GCN2 modulators for the treatment of cancer

Abstract

For example, when the subject has advanced head and neck squamous cell carcinoma, colorectal cancer, non-small cell lung cancer, and urothelial carcinoma of the bladder, a method for treating an advanced solid tumor in a subject in need of treatment of the advanced solid tumor is provided herein. Also provided herein is a method for treating a blood cancer, such as acute myeloid leukemia, in a subject in need of treatment of the blood cancer.

Description

Technical Field

[0001] Cross - Reference to Related Applications

[0001] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 345,727, filed May 25, 2022; U.S. Provisional Patent Application No. 63 / 440,297, filed January 20, 2023; U.S. Provisional Patent Application No. 63 / 443,269, filed February 3, 2023; and U.S. Provisional Patent Application No. 63 / 455,861, filed March 30, 2023, each of which is hereby incorporated by reference in its entirety.

Background Art

[0002] Background

[0002] Cancer is the most common cause of death worldwide, accounting for nearly 10 million deaths in 2020 (World Health Organization). Targeted therapies and immunotherapies have expanded the scope of treatment for solid tumors by significantly improving the prognosis. However, tumor recurrence, drug resistance, and drug intolerance continue to be major challenges in the management of advanced cancer (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” Ecancermedicalscience, 2012, 6:ed16). Cancer cells are often exposed to various stresses in their microenvironment, such as hypoxia, low pH, and nutrient deprivation. To survive in the harsh 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 [RNA]-like endoplasmic reticulum kinase, double-stranded RNA-dependent protein kinase, general control nondepressible 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 unique stressors and phosphorylate the α-subunit of eukaryotic translation 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). The high-molecular-weight kinase GCN2 senses amino acid deficiency as part of the ISR. Uncharged transfer RNAs accumulate under amino acid starvation 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 global protein synthesis during cellular stress but also promotes the translation of selected mRNAs, including the activating transcription factor 4 (ATF4), which is the main effector of the ISR (Pakos-Zebrucka et al.). Once translated, ATF4 translocates to the nucleus and drives the expression of genes involved in stress adaptation, such as autophagy, the 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 tumor cell survival under nutrient depletion (Ye et al.). GCN2 / ATF4 expression is elevated in primary human liver, breast, lung, and head and neck tumors, and GCN2 activation has been observed in colon, breast, and lung tumor samples compared to normal tissues.

[0003]

[0003] ISR activation plays a dual role in cell fate determination. During acute stress conditions, ISR can promote adaptation, and during chronic stress conditions, this pathway can shift to apoptosis, which results in increased phosphorylation of eIF2α over a long period (Wortel et al., “Surviving Stress: Modulation of ATF4-Mediated Stress Responses in Normal and Malignant Cells,” Trends Endocrinol Metabol., 2017, 28(11):794-806). Long-term activation of ISR can be detrimental to cell growth by reducing protein synthesis or activating the apoptotic pathway (Wortel et al.; Harding et al., “Ppplr14 gene knockout reveals an essential role for translation initiation factor 2 alpha (eIF2alpha) dephosphorylation in mammalian development,” Proc Natl Acad Sci USA, 2009, 106(6);1832-1837; Muench, “The different axes of the mammalian mitochondrial unfolded protein response,” BMCBiology, 2018;16(1):81). Persistent ISR activation as a result of mutations in eIF2α phosphatases has been shown to have harmful effects on embryonic development due to inhibition of protein synthesis (Harding et al., “Ppplr14 gene knockout reveals an essential role for translation initiation factor 2 alpha (eIF2alpha) dephosphorylation in mammalian development,” Proc Natl Acad Sci USA, 2009, 106(6); 1832-1837).GCN2 activation may also have an anti-proliferative effect through suppression of general protein synthesis and induction of cell cycle arrest that impede cell growth during nutrient deprivation (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, continuous activation of the GCN2 pathway may suppress protein synthesis and cell growth, thereby inhibiting tumor growth.

[0004]

[0004] Accordingly, there remains an unmet need to develop a new therapeutic approach that utilizes either modulation, activation, or inhibition of the GCN2 pathway for the treatment of various cancers (e.g., advanced solid tumors and hematological cancers).

Summary of the Invention

Means for Solving the Problems

[0005] Summary

[0005] In one aspect, a method of treating an advanced solid tumor in a subject in need of treatment of an advanced solid tumor, the method comprising administering to the subject an effective amount of a compound of formula (I)

Chemical formula

[0006]

[0006] In certain embodiments, the advanced solid tumor is selected from the group consisting of head and neck squamous cell carcinoma, colorectal cancer, non-small cell lung cancer, renal cell carcinoma, and urothelial carcinoma of the bladder.

[0007]

[0007] In certain embodiments, the advanced solid tumor is selected from the group consisting of sarcoma, colorectal cancer, head and neck cancer, and prostate cancer.

[0008]

[0008] In another aspect, a method of treating a blood cancer in a subject in need of treatment for the blood cancer, the method comprising administering to the subject an effective amount of a compound of formula (I)

Chemical formula

[0009]

[0009] In certain embodiments, the blood cancer is leukemia. In certain embodiments, the blood cancer is acute myeloid leukemia.

[0010]

[0010] In some embodiments, the blood cancer is resistant to B cell lymphoma inhibitors. In certain embodiments, the blood cancer is resistant to venetoclax.

[0011]

[0011] 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 advanced solid tumors or blood cancers. In some embodiments, the ISR activation is GCN2-dependent.

[0012] In some embodiments, administering 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, administering 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, administering 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, administering an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, reduces progenitor cells and mature myeloid cells limited to the myeloid lineage in a subject. In some embodiments, administering an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, modifies 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 the activity of HIF and / or E2F transcription factor 1 (E2F1)-driven transcription 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, increases the transcriptional activity of ATF4 and / or (Jun oncogene AP-1 transcription factor subunit) JUN in advanced solid tumors or blood cancers.

[0013]

[0013] 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, daily. 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.

[0014]

[0014] 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.

[0015]

[0015] In certain embodiments, the subject has been previously administered at least 1 and no more than 5 prior treatment lines.

[0016]

[0016] 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.

[0017]

[0017] 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 the group consisting of immune checkpoint inhibitors, epidermal growth factor receptor (EGFR) inhibitors, anti-angiogenic agents, venetoclax, fluorouracil, and combinations thereof.

[0018]

[0018] In some embodiments, the second therapeutic agent is selected from the group consisting of anti-vascular endothelial growth factor receptor (VEGFR) antibodies, fluorouracil, phosphoinositide 3-kinase alpha (PI3Kα) inhibitors, mitogen-activated protein kinase kinase 1 / 2 (MEK1 / 2) inhibitors, and hypoxia-inducible factor (HIF) inhibitors.

[0019]

[0019] In some embodiments, the second therapeutic agent is venetoclax.

[0020]

[0020] 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 the compound of formula (I) administered alone, or a pharmaceutically acceptable salt thereof, or venetoclax.

[0021]

[0021] In some embodiments, the second therapeutic agent is an anti-VEGFR antibody.

[0022]

[0022] In some embodiments, the second therapeutic agent is a HIF inhibitor. In certain embodiments, the second therapeutic agent is belzutifan.

[0023]

[0023] In some embodiments, the second therapeutic agent is 5-fluorouracil.

[0024]

[0024] In some embodiments, the second therapeutic agent is a PI3Kα inhibitor. In some embodiments, the second therapeutic agent is alpelisib.

[0025]

[0025] In some embodiments, the second therapeutic agent is a MEK1 / 2 inhibitor. In some embodiments, the second therapeutic agent is trametinib.

[0026]

[0026] In some embodiments, the second therapeutic agent is an EGFR inhibitor. In some embodiments, the second therapeutic agent is selected from osimertinib and dacomitinib.

[0027]

[0027] In certain embodiments, the subject is human. In certain embodiments, the subject is an adult human.

Brief Description of the Drawings

[0028] Brief Description of the Drawings

Figure 1

[0028] Figure 1 is a graph showing the inhibitory effect of HC-7366 on double-stranded RNA-dependent protein kinase, general control non-derepressible 2 (GCN2) activity in a biochemical assay. The data are presented as a plot of HC-7366 concentration versus % vehicle, where % vehicle represents the DMSO control.

Figure 2

[0029] Figure 2 is a graph showing the effect of HC-7366 on activating transcription factor 4 (ATF4) activation in HT1080 cells using an activating transcription factor 4 (ATF4) activity assay.

Figure 3

[0030] Figure 3 is a graph showing the results of an ATF4 activity assay for HT1080 cells treated with HC-7366 and halofuginone, demonstrating the inhibitory effect of HC-7366 on GCN2 in the ATF4 assay of Figure 2.

Figure 4

[0031] Figure 4 is a graph showing the effect of HC-7366 on the viability of MOLM-16 cells using the CellTiter-Glo® (CTG) assay.

Figure 5

[0032] Figure 5 is a Western blot showing the expression of various proteins including GCN2 and ATF4 in HT1080 cells after treatment with HC-7366 with or without halofuginone at the various concentrations shown in the figure.

Figure 6

[0033] Figure 6 is a graph comparing the changes in mean 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 of HC-7366 twice daily, 1 mg / kg of HC-7366 twice daily, 3 mg / kg of HC-7366 twice daily, 0.6 mg / kg of HC-7366 once daily, 2 mg / kg of HC-7366 once daily, or 6 mg / kg of HC-7366 once daily.

Figure 7

[0034] Figure 7 is a graph comparing the changes in mean 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 of HC-7366 twice daily, 1 mg / kg of HC-7366 twice daily, or 3 mg / kg of HC-7366 twice daily.

Figure 8

[0035] Figure 8 is a graph comparing the changes in mean 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 of HC-7366 twice daily, 1 mg / kg of HC-7366 twice daily, 3 mg / kg of HC-7366 twice daily, 10 mg / kg of HC-7366 twice daily, or 30 mg / kg of HC-7366 twice daily.

Figure 9

[0036] Figure 9 is a graph comparing the changes 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 of HC-7366 twice daily, 1 mg / kg of HC-7366 twice daily, or 3 mg / kg of HC-7366 twice daily.

Figure 10

[0037] Figure 10 is a graph comparing the changes 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) in PBS (final pH 7.4)) twice daily, 0.3 mg / kg of HC-7366 twice daily, 1 mg / kg of HC-7366 twice daily, 3 mg / kg of HC-7366 twice daily, 10 mg / kg of HC-7366 twice daily, or 30 mg / kg of HC-7366 twice daily.

Figure 11

[0038] Figure 11 is a graph comparing the changes 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) in PBS (final pH 7.4)) twice daily, 0.3 mg / kg of HC-7366 twice daily, 1 mg / kg of HC-7366 twice daily, 3 mg / kg of HC-7366 twice daily, or 10 mg / kg of HC-7366 twice daily.

Figure 12

[0039] Figure 12 is a graph comparing the changes 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) in PBS (final pH 7.4)) twice daily, 0.3 mg / kg of HC-7366 twice daily, 1 mg / kg of HC-7366 twice daily, or 3 mg / kg of HC-7366 twice daily.

Figure 13

[0040] Figure 13 is a graph comparing the changes 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) in PBS (final pH 7.4)) twice daily, 0.3 mg / kg of HC-7366 twice daily, 1 mg / kg of HC-7366 twice daily, 3 mg / kg of HC-7366 twice daily, 10 mg / kg of HC-7366 twice daily, or 30 mg / kg of HC-7366 twice daily.

Figure 14

[0041] Figure 14 is a graph comparing the changes in average 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) in PBS (final pH 7.4)) twice daily, 0.3 mg / kg of HC-7366 twice daily, 1 mg / kg of HC-7366 twice daily, 3 mg / kg of HC-7366 twice daily, 10 mg / kg of HC-7366 twice daily, or 30 mg / kg of HC-7366 twice daily.

Figure 15

[0042] Figure 15 is a graph comparing the changes in average 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) in PBS (final pH 7.4)) twice daily, 3 mg / kg of HC-7366 twice daily, or 30 mg / kg of HC-7366 twice daily.

Figure 16

[0043] Figure 16 is a graph comparing the changes 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) in PBS (final pH 7.4)) twice daily, 0.25 mg / kg of HC-7366 twice daily, 0.5 mg / kg of HC-7366 twice daily, 1 mg / kg of HC-7366 twice daily, or 2 mg / kg of HC-7366 twice daily.

Figure 17

[0044] Figure 17 is a graph comparing the changes 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 of HC-7366 twice daily, 1 mg / kg of HC-7366 twice daily, 3 mg / kg of HC-7366 twice daily, 10 mg / kg of HC-7366 twice daily, or 30 mg / kg of HC-7366 twice daily.

Figure 18

[0045] Figure 18 is a graph comparing the changes 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 of HC-7366 twice daily, or 30 mg / kg of HC-7366 twice daily.

Figure 19

[0046] Figure 19 is a graph comparing the changes 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 of HC-7366 twice daily, 1 mg / kg of HC-7366 twice daily, 3 mg / kg of HC-7366 twice daily, 10 mg / kg of HC-7366 twice daily, or 30 mg / kg of HC-7366 twice daily.

Figure 20

[0047] Figure 20 is a graph comparing the changes 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 of HC-7366 twice daily, 1 mg / kg of HC-7366 twice daily, 3 mg / kg of HC-7366 twice daily, 10 mg / kg of HC-7366 twice daily, or 30 mg / kg of HC-7366 twice daily.

Figure 21

[0048] Figure 21 is an image of an immunohistochemistry (IHC) assay of ASNS expressed in KG-1 tumors recovered from the treatment groups described in Figure 17. The tumors were recovered on day 27 after the start of treatment (end of the study).

Figure 22

[0049] Figure 22 is a plot showing the total intensity per unit area of the immunohistochemistry assay described in Figure 21. ****p < 0.0001, one-way ANOVA.

Figure 23

[0050] Figure 23 is an image of an IHC assay of PSAT1 expressed in KG-1 tumors recovered from the treatment groups described in Figure 17. The tumors were recovered on day 27 after the start of treatment (end of the study).

Figure 24

[0051] Figure 24 is a plot showing the total intensity per unit area of the immunohistochemistry assay described in Figure 23. *p < 0.05, ****p < 0.0001, one-way ANOVA.

Figure 25

[0052] Figure 25 is an image of an IHC assay of PHGDH expressed in KG-1 tumors recovered from the treatment groups described in Figure 17. The tumors were recovered on day 27 after the start of treatment (end of the study).

Figure 26

[0053] Figure 26 is a plot showing the total intensity per unit area of the immunohistochemistry assay described in Figure 25. ****p < 0.0001, one-way ANOVA.

Figure 27

[0054] Figure 27 is an image of an IHC assay of S100A8 / A9 expressed in KG-1 tumors recovered from the treatment groups described in Figure 17. The tumors were recovered on day 27 after the start of treatment (end of the study).

Figure 28

[0055] Figure 28 is a plot showing the proportion of S100A8 / A9+ cells in the tumors described in Figure 27. *p < 0.05, **p < 0.01, one-way ANOVA.

Figure 29

[0056] Figure 29 is an image of an IHC assay of ASNS expressed in DLD-1 tumors recovered from the treatment groups described in Figure 10. The tumors were recovered on day 20 (end of the test) after the start of treatment.

Figure 30

[0057] Figure 30 is a plot showing the % positive per area of the IHC assay described in Figure 29. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA.

Figure 31

[0058] Figure 31 is an image of an IHC assay of PSAT1 expressed in DLD-1 tumors recovered from the treatment groups described in Figure 10. The tumors were recovered on day 20 (end of the test) after the start of treatment.

Figure 32

[0059] Figure 32 is a plot showing the % positive per area of the IHC assay described in Figure 31. ****p < 0.0001, one-way ANOVA.

Figure 33

[0060] Figure 33 is an image of an IHC assay of PUMA expressed in DLD-1 tumors recovered from the treatment groups described in Figure 10. The tumors were recovered on day 20 (end of the test) after the start of treatment.

Figure 34

[0061] Figure 34 is a plot showing the % positive per area of the IHC assay described in Figure 33. ****p < 0.0001, one-way ANOVA.

Figure 35

[0062] Figure 35 is an image of an IHC assay of HIf1α and HIf2α expressed in DLD-1 tumors recovered from the treatment groups described in Figure 10.

Figure 36

[0063] Figure 36 is a plot showing the expression of HIf1α (% positive cells) in the immunohistochemistry assay described in Figure 35. *p < 0.05, ***p < 0.001, ****p < 0.0001, one-way ANOVA.

Figure 37

[0064] Figure 37 is a plot showing the expression (% positive cells) of HIf2α in the immunohistochemistry assay described in Figure 35. ***p < 0.001, ****p < 0.0001, one-way ANOVA.

Figure 38

[0065] Figure 38 is an image of an IHC assay of HIF1α and HIF2α expressed 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 of HC-7366 twice daily.

Figure 39

[0066] Figure 39 is a plot showing the expression (% positive cells) of HIf1α in the immunohistochemistry assay described in Figure 38. **p < 0.01, one-way ANOVA.

Figure 40

[0067] Figure 40 is an image of an IHC assay of HIf1α and HIf2α expressed in FaDu tumors recovered from the treatment groups described in Figure 13 and the treatment group administered 30 mg / kg of HC-7366 twice daily.

Figure 41

[0068] Figure 41 is a plot showing the expression (% positive cells) of HIf1α in the immunohistochemistry assay described in Figure 40. **p < 0.01, one-way ANOVA.

Figure 42

[0069] Figure 42 is a plot showing the expression (% positive cells) of HIf2α in the immunohistochemistry assay described in Figure 40. ***p < 0.001, ****p < 0.0001 one-way ANOVA.

Figure 43

[0070] Figure 43 is a graph comparing the changes 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)), twice daily with 3 mg / kg of HC-7366, twice daily with 30 mg / kg of HC-7366, once daily with 50 mg / kg of venetoclax, twice daily with 3 mg / kg of HC-7366 + once daily with 50 mg / kg of venetoclax, or twice daily with 30 mg / kg of HC-7366 + once daily with 50 mg / kg of venetoclax.

Figure 44

[0071] Figure 44 is an image of an IHC assay of ASNS expressed in MV4-11 tumors recovered from the treatment groups described in Figure 43.

Figure 45

[0072] Figure 45 is a plot showing the expression of ASNS (% positive per area) in the immunohistochemistry assay described in Figure 44. ***p < 0.001, ****p < 0.0001, one-way ANOVA.

Figure 46

[0073] Figure 46 is an image of an IHC assay of PHGDH expressed in MV4-11 tumors recovered from the treatment groups described in Figure 43.

Figure 47

[0074] Figure 47 is a plot showing the expression of PHGDH (% positive per area) in the immunohistochemistry assay described in Figure 46. **p < 0.01, ****p < 0.0001, one-way ANOVA.

Figure 48

[0075] Figure 48 is an image of an IHC assay of PUMA expressed in MV4-11 tumors recovered from the treatment groups described in Figure 43.

Figure 49

[0076] Figure 49 is a plot showing the expression of PUMA (% positive per area) in the immunohistochemistry assay described in Figure 48. *p < 0.05, ****p < 0.0001, one-way ANOVA.

Figure 50

[0077] Figure 50 is an image of an IHC assay of S100A8 / A9 expressed in MV4-11 tumors recovered from the treatment groups described in Figure 43.

Figure 51

[0078] Figure 51 is a plot showing the percentage of S100A8 / A9+ cells in the tumors described in Figure 50. *p<0.05, **p<0.01, one-way ANOVA.

Figure 52

[0079] Figure 52 is an image of an IHC assay of HIf2α and GLUT1 expressed in 786-O tumors recovered 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 of HC-7366 twice daily, DC-101 15 mg / kg twice weekly, or 2 mg / kg of HC-7366 twice daily + DC-101 15 mg / kg twice weekly.

Figure 53

[0080] Figure 53 is a plot showing the expression of HIf2α (% positive cells) in the immunohistochemical assay described in Figure 52. *p<0.05, ****p<0.0001, one-way ANOVA.

Figure 54

[0081] Figure 54 is a plot showing the expression of GLUT1 (% positive per area) in the immunohistochemical assay described in Figure 52. ****p<0.0001, one-way ANOVA.

Figure 55

[0082] Figure 55 is an image of an IHC assay of HIf2α and GLUT1 expressed in A498 tumors recovered from A498 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 of HC-7366 twice daily, DC-101 15 mg / kg twice weekly, or 2 mg / kg of HC-7366 twice daily + DC-101 15 mg / kg twice weekly.

Figure 56

[0083] Figure 56 is a plot showing the expression (% positive cells) of HIf2α in the immunohistochemical assay described in Figure 55. ***p < 0.001, one-way ANOVA.

Figure 57

[0084] Figure 57 is a plot showing the expression (% positive per area) of GLUT1 in the immunohistochemical assay described in Figure 55.

Figure 58

[0085] Figure 58 is a graph comparing the changes in tumor volume over time in 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, 3 mg / kg of HC-7366 twice daily, 0.1 mg / kg of PT-2977 (belzutifan) twice daily, or 3 mg / kg of HC-7366 twice daily + 0.1 mg / kg of PT-2977 (belzutifan) twice daily.

Figure 59

[0086] Figure 59 is an image of the IHC assay for HIF1α in head and neck tumors (102-101) from human patients treated with 10 mg of HC-7366, colorectal tumors (102-201) from human patients treated with 10 mg of HC-7366, and colorectal tumors (101-201) from human patients treated with 20 mg of HC-7366.

Figure 60

[0087] Figure 60 is a plot showing the expression (% total cells) of HIf2α+ cells in the immunohistochemical assay described in Figure 59.

Figure 61

[0088] Figure 61 is a graph comparing the changes 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) in PBS (final pH 7.4)) twice daily, 3 mg / kg of HC-7366 twice daily, 30 mg / kg of HC-7366 twice daily, 20 mg / kg of DC-101 twice weekly, 20 mg / kg of DC-101 twice weekly + 3 mg / kg of HC-7366 twice daily, or 20 mg / kg of DC-101 twice weekly + 30 mg / kg of HC-7366 twice daily.

Figure 62

[0089] Figure 62 is a graph comparing the changes 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) in PBS (final pH 7.4)) twice daily, 3 mg / kg of HC-7366 twice daily, 30 mg / kg of HC-7366 twice daily, 75 mg / kg of 5-fluorouracil once weekly, 75 mg / kg of 5-fluorouracil once weekly + 3 mg / kg of HC-7366 twice daily, or 75 mg / kg of 5-fluorouracil once weekly + 30 mg / kg of HC-7366 twice daily.

Figure 63

[0090] Figure 63 is a graph comparing the changes in average 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) in PBS (final pH 7.4)) twice daily, 3 mg / kg of HC-7366 twice daily, 30 mg / kg of HC-7366 twice daily, 50 mg / kg of alpelisib, 50 mg / kg of alpelisib + 3 mg / kg of HC-7366 twice daily, or 50 mg / kg of alpelisib + 30 mg / kg of HC-7366 twice daily.

Figure 64

[0091] Figure 64 is a graph comparing the changes in the average tumor volume over time in HCT116 tumor-bearing mice administered with 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 of HC-7366 twice daily, 30 mg / kg of HC-7366 twice daily, trametinib 1 mg / kg, trametinib 1 mg / kg + 3 mg / kg of HC-7366 twice daily, or trametinib 1 mg / kg + 30 mg / kg of HC-7366 twice daily.

Figure 65

[0092] Figure 65 is a graph showing the effect of HC-7366 on the viability of MOLM-16 GCN2 wild-type cells measured by the CTG assay 24 and 48 hours after treatment with HC-7366 compared to cells treated with vehicle (DMSO).

Figure 66

[0093] Figure 66 is a graph showing the effect of HC-7366 on the viability of MOLM-16 GCN2 CRISPR knockout cells measured by the CTG assay 24 and 48 hours after treatment with HC-7366 compared to cells treated with vehicle (DMSO).

Figure 67

[0094] Figure 67 is a graph showing the expression of GCN2 in MOLM-16 GCN2 wild-type cells and MOLM-16 GCN2 CRISPR knockout cells described in Figures 65 and 66, respectively, after treatment with HC-7366.

Figure 68

[0095] Figure 68 is a graph showing the expression of ATF4 in MOLM-16 GCN2 wild-type cells and MOLM-16 GCN2 CRISPR knockout cells described in Figures 65 and 66, respectively, after treatment with HC-7366.

Figure 69

[0096] Figure 69 is a graph showing the expression of ASNS in MOLM-16 GCN2 wild-type cells and MOLM-16 GCN2 CRISPR knockout cells described in Figures 65 and 66, respectively, after treatment with HC-7366.

Figure 70

[0097] Figure 70 is a graph showing the expression of PSAT1 in MOLM-16 GCN2 wild-type cells and MOLM-16 GCN2 CRISPR knockout cells described in FIGS. 65 and 66 respectively after treatment with HC-7366.

Figure 71

[0098] Figure 71 is a graph showing the effect of HC-7366 on FaDu GCN2 wild-type cell viability measured by CTG assay 72 and 96 hours after treatment with HC-7366 compared to cells treated with vehicle (DMSO).

Figure 72

[0099] Figure 72 is a graph showing the effect of HC-7366 on FaDu GCN2 CRISPR knockout cell viability measured by CTG assay 72 and 96 hours after treatment with HC-7366 compared to cells treated with vehicle (DMSO).

Figure 73

[0100] Figure 73 is a graph showing the expression of pGCN2 in FaDu wild-type cells and FaDu GCN2 CRISPR knockout cells described in FIGS. 71 and 72 respectively after treatment with HC-7366.

Figure 74

[0101] Figure 74 is a graph showing the expression of GCN2 in FaDu wild-type cells and FaDu GCN2 CRISPR knockout cells described in FIGS. 71 and 72 respectively after treatment with HC-7366.

Figure 75

[0102] Figure 75 is a graph showing the expression of ATF4 in FaDu GCN2 wild-type cells and FaDu CRISPR knockout cells described in FIGS. 71 and 72 respectively after treatment with HC-7366.

Figure 76

[0103] Figure 76 is a graph showing the expression of ASNS in FaDu GCN2 wild-type cells and FaDu GCN2 CRISPR knockout cells described in FIGS. 71 and 72 respectively after treatment with HC-7366.

Figure 77

[0104] Figure 77 is a graph showing the absorbance of lysates from HEK293 GCN2 wild-type cells on a sucrose gradient treated with either vehicle (DMSO) or 100 nM HC-7366 for 16 hours. The ratio of polysomes to monosomes is also provided.

Figure 78

[0105] Figure 78 is a graph showing the absorbance of lysates from HEK293 GCN2 CRISPR knockout cells on a sucrose gradient treated with either vehicle (DMSO) or 100 nM HC-7366 for 16 hours. The ratio of polysomes to monosomes is also provided.

Figure 79

[0106] Figure 79 is a gel showing α-puromycin staining of newly synthesized proteins in 100 nM HC-7366.

Figure 80

[0107] Figure 80 is a Western blot showing ISR marker levels after treatment with HC-7366.

Figure 81

[0108] Figure 81 is a graph showing the effect of HC-7366 on CTG-2229 cell viability measured by the CTG assay after 6 days of treatment with HC-7366.

Figure 82

[0109] Figure 82 is a graph showing the effect of HC-7366 on CTG-3680 cell viability measured by the CTG assay after 6 days of treatment with HC-7366.

Figure 83

[0110] Figure 83 is a graph showing the effect of HC-7366 on CTG-3667 cell viability measured by the CTG assay after 6 days of treatment with HC-7366.

Figure 84

[0111] Figure 84 is a graph showing the effect of HC-7366 on CTG-2456 cell viability measured by the CTG assay after 6 days of treatment with HC-7366.

Figure 85

[0112] Figure 85 is a graph showing the effect of HC-7366 on CTG-2457 cell viability measured by CTG assay after 6 days of treatment with HC-7366.

Figure 86

[0113] Figure 86 is a graph showing the effect of HC-7366 on CTG-2454 cell viability measured by CTG assay after 6 days of treatment with HC-7366.

Figure 87

[0114] Figure 87 is a series of t-distribution type probabilistic neighborhood embedding (tSNE) plots showing the distribution of AML stem cells, myeloid-restricted progenitor cells, mature myeloid cells, non-myeloid cells, and other cell types in terminal samples recovered from the bone marrow (BM), whole blood (WB), and spleen (SP) of female NCG immunodeficient mice bearing a primary (P1) human acute myeloid leukemia (AML) xenograft TumorGraft model and treated with vehicle, 1 mg / kg of HC-7366 twice daily for 28 days, 10 mg / kg of HC-7366 twice daily for 28 days, 30 mg / kg of HC-7366 twice daily for 28 days, or 100 mg / kg of venetoclax once daily for 28 days.

Figure 88

[0115] Figure 88 is a graph showing the oxygen consumption rate (pmol / min) over time of MOLM-16 cells treated with vehicle (DMSO), 0.001 μM of HC-7366, 0.1 μM of HC-7366, or 10 μM of HC-7366.

Figure 89

[0116] Figure 89 is a graph showing the extracellular acidification rate (ECAR) (mpH / min) over time of MOLM-16 cells treated with vehicle (DMSO), 0.001 μM of HC-7366, 0.1 μM of HC-7366, or 10 μM of HC-7366.

Figure 90

[0117] Figure 90 is a heatmap showing the amino acid levels in MOLM-16 tumors treated with 0.3 mg / kg of HC-7366, 1 mg / kg of HC-7366, 3 mg / kg of HC-7366, 10 mg / kg of HC-7366, or 30 mg / kg of HC-7366 for 4 days, normalized to the vehicle control.

Figure 91

[0118] Figure 91 is a graph showing the scaled intensity of aspartic acid levels in the treatment groups described in Figure 90.

Figure 92

[0119] Figure 92 is a graph showing the scaled intensity of cysteine levels in the treatment groups described in Figure 90.

Figure 93

[0120] Figure 93 is a graph showing the scaled intensity of methionine levels in the treatment groups described in Figure 90.

Figure 94

[0121] Figure 94 is a heatmap showing the levels of oxidative stress markers in MOLM-16 tumors treated for 4 days with 0.3 mg / kg of HC-7366, 1 mg / kg of HC-7366, 3 mg / kg of HC-7366, 10 mg / kg of HC-7366, or 30 mg / kg of HC-7366, normalized to the vehicle control.

Figure 95

[0122] Figure 95 is a schematic diagram showing the molecular pathways involved in glutathione production.

Figure 96

[0123] Figure 96 is a graph showing the scaled intensity of S-adenosylmethionine (SAM) levels in the treatment groups described in Figure 94.

Figure 97

[0124] Figure 97 is a graph showing the scaled intensity of oxidized glutathione (GSSG) levels in the treatment groups described in Figure 94.

Figure 98

[0125] Figure 98 is a graph showing the scaled intensity of S-adenosylhomocysteine (SAH) levels in the treatment groups described in Figure 94.

Figure 99

[0126] Figure 99 is a graph showing the scaled intensity of ophthamic acid levels in the treatment groups described in Figure 94.

Figure 100

[0127] Figure 100 is a graph showing the scaled intensity of cysteine levels in the treatment groups described in Figure 94.

Figure 101

[0128] Figure 101 is a graph showing the scaled intensity of cystathionine levels in the treatment groups described in Figure 94.

Figure 102

[0129] Figure 102 is a heat map showing the levels of pyrimidine synthetic metabolites in MOLM-16 tumors treated for 4 days with 0.3 mg / kg of HC-7366, 1 mg / kg of HC-7366, 3 mg / kg of HC-7366, 10 mg / kg of HC-7366, or 30 mg / kg of HC-7366, normalized to vehicle control.

Figure 103

[0130] Figure 103 is a graph showing the scaled intensity of orotidine levels in the treatment groups described in Figure 102.

Figure 104

[0131] Figure 104 is a graph showing the scaled intensity of orotic acid levels in the treatment groups described in Figure 102.

Figure 105

[0132] Figure 105 is a graph showing the scaled intensity of dihydroorotic acid levels in the treatment groups described in Figure 102.

Figure 106

[0133] Figure 106 is a graph showing the scaled intensity of UMP levels in the treatment groups described in Figure 102.

Figure 107

[0134] Figure 107 is a heat map showing the levels of aspartic acid metabolic product markers in MOLM-16 tumors treated for 4 days with 0.3 mg / kg of HC-7366, 1 mg / kg of HC-7366, 3 mg / kg of HC-7366, 10 mg / kg of HC-7366, or 30 mg / kg of HC-7366, normalized to vehicle control.

Figure 108

[0135] Figure 108 is a schematic diagram showing the molecular pathways involved in aspartic acid metabolism.

Figure 109

[0136] Figure 109 is a graph showing the scaled intensity of ornithine levels in the treatment groups described in Figure 107.

Figure 110

[0137] Figure 110 is a graph showing the scaled intensity of citrulline levels in the treatment groups described in Figure 107.

Figure 111

[0138] Figure 111 is a graph showing the scaled intensity of asparagine levels in the treatment groups described in Figure 107.

Figure 112

[0139] Figure 112 is a graph showing the scaled intensity of 5-methylthioadenosine (MTA) levels in the treatment groups described in Figure 107.

Figure 113

[0140] Figure 113 is a graph showing the scaled intensity of arginine in the treatment groups described in Figure 107.

Figure 114

[0141] Figure 114 is a graph showing the scaled intensity of aspartic acid in the treatment groups described in Figure 107.

Figure 115

[0142] Figure 115 is a graph showing the changes in tumor amino acid levels in FaDu tumor-bearing mice treated for 4 days with 1 mg / kg of HC-7366 twice daily, 3 mg / kg of HC-7366 twice daily, or 30 mg / kg of HC-7366 twice daily, normalized to the vehicle control. Statistically significant changes are labeled with asterisks (p ≤ 0.05).

Figure 116

[0143] Figure 116 is a graph showing the changes in plasma amino acid levels in FaDu tumor-bearing mice treated for 4 days with 1 mg / kg of HC-7366 twice daily, 3 mg / kg of HC-7366 twice daily, or 30 mg / kg of HC-7366 twice daily, normalized to the vehicle control. Statistically significant changes are labeled with asterisks (p ≤ 0.05).

Figure 117

[0144] Figure 117 is a graph showing the changes in tumor gamma-glutamyl amino acid levels in FaDu tumor-bearing mice treated for 4 days with 1 mg / kg of HC-7366 twice daily, 3 mg / kg of HC-7366 twice daily, or 30 mg / kg of HC-7366 twice daily, normalized to vehicle control. Statistically significant changes are labeled with asterisks (p ≤ 0.05).

Figure 118

[0145] Figure 118 is a graph showing the changes in plasma gamma-glutamyl amino acid levels in FaDu tumor-bearing mice treated for 4 days with 1 mg / kg of HC-7366 twice daily, 3 mg / kg of HC-7366 twice daily, or 30 mg / kg of HC-7366 twice daily, normalized to vehicle control. Statistically significant changes are labeled with asterisks (p ≤ 0.05).

Figure 119

[0146] Figure 119 is a graph showing the changes in tumor urea cycle marker levels in FaDu tumor-bearing mice treated for 4 days with 1 mg / kg of HC-7366 twice daily, 3 mg / kg of HC-7366 twice daily, or 30 mg / kg of HC-7366 twice daily, normalized to vehicle control. Statistically significant changes are labeled with asterisks (p ≤ 0.05).

Figure 120

[0147] Figure 120 is a graph showing the changes in plasma urea cycle marker levels in FaDu tumor-bearing mice treated for 4 days with 1 mg / kg of HC-7366 twice daily, 3 mg / kg of HC-7366 twice daily, or 30 mg / kg of HC-7366 twice daily, normalized to vehicle control. Statistically significant changes are labeled with asterisks (p ≤ 0.05).

Figure 121

[0148] Figure 121 is a graph showing the changes in tumor pyrimidine synthesis marker levels in FaDu tumor-bearing mice treated for 4 days with 0.3 mg / kg of HC-7366 twice daily, 1 mg / kg of HC-7366 twice daily, or 3 mg / kg of HC-7366 twice daily, normalized to vehicle control. Statistically significant changes are labeled with asterisks (p ≤ 0.05).

Figure 122

[0149] Figure 122 is a graph showing changes in tumor oxidative stress marker levels in FaDu tumor-bearing mice treated with 1 mg / kg of HC-7366 twice daily, 3 mg / kg of HC-7366 twice daily, or 30 mg / kg of HC-7366 twice daily for 4 days, normalized to vehicle control. Statistically significant changes are labeled with asterisks (p ≤ 0.05).

Figure 123

[0150] Figure 123 is a heatmap showing intracellular amino acid levels in GCN2 wild-type and GCN2 CRISPR knockout FaDu cells treated with vehicle (DMSO) or 0.1 μM of HC-7366.

Figure 124

[0151] Figure 124 is a graph showing GCN2 expression in GCN2 wild-type and GCN2 CRISPR knockout FaDu cells treated with vehicle (DMSO) or 0.1 μM of HC-7366.

Figure 125

[0152] Figure 125 is a graph showing ATF4 expression in GCN2 wild-type and GCN2 CRISPR knockout FaDu cells treated with vehicle (DMSO) or 0.1 μM of HC-7366.

Figure 126

[0153] Figure 126 is a schematic diagram showing pathway analysis (IPA) of differentially expressed proteins in FaDu tumors, which predicted strong suppression of the oxidative phosphorylation pathway for 3 mg / kg of HC-7366 but not for 30 mg / kg of HC-7366.

Figure 127

[0154] Figure 127 is a heatmap showing fold changes in oxidative phosphorylation proteins in FaDu tumor-bearing mice treated with 3 mg / kg of HC-7366 or 30 mg / kg of HC-7366.

Figure 128

[0155] Figure 128 is a graph comparing the changes 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) in PBS (final pH 7.4)) twice daily, 0.3 mg / kg of HC-7366 twice daily, 1 mg / kg of HC-7366 twice daily, 3 mg / kg of HC-7366 twice daily, 10 mg / kg of HC-7366 twice daily, or 30 mg / kg of HC-7366 twice daily.

Figure 129

[0156] Figure 129 is a chart showing the changes in the expression of upstream regulators of differentially expressed genes in the treatment groups described in Figure 128.

Figure 130

[0157] Figure 130 is a plot showing the results of IHC staining of Ki67-positive cells in tumor sections of the treatment groups described in Figure 128. *p < 0.05, **p < 0.01, one-way ANOVA.

Figure 131

[0158] Figure 131 is a graph comparing the changes in average 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 of HC-7366 twice daily, osimertinib 2.5 mg / kg once daily, or 3 mg / kg of HC-7366 twice daily + osimertinib 2.5 mg / kg once daily.

Figure 132

[0159] Figure 132 is a graph comparing the changes 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 of HC-7366 twice daily, osimertinib 2.5 mg / kg once daily, or 3 mg / kg of HC-7366 twice daily + osimertinib 2.5 mg / kg once daily.

Figure 133

[0160] Figure 133 is a graph comparing the changes in average 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 of HC-7366 twice daily, 15 mg / kg of dacomitinib once daily, or 3 mg / kg of HC-7366 twice daily + 15 mg / kg of dacomitinib once daily.

Figure 134

[0161] Figure 134 is a graph comparing the changes 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 of HC-7366 twice daily, 15 mg / kg of dacomitinib once daily, or 3 mg / kg of HC-7366 twice daily + 15 mg / kg of dacomitinib once daily.

Figure 135

[0162] Figure 135 is a graph comparing the changes in tumor volume over time in MFE280 tumor-bearing mice administered vehicle twice daily, 0.5 mg / kg of HC-7366 twice daily, 2 mg / kg of HC-7366 twice daily, 1 mg / kg of PT-2977 (berzosertib) twice daily, 0.5 mg / kg of HC-7366 twice daily + 1 mg / kg of PT-2977 twice daily, or 2 mg / kg of HC-7366 twice daily + 1 mg / kg of PT-2977 twice daily.

Mode for Carrying Out the Invention

[0029] Detailed Description

[0163] 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 bladder transitional cell carcinoma) in a subject in need of treatment of an advanced solid tumor. The present disclosure also provides methods for treating blood cancers (e.g., acute myeloid leukemia (AML)) in a subject in need of treatment of a blood cancer. 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.

[0030] Definitions

[0164] For ease of understanding of the present invention, a number of terms and phrases are defined below.

[0031]

[0165] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Abbreviations used in this specification have their customary meanings within the fields of chemistry and biology. The chemical structures and formulas shown in this specification are constructed in accordance with the standard rules of chemical valence known in the chemical art.

[0032]

[0166] Throughout this detailed description, when a composition and kit are described as having, including, or comprising specific components, or a process and method are described as having, including, or comprising specific steps, it is additionally contemplated that there exist compositions and kits of the invention consisting essentially of, or consisting of, the recited components, and processes and methods according to the invention consisting essentially of, or consisting of, the recited process steps.

[0033]

[0167] In this application, when an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0034]

[0168] Furthermore, 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 described herein. For example, when a particular compound is referenced, that 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, within this application, embodiments are described and shown so as to enable a clear and accurate description and depiction of their application, but it is intended and recognized that the embodiments can be variously combined or separated without departing from the present teachings and the present invention. For example, it is recognized that all features described and shown herein can be applicable to all aspects of the present invention described and shown herein.

[0035]

[0169] The articles "a" and "an" are used in this disclosure to refer to one or more (i.e., at least one) of the grammatical objects of the article, provided the context is not inappropriate. By way of example, "an element" means one element or more than one element.

[0036]

[0170] The term "and / or" is used in this disclosure to mean either "and" or "or" unless otherwise indicated.

[0037]

[0171] The expression "at least one of" should be understood to include each of the objects recited before that expression individually, as well as various combinations of two or more of the recited objects, unless otherwise understood from the context and use. The expression "and / or" associated with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

[0038]

[0172] The use of the terms "include", "includes", "including", "have", "has", "having", "contain", "contains", or "containing", including their grammatical equivalents, is open-ended and non-limiting unless specifically recited or understood from the context, and is generally understood, for example, not to exclude additional unrecited elements or steps.

[0039]

[0173] When the term "about" is used before a quantitative value, the present invention also includes the specific quantitative value itself unless specifically stated otherwise. As used herein, the term "about" refers to a variation of ±10% from the nominal value unless otherwise indicated or inferred from the context.

[0040]

[0174] At various places in this specification, variables or parameters are disclosed in groups or ranges. It is specifically intended that this detailed description include each and every member of such groups and ranges and any and all individual subcombinations. For example, integers in the range of 0 to 40 are specifically intended to individually disclose 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 individually disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0041]

[0175] Any examples or exemplary language, such as "for example" or "including", used in this specification are intended merely to better illustrate the invention and do not limit the scope of the invention unless claimed. Words in this specification should not be construed as indicating any unclaimed element as essential to the practice of the invention.

[0042]

[0176] In general, compositions that define a ratio are on a weight basis unless otherwise specified. Further, when a variable is not accompanied by a definition, the prior definition of the variable applies.

[0043]

[0177] As used herein, "pharmaceutical composition" or "pharmaceutical formulation" refers to a combination of an active agent and an inert or active carrier that renders the composition particularly suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0044]

[0178] "Pharmaceutically acceptable" means approved by, or approvable by, the federal or state government regulatory authorities or the corresponding authorities in countries outside the United States, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias for use in animals, and more particularly for use in humans.

[0045]

[0179] As used herein, "pharmaceutically acceptable salt" refers to any salt of an acidic or basic group that may be present in a compound of the invention (e.g., a compound of formula (I)) where the salt is compatible with pharmaceutical administration.

[0046]

[0180] As is known to those skilled in the art, the "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, toluene-p-sulfonic 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 per se, can be used in the preparation of salts useful as intermediates in obtaining the compounds described herein and their pharmaceutically acceptable acid addition salts.

[0047]

[0181] Examples of bases include, but are not limited to, alkali metal (e.g., sodium and potassium) hydroxides, alkaline earth metal (e.g., magnesium and calcium) hydroxides, ammonia, and compounds of the formula NW 4+ (wherein W is C 1~4 alkyl) and the like.

[0048]

[0182] 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, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include suitable cations such as Na + , K + , Ca 2+ , NH4+ 、and NW 4+ (wherein, W is C 1~4 which may be an alkyl group), and anions of the compounds of the present invention formulated therewith, and the like.

[0049]

[0183] For therapeutic use, salts of the compounds of the present invention (for example, compounds of formula (I)) are intended to be pharmaceutically acceptable. However, salts of acids and bases that are not pharmaceutically acceptable may also be used, for example, in the preparation or purification of pharmaceutically acceptable compounds.

[0050]

[0184] As used herein, "pharmaceutically acceptable excipient" refers to a substance that aids in the administration of an active agent to a subject and / or absorption by the subject and can be included in the compositions of the present invention without causing significant adverse toxic effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, physiological saline, for example, phosphate buffered saline, emulsions (for example, oil-in-water or water-in-oil emulsions, etc.), lactated Ringer's solution, usually sucrose, usually glucose, binders, extenders, disintegrants, lubricants, coating agents, sweeteners, flavors, salt solutions (for example, Ringer's solution), alcohols, oils, gelatin, carbohydrates, fatty acid esters, and coloring agents, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliaries that do not react detrimentally with the compounds of the present invention, for example, lubricants, preservatives, stabilizers, wetting agents, emulsifying agents, salts for affecting osmotic pressure, buffers, coloring agents, and / or aromatic substances, and the like. For examples of excipients, see Martin, Remington’s Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA (1975).

[0051]

[0185] 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-infinity represents the area under the plasma concentration versus time curve from 0 hours to infinite time. AUC 0-tshows the area under the plasma concentration-time curve from 0 hour to time t. It should be recognized that the AUC value can be determined by methods known in the art.

[0052]

[0186] The "subject" for which administration is contemplated includes, but is not limited to, humans (i.e., males or females of any age group, such as pediatric subjects (e.g., infants, toddlers, minors) 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), cows, 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 human. In certain embodiments, the subject is a non-human animal.

[0053]

[0187] The term "C max " refers to the maximum concentration of the 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 comprising the therapeutic agent (e.g., a pharmaceutical composition described herein).

[0054]

[0188] The term "t max " refers to the time (in hours) at which C max is achieved after administration of the 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).

[0055]

[0189] As used herein, "solid dosage form" means a pharmaceutical dosage in solid form, such as tablets, capsules, granules, powders, sachets, reconstitutable powders, dry powder inhalers and chewables.

[0056]

[0190] As used herein, "administering" means oral administration to a subject, 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 mini-osmotic pump. Administration can be 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, arterial, 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-administering" means that the compositions described herein are administered simultaneously with, immediately before, or immediately 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, can be administered to a patient alone or co-administered. Co-administration means including simultaneous or sequential administration of the compound, either individually or in combination (two or more compounds or agents). Thus, the preparation can optionally also be combined with other active substances (e.g., to reduce metabolic degradation).

[0057]

[0191] As used herein, "fasting state" means that at least 1 hour before a meal or at least 2 hours after a meal has been consumed by the subject.

[0058]

[0192] The terms "disease", "disorder", and "condition" are used interchangeably herein.

[0059]

[0193] As used herein, unless otherwise specified, the terms "treating", "treatment", and "treat" contemplate an action (e.g., a "therapeutic treatment") that reduces the severity of, or delays or slows the progression of, a specified disease, disorder or condition while the subject is suffering from that disease, disorder or condition.

[0060]

[0194] 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 induce a desired biological response, e.g., to treat advanced solid tumors and / or blood cancers. As will be recognized by those skilled in the art, the effective amount of the compounds of the present disclosure can vary depending on the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and factors such as the age, weight, health status, and medical condition of the subject.

[0061] Compound

[0195] The compounds of formula (I) shown below are selective GCN2 modulators (e.g., activation or inhibition of GCN2) and are also known as 6-(3-((5-chloro-2-methoxypyridin)-3-sulfonamido)-2,6-difluorophenyl)-N-methylimidazo[1,5-a]pyrazine-1-carboxamide:

Chem.

[0062]

[0196] The compounds of formula (I) are also referred to as HC-7366 throughout the present disclosure. A method for chemically synthesizing the compounds of formula (I) is described in Example 1.

[0063]

[0197] In one aspect, provided herein is a method of administering a compound of formula (I), or a pharmaceutically acceptable salt thereof, for the treatment of 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 of treatment of advanced solid tumors.

[0064]

[0198] In another aspect, provided herein is a method of administering a compound of formula (I), or a pharmaceutically acceptable salt thereof, for the treatment of blood cancers (e.g., AML) in a subject in need of treatment of blood cancers.

[0065]

[0199] In various embodiments, provided herein is a method of administering a pharmaceutically acceptable salt of a compound of formula (I) for the treatment of 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 of treatment of an advanced solid tumor.

[0066]

[0200] In various embodiments, provided herein is a method of administering a pharmaceutically acceptable salt of a compound of formula (I) for the treatment of blood cancer (e.g., AML) in a subject in need of treatment of blood cancer.

[0067]

[0201] In one embodiment, the pharmaceutically acceptable salt of the compound of formula (I) is a potassium salt. In one embodiment, the potassium salt of the compound of formula (I) is a hydrate. In one embodiment, the potassium salt of the compound of formula (I) is a monohydrate. A method for preparing the potassium salt of the compound of formula (I) is described in Example 2.

[0068] Pharmaceutical Composition

[0202] Provided herein is a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0069]

[0203] In one aspect, provided herein is a method 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 the treatment of 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 of treatment of an advanced solid tumor.

[0070]

[0204] In another aspect, provided herein is a method 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 the treatment of blood cancer (e.g., AML) in a subject in need of treatment of blood cancer.

[0071]

[0205] In another aspect, provided herein is a method of administering a pharmaceutical composition comprising a pharmaceutically acceptable salt of a compound of formula (I) and one or more pharmaceutically acceptable excipients for the treatment of advanced solid tumors (e.g., head and neck squamous cell carcinoma, colorectal cancer, NSCLC, renal cell carcinoma, and bladder transitional cell carcinoma) in a subject in need of treatment of advanced solid tumors.

[0072]

[0206] In another aspect, provided herein is a method of administering a pharmaceutical composition comprising a pharmaceutically acceptable salt of a compound of formula (I) and one or more pharmaceutically acceptable excipients for the treatment of blood cancers (e.g., AML) in a subject in need of treatment of blood cancers.

[0073]

[0207] In various embodiments, provided herein are pharmaceutical compositions comprising an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0074]

[0208] In various embodiments, provided herein are pharmaceutical compositions comprising an effective amount of a pharmaceutically acceptable salt of a compound of formula (I) and one or more pharmaceutically acceptable excipients.

[0075]

[0209] 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 to about 150 mg, about 20 mg to about 150 mg, about 40 mg to about 150 mg, about 75 mg to about 150 mg, about 125 mg to about 150 mg, about 10 mg to about 125 mg, about 10 mg to about 75 mg, about 10 mg to about 40 mg, about 10 mg to about 20 mg, about 20 mg to about 125 mg, about 20 mg to about 75 mg, about 20 mg to about 40 mg, about 40 mg to about 125 mg, about 40 mg to about 75 mg, or 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 about 10 mg to about 150 mg on a free acid equivalent weight basis.

[0076]

[0210] 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 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 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 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 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 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 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 weight basis.

[0077]

[0211] 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 to about 150 mg, about 20 mg to about 150 mg, about 40 mg to about 150 mg, about 75 mg to about 150 mg, about 125 mg to about 150 mg, about 10 mg to about 125 mg, about 10 mg to about 75 mg, about 10 mg to about 40 mg, about 10 mg to about 20 mg, about 20 mg to about 125 mg, about 20 mg to about 75 mg, about 20 mg to about 40 mg, about 40 mg to about 125 mg, about 40 mg to about 75 mg, or about 75 mg to about 125 mg on a free acid 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 to about 150 mg on a free acid weight basis.

[0078]

[0212] 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.

[0079]

[0213] In various embodiments, (i) a compound of formula (I), or a pharmaceutically acceptable salt thereof, in an amount of about 10 mg to about 150 mg on a free acid equivalent weight basis; and (ii) one or more pharmaceutically acceptable excipients are provided herein in pharmaceutical compositions.

[0080]

[0214] In various embodiments, (i) a pharmaceutically acceptable salt of a compound of formula (I) in an amount of about 10 mg to about 150 mg on a free acid equivalent weight basis; and (ii) one or more pharmaceutically acceptable excipients A pharmaceutical composition comprising the same is provided herein.

[0081]

[0215] In another aspect, for the treatment of advanced solid tumors (e.g., head and neck squamous cell carcinoma, colorectal cancer, NSCLC, renal cell carcinoma, and bladder transitional cell carcinoma) in a subject in need of treatment of advanced solid tumors, a compound of formula (I) in an amount of about 10 mg to about 150 mg on a free acid equivalent weight basis, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, a pharmaceutical composition is provided herein.

[0082]

[0216] In another aspect, for the treatment of blood cancer (e.g., AML) in a subject in need of treatment of blood cancer, a compound of formula (I) in an amount of about 10 mg to about 150 mg on a free acid equivalent weight basis, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, a pharmaceutical composition is provided herein.

[0083]

[0217] In another aspect, for the treatment of blood cancer (e.g., AML) in a subject in need of treatment of blood cancer, a pharmaceutically acceptable salt of a compound of formula (I) in an amount of about 10 mg to about 150 mg on a free acid equivalent weight basis, and one or more pharmaceutically acceptable excipients, a pharmaceutical composition is provided herein.

[0084]

[0218] In another aspect, for the treatment of advanced solid tumors (e.g., head and neck squamous cell carcinoma, colorectal cancer, NSCLC, renal cell carcinoma, and bladder transitional cell carcinoma) in a subject in need of treatment of advanced solid tumors, a pharmaceutically acceptable salt of a compound of formula (I) in an amount of about 10 mg to about 150 mg on a free acid equivalent weight basis, and one or more pharmaceutically acceptable excipients, a pharmaceutical composition is provided herein.

[0085]

[0219] 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.

[0086]

[0220] 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.

[0087]

[0221] 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 long period of time, e.g., for 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or indefinitely, e.g., for the lifetime of the subject. In certain embodiments, chronic administration is intended to provide a constant level of the compound in the blood within the therapeutic concentration range over a long period of time.

[0088]

[0222] The pharmaceutical compositions described herein can be provided in unit dosage forms to facilitate accurate dosing. The term “unit dosage form” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with a suitable pharmaceutical excipient. Typical unit dosage forms include pre-filled, pre-measured ampoules or syringes of liquid compositions or, in the case of solid compositions, pills, tablets, capsules, etc.

[0089]

[0223] In certain embodiments, the pharmaceutical compositions provided herein are administered to a patient as a solid dosage form. In certain embodiments, the solid dosage form is a capsule.

[0090]

[0224] 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 comprises one or more of lactose monohydrate, microcrystalline cellulose, croscarmellose sodium, colloidal silicon dioxide, and magnesium stearate. In some embodiments, the capsule comprises each of lactose monohydrate, microcrystalline cellulose, croscarmellose sodium, colloidal silicon dioxide, and magnesium stearate. In some embodiments, the capsule comprises 10 mg, 25 mg, or 100 mg of HC-7366 potassium salt monohydrate on a free acid equivalent weight basis.

[0091]

[0225] The description of the pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, although it will be understood by those skilled in the art that such compositions are generally suitable for administration to all kinds of animals. Modifications to make pharmaceutical compositions suitable for administration to humans suitable for administration to various animals are well understood, and veterinary pharmacologists of ordinary skill in the art can design and / or implement such modifications using routine experimentation. General considerations in 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.

[0092] Methods of Use and Treatment

[0226] Methods for treating solid tumors in a subject in need of treatment of a solid tumor are provided herein. The methods generally comprise 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.

[0093]

[0227] Solid tumors, a compound of formula (I), or a pharmaceutically acceptable salt thereof are contemplated to be useful in treatment, including, but 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 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, advanced epithelial or primary peritoneal cancer; 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 or uterine cancer, hepatoma, hepatocellular carcinoma, liver cancer, gastric or stomach cancer, including gastrointestinal cancer, peritoneal cancer, squamous cell carcinoma of the lung, gastroesophageal cancer, cholangiocarcinoma, gallbladder cancer, colorectal / appendiceal cancer, and squamous cell carcinoma (e.g., squamous cell carcinoma of the epithelium).

[0094]

[0228] In certain embodiments, the advanced solid tumor is selected from the group consisting of squamous cell carcinoma of the head and neck, colorectal cancer, NSCLC, renal cell carcinoma, and transitional cell carcinoma of the bladder.

[0095]

[0229] 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.

[0096]

[0230] In certain embodiments, the advanced solid tumor is selected from the group consisting of sarcoma, colorectal cancer, head and neck cancer, and prostate cancer.

[0097]

[0231] Also provided herein is a method of treating a blood cancer in a subject in need of treatment for a blood cancer. The method generally comprises administering to the subject an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.

[0098]

[0232] In certain embodiments, the blood cancer is selected from the group consisting of multiple myeloma, leukemia, and lymphoma. In certain embodiments, the leukemia is selected from the group consisting of chronic lymphocytic leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, and AML.

[0099]

[0233] In certain embodiments, the blood cancer is leukemia. In certain embodiments, the blood cancer is AML.

[0100]

[0234] In some embodiments, the blood cancer is resistant to B cell lymphoma inhibitors. In certain embodiments, the blood cancer is resistant to venetoclax.

[0101]

[0235] 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 advanced solid tumors or blood cancers. In some embodiments, the ISR activation is GCN2-dependent.

[0102]

[0236] 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 synthetase 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 progenitor cells and mature myeloid cells limited to the myeloid lineage in a subject. In some embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof modifies 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 the activity of HIF and / or E2F1-driven transcription in advanced solid tumors or blood cancers. In some embodiments, HIF and / or E2F1-driven transcription includes the expression of late-to-middle transition genes. In some embodiments, administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof increases the ATF4 and / or JUN transcriptional activity in advanced solid tumors or blood cancers.

[0103]

[0237] 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 weight basis.

[0104]

[0238] 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 weight basis.

[0105]

[0239] 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 mg, 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 a compound of formula (I) or a pharmaceutically acceptable salt thereof, on a free acid equivalent weight basis.

[0106]

[0240] 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.

[0107]

[0241] 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 of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free acid 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 from about 20 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free acid 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 from about 40 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free acid 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 from about 75 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free acid 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 from about 125 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free acid 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 from about 150 mg of the compound of formula (I), or a pharmaceutically acceptable salt thereof, on a free acid basis.

[0108]

[0242] 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 weight basis.

[0109]

[0243] 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.

[0110]

[0244] 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.

[0111]

[0245] In certain embodiments, the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally once daily to a subject 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, the compound of formula (I), or a pharmaceutically acceptable salt thereof, is administered orally once daily to a subject for at least 21 consecutive days.

[0112]

[0246] In certain embodiments, 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.

[0113]

[0247] In certain embodiments, 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.

[0114]

[0248] In certain embodiments, 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, daily.

[0115]

[0249] In certain embodiments, administering an effective amount of the compound of formula (I), or a pharmaceutically acceptable salt thereof, comprises orally administering to the 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.

[0116]

[0250] 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, once daily for 21 consecutive days.

[0117]

[0251] 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 the effective amount to the subject about 1 hour before a meal or about 2 hours after a meal.

[0118]

[0252] 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.

[0119]

[0253] In certain embodiments, the subject has been previously administered at least one prior treatment line. In certain embodiments, the subject has been previously administered less than 5 prior treatment lines. In certain embodiments, the subject has been previously administered 1, 2, 3, or 4 prior treatment lines. In certain embodiments, the subject has not been administered a prior treatment line.

[0120]

[0254] In certain embodiments, the subject has been previously administered at least 1 and no more than 5 prior treatment lines.

[0121]

[0255] As pre-treatment lines, although not limited thereto, surgery, radiotherapy (e.g., external beam radiotherapy or brachytherapy), chemotherapy (e.g., alkylating agents, nitrosoureas, antimetabolites, plant alkaloids and natural products, antitumor antibiotics, hormonal agents, and biological response modifiers), gene therapy, DNA therapy, viral therapy (e.g., oncolytic viral therapy), RNA therapy, adjuvant 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) may be mentioned.

[0122]

[0256] In certain embodiments, the method comprises 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.

[0123]

[0257] In certain embodiments, the methods described herein further comprise administering to the subject an effective amount of a second therapeutic agent. In certain embodiments, the second therapeutic agent is selected from the group consisting of checkpoint inhibitors, EGFR inhibitors, anti-angiogenic agents, venetoclax, fluorouracil, and combinations thereof.

[0124]

[0258] In some embodiments, the second therapeutic agent is selected from the group consisting of anti-VEGFR antibodies, fluorouracil, PI3Kα inhibitors, MEK1 / 2 inhibitors, and hypoxia-inducible factor (HIF) inhibitors.

[0125]

[0259] In some embodiments, the second therapeutic agent is venetoclax.

[0126]

[0260] In some embodiments, the second therapeutic agent is fluorouracil. In some embodiments, the second therapeutic agent is 5-fluorouracil.

[0127]

[0261] 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 the compound of formula (I) administered alone, or a pharmaceutically acceptable salt thereof, or venetoclax alone.

[0128]

[0262] In some embodiments, the degree of ISR activation can be determined by measuring an ISR activation marker (e.g., ASNS, PSAT1, PHGDH, ASS1, and / or PUMA) described herein in a tumor or cancer using the experimental methods described herein.

[0129]

[0263] 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 the group consisting of nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, semipilimab, and dostarlimab.

[0130]

[0264] In certain embodiments, the second therapeutic agent is an EGFR inhibitor. In some embodiments, the EGFR inhibitor is selected from the group consisting of 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.

[0131]

[0265] 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 drug 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.

[0132]

[0266] In certain embodiments, the second therapeutic agent is a VEGFR inhibitor. In certain embodiments, the VEGFR inhibitor is selected from the group consisting of 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.

[0133]

[0267] In some embodiments, the second therapeutic agent is an HIF inhibitor. In certain embodiments, the HIF inhibitor is belzutifan.

[0134]

[0268] In some embodiments, the second therapeutic agent is a PI3Kα inhibitor. In some embodiments, the PI3Kα inhibitor is selected from the group consisting of copanlisib and alpelisib. In certain embodiments, the PI3Kα inhibitor is alpelisib. In some embodiments, the second therapeutic agent is alpelisib.

[0135]

[0269] In some embodiments, the second therapeutic agent is a MEK1 / 2 inhibitor. In some embodiments, the MEK1 / 2 inhibitor is selected from the group consisting of binimetinib, cobimetinib, selumetinib, and trametinib. In certain embodiments, the MEK1 / 2 inhibitor is trametinib. In some embodiments, the second therapeutic agent is trametinib.

[0136]

[0270] In some embodiments, the second therapeutic agent is an EGFR inhibitor. In some embodiments, the second therapeutic agent is selected from osimertinib and dacomitinib.

[0137]

[0271] In another aspect, provided herein is a method of treating a blood cancer in a subject in need of treatment for a blood cancer, the method 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.

[0138]

[0272] In another aspect, provided herein is a method of treating a solid tumor (e.g., a solid tumor / advanced solid tumor described herein) in a subject in need of treatment for a solid tumor, the method 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 the group consisting of an anti-VEGFR2 antibody, 5-fluorouracil, alpelisib, and trametinib.

[0139]

[0273] In another aspect, provided herein is a method of treating a solid tumor (e.g., the solid tumors / advanced solid tumors described herein) in a subject in need of treatment of a solid tumor, the method 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 tumor is selected from renal cell carcinoma and colorectal cancer.

[0140]

[0274] In another aspect, provided herein is a method of treating a solid tumor (e.g., the solid tumors / advanced solid tumors described herein) in a subject in need of treatment of a solid tumor, the method 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 tumor is selected from renal cell carcinoma and colorectal cancer.

[0141]

[0275] In another aspect, provided herein is a method of treating a solid tumor (e.g., the solid tumors / advanced solid tumors described herein) in a subject in need of treatment of a solid tumor, the method 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.

[0142]

[0276] In another aspect, provided herein is a method of treating a solid tumor (e.g., the solid tumors / advanced solid tumors described herein) in a subject in need of treatment of a solid tumor, the method 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.

[0143]

[0277] In another aspect, provided herein is a method of treating a solid tumor (e.g., the solid tumors / advanced solid tumors described herein) in a subject in need of treatment of a solid tumor, the method 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.

[0144]

[0278] In another aspect, provided herein is a method of treating a solid tumor (e.g., the solid tumors / advanced solid tumors described herein) in a subject in need of treatment of a solid tumor, the method 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.

[0145]

[0279] In another aspect, provided herein is a method of treating a solid tumor (e.g., a solid tumor / advanced solid tumor as described herein) in a subject in need of treatment of a solid tumor, the method 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.

[0146]

[0280] In another aspect, provided herein is a method of treating a solid tumor (e.g., a solid tumor / advanced solid tumor as described herein) in a subject in need of treatment of a solid tumor, the method comprising administering to the subject any one of the pharmaceutical compositions described herein.

[0147]

[0281] In another aspect, provided herein is a method of treating a blood cancer (e.g., a blood cancer as described herein) in a subject in need of treatment of a blood cancer, the method comprising administering to the subject any one of the pharmaceutical compositions described herein.

[0148]

[0282] In certain embodiments, the subject is a human. In certain embodiments, the subject is an adult human.

Example

[0149] Example

[0283] The following examples are presented to enable a more complete understanding of the disclosure described herein. The synthetic and biological examples described in this application are provided to illustrate the compounds, pharmaceutical compositions, and methods provided herein and should in no way be construed as limiting their scope.

[0150] 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))

Chemical formula

[0284] 2,5-Dibromopyrazine (10 g, 42 mmol, 1 equivalent), ethyl 2-[(diphenylmethylene)amino]acetate (11.8 g, 44 mmol, 1.05 equivalents), tetrabutylammonium bromide (TBAB) (13.6 g, 42 mmol, 1 equivalent), and K 2 CO 3 (17.4 g, 126 mmol, 3 equivalents) were stirred in an oil bath at 100 °C overnight. The reaction mixture was cooled and filtered. The filtrate was diluted with 200 mL of water. The resulting solution was extracted with 2 × 200 mL of ethyl acetate, and the organic layers were combined. The resulting mixture was washed with 2 × 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 obtain ethyl 2-(5-bromopyrazin-2-yl)-2-[(diphenylmethylene)amino]acetate (8 g, 45% yield) as a yellow solid. LCMS (ES, m / z): [M+H] + : 424

[0151] 1-b: Synthesis of ethyl 2-amino-2-(5-bromopyrazin-2-yl)acetate

[0285] In a 250 mL round-bottom flask, ethyl 2-(5-bromopyrazin-2-yl)-2-[(diphenylmethylene)amino]acetate (8 g, 18.8 mmol, 1 equiv), tetrahydrofuran (THF) (10 mL), and HCl (aqueous, 1 M) (20 mL) were charged. The resulting solution was stirred at 25 °C for 30 minutes. The formed solution was diluted with 50 mL of water and extracted with 2 × 50 mL of dichloromethane. The aqueous layer was adjusted to pH 8 with NH 3 .H 2 O and further extracted with 3 × 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. LCMS (ES, m / z): [M+H] + : 260

[0152] 1-c: Synthesis of ethyl 6-bromoimidazo[1,5-a]pyrazine-1-carboxylate

[0286] In a 50 mL round-bottom flask, ethyl 2-amino-2-(5-bromopyrazin-2-yl)acetate (4.2 g, 0.02 mol, 1 equiv) and triethyl orthoformate (20 mL) were charged. The resulting solution was stirred in an oil bath at 80 °C for 2 hours. 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 by air drying. LCMS (ES, m / z): [M+H] + : 270

[0153] 1-d: Synthesis of 2,4-difluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline

[0287] 3-Bromo-2,4-difluoroaniline (10 g, 48 mmol, 1 equiv), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (Pd(dppf)Cl 2(3.5 g, 4.8 mmol, 0.1 eq), bis(pinacolato)diboron (18.3 g, 72 mmol, 1.5 eq) and potassium acetate (KOAc) (14.2 g, 144.2 mmol, 3 eq) were dissolved in dioxane (240 mL). The resulting solution was stirred overnight at 100 °C in an oil bath. The reaction mixture was cooled and the solid was removed by filtration. The filtrate was concentrated, diluted with dichloromethane (DCM) (100 mL), and then washed with 2 × 100 mL of water and 100 mL of brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was applied to a silica gel column and eluted with ethyl acetate / petroleum ether (1 / 10). 2,4-Difluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (8 g, 65% yield) was isolated as a yellow solid. LCMS(ES, m / z): [M+H] + : 256

[0154] 1-e: Synthesis of ethyl 6-(3-amino-2,6-difluorophenyl)imidazo[1,5-a]pyrazine-1-carboxylate

[0288] Dioxane (10 mL) and H 2 O (2 mL) of ethyl 6-bromoimidazo[1,5-a]pyrazine-1-carboxylate (500 mg, 1.9 mmol, 1 eq), 2,4-difluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (708 mg, 2.8 mmol, 1.5 eq), Pd(dppf)Cl 2 (135 mg, 0.2 mmol, 0.1 eq), K 2 CO 3 (767 mg, 5.6 mmol, 3 eq) were stirred at 60 °C for 1 hour in an oil bath. The reaction mixture was cooled, diluted with water (20 ml), and extracted with 3 × 20 mL of dichloromethane. The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was applied to a silica gel column and eluted with ethyl acetate / PE (1 / 2). Ethyl 6-(3-amino-2,6-difluorophenyl)imidazo[1,5-a]pyrazine-1-carboxylate (200 mg, 34% yield) was isolated as a brown solid. LCMS (ES, m / z): [M+H] + : 319

[0155] 1-f: Synthesis of ethyl 6-[3-(5-chloro-2-methoxypyridine-3-sulfonamido)-2,6-difluorophenyl]imidazo[1,5-a]pyrazine-1-carboxylate

[0289] 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), 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-preparative HPLC under the following conditions: column, WelFlash™ C18-I, spherical C18 20 - 40 μm; mobile phase: 0.1% formic acid / 5 - 70% MeCN over 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, 97% yield) was isolated as a yellow solid. LCMS (ES, m / z): [M+H] + : 524

[0156] 1-g: Synthesis of 6-[3-(5-chloro-2-methoxypyridine-3-sulfonamido)-2,6-difluorophenyl]imidazo[1,5-a]pyrazine-1-carboxylic acid

[0290] 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), H 2O (2 mL) and LiOH (27 mg, 1.1 mmol, 3 eq) were stirred in an oil bath at 60 °C for 1 h. After concentration, the crude product was purified by flash-preparative high-performance liquid chromatography (HPLC) under the following conditions: column, WelFlash™ C18-I, spherical C18 20 - 40 μm; mobile phase: 5 - 60% acetonitrile (MeCN) / 0.1% ammonia over 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, 90% yield) was isolated as a yellow solid. LCMS (ES, m / z): [M+H] + : 496

[0157] Synthesis of 6-[3-(5-Chloro-2-methoxypyridine-3-sulfonamido)-2,6-difluorophenyl]-N-methylimidazo[1,5-a]pyrazine-1-carboxamide

[0291] 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 eq) in N,N-dimethylformamide (DMF) (4 mL) was treated with diisopropylethylamine (DIEA) (133 mg, 1 mmol, 3 eq), methylamine hydrochloride (16 mg, 0.5 mmol, 1.5 eq) and 1-[bis(dimethyl)methylene]-1H-1,2,3-triazolopyridinium 3-oxide hexafluorophosphate (HATU) (195 mg, 0.5 mmol, 1.5 eq). The resulting solution was stirred for 1 h and then concentrated. The crude product was purified by flash-preparative HPLC under the following conditions: column, WelFlash™ 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. Liquid Chromatography / Mass Spectrometry (LCMS) (ES, m / z): [M+H] + : 509 1 H Nuclear Magnetic Resonance Spectroscopy (NMR) (300 MHz, DMSO-d 6 ) 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).

[0158] Example 2: Preparation of the Potassium Salt of the Compound of Formula (I)

Chem.

[0292] To a stirred mixture of 6-(3-((5-chloro-2-methoxypyridin)-3-sulfonamido)-2,6-difluorophenyl)-N-methylimidazo[1,5-a]pyrazine-1-carboxamide (prepared in Example 1) in aqueous isopropyl alcohol (IPA) was slowly added 1.1 equivalents of aqueous KOH solution, and the solution was heated. The resulting mixture was cooled, the solid was recovered, washed with IPA / water, and then dried under heat and vacuum to obtain potassium ((5-chloro-2-methoxypyridin-3-yl)sulfonyl)(2,4-difluoro-3-(1-(methylcarbamoyl)imidazo[1,5-a]pyrazin-6-yl)phenyl)amide. 19 F NMR (400 MHz, d6-DMSO): -129.62 and -127.72 ppm.

[0159] Example 3: Biochemical Assay

[0293] The GCN2 protein was obtained from Carna Biosciences (GCN2 catalog #: 05-153). This 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 in a 384-well white assay plate. HC-7366 was serially diluted to 11 concentrations by 3-fold dilution in dimethyl sulfoxide (DMSO), and 10 nL of the stock 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 (catalog #PV4809). This protein was diluted to 2-fold concentration of 200 nM with 300 mM ATP in the presence of 2 mM DTT in assay buffer (final concentrations of 100 nM GFP-eIF2α and 150 μM ATP), and 5 μL aliquots were added to each well containing the GCN2 protein and HC-7366. The plate was incubated at 25 °C for 1.5 h with shaking at 1250 rpm in the dark. Tb-anti-P-eIF2α (ThermoFisher catalog #PV4810) was diluted to a concentration of 4 nM with 20 mM ethylenediaminetetraacetic acid (EDTA) in time-resolved fluorescence energy transfer (TR-FRET) dilution buffer (ThermoFisher catalog #PV3574) (final concentrations of 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 with shaking at 600 rpm in the dark. The FRET signal from the plate was read on an Envision (PerkinElmer) plate reader: Label 1: Excitation: 340 nm, bandwidth 30 nm; Emission: 495 nm, bandwidth 10 nm. Delay time: 100 μs. Integration time: 400 μs. Flash number: 30. Label 2: Excitation: 340 nm, bandwidth 30 nm; Emission: 520 nm, bandwidth 25 nm. Delay time: 100 μs. Integration time: 400 μs. Flash number: 30

[0160]

[0294] As shown in Figure 1, the data was analyzed using XLfit. The graph shows the data presented as % vehicle, where the vehicle is the DMSO control.

[0161]

[0295] Figure 1 shows the experimental results for the above-described GCN2 biochemical assay HC-7366 that demonstrates the GCN2 inhibitory activity of the compound.

[0162] Example 4: Cell-based Assay

[0296] HEK293-ATF4-Luc cells were cultured in culture medium at 1.5e 5 cells / mL, and 25 μL of the cell suspension was added into 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 adhesion. 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 HC-7366 treatment, the cells were treated with either DMSO or 12.5 nM halofuginone for 6 hours to activate GCN2. After 6 hours, 25 μL of One-Glo reagent (Promega, catalog #: E6120) was added into each well to be detected (1:1 with the culture medium). Then, the plate was placed at room temperature for 10 minutes, and then the luciferase luminescence was read on EnVision (PerkinElmer). The data was analyzed by XLfit (v5.3.1.3) according to Equation 201.

[0163]

[0297] Figure 2 shows the experimental results for HC-7366 in the above-described ATF4 activity assay for cells treated with HC-7366 alone, demonstrating the effect of HC-7366 on ATF4 activation in cells. The concentrations showing ATF4 activation were comparable to the concentrations at which ATF4 protein expression was observed in HT1080 cells in Figure 5. n = 1 or n = 2 represents different runs performed on the same day. Figure 3 shows the experimental results for HC-7366 for cells treated with HC-7366 and halofuginone, demonstrating the GCN2 inhibitory effect of GCN2 in cells in the above-described ATF4 activity assay.

[0164] CellTiter-Glo® (CTG) viability assay in MOLM-16 cells

[0298] MOLM-16 cells were obtained from DSMZ (#ACC555) and cultured in RPMI 1640 (Gibco, #11875119) with 20% FBS (Invitrogen, #10099141C) in a cell culture incubator (Thermo Scientific) set at 37°C - 5% CO 2 ₂, 95% relative humidity. 30 μL of cell suspension (2000 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 diluted to 10 concentrations by 3-fold dilution in a 384pp-plate using a TECAN EVO200. 40 nL of the HC-7366 stock solution was transferred to the 384-well cell culture plate using an Echo550 to give a final concentration in the range of 1 - 10 μ. The plate was incubated in the incubator at 37°C, 5% CO 2 ₂ for 72 hours. Viability was assessed by adding 30 μL of CellTiter-Glo® 2.0 assay (Promega, G9243) and adding it to each well. The plate was then incubated at room temperature (RT) for 30 minutes. The luminescence signal was read by an Envision (PerkinElmer) plate reader. The data was analyzed by XLfit (v5.3.1.3), equation 201.

[0165]

[0299] Figure 4 shows the experimental results for HC-7366 in the above cell viability assay.

[0166] Example 5: Western blot analysis

[0300] Cell lysates were obtained from HT1080 cells treated with HC-7366 (0 - 10 μM) for 30 minutes, followed by treatment with either DMSO or 100 nM halofuginone to activate GCN2, and subjected to Western blot analysis. Reagents Fisher Bioreagent (Catalog #: BP2471-1) 10× Tris-buffered saline (TBS) Fisher Tween 20 (Catalog #: BP337-100 Lot 10817) BioRad blotting grade blocker (milk) (Catalog #: 170-6404) Pierce SuperSignal West Femto (Catalog #: 34095) Primary antibodies Anti-phospho-GCN2-T899 rabbit monoclonal, Abcam Catalog #: ab75836 Anti-GCN2 rabbit monoclonal, Abcam Catalog #ab134053 Anti-p-PERK rabbit antibody, Eli Lilly Anti-PERK rabbit monoclonal, Cell Signaling Technology Catalog #: 3192 Anti-p-PKR rabbit polyclonal, ThermoFisher Scientific Catalog #44-668G Anti-PKR rabbit polyclonal, ThermoFisher Scientific Catalog #: 700286 Anti-p-eIF2α S51 rabbit monoclonal, Cell Signaling Technology Catalog #: 3398 Anti-eIF2α rabbit, Cell Signaling Technology Catalog #: 9722 Anti-ATF4 Rabbit Monoclonal, Cell Signaling Technology Catalog # : 11815 Anti-β-actin Mouse Monoclonal - Sigma Catalog # : A5441 Jackson Immunoresearch secondary antibody 1:5000 in 5% milk HRP Goat anti-Rabbit, Catalog # : 111 - 035 - 046 Lot 105262 HRP Goat anti-Mouse, Catalog # : 115 - 035 - 071 Lot 100214

[0167] Sample Preparation

[0301] HT1080 cells were harvested and counted, and diluted to the desired density. 2 mL of cell suspension was added into each well of a 6-well cell culture plate. The plate was transferred to an incubator at 37 °C with 5% CO 2 overnight. The cells were treated as described above. The medium was aspirated, and the cells were washed with ice-cold phosphate-buffered saline (PBS). Ice-cold 1× radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with protease and phosphatase inhibitors was added to the cell pellet on ice. After 30 minutes on ice, the cell lysate was centrifuged at 4 °C and 12,000 rpm for 15 minutes, and the supernatant was collected. The protein concentration was determined using a BCA kit. The cell lysate was mixed with 5× sodium dodecyl sulfate (SDS) loading buffer and denatured at 95 °C for 5 minutes.

[0168] Western Blot

[0302] 30 μg of protein was loaded into a 4 - 12% bis - tris gel in 1×MOPS buffer and run at 120 V for 120 minutes. After completion of electrophoresis, the separated proteins were transferred to a nitrocellulose membrane using a transblot system at 300 mA for 90 minutes, and then the membrane was blocked with (Tris - buffered saline) TBS buffer (5% bovine serum albumin (BSA)) for 1 hour at room temperature. The membrane was exposed to the primary antibody in TBS buffer (1% BSA) in a cold room overnight. Then, the membrane was washed 3×10 minutes with TBS - Tween at R / T, and incubated with the secondary antibody in TBS buffer (1% BSA) at RT for 1 hour. The membrane was washed 3×10 minutes with TBS - Tween at R / T, and imaging was acquired using a Li - COR imaging system.

[0169]

[0303] Figure 5 shows the experimental results for HC - 7366 in the above Western blot analysis. HC - 7366 showed activation of the GCN2 pathway at low concentrations. Halofuginone acts as a GCN2 agonist by inhibiting prolyl - tRNA synthetase. Cells treated with halofuginone showed activation of GCN2 and ATF4. The GCN2 modulator HC - 7366 dose - dependently inhibited halofuginone - induced p - GCN2 and ATF4. This result demonstrates the GCN2 - regulatory property of HC - 7366, which acts as an inhibitor of pGCN2 and ATF4 in the presence of halofuginone but activates the GCN2 pathway by itself.

[0170] Example 6: In Vivo Tumor Growth Inhibition (TGI) Test

[0304] The effect of HC - 7366 on xenograft tumor growth was investigated in several models listed below. 1. Test Design 1.1 Animals Cell lines used as well as mouse species and strains:

[0305] 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 (1×10 7 cells) in RPMI 1640 without serum for tumor growth on the right flank. Treatment was initiated when the average tumor size reached approximately 220 mm 3 on day 14 after cell inoculation and continued until the end of the test by twice-daily oral gavage of HC-7366 at the listed doses (vehicle, 0.25 mg / kg of HC-7366, 0.5 mg / kg of HC-7366, 1 mg / kg of HC-7366, or 2 mg / kg of HC-7366, Figure 16).

[0171]

[0306] HT1080 (human fibrosarcoma cells): Female 6 - 8 week-old BALB / c nude mice were subcutaneously inoculated with HT1080 tumor cells (1×10 7 cells) in 0.1 ml of EMEM medium without serum for tumor growth on the right flank. Treatment was initiated when the average tumor size reached approximately 109 mm 3 on day 4 after inoculation and continued until the end of the test by oral gavage of HC-7366 at the listed doses (vehicle, 0.3 mg / kg of HC-7366 BID (twice a day), 1 mg / kg of HC-7366 BID, 3 mg / kg of HC-7366 BID, 0.6 mg / kg of HC-7366 QD (once a day), 2 mg / kg of HC-7366 QD, or 6 mg / kg of HC-7366 QD, Figures 6 - 7).

[0172]

[0307] LoVo (human colorectal cancer cells): Female 6 - 8 week-old BALB / c nude mice were subcutaneously inoculated with LoVo tumor cells (5×10 6 cells) in 0.1 ml of EMEM medium without serum for tumor growth on the right flank. Treatment was initiated when the average tumor size reached approximately 243 mm 3 on day 16 after inoculation and continued until the end of the test by twice-daily oral gavage of HC-7366 at the listed doses (vehicle, 0.3 mg / kg of HC-7366, 1 mg / kg of HC-7366, 3 mg / kg of HC-7366, 10 mg / kg of HC-7366, or 30 mg / kg of HC-7366, Figures 8 - 9).

[0173]

[0308] DLD-1 (human colorectal cancer cells): DLD-1 tumor cells (5×10 6 cells) in 0.1 ml of RPMI 1640 medium without serum for tumor growth were subcutaneously inoculated on the right flank of female 6 - 8 week-old BALB / c nude mice. Treatment was initiated when the average tumor size reached approximately 195 mm 3 on the 7th day after inoculation, and continued until the end of the test by twice-daily oral gavage of HC-7366 at the listed doses (vehicle, 0.3 mg / kg of HC-7366, 1 mg / kg of HC-7366, 3 mg / kg of HC-7366, 10 mg / kg of HC-7366, or 30 mg / kg of 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).

[0174]

[0309] FaDu (human head and neck cancer cells): FaDu tumor cells (5×10 6 cells) in 0.1 ml of EMEM medium without serum for tumor growth were subcutaneously inoculated on the right flank of female 6 - 8 week-old BALB / c nude mice. Treatment was initiated when the average tumor size reached approximately 194 mm 3 on the 21st day after inoculation, and continued until the end of the test by twice-daily oral gavage of HC-7366 at the listed doses (vehicle, 0.3 mg / kg of HC-7366, 1 mg / kg of HC-7366, 3 mg / kg of HC-7366, 10 mg / kg of HC-7366, or 30 mg / kg of HC-7366, Figures 11 - 13).

[0175]

[0310] KG-1 (human AML cancer cells): KG1 tumor cells (5×10 6 cells) in RPMI 1640 without serum for tumor growth were subcutaneously inoculated on the right flank of female 6 - 8 week-old NOD SCID mice. On the 20th day after cell inoculation, the average tumor size reached approximately 194 mm 3Treatment was initiated when [the specified condition] was reached and continued until the end of the study by twice-daily oral gavage of HC-7366 at the listed doses (vehicle, 0.3 mg / kg of HC-7366, 1 mg / kg of HC-7366, 3 mg / kg of HC-7366, 10 mg / kg of HC-7366, or 30 mg / kg of HC-7366, Figure 17).

[0176]

[0311] Kasumi-1 (human AML cancer cells): Female 6 - 8-week-old CB17 SCID mice were subcutaneously inoculated with KG1 tumor cells (1×10 7 cells) in RPMI 1640 without serum for tumor growth on the right flank. On day 29 after cell inoculation, when the average tumor size reached approximately 151 mm 3 treatment was initiated and continued until the end of the study by twice-daily oral gavage of HC-7366 at the listed doses (vehicle, 3 mg / kg of HC-7366, or 30 mg / kg of HC-7366, Figure 18).

[0177]

[0312] OCI-AML2 (human AML cancer cells): Female 6 - 8-week-old NOD SCID mice were subcutaneously inoculated with OCI-AML2 tumor cells (5×10 6 cells) in RPMI 1640 without serum for tumor growth on the right flank. On day 7 after cell inoculation, when the average tumor size reached approximately 149 mm 3 treatment was initiated and continued until the end of the study by twice-daily oral gavage of HC-7366 at the listed doses (vehicle, 0.3 mg / kg of HC-7366, 1 mg / kg of HC-7366, 3 mg / kg of HC-7366, 10 mg / kg of HC-7366, or 30 mg / kg of HC-7366, Figure 19).

[0178]

[0313] MV4-11 (human AML cancer cells): Female 6 - 8-week-old BALB / c nude mice were subcutaneously inoculated with MV4-11 tumor cells (1×10 7 cells) in RPMI 1640 without serum for tumor growth on the right flank. On day 13 after cell inoculation, when the average tumor size reached approximately 150 mm 3Treatment was initiated when the [specific condition] was reached and continued until the end of the test by twice-daily oral gavage of HC-7366 at the listed doses (vehicle, 0.3 mg / kg of HC-7366, 1 mg / kg of HC-7366, 3 mg / kg of HC-7366, 10 mg / kg of HC-7366, or 30 mg / kg of HC-7366, Figure 20). For the venetoclax combination test, venetoclax was dissolved in 0.5% carboxymethylcellulose (CMC) and co-administered once daily at a dose of 50 mg / kg (see Example 8).

[0179]

[0314] 786-O (human renal cell carcinoma cancer cells): 786-O tumor cells (5×10 6 cells) in 0.1 ml of RPMI 1640 medium without serum for tumor growth were subcutaneously inoculated on the right flank of female 6- to 8-week-old BALB / c nude mice. Treatment was initiated when the average tumor size reached approximately 189 mm 3 after 3 weeks of inoculation and continued until the end of the test by twice-daily oral gavage of HC-7366 at the listed doses (vehicle or 2 mg / kg of HC-7366, Figures 52-54). For the DC101 combination test, DC101 (mouse monoclonal anti-VEGFR2 antibody) was administered by intraperitoneal injection at 15 mg / kg twice a week.

[0180]

[0315] A498 (human renal cell carcinoma cancer cells): A498 tumor cells (5×10 6 cells) in 0.1 ml of MEM medium without serum for tumor growth were subcutaneously inoculated on the right flank of female 6- to 8-week-old NOD SCID mice. Treatment was initiated when the average tumor size reached approximately 349.44 mm 3 after 4 weeks of inoculation and continued until the end of the test by twice-daily oral gavage of HC-7366 at the listed doses (vehicle or 2 mg / kg of HC-7366, Figures 55-57). DC101 was administered by intraperitoneal injection at 15 mg / kg twice a week.

[0181]

[0316] LNCaP (human prostate cancer cells): LNCaP tumor cells (1×10 7 cells) were subcutaneously inoculated in the right flank of male 6-8 week-old NOD SCID mice in 0.1 ml of Matrigel (1:1) in RPMI 1640 with 10% fetal bovine serum (FBS) for tumor growth. Treatment was initiated when the average tumor size reached approximately 207 mm 3 on day 14 after inoculation and continued until the end of the study by twice-daily oral gavage of HC-7366 at the listed doses (vehicle, 0.3 mg / kg of HC-7366, 1 mg / kg of HC-7366, 3 mg / kg of HC-7366, 10 mg / kg of HC-7366, or 30 mg / kg of HC-7366 twice daily, Figure 14).

[0182]

[0317] TM00298 (human prostate cancer cells): Male 6-8 week-old NSG mice were subcutaneously inoculated 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 study by twice-daily oral gavage of HC-7366 at the listed doses (vehicle, 3 mg / kg of HC-7366, or 30 mg / kg of HC-7366, Figure 15).

[0183]

[0318] NCI-H1975 (human non-small cell lung cancer cells): Female 7-8 week-old athymic nude Foxn1nu mice were subcutaneously inoculated in the right flank with NCI-H1975 tumor cells (2.5×10 6 cells) in 0.1 ml of RPMI 1640 medium without serum for tumor growth. HC-7366 was administered at 3 mg / kg as a single agent, osimertinib at 2.5 mg / kg, and dacomitinib at 15 mg / kg. HC-7366 was combined with either osimertinib or dacomitinib. The vehicle for the groups 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 given twice daily by oral gavage, while osimertinib or dacomitinib was administered once daily by oral gavage (Figures 131 and 133).

[0184]

[0319] HCT116 (human colorectal cancer cells): HCT116 tumor cells (5 × 10 6 cells) in 0.1 ml of McCoy's 5A medium without serum for tumor growth were subcutaneously inoculated on the right flank of female 7 - 8 - week - old athymic nude Foxn1nu mice. 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 either trametinib or alpelisib. Treatment was started when the average tumor size reached approximately 270 mm 3 on day 18 after inoculation and continued until the end of the test (Figures 63 - 64).

[0185]

[0320] MFE280 (human endometrial cancer cells): MFE280 tumor cells (10 × 10 6 cells) in 0.2 ml of PBS for tumor growth were subcutaneously inoculated on the right flank of female 7 - 8 - week - old athymic nude Foxn1nu mice. 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 at 0.5 or 2 mg / kg was combined with PT2977. Treatment was started when the average tumor size reached approximately 191 mm 3 on day 28 after inoculation and continued until the end of the test (Figure 135).

[0186]

[0321] 1.1.2 Supplier: Beijing AniKeeper Biotech Co., Ltd.

[0187]

[0322] 1.1.3 Age: 6 - 8 weeks old

[0188]

[0323] 1.1.4 Gender: Female

[0189]

[0324] 1.1.5 Body weight: 16 - 22 g

[0190] 1.2 Animal maintenance

[0325] 1.2.1 Quarantine: The animals were quarantined for 7 days before the test. The overall health status of the animals was evaluated by a veterinarian, and a detailed physical examination was conducted. Animals with abnormalities were excluded before the test. General procedures for animal care and breeding followed the standard operating procedures (SOP) of the Commission on Life Sciences, National Research Council of the standard book.

[0191]

[0326] 1.2.2 Breeding: General procedures for animal care and breeding followed the standard operating procedures (SOP) of the Commission on Life Sciences, National Research Council of the standard book. All procedures related to animal handling, care, and animal research methods used in this test were carried out in accordance with the guidelines approved by the Institute of Animal Care and Use Committee (IACUC) of Pharmaron following the guidance of the American Association for Accreditation of Laboratory Animal Care International (AAALAC). The mice were kept at a constant temperature and humidity in a laminar flow room, with 3 - 5 mice in each cage. The animals were 3 housed in polycarbonate cages of size 300×180×150 mm, in a room with environmental monitoring, maintained at a temperature of (22 ± 3) °C and a relative humidity of 40% - 80% with sufficient ventilation. Fluorescent lights provided approximately 12 hours of light per day. The bedding was cork, which was changed once a week.

[0192]

[0327] 1.2.4 Animal ID: An identification number was assigned to each animal; the following identification method was applied. Information such as the test number, group, gender, dose, animal number, start date, test responsible person, and phone number was displayed on each cage card. Individual animals were identified by ear tags.

[0193]

[0328] 1.2.4 Diet: The animals had free access to irradiated and sterilized dry pellet food throughout the test period.

[0194]

[0329] 1.2.5 Water: Sterile drinking water in the bottles was made freely available to all animals during the quarantine and test periods. The bottles and stoppers with attached sipper tubes were autoclaved before use. Water samples from the animal facility were analyzed, the results of the water analysis were maintained in the facility records, and were reviewed by a veterinarian, or designee, to ensure the absence of known contaminants that could interfere with the test or affect its outcome.

[0195] 1.3 Groups and Treatments

[0330] Groups and treatments were initiated when the average tumor volume reached approximately 100 - 250 mm 3 Mice were assigned to each group based on tumor volume and body weight such that the average starting tumor size was the same for each treatment group.

[0196] 1.4 Vehicle

[0331] Vehicle for HC - 7366: Unless otherwise indicated, 5% (v / v) DMSO and 95% (v / v) Captisol (20% w / v) in PBS (final pH 7.4).

[0197] 2. Experimental Methods and Measurement Parameters 2.1 Method of Tumor Inoculation

[0332] Each mouse was inoculated subcutaneously in situ on the right flank or in the mammary fat pad with 0.1 ml total volume of tumor cells for tumor growth. Treatments were initiated when the average tumor size reached approximately 100 - 250 mm 3 Animals were randomized based on tumor volume into groups of 8 - 10 animals typically including a vehicle control and treatment groups at various dose levels.

[0198] 2.2 Measurement Parameters

[0333] For routine monitoring, all test animals were monitored for tumor growth as well as behavior, e.g., locomotor ability, food and water consumption (by cage - side observation only), body weight (BW), eye / coat shine, and any other adverse effects. Any deaths and / or abnormal clinical signs were recorded.

[0199]

[0334] 2.2.1 Body weight (BW): The body weights of all animals were measured and recorded three times a week throughout the test. The change in body weight was expressed as a percentage and calculated using the following formula: BW change (%) = (BW X日目 / BW 0日目 ) × 100

[0200]

[0335] 2.2.2 Tumor measurements: Tumor size was measured three times a week using calipers and recorded. Tumor volume (mm 3 ) was estimated using the formula: TV = a × b 2 / 2 (where "a" and "b" are the major and minor diameters of the tumor, respectively). Tumor volume was calculated using the formula [tumor volume (mm 3 ) = π / 6 × length × width 2 ).

[0201]

[0336] %T / C (the ratio of tumor volume in the treatment group to tumor volume in the control group) was calculated using the formula tumor growth inhibition (TGI) = (1 - ΔT / ΔC) × 100% when ΔT > 0. ΔT, the average tumor volume in the drug treatment group on the observation day of the test - the average tumor volume in the drug treatment group on the first day of dosing; ΔC, the average tumor volume in the control group on the observation day of the test - the average tumor volume in the control group on the first day of dosing. Regression was calculated using the formula = 100 × ΔT / T initial value when ΔT < 0. Animals with a tumor volume of 0 mm 3 (no measurable tumor) for three consecutive measurements were considered complete responders, and animals with a 50% tumor regression were considered partial responders.

[0202] 2.8 Data acquisition and statistical analysis

[0337] 2.8.1 Data acquisition: The measurements and observations required by the protocol were manually recorded in the appropriate forms.

[0203]

[0338] 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 were calculated for all measurement parameters as per the study design. One - way analysis of variance (ANOVA) was applied between groups.

[0204]

[0339] HC-7366 demonstrated potent single-agent activity in solid tumors, including regression and complete responses. HC-7366 showed up to 80% tumor growth inhibition at both 1 and 3 mg / kg in HT1080 fibrosarcoma model tumor-bearing mice. HC-7366 showed 94% tumor growth inhibition at both 1 and 3 mg / kg in LoVo colorectal model tumor-bearing mice. HC-7366 showed approximately 78% tumor growth inhibition at 1 mg / kg and 3 mg / kg in the DLD-1 colorectal tumor model. HC-7366 showed 33% tumor regression at 1 mg / kg and significant TGI at all doses in FaDu head and neck squamous model tumor-bearing mice. HC-7366 showed approximately 61 - 65% tumor growth inhibition at ≥3 mg / kg in the LNCaP androgen receptor-positive prostate tumor model. HC-7366 showed approximately 70% tumor growth inhibition at both 3 mg / kg and 30 mg / kg in the TM00298 androgen receptor-positive patient-derived prostate tumor model. All treatments were well tolerated by body weight.

[0205]

[0340] Figures 6 - 15 and 135 show solid tumor growth inhibition for HC-7366 in the above TGI test.

[0206]

[0341] HC-7366 demonstrated potent single-agent activity in the AML model. 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 and 3 mg / kg. HC-7366 showed 73% and 77% TGI in Kasumi-1 at 3 and 30 mg / kg, respectively. HC-7366 showed approximately 47% and 51% TGI in OCI-AML2 at 10 and 30 mg / kg, respectively. HC-7366 showed 45% TGI in MV4-11 at 30 mg / kg.

[0207]

[0342] Figures 16 to 20 show the inhibition of AML tumor growth for HC-7366 in the above TGI test. The FAB classification of each AML is shown. HC-7366 showed a robust response in the M0 to M2 subtypes of AML.

[0208] Example 7: Tumor Pharmacodynamic Analysis Immunohistochemistry

[0343] Immunohistochemical analysis was performed on the tumors collected at the end of the test described in Example 6. Formalin-fixed paraffin-embedded samples were sectioned at a thickness of 5 μm 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 cover-slipped using Mowiol anti-fade mounting media (Sigma #D2522).

[0209] Image Analysis for Immunohistochemistry

[0344] Slides were imaged using an Aperio Versa 200 (Leica Biosystems) whole slide scanner with a 10x / 0.32NA objective lens and data were analyzed using a custom-written macro in ImageJ / FIJI (NIH). Briefly, the 150-μm outer edge of tumor sections and necrotic regions (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 tumor tissue was measured using the Gaussian blurred DAPI channel. Immunostaining scans were processed to remove background, and then the total intensity values above an appropriate threshold level were measured and normalized to the tissue area. All graphing and statistical processing (one-way ANOVA and pairwise comparisons) were performed using Prism GraphPad.

[0210] S100A8 and S100A9 immunohistochemical staining and analysis

[0345] Slides were deparaffinized, rehydrated, and heat-mediated antigen retrieval was performed using AR6 buffer (Akoya). Slides were blocked with Roche diagnostics antibody diluent (Fisher) and stained with the primary antibody at 110 rpm for 1 hour. After washing, a horseradish peroxidase (HRP)-conjugated secondary antibody was added at 110 rpm for 10 minutes. After washing, OPAL detection chromogens (Akoya) were added for a 10-minute static incubation. 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 deconvolution, cell segmentation, and phenotyping were performed using Inform Tissue Finder software (Akoya). Imaging data were converted to.csv files and imported into Flowjo for analysis of the percentage of S100A8 / A9-positive cells. The primary antibody used was rabbit anti-human / mouse S100A8 / A9 (clone RM1038, Abcam ab288715, conjugated to Opal570).

[0211]

[0346] The HC-7366-treated KG-1 tumors were evaluated for ISR markers by IHC using the above method at the end of the experiment (day 27). HC-7366 strongly induced ISR in KG-1 tumors. HC-7366 strongly induced ASNS (Figs. 21 and 22), PSAT1 (Figs. 23 and 24), and PHGDH (Figs. 25 and 26). Maximum induction was observed with 1 and 3 mg / kg doses of HC-7366. HC-7366 strongly reduced the protein levels of S100A8 / A9 at 1 and 3 mg / kg doses in KG-1 tumors (Figs. 27 and 28). The PUMA level did not change with HC-7366 in this model.

[0212]

[0347] The HC-7366-treated FaDu tumors were evaluated for HIF1α and HIF2α by IHC using the above method at the end of the experiment. HC-7366 significantly reduced both HIF1α and HIF2α protein levels in FaDu tumors. The expression patterns of HIF1α and HIF2α were mutually exclusive (Figs. 40 - 42).

[0213]

[0348] The HC-7366-treated DLD-1 tumors were evaluated for ASNS, PSAT1, PUMA, HIF1α, and HIF2α by IHC using the above method at the end of the experiment (day 20). HC-7366 strongly induced ASNS, PSAT1, and PUMA in a bell-shaped manner. HC-7366 significantly reduced both HIF1α and HIF2α protein levels in FaDu tumors (Figs. 29 - 39). HIF2α was expressed at extremely low levels in the tumor model. HC-7366 strongly activated GCN2 / ISR in the tumors.

[0214] Example 8: Combination of HC-7366 and Venetoclax in MV4-11 Cells

[0349] The MV4-11 tumors were harvested at the end of the Venetoclax combination test described in Example 6. Tumor growth curves of MV4-11 tumors treated with single-agent HC-7366 and Venetoclax or in combination are provided (Figure 43). HC-7366 and Venetoclax showed a robust combination benefit resulting in regression. HC-7366 had a 34% TGI as a single agent. Venetoclax monotherapy showed a 62% TGI. However, HC-7366 showed a 26% tumor regression when combined with Venetoclax at a GCN2 activation dose of 3 mg / kg.

[0215]

[0350] Immunohistochemical analysis was performed on the tumors according to the procedure described in Example 7. Immunohistochemical ISR marker analysis of the tumors at the end of the study showed that HC-7366 at 3 mg / kg caused strong activation of ISR markers such as ASNS (Figures 44 and 45), PHGDH (Figures 46 and 47), and PUMA (Figures 48 and 49), which were further increased in combination with Venetoclax, suggesting superactivation of ISR. S100A8 / A9 levels were significantly reduced by monotherapy with both HC-7366 and Venetoclax, and the combination of Venetoclax and HC-7366 further robustly decreased S100A8 / A9 levels (Figures 50 and 51). PSAT1 levels did not change.

[0216] Example 9: Combination of HC-7366 and anti-VEGFR antibody in 786-O, A498, and DLD-1 tumors

[0351] The 786-O and A498 tumors were harvested at the end of the DC101 combination test described in Example 6. IHC analysis was performed on the tumors according to the procedure described in Example 7.

[0217]

[0352] The HC-7366-treated 786-O tumors were evaluated for HIF2α and GLUT1 by IHC. HIF2α was significantly decreased with both single-agent HC-7366 and DC101 treatment and in combination. GLUT1 levels were significantly reduced with single-agent HC-7366 or in combination with DC101 (Figs. 52-54). The tumor model did not express HIF1α.

[0218]

[0353] The HC-7366-treated A498 tumors were evaluated for HIF2α and GLUT1 by IHC. HIF2α was significantly increased with single-agent DC101 treatment. No significant change in GLUT1 levels was observed in any of the groups (Figs. 55-57). The tumor model did not express HIF1α.

[0219]

[0354] The effect of the combination of DC101 with HC-7366 in DLD-1 tumor-bearing mice was evaluated using a protocol similar to that described in Example 6. Mice were treated by oral gavage twice daily with 3 or 30 mg / kg of HC-7366. DC101 was administered via intraperitoneal injection at 20 mg / kg twice weekly and twice daily. HC-7366 inhibited tumor growth in the DLD-1 colorectal model at 3 and 30 mg / kg, with TGI% values of approximately 65% and approximately 21%, respectively, while DC101 yielded a TGI% value of approximately 55%. A significant combination benefit was observed when 3 mg / kg of HC-7366 was combined with DC101 (approximately 76% tumor growth inhibition), but no combination benefit was observed at 30 mg / kg (Fig. 61).

[0220] Example 10: Combination of HC-7366 and belzutifan (PT-2977)

[0355] Female 6- to 8-week-old BALB / c nude mice were subcutaneously inoculated on the right flank with 786-O tumor cells (5×10 6 cells) in 0.1 ml of RPMI 1640 medium without serum for tumor growth (see Example 6). Three weeks after inoculation, the average tumor size was approximately 200 mm 3Treatment was initiated when this was reached and continued by twice-daily oral gavage of HC-7366 at the listed dose until the end of the study. PT-2977 was administered by twice-daily oral gavage at 0.1 mg / kg until the end of the study.

[0221]

[0356] Provide the tumor growth curves of 786-O tumors treated with monotherapy HC-7366 and velzufant or in combination (Figure 58). HC-7366 and PT-2977 as monotherapies showed comparable antitumor efficacy in this model, with TGI% values of approximately 78% and approximately 70%, respectively. A significant combined antitumor benefit was observed when HC-7366 was combined with PT2977, resulting in an average tumor regression of approximately 50%.

[0222]

[0357] Female 7- to 8-week-old athymic nude-Foxn1nu mice were subcutaneously inoculated on the right flank with MFE280 tumor cells (10×10 6 cells) in 0.2 ml of PBS for tumor growth (see Example 6). Monotherapy 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 was combined with PT2977 at 0.5 or 2 mg / kg. Treatment was initiated when the average tumor size reached approximately 191 mm 3 and continued until the end of the study.

[0223]

[0358] Provide the tumor growth curves of MFE280 tumors treated with monotherapy HC-7366 and velzufant or in combination (Figure 135). Monotherapy HC-7366 showed tumor growth inhibition of 45% and 53% at 0.5 mg / kg and 2 mg / kg, respectively. PT2977 showed tumor growth inhibition of 53% at 1 mg / kg. HC-7366 0.5 mg / kg + PT2977 1 mg / kg showed tumor growth inhibition of 97%. HC-7366 2 mg / kg + PT2977 1 mg / kg showed tumor regression of 17%. Treatment was well tolerated as determined by body weight measurements.

[0224] Example 11: Combination of HC-7366 and 5-fluorouracil

[0359] 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 by oral gavage twice daily with 3 or 30 mg / kg of HC-7366. 5-Fluorouracil was administered via intravenous injection twice daily at 75 mg / kg once a week. HC-7366 inhibited tumor growth in the DLD-1 colorectal model at 3 and 30 mg / kg, with TGI% values of approximately 65% and approximately 21%, respectively, while 5-fluorouracil resulted in a TGI% value of approximately 68%. A significant combination benefit was observed when 3 mg / kg of HC-7366 was combined with DC101 (approximately 88% tumor growth inhibition; tumor stasis), but no combination benefit was observed at 30 mg / kg (Figure 62).

[0225] Example 12: Combination of HC-7366 and PI3Kα or MEK1 / 2 inhibitors

[0360] HCT-116 tumor growth was monitored in the alpelisib and trametinib combination test described in Example 6.

[0226]

[0361] HC-7366 showed a tendency towards efficacy (approx. 38% TGI) at a dose of 3 mg / kg in the HCT116 colorectal cancer model, while 30 mg / kg of HC-7366 did not reduce tumor growth. A significant combination benefit was observed when alpelisib was co-administered with HC-7366 at either dose (Figure 63).

[0227]

[0362] HC-7366 did not show efficacy in HCT116 at either 3 or 30 mg / kg, but had a significant combination benefit when trametinib was co-administered with 3 mg / kg of HC-7366 (Figure 64).

[0228]

[0363] All treatments were well tolerated as measured by body weight.

[0229] Example 13: Combination of HC-7366 and EGFR inhibitors

[0364] The growth of NCI-H1975 tumors was monitored in the osimertinib and dacomitinib combination test described in Example 6.

[0230]

[0365] HC-7366 showed significant antitumor efficacy at 3 mg / kg, with a TGI of 59%, while osimertinib produced a TGI value of 95% at 2.5 mg / kg, with a partial response in 1 / 10 animals. HC-7366 + osimertinib showed a combination benefit resulting in an average tumor regression of approximately 90%, with a complete response in 2 / 10 animals and a partial response in 8 / 10 animals (Figure 131).

[0231]

[0366] Dacomitinib alone produced tumor stasis followed by tumor growth at 15 mg / kg, with a TGI% value of 36% at the end of the test, which was not statistically significant. HC-7366 + dacomitinib showed a combination benefit, with a TGI% value of approximately 99% and a partial response in 1 / 10 animals (Figure 133). Animals in the dacomitinib-containing groups showed significant weight loss. As a result, the animals in these groups were given a drug withdrawal day. Treatment-related weight loss was not observed in the HC-7366 and vehicle groups.

[0232] Example 14: GCN2-dependence of the HC-7366-mediated effect CTG assay

[0367] MOLM-16 cells were obtained from DSMZ (#ACC555) and cultured in RPMI 1640 (Gibco, #11875119) with 20% FBS (Invitrogen, #10099141C) at 37 °C and 5% CO 2They were cultured. MOLM-16 GCN2 CRISPR knockout cells (sgGCN2) were generated and compared with control cells (sg control). For the survival rate experiment, the cells were plated in a 384-well cell culture plate and incubated overnight. The compounds were diluted to final concentrations in the range of 1 - 10 μM at 8 concentrations by 3-fold dilution in a 384pp plate using TECAN EVO200. The plate was incubated for 24 or 48 hours, and the survival rate was assessed by adding CellTiter-Glo® 2.0 assay to each well according to the manufacturer's protocol (Promega, G9243). Then, the plate was incubated at RT for 30 minutes. The luminescence signal was read by an Envision (PerkinElmer) plate reader and analyzed by Prism.

[0233]

[0368] 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% CO 2 They were cultured. FaDu GCN2 CRISPR knockout cells (sgGCN2) or control cells (sg control) were generated. For the survival rate experiment, the cells were plated in a 384-well cell culture plate and incubated overnight. The compounds were diluted to final concentrations in the range of 1 - 10 μM at 10 concentrations by 3-fold dilution in a 384pp plate using TECAN EVO200. The plate was incubated for 72 or 96 hours, and the survival rate was assessed by adding CellTiter-Glo® 2.0 assay to each well according to the manufacturer's protocol (Promega, G9243). Then, the plate was incubated at RT for 30 minutes. The luminescence signal was read by an Envision (PerkinElmer) plate reader and analyzed by Prism.

[0234]

[0369] HC-7366 reduced in vitro viability and GCN2-dependently induced the ISR in MOLM-16 cells. HC-7366 potently reduced viability at lower concentrations in a U-shaped manner (Figure 65). This response was reversed in GCN2 CRISPR knockout cells (Figure 66).

[0235]

[0370] HC-7366 reduced in vitro viability and GCN2-dependently induced the ISR in FaDu cells. HC-7366 potently reduced viability in FaDu control cells (Figure 71). This response was reversed in GCN2 CRISPR knockout cells (Figure 72).

[0236] Jess protein analysis

[0371] Cell lysates were obtained from sg control or sgGCN2 cells of MOLM-16 treated with HC-7366 (0, 0.01, 0.1, and 10 μM) for 24 hours. Ice-cold 1× radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with protease and phosphatase inhibitors was added to the cell pellet on ice. After 30 minutes on ice, the cell lysates were centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA kit. The cell lysates were mixed with 5× sodium dodecyl sulfate (SDS) loading buffer and denatured at 95°C for 5 minutes. 1 μg of the lysate was run on a Jess capillary, and the results were analyzed by compass simple western software.

[0237]

[0372] Cell lysates were obtained from FaDu sg control or sgGCN2 cells treated with HC-7366 (0, 0.01, 0.1, and 1 μM) for 72 hours. Ice-cold 1× radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with protease and phosphatase inhibitors was added to the cell pellet on ice. After 30 minutes on ice, the cell lysates were centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatants were collected. Protein concentrations were determined using a BCA kit. The cell lysates were mixed with 5× SDS loading buffer and denatured at 95°C for 5 minutes. 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-GCN2T899 (Abcam #ab75836), GCN2 (Cell Signaling #3302), ATF4 (Cell Signaling #11815), and ASNS (Proteintech #14681). The anti-rabbit secondary antibody was from ProteinSimple #042-206.

[0238]

[0373] Protein expression of GCN2 in wild-type MOLM-16 cells (WT, sg control) and CRISPR knockout (sgGCN2) cells was measured by JESS as described above (Figure 67). HC-7366-mediated activation of the ISR was assessed by observing the levels of ATF4 (Figure 68) and its downstream targets ASNS (Figure 69) and PSAT1 (Figure 70), which showed a GCN2-dependent increase.

[0239]

[0374] Protein expression of GCN2 in wild-type (WT) FaDu cells (WT, sg control) and CRISPR knockout (sgGCN2) cells was measured by JESS as described above (Figures 73 and 74). HC-7366-mediated activation of the ISR was assessed 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.

[0240] Polysome profiling

[0375] Polysome profiling was performed in HEK293 GCN2 WT or HEK293 GCN2 knockout cells as previously described by Johannes and Sarnow et al., RNA, 1998. Cells were treated with HC-7366 for 16 hours and then harvested for polysome profiling.

[0241]

[0376] The effect of HC-7366 on protein synthesis was evaluated by polysome profiling of GCN2 WT and CRISPR knockout HEK293 cells. HC-7366 reduced the ratio of polysomes to monosomes, which reflects reduced translation in GCN2 WT cells but not in GCN2 knockout cells (Figures 77 and 78). HC-7366 potently reduced puromycin labeling of newly synthesized proteins at 100 nM, which activates the GCN2 / ISR pathway as measured by increased levels of p-eIF2α and ATF4 (Figures 79 and 80).

[0242]

[0377] HC-7366 reduced in vitro viability and induced the ISR in a GCN2-dependent manner.

[0243] Example 15: Ex vivo patient-derived xenograft (PDX) assay using HC-7366

[0378] HC-7366 (4.57 - 10,000 nM) was evaluated ex vivo in 2D cell culture in 30 patient-derived Champions human AML models. PDX models were treated with DMSO or HC-7366 for 6 days and then cell viability was assayed using a plate-based luminescence assay (Cell Titer Glo). HC-7366 potently reduced the viability of primary AML patient-derived xenograft (PDX) cells.

[0244]

[0379] 11 PDX models were sensitive responders and the EC 50 was < 100 nM. 12 PDX modes were moderate responders and the EC 50was 100 - 350 nM. Five PDX models were resistant responders, and EC 50 was 924 - 2253 nM. Only two models did not show a response to HC - 7366. EC 50 values were calculated relative to the bottom of the curve and are provided below. Survival curves are provided 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).

[0245]

Table 1

[0246] Example 16: AML PDX In Vivo Test

[0380] Sub - lethal irradiated NCG mice were inoculated with 2×10 6 human AML cells of model CTG - 2229. Mice were monitored after AML inoculation and human AML engraftment was assessed using huCD45 / muCD45 / huCD33 / huCD3 antibodies and BD TruCount™ beads. If individual animals had ≥20% viable huCD45 cells in the bone marrow, they were randomized into the treatment cohort. For the efficacy study, animals were dosed with vehicle control, HC - 7366 at 1 mg / kg, 10 mg / kg or 30 mg / kg PO / BID×28, or venetoclax at 100 mg / kg PO / QD×28 (n = 13 / group). 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 adverse treatment - related side effects. Body weight was measured twice a week.

[0247]

[0381] 1 mg / kg of HC - 7366 limited myeloid - restricted progenitor cells (CD34 + CD33 + ) and mature myeloid cells (CD34 - CD33 +) was significantly decreased. HC-7366 was most prominent in the bone marrow compared to venetoclax.

[0248] Example 17: HC-7366-mediated effects on oxygen consumption rate (OCR) and metabolomics

[0382] MOLM-16 cells were treated with 0.001, 0.1 or 10 μM of HC-7366 for 16 hours, and Seahorse XF glycolysis stress test (ECAR; extracellular acidification rate) and Seahorse XF Mito stress test (OCR; oxygen consumption rate) (Agilent technologies, CA, USA) were performed.

[0249]

[0383] HC-7366 significantly reduced OCR (Figure 88) and glycolysis (Figure 89), and the strongest effect was observed at the activating 0.1 μM concentration. Collectively, these results indicate that HC-7366 reduces both mitochondrial oxygen consumption rate and glycolysis, leading to a reduction in ATP production.

[0250] Example 18: HC-7366-mediated effects on metabolomics in a tumor xenograft model

[0384] MOLM-16 xenograft tumors were treated with vehicle or HC-7366 (0.3, 1, 3, 10 or 30 mg / kg) for 4 days, and rapidly frozen tumors were harvested for metabolomic analysis (n = 12). Metabolic analysis was performed on the LC / MS / MS and Polar LC platforms at Metabolon. Data were normalized to the vehicle group, and Welch's two-sample t-test was performed for statistical analysis. Heatmaps show metabolite levels normalized to vehicle controls (Figures 90, 94, 102, and 107).

[0251]

[0385] HC-7366 significantly altered multiple metabolites in MOLM-16 tumors.

[0252]

[0386] HC-7366 significantly increased the levels of most amino acids. Some amino acid levels were decreased by HC-7366; these included aspartic acid and cysteine (Figures 90 - 93). HC-7366 significantly reduced glutathione levels (GSH and GSSG), improved a number of γ-glutamyl-amino acids, suggesting that HC7366-treated tumors attempted to increase glutathione production in potential response to increased oxidative stress in the cells (Figures 94 - 101). HC-7366 significantly reduced the metabolites involved in pyrimidine synthesis (Figures 102 - 106). HC-7366 significantly reduced aspartic acid metabolism and polyamine-related metabolites (putrescine, spermine, 5-methylthioadenosine (MTA)) at all doses (Figures 107 - 114). Generally, most of the metabolite changes occurred at the 1 and 3 mg / kg doses.

[0253]

[0387] FaDu tumor-bearing mice were orally treated twice a day for 3 days with vehicle or HC-7366 (1, 3, or 30 mg / kg), an AM dose was administered on the morning of day 4, and tumors and plasma were collected 1 hour after administration. Tumor and plasma samples were extracted and divided into equal amounts for analysis on LC / MS / MS and Polar LC platforms. Proprietary software was used to match the ions to a standard in-house library (Metabolon) for metabolite identification and metabolite quantification by peak area integration. Proteome analysis of the tumors was performed at MS Bioworks. 3 μg equivalents of peptides from each pooled fraction were analyzed by nano LC-MS / MS on a Waters M-Class HPLC system connected to a ThermoFisher Fusion Lumos mass spectrometer. The histograms show the metabolite levels of the treatment groups normalized to the vehicle control (Figures 115 - 122).

[0254]

[0388] HC-7366 significantly increased the levels of several amino acids in both tumor and plasma. HC-7366 also significantly increased the levels of several gamma-glutamyl amino acids in both tumor and plasma. HC-7366 significantly increased the levels of arginine, arginosuccinic acid, and citrulline in the tumor and significantly increased the urea level in plasma. HC-7366 significantly reduced the pyrimidine synthetic metabolites in the tumor. HC-7366 significantly reduced the level of oxidized glutathione in the tumor but did not alter the level of reduced glutathione.

[0255] Example 19: HC-7366-mediated effects on metabolomics in cells

[0389] Wild-type and GCN2 CRISPR knockout FaDu cells were treated with 0.1 μM for 9 hours and metabolomics was evaluated.

[0256]

[0390] Treatment of wild-type FaDu cells with 0.1 μM of HC-7366 for 9 hours resulted in a GCN2-dependent improvement in the levels of many amino acids. 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 supported the in vivo FaDu data showing an improvement in free amino acids in tumor and plasma (see Example 18) and emphasized that GCN2 mediates this effect during HC-7366 treatment. GCN2 wild-type cells and GCN2 CRISPR knockout cells were treated with 0.1 μM of HC-7366, which induced ATF4 in a GCN2-dependent manner (Figures 123 - 125).

[0257] Example 20: Effect of HC-7366 on oxidative phosphorylation in the FaDu xenograft model

[0391] FaDu tumor-bearing mice were treated with HC-7366 at 3 and 30 mg / kg twice a day for 4 days. Tumors were harvested for non-targeted proteome analysis.

[0258]

[0392] Pathway analysis (IPA) of the proteins with expression changes in FADU tumors predicted a strong regression of the oxidative phosphorylation pathway with 3 mg / kg of HC-7366, but not with 30 mg / kg (Figure 126).

[0259]

[0393] Some protein subunits of the electron transport chain were decreased in the 3 mg / kg treatment group (versus vehicle). Oxidative phosphorylation proteins showed little change (versus vehicle) after treatment with 30 mg / kg, and some increased (Figure 127). The data suggest that the activating dose of 3 mg / kg of HC-7366 inhibits mitochondrial respiration in tumors and represents a possible mechanism of action specific to this dose.

[0260] Example 21: RNAseq analysis of DLD-1 tumor xenograft models treated with HC-7366

[0394] DLD-1 tumor-bearing mice were treated with HC-7366 at 0.3, 1, 3, 10, and 30 mg / kg twice a day for 14 days. Tumors were harvested on days 4, 7, and 14 for RNAseq analysis (Figure 128).

[0261]

[0395] Analysis of upstream regulators of the genes with expression changes showed that ATF4 was significantly induced with all doses of HC-7366. Certain transcription factors were associated with efficacy. These included ARID1A, SMAD4, and E2F1 (Figure 129). The analysis showed a reduction in the HIF1α, SMAD4, and E2F1 signatures, while showing an improvement in the ATF4 and ARID1A signatures.

[0262]

[0396] IHC staining of Ki67-positive cells in DLD-1 tumor sections showed that HC-7366 significantly reduced Ki67-positive cancer cells on day 4 at the most effective doses of 1 and 3 mg / kg, but not at the less effective doses of 0.3, 10, or 30 mg / kg (Figure 130).

[0263] Example 22: Multicenter Open-Label Phase 1a / b Trial of a Compound of Formula (I) in Subjects with Progressive Solid Tumors

[0397] A human first-in-human, multi-site, open-label, Phase 1a / b dose-escalation and dose-expansion study was conducted to establish the maximum tolerated dose (MTD) and 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-escalation 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 subjects with 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 if the study selection criteria were met 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 conventional 3+3 design. The starting dose level was 10 mg QD and, if safety permitted, escalated to 20, 40, 75, 125, and 150 mg QD. All doses were administered in a fasting state with water at least 1 hour before a meal or at least 2 hours after a meal. The Phase 1b dose-expansion part included cohort expansion at up to 2 maximum dose levels selected from the dose-escalation data by the Safety Monitoring Committee (SMC) to obtain additional safety and preliminary efficacy information. Each cohort in Phase 1b enrolled 15 subjects. The study can be expanded to a Phase 2 study via protocol amendment to assess the doses and (one or more) tumor types selected as most appropriate for further clinical development in Phase 1a / b. Subjects were administered until the first occurrence of any of unacceptable toxicity, disease progression according to immune-related Response Evaluation Criteria in Solid Tumor (iRECIST), discontinuation of treatment for reasons permitted by other protocols (e.g., subject refusal), any other administrative reasons, or after 2 years of treatment. For scheduling purposes, administration was performed in 3-week cycles, computerized tomography (CT) scans were performed once every 6 weeks, and the first post-baseline scan was performed after 6 weeks of administration (pre-cycle 3).Subjects spent Cycle 1 Day 1 (C1D1) in the clinic and then stayed overnight for safety monitoring and pharmacokinetic (PK) sampling. Subjects were hospitalized for the first two doses: administration on C1D1 and Cycle 1 Day 2 (C1D2) (to the extent permitted by each facility's COVID-19 restrictions). After the first hospitalization at the start of the trial, subjects attended the outpatient clinic on Days 8, 15, and 21 of Cycle 1 and thereafter for physical and laboratory assessments, adverse event (AE) and dosing compliance monitoring, and on the first day of each cycle for PK C3 - C6; the end-of-treatment visit was also self-reported in the outpatient clinic. The overnight stay for Cycle 1 Day 21 (C1D21) - C2D1 was optional. Subjects who discontinued before the first baseline post-CT scan for reasons other than DLT before completion of the disease progression, treatment-related AE, or dose-limiting toxicity (DLT) assessment period were replenished to ensure appropriate safety assessments at each dose level.

[0264] (1) Dose escalation scheme

[0398] There are six dose escalation levels:

[0265]

Table 2

[0266]

[0399] The dose escalation followed the conventional 3 + 3 design. At least 3 subjects were continuously enrolled in each cohort and expanded up to 6 subjects in each cohort if necessary to determine the DLT. For each cohort, the first subject was a monitored subject. The monitored subject was administered and followed up for 4 days to assess safety and tolerability. If it was determined to be safe and well tolerated, the remainder of the cohort (N = 2) was enrolled. If no treatment-related DLT was observed in the first 21 days (DLT evaluation period) in the first 3 subjects of the cohort, the next cohort was enrolled. If a treatment-related DLT was observed in 1 out of 3 subjects, the same cohort was expanded up to 6 subjects. If a treatment-related DLT was observed in 1 out of 6 subjects, the next cohort was enrolled. If treatment-related DLTs were observed in ≥2 subjects at any point between cohorts and the MTD was exceeded, additional enrollment within the cohort was interrupted and dose escalation was stopped. The MTD was defined as the dose level at which treatment-related DLTs were observed in ≥33% of the subjects in the cohort (i.e., ≥2 out of 6) at levels below that level. If there were 2 DLTs at dose level 1, dose level -1 was enrolled. Before applying the dose escalation rule, 3 subjects at a given dose level had to receive at least 75% of the planned dose and be evaluated for toxicity unless no DLT was observed in 1 or more subjects within the first 21 days. If no DLT was observed in the first 3 DLT-evaluable subjects in the cohort during the DLT evaluation period, the next cohort was enrolled. If a subject did not receive at least 75% of the planned dose for any reason other than DLT (i.e., lost to follow-up), the subject was replaced.

[0267]

[0400] The determination of dose escalation was made based on all observations in all subjects in each cohort. DLT-evaluable subjects included those in whom AEs related to the disease but not related to the treatment and serious AEs were observed. Even if the predetermined criteria were not met, the principal investigator and the sponsor could jointly decide to add additional subjects at a given dose level (≤6 evaluable subjects per cohort) based on all available data or not to escalate the dose.

[0268]

[0401] The dosing escalation guidelines for consecutive cohorts are summarized below:

[0269]

Table 3

[0270] (2) Dose-limiting toxicity

[0402] Dose-limiting toxicity was defined as one of the following events occurring during the DLT evaluation period (i.e., up to the end of cycle 1, 21 days after the first administration of HC-7366):

[0271]

[0403] Non-hematological: · Any grade 3 or higher non-hematological toxicity according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) v5.0, except for the following: 〇 Grade >3 nausea or vomiting that resolves to grade 1 or 2 within 72 hours with optimal medical management. 〇 Grade 3 fatigue <5 days. 〇 Isolated grade 3 clinical laboratory value abnormalities that are not associated with clinical signs or symptoms and resolve within 3 days with appropriate maximal medical intervention. · Grade ≥2 cardiotoxicity. · Grade ≥2 pituitary toxicity (hypopituitarism). · Any symptomatic congestive heart failure, a reduction in left ventricular ejection fraction of 50% or less based on multigated acquisition scan or echocardiogram, or a corrected QT interval (QTc) prolongation >500 msec. · Any treatment-related grade 3 or higher non-hematological clinical laboratory value abnormalities, except for the following: 〇 Grade 3 or higher amylase or lipase not associated with symptoms or clinical signs of pancreatitis. 〇 Grade 3 or higher electrolyte abnormalities that persist for up to 72 hours, are not clinically complex, resolve spontaneously, or respond to conventional medical intervention. ·Delay in the start of Cycle 2 treatment for > 2 weeks due to unrelieved treatment-related grade ≥ 3 non-hematological toxicity.

[0272]

[0404] Hematological: ·Grade 4 thrombocytopenia for ≥ 7 days or grade 3 or 4 thrombocytopenia associated with bleeding or requiring platelet transfusion. ·Grade 4 neutropenia for ≥ 7 days. ·Neutropenic fever of any grade (> 38.5°C). ·Grade 4 anemia or grade 3 or 4 anemia requiring transfusion. ·Note: Grade 3 or 4 lymphopenia is not considered a DLT but is recorded as part of the overall safety assessment.

[0273]

[0405] Other: ·More than 25% of the specified dose is missing due to any unrelieved treatment-related toxicity.

[0274]

[0406] AE is considered related to the investigational drug unless there is a clear relationship with underlying disease or recognized comorbidity.

[0275]

[0407] Any subject in whom treatment-related DLT is observed is immediately excluded from the study treatment. Additional subject cohorts are not enrolled at subsequent dose levels until all subjects at the first (or pre-) dose level have completed all planned treatments for Cycle 1 (defined as 3 weeks of oral QD dosing at the assigned dose level) and Cycle 2 can be started with a delay of 2 weeks or less.

[0276] (3) Recommended Phase 2 Dose

[0408] The dosing duration of RP2D and HC-7366 was determined in consultation with the sponsor and the SMC considering the available data on safety, PK, and efficacy. The RP2D did not exceed the MTD. If the MTD was not reached at the end of the Phase 1a / b study, the RP2D was determined by the SMC in consultation with the sponsor after review of the study data.

[0277] (4) Number of subjects

[0409] Up to 66 subjects (Phase 1a: N = up to 36 subjects; Phase 1b: N = 30 subjects).

[0278] (5) Diagnostic and selection criteria

[0410] Selection criteria: Each subject must 1. Sign an informed consent form prior to any study-specific procedures or treatments. 2. Be ≥ 18 years old (male or female) at the time of consent. 3. Have one of the following tumor types with quantitative characteristics and have received at least 1 and no more than 5 prior treatment lines: a. SCCHN b. CRC c. NSCLC d. TCC e. Other solid tumors (e.g., cancer of unknown primary) excluding rapidly progressive neoplasms (e.g., pancreatic cancer, glioblastoma, hepatocellular carcinoma). Subjects do not need to have progressed through all possible available treatments with known clinical benefit for their respective cancers to participate in this study. Subjects with SCCHN, CRC, NSCLC, and TCC are preferred and should constitute at least 50% of the overall enrolled population. Enrollment of all others is capped at 18 subject slots with SCCHN, CRC, NSCLC, and TCC when combined reach 50%. 4. Have at least one radiologically measurable lesion as defined by Response Evaluation Criteria in Solid Tumors (RECIST) v1.1, imaged by CT scan or magnetic resonance imaging, with a longest diameter of at least 10 mm or a short axis of at least 15 mm for lymph nodes, obtained by imaging within 28 days prior to the study treatment. Tumor lesions located within a previously irradiated area are considered measurable if progression has been demonstrated in such lesions 5. Except for stable sensory neuropathy (≤ Grade 2) and alopecia, all prior treatment-related toxicities must have resolved to Grade 1 severity or less. If the subject has undergone major surgery or received > 30 Gy of radiation therapy, the subject must have recovered from the toxicities and / or complications from the intervention. 6. If the subject has been previously treated with an immune checkpoint inhibitor, at least 4 weeks must have elapsed since the last dose, and the toxicities must have resolved as described above. 7. The subject must have at least one biopsy - viable lesion at baseline. Biopsies in this clinical trial will follow the American Society of Clinical Oncology’s Ethical Framework for Including Research Biopsies in Oncology Clinical Trials. If there are suitable and accessible lesions, no biopsy contraindications, minimal risk of complications, and a decision based on positive information, the subject is willing to provide fresh tissue for biomarker analysis and for assessment of biomarker status based on the adequacy of tissue sample quality. Two biopsies are required: at baseline (within 15 days before Day 1 of the trial) and at the time of the first efficacy assessment CT scan on Cycle 3 / Day 1 (+ 7 days). Newly obtained biopsy specimens are preferably storage samples, and formalin - fixed paraffin - embedded block specimens are preferably slides. 8. Must have an Eastern Cooperative Oncology Group performance status of 0 or 1 and maintain it between screening and the start of dosing on Day 1. 9. Must not have dysphagia that interferes with oral administration compliance. 10. Must not have had > 10% weight loss in the prior 4 weeks. 11. Must have a serum albumin level > 3 g / DL. 12. Must have a life expectancy of at least 3 months as determined by the treating physician. 13. On Day 1, the subject must have appropriate organ function defined by meeting all of the following criteria: a. Total bilirubin ≤ 1.5 × upper limit of normal (ULN) or, for subjects with total bilirubin level > 1.5 × ULN, direct bilirubin ≤ ULN. b. Aspartate aminotransferase and alanine aminotransferase ≤ 2.5 × ULN or, for subjects with known liver metastases, ≤ 5 × ULN. 14. On Day 1, the subject must have appropriate renal function defined by creatinine ≤ 1.5 × ULN and creatinine clearance ≥ 60 mL / min according to the following Cockcroft Gault formula

Number

[0279]

[0411] Exclusion criteria: Subjects must not meet any of the following criteria: 1. Those who have had prior chemotherapy, targeted small molecule therapy, or radiation therapy within 2 weeks before the first dose of the investigational medicinal product for the study treatment, or have not recovered from adverse reactions caused by previously administered drugs or major surgery. 2. Currently participating in, receiving, or within 4 weeks of the first dose of a treatment drug, participating in the study of the test drug, receiving the test treatment, or using the test device. 3. Having a diagnosis of immunodeficiency within 7 days before the first dose of the test treatment drug, or receiving systemic steroid therapy or any other form of immunosuppressive therapy. The use of physiological doses of corticosteroids can be approved after consultation with the study sponsor. 4. Having a known history of active tuberculosis. 5. Having a known history of human immunodeficiency virus (HIV) (HIV 1 / 2 antibody). 6. Having a known active hepatitis B (e.g., hepatitis B surface antigen reactive) or hepatitis C (e.g., hepatitis C virus ribonucleic acid [RNA] [quantitative]) infection. 7. Diagnosed with severe acute respiratory syndrome coronavirus 2 infection, which is confirmed by real-time polymerase chain reaction (PCR) testing according to each facility's guidelines at the time of screening and is positive by PCR within 7 days before the first dose of the test treatment drug. 8. Having a history of clinically severe autoimmune disease or a history of organ transplantation. 9. Having a history of photosensitive skin disorders including retinitis or (but not limited to) erythema multiforme, atopic eczema, psoriasis, viral exanthema, pemphigus, and dermatitis herpetiformis. 10. Having a known additional malignancy that has progressed or required active treatment within the past 5 years. Exceptions include basal cell carcinoma or squamous cell carcinoma of the skin, superficial bladder cancer, or cervical intraepithelial neoplasia that has received curative treatment. Subjects with other malignancies are eligible if the subject has been cured by surgery alone or surgery and radiotherapy and has remained disease-free for at least 5 years. 11. Known active central nervous system metastases and / or carcinomatous meningitis. Subjects with previously treated brain metastases may participate provided that the subject is stable (no evidence of disease progression by imaging for at least 4 years prior to the first dose of study treatment, and any neurological symptoms have returned to baseline), has no evidence of new or expanding brain metastases, and has 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. 12.Interstitial lung disease, history of pneumonia within 12 months prior to screening, or current pneumonia. 13. Active infection requiring systemic therapy. 14. Has history or current evidence of any condition, treatment, or laboratory abnormality that, in the opinion of the Treatment Investigator, may confound the results of the study, prevent the subject's participation for the entire duration of the study, or make the subject's participation in the study inadvisable. 15. Has clinically significant cardiovascular disease, e.g., unstable angina, myocardial infarction, or acute coronary syndrome, symptomatic or uncontrolled arrhythmias, congestive heart failure, baseline electrocardiogram abnormalities (ECG) including, but not limited to, QTc prolongation to greater than 470 ms, or any class III or IV cardiac disease as defined by the New York Heart Association Functional Classification. 16. Has an overt or subclinical disorder of the exocrine pancreas (e.g., acute or chronic pancreatitis of any etiology) or a chronic (including autoimmune) gastrointestinal disorder, e.g., Crohn's disease, ulcerative colitis, rheumatoid arthritis, lupus erythematosus, scleroderma, Sjogren's syndrome, and polyarteritis nodosa. 17. Have a known psychiatric or substance abuse disorder that would prevent them from providing informed consent or cooperating with the requirements of the study. 18. Pregnant or lactating, or expecting to conceive, within the planned duration of the study, from the screening visit through 90 days after the last dose of study drug. 19.A first-degree relative of the study investigator, staff, or sponsor.

[0280] (6) Test drug, dosage, and administration method

[0412] HC-7366 potassium salt monohydrate capsules.

[0281]

[0413] Dosage levels: 10, 20, 40, 75, 125, and 150 mg QD.

[0282]

[0414] Administration route: Oral, using water on an empty stomach.

[0283] (7) Duration of subject participation in the study

[0415] Each subject was treated for up to 2 years and followed up for up to 2 years.

[0284]

[0416] Subjects continued administration until the first occurrence of any of the following: unacceptable toxicity, disease progression documented by iRECIST, discontinuation of treatment for reasons permitted by other protocols (e.g., subject request), any other administrative reason, or after 2 years of treatment. Treatment of subjects beyond 2 years was at the discretion of the physician and the study sponsor in consultation with the subject.

[0285] (8) Endpoints Primary endpoints:

[0417] The primary endpoints of this study are as follows: · Determination of MTD and RP2D. · Safety and tolerability 〇 Incidence of DLT 〇 Number and severity of adverse events (TEAE) and treatment-related TEAE occurring under treatment according to NCI-CTCAE v5.0. 〇 Incidence of TEAE leading to early discontinuation. 〇 Incidence of abnormal clinical test values based on hematological grades of NCI-CTCAE, blood biochemistry tests, and urine test results. 〇 Incidence of abnormalities observed in 12 induction ECG parameters. 〇 Incidence of abnormalities observed in vital sign measurements.

[0286] Secondary endpoints:

[0418] Secondary PK endpoints for this study may include, as appropriate, the following: · Area under the plasma concentration-time curve from time zero to the end. · Measurable concentration (AUC 0-last ). · Area under the plasma concentration-time curve from time zero to 24 hours after dosing (AUC 0-24 ). · Area under the plasma concentration-time curve extrapolated from time zero to infinite time (AUC 0-∞ ). · Area under the plasma concentration-time curve over the dosing interval (AUC 0-t ). · Maximum observed plasma concentration (C max ). · Time of maximum observed plasma concentration (t max ). · Apparent total clearance (CL / F). · Apparent volume of distribution during the terminal phase (V z / F). · Apparent terminal phase elimination half-life (t 1 / 2 ). · Cumulative ratio based on AUC 0-t (AR AUC ). · Linearity ratio (LR).

[0287]

[0419] The secondary efficacy endpoints for this study are as follows: · Overall response rate (ORR). · Duration of response (DOR). · Time to treatment failure (TTF). · Progression-free survival (PFS). · Overall survival (OS).

[0288]

[0420] Note: The ORR, DOR, and PFS were assessed using both the RECIST v1.1 and iRECIST criteria.

[0289] Exploratory endpoints: · PD markers: Levels of ctDNA and CTC. Explore molecular analysis using whole exome sequencing or RNA sequencing. · Immunophenotyping in blood samples (serum and cellular markers, RNA, cytokines) including markers of stress and immune activation. · Local antitumor effects (microscopic lesions, apoptosis / necrosis) and microenvironment changes in tumor biopsies. · CYP3A inducibility of HC-7366, ratio of 4β-hydroxy cholesterol to total cholesterol at C1D1 and C1D21 as endogenous markers of CYP3A induction.

[0290] Statistical methods:

[0421] Since this is an exploratory study to characterize the MTD, RP2D, safety / tolerability, and preliminary PD, formal hypothesis testing will not be performed. Descriptive statistics of the parameters of interest will be presented for each dose level. Safety data will be presented in tabular form by whole organ class and for each cohort dose level by preferred term, severity, and frequency of events.

[0291] Determination of sample size:

[0422] The maximum sample size for Part 1a of the study was 36 subjects. Fewer than 6 subjects were treated at each dose level. The sample size for Phase 1b was 30 subjects, and 15 subjects in each of 2 dose cohorts were selected for expansion.

[0292] Pharmacokinetics:

[0423] Non-compartmental PK analysis was performed using a software platform validated for individual plasma concentration data, e.g., Phoenix WinNonlin. HC-7366 plasma concentrations and PK parameters were listed and summarized using descriptive statistics. Dose proportionality can be assessed graphically as appropriate, in conjunction with a power model.

[0293] Pharmacodynamics:

[0424] Pharmacodynamic biomarker data, such as integrated stress response activation markers and immune-related changes, were listed and summarized as appropriate using descriptive statistics. Figures showing the relationships between PD, PK, and clinical efficacy data were created as appropriate.

[0294] General considerations:

[0425] Descriptive statistics and tabular / graphical presentations were performed and presented for each dose level (cohort). Statistics included, but were not limited to, counts, percentages, rates, means, medians, ranges, and variances, and all statistical analyses were exploratory.

[0295]

[0426] The demographic and baseline characteristics of the subjects, including age, sex, race, ethnicity, weight, baseline disease diagnosis, and medical status, were summarized using descriptive statistics for each dose level.

[0296] Safety:

[0427] Safety parameters were listed and summarized using descriptive statistics. Safety variables included the incidence of TEAE, laboratory data, vital signs, 12-lead ECG results, and physical examination findings. All safety analyses were based on the safety analysis population.

[0297] Efficacy:

[0428] The efficacy parameters are ORR, DOR, TTF, PFS, and OS. ORR is defined as the number of subjects with a confirmed response of complete response (CR) or partial response (PR) divided by the total number of treated subjects with measurable disease at baseline. DOR is defined as the time from the first observation of PR or CR to the time of radiologically documented progression. Tumor response status is defined according to RECIST v1.1 and iRECIST. To conduct an assessment of antitumor activity, the best overall response and ORR are tabulated by overall frequency distribution. The median DOR is summarized using the Kaplan-Meier method for subjects with a confirmed response; PFS is summarized in the same manner. A listing of individual tumor measurements, tumor burden, and % change in tumor burden is provided. The change in tumor burden is presented graphically by a waterfall plot. The analysis of TTF, PFS, and OS is the same as that of DOR.

[0298] Interim analysis:

[0429] Dose-limiting toxicity is reviewed by the SMC when the planned number of subjects has completed its DLT observation period using the dose-escalation rule. The SMC reviews the available clinical, PK, and / or PD data as appropriate.

[0299]

[0430] Tumor biopsies collected from colorectal and head and neck cancer trial patients at the time of screening and in cycle 3 were analyzed by IHC for HIF1α expression. Strong inhibition of HIF1α expression was observed in patients at the lowest starting doses of 10 mg and 20 mg in cycle 3 (Figures 59 and 60).

[0300]

[0431] Multiplex immunohistochemistry (IHC) and imaging of patient biopsies: Slides were deparaffinized, rehydrated, and heat-mediated antigen retrieval was performed using AR9 buffer (Akoya). Slides were blocked with Roche diagnostics antibody diluent (Fisher) and stained with primary antibodies at 110 rpm for 1 hour. HRP-conjugated secondary antibody was added at 110 rpm for 10 minutes, followed by addition of OPAL detection dyes (Akoya) for 10 minutes. For multiplex staining, this process was repeated starting at the antigen retrieval step. At the completion of the multiplex staining rounds, slides were counterstained with DAPI and coverslipped using Vectashield vibrance mounting media (Vector H-1700). Multispectral images were captured on a Polaris imaging system (Akoya), and spectral deconvolution and cell segmentation were performed using Phenochart and Inform Tissue Finder software (Akoya). Imaging data were converted to.csv files and imported into Flowjo for immunophenotyping. The primary antibodies used were rabbit anti-human HIF1α (polyclonal, Novus NB100-122, paired with Opal690) and rabbit anti-human HIF2α (polyclonal, Novus NB100-479, paired with Opal570). The secondary antibody used was HRP-goat anti-rabbit (Jackson Immunoresearch 111-035-144).

[0301] Incorporation by reference

[0432] This application refers to various issued patents, published patent applications, academic papers, and / or other publications, all of which are incorporated herein by reference. In the event of any conflict between any of the incorporated references and this specification, this specification shall prevail. Additionally, any particular embodiment of the present disclosure that falls within the scope of the prior art may be explicitly excluded from any one or more of the claims. Such embodiments are expected to be known to those skilled in the art, and thus they may be excluded even if not explicitly stated as such in this specification. Any particular embodiment of the present disclosure may be excluded from any claim for any reason, whether or not related to the existence of the prior art.

[0302] equivalent

[0433] The present invention may be embodied in other specific forms without departing from its gist or essential characteristics. Accordingly, the above-described embodiments should be considered illustrative rather than restrictive of the invention described herein in every respect. Therefore, the scope of the present invention is indicated by the appended claims rather than by the above detailed description, and it is intended that all modifications falling within the meaning and scope of the equivalence of the claims be included therein.

Claims

1. The compound of formula (I) in an effective amount 【Chemistry 1】 A pharmaceutical composition for use in a method of treating an advanced solid tumor in a subject requiring treatment for an advanced solid tumor, or a method of treating a hematological malignancy in a subject requiring treatment for a hematological malignancy, wherein the method comprises administering the pharmaceutical composition to the subject.

2. Administering the pharmaceutical composition activates the integrated stress response pathway (ISR) in the advanced solid tumor or hematological malignancy, selectively. The aforementioned ISR activation is GCN2-dependent. The pharmaceutical composition according to claim 1.

3. Administering the aforementioned pharmaceutical composition means i. Inducing the expression of ASNS, PSAT1, PHGDH, and / or PUMA in the advanced solid tumor or hematological malignancy; ii. To reduce the protein levels of S100A8 / A9, HIF1α, HIF2α, and / or GLUT1 in the advanced solid tumor or hematological malignancy; iii. To reduce mitochondrial respiration and / or glycolysis in the aforementioned advanced solid tumors or hematological malignancies; iv. Decreases the myeloid-specific progenitor cells and mature myeloid cells in the subject; v. Altering metabolites involved in amino acid metabolism, oxidative stress, the urea cycle, and / or pyrimidine biosynthesis in the aforementioned advanced solid tumors or hematological malignancies; vi. To reduce the proteins involved in oxidative phosphorylation in the advanced solid tumors or hematological malignancies; vii. To reduce the activity of HIF and / or E2F1 drive transcription in the advanced solid tumor or hematological malignancy; and / or, viiii. To increase ATF4 and / or JUN transcriptional activity in the aforementioned advanced solid tumors or hematological malignancies, The pharmaceutical composition according to claim 1.

4. A subject is administered 10 mg to 150 mg of the compound, or a pharmaceutically acceptable salt thereof, on a free acid weight basis, and optionally, It is administered orally and / or once daily, and optionally, It is administered once daily for 21 consecutive days, and is optional. It is administered approximately one hour before or two hours after a meal. The pharmaceutical composition according to claim 1.

5. The pharmaceutical composition according to claim 1, wherein the subject is in a fasted state.

6. The pharmaceutical composition according to claim 1, wherein the subject has been administered at least one and five or fewer prior treatment lines in advance.

7. The aforementioned pharmaceutically acceptable salt is a potassium salt, which can be optionally selected. It is a potassium salt hydrate or monohydrate. The pharmaceutical composition according to claim 1.

8. The method further comprises administering an effective amount of a second therapeutic agent to the subject, optionally, The second therapeutic agent is selected from the group consisting of immune checkpoint inhibitors, EGFR inhibitors, anti-angiogenic agents, venetoclax, fluorouracil, and combinations thereof, or The second therapeutic agent is selected from the group consisting of anti-VEGFR antibodies, fluorouracil, PI3Kα inhibitors, MEK1 / 2 inhibitors, and hypoxia-inducible factor (HIF) inhibitors, and is optionally selected to be an anti-VEGFR antibody, an HIF inhibitor, a PI3Kα inhibitor, a MEK1 / 2 inhibitor, or an EGFR inhibitor. The pharmaceutical composition according to claim 1.

9. The pharmaceutical composition according to claim 8, wherein the second therapeutic agent is berzutifan, 5-fluorouracil, alpelisib, trametinib, osimertinib, dacomitinib, or venetoclax.

10. The second therapeutic agent is venetoclax, and optionally, Administering the aforementioned pharmaceutical composition and venetoclax activates the integrated stress response pathway (ISR) in the advanced solid tumor or hematological malignancy to a greater extent than the pharmaceutical composition or venetoclax administered alone. The pharmaceutical composition according to claim 9.

11. The aforementioned subjects are humans, and are selected arbitrarily. Adult human, The pharmaceutical composition according to claim 1.

12. The pharmaceutical composition according to claim 1, wherein the advanced solid tumor is selected from the group consisting of head and neck squamous cell carcinoma, colorectal cancer, non-small cell lung cancer, renal cell carcinoma, and bladder transitional cell carcinoma.

13. The blood cancer is leukemia, and optionally, It is acute myeloid leukemia, and it is performed randomly. The aforementioned hematological cancer is resistant to B-cell lymphoma inhibitors. The pharmaceutical composition according to claim 1.

14. The pharmaceutical composition according to claim 1 to 11 or 13, wherein the hematological cancer is resistant to venetoclax.

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