Compounds, compositions, and methods for suppressing toxic endoplasmic reticulum stress

Abstract

To provide compounds, compositions, and methods for suppressing toxic endoplasmic reticulum stress.SOLUTION: The present invention provides, inter alia, a compound having the following structure. There are also provided compositions containing a pharmaceutically acceptable carrier and one or more compounds according to the present invention. There are further provided methods for treating or ameliorating the effects of a disorder in a subject, methods of suppressing the toxicity of endoplasmic reticulum (ER) stress in a subject, methods of treating or ameliorating the effects of a disease involving axon degeneration in a subject, and methods for treating or ameliorating the effects of a neurodegenerative disease.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 637,242, filed March 1, 2018, which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION

[0002] The present invention relates, inter alia, to a structure [ka] Also provided are pharmaceutical compositions containing the compounds of the invention, and methods of using such compounds and compositions. Government financial support

[0003] This invention was made with government support under Grant No. XW81XWH-16-1-0204 awarded by the DOD. The U.S. Government has certain rights in this invention. [Background technology]

[0004] Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that targets spinal motor neurons, cells that control essential daily functions such as movement, breathing, and feeding. Like many other neurodegenerative disorders, ALS is characterized by the accumulation of misfolded proteins. Neurons initiate the unfolded protein response (UPR) to refold or eliminate misfolded proteins. If these attempts fail, the UPR transitions to the endoplasmic reticulum (ER) stress pathway, which drives cells to apoptotic cell death. Recent studies have implicated the UPR and ER stress in the pathogenesis of familial and sporadic ALS.

[0005] Markers of ER stress are some of the first pathological features detected in in vitro and in vivo models of ALS, including stem cell-derived motor neurons and mice carrying ALS-associated mutations. Persistent upregulation of ER stress markers was observed in transgenic hSOD1 mice. G93A This observation has been reported in spinal motor neurons derived from mouse embryonic stem (ES) cells (see Example 12). These mice overexpress mutant forms of human SOD1 causally associated with ALS, and their phenotype recapitulates many aspects of patient pathology, including neuromuscular denervation and progressive paralysis. ALS-causing mutations in SOD1 generally do not result in loss or gain of SOD1 function, but instead promote the accumulation of misfolded SOD1 species and the formation of insoluble proteinaceous aggregates in motor neurons, which likely underlie the increase in ER stress markers in these cells. Similar aggregates have been observed across ALS models with multiple disease-causing mutations, including TDP-43, FUS, OPTN, and UBQLN2 (Blokhuis et al., 2013). Furthermore, wild-type spinal motor neurons also Compared with other spinal neuron subtypes, spinal motor neurons were found to be highly sensitive to ER stress-inducing compounds such as cyclopiazonic acid (CPA) (Figure 2C). Collectively, these observations provide insight into why spinal motor neurons are selectively vulnerable to ALS while other neurons are spared.

[0006] Ca in the ER 2+ Prevents Ca uptake, thereby 2+ Based on the susceptibility of motor neurons to CPA, a sarco / ER calcium ATPase (SERCA) pump inhibitor that blocks the activity of dependent protein folding chaperones and rapidly induces ER stress (Figure 26), we screened a library of small molecule compounds for those that could reverse ER stress-induced neurodegeneration (Figure 27).

[0007] Among the hits from this screen of over 100 biologically active small molecules were broad-spectrum kinase inhibitors, including Go6976, sunitinib, K252a (Figure 2A), and kempauron (see Example 12). Preliminary analysis suggested the mitogen-activated protein (MAP) kinases MLK3 and HGK as potential functional targets of these rescue compounds. These findings were consistent with previous reports that the IRE1a branch of the ER stress pathway ultimately activates the JNK pathway, which is associated with many neurodegenerative disease models. Previous studies have also shown that CPA induces IRE1a activity and promotes the phosphorylation of c-jun, which is immediately downstream of JNK (see Example 12).

[0008] Four kinase inhibitor hits, as well as alsterpaullone, a structural analog of kenpaullone that was found to be more potent than kenpaullone, were compared to known targets by mining a publicly available database of the catalytic activity of 300 kinases (Anastassiadis et al., 2011) in the presence of 0.5 μM of various kinase inhibitors and performing a two-way hierarchical clustering analysis (Figure 1).

[0009] Kinases whose activity was potently inhibited by all five neuroprotective compounds (<15% of kinase activity remained at 1 μM inhibitor concentration) included the MAP kinase HGK (MAP4K4) and the MAPK-related kinase NUAK1. Ablation of NUAK1 protects cortical neurons in a mouse model of tauopathy, suggesting that NUAK1 may also modulate the neurotoxic effects of CPA in motor neurons (Lasagna-Reeves et al., 2016). However, the potent and selective NUAK1 inhibitor WZ4003 (IC for NUAK1) 50It was found that WZ4003 (IC = 20 nM) failed to provide rescue from CPA toxicity across a six-point dilution series (0.1-10 µM). At concentrations of 1 µM and above, WZ4003 potentiated CPA-induced neurodegeneration and was toxic to motor neurons even in the absence of CPA (Figure 3). Similarly, SU6656 (10.9% activity remaining at 0.5 µM) and HTH-01-051 (IC 50 Other selective NUAK1 inhibitors, including NUAK1 inhibitors (<100 nM, see Banerjee et al., 2014), not only failed to rescue CPA toxicity but also further enhanced its toxicity at higher doses (Figure 3). These results suggest that NUAK1 activity is essential for motor neuron survival under conditions of ER stress and possibly under basal conditions. That NUAK1 is a common target of hit rescue compounds is likely due to its structural similarity or sequence homology with the functional targets of these compounds.

[0010] On the other hand, Yang et al. (2013) reported that HGK is a hybrid of compounds that can rescue ES-derived spinal motor neurons from death induced by neurotrophic factor withdrawal. We reasoned that HGK may be the primary functional target of kempauron, which was identified as a hit in a high-throughput screen. Later, Larhammar et al. (2017) provided functional evidence that HGK, in combination with two other MAP4 kinases, TNIK and MINK1, mediates neurodegeneration after neurotrophic factor withdrawal in mouse dorsal root ganglion neurons. To further assess the role of HGK in motor neurons under ER stress, we tested the selective HGK inhibitor PF-6260933 (Ammirati et al., 2015) in a six-point dilution series (0.01 μM–5 μM) and found that HGK protected against CPA toxicity in a dose-dependent manner (Figure 3). The HGK inhibitor GNE-495 also provided neuroprotection (Figure 3). This finding provided preliminary support for the role of HGK in ER stress-induced neurodegeneration.

[0011] Another common target of K252a, Go6976, and alsterpaullone is the MAPK3 kinase MLK3. While kenpaullone and sunitinib only modestly inhibited MLK3 activity (49.3% and 68.4% of residual MLK3 activity at 0.5 μM, respectively; see Anastassiadis et al., 2011), the literature provides strong support for the role of MLK3 in many neurodegenerative conditions. Several additional compounds, including CEP1347, NU6140, bosutinib, JAK3 inhibitor IV, and a Syk inhibitor, which potently (<15% residual activity) but nonselectively inhibited MLK3 activity, were found to result in dose-dependent rescue from CPA toxicity in human ES motor neurons (Figure 3).

[0012] Based on these findings, we hypothesized that the protective effects of hit kinase inhibitors from the CPA survival screen were likely due to their inhibition of HGK and / or MLK3 activity. An ideal compound would 1) potently inhibit both HGK and MLK3, 2) completely reverse CPA-mediated neurotoxicity at low (nanomolar to micromolar) doses, 3) be soluble in aqueous vehicles, 4) be permeable to the blood-brain barrier and orally bioavailable, and 5) be amenable to further structural modifications to optimize its use in vivo. However, none of the hit compounds from the screen met these criteria. Neither Go6976 nor sunitinib were able to provide complete rescue from CPA toxicity at any of the doses tested. Meanwhile, K-252a is not a suitable lead compound due to both its broad kinase inhibition and its recalcitrance to analog synthesis. Further dampening enthusiasm for this scaffold was the observation that CEP-1347 (Figure 2A), a structurally related, nonspecific staurosporine-based kinase inhibitor, was ineffective as a neuroprotectant in clinical trials for Parkinson's disease. Kenpaullone, while highly selective, is insoluble at the concentrations we found effective in the in vitro CPA assay and is therefore unsuitable for in vivo use.

[0013] Through literature research, we identified the small molecule URMC-099 as a promising lead compound (compound 1) (Figure 2A). Compound 1 is orally bioavailable, penetrates the brain, and inhibits MLK3 (IC 50 Compound 1 potently binds to and inhibits both CPA (=14 nM) and HGK (0.54% residual enzyme activity upon 1 μM treatment), making it suitable for analog synthesis (Figure 28). The ability of compound 1 to rescue CPA toxicity in human ALS stem cell-derived motor neurons in a six-point dose-response assay (0.01 μM to 3 μM) was tested, and it was found that compound 1 exhibited a protective effect at a starting concentration of 100 nM, but completely reversed CPA-induced neurodegeneration at 1 μM (Figure 2B).

[0014] Next, it was determined whether the protective effect of compound 1 after CPA treatment was mediated by HGK and MLK3. Prolonged activation of the ER stress pathway activates the JNK kinase cascade, ultimately leading to apoptosis (see Sano et al., 2013). The MAP kinases JNK1-3 directly phosphorylate and activate c-jun, a transcription factor that promotes the expression of proapoptotic factors such as caspase 3. We have previously shown that CPA induces c-jun phosphorylation in ES MNs, which are particularly vulnerable to ER stress-mediated neurodegeneration, but not in resistant co-cultured ES-derived interneurons (see Example 12). Compound 1 was observed to potently inhibit both c-jun phosphorylation and caspase 3 cleavage in CPA-treated MNs (Figure 4), indicating that compound 1 prevents the induction of apoptosis. Next, we assessed the phosphorylation status of the MAPKs JNK, immediately upstream of c-jun, and MKK4, immediately upstream of JNK. Changes in MKK4 and JNK phosphorylation reflect the activity of upstream kinases, including HGK (MAP4K) and / or MLK3 (MAP3K), which are putative targets of compound 1. We observed that CPA treatment upregulated JNK and MKK4 phosphorylation, and addition of compound 1 strongly attenuated JNK and MKK4 phosphorylation (Fig. 4 ), further supporting the idea that compound 1 acts on HGK and / or MLK3 to prevent activation of downstream apoptotic pathways.

[0015] Although these early results were promising, compound 1 required further optimization before it could be considered a potential clinical candidate. Microsomal stability studies showed that compound 1 had a short half-life (T 1 / 2 = 7.8 min) and an in vivo half-life of approximately 2-3 hours in mice. Furthermore, compound 1 is nonselective, inhibits several kinases, and has a cellular potency of >100 nM; therefore, more selective and potent derivatives with improved PK properties are desirable and potentially obtainable. Summary of the Invention [Problem to be solved by the invention]

[0016] Thus, there is a need for the exploration of various analogs of Compound 1, as well as compositions and methods for inhibiting toxic endoplasmic reticulum (ER) stress. The present invention is directed to meeting these and other needs. [Means for solving the problem]

[0017] Without being bound by theory, we docked compound 1 into the crystal structure of HGK (PDB ID: 5DI1) using Glide (Figure 5A) to aid in the design of potential analogs. From the docking pose and the depiction of binding site interactions (see Figure 5B), we found that 1) the 7-azaindole moiety binds to the hinge region of the kinase and is essential for activity, 2) the piperazine moiety extends into the solvent-exposed region and can be modified to improve the physical properties of the molecule and enhance its stability, and 3) the side chain can be modified to acquire additional interactions in the binding pocket and increase kinase specificity.

[0018] Thus, one embodiment of the present invention is a compound according to formula (I) [ka] [In the formula, R1 is alkyl-heterocycloalkyl or alkyl-heterobicycloalkyl, where the heterocycloalkyl or heterobicycloalkyl is optionally substituted with one or more atoms or groups selected from alkyl, hydroxy, alcohol, or heterocycloalkyl; R2 is selected from phenyl, indolyl, pyridyl, dibenzofuranyl, benzodioxane, benzothiophenyl, pyrimidinyl, pyrazole, thienyl, indazole, or furanyl, any of which is optionally substituted with one or more atoms or groups selected from alkyl, alkoxy, amino, amido, cyano, ether, nitro, hydroxy, halogen, alkyl-heterocycloalkyl, sulfonyl, ketone, haloalkyl, haloalkoxy, alkylphenyl, carbamate, carboxylic acid, or urea; or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof; However, the compound is [ka] None of the above.

[0019] Another embodiment of the present invention is [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0020] Another embodiment of the present invention is [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0021] Another embodiment of the present invention is [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0022] Another embodiment of the present invention is [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0023] Another embodiment of the present invention is a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and one or more compounds according to formula (I). [ka] [In the formula, R1 is alkyl-heterocycloalkyl or alkyl-heterobicycloalkyl, where the heterocycloalkyl or heterobicycloalkyl is optionally substituted with one or more atoms or groups selected from alkyl, hydroxy, alcohol, or heterocycloalkyl; R2 is selected from phenyl, indolyl, pyridyl, dibenzofuranyl, benzodioxane, benzothiophenyl, pyrimidinyl, pyrazole, thienyl, indazole, or furanyl, any of which is optionally substituted with one or more atoms or groups selected from alkyl, alkoxy, amino, amido, cyano, ether, nitro, hydroxy, halogen, alkyl-heterocycloalkyl, sulfonyl, ketone, haloalkyl, haloalkoxy, alkylphenyl, carbamate, carboxylic acid, or urea, or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof; However, the compound is [ka] None of the above.

[0024] A further embodiment of the invention is a kit comprising a compound or a pharmaceutical composition according to the invention together with instructions for use of the compound or pharmaceutical composition, respectively.

[0025] Another embodiment of the present invention is a method for treating or ameliorating the effects of a disorder in a subject in need thereof. The method comprises administering to the subject an effective amount of one or more compounds having the structure of Formula (I). is multiple compounds [ka] [In the formula, R1 is alkyl-heterocycloalkyl or alkyl-heterobicycloalkyl, where the heterocycloalkyl or heterobicycloalkyl is optionally substituted with one or more atoms or groups selected from alkyl, hydroxy, alcohol, or heterocycloalkyl; R2 is selected from phenyl, indolyl, pyridyl, dibenzofuranyl, benzodioxane, benzothiophenyl, pyrimidinyl, pyrazole, thienyl, indazole, or furanyl, any of which is optionally substituted with one or more atoms or groups selected from alkyl, alkoxy, amino, amido, cyano, ether, nitro, hydroxy, halogen, alkyl-heterocycloalkyl, sulfonyl, ketone, haloalkyl, haloalkoxy, alkylphenyl, carbamate, carboxylic acid, or urea; or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof; However, the compound is [ka] None of the above.

[0026] A further embodiment of the present invention is a method for treating or ameliorating the effects of a disorder in a subject in need thereof, comprising administering to the subject a pharmaceutically acceptable carrier or diluent and one or more compounds having the structure of Formula (I): [ka] [In the formula, R1 is alkyl-heterocycloalkyl or alkyl-heterobicycloalkyl, where the heterocycloalkyl or heterobicycloalkyl is optionally substituted with one or more atoms or groups selected from alkyl, hydroxy, alcohol, or heterocycloalkyl; R2 is selected from phenyl, indolyl, pyridyl, dibenzofuranyl, benzodioxane, benzothiophenyl, pyrimidinyl, pyrazole, thienyl, indazole, or furanyl, any of which is optionally substituted with one or more atoms or groups selected from alkyl, alkoxy, amino, amido, cyano, ether, nitro, hydroxy, halogen, alkyl-heterocycloalkyl, sulfonyl, ketone, haloalkyl, haloalkoxy, alkylphenyl, carbamate, carboxylic acid, or urea; or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof; However, the compound is [ka] None of the above.

[0027] Another embodiment of the present invention is a method of inhibiting the toxicity of endoplasmic reticulum (ER) stress in a subject in need thereof, the method comprising administering to the subject one or more compounds having the structure of Formula (I): [ka] [In the formula, R1 is alkyl-heterocycloalkyl or alkyl-heterobicycloalkyl, where the heterocycloalkyl or heterobicycloalkyl is optionally substituted with one or more atoms or groups selected from alkyl, hydroxy, alcohol, or heterocycloalkyl; R2 is selected from phenyl, indolyl, pyridyl, dibenzofuranyl, benzodioxane, benzothiophenyl, pyrimidinyl, pyrazole, thienyl, indazole, or furanyl, any of which is optionally substituted with one or more atoms or groups selected from alkyl, alkoxy, amino, amido, cyano, ether, nitro, hydroxy, halogen, alkyl-heterocycloalkyl, sulfonyl, ketone, haloalkyl, haloalkoxy, alkylphenyl, carbamate, carboxylic acid, or urea; or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof; However, the compound is [ka] None of the above.

[0028] A further embodiment of the present invention is a method of treating or ameliorating the effects of a disease involving axonal degeneration in a subject in need thereof, the method comprising administering to the subject one or more compounds having the structure of Formula (I): [ka] [In the formula, R1 is alkyl-heterocycloalkyl or alkyl-heterobicycloalkyl, where the heterocycloalkyl or heterobicycloalkyl is optionally substituted with one or more atoms or groups selected from alkyl, hydroxy, alcohol, or heterocycloalkyl; R2 is selected from phenyl, indolyl, pyridyl, dibenzofuranyl, benzodioxane, benzothiophenyl, pyrimidinyl, pyrazole, thienyl, indazole, or furanyl, any of which is optionally substituted with one or more atoms or groups selected from alkyl, alkoxy, amino, amido, cyano, ether, nitro, hydroxy, halogen, alkyl-heterocycloalkyl, sulfonyl, ketone, haloalkyl, haloalkoxy, alkylphenyl, carbamate, carboxylic acid, or urea; or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof; However, the compound is [ka] None of the above.

[0029] A further embodiment of the present invention is a method for treating or ameliorating the effects of a neurodegenerative disease in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds having the structure of Formula (I): [ka] [In the formula, R1 is alkyl-heterocycloalkyl or alkyl-heterobicycloalkyl, where the heterocycloalkyl or heterobicycloalkyl is optionally substituted with one or more atoms or groups selected from alkyl, hydroxy, alcohol, or heterocycloalkyl; R2 is selected from phenyl, indolyl, pyridyl, dibenzofuranyl, benzodioxane, benzothiophenyl, pyrimidinyl, pyrazole, thienyl, indazole, or furanyl, any of which is optionally substituted with one or more atoms or groups selected from alkyl, alkoxy, amino, amido, cyano, ether, nitro, hydroxy, halogen, alkyl-heterocycloalkyl, sulfonyl, ketone, haloalkyl, haloalkoxy, alkylphenyl, carbamate, carboxylic acid, or urea; or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof; However, the compound is [ka] None of the above.

[0030] This application contains at least one photograph taken in color. Copies of this patent application together with the color photograph(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0031] [Figure 1] FIG. 1 shows the results of Spotfire analysis of 300 kinases to rank target kinases.

[0032] [Figure 2] Figure 2A shows the structures of CEP-1347, K-252a, and compound 1 (URMC-099).

[0033] FIG. 2B shows the rescue effect of Compound 1 after ER stress induced by treatment of human ALS iPS-MNs with 33 μM CPA.

[0034] FIG. 2C shows the structure of the CPA.

[0035] [Figure 3]Figure 3 shows the pharmacological inhibition of predicted targets of certain kinase inhibitors. Neither the selective NUAK1 inhibitors WZ4003 nor HTG-01-015 provided neuroprotection against CPA. In contrast, inhibition of HGK by the selective inhibitors PF626093 and GNE-495, and inhibition of MLK3 by the nonselective inhibitor bosutinib, were neuroprotective. These results suggest that MLK3 and HGK may be targets of Compound 1, but not NUAK1.

[0036] [Figure 4A] Figure 4A shows the expression of various putative downstream targets of Compound 1 in the context of ER stress. Mouse ES-induced motor neurons were treated with CPA, CPA + Compound 1, or vehicle control (DMSO) for 4 hours. Cells were then lysed and proteins were extracted. Protein extracts were analyzed by Western blot for the expression of activated forms of putative downstream targets of Compound 1, including phosphorylated MKK4 (a kinase immediately downstream of Compound 1 target MLK3), phosphorylated JNK (a kinase immediately downstream of MKK4), phosphorylated c-jun (a transcription factor immediately downstream of JNK), and cleaved caspase 3 (a widely recognized marker of apoptotic cell death). All were upregulated by CPA, indicating that CPA causes motor neuron degeneration via apoptosis driven by the JNK pathway. Meanwhile, addition of Compound 1 attenuated the activation of these pathway components, confirming that Compound 1 targets upstream kinases within this pathway.

[0037] [Figure 4B]Figure 4B shows immunostained mouse ES-induced motor neurons. ES-induced motor neurons were treated with CPA + / - Compound 1 in the same manner as shown in Figure 4A to determine whether modulation of Compound 1 target activity occurs in motor neurons or other contaminating cell types. The resulting images show that CPA treatment potently and selectively induces c-jun phosphorylation in motor neurons, whereas Compound 1 completely attenuates it. Motor neurons are indicated by Islet1-2 immunoreactivity.

[0038] [Figure 5] Figure 5A shows the docking pose of compound 1 in the HGK (MAP4K4) crystal structure (PDB ID: 5DI1).

[0039] Figure 5B shows the ligand interaction diagram for compound 1 with HGK corresponding to the docking pose (arrows indicate hydrogen bonds).

[0040] [Figure 6] Figure 6 shows that compound 1 analogs, such as 7aa, increase the protection of motor neurons against CPA. Human embryonic stem cell-derived motor neurons were treated with 33 μM CPA and increasing concentrations of compound 1 or its analogs. The number of surviving motor neurons was normalized to vehicle-treated controls.

[0041] [Figure 7] Figure 7A shows the correlation between % motor neuron viability and % MLK1 (R2 = 0.40) or % MLK3 (R2 = 0.52) activity. Human embryonic stem cell-derived motor neurons were treated with 33 μM CPA and 1.0 μM compound 1 or analogs 7a-7az. % MN viability was based on the amount of cells showing significant growth relative to the DMSO control. % MLK1 or MLK3 activity remaining is the mean % enzyme activity relative to the vehicle control with 0.1 μM compound treatment in the presence of 10 μM ATP (Reaction Biology Corp.).

[0042] Figure 7B shows the % motor neuron survival and HGK (R 2 = 0.84) shows the correlation of % activity. Human embryonic stem cell-derived motor neurons were treated with 33 μM CPA and 1.0 μM compound 1 or analogs 7a-7az. % MN viability was based on the amount of cells showing significant growth relative to vehicle control. % HGK activity remaining is the mean % enzyme activity relative to DMSO control with 0.025 μM compound treatment in the presence of 10 μM ATP (Reaction Biology Corp.).

[0043] [Figure 8] Figure 8 shows the results of inhibition of 60 selected kinases by Compound 1 and its analog, Compound 7ad. Despite providing similar levels of protection against CPA in the motor neuron survival assay, Compound 7ad inhibited the activity of fewer kinases than Compound 1, thereby highlighting the putative functional target of Compound 1 and its analogs in mediating cell survival.

[0044] [Figure 9] Figure 9 shows that two GCK inhibitors, compound 17 and NG25 (both from Tan et al., 2015), are unable to rescue motor neurons from CPA-induced ER stress.

[0045] [Figure 10-1] Figure 10A shows the generation of HGK knockout by CRISPR.Two guide RNAs are designed to target the genomic region surrounding the ATP-binding domain of HGK.CRISPR-mediated excitation of this region can lead to double-strand break repair by non-homologous end joining.

[0046] Figure 10B shows validation of HGK knockout clones by Western blot for HGK protein. No detectable HGK protein remained in the three knockout clones tested.

[0047] [Figure 10-2] Figure 10C shows motor neuron (MN) survival after CPA dose-response treatment in WT (blue) and HGK knockout (orange) mice. Motor neurons were purified by magnetic-activated cell sorting (MACS) and treated with various doses of CPA 24 hours after plating and then imaged 48 hours later.

[0048] Figure 10D shows that HGK knockout provides modest but statistically significant (p<0.0001, 2-way ANOVA) neuroprotection early (<8 h) after CPA exposure. MACS-purified motor neurons were treated with 10 μM CPA for 0–24 h and imaged at 24 h. The greatest difference in survival between genotypes occurred between 3–8 h of CPA exposure but disappeared at later time points.

[0049] [Figure 10-3] Figure 10E shows that HGK knockout modestly reduces MAPK pathway activation (p<0.01, two-way ANOVA). Expression of phosphorylated (activated) c-jun, a putative target downstream of compound 1, was monitored by FRET during CPA exposure (10 μM). c-jun was moderately but significantly less activated by CPA in HGK knockout compared with wild-type motor neurons.

[0050] [Figure 11-1] Figure 11 shows the results of a microsomal stability assay of compound 1 and its analogs (7r, 7v, 7w, 7y, 7aa, 7ad, 7ah, 7al, 7as, 7at, 12e, 12f, 12g, 12h, 12i, 12j, 12k, 12l). Their structures and half-lives in mouse liver microsomes are shown. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above. [Figure 11-5] Same as above. [Figure 11-6] Same as above. [Figure 11-7] Same as above. [Figure 11-8] Same as above. [Figure 11-9] Same as above. [Figure 11-10] Same as above.

[0051] [Figure 12] Figure 12 shows that the addition of 2-hydroxypropyl-β-cyclodextrin can increase the solubility of Compound 1 and its analogs. Compound 1 or DMSO (dilutions of Compound 1 and its analogs) was mixed with various vehicle compositions at concentrations corresponding to in vivo doses (2–10 mg / kg). Vehicle 1 (55% saline, 40% PEG-400, 5% DMSO) has previously been used for in vivo delivery of Compound 1 (Goodfellow VS et al., 2013). These mixtures were then read spectrophotometrically at 540 nM to assess optical density, a measure of mixture clarity and an indicator of precipitate formation. Vehicles 1 and 2 did not form precipitates in the presence of DMSO alone, but did form precipitates in the presence of Compound 1. At higher concentrations, these precipitates were visible to the eye. Addition of 11% 2-hydroxypropyl-β-cyclodextrin to the components of Vehicle 1 was sufficient to solubilize Compound 1 (Vehicle 3).

[0052] [Figure 13] Figure 13A shows the half-lives of compounds 1 and 12k in a liver microsomal stability assay. The half-life of optimized compound 12k (>120 min) is significantly longer than that of compound 1 (15 min), indicating that 12k has greater metabolic stability.

[0053] Figure 13B shows the concentrations (ng / mL) of Compound 1 and 12k in plasma and brain samples from mice intravenously (IV) injected with 10 mg / kg of either compound. Drug concentrations were analyzed by LC-MS. One hour after injection, the level of Compound 1 was below the detection limit of the LC-MS assay, while 12k was still detectable. Furthermore, the amount of 12k in the brain exceeded that in plasma, indicating that 12k is a highly permeable blood-brain barrier agent.

[0054] [Figure 14] Figure 14 shows the expression of phosphorylated (activated) c-jun in the olfactory bulb of wild-type mice after treatment with Compound 1 and its analogs 12h and 12k. 10 mg / kg of the compound was delivered intraperitoneally (IP), orally, or subcutaneously (SC). Animals were sacrificed 1 hour after treatment. Their olfactory bulbs were excised and lysed for Western blot analysis. Phospho-cjun expression was normalized to the housekeeping protein GAPDH. Compared to untreated animals, phospho-cjun levels were reduced by nearly all treatment conditions, indicating that Compound 1 and its analogs engage the same targets in vivo as they do in vitro.

[0055] [Figure 15-1] 15A-15K show the derivation and characterization of new transgenic mouse ESC lines used to design a two-color motor neuron toxicity screen. [Figure 15-2] Same as above. [Figure 15-3] Same as above. [Figure 15-4] Same as above. [Figure 15-5] Same as above. [Figure 15-6] Same as above. [Figure 15-7] Same as above.

[0056] FIG. 15A shows Hb9::RFP hSOD1 stained for motor neuron (MN) transcription factors Hb9 and Islet1 / 2. WTand Hb9::GFP hSOD1 G93A Confocal micrographs of embryoid bodies from .

[0057] Figure 15B shows overlap measurements between fluorescent reporter proteins (FPs) and transcription factors (TFs). Bars indicate the mean and error bars indicate the SEM.

[0058] FIG. 15C shows a schematic 96-well plate with a representative photomicrograph showing two-color culture.

[0059] Figure 15D shows the Trophos Plate Runner HD Representative whole-well images of live cells acquired in are shown.

[0060] FIG. 15E shows an enlarged view of FIG. 15D and the results of automated image analysis using Metamorph software.

[0061] FIG. 15F shows a "healthy cell criterion" that uses the presence of significant neurites to distinguish live cells from fluorescent debris.

[0062] Figure 15G shows MN viability in transgenic hSOD1 WT and G93A cell lines expressing various fluorescent reporters. Viability was assessed in moderate (G+F+I) and low (F+I) neurotrophic carriers in mixed red-green cultures (red WT#1 + green G93A#1, N=6) or individual cultures (green WT#1, green G93A#2, N=3). Bars indicate mean, error bars indicate SEM, ** p<0.01 *** p<0.001 is indicated.

[0063] Figure 15H shows the results of the small molecule screen at 48 hours post-exposure; dark blue data points represent normalized G93A / WT survival ratios relative to compound-exposed wells, while light blue and yellow data points represent G93A / WT survival ratios relative to controls. Circles indicate promising lead compounds confirmed after secondary screening, presented in Figure 15I.

[0064] FIG. 15I shows a list of promising lead compounds after secondary screening based on the results shown in FIG. 15H.

[0065] Figure 15J shows the results of the small molecule screen at 48 hours of exposure; red and green data points represent Hb9::RFP-hSDO1WT (red) and Hb9::GFP-hSDO1 exposed to the compounds. G93A (Green) indicates viability relative to cells, and light blue, yellow, blue, and magenta data points indicate viability relative to controls. Circles indicate promising lead compounds confirmed after secondary screening, presented in Figure 15K.

[0066] FIG. 15K shows a list of promising lead compounds after secondary screening based on the results shown in FIG. 15J.

[0067] [Figure 16-1] 16A-16J show the characterization of cyclopiazonic acid and its effects on motor neuron and interneuron survival, as well as calcium homeostasis. [Figure 16-2] Same as above. [Figure 16-3] Same as above. [Figure 16-4] Same as above. [Figure 16-5] Same as above. [Figure 16-6] Same as above.

[0068] Figure 16A shows Hb9::RFP hSOD1 WT and Hb9::GFP hSOD1 G93A 1 shows a dose-response curve for cyclopiazonic acid (CPA) indicating MN viability.

[0069] Figure 16B shows the normalized G93A / WT survival ratio. Bars indicate the mean, error bars indicate SEM, ** p<0.01 (N=10).

[0070] FIG. 16C shows a representative image from whole-well imaging and Hb9::RFP hSOD1WT and Hb9::GFP hSOD1 in the same well exposed to CPA. G93A A magnified view showing the MN is shown.

[0071] FIG. 16D shows Hb9::GFP hSOD1 stained for Tuj-1 (red) in control and CPA-exposed cells. G93A Micrographs of the cultures are shown.

[0072] FIG. 16E shows photomicrographs of mixed Hb9::GFP (green) / Ptf1α::td (Tomato Red) cultures exposed to ctrl and CPA.

[0073] Figures 16F and 16G show the effects of ctrl and CPA on MN, Tuj-1 + , and Ptf1α + Neuronal viability was quantified at 48 hours (N=3 for both analyses). Bars indicate mean, error bars indicate SEM, ** p<0.01 *** p<0.001 is indicated.

[0074] FIG. 16H shows a photomicrograph of unpurified Hb9::GFP MN cultures stained for SERCA2 (red), Tuj-1 (magenta), and DAPI (blue).

[0075] Figure 16I shows the cytoplasmic Ca ++ CPA-induced changes in purified hSOD1 WT and hSOD1 G93A Figure 1 shows that the NF-kappa B signal was assessed by calculating the 340 / 380 nm ratio in MN cultures (N=5 for WT, N=4 for G93A). Bars indicate the mean and error bars indicate SEM.

[0076] Figure 16J shows the slope of the decrease in Fura-2 ratio (Tau) measured in cultures exposed to kainic acid (KA) in the absence or presence of CPA. Bars indicate the mean, and error bars indicate the SEM.

[0077] [Figure 17A] 17A-17G show the characterization of ER stress markers in motor neuron cultures treated with cyclopiazonic acid. [Figure 17B] Same as above. [Figure 17C] Same as above. [Figure 17D] Same as above. [Figure 17E] Same as above. [Figure 17F] Same as above. [Figure 17G] Same as above.

[0078] Figure 17A shows a histogram showing that qPCR results specifically point to genes of interest early after CPA exposure. G93A Extracted from MN (N=3). Bars indicate mean, error bars indicate SEM.

[0079] Figure 17B shows histograms and inverted gel images showing XBP1 splicing in hSDO1G93A 967 MN at various time points after CPA exposure (N=3). Bars indicate the mean ratio s / u, and error bars indicate SEM.

[0080] FIG. 17C shows immunoblots of the expression of ER stress-related proteins and their loading controls at various time points after CPA exposure.

[0081] FIG. 17D shows hSDO1 treated with CPA. G93A Immunoblots of SOD1 expression and loading controls in cell lysates are shown. The middle lane shows panSOD1 expression. The bottom lane shows lysates immunoprecipitated using antibodies specific for misfolded hSOD1 species (C4F6 and B8H10 clones).

[0082] Figures 17E and 17F show phospho-c-jun expression in MN cultures (N=5) treated with ctrl and CPA. + Histograms and confocal micrographs of MNs are shown. Bars indicate mean, error bars indicate SEM. ** p<0.01 is indicated.

[0083] Figure 17G shows Ptf1α + Histograms of the differential effects of CPA exposure on ::tdTomato interneurons (INs) and Hb9::GFP motor neurons (MNs) are shown, with bars indicating the mean ratio MN / INs (N = 3 technical replicates) and error bars indicating SEM.

[0084] [Figure 18A] Figures 18A and 18B show selected results from the ER stress rescue screen, specifically those related to motor neuron viability and neurite outgrowth. Compounds are subcategorized and color-coded based on their putative mechanism of action. Bars indicate the mean, and error bars indicate SEM (N = 3-6 culture wells / compound). The cutoff level for further analysis is indicated by the gray line. [Figure 18B] Same as above.

[0085] [Figure 19A] 19A-19F show the characterization of rescue compounds, including their effects on c-jun phosphorylation in mouse motor neurons and viability in human motor neurons. [Figure 19B] Same as above. [Figure 19C] Same as above. [Figure 19D] Same as above. [Figure 19E] Same as above. [Figure 19F] Same as above.

[0086] Figure 19A shows dose-response curves for lead compounds from the rescue screen (GO6976 N=6 culture wells, K252-a N=2 independent cultures, Kenpaullon N=3 independent cultures, TUDCA N=2 independent cultures). Bars indicate mean, error bars indicate SEM.

[0087] Figures 19B and 19C show histochemical analyses of the effects of rescue compounds on phospho-c-jun expression in CPA-treated MN cultures 2 hours after exposure. The figures show grams and confocal micrographs. Bars indicate the mean, error bars indicate SEM, *** p<0.001 is indicated.

[0088] Figure 19D shows SOD1 + / + and isogenically engineered SOD1 + / A4V FACS-purified Hb9::GFP differentiated from hESC lines + Histograms are shown of the effect of CPA in the absence or presence of rescue compounds in human MN (N=9 for CPA vs. CTRL for both genotypes, N=3 for rescue compounds for both genotypes). Bars represent the mean, error bars represent SEM, * p<0.05, ** p<0.01, *** p<0.001 (The effect of CPA on SOD1 as indicated by the line) + / + and SOD1 + / A4V MN; the effects of rescue compounds were compared on CPA by genotype).

[0089] Figure 19E shows calcein + Representative cropped whole-well images of cells are shown.

[0090] Figure 19F is a schematic of the putative signaling pathways for CPA and targets for rescue compounds. Numbers in superscript indicate supporting references ( 1 Sakaki et al. 2008, 2 Yamada et al. 1998,3 Roux et al. 2002, 4 Yang et al. 2013, 5 Ozcan et al. 2006; 6 Uppala et al. 2017).

[0091] [Figure 20-1] 20A-20F show the characterization of the trophic action of TUDCA in vitro and its effects on muscle denervation in vivo in a mouse model of ALS. [Figure 20-2] Same as above. [Figure 20-3] Same as above. [Figure 20-4] Same as above. [Figure 20-5] Same as above.

[0092] Figures 20A and 20B show the effect of TUDCA on MN viability and neurite outgrowth compared to canonical neurotrophic factors (N=3 for CT-1, N=4 for other groups). Bars indicate mean, error bars indicate SEM, * p<0.05, ** p<0.01, *** p<0.001 is shown (compared to control).

[0093] Figure 20C shows Hb9::GFP + Representative cropped whole-well images of cells are shown.

[0094] Figure 20D shows hSOD1 G93A Schematic diagram of the experimental design for the in vivo study of TUDCA on muscle denervation in mice.

[0095] Figure 20E shows a scatter plot of NMJ innervation in the TA (N=4 for WT+TUDCA, N=6 for G93A+TUDCA, N=5 for G93A+vehicle). Data points represent individual animals, horizontal lines represent the mean, * p<0.05, ** p<0.01, ***p<0.001.

[0096] FIG. 20F shows hSOD1 treated with vehicle and TUDCA. G93A Confocal micrographs of TA NMJs (α-btx red, VAChT green) from a mouse are shown. Circles indicate denervated NMJs.

[0097] [Figure 21A] Figures 21A-21K show the evaluation of newly derived transgenic ES lines, biochemical analysis of normal and misfolded SOD1 expression and pEif2α levels in differentiated cells, and titration of neurotrophic carriers in motor neuron cultures. [Figure 21B] Same as above. [Figure 21C] Same as above. [Figure 21D] Same as above. [Figure 21E] Same as above. [Figure 21F] Same as above. [Figure 21G] Same as above. [Figure 21H] Same as above. [Figure 21I] Same as above. [Figure 21J] Same as above. [Figure 21K] Same as above.

[0098] FIG. 21A shows a light micrograph of a typical ES colony from a newly derived reporter line.

[0099] FIG. 21B shows the sequencing results confirming the presence of wild-type or mutated human SOD1.

[0100] Figures 21C, 21D, and 21E show light micrographs and histograms of reporter validation in differentiated new ES lines (two lines per reporter represent two separate rounds of differentiation, and results are presented as mean values / reporter). Bars indicate mean values, and error bars indicate SEM.

[0101] Figures 21F and 21G show Hb9::GFP hSOD1 WT or Hb9::GFP hSOD1 G93A Assessment of motor neuron viability and neurite outgrowth in individual cultures of cells (N=3 for both genotypes) is shown. Bars indicate mean and error bars indicate SEM.

[0102] Figure 21H shows a cropped image of an immunoblot displaying the expression of total and misfolded (C4F6) SOD1 in untreated motor neuron cultures. G93A P100 spinal cord from a mouse was used as a control. The dashed line indicates where the bottom figure was cropped.

[0103] FIG. 21I shows an immunoblot demonstrating the specificity of the C4F6 and B8H10 antibodies for misfolded mutant SOD1.

[0104] Figures 21J and 21K show histograms showing dose-response curves for motor neuron viability in cultures treated with combinations of forskolin (F, range: 100 µM to 0.78125 µM), IBMX (I, range: 1 mM to 7.8125 µM), and GDNF (G, range: 50 ng / mL to 0.128 pg / mL), with high-medium vehicle used as a control (NTF) (N = 6 culture wells / condition). Bars indicate mean, error bars indicate SEM.

[0105] [Figure 22A] 22A-22F show additional results from a primary toxicity screen and calcium imaging experiments in enriched motor neuron cultures treated with CPA. [Figure 22B] Same as above. [Figure 22C] Same as above. [Figure 22D] Same as above. [Figure 22E] Same as above. [Figure 22F] Same as above.

[0106] Figure 22A shows the results of the small molecule screen at 48 hours post-exposure; dark blue data points represent normalized G93A / WT survival ratios relative to compound-exposed wells, while light blue and yellow data points represent G93A / WT survival ratios relative to controls. Circles indicate promising lead compounds confirmed after secondary screening, which are presented in the table below.

[0107] Figure 22B shows the results of the small molecule screen at 48 hours of exposure; red and green data points represent Hb9::RFP-hSDO1 cells exposed to the compounds. WT (red) and Hb9::GFP-hSDO1 G93A (Green) indicates viability relative to the cells, and light blue, yellow, blue, and magenta data points indicate viability relative to the control. Circles indicate promising lead compounds identified after secondary screening, and these are presented in the table below.

[0108] Figures 22C and 22E show the Fura-2 signal ratio 340 / 380 in enriched motor neuron cultures subjected to a kainate pulse either before or after CPA exposure (N=5 for WT and N=4 for G93A). Data points represent the mean, and error bars represent the SEM.

[0109] FIG. 22D shows hSOD1 exposed to kainate. G93A Representative still images from a video of a motor neuron are shown. The image shows a Fura-2 signal ratio of 340 / 380, and the signals were displayed using the image lookup table function in FIJI (NIH). The arrow indicates Hb9::RFP. + Cells are shown.

[0110] Figure 22F shows Hb9::RFP-hSDO1 WT (red) and Hb9::RFP-hSDO1 WT 1 shows histograms of measurements of peak kainate and CPA-induced Fura-2 signal ratio 340 / 380 in .

[0111] [Figure 23-1] Figures 23A-23F show the assessment of CPA toxicity in two independent paired cell lines in mixed and FACS-sorted motor neuron cultures. [Figure 23-2] Same as above. [Figure 23-3] Same as above. [Figure 23-4] Same as above.

[0112] Figures 23A, 23B, 23C, and 23D show dose-response curves for survival ratios in cultures treated with CPA with two different combinations of reporter cell lines. Cultures were either unpurified or purified using FACS (Figure 23A: N=5, Figure 23B: N=4, Figure 23C: N=6 culture wells, Figure 23D: N=2). Bars indicate the mean, and error bars indicate SEM.

[0113] Figures 23E and 23F show scatter plots of representative gates for FACS purification of the reporter system.

[0114] [Figure 24A] 24A-24L show the generation of human isogenic cell lines to study aspects of ALS pathogenesis in vitro. [Figure 24B] Same as above. [Figure 24C] Same as above. [Figure 24D] Same as above. [Figure 24E] Same as above. [Figure 24F] Same as above. [Figure 24G] Same as above. [Figure 24H] Same as above. [Figure 24I] Same as above. [Figure 24J] Same as above. [Figure 24K] Same as above. [Figure 24L] Same as above.

[0115] FIG. 24A shows a diagram of the gene targeting strategy for introducing the SOD1A4V mutant allele into the SOD1 locus of HUES3 Hb9::GFP.

[0116] FIG. 24B shows the sequencing of the SOD1 locus.

[0117] FIG. 24C shows that PCR-RFLP analysis for the unique PshAI restriction site confirmed the correct target.

[0118] Figures 24D and 24E show that qRT-PCR and immunoblot assays for SOD1 expression in target stem cell lines demonstrated reduced levels of SOD1 transcript and protein levels.

[0119] Figure 24F shows a FACS plot showing a distinct population of differentiated cells expressing the GFP reporter. Differentiated neurons not exposed to the MN patterning molecules RA and SAG were used as a negative control to gate for green fluorescence.

[0120] Figure 24G shows Hb9::GFP + We show that the cells express the transcription factor Islet1, which confirms their spinal MN identity.

[0121] Figures 24H and 24I show that cell viability assays in mixed cultures showed increased SOD1 activity compared to isogenic controls. A4V Representative images of human nuclei and Tuj1 staining at days 3 and 30 show that isogenic human ES MNs expressing the variants show a trend towards reduced viability (P=0.09, n=4).

[0122] FIG. 24J shows short-term assessment (5 days post-dissociation) of MN viability and neurite outgrowth in a FACS-purified human isogenic line, assessed by calcein labeling in live cultures.

[0123] Figure 24K shows a histogram of neurite outgrowth in purified isogenic human motor neuron cultures. Cells were grown under control conditions, treated with CPA, or pretreated with rescue compound followed by treatment with CPA (N=9 for CPA vs. CTRL for both genotypes, N=3 for rescue compound for both genotypes). Bars represent mean, error bars represent SEM, * p<0.05, ** p<0.01, *** p<0.001 indicated (effect of CPA compared between WT vs. ALS MN as indicated by the lines; effect of rescue compounds compared to CPA by genotype).

[0124] Figure 24L shows purified human SOD1 + / A4V 1 shows histograms showing dose-response survival curves for CPA toxicity in ES MN. Bars indicate mean and error bars indicate SEM (N=3 culture wells / concentration).

[0125] [Figure 25A] Figures 25A-25H show c-jun phosphorylation and calcium homeostasis in CPA-treated mixed cultures of Hb9::GFP+ motor neurons and Ptf1a::tdTomato+ interneurons. Miniature screen for CPA rescue effects of conjugated bile acids, and survival and rescue effects of TUDCA in the presence of high and low neurotrophic carriers. [Figure 25B] Same as above. [Figure 25C] Same as above. [Figure 25D] Same as above. [Figure 25E] Same as above. [Figure 25F] Same as above. [Figure 25G] Same as above. [Figure 25H] Same as above.

[0126] Figure 25A shows Hb9::GFP +Confocal micrographs of pc-jun (blue) expression in motor neurons (green) and Ptf1::tdTomato interneurons (red) are shown.

[0127] Figure 25B shows a histogram (N=5 for WT, N=4 for G93A) showing the Fura-2 signal ratio 340 / 380 in motor neurons relative to other neurons in the culture. Bars indicate the mean, and error bars indicate the SEM.

[0128] Figures 25C and 25D show Hb9::GFP-hSDO1 cells treated with CPA. G93A Figure 1 shows a mini-screen of the rescue effects of taurine-conjugated bile acids in motor neurons (N=3-6 culture wells / compound). Data points represent mean values.

[0129] Figures 25E and 25F show CPA-treated Hb9::GFP-hSDO1 in a low neurotrophic carrier. G93A Histograms showing the rescue effects of TUDCA and canonical neurotrophic factors on viability and neurite outgrowth in developing motor neurons (N=3). Bars indicate the mean, and error bars indicate SEM.

[0130] Figure 25G shows Hb9::GFP-hSDO1 in the presence of a high neurotrophic carrier. G93A The effect of TUDCA on viability and neurite outgrowth in developing motor neurons (N=3) is shown. Bars indicate the mean, and error bars indicate the SEM.

[0131] Figure 25H shows CPA-treated Hb9::GFP-hSDO1 cells under high neurotrophic carrier conditions. G93A The effect of TUDCA on viability and neurite outgrowth in developing motor neurons (N=3) is shown. Bars indicate the mean, and error bars indicate the SEM.

[0132] [Figure 26]FIG. 26 shows that CPA prevents Ca 2+ uptake in the ER, thereby blocking the activity of Ca 2+ -dependent protein folding chaperones and rapidly inducing ER stress.

[0133] [Figure 27] FIG. 27 shows a schematic of the screening process to identify ALS-associated stress compounds that also rescue CPA toxicity.

[0134] [Figure 28] FIG. 28 shows potential modifications for synthesizing Compound 1 analogs.

[0135] [Figure 29] FIG. 29 shows a schematic of an in vitro assay for testing the metabolic stability of the synthesized analogs.

[0136] [Figure 30] FIG. 30 shows a schematic diagram of the evaluation process for the in vivo pharmacokinetics of the synthesized analogs.

[0137] [Figure 31A] Figure 31A shows the inhibition profile of Compound 1 and analogs, demonstrating potent inhibition of certain MAP4K kinases, including HGK, TNIK, and MINK1. Amino acid sequence alignments of the MAP4K4 catalytic domains of HGK, TNIK, and MINK1 are shown.

[0138] [Figure 31B] FIG. 31B is a schematic diagram of the possible mechanism of action for the neuroprotective effects of Compound 1 and its analogs in CPA-treated ES MNs.

[0139] [Figure 31C] FIG. 31C is a schematic diagram showing the procedure for attenuating the expression of all three kinases (HGK, TNIK, and MINK1).

[0140] [Figure 32] Figure 32 shows the ranked R-squared values ​​(Pearson's r) indicating the correlation between inhibition of a given kinase and motor neuron survival. Compound 1 analogs were compared for their ability to inhibit 60 different known targets of Compound 1 and promote motor neuron survival after CPA treatment. MAP4K (highlighted in green) is one of the highest ranked kinases. **P<0.01; *P<0.05.

[0141] [Figure 33] Figure 33 shows that CRISPR knockout of MAP4K is neuroprotective and has additive effects in motor neurons, demonstrating that they are functional targets of Compound 1 and its neuroprotective analogs.

[0142] [Figure 34] FIG. 34A shows the pharmacokinetic results of Compound 1 administered once at 10 mg / kg IP or OG and assessed over time in brain and plasma.

[0143] FIG. 34B shows the pharmacokinetic results of compound 12k administered once at 10 mg / kg IP or OG and assessed over time in brain and plasma.

[0144] [Figure 35A] Figure 35A shows the differences between Compound 1 and Compound 12k by comparing their pharmacokinetic results in the following settings: brain vs. plasma, concentrations calculated in [ng / mL or ng / g], administered once at 10 mg / kg IP or OG and assessed over time.

[0145] [Figure 35B] Figure 35B shows the differences between Compound 1 and Compound 12k by comparing their pharmacokinetic results in the following settings: brain vs. plasma, concentrations calculated in [nM], administered once at 10 mg / kg IP or OG and assessed over time.

[0146] [Figure 35C] FIG. 35C shows the estimates in [nM] compared to in vitro activity, where compounds are dosed once at 10 mg / kg IP and evaluated over time.

[0147] [Figure 36] FIG. 36 shows the brain / plasma ratio over time, where compounds are administered once at 10 mg / kg IP or OG and evaluated over time.

[0148] [Figure 37A] FIG. 37A shows the comparative results of Compound 1 versus Compound 12k delivered os in drinking water for 7 days and evaluated at the 7 day time point.

[0149] [Figure 37B] FIG. 37B shows the comparative results of Compound 1 versus Compound 12k delivered os in drinking water for 15 days and evaluated at the 15 day time point.

[0150] [Figure 38-1] 38A-38C show that compound 12k exhibits greater MAP4K inhibition than compound 1. [Figure 38-2] Same as above.

[0151] FIG. 38A shows the global kinase inhibition profile of Compound 1, separated by kinase family.

[0152] Figure 38B shows the global kinase inhibition profile of compound 12k, separated by kinase family.

[0153] Figure 38C shows the fold change in inhibition of a given kinase by compound 12k compared to compound 1. MAP4K (green) is one of the most enhanced targets of 12k.

[0154] [Figure 39]FIG. 39 shows that Compound 1 and its analogs suppress the release of the inflammatory cytokine TNFα by microglia.

[0155] [Figure 40] FIG. 40 shows that the functional targets of Compound 1 and analogs differ with respect to inflammation versus neurodegeneration.

[0156] [Figure 41A] Figures 41A-41C show that HGK inhibitors are neuroprotective and act via the JNK pathway. [Figure 41B] Same as above. [Figure 41C] Same as above.

[0157] Figure 41A shows the results of two-way hierarchical clustering of neuroprotective kinase targets hits from the previous viability screen, showing that HGK and NUAK1 are shared targets of these compounds. MAP4 kinase is underlined.

[0158] Figure 41B shows the maximum efficacy of hits from Figure 41A (gray) compared to more specific HGK and NUAK1 inhibitors across the dilution series. Bars represent the mean ± SEM of wild-type motor neurons treated with CPA (33 μM) + test compound normalized to vehicle control. n = 3 replicate wells from a 96-well plate. The dashed line represents the mean viability of cells treated with CPA alone.

[0159] Figure 41C shows Western blot results of mixed motor neuron cultures treated with vehicle, CPA, or CPA + Compound 1 (0.5 μM). Samples were collected 4 hours after treatment. N = samples from three separate motor neuron differentiations that were treated and lysed separately. Cells from each differentiation were divided into three samples, each with one treatment condition. All samples were run simultaneously on the same blot. All band intensities were normalized to GAPDH to control for loading, and then DMSO and CPA + Compound 1 values ​​were normalized to the CPA value for each n. One-way ANOVA was used to assess the overall effect of treatment on JNK phosphorylation (P = 0.0009; F = 28.33; R = 0.9042) and c-Jun phosphorylation (P < 0.0001; F = 101.9; R = 0.9714). Data represent mean CPA + Compound 1 values, log2 fold change relative to CPA alone ± SEM. Bonferroni's multiple comparison test was used to compare CPA vs. CPA + Compound 1 conditions for phospho-JNK and phospho-c-Jun; ** P < 0.01; *** P<0.001.

[0160] [Figure 42A] Figures 42A-42C show the in vitro and in vivo characterization of Compound 1 analogs. [Figure 42B] Same as above. [Figure 42C] Same as above.

[0161] Figure 42A provides a summary of the improvements in potency (red) and efficacy (blue) across the initially generated 49 Compound 1 analogs.

[0162] FIG. 42B shows the SOD1 activity of SOD1 cells treated with CPA (33 μM) and compounds 1, 12f, and 12k across a dilution series (log μM). A4VFigure 1 shows viability curves for mutant ALS motor neurons. Compound 12f shows improved potency and efficacy compared to 1, but becomes rapidly toxic above 0.5 μM. Compound 12k shows improved potency and efficacy compared to 1, and is less toxic than 12f at higher doses. Data are mean ± SEM of test compound-treated motor neurons normalized to CPA (33 μM) + vehicle control. n = 3 replicate wells from a 96-well plate for each test compound dose.

[0163] Figure 42C shows SOD1 cells treated with vehicle (DMSO) alone (top left); CPA (33 μM) alone (top right); CPA + Compound 1 (0.5 μM) (bottom left); or CPA + Compound 12k (0.1 μM) (bottom right). A4V Representative fields from whole-well images of motor neurons are shown.

[0164] [Figure 43A] Figures 43A-43B show full dose-response curves for specific NUAK1 and HGK inhibitors. Data are means of wild-type motor neurons treated with test compound ± SEM normalized to CPA + vehicle. n = 3 replicate wells from a 96-well plate. [Figure 43B] Same as above.

[0165] Figure 43A shows eight-point dilution curves for the NUAK1 inhibitors HTH-01-015 and WZ4003. The mean without CPA is included to show the intrinsic toxicity of the compounds.

[0166] Figure 43B shows eight-point dilution curves for the HGK inhibitors URMC-099 (compound 1), PF-6260933, and GNE-495.

[0167] [Figure 44]Figure 44 shows that compound 12k maintains its ability to suppress microglial cytokine release. Microglial N9 cells were treated with increasing concentrations of LPS plus compound 1, 12k, or 12e. TNFα release was measured by quantitative ELISA from cell culture medium. Compound 1 showed significant inhibition of TNFα release at 0.1 μM across all concentrations of LPS. Compound 12k was approximately 5-fold less potent than compound 1 in this assay, yet still successfully suppressed TNFα release. Compound 12e, which potently inhibits HGK but has little activity at MLK3, did not inhibit TNFα release at 0.5 μM except at the highest concentration of LPS, suggesting that inhibition of TNFα release may not be related to HGK inhibition but may instead be related to inhibition of other kinases, such as MLK3. Data are mean ± SEM. n = 2 replicate wells of a 96-well plate. The overall effects of Compound 1 analogs versus LPS concentrations were compared by two-way ANOVA (significant effects of Compound 1 analogs, LPS concentrations, and interactions, all P<0.0001). The effects of individual Compound 1 analogs at various LPS concentrations relative to LPS alone (vehicle, gray bars) were assessed by Bonferroni's multiple comparison test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0168] [Figure 45] Figure 45 shows markers of target association for compound 12k.

[0169] [Figure 46A] FIG. 46A shows the weight change in mice given compound 12k versus controls.

[0170] [Figure 46B] FIG. 46B shows the change in grip strength in mice given compound 12k versus controls. DETAILED DESCRIPTION OF THE INVENTION

[0171] The present invention provides new analogs of compound 1. Some of the analogs have improved microsomal stability and solubility, while maintaining good potency in inhibiting toxic ER stress. Thus, one embodiment of the present invention provides compounds according to formula (I): [ka] [In the formula, R1 is alkyl-heterocycloalkyl or alkyl-heterobicycloalkyl, where the heterocycloalkyl or heterobicycloalkyl is optionally substituted with one or more atoms or groups selected from alkyl, hydroxy, alcohol, or heterocycloalkyl; R2 is selected from phenyl, indolyl, pyridyl, dibenzofuranyl, benzodioxane, benzothiophenyl, pyrimidinyl, pyrazole, thienyl, indazole, or furanyl, any of which is optionally substituted with one or more atoms or groups selected from alkyl, alkoxy, amino, amido, cyano, ether, nitro, hydroxy, halogen, alkyl-heterocycloalkyl, sulfonyl, ketone, haloalkyl, haloalkoxy, alkylphenyl, carbamate, carboxylic acid, or urea; or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof; However, the compound is [ka] None of the above.

[0172] In one aspect of this embodiment, the compound has the structure of formula (II) [ka] [In the formula, R 1a and R 1b together with the nitrogen atom to which they are attached, form C 5~6 Form a heterocycloalkyl, where C 5~6heterocycloalkyl is optionally substituted with one or more atoms or groups selected from alkyl, hydroxy, alcohol, or heterocycloalkyl; R2 is selected from phenyl, indolyl, pyridyl, dibenzofuranyl, benzodioxane, benzothiophenyl, pyrimidinyl, pyrazole, thienyl, indazole, or furanyl, any of which is optionally substituted with one or more atoms or groups selected from alkyl, alkoxy, amino, amido, cyano, ether, nitro, hydroxy, halogen, alkyl-heterocycloalkyl, sulfonyl, ketone, haloalkyl, haloalkoxy, alkylphenyl, carbamate, carboxylic acid, or urea; or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof, However, the compound is [ka] None of the above.

[0173] In another aspect of this embodiment, the compound is [ka] [ka] [ka] [ka] [ka] and combinations thereof, or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0174] Preferably, the compound is [ka] and combinations thereof, or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0175] More preferably, the compound is [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0176] More preferably, the compound is [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0177] More preferably, the compound is [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0178] More preferably, the compound is [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0179] Another embodiment of the present invention is a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and one or more compounds according to formula (I). [ka] [In the formula, R1 is alkyl-heterocycloalkyl or alkyl-heterobicycloalkyl, where the heterocycloalkyl or heterobicycloalkyl is optionally substituted with one or more atoms or groups selected from alkyl, hydroxy, alcohol, or heterocycloalkyl; R2 is selected from phenyl, indolyl, pyridyl, dibenzofuranyl, benzodioxane, benzothiophenyl, pyrimidinyl, pyrazole, thienyl, indazole, or furanyl, any of which is optionally substituted with one or more atoms or groups selected from alkyl, alkoxy, amino, amido, cyano, ether, nitro, hydroxy, halogen, alkyl-heterocycloalkyl, sulfonyl, ketone, haloalkyl, haloalkoxy, alkylphenyl, carbamate, carboxylic acid, or urea; or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof, However, the compound is [ka] None of the above.

[0180] Suitable preferred compounds for use in the pharmaceutical compositions of the present invention are disclosed above in Formulas (I)-(II), including the specific compounds identified above.

[0181] A further embodiment of the present invention is a kit comprising a compound or pharmaceutical composition disclosed herein together with instructions for use of the compound or pharmaceutical composition, respectively.

[0182] The kits can also include suitable storage containers, e.g., ampoules, vials, tubes, etc., for each compound of the invention (which may be, e.g., in the form of a pharmaceutical composition) and other reagents, e.g., buffers, balanced salt solutions, etc., for use in administering the active agent to a subject. The compounds and / or pharmaceutical compositions of the invention, and other reagents, can be presented in the kits in any convenient form, e.g., in solution or powder form. The kits can further include a packaging container, optionally having one or more portions for housing the compounds and / or pharmaceutical compositions, and other optional reagents.

[0183] Another embodiment of the present invention is a method for treating or ameliorating the effects of a disorder in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds having the structure of Formula (I): [ka] [In the formula, R1 is alkyl-heterocycloalkyl or alkyl-heterobicycloalkyl. wherein the heterocycloalkyl or heterobicycloalkyl is optionally substituted with one or more atoms or groups selected from alkyl, hydroxy, alcohol, or heterocycloalkyl; R2 is selected from phenyl, indolyl, pyridyl, dibenzofuranyl, benzodioxane, benzothiophenyl, pyrimidinyl, pyrazole, thienyl, indazole, or furanyl, any of which is optionally substituted with one or more atoms or groups selected from alkyl, alkoxy, amino, amido, cyano, ether, nitro, hydroxy, halogen, alkyl-heterocycloalkyl, sulfonyl, ketone, haloalkyl, haloalkoxy, alkylphenyl, carbamate, carboxylic acid, or urea; or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof; However, the compound is [ka] None of the above.

[0184] As used herein, the terms "treat," "treating," "treatment," and grammatical variations thereof refer to subjecting an individual subject, e.g., a patient, to a protocol, regimen, process, or therapy that is desired to produce a physiological response or outcome.In particular, the methods and compositions of the present invention can be used to slow the onset of disease symptoms, or delay the onset of a disease or condition, or stop the progression of disease development.However, treating does not require that the desired physiological response or outcome be achieved in every subject or subject group, e.g., a patient population, because each treated subject may not respond to a particular treatment protocol, regimen, process, or therapy.Therefore, a given subject or subject group, e.g., a patient population, may not respond to treatment or may respond inappropriately.

[0185] As used herein, the terms "ameliorate," "ameliorating," and grammatical variations thereof mean to reduce the severity of symptoms of a disease in a subject.

[0186] As used herein, the term "administering" means oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal or subcutaneous administration, or implantation of a sustained release device, such as a mini-osmotic pump, to a subject. Administration can also be parenteral, and Administration can be by any route, including transmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal). Parenteral administration can include, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other delivery methods include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

[0187] As used herein, a "subject" is a mammal, preferably a human. In addition to humans, classes of mammals within the scope of the present invention include, for example, agricultural animals, veterinary animals, laboratory animals, etc. Some examples of agricultural animals include cows, pigs, horses, goats, etc. Some examples of veterinary animals include dogs, cats, etc. Some examples of laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc.

[0188] Preferred compounds and pharmaceutical compositions suitable for use in this method are as disclosed in Formulas (I)-(II) above, including the specific compounds identified above.

[0189] In certain embodiments, the disorder is a disease involving endoplasmic reticulum (ER) stress.

[0190] In certain embodiments, the disorder is a disease characterized by abnormal kinase levels in a subject. Non-limiting examples of diseases characterized by abnormal kinase levels in a subject according to the present invention include chronic myeloid leukemia (CML), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), acute promyelocytic leukemia, acute megakaryoblastic leukemia, childhood leukemia, familial chronic lymphocytic leukemia, left-right axis dysplasia, malignant melanoma, head and neck cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, liver cancer, testicular cancer, stomach cancer, gastrointestinal cancer, glioma, thyroid cancer, and ovarian cancer. Cancer, endometrial cancer, colon cancer, colorectal cancer, large cell lymphoma, soft tissue sarcoma, inflammatory myofibroblastic tumor, hereditary hemorrhagic telangiectasia type 2 (Osler-Rendu-Weber syndrome 2), intestinal bleeding, arterial hypertension, arteriovenous malformation, fibrodysplasia ossificans progressiva, skeletal malformation, extraskeletal bone formation, addiction, hypertension, myocardial infarction, distal intermedius (acromesomelic) dysplasia, ataxia, telangiectasia, Seckel syndrome, heart failure, juvenile polyposis syndrome, Brachydactyly type A2 (hand malformation), distal intermedius chondrodysplasia (bone malformation), vulvar anomalies, primary pulmonary hypertension (PPH1), cardio-facial-cutaneous syndrome, juvenile midline carcinoma, X-linked agammaglobulinemia, cardiac arrhythmias, somatic melanoma, familial melanoma, sarcoma, head and neck squamous cell carcinoma, epithelial tumors, cardiomegaly, Rett syndrome, X-linked infantile spasms syndrome, Li-Fraumeni syndrome, circadian disorders, ductal carcinoma, mammary hyperplasia, cone-rod dystrophy (CORD) ) type 5, cone-rod dystrophy (CORD) type 6, Leber congenital amaurosis type 1 (LCA1), epilepsy, myotonia, muscle wasting, cataracts, hypogonadism, endocrine deficiency, male pattern baldness, Down syndrome (DS), glioblastoma, hepatocellular carcinoma, Pfeiffer syndrome, Kallmann syndrome 2, stem cell leukemia-lymphoma syndrome (SCLL), myeloproliferative disorders, Apert syndrome, Jackson-Weiss syndrome, Crouzon syndrome, Beare-Stevenson gyrus cutisgyrata syndrome, achondroplasia, hypochrondroplasia, thanatophoric dysplasia, Adelaide craniosynostosis, San Diego dysplasia, Muenke syndrome, pituitary adenoma, myelodysplasia, infantile capillary hemangioma, idiopathic myelofibrosis, neuroblastoma, kidney cancer, papillary carcinoma, hyper-IgM syndrome, incontinentia pigmenti, anhidrotic ectodermal dysplasia, rheumatoid arthritis, acanthosis nigricans, pineal hyperplasia, polycystic ovary syndrome, atypical migraine, diabetic hyperlipidemia, elfinism, bacteria-induced macrophage apoptosis, febrile bacterial infection, uterine leiomyosarcoma, post-transplant lymphoproliferative disorder, myeloproliferative disorder (MPD), polycythermia vera vera), brain tumor, gastrointestinal stromal tumor (GIST), mastocytosis, varicella, T-cell leukemia, Williams-Beuren syndrome, Peutz-Jeghers syndrome, systemic lupus erythematosus, autosomal dominant thrombocytopenia, retinitis pigmentosa, hereditary papillary renal carcinoma, Müllerian duct syndrome type II, familial hypertrophic cardiomyopathy, myasthenia gravis, progressive hearing loss, polycystic kidney disease, Ewing's tumor, non-syndromic spermatogenesis Mental retardation type 30 (MRX30), idiopathic hypereosinophilic syndrome, spina bifida, Wolcott-Rallison syndrome (WRS), hepatic glycogen storage disease, liver cirrhosis, hematopoietic malignancies, Carney complex tumors, cardiac contractility, diabetic nephropathy, diabetic retinopathy, diabetic vascular complications, autism, dominant spinocerebellar ataxia type 14, pain sensation, osteoarthritis of cartilage, bladder cancer, nasopharyngeal carcinoma, undifferentiated large cell leukemia, familial medullary thyroid carcinoma (FMTC), multiple endocrine neoplasia (multiple These include Myeloacanthosis nigricans (MEN2A), Myeloacanthosis nigricans (MEN2B), pheochromocytoma, papillary thyroid carcinoma, Hirschsprung's disease, Oguchi's type 2 disease, HPC1, blood clots, angina pectoris, renal oncocytoma, pulmonary adenoma, dominant brachydactyly type B, recessive Robinow syndrome (RRS), Coffin-Lowry syndrome, CNS tumors, Loeys-Dietz syndrome, esophageal cancer, hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome), Marfan syndrome type II, venous dysplasia, astrocytoma, hypertrophic and dilated cardiomyopathy, tibial muscular dystrophy, anhidrosis, pseudohypoaldosteronism type II, and chronic arthritis.

[0191] In one aspect of this embodiment, the disorder may include, but is not limited to, traumatic brain injury, stroke, ischemia, bipolar disorder, heart disease, atherosclerosis, type 1 diabetes, type 2 diabetes, obesity, cancer, autoimmune disease, and neurodegenerative disease.

[0192] In one aspect of this embodiment, the disorder is a neurodegenerative disease. Non-limiting examples of neurodegenerative diseases according to the present invention include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Friedreich's ataxia, multiple sclerosis, Huntington's disease, transmissible spongiform encephalopathies, Charcot-Marie-Tooth disease, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy, chronic traumatic encephalopathy (CTE), polyglutamine diseases, prion diseases, glaucoma, and hereditary spastic paraplegia.

[0193] In certain embodiments, the disorder is amyotrophic lateral sclerosis (ALS).

[0194] In another aspect of this embodiment, the method includes administering to a subject one or more compounds or pharmaceutical compositions of the present invention together with an effective amount of one or more additional therapeutic agents, such as 5-hydroxytryptophan, Activase, AFQ056 (Novartis Corp., New York, NY), Aggrastat, Albendazole, alpha-lipoic acid / L-acetylcarnitine, Alteplase, Amantadine (Symmetrel), Amlodipine, Ancrod, Apomorphine (Apokyn), Arimoclomol, Ali Arixtra, Armodafinil, Ascorbic Acid, Ascriptin, Aspirin, Atenolol, Avonex, Baclofen (Lioresal), Banzel, Benztropine (Cogentin), Betaseron, BGG492 (Novartis) Corp., New York, NY), botulinum toxin, Bufferin, Carbatrol®, carbidopa / levodopa immediate release (Sinemet), carbidopa / levodopa orally disintegrating tablets (Parcopa), carbidopa / levodopa / entacapone (Stalevo), CERE-110: adeno-associated viral delivery of NGF (Ceregene, San Diego, CA), cerebrolysin, CinnoVex, citalopram, citicoline, clobazam, clonazepam, clopidogrel, clozapine (Clozaril), coenzyme Q, creatine, dabigatran, dalteparin, dapsone, dab Davunetide, Deferiprone, Depakene®, Depakote ER®, Depakote®, Desmoteplase, Diastat, Diazepam, Digoxin, Dilantin®, Dimebon, Dipyridamole, Divalproex (Depakote), Donepezil (Aricept), EGb 761, Eldepryl, ELND002 (Elan Pharmaceuticals, Dublin, Ireland, Enalapril, Enoxaparin, Entacapone (Comtan), Epoetin Alfa, Eptifibatide, Erythropoietin, Escitalopram, Eslicarbazepine Acetate, Esmolol, Ethosuximide, Ethyl-EPA (Miraxion™), Exenatide, Extavia, Ezogabine, Felbamate, Felbatol® , Fingolimod (Gilenya), Fluoxetine (Prozac), Fondaparinux, Fragmin, Frisium, Gabapentin, Gabitril®, Galantamine, Glatiramer (Copaxone), Haloperidol (Haldol), Heparin, Human Chorionic Gonadotropin (hCG), Idebenone, Inovelon®, Insulin, Interferon beta 1a, Interferon beta 1b, Iofulpane 123I (DATSCAN®), IPX066 (ImpaxLaboratories Inc., Hayward, CA), JNJ-26489112 (Johnson and Johnson, New Brunswick, NJ), Keppra®, Klonopin, Lacosamide, L-alphaglycerylphosphorylcholine, Lamictal®, Lamotrigine, Levetiracetam, Liraglutide, Lisinopril, Lithium Carbonate, Lopressor, Lorazepam, Losartan, Lovenox, Lu AA24493, Luminal, LY450139 (Eli Lilly, Indianapolis, Indiana, Lyrica, Masitinib, Mecobalamin, Memantine, Methylprednisolone, Metoprolol Tartrate, Minitran, Minocycline, Mirtazapine, Mitoxantrone (Novantrone), Mysoline®, Natalizumab (Tysabri), Neurontin®, Niacinamide, Nitro-Bid, Nitro-Dur, Nitroglycerin, Nitrolingual, Nitromist, Nitrostat, Nitro-Time, Norepinephrine (NOR), Carbamazepine, Octreotide, Onfi®, Oxcarbazepine, Oxybutinin chloride, PF-04360365 (Pfizer, New New York, NY, Phenobarbital, Phenytek®, Phenytoin, Piclozotan, Pioglitazone, Plavix, Potigal iga, Pramipexole (Mirapex), Pramlintide, Prednisone, Primidone, Prinivil, Probenecid, Propranolol, PRX-00023 (EPIX Pharmaceuticals) Inc., PXT3003, quinacrine, ramelteon, rasagiline (Azilect), Rebif, ReciGen, remacemide, resveratrol, retavase, reteplase, riluzole (Rilutek), rivastigmine (Exelon), ropinirole (Requip), rotigotine (Neupro), rufinamide, Sabril, safinamide (EMD Serono, Rockland, MA), Salagen, Sarafem, Selegiline (l-deprenyl, Eldepryl), SEN0014196 (Siena Biotech, Siena, Italy), sertraline (Zoloft), simvastatin, nitroprussiate sodium (NPS), sodium phenylbutyrate, Stanback Headache Powder Powder), Tacrine (Cognex), Tamoxifen, Tauroursodeoxycholic Acid (TUDCA), Tegretol®, Tenecteplase, Tenormin, Tetrabenazine (Xenazine), THR-18 (Thrombotech Ltd.), Tiagabine, Tideglusib, Tirofiban, Tissue Plasminogen Activator (tPA), Tizanidine (Zanaflex), TNKase, Tolcapone (Tasmar), Tolterodine, Topamax®, Topiramate, Trihexyphenidyl (formerly Artane), Tri Further steps include co-administering leptal®, ursodiol, valproic acid, valsartan, varenicline (Pfizer), Vimpat, vitamin E, warfarin, Zarontin®, Zestril, Zonegran®, zonisamide, Zydis selegiline HCl orally disintegrating tablets (Zelapar), and combinations thereof.

[0195] For example, to treat or ameliorate the effects of Alzheimer's disease, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the present invention, as well as, for example, one or more of the following: donepezil (Aricept), rivastigmine (Exelon), galantamine (Razadyne), tacrine (Cognex), memantine (Namenda), vitamin E, CERE-110: adeno-associated viral delivery of NGF (Ceregene), LY450139 (Eli Lilly), exenatide, varenicline (Pfizer), PF-04360365 (Pfizer), resveratrol, and donepezil (Eisai Korea).

[0196] To treat or ameliorate the effects of Parkinson's disease, a subject may receive an effective amount of one or more compounds or pharmaceutical compositions of the present invention and, for example, one or more of the following: carbidopa / levodopa immediate release tablets (Sinemet), carbidopa / levodopa orally disintegrating tablets (Parcopa), carbidopa / levodopa / entacapone (Stalevo), ropinirole (Requip), pramipexole (Mirapex), rotigotine (Neupro), apomorphine (Apokyne), or the like. ), selegiline (l-deprenyl, eldepryl), rasagiline (Azilect), Zydis selegiline HCl orally disintegrating tablets (Zelapar), entacapone (Comtan), tolcapone (Tasmar), amantadine (Symmetrel), trihexyphenidyl (formerly Artene), benztropine (Cogentin), IPX066 (Impax Laboratories Inc.), rasagiline (Teva Neuroscience, Inc.), iofulpane 123I (DATSCAN®), safinamide (EMD Serono), and pioglitazone may be administered.

[0197] To treat or ameliorate the effects of amyotrophic lateral sclerosis, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the invention, as well as, for example, one or more of the following: riluzole (Rilutek), lithium carbonate, arimoclomol, creatine, tamoxifen, mecobalamin, memantine (Ebixa), and tauroursodeoxycholic acid (TUDCA).

[0198] To treat or ameliorate the effects of Friedreich's ataxia, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the invention, as well as one or more of the following, for example, idebenone, coenzyme Q, 5-hydroxytryptophan, propranolol, enalapril, lisinopril, digoxin, erythropoietin, Lu AA24493, deferiprone, varenicline, IVIG, pioglitazone, and EGb 761.

[0199] To treat or ameliorate the effects of multiple sclerosis, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the invention, as well as, for example, one or more of the following: Avonex, Betaseron, Extavia, Rebif, glatiramer (Copaxone), fingolimod (Gilenya), natalizumab (Tysabri), mitoxantrone (Novantrone), baclofen (Lioresal), tizanidine (Zanaflex), methylprednisolone, Synovex, Resigen, masitinib, prednisone, interferon beta 1a, interferon beta 1b, and ELND002 (Elan Pharmaceuticals).

[0200] To treat or ameliorate the effects of Huntington's disease, a subject may receive an effective amount of one or more compounds or pharmaceutical compositions of the present invention, as well as other steroids, such as one or more of the following: tetrabenazine (Xenazine), haloperidol (Haldol), clozapine (Clozaril), clonazepam (Klonopin), diazepam (Valium), escitalopram (Lexapro), fluoxetine (Prozac, Sarafem), sertraline (Zoloft), valproic acid (Depakene), divalproex (Depakote), lamotrigine (Lamictal), Dimebon, AFQ056 (Novartis), ethyl-EPA (Miraxion™), SEN0014196 (Siena Biotech), sodium phenylbutyrate, citalopram, ursodiol, minocycline, remacemide, and mirtazapine may be administered.

[0201] To treat or ameliorate the effects of a transmissible spongiform encephalopathy, a subject can be administered an effective amount of one or more compounds or pharmaceutical compositions of the present invention, and, for example, quinacrine.

[0202] To treat or ameliorate the effects of Charcot-Marie-Tooth disease, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the invention and one or more of the following, for example, ascorbic acid and PXT3003.

[0203] To treat or ameliorate the effects of Lewy body dementia, a subject is administered an effective amount of one of the compounds of the present invention. One or more compounds or pharmaceutical compositions may be administered, as well as one or more of the following, for example, Aricept, galantamine, memantine, armodafinil, donepezil, and ramelteon.

[0204] To treat or ameliorate the effects of corticobasal degeneration, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the present invention and one or more of the following, for example, davunetide and coenzyme Q10.

[0205] To treat or ameliorate the effects of progressive supranuclear palsy, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the invention, as well as one or more of the following, for example, tideglusib, rasagiline, alpha-lipoic acid / L-acetylcarnitine, riluzole, niacinamide, and rivastigmine.

[0206] To treat or ameliorate the effects of hereditary spastic paraplegia, a subject may be administered an effective amount of one or more compounds or pharmaceutical compositions of the present invention, as well as one or more of the following, for example, baclofen, tizanidine, oxybutynin chloride, tolterodine, and botulinum toxin.

[0207] In the present invention, one or more compounds or pharmaceutical compositions may be co-administered to a subject in need thereof either together in the same composition, simultaneously in separate compositions, or as separate compositions administered at different times, as deemed most appropriate by a physician.

[0208] A further embodiment of the present invention is a method for treating or ameliorating the effects of a disorder in a subject in need thereof, comprising administering to the subject a pharmaceutically acceptable carrier or diluent and one or more compounds having the structure of Formula (I): [ka] [In the formula, R1 is alkyl-heterocycloalkyl or alkyl-heterobicycloalkyl, where the heterocycloalkyl or heterobicycloalkyl is optionally substituted with one or more atoms or groups selected from alkyl, hydroxy, alcohol, or heterocycloalkyl; R2 is selected from phenyl, indolyl, pyridyl, dibenzofuranyl, benzodioxane, benzothiophenyl, pyrimidinyl, pyrazole, thienyl, indazole, or furanyl, any of which is optionally substituted with one or more atoms or groups selected from alkyl, alkoxy, amino, amido, cyano, ether, nitro, hydroxy, halogen, alkyl-heterocycloalkyl, sulfonyl, ketone, haloalkyl, haloalkoxy, alkylphenyl, carbamate, carboxylic acid, or urea; or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof; However, the compound is [ka] None of the above.

[0209] Preferred pharmaceutical compositions suitable for use in this method are as disclosed in formulas (I)-(II) above, including pharmaceutical compositions containing the specific compounds identified above. Suitable preferred subjects that can be treated according to this method are as disclosed above. In this embodiment, the method can be used to treat the above-mentioned disorders, including diseases involving endoplasmic reticulum (ER) stress and diseases characterized by abnormal kinase levels in a subject.

[0210] In another aspect of this embodiment, the method further comprises co-administering to the subject an effective amount of one or more additional therapeutic agents disclosed herein.

[0211] Another embodiment of the present invention is a method of inhibiting the toxicity of endoplasmic reticulum (ER) stress in a subject in need thereof, the method comprising administering to the subject one or more compounds having the structure of Formula (I): [ka] [In the formula, R1 is alkyl-heterocycloalkyl or alkyl-heterobicycloalkyl, where the heterocycloalkyl or heterobicycloalkyl is optionally substituted with one or more atoms or groups selected from alkyl, hydroxy, alcohol, or heterocycloalkyl; R2 is phenyl, indolyl, pyridyl, dibenzofuranyl, benzodioxane, benzothiophenyl, pyrimidinyl, pyrazole, thienyl, indazole, or furanyl any of which is optionally substituted with one or more atoms or groups selected from alkyl, alkoxy, amino, amido, cyano, ether, nitro, hydroxy, halogen, alkyl-heterocycloalkyl, sulfonyl, ketone, haloalkyl, haloalkoxy, alkylphenyl, carbamate, carboxylic acid, or urea; or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof; However, the compound is [ka] None of the above.

[0212] As used herein, "endoplasmic reticulum (ER) stress toxicity" refers to several physiological and pathological conditions that can disrupt proper ER function, thereby causing ER stress, thereby significantly impairing protein folding and thus posing a risk of proteotoxicity, as well as various pharmacological agents, such as cyclopiazonic acid (CPA). Assays for ER stress are disclosed herein, for example, in the Examples section.

[0213] Suitable preferred compounds for use in this method are as disclosed in formulas (I)-(II) above, including the specific compounds identified above. Suitable preferred subjects that can be treated according to this method are as disclosed above. In this embodiment, the method can be used to treat the disorders described above, including neurodegenerative diseases.

[0214] In another aspect of this embodiment, the method further comprises co-administering to the subject an effective amount of one or more additional therapeutic agents disclosed herein.

[0215] As used herein, the terms "inhibit," "inhibiting," and grammatical variations thereof refer to changes such as reducing or lowering the occurrence of toxic ER stress. In this embodiment, "contacting" refers to bringing a compound and, if necessary, one or more additional therapeutic agents into close proximity with cells that require such modulation. This can be achieved using conventional techniques for delivering drugs to a subject or in an in vitro setting, for example, by providing a compound and, if necessary, other therapeutic agents to the culture medium in which the cells are placed.

[0216] In one aspect of this embodiment, the subject is a mammal. Preferably, the mammal is selected from the group consisting of humans, veterinary animals, and agricultural animals. More preferably, the mammal is a human.

[0217] A further embodiment of the present invention is a method of treating or ameliorating the effects of a disease involving axonal degeneration in a subject in need thereof, the method comprising administering to the subject one or more compounds having the structure of Formula (I): [ka] [In the formula, R1 is alkyl-heterocycloalkyl or alkyl-heterobicycloalkyl, where the heterocycloalkyl or heterobicycloalkyl is optionally substituted with one or more atoms or groups selected from alkyl, hydroxy, alcohol, or heterocycloalkyl; R2 is selected from phenyl, indolyl, pyridyl, dibenzofuranyl, benzodioxane, benzothiophenyl, pyrimidinyl, pyrazole, thienyl, indazole, or furanyl, any of which is optionally substituted with one or more atoms or groups selected from alkyl, alkoxy, amino, amido, cyano, ether, nitro, hydroxy, halogen, alkyl-heterocycloalkyl, sulfonyl, ketone, haloalkyl, haloalkoxy, alkylphenyl, carbamate, carboxylic acid, or urea; or an N-oxide, crystalline form, hydrate, or pharmaceutically acceptable salt thereof; However, the compound is [ka] None of the above.

[0218] As used herein, "axonal degeneration" refers to a hallmark event that disrupts the functional connectivity of neural circuits and is a key feature in many neurodegenerative conditions, including stroke, glaucoma, and motor neuropathy.

[0219] Suitable preferred compounds for use in this method are as disclosed in formulas (I)-(II) above, including the specific compounds identified above. Suitable preferred subjects that can be treated according to this method are as disclosed above. In this embodiment, the method can be used to treat the disorders described above.

[0220] A further embodiment of the present invention is a method for treating or ameliorating the effects of a neurodegenerative disease in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds having the structure of Formula (I): [ka] [In the formula, R1 is alkyl-heterocycloalkyl or alkyl-heterobicycloalkyl, where the heterocycloalkyl or heterobicycloalkyl is optionally substituted with one or more atoms or groups selected from alkyl, hydroxy, alcohol, or heterocycloalkyl; R2 is selected from phenyl, indolyl, pyridyl, dibenzofuranyl, benzodioxane, benzothiophenyl, pyrimidinyl, pyrazole, thienyl, indazole, or furanyl, any of which may be alkyl, alkoxy, amino, amido, cyano, acetonitrile, or acetonitrile; optionally substituted with one or more atoms or groups selected from ter, nitro, hydroxy, halogen, alkyl-heterocycloalkyl, sulfonyl, ketone, haloalkyl, haloalkoxy, alkylphenyl, carbamate, carboxylic acid, or urea; or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof; However, the compound is [ka] None of the above.

[0221] Preferred compounds suitable for use in this method are as disclosed in Formulas (I)-(II) above, including the specific compounds identified above. In this embodiment, the method can be used to treat the disorders described above.

[0222] Suitable preferred subjects are as disclosed herein. In this embodiment, the method can be used to treat the neurodegenerative disorders mentioned above.

[0223] In one aspect of this embodiment, the method further comprises co-administering to the subject an effective amount of one or more therapeutic agents disclosed herein.

[0224] A further embodiment of the present invention comprises: [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0225] A further embodiment of the present invention comprises: [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0226] A further embodiment of the present invention comprises: [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0227] A further embodiment of the present invention comprises: [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0228] A further embodiment of the present invention is a composition comprising a pharmaceutically acceptable carrier or diluent, and [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0229] A further embodiment of the present invention is a composition comprising a pharmaceutically acceptable carrier or diluent, and [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0230] A further embodiment of the present invention is a composition comprising a pharmaceutically acceptable carrier or diluent, and [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0231] A further embodiment of the present invention is a composition comprising a pharmaceutically acceptable carrier or diluent, and [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0232] A further embodiment of the present invention is a method for treating or ameliorating the effects of a disorder in a subject in need thereof. The method comprises administering to the subject an effective amount of [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0233] A further embodiment of the present invention is a method for treating or ameliorating the effects of a disorder in a subject in need thereof. The method comprises administering to the subject an effective amount of [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0234] A further embodiment of the present invention is a method for treating or ameliorating the effects of a disorder in a subject in need thereof. The method comprises administering to the subject an effective amount of [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0235] A further embodiment of the present invention is a method for treating or ameliorating the effects of a disorder in a subject in need thereof. The method comprises administering to the subject an effective amount of [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0236] A further embodiment of the present invention is a method of inhibiting the toxicity of endoplasmic reticulum (ER) stress in a subject in need thereof, the method comprising administering to the subject: [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0237] A further embodiment of the present invention is a method of inhibiting the toxicity of endoplasmic reticulum (ER) stress in a subject in need thereof, the method comprising administering to the subject: [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0238] A further embodiment of the present invention is a method of inhibiting the toxicity of endoplasmic reticulum (ER) stress in a subject in need thereof, the method comprising administering to the subject: [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0239] A further embodiment of the present invention is a method of inhibiting the toxicity of endoplasmic reticulum (ER) stress in a subject in need thereof, the method comprising administering to the subject: [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0240] A further embodiment of the present invention is a method of treating or ameliorating the effects of a disease involving axonal degeneration in a subject in need thereof. The method comprises administering to the subject: [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0241] A further embodiment of the present invention is a method of treating or ameliorating the effects of a disease involving axonal degeneration in a subject in need thereof. The method comprises administering to the subject: [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0242] A further embodiment of the present invention is a method of treating or ameliorating the effects of a disease involving axonal degeneration in a subject in need thereof. The method comprises administering to the subject: [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0243] A further embodiment of the present invention is a method of treating or ameliorating the effects of a disease involving axonal degeneration in a subject in need thereof. The method comprises administering to the subject: [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0244] A further embodiment of the present invention is a method for treating or ameliorating the effects of a neurodegenerative disease in a subject in need thereof. The method comprises administering to the subject an effective amount of [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0245] A further embodiment of the present invention is a method for treating or ameliorating the effects of a neurodegenerative disease in a subject in need thereof. The method comprises administering to the subject an effective amount of [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0246] A further embodiment of the present invention is a method for treating or ameliorating the effects of a neurodegenerative disease in a subject in need thereof. The method comprises administering to the subject an effective amount of [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0247] A further embodiment of the present invention is a method for treating or ameliorating the effects of a neurodegenerative disease in a subject in need thereof. The method comprises administering to the subject an effective amount of [ka] or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0248] As used herein, "pharmaceutically acceptable salts" refers to salts of the compounds of the present invention that are pharmaceutically acceptable, as defined herein, and that possess the desired pharmacological activity. Such salts include salts prepared with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or with organic acids such as acetic acid, propionic acid, hexanoic acid, heptanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, o-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, and the like. Pharmaceutically acceptable salts include acid addition salts formed with benzoic acid, p-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like. Pharmaceutically acceptable salts also include base addition salts that can be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.

[0249] In the present invention, an "effective amount" or "therapeutically effective amount" of a compound or pharmaceutical composition is an amount of such compound or composition sufficient to produce a beneficial or desired result, as described herein, when administered to a subject. Effective dosage forms, methods of administration, and dosages can be determined empirically, and such determinations are made by those skilled in the art. It will be understood by those skilled in the art that dosages will vary with the route of administration, excretion rate, duration of treatment, the identity of any other drugs administered, the age, size, and species of the subject, and similar factors well known, for example, in the fields of medicine and veterinary medicine. Generally, an appropriate dose of a compound or pharmaceutical composition according to the present invention is the amount of the compound or composition that is the lowest dose effective to produce the desired effect with no or minimal side effects. An effective dose of a compound or pharmaceutical composition according to the present invention can be administered as two, three, four, five, six, or more sub-doses administered separately at appropriate intervals throughout the day.

[0250] Suitable non-limiting examples of dosages of compounds or pharmaceutical compositions according to the invention, or compositions comprising such compounds, are from about 1 mg / kg to about 1000 mg / kg, e.g., from about 1 mg / kg to about 100 mg / kg, including from about 5 mg / kg to about 50 mg / kg. Other representative dosages of compounds or pharmaceutical compositions of the invention include about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, Including about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 250 mg / kg, about 300 mg / kg, about 400 mg / kg, about 500 mg / kg, about 600 mg / kg, about 700 mg / kg, about 800 mg / kg, about 900 mg / kg, or about 1000 mg / kg.

[0251] The compounds or pharmaceutical compositions of the present invention can be administered in any desired effective manner, such as for oral ingestion, or as ointments or eye drops for topical administration to the eye, or for other administration in any suitable manner, such as parenteral or intraperitoneal, subcutaneous, topical, intradermal, inhalation, pulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. Furthermore, the compounds or pharmaceutical compositions of the present invention can be administered in conjunction with other treatments. The compounds or pharmaceutical compositions of the present invention can be encapsulated or otherwise protected from gastric juices or other secretions, if desired.

[0252] The pharmaceutical compositions of the present invention are pharmaceutically acceptable and comprise one or more active ingredients in admixture with one or more pharmaceutically acceptable carriers or diluents, and optionally one or more other compounds, drugs, ingredients and / or materials. Regardless of the selected route of administration, the compounds / pharmaceutical compositions of the present invention are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. See, for example, Remington, The Science and Practice of Pharmacy (21 st Edition, Lippincott Williams and Wilkins, Philadelphia, PA. More generally, "pharmaceutically acceptable" means generally safe, non-toxic, and useful in the preparation of biologically or otherwise undesirable compositions, including those acceptable for veterinary and human medicine.

[0253] Pharmaceutically acceptable carriers and diluents are well known in the art (see, e.g., Remington, The Science and Practice of Pharmacy (21 st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.) and The National Formulary (American Pharmaceutical Association, Washington, DC), which may include sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starch, cellulose preparations, calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate, and calcium hydrogen phosphate), sodium citrate, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, Pharmaceutically acceptable carriers or diluents used in the compositions of the invention include pharmaceutically acceptable carriers or diluents such as glycerides, propylene glycol, and polyethylene glycol), organic esters (e.g., ethyl oleate and triglycerides), biodegradable polymers (e.g., polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils (e.g., corn germ, olive, castor, sesame, cottonseed, and peanut), cocoa butter, waxes (e.g., suppository wax), paraffin, silicones, talc, silicylate, and the like. Each pharmaceutically acceptable carrier or diluent used in the compositions of the invention must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject. Carriers or diluents suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable carriers or diluents for a selected dosage form and method of administration can be determined using skills in the art.

[0254] The pharmaceutical compositions of the present invention may contain additional components and / or materials commonly used in such compositions, if necessary. These components and materials are well known in the art and include: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, hydroxypropylmethylcellulose, sucrose, and acacia; (3) humectants, such as glycerol; (4) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, and certain silicates. , sodium starch glycolate, cross-linked sodium carboxymethylcellulose, and sodium carbonate, (5) solution retarders, such as paraffin, (6) absorption enhancers, such as quaternary ammonium compounds, (7) wetting agents, such as cetyl alcohol and glycerol monostearate, (8) absorbents, such as kaolin and bentonite clay, (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, and sodium lauryl sulfate, (10) suspending agents, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, and tragacanth, (11) buffering agents, (12) excipients, such as lactose, milk sugar, polyethylene glycol, animal and vegetable fats, oils, waxes, paraffin, cocoa butter, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc, salicylate, zinc oxide, aluminum hydroxide aluminum, calcium silicate, and polyamide powder, (13) inert diluents, such as water or other solvents, (14) preservatives, (15) surface-active agents, (16) dispersing agents, (17) controlled-release or absorption-retarding agents, such as hydroxypropyl methylcellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monosterate, gelatin, and waxes, (18) opacifying agents, (19) adjuvants, (20) wetting agents, (21) emulsifying and suspending agents,(22) solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed, peanut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, (23) propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane, (24) antioxidants, (25) agents that render the formulation isotonic with the blood of the intended recipient, such as sugars and sodium chloride, (26) thickening agents, (27) coating materials, such as lecithin, and (28) sweetening agents, flavoring agents, coloring agents, perfumes, and preservatives. Each such ingredient or material must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Ingredients and materials suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable ingredients and materials for a selected dosage form and administration method can be determined using techniques in the art.

[0255] The compound or pharmaceutical composition suitable for oral administration may be in the form of a capsule, cachet, pill, tablet, powder, granule, aqueous or non-aqueous liquid solution or suspension, oil-in-water or water-in-oil liquid emulsion, elixir or syrup, troche, bolus, electuary, or paste. These formulations can be prepared by methods known in the art, for example, using conventional pan-coating, mixing, granulating, or lyophilizing processes.

[0256] Solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.) may, for example, comprise the active ingredient in one or more pharmaceutically acceptable carriers or diluents, and optionally one or more fillers, extenders, binders, humectants, disintegrants, dissolution retardants, or the like. The solid dosage forms may be prepared by mixing with an agent, an absorption enhancer, a wetting agent, an absorbent, a lubricant, and / or a colorant. Similar types of solid compositions can also be used as fillers in soft and hard-filled gelatin capsules using appropriate excipients. Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using suitable binders, lubricants, inert diluents, preservatives, disintegrants, surface active agents, or dispersing agents. Molded tablets can be made by molding in a suitable machine. Tablets and other solid dosage forms, such as sugar-coated tablets, capsules, pills, and granules, can be optionally scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They can also be formulated to provide sustained or controlled release of the active ingredient contained therein. They can be sterilized, for example, by filtration through a bacteria-retaining filter. These compositions may optionally contain opacifying agents and may be of a composition such that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. The active ingredient(s) may also be in microencapsulated form.

[0257] The liquid dosage form for oral administration includes pharmaceutically acceptable emulsion, microemulsion, solution, suspension, syrup and elixir.Liquid dosage form can contain suitable inert diluent that is commonly used in the art.In addition to inert diluent, oral composition can also contain adjuvants, such as wetting agent, emulsifier and suspending agent, sweetener, flavoring agent, coloring agent, flavoring agent and preservative.Suspension can contain suspending agent.

[0258] The composition for rectal or vaginal administration can be presented as a suppository, which can be prepared by mixing one or more active ingredients with one or more suitable non-irritating carriers that are solid at room temperature but become liquid at body temperature, and thus melt in the rectum or vaginal cavity and release the active compound.The composition suitable for vaginal administration also includes pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such pharmaceutically acceptable carriers, as known in the art.

[0259] Dosage forms for topical or transdermal administration include powder, spray, ointment, paste, cream, lotion, gel, solution, patch, droplets and inhalant.The active agent / compound can be mixed with a suitable pharmaceutically acceptable carrier or diluent under sterile conditions.Ointment, paste, cream and gel can contain excipients.Powder and spray can contain excipients and propellants.

[0260] Compositions suitable for parenteral administration contain one or more drugs / compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted immediately before use into sterile injectable solutions or dispersions, and may contain appropriate antioxidants, buffers, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Proper fluidity can be maintained, for example, by the use of coating materials, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. These compositions can also contain suitable adjuvants, such as wetting agents, emulsifying agents, and dispersing agents. It may be desirable for the composition to contain an isotonic agent. In addition, prolonged absorption of injectable pharmaceutical forms can be achieved by including an agent that delays absorption.

[0261] In some cases, in order to prolong the effect of a drug (e.g., a pharmaceutical formulation), it is desirable to slow its absorption from subcutaneous or intramuscular injection. This can be achieved by the use of a liquid suspension of crystalline or amorphous material with poor water solubility.

[0262] The absorption rate of the active agent / drug then depends on its dissolution rate, which may vary depending on the crystal size and crystalline form. Alternatively, delayed absorption of parenterally administered drugs / drugs can be achieved by dissolving or suspending the active agent / drug in an oil vehicle. Injectable depot forms can be prepared by forming microencapsulated matrices of the active ingredient in biodegradable polymers. The release rate of the active ingredient can be controlled depending on the ratio of the active ingredient to the polymer and the properties of the specific polymer used. Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. Injectable materials can be sterilized, for example, by filtration through a bacteria-retaining filter.

[0263] The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier or diluent, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.

[0264] In the above embodiment, the following definitions apply:

[0265] The term "aliphatic," as used herein, refers to a group composed of carbon and hydrogen and not containing an aromatic ring. Thus, aliphatic groups include alkyl, alkenyl, alkynyl, and carbocyclyl groups. Furthermore, unless otherwise specified, the term "aliphatic" is intended to include both "unsubstituted aliphatic" and "substituted aliphatic," where substituted aliphatic refers to an aliphatic moiety having a substituent replacing a hydrogen on one or more carbons of the aliphatic group. Such substituents may include, for example, halogen, deuterium, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, aromatic, or heteroaromatic moieties.

[0266] The term "alkyl" refers to the radical of a saturated aliphatic group not having a ring structure, including straight-chain and branched-chain alkyl groups. In certain embodiments, a straight-chain or branched-chain alkyl has six or fewer carbon atoms in its backbone (e.g., C1-C6 for straight chain, C3-C6 for branched chain). Such substituents include all substituents contemplated for aliphatic groups, as discussed below, except where stability would be prohibitive.

[0267] The term "alkenyl," as used herein, refers to an aliphatic group containing at least one double bond and, unless otherwise specified, is intended to include both "unsubstituted alkenyl" and "substituted alkenyl," where substituted alkenyl refers to an alkenyl moiety having substituents replacing a hydrogen on one or more carbons of the alkenyl group. Such substituents include all substituents contemplated for aliphatic groups, as discussed below, except where stability would be prohibitive. For example, substitution of alkenyl groups with one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.

[0268] Furthermore, unless otherwise specified, the term "alkyl," as used throughout the specification, examples, and claims, is intended to include both "unsubstituted alkyls" and "substituted alkyls," which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Indeed, unless otherwise specified, the term "alkyl" as used herein refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. All groups listed are intended to include both substituted and unsubstituted options.

[0269] The term “C x~y " when used with chemical moieties such as alkyl and cycloalkyl, is meant to include groups containing x to y carbons in the chain. For example, the term "C x~y "Alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups including straight-chain alkyl and branched-chain alkyl groups containing x to y carbons in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-tirfluoroethyl.

[0270] The term "aryl," as used herein, includes substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon. Preferably, the ring is 3- to 8-membered, more preferably 6-membered. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings, where two or more carbons are common to two adjacent rings, and at least one of the rings is aromatic; for example, the other cyclic ring may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0271] The term "alkyl-aryl" refers to an alkyl group substituted with at least one aryl group.

[0272] The term "alkyl-heteroaryl" refers to an alkyl group substituted with at least one heteroaryl group.

[0273] The term "alkenyl-aryl" refers to an alkenyl group substituted with at least one aryl group.

[0274] The term "alkenyl-heteroaryl" refers to an alkenyl group substituted with at least one heteroaryl group.

[0275] The term "amino," as used herein, alone or in combination, refers to -NRR', where R and R' are independently selected from hydrogen, alkyl, acyl, heteroalkyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl, any of which may themselves be optionally substituted. Additionally, R and R' can combine to form a heterocycloalkyl, any of which may themselves be optionally substituted.

[0276] The term "amide," as used herein, alone or in combination, refers to a group consisting of -R n E(O) x"NR'2," where R and R' are independently selected from hydrogen, alkyl, acyl, heteroalkyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl, any of which may themselves be optionally substituted. Examples include carboxamide (n=1, E=C, x=1), phosphoramide (n=2, E=P, x=1), and sulfonamide (n=1, E=S, x=2).

[0277] The terms "benzo" and "benz," as used herein, alone or in combination, refer to the divalent radical CH= derived from benzene. Examples include benzothiophene and benzimidazole.

[0278] The term "carbamate," as used herein, alone or in combination, can be attached to the parent molecular moiety from either the nitrogen or the acid terminus, as defined herein. "carbamyl" refers to an optionally substituted ester of carbamic acid (-NRC(O)O-). The term "O-carbamyl," as used herein, alone or in combination, refers to the group -OC(O)NRR', and the term "N-carbamyl," as used herein, alone or in combination, refers to the group -ROC(O)NR'-. R and R' are as defined herein or by the designated "R" group specifically recited.

[0279] The term "carbonyl," as used herein, when alone includes formyl [-C(O)H] and in combination is a -C(O)- group.

[0280] The terms "carboxyl" or "carboxy," as used herein, refer to -C(O)OH or the corresponding "carboxylate" anion present, for example, in a carboxylic acid salt. An "O-carboxy" group refers to a RC(O)O- group, where R is as defined herein. A "C-carboxy" group refers to a -C(O)OR group, where R is as defined herein.

[0281] The term "cyano," as used herein, alone or in combination, refers to --CN.

[0282] The term "cycloalkyl," or alternatively, "carbocycle," as used herein, alone or in combination, refers to a saturated or partially saturated monocyclic, bicyclic, or tricyclic alkyl group, wherein each cyclic moiety contains 3 to 12 carbon atom ring members and may be a benzo-fused ring system, optionally substituted as defined herein. In certain embodiments, the cycloalkyl contains 5 to 7 carbon atoms. Examples of such cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, tetrahydronaphthyl, indanyl, octahydronaphthyl, 2,3-dihydro-1H-indenyl, adamantyl, and the like. "Bicyclic" and "tricyclic," as used herein, are intended to include both fused ring systems, e.g., decahydronaphthalene, octahydronaphthalene, and polycyclic (multi-center) saturated or partially unsaturated types. The latter type of isomer is commonly exemplified by bicyclo[1,1,1]pentane, camphor, adamantane, and bicyclo[3,2,1]octane.

[0283] The terms "carbocycle," "carbocyclyl," and "carbocyclic," as used herein, refer to a non-aromatic saturated or unsaturated ring in which each atom of the ring is carbon. Preferably, the carbocycle contains 3 to 10 atoms, more preferably 3 to 8 atoms, including, for example, 5 to 7 atoms, such as 6 atoms. The term "cabocycle" also includes bicyclic, tricyclic, and other polycyclic ring systems, including an adamantyl ring system.

[0284] The term "heteroatom," as used herein, means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur, more preferably nitrogen and oxygen.

[0285] The term "heteroalkyl," as used herein, alone or in combination, refers to a stable straight-chain, branched-chain, or cyclic hydrocarbon radical, or combination thereof, which is fully saturated or contains 1 to 3 degrees of unsaturation and consists of the stated number of carbon atoms and 1 to 3 heteroatoms selected from O, N, and S, where the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N, and S may be placed at any interior position of the heteroalkyl group. For example, up to two heteroatoms may be consecutive, such as -CH2-NH-OCH3. It may be possible.

[0286] The term "heteroaryl" includes substituted or unsubstituted aromatic single ring structures, preferably 3- to 8-membered rings, more preferably 5- to 7-membered rings, and even more preferably 5- to 6-membered rings, which ring structures contain at least one heteroatom, preferably 1-4 heteroatoms, and more preferably 1 or 2 heteroatoms. The term "heteroaryl" also includes polycyclic ring systems having two or more cyclic rings, where two or more carbons are common to two adjacent rings, and at least one of the rings is heteroaromatic, e.g., the other cyclic ring may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0287] The terms "heterocycloalkyl" and, interchangeably, "heterocycle," as used herein, alone or in combination, refer to a saturated, partially unsaturated, or fully unsaturated monocyclic, bicyclic, or tricyclic heterocyclic group containing at least one heteroatom as a ring member, respectively, where each said heteroatom can be independently selected from N, O, and S. Furthermore, a heterocycloalkyl can contain one or two C(O), S(O), or S(O) groups as ring members. In certain embodiments, the heterocycloalkyl contains one to four heteroatoms as ring members. In further embodiments, the heterocycloalkyl contains one to two heteroatoms as ring members. In certain embodiments, the heterocycloalkyl contains three to eight ring members in each ring. In further embodiments, the heterocycloalkyl contains three to seven ring members in each ring. In still further embodiments, the heterocycloalkyl contains five to six ring members in each ring. "Heterocycloalkyl" and "heterocyclyl" are intended to include sulfone, sulfoxide, N-oxide of a tertiary nitrogen ring member, and carbocyclic fused and benzofused ring systems; further, both terms include systems in which a heterocyclic ring is fused to an aryl group or an additional heterocyclic group, as defined herein. Examples of heterocyclic groups include aziridinyl, azetidinyl, 1,3-benzodioxolyl, dihydroisoindolyl, dihydroisoquinolinyl, dihydrocinnolinyl, dihydrobenzodioxinyl, dihydro[1,3]oxazolo[4,5-b]pyridinyl, benzothiazolyl, dihydroindolyl, dihydropyridinyl, 1,3-dioxanyl, 1,4-dioxanyl, 1,3-dioxolanyl, isoindolinyl, morpholinyl, piperazinyl, pyrrolidinyl, tetrahydropyridinyl, piperidinyl, thiomorpholinyl, and the like. A heterocyclic group may be optionally substituted unless specifically prohibited.

[0288] The term "hydroxy," as used herein, alone or in combination, refers to --OH.

[0289] The terms "halo" and "halogen" are used interchangeably herein to mean halogen and include chloro, fluoro, bromo, and iodo.

[0290] The term "haloalkoxy," as used herein, alone or in combination, refers to a haloalkyl group attached to the parent molecular moiety through an oxygen atom. Haloalkoxy includes perhaloalkoxy. The term "perhaloalkoxy" refers to an alkoxy group in which all of the hydrogen atoms are replaced by halogen atoms. An example of a perhaloalkoxy is perfluoromethoxy.

[0291] The term "haloalkyl," as used herein, alone or in combination, refers to an alkyl group having the meaning as defined above, in which one or more hydrogens are replaced with halogen. "Haloalkyl" refers to an alkyl radical. Specifically, it includes monohaloalkyl, dihaloalkyl, polyhaloalkyl, and perhaloalkyl radicals. For example, a monohaloalkyl radical can have an iodo, bromo, chloro, or fluoro atom within the radical. Dihalo and polyhaloalkyl radicals can have two or more of the same halo atoms or a combination of different halo radicals. Examples of haloalkyl radicals include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. "Haloalkylene" refers to a haloalkyl group that is bonded at two or more positions. Examples of haloalkyl radicals include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. "Haloalkylene" refers to a haloalkyl group that is bonded at two or more positions. Examples include fluoromethylene (-CFH-), difluoromethylene (-CF2-), chloromethylene (-CHCl-), and the like. The term "perhaloalkyl," as used herein, alone or in combination, refers to an alkyl group in which all of the hydrogen atoms are replaced by halogen atoms. Examples include perfluoromethyl.

[0292] The term "alcohol" refers to an organic compound in which a hydroxyl functional group (-OH) is attached to a saturated carbon atom.

[0293] The term "ketone" refers to an organic compound having the structure RC(=O)R', where neither R nor R' can be a hydrogen atom.

[0294] The term "ester" refers to an organic compound having the structure RC(=O)OR', where neither R nor R' can be a hydrogen atom.

[0295] The term "ether," as used herein, alone or in combination, refers to an oxy group bridging two moieties joined at a carbon atom.

[0296] The term "nitro," as used herein, alone or in combination, refers to --NO.sub.2.

[0297] The term "polyyne" refers to an organic compound having alternating single and triple bonds, ie, a series of consecutive alkynes, (-C≡C-)n, where n is greater than 1.

[0298] The terms "thia" and "thio," as used herein, alone or in combination, refer to an -S- group or ether, in which the oxygen has been replaced by sulfur. The oxidized derivatives of the thio group, namely, sulfinyl and sulfonyl, are included in the definition of thia and thio. The term "sulfanyl," as used herein, alone or in combination, refers to -S-. The term "sulfinyl," as used herein, alone or in combination, refers to -S(O)-. The term "sulfonyl," as used herein, alone or in combination, refers to -S(O)-.

[0299] The terms "urea" or "carbamide," as used herein, alone or in combination, refer to --NHC(O)NH--.

[0300] The term "substituted" refers to substituents replacing a hydrogen on one or more carbons of the backbone. " refers to a moiety having the formula: ". It is understood that "substituted" or "substituted with" includes the implicit proviso that such substitution is made in accordance with the allowed valences of the substituted atom and substituent, and that the substitution results in a stable compound that is not naturally subject to transformation, e.g., by rearrangement, cyclization, elimination, and the like. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In one broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. The permissible substituents for appropriate organic compounds can be one or more and the same or different. For purposes of this invention, heteroatoms, such as nitrogen, can have hydrogen substituents that satisfy the valence of the heteroatom and / or any permissible substituent of organic compounds described herein. Substituents can include any of the substituents described herein, such as halogen, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. Those skilled in the art will understand that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.

[0301] As noted above, unless specifically described as "unsubstituted," references herein to chemical moieties are understood to include substituted variants. For example, reference to an "aryl" group or moiety implicitly includes both substituted and unsubstituted variants.

[0302] As used herein, the term "furanyl" means any compound or chemical group containing the following structure: [ka]

[0303] As used herein, the term "indolyl" means any compound or chemical group containing the following structure: [ka]

[0304] As used herein, the term "indazole" means any compound or chemical group containing the following structure: [ka]

[0305] As used herein, the term "pyridyl" means any compound or chemical group containing the following structure: [ka]

[0306] As used herein, the term "pyrimidinyl" means any compound or chemical group that contains the following structure: [ka]

[0307] As used herein, the term "pyrazole" means any compound or chemical group containing the following structure: [ka]

[0308] As used herein, the term "thienyl" or "thiophene" means any compound or chemical group containing the following structure: [ka]

[0309] As used herein, the term "benzothiophenyl" refers to any benzothiophenyl containing the structure: "N" means any compound or chemical group. [ka]

[0310] As used herein, the term "benzodioxane" means any compound or chemical group containing the following structure: [ka]

[0311] As used herein, the term "dibenzofuranyl" means any compound or chemical group containing the following structure: [ka]

[0312] The disclosure of compound herein is understood to encompass all stereoisomers of the compound.As used herein, the term "stereoisomer" refers to compounds that are made up of the same atoms bonded by the same bond, but have different three-dimensional structures that cannot be interchanged.Third-dimensional structures are called configurations.Stereoisomers include enantiomers and diastereomers.

[0313] The term "racemate" or "racemic mixture" refers to a mixture of equal enantiomeric portions. The term "chiral center" refers to a carbon atom to which four different groups are attached. The term "enantiomerically enriched," as used herein, refers to an increase in the amount of one enantiomer relative to the other enantiomer.

[0314] It is recognized that the compounds of the present invention can exist and be isolated in optically active racemic forms to the extent that they have chiral centers. Some compounds may exhibit polymorphism. The present invention encompasses any racemic, optically active diastereomeric, polymorphic or stereoisomeric form of the compounds of the present invention, or mixtures thereof, that have the useful properties described herein, and it should be understood that methods for preparing optically active forms (e.g., by resolving racemic forms by recrystallization techniques, by synthesis from optically active starting materials, by chiral synthesis, or by chromatographic separation using chiral stationary phases) are well known in the art.

[0315] Examples of methods for obtaining optically active materials are known in the art and include at least the following: i) Physical Separation of Crystals - Macroscopic crystals of the individual enantiomers are manually separated. This technique can be used when crystals of the separate enantiomers exist, i.e., when the material is a conglomerate and the crystals are visually distinct. ii) Simultaneous crystallization--a technique in which individual enantiomers are crystallized separately from a solution of the racemate, which is only possible if the racemate is a conglomerate in the solid state. iii) Enzymatic resolution - a technique in which a racemate is partially or completely separated by the difference in the rate at which the enantiomers react with an enzyme. iv) Enzymatic Asymmetric Synthesis--a synthetic technique in which at least one step of the synthesis uses an enzymatic reaction to yield an enantiomerically pure or enriched synthetic precursor of a desired enantiomer. v) Chemical Asymmetric Synthesis—a synthetic technique in which a desired enantiomer is synthesized from an achiral precursor under conditions that result in asymmetry (i.e., chirality) in the product (which can be achieved using chiral catalysts or chiral auxiliaries as disclosed in more detail herein). vi) Diastereomeric Separation -- a technique in which a racemic compound is reacted with an enantiomerically pure reagent (chiral auxiliary) that converts the individual enantiomers into diastereomers. The resulting diastereomers are then separated by chromatography or crystallization due to their now more distinct structural differences, and the chiral auxiliary is later removed to yield the desired enantiomer. vii) Primary and secondary asymmetric transformations - techniques in which either the diastereomers from the racemate reach equilibrium and the diastereomer from the desired enantiomer predominates in solution, or the equilibrium is disturbed by preferential crystallization of the diastereomer from the desired enantiomer, thus ultimately converting essentially all of the material from the desired enantiomer to the crystalline diastereomer, which is then released from the diastereomer. viii) Kinetic Resolution--This technique refers to the partial or complete resolution of a racemate (or further resolution of a partially resolved compound) achieved by the heterogeneity of the reaction rates of enantiomers with chiral non-racemic reagents or catalysts under kinetic conditions. ix) Enantiospecific synthesis from non-racemic precursors--a synthetic technique in which the desired enantiomer is obtained from non-chiral starting materials and in which stereochemical integrity is not, or is only minimally, compromised throughout the synthetic process. x) Chiral liquid chromatography - a technique in which the enantiomers of a racemate are separated in a liquid mobile phase by their different interactions with a stationary phase. The stationary phase can be made from a chiral material, or the mobile phase can contain additional chiral materials that cause the different interactions. xi) Chiral Gas Chromatography--A technique in which the racemate is volatilized and the enantiomers are separated by their different interactions in the gaseous mobile phase using a column containing a fixed non-racemic chiral adsorbent phase. xii) Extraction using chiral solvents - a technique in which enantiomers are separated by the preferential dissolution of one enantiomer in a particular chiral solvent. xiii) Transport through chiral membranes - a technique in which a racemate is placed in contact with a thin film barrier. The barrier typically separates two miscible fluids, one containing the racemate, and a driving force such as concentration or a pressure difference causes preferential transport across the membrane barrier. Separation occurs as a result of the non-racemic chiral nature of the membrane, which allows only one enantiomer of the racemate to pass through.

[0316] Stereoisomers can also be separated by conventional techniques known to those skilled in the art, including fractional crystallization of bases or their salts, or chromatographic techniques, such as LC or flash chromatography. The (+) enantiomer can be separated by the methods described by J. Jacques, using techniques and procedures well known in the art, such as those described by W. et al., "Enantiomers, Racemates, and Resolutions," John Wiley and Sons, Inc., 1981; The (-) enantiomer can be separated using chiral chromatography, for example, using a suitable organic solvent such as ethanol / acetonitrile and a Chiralpak AD pack, 20 micron.

[0317] The following examples are provided to further illustrate the method of the present invention. These examples are merely illustrative and are not intended to limit the scope of the invention in any way. [Example]

[0318] The present invention is further illustrated by the following examples, which are provided for illustrative purposes and are not intended to limit the invention in any way. Those of ordinary skill in the art will readily recognize a variety of noncritical parameters that can be changed or modified to yield essentially the same results. Example 1 Methods and Materials chemicals

[0319] Starting materials were purchased from Sigma-Aldrich, Fisher Scientific, Ark Pharm, Oakwood Chemical, Cambridge Isotope Laboratory, or AK Scientific and used as received unless otherwise indicated. All solvents were reagent grade. Chromatography

[0320] Column chromatography was performed on a Teledyne ISCO CombiFlash® Rf+ using RediSep® normal-phase silica flash columns. Thin-layer chromatography (TLC) was performed on Silicycle SiliaPlate™ glass TLC plates (250 μm, 20 × 20 cm). Where indicated, compounds were purified on a Phenomenex Gemini NX-C column using a Gilson HPLC equipped with a GX-271 liquid handler. 18 Purification was carried out by preparative HPLC on a column (250 x 21.2 mm, particle size: 5 μm, pore size: 110 Å). spectroscopy

[0321] 1 H NMR spectra were recorded at ambient temperature using a 400 MHz or 500 MHz spectrophotometer as indicated. Chemical shifts are reported in ppm relative to the residual solvent peak ( 1H NMR: DMSO-d6, δ 2.50; chloroform-d, δ 7.26; methanol-d4, δ 3.31). The following abbreviations are used to indicate multiplicity: s (singlet), d (doublet), t (triplet), q (quartet), hept (septet), m (multiplet), and br (broad). High-resolution mass spectra (HRMS) were acquired on a time-of-flight spectrometer by atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI), as indicated, and obtained by peak matching. All reactions were performed under an atmosphere of nitrogen or argon in flame-dried glassware under argon unless otherwise indicated. Aqueous solutions were prepared from nanopure water with a resistivity greater than 18 MΩ cm. All reagents were commercially available unless otherwise indicated. software

[0322] Molecular docking, molecular modeling, and visualization were performed using Glide (2012-2016 version, Schrodinger) and Schrodinger Maestro (2017-1 version). All chemical structures were drawn using ChemDraw Professional 16.0 (Perkin Elmer). All microsomes were analyzed using Glide software. Stability curves were generated using Prism7 (GraphPad Software). Example 2 Synthesis of piperazine analogues

[0323] A general route to compounds of formula (I) through (II) follows the synthesis exemplified below, where the efficacy mediator group was added in the final step instead of earlier. The preparation of each compound is also provided. [ka] Scheme 1: General synthetic scheme for compounds 1 and 7a-7az. Preparation of 4-(1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (compound 3) [ka]

[0324] 5-Bromo-1H-pyrrolo[2,3-b]pyridine (2, 3.94 g, 20 mmol, 1.0 eq) and 4-formylphenylboronic acid (3.30 g, 22 mmol, 1.1 eq) were added to a pressure tube, and acetonitrile (80 mL) and 1,4-dioxane (20 mL) were added. The reaction mixture was degassed and kept under argon. Pd(PPh)Cl (0.70 g, 1.0 mmol, 5 mol%) was added, followed by aqueous NaCO (2.0 M, 50 mL). The reaction mixture was stirred for 5 minutes, transferred to an oil bath, and stirred at 130 °C overnight. After cooling to room temperature, the reaction mixture was partitioned between ethyl acetate and brine, separated, and the aqueous layer was extracted with ethyl acetate (3x). The combined organic layers were dried over anhydrous sodium sulfate and filtered through Celite. The solvent was evaporated and the crude product was purified by column chromatography (0-100% EtOAc in hexanes) to give 4-(1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (3, 2.15 g, 48% yield) as a colorless solid. 1 H NMR (400 MHz, DMSO-d6) δ 11.80 (s, 1H), 10.06 (s, 1H), 8.63 (d, J = 2.2 Hz, 1H), 8.35 (d, J = 2.2 Hz, 1H), 8.05 - 7.94 (m, 4H), 7.55 (d, J = 3.5 Hz, 1H), 6.54 (d, J = 3.4 Hz, 1H) ppm.HRMS(APCI + , m / z):C 14 H 11 NO[M+H + Calculated value for: 22 3.0872, Actual value: 223.0872. Preparation of 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 4) [ka]

[0325] 4-(1H-Pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (3, 2.15 g, 9.66 mmol, 1.0 eq) was suspended in dichloromethane (ca. 0.1 M), 1-methylpiperazine (1.93 g, 2.14 mL, 19.3 mmol, 2.0 eq) and Na(OAc)3BH (3.07 g, 14.5 mmol, 1.5 eq) were added, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was partitioned between dichloromethane and brine, the layers were separated, and the aqueous layer was extracted with dichloromethane. The combined organic layers were washed with brine, dried over sodium sulfate, filtered over Celite, and the solvent was evaporated to give 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (4, 2.2 grams, 74% yield) as a colorless solid. The product was used in the next step without further purification. 1 H NMR (400 MHz, methanol-d4) δ 8.43 (d, J = 2.1 Hz, 1H), 8.21 (d, J = 2.1 Hz, 1H), 7.64 (d, J = 8.2 Hz, 2H), 7.45 (d, J = 8.2 Hz, 2H), 7.42 (d, J = 3.5 Hz, 1H), 6.55 (d, J = 3.5 Hz, 1H), 3.64 (s, 2H), 2.71 (bs, 4H), 2.63 (bs, 4H), 2.44 (s, 3H) ppm.HRMS(APCI + , m / z):C 19 H 23 N4[M+H + ] Calculated value: 307.1923, measured value: 307.1918. Preparation of 3-iodo-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 5) [ka]

[0326] 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (4, 2.2 g, 7.18 mmol, 1.0 eq) was suspended in acetone (0.015 M, 480 mL), N-iodosuccinimide (1.78 g, 7.9 mmol, 1.1 eq) was added, and the reaction mixture was stirred at room temperature overnight. The solvent was evaporated and the crude product was purified by column chromatography (0-10% MeOH in DCM containing 1% EtN) to give 3-iodo-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (4, 2.2 g, 7.18 mmol, 1.0 eq). To this was obtained (5, 2.3 g, 74% yield)-1H-pyrrolo[2,3-b]pyridine (5, 2.3 g, 74% yield) as a light brown solid. 1 H NMR (400 MHz, methanol-d4) δ 8.46 (d, J = 2.1 Hz, 1H), 7.88 (d, J = 2.1 Hz, 1H), 7.64 (d, J = 8.2 Hz, 2H), 7.57 (s, 1H), 7.47 (d, J = 8.2 Hz, 2H), 3.71 (s, 2H), 3.35 (s, 1H), 3.21 (bs, 8H), 2.80 (s, 3H) ppm.HRMS(APCI + , m / z):C 19 H 22 N4I[M+H + ] Calculated value: 433.0889, measured value: 433.0883. Preparation of tert-butyl 3-iodo-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine-1-carboxylate (Compound 6) [ka]

[0327] 3-Iodo-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (5, 2.3 g, 5.32 mmol, 1.0 eq) was dissolved in a mixture of THF (250 mL) and DMF (20 mL). BocO (3.48 g, 15.96 mmol, 3.0 eq) and DMAP (0.97 g, 7.98 mmol, 1.5 eq) were added and the mixture was stirred at room temperature overnight. Water was added and the reaction mixture was extracted with ethyl acetate (3x). The combined organic layers were washed with brine, dried over sodium sulfate, filtered and the solvent was evaporated. The crude material was purified by column chromatography on silica (0-10% MeOH in DCM containing 1% EtN) to give tert-butyl 3-iodo-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine-1-carboxylate as a light brown solid (6, 2.0 g, 71% yield). 1 H NMR (400 MHz, chloroform-d) δ 8.73 (d, J = 2.1 Hz, 1H), 7.84 (d, J = 2.1 Hz, 1H), 7.82 (s, 1H), 7.59 (d, J = 8.2 Hz, 2H), 7.45 (d, J = 8.2 Hz, 2H), 3.58 (s, 2H), 2.53 (bs, 4H), 2.47 (bs, 4H), 2.30 (s, 3H), 1.69 (s, 9H) ppm.HRMS(APCI + , m / z):C 24 H 30 N4O2I[M+H + ] Calculated value: 533.1413, measured value: 533.1423. General procedure for Suzuki coupling and in situ deprotection [ka] Scheme 2: General synthetic scheme for Suzuki coupling and in situ deprotection.

[0328] tert-Butyl 3-iodo-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine-1-carboxylate (6, 26.2 mg, 0.05 mmol, 1.0 eq) was dissolved in 1,4-dioxane (2.0 mL) in a 1-dram glass vial with a Teflon-lined cap. Boronic acid or boronic ester (0.075 mmol, 1.5 eq) was added, followed by tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4, 5.8 mg, 5 mol%). The reaction mixture was degassed, aqueous Na2CO3 (2.0 M, 0.5 mL) was added, and the mixture was stirred at 100 °C overnight. The reaction mixture was filtered over Celite, and the Celite was washed with ethyl acetate. The solvent was evaporated, and the crude product was purified by preparative HPLC purification to give pure material. Preparation of 3-(1H-indol-5-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 1) [ka]

[0329] Compound 1 was prepared using 5-indolylboronic acid (12.1 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 3-(1H-indol-5-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (1, 14.8 mg, 70% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.61 (d, J = 2.0 Hz, 1H), 8.52 (d, J = 2.0 Hz, 1H), 7.86 (dd, J = 1.7, 0.7 Hz, 1H), 7.70 (d, J = 8.2 Hz, 2H), 7.67 (s, 1H), 7.50 (m, 3H), 7.44 (dd, J = 8.4, 1.7 Hz, 1H), 7.28 (d, J = 3.2 Hz, 1H), 6.51 (dd, J = 3.2, 0.9 Hz, 1H), 3.84 (s, 2H), 3.00 - 2.80 (bs, 8H), 2.87 (s, 3H) ppm.Spectral data was consistent with data reported in the literature. Preparation of 3-(1H-indol-2-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 7a) [ka]

[0330] Compound 7a was prepared using 2-boronic acid pinacol ester (18.2 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 3-(1H-indol-2-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7a, 14.1 mg, 67% yield). 1H NMR (400 MHz, methanol-d4) δ 8.54 (d, J = 2.1 Hz, 1H), 8.49 (d, J = 2.1 Hz, 1H), 7.82 (s, 1H), 7.65 (d, J = 8.2 Hz, 2H), 7.54 (dt, J = 7.8, 1.0 Hz, 1H), 7.41 (d, J = 8.1 Hz, 2H), 7.42 - 7.35 (m, 1H), 7.07 (ddd, J = 8.0, 7.2, 1.1 Hz, 1H), 7.00 (ddd, J = 8.0, 7.1, 1.1 Hz, 1H), 6.78 (d, J = 1.0 Hz, 1H), 3.56 (s, 2H), 2.75 - 2.40 (bs, 8H), 2.34 (s, 3H) ppm.HRMS(APCI + , m / z):C 27 H 28 N5[M+H + ] Calculated value: 422.2339, measured value: 422.2336. Preparation of 3-(6-ethoxypyridin-3-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 7b) [ka]

[0331] Compound 7b was prepared using 6-ethoxy-3-pyridinylboronic acid (12.5 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 3-(6-ethoxypyridin-3-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7b, 16.2 mg, 76% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.48 (d, J = 2.1 Hz, 1H), 8.42 (dd, J = 2.6, 0.8 Hz, 1H), 8.33 (d, J = 2.1 Hz, 1H), 7.99 (dd, J = 8.6, 2.5 Hz, 1H), 7.66 (s, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 6.88 (dd, J = 8.6, 0.8 Hz, 1H), 4.34 (q, J = 7.0 Hz, 2H), 2.80 - 2.45 (bs, 8H), 2.40 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H) ppm.HRMS(APCI + , m / z):C 26 H 30 NO[M+H + ] Calculated value: 428.2450, measured value: 428.2449. Preparation of 3-(dibenzo[b,d]furan-4-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 7c) [ka]

[0332] Compound 7c was prepared using 4-(dibenzofuranyl)boronic acid (15.9 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 3-(dibenzo[b,d]furan-4-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7c, 12.6 mg, 58% yield). 1H NMR (400 MHz, methanol-d4) δ 8.57 (s, 2H), 8.18 (s, 1H), 8.12 (dt, J = 7.7, 1.2 Hz, 1H), 8.00 (dd, J = 7.7, 1.2 Hz, 1H), 7.94 (dd, J = 7.6, 1.2 Hz, 1H), 7.72 (d, J = 8.2 Hz, 2H), 7.70 - 7.65 (m, 2H), 7.57 - 7.48 (m, 4H), 3.69 (s, 2H), 3.05 - 2.75 (bs, 4H), 2.85 - 2.55 (bs, 4H), 2.59 (s, 3H) ppm.HRMS(APCI + , m / z):C 31 H 29 NO[M+H + ] Calculated value: 473.2341, measured value: 473.2345. Preparation of 3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 7d) [ka]

[0333] Compound 7d was prepared using 1,4-benzodioxane-6-boronic acid (13.5 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7d, 10.2 mg, 46% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.45 (d, J = 2.1 Hz, 1H), 8.34 (d, J = 2.1 Hz, 1H), 7.61 (d, J = 8.2 Hz, 2H), 7.55 (s, 1H), 7.43 (d, J = 8.2 Hz, 2H), 7.17 - 7.10 (m, 2H), 6.91 (d, J = 8.8 Hz, 1H), 4.27 (s, 4H), 3.60 (s, 2H), 2.77 - 2.46 (bs, 8H), 2.39 (s, 3H) ppm.HRMS(APCI + , m / z):C 27 H 29 N4O2[M+H + ] Calculated value: 441.2291, measured value: 441.2287. Preparation of 4-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)aniline (Compound 7e) [ka]

[0334] Compound 7e was prepared using 4-aminophenylboronic acid pinacol ester (17.0 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 4-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)aniline (7e, 14.4 mg, 73% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.43 (d, J = 2.1 Hz, 1H), 8.34 (d, J = 2.1 Hz, 1H), 7.61 (d, J = 8.2 Hz, 2H), 7.50 (s, 1H), 7.46 -7.39 (m, 4H), 6.84 (d, J = 8.5 Hz, 2H), 3.62 (s, 2H), 2.79 (bs, 4H), 2.63 (bs, 4H), 2.49 (s, 3H) ppm.HRMS(APCI + , m / z):C 25 H28 N5[M+H + Calculated value for : 3 98.2345, Actual value: 398.2344. Preparation of 3-(benzo[b]thiophen-3-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 7f) [ka]

[0335] Compound 7f was prepared using benzo[b]thien-3-ylboronic acid (14.0 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 3-(benzo[b]thiophen-3-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7f, 16.0 mg, 73% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.50 (d, J = 2.1 Hz, 1H), 8.17 (d, J = 2.1 Hz, 1H), 7.97 - 7.87 (m, 2H), 7.71 (s, 1H), 7.63 (s, 1H), 7.55 (d, J = 8.2 Hz, 2H), 7.44 - 7.33 (m, 4H), 3.55 (s, 2H), 2.71 - 2.45 (bs, 8H), 2.37 (s, 3H) ppm.HRMS(APCI + , m / z):C 27 H 27 N4S[M+H + ] Calculated value: 439.1956, measured value: 439.1955. Preparation of 3-(benzo[b]thiophen-2-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 7g) [ka]

[0336] Compound 7g was prepared using benzo[b]thien-2-ylboronic acid (14.0 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC purification to give 3-(benzo[b]thiophen-2-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7g, 15.9 mg, 73% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.52 (d, J = 2.1 Hz, 1H), 8.50 (d, J = 2.1 Hz, 1H), 7.83 - 7.74 (m, 3H), 7 .64 (d, J = 8.2 Hz, 2H), 7.57 (d, J = 0.7 Hz, 1H), 7.43 (d, J = 8.2 Hz, 2H), 7.32 (ddd, J = 7.9, 7.2, 1.2 Hz, 1H), 7.26 (ddd, J = 8.4, 7.2, 1.2 Hz, 1H), 3.59 (s, 2H), 2.72 - 2.48 (bs, 8H), 2.38 (s, 3H) ppm.HRMS(APCI + , m / z):C 27 H 27 N4S[M+H + ] Calculated value: 439.1956, measured value: 439.1959. Preparation of 3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)aniline (compound 7h) [ka]

[0337] Compound 7h was prepared using 3-aminophenylboronic acid hydrochloride (13.3 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)aniline (7h, 14.9 mg, 75% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.47 (d, J = 2.1 Hz, 1H), 8.45 (d, J = 2.1 Hz, 1H), 7.64 (d, J = 8.2 Hz, 2H), 7.61 (s, 1H), 7.44 (d, J = 8.2 Hz, 2H), 7.19 (t, J = 7.8 Hz, 1H), 7.11 (t, J = 2.0 Hz, 1H), 7.03 (ddd, J = 7.6, 1.7, 1.0 Hz, 1H), 6.68 (ddd, J = 7.8, 2.3, 1.0 Hz, 1H), 3.60 (s, 2H), 2.58 (bs, 8H), 2.34 (s, 3H) ppm.HRMS(APCI + , m / z):C 25 H 28 N5[M+H + ] Calculated value: 398.2345, measured value: 398.2337. Preparation of 2-fluoro-5-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid (compound 7i) [ka]

[0338] Compound 7i was prepared by Suzuki coupling and i using 3-carboxy-4-fluorophenylboronic acid (13.8 mg, 0.075 mmol, 1.5 eq) as described above. Prepared according to the general procedure for in situ deprotection. The crude product was purified by preparative HPLC to give 2-fluoro-5-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid (7i, 8.9 mg, 40% yield). 1 H NMR (500 MHz, methanol-d4) δ 8.56 (d, J = 2.0 Hz, 1H), 8.53 (d, J = 2.0 Hz, 1H), 8.24 (dd, J = 6.8, 2.5 Hz, 1H), 7.95 (ddd, J = 8.6, 4.5, 2.5 Hz, 1H), 7.81 (s, 1H), 7.75 (d, J = 8.2 Hz, 2H), 7.55 (d, J = 8.2 Hz, 2H), 7.32 (dd, J = 10.6, 8.6 Hz, 1H), 3.96 (s, 2H), 3.38 (bs, 4H), 3.05 (bs, 4H), 2.91 (s, 3H) ppm.HRMS(APCI + , m / z):C 26 H 26 FN4O2[M+H + ] Calculated value: 445.2040, measured value: 445.2035. Preparation of 3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid (compound 7j) [ka]

[0339] Compound 7j was prepared using 3-carboxyphenylboronic acid (12.5 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid (7j, 12.0 mg, 56% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.62 - 8.56 (m, 2H), 8.36 (s, 1H), 8.02 - 7.94 (m, 2H), 7.85 (s, 1H), 7.76 (d, J = 8.0 Hz, 2H), 7.60 (d, J = 7.8 Hz, 1H), 7.56 (d, J = 8.0 Hz, 2H), 3.97 (s, 2H), 3.39 (bs, 4H), 3.05 (bs, 4H), 2.91 (s, 3H) ppm.HRMS(APCI + , m / z):C 26 H 27 N4O2[M+H + ] Calculated value: 427.2134, measured value: 427.2137. Preparation of 5-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine (compound 7k) [ka]

[0340] Compound 7k was prepared using 2-aminopyrimidine-5-boronic acid (11.0 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC purification to give 5-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-2-amine (7k, 16.4 mg, 82%). 1H NMR (400 MHz, methanol-d4) δ 8.87 (s, 2H), 8.58 (d, J = 2.1 Hz, 1H), 8.46 (d, J = 2.1 Hz, 1H), 7.87 (s, 1H), 7.76 (d, J = 8.2 Hz, 2H), 7.54 (d, J = 8.2 Hz, 2H), 3.93 (s, 2H), 3.37 (bs, 4H), 3.01 (bs, 4H), 2.90 (s, 3H) ppm.HRMS(APCI + , m / z):C 23 H 26 N7[M+H + ] Calculated value: 400.2250, measured value: 400.2250. Preparation of 3-methyl-5-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2-amine (Compound 7l) [ka]

[0341] Compound 7l was prepared using 2-amino-3-methylpyridine-5-boronic acid pinacol ester (18.3 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC purification to give 3-methyl-5-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2-amine (7l, 8.2 mg, 40% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.55 (s, 1H), 8.42 (d, J = 2.0 Hz, 1H), 8.24 (dd, J = 2.2, 1.1 Hz, 1H), 8.14 - 8.08 (m, 1H), 7.83 (s, 1H), 7.73 (d, J = 8.2 Hz, 2H), 7.51 (d, J = 8.1 Hz, 2H), 3.79 (s, 2H), 3.04 - 2.68 (bs, 8H), 2.87 (s, 3H), 2.37 (s, 3H) ppm. HRMS (APCI) + , m / z):C 25 H 29 N6[M+H + ] Calculated value: 413.2454, measured value: 413.2457. Preparation of 5-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2-amine (Compound 7m) [ka]

[0342] Compound 7m was prepared using 2-aminopyridine-5-boronic acid pinacol ester (16.5 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC purification to give 5-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)pyridin-2-amine (7m, 15.2 mg, 76% yield). 1H NMR (400 MHz, methanol-d4) δ 8.60 (d, J = 2.0 Hz, 1H), 8.52 (d, J = 2.0 Hz, 1H), 8.35 (dd, J = 9.3, 2.2 Hz, 1H), 8.22 (dd, J = 2.2, 0.7 Hz, 1H), 7.87 (s, 1H), 7.80 (d, J = 8.2 Hz, 2H), 7.59 (d, J = 8.2 Hz, 2H), 7.16 (dd, J = 9.2, 0.7 Hz, 1H), 4.10 (s, 2H), 3.46 (bs, 4H), 3.20 (s, 4H), 2.93 (s, 3H) ppm.HRMS(APCI + , m / z):C 24 H 27 N6[M+H + ] Calculated value: 399.2297, measured value: 399.2307. Preparation of 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine (compound 7n) [ka]

[0343] Compound 7n was prepared using 4-pyridinylboronic acid (10.3 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine (7n, 8.7 mg, 45% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.74 (d, J = 2.0 Hz, 1H), 8.68–8.64 (m, 3H), 8. 57 (s, 1H), 8.43 (d, J = 7.1 Hz, 2H), 7.83 (d, J = 8.2 Hz, 2H), 7.60 (d, J = 8.2 Hz, 2H), 4.04 (s, 2H), 3.43 (bs, 4H), 3.13 (bs, 4H), 2.92 (s, 3H) ppm.HRMS(APCI + , m / z):C 24 H 26 N5[M+H + ] Calculated value: 384.2188, measured value: 384.2194. Preparation of 4-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (Compound 7o) [ka]

[0344] Compound 7o was prepared using 4-cyanophenylboronic acid (11.0 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 4-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (7o, 14.4 mg, 71% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.56 (d, J = 2.1 Hz, 1H), 8.54 (d, J = 2.1 Hz, 1H), 7.97 - 7.91 (m, 3H), 7.83 - 7.77 (m, 2H), 7.75 (d, J = 8.2 Hz, 2H), 7.54 (d, J = 8.2 Hz, 2H), 3.89 (s, 2H), 3.35 (bs, 4H), 2.96 (bs, 4H), 2.89 (s, 3H) ppm.HRMS(APCI + , m / z):C 26 H 26 N5[M+H + ] Calculated value: 408.2188, measured value: 408.2198. Preparation of 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-(3-nitrophenyl)-1H-pyrrolo[2,3-b]pyridine (compound 7p) [ka]

[0345] Compound 7p was prepared using 3-nitrophenylboronic acid (12.5 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-(3-nitrophenyl)-1H-pyrrolo[2,3-b]pyridine (7p, 15.4 mg, 72% yield). ) was obtained. 1 H NMR (400 MHz, methanol-d4) δ 8.59 (d, J = 2.0 Hz, 1H), 8.58 (d, J = 2.0 Hz, 1H), 8.54 (t, J = 2.0 Hz, 1H), 8.15 (dddd, J = 7.7, 4.3, 2.1, 1.0 Hz, 2H), 7.96 (s, 1H), 7.77 (d, J = 8.2 Hz, 2H), 7.71 (t, J = 8.0 Hz, 1H), 7.58 (d, J = 8.2 Hz, 2H), 4.08 (s, 2H), 3.44 (bs, 4H), 3.17 (bs, 4H), 2.93 (s, 3H) ppm.HRMS(APCI + , m / z):C 25 H 26 N5O2[M+H + ] Calculated value: 428.2087, measured value: 428.2081. Preparation of 3-(4-isopropoxyphenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 7q) [ka]

[0346] Compound 7q was prepared using 4-isopropoxyphenylboronic acid (13.5 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 3-(4-isopropoxyphenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7q, 18.2 mg, 83% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.61 (d, J = 2.0 Hz, 1H), 8.56 (d, J = 2.0 Hz, 1H), 7.75 (d, J = 8.2 Hz, 2H), 7.69 (s, 1H), 7.64 - 7.58 (m, 2H), 7.55 (d, J = 8.2 Hz, 2H), 7.08 - 6.98 (m, 2H), 4.65 (septet, J = 6.0 Hz, 1H), 3.94 (s, 2H), 3.37 (bs, 4H), 3.02 (bs, 4H), 2.90 (s, 3H), 1.35 (d, J = 6.0 Hz, 6H) ppm.HRMS(APCI + , m / z):C 28 H 33 NO[M+H + ] Calculated value: 441.2654, measured value: 441.2646. Preparation of 3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenol (compound 7r) [ka]

[0347] Compound 7r was prepared using 3-hydroxyphenylboronic acid (10.3 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenol (7r, 9.8 mg, 49% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.68 (d, J = 1.9 Hz, 1H), 8.60 (s, 1H), 7.80 - 7.73 (m, 3H), 7.58 (d, J = 8.2 Hz, 2H), 7.30 (t, J = 7.8 Hz, 1H), 7.24 - 7.17 (m, 1H), 7.16 (t, J = 2.0 Hz, 1H), 6.78 (ddd, J = 8.1, 2.5, 1.0 Hz, 1H), 4.04 (s, 2H), 3.42 (bs, 4H), 3.13 (bs, 4H), 2.92 (s, 3H) ppm.HRMS(APCI + , m / z):C 25 H 27 NO[M+H + ] Calculated value: 399.2185, measured value: 399.2180. Preparation of 3-(1-methyl-1H-pyrazol-4-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 7s) [ka]

[0348] Compound 7s was prepared using 1-methylpyrazole-4-boronic acid pinacol ester (16.1 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 3-(1-methyl-1H-pyrazol-4-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7s, 14.8 mg, 77% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.62 (d, J = 2.0 Hz, 1H), 8.31 (d, J = 2.0 Hz, 1H), 7.80 (s, 1H), 7.76 (d, J = 8.2 Hz, 2H), 7.64 (d, J = 2.0 Hz, 1H), 7.58 (d, J = 8.2 Hz, 2H), 6.60 (d, J = 2.1 Hz, 1H), 4.14 (s, 2H), 3.95 (s, 3H), 3.48 (bs, 4H), 3.25 (bs, 4H), 2.94 (s, 3H) ppm.HRMS(APCI + , m / z):C 23 H 27 N6[M+H + ] Calculated value: 387.2297, measured value: 387.2303. Preparation of 4-((5-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)thiophen-2-yl)methyl)morpholine (Compound 7t) [ka]

[0349] Compound 7t was prepared using 5-(morpholinomethyl)-2-thiopheneboronic acid pinacol ester (24.4 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC purification to give 4-((5-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)thiophen-2-yl)methyl)morpholine (7t, 10.0 mg, 41% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.57 (d, J = 2.1 Hz, 1H), 8.52 (d, J = 2.1 Hz, 1H), 7.84 (s, 1H), 7.75 (d, J = 8.2 Hz, 2H), 7.57 (d, J = 8.2 Hz, 2H), 7.42 (d, J = 3.7 Hz, 1H), 7.37 (d, J = 3.7 Hz, 1H), 4.64 (s, 2H), 4.06 (bs, 4H), 4.01 (s, 2H), 3.80 (bs, 4H), 3.41 (bs, 4H), 3.09 (bs, 4H), 2.92 (s, 3H) ppm.HRMS(APCI + , m / z):C 28 H 34 N5OS[M+H + ] Calculated value: 488.2484, measured value: 488.2475. Preparation of 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-(6-(methylsulfonyl)pyridin-3-yl)-1H-pyrrolo[2,3-b]pyridine (compound 7u) [ka]

[0350] Compound 7u was prepared using 6-(methylsulfonyl)pyridine-3-boronic acid (15.0 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-(6-(methylsulfonyl)pyridin-3-yl)-1H-pyrrolo[2,3-b]pyridine (7u, 11.0 mg, 48% yield). 1 H NMR (400 MHz, methanol-d4) δ 9.14 (dd, J = 2.2, 0.8 Hz, 1H), 8.59 (q, J = 2.1 Hz, 2H), 8.46 (dd, J = 8.2, 2.2 Hz, 1H), 8.15 (dd, J = 8.2, 0.8 Hz, 1H), 8.08 (s, 1H), 7.78 (d, J = 8.2 Hz, 2H), 7.57 (d, J = 8.2 Hz, 2H), 4.02 (s, 2H), 3.41 (bs, 4H), 3.27 (s, 3H), 3.11 (bs, 4H), 2.92 (s, 3H) ppm.HRMS(APCI + , m / z):C 25 H 28 N5O2S[M+H + ] Calculated value: 462.1964, measured value: 462.1968. Preparation of 5-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-indazole (Compound 7v) [ka]

[0351] Compound 7v was prepared using 1H-indazole-5-boronic acid (12.1 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC purification to give 5-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-indazole (7v, 8.4 mg, 40% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.67 (d, J = 1.9 Hz, 1H), 8.58 (d, J = 1.9 Hz, 1H), 8.13 - 8.09 (m, 2H), 7.79 (s, 1H), 7.80 - 7.73 (m, 3H), 7.67 (dt, J = 8.7, 0.9 Hz, 1H), 7.55 (d, J = 8.3 Hz, 2H), 3.95 (s, 2H), 3.38 (bs, 4H), 3.03 (bs, 4H), 2.90 (s, 3H) ppm.HRMS(APCI + , m / z):C 26 H 27 N6[M+H + ] Calculated value: 423.2297, measured value: 423.2298. Preparation of 3-(furan-3-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 7w) [ka]

[0352] Compound 7w was prepared using 3-furanylboronic acid (7w, 8.4 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC purification to give 3-(furan-3-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)furan. (phenyl)-1H-pyrrolo[2,3-b]pyridine (12.9 mg, yield 69%) was obtained. 1 H NMR (500 MHz, methanol-d4) δ 8.58 (s, 2H), 8.08 (t, J = 1.1 Hz, 1H), 7.80 (d, J = 8.2 Hz, 2H), 7.76 (s, 1H), 7.62 (t, J = 1.7 Hz, 1H), 7.58 (d, J = 8.2 Hz, 2H), 6.87 (dd, J = 1.8, 1.1 Hz, 1H), 4.00 (s, 2H), 3.41 (bs, 4H), 3.07 (bs, 4H), 2.92 (s, 3H) ppm.HRMS(APCI + , m / z):C 23 H 25 NO[M+H + ] Calculated value: 373.2028, measured value: 373.2036. Preparation of 1-(4-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)ethan-1-one (compound 7x) [ka]

[0353] Compound 7x was prepared using 4-acetylphenylboronic acid (12.3 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 1-(4-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)ethan-1-one (7x, 15.1 mg, 71% yield). 1 H NMR (500 MHz, methanol-d4) δ 8.63 (d, J = 2.0 Hz, 1H), 8.58 (d, J = 2.0 Hz, 1H), 8.11 (d, J = 8.4 Hz, 2H), 7.95 (s, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.78 (d, J = 8.2 Hz, 2H), 7.57 (d, J = 8.2 Hz, 2H), 4.01 (s, 2H), 3.41 (bs, 4H), 3.09 (bs, 4H), 2.92 (s, 3H), 2.65 (s, 3H) ppm.HRMS(APCI + , m / z):C 27 H 29 NO[M+H + ] Calculated value: 425.2341, measured value: 425.2347. Preparation of 3-(3-fluorophenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 7y) [ka]

[0354] Compound 7y was prepared using 3-fluorophenylboronic acid (10.5 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 3-(3-fluorophenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7y, 12.8 mg, 64% yield). 1 H NMR (500 MHz, methanol-d4) δ 8.59 (d, J = 2.0 Hz, 1H), 8.58 (d, J = 2.2 Hz, 1H), 7.85 (s, 1H), 7.78 (d, J = 8.2 Hz, 2H), 7.61 - 7.55 (m, 3H), 7.52 - 7.44 (m, 2H), 7.11 - 7.02 (m, 1H), 4.02 (s, 2H), 3.41 (bs, 4H), 3.09 (bs, 4H), 2.92 (s, 3H) ppm.HRMS(APCI + , m / z):C 25 H 26 N4F[M+H + ] Calculated value: 401.2142, measured value: 401.2146. Preparation of 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-(4-(trifluoromethoxy)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 7z) [ka]

[0355] Compound 7z was prepared using 4-(trifluoromethoxy)phenylboronic acid (15.4 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-(4-(trifluoromethoxy)phenyl)-1H-pyrrolo[2,3-b]pyridine (7z, 17.4 mg, 75% yield). 1 H NMR (500 MHz, methanol-d4) δ 8.62 (d, J = 2.0 Hz, 1H), 8.59 (d, J = 2.0 Hz, 1H), 7.85 (s, 1H), 7.86 - 7.80 (m, 2H), 7.79 (d, J = 8.2 Hz, 2H), 7.58 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 4.05 (s, 2H), 3.43 (bs, 4H), 3.13 (bs, 4H), 2.92 (s, 3H) ppm.HRMS(APCI + , m / z):C 26 H 26 F3N4O[M+H +] Calculated value: 467.2059, measured value: 467.2062. Preparation of 4-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-indazole (compound 7aa) [ka]

[0356] Compound 7aa was prepared using 1H-indazole-5-boronic acid (12.5 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 4-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-indazole (7aa, 15.6 mg, 74% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.62 (d, J = 2.0 Hz, 1H), 8.52 (d, J = 2.0 Hz, 1H), 8.18 (s, 1H), 7.94 (s, 1H), 7.73 (d, J = 8.2 Hz, 2H), 7.58 - 7.51 (m, 4H), 7.44 (dd, J = 6.2, 1.7 Hz, 1H), 3.97 (s, 2H), 3.38 (bs, 4H), 3.05 (bs, 4H), 2.90 (s, 3H) ppm.HRMS(APCI + , m / z):C 26 H 27 N6[M+H + ] Calculated value: 423.2297, measured value: 423.2303. Preparation of 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-(3-(trifluoromethyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 7ab) [ka]

[0357] Compound 7ab was prepared using 3-(trifluoromethyl)phenylboronic acid (14.2 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-(3-(trifluoromethyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7ab, 17.0 mg, 75% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.59 (d, J = 2.0 Hz, 1H), 8.54 (d, J = 2.0 Hz, 1H), 8.00 (d, J = 7.8 Hz, 1H), 7.97 (s, 1H), 7.89 (s, 1H), 7.76 (d, J = 8.2 Hz, 2H), 7.68 (t, J = 7.7 Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.57 (d, J = 8.2 Hz, 2H), 4.03 (s, 2H), 3.42 (bs, 4H), 3.12 (bs, 4H), 2.92 (s, 3H) ppm.HRMS(APCI + , m / z):C 26 H 26 F3N4[M+H + ] Calculated value: 451.2110, measured value: 451.2115. Preparation of 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-phenyl-1H-pyrrolo[2,3-b]pyridine (compound 7ac) [ka]

[0358] Compound 7ac was prepared using phenylboronic acid (9.6 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-phenyl-1H-pyrrolo[2,3-b]pyridine (7ac, 16.2 mg, 85% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.69 (d, J = 1.9 Hz, 1H), 8.61 (d, J = 1.9 Hz, 1H), 7.82 (s, 1H), 7.78 (d, J = 8.2 Hz, 2H), 7.76 - 7.69 (m, 2H), 7.59 (d, J = 8.3 Hz, 2H), 7.53 - 7.44 (m, 2H), 7.39 - 7.29 (m, 1H), 4.08 (s, 2H), 3.45 (bs, 4H), 3.17 (bs, 4H), 2.93 (s, 3H) ppm.HRMS(APCI + , m / z):C 25 H 27 N4[M+H + ] Calculated value: 383.2236, measured value: 383.2236. Preparation of 3-(3-chlorophenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 7ad) [ka]

[0359] Compound 7ad was prepared using 3-chlorophenylboronic acid (11.7 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 3-(3-chlorophenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7ad, 18.4 mg, 88% yield).1 H NMR (400 MHz, methanol-d4) δ 8.60 (d, J = 1.3 Hz, 2H), 7.85 (s, 1H), 7.79 (d, J = 8.2 Hz, 2H), 7.71 (t, J = 1.9 Hz, 1H), 7.66 (dt, J = 7.8, 1.3 Hz, 1H), 7.61 (d, J = 8.2 Hz, 2H), 7.46 (t, J = 7.9 Hz, 1H), 7.32 (ddd, J = 8.1, 2.1, 1.0 Hz, 1H), 4.17 (s, 2H), 3.49 (bs, 4H), 3.29 (bs, 4H), 2.94 (s, 3H) ppm.HRMS(APCI + , m / z):C 25 H 26 ClN4 [M+H + ] Calculated value: 417.1846, measured value: 417.1843. Preparation of 3-(2,4-dichlorophenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 7ae) [ka]

[0360] Compound 7ae was prepared using 2,4-dichlorophenylboronic acid (14.3 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 3-(2,4-dichlorophenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7ae, 14.9 mg, 66% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.61 (d, J = 2.0 Hz, 1H), 8.32 (d, J = 2.0 Hz, 1H), 7.77 (s, 1H), 7.75 (d, J = 8.3 Hz, 2H), 7.64 (d, J = 2.2 Hz, 1H), 7.58 (dd, J = 8.3, 1.4 Hz, 3H), 7.44 (dd, J = 8.3, 2.2 Hz, 1H), 4.16 (s, 2H), 3.49 (bs, 4H), 3.27 (bs, 4H), 2.94 (s, 3H) ppm.HRMS(APCI + , m / z):C 25 H 25 Cl2N4[M+H + ] Calculated value: 451.1456, measured value: 451.1447. Preparation of 3-(2,5-dimethylphenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 7af) [ka]

[0361] Compound 7af was dissolved in 2,5-dimethylphenylboronic acid (11.2 mg, 0.075 mmHg). The crude product was purified by preparative HPLC purification to give 3-(2,5-dimethylphenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7af, 14.0 mg, 68% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.65 (d, J = 1.9 Hz, 1H), 8.36 (d, J = 1.9 Hz, 1H), 7.75 (d, J = 8.2 Hz, 2H), 7.63 (s, 1H), 7.60 (d, J = 8.2 Hz, 2H), 7.25 (d, J = 7.8 Hz, 1H), 7.21 (s, 1H), 7.13 (dd, J = 7.4, 1.7 Hz, 1H), 4.18 (s, 2H), 3.50 (bs, 4H), 3.30 (bs, 4H), 2.94 (s, 3H), 2.35 (s, 3H), 2.26 (s, 3H) ppm.HRMS(APCI + , m / z):C 27 H 31 N4[M+H + ] Calculated value: 411.2549, measured value: 411.2556. Preparation of 1-methyl-6-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-indazole (Compound 7ag) [ka]

[0362] Compound 7ag was prepared using 1-methyl-1H-indazole-6-boronic acid (13.2 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 1-methyl-6-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-indazole (7ag, 13.2 mg, 60% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.67 (d, J = 2.0 Hz, 1H), 8.58 (d, J = 2.0 Hz, 1H), 8.01 (d, J = 0.9 Hz, 1H), 7.88 (s, 1H), 7.84 (dd, J = 8.4, 0.9 Hz, 1H), 7.81 (d, J = 1.2 Hz, 1H), 7.76 (d, J = 8.2 Hz, 2H), 7.60 - 7.52 (m, 3H), 4.11 (s, 3H), 3.99 (s, 2H), 3.40 (bs, 4H), 3.09 (bs, 4H), 2.91 (s, 3H) ppm.HRMS(APCI +, m / z):C 27 H 29 N6[M+H + ] Calculated value: 437.2454, measured value: 437.2449. Preparation of 3-(1-benzyl-1H-pyrazol-4-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 7ah) [ka]

[0363] Compound 7ah was prepared using 1-benzyl-1H-pyrazole-4-boronic acid (15.2 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 3-(1-benzyl-1H-pyrazol-4-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7ah, 14.3 mg, 62% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.61 (d, J = 2.0 Hz, 1H), 8.57 (d, J = 2.0 Hz, 1H), 8.19 (d, J = 0.8 Hz, 1H), 7.94 (d, J = 0.8 Hz, 1H), 7.79 (d, J = 8.2 Hz, 2H), 7.75 (s, 1H), 7.57 (d, J = 8.2 Hz, 2H), 7.39 - 7.26 (m, 5H), 5.42 (s, 2H), 4.01 (s, 2H), 3.41 (bs, 4H), 3.09 (bs, 4H), 2.92 (s, 3H) ppm.HRMS(APCI + , m / z):C 29 H 31 N6[M+H + ] Calculated value: 463.2610, measured value: 463.2612. Preparation of 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (compound 7ai) [ka]

[0364] Compound 7ai was prepared using 1,3,5-trimethyl-1H-pyrazole-4-boronic acid pinacol ester (17.7 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-(1,3,5-trimethyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridine (7ai, 7.6 mg, 37% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.59 (d, J = 2.0 Hz, 1H), 8.16 (d, J = 2.0 Hz, 1H), 7.70 (d, J = 8.2 Hz, 2H), 7.56 - 7 .50 (m, 3H), 3.93 (s, 2H), 3.88 (s, 3H), 3.37 (bs, 4H), 3.01 (bs, 4H), 2.90 (s, 3H), 2.28 (s, 3H), 2.22 (s, 3H) ppm.HRMS(APCI + , m / z):C 25 H 31 N6[M+H + ] Calculated value: 415.2610, measured value: 415.2604. Preparation of 3-(3,5-bis(trifluoromethyl)phenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 7aj) [ka]

[0365] Compound 7aj was prepared using 3,5-bis(trifluoromethyl)phenylboronic acid (19.3 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 3-(3,5-bis(trifluoromethyl)phenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7aj, 11.2 mg, 43% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.58 (d, J = 2.0 Hz, 1H), 8.44 (d, J = 2.0 Hz, 1H), 8.29 - 8.23 ​​(m, 2H), 8.02 (s, 1H), 7.86 (s, 1H), 7.74 (d, J = 8.2 Hz, 2H), 7.56 (d, J = 8.2 Hz, 2H), 3.96 (s, 2H), 3.38 (bs, 4H), 3.04 (bs, 4H), 2.90 (s, 3H) ppm.HRMS(APCI + , m / z):C 27 H 25 F6N4[M+H + ] Calculated value: 519.1983, measured value: 519.1983. Preparation of 1-methyl-3-(3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)urea (Compound 7ak) [ka]

[0366] Compound 7ak was prepared using (3-(3-methylureido)phenyl)boronic acid (13a, 14.6 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by HPLC. Purification by preparative HPLC purification afforded 1-methyl-3-(3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)urea (7ak, 6.4 mg, 14% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.70 (d, J = 2.0 Hz, 1H), 8.57 (d, J = 2.0 Hz, 1H), 7.96 (t, J = 1.8 Hz, 1H), 7.81 - 7.75 (m, 3H), 7.54 (d, J = 8.2 Hz, 2H), 7.39 - 7.31 (m, 2H), 7.18 (dt, J = 7.3, 2.0 Hz, 1H), 3.91 (s, 2H), 3.35 (bs, 4H), 2.98 (bs, 4H), 2.89 (s, 3H), 2.80 (s, 3H) ppm.HRMS(APCI + , m / z):C 27 H 31 NO[M+H + ] Calculated value: 455.2559, measured value: 455.2559. Preparation of methyl (3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)carbamate (compound 7al) [ka]

[0367] Compound 7al was prepared using (3-((methoxycarbonyl)amino)phenyl)boronic acid (13b, 14.6 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC purification to give methyl (3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)carbamate (7al, 8.5 mg, 19% yield). 1H NMR (400 MHz, methanol-d4) δ 8.74 (d, J = 2.0 Hz, 1H), 8.59 (d, J = 2.0 Hz, 1H), 7.98 (s, 1H), 7.83 - 7.78 (m, 3H), 7.56 (d, J = 8.2 Hz, 2H), 7.41 - 7.37 (m, 2H), 7.36 - 7.27 (m, 1H), 3.97 (s, 2H), 3.77 (s, 3H), 3.38 (bs, 4H), 3.04 (bs, 4H), 2.91 (s, 3H) ppm.HRMS(APCI + , m / z):C 27 H 30 N5O2[M+H + ] Calculated value: 456.2400, measured value: 456.2400. Preparation of 1-methyl-3-(4-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)urea (Compound 7am) [ka]

[0368] Compound 7am was prepared using (4-(3-methylureido)phenyl)boronic acid (13c, 14.6 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC purification to give 1-methyl-3-(4-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)urea (7am, 5.1 mg, 22% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.63 (d, J = 2.0 Hz, 1H), 8.56 (d, J = 2.0 Hz, 1H), 7.76 (s, 1H), 7.74 (d, J = 4.6 Hz, 2H), 7.65 - 7.60 (m, 2H), 7.55 (d, J = 8.1 Hz, 2H), 7.52 - 7.45 (m, 2H), 3.92 (s, 2H), 3.36 (bs, 4H), 2.99 (bs, 4H), 2.90 (s, 3H), 2.80 (s, 3H) ppm.HRMS(APCI + , m / z):C 27 H 31 NO[M+H + ] Calculated value: 455.2559, measured value: 455.2566. Preparation of methyl (4-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)carbamate (Compound 7an) [ka]

[0369] Compound 7an was prepared using (4-((methoxycarbonyl)amino)phenyl)boronic acid (13d, 14.6 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC purification to give (4-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)carbamate (7an, 7.6 mg, 33% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.68 (d, J = 1.9 Hz, 1H), 8.59 (d, J = 2.0 Hz, 1H), 7.82 - 7.72 (m, 3H), 7.65 (d, J = 8.7 Hz, 2H), 7.61 - 7.51 (m, 4H), 4.04 (s, 3H), 3.76 (s, 2H), 3.43 (bs, 4H), 3.13 (bs, 4H), 2 0.92 (s, 3H) ppm. HRMS (APCI + , m / z):C27 H 30 N5O2[M+H + ] Calculated value: 456.2400, measured value: 456.2397. Preparation of 3-(4-((4-methoxybenzyl)oxy)phenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 7ao) [ka]

[0370] Compound 7ao was prepared using 4-(4-methoxybenzyloxy)phenylboronic acid (19.4 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC purification to give 3-(4-((4-methoxybenzyl)oxy)phenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7ao, 20.4 mg, 79% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.69 (dd, J = 7.3, 1.9 Hz, 2H), 8.59 (d, J = 1.8 Hz, 1H), 7.77 (dd, J = 8.3, 1.6 Hz, 2H), 7.72 (d, J = 8.2 Hz, 1H), 7.66 - 7.51 (m, 3H), 7.37 (dd, J = 8.7, 6.9 Hz, 2H), 7.28 - 7.03 (m, 1H), 6.98 - 6.77 (m, 3H), 5.05 (s, 2H), 4.06 (s, 2H), 3.80 (s, 3H), 3.44 (bs, 4H), 3.16 (bs, 4H), 2.92 (s, 3H) ppm.HRMS(APCI + , m / z):C 33 H 35 N4O2[M+H + ] Calculated value: 519.2760, measured value: 519.2755. Preparation of 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (compound 7ap) [ka]

[0371] Compound 7ap was subjected to Suzuki coupling and in situ hybridization as described above using 3,4,5-trimethoxyphenylboronic acid (15.9 mg, 0.075 mmol, 1.5 eq). Prepared according to the general procedure for in situ deprotection. The crude product was purified by preparative HPLC purification to give 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl) 19.2 mg of 7ap (81% yield) was obtained. 1 H NMR (400 MHz, methanol-d4) δ 8.67 (d, J = 1.9 Hz, 1H), 8.60 (d, J = 1.9 Hz, 1H), 7.81 (s, 1H), 7.78 (d, J = 8.2 Hz, 2H), 7.59 (d, J = 8.2 Hz, 2H), 6.96 (s, 2H), 4.10 (s, 2H), 3.92 (s, 6H), 3.82 (s, 3H), 3.46 (bs, 4H), 3.20 (bs, 4H), 2.93 (s, 3H) ppm.HRMS(APCI + , m / z):C 28 H 33 N4O3[M+H + ] Calculated value: 473.2553, measured value: 473.2554. Preparation of 3-(4-methoxy-3-methylphenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 7aq) [ka]

[0372] Compound 7aq was subjected to Suzuki coupling and in situ hybridization as described above using 4-methoxy-3-methylphenylboronic acid (12.4 mg, 0.075 mmol, 1.5 eq). Prepared according to the general procedure for in situ deprotection. The crude product was purified by preparative HPLC to give 3-(4-methoxy-3-methylphenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7aq, 17.2 mg, 81% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.69 (d, J = 1.9 Hz, 1H), 8.59 (d, J = 1.9 Hz, 1H), 7.77 (d, J = 8.2 Hz, 2H), 7.72 (s, 1H), 7.59 (d, J = 8.2 Hz, 2H), 7.51 (dd, J = 8.4, 2.3 Hz, 1H), 7.48 - 7.44 (m, 1H), 7.03 (d, J = 8.4 Hz, 1H), 4.03 (s, 2H), 3.88 (s, 3H), 3.43 (bs, 4H), 3.12 (bs, 4H), 2.92 (s, 3H), 2.28 (s, 3H) ppm.HRMS(APCI + , m / z):C 27 H 31 NO[M+H + ] Calculated value: 427.2498, measured value: 427.2501. Preparation of 3-(3-(benzyloxy)phenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 7ar) [ka]

[0373] Compound 7ar was prepared using 3-(benzyloxy)phenylboronic acid (17.1 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 3-(3-(benzyloxy)phenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7ar, 15.4 mg, 63% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.58 (s, 2H), 7.79 (s, 1H), 7.74 (d, J = 8.2 Hz, 2H), 7.58 (d, J = 8.2 Hz, 2H), 7.49 - 7.43 (m, 2H), 7.43 - 7.22 (m, 6H), 7.06 - 6.96 (m, 1H), 5.17 (s, 2H), 4.06 (s, 2H), 3.43 (bs, 4H), 3.15 (bs, 4H), 2.92 (s, 3H) ppm.HRMS(APCI + , m / z):C 32 H 33 NO[M+H + ] Calculated value: 489.2654, measured value: 489.2657. Preparation of 3-(2-chlorophenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 7as) [ka]

[0374] Compound 7as was prepared using 2-chlorophenylboronic acid (17.1 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 3-(2-chlorophenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7as, 16.6 mg, 80% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.60 (d, J = 2.0 Hz, 1H), 8.32 (d, J = 2.0 Hz, 1H), 7.74 (s, 1H), 7.72 (d, J = 8.2 Hz, 2H), 7.59 (ddd, J = 7.7, 4.0, 1.7 Hz, 2H), 7.55 (d, J = 8.2 Hz, 2H), 7.43 (td, J = 7.5, 1.7 Hz, 1H), 7.38 (td, J = 7.5, 2.0 Hz, 1H), 4.01 (s, 2H), 3.41 (bs, 4H), 3.10 (bs, 4H), 2.91 (s, 3H) ppm.HRMS(APCI + , m / z):C 25 H 26 N4Cl[M+H + ] Calculated value: 417.1846, measured value: 417.1849. Preparation of 3-(2-methoxyphenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 7at) [ka]

[0375] Compound 7at was prepared using 2-methoxyphenylboronic acid (11.4 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to give 3-(2-methoxyphenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7at, 17.1 mg, 83% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.61 (d, J = 1.9 Hz, 1H), 8.58 (d, J = 1.9 Hz, 1H), 7.80 (s, 1H), 7.75 (d, J = 8.2 Hz, 2H), 7.61 - 7.54 (m, 3H), 7.38 (ddd, J = 8.3, 7.4, 1.7 Hz, 1H), 7.16 (dd, J = 8.3, 1.1 Hz, 1H), 7.08 (td, J = 7.4, 1.1 Hz, 1H), 4.02 (s, 2H), 3.86 (s, 3H), 3.42 (s, 4H), 3.11 (s, 4H), 2.92 (s, 3H) ppm.HRMS(APCI + , m / z):C 26 H 29 NO[M+H + ] Calculated value: 413.2341, measured value: 413.2337. Preparation of 1-(3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)-3-phenylurea (compound 7au) [ka]

[0376] Compound 7au was prepared using (3-(3-phenylureido)phenyl)boronic acid (13e, 19.2 mg, 0.075 mmol, 1.5 eq) following the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC purification to give 1-(3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl. The resulting product was 7au (5.4 mg, 21% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.61 (d, J = 2.0 Hz, 1H), 8.51 (d, J = 2.0 Hz, 1H), 8.08 (s, 1H), 7.78 - 7.71 (m, 3H), 7.60 (d, J = 6.9 Hz, 2H), 7.49 (d, J = 8.0 Hz, 2H), 7.46 - 7.40 (m, 1H), 7.41 - 7.36 (m, 2H), 7.36 - 7.27 (m, 2H), 7.24 - 7.17 (m, 1H), 3.76 (s, 2H), 3.13 (bs, 8H), 2.85 (s, 3H) ppm.HRMS(APCI + , m / z):C 32 H 33 NO[M+H + ] Calculated value: 517.2716, measured value: 517.2713. Preparation of 2-fluoro-N-(furan-2-ylmethyl)-5-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzamide (compound 7av) [ka] Scheme 3: Synthesis of compound 7av.

[0377] 2-Fluoro-5-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid (7i, 3.3 mg, 7.4 μmol), EDC HCl (2.8 mg, 14.8 μmol, 2.0 eq), and HOBT (2.0 mg, 14.8 μmol, 2.0 eq) were dissolved in DMF (1.0 mL). Furfurylamine (1.4 mg, 1.3 μL, 14.8 μmol, 2.0 eq) and EtN (1.5 mg, 2.0 μL, 14.8 μmol, 2.0 eq) were added, and the reaction mixture was stirred at room temperature overnight. The solvent was evaporated and the crude mixture was purified by preparative HPLC purification to give 2-fluoro-N-(furan-2-ylmethyl)-5-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzamide (7av, 3.8 mg, 98% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.54 (d, J = 2.0 Hz, 1H), 8.52 (d, J = 2.0 Hz, 1H), 8.08 (dd, J = 6.9, 2.4 Hz, 1H), 7.88 (ddd, J = 8.6, 4.8, 2.4 Hz, 1H), 7.78 (s, 1H), 7.73 (d, J = 8.2 Hz, 2H), 7.52 (d, J = 8.2 Hz, 2H), 7.44 (dd, J = 1.9, 0.9 Hz, 1H), 7.32 (dd, J = 10.7, 8.6 Hz, 1H), 6.37 (dd, J = 3.2, 1.9 Hz, 1H), 6.33 (dd, J = 3.2, 0.9 Hz, 1H), 4.61 (s, 2H), 3.84 (s, 2H), 2.97 - 2.84 (m, 8H), 2.88 (s, 3H) ppm.HRMS(APCI + , m / z):C 31 H 31 FN5O2[M+H + ] Calculated value: 524.2462, measured value: 524.2452. Preparation of 1-cyclohexyl-3-(4-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)urea (Compound 7aw) [ka] Scheme 4: Synthesis of compound 7aw.

[0378] 4-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)aniline (7e, 10 mg, 0.025 mmol, 2.5 eq) was dissolved in DCM (0.5 mL) and triethylamine (10 mg, 14 μL, 0.1 mmol, 10 eq) was added. The solution was added dropwise to a solution of triphosgene (3.0 mg, 0.01 mmol, 1.0 eq) in DCM (0.5 mL) and stirred for 5 min at room temperature. The reaction mixture was added to a solution of cyclohexylamine (19.8 mg, 23 μL, 0.2 mmol, 20.0 eq) in DCM (0.5 mL) and stirred overnight. The solvent was evaporated and the crude mixture was purified by preparative HPLC purification to give 1-cyclohexyl-3-(4-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)urea (7aw, 2.8 mg, 54% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.53 (m, 2H), 7.72 (d, J = 8.2 Hz, 2H), 7.68 (s, 1H), 7.64 - 7.59 (m, 2H), 7.52 (d, J = 8.2 Hz, 2H), 7.49 - 7.44 (m, 2H), 3.82 (s, 2H), 3.65 - 3.54 (m, 1H), 3.00 - 2.74 (bs, 8H), 2.88 (s, 3H), 2.00 - 1.90 (m, 2H), 1.84 - 1.71 (m, 2H), 1.65 (d, J = 12.9 Hz, 1H), 1.48 - 1.34 (m, 2H), 1.36 - 1.16 (m, 3H) ppm.HRMS(APCI + , m / z):C 32 H 39 NO[M+H + ] Calculated value: 523.3185, measured value: 523.3182. Preparation of (R)-1-(3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)-3-(1-phenylethyl)urea (Compound 7ax) [ka] Scheme 5: Synthesis of compound 7ax.

[0379] 3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)aniline (7h, 16 mg, 0.04 mmol, 2.5 eq) was dissolved in DCM (1.0 mL) and triethylamine (16.2 mg, To the resulting mixture was added triphosgene (4.75 mg, 0.016 mmol, 1.0 eq). The solvent was added dropwise to a solution of triphosgene (4.75 mg, 0.016 mmol, 1.0 eq) in DCM (1.0 mL) and stirred at room temperature for 5 min. The reaction mixture was added to a solution of (R)-(+)-α-methylbenzylamine (38.8 mg, 41 μL, 0.32 mmol, 20.0 eq) in DCM (1.0 mL) and stirred overnight. The solvent was evaporated and the crude mixture was purified by preparative HPLC to give (R)-1-(3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)-3-(1-phenylethyl)urea (7ax, 6.7 mg, 31% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.65 (d, J = 2.0 Hz, 1H), 8.54 (d, J = 2.1 Hz, 1H), 7.94 (s, 1H), 7.78 - 7.72 (m, 3H), 7.51 (d, J = 8.0 Hz, 2H), 7.42 - 7.28 (m, 6H), 7.23 (t, J = 7.0 Hz, 1H), 7.18 - 7.11 (m, 1H), 4.95 (q, J = 7.0 Hz, 1H), 3.83 (s, 2H), 3.14 - 2.50 (bs, 8H), 2.88 (s, 3H), 1.50 (d, J = 7.0 Hz, 3H) ppm.HRMS(APCI + , m / z):C 34 H 37 NO[M+H + ] Calculated value: 545.3029, measured value: 545.3027. Preparation of 1-(furan-2-ylmethyl)-3-(3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)urea (compound 7ay) [ka] Scheme 6: Synthesis of compound 7ay.

[0380] 3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)aniline (7h, 16 mg, 0.04 mmol, 2.5 eq) was dissolved in DCM (1.0 mL) and triethylamine (16.2 mg, 22 μL, 0.16 mmol, 10 eq) was added. The solution was added dropwise to a solution of triphosgene (4.75 mg, 0.016 mmol, 1.0 eq) in DCM (1.0 mL) and stirred at room temperature for 5 min. The reaction mixture was added to a solution of furfurylamine (31.1 mg, 28.3 μL, 0.32 mmol, 20.0 eq) in DCM (1.0 mL) and stirred overnight. The solvent was evaporated and the crude mixture was purified by preparative HPLC purification to give 1-(furan-2-ylmethyl)-3-(3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)urea (7ay, 8.6 mg, 41% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.74 (d, J = 2.0 Hz, 1H), 8.69 - 8.66 (m, 2H), 7.82 - 7.78 (m, 2H), 7.74 (s, 1H), 7.65 (d, J = 7.9 Hz, 2H), 7.41 - 7.34 (m, 4H), 6.35 (dd, J = 3.2, 1.9 Hz, 1H), 6.28 (dd, J = 3.2, 0.8 Hz, 1H), 4.41 (s, 2H), 3.89 (s, 2H), 3.21 (bs, 4H), 3.11 (bs, 4H), 2.89 (s, 3H) p pm.HRMS(APCI + , m / z):C 31 H 33 N6O2[M+H + ] Calculated value: 521.2665, measured value: 521.2670. Preparation of 1-(2-methoxyethyl)-3-(3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)urea (Compound 7az) [ka] Scheme 7: Synthesis of compound 7az.

[0381] 3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)aniline (7h, 16 mg, 0.04 mmol, 2.5 eq) was dissolved in DCM (1.0 mL) and triethylamine (16.2 mg, 22 μL, 0.16 mmol, 10 eq) was added. The solution was added dropwise to a solution of triphosgene (4.75 mg, 0.016 mmol, 1.0 eq) in DCM (1.0 mL) and stirred at room temperature for 5 min. The reaction mixture was added to a solution of 2-methoxyethan-1-amine (24.0 mg, 27.8 μL, 0.32 mmol, 20.0 eq) in DCM (1.0 mL) and stirred overnight. The solvent was evaporated and the crude mixture was purified by preparative HPLC purification to give 1-(2-methoxyethyl)-3-(3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)urea (7az, 4.0 mg, 20% yield). 1 H NMR (400 MHz, methanol-d4) δ 8.78 (d, J = 2.0 Hz, 1H), 8.60 (d, J = 2.0 Hz, 1H), 8.48 (d, J = 2.0 Hz, 1H), 7.81 (s, 1H), 7.76 (s, 1H), 7.61 - 7.53 (m, 4H), 7.45 - 7.25 (m, 4H), 3.54 (s, 2H), 3.50 (s, 2H), 3.47 (s, 3H), 3.27 - 3.12(bs, 8H), 2.87 (s, 3H) ppm.HRMS(APCI + , m / z):C 29 H 35 N6O2[M+H + ] Calculated value: 499.2821, measured value: 499.2827. Example 3 Synthesis of compounds 9a-9c

[0382] A general route to compounds 9a-9c follows the synthesis shown below, and the preparation of each compound is also provided. [ka] Scheme 8: General synthetic scheme for compounds 9a-9c.

[0383] 5-Bromo-3-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine 1 (8, 1.0 g, 2.1 mmol, 1.0 eq) was dissolved in 1,4-dioxane (20 mL). Boronic acid (2.3 mmol, 1.1 eq) was added, followed by Pd(PPh3)2Cl2 (70 mg, 0.1 mmol, 5 mol%). The reaction mixture was degassed by sonication under argon. Aqueous Na2CO3 was added, and the reaction mixture was stirred at 45 °C until complete (typically 8 h). The reaction mixture was partitioned between EtOAc and brine, the layers were separated, and the aqueous layer was extracted with EtOAc (2x). The combined organic layers were dried over Na2SO4, filtered, and the solvent was evaporated. The crude material was purified by column chromatography on silica (0-50% EtOAc in hexanes) to give the desired products (9a-c). Preparation of 4-(5-bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-indazole (compound 9a) [ka]

[0384] Following the general procedure described above, 4-(5-bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-indazole (9a) was isolated as a light brown solid (461 mg, 47% yield). 1 H NMR (400 MHz, chloroform-d) δ 10.29 (bs, 1H), 8.53 (d, J = 2.2 Hz, 1H), 8.19 - 8.13 (m, 2H), 8.13 - 8.10 (m, 2H), 8.05 (s, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.49 (dd, J = 8.4, 6.9 Hz, 1H), 7.37 - 7.31 (m, 2H), 7.29 (dd, J = 6.9, 1.0 Hz, 1H), 2.41 (s, 3H) ppm.HRMS(APCI + , m / z):C 21 H 16 N4O2SBr[M+H + ] Calculated value: 467.0177, measured value: 467.0176. Preparation of 5-bromo-3-(3-fluorophenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (compound 9b) [ka]

[0385] Following the general procedure described above, 5-bromo-3-(3-fluorophenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (9b) was isolated as a colorless solid (617 mg, 67% yield). 1 H NMR (400 MHz, chloroform-d) δ 8.50 (d, J = 2.1 Hz, 1H), 8.19 (d, J = 2.1 Hz, 1H), 8.09 (d, J = 8.4 Hz, 2H), 7.90 (s, 1H), 7.44 (td, J = 8.0, 5.9 Hz, 1H), 7.36 - 7.28 (m, 3H), 7.27 - 7.21 (m, 1H), 7.08 (tdd, J = 8.4, 2.6, 1.0 Hz, 1H), 2.39 (s, 3H) ppm.HRMS(APCI + , m / z):C 20 H 15 N2O2FSBr[M+H + ] Calculated value: 445.0022, measured value: 445.0020. Preparation of 5-bromo-3-(3-chlorophenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (compound 9c) [ka]

[0386] Following the general procedure described above, 5-bromo-3-(3-chlorophenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (9c) was isolated as a colorless solid (850 mg, 88% yield). 1 H NMR (400 MHz, chloroform-d) δ 8.50 (d, J = 2.1 Hz, 1H), 8.18 (d, J = 2.1 Hz, 1H), 8.09 (d, J = 8.4 Hz, 2H), 7.89 (s, 1H), 7.56 - 7.50 (m, 1H), 7.46 - 7.40 (m, 2H), 7.40 - 7.34 (m, 1H), 7.31 (d, J = 7.8 Hz, 2H), 2.39 (s, 3H) ppm.HRMS(APCI + , m / z):C 20 H 15 N2O2SBrCl[M+H + ] Calculated value: 460.9726, measured value: 460.9715. Example 4 Synthesis of compounds 10a-10c

[0387] A general route to compounds 10a-10c follows the synthesis shown below, and the preparation of each compound is also provided. [ka] Scheme 9: General synthetic scheme for compounds 10a-10c.

[0388] The substrates (9a-c, 1.0 mmol, 1.0 eq) were dissolved in 1,4-dioxane (40 mL) in a pressure tube, and 4-formylphenylboronic acid (225 mg, 1.5 mmol, 1.5 eq) was added. The reaction mixture was degassed by sonication under argon, and Pd(PPh3)4 (116 mg, 0.1 mmol, 10 mol%) was added. After adding aqueous Na2CO3 (12.5 mL, 2.0 M), the reaction mixture was stirred at 100 °C overnight. The crude mixture was partitioned between EtOAc and brine, the layers were separated, and the aqueous layer was diluted with EtOAc. The resulting mixture was extracted with HCl (3x). The combined organic layers were dried over NaSO and filtered over Celite. The solvent was evaporated and the crude material was purified by column chromatography on silica (0-100% EtOAc in hexanes) to give the desired products (10a-c). Preparation of 4-(3-(1H-indazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (compound 10a) [ka]

[0389] Following the general procedure described above, 4-(3-(1H-indazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (10a) was isolated as a light brown solid (350 mg, 71% yield). 1 H NMR (400 MHz, chloroform-d) δ 10.37 (bs, 1H), 10.06 (s, 1H), 8.78 (d, J = 2.2 Hz, 1H), 8.23 ​​- 8.17 (m, 3H), 8.14 (d, J = 1.1 Hz, 1H), 8.10 (s, 1H), 7.96 (d, J = 8.3 Hz, 2H), 7.72 (d, J = 8.2 Hz, 2H), 7.59 - 7.51 (m, 2H), 7.40 - 7.32 (m, 3H), 2.41 (s, 3H) ppm.HRMS(APCI + , m / z):C 28 H21 N4O3S[M+H + ] Calculated value: 493.1334, measured value: 493.1329. Preparation of 4-(3-(3-fluorophenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (compound 10b) [ka]

[0390] Following the general procedure described above, 4-(3-(3-fluorophenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (10b) was isolated as a colorless solid (466 mg, 99% yield). 1 H NMR (400 MHz, chloroform-d) δ 10.08 (s, 1H), 8.74 (d, J = 2.1 Hz, 1H), 8.26 (d, J = 2.1 Hz, 1H), 8.16 (d, J = 8.4 Hz, 2H), 7.99 (d, J = 8.4 Hz, 2H), 7.96 (s, 1H), 7.74 (d, J = 8.2 Hz, 2H), 7.47 (td, J = 7.9, 5.8 Hz, 1H), 7.40 (dt, J = 7.7, 1.3 Hz, 1H), 7.36 - 7.28 (m, 3H), 7.10 (tdd, J = 8.4, 2.6, 1.1 Hz, 1H), 2.40 (s, 3H) ppm.HRMS(APCI + , m / z):C 27 H 20 N2O3FS[M+H + ] Calculated value: 471.1179, measured value: 471.1185. Preparation of 4-(3-(3-chlorophenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (compound 10c) [ka]

[0391] Following the general procedure described above, the starting material was increased to 2.0 mmol, and 4-(3-(3-chlorophenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (10c) was isolated as a colorless solid (730 mg, 87% yield). 1 H NMR (400 MHz, chloroform-d) δ 10.08 (s, 1H), 8.74 (d, J = 2.1 Hz, 1H), 8.24 (d, J = 2.1 Hz, 1H), 8.16 (d, J = 8.4 Hz, 2H). 7.99 (d, J = 8.2 Hz, 2H), 7.95 (s, 1H), 7.74 (d, J = 8.2 Hz, 2H), 7.59 (t, J = 1.8 Hz, 1H), 7.50 (dt, J = 7.6, 1.5 Hz, 1H), 7.43 (t, J = 7.7 Hz, 1H), 7.38 (ddd, J = 7.9, 2.0, 1.3Hz, 1H), 7.36 - 7.29 (m, 2H), 2.40 (s, 3H) ppm.HRMS(APCI + , m / z):C 27 H 20 N2O3ClS[M+H + ] Calculated value: 487.0883, measured value: 487.0878. Example 5 Synthesis of compounds 12a-12l

[0392] A general route to compounds 12a-12l follows the synthesis illustrated below. The preparation of each compound is also provided. [ka] Scheme 10: General synthetic scheme for compounds 12a-12l.

[0393] The detailed synthesis of bicyclic piperazine analogs 12e and 12f is illustrated in the scheme below. [ka] Scheme 11: Synthesis of compounds 12e and 12f.

[0394] The synthesis of analogs 12g-12l and their half-lives in mouse liver microsomes are shown in the scheme below. [ka] Scheme 12: (A) Synthesis of analogs 12g-12l; (B) Structures of analogs 12g-12l and their half-lives in mouse liver microsomes.

[0395] Aldehydes (10a-c) or 11 (Goodfellow VS et al., 2013)) (0.5 mmol, 1.0 eq) was dissolved in dichloromethane, and the corresponding amine (1.0 mmol, 2.0 eq) and Na(OAc)3BH (159 mg, 0.75 mmol, 1.5 eq) were added. The reaction mixture was stirred overnight at room temperature and partitioned between dichloromethane and brine. The aqueous layer was extracted with dichloromethane (2x), and the combined organic layers were dried over Na2SO4, filtered, and the solvent was evaporated. The crude product was used in the deprotection step without further purification.

[0396] The intermediate was dissolved in acetone (20 mL), methanol (30 mL), and aqueous NaOH (2 The resulting mixture was dissolved in a 1.0 M aqueous NaOH (1.0 M, 15 mL) and stirred at 65° C. for 3 h. The reaction mixture was partitioned between ethyl acetate and aqueous NaOH (1.0 M). The layers were separated and the aqueous layer was extracted with ethyl acetate (2×). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and the solvent was evaporated. The crude material was purified by preparative HPLC purification to give the products (12a-1). Preparation of 3-(4-(3-(1H-indol-5-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzyl)-8-oxa-3-azabicyclo[3.2.1]octane (compound 12a) [ka]

[0397] Following the general procedure described above, starting from 4-(3-(1H-indol-5-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (11, 100 mg, 0.2 mmol) and 8-oxa-3-azabicyclo[3.2.1]octane (45 mg, 0.4 mmol, 2.0 eq), 3-(4-(3-(1H-indol-5-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzyl)-8-oxa-3-azabicyclo[3.2.1]octane (12a) was isolated as a light brown solid (37.2 mg, 43% yield over two steps). 1 H NMR (500 MHz, methanol-d4) δ 8.42 (d, J = 2.1 Hz, 1H), 8.40 (d, J = 2.1 Hz, 1H), 7.83 (d, J = 1.5 Hz, 1H), 7.55 (s, 1H), 7.52 (d, J = 8.1 Hz, 2H), 7.47 (d, J = 8.4 Hz, 1H), 7.46 - 7.39 (m, 1H), 7.33 (d, J = 7.9 Hz, 2H), 7.24 (d, J = 3.1 Hz, 1H), 6.49 (dd, J = 3.1, 0.8 Hz, 1H), 4.20 (dd, J = 4.7, 2.3 Hz, 2H), 3.41 (s, 2H), 2.52 (d, J = 11.5 Hz, 2H), 2.25 (dd, J = 11.4, 2.1 Hz, 2H), 2.00 - 1.90 (m, 3H), 1.86 - 1.73 (m, 2H) ppm. HRMS (APCI + , m / z):C 32 H 33 NO[M+H + ] Calculated value: 517.2716, measured value: 517.2713. Preparation of 3-(1H-indol-5-yl)-5-(4-((8-methyl-3,8-diazabicyclo[3.2.1]octan-3-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 12b) [ka]

[0398] Following the general procedure described above, starting from 4-(3-(1H-indol-5-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (11, 67.1 mg, 0.137 mmol) and 8-methyl-3,8-diazabicyclo[3.2.1]octane (16b, 55.5 mg, 0.44 mmol, 3.2 eq), 3-(1H-indol-5-yl)-5-(4-((8-methyl-3,8-diazabicyclo[3.2.1]octan-3-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (12b) was isolated as a light brown solid (2.6 mg, 4% yield over two steps). 1 H NMR (400 MHz, methanol-d4) δ 8.57 (d, J = 2.0 Hz, 1H), 8.51 (d, J = 2.0 Hz, 1H), 7.87 (dd, J = 1.6, 0.7 Hz, 1H). 7.70 (d, J = 8.1 Hz, 2H), 7.65 (s, 1H), 7.56 - 7.38 (m, 5H), 7.27 (d, J = 3.2 Hz, 1H), 3.88 (s, 2H), 3.70 (s, 2H), 2.95 (d, J = 12.6 Hz, 2H), 2.80 (s, 3H), 2.57 (d, J = 13.0 Hz, 2H), 2.23 (s, 4H) ppm.HRMS(APCI + , m / z):C 29 H 30 N5[M+H + ] Calculated value: 448.2501, measured value: 448.2505. Preparation of 3-(1H-indol-5-yl)-5-(4-((3-methyl-3,8-diazabicyclo[3.2.1]octan-8-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 12c) [ka]

[0399] Following the general procedure described above, starting from 4-(3-(1H-indol-5-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (11, 60.2 mg, 0.122 mmol) and 3-methyl-3,8-diazabicyclo[3.2.1]octane (16a, 36.6 mg, 0.29 mmol, 2.4 eq), 3-(1H-indol-5-yl)-5-(4-((3-methyl-3,8-diazabicyclo[3.2.1]octan-8-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (12c) was isolated as a light brown solid (9.6 mg, 18% yield over two steps). 1 H NMR (400 MHz, methanol-d4) δ 8.74 (d, J = 2.0 Hz, 1H), 8.60 (d, J = 2.0 Hz, 1H), 7.88 (dd, J = 1.7, 0.8 Hz, 1H), 7.86 - 7.79 (m, 2H), 7.74 (s, 1H), 7.68 - 7.64 (m, 3H), 7.54 - 7.50 (m, 1H), 7.48 - 7.43 (m, 2H), 4.12 (s, 2H), 3.84 (bs, 4H), 3.29 (d, J = 2.7 Hz, 2H), 3.10 (bs, 4H), 2.69 (s, 3H) ppm.HRMS(APCI + , m / z):C 29 H 30 N5[M+H + ] Calculated value: 448.2501, measured value: 448.2503. 4-(5-(4-((8-methyl-3,8-diazabicyclo[3.2.1]octane-3 Preparation of (1H-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-indazole (Compound 12d) [ka]

[0400] Following the general procedure described above, starting from 4-(3-(1H-indazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (10a, 49.2 mg, 0.1 mmol) and 8-methyl-3,8-diazabicyclo[3.2.1]octane (16b, 39.1 mg, 0.31 mmol, 3.1 eq), 4-(5-(4-((8-methyl-3,8-diazabicyclo[3.2.1]octan-3-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-indazole (12d) was isolated as a light brown solid (11.7 mg, 26% yield over two steps). 1 H NMR (400 MHz, methanol-d4) δ 8.61 (d, J = 2.0 Hz, 1H), 8.55 (d, J = 2.0 Hz, 1H), 8.18 (d, J = 0.9 Hz, 1H), 7.95 (s, 1H), 7.67 (d, J = 8.2 Hz, 2H), 7.55 - 7.50 (m, 2H), 7.47 (d, J = 8.2 Hz, 2H), 7.43 (dd, J = 6.5, 1.4 Hz, 1H), 3.88 (s, 2H), 3.70 (s, 2H), 2.94 (dd, J = 13.1, 2.8Hz, 2H), 2.80 (s, 3H), 2.67 - 2.62 (m, 2H), 2.27 - 2.19 (bs, 4H) ppm.HRMS(APCI + , m / z):C 28 H 29 N6[M+H + ] Calculated value: 449.2454, measured value: 449.2462. Preparation of 4-(5-(4-((3-methyl-3,8-diazabicyclo[3.2.1]octan-8-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-indazole (Compound 12e) [ka]

[0401] According to the general procedure described above, starting from 4-(3-(1H-indazol-4-yl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (10a, 49.2 mg, 0.1 mmol) and 3-methyl-3,8-diazabicyclo[3.2.1]octane (16a, 34.0 mg, 0.27 mmol, 2.7 eq), 4-(5-(4-((3-methyl-3,8-diazabicyclo[3.2.1]octane-8- (yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-indazole (12e) was isolated as a light brown solid (12.0 mg, 27% yield over two steps). 1 H NMR (400 MHz, methanol-d4) δ 8.60 (d, J = 2.1 Hz, 1H), 8.45 (d, J = 2.1 Hz, 1H), 8.18 (d, J = 0.7 Hz, 1H), 7.90 (s, 1H), 7.77 (d, J = 8.2 Hz, 2H), 7.62 (d, J = 8.2 Hz, 2H), 7.54 - 7.51 (m, 2H), 7.43 (dd, J = 5.6, 2.3 Hz, 1H), 4.09 (s, 2H), 3.80 (s, 2H), 3.18 (d, J = 11.9 Hz, 2H), 2.94 (d, J = 12.6 Hz, 2H), 2.61 (s, 3H), 2.45 - 2.36 (m, 2H), 2.14 (d, J = 8.6 Hz, 2H) ppm.HRMS(APCI + , m / z):C 28 H 29 N6[M+H + ] Calculated value: 449.2454, measured value: 449.2455. Preparation of 3-(3-chlorophenyl)-5-(4-((3-methyl-3,8-diazabicyclo[3.2.1]octan-8-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (compound 12f) [ka]

[0402] Following the general procedure described above, starting from 4-(3-(3-chlorophenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (10c, 48.6 mg, 0.1 mmol) and 3-methyl-3,8-diazabicyclo[3.2.1]octane (16a, 25.2 mg, 0.2 mmol, 2.0 eq), 3-(3-chlorophenyl)-5-(4-((3-methyl-3,8-diazabicyclo[3.2.1]octan-8-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (12f) was isolated as a pale yellow solid (18.0 mg, 41% yield over two steps). 1 H NMR (400 MHz, methanol-d4) δ 8.54 (d, J = 2.1 Hz, 1H), 8.46 (d, J = 2.1 Hz, 1H), 7.81 - 7.75 (m, 3H), 7.70 (t, J = 1.9 Hz, 1H), 7.68 - 7.61 (m, 3H), 7.45 (t, J = 7.9 Hz, 1H), 7.30 (ddd, J = 8.0, 2.1, 1.0 Hz, 1H), 4.11 (s, 2H), 3.82 (s, 2H), 3.25 - 3.16 (m, 2H), 3.04 - 2.94 (m, 2H), 2.62 (s, 3H), 2.42 (dd, J = 9.6, 4.7 Hz, 2H), 2.17 (t, J = 7.3 Hz, 2H) ppm.HRMS(APCI + , m / z):C 27 H 28 N4Cl[M+H + ] Calculated value: 443.2002, measured value: 443.2005. Preparation of 5-(4-([1,4'-bipiperidine]-1'-ylmethyl)phenyl)-3-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 12g) [ka]

[0403] Following the general procedure described above, starting from 4-(3-(3-fluorophenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (10b, 26.0 mg, 0.055 mmol) and 4-piperidinopiperidine (18.6 mg, 0.11 mmol, 2.0 eq), 5-(4-([1,4'-bipiperidin]-1'-ylmethyl)phenyl)-3-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridine (12g) was isolated as an off-white solid (6.2 mg, 24% yield over two steps). 1 H NMR (400 MHz, methanol-d4) δ 8.58 (d, J = 2.0 Hz, 1H), 8.56 (d, J = 2.1 Hz, 1H), 7.86 (d, J = 8.2 Hz, 2H), 7.82 (s, 1H), 7.66 (d, J = 8.2 Hz, 2H), 7.57 - 7.53 (m, 1H), 7.52 - 7.42 (m, 2H), 7.04 (tdd, J = 8.2, 2.6, 1.0 Hz, 1H), 4.43 (s, 2H), 3.72 (d, J = 12.9 Hz, 2H), 3.63 - 3.47 (m, 4H), 3.23 - 3.12 (m, 4H), 3.03 (t, J = 11.3 Hz, 2H), 2.41 (d, J = 13.5 Hz, 2H), 2.20 - 2.08 (m, 3H), 2.04 - 1.94 (m, 2H), 1.86 - 1.73 (m, 2H), 1.64 - 1.46 (m, 1H), 1.42 - 1.26 (m, 2H) ppm.HRMS(APCI + , m / z):C30 H 34 N4F[M+H + ] Calculated value: 469.2768, measured value: 469.2771. Preparation of 2-(4-(4-(3-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzyl)piperazin-1-yl)ethan-1-ol (compound 12h) [ka]

[0404] Following the general procedure described above, 4-(3-(3-fluorophenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (10b, 318 mg, 0.675 mmol) and 1-(2-hydroxyethyl)piperazine (176 mg, 16 Starting with 6 μL, 1.35 mmol, 2.0 eq) of 2-(4-(4-(3-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzyl)piperazin-1-yl)ethan-1-ol (12h) was isolated as an off-white solid (153.7 mg, 53% yield over two steps). 1 H NMR (400 MHz, methanol-d4) δ 8.62 (d, J = 2.0 Hz, 1H), 8.60 (d, J = 2.0 Hz, 1H), 7.85 (s, 1H), 7.80 (d, J = 8.2 Hz, 2H), 7.62 (d, J = 8.2 Hz, 2H), 7.55 (dt, J = 7.8, 1.3 Hz, 1H), 7.51 - 7.42 (m, 2H), 7.05 (dddd, J = 9.0, 8.2, 2.6, 1.1 Hz, 1H), 4.22 (s, 2H), 3.95 - 3.85 (m, 2H), 3.58 (bs, 4H), 3.34 (bs, 6H) ppm.HRMS(APCI + , m / z):C 26 H 28 N4OF[M+H +] Calculated value: 431.2247, measured value: 431.2250. Preparation of (R)-1-(4-(3-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzyl)pyrrolidin-3-ol (compound 12i) [ka]

[0405] Following the general procedure described above, starting from 4-(3-(3-fluorophenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (10b, 318 mg, 0.675 mmol) and (R)-3-pyrrolidinol (118 mg, 109 μL, 1.35 mmol, 2.0 eq), (R)-1-(4-(3-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzyl)pyrrolidin-3-ol (12i) was isolated as an off-white solid (113 mg, 43% yield over two steps). 1 H NMR (400 MHz, methanol-d4) δ 8.56 (d, J = 2.0 Hz, 1H), 8.53 (d, J = 2.0 Hz, 1H), 7.84 - 7.77 (m, 3H), 7.68 - 7.61 (m, 2H), 7.52 (dt, J = 7.8, 1.3 Hz, 1H), 7.50 - 7.38 (m, 2H), 7.02 (dddd, J = 9.0, 8.2, 2.6, 1.1 Hz, 1H), 4.66 - 4.34 (m, 3H), 3.82 - 3.42 (m, 2H), 3.41 - 3.20 (m, 2H), 2.49 - 1.92 (m, 2H) ppm.HRMS(APCI + , m / z):C 24 H 23 N3OF[M+H + ] Calculated value: 388.1825, measured value: 388.1824. Preparation of 5-(4-((4-(tert-butyl)piperazin-1-yl)methyl)phenyl)-3-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 12j) [ka]

[0406] Following the general procedure described above, starting from 4-(3-(3-fluorophenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (10b, 32.8 mg, 0.055 mmol) and 1-tert-butylpiperazine (15.7 mg, 0.11 mmol, 2.0 eq), 5-(4-((4-(tert-butyl)piperazin-1-yl)methyl)phenyl)-3-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridine (12j) was isolated as an off-white solid (9.8 mg, 40% yield over two steps). 1 H NMR (400 MHz, methanol-d4) δ 8.63 (d, J = 1.9 Hz, 1H), 8.60 (bs, 1H), 7.85 (s, 1H), 7.83 - 7.78 (m, 2H), 7.65 - 7.60 (m, 2H), 7.56 (dt, J = 7.8, 1.3 Hz, 1H), 7.52 - 7.42 (m, 2H), 7.05 (dddd, J = 9.0, 8.2, 2.6, 1.0 Hz, 1H), 4.22 (s, 2H), 3.89 - 3.04 (m, 8H), 1.44 (s, 9H) ppm.HRMS(APCI + , m / z):C 28 H 32 N4F[M+H + ] Calculated value: 443.2611, measured value: 443.2610. Preparation of 2-(4-(4-(3-(3-chlorophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzyl)piperazin-1-yl)ethan-1-ol (compound 12k) [ka]

[0407] Following the general procedure described above, starting from 4-(3-(3-chlorophenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (10c, 418 mg, 0.86 mmol) and 1-(2-hydroxyethyl)piperazine (224 mg, 211 μL, 1.72 mmol, 2.0 eq), 2-(4-(4-(3-(3-chlorophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzyl)piperazin-1-yl)ethan-1-ol (12k) was isolated as an off-white solid (139.9 mg, 36% yield over two steps). 1 H NMR (400 MH z, methanol-d4) δ 8.57 (d, J = 2.0 Hz, 1H), 8.54 (d, J = 2.0 Hz, 1H), 7.80 (s, 1H), 7.76 (d, J = 8.3 Hz, 2H), 7.65 (t, J = 1.9 Hz, 1H), 7.64 - 7.57 (m, 3H), 7.42 (t, J = 7.9 Hz, 1H), 7.29 (ddd, J = 8.0, 2.1, 1.0 Hz, 1H), 4.27 (s, 2H), 3.93 - 3.86 (m, 2H), 3.62 (s, 4H), 3.41 (s, 4H), 3.35 - 3.31 (m, 2H) ppm.HRMS(APCI + , m / z):C 26 H 28 N4OCl[M+H + ] Calculated value: 447.1952, measured value: 447.1954. Preparation of (R)-1-(4-(3-(3-chlorophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzyl)pyrrolidin-3-ol (Compound 12l) [ka]

[0408] Following the general procedure described above, starting from 4-(3-(3-chlorophenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (10c, 177 mg, 0.86 mmol) and (R)-3-pyrrolidinol (150 mg, 139 μL, 1.72 mmol, 2.0 eq), (R)-1-(4-(3-(3-chlorophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzyl)pyrrolidin-3-ol (12l) was isolated as an off-white solid (177 mg, 51% yield over two steps). 1 H NMR (400 MHz, methanol-d4) δ 8.52 (d, J = 2.0 Hz, 1H), 8.42 (d, J = 2.0 Hz, 1H), 7.78 - 7.71 (m, 3H), 7.64 - 7.60 (m, 3H), 7.58 (ddd, J = 7.7, 1.7, 1.1 Hz, 1H), 7.39 (t, J = 7.9 Hz, 1H), 7.25 (ddd, J = 8.0, 2.2, 1.0 Hz, 1H), 4.58 (s, 1H), 4.54 - 4.32 (m, 2H), 3.82 - 3.39 (m, 4H), 2.50 - 1.96 (m, 2H) ppm. HRMS (APCI + , m / z):C 24 H 23 N3OCl[M+H + ] Calculated value: 404.1530, measured value: 404.1529. Example 6 Synthesis of boronic acids 13a–13e

[0409] A general route to compounds 13a-13e follows the synthesis exemplified below: The preparation of each compound is also provided. [ka] Scheme 13: General synthetic scheme for compounds 13a-13e.

[0410] Boronic acid (3-aminophenylboronic acid or 4-aminophenylboronic acid, 87 mg, 0.5 mmol, 2.5 eq) was suspended in DCM (4 mL) and triethylamine (202 mg, 277 μL, 2.0 mmol, 10 eq) was added. The resulting solution was added dropwise to a solution of triphosgene (59 mg, 0.2 mmol, 1.0 eq) in DCM (6 mL) and stirred at room temperature for 5 min. An amine (0.6 mmol, 3 eq) or an alcohol (excess) was added dropwise and the mixture was stirred at room temperature overnight. The solvent was evaporated, toluene was added, and the solvent was evaporated again. The crude material was analyzed by NMR and used in the next step without further purification. Preparation of (3-(3-methylureido)phenyl)boronic acid (compound 13a) [ka]

[0411] Following the general procedure described above, with 3-aminophenylboronic acid and methylamine, (3-(3-methylureido)phenyl)boronic acid (13a) was isolated as an off-white solid and used in the next step without further purification. 1 H NMR (400 MHz, methanol-d₄) δ 7.49 (bs, 1H), 7.38 (bs, 1H), 7.22 (d, J = 5.3 Hz, 2H), 2.77 (s, 3H). Preparation of (3-((methoxycarbonyl)amino)phenyl)boronic acid (compound 13b) [ka]

[0412] Following the general procedure described above, with 3-aminophenylboronic acid and methanol, (3-((methoxycarbonyl)amino)phenyl)boronic acid (13b) was isolated as an off-white solid and used in the next step without further purification. 1 H NMR (400 MHz, methanol-d4) δ 7.59 (bs, 1H), 7.48 (bs, 1H), 7.26 (d, J = 5.2 Hz, 2H), 3.73 (s, 3H). Preparation of (4-(3-methylureido)phenyl)boronic acid (compound 13c) [ka]

[0413] Following the general procedure described above, with 4-aminophenylboronic acid and methylamine, (4-(3-methylureido)phenyl)boronic acid (13c) was isolated as an off-white solid and used in the next step without further purification. 1 H NMR (400 MHz, methanol-d4) δ 7.62 (d, J = 8.5 Hz, 2H), 7.37 (d, J = 8.5 Hz, 2H), 2.77 (s, 3H). Preparation of (4-((methoxycarbonyl)amino)phenyl)boronic acid (compound 13d) [ka]

[0414] Following the general procedure described above, with 4-aminophenylboronic acid and methanol, (4-((methoxycarbonyl)amino)phenyl)boronic acid (13d) was isolated as an off-white solid and used in the next step without further purification. 1 H NMR (400 MHz, methanol-d4) δ 7.65 (d, J = 8.6 Hz, 1H), 7.43 (d, J = 8.6 Hz, 2H), 3.74 (s, 3H). Preparation of (3-(3-phenylureido)phenyl)boronic acid (compound 13e) [ka]

[0415] Following the general procedure described above, with 3-aminophenylboronic acid and aniline, (3-(3-phenylureido)phenyl)boronic acid (13e) was isolated as an off-white solid and used in the next step without further purification. 1 H NMR (400 MHz, methanol-d4) δ 7.62 (bs, 1H), 7.51 (d, J = 7.4 Hz, 1H), 7.34 - 7.23 (m, 2H), 7.08 (dd, J = 8.5, 7.4 Hz, 2H), 6.71 (dt, J = 7.7, 1.1 Hz, 2H), 6.67 (tt, J = 7.4, 1.1 Hz, 1H). Example 7 Synthesis of bicyclic piperazines 16a and 16b

[0416] A general route to compounds 16a and 16b follows the synthesis exemplified below: The preparation of each compound is also provided. [ka] Scheme 14: General synthetic scheme for compounds 16a and 16b.

[0417] Boc-protected bicyclic piperazine (14a-b; 250 mg, 1.18 mmol, 1.0 eq) was dissolved in a mixture of THF / MeOH (1:1). Formaldehyde (37% in HO, 287 μL, 3.53 mmol, 3.0 eq) was added, followed by Na(OAc)BH (274.5 mg, 1.30 mmol, 1.1 eq), and the reaction mixture was stirred overnight. The solvent was evaporated, and the crude product was purified by column chromatography on silica (0-20% MeOH in DCM). The product was visualized on a TLC plate using KMnO stain.

[0418] To remove the Boc group, products 15a-b were dissolved in DCM, excess TFA was added, and the reaction mixture was stirred overnight at room temperature. The solvent was evaporated, toluene was added, and the solvent was evaporated again. The crude products 16a-b were used in the next step without further purification, and the spectroscopic data were consistent with those reported in the literature (Paliulis O et al., 2007). Example 8 HGK (MAP4K4) is a prominent biologically relevant target of compound 1 and its analogs

[0419] With the analogs synthesized above, individual viability assays were performed on CPA-treated (33 μM) human embryonic stem cell-derived motor neurons (HB9::GFP reporter and SOD1 A4V mutant), which would allow for real-time feedback and optimization of the assay. Viability assays were then performed on all analogs at once to generate a data set with low variability. All compounds were evaluated at concentrations ranging from 0.1 to 3.0 μM (5-point dilutions) in three technical replicates and two biological replicates.

[0420] To assess the ability of analogs to inhibit HGK phosphorylation, analogs were subjected to a high-throughput radiolabeled ATP assay from Reaction Biology Corp. at doses of 0.025 μM or 0.1 μM in the presence of 10 μM ATP. Staurosporine was used as a control, with 10 doses of IC starting at 20 μM. 50 Analogs were tested in four-fold serial dilutions. In this way, we were able to test not only whether the analogs bind to HGK but also whether they inhibit its kinase activity. Approximately 45% of the analogs tested were found to inhibit at least half of HGK activity at a dose of 0.025 μM, thereby validating the computational analog design. The ability of the analogs to inhibit the phosphorylation of MLK1 and MLK3 was also evaluated at a dose of 0.1 μM in the presence of 10 μM ATP. Although the overall inhibition of MLK1 and MLK3 was lower, approximately 40% of the analogs tested still inhibited at least half of MLK3 activity at a dose of 0.1 μM.

[0421] Next, we evaluated the correlation between the degree to which each analog inhibited enzyme activity and the degree to which it rescued CPA toxicity. Overall, a moderate correlation was found between % motor neuron survival after CPA treatment and % MLK1 activity (R 2 =0.40) or MLK3 activity % (R 2 = 0.52) (see Figure 7A), suggesting that MLK1 and MLK3 are not biologically relevant targets of compound 1 and its analogs (7a–7az) in the context of ER stress and CPA-induced cell death pathways.

[0422] A very strong correlation was found between % motor neuron survival and % HGK activity (R 2 = 0.84) (see Figure 7B), suggesting that HGK or closely related members of the MAP4K kinase family may be biologically relevant targets of compound 1 and its analogs (7a–7az) in the context of CPA-induced ER stress and motor neuron degeneration.

[0423] A CRISPR strategy was developed to completely ablate HGK expression in mouse ES cells (Figure 10A). The ES line used contains a transgene expressing the cell surface marker CD14 under the control of the motor neuron-specific HB9 promoter; therefore, motor neurons can be purified from mixed differentiation cultures by downstream assay magnetic-activated cell sorting (MACS). The CRISPR strategy uses two guide RNAs targeting genomic regions upstream and downstream of the ATP-binding moiety of HGK, which mediates its kinase activity. Double-strand break repair, achieved in this scenario by non-homologous end joining, generally results in a frameshift and complete ablation of expression. HGK ablation was confirmed in differentiated motor neurons by Western blotting (Figure 10B).

[0424] CRISPR-mediated HGK ablation did not confer neuroprotection in our typical 48-hour dose-response assay with increasing doses of CPA (FIG. 10C), in which motor neurons were MACS-purified and treated with CPA for 48 hours before imaging.

[0425] By modulating our CPA treatment paradigm so that MACS-purified WT or HGK knockout motor neurons were treated with CPA for very short periods (1–24 h), we were able to observe that HGK ablation conferred modest but highly statistically significant neuroprotection against CPA (Figure 10D). Thus, HGK plays an early role in the cell death cascade after CPA treatment; however, the fact that the effects of its ablation are undetectable at later time points suggests that there may be other redundant kinases that ultimately take over in its absence. We suggest that other structurally homologous MAP4K kinases, such as TNIK and MINK1, may act redundantly with HGK to mediate neurodegeneration in the context of ER stress, and that compound 1 and its analogs can target all of these relevant MAP4Ks to promote neuronal survival (Figure 31A).

[0426] To better understand the biologically relevant targets of compound 1 and its analogs, we performed a second round of cell-free kinase assays. In this expanded assay, we selected 10 analogs ranging from moderately to highly protective in the CPA assay. These analogs were evaluated for their ability to inhibit 60 selected kinases at 0.1 μM and 0.5 μM. This list of 60 kinases was derived from known targets of compound 1 (Goodfellow et al., 2013), known targets of other kinase inhibitors that showed rescue, and related targets from the literature. We confirmed the expression of each kinase in ES MN using RNA-seq data (see Example 12).

[0427] There was no overall trend toward increased target selectivity among analogs to compound 1, indicating that the process of analog generation itself did not improve selectivity, and that neither selectivity nor promiscuity was tracked by neuroprotection in the CPA viability assay. However, compound 7ad was similarly neuroprotective relative to compound 1, demonstrating high target selectivity among the 60 kinases tested: at 0.5 μM, compound 1 potently inhibited 27 of the 60 kinases (<20% residual activity), while compound 7ad potently inhibited 12 of the 60 kinases (Figure 8).

[0428] Comparing the targets of relatively selective neuroprotective analogs with those of compound 1 highlights the targets most likely to attenuate neurodegeneration in the CPA viability assay. In addition to HGK, selective compound 7ad most potently inhibits closely related MAP4 kinases, including TNIK, MINK1, KHS (MAP4K5), and GCK (MAP4K2) (Figure 8). These findings are consistent with our analysis of shared targets of hit compounds from our preliminary screen (Figure 3), in which HGK, MINK, KHS, and GCK were among the most highly ranked targets (TNIK activity was not assessed in existing publicly available databases; Anastassiadis et al., 2011).

[0429] Overall, we observed a modest correlation between MAP4K inhibition and neuroprotection for compound 1 and its analogs at 0.5 μM and 0.1 μM (Figure 32), but meaningful correlations were likely obscured by the fact that even moderately neuroprotective compounds still robustly inhibited MAP4Ks at these concentrations. Furthermore, the high degree of structural homology between the kinase domains of MAP4K family members makes it difficult to parse whether inhibition of individual MAP4Ks is functionally relevant to survival in the CPA assay or is merely incidental.

[0430] Thus, there are few compounds that are selective for individual MAP4Ks over other MAP4s; however, we have found that GCK-selective inhibitors (Tan et al. We found that potential functional targets of compound 1 analogs were limited to HGK, MINK1, TNIK, or KHS, or some combination of these, because compounds 17 and NG-25 (see Fig. 9 in Fig. 3) failed to rescue MN in the CPA viability assay (Fig. 33).

[0431] Furthermore, recent studies from Larhammar et al. (2017) show that compounds inhibiting HGK, TNIK, and MINK1 are neuroprotective in primary dorsal root ganglion neurons subjected to neurotrophic factor withdrawal, and attenuation of the expression of all three kinases collectively, rather than individually, reflects the compound's effect (Figure 31C). These findings argue that the roles of HGK, TNIK, and MINK1 in this neurodegeneration assay are redundant, and the effects of inhibiting a single kinase can be overcome by the activity of the remaining kinases. Therefore, combined inhibition of multiple MAP4 kinases may underlie the neuroprotective effect of compound 1 and its analogs in CPA-treated ES MNs (Figure 31B). Example 9 Metabolic stability of analogs

[0432] To test the metabolic stability of the analogs, an in vitro microsomal stability assay was performed. In this assay, analogs that showed good rescue properties in the ER stress assay were incubated with mouse liver microsomes in a buffer containing an NADPH-regenerating system. Aliquots were taken over a 2-hour period and analyzed by HP using an internal standard. Compound stability over time was assessed by analysis by LC-MS (Figure 29). Methods and Materials

[0433] Microsome stability assays were performed in a 96-well format (Perkin-Elmer, StorPlate-96U, PP, 96-well, round-bottom). To each well, 182.2 μL of phosphate buffer (pH 7.4, 100 mM) was added, followed by 10 μL of NADPH-regenerating system solution A and 2 μL of NADPH-regenerating system solution B (Corning Gentest 3P NADPH-regenerating system solutions A (#451220) and B (#451200)). A stock solution of the compound to be analyzed (0.8 μL, 5 mM) or ethoxycoumarin (positive control, 0.8 μL, 5 mM) was added, and the mixture was warmed to 37°C for 5 minutes. Mouse microsomes (CD-1, 5 μL, thawed in a 37°C water bath before use, 20 mg / mL, Life Technologies) were added. At selected time points (0, 15, 30, 45, 60, and 120 min), aliquots (15 μL) were withdrawn from the plate and quenched into cold acetonitrile (60 μL) containing an internal standard (5 μM) in a 96-well plate. Samples were centrifuged at 13,000 rpm for 10 min at 4°C. Supernatant (40 μL) was withdrawn and transferred to a sample vial containing an insert. Samples were analyzed by LC-MS.

[0434] LC-MS analysis was performed on a platform including a Thermo Scientific Dionex Ultimate 3000 and a Bruker amaZon SL equipped with an electrospray ionization source controlled by a Bruker Hystar 3.2. Chromatographic separation was performed by injecting 5 μL of sample onto an Agilent Eclipse Plus C18 column (2.1 × 50 mm, 3.5 μm) maintained at 20 °C. The flow rate was maintained at 400 μL / min. The initial flow conditions were 60% solvent A (water containing 0.1% acetic acid) and 40% solvent B (methanol containing 0.1% acetic acid). Solvent B increased to 60% by 0.25 min and to 70% by 6.75 min. Solvent B increased to 95% by 7.00 min and decreased to the original initial condition (40%) by 8.00 min, for a total run time of 9.00 min. result

[0435] First, 11 selected analogs were analyzed for their stability in mouse liver microsomes (Figure 11). Compounds with a half-life of less than 30 minutes in microsomes are generally not ideal drug candidates due to their high clearance rate. In accordance with previously published studies, we found that Compound 1 has a relatively short half-life (T 1 / 2 Of the 11 analogs initially evaluated, only three (7y, 7ad, and 7at) showed promising half-lives (>30 min) in mouse liver microsomes.

[0436] Next, compound modifications were carried out to improve half-life. Computer docking experiments showed that the methylpiperazine moiety was solvent-exposed. Therefore, we modified the piperazine group of the analogs to increase their metabolic stability while maintaining (or improving) their efficacy in ER stress assays.

[0437] Introduction of a bicyclic methylpiperazine into one of the most potent analogs (see Scheme 11) extended the half-life from 9 minutes (compound 7aa) to 13 minutes (compound 12e). To further enhance the microsomal stability of our analogs, we introduced the same bicyclic piperazine into one of the analogs from the initial series with a half-life of >30 minutes (Scheme 11, compound 12f). In this case, the half-life extended from 38 minutes (compound 7ad) to 158 minutes (compound 12f), a significant improvement. Compound 12f was more potent in the ER stress assay, showing complete rescue at 0.3 μM but slightly higher toxicity at concentrations >0.5 μM. To reduce this increased toxicity while maintaining potency and microsomal stability, we synthesized another series of analogs (Scheme 12) containing m-chlorophenyl or m-fluorophenyl substituents at the 3-position of the 7-azaindole scaffold.

[0438] The potency of these compounds was evaluated in an ER stress assay, and their stability was assessed in a microsomal stability assay. Two of these compounds, 12g and 12j, had shorter-than-desired half-lives or showed reduced potency in the ER stress assay. The remaining four compounds (12h, 12i, 12k, and 12l) potently rescued motor neurons from CPA-induced ER stress and were able to completely abolish the effects of CPA treatment at concentrations <0.3 μM. Among these highly potent analogs, 12h and 12k were the most promising candidates for further in vivo studies due to their improved stability in mouse liver microsomes (Figure 11; Figure 13A). Example 10 Pharmacokinetic evaluation of compounds Improving the solubility of Compound 1 and its analogs

[0439] A previous publication described a basic formulation for in vivo delivery of Compound 1, consisting of 55% saline, 40% PEG-400, and 5% DMSO. Unexpectedly, the inventors observed that in this formulation, Compound 1 was highly insoluble in cell culture medium, indicating that the formulation was not suitable for in vivo experiments. To optimize the vehicle formulation, the inventors developed a 96-well format spectrophotometric assay that could enable rapid screening of the effects of pH and solubilizing agents at various concentrations on Compound 1 and its analogs ( FIG. 30 ).

[0440] Compounds diluted in various vehicle formulations were added to clear cell culture media in ratios consistent with our in vivo dosing scheme. Plates were then read spectrophotometrically at 540 nM, a wavelength at which the compound does not absorb light, to measure optical density and determine whether a light-interfering precipitate formed. We found that the addition of 2-hydroxypropyl-β-cyclodextrin, a cyclic sugar oligomer with a well-characterized safety profile, was sufficient to completely solubilize Compound 1 and its analogs (Figure 12). Animal treatment

[0441] All studies were performed on 6-week-old male C57BL6 / J mice housed at the Columbia University Medical Center facility under a 12-hour light / dark cycle with food and water available ad libitum. All animal procedures were approved by the Columbia University IACUC. Compound 1 and its analogs were formulated in a vehicle composed of 50% ddH2O + 40% PEG-400 + 5% DMSO containing 20% ​​2-hydroxypropyl-β-cyclodextrin (11% final concentration). Animals were weighed and injected intraperitoneally (IP) once at the indicated dose (10 or 20 mg / kg). After 30 minutes, animals were deeply anesthetized with 2.5% avertin. Whole blood was collected by cardiac puncture into tubes containing 0.5 M EDTA as an anticoagulant and centrifuged at 845 rcf for 10 minutes at 4°C to isolate plasma. After cardiac puncture, the animals were decapitated, and the brains were excised and weighed. Brain samples were homogenized in 5 volumes of ddH2O using a glass Dounce homogenizer. Brain and plasma samples were snap-frozen in liquid nitrogen and stored at -80°C before further processing. Plasma compound concentration

[0442] Plasma samples were transferred to Eppendorf tubes (50 μL per sample) and diluted with acetonitrile (200 μL). Samples were sonicated for 1 minute and then centrifuged at 16,000 rpm for 10 minutes. The supernatant was transferred to another Eppendorf tube, and the solvent was allowed to evaporate overnight. Samples were reconstituted in a mixture of 40 μL of acetonitrile and 10 μL of water, transferred to sample vials containing inserts, and analyzed by LC-MS. Compound concentration in the brain

[0443] The homogenized brain samples (250 μL) were transferred to Eppendorf tubes and diluted with acetonitrile (750 μL). The samples were sonicated for 1 minute and then centrifuged at 16,000 rpm for 10 minutes. The supernatant (900 μL) was transferred to another Eppendorf tube, and the solvent was allowed to evaporate overnight. The samples were reconstituted in a mixture of 80 μL of acetonitrile and 20 μL of water, transferred to sample vials with inserts, and analyzed by LC-MS. LC-MS analysis

[0444] LC-MS analysis was performed on a platform including a Thermo Scientific Dionex Ultimate 3000 and a Bruker amaZon SL equipped with an electrospray ionization source controlled by a Bruker Hystar 3.2. Compound concentrations in each plasma sample were determined using a calibration curve. Chromatographic separation was performed by injecting 10 μL of sample onto an Agilent Eclipse Plus C18 column (2.1 × 50 mm, 3.5 μm) maintained at 20 °C. The flow rate was maintained at 400 μL / min. The initial flow conditions were 60% solvent A (water containing 0.1% acetic acid) and 40% solvent B (methanol containing 0.1% acetic acid). Solvent B was increased to 60% over 0.25 min and reached 70% by 6.75 min. Solvent B was increased to 95% by 7.00 min and decreased back to the original initial condition (40%) by 8.00 min for a total run time of 9.00 min. result

[0445] The pharmacokinetic results of selected compounds (12h, 12i, 12k, 12l) at various dosages in brain and plasma are summarized in the following table. A comparison between Compound 1 and Compound 12k is further shown and discussed in at least Figure 13B, Figures 34A-34B, Figures 35A-35C, Figure 36, Figures 37A-37B, and Example 13. [Table 1] [Table 2] Example 11 Bioavailability of the compound and best delivery route

[0446] We observed in vitro that phospho-cjun expression was strongly upregulated by CPA treatment and strongly attenuated by Compound 1, and that it may be a downstream effector of Compound 1's kinase target(s) (Figures 4A and 4B). Therefore, we selected phospho-cjun as a marker of target association for further in vivo studies. c-jun is highly expressed throughout the murine central nervous system, particularly in the olfactory bulb. Eight-week-old wild-type C57BL / 6j mice (Jackson) were treated with 10 mg / kg of Compound 1, 12h, or 12k delivered by oral gavage, intraperitoneal injection, or subcutaneous injection (Figure 14). The animals were then sacrificed 1 hour later, and their olfactory bulbs were partially dissected and lysed for further Western blot analysis. Samples were probed with an antibody specific for phosphorylated c-jun (at serine 63; Cell Signaling) and normalized to the housekeeping gene GAPDH. Nearly all drug-treated samples showed reduced Cjun phosphorylation compared with untreated controls, indicating that the drugs engage the same targets in vivo as they do in vitro. Furthermore, the reduced Cjun phosphorylation in samples from animals treated by oral gavage indicates that both 12h and 12k are orally bioavailable. Example 12 Sensitive two-color motor neuron screen reveals ALS-associated stressors and neuroprotective agents

[0447] Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that targets neurons in the motor cortex, brainstem, and spinal cord. Mutations that cause ALS have been found in dozens of diverse genes, yet the reasons for differentiated motor neuron susceptibility remain poorly understood. We developed a sensitive two-color culture system to study the cell-autonomous mechanisms involved in ALS motor neuron degeneration. Screening of a library of bioactive compounds identified cyclopiazonic acid (CPA), an inhibitor of the endoplasmic reticulum-expressed calcium pump (SERCA), as a stressor to which motor neurons expressing human SOD1 carrying the ALS-causing G93A mutation are highly sensitive. Importantly, motor neurons We found that motor neurons are significantly more sensitive to CPA and to the induction of endoplasmic reticulum (ER) stress and the unfolded protein response pathway than spinal interneurons. A secondary screen identified compounds that rescue motor neurons from CPA-induced degeneration, including three kinase inhibitors and tauroursodeoxycholic acid (TUDCA), an FDA-approved bile acid derivative. TUDCA inhibits hSOD1. G93A Treatment of mice expressing SOD1 ameliorated early muscle denervation. The neuroprotective effects of the identified compounds were confirmed by the SOD1 A4V This was validated using human motor neurons differentiated from an isogenic embryonic stem cell line harboring the 17 mutation. Overall, our results highlight the translational potential of the developed in vitro drug discovery platform, which can serve as a valuable tool for identifying novel drug targets in the treatment of neurodegenerative diseases.

[0448] Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disease that preferentially targets neurons in the motor cortex and motor neurons in the brainstem and spinal cord. Despite the discovery of >40 mutations that cause familial ALS (Peters et al. 2015), the pathogenic processes leading to motor neuron degeneration and the reasons for the susceptibility of differentiated neuronal subtypes to widely expressed mutant proteins remain poorly understood.

[0449] Effective modeling of ALS is hindered by the limited accessibility of motor neurons in patients and animal models, as well as by the fact that ALS is a late-onset, relatively slowly progressive disease. The development of stem cell technologies, which facilitate the large-scale production of spinal motor neurons carrying disease-causing mutations, has circumvented the initial challenge and enabled biochemical analysis and drug screening in a relevant cellular context (Amoroso et al. al. 2013;Dimos et al. 2008;Hoing et al. 2012;Wichterle et al. 2002;Yang et al. 2013). However, stem cell-derived motor neurons are transcriptionally and electrophysiologically immature and most closely resemble embryonic or early postnatal motor neurons (Patterson et al. 2012;Stein et al. 2014;Miles et al. 2004). Most importantly, stem cell-derived motor neurons carrying ALS-causing mutations do not exhibit key features of motor neuron disease, such as mutant protein aggregation (Bosco et al. 2010;Kiskinis et al. 2014;Wang et al. 2009).

[0450] Despite their relatively immature state, several studies have reported differences in the viability, physiology, and biochemistry of ALS motor neurons derived from cultured human and mouse hepatocytes (Kiskinis et al. 2014; Alami et al. 2014; Devlin et al. 2015; Di Giorgio et al. al. 2008;Di Giorgio et al. 2007;Donnelly et al. 2013;Egawa et al. 2012;Naujock et al. 2016;Sareen et al. 2013;Sivadasan (Wainger et al. 2014; Wainger et al. 2016). These phenotypes may result from stressful in vitro environments that induce premature or aberrant manifestations of pathological processes in cultured cells, yet the nature of these culture-related stressors remains ill-defined. Understanding which specific stressors enhance disease-related motor neuron pathology will enable the development of more faithful models of ALS—better tools for understanding disease onset and progression and for screening neuroprotective drugs.

[0451] Here we developed a highly sensitive motor neuron viability assay and used it to induce ALS-like motor neuron degeneration in transgenic mice. Human SOD1 transgene carrying the G93A mutation (hSOD1 G93A A library of bioactive compounds was screened for stressors that sensitize motor neurons to overexpression of SERCA (Gurney et al. 1994). The screen identified cyclopiazonic acid (CPA), an inhibitor of the endoplasmic reticulum-expressed calcium pump (SERCA) (Goeger et al. 1988), as a compound to which ALS motor neurons are highly sensitive. We demonstrate that CPA rapidly induces endoplasmic reticulum (ER) stress and the unfolded protein response pathway in cultured motor neurons. The accumulation of misfolded proteins is a hallmark of many neurodegenerative diseases and has been reported in animal and stem cell-based models of ALS (Kiskinis et al. 1994). al. 2014;Hetz et al. 2009;Saxena et al. 2009;Saxena et al. 2013), as well as in post-mortem spinal cord samples from ALS patients (Hetz et al. 2009;Atkin et al. 2006; Atkin et al. 2008), and has been described in association with ER stress activation.

[0452] Taking advantage of this accelerated motor neuron degeneration paradigm, we performed a second screen to identify compounds that protect spinal motor neurons against CPA toxicity. We demonstrate that kenpaullone, a protein kinase inhibitor recently shown to protect motor neurons from neurotrophic factor withdrawal (Yang et al. 2013; Liu et al. 2016), also protects motor neurons in the CPA assay. In addition to kenpaullone, we identified several other protective compounds, including additional kinase inhibitors and the bile acid component, tauroursodeoxycholic acid (TUDCA). The rescue effects of these compounds in mouse hepatocyte-derived motor neurons were also reproduced in human stem cell-derived motor neurons. Furthermore, we demonstrate that TUDCA inhibits the proliferation and differentiation of transgenic hSOD1 neurons at early symptomatic stages of the disease. G93A It was found to reduce denervation of the neuromuscular junction in mice.

[0453] Collectively, these findings highlight the translational potential of the developed stem cell-based motor neuron viability assay. In identifying the ER stress-inducing compound CPA as preferentially toxic to motor neurons, our initial stressor screen independently rediscovered a pathway known to be involved in ALS pathology (Kiskinis et al. 2014; Saxena et al. 2009; Saxena et al. 2013; Nishitoh et al. 2008). Furthermore, hSOD1 G93A The protective effect of TUDCA in mice provides proof-of-principle that compounds arising from in vitro screening platforms can be seamlessly transferred to in vivo models of ALS. hSOD1 G93A Subtle defects in survival and neurite outgrowth of motor neurons derived from hepatocytes expressing β-actin

[0454] To gain insight into the pathological processes involved in the development of motor neuron degeneration in animals expressing mutant SOD1 proteins, we developed a two-color motor neuron viability assay. We reasoned that discovering the mechanisms involved in the increased susceptibility of motor neurons to ALS-causing mutations would require a robust, sensitive, and scalable system capable of distinguishing motor neuron cell-autonomous phenotypes from non-cell-autonomous phenotypes associated with more advanced stages of the disease (Di Giorgio et al. 2008; Di Giorgio et al. 2007; Boillee et al. 2006; Kang et al. 2013; Nagai et al. 2007).

[0455] Distinguish between wild-type (WT) and mutant motor neurons and minimize well-to-well variability To minimize the number of genes involved and increase the scalability, we used hSOD1 WT (control) and hSOD1 G93A (mutant) transgene-containing mice (Gurney et al. We generated new pluripotent embryonic stem cell (ESC) lines by crossing mice expressing eGFP (Wichterle et al. 2002) or tagRFP under the control of the motor neuron-specific Hb9 (Mnx1) promoter (Figure 15A; Figure 21A, and Figure 21B). Immunostaining with antibodies against Hb9 and the motor neuron transcription factor Isl1 confirmed that the new cell lines differentiated into motor neurons with comparable efficiency (Figure 15A and Figure 15B, Figures 21C-21E). We next sought to determine whether the new mutant cell lines recapitulated known ALS phenotypes. Using immunohistochemistry and biochemistry (data not shown), we did not observe large SOD1 aggregates within the mutant motor neurons, which are typically observed in postmortem tissue from ALS patients (Grad et al. 2014). However, immunoprecipitation with an antibody that specifically recognizes a conformational epitope on misfolded human SOD1 revealed the presence of a misfolded SOD1 species (Fig. 21H), which has already been implicated in ALS pathogenesis (Gros-Louis et al. 2010).

[0456] Newborn motor neurons are highly dependent on neurotrophic factors for their survival and neurite outgrowth (Henderson et al. 1994; Oppenheim et al. 1992; Sendtner et al. 1991). To determine whether growing motor neurons under suboptimal conditions results in preferential degeneration in mutant over WT motor neurons, we cultured both genotypes in medium supplemented with reduced amounts of neurotrophic factors and assessed motor neuron survival and neurite outgrowth. The most supportive medium contained neurotrophic factors with documented effects on motor neuron survival and neurite outgrowth: glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and insulin-like growth factor-1 (IGF-1). We also evaluated the effects of IBMX and forskolin, two compounds previously shown to increase cyclic AMP (cAMP) levels and act synergistically with neurotrophins to support primary motor neuron survival (Hanson et al. 1998). IBMX and forskolin alone supported motor neuron survival over the first 24 hours after plating, but their effects rapidly diminished over the following 24–48 hours (Figure 21J). In contrast, GDNF alone provided robust support for motor neuron survival for up to 1 week in culture, an effect that could be enhanced by cAMP-elevating compounds (Figure 21K). Based on these results, we evaluated three different culture conditions: low neurotrophic support (10 μM forskolin + 100 μM IBMX), medium neurotrophic support (10 μM forskolin + 100 μM IBMX + 0.4 ng / ml GDNF), and strong neurotrophic support (10 μM forskolin + 100 μM IBMX + 10 ng / ml BDNF, CNTF, GDNF, and IGF-1) (Figures 21F and 21G).

[0457] Cell viability and neurite outgrowth were assessed by automated live imaging (Trophos Plate Runner) of fluorescent motor neurons (Figures 15D and 15E). To distinguish live cells from GFP-positive dead cells or fluorescent debris, we used Metamorph software (Molecular Devices) and applied a "healthy cell criterion" in which only cells with neurite lengths equal to five times the average cell body diameter were included in the analysis (Figure 15F). Quantification of the number of live motor neurons over the course of 6 days (Figures 21F and 21G) revealed a modest but consistent 15-20% reduction in mutant motor neuron viability and neurite outgrowth relative to controls. This difference was highly and moderately sustained and observed in mixed red-green cultures or individual cultures of each genotype (Figure 15G). Collectively, these findings suggest that neurotrophic factor withdrawal significantly alters the ability of ESC-derived hSOD to differentiate. 1 G93A These results demonstrate that expressing EGFR-1 does not enhance the survival defect detected in EGFR-1-dependent motoneurons. A two-color screen for stressors that induce preferential degeneration of mutant motor neurons

[0458] To identify pathways involved in mutant motor neuron degeneration, we designed a stressor assay in which RFP-expressing control motor neurons were mixed with GFP-expressing mutant neurons in equal proportions and cultured in 96-well plates under moderate neurotrophic support (Figure 15C). We reasoned that by analyzing motor neuron viability in mixed cultures containing other ventral spinal cord neurons and glial cells from both genotypes, our study could focus on cell-autonomous changes in motor neuron viability. Therefore, the effects of any non-cell-autonomous factors, such as ALS astrocyte toxicity (Kiskinis et al. 2014; Nagai et al. 2007), which may equally affect the viability of both genotypes, could be minimized.

[0459] Plated motor neurons were allowed to recover and extend neurites for 24 hours before treatment with a library of 1275 bioactive small molecules (Tocris Screen Mini and Custom Collection, Compounds were added at a final concentration of 10 μM using an automated robot-assisted liquid handling platform (Tecan), and cells were analyzed by live imaging 48 and 72 hours later. We measured the following parameters: the number of live fluorescent motor neurons ("motor neuron viability," Figure 15J), the average neurite outgrowth per cell ("total neurite outgrowth," Figure 22B), the GFP / RFP motor neuron ratio normalized to the GFP / RFP ratio of motor neurons in control wells ("normalized survival ratio," Figure 15H), and the GFP / RFP average neurite outgrowth ratio ("preferential neurite outgrowth," Figure 22A). To identify stressors that preferentially target mutant motor neurons, we first excluded all compounds that were clearly toxic to both control and mutant motor neurons (average survival rates of mutant and control cells were <25%). Among the remaining compounds, the subset that preferentially reduced mutant motor neuron viability and yielded ratios corresponding to a difference in viability of at least 50% included membrane receptor agonists and antagonists, ion pump and channel inhibitors, antimitotic drugs, and general proapoptotic agents (Figures 15H and 15I).

[0460] One of the selective stressors we identified was cyclopiazonic acid (CPA), a mycotoxin that reversibly blocks the sarcoplasmic reticulum-associated calcium ATPase (SERCA). SERCA is involved in sequestration of calcium from the cytoplasm to the endoplasmic reticulum (ER) (Goeger et al. 1988). Inhibition of SERCA reduces calcium concentrations in the ER, leading to protein folding dysfunction, activation of the unfolded protein response, ER stress, and apoptosis (Doutheil et al. 1997). We titrated CPA to establish an effective concentration range (6.25–12.5 μM) within which mutant motor neurons exhibited reduced survival compared to control motor neurons (Figures 16A–16C, 23A, and 23B). In all subsequent experiments, motor neurons were treated with 7.5 μM CPA unless otherwise indicated.

[0461] To further explore the effects of CPA, we examined whether it acts directly on motor neurons or indirectly through other cells in culture. We found that motor neurons purified by fluorescence-activated cell sorting (FACS) (Figures 23C-23F) were as sensitive to CPA as motor neurons in mixed cultures, indicating that CPA acts directly on motor neurons rather than on other cell types that subsequently produce secondary toxins (Di Giorgio et al. 2008; Di Giorgio et al. 2007; Nagai et al. 2007; Haidet-Phillips et al. 2011). These findings also suggest that other cell types present in the mixed cultures do not confer protection to CPA-treated motor neurons.

[0462] Degeneration of spinal motor neurons and neurons within the motor cortex, preferentially over other types of nerve cells, is a hallmark of ALS. To determine whether motor neurons are more sensitive to CPA treatment than other types of neurons, we investigated the effects of hSOD1 G93AWe analyzed mixed cultures composed of GFP-expressing spinal motor neurons, interneurons, and other cell types. Immunostaining of viable cells revealed survival of GFP-expressing motor neurons, with a reduction of the pan-neuronal marker Tuj1 by approximately 71%, while all GFP-expressing motor neurons were reduced by approximately 71%. - Tuj-1 + Non-motor neuron survival was reduced by only about 23% (FIGS. 16D and 16F). G93A Given this considerable difference in CPA sensitivity between various neuronal subtypes in mixed cultures derived from ESC lines, we sought to determine whether this was also the case in cultures derived from WT lines.

[0463] In this analysis, we generated a new ESC line expressing tdTomato in dI4 and dIL dorsal spinal cord inhibitory interneurons induced from Ptf1α-expressing progenitor cells (Hoang et al. 2017, unpublished data). We differentiated this line under conditions that promoted detailed dorsal interneuron identity. FACS-purified tdTomato-expressing interneurons were co-cultured with FACS-purified GFP-expressing spinal motor neurons (Figures 16E and 16G). Quantification of RFP-to-GFP-positive neurons revealed that CPA treatment reduced dorsal spinal cord interneuron viability by only approximately 17%, compared with approximately 70% of co-cultured spinal cord motor neuron viability. Together, these data demonstrate increased sensitivity of spinal cord motor neurons to CPA compared with other ventral or dorsal spinal cord neurons. Effect of CPA on cytoplasmic calcium levels

[0464] CPA is a reversible inhibitor of the SERCA pump, which is important for sequestration of cytosolic calcium into the endoplasmic reticulum. Immunostaining revealed that cultured ESC-derived motor neurons express high levels of the SERCA2 isoform (ATP2A2) (Figure 16H). To study the effects of CPA on calcium handling in motor neurons, we performed a series of imaging experiments to visualize free cytosolic calcium and measured it by radiometric analysis of the calcium indicator Fura-2. To avoid crosstalk between GFP and Fura-2 fluorescence, experiments were performed in sorted Hb9::tagRFP-expressing motor neurons. First, we characterized global calcium handling in control and mutant motor neurons 3 days after plating by measuring calcium transients evoked by the inotropic glutamate receptor agonist kainate (KA) (Carriedo et al. 2000; Carriedo et al. 1996). Exposure of cells to a 5-second pulse of 100 μM KA induced a rapid rise in cytosolic calcium in ESC-derived motor neurons, followed by a sequestration period in which calcium returned to baseline levels (Figures 22C and 22D), similar to primary motor neuron cultures (Carriedo et al. 2000). Motor neurons that failed to return to baseline within the 60-second imaging window were removed from further analysis. After a 5-minute recovery period, cells were exposed to 7.5 μM CPA and imaged for an additional 20 minutes. Continuous CPA exposure induced slow calcium transients in both genotypes (Figure 16I), eventually returning to near-baseline levels toward the end of the imaging sequence. Cells were allowed to recover for an additional 5 minutes and then subjected to a second KA pulse in the presence of CPA, which caused a rapid calcium transient returning to baseline (Figure 22E). This led us to conclude that calcium sequestration within the ER was blocked. We examined how cells handle cytosolic calcium elevations when CPA is disrupted. CPA significantly slowed but did not eliminate KA-induced calcium sequestration (Fig. 16J and Fig. 22E), indicating that although motor neurons normally rely on the ER to sequester cytosolic calcium, loss of this compartment allows them to use other sequestration pathways. This difference was not due to repeated stimulation with KA, as parallel experiments with multiple consecutive KA pulses in the absence of CPA did not reveal any impairment in the rate of calcium sequestration (data not shown). When comparing control and mutant motor neurons, we did not observe any difference between genotypes in the rate of KA-induced calcium sequestration (Tau) in the absence or presence of CPA (Fig. 16J), indicating that CPA relatively affects calcium handling.

[0465] Elevated cytosolic calcium can activate numerous intracellular signaling processes, including cell death pathways ( De Stefani et al. 2012 ; Jayaraman et al. 1997 ; Pinton et al. 2008 ), and its involvement in calcium dysregulation has been suggested in many neurodegenerative conditions, including ALS ( Kiskinis et al. 2014 ; Bernard-Marissal et al. 2012 ; Jaiswal et al. 2009 ; Jaiswal et al. 2009 ; Kawamata et al. 2010 ; Kim et al. 2012 ; Rothstein et al. 1990 ; Takuma et al. 2010 ). 1999;Tradewell et al. 2011;von Lewinski and Keller 2005). Therefore, improving calcium sequestration in the ER or inactivating degenerative pathways that promote calcium signaling could potentially counteract CPA toxicity. Therefore, we evaluated a panel of eight compounds with known effects on cytosolic calcium handling and / or signaling. These included BAPTA-am, a cell-permeable calcium chelator; dantrolene, an inhibitor of the ryanodine receptor, which releases calcium from ER stores into the cytoplasm; three inhibitors of calpain, a family of calcium-dependent cysteine ​​proteases; and three inhibitors of the calcium-activated kinase CaMKK / II. Notably, these treatments did not improve motor neuron viability or neurite retraction caused by CPA exposure (Figures 18A–18B). These data, combined with the observation that CPA-induced cytosolic calcium levels were essentially equal in vulnerable and resistant nonmotor neurons (Figure 25B), suggested that cytosolic calcium overload is unlikely to be the primary cause of CPA-induced motor neuron death. Spinal motor neurons are sensitive to activation of the ER stress pathway

[0466] In addition to its effects on cytosolic calcium, CPA treatment has been shown to decrease calcium levels in the ER, leading to the activation of ER stress pathways (Doutheil et al. 1997). These pathways are initiated by binding immunoglobulin protein (BiP; heat shock protein 70, HSP70; 78 kDa glucose-regulated protein, GRP-78), an ER-resident chaperone that translocates from its binding site on the ER membrane-bound stress sensor after detecting unfolded proteins in the ER lumen. Unbound BiP mediates the activation of three distinct ER stress-related signaling pathways: PERK, ATF-6, and IRE1α, hereafter referred to as the unfolded protein response (UPR, Figure 19F) (Gardner et al. 2013; Hetz 2012; Lurlaro and Munoz-Pinedo 2016; Kozutsumi et al. 1988).

[0467] To assess the activation of these pathways in motor neurons exposed to CPA, we used RT-PCR to measure the activity of hSOD1 G93A The expression levels of 15 stress-related genes were examined at three time points after CPA treatment in motor neurons (Figure 17A We observed a rapid increase in the expression of Bip and the key downstream effector Chop (Ddit3). Bip increased 2-fold 1 hour after CPA exposure and continued to increase approximately 5-fold by 8 hours. Chop was induced 4-fold at 1 hour of CPA treatment, reaching a 16-fold induction at 4 and 8 hours. Other genes with >2-fold induction included: p58IPK255 (6.5-fold at 8 hours), ER stress-induced protein kinase; growth arrest and DNA damage-induced protein 45 alpha (Gadd45a; 5-fold at 4 hours), and pre-symptomatic SOD1. G93A It has recently been shown to be upregulated in the spinal cord of mice (Saxena et al. 2009); Erdj4, a Bip cofactor involved in ER-associated protein degradation (ERAD) (5-fold increase at 4-8 hours); Atf4, a downstream mediator of the PERK axis of UPR (3-fold increase at 8 hours); calreticulin, hSOD1 G93A These included ER-associated chaperones (3-fold at 8 h), which have been implicated in non-mediated motor neuron degeneration in mice (Bernard-Marissal et al. 2012); and Nrf2, a PERK substrate (2-fold at 8 h). Overall, these expression changes indicated a robust activation of multiple axes of the unfolded protein response (UPR) in motor neurons exposed to CPA (Figure 19F).

[0468] Western blot analysis of protein extracts from control and mutant motor neurons exposed to CPA for 1, 2, 4, 8, and 24 h confirmed early activation of the PERK pathway (increased phosphorylation of Eif2α, already detectable 1 h after CPA exposure) and the accumulation of an active truncated form of ATF-6, detectable at 8 h in both genotypes (Figure 17C). Activation of the IRE1α branch was assessed by qPCR analysis of X-box binding protein 1 (XBP1) splicing, which was induced by 1 h after CPA treatment and peaked at 4 h after exposure (Figure 17B). We also assessed activation of the IRE1α branch by immunocytochemical analysis of c-jun phosphorylation (Urano et al. 2000), which peaked 2 h after CPA treatment (Figures 17E and 17F).

[0469] Activation of the PERK and ATF-6 branches of the UPR / ER stress pathway is associated with protective cellular responses aimed at attenuating protein synthesis and accelerating the clearance of misfolded proteins (Hetz 2012; Lurlaro and Munoz-Pinedo 2016). Therefore, we sought to examine the effect of CPA treatment on the accumulation of misfolded SOD1 protein in cultured motor neurons (Bosco et al. 2010; Hetz et al. al. 2009;Saxena et al. 2009;Nishitoh et al. (Gros-Louis et al. 2010). We treated mutant motor neurons with CPA or vehicle and immunoprecipitated misfolded SOD1 using two different conformation-specific hSOD1 antibodies. Western blot analysis revealed an unexpected CPA-dependent increase in the accumulation of misfolded SOD1, indicating that CPA treatment impairs the clearance of misfolded SOD1 protein (Figure 17D).

[0470] To strengthen the link between ER stress activation and motor neuron degeneration, we extended our studies to include two additional compounds that activate ER stress: thapsigargin, a drug that acts similarly to CPA by inhibiting the SERCA pump but is irreversible (Lytton et al. 1991), and tunicamycin, a drug that triggers ER stress in a calcium-independent manner by inhibiting protein N-glycosylation in the ER (Oslowski et al. 2011). We exposed cocultures of GFP-expressing motor neurons and tdTomato-expressing dorsal interneurons to increasing concentrations of thapsigargin and tunicamycin, and found that, as in the case of CPA, spinal motor neurons were selective for both compounds. Therefore, the motor neuron toxic effect of CPA appears to be related to its activation of the ER stress pathway, and the effect does not appear to be related to calcium levels or other off-target effects (Figure 16G). Compounds that protect motor neurons from CPA-induced degeneration

[0471] Understanding that motor neurons are more sensitive to activation of the UPR / ER stress pathway than other spinal cord neurons, we set out to screen candidate molecules aimed at identifying compounds that could protect motor neurons from CPA toxicity. ER stress has been implicated in the pathogenesis of ALS and many other neurodegenerative conditions (Hetz and Saxena 2017). We screened a panel of >100 compounds compiled from an in-house library and supplemented with compounds derived from literature research (Table 3). This panel included compounds that modulate various branches of ER stress, affect calcium sequestration, act as neurotrophic factors, and have previously been shown to promote motor neuron survival. Mixed motor neuron cultures were pretreated with rescue compounds for 45 minutes before exposure to 7.5 μM CPA. Viability and neurite outgrowth were assessed 24 and 48 hours later. The screen yielded several compounds that protect >50% of motor neurons that die in response to CPA (Figures 18A-18B): the c-jun N-terminal kinase (JNK) inhibitor SP600125; the tyrosine kinase inhibitor sunitinib; and the broad-spectrum kinase inhibitors Ro31-8220 mesylate, kenpaullone, GO6976, H-7, and K252a (Sakaki et al. 2008; Kase et al. 1987; Roux et al. 2002). Compounds that rescued >50% of neurite outgrowth included the neurotrophic factor calcidotrophin-1; p38 inhibitors SB293063 and SB203580; SP600125; sunitinib; bile acids TCA, TGCA, and TUDCA; and kinase inhibitors Ro31-8220 mesylate, GO6976, Kenpaullone, H-7, and K252a (Figure 18B). Overall, GO6976, Kenpaullone, K252a, and TUDCA appeared to be the most promising candidates due to their potent pro-survival effects (GO6976, Kenpaullone, K252a) or potent neurite outgrowth promotion (TUDCA) at low concentrations and were selected for further characterization.

[0472] Kenpaullone, a protein kinase inhibitor, recently emerged as a hit from a screen for compounds that rescue neurotrophic factor deprivation in ESC-derived motor neurons (Yang et al. 2013). We found that it also effectively prevented motor neuron degeneration in response to CPA. We identified two additional kinase inhibitors, the staurosporine analog GO6976 and K252a, that provided equivalent rescue of motor neurons but were active at lower concentrations than Kenpaullone (0.5-2 μM compared to 5-10 μM for Kenpaullone) (Figure 19A). We also determined that TUDCA, an acid-base amphoteric bile acid moiety that functions as a chemical chaperone, rescues neurite outgrowth (Figure 25F) but exhibits only a modest effect on motor neuron degeneration (Figure 19A).

[0473] One advantage of the stem cell-derived motor neuron viability assay is that it can be easily extended to human motor neurons. To adapt the assay to human cells, we generated a new isogenic pair of ESC lines derived from a human ESC line expressing GFP under the control of the Hb9 motor neuron promoter (HUES3 HB9::GFP, (Di Giorgio et al. 2008)). The ALS-causing A4V mutation was introduced into a single allele of the SOD1 gene using zinc finger nuclease (ZFN)-based genome engineering to recapitulate the human patient genotype (Figures 24A-24L). The cell line pair was constructed using previously published protocols (Amoroso et al. 2013; Maury et al. 2015), were used in parallel to differentiate into motor neurons and characterized for viability and neurite outgrowth under increasing concentrations of CPA. Although human motor neurons are less sensitive to CPA than mouse motor neurons (Figure 24J), we observed a significant decrease in the viability and neurite outgrowth of mutant human SOD1 neurons exposed to 33 μM CPA (approximately 86% loss) compared to control neurons (approximately 71% loss). A4VA significantly increased degeneration of motor neurons was detected, thereby recapitulating the genotype-dependent effects of CPA in mouse motor neurons (FIGS. 19D and 19E).

[0474] Next, we used an assay to test whether compounds protective for mouse motoneurons could also protect human motoneurons exposed to 33 μM CPA. Notably, all tested protective compounds identified in the mouse motoneuron screen were also effective in protecting human motoneurons against CPA. Pretreatment of human motoneurons with Kenpaullone prevented 42% of CPA-induced cell death in WT motoneurons and reduced SOD1 expression. A4V GO6976 rescued 31% of cell death in WT motor neurons (Fig. 19D), but had no significant effect on neurite outgrowth (Fig. 24I). GO6976 rescued 45% of cell death in WT motor neurons and SOD1 A4V K252a rescued 34% of the cell death in motoneurons (Fig. 19D) and also significantly rescued the loss of neurite outgrowth (Fig. 24K). K252a was the most promising compound overall, reversing 56% of cell death in WT and 34% of SOD1 A4V TUDCA rescued 100% of cell death in motor neurons (Figures 19D and 19E) and significantly affected neurite outgrowth for the latter group. Finally, TUDCA inhibited WT and SOD1 A4V It reduced motor neuron cell death by 34% and 9%, respectively, without substantially affecting neurite outgrowth (FIG. 19D, FIG. 24K). Table 3. Screen for rescue of CPA toxicity. List of compounds tested for their ability to reverse CPA-induced MN toxicity and / or neurite retraction. Compounds are subdivided based on their putative pathway or mechanism of action. Rescue activity is expressed as % reversal of CPA effects on viability and neurite outgrowth for the best time point (24 or 48 hours post-CPA) and concentration tested. Values ​​represent either the average of replicate culture wells or from independent experiments for compounds evaluated in follow-up experiments. [Table 3-1] [Table 3-2] The dietary supplement TUDCA is neuroprotective, reverses CPA-induced neurite retraction, and delays ALS-associated muscle denervation in vivo

[0475] TUDCA is an FDA-approved dietary supplement, and its effects on various pathological conditions have been the focus of numerous clinical trials (NCT00877604, NCT022186 19, NCT00771901, NCT01829698). TUDCA is generally safe, has few side effects, and exhibits good blood-brain barrier penetration when administered subcutaneously or orally (Kaemmerer et al. 2001). Because neuromuscular junction (NMJ) denervation is one of the earliest phenotypes observed in mouse models of ALS (Azzouz et al. 1997; Maselli et al. 1993; Tsujihata et al. 1984; Sharma et al. 2016), we reasoned that TUDCA might promote the maintenance of motor axon terminal integrity and delay the denervation process.

[0476] To compare the efficacy of TUDCA with its analogs, we screened it in parallel with 10 conjugated bile acids in CPA-treated motor neurons. Taurine-glycine conjugated cholic acid (TGCA) was consistent with TUDCA's modest effect on motor neuron viability but worked at lower concentrations; however, it had less effect than TUDCA on neurite outgrowth (Figures 25C and 25D). Taurine-conjugated cholic acid, TCA, showed similar effects to TUDCA on both motor neuron viability and neurite outgrowth, but did not offer any advantages for drug development. Therefore, we decided to proceed with TUDCA for further in vivo experiments.

[0477] To test the ability of TUDCA to preserve motor axons in an ALS model in vivo, we designed a small-scale study to evaluate denervation of the fatigue-prone hindlimb tibialis anterior (TA) muscle (Figure 20D). We have previously determined that TA motor neurons in B6.Cg-Tg(SOD1*G93A)1Gur / J mice undergo a period of ER stress starting at P30, followed by muscle denervation extending to P50 (Kaplan et al. 2014). During this period, the TA muscle experiences 25-40% denervation before becoming substantially atrophied at later time points. To target this window of ER stress-related neurodegeneration, we used hSOD1 G93A Mice were treated with subcutaneous TUDCA injections every 3 days between P30 and P50. At the end of the experiment, we counted total NMJs by staining acetylcholine receptors on the TA muscle with Alexa Fluor 488-conjugated α-bungarotoxin and assessed their innervation by staining motor axons with an antibody against vesicular acetylcholine transferase (VAChT) (Figure 20F). Despite the fact that mice received only seven injections over the course of 21 days of treatment, we found significantly higher levels of TUDCA-treated hSOD1 compared to untreated animals. G93A We observed a modest but statistically significant increase in NMJ innervation in mice (FIG. 20E). Consideration

[0478] Modeling degenerative diseases in cell culture systems opens new opportunities for investigating the pathological processes associated with disease-causing mutations and for screening novel therapeutic agents. However, adult-onset degenerative diseases in which the causative mutations result in subtle changes in cell function or mild viability or morphological phenotypes are difficult to model in the kind of short-term culture systems that are amenable to high-throughput drug screening. The inventors reasoned that the discovery of stressors that induce and accelerate phenotypic changes in motor neurons in vitro could provide insight into the molecular pathways involved in motor neuron degeneration and could lead to the discovery of novel motor neuron-protective compounds.

[0479] ALS-causing mutations are not clearly toxic to spinal motor neurons: patients do not show any obvious motor defects during the presymptomatic stage of the disease, which typically lasts for decades. Even in an invasive mouse model of ALS caused by overexpression of mutant hSOD1, motor neuron death is not observed until adulthood (Chiu et al. 1995), suggesting that the effects of ALS mutations are cumulative or that they progress with age and development. These results suggest that stressors are potentiated by ER stress and / or environmentally related stressors. To identify the stressors involved in mutant motor neuron degeneration, we designed a highly sensitive, endogenously controlled viability assay. The coculture setting reduces the influence of non-cell-autonomous virulence factors, allowing us to focus on the inherently increased vulnerability of motor neurons expressing disease-causing mutant SOD1 proteins. Using this platform, we identified CPA as a compound that is more toxic to mutant than control ESC-derived motor neurons. Perhaps an even more intriguing observation was that motor neurons were significantly more sensitive to CPA and other ER stress-inducing compounds than other types of spinal cord neurons.

[0480] CPA inhibits the SERCA pump and affects several pathways and cellular processes already associated with ALS (Kiskinis et al. 2014;Hetz et al. 2009;Saxena et al. 2009;Saxena et al. 2013;Nishitoh et al. 2008;Bernard-Marissal et al. 2012;Jaiswal et al. 2009;Jaiswal et al. 2009;Takuma et al. 1999;von Lewinski and Keller 2005;Kikuchi et al. 2006;Parone et al. 2013). We have shown that CPA treatment (Peters et al. 2015) disrupts calcium handling; (Amoroso et al. 2013) triggers ER stress and the unfolded protein response; and (Dimos et al. 2008) disrupts hSOD1. G93A We show that SERCA inhibition results in the accumulation of mutant SOD1 in ESC-derived motor neurons. This last finding suggests that mutant SOD1 is normally cleared via a constitutive pathway, which is impaired after SERCA inhibition. Misfolded forms of SOD1 have been observed in human postmortem tissue samples and in several ALS models, but the exact mechanism through which they perpetuate motor neuron death is unclear (Gros-Louis et al. 2010; Kikuchi et al. 2006). Even though SOD1 is translated on free cytoplasmic ribosomes, previous studies have shown that mutant forms can be detected within mitochondria and the ER (Kikuchi et al. 2010). et al. 2006;Vande Velde et al. 2008).

[0481] To analyze the role of abnormal calcium handling in inducing ER stress in CPA-mediated neurodegeneration, we tested a series of pharmacological agents that modulate intracellular calcium levels or induce ER stress via calcium-independent pathways. Based on these studies, we concluded that calcium mishandling is unlikely to drive the acute degenerative process observed in response to CPA. Therefore, we focused on the unfolded protein response and ER stress-related and downstream pathways.

[0482] Screening of candidate neuroactive compounds identified several potent drugs capable of reversing the deleterious effects of CPA. Two classes of compounds were of particular interest: kinase inhibitors and bile acid derivatives. Kinase inhibitors demonstrated remarkable ability to protect motor neurons from CPA toxicity. One compound, kenpaullone, has previously been shown to protect motor neurons from neurotrophic deprivation (Yang et al. 2013). It has also been demonstrated to improve survival and reverse electrophysiological defects in motor neurons derived from human stem cells from patients with mutations in the FUS gene (Liu et al. 2016). In addition to kenpaullone, we identified two staurosporine analogs, K252a and GO6976, which have previously been reported to increase neuronal survival in several in vitro neurodegeneration models (Roux et al. 2002; Jeohn et al. 2000). However, However, these inhibitors will require further optimization. Kenpaullone is insoluble in aqueous solution at its most effective concentration, precluding further in vivo studies without extensive attempts to reformulate its vehicle composition. K252a and GO6976 are more potent and soluble than Kenpaullone in our viability assays (data not shown); however, these compounds are broad-spectrum inhibitors, each targeting >100 different kinases, and the identity of the targets relevant to motor neuron degeneration remains ill-defined. We are currently working to identify these targets and develop more specific analogs for further in vivo testing.

[0483] The second class of neuroprotective compounds emerging from our screening was a derivative of mammalian bile acids, which have been widely used in traditional Tibetan and Chinese herbal medicine. This class of compounds protected <30% of dying motor neurons, but it completely restored neurite outgrowth. In contrast to kinase inhibitors not approved for human use, tauroursodeoxycholic acid (TUDCA) is a widely available dietary supplement, and its analog, UDCA, is a water-soluble, FDA-approved drug for treating itch and liver disease. TUDCA has previously been shown to have beneficial effects in mouse models of Huntington's disease and Alzheimer's disease (Keene et al. 2002; Keene et al. 2001; Nunes et al. 2012). TUDCA has also been shown to reduce the expression of markers of the unfolded protein response in mouse models of type 2 diabetes, in part by acting as a chaperone for misfolded proteins (Ozcan et al. 2006; Uppala et al. 2017). We extend these studies by demonstrating that TUDCA preserves the integrity of the neuromuscular junction in a mouse model of ALS, raising the possibility that TUDCA alone or in combination with other treatments may delay the onset or progression of motor disease.

[0484] Our findings add to the mounting evidence that ER stress contributes to motor neuron cell death in ALS, and we describe a scalable stem cell-based system for probing both the cell-autonomous effects of ER stress and means to reverse them. This system can be readily adapted to other neurodegenerative conditions characterized by ER stress, such as Parkinson's disease, Huntington's disease, prion diseases, and Alzheimer's disease (Hetz and Saxena 2017). Most importantly, we adapted the assay to test neuroprotective compounds directly on human motor neurons, thereby accelerating the preclinical evaluation of drugs that modulate motor neuron survival by maximizing their potential efficacy in human patients. material and method Derivation of mouse transgenic embryonic stem cell lines

[0485] Heterozygous Tg(Hlxb9-GFP)1Tmj or Tg(Hlxb9-tagRFP) reporter mice were crossed with mice expressing a mutant form of human SOD1 (B6.Cg-Tg(SOD1*G93A)1Gur / J) or a wild-type form (B6SJL-Tg(SOD1)2Gur / J). Blastocysts were collected at embryonic day 3.5. Mouse embryonic stem cell (ESC) lines were derived as previously described (5). New lines were genotyped and sequenced to confirm the presence of both the transgene and the G93A point mutation.

[0486] For interneuron differentiation, a mouse embryonic stem cell line was derived from Ptf1α::cre mice (kidneys provided by Dr. Kaltschmidt) crossed with Rosa-LSL-tdTomato fluorescent reporter mice (Kawaguchi et al. 2002;Madisen et al. 2010). Generation of isogenic human embryonic stem cell lines by gene targeting

[0487] To extrapolate the results from the mouse assay, we investigated the SOD1 expression level by introducing the A4V mutation into the wild-type SOD1 locus of the human embryonic stem cell line HUES3 Hb9::GFP. + / A4V and SOD1 + / + An independent collection of isogenic cell lines was generated (Di Giorgio et al. 2008). Again using a two-step nuclease-mediated gene targeting strategy (Maeder et al. 2008), we transfected SOD1 A4V The mutation was introduced into the HUES3 Hb9::GFP genetic background (Figure 24A). After confirming the desired SOD1 gene editing (Figures 24B and 24C), we performed a transfection of the SOD1 gene. + / A4V and SOD1 + / + Differentiation of HUES3 Hb9::GFP cells into cultures containing spinal motor neurons. GFP of both genotypes + Motor neurons could be isolated using FACS (Figure 24D), and SOD1 transcription and protein expression in these cells was confirmed (Figures 24F and 24G). The new system was further validated by staining for the motor neuron transcription factor Islet1 (Figure 24E) and assessing survival in short-term and long-term cultures (Figures 24H and 24I). Mouse and human motor neuron differentiation

[0488] Motor neuron differentiation of transgenic mouse ESCs was performed as previously described (Wichterle et al. 2002). Briefly, cells were dissociated on day 6 of differentiation and plated onto surfaces coated with polyornithine (Sigma, 100 μg / ml) and laminin (4 μg / ml). Cells were cultured in the presence of the cAMP-elevating compounds forskolin (10 μM) and IBMX (100 μM) in combination with a variety of neurotrophic factors (GDNF, BDNF, CNTF, IGF-1) as described in the text (Figures 21G, 21K, and 21L). In the majority of all experimental mouse cultures containing motor neurons, interneuron and glial precursor cells were used (referred to as "motor neuron cultures"), and in some experiments, motor neurons were purified by FACS (see below).

[0489] For differentiation into dI4 interneurons, dissociate Ptf1α-tdTomato ESCs and culture 8.0 × 10 cells. 5 The cells were cultured in suspension as embryoid bodies (EBs) at a density of 10 cells / 10 cm culture-treated Petri dish. On day 2 of differentiation, EBs were collected, spun down, and split 1:4 into new Petri dishes supplemented with 1 μM RA. Medium was changed on days 4 and 6 of differentiation. The endpoint of dl4 IN differentiation was day 8, and EBs were collected for coculture studies.

[0490] Differentiation of the human isogenic HUES3 ESC HB9::GFP reporter line into motor neurons was performed as previously described (Maury et al. 2015). Cells were dissociated on day 16 of differentiation, FACS sorted, and plated onto polyornithine- and laminin-coated surfaces as described above. Serum-free human motor neuron plating medium was supplemented with the antimitotic UFdU and the neurotrophic factors GDNF, BDNF, CNTF, and IGF1 (all at 10 ng / mL) as previously described (Johnson-Kerner et al. 2015).

[0491] All cell lines used were routinely tested for mycoplasma. Flow cytometry-assisted cell sorting (FACS)

[0492] The cells were then imaged using a five-laser ARIA-IIu laser, configured with a 100 μm ceramic nozzle. Cells were sorted based on GFP or RFP expression using a ROU Cell Sorter (BD BioSciences) operated at 20 psi. Two-color ESC-motor neuron co-culture assay

[0493] Dissociated fluorescent Hb9::RFP-hSOD1 WT The cells were counted using a hemocytometer, and the same number of Hb9::GFP-hSOD1 G93A The cells were mixed with motor neurons so that 500 fluorescent cells of each genotype were plated per well. Cells were plated onto coated 96-well plates in medium containing FSK and IBMX (low nutrient support, a positive control for cell death) or FSK, IBMX, and 250 pg / mL GDNF (moderate nutrient support, a positive control for survival). Automated Image Analysis

[0494] Living GFP + Images of all cells in the wells are displayed in Plate Runner HD Images were acquired using a Trophos system. Images were analyzed using Metamorph software (Molecular Devices) to assess the number of "healthy" fluorescent cells showing growth >5x the cell body diameter (assessment of overall viability) and the average neurite length per cell (assessment of neurite outgrowth). These parameters were used consistently throughout the study. Because the endogenous Hb9::GFP reporter in the human system was not bright enough to be detected by our automated imaging platform, cells were treated with the live cell dye calcein-AM (1.33 μM) for 10 min immediately before imaging, followed by quenching with a 10% solution of hemoglobin in PBS. Small molecule screening

[0495] Approximately 1300 bioactive compounds from the Tocris Mini Screen and Custom collection were added to each screening plate at a final concentration of 10 μM. The final concentration of DMSO was 0.5%. Survival ratios were calculated as the ratio of surviving GFP cells 48 and 72 hours after compound exposure. + The number of cells was measured using RFP + The ratio was calculated by dividing by the number of cells. We consistently observed that the ratio of surviving untreated G93A mutant motor neurons to untreated wild-type motor neurons was approximately 0.83; all data were normalized to this ratio to detect compounds that act synergistically across genotypes. Compounds that resulted in a G93A to WT survival ratio of <0.67, corresponding to a 50% difference in survival, were retested in a five-point 2-fold dilution series. All hit compounds were visually evaluated, and wells with obvious artifacts such as autofluorescence and excessive cell clumping were removed. Compounds that were generally toxic, i.e., compounds that resulted in lower survival rates than controls with low nutrient support for both genotypes, were rescreened in a 1:10 dilution series. Compounds that reproduced the effects of the primary screen were then tested in an independent set of cell lines and in co-mixed and FACS-purified co-cultures to confirm cell-autonomous effects. ER Stress Rescue Screen

[0496] Dissociated fluorescent Hb9::GFP-hSOD1G93A 554 cells were counted and plated into coated 96-well plates at a density of 1000 fluorescent cells / well in medium containing FSK, IBMX, and GDNF. Cells were incubated for 45 minutes with rescue compounds or medium + 0.5% DMSO as a control. Rescue compounds were selected from an initial two-color screen or from a literature search focused on compounds with documented effects on ER stress in other models. Cells were then exposed to 7.5 μM CPA or medium + 0.5% DMSO as a control. Cells were imaged and scored as described in the previous section. Human ESC-derived HB9::GFP-SOD1+ / + and HB9::GFP-SOD1 + / A4V Motor neurons were plated separately in 96-well plates at 2000 cells / well as described in the previous section, and test compounds were added to serum-free human motor neuron plating medium supplemented with 10 ng / mL BDNF, GDNF, CNTF, and IGF1 as described above, 33 μM CPA (or DMSO control), and various rescue compounds. were added simultaneously. immunocytochemistry

[0497] Live cultures were pre-fixed with 4% PFA on ice for 2 minutes by adding the fixative directly to the medium, followed by an additional 15 minutes by replacing the well contents with 4% PFA and incubating at 4°C. Fixed cultures were blocked for 1 hour at room temperature with 0.01M PBS containing 0.3% Triton-X and 20% donkey serum. Primary antibodies were diluted in blocking solution and incubated overnight at 4°C. Cultures were washed three times with 0.01M PBS containing 0.3% Triton-X and then incubated with secondary antibodies (Alexa donkey488 / 555 / 647) for 60 minutes at room temperature. The following primary antibodies were used: mouse Hb9 and mouse islet1 / 2, Hybridoma Bank (1:100); Tuj-1, Neuromics or EMD Millipore (1:500); and phospho-c-jun, Cell Signalling (1:100) and SERCA2, Pierce (2A7A1, 1:100). Slides were examined with either a Zeiss AxioObserver, Zeiss LSM Meta 510, or Zeiss LSM 880 Axioobserver Z1 confocal microscope equipped with a Coolsnap HQ2 camera (Photometrics). Images were processed in Adobe Photoshop (Creative suite 6, Adobe Systems) for presentation purposes, with occasional cropping and changes made to the entire image and from there to all images in equal proportions. biochemistry

[0498] On day 6, EBs were lysed in TNG-T lysis buffer (36) containing protease (Complete Mini) and phosphatase (PhoStop) inhibitors for 30 min, followed by mechanical trituration with a 26G syringe. For immunoprecipitation, C4F6 and B8H10 antibodies (MediMabs) were coupled to Protein-G Dynabeads and used to immunoprecipitate misfolded hSOD1 as described by Gros-Louis et al. 2010. A control IgG antibody was used as a negative control (Figure 21I). A pan-SOD1 antibody (Novus Bio) was used for immunoblotting, and 5% of the input amount was used as a loading control. The following antibodies were used for Western blots: Caspase-3 (1:1000), CHOP (1:500), phospho-Eif2α (1:1000) (Cell Signaling Technologies), SOD1 and ATF-6 (1:200, Novus Biologicals), and α-tubulin (1:50000, AbCam). Representative gels are cropped from scanned images of the original film. Individual bands not relevant to the present study are indicated by dashed lines in the figures. Band intensities were measured with Image J (NIH) using the Analyze-Gels application. Quantitative PCR

[0499] Samples were lysed in TRIzol and frozen at -80°C until further processing. RNA was purified using Qiagen RNeasy and Qiashredder according to the manufacturer's protocol. RNA was extracted using the Qiagen Mini Kit. 1–2 μg of total RNA was used per reverse transcription reaction, and the reactions were performed using a TaqMan RT kit (Applied Biosystems, Grand Island, NY, USA). Primer pairs were designed for the target transcripts using Primer Express 3.0 (Applied Biosystems). Quantitative PCR reactions were performed using Power SYBR Green PCR Master Mix (Applied Biosystems). Reactions were run and analyzed on a ViiA 7 (Life Technologies) qPCR instrument using the absolute quantitation setting. XBP1 splicing

[0500] PCR was performed in a 50 μL jumpstart Taq (Sigma-Aldrich, product number D9307) reaction containing 10 pmol of XBP-1-specific primers to detect splicing (forward: 5'-GAATGCCCAAAAGGATATCAGACTC-3' & reverse: 5'-GGCCTTGTGGTTGAGAACCAGGAG-3'). PCR conditions were as follows: 1 cycle at 94°C for 1 minute; 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute; and 1 cycle at 72°C for 1 minute. PCR products were run on a 2.5% agarose gel containing ethidium bromide for 30 minutes. Bands were observed and quantified using Syngene G:Box and Genesis software. Band intensity was measured with Image J (NIH) using the Analyze-Gels application. Calcium imaging

[0501] Hb9::RFP motor neurons were dissociated on day 6 of differentiation and cultured on glass coverslips. Three days after dissociation, coverslips were incubated with 5 μM Fura-2 AM, a ratiometric calcium indicator dye (Life Sciences, USA), for 30 minutes at room temperature. After washing, coverslips were mounted on a Nikon Eclipse TE 3500 inverted microscope equipped with a 40× 1.30 NA objective (Nikon, USA), a pco.EDGE CMOS camera (pco, Germany), a Lambda LS light source, and a Lambda LS-2 filter wheel with 340 nm and 380 nm excitation filters (both Sutter, USA). Images were first acquired using brightfield and fluorescence filters to identify cells to be analyzed in subsequent steps. Drug application was performed using a custom-built focal application system positioned approximately 100 μm from the field of view. A 5-second baseline for the Fura-2 signal was then acquired, followed by a 1-second pulse of 100 μ...

Claims

1. 【Chemical 294】 or an N-oxide, hydrate or pharmaceutically acceptable salt thereof, selected from the group consisting of: 【Request 2】 【Chemical 295】 2. The compound of claim 1, wherein:

3. 【Chemical 296】 2. The compound of claim 1, wherein:

4. 【Chemical 297】 2. The compound of claim 1, wherein:

5. 【Chemical 298】 2. The compound of claim 1, wherein:

6. 【Chemical 299】 2. The compound of claim 1, wherein:

7. A pharmaceutical composition comprising the compound according to any one of claims 1 to 6, or an N-oxide, hydrate or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

8. A kit comprising a compound according to any one of claims 1 to 6, or an N-oxide, hydrate or pharmaceutically acceptable salt thereof, together with instructions for use of said compound.

9. A kit comprising the pharmaceutical composition of claim 7 together with instructions for use of said pharmaceutical composition.

10. A composition for treating or ameliorating the effects of a disorder in a subject in need thereof, said composition comprising one or more compounds, said one or more compounds comprising: 【Chemistry 300】 or an N-oxide, hydrate or pharmaceutically acceptable salt thereof.

11. The composition described in claim 10, wherein the disorder is a disease involving endoplasmic reticulum (ER) stress.

12. The composition described in claim 10, wherein the disorder is a disease characterized by abnormal kinase levels in the subject.

13. The composition described in claim 10, wherein the disorder is a disease accompanied by axonal degeneration.

14. The composition of claim 10, wherein the disorder is selected from the group consisting of traumatic brain injury, stroke, ischemia, bipolar disorder, heart disease, atherosclerosis, type 1 diabetes, type 2 diabetes, obesity, cancer, autoimmune disease, and neurodegenerative disease.

15. The composition of claim 14, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Friedreich's ataxia, multiple sclerosis, Huntington's disease, transmissible spongiform encephalopathy, Charcot-Marie-Tooth disease, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy, chronic traumatic encephalopathy (CTE), polyglutamine disease, prion disease, glaucoma, and hereditary spastic paraplegia.

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