New treatments

Abstract

The present invention relates to PI3K alpha activating compounds and pharmaceutical compositions comprising same. The present invention further relates in particular to the treatment of disorders susceptible to treatment by PI3K alpha activation.

Description

[Technical Field]

[0001] The present invention relates to PI3Kα activating compounds and pharmaceutical compositions comprising same. The present invention further relates, inter alia, to the treatment of disorders treatable by PI3Kα activation. [Background technology]

[0002] Compared with the creation of protein kinase inhibitors and lipid kinase inhibitors, the creation of pharmacological activators that utilize the beneficial activities of these enzymes, such as tissue regeneration and protection, wound healing, immune stimulation, and metabolic enhancement, has been limited to date. The extended family of lipid kinases, and in particular the important subgroup, PI 3-kinase (PI3K), play important roles in these therapies, yet no small molecule activators for this class of enzymes have been reported.

[0003] Class IA PI 3-kinases (PI3Ks) signal downstream of tyrosine kinases, G protein-coupled receptors, and small GTPases, regulating cell growth, proliferation, and migration. These PI3Ks consist of a p110 catalytic subunit (p110α, β, or δ) complexed with a p85 regulatory subunit, which recruits these PI3Ks to activated receptor complexes at the plasma membrane. While p110α and p110β show broad tissue distribution, p110δ is highly enriched in leukocytes. PI3K signaling and its downstream effectors, AKT and mTORC1, are known to be overactivated in cancer and immune dysregulation. This has led to extensive development of PI3K pathway inhibitors, and several PI3K inhibitors are now approved for clinical use.

[0004] As discussed further elsewhere herein, substantial evidence suggests that activation of the PI3K / AKT pathway may also be beneficial in therapeutic settings, including disease-associated cell protection and tissue regeneration. Indeed, PI3K / AKT inhibition attenuates the protective effects of growth factors and various other drugs and therapies in cell / tissue injury models, including neurons, cardiomyocytes, muscle, lung epithelial cells, and retinal cells (see Borges, GA et al. Regen Med 15, 1329-1344 (2020); Matsuda, S. et al. International journal of oncology 49, 1785-1790 (2016); Koh, SH & Lo, EH J Clin Neurol 11, 297-304 (2015); Zhang, Z. et al. Mol Med Rep 18, 3547-3554 (2018)). These include protection from ischemia-reperfusion injury (IRI) through reoxygenation (e.g., in neurons after stroke and cardiomyocytes during cardiac arrest), protection from ionizing radiation, promotion of tissue and wound healing, and neuroprotection / regeneration. Given its critical role in insulin signaling, activation of PI3Kα may also overcome insulin resistance in obesity and type 2 diabetes, as evidenced by cell-based studies using a genetically activated PI3Kα allele and by attenuated several features of diabetic cardiomyopathy in type 2 diabetic mice with cardiac-selective increases in PI3Kα via adenoviral gene therapy. PI3K activation has also been shown to improve in vitro fertilization success rates by activating cryopreserved ovarian tissue and dormant follicles from primary ovarian failure ex vivo.Genetic strategies for PI3K / ACT activation that have been tested in tissue regeneration include expression of activated alleles of PI3Kα (Prakoso, D. et al. Am J Physiol Heart Circ Physiol 318, H840-H852 (2020)) or AKT (Chen, S. et al. Front Endocrinol (Lausanne) 8, 21 (2017)), or inactivation by genetic deletion or knockdown of PTEN, a lipid phosphatase that downregulates PI3K signaling (Borges, GA et al. Regen Med 15, 1329-1344 (2020); Park, KK et al. Science (New York, NY 322, 963-966 (2008); Liu, K. et al. Nature Neuroscience 13, 1075-1081 (2010); Ohtake, Y., Hayat, U. & Li, S. Neural Regen Res 10, 1363-1368 (2015)). The positive effects of PI3K pathway activation in this context are thought to stem from enhanced cell survival and proliferation, as well as the potential activation of tissue-resident stem cells (Koh, SH & Lo, EH J Clin Neurol 11, 297-304 (2015);Wang, G. et al. The EMBO journal 37 (2018)).

[0005] Using isoform-selective PI3K inhibitors, it was shown that PI3Kα is a key mediator of protection from ischemia-reperfusion by insulin or ischemic preconditioning in cardiomyocytes (Rossello, X. et al. Basic Res Cardiol 112, 66 (2017)). Genetic PI3Kα activation also mediates axon regeneration in neurons (Nieuwenhuis, B. et al. EMBO molecular medicine 12, e11674 (2020)).

[0006] To date, few non-genetic PI3K / AKT activators have been developed. These include cell-permeable p85-binding phosphopeptides that activate the p85 / p110 complex, the AKT-activating small molecules SC79 and MX-2043, and a series of PTEN inhibitors. However, these PI3K activators all have poor drug properties, unclear mechanisms of PI3K pathway activation, and do not target PI3K in an isoform-selective manner.

[0007] As a result, it is particularly desirable to identify compounds that can activate PI3Kα in a PI3K isoform-selective manner in cells and tissues in order to provide both useful biological probes and therapeutic methods for diseases that can be treated by PI3Kα activation, such as those described above.

[0008] The present inventors have discovered the first small molecule compounds that directly and allosterically activate PI3K. Similar to the discoveries of wortmannin and LY294002, which enabled the first pharmacological investigation of the cellular effects of PI3K inhibition, this discovery provides a chemical tool for investigating the consequences of direct PI3Kα activation in basic and translational research. In addition to furthering our understanding of the molecular mechanisms of allosteric PI3Kα activation, the present compounds facilitate controlled signaling studies to gain a more quantitative understanding of PI3Kα signaling and elucidate PI3Kα-specific signaling in cells. The present inventors also provide proof-of-concept for PI3Kα activation as a therapeutic approach. Thus, the present inventors also provide proof-of-concept for the use of the present compounds in therapy.

[0009] At the biochemical level, our studies demonstrate that disruption of the inhibitory contact between p85α and p110α by the compounds of the present invention is key to PI3Kα activation. The structural changes induced by the compounds of the present invention bear similarities, but do not completely overlap, with the dynamic structural changes observed upon PI3Kα activation by natural ligands (e.g., pY, which represents the tyrosine-phosphorylated docking site for PI3Kα on receptors and related molecules) or oncogenic PIK3CA mutations, indicating a unique biochemical activation mechanism of action for the compounds of the present invention.

[0010] At the cellular level, PI3Kα signaling induced by the compounds of the present invention and insulin showed similar overall kinetics, including effective downregulation with prolonged exposure, even in the continuous presence of the ligand. This indicates that PI3Kα signaling induced by the compounds of the present invention remains subject to endogenous feedback mechanisms operating within the PI3K pathway. Such transient PI3K activation may temporarily and effectively enhance endogenous protective and regenerative mechanisms. Consequently, biological administration of the compounds of the present invention is likely to result in transient PI3K pathway activation, which is governed by the chemical properties and turnover of the compounds and is distinct from the sustained effects on signaling brought about by constitutive, oncogenic PIK3CA activation. This mechanism of action may alleviate concerns that PI3Kα activators induce or promote cancer. Furthermore, PIK3CA H1047R Studies have shown that mutant PIK3CA alone is only a weak oncogene, as mice constitutively expressing hotspot mutations do not develop cancer within a year. Similarly, people with rare mosaic genetic activation of PIK3CA are not predisposed to cancer in adulthood. These data suggest that short-term, transient pharmacological activation of PI3Kα is unlikely to promote cancer.

[0011] In summary, we have now identified a novel class of PI3Kα-activating compounds that have applications as biochemical probes. Furthermore, we have used the compounds of the present invention to demonstrate the therapeutic potential of allosteric PI3Kα activation, including tissue regeneration (e.g., nerve regeneration) and tissue protection (e.g., cardioprotection from ischemia-reperfusion injury). Summary of the Invention

[0012] The present invention provides compounds ("Compounds of the Invention"), including, inter alia, compounds of formula (I) as defined herein, as well as tautomers, N-oxides, pharmaceutically acceptable salts, and solvates thereof.

[0013] The present invention also provides pharmaceutical compositions comprising a compound of the present invention in association with one or more pharmaceutically acceptable carriers.

[0014] The present invention also provides a compound of the invention or a pharmaceutical composition of the invention for use as a pharmaceutical, in particular for use in a method for treating and / or preventing disorders susceptible to treatment by PI3Kα activation.

[0015] The present invention also provides the use of a compound of the invention or a pharmaceutical composition of the invention for the manufacture of a medicament, in particular a medicament for use in a method for the treatment and / or prevention of disorders susceptible to treatment by PI3Kα activation.

[0016] The present invention also provides methods of treatment, particularly methods of treating and / or preventing disorders susceptible to treatment by PI3Kα activation in a patient in need thereof, which comprise administering to the patient a compound of the invention, or a pharmaceutical composition of the invention. [Brief explanation of the drawings]

[0017] [Figure 1]Biochemical mechanism of PI3Kα activation by UCL-TRO-1938. a. Structure of UCL-TRO-1938 (referred to as 1938 in the text). b. Selectivity of 1938 for PI3Kα over PI3Kβ and PI3Kδ. c. Enzyme kinetics upon ATP titration of PI3Kα with and without 1938 and pY (calculated using the kcat function in Prism 8). d. Membrane binding of PI3Kα shown as FRET signal (I-I0). I: fluorescence intensity at 520 nm; I0: fluorescence intensity at 520 nm in the absence of enzyme. e. Effect of 1938 on PI3Kα catalytic activity in the presence of saturating doses of pY. f. Effect of 1938 on the catalytic activity of oncogenic mutants of PI3Kα. g. Effect of 1938 on membrane binding of oncogenic mutants of PI3Kα. h. Effect of the PI3Kα-selective inhibitor BYL719 on 1938-activated PI3Kα. i. Effect of 1938 on the IC50 of BYL719 against PI3Kα. Data are shown as mean ± SEM, n = 3 (b, c, e, i), n = 2 (d, h), or n = 4 (f, g) independent experiments. Statistical analysis was performed by two-way ANOVA followed by Tukey's multiple comparison test (b, f, g) or Sidak's multiple comparison test (h); one-way ANOVA followed by Dunnett's multiple comparison test (e). [Figure 2] Structural mechanism of PI3Kα activation by 1938. a. Structural changes induced by 1938 in full-length p110α / p85α assessed by HDX-MS are highlighted on the structure of p110α (gray) / niSH2-p85α (green) (pdb:4ZOP). The surface model is shown in Figure 10 / Extended Data Figure 2. [Figure 3]1938 activates PI3Kα pathway signaling in cells. a. Intracellular PIP3 and PI(3,4)P2 production. a. Kinetic data from total internal fluorescence (TIRF) microscopy of PIK3CA-WT and PIK3CA-KO A549 cells expressing the PIP3 reporter EGFP-PH-ARNOI303x2 and treated with DMSO, 1938 (5 μM) + / - BYL719 (0.5 μM). Individual single-cell traces are shown, with the mean intensity value (F(t)) at each time point relative to the mean intensity at the start (F0). Data are from a single experiment (1938 / BYL719:WT (n=11), KO (n=8); DMSO / BYL719:WT (n=14), KO (n=4)). aii. Representative TIRF microscopy images of PIK3CA-WT A549 cells stimulated with 1938 and subsequently neutralized with BYL719. Images were obtained from the cells in the experiment in ai. above, where 1938 was added at t = 27 min, followed by BYL719 at t = 87 min. Images were taken 1.3 min before 1938 addition, 2.3 min after 1938 addition, and 3.3 min after BYL719 addition. Individual pixel values ​​were normalized and scaled to the average intensity observed at all time points before stimulation (F baseline). Scale bar: 11 µm. aiii. TIRF microscopy data from HeLa cells expressing the EGFP-tagged PIP3 reporter PH-ARNO-I303Ex2 (ARNO) or the PI(3,4)P2 reporter mCherry-cPH-TAPP1x3. Overlay plots (mean ± SEM) were generated by scaling the minimum and maximum normalized fluorescence intensity for each time point (Fn(t)). Data for the PIP3 reporter are representative of two experiments (29 (DMSO / 1938) and 20 (BYL719 / DMSO) single cells). Data for the PI(3,4)P2 reporter are representative of four experiments (78 (DMSO / 1938) and 33 (BYL719 / DMSO) single cells). b. Dose-dependent 1938 induction of pAKTS473 in PI3Kα-WT and PI3Kα-null MEFs (detected by automated Western blotting). BYL: BYL719, TGX: TGX-221, IC: IC87144. All PI3K inhibitors were used at 5 μM.Representative blots are shown (n=2 / experiment). c. Kinetic analysis of 1938-induced pAKTS473 in A549 cells by saturating concentrations of 1938 (with or without BYL719) or insulin (detected by automated Western blotting). Representative blots are shown (n=3 / experiment). d. pAKTS473 production in A549 cells by 1938 compared to insulin (measured by ELISA). e. Left panel: Time course analysis of PI3K / ACT / mTORC1 signals in A549 cells induced by insulin or 1938 (detected by ECL Western blotting). Right panel: Quantification of pAKTS473 / vinculin signal ratios expressed as fold-change relative to DMSO-only control treatment. f. In vitro selectivity profile of 1938 for 133 protein kinases and 7 lipid kinases. Figure 3g. Phosphoproteome analysis of PI3Kα-WT and PI3Kα-KO MEFs stimulated with 1938 or insulin (n=4 / independent experiments). gi. Heatmap: Phosphorylation sites significantly altered by stimulation versus DMSO treatment. Green boxes: significantly upregulated phosphorylation sites; magenta boxes: significantly downregulated phosphorylation sites; white crosses: phosphorylation sites not detected in comparison. gii. Volcano plot of phosphorylation sites differentially regulated by 1938 in PI3Kα-WT or PI3Kα-KO MEFs (relative to DMSO-treated cells of the same genotype). Venn diagram: Overlap of the number of phosphorylation sites identified and regulated by 1938 in PI3Kα-WT MEFs with previously identified sites annotated by PhosphoSitePlus as regulated by insulin, IGF-1, LY294002, or MK2206. giii. Venn diagram showing overlap of phosphorylation sites regulated by 1938 and insulin in PI3Kα-WT MEFs. [Figure 4]1938 activates PI3Kα-dependent cell biological responses. PI3Kα-WT MEFs and PI3Kα-KO MEFs were stimulated with 1938 (with or without BYL719), insulin, or FBS, followed by measurement of the effects on (a) cellular metabolic activity (assessed by measuring cellular ATP content with CellTiter-Glo®), (b) cell cycle progression (measured by EdU incorporation), or (c) cell number (assessed by crystal violet staining). Data are presented as mean ± SEM (n = 2 / independent experiments) (a-c). [Figure 5]Disease-related biological activity of 1938. Effect of 1938 on ischemia-reperfusion injury in isolated rat hearts. Hearts were subjected to 45 minutes of global ischemia followed by 2 hours of reperfusion. DMSO (0.1%) or 1938 (5 μM) was administered during the first 15 minutes of perfusion. Left panel: Two representative tetrazolium-stained slices of a heart after ischemia and reperfusion. Red: live tissue; white: infarcted tissue. Representative cardiac electrical activity (measured by ECG) is shown for hearts treated with DMSO or 1938. b. Infarct size measured at the end of 2 hours of reperfusion in hearts treated with DMSO (n = 6) or 1938 (n = 6). c. pAKTS437 in hearts treated with DMSO (n = 5), 1938 (n = 6), or insulin (n = 2). One-way ANOVA with Tukey post-test. d. Effect of 1938 on neuronal regeneration in vitro and in vivo. DRG cultures were stimulated with 1938 in the presence or absence of BYL719 for 72 hours, after which neurite length was measured. Representative images of neurons stained with anti-β-III tubulin after 72 hours are shown. Scale bar = 1000 μm. Quantified data represent the mean ± SEM, n = 3 independent experiments. e. Diagram of sciatic nerve crush injury (i), with arrowheads indicating the resulting lesion (ii). Following injury induction, (iii) a single injection of dH2O or 1938 (2 μl of a 5 μM solution of 1938 in sterile dH2O) was performed proximal to the injury, and (iv) a minipump was implanted for continuous administration of dH2O or 1938 (100 μM solution of 1938 in dH2O at 0.11 μl / h) for 21 days. Histological and functional analyses were performed. f. Motor unit number estimation (MUNE) electrophysiological recording from the tibialis anterior (TA) muscle. g. Compound muscle action potential (CMAP) recording from the TA muscle after nerve stimulation proximal to the crush site. CMAP recovery is shown as a percentage of the contralateral side. h. Total number of choline acetyltransferase (ChAT)-positive motor axons in a cross section of the distal common peroneal nerve innervating the TA muscle. i. Representative immunohistochemistry image of a cross section of the distal common peroneal nerve from a 1938-treated animal, showing ChAT- and neurofilament-positive motor axons with a histology typical of normal tissue. Scale bar = 50 μm.j. Percentage of neuromuscular junctions (NMJs) reinnervated by axons of the target TA muscle, as revealed by double staining for α-bungarotoxin (α-BTX) and neurofilament (NF). k. Representative immunohistochemistry image of the TA muscle from a 1938-treated animal showing postsynaptic NMJ structures and associated neurofilament-positive neurons stained with α-BTX. Scale bar = 20 μm. l. Quantification of total axons (neurofilament) and motor axons (ChAT) in the sciatic nerve 3 mm and 6 mm distal to the injury site at 21 days. CP = location of distal common peroneal nerve cross section. Two-tailed Student's t-test, * = p < 0.05, ** = p < 0.01. All data are for the 21-day endpoint. [Figure 6] Cell death-inducing effect of PI3K activators in lung cancer cells. This shows the ability of PI3K activators to induce cell death in lung cancer cells in the presence and absence of a PI3Kα-selective inhibitor. [Figure 7] Ability of PI3K activators to induce cell death in lung cancer cells. This figure shows the ability of PI3K activators to induce cell death in lung cancer cells in the presence and absence of PI3K pathway inhibitors. [Figure 8] Cell death induction by short-term exposure to PI3Kα activators. The ability of TRO-1938 to induce cell death in H460, H1975, and U87-MG cancer cell lines after short-term exposure is shown. [Figure 9] Extended Data Figure 1. Activation of class IA PI3K isoforms by a range of concentrations of pY, as described in more detail in Example 1. [Figure 10] Extended Data Figure 2. Surface model of full-length p110α / p85α showing changes induced by 1938 assessed by HDX-MS. [Figure 11] In an in vivo model of IRI in mice (left panel), 1938 provides significant cardioprotection, accompanied by increased pAKTS473 levels in the hearts of these mice (right panel). [Figure 12]Extended Data Figure 4. TIRF microscopy data of HeLa cells expressing the EGFP-tagged PIP3 reporter PH-ARNO-1303Ex2 (ARNO) or the PI(3,4)P2 reporter mCherry-cPH-TAPP1x3, as detailed in Example 4. [Figure 13] Extended Data Figure 5. Time course analysis of pAKTS473 and pS6S240 / 44 in MCF10A cells induced with 1938 in the presence and absence of BYL719, as detailed in Example 4. [Figure 14] Extended Data Figure 6. In vitro kinase inhibition profile of 1938 (1 μM) against 133 protein kinases and 7 lipid kinases, as detailed in Example 5. [Figure 15] Extended Data Figure 7. Effect of 1938 on the in vitro kinase activity of the PI3K-related kinases ATM and mTORC1 (mTOR / RAPTOR / LST8 complex), as detailed in Example 5. [Figure 16] Extended Data Figure 8. a. Experimental design and workflow of the phosphoproteomics experiment, as detailed in Example 5. b. Validation of phosphoproteomics conditions, as detailed in Example 5. c. Plot showing how insulin stimulation induces phosphorylation of predicted PI3K targets in PI3Kα-WT MEFs, as detailed in Example 5. d. Plot showing high experimental reproducibility of the phosphoproteomics experiment. [Figure 17] Extended Data Figure 9. Plot showing the effect of stimulating PI3Kα-WT and PI3Kα-KO MEFs with 1938 for 24, 48 or 72 hours, as detailed in Example 6. [Figure 18] Extended Data Figure 10. Western blot showing pAKTS473 induction in primary adult rat cardiomyocytes, as detailed in Example 7. [Figure 19]Extended Data Figure 11. Left. Control experiment examining the biological activity of 1938 after freezing. Right. Blot showing induction of pAKTS473 in exposed sciatic nerves injected with vehicle or 1938, or bathed in vehicle or 1938 solution, as detailed in Example 7. [Figure 20] a. Structural changes induced by 1938 in full-length p110α / p85α assessed by HDX-MS highlighted on the structure of p110α / ninterSH2-p85α (pdb:4ZOP). bi, bii. Crystal structure binding mode of 1938 to p110α. biii. Comparison of 1938 binding in the pocket on p110α, the region of p110β analogous to the p110α pocket, and the analogous pocket of p110δ. biv. Comparative activation of WT and mutant PI3Kα by pY and 1938. The mutant incorporates mutations in p110α residues near the pocket accommodating 1938. [Figure 21] a) MEFs were stimulated with 1938 (5 μM), PDGF (20 ng / ml), or insulin (100 nM) for 2 minutes, followed by lipid extraction and PIP3 measurement by mass spectrometry. b) MEFs were stimulated with increasing amounts of 1938 or PDGF for 2 minutes, followed by lipid extraction and PIP3 measurement by mass spectrometry. c) A549 cells were stimulated with increasing amounts of 1938 or insulin, or 10 ng / ml PDGF for 2 minutes, followed by lipid extraction and PIP3 measurement by mass spectrometry. [Figure 22](Updated version of Figure 1) Biochemical mechanism of PI3Kα activation by UCL-TRO-1938. a. Structure of UCL-TRO-1938 (referred to as 1938 in the text). b. Selectivity of 1938 for PI3Kα over PI3Kβ and PI3Kδ. c. Enzyme kinetics upon ATP titration of PI3Kα with and without 1938 and pY (calculated using the kcat function in Prism 8). d. Membrane binding of PI3Kα shown as FRET signal (I-I0). I: fluorescence intensity at 520 nm; I0: fluorescence intensity at 520 nm in the absence of enzyme. e. Effect of 1938 on PI3Kα catalytic activity in the presence of saturating doses of pY. f. Effect of 1938 on the catalytic activity of oncogenic mutants of PI3Kα. g. Effect of 1938 on membrane binding of oncogenic mutants of PI3Kα. h. Effect of the PI3Kα-selective inhibitor BYL719 on 1938-activated PI3Kα. i. Effect of 1938 on the IC50 of BYL719 against PI3Kα. Data are shown as mean ± SEM, n = 3 (b, c, e, i), n = 2 (d, h), or n = 4 (f, g) independent experiments. Statistical analysis was performed by two-way ANOVA followed by Tukey's multiple comparison test (b, f, g) or Sidak's multiple comparison test (h); one-way ANOVA followed by Dunnett's multiple comparison test (e). MODE FOR CARRYING OUT THE INVENTION

[0018] definition The compounds of the present invention may have asymmetric centers. Compounds of the present invention containing an asymmetrically substituted atom can be isolated in optically active or racemic forms. Methods for preparing optically active forms, such as by resolution of racemates or synthesis from optically active starting materials, are well known in the art. Geometric isomers of double bonds, such as olefinic bonds, may also be present in the compounds described herein, and all such stable isomers are contemplated. Cis and trans geometric isomers of the compounds are described and may be isolated as a mixture of isomers or as separate isomers. Unless a specific stereochemistry or isomeric form is specifically indicated, all chiral, diastereomeric, racemic, and all geometric isomeric forms of a structure are intended. Thus, the compounds of the present invention include all possible chiral, diastereomeric, racemic, and all possible geometric isomers thereof. When no specific reference is made to the configuration (cis, trans, R, S, etc.) of a compound (or an asymmetric carbon), any one isomer or a mixture of two or more isomers is intended. Limitations to particular asymmetric forms of a compound are intended only where explicitly stated.

[0019] In the preparation process, racemates, enantiomers, or diastereomers can be used as starting materials.When enantiomeric or diastereomeric products are prepared, they can be separated by conventional methods, such as chromatography or fractional crystallization.The prepared compounds can be in the form of free form or hydrate.

[0020] The compounds of the present invention include both (a) compounds in which all of the atoms contained therein are in their natural isotopic form ("natural isotopic forms of the compounds"); and (b) compounds in which one or more atoms contained therein are in their non-natural isotopic form ("non-natural isotopic forms of the compounds"). For example, compounds containing isotopic substitution, isotopic enrichment, or removal. Thus, non-natural isotopic forms of the compounds include compounds containing deuterium ( 2 H or D), carbon 11 ( 11 C), carbon-13( 13 C), carbon-14( 14 C), nitrogen 13(13 N), nitrogen-15( 15 N), oxygen-15( 15 O), oxygen-17( 17 O), oxygen-18( 18 O), phosphorus 32 ( 32 P), sulfur 35( 35 S), chlorine 36( 36 Cl), chlorine 37( 37 Cl), fluorine 18( 18 F) Iodine-123( 123 I), iodine-125( 125 It may contain one or more artificial or uncommon isotopes, such as I), or may contain an increased proportion of said isotopes in one or more atoms compared to the proportion predominant in nature.

[0021] Non-naturally occurring variant isotopic forms of compounds comprising radioactive isotopes can be used, for example, in drug and / or substrate tissue distribution studies. The radioactive isotope tritium, i.e. 3 H and carbon-14, i.e. 14 C is particularly useful for this purpose in view of the ease of incorporation and the ease of preparation of detection means. 2 Non-naturally occurring variant isotopic forms incorporating H or D may offer certain therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances. 11 C. 18 F, 15 O and 13 Non-natural variants incorporating positron-emitting isotopes such as N can also be prepared and may be useful in positron emission tomography (PET) studies to examine substrate receptor occupancy.

[0022] The following are definitions of terms used herein. The first definition provided for a group or term herein applies to that group or term throughout the specification, individually or as part of another group, unless otherwise specified. Preferably, the molecular weight of the compounds of the present invention is less than about 500, 550, 600, 650, 700, 750, or 800 grams / mole.

[0023] Preferably, the molecular weight is less than about 800 grams per mole. More preferably, the molecular weight is less than about 750 grams per mole. Even more preferably, the molecular weight is less than about 700 grams per mole.

[0024] As used herein, the term "substituted" means that any one or more hydrogens on the designated atom are replaced with a selection from the indicated group, provided that the normal valence of the designated atom is not exceeded and that the substitution results in a stable compound (e.g., avoiding unstable acetals or similar groups).

[0025] Any variable (e.g., R4, R b When any group (e.g., aryl, aryl, aryl) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is defined as being substituted with 0 to 3 R4, then that group is also optionally substituted with 3 R4 groups, and each occurrence of R4 is independently selected from the definitions of R4. Also, combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0026] When a bond to a substituent is shown to cross the bond connecting two atoms in a ring, then such substituent may be bonded to any atom on the ring. When a substituent is listed without indicating the atom to which it is attached to the remainder of the compound of a given formula, then the substituent may be bonded through any atom in that substituent. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0027] As used herein, "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, "C1-C6 alkyl" is intended to include C1, C2, C3, C4, C5, and C6 alkyl groups. Further, for example, "C1-C6 alkyl" refers to an alkyl having 1 to 6 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, n-hexyl, 2-methylbutyl, 2-methylpentyl, 2-ethylbutyl, 3-methylpentyl, and 4-methylpentyl.

[0028] The term "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human beings and animals, particularly human tissues, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0029] The compounds of the present invention include pharmaceutically acceptable salts, particularly pharmaceutically acceptable salts of compounds of Formula (I), as well as solvates, N-oxides, and tautomers thereof. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds, in which the parent compound (e.g., of Formula (I)) is modified by making a pharmaceutically acceptable acid or base salt thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic groups such as amines; and alkali or organic acid salts of acidic groups such as carboxylic acids. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; and organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, and the like.

[0030] The pharmaceutically acceptable salts of the present invention can be synthesized from a parent compound (e.g., of Formula (I)) that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water, an organic solvent (generally, a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile is preferred), or a mixture thereof. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, p. 1418, the disclosure of which is incorporated herein by reference.

[0031] Non-pharmaceutically acceptable salt forms of the compounds of the present invention may be used in the preparation of the non-salt form or the pharmaceutically acceptable salt form. Thus, the present invention also encompasses non-pharmaceutically acceptable salts of the compounds of formula (I).

[0032] The compounds of the present invention include solvates (particularly solvates of the compounds of formula (I), as well as salts, N-oxides and tautomers thereof). Solvates include hydrates, and are preferably hydrates. Solvation methods are generally known in the art.

[0033] The compounds of the present invention include tautomers (particularly tautomers of the compounds of formula (I), as well as salts and solvates thereof). Some compounds of the present invention exist in multiple tautomers in which hydrogen atoms are transposed to other parts of the molecule, resulting in a rearrangement of the chemical bonds between the atoms of the molecule. It should be understood that all tautomers, to the extent that they may exist, are included in the compounds of the present invention.

[0034] When nitrogen atoms (e.g., amines) are present on compounds of the invention (e.g., of formula (I)), they can be converted to N-oxides by treatment with an oxidizing agent (e.g., MCPBA and / or hydrogen peroxide) to provide other compounds of the invention. Thus, all depicted nitrogen atoms are considered to cover both the depicted nitrogen and its N-oxide (N→O) derivative. Thus, the compounds of the invention encompass N-oxides, particularly N-oxides of compounds of formula (I), as well as salts, solvates, and tautomers thereof.

[0035] For the avoidance of doubt, many compounds of the present invention may simultaneously exist in more than one form: tautomers, N-oxides, pharmaceutically acceptable salts, and solvates of a particular parent compound (e.g., compounds of Formula (I)), and all such possible compounds are encompassed within the definition of "compounds of the present invention." Accordingly, compounds of the present invention (explicitly, to the extent chemically possible in view of the associated Formula (I)) include any one of the following: (1) compounds of Formula (I) (which may also be referred to herein as the "free base form" of the compound); (2) tautomers of Formula (I); (3) N-oxides of Formula (I); (4) pharmaceutically acceptable salts of Formula (I); (5) solvates of Formula (I); (6) N-oxides of tautomers of Formula (I); (7) pharmaceutically acceptable salts of tautomers of Formula (I); (8) solvates of tautomers of Formula (I); (9) compounds of Formula (I) (10) a solvate of the N-oxide of the tautomer of formula (I); (11) a solvate of a pharmaceutically acceptable salt of the tautomer of formula (I); (12) a solvate of a pharmaceutically acceptable salt of the N-oxide of the tautomer of formula (I); (13) a pharmaceutically acceptable salt of the N-oxide of formula (I); (14) a solvate of the N-oxide of formula (I); (15) a solvate of a pharmaceutically acceptable salt of the N-oxide of the tautomer of formula (I); and (16) a solvate of a pharmaceutically acceptable salt of formula (I).

[0036] After their preparation, the compounds of the present invention are preferably isolated and purified to obtain compositions containing greater than or equal to 99% by weight of the compound ("substantially pure"), which can then be used or formulated as described herein. Such "substantially pure" compounds are also part of the present invention.

[0037] By "stable compound" and "stable structure" is meant a compound that is sufficiently robust to be isolated to a useful degree of purity from a reaction mixture and formulated into an efficacious therapeutic agent.

[0038] Purely for the avoidance of doubt, it should be noted that wherever a compound is disclosed as being of a particular structure (e.g., a particular general chemical formula such as Formula (I)), or any of its tautomers, N-oxides, pharmaceutically acceptable salts, or solvates, further disclosed embodiments of that compound (e.g., "a compound according to...") continue to encompass such tautomers, N-oxides, pharmaceutically acceptable salts, or solvates, unless expressly stated to the contrary, and reciting additional limitations to the recited structure will continue to encompass such tautomers, N-oxides, pharmaceutically acceptable salts, or solvates.

[0039] As used herein, "treating and / or preventing" or "treatment and / or prevention" of a disease state in a mammal, particularly a human, includes: (a) preventing the disease state from occurring in a mammal, particularly when the mammal is susceptible to the disease state but has not yet been diagnosed as having the disease state; (b) inhibiting the disease state, i.e., slowing or preventing its onset; and / or (c) alleviating the disease state, i.e., causing regression of the disease state or a reduction in associated symptoms.

[0040] The term "therapeutically effective amount" is intended to include an amount of a compound effective to achieve a desired effect in the treatment and / or prevention of a disease state. A desired effect is typically a clinically significant and / or measurable effect, such as (a) preventing the occurrence of a disease state in a mammal, particularly when the mammal is susceptible to the disease state but has not yet been diagnosed with the disease state; (b) inhibiting the disease state, i.e., delaying or preventing its onset; and / or (c) alleviating the disease state, i.e., causing regression of the disease state or the alleviation of associated symptoms. A therapeutically effective amount can be an amount sufficient to achieve a desired effect when the compound is administered alone or, alternatively, when administered in combination with one or more additional APIs that are additional compounds of the invention or that are different from the compounds of the invention. Furthermore, a therapeutically effective amount is typically an amount sufficient to activate PI3Kα, and also an amount sufficient to activate PI3Kα when administered alone or in combination with one or more additional APIs (which may also activate PI3Kα or, alternatively, may exert their pharmacological effects through a different mechanism). Thus, a "therapeutically effective amount" is intended to include an amount of a combination of compounds, each a compound of the invention, that is effective to activate PI3K alpha. The combination of compounds is preferably a synergistic combination.

[0041] Synergy occurs when the effect of compounds administered in combination (in this case, activation of PI3Kα) is greater than the additive effect of the compounds administered alone as single agents, as described, for example, in Chou and Talalay, Adv. Enzyme Regul. 1984, 22: 27-55. Generally, synergy is most evident when the concentrations of the compounds are suboptimal. Synergy can be characterized by lower cytotoxicity or other beneficial effects of the combination compared to the individual components.

[0042] For the avoidance of doubt, the "therapeutically effective amount" described herein can be achieved by any suitable administration regimen, including, but not limited to, the exemplary administration regimens described elsewhere herein. Thus, for example, references herein to administering a therapeutically effective amount of a compound by a particular route of administration include achieving that therapeutically effective amount by a single dose or by multiple doses administered by the specified route of administration. For example, orally administering a therapeutically effective amount includes orally administering a single dose or any multiple doses, provided that the therapeutically effective amount is achieved by oral administration.

[0043] The present invention further includes compositions comprising one or more compounds of the present invention and one or more pharmaceutically acceptable carriers.

[0044] "Pharmaceutically acceptable carrier" refers to a vehicle generally recognized in the art for delivering a biologically active agent to an animal, particularly a mammal. Pharmaceutically acceptable carriers are formulated according to many factors well within the purview of those skilled in the art. These include, but are not limited to, the type and nature of the active agent being formulated; the subject to whom the agent-containing composition will be administered; the intended route of administration of the composition; and the targeted therapeutic indication. Pharmaceutically acceptable carriers include aqueous and non-aqueous liquid media, as well as various solid and semi-solid dosage forms. Such carriers may contain many different components and additives in addition to the active agent; such additional components are included in the formulation for various reasons well known to those skilled in the art, such as for stabilizing the active agent, as a binder, etc. Descriptions of suitable pharmaceutically acceptable carriers and the factors involved in their selection can be found in a variety of readily available sources, such as Remington's Pharmaceutical Sciences, 17th ed., 1985, which is incorporated herein by reference in its entirety. DETAILED DESCRIPTION OF THE INVENTION

[0045] Compounds of the Invention The present invention provides (a) a compound of formula (I), or (b) a tautomer, N-oxide, pharmaceutically acceptable salt, or solvate thereof: [ka] During the ceremony: X is a bond or NH; Y is a bond or NH; provided that at least one of X and Y is NH; R 1 is H, F or CH3; R 2 are H, F, Cl, Br, -COR 4 , -SO2R 5 , -SOR 5 , -CN, -NO, -NO2 or -NR 6 3 + and; R 3 H, CH3, 0-3 R 7 , -COR 4 , -SO2R 5 , -SOR 5 , -CN, -NO, -NO2 or -NR 6 3 + is a C2-C6 alkyl substituted with; R 4 H, 0 to 3 R 7 , -OH, -OR 8 , -NH2, -NHR 8 or -NR 8 C1-C6 alkyl substituted with 2; R 5 is an R from 0 to 3 7 , -OH, -OR 8 , -NH2, -NHR 8 or -NR 8 C1-C6 alkyl substituted with 2; R 6 are independently selected from C1-C3 alkyl; R 7 is independently selected from O-C1-C3 alkyl, F, or Cl; R 8is an R from 0 to 3 7 C1-C6 alkyl substituted with; Ring A is selected from rings in Group I, Group II, Group III, Group IV and Group V; * indicates a bond to X; Group I is Group I-1, i.e., [ka] and: Group II is Group II-1, i.e., [ka] and: Group III is Group III-1, i.e., [ka] and: Group IV is Group IV-1, i.e., [ka] JPEG2024535128000007.jpg220170JPEG2024535128000008.jpg198170 Group V is group V-1, i.e., [ka] and: Ring B is selected from Group IA, Group IIA, Group IIA, Group IVA, and Group VA; $ indicates the bond to Y; Group IA is group IA-1, i.e. [ka] and: Group IIA is Group IIA-1, i.e., [ka] and: Group IIIA includes Group IIIA-1, i.e., [ka] and: Group IVA is Group IVA-1, i.e., [ka] and: The group VA is the group VA-1, i.e. [ka] JPEG2024535128000015.jpg167170: During the ceremony: Q and T are each selected from CH or N, provided that at most one of Q and T may be N; V is CH or N; W is CH2, O, NR y , S, S(O) or S(O)2; Z is C(O), S(O), or S(O); R a is C1-C6 alkyl substituted with 0-3 substituents independently selected from O-C1-C3 alkyl, F and Cl; R b are independently selected from C1-C6 alkyl, F and Cl; R c is C1-C6 alkyl substituted with 0-3 substituents independently selected from O-C1-C3 alkyl, F and Cl; R d is independently selected from C1-C6 alkyl substituted with 0-3 substituents independently selected from O-C1-C3 alkyl, F, and Cl; or phenyl substituted with 0-3 substituents independently selected from O-C1-C3 alkyl, F, and Cl; R e is C1-C6 alkyl substituted with 0-3 substituents independently selected from O-C1-C3 alkyl, F and Cl; R f is C1-C6 alkyl substituted with 0-3 substituents independently selected from O-C1-C3 alkyl, F and Cl; R g is H or C1-C3 alkyl; R h is C1-C6 alkyl substituted with 0-3 substituents independently selected from O-C1-C3 alkyl, F and Cl; R i is C1-C6 alkyl substituted with 0-3 substituents independently selected from O-C1-C3 alkyl, F and Cl; R j is H or C1-C3 alkyl R k is C1-C6 alkyl substituted with 0-3 substituents independently selected from O-C1-C3 alkyl, F and Cl; R l is H or C1-C3 alkyl; R m is C1-C6 alkyl substituted with 0-3 substituents independently selected from O-C1-C3 alkyl, F and Cl; R n is H or C1-C3 alkyl R o is independently selected from C1-C6 alkyl substituted with 0-3 substituents independently selected from O-C1-C3 alkyl, F, and Cl; or phenyl substituted with 0-3 substituents independently selected from O-C1-C3 alkyl, F, and Cl; R p are independently selected from C1-C6 alkyl, F, Cl and Br R q is C1-C6 alkyl substituted with 0-3 substituents independently selected from O-C1-C3 alkyl, F and Cl; R r is H or C1-C6 alkyl substituted with 0-3 substituents independently selected from O-C1-C3 alkyl, F and Cl; Rs is H or C1-C3 alkyl R t is C1-C6 alkyl substituted with 0-3 substituents independently selected from O-C1-C3 alkyl, F and Cl; R u are independently selected from C1-C6 alkyl, F and Cl; R v is phenyl substituted with 0-2 substituents selected from C1-C6 alkyl, F and Cl; R w is H or C1-C3 alkyl; R x are independently selected from H or C1-C3 alkyl; R y is C1-C6 alkyl substituted with 0-3 substituents independently selected from H, O-C1-C3 alkyl, F, and Cl; benzyl substituted with 0-3 substituents independently selected from C1-C6 alkyl, F, and Cl; or C3-C6 cycloalkyl substituted with 0-3 substituents independently selected from C1-C6 alkyl, O-C1-C3 alkyl, F, and Cl; n is 1 to 3; p is 0 to 2; q is 1 or 2; r is 0 to 2; s is 1 to 3; t is 0 to 2; u is 2 to 3; v is 1 to 3; and: X is NH; Y is NH; R 2 is H; and when ring A is a ring in group I or group II: ring B is a ring in group IA, group IIA, group IIIA, group IVA or group VA X is NH; Y is NH; R 2 is H; and when ring A is a ring in group III: ring B is a ring in group IA, group IIA or group IIIA; X is NH; Y is NH; R 2is H; and when ring A is a ring in group IV: ring B is a ring in group IA or group IIA; X is NH; Y is NH; R 2 is H; and ring A is a ring in group V: ring B is a ring in group IA; X is NH; Y is NH; R 2 F, Cl, Br, -COR 4 , -SO2R 5 , -SOR 5 , -CN, -NO, -NO2 or -NR 6 3 + when ring A is a ring in group I or group II and ring B is a ring in group IA; When X is NH and Y is NH: R 2 is H; ring A is a ring within Group I, Group II and Group III; and ring B is a ring within Group IA; and When X is NH and Y is a bond: R 2 is H; ring A is a ring within Group I, Group II and Group III; and ring B is a ring within Group IA, Group IIA, and Group IIIA; and with the proviso that the compound of formula (I) is not (a) a compound selected from the group consisting of: [ka]

[0046] In a preferred embodiment, X is NH and Y is NH.

[0047] In another embodiment, X is a bond and Y is NH. In yet another embodiment, X is NH and Y is a bond.

[0048] Preferably, R 1 is H or F. For example, R 1 is H. Alternatively, R 1 is F.

[0049] Preferably, R2 are H, F, Cl, Br, -COR 4 , -SO2R 5 , -CN, -NO2 or -NR 6 3 + More preferably, R 2 are H, F, Cl, Br, -COR 4 , -SO2R 5 or -CN. More preferably, R 2 is H, F, Cl, or Br. Even more preferably, R 2 is H or F. Most preferably, R 2 is H.

[0050] Preferably, R 3 H, CH3, 0-3 R 7 C2-C6 alkyl substituted with -COR 4 , -SO2R 5 , -CN, -NO2 or -NR 6 3 + More preferably, R 3 H, CH3, 0-3 R 7 C2-C6 alkyl substituted with -COR 4 , -SO2R 5 or -CN. Even more preferably, R 3 is H, CH3, or 0-3 R 7 More preferably, R 3 is H, CH, or C-C alkyl. Even more preferably, R 3 is H or CH3. Most preferably, R 3 is H.

[0051] Preferably, R 4 -OR 8 , -NH2, -NHR 8 or -NR 8 2 are independently selected.

[0052] Preferably, R 5 -OR 8 , -NH2, -NHR8 or -NR 8 2 are independently selected.

[0053] Preferably, R 6 are independently selected from C1-C2 alkyl. Most preferably, R 6 is CH3.

[0054] R with 0 to 3 alkyl groups 7 If replaced by R 7 The number of groups is 0 (i.e., the alkyl group is unsubstituted), 1, 2, or 3, preferably 0, 1, or 2, more preferably 0 or 1, and most preferably 0 (i.e., the alkyl group is unsubstituted).

[0055] Preferably, R 7 is independently selected from O-C1-C2 alkyl, F or Cl. More preferably, R 7 are independently selected from OCH3, F or Cl.

[0056] Preferably, R 8 is an R from 0 to 3 7 More preferably, R 8 are independently selected from C1-C3 alkyl. More preferably, R 8 are independently selected from C1-C2 alkyl. Most preferably, R 8 is CH3.

[0057] So, in the preferred combination (combination A1): R 2 are H, F, Cl, Br, -COR 4 , -SO2R 5 , or -CN; R 3 H, CH3, 0-3 R 7 C2-C6 alkyl substituted with -COR 4 , -SO2R 5 , or -CN; R 4and R 5 -OR 8 , -NH2, -NHR 8 or -NR 8 2 are independently selected from R 6 are independently selected from C1-C2 alkyl; R 7 are independently selected from OCH3, F, or Cl; and R 8 are independently selected from C1-C3 alkyl.

[0058] A more favorable combination (combination A2) is: R 2 is H, F, Cl, or Br; and R 3 is H, CH3 or C2-C6 alkyl.

[0059] An even more favorable combination (combination A3) is: R 2 is H or F; and R 3 is H or CH3.

[0060] An even more preferred combination (combination A3): R 2 is H or F; And R 3 is H.

[0061] In the compounds of formula (I), in a particularly preferred embodiment for ring A, group I is group I-2, i.e., [ka] is.

[0062] More preferably, Group I is Group I-3, i.e. [ka] is.

[0063] Most preferably, Group I is Group I-4, i.e. [ka] is.

[0064] In some embodiments, Group II is Group II-2, i.e., [ka] is.

[0065] In another embodiment, Group II is Group II-2', i.e. [ka] is.

[0066] In a preferred embodiment, Group II is Group II-3, i.e. [ka] is.

[0067] In a particularly preferred embodiment, Group II is Group II-4, i.e. [ka] is.

[0068] More preferably, Group II is Group II-5, i.e. [ka] is.

[0069] Most preferably, Group II is Group II-6, i.e. [ka] is.

[0070] In one embodiment, Group III is Group III-2, i.e., [ka] is.

[0071] In another embodiment, Group III is Group III-3, i.e., [ka] is.

[0072] In a preferred embodiment, Group III is Group III-4, i.e. [ka] is.

[0073] In a particularly preferred embodiment, Group III is Group III-5, i.e. [ka] is.

[0074] In a most preferred embodiment, Group III is Group III-6, i.e. [ka] is.

[0075] In some embodiments, Group IV is Group IV-2, i.e., [ka] JPEG2024535128000032.jpg208170JPEG2024535128000033.jpg77170.

[0076] In a preferred embodiment, Group IV is Group IV-3, i.e. [ka] JPEG2024535128000035.jpg75170.

[0077] In a preferred embodiment, Group IV is Group IV-4, i.e. [ka] is.

[0078] In a particularly preferred embodiment, Group IV is Group IV-5, i.e. [ka] is.

[0079] In a most preferred embodiment, Group IV is Group IV-6, i.e. [ka] is.

[0080] In a preferred embodiment, Group V is Group V-2, i.e., [ka] is.

[0081] In the most preferred embodiment, Group V is Group V-3, i.e. [ka] is.

[0082] In the compounds of formula (I), for ring B, in a particularly preferred embodiment, group IA is group IA-2, i.e. [ka] is.

[0083] Most preferably, Group IA is Group IA-3, i.e. [ka] is.

[0084] In a preferred embodiment, Group IIA is Group IIA-2, i.e. [ka] is.

[0085] In a particularly preferred embodiment, Group IIA is Group IIA-3, i.e. [ka] is.

[0086] Most preferably, Group IIA is Group IIA-4, i.e. [ka] is.

[0087] In some embodiments, Group IIIA is Group IIIA-2, i.e., [ka] is.

[0088] In a preferred embodiment, Group IIIA is Group IIIA-3, i.e. [ka] is.

[0089] Most preferably, Group IIIA is Group IIIA-4, i.e. [ka] is.

[0090] In some embodiments, Group IVA is Group IVA-2, i.e., [ka] is.

[0091] In a preferred embodiment, Group IVA is Group IVA-3, i.e. [ka] is.

[0092] In a particularly preferred embodiment, Group IVA is Group IVA-4, i.e. [ka] is.

[0093] In a particularly preferred embodiment, Group IVA is Group IVA-5, i.e. [ka] is.

[0094] In a most preferred embodiment, Group IVA is Group IVA-6, i.e. [ka] is.

[0095] In some embodiments, group VA is group VA-2, i.e., [ka] JPEG2024535128000055.jpg114170.

[0096] In a preferred embodiment, group VA is group VA-3, i.e. [ka] JPEG2024535128000057.jpg107170.

[0097] In a more preferred embodiment, group VA is group VA-4, i.e. [ka] JPEG2024535128000059.jpg131170.

[0098] Most preferably, group VA is group VA-5, i.e. [ka] is.

[0099] Therefore, a preferred combination (combination B1) is as follows: Group I is Group I-1; Group II is Group II-1; Group III is Group III-1; Group IV is Group IV-2; Group V, if present, is group V-2; Group IA is Group IA-1; Group IIA is Group IIA-1; Group IIIA is Group IIIA-1; Group IVA is Group IVA-1; and Group VA, if present, is group VA-2.

[0100] Another preferred combination (combination B2) is: Group I is Group I-1; Group II is Group II-1; Group III is Group III-1; Group IV is Group IV-3; Group V, if present, is group V-2; Group IA is Group IA-1; Group IIA is Group IIA-1; Group IIIA is Group IIIA-1; Group IVA is Group IVA-1; and Group VA, if present, is group VA-3.

[0101] Another preferred combination (combination B3) is: Group I is Group I-1; Group II is Group II-1; Group III is Group III-1; Group IV is Group IV-4; Group V, if present, is group V-2; Group IA is Group IA-1; Group IIA is Group IIA-1; Group IIIA is Group IIIA-1; Group IVA is Group IVA-1; and Group VA, if present, is group VA-3.

[0102] Another preferred combination (combination B4) is: Group I is Group I-1; Group II is Group II-1; Group III is Group III-1; Group IV is Group IV-5; Group V, if present, is group V-2; Group IA is Group IA-1; Group IIA is Group IIA-1; Group IIIA is Group IIIA-1; Group IVA is Group IVA-1; and Group VA, if present, is group VA-3.

[0103] Another preferred combination (combination B5) is: Group I is Group I-1; Group II is Group II-2; Group III is Group III-2; Group IV is Group IV-5; Group V, if present, is group V-2; Group IA is Group IA-1; Group IIA is Group IIA-2; Group IIIA is Group IIIA-2; Group IVA is Group IVA-2; and Group VA, if present, is group VA-3.

[0104] Another preferred combination (combination B6) is: Group I is Group I-1; Group II is Group II-2'; Group III is Group III-2; Group IV is Group IV-5; Group V, if present, is group V-2; Group IA is Group IA-1; Group IIA is Group IIA-2; Group IIIA is Group IIIA-2; Group IVA is Group IVA-2; and Group VA, if present, is group VA-3.

[0105] Another preferred combination (combination B7) is: Group I is Group I-1; Group II is Group II-3; Group III is Group III-3; Group IV is Group IV-5; Group V, if present, is group V-2; Group IA is Group IA-1; Group IIA is Group IIA-3; Group IIIA is Group IIIA-2; Group IVA is Group IVA-3; and Group VA, if present, is group VA-3.

[0106] Another preferred combination (combination B8) is: Group I is Group I-1; Group II is Group II-3; Group III is Group III-4; Group IV is Group IV-5; Group V, if present, is group V-2; Group IA is Group IA-1; Group IIA is Group IIA-3; Group IIIA is Group IIIA-2; Group IVA is Group IVA-4; and Group VA, if present, is group VA-3.

[0107] Another preferred combination (combination B9) is: Group I is Group I-1; Group II is Group II-3; Group III is Group III-4; Group IV is Group IV-5; Group V, if present, is group V-2; Group IA is Group IA-1; Group IIA is Group IIA-3; Group IIIA is Group IIIA-2; Group IVA is Group IVA-4; and Group VA, if present, is group VA-3.

[0108] Another preferred combination (combination B10) is: Group I is Group I-2; Group II is Group II-4; Group III is Group III-5; Group IV is Group IV-6; Group V, if present, is group V-2; Group IA is Group IA-2; Group IIA is Group IIA-4; Group IIIA is Group IIIA-3; Group IVA is Group IVA-5; and Group VA, if present, is group VA-3.

[0109] Another preferred combination (combination B11) is: Group I is Group I-3; Group II is Group II-5; Group III is Group III-5; Group IV is Group IV-6; Group V, if present, is group V-3; Group IA is Group IA-3; Group IIA is Group IIA-4; Group IIIA is Group IIIA-3; Group IVA is Group IVA-5; and Group VA, if present, is group VA-4.

[0110] Another preferred combination (combination B12) is: Group I is Group I-4; Group II is Group II-6; Group III is Group III-6; Group IV is Group IV-6; Group V, if present, is group V-3; Group IA is Group IA-3; Group IIA is Group IIA-4; Group IIIA is Group IIIA-4; Group IVA is Group IVA-6; and Group VA, if present, is group VA-5.

[0111] In the compound of formula (I), each substituent on ring A and ring B can be more narrowly defined as follows.

[0112] Preferably, Q is selected from CH or N.

[0113] Preferably, T is CH.

[0114] Preferably, W is CH, O, NR y or S(O)2. Most preferably, W is CH2, O or NR y is.

[0115] Preferably, Z is C(O) or S(O). Most preferably, Z is C(O).

[0116] Preferably, R a is C1-C3 alkyl substituted with 0 to 1 substituents selected from O-C1-C3 alkyl, F and Cl. More preferably, R a is C1-C3 alkyl substituted with 0 to 1 substituents selected from O-C1-C3 alkyl. Most preferably, R a is CH3, CH2CH3, or CH2OCH3.

[0117] Preferably, R b are independently selected from C1-C3 alkyl, F and Cl. More preferably, R b are independently selected from C1-C3 alkyl. Most preferably, R b is CH3.

[0118] Preferably, R c is C1-C3 alkyl substituted with 0 to 3 substituents independently selected from O-C1-C3 alkyl, F, and Cl. More preferably, R c is C1-C3 alkyl. Most preferably, R c is CH3.

[0119] Preferably, R d is independently selected from C1-C4 alkyl substituted with 0-1 substituents independently selected from O-C1-C3 alkyl, F, and Cl; or phenyl substituted with 0-1 substituents independently selected from O-C1-C3 alkyl, F, and Cl. More preferably, R d is independently selected from C1-C4 alkyl or phenyl. Most preferably, R d is independently selected from CH(CH3)2 or C(CH3)3.

[0120] Preferably, R e is C1-C3 alkyl substituted with 0 to 1 substituents independently selected from O-C1-C3 alkyl, F, and Cl. More preferably, Re is C1-C3 alkyl. Most preferably, R e is CH3.

[0121] Preferably, R f is C1-C3 alkyl substituted with 0 to 1 substituents independently selected from O-C1-C3 alkyl, F, and Cl. More preferably, R f is C1-C3 alkyl. Even more preferably, R f is CH3, CH2CH3, or CH(CH3)2.

[0122] Preferably, R g is H or CH3. Most preferably, R g is H.

[0123] Preferably, R h is C1-C3 alkyl substituted with 0 to 1 substituents independently selected from O-C1-C3 alkyl, F, and Cl. More preferably, R h is C1-C3 alkyl. Most preferably, R h is CH3.

[0124] Preferably, R i is C1-C3 alkyl substituted with 0 to 1 substituents independently selected from O-C1-C3 alkyl, F, and Cl. More preferably, R i is C1-C3 alkyl. Most preferably, R i is CH3.

[0125] Preferably, R j is H or CH3. Most preferably, R j is H.

[0126] Preferably, R k is C1-C3 alkyl substituted with 0 to 1 substituents independently selected from O-C1-C3 alkyl, F, and Cl. More preferably, R k is C1-C3 alkyl. Most preferably, Rk is CH3.

[0127] Preferably, R l is H or CH3. Most preferably, R l is H.

[0128] Preferably, R m is C1-C4 alkyl substituted with 0 to 1 substituents independently selected from O-C1-C3 alkyl, F, and Cl. More preferably, R m is C1-C4 alkyl. Even more preferably, R m is C1-C3 alkyl. Most preferably, R m is CH3.

[0129] Preferably, R n is H or CH3. Most preferably, R n is H.

[0130] Preferably, R o is independently selected from C1-C3 alkyl substituted with 0-1 substituents independently selected from O-C1-C3 alkyl, F, and Cl; or phenyl substituted with 0-1 substituents independently selected from O-C1-C3 alkyl, F, and Cl. More preferably, R o is independently selected from O-C1-C3 alkyl, C1-C3 alkyl substituted with 0-1 substituents independently selected from F and Cl; or phenyl. Even more preferably, R o is independently selected from C1-C3 alkyl substituted with 0-1 substituents independently selected from O-C1-C3 alkyl; or phenyl. More preferably, R o is independently selected from C1-C3 alkyl or phenyl. Even more preferably, R o are independently selected from C1-C3 alkyl. Most preferably, R o are independently selected from CH3, CH2CH3, or CH(CH3)2.

[0131] Preferably, R p are independently selected from C1-C3 alkyl, F, Cl and Br. More preferably, R p are independently selected from C1-C3 alkyl. Most preferably, R p is CH3.

[0132] Preferably, R q is C1-C3 alkyl substituted with 0 to 1 substituents independently selected from O-C1-C3 alkyl, F, and Cl. More preferably, R q is C1-C3 alkyl substituted with 0-1 substituents independently selected from O-C1-C3 alkyl. Most preferably, R q is CH3, CH2CH3, or CH2OCH3.

[0133] Preferably, R r is H or C1-C3 alkyl substituted with 0-1 substituents independently selected from O-C1-C3 alkyl, F and Cl. More preferably, R r is H or C1-C3 alkyl. Even more preferably, R r is C1-C3 alkyl. Most preferably, R r is CH3.

[0134] Preferably, R s is H or CH3. Most preferably, R s is H.

[0135] Preferably, R t is C1-C4 alkyl substituted with 0 to 1 substituents independently selected from O-C1-C3 alkyl, F, and Cl. More preferably, R t is C1-C4 alkyl. Even more preferably, R t is C3-C4 alkyl. Most preferably, R t is C(CH3)3.

[0136] Preferably, R uare independently selected from C1-C3 alkyl, F and Cl. More preferably, R u are independently selected from C1-C3 alkyl. Most preferably, R u is CH3.

[0137] Preferably, R v is phenyl substituted with 0 to 1 substituents selected from C1-C3 alkyl, F and Cl. More preferably, R v is phenyl substituted with 0 to 1 substituents selected from C1-C3 alkyl. Most preferably, R v is phenyl.

[0138] Preferably, R w is H or CH3. Most preferably, R w is H.

[0139] Preferably, R x is independently selected from H or CH3. Most preferably, R x is H.

[0140] Preferably, R y is C1-C3 alkyl substituted with 0-1 substituents selected from H, O-C1-C3 alkyl, F, and Cl; benzyl substituted with 0-1 substituents selected from C1-C3 alkyl, F, and Cl; or C3-C5 cycloalkyl substituted with 0-1 substituents selected from C1-C3 alkyl, O-C1-C3 alkyl, F, and Cl. More preferably, R y is H, C1-C3 alkyl substituted with 0-1 substituents selected from O-C1-C3 alkyl; benzyl substituted with 0-1 substituents selected from C1-C3 alkyl; or C3-C5 cycloalkyl substituted with 0-1 substituents selected from C1-C3 alkyl. Even more preferably, R yis C1-C3 alkyl substituted with 0 to 1 substituents selected from O-C1-C3 alkyl; benzyl substituted with 0 to 1 substituents selected from C1-C3 alkyl; or C3-C5 cycloalkyl substituted with 0 to 1 substituents selected from C1-C3 alkyl. More preferably, R y is C1-C3 alkyl substituted with 0-1 substituents selected from O-C1-C3 alkyl; benzyl; or cyclopropyl. Even more preferably, R y is C1-C3 alkyl substituted with 0-1 substituents selected from O-C1-C3 alkyl; or cyclopropyl. More preferably, R y is C1-C3 alkyl. Most preferably, R y is CH3, CH2CH3, or CH(CH3)2.

[0141] Preferably, n is 2 or 3. Most preferably, n is 2.

[0142] Preferably, p is 0 or 1. More preferably, p is 0.

[0143] Preferably, q is 1.

[0144] Preferably, r is 1 or 2. Most preferably, r is 2.

[0145] Preferably, s is 1 or 2. Most preferably, s is 2.

[0146] Preferably, t is 0 or 1. More preferably, t is 0.

[0147] Preferably, u is 3.

[0148] Preferably, v is 2 or 3. Most preferably, v is 2.

[0149] So, in the preferred combination (combination C1): Q is CH or N; T is CH; W is CH2, O, NR y , or S(O)2; Z is C(O) or S(O)2; R a is C1-C3 alkyl substituted with 0-1 substituents selected from O-C1-C3 alkyl, F and Cl; R b are independently selected from C1-C3 alkyl, F and Cl; R c is C1-C3 alkyl substituted with 0-3 substituents independently selected from O-C1-C3 alkyl, F and Cl; R d is independently selected from C1-C4 alkyl substituted with 0-1 substituents independently selected from O-C1-C3 alkyl, F, and Cl; or phenyl substituted with 0-1 substituents independently selected from O-C1-C3 alkyl, F, and Cl; R e is C1-C3 alkyl substituted with 0 to 1 substituents independently selected from O-C1-C3 alkyl, F, and Cl; R f is C1-C3 alkyl substituted with 0-1 substituents independently selected from O-C1-C3 alkyl, F and Cl; R g is H or CH3; R h is C1-C3 alkyl substituted with 0-1 substituents independently selected from O-C1-C3 alkyl, F and Cl; R i is C1-C3 alkyl substituted with 0-1 substituents independently selected from O-C1-C3 alkyl, F and Cl; R j is H or CH3; R k is C1-C3 alkyl having 0-1 substituents independently selected from O-C1-C3 alkyl, F, and Cl; Rl is H or CH3C1-C3 alkyl; R m is C1-C4 alkyl substituted with 0-1 substituents independently selected from O-C1-C3 alkyl, F and Cl; R n is H or CH3; R o is independently selected from C1-C3 alkyl substituted with 0-1 substituents independently selected from O-C1-C3 alkyl, F, and Cl; or phenyl substituted with 0-1 substituents independently selected from O-C1-C3 alkyl, F, and Cl; R p are independently selected from C1-C3 alkyl, F, Cl, and Br; R q is C1-C3 alkyl substituted with 0-1 substituents independently selected from O-C1-C3 alkyl, F and Cl; R r is H or C1-C3 alkyl substituted with 0-1 substituents independently selected from O-C1-C3 alkyl, F and Cl; R s is H or CH3; R t is C1-C4 alkyl substituted with 0-1 substituents independently selected from O-C1-C3 alkyl, F and Cl; R u are independently selected from C1-C3 alkyl, F and Cl; R v is phenyl substituted with 0-1 substituents selected from C1-C3 alkyl, F and Cl; R w is H or CH3; R x is independently selected from H or CH3; R yis C1-C3 alkyl substituted with 0-1 substituents selected from H, O-C1-C3 alkyl, F, and Cl; or benzyl substituted with 0-1 substituents selected from C1-C3 alkyl, F, and Cl; or C3-C5 cycloalkyl substituted with 0-1 substituents selected from C1-C3 alkyl, O-C1-C3 alkyl, F, and Cl; n is 2 or 3; p is 0 or 1; q is 1; r is 1 or 2; s is 1 or 2; t is 0 or 1; u is 2 or 3; and v is 2 or 3.

[0150] A more favorable combination (combination C2) is: Q and T are as defined in combination C1; W is CH2, O or NR y and; Z is C(O); R a is C1-C3 alkyl substituted with 0-1 substituents selected from O-C1-C3 alkyl; R b are independently selected from C1-C3 alkyl; R c is C1-C3 alkyl; R d is independently selected from C1-C4 alkyl or phenyl; R e is C1-C3 alkyl; R f is C1-C3 alkyl; R g is H; R h is C1-C3 alkyl; R i is C1-C3 alkyl; R j is H; Rk is C1-C3 alkyl; R l is H; R m is C1-C4 alkyl; R n is H; R o is independently selected from C1-C3 substituted with 0-1 substituents independently selected from O-C1-C3 alkyl, F, and Cl; or phenyl; R p are independently selected from C1-C3 alkyl; R q is C1-C3 alkyl substituted with 0-1 substituents selected from O-C1-C3 alkyl; R r is H or C1-C3 alkyl; R s is H or CH3; R t is C1-C4 alkyl; R u are independently selected from C1-C3 alkyl; R v is phenyl substituted with 0-1 substituents selected from C1-C3 alkyl; R w is H; R x is H; and R y is H, C1-C3 alkyl substituted with 0-1 substituents selected from O—C1-C3 alkyl; or benzyl substituted with 0-1 substituents selected from C1-C3 alkyl; or C3-C5 cycloalkyl substituted with 0-1 substituents selected from C1-C3 alkyl; n is 2; p is 0 or 1; q is 1; r is 2; s is 2; t is 0 or 1; u is 2 or 3; and v is 2.

[0151] A more favorable combination (combination C3) is: Q and T are as defined in combination C1; W, Z, R a , R b , R c , R d , R e , R f , R g , R h , R i , R j , R k , R l , R m , R n , R p , R q , R r , R s , R t , R u , R w , R x , n, p, q, r, s, t, u, and v are as defined in combination C2; R o is independently selected from C1-C3 alkyl substituted with 0-1 substituents independently selected from O-C1-C3 alkyl; or phenyl; R v is phenyl; R y is C1-C3 alkyl substituted with 0-1 substituents selected from O-C1-C3 alkyl; or benzyl substituted with 0-1 substituents selected from C1-C3 alkyl; or C3-C5 cycloalkyl substituted with 0-1 substituents selected from C1-C3 alkyl.

[0152] In a more preferred combination (combination C4); Q and T are as defined in combination C1; W, Z, R a , R b , R c , R d , R e , R f , Rg , R h , R i , R j , R k , R l , R m , R n , R p , R q , R r , R s , R t , R u , R w , R x , n, p, q, r, s, t, u, and v are as defined in combination C2; R o and R v is as defined in combination C3; and R y is C1-C3 alkyl substituted with 0-1 substituents selected from O—C1-C3 alkyl; or benzyl; or cyclopropyl.

[0153] An even more preferred combination (combination C5): Q and T are as defined in combination C1; W, Z, R a , R b , R c , R d , R e , R f , R g , R h , R i , R j , R k , R l , R m , R n , R p , R q , R r , R s , R t , R u , R w , R x , n, p, q, r, s, t, u, and v are as defined in combination C2; R o and R v is as defined in combination C3; and R y is C1-C3 alkyl substituted with 0-1 substituents selected from O—C1-C3 alkyl; or cyclopropyl.

[0154] In an even more preferred combination (combination C6): Q and T are as defined in combination C1; W, Z, R b , R c , R e , R f , R g , R h , R i , R j , R k , R l , R n , R p , R u , R w , R x , n, p, q, r, s, t, and v are as defined in combination C2; R v is as defined in combination C3; R a is CH3, CH2CH3, or CH2OCH3; R d is CH(CH3)2 or C(CH3)3; R m is C1-C3 alkyl; R o is independently selected from C1-C3 alkyl or phenyl; R q is CH3, CH2CH3, or CH2OCH3; R r is C1-C3 alkyl; R s is H; R t is C3-C4 alkyl; R y is C1-C3 alkyl; and u is 3.

[0155] An even more preferred combination (combination C7): Q and T are as defined in combination C1; W, Z, R b , R c , R e , R f , R g , R h , R i , R j , R k , R l , R n , R p , R u , R w , R x , n, p, q, r, s, t, and v are as defined in combination C2; R v is as defined in combination C3; R a , R d , R m , R q , R r , R s , R t , R y and u is as defined in combination C6; and R o are independently selected from C1-C3 alkyl.

[0156] An even more preferred combination (combination C8): Q and T are as defined in combination C1; W, Z, R b , R c , R e , R f , R g , R h , R i , R j , R k , R l , R n , R p , R u , R w , R x , n, p, q, r, s, t, and v are as defined in combination C2; Rv is as defined in combination C3; R a , R d , R m , R q , R r , R s , R t , and u are as defined in combination C6; R o is independently selected from CH3, CH2CH3, or CH(CH3)2; and R y is CH3, CH2CH3, or CH(CH3)2.

[0157] In an even more preferred combination (combination C9): Q and T are as defined in combination C1; W, Z, R g , R j , R l , R n , R w , R x , n, p, q, r, s, t, and v are as defined in combination C2; R v is as defined in combination C3; R a , R d , R q , R s , and u is as defined in combination C6; R o and R y is as defined in combination C9; R b is CH3; R c is CH3; R e is CH3; R f is CH3, CH2CH3, or CH(CH3)2; R h is CH3; R i is CH3; Rk is CH3; R m is CH3; R p is CH3; R r is CH3 R u is CH3; and R t is C(CH3)3.

[0158] In the most favorable combination (combination C10): Q and T are as defined in combination C1; W, Z, R g , R j , R l , R n , R w , R x , n, q, r, s, and v are as defined in combination C2; R v is as defined in combination C3; R a , R d , R q , R s , and u is as defined in combination C6; R o and R y is as defined in combination C7; R c , R e , R f , R i , R k , R m , R p , R r , R u , and R t is as defined in combination C10; and P is 0; and t is 0.

[0159] In the above combination C10, p and t are both 0, so R b and R pIt will be understood that the definition of is omitted.

[0160] In the present invention, the preferred combinations of the above are as follows: A combination of combination A1, combination B1, and combination C1; a combination of combination A2, combination B1, and combination C1; a combination of combination A3, combination B1, and combination C1; a combination of combination A4, combination B1, and combination C1; a combination of combination A1, combination B2, and combination C2; a combination of combination A2, combination B2, and combination C2; a combination of combination A3, combination B2, and combination C2; a combination of combination A4, combination B2, and combination C2; a combination of combination A1, combination B3, and combination C3; a combination of combination A2, combination B3, and combination C3; a combination of combination A3, combination B3, and combination C3; a combination of combination A4, combination B3, and combination C3; a combination of combination A1, combination B4, and combination C4; a combination of combination A2, combination B4, and combination C4; a combination of combination A3, combination B4, and combination C4; The combination of combination A4, combination B4, and combination C4; the combination of combination A1, combination B5, and combination C5; the combination of combination A2, combination B5, and combination C5; the combination of combination A3, combination B5, and combination C5; the combination of combination A4, combination B5, and combination C5; the combination of combination A1, combination B6, and combination C6; the combination of combination A2, combination B6, and combination C6; the combination of combination A3, combination B6, and combination C6; the combination of combination A4, combination B6, and combination C6; the combination of combination A1, combination B7, and combination C7; the combination of combination A2, combination B7, and combination C7; the combination of combination A3, combination B7, and combination C7; the combination of combination A4, combination B7, and combination C7; the combination of combination A1, combination B8, and combination C8; the combination of combination A2, combination B8, and combination C8;A combination of combination A3, combination B8, and combination C8; a combination of combination A4, combination B8, and combination C8; a combination of combination A1, combination B9, and combination C9; a combination of combination A2, combination B9, and combination C9; a combination of combination A3, combination B9, and combination C9; a combination of combination A4, combination B9, and combination C9; a combination of combination A1, combination B10, and combination C10; a combination of combination A2, combination B10, and combination C10; a combination of combination A3, combination B10, and combination C10; a combination A4, The combination of combination B10 and combination C10; the combination of combination A1, combination B11, and combination C10; the combination of combination A2, combination B11, and combination C10; the combination of combination A3, combination B11, and combination C10; the combination of combination A4, combination B11, and combination C10; the combination of combination A1, combination B12, and combination C10; the combination of combination A2, combination B12, and combination C10; the combination of combination A3, combination B12, and combination C10; and the combination of combination A4, combination B12, and combination C10.

[0161] In the above combinations, it will be understood that the variables in the combinations C1 to C10 are ignored unless the variables in the combinations C1 to C10 are present in any of the combinations B1 to B12.

[0162] In a preferred embodiment, the compound of the present invention is (a) a compound of formula (Ia) or (b) a tautomer, N-oxide, pharmaceutically acceptable salt, or solvate thereof: [ka] In the formula, R 1 , R 2 , R 3, wherein ring A and ring B are as defined above (including any of the combinations defined above), and * and $ Each of the indicates a bond to the associated NH group.

[0163] In a more preferred embodiment, the compound of the present invention is (a) a compound of formula (Ib) or (b) a tautomer, N-oxide, pharmaceutically acceptable salt, or solvate thereof: [ka] In the formula, R 1 , R 2 , wherein ring A and ring B are as defined above (including any of the combinations defined above), and * and $ Each of the indicates a bond to the associated NH group.

[0164] It will be understood that in the present invention, a balance between the properties of ring A and ring B is important to obtain good results. In the present invention, the groups for ring A and ring B are listed in order of preference, i.e., group I is the most preferred group for ring A, while group V is the least preferred group listed, and group IA is the most preferred group for ring B, while group VA is the least preferred group listed. As defined by the present invention, it is permissible to combine a more preferred ring A group with a less preferred ring B group, and vice versa. It is also possible to combine a more preferred ring A group with a more preferred ring B group. However, it is not permissible to combine the least preferred ring A group with the least preferred ring B group.

[0165] It should be noted that, in general, it is preferred that each of Ring A and Ring B (especially Ring B) be relatively electron-rich. If Ring A or Ring B has an electron-withdrawing substituent, it is generally preferred that it be further substituted with an electron-donating substituent.

[0166] When ring A is selected from rings in Group I, Group II, Group III, Group IV and Group V defined above; and ring B is selected from rings in Group IA, Group IIA, Group IIA, Group IVA and Group VA defined above: The following are preferred: X is NH; Y is NH; R 2 is H; and when ring A is a ring in group I: ring B is a ring in group IA, group IIA, group IIIA, group IVA or group VA; and X is NH; Y is NH; R 2 is H; and ring A is a ring in group II: ring B is a ring in group IA, group IIA, group IIIA or group IVA.

[0167] The following are more preferred (independently or in combination with the preferred bullet points above): X is NH; Y is NH; R 2 is H; and ring A is a ring in group III: ring B is a ring in group IA or group IIA.

[0168] In preferred embodiments, ring A is a ring within Group I, Group II, Group III, and Group IV as defined above; and ring B is a ring within Group IA, Group IIA, Group IIIA, and Group IVA as defined above; and X is NH; Y is NH; R 2 is H; and when ring A is a ring in group I or group II: ring B is a ring in group IA, group IIA, group IIIA or group IVA; X is NH; Y is NH; R 2 is H; and when ring A is a ring in group III: ring B is a ring in group IA, group IIA or group IIIA; X is NH; Y is NH; R 2 is H; and ring A is a ring in group IV: ring B is a ring in group IA.

[0169] In this embodiment, the following are further preferred: X is NH; Y is NH; R2 is H; and when ring A is a ring in group I: ring B is a ring in group IA, group IIA, group IIIA or group IVA; and X is NH; Y is NH; R 2 is H; and ring A is a ring in group II: ring B is a ring in group IA, group IIA or group IIIA.

[0170] In this embodiment, the following are also preferred (alone or in combination with the above preferred bullet points): X is NH; Y is NH; R 2 is H; and ring A is a ring in group III: ring B is a ring in group IA or group IIA.

[0171] In this embodiment, the following are even more preferred: X is NH; Y is NH; R 2 is H; and when ring A is a ring in group II: ring B is a ring in group IA or group IIA; and X is NH; Y is NH; R 2 is H; and ring A is a ring in group III: ring B is a ring in group IA or group IIA.

[0172] In another preferred embodiment, ring A is a ring within Group I, Group II, and Group III as defined above; and ring B is a ring within Group IA, Group IIA, and Group IIIA as defined above; and X is NH; Y is NH; R 2 is H; and when ring A is a ring in group I: ring B is a ring in group IA, group IIA or group IIIA; X is NH; Y is NH; R 2 is H; and when ring A is a ring in group II: ring B is a ring in group IA or group IIA; X is NH; Y is NH; R 2 is H; and ring A is a ring in group III: ring B is a ring in group IA.

[0173] In another preferred embodiment, ring A is a ring within Group I or Group II as defined above; and ring B is a ring within Group IA or Group IIA as defined above; and X is NH; Y is NH; R 2 is H; and when ring A is a ring in group I: ring B is a ring in group IA or group IIA; X is NH; Y is NH; R 2 is H; and ring A is a ring in group II: ring B is a ring in group IA.

[0174] In another preferred embodiment, ring A is a ring within group I as defined above; and ring B is a ring within group IA as defined above.

[0175] The following are also generally preferred: X is NH; Y is NH; and R 2 F, Cl, Br, -COR 4 , -SO2R 5 , -SOR 5 , -CN, -NO, -NO2 or -NR 6 3 + When: Ring A is a ring in group I and ring B is a ring in group IA.

[0176] The following are also generally preferred: When X is a bond and Y is NH: R 2 is H; ring A is a ring in Group I or Group II; and ring B is a ring in Group IA.

[0177] The following are also generally preferred: When X is NH and Y is a bond: R 2 is H; ring A is a ring in Group I or Group II; and ring B is a ring in Group IA or Group IIA.

[0178] The following are generally more preferred: When X is NH and Y is a bond: R 2is H; ring A is a ring in Group I or Group II; and ring B is a ring in Group IA or Group IIA.

[0179] It will be understood that each of the above preferred embodiments can be combined with each of the specific combinations defined herein (i.e., combination A1, combination B1, combination C1, and combinations thereof). Any combination defined herein may be combined with any group (i.e., group V or VA) or substituent (i.e., R n , R w , R x ), those groups or substituents are disregarded.

[0180] For the avoidance of doubt, it will be understood that each and every bullet point set forth herein as defining a combination of Ring A and Ring B is considered to be a separate (i.e., separable) embodiment of the present invention.

[0181] In particularly preferred embodiments, the present invention provides (a) a compound of formula (Ia) or (b) a tautomer, N-oxide, pharmaceutically acceptable salt, or solvate thereof: [ka] In the formula: R 1 is H or F; R 2 is H or F; R 3 is H or CH3; Ring A is selected from rings from within Group I, Group II, Group III and Group IV, where * indicates a bond to NH; Group I is: [ka] and; Group II is: [ka] and; Group III is: [ka] and; Group IV is: [ka] and; Ring B is selected from rings in Group IA, Group IIA, Group IIA and Group IVA; $ indicates a bond to NH; Group IA is [ka] and: Group IIA [ka] and: Group IIIA [ka] and: Group IIIVA is [ka] and: During the ceremony: W is CH2, O, or NR y and; Z is C(O); R a is C1-C3 alkyl substituted with 0-1 substituents selected from O-C1-C3 alkyl; R b are independently selected from C1-C3 alkyl; R c is C1-C3 alkyl; R d is independently selected from C1-C4 alkyl or is phenyl; R e is C1-C3 alkyl; R is C1-C3 alkyl; R g is H; R h is C1-C3 alkyl; R i is C1-C3 alkyl; R j is H; R k is C1-C3 alkyl; R l is H; R o is independently selected from C1-C3 alkyl substituted with 0-1 substituents selected from O—C1-C3 alkyl; or phenyl; R p are independently selected from C1-C3 alkyl R q is C1-C3 alkyl substituted with 0-1 substituents selected from O-C1-C3 alkyl; R r is H or C1-C3 alkyl; R s is H or C1-C3 alkyl; R t is C1-C4 alkyl; R u are independently selected from C1-C3 alkyl; R y is C1-C3 alkyl substituted with 0-1 substituents selected from O-C1-C3 alkyl; or cyclopropyl; n is 2; p is 0 or 1; q is 1; r is 2; s is 2; t is 0 or 1; u is 2 to 3; v is 2; and: ·R 2 is H; and when ring A is a ring in group I: ring B is a ring in group IA, group IIA, group IIIA or group IVA; ·R2 is H; and when ring A is a ring in group II: ring B is a ring in group IA, group IIA or group IIIA; ·R 2 is H; and when ring A is a ring in group III: ring B is a ring in group IA, group IIA or group IIIA; ·R 2 is H; and when ring A is a ring in group IV: ring B is a ring in group IA; ·R 2 is F: Ring A is a ring in Group I or Group II and Ring B is a ring in Group IA; With the proviso that the compound of formula (Ia) is not (a) a compound selected from the following, or (b) a tautomer, N-oxide, pharmaceutically acceptable salt, or solvate thereof: [ka]

[0182] In a further particularly preferred embodiment, the present invention provides (a) a compound of formula (Ib) or (b) a tautomer, N-oxide, pharmaceutically acceptable salt, or solvate thereof: [ka] During the ceremony: R 1 is H or F; R 2 is H or F; Ring A is selected from rings in Group I, Group II, Group III and Group IV, where * indicates a bond to NH; Group I is: [ka] and; Group II is: [ka] and; Group III is: [ka] and; Group IV is: [ka] and; Ring B is selected from rings in Group IA, Group IIA, Group IIA and Group IVA; $ indicates a bond to NH; Group IA is: [ka] and; Group IIA is: [ka] and; Group IIIA is: [ka] and; Group IIIVA is: [ka] and; During the ceremony: W is CH2, O, or NR y and; Z is C(O); R a is CH3, CH2CH3, or CH2OCH3; R b is C1-C3 alkyl; R c is C1-C3 alkyl; R d is CH(CH3)2 or C(CH3)3; R e is C1-C3 alkyl; R f is C1-C3 alkyl; R g is H; R h is C1-C3 alkyl; Ri is C1-C3 alkyl; R j is H; R o is independently selected from CH3, CH2CH3, or CH(CH3)2; R q is CH3, CH2CH3, or CH2OCH3; R r is C1-C3 alkyl; R s is H; R y is CH3, CH2CH3, or CH(CH3)2; n is 2; r is 2; s is 2; v is 2; and: ·R 2 is H; and when ring A is a ring in group I: ring B is a ring in group IA, group IIA, group IIIA or group IVA; ·R 2 is H; and when ring A is a ring in group II: ring B is a ring in group IA or group IIA; and ·R 2 is H; and when ring A is a ring in group III: ring B is a ring in group IA or group IIA, ·R 2 is H; and when ring A is a ring in group IV: ring B is a ring in group IA; ·R 2 is F: Ring A is a ring in Group I or Group II and Ring B is a ring in Group IA; with the proviso that the compound of formula (Ib) is not (a) a compound selected from the following, or (b) a tautomer, N-oxide, pharmaceutically acceptable salt, or solvate thereof: [ka]

[0183] Preferably, the compound of the present invention is a compound selected from the following, or a tautomer, N-oxide, pharmaceutically acceptable salt, or solvate thereof: [ka] JPEG2024535128000084.jpg230170JPEG2024535128000085.jpg222170JPEG202 4535128000086.jpg213170JPEG2024535128000087.jpg222170JPEG2024535128 000088.jpg237170JPEG2024535128000089.jpg217170JPEG2024535128000090. jpg247170JPEG2024535128000091.jpg216170JPEG2024535128000092.jpg87170

[0184] Preferably, the compound of the present invention is a compound selected from the following, or a tautomer, N-oxide, pharmaceutically acceptable salt, or solvate thereof: [ka] JPEG2024535128000094.jpg216170JPEG2024535128000095.jpg216170JPEG2024535128000096.jpg242170JPEG2024535128000097.jpg225170

[0185] More preferably, the compound of the present invention is a compound selected from the following, or a tautomer, N-oxide, pharmaceutically acceptable salt, or solvate thereof: [ka] JPEG2024535128000099.jpg240170JPEG2024535128000100.jpg172170

[0186] Most preferably, the compounds of the present invention are: [ka] or a tautomer, N-oxide, pharmaceutically acceptable salt, or solvate thereof.

[0187] Dosage, Formulation and Route When used in a treatment method, the compound of the present invention can be administered alone, but is generally administered with a pharmaceutical carrier selected based on the selected administration route and standard pharmaceutical practice.Thus, preferably, the compound of the present invention can be included in a pharmaceutical composition.

[0188] The compounds of the present invention may be administered intravenously (bolus or infusion), intraperitoneally, subcutaneously, or intramuscularly, all in dosage forms well known to those of ordinary skill in the pharmaceutical arts.

[0189] Alternatively, the compound of the present invention may be contained in a pharmaceutical composition suitable for oral administration.One exemplary example of such a pharmaceutical composition is a solid dosage form suitable for oral administration (for example, tablet, capsule (each including sustained release formulation or timed release formulation), tablet, powder or granule).Another exemplary example of such a pharmaceutical composition is a liquid dosage form suitable for oral administration (for example, elixir, tincture, suspension, syrup, solution or emulsion).

[0190] Alternatively, the compounds of the invention may be administered in pharmaceutical compositions suitable for topical administration. Exemplary pharmaceutical compositions suitable for topical administration include creams, gels, or lotions.

[0191] Alternatively, the compounds of the present invention may be administered in pharmaceutical compositions adapted for buccal administration.

[0192] Alternatively, the compounds of the present invention may be administered in pharmaceutical compositions adapted for nasal administration.

[0193] Alternatively, the compounds of the invention may be administered in a pharmaceutical composition suitable for ophthalmic administration.

[0194] Alternatively, the compounds of the present invention may be administered in pharmaceutical compositions adapted for rectal administration.

[0195] Alternatively, the compounds of the present invention may be administered in pharmaceutical compositions adapted for vaginal administration.

[0196] Alternatively, the compounds of the present invention may be administered in a form suitable for inhalation or insufflation.

[0197] In certain embodiments, the compounds of the present invention can be administered locally.For example, the compounds of the present invention can be administered locally in a form suitable for wound healing.In another example, the compounds of the present invention can be administered locally in a form suitable for treating peripheral nerve injury.

[0198] In another particular embodiment, the compounds of the invention may be administered in a form suitable for direct application to exposed nerves, for example, in the treatment of peripheral nerve injury.

[0199] In another particular embodiment, the compounds of the present invention may be administered in a form suitable for transdermal administration to a site adjacent to a nerve, for example, in the treatment of peripheral nerve injury.

[0200] In another particular embodiment, the compounds of the invention can be administered by injection into a nerve, for example, in the treatment of peripheral nerve injury.

[0201] In another particular embodiment, the compounds of the present invention can be administered by controlled local delivery to an area adjacent to a nerve, for example, in the treatment of peripheral nerve injury. Suitable means for providing controlled local delivery include the use of biomaterials and minipumps for drug delivery.

[0202] In another particular embodiment, the compounds of the invention can be administered by injection into the brain or spinal cord, for example, in the treatment of CNS injuries.

[0203] In another particular embodiment, the compounds of the invention can be administered by intrathecal administration, for example, in the treatment of CNS injuries.

[0204] In another specific embodiment, the compounds of the present invention can be administered by controlled local delivery to the nervous system, for example, in the treatment of CNS injury. Suitable means for providing controlled local delivery include the use of biomaterials and minipumps for drug delivery.

[0205] In another particular embodiment, the compounds of the present invention can be administered intravenously with a stent, for example, in the treatment of post-stroke or heart attack.

[0206] The dosage regimen for the compounds of the present invention will, of course, vary depending on known factors such as the pharmacodynamic properties of the particular agent and its mode and route of administration, the species, age, sex, health, condition, and weight of the recipient, the nature and extent of the condition, type of concurrent treatment, frequency of treatment, route of administration, the patient's renal and hepatic function, and the desired effect. A physician or veterinarian can determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of thromboembolic disease.

[0207] As a general guideline, the daily oral dosage of each active ingredient, when used for the indicated effects, is about 0.001 to 1000 mg / kg of body weight per day, preferably about 0.01 to 100 mg / kg of body weight, and most preferably about 1.0 to 20 mg / kg / day.

[0208] Intravenously, the most preferred doses will be in the range of about 1 to about 10 mg / kg / minute during a constant rate infusion. The compounds of the present invention may be administered once daily, or the total daily dosage may be administered in divided doses of two, three, or four times daily.

[0209] The compounds of the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes using transdermal skin patches. When administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.

[0210] The compounds are typically administered in admixture with a suitable pharmaceutical diluent, excipient, or carrier (collectively referred to herein as a pharmaceutical carrier) appropriately selected for the intended form of administration, i.e., oral tablet, capsule, elixir, syrup, etc., and consistent with conventional pharmaceutical practice.

[0211] For example, when administered orally in tablet or capsule form, the active ingredient can be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as lactose, starch, sucrose, glucose, methylcellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, etc.; when administered orally in liquid form, the oral drug component can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, etc. Furthermore, if desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose and β-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, and sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, etc. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Disintegrants include, without being restricted thereto, starch, methylcellulose, agar, bentonite, xanthan gum and the like.

[0212] The compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, multilamellar vesicles, etc. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, phosphatidylcholines, and the like.

[0213] The compounds of the present invention may also be conjugated with soluble polymers as targetable drug carriers. Such polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds of the present invention may be conjugated with classes of biodegradable polymers useful for achieving controlled drug release, such as polyacetic acid, polyglycolic acid, copolymers of polyacetic acid and polyglycolic acid, polyepsiloncaprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphiphilic block copolymers of hydrogels.

[0214] Dosage forms (pharmaceutical compositions) suitable for administration may contain from about 1 milligram to about 100 milligrams of active ingredient per dosage unit. In these pharmaceutical compositions, the active ingredient is typically present in an amount of about 0.5 to 95% by weight based on the total weight of the composition.

[0215] Gelatin capsules may contain the active ingredient and powdered carriers such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Compressed tablets can also be prepared using similar diluents. Both tablets and capsules can be prepared as sustained-release formulations that release the drug over several hours. Compressed tablets can be sugar-coated or film-coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated to selectively disintegrate in the gastrointestinal tract.

[0216] Liquid dosage forms for oral administration may contain coloring and flavoring to increase patient acceptance.

[0217] Generally, suitable carriers for parenteral solutions include water, a suitable oil, saline, aqueous dextrose (glucose) and related sugar solutions, and glycols such as propylene glycol and polyethylene glycol. Solutions for parenteral administration preferably contain a water-soluble salt of the active ingredient, suitable stabilizers, and, if necessary, buffer substances. Antioxidants such as sodium bisulfite, sodium sulfite, and ascorbic acid, either alone or in combination, are suitable stabilizers. Citric acid and its salts, and sodium EDTA are also used. Parenteral solutions may also contain preservatives such as benzalkonium chloride, methylparaben or propylparaben, and chlorobutanol.

[0218] Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.

[0219] Compounds / Compositions for Pharmaceutical Use In one embodiment, the compound or pharmaceutical composition of the invention is for use as a medicament.

[0220] Compounds / Compositions for Use in Methods of Treating and / or Preventing Disorders Susceptible to Treatment by PI3K Alpha Activation In another embodiment, the compound or pharmaceutical composition of the invention is for use in a method for treating and / or preventing a disorder susceptible to treatment by PI3Kα activation.

[0221] Thus, in one embodiment, the compound or pharmaceutical composition of the invention is for use in a method for protecting tissue from ischemia-reperfusion injury, i.e., toxicity induced by reoxygenation following interruption of the blood supply to the tissue. Specific examples of this embodiment include application in methods for protecting tissue from ischemia-reperfusion injury in stroke patients undergoing tPA / mechanical thrombectomy (Koh, SH & Lo, EH, J Clin Neurol 11, 297-304 (2015); Khan, H. et al. Brain research, 147399 (2021)), patients with myocardial infarction (heart attack) who receive percutaneous coronary intervention (angioplasty with stent) (Koh, SH & Lo, EH, J Clin Neurol 11, 297-304 (2015); Rossello, X. et al. Basic Res Cardiol 112, 66 (2017); Wang, G. et al. The EMBO journal 37 (2018)), or re-oxygenation of other organs, such as kidney (Zhang, G. et al., Kidney Blood Press Res 43, 904-913 (2018); Xiang, H. et al. Clinical and experimental pharmacology & physiology 47, 1030-1040 (2020)).

[0222] In another embodiment, the compound or pharmaceutical composition of the present invention is for use in a method for protecting the human or animal body from ionizing radiation (Chauhan, A. et al. Scientific reports 11, 1720 (2021)).

[0223] In another embodiment, the compounds or pharmaceutical compositions of the present invention are useful in wound healing (including healing from skin-related injuries including burns (Gan, D. et al. Front Pharmacol 12, 631102 (2021); Sugita, H. et al. Am J Physiol Endocrinol Metab 288, E585-591 (2005); Park, KK et al. Science (New York, NY 322, 963-966 (2008)) and diabetic foot ulcers), regeneration of airway / pulmonary epithelium in childhood wheezing and asthma (Iosifidis, T. et al. JCI insight 5 (2020)), and reducing the need for corneal transplants to repair endothelial diseases in the eye / cornea, most commonly resulting from endothelial dysfunction (Sabater, AL et al. Invest Ophthalmol Vis Sci 58, 745-754 (2017)).

[0224] In another embodiment, the compound or pharmaceutical composition of the present invention is for use in a method for metabolic sensitization by overcoming insulin resistance in obesity and type 2 diabetes (Foukas, LC et al. Nature 441, 366-370 (2006); Knight, ZA et al. Cell 125, 733-747 (2006); Frevert, EU & Kahn, BB Molecular and cellular biology 17, 190-198 (1997); Cichy, SB et al. J Biol Chem 273, 6482-6487 (1998); Prakoso, D. et al. Am J Physiol Heart Circ Physiol 318, H840-H852 (2020)).

[0225] In another embodiment, the compound or pharmaceutical composition of the present invention is for use in a method for treating cancer by inducing cancer cell death through overactivation of the PI3K pathway in cells with overactivated PI3K, particularly in cells with overactivated PI3K, which is frequently found in treatment-resistant cancers (Klippel, A. et al. Molecular and cellular biology 18, 5699-5711 (1998); Chen, Z. et al. Nature 521, 357-361 (2015); Shojaee, S. et al. Nat Med 22, 379-387 (2016); Muschen, M. Nat Rev Cancer 18, 103-116 (2018)). (2009)).

[0226] In another embodiment, the compound or pharmaceutical composition of the present invention is for use in a method for neuroprotection / regeneration (Arnes, M. et al. Mol Biol Cell 31, 244-260 (2020); Cuesto, G. et al. J Neurosci 31, 2721-2733 (2011); Asua, D. et al. Neuroscience 370, 81-87 (2018)), including protection from neuronal damage during cancer treatment (Matsuda, S. et al. International journal of oncology 49, 1785-1790 (2016)). Genetic PI3Kα activation mediates axonal regeneration in neurons (Nieuwenhuis, B. et al. EMBO Mol Med 12, e11674 (2020)).

[0227] In another embodiment, the compound or pharmaceutical composition of the invention is for use in a method for the treatment of traumatic optic neuropathy, e.g., retinal ganglion cell survival and axonal regeneration (Morgan-Warren, PJ et al. Invest Ophthalmol Vis Sci 54, 6903-6916 (2013)).

[0228] In another embodiment, the compound or pharmaceutical composition of the present invention is for use in a method for neuroprotection / regeneration after CNS injury, including traumatic brain injury (Minnich, J. et al. Restor Neurol Neurosci 28, 293-309 (2010)), spinal cord injury (Zhu, S. et al. Cell Prolif 53, e12860 (2020)), hypoxia / ischemia / stroke (Houlton, J. et al. Front Neurosci 13, 790 (2019); Larpthaveesarp, A. et al. Brain Sci 5, 165-177 (2015)), and nerve root injury (Wang, R. et al. Nat Neurosci 11, 488-496 (2008)).

[0229] In another embodiment, the compound or pharmaceutical composition of the present invention is used to treat Parkinson's disease (Yang, L., Wang, H., Liu, L. & Xie, A. Front Neurosci 12, 73 (2018); Jha, SK et al. Int J Mol Cell Med 4, 67-86 (2015)), Alzheimer's disease (Nagahara, AH et al. Nat Med 15, 331-337 (2009)), Huntington's disease (Simmons, DA et al. Proc Natl Acad Sci USA 106, 4906-4911 (2009)), ALS (Yin, X. et al. Mol Cell Neurosci 68, 303-313 (2015)), Rett syndrome (Castro, J., Mellios, N. & Sur, M. Curr Opin Neurol 26, 154-159 (2015)), or other conditions. (2013)) and neurodegenerative diseases (Allen, SJ et al. Pharmacol Ther 138, 155-175 (2013); Rai, SN et al. Neurotox Res 35, 775-795 (2019); Nagahara, AH & Tuszynski, MH Nat Rev Drug Discov 10, 209-219 (2011)).

[0230] In another embodiment, the compound or pharmaceutical composition of the invention is for use in a method for protecting / regenerating enteric neurons, for example in the treatment of gastrointestinal motility disorders as a result of diabetes (Anitha, M. et al. J Clin Invest 116, 344-356 (2006)).

[0231] Thus, non-limiting examples of disorders that are susceptible to treatment by PI3Kα activation and that can be treated and / or prevented using the compounds or pharmaceutical compositions of the present invention include ischemia-reperfusion injury; ionizing radiation injury; tissue injury (e.g., to promote tissue regeneration); childhood asthma; asthma; ocular / corneal endothelial diseases; obesity; type 2 diabetes; cancer (e.g., cancers that exhibit excessive PI3K activity, particularly in treatment-resistant cancers); neuronal injury; traumatic optic neuropathy; central nervous system injury (e.g., traumatic brain injury, spinal cord injury, hypoxia, ischemia, stroke); neurodegenerative diseases (Parkinson's disease, Alzheimer's disease, Huntington's disease, ALS, and Rett syndrome); and gastrointestinal motility disorders (e.g., as a result of diabetes).

[0232] In a particularly preferred embodiment of the invention, the disorder susceptible to treatment with PI3Kα activation is a peripheral neuropathy.

[0233] Use for the production of medicines The present invention provides the use of a compound or pharmaceutical composition according to the invention for the manufacture of a medicament, preferably for use in a method for the treatment and / or prevention of a disorder susceptible to treatment by PI3Kα activation.

[0234] It will be understood that all preferred disclosures provided above in relation to a compound or pharmaceutical composition according to the invention for use according to the invention are equally contemplated as preferred in the context of the use of a compound or pharmaceutical composition according to the invention for the manufacture of a medicament according to the invention.

[0235] Treatment method The present invention also provides methods of treatment, particularly methods of treating and / or preventing disorders susceptible to treatment by PI3Kα activation in a patient in need thereof, comprising administering to the patient a compound of the present invention, or a pharmaceutical composition of the present invention.

[0236] It will be understood that all preferred disclosures provided above in relation to compounds or pharmaceutical compositions according to the invention for use according to the invention are likewise contemplated as preferred in the context of methods of treatment according to the invention.

[0237] Use of the compound The present invention provides the use of the compounds of the invention as biochemical probes.

[0238] PI3K activation has also been shown to improve the success rate of in vitro fertilization by ex vivo activation of dormant follicles from cryopreserved ovarian tissue. Therefore, the present invention further provides the use of the compounds of the present invention in in vitro fertilization techniques.

[0239] PI3K activation is also believed to be useful in tissue preservation for organ transplants, such as kidney and liver transplants, by extending the lifespan of donor organs and improving transplant outcomes. Therefore, the present invention further provides use of the compounds of the present invention in in vitro tissue preservation for organ transplantation. [Example]

[0240] Example 1 PI3Kα activation data of an example of the present invention. Determination of EC50 values EC50 values ​​were determined by quantifying phosphorylation of Akt on the S473 residue of Akt in A549 human adenocarcinoma cells using an enzyme-linked immunoadsorbent assay (ELISA, R&D Systems, DYC887BE). A549 cells were seeded in 96-well plates (50,000 cells / well) in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomyosin. After 24 hours of incubation, cells were starved in serum-free DMEM for 24 hours and then treated with a compound concentration-response curve for 15 minutes. Experimental compounds were diluted in DMSO and a 10-point 1:3 concentration-response curve was prepared in a polypropylene V-bottom plate (SLS MIC9050). The concentration-response curve was diluted 3-fold in serum-free DMEM. A standard 8-point concentration-response curve was performed at 50, 16.7, 5.6, 1.9, 0.6, 0.2, 0.07, and 0.02 μM using serum-free DMEM containing 0.5% DMSO as a negative control. Compound-induced Akt phosphorylation was normalized to the DMSO negative control, and the E of insulin-induced Akt phosphorylation after 15 minutes of treatment (1 μM insulin) was calculated. max was normalized to

[0241] result Each compound disclosed herein exhibits PI3Kα activation. The table below (Table 1) shows the average EC50 for each compound and an indication of whether the compound achieves a 50% or greater response at 10 μM (preferred), or whether a less than 50% response is observed at 10 μM.

[0242] [Table 1] JPEG2024535128000103.jpg204170JPEG2024535128000104.jpg211170JPEG2024535128000105.jpg215170JPEG202 4535128000106.jpg208170JPEG2024535128000107.jpg201170JPEG2024535128000108.jpg210170JPEG20245351280 00109.jpg212170JPEG2024535128000110.jpg201170JPEG2024535128000111.jpg204170JPEG2024535128000112.j pg210170JPEG2024535128000113.jpg216170JPEG2024535128000114.jpg184170JPEG2024535128000115.jpg191170 JPEG2024535128000116.jpg212170JPEG2024535128000117.jpg193170JPEG2024535128000118.jpg183170JPEG202 4535128000119.jpg202170JPEG2024535128000120.jpg225170JPEG2024535128000121.jpg195170JPEG20245351280 00122.jpg230170JPEG2024535128000123.jpg187170JPEG2024535128000124.jpg213170JPEG2024535128000125.j pg208170JPEG2024535128000126.jpg232170JPEG2024535128000127.jpg193170JPEG2024535128000128.jpg103170

[0243] Example 2 In vitro characterization of the activity of 1938 as a selected exemplary compound of the present invention 1938 is an allosteric, non-ATP-competitive, PI3Kα isoform-selective activator. We tested the effect of 1938 on the in vitro lipid kinase activity of p85α complexed with p110α, p110β, or p110δ (further referred to as PI3Kα, PI3Kβ, and PI3Kδ). As a positive control, we used a diphosphorylated phosphopeptide (a peptide derived from the PDGF receptor phosphorylated at Tyr-740 and Tyr-751, hereafter referred to as pY peptide) to mimic the binding of the SH2 domain of p85α to tyrosine-phosphorylated peptides of membrane-bound receptors and adaptor proteins known to activate the PI3Kα complex. Unlike pY, which activates all class IA PI3K isoforms (Fig. 9 / Extended Data Fig. 1), we found that 1938 concentration-dependently activated PI3Kα but not PI3Kβ or PI3Kδ (Fig. 1b / Fig. 22b). Enzyme kinetic assays showed that 1938, like pY, increased the turnover rate (Kcat) and maximum reaction velocity (Vmax) of PI3Kα (Figure 1c / Figure 22c). While pY did not affect the Km of PI3Kα for ATP, 1938 modestly reduced this parameter for PI3Kα at 1 μM and 10 μM, but not at 30 μM (Figure 1c). 1938 also increased PI3Kα binding to lipid membranes to approximately half the maximal level induced by pY (Figure 1d / Figure 22d). Combination of 1938 with saturating concentrations of pY (Figure 1e, left panel / Figure 22e, left panel) resulted in synergistic PI3Kα activation (Figure 1e, right panel / Figure 22e, right panel), indicating that 1938 activates PI3Kα via a different mechanism or enhances activation events beyond those induced by pY. The combination of 1938 and pY did not further increase PI3Kα membrane association beyond that induced by pY (Fig. 1d / Fig. 22d), so this synergistic effect is unlikely to involve alterations in membrane binding.

[0244] Next, we tested the effects of 1938 on various oncogenic mutants of p110α, each of which has been shown to activate the p85α / p110α complex by different mechanisms (Burke, JE et al. Proc Natl Acad Sci USA 109, 15259-15264 (2012)) (summarized in Table 2). 1938 activated the G106V and N345K mutants to levels comparable to those stimulated by pY. The E545K mutant was insensitive to pY stimulation but, as previously shown, was further activated by 1938 (Fig. 1f / Fig. 22f). Co-stimulation of 1938 with pY resulted in an additive activation effect on the G106V mutant but not on the N345K or E545K mutants (Fig. 1f / Fig. 22f). 1938 did not increase membrane binding of G106V but did increase it for N345K (to a level similar to that induced by pY) and E545K (whose membrane binding was not increased by pY, as previously reported) (Fig. 1g / Fig. 22g). Together, these data suggest that 1938 enhances multiple steps in the PI3Kα catalytic cycle and does not specifically mimic the mechanism of activation conferred by any one of the oncogenic p110α mutations tested (Table 2).

[0245] [Table 2]

[0246] Next, we examined the effect of BYL719, an ATP-competitive PI3Kα-selective inhibitor, on 1938-activated PI3Kα. In vitro PI3Kα activity stimulated by 25–50 μM 1938 was completely inhibited by 500 nM BYL719 (Fig. 1h / Fig. 2h). The IC values ​​of 10–20 nM BYL719 on PI3Kα inhibition in the presence of 1938 (10 μM) and ATP (200 μM) were 50 Values ​​are the IC reported previously for PI3Kα in the absence of 1938. 50These data indicate that 1938 does not compete with ATP binding on PI3Kα, and that 1938-activated PI3Kα is completely inhibited by BYL719.

[0247] Example 3 HDX-MS studies of 1938 as selected exemplary compounds of the present invention. 1938 activates PI3Kα by blocking inhibitory contacts between p85α and p110α We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the binding sites and structural changes of soluble native proteins, which are key to understanding the molecular mechanisms underlying the physiological activation of class IA PI3K. Binding of the complex to diphosphorylated motifs present in membrane-resident receptors and cytoplasmic adaptors releases the inhibitory interaction between p85 and p110, leading to the deinhibition of the autoinhibited p85 / p110 complex, resulting in the activation of class IA PI3K. These events include the release of the p85α-nSH2 domain on the p110α-helical domain, the disruption of the inhibitory interaction between p85α-iSH2 / p110α-C2 domain, the relative movement of the p85 adaptor domain of p110α toward residues in the catalytic subunit, and the interaction of the p110α kinase domain with lipid membranes.

[0248] HDX-MS of 1938 incubated with PI3Kα showed that a small loop consisting of amino acids (AA) 1001–1016 of p110α was protected by 1938 binding, suggesting that this region is a potential 1938-binding site on p110α (Fig. 2a; a surface model is shown in Extended Data Fig. 2). We also observed that 1938 binds outside the ATP-binding site, at the interface between the p85α-iSH2 domain and the p110α-C2 and -kinase domains (Fig. 2a). Increased solvent exchange rates were also observed in several additional regions of p85α / p110α, namely the p85α-iSH2 domain (AA 550-570) and multiple regions of p110α (from the N-terminus to the C-terminus): AA 444-474 (the interface between the p85α-iSH2 and p110α-C2 domains), and AA 848-859 (the ATP-binding site). These observed structural changes suggest that 1938 likely activates PI3Kα by disrupting inhibitory contacts between the p85α-iSH2 domain and the p110α-C2 domain, causing movement of the p110α ABD / RBD linker and kinase domain, enhancing catalytic activity. Additionally, HDX-MS experiments were performed with BYL719 and a mixture of BYL719 and 1938. BYL719 strongly protected the 848-858 kinase domain linker region and the 767-781 region, resulting in a characteristic ATP-competitive footprint on PI3Kα, as previously reported (see PMID: 28381646). However, when the two compounds were combined, the footprints of PI3Kα were integrated, with protection in the kinase linker region, protection in the 1002-1016 pocket, and exposure at the p85α-iSH2 interface, suggesting that PI3Kα can simultaneously accept both ligands.

[0249] Example 4 Cell-based characterization of the activity of 1938 as selected exemplary compounds of the present invention. 1938 induces PI3Kα pathway activation in cells Class I PI3K converts the plasma membrane lipid PtdIns(4,5)P2 to PtdIns(3,4,5)P3 (or PIP3), which is then converted to PtdIns(3,4)P2 by the action of 5-phosphatase. Therefore, we used live imaging of cells expressing fluorescent biosensors that selectively bind these lipids to test whether stimulation of cells with 1938 results in the production of PIP3 and PI(3,4)P2. Within 3 min of serum-starved A549 cells, 1938 induced plasma membrane-associated PIP3 production, which was completely and rapidly neutralized by the addition of BYL719 (Figure 3ai, ii). 1938 did not increase PIP3 signaling in PIK3CA-null A549 cells (Figure 3ai). In serum-starved HeLa cells, 1938 also induced an acute, but more transient, BYL719-dependent burst of PIP3 production compared to A549 cells, followed by a return to intermediate levels (Figure 3aiii). This PIP3 peak was followed by membrane-associated PI(3,4)P2 production (Figure 3aiii), the timing of which was consistent with the known mechanism by which PI(3,4)P2 is produced in cells from PIP3 via the action of 5-phosphatases. Similar kinetics of PIP3 and PI(3,4)P2 production have previously been reported in insulin-stimulated HeLa cells. The slight increase in signal after the addition of BYL719 and 1938 in HeLa cells (Figure 3aiii, lower panel) represents a nonspecific response to medium addition seen only in these cells (Figure 12 / Extended Data Figure 4).

[0250] Next, we monitored the activation of AKT, the most well-known PI3K effector, stimulated by intracellular PIP3 / PI(3,4)P2 production. Treatment with 1938 for 15 min increased pAKT in a concentration-dependent manner in mouse embryonic fibroblasts (MEFs). S473 Although the levels of pAKT were elevated in PI3Kα-null MEFs (Fig. 3b), S473As previously reported, PI3Kα-null MEFs still respond to insulin, but the insulin-stimulated pAKT upregulation in response to the PI3Kβ-selective inhibitor TGX-221 was not observed. S473 This response was PI3Kβ-dependent, as indicated by the sensitivity of 1938 to PI3Kα (Fig. 3b). In PI3Kα wild-type MEFs (Fig. 3b), A549 cells (Fig. 3c), and MCF10A cells (Fig. 13 / Extended Data Fig. 5), co-treatment with BYL719 completely blocked 1938-induced AKT phosphorylation. At 15 min, 1938 treatment significantly reduced pAKT-induced EC20 expression in both mouse (MEFs; Fig. 3b) and human (A549) cells. 50 The E of 1938 was below 2–4 μM (Fig. 3d). Dose titration of 1938 and insulin in A549 cells demonstrated that in these cells, 1938 can overactivate the PI3K pathway, as measured by AKT phosphorylation, beyond the saturating dose of insulin; i.e., at doses of 5–10 μM, the E of 1 μM insulin was significantly higher than that of 1 μM insulin. max The effect of 1938 (5 μM) on pAKT expression in MEFs and A549 cells was found to be approximately 200% of that observed in MEFs (Fig. 3d). S473 The induction of 1938 was rapid (5 min; Fig. 3c, e; Fig. 13 / Extended Data Fig. 5), reaching peak activation at 30 min and persisting for several hours before returning to slightly higher levels than baseline 24 or 48 h after stimulation (Fig. 3e). Similar observations were made for mTORC1 pathway activation, measured by phosphorylation of S6 (Ser240 / 44) and 4EBP1 (Ser65) (Fig. 3e). Interestingly, the pattern and kinetics of Akt / mTORC1 pathway activation were overall similar to those induced by insulin (Fig. 3c, e), suggesting that 1938-mediated PI3K pathway activation follows an endogenous cellular feedback mechanism known to operate in the PI3K signaling pathway. In summary, our results demonstrate that 1938 activates both proximal and distal signaling in a dose-dependent and PI3Kα-dependent manner in rodent and human cells, demonstrating its ability to directly activate PI3Kα signaling in cells.

[0251] Example 5 Unbiased evaluation of the signal induced by 1938 as a selected exemplary compound of the invention. Unbiased evaluation of 1938-induced signals Given that the structure of 1938 contains a pyridine core, a known scaffold for multiple kinase inhibitors, we next tested the effect of 1938 on the in vitro activity of a panel of 133 protein kinases and seven lipid kinases (data presented as KinMap (Eid, S. et al. BMC Bioinformatics 18, 16 (2017)) (Figure 3f) or as a waterfall plot (Figure 14 / Extended Data Figure 6)). At a concentration of 1 μM, 1938 inhibited 13 protein kinases by 25–50%, with only two protein kinases, LCK and BRK, inhibited by more than 50% (58% and 56%, respectively). It is important to note that LCK and BRK were tested in vitro in the presence of 50 μM and 75 μM ATP, respectively; if 1938 acts as an ATP-competitive inhibitor of these kinases, we would expect the inhibition by 1938 to be significantly lower in cells where ATP concentrations are known to be 1–10 mM.

[0252] 1938 did not affect the activity of other PI3K isoforms in the panel [PI3Kβ (PIK3CB), PI3Kγ (PIK3CG), PI3Kδ (PIK3CD), PI3K-C2α (PIK3C2A), and Vps34 (PIK3C3)] or the PI3K-related kinases PI4Kβ, mTOR, and DNA-PK (Figure 3f). In separate in vitro assays, 1938 did not affect the activity of the PI3K-related kinases ATM (Figure 15 / Extended Data Figure 7) and mTORC1 (Figure 14 / Extended Data Figure 7; tested as an mTOR / RAPTOR / LST8 complex; note that mTOR activity in the Thermofisher screen (Figure 3f; Figure 14 / Extended Data Figure 6) was tested using the mTOR monomer without a binding partner).

[0253] Next, we used phosphoproteomics to examine the effects of 1938 on cell signaling in an unbiased manner. PI3Kα-WT and PI3Kα-KO MEFs were treated with 1938 or insulin for 15 minutes or 4 hours (Fig. 16 / Extended Data Fig. 8a, b). Phosphorylation sites showing a greater than two-fold change relative to DMSO and an adjusted p-value <0.05 were defined as significantly regulated. We quantified 10,611 phosphorylation sites from 3,093 proteins, including 9,100 pSer residues, 1,420 pThr residues, and 91 pTyr residues (Fig. 16 / Extended Data Fig. 8a). Consistent with the data shown in Fig. 3a, b, 1938 had little effect on signaling in PI3Kα-KO MEFs (Fig. 3gi, ii; Fig. 16 / Extended Data Fig. 8b), and paxillin (pPXN) was significantly down-regulated. S322 ) was the only phosphorylation site that changed (downregulated by 15 min of 1938 treatment but unaffected by 4 h of stimulation; Fig. 3gi, ii). In PI3Kα-WT MEFs, 1938 induced the differential phosphorylation of 27 and 50 peptides after 15 min and 4 h of treatment, respectively, the majority of which were upregulated (Fig. 3gi, ii). The upregulated phosphorylation sites included pAKT1S1, a well-known component of the PI3K pathway. T247 (also known as PRAS40) and pGSK3B S9(Fig. 3gi). Compared with vehicle-treated cells, insulin treatment of PI3Kα-WT MEFs induced phosphorylation of 11 and 18 sites after 15 min and 4 h, respectively (Fig. 16 / Extended Data Fig. 8c). At both time points, significant overlap was observed between the phosphorylation sites in PI3Kα-WT MEFs regulated by 1938 and insulin (Fig. 3giii). The majority of phosphorylation sites upregulated by 1938 and insulin after 4 h were similar to those upregulated by 15 min of 1938 treatment (Fig. 3gi). Furthermore, approximately half of the phosphorylation sites regulated by 1938 were previously reported (by PhosphoSitePlus) to be regulated by insulin, IGF-1, PI3K inhibition, or AKT inhibition, and some were associated with regulation by mTOR or PDK1 (Fig. 3gi, ii). Notably, pSPCC1L S923 , pMSN S384 and pMAPK3 Y205 Several phosphorylation sites upregulated by 1938 in PI3Kα-WT MEFs but not in PI3Kα-KO MEFs, including the top hits from et al., have not previously been linked to PI3K signaling by PhosphoSitePlus ( Figure 3gi ), highlighting the utility of 1938 as a tool compound to discover novel signaling pathways downstream of PI3Kα.

[0254] Example 6 In vitro evaluation of PI3Kα-dependent cell biological responses induced by 1938 as a selected exemplary compound of the present invention. 1938-induced PI3Kα-dependent cell biological responses The role of PI3Kα in activating anabolic metabolism, cell cycle progression, and cell proliferation is well established. In PI3Kα-WT MEFs, metabolic activity, as measured by ATP content using the CellTiterGlo assay, was increased by 1938 in a dose-dependent manner, with an EC of 1938. 50At concentrations of 1938 above 7.5 μM, ATP levels were significantly reduced in both PI3Kα-WT and PI3Kα-KO MEFs (Fig. 4a), but not in PI3Kα-KO MEFs. After 24 h of incubation, 1938 at concentrations above 7.5 μM significantly reduced ATP levels in both PI3Kα-WT and PI3Kα-KO MEFs, indicating a PI3Kα-independent effect of 1938 at these concentrations (Fig. 4a). With longer incubations (48 and 72 h), these non-PI3Kα-dependent effects of 1938 were observed at concentrations ≥2 μM (Fig. 17 / Extended Data Fig. 9).

[0255] In addition to increasing metabolic activity, 1938 treatment of PI3Kα-WT MEFs also induced cell cycle progression (measured by EdU incorporation; Figure 4b) and increased cell number (measured by crystal violet staining; Figure 4c). These biological effects were not observed in PI3Kα-KO MEFs and were completely neutralized by co-treatment with BYL719 in PI3Kα-WT MEFs (Figure 4b, c). Interestingly, unlike 72-hour treatment with 1938, incubation with insulin under the same conditions did not result in an increase in cell number (Figure 4c), suggesting differential cellular activity of these agents, as suggested by the proteomic data (Figure 3f).

[0256] Example 7 Ex vivo and in vivo evaluation of disease-related biological responses induced by 1938 as a selected exemplary compound of the present invention. Therapeutic potential of pharmacological PI3Kα activation Myocardial infarction (MI) is a significant cause of morbidity and mortality in patients with coronary artery disease. Despite the development of novel antiplatelet and antithrombotic agents, timely reperfusion via catheter-based percutaneous coronary intervention remains fundamental for cardiac tissue salvage. Paradoxically, such reperfusion can lead to ischemia-reperfusion injury (IRI), tissue damage that occurs following the restoration of blood supply after a period of blood deprivation, which is also observed in stroke treatments using intra-arterial devices. Finding ways to mitigate IRI is essential to improve the long-term prognosis of patients with MI. Ischemic preconditioning, an experimental method for protecting the heart from IRI, leads to the activation of kinases such as MEK / ERK1 / 2 and PI3K / AKT as part of the so-called reperfusion injury salvage kinase (RISK) pathway, a cardioprotective pathway induced by the majority of cardioprotective agents, including insulin, a canonical activator of the PI3K / AKT pathway. Using a PI3Kα inhibitor, we have previously shown that activation of PI3Kα is necessary and sufficient for cardioprotection.

[0257] In Langendorff ex vivo perfused rat hearts, an established experimental model of IRI, 1938 was found to be a fast-acting agonist that substantially protected tissue from IRI when administered during the first 15 minutes of reperfusion. This was evidenced by increased tissue viability and reduced infarct size (representative images shown in Figure 5a; quantified in Figure 5b) as well as increased functionality (assessed by ECG; Figure 5a), both of which were associated with increased production of pAKTS473 (Figure 5c; all data shown in Figure 18 / Extended Data Figure 10). 1938 also provided significant cardioprotection in an in vivo model of IRI in mice (left panel of Figure 11), downregulating pAKTS expression in the hearts of these mice. S473 There was a corresponding increase in the levels of PI3Kα (right panel of Figure 11). The observed rapid activation of PI3Kα suggests that the therapeutic application of direct PI3Kα activators may be cardioprotective and clinically feasible in patients undergoing emergency coronary revascularization after myocardial infarction.

[0258] Activation of the PI3K pathway is widely associated with neuroprotection and nerve regeneration, and recently, genetic approaches have demonstrated the positive role of PI3Kα in axon regeneration. Currently, there are no clinical-stage small molecule therapies routinely used to promote neuronal regeneration following peripheral nerve, spinal cord, or optic nerve injury. Therefore, we explored the potential of PI3Kα activation in these settings. In isolated adult rat dorsal root ganglion (DRG) cultures, an in vitro model of nerve regeneration, 1938 significantly increased neurite outgrowth in a dose-dependent manner, with higher 1938 concentrations doubling the total neurite length measured after 72 hours (Figure 5d).

[0259] Inspired by these observations, we next tested 1938 in a rat sciatic nerve crush model, an in vivo model of peripheral nerve injury and regeneration. Exploratory experiments demonstrated induction of pAKT by direct injection of 1938 or by bathing the exposed sciatic nerve in 1938 solution (Figure 19 / Extended Data Figure 11), indicating that local administration of 1938 results in activation of the PI3K pathway in this tissue. Immediately after nerve crush (Figure 5e, i-ii), 1938 was delivered via a single intrathecal injection into the proximal crush site (Figure 5e, iii) and via a minipump implanted adjacent to the nerve (Figure 5e, iv) and loaded with 1938 solution for the duration of the experiment. Analysis was performed 3 weeks after injury.

[0260] Electrophysiological recordings from the tibialis anterior muscle during nerve stimulation proximal to the injury site demonstrated that 1938 treatment enhanced electrophysiological recovery, as indicated by an increase in motor unit number estimate (MUNE) (Figure 5f) and recovery of the compound muscle action potential (CMAP) (Figure 5g). This correlated with (1) an increase in the number of choline acetyltransferase (ChAT)-positive motor axons in 1938-treated animals, whose neurites were grouped within normal myofascial nerve structures [Figure 5h; assessed in a distal nerve section of the common peroneal branch of the sciatic nerve, proximal to the reinnervation point of the tibialis anterior muscle] (Figure 5i); and (2) histological analysis demonstrating that a portion of the neuromuscular junction of the tibialis anterior muscle (Figure 5j) was innervated, with α-bungarotoxin (α-BTX) staining revealing a characteristic distribution of postsynaptic acetylcholine receptors and neurofilament immunoreactivity (representative example shown in Figure 5k). The 21-day analysis represents an early time point in terms of regeneration, and low levels of initial muscle reinnervation are expected in untreated animals. Histological detection of motor axons in the distal nerve and neuromuscular junction (NMJ) corresponded to improved electrophysiological reinnervation of the tibialis anterior muscle. Histological analysis of nerve sections closer to the injury site (3 mm and 6 mm distal to the crush site) revealed similar numbers of neurofilament- and ChAT-positive axons in the treated and control groups (Figure 5l). This suggests that the functional improvement in muscle reinnervation following 1938 treatment is due to enhanced spontaneous neuronal regeneration rather than a change in the total number of regenerating neurites.

[0261] Taken together, these data demonstrate the therapeutic potential of short-term, localized pharmacological PI3Kα activation in tissue protection and regeneration in preclinical studies.

[0262] Example 8 Evaluating the suitability of PI3Kα activation in cancer therapy. Therapeutic potential of PI3Kα activation in cancer treatment The PI3Kα activator developed by the inventors was able to induce cell death in multiple cancer cell lines derived from various tissues under nutrient- and oxygen-deprived conditions, but showed no cytotoxicity to primary rat neurons, human endothelial cells (HUVECs), and the immortalized but untransformed MCF-10A breast epithelial cell line (Table 3; Figures 6 and 7). This cell death was assessed by propidium iodide staining / FACS and could be partially neutralized by the PI3Kα inhibitor BYL719 (Figures 6 and 7) or PI3K pathway inhibitors (Figures 6 and 7). PI3Kα-induced overactivation cell death was observed at 72 hours after continuous exposure, and cytotoxic responses could be observed after 72 hours with only 0.5–1 hour of drug exposure (Figure 8), demonstrating the potential of pulsed drug administration in vivo.

[0263] [Table 3]

[0264] Example 9 Supplementary crystallographic studies on 1938 as selected exemplary compounds of the present invention. 1938 activates PI3Kα by blocking inhibitory contacts between p85α and p110α To understand how 1938 interacts with PI3Kα, we attempted to crystallize PI3Kα in the presence of 1938. First, we used a construct containing full-length p110α and a truncated niSH2 p85α (p110α M232K L223K / p85α 307-593). PI3Kα crystals diffracting to 2.2A resolution were obtained, but no compound was visible when co-crystallized or when soaking the compound in preformed crystals (PDB: 7PG5). Co-crystallizing PI3Kα with both 1938 and BYL719 yielded crystals in which only the BYL719 density was visible (2.5A resolution, PDB: 7PG6). Therefore, we used a construct containing only the p110α catalytic subunit, lacking the adaptor-binding domain and lipid-binding surface of the kinase domain (p110α 105-1048). Although cocrystallization of p110α with 1938 did not yield crystals, we soaked 1938 in preformed crystals and were able to observe the density of 1938. Apo p110α 105-1048 yielded crystals that diffracted up to 2.4 A, and p110α 105-1048 soaked with 1938 yielded crystals that diffracted up to 2.5 A.

[0265] Consistent with the HDX-MS results, the crystal structure shows that 1938 binds to a pocket bounded by residues E365, I459, L540, D603, C604, N605, Y641, S1003, L1006, G1007, and F1016 (Figure 20bi, ii). The core pyridine nitrogen of 1938 is sufficiently basic that it is primarily protonated at physiological pH, and this NH + makes a critical interaction with the side chain of D603. In this protonated state, the molecule cannot form the donor-acceptor motif characteristic of canonical protein kinase inhibitors. The acetylated indoline of 1938 sits in a pocket consisting of L1006, F1016, and I459, and makes a face-to-face interaction with F1016. Upon binding of 1938, F1016 moves out of the pocket to accommodate the ligand. The piperazine is surrounded by E365 and L540 and faces toward the solvent.

[0266] Upon compound binding, a global conformational shift is observed. Both the C2 domain and the helical domain are distant from the kinase domain. The loop (1002-1016), identified by HDX-MS as protected by compound binding, is farther from the activator binding site in the p110α-1938 structure than in the apo. In addition, the α-helix 1016-1026 also moves farther away upon compound binding. The 1938 binding site is close to the E542 and E545 hotspots (approximately 10 Å). This region is important for the inhibition of p110α by the nSH2 domain of p85α, so it is possible that 1938 weakens the inhibitory effect of p85α on p110α, contributing to enzyme activation.

[0267] Interestingly, the structure of p110α-1938 highlights a potential reason for its lack of activity against both PI3Kβ and PI3Kδ isoforms. Comparing the structure of the 1938 binding pocket in p110α with the analogous regions in p110β (PDB: 2Y3A) and p110δ (PDB: 6PYU) indicates that p110β and p110δ do not possess a pocket capable of accommodating 1938 (Figure 20biii). Preliminary structure-activity relationship analysis confirmed key components of the compound's binding mode. Replacing the core pyridine with a 2,4-pyrimidine (compound 2152) reduced activity by more than 95%, consistent with the suggestion that the corresponding nitrogen is no longer protonated at physiological pH and is therefore unable to form the critical interaction with D603. Compounds 1887 and 1889 exhibited comparable activity to 1938, demonstrating their tolerance to piperazine modifications and substitutions (Table 4). However, complete removal of the piperazine (compound 2016) reduces activity by more than 90%. Compound 2016 is also less soluble than 1938. The crystal structure shows that the piperazine faces toward the solvent, suggesting that the presence of the piperazine or tri-O-methyl-substituted phenyl may be important in displacing water molecules and maintaining hydrophobic interactions between L1006 and F1016. The indoline is required for edge-to-face and hydrophobic interactions between F1016 and L1006R. The carbonyl group forms an internal hydrogen bond with the NH group connecting the indoline and pyridine, holding the indoline in a favorable orientation for interaction with F1016. Replacing the acetylated indoline with a pyrimidine (compound 2106) reduces activity by more than 95%, which may be due to less favorable end-to-end interactions with F1016 and increased flexibility of the pyridine.

[0268] Therefore, we performed mutagenesis to generate 1938-resistant mutants. Based on the crystal structure and SAR data, we generated the following mutants: D603K, D603A, 603DCN_AAA605 triple mutant, D603A / F1016S double mutant, and L1006R, F1016S, L1006R / F1016S double mutant. The basal activities of the D603K, D603A, DCN_AAA triple mutant, D603A / F1016S, and L1006R / F1016S double mutant were comparable to those of the WT, while the basal activities of L1006R (p<0.0001) and F1016S (p=0.0056) were significantly higher than those of the WT. All mutants were further activated by pY stimulation, yet all mutants were resistant to activation by 1938 (Figure 20biv).

[0269] [Table 4]

[0270] Example 10 Additional cell-based characterization of the activity of 1938 as a selected exemplary compound of the present invention. 1938 induces activation of the PI3Kα pathway in cells Class I PI3K phosphorylates the plasma membrane lipid PtdIns(4,5)P2 to generate PtdIns(3,4,5)P3 (or PIP3), which is then converted to PtdIns(3,4)P2 by the action of 5-phosphatase. Treatment of MEF (mouse embryonic fibroblast) cells with 1938 resulted in a very rapid (within 30 seconds) increase in PIP3 levels, as assessed by mass spectrometry, which reached a maximum at 5 minutes and maintained this maximum level for up to 40 minutes (Figure 21a). At 2 minutes, the PIP3 levels induced by 1938 were comparable to those induced by insulin but lower than those induced by PDGF. The observation that the PIP3 levels induced by these growth factors differ is consistent with the idea that PI3Kα is the sole mediator of PIP3 production downstream of insulin. This contrasts with PDGF, which activates both PI3Kα and PI3Kβ, suggesting that the latter PI3K isoform contributes significantly to acute PDGF-stimulated PIP3 production in MEFs, correlating with the higher PIP3 levels induced by this agonist compared with insulin and 1938 (Figure 21a). In the same experiment as Figure 21a, a clear PI(3,4)P2 signal was detected in MEFs upon PDGF stimulation, but not with 1938 (5 μM). This is consistent with the relatively low PI3K activation by 1938 compared with high doses of PDGF, as demonstrated by the experiments described below, as well as the higher threshold for detection of PI(3,4)P2 compared with PIP3 by mass spectrometry (primarily due to background contamination, as discussed by Malek et al.).

[0271] When tested at different doses at a fixed 2-minute time point, PIP3 induction by 1938 in MEFs was significantly lower than the PIP3 levels induced by 1 ng / ml or 3 ng / ml PDGF, and was significantly lower than that of ECs. 50We found that the PIP3 response to 1938 was ~5 μM and plateaued around 10 μM (Figure 21b). These maximum 1938-induced PIP3 levels were below the levels required to produce sufficient PI(3,4)P2 detectable by mass spectrometry. This conclusion is supported by the observation that the substantial levels of PIP3 induced by lower doses of PDGF (e.g., 0.5 ng / ml) were also insufficient to produce detectable levels of PI(3,4)P2 by mass spectrometry. Similar to what was observed in MEFs, stimulation of A549 cells with a range of 1938 doses for 2 min revealed that the PIP3 response to 1938 was also maximal at 10 μM (Figure 21c). In these cells, a strong PIP3 response was also observed with insulin, but not with PDGF, consistent with the absence of this receptor in epithelial cells, including A549.

[0272] method PI3K protein expression and purification Full-length p110α was expressed in complex with full-length p85α (for biochemistry and HDX-MS) or truncated p85-niSH2 protein (for crystallography). A p110α construct lacking the adaptor-binding domain and the lipid-binding surface (105-1048) was also used for crystallography (Chen et al. (2014) Protein Sci 23: 1332-1340).

[0273] Expression and purification of full-length p85α (LMB-MRC plasmid OP809) and complexed p110α (LMB-MRC plasmid OP831) were performed as described (Burke, JE et al. Proc Natl Acad Sci USA 109, 15259-15264 (2012)). Oncogenic mutants G106V (LMB-MRC plasmid JB35), N345K (LMB-MRC plasmid OP661), and E545K (LMB-MRC plasmid OP663) were also purified using this protocol. Briefly, 1.0 × 10 6Ten liters of Spodoptera frugiperda (Sf9) cell cultures at a density of 100 cells / ml were co-infected with a virus encoding p85α [LMB-MRC plasmid LOP809] and a virus encoding p110α carrying an N-terminal 6xHis tag followed by a tobacco etch virus (TEV) protease site [LMB-MRC plasmid OP831]. After 48 hours of infection at 27°C, cells were harvested and washed with PBS. The cell pellet was suspended in lysis buffer (20 mM Tris pH 8.0, 300 mM NaCl, 5% glycerol, 10 mM imidazole pH 8.0, 2 mM β-mercaptoethanol, 1 tablet of EDTA-free protease inhibitor (Roche) per 50 ml of buffer) and sonicated at 15-second intervals for 7 minutes at 4°C, followed by a 15-second rest period. The cell lysate was then centrifuged at 45,000 g for 45 minutes at 4°C. The supernatant was filtered through a 0.45 μM filter and then loaded onto two 5 ml HisTrap FF (Cytiva) columns (equilibrated with NiNTA Buffer [20 mM Tris pH 8.0, 300 mM NaCl, 5% glycerol, 10 mM imidazole pH 8.0, 2 mM β-mercaptoethanol]) at a flow rate of 3 ml / min. The column was then washed with a 20 mM imidazole wash, and the protein was eluted with a gradient to NiNTA B Buffer (20 mM Tris pH 8.0, 300 mM NaCl, 5% glycerol, 200 mM imidazole pH 8.0, 2 mM β-mercaptoethanol). Fractions containing PI3Kα were pooled and diluted 1:2 with salt dilution buffer (20 mM Tris pH 8.0, 1 mM DTT) and adjusted to 100 mM NaCl. This solution was passed through a HiTrap Heparin (Cytiva) column equilibrated with Hep A Buffer (20 mM Tris pH 8.0, 100 mM NaCl, 2 mM β-mercaptoethanol) at a rate of 3 ml / min. PI3Kα was eluted using a gradient to Hep B Buffer (20 mM Tris pH 8.0, 1 M NaCl, 2 mM β-mercaptoethanol).Protein-containing fractions were pooled and concentrated to 8 mg / ml, then loaded onto a Superdex 200 16 / 60 column equilibrated with gel filtration buffer (20 mM HEPES pH 7.4, 100 mM NaCl, 2 mM TCEP) and operated at 1 ml / min at 4°C. PI3Kα-containing fractions were pooled and concentrated to 2.5 mg / ml, then flash-frozen in liquid nitrogen and stored at -80°C.

[0274] The complex of p110α and p85α-niSH2 was expressed and purified as follows: Sf9 insect cells were cultured in Insect-XPRESS with L-Glutamine medium (Lonza BE12-730Q) at 27°C, and baculovirus encoding both p110α and p85α-niSH2 [LMB-MRC plasmid GM129] was transfected into 1.6–1.8 × 10 cells. 6Cells were infected at a density of 1000 cells / ml. After 48 hours of incubation, cells were harvested, washed with PBS, flash-frozen in liquid nitrogen, and stored at -80°C. For purification, the cell pellet was suspended in 100 ml of lysis buffer (20 mM Tris, 150 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol, 0.02% CHAPS, pH 8.0) and EDTA-free protease inhibitor tablets (Roche, 1 tablet per 50 ml of solution) and 500 μl of DNAse I were added. The suspension was sonicated on ice for 10 seconds, 10 seconds on, 10 seconds off. The lysate was then centrifuged at 35,000 rpm in a Ti45 rotor for 45 minutes (4°C). The sample was loaded onto a StrepTrap (Cytiva) column in S300 buffer (20 mM Tris, 300 mM NaCl, 5% glycerol, 2 mM TCEP, pH 8.0). Once loaded, the column was washed with buffer A (20 mM Tris, 100 mM NaCl, 5% glycerol, 1 mM TCEP, pH 8.0). The column was eluted with a gradient of 1–100% buffer B (buffer A containing 5 mM d-desthiobiotin). Fractions from the p110α / p85α-niSH2 peak were pooled and cleaved with TEV protease (0.8 mg / ml) at a 1:10 ratio overnight at 4°C. The protein was loaded onto a 5 ml HiTrap Heparin HP column (Cytiva), washed with buffer A, and eluted with a gradient of 1–100% buffer C (20 mM Tris, 1 M NaCl, 1 mM TCEP, pH 8.0). Fractions were collected, concentrated, and loaded onto a Superdex 200 26 / 60 HiLoad gel filtration column (Cytiva) and eluted with 20 mM Tris, 200 mM NaCl, 2 mM TCEP, 1% betaine, 1% ethylene glycol, 0.02% CHAPS, pH 7.2. Peak fractions were pooled and concentrated to 10–13 mg / ml using Amicon Ultra-15 Centrifugal filters 100K (Millipore). The protein was flash-frozen in liquid nitrogen and stored at -80°C.The purity of the protein was confirmed by SDS-PAGE.

[0275] Expression and purification of truncated human p110α (105-1048) was performed as follows: Sf9 insect cells (9 L) were cultured in Insect-XPRESS with L-Glutamine medium (Lonza BE12-730Q) at 27°C, and 1.6 × 10 baculovirus encoding the p110α subunit [LMB-MRC plasmid OP798] was transfected. 6Cells were infected at a density of 1000 cells / ml. After 48 hours of incubation, the cells were harvested, flash-frozen in liquid nitrogen, and stored at -80°C. For purification, the cell pellet was suspended in 360 ml of lysis buffer (20 mM Tris, 150 mM NaCl, 5% glycerol, 1 mM TCEP, pH 8.0) and supplemented with EDTA-free protease inhibitor tablets (1 tablet per 50 ml of solution), 0.5 mM PEFA, and 36 μl Piece® Universal Nuclease for Cell Lysis. The suspension was sonicated on ice for 5 minutes. The lysate was then centrifuged at 35,000 rpm in a Ti45 rotor for 35 minutes at 4°C. The sample was filtered through a 5 μm filter and loaded onto a StrepTrap (Cytiva) column equilibrated with lysis buffer. Once the sample was loaded, the column was washed with 20 mM Tris, 300 mM NaCl, 5% glycerol, 1 mM TCEP, pH 8.0, followed by 20 mM Tris, 150 mM NaCl, 5% glycerol, 1 mM TCEP, pH 8.0. Five ml of a 0.14 mg / ml TEV solution was then added to the column and allowed to stand overnight at 4°C for cleavage. The protein was loaded onto a 5 ml HiTrap Heparin HP column (Cytiva) equilibrated with 20 mM Tris, 150 mM NaCl, 5% glycerol, 1 mM TCEP, pH 8.0, and eluted with a 1–100% gradient of 20 mM Tris, 1 M NaCl, 1 mM TCEP, pH 8.0. Fractions were collected, concentrated, and loaded onto a Superdex 200 16 / 60 HiLoad gel filtration column (Cytiva) and eluted with 50 mM Tris, 100 mM NaCl, 2% ethylene glycol, 1 mM TCEP, pH 8.0. Peak fractions were pooled and concentrated to 5.83 mg / ml using Amicon Ultra-15 Centrifugal filters 50K (Millipore). Protein was flash-frozen in liquid nitrogen and stored at -80°C. Protein purity was confirmed by SDS-PAGE.

[0276] Full-length p110β / p85α and p110δ / p85α were cloned and expressed in a similar manner, but with a streptavidin tag instead of the His tag. 6 Five liters of Spodoptera frugiperda (Sf9) cell cultures at a density of 1000 cells / ml were co-infected with both viruses encoding p85α and viruses encoding p110β / δ carrying an N-terminal streptotag followed by a tobacco etch virus (TEV) protease site (plasmid OP832 for p110β, plasmid OP833 for p110δ, and plasmid OP809 for p85α). After 48 hours of infection at 27°C, cells were harvested and washed with PBS. The cell pellet was suspended in lysis buffer (20 mM Tris pH 8.0, 150 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol, 1 tablet of EDTA-free protease inhibitor (Roche) per 50 ml of buffer) and sonicated at 15-second intervals for 7 minutes at 4°C, followed by a 15-second rest period. The cell lysate was then centrifuged at 45,000 g for 45 minutes at 4°C. The supernatant was filtered through a 0.45 μM filter and then loaded onto a 1x5 ml StrepTap No. 1 (GE Healthcare) column (equilibrated with 100S Buffer [20 mM Tris pH 8.0, 100 mM NaCl, 5% glycerol, 1 mM TCEP]) at a flow rate of 3 ml / min. The column was then washed with 70 ml of 100S Buffer, 80 ml of S300 Buffer (20 mM Tris pH 8.0, 300 mM NaCl, 5% glycerol, 1 mM TCEP), and 50 ml of S100 Buffer. Five ml of 0.1 mg / ml His6TEV protease (p30) in S100 Buffer was injected onto the column and incubated at 4°C for 4 hours.

[0277] Fluorescence polarization assay PIP3 production was measured using a fluorescence polarization assay (#K-1100; Echelon Biosciences, Salt Lake City, UT, USA) in a 384-well microtiter plate. PI3Kα, liposomes, and ATP were all diluted in reaction buffer (20 mM HEPES, 50 mM NaCl, 50 mM KCl, 3 mM MgCl2, 1 mM EGTA, 1 mM TCEP, pH 7.4) and added to the microtiter plate to achieve final reaction concentrations of 10 nM PI3Kα, 75 μg / ml liposomes, and 10 μM ATP. Reactions were carried out at room temperature for 45 minutes, quenched with the PIP3 detector and TAMRA probe, and then read on a Hidex Sense plate reader using polarization filters at λ544 ± 20 and λ590 ± 20. Data were normalized to the minimum and maximum PIP3 production values ​​measured by the TAMRA probe alone and the TAMRA Plus detector, respectively.

[0278] Microscale thermophoresis MST experiments were performed using an automated Monolith NT.115 (NanoTemper Technologies, Munich, Germany). Fluorescent labeling of PI3Kα with NT647 dye was performed using the RED-NHS protein labeling kit (NanoTemper Technologies, Munich, Germany) according to the manufacturer's protocol. PI3Kα was diluted to a final concentration of 2.5 nM in reaction buffer (20 mM HEPES, 100 mM NaCl, 0.1% Tween-20, 2 mM TCEP, pH 7.4). Compounds were serially diluted in neat DMSO and added to the enzyme to a final concentration of 3% DMSO. In a premium-processed capillary, 80% infrared laser power and 10% LED intensity were used. Data were analyzed using NanoTemper Analysis software, and ΔF was calculated. norm value (ΔF norm = Fhot / F cold ) was used to define the bonds in the compounds.

[0279] ADP-Glo TM Kinase assay Kinase reactions were performed using the ADP-Glo ​​Kinase Assay Kit (Promega Corporation). The enzyme, substrate, and compounds were diluted in reaction buffer (20 mM HEPES, 50 mM NaCl, 50 mM KCl, 3 mM MgCl2, 1 mM EGTA, 1 mM TCEP, pH 7.4). The final concentrations of PI3Kα and PI3Kδ used were 25 nM and 50 nM for PI3Kβ, respectively. Liposomes (5% brain PI(4,5)P2, 20% brain phosphatidylserine, 45% brain phosphatidylethanolamine, 15% brain phosphatidylcholine, 10% cholesterol, 5% sphingomyelin (Avanti Polar Lipids)) were used at a final concentration of 1 mg / ml. The pY sequence was ESDGG(pY)MDMSKDESID(pY)VPMLDMKGDIKYADIE.

[0280] Reaction mixtures for compound profiling contained 2 μl of PI3K enzyme, 2 μl of compound and / or pY, and 2 μl of liposomal substrate mixed with ATP. ATP was used at a final concentration of 500 μM for PI3Kα and PI3Kβ, and 200 μM for PI3Kδ, unless otherwise specified. The final DMSO concentration in the assay was 1%. Experiments were performed in 384 white polystyrene plates (Corning #3824) at room temperature for 3 hours, after which the reaction was terminated by the addition of 6 μl of ADP-Glo ​​R1. After incubating the plates for 45 minutes, 12 μl of ADP-Glo ​​R2 was added and incubated for an additional 60 minutes in the dark. Luminescence was read using a Sense (Hidex) plate reader. Compound data were normalized to the DMSO negative control without enzyme and expressed as maximum activation (E). max All analyses were performed using GraphPad Prism 7.

[0281] To characterize the effects of 1938 on PI3K enzymology in vitro, all reactions were performed at room temperature in 384-well white polystyrene plates (Corning #3574). The final DMSO concentration in the assay ranged from 0.5% to 1.8%. The reaction mixture contained 2 μl of PI3K enzyme, 2 μl of compound and / or pY, and 2 μl of liposomal substrate mixed with ATP. ATP was used at a final concentration of 200 μM unless otherwise specified. The enzyme and compound were preincubated for 10 min before adding the substrate. The reaction proceeded for 45 min at room temperature and then terminated by adding 6 μl of ADP-Glo ​​R1. After incubating the plate for 60 min, 12 μl of ADP-Glo ​​R2 was added and incubated for an additional 60 min in the dark. Data for enzyme kinetic calculations were expressed as rate (pmol ADP produced / sec). An ADP-ATP standard curve was performed according to the manufacturer's instructions, and all analyses were performed using GraphPad Prism 8.

[0282] FRET membrane binding assay Membrane binding assays were performed as previously described (Burke, JE et al. Proc Natl Acad Sci USA 109, 15259-15264 (2012)). Briefly, liposomes were prepared with 5% (w / v) brain PtdIns(4,5)P2, 20% brain phosphatidylserine, 35% brain phosphatidylethanolamine, 15% brain phosphatidylcholine, 10% cholesterol, 5% sphingomyelin, and 10% dansyl-phosphatidylserine (Avanti Polar Lipids). PI3Kα was used at a final concentration of 0.5 μM. Protein solutions were preincubated with 10 μM pY or compounds for 10 min before adding liposomes. Liposomes were used at a final concentration of 50 μg / ml. The reaction mixture contained 5 μl of enzyme, 2 μl of compound, and 3 μl of liposomes, all diluted in 30 mM HEPES, 50 mM NaCl, pH 7.4. The reaction proceeded for 10 minutes at room temperature on an orbital shaker at 200 rpm in a 384-well black polystyrene plate (Corning #3544). FRET signals were measured using a PHERAStar (BMG) with excitation filters at 280 nm and emission filters at 350 and 520 nm for dansyl-PS FRET emission. FRET signals are expressed as I-I, where I is the intensity at 520 nm and I is the intensity at 520 nm of the solution in the absence of protein.

[0283] HDX-MS Sample preparation: HDX-MS experiments were performed as described in (Anandapadamanaban, M. et al. Science (New York, NY 366, 203-210 (2019)). Briefly, in the absence of compound, 5 μM PI3Kα was incubated with 300 μM 1938, 100 μM BYL719, or both 1938 and BYL719 in protein dilution buffer (50 mM Tris pH 7.5, 150 mM NaCl, 2 mM TCEP) containing 1% DMSO. 5 μl of PI3Kα, with or without compound, was diluted in 45 μl of DO buffer (50 mM Tris pH 7.5, 150 mM NaCl, 2 mM TCEP, 1% DMSO, with or without 50 μM 1938, 90.6% DMSO). The exchange experiments were performed in triplicate with three separate protein preparations (D2O) and five time points (0.3 s, 3 s, 30 s, 300 s, and 3000 s, with the 0.3 s time point being a 3 s time point performed at 0°C). The samples were quenched with 20 μl of ice-cold quench solution (2 M guanidinium chloride, 2.4% formic acid), snap-frozen in liquid nitrogen, and then stored at -80°C. A total of three biological replicates were performed; three separate protein preparations were used, each with triplicate exchange experiments. The results shown are from one representative biological replicate. Data acquisition and analysis were performed as follows: each sample was thawed and injected into an M-Class Acquity UPLC with HDX technology (Waters) maintained at 0.1°C. Proteins were digested in-line using an Enzymate Pepsin Column (Waters, 186007233) at 15°C for 2 minutes. Peptides were then eluted onto an Acquity UPLC BEH C18 column (Waters, 186002346) equilibrated with pepsin-A buffer (0.1% formic acid) and separated using a 3-43% gradient of pepsin-B buffer (0.1% formic acid, 99% acetonitrile) over 16 minutes. Data were collected in the m / z range of 50-2000 on a Waters Cyclic IMS equipped with an electrospray ion source. Data were collected in HDMSe mode. Cyclic IMS was performed in a single pass.A "blank" sample of protein dilution buffer containing quenching was run between samples to routinely monitor peptide carryover. Five replicates were used to identify undeuterated peptides. Criteria used for peptide inclusion in the HDX-MS dataset were: minimum intensity 5000, minimum sequence length 5, maximum sequence length 25, minimum 3 fragment ions, minimum 0.1 product per amino acid, minimum score 6.62, maximum MH+ error 10 ppm, identification in at least two datasets, and retention time RSD less than 10%. Data were analyzed using the Protein Lynx Global Server (Waters) and DynamX (Waters). All peptides were manually inspected for sufficient quality of EX1 kinetics and peptide envelopes. Data quality, experimental design, and data reporting met the criteria determined by the HDX-MS community (Masson, GR et al. Nat Methods 16, 595-602 (2019)). Acquisition files were generated using Baryonyx.

[0284] Kinase profiling (mTORC1 and ATM kinase assays) 1988 at 1 μM was used to counterscreen 133 protein kinases and 7 lipid kinases using Adapta, Lantha, and Z-LYTE assays (SelectScreen Kinase Profiling Service; Thermofisher - experimental details of these assays can be found here: https: / / www.thermofisher.com / uk / en / home / industrial / pharma-biopharma / drug-discovery-development / target-and-lead-identification-and-validation / kinasebiology / kinase-activity-assays.html). Tree representation in KinMap (www.cellsignal.com), courtesy of Cell Signaling Technology, Inc. The mTORC1 (mTOR / RAPTOR / LST8) protein complex and ATM kinase and substrate were prepared as described (Anandapadamanaban, M. et al. Science (New York, NY 366, 203-210 (2019); Baretic, D. et al. Sci Adv 3, e1700933 (2017)). 1938 was screened using SuperSep Phos-Tag 50 μmol / L 100 x 100 x 6.6 mm 17-well (192-18001 / 199-18011) gels. For the ATM assay, 100 nM ATM was incubated at 5 μM in the absence or presence of 200 μM 1938 in ATM kinase buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 10% glycerol, 2 mM trichloroethylene, 5 mM MgCl2). The cells were incubated with GST-p53 and 1 mM ATP for 30 min at 30°C. As a positive control for ATM activation, the same reaction was performed with 100 nM ATM, 5 μM GST-p53, and 1 mM ATP in the presence of 100 nM of the Mre11-Rad50-Nbs1 (MRN) complex, a known ATM activator.For the mTORC1 assay, 50 nM mTORC1 complex (mTOR / LST8 / RAPTOR) was incubated with 15 μM 4E-BP1, 10 mM MgCl2, and 250 μM ATP in the absence or presence of 200 μM 1938 for 3 h at 30 °C. As a "positive" control, 150 nM mTORC1 complex (mTOR / LST8 / RAPTOR) was incubated with 15 μM 4E-BP1, 10 mM MgCl2, and 250 μM ATP for 3 h at 30 °C. The kinase reaction was quenched by adding SDS-PAGE loading buffer (according to the manufacturer's instructions) and freezing at -20 °C. The reaction was then run on a Phos-tag gel at 150 V for 90 min. The gel was then visualized with InstantBlue. TM The kinase assays were performed in triplicate and stained with Coomassie stain and quantified using BioRad Image Lab Software.

[0285] Co-crystallization of p110α / p85α niSH2-compound complexes An initial screening of approximately 2,000 conditions was performed using an LMB robotic crystallization setup (Stock, D. et al. Prog Biophys Mol Biol 88, 311-327 (2005)). p110α / p85α niSH2 was preincubated with 100 μM BYL719 for 1 hour, or preincubated with 100 μM BYL719 for 1 hour followed by incubation with 500 μM 1938 for 1 hour. Sitting drops were set up in a 96-well MRC plate by mixing 100 nl of the reservoir with 100 nl of protein solution (10 mg / ml). Initial crystals were obtained in 0.2 M KSCN, 0.1 M sodium cacodylate, and 8-30% PEG 2K, PEG 4K, PEG 5K, or PEG 6K (w / v), or in 80 mM KSCN, 30% PEG 1K (w / v), 150 mM MES, pH 6.0. For optimization, 1 μl of 5.5 mg / ml protein was mixed with 1 μl of reservoir, and the best diffracting crystals were obtained in 16% PEG 1K (w / v), 150 nM KSCN, 150 mM MES pH 6.0, 9% PEG 4K (w / v), 180 mM KSCN, 100 mM sodium cacodylate, 10% PEG 5K MME (w / v), 160 nM KSCN, and 100 mM sodium cacodylate. Crystals were also soaked in 10 mM 1938 for 1–20 h.

[0286] Crystallization of p110α-compound complexes All crystallization experiments were performed at 20 °C. An initial screening of approximately 2,300 conditions was performed using the LMB robotic crystallization setup. p110α was preincubated for 1 hour with 500 μM 1938 or 1% DMSO. Sitting drops were set up in a 96-well MRC plate by mixing 100 nL of protein solution (5.8 mg / ml) with a 100 nL reservoir. ApoE crystals were obtained from 12.5% ​​(w / v) PEG 4K, 20% (v / v) 1,2,6-hexanetriol, 40 mM polyamines, 0.1 M MOPSO / bis-tris pH 6.5; and 12.5% ​​(w / v) PEG 4K, 20% (v / v) 1,2,6-hexanetriol, 90 mM LiNaK, 0.1 M MOPSO / bis-tris pH 6.5. To optimize 1938, crystallization experiments were performed in a 96-well MRC plate by adding dropwise the following mixtures: 200 nL of 5.8 mg / mL protein and 200 nL of reservoir solution, or 500 nL of 5.8 mg / mL protein and 500 nL of reservoir solution, containing varying concentrations of PEG, 1,2,6-hexanetriol, polyamine, or LiNaK. Crystals were formed only under apo conditions. These apo crystals were soaked in 20 mM 1938 (20% DMSO) for 1.5–2 hours. For data collection, apo crystals were obtained under conditions containing 12.5% ​​(w / v) PEG 4K, 20% (v / v) 1,2,6-hexanetriol, 90 mM LiNaK, and 0.1 M MOPSO / bis-tris pH 6.5. Crystals were soaked in a solution containing 12.5% ​​(w / v) PEG 4K, 20% (v / v) 1,2,6-hexanetriol, 50 mM polyamine, and 0.1 M MOPSO / bis-tris pH 6.5 at 1938. The collected crystals were freeze-cooled in liquid nitrogen before data collection.

[0287] X-ray crystal structure analysis of p110α 105-1048 X-ray diffraction data were collected from single crystals of p110α 105-1048 alone and soaked with 1938 using a synchrotron X-ray source. Images were processed using automated image processing in Xia. Initial phases were determined by molecular replacement using Phaser in the CCP4 suite, using an initial model from PDB entry 4TUU. The model density was manually adjusted using COOT, and the structure was refined using PHENIX. The structure in the presence of 1938 revealed density for a pocket with a wall consisting of residues E365, I459, L540, D603, C604, N605, Y641, S1003, L1006, G1007, and F1016. This pocket has not previously been occupied by any ligand in any structure of p110α. The binding mode is consistent with previous HDX-MS results. A 3D model was constructed using PHENIX ELBOW from the 1938 chemical structure, and this model closely matched the density of crystals soaked in 1938. This pocket was empty in structures obtained from crystals not soaked in 1938. The protein / ligand complex was manually prepared and refined using COOT and PHENIX. Representations of the complex were created using PyMOL and Chimera.

[0288] Wes TM Detection of protein phosphorylation using Experiments using A549 cells and MEFs were performed separately using slightly different protocols. Briefly, A549 cells were seeded at 200,000 cells per well in 24-well plates in DMEM (10% FBS + 1% P / S) and allowed to adhere overnight. The following day, cells were washed once with PBS before adding serum-free DMEM for 24 hours. Cells were pretreated with PI3Kα inhibitor (BYL719, 500 nM) or 0.1% DMSO for 15 minutes at 37°C and 5% CO2 before compound addition. Cells were washed with cold RIPA buffer (Thermo, supplemented with protease inhibitors and phosphatase inhibitors (Roche)). MEFs were seeded at 50 cells / well in 12-well plates and allowed to adhere overnight. The next day, cells were serum-deprived for 4 hours before being treated with 1 μM insulin or 1938 (0.2–30 μM, final DMSO concentration 0.5%) for 1 hour at 37°C, 5% CO2. Cells were then washed with cold PBS and lysed in 50 mM Tris.HCl pH 7.4, 1% Triton-X100, 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 2 mM EGTA, 10 mM Na4P2O7, and Merck's protease / phosphatase inhibitor cocktail. Lysates were collected and centrifuged at 15,000 rpm for 15 minutes at 4°C. The supernatants were then stored at -80°C. Western blotting was performed using Western blotting software. TM The assay was performed using ProteinSimple according to the manufacturer's instructions. Antibodies to pAKT-S473 (CST #4060) and total AKT (CST #9272) were used at a dilution of 1:50. Antibodies to β-actin (CST #4970) were used at a dilution of 1:100.

[0289] Detection of AKT phosphorylation by ELISA A549 cells were seeded at 50,000 cells per well in a 96-well plate in DMEM (10% FBS + 1% P / S). The following day, cells were washed once with PBS and then cultured for 24 hours with serum-free DMEM. Compounds solubilized at 10 mM in DMSO were diluted 1:3 and an 8-point concentration-response curve was generated in DMSO. The concentration-response curve was transferred to an intermediate plate using a BRAVO liquid handler (Agilent) and diluted with serum-free DMEM. The intermediate plate was used to process the cell plate using the BRAVO liquid handler. The highest concentration of the compound concentration-response curve was 50 μM, and the final well concentration was 0.5% DMSO. The cell plate was incubated at 37°C and 5% CO2 for 15 minutes, then washed with ice-cold PBS, lysed in Lysis Buffer 6 (R&D Systems #895561), and stored frozen at -80°C. pAKT-S473 levels were measured using a phospho-AKT (S473) pan-specific Duoset IC ELISA (R&D Systems #DYC887BE) in 96-well white high-binding plates (Corning #3922) according to the manufacturer's instructions. End-point luminescence was measured using a Sense (Hidex) plate reader. Compound data were normalized to the negative DMSO control and expressed as the maximum insulin-induced activation (E). max The data were expressed as a percentage of the internal insulin control (1 μM), which corresponds to the EC 50 Data were determined by variable slope (four parameter) nonlinear regression using Prism 7 (Graphpad).

[0290] cell culture Immortalized PI3Kα-WT and PI3Kα-KO MEFs were generated as previously described (Foukas, LC et al. Proc Natl Acad Sci USA 107, 11381-11386 (2010)). MEFs were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin and then starved in serum-free DMEM containing 1% penicillin-streptomycin at 37°C and 5% CO2. A549 cells were cultured in DMEM Glutamax (Gibco #31966021) supplemented with 10% FBS and 1% penicillin-streptomycin, or in RPMI 1640 medium supplemented with 10% FBS, 1 mM sodium pyruvate, and 1% penicillin-streptomycin. For starvation experiments, A549 cells were cultured in serum-free RPMI containing 1 mM sodium pyruvate and 1% penicillin-streptomycin. All cell cultures were routinely tested negative for mycoplasma.

[0291] Generation of PIK3CA-deficient A549 cells by CRISPR / Cas9 gene targeting Pooled PIK3CA null A549 cells were generated by Synthego Corporation. Briefly, the PIK3CA gene was targeted with a synthetic ribonucleoprotein (RNP) complex containing the following single guide RNA (sgRNA) sequence: 5'-CUCUACUAUGAGGUGAAUUG-3' (located within PIK3CA exon 3). In parallel, control cultures were exposed to Cas9 protein without the sgRNA (hereafter referred to as "WT cultures"). Single-cell clones were established from both the WT and target cultures by limiting dilution, ensuring a maximum of one cell seeded per well of a 96-well plate. To facilitate recovery, subcloned cells in 96 wells were cultured in a 1:1 mixture of standard A549 complete medium and conditioned medium. Conditioned medium was prepared by centrifugation of WT cultures at 1000 x g for 10 minutes, filtration through a 0.22 µm PES filter, and storage at 4°C (-80°C for storage longer than 2 weeks). The medium was replenished every 2–3 days, as gently as possible to avoid dislodging the cells. When the cells reached subconfluence, they were cultured in 24-well plates and 25 cm 2 The cells were expanded into flasks, and genotyped and cell banked.

[0292] For genotyping, genomic DNA was extracted from replicate cells grown in 24-well plates using 50 μl of QuickExtract solution (Cambridge Bioscience #QE0905T) and the following thermocycling conditions: 68°C for 15 minutes, 95°C for 10 minutes, and a 4°C hold. The edited locus was amplified by standard PCR using GoTAQ G2 MasterMix (2X) (Promega #M7822) with 2 μl of QuickExtract-treated genomic DNA and the following primers: F 5'-TCTACAGAGTTCCCTGTTGC-3'; R 5'-AGCACTCAACTATATCTTGTCAGT-3'. Annealing and extension were performed at 55°C for 30 seconds and 72°C for 30 seconds, respectively. PCR reactions were performed using ExoSAP-IT Express (Thermo Fisher Scientific #75001.1.ML) according to the manufacturer's instructions at 37°C for 30 minutes, followed by cleanup at 80°C for 1 minute. The cleaned-up reactions were subjected to Sanger sequencing (Eurofins Genomics). Subsequent analysis of Sanger sequencing traces was performed using Synthego's open-source ICE tool. All predicted knockout (KO) clones were then verified by Western blotting of PIK3CA protein using two complementary antibodies (CST #4249 and #4255; each diluted 1:1000 in 1X TBS / T containing 3% BSA). Clones showing complete loss of expression were retained for further experimental studies.

[0293] Mass spectrometry-based phosphoproteomics PI3Kα-WT MEFs and PI3Kα-KO MEFs grown in 15-cm dishes were serum-deprived overnight in DMEM supplemented with 1% penicillin-streptomycin and stimulated for 15 min or 4 h by the addition of 0.05% DMSO, 5 mM 1938 in final 0.05% DMSO, or 100 nM insulin (Sigma, I5016). Cells were lysed in 500 μl of urea lysis buffer [50 mM triethylammonium bicarbonate, 8 M urea, cCompleteTMThe lysate was lysed in 1:50 dilution of EDTA-free protease inhibitor cocktail (Roche, 11873580001), one PhosSTOP tablet (Roche, 4906845001), and 1 mM sodium orthovanadate. The lysate was sonicated on ice for ~10 minutes until clear. Protein concentration was measured using a BCA protein assay (Pierce #23227). 300 μg of protein was reduced with 5 mM Tris(2-carboxyethyl)phosphine hydrochloride (Sigma, C4706) for 20 minutes at 37°C and alkylated with 10 mM 2-chloroacetamide (Sigma, 22790) for 20 minutes at room temperature in the dark. Protein was digested with LysC for 3.5 hours at 30°C. The sample was then diluted with 50 mM triethylammonium bicarbonate (Sigma, T7408) to a 1.5 M urea concentration, and peptide digestion with trypsin was performed overnight at 37°C. The digestion reaction was quenched by adding 10% trifluoroacetic acid (EMD Millipore 302031-M) to a final pH of 2.0. Sample desalting was performed using a 35–350 μg C18 column (HMM S18V; The Nest Group, Inc., Southborough, MA, USA) according to the manufacturer's specifications. Phosphorylation enrichment was performed using TiO2 (Hichrome Titansphere TiO2, 10 μm capacity, 100 mg, GL Sciences #5020-75010). After loading the peptides onto the TiO2, the beads were washed sequentially with 1 M glycolic acid (Sigma #124737) / 80% acetonitrile / 5% trifluoroacetic acid, followed by 80% acetonitrile / 0.2% trifluoroacetic acid and 20% acetonitrile, before elution with 5% ammonium hydroxide. The concentrated sample was desalted using a 7-70 μg C18 column (HUM S18V; The Nest Group, Inc., Southborough, MA, USA) according to the manufacturer's specifications.nLC-MS / MS was performed on a Q-Exactive Orbitrap Plus coupled to an Easy-nLC 1000 (Thermo Scientific) and connected to a NANOSPRAY FLEX ion source. Fifty percent of each sample was analyzed as a 10 μl injection. Peptides were separated using a linear gradient from 5% to 30% acetonitrile / 0.1% formic acid over 180 min at a flow rate of 250 nl / min on a 75 μm diameter, 27 cm fused silica emitter packed with Reprosil-Pur 200 C18-AQ, 2.4 μm resin (Dr. Maisch, Ammerbuch-Entringen, Germany). Peptides were ionized by electrospray ionization via a micro-tee integrated into the nanospray source, with a voltage of 1.9 kV applied immediately before the analytical column. The ion transmission tube was heated to 320 °C and the S-lens was set to 60%. Precursor ions were measured in data-dependent mode on the Orbitrap analyzer at a resolution of 70,000 and a target of 3e6 ions. The 10 most intense ions from each MS1 scan were isolated, fragmented in the HCD cell, and measured in the Orbitrap at a resolution of 17,500.

[0294] Peptide identification, quantification, and statistical analysis of phosphoproteomics data Raw data were analyzed using MaxQuant84 (version 1.5.5.1) and searched against the mouse UniProt database (http: / / www.uniprot.org / , downloaded December 4, 2018) using default settings. Cysteine ​​carbamidomethylation was considered a fixed modification, while methionine oxidation, protein N-terminal acetylation, and phosphorylation (STY) were considered variable modifications. Enzyme specificity was set to trypsin, allowing a maximum of two missed cleavages. To ensure reliable identification, peptide-spectrum matches, peptides, and proteins were filtered at a false discovery rate (FDR) of less than 1%. Label-free quantification in MaxQuant was performed using an LFQ minimum ratio count of 2, Fast LFQ, and the "skip normalization" option. The "match between runs" function was selected with a 0.7-minute match time window and a 20-minute alignment time window. The "phospho(STY)Sites.txt" MaxQuant output file was processed with an in-house R script, and the accession number and corresponding phosphosite for each protein were merged to yield "Annotated_PhosphoSite.txt." This file, along with the "evidence.txt" MaxQuant output file and the experimental design "annotation.csv" file, were further processed by removing contaminants and reverse sequences, log2 data transformation, and removing phosphorylation sites with significant values ​​of 0 or 1 across all runs. High experimental reproducibility was observed, as evidenced by the average Pearson correlation coefficient r = 0.862 across biological replicates (Figure 16 / Extended Data Figure 8d). The quantified phosphopeptides were analyzed within the model-based statistical framework MSstats (version 3.20.0, run through RStudio (version 1.2.5042, R version 4.0.0)). Data were log2 transformed and quantile normalized, and a linear mixed-effects model was fitted to the data. Between-group comparison functions were used to test for differences in abundance between conditions.P values ​​were adjusted to control for FDR using the Benjamini-Hochberg procedure (Benjamini, Y. & Hochberg, JR Stat Soc B 57, 289-300 (1995)).

[0295] Mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium via the PRIDE24 partner repository under dataset identifier PXD027993. Reviewer account details (username: reviewer_pxd027993@ebi.ac.uk; password: FSaiKH6M).

[0296] Total internal reflection fluorescence (TIRF) microscopy of phosphorylated inositide reporters TIRF microscopy allows selective imaging of the small cell volume, including the plasma membrane, directly adjacent to the cell-seeded coverslip. HeLa or A549 cells were seeded at a density of 5,000 cells / well onto 8-well chamber slides (glass bottom, refractive index 1.55; Thermo Fisher Scientific #155409) coated with Matrigel (Corning #354230; diluted 1:50 with Opti-MEM). The next day, cells were transfected with 50 ng (A549) or 10 ng (HeLa) of the PIP3 reporter plasmid (GFP-PH-ARNOI303Ex2) (Goulden, B.D. et al. J Cell Biol 218, 1066-1079 (2019)) using FuGENE® HD Transfection Reagent (Promega #E2311) at a 3:1 Fugene:DNA ratio according to the manufacturer's instructions. To ensure low but uniform expression of the reporter in HeLa cells and to aid in identifying the critical TIRF angle for imaging, we also co-transfected these cells with 200 ng of iRFP-tagged Paxillin plasmid (generated by conventional restriction enzyme-based subcloning from the mCherry-Paxillin plasmid, Addgene #50526). In separate experiments, HeLa cells were also transfected with 10 ng or 50 ng of the PI(3,4)P2 reporter mCherry-cPH-TAPP1x3 (Goulden, B.D. et al. J Cell Biol 218, 1066-1079 (2019)); the use of 50 ng of this reporter facilitated visualization in the TIRF field but did not alter the kinetics of the response, so the results from both experiments were pooled.

[0297] After a further 24 hours post-transfection, cells were incubated with 150 μl of serum-free Fluorobrite. TMTime-lapse imaging was performed on a 3i Spinning Disk confocal microscope equipped with a TIRF sCMOS Prime95B (Photometric) sensor, switching to DMEM (Thermo Fisher Scientific #A1896701; supplemented with 2 mM L-glutamine and 1% penicillin-streptomycin) for 3 hours. A 100x 1.45NA planar apochromatic oil-immersion TIRF objective was used, with a laser illumination beam (40-50% output) at the critical angle of TIRF. Images were acquired by epifluorescence (300-500 msec exposure) using single bandpass filters (445 / 20 nm and 525 / 30 nm). Images were acquired in sequential mode without binning, using Slidebook 6.0, at 2- or 3-minute imaging speeds. Each treatment was added at 2x-5x concentrations to the same imaging medium at the specified times, ensuring that the final concentration was correct and thoroughly mixed with the existing medium solution. BYL719 (Advanced ChemBlocks Inc #R16000) was used at a high concentration of 0.5 μM (to achieve pan-class I PI3K inhibition).

[0298] Image analysis of total reporter intensity was performed using the Fiji open-source image analysis package. Regions of interest (ROIs) corresponding to individual cell footprints at each time point were defined using a minimum intensity projection method, selecting only pixels present across all time points. After pre-subtraction of camera noise (rolling ball method, radius = 500 pixels) and xy-drift correction, intensity levels over time were measured. These analyses were performed using custom-written FIJI / ImageJ macros. Another macro was used to normalize all pixels to the mean intensity (Ft / Fb) before treatment and generate scaled images. All other quantification was performed using the open-source software R / RStudio. All macros and analysis scripts are provided via the Open Science Framework (https: / / osf.io / gzxfm / ?view_only=8de666831f5b444087a0ab7c6cf3a636).

[0299] CellTiter-Glo® Cell Assay MEFs were seeded at 5000 cells per well in 96-well plates in DMEM supplemented with 10% FBS and 1% P / S and allowed to adhere overnight. The following day, cells were serum-starved for 4 hours before compound treatment with fresh serum-free DMEM. DMSO-solubilized compounds were diluted 1:2 in a 12-point concentration-response curve in DMSO. An intermediate plate was prepared by transferring 4 μl of compound in DMSO to 96 μl of serum-free DMEM medium. This was then used to treat the cell plate by transferring 12.5 μl of the solution from the intermediate plate to 87.5 μl of serum-free DMEM. Compound concentration-response curves were performed with a maximum concentration of 30 μM and a final well concentration of 0.5% DMSO. Cell plates were then incubated at 37°C, 5% CO2 for 24, 48, or 72 hours. Cell viability was then measured using CellTiter-Glo® reagent (Promega #G7571) according to the manufacturer's instructions. End-point luminescence was measured using a CLARIOstar (BMG). Compound data was analyzed using GraphPad Prism 8.

[0300] Measurement of cell proliferation by crystal violet staining MEFs were seeded at 5000 cells per well in 96-well plates in DMEM supplemented with 10% FBS and 1% P / S and allowed to adhere overnight. The next day, cells were serum-deprived for 5 hours with fresh serum-free DMEM before adding compounds. After different time points, cells were quickly washed with distilled water and then fixed and stained with a solution of 0.5% crystal violet (Sigma-Aldrich cat#C0775) in 20% methanol (v:v) (Feoktistova, M. et al. Cold Spring Harb Protoc 2016, pdb prot087379 (2016)). Briefly, after 20 minutes of incubation on a rocking platform at room temperature, fixed and stained cells were washed three times with distilled water, and the plate was air-dried overnight. Next, 200 μl of methanol was added to each well, and the plate was incubated on a bench rocker for 20 minutes at room temperature. The optical density was then measured over 570 time points using a plate reader.

[0301] Measurement of cell cycle progression by Edu staining We used the Click-IT EdU strategy according to the manufacturer's instructions (Sigma-Aldrich #BCK-FC488-50). Briefly, MEFs were seeded at 50,000 cells per well in 6-well plates in DMEM supplemented with 10% FBS and 1% P / S and allowed to adhere overnight. The following day, cells were serum-deprived for 5 hours with fresh serum-free DMEM before adding compounds. At different time points, cells were pulsed with 10 μM EdU for 3 hours, then harvested by trypsinization, fixed with 3.7% FA in PBS for 15 minutes in the dark, washed with 3% BSA, and permeabilized with 1x saponin-based permeabilization buffer for 20 minutes in the dark. EdU was then detected using the FAM-azide assay cocktail for 30 minutes in the dark. Cells were washed twice with 1x saponin-based permeabilization buffer and analyzed using a flow cytometer (Novocyte Advanteon Flow Cytometer, Agilent).

[0302] animal Adult (≥200 g) Wistar rats (Charles River, UK) were housed 4–5 per cage and maintained on a 14:10 h light:dark cycle with free access to food and water. All experiments were performed in accordance with the UK Animals Act (1986) and the European Communities Council Directive (86 / 609 / EEC) and with the approval of the University College London Animal Welfare and Ethical Review Board.

[0303] In vivo ischemia-reperfusion injury model in mice Male C57 / BL6 mice weighing 25–30 g were used. Animals received humane care in accordance with the UK Home Office Guide for the Operation of the Animals (Scientific Procedures) Act 1986, Project Licence PPL70 / 15358.

[0304] Animals were anesthetized intraperitoneally with 100 mg / kg pentobarbital sodium. Mice were tracheotomized and ventilated with room air using a small animal ventilator (MinVent, Type 845, Hugo Sachs Elektronik, Harvard Apparatus). Then, the mice were placed on a heating pad, and rectal temperature was monitored and maintained at ~37°C using a temperature controller. During the experiment, both electrocardiogram and heart rate were continuously recorded using a PowerLab (Adinstrument, USA). A thoracotomy was performed between the third and fourth ribs to expose the heart. A suture was placed around the left anterior descending coronary artery (LAD), followed by placement of a snare to allow occlusion and patency of the LAD. A cannula was placed in the left external jugular vein for drug administration.

[0305] A snare was tightened to occlude the LAD coronary artery, and the heart was subjected to 40 minutes of ischemia, as evidenced by ST-segment elevation on the electrocardiogram and a change in cardiac color. After 40 minutes, the snare was loosened, and the heart was reperfused for the next 120 minutes. 15 minutes before reperfusion, 50 μl of DMSO vehicle or 10 mg / kg of 1938 compound in DMSO was slowly infused via the jugular vein. The experimenters were blinded to the treatment groups.

[0306] After 120 minutes of reperfusion, the chest was opened again, the heart was excised, and the thoracic aorta was tubularly removed. The intracardiac blood was flushed with saline. The LAD coronary artery was then re-occluded with the suture that had been left loose after ischemia. The heart was then infused with 2% Evans blue to visualize the area at risk. These hearts were frozen at -80°C for 10 minutes and then cut into 5–6 0.5 mm-thick slices. These cardiac slices were incubated in triphenyltetrazolium chloride (10 mg / ml) solution at pH 7.4 at 37°C for 15 minutes to separate necrotic tissue (white areas) from viable tissue (red staining). The slices were then transferred to 10% formalin solution and fixed overnight. The cardiac slices, excluding the right ventricular wall, were scanned using a Cannon digital scanner. The total myocardial area, non-ischemic area (stained with Evans blue), and infarcted area (i.e., white areas) of each slice were measured using Image-J software. The "area at risk" was calculated by subtracting the non-ischemic area (blue area) from the total slice area and expressed as a percentage of the left ventricle. The "infarct size" was calculated as the ratio of the infarct area to the area at risk. Four mice died before reperfusion during the experiment (three in the DMSO group and one in the 1938 group) and were therefore excluded from analysis.

[0307] Tissue samples were analyzed by Western blotting as follows. Mice anesthetized and intubated as described above were injected via the jugular vein with 50 μl of DMSO vehicle or 10 mg / kg of 1938 compound in DMSO. After 15 minutes, the chest was opened, the hearts were removed, and freeze-clamped in liquid nitrogen. The hearts were then crushed using a pestle and mortar in lysis buffer [100 mM Tris.HCl, 300 mM NaCl, 1% IGEPAL, pH 7.4, supplemented with protease inhibitor (Product No. 78438; Thermo Fisher Scientific) and phosphatase inhibitor (Product No. 78427; Thermo Fisher Scientific)] and sonicated five times for 3 seconds on ice. The supernatant was then collected and analyzed by NuPAGE. TMAfter adding LDS Sample Buffer (4X) (Thermo Fisher Scientific), samples were boiled and stored at -80°C until SDS-polyacrylamide gel electrophoresis (SDS-PAGE). 20 μg of protein per well was loaded onto a 10% NuPAGE Bis-Tris gel (Invitrogen), separated by SDS-PAGE, and transferred to a PVDF membrane (Millipore) for Western blot analysis. The membrane was incubated with primary antibody in 5% BSA / TBS-0.1% Tween-20 overnight at 4°C, washed three times for 10 minutes with TBS-0.1% Tween, and then incubated with secondary antibody in 5% BSA / TBS-0.1% Tween for 1 hour and washed three times for 10 minutes with TBS-0.1% Tween. The antibodies used were a mouse monoclonal antibody against β-actin (Santa Cruz; sc-47778; used at 1:2000), a mouse monoclonal antibody against total Akt (Cell Signaling Technology; CST2920; used at 1:1000), and a rabbit antibody against phosphorylated Akt Thr308 (CST2965; used at 1:1000) or phosphorylated Akt Ser473 (CST9271; used at 1:1000) from Cell Signaling Technology. Secondary antibodies were IRDye 680LT goat anti-mouse and IRDye 800CW goat anti-rabbit (LI-COR Biosciences). Protein visualization and quantification were performed using the Odyssey Imaging System (LI-COR Biosciences).

[0308] Quantification of neurite outgrowth Dorsal root ganglion (DRG) neurons were isolated from adult male (>250 g) Wistar rats as described previously, and DRGs from each rat were cultured separately (Rayner, MLD et al. Anatomical Record 301, 1628-1637 (2018)). After culling under schedule 1 (elevated CO2), the spinal column was removed and stored on ice in PBS. The umbilical cord tissue was removed to expose the DRG and roots within the intervertebral foramen. The DRG were then removed using forceps and a scalpel under a dissecting microscope (Olympus SZ40). The DRG were manually cleaned by removing the roots, capsule, and capillaries with forceps and placed in DMEM supplemented with P / S. The DRG were treated with 0.125% collagenase type IV solution at 37°C for 90 minutes, then mechanically dissociated by trituration using a 1 ml pipette. The collagenase solution was removed by centrifugation twice at 400 × g for 5 min in complete DMEM (DMEM supplemented with 1% P / S and 10% FBS), and the DRG cell pellet was then resuspended in complete DMEM supplemented with 0.01 mM cytosine arabinoside. DRG were then cultured on a 75 cm plate coated with 0.1 mg / ml poly-D-lysine. 2DRGs were plated into flasks and incubated at 37°C, 5% CO2. After 24 hours, DRGs were trypsinized, resuspended, and centrifuged at 190 x g for 4 minutes to remove trypsin. The resulting cell pellet was mechanically suspended in Neurobasal-A medium (Gibco #10888022) supplemented with B-27 (Gibco #17504044), 2 mM L-glutamine (Merck #G7513), and 1% penicillin / streptomycin. DRGs were plated at a density of 1,000 cells / well in 0.1 mg / ml poly-D-lysine-coated, black-walled, clear-bottom 384-well plates (Greiner 781090). Prior to treatment, cells were washed to a uniform volume with supplemented Neurobasal-A medium using a BRAVO liquid handler (Agilent). 1938 solubilized at 3 mM in DMSO was diluted 1:3 in DMSO for an 8-point concentration-response curve. Using a BRAVO liquid handler, the drugs in the concentration-response curve were transferred to the middle plate and diluted in supplemented Neurobasal-A medium. The middle plate was then used to process the cell plate using a BRAVO liquid handler (final concentration of 0.1% DMSO in DRG culture medium). The PI3Kα inhibitor BYL-719 (final concentration 500 nM in DRG culture medium) or vehicle (0.005% DMSO in supplemented Neurobasal-A medium; total concentration 0.105% DMSO in DRG culture medium) was added 15 min before the addition of a concentration-response curve (1938). After 72 h of incubation at 37°C and 5% CO2, cells were fixed by adding 4% paraformaldehyde for 20 min. Wells were washed three times with PBS containing 0.05% Tween-20 (PBST) and then permeabilized with PBS containing 0.1% Triton X-100. Wells were washed three more times with PBST and then blocked with fish skin gelatin / PBST for 1 h at room temperature. The wells were then incubated with a primary antibody against the β-III tubulin neuronal marker (abcam). The cells were incubated with anti-rabbit Alexafluor-488 (1:2000, A-11008) overnight at 4°C. The next day, the cells were washed three times with PBST using a BRAVO liquid handler and then incubated with anti-rabbit Alexafluor-488 (1:2000, A-11008) for 1 hour at room temperature.Cells were washed three times with PBST using a BRAVO liquid handler and then stained with Hoechst 33342 nucleic acid stain (Thermo Scientific #62249; 1:2000) for 20 minutes in the dark. Cells were washed three more times with PBST and three times with PBS, and the cell plates were stored at 4°C in the dark before imaging. Images were acquired using a 20x water objective with an Opera (PerkinElmer) high-content screening system. Images of cell nuclei and β-III tubulin-positive cells were captured using excitation / emission wavelengths of λ380 / 455 and λ490 / 518, respectively. Nine fields were captured per well and analyzed using CSIRO Neurite Analysis 2 logarithm in Columbus analysis software (PerkinElmer). Neurites were defined using the following parameters: smoothing window 0 pixels (px), linear window 15 px, contrast >1.5, diameter ≥3 px, gap closure distance ≤17 px, gap closure quality 0, deverb length ≤40 px, body thickening 1 px, and tree length ≤0 px. Each experiment was performed in quadruplicate, and data are expressed as the mean ± standard error of the mean for three biological replicates (n = 3). Variable slope nonlinear regression (4 parameters) was performed in Prism 7. Representative images of whole wells were taken using a Cytation 3 (Biotek) imaging plate reader with a 10x objective. Image montages were captured before stitching and deconvolution using Gen 5 software (Biotek). Images of cell nuclei and β-III tubulin-positive cells were captured using excitation / emission wavelengths of λ380 / 455 and λ490 / 518, respectively.

[0309] Control experiments for the neuroclastic assay Experiments to investigate the stability of 1938 in aqueous solution and the biological activity of 1938 on the sciatic nerves of exposed rats were carried out as follows.

[0310] Lyophilized 1938 was solubilized to 100 μM in autoclaved dH2O. Solubilization required 25 minutes of sonication at 30°C before passing through a 0.22 μm filter. Aliquots of 1938 (5 μM and 100 μM) or vehicle were frozen at -20°C for later use on separate experimental days. Aliquots of 100 μM TRO-1938 and vehicle were thawed and tested for activity on A549 cells (Figure 17 / Extended Data Figure 9, left panel). Cells were seeded at 20,000 cells / well in 24-well plates in DMEM+Glutamax supplemented with 10% FBS and 1% Pen / Strep. Prior to treatment, cells were washed and incubated in serum-free DMEM+Glutamax. Cells were treated with a 1:3 dose response of 1938 diluted in serum-free DMEM + Glutamax, starting at 10 μM, for 15 minutes at 37°C. Cells were then washed with ice-cold PBS and lysed in RIPA buffer supplemented with protease and phosphatase inhibitors. Lysates were analyzed by automated Western blot (Wes) (Figure 17 / Extended Data Figure 9; left panel).

[0311] In a test experiment to evaluate whether 1938 can induce pAkt production in exposed rat sciatic nerves, adult male Sprague-Dawley rats (≥250 g; n = 2) were anesthetized with isoflurane. The sciatic nerve in the left leg was exposed and injected with 2 μl of vehicle (sterile dH2O) or 1938 (5 μM in sterile dH2O). Meanwhile, the sciatic nerve in the right leg was exposed and injected with 250 μl of vehicle (sterile dH2O) or 1938 (5 μM in sterile dH2O). Each animal received one vehicle and one compound treatment. Following treatment, the applied nerve was washed with sterile PBS and allowed to sit for 30 minutes before culling the animal via sodium pentobarbital injection in accordance with local regulations. The nerve was then harvested, washed with fresh 4°C PBS, and stored in a fresh vial before snap-freezing in liquid nitrogen. Frozen sciatic nerves were homogenized using a mortar and pestle in RIPA buffer supplemented with protease and phosphatase inhibitors. The crude lysates were then centrifuged at 10,000 x g for 10 minutes at 4°C. The supernatants were collected and stored at -80°C before automated Western blot (Wes) analysis of pAkt and controls (Fig. 17 / Extended Data Fig. 9, right panel).

[0312] Rat sciatic nerve crush injury and 1938 treatment Adult female Sprague Dawley rats (230–280 g, n = 10, Charles River, UK) were anesthetized with isoflurane inhalation in an induction chamber (5% isoflurane in O2, 0.8 l / min). Anesthesia was maintained with 1.5–2.5% isoflurane inhalation, and the left sciatic nerve was exposed at the mid-thigh.

[0313] The nerve was crushed using fully closed sterile type 4 forceps (TAAB) with constant pressure for 15 seconds. This was repeated two more times at the same location, with a 45° rotation between each crush. The injury site was marked with 10 / 0 epineural non-absorbable suture (Ethicon). After injury, frozen aliquots of 1938 solution and vehicle were thawed. A single 2 μl injection of 1938 solution (5 μM in sterile H2O) or vehicle (sterile dH2O) was administered proximal to the crush site using a 10 μl Hamilton syringe. An osmotic minipump (Alzet 1004, Charles River, UK) was also implanted adjacent to the nerve, between the myenteric layers, with the outlet closest to the crush site and loaded with 1938 solution (100 μM in sterile H2O) or vehicle (sterile H2O). Animals were randomly assigned to groups (n = 5 per group), blinded to the conditions, and functional and histological analyses were performed by a single investigator. The muscle layer was closed with 4 / 0 sutures (Ethicon), and the skin was closed with wound clips (Clay Adams). Animals were allowed to recover for 21 days.

[0314] Functional evaluation of muscle regeneration At the experimental endpoint (day 21), rats were anesthetized, and the sciatic nerve was exposed as described above. Reference, ground, and recording electrodes (Natus, Ambu Neuroline) were attached to the tail, the lumbar spine, and the tibialis anterior muscle, respectively. A microchannel neurointerface (MNI) was placed approximately 2 mm proximal to the injury site and used to stimulate the nerve. The MNI was fabricated using a previously published protocol (Lancashire, HT et al. J Neural Eng 13, 034001 (2016)). The MNI electrode impedance was 27.1 ± 19.8 kΩ at 1 kHz. Compound muscle action potentials (CMAPs) were obtained by stimulating the sciatic nerve with 100 μsec square-wave pulses at intensities ranging from 1 to 10 mA. Stimulation was increased in 0.2 mA steps until muscle response amplitude no longer increased. CMAP amplitude was measured peak-to-peak and recorded in triplicate on both the ipsilateral and contralateral sides. The CMAP with the largest amplitude was selected for analysis.

[0315] Motor unit number estimates (MUNEs) were calculated using a modified multipoint stimulation method (Shefner, JM et al. Muscle & nerve 34, 603-607 (2006); Jacobsen, AB et al. J Vis Exp (2018); Arnold, WD et al. J Vis Exp (2015)). To obtain a minimal response, incremental responses were obtained by administering submaximal stimulation of 100 μsec duration at a frequency of 1 Hz, increasing the stimulation intensity by 0.02 mA increments. Initial responses were obtained with stimulation intensities ranging from 0.21 mA to 0.70 mA. If no initial responses occurred between these intensities, the stimulation electrode was adjusted to increase or decrease the stimulation intensity as needed. Additional single motor unit potentials (SMUPs) were elicited by stimulating in 0.02 mA increments, resulting in a minimum of four additional increments. The stimulating and recording electrode positions were varied to record SMUPs from different muscle regions. This process was repeated at least three times. MUNE was quantified by dividing CMAP by the mean value of SMUP.

[0316] Harvesting and processing of the sciatic nerve After electrophysiological recordings, animals were culled by sodium pentobarbital injection according to local regulations. The sciatic nerve, including the common peroneal branch, and the tibialis anterior muscle were harvested and placed in 4% paraformaldehyde (PFA). After 15 minutes, the muscle was transferred to phosphate-buffered saline (PBS) and stored at 4°C until processing. Nerve samples were fixed overnight in 4% PFA at 4°C before being transferred to PBS. The nerve samples were separated into the sciatic nerve, including the crush site, and the common peroneal branch for sectioning. The nerve samples were immersed in 30% sucrose overnight at 4°C and snap-frozen in Neg-50 cryosection medium (Thermo Scientific) using liquid nitrogen-cooled isopentane. Transverse sections (10 μm) were cut from the distal segment of the common peroneal nerve using a cryostat (HM535, Thermo Scientific). Transverse frozen sections (15 μm) were cut from the sciatic nerve 3 mm and 6 mm distal to the crush site and attached to glass slides (Superfrost Plus, Thermo Fisher Scientific) for immunofluorescence staining.

[0317] For immunofluorescence staining, all washes and dilutions were performed using immunostaining buffer (PBS containing 0.002% sodium azide and 0.3% Triton-X 100). For antigen removal, slides were heated at 37°C for 20 minutes and blocked with 5% normal horse serum for 40 minutes. The sections were then incubated with primary antibodies overnight at 4°C, followed by secondary antibodies at room temperature for 45 minutes. The following antibodies were used: mouse anti-neurofilament (Biolegend 835604, 1:500), goat anti-choline acetyltransferase (Millipore AB144P, 1:50), DyLight anti-mouse IgG 549 (Vector DI-2549, 1:300), and DyLight anti-goat IgG 488 (Vector DI-1488, 1:300). Slides were coverslipped with Vectashield Hardset mounting medium (Vector, H-1400).

[0318] Fluorescence microscopy (Zeiss AxiolabA1, Axiocam Cm1) was performed to quantify motor axons (ChAT) in the distal segment of the common peroneal nerve. For analysis of sciatic nerve sections, confocal tile scans (Zeiss LSM 710, 20x magnification) were performed on each transverse section at 3 and 6 mm distal to the crush injury. Volocity™ software (Perkin Elmer, Waltham, MA) was used to quantify all neurofilament-positive axons.

[0319] Muscle harvesting and processing Tibialis anterior muscles were fixed in 4% PFA for up to 15 minutes, embedded at optimal cutting temperature (OCT), and then either snap-frozen in liquid nitrogen-cooled isopentate or left in immunostaining buffer until ready for processing. Transverse 20 μm cryosections were taken at 300 μm intervals. A minimum of 20 sections from each sample were taken across the entire muscle cross-section and mounted on glass slides for immunofluorescence staining.

[0320] All washes and dilutions were performed using immunostaining buffer (PBS containing 0.002% sodium azide and 0.3% Triton-X100). Slides were heated to 42°C with 20 μg / ml proteinase K for 30 minutes and blocked with 10% goat serum for 40 minutes at room temperature. After washing, sections were incubated with primary antibody (neurofilament, Biolegend 835604, 1:500), washed, and then incubated with DyLight anti-mouse IgG 488 (Vector DI-2488, 1:300) and α-bungarotoxin (Alexa 594 conjugate, ThermoFisher Scientific, 1:1000). Sections were mounted using Vectashield Hardset mounting medium.

[0321] The percentage of reinnervated motor endplates was quantified by measuring the percentage of motor endplates that co-stained with neurofilament (α-bungarotoxin) using a fluorescence microscope (Zeiss AxiolabA1, Axiocam Cm1). For each sample, a minimum of 20 non-overlapping regions across the muscle cross section were analyzed.

[0322] For statistical analysis, data from 1938-treated animals were compared with data from vehicle-treated animals using an unpaired t = test (Graphpad Prism 8.0.0).

[0323] statistical methods Statistical methods for different types of experiments are included in each experimental section above.

[0324] compound synthesis experiment Chemicals and solvents were commonly used without further purification. Chromatographic purification was performed using a Biotage Isolera Purification system (Uppsala, Sweden) with prepacked SNAP columns. Microwave-assisted reactions were performed in sealed vials with Biotage Initiator TMThe synthesis was carried out using a microwave synthesizer. Deuterated solvents were obtained from Sigma-Aldrich.

[0325] NMR spectra were recorded on a Bruker 400 MHz or 500 MHz spectrometer. Chemical shifts are given in ppm relative to the solvent peak, and coupling constants (J) are reported in Hz.

[0326] LCMS spectra were obtained using one of the following methods: LCMS Method A: A Waters LCMS system (Waters Micromass ZQ mass spectrometer connected to a Waters 2000 Series HPLC) was used. Analysis was performed using a Gemini column (3.0 μM, NX-C18, 110A, 50 x 4.6 mm). Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of 0.1% formic acid in HPLC-grade acetonitrile. A 5.0-minute gradient was run from 99% mobile phase A to 100% mobile phase B at a flow rate of 1.00 mL min-1.

[0327] LCMS Method B: Agilent LCMS system (Agilent 6140 Series Quadrupole Mass Spectrometer with Multimode Light Source connected to Agilent 1200 Series HPLC). A kinetic column (2.6 μM, EVO, C18, 100A, 50 x 2.1 mm) was used for the analysis. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in HPLC-grade acetonitrile. A 5.5-minute gradient was run from 99% mobile phase A to 100% mobile phase B at a flow rate of 1.00 mL min-1.

[0328] LCMS Method C: Shimadzu LCMS2020 system. A Waters X-Bridge™ column (2.5 μM, MS C18, 100A, 50 x 3.0 mm) was used for the analysis. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in HPLC-grade acetonitrile. A 4.0-minute gradient was run from 99% mobile phase A to 100% mobile phase B at a flow rate of 1.00 mL min-1.

[0329] [ka]

[0330] Intermediate A1 [ka]

[0331] 1-(7-aminoindolin-1-yl)propan-1-one Step a: To propanoyl chloride (0.72 mL, 8.23 ​​mmol) in chloroform (10 mL) was added triethylamine (0.63 mL, 4.53 mmol) and 5-bromo-7-nitro-indoline (500 mg, 2.06 mmol), and the reaction mixture was stirred at rt for 18 h. Purification by Biotage Isolera afforded 1-(5-bromo-7-nitroindolin-1-yl)propan-1-one (571 mg, 92% yield). 1 H NMR (CDCl3, 500 MHz): 7.77 (s, 1H), 7.52 (s, 1H), 4.23 (t, J = 8.1 Hz, 2H), 3.22 (t, J = 8.1 Hz, 2H), 2.49 (q, J = 7.4 Hz, 2H), 1.22 (t, J = 7.4 Hz, 3H).

[0332] Step b: To a stirred solution of 1-(5-bromo-7-nitro-indolin-1-yl)propan-1-one (300 mg, 1 mmol) in methanol (4 mL) was added ammonium formate (632 mg, 10.03 mmol) and Pd / C (11 mg, 0.10 mmol), the reaction mixture was stirred at rt for 18 h, the reaction mixture was filtered through Celite, and the filtrate was concentrated in vacuo under reduced pressure to give the title compound (176 mg, 92% yield). 1H NMR (CDCl3, 500 MHz): 6.97 (t, J = 7.7 Hz, 1H), 6.66 (d, J = 8.1 Hz, 1H), 6.62 (d, J = 8.0 Hz, 1H), 4.79 (br s, 2H), 4.07 (t, J = 7.8 Hz, 2H), 3.06 (t, J = 7.8 Hz, 2H), 2.58 (q, J = 7.4 Hz, 2H), 1.28 (t, J = 7.5 Hz, 3H).

[0333] Intermediate A2: [ka]

[0334] 1-(7-aminoindolin-1-yl)-2-methylpropan-1-one Intermediate A1 was synthesized using the procedure described above, but in step a, isobutyryl chloride. 1 H NMR (CDCl3, 400 MHz): 6.97 (t, J = 7.4 Hz, 1H), 6.66 (dd, J = 7.3 and 1.0 Hz, 1H), 6.60 (dd, J = 8.0 and 1.0 Hz, 1H), 4.70 (br s, 2H), 4.12 (t, J = 7.7 Hz, 2H), 3.06 (t, J = 7.7 Hz, 2H), 2.94 (sept, J = 7.0 Hz, 1H), 1.29 (s, 3H), 1.27 (s, 3H).

[0335] Intermediate A3: [ka]

[0336] 1-(7-aminoindolin-1-yl)-2-methoxyethan-1-one Intermediate A1 was synthesized using the procedure described above, but using methoxyacetyl chloride in step a. 1H NMR (CDCl3, 400 MHz): 6.97 (t, J = 7.7 Hz, 1H), 6.65 (d, J = 8.1 Hz, 1H), 6.58 (d, J = 8.0 Hz, 1H), 4.26 (s, 2H), 4.03 (t, J = 7.7 Hz, 2H), 3.50 (s, 3H), 3.05 (t, J = 7.7 Hz, 2H).

[0337] Intermediate A4: [ka]

[0338] 1-(7-aminoindolin-1-yl)-3-methoxypropan-1-one Intermediate A1 was synthesized using the procedure described above, using 3-methoxypropanoyl chloride in step a. 1 H NMR (CDCl3, 400 MHz): 6.95 (t, J = 7.6 Hz, 1H), 6.64 (d, J = 7.2 Hz, 1H), 6.58 (d, J = 8.0 Hz, 1H), 4.11 (t, J = 7.7 Hz, 2H), 3.79 (t, J = 6.5 Hz, 2H), 3.38 (s, 3H), 3.03 (t, J = 7.7 Hz, 2H), 2.81 (t, J = 6.5 Hz, 2H).

[0339] [ka]

[0340] Intermediate B1 [ka]

[0341] 1-(7-((2-chloropyridin-4-yl)amino)indolin-1-yl)ethan-1-one A mixture of 2-chloro-4-iodopyridine (1 g, 4.2 mmol), 1-(7-aminoindolin-1-yl)ethanone (662 mg, 3.8 mmol), XANTPHOS (145 mg, 0.25 mmol), palladium acetate (38 mg, 0.17 mmol), and cesium carbonate (2.71 g, 8.35 mmol) in 1,4-dioxane (4 mL) was heated in a microwave at 80 °C for 1 h. After cooling to room temperature, the mixture was filtered through Celite and washed with ethyl acetate. The filtrate was concentrated under reduced pressure and triturated with diethyl ether / petrol to give the title compound as a yellow solid (887 mg, 84%). 1 H NMR (CDCl3, 400 MHz) δ 8.67 (s, 1H), 7.98 (d, J = 5.7 Hz, 1H), 7.27 (d, J = 8.1 Hz, 1H), 7.18 (t, J = 7.7 Hz, 1H), 7.00 (dq, J = 7.2, 1.1 Hz, 1H), 6.82 (d, J = 2.1 Hz, 1H), 6.70 (dd, J = 5.8, 2.1 Hz, 1H), 4.13 (t, J = 7.7 Hz, 2H), 3.15 (t, J = 7.8 Hz, 2H), 2.35 (s, 3H). LC-MS method A; RT 2.21; m / z [M+H] + 288.0.

[0342] Intermediate B2 [ka]

[0343] 2-chloro-N-(1-methyl-1H-pyrazol-3-yl)pyridin-4-amine Intermediate B1 was synthesized using the procedure described above using 2-chloro-4-iodopyridine and 3-amino-1-methyl-1H-pyrazole. 1H NMR (CDCl3, 400 MHz) δ 8.05 (d, J = 5.8 Hz, 1H), 7.29 (d, J = 2.3 Hz, 1H), 7.10 (d, J = 2.1 Hz, 1H), 6.87 (dd, J = 5.8, 2.1 Hz, 1H), 6.58 (s, 1H), 5.98 (d, J = 2.3 Hz, 1H), 3.85 (s, 3H).

[0344] Intermediate B3 [ka]

[0345] 2-Chloro-N-phenylpyridin-4-amine Intermediate B1 was synthesized using the procedure described above using 2-chloro-4-iodopyridine and aniline. 1 H NMR (DMSO-d6, 400 MHz) δ 9.08 (s, 1H), 7.98 (d, J = 5.8 Hz, 1H), 7.43 - 7.35 (m, 2H), 7.21 (dd, J = 7.5, 1.1 Hz, 2H), 7.11 (td, J = 7.3, 1.2 Hz, 1H), 6.85 (dd, J = 5.8, 2.1 Hz, 1H), 6.82 (d, J = 2.1 Hz, 1H).

[0346] Intermediate B4 [ka]

[0347] 2-chloro-N-(1-methyl-1H-pyrazol-4-yl)pyridin-4-amine Intermediate B1 was synthesized using the procedure described above using 2-chloro-4-iodopyridine and 1-methylpyrazol-4-amine. 1H NMR (CDCl3, 400 MHz) δ 7.95 (d, J = 5.8 Hz, 1H), 7.42 (s, 1H), 7.37 (s, 1H), 6.58 (d, J = 2.2 Hz, 1H), 6.50 (dd, J = 5.8, 2.0 Hz, 1H), 5.92 (s, 1H), 3.92 (s, 3H).

[0348] Intermediate B5 [ka]

[0349] 1-(5-((2-chloropyridin-4-yl)amino)indolin-1-yl)ethan-1-one Intermediate B1 was synthesized using the procedure described above using 2-chloro-4-iodopyridine and 1-(5-aminoindolin-1-yl)ethanone. 1 H NMR (CDCl3, 500 MHz) δ 8.24 - 8.20 (m, 1H), 8.00 (d, J = 5.7 Hz, 1H), 7.05 - 6.96 (m, 3H), 6.90 (q, J = 1.9 Hz, 1H), 6.70 (d, J = 2.1 Hz, 1H), 6.60 (dd, J = 5.8, 2.1 Hz, 1H), 4.11 (t, J = 8.5 Hz, 2H), 3.23 (t, J = 8.4 Hz, 2H), 2.25 (s, 3H).

[0350] Intermediate B6 [ka]

[0351] 1-(6-((2-chloropyridin-4-yl)amino)indolin-1-yl)ethan-1-one Intermediate B1 was synthesized using the procedure described above using 2-chloro-4-iodopyridine and 1-(6-aminoindolin-1-yl)ethanone. 1H NMR (MeOD, 500 MHz) δ 8.04 (s, 1H), 7.88 (d, J = 5.8 Hz, 1H), 7.23 (d, J = 7.9 Hz, 1H), 6.89 (d, J = 8.0 Hz, 1H), 6.82 (d, J = 10.1 Hz, 2H), 4.18 (t, J = 8.3 Hz, 2H), 3.19 (d, J = 8.5 Hz, 2H), 2.24 (s, 3H).

[0352] Intermediate B14 [ka]

[0353] 2-((2-chloropyridin-4-yl)amino)-N-methylbenzamide Intermediate B1 was synthesized using the procedure described above using 2-chloro-4-iodopyridine and 2-amino-N-methyl-benzamide. 1 H NMR (CDCl3, 400 MHz) δ 9.67 (s, 1H), 8.10 (d, J = 5.8 Hz, 1H), 7.54 (dd, J = 8.2, 1.2 Hz, 1H), 7.50 - 7.44 (m, 2H), 7.08 - 7.02 (m, 2H), 6.87 (dd, J = 5.7, 2.1 Hz, 1H), 6.22 (s, 1H), 3.01 (d, J = 4.8 Hz, 3H).

[0354] Intermediate B15 [ka]

[0355] 1-(7-((2-chloro-6-methylpyridin-4-yl)amino)indolin-1-yl)ethan-1-one Intermediate B1 was synthesized using the procedure described above using 2,4-dichloro-6-picoline and 1-(7-aminoindolin-1-yl)ethanone. 1H NMR (CDCl3, 400 MHz) δ 9.00 (s, 1H), 7.75 (dd, J = 8.2, 1.1 Hz, 1H), 7.14 (dd, J = 8.2, 7.3 Hz, 1H), 6.90 (dq, J = 7.4, 1.1 Hz, 1H), 6.62 - 6.58 (m, 1H), 6.56 (d, J = 1.5 Hz, 1H), 4.09 (t, J = 7.8 Hz, 2H), 3.13 - 3.06 (m, 2H), 2.39 (s, 3H), 2.36 (s, 3H).

[0356] Intermediate B16 [ka]

[0357] 1-(7-((2-chloropyridin-4-yl)amino)indolin-1-yl)-2-methylpropan-1-one Intermediate B1 was synthesized using the procedure described above using 2-chloro-4-iodopyridine and Intermediate A2. 1 H NMR (CDCl3, 400 MHz): 8.50 (s, 1H), 8.00 (d, J = 5.8 Hz, 1H), 7.19 (t, J = 7.4 Hz, 1H), 7.14 (d, J = 7.5 Hz, 1H), 7.03 (dd, J = 7.3 and 1.0 Hz, 1H), 6.82 (d, J = 2.0 Hz, 1H), 6.70 (dd, J = 5.8 and 2.1 Hz, 1H), 4.19 (t, J = 7.7 Hz, 2H), 3.15 (t, J = 7.7 Hz, 2H), 2.94 (sept, J = 6.7 Hz, 1H), 1.29 (s, 3H), 1.27 (s, 3H).

[0358] Intermediate B17 [ka]

[0359] 1-(7-((2-chloropyridin-4-yl)amino)indolin-1-yl)-2-methoxyethan-1-one Intermediate B1 was synthesized using the procedure described above using 2-chloro-4-iodopyridine and Intermediate A3. 1 H NMR (CDCl3, 400 MHz): 8.50 (s, 1H), 8.00 (d, J = 5.8 Hz, 1H), 7.31 (d, J = 7.6 Hz, 1H), 7.19 (t, J = 7.4 Hz, 1H), 7.04 (d, J = 6.2 Hz, 1H), 6.84 (d, J = 2.0 Hz, 1H), 6.73 (dd, J = 5.8 and 2.1 Hz, 1H), 4.30 (s, 2H), 4.14 (t, J = 7.7 Hz, 2H), 3.50 (s, 3H), 3.17 (t, J = 7.8 Hz, 2H)

[0360] Intermediate B18 [ka]

[0361] 1-(7-((2-chloro-3-fluoropyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized as described for intermediate B1 using 2-chloro-3-fluoro-4-iodo-pyridine and 1-(7-aminoindolin-1-yl)ethanone. 1 H NMR (CDCl3, 500 MHz): 9.04 (s, 1H), 7.82 (d, J = 5.6 Hz, 1H), 7.27 (d, J = 8.1 Hz, 1H), 7.20 (t, J = 7.5 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 6.97 (t, J = 5.8 Hz, 1H), 4.16 (t, J = 7.9 Hz, 2H), 3.18 (t, J = 7.9 Hz, 2H), 2.38 (s, 3H).

[0362] Intermediate B19 [ka]

[0363] 1-(7-((2-chloropyridin-4-yl)amino)-5-methylindolin-1-yl)ethan-1-one Synthesized using 2-chloro-4-iodopyridine and 1-(7-amino-5-methyl-indolin-1-yl) as described for intermediate B1. 1 H NMR (CDCl3, 500 MHz): 8.75 (s, 1H), 8.00 (d, J = 5.8 Hz, 1H), 7.09 (s, 1H), 6.84-6.83 (m, 2H), 6.72 (dd, J = 5.8 and 2.1 Hz, 1H), 4.12 (t, J = 7.7 Hz, 2H), 3.11 (t, J = 7.8 Hz, 2H), 2.36 (s, 3H), 2.35 (s, 3H).

[0364] Intermediate B20 [ka]

[0365] 1-(7-((2-chloropyridin-4-yl)amino)indolin-1-yl)propan-1-one Intermediate B1 was synthesized using 2-chloro-4-iodopyridine (and Intermediate A1) as described above. 1H NMR (CDCl3, 500 MHz): 8.69 (s, 1H), 7.98 (d, J = 5.8 Hz, 1H), 7.27 (d, J = 5.6 Hz, 1H), 7.16 (t, J = 7.5 Hz, 1H), 7.00 (d, J = 7.3 Hz, 1H), 6.81 (d, J = 1.9 Hz, 1H), 6.70 (dd, J = 5.8 and 2.0 Hz, 1H), 4.12 (t, J = 7.8 Hz, 2H), 3.13 (t, J = 7.8 Hz, 2H), 2.58 (q, J = 7.4 Hz, 2H), 1.27 (t, J = 7.4 Hz, 3H).

[0366] Intermediate B21 [ka]

[0367] 1-(7-((2-chloropyridin-4-yl)amino)indolin-1-yl)-3-methoxypropan-1-one Intermediate B1 was synthesized using the procedure described above using 2-chloro-4-iodopyridine and Intermediate A4. 1 H NMR (CDCl3, 400 MHz): 8.55 (s, 1H), 8.00 (d, J = 5.8 Hz, 1H), 7.29 (d, J = 5.8 Hz, 1H), 7.20 (t, J = 7.5 Hz, 1H), 7.03 (d, J = 8.1 Hz, 1H), 6.83 (d, J = 2.0 Hz, 1H), 6.71 (dd, J = 5.8 and 2.1 Hz, 1H), 4.22 (t, J = 7.8 Hz, 2H), 3.82 (t, J = 6.2 Hz, 2H), 3.40 (s, 3H), 3.14 (t, J = 7.7 Hz, 2H), 2.84 (t, J = 6.2 Hz, 2H).

[0368] Intermediate B22 [ka]

[0369] 2-chloro-N-(1-ethyl-3-phenyl-1H-pyrazol-5-yl)pyridin-4-amine Intermediate B1 was synthesized using the procedure described above using 2-chloro-4-iodopyridine and 2-methyl-5-phenyl-pyrazol-3-amine. 1 H NMR (CDCl3, 400 MHz): 8.07 (d, J = 5.7 Hz, 1H), 7.77 (d, J = 7.1 Hz, 2H), 7.41 (t, J = 7.2 Hz, 2H), 7.33 (t, J = 2.0 Hz, 1H), 6.54 (dd, J = 5.7 and 2.2 Hz, 1H), 6.45 (s, 1H), 6.06 (br s, 1H), 3.76 (s, 3H).

[0370] Intermediate B25 [ka]

[0371] N-(3-(tert-butyl)-1-methyl-1H-pyrazol-5-yl)-2-chloropyridin-4-amine Synthesized as described for intermediate B1 using 2-chloro-4-iodopyridine and 5-tert-butyl-2-methyl-pyrazol-3-amine. 1 H NMR (CDCl3, 400 MHz): 8.06 (d, J = 5.7 Hz, 1H), 6.56 (d, J = 2.1 Hz, 1H), 6.47 (d, J = 2.2 Hz, 1H), 5.99 (s, 1H), 4.75 (s, 1H), 3.66 (s, 3H), 1.31 (s, 9H).

[0372] Intermediate B27 [ka]

[0373] 2-chloro-N-(3-isopropyl-1-methyl-1H-pyrazol-5-yl)pyridin-4-amine Synthesized as described for intermediate B1 using 2-chloro-4-iodopyridine and 5-isopropyl-2-methyl-pyrazol-3-amine. 1 H NMR (CDCl3, 400 MHz): 8.04 (d, J = 5.7 Hz, 1H), 6.56 (d, J = 2.0 Hz, 1H), 6.48 (dd, J = 5.7 and 2.2 Hz, 1H), 5.95 (s, 1H), 3.65 (s, 3H), 2.94 (sept, J = 6.9 Hz, 1H), 1.27 (s, 3H), 1.26 (s, 3H).

[0374] Intermediate B28 [ka]

[0375] 3-((2-chloropyridin-4-yl)amino)-N-methylbenzamide Intermediate B1 was synthesized using the procedure described above using 2-chloro-4-iodopyridine and 3-amino-N-methylbenzamide. 1 H NMR (MeOD, 400 MHz): 7.96 (d, J = 6.6 Hz, 1H), 7.69 (t, J = 1.8 Hz, 1H), 7.59-7.57 (m, 1H), 7.50 (t, J = 7.7 Hz, 1H), 7.42-7.39 (m, 1H), 6.93-6.91 (m, 2H), 2.95 (s, 3H).

[0376] Intermediate B29 [ka]

[0377] 4-((2-chloropyridin-4-yl)amino)-N-methylbenzamide Intermediate B1 was synthesized using the procedure described above using 2-chloro-4-iodopyridine and 4-amino-N-methylbenzamide. 1 H NMR (MeOD, 400 MHz): 8.01 (d, J = 5.8 Hz, 1H), 7.86 (d, J = 8.8 Hz, 2H), 7.30 (d, J = 8.8 Hz, 2H), 7.02-6.99 (m, 2H), 2.95 (s, 3H).

[0378] Intermediate B30 [ka]

[0379] N-(3-(1H-imidazol-1-yl)phenyl)-2-chloropyridin-4-amine Intermediate B1 was synthesized using the procedure described above using 2-chloro-4-iodopyridine and 3-imidazol-1-ilaniline. 1 H NMR (MeOD, 400 MHz): 8.15 (s, 1H), 7.98 (d, J = 6.4 Hz, 1H), 7.58 (s, 1H), 7.54 (t, J = 8.1 Hz, 1H), 7.38 (t, J = 2.0 Hz, 1H), 7.34-7.32 (m, 1H), 7.31-7.28 (m, 1H), 7.16 (s, 1H), 6.97-6.95 (m, 2H).

[0380] Intermediate B31 [ka]

[0381] 2-chloro-N-(3-(methylsulfonyl)phenyl)pyridin-4-amine Intermediate B1 was synthesized using the procedure described above using 2-chloro-4-iodopyridine and 3-methylsulfonylaniline. 1H NMR (MeOD, 400 MHz): 8.01 (d, J = 6.6 Hz, 1H), 7.75 (t, J = 1.7 Hz, 1H), 7.69-7.67 (m, 1H), 7.64 (t, J = 7.7 Hz, 1H), 7.58-7.56 (m, 1H), 6.96-6.95 (m, 2H), 3.15 (s, 3H).

[0382] Intermediate B32 [ka]

[0383] N-(2-chloropyridin-4-yl)pyrimidin-4-amine Intermediate B1 was synthesized using the procedure described above using 2-chloro-4-iodopyridine and pyrimidin-4-amine. 1 H NMR (MeOD, 400 MHz): 8.78 (d, J = 0.6 Hz, 1H), 8.39 (d, J = 6.0 Hz, 1H), 8.16 (d, J = 5.8 Hz, 1H), 8.08 (d, J = 1.9 Hz, 1H), 7.62 (dd, J = 5.8 and 2.0 Hz, 1H), 6.91 (dd, J = 6.0 and 1.2 Hz, 1H).

[0384] Intermediate B33 [ka]

[0385] 1-(7-((2-chloro-3-methylpyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized as described for intermediate B1 using 2-chloro-4-iodo-3-methyl-pyridine and 1-(7-aminoindolin-1-yl)ethanone. 1H NMR (CDCl3, 400 MHz): 8.65 (s, 1H), 7.87 (d, J = 5.7 Hz, 1H), 7.23 (d, J = 8.1 Hz, 1H), 7.17 (t, J = 7.2 Hz, 1H), 7.00 (dd, J = 7.2 and 1.1 Hz, 1H), 6.84 (d, J = 5.7 Hz, 1H), 4.14 (t, J = 7.8 Hz, 2H), 3.16 (t, J = 7.8 Hz, 2H), 2.42 (s, 3H), 2.36 (s, 3H).

[0386] [ka]

[0387] Intermediate C1 [ka] 4-chloro-N-(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)pyridin-2-amine

[0388] A 20 mL microwave vial evacuated and backfilled with argon was charged with 2-methoxy-4-(4-methylpiperazin-1-yl)aniline (0.60 g, 2.71 mmol), Pd(OAc) (12 mg, 0.054 mmol), cesium carbonate (1.76 g, 5.41 mmol), and XANTPHOS (47 mg, 0.08 mmol). A solution of 2,4-dichloropyridine (0.8 g, 5.42 mmol) in 1,4-dioxane (12 mL) was added. The resulting suspension was heated in a microwave at 100 °C for 2 h. The reaction mixture was poured into water (100 mL) and extracted with DCM (3 x 50 mL). The combined organics were washed with brine (50 mL), separated, dried (MgSO), and concentrated under reduced pressure. Purification by Biotage Isolera gave the title compound as an off-white solid (0.95 g, 63%).

[0389] [ka]

[0390] Intermediate D1 [ka]

[0391] 2-(2-Methoxyethoxy)-4-(4-methylpiperazin-1-yl)aniline Step a: To a stirred solution of 2-bromoethyl methyl ether (0.3 mL, 3.18 mmol) and potassium carbonate (880 mg, 6.37 mmol) in DMF (6 mL) was added 5-fluoro-2-nitro-phenol (500 mg, 3.18 mmol), and the reaction mixture was stirred at rt for 18 h. The reaction mixture was then diluted with water, and the resulting precipitate was collected by vacuum filtration to give 4-fluoro-2-(2-methoxyethoxy)-1-nitro-benzene (210 mg, 31% yield). 1 H NMR (CDCl3, 400 MHz): 7.96 (dd, J = 9.1 and 6.0 Hz, 1H), 6.85 (dd, J = 10.3 and 2.5 Hz, 1H), 6.78-6.73 (m, 1H), 4.26 (t, J = 4.7 Hz, 2H), 3.83 (t, J = 4.7 Hz, 2H), 3.48 (s, 3H).

[0392] Step b: To a stirred solution of potassium carbonate (270 mg, 1.95 mmol) and 1-methylpiperazine (0.11 mL, 0.98 mmol) in DMF (5 mL) was added 4-fluoro-2-(2-methoxyethoxy)-1-nitro-benzene (210 mg, 0.98 mmol), and the reaction mixture was stirred at rt for 18 h. The reaction mixture was then diluted with water, and the resulting precipitate was collected by vacuum filtration to give 1-[3-(2-methoxyethoxy)-4-nitro-phenyl]-4-methyl-piperazine (253 mg, 88% yield). 1H NMR (CDCl3, 400 MHz): 7.99 (d, J = 9.3 Hz, 1H), 6.44 (dd, J = 9.3 and 2.6 Hz, 1H), 6.42 (d, J = 2.5 Hz, 1H), 4.25 (t, J = 4.8 Hz, 2H), 3.84 (t, J = 5.0 Hz, 2H), 3.50 (s, 3H), 3.41 (t, J = 5.1 Hz, 4H), 2.56 (t, J = 5.2 Hz, 4H), 2.37 (s, 3H).

[0393] Step c: To a stirred solution of 1-[3-(2-methoxyethoxy)-4-nitrophenyl]-4-methyl-piperazine (253 mg, 0.86 mmol) in methanol (5 mL) was added ammonium formate (270 mg, 4.28 mmol) and palladium on carbon (9 mg, 0.09 mmol), and the reaction mixture was stirred at rt for 18 h. The reaction mixture was then filtered through Celite, and the filtrate was concentrated under reduced pressure to give the title compound (165 mg, 73% yield). 1 H NMR (CDCl3, 400 MHz): 6.65 (d, J = 8.4 Hz, 1H), 6.54 (d, J = 2.5 Hz, 2H), 6.54 (dd, J = 8.4 and 2.5 Hz, 1H), 4.13 (t, J = 4.7 Hz, 2H), 3.74 (t, J = 4.8 Hz, 2H), 3.60 (br s, 2H), 3.07 (t, J = 4.9 Hz, 4H), 2.58 (t, J = 5.0 Hz, 4H), 2.35 (s, 3H).

[0394] Intermediate D2 [ka]

[0395] 2-Isopropoxy-4-(4-methylpiperazin-1-yl)aniline Intermediate D1 was synthesized using the procedure described above, using 2-iodopropane in step a. 1H NMR (CDCl3, 400 MHz): 6.65 (d, J = 8.4 Hz, 1H), 6.52 (d, J = 2.5 Hz, 1H), 6.42 (dd, J = 8.4 and 2.5 Hz, 1H), 4.50 (sept, J = 6.1 Hz, 1H), 3.06 (t, J = 4.9 Hz, 4H), 2.58 (t, J = 5.0 Hz, 4H), 2.34 (s, 3H), 1.35 (s, 3H), 1.33 (s, 3H).

[0396] Intermediate D3 [ka]

[0397] 2-ethoxy-4-(4-methylpiperazin-1-yl)aniline Intermediate D1 was synthesized using the procedure described above, but using 2-iodoethane in step a. 1 H NMR (CDCl3, 400 MHz): 6.64 (d, J = 8.4 Hz, 1H), 6.51 (d, J = 2.4 Hz, 1H), 6.41 (dd, J = 8.4 and 2.5 Hz, 1H), 4.04 (qu, J = 7.0 Hz, 2H), 3.54 (br s, 2H), 3.07 (t, J = 4.9 Hz, 4H), 2.58 (t, J = 5.0 Hz, 4H), 2.35 (s, 3H), 1.42 (t, J = 7.0 Hz, 3H).

[0398] [ka]

[0399] Intermediate E1 [ka]

[0400] 2-Methoxy-4-(4-(2-methoxyethyl)piperazin-1-yl)aniline Step a: To a stirred solution of potassium carbonate (808 mg, 5.84 mmol) and 1-Boc-piperazine (544 mg, 2.92 mmol) in DMF (5 mL) was added 4-fluoro-2-methoxy-1-nitro-benzene (500 mg, 2.92 mmol), and the reaction mixture was stirred at rt for 18 h. The reaction mixture was then diluted with water, and the resulting precipitate was collected by vacuum filtration to give tert-butyl 4-(3-methoxy-4-nitro-phenyl)piperazine-1-carboxylate (222 mg, 23% yield). 1 H NMR (CDCl3, 500 MHz): 8.04 (d, J = 9.3 Hz, 1H), 6.44 (dd, J = 9.4 and 2.5 Hz, 1H), 6.34 (d, J = 2.4 Hz, 1H), 3.98 (s, 3H), 3.63 (t, J = 5.0 Hz, 4H), 3.42 (t, J = 5.4 Hz, 4H), 1.52 (s, 9H).

[0401] Step b: To a solution of tert-butyl 4-(3-methoxy-4-nitro-phenyl)piperazine-1-carboxylate (222 mg, 0.66 mmol) in DCM (10 mL) was added HCl (4 M in dioxane) (1 mL, 0.66 mmol), and the reaction mixture was stirred at rt for 18 h. The reaction mixture was filtered to give 1-(3-methoxy-4-nitro-phenyl)piperazine hydrochloride (178 mg, 0.7234 mmol, 99% yield). 1 H NMR (MeOD, 400 MHz): 7.94 (d, J = 9.0 Hz, 1H), 6.67-6.63 (m, 2H), 3.96 (s, 3H), 3.70-3.68 (m, 4H), 3.39-3.37 (m, 4H).

[0402] Step c: To a stirred solution of 2-bromoethyl methyl ether (0.08 mL, 0.83 mmol) and potassium carbonate (231 mg, 1.67 mmol) in DMF (3 mL) was added 1-(3-methoxy-4-nitro-phenyl)piperazine (198 mg, 0.83 mmol), and the reaction mixture was stirred at rt for 18 h. The reaction mixture was then diluted with EtOAc, and the organics were washed with water and brine before being dried (MgSO4) and concentrated in vacuo. The residue was purified by flash silica chromatography to give 1-(2-methoxyethyl)-4-(3-methoxy-4-nitro-phenyl)piperazine (165 mg, 67% yield). 1 H NMR (CDCl3, 400 MHz): 8.02 (d, J = 9.4 Hz, 1H), 6.44 (dd, J = 9.4 and 2.6 Hz, 1H), 6.33 (d, J = 2.5 Hz, 1H), 3.97 (s, 3H), 3.58 (t, J = 5.4 Hz, 2H), 3.45 (t, J = 5.1 Hz, 4H), 3.40 (s, 3H), 2.69-2.65 (m, 6H).

[0403] Step d: To a stirred solution of 1-(2-methoxyethyl)-4-(3-methoxy-4-nitro-phenyl)piperazine (165 mg, 0.56 mmol) in methanol (10 mL) was added ammonium formate (176 mg, 2.79 mmol) and palladium on carbon (6 mg, 0.06 mmol), and the reaction mixture was stirred at rt for 18 h. The reaction mixture was then filtered through Celite, and the filtrate was concentrated in vacuo to give 2-methoxy-4-[4-(2-methoxyethyl)piperazin-1-yl]aniline (107 mg, 72% yield). 1H NMR (CDCl3, 400 MHz): 6.64 (d, J = 8.3 Hz, 1H), 6.49 (d, J = 2.4 Hz, 1H), 6.41 (dd, J = 8.3 and 2.5 Hz, 1H), 3.83 (s, 3H), 3.69 (t, J = 5.0 Hz, 2H), 3.36 (s, 3H), 3.21 (t, J = 4.6 Hz, 4H), 3.07 (t, J = 4.7 Hz, 4H), 2.60 (t, J = 5.2 Hz, 2H).

[0404] Intermediate E2 [ka]

[0405] 2-Methoxy-4-(4-(4-methylbenzyl)piperazin-1-yl)aniline Intermediate E1 was synthesized using the procedure described above, but using 1-(bromomethyl)-4-methylbenzene in step c. 1 H NMR (CDCl3, 400 MHz): 7.25 (d, J = 7.9 Hz, 2H), 7.16 (d, J = 7.8 Hz, 2H), 6.65 (d, J = 8.3 Hz, 2H), 6.53 (d, J = 2.4 Hz, 1H), 6.43 (dd, J = 8.4 and 2.5 Hz, 2H), 3.85 (s, 2H), 3.56 (s, 3H), 3.08 (t, J = 4.8 Hz, 4H), 2.63 (t, J = 5.0 Hz, 4H), 2.37 (s, 3H).

[0406] [ka]

[0407] Intermediate F1 [ka]

[0408] 3-(2-chloropyridin-4-yl)-N,N-dimethylbenzamide A mixture of 2-chloro-4-iodopyridine (200 mg, 0.84 mmol), N,N-dimethylbenzamido-3-boronic acid (0.15 mL, 0.75 mmol), bis(triphenylphosphine)palladium(II) dichloride (29 mg, 0.04 mmol), and sodium carbonate (2 M aqueous solution) (1.67 mL, 3.34 mmol) in MeCN (3 mL) was heated in a microwave at 120 °C for 45 min. After cooling to rt, the mixture was diluted with ethyl acetate (20 mL) and washed with water (20 mL). The organics were separated, dried, concentrated under reduced pressure, and purified on a Biotage Isolera to give the title compound (128 mg, 59% yield). 1 H NMR (CDCl3, 400 MHz) δ 8.43 (d, J = 5.2 Hz, 1H), 7.69 - 7.61 (m, 2H), 7.56 - 7.47 (m, 3H), 7.43 (dd, J = 5.1, 1.7 Hz, 1H), 3.14 (s, 3H), 3.01 (s, 3H).

[0409] Example 1 (1902) [ka]

[0410] N4-phenyl-N2-(3,4,5-trimethoxyphenyl)pyridine-2,4-diamine A mixture of Intermediate B3 (72 mg, 0.35 mmol), 3,4,5-trimethoxyaniline (64 mg, 0.35 mmol), XANTPHOS (12 mg, 0.02 mmol), palladium acetate (3 mg, 0.01 mmol), and cesium carbonate (226 mg, 0.70 mmol) in DMA (4 mL) was heated in a microwave at 150 °C for 1 h. After cooling to rt, the mixture was diluted with ethyl acetate (10 mL) and washed with water (10 mL) and brine (3 x 10 mL). The organics were separated, dried, concentrated in vacuo, and purified by Biotage Isolera to give the title compound.1 H NMR (MeOD, 500 MHz) δ 7.75 (d, J = 6.0 Hz, 1H), 7.35 - 7.28 (m, 2H), 7.21 - 7.16 (m, 2H), 7.04 (tt, J = 7.4, 1.2 Hz, 1H), 6.67 (s, 2H), 6.48 (d, J = 2.1 Hz, 1H), 6.38 (dd, J = 6.0, 2.1 Hz, 1H), 3.79 (s, 6H), 3.71 (s, 3H). LCMS method A; RT 1.66; m / z [M+H] + 352.1.

[0411] Example 2 (1903) [ka]

[0412] N4-(1-methyl-1H-pyrazol-3-yl)-N2-(3,4,5-trimethoxyphenyl)pyridine-2,4-diamine The title compound was synthesized using the procedure described in Example 1 using intermediate B2 and 3,4,5-trimethoxyaniline. 1 H NMR (MeOD, 400 MHz) δ 7.74 (d, J = 6.0 Hz, 1H), 7.44 (d, J = 2.3 Hz, 1H), 6.94 (d, J = 2.0 Hz, 1H), 6.69 (s, 2H), 6.49 (dd, J = 6.0, 2.1 Hz, 1H), 5.92 (d, J = 2.3 Hz, 1H), 3.82 (s, 6H), 3.78 (s, 3H), 3.73 (s, 3H). LCMS method A; RT 1.41; m / z [M+H] + 356.1.

[0413] Example 3 (1904) [ka]

[0414] N2-(2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)-N4-(1-methyl-1H-pyrazol-3-yl)pyridine-2,4-diamine A mixture of Intermediate B2 (73 mg, 0.35 mmol), 2-methoxy-4-(4-methylpiperazin-1-yl)aniline (77 mg, 0.35 mmol), XANTPHOS (12 mg, 0.02 mmol), palladium acetate (3 mg, 0.01 mmol), and cesium carbonate (226 mg, 0.70 mmol) in DMA (4 mL) was heated in a microwave at 150 °C for 1 h. After cooling to room temperature, the mixture was filtered through an SCX cartridge, washed first with DCM and then with ammonia in methanol (7 M). The ammonia layer was concentrated under reduced pressure and purified on a Biotage Isolera to give the title compound. 1 H NMR (MeOD, 400 MHz) δ 7.57 (d, J = 6.6 Hz, 1H), 7.46 (d, J = 2.3 Hz, 1H), 7.24 (d, J = 8.6 Hz, 2H), 6.92 (d, J = 2.1 Hz, 1H), 6.70 (d, J = 2.5 Hz, 1H), 6.60 (dd, J = 8.6, 2.6 Hz, 1H), 6.53 (dd, J = 6.7, 2.2 Hz, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.24 (t, J = 5.1 Hz, 4H), 2.64 (t, J = 5.1 Hz, 4H), 2.37 (s, 3H). LCMS method A; RT 0.84; m / z [M+H] + 394.2.

[0415] Example 4 (1905) [ka]

[0416] N4-(1-methyl-1H-pyrazol-4-yl)-N2-(3,4,5-trimethoxyphenyl)pyridine-2,4-diamine Synthesized according to the procedure described in Example 1 using Intermediate B4 and 3,4,5-trimethoxyaniline. 1 H NMR (MeOD, 400 MHz) δ 7.20 (d, J = 6.8 Hz, 2H), 7.00 (d, J = 0.9 Hz, 1H), 6.19 (s, 2H), 5.88 (dd, J = 6.4, 2.2 Hz, 1H), 5.78 (d, J = 2.2 Hz, 1H), 3.45 (s, 3H), 3.38 (s, 6H), 3.31 (s, 3H). LCMS method A; RT 1.25; m / z [M+H] + 356.1.

[0417] Example 5 (1906) [ka]

[0418] 1-(5-((2-((3,4,5-trimethoxyphenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using Intermediate B5 and 3,4,5-trimethoxyaniline. 1 H NMR (CDCl3, 400 MHz) δ 8.15 (d, J = 8.3 Hz, 1H), 7.71 (d, J = 6.2 Hz, 1H), 6.99-6.96 (m, 3H), 6.48 (s, 3H), 6.30 (dd, J = 6.4, 2.2 Hz, LCMS method A; RT 1.62; m / z [M+H] + 435.1.

[0419] Example 6 (1907) [ka]

[0420] 1-(6-((2-((3,4,5-trimethoxyphenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using Intermediate B6 and 3,4,5-trimethoxyaniline. 1 H NMR (CDCl3, 400 MHz) δ 8.04 (d, J = 2.0 Hz, 1H), 7.71 (d, J = 6.3 Hz, 1H), 7.64 (s, 1H), 7.10 (d, J = 7.9 Hz, 1H), 6.87 (dd, J = 7.9, 2.1 Hz, 1H), 6.71 (s, 1H), 6.49 (s, 2H), 6.35 (dd, J = 6.3, 2.1 Hz, 1H), 6.28 (d, J = 2.1 Hz, 1H), 4.09 (t, J = 8.4 Hz, 2H), 3.82 (s, 3H), 3.80 (s, 6H), 3.18 (t, J = 8.4 Hz, 2H), 2.21 (s, 3H). LCMS method A; RT 1.60; m / z [M+H] + 435.2.

[0421] Example 7 (1912) [ka]

[0422] 1-(7-((2-((2-methoxy-4-morpholinophenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized using the procedure described in Example 3 using Intermediate B1 and 2-methoxy-4-morpholinoaniline. 1H NMR (CDCl3, 400 MHz) δ 8.78 (s, 1H), 7.94 (s, 1H), 7.64 (d, J = 6.5 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.21 (dd, J = 8.2, 1.3 Hz, 1H), 7.12 (dd, J = 8.1, 7.2 Hz, 1H), 6.99 (dd, J = 7.4, 1.2 Hz, 1H), 6.54 - 6.42 (m, 2H), 6.29 (dd, J = 6.6, 2.2 Hz, 1H), 6.09 (s, 1H), 4.18 - 4.07 (m, 2H), 3.93 - 3.84 (m, 4H), 3.83 (s, 3H), 3.19 - 3.06 (m, 6H), 2.34 (s, 3H). LCMS method A; RT 1.76; m / z [M+H] + 460.2.

[0423] Example 8 (1934) [ka]

[0424] 2-((2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)-N-methylbenzamide Synthesized using the procedure described in Example 3 using intermediate B14 and 2-methoxy-4-(4-methylpiperazin-1-yl)aniline. 1H NMR (CDCl3, 400 MHz) δ 9.71 (s, 1H), 7.67 (s, 1H), 7.48 (d, J = 7.8 Hz, 1H), 7.40 (dt, J = 15.4, 8.0 Hz, 2H), 7.06 (t, J = 7.4 Hz, 1H), 6.53 - 6.40 (m, 3H), 6.28 (s, 1H), 6.20 (s, 1H), 3.83 (s, 2H), 3.29 (s, 4H), 2.98 (d, J = 4.8 Hz, 3H), 2.72 (s, 4H), 2.46 (s, 3H). LCMS method A; RT 0.90; m / z [M+H] + 447.1.

[0425] Example 9 (1935) [ka]

[0426] 2-((2-((2-methoxy-4-morpholinophenyl)amino)pyridin-4-yl)amino)-N-methylbenzamide Synthesized according to the procedure described in Example 3 using intermediate B14 and 2-methoxy-4-morpholinoaniline. 1 H NMR (CDCl3, 400 MHz) δ 7.91 (d, J = 5.8 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.50 (dd, J = 8.4, 1.2 Hz, 1H), 7.44 (dd, J = 7.8, 1.6 Hz, 1H), 7.36 (ddd, J = 8.5, 7.3, 1.6 Hz, 1H), 6.95 (td, J = 7.5, 1.2 Hz, 1H), 6.81 (s, 1H), 6.55 - 6.46 (m, 3H), 6.38 (d, J = 2.0 Hz, 1H), 6.26 (s, 1H), 3.91 - 3.86 (m, 4H), 3.84 (s, 3H), 3.17 - 3.10 (m, 4H), 2.98 (d, J = 4.8 Hz, 3H). LCMS method A; RT 1.42; m / z [M+H]+ 434.1.

[0427] Example 10 (1936) [ka]

[0428] 1-(7-((2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-6-methylpyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 3 using Intermediate B15 and 2-methoxy-4-(4-methylpiperazin-1-yl)aniline. 1 H NMR (MeOD, 400 MHz) δ 7.29 (d, J = 8.2 Hz, 1H), 7.10 - 7.00 (m, 2H), 6.90 (d, J = 7.4 Hz, 1H), 6.61 (d, J = 2.6 Hz, 1H), 6.49 (dd, J = 8.6, 2.5 Hz, 1H), 6.09 (d, J = 1.9 Hz, 1H), 5.93 (s, 1H), 4.09 (dd, J = 7.3, 3.6 Hz, 2H), 3.78 (s, 3H), 3.35 (d, J = 0.9 Hz, 3H), 3.18 (t, J = 5.0 Hz, 4H), 3.05 (t, J = 7.5 Hz, 2H), 2.61 (q, J = 4.3 Hz, 4H), 2.34 (s, 3H), 2.30 (s, 3H). LCMS method A; RT 1.24; m / z [M+H] + 487.2.

[0429] Example 11 (1937) [ka]

[0430] 1-(7-((2-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized using the procedure described in Example 3 using Intermediate B1 and 4-(4-methylpiperazin-1-yl)aniline. 1 H NMR (CDCl3, 400 MHz) δ 8.34 (s, 1H), 7.78 (d, J = 6.0 Hz, 1H), 7.25 (d, J = 8.1 Hz, 1H), 7.21 - 7.14 (m, 2H), 7.10 (t, J = 7.7 Hz, 1H), 6.96 - 6.87 (m, 4H), 6.34 - 6.25 (m, 2H), 4.10 (t, J = 7.7 Hz, 2H), 3.18 (t, J = 5.0 Hz, 4H), 3.11 (t, J = 7.6 Hz, 2H), 2.60 (t, J = 5.0 Hz, 4H), 2.37 (s, 3H), 2.32 (s, 3H). LCMS method A; RT 1.20; m / z [M+H] + 443.2.

[0431] Example 12 (1938) [ka]

[0432] 1-(7-((2-((4-(4-ethylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized using the procedure described in Example 3 using Intermediate B1 and 4-(4-ethylpiperazin-1-yl)aniline. 1H NMR (CDCl3, 400 MHz) δ 8.38 (s, 1H), 7.76 (d, J = 6.0 Hz, 1H), 7.24 (s, 1H), 7.19 - 7.13 (m, 2H), 7.13 - 7.07 (m, 1H), 6.94 - 6.88 (m, 3H), 6.32 - 6.24 (m, 2H), 4.12 (q, J = 7.4 Hz, 2H), 3.23 - 3.16 (m, 4H), 3.11 (t, J = 7.7 Hz, 2H), 2.64 (dd, J = 6.5, 3.5 Hz, 4H), 2.50 (q, J = 7.2 Hz, 2H), 2.33 (s, 3H), 1.15 (t, J = 7.4, 3H). LCMS method A; RT 1.31; m / z [M+H] + 457.1.

[0433] Example 13 (1941) [ka]

[0434] 1-(7-((2-((2-methoxy-4-(piperidin-1-yl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 3 using Intermediate B1 and 2-methoxy-4-(1-piperidyl)aniline. 1H NMR (CDCl3, 400 MHz) δ 8.65 (s, 1H), 7.67 (d, J = 6.4 Hz, 1H), 7.59 (s, 1H), 7.33 (d, J = 8.6 Hz, 1H), 7.22 (dd, J = 8.1, 1.2 Hz, 1H), 7.11 (t, J = 7.7 Hz, 1H), 6.96 (dd, J = 7.3, 1.2 Hz, 1H), 6.57 - 6.44 (m, 2H), 6.28 (dd, J = 6.4, 2.1 Hz, 1H), 6.13 (d, J = 2.1 Hz, 1H), 4.11 (t, J = 7.8 Hz, 2H), 3.82 (s, 3H), 3.16 - 3.08 (m, 6H), 2.33 (s, 3H), 1.73 (p, J = 5.7 Hz, 4H), 1.63 - 1.55 (m, 2H). LCMS method A; RT 1.52; m / z [M+H] + 458.2.

[0435] Example 14 (1942) [ka]

[0436] 1-(7-((2-((4-(4-isopropylpiperazin-1-yl)-2-methoxyphenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized using the procedure described in Example 3 using Intermediate B1 and 4-(4-isopropylpiperazin-1-yl)-2-methoxy-aniline. 1H NMR (MeOD, 400 MHz) δ 7.53 (d, J = 6.9 Hz, 1H), 7.25 - 7.18 (m, 2H), 7.15 (dd, J = 8.1, 5.1 Hz, 2H), 6.71 (d, J = 2.5 Hz, 1H), 6.61 (dd, J = 8.7, 2.5 Hz, 1H), 6.37 (dd, J = 6.9, 2.2 Hz, 1H), 6.11 (s, 1H), 4.19 (t, J = 7.8 Hz, 2H), 3.84 (s, 3H), 3.33 - 3.30 (m, 4H), 3.16 (t, J = 7.7 Hz, 2H), 2.93 - 2.90 (m, 5H), 2.34 (s, 3H), 1.22 (d, J = 6.6 Hz, 6H). LCMS method A; RT 1.15; m / z [M+H] + 501.2.

[0437] Example 15 (1943) [ka]

[0438] 1-(7-((2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)-2-methylpropan-1-one Synthesized using the procedure described in Example 3 using intermediate B16 and 2-methoxy-4-(4-methylpiperazin-1-yl)aniline. 1H NMR (CDCl3, 500 MHz) δ 8.80 (s, 1H), 8.47 (s, 1H), 7.52 (d, J = 6.8 Hz, 1H), 7.16 (dd, J = 15.4, 8.3 Hz, 2H), 7.09 (t, J = 7.7 Hz, 1H), 7.00 (dd, J = 7.2, 1.3 Hz, 1H), 6.50 - 6.43 (m, 2H), 6.23 (dd, J = 6.9, 2.2 Hz, 1H), 5.97 (d, J = 2.1 Hz, 1H), 4.15 (t, J = 7.7 Hz, 2H), 3.81 (s, 3H), 3.23 (t, J = LCMS method A; RT 1.35; m / z [M+H] + 501.3.

[0439] Example 16 (1944) [ka]

[0440] 1-(7-((2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-3-methylpyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 3 using Intermediate B33 and 2-methoxy-4-(4-methylpiperazin-1-yl)aniline. 1H NMR (CDCl3, 500 MHz) δ 8.71 (s, 1H), 7.78 (d, J = 6.3 Hz, 1H), 7.72 (d, J = 9.0 Hz, 1H), 7.17 - 7.10 (m, 2H), 6.95 (dd, J = 6.8, 1.6 Hz, 1H), 6.91 (s, 1H), 6.56 - 6.51 (m, 3H), 4.12 (t, J = 7.7 Hz, 2H), 3.86 (s, 3H), 3.37 (t, J = 5.0 Hz, 4H), 3.13 (t, J = 7.7 Hz, 2H), 2.93 (t, J = 4.9 Hz, 4H), 2.56 (s, 3H), 2.34 (s, 3H), 2.17 (s, 3H). LCMS method A; RT 1.24; m / z [M+H] + 487.2.

[0441] Example 17 (1950) [ka]

[0442] 2-Methoxy-1-(7-((2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 3 using intermediate B17 and 2-methoxy-4-(4-methylpiperazin-1-yl)aniline. 1H NMR (CDCl3, 500 MHz) δ 8.80 (s, 1H), 8.76 (s, 1H), 7.53 (d, J = 6.9 Hz, 1H), 7.20 - 7.09 (m, 3H), 7.03 (dd, J = 7.1, 1.3 Hz, 1H), 6.51 - 6.44 (m, 2H), 6.26 (dd, J = 6.9, 2.2 Hz, 1H), 5.97 (d, J = 2.2 Hz, 1H), 4.26 (s, 2H), 4.10 (t, J = 7.7 Hz, 2H), 3.81 (s, 3H), 3.46 (s, 3H), 3.26 (t, J = 5.1 Hz, 4H), 3.13 (t, J = 7.7 Hz, 2H), 2.68 (t, J = 5.0 Hz, 4H), 2.42 (s, 3H). LCMS method A; RT 1.10; m / z [M+H] + 503.2.

[0443] Example 18 (1951) [ka]

[0444] 1-(7-((3-fluoro-2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized using the procedure described in Example 3 using intermediate B18 and 2-methoxy-4-(4-methylpiperazin-1-yl)aniline. 1H NMR (DMSO-d6, 400 MHz) δ 8.85 (s, 1H), 7.88 (d, J = 8.7 Hz, 1H), 7.58 (d, J = 5.6 Hz, 1H), 7.18 (d, J = 4.2 Hz, 3H), 7.03 (t, J = 4.3 Hz, 1H), 6.64 (d, J = 2.6 Hz, 1H), 6.49 - 6.38 (m, 2H), 4.16 (t, J = 7.7 Hz, 2H), 3.84 (s, 3H), 3.10 (q, J = 5.8 Hz, 6H), 2.47 (d, J = 4.9 Hz, 4H), 2.31 (s, 3H), 2.24 (s, 3H). LCMs Method A; RT 1.14; m / z [M+H] + 491.1.

[0445] Example 19 (1958) [ka]

[0446] 1-(7-((2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)-5-methylindolin-1-yl)ethan-1-one Synthesized using the procedure described in Example 3 using intermediate B19 and 2-methoxy-4-(4-methylpiperazin-1-yl)aniline. 1H NMR (CDCl3, 500 MHz) δ 9.15 (s, 1H), 8.91 (s, 1H), 7.50 (d, J = 7.0 Hz, 1H), 7.15 (d, J = 8.6 Hz, 1H), 6.94 (s, 1H), 6.80 (s, 1H), 6.52 - 6.45 (m, 2H), 6.22 (dd, J = 7.0, 2.2 Hz, 1H), 6.01 (d, J = 2.2 Hz, 1H), 4.09 (t, J = 7.8 Hz, 2H), 3.82 (s, 3H), 3.24 (t, J = 5.0 Hz, 4H), 3.08 (t, J = 7.7 Hz, 2H), 2.65 (t, J = 5.2 Hz, 4H), 2.41 (s, 3H), 2.31 (s, 3H), 2.28 (s, 3H). LCMS method A; RT 1.25; m / z [M+H] + 487.2.

[0447] Example 20 (1959) [ka]

[0448] 1-(7-((2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)propan-1-one Synthesized according to the procedure described in Example 3 using intermediate B20 and 2-methoxy-4-(4-methylpiperazin-1-yl)aniline. 1H NMR (CDCl3, 500 MHz) δ 9.07 (s, 1H), 8.98 (s, 1H), 7.51 (d, J = 6.9 Hz, 1H), 7.16 (dd, J = 8.4, 3.4 Hz, 2H), 7.09 (t, J = 7.6 Hz, 1H), 7.00 (d, J = 7.2 Hz, 1H), 6.50 (d, J = 2.5 Hz, 1H), 6.47 (dd, J = 8.5, 2.6 Hz, 1H), 6.24 (dd, J = 6.9, 2.2 Hz, 1H), 5.96 (d, J = 2.3 Hz, 1H), 4.11 (t, J = 7.7 Hz, 2H), 3.82 (s, 3H), 3.25 (t, J = 5.0 Hz, 4H), 3.12 (t, J = 7.7 Hz, 2H), 2.67 (t, J = 4.9 Hz, 4H), 2.57 (q, J = 7.4 Hz, 2H), 2.42 (s, 3H), 1.28 - 1.23 (m, 3H). LCMS method A; RT 1.26; m / z [M+H] + 487.2.

[0449] Example 21 (1961) [ka]

[0450] 3-Methoxy-1-(7-((2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)propan-1-one Synthesized according to the procedure described in Example 3 using intermediate B21 and 2-methoxy-4-(4-methylpiperazin-1-yl)aniline. 1H NMR (CDCl3, 500 MHz) δ 7.79 (d, J = 6.3 Hz, 1H), 7.43 (d, J = 8.6 Hz, 1H), 7.11 - 7.05 (m, 2H), 6.99 (t, J = 7.6 Hz, 1H), 6.53 (d, J = 2.5 Hz, 1H), 6.49 (dd, J = 8.7, 2.5 Hz, 1H), 6.24 (dd, J = 6.4, 2.3 Hz, 1H), 6.05 (d, J = 2.3 Hz, 1H), 4.18 (t, J = 8.6 Hz, 2H), 3.83 (s, 3H), 3.81 - 3.76 (m, 2H), 3.21 (t, J = 5.0 Hz, 4H), 3.13 (t, J = 8.6 Hz, 2H), 2.93 - 2.89 (m, 2H), 2.65 (t, J = 5.1 Hz, 4H), 2.40 (s, 3H), 2.18 (s, 3H). LCMS method A; RT 1.16; m / z [M+H] + 485.1.

[0451] Example 22 (1968) [ka]

[0452] 1-(7-((2-((3-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized using the procedure described in Example 3 using Intermediate B1 and 3-(4-methylpiperazin-1-yl)aniline. 1H NMR (CDCl3, 500 MHz) δ 8.83 (s, 1H), 8.03 (s, 1H), 7.68 (d, J = 6.5 Hz, 1H), 7.25 - 7.17 (m, 2H), 7.13 (t, J = 7.7 Hz, 1H), 6.99 (dd, J = 7.2, 1.2 Hz, 1H), 6.77 (t, J = 2.2 Hz, 1H), 6.75 - 6.70 (m, 1H), 6.67 (dd, J = 8.3, 2.4 Hz, 1H), 6.42 (d, J = 2.1 Hz, 1H), 6.31 (dd, J = 6.5, 2.1Hz, 1H), 4.12 LCMS method A; RT 1.16; m / z [M+H] + 443.2.

[0453] Example 23 (1971) [ka]

[0454] N2-(2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)-N4-phenylpyridine-2,4-diamine A mixture of palladium acetate (2 mg, 0.01 mmol) and cesium carbonate (118 mg, 0.36 mmol), A mixture of Intermediate C1 (60 mg, 0.18 mmol), aniline (0.02 mL, 0.18 mmol), and XANTPHOS (6 mg, 0.01 mmol) in 1,4-dioxane (2 mL) was heated in a microwave oven at 100 °C for 30 min. After cooling to room temperature, the mixture was filtered through an SCX cartridge and washed first with DCM and then with ammonia in methanol (7 M). The ammonia layer was concentrated under reduced pressure and purified on a Biotage Isolera to give the title compound (20 mg, 28% yield). 1H NMR (CDCl3, 400 MHz) δ 8.53 (s, 1H), 7.50 (d, J = 6.9 Hz, 1H), 7.34 (t, J = 7.8 Hz, 2H), 7.18 (t, J = 7.4 Hz, 1H), 7.13 (dd, J = 8.1, 2.2 Hz, 3H), 6.85 (s, 1H), 6.47 (d, J = 8.1 Hz, 2H), 6.22 (dd, J = 6.9, 2.3 Hz, 1H), 5.88 (d, J = 2.2 Hz, 1H), 3.80 (s, 3H), 3.28 (t, J = 5.1 Hz, 4H), 2.86 - 2.78 (m, 4H), 2.49 (s, 3H). LCMS method A; RT 1.09; m / z [M+H] + 390.2.

[0455] Example 24 (1989) [ka]

[0456] 1-(5-((2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using Intermediate B5 and 2-methoxy-4-(4-methylpiperazin-1-yl). 1H NMR (CDCl3, 400 MHz) δ 8.13 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 6.8 Hz, 1H), 7.37 (s, 1H), 7.19 (d, J = 8.5 Hz, 1H), 7.00 - 6.92 (m, 2H), 6.51 - 6.40 (m, 2H), 6.32 (dd, J = 6.8, 2.1 Hz, 1H), 5.99 (d, J = 2.1 Hz, 1H), 4.07 (t, J = 8.5 Hz, 2H), 3.80 (s, 3H), 3.23 (t, J = 5.0 Hz, 4H), 3.14 (t, J = 8.4 Hz, 2H), 2.66 (q, J = 5.4 Hz, 4H), 2.42 (s, 3H), 2.23 (s, 3H). LCMS method A; RT 1.03; m / z [M+H] + 473.1.

[0457] Example 25 (1992) [ka]

[0458] 1-(7-((2-((4-(4-methylpiperazin-1-yl)-2-(trifluoromethyl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized using the procedure described in Example 3 using Intermediate B1 and 4-(4-methylpiperazin-1-yl)-2-(trifluoromethyl)aniline. 1H NMR (CDCl3, 400 MHz) δ 8.57 (s, 1H), 7.74 (d, J = 6.3 Hz, 1H), 7.46 (d, J = 8.9 Hz, 1H), 7.20 (d, J = 8.3 Hz, 1H), 7.15 (d, J = 2.9 Hz, 1H), 7.09 (t, J = 7.7 Hz, 1H), 7.05 (dd, J = 8.9, 2.9 Hz, 1H), 6.95 (dd, J = 7.2, 1.2 Hz, 1H), 6.35 (dd, J = 6.3, 2.1 Hz, 1H), 6.01 (s, 1H), 4.11 (t, J = 7.8 Hz, 2H), 3.29 - 3.19 (m, 4H), 3.12 (t, J = 7.7 Hz, 2H), 2.64 - 2.57 (m, 4H), 2.39 (s, 3H), 2.33 (s, 3H). LCMS method A; RT 1.20; m / z [M+H] + 511.2.

[0459] Example 26 (1993) [ka]

[0460] 1-(7-((2-((2-methoxy-4-(4-(4-methylbenzyl)piperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 3 using Intermediate B1 and Intermediate E2. 1H NMR (CDCl3, 400 MHz) δ 8.85 (s, 1H), 7.59 (d, J = 6.6 Hz, 1H), 7.26 (d, J = 8.0 Hz, 2H), 7.20 - 7.14 (m, 3H), 7.10 (t, J = 7.7 Hz, 1H), 6.99 (d, J = 7.3 Hz, 1H), 6.49 (d, J = 2.5 Hz, 1H), 6.46 (dd, J = 8.6, 2.5 Hz, 1H), 6.27 (dd, J = 6.7, 2.2 Hz, 1H), 6.05 (s, 1H), 4.12 (t, J = 7.8 Hz, 2H), 3.81 (s, LCMS method A; RT 1.44; m / z [M+H] + 563.3.

[0461] Example 27 (1994) [ka]

[0462] 1-(7-((2-((2-methoxy-4-(4-(2-methoxyethyl)piperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 3 using Intermediate B1 and Intermediate E1. 1H NMR (CDCl3, 400 MHz) δ 8.26 (s, 1H), 7.83 (d, J = 5.9 Hz, 1H), 7.56 (d, J = 8.5 Hz, 1H), 7.30 - 7.27 (m, 1H), 7.10 (dd, J = 8.2, 7.3 Hz, 1H), 6.98 (s, 1H), 6.91 (dt, J = 7.3, 1.1 Hz, 1H), 6.55 - 6.49 (m, 2H), 6.31 (dd, J = 5.9, 2.0 Hz, 1H), 6.26 (d, J = 2.0 Hz, 1H), 4.13 - 4.05 (m, 2H), 3.83 (s, 3H), 3.57 (t, J = 5.6 Hz, 2H), 3.39 (s, 3H), 3.22 - 3.16 (m, 4H), 3.10 (t, J = 7.7 Hz, 2H), 2.73 - 2.62 (m, 6H), 2.32 (s, 3H). LCMS method A; RT 1.19; m / z [M+H] + 517.2.

[0463] Example 28 (1995) [ka]

[0464] N2-(2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)-N4-(1-methyl-3-phenyl-1H-pyrazol-5-yl)pyridine-2,4-diamine Synthesized according to the procedure described in Example 3 using intermediate B22 and 2-methoxy-4-(4-methylpiperazin-1-yl)aniline. 1H NMR (CDCl3, 400 MHz) δ 7.76 - 7.71 (m, 3H), 7.43 - 7.36 (m, 3H), 7.35 - 7.28 (m, 1H), 6.72 (s, 1H), 6.49 - 6.42 (m, 2H), 6.35 (s, 1H), 6.23 (dd, J = 6.3, 2.1 Hz, 1H), 5.99 (d, J = 2.1 Hz, 1H), 3.79 (s, 3H), 3.75 (s, 3H), 3.16 (t, J = 5.1 Hz, 4H), 2.63 (t, J = 4.8 Hz, 4H), 2.39 (s, 3H). LCMS method A; RT 1.20; m / z [M+H] + 470.1.

[0465] Example 29 (1998) [ka]

[0466] N4-(3-(tert-butyl)-1-methyl-1H-pyrazol-5-yl)-N2-(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)pyridine-2,4-diamine Synthesized according to the procedure described in Example 3 using Intermediate B25 and 2-methoxy-4-(4-methylpiperazin-1-yl)aniline. 1H NMR (CDCl3, 400 MHz) δ 7.75 (s, 1H), 7.65 (d, J = 6.3 Hz, 1H), 7.29 (d, J = 8.6 Hz, 1H), 6.88 (s, 1H), 6.48 (d, J = 2.5 Hz, 1H), 6.45 (dd, J = 8.6, 2.6 Hz, 1H), 6.27 (d, J = 6.1 Hz, 1H), 5.94 (d, J = 2.1 Hz, 1H), 5.87 (s, 1H), 3.79 (s, 3H), 3.65 (s, 3H), 3.22 - 3.14 (m, 4H), 2.65 - 2.60 (m, 4H), 2.39 (s, 3H), 1.26 (s, 9H). LCMS method A; RT 1.25; m / z [M+H] + 450.2.

[0467] Example 30 (2000) [ka]

[0468] N4-(3-isopropyl-1-methyl-1H-pyrazol-5-yl)-N2-(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)pyridine-2,4-diamine Synthesized according to the procedure described in Example 3 using intermediate B27 and 2-methoxy-4-(4-methylpiperazin-1-yl)aniline. 1H NMR (CDCl3, 400 MHz) δ 7.73 (d, J = 6.1 Hz, 1H), 7.36 (d, J = 8.5 Hz, 1H), 6.53 - 6.42 (m, 2H), 6.19 (d, J = 5.8 Hz, 1H), 5.95 (d, J = 1.9 Hz, 1H), 5.86 (s, 1H), 3.81 (s, 3H), 3.65 (s, 3H), 3.20 (t, J = 5.0 Hz, 4H), 2.90 (p, J = 6.9 Hz, 1H), 2.65 (d, J = 6.3 Hz, 4H), 2.40 (s, 3H), 1.26 (s, 6H). LCMS method A; RT 1.16; m / z [M+H] + 436.2

[0469] Example 31 (2007) [ka]

[0470] 1-(7-((2-((2-ethoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 3 using Intermediate B1 and Intermediate D3. 1H NMR (CDCl3, 400 MHz) δ 9.12 (s, 1H), 9.03 (s, 1H), 7.50 (d, J = 7.0 Hz, 1H), 7.19 - 7.07 (m, 3H), 7.05 - 6.98 (m, 1H), 6.53 - 6.45 (m, 2H), 6.24 (dd, J = 7.0, 2.2 Hz, 1H), 6.00 (d, J = 2.2 Hz, 1H), 4.12 (t, J = 7.8 Hz, 2H), 4.04 (q, J = 7.0 Hz, 2H), 3.27 (t, J = 5.1 Hz, 4H), 3.14 (t, J = 7.7 Hz, 2H), 2.73 - 2.71 (m, 4H), 2.45 (s, 3H), 2.33 (s, 3H), 1.39 (t, J = 7.0 Hz, 3H). LCMS method A; RT 1.23; m / z [M+H] + 487.2.

[0471] Example 32 (2008) [ka]

[0472] 1-(7-((2-((2-isopropoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 3 using Intermediate B1 and Intermediate D2. 1H NMR (CDCl3, 400 MHz) δ 8.99 (s, 1H), 8.84 (s, 1H), 7.52 (d, J = 6.8 Hz, 1H), 7.18 (d, J = 8.2 Hz, 2H), 7.11 (t, J = 7.6 Hz, 1H), 7.00 (dd, J = 7.3, 1.2 Hz, 1H), 6.54 - 6.45 (m, 2H), 6.25 (dd, J = 6.9, 2.2 Hz, 1H), 6.03 (d, J = 2.2 Hz, 1H), 4.50 (p, J = 6.1 Hz, 1H), 4.12 (t, J = 7.8 Hz, 2H), 3.23 (t, J LCMS method A; RT 1.34; m / z [M+H] + 501.2.

[0473] Example 33 (2009) [ka]

[0474] 1-(7-((2-((2-(2-methoxyethoxy)-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 3 using Intermediate B1 and Intermediate D1. 1H NMR (CDCl3, 400 MHz) δ 8.93 (s, 1H), 8.47 (s, 1H), 7.57 (d, J = 6.8 Hz, 1H), 7.27 - 7.17 (m, 2H), 7.10 (dd, J = 8.1, 7.3 Hz, 1H), 6.99 (dd, J = 7.4, 1.1 Hz, 1H), 6.55 (d, J = 2.6 Hz, 1H), 6.50 (dd, J = 8.7, 2.6 Hz, 1H), 6.26 (dd, J = 6.8, 2.2 Hz, 1H), 6.07 (d, J = 2.1 Hz, 1H), 4.17 - 4.05 (m, 4H), 3.78 - 3.71 (m, 2H), 3.39 (s, 3H), 3.22 (t, J = 5.1 Hz, 4H), 3.13 (t, J = 7.8 Hz, 2H), 2.66 - 2.58 (m, 4H), 2.41 (s, 3H), 2.34 (s, 3H). LCMS method A; RT 1.27; m / z [M+H] + 517.2.

[0475] Example 34 (2010) [ka]

[0476] 1-(7-((2-((3-phenoxyphenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using intermediate B1 and 3-phenoxyaniline. 1H NMR (CDCl3, 400 MHz) δ 8.38 (s, 1H), 7.86 (d, J = 5.8 Hz, 1H), 7.37 - 7.31 (m, 2H), 7.29 (d, J = 1.1 Hz, 1H), 7.23 (t, J = 8.1 Hz, 1H), 7.13 - 7.07 (m, 2H), 7.07 - 6.97 (m, 4H), 6.92 (dq, J = 7.4, 1.1 Hz, 1H), 6.67 (s, 1H), 6.61 (ddd, J = 8.1, 2.3, 0.9 Hz, 1H), 6.45 (d, J = 2.0 Hz, 1H), 6.37 (dd, LCMS method A; RT 2.13; m / z [M+H] + 437.2.

[0477] Example 35 (2011) [ka]

[0478] 4-((4-((1-acetylindolin-7-yl)amino)pyridin-2-yl)amino)benzamide Synthesized according to the procedure described in Example 1 using intermediate B1 and 4-aminobenzamide. 1H NMR (CDCl3, 400 MHz): 8.53 (br s, 1H), 7.93 (d, J = 5.9 Hz, 1H), 7.76 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 8.8 Hz, 2H), 7.31 (d, J = 8.6 Hz, 1H), 7.17 (t, J = 7.4 Hz, 1H), 7.02 (br s, 1H), 6.98 (d, J = 8.3 Hz, 1H), 6.49 (s, 1H), 6.47 (d, J = 5.9 Hz, 1H), 5.75 (br s, 2H), 4.14 (t, J = 7.8 Hz, 2H), 3.15 (t, J = 7.8 Hz, 2H), 2.36 (s, 3H). LCMS method A; RT 1.49; m / z [M+H] + 388.2.

[0479] Example 36 (2013) [ka]

[0480] 1-(7-((2-((4-phenoxyphenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using intermediate B1 and 4-phenoxyaniline. 1H NMR (CDCl3, 500 MHz) δ 8.40 (s, 1H), 7.84 (d, J = 5.9 Hz, 1H), 7.33 (dd, J = 8.5, 7.2 Hz, 2H), 7.27 - 7.24 (m, 3H), 7.15 - 7.06 (m, 2H), 7.03 - 6.96 (m, 4H), 6.94 (d, J = 7.2 Hz, 1H), 6.71 (s, 1H), 6.36 (dd, J = 6.0, 2.0 Hz, 1H), 6.33 (d, J = 2.0 Hz, 1H), 4.12 (td, J = 7.4, 3.0 Hz, 2H), 3.12 (t, J = 7.7 Hz, 2H), 2.34 (s, 3H). LCMS method A; RT 2.08; m / z [M+H] + 437.2.

[0481] Example 37 (2014) [ka]

[0482] 1-(7-((2-((4-(benzyloxy)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using intermediate B1 and 4-benzyloxyaniline. 1H NMR (CDCl3, 400 MHz) δ 8.50 (s, 1H), 7.76 (d, J = 6.2 Hz, 1H), 7.47 - 7.43 (m, 2H), 7.43 - 7.38 (m, 2H), 7.37 - 7.31 (m, 1H), 7.23 (dd, J = 8.2, 1.1 Hz, 1H), 7.20 - 7.16 (m, 2H), 7.09 (dd, J = 8.1, 7.3 Hz, 2H), 6.97 - 6.91 (m, 3H), 6.32 (dd, J = 6.1, 2.1 Hz, 1H), 6.23 (d, J = 2.0 Hz, 1H), 5.06 (s, 2H), 4.14 - 4.06 (m, 2H), 3.11 (t, J = 7.7 Hz, 2H), 2.33 (s, 3H). LCMS method A; RT 2.09; m / z [M+H] + 451.2.

[0483] Example 38 (2015) [ka]

[0484] 1-(7-((2-((4-morpholinophenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 3 using Intermediate B1 and N-(4-aminophenyl)morpholine. 1H NMR (CDCl3, 500 MHz) δ 8.47 (s, 1H), 7.78 (d, J = 6.1 Hz, 1H), 7.28 - 7.24 (m, 1H), 7.23 - 7.17 (m, 2H), 7.16 - 7.08 (m, 1H), 6.96 (dd, J = 7.3, 1.1 Hz, 2H), 6.93 - 6.89 (m, 2H), 6.34 (dd, J = 6.1, 2.1 Hz, 1H), 6.26 (d, J = 2.0 Hz, 1H), 4.13 (t, J = 7.7 Hz, 2H), 3.95 - 3.86 (m, 4H), 3.20 - 3.10 (m, 6H), 2.35 (s, 3H). LCMS method A; RT 1.68; m / z [M+H] + 430.1.

[0485] Example 39 (2016) [ka]

[0486] 1-(7-((2-(phenylamino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using intermediate B1 and aniline. 1 H NMR (CDCl3, 400 MHz): 8.38 (br s, 1H), 7.85 (d, J = 5.9 Hz, 1H), 7.32-7.27 (m, 3H), 7.25-7.24 (m, 2H), 7.12 (t, J = 7.4 Hz, 1H), 7.01 (t, J = 7.0 Hz, 1H), 6.92 (dd, J = 7.3 and 1.0 Hz, 1H), 6.68 (br s, 1H), 6.43 (d, J = 1.8 Hz, 1H), 6.36 (dd, J = 5.9 and 2.0 Hz, 1H), 4.10 (t, J = 7.6 Hz, 2H), 3.11 (t, J = 7.8 Hz, 2H), 2.33 (s, 3H). LCMS method A; RT 1.76; m / z [M+H]+ 345.2.

[0487] Example 40 (2017) [ka]

[0488] 1-(7-((2-((4-(tert-butyl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using Intermediate B1 and 4-(tert-butyl)aniline. 1 H NMR (CDCl3, 400 MHz): 8.42 (br s, 1H), 7.79 (d, J = 6.0 Hz, 1H), 7.32 (d, J = 8.7 Hz, 2H), 7.26 (d, J = 7.3 Hz, 1H), 7.17 (d, J = 8.7 Hz, 2H), 7.11 (t, J = 7.4 Hz, 1H), 6.93 (dd, J = 7.3 and 1.0 Hz, 1H), 6.90 (m, 1H), 6.38 (d, J = 1.9 Hz, 1H), 6.33 (dd, J = 6.0 and 2.1 Hz, 1H), 4.11 (t, J = 7.8 Hz, 2H), 3.11 (t, J = 7.8 Hz, 2H), 2.33 (s, 3H), 1.32 (s, 9H). LCMS method A; RT 2.13; m / z [M+H] + 401.2.

[0489] Example 41 (2018) [ka]

[0490] 4-((4-((1-acetylindolin-7-yl)amino)pyridin-2-yl)amino)benzonitrile Synthesized according to the procedure described in Example 1 using intermediate B1 and 4-aminobenzonitrile. 1H NMR (CDCl3, 400 MHz): 8.47 (br s, 1H), 7.95 (d, J = 5.8 Hz, 1H), 7.52 (d, J = 8.9 Hz, 2H), 7.43 (d, J = 9.0 Hz, 2H), 7.29 (d, J = 7.9 Hz, 1H), 7.15 (t, J = 7.4 Hz, 1H), 6.96 (dd, J = 7.2 and 1.0 Hz, 1H), 6.60 (br s, 1H), 6.48 (dd, J = 5.8 and 2.0 Hz, 1H), 6.39 (d, J = 1.8 Hz, 1H), 4.12 (t, J = 7.8 Hz, 2H), 3.13 (t, J = 7.8 Hz, 2H), 2.34 (s, 3H). LCMS method A; RT 1.67; m / z [M+H] + 370.2.

[0491] Example 42 (2019) [ka]

[0492] 4-((4-((1-acetylindolin-7-yl)amino)pyridin-2-yl)amino)-N-methylbenzamide Synthesized according to the procedure described in Example 1 using intermediate B1 and 4-amino-N-methylbenzamide. 1H NMR (CDCl3, 400 MHz): 8.43 (br s, 1H), 7.91 (d, J = 5.8 Hz, 1H), 7.68 (d, J = 8.8 Hz, 2H), 7.32 (d, J = 8.7 Hz, 2H), 7.29 (d, J = 7.7 Hz, 1H), 7.14 (t, J = 7.4 Hz, 1H), 6.94 (d, J = 8.2 Hz, 1H), 6.69 (br s, 1H), 6.45-6.42 (m, 2H), 6.06 (br d, J = 4.9 Hz, 1H), 4.11 (t, J = 7.8 Hz, 2H), 3.12 (t, J = 7.7 Hz, 2H), 3.00 (d, J = 4.8 Hz, 3H), 2.33 (s, 3H). LCMS method A; RT 1.44; m / z [M+H] + 402.2.

[0493] Example 43 (2020) [ka]

[0494] 1-(7-((2-(quinolin-2-ylamino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using intermediate B1 and quinolin-2-amine. 1H NMR (CDCl3, 400 MHz): 8.85 (br s, 1H), 8.15 (br, s, 1H), 7.99 (d, J = 8.8 Hz, 1H), 7.90 (d, J = 6.0 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.69 (d, 8.0 Hz, 1H), 7.63 (t, J = 8.4 Hz, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.39-7.30 (m, 4H), 7.06 (d, J = 8.2 Hz, 1H), 6.51 (dd, J = 6.1 and 2.2 Hz, 1H), 4.16 (t, J = 7.8 Hz, 2H), 3.18 (t, J = 7.8 Hz, 2H), 2.38 (s, 3H). LCMS method A; RT 2.04; m / z [M+H] + 396.2.

[0495] Example 44 (2021) [ka]

[0496] 1-(7-((2-(isoquinolin-3-ylamino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using intermediate B1 and isoquinolin-3-amine. 1H NMR (CDCl3, 400 MHz): 8.86 (s, 1H), 8.64 (br s, 1H), 7.99 (s, 1H), 7.82 (d, J = 6.0 Hz, 1H), 7.75 (d, J = 8.9 Hz, 1H), 7.66 (d, J = 8.3 Hz, 1H), 7.50 (t, J = 6.8 Hz, 1H), 7.33-7.27 (m, 2H), 7.19-7.13 (m, 2H), 6.92 (d, J = 7.3 Hz, 2H), 6.38 (dd, J = 6.2 and 2.1 Hz, 1H), 4.06 (t, J = 7.8 Hz, 2H), 3.07 (t, J = 7.8 Hz, 2H), 2.28 (s, 3H). LCMS method A; RT 2.00; m / z [M+H] + 396.2.

[0497] Example 45 (2022) [ka]

[0498] 1-(7-((2-(quinolin-3-ylamino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized using the procedure described in Example 1 using intermediate B1 and quinolin-3-amine. 1H NMR (CDCl3, 400 MHz): 8.69 (d, J = 2.7 Hz, 1H), 8.36 (s, 1H), 8.29 (d, J = 2.4 Hz, 1H), 7.94-7.90 (m, 2H), 7.66 (d, J = 9.4 Hz, 1H), 7.49-7.40 (m, 2H), 7.26 (d, J = 8.0 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H), 6.86 (d, J = 8.3 Hz, 1H), 6.63 (br s, 1H), 6.40-6.37 (m, 2H), 4.04 (t, J = 7.8 Hz, 2H), 3.05 (t, J = 7.8 Hz, 2H), 2.26 (s, 3H). LCMS method A; RT 1.67; m / z [M+H] + 396.1.

[0499] Example 46 (2023) [ka]

[0500] 1-(7-((2-((1-methyl-3-phenyl-1H-pyrazol-5-yl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using intermediate B1 and 2-methyl-5-phenyl-pyrazol-3-amine. 1H NMR (CDCl3, 400 MHz): 8.31 (br s, 1H), 7.78 (d, J = 5.9 Hz, 1H), 7.69 (d, J = 7.1 Hz, 2H), 7.32 (t, J = 7.3 Hz, 1H), 7.23 (d, J = 7.4 Hz, 1H), 7.17 (s, 1H), 7.01 (d, J = 7.4 Hz, 1H), 6.83 (d, J = 7.3 Hz, 1H), 6.34-6.32 (m, 2H), 6.08 (d, J = 1.8 Hz, 1H), 5.23 (s, 1H), 4.02 (t, J = 7.8 Hz, 2H), 3.72 (s, 3H), 3.03 (t, J = 7.7 Hz, 2H), 2.24 (s, 3H). LCMS method A; RT 1.78; m / z [M+H] + 425.2

[0501] Example 47 (2026) [ka]

[0502] 3-((4-((1-acetylindolin-7-yl)amino)pyridin-2-yl)amino)-N-methylbenzamide Synthesized according to the procedure described in Example 1 using intermediate B1 and 3-amino-N-methyl-benzamide. 1H NMR (CDCl3, 400 MHz): 8.44 (s, 1H), 7.88 (d, J = 6.1 Hz, 1H), 7.72 (br s, 1H), 7.44-7.41 (m, 1H), 7.33-7.29 (m, 3H), 7.14 (t, J = 7.4 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 6.71 (br s, 1H), 6.40-6.38 (m 2H), 6.22 (br d, J = 3.4 Hz, 1H), 4.10 (t, J = 7.8 Hz, 2H), 3.11 (t, J = 7.8 Hz, 2H), 3.01 (d, J = 4.8 Hz, 3H), 2.33 (s, 3H). LCMS method A; RT 1.48; m / z [M+H] + 402.2.

[0503] Example 48 (2027) [ka]

[0504] 1-(7-((2-((1-methyl-1H-indazol-3-yl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using intermediate B1 and 1-methylindazol-3-amine. 1H NMR (CDCl3, 400 MHz): 8.72 (br s, 1H), 7.82 (d, J = 6.2 Hz, 2H), 7.62 (s, 1H), 7.46-7.40 (m, 2H), 7.31 (s, 1H), 7.27 (s, 1H), 7.18 (t, J = 7.6 Hz, 1H), 7.12 (t, J = 7.9 Hz, 1H), 7.00 (dd, J = 7.4 and 0.8 Hz, 1H), 6.41 (dd, J = 6.2 and 2.2 Hz, 1H), 4.15 (t, J = 7.8 Hz, 2H), 3.96 (s, 3H), 3.16 (t, J = 7.7 Hz, 2H), 2.37 (s, 3H). LCMS method A; RT 1.93; m / z [M+H] + 399.2.

[0505] Example 49 (2028) [ka]

[0506] 1-(7-((2-(quinolin-6-ylamino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using intermediate B1 and quinolin-6-amine. 1H NMR (CDCl3, 400 MHz): 8.68 (dd, J = 4.2 and 1.7 Hz, 1H), 8.38 (br s, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 9.1 Hz, 1H), 7.87 (d, J = 5.9 Hz, 1H), 7.77 (d, J = 2.5 Hz, 1H), 7.47 (dd, J = 9.0 and 2.5 Hz, 1H), 7.28-7.25 (m, 2H), 7.07 (t, J = 7.4 Hz, 1H), 6.86 (d, J = 7.3 Hz, 1H), 6.79 (br s, 1H), 6.44 (d, J = LCMS method A; RT 1.40; m / z [M+H] + 396.2.

[0507] Example 50 (2029) [ka]

[0508] N2-[2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl]-N4-(4-quinolyl)pyridine-2,4-diamine Quinolin-4-amine (33 mg, 0.23 mmol), Pd(OAc)2 (2.7 mg, 0.012 mmol), and XANTPHOS (10 mg, 0.018 mmol) were placed in a microwave vial, and Intermediate C1 (50 mg, 0.15 mmol) in DMA (1 mL) was added. The vial was sealed, evacuated, and purged with argon three times. Cesium carbonate (98 mg, 0.30 mmol) in water (0.5 mL) was added (the vial was evacuated and purged with argon), and the reaction mixture was heated in a microwave at 150 °C for 1 h. The reaction mixture was diluted with ethyl acetate and washed with water (3 x 25 mL) and brine (25 mL). The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure. The resulting residue was purified by flash chromatography (0-50% 90 / 10 / 1 DCM / MeOH / NH4OH). The appropriate fractions were combined, and the solvent was removed under reduced pressure. After further purification by HPLC (XBridge column, 0.1% NH4OH modifier), evaporation of the solvent from the appropriate fractions in a Genevac afforded the title compound (9 mg, 9%). 1 H NMR (CDCl3, 400 MHz): 8.70 - 8.79 (1H, m), 8.04 - 8.17 (2H, m), 7.88 - 7.98 (1H, m,), 7.69 - 7.80 (1H, m), 7.50 - 7.62 (2H, m), 7.31 - 7.37 (1H, m), 6.45 - 6.73 (6H, m), 3.87 (3H, s), 3.21 (4H, br. s.), 2.55 - 2.73 (4H, m), 2.39 (3H, s). LCMS method B; RT = 0.67; m / z [M+H] + 441.2.

[0509] Example 51 (2031) [ka]

[0510] N4-(benzo[d]thiazol-5-yl)-N2-(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)pyridine-2,4-diamine Synthesized using the procedure described in Example 50 using intermediate C1 and benzo[d]thiazol-5-amine. 1 H NMR (CDCl3, 400 MHz): 8.97 - 9.07 (1H, m), 7.93 - 8.00 (2H, m), 7.90 (1H, d, J = 8.5 Hz), 7.58 - 7.70 (1H, m), 6.47 - 6.63 (3H, m), 6.28 - 6.43 (2H, m), 6.05 (1H, s), 3.80 - 3.93 (3H, m), 3.07 - 3.25 (4H, m), 2.53 - 2.68 (4H, m), 2.38 (3H, s). LCMS method B; RT = 1.19; + 447.2.

[0511] Example 52 (2038) [ka]

[0512] N2-(2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)-N4-(4-(methylsulfonyl)phenyl)pyridine-2,4-diamine Synthesized using the procedure described in Example 50 using intermediate C1 and 4-(methylsulfonyl)aniline. 1H NMR (CDCl3, 400 MHz): 8.00 - 8.07 (1H, m), 7.80 - 7.87 (2H, m), 7.51 - 7.59 (1H, m), 7.16 - 7.25 (2H, m), 6.48 - 6.60 (3H, m), 6.41 - 6.47 (1H, m), 6.37 - 6.41 (1H, m), 6.25 - 6.33 (1H, m), 3.85 (3H, s), 3.12 - 3.28 (4H, m), 3.05 (3H, s), 2.52 - 2.67 (4H, m), 2.37 (3H, s). LCMS method B; RT = 1.08; m / z [M+H] + 468.2.

[0513] Example 53 (2039) [ka]

[0514] N2-(2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)-N4-(pyrimidin-4-yl)pyridine-2,4-diamine Synthesized using the procedure described in Example 50 using intermediate C1 and pyrimidin-4-amine. 1 H NMR (CDCl3, 400 MHz): 8.72 - 8.80 (1H, m), 8.34 - 8.41 (1H, m), 8.04 - 8.11 (1H, m), 7.56 - 7.63 (1H, m), 7.28 - 7.34 (1H, m), 6.89 - 6.94 (1H, m), 6.78 - 6.83 (1H, m), 6.73 - 6.77 (1H, m), 6.64 - 6.69 (1H, m), 6.50 - 6.57 (2H, m), 3.84 (1H, s), 3.09 - 3.29 (4H, m), 2.52 - 2.70 (4H, m), 2.37 (3H, s). LCMS method B; RT = 0.94; m / z [M+H] + 392.2.

[0515] Example 54 (2043) [ka]

[0516] N2-(2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)-N4-(3-(methylsulfonyl)phenyl)pyridine-2,4-diamine Synthesized using the procedure described in Example 50 using intermediate C1 and 3-methylsulfonylaniline. 1 H NMR (DMSO-d6, 400 MHz): 8.88 (1H, s), 7.81 (1H, d, J = 5.5 Hz), 7.50 - 7.61 (4H, m), 7.40 - 7.49 (2H, m), 6.60 (1H, d, J = 2.3 Hz), 6.45 (1H, dd, J = 8.7, 2.4 Hz), 6.31 - 6.38 (2H, m), 3.79 (3H, s), 3.19 (3H, s), 3.06 - 3.14 (4H, m), 2.42 - 2.48 (4H, m), 2.22 (3H, s). LCMS method B; RT = 1.07; m / z [M+H] + 468.2.

[0517] Example 55 (2045) [ka]

[0518] N2-(2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)-N4-(pyridin-4-yl)pyridine-2,4-diamine Synthesized using the procedure described in Example 50 using intermediate C1 and pyridin-4-amine. 1H NMR (CDCl3, 400 MHz): 8.37 - 8.47 (2H, m), 8.07 (1H, d, J = 5.8 Hz), 7.50 - 7.62 (1H, m), 6.92 - 7.04 (2H, m), 6.52 - 6.60 (3H, m), 6.49 (1H, dd, J = 5.8, 2.0 Hz), 6.43 (1H, d, J = 1.8 Hz), 6.19 (1H, s), 3.87 (3H, s), 3.16 - 3.26 (4H, m), 2.56 - 2.68 (4H, m), 2.39 (3H, s). LCMS method C; RT = 0.31; m / z [M+H] + 391.1.

[0519] Example 56 (2046) [ka]

[0520] N-(2-methoxy-5-((2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)phenyl)acetamide Synthesized using the procedure described in Example 50 using intermediate C1 and N-(5-amino-2-methoxyphenyl)acetamide. 1H NMR (CDCl3, 400 MHz): 8.20 - 8.31 (1H, m), 7.85 - 7.91 (1H, m), 7.78 (1H, br. s.), 7.59 (1H, d, J = 8.5 Hz), 6.91 (1H, dd, J =, 8.7, 2.4 Hz), 6.81 - 6.87 (1H, m), 6.55 - 6.64 (2H, m), 6.52 (1H, dd, J = 8.5, 2.5 Hz), 6.25 (1H, dd, J = 5.9, 1.9 Hz), 6.20 (1H, s), 5.81 (1H, br. s.), 3.91 (3H, s), 3.85 (3H, s), 3.11 - 3.28 (4H, m), 2.56 - 2.71 (4H, m), 2.38 (3H, s), 2.23 (3H, s). LCMS method B; RT = 1.16; m / z [M+H] + 477.2.

[0521] Example 57 (2047) [ka]

[0522] N2-(2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)-N4-(quinolin-6-yl)pyridine-2,4-diamine Synthesized using the procedure described in Example 50 using intermediate C1 and quinolin-6-amine. 1H NMR (CDCl3, 400 MHz): 8.83 (1H, dd, J = 4.3, 1.83 Hz), 8.09 - 7.99 (3H, m), 7.60 - 7.50 (3H, m), 7.39 (1H, dd, J = 8.3, 4.3 Hz), 6.57 - 6.51 (3H, m), 6.46 - 6.41 (2H, m), 6.19 (1H, s), 3.86 - 3.86 (3H, m), 3.21 - 3.17 (4H, m), 2.63 - 2.59 (4H, m), 2.39 - 2.38 (3H, m). LCMS method B; RT = 1.06; m / z [M+H] + 441.2.

[0523] Example 58 (2049) [ka]

[0524] 3-((2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)-N-methylbenzamide Synthesized using the procedure described in Example 50 using intermediate C1 and 3-amino-N-methylbenzamide. 1 H NMR (CDCl3, 400 MHz): 7.97 (1H, d, J = 6.0 Hz), 7.59 - 7.56 (2H, m), 7.39 - 7.37 (2H, m), 7.29 - 7.28 (1H, m), 6.57 - 6.48 (3H, m), 6.35 - 6.32 (2H, m), 6.14 (1H, d, J = 3.8 Hz), 6.01 (1H, s), 3.86 - 3.85 (3H, m), 3.21 - 3.17 (4H, m), 3.05 - 3.03 (3H, m), 2.63 - 2.59 (4H, m), 2.38 (3H, s). LCMS method B; RT = 1.12; m / z [M+H] + 447.2.

[0525] Example 59 (2052) [ka]

[0526] N2-(2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)-N4-(3-(pyridin-2-yl)phenyl)pyridine-2,4-diamine Synthesized using the procedure described in Example 50 using intermediate C1 and 3-(pyridin-2-yl)aniline. 1 H NMR (CDCl3, 400 MHz): 8.73 - 8.71 (1H, m), 7.97 - 7.95 (1H, m), 7.88 (1H, t, J = 2.0 Hz), 7.81 - 7.67 (3H, m), 7.58 - 7.55 (1H, m), 7.44 (1H, t, J = 7.9 Hz), 7.28 (1H, s), 6.58 - 6.54 (2H, m), 6.42 - 6.34 (3H, m), 6.02 (1H, s), 3.84 (3H, s), 3.17 - 3.12 (4H, m), 2.62 - 2.58 (4H, m), 2.38 - 2.38 (3H, m). LCMS method B; RT = 1.21; m / z [M+H] + 467.2.

[0527] Example 60 (2054) [ka]

[0528] N-(3-((2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)phenyl)methanesulfonamide Synthesized using the procedure described in Example 50 using intermediate C1 and N-(3-aminophenyl)methanesulfonamide. 1H NMR (DMSO-d6, 400 MHz): 9.70 (1H, br. s.), 8.57 (1H, s), 7.75 (1H, d, J = 6.3 Hz), 7.57 (1H, d, J = 8.5 Hz), 7.39 (1H, s), 7.22 (1H, t, J = 8.0 Hz), 7.01 (1H, s), 6.85 - 6.94 (1H, m), 6.79 (1H, dd, J = 8.0, 1.3), 6.60 (1H, d, J = 2.5 Hz), 6.45 (1H, dd, J = 8.7, 2.4 Hz), 6.24 - 6.34 (2H, m), 3.78 (3H, m / z), 3.04 - 3.15 (4H, m), 2.98 (3H, s), 2.40 - 2.48 (4H, m), 2.23 (3H, s). LCMS method B; RT = 1.09; + 483.2.

[0529] Example 61 (2055) [ka]

[0530] 4-((2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)-N-methylbenzamide Synthesized using the procedure described in Example 50 using intermediate C1 and 4-amino-N-methylbenzamide. 1H NMR (CDCl3, 400 MHz): 7.99 (1H, d, J = 5.5 Hz), 7.72 (2H, d, J = 8.8 Hz), 7.56 (1H, d, J = 8.5 Hz), 7.14 (2H, d, J = 8.8 Hz), 6.56-6.50 LCMS method B; RT = 1.15; m / z [M+H] + 447.3.

[0531] Example 62 (2056) [ka]

[0532] N2-(2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)-N4-(quinoxalin-5-yl)pyridine-2,4-diamine Synthesized using the procedure described in Example 50 using intermediate C1 and quinoxalin-5-amine. 1 H NMR (CDCl3, 400 MHz): 8.90 (1H, d, J = 1.8 Hz), 8.73 (1H, d, J = 1.8 Hz), 8.20 (1H, br s), 8.08 (1H, d, J = 5.8 Hz), 7.72-7.62 (4H, m), 6.70 (1H, dd, J = 5.8, 2.0 Hz), 6.65 (1H, d, J = 2.0 Hz), 6.61 (1H, br s), 6.58-6.56 (2H, m), 3.87 (3H, s), 3.20 (4H, t, J = 5.0 Hz), 2.62 (4H, t, J = 5.0 Hz), 2.38 (3H, s). LCMS method B; RT = 1.15; m / z [M+H] + 447.3.

[0533] Example 63 (2057) [ka]

[0534] N4-(1H-indol-4-yl)-N2-(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)pyridine-2,4-diamine A mixture of Intermediate C1 (75 mg, 0.2 mmol), 1H-indol-4-amine (33 mg, 0.25 mmol), XANTPHOS (8 mg, 0.014 mmol), and cesium carbonate (147 mg, 0.45 mmol) in DMA (1.5 mL) was degassed by bubbling argon for 10 minutes. Pd(OAc)2 (2 mg, 0.01 mmol) was then added, and the reaction was heated at 150 °C for 90 minutes. The reaction was diluted with MeOH and eluted with MeOH, then loaded directly onto a 5 g SCX cartridge eluted with 2 M methanolic ammonia. The basic fractions were combined and concentrated under reduced pressure. Purification by flash chromatography (0–10% MeOH in DCM) afforded the title compound as a brown solid (13 mg, 13% yield). 1 H NMR (CDCl3, 400 MHz): 8.32 (1H, br s), 7.92 (1H, d, J = 5.8 Hz), 7.59 (1H, d, J = 8.6 Hz), 7.21 (2H, m), 7.16 (1H, t, J = 8.0 Hz), 7.03 (1H, m), 6.59-6.53 (2H, m), 6.50-6.47 (2H, m), 6.34 (1H, dd, J = 5.8, 2.0 Hz), 6.30 (1H, d, J = 1.8 Hz), 6.08 (1H, br s), 3.83 (3H, s), 3.16 (4H, m), 2.60 (4H, m), 2.37 (3H, s). LCMS method B; RT = 1.01; m / z [M+H] + 429.3.

[0535] Example 64 (2061) [ka]

[0536] N2-(2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)-N4-(pyridin-3-yl)pyridine-2,4-diamine Synthesized using the procedure described in Example 50 using intermediate C1 and pyridin-3-amine. 1 H NMR (CDCl3, 400 MHz): 8.48 (1H, d, J = 2.5 Hz), 8.33 (1H, dd, J = 4.7, 1.5 Hz), 7.99 (1H, d, J = 5.7 Hz), 7.58 (1H, d, J = 8.6 Hz), 7.53 (1H, ddd, J = 8.3, 2.6, 1.7 Hz), 7.26 - 7.24 (1H, m), 6.57 (1H, d, J = 2.3 Hz), 6.54 (1H, dd, J = 8.6, 2.5 Hz), 6.51 (1H, s), 6.33 (1H, dd, J = 5.9, 2.1 Hz), 6.28 (1H, d, J = 2.0 Hz), 5.88 (1H, s), 3.86 (3H, s), 3.23 – 3.17 (4H, m), 2.64 – 2.59 (4H, m), 2.59 (3H, s). LCMS method B; RT = 0.57; + 391.0.

[0537] Example 65 (2063) [ka]

[0538] N4-(3-(1H-imidazol-1-yl)phenyl)-N2-(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)pyridine-2,4-diamine Synthesized using the procedure described in Example 50 using intermediate C1 and 3-(1H-imidazol-1-yl)aniline. 1H NMR (DMSO-d6, 400 MHz): 8.71 (1H, s), 8.23 ​​- 8.19 (1H, m), 7.78 (1H, d, J = 5.7 Hz), 7.68 (1H, t, J = 1.4 Hz), 7.55 (1H, d, J = 8.7 Hz), 7.48 (1H, s), 7.41 (1H, t, J = 8.1 Hz), 7.33 (1H, t, J = 2.1 Hz), 7.17 (1H, ddd, J = 8.0, 2.3, 0.7 Hz), 7.11 (1H, t, J = 1.0 Hz), 7.09 (1H, ddd, J = 8.2, 2.0, 0.8 Hz), 6.58 (1H, d, J = 2.7 Hz), 6.41 (1H, d, J = 2.0 Hz), 6.37 (1H, dd, J = 8.6, 2.7 Hz), 6.32 (1H, dd, J = 5.7, 2.1 Hz), 3.76 (3H, s), 3.11 - 3.02 (4H, m), 2.48 - 2.41 (4H, m), 2.22 (3H, s). LCMS method C; RT = 0.35; m / z [M+H] + 456.2.

[0539] Example 66 (2064) [ka]

[0540] N4-(3-(4H-1,2,4-triazol-4-yl)phenyl)-N2-(2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)pyridine-2,4-diamine Synthesized using the procedure described in Example 50 using intermediate C1 and 3-(1,2,4-triazol-4-yl)aniline. 1H NMR (DMSO-d6, 400 MHz): 9.10 (2H, s), 8.77 (1H, s), 7.79 (1H, d, J = 5.7 Hz), 7.58 (1H, d, J = 8.7 Hz), 7.49 - 7.43 (2H, m), 7.42 (1H, t, J = 2.2 Hz), 7.21 (1H, ddd, J = 8.0, 2.2, 0.7 Hz), 7.15 (1H, ddd, J = 8.2, 2.2, 0.8 Hz), 6.57 (1H, d, J = 2.7 Hz), 6.43 (1H, d, J = 2.0 Hz), 6.38 - 6.31 (2H, m), 3.76 (3H, s), 3.10 - 3.02 (4H, m), 2.47 - 2.41 (4H, m), 2.22 (3H, s). LCMS method B; RT = 1.04; m / z [M+H] + 416.0.

[0541] Example 67 (2066) [ka]

[0542] 2-((2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)nicotinonitrile Synthesized using the procedure described in Example 50 using intermediate C1 and 2-aminonicotinonitrile. 1H NMR (CDCl3, 400 MHz): 8.43 - 8.54 (1H, m), 8.06 - 8.17 (1H, m), 7.86 (1H, dd, J = 7.8, 1.8 Hz), 7.73 (1H, d, J = 9.3 Hz), 7.24 (1H, d, J = 1.8 Hz), 6.97 - 7.05 (2H, m), 6.93 (1H, dd, J = 7.5, 5.0 Hz), 6.68 (1H, br. s.), 6.56 - 6.62 (2H, m), 3.88 (3H, s), 3.15 - 3.33 (4H, m), 2.59 - 2.70 (4H, m), 2.40 (3H, s). LCMS method B; RT = 1.10; m / z [M+H] + 416.0.

[0543] Example 68 (2067) [ka]

[0544] 1-(7-((2-(quinolin-5-ylamino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized using the procedure described in Example 1 using intermediate B1 and quinolin-5-amine. 1H NMR (CDCl3, 400 MHz): 8.87 (dd, J = 4.2 and 1.6 Hz, 1H), 8.36 (d, J = 9.2 Hz, 1H), 8.31 (br s, 1H), 7.85 (d, J = 8.5 Hz, 1H), 7.78 (d, J = 6.0 Hz, 1H), 7.60 (t, J = 7.5 Hz, 1H), 7.49 (dd, J = 7.5 and 1.0 Hz, 1H), 7.34 (dd, J = 8.5 and 4.2 Hz, 1H), 7.07-7.02 (m, 2H), 6.87 (t, J = 7.4 Hz, 1H), 6.78 (dd, J = 7.2 and 0.9 Hz, 1H). Hz, 1H), 6.29 (dd, J = 6.0 and 2.0 Hz, 1H), 6.07 (d, J = 1.9 Hz, 1H), 3.98 (t, J = 7.8 Hz, 2H), 2.99 (t, J = 7.7 Hz, 2H), 2.21 (s, 3H). LCMS method A; RT = 1.34; m / z [M+H] + 396.2.

[0545] Example 69 (2069) [ka]

[0546] 1-(7-((2-((3-ethynylphenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using Intermediate B1 and 3-aminophenylacetylene. 1H NMR (CDCl3, 400 MHz): 8.54 (br s, 1H), 8.21 (d, J = 5.8 Hz, 1H), 7.31 (d, J = 8.1 Hz, 1H), 7.17 (t, J = 7.4 Hz, 1H), 7.11 (t, J = 7.8 Hz, 1H), 7.06 (d, J = 2.0 Hz, 1H), 6.99-6.96 (m, 2H), 6.88 (t, J = 2.0 Hz, 1H), 6.75 (dd, J = 8.2 and 2.4 Hz, 1H), 6.68-6.65 (m, 1H), 4.12 (t, J = 7.8 Hz, 2H), 3.68 (br s, 1H), 3.14 (t, J = 7.8 Hz, 2H), 2.35 (s, 3H). LCMS method A; RT = 1.59; m / z [M+H] + 369.2.

[0547] Example 70 (2070) [ka]

[0548] 1-(7-((2-((3-isopropyl-1-methyl-1H-pyrazol-5-yl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using intermediate B1 and isopropyl-2-methyl-pyrazol-3-amine. 1H NMR (CDCl3, 400 MHz): 8.38 (br s, 1H), 7.73 (d, J = 5.9 Hz, 1H), 7.14 (d, J = 9.0 Hz, 1H), 7.02 (t, J = 7.3 Hz, 1H), 6.85 (dd, J = 7.3 and 1.0 Hz, 1H), 6.28 (dd, J = 6.0 and 2.1 Hz, 1H), 6.04 (d, J = 1.8 Hz, 1H), 5.83 (s, 1H), 4.03 (t, J = 7.8 Hz, 2H), 3.60 (s, 1H), 3.04 (t, J = 7.7 Hz, 2H), 2.25 (s, 3H), 1.19 (s, 3H), 1.17 (s, 3H). LCMS method A; RT = 1.77; m / z [M+H] + 391.2

[0549] Example 71 (2071) [ka]

[0550] 1-(7-((2-(isoquinolin-8-ylamino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using intermediate B1 and isoquinolin-8-amine. 1H NMR (CDCl3, 400 MHz): 9.43 (s, 1H), 8.48 (d, J = 5.7 Hz, 1H), 8.30 (br s, 1H), 7.82 (d, J = 6.0 Hz, 1H), 7.61-7.54 (m, 3H), 7.48 (d, J = 7.6 Hz, 1H), 7.12-7.10 (m, 2H), 6.93 (t, J = 7.4 Hz, 1H), 6.80 (d, J = 7.3 Hz, 1H), 6.34 (dd, J = 6.0 and 2.0 Hz, 1H), 6.20 (d, J = 1.9 Hz, 1H), 4.00 (t, J = 7.8 Hz, 2H), 3.01 (t, J = 7.8 Hz, 2H), 2.22 (s, 3H). LCMS method A; RT = 1.30; m / z [M+H] + 396.1

[0551] Example 72 (2072) [ka]

[0552] 1-(7-((2-((1-acetylindolin-5-yl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using intermediate B1 and 1-acetyl-5-amino-2,3-dihydro(1H)indole. 1H NMR (CDCl3, 400 MHz): 8.69 (br s, 1H), 8.13 (d, J = 8.6 Hz, 1H), 7.70 (d, J = 6.2 Hz, 1H), 7.64 (br s, 1H), 7.22 (d, J = 8.1 Hz, 1H), 7.15 (s, 1H), 7.12 (t, J = 7.4 Hz, 1H), 7.00 (dd, J = 8.6 and 2.2 Hz, 1H), 6.96 (d, J = 7.3 Hz, 1H), 6.32-6.28 (m, 2H), 4.13-4.05 (m, 4H), 3.20-3.10 (m, 4H), 2.32 (s, 3H), 2.22 (s, 3H). LCMS method A; RT = 1.65; m / z [M+H] + 428.1.

[0553] Example 73 (2073) [ka]

[0554] 1-(7-((2-((1-acetylindolin-6-yl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using intermediate B1 and 1-acetyl-6-amino-2,3-dihydro-1H-indole. 1H NMR (CDCl3, 400 MHz): 8.69 (br s, 1H), 8.13 (s, 1H), 7.79-7.69 (m, 2H), 7.32 (d, J = 8.2 Hz, 1H), 7.10 (t, J = 7.5 Hz, 2H), 6.98 (dd, J = 7.9 and 1.5 Hz, 1H), 6.94 (d, J = 7.3 Hz, 1H), 6.37 (d, J = 1.6 Hz, 1H), 6.30 (dd, J = 6.3 and 1.9 Hz, 1H), 4.12-4.06 (m, 4H), 3.18-3.09 (m, 4H), 2.33 (s, 3H), 2.24 (s, 3H). LCMS method A; RT = 1.73; m / z [M+H] + 428.1.

[0555] Example 74 (2079) [ka]

[0556] 1-(7-((2-((5-phenylpyridin-2-yl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using intermediate B1 and 2-amino-5-phenylpyridine. 1H NMR (CDCl3, 400 MHz): 9.61 (br s, 1H), 8.42 (s, 1H), 7.87 (dd, J = 8.6 and 2.4 Hz, 1H), 7.71 (d, J = 8.7 Hz, 1H), 7.63 (d, J = 7.0 Hz, 1H), 7.61 (br s, 1H), 7.53 (d, J = 7.0 Hz, 2H), 7.47 (t, J = 7.2 Hz, 2H), 7.45-7.33 (m, 4H), 7.10 (d, J = 7.3 Hz, 1H), 6.47 (dd, J = 7.0 and 2.3 Hz, 1H), 4.17 (t, J = 7.8 Hz, 2H), 3.18 (t, J = 7.8 Hz, 2H), 2.38 (s, 3H). LCMS method A; RT = 2.25; m / z [M+H] + 422.1.

[0557] Example 75 (2101) [ka]

[0558] 4-((4-((1-acetylindolin-7-yl)amino)pyridin-2-yl)amino)-N,N-dimethylbenzamide Synthesized according to the procedure described in Example 1 using intermediate B1 and 4-amino-N,N-dimethyl-benzamide. 1H NMR (CDCl3, 400 MHz): 8.50 (s, 1H), 7.86 (d, J = 6.0 Hz, 1H), 7.38 (d, J = 8.7 Hz, 2H), 7.29 (d, J = 8.7 Hz, 2H), 7.26 (s, 1H), 7.13 (t, J = 7.4 Hz, 1H), 7.00 (br s, 1H), 6.94 (dd, J = 7.3 and 1.0 Hz, 1H), 6.43 (d, J = 1.8 Hz, 1H), 6.40 (dd, J = 6.0 and 2.0 Hz, 1H), 4.11 (t, J = 7.8 Hz, 2H), 3.12 (t, J = 7.7 Hz, 2H), 3.06 (br s, 6H), 2.33 (s, 3H). LCMS method A; RT = 1.53; m / z [M+H] + 416.2.

[0559] Example 76 (2102) [ka]

[0560] N4-(3-(tert-butyl)-1-methyl-1H-pyrazol-5-yl)-N2-(4-(4-ethylpiperazin-1-yl)phenyl)pyridine-2,4-diamine Synthesized according to the procedure described in Example 1 using intermediate B25 and 4-(4-ethylpiperazin-1-yl)aniline. 1H NMR (CDCl3, 400 MHz): 7.70 (d, J = 5.2 Hz, 1H), 7.17 (d, J = 8.9 Hz, 2H), 6.97 (d, J = 8.8 Hz, 2H), 6.29-6.27 (m, 1H), 6.06-6.05 (d, J = 2.2 Hz, 1H), 5.97 (s, 1H), 3.62 (s, 3H), 3.22 (t, J = 5.6 Hz, 4H), 2.79 (t, J = 5.0 Hz, 4H), 2.64 (q, J = 7.8 Hz, 2H), 1.27 (s, 9H), 1.20 (t, J = 7.2 Hz, 3H). LCMS method A; RT = 1.15; m / z [M+H] + 434.2.

[0561] Example 77 (2103) [ka]

[0562] N2-(4-(4-ethylpiperazin-1-yl)phenyl)-N4-(3-isopropyl-1-methyl-1H-pyrazol-5-yl)pyridine-2,4-diamine Synthesized according to the procedure described in Example 1 using intermediate B27 and 4-(4-ethylpiperazin-1-yl)aniline. 1H NMR (MeOD, 400 MHz): 7.69 (d, J = 6.2 Hz, 1H), 7.18 (d, J = 8.8 Hz, 2H), 6.97 (d, J = 8.9 Hz, 2H), 6.27 (dd, J = 6.2 and 2.0 Hz, 1H), 6.07 (d, J = 2.0 Hz, 1H), 5.94 (s, 1H), 3.22 (t, J = 4.8 Hz, 4H), 2.86 (sept, J = 8.2 Hz, 1H), 2.79 (t, J = 5.0 Hz, 4H), 2.64 (q, J = 7.3 Hz, 2H), 1.23 (s, 3H), 1.22 (s, 3H), 1.20 (t, J = 7.3 Hz, 3H). LCMS method A; RT = 1.13; m / z [M+H] + 420.2.

[0563] Example 78 (2104) [ka]

[0564] 3-((2-((4-(4-ethylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)-N-methylbenzamide Synthesized according to the procedure described in Example 1 using intermediate B28 and 4-(4-ethylpiperazin-1-yl)aniline. 1H NMR (MeOD, 400 MHz): 7.71 (t, J = 1.8, 1H), 7.62 (d, J = 6.7 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.46 (t, J = 7.9 Hz, 1H), 7.36-7.33 (m, 1H), 7.19 (d, J = 8.9 Hz, 2H), 7.01 (d, J = 9.0 Hz, 2H), 6.45 (dd, J = 6.7 and 2.2 Hz, 1H), 6.34 (d, J = 2.1 Hz, 1H), 3.25 (t, J = 4.9 Hz, 4H), 2.93 (s, 3H), 2.82 (t, J = 4.9 Hz, 4H), 2.67 (q, J = 7.3 Hz, 2H), 1.21 (t, J = 7.2 Hz, 3H). LCMS method A; RT = 0.94; m / z [M+H] + 431.2.

[0565] Example 79 (2105) [ka]

[0566] 4-((2-((4-(4-ethylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)-N-methylbenzamide Synthesized according to the procedure described in Example 1 using intermediate B29 and 4-(4-ethylpiperazin-1-yl)aniline. 1H NMR (MeOD, 400 MHz): 7.78 (d, J = 8.7 Hz, 2H), 7.71 (d, J = 6.2 Hz, 1H), 7.22 (dd, J = 8.7 and 3.4 Hz, 4H), 6.97 (d, J = 9.0 Hz, 2H), 6.47-6.44 LCMS method A; RT = 1.13; m / z [M+H] + 431.2.

[0567] Example 80 (2106) [ka]

[0568] N2-(4-(4-ethylpiperazin-1-yl)phenyl)-N4-(pyrimidin-4-yl)pyridine-2,4-diamine Synthesized according to the procedure described in Example 1 using intermediate B32 and 4-(4-ethylpiperazin-1-yl)aniline. 1 H NMR (MeOD, 400 MHz): 8.66 (s, 1H), 8.29 (d, J = 6.0 Hz, 1H), 7.86 (d, J = 5.9, 1H), 7.47 (d, J = 1.6 Hz, 1H), 7.29 (d, J = 8.9 Hz, 2H), 6.98 (d, J = 9.1 Hz, 2H), 6.95 (d, J = 1.9 Hz, 1H), 6.86 (dd, J = 6.0 and 1.1 Hz, 1H), 3.23 (t, J = 4.6 Hz, 4H), 2.85 (t, J = 5.0 Hz, 4H), 2.69 (q, J= 7.3 Hz, 2H), 1.21 (t, J = 7.3 Hz, 3H). LCMS method A; RT = 0.90; m / z [M+H] + 376.2.

[0569] Example 81 (2107) [ka]

[0570] N4-(3-(1H-imidazol-1-yl)phenyl)-N2-(4-(4-ethylpiperazin-1-yl)phenyl)pyridine-2,4-diamine Synthesized according to the procedure described in Example 1 using intermediate B30 and 4-(4-ethylpiperazin-1-yl)aniline. 1 H NMR (MeOD, 400 MHz): 8.12 (s, 1H), 7.69 (d, J = 6.4 Hz, 1H), 7.55 (s, 1H), 7.48 (t, J = 8.2 Hz, 1H), 7.40 (t, J = 2.1 Hz, 1H), 7.26 (dd, J = 8.2 and 2.2 Hz, 1H), 7.20-7.17 (m, 4H), 6.91 (d, J = 9.0 Hz, 2H), 3.22 (t, J = 4.7 Hz, 4H), 2.85 (t, J = 5.0 Hz, 4H), 2.71 (q, J = 7.4 Hz, 2H), 1.22 (t, J = 7.2 Hz, 3H). LCMS method A; RT = 0.70; m / z [M+H] + 440.2.

[0571] Example 82 (2108) [ka]

[0572] N2-(4-(4-ethylpiperazin-1-yl)phenyl)-N4-(3-(methylsulfonyl)phenyl)pyridine-2,4-diamine Synthesized according to the procedure described in Example 1 using intermediate B31 and 4-(4-ethylpiperazin-1-yl)aniline. 1H NMR (MeOD, 400 MHz): 7.76-7.73 (m, 2H), 7.59-7.56 (m, 2H), 7.46 (d, J = 6.9 Hz, 1H), 7.21 (d, J = 8.5 Hz, 2H), 7.00 (d, J = 8.8 Hz, 2H), 6.45-6.42 (m, 2H), 3.25 (t, J = 4.8 Hz, 4H), 3.10 (s, 3H), 2.86 (t, J = 4.9 Hz, 4H), 2.71 (q, J = 7.3 Hz, 2H), 1.22 (t, J = 7.3 Hz, 3H). LCMS method A; RT = 1.04; m / z [M+H] + 452.1.

[0573] Example 83 (2109) [ka]

[0574] 1-(5-((4-((3-(tert-butyl)-1-methyl-1H-pyrazol-5-yl)amino)pyridin-2-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using intermediate B25 and 1-acetyl-5-amino-2,3-dihydro(1H)indole. 1 H NMR (MeOD, 400 MHz): 8.00 (d, J = 8.6 Hz, 1H), 7.74 (d, J = 5.8 Hz, 1H), 7.24 (s, 1H), 7.04 (d, J = 8.5 Hz, 1H), 6.25 (dd, J = 5.9 and 1.7 Hz, 1H), 6.07 (d, J = 1.8 Hz, 1H), 5.97 (s, 1H), 4.11 (t, J = 8.3 Hz, 2H), 3.62 (s, 3H), 3.17 (t, J = 8.4 Hz, 2H), 2.21 (s, 3H), 1.27 (s, 9H). LCMS method A; RT = 1.63; m / z [M+H] + 405.2.

[0575] Example 85 (2110) [ka]

[0576] 1-(5-((4-((3-isopropyl-1-methyl-1H-pyrazol-5-yl)amino)pyridin-2-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using intermediate B27 and 1-acetyl-5-amino-2,3-dihydro(1H)indole. 1 H NMR (MeOD, 400 MHz): 7.99 (d, J = 8.6 Hz, 1H), 7.74 (d, J = 6.0 Hz, 1H), 7.25 (s, 1H), 7.05 (d, J = 8.4 Hz, 1H), 6.25 (dd, J = 6.0 and 2.0 Hz, 1H), 6.09 (d, J = 1.9 Hz, 1H), 5.94 (s, 1H), 4.10 (t, J = 8.4 Hz, 2H), 3.62 (s, 3H), 3.16 (t, J = 8.4 Hz, 2H), 2.87 (sept, J = 6.9 Hz, 1H), 2.20 (s, 3H), 1.24 (s, 3H), 1.22 (s, 3H). LCMS method A; RT = 1.61; m / z [M+H] + 391.2.

[0577] Example 86 (2111) [ka]

[0578] 3-((2-((1-acetylindolin-5-yl)amino)pyridin-4-yl)amino)-N-methylbenzamide Synthesized according to the procedure described in Example 1 using intermediate B28 and 1-acetyl-5-amino-2,3-dihydro(1H)indole. 1H NMR (MeOD, 400 MHz): 8.00 (d, J = 8.6 Hz, 1H), 7.73 (d, J = 6.1 Hz, 1H), 7.68 (t, J = 1.7 Hz, 1H), 7.47 (d, J = 7.7 Hz, 1H), 7.41 (t, J = 7.8 Hz, 1H), 7.32 (d, J = 8.8 Hz, 1H), 7.23 (s, 1H), 7.06 (dd, J = 8.6 and 1.9 Hz, 1H), 6.45-6.41 (m, 2H), 4.07 (t, J = 8.4 Hz, 2H), 3.14 (t, J = 8.4 Hz, 2H), 2.94 (s, 3H), 2.19 (s, 3H). LCMS method A; RT = 1.46; m / z [M+H] + 402.2.

[0579] Example 87 (2112) [ka]

[0580] 4-((2-((1-acetylindolin-5-yl)amino)pyridin-4-yl)amino)-N-methylbenzamide Synthesized according to the procedure described in Example 1 using intermediate B29 and 1-acetyl-5-amino-2,3-dihydro(1H)indole. 1 H NMR (MeOD, 400 MHz): 7.99 (d, J = 8.6 Hz, 1H), 7.79-7.76 (m, 3H), 7.27 (s, 1H), 7.20 (d, J = 8.7 Hz, 2H), 7.06 (dd, J = 8.6 and 2.0 Hz, 1H), 6.51 (d, J = 1.9 Hz, 1H), 6.45 (dd, J = 6.0 and 2.0 Hz, 1H), 4.08 (t, J = 8.4 Hz, 2H), 3.15 (t, J = 8.3 Hz, 2H), 2.90 s, 3H), 2.19 (s, 3H). LCMS method A; RT = 1.42; m / z [M+H] + 402.2.

[0581] Example 88 (2115) [ka]

[0582] 1-(5-((4-((3-(methylsulfonyl)phenyl)amino)pyridin-2-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using intermediate B31 and 1-acetyl-5-amino-2,3-dihydro(1H)indole. 1 H NMR (MeOD, 400 MHz): 7.92 (d, J = 8.6 Hz, 1H), 7.70 (d, J = 6.0 Hz, 1H), 7.62 (s, 1H), 7.46-7.45 (m, 2H), 7.38-7.35 (m, 1H), 7.17 (s, 1H), 6.98 (dd, J = 8.6 and 2.1 Hz, 1H), 6.38 (d, J = 1.9 Hz, 1H), 6.33 (dd, J = 6.0 and 2.0 Hz, 1H), 4.01 (t, J = 8.3 Hz, 2H), 3.09 (t, J = 8.3 Hz, 2H), 3.00 (s, 3H), 2.11 (s, 3H). LCMS method A; RT = 1.45; m / z [M+H] + 423.1.

[0583] Example 89 (2116) [ka]

[0584] 4-((4-((3-(tert-butyl)-1-methyl-1H-pyrazol-5-yl)amino)pyridin-2-yl)amino)-N-methylbenzamide Synthesized according to the procedure described in Example 1 using intermediate B25 and 4-amino-N-methylbenzamide. 1H NMR (MeOD, 400 MHz): 7.81 (d, J = 5.9 Hz, 1H), 7.67 (d, J = 8.8 Hz, 2H), 7.44 (d, J = 8.7 Hz, 2H), 6.28 (dd, J = 5.9 and 2.0 Hz, 1H), 6.15 (d, J = 1.9 Hz, 1H), 5.98 (s, 1H), 3.60 (s, 3H), 2.86 (s, 3H), 1.26 (s, 9H). LCMS method A; RT = 1.48; m / z [M+H] + 379.2.

[0585] Example 90 (2117) [ka]

[0586] 4-((4-((3-isopropyl-1-methyl-1H-pyrazol-5-yl)amino)pyridin-2-yl)amino)-N-methylbenzamide Synthesized according to the procedure described in Example 1 using intermediate B27 and 4-amino-N-methylbenzamide. 1 H NMR (MeOD, 400 MHz): 7.86 (d, J = 5.9 Hz, 1H), 7.72 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 8.8 Hz, 2H), 6.34 (dd, J = 5.9 and 2.0 Hz, 1H), 6.20 (d, J = LCMS method A; RT = 1.34; m / z [M+H] + 365.2.

[0587] Example 91 (2347) [ka]

[0588] 1-(7-((2-((2-methoxy-4-(morpholine-4-carbonyl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using Intermediate B1 and (4-amino-3-methoxyphenyl)(morpholino)methanone. 1 H NMR (DMSO-d6, 400MHz) δ 8.82 (bs, 1H), 8.13 (bs, 1H), 7.79 (d, J = 6Hz, 1H), 7.20 (m, J = 8Hz, 2H), 7.03 (s, 2H), 6.95 (dd, J = 6Hz, 1H), 6.48 (bs, 1H), 6.33 (d, J = 6Hz, 1H), 4.14 (t, J = 8Hz, 2H), 3.85 (s, 3H), 3.61 (m, J = 5Hz, 4H), 3.52 (bs, 4H), 3.08 (t, J = 8Hz, 2H), 2.28 (s, 3H). LCMS method C; RT = 2.02; m / z [M+H] + 488.2.

[0589] Example 92 (2348) [ka]

[0590] 1-(7-((2-((4-(morpholine-4-carbonyl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using Intermediate B1 and (4-aminophenyl)(morpholino)methanone. 1H NMR (CDCl3, 400MHz) δ 8.39 (s, 1H), 7.92 (t, J = 4 Hz, 1H), 7.38-7.28 (m, 5H), 7.13 (t, J = 8 Hz, 1H), 6.95-6.93 (m, 1H), 6.53 (s, 1H), 6.428 (t, J = 4 Hz, 2H), 4.11 (t, J = 8 Hz, 2H), 3.70-3.66 (br m, 9H), 3.12 (t, J = 8 Hz, 2H), 2.33 (s, 3H). LCMS method C; RT = 1.89; m / z [M+H] + 458.2.

[0591] Example 93 (2349) [ka]

[0592] 4-((4-((1-acetylindolin-7-yl)amino)pyridin-2-yl)amino)-3-methoxy-N-methylbenzamide Synthesized according to the procedure described in Example 1 using Intermediate B1 and 4-amino-3-methoxy-N-methylbenzamide. 1 H NMR (CDCl3, 400MHz) δ 9.44 (s, 1H), 9.07 (s, 1H), 7.53-7.35 (m, 3H), 7.14-7.05 (m, 5H), 6.31 (dd, J = 2 Hz, J = 2Hz, 1H), 6.16 (d, J = 2 m / z [M+H] LCMS method C; RT = 1.96; m / z [M+H] + 432.1.

[0593] Example 94 (2350) [ka]

[0594] 1-(7-((2-((2-methyl-4-morpholinophenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using Intermediate B1 and 2-methyl-4-morpholinoaniline. 1 H NMR (DMSO-d6, 400MHz) δ 8.65 (bs, 1H), 7.9 (bs, 1H), 7.64 (d, J = 6Hz, 1H), 7.17-7.10, (m, 3H), 7.00 (d, J = 7Hz, 1H), 6.81 (d, J = 3Hz, 1H), 6.75 (dd, J = 6Hz, 1H), 6.16 (dd, J = 4Hz, 1H), 5.91 (s, 1H), 4.11 (t, J= 8Hz, 2H), 3.73 (m, 3H), 3.06 (m, 7H), 2.25 (s, 4H), 2.13 (s, 3H). LCMS method C; RT = 2.25; m / z [M+H] + 444.2.

[0595] Example 95 (2351) [ka]

[0596] 1-(7-((2-((2-methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using Intermediate B1 and 2-methyl-4-(4-methylpiperazin-1-yl)aniline. 1H NMR (DMSO-d6, 400MHz) δ 9.80 (bs, 1H), 9.60 (bs, 1H), 9.26 (bs, 1H), 7.56 (d, J = 7Hz, 1H), 7.16 (m, 4H), 7.00 (d, J = 2Hz, 1H), 6.91 (dd, 1H), 6.38 (dd, J = 6hz, 1H), 5.88 (d, J=2Hz, 1H), 4.11 (t, J = 8Hz, 2H), 3.84 (bs, 4H), 3.51 (bs, 3H), 3.09 (t, J = 8Hz, 2H), 2.87 (s, 3H), 2.22 (s, 3H), 2.13 (s, 3H). LCMS Method C; RT = 1.55; m / z [M+H] + 457.2.

[0597] Example 96 (2353) [ka]

[0598] 1-(7-((2-((2-methoxy-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using Intermediate B1 and 2-methoxy-4-(2-oxa-6-azaspiro[3.3]heptan-6-yl)aniline. 1H NMR (DMSO-d6, 400MHz) δ 8.39 (s, 1H), 7.68 (d, J = 8Hz, 1H), 7.40 (d, J = 7 Hz, 1H), 7.26 (s, 1H), 7.15-7.09 (m, 2H), 9.96-6.94 (m, 1H), 6.12-6.09 (m, 2H), 6.03 (s, 1H), 6.02-5.96 (m, 1H), 4.72 (s, 4H), 4.11 (t, J = 8 Hz, 2H), 3.94 (s, 4H), 3.74 (s, 3H), 3.04 (t, J = 8 Hz, 2H), 2.26 (s, 3H). LCMS Method C; RT = 2.21; m / z [M+H] + 472.2.

[0599] Example 97 (2354) [ka]

[0600] 1-(7-((2-((4-(4-cyclopropylpiperazin-1-yl)-2-methoxyphenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using Intermediate B1 and 4-(4-cyclopropylpiperazin-1-yl)-2-methoxyaniline. 1H NMR (MeOD, 400 MHz) δ 7.49 (d, J = 7.3 Hz, 1H), 7.25 (d, J = 4.4 Hz, 2H), 7.20 (d, J = 4.4 Hz, 1H), 7.16 (d, J = 8.6 Hz, 1H), 6.78 (d, J = 2.5 Hz, 1H), 6.67 (d, J = 8.7 Hz, 1H), 6.43 (dd, J = 7.4, 2.2 Hz, 1H), 6.10 (s, 1H), 4.21 (t, J = 7.8 Hz, 2H), 3.86 (s, 3H), 3.56 (s, 4H), 3.50 (t, J = 5.0 Hz, 2H), 3.41 (t, J = 5.1 Hz, 2H), 3.18 (t, J = 7.8 Hz, 2H), 2.87 (s, 1H), 2.35 (s, 3H). LCMS method C; RT = 1.59; m / z [M+H] + 499.3.

[0601] Example 98 (2355) [ka]

[0602] 1-(7-((2-(quinolin-4-ylamino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using intermediate B1 and quinolin-4-amine. 1H NMR (CDCl3, 400MHz) δ 8.71 (d, J = 5 Hz 1H), 8.57 (s, 1H), 8.06 (d, J = 8 Hz 1H), 8.02-7.98 (m, 2H), 7.70 (t, J = 6 Hz, 1H), 7.55-7.51 (m, 2H), 7.14 (t, J = 8 Hz, 2H), 6.96 (d, J = 3 Hz 1H), 6.68 (s, 1H), 6.68-6.54 (m, 1H), 4.12 (t, J = 8 Hz, 2H), 3.13 (t, J = 8 Hz, 2H), 3.01(s, 2H), 2.94(s, 2H), 2.34 (s, 3H), 2.08 (s, 3H). LCMS method C; RT = 1.79; m / z [M+H] + 396.1.

[0603] Example 99 (2356) [ka]

[0604] 1-(7-((2-((2-methoxyphenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using Intermediate B1 and 2-methoxyaniline. 1 H NMR (CDCl3, 400MHz) δ 8.21 (bs, 1H), 7.94-7.91 (m, 2H), 7.32 (d, J = 8 Hz, 1H), 7.13 (t, J = 8 Hz, 1H), 6.93-6.87 (m, 4H), 6.78 (bs, m / z [M+H] + 375.1.

[0605] Example 100 (2357) [ka]

[0606] 4-((4-((1-acetylindolin-7-yl)amino)pyridin-2-yl)amino)-3-methoxybenzonitrile Synthesized according to the procedure described in Example 1 using Intermediate B1 and 4-amino-3-methoxybenzonitrile. 1 H NMR (CDCl3, 400MHz) δ 8.46 (bs, 1H), 8.39 (d, J = 8Hz, 1H), 7.98 (d, J = 6Hz, 1H), 7.32 (d, J = 7Hz, 1H), 7.26 (m, 1H), 7.16 (t, J = 7Hz, 1H), 7.09 (bs, 1H), 7.02 (d, J = 2Hz, 1H), 6.96 (dd, J = 6Hz, 1H), 6.49 (dd, 1H), 6.38 (d, 1H), 4.12 (m, 2H), 3.91 (s, 3H), 3.13 (t, J = 8Hz, 2H), 2.34 (s, 3H). LCMS method C; RT = 2.15; m / z [M+H] + 400.1.

[0607] Example 101 (2358) [ka]

[0608] N-(3-((4-((1-acetylindolin-7-yl)amino)pyridin-2-yl)amino)phenyl)methanesulfonamide Synthesized according to the procedure described in Example 1 using Intermediate B1 and N-(3-aminophenyl)methanesulfonamide. 1H NMR (DMSO-d6, 400MHz) δ 9.528 (bs, 1H), 8.73-8.57 (bd, 1H), 7.82 (d, J = 6Hz, 1H), 7.47 (t, J = 2Hz, 1H), 7.42 (dd, J = 8Hz, 1H), 7.24-7.11 (m, 3H), 7.01 (dd, J = 7Hz, 1H), 6.93 (t, J = 8Hz, 1H), 6.68 (dd, J = 7Hz, 1H), 6.46 (t, 1H), 6.31 (s, 1H), 6.25 (dd, J = 4Hz, 1H), 4.15 (t, J = 8Hz, 2H), 3.08 (t, J = 8Hz, 2H), 3.08 (s, 3H), 2.28 (s, 3H). LCMS method C; RT = 2.07; m / z [M+H] + 438.1.

[0609] Example 102 (2359) [ka]

[0610] 1-(7-((2-((3-(methylsulfonyl)phenyl)amino)pyridin-4-yl)amino)indolin-1-yl)ethan-1-one Synthesized according to the procedure described in Example 1 using Intermediate B1 and 3-(methylsulfonyl)aniline. 1H NMR (CDCl3, 400MHz) δ 8.54 (bs, 1H), 7.95-7.93 (m, 2 H), 7.62-7.59 (m, 1H) 7.49-7.46 (m, 2H), 7.43 (d, J = 8 Hz, 1H), 7.32 (d, J = 8Hz, 1H), 7.18 (t, J = 8 Hz, 1H), 6.94 (d, J = 7 Hz, 1H), 6.49 (bs, 1H), 6.45 (dd, J = 2 Hz, J = 2Hz, 1H), 6.39 (d, J = 2 Hz, 1H), 4.11 (t, J = 8 Hz, 2H), 3.12 (t, J = 8 Hz, 2H), 3.05 (s, 3H), 2.34 (s, 3H). LCMS method C; RT = 1.93; m / z [M+H] + 423.1.

[0611] Example 103 (1940) [ka]

[0612] 3-(2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyridin-4-yl)-N,N-dimethylbenzamide Synthesized according to the procedure described in Example 3 using intermediate F1 and 2-methoxy-4-(4-methylpiperazin-1-yl)aniline. 11H NMR (CDCl3, 400 MHz) δ 8.23 (dd, J = 5.3, 0.8 Hz, 1H), 7.77 - 7.71 (m, 1H), 7.61 (dt, J = 7.1, 1.6 Hz, 2H), 7.51 - 7.40 (m, 2H), 6.93 - 6.86 (m, 2H), 6.69 (s, 1H), 6.57 (d, J = 8.1 Hz, 2H), 3.87 (s, 3H), 3.23 - 3.18 (m, 4H), 3.14 (s, 3H), 3.00 (s, 3H), 2.61 (dd, J = 6.2, 3.8 Hz, 4H), 2.37 (s, 3H). LCMS method A; RT 1.08; m / z [M+H] + 446.2 .

Claims

1. (a) a compound of formula (I) or (b) a tautomer, N-oxide, pharmaceutically acceptable salt, or solvate thereof; 【Chemical 1】 During the ceremony: X is a bond or NH; Y is a bond or NH; with the proviso that at least one of X and Y is NH; R 1 is H, F or CH 3 and R 2 are H, F, Cl, Br, -COR 4 , -SO 2 R 5 , -SOR 5 , -CN, -NO, -NO 2 or -NR 6 3 + and R 3 H, CH 3 , 0 to 3 R 7 , -COR 4 , -SO 2 R 5 , -SOR 5 , -CN, -NO, -NO 2 or -NR 6 3 + C is replaced by 2 -C 6 is alkyl; R 4 is H, 0 to 3 R 7 , -OH, -OR 8 , -NH 2 , -NHR 8 or -NR 8 2 C is replaced by 1 -C 6 independently selected from alkyl; R 5 is an R from 0 to 3 7 , -OH, -OR 8 , -NH 2 , -NHR 8 or -NR 8 2 C is replaced by 1 -C 6 independently selected from alkyl; R 6 is C 1 -C 3 independently selected from alkyl; R 7 is O.C. 1 -C 3 independently selected from alkyl, F, or Cl; R 8 is an R from 0 to 3 7 C is replaced by 1 -C 6 independently selected from alkyl; Ring A is selected from rings in Group I, Group II, Group III, Group IV and Group V, where * indicates a bond to X; Group I is 【Chemistry 2】 and: Group II is 【Chemistry 3】 and: Group III is 【Chemistry 4】 and: Group IV is 【Chemistry 5】 【change】 【change】 and: Group V is 【Chemistry 6】 and: Ring B is selected from rings in Group IA, Group IIA, Group IIA, Group IVA, and Group VA; $ indicates a bond to Y; Group IA is 【Chemistry 7】 and: Group IIA is 【Chemistry 8】 and: Group IIIA is 【Chemistry 9】 and: Group IVA is 【Chemistry 10】 and: Group VA is 【Chemistry 11】 【change】 and: During the ceremony: Q and T are each selected from CH or N, provided that at most one of Q and T may be N; V is CH or N; W is CH 2 , O, N.R. y , S, S(O) or S(O) 2 and Z is C(O), S(O) or S(O) 2 and R a is O.C. 1 -C 3 C substituted with 0 to 3 substituents independently selected from alkyl, F and Cl 1 -C 6 is alkyl; R b is C 1 -C 6 independently selected from alkyl, F, and Cl; R c is O.C. 1 -C 3 C substituted with 0 to 3 substituents independently selected from alkyl, F and Cl 1 -C 6 is alkyl; R d is O.C. 1 -C 3 C substituted with 0 to 3 substituents independently selected from alkyl, F and Cl 1 -C 6 Alkyl; or OC 1 -C 3 independently selected from phenyl substituted with 0-3 substituents independently selected from alkyl, F and Cl; R e is O.C. 1 -C 3 C substituted with 0 to 3 substituents independently selected from alkyl, F and Cl 1 -C 6 is alkyl; R f is O.C. 1 -C 3 C substituted with 0 to 3 substituents independently selected from alkyl, F and Cl 1 -C 6 is alkyl; R g is H or C 1 -C 3 is alkyl; R h is O.C. 1 -C 3 C substituted with 0 to 3 substituents independently selected from alkyl, F and Cl 1 -C 6 is alkyl; R i is O.C. 1 -C 3 C substituted with 0 to 3 substituents independently selected from alkyl, F and Cl 1 -C 6 is alkyl; R j is H or C 1 -C 3 It is an alkyl R k is O.C. 1 -C 3 C substituted with 0 to 3 substituents independently selected from alkyl, F and Cl 1 -C 6 is alkyl; R l is H or C 1 -C 3 is alkyl; R m is O.C. 1 -C 3 C substituted with 0 to 3 substituents independently selected from alkyl, F and Cl 1 -C 6 is alkyl; R n is H or C 1 -C 3 is alkyl; R o is O.C. 1 -C 3 C substituted with 0 to 3 substituents independently selected from alkyl, F and Cl 1 -C 6 Alkyl; or OC 1 -C 3 independently selected from phenyl substituted with 0-3 substituents independently selected from alkyl, F and Cl; R p is C 1 -C 6 independently selected from alkyl, F, Cl, and Br; R q is O.C. 1 -C 3 C substituted with 0 to 3 substituents independently selected from alkyl, F and Cl 1 -C 6 is alkyl; R r is H, or OC 1 -C 3 C substituted with 0 to 3 substituents independently selected from alkyl, F and Cl 1 -C 6 is alkyl; R s is H or C 1 -C 3 is alkyl; R t is O.C. 1 -C 3 C substituted with 0 to 3 substituents independently selected from alkyl, F and Cl 1 -C 6 is alkyl; R u is C 1 -C 6 independently selected from alkyl, F, and Cl; R v is C 1 -C 6 phenyl substituted with 0-2 substituents selected from alkyl, F and Cl; R w is H or C 1 -C 3 is alkyl; R x is H or C 1 -C 3 independently selected from alkyl; R y H,OC 1 -C 3 C substituted with 0 to 3 substituents independently selected from alkyl, F and Cl 1 -C 6 Alkyl; C 1 -C 6 benzyl substituted with 0-3 substituents independently selected from alkyl, F and Cl; or C 1 -C 6 Alkyl, OC 1 -C 3 C substituted with 0 to 3 substituents independently selected from alkyl, F and Cl 3 -C 6 is cycloalkyl; n is 1 to 3; p is 0 to 2; q is 1 or 2; r is 0 to 2; s is 1 to 3; t is 0 to 2; u is 2 to 3; v is 1 to 3; and: X is NH; Y is NH; R 2 is H; and when ring A is a ring in group I or group II: ring B is a ring in group IA, group IIA, group IIIA, group IVA or group VA; X is NH; Y is NH; R 2 is H; and when ring A is a ring in group III: ring B is a ring in group IA, group IIA or group IIIA; X is NH; Y is NH and R 2 is H; and when ring A is a ring in group IV: ring B is a ring in group IA or group IIA; X is NH; Y is NH; R 2 is H; and ring A is a ring in group V: ring B is a ring in group IA; X is NH; Y is NH; and R 2 F, Cl, Br, -COR 4 , -SO 2 R 5 , -SOR 5 , -CN, -NO, -NO 2 or -NR 6 3 + when: Ring A is a ring in Group I or Group II and Ring B is a ring in Group IA; When X is a bond and Y is NH: R 2 is H; ring A is a ring within Group I, Group II and Group III; and ring B is a ring within Group IA; and When X is NH and Y is a bond: R 2 is H; ring A is a ring within Group I, Group II and Group III; and ring B is a ring within Group IA, Group IIA, and Group IIIA; with the proviso that the compound of formula (I) is not (a) a compound selected from the group consisting of: The compound or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof. 【Chemistry 12】

2. 10. The compound of claim 1 , wherein: X is NH; Y is NH; R 2 is H; and when ring A is a ring in group I: ring B is a ring in group IA, group IIA, group IIIA, group IVA or group VA; and X is NH; Y is NH; R 2 is H; and when ring A is a ring in group II: ring B is a ring in group IA, group IIA, group IIIA or group IVA, compound.

3. 3. A compound according to claim 1 or 2, wherein: X is NH; Y is NH; R 2 is H or F; R 3 is H or CH 3 and Q is selected from CH or N; T is CH; W is CH 2 , O, or NR y and Z is C(O); R a But, O.C. 1 -C 3 C substituted with 0 to 1 substituents selected from alkyl 1 -C 3 is alkyl; R b But C 1 -C 3 independently selected from alkyl; R c C 1 -C 3 is alkyl; R d But C 1 -C 4 independently selected from alkyl or phenyl; R e C 1 -C 3 is alkyl; R f C 1 -C 3 is alkyl; R g is H; R h C 1 -C 3 is alkyl; R i C 1 -C 3 is alkyl; R j is H; R k C 1 -C 3 is alkyl; R l is H; R m C 1 -C 4 is alkyl; R n is H; R o But, O.C. 1 -C 3 C substituted with 0 to 1 substituents selected from alkyl 1 -C 3 alkyl; or phenyl; R p But C 1 -C 3 independently selected from alkyl; R q But, O.C. 1 -C 3 C substituted with 0 to 1 substituents selected from alkyl 1 -C 3 is alkyl; R r But H or C 1 -C 3 is alkyl; R t C 1 -C 4 is alkyl; R u But C 1 -C 3 independently selected from alkyl; R v is phenyl; R w is H; R x is H; R y But, O.C. 1 -C 3 C substituted with 0 to 1 substituents selected from alkyl 1 -C 3 Alkyl; or C 1 -C 3 benzyl substituted with 0 to 1 substituents selected from alkyl; or C 1 -C 3 C substituted with 0 to 1 substituents selected from alkyl 3 -C 5 is cycloalkyl; n is 2; p is 0 or 1; q is 1; r is 2; s is 2; t is 0 or 1; and v is 2, compound.

4. 4. The compound of claim 3, wherein: R 1 is H or F; and / or R y But, O.C. 1 -C 3 C substituted with 0 to 1 substituents selected from alkyl 1 -C 3 alkyl; or benzyl; or cyclopropyl, compound.

5. 5. The compound of claim 4, wherein: R y But, O.C. 1 -C 3 C substituted with 0 to 1 substituents selected from alkyl 1 -C 3 alkyl; or cyclopropyl, compound.

6. 10. The compound of claim 1, wherein: Ring A is a ring within Group I, Group II, Group III and Group IV; and Ring B is a ring within Group IA, Group IIA, Group IIIA, and Group IVA; and X is NH; Y is NH; R 2 is H; and when ring A is a ring in Group I or Group II: ring B is a ring in Group IA, Group IIA, Group IIIA or Group IVA; X is NH; Y is NH; R 2 is H; and when ring A is a ring in group III: ring B is a ring in group IA, group IIA or group IIIA; X is NH; Y is NH; R 2 is H; and when ring A is a ring in group IV: ring B is a ring in group IA, compound.

7. 7. The compound of claim 6, wherein: X is NH; Y is NH; R 2 is H; and when ring A is a ring in Group I: ring B is a ring in Group IA, Group IIA, Group IIIA or Group IVA; and X is NH; Y is NH; R 2 is H; and when ring A is a ring in group II: ring B is a ring in group IA, group IIA or group IIIA, compound.

8. 8. A compound according to claim 6 or claim 7, wherein: Group IV is: 【Chemistry 13】 That is, compound.

9. (a) a compound of formula (Ia) or (b) a tautomer, N-oxide, pharmaceutically acceptable salt, or solvate thereof; 【Chemistry 14】 During the ceremony: R 1 is H or F; R 2 is H or F; R 3 is H or CH 3 and Ring A is selected from rings in Group I, Group II, Group III and Group IV, where * indicates a bond to NH; Group I is 【Chemistry 15】 and: Group II is 【Chemistry 16】 and: Group III is 【Chemistry 17】 and: Group IV is 【Chemistry 18】 and: Ring B is selected from rings in Group IA, Group IIA, Group IIA and Group IVA; $ indicates a bond to NH; Group IA is 【Chemistry 19】 and: Group IIA is 【Chemistry 20】 and: Group IIIA is 【Chemical 21】 and: Group IIIVA is 【Chemical 22】 and: During the ceremony: W is CH 2 , O, or NR y and Z is C(O); R a is O.C. 1 -C 3 C substituted with 0 to 1 substituents selected from alkyl 1 -C 3 is alkyl; R b is C 1 -C 3 independently selected from alkyl; R c is C 1 -C 3 is alkyl; R d is C 1 -C 4 selected from alkyl or phenyl; R e is C 1 -C 3 is alkyl; R f is C 1 -C 3 is alkyl; R g is H; R h is C 1 -C 3 is alkyl; R i is C 1 -C 3 is alkyl; R j is H; R k is C 1 -C 3 is alkyl; R l is H; R o is O.C. 1 -C 3 C substituted with 0 to 1 substituents selected from alkyl 1 -C 3 alkyl; or phenyl; R p is C 1 -C 3 independently selected from alkyl R q is O.C. 1 -C 3 C substituted with 0 to 1 substituents selected from alkyl 1 -C 3 is alkyl; R r is H or C 1 -C 3 is alkyl; R s is H or C 1 -C 3 is alkyl; R t is C 1 -C 4 is alkyl; R u is C 1 -C 3 independently selected from alkyl; R y is O.C. 1 -C 3 C substituted with 0 to 1 substituents selected from alkyl 1 -C 3 alkyl; or cyclopropyl; n is 2; p is 0 or 1; q is 1; r is 2; s is 2; t is 0 or 1; u is 2 to 3; v is 2; and: ・R 2 is H; and when ring A is a ring in group I: ring B is a ring in group IA, group IIA, group IIIA or group IVA; ・R 2 is H; and when ring A is a ring in group II: ring B is a ring in group IA, group IIA or group IIIA; ・R 2 is H; and when ring A is a ring in group III: ring B is a ring in group IA, group IIA or group IIIA; ・R 2 is H; and when ring A is a ring in group IV: ring B is a ring in group IA; ・R 2 is F: Ring A is a ring in Group I or Group II and Ring B is a ring in Group IA; with the proviso that the compound of formula (Ia) is not (a) a compound selected from the group consisting of: The compound or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof. 【Chemical 23】

10. 10. The compound of claim 9, wherein: Group I is: 【Chemistry 24】 and and / or Group II is: 【Chemistry 25】 and and / or Group III is: 【Chemical 26】 and and / or Group IV is: 【Chemical 27】 and and / or Group IA is: 【Chemical 28】 and / or Group IIA 【Chemical 29】 and / or Group IIIA 【Chemistry 30】 and / or Group IVA 【Chemical 31】 That is, compound.

11. 11. A compound according to claim 9 or claim 10, wherein: R 3 is H; and / or R a But CH 3 , C.H. 2 CH 3 , or CH 2 OCH 3 and / or R d But CH(CH 3 ) 2 or C(CH 3 ) 3 and / or R o But CH 3 , C.H. 2 CH 3 , or CH(CH 3 ) 2 and / or R q But CH 3 , C.H. 2 CH 3 , or CH 2 OCH 3 and / or R r C 1 -C 3 is alkyl; and / or R s is H; and / or R y But CH 3 , C.H. 2 CH 3 , or CH(CH 3 ) 2 That is, compound.

12. 11. A compound according to claim 9 or claim 10, wherein: ・R 2 is H; and when ring A is a ring in group II: ring B is a ring in group IA or group IIA; and ・R 2 is H; and when ring A is a ring in group III: ring B is a ring in group IA or group IIA, compound.

13. (a) a compound of formula (Ib) or (b) a tautomer, N-oxide, pharmaceutically acceptable salt, or solvate thereof; 【Chemical 32】 During the ceremony: R 1 is H or F; R 2 is H or F; Ring A is selected from rings in Group I, Group II, Group III and Group IV, where * indicates a bond to NH; Group I is: 【Chemical 33】 and Group II is: 【Chemical 34】 and Group III is 【Chemistry 35】 and: Group IV is: 【Chemical 36】 and Ring B is selected from rings in Group IA, Group IIA, Group IIA and Group IVA; $ indicates a bond to NH; Group IA is: 【Chemical 37】 indicates; Group IIA is: 【Chemical Formula 38】 indicates; Group IIIA is: 【Chemical 39】 indicates; Group IIIVA is: 【Chemistry 40】 indicates; During the ceremony: W is CH 2 , O, or NR y and Z is C(O); R a is CH 3 , C.H. 2 CH 3 , or CH 2 OCH 3 and R b is C 1 -C 3 is alkyl; R c is C 1 -C 3 is alkyl; R d is CH(CH 3 ) 2 or C(CH 3 ) 3 and R e is C 1 -C 3 is alkyl; R f is C 1 -C 3 is alkyl; R g is H; R h is C 1 -C 3 is alkyl; R i is C 1 -C 3 is alkyl; R j is H; R o is CH 3 , C.H. 2 CH 3 , or CH(CH 3 ) 2 are independently selected from R q is CH 3 , C.H. 2 CH 3 , or CH 2 OCH 3 and R r is C 1 -C 3 is alkyl; R s is H; R y is CH 3 , C.H. 2 CH 3 , or CH(CH 3 ) 2 and n is 2; r is 2; s is 2; v is 2; and: ・R 2 is H; and when ring A is a ring in group I: ring B is a ring in group IA, group IIA, group IIIA or group IVA; ・R 2 is H; and when ring A is a ring in group II: ring B is a ring in group IA or group IIA; and ・R 2 is H; and when ring A is a ring in group III: ring B is a ring in group IA or group IIA, ・R 2 is H; and when ring A is a ring in group IV: ring B is a ring in group IA; ・Katsu R 2 is F: Ring A is a ring in Group I or Group II and Ring B is a ring in Group IA; with the proviso that the compound of formula (Ib) is not (a) a compound selected from the group consisting of: The compound or a tautomer, N-oxide, pharmaceutically acceptable salt or solvate thereof. 【Chemistry 41】

14. 14. The compound of claim 13, wherein: Group I is: 【Chemistry 42】 and / or Group II is: 【Chemistry 43】 That is, is a compound.

15. 2. The compound of claim 1, wherein formula (I) is selected from the following: 【Chemical 44】 【change】 【change】 【change】 【change】

16. 16. The compound of claim 15, wherein formula (I) is selected from: 【Chemistry 45】 【change】 【change】

17. 17. The compound of claim 16, wherein formula (I) is: 【Chemistry 46】

18. 10. A pharmaceutical composition comprising a compound of claim 1 in combination with one or more pharmaceutically acceptable carriers.

19. 19. A pharmaceutical composition according to claim 18 for use as a medicament.

20. 19. The pharmaceutical composition of claim 18 for use in a method for the treatment and / or prevention of disorders susceptible to treatment by PI3K alpha activation.

21. 19. The pharmaceutical composition of claim 18 for use in a method for treating and / or preventing peripheral nerve injury.

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