrN5: 440.1 (M+H + ), Actual value: 440.1.

[0189] [C98] 1-Benzyl-N-((5-bromo-1H-indol-3-yl)methyl)-1H-benzo[d]imidazol-2-amine (AJ2-66): Synthesized according to general scheme 3 and purified by biotage (hexane / ethyl acetate; 6:4) to afford AJ2-66 as a brown solid (48 mg, 63%). 1 H NMR (400 MHz, CDCl3) δ 9.64 - 9.52 (m, 1H), 7.38 - 7.30 (m, 2H), 7.09 (dd, J = 5.0, 1.9 Hz, 3H), 7.03 - 6.97 (m, 3H), 6.94 (ddd, J = 6.6, 4.9, 2.3 Hz, 4H), 6.83 (d, J = 2.3 Hz, 1H), 4.92 (s, 2H), 4.51 (s, 2H). 13C NMR (101 MHz, CDCl3) δ 153.72, 140.70, 135.15, 134.96, 134.32, 129.16, 128.24, 128.10, 126.53, 124.88, 124.68, 121.86, 121.07, 120.48, 115.77, 113.02, 112.76, 111.71, 107.89, 45.80, 39.24.LCMS C 23 H 20 Calculated for BrN4: 431.0 (M+H + ), Actual value: 431.0.

[0190] [C99]

[0191] (R)-1-(1H-Benzo[d]imidazol-2-yl)-N-((9-ethyl-9H-carbazol-3-yl)methyl)ethan-1-amine (AJ2-67): Synthesized according to general procedure 1 and purified by biotage (hexane / ethyl acetate; 6:4) to afford AJ2-67 as a brown solid (34 mg, 63%). 1 H NMR (400 MHz, CDCl3) δ 8.02 - 7.92 (m, 2H), 7.71 (dd, J = 6.1, 3.2 Hz, 2H), 7.53 (td, J = 7.6, 7.0, 1.2 Hz, 1H), 7.41 (d, J = 8.2 Hz, 1H), 7.37 - 7.24 (m, 4H), 7.20 (d, J = 8.3 Hz, 1H), 4.40 (q, J = 6.6 Hz, 1H), 4.26 (q, J = 7.1 Hz, 2H), 4.01 - 3.83 (m, 2H), 1.61 (d, J = 6.7 Hz, 3H), 1.38 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 158.50, 140.27, 139.30, 129.82, 126.30, 125.77, 123.01, 122.78, 122.39, 120.55, 120.13, 118.88, 108.59, 108.41, 52.50, 52.31, 37.53, 21.83, 13.83.LCMS C 24 H 25 Calculated value for N4: 369.2 (M+H + ), Actual value: 369.2.

[0192] [C100] N-((9-Ethyl-9H-carbazol-3-yl)methyl)-1-(4,4,4-trifluorobutyl)-1H-benzo[d]imidazol-2-amine (AJ2-68): Synthesized according to Scheme 1 and general procedure 5 and purified by biotage (hexane / ethyl acetate; 6:4) to afford AJ2-68 as a brown solid (34 mg, 63%). 1 H NMR (400 MHz, CDCl3) δ 8.02 (d, J = 1.7 Hz, 1H), 7.96 (dt, J = 7.8, 1.0 Hz, 1H), 7.50 - 7.36 (m, 3H), 7.34 - 7.24 (m, 2H), 7.21 - 7.10 (m, 1H), 7.07 (td, J = 7.6, 1.3 Hz, 1H), 7.00 (td, J = 7.6, 1.2 Hz, 1H), 6.97 - 6.89 (m, 1H), 4.81 (s, 2H), 4.25 (q, J = 7.2 Hz, 2H), 3.82 (t, J = 7.0 Hz, 2H), 2.10 - 1.98 (m, 2H), 1.98 - 1.84 (m, 2H), 1.32 (t, J = 7.2 Hz, 3H). 19 F NMR (376 MHz, CDCl3) δ -65.84 (t, J = 10.4 Hz). LCMS C 26 H 26 Calculated value for F3N4: 451.2 (M+H +), Actual value: 451.2.

[0193] [C101] Methyl 3-(((1H-benzo[d]imidazol-2-yl)amino)methyl)-1H-indole-5-carboxylate (AJ2-69): Synthesized according to general procedure 1 and purified by biotage (hexane / ethyl acetate; 3:7) to afford AJ2-69 as a brown solid (30 mg, 23%). 1 H NMR (400 MHz, MeOD) δ 8.30 (dd, J = 1.7, 0.7 Hz, 1H), 7.69 (dd, J = 8.6, 1.7 Hz, 1H), 7.34 - 7.26 (m, 2H), 7.14 (dd, J = 5.8, 3.2 Hz, 3H), 6.92 (dd, J = 5.8, 3.2 Hz, 2H), 4.69 - 4.62 (m, 2H), 3.70 (s, 3H). LCMS C 18 H 17 Calculated value for N4O2: 321.1 (M+H + ), Actual value: 321.1.

[0194] [C102] 3-(((1H-Benzo[d]imidazol-2-yl)amino)methyl)-1H-indole-5-carboxylic acid (AJ2-70): To a solution of methyl 3-(((1H-benzo[d]imidazol-2-yl)amino)methyl)-1H-indole-5-carboxylate (AJ2-69) (0.078 mmol) in tetrahydrofuran (1 mL) and water (1 mL) was added lithium hydroxide (0.023 mmol) at 0 °C and the reaction mixture was stirred at room temperature for 6 h. Upon completion, the reaction mixture was neutralized with 1 N HCl (pH 6-7) and the solid was filtered to afford AJ2-70 as a brown solid (6 mg, 42%). 1H NMR (400 MHz, DMSO-d6) δ 11.26 (s, 1H), 8.40 (s, 1H), 7.72 (d, J = 8.6 Hz, 1H), 7.45 (d, J = 2.3 Hz, 1H), 7.41 (d, J = 8.6 Hz, 1H), 7.18 - 7.09 (m, 2H), 7.05 (s, 1H), 6.86 (dd, J = 5.8, 3.2 Hz, 2H), 4.66 (d, J = 5.6 Hz, 2H). 13 LCMS C 17 H 15 Calculated value for N4O2: 307.1 (M+H + ), Actual value: 307.1.

[0195] [C103] N-((1H-Benzo[g]indol-3-yl)methyl)-1H-benzo[d]imidazol-2-amine (AJ2-71): Synthesized according to general procedure 1 and purified by biotage (hexane / ethyl acetate; 2:8) to afford AJ2-71 as an off-white solid (34 mg, 63%). 1 H NMR (400 MHz, DMSO) δ 11.88 (s, 1H), 10.63 (s, 1H), 8.34 (d, J = 8.2 Hz, 1H), 7.91 (d, J = 8.1 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.57 - 7.50 (m, 1H), 7.50 - 7.36 (m, 3H), 7.16 (s, 2H), 6.86 (t, J = 7.7 Hz, 3H), 4.72 (d, J = 5.8 Hz, 2H). LCMS C 20 H 17 Calculated value for N4: 313.1 (M+H + ), Actual value: 313.1.

[0196] [chemical 104] N-((1H-Benzo[g]indol-3-yl)methyl)-1-methyl-1H-benzo[d]imidazol-2-amine (AJ2-72): Synthesized according to general procedure 1 and purified by biotage (hexane / ethyl acetate; 3:7) to afford AJ2-62 as a brown solid (34 mg, 63%). 1 H NMR (400 MHz, DMSO-d6) δ 11.89 (d, J = 2.6 Hz, 1H), 8.35 (dd, J = 8.2, 1.1 Hz, 1H), 7.91 (dd, J = 8.3, 1.2 Hz, 1H), 7.82 (d, J = 8.6 Hz, 1H), 7.52 (ddd, J = 8.2, 6.8, 1.3 Hz, 1H), 7.48 - 7.43 (m, 2H), 7.39 (ddd, J = 8.1, 6.9, 1.3 Hz, 1H), 7.29 - 7.23 (m, 1H), 7.14 (dd, J = 7.9, 1.2 Hz, 1H), 7.03 - 6.89 (m, 3H), 4.82 (d, J = 5.4 Hz, 2H), 3.50 (s, 3H). LCMS C 21 H 19 Calculated value for N4: 327.1 (M+H + ), Actual value: 327.1.

[0197] [C105] N-((9-Ethyl-9H-carbazol-3-yl)methyl)quinolin-3-amine (AJ2-73): Synthesized according to general procedure 1 and purified by biotage (hexanes / ethyl acetate; 6:4) to afford AJ2-73 as an off-white solid (76 mg, 71%). 1H NMR (400 MHz, CDCl3) δ 8.54 (dd, J = 2.9, 1.0 Hz, 1H), 8.16 (d, J = 1.8 Hz, 1H), 8.13 (dd, J = 7.8, 1.0 Hz, 1H), 8.00 (dd, J = 6.2, 3.2 Hz, 1H), 7.62 (dd, J = 6.3, 3.3 Hz, 1H), 7.52 (ddt, J = 9.4, 7.0, 1.4 Hz, 2H), 7.47 - 7.41 (m, 4H), 7.31 - 7.24 (m, 1H), 7.14 (d, J = 2.6 Hz, 1H), 4.58 (t, J = 3.1 Hz, 2H), 4.47 (s, 1H), 4.39 (qd, J = 7.4, 2.6 Hz, 2H), 1.46 (td, J = 7.1, 1.2 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 143.45, 142.18, 141.63, 140.34, 139.53, 129.57, 129.03, 128.46, 126.91, 126.07, 125.92, 125.56, 124.92, 123.26, 122.65, 120.52, 119.77, 118.97, 110.38, 108.75, 108.62, 48.53, 37.65, 13.84.LCMS C 24 H 22 Calculated value for N3: 352.2 (M+H + ), Actual value: 352.2.

[0198] [chemical 106] N-((9-Ethyl-9H-carbazol-3-yl)methyl)-N-(quinolin-3-yl)butyramide (AJ2-74): Synthesized according to general procedure 1 and general procedure 4 and purified by PTLC (hexanes / ethyl acetate; 6:4) to afford AJ2-62 as a brown solid (17 mg, 68%). 1H NMR (400 MHz, CDCl3) δ 8.61 - 8.32 (m, 1H), 8.02 (d, J = 8.5 Hz, 1H), 7.92 (d, J = 7.7 Hz, 1H), 7.85 (d, J = 1.2 Hz, 1H), 7.71 - 7.57 (m, 3H), 7.46 (ddd, J = 8.1, 6.8, 1.1 Hz, 1H), 7.41 - 7.28 (m, 2H), 7.21 - 7.16 (m, 1H), 7.10 (ddd, J = 7.9, 7.0, 1.1 Hz, 1H), 5.10 (s, 2H), 4.26 (q, J = 7.2 Hz, 2H), 2.06 - 1.92 (m, 2H), 1.61 (q, J = 7.4 Hz, 2H), 1.34 (t, J = 7.2 Hz, 3H), 0.77 (t, J = 7.4 Hz, 3H). LCMS C 28 H 28 Calculated for NO: 422.2 (M+H + ), Actual value: 422.1.

[0199] [C107] 1-(2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethyl)-N-(3,4-dimethoxybenzyl)-1H-benzo[d]imidazol-2-amine (AJ2-75): Synthesized according to general procedure 1 and general procedure 5 and purified by PTLC (hexanes / ethyl acetate; 6:4) to afford AJ2-75 as a brown solid (12 mg, 64%). 1H NMR (600 MHz, CDCl3) δ 7.43 (dd, J = 7.7, 4.7 Hz, 1H), 7.07 (dt, J = 8.4, 4.2 Hz, 1H), 7.05 - 6.98 (m, 2H), 6.97 - 6.88 (m, 2H), 6.76 (dd, J = 8.1, 4.7 Hz, 1H), 4.60 (d, J = 4.6 Hz, 3H), 3.79 (t, J = 5.3 Hz, 6H), 3.66 (q, J = 6.4, 5.8 Hz, 2H), 1.81 (tt, J = 12.3, 5.1 Hz, 5H), 1.37 (q, J = 6.3 Hz, 2H). 13 C NMR (151 MHz, CDCl3) δ 153.55, 149.17, 148.66, 142.23, 133.86, 131.05, 121.69, 120.40, 119.97, 116.77, 111.55, 111.21, 107.23, 82.47, 69.66, 55.97, 55.94, 47.66, 36.82, 32.25, 26.66, 13.17.LCMS C 23 H 26 Calculated value for NO: 404.2 (M+H + ), Actual value: 404.2.

[0200] [C108] N-((7-Ethyl-7H-benzo[c]carbazol-10-yl)methyl)-1H-benzo[d]imidazol-2-amine (AJ2-76): Synthesized according to general procedure 1 and purified by biotage (hexane / ethyl acetate; 2:8) to afford AJ2-76 as a brown solid (27 mg, 56%). 1H NMR (400 MHz, CDCl3) δ 8.59 (d, J = 8.3 Hz, 1H), 8.41 (s, 1H), 7.91 (dt, J = 8.1, 0.9 Hz, 1H), 7.81 (d, J = 8.9 Hz, 1H), 7.64 - 7.54 (m, 1H), 7.52 (d, J = 8.9 Hz, 1H), 7.38 - 7.40 (m, 1H), 7.35 - 7.30 (m, 2H), 7.16 (dd, J = 5.9, 3.2 Hz, 2H), 6.94 (dd, J = 5.8, 3.2 Hz, 2H), 4.69 (s, 2H), 4.32 (q, J = 7.2 Hz, 2H), 1.33 (t, J = 7.2 Hz, 3H). LCMS C 26 H 23 Calculated value for N4: 391.1 (M+H + ), Actual value: 391.1.

[0201] [C109] N-((7-ethyl-7H-benzo[c]carbazol-10-yl)methyl)-1-methyl-1H-benzo[d]imidazol-2-amine (AJ2-77): Synthesized according to general procedure 1 and purified by biotage (hexane / ethyl acetate; 3:7) to afford AJ2-77 as a brown solid (48 mg, 66%). 1H NMR (400 MHz, CDCl3) δ 8.78 (dd, J = 8.4, 1.1 Hz, 1H), 8.67 (d, J = 1.6 Hz, 1H), 8.03 (dt, J = 8.1, 0.8 Hz, 1H), 7.94 (d, J = 8.9 Hz, 1H), 7.74 - 7.70 (m, 1H), 7.67 (d, J = 8.9 Hz, 1H), 7.64 - 7.58 (m, 2H), 7.58 - 7.47 (m, 2H), 7.21 - 7.17 (m, 1H), 7.16 - 7.05 (m, 2H), 5.00 (d, J = 5.2 Hz, 2H), 4.51 (q, J = 7.2 Hz, 3H), 3.45 (s, 3H), 1.50 (t, J = 7.2 Hz, 3H). LCMS C 27 H 25 Calculated value for N4: 405.2 (M+H + ), Actual value: 405.1.

[0202] [C110] N-((9-(4-(tert-butyl)phenyl)-9H-carbazol-3-yl)methyl)-1-methyl-1H-benzo[d]imidazol-2-amine (AJ2-78): Synthesized according to general procedure 1 and purified by biotage (hexane / ethyl acetate; 3:7) to afford AJ2-78 as a brown solid (65 mg, 72%). 1H NMR (400 MHz, CDCl3) δ 8.22 (dd, J = 1.7, 0.7 Hz, 1H), 8.16 (dt, J = 7.8, 1.0 Hz, 1H), 7.65 - 7.61 (m, 2H), 7.58 (dt, J = 7.8, 1.0 Hz, 1H), 7.54 - 7.47 (m, 3H), 7.47 - 7.41 (m, 3H), 7.34 - 7.29 (m, 1H), 7.17 (ddd, J = 7.8, 5.0, 3.6 Hz, 1H), 7.13 - 7.08 (m, 2H), 4.94 (d, J = 5.3 Hz, 2H), 4.35 (t, J = 5.3 Hz, 1H), 3.51 (s, 3H), 1.46 (s, 9H). LCMS C 31 H 31 Calculated value for N4: 459.2 (M+H + ), Actual value: 459.1.

[0203] [C111] N-((6-(3,5-Dimethylisoxazol-4-yl)-9-ethyl-9H-carbazol-3-yl)methyl)-1H-benzo[d]imidazol-2-amine (AJ2-79): Synthesized according to general procedure 1 according to general synthesis scheme 2 and purified by biotage (hexane / ethyl acetate; 3:7) to afford AJ2-79 as a brown solid (10 mg, 52%). 1 H NMR (400 MHz, CDCl3) δ 8.04 - 7.96 (m, 1H), 7.75 (d, J = 1.6 Hz, 1H), 7.38 - 7.35 (m, 2H), 7.26 - 7.20 (m, 2H), 7.14 (dd, J = 5.8, 3.2 Hz, 2H), 6.91 (dd, J = 5.8, 3.2 Hz, 2H), 4.67 (s, 2H), 4.25 (q, J = 7.2 Hz, 2H), 2.30 (s, 3H), 2.18 (s, 3H), 1.34 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 164.97, 159.15, 154.73, 139.87, 139.56, 128.61, 127.13, 125.69, 122.90, 122.81, 121.16, 120.92, 120.85, 119.38, 117.28, 112.32, 109.10, 108.89, 53.45, 47.67, 37.76, 13.88, 11.55, 10.87.LCMS C 27 H 26 Calculated for NO: 436.2 (M+H + ), Actual value: 436.1.

[0204] [C112] N-((6-(3,5-Dimethylisoxazol-4-yl)-9-ethyl-9H-carbazol-3-yl)methyl)-1-methyl-1H-benzo[d]imidazol-2-amine (AJ2-80): Synthesized according to general procedure 1 according to general procedure scheme 2 and purified by biotage (hexane / ethyl acetate; 3:7) to afford AJ2-80 as a brown solid (34 mg, 62%). 1 H NMR (400 MHz, CDCl3) δ 8.19 (d, J = 1.7 Hz, 1H), 7.96 (d, J = 1.7 Hz, 1H), 7.65 - 7.54 (m, 2H), 7.53 - 7.42 (m, 2H), 7.36 (dt, J = 8.4, 1.3 Hz, 1H), 7.16 (ddd, J = 7.8, 4.1, 1.5 Hz, 1H), 7.10 (dt, J = 4.1, 1.1 Hz, 2H), 4.95 (d, J = 5.1 Hz, 2H), 4.43 (dt, J = 8.3, 6.6 Hz, 3H), 3.50 (s, 3H), 2.46 (s, 3H), 2.33 (s, 3H), 1.55 - 1.42 (m, 3H). LCMS C 28 H 28 Calculated for NO: 450.2 (M+H + ), Actual value: 450.2.

[0205] [C113] N-((6-Bromo-9-ethyl-9H-carbazol-3-yl)methyl)-1-methyl-1H-benzo[d]imidazol-2-amine (AJ2-81): Synthesized according to general procedure 1 and purified by biotage (hexane / ethyl acetate; 3:7) to afford AJ2-81 as a brown solid (85 mg, 76%). 1 H NMR (400 MHz, CDCl3) δ 8.21 (d, J = 1.9 Hz, 1H), 8.11 (dd, J = 1.7, 0.7 Hz, 1H), 7.63 - 7.54 (m, 3H), 7.42 (dd, J = 8.4, 0.6 Hz, 1H), 7.31 (d, J = 8.7 Hz, 1H), 7.22 - 7.14 (m, 1H), 7.14 - 7.09 (m, 2H), 4.92 (d, J = 5.0 Hz, 2H), 4.37 (q, J = 7.2 Hz, 3H), 3.51 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H).LCMS C 23 H 22 Calculated for BrN4: 433.0 (M+H + ), Actual value: 433.0.

[0206] [C114] N-((6-(benzo[d][1,3]dioxol-5-yl)-9-ethyl-9H-carbazol-3-yl)methyl)-1-methyl-1H-benzo[d]imidazol-2-amine (AJ2-82): Synthesized according to general scheme 2 and general procedure 1 and purified by biotage (hexane / ethyl acetate; 2:8) to afford AJ2-82 as a brown solid (13 mg, 52%). 1H NMR (400 MHz, CDCl3) δ 7.96 (d, J = 1.8 Hz, 1H), 7.88 (d, J = 1.6 Hz, 1H), 7.50 - 7.38 (m, 2H), 7.35 (dd, J = 8.4, 1.7 Hz, 1H), 7.18 - 7.10 (m, 3H), 7.06 (dt, J = 7.7, 1.2 Hz, 1H), 7.01 - 6.93 (m, 2H), 6.87 (d, J = 7.7 Hz, 1H), 6.84 - 6.77 (m, 1H), 5.22 (s, 2H), 4.77 (s, 3H), 4.09 (q, J = 6.9 Hz, 2H), 3.25 (s, 3H), 1.21 (s, 3H).LCMS C 30 H 27 Calculated value for N4O2: 475.2 (M+H + ), Actual value: 475.1.

[0207] [C115] 3-(9-Ethyl-6-(((1-methyl-1H-benzo[d]imidazol-2-yl)amino)methyl)-9H-carbazol-3-yl)phenol (AJ2-83): Synthesized according to general scheme 2 and general procedure 1 and purified by biotage (hexane / ethyl acetate; 3:7) to afford AJ2-83 as an off-white solid (35 mg, 62%). 1 H NMR (600 MHz, DMSO) δ 9.56 (s, 1H), 8.40 (s, 1H), 8.30 (s, 1H), 7.76 - 7.62 (m, 2H), 7.59 (s, 2H), 7.35 - 7.27 (m, 2H), 7.21 (dt, J = 22.1, 7.7 Hz, 4H), 6.96 (dt, J = 23.9, 7.9 Hz, 2H), 6.78 (d, J = 8.1 Hz, 1H), 4.93 - 4.71 (m, 2H), 4.61 - 4.29 (m, 2H), 3.59 (s, 3H), 1.32 (t, J = 7.4 Hz, 3H). 13C NMR (151 MHz, DMSO) δ 158.29, 155.72, 143.10, 142.97, 139.84, 139.70, 135.86, 131.73, 131.20, 130.31, 126.42, 125.08, 123.15, 122.64, 120.68, 120.03, 118.76, 118.70, 118.04, 115.41, 114.05, 113.94, 109.88, 109.48, 107.69, 46.81, 37.56, 28.76, 14.18. LCMS C 29 H 27 Calculated for NO: 447.2 (M+H + ), Actual value: 447.1.

[0208] [C116] 2-((9-Ethyl-2,3,4,9-tetrahydro-1H-carbazol-6-yl)methyl)isoindoline-1,3-dione (AJ2-83): Synthesized according to general procedure 4 and purified by biotage (hexane / ethyl acetate; 6:4) to afford AJ2-85 as a yellow solid (45 mg, 62%). 1 H NMR (400 MHz, CDCl3) δ 7.72 (dd, J = 5.5, 3.1 Hz, 2H), 7.56 (dd, J = 5.5, 3.0 Hz, 2H), 7.49 (d, J = 1.7 Hz, 1H), 7.19 (dd, J = 8.4, 1.7 Hz, 1H), 7.10 (dd, J = 8.4, 0.7 Hz, 1H), 4.85 (s, 2H), 3.93 (q, J = 7.2 Hz, 2H), 2.67 - 2.51 (m, 4H), 1.91 - 1.79 (m, 2H), 1.74 (dtd, J = 11.2, 5.9, 2.3 Hz, 2H), 1.18 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 168.23, 135.63, 135.17, 133.74, 132.35, 127.42, 126.76, 123.14, 121.66, 118.53, 109.48, 108.54, 42.35, 37.43, 23.26, 23.22, 22.06, 21.11, 15.47, 15.31.LCMS C 23 H 23 Calculated value for N2O2: 359.1 (M+H + ), Actual value: 359.1.

[0209] [C117] 3-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)-N-cyclopentyl-N-((9-ethyl-9H-carbazol-2-yl)methyl)propanamide (AJ2-86): Synthesized according to general procedure 2 and purified by biotage (hexanes / ethyl acetate; 6:4) to afford AJ2-86 as a colorless oil (14 mg, 62%). Note: Rotamers were observed. 1 H NMR (400 MHz, CDCl3) δ 8.13 (d, J = 7.9 Hz, 1H), 7.95 - 7.91 (m, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.48 - 7.38 (m, 3H), 7.32 - 7.19 (m, 3H), 5.03 - 4.95 (m, 1H), 4.76 (s, 1H), 4.71 - 4.65 (m, 2H), 4.42 - 4.34 (m, 3H), 2.30 (t, J = 7.5 Hz, 1H), 2.08 (t, J = 7.4 Hz, 3H), 1.99-188 (m, 4H), 1.90 - 1.79 (m, 4H), 1.69 (td, J = 8.0, 6.6, 3.3 Hz, 3H), 1.58 (q, J = 7.4, 6.6 Hz, 6H), 1.48 (d, J = 7.2 Hz, 3H). Note: Rotamers observed. LCMS C 28 H 33 Calculated for NO: 441.2 (M+H+ ), Actual value: 441.1.

[0210] [C118] N-((9-Ethyl-6-methoxy-9H-carbazol-3-yl)methyl)-1-methyl-1H-benzo[d]imidazol-2-amine (AJ2-87): Synthesized according to general procedure 1 and purified by biotage (hexane / ethyl acetate; 4:6) to afford AJ2-87 as a yellow solid (72 mg, 64%). 1 H NMR (400 MHz, CDCl3) δ 7.99 (d, J = 1.7 Hz, 1H), 7.46 (dd, J = 7.3, 1.6 Hz, 2H), 7.42 (dt, J = 8.3, 1.5 Hz, 1H), 7.25 (dd, J = 8.4, 1.5 Hz, 1H), 7.22 (d, J = 8.8 Hz, 1H), 7.09 - 7.01 (m, 2H), 7.01 - 6.94 (m, 2H), 4.78 (d, J = 4.1 Hz, 2H), 4.37 (s, 1H), 4.23 (qd, J = 7.2, 1.4 Hz, 2H), 3.83 (s, 3H), 3.34 (s, 3H), 1.31 (t, J = 7.2 Hz, 3H). 13 C NMR (101 MHz, CDCl3) δ 154.38, 153.65, 142.29, 140.02, 135.35, 135.02, 128.37, 126.24, 122.94, 122.90, 121.25, 120.35, 119.60, 116.50, 115.21, 109.33, 108.78, 107.05, 103.34, 56.16, 48.27, 37.72, 28.24, 13.90.LCMS C 24 H 25 Calculated for NO: 385.2 (M+H + ), Actual value: 384.1.

[0211] [C119] N-((5-Methoxy-1H-indol-3-yl)methyl)-1-methyl-1H-benzo[d]imidazol-2-amine (AJ2-88): Synthesized according to general procedure 1 and purified by biotage (hexane / ethyl acetate; 2:8) to afford AJ2-87 as a yellow solid (72 mg, 64%). 1 H NMR (400 MHz, CDCl3) δ 8.43 (s, 1H), 7.47 (dt, J = 7.8, 1.0 Hz, 1H), 7.20 - 7.16 (m, 1H), 7.14 (d, J = 2.5 Hz, 1H), 7.08 (dd, J = 2.5, 1.6 Hz, 1H), 7.08 - 7.03 (m, 1H), 7.02 - 6.98 (m, 2H), 6.80 (dd, J = 8.8, 2.5 Hz, 1H), 4.85 - 4.73 (m, 2H), 4.13 (s, 1H), 3.69 (s, 3H), 3.34 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 154.43, 154.35, 142.28, 134.98, 131.50, 127.19, 124.22, 121.28, 119.67, 116.43, 112.92, 112.86, 112.18, 107.09, 100.61, 55.88, 39.39, 28.24.LCMS C 18 H 19 Calculated for NO: 307.1 (M+H + ), Actual value: 307.1.

[0212] [C120] N-((9-Ethyl-6-(4-methylpentyl)-9H-carbazol-3-yl)methyl)-1-methyl-1H-benzo[d]imidazol-2-amine (AJ2-89): Synthesized according to general scheme 4 followed by general procedure 1 and purified by biotage (hexane / ethyl acetate; 4:6) to afford AJ2-89 as a yellow solid (68 mg, 72%). 1H NMR (600 MHz, CDCl3) δ 8.11 (s, 1H), 7.92 (s, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.51 (d, J = 8.3 Hz, 1H), 7.39 ‐ 7.32 (m, 3H), 7.20 (t, J = 7.6 Hz, 1H), 7.13 (d, J = 7.6 Hz, 1H), 7.06 (d, J = 7.8 Hz, 1H), 4.89 (d, J = 5.1 Hz, 2H), 4.79 (s, 1H), 4.34 (q, J = 7.2 Hz, 2H), 3.38 (s, 3H), 2.83 (t, J = 7.8 Hz, 2H), 1.80 ‐ 1.74 (m, 2H), 1.69 ‐ 1.64 (m, 1H), 1.44 (t, J = 7.2 Hz, 3H), 1.40 ‐ 1.30 (m, 2H), 1.01 ‐ 0.93 (m, 6H). 13 C NMR (151 MHz, CDCl3) δ 154.55, 142.38, 139.75, 138.83, 135.09, 133.65, 128.64, 126.68, 126.06, 123.00, 122.75, 121.21, 120.24, 119.90, 119.54, 116.39, 108.55, 108.32, 107.08, 48.24, 38.80, 37.63, 36.35, 30.36, 28.18, 28.05, 22.76, 13.90.LCMS C 29 H 35 Calculated value of N4: 439.2 (M+H + ), actual measured value: 439.2.

[0213]

Chemistry 121

[0214] [C122] N-((1H-indol-5-yl)methyl)-N-(2-(azepan-1-yl)-2-phenylethyl)-3-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)propanamide (AJ2-91): Synthesized according to general procedure 1 followed by general procedure 2 and purified by PTLC (hexane / ethyl acetate; 7:3) to afford AJ2-91 as a colorless oil (14 mg, 46%). 1 H NMR (400 MHz, CDCl3) δ 8.25 (s, 1H), 8.18 (s, 1H), 7.33 (d, J = 1.5 Hz, 1H), 7.29 - 7.16 (m, 10 H), 6.95 (dd, J = 8.4, 1.7 Hz, 1H), 6.78 (dd, J = 8.3, 1.7 Hz, 1H), 6.43 (q, J = 2.8 Hz, 2H), 4.40 - 4.30 (m, 2H), 4.08 (dd, J = 13.2, 6.9 Hz, 1H), 3.95 (t, J = 7.4 Hz, 1H), 3.89 - 3.85 (m, 1H), 3.75 ‐ 3.60 (m, 1H), 3.39 ‐ 3.26 (m, 1H), 2.68 ‐ 2.58 (m, 2H), 2.51 (qt, J = 9.9, 5.5 Hz, 4H), 2.00 (t, J = 7.6 Hz, 2H), 1.97 ‐ 1.85 (m, 5H), 1.74 (td, J = 7.9, 7.4, 2.8 Hz, 3H), 1.54 ‐ 1.50 (m, 4H). Note: Rotamers observed. LCMS C 31 H 38 Calculated for NO: 496.30 (M+H + ), Actual value: 496.1.

[0215] [C123] 1-(2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethyl)-N-((1-phenyl-1H-pyrazol-4-yl)methyl)-1H-benzo[d]imidazol-2-amine (AJ2-92): Synthesized according to general procedure 1 followed by general procedure 5 and purified by PTLC (hexane / ethyl acetate; 5:5) to afford AJ2-92 as a yellow sticky liquid (8 mg, 52%). 1H NMR (600 MHz, CDCl3) δ 7.95 (s, 1H), 7.69 (s, 1H), 7.60 - 7.55 (m, 2H), 7.46 (d, J = 7.9 Hz, 1H), 7.39 - 7.32 (m, 2H), 7.23 - 7.17 (m, 1H), 7.10 - 7.07 (m, 1H), 7.04 - 6.98 (m, 2H), 4.68 (s, 1H), 4.62 (s, 2H), 3.66 (t, J = 7.3 Hz, 2H), 1.86 - 1.77 (m, 5H), 1.36 (t, J = 7.0 Hz, 2H). 13 C NMR (151 MHz, CDCl3) δ 153.38, 142.28, 140.82, 139.99, 133.92, 129.47, 126.59, 126.37, 121.71, 120.88, 120.05, 119.10, 116.88, 107.31, 82.44, 69.65, 38.00, 36.82, 32.24, 26.68, 13.14.LCMS(ESI) C 24 H 24 Calculated value for N7: 410.2 (M+H + ), Actual value: 410.1.

[0216] [C124] 1-(2-((benzo[d][1,3]dioxol-5-ylmethyl)amino)-1H-benzo[d]imidazol-1-yl)butan-1-one (AJ2-CP53): Synthesized according to Scheme 1 and general procedure 4 and purified by PTLC (hexane / ethyl acetate 5:5) to afford AJ2-CP53 as an off-white solid (18 mg, 45%). 1H NMR (400 MHz, CDCl3) δ 8.11 (d, J = 5.8 Hz, 1H), 7.44 (dd, J = 8.2, 1.1 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.24 (dd, J = 7.8, 1.1 Hz, 1H), 7.07 (ddd, J = 8.6, 7.5, 1.3 Hz, 1H), 6.91 (d, J = 1.7 Hz, 1H), 6.89 - 6.84 (m, 1H), 6.78 (d, J = 7.9 Hz, 1H), 5.95 (s, 2H), 4.67 (d, J = 5.4 Hz, 2H), 3.00 (t, J = 7.2 Hz, 2H), 1.87 (h, J = 7.3 Hz, 2H), 1.10 (t, J = 7.4 Hz, 3H). 19 H 20 Calculated for N3O3: 338.1 (M+H + ), Actual value: 338.1.

[0217] Example 3. Fragment-based chemical proteomics approach to developing SLC inhibitors Chemical probes offer a valuable method for directly exploring the relationship between protein function and disease, complementing genetic approaches by providing reversible and gradual increases and decreases in protein activity, as well as novel functional outcomes in a variety of cases. Chemical probes are typically prepared by screening large chemical libraries (approximately 10 6 These compounds are discovered by high-throughput screening (HTS) of various types of compounds. Although HTS has had some success, this approach continues to face significant technical hurdles that limit its general utility. First, it is often performed with large libraries of structurally sophisticated compounds (molecular weight 400-600 Da), which can be difficult to optimize due to suboptimal ligand efficiency. 42Furthermore, the vast number of possible atomic combinations makes such large molecular weight compound libraries inefficient and incomplete for exploring the biologically relevant ("druggable") chemical space of the entire human proteome. 43、44 Fragment-based ligand discovery (FBLD) has the potential to overcome some of the limitations of traditional HTS by assaying relatively small libraries (approximately 1000) of low molecular weight compounds (<300 Da) for binding to protein targets. 42、43 FBLD imposes a low molecular weight restriction on compound screening, reducing the total number of possible atomic combinations by several orders of magnitude compared to the conventional molecular weight cutoff value (approximately 500 Da) used in HTS. 45 Thus, fragment screening allows exploring a large portion of the small molecule-protein interaction space with a much smaller and simpler compound library that is more ligand-efficient than HTS hits. 43 Fragment screening typically produces higher hit rates than HTS, but the low affinity of these hit compounds has so far limited FBLD to the study of purified protein targets where the ligand-protein interaction can be characterized by biophysical methods (e.g., NMR, X-ray).

[0218] To address these limitations, we developed a powerful approach, intracellular fragment-based ligand mapping (FbLMiC, Figure 1A), that integrates fragment-based ligand discovery and chemical proteomics to comprehensively survey ligandable proteins and their ligandable sites. 46、47In this approach, small-molecule probes, called fully functionalized fragment (FFF) probes, contain 1) a structurally minimal "constant" region with a photoactivatable diazirine group and alkyne handle, which together enable UV-induced covalent modification and detection, enrichment, and identification of protein targets conjugated with compounds; and 2) a "variable" recognition region consisting of structurally diverse small-molecule fragments (MW <300 Da) to facilitate interactions with subsets of the proteome (Figure 1B-C). The notable advantages of FbLMiCs are: 1) the ability to capture and identify probe-protein interactions from live cells, preserving labile interactions that may be disrupted by cell lysis; 2) the FFF probes interact at functional protein sites (e.g., active sites, allosteric sites, and protein-protein interaction sites); 3) covalent capture allows for efficient enrichment and identification of low-abundance and low-affinity proteins; and 4) FbLMiC-guided medicinal chemistry allows for optimization of fragments into higher-affinity ligands. The platform has outstanding proteomic coverage, including an unprecedented and ever-growing ligandability map of over 4000 human proteins, including proteins that fall outside traditional "druggable" categories (e.g., adaptor proteins, transcription factors), the majority of which currently lack chemical probes (Figure 1C).

[0219] Using our FbLMiC platform, we directly identified over 100 SLC-FFF interactions in human cell lines and primary immune cells. Furthermore, we demonstrated that these FFF-SLC interactions could serve as a starting point for the development of SLC inhibitors. 46 For example, we recently identified a coumarin-based FFF (FFF 3) that highly binds to the acylcaritin transporter SLC25A20. 46Using FbLMiC-assisted medicinal chemistry, we developed a first-in-class inhibitor (CP22) for SLC25A20 and used it to characterize SLC25A20 function in fatty acid metabolism. Using FFF3 and the structurally similar CP26 and CP22 inhibitors that do not bind to SLC25A20, we demonstrated that 1) pharmacological inhibitors bind to the intermembrane side of the SLC25A20 transport domain (Figure 2C), 2) blockade of SLC25A20 transport led to the accumulation of long-chain acylcarnitines (>C14) (Figure 2D), suggesting that these are major SLC25A20 substrates, and 3) inhibition of SLC25A20 transport inhibited fatty acid oxidation. As described in the next section, we used FbLMiC to develop a first-generation SLC25A4 inhibitor. Here, we propose to use these chemical probes to investigate the mechanisms by which SLC15A4 promotes cytokine production in immune cells and to evaluate its potential as a therapeutic target for autoimmune conditions such as lupus.

[0220] Previous studies have revealed that SLC15A4 plays a unique and important role in the production of IFN-I and other inflammatory cytokines in pDCs and the pathogenesis of autoimmune conditions, raising the possibility of SLC15A4 as a therapeutic target for such disorders. However, SLC15A4 has not yet been druggable, and no inhibitors have been disclosed. This application describes a feasible chemoproteomic strategy to develop first-in-class inhibitors, intracellular target binding capacity, and structurally similar but inactive control compounds that block SLC15A4 trafficking and suppress IFN-I production in human and mouse pDCs. Currently, our lead inhibitors are IC15A4-dependent in primary human pDCs. 50 At approximately 200 nM, it can suppress IFN-I production.

[0221] Chemoproteomic development of SLC15A4 chemical probes. To identify small molecule fragments that could serve as first-line candidates for SLC15A4 inhibitor development, we used FbLMiC to interrogate our previously published dataset.46、47 A small-scale (approximately 30) in-house library of FFFs in human peripheral blood mononuclear cells (PBMCs) (20 and 200 mM) was screened via multiplexed proteomics as previously described. 47Specifically (Figure 1A), freshly isolated PBMCs from healthy donor blood were treated with FFF for 30 min, exposed to UV irradiation to capture fragment-bound proteins, lysed, and conjugated to biotin-azide tags via copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC, or "click chemistry"); the fragment-labeled proteins were enriched on streptavidin-coated beads. The enriched proteins were trypsinized, and the resulting peptides were labeled with tandem mass tags (TMTs) for quantification. These peptides were analyzed by LC-MS / MS / MS to identify and quantify proteins by their MS1 / MS2 and MS3 signals, respectively. Target candidates were defined as proteins enriched (>5-fold) by the hit FFF over the control FFF (methyl fragment). We identified several fragments that significantly enriched endogenous SLC15A4 (Figure 3A). To prioritize potential lead compounds that inhibit SLC15A4 function, we investigated whether these compounds modulated IFN-I production in TLR-stimulated human pDCs. Human pDCs were isolated from PBMCs using a commercially available negative selection kit (Miltenyi) and treated with CpG-A DNA (4 μg / mL) for 1 hour. Subsequently, FFF (20 μM) was added and incubated overnight at 370°C. The next day, supernatants were collected and IFN-α levels were quantified by ELISA. FFF5 significantly suppressed IFN-I levels (Figure 4), whereas the other compounds only moderately suppressed them (not shown). Furthermore, we identified a structurally similar probe (FFF6) that neither bound to SLC15A4 nor suppressed IFN-I. We next synthesized "non-clickable" versions of FFF5 and FFF6 and confirmed the high-stoichiometric ligand-protein interaction by competition experiments using excess "non-clickable" competitors of FFF5+ / - in both fluorescent gel-based (Figure 3B) and MS-based (Figure 3C) experiments. We identified 14 proteins that were substantially enriched by FFF5 (20 μM) and competed (>4-fold) by treatment with excess 5-comp, some of which were also competed by the inactive control 6-comp.Among the most competitive targets was SLC15A4, which was not competed for by the inactive 6-comp, indicating that it is a highly stoichiometric target of FFF5 and 5-comp (Figure 2C-D). Furthermore, we observed no evidence that 5 interacts with SLC15A3 in any of our proteomic studies. We then demonstrated that FFF5 inhibited SLC15A3 in a dose-dependent manner (IC). 50 IFN-I production was suppressed to approximately 1 mM (Figure 4A), confirming that both FFF5 and 5-comp, but not the inactive analogue 6, could suppress inflammatory cytokine production in both human and mouse pDCs (isolates described in Aim 1) (Figures 4B-D).

[0222] Development of an SLC15A4-Transport Luciferase Reporter Assay. To assess whether FFF5 and related compounds inhibit SLC15A4 transport, we generated a reporter cell system based on SLC15A4 transport. The precise substrate range of endolysosomal SLC15A4 has not been established. However, several studies have suggested that SLC15A4 is a transporter of bacterial peptidoglycans, such as MDP and Tri-DAP, which are ligands for the immune sensors NOD1 and NOD2. 27-29、48 . Recently, SLC15A3 49 and SLC15A4 50 It has been shown that disruption of the dileucine motif (DE)-XXXL-(L / I) or DXXLL in NOD can result in successful targeting to the cell membrane. Given that NOD signals through the NFκB pathway, we sought to develop an NFκB reporter assay as a strategy to measure SLC15A4 trafficking in cells.

[0223] Specifically, both wild-type (WT) human SLC15A4 and dileucine mutants (L14A, L15A, L318A, and V319A) were cloned in-frame with mCherry using a (GGGGS)3 linker in the pLPC lentiviral backbone. The lentiviral vectors were packaged into psPAX2 and pMD2.G packaging plasmids and used to generate stable cell lines expressing either SLC15A4WT or membrane-translocated SLC15A4 mutants in A549 cells (Figure 5A). Subsequently, stable reporter cells were generated from these SLC15A4 cell lines using the Promega Dual-Luciferase Reporter System (pGLA4.32[luc2P / NFB-RE / Hygro]), which contains five copies of the NF-κB response element. We confirmed luciferase production upon exposure to MDP or Tri-DAP NOD ligands (Figure 6B-C), which was suppressed by exposure to triptolide (an NFκB inhibitor; Figure 5C), ML130 (a NOD1 inhibitor; not shown), or GSK717 (a NOD2 inhibitor; not shown). Furthermore, FFF5, but not FFF6, blocked MDP-induced luciferase, both of which indicated that FFF5, but not FFF6, blocked SLC15A4-mediated transport.

[0224] Preliminary Structure-Activity Relationship (SAR) Studies. Without structure or prior art, we set out to develop a robust and straightforward synthetic strategy that would provide access to a large number of diverse scaffolds in a few simple synthetic steps from readily available starting materials, enabling rapid synthetic exploration of chemicals that might enhance SLC15A4 inhibitory activity and, if necessary, improve PK properties for in vivo testing (Objective 2). To this end, we divided 5-comp into three major chemical domains: benzimidazole-purple, butanoyl-red, and benzodioxole / aryl-green (Figure 6A). In preliminary studies, we synthesized 18 analogs of 5-comp (7–24, Figure 6B) via straightforward synthetic routes. That is, readily available aryl aldehydes (green) can be coupled to benzimidazoles (Int-1A, upper pathway) or other aromatic-containing amines (Int-1B, lower pathway) using standard reductive amination conditions to give intermediates Int-A2 / Int-B2, which can then be used to diversify at the N1 position of the benzimidazole by treatment with a wide variety of acyl chlorides or coupling to acids (3) or alkylation reactions. We first tested the cytotoxicity of the analogs at 10 M in primary human pDCs and found no signs of significant toxic effects. We next examined the ability of 7-24 to 1) suppress IFN-I production in TLR7(CpG)-stimulated human pDCs; and 2) block SLC15A4-mediated MDP transport (Figure 7D). Nearly all substitutions at the benzimidazole ring (21–24) inhibited activity, whereas various substitutions at the butanoyl (red, 16–20) and benzodioxole (green, 7–15) positions were better tolerated. Specifically, we found that the 5-bromoindole analog 8 was the most potent in both assays, with an IFN-I inhibitory IC 50We observed a 190 nM (Figure 6E), a roughly 5-fold improvement over 5-comp. We also identified several analogs (13–17, 20–24) that showed no activity, providing valuable additional control compounds for functional studies. Notably, we observed a strong correlation of activity for each compound in both assays: analogs that suppressed IFN-I production also blocked MDP transport to a similar extent (Figure 6D). The concordance between the assays suggests a mechanism by which inhibitor interaction with SLC15A4 similarly affects both activities, thus consistent with the hypothesis that transport function is mechanistically linked to subsequent TLR signaling in pDCs.

[0225] Incorporation by Reference All US patents and US patent application publications cited herein are hereby incorporated by reference.

[0226] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein which equivalents are encompassed by the following claims.

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Claims

1. A compound of the formula: or a pharmaceutically acceptable salt thereof. 【Chemistry 1】

2. Claim 1 or a pharmaceutically acceptable salt of said compound, and a pharmaceutically acceptable carrier.

3. Claims for the treatment of pDC-mediated conditions. 2 The pharmaceutical composition described in

4. 10. The method of claim 9, wherein the pDC-mediated condition is lupus, Crohn's disease, irritable bowel syndrome (IBS), irritable bowel disease (IBD), psoriasis, dermatomyositis, Sjogren's syndrome, or type I interferon-driven interferonopathy. 3 The pharmaceutical composition described in

5. 10. The method of claim 1, wherein the pDC-mediated condition is multiple sclerosis (MS). 3 The pharmaceutical composition described in

6. Claims for the treatment of conditions mediated by B cells, macrophages or monocytes. 2 The pharmaceutical composition described in

Images