Compounds, Compositions, and Methods for Suppressing 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 Mar. 1, 2018, which is incorporated herein by reference in its entirety. Field of the Invention

[0002] The present invention relates, inter alia, to structures

Chem.

[0003] This invention was made with government support under award number XW81XWH - 16 - 1 - 0204 from the DOD. The United States government has certain rights in this invention.

Background Art

[0004] Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that targets spinal motor neurons, which are cells that control essential daily functions such as movement, breathing, and eating. ALS, like many other neurodegenerative disorders, is characterized by the accumulation of misfolded proteins Neurons activate the unfolded protein response (UPR) to refold or eliminate misfolded proteins, but when these attempts fail, the UPR shifts to the endoplasmic reticulum (ER) stress pathway that leads the cell to apoptotic cell death. Recent studies have implicated 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 previously 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 were also found to be highly sensitive to ER stress-inducing compounds such as cyclopiazonic acid (CPA) compared to other spinal neuron subtypes (Figure 2C). Together, these observations provide insight into the selective vulnerability of spinal motor neurons 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 screening of over 100 biologically active small molecules were a broad range of kinase inhibitors, including Go6976, sunitinib, K252a (Figure 2A), and kenpaullone (see Example 12). In a preliminary analysis, mitogen-activated protein (MAP) kinase MLK3 and HGK were suggested as potential functional targets of these rescue compounds. These findings were consistent with past reports that the IRE1a branch of the ER stress pathway ultimately activates the JNK pathway, which is associated with many neurodegenerative disease models. Past studies have also shown that CPA induces IRE1a activity and promotes phosphorylation of c-jun, which is immediately downstream of JNK (see Example 12).

[0008] For four kinase inhibitor hit compounds and alsterpaullone, a structural analog of kenpaullone that was found to be more potent than kenpaullone, publicly available data on the catalytic activity of 300 kinases (Anastassiadis et al., 2011) were mined in the presence of various kinase inhibitors at 0.5 μM, and known targets were compared by performing binary hierarchical clustering analysis (Figure 1).

[0009] Kinases whose activity was strongly inhibited by all five neuroprotective compounds (kinase activity remaining < 15% at 1 μM inhibitor concentration) included MAP kinase HGK (MAP4K 4) and the kinase NUAK1, which is related to MAPK. 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, WZ4003, a potent and selective NUAK1 inhibitor (IC for NUAK1 50= 20 nM) was found to be unable to rescue from CPA toxicity across a 6-point dilution series (0.1 - 10 μM). At 1 μM and higher concentrations, WZ4003 enhanced CPA-induced neurodegeneration and was toxic to motor neurons even in the absence of CPA (Figure 3). Similarly, other selective NUAK1 inhibitors, including SU6656 (remaining activity 10.9% at 0.5 μM) and HTH-01-051 (IC 50 = 100 nM, see Banerjee et al., 2014), were not only unable to rescue from CPA toxicity but also enhanced their toxicity at higher doses (Figure 3). These results suggest that NUAK1 activity is essential for the survival of motor neurons in the context of ER stress and possibly under basal conditions. The fact that NUAK1 is a common target of hit-rescue compounds is highly likely due to their structural similarity or sequence homology to the functional target of these compounds.

[0010] On the other hand, Yang et al. (2013) inferred that HGK is the main functional target of kenpaullone, which was identified as a hit in a high-throughput screening for compounds that can rescue ES-derived spinal motor neurons from death induced by neurotrophin withdrawal. Subsequently, Larhammar et al. (2017) provided functional evidence that HGK, in combination with two other MAP4 kinases, TNIK and MINK1, mediates neurodegeneration after neurotrophin withdrawal in mouse dorsal root ganglion neurons. To further assess the role of HGK in motor neurons in the context of ER stress, the selective HGK inhibitor PF-6260933 (Ammirati et al., 2015) was tested in a 6-point dilution series (0.01 μM - 5 μM), and it was found that HGK protected from CPA toxicity in a dose-dependent manner (Figure 3). The HGK inhibitor GNE-495 similarly 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. Although kenpaullone and sunitinib only slightly inhibited MLK3 activity (remaining MLK3 activity at 0.5 μM was 49.3% and 68.4%, respectively, see Anastassiadis et al., 2011), that literature provides strong support for the role of MLK3 in many neurodegenerative situations. Several additional compounds, including CEP1347, NU6140, bosutinib, JAK3 inhibitor IV, and Syk inhibitor, which potently (remaining activity < 15%), but non-selectively inhibit MLK3 activity, were found to provide dose-dependent rescue from CPA toxicity in human ES motor neurons (Figure 3).

[0012] Based on these findings, it is hypothesized that the protective effects of hit kinase inhibitors from the CPA survival screen were likely due to inhibition of their HGK and / or MLK3 activity. The ideal compound should 1) potently inhibit both HGK and MLK3, 2) completely reverse CPA-mediated neurotoxicity at low (nanomolar to micromolar) doses, 3) be soluble in an aqueous vehicle, 4) be able to penetrate the blood-brain barrier and be orally bioavailable, and 5) be suitable for further structural modifications to optimize its use in vivo. However, the hit compounds from that screen did not meet these criteria. Neither Go6976 nor sunitinib was able to provide complete rescue from CPA toxicity at any of the test doses. On the other hand, K-252a is not a suitable lead compound due to both its broad kinase inhibition and its refractoriness to analog synthesis. Further dampening enthusiasm for this scaffold was the observation that the structurally related non-specific staurosporine-based kinase inhibitor CEP-1347 (Figure 2A) was ineffective as a neuroprotective agent in a clinical trial for Parkinson's disease. Kenpaullone is highly selective but insoluble at the concentrations at which the inventors found it to be effective in in vitro CPA assays and is therefore not suitable for in vivo use.

[0013] Through literature research, the inventors of the present invention identified the small molecule URMC-099 as a promising lead compound (Compound 1) (Figure 2A). Compound 1 is orally bioavailable, penetrates the brain, and strongly binds to and inhibits both MLK3 (IC 50 = 14 nM) and HGK (residual enzyme activity of 0.54% at 1 μM treatment), and is suitable for analog synthesis (Figure 28). The ability of Compound 1 to provide rescue from CPA toxicity in human ALS stem cell-derived motor neurons in a 6-point dose-response assay (0.01 μM to 3 μM) was tested, and it was found that Compound 1 showed 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. When the activation of the ER stress pathway is prolonged, the JNK kinase cascade is activated, ultimately leading to apoptosis (see Sano et al., 2013). MAP kinases JNK1-3 directly phosphorylate and activate c-jun, a transcription factor that promotes the expression of apoptosis-promoting factors such as caspase 3. CPA induces phosphorylation of c-jun in ES MNs, which are particularly vulnerable to ER stress-mediated neurodegeneration, but not in resistant co-cultured ES-derived interneurons, as already shown (see Example 12). Compound 1 was observed to strongly suppress both c-jun phosphorylation and caspase 3 cleavage in MNs treated with CPA (Figure 4), indicating that Compound 1 prevents the induction of apoptosis. Next, the inventors evaluated the phosphorylation status of MAPK 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. CPA treatment upregulated JNK and MKK4 phosphorylation, and it was observed that the addition of Compound 1 strongly attenuated JNK and MKK4 phosphorylation (Figure 4), further supporting the idea that Compound 1 acts on HGK and / or MLK3 to prevent activation of the downstream apoptosis pathway.

[0015] These initial results were promising, but Compound 1 required further optimization before it could be considered a potential clinical candidate. In microsomal stability studies, Compound 1 was shown to have a short half-life (T 1 / 2 = 7.8 minutes) in mouse liver microsomes and an in vivo half-life of approximately 2-3 hours in mice. Furthermore, Compound 1 is non-selective, inhibits several kinases, and has a cellular potency >100 nM. Therefore, more selective and potent derivatives with improved PK properties are preferred and may be 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 any particular theory, the inventors have used the following techniques to aid in the design of potential analogs: To investigate this, compound 1 was docked into the crystal structure of HGK (PDB ID: 5DI1) using Glide (Figure 5A). From the docking pose and the depiction of binding site interactions (see Figure 5B), it was 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, amide, 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, provided that the compound is,

Chemical formula

[0019] Another embodiment of the present invention is,

Chemical formula

[0020] Another embodiment of the present invention is,

Chemical formula

[0021] Another embodiment of the present invention is,

Chemical formula

[0022] Another embodiment of the present invention is,

Chemical formula

[0023] Another embodiment of the present invention is a pharmaceutical composition. This pharmaceutical composition comprises a pharmaceutically acceptable carrier or diluent, and one or more compounds according to formula (I) [Chemical formula] [wherein, 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, amide, 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, provided that the compound is [Chemical formula] none of.

[0024] A further embodiment of the present invention is a kit. This kit comprises a compound or pharmaceutical composition according to the present invention, together with instructions for use for 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) [Chemical formula] [wherein 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, amide, 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, provided that the compound is [Chemical formula] none of which.

[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. The method comprises administering to the subject a pharmaceutically acceptable carrier or diluent, and one or more compounds having the structure of formula (I) [Chemical formula] [wherein 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, amide, 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, in an effective amount comprising the step of administering a pharmaceutical composition, provided that the compound is

Chemical formula

[0027] Another embodiment of the present invention is a method of suppressing the toxicity of endoplasmic reticulum (ER) stress in a subject in need thereof. This method comprises administering to the subject one or more compounds having the structure of formula (I)

Chemical formula

Chemical formula

[0028] A further embodiment of the present invention is a method of treating or ameliorating the effects of a disease associated with axonal degeneration in a subject in need thereof. This method comprises administering to the subject one or more compounds having the structure of formula (I)

Chemical formula

Chemical formula

[0029] A further embodiment of the present invention is a method for treating or ameliorating the effects of neurodegenerative diseases in a subject in need thereof. This method comprises administering to the subject an effective amount of one or more compounds having the structure of formula (I)

Chemical formula

Chemical Structure

[0030] This application document contains at least one photograph taken in color. A copy of this patent application will be provided by the authority together with the color photograph upon request and payment of the necessary fees.

Brief Description of the Drawings

[0031]

Figure 1

[0032]

Figure 2

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

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

[0035]

Figure 3

[0036]

Figure 4A

[0037]

Figure 4B

[0038]

Figure 5

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

[0040]

Figure 6

[0041]

Figure 7

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

[0043]

Figure 8

[0044]

Figure 9

[0045]

Figure 10-1

[0046] Figure 10B shows the verification of HGK knockout clones by Western blot for the HGK protein. Detectable HGK protein was not left in the three knockout clones tested.

[0047]

Figure 10-2

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

[0049]

Figure 10-3

[0050]

Figure 11-1

Figure 11-2

Figure 11-3

Figure 11-4

Figure 11-5

Figure 11-6

Figure 11-7

Figure 11-8

Figure 11-9

Figure 11-10

[0051]

Figure 12

[0052]

Figure 13

[0053] Figure 13B shows the concentrations (ng / mL) of Compounds 1 and 12k in plasma and brain samples of mice intravenously (IV) injected with either compound at 10 mg / kg. Drug concentrations were analyzed by LC-MS. The level of Compound 1 at 1 hour after injection 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 blood-brain barrier penetrant.

[0054]

Figure 14

[0055]

Figure 15-1

Figure 15-2

Figure 15-3

Figure 15-4

Figure 15-5

Figure 15-6

Figure 15-7

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

[0057] Figure 15B shows measurements of overlap between the fluorescent reporter protein (FP) and the transcription factor (TF). Bars indicate the mean and error bars indicate SEM.

[0058] Figure 15C shows a schematic 96-well plate with representative micrographs showing two-color cultures.

[0059] Figure 15D shows representative whole-well images of live cells obtained with a HD Trophos Plate Runner.

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

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

[0062] Figure 15G shows MN survival rates in transgenic hSOD1 WT and G93A cell lines expressing various fluorescent reporters. Survival rates were evaluated with 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 the mean and error bars indicate SEM, ** p<0.01 *** p<0.001.

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

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

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

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

[0067]

Figure 16-1

Figure 16-2

Figure 16-3

Figure 16-4

Figure 16-5

Figure 16-6

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

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

[0070] Figure 16C shows a representative image from whole-well imaging and magnified views of Hb9::RFP hSOD1WT and Hb9::GFP hSOD1 MNs in the same well exposed to CPA. G93A

[0071] Figure 16D shows micrographs of ctrl and CPA-exposed cultures stained for Tuj-1 (red) in Hb9::GFP hSOD1. G93A

[0072] Figure 16E shows micrographs of ctrl and CPA-exposed mixed Hb9::GFP (green) / Ptf1α::tdTomato (red) cultures.

[0073] Figures 16F and 16G show the survival rates of ctrl and CPA-exposed MNs, Tuj-1, + , and Ptf1α neurons quantified at 48 h (N = 3 for both analyses). Bars represent the mean and error bars represent SEM, + p < 0.01 ** p < 0.001. ***

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

[0075] Figure 16I shows that changes induced by CPA in cytoplasmic Ca ++ were evaluated by calculating the ratio of 340 / 380 nm in Fura-2-labeled purified hSOD1 WT and hSOD1 G93A MN cultures (N = 5 for WT, N = 4 for G93A). Bars represent the mean and error bars represent SEM.

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

[0077]

Figure 17A

Figure 17B

Figure 17C

Figure 17D

Figure 17E

Figure 17F

Figure 17G

[0078] Figure 17A shows a histogram indicating that the qPCR results specifically indicate the gene of interest early after CPA exposure. RNA was extracted from non - purified hSDO1 G93A MN (N = 3). Bars indicate the mean and error bars indicate SEM.

[0079] Figure 17B shows a histogram and a reverse gel image indicating 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] Figure 17C shows an immunoblot of the expression of ER stress - related proteins and their loading controls at various time points after CPA exposure.

[0081] Figure 17D shows an immunoblot of SOD1 expression and its loading control in the lysates of hSDO1 G93A cells treated with CPA. The middle lane shows panSOD1 expression. The lower lane shows the lysates immunoprecipitated using an antibody specific for misfolded hSOD1 species (C4F6 and B8H10 clones).

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

[0083] Figure 17G shows the differential effect of CPA exposure on Ptf1α + ::tdTomato-expressing interneurons (IN) and Hb9::GFP motor neurons (MN) as a histogram, bars indicate the mean ratio MN / IN (N = 3 technical replicates), error bars indicate SEM.

[0084]

Figure 18A

Figure 18B

[0085]

Figure 19A

Figure 19B

Figure 19C

Figure 19D

Figure 19E

Figure 19F

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

[0087] Figures 19B and 19C show a histogram and confocal micrograph of the effect of rescue compounds on phospho-c-jun expression in MN cultures treated with CPA 2 hours after exposure. Bars indicate the mean and error bars indicate SEM, *** showing p < 0.001.

[0088] Figure 19D + / + and isogenic genetically modified SOD1 + / A4V histogram of the effect of CPA in FACS-purified Hb9::GFP + human MNs differentiated from hESC lines in the absence or presence of rescue compounds (N = 9 for CPA vs CTRL for both genotypes, N = 3 for rescue compounds for both genotypes). Bars indicate the mean and error bars indicate SEM, * showing p < 0.05, ** p < 0.01, *** p < 0.001 (the effect of CPA is compared between SOD1 + / + and SOD1 + / A4V MNs as indicated by the lines; the effect of the rescue compounds is compared for each genotype with respect to CPA).

[0089] Figure 19E + shows representative cropped full-well images of calcein

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

[0091]

Figure 20-1

Figure 20-2

Figure 20-3

Figure 20-4

Figure 20-5

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

[0093] Figure 20C shows a representative cropped full-well image of Hb9::GFP + cells.

[0094] Figure 20D shows a schematic diagram of the experimental design for the in vivo test of TUDCA on denervation in hSOD1 G93A mice.

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

[0096] Figure 20F shows confocal microscopy images (α-btx red, VAChT green) of the TA NMJ of hSOD1 mice treated with vehicle and TUDCA. The circles indicate denervated NMJs. G93A The circles indicate denervated NMJs.

[0097]

Figure 21A

Figure 21B

Figure 21C

Figure 21D

Figure 21E

Figure 21F

Figure 21G

Figure 21H

Figure 21I

Figure 21J

Figure 21K

[0098] Figure 21A shows an optical microscopy image of a typical ES colony from the newly derived reporter system.

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

[0100] Figures 21C, 21D, and 21E show optical microscopy images and histograms of reporter validation in the newly differentiated ES lines (the two lines for each reporter were differentiated in two separate rounds and the results are presented as mean / reporter). Bars indicate the mean and error bars indicate SEM.

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

[0102] Figure 21H shows a cropped image of an immunoblot depicting the expression of total and misfolded (C4F6) SOD1 in untreated motor neuron cultures. P100 spinal cord from WT or SOD1 G93A mice was used as a control. Dashed lines indicate where the lower figure was cropped.

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

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

[0105]

Figure 22A

Figure 22B

Figure 22C

Figure 22D

Figure 22E

Figure 22F

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

[0107] Figure 22B shows the results of the small molecule screen at 48 hours post-exposure; red and green data points indicate the survival rates for Hb9::RFP-hSDO1 WT (red) and Hb9::GFP-hSDO1 G93A (green) cells, and light blue, yellow, blue, and magenta data points indicate the survival rates relative to the control. Circles indicate promising lead compounds identified after secondary screening, which 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 indicate the mean and error bars indicate SEM.

[0109] Figure 22D shows representative still images from a video of hSOD1 G93A motor neurons exposed to kainate. The images show the Fura-2 signal ratio 340 / 380, and the signal is displayed using the image lookup table function in FIJI (NIH). Arrows indicate Hb9::RFP + cells.

[0110] Figure 22F shows a histogram of the measured peak kainate- and CPA-induced Fura-2 signal ratios 340 / 380 in Hb9::RFP-hSDO1 WT (red) and Hb9::RFP-hSDO1 WT .

[0111]

Figure 23-1

Figure 23-2

Figure 23-3

Figure 23-4

[0112] Figures 23A, 23B, 23C, and 23D show dose - response curves for the survival ratio in cultures treated with CPA in two different combinations of reporter cell lines. The 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 representative gating scatter plots for FACS purification of the reporter system.

[0114]

Figure 24A

Figure 24B

Figure 24C

Figure 24D

Figure 24E

Figure 24F

Figure 24G

Figure 24H

Figure 24I

Figure 24J

Figure 24K

Figure 24L

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

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

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

[0118] Figures 24D and 24E show that qRT-PCR and immunoblot assays regarding SOD1 expression in the target stem cell line demonstrated reduced levels of SOD1 transcription and protein levels.

[0119] Figure 24F shows a FACS plot showing a completely different 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 for the gate for green fluorescence.

[0120] Figure 24G + shows that Hb9::GFP

[0121] Figures 24H and 24I show that cell viability assays in mixed cultures show a tendency of reduced viability in isogenic human ES MN expressing the SOD1 variant compared to the isogenic control A4V Representative images of human nuclei and Tuj1 staining at day 3 and day 30 (P = 0.09, n = 4).

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

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

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

[0125]

Figure 25A

Figure 25B

Figure 25C

Figure 25D

Figure 25E

Figure 25F

Figure 25G

Figure 25H

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

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

[0128] Figures 25C and 25D show Hb9::GFP-hSDO1 treated with CPA G93A A small screen regarding the rescue effect of bile acids conjugated with taurine on motor neurons is shown (N = 3 - 6 culture wells / compound). Data points indicate the mean value.

[0129] Figures 25E and 25F show Hb9::GFP-hSDO1 treated with CPA on a low neurotrophic carrier G93A A histogram showing the rescue effects of TUDCA and canonical neurotrophic factors on survival rate and neurite outgrowth in motor neuron growth is shown (N = 3). Bars indicate the mean and error bars indicate SEM.

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

[0131] Figure 25H shows Hb9::GFP-hSDO1 treated with CPA under a high neurotrophic carrier G93A The effect of TUDCA on survival rate and neurite outgrowth in motor neuron growth is shown (N = 3). Bars indicate the mean and error bars indicate SEM.

[0132]

Figure 26

[0133]

Figure 27

[0134]

Figure 28

[0135]

Figure 29

[0136]

Figure 30

[0137]

Figure 31A

[0138]

Figure 31B

[0139]

Figure 31C

[0140]

Figure 32

[0141]

Figure 33

[0142]

Figure 34

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

[0144]

Figure 35A

[0145]

Figure 35B

[0146]

Figure 35C

[0147]

Figure 36

[0148]

Figure 37A

[0149]

Figure 37B

[0150]

Figure 38-1

Figure 38-2

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

[0152] Figure 38B shows the comprehensive 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

[0155]

Figure 40

[0156]

Figure 41A

Figure 41B

Figure 41C

[0157] [[ID=②]] Figure 41A shows that the results of the two - tiered clustering of the kinase targets of neuroprotection were hit from the previous survival screen, and that HGK and NUAK1 are the shared targets of these compounds. MAP4 kinase is underlined.

[0158] Figure 41B shows the maximum efficacy of the 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 the test compound normalized to CPA (33 μM)+vehicle control. n = 3 replicate wells from a 96 - well plate. The dashed line represents the mean survival rate in cells treated with CPA alone.

[0159] Figure 41C shows the results of Western blot 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 the differentiation of 3 separate motor neurons treated and lysed separately. Cells from each differentiation were split into 3 samples, each with 1 treatment condition. All samples were run simultaneously on the same blot. All band intensities were normalized to GAPDH to control for loading and then the values of DMSO and CPA + compound 1 were normalized to the CPA values for each n. One-way ANOVA was used to evaluate the overall effect of treatment on JNK phosphorylation (P = 0.0009; F = 28.33; R2 = 0.9042) and c-Jun phosphorylation (P < 0.0001; F = 101.9; R2 = 0.9714). Data are represented as mean CPA + compound 1 values, log2 fold change ± SEM relative to CPA alone. Bonferroni's multiple comparison test was used to compare the CPA vs CPA + compound 1 states for phospho-JNK and phospho-c-Jun; ** P < 0.01; *** P < 0.001.

[0160]

Figure 42A

Figure 42B

Figure 42C

[0161] Figure 42A provides an overview of the improvement in efficacy (red) and potency (blue) in the first 49 compound 1 analogs generated.

[0162] Figure 42B shows SOD1 treated with CPA (33 μM) and compounds 1, 12f, and 12k across a dilution series (log μM) A4VSurvival curves for mutant ALS motor neurons are shown. Compound 12f shows improved potency and efficacy compared to 1 but becomes acutely 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 motor neurons treated with test compounds normalized to CPA (33 μM) + vehicle control. n = 3 replicate wells from 96-well plates for each test compound dose.

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

[0164]

Figure 43A

Figure 43B

[0165] Figure 43A shows an 8-point dilution curve for 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 an 8-point dilution curve for HGK inhibitors URMC-099 (compound 1), PF-6260933, and GNE-495.

[0167]

Figure 44

[0168]

Figure 45

[0169]

Figure 46A

[0170]

Figure 46B

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, and moreover, maintain good inhibitory efficacy against toxic ER stress. Accordingly, one embodiment of the present invention is a compound according to formula (I) [Chemical formula] [wherein,[[]END]] 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, amide, cyano, ether, nitro, hydroxy, halogen, alkyl-heterocycloalkyl, sulfonyl, ketone, haloalkyl, haloalkoxy, alkylphenyl, carbamate, carboxylic acid, or urea], or its N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt, provided that the compound is [Chemical formula] none of which.

[0172] In one aspect of this embodiment, the compound has the structure of formula (II) [Chemical formula] [wherein,[[]END]] R 1a and R 1b together with the nitrogen atom to which they are attached form a C 5~6 heterocycloalkyl, where C 5~6The heterocycloalkyl 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, amide, cyano, ether, nitro, hydroxy, halogen, alkyl - heterocycloalkyl, sulfonyl, ketone, haloalkyl, haloalkoxy, alkylphenyl, carbamate, carboxylic acid, or urea, or has its N - oxide, crystalline form, hydrate or pharmaceutically acceptable salt, provided that the compound is

Chemical Structure

[0173] In another aspect of this embodiment, the compound is

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

[0174] Preferably, the compound is

Chemical Structure

[0175] More preferably, the compound is

Chemical formula

[0176] More preferably, the compound is

Chemical formula

[0177] More preferably, the compound is

Chemical formula

[0178] More preferably, the compound is

Chemical formula

[0179] Another embodiment of the present invention is a pharmaceutical composition. This pharmaceutical composition comprises a pharmaceutically acceptable carrier or diluent, and one or more compounds of formula (I)

Chemical formula

Chemical formula

[0180] Suitable preferred compounds for use in the pharmaceutical compositions of the present invention are those disclosed in the previous formulas (I)-(II) including the specific compounds identified previously.

[0181] A further embodiment of the present invention is a kit. This kit contains the compounds or pharmaceutical compositions disclosed herein, together with instructions for use for the use of the compounds or pharmaceutical compositions, respectively.

[0182] The kit can also include each compound of the present invention (which may be in the form of a pharmaceutical composition, for example) and other reagents for use in administering the active agent to a subject, such as storage containers suitable for buffers, balanced salt solutions, etc., such as ampoules, vials, tubes, etc. The compounds and / or pharmaceutical compositions of the present invention, as well as other reagents, can be presented in the kit in any convenient form, such as in solution or powder form, for example. The kit can further include a packaging container that optionally has one or more compartments for containing the compound and / or pharmaceutical composition, as well as other reagents as needed.

[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. This method comprises administering to the subject an effective amount of one or more compounds having the structure of formula (I)

Chemical formula

Chemical formula

[0184] As used herein, the terms "treating," "treatment," "treat" and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or therapy that is desired to bring about a physiological response or outcome in that subject, e.g., a patient. In particular, the methods and compositions of the present invention can be used to slow the onset of symptoms of a disease, or delay the occurrence of a disease or condition, or halt the progression of disease onset. However, since each subject treated may fail to respond to a particular treatment protocol, regimen, process or therapy, treating does not require that a desired physiological response or outcome be achieved in every subject or population of subjects, e.g., a patient population. Thus, a given subject or population of subjects, e.g., a patient population, may fail to respond or may respond inadequately to treatment.

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

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

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

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

[0189] In certain embodiments, the disorder is a disease associated with 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 myelogenous leukemia (CML), acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), acute promyelocytic leukemia, acute megakaryoblastic leukemia, childhood leukemia, familial chronic lymphocytic leukemia, left-right axis formation abnormalities, 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, ovarian 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, progressive osseous heteroplasia, skeletal abnormalities, extraskeletal bone formation, addictions, hypertension, myocardial infarction, acromesomelic dysplasia, ataxia, telangiectasia, Zeekel syndrome, heart failure, juvenile polyposis syndrome, brachydactyly type A2 (hand formation abnormality), acromesomelic chondrodysplasia (bone formation abnormality), vulvar abnormalities, primary pulmonary hypertension (PPH1), heart-face-skin syndrome, juvenile midline carcinoma, X-linked agammaglobulinemia, cardiac arrhythmia, somatic melanoma, familial melanoma, sarcoma, head and neck squamous cell carcinoma, epithelial tumor, cardiac hypertrophy, Rett syndrome, X-linked infantile spasm syndrome, Li-Fraumeni syndrome, circadian disorder, ductal carcinoma of the breast, breast hyperplasia, cone-rod dystrophy (CORD) type 5, cone-rod dystrophy (CORD) type 6, Leber congenital amaurosis type 1 (LCA1), epilepsy, dystonia, muscle wasting, cataract, hypogonadism, endocrine deficiency, male pattern baldness, Down syndrome (DS), glioblastoma, hepatocellular carcinoma, Fife syndrome, Kallmann syndrome 2, stem cell leukemia lymphoma syndrome (SCLL), myeloproliferative disorders, Apert syndrome, Jackson-Weiss syndrome, Crouzon syndrome, Beare-Stevenson cutisGyrate atrophy syndrome, achondrogenesis, hypochondroplasia, thanatophoric dysplasia, Apert craniosynostosis, San Diego dysplasia, Muenke syndrome, pituitary adenoma, spinal cord malformation, infantile hemangioma, idiopathic myelofibrosis, neuroblastoma, kidney cancer, papillary carcinoma, hyper IgM syndrome, dyschromatosis, anhidrotic ectodermal dysplasia, rheumatoid arthritis, melanoma, pineal hyperplasia, polycystic ovary syndrome, atypical migraine, diabetic hyperlipidemia, Morquio syndrome, macrophage apoptosis induced by bacteria, febrile bacterial infection, uterine leiomyosarcoma, post-transplant lymphoproliferative disorder, myeloproliferative disease (MPD), polycythermia vera, brain tumor, gastrointestinal stromal tumor (GIST), mastocytosis, vitiligo, 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 tumor, non-symptomatic sperm Mental retardation type 30 (MRX30), idiopathic eosinophilia syndrome, split spine, Wolcott-Rallison syndrome (WRS), hepatic glycogenosis, cirrhosis, hematopoietic malignancies, Carney complex tumors, cardiac contractility, diabetic nephropathy, diabetic retinopathy, diabetic vascular complications, autism, autosomal dominant spinocerebellar ataxia type 14, pain sensation, osteoarthritis cartilage, bladder cancer, nasopharyngeal carcinoma, undifferentiated large cell leukemia, familial medullary thyroid carcinoma (FMTC), multiple endocrine neoplasia type IIA (MEN2A), MEN2B, pheochromocytoma, papillary thyroid carcinoma, Hirschsprung disease, type 2 microphthalmia, HPC1, blood coagulation, angina, renal oncocytoma, lung adenocarcinoma, autosomal dominant brachydactyly type B, autosomal recessive Robinow syndrome (RRS), Coffin-Lowry syndrome, CNS tumors, Loeys-Dietz syndrome, esophageal cancer, hereditary non-polyposis colorectal cancer (HNPCC, Lynch syndrome), Marfan syndrome type II, venous malformation, astrocytoma, hypertrophic and dilated cardiomyopathy, tibial muscular dystrophy, anhidrosis, pseudohypoaldosteronism type II, and chronic arthritis are included.

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

[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 encephalopathy, Charcot-Marie-Tooth disease, Lewy body dementia, corticobasal degeneration, progressive supranuclear palsy, chronic traumatic encephalopathy (CTE), polyglutamine disease, prion disease, 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 comprises administering to the subject, together with one or more compounds or pharmaceutical compositions of the present invention, 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, Arixtra, Armodafinil, Ascorbic acid, Ascriptin, Aspirin, Atenolol, Avonex, Baclofen (Lioresal), Banzel, Benztropine (Cogentin), Betaseron, BGG492 (Novartis Corp., New York, NY), Botulinum toxin, Bufferin, Carbatrol (registered trademark), Carbidopa / Levodopa immediate release (Sinemet), Carbidopa / Levodopa orally disintegrating tablets (Parcopa), Carbidopa / Levodopa / Entacapone (Stalevo), CERE-110: Adeno-associated virus delivery of NGF (Ceregene, San Diego, CA), Cerebrolysin, CinnoVex, Citalopram, Citicoline, Clobazam, Clonazepam, Clopidogrel, Clozapine (Clozaril), Coenzyme Q, Creatine, Dabigatran, Dalteparin, Dapsone, Dub Davunetide, Deferiprone, Depakene (registered trademark), Depakote ER (registered trademark), Depakote (registered trademark), Desmoteplase, Diastat, Diazepam, Digoxin, Dilantin (registered trademark), 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 (trademark)), Exenatide, Extavia, Ezogabine, Felbamate, Felbatol (registered trademark), Fingolimod (Gilenya), Fluoxetine (Prozac), Fondaparinux, Fragmin, Frisium, Gabapentin, Gabitril (registered trademark), Galantamine, Glatiramer (Copaxone), Haloperidol (Haldol), Heparin, Human chorionic gonadotropin (hCG), Idebenone, Inovelon (registered trademark), Insulin, Interferon beta-1a, Interferon beta-1b, Ioflupane 123I (DATSCAN (registered trademark)), IPX066 (ImpaxLaboratories Inc., Hayward, CA), JNJ-26489112 (Johnson and Johnson, New Brunswick, NJ), Keppra (registered trademark), Klonopin, Lacosamide, L-alpha glyceryl phosphorylcholine, Lamictal (registered trademark), 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 (registered trademark), Natalizumab (Tysabri), Neurontin (registered trademark), Niacinamide, Nitro-Bid, Nitro-Dur, Nitroglycerin, Nitrolingual, Nitromist, Nitrostat, Nitro-Time, Norepinephrine (NOR), Carbamazepine, Octreotide, Onfi (registered trademark), Oxcarbazepine, Oxybutinin chloride, PF-04360365 (Pfizer, New York, NY), Phenobarbital, Phenytek (registered trademark), Phenytoin, Piclotan, Pioglitazone, Plavix, Potiga (Pot iga), Pramipexole (Mirapex), Pramlintide, Prednisone, Primidone, Prinivil, Probenecid, Propranolol, PRX-00023 (EPIX Pharmaceuticals Inc.), PXT3003, Quinacrine, Ramelteon, Rasagiline (Azilect), Rebif, ReciGen, Remacemide, Resveratrol, Retavase, Reteprase, Riluzole (Rilutek), Rivastigmine (Exelon), Ropinirole (Requip), Rotigotine (Neupro), Rufinamide, Sabril, Safinamide (EMD Serono, Rockland, MA), Salagen, Sarafem, Selegiline (l-deprenyl, Eldepril), SEN0014196 (Siena Biotech, Siena, Italy), Sertraline (Zoloft), Simvastatin, Sodium Nitroprussiate (NPS), Sodium Phenylbutyrate, Stanback Headache 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), Trileptal®, 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 improve 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, and one or more of the following, for example, Donepezil (Aricept), Rivastigmine (Exelon), Galantamine (Razadyne), Tacrine (Cognex), Memantine (Namenda), Vitamin E, CERE-110: Adeno-associated virus 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 be administered an effective amount of one or more compounds or pharmaceutical compositions of the present invention, and one or more of, for example, 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 (Apokyn ), selegiline (l-deprenyl, Eldepryl, rasagiline (Azilect), Zydis selegiline HCL orally disintegrating tablets (Zelapar), entacapone (Comtan), tolcapone (Tasmar), amantadine (Symmetrel), trihexyphenidyl (formerly Artane), benztropine (Cogentin), IPX066 (Impax Laboratories Inc.), rasagiline (Teva Neuroscience, Inc.), ioflupane 123I (DATSCAN®), safinamide (EMD Serono), and pioglitazone.

[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 present invention, and one or more of, for example, the following: riluzole (Rilutek), lithium carbonate, alimemazine, 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 present invention, and one or more of, for example, the following: 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 present invention, and one or more of, for example, Avonex, Betaseron, Extavia, Rebif, Glatiramer (Copaxone), Fingolimod (Gilenya), Natalizumab (Tysabri), Mitoxantrone (Novantrone), Baclofen (Lioresal), Tizanidine (Zanaflex), Methylprednisolone, Synacthen, Recigen, 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 be administered an effective amount of one or more compounds or pharmaceutical compositions of the present invention, and one or more of, for example, Tetrabenazine (Xenazine), Haloperidol (Haldol), Clozapine (Clozaril), Clonazepam (Klonopin), Diazepam (Valium), Escitalopram (Lexapro), Fluoxetine (Prozac, Sarafem), Sertraline (Zoloft), Valproic acid (Depakene), Divalproex (Depakote), Lamotrigine (Lamictal), Dimembon, AFQ056 (Novartis), Ethyl-EPA (Miraxion™), SEN0014196 (Siena Biotech), Sodium phenylbutyrate, Citalopram, Ursodiol, Minocycline, Remacemide, and Mirtazapine.

[0201] To treat or ameliorate the effects of transmissible spongiform encephalopathy, a subject may 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 present invention, and one or more of, for example, ascorbic acid and PXT3003.

[0203] To treat or ameliorate the effects of Lewy body dementia, 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, for example, Aricept, galantamine, memantine, modafinil, 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, for example, dabenetide 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 present invention, and one or more of, 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, and one or more of, for example, baclofen, tizanidine, oxybutynin chloride, tolterodine, and botulinum toxin.

[0207] In the present invention, one or more compounds or pharmaceutical compositions can be co - administered to a subject in need thereof, as the same composition together, as separate compositions simultaneously, or as separate compositions at different times, when considered 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. The method comprises administering to the subject a pharmaceutically acceptable carrier or diluent, and one or more compounds having the structure of formula (I) [Chemical Formula] [wherein 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, amide, 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, in an effective amount of a pharmaceutical composition, provided that the compound is [Chemical Formula] none of which.

[0209] Preferred pharmaceutical compositions suitable for use in this method include those disclosed in the previous formulas (I)-(II) containing the specific compounds identified above. Suitable preferred subjects that can be treated according to this method are as previously disclosed. In this embodiment, the method can be used to treat the above-mentioned disorders including diseases associated with 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 the step of co-administering to the subject an effective amount of one or more additional therapeutic agents disclosed herein.

[0211] Another embodiment of the invention is a method of suppressing the toxicity of endoplasmic reticulum (ER) stress 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) [Chemical formula] [wherein, 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, amide, 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, comprising the step of administering an effective amount of a kinase inhibitor, provided that the compound is [Chemical formula] none of.

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

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

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

[0215] As used herein, the terms "suppress", "suppressing" and their grammatical variations mean a change such as reducing or lowering the occurrence of toxic ER stress. In this embodiment, "contacting" means bringing the compound and optionally one or more additional therapeutic agents into proximity to cells that require such modulation. This can be achieved using conventional techniques for delivering drugs to a subject or in an in vitro situation, for example, by providing the compound and optionally 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 associated with axonal degeneration in a subject in need thereof. The method comprises administering to the subject an effective amount of a kinase inhibitor comprising one or more compounds having the structure of formula (I) [Chemical formula] [wherein, 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, amide, 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, provided that the compound is [Chemical formula] none of.

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

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

[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. This method comprises administering to the subject an effective amount of one or more compounds having the structure of formula (I)

Chemical formula

Chemical formula

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

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

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

[0224] A further embodiment of the present invention is

Chemical formula

[0225] A further embodiment of the present invention is

Chemical formula

[0226] A further embodiment of the present invention is

Chemical formula

[0227] A further embodiment of the present invention is

Chemical formula

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

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

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

[0231] A further embodiment of the present invention is a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent, and [Chemical formula] a compound having the structure of, 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 [Chemical formula] administering a compound having the structure of, 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

Chemical formula

[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

Chemical formula

[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

Chemical formula

[0236] A further embodiment of the present invention is a method for suppressing the toxicity of endoplasmic reticulum (ER) stress in a subject in need thereof. The method comprises administering to the subject

Chemical formula

[0237] A further embodiment of the present invention is a method for suppressing the toxicity of endoplasmic reticulum (ER) stress in a subject in need thereof. This method comprises administering to the subject, [Chemical formula] an effective amount of a kinase inhibitor comprising a compound having the structure of, or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0238] A further embodiment of the present invention is a method for suppressing the toxicity of endoplasmic reticulum (ER) stress in a subject in need thereof. This method comprises administering to the subject, [Chemical formula] an effective amount of a kinase inhibitor comprising a compound having the structure of, or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0239] A further embodiment of the present invention is a method for suppressing the toxicity of endoplasmic reticulum (ER) stress in a subject in need thereof. This method comprises administering to the subject, [Chemical formula] an effective amount of a kinase inhibitor comprising a compound having the structure of, or an N-oxide, crystalline form, hydrate or pharmaceutically acceptable salt thereof.

[0240] A further embodiment of the present invention is a method for treating or improving the effect of a disease associated with axonal degeneration in a subject in need thereof. This method comprises administering to the subject, [Chemical formula] an effective amount of a kinase inhibitor comprising a compound having the structure of, 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 associated with axonal degeneration in a subject in need thereof. The method comprises administering to the subject an effective amount of a kinase inhibitor comprising a compound having the structure of

Chemical formula

[0242] A further embodiment of the present invention is a method of treating or ameliorating the effects of a disease associated with axonal degeneration in a subject in need thereof. The method comprises administering to the subject an effective amount of a kinase inhibitor comprising a compound having the structure of

Chemical formula

[0243] A further embodiment of the present invention is a method of treating or ameliorating the effects of a disease associated with axonal degeneration in a subject in need thereof. The method comprises administering to the subject an effective amount of a kinase inhibitor comprising a compound having the structure of

Chemical formula

[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

Chemical formula

[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

Chemical formula

[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

Chemical formula

[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

Chemical formula

[0248] As used herein, "pharmaceutically acceptable salt" means a salt of a compound of the present invention that is pharmaceutically acceptable as defined herein and has the desired pharmacological activity. Such salts include acid addition salts formed using inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., or 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, 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, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, etc. Pharmaceutically acceptable salts also include base addition salts that can be formed when the 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, etc.

[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 that, when administered to a subject, is sufficient to produce a beneficial or desired result, as described herein. Effective dosage forms, methods of administration, and dosages can be determined empirically, and such determinations are made by those of ordinary skill in the art. Dosages will vary with the route of administration, rate of excretion, duration of treatment, identity of any other drugs being administered, age, size, and species of the subject, as well as similar factors well known in the fields of, for example, medicine and veterinary medicine, as will be understood by those of ordinary skill in the art. Generally, an appropriate dosage of a compound or pharmaceutical composition according to the present invention is an amount of the compound or composition that is effective to produce the desired effect without or with minimal accompanying side effects, at the lowest dosage effective for this purpose. The effective dosage of a compound or pharmaceutical composition according to the present invention can be administered as two, three, four, five, six, or more sub-dosages administered separately at appropriate intervals throughout the day.

[0250] Suitable non-limiting examples of dosages of a compound or pharmaceutical composition according to the present invention, or a composition containing such a compound, include from about 1 ng / kg to about 1000 mg / kg, such as 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 the compounds or pharmaceutical compositions of the present 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, 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 for oral ingestion, or as an ointment or eye drops for topical administration to the eye, or for other administration by any suitable route such as parenteral, intraperitoneal, subcutaneous, topical, intradermal, inhalation, intratracheal, rectal, intravaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal or intralymphatic. Further, the compounds or pharmaceutical compositions of the present invention can be administered in combination with other treatments. The compounds or pharmaceutical compositions of the present invention can be encapsulated, if desired, or otherwise protected from gastric juice or other secretions.

[0252] The pharmaceutical compositions of the present invention are pharmaceutically acceptable and contain 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. 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, regardless of the selected route of administration. 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 for the preparation of compositions that are biologically and otherwise desirable, including those acceptable for veterinary use and for use in human pharmaceuticals.

[0253] Pharmaceutically acceptable carriers and diluents are well known in the art (e.g., Remington, The Science and Practice of Pharmacy (21 st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.) and The National Formulary (American Pharmaceutical (see Association, Washington, D.C.), which includes sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starch, cellulose preparations, calcium phosphate (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), alcohol (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), organic esters (e.g., ethyl oleate and tryglyceride), biodegradable polymers (e.g., polylactide - polyglycolide, poly(orthoester), and poly(anhydride)), elastomeric matrix, liposomes, microspheres, oils (e.g., corn, germ, olive, castor, sesame, cottonseed, and peanut), cocoa butter, waxes (e.g., suppository wax), paraffin, silicone, talc, silicylate, etc. Each pharmaceutically acceptable carrier or diluent used in the compositions of the present 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 the selected dosage form and intended route of administration are well known in the art, and the carriers or diluents acceptable for the selected dosage form and method of administration can be determined using the techniques of the art.

[0254] The pharmaceutical composition of the present invention can, if necessary, contain additional components and / or materials commonly used in such compositions. 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 carboxymethyl cellulose, 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, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose, and sodium carbonate; (5) dissolution retardants such as paraffin; (6) absorption promoters 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, lactitol, 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, calcium silicate, and polyamide powder; (13) inert diluents such as water or other solvents; (14) preservatives; (15) surfactants; (16) dispersing agents; (17) controlled release agents or absorption delaying agents such as hydroxypropylmethylcellulose, 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, tetrahydrofurfuryl alcohol, fatty acid esters of polyethylene glycol and sorbitan, (23) propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane, (24) antioxidants, (25) agents for making 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, perfuming agents and preservatives are included. Such components or materials must be "acceptable" in the sense that they are compatible with the other components of the formulation and not harmful to the subject. Components and materials suitable for the selected dosage form and intended route of administration are well known in the art, and those acceptable for the selected dosage form and method of administration can be determined using the techniques of the art.,

[0255] Compounds or pharmaceutical compositions suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules, solutions or suspensions of aqueous or non-aqueous liquids, water-in-oil or oil-in-water liquid emulsions, elixirs or syrups, lozenges, boluses, pastilles, or pastes. These formulations can be prepared by methods known in the art, such as by using conventional pan coating, mixing, granulation or freeze-drying processes.

[0256] Solid dosage forms (such as capsules, tablets, pills, dragees, powders, granules, etc.) for oral administration may contain, for example, the active ingredient, one or more pharmaceutically acceptable carriers or diluents, and optionally one or more fillers, extenders, binders, humectants, disintegrants, dissolution retardants It can be prepared by mixing with a medicament, an absorption promoter, a wetting agent, an absorbent, a lubricant, and / or a coloring agent. 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 compressing or molding, optionally with one or more auxiliary components. Compressed tablets can be prepared using appropriate binders, lubricants, inert diluents, preservatives, disintegrants, surfactants or dispersing agents. Molded tablets can be made by molding with an appropriate machine. Tablets, and other solid dosage forms, such as sugar-coated tablets, capsules, pills and granules, can be prepared, if necessary, with indentations or using 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(s) contained therein. They can be sterilized, for example, by filtration through a bacteria-retaining filter. These compositions can also optionally contain opacifying agents and can be of a composition such that the active ingredient is released only or preferentially in a particular part of the gastrointestinal tract, optionally in a delayed manner. The active ingredient can also be in microencapsulated form.

[0257] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. The liquid dosage forms can contain appropriate inert diluents commonly used in the art. Oral compositions can also contain, in addition to the inert diluent, adjuvants such as wetting agents, emulsifying agents and suspending agents, sweetening agents, flavoring agents, coloring agents, perfuming agents, as well as preservatives. Suspensions can contain suspending agents.

[0258] Compositions for rectal or vaginal administration can be presented as suppositories, 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 liquid at body temperature and thus melt within the rectal or vaginal cavity to release the active compound. Compositions suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such pharmaceutically acceptable carriers, as is known to be suitable in the art.

[0259] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants. The active agent / compound can be mixed with a suitable pharmaceutically acceptable carrier or diluent under sterile conditions. Ointments, pastes, creams and gels can contain excipients. Powders and sprays can contain excipients and propellants.

[0260] Compositions suitable for parenteral administration include 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 into injectable sterile solutions or dispersions immediately before use, and can contain suitable antioxidants, buffers, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Suitable 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 isotonic agents. Furthermore, extended absorption of injectable pharmaceutical forms can be achieved by including agents that delay absorption.

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

[0262] Next, the absorption rate of the active agent / drug varies according to its dissolution rate, which in turn can vary according to the crystal size and crystal form. Alternatively, delayed absorption of a parenterally administered agent / drug can be achieved by dissolving or suspending the active agent / drug in an oily vehicle. Injectable depot forms can be created by forming a microencapsulation matrix of the active ingredient in a biodegradable polymer. The release rate of the active ingredient can be controlled according to the ratio of the active ingredient to the polymer and the properties of the specific polymer used. Also, depot injectable formulations are 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 formulation can be presented in single-dose or multi-dose sealed containers, for example, ampoules and vials, and stored in a lyophilized state that requires only the addition of a sterile liquid carrier or diluent, such as water for injection, immediately prior to use. Immediate injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the aforementioned types.

[0264] In the previous embodiments, the following definitions apply.

[0265] As used herein, the term "aliphatic" refers to a group composed of carbon and hydrogen that does not contain an aromatic ring. Thus, aliphatic groups include alkyl, alkenyl, alkynyl, and carbocyclic groups. Further, unless otherwise specified, the term "aliphatic" is intended to include both "unsubstituted aliphatic" and "substituted aliphatic", and substituted aliphatic refers to an aliphatic moiety having a substituent that replaces one or more hydrogens on one or more carbons of the aliphatic group. Such substituents can 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, amide, amidine, imine, cyano, nitro, azide, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, aromatic, or heteroaromatic moieties.

[0266] The term "alkyl" refers to the radical of a saturated aliphatic group having no ring structure, including straight-chain alkyl groups and branched-chain alkyl groups. In certain embodiments, straight-chain or branched-chain alkyl has 6 or fewer carbon atoms in its backbone (e.g., C1-C6 for straight-chain and C3-C6 for branched-chain). Such substituents include all substituents contemplated for aliphatic groups as discussed below, except where stability is prohibitive.

[0267] As used herein, the term "alkenyl" refers to an aliphatic group containing at least one double bond and is intended to include both "unsubstituted alkenyl" and "substituted alkenyl" unless otherwise specified. Substituted alkenyl refers to an alkenyl moiety having substituents that replace one or more hydrogens on one or more carbons of the alkenyl group. Such substituents include, except where stability is prohibitive, all substituents contemplated for aliphatic groups as discussed below. For example, substitution of an alkenyl group by one or more alkyl, carbocyclic, aryl, heterocyclic, or heteroaryl groups is contemplated.

[0268] Furthermore, unless otherwise specified, the term "alkyl" as used throughout this specification, the examples, and the claims is intended to include both "unsubstituted alkyl" and "substituted alkyl". Substituted alkyl refers to an alkyl moiety having substituents that replace one or more hydrogens on one or more carbons of the hydrocarbon backbone. In fact, unless otherwise specified, all groups listed throughout this specification are intended to include both substituted and unsubstituted options.

[0269] The term "C x~y ", when used with a chemical moiety such as alkyl and cycloalkyl, means including a group containing x to y carbons in the chain. For example, the term "C x~y alkyl" refers to a substituted or unsubstituted saturated hydrocarbon group including linear alkyl and branched alkyl groups containing x to y carbons in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl.

[0270] As used herein, the term "aryl" includes substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon. Preferably, the ring is a 3- to 8-membered ring, more preferably a 6-membered ring. 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] As used herein, the term "amino", 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 be optionally substituted itself. Further, R and R' may combine to form a heterocycloalkyl, any of which may be optionally substituted.

[0276] As used herein, the term "amide", alone or in combination, refers to -R n E(O) xRefers to NR’2, where R and R’ are independently selected from hydrogen, alkyl, acyl, heteroalkyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl, any of which may optionally be substituted. By way of example, carboxamide (n = 1, E = C, x = 1), phosphoramide (n = 2, E = P, x = 1), and sulfonamide (n = 1, E = S, x = 2) may be mentioned.

[0277] The terms “benzo” and “benz” as used herein, alone or in combination, refer to the divalent radical C6H4= derived from benzene. By way of example, benzothiophene and benzimidazole may be mentioned.

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

[0279] The term “carbonyl” as used herein, alone includes formyl [—C(O)H], and in combination is the —C(O)— group.

[0280] The term “carboxyl” or “carboxy” as used herein refers to —C(O)OH, or the corresponding “carboxylate” anion present, for example, as a carboxylate salt. The “O-carboxy” group refers to the RC(O)O— group, where R is as defined herein. The “C-carboxy” group refers to the —C(O)OR group, where R is as defined herein.

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

[0282] As used herein, the term "cycloalkyl" or alternatively "carbocycle" alone or in combination refers to a saturated or partially saturated monocyclic, bicyclic or tricyclic alkyl group, where each cyclic moiety contains ring members of 3 to 12 carbon atoms 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 such as decahydronaphthalene, octahydronaphthalene, as well as polycyclic (polycentric) saturated or partially unsaturated types. Isomers of the latter type are generally exemplified by bicyclo[1,1,1]pentane, camphor, adamantane, and bicyclo[3,2,1]octane.

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

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

[0285] As used herein, the term "heteroalkyl", alone or in combination, refers to a stable straight-chain, branched-chain, or cyclic hydrocarbon radical, or combinations thereof, that is fully saturated or contains from 1 to 3 degrees of unsaturation and consists of the stated number of carbon atoms and from 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 internal position of the heteroalkyl group. For example, up to 2 heteroatoms may be consecutive, such as in -CH2-NH-OCH3. and may be.

[0286] The term "heteroaryl" includes substituted or unsubstituted aromatic monocyclic structures, preferably 3- to 8-membered rings, more preferably 5- to 7-membered rings, even more preferably 5- to 6-membered rings, the ring structure of which contains at least 1 heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The term "heteroaryl" also includes polycyclic ring systems having 2 or more cyclic rings, where 2 or more carbons are common to 2 adjacent rings and at least 1 of the rings is heteroaromatic, and for example, 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, each independently 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, where each said heteroatom can be independently selected from N, O, and S. Further, heterocycloalkyl can contain one or two C(O), S(O), or S(O)2 groups as ring members. In certain embodiments, said heterocycloalkyl contains 1 to 4 heteroatoms as ring members. In further embodiments, said heterocycloalkyl contains 1 to 2 heteroatoms as ring members. In certain embodiments, said heterocycloalkyl contains 3 to 8 ring members in each ring. In further embodiments, said heterocycloalkyl contains 3 to 7 ring members in each ring. In still further embodiments, said heterocycloalkyl contains 5 to 6 ring members in each ring. "Heterocycloalkyl" and "heterocyclyl" are intended to include sulfones, sulfoxides, N-oxides of tertiary nitrogen ring members, as well as carbocyclic fused and benzo-fused ring systems, and further, both of those terms together also include systems where the ring of the heterocycle is fused to an aryl group or a further heterocyclic group as defined herein. Examples of heterocyclic groups include aziridinyl, azetidinyl, 1,3-benzodioxolyl, dihydroisoindolyl, dihydroisoquinolinyl, dihydrosinnolinyl, 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, etc. The heterocyclic group may be optionally substituted as necessary, 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 and mean halogen and include chloro, fluoro, bromo, and iodo.

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

[0291] As used herein, the term "haloalkyl" refers to an alkyl having the meaning as defined above, either alone or in combination, in which one or more hydrogens are replaced by halogen. Refers to a haloalkyl radical. Specifically, it includes monohaloalkyl, dihaloalkyl, polyhaloalkyl, and perhaloalkyl radicals. As an example, a monohaloalkyl radical can have an iodine, bromo, chloro, or fluoro atom within the radical. Dihalo and polyhaloalkyl radicals can have a combination of two or more of the same or different halo atoms. 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 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 bonded at two or more positions. Examples include fluoromethylene (-CFH-), difluoromethylene (-CF2-), chloromethylene (-CHCl-), etc. The term "perhaloalkyl", as used herein, alone or in combination, refers to an alkyl group in which all hydrogen atoms are replaced by halogen atoms. An example is perfluoromethyl.

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

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

[0294] The term "ester" means 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 linked at a carbon atom.

[0296] The term "nitro", as used herein, alone or in combination, refers to -NO2.

[0297] The term "polyine" means an organic compound having alternating single and triple bonds, i.e., 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 an ether in which oxygen is replaced by sulfur. Oxidation derivatives of the thio group, i.e., sulfinyl and sulfonyl, are included in the definitions 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)2-.

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

[0300] The term "substituted" refers to a substituent that replaces hydrogen on one or more carbons of the backbone Refers to the part having. "Substituted" or "substituted with" means that such substitution is carried out in accordance with the acceptable valences of the atoms being substituted and the substituents, and that the substitution results in a stable compound that does not naturally undergo conversions such as rearrangement, cyclization, elimination, etc. It will be understood that when used herein, the term "substituted" is intended to include all acceptable substituents of an organic compound. In a broad aspect, acceptable substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic aromatic and non-aromatic substituents of an organic compound. The acceptable substituents for a suitable organic compound may be one or more, and may be the same or different. For the purposes of the present invention, a heteroatom, such as nitrogen, can have a hydrogen substituent that satisfies the valence of the heteroatom and / or any acceptable substituent of the 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, amide, amidine, imine, cyano, nitro, azide, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. It will be understood by those skilled in the art that portions substituted on a hydrocarbon chain may themselves be substituted where appropriate.

[0301] As already described, references herein to chemical moieties are understood to include substituted variants unless specifically described as "unsubstituted". For example, a 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.

Chemical formula

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

Chem.

[0304] As used herein, the term "indazolyl" means any compound or chemical group containing the following structure.

Chem.

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

Chem.

[0306] As used herein, the term "pyrimidinyl" means any compound or chemical group containing the following structure.

Chem.

[0307] As used herein, the term "pyrazolyl" means any compound or chemical group containing the following structure.

Chem.

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

Chem.

[0309] As used herein, the term "benzothiophenyl" means any compound or chemical group containing the following structure.

Chem.

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

Chem.

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

Chem.

[0312] The disclosure of a compound herein is understood to include all stereoisomers of that compound. As used herein, the term "stereoisomer" refers to a compound that is made up of the same atoms bonded by the same bonds but has different three-dimensional structures that are not interconvertible. The three-dimensional structure is called the configuration. Stereoisomers include enantiomers and diastereomers.

[0313] The term "racemate" or "racemic mixture" refers to a mixture of equal enantiomeric parts. 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 the amount of one enantiomer being greater compared to the other enantiomer.

[0314] The compounds of the present invention exist in a racemic form that is optically active to the extent of having chiral centers and are recognized as isolable. Some compounds may exhibit polymorphs. The present invention encompasses any racemate, optically active diastereomer, polymorph or stereoisomeric form of the compounds of the present invention having the useful properties described herein, or mixtures thereof, and how to prepare the optically active forms (e.g., by resolution of the racemic form by recrystallization techniques, by synthesis from optically active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) is to be understood as 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 - a technique in which macroscopic crystals of individual enantiomers are separated by hand. This technique can be used when crystals of the separate enantiomers are present, i.e., when the material is an aggregate and the crystals are visually distinguishable. ii) Simultaneous crystallization - a technique in which individual enantiomers are crystallized separately from a solution of the racemate, which is only possible when the racemate is an aggregate in the solid state. iii) Enzymatic resolution - a technique for partially or completely separating a racemate by the difference in the rates 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 obtain an enantiomerically pure or enriched synthetic precursor of the desired enantiomer. v) Chemical asymmetric synthesis - a synthetic technique in which the desired enantiomer is synthesized from an achiral precursor under conditions that generate asymmetry (i.e., chirality) in the product (which can be achieved using a chiral catalyst or chiral auxiliary as disclosed in more detail herein). vi) Diastereomer 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 based on their more distinct structural differences at that point, and later the chiral auxiliary is removed to obtain the desired enantiomer. vii) Primary and secondary asymmetric transformation - A technique in which the diastereomers from a racemate reach equilibrium, and the diastereomers from the desired enantiomer become dominant in solution or the equilibrium is disrupted by the preferential crystallization of the diastereomers from the desired enantiomer, so that ultimately in principle all the material is converted into crystalline diastereomers from the desired enantiomer. The desired enantiomer is then released from the diastereomer. viii) Kinetic resolution - This technique refers to the achievement of partial or complete resolution of a racemate (or further resolution of a partially resolved compound) due to the non-uniform reaction rates of enantiomers with a chiral non-racemic reagent or catalyst under kinetic conditions. ix) Enantioselective synthesis from non-racemic precursors - A synthetic technique in which the desired enantiomer is obtained from achiral starting materials and the stereochemical integrity is not impaired or only minimally impaired throughout the synthetic process. x) Chiral liquid chromatography - A technique in which the enantiomers of a racemate are separated in the liquid mobile phase by their different interactions with the stationary phase. The stationary phase can be made from a chiral material, or the mobile phase can contain an additional chiral material that induces different interactions. xi) Chiral gas chromatography - A technique in which a racemate is volatilized and the enantiomers are separated in the gas mobile phase by their different interactions using a column containing a fixed non-racemic chiral adsorbent phase. xii) Extraction using a chiral solvent - A technique in which the enantiomers are separated by the preferential dissolution of one enantiomer in a specific chiral solvent. xiii) Transport through chiral membranes - A technique where 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 pressure difference causes preferential transport through the membrane barrier. The separation results from the non-racemic chiral nature of the membrane that 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 chromatography techniques such as LC or flash chromatography. The (+) enantiomer can be separated from the (-) enantiomer using techniques and procedures well-known in the art, such as those described by J. Jacques, et al., ”Enantiomers, Racemates, and Resolutions”, John Wiley and Sons, Inc., 1981. For example, enantiomers can also be separated using chiral chromatography with a suitable organic solvent, such as ethanol / acetonitrile, and a Chiralpak AD column, 20 microns. (-) enantiomer.

[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 present invention in any way.

Examples

[0318] The present invention is further illustrated by the following examples provided for illustrative purposes and not limiting the present invention in any way. Those skilled in the art will readily recognize various non-essential parameters that can be changed or modified to produce 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 of reagent grade. Chromatography

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

[0321] 1 1H NMR spectra were recorded at ambient temperature using a 400 MHz or 500 MHz spectrometer as indicated. Chemical shifts were reported in ppm relative to the residual solvent peak ( 11H 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), 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 were obtained by peak matching. All reactions were carried out under an atmosphere of nitrogen or argon in glassware dried by heating under argon unless otherwise indicated. Aqueous solutions were prepared from nanopure water with a resistivity greater than 18 MΩ·cm. Unless otherwise indicated, all reagents were commercially available. Software

[0322] Molecular docking, molecular modeling, and visualization were performed using Glide (versions 2012 - 2016, Schrodinger) and Schrodinger Maestro (version 2017 - 1). All chemical structures were drawn using ChemDraw Professional 16.0 (Perkin Elmer). All microsomes Stability curves were generated using Prism 7 (GraphPad Software). (Example 2) Synthesis of Piperazine Analogs

[0323] The general route to obtain the compounds from formula (I) to (II) follows the synthesis exemplified below. The potency mediator group was added in the final step instead of earlier. The preparation of each compound is also provided. [Chemical Structure Diagram] 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) [Chemical Structure Diagram]

[0324] 5-Bromo-1H-pyrrolo[2,3-b]pyridine (2.394 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(PPh3)2Cl2 (0.70 g, 1.0 mmol, 5 mol%) was added, and then aqueous Na2CO3 (2.0 M, 50 mL) was added. 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 (3×). 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 (EtOAc in 0 - 100% hexane) 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.H RMS(APCI + 、m / z):C 14 H 11 N2O[M+H + Calculated for: 223.0872, Found: 223.0872. Preparation of 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 4)

Chemical Structure

[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 at room temperature overnight. 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 through 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 g, 74% yield) as a colorless solid. The product was used in the next step without further purification. 1 1H 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: 307.1923, found: 307.1918. Preparation of 3-iodo-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 5)

Chemical Structure

[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% Et3N) to give 3-iodo-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-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 for: 433.0889, found: 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)

Chemical Structure

[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). Boc2O (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 (3×). 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% Et3N) 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, yield 71%). 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: 533.1413, found: 533.1423. General procedure for Suzuki coupling and in situ deprotection

Chemical formula

[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-drum glass vial equipped with a Teflon®-lined cap. A 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 through celite, and the celite was washed with ethyl acetate. The solvent was evaporated, and the crude product was purified by preparative HPLC to afford the 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)

Chemical Structure

[0329] Compound 1 was prepared using 5-indolylboronic acid (12.1 mg, 0.075 mmol, 1.5 eq) according to the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to afford 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 the 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) [Chemical formula]

[0330] Compound 7a was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above, using 2-borononic acid pinacol ester (18.2 mg, 0.075 mmol, 1.5 eq). 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). 11H 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 for: 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)

Chemical Structure

[0331] Compound 7b was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 6-ethoxy-3-pyridinylboronic acid (12.5 mg, 0.075 mmol, 1.5 eq). 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). 11H 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 N5O [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)

Chemical Structure

[0332] Compound 7c was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 4-(dibenzofuranyl)boronic acid (15.9 mg, 0.075 mmol, 1.5 eq). 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). 11H 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 N4O [M+H + Calculated for: 473.2341, Found: 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)

Chemical Structure

[0333] Compound 7d was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 1,4-benzo dioxane-6-boronic acid (13.5 mg, 0.075 mmol, 1.5 eq). 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 1H 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 for: 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)

Chemical Structure

[0334] Compound 7e was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 4-aminophenylboronic acid pinacol ester (17.0 mg, 0.075 mmol, 1.5 eq). 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 Calculated value for N5[M+H + : 398.2345, measured 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)

Chemical Structure

[0335] Compound 7f was prepared using benzo[b]thiophene-3-ylboronic acid (14.0 mg, 0.075 mmol, 1.5 eq) according to the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to obtain 3-(benzo[b]thiophen-3-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7f, 16.0 mg, yield 73%). 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)

Chemical Structure

[0336] Compound 7g was prepared using benzo[b]thiophen-2-ylboronic acid (14.0 mg, 0.075 mmol, 1.5 eq) according to 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-2-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7g, 15.9 mg, yield 73%). 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 for: 439.1956, found: 439.1959. Preparation of 3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)aniline (Compound 7h)

Chemical Structure

[0337] Compound 7h was prepared using 3-aminophenylboronic acid hydrochloride (13.3 mg, 0.075 mmol, 1.5 eq) according to 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: 398.2345, found: 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)

Chemical Structure

[0338] Compound 7i was prepared using 3-carboxy-4-fluorophenylboronic acid (13.8 mg, 0.075 mmol, 1.5 eq) according to the general procedure for Suzuki coupling and in situ deprotection as described above. 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)

Chemical Structure

[0339] Compound 7j was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 3-carboxyphenylboronic acid (12.5 mg, 0.075 mmol, 1.5 eq). 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)

Chemical Structure

[0340] Compound 7k was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 2-aminopyrimidine-5-boronic acid (11.0 mg, 0.075 mmol, 1.5 eq). The crude product was purified by preparative HPLC 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%). 11H 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)

Chemical Structure

[0341] Compound 7l was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 2-amino-3-methylpyridine-5-boronic acid pinacol ester (18.3 mg, 0.075 mmol, 1.5 eq). The crude product was purified by preparative HPLC 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 1H 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 for: 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)

Chemical Structure

[0342] Compound 7m was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 2-aminopyridine-5-boronic acid pinacol ester (16.5 mg, 0.075 mmol, 1.5 eq). The crude product was purified by preparative HPLC 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, yield 76%). 11H 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 for: 399.2297, Found: 399.2307. Preparation of 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridine (Compound 7n)

Chemical Structure

[0343] Compound 7n was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 4-pyridinylboronic acid (10.3 mg, 0.075 mmol, 1.5 eq). 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 1H 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)

Chemical Structure

[0344] Compound 7o was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 4-cyanophenylboronic acid (11.0 mg, 0.075 mmol, 1.5 eq). The crude product was purified by preparative HPLC to obtain 4-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzonitrile (7o, 14.4 mg, yield 71%). 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)

Chem.

[0345] Compound 7p was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 3-nitrophenylboronic acid (12.5 mg, 0.075 mmol, 1.5 eq). 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, yield 72%). 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: 428.2087, found: 428.2081. Preparation of 3-(4-isopropoxyphenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 7q)

Chem.

[0346] Compound 7q was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 4-isopropoxyphenylboronic acid (13.5 mg, 0.075 mmol, 1.5 eq). 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 N4O[M+H + Calculated for: 441.2654, Found: 441.2646. Preparation of 3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenol (Compound 7r)

Chemical Structure

[0347] Compound 7r was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 3-hydroxyphenylboronic acid (10.3 mg, 0.075 mmol, 1.5 eq). 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 N4O[M+H + calculated: 399.2185, found: 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)

Chemical Structure

[0348] Compound 7s was prepared using 1-methylpyrazole-4-boronic acid pinacol ester (16.1 mg, 0.075 mmol, 1.5 eq) according to 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: 387.2297, found: 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)

Chemical Structure

[0349] Compound 7t was prepared using 5-(morpholinomethyl)-2-thiopheneboronic acid pinacol ester (24.4 mg, 0.075 mmol, 1.5 eq) according to 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-(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, yield 41%). 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)

Chemical Structure

[0350] Compound 7u was prepared using 6-(methylsulfonyl)pyridine-3-boronic acid (15.0 mg, 0.075 mmol, 1.5 eq) according to 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, yield 48%). 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)

Chemical Structure

[0351] Compound 7v was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 1H-indazole-5-boronic acid (12.1 mg, 0.075 mmol, 1.5 eq). The crude product was purified by preparative HPLC 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: 423.2297, found: 423.2298. Preparation of 3-(furan-3-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 7w)

Chemical Structure

[0352] Compound 7w was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 3-furanylboronic acid (7w, 8.4 mg, 0.075 mmol, 1.5 eq). The crude product was purified by preparative HPLC to give 3-(furan-3-yl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (12.9 mg, 69% yield). 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 N4O[M+H + calculated value: 373.2028, found 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)

Chemical Structure

[0353] Compound 7x was prepared using 4-acetylphenylboronic acid (12.3 mg, 0.075 mmol, 1.5 eq) according to 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 N4O[M+H + Calculated value for: 425.2341, Found: 425.2347. Preparation of 3-(3-fluorophenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 7y)

Chemical Structure

[0354] Compound 7y was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 3-fluorophenylboronic acid (10.5 mg, 0.075 mmol, 1.5 eq). 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: 401.2142, found: 401.2146. Preparation of 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-(4-(trifluoromethoxy)phenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 7z)

Chemical Structure

[0355] Compound 7z was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 4-(trifluoromethoxy)phenylboronic acid (15.4 mg, 0.075 mmol, 1.5 eq). 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 for: 467.2059, Found: 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)

Chemical Structure

[0356] Compound 7aa was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 1H-indazole-5-boronic acid (12.5 mg, 0.075 mmol, 1.5 eq). 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: 423.2297, found: 423.2303. Preparation of 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-(3-(trifluoromethyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 7ab)

Chemical Structure

[0357] Compound 7ab was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 3-(trifluoromethyl)phenylboronic acid (14.2 mg, 0.075 mmol, 1.5 eq). 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: 451.2110, found: 451.2115. Preparation of 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-phenyl-1H-pyrrolo[2,3-b]pyridine (Compound 7ac)

Chemical Structure

[0358] Compound 7ac was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using phenylboronic acid (9.6 mg, 0.075 mmol, 1.5 eq). 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: 383.2236, found: 383.2236. Preparation of 3-(3-chlorophenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 7ad)

Chemical Structure

[0359] Compound 7ad was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 3-chlorophenylboronic acid (11.7 mg, 0.075 mmol, 1.5 eq). 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 1H 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: 417.1846, found: 417.1843. Preparation of 3-(2,4-dichlorophenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 7ae)

Chemical Structure

[0360] Compound 7ae was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 2,4-dichlorophenylboronic acid (14.3 mg, 0.075 mmol, 1.5 eq). 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 1H 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 for: 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)

Chemical Structure

[0361] Compound 7af was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 2,5-dimethylphenylboronic acid (11.2 mg, 0.075 mmol, 1.5 eq). The crude product was purified by preparative HPLC 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)

Chemical Structure

[0362] Compound 7ag was prepared using 1 - methyl - 1H - indazole - 6 - boronic acid (13.2 mg, 0.075 mmol, 1.5 eq) according to the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to obtain 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, yield 60%). 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 for []: 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)

Chemical formula

[0363] Compound 7ah was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 1-benzyl-1H-pyrazole-4-boronic acid (15.2 mg, 0.075 mmol, 1.5 eq). 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, yield 62%). 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) [Chemical Structure]

[0364] Compound 7ai was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above, using 1,3,5-trimethyl-1H-pyrazole-4-boronic acid pinacol ester (17.7 mg, 0.075 mmol, 1.5 eq). 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, yield 37%). 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) [Chemical Structure]

[0365] Compound 7aj was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 3,5-bis(trifluoromethyl)phenylboronic acid (19.3 mg, 0.075 mmol, 1.5 eq). 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: 519.1983, found: 519.1983. Preparation of 1-methyl-3-(3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine-3-yl)phenyl)urea (Compound 7ak)

Chemical Structure

[0366] Compound 7ak was prepared using (3-(3-methylureido)phenyl)boronic acid (13a, 14.6 mg, 0.075 mmol, 1.5 eq) according to 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-3-(3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)urea (7ak, 6.4 mg, yield 14%). 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 N6O[M+H + Calculated value for: 455.2559, Found: 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)

Chemical Structure

[0367] Compound 7al was prepared using (3-((methoxycarbonyl)amino)phenyl)boronic acid (13b, 14.6 mg, 0.075 mmol, 1.5 eq) according to the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC 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, yield 19%). 1 H 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)

Chemical Structure

[0368] Compound 7am was prepared using (4-(3-methylureido)phenyl)boronic acid (13c, 14.6 mg, 0.075 mmol, 1.5 eq) according to 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-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[[ID=II]] 31 N6O[M+H + calculated value: 455.2559, found 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)

Chemical Structure

[0369] Compound 7an was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using (4-((methoxycarbonyl)amino)phenyl)boronic acid (13d, 14.6 mg, 0.075 mmol, 1.5 eq). 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)phenyl)carbamate (7an, 7.6 mg, yield 33%). 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), 2.92 (s, 3H) ppm. HRMS(APCI + , m / z): C 27 H 30 N5O2[M+H + calculated: 456.2400, found: 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)

Chemical Structure

[0370] Compound 7ao was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 4-(4-methoxybenzyloxy)phenylboronic acid (19.4 mg, 0.075 mmol, 1.5 eq). The crude product was purified by preparative HPLC 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: 519.2760, found: 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)

Chemical Structure

[0371] Compound 7ap was prepared according to the general procedure for Suzuki coupling and in situ as described above using 3,4,5-trimethoxyphenylboronic acid (15.9 mg, 0.075 mmol, 1.5 eq). It was prepared according to the general procedure for situ deprotection. The crude product was purified by preparative HPLC to obtain 5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (7ap, 19.2 mg, yield 81%). 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)

Chemical Structure

[0372] Compound 7aq was prepared as described above using 4-methoxy-3-methylphenylboronic acid (12.4 mg, 0.075 mmol, 1.5 eq) in the Suzuki coupling and in situ deprotection general procedure. The crude product was purified by preparative HPLC to obtain 3-(4-methoxy-3-methylphenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7aq, 17.2 mg, yield 81%). 11H 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 N4O [M+H + Calculated for: 427.2498, Found: 427.2501. Preparation of 3-(3-(Benzyloxy)phenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 7ar)

Chemical Structure

[0373] Compound 7ar was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 3-(benzyloxy)phenylboronic acid (17.1 mg, 0.075 mmol, 1.5 eq). 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 1H 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 N4O [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)

Chemical Structure

[0374] Compound 7as was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 2-chlorophenylboronic acid (17.1 mg, 0.075 mmol, 1.5 eq). 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 + and 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)

Chemical Structure

[0375] Compound 7at was prepared according to the general procedure for Suzuki coupling and in situ deprotection as described above using 2-methoxyphenylboronic acid (11.4 mg, 0.075 mmol, 1.5 eq). The crude product was purified by preparative HPLC to obtain 3-(2-methoxyphenyl)-5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridine (7at, 17.1 mg, yield 83%). 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 N4O [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)

Chemical Structure

[0376] Compound 7au was prepared using (3-(3-phenylureido)phenyl)boronic acid (13e, 19.2 mg, 0.075 mmol, 1.5 eq) according to the general procedure for Suzuki coupling and in situ deprotection as described above. The crude product was purified by preparative HPLC to obtain 1-(3-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)-3-phenylurea (7au, 5.4 mg, yield 21%). 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 N6O [M+H +Calculated value regarding : 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) [Chemical formula] 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 Et3N (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 to obtain 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, yield 98%). 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. 1-Cyclohexyl-3-(4-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)phenyl)urea( Preparation of compound 7aw)

Chemical formula

[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) dissolved in DCM (0.5 mL), and the mixture was stirred at room temperature for 5 minutes. The reaction mixture was added to a solution of cyclohexylamine (19.8 mg, 23 μL, 0.2 mmol, 20.0 eq) dissolved in DCM (0.5 mL), and the mixture was stirred overnight. The solvent was evaporated, and the crude mixture was purified by preparative HPLC to obtain 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, yield 54%). 11H 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 N6O [M+H + calculated value: 523.3185, measured value: 523.3182. (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) Preparation

Chemical Structure

[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, 22 μL, 0.16 mmol, 10 eq) was added. 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 the mixture was stirred at room temperature for 5 minutes. 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 the mixture was stirred overnight. The solvent was evaporated, and the crude mixture was purified by preparative HPLC to obtain (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, yield 31%). 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 N6O[M+H + Calculated value for: 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) [Chemical formula] 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) dissolved in DCM (1.0 mL), and the mixture was stirred at room temperature for 5 minutes. The reaction mixture was added to a solution of furfurylamine (31.1 mg, 28.3 μL, 0.32 mmol, 20.0 eq) dissolved in DCM (1.0 mL), and the mixture was stirred overnight. The solvent was evaporated, and the crude mixture was purified by preparative HPLC to obtain 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, yield 41%). 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) ppm. 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)

Chemical formula

[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) dissolved in DCM (1.0 mL), and the mixture was stirred at room temperature for 5 minutes. 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) dissolved in DCM (1.0 mL), and the mixture was stirred overnight. The solvent was evaporated, and the crude mixture was purified by preparative HPLC to obtain 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, yield 20%). 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] The general route for obtaining Compounds 9a - 9c follows the synthesis shown below. The preparation of each compound is also provided. [Chemical formula] Scheme 8: General synthesis 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 (2×). 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 hexane) to give the desired product (9a - c). Preparation of 4 - (5 - bromo - 1 - tosyl - 1H - pyrrolo[2,3 - b]pyridin - 3 - yl) - 1H - indazole (Compound 9a) [Chemical formula]

[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 for: 467.0177, found value: 467.0176. Preparation of 5-bromo-3-(3-fluorophenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (Compound 9b)

Chemical Structure

[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)

Chemical Structure

[0386] According to the above general procedure, 5-Bromo-3-(3-chlorophenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridine (9c) was isolated as a colorless solid (850 mg, yield 88%). 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] The general route to obtain compounds 10a - 10c follows the synthesis shown below. The preparation of each compound is also provided.

Chem.

[0388] The substrate (9a - c, 1.0 mmol, 1.0 eq) was 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 extracted with EtOAc (3×). The combined organic layers were dried over Na2SO4 and filtered through celite. The solvent was evaporated, and the crude material was purified by column chromatography on silica (0 - 100% EtOAc in hexane) 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)

Chem.

[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 H 21 N4O3S[M+H + Calculated value for: 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) [Chemical formula]

[0390] According to the above general procedure, 4-(3-(3-Fluorophenyl)-1-tosyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (10b) was isolated as a colorless solid (466 mg, yield 99%). 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)

Chemical Structure

[0391] According to the above general procedure, the starting materials were 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, yield 87%). 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, 1.3 Hz, 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] The general route for obtaining compounds 12a - 12l follows the synthesis exemplified below. The preparation of each compound is also provided. [Chemical formula] Scheme 10: General synthetic scheme for compounds 12a - 12l.

[0393] The detailed synthesis of bicyclic piperazine analogs 12e and 12f is exemplified in the scheme below. [Chemical formula] 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. [Chemical formula] 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) were 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 at room temperature overnight and partitioned between dichloromethane and brine. The aqueous layer was extracted with dichloromethane (2×), 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 a mixture of acetone (20 mL), methanol (30 mL), and aqueous NaOH (2.0 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 to give the products (12a - l). 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) [Chemical formula]

[0397] Following the general procedure described above, 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) were used as starting materials to isolate 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) as a pale brown solid (37.2 mg, 43% yield, over 2 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 N6O[M+H + Calculated value for: 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)

Chemical Structure

[0398] Following the general procedure described above, using 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) as starting materials, 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, yield 4%, over 2 steps). 11H 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)

Chemical Structure

[0399] Following the above general procedure, using 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) as starting materials, 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, yield 18%, over 2 steps). 11H 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 for: 448.2501, Found: 448.2503. Preparation of 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 (Compound 12d)

Chemical Structure

[0400] Following the general procedure above, using 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) as starting materials, 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 pale brown solid (11.7 mg, yield 26%, over 2 steps). 11H 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, 2.8 Hz, 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 for: 449.2454, Found: 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)

Chemical Structure

[0401] Following the general procedure above, using 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) as starting materials, 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 (12e) was isolated as a pale brown solid (12.0 mg, yield 27%, over 2 steps). 11H 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 for: 449.2454, Found: 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)

Chemical Structure

[0402] Following the general procedure described above, using 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) as starting materials, 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 2 steps). 11H 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'-bipiperidin]-1'-ylmethyl)phenyl)-3-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridine (Compound 12g)

Chemical Structure

[0403] Following the above general procedure, using 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) as starting materials, 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, yield 24%, over 2 steps). 11H 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): C 30 H 34 N4F [M+H + Calculated value for: 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)

Chemical Structure

[0404] Following the general procedure described above, using 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, 166 μL, 1.35 mmol, 2.0 eq) as starting materials, 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 2 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 for: 431.2247, Found: 431.2250. Preparation of (R)-1-(4-(3-(3-fluorophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzyl)pyrrolidin-3-ol (Compound 12i)

Chemical Structure

[0405] Following the general procedure described above, using 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) as starting materials, (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 2 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: 388.1825, found: 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)

Chemical Structure

[0406] Following the general procedure described above, 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) were used as starting materials, and 5-(4-((4-(tert-butyl)piperazine-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 2 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: 443.2611, found: 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)

Chemical Structure

[0407] Following the general procedure described above, using 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) as starting materials, 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, yield 36%, over 2 steps). 1 H NMR (400 MHz, 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 for: 447.1952, found 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)

Chemical Structure

[0408] Following the general procedure above, using 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) as starting materials, (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 2 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 for: 404.1530, found: 404.1529. (Example 6) Synthesis of boronic acids 13a - 13e

[0409] The general route to obtain compounds 13a - 13e follows the synthesis exemplified below. The preparation of each compound is also provided.

Chemical Structure

[0410] Boric 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) dissolved in DCM (6 mL), and the mixture was stirred at room temperature for 5 minutes. Amine (0.6 mmol, 3 eq) or 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) [Chemical formula]

[0411] According to the above general procedure, (3-(3-methylureido)phenyl)boronic acid (13a) was isolated as an off-white solid using 3-aminophenylboronic acid and methylamine, 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) [Chemical formula]

[0412] According to the above general procedure, (3-((methoxycarbonyl)amino)phenyl)boronic acid (13b) was isolated as an off-white solid using 3-aminophenylboronic acid and methanol, 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). (4-(3-Methylureido)phenyl)boronic acid (Compound 13c) Preparation

Chem.

[0413] Following the above general procedure, (4-(3-Methylureido)phenyl)boronic acid (13c) was isolated as an off-white solid from 4-aminophenylboronic acid and methylamine 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). (4-((Methoxycarbonyl)amino)phenyl)boronic acid (Compound 13d) Preparation

Chem.

[0414] Following the above general procedure, (4-((Methoxycarbonyl)amino)phenyl)boronic acid (13d) was isolated as an off-white solid from 4-aminophenylboronic acid and methanol 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). (3-(3-Phenylureido)phenyl)boronic acid (Compound 13e) Preparation

Chem.

[0415] Following the general procedure described above, (3-(3-phenylureido)phenyl)boronic acid (13e) was isolated as an off-white solid from 3-aminophenylboronic acid and aniline 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] The general route to obtain compounds 16a and 16b follows the synthesis exemplified below. The preparation of each compound is also provided. [Chemical formula] 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 H2O, 287 μL, 3.53 mmol, 3.0 eq) was added, followed by Na(OAc)3BH (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 KMnO4 staining.

[0418] To remove the Boc group, the product (15a - b) was 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 product (16a - b) was used in the next step without further purification, and the spectroscopic data was matched with the data 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 synthesized analogs above, individual viability assays were performed on human embryonic stem cell-derived motor neurons (HB9::GFP reporter, and SOD1 A4V mutation) treated with CPA (33 μM), which is thought to enable real-time feedback and optimization of this assay. Next, viability assays were performed with all analogs at once to generate a low-variability data set. All compounds were evaluated at concentrations ranging from 0.1 - 3.0 μM (5-point dilution) with 3 technical replicates and 2 biological replicates.

[0420] To evaluate the ability of the analogs to inhibit HGK phosphorylation, the 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, starting at 20 μM for 10 dose IC 50 tested in 4-fold serial dilutions. Thus, it was possible to test not only whether the analogs bind to HGK but also whether they interfere with its kinase activity. Approximately 45% of the analogs tested were found to inhibit at least half of the HGK activity at a dose of 0.025 μM, thereby validating the computer-aided 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. The overall inhibition of MLK1 and MLK3 was lower, but approximately 40% of the analogs tested still inhibited at least half of the MLK3 activity at a dose of 0.1 μM.

[0421] Next, the inventors evaluated the correlation between the degree to which each analog suppresses enzyme activity and the degree to which it rescues CPA toxicity. Overall, a moderate correlation was observed between the percentage of 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 the CPA - induced cell death pathway.

[0422] A very strong correlation was observed between the percentage of motor neuron survival after CPA treatment and HGK activity% (R 2 = 0.84) (see Figure 7B), suggesting that HGK or a member of the closely related MAP4K kinase family may be a biologically relevant target 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, and the cell surface marker CD 14 is expressed under the control of the motor neuron - specific HB9 promoter, and thus, motor neurons can be purified from the mixed differentiation culture by magnetic - activated cell sorting (MACS) for downstream assays. In the CRISPR strategy, two guide RNAs targeting genomic regions upstream and downstream of the ATP - binding portion of HGK that mediates its kinase activity are used. Double - strand break repair is achieved by non - homologous end joining in this scenario, which 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 that increases the dose of CPA (Figure 10C). In this assay, motor neurons are imaged after being MACS-purified and treated with CPA for 48 hours.

[0425] By modulating our CPA treatment paradigm such that MACS-purified WT or HGK knockout motor neurons are treated with CPA for a very short period (1 - 24 hours), we were able to observe that HGK ablation confers modest but highly statistically significant neuroprotection against CPA (Figure 10D). Thus, HGK plays an early role in the cell death cascade following CPA treatment; however, the fact that the effect of its ablation is not detectable at later time points suggests that there may be other redundant kinases that ultimately take over its role in its absence. We propose 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 may target all of these related MAP4Ks to promote neuronal survival (Figure 31A).

[0426] To better understand the biologically relevant targets of compound 1 and its analogs, a second round of cell-free kinase assays was performed. In this extended assay, the inventors selected 10 analogs ranging from moderately to very potently protective in the CPA assay. These analogs were evaluated for their ability to inhibit 60 kinases selected at 0.1 μM and 0.5 μM. The list of these 60 kinases was derived from known targets of compound 1 (Goodfellow et al 2013), known targets of other kinase inhibitors that showed rescue, and relevant targets from the literature, known targets of other kinase inhibitors that were protective in the CPA survival assay, and relevant targets from the literature. RNAseq data was used to confirm the expression of each kinase in ES MN (see Example 12).

[0427] Among the analogs to compound 1, there was no overall trend towards increased target selectivity, the process of analog generation itself did not improve selectivity, and neither selectivity nor promiscuity was tracked by neuroprotection in the CPA survival assay. However, compound 7ad was similarly neuroprotective against compound (1) and, at the same time, showed high target selectivity among the 60 kinases tested: at 0.5 μM, compound 1 potently inhibited 27 out of 60 kinases (<20% residual activity), while compound 7ad potently inhibited 12 out of 60 kinases (Figure 8).

[0428] By comparing the targets of the relatively selective neuroprotective analogs to those of compound 1, it is emphasized that the targets are most likely to reduce neurodegeneration in the CPA survival assay. In addition to HGK, the selective compound 7ad most potently inhibits closely related MAP4 kinases including TNIK, MINK1, KHS (MAP4K5), and GCK (MAP4K2) (Figure 8). These findings are also consistent with the inventors' analysis of the shared targets of the hit compounds from their preliminary screen (Figure 3), HGK, MINK, KHS, And GCK was among the most highly ranked targets (TNIK activity was not evaluated in existing publicly available databases; Anastassiadis et al., 2011).

[0429] Overall, the inventors 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 the meaningful correlation was likely obscured by the fact that moderately neuroprotective compounds still robustly inhibited MAP4K at these concentrations. Furthermore, the high structural homology between the kinase domains of MAP4K family members makes it difficult to analyze 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 more selective for an individual MAP4K over the others; however, the inventors found that GCK-selective inhibitors (Compound 17 and NG-25 in Tan et al., 2015; Figure 9) were unable to rescue MN in the CPA survival assay, so the potential functional targets of the Compound 1 analogs were limited to HGK, MINK1, TNIK, or KHS, or some combination of these (Figure 33).

[0431] Furthermore, recent studies from Larhammar et al (2017) have shown that compounds that inhibit HGK, TNIK, and MINK1 are neuroprotective in primary dorsal root ganglion neurons subjected to neurotrophin withdrawal, and that attenuating the expression of all three kinases together, rather than individually, reflects the effect of the compounds (Figure 31C). These argue that the roles of HGK, TNIK, and MINK1 in their neurodegeneration assay are redundant and that the effect of excluding a single kinase can be overcome by the activity of the remaining kinases. Thus, inhibition of a combination of multiple MAP4 kinases has the potential to underlie the neuroprotective effects of Compound 1 and its analogs in ES MNs treated with CPA (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 excellent rescue properties in the ER stress assay were incubated with mouse liver microsomes in a buffer containing a NADPH regenerating system. Aliquots were taken over 2 hours and analyzed by HPLC-MS using an internal standard to evaluate compound stability over time (Figure 29). Methods and Materials

[0433] The microsomal stability assay was performed in a 96-well format (Perkin-Elmer, StorPlate-96U, PP, 96 wells, round bottom). To each well, phosphate buffer (182.2 μL, pH 7.4, 100 mM) was added, followed by NADPH regeneration system solution A (10 μL) and NADPH regeneration system solution B (2 μL) (Corning Gentest 3P NADPH regeneration system solution A (#451220) and B (#451200)). A stock solution (0.8 μL, 5 mM) of the compound to be analyzed 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 minutes), an aliquot (15 μL) was withdrawn from the plate and quenched by adding it to cold acetonitrile (60 μL) containing an internal standard (5 μM) in a 96-well plate. The samples were centrifuged at 13,000 rpm for 10 minutes at 4 °C. The supernatant (40 μL) was withdrawn and transferred to a sample vial containing an insert. The samples were analyzed by LC-MS.

[0434] LC-MS analysis was performed on a platform consisting of a Thermo Scientific Dionex Ultimate3000 and a Bruker amaZon SL equipped with an electrospray ionization source controlled by Bruker Hystar3.2. Chromatographic separation was performed by injecting 5 μL of the 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 mobile phase 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% in 0.25 minutes and to 70% by 6.75 minutes. Solvent B was increased to 95% by 7.00 minutes and decreased to the original initial conditions (40%) by 8.00 minutes, and the total run time was 9.00 minutes. Results

[0435] First, the 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 rates. Following a previously published study, the inventors found that Compound 1 has a relatively short half-life (T 1 / 2 = 15 minutes) in mouse liver microsomes. Of the 11 analogs initially evaluated, only three analogs (7y, 7ad, and 7at) showed promising half-lives (> 30 minutes) in mouse liver microsomes.

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

[0437] The introduction of 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 the inventors' analogs, the same bicyclic piperazine was introduced into one of the analogs from the initial series with a half-life > 30 minutes (Scheme 11, Compound 12f). In this case, the half-life extended from 38 minutes (Compound 7ad) to 158 minutes (Compound 12f), which was a significant improvement. Compound 12f was more potent in the ER stress assay, showing complete rescue at 0.3 μM, but also showed slightly higher toxicity at concentrations > 0.5 μM. To reduce this increased toxicity while maintaining potency and microsomal stability, the inventors synthesized another series of analogs (Scheme 12) containing an m-chlorophenyl or m-fluorophenyl substituent at the 3-position of the scaffold of 7-azaindole.

[0438] The potencies of these compounds were evaluated in an ER stress assay, and their stabilities were evaluated in a microsomal stability assay. Two of these compounds, 12g and 12j, had a shorter half-life than desired or showed a decrease in potency in the ER stress assay. The remaining four compounds (12h, 12i, 12k, and 12l) were able to potently rescue motor neurons from CPA-induced ER stress and completely abrogate the effects of CPA treatment at a concentration of <0.3 μM. Among these very 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 the compounds Improvement of the solubility of compound 1 and its analogs

[0439] The previous publication described a basic formulation for in vivo delivery of compound 1 composed 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 solubilizers at various concentrations on compound 1 and its analogs (Figure 30).

[0440] Compounds diluted in various vehicle formulations were added to clear cell culture medium at ratios consistent with the inventors' in vivo dosing scheme. The plates were then read on a spectrophotometer at a wavelength of 540 nM at which the compounds do not absorb light to measure the optical density and determine whether precipitates that interfere with light were formed. The inventors found that adding 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). Treatment of Animals

[0441] All studies were performed in 6-week-old male C57BL6 / J mice housed at the Columbia University Medical Center facility on a 12-hour light / dark cycle with ad libitum access to food and water. 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 (final concentration of 11%). Animals were weighed and injected intraperitoneally (IP) once at the indicated dose (10 or 20 mg / kg). Thirty minutes later, the 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' heads were removed, 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 until further processing. Compound Concentration in Plasma

[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 evaporated overnight. Samples were reconstituted in a mixture of 40 μL of acetonitrile and 10 μL of water, transferred to sample vials with inserts, and analyzed by LC-MS. Compound Concentration in Brain

[0443] The homogenized brain sample (250 μL) was transferred to an Eppendorf tube and diluted with acetonitrile (750 μL). The sample was 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 evaporated overnight. The sample was reconstituted in a mixture of 80 μL of acetonitrile and 20 μL of water, transferred to a sample vial with an insert, and analyzed by LC-MS. LC-MS analysis

[0444] LC-MS analysis was performed on a platform including a Thermo Scientific Dionex Ultim ate 3000 and a Bruker amaZon SL equipped with an electrospray ionization source controlled by Bruker Hystar 3.2. The compound concentration in each plasma sample was determined using a calibration curve. Chromatographic separation was performed by injecting 10 μL of the 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 mobile phase 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 minutes and to 70% by 6.75 minutes. Solvent B was increased to 95% by 7.00 minutes and decreased back to the original initial state (40%) by 8.00 minutes, and the total run time was 9.00 minutes. Results

[0445] The pharmacokinetic results of the selected compounds (12h, 12i, 12k, 12l) at various dosages in the 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 Figures 13B, 34A - 34B, 35A - 35C, 36, 37A - 37B, and Example 13.

Table 1

Table 2

[0446] The inventors observed in vitro that phospho-cjun expression was strongly upregulated by CPA treatment and strongly attenuated by compound 1, and that compound 1 could be a downstream effector of the kinase target(s) (Figure 4A and Figure 4B). Therefore, the inventors selected phospho-cjun as a marker of target engagement 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 delivered by gavage, intraperitoneal injection, or subcutaneous injection at 12 h or 12 k (Figure 14). The animals were then sacrificed 1 hour later, their olfactory bulbs were partially excised, 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. Almost all drug-treated samples showed a reduction in cjun phosphorylation compared to untreated controls, indicating that the drug binds to the same target in vivo as it does in vitro. Furthermore, the reduction in cjun phosphorylation in samples from animals treated by gavage indicates that both 12 h and 12 k are orally bioavailable. (Example 12) Sensitive two-color motor neuron screening reveals ALS-related stressors and neuroprotective agents

[0447] Amyotrophic lateral sclerosis is a neurodegenerative disease that targets neurons in the motor cortex, brainstem, and spinal cord. Mutations that cause ALS have been discovered in dozens of diverse genes, yet the reasons for the differential motor neuron susceptibility remain poorly understood. The inventors 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 calcium pump (SERCA) expressed in the endoplasmic reticulum, as a stressor to which motor neurons expressing human SOD1 with the G93A mutation that causes ALS are highly sensitive. Importantly, 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 cord interneurons. Secondary screening identified compounds that rescue motor neurons from CPA-induced degeneration, including three kinase inhibitors and tauroursodeoxycholic acid (TUDCA), an FDA-approved bile acid derivative. Treatment of mice expressing hSOD1 with TUDCA improved early denervation. The neuroprotective effects of the identified compounds were verified using human motor neurons differentiated from isogenic embryonic stem cell lines carrying the SOD1 G93A mutation. Overall, the inventors' results highlight the translational potential of the developed in vitro drug discovery platform, which can serve as a valuable tool for identifying new drug targets in the treatment of neurodegenerative diseases. inhibitor as a stressor to which motor neurons expressing human SOD1 with the G93A mutation that causes ALS are highly sensitive. G93A expressing mice improved early denervation. A4V G93A mutant human motor neurons differentiated from isogenic embryonic stem cell lines. Overall, the inventors' results highlight the translational potential of the developed in vitro drug discovery platform, which can serve as a valuable tool for identifying new drug targets in the treatment of neurodegenerative diseases.

[0448] Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disease that preferentially targets neurons within the motor cortex as well as motor neurons within 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 differential neuron subtype susceptibility to the widely expressed mutant proteins remain poorly understood.

[0449] Effective modeling of ALS is hampered by the limited accessibility of motor neurons in patients and animal models, as well as the fact that ALS is a late-onset and relatively slowly progressing disease. The development of stem cell technologies that facilitate the large-scale production of spinal motor neurons with disease-causing mutations has circumvented the initial challenges and enabled biochemical analysis and drug screening in the context of relevant cells (Amoroso et 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 with ALS-causing mutations do not exhibit important features of motor neuron disease, such as aggregation of mutant proteins (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 survival, 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. 2008; Di Giorgio et al. 2007; Donnelly et al. 2013; Egawa et al. 2012; Naujock et al. 2016; Sareen et al. 2013; Sivadasan (et al. 2016; Wainger et al. 2014). These phenotypes may be obtained from a stressful in vitro environment that causes premature or abnormal symptoms of the pathological process in cultured cells, yet the nature of these culture-related stressors remains poorly defined. Understanding which specific stressors enhance the pathology of disease-related motor neurons could enable the development of a more faithful model of ALS, i.e., a better tool for understanding the onset and progression of the disease and for screening neuroprotective drugs.

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

[0452] Using this accelerated motor neuron degeneration paradigm, the inventors performed a second screening to identify compounds that protect spinal motor neurons against CPA toxicity. The inventors demonstrated that kenpaullone, a protein kinase inhibitor that has recently been 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, the inventors identified several other protective compounds, including additional kinase inhibitors and the bile acid component, tauroursodeoxycholic acid (TUDCA). The rescue effects of these compounds in motor neurons derived from mouse hepatocytes were recapitulated in motor neurons derived from human stem cells. Furthermore, the inventors found that TUDCA reduces denervation of the neuromuscular junction in transgenic hSOD1 G93A mice at the early symptomatic stage of the disease.

[0453] Collectively, these findings highlight the translational potential of the developed stem cell-based motor neuron survival assay. When identifying that the compound CPA, which induces ER stress, is preferentially toxic to motor neurons, the inventors' initial stress screening rediscovered, independently, pathways known to be involved in ALS pathology (Kiskinis et al. 2014; Saxena et al. 2009; Saxena et al. 2013; Nishitoh et al. 2008). Furthermore, the protective effect of TUDCA in hSOD1 G93A mice represents a proof of principle that compounds arising from in vitro screening platforms can seamlessly translate to in vivo models of ALS. hSOD1 G93A and the slight defects in the survival and neurite outgrowth of hepatocyte-derived motor neurons expressing

[0454] To gain insight into the pathological processes involved in the onset of motor neuron degeneration in animals expressing mutant SOD1 protein, the inventors developed a two-color motor neuron survival assay. The inventors reasoned that the discovery of mechanisms involved in the increased sensitivity of motor neurons to mutations that cause ALS would require a robust, sensitive, scalable system that could distinguish the cell-autonomous motor neuron phenotype from the non-cell-autonomous phenotype 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] To distinguish wild-type (WT) from mutant motor neurons, minimize well-to-well variability, and increase scalability, the inventors used mice containing the hSOD1 WT (control) and hSOD1 G93A (mutant) transgenes (Gurney et By crossing with mice expressing eGFP (Wichterle et al. 2002) or tagRFP under the control of the motor neuron-specific Hb9 (Mnx1) promoter (al. 1994), a new pluripotent embryonic stem cell (ESC) line was generated (Figure 15A; Figures 21A and 21B). Immunostaining with an antibody against Hb9 and the motor neuron transcription factor Isl1 confirmed that the new cell line differentiates into motor neurons with equivalent efficiency (Figures 15A and 15B, Figures 21C-21E). Next, the inventors sought to determine whether the new mutant cell line recapitulates the known ALS phenotype. Using immunohistochemistry and biochemistry (data not shown), the inventors did not observe large SOD1 aggregates within 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 the conformational epitope on misfolded human SOD1 revealed the presence of misfolded SOD1 species (Figure 21H), which has already been suggested to be involved in the etiology of ALS (Gros-Louis et al. 2010).

[0456] Newborn motor neurons are highly dependent on neurotrophic factors for their survival and for neurite outgrowth (Henderson et al. 1994; Oppenheim et al. 1992; Sendtner et al. 1991). To determine whether growing motor neurons under suboptimal conditions would result in preferential degeneration in mutants over WT motor neurons, we cultured both genotypes in media supplemented with reduced amounts of neurotrophic factors and evaluated motor neuron survival and neurite growth. The most supportive media 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 two compounds, IBMX and forskolin, which we have previously shown increase the levels of cyclic AMP (cAMP) 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 declined over the subsequent 24-48 hours (Figure 21J). In contrast, GDNF alone provided robust support for motor neuron survival up to 1 week in culture, and its effects 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 of BDNF, CNTF, GDNF, and IGF-1) (Figure 21F and Figure 21G).

[0457] Cell viability and neurite outgrowth were evaluated by automated live imaging of fluorescent motor neurons (Trophos Plate Runner) (Figures 15D and 15E). To distinguish live cells from GFP-positive dead cells or fluorescent debris, the inventors applied a “healthy cell criterion” using Metamorph software (Molecular Devices), in which only cells with neurite lengths equal to 5 times the average cell body diameter were included in the analysis (Figure 15F). Quantification of the number of live motor neurons over a 6-day period (Figures 21F and 21G) revealed a modest but consistent 15–20% reduction in mutant motor neuron viability and neurite growth relative to controls. This difference was highly and moderately supported and observed in red–green mixed cultures or individual cultures of each genotype (Figure 15G). Collectively, these findings indicate that removal of neurotrophic factors does not enhance the survival defects detected in ESC-derived hSOD1 G93A expressing motor neurons. Two-color screen for stressors that induce preferential degeneration of mutant motor neurons

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

[0459] The plated motor neurons were recoverable and able to extend neurites over a 24-hour period prior to treatment with a library of 1275 bioactive small molecules (Tocris Screen Mini and Custom Collection, Tocris). 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 at 48 and 72 hours. The inventors measured the following parameters: the number of live fluorescent motor neurons (“motor neuron survival rate,” Figure 15J), the average neurite outgrowth per cell (“total neurite growth,” 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 growth ratio (“selective neurite growth,” Figure 22A). To identify stressors that preferentially target mutant motor neurons, the inventors first excluded all compounds that were clearly toxic to both control and mutant motor neurons (average survival rate < 25% of mutant and control cells). Among the remaining compounds, the subset that preferentially decreased the survival rate of mutant motor neurons and resulted in a ratio corresponding to at least a 50% difference in survival rate included agonists and antagonists of membrane receptors, ion pump and channel inhibitors, anti-mitotic agents, and general apoptosis promoters (Figures 15H and 15I).

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

[0461] To further investigate the effects of CPA, the inventors examined whether it acts directly on motor neurons or indirectly through other cells in the culture. The inventors found that motor neurons purified by fluorescence-activated cell sorting (FACS) (Figures 23C - 23F) are sensitive to CPA as motor neurons in mixed cultures, indicating that CPA acts directly on motor neurons rather than on other cell types that may generate secondary toxins later (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 mixed cultures do not provide protection to motor neurons treated with CPA.

[0462] The preferential degeneration of spinal motor neurons and neurons within the motor cortex over other types of neurons is a feature of ALS. To determine whether motor neurons are more sensitive to CPA treatment than other types of neurons, the inventors used hSOD1 G93AA mixed culture composed of expressing spinal motor neurons, interneurons, and other cell types was analyzed. Immunostaining of viable cells revealed the survival of motor neurons expressing GFP, with the pan-neuron marker Tuj1 reduced by approximately 71%, while all GFP - Tuj-1 + The survival of non-motor neurons was only reduced by approximately 23% (Figures 16D and 16F). hSOD1 G93A Since there was such a significant difference in CPA sensitivity among various neuron subtypes in the mixed culture induced from the ESC line, we tried to examine whether the same was true for the culture induced from the WT line.

[0463] In this analysis, we generated a new ESC line that expresses tdTomato in dI4 and dIL dorsal spinal inhibitory interneurons induced from Ptf1α-expressing progenitor cells (Hoang et al. 2017, data not shown). We differentiated this line under conditions that promoted the detailed identity of dorsal interneurons. FACS-purified tdTomato-expressing interneurons were co-cultured with FACS-purified GFP-expressing spinal motor neurons (Figures 16E and 16G). Quantification of RFP- versus GFP-positive neurons revealed that CPA treatment reduced the survival rate of co-cultured spinal motor neurons by approximately 70%, compared to only approximately 17% reduction in the survival rate of dorsal spinal interneurons. Combining these data, it became clear that the sensitivity of spinal motor neurons to CPA was increased compared to other ventral or dorsal spinal neurons. Effect of CPA on cytoplasmic calcium levels

[0464] CPA is a reversible inhibitor of the SERCA pump, which is important for the sequestration of cytoplasmic 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 effect of CPA on calcium handling in motor neurons, the inventors performed a series of imaging experiments to visualize free cytoplasmic calcium and measure it by radiometric analysis of the calcium indicator Fura-2. To avoid crosstalk between GFP and Fura-2 fluorescence, the experiments were performed on sorted Hb9::tagRFP-expressing motor neurons. First, the inventors characterized the overall calcium handling in control and mutant motor neurons three days after plating by measuring the calcium transients induced by the ionotropic glutamate receptor agonist kainic acid (KA) (Carriedo et al. 2000; Carriedo et al. 1996). Exposure of cells to a 5-second pulse of 100 μM KA induced a rapid increase in cytoplasmic calcium in ESC-derived motor neurons, followed by a period of sequestration during which the calcium returned to baseline levels (Figures 22C and 22D), which was similar to primary motor neuron cultures (Carriedo et al. 2000). Motor neurons that were unable to return to baseline within a 60-second imaging window were excluded from further analysis. After a 5-minute recovery period, the 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), and ultimately returned to levels near baseline towards the end of the imaging sequence. The cells were allowed to recover for an additional 5 minutes, after which the second K The A pulse was applied in the presence of CPA, which caused a rapid calcium transient that returned to baseline (Figure 22E). This allowed the inventors to examine how cells handle an increase in cytoplasmic calcium when calcium sequestration within the ER is disrupted. CPA significantly slows, but does not abolish, KA-induced calcium sequestration (Figures 16J and 22E), indicating that motor neurons, which normally rely on the ER to sequester cytoplasmic calcium, can use alternative sequestration pathways upon loss of this compartment. This difference was not due to the stimulation repeated by KA, as parallel experiments with multiple consecutive KA pulses in the absence of CPA revealed no impairment in the rate of calcium sequestration (data not shown). When comparing control and mutant motor neurons, the inventors observed no genotype-to-genotype differences in the rate (Tau) of KA-induced calcium sequestration, either in the absence or presence of CPA (Figure 16J), indicating that CPA has a relatively minor impact on calcium handling.

[0465] Elevated cytoplasmic calcium can activate a number of intracellular signaling processes, including cell death pathways (De Stefani et al. 2012; Jayaraman et al. 1997; Pinton et al. 2008), and its dysregulation has been implicated 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. 1999; Tradewell et al. 2011; von Lewinski and Keller 2005). Thus, improvement of calcium sequestration into the ER or inactivation of the degenerative pathways that promote calcium signaling potentially counteracts CPA toxicity. Thus, the inventors evaluated a panel of eight compounds known to have an impact on the handling and / or signaling of cytoplasmic calcium. These included BAPTA-am, a cell-permeable calcium chelator; dantrolene, an inhibitor of the ryanodine receptor that releases calcium from the 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. In particular, these treatments did not improve motor neuron survival or neurite retraction induced by CPA exposure (Figs. 18A-18B). These data, combined with the observation that CPA-induced cytoplasmic calcium levels are essentially equal in vulnerable motor neurons and resistant non-motor neurons (Fig. 25B), suggest that cytoplasmic 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 effect on cytoplasmic calcium, CPA treatment has been shown to reduce calcium levels in the ER, leading to activation of the ER stress pathway (Doutheil et al. 1997). These pathways are initiated by the 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 upon detection of unfolded proteins in the ER lumen. Unbound BiP is involved in the activation of three separate signaling pathways associated with ER stress: the PERK, ATF-6, and IRE1α pathways, which are 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 evaluate the activation of these pathways in motor neurons exposed to CPA, we used RT-PCR to examine the expression levels of 15 stress-related genes at three time points after CPA treatment in motor neurons (Figure 17A). We observed a rapid increase in the expression of Bip and the important downstream effector Chop (Ddit3). Bip increased two-fold 1 hour after CPA exposure and continued to increase to approximately five-fold by 8 hours. Chop was induced four-fold at 1 hour of CPA treatment and reached 16-fold induction at 4 and 8 hours. Other genes with > two-fold induction were: p58IPK 255 (6.5-fold at 8 hours), ER stress-inducible protein kinase; growth arrest and DNA damage-inducible protein 45 alpha (Gadd45a; five-fold at 4 hours), pre-onset SOD1 G93A which has recently been shown to be upregulated in the spinal cord of mice (Saxena G93A ​et al. 2009); Bip cofactors involved in ER-associated protein degradation (ERAD) (5-fold in 4 - 8 hours); Atf4, a downstream mediator of the PERK axis of the UPR (3-fold in 8 hours); calreticulin, hSOD1 G93A ER-related chaperones associated with non-mediated motor neuron degeneration in mice (3-fold in 8 hours) (Bernard-Marissal et al. 2012); as well as Nrf2, a PERK substrate (2-fold in 8 hours) were included. Overall, these changes in expression indicated strong 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 hours confirmed early activation of the PERK pathway (increased phosphorylation of Eif2α already detectable 1 hour after CPA exposure) and accumulation of the active cleaved form of ATF-6 detectable at 8 hours in both genotypes (Figure 17C). Activation of the IRE1α branch was evaluated by qPCR analysis of X-box binding protein 1 (XBP1) splicing, which was already induced by 1 hour after CPA treatment and reached a peak at 4 hours after exposure (Figure 17B). The inventors also evaluated activation of the IRE1α branch by immunocytochemical analysis of c-jun phosphorylation (Urano et al. 2000), which reached a peak 2 hours 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 a protective cellular response aimed at attenuating protein synthesis and accelerating the clearance of misfolded proteins (Hetz 2012; Lurlaro and Munoz-Pinedo 2016). Therefore, the inventors attempted 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. 2009; Saxena et al. 2009; Nishitoh et al. 2008; Gros-Louis et al. 2010). The inventors 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, the inventors extended their studies to include two additional compounds that activate ER stress: thapsigargin, a drug that acts like 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). The inventors exposed co-cultures of GFP-expressing motor neurons and tdTomato-expressing dorsal interneurons to increasing concentrations of thapsigargin and tunicamycin and observed that, as in the case of CPA, spinal motor neurons are selectively sensitive to both compounds. Thus, the motor neuron toxic effects of CPA appear to be related to its activation of the ER stress pathway and not related to calcium levels or other off-target effects (Figure 16G). Compounds that protect motor neurons from CPA-induced degeneration

[0471] ​Since it was understood that motor neurons are more sensitive to activation of the UPR / ER stress pathway than other spinal cord neurons, the inventors sought to establish pre-screened candidate molecules aimed at identifying compounds that can protect motor neurons from CPA toxicity. This is because ER stress has been implicated in the etiology of ALS as well as many other neurodegenerative conditions (Hetz and Saxena 2017). The inventors screened a panel of >100 compounds supplemented with compounds curated from the in-house library and arising from literature searches (Table 3). This panel included compounds that modulate various branches of ER stress, compounds that affect calcium sequestration, compounds that act as neurotrophic factors, and compounds that have already been shown to promote motor neuron survival. Mixed motor neuron cultures were pre-treated with rescue compounds for 45 minutes prior to exposure to 7.5 μM CPA. Survival and neurite outgrowth were evaluated at 24 and 48 hours. Screening resulted in several compounds that protected more than 50% of the 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; the broad 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 more than 50% of neurite outgrowth included the neurotrophic factor cardiotrophin-1; the p38 inhibitors SB293063 and SB203580; SP600125; sunitinib; the bile acids TCA, TGCA, and TUDCA; and the 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 survival-promoting effects (GO6976, kenpaullone, K252a) or potent neurite outgrowth promotion (TUDCA) at their low concentrations and were selected for further characterization.

[0472] Kenpaullone, a protein kinase inhibitor, recently emerged as a hit from the screening of compounds that rescue the removal of neurotrophic factors in ESC-derived motor neurons (Yang et al. 2013). The inventors have found that it is also effective in preventing motor neuron degeneration in response to CPA. The inventors have identified two additional kinase inhibitors, the staurosporine analog GO6976 and K252a, which provide an equivalent rescue in motor neurons but are active at lower concentrations (0.5 - 2 μM compared to 5 - 10 μM of kenpaullone) (Figure 19A). The inventors have also determined that TUDCA, an amphoteric bile acid component that functions as a chemical chaperone, rescues neurite outgrowth (Figure 25F) but shows only a moderate effect on motor neuron degeneration (Figure 19A).

[0473] One advantage of the stem cell-derived motor neuron survival assay is that it can be easily expanded in human motor neurons. To adapt the assay to human cells, the inventors 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 A4V mutation that causes ALS was introduced into a single allele of the SOD1 gene using genome engineering based on zinc finger nucleases (ZFN) to recapitulate the human patient genotype (Figures 24A - 24L). Pairs of cell lines were differentiated into motor neurons in parallel using a previously published protocol (Amoroso et al. 2013; Maury et al. 2015) and characterized for survival and neurite outgrowth in the presence of increasing concentrations of CPA. Human motor neurons are not as sensitive to CPA as mouse motor neurons (Figure 24J), but the inventors found that mutant human SOD1 exposed to 33 μM CPA (about 86% loss) compared to control neurons (about 71% loss) A4V ​Significantly increased degeneration of motor neurons was detected, thereby reproducing the genotype-dependent effect of CPA in mouse motor neurons (Figures 19D and 19E).

[0474] Next, the inventors used an assay to test whether a compound protective against mouse motor neurons would be able to protect human motor neurons exposed to 33 μM CPA. Notably, all tested protective compounds identified in the mouse motor neuron screening were also effective in protecting human motor neurons against CPA. Pretreatment of human motor neurons with kenpaullone rescued 42% of CPA-induced cell death in WT motor neurons and A4V 31% in SOD1 motor neurons (Figure 19D), but had no significant effect on neurite outgrowth (Figure 24I). GO6976 rescued 45% of cell death in WT motor neurons and A4V 34% in SOD1 motor neurons (Figure 19D) and significantly rescued the decrease in neurite outgrowth (Figure 24K). K252a was the most promising compound overall, rescuing 56% of cell death in WT and A4V 100% of cell death in SOD1 motor neurons (Figures 19D and 19E) and had a significant effect on neurite outgrowth in the latter group. Finally, TUDCA reduced cell death in WT and SOD1 A4V motor neurons by 34% and 9%, respectively, and had no substantial effect on neurite outgrowth (Figures 19D, 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 the putative pathway or mechanism of action. Rescue effects are expressed as the % reversal of the CPA effect on survival and neurite outgrowth, for the best time point (24 or 48 h after CPA) and concentration tested. Values represent either the mean of replicate culture wells or from independent experiments for compounds evaluated in the tracking experiment.

Table 3-1

Table 3-2

[0475] TUDCA is an FDA-approved nutritional supplement, and its effects on various pathological conditions have been the focus of numerous clinical trials (NCT00877604, NCT02218619, NCT00771901, NCT01829698). TUDCA is generally safe, has few side effects, and shows good blood-brain barrier penetration when administered subcutaneously or orally (Kaemmerer et al. 2001). Since denervation of the neuromuscular junction (NMJ) is one of the earliest phenotypes observed in a mouse model of ALS (Azzouz et al. 1997; Maselli et al. 1993; Tsujihata et al. 1984; Sharma et al. 2016), the inventors inferred that TUDCA could promote the maintenance of the integrity of motor axon terminals and delay the denervation process.

[0476] To compare the efficacy of TUDCA with its analogs, the inventors screened it in parallel with 10 conjugated bile acids in CPA-treated motor neurons. Taurocholic acid (TGCA) was consistent with the modest effect of TUDCA on motor neuron survival but acted at lower concentrations; however, it had a smaller effect on neurite outgrowth than TUDCA (Figures 25C and 25D). Taurocholic acid (TCA) showed similar effects to TUDCA on both motor neuron survival and neurite outgrowth but did not provide any advantages for drug development. Therefore, the inventors 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, the inventors designed a small study to evaluate denervation of the easily fatigued hindlimb anterior tibialis muscle (TA) (Figure 20D). The inventors had already determined that TA motor neurons in B6.Cg-Tg(SOD1*G93A)1Gur / J mice undergo an ER stress period starting at P30, followed by muscle denervation extending to P50 (Kaplan et al. 2014). During this period, the TA muscle exhibits 25 - 40% denervation before becoming substantially atrophied at later time points. To target this window of ER stress-related neurodegeneration, the inventors used hSOD1 G93A mice and treated them subcutaneously with TUDCA injections every three days between P30 and P50. At the end of the experiment, the inventors counted all NMJs by staining acetylcholine receptors on the TA muscle with Alexa Fluor488-conjugated α-bungarotoxin and evaluated their innervation by staining motor axons with an antibody against vesicular acetylcholine transferase (VAChT) (Figure 20F). Despite the fact that the mice received only 7 injections over the course of 21 days of treatment, the inventors observed a modest but statistically significant increase in NMJ innervation in TUDCA-treated hSOD1 G93A mice compared to untreated animals (Figure 20E). Discussion

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

[0479] The mutations that cause ALS are not clearly toxic to spinal motor neurons: patients show no obvious motor deficits during the pre-onset stage of the disease, which typically lasts for decades. Nor is motor neuron death observed until adulthood in the aggressive mouse model of ALS caused by overexpression of mutant hSOD1 (Chiu et al. 1995), indicating that the effects of the ALS mutations accumulate or are enhanced by age and / or environment-related stressors. Mutant motor To identify stressors involved in the degeneration of mutant motor neurons, the inventors designed a highly sensitive, intrinsically controlled survival assay. The co-culture setting reduces the influence of non-cell-autonomous toxic factors and focuses on the intrinsic increasing vulnerability of motor neurons expressing the disease-causing mutant SOD1 protein. Using this platform, the inventors identified CPA as a compound that is toxic to mutants relative to control ESC-derived motor neurons. Perhaps even more interesting was the observation that motor neurons were considerably more sensitive to CPA and other ER stress-inducing compounds than other types of spinal 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). The inventors 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) results in the accumulation of mutant SOD1 in hSOD1 G93A ESC-derived motor neurons. This last finding suggests that mutant SOD1 is normally cleared via the constitutive pathway, which is impaired after SERCA inhibition. Mutant SOD1 in its misfolded form has been observed in human postmortem tissue samples and in several ALS models, but the exact mechanism through which it perpetuates motor neuron death is not clear (Gros-Louis et al. 2010; Kikuchi et al. 2006). Even though SOD1 is translated on free cytoplasmic ribosomes, previous studies have shown that the mutant form can be detected within mitochondria and the ER (Kikuchi et al. 2006; Vande Velde et al. 2008).

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

[0482] Screening of candidate neuroactive compounds identified several potent drugs that can reverse the detrimental effects of CPA. Two classes of compounds were of particular interest: kinase inhibitors and bile acid derivatives. Kinase inhibitors showed a remarkable ability to protect motor neurons from CPA toxicity. One compound, kenpaullone, has already 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 human stem cell-derived motor neurons from patients with mutations in the FUS gene (Liu et al. 2016). In addition to kenpaullone, the inventors identified two staurosporine analogs, K252a and GO6976, which have already been reported to increase neuron survival in several in vitro neurodegeneration models (Roux et al. 2002; Jeohn et al. 2000). However, these inhibitors will require further optimization. Kenpaullone Insoluble in aqueous solution at its most effective concentration, further in vivo studies without extensive attempts to reformulate its vehicle composition are excluded. K252a and GO6976 are more potent and more soluble than kempaurone in our survival assays (data not shown); however, these compounds are broad-spectrum inhibitors, each targeting >100 different kinases, and the identity of the targets associated with motor neuron degeneration remains ill-defined. We are currently conducting studies to identify these targets and develop more specific analogs for further in vivo testing.

[0483] A second class of neuroprotective compounds resulting from our screening were derivatives of mammalian bile acids that have been widely used in traditional Tibetan and Chinese medicine. This class of compounds protected <30% of dying motor neurons, which fully 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 itching and liver disease. TUDCA has already been shown to have beneficial effects in mouse models of Huntington's and Alzheimer's diseases (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 a mouse model of type 2 diabetes, in part by acting as a chaperone for misfolded proteins (Ozcan et al. 2006; Uppala et al. 2017). We extended these studies by demonstrating that TUDCA preserves the integrity of the neuromuscular junction in a mouse model of ALS and raises the possibility that TUDCA alone or in combination with other treatments may delay the onset or progression of motor disease.

[0484] The inventors' findings add to a mountain of evidence that ER stress is involved in motor neuron cell death in ALS, and the inventors describe a scalable stem cell-based system to probe 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 disease, and Alzheimer's disease (Hetz and Saxena 2017). Most importantly, the inventors adapted the assay to directly test neuroprotective compounds on human motor neurons, thereby accelerating the preclinical evaluation of drugs that modulate motor neuron survival by maximizing the likelihood of being effective in human patients. Materials and Methods 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 either the mutant (B6.Cg-Tg(SOD1*G93A)1Gur / J) or WT (B6SJL-Tg(SOD1)2Gur / J) form of human SOD1. 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, mouse embryonic stem cell lines were 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, the inventors introduced the A4V mutation into the wild-type SOD1 locus of the human embryonic stem cell line HUES3 Hb9::GFP, generating an independent collection of isogenic cell lines (Di Giorgio et al. 2008). Using the two-step nuclease-mediated gene targeting strategy (Maeder et al. 2008) again, the inventors introduced the SOD1 mutation into the HUES3 Hb9::GFP genetic background (Figure 24A). After verifying the desired SOD1 gene editing (Figures 24B and 24C), the inventors differentiated SOD and SOD1 HUES3 Hb9::GFP cells into cultures containing spinal motor neurons. GFP motor neurons of both genotypes could be isolated using FACS (Figure 24D), and SOD1 transcription and protein expression within these cells were confirmed (Figures 24F and 24G). The new system was further verified by staining for the motor neuron transcription factor Islet1 (Figure 24E) and assessing survival rates in short- and long-term cultures (Figures 24H and 24I). Mouse and human motor neuron differentiation + / A4V and SOD1 + / + isogenic cell lines (Di Giorgio et al. 2008). Using the two-step nuclease-mediated gene targeting strategy (Maeder et al. 2008) again, the inventors introduced the SOD1 A4V mutation into the HUES3 Hb9::GFP genetic background (Figure 24A). After verifying the desired SOD1 gene editing (Figures 24B and 24C), the inventors introduced the SOD + / A4V and SOD1 + / + HUES3 Hb9::GFP cells were differentiated into cultures containing spinal motor neurons. GFP motor neurons of both genotypes could be isolated using FACS (Figure 24D), and SOD1 transcription and protein expression within these cells were confirmed (Figures 24F and 24G). The new system was further verified by staining for the motor neuron transcription factor Islet1 (Figure 24E) and assessing survival rates in short- and long-term cultures (Figures 24H and 24I). Mouse and human motor neuron differentiation + motor neurons could be isolated using FACS (Figure 24D), and SOD1 transcription and protein expression within these cells were confirmed (Figures 24F and 24G). The new system was further verified by staining for the motor neuron transcription factor Islet1 (Figure 24E) and assessing survival rates in short- and long-term cultures (Figures 24H and 24I). Mouse and human motor neuron differentiation

[0488] The differentiation of transgenic mouse ESCs into motor neurons was performed as described above (Wichterle et al. 2002). Briefly, cells were dissociated on day 6 of differentiation and plated on 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 various combinations of neurotrophic factors (GDNF, BDNF, CNTF, IGF-1) as described in the text (Figure 21G, Figure 21K, and Figure 21L). In most of all experimental mouse cultures containing motor neurons, interneurons and glial progenitor 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, Ptf1α-tdTomato ESCs were dissociated and cultured as a suspension in embryoid bodies (EBs) at a density of 8.0×10 5 cells / 10 cm culture-treated Petri dish. On day 2 of differentiation, the EBs were collected, centrifuged, and split 1:4 into new Petri dishes supplemented with 1 μM RA. The medium was changed on days 4 and 6 of differentiation. The endpoint of dI4 IN differentiation was day 8, and the EBs were collected for co-culture studies.

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

[0491] All cell lines used were regularly 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 wells of cells 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%. The survival ratio was calculated by dividing the number of surviving GFP + cells by the number of RFP + cells at 48 and 72 hours after exposure to the compound. The inventors 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 with respect to genotype. Compounds that resulted in a survival ratio of G93A to WT of <0.67-fold, corresponding to a 50% difference in survival rate, were retested in a 5-point 2-fold dilution series. All hit compounds were visually evaluated, and wells with obvious artifacts such as autofluorescence and excessive cell c...

Claims

1. 【Fig. 296】 A compound selected from the group consisting of these and combinations thereof, or an N-oxide, hydrate or pharmaceutically acceptable salt thereof.

2. 【Fig. 297】 The compound according to claim 1, or an N-oxide, hydrate or pharmaceutically acceptable salt thereof.

3. 【Fig. 298】 The compound according to claim 1, or an N-oxide, hydrate or pharmaceutically acceptable salt thereof.

4. 【Fig. 299】 The compound according to claim 1, or an N-oxide, hydrate or pharmaceutically acceptable salt thereof.

5. 【Fig. 300】 The compound according to claim 1, or an N-oxide, hydrate or pharmaceutically acceptable salt thereof.

6. 【Fig. 301】 The compound according to claim 1, or an N-oxide, hydrate or pharmaceutically acceptable salt thereof.

7. 【Fig. 302】 The compound according to claim 1, or an N-oxide, hydrate or pharmaceutically acceptable salt thereof.

8. 【Fig. 303】 The compound according to claim 1, or an N-oxide, hydrate or pharmaceutically acceptable salt thereof.

9. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and one or more compounds, wherein the one or more compounds are 【Chemical 308】 A pharmaceutical composition having a structure selected from the group consisting of these and combinations thereof, or an N-oxide, hydrate or pharmaceutically acceptable salt thereof.

10. The one or more compounds have the following structure: 【Chemical 309】 The pharmaceutical composition according to claim 9, or an N-oxide, hydrate or pharmaceutically acceptable salt thereof.

11. The one or more compounds have the following structure: 【Chemical 310】 The pharmaceutical composition according to claim 9, or an N-oxide, hydrate or pharmaceutically acceptable salt thereof.

12. The one or more compounds have the following structure: 【Chemical 311】 The pharmaceutical composition according to claim 9, or an N-oxide, hydrate or pharmaceutically acceptable salt thereof.

13. The one or more compounds have the following structure: 【Chemical 312】 The pharmaceutical composition according to claim 9, or an N-oxide, hydrate or pharmaceutically acceptable salt thereof.

14. The one or more compounds have the following structure: 【Chemical 313】 The pharmaceutical composition according to claim 9, or an N-oxide, hydrate or pharmaceutically acceptable salt thereof.

15. The one or more compounds have the following structure: 【Chemical 314】 The pharmaceutical composition according to claim 9, or an N-oxide, hydrate or pharmaceutically acceptable salt thereof.

16. The one or more compounds have the following structure: 【Chemical 315】 The pharmaceutical composition according to claim 9, having, or its N-oxide, hydrate or pharmaceutically acceptable salt.

17. A kit comprising the pharmaceutical composition according to any one of claims 9 to 16, together with instructions for use of the pharmaceutical composition.

18. A composition for treating or ameliorating the effects of a disorder in a subject in need thereof, the composition comprising one or more compounds, the one or more compounds being 【Chemical 321】 as well as combinations thereof, or a composition selected from the group consisting of its N-oxide, hydrate or pharmaceutically acceptable salt.

19. The composition according to claim 18, wherein the disorder is a disease associated with endoplasmic reticulum (ER) stress.

20. The composition according to claim 18, wherein the disorder is a disease characterized by abnormal kinase levels in the subject.

21. The composition according to claim 18, wherein the disorder is a disease associated with axonal degeneration.

22. The composition according to claim 18, 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 diseases, and neurodegenerative diseases.

23. The composition according to claim 22, 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, Lewy body dementia, corticobasal degeneration, progressive supranuclear palsy, chronic traumatic encephalopathy (CTE), polyglutamine disease, prion disease, glaucoma, and hereditary spastic paraplegia.

24. The composition according to claim 18, wherein the disorder is amyotrophic lateral sclerosis (ALS).

25. The disease characterized by the abnormal kinase level in the subject is chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), acute promyelocytic leukemia, acute megakaryoblastic leukemia, childhood leukemia, familial chronic lymphocytic leukemia, left-right axis formation abnormality, 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, ovarian 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, progressive osseous heteroplasia, skeletal malformation, extraskeletal bone formation, pica, hypertension, myocardial infarction, distal intermediate limb malformation, ataxia, telangiectasia, Zwicker syndrome, heart failure, juvenile polyposis syndrome, brachydactyly type A2 (hand formation abnormality), distal intermediate limb chondrodysplasia (bone formation abnormality), vulvar abnormality, primary pulmonary hypertension (PPH1), heart-face-skin syndrome, juvenile-onset medullary carcinoma, X-linked agammaglobulinemia, cardiac arrhythmia, somatic melanoma, familial melanoma, sarcoma, head and neck squamous cell carcinoma, epithelial tumor, cardiac hypertrophy, Rett syndrome, X-linked infantile spasm syndrome, Li-Fraumeni syndrome, circadian disorder, ductal carcinoma, breast hyperplasia, cone-rod dystrophy (CORD) type 5, cone-rod dystrophy (CORD) type 6, Leber congenital amaurosis type 1 (LCA1), epilepsy, myotonia, muscle wasting, cataract, hypogonadism, endocrine deficiency, male pattern baldness, Down syndrome (DS), glioblastoma, hepatocellular carcinoma, Pfeiffer syndrome, Kallmann syndrome 2, stem cell leukemia lymphoma syndrome (SCLL), myeloproliferative disorder, Apert syndrome, Jackson-Weiss syndrome, Crouzon syndrome, Beare-Stevenson cutis gyrata syndrome, achondrogenesis, hypochondrogenesis, thanatophoric dysplasia, Aderlad type craniosynostosis, San Diego type dysplasia, Muenke syndrome, pituitary adenoma, spinal dysraphism, infantile hemangioma, idiopathic myelofibrosis, neuroblastoma, kidney cancer, papillary carcinoma, hyper-IgM syndrome, dyschromatosis, anhidrotic ectodermal dysplasia, rheumatoid arthritis, epidermal nevus, pineal hyperplasia, polycystic ovary syndrome, atypical migraine, diabetic hyperlipidemia, Morquio syndrome, bacterially induced macrophage apoptosis, febrile bacterial infection,Uterine leiomyosarcoma, post-transplant lymphoproliferative disorder, myeloproliferative disease (MPD), polycythemia vera, brain tumor, gastrointestinal stromal tumor (GIST), mastocytosis, vitiligo, 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 tumor, non-syndromic mental retardation type 30 (MRX30), idiopathic hypereosinophilic syndrome, split spine, Wolcott-Rallison syndrome (WRS), hepatic glycogenosis, cirrhosis, hematopoietic malignancy, Carney complex tumor, cardiac contractility, diabetic nephropathy, diabetic retinopathy, diabetic vascular complications, autism, autosomal dominant spinocerebellar ataxia type 14, pain sensation, osteoarthritis cartilage, bladder cancer, nasopharyngeal carcinoma, undifferentiated large cell leukemia, familial medullary thyroid carcinoma (FMTC), multiple endocrine neoplasia type IIA (MEN2A), MEN2B, pheochromocytoma, papillary thyroid cancer, Hirschsprung disease, type 2 microphthalmia, HPC1, blood coagulation, angina pectoris, renal oncocytoma, lung adenocarcinoma, autosomal dominant brachydactyly type B, autosomal recessive Robinow syndrome (RRS), Coffin-Lowry syndrome, CNS tumor, Loeys-Dietz syndrome, esophageal cancer, hereditary non-polyposis colorectal cancer (HNPCC, Lynch syndrome), Marfan syndrome type II, venous malformation, astrocytoma, hypertrophic and dilated cardiomyopathy, tibial muscular dystrophy, anhidrosis, pseudohypoaldosteronism type II, and chronic arthritis, the composition according to claim 20, selected from the group consisting of.

26. The composition comprises 5-hydroxytryptophan, Activase, AFQ056 (Novartis), Aggrastat, Albendazole, alpha-lipoic acid / L-acetylcarnitine, Alteplase, Amantadine (Symmetrel), Amlodipine, Ancrod, Apomorphine (Apokyn), Arimoclomol, Arixtra, Armodafinil, Ascorbic acid, Ascriptin, Aspirin, Atenolol, Avonex, Baclofen (Lioresal), Banzel, Benztropine (Cogentin), Betaseron, BGG492 (Novartis Corp.), Botulinum toxin, Bufferin, Carbator (registered trademark), Carbidopa / Levodopa immediate release (Sinemet), Carbidopa / Levodopa orally disintegrating tablets (Parcopa), Carbidopa / Levodopa / Entacapone (Stalevo), CERE-110: Adeno-associated virus delivery of NGF (Ceregene), Cerebrolysin, Cinnovex, Citalopram, Citicoline, Clobazam, Clonazepam, Clopidogrel, Clozapine (Clozaril), Coenzyme Q, Creatine, Dabigatran, Dalteparin, Dapsone, Davunetide, Deferiprone, Depakene (registered trademark), DepakoteER (Registered Trademark), Depakote (Registered Trademark), Desmoteplase, Diastat, Diazepam, Digoxin, Dilantin (Registered Trademark), Dimebon, Dipyridamole, Divalproex (Depakote), Donepezil (Aricept), EGb 761, Eldepryl, ELND002 (Elan Pharmaceuticals), Enalapril, Enoxaparin, Entacapone (Comtan), Epoetin Alfa, Eptifibatide, Erythropoietin, Escitalopram, Eslicarbazepine Acetate, Esmolol, Ethosuximide, Ethyl - EPA (Miraxion (Trademark)), Exenatide, Extavia, Ezogabine, Felbamate, Felbatol (Registered Trademark), Fingolimod (Gilenya), Fluoxetine (Prozac), Fondaparinux, Fragmin, Frisium, Gabapentin, Gabitril (Registered Trademark), Galantamine, Glatiramer (Copaxone), Haloperidol (Haldol), Heparin, Human Chorionic Gonadotropin (hCG), Idebenone, Inovelon (Registered Trademark), Insulin, Interferon Beta 1a, Interferon Beta 1b, Ioflupane 123I (DATSCAN (Registered Trademark)), IPX066 (Impax Laboratories Inc.), JNJ - 26489112 (Johnson andJohnson, Keppra (registered trademark), Klonopin, Lacosamide, L-alpha glyceryl phosphorylcholine, Lamictal (registered trademark), Lamotrigine, Levetiracetam, Liraglutide, Lisinopril, Lithium carbonate, Lopressor, Lorazepam, Losartan, Lovenox, Lu AA24493, Luminal, LY450139 (EliLilly, Lyrica, Masitinib, Mecobalamin, Memantine, Methylprednisolone, Metoprolol Tartrate, Minitran, Minocycline, Mirtazapine, Mitoxantrone (Novantrone), Mysoline (registered trademark), Natalizumab (Tysabri), Neurontin (registered trademark), Niacinamide, Nitro-Bid, Nitro-Dur, Nitroglycerin, Nitrolingual, Nitromist, Nitrostats, Nitro-Time, Norepinephrine (NOR), Carbamazepine, Octreotide, Onfi (registered trademark), Oxcarbazepine, Oxybutynin Chloride, PF-04360365 (Pfizer), Phenobarbital, Phenyted (registered trademark), Phenytoin, Piclotan, Pioglitazone, Plavix, Potiga, Pramipexole (Mirapex), Pramlintide, Prednisone, Primidone, Prinivil, Probenecid, Propranolol, PRX-00023 (EPIX PharmaceuticalsInc.), PXT3003, Quinacrine, Ramelteon, Rasagiline (Azilect), Rebif, Recigen, Remacemide, Resveratrol, Retavase, Reteplease, Riluzole (Rilutek), Rivastigmine (Exelon), Ropinirole (Requip), Rotigotine (Neupro), Rufinamide, Sabril, Safinamide (EMD Serono), Salagen, Sarafem, Selegiline (l-deprenyl, Eldepril), SEN0014196 (Siena Biotech), Sertraline (Zoloft), Simvastatin, Sodium Nitroprusside (NPS), Sodium Phenylbutyrate, Stanback Headache Powder, Tacrine (Cognex), Tamoxifen, Tauroursodeoxycholic Acid (TUDCA), Tegretol (registered trademark), Tenecteplase, Tenormin, Tetrabenazine (Xenazine), THR-18 (ThrombotechLtd.), tiagabine, tideglusib, tirofiban, tissue plasminogen activator (tPA), tizanidine (Zanaflex), TNKase, tolcapone (Tasmar), tolterodine, Topamax (registered trademark), topiramate, trihexyphenidyl (formerly Artane), Trileptal (registered trademark), ursodiol, valproic acid, valsartan, varenicline (Pfizer), Vimpat, vitamin E, warfarin, Zarontin (registered trademark), Zestril, Zonegran (registered trademark), zonisamide, Zydis selegiline HCL orally disintegrating tablets (Zelapar), and combinations thereof, the composition according to claim 18, characterized in that it is administered in combination with one or more additional therapeutic agents selected from the group consisting of

27. The composition according to claim 18, wherein the subject is a mammal.

28. The composition according to claim 27, wherein the mammal is selected from the group consisting of humans, veterinary animals, and agricultural animals.

29. The composition according to claim 18, wherein the subject is a human.

30. A pharmaceutical composition for treating or ameliorating the effects of a disorder in a subject in need thereof, the one or more compounds being 【Chemical 326】 A pharmaceutical composition selected from the group consisting of those combinations, or their N-oxides, hydrates or pharmaceutically acceptable salts.

31. The one or more compounds have the following structure: 【Chemical 327】 , or its N-oxide, hydrate or pharmaceutically acceptable salt, the pharmaceutical composition according to claim 30.

32. The one or more compounds have the following structure: 【Chemical 328】 , or its N-oxide, hydrate or pharmaceutically acceptable salt, the pharmaceutical composition according to claim 30.

33. The one or more compounds have the following structure: 【Chemical 329】 , or its N-oxide, hydrate or pharmaceutically acceptable salt, the pharmaceutical composition according to claim 30.

34. The one or more compounds have the following structure: 【Chemical 330】 , or its N-oxide, hydrate or pharmaceutically acceptable salt, the pharmaceutical composition according to claim 30.

35. The one or more compounds have the following structure: 【Chemical 331】 , or its N-oxide, hydrate or pharmaceutically acceptable salt, the pharmaceutical composition according to claim 30.

36. The one or more compounds have the following structure: 【Chemical 332】 , or its N-oxide, hydrate or pharmaceutically acceptable salt, the pharmaceutical composition according to claim 30.

37. The one or more compounds have the following structure: 【Chemical 333】 , or its N-oxide, hydrate or pharmaceutically acceptable salt, the pharmaceutical composition according to claim 30.

38. The disorder is a disease associated with endoplasmic reticulum (ER) stress, the pharmaceutical composition according to claim 30.

39. The disorder is a disease characterized by abnormal kinase levels in the subject, the pharmaceutical composition according to claim 30.

40. The disorder is a disease associated with axonal degeneration, the pharmaceutical composition according to claim 30.

41. 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 diseases, and neurodegenerative diseases, the pharmaceutical composition according to claim 30.

42. The pharmaceutical composition according to claim 41, 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, Lewy body dementia, corticobasal degeneration, progressive supranuclear palsy, chronic traumatic encephalopathy (CTE), polyglutamine disease, prion disease, glaucoma, and hereditary spastic paraplegia.

43. The pharmaceutical composition according to claim 30, wherein the disorder is amyotrophic lateral sclerosis (ALS).

44. The disease characterized by the abnormal kinase level in the subject is chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), acute promyelocytic leukemia, acute megakaryoblastic leukemia, pediatric leukemia, familial chronic lymphocytic leukemia, left-right axis formation abnormality, 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, ovarian 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, progressive osseous heteroplasia, skeletal malformation, extraskeletal bone formation, pica, hypertension, myocardial infarction, distal intermediate limb malformation, ataxia, telangiectasia, Zwicker syndrome, heart failure, juvenile polyposis syndrome, brachydactyly type A2 (hand formation abnormality), distal intermediate limb cartilage dysplasia (bone formation abnormality), vulvar abnormality, primary pulmonary hypertension (PPH1), heart-face-skin syndrome, juvenile-onset medulloblastoma, X-linked agammaglobulinemia, cardiac arrhythmia, somatic melanoma, familial melanoma, sarcoma, head and neck squamous cell carcinoma, epithelial tumor, cardiac hypertrophy, Rett syndrome, X-linked infantile spasm syndrome, Li-Fraumeni syndrome, circadian disorder, ductal carcinoma in situ, breast hyperplasia, cone-rod dystrophy (CORD) type 5, cone-rod dystrophy (CORD) type 6, Leber congenital amaurosis type 1 (LCA1), epilepsy, dystonia, muscle wasting, cataract, hypogonadism, endocrine deficiency, male pattern baldness, Down syndrome (DS), glioblastoma, hepatocellular carcinoma, Pfeiffer syndrome, Kallmann syndrome 2, stem cell leukemia lymphoma syndrome (SCLL), myeloproliferative disorder, Apert syndrome, Jackson-Weiss syndrome, Crouzon syndrome, Beare-Stevenson cutis gyrata syndrome, achondrogenesis, hypochondrogenesis, thanatophoric dysplasia, Aderlad type craniosynostosis, San Diego type dysplasia, Muenke syndrome, pituitary adenoma, spinal dysraphism, infantile hemangioma, idiopathic myelofibrosis, neuroblastoma, kidney cancer, papillary carcinoma, hyper IgM syndrome, incontinentia pigmenti, anhidrotic ectodermal dysplasia, rheumatoid arthritis, epidermal nevus, pineal hyperplasia, polycystic ovary syndrome, atypical migraine, diabetic hyperlipidemia, Morquio syndrome, bacteria-induced macrophage apoptosis, febrile bacterial infection,Uterine leiomyosarcoma, post-transplant lymphoproliferative disorder, myeloproliferative disease (MPD), polycythemia vera, brain tumor, gastrointestinal stromal tumor (GIST), mastocytosis, vitiligo, 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 tumor, non-syndromic mental retardation type 30 (MRX30), idiopathic hypereosinophilic syndrome, split spine, Wolcott-Rallison syndrome (WRS), hepatic glycogenosis, cirrhosis, hematopoietic malignancies, Carney complex tumors, cardiac contractility, diabetic nephropathy, diabetic retinopathy, diabetic vascular complications, autism, autosomal dominant spinocerebellar ataxia type 14, pain sensation, osteoarthritis cartilage, bladder cancer, nasopharyngeal carcinoma, undifferentiated large cell leukemia, familial medullary thyroid carcinoma (FMTC), multiple endocrine neoplasia type IIA (MEN2A), MEN2B, pheochromocytoma, papillary thyroid carcinoma, Hirschsprung disease, type 2 microphthalmia, HPC1, blood coagulation, angina pectoris, renal oncocytoma, lung adenocarcinoma, autosomal dominant brachydactyly type B, autosomal recessive Robinow syndrome (RRS), Coffin-Lowry syndrome, CNS tumors, Loeys-Dietz syndrome, esophageal cancer, hereditary non-polyposis colorectal cancer (HNPCC, Lynch syndrome), Marfan syndrome type II, venous malformation, astrocytoma, hypertrophic and dilated cardiomyopathy, tibial muscular dystrophy, anhidrosis, pseudohypoaldosteronism type II, and chronic arthritis, the pharmaceutical composition according to claim 43, selected from the group consisting of.

45. The pharmaceutical composition comprises 5-hydroxytryptophan, Activase, AFQ056 (Novartis), Aggrastat, Albendazole, alpha-lipoic acid / L-acetylcarnitine, Alteplase, Amantadine (Symmetrel), Amlodipine, Ancrod, Apomorphine (Apokyn), Arimoclomol, Arixtra, Armodafinil, Ascorbic acid, Ascriptin, Aspirin, Atenolol, Avonex, Baclofen (Lioresal), Banzel, Benztropine (Cogentin), Betaseron, BGG492 (Novartis Corp.), Botulinum toxin, Bufferin, Carbator (registered trademark), Carbidopa / Levodopa immediate release (Sinemet), Carbidopa / Levodopa orally disintegrating tablets (Parcopa), Carbidopa / Levodopa / Entacapone (Stalevo), CERE-110: Adeno-associated virus delivery of NGF (Ceregene), Cerebrolysin, Cinnovex, Citalopram, Citicoline, Clobazam, Clonazepam, Clopidogrel, Clozapine (Clozaril), Coenzyme Q, Creatine, Dabigatran, Dalteparin, Dapsone, Davunetide, Deferiprone, Depakene (registered trademark), DepakoteER (Registered Trademark), Depakote (Registered Trademark), Desmoteplase, Diastat, Diazepam, Digoxin, Dilantin (Registered Trademark), Dimebon, Dipyridamole, Divalproex (Depakote), Donepezil (Aricept), EGb 761, Eldepryl, ELND002 (Elan Pharmaceuticals), Enalapril, Enoxaparin, Entacapone (Comtan), Epoetin Alfa, Eptifibatide, Erythropoietin, Escitalopram, Eslicarbazepine Acetate, Esmolol, Ethosuximide, Ethyl-EPA (Miraxion (Trademark)), Exenatide, Extavia, Ezogabine, Felbamate, Felbatol (Registered Trademark), Fingolimod (Gilenya), Fluoxetine (Prozac), Fondaparinux, Fragmin, Frisium, Gabapentin, Gabitril (Registered Trademark), Galantamine, Glatiramer (Copaxone), Haloperidol (Haldol), Heparin, Human Chorionic Gonadotropin (hCG), Idebenone, Inovelon (Registered Trademark), Insulin, Interferon Beta 1a, Interferon Beta 1b, Ioflupane 123I (DATSCAN (Registered Trademark)), IPX066 (Impax Laboratories Inc.), JNJ-26489112 (Johnson andJohnson, Keppra (registered trademark), Klonopin, Lacosamide, L-alpha glyceryl phosphorylcholine, Lamictal (registered trademark), Lamotrigine, Levetiracetam, Liraglutide, Lisinopril, Lithium carbonate, Lopressor, Lorazepam, Losartan, Lovenox, Lu AA24493, Luminal, LY450139 (EliLilly, Lyrica, Masitinib, Mecobalamin, Memantine, Methylprednisolone, Metoprolol Tartrate, Minitran, Minocycline, Mirtazapine, Mitoxantrone (Novantrone), Mysoline (registered trademark), Natalizumab (Tysabri), Neurontin (registered trademark), Niacinamide, Nitro-Bid, Nitro-Dur, Nitroglycerin, Nitrolingual, Nitromist, Nitrostats, Nitro-Time, Norepinephrine (NOR), Carbamazepine, Octreotide, Onfi (registered trademark), Oxcarbazepine, Oxybutynin Chloride, PF-04360365 (Pfizer), Phenobarbital, Phenyteck (registered trademark), Phenytoin, Piclotan, Pioglitazone, Plavix, Potiga, Pramipexole (Mirapex), Pramlintide, Prednisone, Primidone, Prinivil, Probenecid, Propranolol, PRX-00023 (EPIX PharmaceuticalsInc.), PXT3003, Quinacrine, Ramelteon, Rasagiline (Azilect), Rebif, Recigen, Remacemide, Resveratrol, Retavase, Reteplease, Riluzole (Rilutek), Rivastigmine (Exelon), Ropinirole (Requip), Rotigotine (Neupro), Rufinamide, Sabril, Safinamide (EMD Serono), Salagen, Sarafem, Selegiline (l - Deprenyl, Eldepril), SEN0014196 (Siena Biotech), Sertraline (Zoloft), Simvastatin, Sodium Nitroprusside (NPS), Sodium Phenylbutyrate, Stanback Headache Powder, Tacrine (Cognex), Tamoxifen, Tauroursodeoxycholic Acid (TUDCA), Tegretol (registered trademark), Tenecteplase, Tenormin, Tetrabenazine (Xenazine), THR - 18 (ThrombotechLtd.), tiagabine, tideglusib, tirofiban, tissue plasminogen activator (tPA), tizanidine (Zanaflex), TNKase, tolcapone (Tasmar), tolterodine, Topamax (registered trademark), topiramate, trihexyphenidyl (formerly Artane), Trileptal (registered trademark), ursodiol, valproic acid, valsartan, varenicline (Pfizer), Vimpat, vitamin E, warfarin, Zarontin (registered trademark), Zestril, Zonegran (registered trademark), zonisamide, Zydis selegiline HCl orally disintegrating tablets (Zelapar), and combinations thereof, and is administered in combination with one or more additional therapeutic agents selected from the group consisting of the pharmaceutical composition according to claim 30, characterized in that.

46. The pharmaceutical composition according to claim 30, wherein the subject is a mammal.

47. The pharmaceutical composition according to claim 46, wherein the mammal is selected from the group consisting of humans, veterinary animals, and agricultural animals.

48. The pharmaceutical composition according to claim 30, wherein the subject is a human.

49. A composition for suppressing the toxicity of endoplasmic reticulum (ER) stress in a subject in need thereof, the composition comprising a kinase inhibitor comprising one or more compounds, wherein the one or more compounds are 【Chemical 339】 【Chemical 340】 and combinations thereof, or N - oxides, hydrates or pharmaceutically acceptable salts thereof.

50. A composition for treating or improving the effects of a disease associated with axonal degeneration in a subject in need thereof, the composition comprising a kinase inhibitor comprising one or more compounds, wherein the one or more compounds are 【Chemical 341】 【Chemical 342】 and combinations thereof, or N - oxides, hydrates or pharmaceutically acceptable salts thereof.

51. A composition for treating or improving the effects of a neurodegenerative disease in a subject in need thereof, the composition comprising one or more compounds, wherein the one or more compounds are 【Chemical 343】 【Chemical 344】 and combinations thereof, or N - oxides, hydrates or pharmaceutically acceptable salts thereof.

52. The one or more compounds have the following structure: 【Chemical 345】 or an N - oxide, hydrate or pharmaceutically acceptable salt thereof. The composition according to any one of claims 49 - 51.

53. The one or more compounds have the following structure: 【Chemical 346】 The composition according to any one of claims 49 to 51, having, or its N-oxide, hydrate or pharmaceutically acceptable salt.

54. The one or more compounds have the following structure: 【Chemical 347】 The composition according to any one of claims 49 to 51, having, or its N-oxide, hydrate or pharmaceutically acceptable salt.

55. The one or more compounds have the following structure: 【Chemical 348】 The composition according to any one of claims 49 to 51, having, or its N-oxide, hydrate or pharmaceutically acceptable salt.

56. The one or more compounds have the following structure: 【Chemical 349】 The composition according to any one of claims 49 to 51, having, or its N-oxide, hydrate or pharmaceutically acceptable salt.

57. The one or more compounds have the following structure: 【Chemical 350】 The composition according to any one of claims 49 to 51, having, or its N-oxide, hydrate or pharmaceutically acceptable salt.

58. The one or more compounds have the following structure: 【Chemical 351】 The composition according to any one of claims 49 to 51, having, or its N-oxide, hydrate or pharmaceutically acceptable salt.

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