Compositions and methods for treating cancer

Abstract

To provide compounds that are capable of penetrating to the blood brain barrier to modulate the activity of EGFR tyrosine kinase, pharmaceutical compositions, and a method for treating cancer.SOLUTION: There is provided compounds with specific structures having quinazoline moieties exemplified below. The disclosure further relates to methods of treating Glioblastoma and other EGFR mediated cancers. The disclosure further relates to methods of treating Glioblastoma and other EGFR mediated cancers that have been determined to have altered glucose metabolism in the presence of inhibitors. The present disclosure also provides methods of administering to a subject a glucose metabolism inhibitor and a cytoplasmic p53 stabilizer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 819,322, filed March 15, 2019, and U.S. Provisional Application No. 62 / 904,241, filed September 23, 2019, the entire contents of which are hereby incorporated by reference in their entireties. [Background technology]

[0002] Glioblastoma (glioblastoma multiforme, GBM) accounts for the majority of primary malignant brain tumors in adults. Amplification and mutation of the epidermal growth factor receptor (EGFR) gene are characteristic genetic abnormalities occurring in GBM (Sugawa, et al. (1990) Proc. Natl. Acad. Sci. 87:8602-8606; Ekstrand, et al. (1992) Proc. Natl. Acad. Sci. 89:4309-4313). A series of promising therapeutic agents targeting EGFR or its mutant, constitutively activated form, ΔEGFR, for the treatment of GBM, including tyrosine kinase inhibitors (TKIs), monoclonal antibodies, vaccines, and RNA-based agents, are currently under development or in clinical trials. However, its clinical effectiveness has been limited so far by both prior and acquired drug resistance (Taylor, et al. (2012) Curr. Cancer Drug Targets. 12:197-209). A major limitation is the poor brain penetration of current therapeutic agents, such as erlotinib, lapatinib, gefitinib, and afatinib (Razier, et al. (2010) Neuro-Oncology 12:95-103; Reardon, et al. (2015) Neuro-Oncology 17:430-439; Thiessen, et al. (2010) Cancer Chemother. Pharmacol. 65:353-361).

[0003] Molecularly targeted therapeutics have revolutionized cancer treatment, paving the way for modern precision medicine. However, despite clear and accessible genetic alterations, targeted agents have not been effective in patients with glioblastoma (GBM). This is due, in large part, to insufficient CNS penetration of most targeted agents to the level required for tumor killing, which may induce powerful adaptive mechanisms that promote treatment resistance. While drug combinations that inhibit both primary and compensatory signaling pathways are attractive, these combination strategies are limited by high toxicity, leading to subthreshold dosing of each agent.

[0004] Alternative therapeutic approaches target oncogenic drivers to modulate functional properties important for tumor survival, but cells often lack the ability to target specific genes through orthogonal second hits. 6 This "synthetic lethal" strategy may be particularly attractive when the functional network(s) regulated by an oncogene overlaps with tumor cell death pathways. In certain instances, oncogenic signaling promotes glucose metabolism, suppressing intrinsic apoptosis and promoting survival. Inhibiting oncogenic drivers with targeted therapeutics can induce the intrinsic apoptotic machinery as a direct result of reduced glucose consumption. The intersecting nature of these tumorigenic pathways may provide therapeutic opportunities for rational combinatorial therapy, which requires further investigation.

[0005] In view of the above, there remains a clinical need for brain-penetrating chemotherapeutic agents for the treatment of glioblastoma and other cancers. Summary of the Invention

[0006] In one aspect, the present disclosure provides a compound of Formula I or Formula I * The compound [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, Z is aryl or heteroaryl; R 2a and R 2b are each independently selected from hydrogen, alkyl, halo, CN, and NO; R 3 is hydrogen, alkyl, or acyl; R 4 is an alkoxy; R 5 is alkyl, and R 7 and R 8 are each independently selected from hydrogen, alkyl, e.g., alkoxyalkyl, aralkyl, etc., or arylacyl; R 11 But hydrogen, alkyl, halo, CN, NO2, OR 7 , cycloalkyl, heterocyclyl, aryl, or heteroaryl; R 12 But hydrogen, alkyl, halo, CN, NO2, OR 8 , cycloalkyl, heterocyclyl, aryl, or heteroaryl, or R 11 and R 12 together complete a carbocyclic or heterocyclic ring.

[0007] In certain aspects, the present disclosure provides methods for inhibiting EGFR or ΔEGFR, comprising administering to a subject an amount of a compound of the present disclosure.

[0008] In certain aspects, the present disclosure provides methods for treating cancer, comprising administering to a subject in need thereof an amount of a compound of the present disclosure, hi some embodiments, the cancer is glioblastoma multiforme.

[0009] In certain aspects, the present disclosure provides methods for treating cancer, comprising administering to a subject a glucose metabolism inhibitor and a cytoplasmic p53 stabilizer, wherein the glucose metabolism inhibitor is a compound of the present disclosure. In some embodiments, the cancer is glioblastoma multiforme.

[0010] In certain aspects, the present disclosure provides compounds of Formula I or Formula I* The present invention provides a method for making the compound of formula (I). [Brief explanation of the drawings]

[0011] [Figure 1A] This shows that inhibition of EGFR-driven glucose metabolism induces minimal cell death but activates GBM cells toward apoptosis. Percent change in 18F-FDG uptake in 19 patient-derived GBM glioma spheres after 4 hours of erlotinib treatment compared to vehicle is shown. "Metabolic responders" (blue) are samples that show a significant decrease in 18F-FDG uptake compared to vehicle, while "non-responders" (red) show no significant decrease. [Figure 1B] Figure 1 shows that inhibition of EGFR-driven glucose metabolism induces minimal cell death but activates GBM cells toward apoptosis. Figure 2 shows the percent change in glucose consumption and lactate production with 12 hours of erlotinib treatment compared to vehicle. Measurements were performed using a Nova Biomedical BioProfile Analyzer. [Figure 1C] Figure 1 shows that inhibition of EGFR-driven glucose metabolism induces minimal cell death but activates GBM cells toward apoptosis. Annexin V staining of metabolic responders (blue, n=10) or non-responders (red, n=9) after 72 hours of treatment with erlotinib is shown. [Figure 1D] Figure 1 shows that inhibition of EGFR-driven glucose metabolism induces minimal cell death but activates GBM cells toward apoptosis. Figure 2 shows the percent change in activation, as measured by cytochrome c release, following exposure to each BH3 peptide (BIM, BID, or PUMA) in metabolic responders or non-responders treated with erlotinib for 24 hours compared to vehicle control. [Figure 1E]Inhibition of EGFR-driven glucose metabolism induces minimal cell death but activates GBM cells toward apoptosis. Left: Immunoblot of whole cell lysates from GFP control or HK301 cells overexpressing GLUT1 and GLUT3 (GLUT1 / 3). Right: Changes in glucose consumption or lactate production in HK301-GFP or HK301-GLUT1 / 3 after 12 hours of erlotinib treatment. Values ​​are relative to the solvent control. [Figure 1F] These results demonstrate that inhibition of EGFR-driven glucose metabolism induces minimal cell death but activates GBM cells toward apoptosis. The use of HK301-GFP or HK301-GLUT1 / 3 cells is shown. Erlotinib concentration in all experiments was 1 μM. Comparisons were performed using a two-tailed, unpaired Student's t-test. Data represent the mean ± sem values ​​of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 2A] Cytoplasmic p53 binds to EGFR, resulting in intrinsic apoptosis. Immunoblots of the indicated proteins are shown in two responders (HK301 and HK336) expressing CRISPR / CAS9 proteins with control guide RNA (sg control) or p53 guide RNA (p53KO). [Figure 2B] Figure 1 shows that cytoplasmic p53 binds to EGFR and causes intrinsic apoptosis. Figure 2 shows the percent change in activation, as measured by cytochrome c release, after exposure to BIM peptide in sg control and p53KO cells treated with erlotinib for 24 hours compared to vehicle control. [Figure 2C] 1 shows that cytoplasmic p53 binds to EGFR to cause intrinsic apoptosis. Immunoblots of the indicated proteins in HK301 sg control, p53KO, p53KO+p53cyto, and p53KO+p53wt. [Figure 2D]This shows that cytoplasmic p53 binds to EGFR and induces intrinsic apoptosis. Immunofluorescence of p53 protein combined with DAPI staining is shown to reveal protein localization in HK301 sg control, p53KO+p53cyto, and p53KO+p53wt (scale bar = 20 μm). Gliomaspheres were first dissociated into single cells and then attached to 96-well plates using a Cell-Tak (Corning) according to the manufacturer's instructions. Adherent cells were then fixed with ice-cold methanol for 10 minutes and then washed three times with PBS. The cells were then incubated with a blocking solution containing 10% FBS and 3% BSA in PBS for 1 hour, followed by overnight incubation with p53 antibody (Santa Cruz, SC-126, 1:50 dilution) at 4°C. The next day, cells were incubated with secondary antibody (Alexa Fluor 647, 1:2000 dilution) for 1 hour, stained with DAPI for 10 minutes, and then imaged using a Nikon TI Eclipse microscope equipped with a Cascade II fluorescence camera (Roper Scientific). Cells were imaged at 461 nM and 647 nM emission and then processed using NIS-Elements AR analysis software. [Figure 2E] Figure 1 shows that cytoplasmic p53 binds to EGFR to induce intrinsic apoptosis. Figure 1 shows changes in the levels of the indicated mRNAs after 24 hours of 100 nM doxorubicin treatment in HK301 sg control, p53KO, p53KO+p53cyto, and p53KO+p53wt cells. Levels were normalized to the corresponding DMSO-treated cells. [Figure 2F] Figure 2 shows that cytoplasmic p53 binds to EGFR and drives intrinsic apoptosis. Data similar to Figure 2B are shown for HK301 sg control, p53KO, p53KO+p53cyto, and p53KO+p53wt. [Figure 2G]Figure 2B shows that cytoplasmic p53 binds to EGFR and drives intrinsic apoptosis. Data similar to Figure 2E are shown for HK301 sg control, p53KO, p53KO+p53R175H, p53KO+p53R273H, and p53KO+p53NES. [Figure 2H] These results demonstrate that cytoplasmic p53 binds to EGFR and induces intrinsic apoptosis. Similar data to Figures 2B and 2F are shown for HK301 sg control, p53KO, p53KO+p53R175H, p53KO+p53R273H, and p53KO+p53NES. The erlotinib concentration in all experiments was 1 μM. Comparisons were performed using a two-tailed, unpaired Student's t-test. Data represent the mean ± sem values ​​of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 3A] Figure 1 shows that Bcl-xL prevents GBM cell death by binding to and sequestering cytoplasmic p53 in EGFRi metabolic responders. Immunoprecipitation of p53 in two metabolic responders (HK301 and GBM39) 24 hours after erlotinib treatment is shown. Immunoprecipitations were probed with the indicated antibodies. Below are the corresponding pre-immunoprecipitation lysates (input). [Figure 3B] We show that Bcl-xL prevents GBM cell death by binding to and sequestering cytoplasmic p53 in EGFRi metabolic responders. Data similar to Figure 3A are shown, but in two non-responders (HK393 and HK254). [Figure 3C] Figure 3 shows that Bcl-xL prevents GBM cell death by binding to and sequestering cytoplasmic p53 in EGFRi metabolic responders. Data similar to Figure 3A and Figure 3B are shown for HK301-GFP and HK301-GLUT1 / 3. Immunoblots with indicated inputs are shown on the right. [Figure 3D]Figure 1 shows that Bcl-xL prevents GBM cell death by binding to and sequestering cytoplasmic p53 in EGFRi metabolic responders.HK301 were treated with erlotinib, WEHI-539, or both for 24 hours, and then immunoprecipitation and immunoblotting were performed as described above. [Figure 3E] Figure 1 shows that Bcl-xL prevents GBM cell death by binding to and sequestering cytoplasmic p53 in EGFRi metabolic responders.Annexin V staining of two responders (GBM39 and HK301) and a non-responder (HK393) 72 hours after treatment with erlotinib, WEHI-539, or both is shown. [Figure 3F] Figure 1 shows that Bcl-xL prevents GBM cell death by binding to and sequestering cytoplasmic p53 in EGFRi metabolic responders. Annexin V staining of HK301-GFP and HK301-GLUT1 / 3 is shown 72 hours after treatment with erlotinib, WEHI-539, or both. The concentrations of erlotinib and WEHI-539 in all experiments were 1 μM and 5 μM, respectively. Comparisons were performed using a two-tailed, unpaired Student's t-test. Data represent the mean ± sem of three independent experiments. *p<0.05, **p<0.01. [Figure 4A] Illustrates the synergistic lethality of combined targeting of EGFR and p53. Shows a list of mutations in genes involved in regulating EGFR and p53 across 273 GBM samples. Genetic mutations (amplification / mutation) in EGFR are mutually exclusive with genetic mutations in p53. As shown, EGFR mutations are on the left side of the table, while most mutations in p53 are on the right side. [Figure 4B] Figure 1 shows the synergistic lethality of combined targeting of EGFR and p53. Figure 2 shows a table showing the significant association between mutations in EGFR and mutations in genes involved in the p53 pathway. [Figure 4C]Figure 1 shows the synergistic lethality of combined targeting of EGFR and p53. Annexin V staining of metabolic responders (left: HK301) and non-responders (right: GS017) treated with various concentrations of erlotinib, Nutlin, and the combination, represented in a dose-escalation matrix. [Figure 4D] Figure 4C shows the synergistic lethality of combined targeting of EGFR and p53. Dose escalation of erlotinib and Nutlin as described in Figure 4C was performed across 10 metabolic responders and 6 non-responders, and then a synergy score was calculated (see Materials and Methods). [Figure 4E] Figure 1 shows the synergistic lethality of combined targeting of EGFR and p53. Figure 2 shows Annexin V staining of HK301-GFP and HK301 GLUT1 / 3 after 72 hours of treatment with erlotinib, nutlins, or both. [Figure 4F] Figure 4B shows the synergistic lethality of combined targeting of EGFR and p53. Same as Figure 4E, but in HK301-sg control and HK301-p53KO. [Figure 4G] Figure 1 shows the synergistic lethality of combined targeting of EGFR and p53. HK301 treated with erlotinib, Nutlin, or the combination for 24 hours is shown. Immunoprecipitations were performed with an immunoglobulin G control antibody or an anti-p53 antibody, and then probed with the indicated antibodies. At the bottom are the corresponding pre-immunoprecipitation lysates (input). All data are representative (mean ± SEM) of at least n=3 independent experiments. Unless otherwise stated, the concentrations of erlotinib and Nutlin in all experiments were 1 μM and 2.5 μM, respectively. **p<0.01, ***p<0.001, ****p<0.0001. [Figure 5A] Figure 1 shows that modulation of glucose metabolism activates EGFRi non-responders toward p53-mediated cell death. Figure 2 shows the percent change in 18F-FDG uptake compared to vehicle in HK393 and HK254 cells 4 hours after treatment with erlotinib, 2DG, or pictilisib. [Figure 5B]Figure 1 shows that modulation of glucose metabolism activates EGFRi non-responders toward p53-mediated cell death. Figure 1 shows the percent change in activation, as measured by cytochrome c release, after exposure to BIM peptide in HK393 and HK254 cells 24 hours after erlotinib, 2DG, or pictilisib compared to vehicle control. [Figure 5C] Figure 5B shows that modulation of glucose metabolism activates EGFRi non-responders toward p53-mediated cell death. Data similar to Figure 5B are shown in HK393 sg control and p53KO cells. [Figure 5D] This shows that modulation of glucose metabolism activates EGFRi non-responders toward p53-mediated cell death. Immunoprecipitation of p53 in HK393 and HK254 cells 24 hours after 2DG or pictilisib treatment is shown. Immunoprecipitations were probed with the indicated antibodies. The bottom panel shows the corresponding pre-immunoprecipitation lysates (input). [Figure 5E] Figure 1 shows that modulation of glucose metabolism activates EGFRi non-responders towards p53-mediated cell death. Figure 2 shows synergy scores for various drugs (erlotinib, 2DG, and pictilisib) in combination with Nutlins in HK393 and HK254. [Figure 5F] This shows that modulation of glucose metabolism activates EGFRi non-responders toward p53-mediated cell death. Annexin V staining of HK393 sg control and HK393 p53KO cells after 72 hours of treatment with 2DG, pictilisib, 2DG + Nutlin, or pictilisib + Nutlin is shown. Unless otherwise stated, the concentrations of erlotinib, 2DG, pictilisib, and Nutlin in all experiments were 1 μM, 1 mM, 1 μM, and 2.5 μM, respectively. Comparisons were performed using a two-tailed, unpaired Student's t-test. Data represent the mean ± sem of three independent experiments. *p<0.05, **p<0.01, ***p<0.001. [Figure 6A]Figure 1 shows that combined targeting of EGFR-driven glucose uptake and p53 suppresses tumor growth in vivo. Figure 2 shows 18F-FDG PET / CT images of GBM39 intracranial xenografts before and 15 hours after erlotinib treatment (75 mg / kg). [Figure 6B] Figure 6B shows that combined targeting of EGFR-driven glucose uptake and p53 suppresses tumor growth in vivo. GBM39 intracranial xenografts were treated daily with vehicle (n = 5), 75 mg / kg erlotinib (n = 7), 50 mg / kg idasanutlin (n = 5), or the combination (n = 12), followed by tumor burden assessment on the indicated days using secreted Gaussia luciferase (see Materials and Methods). Comparisons between Figures 6B and 6D used the dataset from the last measurement, and comparisons were performed using a two-tailed, unpaired t-test. [Figure 6C] Figure 6 shows that combined targeting of EGFR-driven glucose uptake and p53 suppresses tumor growth in vivo. Data similar to Figure 6A, but in HK393 intracranial xenografts. [Figure 6D] Figure 6 shows that combined targeting of EGFR-driven glucose uptake and p53 suppresses tumor growth in vivo. Data similar to Figure 6B are shown, but in HK393 intracranial xenografts (n = 7 in all groups). Comparison of Figure 6B and Figure 6D used the data set from the last measurement, and comparison was performed using a two-tailed unpaired t-test. [Figure 6E] Figure 6B shows that combined targeting of EGFR-driven glucose uptake and p53 suppresses tumor growth in vivo. [Figure 6F] Figure 6C shows that combined targeting of EGFR-driven glucose uptake and p53 suppresses tumor growth in vivo. [Figure 6G]Figure 1 shows that combined targeting of EGFR-driven glucose uptake and p53 suppresses tumor growth in vivo. Figure 2 shows the survival rate of metabolic responder HK336 after 25 days of designated treatment followed by a drug holiday (n=7 in all groups). [Figure 6H] Figure 1 shows that combined targeting of EGFR-driven glucose uptake and p53 suppresses tumor growth in vivo. Survival of non-responder GS025 after 25 days of designated treatment followed by a drug holiday is shown (n=9 in all groups). Data represent mean ± sem values. **p<0.01. [Figure 7A] Figure 1 shows the characterization of GBM cell lines after EGFR inhibition. Figure 1 shows the percent change in 18F-FDG uptake in two metabolic responders (HK301 and GBM39) compared to vehicle upon erlotinib treatment at the indicated times. [Figure 7B] Figure 1 shows characterization of GBM cell lines after EGFR inhibition. Immunoblots of indicated proteins in metabolic responders (HK301) and non-responders (HK217) after gene knockdown of EGFR by siRNA. [Figure 7C] Figure 1 shows the characterization of GBM cell lines after EGFR inhibition. Figure 2 shows the percent change in 18F-FDG uptake in HK301 and HK217 after genetic knockdown of EGFR. [Figure 7D]

[0023] Figure 1 shows the characterization of GBM cell lines after EGFR inhibition. Figure 2 shows the change in glucose consumption in three metabolic responders (HK301, GBM39, HK390) and three non-responders (HK393, HK217, HK254) following 12 hours of erlotinib treatment. Measurements were performed using a Nova Biomedical BioProfile Analyzer. [Figure 7E]Figure 1 shows the characterization of GBM cell lines after EGFR inhibition. Figure 2 shows the changes in lactate production in three metabolic responders (HK301, GBM39, HK390) and three non-responders (HK393, HK217, HK254) following 12 hours of erlotinib treatment. Measurements were performed using a Nova Biomedical BioProfile Analyzer. [Figure 7F] Figure 1 shows characterization of GBM cell lines after EGFR inhibition. Basal ECAR measurements are shown for two responders (HK301 and GBM39, blue) and two non-responders (HK217 and HK393, red) after 12 hours of erlotinib treatment. [Figure 7G] Characterization of GBM cell lines following EGFR inhibition. Changes in glutamine consumption measured with a Nova Biomedical BioProfile Analyzer after 12 hours of erlotinib treatment are shown. The erlotinib concentration in all experiments was 1 μM. Comparisons were performed using a two-tailed, unpaired Student's t-test. Data represent the mean ± sem values ​​of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 8] A shows that changes in downstream signaling following EGFR inhibition correlate with metabolic responses. Immunoblots of the indicated proteins in metabolic responders 4 hours after erlotinib treatment are shown. B shows that changes in downstream signaling following EGFR inhibition correlate with metabolic responses. Immunoblots of the indicated proteins in metabolic non-responders 4 hours after erlotinib treatment are shown. [Figure 9]A shows genetic characterization of patient-derived GBM cell lines. Genetic backgrounds across a panel of GBM lines are shown. B shows genetic characterization of patient-derived GBM cell lines. Fluorescence in situ hybridization (FISH) of HK390, HK336, HK254, and HK393 cells, demonstrating EGFR polysomy, was performed using a commercially available fluorescently labeled dual-color EGFR (red) / CEP 7 (green) probe (Abbott-Molecular). FISH hybridization and analysis of cell lines were performed according to the manufacturer's recommended protocol. Cells were counterstained with DAPI, and fluorescent probe signals were imaged under a Zeiss (Axiophot) fluorescence microscope equipped with dual-color and triple-color filters. [Figure 10A] Figure 1 shows that EGFR inhibition shifts the apoptotic balance in metabolic responders. Immunoblots of the indicated proteins in metabolic responders (GBM39, HK301, and HK336) and non-responders (HK217, HK393, and HK254) 24 hours after erlotinib treatment. [Figure 10B] This shows that EGFR inhibition shifts the apoptotic balance in metabolic responders. An example of dynamic BH3 profiling analysis in a metabolic responder (HK301) is shown. Left: Rate of cytochrome c release is measured after exposure to various peptides at the indicated concentrations. Right: Rate of activation is obtained by calculating the difference in cytochrome c release between vehicle-treated and erlotinib-treated cells. The erlotinib concentration in all experiments was 1 μM. [Figure 11](A) GLUT1 / 3 overexpression rescues glucose hypometabolism caused by EGFR inhibition. Changes in glucose consumption and lactate production in HK301-GFP and HK301 GLUT1 / 3 following 12 hours of erlotinib treatment are shown. Measurements were performed using a Nova Biomedical BioProfile Analyzer. (B) GLUT1 / 3 overexpression rescues glucose hypometabolism caused by EGFR inhibition. Left: Immunoblot of whole cell lysates from GFP control or GBM39 cells overexpressing GLUT1 and GLUT3 (GLUT1 / 3). Right: Changes in glucose consumption or lactate production in GBM39-GFP or GBM39-GLUT1 / 3 after 12 hours of erlotinib treatment. Values ​​are relative to the solvent control. (C) GLUT1 / 3 overexpression rescues glucose hypometabolism caused by EGFR inhibition. Similar data as in A, but for GBM39-GFP and GBM39-GLUT1 / 3. Erlotinib concentration in all experiments was 1 μM. Comparisons were performed using a two-tailed, unpaired Student's t-test. Data represent the mean ± sem of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 12A] Figure 1 shows that cytoplasmic p53 is required for EGFRi-mediated apoptosis activation. Figure 2 shows the percent change in 18F-FDG uptake in HK301 sg control and p53KO cells after 4 hours of erlotinib treatment (mean ± sd, n = 3). [Figure 12B] Figure 1 shows that cytoplasmic p53 is required for EGFRi-mediated apoptosis activation.Figure 2 shows the relative mRNA levels of p53-regulated genes in HK301 (metabolic responder) cells 24 hours after treatment with 1 μM erlotinib or 100 nM doxorubicin. [Figure 12C]This shows that cytoplasmic p53 is required for EGFRi-mediated apoptosis activation. HK301 cells were infected with a p53-luciferase reporter system and p53 activity was measured 24 hours after treatment with 1 μM erlotinib (mean ± SD, n=3). Results are representative of two independent experiments. [Figure 12D] 1 shows that cytoplasmic p53 is required for EGFRi-mediated apoptosis activation. Immunoblots of the indicated proteins in HK336 sg control, p53KO, p53KO+p53cyto, and p53KO+p53wt. [Figure 12E] This shows that cytoplasmic p53 is required for EGFRi-mediated apoptosis activation. Immunofluorescence of p53 protein combined with DAPI staining to reveal protein localization in HK336 sg control, p53KO+p53cyto, and p53KO+p53wt cells is shown (scale bar = 20 μm). Immunofluorescence was performed as described above. [Figure 12F] This shows that cytoplasmic p53 is required for EGFRi-mediated apoptosis activation. Changes in the levels of the indicated mRNAs after 24 hours of 100 nM doxorubicin treatment in HK336 sg control, p53KO, p53KO+p53cyto, and p53KO+p53wt cells are shown (mean ± SD, n = 3). Levels were normalized to the corresponding DMSO-treated cells. [Figure 12G] Figure 1 shows that cytoplasmic p53 is required for EGFRi-mediated apoptosis activation. Percent changes in apoptosis activation, measured by cytochrome c release, after exposure to BIM peptide in HK336 sg control, p53KO, p53KO+p53cyto, and p53KO+p53wt cells treated with erlotinib for 24 hours compared to vehicle control are shown (mean ± SD, n=2). Results are representative of two independent experiments. [Figure 12H]1 shows that cytoplasmic p53 is required for EGFRi-mediated apoptosis activation. Immunoblots of the indicated proteins in HK301 sg control, p53KO, p53KO+p53R175H, p53KO+p53R273H, and p53KO+p53NES. [Figure 12I] Figure 1 shows that cytoplasmic p53 is required for EGFRi-mediated apoptosis activation. Figure 2 shows the percent change in activation in HK301 after 24 hours of erlotinib treatment with or without PFTμ pretreatment (10 μM, 2 hours). Results are representative of two independent experiments. [Figure 13A] Figure 1 shows that inhibition of EGFR-driven glucose metabolism induces Bcl-xL dependence via cytoplasmic p53 function. Figure 2 shows the percent change in activation, as measured by cytochrome c release, after exposure to BAD and HRK peptides in metabolic responders (HK301 and HK336) or non-responders (HK229) treated with erlotinib compared to vehicle controls. [Figure 13B] Inhibition of EGFR-driven glucose metabolism induces Bcl-xL dependence via cytoplasmic p53 function. Left: Immunoprecipitation of p53 in GBM39-GFP and GBM39-GLUT1 / 3 cells 24 hours after erlotinib treatment (immunoprecipitations were probed with the indicated antibodies). Right: Corresponding pre-immunoprecipitation lysates (input). [Figure 13C] Figure 1 shows that inhibition of EGFR-driven glucose metabolism induces Bcl-xL dependence via cytoplasmic p53 function. Annexin V staining of HK301 (left) sg control, p53KO, p53KO+p53cyto, and p53KO+p53wt, and HK336 (right) sg control, p53KO, p53KO+p53cyto, and p53KO+p53wt after 72 hours of treatment with erlotinib, WEHI-539, or the combination is shown. [Figure 13D]Figure 13 shows that inhibition of EGFR-driven glucose metabolism induces Bcl-xL dependence via cytoplasmic p53 function. Data similar to Figure 13C are shown for GBM39-GFP and GBM39-GLUT1 / 3. The concentration of erlotinib and WEHI-539 in all experiments was 1 μM. Comparisons were performed using a two-tailed, unpaired Student's t-test. Data represent the mean ± sem of three independent experiments. *p<0.05, **p<0.01, ***p<0.001. [Figure 14A] Figure 1 shows that inhibition of EGFR-regulated glucose metabolism and p53 activation promotes intrinsic apoptosis in GBM. Immunoblots of the indicated proteins in two metabolic responders (HK301 and GBM39) 24 hours after erlotinib, Nutlin, or the combination are shown. [Figure 14B] Figure 1 shows that inhibition of EGFR-regulated glucose metabolism and p53 activation promotes intrinsic apoptosis in GBM. Figure 2 shows Annexin V staining of HK301 and HK217 after genetic knockdown of EGFR followed by 72 hours of Nutlin treatment. [Figure 14C] These results demonstrate that inhibition of EGFR-regulated glucose metabolism and p53 activation promote intrinsic apoptosis in GBM. Detection of BAX oligomerization in HK301-GFP and HK301-GLUT1 / GLUT3 is shown. After 24 hours of the indicated treatments, cells were harvested, cultured in 1 mM BMH to promote protein cross-linking, and immunoblotted with the indicated antibodies. The bottom image shows immunoblot of cytosolic cytochrome c after cell fractionation. [Figure 14D] Figure 1 shows that inhibition of EGFR-regulated glucose metabolism and p53 activation promotes intrinsic apoptosis in GBM. Top: Immunoblots of the indicated proteins in HK301-GFP and HK301-HA-BclxL. Bottom: Annexin V staining of HK301-GFP and HK301-HA-BclxL after 72 hours of treatment with erlotinib, nutlin, or the combination. [Figure 14E]Figure 1 shows that inhibition of EGFR-regulated glucose metabolism and p53 activation promotes intrinsic apoptosis in GBM. Figure 2 shows Annexin V staining of HK301 after 72 hours ± PFTμ pretreatment (10 μM, 2 hours) with erlotinib, Nutlin, or the combination. [Figure 14F] Figure 1 shows that inhibition of EGFR-regulated glucose metabolism and p53 activation promotes intrinsic apoptosis in GBM. Figure 2 shows Annexin V staining of HK301 sg control, p53KO, p53KO+p53R175H, p53KO+p53R273H, and p53KO+p53NES after 72 hours of treatment with erlotinib, Nutlin, or the combination. [Figure 14G] These results demonstrate that inhibition of EGFR-regulated glucose metabolism and p53 activation promote intrinsic apoptosis in GBM. Similar data to Figure 14F are shown for HK301 sg control, p53KO, p53KO+p53cyto, and p53KO+p53wt. Drug concentrations in all experiments are as follows: erlotinib (1 μM), Nutlin (2.5 μM). Comparisons were performed using a two-tailed, unpaired Student's t-test. Data represent the mean ± sem values ​​of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 14H] These results demonstrate that inhibition of EGFR-regulated glucose metabolism and p53 activation promote intrinsic apoptosis in GBM. Similar data to Figure 14G are shown for HK336 sg control, p53KO, p53KO+p53cyto, and p53KO+p53wt. Drug concentrations in all experiments are as follows: erlotinib (1 μM), Nutlin (2.5 μM). Comparisons were performed using a two-tailed, unpaired Student's t-test. Data represent the mean ± sem values ​​of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 15A]Figure 1 shows that inhibition of glucose metabolism in metabolic responders and non-responders promotes intrinsic apoptosis. Figure 2 shows the percent change in activation, as measured by cytochrome c release, after exposure to BIM peptide in metabolic responder HK301 24 hours after erlotinib or 2DG treatment compared to vehicle control. [Figure 15B] Figure 1 shows that inhibition of glucose metabolism promotes intrinsic apoptosis in metabolic responders and non-responders. Left: Immunoprecipitation of p53 in HK301 cells 24 hours after 2DG treatment (immunoprecipitations were probed with the indicated antibodies). Right: Corresponding lysates before immunoprecipitation (input). [Figure 15C] 1 shows that inhibition of glucose metabolism promotes intrinsic apoptosis in metabolic responders and non-responders. 1 shows OCR and ECAR measurements of HK301 cells after exposure to oligomycin and rotenone. [Figure 15D] Figure 1 shows that inhibition of glucose metabolism in metabolic responders and non-responders promotes intrinsic apoptosis. Figure 2 shows Annexin V staining of HK301 after 72 hours of treatment with Nutlins, erlotinib, 2DG, oligomycin, rotenone as individual agents, or in combination with Nutlins. [Figure 15E] Figure 1 shows that inhibition of glucose metabolism in metabolic responders and non-responders promotes intrinsic apoptosis. Immunoblots of the indicated proteins in two non-responders (HK254 and HK393) 4 hours after erlotinib or pictilisib treatment are shown. [Figure 15F]These results demonstrate that inhibition of glucose metabolism in metabolic responders and non-responders promotes intrinsic apoptosis. Immunoprecipitation of p53 in HK254 cells 24 hours after pictilisib or 2DG treatment is shown. Immunoprecipitations were probed with the indicated antibodies. The corresponding pre-immunoprecipitation lysates (input) are shown at the bottom. Drug concentrations in all experiments were as follows: erlotinib (1 μM), Nutlin (2.5 μM), 2DG (3 mM for HK301 and 1 mM for HK254), oligomycin (1 μM), rotenone (1 μM), and pictilisib (1 μM). Comparisons were performed using a two-tailed, unpaired Student's t-test. Data represent the mean ± sem of three independent experiments. ****p<0.0001. [Figure 16A] We show that direct inhibition of glycolysis by 2DG (a hexokinase inhibitor) or cytochalasin B (a glucose transporter inhibitor) unexpectedly synergizes with p53 activation (by Nutlin). [Figure 16B] 1 shows that low glucose (0.25 mM) results in synergistic cell killing through BCL-xL inhibition with navitoclax (ABT-263). [Figure 16C] Figure 1 shows that low glucose (0.25 mM) results in synergistic cell killing due to BCL-xL inhibition by Nutlins. [Figure 17]

[0033] Figure 1 shows a comparison between metabolic responders and metabolic non-responders to the EGFRi inhibitor erlotinib. The combination of erlotinib and Nutlins results in unexpected synergistic synthetic lethality in metabolic responders but not in non-responders. [Figure 18A] The enantiomeric purity of synthetic intermediate 5 measured using chiral SFC (Chiralpak AD-3 column, 40% MeOH) is shown. [Figure 18B] The enantiomeric purity of synthetic intermediate (S)-5 measured using chiral SFC (Chiralpak AD-3 column, 40% MeOH) is shown. [Figure 18C]The enantiomeric purity of synthetic intermediate (R)-5 measured using chiral SFC (Chiralpak AD-3 column, 40% MeOH) is shown. [Figure 18D] 1 shows the enantiomeric purity of Mosher ester derivative 5 measured using chiral SFC (Chiralpak AD-3 column, 40% MeOH). [Figure 19] 1 shows the activity of erlotinib and exemplary compounds of the present disclosure in a PC9 lung cancer EGFR mutant mouse model. [Figure 20] Specific metabolites of exemplary compounds of the present disclosure are shown. DETAILED DESCRIPTION OF THE INVENTION

[0012] Gliomas are the most common form of brain tumor, and glioblastoma multiforme (GBM) is the most malignant form, causing 3-4% of all cancer-related deaths (Louis et al. (2007) Acta. Neuropathol. 114:97-109). The World Health Organization defines GBM as a grade IV cancer characterized by malignant, mitotically active, and prone to necrosis. The prognosis for GBM is extremely poor, with a 5-year survival rate of 4-5% and a median survival rate of 12.6 months (McLendon et al. (2003) Cancer. 98:1745-1748). This may be due to inherent treatment limitations, such as a high mean age at onset, tumor location, and a current incomplete understanding of tumor pathophysiology (Louis et al. (2007) Acta. Neuropathol. 114:97-109). The current standard of care for GBM includes tumor resection combined with radiation therapy and chemotherapy, but in recent years, few significant improvements have been made to improve survival rates (Stewart, et al. (2002) Lancet. 359:1011-1018.).

[0013] The standard chemotherapy drug for GBM is temozolomide (TMZ), a brain-permeable alkylating agent that methylates purines (A or G) in DNA to induce apoptosis (Stupp, et al. (2005) N. Engl. J. Med. 352:987-996). However, the use of TMZ has the disadvantage that it poses a significant risk from DNA damage in healthy cells and that GBM cells can quickly develop resistance to the drug (Carlsson, et al. (2014) EMBO. Mol. Med. 6:1359-1370). Therefore, there is an urgent need for alternative chemotherapy options.

[0014] EGFR, along with ERBB2, ERBB3, and ERBB4, is a member of the HER superfamily of receptor tyrosine kinases. A common factor in GBM progression is EGFR amplification, which has been found in approximately 40% of all GBM cases (Hynes et al. (2005) Nat. Rev. Cancer. 5:341-354; Hatanpaa et al. (2010) Neoplasia. 12:675-684). In addition, EGFR amplification is associated with the presence of EGFR protein variants, and 68% of EGFR mutations have deletions in the N-terminal ligand-binding domain between amino acids 6 and 273. These deletions in the ligand-binding domain of EGFR can lead to ligand-independent activation (Yamazaki et al. (1990) Jpn. J. Cancer Res. 81:773-779).

[0015] Small molecule tyrosine kinase inhibitors (TKIs) are the most clinically advanced EGFR-targeted therapeutics, with both reversible and irreversible inhibitors undergoing clinical trials. Examples of reversible and irreversible inhibitors include erlotinib, gefitinib, lapatinib, PKI166, canertinib, and pelitinib (Mischel et al. (2003) Brain Pathol. 13:52-61). Mechanistically, these TKIs compete with ATP for binding to the tyrosine kinase domain of EGFR. However, these EGFR-specific tyrosine kinase inhibitors are relatively ineffective against gliomas, with response rates of erlotinib reaching only about 25% (Mischel et al. (2003) Brain Pathol. 13:52-61; Gan et al. (2009) J. Clin. Neurosci. 16:748-54). Although TKIs are well tolerated and demonstrate some antitumor activity in GBM patients, the recurring problem of resistance to receptor blockade limits their effectiveness (Learn et al. (2004) Clin. Cancer. Res. 10:3216-3224; Rich et al. (2004) Nat. Rev. Drug Discov. 3:430-446). In addition, recent studies have shown that posttreatment brain plasma concentrations of gefitinib and erlotinib are only 6-11% of the starting dose, suggesting that these compounds may not cross the blood-brain barrier (Karpel-Massler et al. (2009) Mol. Cancer Res. 7:1000-1012), as shown in Table 1. Therefore, insufficient target delivery may be another cause of disappointing clinical outcomes. [Table 1]

[0016] Given this evidence, there remains an unmet clinical need for potent tyrosine kinase inhibitors that have the ability to cross the blood-brain barrier and therapeutically inhibit EGFR and its isoforms.

[0017] Furthermore, we demonstrate that crosstalk between oncogenic signaling pathways and metabolic pathways creates opportunities for novel combination therapies in GBM. More specifically, we discovered that acute inhibition of EGFR-driven glucose uptake induces minimal cell death and further lowers the apoptotic threshold in patient-derived GBM cells, "activating" them toward apoptosis. Unexpectedly, our mechanistic studies revealed that Bcl-xL inhibits cytoplasmic p53 from triggering intrinsic apoptosis, leading to tumor survival. Pharmacological stabilization of p53 (e.g., with the brain-penetrant small molecule idasanutlin) allows p53 to engage the intrinsic apoptotic machinery, promoting synergistic lethality with targeted EGFR-driven glucose uptake in GBM xenografts. Notably, we also demonstrated, for example, noninvasive positron emission tomography (PET) imaging. 18 F-fluorodeoxyglucose ( 18 We found that rapid changes in F-FDG (F-FDG) uptake could predict sensitivity to combinations in vivo.

[0018] The present inventors identify, inter alia, important causal relationships between oncogene signaling, glucose metabolism, and cytoplasmic p53 that can be exploited for combination therapy in GBM and other malignancies.

[0019] Compounds of the Disclosure In one aspect, the present disclosure provides a compound of Formula I or Formula I * The compound [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, Z is aryl or heteroaryl; R 2a and R 2b are each independently selected from hydrogen, alkyl, halo, CN, and NO; R 3is hydrogen, alkyl, or acyl; R 4 is an alkoxy; R 5 is alkyl, and R 7 and R 8 are each independently selected from hydrogen, alkyl, e.g., alkoxyalkyl, aralkyl, etc., or arylacyl; R 11 But hydrogen, alkyl, halo, CN, NO2, OR 7 , cycloalkyl, heterocyclyl, aryl, or heteroaryl; R 12 But hydrogen, alkyl, halo, CN, NO2, OR 8 , cycloalkyl, heterocyclyl, aryl, or heteroaryl, or R 11 and R 12 together complete a carbocyclic or heterocyclic ring.

[0020] Formula I or Formula I * In certain preferred embodiments of 2a and R 2b At least one of Formula I or Formula I is not H. * In certain such embodiments, R 2a is hydrogen, R 2b is selected from alkyl, halo, CN, and NO2. * In other such embodiments, R 2b is hydrogen, R 2a is selected from alkyl, halo, CN, and NO2.

[0021] Formula I or Formula I * In certain embodiments, the compound is a compound of formula (IVa) or formula (IVb): [ka] or a pharmaceutically acceptable salt thereof; During the ceremony, R 6Each instance of is independently selected from alkyl, alkoxy, OH, CN, NO2, halo, alkenyl, alkynyl, aralkyloxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

[0022] Formula I, I * In certain embodiments of Iva, Iv, and IVb, R 11 is hydrogen. In another preferred embodiment, R 11 is OR 7 is.

[0023] Formula I, I * In certain embodiments of Iva, Iv, and IVb, R 7 is hydrogen. In other embodiments, R 7 is alkyl. In still other embodiments, R 7 is alkoxyalkyl. In still other embodiments, R 7 is an aryl acyl.

[0024] Formula I, I * In certain embodiments of Iva, Iv, and IVb, R 12 is heteroaryl, such as furanyl, etc. In certain embodiments, heteroaryl is alkyl, alkoxy, OH, CN, NO, halo, [ka] In another preferred embodiment, R 12 is OR 8 is.

[0025] Formula I, I * In certain embodiments of Iva, Iv, and IVb, R 8 is hydrogen. In other embodiments, R 8 is alkyl. In still other embodiments, R 8 is alkoxyalkyl. In certain embodiments, R 8 teeth, [ka] is alkyl substituted with

[0026] Formula I, I * In certain preferred embodiments of Iva, Iv, and IVb, R 11 and R 12 are linked to form a carbocyclic or heterocyclic ring, such as a 5-, 6-, or 7-membered carbocyclic or heterocyclic ring. In certain embodiments, the carbocyclic or heterocyclic ring is substituted with hydroxyl, alkyl (e.g., methyl), or alkenyl (e.g., vinyl).

[0027] Formula I, I * In certain embodiments of Iva, Iva, and IVb, the compound is a compound of formula Ia, Ib, Ic, or Id: [ka] or a pharmaceutically acceptable salt thereof; During the ceremony, X is O, S, or NH; Z is aryl or heteroaryl; R 1 is hydrogen or alkyl, R 2a and R 2b are each independently selected from hydrogen, alkyl, halo, CN, and NO; R 3 is hydrogen, alkyl, or acyl; R 4 is an alkoxy; R 5 is alkyl, n is 0 to 3.

[0028] In certain embodiments of formula Ia, Ib, Ic, or Id, R 2a or R 2bis selected from alkyl, halo, CN, and NO. In certain preferred embodiments of Formula Ia, Ib, Ic, or Id, Z is phenyl. In certain preferred embodiments of Formula Ia, Ib, Ic, or Id, X is O. In certain preferred embodiments of Formula Ia, Ib, Ic, or Id, n is 1.

[0029] In certain embodiments of Formula Ia, Ib, Ic, or Id, the compound is a compound of Formula (IIa) or Formula (IIb): [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, R 6 Each instance of is independently selected from alkyl, alkoxy, OH, CN, NO2, halo, alkenyl, alkynyl, aralkyloxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

[0030] In certain embodiments, in formula R 1 is the formula A, [ka] is expressed as During the ceremony, R 7a and R 7b are each independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, or R 7a and R 7b are joined to form a heterocyclyl, y is 0 to 3.

[0031] In certain embodiments of Formula IIa or IIb, R 1 is alkyl (e.g., methyl or ethyl). In certain embodiments, R 1is substituted with heterocyclyl (e.g., morpholinyl, piperidinyl, pyrrolodinyl, or piperazinyl, such as N-methylpiperazinyl). 1 is substituted with amino (e.g., dimethylamino). In other embodiments, R 1 is alkyl substituted with hydroxyl. In certain preferred embodiments, R 1 is in the S configuration. In other embodiments, R 1 is in the R configuration.

[0032] In certain preferred embodiments of formula IIa or IIb, R 3 is hydrogen. In other embodiments, R 3 is acyl. In certain embodiments, R 3 is alkyl acyl. In certain embodiments, R 3 is alkyloxyacyl. In certain embodiments, R 3 is acyloxyalkyl. In certain embodiments, R 3 teeth [ka] and R 9 is alkyl.

[0033] In certain embodiments of formula IIa or IIb, Z is one or more R 6 is an aryl or heteroaryl optionally substituted with 6 Each instance of is independently selected from alkyl, alkoxy, OH, CN, NO, halo, alkenyl, alkynyl, aralkyloxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl. In certain preferred embodiments, Z is selected from 1, 2, 3, 4, or 5 R 6 In certain embodiments, each R 6is independently selected from halo, alkyl, alkynyl, or arylalkoxy. In even more preferred embodiments, Z is 2-fluoro-3-chlorophenyl, 2-fluorophenyl, 2,3-difluorophenyl, 2,4-difluorophenyl, 2,5-difluorophenyl, 2,6-difluorophenyl, 2,4,6-trifluorophenyl, pentafluorophenyl, 2-fluoro-3-bromophenyl, 2-fluoro-3-ethynylphenyl, and 2-fluoro-3-(trifluoromethyl)phenyl. In other even more preferred embodiments, Z is 3-ethynylphenyl. In yet other even more preferred embodiments, Z is 3-chloro-4-((3-fluorobenzyl)oxy)benzene. In yet other even more preferred embodiments, Z is 3-chloro-2-(trifluoromethyl)phenyl. In yet other even more preferred embodiments, Z is 3-bromophenyl. In yet other even more preferred embodiments, Z is 2-fluoro-5-bromophenyl. In still other even more preferred embodiments, Z is 2,6-difluoro-5-bromophenyl. [ka] One R selected from 6 is substituted with R 9 and R 10 is independently selected from alkyl.

[0034] In certain embodiments of Formula IIIa or IIIb, the compound is a compound of formula (IIIa): [ka] and Each R 6 is independently selected from fluoro, chloro, or bromo.

[0035] In certain embodiments of Formula IIIa or IIIb, the compound is a compound of formula (IIIb): [ka] and Each R 6 is independently selected from fluoro, chloro, or bromo.

[0036] In certain embodiments of formula IIIa or IIIb, the compound is a compound of formula (IIIc): [ka] and Each R 6 is independently selected from fluoro, chloro, or bromo.

[0037] In certain embodiments of formula Ia, Ib, Ic, Id, IIa, IIb, IIIa, IIIb, or IIIc, R 2a is halo (e.g., fluoro). In other preferred embodiments, R 2a is hydrogen.

[0038] In certain embodiments of formula Ia, Ib, Ic, Id, IIa, IIb, IIIa, IIIb, or IIIc, R 2b is halo (e.g., fluoro). In other preferred embodiments, R 2b is hydrogen.

[0039] In certain embodiments of Formula Ia, Ib, Ic, Id, IIa, IIb, IIIa, IIIb, or IIIc, the compound is [ka] or a pharmaceutically acceptable salt thereof.

[0040] In certain embodiments of Formula Ia, Ib, Ic, Id, IIa, IIb, IIIa, IIIb, or IIIc, the compound is [ka] JPEG0007789832000017.jpg67168, or a pharmaceutically acceptable salt thereof.

[0041] In certain embodiments of Formula I, the compound is [ka] or a pharmaceutically acceptable salt thereof.

[0042] In certain embodiments of Formula Ia, Ib, Ic, Id, IIa, IIb, IIIa, IIIb, or IIIc, the compound is [ka] or a pharmaceutically acceptable salt thereof.

[0043] Treatment method In certain aspects, the present disclosure provides methods for inhibiting EGFR or ΔEGFR, comprising administering to a subject an amount of a compound of the present disclosure.

[0044] In certain aspects, the present disclosure provides a method for treating cancer, comprising administering to a subject in need of cancer treatment an amount of a compound of the present disclosure. In certain embodiments, the cancer is bladder cancer, bone cancer, brain cancer, breast cancer, heart cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, fibrosarcoma, gastric cancer, gastrointestinal cancer, head, spine, and neck cancer, Kaposi's sarcoma, kidney cancer, leukemia, liver cancer, lymphoma, melanoma, multiple myeloma, pancreatic cancer, penile cancer, testicular germ cell cancer, thymoma cancer, thymic cancer, lung cancer, ovarian cancer, or prostate cancer. In certain embodiments, the cancer is glioma, astrocytoma, or glioblastoma. In certain embodiments, the cancer is glioblastoma. In certain embodiments, the cancer is glioblastoma multiforme. In certain embodiments, the method inhibits cancer cell proliferation.

[0045] In certain aspects, the present disclosure provides a method for treating cancer in a subject, the method comprising administering to the subject a glucose metabolism inhibitor and an additional agent, wherein the glucose metabolism inhibitor is a compound of the present disclosure or a pharmaceutically acceptable salt thereof, and the additional agent is a cytoplasmic p53 stabilizer. In certain embodiments, the cancer is bladder cancer, bone cancer, brain cancer, breast cancer, heart cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, fibrosarcoma, gastric cancer, gastrointestinal cancer, head, spine, and neck cancer, Kaposi's sarcoma, kidney cancer, leukemia, liver cancer, lymphoma, melanoma, multiple myeloma, pancreatic cancer, penile cancer, testicular germ cell cancer, thymoma cancer, thymic cancer, lung cancer, ovarian cancer, or prostate cancer. In certain embodiments, the cancer is glioma, astrocytoma, or glioblastoma. In certain embodiments, the cancer is glioblastoma. In certain embodiments, the cancer is glioblastoma multiforme. In certain embodiments, the method inhibits cancer cell proliferation. In certain embodiments, the cancer is relapsed or refractory, hi other embodiments, the cancer is untreated.

[0046] In certain embodiments, the subject: a. obtaining a first blood sample from a subject; b. implementing a ketogenic diet on the subject; c. Obtaining a second blood sample from the subject after implementing a ketogenic diet for a period of time; d. measuring the glucose level in the first blood sample and in the second blood sample; e. comparing the glucose level in the second blood sample with the glucose level in the first blood sample; and f. determining that the subject is susceptible if the glucose level in the second blood sample is decreased compared to the glucose level in the first blood sample; It has been confirmed that the method includes the steps of:

[0047] In certain embodiments, the decrease in glucose levels between the second blood sample and the control blood sample is about 0.15 mM or more. In certain embodiments, the decrease in glucose levels between the second blood sample and the control blood sample is about 0.20 mM or more. In certain embodiments, the decrease in glucose levels between the second blood sample and the control blood sample is in the range of 0.15 mM to 2.0 mM. In certain embodiments, the decrease in glucose levels between the second blood sample and the control blood sample is in the range of 0.25 mM to 1.0 mM.

[0048] In certain embodiments, the cytoplasmic p53 stabilizer is an MDM2 inhibitor. In certain embodiments, the MDM2 inhibitor is a nutlin. In certain embodiments, the MDM2 inhibitor is nutlin-3 or idasanutlin. In certain embodiments, the subject is administered 50 mg to 1600 mg of idasanutlin. In certain embodiments, the subject is administered 100 mg of idasanutlin. In certain embodiments, the subject is administered 150 mg of idasanutlin. In certain embodiments, the subject is administered 300 mg of idasanutlin. In certain embodiments, the subject is administered 400 mg of idasanutlin. In certain embodiments, the subject is administered 600 mg of idasanutlin. In certain embodiments, the subject is administered 1600 mg of idasanutlin. In other embodiments, the MDM2 inhibitor is RO5045337, RO5503781, RO6839921, SAR405838, DS-3032, DS-3032b, or AMG-232.

[0049] In certain embodiments, the cytoplasmic p53 stabilizer is a BCL-2 inhibitor. In certain embodiments, the BCL-2 inhibitor is antisense oligodeoxynucleotide G3139, mRNA antagonist SPC2996, venetoclax (ABT-199), GDC-0199, obatoclax, paclitaxel, navitoclax (ABT-263), ABT-737, NU-0129, S 055746, or APG-1252.

[0050] In certain embodiments, the cytoplasmic p53 stabilizer is a Bcl-xL inhibitor, ie, WEHI 539, ABT-263, ABT-199, ABT-737, sabutoclax, AT101, TW-37, APG-1252, or gambogic acid.

[0051] In certain embodiments, the glucose metabolism inhibitor and the cytoplasmic p53 stabilizer are administered in the same composition, while in other embodiments, the glucose metabolism inhibitor and the cytoplasmic p53 stabilizer are administered in separate compositions.

[0052] In certain embodiments, the method further comprises administering another therapeutic agent.

[0053] Glioma type and stage Primary malignant brain tumors, tumors that originate in the brain or spine, are collectively known as gliomas. Glioma is not a specific type of cancer, but a term used to describe tumors that originate in glial cells. Examples of primary malignant brain tumors include astrocytoma, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, diffuse astrocytoma, anaplastic astrocytoma, GBM, ganglioglioma, oligodendroglioma, and ependymoma. According to the WHO classification of brain tumors, astrocytomas are classified into four grades determined by the underlying pathology. Characteristics used to classify gliomas include mitoses, cellular or nuclear atypia, and vascular proliferation, as well as necrosis with pseudopalisading characteristics. Malignant (or high-grade) gliomas include anaplastic glioma (WHO grade III) and glioblastoma multiforme (GBM, WHO grade IV). These are the most invasive brain tumors with the poorest prognosis.

[0054] GBM is the most common, complex, treatment-resistant, and fatal type of brain cancer, accounting for 45% of all brain cancers, with nearly 11,000 men, women, and children diagnosed each year. GBM (also known as grade 4 astrocytoma and glioblastoma multiforme) is the most common type of malignant (cancerous) primary brain tumor. GBM is highly invasive for many reasons. First, glioblastoma cells grow rapidly because they secrete substances that promote a rich blood supply. Glioblastoma cells also have the ability to invade and infiltrate normal brain tissue over long distances by sending out microscopic tendrils along with normal cells. Two types of glioblastoma are known. The first, GBM, is the most common form, grows rapidly, and can cause early symptoms. The second, glioblastoma, is less common, accounting for approximately 10% of all GBM cases. Secondary GBMs develop from low-grade diffuse or anaplastic astrocytomas and are more frequently found in younger patients. Secondary GBMs are preferentially located in the frontal lobe and have a better prognosis.

[0055] GBM is typically treated with a combined multimodal treatment plan that includes surgical removal of the tumor, radiation therapy, and chemotherapy. First, as much of the tumor as possible is removed during surgery. The tumor's location in the brain often determines how much of the tumor can be safely removed. After surgery, radiation therapy and chemotherapy are used to slow the growth of remaining tumor cells. The oral chemotherapy drug temozolomide is most often used for six weeks, then monthly. An additional drug, bevacizumab (known as Avastin®), is also used during treatment. This drug attacks the tumor's ability to replenish its blood supply, often slowing or even stopping tumor growth.

[0056] New clinical trial therapies are also used, which may involve adding a therapy to standard of care or substituting part of the standard care with another therapy that may work better. Some of these therapies include immunotherapy, such as vaccine immunotherapy, or low-dose electrical pulses to the area of ​​the brain where the tumor is located, and spherical nucleic acid (SNA) nanotherapy, such as NU-0129. In some embodiments, the methods of the present disclosure are used in combination with one or more of the above therapies.

[0057] Embodiments of the methods and compositions discussed herein are also believed to be applicable to other types of cancer, including, but not limited to, lung cancer, non-CNS cancers, CNS cancers, and CNS metastases, such as brain metastases, leptomeningeal metastases, choroidal metastases, spinal metastases, and other metastases.

[0058] Cytoplasmic p53 stabilizer The inventors have demonstrated that pharmacological p53 stabilization, e.g., with a CNS-permeable small molecule, is synergistically lethal with inhibition of EGFR-driven glucose uptake in a patient-derived primary GBM model. The inventors are the first to demonstrate that non-transcriptional functions of p53 may play an important role in stimulating intrinsic apoptosis in metabolic responders. Therefore, the therapeutic methods described herein involve the administration of cytoplasmic p53 stabilizer(s) in combination with a glucose metabolism inhibitor. The cytoplasmic p53 stabilizer(s) and glucose metabolism inhibitor can be administered in the same composition or in separate compositions, either simultaneously or sequentially. It is contemplated that a single p53 stabilizer will be used in some embodiments, while two or more p53 stabilizers will be used in other embodiments. For example, Nutlin and ABT 737 (BCL-2 and BCL-X) can be combined to inhibit the proliferation and proliferation of BCL-X. LIt has been reported that a combination of p53 stabilizing agents (which bind to p53) promotes cell death by synergistically targeting the balance of pro- and anti-apoptotic proteins at the mitochondrial level (Hoe et al. 2014. Nature Reviews. Vol. 13. pp. 217). As intended herein, a cytoplasmic p53 stabilizer is any small molecule, antibody, peptide, protein, nucleic acid, or derivative thereof that can directly or indirectly pharmacologically stabilize or activate p53. Stabilization of cytoplasmic p53 results in the activation of cells, such as cancer cells, toward apoptosis.

[0059] MDM2 antagonists Intracellular p53 protein levels are tightly controlled and maintained low by its negative regulator, the E3 ubiquitin protein ligase MDM2. In embodiments of the disclosed methods or compositions, the cytoplasmic p53 stabilizer is an MDM2 antagonist / inhibitor. In some embodiments, the MDM2 antagonist is a nutlin. In further embodiments, the nutlin is nutlin-3 or idasanutlin. In other embodiments, the MDM2 antagonist is RO5045337 (also known as RG7112), RO5503781, RO6839921, SAR405838 (also known as MI-773), DS-3032, DS-3032b, or AMG-232, or any other MDM2 inhibitor.

[0060] Other compounds within the scope of the present methods that are known to bind to MDM-2 include Ro-2443, MI-219, MI-713, MI-888, DS-3032b, benzodiazepinediones (e.g., TDP521252), sulfonamides (e.g., NSC279287), chromenotriazolopyrimidines, morpholinones and piperidinones (AM-8553), terphenyls, chalcones, pyrazoles, imidazoles, imidazole-indoles, isoindolinones, pyrrolidinones (e.g., PXN822), priaxones, piperidines, naturally derived prenylated xanthones, SAH-8 (stapled peptide), sMTide-02, sMTide-02a (stapled peptide), ATSP-7041 (stapled peptide), and spiroligomers (α-helix mimetics). Other compounds known to induce protein folding of MDM2 include PRIMA-1MET (also known as APR-246), Aprea 102-105, PK083, PK5174, PK5196, PK7088, benzothiazole, stictic acid, and NSC319726.

[0061] BCL-2 inhibitors In further embodiments of the methods and compositions, the cytoplasmic p53 stabilizer is a BCL-2 inhibitor, hi some embodiments, the BCL-2 inhibitor is, for example, the antisense oligodeoxynucleotide G3139, the mRNA antagonist SPC2996, venetoclax (ABT-199), GDC-0199, obatoclax, paclitaxel, navitoclax (ABT-263), ABT-737, NU-0129, S 055746, APG-1252, or any other BCL-2 inhibitor.

[0062] Bcl-xL inhibitors In still further embodiments of the methods and compositions, the cytoplasmic p53 stabilizer is a Bcl-xL inhibitor, hi some embodiments, the Bcl-xL inhibitor is, for example, WEHI 539, ABT-263, ABT-199, ABT-737, sabutoclax, AT101, TW-37, APG-1252, gambogic acid, or any other Bcl-xL inhibitor.

[0063] Evaluation method Glucose uptake test In embodiments of the disclosed methods and compositions, subjects with GBM or cancer are classified as either "metabolic responders" or "metabolic non-responders" (i.e., identified as susceptible to glucose metabolic inhibitors). In certain embodiments, the subject is classified and then administered a therapeutic agent comprising a glucose metabolic inhibitor and a cytoplasmic p53 stabilizer. Thus, the present disclosure provides methods for assessing cancer, classifying subjects, and determining a subject's susceptibility to a therapeutic agent, including analysis of glucose metabolism, glycolysis, or glucose uptake. Methods for classifying subjects as metabolic responders are described in detail in Example 1. Techniques for monitoring glycolysis and glucose uptake are provided by T. TeSlaa and MATeitell. 2014. Methods in Enzymology, Volume 542, pp. 92-114 (incorporated herein by reference).

[0064] Glycolysis is the intracellular biochemical conversion of one molecule of glucose to two molecules of pyruvate, with the concomitant production of two molecules of ATP. Pyruvate is a metabolic intermediate with several potential fates, including entering the tricarboxylic acid (TCA) cycle in mitochondria to generate NADH and FADH2. Alternatively, pyruvate can be converted to lactate in the cytosol by lactate dehydrogenase (NADH to NAD +(While glucose is simultaneously regenerated). Increased flux through glycolysis supports cancer cell proliferation, for example, by providing additional energy in the form of ATP, as well as glucose-derived metabolic intermediates for the biosynthesis of nucleotides, lipids, and proteins. Warburg (Oncologia. 1956;9(2):75-83) was the first to observe that proliferating tumor cells increase aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen), in contrast to non-malignant cells, which primarily respire when oxygen is available. This mitochondrial bypass, termed the Warburg effect, occurs in rapidly proliferating cells, including cancer cells, activated lymphocytes, and pluripotent stem cells. The Warburg effect can be demonstrated using positron emission tomography (PET) scans to detect the effects of fluorinated glucose analogs, e.g., 18 It is used in clinical diagnostic tests to confirm increased cellular uptake of F-deoxyglucose and other substances.

[0065] Therefore, glycolysis represents a target for therapeutic and diagnostic methods. In the context of this method, measuring glucose uptake and lactate excretion by malignant cells can be useful for detecting shifts in glucose catabolism and / or sensitivity to glucose metabolism inhibitors. Detecting such shifts is important in methods for treating GBM, reducing the risk of ineffective treatment, and reducing the likelihood of tumor survival. For the purposes of this disclosure, 18 In certain embodiments, F-deoxyglucose PET serves as a rapid, noninvasive functional biomarker for predicting susceptibility to p53 activation. This noninvasive analysis may be particularly useful in malignant brain tumors, where pharmacokinetic / pharmacodynamic assessments are extremely challenging and impractical. In some cases, delayed imaging protocols (41) and parametric response maps (PRMs) using MRI fusion can be used to predict tumor sensitivity. 18 It may be useful to quantify changes in F-FDG uptake (42).

[0066] In certain embodiments, the methods may involve measuring glucose uptake and lactate production. For cells in culture, glycolytic flux can be quantified by measuring glucose uptake and lactate efflux. Glucose uptake into cells is via glucose transporters (Glut1-Glut4), whereas lactate efflux at the cell membrane is via monocarboxylate transporters (MCT1-MCT4).

[0067] Extracellular glucose and lactate Methods for detecting glucose uptake and lactate excretion include, for example, extracellular glucose or lactate kits, extracellular bioanalyzers, ECAR measurements, [H]-2-DG or [C]-2-DG uptake, 18 These include FDG uptake and 2-NBDG uptake.

[0068] Commercial kits and instruments are available to quantify glucose and lactate levels in cell culture media. Kit detection methods are generally colorimetric or fluorometric and are compatible with standard laboratory equipment, such as spectrophotometers. BioProfile Analyzers (e.g., Nova Biomedical) or Biochemistry Analyzers (e.g., YSI Life Sciences) can measure both glucose and lactate levels in cell culture media. GlucCell (Cesco BioProducts) can measure only glucose levels in cell culture media. Although each commercially available method has a different detection protocol, collection of the media for analysis is identical.

[0069] Extracellular acidification rate Glycolysis can also be measured by measuring the extracellular acidification rate (ECAR) of the surrounding medium, which is primarily due to the excretion of lactate per unit time after the conversion of pyruvate to lactate. The Seahorse Extracellular Flux (XF) Analyzer (Seahorse Bioscience) is a device for simultaneously measuring glycolysis and oxidative phosphorylation (via oxygen consumption) in the same cell.

[0070] Glucose analogue uptake Certain embodiments of the disclosed methods include the use of glucose analogs. As is well known to those skilled in the art, glucose uptake rates can be determined using cells by adding a labeled isoform of glucose to the cell culture medium and then measuring the intracellular levels after any period of time. Exemplary types of glucose analogs for these tests include radioactive glucose analogs, such as 2-deoxy-D-[1,2-3H]-glucose, 2-deoxy-D-[1-14C]-glucose, or 2-deoxy-2-( 18 F)-fluoro-D-glucose ( 18 Radioactive glucose analogues, such as 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG), are used for measuring uptake of the radioactive glucose analogues. While a scintillation counter is required to measure uptake of the radioactive glucose analogues, 2-NBDG uptake is typically measured using flow cytometry or a fluorescence microscope. In some embodiments, glucose uptake is measured using radioactively labeled glucose, 2-deoxy-2-[fluorine-18]fluoro-D-glucose ( 18 In a further embodiment, the uptake of F-FDG is measured. 18 Detection of F-FDG is by positron emission tomography (PET). In some embodiments, a biopsy is taken from a GBM tumor. 18 A detailed description of an example of the measurement of F-FDG is provided in the Examples below.

[0071] In certain aspects, the methods can involve comparing glucose uptake in a biological sample, such as a tumor sample, to a control. The fold increase or decrease can be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more, or any range derivable therein. Alternatively, the difference in expression between the sample and reference may be expressed as a percentage decrease or increase, such as at least or at most a 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000% difference, or any range derivable therein.

[0072] Other ways to express relative expression levels are: 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03. 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 Normalized or relative numbers such as 1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, or any range derivable therein, are used. In some embodiments, the level may be compared to a control.

[0073] Algorithms, such as weighted voting programs, may be used to facilitate the determination of biomarker levels. Additionally, other clinical evidence may be combined with biomarker-based testing to reduce the risk of false positives. In some embodiments, other cytogenetic assessments may be considered.

[0074] Synthesis method In another aspect, the present disclosure provides a method for preparing a compound according to Scheme 1 or Scheme 2, [ka] [ka] According to formula I, I *or a pharmaceutically acceptable salt thereof, During the ceremony, X is O, S, or NH; Z is aryl or heteroaryl; R 1 is alkyl, R 2a and R 2b are each independently selected from hydrogen, alkyl, halo, CN, and NO; R 3 is hydrogen, alkyl, or acyl; R 4 is an alkoxy; R 5 is alkyl, R 21 is an alkyl substituted with a leaving group, e.g., haloalkyl or sulfonylalkyl; B is a base, Nu is a nitrogen-containing heterocycle (e.g., having at least one N-H bond), aminoalkyl, or hydroxyalkyl; Sv 1 is the solvent, n is 0 to 3.

[0075] In certain preferred embodiments, R 21 is sulfonylalkyl (e.g., CH3S(O)2OCH2-).

[0076] In certain embodiments, B is a nitrogen base (eg, triethylamine or diisopropylethylamine).

[0077] In certain embodiments, Nu is a nitrogen-containing heterocycle having at least one N-H bond (e.g., morpholine, N-methylpiperazine, piperidine, or pyrrolidine). In other embodiments, Nu is an aminoalkyl (e.g., dimethylamine).

[0078] In certain embodiments, the solvent is an aprotic solvent (eg, dimethylformamide).

[0079] In certain preferred embodiments, the method comprises Scheme 3 or 4: [ka] [ka] The method further comprises the steps of During the ceremony, R 22 is alkyl or hydroxyalkyl, R 23a and R 23b are each alkyl, R 24 is aminoaryl or aminoheteroaryl, Sv 2 is an acid.

[0080] In certain preferred embodiments, R 22 is hydroxyalkyl.

[0081] In certain embodiments, R 23a and R 23b are each methyl.

[0082] In certain embodiments, R 24 is aminoaryl. In other embodiments, R 24 is aminoheteroaryl.

[0083] In certain embodiments, Sv 2 is an alkyl acid (e.g., acetic acid).

[0084] In certain preferred embodiments, the steps of Scheme 3 or 4 are carried out at a temperature ranging from 115 to 150° C. In certain embodiments, the steps are carried out at a temperature ranging from 125 to 130° C. In certain embodiments, the steps further comprise treatment with a base, such as ammonium hydroxide.

[0085] In certain embodiments, the method further comprises a purification step, hi certain embodiments, the purification step comprises column chromatography, preparative thin layer chromatography, or high performance liquid chromatography.

[0086] definition Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the terms used in connection with, and techniques related to, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.

[0087] The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, e.g., "Principles of Neural Science," McGraw-Hill Medical, New York, NY (2000); Motulsky, "Intuitive Biostatistics," Oxford University Press, Inc. (1995); Lodish et al., "Molecular Cell Biology, 4th ed.", W.H. Freeman & Co., New York (2000); Griffiths et al., "Introduction to Genetic Analysis, 7th ed.", W.H. Freeman & Co., NY (1999); and Gilbert et al., "Developmental Biology, 6th ed.", Sinauer Associates, Inc., Sunderland, MA (2000).

[0088] Chemical terms used herein, unless otherwise defined herein, are used in accordance with conventional usage in the art, as exemplified by "The McGraw-Hill Dictionary of Chemical Terms," ​​Parker S., Ed., McGraw-Hill, San Francisco, CA (1985).

[0089] All of the above, and any other publications, patents, and published patent applications referenced in this application, are expressly incorporated herein by reference. In the case of conflict, the present specification, including specific definitions thereof, will control.

[0090] The term "drug" is used herein to refer to a compound (e.g., an organic or inorganic compound, a mixture of compounds, etc.), a biological macromolecule (e.g., a nucleic acid, an antibody (including a portion thereof), as well as a humanized, chimeric, and human antibody, and a monoclonal antibody, a protein or a portion thereof, e.g., a peptide, a lipid, a carbohydrate, etc.), or an extract obtained from a biological material, such as a cell or tissue of a bacterium, a plant, a fungus, or an animal (especially a mammal). Drugs include, for example, drugs whose structure is known and drugs whose structure is unknown. The ability of such drugs to inhibit AR or promote AR degradation may make such drugs suitable as "therapeutic agents" in the methods and compositions of the present disclosure.

[0091] The terms "patient," "subject," or "individual" are used interchangeably and refer to either a human or non-human animal. These terms include mammals, such as humans, primates, livestock animals (including cows, pigs, etc.), companion animals (e.g., dogs, cats, etc.), and rodents (e.g., mice and rats).

[0092] "Treating" a condition or patient means taking steps to obtain beneficial or desired results, including clinical results. As used herein, and as well understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or symptoms, whether detectable or undetectable, reduction in the severity of disease, stable (i.e., not worsening) disease, prevention of disease metastasis, delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or complete). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0093] The term "preventing," when used in connection with a condition, such as local recurrence (e.g., pain), a disease, such as cancer, a complex syndrome, such as heart failure, or any other medical condition, is art-recognized and well understood in the art, and includes administration of a composition that reduces the frequency of symptoms of the medical condition or delays the onset of the medical condition in a subject compared to subjects not receiving the composition. Thus, preventing cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic therapy compared to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population compared to an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0094] "Administering" a substance, compound, or agent to a subject, or "administering" a substance, compound, or agent to a subject, can be carried out using one of a variety of methods well known to those of skill in the art. For example, a compound or agent can be administered intravenously, intraarterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through the skin's channels). A compound or agent can also be conveniently introduced using rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, that provide slow, sustained, or controlled release of the compound or agent. Administering can also be carried out once, multiple times, and / or more than one or more times, e.g., over an extended period of time.

[0095] The appropriate method of administering a substance, compound, or agent to a subject also depends, for example, on the age and / or physical condition of the subject, and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability, and toxicity). In some embodiments, the compound or agent is orally administered to the subject, for example, by ingestion. In some embodiments, the orally administered compound or agent is a sustained-release or time-release formulation or is administered using a device for such sustained-release or time-release.

[0096] As used herein, the phrase "co-administration" refers to any form of administration of two or more different therapeutic agents in which a previously administered therapeutic agent is administered while the second agent is still effective in the body (e.g., the two agents are effective in the patient simultaneously, which may involve a synergistic effect of the two agents). For example, the different therapeutic compounds can be administered either in the same formulation or in separate formulations, either simultaneously or sequentially. Thus, an individual receiving such treatment can benefit from the combined effects of the different therapeutic agents.

[0097] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or agent is an amount of the drug or agent that exhibits the desired therapeutic effect when administered to a subject. The full therapeutic effect need not necessarily occur in a single dose, but may occur only after the administration of successive doses. Thus, a therapeutically effective amount may be administered in one or more doses. The precise effective amount required by a subject will depend, for example, on the subject's size, health, and age, as well as the nature and severity of the condition being treated, such as cancer or MDS. One of ordinary skill in the art can readily determine the effective amount in any given situation by routine experimentation.

[0098] As used herein, the term "optional" or "optionally" means that the event or circumstance described following the term may or may not occur, and that the description includes instances in which the event or circumstance occurs as well as instances in which the event or circumstance does not occur. For example, "optionally substituted alkyl" means that the alkyl is not substituted, in addition to alkyl that may be substituted.

[0099] It is understood that one skilled in the art can select substituents and substitution patterns on the compounds of the present invention to provide chemically stable compounds that can be readily synthesized from readily available starting materials using techniques well known in the art as well as the methods described below. It is understood that when a substituent is itself substituted with more than one group, these multiple groups can be on the same carbon or on different carbons, so long as a stable structure is provided.

[0100] As used herein, the term "optionally substituted" means that one to six hydrogen radicals in any structure are replaced with the radical of a specified substituent, including, but not limited to, hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH-O-alkyl, -OP(O)(O-alkyl), or -CH-OP(O)(O-alkyl). Preferably, "optionally substituted" means that one to four hydrogen radicals in any structure are replaced with the above-listed substituents. More preferably, one to three hydrogen radicals are replaced with the above-listed substituents. It is understood that the substituents may be further substituted.

[0101] As used herein, the term "alkyl" refers to a C-C 10 Straight chain alkyl group or C1-C 10 "Alkyl" refers to saturated aliphatic groups, including, but not limited to, branched alkyl groups. Preferably, "alkyl" refers to C1-C6 straight chain alkyl groups or C1-C6 branched chain alkyl groups. Most preferably, "alkyl" refers to C1-C4 straight chain alkyl groups or C1-C4 branched chain alkyl groups. Examples of "alkyl" include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neo-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl, or 4-octyl. "Alkyl" groups may be optionally substituted.

[0102] The term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-. The term "acylamino" is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-.

[0103] The term "acyloxy" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

[0104] The term "alkoxy" means an alkyl group having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like.

[0105] The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl.

[0106] The term "alkyl" refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight-chain alkyl or branched-chain alkyl group has 30 or fewer carbon atoms in its backbone (e.g., C in a straight chain). 1-30 , C in branched chain 3-30 ), and more preferably has 20 or fewer carbon atoms.

[0107] Furthermore, the term "alkyl," as used throughout the specification, examples, and claims, is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups, for example, trifluoromethyl and 2,2,2-trifluoroethyl.

[0108] The term "C" when used with a chemical moiety, such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, x-y " or "Cx -C y " means to include groups containing x to y carbons in the chain. C0 alkyl represents a hydrogen if the group is in a terminal position, or a bond if it is internal. C 1-6 Alkyl groups, for example, contain 1 to 6 carbon atoms in the chain.

[0109] The term "alkylamino," as used herein, means an amino group substituted with at least one alkyl group.

[0110] The term "alkylthio," as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.

[0111] The term "amide," as used herein, refers to the group, [ka] , which means In the formula, R 9 and R 10 each independently represents hydrogen or a hydrocarbyl group, or R 9 and R 10 together with the N atom to which they are attached complete a heterocycle with 4 to 8 atoms in the ring structure.

[0112] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted amines and their salts, and substituted amines and their salts, e.g., [ka] , and In the formula, R 9 , R 10 , and R 10’ each independently represents hydrogen or a hydrocarbyl group, or R 9 and R 10 together with the N atom to which they are attached complete a heterocycle with 4 to 8 atoms in the ring structure.

[0113] The term "aminoalkyl," as used herein, refers to an alkyl group substituted with an amino group.

[0114] The term "aralkyl," as used herein, refers to an alkyl group substituted with an aryl group.

[0115] The term "aryl," as used herein, includes substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon. Preferably, the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, at least one of the rings being aromatic, for example, the other cyclic ring may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0116] The term "carbamate" is art-recognized and refers to the group [ka] , which means In the formula, R 9 and R 10 independently represent hydrogen or a hydrocarbyl group.

[0117] The term "carbocyclylalkyl," as used herein, refers to an alkyl group substituted with a carbocyclic group.

[0118] The term "carbocycle" includes 5- to 7-membered monocyclic rings and 8- to 12-membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated, and aromatic rings. Carbocycles include bicyclic molecules in which one, two, or three atoms are shared between the two rings. The term "fused carbocycle" refers to a bicyclic carbocycle in which each ring shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated, and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Valence permitting, any combination of saturated, unsaturated, and aromatic bicyclic rings is included in the definition of carbocycle. Exemplary "carbocycles" include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene, and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene, and bicyclo[4.1.0]hept-3-ene. A "carbocycle" may be substituted at any one or more positions that can have a hydrogen atom.

[0119] The term "carbocyclylalkyl," as used herein, refers to an alkyl group substituted with a carbocyclic group.

[0120] The term "carbonate" is art-recognized and refers to the group -OCO2-.

[0121] The term "carboxy," as used herein, means a group of the formula -CO2H.

[0122] The term "ester" as used herein refers to a group -C(O)OR 9 In the formula, R9 represents a hydrocarbyl group.

[0123] The term "ether," as used herein, refers to a hydrocarbyl group linked to another hydrocarbyl group through an oxygen. Thus, the ether substituent of a hydrocarbyl group can be hydrocarbyl-O-. Ethers can be either symmetrical or asymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include "alkoxyalkyl" groups, where the alkoxyalkyl group can be represented by the general formula alkyl-O-alkyl.

[0124] The terms "halo" and "halogen," as used herein, mean halogens and include chloro, fluoro, bromo, and iodo.

[0125] The terms "hetaralkyl" and "heteroaralkyl," as used herein, refer to an alkyl group substituted with a hetaryl group.

[0126] The terms "heteroaryl" and "hetaryl" include substituted or unsubstituted aromatic monocyclic ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, whose ring structures contain at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heteroaryl" and "hetaryl" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, at least one of the rings being heteroaromatic, for example, while the other cyclic ring may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0127] The term "heteroatom," as used herein, means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0128] The term "heterocyclylalkyl," as used herein, refers to an alkyl group substituted with a heterocycle group.

[0129] The terms "heterocyclyl," "heterocycle," and "heterocyclic" refer to a substituted or unsubstituted non-aromatic ring system, preferably a 3- to 10-membered ring, more preferably a 3- to 7-membered ring, whose ring system contains at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heterocyclyl" and "heterocyclic" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, where at least one of the rings is heterocyclic, e.g., the other cyclic ring can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0130] The term "hydrocarbyl," as used herein, refers to a group that has no =0 or =5 substituents, typically has at least one carbon-hydrogen bond and a primary carbon backbone, but may optionally contain heteroatoms, and is bonded through a carbon atom. Thus, groups such as methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered hydrocarbyl for purposes of this application, while substituents such as acetyl (which has an =0 substituent on the linking carbon) and ethoxy (which is linked through an oxygen rather than a carbon) are not. Hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbocyclic, heterocyclic, alkyl, alkenyl, alkynyl, and combinations thereof.

[0131] The term "hydroxyalkyl," as used herein, refers to an alkyl group substituted with a hydroxy group.

[0132] The term "lower," when used in conjunction with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is meant to include groups having 10 or fewer atoms, preferably 6 or fewer atoms, in the substituent. For example, "lower alkyl" refers to an alkyl group containing 10 or fewer carbon atoms, preferably 6 or fewer carbon atoms. In certain embodiments, each acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituent defined herein is a lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether present alone or in combination with other substituents, such as in the detailed description of hydroxyalkyl and aralkyl (where, for example, atoms in the aryl group are not counted when counting the carbon atoms in the alkyl substituent).

[0133] The terms "polycyclyl," "polycycle," and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl) in which two or more atoms are common to two adjacent rings, e.g., the rings are "fused rings." Each of the rings of a polycycle can be substituted or unsubstituted. In certain embodiments, each ring of a polycycle contains 3 to 10 atoms, preferably 5 to 7 atoms, within the ring.

[0134] The term "sulfate" is art-recognized and refers to the group -OSO3H or a pharmaceutically acceptable salt thereof.

[0135] The term "sulfonamide" is art-recognized and has the general formula: [ka] means a group represented by In the formula, R 9 and R 10 independently represent hydrogen or hydrocarbyl.

[0136] The term "sulfoxide" is art-recognized and refers to the group --S(O)--.

[0137] The term "sulfonate" is art-recognized and refers to the group SO3H or a pharmaceutically acceptable salt thereof.

[0138] The term "sulfone" is art-recognized and refers to the group -S(O)2-.

[0139] The term "substituted" refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It is understood that "substituted" or "substituted with" includes the implicit proviso that such substitution, subject to the permissible valence of the replacing atom and substituent, results in a stable compound that does not spontaneously undergo transformation, for example, by rearrangement, cyclization, elimination, and the like. As used herein, the term "substituted" is construed to include all permissible substituents of organic compounds. In the broadest sense, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different, for appropriate organic compounds. For purposes of this invention, heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituent of organic compounds described herein that satisfies the valence of the heteroatom. Substituents can include any of the substituents described herein, such as halogen, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. Those skilled in the art will understand that moieties substituted on the hydrocarbon chain can themselves be substituted, if desired.

[0140] The term "thioalkyl," as used herein, refers to an alkyl group substituted with a thiol group.

[0141] The term "thioester," as used herein, refers to the group -C(O)SR 9 or -SC(O)R 9 This means that In the formula, R 9 represents a hydrocarbyl.

[0142] The term "thioether," as used herein, is equivalent to an ether, where the oxygen has been replaced with a sulfur.

[0143] The term "urea" is art-recognized and has the general formula: [ka] , which can be expressed as In the formula, R 9 and R 10 independently represent hydrogen or hydrocarbyl.

[0144] The term "modulate" as used herein includes the inhibition or suppression of a function or activity (such as, for example, cell proliferation) as well as the enhancement of a function or activity.

[0145] The phrase "pharmaceutically acceptable" is art-recognized. In certain embodiments, this term includes compositions, excipients, adjuvants, polymers, and other materials and / or dosage forms that are suitable for contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the bounds of sound medical common sense, commensurate with a reasonable benefit / risk ratio.

[0146] "Pharmaceutically acceptable salt" or "salt" is used herein to mean an acid addition salt or a base addition salt that is suitable or compatible for the treatment of patients.

[0147] The term "pharmaceutically acceptable acid addition salt," as used herein, refers to any non-toxic organic or inorganic salt of any base compound represented by Formula I. Exemplary inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric, and phosphoric acids, as well as metal salts such as disodium hydrogen orthophosphate and potassium hydrogen sulfate. Exemplary organic acids which form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic, and salicylic acids, as well as sulfonic acids such as p-toluenesulfonic and methanesulfonic acids. Either mono- or dibasic acid salts may be formed, and such salts may exist in either hydrated, solvated, or substantially anhydrous form. In general, the acid addition salts of the compounds of formula I are more soluble in water and various hydrophilic organic solvents and generally have a higher melting point than their free base forms. The selection of appropriate salts is well known to those skilled in the art. Other pharmaceutically unacceptable salts, such as oxalates, may be used, for example, for the isolation of the compounds of formula I for laboratory use or for subsequent conversion to pharmaceutically acceptable acid addition salts.

[0148] The term "pharmaceutically acceptable base addition salt," as used herein, refers to any non-toxic organic or inorganic base addition salt of any acid compound represented by Formula I or any of its intermediates. Exemplary inorganic bases which form suitable salts include lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or barium hydroxide. Exemplary organic bases which form suitable salts include aliphatic, alicyclic, or aromatic organic amines, such as methylamine, trimethylamine, and picoline, or ammonia. The selection of appropriate salts is within the skill of the art.

[0149] Many of the compounds useful in the methods and compositions of the present disclosure have at least one asymmetric center in their structure. This asymmetric center may exist in either the R or S configuration, and the RS designation is used in accordance with the rules set forth in Pure Appl. Chem. (1976), 45, 11-30. This disclosure contemplates all stereoisomeric forms of the compounds, salts, prodrugs, or mixtures thereof, including enantiomeric and diastereomeric forms (including mixtures of all possible stereoisomers). See, for example, WO 01 / 062726.

[0150] Furthermore, certain compounds containing alkenyl groups may exist as Z (zusammen) or E (entgegen) isomers, and in each case the present disclosure includes both mixtures of the individual isomers and the individual isomers individually.

[0151] Some of the compounds may also exist in tautomeric forms, and such forms (even though not explicitly indicated in the formulae given herein) are meant to be included within the scope of the present disclosure.

[0152] "Prodrug" or "pharmaceutically acceptable prodrug" refers to a compound that is metabolized, e.g., hydrolyzed or oxidized, in a host after administration to form a compound of the present disclosure (e.g., a compound of Formula I). ​​Typical examples of prodrugs include compounds that have a biologically labile or cleavable (protecting) group on a functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce the active compound. Examples of prodrugs using esters or phosphoramidates as biologically labile or cleavable (protecting) groups are disclosed in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. The prodrugs of the present disclosure are metabolized to produce a compound of Formula I. The present disclosure includes within its scope prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in "Design of Prodrugs," Ed. H. Bundgaard, Elsevier, 1985.

[0153] The phrase "pharmaceutically acceptable carrier," as used herein, means a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is useful in formulating a drug for pharmaceutical or therapeutic use.

[0154] The terms "Log of Solubility," "LogS," or "logS," as used herein, are used in the art to quantify the aqueous solubility of a compound. The aqueous solubility of a compound significantly affects its absorption and distribution characteristics. Poor solubility often results in poor absorption. The LogS value is the unitless logarithm (base 10) of solubility measured in moles per liter.

[0155] Pharmaceutical Composition The compositions and methods of the present invention may be used to administer treatment to an individual in need thereof. In certain embodiments, the individual is a mammal, such as a human or non-human mammal. When administered to an animal, such as a human, the composition or compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions, such as water or buffered saline, or other solvents or vehicles, such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are intended for human administration, particularly for invasive administration routes (i.e., routes that bypass or diffuse through an epithelial barrier, such as injection or implantation), the aqueous solutions are pyrogen-free or substantially pyrogen-free. Excipients may be selected, for example, to achieve delayed release of the drug or to selectively target one or more cells, tissues, or organs. The pharmaceutical compositions may be in dosage unit form, such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, lyophilized formulations for reconstitution, powders, liquids, syrups, suppositories, injections, etc. The compositions may also be included in transdermal delivery systems, such as skin patches. The compositions may also be included in liquids suitable for topical administration, such as lotions, creams, or ointments.

[0156] A pharmaceutically acceptable carrier may contain a physiologically acceptable agent that acts, for example, to stabilize, improve the solubility, or enhance the absorption of a compound, such as a compound of the present invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose, or dextran; antioxidants, such as ascorbic acid or glutathione; chelating agents; low-molecular-weight proteins; or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier containing a physiologically acceptable agent depends, for example, on the route of administration of the composition. The formulation or pharmaceutical composition may be a self-emulsifying or self-microemulsifying drug delivery system. The pharmaceutical composition (formulation) may also be a liposome or other polymer matrix, which may, for example, contain a compound of the present invention therein. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively easy to prepare and administer.

[0157] The phrase "pharmaceutically acceptable" is used herein to mean compounds, substances, compositions and / or dosage forms that are suitable, within the bounds of sound medical common sense, for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0158] The phrase "pharmaceutically acceptable carrier," as used herein, means a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients in the formulation and not harmful to the patient. Some examples of substances that can function as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; and (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil. (10) glycols, such as propylene glycol, (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) esters, such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) phosphate buffer, and (21) other non-toxic, compatible substances used in pharmaceutical formulations.

[0159] Pharmaceutical compositions (formulations) can be administered to a subject by any of a number of routes of administration, including, for example, orally (e.g., solutions such as aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, or pastes for application to the tongue), absorption through the oral mucosa (e.g., sublingually), subcutaneously, transdermally (e.g., as a patch applied to the skin), and topically (e.g., as a cream, ointment, or spray applied to the skin). The compounds may also be formulated for inhalation. In certain embodiments, the compounds may be conveniently dissolved or suspended in sterile water. Details regarding suitable routes of administration and compositions suitable for those routes of administration can be found, for example, in U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970, and 4,172,896, as well as the patents cited therein.

[0160] The formulations may be conveniently presented in unit dosage form and may be prepared by any method well known in the pharmaceutical arts. The amount of active ingredient that may be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. The amount of active ingredient that may be combined with a carrier material to produce a single dosage form will generally be the amount of compound that produces a therapeutic effect. Generally, this amount will range from about 1% to about 99%, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%, of one hundred percent of the active ingredient.

[0161] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0162] Formulations of the present invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (flavor-based, typically using sucrose and gum arabic or tragacanth), lyophilized tablets, powders, granules, or solutions or suspensions in aqueous or non-aqueous solutions, or oil-in-water or water-in-oil liquid emulsions, or elixirs or syrups, or pastilles (using inert bases such as gelatin and glycerin, or sucrose and gum arabic), and / or mouthwashes, each containing a predetermined amount of a compound of the present invention as an active ingredient. The compositions or compounds may also be administered as a bolus, electuary, or paste.

[0163] To prepare solid dosage forms for oral administration (such as capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules, and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or gum arabic; (3) humectants, such as glycerol; (4) disintegrants, such as agar, calcium carbonate, The pharmaceutical composition may be mixed with any of the following: potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarders, such as paraffin; (6) absorption enhancers, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin clay and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as modified and unmodified cyclodextrins; and (11) coloring agents. For capsules (including sprinkle capsules and gelatin capsules), the pharmaceutical composition may also contain a buffer. Solid compositions of a similar type may also be employed as fillers for soft and hard-filled gelatin capsules, using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols.

[0164] Tablets may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surfactants, or dispersing agents. Molded tablets may be prepared by molding, in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.

[0165] Tablets and other solid dosage forms of pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills, and granules, can optionally be obtained or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical manufacturing art. They can also be formulated to provide sustained or controlled release of the active ingredient therein, for example, using various ratios of hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres that provide the desired release characteristics. They can also be sterilized, for example, by filtration through a bacteria-retaining filter or by mixing with a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions can also optionally contain opacifying agents, and can be composed to release the active ingredient(s) only, or preferentially, in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

[0166] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophilized solutions for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs.In addition to active ingredients, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, cyclodextrin and its derivatives, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, etc., and mixtures thereof.

[0167] Besides inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming agents, and preservatives.

[0168] Suspensions may contain, in addition to the active compound, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0169] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

[0170] The ointments, pastes, creams, and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0171] Powders and sprays may contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures of these substances. Sprays may additionally contain conventional propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0172] Transdermal patches have the additional advantage of providing controlled delivery of the compound of the present invention to the body.Such dosage forms can be prepared by dissolving or dispersing the active compound in a suitable medium.Absorption enhancers can also be used to increase the flux of the compound across the skin.The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

[0173] The phrases "parenteral administration" and "administering parenterally," as used herein, refer to modes of administration other than enteral and topical administration, generally by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intradural, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents, or sterile powders which can be reconstituted into a sterile injectable solution or dispersion immediately before use.

[0174] Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate, etc. Suitable fluidity can be maintained, for example, by using a coating agent such as lecithin, by maintaining a predetermined particle size in the case of dispersions, or by using surfactants.

[0175] These compositions may also contain auxiliary agents, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like, in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0176] In some cases, in order to prolong the effect of drug, it is desirable to delay the absorption of drug from subcutaneous injection or intramuscular injection.This can be achieved by using the liquid suspension of crystalline or amorphous substance that is poorly soluble in water.Therefore, the absorption rate of drug is determined by its dissolution rate, and can also be determined by the size and crystalline state of crystals.Alternatively, the delayed absorption of drug dosage form administered parenterally can be achieved by dissolving or suspending drug in oil medium.

[0177] Injectable depot dosage forms are prepared by forming microencapsulated matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the drug:polymer ratio and the nature of the particular polymer employed. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0178] For use in the methods of the present invention, the active compound may be administered per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0179] Methods of introduction may also be provided by rechargeable or biodegradable devices. Recently, a variety of sustained-release polymeric devices have been developed and tested in vivo for the controlled delivery of drugs, including protein biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, may be used to form implants for the sustained release of compounds to specific target sites.

[0180] The actual dosage level of the active ingredient in the pharmaceutical composition may be varied to obtain an amount of the active ingredient which is not toxic to the patient and which produces the desired therapeutic effect for a particular patient, composition, and method of administration.

[0181] The selected dosage level will depend on a variety of factors, including the activity of the individual compound or combination of compounds used or their esters, salts, or amides, the route of administration, the duration of administration, the excretion rate of the individual compound(s) used, the duration of treatment, other drugs, compounds, and / or substances used in combination with the individual compound(s) used, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical arts.

[0182] A physician or veterinarian of ordinary skill can easily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can start a dose of the pharmaceutical composition or compound at a level below that required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. A "therapeutically effective amount" refers to a concentration of the compound sufficient to induce the desired therapeutic effect. It is generally understood that the effective amount of a compound varies depending on the subject's weight, sex, age, and medical history. Other factors that affect the effective amount include, but are not limited to, the severity of the patient's symptoms, the disorder being treated, the stability of the compound, and, if necessary, other types of therapeutic agents administered together with the compound of the present invention. Multiple administrations of the drug may deliver a larger total dose. Methods for confirming efficacy and dosage are well known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882 (incorporated herein by reference)).

[0183] Generally, a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose capable of producing a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0184] If desired, the effective daily dose of the active compound may be administered in 1, 2, 3, 4, 5, 6, or more separate doses administered at appropriate intervals throughout the day, optionally in unit dosage forms. In certain embodiments of the invention, the active compound may be administered twice or three times daily. In a preferred embodiment, the active compound is administered once daily.

[0185] Patients receiving this treatment are generally primates, especially humans, and any animal in need of treatment, including other mammals, e.g., horses, cows, pigs, sheep, cats, and dogs, poultry, and pets.

[0186] In certain embodiments, the compounds of the invention may be used alone or may be administered in combination with another type of therapeutic agent.

[0187] The present disclosure includes the use of pharmaceutically acceptable salts of the compounds of the present invention in the compositions and methods of the present invention. In certain embodiments, contemplated salts of the present invention include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkyl ammonium salts. In certain embodiments, contemplated salts of the present invention include, but are not limited to, L-arginine, benethamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the present invention include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts.In certain embodiments, contemplated salts of the present invention include 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptan-1,2-one ... Acid salts of acetic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, mandelic acid, methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid.

[0188] Pharmaceutically acceptable acid addition salts may also exist as various solvates, such as solvates with water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates may also be prepared. The source of such solvates may be that inherent in the solvent of crystallization, preparation, or crystallization, or that accompanying such solvent.

[0189] Wetting agents, emulsifying agents, and lubricating agents, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening agents, flavoring and perfuming agents, preservatives, and antioxidants may also be included in the compositions.

[0190] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbic acid palmitate, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc. [Example]

[0191] The invention will now be generally described, which will be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to be limiting thereof.

[0192] Example 1: Preparation of exemplary compounds of the JGK series General Procedure: JGK series compounds can be prepared using the methods described below or any other suitable method. JGK series compounds are sometimes referred to herein using the prefix JCN. All reactions were routinely carried out under an inert atmosphere of argon. Unless otherwise noted, raw materials were obtained from commercial suppliers and used without purification. All solvents were purified and dried using standard techniques immediately before use. THF and EtO were freshly distilled from sodium and benzophenone. Methylene chloride, toluene, and benzene were purified by refluxing with CaH. Reactions were confirmed by thin-layer chromatography (Kieselgel 60 F254, Merck). Spots were detected by charring after immersion in p-anisaldehyde or phosphomolybdic acid solutions, visually observed under UV light. For aqueous workups, all organic solutions were dried over anhydrous magnesium sulfate, filtered, and then rotary evaporated with a water pump. The crude compound was purified by column chromatography using silica gel (SilicaFlash P60, 230-400 mesh, SiliCycle Inc.). Proton ( 1 H) and carbon ( 13 NMR spectra of α- and β-hydroxybenzoates (C) were obtained. Chemical shifts are reported in ppm using Me4Si or CHCl3 as an internal standard. Splitting patterns are indicated as follows: s, singlet; d, doublet; t, triplet; m, multiplet; and b, broad. High-resolution mass spectrometry data were obtained using a Thermo Fisher Scientific Exactive Plus equipped with an IonSense ID-CUBE DART source.

[0193] Preparation of JGK001 and JGK003 [ka] [JGK001] To a solution of erlotinib (134 mg, 0.3406 mmol) in anhydrous methanol (5.0 mL) was added di-tert-butyl dicarbonate (228 mg, 1.7029 mmol) in one portion at room temperature. After stirring at the same temperature for 48 h, the mixture was concentrated under reduced pressure. The reaction mixture was diluted with H2O (30 mL) and EtOAc (30 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (2 × 30 mL). The combined organic layers were washed successively with H2O and saturated brine, then dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, hexane / EtOAc, 3 / 1) to give JGK001 (156 mg, 73%). 1 H NMR (400 MHz, CDCl3) δ 7.86 (s, 1 H), 7.43 (s, 1 H), 7.20-7.23 (m, 2 H), 7.16 (td, J = 1.2, 7.6 Hz, 1 H), 7.09 (d, J = 8.0 Hz, 1 H), 4.16-4.25 (m, 4 H), 3.80 (t, J = 5.2 Hz, 2 H), 3.77 (t, J = 5.2 Hz, 2 H), 3.45 (s, 3 H), 3.44 (s, 3 H), 3.44 (s, 3 H), 3.03 (s, 1 H), 1.55 (s, 9 H), 1.11 (s, 9 H); 13 C NMR (100 MHz, CDCl3) δ 152.1, 151.5, 149.2, 148.8, 145.7, 142.7, 130.6, 128.9, 127.4, 125.3, 122.6, 121.5, 114.7, 111.4, 108.0, 90.7, 83.7, 83.3, 82.9, 76.8, 70.9, 70.7, 68.8, 68.7, 59.2, 59.1, 55.0, 28.2, 27.3 + Actual measured value 626.3061 [C 33 H 43 Calculated value for N3O9 625.2993].

[0194] [JGK003] To a solution of erlotinib (101 mg, 0.2567 mmol) in absolute ethanol (2.6 mL) was added di-tert-butyl dicarbonate (172 mg, 1.2836 mmol) in one portion at room temperature. After stirring at the same temperature for 48 h, the mixture was concentrated under reduced pressure. The reaction mixture was diluted with HO (30 mL) and EtOAc (30 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (2 × 30 mL). The combined organic layers were washed successively with HO and saturated brine, then dried over anhydrous MgSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, hexane / EtOAc, 3 / 1) to give JGK003 (117 mg, 71%). 1 H NMR (400 MHz, CDCl3) δ 7.86 (s, 1 H), 7.43 (s, 1 H), 7.34 (s, 1 H), 7.21-7.24 (m, 2 H), 7.16 (d, J = 7.6 Hz, 1 H), 7.10 (d, J = 7.6 Hz, 1 H), 4.17-4.25 (m, 4 H), 3.61-3.82 (m, 6 H), 3.46 (s, 3 H), 3.45 (s, 3 H), 3.02 (s, 1 H), 1.55 (s, 9 H), 1.23 (t, J = 6.8 Hz, 3 H), 1.12 (s, 9 H); 13 C NMR (100 MHz, CDCl3) δ 152.0, 151.4, 149.2, 148.9, 145.5, 143.0, 130.8, 128.8, 127.3, 125.3, 122.5, 121.7, 114.7, 111.3, 107.9, 89.3, 83.7, 83.2, 82.8, 76.7, 70.9, 70.7, 68.8, 68.6, 63.0, 59.2, 59.1, 28.2, 27.4, 14.6 + Actual measured value 640.3211 [C 34 H 45 Calculated value for N3O9 639.3150].

[0195] Preparation of JGK002 Solid erlotinib (165 mg, 0.4194 mmol) was added to acetic anhydride (5.0 mL). After heating with stirring at 90 °C (bath temperature) for 3 days, the reaction mixture was cooled to room temperature, neutralized with saturated aqueous NaHCO3 (20 mL), and diluted with EtOAc (20 mL). [ka] The layers were separated and the aqueous layer was extracted with EtOAc (2 × 30 mL). The combined organic layers were washed successively with HO and saturated brine, then dried over anhydrous MgSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, hexane / EtOAc, 1 / 1 to 1 / 3) to give JGK002 (161 mg, 88% isolated yield). 1 H NMR (400 MHz, CDCl3) δ 9.06 (s, 1 H,), 7.45 (t, J = 1.6 Hz, 1 H,), 7.37-7.39 (m, 2 H), 7.36 (s, 1 H), 7.30-7.34 (m, 1 H), 7.15 (s, 1 H), 4.32 (t, J = 4.8 Hz, 2 H), 4.16 (t, J = 4.8 Hz, 2 H), 3.86 (t, J = 4.8 Hz, 2 H), 3.79 (t, J = 4.8 Hz, 2 H), 3.46 (s, 3 H), 3.45 (s, 3 H), 3.06 (s, 1 H), 2.14 (s, 3 H); 13 C NMR (100 MHz, CDCl3) δ 170.5, 158.8, 156.1, 153.5, 151.1, 150.8, 141.0, 130.9, 130.3, 129.3, 127.5, 123.4, 117.2, 107.9, 103.1, 82.4, 78.4, 70.6, 70.3, 68.9, 68.7, 59.3, 59.3, 23.7;HRMS-ESI [M+H] + Actual measured value 436.1811 [C 24 H 25 Calculated value for N3O5: 435.1788].

[0196] Preparation of JGK010, JGK032 - General procedure for substitution with aniline analogues [ka] [Cyclization] To a solution of diol 2 (530 mg, 2.6959 mmol) in DMF (13.5 mL, 0.2 M), potassium carbonate (1490 mg) was added in one portion, followed by the continuous dropwise addition of 1-bromo-2-chloroethane (1.3 mL) at room temperature under Ar. After heating with stirring at 60 °C (bath temperature) for 24 h, the reaction mixture was cooled to room temperature and quenched with HO (50 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (50 mL). The combined organic layers were washed successively with HO and saturated brine, then dried over anhydrous MgSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, hexane / EtOAc, 6 / 1 to 3 / 1) to give fused-chloroquinazoline 3 (404 mg, 67%). 1 H NMR (400 MHz, CDCl3) δ 8.84 (s, 1H), 7.64 (s, 1H), 7.47 (s, 1H), 4.43-4.45 (m, 2H), 4.39-4.42 (m, 2H). [Known compound; Chilin, A. et al. J. Med. Chem. 2010, 53, 1862-1866]

[0197] [JGK010] To a solution of fused-chloroquinazoline 3 (114 mg, 0.5120 mmol) in DMF (2.6 mL) was added 3-chloro-2-fluoroaniline (0.10 mL) dropwise at room temperature. After heating with stirring at 60 °C (bath temperature) for 24 h, the reaction mixture was cooled to room temperature and then diluted with EtO (30.0 mL) to give a white suspension. The resulting white solid was washed successively with EtO (2 × 50 mL) and then collected to give JGK010 (140 mg, 82%). 1H NMR (400 MHz, CDCl3) δ 8.68 (s, 1 H), 8.59 (ddd, J = 3.2, 6.8, 6.8 Hz, 1 H), 7.39 (s, 1 H), 7.34 (s, 1 H), 7.29 (s, 1 H), 7.10-7.18 (m, 2 H), 4.38-4.43 (m, 4 H); 1 H NMR (500 MHz, DMSO-d6) δ 11.78 (s, 1 H), 8.79 (s, 1 H), 8.45 (s, 1 H), 7.62 (t, J = 7.0 Hz, 1 H), 7.50 (t, J = 7.0 Hz, 1 H), 7.43 (s, 1 H), 7.34 (t, J = 8.0 Hz, 1 H), 4.46-4.53 (m, 2 H), 4.40-4.52 (m, 2 H); 13 C NMR (125 MHz, DMSO-d6) δ 159.8, 154.0, 152.2, 149.9, 145.7, 135.2, 129.9, 128.1, 126.4, 125.8, 120.9, 111.3, 108.1, 105.8, 65.5, 64.6;HRMS-ESI [M+H] + Actual measured value 332.0551 [C 16 H 11 Calculated value for ClFN3O2: 331.0518].

[0198] Preparation of JGK005 [ka] To a solution of fused-chloroquinazoline 3 (14 mg, 0.0628 mmol) in CHCN (2.0 mL) was added 3-ethynylaniline (0.05 mL) dropwise at room temperature. After heating at 80 °C (bath temperature) with stirring for 12 h, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, hexane / EtOAc, 3 / 1) to give JGK005 (10 mg, 52%). 11H NMR (400 MHz, DMSO-d6) δ 9.45 (s, 1 H), 8.43 (s, 1 H), 8.04 - 8.05 (m, 2 H), 7.87 - 7.90 (m, 1 H), 7.34 (t, J = 7.9 Hz, 1 H), 7.14 - 7.16 (m, 2 H), 4.35 - 4.39 (m, 4 H), 4.14 (s, 1 H); 13 13C NMR (100 MHz, DMSO-d6) δ 156.8, 153.3, 149.5, 146.5, 144.1, 140.2, 129.3, 126.7, 124.9, 122.7, 122.1, 113.0, 110.4, 108.8, 84.0, 80.9, 64.9, 64.6;HRMS-ESI [M+H] + Found 304.1079 [C 18 H 13 for C15H11N3O2 calcd 303.1002].

[0199] JGK025

Chemical Structure

[0200] JGK026 [ka] The preparation of JGK026 followed the general procedure. JGK026 (22%). 1 H NMR (400 MHz, DMSO-d6) δ 11.09 (s, 1 H), 8.74 (s, 1 H), 8.18 (s, 1 H), 7.39-7.51 (m, 2 H), 7.30 (s, 1 H), 7.23-7.29 (m, 1 H), 4.45-4.49 (m, 2 H), 4.40-4.44 (m, 2 H); 13 C NMR (125 MHz, MeOD) δ 159.8, 158.1 (J = 239.6 Hz), 153.7 (J = 243.2 Hz), 152.1, 150.1, 145.7, 135.7, 126.0, 117.9, 117.8, 116.0, 110.9, 108.2, 106.2, 65.5, 64.6;HRMS-ESI [M+H] + Actual measured value 316.0893 [C 16 H 11 Calculated value for F2N3O2 315.0813].

[0201] JGK027 [ka] The preparation of JGK027 followed the general procedure. JGK027 (6%). 1 H NMR (400 MHz, DMSO-d6) δ 11.23 (bs, 1 H), 8.75 (s, 1 H), 8.29 (s, 1 H), 7.91 (s, 1 H), 7.46-7.55 (m, 1 H), 7.29 (t, J = 8.1 Hz, 2 H), 4.46-4.50 (m, 2 H), 4.40-4.46 (m, 2 H); 13C NMR (125 MHz, MeOD) δ 163.6, 160.7, 160.4, 158.4, 152.5, 151.5, 146.2, 130.6, 115.6, 113.5, 113.4, 111.9, 109.3, 108.2, 66.2, 65.3;HRMS-ESI [M+H] + Actual measured value 316.0889 [C 16 H 11 Calculated value for F2N3O2 315.0813].

[0202] JGK028 [ka] The preparation of JGK028 followed the general procedure. JGK028 (41%). 1 H NMR (500 MHz, MeOD) δ 8.64 (s, 1 H), 8.03 (s, 1 H), 7.31-7.38 (m, 2 H), 7.24-7.31 (m, 2 H), 4.50-4.55 (m, 2 H), 4.44-4.50 (m, 2 H); 13 C NMR (125 MHz, MeOD) δ 160.1, 152.8, 150.9 (J = 245.6 Hz), 149.0, 146.2, 145.9 (J = 249.7 Hz), 134.6, 126.0, 124.0, 123.1, 116.2, 109.8, 107.8, 105.0, 65.2, 64.2;HRMS-ESI [M+H] + Actual measured value 316.0884 [C 16 H 11 Calculated value for F2N3O2 315.0813].

[0203] JGK029 [ka] The preparation of JGK029 followed the general procedure. JGK029 (52%). 1H NMR (500 MHz, MeOD) δ 8.60 (s, 1 H), 7.98 (s, 1 H), 7.29 (s, 1 H), 7.07-7.13 (m, 2 H), 4.50-4.53 (m, 2 H), 4.44-4.48 (m, 2 H); 13 C NMR (125 MHz, DMSO-d6) δ 161.7, 160.3, 158.6, 158.1, 153.2, 150.3, 144.4, 143.7, 117.2, 113.8, 113.0, 112.3, 109.5, 101.5, 65.3, 64.5;HRMS-ESI [M+H] + Actual measured value 334.0794 [C 16 H 10 Calculated value for F3N3O2 333.0719].

[0204] JGK017 [ka] The preparation of JGK017 followed the general procedure. JGK017 (5%). 1 H NMR (500 MHz, CDCl3) δ 8.59 (s, 1 H), 8.15 (d, J = 8.3 Hz, 1 H), 7.49 (t, J = 8.1 Hz, 1 H), 7.38 (s, 1 H), 7.34 (d, J = 7.9 Hz, 1 H), 7.21 (s, 1 H), 4.41-4.42 (m, 2 H), 4.38-4.40 (m, 2 H); 13 C NMR (125 MHz, CDCl3) δ 156.4, 153.2, 149.7, 146.7, 144.5, 138.4, 133.5, 132.2, 128.2, 125.4, 119.9, 119.7, 114.3, 110.4, 105.7, 64.5, 64.3.

[0205] Preparation of JGK004 [ka] Benzoylation: To a cooled (0 °C) solution of diol 2 (205 mg, 1.0428 mmol) in anhydrous CHCl (5.2 mL, 0.2 M), pyridine (0.5 mL) and benzoyl chloride (0.7 mL) were added dropwise in succession under Ar. After stirring at room temperature for 12 h, the reaction mixture was quenched with saturated aqueous NHCl (20 mL) and then diluted with CHCl (20 mL). The layers were separated, and the aqueous layer was extracted with CHCl (2 × 50 mL). The combined organic layers were washed successively with HO and saturated brine, then dried over anhydrous MgSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, hexane / EtOAc, 10 / 1) to give benzoylchloroquinazoline 2-(2) (220 mg, 52%). 1 H NMR (400 MHz, CDCl3) δ 9.07 (s, 1 H), 8.31 (s, 1 H), 8.16 (s, 1 H), 8.04-8.07 (m, 4 H), 7.53-7.58 (m, 2 H), 7.34-7.39 (m, 4 H).

[0206] [JGK004] To a solution of benzoylchloroquinazoline 2-(2) (180 mg, 0.444 mmol) in CH3CN (3.0 mL) was added 3-chloro-2-fluoroaniline (0.06 mL, 0.533 mmol) dropwise at room temperature. After heating with stirring at 80 °C (bath temperature) for 15 h, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, hexane / EtOAc, 3 / 1) to give JGK004 (109 mg, 48%). 1 H NMR (400 MHz, CDCl3) δ 8.85 (s, 1 H), 8.48-8.53 (m, 1 H), 8.08 (t, J = 7.2 Hz, 4 H), 7.99 (d, J = 3.2 Hz, 2 H), 7.51-7.59 (m, 3 H), 7.39 (dd, J = 8.4, 16.0 Hz, 4 H), 7.16-7.21 (m, 2 H); 13C NMR (125 MHz, CDCl3) δ 164.2, 163.7, 156.6, 155.1, 150.6, 149.1, 148.6, 147.5, 142.1, 134.1, 134.1, 130.3, 130.2, 128.6, 128.6, 128.1, 128.0, 127.9, 127.8, 125.3, 124.6, 124.5, 122.9, 121.6, 121.0, 120.9, 114.4, 113.3 + Actual measured value 514.0963 [C 28 H 17 Calculated value for ClFN3O4: 513.0886].

[0207] Preparation of JGK006 [ka] To a solution of benzoylchloroquinazoline 2-(2) (100 mg, 0.247 mmol) in CHCN (3.0 mL) was added 3-ethynylaniline (0.05 mL, 0.430 mmol) dropwise at room temperature. After heating with stirring at 50 °C (bath temperature) for 24 h, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, hexane / EtOAc, 4 / 1) to give JGK006 (48 mg, 40%). 1 H NMR (400 MHz, CDCl3) δ 8.71 (s, 1 H), 8.03 (d, J = 8.0 Hz, 2 H), 7.96 (s, 1 H), 7.90-7.95 (m, 2 H), 7.79 (s, 1 H), 7.62-7.75 (m, 3 H), 7.55 (t, J = 7.3 Hz, 1 H), 7.48 (t, J = 7.5 Hz, 1 H), 7.37 (t, J = 7.4 Hz, 2 H), 7.22-7.31 (m, 4 H), 3.04 (s, 1 H); 13C NMR (100 MHz, CDCl3) δ 164.8, 163.9, 156.7, 155.3, 148.9, 146.9, 141.3, 138.1, 134.1, 134.0, 130.3, 130.1, 128.9, 128.6, 128.5, 128.1, 128.0, 127.9, 124.8, 122.7, 122.4, 122.1, 115.1, 113.1, 83.3;HRMS-ESI [M+H] + Actual measured value 486.1443 [C 30 H 19 Calculated value for N3O4: 485.1370

[0208] Preparation of JGK032 [ka] A 1.0 M hydrogen chloride solution was prepared by adding hydrogen chloride solution (0.1 mL, 4.0 M in dioxane, 0.4 mmol) to THF (0.3 mL) at room temperature. To a solution of JGK010 (6.1 mg, 0.01839 mmol) in MeOH, the hydrogen chloride solution (0.030 mL, 0.030 mmol) prepared above was added dropwise at room temperature. After stirring at the same temperature for 10 seconds, the reaction mixture was concentrated under reduced pressure to give JGK032 (6.7 mg, 99%). 1 H NMR (500 MHz, DMSO-d6) δ 11.63 (s, 1 H), 8.81 (s, 1 H), 8.36 (s, 1 H), 7.63 (ddd, J = 1.6, 6.9, 8.3 Hz, 1 H), 7.39 (s, 1 H), 7.35 (ddd, J = 1.1, 8.1, 16.2 Hz, 1 H), 4.49-4.51 (m, 2 H), 4.43-4.45 (m, 2 H).

[0209] Preparation of JGK012 [ka] To solid JGK010 (39 mg, 0.1176 mmol) was added acetic anhydride (5.0 mL). After heating at 80 °C (bath temperature) with stirring for 12 h, the reaction mixture was cooled to room temperature, neutralized with saturated aqueous NaHCO (20 mL), and diluted with EtOAc (20 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (2 × 30 mL). The combined organic layers were washed successively with HO and saturated brine, then dried over anhydrous MgSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, hexane / EtOAc, 2 / 1 to 1 / 1) to give JGK012 (37 mg, 84% isolated yield). 1 H NMR (400 MHz, CDCl3) δ 9.01 (s, 1 H), 7.49 (s, 1 H), 7.44 (s, 1 H), 7.31-7.41 (m, 2 H), 7.09 (t, J = 8.0 Hz, 1 H), 4.41-4.43 (m, 2 H), 4.37-4.40 (m, 2 H), 2.15 (s, 3 H); 13 C NMR (125 MHz, CDCl3) δ 170.2, 159.2, 153.3, 151.3, 149.7, 145.8, 130.6, 129.8, 129.7, 124.7, 122.5, 122.4, 117.7, 113.6, 109.2, 64.5, 64.2, 22.9;HRMS-ESI [M+H] + Actual measured value 374.0701 [C 18 H 13 Calculated value for ClFN3O3 373.0623].

[0210] Preparation of JGK015 [ka] To a solution of L-amino acid analog A (227 mg, 0.7712 mmol) in DMF (3.0 mL) was added fused-chloroquinazoline 3 (117 mg, 0.5932 mmol) in one portion at room temperature. After heating with stirring at 35 °C (bath temperature) for 12 h, the reaction mixture was cooled to room temperature and then diluted with saturated brine (30.0 mL) and EtOAc (30.0 mL) to give a yellow suspension. The layers were separated and the aqueous layer was extracted with EtOAc (2 × 50 mL). The combined organic layers were concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, CHCl / MeOH, 40 / 1 to 10 / 1) to give JGK015 (261 mg, 92%). 1 H NMR (500 MHz, CDCl3) δ 8.57 (s, 1 H), 7.64 (s, 1 H), 7.61 (d, J = 8.0 Hz, 2 H), 7.37 (s, 1 H), 7.27 (s, 1 H), 7.08 (d, J = 8.5 Hz, 2 H), 5.09 (d, J = 7.5 Hz, 1 H), 4.54 (dd, J = 6.0, 13.5 Hz, 1 H), 4.31-4.33 (m, 2 H), 4.27-4.29 (m, 2 H), 3.68 (s, 3 H), 3.06 (dd, J = 5.6, 14.0 Hz, 1 H), 3.00 (dd, J = 6.1, 13.8 Hz, 1 H), 1.39 (s, 9 H); 13 C NMR (125 MHz, CDCl3) δ 172.4, 156.5, 155.2, 153.6, 149.1, 146.3, 143.8, 137.6, 131.6, 129.7, 121.7, 113.8, 110.3, 106.6, 80.0, 64.4, 64.2, 54.4, 52.2, 37.6, 28.3;HRMS-ESI [M+H] + Actual measured value 481.2082 [C 25 H 28 Calculated value for N4O6 480.2003].

[0211] Preparation of JGK016 and JGK023 [ka] [JGK016 (Boc deprotection)] To a solution of JGK015 (121 mg, 0.251 mmol) in anhydrous CHCl (5 mL, 0.05 M) was added trifluoroacetic acid (1.0 mL) dropwise at room temperature. After stirring at the same temperature for 5 h, the reaction mixture was quenched with saturated aqueous NaHCO (20 mL) and then diluted with CHCl (20 mL). The layers were separated, and the aqueous layer was extracted with CHCl (2 × 30 mL). The combined organic layer was washed successively with H0 and saturated brine, then dried over anhydrous MgSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, CHCl / MeOH, 20 / 1) to give JGK016 (74 mg, 77%). 1 H NMR (400 MHz, DMSO-d6) δ 10.5 (s, 1 H), 8.68 (s, 1 H), 8.43 (s, 2 H), 8.20 (s, 1 H), 7.68 (d, J = 8.4 Hz, 2 H), 7.26 (d, J = 8.4 Hz, 2 H), 7.23 (s, 1 H), 4.44-4.46 (m, 2 H), 4.39-4.41 (m, 2 H), 3.68 (s, 3 H), 3.03-3.13 (m, 2 H); 1 H NMR (400 MHz, CD3OD) δ 8.26 (s, 1 H), 7.73 (s, 1 H), 7.60 (d, J = 8.4 Hz, 2 H), 7.17 (d, J = 8.4 Hz, 2 H), 7.08 (s, 1 H), 4.31-4.35 (m, 4 H), 3.72 (t, J = 6.6 Hz, 1 H), 3.68 (s, 3 H), 3.27-3.29 (m, 1 H), 3.01 (dd, J = 5.9, 13.6 Hz, 1 H), 2.89 (dd, J = 7.0, 13.5 Hz, 1 H); 13C NMR (100 MHz, CD3OD) δ 174.4, 157.4, 152.6, 149.7, 145.0, 144.2, 137.5, 132.8, 129.2, 122.8, 122.3, 111.5, 110.1, 107.9, 64.5, 64.1, 55.2, 51.0, 39.5;HRMS-ESI [M+H] + Actual measured value 381.1553 [C 20 H 20 Calculated value for N4O4 380.1479].

[0212] [JGK023 (hydrolyzed)] To a cooled (0 °C) solution of JGK016 (42 mg, 0.1104 mmol) in THF / HO (3:1, 4.0 mL total) was added lithium hydroxide (14 mg) in one portion. After stirring at room temperature for 2 h, the reaction mixture was neutralized with 1 N HCl and then diluted with EtOAc (20 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (100 mL). The combined organic layers were washed successively with HO and saturated brine, then dried over anhydrous MgSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, CHCl / MeOH, 30 / 1 to 15 / 1) to give JGK023 (25 mg, 62%). 1 H NMR (400 MHz, CDCl3) δ 8.62 (s, 1 H), 8.12 (s, 1 H), 7.71 (d, J = 8.4 Hz, 2 H), 7.40 (d, J = 8.4 Hz, 2 H), 7.24 (s, 1 H), 4.48-4.50 (m, 2 H), 4.42-4.44 (m, 2 H), 4.28 (t, J = 6.8 Hz, 1 H), 3.32-3.37 (m, 1 H), 3.20 (dd, J = 7.6, 14.8 Hz, 1 H); 13C NMR (125 MHz, CDCl3) δ 169.7, 159.1, 152.4, 148.8, 146.0, 136.1, 134.1, 133.1, 129.7, 129.7, 124.8, 124.8, 110.0, 108.1, 104.9, 65.2, 64.2, 53.6, 35.4;HRMS-ESI [M+H] + Actual measured value 367.1334 [C 19 H 18 Calculated value for N4O4 366.1322].

[0213] Preparation of JGK020 [ka] To a solution of chloroquinazoline 2 (104 mg, 0.5294 mmol) in isopropyl alcohol (5.3 mL) was added amino acid (187 mg) dropwise at room temperature. After heating with stirring at 50 °C (bath temperature) for 12 h, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, hexane / EtOAc, 5 / 1 to 3 / 1) to give JGK020 (128 mg, 53%). 1 H NMR (400 MHz, DMSO-d6) δ 10.68 (s, 1 H), 10.22 (br, 1 H), 8.64 (s, 1 H), 7.91 (s, 1 H), 7.53 (d, J = 8.4 Hz, 2 H), 7.26-7.31 (m, 4 H), 4.12-4.18 (m, 1 H), 3.59 (s, 3 H), 2.98 (dd, J = 5.2, 14.0 Hz, 1 H), 2.84 (dd, J = 10.0, 13.2 Hz, 1 H), 1.30 (s, 9 H); 13 C NMR (125 MHz, DMSO-d6) δ 173.0, 158.2, 155.9, 155.6, 148.7, 148.4, 136.1, 135.8, 129.7, 124.7, 107.6, 107.3, 103.3, 78.8, 55.7, 52.3, 36.3, 28.6;HRMS-ESI [M+H]+ Actual measured value 455.1920 [C 23 H 26 Calculated value for N4O6: 454.1846].

[0214] JGK014 [ka] JGK014 (26%). 1 H NMR (400 MHz, CDCl3) δ 8.62 (s, 1 H), 7.66 (d, J = 8.4 Hz, 2 H), 7.35 (s, 1 H), 7.16 (d, J = 8.4 Hz, 2 H), 4.99 (d, J = 7.6 Hz, 1 H), 4.56-4.62 (m, 1 H), 4.36-4.42 (m, 4 H), 3.73 (s, 3 H), 3.03-3.15 (m, 2 H), 1.43 (s, 9 H); 13 C NMR (125 MHz, CDCl3) δ 172.4, 156.5, 155.2, 153.6, 149.1, 146.3, 143.8, 137.6, 131.6, 129.7, 121.7, 113.8, 110.3, 106.6, 80.0, 64.4, 64.2, 54.4, 52.2, 37.6, 28.3;HRMS-ESI [M+H] + Actual measured value 481.2080 [C 25 H 28 Calculated value for N4O6 480.2003].

[0215] JGK021 [ka] JGK021 was prepared according to the synthesis method for JGK023. 1H NMR (400 MHz, CDCl3) δ 8.62 (s, 1 H), 8.12 (s, 1 H), 7.71 (d, J = 8.4 Hz, 2 H), 7.40 (d, J = 8.4 Hz, 2 H), 7.24 (s, 1 H), 4.48-4.50 (m, 2 H), 4.42-4.44 (m, 2 H), 4.28 (t, J = 6.8 Hz, 1 H), 3.32-3.37 (m, 1 H), 3.20 (dd, J = 7.6, 14.8 Hz, 1 H); 13 C NMR (125 MHz, CDCl3) δ 169.7, 159.1, 152.4, 148.8, 146.0, 136.1, 134.1, 133.1, 129.7, 129.7, 124.8, 124.8, 110.0, 108.1, 104.9, 65.2, 64.2, 53.6, 35.4;HRMS-ESI [M+H] + Actual measured value 367.1347 [C 19 H 18 Calculated value for N4O4 366.1322].

[0216] Preparation of JGK022 [ka] To a solution of diol X (121 mg, 0.6155 mmol) in DMF (3.0 mL) was added 3-chloro-2-fluoroaniline (0.14 mL) dropwise at room temperature. After heating with stirring at 60 °C (bath temperature) for 3 days, the reaction mixture was cooled to room temperature and then diluted with EtO (30.0 mL) to give a white suspension. The resulting white solid was washed successively with EtO (3 × 50 mL) and CHCl (2 × 30 mL) before being collected to give JGK022 (132 mg, 70%). 1H NMR (400 MHz, DMSO-d6) δ 11.16 (br, 1 H), 10.43 (br, 1 H), 8.68 (s, 1 H), 7.91 (s, 1 H), 7.58 (t, J = 7.1 Hz, 1 H), 7.48 (t, J = 6.8 Hz, 1 H), 7.40 (s, 1 H), 7.30 (t, J = 8.1 Hz, 1 H); 13 C NMR (125 MHz, DMSO-d6) δ 159.1, 156.4, 154.1, 152.1, 149.1, 148.3, 135.1, 129.7, 128.1, 126.8, 125.7, 120.8, 107.4, 106.9, 102.9;HRMS-ESI [M+H] + Actual measured value 306.0437 [C 14 Calculated value for H9ClFN3O2 305.0361].

[0217] Preparation of JGK018 [ka] To a solution of 11 (500 mg, 2.134 mmol) in [chlorinated]thionyl chloride (7.5 mL, 0.28 M) was added dropwise dimethylformamide (0.15 mL). After heating with stirring at 80 °C (bath temperature) for 2 h, the reaction mixture was cooled to room temperature and then concentrated under reduced pressure. The residue was washed successively with EtO (200 mL) and used immediately in the next step.

[0218] To a solution of the chloroquinazoline 12 prepared above in anhydrous DMF (11 mL, 0.2 M) was added 3-chloro-2-fluoroaniline (0.50 mL, 4.548 mmol) dropwise under Ar at room temperature. After stirring at the same temperature for 1 h, the reaction mixture was diluted with EtO (100.0 mL) to give a white suspension. The resulting white solid was washed successively with EtO (2 × 50 mL) and then collected to give JGK018 (525 mg, 68%). Spectroscopic data were consistent with Zhang, X. et al. J. Med. Chem. 2015, 58, 8200-8215.

[0219] Preparation of 13 [ka] [Acetyl deprotection] To JGK018 (550 mg, 1.520 mmol) was added dropwise ammonia solution (8.0 mL, 7N in methanol). After heating in a sealed tube with stirring at 50 °C (bath temperature) for 2 h, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting white solid was washed successively with EtO (2 × 50 mL) and then collected to give 13 (394 mg, 81%). The obtained spectroscopic data were consistent with those of Zhang, X. et al. J. Med. Chem. 2015, 58, 8200-8215.

[0220] Preparation of 14 [ka] To a cooled (0 °C) solution of 13 (113 mg, 0.353 mmol) in anhydrous DMF (2 mL) under Ar, triethylamine (0.25 mL, 1.767 mmol) was added dropwise, followed by di-tert-butyl dicarbonate (62 mg, 0.459 mmol) in anhydrous DMF (2 mL). After stirring at room temperature for 3 days, the reaction mixture was quenched with saturated aqueous HO (10 mL) and diluted with EtOAc (10 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (2 × 50 mL). The combined organic layers were washed successively with HO and saturated brine, then dried over anhydrous MgSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, hexane / EtOAc, 5 / 1 to 3 / 1) to give 14 (42 mg, 28% isolated yield). 1 H NMR (400 MHz, CDCl3) δ 8.69 (s, 1 H), 8.39-8.45 (m, 1 H), 7.64 (s, 1 H), 7.44 (s, 1 H), 7.11-7.16 (m, 2 H), 3.90 (s, 3 H), 1.59 (s, 9 H); 13C NMR (125 MHz, CDCl3) δ 156.2 (J = 39.5 Hz), 154.9, 151.3, 150.3, 149.5 (J = 224.1 Hz), 140.4, 128.1 (J = 9.6 Hz), 124.8, 124.4 (J = 4.8 Hz), 121.5, 120.8 (J = 51.2 Hz), 113.5, 109.0, 108.8, 84.6, 56.2, 27.6;

[0221] Preparation of C [ka] A in anhydrous CH2Cl2 (2 mL) 1 To a solution of (56 mg, 0.1904 mmol) under Ar at room temperature, triethylamine (0.08 mL, 0.5712 mmol) was added dropwise, followed by the addition of chloroacetyl chloride (0.05 mL, 0.6286 mmol). After stirring at the same temperature for 1 h, the reaction mixture was quenched with saturated aqueous NH4Cl (20 mL) and then diluted with CHCl (20 mL). The layers were separated, and the aqueous layer was extracted with CHCl (2 × 30 mL). The combined organic layers were washed successively with HO and saturated brine, then dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude product was used in the next step without further purification.

[0222] Preparation of JGK031 [ka] [Alkylation] To a cooled (0 °C) solution of 14 (44 mg, 0.1058 mmol) in DMF (2.0 mL) at room temperature, potassium carbonate (73 mg, 0.528 mmol) was added in one portion, followed by the dropwise addition of the above-prepared C (0.1904 mmol) in DMF (2.0 mL). After heating with stirring at 40 °C (bath temperature) for 3 days, the reaction mixture was quenched with HO (10 mL) and diluted with EtOAc (10 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (2 × 50 mL). The combined organic layer was washed successively with HO and saturated brine, then dried over anhydrous MgSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, hexane / EtOAc, 100 / 1 to 30 / 1) to give the alkylated product 14-(2) (43 mg, 55% isolated yield). 1 H NMR (400 MHz, CDCl3) δ 8.16 (s, 1 H), 7.48 (d, J = 8.4 Hz, 2 H), 7.44 (s, 1 H), 6.98-7.13 (m, 5 H), 6.34 (m, 1 H), 4.95 (d, J = 7.4 Hz, 1 H), 4.54 (d, J = 6.5 Hz, 1 H), 4.48 (s, 2 H), 3.69 (s, 6 H), 2.95-3.13 (m, 2 H), 1.53 (s, 9 H), 1.40 (s, 9 H).

[0223] [Deprotection] To a solution of the alkylated product 14-(2) (26 mg, 0.0348 mmol) in anhydrous MeOH (5.0 mL), 0.5 N hydrochloride solution (0.5 mL) was added dropwise. After stirring at the same temperature for 24 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (reverse-phase silica gel, MeOH or MeOH / HO, 10 / 1) to give JGK031 (12 mg, 61%). 1H NMR (400 MHz, MeOD) δ 8.20 (s, 1 H), 7.67 (s, 1 H), 7.49 (t, J = 7.9 Hz, 2 H), 7.24-7.30 (m, 1 H), 7.11-7.21 (m, 4 H), 3.94 (s, 3 H), 3.59 (s, 3 H), 2.83-3.01 (m, 2 H);HRMS-ESI [M+H] + Actual measured value 554.1631 [C 27 H 25 Calculated value for ClFN5O5: 553.1522].

[0224] Preparation of JGK033 [ka] To a solution of JGK031 (5 mg, 0.009026 mmol) in anhydrous THF (6 mL) and HO (2 mL) was added lithium hydroxide·HO (3 mg) in one portion. After stirring at room temperature for 2 h, the reaction mixture was neutralized with 1N hydrochloride solution and then concentrated under reduced pressure. The residue was purified by column chromatography (reverse-phase silica gel, MeOH / HO, 5 / 1) to give JGK033 (4.4 mg, 90%). 1 H NMR (400 MHz, MeOD) δ 7.16 (s, 1 H), 6.32 (s, 1 H), 6.03 (d, J = 8.2 Hz, 2 H), 5.89-5.98 (m, 2 H), 5.59-5.79 (m, 4 H), 4.00 (s, 2 H), 2.51 (s, 3 H), 2.46 (t, J = 5.6 Hz, 1 H); 13 C NMR (125 MHz, MeOD) δ 163.9, 159.4, 157.2, 154.3, 152.1, 149.5, 137.1, 135.4, 129.7, 126.9, 124.6, 121.5, 120.3, 108.0, 106.9, 97.8, 56.6, 53.5, 35.6;HRMS-ESI [M+H] + Actual measured value 540.1435 [C 26 H 23Calculated value for ClFN5O5: 539.1366].

[0225] Preparation of JGK008 [ka] To a solution of lapatinib (326 mg, 0.561 mmol) in anhydrous MeOH (5.6 mL) and CHCl (5.6 mL), di-tert-butyl dicarbonate (378 mg, 2.819 mmol) was added dropwise in one portion under Ar. After stirring at room temperature for 2 days, the reaction mixture was quenched with saturated aqueous HO (10 mL) and then diluted with CHCl (10 mL). The layers were separated, and the aqueous layer was extracted with CHCl (5 × 50 mL). The combined organic layers were washed successively with HO and saturated brine, then dried over anhydrous MgSO, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, hexane / EtOAc, 3 / 1 to 1 / 1) to give JGK008 (119 mg, 31% isolated yield). 1 H NMR (500 MHz, CDCl3) δ 8.71 (bs, 1 H), 8.64 (s, 1 H), 8.44 (s, 1 H), 7.85-7.95 (m, 3 H), 7.68 (d, J = 7.8 Hz, 1 H), 7.32-7.37 (m, 1 H), 7.21 (dd, J = 8.4, 10.8 Hz, 2 H), 6.98-7.03 (m, 1 H), 6.96 (d, J = 8.9 Hz, 1 H), 6.39 (d, J = 47.1 Hz, 1 H), 5.13 (s, 2 H), 4.53 (d, J = 32.2 Hz, 2 H), 3.99 (t, J = 7.3 Hz, 2 H), 3.39 (d, J = 66.1 Hz, 2 H), 2.89 (s, 3 H), 1.49 (s, 9 H); 13C NMR (125 MHz, CDCl3) δ 163.9, 162.0, 158.0, 154.2, 152.7, 151.7, 151.0, 139.1 (d, J = 7.3 Hz), 132.4, 130.1 (d, J = 8.1 Hz), 129.1, 128.6, 128.2, 125.1, 123.1, 122.4, 122.3, 115.4, 114.9 (d, J = 21.0 Hz), 114.1 (d, J = 13.9 Hz), 113.9, 111.5, 110.9, 107.7, 81.3, 70.9, 45.0, 43.6, 42.3, 41.4, 41.2, 28.4;HRMS-ESI [M+H] + Actual measured value 681.1946 [C 34 H 34 Calculated value for ClFN4O6S 680.1866].

[0226] Preparation of JGK011 [ka] To solid lapatinib (68 mg, 0.353 mmol) was added acetic anhydride (5.0 mL) under Ar. After stirring at room temperature for 2 days, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, hexane / EtOAc, 3 / 1 to 1 / 3) to give JGK002 (48 mg, 62% isolated yield). 11H NMR (500 MHz, CDCl3) δ 9.18 (s, 1 H), 8.10 - 8.17 (m, 3 H), 7.50 (d, J = 2.5 Hz, 1 H), 7.27 - 7.36 (m, 2 H), 7.18 (dd, J = 7.6, 13.8 Hz, 2 H), 6.97 - 7.03 (m, 2 H), 6.79 (d, J = 3.3 Hz, 1 H), 6.44 (d, J = 3.3 Hz, 1 H), 5.14 (s, 2 H), 4.66 (s, 2 H), 3.86 (t, J = 6.6 Hz, 2 H), 3.30 (t, J = 6.6 Hz, 2 H), 2.95 (s, 3 H), 2.33 (s, 3 H), 2.23 (s, 3 H); 13 13C NMR (125 MHz, CDCl3) δ 171.4, 171.2, 163.9, 162.2, 162.0, 154.0, 153.7, 152.5, 151.0, 138.5 (J = 7.3 Hz), 134.2, 130.8, 130.3, 130.2, 129.9, 129.1, 127.4, 123.9, 122.4 (J = 2.9 Hz), 121.8, 118.0, 115.1, 115.0, 114.0 (J = 5.2 Hz), 113.8, 111.1, 108.9, 70.1 (J = 1.7 Hz), 52.3, 47.0, 41.4, 40.7, 23.7, 21.8; HRMS-ESI [M+H] + Observed 665.1628 [C 33 H 30 for ClFN4O6S calcd 664.1553].

[0227] Example 2: Preparation of Further Exemplary Compounds of the JGK Series General Chemical Information All chemicals, reagents, and solvents, when available, were purchased from commercial sources and used as received. When necessary, reagents and solvents were purified and dried by standard methods. Air-sensitive and moisture-sensitive reactions were carried out in oven-dried glassware under an inert atmosphere of argon. Microwave-irradiated reactions were carried out in a single-mode reactor, a CEM Discover microwave synthesizer. Room-temperature reactions were carried out at ambient temperature (approximately 23 °C). All reactions were carried out on pre-coated Merck 60 F plates, with spots visualized using UV light (λ = 254, 365 nm) or alkaline KMnO4 solution. 254 Thin layer chromatography (TLC) was performed using silica gel plates. Flash column chromatography (FC) was performed using SiO260 (particle size 0.040-0.063 mm, 230-400 mesh). Concentration under reduced pressure (vacuum) was performed by rotary evaporation at 25-50°C. The purified compounds were further dried under high vacuum or in a desiccator. The yields corresponded to the purified compounds and were not further optimized. Proton nuclear magnetic resonance ( 1 H NMR spectra were recorded using a Bruker spectrometer operating at 300, 400, or 500 MHz. Carbon NMR ( 13 C NMR spectra were recorded using a Bruker spectrometer (either 400 or 500 MHz). NMR chemical shifts (δ ppm) were referenced to the residual solvent signal. 1 H NMR data are reported as follows: chemical shift (ppm); multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet / complex pattern, td = triple doublet, ddd = double doublet, br = broad signal); coupling constant (J) (Hz), integration. 13C NMR spectral data are reported based on chemical shifts and, where applicable, coupling constants. High-resolution mass spectrometry (HRMS) spectra were recorded using a Thermo Fisher Scientific Exactive Plus equipped with an IonSense ID-CUBE DART source mass spectrometer. Compounds 4-chloro-7,8-dihydro[1,4]dioxino[2,3-g]quinazoline (1), 4-chloroquinazoline-6,7-diol (2), and JGK010 were prepared as previously reported.

[0228] General Procedure A for the Synthesis of 4-Anilinoquinazoline Compounds JGK035-JGK041 and JKG043 A mixture of 4-chloroquinazoline (1 equiv.) in iPrOH (0.1–0.3 M) was treated with aniline (1 equiv.), and the mixture was heated under microwave irradiation (60 W) at 80 °C for 15–20 min. The mixture was cooled to 23 °C, treated with additional aniline (1 equiv.), and again subjected to microwave irradiation (80 °C, 60 W, 15–20 min). The mixture was either concentrated under reduced pressure or precipitated, and 4-anilinoquinazoline hydrochloride was isolated by filtration (washed with cold iPrOH). The residue was suspended in saturated aqueous NaHCO3 and extracted with CHCl2 (3x). The combined organic extracts were washed with water and brine, then dried (NaSO4), filtered, and concentrated. Purification by FC (eluting with a gradient of CHCl2 / EtOAc or hexane / EtOAc) typically afforded the desired product as a white to off-white or pale yellow solid.

[0229] N-(2-Fluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK035). [ka] Compound JGK035 was prepared from 4-chloroquinazoline 1 (51 mg, 0.23 mmol) and 2-fluoroaniline (40 μL, 0.48 mmol) in iPrOH (1.5 mL) according to general procedure A. FC (CHCl / EtOAc 10:1 to 10:4) afforded JGK035 (56 mg, 82%) as a white solid. 1 H NMR (500 MHz, CDCl3): δ 8.68 (s, 1H), 8.64 (td, J = 8.2, 1.7 Hz, 1H), 7.38 (s, 1H), 7.36 (br, 1H), 7.31 (s, 1H), 7.22 (t, J = 7.5 Hz, 1H), 7.17 (ddd, J = 11.2, 8.3, 1.5 Hz, 1H), 7.10 - 7.05 (m, 1H), 4.44 - 4.37 ppm (m, 4H). 13 C NMR (126 MHz, CDCl3): δ 156.08, 153.60, 153.50 (d, J = 242.7 Hz), 149.52, 146.65, 144.34, 127.31 (d, J = 9.5 Hz), 124.66 (d, J = 3.7 HRMS (DART): m / z [M - H] - C 16 H 11 FN3O2 - , calculated value 296.0841; measured value 296.0841.

[0230] 4-chloro(7,7,8,8- 2 H4)-7,8-Dihydro[1,4]dioxino[2,3-g]quinazoline (3). [ka] A solution of compound 2 (193 mg, 0.98 mmol) in dry DMF (4.8 mL) was treated with Cs2CO3 (788 mg, 2.42 mmol) and stirred for 5 min to give 1-bromo-2-chloro( 2 H4) The mixture was treated dropwise with ethane (270 μL, 3.16 mmol). The mixture was stirred at 23 °C for 1 h and then at 70 °C for 18 h. After the mixture was cooled to 23 °C, all volatiles were removed under reduced pressure. The residue was dissolved in CHCl (40 mL), then washed with water (2 × 13 mL), brine (13 mL), dried (NaSO), filtered, and evaporated. Purification using FC (CHCl / EtOAc 1:0 to 10:1.5) afforded the title compound 3 (109 mg, 49%) as a white fluffy solid. 1 H NMR (400 MHz, CDCl3): δ 8.84 (s, 1H), 7.64 (s, 1H), 7.47 ppm (s, 1H). 13 C NMR (101 MHz, CDCl3): δ 160.19, 152.52, 151.54, 147.93, 146.06, 120.10, 113.72, 110.83 ppm (two upfield carbons not observed). HRMS (DART): m / z [M + H] + C 10 H4D4ClN2O2 + Calculated value: 227.0520; measured value: 227.0516.

[0231] N-(3-chloro-2-fluorophenyl)(7,7,8,8- 2 H4)-7,8-Dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK036). [ka] Compound JGK036 was prepared from 4-chloroquinazoline 3 (55 mg, 0.24 mmol) and 3-chloro-2-fluoroaniline (52 μL, 0.47 mmol) in iPrOH (1.2 mL) according to general procedure A. JGK036·HCl was isolated from the crude reaction mixture by filtration, and after basification and extraction, pure JGK036 (67 mg, 82%) was obtained as a pale yellow solid. 1 H NMR (500 MHz, DMSO-d6): δ 9.62 (s, 1H), 8.34 (s, 1H), 7.93 (s, 1H), 7.53 - 7.43 (m, 2H), 7.27 (td, J = 8.1, 1.3 Hz, 1H), 7.19 ppm (s, 1H). 13 C NMR (126 MHz, DMSO-d6): δ 157.17, 153.10, 152.45 (d, J = 249.2 Hz), 149.28, 146.04, 143.68, 128.21 (d, J = 12.0 Hz), 127.27, 127.03, 124.87 (d, J = 4.7 Hz), 120.11 (d, J = 16.7 Hz), 112.48, 109.64, 108.35, 63.50 (m, 2C's). HRMS (DART): m / z [M + H] + C 16 H8D4ClFN3O2 + Calculated value: 336.0848; measured value: 336.0841.

[0232] N-(3-Bromo-2-fluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK037). [ka] Compound JGK037 was prepared from 4-chloroquinazoline 1 (100 mg, 0.45 mmol) and 3-bromo-2-fluoroaniline (100 μL, 0.89 mmol) in iPrOH (1.5 mL) according to general procedure A. FC (CHCl / EtOAc 10:0 to 10:3) afforded JGK037 (150 mg, 89%) as a pale yellow solid. 1 H NMR (500 MHz, CDCl3): δ 8.68 (s, 1H), 8.65 (ddd, J = 8.3, 7.4, 1.5 Hz, 1H), 7.39 (s, 1H), 7.35 (br, 1H), 7.29 (s, 1H), 7.29 - 7.24 (m, 1H), 7.11 (td, J = 8.2, 1.6 Hz, 1H), 4.44 - 4.38 ppm (m, 4H). 13 C NMR (126 MHz, CDCl3): δ 155.89, 153.37, 150.15 (d, J = 242.2 Hz), 149.70, 146.75, 144.53, 128.65 (d, J = 10.5 Hz), 127.24, 125.31 (d, J = 4.7 Hz), 121.79, 114.53, 110.59, 108.59 (d, J = 19.4 Hz), 105.93, 64.70, 64.51 ppm. HRMS (DART): m / z [M - H] - C 16 H 10 BrFN3O2 - Calculated value: 373.9946; Measured value: 373.9946.

[0233] N-{2-fluoro-3-[(triethylsilyl)ethynyl]phenyl}-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (4). [ka] A single dram vial was charged with JGK010 (75 mg, 0.23 mmol), XPhos (19.7 mg, 0.041 mmol), Cs2CO3 (195 mg, 0.60 mmol), and [PdCl2·(MeCN)2] (3.6 mg, 0.014 mmol). The vial was evacuated and backfilled with argon (repeated at least twice). Dry acetonitrile (1 mL) was added, and the orange-yellow suspension was stirred at 23 °C for 25 min before ethynyltriethylsilane (150 μL, 0.84 mmol) was injected. The tube was sealed, and the reaction mixture was stirred at 95 °C in a preheated oil bath for 3.5 h. The suspension was allowed to reach 23 °C before being diluted with EtOAc, filtered through a silica gel pad (rinsing with EtOAc), and evaporated. Purification using FC (SiO2, hexane / EtOAc 8:2 to 4:6) afforded the title compound 4 (48 mg, 49%) as a yellow foamy solid. 1 H NMR (500 MHz, CDCl3): δ 8.681 (td, J = 8.1, 1.9 Hz, 1H), 8.678 (s, 1H), 7.382 (s, 1H), 7.376 (br, 1H), 7.28 (s, 1H), 7.21 - 7.12 (m, 2H), 4.44 - 4.38 (m, 4H), 1.07 (t, J = 7.9 Hz, 9H), 0.71 ppm (q, J = 7.9 Hz, 6H). 13 C NMR (126 MHz, CDCl3): δ 155.95, 153.81 (d, J = 248.0 Hz), 153.44, 149.62, 146.66, 144.47, 127.68, 127.60, 124.15 (d, J = 4.5 Hz), 122.79, 114.49, 111.77 (d. [M+H] + C 24 H 27 N3O2Si +Calculated value: 436.1851; measured value: 436.1831.

[0234] N-(3-ethynyl-2-fluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK038). [ka] A mixture of compound 4 (40 mg, 0.09 mmol) in wet THF (0.9 mL) was treated dropwise with a 1 M solution of TBAF in THF (450 μL, 0.45 mmol), and the mixture was stirred at 23 °C for 18 h. Water (10 mL) was added, and the mixture was extracted with EtOAc (3 × 15 mL). The combined organic solution was washed with brine (20 mL), dried (NaSO), filtered, and evaporated. Purification by FC (SiO, hexane / EtOAc 7:3 → 3:7) followed by a second FC (SiO, CHCl / EtOAc 1:0 → 6:4) gave JGK038 (19 mg, 64%) as an off-white solid. 1 H NMR (500 MHz, CDCl3): δ 8.69 (td, J = 8.0, 1.8 Hz, 1H), 8.67 (s, 1H), 7.38 (s, 1H), 7.36 (br, 1H), 7.29 (s, 1H), 7.24 - 7.15 (m, 2H), 4.43 - 4.38 (m, 4H), 3.34 ppm (s, 1H). 13 C NMR (126 MHz, CDCl3): δ 155.94, 154.04 (d, J = 248.8 Hz), 153.39, 149.65, 146.70, 144.47, 127.81, 127.68 (d, J = 9.1 Hz), 124.30 (d, J = 4.7 Hz), 123.47, 114.49, 110.58, 110.50 (d, J = 14.3 Hz), 105.99, 82.95 (d, J = 3.5 Hz), 76.70 (d, J = 1.6 Hz), 64.69, 64.50 ppm. HRMS (DART): m / z [M + H] + C18 H 13 FN3O2 + Calculated value: 322.0986; measured value: 322.0981.

[0235] N-[2-Fluoro-3-(trifluoromethyl)phenyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK039). [ka] Compound JGK039 was prepared from 4-chloroquinazoline 1 (37 mg, 0.17 mmol) and 2-fluoro-3-(trifluoromethyl)aniline (42 μL, 0.33 mmol) in iPrOH (1.5 mL) according to general procedure A. FC (CHCl / EtOAc 1:0 to 10:3) afforded JGK039 (35 mg, 58%) as an off-white solid. 1 H NMR (500 MHz, CDCl3): δ 9.00 - 8.92 (m, 1H), 8.70 (s, 1H), 7.42 (br, 1H), 7.40 (s, 1H), 7.35 - 7.28 (m, 2H), 7.30 (s, 1H), 4.46 - 4.38 ppm (m, 4H). 13 C NMR (126 MHz, CDCl3): δ 155.77, 153.24, 150.27 (d, J = 252.0 Hz), 149.81, 146.80, 144.66, 128.62 (d, J = 8.5 Hz), 126.34, 124.44, 124.40, 122.66 (q, J = 272.4 Hz), 120.41 (q, J = 4.6 Hz), 114.58, 110.55, 105.86, 64.70, 64.51 ppm. HRMS (DART): m / z [M - H] - C 17 H 10 F4N3O2 - Calculated value: 364.0715; measured value: 364.0712.

[0236] 4-Chloro-8,9-dihydro-7H-[1,4]dioxepino[2,3-g]quinazoline (5). [ka] A solution of compound 2 (100 mg, 0.51 mmol) in dry DMF (10 mL) was treated with CsCO (460 mg, 1.41 mmol), stirred for 15 min, and treated dropwise with 1,3-dibromopropane (135 μL, 1.33 mmol). The mixture was stirred at 23 °C for 1 h and then at 65 °C for 18 h. After cooling to 23 °C, all volatiles were removed under reduced pressure. The residue was suspended in CHCl (20 mL), washed with water (2 × 5 mL), dried (NaSO), filtered, and evaporated. Purification using FC (hexane / CHCl 1:10 → 0:1 → CHCl / EtOAc 10:1.5) followed by a second FC (hexane / EtOAc 10:1 → 10:3) gave the title compound 5 (41 mg, 34%) as a white solid. 1 H NMR (500 MHz, CDCl3): δ 8.87 (s, 1H), 7.75 (s, 1H), 7.55 (s, 1H), 4.46 (t, J = 5.8 Hz, 2H), 4.40 (t, J = 6.0 Hz, 2H), 2.34 ppm (quint, J = 5.9 Hz, 2H). 13 C NMR (126 MHz, CDCl3): δ 160.57, 158.86, 153.10, 153.03, 148.88, 120.83, 118.19, 115.64, 70.51, 70.33, 30.51 ppm. HRMS (DART): m / z [M + H] + C 11 H 10 ClN2O2 + Calculated value: 237.0425; measured value: 237.0416.

[0237] N-(3-chloro-2-fluorophenyl)-8,9-dihydro-7H-[1,4]dioxepino[2,3-g]quinazolin-4-amine (JGK040). [ka] Compound JGK040 was prepared from 4-chloroquinazoline 5 (33 mg, 0.14 mmol) and 3-chloro-2-fluoroaniline (32 μL, 0.29 mmol) in iPrOH (1.5 mL) according to general procedure A. FC (CHCl / EtOAc 1:0 to 10:3.5) afforded JGK040 (34 mg, 70%) as a white solid. 1 H NMR (500 MHz, DMSO-d6): δ 9.72 (s, 1H), 8.39 (s, 1H), 8.08 (s, 1H), 7.53 - 7.45 (m, 2H), 7.29 (s, 1H), 7.27 (td, J = 8.1, 1.3 Hz, 1H), 4.32 (t, J = 5.5 Hz, 2H), 4.29 (t, J = 5.6 Hz, 2H), 2.22 ppm (quint, J = 5.6 Hz, 2H). 13 C NMR (126 MHz, DMSO-d6): δ 157.43, 156.67, 153.85, 152.48 (d, J = 249.3 Hz), 150.74, 147.29, 128.05 (d, J = 12.0 Hz), 127.41, HRMS (DART): m / z [M + H] + C 17 H 14 ClFN3O2 + Calculated value: 346.0753; measured value: 346.0740.

[0238] 8-Chloro-2H-[1,3]dioxolo[4,5-g]quinazoline (6). [ka] A solution of compound 2 (100 mg, 0.51 mmol) in dry DMF (3.4 mL) was treated with CsCO (335 mg, 1.03 mmol) and stirred at 23 °C for 15 min. The mixture was treated dropwise with chloroiodomethane (130 μL, 1.79 mmol) and stirred for 1 h, then at 70 °C for 17 h. The mixture was cooled to 23 °C and all volatiles were removed under reduced pressure. The residue was suspended in CHCl (30 mL), washed with water (2 × 7 mL), dried (NaSO), filtered, and evaporated. Purification using FC (hexane / CHCl 3:10 → 0:1 → CHCl / EtOAc 10:2) gave the title compound 6 (38 mg, 36%) as a white fluffy solid. 1 H NMR (400 MHz, CDCl3): δ 8.85 (s, 1H), 7.49 (s, 1H), 7.32 (s, 1H), 6.21 ppm (s, 2H). 13 C NMR (126 MHz, CDCl3): δ 159.82, 154.89, 152.79, 150.94, 149.78, 121.23, 105.23, 102.89, 101.12 ppm. HRMS (DART): m / z [M + H] + C9H6ClN2O2 + Calculated value: 209.0112; measured value: 209.0104.

[0239] N-(3-chloro-2-fluorophenyl)-2H-[1,3]dioxolo[4,5-g]quinazolin-8-amine (JGK041). [ka] Compound JGK041 was prepared from 4-chloroquinazoline 6 (35 mg, 0.17 mmol) and 3-chloro-2-fluoroaniline (38 μL, 0.35 mmol) in iPrOH (1.5 mL) according to general procedure A. FC (CHCl / EtOAc 1:0 to 1:1) afforded JGK041 (35 mg, 66%) as a pale yellow solid. 1H NMR (500 MHz, DMSO-d6): δ 9.53 (s, 1H), 8.37 (s, 1H), 7.84 (s, 1H), 7.53 - 7.44 (m, 2H), 7.27 (td, J = 8.1, 1.3 Hz, 1H), 7.20 (s, 1H), 6.25 ppm (s, 2H). 13 C NMR (126 MHz, DMSO-d6): δ 157.37, 153.10, 152.60, 152.43 (d, J = 248.9 Hz), 148.56, 147.28, 128.30 (d, J = 11.9 Hz), 127.17, 126.90, 124.88 (d, J = 4.8 Hz), 120.12 (d, J = 16.4 Hz), 109.82, 104.59, 102.38, 98.77 ppm. HRMS (DART): m / z [M + H] + C 15 H 10 ClFN3O2 + Calculated value: 318.0440; measured value: 318.0435.

[0240] [(3-chloro-2-fluorophenyl)(7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-yl)amino]methyl acetate (JGK043). [ka] A mixture of JGK010 (50 mg, 0.15 mmol) in dry THF (0.5 mL) was treated dropwise with a 1 M solution of LiHMDS in THF (150 μL, 0.15 mmol) at 0 °C. After stirring at that temperature for 15 min, the mixture was added dropwise to a solution of chloromethyl acetate (55 μL, 0.57 mmol) in dry THF (0.5 mL). The flask originally containing the JGK010 solution was rinsed with 0.5 mL of dry THF before adding it to the reaction mixture. After stirring at 0 °C for 2 h, stirring at 23 °C was continued for 22 h. Saturated aqueous NaHCO (10 mL) was added, and the mixture was extracted with EtOAc (3 × 10 mL). The combined organic solution was dried (Na SO ), filtered, and evaporated under reduced pressure. Purification using FC (CH2Cl2 / EtOAc 1:0->1:1) gave JGK043 (30 mg, 49%) as a pale yellow solid. 1 H NMR (500 MHz, CDCl3): δ 7.93 (s, 1H), 7.88 (s, 1H), 7.08 - 6.97 (m, 2H), 6.94 (td, J = 7.2, 2.1 Hz, 1H), 6.82 (s, 1H), 5.73 (s, 2H), 4.40 - 4.29 (m, 4H), 2.12 ppm (s, 3H). 13 C NMR (126 MHz, CDCl3): δ 170.33, 152.96, 150.10 (d, J = 245.6 Hz), 149.37, 148.05, 143.24, 140.17 (d, J = 13.1 Hz), 131.89, 124.17, 124.12 (d, J=4.8 Hz), 122.59 (d, J=2.4 Hz), 121.33 (d, J=17.1 Hz), 115.41, 114.31, 102.24, 71.19, 65.07, 64.23, 20.85 ppm. HRMS (DART): m / z [M + H] + C 19 H 16 Calculated for ClFN3O4+, 404.0808; Found, 404.0792.

[0241] Example 3: Preparation of further exemplary compounds of the JGK series General Procedures: All chemicals, reagents, and solvents, when available, were purchased from commercial sources and used as received. When necessary, reagents and solvents were purified and dried by standard methods. Air-sensitive and moisture-sensitive reactions were carried out in oven-dried glassware under an inert atmosphere of argon. Microwave-irradiated reactions were carried out in a single-mode reactor, a CEM Discover microwave synthesizer. Room temperature (RT) reactions were carried out at ambient temperature (approximately 23 °C). All reactions were carried out on pre-coated Merck 60 F plates, with spots visualized using UV light (λ = 254, 365 nm) or alkaline KMnO4 solution. 254 Thin layer chromatography (TLC) was performed using silica gel plates. Flash column chromatography (FC) was performed using SiO260 (particle size 0.040-0.063 mm, 230-400 mesh). Concentration under reduced pressure (vacuum) was performed by rotary evaporation at 25-50°C. The purified compounds were further dried under high vacuum or in a desiccator. The yields corresponded to the purified compounds and were not further optimized. Proton nuclear magnetic resonance ( 1 H NMR spectra were recorded using a Bruker spectrometer operating at 300, 400, or 500 MHz. Carbon NMR ( 13 C NMR spectra were recorded using a Bruker spectrometer (either 400 or 500 MHz). NMR chemical shifts (δ ppm) were referenced to the residual solvent signal. 1 H NMR data are reported as follows: chemical shift (ppm); multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet / complex pattern, td = triple doublet, ddd = double doublet, br = broad signal); coupling constant (J) (Hz), integration. 13C NMR spectral data are reported based on chemical shifts and, where applicable, coupling constants. High-resolution mass (HRMS) spectra were recorded using a Thermo Fisher Scientific Exactive Plus equipped with an IonSense ID-CUBE DART source mass spectrometer or a Waters LCT Premier mass spectrometer equipped with an ACQUITY UPLC with an autosampler.

[0242] 3-[(7,8-Dihydro[1,4]dioxino[2,3-g]quinazolin-4-yl)amino]-2-fluorobenzonitrile (JGK044). [ka] 1 H NMR (500 MHz, CDCl3): δ = 9.06 - 8.98 (m, 1H), 8.69 (s, 1H), 7.41 (s, 1H), 7.39 (br, 1H), 7.35 - 7.31 (m, 2H), 7.30 (s, 1H), 4.45 - 4.37 ppm (m, 4H). 13 C NMR (126 MHz, CDCl3): δ = 155.63, 153.63 (d, J = 254.6 Hz), 153.04, 149.94, 146.80, 144.76, 128.60 (d, J = 7.8 Hz), 127.48, 126.58, 125.31 (d, J = 4.5 Hz), 114.56, 113.80, 110.45, 105.83, 101.30 (d, J = 13.9 Hz), 64.70, 64.51 ppm. HRMS (ESI): m / z [M + H] + C 17 H 12 FN4O2 + Calculated value: 323.0939; measured value: 323.0927.

[0243] 3-[(7,8-Dihydro[1,4]dioxino[2,3-g]quinazolin-4-yl)amino]benzonitrile (JGK045). [ka] 1 H NMR (500 MHz, DMSO-d6): δ = 9.68 (s, 1H), 8.52 (s, 1H), 8.46 (t, J = 1.9 Hz, 1H), 8.18 (ddd, J = 8.2, 2.3, 1.2 Hz, 1H), 8.08 (s, 1H), 7.58 (t, J = 7.9 Hz, 1H), 7.53 (dt, J = 7.6, 1.4 Hz, 1H), 7.22 (s, 1H), 4.49 - 4.36 ppm (m, 4H). 13 C NMR (126 MHz, DMSO-d6): δ = 156.24, 152.69, 149.31, 146.15, 143.80, 140.52, 129.87, 126.35, 125.96, 124.15, 118.93, 112.66, 111.23, 109.96, 108.30, 64.52, 64.19 ppm. HRMS (DART): m / z [M + H] + C 17 H 13 N4O2 + Calculated value: 305.1033; measured value: 305.1018.

[0244] Ethyl (3-chloro-2-fluorophenyl) 7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-ylcarbamate (JGK047). [ka] 1H NMR (500 MHz, CDCl3): δ = 8.96 (s, 1H), 7.483 (s, 1H), 7.477 (s, 1H), 7.37 (dd, J = 8.1, 6.7 Hz, 2H), 7.08 (td, J = 8.1, 1.5 Hz, 1H), 4.45 - 4.38 (m, 4H), 4.27 (q, J = 7.1 Hz, 2H), 1.22 ppm (t, J = 7.1 Hz, 3H). 13 C NMR (126 MHz, CDCl3): 158.98, 154.43 (d, J = 250.7 Hz), 153.90, 153.41, 151.17, 149.68, 145.61, 130.12, 129.85 (d, J = 12.4 Hz), HRMS (ESI): m / z [M + H] + C 19 H 16 ClFN3O4 + Calculated value: 404.0808; measured value: 404.0800.

[0245] (±)-4-(3-Bromo-2-fluoroanilino)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-7-ol ((±)-JGK050). [ka] 1H NMR (500 MHz, DMSO-d6): δ = 9.61 (s, 1H), 8.34 (s, 1H), 7.91 (s, 1H), 7.72 (d, J = 5.4 Hz, 1H), 7.60 (t, J = 7.1 Hz, 1H), 7.55 (t, J = 7.5 Hz, 1H), 7.21 (t, J = 8.2 Hz, 1H), 7.19 (s, 1H), 5.70 - 5.59 (m, 1H), 4.27 (d, J = 11.0 Hz, 1H), 4.17 ppm (d, J = 10.6 Hz, 1H). 13 C NMR (126 MHz, DMSO-d6): δ = 157.18, 153.38 (d, J = 247.4 Hz), 153.13, 148.78, 146.14, 141.92, 130.12, 128.05 (d, J = 13.8 Hz), 127.78, 125.45 (d, J = 4.4 Hz), 111.95, 109.87, 108.71, 108.55 (d, J = 20.3 Hz), 88.63, 67.23 ppm. HRMS (DART): m / z [M + H] + C 16 H 12 BrFN3O3 + Calculated value: 392.0041; measured value: 392.0030.

[0246] Diastereoisomeric mixture of (±)-cis- and (±)-trans-N-(3-bromo-2-fluorophenyl)-7,8-dimethyl-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine ((±)-cis / trans-JGK051). [ka] 1H NMR (500 MHz, CDCl3; (±)-cis / trans 2:1): δ = 8.68 (s, 1H), 8.68 - 8.63 (m, 1H), 7.37 (s, 1H), 7.35 (br, 1H), 7.28 (s, 1H), 7.28 - 7.24 (m, 1H), 7.10 (td, J = 8.2, 1.6 Hz, 1H), 4.52 - 4.39 (m, 1.3H), 4.09 - 3.98 (m, 0.7H), 1.453 (d, J = 6.1 Hz, 1.1H), 1.451 (d, J = 6.1 Hz, 1.1H), 1.369 (d, J = 6.6 Hz, 1.9H), 1.368 ppm (d, J = 6.6 Hz, 1.9H). 13 C NMR (126 MHz, CDCl3; (±)-cis / trans 2:1): δ = 155.87, 155.84, 153.19, 150.12 (d, J = 242.5 Hz), 150.09 (d, J = 242.2 Hz), 149.87, 148.89, 146.77, 144.64, 143.63, 128.73 (d, J = 10.0 Hz), 127.15, 127.12, 125.31 (d, J = 4.7 Hz), 121.71, 121.70, 114.30, 113.93, 110.54, 110.47, 108.57 (d, J = 19.4 Hz), 105.66, 105.28 ppm. HRMS (DART): m / z [M + H] + C 18 H 16 BrFN3O2 + Calculated value: 404.0404; measured value: 404.0393.

[0247] (±)-cis-N-(3-bromo-2-fluorophenyl)-7,8-dimethyl-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine ((±)-JGK052). [ka] 1H NMR (500 MHz, CDCl3): δ = 8.68 (s, 1H), 8.66 (ddd, J = 8.6, 7.4, 1.6 Hz, 1H), 7.38 (s, 1H), 7.35 (br, 1H), 7.28 (s, 1H), 7.30 - 7.23 (m, 1H), 7.10 (td, J = 8.2, 1.5 Hz, 1H), 4.50 - 4.41 (m, 2H), 1.369 (d, J = 6.6 Hz, 3H), 1.368 ppm (d, J = 6.5 Hz, 3H). 13 C NMR (126 MHz, CDCl3): δ = 155.85, 153.19, 150.11 (d, J = 242.1 Hz), 148.89, 146.77, 143.63, 128.73 (d, J = 10.2 Hz), 127.14, 125.31 HRMS (ESI): m / z [M + H] + C 18 H 16 BrFN3O2 + Calculated value: 404.0404; measured value: 404.0416.

[0248] N-(3-bromo-4-chloro-2-fluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK053). [ka] 1 H NMR (500 MHz, DMSO-d6): δ = 9.70 (s, 1H), 8.35 (s, 1H), 7.94 (s, 1H), 7.61 (dd, J = 8.8, 7.7 Hz, 1H), 7.55 (dd, J = 8.7, 1.5 Hz, 1H), 7.20 (s, 1H), 4.47 - 4.35 ppm (m, 4H). 13C NMR (126 MHz, DMSO-d6): δ = 157.03, 154.14 (d, J = 249.5 Hz), 153.01, 149.36, 146.08, 143.74, 130.75, 127.77 (d, J = 2.9 Hz), 126.80 (d, J = 13.4 Hz), 125.37 (d, J = 3.8 Hz), 112.50, 110.15 (d, J = 22.5 Hz), 109.66, 108.39, 64.51, 64.14 ppm. HRMS (DART): m / z [M + H] + C 16 H 11 BrClFN3O2 + Calculated value: 409.9702; measured value: 409.9697.

[0249] N-(3,4-Dibromo-2-fluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK054). [ka] 1 H NMR (500 MHz, DMSO-d6): δ = 9.65 (s, 1H), 8.34 (s, 1H), 7.92 (s, 1H), 7.67 (d, J = 8.7 Hz, 1H), 7.55 (t, J = 8.2 Hz, 1H), 7.20 (s, 1H), 4.45 - 4.35 ppm (m, 4H). 13 C NMR (126 MHz, DMSO-d6): δ = 156.95, 153.98 (d, J = 249.1 Hz), 152.99, 149.35, 146.09, 143.74, 128.50 (d, J = 3.7 Hz), 128.14, 127.21 (d, J = 13.7 Hz), 120.96, 112.51, 112.33, 109.68, 108.36, 64.51, 64.14 ppm. HRMS (DART): m / z [M + H] + C 16 H 11Br2FN3O2 + Calculated value: 453.9197; measured value: 453.9191.

[0250] N-(5-Bromo-2-fluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK055). [ka] 1 H NMR (500 MHz, CDCl3): δ = 8.99 (dd, J = 7.3, 2.5 Hz, 1H), 8.72 (s, 1H), 7.38 (s, 1H), 7.36 (br, 1H), 7.27 (s, 1H), 7.16 (ddd, J = 8.7, 4.6, 2.5 Hz, 1H), 7.04 (dd, J = 10.9, 8.7 Hz, 1H), 4.44 - 4.36 ppm (m, 4H). 13 C NMR (126 MHz, CDCl3): δ = 155.59, 153.35, 152.16 (d, J = 243.1 Hz), 149.69, 146.67, 144.52, 128.75 (d, J = 10.5 Hz), 126.16 (d, J = 7.6 HRMS (DART): m / z [M + H] + C 16 H 12 BrFN3O2 + Calculated value: 376.0091; measured value: 376.0077.

[0251] N-(3-Bromo-2,6-difluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK056). [ka] 1 H NMR (500 MHz, DMSO-d6): δ = 9.60 (s, 1H), 8.32 (s, 1H), 7.94 (s, 1H), 7.74 (td, J = 8.1, 5.5 Hz, 1H), 7.28 (t, J = 9.3 Hz, 1H), 7.21 (s, 1H), 4.44 - 4.38 ppm (m, 4H). 13 C NMR (126 MHz, DMSO-d6): δ = 157.78 (dd, J = 248.8, 3.3 Hz), 157.37, 155.01 (dd, J = 247.9, 4.9 Hz), 153.08, 149.47, 146.04, 143.86, 130.76 (d, J = 9.3 Hz), 117.30 (t, J = 17.5 Hz), 113.30 (dd, J = 21.8, 3.0 Hz), 112.56, 109.45, 108.28, 103.55 (dd, J = 20.4, 3.6 Hz), 64.52, 64.14 ppm. HRMS (ESI): m / z [M + H] + C 16 H 11 BrF2N3O2 + Calculated value: 393.9997; measured value: 394.0008.

[0252] N-(3-Bromo-2,4-difluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK057). [ka] 1 H NMR (500 MHz, CDCl3): δ = 8.64 (s, 1H), 8.51 (td, J = 9.0, 5.6 Hz, 1H), 7.38 (s, 1H), 7.29 (s, 1H), 7.23 (br, 1H), 7.04 (ddd, J = 9.2, 7.8, 2.1 Hz, 1H), 4.45 - 4.37 ppm (m, 4H). 13C NMR (126 MHz, CDCl3): δ = 156.10, 155.80 (dd, J = 246.6, 3.5 Hz), 153.28, 151.25 (dd, J = 245.1, 4.0 Hz), 149.74, 146.56, 144.53, 124.39 (dd, J = 10.8, 3.4 Hz), 122.72 (dd, J = 8.3, 1.8 Hz), 114.42, 111.49 (dd, J = 22.5, 3.9 Hz), 110.34, 105.98, 97.86 (dd, J = 25.7, 22.9Hz), 64.69, 64.50 ppm. HRMS (ESI): m / z [M + H] + C 16 H 11 BrF2N3O2 + Calculated value: 393.9997; Measured value: 394.0013.

[0253] N-(3-bromo-5-chloro-2-fluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK058). [ka] 1 H NMR (500 MHz, CDCl3): δ = 8.88 (dd, J = 6.6, 2.6 Hz, 1H), 8.73 (s, 1H), 7.41 (s, 1H), 7.37 (br, 1H), 7.26 (s, 1H), 7.28 - 7.23 (m, 1H), 4.44 - 4.39 ppm (m, 4H). 13C NMR (126 MHz, CDCl3): δ = 155.45, 153.13, 149.88, 148.60 (d, J = 241.7 Hz), 146.76, 144.72, 130.30 (d, J = 4.4 Hz), 129.26 (d, J = 10.8 HRMS (ESI): m / z [M + H] + C 16 H 11 BrClFN3O2 + Calculated value: 409.9702; measured value: 409.9713.

[0254] (±)-trans-N-(3-bromo-2-fluorophenyl)-7,8-dimethyl-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine ((±)-JGK059). [ka] 1 H NMR (500 MHz, CDCl3): δ = 8.68 (s, 1H), 8.66 (ddd, J = 8.6, 7.3, 1.6 Hz, 1H), 7.376 (s, 1H), 7.375 (br, 1H), 7.28 (s, 1H), 7.28 - 7.24 (m, 1H), 7.10 (td, J = 8.2, 1.6 Hz, 1H), 4.08 - 3.98 (m, 2H), 1.451 (d, J = 6.1 Hz, 3H), 1.448 ppm (d, J = 6.1 Hz, 3H). 13C NMR (126 MHz, CDCl3): δ = 155.90, 153.12, 150.12 (d, J = 242.5 Hz), 149.90, 146.59, 144.65, 128.70 (d, J = 10.2 Hz), 127.18, 125.30 HRMS (ESI): m / z [M + H] + C 18 H 16 BrFN3O2 + Calculated value: 404.0404; measured value: 404.0405.

[0255] N-(3,4-Dichloro-2-fluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK060). [ka] 1 H NMR (500 MHz, CDCl3): δ = 8.67 (s, 1H), 8.59 (t, J = 8.6 Hz, 1H), 7.40 (s, 1H), 7.38 (br, 1H), 7.33 (dd, J = 9.1, 2.1 Hz, 1H), 7.31 (s, 1H), 4.45 - 4.38 ppm (m, 4H). 13 C NMR (126 MHz, CDCl3): δ = 155.84, 153.08, 149.98 (d, J = 246.3 Hz), 149.88, 146.42, 144.67, 127.55, 127.19 (d, J = 10.0 Hz), 125.30 (d, J = 4.1 Hz), 121.05, 120.47 (d, J = 18.2 Hz), 114.36, 110.43, 105.97, 64.71, 64.51 ppm. HRMS (ESI): m / z [M + H] + C 16H 11 Cl2FN3O2 + Calculated value: 366.0207; Measured value: 366.0207.

[0256] N-(3-Bromo-2,5-difluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK061). [ka] 1 H NMR (500 MHz, DMSO-d6): δ = 9.65 (s, 1H), 8.40 (s, 1H), 7.93 (s, 1H), 7.63 - 7.54 (m, 2H), 7.21 (s, 1H), 4.45 - 4.37 ppm (m, 4H). 13 C NMR (126 MHz, DMSO-d6): δ = 157.29 (d, J = 243.5 Hz), 156.84, 152.93, 149.97 (d, J = 242.9 Hz), 149.43, 146.16, 143.81, 129.22 - 128.44 (m), 116.30 (d, J = 26.7 Hz), 113.99 (d, J = 25.7 Hz), 112.53, 109.73, 108.76 (dd, J = 22.5, 12.5 Hz), 108.33, 64.52, 64.15 ppm. HRMS (DART): m / z [M + H] + C 16 H 11 BrF2N3O2 + Calculated value: 393.9997; measured value: 393.9988.

[0257] (±)-N-(3-Bromo-2-fluorophenyl)-7-ethenyl-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine ((±)-JGK062). [ka] 1H NMR (500 MHz, CDCl3): δ = 8.68 (s, 1H), 8.65 (ddd, J = 8.2, 7.3, 1.5 Hz, 1H), 7.40 (s, 1H), 7.37 (br, 1H), 7.35 (s, 1H), 7.27 (ddd, J = 8.0, 6.4, 1.5 Hz, 1H), 7.10 (td, J = 8.2, 1.6 Hz, 1H), 5.95 (ddd, J = 17.3, 10.7, 5.8 Hz, 1H), 5.60 (dt, J = 17.3, 1.2 Hz, 1H), 5.48 (dt, J = 10.7, 1.1 Hz, 1H), 4.82 - 4.74 (m, 1H), 4.42 (dd, J = 11.5, 2.5 Hz, 1H), 4.09 ppm (dd, J = 11.6, 8.1 Hz, 1H). 13 C NMR (126 MHz, CDCl3): δ = 155.90, 153.38, 150.14 (d, J = 242.4 Hz), 149.12, 146.70, 144.12, 131.48, 128.64 (d, J = 10.3 Hz), 127.24, 125.30 (d, J = 4.7 Hz), 121.76, 120.43, 114.29, 110.69, 108.58 (d, J = 19.3 Hz), 106.06, 74.03, 67.84 ppm. HRMS (DART): m / z [M + H] + C 18 H 14 BrFN3O2 + Calculated value: 402.0248; measured value: 402.0233.

[0258] Example 4 : Classification of EGFRi metabolic responders and EGFRi metabolic non-responders We characterized changes in glucose consumption in 19 patient-derived GBM cell lines following acute EGFR inhibition. Cells were cultured as gliomaspheres in supplemented serum-free medium, as opposed to serum-based culture conditions, which preserve many of the molecular features of patient tumors. Treatment with the EGFR tyrosine kinase inhibitor (EGFRi) erlotinib demonstrated that EGFR inhibition resulted in a decrease in radiolabeled glucose uptake ( 18 We identified a subset of GBMs with significantly reduced F-FDG, hereafter referred to as "metabolic responders" ( Figure 1 A and Figure 7 A) Silencing EGFR using siRNA confirmed that the reduced glucose uptake was not due to an off-target effect of erlotinib ( Figure 7 B. Figure 7 C) In EGFRi-treated cells 18 The decrease in F-FDG uptake was associated with a decrease in lactate production, glucose consumption, and extracellular acidification rate (ECAR), while glutamine levels remained unchanged ( Figure 1 B and Figure 7 D~ Figure 7 G). Finally, decreased glucose utilization correlated with alterations in RAS-MAPK signaling and PI3K-AKT-mTOR signaling, each of which may regulate glucose metabolism in GBM and other cancers ( Figure 8 A).

[0259] In contrast, all "non-responder" GBMs (i.e., those treated with EGFRi or siRNA) 18 No change in F-FDG uptake)( Figure 1 A, and Figure 7 B. Figure 7 C) Despite strong EGFR inhibition, no changes were observed in glucose consumption, lactate production, or ECAR ( Figure 1 B. Figure 7 D~ Figure 7 G, and Figure 8 B). Furthermore, RAS-MAPK signaling and PI3K-AKT-mTOR signaling were not significantly affected in these cells ( Figure 8B). Notably, while all metabolic responders had mutations (copy number gains, mutations) in EGFR, six GBM lines that did not show metabolic responses also contained EGFR mutations and / or copy number gains ( Figure 9 A. Figure 9 B). Collectively, these data demonstrate two key points: first, acute inhibition of EGFR rapidly reduces glucose utilization in a subset of primary GBM cells, and second, EGFR genetic mutations do not independently predict which GBMs will demonstrate a metabolic response to EGFRi.

[0260] Example 5 : Activates EGFRi metabolic responders toward apoptosis These findings suggest that altered glucose metabolism can induce the expression of pro-apoptotic factors and promote intrinsic apoptosis, and that reduced glucose uptake in response to EGFRi may stimulate the intrinsic apoptotic pathway. Indeed, acute erlotinib treatment promoted the expression of the pro-apoptotic BH3-only proteins BIM and PUMA only in metabolic responder cultures. Figure 10 A). However, Annexin V staining revealed that metabolic responders exhibited at most modest (approximately 17%) (although significantly higher) apoptosis compared with non-responders (approximately 3%) after 72 hours of erlotinib exposure ( Figure 1 C).

[0261] Given the low apoptosis levels despite the significant induction of pro-apoptotic factors, we asked whether perturbing glucose uptake with EGFRi would activate GBM cells toward apoptosis, thereby increasing apoptotic propensity without inducing significant cell death. To test this, we treated both metabolic responders and metabolic non-responders with erlotinib for 24 hours and then performed dynamic BH3 profiling to quantify changes in apoptotic activation. Figure 10B). Using multiple BH3 peptides (e.g., BIM, BID, and PUMA), we observed a significant increase in apoptotic activation (measured by changes in cytochrome c release compared to vehicle) in erlotinib-treated metabolic responders ( Figure 1 D - dark grey bars). Importantly, activation in metabolic responders was significantly higher compared to activation in metabolic non-responders ( Figure 1 D - light grey bars), supporting the premise that reduced glucose uptake by EGFRi induces activation of apoptosis in GBM.

[0262] We tested whether reduced glucose uptake is required for EGFRi-mediated apoptosis activation by determining whether rescuing glucose consumption should alleviate these effects. To test this, we ectopically expressed glucose transporters 1 (GLUT1) and 3 (GLUT3) in two metabolic responders (HK301 and GBM39). Forced expression of GLUT1 and GLUT3 (GLUT1 / 3) rescued the EGFRi-mediated reduction in glucose uptake and lactate production in both cell lines (Figure 1). Figure 1 E, and Figure 11 A~ Figure 11 C), and importantly, apoptosis activation in response to EGFRi was significantly suppressed ( Figure 1 F). Collectively, these data indicate that EGFRi-mediated inhibition of glucose consumption, while insufficient to induce significant cell death, lowers the apoptotic threshold, potentially making GBM cells more susceptible to agents that exploit this activated state.

[0263] Example 6 : Cytoplasmic p53 is required for apoptosis activation by EGFRi We investigated the mechanism by which GBM cells are activated toward apoptosis by EGFRi. Inhibiting oncogene-driven glucose metabolism synergistically renders GBM cells susceptible to cytoplasmic p53-dependent apoptosis. Reduced glucose metabolic flux in GBM cells through targeted oncogenic signaling (e.g., EGFRi) leads to cytoplasmic p53 engaging ("activating") the intrinsic apoptotic pathway. However, Bcl-xL inhibits cytoplasmic p53-mediated cell death. Pharmacological p53 stabilization overcomes this apoptotic inhibition, resulting in synergistic lethality with the combined targeting of oncogene-driven glucose metabolism in GBM.

[0264] In activated cells, the majority of anti-apoptotic Bcl-2 family proteins (e.g., Bcl-2, Bcl-xL, and Mcl-1) associate with pro-apoptotic BH3 proteins (e.g., BIM, BID, PUMA, BAD, NOXA, HRK), and cell survival is therefore dependent on these interactions. The tumor suppressor protein p53 is known to prevent cell death by upregulating pro-apoptotic proteins that subsequently require binding by anti-apoptotic Bcl-2 proteins. To investigate whether p53 is required for EGFRi-induced activation, we used CRISPR / CAS-9 to transfect two metabolic responders (HK301 and HK336, Figure 2 A) p53 was silenced in p53KO cells (hereafter referred to as p53KO). The change in glucose consumption induced by EGFRi was not affected in p53KO cells ( Figure 12 A) BH3 profiling revealed that p53KO nearly abolished erlotinib-induced apoptosis activation in both HK301 and HK336 cells ( Figure 2 B).

[0265] Since p53 transcriptional activity has been shown to be enhanced under glucose restriction, we investigated whether p53-mediated transcription is induced by EGFRi. However, erlotinib did not increase the expression of p53-regulated genes (e.g., p21, MDM2, PIG3, TIGAR) ( Figure 12 B) or did not induce p53-luciferase reporter activity in HK301 metabolic responder cells ( Figure 12 C). These data indicate that while p53 is required for activation by EGFRi, its transcriptional activity may not be essential.

[0266] In addition to the well-described nuclear functions of p53, p53 can localize in the cytoplasm, where p53 can directly participate in the intrinsic apoptotic pathway. To assess whether cytoplasmic p53 is important for apoptosis activation by EGFRi, we investigated the role of p53 mutants with defective nuclear localization signals (p53 cyto ) was stably transfected into HK301 p53KO glioma spheres and HK336 p53KO glioma spheres. As expected, p53 cyto is expressed ( Figure 2 C and Figure 12 D), confined to the cytoplasm ( Figure 2 D and Figure 12 E), but did not have transcriptional activity ( Figure 2 Conversely, wild-type p53 (p53 wt ) showed localization similar to that in parental cells and rescued transcription of p53-regulated genes ( Figure 2 C~ Figure 2 E, and Figure 12 E~ Figure 12 G). Notably, p53 cyto Stable introduction of p53 inhibited erlotinib-induced activation in both HK301 p53KO and HK336 p53KO cells. wt Significantly regenerated to a level equivalent to that of Figure 2 F and Figure 12G), the cytoplasmic function of p53 was shown to be required for EGFRi-mediated activation. By sex Furthermore, p53 mutations restricted to the nucleus (p53 NES ) into HK301 p53KO cells failed to induce EGFRi-mediated apoptosis activation ( Figure 2 G. Figure 2 H, and Figure 12 H). Finally, pharmacological inhibition of cytoplasmic p53 activity by pifithrin-μ (PFTμ) was significantly attenuated by activation by erlotinib ( Figure 12 I). Collectively, these results indicate that cytoplasmic p53 is involved in the intrinsic apoptotic machinery following EGFRi in GBM.

[0267] Previous studies have shown that human tumor-derived p53 mutations, particularly those within the DNA-binding domain, impair cytoplasmic function in addition to impaired transactivation. Therefore, we tested whether stable expression of two of these "hotspot" p53 mutations, R175H or R273H, in HK301 p53KO could impair EGFR-mediated activation. Figure 12 H). As expected, it is transcriptionally deficient ( Figure 2 G) Both mutations, consistent with reduced cytoplasmic activity, prevented EGFRi from activating apoptosis ( Figure 2 H). Thus, consistent with previous findings, oncogenic mutations within the DNA-binding domain of p53 result in "dual hits," thereby inhibiting both transactivation and cytoplasmic function (the latter being affected by apoptotic activation by EGFRi).

[0268] Example 7 Inhibition of EGFR-driven glucose uptake leads to exploitable Bcl-xL dependence Bcl-xL can sequester cytoplasmic p53 and prevent p53-mediated apoptosis, resulting in an activated apoptotic state and a dependency on Bcl-xL for survival. Indeed, BH3 profiling revealed a dependency on Bcl-xL for cell survival in EGFRi metabolic responders ( Figure 13 A). Therefore, we hypothesized that reduced glucose consumption by EGFRi may result in the sequestration of cytoplasmic p53 by Bcl-xL. To investigate this, we performed co-immunoprecipitation to examine the dynamics of p53-Bcl-xL interaction in response to EGFRi in both responders (n=2) and non-responders (n=2). Importantly, we found that metabolic non-responders ( Figure 3 B) but not in metabolic responders ( Figure 3 In A), we observed significantly increased interaction between Bcl-xL and p53 after erlotinib treatment, suggesting that inhibition of EGFR-dependent glucose consumption results in sequestration of p53 by Bcl-xL. Consistent with this interpretation, ectopic expression of GLUT1 / 3, which rescues EGFR-mediated reductions in glucose uptake and apoptotic activation, prevented p53 from binding to Bcl-xL ( Figure 3 C and Figure 13 B). These findings strongly suggest that EGFRi-mediated inhibition of glucose uptake activates GBM cells toward apoptosis by promoting the interaction between cytoplasmic p53 and Bcl-xL.

[0269] Release of p53 from Bcl-xL allows p53 to directly activate BAX, leading to cytochrome c release and cell death. After we recognized the high binding between Bcl-xL and p53 in response to EGFRi in metabolic responders, we asked ourselves whether the displacement of p53 from Bcl-xL induces apoptosis. To test this, we treated metabolic responders (HK301) with erlotinib and the specific Bcl-xL inhibitor WEHI-539. Addition of WEHI-539 inhibited the binding of Bcl-xL to p53 under erlotinib treatment ( Figure 3 D), resulting in synergistic lethality in HK301 and GBM39 cells ( Figure 3 E). Notably, cytoplasmic p53 was sufficient for the combinatorial effect in EGFRi metabolic responder cells ( Figure 13 C). However, WEHI-539 did not enhance apoptosis in erlotinib-treated non-responders (HK393), suggesting that EGFRi-mediated glucose uptake and the subsequent reduction in binding between p53 and Bcl-xL are necessary to generate a dependency on Bcl-xL for survival ( Figure 13 E) This is supported by the fact that forced expression of GLUT1 / 3 significantly reduced cell death caused by the drug combination ( Figure 3 F and Figure 13 D). Collectively, these findings indicate that by sequestering cytoplasmic p53, Bcl-xL reduces GBM cell death in response to EGFRi-mediated inhibition of glucose uptake ( Figure 12 G).

[0270] Example 8 : Combined targeting of EGFR and p53 is synergistic in EGFRi metabolic responders Mechanistic studies revealed potential therapeutic opportunities in EGFR-driven GBM that depend on functional p53. Although the p53 signaling axis is one of three central pathways altered in GBM, analysis of the TCGA GBM dataset showed that p53 mutations are mutually exclusive with EGFR mutations ( Figure 8 A and Figure 8 B). Conversely, in patients with EGFR mutations or gains, the p53 pathway can be suppressed by MDM2 amplification and / or deletion of p14 ARF at the CDKN2A locus, a negative regulator of MDM2. Given these relationships and the requirement of p53 for activation under reduced glucose uptake by EGFRi, we hypothesized that stabilization of p53 by MDM2 inhibition may have a similar therapeutic effect to Bcl-xL antagonism. We found that pairing Nutlin (a well-characterized inhibitor of MDM2) with erlotinib resulted in significant synergistic lethality in metabolic responder glioma spheroids. Over 90% of HK301 cells underwent apoptosis with the combination of erlotinib and Nutlin ( Figure 4 C). Notably, we found that metabolic non-responders (GS017, Figure 4 We did not observe synergy between these agents in GBM cells (C). We then tested this combination across our panel of primary GBM cells (all p53 wild-type) and found that synergistic lethality was only observed in GBM cells that exhibited a metabolic response to EGFRi ( Figure 4 D and Figure 14 A) Genetic knockdown of EGFR confirmed that the synergistic effect was only in metabolic responders ( Figure 14 B). Importantly, forced expression of GLUT1 / 3 significantly reduced BAX oligomerization, cytochrome c release, and apoptosis induced by the combination of erlotinib and Nutlin ( Figure 4 E and Figure 14 C) Supporting the concept that inhibition of glucose metabolism by EGFRi is required for the synergistic effect of the combination of erlotinib and Nutlin.

[0271] We then investigated the role of p53 in inducing cell death in combination with erlotinib and Nutlin. As expected, CRISPR / CAS-9 targeting of p53 in two EGFRi metabolic responders (HK301 and HK336) completely alleviated sensitivity to the drug combination ( Figure 4 F). Similarly, ectopic expression of Bcl-xL significantly suppressed cell death induced by the combined treatment, consistent with an important function of Bcl-xL in counteracting p53-mediated apoptosis. Figure 14 D). Furthermore, similar to the results of Bcl-xL inhibition (e.g., WEHI-539), the addition of Nutlin released p53 from Bcl-xL under erlotinib treatment ( Figure 4 G). These data are consistent with previous observations that p53 stabilization can promote cytoplasmic p53-mediated apoptosis. Supporting the suggestion that cytoplasmic p53 activity is required for EGFRi- and nutlin-induced apoptosis in metabolic responders, inhibiting cytoplasmic p53 activity with PFTμ significantly reduced the synergistic effect of the combination ( Figure 14 E), whereas p53, a p53 mutation restricted to the nucleus, NES In HK301 cells containing α-glucan, apoptosis induced by erlotinib and nutlin could not be enhanced ( Figure 14 Finally, the cancer "hotspot" mutations R175H and R273H (which have both transactivation and cytoplasmic defects) were completely unaffected by the drug combination (F). Figure 14 F).

[0272] Although cytoplasmic p53 is desirable for promoting cell death by drug combinations, we observed that in some instances, both transcription-dependent and transcription-independent functions of p53 are required for optimal execution of synergistic apoptosis by Nutlin ( Figure 14F). These results are consistent with reports that while the transcription-independent function of p53 can alone execute intrinsic apoptosis, in other situations its transcription-dependent function may be required to promote cytoplasmic p53-mediated cell killing. Collectively, the results described herein demonstrate that combined targeting of EGFR-driven glucose metabolism and p53 can induce significant synergistic cell death in primary GBM, and that the combined targeting is dependent on the cytoplasmic function of p53.

[0273] Example 9 : Regulation of glucose metabolism primes EGFRi non-responders for p53-mediated cell death Based on these data, the inventors proposed a model in which EGFRi-mediated reduction in glucose metabolism activates the apoptotic machinery, resulting in a synergistic effect with pro-apoptotic stimuli such as p53 activation. Synergy exists between the induction of cellular stress by EGFR inhibitors, the reduction of glucose uptake, and the activation of cells toward apoptosis, and the stabilization of p53 by BCL-2 antagonists. EGFR inhibition can rapidly reduce glycolysis through cellular stress. This creates a tumor-specific vulnerability in which intrinsic apoptosis can be significantly enhanced by 1) p53 activation (e.g., with Nutlin, an analog described herein, or others) and 2) BCL-2 inhibition (with any of several agents described herein, such as ABT-263 (Navitoclax)).

[0274] A logical prediction of this model is that direct inhibition of glucose metabolism should phenocopy the effects of EGFRi. Excitement Interestingly, inhibition of oxidative phosphorylation by oligomycin (complex V / ATP synthase) or rotenone (complex I) did not synergize with Nutlin treatment in HK301 glioma spheres ( Figure 15 C and Figure 15 D). Therefore, a reduction in glucose metabolic flux alone, but not oxidative metabolism, appears to be sufficient for the synergistic sensitivity to p53 activation.

[0275] This led us to investigate whether modulating glucose consumption in EGFRi non-responders would result in similar p53-dependent vulnerability. To investigate this, we tested whether direct inhibition of glucose uptake by 2DG or targeted PI3K (a well-characterized driver of glucose metabolism) induced apoptosis activation in two EGFRi metabolic non-responders ( Figure 5 A). In contrast to erlotinib treatment, acute inhibition of PI3K by pictilisib inhibited PI3K-AKT-mTOR signaling ( Figure 15 E) In HK393 and HK254 cells 18 F-FDG uptake was significantly reduced ( Figure 5 B). The reduction in glucose consumption by pictilisib was associated with significantly greater apoptosis activation, and as expected, 2DG fully reflected these effects ( Figure 5 B and Figure 5 C). Therefore, EGFRi metabolic non-responders can be activated toward apoptosis after inhibition of glucose uptake. Importantly, CRISPR / CAS-9 targeting of p53 in HK393 significantly suppressed 2DG- or pictilisib-mediated activation ( Figure 5 D). Furthermore, p53-dependent activation was associated with increased binding of Bcl-xL and p53, indicating that sequestration of p53 by Bcl-xL inhibits apoptosis ( Figure 5 E and Figure 15 F). Consistent with this interpretation, combining 2DG or pictilisib with Nutlin resulted in significant p53-dependent synergistic killing in EGFRi non-responder cells. Figure 5 F and Figure 5G). Collectively, these data demonstrate that acute inhibition of glucose metabolism, either directly or with targeted therapy, promotes p53-dependent apoptosis activation in GBM, rendering them vulnerable to increased cell killing.

[0276] Example 10 : Combination therapeutic strategies and non-invasive biomarkers for targeting GBM in vivo Results obtained in cell culture indicate that the combined targeting of oncogene-driven glucose metabolism and p53 exhibits synergistic activity in primary GBM. Based on this, we investigated whether this approach could be effective in orthotopic GBM xenograft models. In these studies, we employed idasanutlin, a potent MDM2 inhibitor currently in clinical trials for a number of malignancies. Ta.

[0277] Next, because the alteration of glucose metabolism by oncogene inhibition is necessary for synergistic sensitivity to p53 activation, we hypothesized that a rapid decrease in in vivo glucose uptake after administration of EGFRi ( 18 We reasoned that F-FDG PET) could serve as a noninvasive predictive biomarker for the therapeutic effect of erlotinib + idasanutlin combination treatment ( Figure 6 A). We demonstrated that acute erlotinib treatment (75 mg / kg) significantly increased the tumor growth rate in orthotopic xenografts of EGFR metabolic responder glioma spheres (GBM39). 18 We observed a rapid decline in F-FDG uptake (15 hours after erlotinib administration) Figure 6B) We tested the individual agents and the combination of erlotinib (75 mg / kg) and idasanutlin (50 mg / kg) in daily treatment in separate groups of mice. Compared to single-agent controls, we observed synergistic growth inhibition (measured by secreted Gaussia luciferase) in GBM39 intracranial tumor-bearing mice with minimal toxicity. Figure 6B)In contrast, orthotopic xenografts of metabolic non-responders (HK393) showed no significant improvement in response to acute EGFRi. 18 No changes in F-FDG uptake were observed ( Figure 6D) , nor was synergistic activity demonstrated by the combination of erlotinib and idasanutlin ( Figure 6 E). Therefore, a noninvasive method for measuring rapid changes in glucose uptake by EGFRi is 18 F-FDG PET was effective in predicting subsequent synergistic sensitivity to the combination of erlotinib and idasanutlin.

[0278] Finally, we evaluated the effect of the drug combination on overall survival in orthotopic xenografts of either two EGFRi metabolic responders (GBM39 and HK336) or two non-responders (HK393 and GS025). All tumors were p53 wild-type ( Figure 9 A). Following evidence of tumor growth (measured by Gaussia luciferase), mice were treated with vehicle, erlotinib, idasanutlin, or the combination for up to 25 days. The drug combination resulted in a significant prolongation of survival only in EGFRi metabolic responder GBM tumors ( Figure 10 F~ Figure 10 I). Collectively, these data demonstrate that combined targeting of EGFR and p53 synergistically inhibits growth and prolongs survival in a subset of p53 wild-type GBM orthotopic xenografts. Importantly, 18 F-FDG PET was useful as a noninvasive predictive biomarker of sensitivity to this new combination treatment strategy.

[0279] Example 11 Direct inhibition of glycolysis by 2DG or cytochalasin B We tested how direct inhibition of glycolysis with a hexokinase inhibitor (2DG) and a glucose transporter inhibitor (cytochalasin B) affected p53 activation by Nutlin. Figure 16The results shown in Figure 1 demonstrate that low glucose (0.25 mM) results in synergistic cell killing due to BCL-xL inhibition by navitoclax or nutlin. Cell death was measured using Annexin V staining in gliomasphere samples treated for 72 hours with the glycolysis inhibitor 2DG or cytochalasin B, either as single agents or in combination with the p53 activator nutlin. The same effect was reproduced by culturing gliomaspheres under low glucose conditions (0.25 mM) and treating them with nutlin or navitoclax (ABT-263) for 72 hours.

[0280] Example 12 :Experimental procedure mouse Female NOD scid gamma (NSG) mice (6–8 weeks old) were purchased from the University of California, Los Angeles (UCLA) Medical Center animal breeding facility. Male CD-1 mice (6–8 weeks old) were purchased from Charles River. All mice were maintained under defined, pathogen-free conditions in the AAALAC-approved animal facility at the Division of Laboratory Animals (DLAM) at UCLA. All animal experiments were performed with approval from the UCLA Office of Animal Resource Oversight (OARO).

[0281] Patient-derived GBM cells All patient tissues for obtaining GBM cell cultures were obtained through explicit informed consent using UCLA Institutional Review Board (IRB) protocol 10-00065, as previously described. 12After primary GBM cells were established, they were maintained under gliomasphere conditions consisting of DMEM / F12 (Gibco), B27 (Invitrogen), penicillin-streptomycin (Invitrogen), and Glutamax (Invitrogen) supplemented with heparin (5 μg / mL, Sigma), EGF (50 ng / mL, Sigma), and FGF (20 ng / mL, Sigma). All cells were grown at 37°C, 20% O2, and 5% CO2, and were routinely monitored and tested negative for the presence of mycoplasma using a commercially available kit (MycoAlert, Lonza). At the time of the experiment, most HK lines used were at passages 20–30 (exceptions: HK385 p8 and HK336 p15), while the GS and GBM39 lines were at passages less than 10. All cells were characterized by short tandem repeat (STR) analysis.

[0282] Reagents and antibodies For in vitro studies, chemical inhibitors obtained from the following sources were dissolved in DMSO: erlotinib (Chemietek), Nutlin-3A (Selleck Chemicals), WEHI-539 (APExBIO), pictilisib (Selleck Chemicals), oligomycin (Sigma), and rotenone (Sigma). 2DG (Sigma) was dissolved in medium freshly before use. Antibodies used for immunoblotting were from the following suppliers: β-actin (Cell Signaling, 3700), tubulin (Cell Signaling, 3873), p-EGFR Y1086 (Thermo Fischer Scientific, 36-9700), t-EGFR (Millipore, 06-847), t-AKT (Cell Signaling, 4685), p-AKT T308 (Cell Signaling, 13038), p-AKT S473 (Cell Signaling, 4060), t-ERK (Cell Signaling, 4695), p-ERK T202 / Y204 (Cell Signaling, 4370), t-S6 (Cell Signaling, 2217), p-S6 S235 / 236 (Cell Signaling, 4858), t-4EBP1 (Cell Signaling, 4858), and t-AKT T308 (Cell Signaling, 13038). Signaling, 9644), p-4EBP1 S65 (Cell Signaling 9451), Glut3 (Abcam, ab15311), Glut1 (Millipore, 07-1401), p53 (Santa Cruz Biotechnology, SC-126), BAX (Cell Signaling, 5023), BIM (Cell Signaling, 2933), Bcl-2 (Cell Signaling, 2870), Bcl-xL (Cell Signaling, 2764), Mcl-1 (Cell Signaling, 5453), cytochrome c (Cell Signaling, 4272), and cleaved caspase 3 (Cell Signaling, 9661) were obtained from Cell Signaling. Antibodies used for immunoprecipitation were obtained from the sources listed: p53 (Cell Signaling, 12450) and Bcl-xL (Cell Signaling, 2764).Secondary antibodies were obtained from the listed suppliers: anti-rabbit IgG HRP-conjugated (Cell Signaling, 7074) and anti-mouse IgG HRP-conjugated (Cell Signaling, 7076). All immunoblot antibodies were used at a dilution of 1:1000, except for β-actin and tubulin, which were used at 1:10,000. Immunoprecipitation antibodies were diluted according to the manufacturer's instructions (p53 1:200, Bcl-xL 1:100). Secondary antibodies were used at a dilution of 1:5000.

[0283] 18 F-fluorodeoxyglucose (18F-FDG) uptake assay. 5 x 10 cells 4 Cells were seeded at 1000 cells / ml and then treated with the indicated drugs at the indicated time points. Following the appropriate treatment, cells were harvested and 18 The cells were resuspended in glucose-free DMEM / F12 (USBiological) containing F-FDG (radioactivity 1 μCi / mL). Cells were incubated at 37°C for 1 hour and then washed three times with ice-cold PBS. The radioactivity of each sample was then measured using a gamma counter.

[0284] Glucose, glutamine, and lactate measurements Cellular glucose consumption and lactate production were measured using a Nova Biomedical BioProfile Basic Analyzer. Briefly, cells were cultured in 2 mL of glioma sphere and appropriate drug conditions, 1 x 10 5 Cells were seeded at 0.1 cells / ml (n=5). 12 hours after drug treatment, 1 ml of medium was removed from each sample and analyzed using a Nova BioProfile analyzer. Measurements were normalized to cell number.

[0285] Annexin V apoptosis assay Cells were harvested and analyzed by Annexin V and PI staining according to the manufacturer's protocol (BD Biosciences). Briefly, 5 × 10 cells were cultured at 4°C for 1 hour. 4Cells were seeded at 2000 cells / ml and then treated with the appropriate drug. After the indicated time points, cells were harvested, trypsinized, washed with PBS, and stained with Annexin V and PI for 15 minutes. Samples were then analyzed using a BD LSRII flow cytometer.

[0286] Immunoblot Cells were harvested and lysed in RIPA buffer (Boston BioProducts) containing Halt Protease and Phosphatase Inhibitor (Thermo Fischer Scientific). Lysates were centrifuged at 14,000 × g at 4°C for 15 minutes. Protein samples were then boiled in NuPAGE LDS Sample Buffer (Invitrogen) and NuPAGE Sample Reducing Agent (Invitrogen) before separation using SDS-PAGE on 12% Bis-Tris gels (Invitrogen) and transfer to nitrocellulose membranes (GE Healthcare). Immunoblotting was performed according to the antibody manufacturer's specifications and as previously described. Membranes were stained using the SuperSignal system (Thermo Fischer Scientific).

[0287] Immunoprecipitation Cells were harvested, washed once with PBS, and incubated in IP lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol) at 4°C for 15 minutes. Next, 300–500 μg of each sample was precleared with Protein A / G Plus Agarose Beads (Thermo Fischer Scientific) for 1 hour. After preclearing, the samples were incubated overnight with antibody-bead complexes according to the manufacturer's specifications and as previously described. The samples were then centrifuged at 1000 g for 1 minute, and the beads were washed five times with 500 μL of IP lysis buffer. Protein was eluted from the beads by boiling at 95°C for 5 minutes in 2× LDS Sample Buffer (Invitrogen). Samples were analyzed by immunoblotting as previously described. Immunoprecipitation antibodies were diluted according to the manufacturer's instructions (1:200 for p53 and 1:100 for Bcl-xL).

[0288] Dynamic BH3 Profiling GBM glioma spheres were first dissociated into a single cell suspension containing TrypLE (Gibco) and then resuspended in MEB buffer (150 mM mannitol, 10 mM HEPES-KOH, 50 mM KCl, 0.02 mM EGTA, 0.02 mM EDTA, 0.1% BSA, 5 mM succinate). 50 μl of cell suspension (3 × 10 4Cells (1000 cells / well) were seeded into wells of a 96-well plate containing 50 μL of MEB buffer containing 0.002% digitonin and the indicated peptide. The plates were then incubated at 25°C for 50 minutes. Cells were then fixed with 4% paraformaldehyde for 10 minutes and neutralized with N2 buffer (1.7 M Tris, 1.25 M glycine pH 9.1) for 5 minutes. Samples were stained overnight with 20 μL of staining solution (10% BSA, 2% Tween 20 in PBS) containing DAPI and anti-cytochrome c (BioLegend). The following day, cytochrome c release was quantified using a BD LSRII flow cytometer. Measurements were normalized to appropriate controls (DMSO and inactive PUMA2A peptide) that do not stimulate cytochrome c release. Delta activation refers to the difference in the amount of cytochrome c release between vehicle-treated and drug-treated cells.

[0289] BAX oligomerization 7.5×10 5 Cells were treated with the indicated drugs. 24 hours after treatment, cells were harvested, washed once with ice-cold PBS, and resuspended in 1 mM bismaleimidohexane (BMH) in PBS for 30 minutes. Cells were then pelleted and lysed for immunoblotting as described above.

[0290] Cytochrome c detection 1 x 10 5 million cells 5 Cells were seeded at a concentration of 0.1 cells / mL and then treated with the indicated drugs. 24 hours after treatment, cells were harvested and washed once with ice-cold PBS. Subcellular fractionation was then performed using a mitochondrial isolation kit (Thermo Fischer Scientific, 89874). Both the cytosolic and mitochondrial fractions were subjected to immunoblotting, followed by detection of cytochrome c using a 1:1000 dilution of cytochrome c antibody (Cell Signaling, 4272).

[0291] Example 13 : Exemplary design rationale for specific compounds in the JGK series Certain compounds of the present disclosure were designed according to Scheme 1. [ka]

[0292] Example 14 Preparation of further exemplary compounds of the JGK series Exemplary compounds of the present disclosure were prepared according to the following methods.

[0293] [ka] Scheme 1. Synthesis of monoprotected quinazoline intermediate 3.

[0294] [ka] Scheme 2. Synthesis of JGK063–JGK070.

[0295] [ka] Scheme 3. Synthesis of JGK068S ((S)-JGK068). Synthesis was carried out in the same manner as for the racemic sample of JGK068 ((±)-JGK068), but using enantiomerically pure (S)-(-)-glycidol. (R)-(+)-glycidol (not shown) was used to prepare the other enantiomer, JGK068R ((R)-JGK068).

[0296] [ka] Scheme 4. Using chiral SFC (Chiralpak AD-3 column, 40% MeOH), the Mosher ester derivative of 5 ( Figure 18 )of 19 The enantiomeric purity of synthetic intermediate 5 was determined by comparing the F NMR spectra.

[0297] [ka] Scheme 5. Synthesis of JGK071.

[0298] [ka] Scheme 6. Synthesis of JGK072.

[0299] [ka] Scheme 7. Synthesis of JGK076–JGK080.

[0300] [ka] Scheme 8. Synthesis of JGK086–JGK090.

[0301] General Chemical Information All chemicals, reagents, and solvents, when available, were purchased from commercial sources and used as received. When necessary, reagents and solvents were purified and dried by standard methods. Air-sensitive and moisture-sensitive reactions were carried out in oven-dried glassware under an inert atmosphere of argon. Microwave-irradiated reactions were carried out in a single-mode reactor, a CEM Discover microwave synthesizer. Room temperature (RT) reactions were carried out at ambient temperature (approximately 23 °C). All reactions were carried out on pre-coated Merck 60 F plates, with spots visualized using UV light (λ = 254, 365 nm) or alkaline KMnO4 solution. 254 Thin layer chromatography (TLC) was performed using silica gel plates. Flash column chromatography (FC) was performed using SiO260 (particle size 0.040-0.063 mm, 230-400 mesh). Preparative thin layer chromatography (PTLC) was performed using Merck 60 F 254Silica gel plates (20 x 20 cm, 210-270 mm) or Analtech silica gel GF TLC plates (20 x 20 cm, 1000 mm) were used. Concentration under reduced pressure (vacuum) was performed by rotary evaporation at 23-50 °C. Purified compounds were further dried under high vacuum or in a desiccator. Yields corresponded to the purified compounds and were not further optimized. Proton nuclear magnetic resonance ( 1 H NMR spectra were recorded using a Bruker spectrometer operating at 300, 400, or 500 MHz. Carbon NMR ( 13 C NMR spectra were recorded using a Bruker spectrometer (either 400 or 500 MHz). NMR chemical shifts (δ ppm) were referenced to the residual solvent signal. 1 H NMR data are reported as follows: chemical shift (ppm); multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet / complex pattern, td = triple doublet, ddd = double doublet, br = broad signal); coupling constant (J) (Hz), integration. 13 C NMR spectral data are reported based on chemical shifts and, where applicable, coupling constants. High-resolution mass (HRMS) spectra were recorded using a Thermo Fisher Scientific Exactive Plus equipped with an IonSense ID-CUBE DART source mass spectrometer or a Waters LCT Premier mass spectrometer equipped with an ACQUITY UPLC with an autosampler.

[0302] General Procedure (GP). GP-1: Nucleophilic substitution of quinazolinyl mesylate with secondary amines. A mixture of quinazolinyl mesylate (1 equiv.) in DMF (0.05 M) was treated with secondary amine (5 equiv.) and triethylamine (2 equiv.), and the mixture was stirred at 85 °C for 24 h. The mixture was cooled to 23 °C and evaporated. The residue was dissolved in EtOAc (20 mL), washed with 10 mM NaOH (4 × 5 mL), brine (5 mL), dried (NaSO), filtered, and evaporated. Purification by FC or PTLC typically afforded the desired product as an off-white, brittle foam.

[0303] GP-2: Nucleophilic aromatic substitution of 4-chloroquinazoline with aniline. A mixture of 4-chloroquinazoline (1 equiv.) in acetonitrile (0.1 M) was treated with aniline (2 equiv.) and a 4 M solution of HCl in dioxane (1 equiv.). The mixture was heated at 80 °C for 30 min under microwave irradiation. The mixture was either concentrated under reduced pressure or precipitated, and 4-anilinoquinazoline hydrochloride was isolated by filtration (washed with EtO). The residue was suspended in saturated aqueous NaHCO and extracted with CHCl (3x). The combined organic extracts were washed with water and brine, then dried (NaSO), filtered, and concentrated. Purification by FC (eluting with a gradient of CHCl / EtOAc or hexane / EtOAc) typically afforded the desired product as a white to off-white or pale yellow solid.

[0304] 4-(3-Bromo-2-fluoroanilino)quinazoline-6,7-diyl bis(2,2-dimethylpropionic acid) (1). [ka] 4-Chloroquinazoline-6,7-diyl bis(2,2-dimethylpropionic acid) in iPrOH (450 mL) 1A mixture of 1 (41.08 g, 113 mmol) and 3-bromo-2-fluoroaniline (17.05 mL, 152 mmol) was treated with 3-bromo-2-fluoroaniline (17.05 mL, 152 mmol) and stirred at 80 °C for 3.5 h. The mixture was cooled to 23 °C and evaporated. The residue was resuspended several times in hexane (50 mL), concentrated, and then dried under high vacuum. The residue was recrystallized from EtOH to give a yellow solid, which was suspended in saturated aqueous NaHCO3 (1 L) and extracted with DCM (3 × 550 mL). The combined organics were washed with water (400 mL), brine (400 mL), dried (MgSO4), filtered, and evaporated to give the title compound 1 (35.057 g, 60%) as a yellow, brittle foam.

[0305] 1 H NMR (500 MHz, CDCl3): δ = 8.76 (s, 1H), 8.46 (t, J = 7.5 Hz, 1H), 7.72 (s, 1H), 7.68 (s, 1H), 7.56 (br, 1H), 7.32 (ddd, J = 8.0, 6.4, 1.5 Hz, 1H), 7.11 (td, J = 8.2, 1.5 Hz, 1H), 1.40 (s, 9H), 1.39 ppm (s, 9H). 13 C NMR (126 MHz, CDCl3): δ = 176.13, 175.55, 156.71, 154.96, 150.69 (d, J CF = 243.7 Hz), 148.75, 147.83, 142.45, 128.27, 127.86 (d, J CF HRMS (DART): m / z [M + H] + C 24 H 26 BrFN3O4 + Calculated value: 518.1085; measured value: 518.1072.

[0306] 4-(3-Bromo-2-fluoroanilino)quinazoline-6,7-diol (2). [ka] A stirred slurry of 1 (34.988 g, 67.5 mmol) was treated with a 7 M solution of NH in MeOH (241 mL, 1.69 mol) at 0 °C. The mixture was stirred at 0 °C for 15 min and then at 23 °C for 4.5 h. The mixture was evaporated, and the residue was suspended in water (400 mL), stirred overnight, and filtered. The residue was washed with water (500 mL), acetonitrile (100 mL), DCM (4 × 150 mL), EtO (2 × 150 mL), and then dried in a desiccator to give the title compound 2 (23.68 g, quant.) as a pale yellow powder.

[0307] 1 H NMR (500 MHz, DMSO-d6): δ = 8.18 (s, 1H), 7.59 - 7.47 (m, 2H), 7.51 (s, 1H), 7.16 (t, J = 8.0 Hz, 1H), 6.87 ppm (s, 1H). 13 C NMR (126 MHz, DMSO-d6): δ = 156.43, 156.12, 153.06 (d, J CF = 246.7 Hz), 151.34, 148.39, 146.80, 129.23, 129.01, 127.12, 125.23 (d, J CF = 4.3 Hz), 108.47, 108.32, 107.09, 103.04 ppm. HRMS (DART): m / z [M + H] + C 14 H 10 BrFN3O2 + Calculated value: 349.9935; measured value: 349.9923.

[0308] 4-(3-Bromo-2-fluoroanilino)-7-hydroxyquinazolin-6-yl 2,2-dimethylpropanoate (3). [ka] A stirred suspension of 2 (3500 mg, 10.0 mmol) in DMF (52.6 mL) was treated with EtN (5.57 mL, 40.0 mmol), cooled to −40 °C, and treated dropwise with PivO (3.14 mL, 15.5 mmol). The mixture was stirred at −40 °C for 1 h, then removed from the cooling bath and stirring continued for 2.5 h. The reaction mixture was diluted with DCM (500 mL), washed with 10% citric acid (2 × 50 mL), dried (NaSO), filtered, and evaporated. FC (DCM / EtOAc 1:1 → 0:1) gave a solid that was redissolved in EtOAc (750 mL), washed with half-saturated aqueous NHCl (4 × 75 mL), dried (NaSO), filtered, and evaporated to give the title compound 3 (2.844 g, 66%) as a beige-yellow solid.

[0309] 1 H NMR (500 MHz, DMSO-d6): δ = 11.00 (br, 1H), 9.70 (s, 1H), 8.39 (s, 1H), 8.14 (s, 1H), 7.59 (ddd, J = 8.0, 6.2, 1.6 Hz, 1H), 7.53 (ddd, J = 8.3, 7.1, 1.6 Hz, 1H), 7.21 (td, J = 8.1, 1.2 Hz, 1H), 7.17 (s, 1H), 1.36 ppm (s, 9H). 13 C NMR (126 MHz, DMSO-d6): δ = 175.93, 157.68, 154.61, 154.53, 153.34 (d, J CF = 247.3 Hz), 149.80, 139.65, 130.14, 127.92 (d, J CF HRMS (DART): m / z [M + H] + C19 H 18 BrFN3O3 + Calculated value: 434.0510; measured value: 434.0489.

[0310] (±)-4-(3-Bromo-2-fluoroanilino)-7-[(oxiran-2-yl)methoxy]quinazolin-6-yl 2,2-dimethylpropanoate ((±)-4). [ka] A mixture of 3 (1350 mg, 3.11 mmol) and PPh3 (2038 mg, 7.77 mmol) in THF (21 mL) was treated with glycidol (495 μL, 7.46 mmol), then cooled to 0 °C and treated with DIAD (1.47 mL, 7.46 mmol) for 10 min. The mixture was stirred at 23 °C for 2.5 h and then concentrated. FC (DCM / EtOAc 9:1 to 4:6) gave the title compound (±)-4 (848 mg, 56%) as an off-white solid.

[0311] 1 H NMR (500 MHz, CDCl3): δ = 8.73 (s, 1H), 8.54 (ddd, J = 8.6, 7.3, 1.6 Hz, 1H), 7.54 (s, 1H), 7.45 (br, 1H), 7.30 (ddd, J = 8.2, 6.4, 1.5 Hz, 1H), 7.28 (s, 1H), 7.11 (td, J = 8.2, 1.6 Hz, 1H), 4.34 (dd, J = 10.8, 3.0 Hz, 1H), 3.99 (dd, J = 10.8, 6.2 Hz, 1H), 3.35 (ddt, J = 6.2, 4.1, 2.8 Hz, 1H), 2.92 (dd, J = 4.8, 4.1 Hz, 1H), 2.74 (dd, J = 4.8, 2.6 Hz, 1H), 1.45 ppm (s, 9H). 13 C NMR (126 MHz, CDCl3): δ = 176.87, 156.46, 155.10, 154.93, 150.41 (d, J CF= 243.3 Hz), 150.27, 140.99, 128.25 (d, J CF = 10.5 Hz), 127.75, 125.28 (d, J CF = 4.7 Hz), 122.22, 114.02, 109.72, 109.49, 108.74 (d, J CF = 19.1 Hz), 70.05, 49.55, 44.56, 39.45, 27.38 ppm. HRMS (DART): m / z [M + H] + C 22 H 22 BrFN3O4 + Calculated value: 490.0772; measured value: 490.0764.

[0312] (±)-N-(3-Bromo-2-fluorophenyl)-7-ethenyl-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine ((±)-JGK062). [ka] A solution of PPh3 (832 mg, 3.17 mmol) and DIAD (624 μL, 3.17 mmol) in THF (23 mL) was stirred at 0° C. for 15 min, then added dropwise to a solution of (±)-8 (1149 mg, 2.73 mmol) in THF (27 mL) at 0° C. for 10 min. The mixture was stirred at 0° C. for 2 h, then evaporated. FC (hexane / EtOAc 9:1 → 4:6) followed by another FC (DCM / EtOAc 1:0 → 6:4) gave the title compound (±)-JGK062 (1115 mg, quant.) as an off-white brittle foam.

[0313] 1H NMR (500 MHz, CDCl3): δ = 8.68 (s, 1H), 8.65 (ddd, J = 8.2, 7.3, 1.5 Hz, 1H), 7.40 (s, 1H), 7.37 (br, 1H), 7.35 (s, 1H), 7.27 (ddd, J = 8.0, 6.4, 1.5 Hz, 1H), 7.10 (td, J = 8.2, 1.6 Hz, 1H), 5.95 (ddd, J = 17.3, 10.7, 5.8 Hz, 1H), 5.60 (dt, J = 17.3, 1.2 Hz, 1H), 5.48 (dt, J = 10.7, 1.1 Hz, 1H), 4.82 - 4.74 (m, 1H), 4.42 (dd, J = 11.5, 2.5 Hz, 1H), 4.09 ppm (dd, J = 11.6, 8.1 Hz, 1H). 13 C NMR (126 MHz, CDCl3): δ = 155.90, 153.38, 150.14 (d, J = 242.4 Hz), 149.12, 146.70, 144.12, 131.48, 128.64 (d, J = 10.3 Hz), 127.24, 125.30 (d, J = 4.7 Hz), 121.76, 120.43, 114.29, 110.69, 108.58 (d, J = 19.3 Hz), 106.06, 74.03, 67.84 ppm. HRMS (DART): m / z [M + H] + C 18 H 14 BrFN3O2 + Calculated value: 402.0248; measured value: 402.0233.

[0314] (±)-[4-(3-Bromo-2-fluoroanilino)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-7-yl]methanol ((±)-5). [ka] A mixture of (±)-4 (842 mg, 1.72 mmol) in MeOH (31 mL) was treated with KCO (482 mg, 3.49 mmol), then stirred at 23 °C for 10.5 h and concentrated. The residue was suspended in half-saturated aqueous NHCl (130 mL) and extracted with EtOAc (3 × 20 mL). The combined organics were washed with water (20 mL), brine (20 mL), then dried (NaSO), filtered, and concentrated to give the title compound (±)-5 (720 mg, quant.) as a yellow solid.

[0315] 1 H NMR (500 MHz, DMSO-d6): δ = 9.59 (s, 1H), 8.34 (s, 1H), 7.95 (s, 1H), 7.59 (ddd, J = 8.0, 6.2, 1.6 Hz, 1H), 7.55 (ddd, J = 8.4, 7.0, 1.6 Hz, 1H), 7.24 - 7.18 (m, 1H), 7.21 (s, 1H), 5.16 (t, J = 5.6 Hz, 1H), 4.49 (dd, J = 11.5, 2.4 Hz, 1H), 4.34 (dtd, J = 7.6, 5.2, 2.3 Hz, 1H), 4.21 (dd, J = 11.5, 7.4 Hz, 1H), 3.76 - 3.64 ppm (m, 2H). 13 C NMR (126 MHz, DMSO-d6): δ = 157.20, 153.35 (d, J CF = 247.5 Hz), 153.10, 148.88, 145.95, 143.39, 130.11, 128.05 (d, J CF = 13.0 Hz), 127.73, 125.44 (d, J CF = 4.4 Hz), 112.33, 109.79, 108.56 (d, J CF = 20.0 Hz), 108.37, 73.78, 65.50, 59.78 ppm. HRMS (DART): m / z [M + H] + C 17 H 14 BrFN3O3 +Calculated value: 406.0197; measured value: 406.0185.

[0316] (±)-[4-(3-Bromo-2-fluoroanilino)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-7-yl]methyl methanesulfonate ((±)-6). [ka] A solution of (±)-5 (688 mg, 1.69 mmol) in THF (14 mL) was treated with EtN (357 μL, 2.56 mmol), then cooled to 0 °C and treated dropwise with MsCl (174 μL, 2.24 mmol). The mixture was stirred at 23 °C for 16 h, then cooled to 0 °C, treated with saturated aqueous NaHCO (120 mL), and extracted with DCM (3 × 120 mL). The combined organics were washed with water (100 mL), brine (100 mL), dried (NaSO), filtered, and evaporated. FC (DCM / EtOAc 8:2 to 3:7) afforded the title compound (±)-6 (496 mg, 61%) as an off-white solid.

[0317] 1 H NMR (500 MHz, CDCl3): δ = 8.69 (s, 1H), 8.60 (ddd, J = 8.5, 7.2, 1.4 Hz, 1H), 7.43 (s, 1H), 7.39 (br, 1H), 7.37 (s, 1H), 7.29 (ddd, J = 8.1, 6.5, 1.5 Hz, 1H), 7.11 (td, J = 8.2, 1.5 Hz, 1H), 4.63 (dtd, J = 7.2, 4.9, 2.5 Hz, 1H), 4.52 (dd, J = 4.9, 0.9 Hz, 2H), 4.49 (dd, J = 11.8, 2.5 Hz, 1H), 4.29 (dd, J = 11.8, 7.1 Hz, 1H), 3.13 ppm (s, 3H). 13 C NMR (126 MHz, CDCl3): δ = 156.02, 153.66, 150.28 (d, J CF= 242.9 Hz), 148.65, 146.80, 143.09, 128.43 (d, J CF = 10.4 Hz), 127.54, 125.32 (d, J CF = 4.7 Hz), 122.01, 114.77, 110.90, 108.66 (d, J CF = 19.4 Hz), 106.44, 71.10, 66.46, 64.77, 38.02 ppm. HRMS (DART): m / z [M + H] + C 18 H 15 BrFN3O5S + Calculated value: 483.9973; measured value: 483.9950.

[0318] (±)-4-(3-Bromo-2-fluoroanilino)-7-{[2-(acetoxy)but-3-en-1-yl]oxy}quinazolin-6-yl 2,2-dimethylpropanoate ((±)-7). [ka] A mixture of 3 (2639 mg, 6.08 mmol) and PPh3 (3986 mg, 15.2 mmol) in THF (41 mL) was treated with racemic 1-hydroxybut-3-en-2-yl acetate. 2 (1.7 mL, 13.7 mmol), then cooled to 0 °C and treated dropwise with DIAD (2.7 mL, 13.7 mmol). The mixture was stirred at 23 °C for 3 h and then concentrated. FC (DCM / EtOAc 1:0 to 6:4) gave crude (±)-7 (5.508 g, approximate yield 60%), which was contaminated with residual PhPO. The material was used in the next step without further purification.

[0319] 1H NMR (400 MHz, CDCl3): δ = 8.74 (s, 1H), 8.53 (t, J = 7.9 Hz, 1H), 7.53 (s, 1H), 7.45 (br, 1H), 7.33 (s, 1H), 7.30 (t, J = 7.7 Hz, 1H), 7.11 (t, J = 8.0 Hz, 1H), 5.90 (ddd, J = 17.0, 10.6, 6.2 Hz, 1H), 5.65 (q, J = 6.0 Hz, 1H), 5.49 - 5.29 (m, 2H), 4.31 - 4.08 (m, 2H), 2.11 (s, 3H), 1.41 ppm (s, 9H). 13 C NMR (126 MHz, CDCl3): δ = 176.51, 170.08, 156.49, 155.24, 154.88, 150.46 (d, J CF = 243.2 Hz), 150.17, 140.90, 132.16, 128.18 (d, J CF = 11.0 Hz), 127.86, 125.31 (d, J CF = 4.8 Hz), 122.27, 119.64, 114.00, 109.56, 109.39, 108.76 (d, J CF = 19.4 Hz), 72.18, 69.81, 39.34, 27.33, 21.19 ppm. HRMS (DART): m / z [M + H] + C 25 H 26 BrFN3O5 + Calculated value: 546.1034; measured value: 546.1018.

[0320] (±)-4-(3-Bromo-2-fluoroanilino)-7-[(2-hydroxybut-3-en-1-yl)oxy]quinazolin-6-ol ((±)-8). [ka] A mixture of crude (±)-7 (5508 mg, contaminated with residual PhPO from the previous step) in MeOH (61 mL) was treated with KCO (4198 mg, 30.4 mmol), then stirred at 23 °C for 1 h and concentrated. The residue was suspended in half-saturated aqueous NHCl (1 L) and extracted with EtOAc (3 × 600 mL). The combined organics were dried (NaSO), filtered, and evaporated. FC (DCM / EtOAc 1:1 → 0:1) afforded the title compound (±)-8 (1154 mg, 45% over two steps) as an off-white solid.

[0321] 1 H NMR (500 MHz, DMSO-d6): δ = 9.46 (s, 1H), 9.40 (br, 1H), 8.33 (s, 1H), 7.71 (s, 1H), 7.59 - 7.52 (m, 2H), 7.203 (s), 7.197 (td, J = 8.1, 1.1 Hz, 1H), 6.01 (ddd, J = 17.4, 10.7, 4.9 Hz, 1H), 5.42 (dt, J = 17.3, 1.9 Hz, 1H), 5.36 (br, 1H), 5.20 (dt, J = 10.6, 1.8 Hz, 1H), 4.49 (br, 1H), 4.20 (dd, J = 9.8, 3.8 Hz, 1H), 3.95 ppm (dd, J = 9.8, 7.5 Hz, 1H). 13 C NMR (126 MHz, DMSO-d6): δ = 156.77, 153.30 (d, J CF = 244.9 Hz), 152.77, 152.31, 146.66, 146.11, 137.61, 129.75, 128.46 (d, J CF = 13.0 Hz), 127.49, 125.38 (d, J CF = 4.3 Hz), 115.58, 109.42, 108.50 (d, J CF = 19.8 Hz), 107.68, 105.14, 72.56, 69.26 ppm. HRMS (DART): m / z [M + H] + C18 H 16 BrFN3O3 + Calculated value: 420.0354; measured value: 420.0340.

[0322] (±)-2-[4-(3-bromo-2-fluoroanilino)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-7-yl]ethan-1-ol ((±)-9). [ka] A mixture of (±)-JGK062 (480 mg, 1.19 mmol) in THF (4.8 mL) was treated with a 0.5 M solution of 9-BBN in THF (4.8 mL, 2.39 mmol), and the mixture was stirred at 68 °C for 16 h. The mixture was cooled to 0 °C, diluted with THF (2.4 mL), treated with 3 N NaOH (3 mL, 8.95 mmol) and 30% HO (474 ​​μL, 8.95 mmol), and stirred at 23 °C for 6 h. The mixture was concentrated to approximately half the original THF volume, diluted with water (100 mL) and brine (40 mL), and extracted with EtOAc (3 × 100 mL). The combined organics were washed with water (70 mL), brine (70 mL), then dried (NaSO), filtered and evaporated to give the title compound (±)-9 (912 mg) as a yellow foam, which was used directly in the next step without further purification.

[0323] 1H NMR (500 MHz, CDCl3): δ = 8.66 (s, 1H), 8.62 (ddd, J = 8.8, 7.4, 1.6 Hz, 1H), 7.35 (s, 1H), 7.33 (br, 1H), 7.2 (ddd, J = 8.0, 6.5, 1.6 Hz, 1H), 7.16 (s, 1H), 7.09 (td, J = 8.2, 1.6 Hz, 1H), 4.50 (dtd, J = 8.4, 6.4, 2.3 Hz, 1H), 4.43 (dd, J = 11.5, 2.3 Hz, 1H), 4.09 (dd, J = 11.5, 8.2 Hz, 1H), 4.01 - 3.91 (m, 2H), 1.95 ppm (td, J = 6.5, 5.3 Hz, 2H). 13 C NMR (126 MHz, CDCl3): δ = 155.84, 153.28, 150.08 (d, J CF = 242.6 Hz), 149.42, 146.47, 144.20, 128.51 (d, J CF = 10.2 Hz), 127.31, 125.30 (d, J CF = 4.7 Hz), 121.69, 113.95, 110.50, 108.58 (d, J CF = 19.2 Hz), 105.83, 71.33, 68.49, 58.23, 33.61 ppm. HRMS (ESI): m / z [M + H] + C 18 H 16 BrFN3O3 + Calculated value: 420.0354; measured value: 420.0370.

[0324] (±)-2-[4-(3-bromo-2-fluoroanilino)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-7-yl]ethyl methanesulfonate ((±)-10). [ka] A solution of (±)-9 (912 mg) in THF (11.9 mL) was treated with EtN (931 mL, 6.68 mmol), cooled to 0 °C, and treated dropwise with MsCl (462 μL, 5.97 mmol). The mixture was stirred at 0 °C for 15 min and then at 23 °C for 21 h. The mixture was cooled to 0 °C, treated dropwise with saturated aqueous NaHCO (120 mL), and extracted with DCM (3 × 120 mL). The combined organics were washed with water (100 mL), brine (100 mL), dried (NaSO), filtered, and evaporated. FC (DCM / EtOAc 9:1 to 4:6) afforded the title compound (±)-10 (112 mg, 19% over two steps) as an off-white brittle foam.

[0325] 1 H NMR (500 MHz, CDCl3): δ = 8.68 (s, 1H), 8.60 (ddd, J = 8.6, 7.3, 1.5 Hz, 1H), 7.44 (br, 1H), 7.42 (s, 1H), 7.35 (s, 1H), 7.29 (ddd, J = 8.1, 6.5, 1.6 Hz, 1H), 7.11 (td, J = 8.2, 1.5 Hz, 1H), 4.60 - 4.48 (m, 3H), 4.44 (dd, J = 11.6, 2.4 Hz, 1H), 4.12 (dd, J = 11.6, 7.6 Hz, 1H), 3.08 (s, 3H), 2.24 - 2.10 ppm (m, 2H). 13 C NMR (126 MHz, CDCl3): δ = 156.03, 153.39, 150.31 (d, J CF = 242.9 Hz), 149.11, 146.54, 143.60, 128.47 (d, J CF = 10.5 Hz), 127.52, 125.32 (d, J CF = 4.6 Hz), 122.02, 114.30, 110.68, 108.66 (d, J CF= 19.2 Hz), 106.32, 69.78, 67.82, 65.05, 37.75, 30.90 ppm. HRMS (ESI): m / z [M + H] + C 19 H 18 BrFN3O5S + Calculated value: 498.0129; measured value: 498.0144.

[0326] (±)-N-(3-Bromo-2-fluorophenyl)-7-[(morpholin-4-yl)methyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine ((±)-JGK063). [ka] Compound (±)-JGK063 was prepared from (±)-6 (20 mg, 0.04 mmol) and morpholine (18 μL, 0.21 mmol) in DMF (826 μL) according to general procedure GP-1. PTLC (DCM / EtOAc 1:9) afforded (±)-JGK063 (15 mg, 76%) as an off-white brittle foam.

[0327] 1 H NMR (500 MHz, CDCl3): δ = 8.67 (s, 1H), 8.63 (ddd, J = 8.6, 7.3, 1.5 Hz, 1H), 7.38 (s, 1H), 7.37 (br, 1H), 7.31 (s, 1H), 7.27 (ddd, J = 8.0, 6.3, 1.5 Hz, 1H), 7.10 (td, J = 8.2, 1.5 Hz, 1H), 4.50 - 4.41 (m, 2H), 4.21 - 4.12 (m, 1H), 3.75 (t, J = 4.7 Hz, 4H), 2.77 (dd, J = 13.4, 5.9 Hz, 1H), 2.69 - 2.54 ppm (m, 5H). 13 C NMR (126 MHz, CDCl3): δ = 155.89, 153.36, 150.15 (d, J CF= 242.5 Hz), 149.35, 146.66, 144.02, 128.60 (d, J CF = 10.4 Hz), 127.27, 125.30 (d, J CF = 4.6 Hz), 121.80, 114.29, 110.63, 108.58 (d, J CF = 19.5 Hz), 106.06, 71.61, 67.18, 67.01, 58.94, 54.56 ppm. HRMS (ESI): m / z [M - H] - C 21 H 19 BrFN4O3 - Calculated value: 473.0630; Measured value: 473.0630.

[0328] (±)-N-(3-Bromo-2-fluorophenyl)-7-[2-(morpholin-4-yl)ethyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine ((±)-JGK064). [ka] Compound (±)-JGK064 was prepared from (±)-10 (35 mg, 0.07 mmol) and morpholine (31 μL, 0.35 mmol) in DMF (1.4 mL) according to general procedure GP-1. PTLC (EtOAc, 0.5% acetonitrile, 1.5% aqueous NH4OH) followed by another PTLC (EtOAc) afforded (±)-JGK064 (25 mg, 73%) as an off-white brittle foam.

[0329] 1H NMR (500 MHz, CDCl3): δ = 8.68 (s, 1H), 8.65 (ddd, J = 8.3, 7.4, 1.5 Hz, 1H), 7.39 (s, 1H), 7.36 (br, 1H), 7.28 (s, 1H), 7.30 - 7.25 (m, 1H), 7.11 (td, J = 8.2, 1.5 Hz, 1H), 4.44 (dd, J = 11.3, 2.3 Hz, 1H), 4.43 - 4.37 (m, 1H), 4.10 (dd, J = 11.3, 7.7 Hz, 1H), 3.73 (t, J = 4.7 Hz, 4H), 2.62 (ddt, J = 12.5, 8.4, 3.9 Hz, 2H), 2.57 - 2.42 (m, 4H), 2.00 - 1.82 ppm (m, 2H). 13 C NMR (126 MHz, CDCl3): δ = 155.86, 153.31, 150.13 (d, J CF = 242.3 Hz), 149.40, 146.67, 144.33, 128.66 (d, J CF = 10.4 Hz), 127.22, 125.33 (d, J CF = 4.6 Hz), 121.75, 114.21, 110.63, 108.58 (d, J CF HRMS (ESI): m / z [M + H] + C 22 H 23 BrFN4O3 + Calculated value: 489.0932; measured value: 489.0935.

[0330] (±)-N-(3-Bromo-2-fluorophenyl)-7-[(piperidin-1-yl)methyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine ((±)-JGK065). [ka] Compound (±)-JGK065 was prepared from (±)-6 (40 mg, 0.08 mmol) and piperidine (41 μL, 0.41 mmol) in DMF (1.65 mL) according to general procedure GP-1. PTLC (EtOAc) afforded (±)-JGK065 (24 mg, 61%) as an off-white brittle foam.

[0331] 1 H NMR (500 MHz, CDCl3): δ = 8.66 (s, 1H), 8.63 (ddd, J = 8.7, 7.3, 1.5 Hz, 1H), 7.369 (s, 1H), 7.368 (br, 1H), 7.30 (s, 1H), 7.26 (ddd, J = 8.1, 6.5, 1.5 Hz, 1H), 7.09 (td, J = 8.2, 1.5 Hz, 1H), 4.46 (dd, J = 11.3, 2.3 Hz, 1H), 4.43 (ddd, J = 8.3, 5.8, 2.0 Hz, 1H), 4.12 (dd, J = 11.2, 7.5 Hz, 1H), 2.71 (dd, J = 13.3, 5.9 Hz, 1H), 2.58 (dd, J = 13.4, 6.2 Hz, 1H), 2.59 - 2.42 (m, 4H), 1.65 - 1.57 (m, 4H), 1.49 - 1.41 ppm (m, 2H). 13 C NMR (126 MHz, CDCl3): δ = 155.86, 153.26, 150.12 (d, J CF = 242.6 Hz), 149.49, 146.62, 144.23, 128.65 (d, J CF = 10.3 Hz), 127.18, 125.27 (d, J CF = 4.5 Hz), 121.76, 114.16, 110.57, 108.56 (d, J CF HRMS (ESI): m / z [M + H] + C 22H 23 BrFN4O2 + Calculated value: 473.0983; measured value: 473.0991.

[0332] (±)-N-(3-Bromo-2-fluorophenyl)-7-[(dimethylamino)methyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine ((±)-JGK066). [ka] Compound (±)-JGK066 was prepared from (±)-6 (45 mg, 0.09 mmol) and a 2 M solution of MeNH in THF (232 μL, 0.46 mmol) in DMF (1.85 mL) according to general procedure GP-1. PTLC (EtOAc, 0.5% acetonitrile, 1.5% aqueous NHOH) afforded (±)-JGK066 (39 mg, 97%) as an off-white brittle foam.

[0333] 1 H NMR (500 MHz, CDCl3): δ = 8.680 (s, 1H), 8.675 (ddd, J = 8.2, 7.5, 1.5 Hz, 1H), 7.39 (s, 1H), 7.38 (s, 1H), 7.37 (br, 1H), 7.27 (ddd, J = 8.0, 6.4, 1.5 Hz, 1H), 7.10 (d, J = 1.6 Hz, 1H), 4.46 - 4.41 (m, 1H), 4.45 (dd, J = 11.8, 2.3 Hz, 1H), 4.12 (dd, J = 11.9, 8.1 Hz, 1H), 2.73 (dd, J = 13.2, 7.1 Hz, 1H), 2.55 (dd, J = 13.1, 5.0 Hz, 1H), 2.38 ppm (s, 6H). 13 C NMR (126 MHz, CDCl3): δ = 155.89, 153.34, 150.07 (d, J CF = 242.3 Hz), 149.38, 146.67, 144.06, 128.70 (d, J CF= 10.4 Hz), 127.16, 125.29 (d, J CF = 4.7 Hz), 121.65, 114.27, 110.67, 108.56 (d, J CF = 19.4 Hz), 106.15, 71.70, 67.20, 59.78, 46.41 ppm. HRMS (ESI): m / z [M + H] + C 19 H 19 BrFN4O2 + Calculated value: 433.0670; measured value: 433.0677.

[0334] (±)-N-(3-Bromo-2-fluorophenyl)-7-[(pyrrolidin-1-yl)methyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine ((±)-JGK067). [ka] Compound (±)-JGK067 was prepared from (±)-6 (35 mg, 0.07 mmol) and pyrrolidine (30 μL, 0.36 mmol) in DMF (1.45 mL) according to general procedure GP-1. PTLC (EtOAc, 1.5% iPrOH, 1.5% NH4OH in water) afforded (±)-JGK067 (31 mg, 93%) as an off-white brittle foam.

[0335] 1H NMR (500 MHz, CDCl3): δ = 8.68 (s, 1H), 8.67 (ddd, J = 8.7, 7.5, 1.6 Hz, 2H), 7.39 (s, 1H), 7.36 (br, 1H), 7.35 (s, 1H), 7.27 (ddd, J = 8.0, 6.4, 1.5 Hz, 2H), 7.10 (td, J = 8.2, 1.5 Hz, 1H), 4.49 - 4.42 (m, 1H), 4.48 (dd, J = 11.6, 2.0 Hz, 1H), 4.15 (dd, J = 11.7, 8.0 Hz, 1H), 2.88 (dd, J = 12.9, 6.5 Hz, 1H), 2.80 (dd, J = 12.6, 5.5 Hz, 1H), 2.72 - 2.60 (m, 4H), 1.90 - 1.79 ppm (m, 4H). 13 C NMR (126 MHz, CDCl3): δ = 155.87, 153.32, 150.09 (d, J CF = 242.6 Hz), 149.45, 146.68, 144.18, 128.71 (d, J CF = 10.3 Hz), 127.15, 125.30 (d, J CF = 4.7 Hz), 121.67, 114.26, 110.65, 108.56 (d, J CF = 19.4 Hz), 106.06, 72.73, 67.35, 56.57, 55.15, 23.75 ppm. HRMS (ESI): m / z [M + H] + C 21 H 21 BrFN4O2 + Calculated value: 459.0826; measured value: 459.0845.

[0336] (±)-N-(3-Bromo-2-fluorophenyl)-7-[(4-methylpiperazin-1-yl)methyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine ((±)-JGK068). [ka] Compound (±)-JGK068 was prepared from (±)-6 (35 mg, 0.07 mmol) and 1-methylpiperazine (40 μL, 0.36 mmol) in DMF (1.45 mL) according to general procedure GP-1. PTLC (EtOAc / iPrOH 85:15, 1.5% NH4OH in water) afforded (±)-JGK068 (29 mg, 82%) as an off-white brittle foam.

[0337] 1 H NMR (500 MHz, CDCl3): δ = 8.68 (s, 1H), 8.64 (ddd, J = 8.3, 7.3, 1.5 Hz, 1H), 7.39 (s, 1H), 7.36 (br d, J = 3.8 Hz, 1H), 7.32 (s, 1H), 7.27 (ddd, J = 8.0, 6.5, 1.6 Hz, 1H), 7.10 (td, J = 8.2, 1.5 Hz, 1H), 4.48 - 4.41 (m, 2H), 4.15 (dd, J = 11.5, 8.6 Hz, 1H), 2.78 (dd, J = 13.4, 6.0 Hz, 1H), 2.661 (dd, J = 13.4, 5.8 Hz, 1H), 2.656 (br, 4H), 2.51 (br, 4H), 2.32 ppm (s, 3H). 13 C NMR (126 MHz, CDCl3): δ = 155.89, 153.35, 150.15 (d, J CF = 242.6 Hz), 149.40, 146.69, 144.11, 128.64 (d, J CF = 10.3 Hz), 127.24, 125.30 (d, J CF = 4.7 Hz), 121.78, 114.27, 110.63, 108.59 (d, J CF HRMS (ESI): m / z [M + H] + C 22 H24 BrFN5O2 + Calculated value: 488.1092; measured value: 488.1109.

[0338] (±)-N-(3-Bromo-2-fluorophenyl)-7-[2-(dimethylamino)ethyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine ((±)-JGK069). [ka] Compound (±)-JGK069 was prepared from a 2 M solution of (±)-10 (32 mg, 0.06 mmol) and MeNH in THF (161 μL, 0.32 mmol) in DMF (1.3 mL) according to general procedure GP-1. PTLC (EtOAc, 5% iPrOH, 1.5% NHOH in water) afforded (±)-JGK069 (19 mg, 66%) as an off-white brittle foam.

[0339] 1 H NMR (500 MHz, CDCl3): δ = 8.67 (s, 1H), 8.63 (ddd, J = 8.7, 7.4, 1.6 Hz, 1H), 7.373 (br, 1H), 7.371 (s, 1H), 7.28 (s, 1H), 7.28 - 7.24 (m, 1H), 7.10 (td, J = 8.2, 1.5 Hz, 1H), 4.42 (dd, J = 11.4, 2.3 Hz, 1H), 4.38 (tdd, J = 7.7, 5.1, 2.3 Hz, 1H), 4.08 (dd, J = 11.3, 7.8 Hz, 1H), 2.56 (t, J = 7.2 Hz, 2H), 2.29 (s, 6H), 1.93 (dq, J = 14.2, 7.4 Hz, 1H), 1.84 ppm (dtd, J = 14.2, 7.5, 5.1 Hz, 1H). 13 C NMR (126 MHz, CDCl3): δ = 155.86, 153.26, 150.14 (d, J CF= 242.4 Hz), 149.42, 146.65, 144.36, 128.67 (d, J CF = 10.5 Hz), 127.18, 125.30 (d, J CF = 4.7 Hz), 121.77, 114.14, 110.60, 108.56 (d, J CF = 19.2Hz), 105.88, 72.19, 68.34, 55.06, 45.58, 29.16 ppm. HRMS (ESI): m / z [M + H] + C 20 H 21 BrFN4O2 + Calculated value: 447.0826; measured value: 447.0820.

[0340] (±)-N-(3-Bromo-2-fluorophenyl)-7-[2-(4-methylpiperazin-1-yl)ethyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine ((±)-JGK070). [ka] Compound (±)-JGK070 was prepared from (±)-10 (32 mg, 0.06 mmol) and 1-methylpiperazine (36 μL, 0.32 mmol) in DMF (1.3 mL) according to general procedure GP-1. PTLC (EtOAc / iPrOH 8:2, 1.5% NH4OH in water) afforded (±)-JGK070 (21 mg, 65%) as an off-white brittle foam.

[0341] 11H NMR (500 MHz, CDCl3): δ = 8.66 (s, 1H), 8.62 (ddd, J = 8.5, 7.3, 1.5 Hz, 1H), 7.373 (br, 1H), 7.367 (s, 1H), 7.29 - 7.24 (m, 1H), 7.28 (s, 1H), 7.09 (td, J = 8.2, 1.5 Hz, 1H), 4.43 (dd, J = 11.4, 2.3 Hz, 1H), 4.37 (tdd, J = 7.7, 5.4, 2.3 Hz, 1H), 4.08 (dd, J = 11.4, 7.9 Hz, 1H), 2.68 - 2.54 (m, 2H), 2.50 (br, 8H), 2.30 (s, 3H), 1.94 (dtd, J = 13.6, 7.5, 6.0 Hz, 1H), 1.86 ppm (dtd, J = 14.2, 7.3, 5.3 Hz, 1H). 13 13C NMR (126 MHz, CDCl3): δ = 155.86, 153.27, 150.16 (d, J CF = 242.5 Hz), 149.41, 146.64, 144.37, 128.64 (d, J CF = 10.3 Hz), 127.22, 125.28 (d, J CF = 4.6 Hz), 121.81, 114.13, 110.60, 108.57 (d, J CF = 19.4 Hz), 105.88, 72.38, 68.36, 55.20, 53.77, 53.25, 46.11, 28.50 ppm. HRMS (ESI): m / z [M + H] + ​​​​​​​​​​​​​​​​​A mixture of 3-fluorobenzene-1,2-diol (7233 mg, 56.5 mmol) in DMF (113 mL) was treated with K2CO3 (19514 mg, 141 mmol), stirred at 23 °C for 10 min, and treated with 1-bromo-2-chloroethane (9.4 mL, 113 mmol). The mixture was stirred at 23 °C for 1 h and then at 95 °C for 16 h. The mixture was cooled to 23 °C, diluted with water (150 mL), and extracted with EtOAc (3 × 150 mL). The combined organics were washed with water (90 mL), brine (90 mL), dried (Na2SO4), filtered, and evaporated. FC (hexane / EtOAc 30:1 to 10:1) gave the title compound 11 (7973 mg, 92%) as a clear, colorless oil.

[0343] 1 H NMR (400 MHz, CDCl3): δ = 6.78 - 6.63 (m, 3H), 4.34 - 4.26 ppm (m, 4H). 13 C NMR (101 MHz, CDCl3): δ = 152.05 (d, J CF = 244.3 Hz), 145.27 (d, J CF = 3.8 Hz), 132.78 (d, J CF = 13.9 Hz), 120.02 (d, J CF = 8.9 Hz), 112.74 (d, J CF = 3.1 Hz), 108.52 (d, J CF = 18.1 Hz), 64.50, 64.45 ppm. HRMS (DART): m / z [M] ·+ C8H7FO2 ·+ Calculated value: 154.0425; measured value: 154.0420.

[0344] 6-Bromo-5-fluoro-2,3-dihydro-1,4-benzodioxin (12). [ka] A solution of 11 (7812 mg, 50.7 mmol) in MeOH (101 mL) was treated with NBS (9022 mg, 50.7 mmol) and heated at 70 °C for 30 min. The mixture was cooled to 23 °C and then concentrated. The residue was dissolved in DCM (700 mL), washed with water (300 mL), dried (MgSO), filtered, and evaporated. FC (hexane / EtOAc 30:1 to 20:1) followed by drying under HV at 100 °C removed all remaining starting material and gave the title compound 12 (8807 mg, 75%) as a clear, colorless oil, containing approximately 15% of the regioisomer, which solidified in the freezer to give an off-white solid.

[0345] 1 H NMR (400 MHz, CDCl3): δ = 6.96 (dd, J = 9.0, 7.0 Hz, 1H), 6.59 (dd, J = 9.0, 2.0 Hz, 1H), 4.35 - 4.24 ppm (m, 4H). 13 C NMR (101 MHz, CDCl3): δ = 148.87 (d, J CF = 245.1 Hz), 144.53 (d, J CF = 3.5 Hz), 133.81 (d, J CF = 14.6 Hz), 123.31, 113.39 (d, J CF = 3.6 Hz), 109.17 (d, J CF = 19.3 Hz), 64.51, 64.34 ppm. HRMS (DART): m / z [M] ·+ C8H6BrFO2 ·+ Calculated value: 231.9530; measured value: 231.9525.

[0346] 5-Fluoro-2,3-dihydro-1,4-benzodioxine-6-carboxylic acid (13). [ka] A mixture of 12 (7.0 g, 30.0 mmol) in THF (108 mL) was cooled to −78 °C and treated dropwise with a 2.5 M solution of nBuLi in hexanes (12.02 mL, 30.0 mmol) over 10 min. The mixture was stirred at −78 °C for 30 min and then transferred via tube onto crushed dry ice (the tube was rinsed with 10 mL of THF). The mixture was warmed to 23 °C and then concentrated. Water (200 mL) and 1 M NaOH (50 mL) were added to the residue, and the aqueous phase was extracted with EtO (3 × 60 mL). The aqueous phase was acidified with 6 M HCl (15 mL) and then extracted with DCM (3 × 150 mL). The combined organic solution was washed with brine (150 mL), dried (MgSO), filtered, and evaporated. FC (hexane / EtOAc 7:3 -> 3:7) afforded the title compound 13 (3591 mg, 60%) as a white solid.

[0347] 1 H NMR (400 MHz, DMSO-d6): δ = 12.90 (br, 1H), 7.33 (dd, J = 8.9, 7.7 Hz, 1H), 6.78 (dd, J = 8.9, 1.7 Hz, 1H), 4.39 - 4.29 ppm (m, 4H). 13 C NMR (101 MHz, DMSO-d6): δ = 164.65 (d, J CF = 3.0 Hz), 151.21 (d, J CF = 257.5 Hz), 148.50 (d, J CF = 4.4 Hz), 132.68 (d, J CF = 13.6 Hz), 122.44 (d, J CF = 1.4 Hz), 112.12 (d, J CF = 3.4 Hz), 111.97 (d, J CF = 7.3 Hz), 64.42, 63.91 ppm. HRMS (DART): m / z [M - H] - C9H6FO4 - Calculated value: 197.0256; measured value: 197.0250.

[0348] Ethyl (5-fluoro-2,3-dihydro-1,4-benzodioxin-6-yl)carbamate (14). [ka] A mixture of 13 (650 mg, 3.28 mmol) in toluene (13.1 mL) was treated with EtN (1.4 mL, 9.84 mmol) and DPPA (780 μL, 3.62 mmol) at 10° C. The mixture was stirred at 23° C. for 30 min and then at 85° C. for 1.5 h. The mixture was cooled to 23° C., then treated with EtOH (5 mL), stirred at 23° C. for 1.5 h, and concentrated. The residue was dissolved in EtO (150 mL), washed with saturated aqueous NaHCO (40 mL), water (40 mL), brine (40 mL), dried (MgSO), filtered, and evaporated. FC (hexane / DCM 7:3 to 1:9) gave the title compound 14 (512 mg, 65%) as a white solid.

[0349] 1 H NMR (500 MHz, CDCl3): δ = 7.42 (br, 1H), 6.64 (dd, J = 9.2, 2.2 Hz, 1H), 6.56 (br, 1H), 4.32 - 4.24 (m, 4H), 4.22 (q, J = 7.1 Hz, 2H), 1.31 ppm (t, J = 7.1 Hz, 3H). 13 C NMR (126 MHz, CDCl3): δ = 153.80, 142.61 (d, J CF = 246.0 Hz), 140.82, 132.66 (d, J CF = 12.4 Hz), 120.36 (d, J CF = 6.9 Hz), 112.36, 111.81 (d, J CF = 3.7 Hz), 64.72, 64.29, 61.61, 14.66 ppm. HRMS (DART): m / z [M + H] + C 11 H 13 FNO4 +Calculated value: 242.0823; measured value: 242.0816.

[0350] 10-Fluoro-7,8-dihydro[1,4]dioxino[2,3-g]quinazoline (15). [ka] A mixture of 14 (450 mg, 1.87 mmol) and HMTA (263 mg, 1.87 mmol) in TFA (5.7 mL) was irradiated in a microwave at 110 °C for 10 min. The mixture was cooled to 23 °C, then diluted with water (60 mL), treated with 6 M NaOH (12 mL), and extracted with DCM (3 × 60 mL). The combined organics were washed with water (50 mL), brine (50 mL), then dried (NaSO), filtered, and evaporated to give a foamy yellow oil.

[0351] A mixture of the oil in 10% KOH in 1:1 dioxane / water (15.5 mL) was treated with [KFe(CN)] (614 mg, 1.87 mmol) and then irradiated in a microwave at 100 °C for 10 min. This procedure was repeated a total of four times (four cycles of adding 1 equivalent of potassium ferricyanide followed by microwave irradiation). The resulting mixture was diluted with water (160 mL) and extracted with DCM (3 × 120 mL). The combined organic solution was washed with water (100 mL), brine (100 mL), dried (NaSO), filtered, and evaporated to give the title compound 15 (330 mg, 86%) as a yellow solid, which was used in the next step without any further purification.

[0352] 1 H NMR (500 MHz, CDCl3): δ = 9.21 (br, 1H), 9.19 (s, 1H), 7.18 (d, J = 2.0 Hz, 1H), 4.53 - 4.41 ppm (m, 4H). 13 C NMR (126 MHz, CDCl3): δ = 158.06 (d, J CF = 2.9 Hz), 153.81 (d, J CF= 1.8 Hz), 145.76 (d, J CF = 2.9 Hz), 144.40 (d, J CF = 256.1 Hz), 138.56 (d, J CF = 11.0 Hz), 136.73 (d, J CF = 10.1 Hz), 119.81 (d, J CF = 2.7 Hz), 106.55 (d, J CF = 4.3 Hz), 64.78, 64.34 ppm. HRMS (DART): m / z [M + H] + C 10 H8FN2O2 + Calculated value: 207.0564; measured value: 207.0563.

[0353] 10-Fluoro-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4(3H)-one (16). [ka] A solution of 15 (306 mg, 1.48 mmol) in AcOH (1 mL) was treated dropwise with a 0.833 M solution of CAN in water (7.12 mL, 5.94 mmol) and stirred at 23 °C for 15 min. The white precipitate was collected by filtration and washed with water (2 × 2 mL), acetonitrile (2 × 2 mL), DCM (2 mL), and EtO (2 mL) to give the first batch of the title compound. The filtrate was neutralized to pH 7 with 1 M NaOH, and the white precipitate was collected by filtration and washed as before to give the second batch of the title compound 16 (81 mg, 25%) as a white solid.

[0354] 1 H NMR (500 MHz, DMSO-d6): δ = 12.19 (br, 1H), 7.98 (d, J = 3.3 Hz, 1H), 7.32 (s, 1H), 4.52 - 4.28 ppm (m, 4H). 13 C NMR (126 MHz, DMSO-d6): δ = 159.31, 144.58 (d, JCF = 251.8 Hz), 144.28, 143.80 (d, J CF = 3.4 Hz), 137.86 (d, J CF = 11.1 Hz), 132.94 (d, J CF = 8.9 Hz), 115.75, 106.62 (d, J CF = 3.7 Hz), 64.57, 64.02 ppm. HRMS (DART): m / z [M + H] + C 10 H8FN2O3 + Calculated value: 223.0513; measured value: 223.0503.

[0355] 4-Chloro-10-fluoro-7,8-dihydro[1,4]dioxino[2,3-g]quinazoline (17). [ka] A stirred suspension of 16 (92 mg, 0.41 mmol) in toluene (1.2 mL) was treated with DIPEA (220 μL, 1.26 mmol), and then POCl (103 μL, 1.12 mmol) was added dropwise at 10 °C. The mixture was stirred at 23 °C for 1 h and then at 90 °C for 5 h before being concentrated. The residue was treated with saturated aqueous NaHCO (10 mL) at 0 °C for 5 min, then diluted with water (5 mL), and extracted with DCM (3 × 7 mL). The combined organic solution was washed with half-saturated aqueous NaHCO (7 mL), brine (7 mL), dried (NaSO), filtered, and evaporated to give the title compound 17 (51 mg, 51%) as a pale brown solid, which was used in the next step without any further purification.

[0356] 1 H NMR (500 MHz, CDCl3): δ = 8.90 (s, 1H), 7.51 (d, J = 2.0 Hz, 1H), 4.55 - 4.43 ppm (m, 4H). 13 C NMR (126 MHz, CDCl3): δ = 160.48 (d, J CF= 4.3 Hz), 152.31, 146.29 (d, J CF = 3.3 Hz), 144.63 (d, J CF = 256.2 Hz), 138.95 (d, J CF = 11.3 Hz), 137.68 (d, J CF = 10.2 Hz), 118.56 (d, J CF = 2.4 Hz), 105.82 (d, J CF = 4.2 Hz), 64.81, 64.41 ppm. HRMS (DART): m / z [M + H] + C 10 H7ClFN2O2 + Calculated value: 241.0175; measured value: 241.0174.

[0357] 5-Fluoro-7-nitro-2,3-dihydro-1,4-benzodioxine-6-carboxylic acid (18). [ka] A mixture of 13 (1500 mg, 7.57 mmol) in AcOH (7.5 mL) was treated with dropwise addition of HSO (2.02 mL) at 10 °C. The vigorously stirred mixture was treated with dropwise addition of 65% HNO (2.6 mL) at 0 °C over 10 min. The resulting mixture was stirred at 0 °C for 30 min and then at 23 °C for 16 h. The mixture was poured into ice water (40 mL), and the white precipitate was collected by filtration (washed with 40 mL of cold water) and dried in a desiccator to give the title compound 18 (1280 mg, 70%) as a white solid.

[0358] 1 H NMR (500 MHz, DMSO-d6): δ = 14.09 (br, 1H), 7.62 (d, J = 1.7 Hz, 1H), 4.52 - 4.40 ppm (m, 4H). 13 C NMR (126 MHz, DMSO-d6): δ = 162.71, 147.16 (d, J CF = 248.7 Hz), 144.72 (d, JCF = 5.1 Hz), 138.15 (d, J CF = 13.7 Hz), 137.10 (d, J CF = 6.6 Hz), 113.44 (d, J CF = 20.3 Hz), 109.52 (d, J CF = 2.3 Hz), 64.97, 64.48 ppm. HRMS (DART): m / z [M - H] - C9H5FNO6 - Calculated value: 242.0106; measured value: 242.0124.

[0359] 7-Amino-5-fluoro-2,3-dihydro-1,4-benzodioxine-6-carboxylic acid (19). [ka] A mixture of 18 (500 mg, 2.06 mmol) and 5% Pd / C (223 mg, 0.10 mmol) in MeOH (21 mL) was stirred under an atmosphere of H for 13.5 h at 23° C. The mixture was filtered through Celite (washed with EtOH) and then evaporated to give the title compound 19 (418 mg, 95%) as a gray solid, which did not appear to be very stable.

[0360] 1 H NMR (500 MHz, DMSO-d6): δ = 8.35 (br, 2H), 6.04 (d, J = 1.9 Hz, 1H), 4.29 - 4.24 (m, 2H), 4.19 - 4.14 ppm (m, 2H). 13 C NMR (126 MHz, DMSO-d6): δ = 167.36 (d, J CF = 2.9 Hz), 151.36 (d, J CF = 252.0 Hz), 148.86 (d, J CF = 7.0 Hz), 145.81 (d, J CF = 5.7 Hz), 122.88 (d, J CF= 15.4 Hz), 97.19 (d, J CF = 2.9 Hz), 95.37 (d, J CF = 10.9 Hz), 64.95, 63.58 ppm. HRMS (DART): m / z [M + H] + C9H9FNO4 + Calculated value: 214.0510; measured value: 214.0508.

[0361] 5-Fluoro-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4(3H)-one (20). [ka] A mixture of 19 (417 mg, 1.96 mmol) in formamide (2.3 mL, 58.7 mmol) was stirred at 120-125 °C for 16 h. The mixture was cooled to 0 °C, treated with water (4 mL), stirred for 30 min, diluted with water (4 mL), and filtered. The residue was washed with cold water (3 × 5 mL) and then dried over Drierite under high vacuum to give the title compound 20 (249 mg, 57%) as an off-white solid.

[0362] 1 H NMR (400 MHz, DMSO-d6): δ = 12.00 (br, 1H), 7.90 (d, J = 3.6 Hz, 1H), 6.93 (d, J = 1.9 Hz, 1H), 4.45 - 4.35 ppm (m, 4H). 13 C NMR (126 MHz, DMSO-d6): δ = 157.64 (d, J CF = 3.0 Hz), 149.70 (d, J CF = 6.0 Hz), 148.45 (d, J CF = 261.3 Hz), 144.60, 142.99, 131.47 (d, J CF = 12.7 Hz), 108.76 (d, J CF = 3.5 Hz), 106.38 (d, J CF= 3.8 Hz), 64.69, 63.98 ppm. HRMS (DART): m / z [M + H] + C 10 H8FN2O3 + Calculated value: 223.0513; measured value: 223.0510.

[0363] 4-Chloro-5-fluoro-7,8-dihydro[1,4]dioxino[2,3-g]quinazoline (21). [ka] A stirred suspension of 20 (90 mg, 0.41 mmol) in toluene (1.2 mL) was treated with DIPEA (215 μL, 1.24 mmol), and then POCl (100 μL, 1.09 mmol) was added dropwise at 10 °C. The mixture was stirred at 23 °C for 1 h and then at 88 °C for 5 h before being concentrated. The residue was treated with saturated aqueous NaHCO (10 mL) at 0 °C, diluted with water (5 mL), and extracted with DCM (3 × 7 mL). The combined organic solution was washed with half-saturated aqueous NaHCO (7 mL), brine (7 mL), dried (NaSO), filtered, and evaporated to give the title compound 21 (96 mg, 99%) as a pale orange-yellow solid, which was used in the next step without any further purification.

[0364] 1 H NMR (500 MHz, CDCl3): δ = 8.83 (s, 1H), 7.35 (d, J = 2.0 Hz, 1H), 4.51 - 4.45 ppm (m, 4H). 13 C NMR (126 MHz, CDCl3): δ = 156.76 (d, J CF = 4.5 Hz), 152.70 (d, J CF = 2.3 Hz), 151.66 (d, J CF = 4.9 Hz), 146.08, 144.51 (d, J CF = 261.8 Hz), 134.04 (d, J CF = 14.0 Hz), 110.85 (d, JCF = 7.7 Hz), 109.43 (d, J CF = 4.0 Hz), 64.89, 64.37 ppm. HRMS (DART): m / z [M + H] + C 10 H7ClFN2O2 + Calculated value: 241.0175; measured value: 241.0176.

[0365] N-(3-Bromo-2-fluorophenyl)-10-fluoro-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK071). [ka] Compound JGK071 was prepared from chloroquinazoline 17 (35 mg, 0.15 mmol) and 3-bromo-2-fluoroaniline according to general procedure GP-2. FC (DCM / EtOAc 1:0 to 8:2) afforded JGK071 (44 mg, 77%) as a white solid.

[0366] 1 H NMR (500 MHz, DMSO-d6): δ = 9.76 (s, 1H), 8.38 (s, 1H), 7.80 (d, J = 1.8 Hz, 1H), 7.62 (ddd, J = 8.0, 6.3, 1.6 Hz, 1H), 7.54 (ddd, J = 8.5, 7.1, 1.6 Hz, 1H), 7.22 (td, J = 8.0, 1.2 Hz, 1H), 4.53 - 4.40 ppm (m, 4H). 13 C NMR (126 MHz, DMSO-d6): δ = 156.93 (d, J CF = 3.7 Hz), 153.44 (d, J CF = 247.5 Hz), 153.12, 144.04 (d, J CF = 250.0 Hz), 143.97 (d, J CF = 3.2 Hz), 137.04 (d, J CF= 10.9 Hz), 135.62 (d, J CF = 9.9 Hz), 130.48, 127.89, 127.62 (d, J CF = 13.1 Hz), 125.51 (d, J CF = 4.5 Hz), 108.58 (d, J CF = 23.4 Hz), 108.51, 103.25 (d, J CF = 3.9 Hz), 64.63, 64.21 ppm. HRMS (DART): m / z [M + H] + C 16 H 11 BrF2N3O2 + Calculated value: 393.9997; measured value: 393.9999.

[0367] N-(3-Bromo-2-fluorophenyl)-5-fluoro-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK072). [ka] Compound JGK072 was prepared from chloroquinazoline 21 (35 mg, 0.15 mmol) and 3-bromo-2-fluoroaniline according to general procedure GP-2. FC (DCM / EtOAc 1:0 to 8:2) afforded JGK072 (47 mg, 82%) as a white solid.

[0368] 1 H NMR (500 MHz, CDCl3): δ = 8.67 (ddd, J = 8.6, 7.2, 1.5 Hz, 1H), 8.62 (s, 1H), 8.52 (dd, J = 19.6, 2.2 Hz, 1H), 7.29 (ddd, J = 8.1, 6.4, 1.5 Hz, 1H), 7.23 (d, J = 2.0 Hz, 1H), 7.10 (td, J = 8.2, 1.6 Hz, 1H), 4.48 - 4.42 ppm (m, 4H). 13 C NMR (126 MHz, CDCl3): δ = 155.27 (d, JCF = 5.2 Hz), 153.90, 150.34 (d, J CF = 243.9 Hz), 149.93 (d, J CF = 6.2 Hz), 145.75 (d, J CF = 250.3 Hz), 144.78, 131.96 (d, J CF = 15.6 Hz), 128.43 (d, J CF = 10.4 Hz), 127.71, 125.20 (d, J CF = 4.7 Hz), 122.48, 109.69 (d, J CF = 3.3 Hz), 108.63 (d, J CF = 19.2 Hz), 101.42 (d, J CF = 7.2 Hz), 64.84, 64.48 ppm. HRMS (DART): m / z [M + H] + C 16 H 11 BrF2N3O2 + Calculated value: 393.9997; measured value: 393.9996.

[0369] Example 15 : Preparation of example compounds of the JGK series [ka] Scheme 1. Synthesis of JGK068S.

[0370] [ka] Scheme 2. Preparation of the (R)-enantiomer JGK068R or the racemic mixture (JGK068) follows the same route as shown in Scheme 1, but employing (R)-glycidol or racemic glycidol, respectively.

[0371] [ka] Scheme 3. One-step synthesis of benzodioxane carbaldehyde (R)-10 from benzaldehyde 1 using chiral glycidyl toluenesulfonate. This route avoids the Mitsunobu reaction in Scheme 1 (preparation of 2 from 1). Compound (R)-10 can be used in the route shown in Scheme 1 to prepare JGK068S.

[0372] General Chemical Information All chemicals, reagents, and solvents, when available, were purchased from commercial sources and used as received. When necessary, reagents and solvents were purified and dried by standard methods. Air-sensitive and moisture-sensitive reactions were carried out in oven-dried glassware under an inert atmosphere of argon. Microwave-irradiated reactions were carried out in a single-mode reactor, a CEM Discover microwave synthesizer. Room temperature (RT) reactions were carried out at ambient temperature (approximately 23 °C). All reactions were carried out on pre-coated Merck 60 F plates, with spots visualized using UV light (λ = 254, 365 nm) or alkaline KMnO4 solution. 254 Thin layer chromatography (TLC) was performed using silica gel plates. Flash column chromatography (FC) was performed using SiO260 (particle size 0.040-0.063 mm, 230-400 mesh). Preparative thin layer chromatography (PTLC) was performed using Merck 60 F 254 Silica gel plates (20 x 20 cm, 210-270 mm) or Analtech silica gel GF TLC plates (20 x 20 cm, 1000 mm) were used. Concentration under reduced pressure (vacuum) was performed by rotary evaporation at 25-50 °C. The purified compounds were further dried under high vacuum or in a desiccator. The yields corresponded to the purified compounds and were not further optimized. Proton nuclear magnetic resonance ( 1 H NMR spectra were recorded using a Bruker spectrometer operating at 300, 400, or 500 MHz. Carbon NMR ( 13C NMR spectra were recorded using a Bruker spectrometer (either 400 or 500 MHz). NMR chemical shifts (δ ppm) were referenced to the residual solvent signal. 1 H NMR data are reported as follows: chemical shift (ppm); multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet / complex pattern, td = triple doublet, ddd = double doublet, br = broad signal); coupling constant (J) (Hz), integration. 13 C NMR spectral data are reported based on chemical shifts and, where applicable, coupling constants. High-resolution mass (HRMS) spectra were recorded using a Thermo Fisher Scientific Exactive Plus equipped with an IonSense ID-CUBE DART source mass spectrometer or a Waters LCT Premier mass spectrometer equipped with an ACQUITY UPLC with an autosampler.

[0373] 5-Formyl-2-hydroxyphenylacetate (1). [ka] A mixture of 3,4-dihydroxybenzaldehyde (100 g, 0.724 mol) in THF (965 mL) was cooled to 0 °C and treated with 10% aqueous NaOH (724 mL, 1.81 mol) over 4–5 min. The reaction mixture was stirred at 0 °C for 15 min, and then acetic anhydride (AcO, 82.1 mL, 0.869 mol) was added dropwise over 20 min. The mixture was stirred at the same temperature for 30 min and then poured into a mixture of EtOAc (1.25 L) and 2 M HCl (1.13 L) at 0 °C. The phases were separated, and the aqueous phase was extracted with EtOAc (4 × 250 mL). The combined organic solution was washed with water (2 × 500 mL) and brine (500 mL), then dried (NaSO), filtered, and evaporated. The residue was treated with a small amount of n-heptane and evaporated (3 ×). Recrystallization from EtOAc (275 mL, washed with EtO) gave the first crop of title compound 1 (66.96 g, 51%) as pale brown crystals. The mother liquor was recrystallized from EtOAc to give a second crop of title compound 1 (29.436 g, 23%) as a pale brown solid. 1 H NMR (500 MHz, CDCl3): δ 9.85 (s, 1H), 7.73 - 7.65 (m, 2H), 7.11 (d, J = 8.8 Hz, 1H), 6.34 (br, 1H), 2.39 (s, 3H). 13 C NMR (126 MHz, CDCl3): δ 190.40, 168.99, 152.96, 138.81, 130.24, 129.72, 124.13, 117.87, 21.09. HRMS (DART): m / z [M + H] + C9H9O4 + Calculated value: 181.0495; measured value: 181.0488.

[0374] 5-Formyl-2-{[(2R)-oxiran-2-yl]methoxy}phenyl acetate ((R)-2). [ka] A mixture of 1 (32.5 g, 0.18 mol) and triphenylphosphine (PPh3, 70.976 g, 0.27 mol) in THF (905 mL) was treated with (S)-glycidol (17.95 mL, 0.27 mol), then cooled to 0 °C and treated with diisopropyl azodicarboxylate (DIAD, 56.8 mL, 0.289 mmol) dropwise over 30 min. The mixture was stirred at 0 °C for an additional 10 min, then removed from the cooling bath, and stirring continued at 23 °C for 15.5 h. Evaporation of all volatiles afforded crude (R)-2 as a brown oil, which was used in the next step without any further purification.

[0375] (3S)-3-(Hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-carbaldehyde ((S)-3). [ka] A mixture of crude (R)-2 in MeOH (1.564 L) was treated with KCO (49.87 g, 0.36 mol) and stirred at 23 °C for 18.5 h, after which the solvent was evaporated. The residue was suspended in half-saturated NHCl (750 mL) and extracted with EtOAc (3 × 500 mL). The combined organics were washed with water (250 mL), brine (250 mL), dried (NaSO), filtered, and evaporated. The crude product was purified by several rounds of flash chromatography (hexane / EtOAc 9:1 → 1:1) as well as precipitation from hexane / EtO 1:1 (to remove triphenylphosphine oxide PhPO) to give the title compound (S)-3 (17.322 g, 49% over two steps) as a white solid. 1H NMR (500 MHz, CDCl3): δ 9.81 (s, 1H), 7.43 (d, J = 1.8 Hz, 1H), 7.41 (dd, J = 8.1, 1.9 Hz, 1H), 7.00 (d, J = 8.2 Hz, 1H), 4.39 (dd, J = 11.4, 2.3 Hz, 1H), 4.31 - 4.25 (m, 1H), 4.20 (dd, J = 11.3, 7.9 Hz, 1H), 3.95 (dd, J = 12.1, 4.3 Hz, 1H), 3.87 (dd, J = 12.1, 4.9 Hz, 1H), 2.18 (br, 1H). 13 C NMR (126 MHz, CDCl3): δ 190.79, 148.76, 143.46, 130.79, 124.46, 118.33, 117.70, 73.31, 65.61, 61.54. HRMS (DART): m / z [M + H] + C 10 H 11 O4 + Calculated value: 195.0652; measured value: 195.0650.

[0376] [(2R)-7-cyano-2,3-dihydro-1,4-benzodioxin-2-yl]methyl acetate ((R)-4). [ka] A mixture of (S)-3 (17.322 g, 0.089 mol) in AcOH (189 mL) was treated with KOAc (22.944 g, 0.234 mol) and stirred at 23 °C for 10 min, followed by treatment with NHOH·HCl (16.233 g, 0.234 mol). The resulting mixture was stirred at 120–125 °C for 18.5 h. The mixture was cooled to 23 °C, poured into water (1 L), and extracted with EtOAc (4 × 250 mL). The combined organic solution was treated with 3.5 M NaOH (400 mL) and saturated aqueous NaHCO (100 mL) to obtain a final pH of approximately 8, and the emulsion was stirred at 23 °C for 1 h. The organic layer was separated, washed with saturated aqueous NaHCO (300 mL), water (300 mL), brine (300 mL), dried (NaSO), filtered, and evaporated. Purification by flash chromatography (hexane / EtOAc 10:1 → 6:4) afforded the title compound (R)-4 (13.513 g, 65%) as a clear, colorless oil. 1 H NMR (500 MHz, CDCl3): δ 7.20 (d, J = 2.0 Hz, 1H), 7.16 (dd, J = 8.4, 2.0 Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 4.43 - 4.38 (m, 1H), 4.36 (dd, J = 11.6, 2.4 Hz, 1H), 4.34 (dd, J = 11.1, 4.5 Hz, 1H), 4.30 (dd, J = 11.6, 4.6 Hz, 1H), 4.11 (dd, J = 11.5, 7.2 Hz, 1H), 2.12 (s, 3H). 13 C NMR (126 MHz, CDCl3): δ 170.64, 147.13, 143.11, 126.28, 121.56, 118.85, 118.32, 105.13, 71.11, 65.45, 62.24, 20.83. HRMS (DART): m / z [M + H] + C 12 H 12 No. 4 + Calculated value: 234.0761; measured value: 234.0759.

[0377] [(2R)-7-cyano-6-nitro-2,3-dihydro-1,4-benzodioxin-2-yl]methyl acetate ((R)-5). [ka] A mixture of (R)-4 (13.345 g, 57.2 mmol) in AcO (74.3 mL) was treated with HSO (3.05 mL, 57.2 mmol), cooled to 0 °C, and treated dropwise with 70% HNO (19.63 mL, 286 mmol) at 0 °C for 35 min. The mixture was stirred at 0 °C for an additional 2 h and then at 23 °C for 3.5 h. The mixture was poured into ice water (850 mL), and the pH was adjusted to approximately 7 with 6 M NaOH (320 mL). Saturated aqueous NaHCO (200 mL) was added, and the mixture was extracted with CHCl (3 × 500 mL). The combined organics were washed with saturated aqueous NaHCO (400 mL), water (400 mL), brine (400 mL), then dried (NaSO), filtered, and evaporated to give the title compound (R)-5 (15.696 g, 99%) as a yellow oil, which was used in the next step without any further purification. 1 H NMR (500 MHz, DMSO-d6): δ 7.96 (s, 1H), 7.80 (s, 1H), 4.73 - 4.67 (m, 1H), 4.58 (dd, J = 11.8, 2.6 Hz, 1H), 4.36 (dd, J = 12.5, 3.7 Hz, 1H), 4.31 (dd, J = 12.5, 5.7 Hz, 1H), 4.27 (dd, J = 11.8, 7.0 Hz, 1H), 2.05 (s, 3H). 13 C NMR (126 MHz, DMSO-d6): δ 170.11, 147.75, 146.26, 142.23, 123.77, 115.21, 115.17, 100.06, 72.00, 64.98, 61.72, 20.52. HRMS (DART): m / z [M + H] + C 12 H 11 N2O6 +Calculated value: 279.0612; measured value: 279.0601.

[0378] (3S)-7-Amino-3-(hydroxymethyl)-2,3-dihydro-1,4-benzodioxine-6-carbonitrile ((S)-6). [ka] A suspension of (R)-5 (15.591 g, 56 mmol) in 1:1 water / ethanol (250 mL) was treated with NaSO (39.266 g, 185 mmol), and the mixture was stirred at 50 °C for 105 min and then heated at 70 °C for 2 h, during which time it was treated with concentrated HCl (73.6 mL, 0.897 mol) in small portions. The mixture was cooled to 23 °C, poured onto ice, and the pH was adjusted to approximately 10 with 6 M NaOH (200 mL) and half-saturated NaHCO (500 mL). The mixture was extracted with EtOAc (3 × 500 mL). The combined organics were washed with water (500 mL), brine (500 mL), then dried (NaSO), filtered, and evaporated to give crude (S)-6 (9.483 g, 82%) as an orange-yellow solid, which was used in the next step without any further purification. 1 H NMR (500 MHz, DMSO-d6): δ 6.92 (s, 1H), 6.29 (s, 1H), 5.50 (br, 2H), 5.04 (t, J = 5.7 Hz, 1H), 4.32 (dd, J = 10.7, 1.6 Hz, 1H), 4.07 - 3.99 (m, 1H), 4.00 (dd, J = 11.2, 8.3 Hz, 1H), 3.64 - 3.51 (m, 2H). 13 C NMR (126 MHz, DMSO-d6): δ 148.50, 147.29, 134.39, 119.04, 118.04, 102.45, 86.72, 73.25, 65.92, 59.77. HRMS (DART): m / z [M + H] + C 10 H 10 N2O3 ·+Calculated value: 206.0686; measured value: 206.0685.

[0379] N'-[(2S)-7-cyano-2-(hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-yl]-N,N-dimethylmethane-imidamide ((S)-7). [ka] A mixture of (S)-6 (9.38 g, 45.5 mmol) in toluene (117 mL) was treated with AcOH (143 μL, 2.5 mmol) and DMF-DMA (13.1 mL, 98.9 mmol), and then the mixture was stirred at 105 °C for 3 h. The progress of the reaction was monitored by collecting evaporated MeOH (approximately 4–5 mL) in a Dean-Stark trap. The mixture was cooled to 23 °C and then evaporated to give crude (S)-7 (14.243 g, quant.) as a viscous brown oil, which was used in the next step without any further purification. 1 H NMR (500 MHz, CDCl3): δ 7.51 (s, 1H), 7.05 (s, 1H), 6.48 (s, 1H), 4.33 (dd, J = 11.2, 2.0 Hz, 1H), 4.23 - 4.17 (m, 1H), 4.13 (dd, J = 11.2, 8.1 Hz, 1H), 3.90 (dd, J = 12.1, 4.2 Hz, 1H), 3.83 (dd, J = 12.1, 4.8 Hz, 1H), 3.07 (s, 3H), 3.05 (s, 3H). 13 C NMR (126 MHz, CDCl3): δ 160.40, 153.78, 147.68, 138.63, 121.08, 118.64, 108.16, 99.31, 73.34, 65.83, 61.67, 40.51, 34.82. HRMS (DART): m / z [M + H] + C 13 H 16 N3O3 + Calculated value: 262.1186; measured value: 262.1183.

[0380] [(7S)-4-(3-bromo-2-fluoroanilino)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-7-yl]methanol ((S)-8). [ka] A mixture of (S)-7 in AcOH (152 mL) was treated with 3-bromo-2-fluoroaniline (6.63 mL, 59.1 mmol), and the mixture was stirred at 125–130 °C for 3 h. The mixture was cooled to 23 °C, poured into ice-water (500 mL), and the pH was adjusted to approximately 9 with saturated aqueous NHOH (185 mL) and half-saturated aqueous NaHCO (200 mL). The mixture was extracted with EtOAc (3 × 400 mL), and the combined organics were washed with half-saturated aqueous NaHCO (400 mL), water (400 mL), brine (400 mL), dried (NaSO), filtered, and evaporated. The residue was dissolved in MeOH (272 mL), treated with KCO (12.579 g, 91 mmol), stirred at 23 °C for 1 h, and evaporated. The residue was suspended in half-saturated aqueous NH4Cl (700 mL) and extracted with EtOAc (3 × 400 mL). The combined organics were washed with water (400 mL), brine (400 mL), dried (Na2SO4), filtered, and evaporated. The orange-yellow residue was suspended in EtOAc and warmed to 65 °C, then slowly cooled to 23 °C overnight. After filtration, the residue was washed with cold hexane (2 × 15 mL) and Et2O (2 × 15 mL) and dried under high vacuum to give the title compound (S)-8 (9.407 g, 50.9% over two steps) as a fine yellow powder. 1H NMR (500 MHz, DMSO-d6): δ 9.69 (s, 1H), 8.33 (s, 1H), 7.99 (s, 1H), 7.59 (ddd, J = 8.0, 6.2, 1.6 Hz, 1H), 7.54 (ddd, J = 8.4, 7.0, 1.6 Hz, 1H), 7.24 - 7.17 (m, 1H), 7.20 (s, 1H), 5.20 (t, J = 5.6 Hz, 1H), 4.49 (dd, J = 11.5, 2.4 Hz, 1H), 4.37 - 4.29 (m, 1H), 4.21 (dd, J = 11.6, 7.4 Hz, 1H), 3.76 - 3.64 (m, 2H). 13 C NMR (126 MHz, DMSO-d6): δ 157.22, 153.38 (d, J CF = 247.0 Hz), 153.09, 148.87, 145.94, 143.37, 130.08, 128.09 (d, J CF = 12.9 Hz), 127.75, 125.43 (d, J CF = 4.5 Hz), 112.29, 109.81, 108.54 (d, J CF = 20.0 Hz), 108.49, 73.77, 65.52, 59.76. HRMS (DART): m / z [M + H] + C 17 H 14 BrFN3O3 + Calculated value: 406.0197; measured value: 406.0185.

[0381] [(7R)-4-(3-bromo-2-fluoroanilino)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-7-yl]methyl methanesulfonate ((R)-9). [ka] A mixture of (S)-8 (9.01 g, 22.2 mmol) and MeN HCl (234 mg, 2.45 mmol) in acetonitrile (148 mL) was treated with EtN (6.18 mL, 44.4 mmol), then cooled to 0–5 °C and treated with a solution of MsCl (2.57 mL, 33.2 mmol) in acetonitrile (17 mL, rinsed with 3 mL) dropwise over 10 min. The mixture was stirred at 0 °C for 1 h. Water (100 mL) was added, and most of the acetonitrile was evaporated under reduced pressure. Additional water (700 mL) was added, and the mixture was extracted with EtOAc (3 × 400 mL). The combined organics were washed with water (400 mL), brine (400 mL), then dried (NaSO), filtered, and evaporated to give the title compound (R)-9 (10.33 g, 96%) as a yellow brittle foam, which was used in the next step without any further purification. 1 H NMR (500 MHz, CDCl3): δ 8.70 (s, 1H), 8.62 (ddd, J = 8.7, 7.3, 1.6 Hz, 1H), 7.44 (s, 1H), 7.362 (s, 1H), 7.360 (br, 1H), 7.29 (ddd, J = 8.1, 6.5, 1.5 Hz, 1H), 7.11 (td, J = 8.2, 1.6 Hz, 1H), 4.67 - 4.61 (m, 1H), 4.54 - 4.51 (m, 2H), 4.50 (dd, J = 11.7, 2.4 Hz, 1H), 4.29 (dd, J = 11.8, 7.1 Hz, 1H), 3.13 (s, 3H).

[0382] (7S)-N-(3-bromo-2-fluorophenyl)-7-[(4-methylpiperazin-1-yl)methyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK068S). [ka] A mixture of (R)-9 in DMF (427 mL) was treated with 1-methylpiperazine (11.83 mL, 107 mmol) and EtN (5.95 mL, 42.7 mmol), then the mixture was stirred at 85 °C for 24 h. The mixture was cooled to 23 °C and evaporated. The residue was dissolved in EtOAc (1.2 L), washed with 0.5 M NaOH (4 × 250 mL), brine (250 mL), dried (NaSO), filtered, and evaporated. Purification by flash chromatography (CHCl / MeOH 1:0 to 8:2) afforded the title compound JGK068S (6.013 g, 58% over two steps) as an off-white, brittle foam. 1 H NMR (500 MHz, CDCl3): δ 8.67 (s, 1H), 8.63 (ddd, J = 8.7, 7.3, 1.6 Hz, 1H), 7.374 (s, 1H), 7.372 (br, 1H), 7.32 (s, 1H), 7.26 (ddd, J = 8.1, 6.5, 1.5 Hz, 1H), 7.09 (td, J = 8.2, 1.6 Hz, 1H), 4.48 - 4.40 (m, 2H), 4.14 (dd, J = 11.8, 8.0 Hz, 1H), 2.77 (dd, J = 13.4, 6.0 Hz, 1H), 2.653 (dd, J = 13.4, 5.8 Hz, 1H), 2.648 (br, 4H), 2.51 (br, 4H), 2.32 (s, 3H).

[0383] [ka] Scheme 4. Synthesis of JGK083S.

[0384] tert-Butyl {[(7S)-4-(3-bromo-2-fluoroanilino)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-7-yl]methyl}piperazine-1-carboxylate ((S)-10). [ka] Example 14Compound (S)-10 was prepared from R-9 (91 mg, 0.188 mmol) and tert-butyl piperazine-1-carboxylate (175 mg, 0.94 mmol) in DMF (3.8 mL) according to general procedure GP-1 in

[23] and stirred at 85° C. for 15 h. PTLC (CHCl / EtOAc 4:6) afforded (S)-10 (50 mg, 46%) as an off-white brittle foam. 1 H NMR (500 MHz, CDCl3): δ 8.68 (s, 1H), 8.65 (ddd, J = 8.3, 7.4, 1.5 Hz, 1H), 7.39 (s, 1H), 7.36 (br, 1H), 7.31 (s, 1H), 7.27 (ddd, J = 8.0, 6.5, 1.5 Hz, 1H), 7.11 (td, J = 8.2, 1.5 Hz, 1H), 4.50 - 4.42 (m, 2H), 4.17 (dd, J = 12.1, 8.2 Hz, 1H), 3.47 (t, J = 5.1 Hz, 4H), 2.78 (dd, J = 13.4, 5.9 Hz, 1H), 2.67 (dd, J = 13.5, 5.9 Hz, 1H), 2.62 - 2.47 (m, 4H), 1.47 (s, 9H). 13 C NMR (126 MHz, CDCl3): δ 155.89, 154.83, 153.39, 150.15 (d, JCF = 242.4 Hz), 149.36, 146.72, 144.02, 128.63 (d, J CF = 10.3 Hz), 127.27, 125.34, 121.78, 114.34, 110.66, 108.60 (d, J CF = 19.5 Hz), 106.06, 79.97, 71.76, 67.18, 58.56, 53.96, 28.57, one carbon signal missing (probably due to overlapping peaks). HRMS (DART): m / z [M + H] + C 26 H 30 BrFN5O4 + Calculated value: 574.1460; measured value: 574.1432.

[0385] (7S)-N-(3-bromo-2-fluorophenyl)-7-[(piperazin-1-yl)methyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK083S). [ka] A mixture of (S)-10 (42 mg, 0.073 mmol) in CHCl (500 μL) and TFA (250 μL) was stirred at 23 °C for 12 h. The mixture was diluted with 1 M HCl (20 mL) and then washed with CHCl (3 × 7 mL). The aqueous phase was diluted with 6 M NaOH (4 mL) to pH > 12 and then extracted with EtOAc (3 × 8 mL). The combined organics were washed with brine (8 mL) and then dried (NaSO), filtered, and evaporated. Purification by PTLC (CHCl / MeOH 8:2) afforded the title compound JGK083S (18 mg, 52%) as a white, brittle foam. 1 H NMR (500 MHz, CDCl3): δ 8.68 (s, 1H), 8.66 (ddd, J = 8.2, 7.3, 1.6 Hz, 1H), 7.39 (s, 1H), 7.35 (br d, J = 4.0 Hz, 1H), 7.32 (s, 1H), 7.27 (ddd, J = 8.1, 6.5, 1.6 Hz, 1H), 7.11 (td, J = 8.2, 1.5 Hz, 1H), 4.50 - 4.42 (m, 2H), 4.19 - 4.13 (m, 1H), 2.93 (t, J = 4.9 Hz, 4H), 2.76 (dd, J = 13.4, 5.9 Hz, 1H), 2.63 (dd, J = 13.4, 6.0 Hz, 1H), 2.63 - 2.50 (m, 4H). 13 C NMR (126 MHz, CDCl3): δ 155.88, 153.35, 150.12 (d, J CF = 242.3 Hz), 149.45, 146.71, 144.17, 128.67 (d, J CF = 10.4 Hz), 127.21, 125.32 (d, JCF = 4.7 Hz), 121.74, 114.30, 110.64, 108.58 (d, J CF = 19.3 Hz), 106.02, 71.70, 67.32, 59.19, 55.54, 46.23. HRMS (DART): m / z [M - H] - C 21 H 20 BrFN5O2 - Calculated value: 472.0790; measured value: 472.0773.

[0386] [(2R)-7-Formyl-2,3-dihydro-1,4-benzodioxin-2-yl]methyl acetate ((R)-10). [ka] A mixture of 1 (150 mg, 0.833 mmol) and (2R)-glycidyl toluenesulfonate (203 mg, 0.891 mmol) in DMF (2 mL) was treated with KCO (181 mg, 1.31 mmol), and the mixture was stirred at 60 °C for 15 h. The mixture was cooled to 23 °C, then water (30 mL) was added, and the mixture was extracted with EtOAc (3 × 15 mL). The combined organic solution was washed with water (15 mL), brine (15 mL), then dried (NaSO), filtered, and evaporated. Purification using preparative thin-layer chromatography (hexane / EtOAc 7:3) afforded the title compound (R)-10 (111 mg, 56%) as a clear, colorless oil. 1H NMR (400 MHz, CDCl3): δ = 9.82 (s, 1H), 7.44 (d, J = 1.8 Hz, 1H), 7.42 (dd, J = 8.2, 1.9 Hz, 1H), 7.00 (d, J = 8.1 Hz, 1H), 4.46 - 4.39 (m, 1H), 4.37 (dd, J = 11.5, 2.4 Hz, 1H), 4.35 (dd, J = 11.7, 4.9 Hz, 1H), 4.31 (dd, J = 11.9, 5.1 Hz, 1H), 4.13 (dd, J = 11.5, 7.1 Hz, 1H), 2.11 (s, 3H). 13 C NMR (101 MHz, CDCl3): δ 190.72, 170.67, 148.57, 143.22, 131.15, 124.38, 118.76, 117.85, 70.94, 65.54, 62.36, 20.83. HRMS (DART): m / z [M + H] + C 12 H 13 O5 + Calculated value: 237.0757; measured value: 237.0745.

[0387] (±)-N-(3-chloro-2-fluorophenyl)-7-[(4-methylpiperazin-1-yl)methyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK075). [ka] 1H NMR (500 MHz, CDCl3): δ 8.68 (s, 1H), 8.61 (td, J = 7.3, 2.2 Hz, 1H), 7.39 (s, 1H), 7.35 (br d, J = 3.4 Hz, 1H), 7.32 (s, 1H), 7.16 (td, J = 8.1, 1.2 Hz, 1H), 7.13 (td, J = 8.2, 1.9 Hz, 1H), 4.49 - 4.41 (m, 2H), 4.15 (dd, J = 11.8, 8.1 Hz, 1H), 2.78 (dd, J = 13.4, 5.9 Hz, 1H), 2.66 (dd, J = 13.4, 5.9 Hz, 1H), 2.64 (br, 4H), 2.48 (br, 4H), 2.31 (s, 3H). 13 C NMR (126 MHz, CDCl3): δ 155.89, 153.35, 149.44, 149.30 (d, J CF = 244.2 Hz), 146.72, 144.15, 128.76 (d, J CF = 9.3 Hz), 124.71 (d, J CF = 4.7 Hz), 124.45, 121.01, 120.85 (d, J CF HRMS (DART): m / z [M + H] + C 22 H 24 ClFN5O2 + Calculated value: 444.1597; measured value: 444.1582.

[0388] (±)-N-(3-Bromo-2-fluorophenyl)-8-[(morpholin-4-yl)methyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK076). [ka] 1H NMR (500 MHz, DMSO-d6): δ 9.62 (s, 1H), 8.33 (s, 1H), 7.94 (s, 1H), 7.59 (ddd, J = 8.0, 6.2, 1.6 Hz, 1H), 7.54 (ddd, J = 8.5, 7.1, 1.6 Hz, 1H), 7.21 (td, J = 8.1, 1.2 Hz, 1H), 7.19 (s, 1H), 4.63 - 4.56 (m, 1H), 4.46 (dd, J = 11.6, 2.5 Hz, 1H), 4.17 (dd, J = 11.6, 7.1 Hz, 1H), 3.59 (t, J = 4.6 Hz, 4H), 2.71 - 2.59 (m, 2H), 2.57 - 2.44 (m, 4H). 13 C NMR (126 MHz, DMSO-d6): δ 157.22, 153.37 (d, J CF = 247.3 Hz), 153.13, 148.75, 146.16, 143.28, 130.14, 128.02 (d, J CF = 13.0 Hz), 127.74, 125.45 (d, J CF = 4.7 Hz), 112.57, 109.61, 108.55 (d, J CF = 19.9 Hz), 108.23, 71.41, 66.29, 66.18, 57.97, 53.89. HRMS (DART): m / z [M - H] - C 21 H 19 BrFN4O3 - Calculated value: 473.0630; measured value: 473.0608.

[0389] (±)-N-(3-Bromo-2-fluorophenyl)-8-[(dimethylamino)methyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK077). [ka] 1H NMR (500 MHz, DMSO-d6): δ 9.62 (s, 1H), 8.33 (s, 1H), 7.94 (s, 1H), 7.59 (ddd, J = 8.0, 6.2, 1.6 Hz, 1H), 7.54 (ddd, J = 8.5, 7.1, 1.6 Hz, 1H), 7.21 (td, J = 8.1, 1.2 Hz, 1H), 7.17 (s, 1H), 4.57 - 4.51 (m, 1H), 4.44 (dd, J = 11.6, 2.5 Hz, 1H), 4.14 (dd, J = 11.7, 7.1 Hz, 1H), 2.58 (s, 1H), 2.57 (s, 1H), 2.25 (s, 6H). 13 C NMR (126 MHz, DMSO-d6): δ 157.22, 153.38 (d, J CF = 247.4 Hz), 153.12, 148.78, 146.16, 143.29, 130.14, 128.02 (d, J CF = 13.1 Hz), 127.75, 125.45 (d, J CF = 4.4 Hz), 112.54, 109.59, 108.55 (d, J CF = 19.8 Hz), 108.20, 71.76, 66.31, 58.73, 45.92. HRMS (DART): m / z [M - H] - C 19 H 17 BrFN4O2 - Calculated value: 431.0524; measured value: 431.0503.

[0390] (±)-N-(3-Bromo-2-fluorophenyl)-8-[(4-methylpiperazin-1-yl)methyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK078). [ka] 1H NMR (500 MHz, DMSO-d6): δ 9.61 (s, 1H), 8.33 (s, 1H), 7.94 (s, 1H), 7.59 (ddd, J = 8.0, 6.2, 1.6 Hz, 1H), 7.54 (ddd, J = 8.5, 7.1, 1.6 Hz, 1H), 7.21 (td, J = 8.1, 1.2 Hz, 1H), 7.19 (s, 1H), 4.60 - 4.53 (m, 1H), 4.44 (dd, J = 11.6, 2.5 Hz, 1H), 4.14 (dd, J = 11.7, 7.1 Hz, 1H), 2.68 - 2.59 (m, 2H), 2.53 (br, 4H), 2.34 (br, 4H), 2.16 (s, 3H). 13 C NMR (126 MHz, DMSO-d6): δ 157.22, 153.38 (d, J CF = 247.4 Hz), 153.12, 148.78, 146.15, 143.29, 130.13, 128.02 (d, J CF = 13.1 Hz), 127.74, 125.45 (d, J CF = 4.5 Hz), 112.55, 109.60, 108.55 (d, J CF = 19.8 Hz), 108.22, 71.57, 66.34, 57.52, 54.68, 53.29, 45.72. HRMS (DART): m / z [M - H] - C 22 H 22 BrFN5O2 - Calculated value: 486.0946; measured value: 486.0928.

[0391] (±)-N-(3-Bromo-2-fluorophenyl)-8-[(pyrrolidin-1-yl)methyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK079). [ka] 1H NMR (500 MHz, DMSO-d6): δ 9.61 (s, 1H), 8.33 (s, 1H), 7.94 (s, 1H), 7.59 (ddd, J = 8.0, 6.3, 1.6 Hz, 1H), 7.54 (ddd, J = 8.5, 7.1, 1.6 Hz, 1H), 7.21 (td, J = 8.0, 1.2 Hz, 1H), 7.19 (s, 1H), 4.57 - 4.49 (m, 1H), 4.46 (dd, J = 11.6, 2.5 Hz, 1H), 4.16 (dd, J = 11.6, 7.1 Hz, 1H), 2.80 (dd, J = 12.8, 6.0 Hz, 1H), 2.73 (dd, J = 12.8, 6.2 Hz, 1H), 2.62 - 2.48 (m, 4H), 1.74 - 1.66 (m, 4H). 13 C NMR (126 MHz, DMSO-d6): δ 157.22, 153.37 (d, J CF = 247.5 Hz), 153.12, 148.79, 146.17, 143.29, 130.13, 128.02 (d, J CF = 12.9 Hz), 127.74, 125.45 (d, J CF = 4.5 Hz), 112.54, 109.58, 108.55 (d, J CF = 20.0 Hz), 108.19, 72.65, 66.32, 55.42, 54.31, 23.23. HRMS (DART): m / z [M - H] - C 21 H 19 BrFN4O2 - Calculated value: 457.0681; measured value: 457.0660.

[0392] (±)-N-(3-Bromo-2-fluorophenyl)-8-[(piperidin-1-yl)methyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK080). [ka] 1 H NMR (500 MHz, DMSO-d6): δ 9.61 (s, 1H), 8.33 (s, 1H), 7.94 (s, 1H), 7.59 (ddd, J = 8.0, 6.3, 1.6 Hz, 1H), 7.54 (ddd, J = 8.4, 7.0, 1.6 Hz, 1H), 7.21 (td, J = 8.1, 1.2 Hz, 1H), 7.18 (s, 1H), 4.59 - 4.52 (m, 1H), 4.44 (dd, J = 11.6, 2.5 Hz, 1H), 4.14 (dd, J = 11.7, 7.1 Hz, 1H), 2.65 - 2.54 (m, 2H), 2.53 - 2.37 (m, 4H), 1.55 - 1.47 (m, 4H), 1.43 - 1.34 (m, 2H). 13 C NMR (126 MHz, DMSO-d6): δ 157.21, 153.37 (d, J CF = 247.1 Hz), 153.11, 148.83, 146.15, 143.32, 130.12, 128.03 (d, J CF = 13.1 Hz), 127.73, 125.45 (d, J CF = 4.5 Hz), 112.53, 109.57, 108.55 (d, J CF = 19.8 Hz), 108.19, 71.63, 66.42, 58.35, 54.74, 25.61, 23.83. HRMS (DART): m / z [M - H] - C 22 H 21 BrFN4O2 - Calculated value: 471.0837; measured value: 471.0814.

[0393] N-(3-bromo-2-fluorophenyl)(7,7,8,8- 2 H4)-7,8-Dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK081). [ka] 1 H NMR (500 MHz, DMSO-d6): δ 9.61 (s, 1H), 8.33 (s, 1H), 7.93 (s, 1H), 7.59 (ddd, J = 7.9, 6.3, 1.6 Hz, 1H), 7.54 (ddd, J = 8.4, 7.1, 1.6 Hz, 1H), 7.21 (td, J = 8.1, 1.2 Hz, 1H), 7.19 (s, 1H). 13 C NMR (126 MHz, DMSO-d6): δ 157.19, 153.37 (d, J CF = 247.2 Hz), 153.10, 149.27, 146.03, 143.67, 130.12, 128.03 (d, J CF = 13.0 Hz), 127.74, 125.44 (d, J CF = 4.2 Hz), 112.47, 109.63, 108.55 (d, J CF = 19.9 Hz), 108.35. HRMS (DART): m / z [M + H] + C 16 H8D4BrFN3O2 + Calculated value: 380.0342; measured value: 380.0327.

[0394] (±)-N-(3-Bromo-2-fluorophenyl)-7-[2-(pyrrolidin-1-yl)ethyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK084). [ka] 1H NMR (500 MHz, DMSO-d6): δ 9.60 (s, 1H), 8.33 (s, 1H), 7.95 (s, 1H), 7.58 (ddd, J = 8.0, 6.2, 1.6 Hz, 1H), 7.53 (ddd, J = 8.4, 7.0, 1.6 Hz, 1H), 7.21 (td, J = 8.1, 1.1 Hz, 2H), 7.20 (s, 1H), 4.50 (dd, J = 11.5, 2.3 Hz, 1H), 4.42 - 4.36 (m, 1H), 4.12 (dd, J = 11.5, 7.7 Hz, 1H), 2.70 - 2.56 (m, 2H), 2.49 - 2.40 (m, 4H), 1.89 - 1.78 (m, 2H), 1.73 - 1.64 (m, 4H). 13 C NMR (126 MHz, DMSO-d6): δ 157.21, 153.33 (d, J CF = 247.5 Hz), 153.13, 148.95, 146.02, 143.37, 130.08, 128.07 (d, J CF = 13.1 Hz), 127.64, 125.48 (d, J CF = 4.6 Hz), 112.26, 109.78, 108.58 (d, J CF HRMS (DART): m / z [M + H] + C 22 H 23 BrFN4O2 + Calculated value: 473.0983; measured value: 473.0976.

[0395] (±)-N-(3-Bromo-2-fluorophenyl)-7-[2-(piperidin-1-yl)ethyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK085). [ka] 1H NMR (500 MHz, DMSO-d6): δ 9.60 (s, 1H), 8.33 (s, 1H), 7.94 (s, 1H), 7.58 (ddd, J = 8.0, 6.2, 1.6 Hz, 1H), 7.53 (ddd, J = 8.4, 7.1, 1.6 Hz, 1H), 7.204 (td, J = 8.2, 1.3 Hz, 1H), 7.198 (s, 1H), 4.51 (dd, J = 11.5, 2.4 Hz, 1H), 4.39 - 4.33 (m, 1H), 4.11 (dd, J = 11.6, 7.8 Hz, 1H), 2.50 - 2.44 (m, 2H), 2.42 - 2.27 (m, 4H), 1.90 - 1.76 (m, 2H), 1.55 - 1.45 (m, 4H), 1.42 - 1.34 (m, 2H). 13 C NMR (126 MHz, DMSO-d6): δ 157.20, 153.33 (d, J CF = 247.4 Hz), 153.12, 148.95, 146.02, 143.39, 130.08, 128.07 (d, J CF = 13.1 Hz), 127.64, 125.48 (d, J CF = 4.5 Hz), 112.25, 109.77, 108.58 (d, J CF HRMS (DART): m / z [M + H] + C 23 H 25 BrFN4O2 + Calculated value: 487.1139; measured value: 487.1137.

[0396] (±)-N-(3-Bromo-2-fluorophenyl)-8-[2-(morpholin-4-yl)ethyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK086). [ka] 1 H NMR (500 MHz, DMSO-d6): δ 9.63 (s, 1H), 8.32 (s, 1H), 7.93 (s, 1H), 7.58 (ddd, J = 8.0, 6.3, 1.5 Hz, 1H), 7.53 (t, J = 7.0 Hz, 1H), 7.21 (td, J = 8.1, 1.2 Hz, 1H), 4.50 (dd, J = 11.5, 2.4 Hz, 1H), 4.47 - 4.40 (m, 1H), 4.10 (dd, J = 11.6, 7.4 Hz, 1H), 3.58 (t, J = 4.7 Hz, 4H), 2.55 - 2.46 (m, 2H), 2.45 - 2.33 (m, 4H), 1.92 - 1.79 (m, 2H). 13 C NMR (126 MHz, DMSO-d6): δ 157.20, 153.33 (d, J CF = 248.3 Hz), 153.06, 148.94, 146.06, 143.26, 130.03, 128.12 (d, J CF = 9.8 Hz), 127.69, 125.44 (d, J CF = 4.4 Hz), 112.47, 109.64, 108.55 (d, J CF HRMS (DART): m / z [M + H] + C 22 H 23 BrFN4O3 + Calculated value: 489.0932; measured value: 489.0926.

[0397] (±)-N-(3-Bromo-2-fluorophenyl)-8-[2-(dimethylamino)ethyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK087). [ka] 1 H NMR (500 MHz, DMSO-d6): δ 9.61 (s, 1H), 8.33 (s, 1H), 7.93 (s, 1H), 7.59 (t, J = 6.9 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H), 7.21 (t, J = 8.1 Hz, 1H), 7.18 (s, 1H), 4.49 (dd, J = 11.6, 2.3 Hz, 1H), 4.45 - 4.38 (m, 1H), 4.09 (dd, J = 11.6, 7.5 Hz, 1H), 2.47 - 2.38 (m, 2H), 2.17 (s, 6H), 1.86 - 1.78 (m, 2H). 13 C NMR (126 MHz, DMSO-d6): δ 157.20, 153.37 (d, J CF = 247.6 Hz), 153.08, 148.99, 146.14, 143.29, 130.10, 128.07 (d, J CF = 15.6 Hz), 127.72, 125.44 (d, J CF = 4.4 Hz), 112.50, 109.58, 108.54 (d, J CF = 19.7 Hz), 108.13, 72.30, 67.36, 54.43, 45.17, 28.27. HRMS (DART): m / z [M + H] + C 20 H 21 BrFN4O2 + Calculated value: 447.0826; measured value: 447.0818.

[0398] (±)-N-(3-Bromo-2-fluorophenyl)-8-[2-(4-methylpiperazin-1-yl)ethyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK088). [ka] 1H NMR (500 MHz, DMSO-d6): δ 9.62 (s, 1H), 8.33 (s, 1H), 7.93 (s, 1H), 7.59 (t, J = 7.1 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H), 7.21 (t, J = 8.0 Hz, 1H), 7.17 (s, 1H), 4.49 (dd, J = 11.5, 2.4 Hz, 1H), 4.45 - 4.38 (m, 1H), 4.10 (dd, J = 11.6, 7.4 Hz, 1H), 2.48 - 2.21 (m, 10H), 2.14 (s, 3H), 1.91 - 1.76 (m, 2H). 13 C NMR (126 MHz, DMSO-d6): δ 157.20, 153.36 (d, J CF = 246.9 Hz), 153.08, 148.98, 146.13, 143.28, 130.09, 128.05 (d, J CF = 11.7 Hz), 127.72, 125.44 (d, J CF = 4.3 Hz), 112.50, 109.58, 108.55 (d, J CF HRMS (DART): m / z [M + H] + C 23 H 26 BrFN5O2 + Calculated value: 502.1248; measured value: 502.1240.

[0399] (±)-N-(3-Bromo-2-fluorophenyl)-8-[2-(pyrrolidin-1-yl)ethyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK089). [ka] 1H NMR (500 MHz, DMSO-d6): δ 9.62 (s, 1H), 8.33 (s, 1H), 7.93 (s, 1H), 7.59 (t, J = 7.2 Hz, 1H), 7.53 (t, J = 7.5 Hz, 1H), 7.21 (t, J = 8.0 Hz, 1H), 7.17 (s, 1H), 4.49 (dd, J = 11.5, 2.4 Hz, 1H), 4.47 - 4.41 (m, 1H), 4.10 (dd, J = 11.5, 7.4 Hz, 1H), 2.68 - 2.53 (m, 2H), 2.50 - 2.40 (m, 4H), 1.89 - 1.81 (m, 2H), 1.73 - 1.65 (m, 4H). 13 C NMR (126 MHz, DMSO-d6): δ 157.20, 153.36 (d, J CF = 246.9 Hz), 153.07, 148.98, 146.13, 143.28, 130.07, 128.10, 127.71, 125.44 (d, J CF = 4.6 Hz), 112.48, 109.60, 108.55 (d, J CF HRMS (DART): m / z [M + H] + C 22 H 23 BrFN4O2 + Calculated value: 473.0983; measured value: 473.0976.

[0400] (±)-N-(3-Bromo-2-fluorophenyl)-8-[2-(piperidin-1-yl)ethyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK090). [ka] 1H NMR (500 MHz, DMSO-d6): δ 9.62 (s, 1H), 8.32 (s, 1H), 7.93 (s, 1H), 7.59 (t, J = 7.1 Hz, 1H), 7.53 (t, J = 7.5 Hz, 1H), 7.21 (td, J = 8.0, 1.2 Hz, 1H), 7.17 (s, 1H), 4.49 (dd, J = 11.5, 2.4 Hz, 1H), 4.44 - 4.37 (m, 1H), 4.10 (dd, J = 11.6, 7.4 Hz, 1H), 2.48 - 2.43 (m, 2H), 2.41 - 2.27 (m, 4H), 1.90 - 1.77 (m, 2H), 1.54 - 1.45 (m, 4H), 1.42 - 1.34 (m, 2H). 13 C NMR (126 MHz, DMSO-d6): δ 157.19, 153.34 (d, J CF = 246.7 Hz), 153.07, 149.00, 146.11, 143.29, 130.07, 128.10, 127.71, 125.44 (d, J CF = 4.3 Hz), 112.48, 109.60, 108.55 (d, J CF HRMS (DART): m / z [M + H] + C 23 H 25 BrFN4O2 + Calculated value: 487.1139; measured value: 487.1133.

[0401] N-(3-Bromo-2-fluorophenyl)-8,9-dihydro-7H-[1,4]dioxepino[2,3-g]quinazolin-4-amine (JGK091). [ka] 1H NMR (500 MHz, CDCl3): δ 8.71 (s, 1H), 8.65 (ddd, J = 8.3, 7.3, 1.5 Hz, 1H), 7.48 (s, 1H), 7.43 (s, 1H), 7.39 (br, 1H), 7.28 (ddd, J = 8.1, 6.5, 1.5 Hz, 1H), 7.11 (td, J = 8.2, 1.6 Hz, 1H), 4.41 (t, J = 5.7 Hz, 1H), 4.38 (t, J = 5.8 Hz, 1H), 2.32 (p, J = 5.8 Hz, 1H). 13 C NMR (126 MHz, CDCl3): δ 157.06, 156.08, 153.93, 151.62, 150.19 (d, J CF = 242.7 Hz), 147.83, 128.53 (d, J CF = 10.4 Hz), 127.39, 125.32 (d, J CF = 4.7 Hz), 121.84, 119.15, 111.47, 110.85, 108.62 (d, J CF = 19.3 Hz), 70.86, 70.51, 31.03. HRMS (DART): m / z [M + H] + C 17 H 14 BrFN3O2 + Calculated value: 390.0248; measured value: 390.0236.

[0402] N-(3-Bromo-2-fluorophenyl)-2H-[1,3]dioxolo[4,5-g]quinazolin-8-amine (JGK092). [ka] 1H NMR (500 MHz, CDCl3): δ 8.69 (s, 1H), 8.58 (ddd, J = 8.3, 7.3, 1.5 Hz, 1H), 7.28 (ddd, J = 8.1, 6.5, 1.6 Hz, 1H), 7.25 (br, 1H), 7.14 (s, 1H), 7.11 (td, J = 8.2, 1.6 Hz, 1H), 6.17 (s, 2H), one proton signal missing (probably masked by chloroform signals). 13 C NMR (126 MHz, CDCl3): δ 156.10, 153.37, 153.22, 150.22 (d, J CF = 242.3 Hz), 149.37, 148.43, 128.67 (d, J CF = 10.4 Hz), 127.28, 125.30 (d, J CF = 4.7 Hz), 121.84, 110.75, 108.64 (d, J CF = 19.4 Hz), 106.29, 102.48, 96.49. HRMS (DART): m / z [M + H] + C 15 H 10 BrFN3O2 + Calculated value: 361.9935; measured value: 361.9925.

[0403] Example 16 :Metabolism test of example compounds of the JGK series Exemplary compounds (10 mM) were incubated in human, dog, mouse, or rat liver microsomes (1 mg / mL) at 37°C for up to 90 minutes. Reactions were stopped by adding acetonitrile. Control (compound-free) microsome experiments were performed in parallel. LCMS experiments were performed on a Waters Xevo G2 QT equipped with a Luna Omega Polar C18, 1.6 m, 2.1 x 30 mm column. Chemical structures of exemplary metabolites were identified. Figure 20 Shown below. [Table 2]

[0404] Incorporation by Reference All publications and patents mentioned in this specification are incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0405] equivalent While specific embodiments of the present invention have been discussed, the above detailed description is illustrative and not limiting. Many variations of the present invention will become apparent to those skilled in the art upon review of this specification and the claims that follow. The full scope of the invention will be apparent by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations. The present invention includes, for example, the following aspects. [Section 1] a compound of formula I or formula I*, [ka] or a pharmaceutically acceptable salt thereof, During the ceremony, Z is aryl or heteroaryl; R 2a and R 2b are each independently selected from hydrogen, alkyl, halo, CN, and NO; R 3 is hydrogen, alkyl, or acyl; R 4 is an alkoxy; R 5 is alkyl, and R 7 and R 8 are each independently selected from hydrogen, alkyl, e.g., alkoxyalkyl, aralkyl, etc., or arylacyl; R 11 But hydrogen, alkyl, halo, CN, NO2, OR 7, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R 12 But hydrogen, alkyl, halo, CN, NO2, OR 8 , cycloalkyl, heterocyclyl, aryl, or heteroaryl, or R 11 and R 12 together complete a carbocyclic or heterocyclic ring, The compound or a pharmaceutically acceptable salt thereof. [Section 2] In the formula, R 2a is hydrogen, R 2b is selected from alkyl, halo, CN, and NO2. [Section 3] In the formula, R 2b is hydrogen, R 2a is selected from alkyl, halo, CN, and NO2. [Section 4] A compound of formula (IVa) or formula (IVb), [ka] 10. The compound of any one of the preceding claims, wherein: R 6 is independently selected from alkyl, alkoxy, OH, CN, NO, halo, alkenyl, alkynyl, aralkyloxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl; The compound or a pharmaceutically acceptable salt thereof. [Section 5] In the formula, R 11

[0013] Item 5. The compound according to any one of items 1 to 4, wherein is hydrogen. [Section 6] In the formula, R 11 is OR 7 Item 5. The compound according to any one of items 1 to 4, wherein [Section 7] In the formula, R 7 Item 7. The compound according to item 6, wherein is hydrogen. [Section 8] In the formula, R 7 Item 7. The compound according to item 6, wherein is alkyl. [Section 9] In the formula, R 7 Item 7. The compound according to item 6, wherein is alkoxyalkyl. [Section 10] In the formula, R 7 Item 7. The compound according to item 6, wherein is aryl acyl. [Section 11] In the formula, R 12

[0023] The compound according to any one of items 1 to 10, wherein is heteroaryl, such as furanyl. [Section 12] wherein the heteroaryl is alkyl, alkoxy, OH, CN, NO2, halo, [ka] Item 12. The compound according to item 11, substituted with: [Section 13] In the formula, R 12 is OR 8 Item 11. The compound according to any one of items 1 to 10, wherein [Section 14] In the formula, R 8 Item 14. The compound according to item 13, wherein is hydrogen. [Section 15] In the formula, R 8 Item 14. The compound according to item 13, wherein is alkoxyalkyl. [Section 16] In the formula, R 8 teeth, [ka] Item 16. The compound according to item 15, wherein the alkyl is substituted with . [Section 17] In the formula, R 8 Item 16. The compound according to item 15, wherein is acyl. [Section 18] In the formula, R 11 and R 12and R are bonded to form a carbocyclic or heterocyclic ring, such as a 5-, 6-, or 7-membered carbocyclic or heterocyclic ring. [Section 19] Item 19. The compound according to item 18, wherein the carbocyclic ring or the hete...

Claims

1. A certain amount of the following compound: 【Chemistry 1】 【change】 10. An oral pharmaceutical composition comprising a compound selected from:

2. The compound is 【Chemistry 2】 2. The oral pharmaceutical composition of claim 1, wherein the compound is a pharmaceutically acceptable salt thereof.

3. The compound is 【Transformation 3】 2. The oral pharmaceutical composition of claim 1, wherein the compound is a pharmaceutically acceptable salt thereof.

4. The compound is 【Chemistry 4】 2. The oral pharmaceutical composition of claim 1, wherein the compound is a pharmaceutically acceptable salt thereof.

5. The oral pharmaceutical composition of any one of claims 1 to 4, wherein the compound crosses the blood-brain barrier.

6. 6. The oral pharmaceutical composition of claim 5, wherein the compound crosses the blood-brain barrier in the range of 72% of plasma to 378% of plasma.

7. The oral pharmaceutical composition according to any one of claims 1 to 6, which is solid or liquid.

8. 8. The oral pharmaceutical composition of claim 7, which is a solid selected from capsules, tablets, pills, dragees, powders, and granules.

9. 9. The oral pharmaceutical composition of claim 8, further comprising an excipient, filler, binder, humectant, disintegrant, dissolution retarder, absorption enhancer, wetting agent, absorbent, buffer, enteric coating, opacifier, sweetener, or flavoring agent.

10. 8. The oral pharmaceutical composition of claim 7, which is a liquid selected from an emulsion, a solution, a suspension, a syrup, and an elixir.

11. 11. An oral pharmaceutical composition according to any one of claims 1 to 10 for use in the treatment of cancer selected from brain cancer, lung cancer, and cancer of the head, spine and neck.

12. An oral pharmaceutical composition according to any one of claims 1 to 10 for use in the treatment of brain tumors.

13. The oral pharmaceutical composition of claim 12, wherein the brain tumor is a glioma, an astrocytoma, or a glioblastoma.

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