EA044668B1 - COMPOSITIONS AND METHODS FOR TREATING CANCER - Google Patents

Description

Related applications

This application claims the benefit of US Provisional Applications No. 62/819332, filed March 15, 2019, and 62/904241, filed September 23, 2019, the contents of which are incorporated herein by reference in their entirety.

State of the art

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 is a characteristic genetic abnormality found 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 number of potential therapeutics targeting EGFR or its mutant constitutively active form, AEGFR, are currently in development or in clinical trials for the treatment of GBM, including tyrosine kinase inhibitors (TKIs), monoclonal antibodies, vaccines, and RNA-based agents. However, to date, their effectiveness in the healthcare setting is limited by both primary and acquired drug resistance (Taylor et al. (2012), Curr. Cancer Drug Targets., 12:197-209). A major limitation is that current treatments such as erlotinib, lapatinib, gefitinib and afatinib have poor penetration into the brain (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).

Molecular targeted therapy has revolutionized cancer treatment and paved the way for modern precision medicine. However, despite clearly identifying actionable genetic changes, targeted drugs have failed to benefit patients with glioblastoma (GBM). This is largely due to the insufficient penetration of most targeted agents into the CNS to levels required for tumor eradication; potentially inducing robust adaptive mechanisms to manage therapeutic resistance. While drug combinations that inhibit both the primary lesion and compensatory signaling pathways have attracted interest, these combination therapy strategies are hampered by increased toxicity resulting in subthreshold dosing of each drug.

An alternative therapeutic approach targets the oncogenic driver to alter an important functional property for tumor survival, rendering cells vulnerable to an orthogonal second hit ⁶ . This synthetic lethal strategy may be particularly attractive when the functional network(s) regulated by the oncogene intersect with tumor cell death pathways. In a specific example, oncogenic signaling stimulates glucose metabolism, suppressing intrinsic apoptosis and promoting survival. Suppression of oncogenic factors using targeted therapy may trigger an intrinsic mechanism of apoptosis as a direct consequence of reduced glucose uptake. The interconnected nature of these oncogenic pathways may provide therapeutic opportunities for rational combination treatments, but this remains to be explored.

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

The essence of the invention

In one aspect of the present invention there are provided compounds selected from the group consisting of

- 1 044668

- 2 044668 or a pharmaceutically acceptable salt thereof.

In certain aspects, the present invention provides methods for treating cancer that can be treated by inhibition of EGFR, comprising administering to a subject in need of treatment for the cancer an amount of a compound of the invention. In some embodiments, the cancer is glioblastoma multiforme.

Brief description of graphic materials

In fig. Figure 1 shows the oral pharmacokinetics of JGK005 at 10 mg/kg and the oral pharmacokinetics of erlotinib at 25 mg/kg. JGK005 has good CNS penetration compared to erlotinib.

In fig. Figure 2 shows the activity of erlotinib (left columns) and JGK005 (right columns) against EGFR mutant glioblastomas HK301 and GBM39, respectively. JGK005 has lower activity than erlotinib in both conditions.

In fig. Figure 3 shows the extracellular EGFR kinase activities of erlotinib and JGK010. Both compounds have an IC ₅₀ value of approximately 8 nM.

In fig. Figure 4 shows the efficacy of erlotinib (left columns), JGK005 (center columns), and JGK010 (right columns) against HK301 and GBM39 cells.

In fig. Figure 5 shows the oral pharmacokinetics of JGK005 at 10 mg/kg and JGK010 at 10 mg/kg.

In fig. Figure 6 shows comparisons of EGFR inhibitors in several primary glioblastoma cell lines. Columns 1-4: GBM39 (EGFRvIII), 5-8: GS100 (EGFRwt/EGFRvIII), 9-12: GS017 (A289T), 1316: GS024 (EGFR polysomy).

In fig. 7A shows the activity of JGK010 in EGFR-mutated lung cancer. In fig. 7B shows JGK010 activity in EGFR Amp epidermoid carcinoma.

In fig. 8A shows the pharmacokinetics of JGK010 when administered orally at 6 mg/kg. In fig. 8B shows the pharmacokinetics of JGK010 when administered orally at 10 mg/kg. In fig. Figure 8C shows the pharmacokinetics of JGK010 when administered intravenously at 6 mg/kg. In fig. 8D shows the pharmacokinetics of JGK010 when administered intraperitoneally at 6 mg/kg.

In fig. 9 shows the activities of erlotinib and exemplary compounds of the invention against EGFR Amp WT+vIII HK301.

In fig. 10 shows the activities of erlotinib and exemplary compounds of the invention against EGFR vIII Amp GBM 39.

In fig. 11 shows the activities of erlotinib and exemplary compounds of the invention against HK301 cells.

In fig. 12 shows the activities of erlotinib and exemplary compounds of the invention against GBM39 cells.

In fig. 13A shows phospho-EGFR vIII inhibition by erlotinib and exemplary compounds of the invention. In fig. 13B shows phospho-EGFR vIII inhibition by erlotinib and exemplary compounds of the invention.

In fig. 14A shows the pharmacokinetics of JGK005. In fig. 14B shows the pharmacokinetics of JGK005.

In fig. 15A shows the pharmacokinetics of JGK038. In fig. 15B shows the pharmacokinetics of JGK038.

In fig. 16A shows the pharmacokinetics of JGK010. In fig. 16B shows the pharmacokinetics of JGK010.

In fig. 17A shows the pharmacokinetics of JGK037. In fig. 17B shows the pharmacokinetics of JGK037.

In fig. 18A shows a comparison of the pharmacokinetics of erlotinib and JGK037 in mouse brain/blood. In fig. 18B shows a comparison of the pharmacokinetics of erlotinib and JGK037 in mouse brain/blood.

In fig. 19 shows the brain penetration of erlotinib and exemplary compounds of the invention.

In fig. 20 shows the effect of treatment with either vehicle or JGK037 on the change in RLU.

In fig. 21A-21F show inhibition of EGFR-induced glucose metabolism, which causes minimal death in animal cells but causes apoptosis in GBM cells. In fig. 21A shows the percentage change in 18F-FDG uptake 4 hours after erlotinib treatment relative to vehicle in 19 patient-derived GBM gliomaspheres. Cells with a metabolic response are samples that show a significant reduction in 18F-FDG uptake compared to vehicle, whereas cells without a metabolic response do not show a significant reduction. In fig. 21B shows the percentage change in glucose consumption and lactate production over 12 hours of erlotinib treatment relative to vehicle. Measurements are made using a Nova Biomedical BioProfile analyzer. In fig. 21C shows Annexin V staining of cells with metabolic response (blue, n=10) or cells without metabolic response (red, n=9) after treatment with erlotinib for 72 hours. FIG. 21D shows the percentage change in priming relative to vehicle control, as determined by cytochrome c release, followed by exposure to each BH3 peptide (BIM, BID, or PUMA) in patients with or without metabolic response treated with erlotinib for 24 hours. fig. 21E left shows an immunoblot of HK301 whole cell lysate,

- 3 044668 overexpressing GFP control or GLUT1 and GLUT3 (GLUT1/3). Right: Changes in glucose consumption or lactate production of HK301-GFP or HK301-GLUT1/3 12 h after erlotinib treatment. Values refer to the carrier control. In fig. 21F shows the use of HK301-GFP or HK301-GLUT1/3 cells. The erlotinib concentration was 1 μM for all experiments. Comparisons were made using a two-tailed unpaired Student's t test. Data represent means ± s.e.m. values. three independent experiments. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

In fig. 22A-22H show the binding of cytoplasmic p53 to EGFR for intrinsic apoptosis. In fig. 22A shows an immunoblot of these proteins in two metabolic response cells (HK301 and HK336) expressing CRISPR/CAS9 protein with control guide RNA (sgCtrl) or p53 guide RNA (P53KO). In fig. 22B shows the percentage change in priming relative to vehicle control as determined by cytochrome c release followed by BIM peptide exposure in sgCtrl and P53KO cells treated with erlotinib for 24 hours. FIG. 22C shows an immunoblot of the indicated proteins in HK301 sgCtrl, p53KO, p53KO+p53 ^cyto and p53KO+p53 ^wl . In fig. 22D shows immunofluorescence of p53 protein in combination with DAPI staining to reveal protein localization in HK301 sgCtrl, p53KO+p53 ^cyto , and p53KO+p53wt (scale bars=20 μm). First, gliomaspheres were separated from individual cells and attached to 96-well plates using Cell-Tak (Corning) according to the manufacturer's instructions. Attached cells were then fixed with ice-cold methanol for 10 min and then washed three times with PBS. Cells were then incubated with blocking solution containing 10% FBS and 3% BSA in PBS for 1 h and then incubated with p53 antibody (Santa Cruz, SC-126, 1:50 dilution) overnight at 4°C. The next day, cells were incubated with secondary antibody (Alexa Fluor 647, 1:2000 dilution) for 1 h and DAPI staining for 10 min and then imaged using a Nikon TI Eclipse microscope equipped with a Cascade II fluorescence camera (Roper Scientific). Cells were imaged with emissions at 461 and 647 nM and then processed using NIS-Elements AR analysis software. In fig. 22E shows changes in the indicated mRNA levels following treatment with 100 nM doxorubicin for 24 hours in HK301 sgCtrl, P53KO, p53KO+p53 ^cyto and p53KO+p53 ^wt . Levels are normalized to DMSO-treated cells. In fig. 22F shows data similar to 22B, but in HK301 sgCtrl, p53KO, P53KO+p53 ^cy * ⁰ and p53KO+p53wt. In fig. 22G shows data similar to 22E, but in HK301 sgCtrl, p53KO, p53KO+p53 ^R175H , p53KO+p53 ^R273H and p53KO+p53 ^NES . FIG. 22H shows data similar to 22B and 22F, but in HK301 sgCtrl, p53KO, p53KO+p53 ^R175H , p53KO+p53 ^R273H and p53KO+p53 ^NES Erlotinib concentration was 1 µM for all experiments. Comparisons were made using a two-tailed unpaired Student's t test. Data represent means ± s.e.m. of three independent experiments. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

In fig. 23A-23F show how Bcl-xL prevents GBM cell death by binding to and sequestering cytoplasmic p53 in cells metabolically responsive to EGFRi. In fig. Figure 23A shows p53 immunoprecipitation in two metabolically responsive cells (HK301 and GBM39) 24 hours after erlotinib treatment. The immunoprecipitate was probed with the indicated antibodies. Below are the corresponding lysates before immunoprecipitation (input). In fig. 23B shows data similar to 23A, but in two cells without a metabolic reaction (HK393 and HK254). In fig. 23C shows data similar to 23A and 23B, but in HK301-GFP and HK301-GLUT1/3. Immunoblots for the indicated inputs are shown on the right. In fig. 23D shows treatment of HK301 for 24 hours with erlotinib, WEHI-539, or both, and immunoprecipitation and immunoblotting were performed as previously described. In fig. 23E shows Annexin V staining in two cells with a metabolic response (GBM39 and HK301) and a cell without a metabolic response (HK393) 72 hours after treatment with erlotinib, WEHI-539, or both. In fig. 23F shows Annexin V staining of HK301-GFP and HK301-GLUT1/3 72 h after treatment with erlotinib, wehi-539, or both. The concentrations of erlotinib and WEHI-539 for all experiments were 1 and 5 μM, respectively. Comparisons were made using a two-tailed unpaired Student's t test. Data represent means ± s.e.m. values. three independent experiments. * p<0.05, ** p<0.01.

In fig. 24A-24G show synergistic lethality of combined targeting of EGFR and p53. In fig. 24A shows a summary of changes in EGFR and genes involved in p53 regulation across 273 GBM samples. Genetic changes in EGFR (amp/mutation) are mutually exclusive compared with changes in p53. As shown, EGFR changes are on the left side of the table, while most p53 changes are on the right. In fig. 24B shows a table indicating a significant association between changes in EGFR and genes involved in the p53 pathway. In fig. 24C shows Annexin V staining of metabolically responsive cells (left: HK301) and non-responsive cells (right: GS017) treated with various concentrations of erlotinib, nutlin, and the combination, presented as a dose titration matrix. In fig. 24D shows dose titration of erlotinib and nutlin, as described in 24C, was performed for 10 patients with metabolic response and 6 patients

- 4,044,668 ents without reaction, and a synergy score was calculated (see Materials and Methods). In fig. 24E shows Annexin V staining of HK301-GFP and HK301 GLUT1/3 72 h after treatment with erlotinib, nutlin, or both. In fig. 24F shows similar data as 24E, but in HK301-sgCtrl and HK301-P53KO. In fig. 24G shows HK301 treated for 24 hours with erlotinib, Nutlin, or a combination. Immunoprecipitation was performed with an immunoglobulin G control antibody or an anti-p53 antibody, and the immunoprecipitate was probed with the indicated antibodies. Below are the corresponding lysates before immunoprecipitation (input). All data are representative of at least n=3 independent experiments, mean ± SEM. Unless otherwise stated, the concentrations of erlotinib and nutlin for all experiments were 1 and 2.5 μM, respectively. ** p<0.01, *** p<0.001, **** p<0.0001

In fig. 25A-25F show modulation of glucose metabolism causing p53-mediated cell death in patients without a metabolic response to EGFRi. In fig. 25A shows the percentage change in 18F-FDG uptake 4 hours after treatment with erlotinib, 2DG or pictilizib compared to vehicle in HK393 and HK254. In fig. 25B shows the percentage change in priming relative to vehicle control, as determined by cytochrome c release, followed by exposure to the BIM peptide in HK393 and HK254, followed by erlotinib, 2DG, or pictilisib for 24 hours. FIG. 25C shows data similar to 25B, but in HK393 sgCtrl and P53KO. In fig. 25D shows p53 immunoprecipitation in HK393 and HK254 24 hours after treatment with 2DG or pictilisib. The immunoprecipitate was probed with the indicated antibodies. Below are the corresponding lysates before immunoprecipitation (input). In fig. Figure 25E shows the synergism of various drugs (erlotinib, 2DG and pictilisib) in combination with Nutlin HK393 and HK254. In fig. 25F shows Annexin V staining of HK393 sgCtrl and HK393 P53KO 72 hours after treatment with 2DG, pictilisib, 2DG+nutlin, or pictilisib+nutlin. Unless otherwise stated, the concentrations of erlotinib, 2DG, pictilizib, and nutlin for all experiments were 1 μM, 1 mM, 1, and 2.5 μM, respectively. Comparisons were made using a two-tailed unpaired Student's t test. Data represent means ± s.e.m. values. three independent experiments. * p<0.05, ** p<0.01, *** p<0.001.

In fig. 26A-26H show combined targeting of EGFR-induced glucose uptake and p53 suppression of tumor growth in vivo. In fig. 26A shows ¹⁸ F-FDG PET-CT imaging of GBM39 intracranial xenografts before and after 15 hours of treatment with erlotinib (75 mg/kg). In fig. 26B shows GBM39 intracranial xenografts treated with vehicle (n=5), 75 mg/kg erlotinib (n=7), 50 mg/kg idasanutlin (n=5), or the combination daily (n=12), and tumor burden was estimated at the indicated days using secreted gaussia luciferase (see Materials and Methods). In fig. 26C shows data similar to 26A, but in HK393 intracranial xenografts. In fig. 26D shows data similar to 26B, but in HK393 intracranial xenografts (n=7 for all groups). In fig. Figure 25E shows the survival rate for 26B. In fig. 26F shows the survival rate for 26C. In fig. 26G shows the percentage survival of HK336 cells with metabolic response following the indicated treatments for 25 days and subsequent drug release (n=7 for all groups). In fig. 26H shows the percentage of cell survival without metabolic reaction of GS025 followed by the indicated treatments for 25 days and subsequent drug release (n=9 for all groups). Comparisons for 26B and 26D used data sets from the most recent measurements and were made using a two-tailed unpaired t test. Data represent means ± sem values ** p < 0.01.

In fig. 27A-27G show the characterization of GBM cell lines after EGFR inhibition. In fig. 27A shows the percentage change in ¹⁸ F-FDG uptake at the indicated time points of erlotinib treatment relative to vehicle in two metabolic response cells (HK301 and GBM39). In fig. 27B shows an immunoblot of the indicated proteins from a metabolically responsive cell (HK301) and a non-metabolic cell (HK217) after genetic knockdown of EGFR using siRNA. In fig. 27C shows the percentage change in 18F-FDG uptake in HK301 and HK217 after genetic knockdown of EGFR. In fig. 27D shows the change in glucose consumption by 12 hours of erlotinib treatment in three cells with metabolic response (HK301, GBM39, HK390) and three cells without metabolic response (HK393, HK217, HK254). Measurements are made using a Nova Biomedical BioProfile analyzer. In fig. 27E shows the change in lactate production by 12 hours of erlotinib treatment in three cells with a metabolic response (HK301, GBM39, HK390) and three cells without a metabolic response (HK393, HK217, HK254). Measurements are made using a Nova Biomedical BioProfile analyzer. In fig. 27F shows baseline ECAR measurements of two cells with a metabolic response (HK301 and GBM39, in blue) and two cells without a metabolic response (HK217 and HK393, in red) 12 hours after treatment with erlotinib. In fig. 27G shows the change in glutamine consumption 12 hours after treatment with erlotinib, measured using the Nova Biomedical BioProfile analyzer. Erlotinib concentrations for all experiments were 1 μM. Comparisons

- 5 044668 were conducted using a two-tailed unpaired Student's t test. Data represent means ± s.e.m. values. three independent experiments. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

In fig. 28A, 28B show changes in downstream signaling following EGFR inhibition, correlating with metabolic response. In fig. 28A shows an immunoblot of the indicated proteins 4 hours after treatment with erlotinib in metabolically responsive cells. In fig. 28B shows an immunoblot of the indicated proteins 4 hours after treatment with erlotinib in cells without a metabolic reaction.

In fig. 29A, 29B show genetic characterization of patient-derived GBM cell lines. In fig. 29A shows the genetic background in a panel of GBM lines. In fig. 29B shows fluorescence in situ hybridization (FISH) of HK390, HK336, HK254 and HK393 demonstrating EGFR polysomy. Fluorescence in situ hybridization (FISH) was performed using a commercially available fluorescently labeled dual-color EGFR (red)/CEP 7 (green) probe (Abbott-Molecular). Hybridization and FISH analysis were performed on cell lines according to protocols suggested by the manufacturer. Cells were counterstained with DAPI, and fluorescent probe signals were visualized under a Zeiss fluorescence microscope (Axiophot) equipped with two- and three-color filters.

In fig. 30A, 30B show a shift by EGFR inhibition of the apoptotic balance in metabolically responsive cells. In fig. 30A shows an immunoblot of the indicated proteins 24 hours after treatment with erlotinib in cells with a metabolic response (GBM39, HK301 and HK336) and cells without a metabolic response (HK217, HK393 and HK254). In fig. 30B shows an example of a BH3 dynamic profiling assay in a metabolic response cell (HK301). Left: The percentage of cytochrome c released is measured after exposure to different peptides at the indicated concentrations. Right: The difference in cytochrome c release between vehicle-treated and erlotinib-treated cells is calculated to obtain the percentage of priming. Erlotinib concentrations for all experiments were 1 μM.

In fig. 31A-31C show overexpression of GLUT1/3, which restores impaired glucose metabolism caused by EGFR inhibition. In fig. 31A shows changes in glucose consumption and lactate production 12 hours after erlotinib treatment in HK301-GFP and HK301 GLUT1/3. Measurements are made using a Nova Biomedical BioProfile analyzer. In fig. 31B, left, shows an immunoblot of whole cell lysate of GBM39 overexpressing GFP control or GLUT1 and GLUT3 (GLUT1/3). Right: Changes in glucose consumption or lactate production of GBM39-GFP or GBM39-GLUT1/3 12 h after erlotinib treatment. Values refer to the carrier control. In fig. 31C shows data similar to 35A, but in GBM39-GFP and GBM39-GLUT1/3, erlotinib concentrations were 1 μM for all experiments. Comparisons were made using a two-tailed unpaired Student's t test. Data represent means ± s.e.m. values. three independent experiments. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

In fig. 32A-32I show cytoplasmic p53 required for EGFRi-mediated priming of apoptosis. In fig. 32A shows the percentage change in ¹⁸ F-FDG uptake 4 hours after erlotinib treatment in HK301 sgCtrl and p53 KO cells (mean±sd, n=3). In fig. 32B shows the relative mRNA levels of p53-regulated genes 24 hours after treatment with 1 μM erlotinib or 100 nM doxorubicin in HK301 (metabolic cell). In fig. 32C shows HK301 cells infected with the 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. In fig. 32D shows an immunoblot of the indicated proteins in HK336 sgCtrl, P53KO, P53KO+p53 ^c ^ ⁰ and p53KO+p53 ^wt . In fig. 32E shows immunofluorescence of p53 protein in combination with DAPI staining to reveal protein localization in HK336 sgCtrl, p53KO+p53 ^cyto and p53KO+p53 ^wt (scale marks=20 µm). Immunofluorescence was performed as described above. In fig. 32F shows changes in the indicated mRNA levels following treatment with 100 nM doxorubicin for 24 hours in HK336 sgCtrl, P53KO, P53KO+p53 ^cyto and p53KO+p53wt (mean±sd, n=3). Levels are normalized to DMSO-treated cells. In fig. 32G shows the percentage change in apoptosis priming relative to vehicle control, as determined by cytochrome release followed by BIM peptide exposure, in HK336 sgCtrl, p53KO, P53KO+p53 ^c ^ ⁰ , and p53KO+p53 ^wt cells treated with erlotinib for 24 h ( mean±sd, n=2). Results are representative of two independent experiments. In fig. 32H shows an immunoblot of the indicated proteins in HK301 sgCtrl, p53KO, p53KO+p53 ^R175H , p53KO+p53 ^R273H and p53KO+p53 ^NES . In fig. 32I shows the percentage change in priming in HK301 24 hours after treatment with erlotinib with or without PFTμ pretreatment (10 μM for 2 hours) (mean±sd, n=2). Results are representative of two independent experiments.

In fig. 33A-33D show inhibition of EGFR-induced glucose metabolism inducing Bcl-xL dependence via cytoplasmic p53 functions. In fig. 33A shows the percentage change in priming relative to vehicle control as determined by cytochrome c release followed by exposure to BAD and HRK peptides in metabolically responsive cells.

- 6 044668 her (HK301 and HK336) or non-metabolic cell (HK229) treated with erlotinib. In fig. 33B left shows p53 immunoprecipitation in GBM39-GFP and GBM39-GLUT1/3 24 hours after erlotinib treatment. The immunoprecipitate was probed with the indicated antibodies. The corresponding lysates before immunoprecipitation (input) are shown on the right. In fig. 33C shows Annexin V staining of HK301 (left) and HK336 (right) sgCtrl, p53KO, p53 KO+p53 ^cyto , and p53KO+p53wt 72 hours after treatment with erlotinib, WEHI-539, or the combination. In fig. 33D shows data similar to 33C, but in GBM39-GFP and GBM39-GLUT1/3, the concentrations of erlotinib and WEHI-539 were 1 μM for all experiments. Comparisons were made using a two-tailed unpaired Student's t test. Data represent means ± s.e.m. of three independent experiments. * p<0.05, ** p<0.01, *** p<0.001.

In fig. 34A-34H show inhibition of EGFR-regulated glucose metabolism, and activation of p53 promotes intrinsic apoptosis in the GBM. In fig. 34A shows an immunoblot of the indicated proteins 24 hours after treatment with erlotinib, nutlin, or the combination in two metabolic cells (HK301 and GBM39). In fig. 34B shows Annexin V staining in HK301 and HK217 after genetic knockdown of EGFR and subsequent treatment with Nutlin for 72 hours. FIG. 34C shows detection of BAX oligomerization in HK301-GFP and HK301-GLUT1/GLUT3. 24 hours after this treatment, cells were harvested and incubated in 1 mM BMN to stimulate protein cross-linking and immunoblotted with the indicated antibodies. Below, the BAC is an immunoblot for cytosolic cytochrome c after cellular fractionation. In fig. 34D top shows an immunoblot of the indicated proteins in HK301-GFP and HK301-HA-BclxL. Bottom: Annexin V staining of HK301-GFP and HK301-HA-BclxL 72 h after treatment with erlotinib, nutlin, or the combination. In fig. 34E shows Annexin V HK301 staining after 72 hours of pretreatment with +/-PFTμ, erlotinib, Nutlin, or the combination (10 μM for 2 hours). In fig. 34F shows Annexin V staining of HK301 sgCtrl, p53KO, p53KO+p53 ^R175H , p53KO+p53 ^R273H , and p53KO+p53 ^NES 72 hours after treatment with erlotinib, nutlin, or the combination. In fig. 34G shows data similar to 34F, but in HK301 sgCtrl, P53KO, P53KO+p53 ^cyto and p53KO+p53wt. Drug concentrations for all experiments are as follows: erlotinib (1 μM), nutlin (2.5 μM). Comparisons were made using a two-tailed unpaired Student's t test. Data represent means ± s.e.m. of three independent experiments. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. In fig. 34H shows data similar to 34G, but in HK336 sgCtrl, p53KO, P53KO+p53 ^cyto and p53KO+p53wt. Drug concentrations for all experiments are as follows: erlotinib (1 μM), nutlin (2.5 μM). Comparisons were made using a two-tailed unpaired Student's t test. Data represent means ± s.e.m. of three independent experiments. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

In fig. 35A-35F show that inhibition of glucose metabolism in metabolic and non-metabolic patients promotes intrinsic apoptosis. In fig. 35A shows the percentage change in priming relative to vehicle control as determined by cytochrome c release followed by exposure to BIM peptides in a HK301 metabolic cell 24 hours after treatment with erlotinib or 2DG. In fig. 35B left shows p53 immunoprecipitation in HK301 24 hours after treatment with 2DG. The immunoprecipitate was probed with the indicated antibodies. The corresponding lysates before immunoprecipitation (input) are shown on the right. In fig. 35C shows OCR and ECAR measurements of HK301 cells after exposure to oligomycin and rotenone. In fig. 35D shows Annexin V staining of HK301 72 hours after treatment with Nutlin, erlotinib, 2DG, oligomycin, rotenone alone or in combination with Nutlin. In fig. 35E shows an immunoblot of the indicated proteins 4 hours after treatment with erlotinib or pictilisib in two cells without a metabolic response (HK254 and HK393). In fig. 35F shows immunoprecipitation of p53 in HK254 24 hours after treatment with pictilisib or 2DG. The immunoprecipitate was probed with the indicated antibodies. Below are the corresponding lysates before immunoprecipitation (input). Drug concentrations for all experiments are 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 made using a two-tailed unpaired Student's t test. Data represent means ± s.e.m. values. three independent experiments. ****p<0.0001.

In fig. 36A-36D show in vivo effectiveness of EGFR inhibition and p53 activation. In fig. 36A shows brain and plasma concentrations of idasanutlin at the indicated time points (n=2 mice/time point) in tumor-free mice. In fig. 36B shows immunohistochemical (IHC) analysis of p53 expression in intracranial tumor-bearing xenografts after 36 hours of treatment with idasanutlin (50 mg/kg). In fig. 36C shows the percentage change in ¹⁸ F-FDG uptake 15 hours after erlotinib treatment in GBM39 (n=3) and HK393 (n=5) intracranial xenografts. In fig. 36D shows the change in body weight of mice following daily treatment with erlotinib (75 mg/kg) or a combination of erlotinib (75 mg/kg) and idasanutlin (50 mg/kg). All processing about

- 7 044668 administered orally. Data represent means ± s.e.m. values. three independent experiments. * p<0.05.

In fig. 37A shows that direct inhibition of glycolysis with 2DG (a hexokinase inhibitor) or cytochalasin B (a glucose transporter inhibitor) unexpectedly acts synergistically with p53 activation (with nutlin). In fig. 37B shows that low glucose (0.25 mM) leads to synergistic cell death through inhibition of BCL-xL by navitoclax (ABT-263). In fig. 37C shows that low glucose (0.25 mM) leads to synergistic cell death through inhibition of BCL-xL by nutlin.

In fig. 38 shows a comparison between patients with a metabolic response to the EGFRi inhibitor, erlotinib, and patients without a metabolic response. The combination of erlotinib and nutlin resulted in unexpected synergistic synthetic lethality in patients with a metabolic response but not in patients without a metabolic response.

In fig. 39A shows the enantiomeric purity of synthetic intermediate 5 as determined by chiral SFC (Chiralpak AD-3 column, 40% MeOH). In fig. 39B shows the enantiomeric purity of the synthetic intermediate (S)-5 as determined by chiral SFC (Chiralpak AD-3 column, 40% MeOH). In fig. 39C shows the enantiomeric purity of the synthetic intermediate (R)-5 as determined by chiral SFC (Chiralpak AD-3 column, 40% MeOH). In fig. 39D shows the enantiomeric purity of Mosher ester derivatives 5 as determined by chiral SFC (Chiralpak AD-3 column, 40% MeOH).

In fig. 40 shows the activities of erlotinib, lapatinib, gefitinib and exemplary compounds of the invention against U87 EGFRwt.

In fig. 41 shows the activities of erlotinib, lapatinib, gefitinib and exemplary compounds of the invention against U87 EGFRviii.

In fig. 42 shows the activities of erlotinib, lapatinib, gefitinib and exemplary compounds of the invention against HK301, a patient-derived EGFRvIII mutant GBM.

In fig. 43 shows the activities of erlotinib, lapatinib, gefitinib and exemplary compounds of the invention against GBM39, a patient-derived EGFRvIII mutant GBM.

In fig. 44 shows the activities of erlotinib, lapatinib, and exemplary compounds of the invention in the EGFRvIII mutant GBM39 mouse model.

In fig. 45A shows the activities of erlotinib and exemplary compounds of the invention in the EGFR mutant lung cancer cell line HCC827. In fig. 45B shows the activities of erlotinib and exemplary compounds of the invention in the EGFR mutant lung cancer cell line PC9. In fig. 45C shows the activities of erlotinib and exemplary compounds of the invention in the mutant lung cancer cell line H838.

In fig. 46 shows the activities of erlotinib and exemplary compounds of the invention in the EGFR mutant PC9 mouse model of lung cancer.

In fig. 47 shows specific metabolites of exemplary compounds of the invention.

In fig. 48A shows the activity of exemplary compounds of the invention against HK301. In fig. 48B shows the activity of exemplary compounds of the invention against GBM39. In fig. 48C shows the anti-NHA activity of exemplary compounds of the invention.

In fig. 49A shows the ADME characteristics of an exemplary compound of the present invention in rats after administration of PO. In fig. 49B shows the ADME characteristics of an exemplary compound of the present invention in rats after administration of PO.

In fig. 50A shows the activity of certain compounds of the invention compared to existing standard of care (ie labpatinib, erlotinib, gefitinib and AZD3759) against HK301 patient-derived EGFRvIII mutant GBM. In fig. 50B shows the activity of certain compounds of the invention compared to existing standard of care (ie labpatinib, erlotinib, gefitinib and AZD3759) against HK301 patient-derived EGFRvIII mutant GBM.

In fig. 51A shows the activity of certain compounds of the invention compared to existing standard of care (ie, labpatinib, erlotinib, gefitinib and AZD3759) against GBM39, a patient-derived EGFRvIII mutant GBM. In fig. 51B shows the activity of certain compounds of the invention compared to existing standard of care (ie, labpatinib, erlotinib, gefitinib and AZD3759) against GBM39, a patient-derived EGFRvIII mutant GBM.

In fig. 52A shows the activity of osimertinib and JGK068S against pEGFRwt. In fig. 52B shows the activity of osimertinib and JGK068S against pEGFRvIII.

In fig. 53A shows the activity of osimertinib and JGK068S against HK301. In fig. 53B shows the activity of osimertinib and JGK068S against GBM39.

In fig. 54A shows the activity of AZD3759, AZD9291 and JGK068S against certain EGFR mutants. In fig. 54B shows the activity of AZD3759, AZD9291 and JGK068S against pEGFR A263P. In fig. 54C

- 8 044668 shows the activity of AZD3759, AZD9291 and JGK068S against pEGFR A289V. In fig. 54D shows the activity of AZD3759, AZD9291 and JGK068S against pEGFR A289D. In fig. 54E shows the activity of AZD3759, AZD9291 and JGK068S against pEGFR G598V.

Detailed Description of the Invention

Gliomas are the most common form of brain tumor, with glioblastoma multiforme (GBM) being the most malignant form, causing 3-4% of all cancer-related deaths (Louis et al. (2007), Ada. Neuropathol., 114:97- 109.). The World Health Organization defines GBM as a grade IV cancer, characterized as malignant, mitotically active, and prone to necrosis. GBM has a very poor prognosis, with a 5-year survival rate of 4-5% and a median survival of GBM of 12.6 months (McLendon et al. (2003), Cancer, 98:1745-1748). This may be explained by unique treatment limitations such as high mean age of onset, tumor location, and poor understanding of tumor pathophysiology (Louis et al. (2007), Acta. Neuropathol., 114:97-109). Treatment of GBM involves tumor resection with concurrent radiation and chemotherapy, and in recent years there have been several notable improvements that increase survival (Stewart et al. (2002), Lancet, 359:1011-1018.).

The standard chemotherapy for GBM is temozolomide (TMZ), which is a brain-permeable alkylating agent that methylates purines (A or G) in DNA and induces apoptosis (Stupp et al. (2005), N. Engl. J. Med. , 352:987-996). However, the use of TMZ has the disadvantages that a significant risk arises 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, additional chemotherapy options are urgently needed.

EGFR is a member of the HER superfamily of receptor tyrosine kinases along with ERBB2, ERBB3 and ERBB4. A common factor in the progression of GBM is the accumulation of EGFR, which is found in almost 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 accumulation is associated with the presence of EGFR protein variants: in 68% of EGFR mutants; there is a deletion in the N-terminal ligand-binding region between amino acids 6 and 273. These deletions in the EGFR ligand-binding domains may result in ligand-independent activation of EGFR (Yamazaki et al. (1990), Jpn. J. Cancer Res., 81 :773-779.).

Small molecule tyrosine kinase inhibitors (TKIs) are the most clinically advanced of the EGFR-targeted therapeutics, and both reversible and irreversible inhibitors are in clinical trials. Examples of reversible inhibitors and irreversible inhibitors include erlotinib, gefitinib, lapatinib, PKI166, canertinib and pelitinib (Mischel et al. (2003), Brain Pathol., 13:52-61). Mechanically, these TKIs compete with ATP for binding to the EGFR tyrosine kinase domain, however, these EGFR-linked tyrosine kinase inhibitors were relatively ineffective against gliomas, with response rates as high as 25% with erlotinib (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 show some antitumor activity in patients with GBM, the recurring problem of resistance to receptor inhibition 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 post-therapy brain plasma concentrations of gefitinib and erlotinib were only 6-11% of the initial dose, suggesting that these compounds may not cross the blood-brain barrier, as shown in Table 1. 1 (Karpel-Massler et al. (2009), Mol. Cancer Res., 7:1000-1012). Thus, insufficient delivery to the target may be another reason for disappointing clinical results.

Table 1

The rate of penetration of current standard medications into the brain

Compound Primary Daily dose Blood plasma (ng/ml) CSF (ng/ml) Rate of penetration of NIA into the brain (%) Afatinib EGFR- mutant NSCLC 50 66.7 0.46 0.7 Alectinib ALK- mutant NSCLC 1200 1.5 (unbound CONC.) 1.3 86.7

- 9 044668

Crizotinib ALK- mutant NSCLC 500 237 0.616 0.26 Erlotinib EGFR- NSCLC mutant EGFR- mutant NSCLC 150 1500 (weekly) 1140 ±937 4445.9 28.7 ± 16.8 51.1 2.77 ±0.45 1.2 Gefitinib EGFR- NSCLC mutant EGFR- mutant NSCLC 250 750-1000 326± 116 1345.9- 5094.4 3.7 ± 1.9 14.7-143.1 1.13 ±0.36 1.07-3.58 Lapatinib HER2+paK breast 1250 1515, 3472 1,3,4,5 0.09, 0.13

In light of these data, there remains an unmet clinical need for potent tyrosine kinase inhibitors that have the ability to cross the blood-brain barrier and treat by inhibiting EGFR and its isoforms.

In addition, we showed that cross-talk between oncogenic signaling and metabolic pathways creates opportunities for new combination therapies for GBM. More specifically, we found that acute inhibition of EGFR-induced glucose uptake causes minimal cell death but lowers the threshold for apoptosis in patient-derived GBM cells and primes the cells for apoptosis. Surprisingly, our mechanism of action studies revealed that Bcl-xL blocks cytoplasmic p53 from triggering intrinsic apoptosis, resulting in tumor survival. Pharmacological stabilization of p53 (eg, with the brain-permeable small molecule idasanutlin) allows p53 to engage the intrinsic machinery of apoptosis, promoting mutually reinforcing lethality with targeted EGFR-induced glucose uptake in GBM xenografts. Notably, the inventors also found that rapid changes in ¹⁸ F-fluorodeoxyglucose ( ¹⁸ F-FDG) uptake, using, for example, non-invasive positron emission tomography, can predict sensitivity to the combination in vivo.

We, among other things, identify a critical link between oncogene signaling, glucose metabolism and cytoplasmic p53, which can be used for combination therapy in GBM and other malignancies.

Compounds of the invention.

In one aspect of the present invention, the following connections are provided.

- 10 044668

- 11 044668

or a pharmaceutically acceptable salt thereof.

Methods of treatment.

In certain aspects, the present invention provides methods for treating cancer that can be treated by inhibition of EGFR, comprising administering to a subject in need of treatment for the cancer an amount of a compound of the invention. 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, stomach cancer, gastrointestinal cancer, head cancer , spine and neck, Kaposi's sarcoma, kidney cancer, leukemia, liver cancer, lymphoma, melanoma, multiple myeloma, pancreatic cancer, penile cancer, testicular germ cell cancer, thymoma carcinoma, thymic carcinoma, lung cancer, ovarian cancer or prostate cancer . In certain embodiments, the cancer is a glioma, astrocytoma, or glioblastoma. In certain embodiments, the cancer is a glioblastoma. In certain embodiments, the cancer is glioblastoma multiforme. In certain embodiments, the method reduces the proliferation of cancer cells.

Types and stages of gliomas.

Primary malignant brain tumors are tumors that start in the brain or spinal cord and are called gliomas. Gliomas are not a specific type of cancer, but are a term used to describe tumors that originate from glial cells. Examples of primary malignant brain tumors include astrocytomas, pilocytic astrocytomas, pleomorphic xanthoastrocytomas, diffuse astrocytomas, anaplastic astrocytomas, GBM, gangliogliomas, oligodendrogliomas, ependymomas. According to the WHO classification of brain tumors, astrocytomas are divided into four grades depending on the underlying pathology. Characteristics used to classify gliomas include mitoses, cellular or nuclear atypia, and vascular proliferation and necrosis with pseudoenclosing features. Malignant (or high-grade) gliomas include anaplastic glioma (WHO grade III) as well as glioblastoma multiforme (GBM; WHO grade IV). These are the most aggressive brain tumors with the worst prognosis.

GBM is the most common, complex, treatment-resistant and deadliest 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 primary brain tumor. They are extremely aggressive for a number of reasons. Firstly, glioblastoma cells multiply rapidly as they secrete substances that stimulate an abundant blood supply. They also have the ability to invade and travel long distances into healthy brains, sending out microscopic tumor tendrils along with healthy cells. There are two known types of glioblastomas. Primary GBM is the most common form; they grow quickly and often cause symptoms early. Secondary glioblastomas are less common, accounting for about 10% of all GBMs. They progress from diffuse low-grade astrocytoma or anaplastic astrocytoma and are more common in younger patients. Secondary GBMs are predominantly located in the frontal lobe and have a better prognosis.

GBM is usually treated with a combination multimodal treatment plan that includes surgical removal of the tumor, radiation therapy, and chemotherapy. First, during surgery, as much of the tumor as possible is removed. The location of the tumor in the brain often determines how safely it can be removed. After surgery, radiation therapy and chemotherapy slow the growth of remaining tumor cells. The oral chemotherapy drug temozolomide is most often used for six weeks and then monthly. Another medicine, bevacizumab (known as Avastin®), is also used during treatment. This drug blocks the tumor's ability to generate blood supply, often slowing or even stopping tumor growth.

New investigational treatments are also used, which may involve adding a treatment to standard therapy or replacing one part of standard therapy with another treatment that may work better. Some of these treatments include immunotherapy, such as vaccine immunotherapy, or low-dose pulses of electricity into the area of the brain where the tumor is located, and nanotherapy using spherical nucleic acids (SNAs), such as NU-0129. In some embodiments, the methods of the present invention are used in combination with one or more of the above-mentioned treatments.

Variants of the methods and compositions discussed herein are also discussed

- 12 044668 as applicable to other types of cancer, including, but not limited to, lung cancer, non-CNS cancer, CNS cancer and CNS metastases such as brain metastases, leptomeningeal metastases, choroidal metastases, spinal cord metastases and others .

Cytoplasmic stabilizers of p53.

We demonstrated that pharmacological stabilization of p53, for example with a CNS-permeable small molecule, was synergistically lethal with inhibition of EGFR-induced glucose uptake in primary patient-derived GBM models. We demonstrate for the first time that non-transcriptional functions of p53 may play a critical role in promoting intrinsic apoptosis in metabolic patients. Accordingly, the methods of treatment described herein include the administration of cytoplasmic stabilizer(s) p53 in combination with inhibitors of glucose metabolism. The p53 cytoplasmic stabilizer(s) and glucose metabolism inhibitors can be administered in the same or different compositions, simultaneously or sequentially.

It is contemplated that in some embodiments, a single p53 stabilizer is used, and in other embodiments, more than one p53 stabilizer is used. For example, the combination of nutlin with ABT 737 (which binds BCL-2 and BCL-XL) has been reported to synergistically affect the balance of proapoptotic and antiapoptotic proteins at the mitochondrial level, thereby promoting cell death (Hoe et al., 2014, Nature Reviews, vol. 13, p. 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 priming of cells such as cancer cells for apoptosis.

MDM2 antagonists.

Levels of p53 protein in cells are tightly controlled and maintained at low levels by its negative regulator, the E3 ubiquitin protein ligase MDM2. In embodiments of the methods or compositions of the present invention, the p53 cytoplasmic stabilizer is an MDM2 antagonist/inhibitor. In some embodiments, the MDM2 antagonist is nutlin. In additional 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.

Other compounds within existing pathways known to bind MDM-2 include Ro-2443, MI-219, MI-713, MI-888, DS-3032b, benzodiazepinediones (eg TDP521252), sulfonamides (eg NSC279287), chromenotriazolopyrimidine , morpholinone and piperidinones (AM-8553), terphenyls, chalcones, pyrazoles, imidazoles, imidazole-indoles, isoindolinone, pyrrolidinone (e.g. PXN822), priaxon, naturally occurring piperidines, SAH-8 (linked peptides) sMTide-02, sMTide- 02a (linked peptides), ATSP-7041 (linked peptide), spiroligomer (α-helix mimic). Other compounds known to induce MDM2 protein folding include PRIMA-1MET (also known as APR-246), Aprea 102-105, PK083, PK5174, PK5196, PK7088, benzothiazoles, stytic acid, and NSC319726.

BCL-2 inhibitors.

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

Bcl-xL inhibitors.

In other embodiments of the methods or compositions of the invention, the p53 cytoplasmic stabilizer is a Bcl-xL inhibitor. In some embodiments, the Bcl-xL inhibitor is, for example, WEHI 539, ABT-263, ABT-199, ABT-737, sabutoclax, AT101, TW-37, APG-1252, gumic acid, or any other Bcl-xL inhibitor.

Assessment methods.

Glucose absorption tests.

In embodiments of the methods and compositions of the present invention, a subject with GBM or cancer is classified as a patient with a metabolic response or a patient without a metabolic response, i.e. defined as susceptible to inhibitors of glucose metabolism. In certain embodiments of the invention, the classification of the subject is carried out before administering to the subject a treatment comprising a glucose metabolism inhibitor and a p53 cytoplasmic stabilizer. Accordingly, the present disclosure provides methods for assessing cancer, classifying a subject, determining the subject's susceptibility to treatment, including analyzing glucose metabolism, glycolysis, or glucose uptake. Methods for classifying a subject as having a metabolic reaction are described in detail in Example 1. Methods for monitoring glycolysis and glucose uptake

- 13 044668 goats provided by T. TeSlaa, M.A. Teitell, 2014, Methods in Enzymology, vol. 542, p. 92-114, incorporated herein by reference.

Glycolysis is the intracellular biochemical conversion of one glucose molecule into two pyruvate molecules with the simultaneous formation of two ATP molecules. Pyruvate is a metabolic intermediate with several potential metabolic pathways, including entry into the tricarboxylic acid (TCA) cycle in mitochondria to produce NADH and FADH ₂ . Alternatively, pyruvate can be converted to lactate in the cytosol by lactate dehydrogenase, with simultaneous reduction of NAD+ from NADH. The increased flux due to glycolysis supports the proliferation of cancer cells by providing, for example, 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) first noted that proliferating tumor cells increase aerobic glycolysis, the conversion of glucose to lactate in the presence of oxygen, in contrast to non-malignant cells, which primarily respire in the presence of oxygen. This mitochondrial bypass, called the Warburg effect, occurs in rapidly proliferating cells, including cancer cells, activated lymphocytes, and pluripotent stem cells. The Warburg effect has been used in clinical diagnostic tests that use positron emission tomography (PET) to detect increased cellular uptake of fluorinated glucose analogs such as %-deoxyglucose.

Thus, glycolysis represents a target for therapeutic and diagnostic methods. In the context of existing techniques, measuring glucose uptake and lactate excretion by malignant cells may be useful in detecting shifts in glucose catabolism and/or susceptibility to inhibitors of glucose metabolism. Detection of such shifts is important for ways to treat GBM, ways to reduce the risk of treatment failure, and ways to reduce the chances of tumor survival. For purposes of the present invention, ¹⁸ F-deoxyglucose PET in some embodiments serves as a rapid, non-invasive functional biomarker to predict sensitivity to p53 activation. This noninvasive assay may be particularly valuable in malignant brain tumors, where pharmacokinetics/pharmacodynamics assessment is extremely difficult and impractical. In some cases, delayed imaging protocols (41) and parametric response maps (PRMs) with NMR fusion may be useful for quantifying changes in tumor 18F-FDG uptake (42).

In certain aspects, the methods may relate to measuring glucose uptake and lactate excretion. For cells in culture, glycolytic flux can be quantified by measuring glucose uptake and lactate excretion. Glucose uptake into the cell occurs through glucose transporters (Glut1-Glut4), while lactate excretion occurs through monocarboxylate transporters (MCT1-MCT4) on the cell membrane.

Extracellular glucose and lactate.

Methods for determining glucose uptake and lactate excretion include, for example, extracellular glucose or lactate array, extracellular bioanalyzer, ECAR measurement, [3H]-2-DG or [14C]-2-DG uptake, 18FDG uptake, or 2-NBDG uptake.

There are commercially available kits and instruments for quantifying glucose and lactate levels in cell culture media. Detection method kits are typically colorimetric or fluorometric and are compatible with standard laboratory equipment such as spectrophotometers. BioProfile analyzers (such as Nova Biomedical) or biochemical analyzers (such as YSI Life Sciences, for example) can measure both glucose and lactate levels in cell culture media. GlucCell (Cesco BioProducts) can only measure glucose levels in cell culture media. Although each commercial method has its own detection protocol, the set of culture media used for the assay is the same.

Rate of extracellular acidification.

Glycolysis can also be determined by measuring the environmental extracellular acidification rate (ECAR), which is primarily associated with the excretion of lactic acid per unit time after its conversion from pyruvate. The Seahorse Extracellular Flux (XF) Analyzer (Seahorse Bioscience) is a tool for measuring glycolysis and oxidative phosphorylation (via oxygen consumption) simultaneously in the same cells.

Absorption of glucose analogue.

Certain embodiments of the methods of the present invention include the use of glucose analogues. As is known to one skilled in the art, to determine the rate of glucose uptake into cells, a labeled glucose isoform can be added to cell culture media and then measured in the cells over a specified period of time. Examples of the types of glucose analogs for these studies include, but are not limited to, radioactive glucose analogs such as 2-deoxy-D-[1,2-3H]-glucose, 2-deoxy-D-[1-14C]-glucose, or 2-deoxy-2-( ^18F )-fluoro-Dglucose (18FDG), or fluorescent glucose analogues such as 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol4-yl)amino] -2-deoxyglucose (2-NBDG). Measuring the absorbance of a radioactive analogue of glucose requires a scintillation counter, whereas the absorbance of 2-NBDG is usually measured using

- 14 044668 flow cytometry or fluorescence microscopy. In some embodiments, glucose uptake is measured by uptake of radiolabeled glucose 2-deoxy-2-[fluoro18]fluoro-D-glucose ( ^18F -FDG). In additional embodiments, detection of 18F-FDG is performed using positron emission tomography (PET). In some embodiments, the biopsy is taken from the GBM tumor. A detailed description of the 18F-FDG measurement example is given in the examples below.

In certain aspects, the methods may relate to comparing the glucose uptake of a biological sample, such as a tumor sample, with a control. The increase or decrease of the factor may be at least or no more than 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 derived therefrom. Alternatively, differences in expression between the sample and the reference may be expressed as a percentage decrease or increase, such as at least or maximum 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 obtained from them.

Other ways of expressing relative expression levels are normalized or relative numbers such as 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, 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 derived from them. In some embodiments, the levels may relate to control.

Algorithms such as weighted voting programs can be used to facilitate the assessment of biomarker levels. Additionally, other clinical data can be combined with the biomarker-based test to reduce the risk of false assessments. In some embodiments, other cytogenetic assessments may be considered.

Definitions.

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

The methods and procedures of the present disclosure are generally performed, unless otherwise indicated, in accordance with conventional methods well known in the art and described in the various general and more specific references cited and discussed herein. See, for example, Principles of Neural Science, McGraw-Hill Medical, New York, N.Y. (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., N.Y. (1999); and Gilbert et al., Developmental Biology, 6th ed., Sinauer Associates, Inc., Sunderland, MA (2000).

Chemical terms used herein, unless otherwise noted, are used in accordance with common usage in the art, as shown in The McGraw-Hill Dictionary of Chemical Terms, Parker S., ed., McGraw-Hill, San Francisco , C.A. (1985).

All of the foregoing and any other publications, patents and published patent applications mentioned in this application are expressly incorporated herein by reference. In the event of a conflict, this description, including its specific definitions, will control.

The term agent is used herein to refer to a chemical compound (such as an organic or inorganic compound, a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof, and humanized, chimeric and human antibodies and monoclonal antibodies, protein or part thereof, e.g. peptide, lipid, carbohydrate), or extract obtained from biological materials such as bacterial cells and cells or tissues of plants, fungi or animals (especially mammals). Agents include, for example, agents whose structure is known and agents whose structure is unknown. The ability of such agents to inhibit AR or promote AR degradation may make them useful as therapeutic agents in the methods and compositions of the present invention.

Patient, subject or individual are used interchangeably and refer to a human or non-human animal. These terms include mammals such as humans, primates, farm animals (including cows, pigs, etc.), pets (eg, dogs, cats, etc.) and rodents (eg, mice and rats).

- 15 044668

Treating a condition or patient means taking steps to obtain beneficial or desired results, including clinical results. As used herein, and as is well known in the art, treatment is an approach to obtain favorable or desired results, including clinical results. Beneficial or desirable clinical outcomes may include, but are not limited to, amelioration or improvement of one or more symptoms or conditions, reduction in the severity of a disease, stabilization (i.e., not worsening) of a disease, preventing the spread of a disease, delaying or slowing the progression of a disease, improvement or temporary alleviation of a disease state and remission (partial or complete), whether detectable or undetectable. Treatment may also mean longer survival than would be expected if no treatment was given.

The term prevention is well recognized in the art, and when used in relation to a condition such as a local relapse (eg, pain), a disease such as cancer, a syndrome complex such as heart failure, or any other medical condition, is well understood in the art. field of art and involves administering a composition that reduces the frequency or delays the onset of symptoms of a medical condition in a subject compared to a subject who does not receive the composition. Thus, cancer prevention involves, for example, reducing the number of detectable cancers in a population of patients receiving prophylactic treatment compared to an untreated control population, and/or delaying the occurrence of detectable cancers in a treated population compared to untreated control populations, for example, statistically and /or a clinically significant amount.

The use or administration of a substance, compound or agent to a subject can be accomplished using one of a variety of methods known to those skilled in the art. For example, the compound or agent can be administered intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by oral administration), intranasally (by inhalation), intraspinal, intracerebral, and transdermally (by absorption, e.g., through the skin duct). The compound or agent can also suitably be administered through rechargeable or biodegradable polymer devices or other devices, such as patches and pumps, or formulations that provide sustained, slow, or controlled release of the compound or agent. Administration may also be performed, for example, once, multiple times and/or over one or more extended periods.

Suitable methods of administering a substance, compound or agent to a subject will also depend, for example, on the age and/or physical condition of the subject and the chemical and biological properties of the compound or agent (eg, solubility, digestibility, bioavailability, stability and toxicity). In some embodiments, the compound or agent is administered orally, for example, to a subject by oral administration. In some embodiments, the orally administered compound or agent is in a sustained or sustained release formulation or is administered using a slow or sustained release device.

As used herein, coadministration refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., two agents are simultaneously effective in a patient such that may involve synergistic action of two agents). For example, different therapeutic compounds may be administered either in the same composition or in separate compositions, simultaneously or sequentially. Thus, an individual who receives such treatment may benefit from the combined effects of various therapeutic agents.

A therapeutically effective amount or therapeutically effective dose of a drug or agent is an amount of the drug or agent that, when administered to a subject, will have the intended therapeutic effect. Full therapeutic effect does not necessarily occur with a single dose and may only occur after a series of doses. Thus, a therapeutically effective amount can be administered in one or more administrations. The exact effective amount required for a subject will depend, for example, on the size, health and age of the subject, as well as the nature and severity of the condition being treated, such as cancer or MDS. One skilled in the art can readily determine the effective amount for a given situation through routine experimentation.

As used herein, the term modulate includes inhibiting or suppressing a function or activity (such as cell proliferation), as well as enhancing a function or activity.

The phrase pharmaceutically acceptable is recognized in the art. In certain embodiments, the term includes compositions, excipients, adjuvants, polymers, and other materials and/or dosage forms that, within the bounds of sound medical judgment, are suitable for use in contact with human and animal tissue without undue toxicity, irritation, allergic reaction, or other problems. or complications commensurate with a reasonable ratio

- 16 044668 wearing benefits/risks.

The term pharmaceutically acceptable salt or salt is used herein to mean an acid addition salt or a base addition salt that is suitable for or compatible with the treatment of patients.

As used herein, the term pharmaceutically acceptable acid addition salt means any non-toxic organic or inorganic salt of any of the basic compounds of the invention. Illustrative inorganic acids that form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that 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 acid and methanesulfonic acid. Mono- or di-acid salts may be formed, and such salts may exist in hydrated, solvated, or substantially anhydrous form. In general, the acid addition salts of the compounds of the invention are more soluble in water and various hydrophilic organic solvents and generally exhibit higher melting points compared to their free base forms. The selection of a suitable salt will be known to one skilled in the art. Other pharmaceutically unacceptable salts, such as oxalates, may be used, for example, to isolate the compounds of the invention for laboratory use or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

The term pharmaceutically acceptable base addition salt as used herein means any non-toxic organic or inorganic base addition salt of any of the acidic compounds of the invention, or any intermediates thereof. Exemplary inorganic bases that form suitable salts include lithium, sodium, potassium, calcium, magnesium or barium hydroxide. Exemplary organic bases that form suitable salts include aliphatic, alicyclic or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The selection of a suitable salt will be known to one skilled in the art.

Many of the compounds useful in the methods and compositions herein have at least one stereogenic center in their structure. This stereogenic center may be present in an R or S configuration, these R and S designations being used in accordance with the rules described in Pure Appl. Chem. (1976), 45, 11-30. The specification covers all stereoisomeric forms, such as enantiomeric and diastereoisomeric forms of compounds, salts, prodrugs or mixtures thereof (including all possible mixtures of stereoisomers). See, for example, WO 01/062726.

In addition, some compounds that contain alkenyl groups can exist as Z (zusammen, together) or E (entgegen, opposite) isomers. In each case, the description includes both the mixture and the individual individual isomers.

Some of the compounds may also exist in tautomeric forms. Such forms, although not expressly set forth in the claims described herein, are intended to be included within the scope of this specification.

A prodrug or pharmaceutically acceptable prodrug refers to a compound that is metabolized, eg, hydrolyzed or oxidized, in the host after administration to form a compound of the present invention. Typical examples of prodrugs include compounds that have biologically labile or cleavable (protecting) groups 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 an ester or phosphoramidate as biologically labile or cleavable (protecting) groups are disclosed in US Pat. Nos. 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. The prodrugs of the present invention are metabolized to form the compound of the invention. The present invention includes within its scope prodrugs of the compounds described herein. Conventional procedures for selecting and preparing suitable prodrugs are described, for example, in Design of Prodrugs, ed. H. Bundgaard, Elsevier, 1985.

The phrase pharmaceutically acceptable carrier as used herein means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material, suitable for the preparation of a medicinal product for medical or therapeutic use.

As used herein, the terms Log solubility, LogS or logS are used in the art to quantify the solubility of a compound in water. The solubility of a compound in water significantly influences its absorption and distribution characteristics. Low solubility hour

- 17 044668 this is accompanied by poor absorption. The LogS value is the logarithm with the unit removed (base 10) of solubility, measured in moles/liter.

Pharmaceutical compositions.

The compositions and methods of the present invention can be used to treat an individual in need thereof. In certain embodiments, the individual is a mammal, such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or compound is preferably administered in the form of a pharmaceutical composition containing, for example, the compounds 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 physiologically buffered saline, or other solvents or carriers such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments of the invention, when such pharmaceutical compositions are intended for administration to humans, especially for invasive routes of administration (i.e., routes such as injection or implantation that allow for survivability or diffusion across an epithelial barrier), the aqueous solution does not contain pyrogens or practically does not contain pyrogens. Excipients may be selected, for example, to provide delayed release of the agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition may be in unit dose form such as a tablet, capsule (including break-open capsule and gelatin capsule), granule, lyophilic reconstitute, powder, solution, syrup, suppository, injection or the like. The composition may also be present in a transdermal delivery system, such as a skin patch. The composition may also be present in a solution suitable for topical application, such as a lotion, cream or ointment.

The pharmaceutically acceptable carrier may contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or increase absorption of a compound, such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose or dextrans, 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, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The drug or pharmaceutical composition may be a self-emulsifying drug delivery system or a self-forming microemulsion drug delivery system. The pharmaceutical composition (drug) may also be a liposome or other polymer matrix, which may include, for example, a compound of the invention. For example, liposomes that contain phospholipids or other lipids are non-toxic, physiologically acceptable and metabolizable carriers that are relatively easy to manufacture and use.

The phrase pharmaceutically acceptable is used herein to mean those compounds, materials, compositions and/or dosage forms that, within the bounds of sound medical judgment, are suitable for use in contact with tissues of humans and animals without excessive toxicity, irritation, allergic reaction or other problem. or complications, commensurate with a reasonable benefit/risk ratio.

The phrase pharmaceutically acceptable carrier as used herein means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid excipient, diluent, excipient, solvent or encapsulating material. Each carrier must be acceptable in the sense that it is compatible with the other ingredients of the formulation and does not cause harm to the patient. Some examples of materials that may serve 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 waxes; (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) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline solution; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

The pharmaceutical composition may be administered to a subject by any of a number of routes of administration, including, for example, oral administration (e.g., drops, as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including break-open capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (for example, sublingually); subcutaneously; transdermally (for example, in the form of a patch applied to the skin

- 18 044668 zhu); and topically (eg, as a cream, ointment, or spray applied to the skin). The compound may also be formulated for inhalation. In certain embodiments of the invention, the compound may be simply dissolved or suspended in sterile water. Details of suitable routes of administration and suitable compositions therefor can be found, for example, in US patent No. 6110973, 5763493, 5731000, 5541231, 5427798, 5358970 and 4172896, as well as in the patents cited therein.

The compositions may be conveniently provided in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that can be combined with the carrier material to form a unit dosage form will vary depending on the organism being treated and the particular route of administration. The amount of active ingredient that can be combined with the carrier material to form a unit dosage form will generally be that amount of the compound that produces a therapeutic effect. In general, of 100%, the amount will range from about 1% to about 99% of the active ingredient, preferably from about 5% to about 70%, most preferably from about 10% to about 30%.

Methods for preparing these formulations or compositions include the step of combining an active compound, such as a compound of the invention, with a carrier and, optionally, one or more additional ingredients. Typically, the formulations are prepared by uniformly and thoroughly combining the compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, molding the product.

The compositions of the invention suitable for oral administration may be in the form of capsules (including pop capsules and gelatin capsules), starch capsules, pills, tablets, lozenges (using a flavored base, usually sucrose and gum arabic or tragacanth), freeze-dried preparations, powders, granules or as a solution or suspension in an aqueous or non-aqueous liquid, or as a liquid oil-in-water or water-in-oil emulsion, or as an elixir or syrup, or as lozenges (using an inert base such as gelatin and glycerin, or sucrose and gum arabic) and/or in the form of mouthwashes and the like, each of which contains 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.

To prepare solid oral dosage forms (capsules (including break-open capsules and gelatin capsules), tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or additives such as starches, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or gum arabic; (3) humectants such as glycerin; (4) disintegrants such as agaragar, calcium carbonate, potato or tapioca starch, alginic acid, some silicates and sodium carbonate; (5) dissolution retarding agents such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; (10) complexing agents such as modified and unmodified cyclodextrins; and (11) dyes. In the case of capsules (including break-open capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of this type can also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using a binder (eg, gelatin or hydroxypropyl methylcellulose), lubricant, inert diluent, preservative, disintegrant (eg, sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surfactant, or dispersing agent. Molded tablets can be prepared by molding in a suitable apparatus a mixture of a powdered compound moistened with an inert liquid diluent.

Tablets and other solid dosage forms of pharmaceutical compositions, such as dragees, capsules (including break-open capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and coatings, such as enteric coatings and other coatings well known in the art. pharmaceutical field. They can also be formulated to provide a slow or controlled release of the active ingredient therein, using, for example, hydroxypropyl methylcellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water or some other sterile injection medium directly

- 19 044668 just before use. These compositions may also optionally contain opacifiers and may be of a composition from which they release the active ingredient(s) only or preferentially in a specific 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 may also be in microencapsulated form, if appropriate, with one or more of the excipients described above.

Liquid dosage forms suitable for oral administration include pharmaceutically acceptable emulsions, lyophilic reconstitutes, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate , benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed, peanut, corn, germ, olive, castor and sesame oils), glycerin, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan fatty acid esters and their mixtures.

In addition to inert diluents, oral care compositions may also include auxiliary agents such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, colors, fragrances and preservatives.

Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth and mixtures thereof.

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

Ointments, pastes, creams and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide or mixtures of them.

Powders and aerosols may contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Aerosols may additionally contain conventional propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

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 a compound through the skin. The rate of such flow can be controlled either by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

The phrases parenteral administration and parenterally administered, as used herein, mean modes of administration other than enteral and topical administration, typically by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal , transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration contain one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions or sterile powders, which can be reconstituted into sterile injectable solutions or dispersions immediately before application, which may contain antioxidants, buffers, bacteriostatic agents, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerin, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain auxiliary substances such as preservatives, wetting agents, emulsifying agents and dispersing agents. Preventing the action of microorganisms

- 20 044668 ganisms can be provided by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenolsorbic acid, etc. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be caused by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

In some cases, to prolong the effect of the drug, it is desirable to slow down the absorption of the drug by subcutaneous or intramuscular injection. This can be accomplished by using a liquid suspension of crystalline or amorphous material having poor solubility in water. The rate of absorption of a drug depends on the rate of its dissolution, which in turn may depend on the crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered dosage form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are prepared by forming microencapsulated matrices of the compounds in question in biodegradable polymers such as polylactide-polyglycolide. Depending on the drug to polymer ratio and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by enclosing the drug in liposomes or microemulsions that are compatible with body tissues.

For use in the methods of the invention, the active compounds may be given alone or in the form of a pharmaceutical composition containing, for example, from 0.1 to 99.5% (more preferably from 0.5 to 90%) of the active ingredient in combination with a pharmaceutical acceptable medium.

Methods of administration may also be provided by rechargeable or biodegradable devices. In recent years, various slow-release polymer devices have been developed and tested in vivo for controlled drug delivery, including protein biopharmaceuticals. Various biocompatible polymers (including hydrogels), including both biodegradable and non-biodegradable polymers, can be used to form an implant for sustained release of the compound at a specific target site.

The actual dosage levels of the active ingredients in the pharmaceutical compositions can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and route of administration, without causing toxicity to the patient.

The dosage level chosen will depend on a variety of factors, including the potency of the particular compound or combination of compounds used or their ester, salt or amide, route of administration, time of administration, rate of elimination of the particular compound(s) used, duration of treatment, other drugs , compounds and/or materials used in combination with the specific compound(s) used, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors well known in the art medicine.

A physician or veterinarian of ordinary skill in the art can readily determine and administer a therapeutically effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian may begin dosages of a pharmaceutical composition or compound at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By therapeutically effective amount is meant a concentration of a compound that is sufficient to achieve the desired therapeutic effect. It is generally believed that the effective amount of a compound will vary depending on the weight, sex, age and medical history of the subject. Other factors that influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, the other type of therapeutic agent administered with the compound of the present invention. A large total dose can be delivered by multiple administrations of the agent. Methods for determining potency and dosage are 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).

In general, a suitable daily dose of the active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such effective dose will generally depend on the factors described above.

If desired, an effective daily dose of the active compound may be administered in one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally in unit dosage forms. In certain embodiments of the present invention, the active compound may be administered two or three times daily. In preferred embodiments, the active compound will be administered once daily.

- 21 044668

The patient receiving such treatment is any animal in need, including primates, in particular humans; and other mammals such as horses, cattle, pigs, sheep, cats and dogs; birds; and pets in general.

In certain embodiments, the compounds of the invention may be administered alone or in conjunction with another type of therapeutic agent.

The present description includes the use of pharmaceutically acceptable salts of the compounds of the invention in the compositions and methods of the present invention. In certain embodiments, the contemplated salts of the present invention include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkylammonium salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, L-arginine, benthamine, benzathine, betaine, calcium hydroxide, choline, dinol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, Nmethylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine and zinc salts. In some embodiments, the provided salts of the present invention include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts. In some embodiments, contemplated salts of the invention include, but are not limited to, 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, dodecylsulfonic acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic 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 salts.

Pharmaceutically acceptable acid addition salts may also exist as various solvates such as water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate may be a crystallization solvent belonging to the preparation or crystallization solvent or incidental to such a solvent.

The compositions may also contain wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coatings, sweeteners, flavors, preservatives and antioxidants.

Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

Examples

The invention as generally described will now be more readily understood with reference to the following examples, which are included only for the purpose of illustrating certain aspects and embodiments of the present invention and are not intended to limit the invention.

Example 1 Preparation of exemplary JGK series compounds.

General procedures. The JGK series compounds can be prepared by the methods described below or by any other suitable method. JGK series connections are sometimes referred to in this document with the prefix JCN. All reactions were typically carried out under an inert atmosphere of argon. Unless otherwise noted, materials were obtained from commercial suppliers and used without purification. All solvents were purified and dried by standard methods immediately before use. THF and Et ₂ O were recently distilled from sodium and benzophenone. Methylene chloride, toluene and benzene were purified by refluxing CaH ₂ . Reactions were checked using thin layer chromatography (Kieselgel 60 F254, Merck). The stains were observed by viewing under UV light and staining by charring after immersion in p-anisaldehyde solution or phosphomolybdic acid solution. During aqueous treatment, all organic solutions were dried over anhydrous magnesium sulfate

- 22 044668 tion and filtered before rotary evaporation at water pump pressure. The crude compounds were purified by silica gel column chromatography (SilicaFlash P60, 230-400 mesh, SiliCycle Inc). NMR spectra of protons (1H) and carbon ( ^13C ) were obtained on a Bruker AV 400 (400/100 MHz) or Bruker AV500 (500/125 MHz) spectrometer. Chemical shifts are presented in ppm units with Me ₄ Si or CHCl ₃ as internal standard. Cleavage patterns are designated: s, singlet; d, doublet; t, triplet; m, multiplet; b, wide. High-resolution mass spectrometry data were acquired using a Thermo Fisher Scientific Exactive Plus with an IonSense ID-CUBE DART source.

Receiving JGK001, JGK003.

kg, 48 h

Erlotinib

RCH

JGK001 (R - Me, 73%)

JGKO03 (R - Et. 71%)

JGK001. To a solution of erlotinib (134 mg, 0.3406 mmol) in anhydrous methanol (5.0 mL) was added ditert-butyl dicarbonate (228 mg, 1.7029 mmol) in one portion at room temperature. After stirring at the same temperature for 48 h and concentrated in vacuo. The reaction mixture was diluted with H ₂ O (30 ml) and EtOAc (30 ml). The layers were separated and the aqueous layer was extracted with EtOAc (2x30 ml). The combined organic layers were washed successively with H ₂ O and brine, dried over anhydrous MgSO ₄ , filtered and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 3/1) to give JGK001 (156 mg, 73%).

1H NMR (400 MHz, CDCl3) δ 7.86 (s, 1H), 7.43 (s, 1H), 7.20-7.23 (m, 2H), 7.16 (td, J=1, 2, 7.6 Hz, 1H), 7.09 (d, J=8.0 Hz, 1H), 4.16-4.25 (m, 4H), 3.80 (t, J=5.2 Hz, 2H), 3.77 (t, J=5.2 Hz, 2H), 3.45 (s, 3H), 3.44 (s, 3H), 3.44 (s, 3H), 3, 03 (s, 1H), 1.55 (s, 9H), 1.11 (s, 9H);

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;

MSHR-IER [M+H]+ detected 626.3061 [calc. for C ₃₃ H ₄₃ N ₃ O ₉ 625.2993].

JGK003. To a solution of erlotinib (101 mg, 0.2567 mmol) in anhydrous ethanol (2.6 mL), di-tert-butyl dicarbonate (172 mg, 1.2836 mmol) was added in one portion at room temperature. After stirring at the same temperature for 48 h and concentrated in vacuo. The reaction mixture was diluted with H ₂ O (30 ml) and EtOAc (30 ml). The layers were separated and the aqueous layer was extracted with EtOAc (2x30 ml). The combined organic layers were washed successively with H ₂ O and brine, dried over anhydrous MgSO ₄ , filtered and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 3/1) to give JGK003 (117 mg, 71%).

1H NMR (400 MHz, CDCl ₃ ) δ 7.86 (s, 1H), 7.43 (s, 1H), 7.34 (s, 1H), 7.21-7.24 (m, 2H), 7.16 (d, J=7.6 Hz, 1H), 7.10 (d, J=7.6 Hz, 1H), 4.17-4.25 (m, 4H), 3.61-3 .82 (m, 6H), 3.46 (s, 3H), 3.45 (s, 3H), 3.02 (s, 1H), 1.55 (s, 9H), 1.23 (t, J=6.8 Hz, 3H), 1.12 (s, 9H);

¹³ C NMR (100 MHz, CDCl ₃ ) 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;

MSVR-IER [M+H] ⁺ found 640.3211 [calc. for C34H45N3O9 639.3150].

Receiving JGK002.

Acetic anhydride (5.0 mL) was added to solid erlotinib (165 mg, 0.4194 mmol). After heating at 90°C (bath temperature) with stirring for 3 days, the reaction mixture was cooled to room temperature, neutralized with saturated aqueous NaHCO ₃ (20 ml) and diluted with EtOAc (20 ml). The layers were separated and the aqueous layer was extracted with EtOAc (2x30 ml). The combined organic layers were washed successively with H ₂ O and brine, dried over anhydrous MgSO ₄ , filtered and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 1/1 to 1/3) to give JGK002 (161 mg, 88% recovery).

- 23 044668

1H NMR (400 MHz, CDCI3) δ 9.06 (s, 1H), 7.45 (t, J=1.6 Hz, 1H), 7.37-7.39 (m, 2H), 7.36 (s, 1H), 7.30-7.34 (m, 1H), 7.15 (s, 1H), 4.32 (t, J=4.8 Hz, 2H), 4.16 (t, J=4.8 Hz, 2H), 3.86 (t, J=4.8 Hz, 2H), 3.79 (t, J=4.8 Hz, 2H), 3.46 (s, 3H) , 3.45 (s, 3H), 3.06 (s, 1H), 2.14 (s, 3H);

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;

MSHR-ESI [M+H]+ detected 436.1811 [calc. for C ₂₄ H ₂₅ N ₃ O ₅ 435.1788].

Receiving JGK010, JGK032. General procedure for substitution with aniline analogues.

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 sequential addition dropwise of 1-bromo-2chloroethane (1.3 ml) at room temperature in an Ar atmosphere. After heating at 60°C (bath temperature) with stirring for 24 hours, the reaction mixture was cooled to room temperature and quenched with H ₂ O (50 ml). The layers were separated and the aqueous layer was extracted with EtOAc (50 ml). The combined organic layers were washed successively with H ₂ O and brine, dried over anhydrous MgSO ₄ , filtered and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 6/1 to 3/1) to give the chloroquinazoline fusion 3 (404 mg, 67%).

1H 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].

JGK010. To a solution of fused chloroquinazoline 3 (114 mg, 0.5120 mmol) in DMF (2.6 mL), 3-chloro-2-fluoroaniline (0.10 mL) was added dropwise at room temperature. After heating at 60°C (bath temperature) with stirring for 24 hours, the reaction mixture was cooled to room temperature and diluted with Et2O (30.0 ml) to obtain a white suspension. The resulting white solid was washed successively with Et ₂ O (2x50 ml) and collected to give JGK010 (140 mg, 82%).

1H NMR (400 MHz, CDCl3) δ 8.68 (s, 1H), 8.59 (ddd, J=3.2, 6.8, 6.8 Hz, 1H), 7.39 (s, 1H) , 7.34 (s, 1H), 7.29 (s, 1H), 7.10-7.18 (m, 2H), 4.38-4.43 (m, 4H);

1H NMR (500 MHz, DMSO-d6) δ 11.78 (s, 1H), 8.79 (s, 1H), 8.45 (s, 1H), 7.62 (t, J=7.0 Hz , 1H), 7.50 (t, J=7.0 Hz, 1H), 7.43 (s, 1H), 7.34 (t, J=8.0 Hz, 1H), 4.46-4 .53 (m, 2H), 4.40-4.52 (m, 2H);

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;

MSVR-IER [M+H]+ detected 332.0551 [calc. for C ₁₆ H ₁₁ ClFN ₃ O ₂ 331.0518].

Receiving JGK005.

To a solution of fused chloroquinazoline 3 (14 mg, 0.0628 mmol) in CH3CN (2.0 mL), 3-ethynylaniline (0.05 mL) was added dropwise at room temperature. After heating at 80°C (bath temperature) with stirring for 12 hours, the reaction mixture was cooled to room temperature and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 3/1) to give JGK005 (10 mg, 52%).

1H NMR (400 MHz, DMSO- _d6 ) δ 9.45 (s, 1H), 8.43 (s, 1H), 8.04-8.05 (m, 2H), 7.87-7.90 (m, 1H), 7.34 (t, J=7.9 Hz, 1H), 7.14-7.16 (m, 2H), 4.35-4.39 (m, 4H), 4, 14 (s, 1H);

¹³ C NMR (100 MHz, DMSO-d ₆ ) 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;

MSHR-ESI [M+H]+ found 304.1079 [calc. for C ₁₈ H ₁₃ N ₃ O ₂ 303.1002].

- 24 044668

JGK025.

Preparation of JGK025 was carried out according to the general procedure; JGK025 (25%).

1H NMR (400 MHz, DMSO-06) δ 11.30 (s, 1H), 8.73 (s, 1H), 8.28 (s, 1H), 7.51-7.58 (m, 1H) , 7.41-7.48 (m, 1H), 7.35 (s, 1H), 7.17-7.23 (m, 1H), 4.44-4.50 (m, 2H), 4 .39-4.44 (m, 2H);

¹³ C NMR (125 MHz, MeOD) δ 161.6 (J=245.9 Hz), 160.0, 157.6 (J=249.6 Hz), 152.1, 150.1, 145.6, 135.4, 130.4, 121.4, 112.4, 110.9, 108.1, 108.1, 105.4, 65.5, 64.6;

MSVR-IER [M+H]+ detected 316.0890 [calc. for C16H11F2N3O2 315.0813].

JGK026.

Preparation of JGK026 was carried out according to the general procedure; JGK026 (22%).

1H NMR (400 MHz, DMSO-db) δ 11.09 (s, 1H), 8.74 (s, 1H), 8.18 (s, 1H), 7.39-7.51 (m, 2H) , 7.30 (s, 1H), 7.23-7.29 (m, 1H), 4.45-4.49 (m, 2H), 4.40-4.44 (m, 2H);

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;

MSHR-IER [M+H]+ found 316.0893 [calc. for C16H11F2N3O2 315.0813].

JGK027.

Preparation of JGK027 was carried out according to the general procedure; JGK027 (6%).

1H NMR (400 MHz, DMSO^) δ 11.23 (bs, 1H), 8.75 (s, 1H), 8.29 (s, 1H), 7.91 (s, 1H), 7.46- 7.55 (m, 1H), 7.29 (t, J=8.1 Hz, 2H), 4.46-4.50 (m, 2H), 4.40-4.46 (m, 2H) ;

¹³ C 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;

MSHR-IER [M+H]+ found 316.0889 [calc. for Q6H11F2N3O2 315.0813].

JGK028.

Preparation of JGK028 was carried out according to the general procedure; JGK028 (41%).

1H NMR (500 MHz, MeOD) δ 8.64 (s, 1H), 8.03 (s, 1H), 7.31-7.38 (m, 2H), 7.24-7.31 (m, 2H), 4.50-4.55 (m, 2H), 4.44-4.50 (m, 2H);

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;

MSVR-IER [M+H]+ detected 316.0884 [calc. for C16H11F2N3O2 315.0813].

JGK029.

Preparation of JGK029 was carried out according to the general procedure; JGK029 (52%).

1H NMR (500 MHz, MeOD) δ 8.60 (s, 1H), 7.98 (s, 1H), 7.29 (s, 1H), 7.07-7.13 (m, 2H), 4 .50-4.53 (m,

- 25 044668

2H), 4.44-4.48 (m, 2H);

¹³ C NMR (125 MHz, DMSOL) 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;

MSHR-ESI [M+H]+ detected 334.0794 [calc. for Ci ₆ Hi ₀ F ₃ N ₃ O ₂ 333.0719].

JGK017.

Preparation of JGK017 was carried out according to the general procedure; JGK017 (5%).

1H NMR (500 MHz, CDCl ₃ ) δ 8.59 (s, 1H), 8.15 (d, J=8.3 Hz, 1H), 7.49 (t, J=8.1 Hz, 1H) , 7.38 (s, 1H), 7.34 (d, J=7.9 Hz, 1H), 7.21 (s, 1H), 4.41-4.42 (m, 2H), 4, 38-4.40 (m, 2H);

¹³ C NMR (125 MHz, CDCl ₃ ) 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.

Receiving JGK004.

Benzoylation. To a cold (0°C) solution of diol 2 (205 mg, 1.0428 mmol) in anhydrous CH ₂ Cl ₂ (5.2 ml, 0.2 M), pyridine (0.5 ml) and benzoyl chloride ( 0.7 ml) in an Ar atmosphere. After stirring at room temperature for 12 hours, the reaction mixture was quenched with saturated aqueous NH ₄ Cl (20 ml) and diluted with CH ₂ Cl ₂ (20 ml). The layers were separated and the aqueous layer was extracted with CH ₂ Cl ₂ (2x50 ml). The combined organic layers were washed successively with H ₂ O and brine, dried over anhydrous MgSO ₄ , filtered and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 10/1) to give benzoylchloroquinazoline 2-(2) (220 mg, 52%).

1H NMR (400 MHz, CDCl ₃ ) δ 9.07 (s, 1H), 8.31 (s, 1H), 8.16 (s, 1H), 8.04-8.07 (m, 4H), 7.53-7.58 (m, 2H), 7.34-7.39 (m, 4H).

JGK004. To a solution of benzoylchloroquinazoline 2-(2) (180 mg, 0.444 mmol) in CH3CN (3.0 mL), 3-chloro-2-fluoroaniline (0.06 mL, 0.533 mmol) was added dropwise at room temperature. After heating at 80°C (bath temperature) with stirring for 15 hours, the reaction mixture was cooled to room temperature and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 3/1) to give JGK004 (109 mg, 48%).

1H NMR (400 MHz, CDCl ₃ ) δ 8.85 (s, 1H), 8.48-8.53 (m, 1H), 8.08 (t, J=7.2 Hz, 4H), 7, 99 (d, J=3.2 Hz, 2H), 7.51-7.59 (m, 3H), 7.39 (dd, J=8.4, 16.0 Hz, 4H), 7.16 -7.21 (m, 2H);

¹³ C NMR (125 MHz, CDCl ₃ ) 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;

MSVR-IER [M+H] ⁺ found 514.0963 [calc. for C ₂₈ H ₁₇ ClFN ₃ O ₄ 513.0886].

Receiving JGK006.

To a solution of benzoylchloroquinazoline 2-(2) (100 mg, 0.247 mmol) in CH ₃ CN (3.0 mL), 3-ethynylaniline (0.05 mL, 0.430 mmol) was added dropwise at room temperature. After heating at 50°C (bath temperature) with stirring for 24 hours, the reaction mixture was cooled to room temperature and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 4/1) to give JGK006 (48 mg, 40%).

- 26 044668 ¹ H NMR (400 MHz, CDCl3) δ 8.71 (s, 1H), 8.03 (d, J=8.0 Hz, 2H), 7.96 (s, 1H), 7.90 -7.95 (m, 2H), 7.79 (s, 1H), 7.62-7.75 (m, 3H), 7.55 (t, J=7.3 Hz, 1H), 7. 48 (t, J=7.5 Hz, 1H), 7.37 (t, J=7.4 Hz, 2H), 7.22-7.31 (m, 4H), 3.04 (s, 1H );

¹³ C NMR (100 MHz, CDCl ₃ ) 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;

MSHR-ESI [M+H]+ detected 486.1443 [calc. for C ₃₀ H ₁₉ N ₃ O ₄ 485.1370]

Receiving JGK032.

A 1.0 M hydrogen chloride solution was prepared by adding a solution of hydrogen chloride (0.1 mL, 4.0 M in dioxane, 0.4 mmol) in THF (0.3 mL) at room temperature. To a solution of JGK010 (6.1 mg, 0.01839 mmol) in MeOH, the hydrogen chloride solution formed above (0.030 mL, 0.030 mmol) was added dropwise at room temperature. After stirring at the same temperature for 10 s, the reaction mixture was concentrated in vacuo to give JGK032 (6.7 mg, 99%).

1H NMR (500 MHz, DMSO-d ₆ ) δ 11.63 (s, 1H), 8.81 (s, 1H), 8.36 (s, 1H), 7.63 (ddd, J=1.6 , 6.9, 8.3 Hz, 1H), 7.39 (s, 1H), 7.35 (ddd, J=1.1, 8.1, 16.2 Hz, 1H), 4.49- 4.51 (m, 2H), 4.43-4.45 (m, 2H).

Receiving JGK012.

Acetic anhydride (5.0 mL) was added to solid JGK010 (39 mg, 0.1176 mmol). After heating at 80°C (bath temperature) with stirring for 12 hours, the reaction mixture was cooled to room temperature, neutralized with saturated aqueous NaHCO ₃ (20 ml) and diluted with EtOAc (20 ml). The layers were separated and the aqueous layer was extracted with EtOAc (2x30 ml). The combined organic layers were washed successively with H2O and brine, dried over anhydrous MgSO ₄ , filtered and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 2/1 to 1/1) to give JGK012 (37 mg, 84% recovery).

1H NMR (400 MHz, CDCl ₃ ) δ 9.01 (s, 1H), 7.49 (s, 1H), 7.44 (s, 1H), 7.31-7.41 (m, 2H), 7.09 (t, J=8.0 Hz, 1H), 4.41-4.43 (m, 2H), 4.37-4.40 (m, 2H), 2.15 (s, 3H) ;

¹³ C NMR (125 MHz, CDCl ₃ ) 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;

MSVR-IER [M+H]+ detected 374.0701 [calc. for C18H1 ₃ ClFN ₃ O ₃ 373.0623].

Receiving JGK015.

To a solution of L-amino acid analogue A (227 mg, 0.7712 mmol) in DMF (3.0 ml), chloroquinazoline fusion 3 (117 mg, 0.5932 mmol) was added in one portion at room temperature. After heating at 35°C (bath temperature) with stirring for 12 hours, the reaction mixture was cooled to room temperature and diluted with brine (30.0 ml) and EtOAc (30.0 ml) to obtain a yellow suspension. The layers were separated and the aqueous layer was extracted with EtOAc (2x50 ml). The combined organic layers were concentrated in vacuo. The residue was purified by column chromatography (silica gel, CH ₂ Cl ₂ /MeOH, 40/1 to 10/1) to obtain JGK015 (261 mg, 92%).

1H NMR (500 MHz, CDCl ₃ ) δ 8.57 (s, 1H), 7.64 (s, 1H), 7.61 (d, J=8.0 Hz, 2H), 7.37 (s, 1H), 7.27 (s, 1H),

- 27 044668

7.08 (d, J=8.5 Hz, 2H), 5.09 (d, J=7.5 Hz, 1H), 4.54 (dd, J=6.0, 13.5 Hz, 1H ), 4.31-4.33 (m, 2H), 4.27-4.29 (m, 2H), 3.68 (s, 3H), 3.06 (dd, J=5.6, 14 ,0 Hz, 1H), 3.00 (dd, J=6.1, 13.8 Hz, 1H), 1.39 (s, 9H);

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;

MSHR-ESI [M+H]+ detected 481.2082 [calc. for C25H28N4O6 480,2003].

Receiving JGK016, JGK023.

JGK016 (Boc protection removal). To a solution of JGK015 (121 mg, 0.251 mmol) in anhydrous CH ₂ Cl ₂ (5 ml, 0.05 M), trifluoroacetic acid (1.0 ml) was added dropwise at room temperature. After stirring at the same temperature for 5 hours, the reaction mixture was quenched with saturated aqueous NaHCO ₃ (20 ml) and diluted with CH ₂ Cl ₂ (20 ml). The layers were separated and the aqueous layer was extracted with CH ₂ Cl ₂ (2x30 ml). The combined organic layers were washed successively with H ₂ O and brine, dried over anhydrous MgSO ₄ , filtered and concentrated in vacuo. The residue was purified by column chromatography (silica gel, CH ₂ Cl ₂ /MeOH, 20/1) to obtain JGK016 (74 mg, 77%).

1H NMR (400 MHz, DMSO-d ₆ ) δ 10.5 (s, 1H), 8.68 (s, 1H), 8.43 (s, 2H), 8.20 (s, 1H), 7, 68 (d, J=8.4 Hz, 2H), 7.26 (d, J=8.4 Hz, 2H), 7.23 (s, 1H), 4.44-4.46 (m, 2H ), 4.39-4.41 (m, 2H), 3.68 (s, 3H), 3.03-3.13 (m, 2H);

1H NMR (400 MHz, CD3OD) δ 8.26 (s, 1H), 7.73 (s, 1H), 7.60 (d, J=8.4 Hz, 2H), 7.17 (d, J =8.4 Hz, 2H), 7.08 (s, 1H), 4.31-4.35 (m, 4H), 3.72 (t, J=6.6 Hz, 1H), 3.68 (s, 3H), 3.27-3.29 (m, 1H), 3.01 (dd, J=5.9, 13.6 Hz, 1H), 2.89 (dd, J=7.0 , 13.5 Hz, 1H);

¹³ C 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;

MSVR-IER [M+H] ⁺ found 381.1553 [calc. for C2oH2oN40 ₄ 380.1479].

JGK023 (hydrolysis). Lithium hydroxide (14 mg) was added in one portion to a cold (0°C) solution of JGK016 (42 mg, 0.1104 mmol) in THF/H2O (3:1, total 4.0 ml). After stirring at room temperature for 2 hours, the reaction mixture was neutralized with 1 N. HCl and 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 H2O and brine, dried over anhydrous MgSO ₄ , filtered and concentrated in vacuo. The residue was purified by column chromatography (silica gel, CH ₂ Cl ₂ /MeOH, 30/1 to 15/1) to give JGK023 (25 mg, 62%).

1H NMR (400 MHz, CDCl3) δ 8.62 (s, 1H), 8.12 (s, 1H), 7.71 (d, J=8.4 Hz, 2H), 7.40 (d, J =8.4 Hz, 2H), 7.24 (s, 1H), 4.48-4.50 (m, 2H), 4.42-4.44 (m, 2H), 4.28 (t, J=6.8 Hz, 1H), 3.32-3.37 (m, 1H), 3.20 (dd, J=7.6, 14.8 Hz, 1H);

¹³ C NMR (125 MHz, CDCl ₃ ) 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;

MSVR-IER [M+H] ⁺ found 367.1334 [calc. for C19H18N4O4 366.1322].

Receiving JGK020.

JGK02Q

An amino acid (187 mg) was added dropwise to a solution of chloroquinazoline 2 (104 mg, 0.5294 mmol) in isopropyl alcohol (5.3 ml) at room temperature. After heating at 50°C (bath temperature) with stirring for 12 hours, the reaction mixture was cooled to room temperature and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 5/1 to 3/1) to give JGK020 (128 mg, 53%).

1H NMR (400 MHz, DMSO-th ₆ ) δ 10.68 (s, 1H), 10.22 (br, 1H), 8.64 (s, 1H), 7.91 (s, 1H), 7 .53 (d, J=8.4 Hz, 2H), 7.26-7.31 (m, 4H), 4.12-4.18 (m, 1H), 3.59 (s, 3H), 2.98 (dd, J=5.2, 14.0 Hz, 1H), 2.84 (dd, J=10.0, 13.2 Hz, 1H), 1.30 (s, 9H);

¹³ C NMR (125 MHz, DMSO-a ₆ ) δ 173.0, 158.2, 155.9, 155.6, 148.7, 148.4, 136.1, 135.8, 129.7, 124 ,7,

- 28 044668

107.6, 107.3, 103.3, 78.8, 55.7, 52.3, 36.3, 28.6;

MSVR-IER [M+H]+ detected 455.1920 [calc. for C23H26N4O6 454.1846].

JGK014.

NHBik

J GK014 ⁰

JGK014 (26%).

1H NMR (400 MHz, CDCl3) δ 8.62 (s, 1H), 7.66 (d, J=8.4 Hz, 2H), 7.35 (s, 1H), 7.16 (d, J =8.4 Hz, 2H), 4.99 (d, J=7.6 Hz, 1H), 4.56-4.62 (m, 1H), 4.36-4.42 (m, 4H) , 3.73 (s, 3H), 3.03-3.15 (m, 2H), 1.43 (s, 9H);

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;

MSVR-IER [M+H] ⁺ found 481.2080 [calc. for C ₂₅ H ₂₈ N ₄ O ₆ 480,2003].

JGK021.

The preparation of JGK021 was carried out according to the synthesis procedure of JGK023.

^1H NMR (400 MHz, CDCl3) δ 8.62 (s, 1H), 8.12 (s, 1H), 7.71 (d, J=8.4 Hz, 2H), 7.40 (d, J=8.4 Hz, 2H), 7.24 (s, 1H), 4.48-4.50 (m, 2H), 4.42-4.44 (m, 2H), 4.28 (t , J=6.8 Hz, 1H), 3.32-3.37 (m, 1H), 3.20 (dd, J=7.6, 14.8 Hz, 1H);

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;

MSVR-IER [M+H] ⁺ found 367.1347 [calc. for C ₁₉ H ₁₈ N ₄ O ₄ 366.1322].

Receiving JGK022.

To a solution of diol X (121 mg, 0.6155 mmol) in DMF (3.0 mL), 3-chloro-2fluoroaniline (0.14 mL) was added dropwise at room temperature. After heating at 60°C (bath temperature) with stirring for 3 days, the reaction mixture was cooled to room temperature and diluted with Et ₂ O (30.0 ml) to obtain a white suspension. The resulting white solid was washed successively with Et ₂ O (3x50 ml) and CH ₂ Cl ₂ (2x30 ml) and collected to give JGK022 (132 mg, 70%).

1H NMR (400 MHz, DMSO-b ₆ ) δ 11.16 (br, 1H), 10.43 (br, 1H), 8.68 (s, 1H), 7.91 (s, 1H), 7.58 (t, J=7.1 Hz, 1H), 7.48 (t, J=6.8 Hz, 1H), 7.40 (s, 1H), 7.30 (t, J=8 ,1 Hz, 1H);

¹³ C NMR (125 MHz, DMSO-a ₆ ) 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;

MSVR-IER [M+H] ⁺ found 306.0437 [calc. for C14H9OTN3O2 305.0361].

Receiving JGK018.

Chlorination. Dimethylformamide (0.15 ml) was added dropwise to a solution of 11 (500 mg, 2.134 mmol) in thionyl chloride (7.5 ml, 0.28 M). After heating at 80°C (bath temperature) with stirring for 2 hours, the reaction mixture was cooled to room temperature and concentrated in vacuo. The residue was washed successively with Et ₂ O (200 ml) and immediately used in the next step.

Substitution. To a solution of chloroquinazoline 12 formed above in anhydrous DMF (11 mL, 0.2 M), 3-chloro-2-fluoroaniline (0.50 mL, 4.548 mmol) was added dropwise at room temperature under Ar atmosphere. After stirring at the same temperature for 1 hour, the reaction mixture was diluted with Et ₂ O (100.0 ml) to obtain a white suspension. The resulting white solid was washed successively with Et ₂ O (2x50 ml) and collected to give JGK018 (525 mg, 68%). The spectroscopic data were consistent with those of Zhang, X. et al., J. Med. Chem., 2015, 55, 8200-8215.

Receipt 13.

Deprotection of acetyl. To JGK018 (550 mg, 1.520 mmol), ammonia solution (8.0 mL, 7N in methanol) was added dropwise. After heating at 50°C (bath temperature) in a sealed tube with stirring for 2 hours, the reaction mixture was cooled to room temperature and concentrated in vacuo. The resulting white solid was washed successively with Et ₂ O (2x50 ml) and collected to give 13 (394 mg, 81%). The obtained spectroscopic data coincided with those

Zhang, X. et al., J. Med. Chem. 2015, 55, 8200-8215.

Receipt 14.

To a cold (0°C) solution of 13 (113 mg, 0.353 mmol) in anhydrous DMF (2 mL), triethylamine (0.25 mL, 1.767 mmol) was added dropwise, followed by dropwise addition of di-tert-butyl dicarbonate (62 mg, 0.459 mmol) in anhydrous DMF (2 ml) under Ar atmosphere. After stirring at room temperature for 3 days, the reaction mixture was quenched with saturated aqueous H ₂ O (10 ml) and diluted with EtOAc (10 ml). The layers were separated and the aqueous layer was extracted with EtOAc (2x50 ml). The combined organic layers were washed successively with H ₂ O and brine, dried over anhydrous MgSO ₄ , filtered and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 5/1 to 3/1) to give 14 (42 mg, 28% recovery).

1H NMR (400 MHz, CDCl3) δ 8.69 (s, 1H), 8.39-8.45 (m, 1H), 7.64 (s, 1H), 7.44 (s, 1H), 7 ,11-7.16 (m, 2H), 3.90 (s, 3H), 1.59 (s, 9H);

¹³ C 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.

Getting S.

To a solution of A ¹ (56 mg, 0.1904 mmol) in anhydrous CH ₂ Cl ₂ (2 ml), triethylamine (0.08 ml, 0.5712 mmol) was added dropwise, followed by the addition of chloroacetyl chloride (0.05 ml, 0.5712 mmol). 6286 mmol) at room temperature in an Ar atmosphere. After stirring at the same temperature for 1 hour, the reaction mixture was quenched with saturated aqueous NH ₄ Cl (20 ml) and diluted with CH ₂ Cl ₂ (20 ml). The layers were separated and the aqueous layer was extracted with CH ₂ Cl ₂ (2x30 ml). The combined organic layers were washed successively with H2O and brine, dried over anhydrous MgSO ₄ , filtered and concentrated in vacuo. The crude product was used in the next step without further purification.

- 30 044668

Receiving JGK031.

Alkylation. To a cold (0°C) solution of 14 (44 mg, 0.1058 mmol) in DMF (2.0 ml), potassium carbonate (73 mg, 0.528 mmol) was added dropwise, followed by dropwise addition of the C formed above (0.1904 mmol) in DMF (2.0 ml) at room temperature. After heating at 40°C (bath temperature) with stirring for 3 days, the reaction mixture was quenched with H ₂ O (10 ml) and diluted with EtOAc (10 ml). The layers were separated and the aqueous layer was extracted with EtOAc (2x50 ml). The combined organic layers were washed successively with H ₂ O and brine, dried over anhydrous MgSO ₄ , filtered and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 100/1 to 30/1) to give the alkylated product 14-(2) (43 mg, 55% yield).

^1H NMR (400 MHz, CDCl ₃ ) δ 8.16 (s, 1H), 7.48 (d, J=8.4 Hz, 2H), 7.44 (s, 1H), 6.98-7 .13 (m, 5H), 6.34 (m, 1H), 4.95 (d, J=7.4 Hz, 1H), 4.54 (d, J=6.5 Hz, 1H), 4 .48 (s, 2H), 3.69 (s, 6H), 2.95-3.13 (m, 2H), 1.53 (s, 9H), 1.40 (s, 9H).

Removing protection. To a solution of the alkylated product 14-(2) (26 mg, 0.0348 mmol) in anhydrous MeOH (5.0 ml), 0.5 N was added dropwise. hydrogen chloride solution (0.5 ml). After stirring at the same temperature for 24 hours, the reaction mixture was concentrated in vacuo. The residue was purified by column chromatography (reverse phase silica gel, MeOH or MeOH/H ₂ O, 10/1) to obtain JGK031 (12 mg, 61%).

^1H NMR (400 MHz, MeOD) δ 8.20 (s, 1H), 7.67 (s, 1H), 7.49 (t, J=7.9 Hz, 2H), 7.24-7, 30 (m, 1H), 7.11-7.21 (m, 4H), 3.94 (s, 3H), 3.59 (s, 3H), 2.83-3.01 (m, 2H) ;

MSHR-ESI [M+H]+ detected 554.1631 [calc. for C2 ₂ H25ClFN5O5 553.1522].

Receiving JGK033.

To a solution of JGK031 (5 mg, 0.009026 mmol) in anhydrous THF (6 ml) and H ₂ O (2 ml) was added lithium hydroxide-H ₂ O (3 mg) in one portion. After stirring at room temperature for 2 hours, the reaction mixture was neutralized with 1 N. hydrogen chloride solution and concentrated in vacuo. The residue was purified by reverse phase column chromatography (reverse phase silica gel, MeOH/H ₂ O, 5/1) to give JGK033 (4.4 mg, 90%).

¹ H NMR (400 MHz, MeOD) δ 7.16 (s, 1H), 6.32 (s, 1H), 6.03 (d, J=8.2 Hz, 2H), 5.89-5, 98 (m, 2H), 5.59-5.79 (m, 4H), 4.00 (s, 2H), 2.51 (s, 3H), 2.46 (t, J=5.6 Hz , 1H);

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;

MSHR-ESI [M+H]+ detected 540.1435 [calc. for C26H23ClFNsO5 539.1366].

Receiving JGK008.

To a solution of lapatinib (326 mg, 0.561 mmol) in anhydrous MeOH (5.6 mL) and CH2Cl2 (5.6 mL), di-tert-butyl dicarbonate (378 mg, 2.819 mmol) was added dropwise in one portion under Ar atmosphere. After stirring at room temperature for 2 days, the reaction mixture was quenched with saturated aqueous H2O (10 ml) and diluted with CH2Cl2 (10 ml). The layers were separated and the aqueous layer was extracted with CH ₂ Cl ₂ (5x50 ml). The combined organic layers are successively

- 31 044668 washed with H2O and brine, dried over anhydrous MgSO ₄ , filtered and concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 3/1 to 1/1) to give JGK008 (119 mg, 31% recovery).

1H NMR (500 MHz, CDCI3) δ 8.71 (bs, 1H), 8.64 (s, 1H), 8.44 (s, 1H), 7.85-7.95 (m, 3H), 7 .68 (d, J=7.8 Hz, 1H), 7.32-7.37 (m, 1H), 7.21 (dd, J=8.4, 10.8 Hz, 2H), 6, 98-7.03 (m, 1H), 6.96 (d, J=8.9 Hz, 1H), 6.39 (d, J=47.1 Hz, 1H), 5.13 (s, 2H ), 4.53 (d, J=32.2 Hz, 2H), 3.99 (t, J=7.3 Hz, 2H), 3.39 (d, J=66.1 Hz, 2H), 2.89 (s, 3H), 1.49 (s, 9H);

¹³ C NMR (125 MHz, CDCI3) 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;

MSVR-IER [M+H]+ found 681.1946 [calc. for C34H34CIFN4O6S 680.1866].

Receiving JGK011.

Acetic anhydride (5.0 mL) was added to solid lapatinib (68 mg, 0.353 mmol) under Ar. After stirring at room temperature for 2 days, the reaction mixture was concentrated in vacuo. The residue was purified by column chromatography (silica gel, hexanes/EtOAc, 3/1 to 1/3) to give JGK002 (48 mg, 62% recovery).

1H NMR (500 MHz, CDCI3) δ 9.18 (s, 1H), 8.10-8.17 (m, 3H), 7.50 (d, J=2.5 Hz, 1H), 7.27 -7.36 (m, 2H), 7.18 (dd, J=7.6, 13.8 Hz, 2H), 6.97-7.03 (m, 2H), 6.79 (d, J =3.3 Hz, 1H), 6.44 (d, J=3.3 Hz, 1H), 5.14 (s, 2H), 4.66 (s, 2H), 3.86 (t, J =6.6 Hz, 2H), 3.30 (t, J=6.6 Hz, 2H), 2.95 (s, 3H), 2.33 (s, 3H), 2.23 (s, 3H );

¹³ C NMR (125 MHz, CDCI3) 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;

MSHR-ESI [M+H]+ found 665.1628 [calc. for C:4 koSIADOA 664.1553].

Example 2: Preparation of additional exemplary JGK series compounds.

General chemical information.

All chemicals, reagents, and solvents were purchased from commercial sources when available and used as received. When necessary, reagents and solvents were purified and dried using standard methods. Air- and moisture-sensitive reactions were carried out under an inert atmosphere of argon in oven-dried glassware. Reactions with microwave irradiation were carried out in a single-mode reactor of a CEM Discover microwave synthesizer. Room temperature reactions were carried out at ambient temperature (approximately 23°C). All reactions were monitored by thin layer chromatography (TLC) on pre-coated Merck 60 F254 silica gel plates spotted with UV light (λ=254, 365 nm) or using an alkaline KMnO ₄ solution. Flash column chromatography (FC) was carried out on SiO ₂ 60 (particle size 0.040-0.063 mm, 230-400 mesh). Concentration under reduced pressure (in vacuum) was carried out on a rotary evaporator at 25-50°C. The purified compounds were further dried in a high vacuum or in a desiccant. The outputs correspond to purified compounds and have not been further optimized. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on Bruker spectrometers (operating at 300, 400 or 500 MHz). Carbon NMR spectra ( ^13C NMR) were recorded on Bruker spectrometers (at 400 or 500 MHz). NMR chemical shifts (δ ppm) were assigned to residual solvent signals. 1H NMR data are presented as follows: chemical shift in ppm; multiplicity (s=singlet, d=doublet, t=triplet, k=quartet, quint=quintet, m=multiple/mixed type, td=triplet of doublets, ddd=doublet of doublets of doublets, br=broad signal); interaction constants (J) in Hz, integration. Data for ¹³ C NMR spectra are presented as a function of chemical shift and, if applicable, interaction constants. High-resolution mass spectra (HRMS) were recorded on a Thermo Fisher Scientific Exactive Plus with the original lonSense ID-CUBE DART mass spectrometer. The compounds 4-chloro-7,8-dihydro[1,4]dioxino[2,3d]quinazoline (1), 4-chloroquinazoline-6,7-diol (2) and JGK010 were prepared as above.

General Procedure A for the synthesis of 4-anilinoquinazoline compounds JGK035-JGK041 and JKG043.

A mixture of 4-chloroquinazoline (1 eq.) in iPrOH (0.1-0.3 m) was treated with aniline (1 eq.) and the mixture was heated at 80°C under microwave conditions (60 W) for 15-20 min. The mixture was cooled to 23°C, treated with additional aniline (1 eq.) and again exposed to microwave radiation (80°C, 60 W, 15-20 min). The mixture was either concentrated under reduced pressure or the precipitated 4-anilinoquinazoline hydrochloride salt was isolated by filtration (washing with cold iPrOH). Remainder

- 32 044668 suspended in sat. aq. NaHCO ₃ solution and extracted with CH2Cl2 (3x). The combined organic extracts were washed with water, brine, dried (Na ₂ SO ₄ ), filtered and concentrated. Purification by PC (elution with gradient CH ₂ Cl ₂ /EtOAc or hexanes/EtOAc) provided the desired products, typically as white to off-white or pale yellow solids.

N-(2-Fluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK035).

Following General Procedure A, 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). PC (CH ₂ Cl ₂ /EtOAc 10:1 > 10:4) provided JGK035 (56 mg, 82%) as a white solid.

1H 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 (lat, 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 Hz), 123.97 (d, J=7.8 Hz), 122.89, 115.06 (d, J=19, 3 Hz), 114.46, 110.62, 106.10, 64.69, 64.51 ppm;

MCBP (DART): m/z [M-Н] ^- calc. for C16H11FN3O2; 296.0841; discovered 296.0841.

₄ -Chloro( ^7,7,8,8-2H4 )-7,8-dihydro[1,4]dioxino[2,3-g]quinazoline (3).

A solution of compound 2 (193 mg, 0.98 mmol) in dry DMF (4.8 ml) was treated with Cs ₂ CO ₃ (788 mg, 2.42 mmol), stirred for 5 min and treated dropwise with 1 -bromo- _2chloro(2H4 ⁾ ethane (270 µl, 3.16 mmol). The mixture was stirred at 23°C for 1 hour and then at 70°C for 18 hours. After cooling the mixture to 23°C, all volatiles were removed in vacuo. The residue was dissolved in CH ₂ Cl ₂ (40 ml), washed with water (2x13 ml), brine (13 ml), dried (Na ₂ SO ₄ ), filtered and evaporated. Purification by PC (CH ₂ Cl ₂ /EtOAc 1:0>10:1.5) provided the title compound 3 (109 mg, 49%) as a white fluffy solid.

^1H NMR (400 MHz, CDCl3) δ 8.84 (s, 1H), 7.64 (s, 1H), 7.47 ppm (s, 1H);

¹³ C NMR (101 MHz, CDCl ₃ ) δ 160.19, 152.52, 151.54, 147.93, 146.06, 120.10, 113.72, 110.83 ppm (two carbons directed into the region strong field, not observed);

MSHR (DART): m/z [M+H]+ calc. for C _w H ₄ D ₄ ClN2O2 ⁺ , 227.0520; discovered 227.0516.

N-(3-Chloro-2-fluorophenyl)( ^7,7,8,8-2H4 )-7,8-dihydro[1,4]dioxino[2,3-g] _quinazolin -4-amine (JGK036 ).

Following General Procedure A, 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). JGK036-HCl was isolated by filtration from the crude reaction mixture, and after alkalization and extraction, pure JGK036 (67 mg, 82%) was obtained as a pale yellow solid.

1H 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-a ₆ ) 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);

MSVR (DART): m/z [M+H] ⁺ calc. for C ₁₆ H ₈ D ₄ ClFN ₃ O ₂ ⁺ , 336.0848; discovered 336.0841.

N-(3-Bromo-2-fluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK037).

After general procedure A, compound JGK037 was prepared from 4-chloroquinazoline 1 (100 mg, 0.45 mmol)

- 33 044668 and 3-bromo-2-fluoroaniline (100 µl, 0.89 mmol) in iPrOH (1.5 ml). PC (CH ₂ Cl ₂ /EtOAc 10:0^10:3) provided JGK037 (150 mg, 89%) as a pale yellow solid.

1H NMR (500 MHz, CDC13) δ 8.68 (s, 1H), 8.65 (ddd, J=8.3, 7.4, 1.5 Hz, 1H), 7.39 (s, 1H) , 7.35 (broad, 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, CDCI3) 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;

MCBP (DART): m/z [M-Н] ^- calc. for C^H^^O/, 373.9946; discovered 373.9946.

N-{2-Fluoro-3-[(triethylsilyl)ethynyl]phenyl}-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (4).

A 1 dram vial was loaded with JGK010 (75 mg, 0.23 mmol), XPhos (19.7 mg, 0.041 mmol), Cs ₂ CO ₃ (195 mg, 0.60 mmol), [PdC1 ₂ -(MeCN) ₂ ] (3.6 mg, 0.014 mmol). The vessel was evacuated and filled again with argon (repeated at least twice). Dry acetonitrile (1 ml) was added and the orange suspension was stirred at 23°C for 25 min, then ethynyltriethylsilane (150 μl, 0.84 mmol) was added. The tube was sealed and the reaction mixture was stirred at 95°C in a preheated oil bath for 3.5 hours. The suspension was allowed to reach 23°C, diluted with EtOAc, filtered through a pad of SiO ₂ (washes with EtOAc) and evaporated. Purification by PC (SiO ₂ ; hexanes/EtOAc 8:2^4:6) provided the title compound 4 (48 mg, 49%) as a yellow foamy solid.

1H NMR (500 MHz, CDCI3) 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 (k, J=7.9 Hz, 6H);

¹³ C NMR (126 MHz, CDCI3) 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, J=14.6 Hz), 110.61, 105.97, 98.65, 98.49 (d, J=3.7 Hz), 64.70, 64.51, 7.63, 4.50 ppm;

MCBP (DART): m/z [M+H]+ calc. for C ₂ 4H ₂₇ N3O ₂ Si ⁺ , 436.1851; 436.1831 discovered.

N-(3-Ethynyl-2-fluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK038).

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 hours Water (10 ml) was added and the mixture was extracted with EtOAc (3x15 ml). The combined organics were washed with brine (20 ml), dried (Na ₂ SO ₄ ), filtered and evaporated. Purification by PC (SiO ₂ ; hexanes/EtOAc 7:3^3:7) followed by a second PC (SiO ₂ ; CH ₂ Cl ₂ /EtOAc 1:0^6:4) provided JGK038 (19 mg, 64%) as an off-white solid.

1H NMR (500 MHz, CDC13) δ 8.69 (td, J=8.0, 1.8 Hz, 1H), 8.67 (s, 1H), 7.38 (s, 1H), 7.36 (lat, 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, CDCI3) 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;

MCBP (DART): m/z [M+H]+ calc. for C ₁₈ H ₁₃ FN ₃ O ₂ ⁺ , 322.0986; discovered 322.0981.

N-[2-Fluoro-3-(trifluoromethyl)phenyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK039).

Following General Procedure A, 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). PC (CH ₂ C1 ₂ /EtOAc 1:0^10:3) provided JGK039 (35 mg, 58%) as an off-white solid.

1H NMR (500 MHz, CDCI3) 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, CDC13) 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 (k, J=272.4 Hz), 120.41 (k, J=4.6 Hz), 114, 58, 110.55, 105.86, 64.70, 64.51 ppm;

- 34 044668

MCBP (DART): m/z [M-I]' calc. for C17H _W F4N ₃ O2; 364.0715; 364.0712 discovered.

4-Chloro-8,9-dihydro-7H-[1,4]dioxepino[2,3-g]quinazoline (5).

A solution of compound 2 (100 mg, 0.51 mmol) in dry DMF (10 ml) was treated with Cs ₂ CO ₃ (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 hour and then at 65°C for 18 hours. After cooling to 23°C, all volatiles were removed in vacuo. The residue was suspended in CH ₂ Cl ₂ (20 ml) and washed with water (2x5 ml), dried (Na ₂ SO ₄ ), filtered and evaporated. Purification with PC (hexanes/CH ₂ Cl ₂ 1:10 >0:1 >CH2Cl2EtOAc 10:1.5) followed by another PC (hexanes/EtOAc 10:1 > 10:3) provided the title compound 5 (41 mg, 34%) as a white solid.

1H NMR (500 MHz, CDCl ₃ ) 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, CDCl ₃ ) δ 160.57, 158.86, 153.10, 153.03, 148.88, 120.83, 118.19, 115.64, 70.51, 70.33 , 30.51 ppm;

MCBP (DART): m/z [M+H]+ calc. for CuH _w ClN2O2 ⁺ , 237.0425; discovered 237.0416.

N-(3-Chloro-2-fluorophenyl)-8,9-dihydro-7H-[1,4]dioxepino[2,3-g]quinazolin-4-amine (JGK040).

Following General Procedure A, 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). PC (CH ₂ Cl ₂ /EtOAc 1:0>10:3.5) provided JGK040 (34 mg, 70%) as a white solid.

1H 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, DMSOC) 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, 127.04, 124.91 (d, J=4.7 Hz), 120.13 (d, J=16.5 Hz), 117.52, 113, 81, 110.77, 70.75, 70.62, 30.80 ppm;

MCBP (DART): m/z [M+H]+ calc. for CnHuClFNY 346.0753; discovered 346.0740.

8-Chloro-2H-[1,3]dioxolo[4,5-g]quinazoline (6).

A solution of compound 2 (100 mg, 0.51 mmol) in dry DMF (3.4 ml) was treated with Cs ₂ CO ₃ (335 mg, 1.03 mmol) and stirred at 23°C for 15 min. The mixture was treated dropwise with chloriodomethane (130 μl, 1.79 mmol), stirred for 1 hour and then at 70°C for 17 hours. After cooling the mixture to 23°C, all volatiles were removed in vacuo. The residue was suspended in CH ₂ Cl ₂ (30 ml), washed with water (2x7 ml), dried (Na ₂ SO ₄ ), filtered and evaporated. Purification by PC (hexanes/CH ₂ Cl ₂ 3:10>0:1 >CH ₂ Cl ₂ /EtOAc 10:2) provided the title compound 6 (38 mg, 36%) as a white fluffy solid.

1H NMR (400 MHz, CDCl ₃ ) δ 8.85 (s, 1H), 7.49 (s, 1H), 7.32 (s, 1H), 6.21 ppm (s, 2H);

¹³ C NMR (126 MHz, CDCl ₃ ) δ 159.82, 154.89, 152.79, 150.94, 149.78, 121.23, 105.23, 102.89, 101.12 ppm;

MCBP (DART): m/z [M+H]+ calc. for C ₉ H ₆ ClN ₂ O ₂ ⁺ , 209.0112; discovered 209.0104.

N-(3-Chloro-2-fluorophenyl)-2H-[1,3]dioxolo[4,5-g]quinazolin-8-amine (JGK041).

Following General Procedure A, 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). PC (CH ₂ Cl ₂ /EtOAc 1:0>1:1) provided 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,

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J=8.1, 1.3 Hz, 1H), 7.20 (s, 1H), 6.25 ppm (s, 2H);

¹³ C NMR (126 MHz, DMSO-d ₆ ) 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;

MCBP (DART): m/z [M+H]+ calc. for C ₁₅ H ₁₀ ClFN ₃ O ₂ ⁺ , 318.0440; discovered 318.0435.

[(3-Chloro-2-fluorophenyl)(7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-yl)amino]methyl acetate (JGK043).

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 for 15 min at this temperature, the mixture was added dropwise to a solution of chloromethyl acetate (55 μL, 0.57 mmol) in dry THF (0.5 mL). The flask, which initially contained the JGK010 solution, was washed with 0.5 ml of dry THF and added to the reaction mixture. After stirring at 0°C for 2 hours, stirring was continued at 23°C for 22 hours. Satur. aq. NaHCO ₃ solution (10 ml) was added and the mixture was extracted with EtOAc (3x10 ml). The combined organics were dried (Na ₂ SO ₄ ), filtered and evaporated in vacuo. Purification by PC (CH ₂ Cl ₂ /EtOAc 1:0^1:1) provided the title compound JGK043 (30 mg, 49%) as a pale yellow solid.

1H NMR (500 MHz, CDCl ₃ ) 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, CDCl ₃ ) 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;

MCBP (DART): m/z [M+H]+ calc. for C ₁₉ H ₁₆ ClFN ₃ O ₄ ⁺ , 404.0808; 404.0792 discovered.

Example 3: Preparation of additional exemplary JGK series compounds.

General procedures. All chemicals, reagents, and solvents were purchased from commercial sources when available and used as received. When necessary, reagents and solvents were purified and dried using standard methods. Air- and moisture-sensitive reactions were carried out under an inert atmosphere of argon in oven-dried glassware. Reactions with microwave irradiation were carried out in a single-mode reactor of a CEM Discover microwave synthesizer. Room temperature (RT) reactions were performed at ambient temperature (approximately 23°C). All reactions were monitored by thin layer chromatography (TLC) on pre-coated Merck 60 F ₂₅₄ silica gel plates spotted with UV light (λ=254, 365 nm) or using an alkaline KMnO ₄ solution. Flash column chromatography (FC) was carried out on SiO2 60 (particle size 0.040-0.063 mm, 230-400 mesh). Concentration under reduced pressure (in vacuum) was carried out on a rotary evaporator at 2550°C. The purified compounds were further dried in a high vacuum or in a desiccant. The outputs correspond to purified compounds and have not been further optimized. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on Bruker spectrometers (operating at 300, 400 or 500 MHz). Carbon NMR spectra ( ^13C NMR) were recorded on Bruker spectrometers (at 400 or 500 MHz). NMR chemical shifts (δ ppm) were assigned to residual solvent signals. 1H NMR data are presented as follows: chemical shift in ppm; multiplicity (s=singlet, d=doublet, t=triplet, k=quartet, quint=quintet, m=multiple/mixed type, td=triplet of doublets, ddd=doublet of doublets of doublets, bg=broad signal); interaction constants (J) in Hz, integration. Data for ¹³ C NMR spectra are presented as a function of chemical shift and, if applicable, interaction constants. High-resolution mass spectra (HRMS) were recorded on a Thermo Fisher Scientific Exactive Plus with an original IonSense ID-CUBE DART mass spectrometer or on a Waters LCT Premier mass spectrometer with ACQUITY UPLC with autosampler.

3-[(7,8-Dihydro[1,4]dioxino[2,3-g]quinazolin-4-yl)amino]-2-fluorobenzonitrile (J GK044).

1H NMR (500 MHz, CDCl ₃ ) 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, CDCl ₃ ) 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;

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MSVR (IER): m/z [M+H]+ calc. for C17H12FN4O2+, 323.0939; discovered 323.0927. 3-[(7,8-Dihydro[1,4]dioxino[2,3-g]quinazolin-4-yl)amino]benzonitrile (JGK045).

1H 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-d ₆ ) 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;

MSVR (DART): m/z [M+H] ⁺ calc. for C ₁₇ H ₁₃ N ₄ O ₂ ⁺ , 305.1033; discovered 305.1018.

Ethyl (3-chloro-2-fluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-ylcarbamate (JGK047).

¹ H NMR (500 MHz, CDCl ₃ ) 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, CDCl ₃ ) δ 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), 128.20, 124.50 (d, J=5.1 Hz), 122.22 (d, J=16.8 Hz), 117 .96, 113.51, 109.68 (d, J=2.0 Hz), 64.73, 64.36, 63.44, 14.43 ppm;

MSVR (IER): m/z [M+H] ⁺ calc. for C ₁₉ H ₁₆ ClFN ₃ O ₄ ⁺ , 404.0808; discovered 404.0800.

(±)-4-(3-Bromo-2-fluoroanilino)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-7-ol ((±)-JGK050).

1H NMR (500 MHz, DMSO-d ₆ ) 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-d ₆ ) 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;

MSVR (DART): m/z [M+H] ⁺ calc. for C ₁₆ H ₁₂ BrFN ₃ O ₃ ⁺ , 392.0041; discovered 392.0030.

Diastereomeric mixture of (±)-cis- and (±)-trans-N-(3-bromo-2-fluorophenyl)-7,8-dimethyl-7,8dihydro[1,4]dioxino[2,3-g]quinazoline -4-amine ((±)-cis/trans-JGK051).

1H NMR (500 MHz, CDCl ₃ ; (±)-cis/trans 2:1) δ 8.68 (s, 1H), 8.68-8.63 (m, 1H), 7.37 (s, 1H ), 7.35 (ush, 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,ZN), 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, CDCl ₃ ; (±)-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;

MSVR (DART): m/z [M+H] ⁺ calc. for C ₁₈ H ₁₆ BrFN ₃ O ₂ ⁺ , 404.0404; discovered 404.0393.

(±)-cis-N-(3-Bromo-2-fluorophenyl)-7,8-dimethyl-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine ((± )-JGK052).

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1H NMR (500 MHz, CDCI3) δ 8.68 (s, 1H), 8.66 (ddd, J=8.6, 7.4, 1.6 Hz, 1H), 7.38 (s, 1H) , 7.35 (ush, 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 (d, J=4.7 Hz), 121.71, 114.30, 110.54, 108.57 (d, J=19.5 Hz ), 105.65, 72.85, 72.58, 14.71, 14.55 ppm;

MSVR (IER): m/z [M+H]+ calc. for C18H1 ₆ BrFNaO2 ⁺ , 404.0404; discovered 404.0416.

N-(3-Bromo-4-chloro-2-fluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK053).

1H NMR (500 MHz, DMSO-d ₆ ) 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);

¹³ C NMR (126 MHz, DMSO-d ₆ ) 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;

MSVR (DART): m/z [M+H] ⁺ calc. for C1 ₆ H _p BrCSH^O2 ⁺ , 409.9702; discovered 409.9697.

N-(3,4-Dibromo-2-fluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK054).

1H NMR (500 MHz, DMSO-d ₆ ) 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-d ₆ ) 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;

MSVR (DART): m/z [M+H] ⁺ calc. for C1 ₆ H _p B^^O2 ⁺ , 453.9197; discovered 453.9191.

K-(5-Bromo-2-fluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (J GK055).

1H NMR (500 MHz, CDCla) δ 8.99 (dd, J=7.3, 2.5 Hz, 1H), 8.72 (s, 1H), 7.38 (s, 1H), 7.36 (ush, 1H), 7.27 (s, 1H), 7.16 (dd, 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, CDCl ₃ ) 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 Hz), 125.06, 117.19 (d, J=3.4 Hz), 116.20 (d, J=20 .9 Hz), 114.52, 110.48, 105.85, 64.68, 64.50 ppm;

MSVR (DART): m/z [M+H] ⁺ calc. for C ₁₆ H ₁₂ BrFN ₃ O ₂ ⁺ , 376.0091; discovered 376.0077.

N-(3-Bromo-2,6-difluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK056).

1H NMR (500 MHz, DMSO-d ₆ ) 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-d ₆ ) δ 157.78 (dd, J=248.8, 3.3 Hz), 157.37, 155.01 (dd, J=247.9, 4.9 Hz),

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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 m .d.;

MSVR (IER): m/z [M+H]+ calc. for C ₁₆ H ₁₁ BrF ₂ N ₃ O ₂ ⁺ , 393.9997; discovered 394.0008.

N-(3-Bromo-2,4-difluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK057).

^1H NMR (500 MHz, CDCl ₃ ) 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 (ush, 1H), 7.04 (ddd, J=9.2, 7.8, 2.1 Hz, 1H), 4.45-4.37 m. d. (m, 4H);

¹³ C NMR (126 MHz, CDCl ₃ ) 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, 22.9 Hz) , 64.69, 64.50 ppm;

MSVR (IER): m/z [M+H] ⁺ calc. for С16Н _p ВШ2^О2 ⁺ , 393.9997; discovered 394.0013.

N-(3-Bromo-5-chloro-2-fluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK058).

1H 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);

¹³ C 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 Hz), 126.08, 121.21, 114.60, 110.49, 108.68 (d, J=20.9 Hz ), 105.71, 64.70, 64.52 ppm;

MSVR (IER): m/z [M+H] ⁺ calc. for C ₁₆ H ₁₁ BrClFN ₃ O ₂ ⁺ , 409.9702; discovered 409.9713.

(±)-trans-N-(3-Bromo-2-fluorophenyl)-7,8-dimethyl-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine ((± )-JGK059).

1H NMR (500 MHz, CDCl ₃ ) δ 8.68 (s, 1H), 8.66 (ddd, J=8.6, 7.3, 1.6 Hz, 1H), 7.376 (s, 1H), 7.375 (ush, 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);

¹³ C NMR (126 MHz, CDCl ₃ ) 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 (d, J=4.5 Hz), 121.74, 113.84, 110.43, 108.57 (d, J=19.2 Hz), 105.31, 75.31, 75.05, 17.23, 17.20 ppm;

MSVR (IER): m/z [M+H] ⁺ calc. for C ₁₈ H ₁₆ BrFN ₃ O ₂ ⁺ , 404.0404; discovered 404.0405.

N-(3,4-Dichloro-2-fluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK060).

1H NMR (500 MHz, CDCl ₃ ) 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, CDCl ₃ ) 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;

MSVR (IER): m/z [M+H] ⁺ calc. for C ₁₆ H ₁₁ Cl ₂ FN ₃ O ₂ ⁺ , 366.0207; discovered 366.0207.

N-(3-Bromo-2,5-difluorophenyl)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK061).

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1H 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-d ₆ ) 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;

MSVR (DART): m/z [M+H] ⁺ calc. for C ₁₆ H ₁₁ BrF ₂ N ₃ O ₂ ⁺ , 393.9997; 393.9988 found.

(±)-N-(3-Bromo-2-fluorophenyl)-7-ethenyl-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine ((±)-JGK062) .

1H NMR (500 MHz, CDCI3) δ 8.68 (s, 1H), 8.65 (ddd, J=8.2, 7.3, 1.5 Hz, 1H), 7.40 (s, 1H) , 7.37 (ush, 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, CDCl ₃ ) 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;

MSHR (DART): m/z [M+H]+ calc. for C ₁₈ H ₁₄ BrFN3O ₂ ⁺ , 402.0248; discovered 402.0233.

Example 4: Bioactivity and Assay Protocol for Exemplary Compounds.

Cell-free EGFR kinase assay was performed using the EGFR Kinase System (Promega #V3831). 13 concentrations at 2-fold dilutions from 250 to 0.03052 nM, no drug control and no enzyme control were used in duplicate for 25 ng of EGFR enzyme per reaction. The ADP-Glo kinase assay (Promega #V6930) was used to measure EGFR activity in the presence of inhibitors.

GI ₅₀ assays were performed using patient-derived glioblastoma cells. 13 concentrations at 2-fold dilutions from 40,000 to 9.77 nM (for GBM lines) or from 4,000 to 0.977 nM (for lung cancer lines (HK031)) were seeded in 384-well plates in quadruplicate at 1,500 cells per well. Cells were incubated for 3 days and then proliferation was assessed using Cell Titer Glo (Promega #G7570). As a reference, erlotinib had a GI of _50,642 nM (HK301) and 2,788 nM (GBM39).

Pharmacokinetic studies were performed on male CD-1 mice aged 8-10 weeks. Mice were administered the doses indicated in duplicate. At specific time points, whole blood was obtained by retroorbital bleeding and brain tissue was collected. Blood samples were centrifuged to obtain blood plasma, and brain tissue was washed and homogenized. Samples were extracted with acetonitrile and the supernatant was dried using high-speed vacuum. Dried samples were dissolved in acetonitrile:water:formic acid (50:50:0.1) and quantified on an Agilent 6400 series triple quadrupole LC/MS.

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table 2

Activity of illustrative compounds

Number Structure ΗΚ301 gi ₅₀ (ηΜ) GBM39 GI ₅₀ (ηΜ) JGK001 ''°γ° ^θγν ^Ν γ°χ zO,/4 _o XX _f N and X Τι ⁰ TH 2214 19670 JGK002 ъ ο ο Η ο ο / \ 5448 19820 JGK003 ^th Thursday 1127 23110 JGK004 - , ·“ - 23383 102690 JGK005 And 8824 20536 JGK006 ъ Λ 11012 51380 JGK007 ι ο¥γΤ = ' 92147 - JGK008 - ω ο X and Ας \__/X ΖΛ -J/ Λ ο 81269 -

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JGK009 JGK010 JGK011 JGK001 2 JGK013 o A DR „ / - fp o o \/ 0^0 ^θΥΛ 1 ο t °<b °4 aІ vrQl >Гь ² h ^z a^ ^z ШШ ° - vJ yy - vJ О ⁴ 6096 780.5 9206 5477 1551 14640 2594 3252 10820 219870 JGK014 ~y / °h h° t\ uch W o^__o 11816 27914

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JGK015 / o o _r 2154 11540 JGK016 o.__r 4052 61593 JGK017 r_r 64235 97624 JGK018 Vo o la X 1071 4543 JGK019 G? 2 O V\= ' ° o o nYa - - JGK020 ^H 0^<^ ^N JX o ^ΗΝ γ4 ηνΑΑ 0 29236 36179

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JGK021 CCQ ^ΗΝ γ% nh ₂ 0 480850 555780 JGK022 II О о zjs ^Ω vy 31116 150164 JGK023 o e Λ J O I 86230 96260 JGK024 O=( )=4 about and A> 156370 124940 JGK025 ll ό O'__o 10659 27706 JGK026 LL bt ^R O\_p 6124 16525 JGK027 O'__p 5807 11837

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- 45 044668

JGK035 . o.__ρ 4040 10721 JGK036 WITH"? Cl 1046 4507 JGK037 d o S' 329.3 1116 JGK038 791.1 2946 JGK039 Λ __o 3614 7820 JGK040 - 1721 7115 JGK041 <XI^ 1658 6042

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JGK042 d ^x o Λ cp ^J 2294 4521 JGK043 745 1778 JGK044 _0 4522 5635 JGK045 ^ь b __0 3940 10939 JGK047 < i° °<Ά Q 316000 - JGK050 f°YYb BUT ' O ' F HN Bl And 1159 3568 JGK051 Όαχ 8253 24140 JGK052 0 0 Λ b 3866 9219 JGK053 ο ΰ - 2778 5277

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JGK054 HN 1 W (Ύ 5723 7697 JGK055 ССу HN B< A 290.1 966.4 JGK056 0%. 418.7 1355.8 JGK057 Coy ^ΗΝ ·-^ 1382.7 9361.5 JGK058 1852.7 12974 JGK059 ΤχΑ ^ΗΝ ·-^ 8110 11218 JGK060 ο Ci□ 2947 3501 JGK061 ΟΧλ Q F 1131 1727

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JGK062 O ----- Ν . f ΥΎ J HN--^ 3784 5856 JGK063 OXCQ _F 816 3431 JGK064 OK M —ς 1213 4005 JGK065 s.„akh< f 3432 7339 JGK066 X z— J ^{1 4} О Λ Gr ? 734 2395 JGK067 α+Οοχ ™Д ^В ' 898 2198 JGK068 '0X0?, --(U 577 1613 JGK068 S 439.7 1212 JGK068 R ώ w o o / 0 / 1396 3384

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JGK069 ώ o o 1 G- / 659 2165 JGK070 b k l 1405 3333 JGK071 F 5749 10256 JGK072 ^{F HN} yb ^Br 5017 12033 JGK073 Q ⁴ D . □a 2055 6073 JGK074 Ό.....3% V 2276 6670 JGK075 / ^2. 2-/o ⁴ E. -- O 1181 4005 JGK076 O~Xk, ΠΝ^Ι,Βγ and 6161 16944

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JGK077 \0N। T ΥΎ ί ¹ _F HN 1 ,.Br V 6844 16733 JGK078 0 o % from 2034 5758 JGK079 Q o,b ast 7214 15709 JGK080 (F>, HN^Jx-Br V 7374 14528 JGK081 ώ st o o Q Q - - JGK082 S T ooo FROM -1 1668 1892 JGK083 S ⁿ O g°oA O _F HN^L, ^Br and 529 841 JGK066 s .a D ₀ XX^ _F HN^A^Br and 301 2633

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Example 5 Protein Binding of Erlotinib and Illustrative Compounds of the Invention.

Below in the table. 3 illustrates the protein binding of erlotinib and several exemplary compounds of the invention. Fu refers to the unrelated fraction. Kpuu refers to the uncoupled distribution coefficient of brain and blood plasma at equilibrium.

Table 3

Protein Binding of Erlotinib and Illustrative Compounds of the Invention

Compound Fu (blood)/Yen (brain) Connected (blood)/(brain) Kpuu (Avg) Erlotinib 4.88% 95.12% 0.0513 2.93% 97.07% AZD3759 5.20% 94.80% 0.802 1.44% 98.56% JGK005 1.35% 98.65% 0.491 1.02% 98.98%

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JGK038 1.30% 98.70% 0.575 0.89% 99.11% JGK028 1.44% 98.56% 1.037 1.41% 98.59% JGK010 1.12% 98.88% 1.045 1.10% 98.90% JGK037 1.70% 98.30% 1,301 1.04% 98.96% JGK042 1.85% 98.15% 1.033 1.14% 98.86% JGK063 8.04% 91.96% 0.341 3.78% 96.22% JGK066 7.04% 92.96% 1.175 3.02% 96.98% JGK068 6.31% 93.69% 1,184 2.11% 97.89% JGK068S 5.96% 94.04% 1.181 1.86% 98.14% JGK068R 5.33% 94.67% 1,046 1.57% 98.43% JGK083S 7.02% 92.98% 0.798 2.42% 97.58%

Example 6: Classification of patients with and without metabolic response to EGFRi.

Changes in glucose uptake with acute EGFR inhibition were characterized in 19 patient-derived GBM cell lines. The cells were cultured in a serum-free medium supplemented with gliomaspheres, which, unlike serum-based culture conditions, retain many of the molecular features of patient tumors. Treatment with the EGFR tyrosine kinase inhibitor (EGFRi) erlotinib identified a subset of GBMs in which uptake of radiolabeled glucose ( ¹⁸ F-FDG) was significantly attenuated by EGFR inhibition; hereafter referred to as metabolic response patients (FIGS. 21A and 27A). Suppression of EGFR using siRNA confirmed that the decrease in glucose uptake was not due to off-target effects of erlotinib (Figures 27B, 27C). Decreased ¹⁸ F-FDG uptake by EGFRi-treated cells was associated with decreased lactate production, glucose consumption, and extracellular acidification rate (ECAR), but glutamine levels remained unchanged (Figures 21B and 27D-27G). Finally, decreased glucose utilization correlated with impairments in RAS-MAPK and PI3K-AKT-mTOR signaling, each of which may regulate glucose metabolism in GBM and other cancers (Figure 28A).

In contrast, in all patients without a metabolic response with GBM (i.e., no change in ¹⁸ F-FDG uptake with EGFRi or siRNA) (Figs. 21A and 27B, 27C), no changes were observed in glucose uptake, lactate production, and ECAR, despite to potent EGFR inhibition (FIGS. 21B, 27D-27G and 28B). Moreover, RAS-MAPK and PI3K-AKT-mTOR signaling was largely unaffected in these cells (Figure 28B). Notably, while all patients with metabolic response had alterations in EGFR (copy number gain, mutation), 6 GBM lines without metabolic response also contained EGFR mutations and/or copy number gain (Fig. 29A, 29B). Together, these data illustrate two key points. First, acute inhibition of EGFR rapidly reduces glucose utilization in a subset of primary GBM cells, and second, genetic changes in EGFR cannot by themselves predict which GBMs have a metabolic response to EGFRi.

Example 7 Cells with a metabolic response to EGFRi are primed for apoptosis.

Impaired glucose metabolism may induce the expression of proapoptotic factors and stimulate intrinsic apoptosis, suggesting that decreased glucose uptake in response to EGFRi will stimulate the intrinsic apoptotic pathway. Indeed, acute treatment with erlotinib promoted the expression of the proapoptotic proteins BH3, BIM, and PUMA only in metabolically responsive cell cultures (Figure 30A). However, Annexin V staining showed that in patients with meta

- 53 044668 pain response was only moderate (~17%), although significantly higher, apoptosis compared to patients without metabolic response (~3%) after 72 hours of exposure to erlotinib (Fig. 21C).

The low level of apoptosis, despite the strong induction of proapoptotic factors, led us to wonder whether impaired glucose uptake by EGFRi primes GBM cells for apoptosis; thus increasing the propensity for apoptosis without causing significant cell death. To investigate this, we treated both metabolically and non-metabolically responsive cells with erlotinib for 24 hours and performed dynamic BH3 profiling to quantify changes in apoptosis priming (Figure 30B). Using several BH3 peptides (eg, BIM, BID and PUMA), we observed a significant increase in apoptotic priming, as determined by changes in cytochrome c release relative to vehicle, in metabolically responsive cells treated with erlotinib (Figure 21D - dark gray bars). Importantly, priming in cells with a metabolic response was significantly higher than priming in cells without a metabolic response (Figure 21D - light gray bars), supporting the proposal that attenuation of glucose uptake by EGFRi triggers priming of apoptosis in the GBM.

We investigated whether reduced glucose uptake is required for priming of apoptosis by EGFRi, testing whether reduced glucose uptake would mitigate these effects. To investigate this, glucose transporters 1 (GLUT1) and 3 (GLUT3) were ectopically expressed in two metabolic response cells (HK301 and GBM39). Increased expression of GLUT1 and GLUT3 (GLUT1/3) restored EGFRi-mediated attenuation of glucose uptake and lactate production in both cell lines (Figures 21E and 31A-31C) and, importantly, markedly suppressed apoptotic priming in response to EGFRi (Figures 21F ). Taken together, these data demonstrate that EGFRi-mediated inhibition of glucose uptake, although insufficient to induce significant cell death, lowers the threshold for apoptosis, potentially rendering GBM cells vulnerable to agents that exploit this primed state.

Example 8: Cytoplasmic p53 is required for priming of apoptosis by EGFRi.

The mechanism by which GBMs are primed for apoptosis by EGFRi has been investigated. Inhibition of oncogene-induced glucose metabolism renders GBM cells synergistically susceptible to cytoplasmic p53-dependent apoptosis. Attenuated glucose metabolic flux in the GBM through targeting oncogenic signaling (eg, EGFRi) results in cytoplasmic p53 being recruited into the intrinsic apoptotic pathway (priming). However, Bcl-xL blocks cell death mediated by cytoplasmic p53. Pharmacological stabilization of p53 overcomes this apoptotic block, resulting in mutually reinforcing lethality with the combined targeting of oncogene-induced glucose metabolism in GBM.

In cells that are in a primed state, anti-apoptotic proteins of the Bcl-2 family (e.g., Bcl-2, Bcl-xL, Mcl-1) are largely loaded with pro-apoptotic BH3 proteins (e.g., BIM, BID, PUMA, BAD, NOXA, HRK); therefore, cell survival depends on these interactions. The tumor suppressor protein p53 is known to activate proapoptotic proteins, which subsequently need to be bound by the antiapoptotic proteins Bcl-2 to prevent cell death. To test whether p53 is required for EGFRi-induced priming, we knocked down p53 using CRISPR/CAS-9 (hereafter referred to as P53KO) in two metabolic responders (HK301 and HK336, Fig. 22A). While the change in glucose uptake by EGFRi was not affected in P53KO cells (Figure 32A), BH3 profiling showed that P53KO nearly abolished erlotinib-induced apoptotic priming in both HK301 and HK336 cells (Figure 22B).

Because p53 transcriptional activity has been shown to be enhanced by glucose limitation, we conducted a study to determine whether p53-mediated transcription is induced by EGFRi. However, erlotinib did not increase the expression of p53-regulated genes (eg, p21, MDM2, PIG3, TIGAR) (Fig. 32B) or induce p53-luciferase reporter activity in metabolic response patient HK301 cells (Fig. 32C). These data indicate that although p53 is required for priming by EGFRi, its transcriptional activity may not be necessary.

In addition to the well-characterized nuclear functions of p53, p53 can localize to the cytoplasm, where it can directly interact with the intrinsic apoptotic pathway. To assess whether cytoplasmic p53 is important for priming apoptosis by EGFRi, we stably introduced a p53 mutant with a defective cellular intranuclear localization signal (p53 ^cyto ) into HK301 and HK336 P53KO gliomaspheres. As expected, p53 ^cyto was expressed (FIGS. 22C and 32D), restricted to the cytoplasm (FIGS. 22D and 32E), and had no transcriptional activity (FIGS. 22E and 32F). In contrast, restoration of wild-type (p53 ^wt ) p53 in HK301 and HK336 P53KO cells showed localization similar to parental cells and rescued transcription of p53-regulated genes (Figures 22C-22E and 32E-32G). Notably, stable administration of p53 ^cyto significantly restored priming by erlotinib in HK301 and HK336 P53KO cells to levels comparable to p53 ^wt (Figures 22F and 32G), indicating

- 54 044668 that the cytoplasmic function of p53 is required for EGFRi-mediated priming. In support of this, introduction of a transcriptionally active (Fig. 22G) but nuclear restricted mutant p53 (p53 ^NES ) into HK301 P53KO cells failed to induce EGFRi-mediated priming of apoptosis (Figs. 22G, 22H and 32H). Finally, pharmacological inhibition of cytoplasmic p53 activity with pifithrin-μ (PFTμ) markedly reduced priming with erlotinib (Figure 32I). Taken together, these results indicate that cytoplasmic p53 engages the intrinsic apoptotic machinery downstream of EGFRi in the GBM.

Previous studies have demonstrated that human tumor-derived p53 mutants, particularly in the DNA-binding domain, have reduced cytoplasmic functions in addition to transactivation deficiencies. We therefore examined whether stable expression of two of these p53 hotspot mutants, R175H or R273H, in HK301 P53KO would reduce EGFRi-mediated priming (Figure 32H). As expected, both mutants lacked transcriptional abilities (Fig. 22G) and, consistent with reduced cytoplasmic activity, were unable to prime apoptosis by EGFRi (Fig. 22H). Therefore, consistent with previous findings, oncogenic mutations in the DNA-binding domain of p53 result in double hits whereby both transactivation and cytoplasmic functions are abolished, the latter having implications for the priming of apoptosis by EGFRi.

Example 9 Inhibition of EGFR-induced glucose uptake creates a usable Bcl-xL dependency.

Bcl-xL can sequester cytoplasmic p53 and prevent p53-mediated apoptosis; thereby creating an apoptosis priming state and Bcl-xL dependence for survival. Indeed, BH3 profiling revealed a dependence on Bcl-xL for cell survival in patients with a metabolic response to EGFRi (Fig. 33A). Thus, we hypothesized that reduction of glucose uptake by EGFRi might lead to sequestration of cytoplasmic p53 by Bcl-xL. To investigate this, we performed coimmunoprecipitation to examine the dynamics of p53-Bcl-xL interactions in response to EGFRi in both responder (n=2) and non-responder (n=2) patients. Importantly, we observed a marked increase in Bcl-xL and p53 interactions following erlotinib treatment in patients with a metabolic response (Figure 23A) but not in patients without a metabolic response (Figure 23B). This suggests that inhibition of EGFR-dependent glucose uptake results in sequestration of p53 by Bcl-xL. Consistent with this interpretation, ectopic expression of GLUT1/3, which restores EGFRi-mediated reduction in glucose uptake and priming of apoptosis, prevents p53 binding to Bcl-xL (FIGS. 23C and 33B). These data strongly suggest that EGFRi-mediated inhibition of glucose uptake primes GBM cells for apoptosis by promoting the interaction between cytoplasmic p53 and Bcl-xL.

The release of p53 from Bcl-xL allows p53 to directly activate BAX, leading to the release of cytochrome c and cell death. Once we observed increased binding between Bcl-xL and p53 in metabolic responders in response to EGFRi, we asked whether displacement of p53 from Bcl-xL causes apoptosis. To investigate this, we treated a metabolic response cell (HK301) with erlotinib and a specific Bcl-xL inhibitor, WEHI-539. The addition of WEHI-539 disrupted the association of Bcl-xL with p53 upon erlotinib treatment (Figure 23D), resulting in synergistic lethality in HK301 and GBM39 cells (metabolic response patients) (Figure 23E). Notably, cytoplasmic p53 was sufficient for combinatorial effects in cells with a metabolic response to EGFRi (Fig. 33C). However, WEHI-539 did not increase apoptosis in non-metabolic patients (HK393) treated with erlotinib, suggesting that attenuation of glucose uptake by EGFRi and subsequent coupling between p53 and Bcl-xL are required to create dependence on Bcl-xL for survival ( Fig. 33E). In support of this, forced expression of GLUT1/3 significantly reduced cell death with the drug combination (Figures 23F and 33D). Together, these observations indicate that Bcl-xL attenuates GBM cell death in response to EGFRi-mediated inhibition of glucose uptake by sequestering cytoplasmic p53 (Figure 32G).

Example 10: Combined targeting of EGFR and p53 is synergistic for patients with metabolic response to EGFRi.

Mechanism of action studies have identified a potential therapeutic opportunity in EGFR-driven GBM that would be dependent on functional p53. While the p53 signaling axis is one of three major pathways altered in GBM, analysis of the TCGA data set for GBM demonstrated that p53 mutations are mutually exclusive with changes in EGFR (Figures 28A and 28B). In contrast, in patients with EGFR mutations or accumulation, the p53 pathway may be suppressed through MDM2 amplification and/or deletions in the MDM2 negative regulator, p14 ARF, at the CDKN2A locus. Given this relationship and the requirement for p53 to prime glucose uptake impaired by EGFRi, we hypothesized that p53 stabilization through MDM2 inhibition may have therapeutic effects similar to Bcl-xL antagonism. Using nutlin, a widely studied MDM2 inhibitor, we found a striking mutually reinforcing lethality in combination with erlotinib in the GBM of a metabolic patient. More than 90% of cells

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HK301 were apoptotic by the combination of erlotinib and nutlin (Fig. 24C). Notably, we did not observe any synergy between these drugs in a patient without a metabolic response (GS017, Fig. 24C). We then tested this combination in our panel of primary GBM cells (all p53 wild type) and found mutually reinforcing lethality only in GBM with a metabolic response to EGFRi (Figures 24D and 34A). Genetic knockdown of EGFR confirmed synergy only in patients with a metabolic response (Fig. 34B). Importantly, artificially induced expression of GLUT1/3 significantly reduced BAX oligerization, cytochrome c release, and apoptosis with the combination of erlotinib and Nutlin (Figures 24E and 34C), supporting the idea that inhibition of glucose metabolism by EGFRi is required for the synergistic effects of the combination of erlotinib and Nutlin. nutlina.

The role of p53 in the induction of cell death by the combination of erlotinib and nutlin was then examined. As expected, targeting p53 with CRISPR/CAS-9 in two EGFRi metabolically responsive cells (HK301 and HK336) completely reduced sensitivity to the drug combination (Figure 24F). Likewise, ectopic expression of Bcl-xL markedly suppressed cell death upon combination treatment, consistent with an important function of Bcl-xL in counteracting p53-mediated apoptosis (Figure 34D). Moreover, similar to the results with Bcl-xL inhibition (eg, WEHI-539), addition of nutlin released p53 from Bcl-xL upon treatment with erlotinib (Figure 24G). These data are consistent with previous observations that stabilization of p53 can stimulate cytoplasmic p53-mediated apoptosis. In support of the proposal that cytoplasmic p53 activity is required for EGFRi and nutlin-induced apoptosis in metabolic patients, blocking cytoplasmic p53 activity with PFTμ significantly reduced the synergistic effects of the combination (Fig. 34E), while HK301 cells containing limited a nuclear p53 mutant, p53 ^NES , was unable to enhance apoptosis by erlotinib and nutlin (Figure 34F). Finally, the cancer hotspot mutants, R175H and R273H, which have both transactivation and cytoplasmic deficiency, were completely insensitive to the drug combination (Fig. 34F).

Although it is desirable for cytoplasmic p53 to promote cell death through drug combinations, we observed in some cases that both transcription-dependent and -independent functions of p53 are required for optimal performance of synergistic apoptosis by nutlin (Figure 34F). These results are consistent with reports that transcription-independent functions of p53 can independently perform intrinsic apoptosis, whereas in other contexts its transcription-dependent functions may be required to promote cytoplasmic p53-mediated cell killing. Taken together, the results described herein indicate that combined targeting of EGFR-induced glucose metabolism and p53 can induce marked synergistic cell death in primary GBM; which depends on the cytoplasmic functions of p53.

Example 11: Modulation of glucose metabolism causes p53-mediated cell death in patients without a metabolic response to EGFRi.

The above data prompted the inventors to propose a model in which EGFRi-mediated attenuation of glucose metabolism triggers the apoptotic mechanism, leading to synergy with pro-apoptotic stimuli such as p53 activation. The synergy is between the induction of cellular stress by EGFR inhibitors, the reduction of glucose uptake and priming of cells for apoptosis, and the stabilization of p53 by BCL-2 antagonists. Inhibition of EGFR can rapidly attenuate glycolysis under cellular stress. This creates a tumor-specific vulnerability in which intrinsic apoptosis can be significantly enhanced by 1) activation of p53 (eg, through nutlin, analogs, or other substances described herein); and 2) inhibition of BCL-2 (by any of several agents as described herein, such as, for example, ABT-263 (Navitoclax).

A logical prediction of this model is that direct inhibition of glucose metabolism should phenocopy the effects of EGFRi. Consistent with this, addition of the metabolic inhibitor glucose 2-deoxyglucose (2DG) stimulated apoptosis priming, p53 binding to Bcl-xL, and synergy with nutlin in HK301 cells (EGFRi metabolic response patient) (FIGS. 40A, 40B, and 40D). Interestingly, inhibition of oxidative phosphorylation by oligomycin (complex V/ATP synthase) or rotenone (complex I) did not act synergistically with nutlin treatment in HK301 gliomaspheres (Figures 35C and 35D). Thus, only a decrease in glucose metabolic flux, but not oxidative metabolism, appears to be sufficient for synergistic sensitivity to p53 activation.

This prompted the inventors to wonder whether modulation of glucose uptake in patients without a metabolic response to EGFRi leads to a similar p53-dependent vulnerability. To investigate this, they tested whether directly inhibiting glucose uptake with 2DG or targeting PI3K (a well-characterized glucose metabolic factor) primed apoptosis in two patients without a metabolic response to EGFRi (Figure 25A). In contrast to erlotinib treatment, acute inhibition of PI3K with pictilisib abolished PI3K-AKTmTOR signaling (Figure 35E) and significantly reduced ¹⁸ F-FDG uptake in HK393 and HK254 cells (Figure 25B). Sleep

- 56 044668 The increase in glucose consumption by pictilisib was associated with significantly higher priming of apoptosis and, as expected, 2DG fully reflected these effects (Figs. 25B and 25C). Therefore, cells without a metabolic response to EGFRi may be primed for apoptosis following inhibition of glucose uptake. Importantly, CRISPR/CAS-9 targeting of p53 in HK393 significantly suppressed priming mediated by 2DG or pictilisib (Figure 25D). Moreover, p53-dependent priming was associated with increased binding of Bcl-xL and p53, indicating disruption of p53 by Bcl-xL to block apoptosis (Figures 25E and 35F). Consistent with this interpretation, combining 2DG or pictilisib with nutlin caused significant p53-dependent synergistic lethality in cells without a metabolic response to EGFRi (Figures 25F and 25G). Together, these data demonstrate that acute inhibition of glucose metabolism, either directly or through targeted therapy, promotes p53-dependent priming of apoptosis in GBM; which creates a targeted vulnerability for enhanced cell killing.

Example 12. Combinatorial therapeutic strategy and non-invasive biomarker for targeting GBM in vivo.

Results obtained in cell culture indicate that combined targeting of oncogene-induced glucose metabolism and p53 has synergistic activity in primary GBM. This prompted us to investigate whether this approach could be effective in orthotopic GBM xenograft models. For these studies, we used a potent MDM2 inhibitor, idasanutlin, which is currently in clinical trials for the treatment of many malignancies. Given the uncertainty of idasanutlin penetration into the CNS, we demonstrated for the first time that idasanutlin can accumulate in the brain of mice with an intact blood-brain barrier (brain: plasma, 0.35) and stabilizes p53 in orthotopic tumor-bearing mice (Figures 41A and 41B).

Further, since impairment of glucose metabolism with oncogene inhibition is required for synergistic sensitivity to p53 activation, we reasoned that the rapid attenuation of glucose uptake in vivo following EGFRi administration, measured by ¹⁸ F-FDG PET, may serve as a noninvasive predictive biomarker for therapeutic efficacy. combination treatment of erlotinib+idasanutlin (Fig. 26A). We observed in orthotopic gliomasphere xenografts from a patient with a metabolic response to EGFR (GBM39) that acute treatment with erlotinib (75 mg/kg) rapidly reduced the uptake of ¹⁸ F-FDG (15 hours after erlotinib administration) (Figs. 26B and 36C). In separate groups of mice, they tested individual drugs and a combination of daily treatment with erlotinib (75 mg/kg) and idasanutlin (50 mg/kg). Relative to the single agent control, we observed synergistic growth inhibition, as measured by secreted gaussia luciferase, in mice bearing the GBM39 intracranial tumor, with minimal toxicity (Figures 26B and 36D). In contrast, orthotopic xenografts from a non-metabolic patient (HK393) showed neither changes in ¹⁸ F-FDG uptake with acute EGFRi (Figures 26D and 36C) nor synergistic activity with the combination of erlotinib and idasanutlin (Figure 26E). In summary, noninvasive 18F-FDG PET used to measure rapid changes in glucose uptake by EGFRi was effective in predicting subsequent synergistic sensitivity to the combination of erlotinib and idasanutlin.

Finally, we assessed the effect of the drug combination on overall survival in orthotopic xenografts of either two patients with a metabolic response to EGFRi (GBM39 and HK336) or two patients without a metabolic response (HK393 and GS025). All tumors were p53 wild type (Fig. 29A). After evidence of tumor growth (as determined by gaussia luciferase), mice received vehicle, erlotinib, idasanutlin, or the combination for up to 25 days. The drug combination resulted in a significant increase in survival only in GBM tumors from patients with a metabolic response to EGFRi (Figures 30F-30I). Together, these data demonstrate that combined targeting of EGFR and p53 synergistically inhibits growth and prolongs survival in a subset of orthotopic GBM xenografts with wild-type p53. Importantly, 18F-FDG PET was a valuable noninvasive predictive biomarker of sensitivity to this novel combination therapeutic strategy.

Example 13: Direct inhibition of glycolysis by 2DG or cytochalasin B.

It was investigated how direct inhibition of glycolysis using a hexokinase inhibitor (2DG) and a glucose transporter inhibitor (cytochalasin B) affects p53 activation by nutlin. The results shown in FIG. 37 demonstrate that low glucose (0.25 mM) results in synergistic cell killing with BCL-xL inhibition by navitoclax or nutlin. Cell death was determined using Annexin V staining in gliomasphere samples treated for 72 h with the glycolytic inhibitors 2DG or cytochalasin B as single agents or in combination with the p53 activator, nutlin. The same effects were reproduced by culturing gliomaspheres under low glucose conditions (0.25 mM) and treating them with nutlin or navitoclax (ABT-263) for 72 h.

Example 14 Experimental Procedures

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Mice.

NOD scid gamma (NSG) females, 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 of age, were purchased from Charles River. All mice were housed under specific pathogen-free conditions in an AAALAC-approved animal facility at the Division of Laboratory Animal Care (DLAM) at UCLA. All animal experiments were performed with the approval of the UCLA Office of Animal Resources Review (OARO).

Patient-derived GBM cells.

All patient tissue for GBM cell culture preparation was obtained on the basis of explicit informed consent using UCLA Institutional Review Board (IRB) protocol: 10-00065. As previously described, primary ¹² GBM cells were established and 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% O ₂ and 5% CO ₂ and were regularly monitored and tested negative for mycoplasma using a commercially available kit (MycoAlert, Lonza). During the experiments, most of the NK lines used were between 20-30 passages (except for HK385 p8, HK336 p15), while the GS and GBM39 lines had less than 10 passages. All cells were authenticated using short tandem repeat (STR) analysis.

Reagents and antibodies.

Chemical inhibitors from the following sources were dissolved in DMSO for in vitro studies: erlotinib (Chemietek), nutlin-ZA (Selleck Chemicals), WEHI-539 (APExBIO), pictilisib (Selleck Chemicals), oligomycin (Sigma), rotenone (Sigma), 2DG (Sigma) was dissolved in the medium before use. Antibodies used for immunoblotting were obtained from the following sources: 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, 9644), p-4EBP1 S65 (Cell Signaling 9451), Glut3 (Abcam, abl5311), 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). Antibodies used for immunoprecipitation were obtained from the following sources: p53 (Cell Signaling, 12450) and Bcl-xL (Cell Signaling, 2764). Secondary antibodies were obtained from the following sources: anti-rabbit IgG HRP-linked (Cell Signaling, 7074) and anti-mouse IgG HRP-linked (Cell Signaling, 7076). All antibodies for immunoblotting were used at a dilution of 1:1000, with the exception of β-actin and tubulin, which were used at a ratio of 1:10,000. Antibodies for immunoprecipitation were diluted according to the manufacturer's instructions (1:200 for p53 and 1:100 for Bcl- xL). Secondary antibodies were used at a dilution of 1:5000.

¹⁸ F-fluorodeoxyglucose (18F-FDG) uptake assay.

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

Measurements of glucose, glutamine and lactate.

Cellular glucose consumption and lactate production were measured using the main program of the Nova Biomedical BioProfile analyzer. Briefly, cells were seeded at 1x105 cells/ml in 2 ml gliomasphere conditions and corresponding drug conditions (n=5). 12 h after drug treatment, 1 ml of medium was removed from each sample and analyzed in a Nova BioProfile analyzer. Measurements were normalized to cell number.

Annexin V apoptosis assay.

Cells were harvested and analyzed for Annexin V and PI staining according to the manufacturer's protocol (BD Biosciences). Briefly, cells were seeded at 5x104 cells/ml and treated with appropriate drugs. At the indicated time points, cells were harvested, trypsinized, washed with PBS, and stained with Annexin V and PI for 15 min. The samples were then analyzed using a BD LSRII flow cytometer.

Immunoblotting.

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 vol. for 15 min at 4°C. Protein samples were then boiled in NuPAGE LDS Sample Buffer (Invitrogen) and NuPAGE Sample Reducing Agent (Invitrogen), separated by SDS-PAGE at 12%

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Bis-Tris gels (Invitrogen) and transferred to a nitrocellulose membrane (GE Healthcare). Immunoblotting was performed according to the antibody manufacturer's directions and as previously mentioned. The membranes were developed using the SuperSignal system (Thermo Fischer Scientific).

Immunoprecipitation.

Cells were harvested, washed once with PBS, and incubated in IP lysis buffer (25 mM TrisHCL pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol) at 4°C for 15 min. 300-500 μg of each sample was then precleared in Protein A/G Plus agarose beads (Thermo Fischer Scientific) for 1 h. After preclearing, samples were then incubated with antibody-bead conjugates overnight according to the manufacturer's directions and as mentioned higher. Samples were then centrifuged at 1000 g for 1 min, and the beads were washed five times with 500 μl of IP lysis buffer. Proteins were eluted from the beads by boiling in 2× LDS sample buffer (Invitrogen) at 95°C for 5 min. Samples were analyzed by immunoblotting as described above. Antibodies for immunoprecipitation were diluted according to the manufacturer's instructions (1:200 for p53 and 1:100 for Bcl-xL).

Dynamic profiling BH3.

GBM gliomaspheres were first separated into single-cell suspensions using TrypLE (Gibco) and 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 (3x104 cells/well) was placed in wells containing 50 μl of MEB buffer containing 0.002% digitonin and the indicated peptides in 96-well plates. The plates were then incubated at 25°C for 50 min. Cells were then fixed with 4% paraformaldehyde for 10 min followed by neutralization with buffer N2 (1.7 M Tris, 1.25 M glycine, pH 9.1) for 5 min. Samples were stained overnight with 20 μl of staining solution (10% BSA, 2% Tween 20 in PBS) containing DAPI and anticytochrome c (BioLegend). The next day, cytochrome c release was quantified using a BD LSRII flow cytometer. Measurements were normalized to appropriate controls that do not promote cytochrome c release (DMSO and inactive PUMA2A peptide). Delta priming refers to the difference in the amount of cytochrome c release between vehicle-treated cells and drug-treated cells.

Oligomerization of VAC.

7.5x105 cells were treated with the indicated drugs. At 24 h post-treatment, cells were harvested, washed once with ice-cold PBS, and resuspended in 1 mM bismaleimidohexane (BMH) in PBS for 30 min. Cells were then pelleted and lysed for immunoblotting as described above.

Detection of cytochrome c 5 million cells were seeded at a concentration of 1x105 cells/ml and treated with the indicated drugs. 24 h 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 cytoplasmic and mitochondrial fractions were immunoblotted and cytochrome c was detected using a 1:1000 dilution of cytochrome c antibody (Cell Signaling, 4272).

Mouse xenograft studies.

For intracranial experiments, GBM39, HK336, HK393, and GS025 cells were injected (4x105 cells per injection) into the right striatum of female NSG mice (6-8 weeks old). The injection coordinates were 2 mm lateral and 1 mm posterior to the bregma at a depth of 2 mm. Tumor weight was monitored using secreted gaussia luciferase, and after three sequential growth measurements, mice were randomized into four treatment groups consisting of matched vehicle, 75 mg/kg erlotinib, 50 mg/kg idasanutlin, or a combination of both drugs. The vehicle consisted of 0.5% methylcellulose in water, which is used to dissolve erlotinib, and a proprietary formulation obtained from Roche, which is used to dissolve idasanutlin. Tumor weight was assessed twice weekly using secreted gaussia luciferase. When possible, mice were treated for 25 days, treatment was stopped, and survival was monitored. Medicines were administered through a gastric tube. Sample sizes were selected based on evaluations of pilot experiments and findings from previous literature ¹² . The investigators were blinded to group allocation or outcome measures. All studies were conducted in accordance with the UCLA OARO protocol guidelines.

Intracranial delayed PET/CT imaging in mice.

Mice were administered the indicated dose of erlotinib at the indicated time, then prewarmed, anesthetized with 2% isoflurane, and given 70 μCi of 18F-FDG intravenously. After 1 h of unconscious uptake, mice were removed from anesthesia but kept warm for an additional 5 h. 6 h after initial 18F-FDG administration, mice were imaged using a G8 PET/CT scanner (Sofie Biosciences). As stated above, quantification was performed by drawing three-dimensional regions of interest (ROIs) using AMIDE software.

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Immunohistochemistry.

Immunohistochemistry was performed on 4-μm sections that were cut from FFPE (formalin-fixed, paraffin-embedded) blocks. Sections were then deparaffinized with xylene and rehydrated with graded ethanol. Antigen retrieval was achieved using a pH 9.5 Nuclear Decloaker (Biocare Medical) in a Decloaking autoclave at 95°C for 40 min. Tissue sections were then treated with 3% hydrogen peroxide (LOT 161509; Fisher Chemical) and Background Sniper (Biocare Medical, Concord, CA, USA) to reduce nonspecific background staining. Primary antibodies to p53 (Cell Signaling, 2527) were used at a dilution of 1:150 for 80 min, followed by detection using a MACH 3 Rabbit HRP-Polymer detection kit (Biocare Medical). Imaging was achieved using VECTOR NovaRED (SK-4800; Vector Laboratories, Inc.) as the chromogen. Finally, the sections were counterstained with Tacha automatic hematoxylin (Biocare Medical).

Quantitative RT-PCR.

RNA was extracted from all cells using a Purelink RNA kit (Invitrogen). cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's instructions. Quantitative PCR (qPCR) was performed on a Roche LightCycler 480 using SYBRGreen Master Mix (Kapa Biosciences). Relative expression values are normalized to the reference gene (GAPDH). Primer sequences are listed (in the 5' to 3' direction): P21 (forward GACTTTGTCACCGAGACACC, reverse GACAGGTCCACATGGTCTTC), PUMA (forward ACGACCTCAAGCCACAGTACG, reverse GTAAGGGCAGGAGTCCCATGATG), GAPDH (forward TGCCATGTAGACCCCTTGAAG, reverse ATGGTACATGACAAGGTGCGG), MDM 2 (forward CTGTGTTCAGTGGCGATTGG, reverse AGGGTCTCTTGTTCCGAAGC ), Tigar (direct GGAAGAGTGCCCTGTGTTTAC, reverse GACTCAAGACTTCGGGAAAGG), PIG3 (direct GCAGCTGCTGGATTCAATTA, reverse TCCCAGTAGGATCCGCCTAT).

Reporter activity of P53.

Cells were first infected with a lentivirus synthesized from a p53 reporter plasmid, which encodes a luciferase under the control of the p53 response element: TACAGAACATGTCTAAGCATGCTGTGCCTTGCCTGGACTTGCCTGGCCTTGCCTTGGG. Infected cells were then plated in a 96-well plate at a concentration of 5000 cells/50 μl and treated with the indicated drugs for 24 h, followed by incubation with 1 mM D-luciferin for 2 h. Bioluminescence was measured using an IVIS Lumina II (Perkin Elmer ).

Genetic manipulation.

Typically, lentivirus used for genetic manipulation was produced by transfecting 293-FT cells (Thermo) using Lipofectamine 2000 (Invitrogen). The virus was harvested 48 h after transfection. The lentiviral vector sgp53 and the control vector sg contained the following guide RNAs, respectively: CCGGTTCATGCCGCCCATGC and GTAATCCTAGCACTTTTAGG. LentiCRISPR-v2 was used as the basis. Glut1 and Glut3 cDNAs were cloned from commercially available vectors and incorporated into a pLenti-GLuc-IRES-EGFP lentiviral backbone containing the CMV promoter (Glut1 was a gift from Wolf Frommer (Addgene #1808544), Glut3 was from OriGene #SC115791, and the lentiviral backbone was from Targeting Systems #GL-GFP). pMIG Bcl-xL was a gift from Stanley Korsmeyer (Addgene #8790 ⁴⁵ ) and cloned into the lentiviral backbone mentioned above (Targeting Systems). Cytoplasmic (KZO5A and R306A) wild-type p53 constructs were a kind gift from R. Agami and G. Lahav. Genes of interest were cloned into a lentiviral vector containing the PGK promoter. Constructs for the p53 DNA binding domain mutants (R175H) and (R273H) and the nuclear mutant (L348A and L350A) were generated using site-directed mutagenesis (New England Biolabs #E0554S) on the wild-type p53 construct.

For EGFR knockdown experiments, siRNA against EGFR (Thermo Fischer Scientific, s563) was transfected into cells using DharmaFECT 4 (Dharmacon). After 48 h, cells were harvested and used for the indicated experiments.

Immunofluorescence.

For immunofluorescence, gliomaspheres were first dissociated from single cells and attached to 96-well plates using Cell-Tak (Corning) according to the manufacturer's instructions. Attached cells were then fixed with ice-cold methanol for 10 min and then washed three times with PBS. Cells were then incubated with blocking solution containing 10% FBS and 3% BSA in PBS for 1 h and then incubated with p53 antibody (Santa Cruz, SC-126, 1:50 dilution) overnight at 4°C. The next day, cells were incubated with secondary antibody (Alexa Fluor 647, 1:2000 dilution) for 1 h and DAPI staining for 10 min, then imaged using a Nikon TI Eclipse microscope equipped with a Cascade II fluorescence camera (Roper Scientific). Cells were imaged with emissions at 461 and 647 nM and then processed using NIS-Elements AR analysis software.

Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements.

For metabolic measurements using OCR and ECAR, gliomaspheres treated with uka

- 60 044668 containing drugs were first dissociated from single cell suspensions and attached to XF24 plates (Seahorse Bioscience) using Cell-Tak (Corning) according to the manufacturer's instructions. Before analysis, unbuffered DMEM was added to the cells and incubated at 37°C for 30 min before OCR and ECAR measurements began. Basic ECAR measurements between control-treated cells and erlotinib-treated cells are shown.

Preparation of samples for mass spectroscopy.

Male CD-1 mice (6-8 weeks old) were administered 50 mg/kg idasanutlin in duplicate via an oral gavage. At 0.5, 1, 2, 4, 6, 8, 12, and 24 h after administration, mice were sacrificed, blood was collected by retroorbital exsanguination, and brain tissue was collected. Whole blood from mice was centrifuged to isolate blood plasma. Idasanutlin was isolated by liquid extraction from blood plasma: 50 μl of blood plasma was added to 2 μl of internal standard and 100 μl of acetonitrile. Mouse brain tissue was washed with 2 ml of cold PBS and homogenized using a tissue homogenizer with 2 ml of freshly prepared cold PBS. Idasanutlin was then isolated and reconstituted in a similar manner by liquid-liquid extraction: 100 μL of brain homogenate was added to 2 μL of internal standard and 200 μL of acetonitrile. After vortex mixing, the samples were centrifuged. The supernatant was removed and evaporated on a rotary evaporator and reconstituted in 100 μl of 50:50 water:acetonitrile.

Detection of idasanutlin using mass spectrometry.

Chromatographic separations were performed on a Phenomenex Kinetex C18 100 x 2.1 mm column (Kinetex) using a 1290 Infinity LC system (Agilent). The mobile phase consisted of solvent A: 0.1% formic acid in Milli-Q water and B: 0.1% formic acid in acetonitrile. Analytes were eluted with a gradient of 5% B (0-4 min), 5-99% B (4-32 min), 99% B (32-36 min), and then returned to 5% B over 12 min until equilibrium was restored between injections. 20 μL was injected into the chromatography system at a solvent flow rate of 0.10 mL/min. Mass spectrometry was performed on a 6460 triple quadrupole LC/MS system (Agilent). Ionization was achieved using electrospray in positive mode, and data acquisition was performed in multiple reaction monitoring (MRM) mode. The MRM transition used to detect idasanutlin was m/z 616.2^421.2 with a fragmenter voltage of 114 V and a collision energy of 20 eV. The analyte signal was normalized to the internal standard and concentrations were determined by comparison with the calibration curve (0.5, 5, 50, 250, 500, 2000 nM). Brain idasanutlin concentrations were adjusted to 1.4% mouse brain weight for the remaining blood in the brain vasculature.

Measurements of secreted gaussia luciferase.

Cells were infected with a lentiviral vector containing the secreted gaussia luciferase (sGluc) reporter gene (Targeting Systems #GL-GFP) and implanted intracranially into the right striatum of mice (4x105 cells/mouse). To measure secreted Gaussia luciferase (sGluc) levels, 6 μl of blood was collected from the tail vein of mice and immediately mixed with 50 mM EDTA to prevent coagulation. Gluc activity was determined by measuring chemiluminescence after injection of 100 μl of 100 μM coelenterazine (Nanolight) into a 96-well plate.

Calculation of synergy points.

1.0x105 GBM cells were seeded in triplicate and treated with erlotinib, nutlin, or a combination at multiple concentrations using a matrix where each drug was added to the cells at six concentrations (0-10 μM). Annexin V staining was measured 72 h after treatment. Using Chalice software, the response of the combination was compared with its individual agents. Combinatorial effects were calculated using a synergy score.

DNA sequencing.

Targeted sequencing was performed on samples HK206, HK217, HK250, HK296 for the following genes BCL11A, BCL11B, BRAF, CDKN2A, CHEK2, EGFR, ERBB2, IDH1, IDH2, MSH6, NF1, PIK3CA, PIK3R1, PTEN, RB1, TP53 using Illumina Miseq. There were 1 to 2 million reads per sample with an average coverage of 230 per gene. Copy number variants were identified for these samples using a genome-wide SNP array. The genetic profile of GBM39 has been previously described in the literature.

Whole exome sequencing was performed on samples HK157, HK229, HK248, HK250, HK254, HK296, HK301, HK336, HK350, HK390, HK393 and performed in SeqWright. The samples were grouped into 2 groups with separate uptake reactions. Nextera rapid capture and library preparation were used, and sequencing was performed on a HiSeq 2500, 2x100 bp. with 100x target coverage, 2 full fast runs, each with 1 normal diploid control. Copy number analysis for these samples was performed using EXCAVATOR software.

Note on TCGA samples.

273 GBM samples from TCGA were analyzed for genetic alterations in the EGFR, p53 and p53 regulated pathways. The co-occurrence of mutations was examined and only significant interactions were shown. Data were analyzed using cBioPortal as previously described.

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Fluorescence in situ hybridization (FISH).

Fluorescence in situ hybridization (FISH) was performed using a commercially available fluorescently labeled dual-color EGFR (red)/CEP 7 (green) probe (Abbott-Molecular). Hybridization and FISH analysis were performed on cell lines according to protocols suggested by the manufacturer. Cells were counterstained with DAPI, and fluorescent probe signals were visualized under a Zeiss fluorescence microscope (Axiophot) equipped with two- and three-color filters.

Statistical analysis.

Comparisons were made using two-tailed unpaired Student's t tests, and p values <0.05 were considered statistically significant. All data from several independent experiments were assumed to have normal variance. Data are means ± standard error of the mean. All statistical analyzes were calculated using Prism 6.0 (GraphPad). For all in vitro and in vivo experiments, no statistical method was used to predetermine sample size, and no samples were excluded. For in vivo tumor measurements, the latest data sets were used for comparisons between groups. As described above, all mice were randomized before the studies.

Example 15: Illustrative Design Guidelines for Certain JFK Series Connections.

Certain compounds of the present invention were developed in accordance with Scheme 1.

JGK037 adding tertiary amines to the dioxane ring

SAR of polar groups attached to the dioxane ring

JGKQ63-JGK072 potent, brain-penetrating low bioavailability and specificity potent, brain-penetrating high bioavailability and specificity

Scheme 1

Example 16 Preparation of additional exemplary JGK series compounds.

Exemplary compounds of the present invention were prepared in accordance with the following methods.

Scheme 1. Synthesis of monoprotected quinazoline intermediate 3

Scheme 2. Synthesis of JGK063 - JGK070

The synthesis was performed in a similar manner as for the racemic sample JGK068 ((±)-JGK068), but with enantiomerically pure (S)-(-)-glycidol. Another enantiomer of JGK068R ((R)-JGK068) was prepared using (R)-(+)-glycidol (not shown).

Scheme 3. Synthesis of JGK068S ((S)-JGK068)

The enantiomeric purity of synthetic intermediate 5 was determined by chiral SFC (Chiralpak AD-3 column, 40% MeOH) and by comparison of the ¹⁹ F NMR spectra of the derivatives

- 62 044668 Mosher ether 5 (Fig. 39).

Scheme 4

Scheme 5. Synthesis of JGK071

Scheme 6. Synthesis of JGK072

Scheme 7. Synthesis of JGK076-JGK080

Scheme 8. Synthesis of JGK086-JGK090

General chemical information.

All chemicals, reagents, and solvents were purchased from commercial sources when available and used as received. When necessary, reagents and solvents were purified and dried using standard methods. Air- and moisture-sensitive reactions were carried out under an inert atmosphere of argon in oven-dried glassware. Re

- 63 044668 actions with microwave irradiation were carried out in a single-mode reactor of the CEM Discover microwave synthesizer. Room temperature (RT) reactions were performed at ambient temperature (approximately 23°C). All reactions were monitored by thin layer chromatography (TLC) on pre-coated Merck 60 F ₂₅₄ silica gel plates spotted with UV light (λ=254, 365 nm) or using an alkaline KMnO ₄ solution. Flash column chromatography (FC) was carried out on SiO ₂ 60 (particle size 0.040-0.063 mm, 230-400 mesh). Preparative thin-layer chromatography (PTLC) was performed with Merck 60 F ₂₅₄ silica gel plates (20x20 cm, 210-270 mm) or Analtech Silica Gel GF TLC plates (20x20 cm, 1000 mm). Concentration under reduced pressure (in vacuum) was carried out on a rotary evaporator at 23-50°C. The purified compounds were further dried in a high vacuum or in a desiccant. The outputs correspond to purified compounds and have not been further optimized. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on Bruker spectrometers (operating at 300, 400 or 500 MHz). Carbon NMR spectra ( ^13C NMR) were recorded on Bruker spectrometers (at 400 or 500 MHz). NMR chemical shifts (δ ppm) were assigned to residual solvent signals. 1H NMR data are presented as follows: chemical shift in ppm; multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, quint=quintet, w=multiple/mixed type, td=triplet of doublets, ddd=doublet of doublets of doublets, bg=broad signal); interaction constants (J) in Hz, integration. Data for ¹³ C NMR spectra are presented as a function of chemical shift and, if applicable, interaction constants. High-resolution mass spectra (HRMS) were recorded on a Thermo Fisher Scientific Exactive Plus with the original lonSense ID-CUBE DART mass spectrometer or on a Waters LCT Premier mass spectrometer with ACQUITY UPLC with autosampler.

General Procedures (GP). GP-1. Nucleophilic substitution of quinazolinyl mesylates with secondary amines. A mixture of quinazolinyl mesylate (1 eq) in DMF (0.05 m) was treated with secondary amine (5 eq) and triethylamine (2 eq) and the mixture was stirred at 85°C for 24 hours. The mixture was cooled to 23°C and evaporated. The residue was dissolved in EtOAc (20 ml), washed with 10 mM NaOH (4x5 ml), brine (5 ml), dried (Na ₂ SO ₄ ), filtered and evaporated. Purification by PC or PTSC provided the required products, usually in the form of off-white, loose foams.

GP-2. Nucleophilic aromatic substitution of 4-chloroquinazoline with anilines. A mixture of 4-chloroquinazoline (1 eq.) in acetonitrile (0.1 m) was treated with aniline (2 eq.) and 4 M HCl in dioxane (1 eq.). The mixture was heated at 80°C under microwave conditions for 30 min. The mixture was concentrated under reduced pressure or the precipitated 4-anilinoquinazoline hydrochloride salt was isolated by filtration (washing with Et ₂ O). The residue was suspended in sat. aq. NaHCO ₃ solution and extracted with CH ₂ Cl ₂ (3x). The combined organic extracts were washed with water, brine, dried (Na ₂ SO ₄ ), filtered and concentrated. Purification by PC (elution with gradient CH ₂ Cl ₂ /EtOAc or hexanes/EtOAc) provided the desired products, typically as white to off-white or pale yellow solids.

4-(3-Bromo-2-fluoroanilino)quinazoline-6,7-diyl-bis(2,2-dimethylpropanoate) (1).

A mixture of 4-chloroquinazoline-6,7-diyl-bis(2,2-dimethylpropanoate)1 (41.08 g, 113 mmol) in iPrOH (450 ml) was treated with 3-bromo-2-fluoroaniline (17.05 ml , 152 mmol) and stirred at 80°C for 3.5 hours. The mixture was cooled to 23°C and evaporated. The residue was suspended several times in hexanes (50 ml) and concentrated and then dried under high vacuum. The residue was recrystallized from EtOH to give a yellow solid, which was suspended in sat. aq. NaHCO ₃ solution (1 L) and extracted with DCM (3x550 ml). The combined organics were washed with water (400 ml), brine (400 ml), dried (MgSO ₄ ), filtered and evaporated to give the title compound 1 (35.057 g, 60%) as a yellow fluffy foam.

1H NMR (500 MHz, CDCl ₃ ) δ 8.76 (s, 1H), 8.46 (t, J=7.5 Hz, 1H), 7.72 (s, 1H), 7.68 (s, 1H), 7.56 (ush, 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, CDCl ₃ ) 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, Jcf=10.8 Hz), 125.29 (d, Jcf=4.7 Hz), 122.70, 122.51, 114.43, 113, 21, 108.84 (d, Jcf=19.4 Hz), 39.54, 39.51, 27.40, 27.32 ppm;

MSHR (DART): m/z [M+H]+ calc. for C ₂₄ H ₂₆ BrFN ₃ O ₄ ⁺ , 518.1085; 518.1072 discovered.

4-(3-Bromo-2-fluoroanilino)quinazoline-6,7-diol (2).

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A stirred slurry of 1 (34.988 g, 67.5 mmol) was treated at 0°C with a 7 M solution of NH ₃ in MeOH (241 ml, 1.69 mol). The mixture was stirred at 0°C for 15 minutes and then at 23°C for 4.5 hours. 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 (4x150 ml), Et ₂ O (2x150 ml) and dried in a desiccant to give the title compound 2 (23.68 g, quant.) as a pale -yellow powder.

1H 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-d ₆ ) δ 156.43, 156.12, 153.06 (d, Jcf=246.7 Hz), 151.34, 148.39, 146.80, 129.23 , 129.01, 127.12, 125.23 (d, Jcf=4.3 Hz), 108.47, 108.32, 107.09, 103.04 ppm;

MSHR (DART): m/z [M+H]+ calc. for C ₁₄ H ₁₀ BrFN ₃ O ₂ ⁺ , 349.9935; discovered 349.9923.

4-(3-Bromo-2-fluoroanilino)-7-hydroxyquinazolin-6-yl-2,2-dimethylpropanoate (3).

A stirred suspension of 2 (3500 mg, 10.0 mmol) in DMF (52.6 ml) was treated with Et ₃ N (5.57 ml, 40.0 mmol), cooled to -40°C and treated dropwise with Piv ₂ O (3.14 ml, 15.5 mmol). The mixture was stirred at -40°C for 1 hour, after which the cooling bath was removed and stirring was continued for 2.5 hours. The reaction mixture was diluted with DCM (500 ml), washed with 10% acetic acid (2x50 ml), dried ( Na ₂ SO ₄ ), filtered and evaporated. PC (DCM/EtOAc 1:1 >0:1) provided a solid, which was redissolved in EtOAc (750 ml) and washed with half-saturation. aq. NH ₄ Cl solution (4x75 ml), dried (Na ₂ SO ₄ ), filtered and evaporated to give the title compound 3 (2.844 g, 66%) as a beige-yellow solid.

1H 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-d ₆ ) 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, Jcf=12.9 Hz), 127.62, 125.47 (d, Jcf=4.4 Hz), 116.36, 111.00, 108.55 ( d, J=20.0 Hz), 107.77, 38.64, 26.93 ppm;

MSVR (DART): m/z [M+H] ⁺ calc. for C ₁₉ H ₁₈ BrFN ₃ O ₃ ⁺ , 434.0510; 434.0489 discovered.

(±)-4-(3-Bromo-2-fluoroanilino)-7-[(oxiran-2-yl)methoxy]quinazolin-6-yl-2,2-dimethylpropanoate ((±)-4).

ABOUT

A mixture of 3 (1350 mg, 3.11 mmol) and PPh ₃ (2038 mg, 7.77 mmol) in THF (21 ml) was treated with glycidol (495 μl, 7.46 mmol), cooled to 0°C and processed with DIAD (1.47 ml, 7.46 mmol) for 10 min. The mixture was stirred at 23°C for 2.5 hours and concentrated. PC (DCM/EtOAc 9:1>4:6) provided the title compound (±)-4 (848 mg, 56%) as an off-white solid.

1H 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 (ush, 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, Jcf=243.3 Hz), 150.27, 140.99, 128, 25 (d, Jcf=10.5 Hz), 127.75, 125.28 (d, Jcf=4.7 Hz), 122.22, 114.02, 109.72, 109.49, 108.74 ( d, Jcf=19.1 Hz), 70.05, 49.55, 44.56, 39.45, 27.38 ppm;

MSVR (DART): m/z [M+H] ⁺ calc. for C ₂₂ H ₂₂ BrFN ₃ O ₄ ⁺ , 490.0772; discovered 490.0764.

- 65 044668 (±)-N-(3-Bromo-2-fluorophenyl)-7-ethenyl-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine ((±) -JGK062).

A solution of PPh ₃ (832 mg, 3.17 mmol) and DIAD (624 μL, 3.17 mmol) in THF (23 ml) was stirred at 0°C for 15 min, and then added dropwise to the solution (±)- 8 (1149 mg, 2.73 mmol) in THF (27 ml) over 10 min. at 0°C. The mixture was stirred at 0° for 2 hours and evaporated. PC (hexanes/EtOAc 9:1^4:6) followed by another PC (DCM/EtOAc 1:0^6:4) provided the title compound (±)-JGK062 (1115 mg, quant.) as dirty -white loose foam.

1H NMR (500 MHz, CDCl ₃ ) δ 8.68 (s, 1H), 8.65 (ddd, J=8.2, 7.3, 1.5 Hz, 1H), 7.40 (s, 1H ), 7.37 (ush, 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;

MSHR (DART): m/z [M+H]+ calc. for C ₁₈ H ₁₄ BrFN ₃ O ₂ ⁺ , 402.0248; discovered 402.0233.

(±)-[4-(3-Bromo-2-fluoroanilino)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-7-yl]methanol ((±)-5).

A mixture of (±)-4 (842 mg, 1.72 mmol) in MeOH (31 ml) was treated with K ₂ CO ₃ (482 mg, 3.49 mmol), stirred at 23°C for 10.5 h and concentrated. The residue was suspended in half sat. aq. NH ₄ Cl solution (130 ml) and extracted with EtOAc (3x20 ml). The combined organics were washed with water (20 ml), brine (20 ml), dried (Na ₂ SO ₄ ), filtered and concentrated to give the title compound (±)-5 (720 mg, quant.) as a yellow solid substances.

1H NMR (500 MHz, DMSO-d ₆ ) 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-d ₆ ) δ 157.20, 153.35 (d, Jcf=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;

MSHR (DART): m/z [M+H]+ calc. for C ₁₇ H ₁₄ BrFN ₃ O ₃ ⁺ , 406.0197; discovered 406.0185.

(±)-[4-(3-Bromo-2-fluoroanilino)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-7-yl]methylmethanesulfonate ((±)-6).

A solution of (±)-5 (688 mg, 1.69 mmol) in THF (14 ml) was treated with Et ₃ N (357 μl, 2.56 mmol), cooled to 0°C and treated dropwise with MsCl ( 174 µl, 2.24 mmol). The mixture was stirred at 23°C for 16 hours, cooled to 0°C, treated with sat. aq. NaHCO3 solution (120 ml) and extracted with DCM (3x120 ml). The combined organics were washed with water (100 ml), brine (100 ml), dried (Na ₂ SO ₄ ), filtered and evaporated. PC (DCM/EtOAc 8:2^3:7) provided the title compound (±)-6 (496 mg, 61%) as an off-white solid.

1H NMR (500 MHz, CDCl ₃ ) δ 8.69 (s, 1H), 8.60 (ddd, J=8.5, 7.2, 1.4 Hz, 1H), 7.43 (s, 1H ), 7.39 (ush, 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 (dd, 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, CDCl ₃ ) δ 156.02, 153.66, 150.28 (d, J _CF = 242.9 Hz), 148.65, 146.80, 143.09, 128.43 ( d, Jcf=10.4 Hz), 127.54, 125.32 (d, Jcf=4.7 Hz), 122.01, 114.77, 110.90, 108.66 (d, Jcf=19, 4 Hz), 106.44, 71.10, 66.46, 64.77, 38.02 ppm;

MSVR (DART): m/z [M+H] ⁺ calc. for C ₁₈ H ₁₅ BrFN ₃ O ₅ S ⁺ , 483.9973; 483.9950 discovered.

- 66 044668 (±)-4-(3-Bromo-2-fluoroanilino)-7-{[2-(acetoxy)but-3-en-1-yl]oxy}quinazolin-6-yl-2,2- dimethylpropanoate ((±)-7).

A mixture of 3 (2639 mg, 6.08 mmol) and PPh ₃ (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), cooled to 0°C and treated dropwise with DIAD (2.7 ml, 13.7 mmol). The mixture was stirred at 23°C for 3 hours and concentrated. PC (DCM/EtOAc 1:0-6:4) provided crude (±)-7 (5.508 g, estimated yield 60%) as an off-white solid that was contaminated with residual Ph ₃ PO. The material was used in the next step without any further purification.

1H NMR (400 MHz, CDCl ₃ ) 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, CDCl ₃ ) δ 176.51, 170.08, 156.49, 155.24, 154.88, 150.46 (d, J _C F = 243.2 Hz), 150.17 , 140.90, 132.16, 128.18 (d, Jcf=11.0 Hz), 127.86, 125.31 (d, Jcf=4.8 Hz), 122.27, 119.64, 114 .00, 109.56, 109.39, 108.76 (d, Jcf=19.4 Hz), 72.18, 69.81, 39.34, 27.33, 21.19 ppm;

MSHR (DART): m/z [M+H]+ calc. for C ₂₅ H ₂₆ BrFN ₃ O ₅ ⁺ , 546.1034; 546.1018 discovered.

(±)-4-(3-Bromo-2-fluoroanilino)-7-[(2-hydroxybut-3-en-1-yl)oxy]quinazolin-6-ol.

A mixture of crude (±)-7 (5508 mg, contaminated with _Ph3PO residue from last step) in MeOH (61 ml) was treated with K2CO3 (4198 mg, 30.4 mmol), stirred at 23°C for 1 h and concentrated. The residue was suspended in half sat. aq. NH ₄ Cl solution (1 L) and extracted with EtOAc (3x600 ml). The combined organics were dried (Na ₂ SO ₄ ), filtered and evaporated. PC (DCM/EtOAc 1:1 ->0:1) provided the title compound (±)-8 (1154 mg, 45% from two steps) as an off-white solid.

^1H 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 (ush, 1H), 5.20 (dt, J=10.6, 1.8 Hz, 1H), 4.49 (ush, 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-d ₆ ) 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, Jcf=13.0 Hz), 127.49, 125.38 (d, Jcf=4.3 Hz), 115.58, 109.42, 108.50 ( d, Jcf=19.8 Hz), 107.68, 105.14, 72.56, 69.26 ppm;

MSHR (DART): m/z [M+H]+ calc. for C ₁₈ H ₁₆ BrFN ₃ O ₃ ⁺ , 420.0354; discovered 420.0340.

(±)-2-[4-(3-Bromo-2-fluoroanilino)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-7-yl]ethan-1-ol (( ±)-9).

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 hours. The mixture was cooled to 0°C, diluted with THF (2.4 ml) and treated with 3N. NaOH (3 ml, 8.95 mmol) and 30% H ₂ O ₂ (474 µl, 8.95 mmol) and stirred at 23°C for 6 hours. The mixture was concentrated to about half the original volume of THF, diluted with water (100 ml) and saline (40 ml) and extracted with EtOAc (3x100 ml). The combined organics were washed with water (70 ml), brine (70 ml), dried (Na ₂ SO ₄ ), filtered and evaporated to give the title compound (±)-9 (912 mg) as a yellow foam which was used at the next stage without additional purification.

1H NMR (500 MHz, CDCl ₃ ) δ 8.66 (s, 1H), 8.62 (ddd, J=8.8, 7.4, 1.6 Hz, 1H), 7.35 (s, 1H ), 7.33 (ush, 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

- 67 044668 (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, Jcf=242.6 Hz), 149.42, 146.47, 144.20, 128.51 (d, Jcf=10.2 Hz), 127.31, 125.30 (d, Jcf=4.7 Hz), 121.69, 113.95, 110.50, 108.58 (d, Jcf=19.2 Hz ), 105.83, 71.33, 68.49, 58.23, 33.61 ppm;

MSVR (IER): m/z [M+H]+ calc. for C18H1 ₆ B^3Oz ⁺ , 420.0354; discovered 420.0370.

(±)-2-[4-(3-Bromo-2-fluoroanilino)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-7-yl]ethylmethanesulfonate ((±)-10 ).

A solution of crude (±)-9 (912 mg) in THF (11.9 ml) was treated with Et ₃ N (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 minutes and then at 23°C for 21 hours. The mixture was cooled to 0°C, treated dropwise with sat. aq. NaHCO ₃ solution (120 ml) and extracted with DCM (3x120 ml). The combined organics were washed with water (100 ml), brine (100 ml), dried (Na ₂ SO ₄ ), filtered and evaporated. PC (DCM/EtOAc 9:1 >4:6) provided the title compound (±)-10 (112 mg, 19% from two steps) as an off-white fluffy foam.

^1H 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, Jcf=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;

MSVR (IER): m/z [M+H] ⁺ calc. for C ₁₉ H ₁₈ BrFN ₃ O ₅ S ⁺ , 498.0129; discovered 498.0144.

(±)-N-(3-Bromo-2-fluorophenyl)-7-[(morpholin-4-yl)methyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin4-amine ((±)-JGK063).

After the general GP-1 procedure, compound (±)-JGK063 was prepared from (±)-6 (20 mg, 0.04 mmol) and morpholine (18 μL, 0.21 mmol) in DMF (826 μL). PTSC (DCM/EtOAc 1:9) provided (±)-JGK063 (15 mg, 76%) as an off-white fluffy foam.

^1H 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 (ush, 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, Jcf=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;

MSVR (IER): m/z [M-N] ^- calc. for C ₂₁ H ₁₉ BrFN ₄ O3 ^- , 473.0630; discovered 473.0630.

(±)-N-(3-Bromo-2-fluorophenyl)-7-[2-(morpholin-4-yl)ethyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazoline4 -amine ((±)-JGK064).

Following the general GP-1 procedure, compound (±)-JGK064 was prepared from (±)-10 (35 mg, 0.07 mmol) and morpholine (31 μL, 0.35 mmol) in DMF (1.4 mL). PTSC (EtOAc, 0.5% acetonitrile, 1.5% aq NH ₄ OH) followed by another PTSC (EtOAc) provided (±)-JGK064 (25 mg, 73%) as an off-white fluffy foam.

^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 (ush, 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),

- 68 044668

2.00-1.82 ppm (m, 2H);

¹³ C NMR (126 MHz, CDCI3) δ 155.86, 153.31, 150.13 (d, Jcf=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 =19 ,2 Hz), 105.87, 72.20, 68.33, 67.06, 54.23, 53.86, 28.15 ppm;

MSVR (IER): m/z [M+H]+ calc. for C ₂₂ H ₂₃ BrFN ₄ O ₃ ⁺ , 489.0932; discovered 489.0935.

(±)-N-(3-Bromo-2-fluorophenyl)-7-[(piperidin-1-yl)methyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin4-amine ((±)-JGK065).

Following the general GP-1 procedure, compound (±)-JGK065 was prepared from (±)-6 (40 mg, 0.08 mmol) and piperidine (41 μL, 0.41 mmol) in DMF (1.65 mL). PTSC (EtOAc) provided (±)-JGK065 (24 mg, 61%) as an off-white fluffy foam.

1H 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 (ush, 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, CDCl ₃ ) 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 =19.4 Hz), 106.00, 71.87, 67.46, 59.34, 55.59, 26.07, 24.20 ppm;

MSVR (IER): m/z [M+H]+ calc. for C ₂₂ H ₂₃ BrFN ₄ O ₂ ⁺ , 473.0983; 473.0991 discovered.

(±)-N-(3-Bromo-2-fluorophenyl)-7-[(dimethylamino)methyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin4-amine ((±) -JGK066).

After the general GP-1 procedure, compound (±)-JGK066 was prepared from (±)-6 (45 mg, 0.09 mmol) and a 2 M solution of Me ₂ NH in THF (232 μl, 0.46 mmol) in DMF (1 .85 ml). PTSC (EtOAc, 0.5% acetonitrile, 1.5% aq NH ₄ OH) provided (±)-JGK066 (39 mg, 97%) as an off-white fluffy foam.

1H 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 (ush, 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, Jcf=242.3 Hz), 149.38, 146.67, 144.06, 128.70 (d, Jcf=10.4 Hz), 127.16, 125.29 (d, Jcf=4.7 Hz), 121.65, 114.27, 110.67, 108.56 (d, Jcf=19.4 Hz ), 106.15, 71.70, 67.20, 59.78, 46.41 ppm;

MSVR (IER): m/z [M+H]+ calc. for Ci ₉ Hi ₉ BrFN ₄ O ₂ ⁺ , 433.0670; 433.0677 discovered.

(±)-N-(3-Bromo-2-fluorophenyl)-7-[(pyrrolidin-1-yl)methyl]-7,8-dihydro[1,4]dioxino[2,3g]quinazolin-4-amine ((±)-JGK067).

Following the general GP-1 procedure, compound (±)-JGK067 was prepared from (±)-6 (35 mg, 0.07 mmol) and pyrrolidine (30 μL, 0.36 mmol) in DMF (1.45 mL). PTSC (EtOAc, 1.5% iPrOH, 1.5% aq NH4OH) produced (±)-JGK067 (31 mg, 93%) as an off-white fluffy foam.

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 (ush, 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, Jcf=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;

- 69 044668

MSVR (IER): m/z [M+H]+ calc. for C ₂₁ H ₂₁ BrFN ₄ O2 ⁺ , 459.0826; discovered 459.0845.

(±)-N-(3-Bromo-2-fluorophenyl)-7-[(4-methylpiperazin-1-yl)methyl]-7,8-dihydro[1,4]dioxino[2,3g]quinazolin-4 -amine ((±)-JGK068).

Following the general GP-1 procedure, compound (±)-JGK068 was prepared from (±)-6 (35 mg, 0.07 mmol) and 1-methylpiperazine (40 μL, 0.36 mmol) in DMF (1.45 mL). PTSC (EtOAc/iPrOH 85:15, 1.5% aq NH4OH) produced (±)-JGK068 (29 mg, 82%) as an off-white fluffy foam.

1H 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 (ush 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 (ush, 4H), 2.32 ppm. (s, 3H);

¹³ C NMR (126 MHz, CDCl ₃ ) 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 =19.2 Hz), 106.07, 71.80, 67.27, 58.43, 55.10, 53.96, 46.06 ppm;

MSVR (IER): m/z [M+H] ⁺ calc. for C ₂₂ H ₂₄ BrFN ₅ O ₂ ⁺ , 488.1092; discovered 488.1109.

(±)-N-(3-Bromo-2-fluorophenyl)-7-[2-(dimetulamino)ethyl]-7,8-dihydro[1,4]dioxuno[2,3-g]quinazolin4-amine (( ±)-JGK069).

After the general GP-1 procedure, compound (±)-JGK069 was prepared from (±)-10 (32 mg, 0.06 mmol) and a 2 M solution of Me ₂ NH in THF (161 μl, 0.32 mmol) in DMF (1 ,3 ml). PTSC (EtOAc, 5% iPrOH, 1.5% aq NH ₄ OH) provided (±)-JGK069 (19 mg, 66%) as an off-white fluffy foam.

1H NMR (500 MHz, CDCl ₃ ) 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 (dk, 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, CDCl ₃ ) 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 =19.2 Hz), 105.88, 72.19, 68.34, 55.06, 45.58, 29.16 ppm;

MSVR (IER): m/z [M+H] ⁺ calc. for C ₂₀ H ₂₁ BrFN ₄ O ₂ ⁺ , 447.0826; 447.0820 discovered.

(±)-N-(3-Bromo-2-fluorophenyl)-7-[2-(4-methylpiperazin-1-yl)ethyl]-7,8-dihydro[1,4]dioxino[2,3g]quinazoline -4-amine ((±)-JGK070).

Following the general GP-1 procedure, compound (±)-JGK070 was prepared from (±)-10 (32 mg, 0.06 mmol) and 1-methylpiperazine (36 μL, 0.32 mmol) in DMF (1.3 mL). PTSC (EtOAc/iPrOH 8:2, 1.5% aq NH ₄ OH) provided (±)-JGK070 (21 mg, 65%) as an off-white fluffy foam.

1H NMR (500 MHz, CDCl ₃ ) 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 (ush, 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 C NMR (126 MHz, CDCl ₃ ) 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;

MSVR (IER): m/z [M+H] ⁺ calc. for C ₂₃ H ₂₆ BrFN ₅ O ₂ ⁺ , 502.1248; 502.1261 discovered.

- 70 044668

5-Fluoro-2,3-dihydro-1,4-benzodioxin (11).

Fω

A mixture of 3-fluorobenzene-1,2-diol (7233 mg, 56.5 mmol) in DMF (113 ml) was treated with K ₂ CO ₃ (19514 mg, 141 mmol), stirred for 10 min at 23°C and treated with 1bromo-2-chloroethane (9.4 ml, 113 mmol). The mixture was stirred at 23°C for 1 hour and then at 95°C for 16 hours. The mixture was cooled to 23°C, diluted with water (150 ml) and extracted with EtOAc (3x150 ml). The combined organics were washed with water (90 ml), brine (90 ml), dried (Na ₂ SO ₄ ), filtered and evaporated. PC (hexanes/EtOAc 30:1^10:1) provided the title compound 11 (7973 mg, 92%) as a clear, colorless oil.

1H NMR (400 MHz, CDCl ₃ ) δ 6.78-6.63 (m, 3H), 4.34-4.26 ppm (m, 4H);

¹³ C NMR (101 MHz, CDCl ₃ ) 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, Jcf=8.9 Hz), 112.74 (d, Jcf=3.1 Hz), 108.52 (d, Jcf=18.1 Hz), 64 .50, 64.45 ppm;

MSVR (DART): m/z [M] ⁺ calc. for C ₈ H ₇ FO ₂ ⁺ , 154.0425; discovered 154.0420.

6-Bromo-5-fluoro-2,3-dihydro-1,4-benzodioxin (12).

F o^D^vg W

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 concentrated. The residue was dissolved in DCM (700 ml), washed with water (300 ml), dried (MgSO ₄ ), filtered and evaporated. PC (hexanes/EtOAc 30:1^20:1) followed by high vacuum drying at 100°C to remove any remaining starting material provided the title compound 12 (8807 mg, 75%, containing about 15% regioisomer) in as a clear, colorless oil that solidified in the freezer to an off-white solid.

1H NMR (400 MHz, CDCl ₃ ) 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, CDCl ₃ ) δ 148.87 (d, Jcf=245.1 Hz), 144.53 (d, Jcf=3.5 Hz), 133.81 (d, Jcf=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;

MSVR (DART): m/z [M] ⁺ calc. for C ₈ H ₆ BrFO ₂ ⁺ , 231.9530; found 231.9525.

5-Fluoro-2,3-dihydro-1,4-benzodioxin-6-carboxylic acid (13).

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) within 10 min. The mixture was stirred at -78°C for 30 min and then transferred via cannula onto crushed dry ice (cannula flushed with 10 ml THF). The mixture was allowed to warm to 23°C and concentrated. Water (200 ml) and 1 M NaOH (50 ml) were added to the residue and aq. the phase was extracted with Et ₂ O (3x60 ml). Vodn. the phase was acidified with 6 M HCl (15 ml) and extracted with DCM (3x150 ml). The combined organics were washed with brine (150 ml), dried (MgSO ₄ ), filtered and evaporated. PC (hexanes/EtOAc 7:3^3:7) provided the title compound 13 (3591 mg, 60%) as a white solid.

1H NMR (400 MHz, DMSO-d ₆ ) 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-d ₆ ) 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, Jcf = 13.6 Hz), 122.44 (d, Jcf = 1.4 Hz), 112.12 (d, Jcf = 3.4 Hz), 111.97 (d, Jcf=7.3 Hz), 64.42, 63.91 ppm;

MSVR (DART): m/z [M -Н] ^- calc. for C9H6FO4 ^- , 197.0256; found 197.0250.

Ethyl (5-fluoro-2,3-dihydro-1,4-benzodioxin-6-yl)carbamate (14).

A mixture of 13 (650 mg, 3.28 mmol) in toluene (13.1 ml) was treated with Et ₃ N (1.4 ml, 9.84 mmol) and at 10°C with DPPA (780 μl, 3. 62 mmol). The mixture was stirred at 23°C for 30 minutes, then at 85°C for 1.5 hours. The mixture was cooled to 23°C, treated with EtOH (5 ml), stirred for 1.5 hours at 23°C and concentrated. The residue was dissolved in Et ₂ O (150 ml), washed with sat. aq. NaHCO ₃ solution (40 ml), water (40 ml), saline solution (40 ml), dried

- 71 044668 (MgSO ₄ ), filtered and evaporated. PC (hexanes/DCM 7:3^1:9) provided the title compound 14 (512 mg, 65%) as a white solid.

1H NMR (500 MHz, CDC^) 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, CDCls) δ 153.80, 142.61 (d, Jcf=246.0 Hz), 140.82, 132.66 (d, Jcf=12.4 Hz), 120.36 ( d, Jcf=6.9 Hz), 112.36, 111.81 (d, Jcf=3.7 Hz), 64.72, 64.29, 61.61, 14.66 ppm;

MSVR (DART): m/z [M+H]+calc. for C ₁₁ H ₁₃ FNO ₄ ⁺ , 242.0823; discovered 242.0816.

10-Fluoro-7,8-dihydro[1,4]dioxino[2,3-g]quinazoline (15).

A mixture of 14 (450 mg, 1.87 mmol) and NMTA (263 mg, 1.87 mmol) in TFA (5.7 ml) was microwaved at 110 °C for 10 min. The mixture was cooled to 23°C, diluted with water (60 ml), treated with 6 M NaOH (12 ml) and extracted with DCM (3x60 ml). The combined organics were washed with water (50 ml), brine (50 ml), dried (Na ₂ SO ₄ ), filtered and evaporated to give a foamy yellow oil.

A mixture of oil in 10% KOH in dioxane/water 1:1 (15.5 ml) was treated with [K ₃ Fe(CN) ₆ ] (614 mg, 1.87 mmol) and microwaved at 100°C for 10 min. This procedure was repeated a total of four times (4 cycles of adding 1 equivalent of potassium ferrocyanide followed by microwave irradiation). The resulting mixture was diluted with water (160 ml) and extracted with DCM (3x120 ml). The combined organics were washed with water (100 ml), brine (100 ml), dried (Na ₂ SO ₄ ), filtered and evaporated to give the title compound 15 (330 mg, 86%) as a yellow solid, which was used in the next stage without any further purification.

1H 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 m.d. (m, 4H);

¹³ C NMR (126 MHz, CDCl3) δ 158.06 (d, Jcf=2.9 Hz), 153.81 (d, Jcf=1.8 Hz), 145.76 (d, Jcf=2.9 Hz ), 144.40 (d, Jcf=256.1 Hz), 138.56 (d, Jcf=11.0 Hz), 136.73 (d, Jcf=10.1 Hz), 119.81 (d, Jcf=2.7 Hz), 106.55 (d, Jcf=4.3 Hz), 64.78, 64.34 ppm;

MSHR (DART): m/z [M+H]+ calc. for C _W H8FN2O2 ⁺ , 207.0564; discovered 207.0563.

10-Fluoro-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4(3H)-one (16).

F

ABOUT

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 (2x2 ml), acetonitrile (2x2 ml), DCM (2 ml) and Et ₂ O (2 ml) to obtain the first batch of the title compound. Vodn. The filtrate was neutralized to pH~7 with 1 M NaOH and the white precipitate was collected by filtration as before, followed by washing to obtain a second batch of title compound 16 (81 mg, 25%) as a white solid.

1H 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, Jcf=3.4 Hz), 137, 86 (d, Jcf=11.1 Hz), 132.94 (d, Jcf=8.9 Hz), 115.75, 106.62 (d, Jcf=3.7 Hz), 64.57, 64, 02 ppm;

MSHR (DART): m/z [M+H]+ calc. for C ₁₀ H ₈ FN ₂ O ₃ ⁺ , 223.0513; discovered 223.0503.

4-Chloro-10-fluoro-7,8-dihydro[1,4]dioxino[2,3-g]quinazoline (17).

A stirred suspension of 16 (92 mg, 0.41 mmol) in toluene (1.2 mL) was treated with DIPEA (220 μL, 1.26 mmol) followed by dropwise addition of POCl ₃ (103 μL, 1.12 mmol) at 10°C. The mixture was stirred at 23°C for 1 hour, then at 90°C for 5 hours and concentrated. The residue was treated with sat. aq. NaHCO ₃ solution (10 ml) at 0°C for 5 min, diluted with water (5 ml) and extracted with DCM (3x7 ml). The combined organics were washed with half sat. aq. NaHCO ₃ solution (7 ml), brine (7 ml), dried (Na ₂ SO ₄ ), filtered and evaporated to give the title compound 17 (51 mg, 51%) as a light brown solid, which was used at the next stage without any

- 72 044668 additional cleaning.

1H NMR (500 MHz, CDCI3) 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, CDCl ₃ ) δ 160.48 (d, J _C F = 4.3 Hz), 152.31, 146.29 (d, J _cf = 3.3 Hz), 144.63 ( d, Jcf=256.2 Hz), 138.95 (d, Jcf=11.3 Hz), 137.68 (d, Jcf=10.2 Hz), 118.56 (d, Jcf=2.4 Hz ), 105.82 (d, Jcf=4.2 Hz), 64.81, 64.41 ppm;

MSHR (DART): m/z [M+H]+ calc. for C ₁₀ H ₇ ClFN ₂ O ₂ ⁺ , 241.0175; discovered 241.0174.

5-Fluoro-7-nitro-2,3-dihydro-1,4-benzodioxin-6-carboxylic acid (18).

F

A mixture of 13 (1500 mg, 7.57 mmol) in AcOH (7.5 ml) was treated dropwise with H ₂ SO ₄ (2.02 ml) at 10°C. The thoroughly mixed mixture was treated dropwise with 65% HNO ₃ (2.6 ml) at 0°C for 10 min. The resulting mixture was stirred at 0°C for 30 minutes and then at 23°C for 16 hours. The mixture was poured into ice water (40 ml) and the white precipitate was collected by filtration (cold water wash, 40 ml) and dried in a desiccant to obtain the title compound 18 (1280 mg, 70%) as a white solid.

1H NMR (500 MHz, DMSO-d ₆ ) 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-d ₆ ) δ 162.71, 147.16 (d, J _cf =248.7 Hz), 144.72 (d, J _cf =5.1 Hz), 138.15 (d, Jcf=13.7 Hz), 137.10 (d, Jcf=6.6 Hz), 113.44 (d, Jcf=20.3 Hz), 109.52 (d, Jcf=2.3 Hz), 64.97, 64.48 ppm;

MSVR (DART): m/z [M-Н] ^- calc. for C ₉ H5FNO ₆ ^- , 242.0106; discovered 242.0124.

-Amino-5-fluoro-2,3-dihydro-1,4-benzodioxin-6-carboxylic acid (19).

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 H ₂ at 23°C for 13.5 hours. The mixture was filtered through celite (EtOH washes) and evaporated to give the title compound 19 (418 mg, 95%) as a gray solid that did not appear to be very stable.

1H NMR (500 MHz, DMSO-d ₆ ) 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-d ₆ ) 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, Jcf=5.7 Hz), 122.88 (d, Jcf=15.4 Hz), 97.19 (d, Jcf=2.9 Hz), 95.37 (d, Jcf=10.9 Hz), 64.95, 63.58 ppm;

MSVR (DART): m/z [M+H] ⁺ calc. for C ₉ H ₉ FNO ₄ ⁺ , 214.0510; discovered 214.0508.

5-Fluoro-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4(3H)-one (20).

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 hours. The mixture was cooled to 0°C and treated with water (4 ml ), stirred for 30 min, diluted with water (4 ml) and filtered. The residue was washed with cold water (3x5 ml) and dried over Drierite under high vacuum to give the title compound 20 (249 mg, 57%) as an off-white solid.

1H 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-d ₆ ) δ 157.64 (d, Jcf=3.0 Hz), 149.70 (d, Jcf=6.0 Hz), 148.45 (d, Jcf=261 .3 Hz), 144.60, 142.99, 131.47 (d, Jcf=12.7 Hz), 108.76 (d, Jcf=3.5 Hz), 106.38 (d, Jcf=3 .8 Hz), 64.69, 63.98 ppm;

MSVR (DART): m/z [M+H] ⁺ calc. for Ci ₀ H ₈ FN ₂ O ₃ ⁺ , 223.0513; discovered 223.0510.

4-Chloro-5-fluoro7,8-dihydro[1,4]dioxino[2,3-g]quinazoline (21).

A stirred suspension of 20 (90 mg, 0.41 mmol) in toluene (1.2 mL) was treated with DIPEA (215 μL, 1.24 mmol) followed by dropwise addition of POCl ₃ (100 μL, 1.09 mmol) at 10°C. The mixture was stirred at 23°C for 1 hour, then at 88°C for 5 hours and concentrated. The residue was treated with sat. aq. NaHCO ₃ solution (10 ml) at 0°C, diluted with water (5 ml) and extracted with DCM (3x7 ml). The combined organics were washed with half sat. aq. NaHCO ₃ solution (7 ml), saline solution (7 ml), dried (Na ₂ SO ₄ ), fillet

- 73 044668 was pickled and evaporated to give the title compound 21 (96 mg, 99%) as a light orange solid, which was used in the next step without any further purification.

¹ H NMR (500 MHz, CDCl ₃ ) 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, CDCl ₃ ) 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, Jcf=261.8 Hz), 134.04 (d, Jcf=14.0 Hz), 110.85 (d, Jcf=7.7 Hz ), 109.43 (d, Jcf=4.0 Hz), 64.89, 64.37 ppm;

MSHR (DART): m/z [M+H]+ calc. for C _w H7C1FN2O2 ⁺ , 241.0175; discovered 241.0176.

N-(3-Bromo-2-fluorophenyl)-10-fluoro-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK071).

Following the general GP-2 procedure, compound JGK071 was prepared from chloroquinazoline 17 (35 mg, 0.15 mmol) and 3-bromo-2-fluoroaniline. PC (DCM/EtOAc 1:0 >8:2) provided JGK071 (44 mg, 77%) as a white solid.

^1H 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-d ₆ ) δ 156.93 (d, J _CF = 3.7 Hz), 153.44 (d, J _cf = 247.5 Hz), 153.12, 144.04 (d, Jcf=250.0 Hz), 143.97 (d, Jcf=3.2 Hz), 137.04 (d, Jcf=10.9 Hz), 135.62 (d, Jcf=9.9 Hz), 130.48, 127.89, 127.62 (d, Jcf=13.1 Hz), 125.51 (d, Jcf=4.5 Hz), 108.58 (d, Jcf=23.4 Hz), 108.51, 103.25 (d, Jcf=3.9 Hz), 64.63, 64.21 ppm;

MSHR (DART): m/z [M+H]+ calc. for C ₁₆ H ₁₁ BrF ₂ N ₃ O2 ⁺ , 393.9997; 393.9999 discovered.

N-(3-Bromo-2-fluorophenyl)-5-fluoro-7,8-dihydro[1,4]dioxuno[2,3-g]quinazolin-4-amine (JGK072).

Following the general GP-2 procedure, compound JGK072 was prepared from chloroquinazoline 21 (35 mg, 0.15 mmol) and 3-bromo-2-fluoroaniline. PC (DCM/EtOAc 1:0>8:2) provided JGK072 (47 mg, 82%) as a white solid.

^1H 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 (dd, 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, Jcf=243.9 Hz), 149.93 (d, Jcf= 6.2 Hz), 145.75 (d, Jcf=250.3 Hz), 144.78, 131.96 (d, Jcf=15.6 Hz), 128.43 (d, Jcf=10.4 Hz ), 127.71, 125.20 (d, Jcf=4.7 Hz), 122.48, 109.69 (d, Jcf=3.3 Hz), 108.63 (d, Jcf=19.2 Hz ), 101.42 (d, Jcf=7.2 Hz), 64.84, 64.48 ppm;

MSHR (DART): m/z [M+H]+ calc. for C ₁₆ H ₁₁ BrF ₂ N ₃ O ₂ ⁺ , 393.9997; 393.9996 discovered.

Example 17 Brain Penetration of Exemplary Compounds of the Invention.

In table Figure 4 discloses the brain-to-plasma percentages and unrelated brain-to-plasma drug ratios of these drugs in tumor-free mice.

- 74 044668

Table 4

Brain Penetration of Exemplary Compounds of the Invention

Compound Penetration into the brain (% of blood plasma) Creep (Avg) Erlotinib 8.50 0.051 JGK005 64.8 0.491 JGK038 84.3 0.575 JGK028 106.2 1.037 JGK010 106.4 1.045 JGK037 212.1 1,301 JGK042 167.6 1.033 JGK063 72.5 0.341 JGK066 274.3 1.175 JGK068 354.5 1,184 JGK068S 378.3 1.181 JGK074 166.2 not detected JGK083S 231.3 0.798

Example 18 Preparation of exemplary JGK series compounds.

Scheme 1. Synthesis of JGK068S

Preparation of the (R)-enantiomer of JGK068R or racemic mixtures (JGK068) occurs in a similar manner as shown in Scheme 1, but uses (R)- or racemic glycidol, respectively.

Scheme 2

One-step synthesis of benzodioxanecarbaldehyde (R)-10 from benzaldehyde 1 by chiral glycidyl tosylate. This method does not include the Mitsunobu reaction in Scheme 1 (obtaining 2 from 1). Compound (R)-10 can be used in the method shown in Scheme 1 to produce JGK068S.

Scheme 3

General chemical information.

All chemicals, reagents, and solvents were purchased from commercial sources when available and used as received. When necessary, reagents and solvents were purified and dried using standard methods. Reactions, sensitive

- 75 044668 to air and moisture, was carried out in an inert atmosphere of argon in oven-dried glassware. Reactions with microwave irradiation were carried out in a single-mode reactor of a CEM Discover microwave synthesizer. Room temperature (RT) reactions were performed at ambient temperature (approximately 23°C). All reactions were monitored by thin layer chromatography (TLC) on pre-coated Merck 60 F ₂₅₄ silica gel plates spotted with UV light (λ=254, 365 nm) or using an alkaline KMnO ₄ solution. Flash column chromatography (FC) was carried out on SiO ₂ 60 (particle size 0.040-0.063 mm, 230-400 mesh). Preparative thin-layer chromatography (PTLC) was performed with Merck 60 F ₂₅₄ silica gel plates (20x20 cm, 210-270 mm) or Analtech Silica Gel GF TLC plates (20x20 cm, 1000 mm). Concentration under reduced pressure (in vacuum) was carried out on a rotary evaporator at 23-50°C. The purified compounds were further dried in a high vacuum or in a desiccant. The outputs correspond to purified compounds and have not been further optimized. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on Bruker spectrometers (operating at 300, 400 or 500 MHz). Carbon NMR spectra ( ^13C NMR) were recorded on Bruker spectrometers (at 400 or 500 MHz). NMR chemical shifts (δ ppm) were assigned to residual solvent signals. 1H NMR data are presented as follows: chemical shift in ppm; multiplicity (s=singlet, b=doublet, t=triplet, q=quartet, quint=KBHHTeT, m=multiple/mixed type, td=triplet of doublets, ddd=doublet of doublets of doublets, br=broad signal); interaction constants (J) in Hz, integration. Data for ¹³ C NMR spectra are presented as a function of chemical shift and, if applicable, interaction constants. High-resolution mass spectra (HRMS) were recorded on a Thermo Fisher Scientific Exactive Plus with an original IonSense ID-CUBE DART mass spectrometer or on a Waters LCT Premier mass spectrometer with ACQUITY UPLC with autosampler.

5-Formyl-2-hydroxyphenylacetate (1).

^Η0 ΌΟ I JL AcO -'' CHO

A mixture of 3,4-dihydroxybenzaldehyde (100 g, 0.724 mol) in THF (965 ml) was cooled to 0°C and treated with 10% aq. NaOH solution (724 ml, 1.81 mol) for 4-5 minutes. After stirring the reaction mixture at 0°C for 15 min, acetic anhydride (Ac ₂ O, 82.1 ml, 0.869 mol) was added dropwise over 20 min. The mixture was stirred for 30 min at the same temperature 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 aq. the phase was extracted with EtOAc (4x250 ml). The combined organics were washed with water (2x500 ml), brine (500 ml), dried (Na ₂ SO ₄ ), filtered and evaporated. The residue was treated with a small amount of n-heptane and evaporated (3x). Recrystallization with EtOAc (275 mL; crystals washed with Et2O) provided the first batch of title compound 1 (66.96 g, 51%) as light brown crystals. Recrystallization of the stock solution from EtOAc provided a second batch of title compound 1 (29.436 g, 23%) as a light brown solid.

¹ H NMR (500 MHz, CDCl ₃ ) δ 9.85 (s, 1H), 7.73-7.65 (m, 2H), 7.11 (d, J=8.8 Hz, 1H), 6 .34 (broad, 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;

MSHR (DART): m/z [M+H]+ calc. for C ₉ H ₉ O ₄ ⁺ , 181.0495; 181.0488 discovered.

5-Formyl-2-{ [(2R)-oxiran-2-yl]methoxy}phenylacetate ((R)-2).

ABOUT

AcO^C^CHO

A mixture of 1 (32.5 g, 0.18 mol) and triphenylphosphine (PPh ₃ , 70.976 g, 0.27 mol) in THF (905 ml) was treated with (S)-glycidol (17.95 ml, 0.27 mol), cooled to 0°C and treated dropwise with diisopropyl azodicarboxylate (DIAD, 56.8 ml, 0.289 mmol) for 30 min. The mixture was stirred for an additional 10 minutes at 0°C, after which the cooling bath was removed and stirring was continued at 23°C for 15.5 hours. All volatiles were evaporated and the crude (R)-2 obtained as a brown oil was used without any additional purification in the next step.

(3S)-3-(Hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-carbaldehyde ((S)-3).

Н<Х X Λ

A mixture of crude (R)-2 in MeOH (1.564 L) was treated with K2CO3 (49.87 g, 0.36 mol) and stirred at 23°C for 18.5 h and then the solvent was evaporated. The residue was suspended in half sat. NH4Cl (750 ml) and extracted with EtOAc (3x500 ml). United organically

- 76 044668 These substances were washed with water (250 ml), saline (250 ml), dried (Na ₂ SO ₄ ), filtered and evaporated. The crude material was purified by multiple rounds of flash chromatography (9:1 to 1:1 hexanes/EtOAc) and also by precipitation from 1:1 hexanes/ _Et2O (to remove triphenylphosphine oxide _Ph3PO ) to give the title compound (S)-3 (17.322 g, 49% from 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 (lat, 1H);

¹³ C NMR (126 MHz, CDCl ₃ ) δ 190.79, 148.76, 143.46, 130.79, 124.46, 118.33, 117.70, 73.31, 65.61, 61.54 ;

MSHR (DART): m/z [M+H]+ calc. ^{for C10H11O4+} _{, 195.0652 ;} found 195.0650.

[(2R)-7-Cyano-2,3-dihydro-1,4-benzodioxin-2-yl]methyl acetate ((R)-4).

ASO

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), stirred at 23°C for 10 min and then treated with NH2OH-HQ (16.233 g, 0.234 mol). The resulting mixture was stirred at 120-125°C for 18.5 hours. The mixture was cooled to 23°C, poured into water (1 L) and extracted with EtOAc (4x250 ml). The combined organics were treated with 3.5 M NaOH (400 ml) and sat. aq. NaHCO ₃ solution (100 ml) to obtain a final pH of ~8, and the emulsion was stirred at 23°C for 1 hour. The organic layer was separated and washed with sat. aq. NaHCO ₃ solution (300 ml), water (300 ml), saline solution (300 ml), dried (Na ₂ SO ₄ ), filtered and evaporated. Purification by flash chromatography (hexanes/EtOAc 10:1 -> 6:4) provided the title compound (R)-4 (13.513 g, 65%) as a clear, colorless oil.

1H 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;

MSVR (DART): m/z [M+H] ⁺ calc. for C ₁₂ H ₁₂ NO ₄ ⁺ , 234.0761; discovered 234.0759.

[(2R)-7-Cyano-6-nitro-2,3-dihydro-1,4-benzodioxin-2-yl]methyl acetate ((R)-5).

NO ₂

ASO

A mixture of (R)-4 (13.345 g, 57.2 mmol) in Ac ₂ O (74.3 ml) was treated with H ₂ SO ₄ (3.05 ml, 57.2 mmol), cooled to 0°C and treated dropwise with 70% HNO ₃ (19.63 mL, 286 mmol) at 0°C for 35 min. The mixture was stirred for another 2 hours at 0°C and then at 23°C for 3.5 hours. The mixture was poured into ice water (850 ml) and the pH was adjusted to ~7 with 6 M NaOH (320 ml). Saturation aq. NaHCO ₃ solution (200 ml) was added and the mixture was extracted with CH ₂ Cl ₂ (3x500 ml). The combined organics were washed with sat. aq. NaHCO ₃ solution (400 ml), water (400 ml), brine (400 ml), dried (Na ₂ SO ₄ ), 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.

1H 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-d ₆ ) 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;

MSVR (DART): m/z [M+H] ⁺ calc. for Ci ₂ H _u N ₂ O ₆ ⁺ , 279.0612; discovered 279.0601.

(3S)-7-Amino-3-(hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-carbonitrile ((S)-6).

NH ₂

BUT

ON

A suspension of (R)-5 (15.591 g, 56 mmol) in water/ethanol 1:1 (250 ml) was treated with Na ₂ S ₂ O ₄ (39.266 g, 185 mmol) and the mixture was stirred at 50°C for 105 min. and then heated to 70°C for 2 hours while simultaneously treating with portions of conc. HCl (73.6 mL, 0.897 mol) at this time. The mixture was cooled to 23°C, poured onto ice and the pH was adjusted to ~10 with 6 M NaOH (200 ml) and half-saturated. NaHCO ₃ (500 ml). The mixture was extracted with EtOAc (3x500 ml). The combined organics were washed with water (500 ml), brine (500 ml), dried (Na ₂ SO ₄ ), 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.

1H NMR (500 MHz, DMSO-d ₆ ) δ 6.92 (s, 1H), 6.29 (s, 1H), 5.50 (br, 2H), 5.04 (t, J=5, 7 Hz, 1H), 4.32

- 77 044668 (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-d ₆ ) δ 148.50, 147.29, 134.39, 119.04, 118.04, 102.45, 86.72, 73.25, 65.92, 59 .77;

MSHR (DART): m/z [M+H]+ calc. for C ₁₀ H ₁₀ N ₂ O ₃ ⁺ , 206.0686; discovered 206.0685.

N'-[(2S)-7-Cyano-2-(hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-yl]-N,N-dimethylmethanimidamide ((S)-7).

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 the mixture was stirred at 105°C for 3 hours. Evaporated MeOH (~4-5 ml) was collected in a Dean-Stark trap to monitor the progress of the reaction. The mixture was cooled to 23°C and 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.

1H NMR (500 MHz, CDCl ₃ ) 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, CDCl ₃ ) 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;

MSVR (DART): m/z [M+H] ⁺ calc. for C ₁₃ H ₁₆ N ₃ O ₃ ⁺ , 262.1186; found 262.1183.

[(7S)-4-(3-Bromo-2-fluoroanshino)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-7-yl]methanol ((S)-8).

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 hours. The mixture was cooled to 23°C, poured into ice water (500 ml) and pH adjusted to ~9 sat. aq. NH4OH solution (185 ml) and half-saturated. aq. NaHCO ₃ solution (200 ml). The mixture was extracted with EtOAc (3x400 ml) and the combined organics were washed with half sat. aq. NaHCO ₃ solution (400 ml), water (400 ml), saline solution (400 ml), dried (Na ₂ SO ₄ ), filtered and evaporated. The residue was dissolved in MeOH (272 ml) and treated with K2CO3 (12.579 g, 91 mmol), stirred at 23°C for 1 hour and evaporated. The residue was suspended in half sat. aq. NH ₄ Cl solution (700 ml) and extracted with EtOAc (3x400 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, heated to 65°C and then allowed to cool slowly to 23°C overnight. Filtration and washing of the residue with cold hexanes (2x15 ml) and Et ₂ O (2x15 ml) and drying under high vacuum provided the title compound (S)-8 (9.407 g, 50.9% from two steps) as crushed yellow powder.

¹ H NMR (500 MHz, DMSO-d ₆ ) 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-b ₆ ) 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;

MSVR (DART): m/z [M+H] ⁺ calc. for C ₁₇ H ₁₄ BrFN ₃ O ₃ ⁺ , 406.0197; discovered 406.0185.

[(7R)-4-(3-Bromo-2-fluoroanilino)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-7-yl]methylmethanesulfonate ((R)-9).

A mixture of (S)-8 (9.01 g, 22.2 mmol) and Me ₃ N-HCl (234 mg, 2.45 mmol) in acetonitrile (148 ml) was treated with Et ₃ N (6.18 ml, 44.4 mmol), cooled to 0-5°C and treated dropwise with a solution of MsCl (2.57 ml, 33.2 mmol) in acetonitrile (17 ml; washed with 3 ml) for 10 min. The mixture was stirred at 0°C for 1 hour. Water (100 ml) was added and most of the acetonitrile was evaporated in vacuo. Additional water (700 ml) was added and the mixture was extracted with EtOAc (3x400 ml). The combined organics were washed with water (400 ml), brine (400 ml), dried (Na ₂ SO ₄ ), filtered and evaporated to obtain the title

- 78 044668 compound (R)-9 (10.33 g, 96%) as a yellow fluffy foam, which was used in the next step without any further purification.

1H NMR (500 MHz, CDCl ₃ ) 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 (broad, 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).

(7S)-N-(3-bromo-2-fluorophenyl)-7-[(4-Methylpiperazin-1-yl)methyl]-7,8-dihydro[1,4]dioxino[2,3g]quinazolin-4 -amine (JGK068S).

A mixture of (R)-9 in DMF (427 ml) was treated with 1-methylpiperazine (11.83 ml, 107 mmol) and Et ₃ N (5.95 ml, 42.7 mmol) and the mixture was stirred at 85°C for 24 hours. The mixture was cooled to 23°C and evaporated. The residue was dissolved in EtOAc (1.2 L) and washed with 0.5 M NaOH (4x250 ml), brine (250 ml), dried (Na2SO4), filtered and evaporated. Purification by flash chromatography (CH ₂ Cl ₂ /MeOH 1:0^8:2) provided the title compound JGK068S (6.013 g, 58% from two steps) as an off-white fluffy foam.

^1H 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 (broad, 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 (lat, 4H), 2.51 (lat, 4H), 2.32 (s, 3H).

Scheme 4. Synthesis of JGK083S tert-Butyl-{[(7S)-4-(3-bromo-2-fluoroanilino)-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-7yl]methyl }piperazine-1-carboxylate ((S)-10).

Following the general procedure of GP-1 of Example 16, compound (S)-10 was prepared from R-9 (91 mg, 0.188 mmol) and tert-butylpiperazine-1-carboxylate (175 mg, 0.94 mmol) in DMF (3.8 ml ) and stirred at 85°C for 15 hours. PTSC (CH ₂ Cl ₂ /EtOAc 4:6) provided (S)-10 (50 mg, 46%) as an off-white fluffy foam.

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 (broad, 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 (possibly due to overlapping peaks);

MSHR (DART): m/z [M+H]+ calc. for C ₂₆ H ₃₀ BrFN ₅ O ₄ ⁺ , 574.1460; discovered 574.1432.

(7S)-N-(3-Bromo-2-fluorophenyl)-7-[(piperazin-1-yl)methyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin4-amine (JGK083S).

A mixture of (S)-10 (42 mg, 0.073 mmol) in CH ₂ Cl ₂ (500 µl) and TFA (250 µl) was stirred at 23°C for 12 hours. The mixture was diluted with 1 M HCl (20 ml) and washed with CH ₂ Cl ₂ (3x7 ml). The aqueous phase was diluted with 6 M NaOH (4 ml) to pH>12 and extracted with EtOAc (3x8 ml). The combined organics were washed with brine (8 ml), dried (Na ₂ SO ₄ ), filtered and evaporated. Purification using PTSCH (CH ₂ Cl ₂ /MeOH 8:2) provided

- 79 044668 of the title compound JGK083S (18 mg, 52%) as a white fluffy foam.

1H NMR (500 MHz, CDCl ₃ ) δ 8.68 (s, 1H), 8.66 (ddd, J=8.2, 7.3, 1.6 Hz, 1H), 7.39 (s, 1H ), 7.35 (lat, 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, CDCl ₃ ) δ 155.88, 153.35, 150.12 (d, J _CF = 242.3 Hz), 149.45, 146.71, 144.17, 128.67 ( d, Jcf=10.4 Hz), 127.21, 125.32 (d, Jcf=4.7 Hz), 121.74, 114.30, 110.64, 108.58 (d, JC=19, 3 Hz), 106.02, 71.70, 67.32, 59.19, 55.54, 46.23;

MSVR (DART): m/z [M - N] - calc. for C ₂₁ H ₂₀ BrFN ₅ O ₂ -, 472.0790; 472.0773 discovered.

[(2R)-7-Formyl-2,3-dihydro-1,4-benzodioxin-2-yl]methyl acetate ((R)-10).

A mixture of 1 (150 mg, 0.833 mmol) and (2R)-glycidyl tosylate (203 mg, 0.891 mmol) in DMF (2 ml) was treated with K2CO3 (181 mg, 1.31 mmol) and the mixture was stirred at 60°C for 15 hours The mixture was cooled to 23°C, water (30 ml) was added and the mixture was extracted with EtOAc (3x15 ml). The combined organics were washed with water (15 ml), brine (15 ml), dried (Na ₂ SO ₄ ), filtered and evaporated. Purification by preparative thin layer chromatography (hexanes/EtOAc 7:3) provided the title compound (R)-10 (111 mg, 56%) as a clear, colorless oil.

1H NMR (400 MHz, CDCl ₃ ) 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, CDCl ₃ ) 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;

MSVR (DART): m/z [M+H] ⁺ calc. for C ₁₂ H ₁₃ O ₅ ⁺ , 237.0757; discovered 237.0745.

(±)-N-(3-Chloro-2-fluorophenyl)-7-[(4-methylpiperazin-1-yl)methyl]-7,8-dihydro[1,4]dioxino[2,3g]quinazolin-4 -amun (JGK075).

1H NMR (500 MHz, CDCl ₃ ) δ 8.68 (s, 1H), 8.61 (td, J=7.3, 2.2 Hz, 1H), 7.39 (s, 1H), 7, 35 (ush 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 (ush, 4H), 2.48 ( ush, 4H), 2.31 (s, 3H);

¹³ C NMR (126 MHz, CDCl ₃ ) δ 155.89, 153.35, 149.44, 149.30 (d, J _CF = 244.2 Hz), 146.72, 144.15, 128.76 ( d, Jcf=9.3 Hz), 124.71 (d, Jcf=4.7 Hz), 124.45, 121.01, 120.85 (d, Jcf=16.1 Hz), 114.30, 110.63, 106.05, 71.81, 67.31, 58.50, 55.17, 54.15, 46.19;

MSHR (DART): m/z [M+H]+ calc. for C ₂₂ H ₂₄ ClFN ₅ O ₂ ⁺ , 444.1597; 444.1582 discovered.

(±)-N-(3-Bromo-2-fluorophenyl)-8-[(morpholin-4-yl)methyl]-7,8-dihydro[1,4]dioxuno[2,3-g]quinazolin4-amine (JGK076).

1H NMR (500 MHz, DMSO-d ₆ ) 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-d ₆ ) δ 157.22, 153.37 (d, JCF=247.3 Hz), 153.13, 148.75, 146.16, 143.28, 130.14 , 128.02 (d, JCF=13.0 Hz), 127.74, 125.45 (d, JCF=4.7 Hz), 112.57, 109.61, 108.55 (d, JCF=19 ,9 Hz), 108.23, 71.41, 66.29, 66.18, 57.97, 53.89;

MSVR (DART): m/z [M-I]' calc. for C ₂₁ H ₁₉ BrFN ₄ O ₃ -, 473.0630; 473.0608 discovered.

- 80 044668 (±)-N-(3-Bromo-2-fluorophenyl)-8-[(dimethylamino)methyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin4-amine ( JGK0 77).

1H NMR (500 MHz, DMSO-d ₆ ) 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-d ₆ ) δ 157.22, 153.38 (d, J _CF = 247.4 Hz), 153.12, 148.78, 146.16, 143.29, 130, 14, 128.02 (d, Jcf=13.1 Hz), 127.75, 125.45 (d, Jcf=4.4 Hz), 112.54, 109.59, 108.55 (d, Jcf= 19.8 Hz), 108.20, 71.76, 66.31, 58.73, 45.92;

MSVR (DART): m/z [M-Н] ^- calc. for C ₁₉ H ₁₇ BrFN ₄ O ₂ ^- , 431.0524; discovered 431.0503.

(±)-N-(3-Bromo-2-fluorophenyl)-8-[(4-methylpiperazin-1-yl)methyl]-7,8-dihydro[1,4]dioxino[2,3g]quinazolin-4 -amine (JGK078).

1H NMR (500 MHz, DMSO-d ₆ ) 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 (ush, 4H), 2.34 (ush, 4H), 2.16 (s, 3H);

¹³ C NMR (126 MHz, DMSO-d ₆ ) δ 157.22, 153.38 (d, J _C F = 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;

MSVR (DART): m/z [MH] ^- calc. for C ₂₂ H ₂₂ BrFN ₅ O ₂ ^- , 486.0946; 486.0928 discovered.

(±)-N-(3-Bromo-2-fluorophenyl)-8-[(pyrrolidin-1-yl)methyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin4-amine (JGK079).

1H NMR (500 MHz, DMSO-d ₆ ) 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-d ₆ ) 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;

MSVR (DART): m/z [M-Н] ^- calc. for C ₂₁ H ₁₉ BrFN ₄ O ₂ ^- , 457.0681; 457.0660 discovered.

(±)-N-(3-Bromo-2-fluorophenyl)-8-[(piperidin-1-yl)mem'yl]-7,8-dighydro[1,4]dioxino[2,3-g]quinazoline4 -amine (JGK080).

1H NMR (500 MHz, DMSO-d ₆ ) 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-d ₆ ) δ 157.21, 153.37 (d, J _CF = 247.1 Hz), 153.11, 148.83, 146.15, 143.32, 130, 12, 128.03 (d, JCF=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;

MSVR (DART): m/z [M-Н] ^- calc. for C ₂₂ H ₂₁ BrFN ₄ O ₂ ^- , 471.0837; 471.0814 discovered.

- 81 044668

N-(3-Bromo- ² -fluorophenyl)( _7,7,8,8-2H4 )-7,8-dihydro[1,4]dioxino[2,3-g]quinazolin-4-amine (JGK081 ).

1H NMR (500 MHz, DMSO-d ₆ ) 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-d ₆ ) 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;

MSHR (DART): m/z [M+H]+ calc. for C ₁₆ H ₈ D ₄ BrFN ₃ O ₂ ⁺ , 380.0342; discovered 380.0327.

(±)-N-(3-Bromo-2-fluorophenyl)-7-[2-(pyrrolidin-1-yl)ethyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazoline4 -amine (JGK084).

¹ H NMR (500 MHz, DMSO-d ₆ ) 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-d ₆ ) 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 =19.8 Hz), 108.44, 71.76, 67.78, 53.63, 51.03, 29.53, 23.16;

MSHR (DART): m/z [M+H]+ calc. for C ₂₂ H ₂₃ BrFN ₄ O ₂ ⁺ , 473.0983; discovered 473.0976.

(±)-N-(3-Bromo-2-fluorophenyl)-7-[2-(piperidin-1-yl)ethyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazoline4 -amine (JGK085).

1H NMR (500 MHz, DMSO-d ₆ ) 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-d ₆ ) δ 157.20, 153.33 (d, J _C F = 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 =20.0 Hz), 108.43, 71.97, 67.80, 54.08, 53.96, 27.69, 25.61, 24.12;

MSHR (DART): m/z [M+H]+ calc. For C ₂₃ H ₂₅ BrFN ₄ O ₂ ⁺ , 487.1139; discovered 487.1137.

(±)-N-(3-Bromo-2-fluorophenyl)-8-[2-(morpholin-4-yl)ethyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazoline4 -amine (JGK086).

1H NMR (500 MHz, DMSO-d ₆ ) 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-d ₆ ) 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 =19.9 Hz), 108.18, 72.30, 67.35, 66.22, 53.53, 53.28, 27.25;

MSHR (DART): m/z [M+H]+ calc. for C ₂₂ H ₂₃ BrFN ₄ O ₃ ⁺ , 489.0932; 489.0926 discovered.

- 82 044668 (±)-N-(3-Bromo-2-fluorophenyl)-8-[2-(dimethylamino)ethyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazoline4- amine (JGK087).

1H NMR (500 MHz, DMSO-d ₆ ) 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, y), 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-d ₆ ) δ 157.20, 153.37 (d, J _C F = 247.6 Hz), 153.08, 148.99, 146.14, 143.29, 130 ,10, 128.07 (d, Jcf=15.6 Hz), 127.72, 125.44 (d, Jcf=4.4 Hz), 112.50, 109.58, 108.54 (d, Jcf =19.7 Hz), 108.13, 72.30, 67.36, 54.43, 45.17, 28.27;

MSHR (DART): m/z [M+H]+ calc. for C ₂₀ H ₂₁ BrFN ₄ O ₂ ⁺ , 447.0826; discovered 447.0818.

(±)-N-(3-Bromo-2-fluorophenyl)-8-[2-(4-methylpiperazin-1-yl)ethyl]-7,8-dihydro[1,4]dioxino[2,3g]quinazoline -4-amine (JGK088).

^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=1.5, 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-d ₆ ) δ 157.20, 153.36 (d, J _CF = 246.9 Hz), 153.08, 148.98, 146.13, 143.28, 130, 09, 128.05 (d, Jcf=11.7 Hz), 127.72, 125.44 (d, Jcf=4.3 Hz), 112.50, 109.58, 108.55 (d, Jcf= 19.8 Hz), 108.13, 72.40, 67.37, 54.78, 53.11, 52.65, 45.76, 27.61;

MSHR (DART): m/z [M+H]+ calc. for C ₂₃ H ₂₆ BrFN ₅ O ₂ ⁺ , 502.1248; discovered 502.1240.

(±)-N-(3-Bromo-2-fluorophenyl)-8-[2-(pyrrolidin-1-yl)ethyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazoline4 -amine (JGK089).

1H NMR (500 MHz, DMSO-d ₆ ) 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, y), 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-d ₆ ) 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, Jcf=4.6 Hz), 112.48, 109.60, 108.55 (d, Jcf=19.8 Hz), 108.15, 72.31, 67.37, 53.57, 50.97, 29.58, 23.14;

MSHR (DART): m/z [M+H]+ calc. for C ₂₂ H ₂₃ BrFN ₄ O ₂ ⁺ , 473.0983; discovered 473.0976.

(±)-N-(3-Bromo-2-fluorophenyl)-8-[2-(piperidin-1-yl)ethyl]-7,8-dihydro[1,4]dioxino[2,3-g]quinazoline4 -amine (JGK090).

1H NMR (500 MHz, DMSO-d ₆ ) 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-d ₆ ) 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 =19.9 Hz), 108, 14, 72.50, 67.40, 54.02, 53.87, 27.70, 25.63, 24.13;

-

Claims (17)

MSHR (DART): m/z [M+H]+ calc. for C ₂₃ H ₂₅ BrFN ₄ O2 ⁺ , 487.1139; discovered 487.1133. N-(3-Bromo-2-fluorophenyl)-8,9-dihydro-7H-[1,4]dioxepino[2,3-g]quinazolin-4-amine (JGK091). ¹ H NMR (500 MHz, CDCl ₃ ) 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 (ush, 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, Jcf=242.7 Hz), 147.83, 128.53 (d, Jcf=10.4 Hz), 127.39, 125.32 (d, Jcf=4.7 Hz), 121.84, 119.15, 111.47, 110.85, 108.62 (d, Jcf= 19.3 Hz), 70.86, 70.51, 31.03; MSVR (DART): m/z [M+H] ⁺ calc. for C _n H _u BrFN3O2 ⁺ , 390.0248; discovered 390.0236. N-(3-Bromo-2-fluorophenyl)-2H-[1,3]dioxolo[4,5-g]quinazolin-8-amine (JGK092). 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 (broad, 1H), 7.14 (s, 1H), 7.11 (td, J=8.2, 1.6 Hz, 1H), 6.17 (s, 2H), the signal of one proton is missing (possibly hidden by the chloroform signal); ¹³ C NMR (126 MHz, CDCl3) δ 156.10, 153.37, 153.22, 150.22 (d, Jcf=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; MSVR (DART): m/z [M+H] ⁺ calc. for C15H _w BrFN3O2 ⁺ , 361.9935; discovered 361.9925. Example 19 Metabolism studies of exemplary JGK series compounds. Exemplary compounds (10 mM) were incubated in human, dog, mouse or rat liver microsomes (1 mg/ml) for up to 90 minutes at 37°C. The reactions were stopped by adding acetonitrile. In parallel, control (compound-free) studies of microsomes were carried out. LCMS studies were carried out on a Waters Xevo G2 QTof instrument equipped with a Luna Omega Polar C18 column, 1.6 m, 2.1x30 mm. The structures of exemplary metabolites are depicted in FIG. 47. Modification Human (%) Dog (%) Mouse (%) Rat (%) 1. Original 67.0 3.5 59.9 70.2 2. Hydroxylation 6.0 0.0 oh oh 4.8 3. N-demethylation 13.7 0.9 5.4 8.2 4. Hydroxylation 4.2 61.9 22.0 21.9 5. Hydroxylation 0.7 oh oh oh oh 0.6 6. N-dealkylation 6.5 oh oh 1.3 2.7 Incorporation by reference. All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent were specifically and separately identified for inclusion by reference. In the event of a conflict, this application, including any definitions herein, will control. Equivalents. Although specific embodiments of the present invention have been discussed, the above description is illustrative and not limiting. Many variations of the invention will become apparent to those skilled in the art upon reading the present specification and the following claims. The entire scope of the invention is to be determined by reference to the claims together with the full scope of their equivalents and the description together with such variations. CLAIM 1. A connection selected from the group consisting of - 84 044668 - 85 044668 or a pharmaceutically acceptable salt thereof. 2. The connection according to claim 1, which is 3. The connection according to claim 1, which is 4. The connection according to claim 1, which is 5. The connection according to claim 1, which is 6. The connection according to claim 1, which is 7. The connection according to claim 1, which is 8. The connection according to claim 1, which is - 86 044668 9. The connection according to claim 1, which is 10. Connection by 11. Connection by claim 1, which is claim 1, which is 12. Connection by 13. Connection by 14. Connection by 15. A compound according to claim 1, which is a pharmaceutically acceptable salt of the following compound: 16. A compound according to claim 1, which is a pharmaceutically acceptable salt of the following compound: - 87 044668 17. Union connection: By claim 1, which is a pharmaceutically acceptable salt of the following co18. Union connection: By claim 1, which is a pharmaceutically acceptable salt of the following co19. Union connection: By claim 1, which is a pharmaceutically acceptable salt of the following co20. Union connection: By claim 1, which is a pharmaceutically acceptable salt of the following co21. Union connection: By claim 1, which is a pharmaceutically acceptable salt of the following co22. Union connection: By claim 1, which is a pharmaceutically acceptable salt of the following co23. Union connection: 24. Union connection: by claim 1, which is a pharmaceutically acceptable claim 1, which is a pharmaceutically acceptable salt salt next next co-