GCN2 modulators for treating cancer

Abstract

Provided herein are methods of treating advanced solid tumors in a subject in need thereof, for example, when the subject has advanced head and neck squamous cell carcinoma, colorectal cancer, non-small cell lung cancer, and bladder transitional cell carcinoma. Also provided herein are methods of treating leukemia (e.g., acute myelogenous leukemia) in a subject in need thereof.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit and priority of U.S. Provisional Application Serial No. 63 / 345,727 (filed May 25, 2022); 63 / 440,297 (filed Jan. 20, 2023); 63 / 443,269 (filed Feb. 3, 2023); and 63 / 455,861 (filed Mar. 30, 2023), the contents of which are hereby incorporated by reference in their entirety. Background of the Invention

[0003] Cancer is a leading cause of death worldwide, resulting in nearly 10 million deaths in 2020 (World Health Organization). Targeted therapies and immunotherapies have broadened the horizons of solid tumor treatment by significantly improving prognosis. However, tumor recurrence, drug resistance, and drug intolerance remain major challenges in advanced cancer management (Wang et al., “Drug resistance and combating drug resistance in cancer,” Cancer Drug Resistance, 2019, 2(2):141-160; Chakraborty et al., “The difficulties in cancer treatment” Ecancermedical science, 2012, 6:ed16). Cancer cells typically experience various stressors in their microenvironment, such as hypoxia, low pH, and nutrient deprivation. To survive in the hostile tumor microenvironment, cancer cells actively utilize adaptive stress pathways, such as the integrated stress response (ISR) (Ye et al., “The GCN2-ATF4 pathway is critical for tumor cell survival and proliferation in response to nutrient deprivation,” EMBO J., 2010, 29(12):2082-2096; Pakos-Zebrucka et al., “The integrated stress response,” EMBO Rep., 2016, 17(10):1374-1395). The ISR consists of four kinases: protein kinase RNA-like endoplasmic reticulum kinase, double-stranded RNA-dependent protein kinase, general control nondepressible kinase 2 (GCN2), and heme-regulated inhibitor (Donnelly et al., “The eIF2α kinases: their structures and functions,” Cell Mol Life Sci., 2013, 70(19):3493-3511).These four kinases sense distinct stressors and phosphorylate the alpha subunit of eukaryotic initiation factor 2 (eIF2α) (Albert et al., “Adaptive Protein Translation by the Integrated Stress Response Maintains the Proliferative and Migratory Capacity of Lung Adenocarcinoma Cells,” Mol Cancer Res., 2019, 17(12):2343-2355). As part of the ISR, the high molecular weight kinase GCN2 senses amino acid deprivation. Under amino acid deprivation, uncharged transfer RNAs accumulate and activate GCN2 (Anda et al., “Activation of Gcn2 in response to different stresses,” PLOS ONE, 2017, 12(8):E0182143). Phosphorylation of eIF2α by ISR kinases such as GCN2 inhibits general protein synthesis during cellular stress but also promotes the translation of selected mRNAs, which includes activating transcription factor 4 (ATF4), a key effector of the ISR (Pakos-Zebrucka et al.). After translation, ATF4 translocates to the nucleus and drives the expression of genes involved in stress adaptation, such as autophagy, antioxidant response, amino acid biosynthesis, and metabolism (Pakos-Zebrucka et al.; Harding et al., “An integrated stress response regulates amino acid metabolism and resistance to oxidative stress,” Mol Cell, 2003, 11(3):619-633). Other factors that activate GCN2 include ultraviolet light, viral infection, and oxidative stress (Costa-Mattioli et al., “The integrated stress response: From mechanism to disease,” Science, 2020, 368(6489):eaat5314). ATF4 is important for tumor cells to maintain amino acid metabolic homeostasis. Activation of the ISR pathway promotes the survival of tumor cells under nutrient deprivation (Ye et al.). GCN2 / ATF4 expression is elevated in primary human liver, breast, lung, and head and neck tumors, and GCN2 activation is observed in colon, breast, and lung tumor samples compared to normal tissues.

[0004] ISR activation plays a dual role in cell fate determination. Under acute stress conditions, ISR can promote adaptation, while under chronic stress conditions, this pathway can be converted to apoptosis, leading to increased eIF2α phosphorylation over a longer period of time (Wortel et al., “Surviving Stress: Modulation of ATF4-Mediated Stress Responses in Normal and Malignant Cells,” Trends Endocrinol Metabol., 2017, 28(11):794-806). By reducing protein synthesis or activating the apoptotic pathway, long-term activation of ISR can be detrimental to cell growth (Wortel et al.; Harding et al., “Ppplr14 gene knockout reveals an essential role for translation initiation factor 2alpha (eIF2alpha) dephosphorylation in mammalian development,” Proc Natl Acad Sci USA, 2009, 106(6);1832-1837; Münch, “The different axes of the mammalian mitochondrial unfolded protein response,” BMC Biology, 2018;16(1):81). Persistent ISR activation due to mutations in eIF2α phosphatases has been shown to have a detrimental effect on embryogenesis (due to inhibition of protein synthesis) (Harding et al., “Ppplr14 gene knockout reveals an essential role for translation initiation factor 2alpha (eIF2alpha) dephosphorylation in mammalian development,” Proc Natl Acad Sci USA, 2009, 106(6);1832-1837).GCN2 activation can also have antiproliferative effects by inhibiting general protein synthesis and inducing cell cycle arrest, thereby preventing cells from growing in the absence of nutrients (Lehman et al., “Translation Upregulation of an Individual p21Cip1 Transcript Variant by GCN2 Regulates Cell Proliferation and Survival under Nutrient Stress,” PLOS Genetics, 2015, 11(6):e1005212). Thus, sustained activation of the GCN2 pathway can inhibit protein synthesis and cell growth, thereby suppressing tumor proliferation.

[0005] Accordingly, there remains an unmet need to develop new therapeutic strategies that utilize modulation (activation or inhibition) of the GCN2 pathway to treat various cancers (such as advanced solid tumors and blood cancers). SUMMARY OF THE INVENTION

[0006] In one aspect, the present disclosure provides methods of treating advanced solid tumors in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula (I)

[0007]

[0008] or a pharmaceutically acceptable salt thereof.

[0009] In certain embodiments, the advanced solid tumor is selected from head and neck squamous cell carcinoma, colorectal cancer, non-small cell lung cancer, renal cell carcinoma, and urothelial carcinoma of the bladder.

[0010] In certain embodiments, the advanced solid tumor is selected from sarcoma, colorectal cancer, head and neck cancer, and prostate cancer.

[0011] In another aspect, the present disclosure provides methods of treating blood cancers in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula (I)

[0012]

[0013] or a pharmaceutically acceptable salt thereof.

[0014] In certain embodiments, the blood cancer is leukemia. In certain embodiments, the blood cancer is acute myeloid leukemia.

[0015] In some embodiments, the blood cancer is resistant to B cell lymphoma inhibitors. In certain embodiments, the blood cancer is resistant to venetoclax.

[0016] In some embodiments, administration of an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof activates the integrated stress response pathway (ISR) in advanced solid tumors or blood cancers. In some embodiments, the ISR activation is GCN2-dependent.

[0017] In some embodiments, administration of an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof induces the expression of asparagine synthetase (ASNS), phosphoserine aminotransferase 1 (PSAT1), phosphoglycerate dehydrogenase (PHGDH), and / or BCL2 binding component 3 (PUMA) in advanced solid tumors or blood cancers. In some embodiments, administration of an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof reduces the protein levels of S100 calcium-binding protein A8 / A9 (S100A8 / A9), hypoxia-inducible factor (HIF) 1α / 2α, and / or glucose transporter type 1 (GLUT1) in advanced solid tumors or blood cancers. In some embodiments, administration of an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof reduces mitochondrial respiration and / or glycolysis in advanced solid tumors or blood cancers. In some embodiments, administration of an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof reduces myeloid-restricted precursors and mature myeloid cells in a subject. In some embodiments, administration of an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof alters metabolites involved in amino acid metabolism, oxidative stress, urea cycle, and / or pyrimidine biosynthesis in advanced solid tumors or blood cancers. In some embodiments, administration of an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof reduces proteins involved in oxidative phosphorylation in advanced solid tumors or blood cancers. In some embodiments, administration of an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof reduces the activity of HIF- and / or E2F transcription factor 1 (E2F1)-driven transcription in advanced solid tumors or blood cancers. In some embodiments, administration of an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof increases the ATF4 and / or (Jun proto-oncogene AP-1 transcription factor subunit) JUN transcriptional activity in advanced solid tumors or blood cancers.

[0018] In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises administering to a subject from about 10 mg to about 150 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis). In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises orally administering to a subject from about 10 mg to about 150 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis). In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises orally administering to a subject from about 10 mg to about 150 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis) per day. In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises orally administering to a subject once daily from about 10 mg to about 150 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis). In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises orally administering to a subject once daily from about 10 mg to about 150 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis) for 21 consecutive days.

[0019] In certain embodiments, the subject is in a fasting state. In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises administering to a subject from about 10 mg to about 150 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis) about 1 hour before a meal or about 2 hours after a meal.

[0020] In certain embodiments, the subject has previously received at least one and no more than five prior lines of therapy.

[0021] In certain embodiments, the pharmaceutically acceptable salt is a potassium salt. In certain embodiments, the potassium salt is a hydrate. In certain embodiments, the potassium salt is a monohydrate.

[0022] In certain embodiments, the method further comprises administering to the subject an effective amount of a second therapeutic agent. In certain embodiments, the second therapeutic agent is selected from immune checkpoint inhibitors, epidermal growth factor receptor (EGFR) inhibitors, anti-angiogenic agents, venetoclax, fluorouracil, and combinations thereof.

[0023] In some embodiments, the second therapeutic agent is selected from anti-vascular endothelial growth factor receptor (VEGFR) antibodies, fluorouracil, phosphoinositide 3-kinase α (PI3Kα) inhibitors, mitogen-activated protein kinase kinase 1 / 2 (MEK1 / 2) inhibitors, and hypoxia-inducible factor (HIF) inhibitors.

[0024] In some embodiments, the second therapeutic agent is venetoclax.

[0025] In some embodiments, administering an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof and venetoclax activates the integrated stress response pathway (ISR) in advanced solid tumors or blood cancers to a greater extent than administering the compound of formula (I) or a pharmaceutically acceptable salt thereof or venetoclax alone.

[0026] In some embodiments, the second therapeutic agent is an anti-VEGFR antibody.

[0027] In some embodiments, the second therapeutic agent is an HIF inhibitor. In certain embodiments, the second therapeutic agent is belzutifan.

[0028] In some embodiments, the second therapeutic agent is 5-fluorouracil.

[0029] In some embodiments, the second therapeutic agent is a PI3Kα inhibitor. In some embodiments, the second therapeutic agent is alpelisib.

[0030] In some embodiments, the second therapeutic agent is a MEK1 / 2 inhibitor. In some embodiments, the second therapeutic agent is trametinib.

[0031] In some embodiments, the second therapeutic agent is an EGFR inhibitor. In some embodiments, the second therapeutic agent is selected from osimertinib and dacomitinib.

[0032] In certain embodiments, the subject is a human. In certain embodiments, the subject is an adult human. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a graph showing the inhibitory effect of HC-7366 on the activity of double-stranded RNA-dependent protein kinase, general control non-derepressible 2 (GCN2) in a biochemical assay. The data are presented as a graph of HC-7366 concentration relative to vehicle %, where vehicle % represents the DMSO control.

[0034] Figure 2 is a graph showing the effect of HC-7366 on the activation of activating transcription factor 4 (ATF4) in HT1080 cells using the activating transcription factor 4 (ATF4) activity assay.

[0035] Figure 3 is a graph showing the results of the ATF4 activity assay of HT1080 cells treated with HC-7366 and febrifugine, demonstrating the inhibitory effect of HC-7366 on GCN2 in the Figure 2 ATF4 assay.

[0036] Figure 4 This is a graph showing the effect of HC-7366 on the viability of MOLM-16 cells using the CellTiter- (CTG) assay.

[0037] Figure 5 This is a Western blot showing the expression of various proteins (including GCN2 and ATF4) in HT1080 cells after treatment with different concentrations (as shown) of HC-7366 (with and without halofuginone).

[0038] Figure 6 This is a graph comparing the change in average tumor volume over time in HT1080 tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) in PBS (final pH 7.4)) twice daily, 0.3 mg / kg HC-7366 twice daily, 1 mg / kg HC-7366 twice daily, 3 mg / kg HC-7366 twice daily, 0.6 mg / kg HC-7366 once daily, 2 mg / kg HC-7366 once daily, or 6 mg / kg HC-7366 once daily.

[0039] Figure 7 This is a graph comparing the change in average tumor volume over time in HT1080 tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) in PBS (final pH 7.4)) twice daily, 0.3 mg / kg HC-7366 twice daily, 1 mg / kg HC-7366 twice daily, or 3 mg / kg HC-7366 twice daily.

[0040] Figure 8 This is a graph comparing the change in average tumor volume over time in LoVo tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) in PBS (final pH 7.4)) twice daily, 0.3 mg / kg HC-7366 twice daily, 1 mg / kg HC-7366 twice daily, 3 mg / kg HC-7366 twice daily, 10 mg / kg HC-7366 twice daily, or 30 mg / kg HC-7366 twice daily.

[0041] Figure 9Graph comparing the change in average tumor volume over time in LoVo tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) (final pH 7.4) in PBS) twice daily, 0.3 mg / kg HC-7366 twice daily, 1 mg / kg HC-7366 twice daily, or 3 mg / kg HC-7366 twice daily.

[0042] Figure 10 Graph comparing the change in average tumor volume over time in DLD-1 tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) (final pH 7.4) in PBS) twice daily, 0.3 mg / kg HC-7366 twice daily, 1 mg / kg HC-7366 twice daily, 3 mg / kg HC-7366 twice daily, 10 mg / kg HC-7366 twice daily, or 30 mg / kg HC-7366 twice daily.

[0043] Figure 11 Graph comparing the change in average tumor volume over time in FaDu tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) (final pH 7.4) in PBS) twice daily, 0.3 mg / kg HC-7366 twice daily, 1 mg / kg HC-7366 twice daily, 3 mg / kg HC-7366 twice daily, or 10 mg / kg HC-7366 twice daily.

[0044] Figure 12 Graph comparing the change in average tumor volume over time in FaDu tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) (final pH 7.4) in PBS) twice daily, 0.3 mg / kg HC-7366 twice daily, 1 mg / kg HC-7366 twice daily, or 3 mg / kg HC-7366 twice daily.

[0045] Figure 13Graph comparing the change in mean tumor volume over time in FaDu tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) (final pH 7.4) in PBS) twice daily, 0.3 mg / kg HC-7366 twice daily, 1 mg / kg HC-7366 twice daily, 3 mg / kg HC-7366 twice daily, 10 mg / kg HC-7366 twice daily, or 30 mg / kg HC-7366 twice daily.

[0046] Figure 14 Graph comparing the change in mean tumor volume over time in LNCaP tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) (final pH 7.4) in PBS) twice daily, 0.3 mg / kg HC-7366 twice daily, 1 mg / kg HC-7366 twice daily, 3 mg / kg HC-7366 twice daily, 10 mg / kg HC-7366 twice daily, or 30 mg / kg HC-7366 twice daily.

[0047] Figure 15 Graph comparing the change in mean tumor volume over time in TM00298 (PDX) tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) (final pH 7.4) in PBS) twice daily, 3 mg / kg HC-7366 twice daily, or 30 mg / kg HC-7366 twice daily.

[0048] Figure 16 Graph comparing the change in tumor volume over time in MOLM-16 tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) (final pH 7.4) in PBS) twice daily, 0.25 mg / kg HC-7366 twice daily, 0.5 mg / kg HC-7366 twice daily, 1 mg / kg HC-7366 twice daily, or 2 mg / kg HC-7366 twice daily.

[0049] Figure 17Graph comparing the change in tumor volume over time in KG-1 tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) in PBS (final pH 7.4)) twice daily, 0.3 mg / kg HC-7366 twice daily, 1 mg / kg HC-7366 twice daily, 3 mg / kg HC-7366 twice daily, 10 mg / kg HC-7366 twice daily, or 30 mg / kg HC-7366 twice daily.

[0050] Figure 18 Graph comparing the change in tumor volume over time in Kasumi-1 tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) in PBS (final pH 7.4)) twice daily, 3 mg / kg HC-7366 twice daily, or 30 mg / kg HC-7366 twice daily.

[0051] Figure 19 Graph comparing the change in tumor volume over time in OCI-AML2 tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) in PBS (final pH 7.4)) twice daily, 0.3 mg / kg HC-7366 twice daily, 1 mg / kg HC-7366 twice daily, 3 mg / kg HC-7366 twice daily, 10 mg / kg HC-7366 twice daily, or 30 mg / kg HC-7366 twice daily.

[0052] Figure 20 Graph comparing the change in tumor volume over time in MV4-11 tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) in PBS (final pH 7.4)) twice daily, 0.3 mg / kg HC-7366 twice daily, 1 mg / kg HC-7366 twice daily, 3 mg / kg HC-7366 twice daily, 10 mg / kg HC-7366 twice daily, or 30 mg / kg HC-7366 twice daily.

[0053] Figure 21 Is Figure 17 Image of immunohistochemical (IHC) assay of ASNS expressed in KG-1 tumors collected from the treatment groups described in

[0054] Figure 22 ShowsFigure 21 Graph of the total intensity per unit area of the immunohistochemical assay described in **** p<0.0001, one-way ANOVA.

[0055] Figure 23 is in from Figure 17 Image of the IHC assay of PSAT1 expressed in KG-1 tumors collected from the treatment groups described in. The tumors were collected on day 27 after the start of treatment (end of the study).

[0056] Figure 24 is showing Figure 23 Graph of the total intensity per unit area of the immunohistochemical assay described in *p<0.05, **** p<0.0001, one-way ANOVA.

[0057] Figure 25 is in from Figure 17 Image of the IHC assay of PHGDH expressed in KG-1 tumors collected from the treatment groups described in. The tumors were collected on day 27 after the start of treatment (end of the study).

[0058] Figure 26 is showing Figure 25 Graph of the total intensity per unit area of the immunohistochemical assay described in **** p<0.0001, one-way ANOVA.

[0059] Figure 27 is in from Figure 17 Image of the IHC assay of S100A8 / A9 expressed in KG-1 tumors collected from the treatment groups described in. The tumors were collected on day 27 after the start of treatment (end of the study).

[0060] Figure 28 is showing Figure 27 Graph of the percentage of S100A8 / A9+ cells in the tumors described in *p<0.05, **p<0.01, one-way ANOVA.

[0061] Figure 29 is in from Figure 10 Image of the IHC assay of ASNS expressed in DLD-1 tumors collected from the treatment groups described in. The tumors were collected on day 20 after the start of treatment (end of the study).

[0062] Figure 30 is showing Figure 29 Graph of the positive % calculated by area of the IHC assay described in *p<0.05, **p<0.01, ***p<0.001, one-way ANOVA.

[0063] Figure 31 is in from Figure 10Images of IHC assays of PSAT1 expression in DLD-1 tumors collected from the treatment groups described in

[0064] Figure 32 show Figure 31 Graphs of the percent positive by area of the IHC assays described in

[0065] Figure 33 from Figure 10 Images of IHC assays of PUMA expression in DLD-1 tumors collected from the treatment groups described in

[0066] Figure 34 show Figure 33 Graphs of the percent positive by area of the IHC assays described in

[0067] Figure 35 from Figure 10 Images of IHC assays of HIf1α and HIf2α expression in DLD-1 tumors collected from the treatment groups described in

[0068] Figure 36 show Figure 35 Graphs of HIf1α expression (percent positive cells) in the immunohistochemical assays described in

[0069] Figure 37 show Figure 35 Graphs of HIf2α expression (percent positive cells) in the immunohistochemical assays described in

[0070] Figure 38 Images of IHC assays of HIF1α and HIF2α expression in DLD-1 tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) in PBS (final pH 7.4)) twice daily or 1 mg / kg HC-7366 twice daily.

[0071] Figure 39 show Figure 38 Graphs of HIf1α expression (percent positive cells) in the immunohistochemical assays described in

[0072] Figure 40 are images of IHC assays of HIf1α and HIf2α expressed in FaDu tumors collected from the treatment groups described in Figure 13 and the treatment group receiving 30 mg / kg HC-7366 administered twice daily.

[0073] Figure 41 is a graph showing Figure 40 HIf1α expression (% positive cells) in the immunohistochemical assay described in **p<0.01, one-way ANOVA.

[0074] Figure 42 is a graph showing Figure 40 HIf2α expression (% positive cells) in the immunohistochemical assay described in ***p<0.001, ****p<0.0001 one-way ANOVA.

[0075] Figure 43 is a graph comparing the change in tumor volume over time in MV4-11 tumors treated twice daily with vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) in PBS (final pH 7.4)), 3 mg / kg HC-7366 twice daily, 30 mg / kg HC-7366 twice daily, 50 mg / kg venetoclax once daily, 3 mg / kg HC-7366 twice daily + 50 mg / kg venetoclax once daily, or 30 mg / kg HC-7366 twice daily + 50 mg / kg venetoclax once daily.

[0076] Figure 44 are images of IHC assays of ASNS expressed in MV4-11 tumors collected from the treatment groups described in Figure 43

[0077] Figure 45 is a graph showing Figure 44 ASNS expression (% positive by area) in the immunohistochemical assay described in ***p<0.001, ****p<0.0001, one-way ANOVA.

[0078] Figure 46 are images of IHC assays of PHGDH expressed in MV4-11 tumors collected from the treatment groups described in Figure 43

[0079] Figure 47 is a graph showing Figure 46 PHGDH expression (% positive by area) in the immunohistochemical assay described in **p<0.01, ****p<0.0001, one-way ANOVA.

[0080] Figure 48 is an IHC assay image of PUMA expressed in MV4-11 tumors collected from the treatment groups described in Figure 43

[0081] Figure 49 is a graph showing Figure 48 PUMA expression (positive % by area) in the immunohistochemical assay described in *p < 0.05, ****p < 0.0001, one-way ANOVA.

[0082] Figure 50 is an IHC assay image of S100A8 / A9 expressed in MV4-11 tumors collected from the treatment groups described in Figure 43

[0083] Figure 51 is a graph showing the percentage of S100A8 / A9+ cells in the tumors described in Figure 50 *p < 0.05, **p < 0.01, one-way ANOVA.

[0084] Figure 52 is an IHC assay image of HIf2α and GLUT1 expressed in 786-O tumors collected from 786-O tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) in PBS (final pH 7.4)) twice daily, 2 mg / kg HC-7366 twice daily, DC-101 15 mg / kg twice weekly, or 2 mg / kg HC-7366 twice daily + DC-101 15 mg / kg twice weekly.

[0085] Figure 53 is a graph showing Figure 52 HIf2α expression (positive cell %) in the immunohistochemical assay described in *p < 0.05, ****p < 0.0001, one-way ANOVA.

[0086] Figure 54 is a graph showing Figure 52 GLUT1 expression (positive % by area) in the immunohistochemical assay described in ****p < 0.0001, one-way ANOVA.

[0087] Figure 55Images of IHC assays of HIf2α and GLUT1 expressed in A498 tumors collected from A498 tumor-bearing mice administered twice daily with vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) (final pH 7.4)), 2 mg / kg HC-7366 twice daily, DC-101 15 mg / kg twice weekly, or 2 mg / kg HC-7366 twice daily + DC-101 15 mg / kg twice weekly.

[0088] Figure 56 Shows Figure 55 Graph of HIf2α expression (% positive cells) in the immunohistochemical assay described in

[0089] Figure 57 Shows Figure 55 Graph of GLUT1 expression (% positive by area) in the immunohistochemical assay described in

[0090] Figure 58 Graph comparing the change in tumor volume over time in 786-O tumor-bearing mice administered twice daily with vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) (final pH 7.4)), 3 mg / kg HC-7366 twice daily, 0.1 mg / kg PT-2977 (belzutifan) twice daily, or 3 mg / kg HC-7366 twice daily + 0.1 mg / kg PT-2977 (belzutifan) twice daily.

[0091] Figure 59 Images of IHC assays of HIF1α in head and neck tumors of human patients treated with 10 mg HC-7366 (102-101), colorectal tumors of human patients treated with 10 mg HC-7366 (102-201), and colorectal tumors of human patients treated with 20 mg HC-7366 (101-201).

[0092] Figure 60 Shows Figure 59 Graph of the expression of HIf2α+ cells (% of total cells) in the immunohistochemical assay described in

[0093] Figure 61Graph comparing the change in mean tumor volume over time in DLD-1 tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) (final pH 7.4)) twice daily, 3 mg / kg HC-7366 twice daily, 30 mg / kg HC-7366 twice daily, 20 mg / kg DC-101 biweekly, 20 mg / kg DC-101 biweekly + 3 mg / kg HC-7366 twice daily, or 20 mg / kg DC-101 biweekly + 30 mg / kg HC-7366 twice daily.

[0094] Figure 62 Graph comparing the change in mean tumor volume over time in DLD-1 tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) (final pH 7.4)) twice daily, 3 mg / kg HC-7366 twice daily, 30 mg / kg HC-7366 twice daily, 75 mg / kg 5-fluorouracil once weekly, 75 mg / kg 5-fluorouracil once weekly + 3 mg / kg HC-7366 twice daily, or 75 mg / kg 5-fluorouracil once weekly + 30 mg / kg HC-7366 twice daily.

[0095] Figure 63 Graph comparing the change in mean tumor volume over time in HCT116 tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) (final pH 7.4)) twice daily, 3 mg / kg HC-7366 twice daily, 30 mg / kg HC-7366 twice daily, 50 mg / kg alpelisib, 50 mg / kg alpelisib + 3 mg / kg HC-7366 twice daily, or 50 mg / kg alpelisib + 30 mg / kg HC-7366 twice daily.

[0096] Figure 64Graph comparing the change in mean tumor volume over time in HCT116 tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) (final pH 7.4)) twice daily, 3 mg / kg HC-7366 twice daily, 30 mg / kg HC-7366 twice daily, trametinib 1 mg / kg, trametinib 1 mg / kg + 3 mg / kg HC-7366 twice daily, or trametinib 1 mg / kg + 30 mg / kg HC-7366 twice daily.

[0097] Figure 65 Graph showing the effect of HC-7366 on MOLM-16 GCN2 wild-type cell viability compared to cells treated with vehicle (DMSO), as measured by CTG assay at 24 and 48 hours after treatment with HC-7366.

[0098] Figure 66 Graph showing the effect of HC-7366 on MOLM-16 GCN2 CRISPR knockout cell viability compared to cells treated with vehicle (DMSO), as measured by CTG assay at 24 and 48 hours after treatment with HC-7366.

[0099] Figure 67 Graph showing the expression of GCN2 in MOLM-16 GCN2 wild-type cells and MOLM-16 GCN2 CRISPR knockout cells, respectively, described in Figure 65 and Figure 66 after treatment with HC-7366.

[0100] Figure 68 Graph showing the expression of ATF4 in MOLM-16 GCN2 wild-type cells and MOLM-16 GCN2 CRISPR knockout cells, respectively, described in Figure 65 and Figure 66 after treatment with HC-7366.

[0101] Figure 69 Graph showing the expression of ASNS in MOLM-16 GCN2 wild-type cells and MOLM-16 GCN2 CRISPR knockout cells, respectively, described in Figure 65 and Figure 66 after treatment with HC-7366.

[0102] Figure 70 Graph showing the expression of ASNS in MOLM-16 GCN2 wild-type cells and MOLM-16 GCN2 CRISPR knockout cells, respectively, described in Figure 65 and Figure 66Graphs of PSAT1 expression in MOLM-16 GCN2 wild-type cells and MOLM-16 GCN2 CRISPR knockout cells, respectively, as described in

[0103] Figure 71 Graph showing the effect of HC-7366 on the viability of FaDu GCN2 wild-type cells compared to cells treated with vehicle (DMSO), as measured by CTG assay at 72 and 96 hours after treatment with HC-7366.

[0104] Figure 72 Graph showing the effect of HC-7366 on the viability of FaDu GCN2 CRISPR knockout cells compared to cells treated with vehicle (DMSO), as measured by CTG assay at 72 and 96 hours after treatment with HC-7366.

[0105] Figure 73 Graph showing the expression of pGCN2 in FaDu wild-type cells and FaDu GCN2 CRISPR knockout cells, respectively, as described in Figure 71 and Figure 72 after treatment with HC-7366.

[0106] Figure 74 Graph showing the expression of GCN2 in FaDu wild-type cells and FaDu GCN2 CRISPR knockout cells, respectively, as described in Figure 71 and Figure 72 after treatment with HC-7366.

[0107] Figure 75 Graph showing the expression of ATF4 in FaDu GCN2 wild-type cells and FaDu CRISPR knockout cells, respectively, as described in Figure 71 and Figure 72 after treatment with HC-7366.

[0108] Figure 76 Graph showing the expression of ASNS in FaDu GCN2 wild-type cells and FaDu GCN2 CRISPR knockout cells, respectively, as described in Figure 71 and Figure 72 after treatment with HC-7366.

[0109] Figure 77 Graph showing the absorbance of lysates of HEK293 GCN2 wild-type cells treated with vehicle (DMSO) or 100 nM HC-7366 on a sucrose gradient for 16 hours. The polyribosome to monomer ratio is also provided.

[0110] Figure 78It is a graph showing the absorbance of lysates of HEK293 GCN2 CRISPR knockout cells treated with vehicle (DMSO) or 100 nM HC-7366 on a sucrose gradient for 16 hours. The polysome to monosome ratio is also provided.

[0111] Figure 79 It is a gel showing α-puromycin staining of newly synthesized proteins in 100 nM HC-7366.

[0112] Figure 80 It is a western blot showing the levels of ISR markers after treatment with HC-7366.

[0113] Figure 81 It is a graph showing the effect of HC-7366 on CTG-2229 cell viability, as measured by CTG assay after treatment with HC-7366 for 6 days.

[0114] Figure 82 It is a graph showing the effect of HC-7366 on CTG-3680 cell viability, as measured by CTG assay after treatment with HC-7366 for 6 days.

[0115] Figure 83 It is a graph showing the effect of HC-7366 on CTG-3667 cell viability, as measured by CTG assay after treatment with HC-7366 for 6 days.

[0116] Figure 84 It is a graph showing the effect of HC-7366 on CTG-2456 cell viability, as measured by CTG assay after treatment with HC-7366 for 6 days.

[0117] Figure 85 It is a graph showing the effect of HC-7366 on CTG-2457 cell viability, as measured by CTG assay after treatment with HC-7366 for 6 days.

[0118] Figure 86 It is a graph showing the effect of HC-7366 on CTG-2454 cell viability, as measured by CTG assay after treatment with HC-7366 for 6 days.

[0119] Figure 87Are a series of t-distributed stochastic neighbor embedding (tSNE) plots showing the distribution of terminal samples of AML stem cells, myeloid-restricted progenitor cells, mature myeloid cells, non-myeloid cells, and other cell types collected from the bone marrow (BM), whole blood (WB), and spleen (SP) of female NCG immunodeficient mice bearing primary (P1) human acute myeloid leukemia (AML) xenograft tumor transplantation models and treated with vehicle, 1 mg / kg HC-7366 twice daily for 28 days, 10 mg / kg HC-7366 twice daily for 28 days, 30 mg / kg HC-7366 twice daily for 28 days, or 100 mg / kg venetoclax once daily for 28 days.

[0120] Figure 88 Is a plot showing the oxygen consumption rate (pmol / min) of MOLM-16 cells treated with vehicle (DMSO), 0.001 μM HC-7366, 0.1 μM HC-7366, or 10 μM HC-7366 over time.

[0121] Figure 89 Is a plot showing the extracellular acidification rate (ECAR) (mpH / min) of MOLM-16 cells treated with vehicle (DMSO), 0.001 μM HC-7366, 0.1 μM HC-7366, or 10 μM HC-7366 over time.

[0122] Figure 90 Is a heatmap showing the amino acid levels (normalized to vehicle control) in MOLM-16 tumors treated with 0.3 mg / kg HC-7366, 1 mg / kg HC-7366, 3 mg / kg HC-7366, 10 mg / kg HC-7366, or 30 mg / kg HC-7366 for 4 days.

[0123] Figure 91 Is a plot showing Figure 90 The proportional intensity of aspartic acid levels in the treatment groups described in

[0124] Figure 92 Is a plot showing Figure 90 The proportional intensity of cysteine levels in the treatment groups described in

[0125] Figure 93 Is a plot showing Figure 90 The proportional intensity of methionine levels in the treatment groups described in

[0126] Figure 94Is a heatmap showing the levels of oxidative stress markers (normalized to vehicle control) in MOLM-16 tumors treated with 0.3 mg / kg HC-7366, 1 mg / kg HC-7366, 3 mg / kg HC-7366, 10 mg / kg HC-7366, or 30 mg / kg HC-7366 for 4 days.

[0127] Figure 95 Is a schematic diagram showing the molecular pathways involved in glutathione production.

[0128] Figure 96 Is showing Figure 94 A graph showing the relative intensities of S-adenosylmethionine (SAM) levels in the treatment groups described in

[0129] Figure 97 Is showing Figure 94 A graph showing the relative intensities of oxidized glutathione (GSSG) levels in the treatment groups described in

[0130] Figure 98 Is showing Figure 94 A graph showing the relative intensities of S-adenosylhomocysteine (SAH) levels in the treatment groups described in

[0131] Figure 99 Is showing Figure 94 A graph showing the relative intensities of ophthalmate levels in the treatment groups described in

[0132] Figure 100 Is showing Figure 94 A graph showing the relative intensities of cysteine levels in the treatment groups described in

[0133] Figure 101 Is showing Figure 94 A graph showing the relative intensities of cystathionine levels in the treatment groups described in

[0134] Figure 102 Is a heatmap showing the levels of pyrimidine anabolic metabolites (normalized to vehicle control) in MOLM-16 tumors treated with 0.3 mg / kg HC-7366, 1 mg / kg HC-7366, 3 mg / kg HC-7366, 10 mg / kg HC-7366, or 30 mg / kg HC-7366 for 4 days.

[0135] Figure 103 Is showing Figure 102 A graph showing the relative intensities of orotidine levels in the treatment groups described in

[0136] Figure 104 Is showing Figure 102 A graph showing the relative intensities of orotic acid levels in the treatment groups described in

[0137] Figure 105 is a graph showing Figure 102 the relative intensities of the levels of dihydroorotate in the treatment groups described in

[0138] Figure 106 is a graph showing Figure 102 the relative intensities of the levels of UMP in the treatment groups described in

[0139] Figure 107 is a heat map showing the levels of aspartate metabolism markers (normalized to vehicle control) in MOLM-16 tumors treated with 0.3 mg / kg HC-7366, 1 mg / kg HC-7366, 3 mg / kg HC-7366, 10 mg / kg HC-7366, or 30 mg / kg HC-7366 for 4 days.

[0140] Figure 108 is a schematic diagram showing the molecular pathways involved in aspartate metabolism.

[0141] Figure 109 is a graph showing Figure 107 the relative intensities of the levels of ornithine in the treatment groups described in

[0142] Figure 110 is a graph showing Figure 107 the relative intensities of the levels of citrulline in the treatment groups described in

[0143] Figure 111 is a graph showing Figure 107 the relative intensities of the levels of asparagine in the treatment groups described in

[0144] Figure 112 is a graph showing Figure 107 the relative intensities of the levels of 5-methylthioadenosine (MTA) in the treatment groups described in

[0145] Figure 113 is a graph showing Figure 107 the relative intensities of the levels of arginine in the treatment groups described in

[0146] Figure 114 is a graph showing Figure 107 the relative intensities of the levels of aspartate in the treatment groups described in

[0147] Figure 115 is a graph showing the changes in tumor amino acid levels (normalized to vehicle control) in FaDu tumor-bearing mice treated twice daily with 1 mg / kg HC-7366, 3 mg / kg HC-7366, or 30 mg / kg HC-7366 for 4 days. Statistically significant changes are marked with asterisks (p < 0.05).

[0148] Figure 116 Graph showing changes in plasma amino acid levels (normalized to vehicle control) in FaDu tumor-bearing mice treated twice daily for 4 days with 1 mg / kg HC-7366, 3 mg / kg HC-7366, or 30 mg / kg HC-7366. Statistically significant changes are marked with an asterisk (p < 0.05).

[0149] Figure 117 Graph showing changes in tumor γ-glutamyl amino acid levels (normalized to vehicle control) in FaDu tumor-bearing mice treated twice daily for 4 days with 1 mg / kg HC-7366, 3 mg / kg HC-7366, or 30 mg / kg HC-7366. Statistically significant changes are marked with an asterisk (p < 0.05).

[0150] Figure 118 Graph showing changes in plasma γ-glutamyl amino acid levels (normalized to vehicle control) in FaDu tumor-bearing mice treated twice daily for 4 days with 1 mg / kg HC-7366, 3 mg / kg HC-7366, or 30 mg / kg HC-7366. Statistically significant changes are marked with an asterisk (p < 0.05).

[0151] Figure 119 Graph showing changes in tumor urea cycle marker levels (normalized to vehicle control) in FaDu tumor-bearing mice treated twice daily for 4 days with 1 mg / kg HC-7366, 3 mg / kg HC-7366, or 30 mg / kg HC-7366. Statistically significant changes are marked with an asterisk (p < 0.05).

[0152] Figure 120 Graph showing changes in plasma urea cycle marker levels (normalized to vehicle control) in FaDu tumor-bearing mice treated twice daily for 4 days with 1 mg / kg HC-7366, 3 mg / kg HC-7366, or 30 mg / kg HC-7366. Statistically significant changes are marked with an asterisk (p < 0.05).

[0153] Figure 121Figure showing changes in tumor pyrimidine synthesis marker levels (normalized to vehicle control) in FaDu tumor-bearing mice treated twice daily for 4 days with 0.3 mg / kg HC-7366, 1 mg / kg HC-7366, or 3 mg / kg HC-7366. Statistically significant changes are marked with asterisks (p < 0.05).

[0154] Figure 122 Figure showing changes in tumor oxidative stress marker levels (normalized to vehicle control) in FaDu tumor-bearing mice treated twice daily for 4 days with 1 mg / kg HC-7366, 3 mg / kg HC-7366, or 30 mg / kg HC-7366. Statistically significant changes are marked with asterisks (p < 0.05).

[0155] Figure 123 Heatmap showing intracellular amino acid levels in GCN2 wild-type and GCN2 CRISPR knockout FaDu cells treated with vehicle (DMSO) or 0.1 μM HC-7366.

[0156] Figure 124 Figure showing GCN2 expression in GCN2 wild-type and GCN2 CRISPR knockout FaDu cells treated with vehicle (DMSO) or 0.1 μM HC-7366.

[0157] Figure 125 Figure showing ATF4 expression in GCN2 wild-type and GCN2 CRISPR knockout FaDu cells treated with vehicle (DMSO) or 0.1 μM HC-7366.

[0158] Figure 126 Schematic diagram showing pathway analysis (IPA) of differentially expressed proteins in FaDu tumors, which predicts strong inhibition of the oxidative phosphorylation pathway by 3 mg / kg HC-7366 but not by 30 mg / kg HC-7366.

[0159] Figure 127 Heatmap showing fold changes in oxidative phosphorylation proteins in FaDu tumor-bearing mice treated with 3 mg / kg HC-7366 or 30 mg / kg HC-7366.

[0160] Figure 128Graph comparing the change in mean tumor volume over time in DLD-1 tumor-bearing mice administered vehicle (e.g., 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) (final pH 7.4) in PBS) twice daily, 0.3 mg / kg HC-7366 twice daily, 1 mg / kg HC-7366 twice daily, 3 mg / kg HC-7366 twice daily, 10 mg / kg HC-7366 twice daily, or 30 mg / kg HC-7366 twice daily.

[0161] Figure 129 is a graph showing Figure 128 the expression changes of upstream regulators of differentially expressed genes in the treatment groups described in

[0162] Figure 130 is a graph showing Figure 128 the IHC staining results of Ki67-positive cells in tumor sections of the treatment groups described in , *p < 0.05, **p < 0.01, one-way ANOVA.

[0163] Figure 131 Graph comparing the change in mean tumor volume over time in NCI-H1975 tumor-bearing mice administered vehicle (e.g., 0.5% methylcellulose (MC), 10 μL / g) twice daily, 3 mg / kg HC-7366 twice daily, osimertinib 2.5 mg / kg once daily, or 3 mg / kg HC-7366 twice daily + osimertinib 2.5 mg / kg once daily.

[0164] Figure 132 Graph comparing the percentage change in body weight over time in NCI-H1975 tumor-bearing mice administered vehicle (e.g., 0.5% MC, 10 μL / g) twice daily, 3 mg / kg HC-7366 twice daily, osimertinib 2.5 mg / kg once daily, or 3 mg / kg HC-7366 twice daily + osimertinib 2.5 mg / kg once daily.

[0165] Figure 133 Graph comparing the change in mean tumor volume over time in NCI-H1975 tumor-bearing mice administered vehicle (e.g., 0.5% MC, 10 μL / g) twice daily, 3 mg / kg HC-7366 twice daily, dacomitinib 15 mg / kg once daily, or 3 mg / kg HC-7366 twice daily + dacomitinib 15 mg / kg once daily.

[0166] Figure 134Graph comparing the percent change in body weight over time in NCI-H1975 tumor-bearing mice administered vehicle (e.g., 0.5% MC, 10 μL / g) twice daily, 3 mg / kg HC-7366 twice daily, dacomitinib 15 mg / kg once daily, or 3 mg / kg HC-7366 twice daily + dacomitinib 15 mg / kg once daily.

[0167] Figure 135 Graph comparing the change in tumor volume over time in MFE280 tumor-bearing mice administered vehicle twice daily, 0.5 mg / kg HC-7366 twice daily, 2 mg / kg HC-7366 twice daily, 1 mg / kg PT-2977 (bemcentinib) twice daily, 0.5 mg / kg HC-7366 twice daily + 1 mg / kg PT-2977 twice daily, or 2 mg / kg HC-7366 twice daily + 1 mg / kg PT-2977 twice daily. Detailed Description of the Invention

[0169] As generally described herein, the present disclosure provides methods for treating advanced solid tumors (e.g., advanced head and neck squamous cell carcinoma, colorectal cancer, non-small cell lung cancer (NSCLC), renal cell carcinoma, and urothelial carcinoma of the bladder) in a subject in need thereof. The present disclosure also provides methods for treating blood cancers (e.g., acute myeloid leukemia (AML)) in a subject in need thereof. The methods described herein generally comprise administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0170] Definitions

[0171] To facilitate understanding of the invention, some terms and phrases are defined below.

[0172] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations used herein have their conventional meanings within the fields of chemistry and biology. The chemical structures and chemical formulas listed herein are constructed according to the standard rules of chemical valence known in the chemical art.

[0173] Throughout the specification, when compositions and kits are described as having, including, or containing particular components, or when processes and methods are described as having, including, or containing particular steps, it is contemplated that, in addition, there are present the compositions and kits of the invention consisting essentially of or consisting of the recited components, and the processes and methods according to the invention consisting essentially of or consisting of the recited processing steps.

[0174] In this application, when it is mentioned that an element or component is included in the list of listed elements or components and / or is selected from the list of listed elements or components, it should be understood that the element or component can be any one of the listed elements or components, or the element or component can be selected from two or more of the listed elements or components.

[0175] In addition, it should be understood that the elements and / or features of the compositions or methods described herein can be combined in various ways without departing from the spirit and scope of the present invention, whether explicitly or implicitly herein. For example, when a particular compound is mentioned, the compound can be used in various embodiments of the compositions of the present invention and / or in the methods of the present invention, unless otherwise understood from the context. In other words, in this application, the embodiments are described and depicted in a manner that enables a clear and concise application to be written and drawn, but it is contemplated and should be understood that the embodiments can be combined or separated in various ways without departing from this teaching and invention. For example, it should be understood that all of the features described and depicted herein are applicable to all aspects of the invention described and depicted herein.

[0176] Unless the context is inappropriate, the articles "a" and "an" as used in this disclosure are used to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. For example, "an element" means one element or more than one element.

[0177] Unless otherwise specified, the term "and / or" as used in this disclosure means "and" or "or".

[0178] It should be understood that, unless otherwise understood according to the context and usage, the expression "at least one" includes each of the objects listed after the expression and various combinations of two or more of the listed objects. Unless otherwise understood from the context, the expression "and / or" in relation to three or more listed objects should be understood to have the same meaning.

[0179] The use of the terms "include", "include", "including", "have", "has", "having", "contain", "contains" or "containing" (including their grammatical equivalents) should generally be understood to be open-ended and non-limiting, e.g., not excluding additional unlisted elements or steps, unless otherwise expressly stated or understood from the context.

[0180] When the term "about" is used before a quantitative value, the present invention also includes the specific quantitative value itself, unless otherwise expressly stated. As used herein, the term "about" means a variation of ±10% relative to the nominal value, unless otherwise stated or inferred from the context.

[0181] Throughout this specification, variables or parameters are disclosed in groups or in ranges. It is specifically intended that the description include every individual sub-combination of the members of these groups and ranges. For example, integers in the range of 0 to 40 are specifically intended to be disclosed individually as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40, and integers in the range of 1 to 20 are specifically intended to be disclosed individually as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.

[0182] Any and all examples or exemplary language used herein, such as "for example" or "including", are only intended to better illustrate the present invention and do not constitute a limitation on the scope of the present invention, unless otherwise required. No language in the specification should be construed as indicating that any unclaimed element is essential for the practice of the present invention.

[0183] Generally, unless otherwise stated, components specified as percentages are by weight. In addition, if a variable is not accompanied by a definition, the previous definition of the variable shall apply.

[0184] As used herein, "pharmaceutical composition" or "pharmaceutical formulation" refers to a combination of an active agent with an inert or active carrier such that the composition is particularly suitable for in vivo or ex vivo diagnostic or therapeutic use.

[0185] "Pharmaceutically acceptable" means approved or approvable by a regulatory agency of the Federal or State government or the corresponding agency in a country other than the United States, or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals and, more particularly, in humans.

[0186] As used herein, "pharmaceutically acceptable salt" refers to a salt of any acidic or basic group that may be present in a compound of the present invention (e.g., a compound of formula (I)), which salt is compatible with pharmaceutical administration.

[0187] As is known to those skilled in the art, "salts" of a compound can be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Other acids (such as oxalic acid), although not pharmaceutically acceptable in themselves, can be used to prepare salts that are used as intermediates for obtaining the compounds described herein and their pharmaceutically acceptable acid addition salts.

[0188] Examples of bases include, but are not limited to, hydroxides of alkali metals (such as sodium and potassium), hydroxides of alkaline earth metals (such as magnesium and calcium), ammonia, and compounds of the formula NW 4+ (wherein W is C 1-4 alkyl), etc.

[0189] Examples of salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, mesylate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, toluenesulfonate, undecanoate, etc. Other examples of salts include the anions of the compounds of the present invention complexed with suitable cations such as Na + , K + , Ca 2+ , NH 4+ and NW 4+ (wherein W can be C 1-4 alkyl), etc.

[0190] For therapeutic use, salts of the compounds of the present invention (e.g., compounds of formula (I)) are considered pharmaceutically acceptable. However, salts of non-pharmaceutically acceptable acids and bases can also find use, for example, in the preparation or purification of pharmaceutically acceptable compounds.

[0191] As used herein, "pharmaceutically acceptable excipient" refers to a substance that aids in the administration and / or absorption of an active agent by a subject and can be included in the compositions of the present invention without causing significant adverse toxicological effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, saline solutions (e.g., phosphate buffered saline solution), emulsions (e.g., oil / water or water / oil emulsions), lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorants, salt solutions (e.g., Ringer's solution), alcohols, oils, gelatin, carbohydrates, fatty acid esters, and coloring agents, etc. Such formulations can be sterilized and, if desired, can be mixed with adjuvants that do not react harmfully with the compounds of the present invention, such adjuvants as lubricants, preservatives, stabilizers, wetting agents, emulsifying agents, salts affecting osmotic pressure, buffers, coloring agents, and / or aromatic substances, etc. For examples of excipients, see Martin, Remington’s Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA (1975).

[0192] The term "AUC" refers to the area under the time / plasma concentration curve after administration of a compound of formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutical composition described herein. AUC 0-无穷大 represents the area under the curve of the plasma concentration relative to time from time 0 to infinity. AUC 0-t represents the area under the curve of the plasma concentration relative to time from time 0 to time t. It should be understood that the AUC value can be determined by methods known in the art.

[0193] The "subjects" covered by the administration include, but are not limited to, humans (i.e., males or females of any age group, such as pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or elderly adults)) and / or non-human animals, such as mammals, such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In certain embodiments, the subject is a human. In certain embodiments, the subject is an adult. In certain embodiments, the subject is a non-human animal.

[0194] The term "C max " refers to the maximum concentration of a therapeutic agent (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof) in the blood (e.g., plasma) after administration of the therapeutic agent or a pharmaceutical composition containing the therapeutic agent (e.g., a pharmaceutical composition described herein).

[0195] The term "t max” refers to the time (in hours) to reach C after administration of a therapeutic agent (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition comprising the therapeutic agent (e.g., a pharmaceutical composition described herein). max The time is measured in hours.

[0196] As used herein, "solid dosage form" refers to a pharmaceutical dosage in solid form, such as tablets, capsules, granules, powders, sachets, reconstitutable powders, dry powder inhalers, and chewable tablets.

[0197] As used herein, "administer" refers to oral administration, administration as a suppository, topical contact, intravenous administration, parenteral administration, intraperitoneal administration, intramuscular administration, intralesional administration, intrathecal administration, intracranial administration, intranasal administration, or subcutaneous administration, or implantation of a sustained release device (e.g., a microosmotic pump) into a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other delivery methods include, but are not limited to, the use of liposomal formulations, intravenous infusions, transdermal patches, etc. "Co-administer" refers to the administration of the compositions described herein either simultaneously with, shortly before, or shortly after the administration of one or more additional therapies (e.g., an anti-cancer agent, a chemotherapeutic agent, or an immunotherapy). The compound of formula (I) or a pharmaceutically acceptable salt thereof may be administered alone or may be co-administered to a patient. Co-administration refers to the inclusion of simultaneous or sequential separate or combined administration of compounds (more than one compound or agent). Thus, when needed, the formulation may also be combined with other active substances (e.g., to reduce metabolic degradation).

[0198] As used herein, "fasting state" refers to at least 1 hour before a subject eats or at least 2 hours after a subject eats.

[0199] The terms "disease", "disorder", and "condition" are used interchangeably herein.

[0200] As used herein, and unless otherwise specified, the terms "treat", "treating", and "treatment" encompass acts that occur when a subject has a particular disease, disorder, or condition, and that act reduces the severity of the disease, disorder, or condition, or slows or retards the progression of the disease, disorder, or condition (e.g., "therapeutic treatment").

[0201] Generally, an "effective amount" of a compound (e.g., a compound of formula (I) or a pharmaceutically acceptable salt thereof) refers to an amount sufficient to elicit a desired biological response, e.g., treating advanced solid tumors and / or blood cancers. Those skilled in the art will understand that the effective amount of the compounds of the present disclosure may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, weight, health, and condition of the subject.

[0202] Compound

[0203] The compound of formula (I) shown below is a selective GCN2 modulator (e.g., activates or inhibits GCN2) and is also known as 6-(3-((5-chloro-2-methoxypyridin)-3-sulfonamido)-2,6-difluorophenyl)-N-methylimidazo[1,5-a]pyrazine-1-carboxamide:

[0204]

[0205] The compound of formula (I) is also referred to as HC-7366 throughout the present disclosure. The method for the chemical synthesis of the compound of formula (I) is described in Example 1.

[0206] In one aspect, provided herein is a method of administering a compound of formula (I) or a pharmaceutically acceptable salt thereof for treating advanced solid tumors (e.g., head and neck squamous cell carcinoma, colorectal cancer, NSCLC, renal cell carcinoma, and urothelial carcinoma of the bladder) in a subject in need thereof.

[0207] In another aspect, provided herein is a method of administering a compound of formula (I) or a pharmaceutically acceptable salt thereof for treating blood cancers (e.g., AML) in a subject in need thereof.

[0208] In various embodiments, provided herein is a method of administering a pharmaceutically acceptable salt of a compound of formula (I) for treating advanced solid tumors (e.g., head and neck squamous cell carcinoma, colorectal cancer, NSCLC, renal cell carcinoma, and urothelial carcinoma of the bladder) in a subject in need thereof.

[0209] In various embodiments, provided herein is a method of administering a pharmaceutically acceptable salt of a compound of formula (I) for treating blood cancers (e.g., AML) in a subject in need thereof.

[0210] In certain embodiments, the pharmaceutically acceptable salt of the compound of formula (I) is a potassium salt. In certain embodiments, the potassium salt of the compound of formula (I) is a hydrate. In certain embodiments, the potassium salt of the compound of formula (I) is a monohydrate. The method for preparing the potassium salt of the compound of formula (I) is described in Example 2.

[0211] Pharmaceutical composition

[0212] The present invention provides pharmaceutical compositions which generally comprise a compound of formula (I) or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0213] In one aspect, the present invention provides methods of administering a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients for treating advanced solid tumors (e.g., head and neck squamous cell carcinoma, colorectal cancer, NSCLC, renal cell carcinoma, and urothelial carcinoma of the bladder) in a subject in need thereof.

[0214] In another aspect, the present invention provides methods of administering a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients for treating blood cancers (e.g., AML) in a subject in need thereof.

[0215] In another aspect, the present invention provides methods of administering a pharmaceutical composition comprising a pharmaceutically acceptable salt of a compound of formula (I) and one or more pharmaceutically acceptable excipients for treating advanced solid tumors (e.g., head and neck squamous cell carcinoma, colorectal cancer, NSCLC, renal cell carcinoma, and urothelial carcinoma of the bladder) in a subject in need thereof.

[0216] In another aspect, the present invention provides methods of administering a pharmaceutical composition comprising a pharmaceutically acceptable salt of a compound of formula (I) and one or more pharmaceutically acceptable excipients for treating blood cancers (e.g., AML) in a subject in need thereof.

[0217] In various embodiments, the present invention provides pharmaceutical compositions which comprise an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients.

[0218] In various embodiments, the present invention provides pharmaceutical compositions which comprise an effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and one or more pharmaceutically acceptable excipients.

[0219] In certain embodiments, the amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the pharmaceutical compositions described herein is from about 10 mg to about 150 mg, from about 20 mg to about 150 mg, from about 40 mg to about 150 mg, from about 75 mg to about 150 mg, from about 125 mg to about 150 mg, from about 10 mg to about 125 mg, from about 10 mg to about 75 mg, from about 10 mg to about 40 mg, from about 10 mg to about 20 mg, from about 20 mg to about 125 mg, from about 20 mg to about 75 mg, from about 20 mg to about 40 mg, from about 40 mg to about 125 mg, from about 40 mg to about 75 mg, or from about 75 mg to about 125 mg (on a free acid equivalent weight basis). In certain embodiments, the amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the pharmaceutical compositions described herein is from about 10 mg to about 150 mg (on a free acid equivalent weight basis).

[0220] In certain embodiments, the amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the pharmaceutical compositions described herein is about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, or about 150 mg (on a free acid equivalent weight basis). In certain embodiments, the amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the pharmaceutical compositions described herein is about 10 mg (on a free acid equivalent weight basis). In certain embodiments, the amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the pharmaceutical compositions described herein is about 20 mg (on a free acid equivalent weight basis). In certain embodiments, the amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the pharmaceutical compositions described herein is about 40 mg (on a free acid equivalent weight basis). In certain embodiments, the amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the pharmaceutical compositions described herein is about 75 mg (on a free acid equivalent weight basis). In certain embodiments, the amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the pharmaceutical compositions described herein is about 125 mg (on a free acid equivalent weight basis). In certain embodiments, the amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the pharmaceutical compositions described herein is about 150 mg (on a free acid equivalent weight basis).

[0221] In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in the pharmaceutical compositions described herein is from about 10 mg to about 150 mg, from about 20 mg to about 150 mg, from about 40 mg to about 150 mg, from about 75 mg to about 150 mg, from about 125 mg to about 150 mg, from about 10 mg to about 125 mg, from about 10 mg to about 75 mg, from about 10 mg to about 40 mg, from about 10 mg to about 20 mg, from about 20 mg to about 125 mg, from about 20 mg to about 75 mg, from about 20 mg to about 40 mg, from about 40 mg to about 125 mg, from about 40 mg to about 75 mg, or from about 75 mg to about 125 mg (on a free acid equivalent weight basis). In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in the pharmaceutical compositions described herein is from about 10 mg to about 150 mg (on a free acid equivalent weight basis).

[0222] In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in the pharmaceutical compositions described herein is about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, or about 150 mg (on a free acid equivalent weight basis). In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in the pharmaceutical compositions described herein is about 10 mg (on a free acid equivalent weight basis). In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in the pharmaceutical compositions described herein is about 20 mg (on a free acid equivalent weight basis). In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in the pharmaceutical compositions described herein is about 40 mg (on a free acid equivalent weight basis). In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in the pharmaceutical compositions described herein is about 75 mg (on a free acid equivalent weight basis). In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in the pharmaceutical compositions described herein is about 125 mg (on a free acid equivalent weight basis). In certain embodiments, the amount of the pharmaceutically acceptable salt of the compound of formula (I) in the pharmaceutical compositions described herein is about 150 mg (on a free acid equivalent weight basis).

[0223] In various embodiments, the present disclosure provides pharmaceutical compositions comprising:

[0224] (i) from about 10 mg to about 150 mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis); and

[0225] (ii) one or more pharmaceutically acceptable excipients.

[0226] In various embodiments, the present disclosure provides pharmaceutical compositions comprising:

[0227] (i) about 10 mg to about 150 mg of a pharmaceutically acceptable salt of a compound of formula (I) (on a free acid equivalent weight basis); and

[0228] (ii) one or more pharmaceutically acceptable excipients.

[0229] In another aspect, the present disclosure provides pharmaceutical compositions comprising about 10 mg to about 150 mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis) and one or more pharmaceutically acceptable excipients for treating advanced solid tumors (e.g., head and neck squamous cell carcinoma, colorectal cancer, NSCLC, renal cell carcinoma, and urothelial carcinoma of the bladder) in a subject in need thereof.

[0230] In another aspect, the present disclosure provides pharmaceutical compositions comprising about 10 mg to about 150 mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis) and one or more pharmaceutically acceptable excipients for treating blood cancers (e.g., AML) in a subject in need thereof.

[0231] In another aspect, the present disclosure provides pharmaceutical compositions comprising about 10 mg to about 150 mg of a pharmaceutically acceptable salt of a compound of formula (I) (on a free acid equivalent weight basis) and one or more pharmaceutically acceptable excipients for treating blood cancers (e.g., AML) in a subject in need thereof.

[0232] In another aspect, the present disclosure provides pharmaceutical compositions comprising about 10 mg to about 150 mg of a pharmaceutically acceptable salt of a compound of formula (I) (on a free acid equivalent weight basis) and one or more pharmaceutically acceptable excipients for treating advanced solid tumors (e.g., head and neck squamous cell carcinoma, colorectal cancer, NSCLC, renal cell carcinoma, and urothelial carcinoma of the bladder) in a subject in need thereof.

[0233] In certain embodiments, the pharmaceutically acceptable salt of the compound of formula (I) is a potassium salt. In certain embodiments, the potassium salt of the compound of formula (I) is a hydrate. In certain embodiments, the potassium salt of the compound of formula (I) is a monohydrate.

[0234] The pharmaceutical compositions described herein can be administered by a variety of routes, including but not limited to oral (enteral) administration, parenteral (by injection) administration, rectal administration, transdermal administration, intradermal administration, intrathecal administration, subcutaneous (SC) administration, intravenous (IV) administration, intramuscular (IM) administration, and intranasal administration. In certain embodiments, the pharmaceutical compositions described herein are administered orally.

[0235] The pharmaceutical compositions described herein can also be administered chronically (“chronic administration”). Chronic administration refers to the administration of a compound or its pharmaceutical composition over a relatively long period of time (e.g., over 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc.), or can be continued indefinitely, e.g., for the remainder of the subject's life. In certain embodiments, chronic administration is intended to provide a constant level of the compound in the blood (e.g., over a relatively long period of time within the therapeutic window).

[0236] The pharmaceutical compositions described herein can be presented in unit dosage forms to facilitate accurate dosing. The term “unit dosage form” refers to physically discrete units suitable as unit doses for human subjects and other mammals, each unit containing a calculated predetermined amount of the active substance combined with a suitable pharmaceutical excipient to produce the desired therapeutic effect. Typical unit dosage forms include prefilled, premeasured ampoules or syringes of liquid compositions, or pills, tablets, capsules, etc. in the case of solid compositions.

[0237] In certain embodiments, the pharmaceutical compositions provided herein are administered to a patient in solid dosage forms. In certain embodiments, the solid dosage form is a capsule.

[0238] In certain embodiments, the pharmaceutical composition is an immediate-release capsule formulation of HC-7366 potassium salt monohydrate. In some embodiments, the capsule is a hard gelatin capsule. In some embodiments, the capsule contains one or more of lactose monohydrate, microcrystalline cellulose, croscarmellose sodium, colloidal silicon dioxide, and magnesium stearate. In some embodiments, the capsule contains each of lactose monohydrate, microcrystalline cellulose, croscarmellose sodium, colloidal silicon dioxide, and magnesium stearate. In some embodiments, the capsule contains 10 mg, 25 mg, or 100 mg of HC-7366 potassium salt monohydrate (on a free acid equivalent weight basis).

[0239] Although the description of the pharmaceutical compositions provided herein is mainly directed to pharmaceutical compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to a wide variety of animals. Modifying a pharmaceutical composition suitable for administration to humans to make it suitable for administration to various animals is readily understandable, and an ordinary veterinary pharmacologist can make such modifications with ordinary experimental designs and / or. General considerations for the formulation and / or manufacture of pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy 21 st ed., Lippincott Williams & Wilkins, 2005.

[0240] Methods of Use and Treatment Methods

[0241] Provided herein are methods of treating solid tumors in a subject in need thereof. The method generally comprises administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof; or a pharmaceutical composition described herein. In certain embodiments, the solid tumor is an advanced solid tumor.

[0242] Solid tumors for which the compound of formula (I) or a pharmaceutically acceptable salt thereof is considered useful for treatment include, but are not limited to, pancreatic cancer; bladder cancer; colorectal cancer; breast cancer, including metastatic breast cancer; prostate cancer, including androgen-dependent and androgen-independent prostate cancer; kidney cancer or renal cancer, including, for example, metastatic renal cell carcinoma; hepatocellular carcinoma; lung cancer, including, for example, NSCLC, bronchioloalveolar carcinoma (BAC), and adenocarcinoma of the lung; ovarian cancer, including, for example, progressive epithelial or primary peritoneal carcinoma; cervical cancer; gastric cancer; esophageal cancer; head and neck cancer, including, for example, squamous cell carcinoma of the head and neck; melanoma; neuroendocrine cancer, including metastatic neuroendocrine tumors; brain tumors, including, for example, glioma, anaplastic oligodendroglioma, adult glioblastoma multiforme, and adult anaplastic astrocytoma; bone cancer; and soft tissue sarcoma, liver cancer, rectal cancer, penile cancer, vulvar cancer, thyroid cancer, salivary gland cancer, endometrial cancer or uterine cancer, hepatoma, hepatocellular carcinoma, liver cancer, gastric cancer or stomach cancer including gastrointestinal cancer, peritoneal cancer, squamous cell carcinoma of the lung, gastroesophageal cancer, biliary tract cancer, gallbladder cancer, colorectal / appendiceal cancer, and squamous cell carcinoma (e.g., epithelial squamous cell carcinoma).

[0243] In certain embodiments, the advanced solid tumor is selected from squamous cell carcinoma of the head and neck, colorectal cancer, NSCLC, renal cell carcinoma, and transitional cell carcinoma of the bladder.

[0244] In certain embodiments, the advanced solid tumor is a sarcoma. In certain embodiments, the advanced solid tumor is colorectal cancer. In certain embodiments, the advanced solid tumor is head and neck cancer. In certain embodiments, the advanced solid tumor is renal cell carcinoma. In certain embodiments, the advanced solid tumor is prostate cancer. In certain embodiments, the advanced solid tumor is NSCLC. In certain embodiments, the advanced solid tumor is endometrial cancer.

[0245] In certain embodiments, the advanced solid tumor is selected from sarcoma, colorectal cancer, head and neck cancer, and prostate cancer.

[0246] Also provided herein are methods of treating blood cancers in a subject in need thereof. The methods generally comprise administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0247] In certain embodiments, the blood cancer is selected from multiple myeloma, leukemia, and lymphoma. In certain embodiments, the leukemia is selected from chronic lymphocytic leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, and AML.

[0248] In certain embodiments, the blood cancer is leukemia. In certain embodiments, the blood cancer is AML.

[0249] In some embodiments, the blood cancer is resistant to B cell lymphoma inhibitors. In certain embodiments, the blood cancer is resistant to venetoclax.

[0250] In some embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof activates the integrated stress response pathway (ISR) in the advanced solid tumor or blood cancer. In some embodiments, the ISR activation is GCN2-dependent.

[0251] In some embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof induces the expression of ASNS, PSAT1, PHGDH, argininosuccinate synthase 1 (ASS1), and / or PUMA in advanced solid tumors or blood cancers. In some embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof reduces the protein level of S100A8 / A9 in advanced solid tumors or blood cancers. In some embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof reduces mitochondrial respiration in advanced solid tumors or blood cancers. In some embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof reduces myeloid-restricted precursors and mature myeloid cells in a subject. In some embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof alters metabolites involved in amino acid metabolism, oxidative stress, urea cycle, and / or pyrimidine biosynthesis in advanced solid tumors or blood cancers. In some embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof reduces proteins involved in oxidative phosphorylation in advanced solid tumors or blood cancers. In some embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof reduces HIF- and / or E2F1-driven transcriptional activity in advanced solid tumors or blood cancers. In some embodiments, HIF- and / or E2F1-driven transcription includes the expression of mid-late transition genes. In some embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof increases ATF4- and / or JUN transcriptional activity in advanced solid tumors or blood cancers.

[0252] In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises administering to a subject from about 10 mg to about 150 mg, from about 15 mg to about 150 mg, from about 20 mg to about 150 mg, from about 25 mg to about 150 mg, from about 30 mg to about 150 mg, from about 35 mg to about 150 mg, from about 40 mg to about 150 mg, from about 45 mg to about 150 mg, from about 50 mg to about 150 mg, from about 55 mg to about 150 mg, from about 60 mg to about 150 mg, from about 65 mg to about 150 mg, from about 70 mg to about 150 mg, from about 75 mg to about 150 mg, from about 80 mg to about 150 mg, from about 85 mg to about 150 mg, from about 90 mg to about 150 mg, from about 95 mg to about 150 mg, from about 100 mg to about 150 mg, from about 105 mg to about 150 mg, from about 110 mg to about 150 mg, from about 115 mg to about 150 mg, from about 120 mg to about 150 mg, from about 125 mg to about 150 mg, from about 130 mg to about 150 mg, from about 135 mg to about 150 mg, from about 140 mg to about 150 mg, or from about 145 mg to about 150 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis).

[0253] In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises administering to a subject from about 10 mg to about 150 mg, from about 15 mg to about 145 mg, from about 20 mg to about 140 mg, from about 25 mg to about 135 mg, from about 30 mg to about 135 mg, from about 35 mg to about 130 mg, from about 40 mg to about 125 mg, from about 45 mg to about 120 mg, from about 50 mg to about 115 mg, from about 55 mg to about 110 mg, from about 60 mg to about 105 mg, from about 65 mg to about 100 mg, from about 70 mg to about 95 mg, from about 75 mg to about 90 mg, or from about 80 to about 85 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis).

[0254] In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises administering to a subject from about 10 mg to about 75 mg, from about 15 mg to about 75 mg, from about 20 mg to about 75 mg, from about 25 mg to about 75 mg, from about 30 mg to about 75 mg, from about 35 mg to about 75 mg, from about 40 mg to about 75 mg, from about 45 mg to about 75 mg, from about 50 mg to about 75, from about 55 mg to about 75 mg, from about 60 mg to about 75 mg, from about 65 mg to about 75 mg, or from about 70 mg to about 75 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis).

[0255] In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises administering to a subject from about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, or about 150 mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis).

[0256] In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises administering to a subject about 10 mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis). In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises administering to a subject about 20 mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis). In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises administering to a subject about 40 mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis). In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises administering to a subject about 75 mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis). In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises administering to a subject about 125 mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis). In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises administering to a subject about 150 mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis).

[0257] In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises administering to a subject at least about 10 mg, at least about 15 mg, at least about 20 mg, at least about 25 mg, at least about 30 mg, at least about 35 mg, at least about 40 mg, at least about 45 mg, at least about 50 mg, at least about 55 mg, at least about 60 mg, at least about 65 mg, at least about 70 mg, at least about 75 mg, at least about 80 mg, at least about 85 mg, at least about 90 mg, at least about 95 mg, at least about 100 mg, at least about 105 mg, at least about 110 mg, at least about 115 mg, at least about 120 mg, at least about 125 mg, at least about 130 mg, at least about 135 mg, at least about 140 mg, or at least about 145 mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis).

[0258] In certain embodiments, a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered orally to a subject. In certain embodiments, a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered orally to a subject daily. In certain embodiments, a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered orally to a subject once daily. In certain embodiments, a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered orally to a subject twice daily.

[0259] In certain embodiments, a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered orally to a subject once daily for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive days. In certain embodiments, a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered orally to a subject once daily for 21 consecutive days.

[0260] In certain embodiments, a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered orally to a subject once daily for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 consecutive days. In certain embodiments, a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered orally to a subject once daily for at least 21 consecutive days.

[0261] In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises administering to a subject from about 10 mg to about 150 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis).

[0262] In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises orally administering to a subject from about 10 mg to about 150 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis).

[0263] In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises orally administering to a subject from about 10 mg to about 150 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis) per day.

[0264] In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises orally administering to a subject once a day from about 10 mg to about 150 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis).

[0265] In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises orally administering to a subject once a day from about 10 mg to about 150 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis) for 21 consecutive days.

[0266] In certain embodiments, the subject is in a fasting state. In certain embodiments, the subject is not in a fasting state. In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises administering an effective amount to the subject about 1 hour before a meal or about 2 hours after a meal.

[0267] In certain embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof comprises administering to the subject from about 10 mg to about 150 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof (on a free acid equivalent weight basis) about 1 hour before a meal or about 2 hours after a meal.

[0268] In certain embodiments, the subject has previously received at least one prior line of therapy. In certain embodiments, the subject has previously received fewer than five prior lines of therapy. In certain embodiments, the subject has previously received one, two, three, or four prior lines of therapy. In certain embodiments, the subject has not previously received prior line therapy.

[0269] In certain embodiments, the subject has previously received at least one and no more than 5 prior-line therapies.

[0270] Prior-line therapies include, but are not limited to, surgery, radiotherapy (e.g., external particle beam radiotherapy or brachytherapy), chemotherapy (e.g., alkylating agents, nitrosoureas, antimetabolites, alkaloids and natural products, antitumor antibiotics, hormonal agents, and biologic response modifiers), gene therapy, DNA therapy, viral therapy (e.g., oncolytic viral therapy), RNA therapy, supportive therapy, and immunotherapy (e.g., immune checkpoint inhibition, adoptive cell therapy (e.g., tumor-infiltrating lymphocyte therapy, engineered T cell receptor therapy, CAR T cell therapy, natural killer cell therapy), or monoclonal antibodies).

[0271] In certain embodiments, the method includes administering a pharmaceutically acceptable salt of a compound of formula (I) in an effective amount. In certain embodiments, the pharmaceutically acceptable salt is a potassium salt. In certain embodiments, the potassium salt is a hydrate. In certain embodiments, the potassium salt is a monohydrate.

[0272] In certain embodiments, the methods described herein further include administering to the subject an effective amount of a second therapeutic agent. In certain embodiments, the second therapeutic agent is selected from checkpoint inhibitors, EGFR inhibitors, anti-angiogenic agents, venetoclax, fluorouracil, and combinations thereof.

[0273] In some embodiments, the second therapeutic agent is selected from anti-VEGFR antibodies, fluorouracil, PI3Kα inhibitors, MEK1 / 2 inhibitors, and hypoxia-inducible factor (HIF) inhibitors.

[0274] In some embodiments, the second therapeutic agent is venetoclax.

[0275] In some embodiments, the second therapeutic agent is fluorouracil. In some embodiments, the second therapeutic agent is 5-fluorouracil.

[0276] In some embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof and venetoclax activates the integrated stress response pathway (ISR) in advanced solid tumors or blood cancers to a greater extent than administering the compound of formula (I) or a pharmaceutically acceptable salt thereof or venetoclax alone.

[0277] In some embodiments, the degree of ISR activation can be determined using the experimental methods described herein by measuring the ISR activation markers described herein (e.g., ASNS, PSAT1, PHGDH, ASS1, and / or PUMA) in a tumor or cancer.

[0278] In certain embodiments, the second therapeutic agent is a checkpoint inhibitor. In certain embodiments, the second therapeutic agent is a PD-1 or PD-L1 inhibitor. In certain embodiments, the second therapeutic agent is selected from nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, and dostarlimab.

[0279] In certain embodiments, the second therapeutic agent is an EGFR inhibitor. In some embodiments, the EGFR inhibitor is selected from erlotinib, gefitinib, afatinib, osimertinib, and dacomitinib. In some embodiments, the EGFR inhibitor is selected from osimertinib and dacomitinib. In certain embodiments, the EGFR inhibitor is osimertinib. In certain embodiments, the EGFR inhibitor is dacomitinib. In some embodiments, the second therapeutic agent is selected from osimertinib and dacomitinib. In certain embodiments, the second therapeutic agent is osimertinib. In certain embodiments, the second therapeutic agent is dacomitinib.

[0280] In certain embodiments, the second therapeutic agent is an anti-angiogenic agent. In certain embodiments, the anti-angiogenic agent is a VEGFR inhibitor. In certain embodiments, the anti-angiogenic agent is an anti-VEGFR antibody. In certain embodiments, the anti-VEGFR antibody is an anti-vascular endothelial growth factor receptor 2 (VEGFR2) antibody. In certain embodiments, the anti-VEGFR2 antibody is ramucirumab or bevacizumab. In certain embodiments, the anti-VEGFR2 antibody is ramucirumab. In certain embodiments, the anti-VEGFR2 antibody is bevacizumab.

[0281] In certain embodiments, the second therapeutic agent is a VEGFR inhibitor. In certain embodiments, the VEGFR inhibitor is selected from sunitinib, axitinib, lenvatinib, tivozanib, pazopanib, cabozantinib, and ramucirumab. In certain embodiments, the second therapeutic agent is an anti-VEGFR antibody. In certain embodiments, the anti-VEGFR antibody is an anti-VEGFR2 antibody. In certain embodiments, the anti-VEGFR2 antibody is ramucirumab or bevacizumab. In certain embodiments, the anti-VEGFR2 antibody is ramucirumab. In certain embodiments, the anti-VEGFR2 antibody is bevacizumab.

[0282] In some embodiments, the second therapeutic agent is a HIF inhibitor. In certain embodiments, the HIF inhibitor is belzutifan.

[0283] In some embodiments, the second therapeutic agent is a PI3Kα inhibitor. In some embodiments, the PI3Kα inhibitor is selected from copanlisib and alpelisib. In certain embodiments, the PI3Kα inhibitor is alpelisib. In some embodiments, the second therapeutic agent is alpelisib.

[0284] In some embodiments, the second therapeutic agent is a MEK1 / 2 inhibitor. In some embodiments, the MEK1 / 2 inhibitor is selected from binimetinib, cobimetinib, selumetinib, and trametinib. In certain embodiments, the MEK1 / 2 inhibitor is trametinib. In some embodiments, the second therapeutic agent is trametinib.

[0285] In some embodiments, the second therapeutic agent is an EGFR inhibitor. In some embodiments, the second therapeutic agent is selected from osimertinib and dacomitinib.

[0286] In another aspect, the present disclosure provides a method for treating a blood cancer in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof; or a pharmaceutical composition described herein, and an effective amount of venetoclax. In some embodiments, the blood cancer is AML.

[0287] In another aspect, the present disclosure provides a method for treating a solid tumor (e.g., the solid tumors / advanced solid tumors described herein) in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof; or a pharmaceutical composition described herein, and an effective amount of a second therapeutic agent selected from an anti-VEGFR2 antibody, 5-fluorouracil, alpelisib, and trametinib.

[0288] In another aspect, the present disclosure provides a method for treating a solid tumor (e.g., the solid tumors / advanced solid tumors described herein) in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof; or a pharmaceutical composition described herein, and an effective amount of a VEGFR inhibitor. In some embodiments, the VEGFR inhibitor is an anti-VEGFR antibody. In some embodiments, the solid tumors are selected from renal cell carcinoma and colorectal cancer.

[0289] In another aspect, the present disclosure provides a method for treating a solid tumor (e.g., the solid tumors / advanced solid tumors described herein) in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof; or a pharmaceutical composition described herein, and an effective amount of an anti-VEGFR antibody. In some embodiments, the anti-VEGFR antibody is an anti-VEGFR2 antibody. In some embodiments, the solid tumors are selected from renal cell carcinoma and colorectal cancer.

[0290] In another aspect, provided herein are methods of treating solid tumors (e.g., the solid tumors / advanced solid tumors described herein) in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof; or a pharmaceutical composition described herein, and an effective amount of a HIF inhibitor. In certain embodiments, the HIF inhibitor is belzutifan. In some embodiments, the solid tumor is renal cell carcinoma. In some embodiments, the solid tumor is endometrial cancer.

[0291] In another aspect, provided herein are methods of treating solid tumors (e.g., the solid tumors / advanced solid tumors described herein) in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof; or a pharmaceutical composition described herein, and an effective amount of 5-fluorouracil. In some embodiments, the solid tumor is colorectal cancer.

[0292] In another aspect, provided herein are methods of treating solid tumors (e.g., the solid tumors / advanced solid tumors described herein) in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof; or a pharmaceutical composition described herein, and an effective amount of a PI3Kα inhibitor. In some embodiments, the PI3Kα inhibitor is alpelisib. In some embodiments, the solid tumor is colorectal cancer.

[0293] In another aspect, provided herein are methods of treating solid tumors (e.g., the solid tumors / advanced solid tumors described herein) in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof; or a pharmaceutical composition described herein, and an effective amount of a MEK1 / 2 inhibitor. In some embodiments, the MEK1 / 2 inhibitor is trametinib. In some embodiments, the solid tumor is colorectal cancer.

[0294] In another aspect, provided herein are methods of treating solid tumors (e.g., the solid tumors / advanced solid tumors described herein) in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof; or a pharmaceutical composition described herein, and an effective amount of an EGFR inhibitor. In some embodiments, the EGFR inhibitor is selected from osimertinib and dacomitinib. In some embodiments, the solid tumor is NSCLC.

[0295] In another aspect, provided herein are methods of treating solid tumors (e.g., the solid tumors / advanced solid tumors described herein) in a subject in need thereof, comprising administering to the subject any one of the pharmaceutical compositions described herein.

[0296] In another aspect, provided herein are methods of treating a blood cancer (e.g., a blood cancer described herein) in a subject in need thereof, comprising administering to the subject any one of the pharmaceutical compositions described herein.

[0297] In certain embodiments, the subject is human. In certain embodiments, the subject is an adult human. Examples

[0298] To more fully understand the disclosure described herein, the following examples are set forth. The synthetic and biological examples described in this application are used to illustrate the compounds, pharmaceutical compositions, and methods provided herein and should not be construed in any way as limiting their scope.

[0299] Example 1: Synthesis of 6-(3-((5-chloro-2-methoxypyridin)-3-sulfonamido)-2,6-difluorophenyl)-N-methylimidazo[1,5-a]pyrazine-1-carboxamide (Compound of Formula (I))

[0300]

[0301] Synthesis of 1-a: Ethyl 2-(5-bromopyrazin-2-yl)-2-[(diphenylmethylene)amino]acetate

[0302] 2,5-Dibromopyrazine (10 g, 42 mmol, 1 equiv), ethyl 2-[(diphenylmethylene)amino]acetate (11.8 g, 44 mmol, 1.05 equiv), tetrabutylammonium bromide (TBAB) (13.6 g, 42 mmol, 1 equiv), and K2CO3 (17.4 g, 126 mmol, 3 equiv) in (N-methyl-2-pyrrolidone) NMP (200 mL) were stirred overnight at 100 °C in an oil bath. The reaction mixture was cooled and filtered. The filtrate was diluted with 200 mL of water. The resulting solution was extracted with 2 x 200 mL of ethyl acetate and the organic layers were combined. The resulting mixture was washed with 2 x 200 ml of water. The mixture was dried over anhydrous sodium sulfate and concentrated. The residue was applied to a silica gel column and eluted with ethyl acetate / petroleum ether (PE) (1 / 10). The collected fractions were combined and concentrated to give ethyl 2-(5-bromopyrazin-2-yl)-2-[(diphenylmethylene)amino]acetate (8 g, 45% yield), which was a yellow solid.

[0303] LCMS (ES, m / z): [M+H] + : 424

[0304] Synthesis of 1-b: Ethyl 2-amino-2-(5-bromopyrazin-2-yl)acetate

[0305] Ethyl 2-(5-bromopyrazin-2-yl)-2-[(diphenylmethylene)amino]acetate (8 g, 18.8 mmol, 1 equiv), tetrahydrofuran (THF) (10 mL), and HCl (aqueous solution, 1 M) (20 mL) were placed in a 250 mL round-bottom flask. The resulting solution was stirred at 25 °C for 30 min. The formed solution was diluted with 50 mL of water and extracted with 2 x 50 mL of dichloromethane. The aqueous layer was adjusted to pH 8 with NH3.H2O and further extracted with 3 x 50 mL of dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate and concentrated. Ethyl 2-amino-2-(5-bromopyrazin-2-yl)acetate (4.7 g, 96% yield) was isolated as a yellow solid and used directly in the next step without further purification.

[0306] LCMS(ES,m / z):[M+H] + :260

[0307] Synthesis of 1-c: Ethyl 6-bromoimidazo[1,5-a]pyrazine-1-carboxylate

[0308] Ethyl 2-amino-2-(5-bromopyrazin-2-yl)acetate (4.2 g, 0.02 mol, 1 equiv) and triethyl orthoformate (20 mL) were placed in a 50 mL round-bottom flask. The resulting solution was stirred in an oil bath at 80 °C for 2 h. The reaction mixture was cooled and the solid was collected by filtration. Ethyl 6-bromoimidazo[1,5-a]pyrazine-1-carboxylate (2.2 g, 50% yield) was obtained as a brown solid after air drying.

[0309] LCMS(ES,m / z):[M+H] + :270

[0310] Synthesis of 1-d: 2,4-Difluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline

[0311] Dissolve 3-bromo-2,4-difluoroaniline (10 g, 48 mmol, 1 equiv), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (Pd(dppf)Cl2) (3.5 g, 4.8 mmol, 0.1 equiv), bis(pinacolato)diboron (18.3 g, 72 mmol, 1.5 equiv) and potassium acetate (KOAc) (14.2 g, 144.2 mmol, 3 equiv) in dioxane (240 mL). Stir the resulting solution in an oil bath at 100 °C overnight. Cool the reaction mixture and filter off the solid. Concentrate the filtrate and dilute with dichloromethane (DCM) (100 mL), then wash with 2 x 100 mL water and 100 mL brine. Dry the organic phase over anhydrous sodium sulfate and concentrate under reduced pressure. Apply the residue to a silica gel column and elute with ethyl acetate / petroleum ether (1 / 10). Isolate 2,4-difluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (8 g, yield 65%), which is a yellow solid.

[0312] LCMS(ES,m / z):[M+H] + :256

[0313] Synthesis of 1-e: Ethyl 6-(3-amino-2,6-difluorophenyl)imidazo[1,5-a]pyrazine-1-carboxylate

[0314] Dissolve ethyl 6-bromoimidazo[1,5-a]pyrazine-1-carboxylate (500 mg, 1.9 mmol, 1 equiv), 2,4-difluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (708 mg, 2.8 mmol, 1.5 equiv), Pd(dppf)Cl2 (135 mg, 0.2 mmol, 0.1 equiv), K2CO3 (767 mg, 5.6 mmol, 3 equiv) in dioxane (10 mL) and H2O (2 mL) and stir at 60 °C in an oil bath for 1 h. Cool the reaction mixture, dilute with water (20 ml), and extract with 3 x 20 mL dichloromethane. Dry the organic layer over anhydrous sodium sulfate and concentrate. Apply the residue to a silica gel column and elute with ethyl acetate / PE (1 / 2). Isolate ethyl 6-(3-amino-2,6-difluorophenyl)imidazo[1,5-a]pyrazine-1-carboxylate (200 mg, yield 34%), which is a brown solid.

[0315] LCMS(ES,m / z):[M+H] + :319

[0316] Synthesis of 1-f: Ethyl 6-[3-(5-chloro-2-methoxypyridine-3-sulfonamido)-2,6-difluorophenyl]imidazo[1, 5-a]pyrazine-1-carboxylate

[0317] Ethyl 6-(3-amino-2,6-difluorophenyl)imidazo[1,5-a]pyrazine-1-carboxylate (150 mg, 0.5 mmol, 1 equiv) in DCM (5 mL) was treated with pyridine (186 mg, 2.3 mmol, 5 equiv), and then with 5-chloro-2-methoxypyridine-3-sulfonyl chloride (137 mg, 0.6 mmol, 1.2 equiv). The resulting solution was stirred overnight. The resulting mixture was concentrated and purified by Flash-Prep-HPLC under the following conditions: column, WelFlashTM C18-I, spherical C18 20 - 40 μm; mobile phase: 0.1% formic acid / 5 - 70% MeCN, within 15 min; detector, 254 and 220 nm. Ethyl 6-[3-(5-chloro-2-methoxypyridine-3-sulfonamido)-2,6-difluorophenyl]imidazo[1,5-a]pyrazine-1-carboxylate (320 mg, yield 97%) was isolated as a yellow solid.

[0318] LCMS(ES,m / z):[M+H] + :524

[0319] Synthesis of 1-g: Ethyl 6-[3-(5-chloro-2-methoxypyridine-3-sulfonamido)-2,6-difluorophenyl]imidazo[1, 5-a]pyrazine-1-carboxylic acid

[0320] Ethyl 6-[3-(5-chloro-2-methoxypyridine-3-sulfonamido)-2,6-difluorophenyl]imidazo[1,5-a]pyrazine-1-carboxylate (200 mg, 0.4 mmol, 1 equiv), methanol (MeOH) (2 mL), THF (2 mL), H2O (2 mL), and LiOH (27 mg, 1.1 mmol, 3 equiv) were stirred in an oil bath at 60 °C for 1 h. After concentration, the crude product was purified by Flash-Prep-high performance liquid chromatography (HPLC) under the following conditions: column, WelFlashTM C18-I, spherical C18 20 - 40 μm; mobile phase: 5 - 60% acetonitrile (MeCN) / 0.1% ammonia water, within 15 min; detector, 254 nm. 6-[3-(5-chloro-2-methoxypyridine-3-sulfonamido)-2,6-difluorophenyl]imidazo[1,5-a]pyrazine-1-carboxylic acid (170 mg, yield 90%) was isolated as a yellow solid.

[0321] LCMS(ES,m / z):[M+H] + :496

[0322] 6-[3-(5-Chloro-2-methoxypyridine-3-sulfonamido)-2,6-difluorophenyl]-N-methylimidazo[1,5-a] Synthesis of Pyrazine-1-carboxamide

[0323] 6-[3-(5-Chloro-2-methoxypyridine-3-sulfonamido)-2,6-difluorophenyl]imidazo[1,5-a]pyrazine-1-carboxylic acid (170 mg, 0.3 mmol, 1 equiv) in N,N-dimethylformamide (DMF) (4 mL) was treated with diisopropylethylamine (DIEA) (133 mg, 1 mmol, 3 equiv), methylamine hydrochloride (16 mg, 0.5 mmol, 1.5 equiv) and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine 3-oxide hexafluorophosphate (HATU) (195 mg, 0.5 mmol, 1.5 equiv). The resulting solution was stirred for 1 hr and then concentrated. The crude product was purified by Flash-Prep-HPLC under the following conditions: column, WelFlashTM C18-I, spherical C18 20 - 40 μm, 120 g; mobile phase: 5 - 60% MeCN / 0.1% formic acid, over 20 min. 6-[3-(5-Chloro-2-methoxypyridine-3-sulfonamido)-2,6-difluorophenyl]-N-methylimidazo[1,5-a]pyrazine-1-carboxamide (43 mg, 25% yield) was isolated as an off-white solid.

[0324] Liquid chromatography / mass spectrometry (LCMS) (ES, m / z): [M + H] + : 509

[0325] 1 1H nuclear magnetic resonance spectroscopy (NMR) (300 MHz, DMSO-d6) δ 10.46 (s, 1H), 9.51 (d, J = 1.6 Hz, 1H), 8.66 (d, J = 5.0 Hz, 2H), 8.51 (d, J = 2.6 Hz, 1H), 8.43 (d, J = 4.9 Hz, 1H), 8.09 (d, J = 2.6 Hz, 1H), 7.42 (td, J = 8.8, 5.8 Hz, 1H), 7.25 (td, J = 9.3, 1.4 Hz, 1H), 3.92 (s, 3H), 2.84 (d, J = 4.7 Hz, 3H).

[0326] Example 2: Preparation of the potassium salt of the compound of formula (I)

[0327]

[0328] To a stirred mixture of 6-(3-((5-chloro-2-methoxypyridin-3-yl)sulfonamido)-2,6-difluorophenyl)-N-methylimidazo[1,5-a]pyrazine-1-carboxamide (prepared as in Example 1) in an aqueous isopropanol solution (IPA) was slowly added 1.1 equivalents of aqueous KOH, and the solution was heated. The resulting mixture was cooled, the solid was collected, washed with IPA / water, and then dried under heat and vacuum to give potassium ((5-chloro-2-methoxypyridin-3-yl)sulfonyl)(2,4-difluoro-3-(1-(methylcarbamoyl)imidazo[1,5-a]pyrazin-6-yl)phenyl)amide.

[0329] 19 19F NMR (400 MHz, d6-DMSO): -129.62 and -127.72 ppm.

[0330] Example 3: Biochemical Assay

[0331] The GCN2 protein was obtained from Carna Biosciences (GCN2 cat#: 05-153). The protein was diluted in assay buffer (ThermoFisher Scientific, #PV6135), 2 mM dithiothreitol (DTT) to obtain a final concentration of 2 nM, and 5 μL was plated into a 384-well white assay plate. HC-7366 was serially diluted 3-fold in dimethyl sulfoxide (DMSO) to 11 concentrations, and 10 nL of the neat solution was plated into a 384-well white assay plate. DMSO was used as a vehicle control. The green fluorescent protein (GFP)-eIF2α protein was obtained from ThermoFisher (cat# PV4809). In the presence of 2 mM DTT, 200 nM of the protein was diluted to 2x concentration (final concentration 100 nM GFP-eIF2α and 150 μM ATP) in assay buffer with 300 mM ATP, and 5 μL aliquots were added to each well containing GCN2 protein and HC-7366. The plate was incubated at 25 °C for 1.5 h in the dark with shaking at 1250 rpm. The Tb-anti-P-eIF2α (ThermoFisher cat# PV4810) was diluted to 4 nM concentration in time-resolved fluorescence energy transfer (TR-FRET) dilution buffer (ThermoFisher cat# PV3574) with 20 mM ethylenediaminetetraacetic acid (EDTA) (final concentration 2 nM Tb-anti-P-eIF2α and 10 nM EDTA). 10 μL of the Tb-anti-P-eIF2α solution was added to the TR-FRET reaction. The plate was incubated at 25 °C for 2 h in the dark with shaking at 600 rpm. The FRET signal from the plate was read on an Envision (PerkinElmer) plate reader:

[0332] Label 1 : Excitation: 340 nm, bandwidth 30 nm; Emission: 495 nm, bandwidth 10 nm. Lag time: 100 μsec. Integration time: 400 μsec. Scintillation: 30.

[0333] Label 2 : Excitation: 340 nm, bandwidth 30 nm; Emission: 520 nm, bandwidth 25 nm. Lag time: 100 μsec. Integration time: 400 μsec. Scintillation: 30

[0334] The data was analyzed using XLfit, as Figure 1 shown. The data shown in the figure is represented as vehicle % (which is the DMSO control).

[0335] Figure 1Describes the experimental results of HC-7366 in the above-mentioned GCN2 biochemical assay, showing the GCN2 inhibitory activity of the compound.

[0336] Example 4: Cell-based assay

[0337] HEK293-ATF4-Luc cells were used at 1.5e 5 cells / mL, and 25 μL of the cell suspension was added to each well of a 384-well cell culture plate (Corning, CLS3570-50EA) as specified and transferred to a 37 °C - 5% CO2 incubator (Thermo Scientific) overnight for cell attachment. HC-7366 was serially diluted to 11 concentrations by 3-fold dilution in DMSO, and 25 nL of the stock solution was plated into the cell plate by Echo550 (Labcyte, Echo550), and then incubated at 37 °C for 30 minutes. After treatment with HC-7366, the cells were treated with DMSO or 12.5 nM halofuginone to activate GCN2 for 6 hours. After 6 hours, 25 μL of One-Glo reagent (Promega, cat#: E6120) was added to each well for detection (1:1 with the medium). Then the plate was placed at room temperature for 10 min, and then the luciferase luminescence was read on an EnVision (PerkinElmer). The data was analyzed by XLfit (v5.3.1.3), equation 20.

[0338] Figure 2 Describes the experimental results of cells treated only with HC-7366 in the above-mentioned ATF4 activity assay, showing the effect of HC-7366 on ATF4 activation in cells. The concentration showing ATF4 activation is consistent with Figure 5 the concentration at which ATF4 protein expression was observed in HT1080 cells in Figure 3 Describes the experimental results of HC-7366 in cells treated with HC-7366 and halofuginone, demonstrating the GCN2 inhibitory effect of GCN2 in cells in the above-mentioned ATF4 activity assay.

[0339] In MOLM-16 cells, CellTiter- (CTG) viability assay

[0340] The MOLM-16 cells were obtained from DSMZ (#ACC 555) and cultured in RPMI 1640 (Gibco, #11875119) with 20% FBS (Invitrogen, #10099141C) in a cell culture incubator (Thermo Scientific) set at 37°C, 5% CO2, and 95% relative humidity. 30 μL of cell suspension (2k cells / well) was added to each well of a 384-well cell culture plate (Corning, #CLS3764-100EA). The plate was then incubated overnight. HC-7366 was serially diluted 3-fold into 10 concentrations in a 384-well plate using a TECAN EVO200. 40 nL of the HC-7366 stock solution was transferred into the 384-well cell culture plate using an Echo550, with a final concentration range of 1 - 10 μM. The plate was incubated in an incubator at 37°C and 5% CO2 for 72 hr. Cell viability was evaluated by adding 30 μL of CellTiter- 2.0 Assay (Promega, G9243) to each well. The plate was then incubated at room temperature (RT) for 30 min. Luminescence signals were read using an Envision (PerkinElmer) plate reader. Data were analyzed by XLfit (v5.3.1.3), equation 201.

[0341] Figure 4 The experimental results of HC-7366 in the above cell viability assay are depicted.

[0342] Example 5: Western Blot Analysis

[0343] Cell lysates were obtained from HT1080 cells treated with HC-7366 (0 to 10 μM) for 30 min, then treated with DMSO or 100 nM halofuginone to activate GCN2, and Western blot analysis was performed.

[0344] Reagents

[0345] Fisher Biological Reagents (cat#: BP2471-1) 10X Tris Buffered Saline (TBS)

[0346] Fisher Tween 20 (cat#: BP337-100 lot 10817)

[0347] BioRad Blotting Grade Blocking Agent (milk) (cat#: 170-6404)

[0348] Pierce SuperSignal West Femto (cat#: 34095)

[0349] Primary antibody

[0350] Anti-phospho-GCN2-T899 rabbit monoclonal, Abcam cat#: ab75836

[0351] Anti-GCN2 rabbit monoclonal, Abcam cat# ab134053

[0352] Anti-p-PERK rabbit antibody, Eli Lilly

[0353] Anti-PERK rabbit monoclonal, Cell Signaling Technology cat#: 3192

[0354] Anti-p-PKR rabbit polyclonal, ThermoFisher Scientific cat# 44-668G

[0355] Anti-PKR rabbit polyclonal, ThermoFisher Scientific cat#: 700286

[0356] Anti-p-eIF2αS51 rabbit monoclonal, Cell Signaling Technology cat#: 3398

[0357] Anti-eIF2α rabbit, Cell Signaling Technology cat#: 9722

[0358] Anti-ATF4 rabbit monoclonal, Cell Signaling Technology cat#: 11815

[0359] Anti-β-actin mouse monoclonal - Sigma cat#: A5441

[0360] Jackson Immunoresearch secondary antibody 1:5000 in 5% milk

[0361] HRP goat anti-rabbit, cat#: 111-035-046 lot 105262

[0362] HRP goat anti-mouse, cat#: 115-035-071 lot 100214

[0363] Sample preparation

[0364] Harvest HT1080 cells, count them, and dilute to the desired density. Add 2 mL of the cell suspension to each well of a 6-well cell culture plate. Transfer the plate to a 37 °C, 5% CO2 incubator overnight. Treat the cells as described above. Aspirate the medium and wash the cells with ice-cold phosphate-buffered saline (PBS). Add ice-cold 1x radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with protease and phosphatase inhibitors to the cell pellet on ice. After 30 min on ice, centrifuge the cell lysate at 4 °C, 12,000 rpm for 15 minutes and collect the supernatant. Determine the protein concentration using a BCA kit. Mix the cell lysate with 5X sodium dodecyl sulfate (SDS) loading buffer and denature at 95 °C for 5 minutes.

[0365] Western blot

[0366] Load 30 μg of protein onto a 4-12% Bis-Tris gel in 1X MOPS buffer and run at 120 V for 120 min. After electrophoresis is complete, transfer the separated proteins to a nitrocellulose membrane using a transblot system for 90 min, 300 mA, and then block the membrane with tris-buffered saline (TBS) buffer (5% bovine serum albumin (BSA)) for 1 hr at room temperature. Expose the membrane to the primary antibody in TBS buffer (1% BSA) in a cold room overnight. Then wash the membrane with TBS-Tween in R / T for 3 x 10 min and incubate with the secondary antibody in TBS buffer (1% BSA) for 1 hr at RT. Wash the membrane with TBS-Tween in R / T for 3 x 10 min and acquire images using a Li-COR imaging system.

[0367] Figure 5 The experimental results of HC-7366 in the above Western blot analysis are depicted. HC-7366 shows activation of the GCN2 pathway at low concentrations. Halofuginone acts as a GCN2 agonist by inhibiting prolyl-tRNA synthetase. Cells treated with halofuginone show activation of GCN2 and ATF4. The GCN2 modulator HC-7366 inhibits halofuginone-induced p-GCN2 and ATF4 in a dose-dependent manner. This result demonstrates the GCN2-modulating property of HC-7366, where it acts as an inhibitor of pGCN2 and ATF4 in the presence of halofuginone but activates the GCN2 pathway itself.

[0368] Example 6: In Vivo Tumor Growth Inhibition (TGI) Study

[0369] The effect of HC-7366 on xenograft tumor growth was investigated in several models listed below.

[0370] 1. Study Design

[0371] 1.1 Animals

[0372] Species and strains of cell lines and mice used :

[0373] MOLM - 16 (human AML cells): Female 6 - 8 - week - old non - obese diabetic / severe combined immunodeficiency (NOD SCID) mice were subcutaneously inoculated with MOLM - 16 tumor cells (1x10 7 ) in serum - free RPMI 1640 under the right flank epithelium for tumor growth. Treatment was started when the average tumor size reached approximately 220 mm 3 on day 14 after cell inoculation and continued until the end of the study by oral gavage of HC - 7366 at the listed doses (vehicle, 0.25 mg / kg HC - 7366, 0.5 mg / kg HC - 7366, 1 mg / kg HC - 7366, or 2 mg / kg HC - 7366, Figure 16 ).

[0374] HT1080 (human fibrosarcoma cells): Female 6 - 8 - week - old BALB / c nude mice were subcutaneously inoculated with HT1080 tumor cells (1×10 7 ) in 0.1 ml of serum - free EMEM medium under the right flank epithelium for tumor growth. Treatment was started when the average tumor size reached approximately 109 mm 3 on day 4 after inoculation and continued until the end of the study by oral gavage of HC - 7366 at the listed doses (vehicle, 0.3 mg / kg HC - 7366 BID (twice a day), 1 mg / kg HC - 7366 BID, 3 mg / kg HC - 7366 BID, 0.6 mg / kg HC - 7366 QD (once a day), 2 mg / kg HC - 7366 QD, or 6 mg / kg HC - 7366 QD, Figures 6-7 ).

[0375] LoVo (human colorectal cancer cells): Female 6 - 8 - week - old BALB / c nude mice were subcutaneously inoculated with LoVo tumor cells (5x10 6 ) in 0.1 ml of serum - free EMEM medium under the right flank epithelium for tumor growth. Treatment was started when the average tumor size reached approximately 243 mm 3 on day 16 after inoculation and continued until the end of the study by oral gavage of HC - 7366 at the listed doses (vehicle, 0.3 mg / kg HC - 7366, 1 mg / kg HC - 7366, 3 mg / kg HC - 7366, 10 mg / kg HC - 7366, or 30 mg / kg HC - 7366, Figures 8-9 ) twice a day.

[0376] DLD-1 (Human colorectal cancer cells): Female 6-8-week-old BALB / c nude mice were subcutaneously inoculated with DLD-1 tumor cells (5×10 6 ) in 0.1 ml of serum-free RPMI 1640 medium under the right flank epithelium for tumor growth. Treatment was started when the average tumor size reached approximately 195 mm 3 on the 7th day after inoculation and continued until the end of the study of intragastric administration of HC-7366 twice a day at the listed doses (vehicle, 0.3 mg / kg HC-7366, 1 mg / kg HC-7366, 3 mg / kg HC-7366, 10 mg / kg HC-7366, or 30 mg / kg HC-7366, Figure 10 ). For the time-course study, tumors were harvested on days 4, 7, and 14. The tumors were then used for IHC and RNAseq experiments (see Example 21).

[0377] FaDu (Human head and neck cancer cells): Female 6-8-week-old BALB / c nude mice were subcutaneously inoculated with FaDu tumor cells (5x10 6 ) in 0.1 ml of serum-free EMEM medium under the right flank epithelium for tumor growth. Treatment was started when the average tumor size reached approximately 194 mm 3 on the 21st day after inoculation and continued until the end of the study of intragastric administration of HC-7366 twice a day at the listed doses (vehicle, 0.3 mg / kg HC-7366, 1 mg / kg HC-7366, 3 mg / kg HC-7366, 10 mg / kg HC-7366, or 30 mg / kg HC-7366, Figures 11-13 ).

[0378] KG-1 (Human AML cancer cells): Female 6-8-week-old NOD SCID mice were subcutaneously inoculated with KG1 tumor cells (5x10 6 ) in serum-free RPMI 1640 under the right flank epithelium for tumor growth. Treatment was started when the average tumor size reached approximately 194 mm 3 on the 20th day after cell inoculation and continued until the end of the study of intragastric administration of HC-7366 twice a day at the listed doses (vehicle, 0.3 mg / kg HC-7366, 1 mg / kg HC-7366, 3 mg / kg HC-7366, 10 mg / kg HC-7366, or 30 mg / kg HC-7366, Figure 17 ).

[0379] Kasumi-1 (human AML cancer cells): Female 6 - 8 week-old CB17 SCID mice were subcutaneously inoculated with KG1 tumor cells (1 x 10 7 ) in serum-free RPMI 1640 under the right flank epithelium for tumor growth. Treatment was started when the average tumor size reached approximately 151 mm 3 on the 29th day after cell inoculation and continued until the end of the study with HC-7366 administered by oral gavage twice a day at the listed doses (vehicle, 3 mg / kg HC-7366, or 30 mg / kg HC-7366, Figure 18 ).

[0380] OCI-AML2 (human AML cancer cells): Female 6 - 8 week-old NOD SCID mice were subcutaneously inoculated with OCI-AML2 tumor cells (5 x 10 6 ) in serum-free RPMI 1640 under the right flank epithelium for tumor growth. Treatment was started when the average tumor size reached approximately 149 mm 3 on the 7th day after cell inoculation and continued until the end of the study with HC-7366 administered by oral gavage twice a day at the listed doses (vehicle, 0.3 mg / kg HC-7366, 1 mg / kg HC-7366, 3 mg / kg HC-7366, 10 mg / kg HC-7366, or 30 mg / kg HC-7366, Figure 19 ).

[0381] MV4-11 (human AML cancer cells): Female 6 - 8 week-old BALB / c nude mice were subcutaneously inoculated with MV4-11 tumor cells (1 x 10 7 ) in serum-free RPMI 1640 under the right flank epithelium for tumor growth. Treatment was started when the average tumor size reached approximately 150 mm 3 on the 13th day after cell inoculation and continued until the end of the study with HC-7366 administered by oral gavage twice a day at the listed doses (vehicle, 0.3 mg / kg HC-7366, 1 mg / kg HC-7366, 3 mg / kg HC-7366, 10 mg / kg HC-7366, or 30 mg / kg HC-7366, Figure 20 ). For the venetoclax combination study, venetoclax was dissolved in 0.5% carboxymethylcellulose (CMC) and co-administered at a dose of 50 mg / kg once a day (see Example 8).

[0382] 786-O (human renal cell carcinoma cancer cells): Female 6 - 8 week-old BALB / c nude mice were subcutaneously inoculated with 786-O tumor cells (5 x 10 in 0.1 ml of serum-free RPMI1640 medium under the right flank epithelium6 ), to allow tumor growth. Treatment was initiated when the average tumor size reached approximately 189 mm three weeks after inoculation 3 and continued until the end of the HC-7366 study, which was administered by oral gavage twice daily at the indicated doses (vehicle or 2 mg / kg HC-7366, Figures 52-54 ). For the DC101 combination study, DC101 (murine monoclonal anti-VEGFR2 antibody) was administered intraperitoneally at 15 mg / kg twice weekly.

[0383] A498 (human renal cell carcinoma cancer cells): Female 6 - 8-week-old NOD SCID mice were inoculated subcutaneously in the right flank with A498 tumor cells (5 x 10 6 ) in 0.1 ml of serum-free MEM medium for tumor growth. Treatment was initiated when the average tumor size reached approximately 349.44 mm four weeks after inoculation 3 and continued until the end of the HC-7366 study, which was administered by oral gavage twice daily at the indicated doses (vehicle or 2 mg / kg HC-7366, Figures 55-57 ). DC101 was administered intraperitoneally at 15 mg / kg twice weekly.

[0384] LNCaP (human prostate cancer cells): Male 6 - 8-week-old NOD SCID mice were inoculated subcutaneously in the right flank with 0.1 ml of LNCaP tumor cells (1 x 10 7 ) in RPMI 1640 with 10% fetal bovine serum (FBS) and Matrigel (1:1) for tumor growth. Treatment was initiated when the average tumor size reached approximately 207 mm 14 days after inoculation 3 and continued until the end of the HC-7366 study, which was administered by oral gavage twice daily at the indicated doses (vehicle, 0.3 mg / kg HC-7366, 1 mg / kg HC-7366, 3 mg / kg HC-7366, 10 mg / kg HC-7366 or 30 mg / kg HC-7366, Figure 14 ).

[0385] TM00298 (human prostate cancer cells): Male 6 - 8-week-old NSG mice were inoculated subcutaneously with 1 mm 3 TM00298 tumor fragments. Treatment was initiated when the average tumor size reached approximately 123 mm 3 and continued until the end of the HC-7366 study, which was administered by oral gavage twice daily at the indicated doses (vehicle, 3 mg / kg HC-7366 or 30 mg / kg HC-7366, Figure 15 ).

[0386] NCI-H1975 (human non-small cell lung cancer cells): Female 7-8-week-old athymic nude-Foxn1nu mice were subcutaneously inoculated with NCI-H1975 tumor cells (2.5 x 10 6 ) in 0.1 ml of serum-free RPMI 1640 medium under the right flank epithelium for tumor growth. The single agent HC-7366 was administered at 3 mg / kg, osimertinib at 2.5 mg / kg, and dacomitinib at 15 mg / kg. HC-7366 was combined with osimertinib or dacomitinib. The vehicle for each group was 0.5% methylcellulose. Treatment was initiated when the average tumor size reached approximately 205 mm 3 on day 22 after inoculation and continued until the end of the study. HC-7366 was administered by oral gavage twice daily, while osimertinib or dacomitinib was administered by oral gavage once daily ( Figure 131 and 133 ).

[0387] HCT116 (human colorectal cancer cells): Female 7-8-week-old athymic nude-Foxn1nu mice were subcutaneously inoculated with HCT116 tumor cells (5 x 10 6 ) in 0.1 ml of serum-free McCoy's 5a medium under the right flank epithelium for tumor growth. The single agent HC-7366 was administered at 3 or 30 mg / kg (twice daily, orally), trametinib at 1 mg / kg (once daily, orally), and alpelisib at 50 mg / kg (once daily, orally). HC-7366 was combined with trametinib or alpelisib. Treatment was initiated when the average tumor size reached approximately 270 mm 3 on day 18 after inoculation and continued until the end of the study ( Figures 63-64 ).

[0388] MFE280 (human endometrial cancer cells): Female 7-8-week-old athymic nude-Foxn1nu mice were subcutaneously inoculated with MFE280 tumor cells (10 × 10 6 ) in 0.2 ml of PBS under the right flank epithelium for tumor growth. The single agent HC-7366 was administered at 0.5 or 2 mg / kg (twice daily, orally), and PT2977 at 1 mg / kg (twice daily, orally). HC-7366 was combined with PT2977 at 0.5 or 2 mg / kg. Treatment was initiated when the average tumor size reached approximately 191 mm 3 on day 28 after inoculation and continued until the end of the study ( Figure 135 ).

[0389] 1.1.2 Supplier : Beijing AniKeeper Biotech Co., Ltd.

[0390] 1.1.3 Age : 6 to 8 weeks

[0391] 1.1.4 Gender : Female

[0392] 1.1.5 Body weight : 16 to 22 g

[0393] 1.2 Animal maintenance

[0394] 1.2.1 Isolation : Animals were quarantined for 7 days before the study. The general health of the animals was evaluated by a veterinarian, and a comprehensive health check was performed. Animals with abnormalities were excluded before the study. The general procedures for animal care and housing conform to the standards of the Standard Operating Procedures (SOP) of the Committee on Life Sciences of the National Research Council.

[0395] 1.2.2 Feeding : The general procedures for animal care and housing conform to the standards of the Committee on Life Sciences of the National Research Council, Standard Operating Procedures (SOP). All procedures related to animal handling, care, and animal research methods used in this study were conducted in accordance with the guidelines approved by Pharmaron's Institutional Animal Care and Use Committee (IACUC) and followed the guidance of the American Association for the Accreditation of Laboratory Animal Care (AAALAC). Mice were kept in a laminar flow room with constant temperature and humidity, with 3 - 5 mice in each cage. Animals were housed in polycarbonate cages with a cage size of 300×180×150 mm 3 , and placed in an environmentally monitored, well - ventilated room where the room temperature was maintained at (22 ± 3°C), and the relative humidity was maintained at 40% - 80%. Fluorescent lights provided illumination for approximately 12 hours per day. The bedding material was cork, which was changed once a week.

[0396] 1.2.3 Animal ID : Each animal was assigned an identification number; the following identification method was applied. Each cage card was marked with information such as study number, group, sex, dose, animal number, start date, study leader, and phone number. Individual animals were identified by ear coding.

[0397] 1.2.4 Diet : Throughout the study period, animals had free access to irradiated and sterilized dry pellet food.

[0398] 1.2.5 Water : During the quarantine and study periods, all animals had free access to bottled sterile drinking water. Before use, the bottles and the stoppers with attached straws were autoclaved. Water samples from the animal facility were analyzed, and the water analysis results were retained in the facility records and reviewed by a veterinarian or designated personnel to ensure the absence of known contaminants that could interfere with or affect the study results.

[0399] 1.3 Grouping and treatment

[0400] When the average tumor volume reaches approximately 100 - 250 mm 3 , grouping and treatment are initiated. Based on tumor volume and body weight, mice are assigned to the appropriate groups such that the average starting tumor size is the same for each treatment group.

[0401] 1.4 Vehicle

[0402] Vehicle for HC - 7366: 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) in PBS (final pH 7.4), unless otherwise specified.

[0403] 2. Experimental methods and measurement parameters

[0404] 2.1 Tumor inoculation method

[0405] Each mouse is inoculated with tumor cells (total volume of 0.1 ml) subcutaneously in the right flank epithelium or orthotopically in the mammary fat pad for tumor growth. When the average tumor size reaches approximately 100 - 250 mm 3 , treatment is initiated. Animals are randomly divided into groups of 8 - 10 animals based on tumor volume, typically including a vehicle control group and treatment groups at different dose levels.

[0406] 2.2 Measurement parameters

[0407] For routine monitoring, all study animals are monitored not only for tumor growth but also for their behavior, such as locomotor activity, food and water consumption (by cage - side inspection only), body weight (BW), dull eyes / hair, and any other abnormal effects. Any deaths and / or abnormal clinical symptoms are recorded.

[0408] 2.2.1 Body weight (BW) : Throughout the study, the body weights of all animals are measured and recorded three times per week.

[0409] The body weight change is expressed as a percentage and calculated using the following formula:

[0410] BW change (%) = (BW 第X天 / BW at 0天 ) × 100

[0411] 2.2.2 Tumor measurement : The tumor size is measured and recorded three times per week using calipers. The tumor volume (mm 3 ) is estimated using the following formula: TV = a × b 2 / 2, where "a" and "b" are the long and short diameters of the tumor, respectively. The tumor volume is calculated using the formula [tumor volume (mm 3 ) = π / 6 x length x width 2 .

[0412] %T / C (the ratio of tumor volume between the treatment group and the control group) was calculated by the formula Tumor Growth Inhibition (TGI) = (1 - ΔT / ΔC) x 100% (if ΔT > 0). ΔT, the mean tumor volume of the drug treatment group on the study observation day – the mean tumor volume of the drug treatment group on the initial dosing day; ΔC, the mean tumor volume of the control group on the study observation day – the mean tumor volume of the control group on the initial dosing day. Regression was calculated using the formula = 100 x ΔT / T initial (if ΔT < 0). Animals with a tumor volume of 0 mm 3 at the time of three consecutive measurements (non-measurable tumor) were considered complete responders, while animals with 50% tumor regression were partial responders.

[0413] 2.8 Data collection and statistical analysis

[0414] 2.8.1 Data collection : The measurements and observations required by the protocol were manually recorded in an appropriate form.

[0415] 2.8.2 Statistical analysis : All statistical tests were performed and the significance level was set at 5% or P < 0.05. Group means and standard deviations of all measured parameters were calculated according to the study design. One-way analysis of variance (ANOVA) was cited between groups.

[0416] HC-7366 demonstrated potent single-agent activity in solid tumors, including regression and complete remission. In HT1080 fibrosarcoma model-bearing mice, HC-7366 showed up to 80% tumor growth inhibition at 1 and 3 mg / kg. In LoVo colorectal model-bearing mice, HC-7366 showed 94% tumor growth inhibition at 1 and 3 mg / kg. In the DLD-1 colorectal tumor model, HC-7366 showed ~78% tumor growth inhibition at 1 mg / kg and 3 mg / kg. In FaDu head and neck squamous model-bearing mice, HC-7366 showed 33% tumor regression at 1 mg / kg and significant TGI at all doses. In the LNCaP androgen receptor-positive prostate tumor model, HC-7366 > showed ~61 - 65% tumor growth inhibition at 3 mg / kg. In the TM00298 androgen receptor-positive patient-derived prostate tumor model, HC-7366 showed ~70% tumor growth inhibition at both 3 mg / kg and 30 mg / kg. All treatments were well tolerated by body weight.

[0417] Figures 6-15 and 135 depict the solid tumor growth inhibition of HC-7366 in the above TGI studies.

[0418] HC-7366 exhibits potent single-agent activity in AML models. HC-7366 showed complete eradication of MOLM-16 xenograft tumors at 2 mg / kg, while other doses showed tumor growth inhibition. HC-7366 showed tumor stasis in KG-1 at 1 mg / kg and 3 mg / kg. HC-7366 showed 73% and 77% TGI in Kasumi-1 at 3 mg / kg and 30 mg / kg, respectively. HC-7366 showed approximately 47% and 51% TGI in OCI-AML2 at 10 mg / kg and 30 mg / kg, respectively. HC-7366 showed 45% TGI in MV4-11 at 30 mg / kg.

[0419] Figures 16-20 Depicted is the AML tumor growth inhibition of HC-7366 in the above TGI studies. The FAB classification of each AML is shown. HC-7366 showed a strong response in the M0-M2 subtypes of AML.

[0420] Example 7: Tumor Pharmacodynamic Analysis

[0421] Immunohistochemistry

[0422] The tumors collected at the end of the study described in Example 6 were subjected to immunohistochemical analysis. Formalin-fixed paraffin-embedded samples were cut into 5-μm thick sections and mounted on Superfrost Plus microscope slides (Fisher Scientific). All immunohistochemical (IHC) staining was performed on a Bond Rx automated stainer (Leica Biosystems) using a conventional TSA amplification detection system. The primary antibodies used were as follows: α-ASNS (Cell Signaling Technology #92479), α-PUMA (Cell Signaling Technology #98672), α-PSAT1 (Proteintech #10501-1-AP), α-PHGDH (Proteintech #14719-1-AP), HIF-1α - Novus Biologicals NB100-479, HIF-2α - Novus Biologicals NB100-122SS, and GLUT-1 - EMB Biosciences 07-1401. TSA-conjugated Alexa488, Alexa568, and Alexa647 fluorophores from Invitrogen (#B40953, #B40956, and #B40958) were used at a 1:500 dilution. The slides were counterstained with DAPI and covered with Mowiol anti-fade mounting medium (Sigma #D2522).

[0423] Image analysis of immunohistochemistry

[0424] The sections were imaged using a 10x / 0.32NA objective on an Aperio Versa 200 (Leica Biosystems) whole-slide scanner, and the data were analyzed using a custom macro in ImageJ / FIJI (NIH). Briefly, the outer margins of the tumor sections and necrotic areas (determined by the absence of 4′,6-diamidino-2-phenylindole (DAPI) signal or the presence of condensed DNA) were excluded from the analysis. The area of the tumor tissue was measured using a Gaussian blur of the DAPI channel. The immunostaining scans were processed to remove the background, and then the total intensity values above an appropriate threshold level were measured and normalized to the tissue area. All plotting and statistics (one-way ANOVA and pairwise comparisons) were performed using Prism graphPad.

[0425] Immunohistochemical staining and analysis of S100A8 and S100A9

[0426] The glass slides were dewaxed, rehydrated, and heat-mediated antigen retrieval was performed using AR6 buffer (Akoya). The slides were blocked with Roche diagnostic antibody diluent (Fisher) and stained with the primary antibody at 110 rpm for 1 hr. After washing, a horseradish peroxidase (HRP)-conjugated secondary antibody was added at 110 rpm for 10 min. After washing, OPAL detection dye (Akoya) was added for static incubation for 10 min. Antigen retrieval was performed after OPAL staining, followed by DAPI staining and slide coverslipping. Multispectral images were captured on a Polaris imaging system (Akoya), and spectral separation, cell segmentation, and phenotypic analysis were performed using Inform Tissue Finder software (Akoya). The imaging data were converted to.csv files and imported into Flowjo to analyze the percentage of S100A8 / A9-positive cells. The primary antibody used was rabbit anti-human / mouse S100A8 / A9 (clone RM1038, Abcam ab288715, paired with Opal 570).

[0427] At the end of the study (day 27), the ISR markers of HC-7366-treated KG-1 tumors were evaluated by IHC using the method described above. HC-7366 potently induced ISR in KG-1 tumors. HC-7366 potently induced ASNS ( Figure 21 and 22 ), PSAT1 ( Figure 23 and 24 ), and PHGDH ( Figure 25 and 26 ). The highest induction was observed at 1 and 3 mg / kg doses of HC-7366. At 1 and 3 mg / kg doses, HC-7366 potently reduced the protein levels of S100A8 / A9 in KG-1 tumors ( Figure 27 and 28 ). In this model, HC-7366 did not alter the PUMA level.

[0428] At the end of the study, HIF1α and HIF2α of HC-7366-treated FaDu tumors were evaluated by IHC using the method described above. HC-7366 significantly reduced the protein levels of HIF1α and HIF2α in FaDu tumors. The expression patterns of HIF1α and HIF2α were mutually exclusive ( Figures 40-42 ).

[0429] At the end of the study (day 20), ASNS, PSAT1, PUMA, HIF1α, and HIF2α of DLD-1 tumors treated with HC-7366 were evaluated by IHC using the above method. HC-7366 potently induced ASNS, PSAT1, and PUMA in a bell-shaped manner. HC-7366 significantly reduced the protein levels of HIF1α and HIF2α in FaDu tumors ( Figures 29-39 ). HIF2α was expressed at very low levels in the tumor model. HC-7366 potently activated GCN2 / ISR in the tumors.

[0430] Example 8: Combination of HC-7366 with Venetoclax in MV4-11 cells

[0431] MV4-11 tumors were collected at the end of the Venetoclax combination study described in Example 6. Tumor growth curves of MV4-11 tumors treated with single agents HC-7366 and Venetoclax or in combination were provided ( Figure 43 ). HC-7366 and Venetoclax showed a strong combination benefit, resulting in tumor regression. HC-7366 had 34% TGI as a single agent. Venetoclax monotherapy showed 62% TGI. However, when combined with Venetoclax, HC-7366 showed 26% tumor regression at a GCN2 activation dose of 3 mg / kg.

[0432] Tumors were analyzed by immunohistochemistry according to the procedure described in Example 7. Analysis of immunohistochemical ISR markers of tumors at the end of the study showed that 3 mg / kg of HC-7366 caused potent activation of ISR markers (such as ASNS ( Figure 44 and 45 ), PHGDH ( Figure 46 and 47 ), and PUMA ( Figure 48 and 49 )) which was further increased when combined with Venetoclax, indicating overactivation of ISR. Treatment with single agents of HC-7366 and Venetoclax both significantly reduced the S100A8 / A9 level, while the combination of Venetoclax with HC-7366 further potently reduced the S100A8 / A9 level ( Figure 50 and 51 ). The PSAT1 level did not change.

[0433] Example 9: Combination of HC-7366 with anti-VEGFR antibody in 786-O, A498, and DLD-1 tumors

[0434] 786-O and A498 tumors were collected at the end of the DC101 combination study described in Example 6. Tumors were analyzed by IHC according to the procedure described in Example 7.

[0435] HIF2α and GLUT1 of 786-O tumors treated with HC-7366 were evaluated by IHC. Treatment with HC-7366 and DC101 alone and in combination significantly reduced HIF2α. Treatment with HC-7366 alone or in combination with DC101 significantly reduced GLUT1 levels ( Figures 52-54 ). The tumor model did not express HIF1α.

[0436] HIF2α and GLUT1 of A498 tumors treated with HC-7366 were evaluated by IHC. HIF2α increased significantly after treatment with DC101 alone. No significant change in GLUT1 levels was observed in any group ( Figures 55-57 ). The tumor model did not express HIF1α.

[0437] The effect of the combination of DC101 and HC-7366 was evaluated in DLD-1 tumor-bearing mice using a protocol similar to that described in Example 6. Mice were treated twice daily by oral gavage with 3 or 30 mg / kg HC-7366. DC101 was administered at 20 mg / kg twice a week and by intraperitoneal injection twice daily. HC-7366 at 3 and 30 mg / kg inhibited tumor growth in the DLD-1 colorectal model, with TGI% values of ~65% and ~21%, respectively, while DC101 resulted in a TGI% value of ~55%. A significant combination benefit (~76% tumor growth inhibition) was observed when 3 mg / kg HC-7366 was combined with DC101, but no combination benefit was observed at 30 mg / kg ( Figure 61 ).

[0438] Example 10: Combination of HC-7366 and bevzutifan (PT-2977)

[0439] Female 6-8-week-old BALB / c nude mice were subcutaneously inoculated in the right flank with 786-O tumor cells (5x10 6 ) in 0.1 ml of serum-free RPMI 1640 medium for tumor growth (see Example 6). Treatment was initiated when the average tumor size reached approximately 200 mm 3 three weeks after inoculation and continued until the end of the study with HC-7366 administered twice daily by oral gavage at the listed doses. PT-2977 was administered by oral gavage at 0.1 mg / kg twice daily until the end of the study.

[0440] Tumor growth curves of 786-O tumors treated with HC-7366 and bevzutifan alone or in combination were provided ( Figure 58)。HC-7366 and PT-2977, as single agents, showed comparable anti-tumor efficacy in this model, with TGI% values of ~78% and ~70%, respectively. A significant combined anti-tumor benefit was observed when HC-7366 was combined with PT2977, resulting in an average tumor regression of ~50%.

[0441] Female 7-8-week-old athymic nude-Foxn1nu mice were subcutaneously inoculated with MFE280 tumor cells (10 x 10 6 ) in 0.2 ml PBS in the right flank subcutis for tumor growth (see Example 6). The single agent HC-7366 was administered at 0.5 or 2 mg / kg (twice daily, orally), and PT2977 was administered at 1 mg / kg (twice daily, orally). HC-7366 at 0.5 or 2 mg / kg was combined with PT2977. Treatment was initiated when the average tumor size reached approximately 191 mm 3 on day 28 after inoculation and continued until the end of the study.

[0442] Tumor growth curves of MFE280 tumors treated with the single agent HC-7366 and bemcentinib or in combination are provided ( Figure 135 ). The single agent HC-7366 showed 45% and 53% tumor growth inhibition at 0.5 mg / kg and 2 mg / kg, respectively. PT2977 showed 53% tumor growth inhibition at 1 mg / kg. HC-7366 0.5 mg / kg + PT2977 1 mg / kg showed 97% tumor growth inhibition. HC-7366 2 mg / kg + PT2977 1 mg / kg showed 17% tumor regression. Treatment tolerance was determined to be good based on body weight measurements.

[0443] Example 11: Combination of HC-7366 with 5-fluorouracil

[0444] The effect of the combination of 5-fluorouracil with HC-7366 in DLD-1 tumor-bearing mice was evaluated using a protocol similar to that described in Example 6. Mice were treated twice daily by oral gavage with 3 or 30 mg / kg HC-7366. 5-fluorouracil was administered intravenously at 75 mg / kg once a week, twice a day. HC-7366 at 3 and 30 mg / kg inhibited tumor growth in the DLD-1 colorectal model, with TGI% values of ~65% and ~21%, respectively, while 5-fluorouracil resulted in a TGI% value of ~68%. A significant combined benefit (~88% tumor growth inhibition; tumor stasis) was observed when 3 mg / kg HC-7366 was combined with DC101, but no combined benefit was observed with 30 mg / kg ( Figure 62 ).

[0445] Example 12: Combination of HC-7366 with PI3Kα or MEK1 / 2 inhibitor

[0446] In the combination study of alpelisib and trametinib described in Example 6, the growth of HCT-116 tumors was monitored.

[0447] In the HCT116 colorectal cancer model, the 3 mg / kg dose of HC-7366 showed a trend of efficacy (~38% TGI), while the 30 mg / kg HC-7366 did not reduce tumor growth. When alpelisib was co-administered with either dose of HC-7366, significant combinatorial benefits were observed ( Figure 63 ).

[0448] HC-7366 did not show efficacy in HCT116 at either 3 or 30 mg / kg, but had significant combinatorial benefits when trametinib was co-administered with the 3 mg / kg dose of HC-7366 ( Figure 64 ).

[0449] All treatments were well tolerated as measured by body weight.

[0450] Example 13: Combination of HC-7366 with EGFR inhibitor

[0451] In the combination study of osimertinib and dacomitinib described in Example 6, the growth of NCI-H1975 tumors was monitored.

[0452] The 3 mg / kg of HC-7366 showed significant anti-tumor efficacy with a TGI of 59%, while 2.5 mg / kg of osimertinib resulted in a TGI value of 95% with a 1 / 10 partial response. HC-7366 + osimertinib showed combinatorial benefits, resulting in an average tumor regression rate of ~90% with 2 / 10 complete responses and 8 / 10 partial responses ( Figure 131 ).

[0453] 15 mg / kg of dacomitinib alone led to tumor stasis followed by tumor growth with a TGI% value of 36% at the end of the study, which was not statistically significant. HC-7366 + dacomitinib showed combinatorial benefits with a TGI% value of ~99% and a 1 / 10 partial response ( Figure 133 ). Animals in the group containing dacomitinib showed significant weight loss. Therefore, animals in these groups were given a drug holiday. No treatment-related weight loss was observed in the HC-7366 and vehicle groups.

[0454] Example 14: GCN2-dependence of the effect mediated by HC-7366

[0455] CTG assay

[0456] MOLM-16 cells were obtained from DSMZ (#ACC 555) and cultured at 37 °C and 5% CO2 in RPMI 1640 (Gibco, #11875119) with 20% FBS (Invitrogen, #10099141C). MOLM-16 GCN2 CRISPR knockout cells (sgGCN2) were generated and compared with control cells (sgControl). For viability assays, cells were plated in 384-well cell culture plates and incubated overnight. Compounds were diluted to 8 concentrations at 3-fold dilutions in 384pp plates using a TECAN EVO200, with final concentrations ranging from 1 - 10 μM. The plates were incubated for 24 or 48 hr, and viability was assessed by adding CellTiter- 2.0 assay to each well according to the manufacturer's protocol (Promega, G9243). The plates were then incubated at RT for 30 min. Luminescence signals were read by an Envision (PerkinElmer) plate reader and analyzed by Prism.

[0457] FaDu cells were obtained from ATCC (#HTB-43) and cultured in Eagle's Minimum Essential Medium (catalog number 30-2003) at 37 °C and 5% CO2. FaDu GCN2 CRISPR knockout cells (sgGCN2) or control cells (sgControl) were generated. For viability assays, cells were plated in 384-well cell culture plates and incubated overnight. Compounds were diluted to 10 concentrations at 3-fold dilutions in 384pp plates using a TECAN EVO200, with final concentrations ranging from 1 - 10 μM. The plates were incubated for 72 or 96 hr, and viability was assessed by adding CellTiter- 2.0 assay to each well according to the manufacturer's protocol (Promega, G9243). The plates were then incubated at room temperature for 30 min. Luminescence signals were read by an Envision (PerkinElmer) plate reader and analyzed by Prism

[0458] HC-7366 decreased viability in vitro and induced the ISR in MOLM-16 cells in a GCN2-dependent manner. HC-7366 potently decreased viability in a U-shaped manner at lower concentrations ( Figure 65 ). This response was reversed in GCN2 CRISPR knockout cells ( Figure 66 ).

[0459] HC-7366 decreased viability in vitro and induced the ISR in FaDu cells in a GCN2-dependent manner. HC-7366 potently decreased viability in FaDu control cells ( Figure 71)。This reaction was reversed in GCN2 CRISPR knockout cells ( Figure 72 )。

[0460] Jess protein analysis

[0461] Cell lysates were obtained from MOLM-16 sgControl or sgGCN2 cells treated with HC-7366 (0, 0.01, 0.1, and 10 μM) for 24 hr. Ice-cold 1x radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with protease and phosphatase inhibitors was added to the cell pellet on ice. After 30 min on ice, the cell lysates were centrifuged at 4°C and 12,000 rpm for 15 min, and the supernatants were collected. Protein concentration was determined using a BCA kit. The cell lysates were mixed with 5X sodium dodecyl sulfate (SDS) loading buffer and denatured at 95°C for 5 min. 1 μg of the lysate was run on a Jess capillary, and the results were analyzed by compass simple western software.

[0462] Cell lysates were obtained from FaDu sgControl or sgGCN2 cells treated with HC-7366 (0, 0.01, 0.1, and 1 μM) for 72 hr. Ice-cold 1x RIPA lysis buffer supplemented with protease and phosphatase inhibitors was added to the cell pellet on ice. After 30 min on ice, the cell lysates were centrifuged at 4°C and 12,000 rpm for 15 min, and the supernatants were collected. Protein concentration was determined using a BCA kit. The cell lysates were mixed with 5X SDS loading buffer, denatured at 95°C for 5 min. 1 μg of the lysate was run on a Jess capillary, and the results were analyzed by compass simple western software. The following primary antibodies were used: p-GCN2 T899 (Abcam#ab75836), GCN2 (Cell Signaling#3302), ATF4 (Cell Signaling#11815), and ASNS (Proteintech#14681). The anti-rabbit secondary antibody was from ProteinSimple#042-206.

[0463] As described above, protein expression of GCN2 in wild-type MOLM-16 cells (WT, sgControl) and CRISPR knockout (sgGCN2) cells was measured by JESS ( Figure 67 )。By observing ATF4 ( Figure 68 ) and its downstream target ASNS ( Figure 69 ) and PSAT1 ( Figure 70) levels to evaluate HC-7366-mediated ISR activation, which showed a GCN2-dependent increase.

[0464] As described above, protein expression of GCN2 in wild-type (WT) FaDu cells (WT, sgControl) and CRISPR knockout (sgGCN2) cells was measured by JESS ( Figure 73 and 74 ). HC-7366-mediated ISR activation was evaluated by observing the levels of ATF4 ( Figure 75 ) and its downstream target ASNS ( Figure 76 ), which showed a GCN2-dependent increase at 0.01 and 0.1 μM concentrations of HC-7366.

[0465] Polysome analysis

[0466] Polysome profiling was performed in HEK 293 GCN2 WT or HEK293 GCN2 knockout cells as previously described in Johannes and Sarnow et al., RNA, 1998. Cells were treated with HC-7366 for 16 h and then harvested for polysome profiling.

[0467] The effect of HC-7366 on protein synthesis was evaluated by polysome profiling of GCN2 WT and CRISPR knockout HEK 293 cells. HC-7366 decreased the polysome-to-monosome ratio, which reflected reduced translation in GCN2 WT cells but not in GCN2 knockout cells ( Figure 77 and 78 ). HC-7366 potently decreased puromycin labeling of newly synthesized proteins at 100 nM, which activated the GCN2 / ISR pathway as measured by increased levels of p-eIF2α and ATF4 ( Figure 79 and 80 ).

[0468] HC-7366 reduces viability in vitro and induces the ISR in a GCN2-dependent manner.

[0469] Example 15: Ex vivo patient-derived xenograft (PDX) study with HC-7366

[0470] HC-7366 (4.57 - 10,000 nM) was evaluated in 30 patient-derived Champions human AML models in ex vivo 2D cell cultures. After treating the PDX models with DMSO or HC-7366 for 6 days, cell viability was measured using a plate-based luminescence assay (CellTiter Glo). HC-7366 potently reduced the viability of primary AML patient-derived xenograft (PDX) cells.

[0471] Eleven PDX models were sensitive responders, with an EC 50 of < 100 nM. Twelve PDX models were intermediate responders, with an EC 50 of 100 - 350 nM. Five PDX models were resistant responders, with an EC 50 of 924 - 2253 nM. Only two models showed no response to HC-7366. EC 50 values were calculated to the bottom of the curve and are shown below. Viability curves for models CTG-2229 ( Figure 81 ), CTG-3680 ( Figure 82 ), CTG-3667 ( Figure 83 ), CTG-2456 ( Figure 84 ), CTG-2457 ( Figure 85 ) and CTG-2454 ( Figure 86 ) are provided.

[0472]

[0473]

[0474] Example 16: AML PDX In Vivo Study

[0475] Sub-lethally irradiated NCG mice were inoculated with 2x10 6 human AML cells of the CTG-2229 type. Mice were monitored after AML inoculation to assess human AML engraftment using huCD45 / muCD45 / huCD33 / huCD3 antibodies and BD TruCountTM beads. When there were ≥ 20% viable huCD45 cells in the bone marrow of individual animals, they were randomized to treatment groups. For the efficacy study, animals were administered (n = 13 / group) vehicle control, HC-7366 at 1 mg / kg, 10 mg / kg or 30 mg / kg PO / BID x 28 or venetoclax at 100 mg / kg PO / QD x 28. Terminal blood, spleen and bone marrow samples were collected for flow cytometry ( Figure 87 ). Tolerability was assessed by weight loss, lethality and clinical signs of treatment-related adverse side effects. Body weight was measured twice weekly.

[0476] 1 mg / kg of HC-7366 significantly reduced myeloid-restricted precursors (CD34 + CD33 + ) and mature myeloid cells (CD34 - CD33 + ) in all tissues. Compared with venetoclax, the effect of HC-7366 was most obvious in the bone marrow.

[0477] Example 17: Effects of HC-7366 on oxygen consumption rate (OCR) and metabolomics

[0478] MOLM-16 cells were treated with 0.001, 0.1, or 10 μM of HC-7366 for 16 hr and subjected to Seahorse XF glycolysis stress test (ECAR; extracellular acidification rate) and Seahorse XF Mito stress test (OCR; oxygen consumption rate) (Agilent technologies, CA, USA).

[0479] HC-7366 significantly decreased OCR( Figure 88 ) and glycolysis( Figure 89 ), and the strongest effect was observed at the activation concentration of 0.1 μM. Collectively, these results indicate that HC-7366 reduces the mitochondrial oxygen consumption rate and glycolysis, leading to decreased ATP production.

[0480] Example 18: Mediated effect of HC-7366 on metabolomics in tumor xenograft models

[0481] MOLM-16 xenograft tumors were treated with vehicle or HC-7366 (0.3, 1, 3, 10, or 30 mg / kg) for four days, and the snap-frozen tumors were collected for metabolomics analysis (n = 12). Metabolic analysis was performed on the Metabolon LC / MS / MS and Polar LC platforms. The data were normalized to the vehicle group, and Welch's two-sample t-test was performed for statistical analysis. The heatmap shows the metabolite levels normalized to the vehicle control( Figure 90 、 94 、102 and 107).

[0482] HC-7366 significantly altered multiple metabolites in MOLM-16 tumors.

[0483] HC-7366 significantly increased the levels of most amino acids. The levels of certain amino acids were decreased with HC-7366; this included aspartic acid and cysteine( Figures 90-93)。HC-7366 significantly reduced glutathione levels (GSH and GSSG) and enhanced a large number of γ-glutamyl amino acids, indicating that tumors treated with HC7366 attempt to increase glutathione production, which may be a response to increased cellular oxidative stress( Figures 94-101 )。HC-7366 significantly reduced metabolites involved in pyrimidine synthesis( Figures 102-106 )。HC-7366 significantly reduced aspartate metabolism and polyamine-related metabolites (putrescine, spermine, 5-methylthioadenosine (MTA)) at all doses( Figures 107-114 )。Overall, the greatest changes in metabolites occurred at 1 and 3 mg / kg doses.

[0484] FaDu tumor-bearing mice were treated with vehicle or HC-7366 (1, 3, or 30 mg / kg) orally twice a day for three days, and an AM dose was administered on the morning of day 4, and tumors and plasma were collected 1 hr after dosing. Tumor and plasma samples were extracted and divided into equal parts for analysis on LC / MS / MS and Polar LC platforms. Ions were matched to an internal standard library using proprietary software for metabolite identification and metabolite quantification by peak area integration (Metabolon). Proteomics analysis of tumors was performed at MSBioworks. Equivalents of 3 μg of peptides from each pooled fraction were analyzed by nano LC-MS / MS using a Waters M-Class HPLC system coupled to a ThermoFisher Fusion Lumos mass spectrometer. Histograms show metabolite levels in treatment groups normalized to vehicle controls( Figures 115-122 )。

[0485] HC-7366 significantly increased the levels of several amino acids in tumors and plasma. HC-7366 also significantly increased the levels of several γ-glutamyl amino acids in tumors and plasma. HC-7366 significantly increased the levels of arginine, arginosuccinate, and citrulline in tumors and significantly increased the level of urea in plasma. HC-7366 significantly reduced pyrimidine synthesis metabolites in tumors. HC-7366 significantly reduced the level of oxidized glutathione in tumors but did not alter the level of reduced glutathione.

[0486] Example 19: Effects of HC-7366 on Cellular Metabolomics

[0487] Wild-type and GCN2 CRISPR knockout FaDu cells were treated with 0.1 μM for 9 hours and metabolomics was evaluated.

[0488] Treatment of wild-type FaDu cells with 0.1 μM HC-7366 for 9 h led to an increase in the levels of many amino acids in a GCN2-dependent manner. The basal levels of Ala, Asp, Glu, Gly, Pro, Thr, and Asn were reduced in GCN2 CRISPR knockout cells, indicating that GCN2 regulates the synthesis of several amino acids after HC-7366 treatment. This result confirmed the in vivo FaDu data (see Example 18), showing an increase in free amino acids in tumors and plasma, and highlighting the role of GCN2 in mediating this effect during HC-7366 treatment. Treatment of GCN2 wild-type cells and GCN2 CRISPR knockout cells with 0.1 μM HC-7366, which induces ATF4 in a GCN2-dependent manner ( Figures 123-125 ).

[0489] Example 20: Effect of HC-7366 on oxidative phosphorylation in the FaDu xenograft model

[0490] FaDu tumor-bearing mice were treated with HC-7366 at 3 and 30 mg / kg, twice daily for four days. Tumors were harvested for untargeted proteomics analysis.

[0491] Pathway analysis (IPA) of differentially expressed proteins in FADU tumors predicted that treatment with 3 mg / kg HC-7366 would strongly inhibit the oxidative phosphorylation pathway, but 30 mg / kg HC-7366 would not ( Figure 126 ).

[0492] Several protein subunits of the electron transport chain were reduced in the 3 mg / kg treatment group (relative to vehicle). Fewer oxidative phosphorylation proteins were altered after treatment with 30 mg / kg (relative to vehicle), and some oxidative phosphorylation proteins increased ( Figure 127 ). The data indicate that the activating dose of 3 mg / kg HC-7366 inhibits mitochondrial respiration in tumors, representing a possible mechanism of action unique to this dose.

[0493] Example 21: RNAseq analysis of the DLD-1 tumor xenograft model treated with HC-7366

[0494] DLD-1 tumor-bearing mice were treated with 0.3, 1, 3, 10, and 30 mg / kg of HC-7366, twice daily for 14 days. Tumors were harvested on days 4, 7, and 14 for RNAseq analysis ( Figure 128 ).

[0495] Analysis of upstream regulators of differentially expressed genes showed that ATF4 was significantly induced at all doses of HC-7366. Certain transcription factors were associated with efficacy. These included ARID1A, SMAD4, and E2F1Figure 129 )。Analysis showed reduced HIF1α, SMAD4, and E2F1 signatures, along with enhanced ATF4 and ARID1A signatures.

[0496] IHC staining of Ki67-positive cells in DLD-1 tumor sections showed that HC-7366 significantly reduced Ki67-positive cancer cells at the most effective doses of 1 and 3 mg / kg on day 4, but not at the lower effective doses of 0.3, 10, or 30 mg / kg( Figure 130 )。

[0497] Example 22: Multicenter, Open-Label, Phase 1a / b Study of a Compound of Formula (I) in Subjects with Advanced Solid Tumors

[0498] A first-in-human, multi-center, open-label, phase 1a / b dose-escalation and dose-expansion study was conducted to determine the maximum tolerated dose (MTD), recommended phase II dose (RP2D), and to evaluate the safety and tolerability of once-daily (QD) oral administration of HC-7366 potassium salt monohydrate in a dose-escalating manner in subjects with advanced solid tumors. Up to 36 subjects were enrolled in the phase 1a dose-escalation part of the study. Approximately 50% of all subjects enrolled in this study had squamous cell carcinoma of the head and neck (SCCHN), colorectal cancer (CRC), non-small cell lung cancer (NSCLC), and transitional cell carcinoma of the bladder (TCC). Subjects with other solid tumor types were eligible, met the specified study selection criteria, and they did not exceed 50% of all enrolled subjects. The study was conducted at approximately 3 to 5 sites in the United States. This phase 1a / b study followed the traditional 3+3 design. The starting dose level was 10 mg QD, and when safety permitted, escalated to 20, 40, 75, 125, and 150 mg QD. All doses were administered in the fasting state, taken with water at least 1 hour before eating or at least 2 hours after eating. The phase 1b dose-expansion part involved cohort expansion at up to 2 dose levels selected from the dose-escalation data of the Safety Monitoring Committee (SMC) to obtain additional safety and preliminary efficacy information. Each cohort in phase 1b enrolled 15 subjects. The study could be expanded to a phase 2 study by protocol amendment that evaluated the doses and tumor types selected in 1a / b as most suitable for further clinical development. Subjects were dosed until unacceptable toxicity occurred, disease progression according to immune-related response evaluation criteria in solid tumors (iRECIST), treatment was discontinued for other protocol-permitted reasons (e.g., subject refusal), any other administrative reason, or 2 years after treatment (whichever occurred first). For scheduling purposes, dosing occurred in 3-week cycles, and computed tomography (CT) scans were performed every 6 weeks, with the first post-baseline scan performed 6 weeks after dosing (before cycle 3). Subjects spent the first cycle / day 1 (C1D1) as outpatients, and subsequently stayed overnight for safety monitoring and pharmacokinetic (PK) sampling. Subjects were hospitalized for the administration of the first 2 doses: C1D1 and cycle 1 / day 2 (C1D2) (permitted according to local coronavirus disease 19 restrictions). After the initial hospitalization stay at the start of the study, subjects visited the outpatient department on days 8, 15, and 21 of cycle 1, and thereafter, on the first day of each cycle for physical and laboratory evaluations, adverse event (AE) and dose compliance monitoring, and PK C3-C6; also in person at the end of the treatment visit. The overnight stay from cycle 1 / day 21 (C1D21) to C2D1 was optional.Subjects who discontinue prior to the first post-baseline CT scan for reasons other than disease progression, treatment-related AEs or dose-limiting toxicity (DLT) before completion of the DLT assessment period will be replaced to ensure adequate safety assessment at each dose level.

[0499] (1) Dose escalation protocol

[0500] There are six dose escalation levels:

[0501]

[0502] Dose escalation follows the traditional 3+3 design. Each cohort is initially enrolled with a minimum of 3 subjects and expanded to 6 subjects per cohort as needed to determine DLT. For each cohort, the first subject is the sentinel subject. The sentinel subject is dosed and followed for 4 days to assess safety and tolerance. If considered safe and well tolerated, the remainder of the cohort (N = 2) is enrolled. If none of the first 3 subjects in the cohort experience a DLT related to the study treatment during the first 21 days (DLT assessment period), the next cohort is enrolled. If 1 of 3 subjects experiences a DLT related to the study treatment, the same cohort is expanded to 6 subjects. If 1 of 6 subjects experiences a DLT related to the study treatment, the next cohort is enrolled. If at any time during the cohort period, ≥2 subjects experience a DLT related to the study treatment, the MTD is exceeded, further enrollment within the cohort is stopped, and dose escalation is stopped. The MTD is defined as the dose level at which ≥33% (i.e., ≥2 out of 6 subjects) of the subjects in the cohort are observed to have a DLT related to the study treatment at lower dose levels. If there are 2 DLTs at dose level 1, dose level -1 is enrolled. Before applying the dose escalation rules, 3 subjects at a given dose level must have received at least 75% of the planned dose and have had a toxicity assessment, unless one or more subjects experience a DLT within the first 21 days. If none of the first 3 DLT-evaluable subjects within the cohort experience a DLT during the DLT assessment period, the next cohort is enrolled. If a subject does not receive at least 75% of the planned dose for any reason other than DLT (i.e., lost to follow-up), that subject is replaced.

[0503] The dose escalation decision is made based on all observations of all subjects in the corresponding cohort. DLT-evaluable subjects include those who experience disease-related AEs and serious AEs but are not treated. Even if pre-defined criteria are not met, the investigator and sponsor may jointly decide, based on all available data, to add additional subjects (not exceeding 6 evaluable subjects per cohort) at a given dose level or not to escalate the dose.

[0504] The dose escalation guidelines for consecutive cohorts are summarized as follows:

[0505]

[0506] (2) Dose-limiting toxicity

[0507] Dose-limiting toxicity (DLT) is defined as the occurrence of one of the following events during the DLT assessment period (i.e., until the end of Cycle 1, 21 days after the first administration of HC-7366):

[0508] Non-hematological:

[0509] · According to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) v5.0,

[0510] Any non-hematological toxicity of grade 3 or higher, except for any of the following:

[0511] o Nausea or vomiting of grade >3 that is managed with best medical practice and resolves to grade 1 or 2 within 72 hours or less.

[0512] o Fatigue of grade 3 <5 days.

[0513] o Isolated grade 3 laboratory abnormalities that are not associated with clinical signs or symptoms and are reversed within 3 days with appropriate maximal medical intervention.

[0514] ● Cardiotoxicity of grade ≥2.

[0515] ● Pituitary toxicity (hypopituitarism) of grade ≥2.

[0516] ● Any symptomatic congestive heart failure, a left ventricular ejection fraction of 50% or lower based on a multi-gated acquisition scan or echocardiogram, or a corrected QT interval (QTc)

[0517] prolonged to more than 500 milliseconds.

[0518] ● Any treatment-related non-hematological laboratory abnormality of grade 3 or higher, except for:

[0519] o Amylase or lipase of grade 3 or higher that is not associated with symptoms or clinical manifestations of pancreatitis.

[0520] o Electrolyte abnormalities of grade 3 or higher that persist for up to 72 hours, are clinically uncomplicated, and resolve spontaneously or respond to routine medical intervention.

[0521] ● Delayed start of Cycle 2 treatment by >2 weeks due to unresolved treatment-related non-hematological toxicity of grade ≥3.

[0522] Hematological:

[0523] ● Grade 4 thrombocytopenia lasting ≥ 7 days, or grade 3 or 4 thrombocytopenia associated with bleeding or the need for platelet transfusion.

[0524] ● Grade 4 neutropenia lasting ≥ 7 days.

[0525] · Neutropenic fever of any grade (> 38.5 °C).

[0526] ● Grade 4 anemia or grade 3 or 4 anemia requiring blood transfusion.

[0527] ● Note: Grade 3 or 4 lymphopenia is not considered a DLT but is recorded as part of the overall safety assessment.

[0528] Other:

[0529] ● More than 25% of the scheduled dose was missed due to any unresolved treatment-related toxicity.

[0530] An AE is considered related to the study drug unless it is clearly related to an underlying disease or a confirmed comorbidity.

[0531] Any subject who experiences a treatment-related DLT will immediately discontinue study treatment. No additional subject cohorts will be enrolled at subsequent dosing levels until all subjects at the initial (or previous) dosing level have completed all planned treatment for Cycle 1 (3-week QD oral dosing at the specified dose level) and are able to initiate Cycle 2 with no more than a 2-week delay.

[0532] (3) Recommended phase 2 dose

[0533] The RP2D and duration of administration of HC-7366 were determined in discussions with the SMC and the sponsor, taking into account the available data on safety, PK, and efficacy. The RP2D does not exceed the MTD. If the MTD is not reached at the end of the Phase 1a / b study, the RP2D will be determined by the SMC and the sponsor after reviewing the study data.

[0534] (4) Number of subjects

[0535] Up to 66 subjects (Phase 1a: N = up to 36 subjects; Phase 1b: N = 30 subjects).

[0536] (5) Diagnostic and inclusion criteria

[0537] Inclusion criteria: Each subject must:

[0538] 1. Sign the informed consent form before any specific study procedure or treatment.

[0539] 2. ≥ 18 years of age (male or female) at consent.

[0540] 3. Have 1 of the following tumor types and meet the criteria, and have received at least 1 and no more than 5 prior lines of therapy:

[0541] a. SCCHN

[0542] b. CRC

[0543] c. NSCLC

[0544] d. TCC

[0545] e. Other solid tumors (e.g., carcinoma of unknown primary), except for rapidly progressing tumors (e.g., pancreatic cancer, glioblastoma, hepatocellular carcinoma).

[0546] Subjects do not need to progress through all available therapies with known clinical benefit for their respective cancers to participate in this study.

[0547] Subjects with SCCHN, CRC, NSCLC, and TCC are prioritized and should comprise at least 50% of the enrolled population as a whole. Recruitment of all other subjects will be capped when they reach 50% in total, in order to reserve 18 slots for subjects with SCCHN, CRC, NSCLC, and TCC.

[0548] 4. According to the Response Evaluation Criteria in Solid Tumors (RECIST) v 1.1, have at least 1 radiologically measurable lesion defined as a lesion with a longest diameter of at least 10 mm by CT scan or magnetic resonance imaging or a lymph node with a short axis of at least 15 mm, and obtained by imaging within 28 days before study treatment. If such a lesion has been confirmed to have progressed, tumor lesions in previously irradiated areas are considered measurable.

[0549] 5. All prior treatment-related toxicities have resolved to grade 1 severity or less, except for stable sensory neuropathy (≤ grade 2) and alopecia. If the subject has undergone major surgery or radiotherapy > 30 Gy, they must have recovered from the toxicities and / or complications caused by the intervention.

[0550] 6. If the subject was previously treated with an immune checkpoint inhibitor, at least 4 weeks must have passed since the last dose, and the toxicities have been resolved as described above.

[0551] 7. Subjects must have at least one biopsy - proven lesion at baseline. Biopsies in this clinical study comply with the American Society of Clinical Oncology's ethical framework for research biopsies in oncology clinical trials. As long as there are suitable and accessible lesions, no biopsy contraindications, minimal risk of complications, and a positive informed decision, subjects are willing to provide fresh tissue for biomarker analysis and the biomarker status is evaluated based on the adequacy of the tissue sample quality. Two biopsies are required: at baseline (within 15 days before Day 1 of the study) and at the time of the first response assessment CT scan at Cycle 3 / Day 1 (+7 days). Newly obtained biopsy specimens are preferred over archived samples, and formalin - fixed, paraffin - embedded block specimens are preferred over slides.

[0552] 8. Eastern Cooperative Oncology Group performance status is 0 or 1 and persists between screening and the start of dosing on Day 1.

[0553] 9. There is no dysphagia that would interfere with oral dosing compliance.

[0554] 10. Have not experienced >10% weight loss in the previous 4 weeks.

[0555] 11. Have a serum albumin level >3 g / DL.

[0556] 12. As determined by the treating physician, the life expectancy is 3 months or longer.

[0557] 13. Have adequate organ function on Day 1, defined as meeting all of the following criteria:

[0558] a. Total bilirubin ≤1.5 × upper limit of normal (ULN), or for subjects with total bilirubin levels >1.5x ULN, direct bilirubin ≤ ULN.

[0559] b. For subjects with known liver metastases, aspartate aminotransferase and alanine aminotransferase ≤2.5 × ULN or ≤5 × ULN.

[0560] 14. Have adequate renal function on Day 1, defined as creatinine ≤1.5 × ULN and creatinine clearance ≥60 mL / min, according to the following Cockcroft Gault formula

[0561]

[0562] 15. Have adequate hematological function on Day 1, defined as meeting all of the following criteria:

[0563] a. Hemoglobin ≥9 g / dL (not corrected by red blood cell transfusions or erythropoietin support).

[0564] b. Absolute neutrophil count ≥ 1.5×10 9 / L.

[0565] c. Platelet count ≥ 100×10 9 / L.

[0566] 16. Have adequate coagulation function on Day 1, as defined by any of the following criteria:

[0567] a. International normalized ratio (INR) < 1.5×ULN or for subjects receiving warfarin or low molecular weight heparin, in the opinion of the investigator, the subject must be clinically stable and show no signs of active bleeding while receiving anticoagulant therapy. If this is the goal of the anticoagulant therapy, the INR of these subjects may exceed 1.5×ULN.

[0568] b. Activated partial thromboplastin time < 1.5×ULN, unless the subject is receiving anticoagulant therapy, provided that the prothrombin time or partial thromboplastin time is within the therapeutic range for the intended use of the anticoagulant.

[0569] 17. Have normal or adequately controlled total endocrine function (pituitary, adrenal, thyroid, pancreas, gonads). Subjects receiving hormone replacement must have their treatment doses stabilized.

[0570] 18. Female subjects of childbearing potential must have a negative urine or serum pregnancy test within 72 hours before receiving the first dose of the study drug. If the urine test is positive or cannot be confirmed as negative, a serum pregnancy test is required.

[0571] 19. Female subjects of childbearing potential must be willing to use appropriate contraception from the time of signing the informed consent form (ICF) until 90 days after the last dose of the study drug.

[0572] 20. Female subjects must agree not to breastfeed and not to donate oocytes from the time of screening, throughout the study treatment period, and for 90 days after the last administration of the study drug.

[0573] 21. Male subjects with a pregnant or breastfeeding partner must agree to abstain from sexual intercourse or use a condom for the duration of the pregnancy or for the duration that their partner is breastfeeding, throughout the study treatment period and for 90 days after the last administration of the study drug.

[0574] 22. Male subjects must not donate sperm during the treatment period and for at least 90 days after the last administration of the study drug.

[0575] 23. Male subjects with female partners of childbearing potential must agree to use a condom with a spermicide during the treatment period and for at least 90 days after the last administration of the study drug.

[0576] 24. Willing and able to comply with pre - arranged visits (including geographical proximity), treatment schedules, laboratory tests, and other study procedures.

[0577] Exclusion criteria: Subjects must not meet any of the following criteria:

[0578] 1. Received prior chemotherapy, targeted small - molecule therapy, or radiotherapy within 2 weeks before the first dose of study treatment, or have not recovered from the adverse reactions caused by previously administered agents or major surgery.

[0579] 2. Currently participating in and receiving study therapy, or have previously participated in a study of a study agent and received study therapy or used a study device within 4 weeks of the first dose of treatment.

[0580] 3. Diagnosed with immunodeficiency or received systemic corticosteroid therapy or any other form of immunosuppressive therapy within 7 days before the first dose of study treatment. Use of physiological doses of corticosteroids may be approved after consultation with the sponsor.

[0581] 4. Have a known history of active tuberculosis.

[0582] 5. Have a known history of human immunodeficiency virus (HIV) (HIV 1 / 2 antibodies).

[0583] 6. Have a known active hepatitis B (e.g., hepatitis B surface antigen reactive) or hepatitis C (e.g., hepatitis C virus ribonucleic acid [RNA] [qualitative]) infection.

[0584] 7. Diagnosed with severe acute respiratory syndrome coronavirus 2 infection confirmed by real - time polymerase chain reaction (PCR) testing according to local guidelines at screening and PCR positive within 7 days before the first dose of study treatment.

[0585] 8. Have a history of clinically severe autoimmune disease or a history of organ transplantation.

[0586] 9. Have a history of retinitis or photosensitive skin disorders, including (but not limited to) erythema multiforme, atopic eczema, psoriasis, viral rashes, pemphigus, and dermatitis herpetiformis.

[0587] 10. There is a known additional malignant tumor that is progressing or has required active treatment within the past 5 years. Exceptions include basal cell carcinoma or squamous cell carcinoma of the skin that has received potentially curative therapy, superficial bladder cancer, or carcinoma in situ of the cervix. Subjects with other malignant tumors are eligible if they have been cured by surgery alone or surgery plus radiotherapy and have been disease-free for at least 5 consecutive years.

[0588] 11. There is a known active central nervous system metastasis and / or carcinomatous meningitis. Subjects with previously treated brain metastases may participate provided they are stable (no signs of disease progression are detected by imaging within at least 4 weeks prior to the first dose of study treatment and any neurological symptoms have returned to baseline), there are no signs of new or enlarging brain metastases, and they have not used systemic steroids for at least 7 days prior to study treatment. This exception does not include carcinomatous meningitis, which is excluded regardless of clinical stability.

[0589] 12. There is a history of interstitial lung disease, pneumonia within 12 months prior to screening, or current pneumonia.

[0590] 13. Have an active infection that requires systemic therapy.

[0591] 14. Have a history or current signs of any condition, therapy, or laboratory abnormality that may confound the study results, interfere with the subject's participation throughout the study duration, or, in the opinion of the treating investigator, is not in the best interest of the subject's participation.

[0592] 15. Have clinically significant cardiovascular disease, such as unstable angina, myocardial infarction, or acute coronary syndrome, symptomatic or uncontrolled arrhythmia, congestive heart failure, baseline electrocardiogram (ECG) abnormalities, including but not limited to QTc prolongation to more than 470 ms, or any class III or IV heart disease as defined by the New York Heart Association functional classification.

[0593] 16. Have overt or potential pancreatic exocrine insufficiency (such as acute or chronic pancreatitis of any etiology) or chronic (including autoimmune) gastrointestinal disorders, such as Crohn's disease, ulcerative colitis, rheumatoid arthritis, lupus, scleroderma, Sjogren's syndrome, and polyarteritis nodosa.

[0594] 17. Have a known mental or substance abuse disorder that interferes with informed consent or compliance requirements of the study.

[0595] 18. Are pregnant, breastfeeding, or expect to become pregnant during the planned study duration, starting from the screening visit until 90 days after the last administration of the study drug.

[0596] 19. Are a first-degree relative of the investigator, study staff, or study sponsor.

[0597] (6) Investigational drug product, dose, and administration method

[0598] HC-7366 potassium salt monohydrate capsules.

[0599] Dose levels: 10, 20, 40, 75, 125, and 150 mg QD.

[0600] Route of administration: Oral with water in the fasting state.

[0601] (7) Duration of subject participation in the study

[0602] Each subject received treatment for up to 2 years and was followed up for up to 2 years.

[0603] Subjects continued dosing until unacceptable toxicity occurred, disease progression was recorded according to iRECIST, treatment was discontinued for reasons permitted by other protocols (e.g., subject request), any other administrative reasons, or after 2 years of treatment (whichever occurred first). Treatment of subjects beyond 2 years was at the discretion of the physician and the sponsor in consultation with the subject.

[0604] (8) Endpoints

[0605] Primary endpoints:

[0606] The primary endpoints of this study are as follows:

[0607] ● Determine the MTD and RP2D.

[0608] ● Safety and tolerability

[0609] o Incidence of DLT.

[0610] o Number and severity of treatment-emergent adverse events (TEAEs) and treatment-related TEAEs according to NCI-CTCAE v 5.0.

[0611] o Incidence of TEAEs leading to early discontinuation.

[0612] o Incidence of laboratory abnormalities by NCI-CTCAE grade based on hematology, serum chemistry, and urine analysis test results

[0613] o Incidence of abnormalities observed in 12-lead electrocardiogram parameters.

[0614] o Incidence of abnormalities observed in vital signs measurements.

[0615] o Incidence of abnormalities observed in vital signs measurements.

[0616] Secondary endpoints:

[0617] The secondary PK endpoints of this study include (as applicable) the following:

[0618] ● Area under the plasma concentration-time curve from time 0 until the last measurable concentration.

[0619] · Measurable concentration (AUC 0-最后 ).

[0620] · Area under the plasma concentration-time curve from time 0 to 24 hours after dosing (AUC

[0621] (AUC 0-24 ).

[0622] · Area under the plasma concentration-time curve extrapolated from time 0 to infinity (AUC 0-

[0623] ∞ ).

[0624] · Area under the plasma concentration-time curve during the dosing interval (AUC 0-t ).

[0625] · Observed maximum plasma concentration (C max ).

[0626] · Time to observed maximum plasma concentration (t max ).

[0627] · Apparent total clearance (CL / F).

[0628] · Apparent volume of distribution at the end of the terminal phase (V z / F).

[0629] · Apparent terminal elimination half-life (t 1 / 2 ).

[0630] · Accumulation rate based on AUC 0-t (AR AUC ).

[0631] · Linear ratio (LR).

[0632] The secondary efficacy endpoints of this study are as follows:

[0633] · Overall response rate (ORR).

[0634] · Duration of response (DOR).

[0635] · Time to treatment failure (TTF).

[0636] · Progression-free survival (PFS).

[0637] · Overall survival (OS).

[0638] Note: The overall response rate, DOR, and PFS were evaluated using the RECIST v1.1 and iRECIST criteria.

[0639] Exploratory endpoints:

[0640] · PD markers: Levels of ctDNA and CTC.

[0641] o Molecular analysis using whole exome sequencing or RNA sequencing will be explored.

[0642] · Immunophenotype in blood samples (serum and cellular markers, RNA, cytokines),

[0643] including markers of stress and immune activation.

[0644] · Local anti-tumor effects (minimal residual disease, apoptosis / necrosis) and changes in the microenvironment in tumor biopsies.

[0645] · CYP3A induction potential of HC-7366, the ratio of 4β-hydroxy cholesterol to total cholesterol on C1D1 and C1D21 as an endogenous marker of CYP3A induction.

[0646] Statistical methods:

[0647] Since this is an exploratory study to describe the MTD, RP2D, safety / tolerance, and preliminary PD, formal hypothesis testing was not performed. Descriptive statistics for the parameters of interest were presented by dose level. Safety data were presented in tabular form by system organ class and preferred term, severity, and event frequency for each cohort dose level.

[0648] Determination of sample size:

[0649] The maximum sample size for the Phase 1a study was 36 subjects. No more than 6 subjects were treated at each dose level. The sample size for Phase 1b was 30 subjects, with 15 subjects each in 2 selected dose cohorts for expansion.

[0650] Pharmacokinetics:

[0651] Non-compartmental PK analysis of individual plasma concentration data was performed using a validated software platform (e.g., Phoenix WinNonlin). Plasma concentrations and PK parameters of HC-7366 were listed and summarized using descriptive statistics. A power model could be used to graphically evaluate dose proportionality as needed.

[0652] Pharmacodynamics:

[0653] Pharmacokinetic biomarker data (e.g., integrated stress response activation markers and immune-related changes) were listed and summarized using descriptive statistics. The figure shows the relationship between PD, PK, and clinical efficacy data generated as needed.

[0654] General considerations:

[0655] Descriptive statistics and tabular / graphical representations were performed and presented by dose level (cohorts). The statistics include, but are not limited to, counts, percentages, ratios, means, medians, ranges, and variability, and any statistical analysis is exploratory.

[0656] Subject demographics and baseline characteristics, including age, sex, race, ethnicity, weight, baseline disease diagnosis, and medical conditions, were summarized by dose level using descriptive statistics.

[0657] Safety:

[0658] Safety parameters were listed and summarized using descriptive statistics. Safety variables include the incidence of TEAE, laboratory data, vital signs, 12-lead ECG results, and physical examination findings. All safety analyses were based on the safety population.

[0659] Efficacy:

[0660] The efficacy parameters were ORR, DOR, TTF, PFS, and OS. ORR was defined as the number of subjects with a response having a confirmed complete response (CR) or partial response (PR) divided by the total number of treated subjects with measurable disease at baseline. DOR was defined as the time from the first observation of PR or CR to radiographically documented progression. Tumor response status was defined according to RECIST v 1.1 and iRECIST. For the assessment of antitumor activity, the best overall response and ORR were tabulated by overall frequency distribution. The median DOR of subjects with a confirmed response was summarized using the Kaplan-Meier method; PFS was summarized similarly. A list of individual tumor measurements, tumor burden, and % change in tumor burden was provided. The change in tumor burden was presented graphically by a waterfall plot. The analysis of TTF, PFS, and OS was the same as that of DOR.

[0661] Interim:

[0662] When the planned number of subjects has completed their DLT observation period, the SMC will review the dose-limiting toxicity using the dose escalation rules. The SMC will review the available clinical, PK, and / or PD data as needed.

[0663] During screening and in Cycle 3, tumor biopsies collected from CRC and HNC patients were analyzed for HIF1α expression by IHC. HIF1α expression was significantly inhibited in patients who received the lowest starting doses of 10 mg and 20 mg observed in Cycle 3 ( Figure 59 and 60 ).

[0664] Multiplex immunohistochemistry (IHC) and patient biopsy imaging: Slides were deparaffinized, rehydrated, and heat-mediated antigen retrieval was performed using AR9 buffer (Akoya). The slides were blocked with Roche Diagnostic antibody diluent (Fisher) and stained with primary antibodies for 1 hour at 110 rpm. The HRP-conjugated secondary antibody was added at 110 rpm for 10 min, followed by staining with OPAL detection dyes (Akoya) for 10 min. For multiplex staining, this process was repeated starting from the antigen retrieval stage. After completion of the multiplex staining rounds, the slides were counterstained with DAPI and coverslipped using Vectashield mounting medium (Vector H-1700). Multispectral images were captured on a Polaris imaging system (Akoya) and spectral separation and cell segmentation were performed using Phenochart and Inform TissueFinder software (Akoya). The imaging data was converted to a.csv file and imported into Flowjo for immunophenotyping analysis. The primary antibodies used were rabbit anti-human HIF1α (polyclonal, Novus NB100-122, paired with Opal 690) and rabbit anti-human HIF2α (polyclonal, Novus NB100-479, paired with Opal 570). The secondary antibody used was HRP-goat anti-rabbit (Jackson Immunoresearch 111-035-144).

[0665] Incorporated by reference

[0666] This application references various published patents, published patent applications, journal articles, and / or other publications, all of which are incorporated herein by reference. If any incorporated reference conflicts with this specification, the specification shall control. Additionally, any specific embodiment of the present disclosure that is part of the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are considered to be known to those of ordinary skill in the art, they may be excluded even if not explicitly recited herein. Any specific embodiment of the present disclosure may be excluded from any claim for any reason, whether or not related to the existence of prior art.

[0667] Equivalents

[0668] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the foregoing embodiments are to be considered in all respects as illustrative and not restrictive of the invention described herein. Thus, the scope of the invention is indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

1. A method for treating advanced solid tumors in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

2. The method according to claim 1, wherein the advanced solid tumor is selected from squamous cell carcinoma of the head and neck, colorectal cancer, non-small cell lung cancer, renal cell carcinoma, and urothelial carcinoma of the bladder.

3. The method according to claim 1, wherein the advanced solid tumor is selected from sarcoma, colorectal cancer, head and neck cancer, and prostate cancer.

4. A method for treating blood cancer in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

5. The method according to claim 4, wherein the blood cancer is leukemia.

6. The method according to claim 4 or 5, wherein the blood cancer is acute myeloid leukemia.

7. The method according to claim 5 or 6, wherein the blood cancer is resistant to B-cell lymphoma inhibitors.

8. The method according to any one of claims 4-7, wherein the blood cancer is resistant to venetoclax.

9. The method according to any one of claims 1-8, wherein administering an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof activates the integrated stress response pathway (ISR) in the advanced solid tumor or blood cancer.

10. The method according to claim 9, wherein the ISR activation is GCN2-dependent.

11. The method according to any one of claims 1-10, wherein administering an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof induces the expression of ASNS, PSAT1, PHGDH, and / or PUMA in the advanced solid tumor or blood cancer.

12. The method according to any one of claims 1-11, wherein administering an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof reduces the protein levels of S100A8 / A9, HIF1α, HIF2α, and / or GLUT1 in the advanced solid tumor or blood cancer.

13. The method according to any one of claims 1-12, wherein administering an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof reduces mitochondrial respiration and / or glycolysis in the advanced solid tumor or blood cancer.

14. The method according to any one of claims 1-13, wherein administering an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof reduces myeloid-restricted precursors and mature myeloid cells in the subject.

15. The method according to any one of claims 1-14, wherein administering an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof alters metabolites involved in amino acid metabolism, oxidative stress, urea cycle, and / or pyrimidine biosynthesis in the advanced solid tumor or blood cancer.

16. The method according to any one of claims 1-15, wherein administering an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof reduces proteins involved in oxidative phosphorylation in the advanced solid tumor or blood cancer.

17. The method according to any one of claims 1-16, wherein administering an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof reduces the activity of HIF- and / or E2F1-driven transcription in the advanced solid tumor or blood cancer.

18. The method according to any one of claims 1-17, wherein administering an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof increases the ATF4 and / or JUN transcriptional activity in the advanced solid tumor or blood cancer.

19. The method according to any one of claims 1-18, wherein administering an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof comprises administering to the subject from about 10 mg to about 150 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof, on a free acid equivalent weight basis.

20. The method according to any one of claims 1-19, wherein administering an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof comprises orally administering to the subject from about 10 mg to about 150 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof, on a free acid equivalent weight basis.

21. The method according to any one of claims 1-20, wherein administering an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof comprises orally administering to the subject from about 10 mg to about 150 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof per day, on a free acid equivalent weight basis.

22. The method according to any one of claims 1-21, wherein administering an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof comprises orally administering to the subject from about 10 mg to about 150 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof once a day, on a free acid equivalent weight basis.

23. The method according to any one of claims 1-22, wherein administering an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof comprises orally administering to the subject from about 10 mg to about 150 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof once a day for 21 consecutive days, on a free acid equivalent weight basis.

24. The method according to any one of claims 1-23, wherein the subject is in a fasting state.

25. The method according to any one of claims 1-24, wherein administering an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof comprises administering to the subject from about 10 mg to about 150 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof about 1 hour before a meal or about 2 hours after a meal, on a free acid equivalent weight basis.

26. The method according to any one of claims 1-25, wherein the subject has previously received at least one and no more than 5 prior lines of therapy.

27. The method according to any one of claims 1-26, wherein the pharmaceutically acceptable salt is a potassium salt.

28. The method according to claim 27, wherein the potassium salt is a hydrate.

29. The method according to claim 27 or 28, wherein the potassium salt is a monohydrate.

30. The method according to any one of claims 1-29, further comprising administering to the subject an effective amount of a second therapeutic agent.

31. The method according to claim 30, wherein the second therapeutic agent is selected from immune checkpoint inhibitors, EGFR inhibitors, anti-angiogenic agents, venetoclax, fluorouracil, and combinations thereof.

32. The method according to claim 30, wherein the second therapeutic agent is selected from anti-VEGFR antibodies, fluorouracil, PI3Kα inhibitors, MEK1 / 2 inhibitors, and hypoxia-inducible factor (HIF) inhibitors.

33. The method according to claim 30, wherein the second therapeutic agent is venetoclax.

34. The method according to claim 33, wherein administering an effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof and venetoclax activates the integrated stress response pathway (ISR) in the advanced solid tumor or blood cancer to a greater extent than administering the compound of formula (I) or a pharmaceutically acceptable salt thereof or venetoclax alone.

35. The method according to claim 30, wherein the second therapeutic agent is an anti-VEGFR antibody.

36. The method according to claim 30, wherein the second therapeutic agent is an HIF inhibitor.

37. The method according to claim 30 or 36, wherein the second therapeutic agent is belzutifan.

38. The method according to claim 30, wherein the second therapeutic agent is 5-fluorouracil.

39. The method according to claim 30, wherein the second therapeutic agent is a PI3Kα inhibitor.

40. The method according to claim 30 or 39, wherein the second therapeutic agent is alpelisib.

41. The method according to claim 30, wherein the second therapeutic agent is a MEK1 / 2 inhibitor.

42. The method according to claim 30 or 41, wherein the second therapeutic agent is trametinib.

43. The method according to claim 30, wherein the second therapeutic agent is an EGFR inhibitor.

44. The method according to claim 30 or 43, wherein the second therapeutic agent is selected from osimertinib and dacomitinib.

45. The method according to any one of claims 1-44, wherein the subject is a human.

46. The method according to any one of claims 1-45, wherein the subject is an adult.

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