Use of quinazoline-based tyrosine kinase inhibitors for the treatment of cancers with NRG1 fusions

Abstract

The present invention provides a method for selecting cancer patients for treatment with a quinazoline-based tyrosine kinase inhibitor, either alone or in combination with an anti-HER2 / HER3 antibody, and a method for treating the selected cancer patients.Cancer patients are selected for treatment if their cancer has NRG1 fusion.The selected patients are then treated with a quinazoline-based tyrosine kinase inhibitor, either alone or in combination with an anti-HER2 / HER3 antibody.

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 967,282, filed January 29, 2020, the entire contents of which are incorporated herein by reference.

[0002] 1. Field The present invention relates generally to the fields of medicine and oncology. More particularly, the present invention relates to methods for selecting cancer patients for treatment with a quinazoline-based tyrosine kinase inhibitor (TKI) or a combination of a quinazoline-based TKI and an anti-HER2 / HER3 antibody, and methods for treating such selected cancer patients. [Background technology]

[0003] 2. Description of Related Technology NRG1 fusions occur in 0.3% of non-small cell lung cancers (NSCLCs) and have been observed in several other cancer types, including gallbladder (0.5%), breast (0.2%), ovarian (0.4%), and colorectal (0.1%) cancers (Jonna et al., 2019). Common NRG1 fusion partners are CD74 (29% of NRG1 fusions), ATP1B1 (10% of NRG1 fusions), and SDC4 (7% of NRG1 fusions) (Jonna et al., 2019). NRG1 binds to the HER3 receptor, leading to preferential heterodimerization with HER2 (Shin et al., 2018; Jung et al., 2015; Fernandez-Cuesta et al., 2014), one of the most potent forms of ERBB family signaling (Holbro et al., 2003). Previous reports have shown that targeting the HER2 / HER3 signaling pathway with a single agent can be effective in inhibiting NRG1 fusion-driven ErbB signaling (Shin et al., 2018; Fernandez-Cuesta et al., 2014; Drilon et al., 2018). However, there are no approved targeted therapies for patients with NRG1 fusions. Summary of the Invention

[0004] overview In one aspect, there is provided a method of treating a patient having cancer, comprising: (a) determining or having determined whether the patient's cancer harbors an NRG1 fusion; (b) selecting or having selected the patient for treatment with a quinazoline-based tyrosine kinase inhibitor (TKI) if the patient's cancer harbors an NRG1 fusion; and (c) administering or having administered to the selected patient a therapeutically effective amount of a quinazoline-based TKI. A method is provided herein, comprising: In some aspects, step (a) comprises: (i) obtaining or having obtained a biological sample from a patient; and (ii) performing or having performed an assay on the biological sample to determine that the patient's cancer has an NRG1 fusion.

[0005] In one aspect, provided herein is a method of treating a patient with cancer, the method comprising administering a therapeutically effective amount of a quinazoline-based TKI to the patient, wherein the cancer has an NRG1 fusion. In one aspect, provided herein is a composition comprising a therapeutically effective amount of a quinazoline-based TKI for use in treating a patient with cancer, wherein the patient's cancer has an NRG1 fusion.

[0006] In one aspect, there is provided a method of selecting a patient having cancer for treatment with a quinazoline-based TKI, comprising: (a) determining or having determined whether the patient's cancer harbors an NRG1 fusion; (b) selecting or having selected the patient for treatment with a quinazoline-based TKI if the patient's cancer harbors an NRG1 fusion. A method is provided herein, comprising: In some aspects, step (a) comprises: (i) obtaining or having obtained a biological sample from a patient; and (ii) performing or having performed an assay on the biological sample to determine that the patient's cancer has an NRG1 fusion. In some aspects, the method comprises: (c) administering or having administered to the selected patient a therapeutically effective amount of a quinazoline-based TKI. Further includes:

[0007] In one aspect, there is provided a method of treating a patient having cancer, comprising: (a) determining or having determined whether the patient's cancer harbors an NRG1 fusion; (b) selecting or having selected the patient for treatment with a quinazoline-based TKI and an anti-HER2 / HER3 antibody if the patient's cancer harbors an NRG1 fusion; and (c) administering or having administered to the selected patient a therapeutically effective amount of a combined quinazoline-based TKI and an anti-HER2 / HER3 antibody. A method is provided herein, comprising: In some aspects, step (a) comprises: (i) obtaining or having obtained a biological sample from a patient; and (ii) performing or having performed an assay on the biological sample to determine that the patient's cancer has an NRG1 fusion.

[0008] In one aspect, provided herein is a method of treating a patient having cancer, the method comprising administering to the patient a therapeutically effective amount of a quinazoline-based TKI and an anti-HER2 / HER3 antibody in combination, wherein the cancer has an NRG1 fusion. In one aspect, provided herein is a composition comprising a therapeutically effective amount of a quinazoline-based TKI and an anti-HER2 / HER3 antibody for use in treating a patient's cancer, wherein the patient's cancer has an NRG1 fusion.

[0009] In one embodiment, there is provided a method of selecting a patient having cancer for treatment with a quinazoline-based TKI and an anti-HER2 / HER3 antibody, comprising: (a) determining or having determined whether the patient's cancer harbors an NRG1 fusion; (b) selecting or having selected the patient for treatment with a quinazoline-based TKI and an anti-HER2 / HER3 antibody if the patient's cancer harbors an NRG1 fusion. A method is provided herein, comprising: In some aspects, step (a) comprises: (i) obtaining or having obtained a biological sample from a patient; and (ii) performing or having performed an assay on the biological sample to determine that the patient's cancer has an NRG1 fusion. In some aspects, the method comprises: (c) administering or having administered to the selected patient a therapeutically effective amount of a combined quinazoline-based TKI and an anti-HER2 / HER3 antibody. Further includes:

[0010] In some aspects of the embodiment, the NRG1 fusion is an NRG1-DOC4 fusion, an NRG1-VAMP2 fusion, an NRG1-CLU fusion, an NRG1-SLC3A2 fusion, an NRG1-CD74 fusion, an NRG1-ATP1B1 fusion, or an NRG1-SDC4 fusion.

[0011] In some aspects of the embodiment, the quinazoline-based TKI is IACS-015285, IACS-015296, IACS-070979, IACS-015293, IACS-070982, IACS-070863, IACS-070864, ​​IACS-070871, IACS-070980, IACS-070968, IACS-070709, IACS-070989, or IACS-052336.

[0012] In some aspects of the embodiment, the method further comprises administering to the patient an anti-HER2 / HER3 antibody. In some aspects, the anti-HER2 / HER3 antibody comprises trastuzumab, pertuzumab, or T-DM1.

[0013] In some aspects of the embodiment, the method further comprises administering to the patient an additional anti-cancer therapy, hi some aspects, the additional anti-cancer therapy is surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, toxin therapy, immunotherapy, or cytokine therapy.

[0014] In some aspects of the embodiment, the cancer is breast cancer, lung cancer, colorectal cancer, neuroblastoma, pancreatic cancer, brain cancer, stomach cancer, skin cancer, testicular cancer, prostate cancer, ovarian cancer, liver cancer, esophageal cancer, cervical cancer, head and neck cancer, melanoma, or glioblastoma. In some aspects, the cancer is breast cancer or lung cancer.

[0015] In some aspects of the embodiment, the patient has previously received at least one round of anti-cancer therapy. In some aspects of the embodiment, the method further comprises reporting the presence of an NRG1 fusion in the patient's cancer. In some aspects, the reporting comprises generating a paper or electronic report. In some aspects, the method further comprises submitting the report to the subject, a physician, a hospital, or an insurance company.

[0016] As used herein, "essentially free" in terms of specific components means that none of the specific components are intentionally incorporated into the composition, and / or only exist as contaminants or only exist in trace amounts.The total amount of the specific components resulting from any unintentional incorporation of the composition is therefore much less than 0.05%, preferably less than 0.01%.The most preferred composition is one in which the amount of the specific components cannot be detected by standard analytical methods.

[0017] As used herein, "a" or "an" may mean one or more. When used in the claims, the words "a" or "an," when used in conjunction with the word "comprising," may mean one or more than one.

[0018] The use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or unless the alternatives are mutually exclusive, although the present disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" may mean at least a second or more.

[0019] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, the variation that exists among study subjects, or a value within 10% of the stated value.

[0020] [The present invention 1001] 1. A method of treating a patient with cancer, comprising: (a) determining or having determined whether the patient's cancer harbors an NRG1 fusion; (b) selecting or having selected the patient for treatment with a quinazoline-based tyrosine kinase inhibitor (TKI) if the patient's cancer harbors an NRG1 fusion; and (c) administering or having administered to said selected patient a therapeutically effective amount of said quinazoline-based TKI. A method comprising: [The present invention 1002] 1. A method of treating a patient with cancer, comprising: administering to said patient a therapeutically effective amount of a quinazoline-based TKI. wherein the cancer has an NRG1 fusion. [The present invention 1003] The method of claim 1001 or 1002, wherein the NRG1 fusion is an NRG1-DOC4 fusion, an NRG1-VAMP2 fusion, an NRG1-CLU fusion, an NRG1-SLC3A2 fusion, an NRG1-CD74 fusion, an NRG1-ATP1B1 fusion, or an NRG1-SDC4 fusion. [The present invention 1004] The method of any one of claims 1001 to 1003, wherein the quinazoline-based TKI is IACS-015285, IACS-015296, IACS-070979, IACS-015293, IACS-070982, IACS-070863, IACS-070864, ​​IACS-070871, IACS-070980, IACS-070968, IACS-070709, IACS-070989, or IACS-052336. [The present invention 1005] administering an anti-HER2 / HER3 antibody to said patient. The method of any one of 1001 to 1004 of the present invention further comprises: [The present invention 1006] 1005. The method of claim 10, wherein said anti-HER2 / HER3 antibody comprises trastuzumab, pertuzumab, or T-DM1. [The present invention 1007] Step (a) (i) obtaining or having obtained a biological sample from said patient; and (ii) performing or having performed an assay on the biological sample to determine that the patient's cancer harbors an NRG1 fusion. Any of the methods 1001 to 1006 of the present invention, comprising: [The present invention 1008] administering to said patient an additional anti-cancer therapy. The method of any one of claims 1001 to 1007, further comprising: [The present invention 1009] 1008. The method of claim 8, wherein said further anti-cancer therapy is surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, toxin therapy, immunotherapy, or cytokine therapy. [The present invention 1010] 1009. The method of any one of claims 1001 to 1009, wherein the cancer is breast cancer, lung cancer, colorectal cancer, neuroblastoma, pancreatic cancer, brain tumor, stomach cancer, skin cancer, testicular cancer, prostate cancer, ovarian cancer, liver cancer, esophageal cancer, cervical cancer, head and neck cancer, melanoma, or glioblastoma. [The present invention 1011] The method of any one of claims 1001 to 1010, wherein the cancer is breast cancer or lung cancer. [The present invention 1012] 1012. The method of any of claims 1001 to 1011, wherein said patient has previously undergone at least one round of anti-cancer therapy. [The present invention 1013] reporting the presence of an NRG1 fusion in the patient's cancer. Any of the methods of 1001 to 1012 of the present invention, further comprising: [The present invention 1014] The method of claim 1013, wherein the reporting step comprises generating a paper or electronic report. [The present invention 1015] submitting said report to said subject, physician, hospital, or insurance company. The method of the present invention 1013 or 1014 further comprising: [The present invention 1016] 1. A method of selecting a patient having cancer for treatment with a quinazoline-based TKI, comprising: (a) determining or having determined whether the patient's cancer harbors an NRG1 fusion; (b) selecting or having selected the patient for treatment with a quinazoline-based TKI if the patient's cancer harbors an NRG1 fusion. A method comprising: [The present invention 1017] Step (a) (i) obtaining or having obtained a biological sample from said patient; and (ii) performing or having performed an assay on the biological sample to determine that the patient's cancer harbors an NRG1 fusion. The method of the present invention 1016, comprising: [The present invention 1018] (c) administering or having administered to said selected patient a therapeutically effective amount of a quinazoline-based TKI. The method of claim 1016 or 1017, further comprising: [The present invention 1019] Any of the methods of claims 1016 to 1018, wherein the NRG1 fusion is an NRG1-DOC4 fusion, an NRG1-VAMP2 fusion, an NRG1-CLU fusion, an NRG1-SLC3A2 fusion, an NRG1-CD74 fusion, an NRG1-ATP1B1 fusion, or an NRG1-SDC4 fusion. [The present invention 1020] Any of the methods of claims 1016 to 1019, wherein the quinazoline-based TKI is IACS-015285, IACS-015296, IACS-070979, IACS-015293, IACS-070982, IACS-070863, IACS-070864, ​​IACS-070871, IACS-070980, IACS-070968, IACS-070709, IACS-070989, or IACS-052336. [The present invention 1021] administering an anti-HER2 / HER3 antibody to said patient. The method of invention 1018 or 1020, further comprising: [The present invention 1022] 1021. The method of claim 1021, wherein said anti-HER2 / HER3 antibody comprises trastuzumab, pertuzumab, or T-DM1. [The present invention 1023] administering to said patient an additional anti-cancer therapy. Any of the methods of inventions 1018 to 1022, further comprising: [The present invention 1024] 1024. The method of claim 1023, wherein said further anti-cancer therapy is surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, toxin therapy, immunotherapy, or cytokine therapy. [The present invention 1025] Any of the methods of claims 1016 to 1024, wherein the cancer is breast cancer, lung cancer, colorectal cancer, neuroblastoma, pancreatic cancer, brain tumor, stomach cancer, skin cancer, testicular cancer, prostate cancer, ovarian cancer, liver cancer, esophageal cancer, cervical cancer, head and neck cancer, melanoma, or glioblastoma. [The present invention 1026] Any of the methods of claims 1016 to 1025, wherein the cancer is breast cancer or lung cancer. [The present invention 1027] 1027. The method of any of claims 1016 to 1026, wherein said patient has previously undergone at least one round of anti-cancer therapy. [The present invention 1028] reporting the presence of an NRG1 fusion in the patient's cancer. Any of the methods of claims 1018 to 1027, further comprising: [The present invention 1029] The method of claim 1028, wherein the reporting step comprises generating a written or electronic report. [The present invention 1030] submitting said report to said subject, physician, hospital, or insurance company. The method of claim 1028 or 1029, further comprising: [The present invention 1031] 1. A method of treating a patient with cancer, comprising: (a) determining or having determined whether the patient's cancer harbors an NRG1 fusion; (b) selecting or having selected the patient for treatment with a quinazoline-based TKI and an anti-HER2 / HER3 antibody if the patient's cancer harbors an NRG1 fusion; and (c) administering or having administered to said selected patient a therapeutically effective amount of a combined quinazoline-based TKI and an anti-HER2 / HER3 antibody. A method comprising: [The present invention 1032] 1. A method of treating a patient with cancer, comprising: administering to the patient a combined therapeutically effective amount of a quinazoline-based TKI and an anti-HER2 / HER3 antibody. wherein the cancer has an NRG1 fusion. [The present invention 1033] The method of any one of claims 1031 to 1032, wherein the NRG1 fusion is an NRG1-DOC4 fusion, an NRG1-VAMP2 fusion, an NRG1-CLU fusion, an NRG1-SLC3A2 fusion, an NRG1-CD74 fusion, an NRG1-ATP1B1 fusion, or an NRG1-SDC4 fusion. [The present invention 1034] Any of the methods of present inventions 1031 to 1033, wherein the quinazoline-based TKI is IACS-015285, IACS-015296, IACS-070979, IACS-015293, IACS-070982, IACS-070863, IACS-070864, ​​IACS-070871, IACS-070980, IACS-070968, IACS-070709, IACS-070989, or IACS-052336. [This invention 1035] The method of any one of claims 1031 to 1034, wherein the anti-HER2 / HER3 antibody comprises trastuzumab, pertuzumab, or T-DM1. [The present invention 1036] Step (a) (i) obtaining or having obtained a biological sample from said patient; and (ii) performing or having performed an assay on the biological sample to determine that the patient's cancer harbors an NRG1 fusion. Any of the methods of the present inventions 1031 to 1035, comprising: [This invention 1037] administering to said patient an additional anti-cancer therapy. Any of the methods of claims 1031 to 1036, further comprising: [The present invention 1038] 1038. The method of claim 1037, wherein said further anti-cancer therapy is surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, toxin therapy, immunotherapy, or cytokine therapy. [This invention 1039] 9. The method of any one of claims 1031 to 1038, wherein the cancer is breast cancer, lung cancer, colorectal cancer, neuroblastoma, pancreatic cancer, brain tumor, stomach cancer, skin cancer, testicular cancer, prostate cancer, ovarian cancer, liver cancer, esophageal cancer, cervical cancer, head and neck cancer, melanoma, or glioblastoma. [The present invention 1040] The method of any one of claims 1031 to 1039, wherein the cancer is breast cancer or lung cancer. [The present invention 1041] 1041. The method of any of claims 1031 to 1040, wherein said patient has previously undergone at least one round of anti-cancer therapy. [The present invention 1042] reporting the presence of an NRG1 fusion in the patient's cancer. Any of the methods of inventions 1031 to 1041, further comprising: [This invention 1043] The method of claim 1042, wherein the reporting step comprises generating a written or electronic report. [This invention 1044] submitting said report to said subject, physician, hospital, or insurance company. The method of any one of claims 1042 to 1043, further comprising: [This invention 1045] 1. A method of selecting a patient having cancer for treatment with a quinazoline-based TKI and an anti-HER2 / HER3 antibody, comprising: (a) determining or having determined whether the patient's cancer harbors an NRG1 fusion; (b) selecting or having selected the patient for treatment with a quinazoline-based TKI and an anti-HER2 / HER3 antibody if the patient's cancer harbors an NRG1 fusion. A method comprising: [The present invention 1046] Step (a) (i) obtaining or having obtained a biological sample from said patient; and (ii) performing or having performed an assay on the biological sample to determine that the patient's cancer harbors an NRG1 fusion. The method of the present invention 1045, comprising: [This invention 1047] (c) administering or having administered to said selected patient a therapeutically effective amount of a combined quinazoline-based TKI and an anti-HER2 / HER3 antibody. The method of any one of claims 1045 to 1046, further comprising: [This invention 1048] Any of the methods of claims 1045 to 1047, wherein the NRG1 fusion is an NRG1-DOC4 fusion, an NRG1-VAMP2 fusion, an NRG1-CLU fusion, an NRG1-SLC3A2 fusion, an NRG1-CD74 fusion, an NRG1-ATP1B1 fusion, or an NRG1-SDC4 fusion. [This invention 1049] Any of the methods of claims 1045 to 1048, wherein the quinazoline-based TKI is IACS-015285, IACS-015296, IACS-070979, IACS-015293, IACS-070982, IACS-070863, IACS-070864, ​​IACS-070871, IACS-070980, IACS-070968, IACS-070709, IACS-070989, or IACS-052336. [The present invention 1050] 1049. The method of any of claims 1045 to 1049, wherein the anti-HER2 / HER3 antibody comprises trastuzumab, pertuzumab, or T-DM1. [This invention 1051] administering to said patient an additional anti-cancer therapy. Any of the methods of claims 1047 to 1050, further comprising: [This invention 1052] 1052. The method of claim 1051, wherein said further anti-cancer therapy is surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, toxin therapy, immunotherapy, or cytokine therapy. [This invention 1053] 1052. The method of any one of claims 1045 to 1052, wherein the cancer is breast cancer, lung cancer, colorectal cancer, neuroblastoma, pancreatic cancer, brain tumor, stomach cancer, skin cancer, testicular cancer, prostate cancer, ovarian cancer, liver cancer, esophageal cancer, cervical cancer, head and neck cancer, melanoma, or glioblastoma. [This invention 1054] The method of any one of claims 1045 to 1053, wherein the cancer is breast cancer or lung cancer. [This invention 1055] 1054. The method of any of claims 1045 to 1054, wherein said patient has previously undergone at least one round of anti-cancer therapy. [The present invention 1056] reporting the presence of an NRG1 fusion in the patient's cancer. Any of the methods of claims 1047 to 1055, further comprising: [This invention 1057] The method of claim 1056, wherein the reporting step comprises generating a written or electronic report. [This invention 1058] submitting said report to said subject, physician, hospital, or insurance company. The method of any one of claims 1056 to 1057, further comprising: Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0021] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Figure 1] Figure 1A: Representative dose-response curves for MDA175-VII (NRG1-DOC4 fusion) treated for 72 hours with the indicated IACS novel quinazoline-based TKIs. Cell viability was determined by Cell Titer Glo assay. Figure 1B: Bar graph of mean ± SEM IC50 values ​​for the MDA17-VII cell line treated for 72 hours with the indicated inhibitors. [Figure 2]Bar graph of mutant / WT EGFR ratio between MDA175-VII cells and Ba / F3 cells expressing WT EGFR. [Figure 3] Figure 3A: Representative dose-response curves for MDA175-VII (NRG1-DOC4 fusion) treated for 72 hours with the indicated anti-HER2 therapy, with or without low-dose treatment of IACS inhibitors. Cell viability was determined by Cell Titer Glo assay. Figure 3B: Bar graph of mean ± SEM IC50 values ​​for MDA17-VII cell lines treated for 72 hours with the indicated inhibitors. Combinations with the anti-HER2 antibody trastuzumab were not included in the bar graph because IC50 values ​​could not be calculated. DETAILED DESCRIPTION OF THE INVENTION

[0022] Detailed Description Provided herein is a method for treating cancer patients with NRG1 fusion.In particular, this method comprises administering quinazoline-based TKI or the combination of quinazoline-based TKI and anti-HER2 / HER3 antibody to cancer patients who are identified as having NRG1 fusion.Furthermore, this method comprises determining whether patient's cancer has NRG1 fusion, thereby identifying and selecting cancer patients who may benefit from the administration of quinazoline-based TKI or the combination of quinazoline-based TKI and anti-HER2 / HER3 antibody.

[0023] I.NRG1 fusion An NRG1 fusion gene comprises at least a portion of the NRG1 gene fused to a sequence from a different chromosomal location. "At least a portion" indicates that the entire NRG1 gene can be present in the fusion, or a portion thereof. The fusion can have coding sequences for at least exons 6, 7, and 8 of NRG1. Another way to define the NRG1 portion in an NRG1 fusion gene is that it contains the EGF-like domain of NRG1. The EGF-like domain is encoded at the 3' end of the gene and is necessary for binding to ErbB-3. An NRG1 fusion retains an in-frame coding region for the EGF-like domain. A portion of the NRG1 gene can be fused to a sequence from a different chromosomal location, such that the sequence is located 5' or 3' to the portion of the NRG1 gene.

[0024] Preferably, the 3' end of the NRG1 gene can be fused to a sequence from a different chromosomal location. In particular, the NRG1 fusion gene is a fusion of the 3' end of the NRG1 gene with the 5' sequence of one of the following genes: DOC4 (teneurin transmembrane protein 4 (TENM4); protein Odd Oz / Ten-M homolog 4; tenascin-M4; Ten-M4; Ten-4; ODZ4; TNM4; Odz, Odd Oz / Ten-M homolog 4 (Drosophila); Odz, Odd Oz^en-M homolog 4; teneurin-4; KIAA1302; Doc4; ETM5; HGNC:29945; Entrez Gene: 26011; Ensembl: ENSG00000149256; OMIM: 610084; and UniProtKB: Q6N022); CD74 (CD74 molecule; CD74 antigen (major histocompatibility complex invariant polypeptide, class II antigen-associated); CD74 molecule, major histocompatibility complex, class II invariant chain; HLA-DR antigen-associated invariant chain; class II antigen-gamma chain; la-associated invariant chain; MHC HLA-DR gamma chain; HLA-DR-gamma; DHLAG; P33; HLA class II histocompatibility antigen gamma chain; la antigen-associated invariant chain; la-gamma; HLADG; HGNC: 1697; Entrez Gene:972; Ensembl:ENSG00000019582; OMIM:142790, and UniProtKB:P04233); TNFRSF10B (TNF receptor superfamily member 10b; tumor necrosis factor receptor superfamily, member 10b; TNF-related apoptosis-inducing ligand receptor 2; death receptor 5; TRAIL-R2; TRAILR2; KILLER; TRICK2; ZTNFR9; DR5; p53-regulated DNA damage-inducible cell death receptor (killer); tumor necrosis factor receptor superfamily member 10B; tumor necrosis factor receptor-like protein ZTNFR9; death domain-containing receptor for TRAIL / Apo-2L; apoptosis-inducing protein TRICK2A / 2B; apoptosis-inducing receptor TRAIL-R2; cytotoxic TRAIL receptor-2; Fas-like protein; TRAIL receptor 2; CD262 antigen; KILLER / DR5; TRICK2A;TRICK2B;TRICKB;CD262;HGNC:11905;Entrez Gene:8795; Ensembl:ENSG00000120889; OMIM:603612; and UniProtKB:014763); CLU (clusterin; testosterone-suppressed prostate message 2; apolipoprotein J; complement-related protein SP-40,40; complement cytolysis inhibitor; complement lysis inhibitor; sulfated glycoprotein 2; Ku70-binding protein 1; NA1 / NA2; TRPM-2; APO-J; APOJ; KUB1; CLI; clusterin (complement lysis inhibitor, SP-40,40, sulfated glycoprotein 2, testosterone-suppressed prostate message 2, apolipoprotein J); senescence-associated gene 4 protein; senescence-associated protein 4; SGP-2; SP-40; TRPM2; AAG4; CLU1; CLU2; SGP2; HGNC:2095; Entrez Gene:1191; Ensembl:ENSG00000120885; OMIM:185430; and UniProtKB:P10909); VAMP2 (vesicle-associated membrane protein 2; synaptobrevin 2; SYB2; vesicle-associated membrane protein 2; synaptobrevin-2; HGNC:12643; Entrez Gene:6844; Ensembl:ENSG00000220205; OMIM:185881; and UniProtKB:P63027); SLC3A2 (solute carrier family 3 member 2; lymphocyte activation antigen 4F2 large subunit; solute carrier family 3 (activator of dibasic and neutral amino acid transport), member 2; monoclonal antibody 4F2, TRA1.10, TROP4, and T4 antigen defined by 3; solute transporter family 3 (amino acid transporter heavy chain), member 2; 4F2 cell surface antigen heavy chain; CD98 heavy chain; 4F2HC; MDU1; antigen defined by monoclonal antibody 4F2, heavy chain; antigen defined by monoclonal antibody 4F2; 4F2 heavy chain antigen; 4F2 heavy chain; CD98 antigen; CD98HC; 4T2HC; NACAE; CD98; 4F2; HGNC: 11026; Entrez Gene: 6520; Ensembl: ENSG00000168003; OMIM: 158070;and UniProtKB:P08195); RBPMS (RNA Binding Protein With Multiple Splicing; Heart and RRM Expressed Sequence; HERMES; RNA-Binding Protein With Multiple Splicing; RBP-MS; HGNC:19097; Entrez Gene:11030; Ensembl:ENSG00000157110; OMIM:601558; and UniProtKB:Q93062); WRN (Werner Syndrome RecQ-like Helicase; DNA Helicase, RecQ-like Type 3; RecQ Protein-Like 2; Exonuclease WRN; RECQL2; RECQ3; Werner Syndrome ATP-Dependent Helicase; Werner Syndrome, RecQ Helicase-Like; Werner Syndrome; EC 3.6.4.12; EC 3.1.-.-; EC 3.6.1; RECQL3; HGNC:12791; Entrez Gene:7486; Ensembl:ENSG00000165392; OMIM:604611 and UniProtKB:Q14191); SDC4 (also known as syndecan 4 (amphiglycan, ryudocan); syndecan proteoglycan 4; ryudocan core protein; amphiglycan; SYND4; ryudocan amphiglycan; syndecan-4; HGNC:10661; Entrez Gene:6385; Ensembl:ENSG00000124145; OMIM:600017; and UniProtKB:P31431); KIF13B; SLECA2; PDE7A; ATP1B1; CDK1; BMPRIB; MCPH1; and RAB2IL1.

[0025] Certain aspects of the present disclosure relate to determining whether a subject has an NRG1 fusion. Detection methods are known in the art, including PCR analysis, nucleic acid sequencing, fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH), and comparative genomic hybridization (CGH).

[0026] Samples suitable for use in the methods described herein include genetic material, such as genomic DNA (gDNA). Genomic DNA is typically extracted from biological samples such as blood or mucosal scrapings from the lining of the oral cavity, but may also be extracted from other biological samples, including urine, tumors, or expectorated sputum. The sample itself is typically considered to include nucleated cells (e.g., blood or oral cells) or tissues removed from a subject, including tumor tissue. Methods and reagents for obtaining, processing, and analyzing samples are known in the art. In some embodiments, the sample is obtained with the assistance of a healthcare provider, for example, to draw blood or obtain a tumor biopsy. In some embodiments, the sample is obtained without the assistance of a healthcare provider, for example, when the sample is obtained non-invasively, such as a sample containing oral cells obtained using a cheek swab or brush, or a mouthwash sample.

[0027] In particular, the patient sample can be any tissue or fluid of the body that contains nucleic acid from the cancer of interest.In certain embodiments, the sample can be a blood sample containing circulating tumor cells or cell-free DNA.In other embodiments, the sample can be tissue such as tumor tissue.Tumor tissue can be fresh frozen or formalin-fixed, paraffin-embedded (FFPE).

[0028] In some cases, biological samples can be processed for DNA isolation. For example, DNA in cell or tissue samples can be separated from other components of the sample. Cells can be harvested from biological samples using standard techniques known in the art. For example, cells can be harvested by centrifuging a cell sample and resuspending the pelleted cells. The cells can be resuspended in a buffered solution such as phosphate-buffered saline (PBS). After centrifuging the cell suspension to obtain a cell pellet, the cells can be lysed to extract DNA, such as gDNA. The sample can be concentrated and / or purified to isolate DNA. All samples obtained from a subject, including those that undergo any type of further processing, are considered to be obtained from the subject. Genomic DNA can be extracted from biological samples using conventional methods, including, for example, phenol extraction. Alternatively, genomic DNA can be extracted using a kit such as the QIAamp® Tissue Kit (Qiagen, Chatsworth, Calif.) or the Wizard® Genomic DNA Purification Kit (Promega).

[0029] If desired, nucleic acid amplification can be achieved using methods known in the art, such as PCR. For example, a sample (e.g., a sample containing genomic DNA) is obtained from a subject. Then, as described herein, the DNA in the sample is examined to determine the identity of NRG1 fusion. NRG1 fusion can be detected by any method described herein, for example, by sequencing, or by hybridization of genes, RNA, or cDNA in genomic DNA to a nucleic acid probe, for example, a DNA probe (including cDNA and oligonucleotide probe) or an RNA probe. The nucleic acid probe can be designed to specifically or preferentially hybridize with a specific NRG1 fusion.

[0030] A set of probes typically refers to a set of primers, usually a primer pair, and / or a detectably labeled probe, used to detect a target genetic variation (e.g., an NRG1 fusion) used in the actionable treatment recommendations of the present disclosure. The primer pair is used in an amplification reaction to define an amplification product corresponding to the NRG1 fusion. The set of amplification products is detected with a matching set of probes. In an exemplary embodiment, the method uses a TaqMan PCR probe used to detect a set of target genetic variations, e.g., an NRG1 fusion. (商標) (Roche Molecular Systems, Pleasanton, Calif.) assays can be used. In one embodiment, the set of probes is a set of primers used to generate amplification products that are detected in a nucleic acid sequencing reaction, e.g., a next-generation sequencing reaction. In these embodiments, for example, AmpliSEQ (商標) (Life Technologies / Ion Torrent, Carlsbad, Calif.) or TruSEQ (商標) (Illumina, San Diego, Calif.) technology can be used.

[0031] Analysis of nucleic acid markers can be performed using techniques known in the art, including, but not limited to, sequence analysis and electrophoretic analysis. Non-limiting examples of sequence analysis include Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing, solid-phase sequencing, sequencing using mass spectrometry such as matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS), and hybridization sequencing. Non-limiting examples of electrophoretic analysis include slab gel electrophoresis, such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis. Additionally, next-generation sequencing methods can be performed using commercially available kits and equipment from companies such as Life Technologies / Ion Torrent PGM or Proton, Illumina HiSEQ or MiSEQ, and Roche / 454 next-generation sequencing systems.

[0032] Other methods of nucleic acid analysis include direct manual sequencing (U.S. Pat. No. 5,288,644); automated fluorescent sequencing; single-strand conformation polymorphism analysis (SSCP); constant denaturing gel electrophoresis (CDGE); two-dimensional gel electrophoresis (2DGE or TDGE); conformational sensitive gel electrophoresis (CSGE); denaturing gradient gel electrophoresis (DGGE); denaturing high-performance liquid chromatography (DHPLC); infrared matrix-assisted laser desorption / ionization (IR-MALDI) mass spectrometry; mobility shift analysis; restriction enzyme analysis; quantitative real-time PCR; heteroduplex analysis; chemical mismatch cleavage (CMC); RNase protection assay; the use of polypeptides that recognize nucleotide mismatches, such as the E. coli mutS protein; allele-specific PCR, and combinations of such methods. See, for example, U.S. Patent Application Publication No. 2004 / 0014095, the entire contents of which are incorporated herein by reference.

[0033] In one example, a method for identifying an NRG1 fusion in a sample comprises contacting nucleic acid from the sample with a nucleic acid probe capable of specifically hybridizing to a nucleic acid encoding an NRG1 fusion, and detecting said hybridization. In certain embodiments, the probe is radioisotope-conjugated ( 3 H, 32 P, or 33 The probe is detectably labeled with, for example, phospholipids (P), a fluorescent agent (rhodamine or fluorescein), or a chromogenic agent. In certain embodiments, the probe is an antisense oligomer, such as a PNA, morpholino-phosphoramidate, LNA, or 2'-alkoxyalkoxy. The probe can be about 8 to about 100 nucleotides, or about 10 to about 75, or about 15 to about 50, or about 20 to about 30. In another aspect, the probe of the present disclosure is provided in a kit for identifying NRG1 fusions in a sample, the kit comprising an oligonucleotide that specifically hybridizes to a particular NRG1 fusion. The kit can further comprise instructions for treating a patient having a tumor containing an NRG1 fusion with a quinazoline-based TKI or a combination of a quinazoline-based TKI and an anti-HER2 / HER3 antibody based on the results of a hybridization test using the kit.

[0034] I. Quinazoline-Based Tyrosine Kinase Inhibitors Previous reports have also disclosed the design of novel quinazoline-based TKIs for inhibiting ErbB family members; however, these inhibitors have not been explored for use in inhibiting NRG fusion cell lines. Exemplary quinazoline-based TKIs can be found, for example, in USSN 62 / 838,702 and USSN 62 / 838,696, each of which is incorporated herein by reference in its entirety.

[0035] Quinazoline-based tyrosine kinase inhibitors have the structural formula (I): TIFF0007734139000001.tif28128, or a salt thereof, wherein: A 1 is C(R 1 ) and N; A 2 is C(R 2 ) and N; A 3 is C(R 3 ) and N; Ar 1 is selected from aryl and heteroaryl, any of which may be selected from one or two R 4 groups, any of which may be substituted with one, two, or three R 5 optionally substituted with a group; R 1 Halo, -CN, -OR 6 , -NR 7a R 7b , -COOR 8 , and -CONR 9a R 9b Selected from; R 2 and R 3 is independently selected from H, alkyl, and alkoxy; Each R 4 are independently selected from alkyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, any of which may be present in combination with one or two R 10 optionally substituted with a group; Each R 5 Halo, -CN, -OR 11 , -NR 12a R 12b , -COOR 13 , and -CONR 14a R 14b are independently selected from; Each R 6 , R 7a , and R 7b are independently selected from H, alkyl, haloalkyl, and C(=O)alkyl; Each R 8 , R 9a, and R 9b are independently selected from H and alkyl; Each R 10 is independently selected from halo, hydroxy, and alkoxy; Each R 11 , R 12a , and R 12b is H, C 1~6 Alkyl, C 1~6 Haloalkyl, and C(=O)C 1~6 independently selected from alkyl; Each R 13 , R 14a , and R 14b is H and C 1~6 alkyl.

[0036] In some cases, the compound has structural formula (II): TIFF0007734139000002.tif28128 or a salt thereof, wherein: Ar 1 is selected from aryl and heteroaryl, any of which may be selected from one or two R 4 groups, any of which may be substituted with one, two, or three R 5 optionally substituted with a group; Each R 4 are independently selected from alkyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, any of which may be present in combination with one or two R 10 optionally substituted with a group; Each R 5 Halo, -CN, -OR 11 , -NR 12a R 12b , -COOR 13 , and -CONR 14a R 14b are independently selected from; Each R 10 is independently selected from halo, hydroxy, and alkoxy; Each R 11 , R 12a , and R 12b is H, C 1~6Alkyl, C 1~6 Haloalkyl, and C(=O)C 1~6 independently selected from alkyl; Each R 13 , R 14a , and R 14b is H and C 1~6 independently selected from alkyl; m and n are independently selected from 1, 2, and 3; Y 1 is selected from —NH— and —O—.

[0037] In some cases, Ar 1 is selected from phenyl and monocyclic heteroaryl, any of which may be selected from one or two R 4 groups, any of which may be substituted with one, two, or three R 5 In some cases, the Ar group is substituted. 1 is selected from phenyl and monocyclic 6-membered heteroaryl, any of which may be selected from one or two R 4 groups, any of which may be substituted with one, two, or three R 5 In some cases, the Ar group is substituted. 1 is selected from phenyl, pyridyl, pyrimidyl, pyridazyl, and pyrazyl, any of which may be selected from one or two R 4 groups, any of which may be substituted with one, two, or three R 5 In some cases, the Ar group is substituted. 1 is phenyl and one or two R 4 and may be substituted with one, two, or three R 5 In some cases, the Ar group is substituted. 1 is selected from pyridyl, pyrimidyl, pyridazyl, and pyrazyl, any of which may contain one or two R 4 groups, any of which may be substituted with one, two, or three R 5 In some cases, the Ar group is substituted. 1is pyridyl and one or two R 4 and may be substituted with one, two, or three R 5 In some cases, the Ar group is substituted. 1 is selected from pyrimidyl, pyridazyl, and pyrazyl, any of which may contain one or two R 4 groups, any of which may be substituted with one, two, or three R 5 In some cases, the Ar group is substituted. 1 is selected from naphthyl and bicyclic heteroaryl, any of which may be selected from one or two R 4 groups, any of which may be substituted with one, two, or three R 5 In some cases, the Ar group is substituted. 1 is a bicyclic heteroaryl, and one or two R 4 and may be substituted with one, two, or three R 5 In some cases, the Ar group is substituted. 1 is a bicyclic 10-membered heteroaryl, and one or two R 4 and may be substituted with one, two, or three R 5 In some cases, the Ar group is substituted. 1 is selected from quinolinyl and isoquinolinyl, any of which may be selected from one or two R 4 groups, any of which may be substituted with one, two, or three R 5 In some cases, the Ar group is substituted. 1 is a bicyclic 9-membered heteroaryl, and one or two R 4 and may be substituted with one, two, or three R 5 In some cases, the Ar group is substituted. 1 is selected from indolyl, benzimidazolyl, benzopyrrolyl, benzoxazolyl, and benzisoxazolyl, any of which may be substituted by one or two R 4 groups, any of which may be substituted with one, two, or three R 5In some cases, the Ar group is substituted. 1 is selected from indolyl, benzimidazolyl, and benzopyrrolyl, any of which may contain one or two R 4 groups, any of which may be substituted with one, two, or three R 5 is substituted with a group.

[0038] In some cases, Ar 1 is one R 4 Optionally, Ar may be substituted with an Ar group. 1 One or two R 4 In some cases, the Ar group is substituted. 1 is one R 4 In some cases, the Ar group is substituted. 1 There are two R 4 In some cases, each R 4 is C 1~6 Alkyl, C 3~7 Cycloalkyl, 4- to 7-membered heterocycloalkyl, C 6~10 aryl, and 6- to 10-membered heteroaryl, any of which may be selected from one or two R 10 Optionally, each R 4 is C 3~7 cycloalkyl and one or two R 10 Optionally, each R 4 is C 1~6 alkyl and one or two R 10 Optionally, each R 4 is C 1~6 alkyl and one or two R 10 Optionally, each R 4 is C 3~7 cycloalkyl and 4- to 7-membered heterocycloalkyl, any of which may be selected from one or two R 10 Optionally, each R 4 is C 6~10aryl and 6- to 10-membered heteroaryl, any of which may be selected from one or two R 10 Optionally, each R 4 is a 6- to 10-membered heteroaryl, and one or two R 10 Optionally, each R 4 is a monocyclic 5- to 7-membered heteroaryl, and one or two R 10 Optionally, each R 4 is selected from pyrrolyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, and isoxazolyl, and one or two R 10 Optionally, each R 4 is oxazolyl and one or two R 10 Optionally, each R 4 is one R 10 Optionally, each R 4 One or two R 10 In some cases, each R 4 is one R 10 In some cases, the R 10 is a halo. In some cases, R 10 is hydroxy. In some cases, R 10 is alkoxy. In some cases, R 10 is C 1~6 In some cases, each R 4 is R 10 In some cases, each R 4 is cyclopropyl. In some cases, each R 4 is cyclobutyl. In some cases, each R 4 is C 1~6 In some cases, each R 4 is methyl. In some cases, each R 4 is hydroxyalkyl. In some cases, each R 4 is hydroxymethyl. In some cases, Ar 1 is R4 is not substituted with a group.

[0039] In some cases, Ar 1 One or two R 5 Optionally, Ar may be substituted with an Ar group. 1 is one R 5 Optionally, Ar may be substituted with an Ar group. 1 One, two, or three R 5 In some cases, the Ar group is substituted. 1 One or two R 5 In some cases, the Ar group is substituted. 1 is one R 5 In some cases, each R 5 is independently selected from halo and cyano. 5 -OR 11 and -NR 12a R 12b In some cases, each R 11 , R 12a , and R 12b is H. In some cases, each R 5 HA-OR 11 In some cases, each R 11 is alkyl. In some cases, each R 11 is C 1~6 In some cases, each R 11 is C 1~6 haloalkyl. In some cases, each R 11 is halomethyl. In some cases, each R 11 is difluoromethyl. In some cases, each R 11 is trifluoromethyl. In some cases, each R 11 , R 12a , and R 12b is C(=O)alkyl. In some cases, each R 11 , R 12a , and R 12b is C(=O)C 1~6 In some cases, each R 5 -COOR13 and -CONR 14a R 14b In some cases, each R 13 , R 14a , and R 14b is H. In some cases, each R 13 , R 14a , and R 14b is alkyl. In some cases, each R 13 , R 14a , and R 14b is C 1~6 In some cases, R 5 HA-COOR 13 In some cases, R 5 -CONR 14a R 14b In some cases, Ar 1 is R 5 is not substituted with an Ar group. 1 teeth, Selected from TIFF0007734139000003.tif43144. In some cases, Ar 1 teeth, Selected from TIFF0007734139000004.tif14128.

[0040] Also provided are embodiments in which any of the above embodiments can be combined with any one or more of these embodiments, provided that the combinations are not mutually exclusive.

[0041] As used herein, two embodiments are "mutually exclusive" when one is defined as something different from the other. For example, an embodiment in which two groups are bonded to form a cycloalkyl is mutually exclusive with an embodiment in which one group is ethyl and the other group is hydrogen. Similarly, an embodiment in which one group is CH2 is mutually exclusive with an embodiment in which the same group is NH.

[0042] below: Also provided is a compound selected from TIFF0007734139000005.tif34140, or a salt thereof.

[0043] The following scheme can be used to make these compounds. Scheme I TIFF0007734139000006.tif151154

[0044] Through a three-step sequence of protection / carboxylation / deprotection, pyridine derivative 101 is converted to isonicotinic acid derivative 102. The bicyclic pyrido[3,4-d]-pyrimidine structure 103 is then formed by condensation with formamide, which is then chlorinated to give dihalo compound 104. Sequential reaction of this intermediate with ArNH and then PMB-NH (PMB = p-methoxybenzyl) affords disubstituted pyrido[3,4-d]-pyrimidine 106. Removal of the PMB group under acidic conditions, followed by coupling of the free primary amine with 2-(diethoxyphosphoryl)acetic acid, affords amide 108. Reaction with 2-(dimethylamino)acetaldehyde (generated in situ from acetal precursor 109) affords butenamide product 110.

[0045] For example, (E)-N-(4-((3-bromo-4-chlorophenyl)amino)pyrido[3,4-d]pyrimidin-6-yl)-4-(dimethylamino)but-2-enamide can be made as follows:

[0046] Process 1 TIFF0007734139000007.tif28128tert-Butyl (6-fluoropyridin-3-yl)carbamate To a solution of 6-fluoropyridin-3-amine (2.8 g, 25 mmol) in 6 mL of MTBE, di-tert-butyl dicarbonate (21.8 g, 100 mmol) was added at room temperature. The mixture was stirred at 45° C. for 16 hours. 1 gram of activated carbon was added, and the mixture was stirred briefly and then filtered. The filtrate was purified by flash column chromatography eluting with PE / EA (2 / 1) to give the title compound (4.8 g, 90.6%) as a white solid. MS (ES+) C10 H 13 FN2O2 theoretical value (requires): 212, measured value: 213 [M+H] + .

[0047] Process 2 TIFF0007734139000008.tif261285-((tert-butoxycarbonyl)amino)-2-fluoroisonicotinic acid To a mixture of the product from the previous step (500 mg, 2.36 mmol), TMEDA (0.88 mL), and MTBE (7 mL) was added a solution of n-BuLi (2.5 M in hexane, 2.36 mL) at -70 °C. After the addition was complete, the mixture was warmed to 10 °C to -15 °C and maintained at this temperature for 3 h. Dry CO2 gas was sparged at -70 °C for 2 h. The mixture was heated to 5 °C, and then water (6 mL) was added. The aqueous phase was collected, and the organic phase was extracted with 1 M NaOH. 6 M HCl was slowly added to the combined aqueous layers to adjust the pH to 2.5-3.0. The resulting mixture was extracted with EtOAc. The organic layer was dried and concentrated. The crude product was washed with a small amount of EtOAc to give the title compound (340 mg, 56.2%) as a white solid. MS (ES+) C 11 H 13 FN2O4 theoretical value: 256, measured value: 257 [M+H] + .

[0048] Process 3 TIFF0007734139000009.tif261285-Amino-2-fluoroisonicotinic acid To a solution of the product of the previous step (1.9 g, 7.4 mmol) in DCM (8 mL) was added CF3COOH (3.5 mL) at 0 °C. The resulting solution was stirred at room temperature for 3 h. The mixture was concentrated in vacuo to give the title compound as a yellow solid (900 mg, 77.8%). MS (ES+) C6H5FN2O2 calculated: 156, found: 157 [M+H] + .

[0049] Process 4 TIFF0007734139000010.tif191286-Fluoropyrido[3,4-d]pyrimidin-4(3H)-one A suspension of the product of the previous step (450 mg, 2.88 mmol) in formamide (5 mL) was heated with stirring at an internal temperature of 140 °C overnight. The mixture was cooled to room temperature, diluted with water (20 mL), and extracted with EtOAc. The organic layer was dried and concentrated. Water (5 mL) was added, and the precipitate that formed was collected by filtration to give the title compound (250 mg, 50.3%) as a yellow solid. MS (ES+) C7H4FN3O, calculated: 165, found: 166 [M+H] + .

[0050] Process 5 TIFF0007734139000011.tif181284-Chloro-6-fluoropyrido[3,4-d]pyrimidine A suspension of the product of the previous step (250 mg, 1.52 mmol) in SOCl (5 mL) and DMF (1 drop) was refluxed for 2 h. The reaction mixture was evaporated to give the title compound, which was used directly in the next step. MS (ES+) C7H3ClFN3 calculated: 183, found: 184 [M+H] + .

[0051] Process 6 TIFF0007734139000012.tif32128N-(3-Bromo-4-chlorophenyl)-6-fluoropyrido[3,4-d]pyrimidin-4-amine A mixture of the product from the previous step (244 mg, 1.33 mmol) and 3-bromo-4-chloroaniline (301 mg, 1.46 mmol) in DMA (3 mL) was stirred at 30 °C for 16 h. The reaction was diluted with water and the pH was adjusted to approximately 8 with saturated Na2CO3. PE was added and the mixture was stirred for 10 min. The solid was removed by filtration to give the title compound (400 mg, 85.5%) as a brown solid. MS (ES+) C 13 H7BrClFN4 Theoretical value: 352, Measured value: 353 [M+H] + .

[0052] Process 7 TIFF0007734139000013.tif45128N 4 -(3-bromo-4-chlorophenyl)-N 6 -(4-Methoxybenzyl)pyrido[3,4-d]pyrimidine-4,6-diamine A mixture of the product from the previous step (365 mg, 1 mmol) and p-methoxybenzylamine (1.37 g, 10 mmol) in DMSO (5 mL) was stirred at 100 °C for 16 h. The reaction was then diluted with HO and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine, dried, and concentrated. The crude material was purified by flash column chromatography eluting with 0% to 100% PE / EtOAc to give the title compound (280 mg, 52.5%) as a yellow solid. MS (ES+) C 21 H 17 BrClNO Theoretical value: 469, Measured value: 470 [M+H] + .

[0053] Process 8 TIFF0007734139000014.tif45128N 4 -(3-Bromo-4-chlorophenyl)pyrido[3,4-d]pyrimidine-4,6-diamine To a solution of the product of the previous step (280 mg, 0.6 mmol) in DCM (3 mL) was added CF3COOH (1 mL). The resulting solution was stirred at room temperature for 16 hours and then evaporated to dryness under vacuum. The residue was taken up in NH4OH (2 mL) and stirred for 5 minutes. The solid was collected by filtration to give the title compound as a yellow solid (160 mg, 76.9%). MS (ES+) C 13 H9BrClN5 Theoretical value: 349, Measured value: 350 [M+H] + .

[0054] Process 9 TIFF0007734139000015.tif32128Diethyl (2-((4-((3-bromo-4-chlorophenyl)amino)pyrido[3,4-d]pyrimidin-6-yl)amino)-2-oxoethyl)phosphonate A mixture of the product from the previous step (150 mg, 0.43 mmol), 2-(diethoxyphosphoryl)acetic acid (126 mg, 0.64 mmol), T3P (409 mg, 0.64 mmol), and Et3N (132 mg, 1.31 mmol) in EtOAc (3 mL) was stirred at 30 °C for 16 h. The reaction was diluted with HO. The solid that formed was removed by filtration and washed with EtOAc to give the title compound as a beige solid (200 mg, 88.5%). MS (ES+) C 19 H 20 BrClNO4P Theoretical value: 527, Measured value: 528 [M+H] + .

[0055] Step 10 TIFF0007734139000016.tif60142(E)-N-(4-((3-bromo-4-chlorophenyl)amino)pyrido[3,4-d]pyrimidin-6-yl)-4-(dimethylamino)but-2-enamide To a solution of 2,2-dimethoxy-N,N-dimethylethan-1-amine (80 mg, 0.6 mmol) in 0.08 mL of HO was added 0.08 mL of 37% HCl. The solution was stirred at 40 °C for 20 h and then cooled to 0 °C. This is called solution A. KOH (90 mg, 1.6 mmol) was dissolved in 0.4 mL of HO and cooled to 0 °C. This is called solution B. To a solution of the product of the previous step (106 mg, 0.2 mmol) in 0.8 mL of THF and 0.4 mL of DMA, LiCl (8 mg, 0.2 mmol) was added at 0 °C under Ar. The mixture was stirred at 0 °C for 15 min. Solution B was added and stirred at 0 °C for 2 min. Solution A was added and stirred for 2 h. H2O (5 mL) and PE (5 mL) were added and the mixture was filtered to also give the title compound as a beige solid (70 mg, 60.9%). MS(ES+)C 19 H 18 BrClNO theoretical value: 460, measured value: 461 [M+H] + . TIFF0007734139000017.tif27159

[0056] In some embodiments, the quinazoline-based tyrosine kinase inhibitor has structural formula (I): TIFF0007734139000018.tif28128, or a salt thereof, wherein: A 1 is C(R 1 ) and N; A 2 is C(R 2 ) and N; A 3 is C(R 3 ) and N; Ar 1 is selected from aryl and heteroaryl, any of which may be selected from one or two R 4 groups, any of which may be substituted with one, two, or three R 5 optionally substituted with a group; R A and R B are independently selected from H and alkyl; R C are H, CH3, and CH2NR 15 R 16 Selected from; R 1 Halo, -CN, -OR 6 , -NR 7a R 7b , -COOR 8 , and -CONR 9a R 9b Selected from; R 2 and R 3 is independently selected from H, alkyl, and alkoxy; Each R 4 are independently selected from alkyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, any of which may be present in combination with one or two R 10 optionally substituted with a group; Each R 5 Halo, -CN, -OR 11 , -NR 12a R 12b , -COOR13 , and -CONR 14a R 14b are independently selected from; Each R 6 , R 7a , and R 7b are independently selected from H, alkyl, haloalkyl, and C(=O)alkyl; Each R 8 , R 9a , and R 9b are independently selected from H and alkyl; Each R 10 is independently selected from halo, hydroxy, and alkoxy; Each R 11 , R 12a , and R 12b is H, C 1~6 Alkyl, C 1~6 Haloalkyl, and C(=O)C 1~6 independently selected from alkyl; Each R 13 , R 14a , and R 14b is H and C 1~6 independently selected from alkyl; R 15 and R 16 is H and C 1~6 alkyl; or R 15 and R 16 are joined together with the nitrogen to which they are both attached to form a 5- to 7-membered heterocycloalkyl; m and n are independently selected from 1, 2, and 3; Y 1 is selected from —NH— and —O—.

[0057] In some embodiments, the quinazoline-based tyrosine kinase inhibitor has structural formula (II): TIFF0007734139000019.tif27128, or a salt thereof, wherein: Ar 1 is selected from aryl and heteroaryl, any of which may be selected from one or two R4 groups, any of which may be substituted with one, two, or three R 5 optionally substituted with a group; R A and R B are independently selected from H and alkyl; R C are H, CH3, and CH2NR 15 R 16 Selected from; R 2 is selected from H, alkyl, and alkoxy; Each R 4 are independently selected from alkyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, any of which may be present in combination with one or two R 10 optionally substituted with a group; Each R 5 Halo, -CN, -OR 11 , -NR 12a R 12b , -COOR 13 , and -CONR 14a R 14b are independently selected from; Each R 10 is independently selected from halo, hydroxy, and alkoxy; Each R 11 , R 12a , and R 12b is H, C 1~6 Alkyl, C 1~6 Haloalkyl, and C(=O)C 1~6 independently selected from alkyl; Each R 13 , R 14a , and R 14b is H and C 1~6 independently selected from alkyl; R 15 and R 16 is H and C 1~6 alkyl; or R 15 and R 16are joined together with the nitrogen to which they are both attached to form a 5- to 7-membered heterocycloalkyl; m and n are independently selected from 1, 2, and 3; Y 1 is selected from —NH— and —O—.

[0058] In some embodiments, the quinazoline-based tyrosine kinase inhibitor has structural formula (III): TIFF0007734139000020.tif28128, or a salt thereof, wherein: Ar 1 is selected from aryl and heteroaryl, any of which may be selected from one or two R 4 groups, any of which may be substituted with one, two, or three R 5 optionally substituted with a group; R A and R B are independently selected from H and alkyl; R C are H, CH3, and CH2NR 15 R 16 Selected from; R 1 Halo, -CN, -OR 6 , -NR 7a R 7b , -COOR 8 , and -CONR 9a R 9b Selected from; R 2 is selected from H, alkyl, and alkoxy; Each R 4 are independently selected from alkyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, any of which may be present in combination with one or two R 10 optionally substituted with a group; Each R 5 Halo, -CN, -OR 11 , -NR 12a R 12b , -COOR 13, and -CONR 14a R 14b are independently selected from; Each R 6 , R 7a , and R 7b are independently selected from H, alkyl, haloalkyl, and C(=O)alkyl; Each R 8 , R 9a , and R 9b are independently selected from H and alkyl; Each R 10 is independently selected from halo, hydroxy, and alkoxy; Each R 11 , R 12a , and R 12b is H, C 1~6 Alkyl, C 1~6 Haloalkyl, and C(=O)C 1~6 independently selected from alkyl; Each R 13 , R 14a , and R 14b is H and C 1~6 independently selected from alkyl; R 15 and R 16 is H and C 1~6 alkyl; or R 15 and R 16 are joined together with the nitrogen to which they are both attached to form a 5- to 7-membered heterocycloalkyl; m and n are independently selected from 1, 2, and 3; Y 1 is selected from —NH— and —O—.

[0059] In some embodiments, the quinazoline-based tyrosine kinase inhibitor has structural formula (IV): TIFF0007734139000021.tif27128, or a salt thereof, wherein: Ar 1 is selected from aryl and heteroaryl, any of which may be selected from one or two R 4groups, any of which may be substituted with one, two, or three R 5 optionally substituted with a group; R A and R B are independently selected from H and alkyl; R C are H, CH3, and CH2NR 15 R 16 Selected from; Each R 4 are independently selected from alkyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, any of which may be present in combination with one or two R 10 optionally substituted with a group; Each R 5 Halo, -CN, -OR 11 , -NR 12a R 12b , -COOR 13 , and -CONR 14a R 14b are independently selected from; Each R 10 is independently selected from halo, hydroxy, and alkoxy; Each R 11 , R 12a , and R 12b is H, C 1~6 Alkyl, C 1~6 Haloalkyl, and C(=O)C 1~6 independently selected from alkyl; Each R 13 , R 14a , and R 14b is H and C 1~6 independently selected from alkyl; R 15 and R 16 is H and C 1~6 alkyl; or R 15 and R 16 are joined together with the nitrogen to which they are both attached to form a 5- to 7-membered heterocycloalkyl; m and n are independently selected from 1, 2, and 3; Y1 is selected from —NH— and —O—.

[0060] In some embodiments, the quinazoline-based tyrosine kinase inhibitor has structural formula (V): TIFF0007734139000022.tif28128, or a salt thereof, wherein: Ar 1 is selected from aryl and heteroaryl, any of which may be selected from one or two R 4 groups, any of which may be substituted with one, two, or three R 5 optionally substituted with a group; R A and R B are independently selected from H and alkyl; R C are H, CH3, and CH2NR 15 R 16 Selected from; R 1 Halo, -CN, -OR 6 , -NR 7a R 7b , -COOR 8 , and -CONR 9a R 9b Selected from; R 2 is selected from H, alkyl, and alkoxy; Each R 4 are independently selected from alkyl, haloalkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, any of which may be present in combination with one or two R 10 optionally substituted with a group; Each R 5 Halo, -CN, -OR 11 , -NR 12a R 12b , -COOR 13 , and -CONR 14a R 14b are independently selected from; Each R 6 , R 7a , and R 7bare independently selected from H, alkyl, haloalkyl, and C(=O)alkyl; Each R 8 , R 9a , and R 9b are independently selected from H and alkyl; Each R 10 is independently selected from halo, hydroxy, and alkoxy; Each R 11 , R 12a , and R 12b is H, C 1~6 Alkyl, C 1~6 Haloalkyl, and C(=O)C 1~6 independently selected from alkyl; Each R 13 , R 14a , and R 14b is H and C 1~6 independently selected from alkyl; R 15 and R 16 is H and C 1~6 alkyl; or R 15 and R 16 are joined together with the nitrogen to which they are both attached to form a 5- to 7-membered heterocycloalkyl; m and n are independently selected from 1, 2, and 3; Y 1 is selected from —NH— and —O—.

[0061] In some cases, Ar 1 is selected from phenyl and monocyclic heteroaryl, any of which may be selected from one or two R 4 groups, any of which may be substituted with one, two, or three R 5 Optionally, Ar may be substituted with an Ar group. 1 is selected from phenyl and monocyclic 6-membered heteroaryl, any of which may be selected from one or two R 4 groups, any of which may be substituted with one, two, or three R 5 Optionally, Ar may be substituted with an Ar group.1 is selected from phenyl, pyridyl, pyrimidyl, pyridazyl, and pyrazyl, any of which may contain one or two R 4 groups, any of which may be substituted with one, two, or three R 5 Optionally, Ar may be substituted with an Ar group. 1 is phenyl and one or two R 4 and may be substituted with one, two, or three R 5 Optionally, Ar may be substituted with an Ar group. 1 is selected from pyridyl, pyrimidyl, pyridazyl, and pyrazyl, any of which may contain one or two R 4 groups, any of which may be substituted with one, two, or three R 5 Optionally, Ar may be substituted with an Ar group. 1 is pyridyl and one or two R 4 and may be substituted with one, two, or three R 5 Optionally, Ar may be substituted with an Ar group. 1 is selected from pyrimidyl, pyridazyl, and pyrazyl, any of which may contain one or two R 4 groups, any of which may be substituted with one, two, or three R 5 Optionally, Ar may be substituted with an Ar group. 1 is selected from naphthyl and bicyclic heteroaryl, any of which may be selected from one or two R 4 groups, any of which may be substituted with one, two, or three R 5 Optionally, Ar may be substituted with an Ar group. 1 is a bicyclic heteroaryl, and one or two R 4 and may be substituted with one, two, or three R 5 Optionally, Ar may be substituted with an Ar group. 1 is a bicyclic 10-membered heteroaryl, and one or two R 4 and may be substituted with one, two, or three R 5Optionally, Ar may be substituted with an Ar group. 1 is selected from quinolinyl and isoquinolinyl, any of which may be selected from one or two R 4 groups, any of which may be substituted with one, two, or three R 5 Optionally, Ar may be substituted with an Ar group. 1 is a bicyclic 9-membered heteroaryl, and one or two R 4 and may be substituted with one, two, or three R 5 Optionally, Ar may be substituted with an Ar group. 1 is selected from indolyl, benzimidazolyl, benzopyrrolyl, benzoxazolyl, and benzisoxazolyl, any of which may be substituted by one or two R 4 groups, any of which may be substituted with one, two, or three R 5 Optionally, Ar may be substituted with an Ar group. 1 is selected from indolyl, benzimidazolyl, and benzopyrrolyl, any of which may contain one or two R 4 groups, any of which may be substituted with one, two, or three R 5 Optionally, Ar may be substituted with an Ar group. 1 is one R 4 Optionally, Ar may be substituted with an Ar group. 1 One or two R 4 In some cases, the Ar group is substituted. 1 is one R 4 In some cases, the Ar group is substituted. 1 There are two R 4 In some cases, each R 4 is C 1~6 Alkyl, C 3~7 Cycloalkyl, 4- to 7-membered heterocycloalkyl, C 6~10 aryl, and 6- to 10-membered heteroaryl, any of which may be selected from one or two R 10 Optionally, each R 4 is C3~7 cycloalkyl and one or two R 10 Optionally, each R 4 is C 1~6 alkyl and one or two R 10 Optionally, each R 4 is C 1~6 alkyl and one or two R 10 Optionally, each R 4 is C 3~7 cycloalkyl and 4- to 7-membered heterocycloalkyl, any of which may be selected from one or two R 10 Optionally, each R 4 is C 6~10 aryl and 6- to 10-membered heteroaryl, any of which may be selected from one or two R 10 Optionally, each R 4 is a 6- to 10-membered heteroaryl, and one or two R 10 Optionally, each R 4 is a monocyclic 5- to 7-membered heteroaryl, and one or two R 10 Optionally, each R 4 is selected from pyrrolyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, and isoxazolyl, and one or two R 10 Optionally, each R 4 is oxazolyl and one or two R 10 Optionally, each R 4 is one R 10 Optionally, each R 4 One or two R 10 In some cases, each R 4 is one R 10 In some cases, the R 10 is a halo. In some cases, R 10is hydroxy. In some cases, R 10 is alkoxy. In some cases, R 10 is C 1~6 In some cases, each R 4 is R 10 In some cases, each R 4 is cyclopropyl. In some cases, each R 4 is cyclobutyl. In some cases, each R 4 is C 1~6 In some cases, each R 4 is methyl. In some cases, each R 4 is hydroxyalkyl. In some cases, each R 4 is hydroxymethyl. In some cases, Ar 1 is R 4 is not substituted with an Ar group. 1 One or two R 5 Optionally, Ar may be substituted with an Ar group. 1 is one R 5 Optionally, Ar may be substituted with an Ar group. 1 One, two, or three R 5 Optionally, Ar may be substituted with an Ar group. 1 One or two R 5 In some cases, the Ar group is substituted. 1 is one R 5 In some cases, each R 5 is independently selected from halo and cyano. 5 -OR 11 and -NR 12a R 12b In some cases, each R 11 , R 12a , and R 12b is H. In some cases, each R 5 HA-OR 11 In some cases, each R 11 is alkyl. In some cases, each R 11 is C 1~6In some cases, each R 11 is C 1~6 haloalkyl. In some cases, each R 11 is halomethyl. In some cases, each R 11 is difluoromethyl. In some cases, each R 11 is trifluoromethyl. In some cases, each R 11 , R 12a , and R 12b is C(=O)alkyl. In some cases, each R 11 , R 12a , and R 12b is C(=O)C 1~6 In some cases, each R 5 -COOR 13 and -CONR 14a R 14b In some cases, each R 13 , R 14a , and R 14b is H. In some cases, each R 13 , R 14a , and R 14b is alkyl. In some cases, each R 13 , R 14a , and R 14b is C 1~6 In some cases, R 5 HA-COOR 13 In some cases, R 5 -CONR 14a R 14b In some cases, Ar 1 is R 5 is not substituted with an Ar group. 1 teeth, Selected from TIFF0007734139000023.tif64139.

[0062] In some cases, Ar 1 teeth, Selected from TIFF0007734139000024.tif41149.

[0063] In some cases, Ar 1 teeth The file is TIFF0007734139000025.tif19128.

[0064] In some cases, m is 1 and n is 1, m is 2 and n is 2, or m is 1 and n is 3. In some cases, m is 1 and n is 1, or m is 2 and n is 2. In some cases, m is 1. In some cases, m is 2. In some cases, m is 3. In some cases, n is 1. In some cases, n is 2. In some cases, n is 3.

[0065] In some cases, Y 1 is -NH-. In some cases, Y 1 is -O-. In some cases, R A and R B is H and C 1~6 alkyl. In some cases, R A is H. In some cases, R A is C 1~6 In some cases, R B is H. In some cases, R B is C 1~6 In some cases, R C is H. In some cases, R C is CH3. In some cases, R C is CH2NR 15 R 16 In some cases, R 15 and R 16 is H and C 1~6 alkyl. In some cases, R 15 and R 16 are independently selected from H and methyl. 15 and R 16 is C 1~6 In some cases, R 15 and R 16 is methyl. In some cases, R 15 and R16 At least one of R is H. 15 and R 16 is H. In some cases, R 15 and R 16 are joined together with the nitrogen to which they are both attached to form a 5- to 7-membered heterocycloalkyl. 15 and R 16 together with the nitrogen to which they are both attached to form a 5- to 7-membered heterocycloalkyl selected from pyrrolidine, piperidine, piperazine, and morpholine.

[0066] In some embodiments, the quinazoline-based tyrosine kinase inhibitor has structural formula (VI): TIFF0007734139000026.tif27128, or a salt thereof, wherein: Ar 1 is selected from aryl and heteroaryl, any of which may be selected from one, two, or three R 5 optionally substituted with a group; Each R 5 Halo, -CN, -OR 11 , -NR 12a R 12b , -COOR 13 , and -CONR 14a R 14b are independently selected from; Each R 11 , R 12a , and R 12b is H, C 1~6 Alkyl, C 1~6 Haloalkyl, and C(=O)C 1~6 independently selected from alkyl; Each R 13 , R 14a , and R 14b is H and C 1~6 alkyl.

[0067] In some cases, Ar 1 is phenyl and one, two, or three R 5In some cases, the R 5 is a halo. In some cases, Ar 1 teeth, Selected from TIFF0007734139000027.tif19128.

[0068] In some embodiments, the quinazoline-based tyrosine kinase inhibitor has structural formula (VII): TIFF0007734139000028.tif28128, or a salt thereof, wherein: Ar 1 is selected from aryl and heteroaryl, any of which may be selected from one, two, or three R 5 optionally substituted with a group; Each R 5 Halo, -CN, -OR 11 , -NR 12a R 12b , -COOR 13 , and -CONR 14a R 14b are independently selected from; Each R 11 , R 12a , and R 12b is H, C 1~6 Alkyl, C 1~6 Haloalkyl, and C(=O)C 1~6 independently selected from alkyl; Each R 13 , R 14a , and R 14b is H and C 1~6 alkyl.

[0069] In some cases, Ar 1 is phenyl and one, two, or three R 5 In some cases, the R 5 is a halo. In some cases, Ar 1 teeth, Selected from TIFF0007734139000029.tif19128.

[0070] In some embodiments, the quinazoline-based tyrosine kinase inhibitor has structural formula (VIII): TIFF0007734139000030.tif27128, or a salt thereof, wherein: Ar 1 is selected from aryl and heteroaryl, any of which may be selected from one, two, or three R 5 optionally substituted with a group; Each R 5 Halo, -CN, -OR 11 , -NR 12a R 12b , -COOR 13 , and -CONR 14a R 14b are independently selected from; Each R 11 , R 12a , and R 12b is H, C 1~6 Alkyl, C 1~6 Haloalkyl, and C(=O)C 1~6 independently selected from alkyl; Each R 13 , R 14a , and R 14b is H and C 1~6 alkyl.

[0071] In some cases, Ar 1 is phenyl and one, two, or three R 5 In some cases, the R 5 is a halo. In some cases, Ar 1 teeth The file is TIFF0007734139000031.tif18128.

[0072] In some cases, each R 5 Halo, -CN, and -OR 11 In some cases, each R 5 is independently selected from halo and —CN. 5 is a halo. In some cases, Ar 1is selected from phenyl and monocyclic heteroaryl, any of which may contain one, two, or three R 5 Optionally, Ar may be substituted with an Ar group. 1 is selected from phenyl, pyridyl, pyrimidyl, pyridazyl, and pyrazyl, any of which may be selected from one, two, or three R 5 Optionally, Ar may be substituted with an Ar group. 1 is phenyl and one, two, or three R 5 In some cases, the Ar group is substituted. 1 One or two R 5 In some cases, the Ar group is substituted. 1 is one R 5 In some cases, the Ar group is substituted. 1 teeth, Selected from TIFF0007734139000032.tif19128.

[0073] In some embodiments, the quinazoline-based tyrosine kinase inhibitor is TIFF0007734139000033.tif211148TIFF0007734139000034.tif204111TIFF0007734139000035.tif208145TIFF0007734139000036.tif228147TIFF0007734139000037.tif207147TIFF0007734139000038.tif208147TIFF0007734139000039.tif244147TIFF0007734139000040.tif235145, or a salt thereof.

[0074] In some embodiments, the quinazoline-based tyrosine kinase inhibitor is TIFF0007734139000041.tif199141, or a salt thereof.

[0075] In some embodiments, the quinazoline-based tyrosine kinase inhibitor is TIFF0007734139000042.tif81128, or a salt thereof.

[0076] In some embodiments, the quinazoline-based tyrosine kinase inhibitor is TIFF0007734139000043.tif84128, or a salt thereof.

[0077] The following schemes can be used to practice the present disclosure.

[0078] Scheme I TIFF0007734139000044.tif128159 Manipulation of the functional groups of starting material 101 via successive Fischer esterification reactions, Williamson ether formation, and nitro group reduction affords functionalized benzene 102. Condensation with DMF dimethyl acetal affords amidine 103, which is converted to chloroquinoline 104 via ring formation with acetonitrile anion followed by chlorination of the intermediate quinolone compound (not shown). Mitsunobu-type coupling of secondary alcohol 105 with phenol 104 affords ether 106. S-coupling with arylamine 107 affords ether 108. N Ar reaction gives the displacement product 108. After removal of the Boc protecting group, the secondary amine 109 is reacted with an acyl chloride to give the amide 107.

[0079] Scheme II The synthesis proceeded as in Scheme I, the difference being the choice of quinazoline starting material 201.

[0080] Scheme III Heterocyclic tosylate 301 is prepared in three steps from Boc-protected hydroxycycloamine 105 (Scheme I). Anthranilic acid analog 302 is converted to a bicyclic arene with formamidine, followed by chloride displacement to form phenolic ether 303. The amide functionality is converted to chloro compound 304 by reaction with phosphorus oxychloride. 301 Reaction with NH2 gives aminoarene 305. Removal of the methoxy group under acidic conditions gives phenol 306. Finally, reaction of the tosylate 301 with a phenol under Williamson ether synthesis conditions gives 307.

[0081] Scheme IV The pyrido[3,4-d]pyrimidine derivative 401 is selectively reacted at the 4-position to give the aniline compound 402. Reaction with hydroxycycloamine 105 (Scheme 1) gives the ether 403. Removal of the Boc protecting group under acidic conditions gives the secondary amine 404, which is then reacted with an acyl chloride to give the amide 405.

[0082] When a range of values ​​is disclosed and the notation "n1... to n2" or "between n1... to n2" is used, where n1 and n2 are numbers, the notation is intended to include the numbers themselves and the range therebetween, unless otherwise specified. The range may be an integer or a continuum between and including the end values. As an example, carbons are added in integer increments, so the range "2 to 6 carbons" is intended to include 2, 3, 4, 5, and 6 carbons. As an example, compare the range "1 to 3 μM (micromolar)," which is intended to include 1 μM, 3 μM, and everything between to any number of significant figures (e.g., 1.255 μM, 2.1 μM, 2.9999 μM, etc.).

[0083] As used herein, the term "about" is intended to qualify the numerical value it modifies and to indicate such value as varying within a range of error. When a specific range of error, such as standard deviation for an average value given in a chart or table of data, is not described, the term "about" should be understood to mean a range that would encompass the described value, and also a range that would be encompassed by rounding up or down to that number, taking into account significant digits.

[0084] The term "acyl," as used herein, alone or in combination, refers to a carbonyl bonded to an alkenyl, alkyl, aryl, cycloalkyl, heteroaryl, heterocycle, or any other moiety where the atom bonded to the carbonyl is carbon. An "acetyl" group refers to a -C(O)CH group. An "alkylcarbonyl" or "alkanoyl" group refers to an alkyl group bonded to the parent molecular moiety through a carbonyl group. Examples of such groups include methylcarbonyl and ethylcarbonyl. Examples of acyl groups include formyl, alkanoyl, and aroyl.

[0085] The term "alkenyl," as used herein, alone or in combination, refers to a straight or branched chain hydrocarbon group having one or more double bonds and containing 2 to 20 carbon atoms. In certain embodiments, said alkenyl is considered to contain 2 to 6 carbon atoms. The term "alkenylene" refers to a carbon-carbon double bond system attached at two or more positions, such as ethenylene [(-CH=CH-), (-C::C-)]. Examples of suitable alkenyl groups include ethenyl, propenyl, 2-methylpropenyl, 1,4-butadienyl, and the like. Unless otherwise specified, the term "alkenyl" can include an "alkenylene" group.

[0086] The term "alkoxy," as used herein, alone or in combination, refers to an alkyl ether group, where the term alkyl is as defined below. Examples of suitable alkyl ether groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, and the like.

[0087] The term "alkyl," as used herein, alone or in combination, refers to a straight- or branched-chain alkyl group containing 1 to 20 carbon atoms. In certain embodiments, the alkyl will contain 1 to 10 carbon atoms. In further embodiments, the alkyl will contain 1 to 8 carbon atoms. The alkyl group can be optionally substituted as defined herein. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, octyl, noyl, and the like. The term "alkylene," as used herein, alone or in combination, refers to a saturated aliphatic group derived from a straight- or branched-chain saturated hydrocarbon bonded at two or more positions, such as methylene (-CH-). Unless otherwise specified, the term "alkyl" can include an "alkylene" group.

[0088] The term "alkylamino," as used herein, alone or in combination, refers to an alkyl group attached to the parent molecular moiety through an amino group. Suitable alkylamino groups can be mono- or di-alkylated, forming groups such as, for example, N-methylamino, N-ethylamino, N,N-dimethylamino, N,N-ethylmethylamino, and the like.

[0089] The term "alkylidene," as used herein, alone or in combination, refers to an alkenyl group in which one carbon atom of the carbon-carbon double bond belongs to the moiety to which the alkenyl group is attached.

[0090] The term "alkylthio," as used herein, alone or in combination, refers to an alkyl thioether (RS-) group, where the term alkyl is as defined above and sulfur may be singly or doubly oxidized. Examples of suitable alkyl thioether groups include methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, iso-butylthio, sec-butylthio, tert-butylthio, methanesulfonyl, ethanesulfinyl, and the like.

[0091] The term "alkynyl," as used herein, alone or in combination, refers to a straight- or branched-chain hydrocarbon group having one or more triple bonds and containing 2 to 20 carbon atoms. In certain embodiments, the alkynyl contains 2 to 6 carbon atoms. In further embodiments, the alkynyl contains 2 to 4 carbon atoms. The term "alkynylene" refers to a carbon-carbon triple bond attached at two positions, such as ethynylene (-C:::C-, -C≡C-). Examples of alkynyl groups include ethynyl, propynyl, hydroxypropynyl, butyn-1-yl, butyn-2-yl, pentyn-1-yl, 3-methylbutyn-1-yl, hexyn-2-yl, and the like. Unless otherwise specified, the term "alkynyl" can include an "alkynylene" group.

[0092] The terms "amido" and "carbamoyl," as used herein, alone or in combination, refer to an amino group, as described below, attached to the parent molecular moiety through a carbonyl group, or vice versa. The term "C-amido," as used herein, alone or in combination, refers to the -C(O)N(RR') group, where R and R' are as defined herein or as defined by a specifically enumerated designated "R" group. The term "N-amido," as used herein, alone or in combination, refers to the RC(O)N(R')- group, where R and R' are as defined herein or as defined by a specifically enumerated designated "R" group. The term "acylamino," as used herein, alone or in combination, encompasses an acyl group attached to the parent moiety through an amino group. An example of an "acylamino" group is acetylamino (CHC(O)NH-).

[0093] The term "amino," as used herein, alone or in combination, refers to --NRR', where R and R' are independently selected from hydrogen, alkyl, acyl, heteroalkyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl, any of which may themselves be optionally substituted. Additionally, R and R' can combine to form a heterocycloalkyl, any of which may also be optionally substituted.

[0094] The term "aryl," as used herein, alone or in combination, means a carbocyclic aromatic system containing one, two, or three rings, and such polycyclic ring systems are fused together. The term "aryl" encompasses aromatic groups such as phenyl, naphthyl, anthracenyl, and phenanthryl.

[0095] The terms "arylalkenyl" or "aralkenyl," as used herein, alone or in combination, refer to an aryl group attached to the parent molecular moiety through an alkenyl group.

[0096] The term "arylalkoxy" or "aralkoxy," as used herein, alone or in combination, refers to an aryl group attached to the parent molecular moiety through an alkoxy group.

[0097] The terms "arylalkyl" or "aralkyl," as used herein, alone or in combination, refer to an aryl group attached to the parent molecular moiety through an alkyl group.

[0098] The terms "arylalkynyl" or "aralkynyl," as used herein, alone or in combination, refer to an aryl group attached to the parent molecular moiety through an alkynyl group.

[0099] The terms "arylalkanoyl" or "aralkanoyl" or "aroyl," as used herein, alone or in combination, refer to an acyl group derived from an aryl-substituted alkanecarboxylic acid, such as benzoyl, naphthoyl, phenylacetyl, 3-phenylpropionyl (hydrocinnamoyl), 4-phenylbutyryl, (2-naphthyl)acetyl, 4-chlorohydrocinnamoyl, and the like.

[0100] The term aryloxy, as used herein, alone or in combination, refers to an aryl group attached to the parent molecular moiety through an oxy.

[0101] The terms "benzo" and "benz," as used herein, alone or in combination, refer to the divalent group CH= derived from benzene. Examples include benzothiophene and benzimidazole.

[0102] The term "carbamate," as used herein, alone or in combination, refers to an ester of carbamic acid (-NHCOO-), which may be attached to the parent molecular moiety through either the nitrogen terminus or the acid terminus, and which may be optionally substituted as defined herein.

[0103] The term "O-carbamyl," as used herein, alone or in combination, refers to --OC(O)NRR', where R and R' are as defined herein.

[0104] The term "N-carbamyl," as used herein, alone or in combination, refers to an ROC(O)NR'- group, with R and R' as defined herein.

[0105] The term "carbonyl," as used herein, when alone includes formyl [-C(O)H] and in combination is a -C(O)- group.

[0106] The terms "carboxyl" or "carboxy," as used herein, refer to -C(O)OH or the corresponding "carboxylate" anion, e.g., in a carboxylic acid salt. An "O-carboxy" group refers to an RC(O)O- group, where R is as defined herein. A "C-carboxy" group refers to an -C(O)OR group, where R is as defined herein.

[0107] The term "cyano," as used herein, alone or in combination, refers to --CN.

[0108] The term "cycloalkyl," or alternatively, "carbocycle," as used herein, alone or in combination, refers to a saturated or partially saturated monocyclic, bicyclic, or tricyclic alkyl group, each ring moiety containing 3 to 12 carbon atom ring members, and optionally a benzo-fused ring system, which may be substituted as defined herein. In certain embodiments, the cycloalkyl will contain 5 to 7 carbon atoms. Examples of such cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, tetrahydronaphthyl, indanyl, octahydronaphthyl, 2,3-dihydro-1H-indenyl, adamantyl, and the like. "Bicyclic" and "tricyclic," as used herein, are intended to include both fused ring systems, e.g., decahydronaphthalene, octahydronaphthalene, and polycyclic (multi-center) saturated or partially unsaturated types. The latter type of isomer is commonly exemplified by bicyclo[1,1,1]pentane, camphor, adamantane, and bicyclo[3,2,1]octane.

[0109] The term "ester," as used herein, alone or in combination, refers to a carboxy group bridging two moieties joined at carbon atoms.

[0110] The term "ether," as used herein, alone or in combination, refers to an oxy group bridging two moieties joined at a carbon atom.

[0111] The terms "halo" or "halogen," as used herein, alone or in combination, refer to fluorine, chlorine, bromine, or iodine.

[0112] The term "haloalkoxy," as used herein, alone or in combination, refers to a haloalkyl group attached to the parent molecular moiety through an oxygen atom.

[0113] The term "haloalkyl," as used herein, alone or in combination, refers to an alkyl group having the above-defined meaning in which one or more hydrogen atoms are replaced by halogen. Monohaloalkyl groups, dihaloalkyl groups, and polyhaloalkyl groups are specifically included. Monohaloalkyl groups, for example, may have an iodine atom, a bromine atom, a chlorine atom, or a fluorine atom within the group. Dihaloalkyl groups and polyhaloalkyl groups may have two or more of the same halo atoms or a combination of different halo groups. Examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. "Haloalkylene" refers to a haloalkyl group bonded at two or more positions. Examples include fluoromethylene (-CFH-), difluoromethylene (-CF2-), chloromethylene (-CHCl-), and the like.

[0114] The term "heteroalkyl," as used herein, alone or in combination, refers to a stable straight or branched chain, or combination thereof, that is fully saturated or contains about one to three unsaturations, and that consists of the specified number of carbon atoms and one to three heteroatoms selected from N, O, and S, where the N and S atoms are optionally oxidized and the N heteroatom is optionally quaternized. The heteroatoms may be placed at any interior position of the heteroalkyl group. For example, up to two heteroatoms may be consecutive, such as -CH-NH-OCH.

[0115] The term "heteroaryl," as used herein, alone or in combination, refers to a 3- to 15-membered unsaturated heteromonocyclic ring or a fused monocyclic, bicyclic, or tricyclic ring system in which at least one of the fused rings is aromatic and contains at least one atom selected from N, O, and S. In certain embodiments, the heteroaryl will contain 1 to 4 heteroatoms as ring members. In further embodiments, the heteroaryl will contain 1 to 2 heteroatoms as ring members. In certain embodiments, the heteroaryl will contain 5 to 7 atoms. The term also encompasses fused polycyclic groups in which a heterocycle is fused to an aryl ring, a heteroaryl ring is fused to another heteroaryl ring, a heteroaryl ring is fused to a heterocycloalkyl ring, or a heteroaryl ring is fused to a cycloalkyl ring. Examples of heteroaryl groups include pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl, pyranyl, furyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, thiadiazolyl, isothiazolyl, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, quinoxalinyl, quinazolinyl, indazolyl, benzotriazolyl, benzodioxolyl, benzopyranyl, benzoxazolyl, benzoxadiazolyl, benzothiazolyl, benzothiadiazolyl, benzofuryl, benzothienyl, chromonyl, coumarinyl, benzopyranyl, tetrahydroquinolinyl, tetrazolopyridazinyl, tetrahydroisoquinolinyl, thienopyridinyl, furopyridinyl, pyrrolopyridinyl, and the like. Exemplary tricyclic heterocyclic groups include carbazolyl, benzidolyl, phenanthrolinyl, dibenzofuranyl, acridinyl, phenanthridinyl, xanthenyl, and the like.

[0116] The terms "heterocycloalkyl" and, interchangeably, "heterocycle," as used herein, alone or in combination, refer to a saturated, partially unsaturated, or fully unsaturated (but non-aromatic), monocyclic, bicyclic, or tricyclic heterocyclic group, each containing at least one heteroatom as a ring member, where each heteroatom may be independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, the heterocycloalkyl will contain 1 to 4 heteroatoms as ring members. In further embodiments, the heterocycloalkyl will contain 1 to 2 heteroatoms as ring members. In certain embodiments, the heterocycloalkyl will contain 3 to 8 ring members in each ring. In further embodiments, the heterocycloalkyl will contain 3 to 7 ring members in each ring. In yet other embodiments, the heterocycloalkyl will contain 5 to 6 ring members in each ring. "Heterocycloalkyl" and "heterocycle" are intended to include sulfone, sulfoxide, N-oxide of tertiary nitrogen ring members, and carbocyclic and benzo-fused ring systems; furthermore, both terms also include systems in which a heterocycle is fused to an aryl group or another heterocyclic group as defined herein. Examples of heterocyclic groups include aziridinyl, azetidinyl, 1,3-benzodioxolyl, dihydroisoindolyl, dihydroisoquinolinyl, dihydrocinnolinyl, dihydrobenzodioxinyl, dihydro[1,3]oxazolo[4,5-b]pyridinyl, benzothiazolyl, dihydroindolyl, dihydropyridinyl, 1,3-dioxanyl, 1,4-dioxanyl, 1,3-dioxolanyl, isoindolinyl, morpholinyl, piperazinyl, pyrrolidinyl, tetrahydropyridinyl, piperidinyl, thiomorpholinyl, and the like. The heterocycle groups may be optionally substituted unless specifically prohibited.

[0117] The term "hydrazinyl," as used herein, alone or in combination, refers to two amino groups joined by a single bond, ie, --NN--.

[0118] The term "hydroxy," as used herein, alone or in combination, refers to --OH.

[0119] The term "hydroxyalkyl," as used herein, alone or in combination, refers to a hydroxy group attached to the parent molecular moiety through an alkyl group.

[0120] The term "imino," as used herein, alone or in combination, refers to =N-.

[0121] The term "iminohydroxy," as used herein, alone or in combination, refers to =N(OH) and =NO-.

[0122] The phrase "in the backbone" refers to the longest continuous or adjacent chain of carbon atoms starting from the point of attachment of a group to a compound of any one of the formulas disclosed herein.

[0123] The term "isocyanato" refers to the -NCO group.

[0124] The term "isothiocyanato" refers to the group --NCS.

[0125] The phrase "linear chain of atoms" refers to the longest straight chain of atoms independently selected from carbon, nitrogen, oxygen, and sulfur.

[0126] The term "lower," as used herein, alone or in combination, means, unless otherwise defined, containing 1 to 6, inclusive, carbon atoms (i.e., C1-C6 alkyl).

[0127] The term "lower aryl," as used herein, alone or in combination, means phenyl or naphthyl, which may be optionally substituted as specified.

[0128] The term "lower heteroaryl," as used herein, alone or in combination, means either (1) a monocyclic heteroaryl containing 5 or 6 ring members, 1 to 4 of which may be heteroatoms selected from N, O, and S, or (2) a bicyclic heteroaryl in which each of the fused rings contains 5 or 6 ring members and between them 1 to 4 heteroatoms selected from N, O, and S.

[0129] The term "lower cycloalkyl," as used herein, alone or in combination, refers to a monocyclic cycloalkyl having 3 to 6 ring members (i.e., C3-C6 cycloalkyl). A lower cycloalkyl may be unsaturated. Examples of lower cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0130] The term "lower heterocycloalkyl," as used herein, alone or in combination, means a monocyclic heterocycloalkyl (i.e., a C3-C6 heterocycloalkyl) having 3 to 6 ring members, 1 to 4 of which may be heteroatoms selected from N, O, and S. Examples of lower heterocycloalkyls include pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, and morpholinyl. A lower heterocycloalkyl may be unsaturated.

[0131] The term "lower amino," as used herein, alone or in combination, refers to --NRR', where R and R' are independently selected from hydrogen and lower alkyl, either of which may be optionally substituted.

[0132] The term "mercaptyl," as used herein, alone or in combination, refers to an RS-group, where R is as defined herein.

[0133] The term "nitro," as used herein, alone or in combination, refers to -NO2.

[0134] The terms "oxy" or "oxa," as used herein, alone or in combination, refer to --O--.

[0135] The term "oxo," as used herein, alone or in combination, refers to =0.

[0136] The term "perhaloalkoxy" refers to an alkoxy group in which all of the hydrogen atoms have been replaced with halogen atoms.

[0137] The term "perhaloalkyl," as used herein, alone or in combination, refers to an alkyl group in which all of the hydrogen atoms have been replaced with halogen atoms.

[0138] The terms "sulfonate," "sulfonic acid," and "sulfonic" as used herein, alone or in combination, refer to the -SO3H group and its anion when sulfonic acid is used in salt formation.

[0139] The term "sulfanyl," as used herein, alone or in combination, refers to --S--.

[0140] The term "sulfinyl," as used herein, alone or in combination, refers to --S(O)--.

[0141] The term "sulfonyl," as used herein, alone or in combination, refers to -S(O)2-.

[0142] The term "N-sulfonamido" refers to an RS(=O)2NR'- group, where R and R' are as defined herein.

[0143] The term "S-sulfonamido" refers to a -S(=O)2NRR' group, where R and R' are as defined herein.

[0144] The terms "thia" and "thio," as used herein, alone or in combination, refer to the -S- group or to ethers where the oxygen is replaced with sulfur. The oxidized derivatives of the thio group, i.e., sulfinyl and sulfonyl, are included in the definition of thia and thio.

[0145] The term "thiol," as used herein, alone or in combination, refers to an SH group.

[0146] The term "thiocarbonyl," as used herein, when alone includes thioformyl-C(S)H and in combination is a -C(S)- group.

[0147] The term "N-thiocarbamyl" refers to an ROC(S)NR'- group, with R and R' as defined herein.

[0148] The term "O-thiocarbamyl" refers to an --OC(S)NRR' group, where R and R' are as defined herein.

[0149] The term "thiocyanato" refers to the group -CNS.

[0150] The term "trihalomethanesulfonamide" refers to a X3CS(O)2NR- group where X is a halogen and R is as defined herein.

[0151] The term "trihalomethanesulfonyl" refers to a X3CS(O)2- group where X is a halogen.

[0152] The term "trihalomethoxy" refers to a X3CO- group where X is a halogen.

[0153] The term "trisubstituted silyl," as used herein, alone or in combination, refers to a silicone group whose three free valences are substituted with a group listed herein under the definition of substituted amino. Examples include trimethylsilyl, tert-butyldimethylsilyl, triphenylsilyl, etc.

[0154] Any definition herein can be used in combination with any other definition to describe a composite structural group.By convention, the last element of any such definition is the one that is attached to the parent moiety.For example, the composite group alkylamido is considered to represent an alkyl group that is attached to the parent molecule via an amide group, and the term alkoxyalkyl is considered to represent an alkoxy group that is attached to the parent molecule via an alkyl group.

[0155] When a group is defined to be "null," what is meant is that the group is absent.

[0156] The term "optionally substituted" means that the preceding group can be substituted or unsubstituted. When substituted, the substituents on the "optionally substituted" group can include, but are not limited to, one or more substituents independently selected from the following groups or a specific specified series of groups, alone or in combination: lower alkyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower heteroalkyl, lower heterocycloalkyl, lower haloalkyl, lower haloalkenyl, lower haloalkynyl, lower perhaloalkyl, lower perhaloalkoxy, lower cycloalkyl, phenyl, aryl, aryloxy, lower alkoxy, lower Haloalkoxy, oxo, lower acyloxy, carbonyl, carboxyl, lower alkylcarbonyl, lower carboxyester, lower carboxyamide, cyano, hydrogen, halogen, hydroxy, amino, lower alkylamino, arylamino, amido, nitro, thiol, lower alkylthio, lower haloalkylthio, lower perhaloalkylthio, arylthio, sulfonate, sulfonic acid, trisubstituted silyl, N3, SH, SCH3, C(O)CH3, CO2CH3, CO2H, pyridinyl, thiophene, furanyl, lower carbamate, and lower urea. When structurally possible, two substituents may be joined together to form a fused 5-, 6-, or 7-membered carbocyclic or heterocyclic ring consisting of 0 to 3 heteroatoms, for example, methylenedioxy or ethylenedioxy. Optionally substituted groups can be unsubstituted (e.g., -CH2CH3), fully substituted (e.g., -CF2CF3), monosubstituted (e.g., -CH2CH2F), or substituted at any level between fully and monosubstituted (e.g., -CH2CF3). When a substituent is recited without qualification as to substitution, both substituted and unsubstituted forms are encompassed. When a substituent is qualified as "substituted," the substituted form is specifically intended. Additionally, various sets of optional substituents for a particular moiety may be defined as necessary; in these cases, the optional substitution is considered as defined and often immediately follows the phrase "optionally substituted with."

[0157] The term R or R', appearing by itself and without a number designation, unless otherwise defined, refers to a moiety selected from hydrogen, alkyl, cycloalkyl, heteroalkyl, aryl, heteroaryl, and heterocycloalkyl, any of which may be optionally substituted. Such R and R' groups should be understood to be optionally substituted as defined herein. Regardless of whether an R group has a number designation, R, R', and R groups with n = (1, 2, 3, ...n) n All R groups, including all substituents, and all terms, should be understood to be independent of all others with respect to selection from a group. If any variable, substituent, or term (e.g., aryl, heterocycle, R, etc.) occurs more than once in a formula or generic structure, its definition at each occurrence is independent of its definition at every other occurrence. Those skilled in the art will further recognize that certain groups can be attached to a parent molecule or can occupy a position within a chain of elements from either end as described. For example, an asymmetric group such as -C(O)N(R)- can be attached to the parent moiety at either the carbon or the nitrogen.

[0158] Asymmetric centers exist in the compounds disclosed herein. These centers are designated by the symbol "R" or "S," depending on the arrangement of substituents around the chiral carbon atom. It should be understood that the present disclosure encompasses all stereochemical isomeric forms, such as diastereomeric, enantiomeric, and epimeric forms, as well as d- and l-isomers, and mixtures thereof. Individual stereoisomers of the compounds can be prepared synthetically from commercially available starting materials containing chiral centers, or by preparing a mixture of enantiomeric products followed by separation, for example, by conversion to a mixture of diastereomers followed by separation or recrystallization, chromatographic techniques, direct separation of enantiomers on a chiral chromatographic column, or any other suitable method known in the art. Starting compounds of a particular stereochemistry are either commercially available or can be prepared and separated by techniques known in the art. Furthermore, the compounds disclosed herein may exist as geometric isomers. The present disclosure includes all cis, trans, syn, anti, entgegen (E), and zusammen (Z) isomers, as well as the appropriate mixtures thereof. Additionally, compounds may exist as tautomers; all tautomers are provided by the present disclosure. Furthermore, the compounds disclosed herein may exist in unsolvated forms as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. In general, solvated forms are considered equivalent to unsolvated forms.

[0159] The term "bond" refers to a covalent bond between two atoms, or between two moieties when the atoms connected by the bond are considered to be part of a larger substructure. A bond may be single, double, or triple unless otherwise specified. A dashed line between two atoms in a drawing of a molecule indicates that an additional bond may or may not be present at that position.

[0160] The compounds disclosed herein may exist as therapeutically acceptable salts. The present disclosure includes the compounds listed above in the form of salts, including acid addition salts. Suitable salts include those formed with both organic and inorganic acids. Such acid addition salts are generally considered pharmaceutically acceptable. However, salts with non-pharmaceutically acceptable salts may be useful in the preparation and purification of the compounds under discussion. Base addition salts may also be formed and may be pharmaceutically acceptable. For a more complete discussion of salt preparation and selection, see Pharmaceutical Salts: Properties, Selection, and Use (Stahl, P. Heinrich. Wiley-VCHA, Zurich, Switzerland, 2002).

[0161] The term "therapeutically acceptable salt," as used herein, refers to a salt or zwitterionic form of a compound disclosed herein that is soluble or dispersible in water or oil and therapeutically acceptable as defined herein. Salts can be prepared during the final isolation and purification of the compound, or can be prepared separately by reacting the free base form of the appropriate compound with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, L-ascorbate, aspartate, benzoate, benzenesulfonate (besylate), bisulfate, butyrate, camphorate, camphorsulfonate, citrate, digluconate, formate, fumarate, gentisate, glutarate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isethionate), lactate, maleate, malonate, DL-mande, and the like. The basic group in the compounds disclosed herein may be methyl, ethyl, propyl, and butyl chloride, bromide, and iodide; dimethyl, diethyl, dibutyl, and diamyl sulfate; decyl chloride, bromide, and iodide, lauryl, myristyl, and steryl chloride; and benzyl bromide and phenethyl bromide. Examples of acids which can be used to form therapeutically acceptable addition salts include inorganic acids such as hydrochloric, hydrobromic, sulfuric, and phosphoric acids, and organic acids such as oxalic, maleic, succinic, and citric acids. Salts can also be formed by coordination of a compound with an alkali metal ion or alkaline earth ion.Thus, the present disclosure contemplates sodium, potassium, magnesium, calcium salts, and the like, of the compounds disclosed herein.

[0162] Base addition salts can be prepared during the final isolation and purification of the compound by reacting the carboxyl group with a suitable base, such as a hydroxide, carbonate, or bicarbonate salt of a metal cation, or with ammonia or an organic primary, secondary, or tertiary amine. Therapeutically acceptable salt cations include lithium, sodium, potassium, calcium, magnesium, and aluminum, as well as non-toxic quaternary amine cations such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine. Other representative organic amines useful for forming base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, and piperazine.

[0163] Although the compound of the present disclosure can be administered as raw chemicals, it can also be provided as pharmaceutical preparations.Therefore, the present disclosure provides pharmaceutical preparations comprising one or more of the specific compounds disclosed herein, or one or more of their pharmaceutically acceptable salts, esters, prodrugs, amides or solvates, together with one or more of their pharmaceutically acceptable carriers and optionally one or more other therapeutic ingredients.Carriers must be "acceptable" in the sense that they are compatible with other ingredients of the formulation and are not harmful to the recipient.Appropriate preparations depend on the route of administration selected.Any well-known technique, carrier and excipient can be used as appropriate and as understood in the art.The pharmaceutical compositions disclosed herein can be prepared by any method known in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, pulverizing, emulsifying, encapsulating, encapsulating or compressing process.

[0164] Formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intraarticular, and intramedullary), intraperitoneal, transmucosal, transdermal, rectal, and topical (including cutaneous, buccal, sublingual, and ocular) administration, although the most suitable route may depend, for example, on the condition and disorder of the recipient. The formulations are conveniently presented in unit dosage form and can be prepared by any of the methods well known in the art of pharmacy. Typically, these methods include the step of combining a compound of the present disclosure or a pharmaceutically acceptable salt, ester, amide, prodrug, or solvate thereof (the "active ingredient") with a carrier which constitutes one or more accessory ingredients. In general, formulations are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired formulation.

[0165] II. Anti-HER2 / HER3 antibody As used herein, "anti-HER2 / HER3 antibody" includes any molecule that interferes with the function of HER2 and / or HER3. Thus, anti-HER2 / HER3 antibodies include anti-HER2 antibodies (e.g., trastuzumab or pertuzumab), anti-HER3 antibodies, and anti-HER2 / HER3 bispecific antibodies (e.g., antibodies disclosed in WO2018 / 182422 or MCLA-128). HER2 / HER3 targeting antibodies can prevent the formation of HER2 / HER2 dimers and / or HER2 / HER3 dimers (e.g., trastuzumab or pertuzumab). In some cases, HER2 / HER3 targeting antibodies can be antibody-drug conjugates (e.g., T-DM1 or U3-1402).

[0166] In certain embodiments, the HER2 / HER3 targeting antibody is selected from the group consisting of trastuzumab (Genentech and Roche), trastuzumab emtansine (T-DM1; Genentech and Roche), pertuzumab (Genentech), ertumaxomab (Fresenius), margetuximab (MacroGenics), MCLA-128 (zenoctuzumab; Merus), MM-111 (Merrimack), MM-121 (Merrimack), CT-P06 (Celltrion), GSK2849330 (GlaxoSmithKline), PF-05280014 (Pfizer), MM-302 (Merrimack), SB3 (Merck & Co), CMAB302 (Shanghai CP Guojian), RG7116 (lemretuzumab; Genentech / Roche), TrasGEX (Glycotope), ARX788 (Ambrx and Zhejiang Medicine), SYD985 (Synthon), FS102 (Bristol-Myers Squibb and f-star), BCD-022 (Biocad), ABP 980 (Amgen), DS-8201a (Daiichi Sankyo), HLX02 (Shanghai Henlius), SAR256212 (Sanofi Oncology), RG7597 (Genentech), U3-1402 (Daiichi Sankyo), or CANMAb (Biocon and Mylan).

[0167] Trastuzumab (CAS 180288-69-1, HERCEPTIN®, huMAb4D5-8, rhuMAb HER2, Genentech) is a humanized IgG1 kappa monoclonal antibody that binds with high affinity and selectivity to the extracellular domain of the human epidermal growth factor receptor 2 protein, HER2 (ErbB2) (U.S. Patent Nos. 5,677,171; 5,821,337; 6,054,297; 6,165,464; 6,339,142; 6,407,213; 6,639,055; 6,719,971; 6,800,738; 7,074,404). Trastuzumab contains human framework regions with the complementarity-determining regions of a murine antibody (4D5) that binds to HER2. Trastuzumab binds to the HER2 antigen, thereby inhibiting the growth of cancerous cells. Trastuzumab has been shown to inhibit the growth of HER2-overexpressing human tumor cells in both in vitro assays and in animals. Trastuzumab is a mediator of antibody-dependent cellular cytotoxicity, or ADCC.

[0168] Trastuzumab emtansine, also known as ado-trastuzumab emtansine and sold under the trade name KADCYLA®, is an antibody-drug conjugate consisting of the humanized monoclonal antibody trastuzumab covalently linked to the cytotoxic agent emtansine (DM1). Trastuzumab alone stops cancer cell growth by binding to the HER2 receptor, but trastuzumab emtansine undergoes receptor-mediated internalization into cells and is catabolized in lysosomes, where DM1-containing catabolites are released and subsequently bind to tubulin, causing mitotic arrest and cell death. Trastuzumab binding to HER2 prevents receptor homodimerization or heterodimerization (HER2 / HER3), ultimately inhibiting the activation of MAPK and PI3K / AKT cell signaling pathways. Because the monoclonal antibody targets HER2, which is overexpressed only in cancer cells, the conjugate specifically delivers the cytotoxic agent DM1 to tumor cells. The conjugate is abbreviated as T-DM1. T-DM1 can be administered at a dose of 2-3 mg / kg, e.g., 3.6 mg / kg. T-DM1 can be administered by intravenous infusion.

[0169] Pertuzumab (CAS Registry Number 380610-27-5, OMNITARG®, 2C4, Genentech) is a recombinant humanized monoclonal antibody that inhibits HER2 dimerization (U.S. Patent Nos. 6,054,297; 6,407,213; 6,800,738; 6,627,196; 6,949,245; 7,041,292). Pertuzumab contains a human IgG1(x) framework sequence. Pertuzumab and trastuzumab target different extracellular regions of the HER2 tyrosine kinase receptor. Pertuzumab binds to an epitope within subdomain 2 of HER2, while the epitope of trastuzumab is located in subdomain 4. Pertuzumab blocks the ability of the HER2 receptor to cooperate with other HER receptor family members, namely, HER1 / EGFR, HER3, and HER4 (U.S. Patent No. 6,949,245). In cancer cells, by interfering with the ability of HER2 to cooperate with other HER family receptors, it blocks cell signaling, which can ultimately lead to the inhibition of cancer cell growth and cancer cell death.

[0170] Further exemplary anti-HER2 / HER3 antibodies include MM-121 / SAR256212, a fully human monoclonal antibody that targets the HER3 receptor and has been reported to be useful in the treatment of non-small cell lung cancer (NSCLC), breast cancer, and ovarian cancer. SAR256212 is an investigational fully human monoclonal antibody that targets the HER3 (ErbB3) receptor. Durigotuzumab (MEHD7945A, RG7597) is a humanized IgG1 monoclonal antibody that targets HER1 and HER3 and is described as useful for head and neck cancer. Margetuximab (MGAH22) is an Fc-optimized monoclonal antibody that targets HER2.

[0171] Antibodies according to the present disclosure can be defined primarily by their binding specificity. Those skilled in the art can determine whether a given antibody falls within the scope of the claims by assessing its binding specificity / affinity using techniques well known to those skilled in the art. Various techniques known to those skilled in the art can be used to determine whether an antibody interacts with a polypeptide or protein. Exemplary techniques include, for example, conventional cross-blocking assays. Cross-blocking can be measured using various binding assays, such as ELISA, biolayer interferometry, or surface plasmon resonance. Other methods include alanine scanning mutation analysis, peptide blot analysis, peptide truncation analysis, high-resolution electron microscopy using single-particle reconstruction, cryoEM, or tomography, crystallographic studies, and NMR analysis.

[0172] The present disclosure includes antibodies that can bind to the same epitope or a part of the epitope.In addition, the present disclosure also includes antibodies that compete with any of the specific exemplary antibodies described herein for binding to a target or a fragment thereof.Using conventional methods known in the art, it can be easily determined whether an antibody binds to the same epitope as a reference antibody or competes with the reference antibody for binding.For example, to determine whether a test antibody binds to the same epitope as a reference antibody, the reference antibody is bound to the target under saturating conditions.Then, the ability of the test antibody to bind to the target molecule is evaluated.If the test antibody can bind to the target molecule after saturating binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope from the reference antibody.On the other hand, if the test antibody cannot bind to the target molecule after saturating binding with the reference antibody, the test antibody may bind to the same epitope as the epitope bound by the reference antibody.

[0173] Two antibodies bind to the same or overlapping epitopes if they each competitively inhibit (block) the binding of the other to the antigen. That is, when measured in a competitive binding assay, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, but preferably 75%, 90%, or even 99%. Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

[0174] Then, further conventional experiments (such as peptide mutation and binding analysis) can be carried out to determine whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding.This type of experiment can be carried out using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.Structural studies using EM or crystallography can also show whether two antibodies that compete for binding recognize the same epitope.

[0175] In another aspect, antibodies can be defined by their variable sequences, including additional "framework" regions. Furthermore, antibody sequences can be varied from these sequences, optionally using methods described in more detail below. For example, nucleic acid sequences can be modified in the following ways: (a) the variable regions can be separated from the light and heavy chain constant domains; (b) the nucleic acid can be varied from those described above but without affecting the residues encoded thereby; (c) the nucleic acid can be varied from those described above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology; (d) the nucleic acid can be modified to a desired concentration, e.g., from about 0.02 M to about 0.15 M at a temperature of about 50° C. to about 70° C. (e) amino acids may vary from those described above by a given percentage of homology, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or (f) amino acids may vary from those described above by allowing for conservative substitutions (described below).

[0176] When comparing polynucleotide and polypeptide sequences, if the nucleotide or amino acid sequences of the two sequences are the same when aligned for maximum matching, as described below, the two sequences are said to be "identical." Comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20 consecutive positions, usually 30 to about 75, 40 to about 50, in which a sequence can be compared with a reference sequence of the same number of consecutive positions after optimally aligning the two sequences.

[0177] Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.) using default parameters.This program incorporates several alignment schemes described in the following references: Dayhoff, MO (1978) A model of evolutionary change in proteins—Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington, DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogeny pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, DG and Sharp, PM (1989) CABIOS 5:151-153; Myers, EW and Muller W. (1988) CABIOS 4:11-17; Robinson, ED (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425;Sneath, PHA and Sokal, RR (1973) Numerical Taxonomy--the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, WJ and Lipman, DJ (1983) Proc. Natl. Acad., Sci. USA 80:726-730.

[0178] Alternatively, optimal alignment of sequences for comparison can be performed by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computer implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.

[0179] One specific example of an algorithm suitable for determining percent sequence identity and percent sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example, with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the present disclosure. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The rearranged nature of antibody sequences and the variable length of each gene necessitates multiple rounds of BLAST searches for a single antibody sequence. Furthermore, manual assembly of different genes is difficult and error-prone. The sequence analysis tool IgBLAST (available on the World Wide Web at ncbi.nlm.nih.gov / igblast / ) reveals matches to germline V, D, and J genes, details of rearrangement junctions, and delineation of Ig V domain framework regions and complementarity-determining regions. IgBLAST can analyze either nucleotide or protein sequences, can process sequences in batches, and allows simultaneous searches against germline gene databases and other sequence databases, minimizing the chance of missing potentially best-matching germline V genes.

[0180] In one example, the cumulative score can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). Extension of word hits in each direction is stopped: until the cumulative alignment score is reduced by an amount X from its maximum achieved value; until the accumulation of one or more negative-scoring residue alignments causes the cumulative score to fall below zero; or until the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11 and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignment, a word length (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0181] For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of word hits in each direction is terminated: until the cumulative alignment score is reduced by an amount X from its maximum achieved value; until the accumulation of one or more negative-scoring residue alignments causes the cumulative score to fall below zero; or until the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.

[0182] In one approach, "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) of up to 20 percent, typically 5-15 percent or 10-12 percent, compared to the reference sequence (which does not contain additions or deletions) due to optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0183] Yet another way to define an antibody is as a "derivative" of any of the described antibodies and antigen-binding fragments thereof. The term "derivative" refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to an antigen but contains one, two, three, four, five, or more amino acid substitutions, additions, deletions, or modifications compared to the "parent" (or wild-type) molecule. Such amino acid substitutions or additions can introduce naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. The term "derivative" encompasses, for example, variants with altered CH1, hinge, CH2, CH3, or CH4 regions to form, for example, antibodies with variant Fc regions that exhibit enhanced or impaired effector or binding properties. The term "derivative" further encompasses amino acids that may have undergone non-amino acid modifications, such as glycosylation (e.g., altered content of mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-glycolneuraminic acid, etc.), acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, etc. In some embodiments, altered glycosylation modulates one or more of the following: antibody solubilization, facilitation of intracellular trafficking and secretion of the antibody, promotion of antibody assembly, conformational integrity, and antibody-mediated effector function. In certain embodiments, altered glycosylation enhances antibody-mediated effector function compared to an antibody lacking the glycosylation. Glycosylation modifications that lead to altered antibody-mediated effector function are well known in the art.

[0184] Derivative antibodies or antibody fragments can be generated with engineered sequences or glycosylation states to confer desired levels of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), or antibody-dependent complement deposition (ADCD) function activity as measured by bead- or cell-based assays or in vivo studies in animal models.

[0185] Derivative antibodies or antibody fragments can be modified by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. In one aspect, an antibody derivative will have similar or identical function as the parent antibody. In another aspect, an antibody derivative will exhibit altered activity compared to the parent antibody. For example, a derivative antibody (or fragment thereof) may bind to its epitope more tightly or be more resistant to proteolysis compared to the parent antibody.

[0186] III. Treatment Methods The present invention provides methods for treating cancer patients using quinazoline-based TKIs, either alone or in combination with anti-HER2 / HER3 antibodies. Such treatment can also be combined with other therapeutic regimens, such as chemotherapy or immunotherapy. Certain aspects of the present invention can be used to select cancer patients for treatment based on the presence of NRG1 fusions in the patient's cancer cells. In various aspects, approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cells comprising the cancer may harbor NRG1 fusions, indicating that the patient is a candidate for treatment. In some aspects, the patient's cancer cells lack mutations at EGFR T790 and / or EGFR C797. In some aspects, the patient's cancer cells lack a mutation in HER2 T798 and / or HER2 C805.

[0187] In certain aspects, the presence of NRG1 fusion is determined by analyzing the genome sample from the subject.In some aspects, the genome sample is isolated from saliva, blood, urine or tumor tissue.In certain aspects, the presence of NRG1 fusion is determined by nucleic acid sequencing (for example, DNA sequencing of tumor tissue or plasma-derived circulating free DNA) or PCR analysis.

[0188] In certain aspects, the quinazoline-based TKI and / or anti-HER2 / HER3 antibody is administered intravenously, subcutaneously, intraosseously, orally, transdermally, sustained-release, controlled-release, delayed-release, as a suppository, or sublingually. In some aspects, the administration of the quinazoline-based TKI and / or anti-HER2 / HER3 antibody includes local, regional, or systemic administration. In certain aspects, the quinazoline-based TKI and / or anti-HER2 / HER3 antibody is administered two or more times, for example, daily, every other day, or weekly. The quinazoline-based TKI and anti-HER2 / HER3 antibody do not need to be administered by the same route or on the same schedule.

[0189] In some aspects, the quinazoline-based TKI is administered before or after the anti-HER2 / HER3 antibody, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 1 month or more apart. In some aspects, the quinazoline-based TKI is administered simultaneously with the anti-HER2 / HER3 antibody.

[0190] As used herein, the term " subject " or " patient " refers to any individual that the subject method is carried out.Generally, patient is human, but as recognized by those skilled in the art, patient can also be animal.Therefore, other animals are included in the definition of patient, including mammals, for example, rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, livestock (including cows, horses, goats, sheep, pigs, etc.), and primates (including monkeys, chimpanzees, orangutans and gorillas).

[0191] "Treatment" and "treating" refer to the administration or application of a therapeutic agent to a subject or the performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit for a disease or health-related condition. For example, treatment can include administering chemotherapy, immunotherapy, radiation therapy, performing surgery, or any combination thereof.

[0192] The methods described herein are useful for inhibiting cell (e.g., tumor cell) survival or proliferation, treating proliferative diseases (e.g., cancer, psoriasis), and treating pathogenic infections. Generally, the terms "cancer" and "cancerous" refer to or describe a physiological state in a mammal that is typically characterized by unregulated cell growth. More specifically, cancers treated in connection with the methods provided herein include, but are not limited to, solid tumors, metastatic cancers, or non-metastatic cancers. In certain embodiments, cancer can occur in the lung, kidney, bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gums, head, liver, nasopharynx, cervix, ovaries, pancreas, prostate, skin, stomach, testes, tongue, or uterus.

[0193] The cancer may be of the following histological types, among others, but not limited to: neoplasia, malignancy; carcinoma; non-small cell lung cancer; kidney cancer; renal cell carcinoma; renal clear cell carcinoma; lymphoma; blastoma; sarcoma; carcinoma, undifferentiated; meningioma; brain tumor; oropharyngeal cancer; nasopharyngeal carcinoma; biliary tract cancer; pheochromocytoma; pancreatic islet cell carcinoma; Li-Fraumeni tumor; thyroid cancer; parathyroid cancer; pituitary tumor; adrenal tumor; osteogenic sarcoma tumor; neuroendocrine tumor; breast cancer; lung cancer; head and neck cancer; prostate cancer; esophageal cancer; tracheal cancer; liver cancer; bladder cancer; gastric cancer; pancreatic cancer; ovarian cancer; uterine cancer; cervical cancer; testicular cancer ;Colon cancer;Rectal cancer;Skin cancer;Giant cell and spindle cell carcinoma;Small cell carcinoma;Small cell lung cancer;Papillary carcinoma;Oral cancer;Oral pharyngeal cancer;Nasopharyngeal cancer;Respiratory cancer;Genitourinary cancer;Squamous cell carcinoma;Lymphoepithelial carcinoma;Basal cell carcinoma;Hair stromal carcinoma;Transitional cell carcinoma;Papillary transitional cell carcinoma;Adenocarcinoma;Gastrointestinal cancer;Gastrinoma, malignant;Bile duct carcinoma;Hepatocellular carcinoma;Mixed hepatocellular and cholangiocarcinoma;Follicle adenocarcinoma;Adenoid cystic carcinoma;Adenomatous intrapolypoid adenocarcinoma;Adenocarcinoma, familial polyposis coli;Solid tumors;Carcinoid tumors, malignant;Bronchioloalveolar carcinoma;Papillary adenocarcinoma;Chromophobe Cancer;Eosinophilic carcinoma;Eosinophilic adenocarcinoma;Basophilic carcinoma;Clear cell adenocarcinoma;Granular cell carcinoma;Follicle adenocarcinoma;Papillary-follicular adenocarcinoma;Non-encapsulated sclerosing carcinoma;Adrenal cortical carcinoma;Endometrioid carcinoma;Cut adnexal carcinoma;Apocrine adenocarcinoma;Sebaceous gland carcinoma;Eurinary gland carcinoma;Mucous epidermoid carcinoma;Cystadenocarcinoma;Papillary cystadenocarcinoma;Papillary serous cystadenocarcinoma;Mucinous cystadenocarcinoma;Mucinous adenocarcinoma;Signet ring cell carcinoma;Invasive ductal carcinoma;Medullary carcinoma;Lobular carcinoma;Inflammatory carcinoma;Paget's disease of the breast;Acinic cell carcinoma;Adenosquamous carcinoma;Adenocarcinoma with squamous metaplasia;Thymoma, malignant;Ovarian stromal tumor, malignant;Theca cell tumor, malignant ;Granulosa cell tumor, malignant;Androblastoma, malignant;Sertoli cell carcinoma;Leydig cell tumor, malignant;Lipocyte tumor, malignant;Paraganglioma, malignant;Extramammary paraganglioma, malignant;Pheochromocytoma;Hemangioangiosarcoma;Malignant melanoma;Amelanotic melanoma;Superficial spreading melanoma;Malignant melanoma in giant pigmented nevus;Lentigo maligna melanoma;Acral lentigo melanoma;Nodular melanoma;Epithelioid cell melanoma;Blue nevus, malignant;Sarcoma;Fibrosarcoma;Fibrous histiocytoma, malignant;Myxosarcoma;Liposarcoma;Leiomyosarcoma;Rhabdomyosarcoma;Embryonal rhabdomyosarcoma;Alveolar rhabdomyosarcoma;Stromatous sarcoma;Mixed tumor, malignant;Mixed Müllerian tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Mesenchymoma, malignant; Brenner tumor, malignant; Phyllodes tumor, malignant; Synovial sarcoma; Mesothelioma, malignant; Dysgerminoma; Embryonal carcinoma; Teratoma, malignant; Ovarian stromatosis, malignant; Chorioepithelioma; Mesonephroma, malignant; Angiosarcoma; Hemangioendothelioma, malignant; Kaposi's sarcoma; Hemangiopericytoma, malignant; Lymphangiosarcoma; Osteosarcoma; Parosteal osteosarcoma; Chondrosarcoma; Chondroblastic, malignant; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's Sarcoma; Odontogenic tumor, malignant; Ameloblastic odontosarcoma; Ameloblastoma, malignant; Ameloblastoma fibrosarcoma; Endocrine or neuroendocrine or hematopoietic carcinoma; Pinealoma, malignant; Chordoma; Cancer of central or peripheral nervous system tissue; Glioma, malignant; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrous astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal tumor; Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Reticulum Membranoblastoma; Olfactory nerve tumor; Meningioma, malignant; Neurofibrosarcoma; Schwannoma, malignant; Granular cell tumor, malignant; B-cell lymphoma; Malignant lymphoma; Hodgkin's disease; Hodgkin's; Low-grade / follicular non-Hodgkin's lymphoma; Paragranuloma; Malignant lymphoma, small lymphoid; Malignant lymphoma, large cell, diffuse; Malignant lymphoma, follicular; Mycosis fungoides; Mantle cell lymphoma; Waldenstrom's macroglobulinemia; Other specified non-Hodgkin's lymphoma; Malignant Histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and hairy cell leukemia.

[0194] The term "therapeutic benefit" or "therapeutically effective," as used throughout this application, refers to anything that promotes or enhances the well-being of a subject in relation to the medical treatment of the condition. This includes, but is not limited to, reducing the frequency or severity of signs or symptoms of a disease. For example, cancer treatment can involve, for example, reducing the invasiveness of a tumor, reducing the rate of cancer growth, or preventing metastasis. Cancer treatment can also refer to extending the survival of a subject with cancer.

[0195] Similarly, a patient's effective response to treatment or a patient's "responsiveness" refers to the clinical or therapeutic benefit that is provided to a patient at risk of or suffering from a disease or disorder. Such benefit can include a cellular or biological response, a complete response, a partial response, a stable disease (no progression or recurrence), or a response with late recurrence. For example, an effective response can be a reduction in tumor size or progression-free survival in a patient diagnosed with cancer.

[0196] Regarding the treatment of neoplastic conditions, depending on the stage of the neoplastic condition, the treatment of the neoplastic condition involves one or a combination of the following therapies: surgery to remove neoplastic tissue, radiation therapy, and chemotherapy.Other therapeutic regimens can be combined with the administration of anti-cancer agents, such as therapeutic compositions and chemotherapeutic agents.For example, patients treated with such anti-cancer agents can also undergo radiation therapy and / or surgery.

[0197] For the treatment of a disease, the appropriate dosage of the therapeutic composition will depend on the type of disease being treated, as defined above, the severity and course of the disease, previous therapy, the patient's medical history and response to the agent, and the discretion of the physician. The agent may be suitably administered to the patient at one time or over a series of treatments.

[0198] Methods and compositions that include combination therapy enhance the therapeutic or protective effect and / or increase the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy. Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect, such as killing cancer cells and / or inhibiting cell hyperproliferation. Tissues, tumors, or cells can be contacted with one or more compositions or pharmacological preparations containing one or more agents, or by contacting the tissues, tumors, and / or cells with two or more different compositions or preparations. It is also contemplated that such combination therapy can be used in conjunction with radiation therapy, surgery, or immunotherapy.

[0199] The combined administration can include simultaneous administration of two or more agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, the subject therapeutic composition and another therapeutic agent can be formulated together in the same dosage form and administered simultaneously. Alternatively, the subject therapeutic composition and another therapeutic agent can be administered simultaneously, in which case both agents are present in separate formulations. In another alternative, a therapeutic agent can be administered immediately followed by another therapeutic agent, or vice versa. In separate administration protocols, the subject therapeutic composition and another therapeutic agent can be administered several days apart, or several hours apart, or several days apart.

[0200] A first anticancer treatment can be administered before, during, or after a second anticancer treatment, or in various combinations. Administration can occur over a period ranging from simultaneous administration to minutes to days to weeks. In embodiments in which a first treatment is provided to a patient separately from a second treatment, there will generally be an effective period between the time of delivery, ensuring that the two compounds can still exert their beneficially combined effect on the patient. In such cases, it is contemplated that the first and second therapies can be provided to the patient within about 12 to 24 or 72 hours of each other, more particularly, within about 6 to 12 hours of each other. In some situations, it may be desirable to extend the period of treatment significantly, where days (2, 3, 4, 5, 6, or 7) to weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between each administration.

[0201] In certain embodiments, a course of treatment is contemplated to last from 1 to 90 days or more (such ranges inclusive). It is contemplated that one agent can be given on any day from day 1 to day 90 (such ranges inclusive), or any combination thereof, and another agent can be given on any day from day 1 to day 90 (such ranges inclusive), or any combination thereof. One or more doses of the agent can be administered to the patient within a single day (24-hour period). Furthermore, it is contemplated that after a course of treatment, there will be a period during which no anti-cancer treatment is administered. This period can last from 1 to 7 days, and / or 1 to 5 weeks, and / or 1 to 12 months or more (such ranges inclusive), depending on the patient's condition, e.g., the patient's prognosis, strength, health, etc. It is expected that the treatment cycle will be repeated as necessary.

[0202] Various combinations can be used. For the following examples, either (a) a quinazoline-based TKI is "A" and an anti-HER2 / HER3 antibody is "B"; or (b) a quinazoline-based TKI, either alone or in combination with an anti-HER2 / HER3 antibody, is "A" and another anti-cancer therapy is "B": TIFF0007734139000048.tif27128.

[0203] Administration of any compound or therapy of the present invention to a patient will follow standard protocols for the administration of such compounds, taking into account the toxicity, if any, of the agent. Thus, in some embodiments, there is a step of monitoring for toxicity that may result from the combination therapy.

[0204] 1.Chemotherapy A wide variety of chemotherapeutic agents can be used in accordance with the present invention. The term "chemotherapy" refers to the use of drugs to treat cancer. The term "chemotherapeutic agent" is used to include compounds or compositions administered in cancer therapy. These agents or drugs are categorized by their mode of activity within cells, for example, whether they affect the cell cycle and at what stage they affect the cell cycle. Alternatively, agents can be characterized based on their ability to directly crosslink DNA, intercalate into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0205] Examples of chemotherapeutic agents include: alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines, such as altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptogens (especially cryptogens) ... tophicins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, e.g., chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquinone, fenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 11 and calicheamicin omega 11); dynemicins, such as dynemicin A; bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, authranycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo- L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, potfiromycin, piriformisin, anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamipiri and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadrenal agents such as mitotane and trilostane; folic acid supplements such as furoic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilon; etoglucide;Gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triazicone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vin Desine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Taxoids, e.g., paclitaxel and docetaxel, gemcitabine; 6-thioguanine; Mercaptopurine; Platinum coordination complexes, e.g., cisplatin, oxaliplatin, and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopterin; Xeloda; Ibandronate; Irinotecan (e.g., CPT-11); Topoisomerase inhibitors RFS 2000; difluoromethylornithine (DFMO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl protein transferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0206] 2. Radiation therapy Other widely used agents that cause DNA damage include gamma rays, X-rays, and / or what are commonly known as directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are also contemplated, such as microwaves, proton beam irradiation (U.S. Patent Nos. 5,760,395 and 4,870,287), and UV irradiation. All of these agents most likely affect a wide range of damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from daily doses of 50-200 roentgens for prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. The dose range for radioisotopes varies widely and depends on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells.

[0207] 3. Immunotherapy Those skilled in the art will understand that additional immunotherapy can be used in combination with or in conjunction with the methods of the present invention. In cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (Rituxan®) is one such example. The immune effector can be, for example, an antibody specific to some marker on the surface of tumor cells. The antibody alone can act as the therapeutic effector, or the antibody can recruit other cells to actually affect cell death. The antibody can also be conjugated to a drug or toxin (such as a chemotherapeutic agent, a radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and simply act as a targeting agent. Alternatively, the effector can be a lymphocyte bearing a surface molecule that interacts either directly or indirectly with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0208] In one aspect of immunotherapy, tumor cells must have some marker suitable for targeting, i.e., not present on the majority of other cells. Many tumor markers exist, any of which may be suitable for targeting in the present invention. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is to combine anti-cancer effects with immunostimulatory effects. There are also immunostimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

[0209] Examples of immunotherapies currently under consideration or in use include immunoadjuvants, such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patents 5,801,005 and 5,739,169; Hui and Hashimoto, Infection Immun., 66(11):5329-5336, 1998; Christodoulides et al., Microbiology, 144(Pt 11):3027-3037, 1998); cytokine therapy, such as interferon α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., Clinical Cancer Res., 4(10):2337-2347, 1998; Davidson et al., J. Immunother., 21(5):389-398, 1998; Hellstrand et al., Acta Oncologica, 37(4):347-353, 1998); gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., Proc. Natl. Acad. Sci. USA, 95(24):14411-14416, 1998; Austin-Ward and Villaseca, Revista Medica de Chile, 126(7):838-845, 1998; U.S. Pat. Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-p185 (Hanibuchi et al., Int. J. Cancer, 78(4):480-485, 1998; U.S. Patent No. 5,824,311). It is contemplated that one or more anti-cancer therapies can be used in conjunction with the antibody therapies described herein.

[0210] In some embodiments, immunotherapy can be adoptive immunotherapy, which involves transferring autoantigen-specific T cells generated ex vivo. T cells used for adoptive immunotherapy can be generated either by expanding antigen-specific T cells or by redirecting T cells through genetic engineering. The isolation and transfer of tumor-specific T cells has been shown to be successful in the treatment of melanoma. Genetic transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) has successfully generated novel T cell specificities. CARs are synthetic receptors consisting of a targeting moiety linked to one or more signaling domains in a single fusion molecule. Generally, the binding moiety of a CAR consists of the antigen-binding domain of a single-chain antibody (scFv), which contains the light chain variable fragment of a monoclonal antibody linked by a flexible linker. Binding moieties based on receptor or ligand domains have also been successfully used. The signaling domain of first-generation CARs is derived from the cytoplasmic region of CD3 zeta or the Fc receptor gamma chain. CARs have successfully redirected T cells against antigens expressed on the surface of tumor cells from a variety of malignancies, including lymphomas and solid tumors.

[0211] In one embodiment, the present application provides a combination therapy for cancer treatment, the combination therapy comprising an adoptive T cell therapy and a checkpoint inhibitor. In one aspect, the adoptive T cell therapy comprises autologous and / or allogeneic T cells. In another aspect, the autologous and / or allogeneic T cells are targeted to a tumor antigen.

[0212] Immunomodulators include immune checkpoint inhibitors, costimulatory molecule agonists, and immune inhibitory molecule antagonists. Immunomodulators can be drugs, such as small molecules, recombinant forms of ligands or receptors, or antibodies, such as human antibodies (e.g., International Patent Publication No. WO2015 / 016718; Pardoll, Nat Rev Cancer, 12(4): 252-264, 2012; both are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or their analogs can be used, particularly chimeric, humanized, or human forms of antibodies. As those skilled in the art will recognize, alternative and / or equivalent names may be used for certain antibodies referred to in this disclosure. Such alternative and / or equivalent names are interchangeable in this disclosure. For example, it is known that lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0213] Costimulatory molecules are ligands that interact with receptors on the surface of immune cells, such as CD28, 4-1BB, OX40 (also known as CD134), ICOS, and GITR. As an example, the complete protein sequence of human OX40 has GenBank accession number NP_003318. In some embodiments, the immunomodulatory agent is an anti-OX40 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human OX40 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present method can be generated using methods well known in the art. Alternatively, art-recognized anti-OX40 antibodies can be used. An exemplary anti-OX40 antibody is PF-04518600 (see, e.g., WO 2017 / 130076). ATOR-1015 is a bispecific antibody that targets CTLA4 and OX40 (see, e.g., WO 2017 / 182672, WO 2018 / 091740, WO 2018 / 202649, WO 2018 / 002339).

[0214] Another costimulatory molecule that can be targeted by the methods provided herein is ICOS, also known as CD278. The complete protein sequence of human ICOS has GenBank accession number NP_036224. In some embodiments, the immune checkpoint inhibitor is an anti-ICOS antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human ICOS antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present method can be generated using methods well known in the art. Alternatively, art-recognized anti-ICOS antibodies can be used. Exemplary anti-ICOS antibodies include JTX-2011 (see, for example, WO 2016 / 154177, WO 2018 / 187191) and GSK3359609 (see, for example, WO 2016 / 059602).

[0215] Yet another costimulatory molecule that can be targeted by the methods provided herein is glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), also known as TNFRSF18 and AITR. The complete protein sequence of human GITR has GenBank accession number NP_004186. In some embodiments, the immunomodulator is an anti-GITR antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human GITR antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present method can be generated using methods well known in the art. Alternatively, art-recognized anti-GITR antibodies can be used. An exemplary anti-GITR antibody is TRX518 (see, for example, WO 2006 / 105021).

[0216] Immune checkpoint proteins that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), CCL5, CD27, CD38, CD8A, CMKLR1, cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), CXCL9, CXCR5, HLA-DRB1, HLA-DQA1, HLA-E, killer cell immunoglobulin (KIR), lymphocyte activation genes These include CD19-3 (also known as CD223, LAG-3), Mer tyrosine kinase (MerTK), NKG7, programmed death 1 (PD-1), programmed death-ligand 1 (PD-L1, also known as CD274), PDCD1LG2, PSMB10, STAT1, T cell immunoreceptor with Ig and ITIM domains (TIGIT), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V domain Ig suppressor of T cell activation (VISTA, also known as C10orf54). In particular, immune checkpoint inhibitors targeting the PD-1 axis and / or CTLA-4 have received widespread FDA approval across a variety of cancer types.

[0217] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand-binding partner. In a specific aspect, the PD-1 ligand-binding partner is PD-L1 and / or PD-L2. In another embodiment, the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In a specific aspect, the PD-L1 binding partner is PD-1 and / or B7-1. In another embodiment, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner. In a specific aspect, the PD-L2 binding partner is PD-1. The antagonist can be an antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art and are described, for example, in U.S. Patent Application Publication Nos. 2014 / 0294898, 2014 / 022021, and 2011 / 0008369, all of which are incorporated herein by reference.

[0218] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion (e.g., an Fc region of an immunoglobulin sequence) of PD-L1 or PD-L2 fused to a constant region). In some embodiments, the PD-1 binding antagonist is AMP-224. MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO (登録商標)Nivolumab, also known as MK-3475, Merck3475, lambrolizumab, KEYTRUDA, is an anti-PD-1 antibody described in WO2006 / 121168. (登録商標) Pembrolizumab, also known as PD-L2-Fc fusion soluble receptor (PD-L2-Fc), is described in WO2009 / 114335. Pembrolizumab, also known as PD-L2-Fc fusion soluble receptor (PD-L2-Fc), is described in WO2010 / 027827 and WO2011 / 066342. ...09 / 114335. Pembrolizumab, also known as PD-L2-Fc fusion soluble receptor (PD-L2-Fc), is described in WO2009 / 101611. Pembrolizumab, also known as PD-L2-Fc fusion soluble receptor (PD-L2

[0219] Another immune checkpoint protein that can be targeted by the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has GenBank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA-4 is similar to the T cell costimulatory protein, CD28; both molecules bind to CD80 and CD68, also known as B7-1 and B7-2, respectively, on antigen-presenting cells. CTLA-4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA-4 is also found in regulatory T cells and may be important for their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for the B7 molecule.

[0220] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, anti-CTLA-4 antibodies disclosed in U.S. Pat. No. 8,119,129; PCT Publication Nos. WO 01 / 14424, WO 98 / 42752, WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab); U.S. Pat. No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA, 95(17): 10067-10071; Camacho et al. (2004) J Clin Oncology, 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res, 58:5301-5304 can be used in the methods disclosed herein. The teachings of each of the above publications are incorporated herein by reference.Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used.For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001 / 014424, WO2000 / 037504, and U.S. Patent No. 8,017,114; all are incorporated herein by reference.

[0221] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO 01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and / or binds to the same epitope on CTLA-4 as the above-described antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-described antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab). Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors such as those described in U.S. Pat. Nos. 5,844,905, 5,885,796, and International Patent Applications WO1995001994 and WO1998042752, which are incorporated herein by reference, and immunoadhesins such as those described in U.S. Pat. No. 8,329,867, which is incorporated herein by reference.

[0222] Another immune checkpoint protein that can be targeted in the methods provided herein is lymphocyte-activation gene 3 (LAG-3), also known as CD223. The complete protein sequence of human LAG-3 has GenBank accession number NP-002277. LAG-3 is found on the surface of activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. LAG-3 acts as an "off" switch when bound to MHC class II on the surface of antigen-presenting cells. Inhibition of LAG-3 activates both effector T cells and inhibitor regulatory T cells. In some embodiments, the immune checkpoint inhibitor is an anti-LAG-3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human LAG-3 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art-recognized anti-LAG-3 antibodies can be used. An exemplary anti-LAG-3 antibody is leratolimab (also known as BMS-986016) or its antigen-binding fragments and variants (see, e.g., WO 2015 / 116539). Other exemplary anti-LAG-3 antibodies include TSR-033 (see, e.g., WO 2018 / 201096), MK-4280, and REGN3767. MGD013 is an anti-LAG-3 / PD-1 bispecific antibody described in WO 2017 / 019846. FS118 is an anti-LAG-3 / PD-L1 bispecific antibody described in WO 2017 / 220569.

[0223] Another immune checkpoint protein that can be targeted in the methods provided herein is V-domain Ig suppressor of T-cell activation (VISTA), also known as C10orf54. The complete protein sequence of human VISTA has GenBank accession number NP_071436. VISTA is found in white blood cells and inhibits the effector function of T cells. In some embodiments, the immune checkpoint inhibitor is an anti-VISTA antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human VISTA antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art-recognized anti-VISTA antibodies can be used. An exemplary anti-VISTA antibody is JNJ-61610588 (also known as onvatilimab) (see, e.g., WO 2015 / 097536, WO 2016 / 207717, WO 2017 / 137830, WO 2017 / 175058). VISTA can also be inhibited by the small molecule CA-170, which selectively targets both PD-L1 and VISTA (see, e.g., WO 2015 / 033299, WO 2015 / 033301).

[0224] Another immune checkpoint protein that can be targeted by the methods provided herein is CD38. The complete protein sequence of human CD38 has GenBank accession number NP_001766. In some embodiments, the immune checkpoint inhibitor is an anti-CD38 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human CD38 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art-recognized anti-CD38 antibodies can be used. An exemplary anti-CD38 antibody is daratumumab (see, e.g., U.S. Patent No. 7,829,673).

[0225] Another immune checkpoint protein that can be targeted by the methods provided herein is T cell immunoreceptor (TIGIT) with Ig and ITIM domains. The complete protein sequence of human TIGIT has GenBank accession number NP_776160. In some embodiments, the immune checkpoint inhibitor is an anti-TIGIT antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Anti-human TIGIT antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present method can be generated using methods well known in the art. Alternatively, art-recognized anti-TIGIT antibodies can be used. An exemplary anti-TIGIT antibody is MK-7684 (see, for example, WO 2017 / 030823, WO 2016 / 028656).

[0226] Other immunosuppressive molecules that can be targeted for immunomodulation include STAT3 and indoleamine 2,3-dioxygenase (IDO). For example, the complete protein sequence of human IDO has GenBank accession number NP_002155. In some embodiments, the immunomodulator is a small molecule IDO inhibitor. Exemplary small molecules include BMS-986205, epacadostat (INCB24360), and navoximod (GDC-0919).

[0227] 4.Surgery Approximately 60% of people with cancer will undergo some type of surgery, including preventive, diagnostic, or staging, curative, and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and this surgery can be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgical surgery (Mohs surgery).

[0228] When cancerous cells, tissues, or tumors are partially or completely removed, a cavity can be formed in the body.Treatment can be achieved by perfusion, direct injection, or local application of additional anti-cancer therapy to the area.Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.These treatments can also vary in dosage.

[0229] 5. Other agents It is contemplated that other agents can be used in combination with certain aspects of the present invention to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, cell adhesion inhibitors, agents that sensitize hyperproliferative cells to apoptosis-inducing factors, or other biological agents. It is believed that increasing intercellular signaling by increasing the number of GAP junctions enhances the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present invention to improve the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors are contemplated to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that sensitize hyperproliferative cells to apoptosis, such as the antibody c225, can be used in combination with certain aspects of the present invention to improve the efficacy of the treatment.

[0230] IV. Kit Various aspects of the present invention contemplate kits containing diagnostic, therapeutic, and / or delivery agents. In some embodiments, the present invention contemplates kits for detecting NRG1 fusions in tumor cells of a patient. In some embodiments, the present invention contemplates kits for preparing and / or administering therapies of the present invention. The kits can include reagents that can be used to administer the active or effective agents of the present invention. The kit reagents can include one or more anti-cancer components of the combination therapy, as well as reagents for preparing, formulating, and / or administering the components of the present invention or performing one or more steps of the methods of the present invention. In some embodiments, the kits can also include suitable container means, such as Eppendorf tubes, assay plates, syringes, bottles, or tubes that will not react with the components of the kit. The containers can be made of a sterilizable material, such as plastic or glass. The kits can further include an instruction sheet outlining the procedural steps of the method and following substantially the same procedures as described herein or known to those of skill in the art. [Example]

[0231] V. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. It will be understood by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and therefore can be considered to constitute preferred modes for its practice. However, those of skill in the art will, in light of the present disclosure, recognize that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result, without departing from the spirit and scope of the invention.

[0232] Example 1 We examined cell viability of the breast cancer cell line, MDA175-VII (NRG1-DOC4 fusion), following treatment with novel quinazoline-based TKIs alone and in combination with anti-HER2 / 3 therapies, including trastuzumab, pertuzumab, and T-DM1. Cell viability was determined by Cell Titer Glo assay. The novel quinazoline-based TKIs potently inhibited cell viability of MDA175-VII (NRG1-DOC4 fusion) cells (Table 1; Figures 1A-1B). These data indicate that the novel quinazoline-based TKIs potently inhibit NRG1 fusions at lower concentrations than other pan-HER inhibitors.

[0233] Furthermore, because inhibition of wild-type (WT) EGFR often leads to off-target adverse events in patients, we performed IC studies on Ba / F3 cells (+10 ng / μL EGF) expressing WT EGFR treated with novel quinazoline-based TKIs. 50 Determine IC values ​​for cells harboring NRG1 fusions 50 The novel quinazoline-based TKIs were selective in inhibiting MDA175-VII (NRG1 fusion) cells (Figure 2).

[0234] Finally, the addition of low-dose quinazoline-based TKIs to anti-HER2 / 3 therapy slightly reduced cell viability compared to anti-HER2 / 3 therapy alone (Table 2; Figures 3A-3B). These preliminary data suggest that these compounds are more potent than other pan-HER inhibitors tested against NRG1 fusions.

[0235] Table 1. Mean IC against MDA175-VII (NRG1-DOC4 fusion) cells treated with IACS inhibitors. 50 value TIFF0007734139000049.tif78164

[0236] Table 2. Mean IC for MDA175-VII (NRG1-DOC4 fusion) cells treated with anti-HER2 antibodies with or without low-dose IACS inhibitors.50 value TIFF0007734139000050.tif51164

[0237] Example 2 Generation of Ba / F3 cells. Ba / F3 cells stably co-expressing WT ErbB2 and WT ErbB3, or WT ErbB3 and WT ErbB4, were generated as previously described. Briefly, retroviral or lentiviral constructs were transfected into Phoenix 293T cells to generate viruses, which were then incubated with the Ba / F3 cell line overnight. The virus was removed, and the cells were cultured in puromycin for 10 days to select for Ba / F3 cell lines stably expressing the retroviral constructs. After selection, the cells were sorted using anti-HER2, anti-HER3, and anti-HER4 antibodies (Biolegend). The cell lines were then re-transduced with lentivirus containing the NRG fusion plasmids listed in Table 3A. The cells were then sorted for NRG1 expression by FACS. IL-3 was then removed from the stable cell lines. The resulting stable cells were used for downstream analyses, including drug screening.

[0238] Drug screening and IC50 determination. Drug screening was performed as previously described. Briefly, cells were plated in technical triplicates at 2000–3000 cells per well in 384-well plates (Greiner Bio-One). Seven different concentrations of quinazoline-based TKIs or DMSO vehicle were added to a final volume of 40 μL per well. After 72 h, 11 μL of Cell Titer Glo (Promega) was added to each well. Plates were incubated for a minimum of 10 min and bioluminescence was measured using a FLUOstar OPTIMA plate reader (BMG LABTECH). Raw bioluminescence values ​​were normalized to cells treated with the DMSO control and plotted in GraphPad Prism. Nonlinear regression was used to fit the normalized data with a variable slope, and IC50 values ​​were determined by interpolation of the concentration at 50% inhibition in GraphPad Prism. 50Drug screens are performed with technical triplicates on each plate and either duplicate or triplicate biological replicates.

[0239] Overexpression model. The overexpression model is generated by lentiviral transduction of the NRG1 fusions in Table 3A. Lentivirus is generated using the Lenti-X cells Lenti-X single shot kit (Takarabio). Lentivirus is generated as described by the manufacturer. The lentivirus is then added to the cell lines in Table 3B. 24 hours after viral transduction, the virus is removed, and the cells are placed in 2 μg / ml puromycin for selection. After 10 days of selection, protein and RNA are harvested from the cell lines, and expression of the NRG1 fusions is determined by Western blotting and RT-PCR, respectively. Stable cell lines expressing the NRG1 fusions are used for downstream analyses, including Western blotting and ELISA.

[0240] Inhibition of HER signaling in overexpressing cell lines was determined by Western blotting and ELISA. Parental and overexpressing (OE) cell lines were plated in 10 cm dishes and treated with increasing doses of quinazoline-based TKIs. Cells were incubated with inhibitors over time, and proteins were harvested using lysis buffer (Cell Signaling). Expression of NRG1 fusions, phosphorylated EGFR, HER2, HER3, and HER4, and total EGFR, HER2, HER3, and HER4 was determined by Western blotting, and blots were exposed using a BioRad Chemidoc imager. To quantify changes in protein expression, proteins from parental and OE-expressing cell lines treated with quinazoline-based TKIs were loaded into ELISA (Cell Signaling), and the ELISA was completed according to the manufacturer's instructions.

[0241] Table 1A. NRG1 fusion plasmids TIFF0007734139000051.tif16164

[0242] Table 1B. Human cell line models TIFF0007734139000052.tif21164

[0243] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes can be made in the methods and method steps or sequence of steps described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related can be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0244] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF0007734139000053.tif128160

Claims

1. A composition comprising a quinazoline-based tyrosine kinase inhibitor (TKI) and an anti-HER2 / HER3 antibody for treating cancer in a patient, wherein the cancer has an NRG1 fusion.

2. The composition of claim 1, wherein the NRG1 fusion is an NRG1-DOC4 fusion, an NRG1-VAMP2 fusion, an NRG1-CLU fusion, an NRG1-SLC3A2 fusion, an NRG1-CD74 fusion, an NRG1-ATP1B1 fusion, or an NRG1-SDC4 fusion.

3. 3. The composition of claim 1 or 2, wherein the quinazoline-based TKI is IACS-015285, IACS-015296, IACS-070979, IACS-015293, IACS-070982, IACS-070863, IACS-070864, ​​IACS-070871, IACS-070980, IACS-070968, IACS-070709, IACS-070989, or IACS-052336.

4. The composition of claim 1, wherein the anti-HER2 / HER3 antibody comprises trastuzumab, pertuzumab, or T-DM1.

5. A composition described in any one of claims 1 to 4, used in combination with an additional anti-cancer therapy.

6. 6. The composition of claim 5, wherein the additional anti-cancer therapy is surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, toxin therapy, immunotherapy, or cytokine therapy.

7. 7. The composition of any one of claims 1 to 6, wherein the cancer is breast cancer, lung cancer, colorectal cancer, neuroblastoma, pancreatic cancer, brain cancer, gastric cancer, skin cancer, testicular cancer, prostate cancer, ovarian cancer, liver cancer, esophageal cancer, cervical cancer, head and neck cancer, melanoma, or glioblastoma.

Images