Methods of increasing immune cell activation and / or treating cancer using dibenzoxazepines

Abstract

The present invention relates to a method of increasing immune cell activation and / or treating cancer in a subject comprising administering to the subject an effective amount of a compound of formula (I): HNOR1ONOR2O # imgabs0 # or a pharmaceutically acceptable salt thereof: HNOR1ONOR2O # imgabs0 #

Description

Technical Field

[0001] The present invention relates to methods and pharmaceutical compositions for increasing immune cell activation, and methods for treating cancer. Background Art

[0002] Cancer is one of the leading causes of death globally. Although progress has been made in the development of cancer therapies, there is still a great need for improved therapies, such as for treating cancers in which tumor resistance is a problem with existing therapies or tumors that are unresponsive to existing therapies.

[0003] One approach that has shown promise in cancer treatment is cancer immunotherapy. Cancer immunotherapy is a form of treatment that involves modulating a patient's immune system to treat cancer. In cancer immunotherapy, antibodies targeting programmed cell death protein-1 (PD-1) have shown particular promise. PD-1 and its ligand PD-L1 are checkpoint regulators that inhibit the immune response of the body against cancer cells. PD-L1 is a transmembrane protein expressed by tumor cells as well as hematopoietic and non-hematopoietic cells. PD-1 is a co-inhibitory receptor mainly expressed by T cells, and is also expressed by B cells, NK cells, and certain myeloid cells. PD-1 encoded by the pdcd1 gene binds to PD-L1 on the tumor surface and prevents cytolysis by immune cells. PD-1 has also been shown to be expressed by tumor cells, including human melanoma cell lines (Kleffel et al., 2015; Li et al., 2019).

[0004] Numerous immune checkpoint inhibitors targeting PD-1 and PD-L1 have been developed, and they have shown promise in treating a variety of malignancies. For example, antibodies that have been approved and / or are in clinical trials for treating various cancers include the anti-PD-1 antibody nivolumab (for metastatic melanoma), pembrolizumab (for treating metastatic melanoma, lymphoma, mesothelioma, and non-small cell lung cancer), anti-PD-L1 antibodies, which include avelumab (for urothelial carcinoma, Merkel cell carcinoma, renal cell carcinoma) and atezolizumab (for urothelial carcinoma, non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), small cell lung cancer (SCLC), and hepatocellular carcinoma (HCC)).

[0005] Although antibodies against PD-1 have shown promising results in the treatment of cancer, the use of antibody-based therapeutic agents has limitations. For example, among melanoma patients who have an initial response to PD-1 inhibitors, 40% of patients will experience progression of existing or new lesions within 3 years. In metastatic melanoma, the long-term survival rate (>3 years) of anti-CTLA-4 is only about 20%, and when combined with a PD-1 antibody, the long-term survival rate (>3 years) is about 30-50%. KEYNOTE-001 showed that the 5-year overall survival rate of advanced melanoma patients treated with anti-PD-1 (pembrolizumab) was 34% among all patients, compared with 41% in untreated patients, and treatment-related adverse events were recorded in 86% of patients. The combination of anti-PD-1 (nivolumab) and anti-CTLA-4 (ipilimumab) increased the response rate to 53%, but serious treatment-related adverse events (≥grade 3) were recorded in 53% of patients, and 21% of patients discontinued treatment. Notably, recent studies have found that 24% of cancer patients receiving immunotherapy develop venous thromboembolism and have a reduced overall survival rate.

[0006] Although safer and more effective immune checkpoint therapies are needed, small molecules offer potential advantages over antibodies, such as favorable pharmacokinetics and drugability, and are suitable for oral formulation and outpatient delivery. This potentially avoids intravenous (i.v.) administration and related risks, as well as greater patient convenience, especially in frail patients. Small molecules are generally cheaper and more stable to produce than antibodies. Although the PD-1 / PD-L1 system is now the main target in immunotherapy, there are no clinically approved small molecule inhibitors of the PD-1 / PD-L1 system.

[0007] Inhibitors of BRAF and MEK have also shown promise in the treatment of cancer. For example, first-line treatment of metastatic melanoma patients with dabrafenib and trametinib results in patients with BRAF V600E or V600KOne-third of patients with mutant melanoma achieve a 5-year survival. However, 30% of patients experience higher grade 3 / 4 toxicities, which often lead to dose reduction and treatment delays. Importantly, resistance can develop after 9-12 months, which may be due to the activation of other signaling pathways or modulation of the immune system. Increased downstream ERK signaling is a resistance mechanism to BRAF / MEK inhibition and has led to various preclinical and clinical initiatives targeting ERK. Additionally, recent studies in mice bearing non-small cell lung cancer have shown that the ERK inhibitor (PD0325901) can enhance the efficacy of anti-PD-1 antibodies. ASN007 is another ERK1 / 2 kinase inhibitor that has shown efficacy in resistant melanoma PDX models. However, among the 62 small molecule therapeutic agents approved by the FDA that target more than 20 different protein kinases, 8 were approved in 2020, and although several are in clinical studies (e.g., ulixertinib NCT03698994, National Cancer Institute; LY3214996 NCT02857270, Eli Lilly; BVD-523 NCT03417739, BioMed Valley Discoveries; LTT462 NCT02711345, Novartis; MK-8353 NCT02972034, Merck), none are ERK inhibitors.

[0008] By combining immune checkpoint inhibitors with targeted therapies, cancer patients can benefit from targeted therapies at least in the short term, while immunotherapy can provide a more durable response.

[0009] What is needed is a non-antibody alternative for treating cancer that targets the immune checkpoint inhibitor system and ERK signaling. SUMMARY OF THE INVENTION

[0010] The inventors have discovered that the compound BT2 (compound of formula (II)) acts both as an immune checkpoint inhibitor and as a targeted inhibitor of ERK signaling. In this regard, the inventors have found that BT2:

[0011] (i) inhibits ERK phosphorylation and increases the expression of JUN (also known as c-Jun) in tumor cells; and

[0012] (ii) reduces the expression of PD-1, increases the expression of JUN, increases the phosphorylation of JNK, and interacts with CD28 in T cells.

[0013] Accordingly, the inventors have inferred that BT2 will be beneficial for increasing immune cell activation and treating cancer through its combined activities as an immune checkpoint inhibitor, as an ERK phosphorylation inhibitor, and as a JUN expression promoter.

[0014] A first aspect provides a method for increasing immune cell activation and / or treating cancer in a subject, which comprises administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0015]

[0016] Wherein:

[0017] R 1 is a straight-chain or branched C1-C6 alkyl; and

[0018] R 2 is a straight-chain or branched C1-C6 alkyl,

[0019] or R 2 is

[0020]

[0021] where q is 1, 2, 3 or 4; and R 3 is a straight-chain or branched C1-C6 alkyl.

[0022] An alternative first aspect provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for increasing immune cell activation and / or treating cancer in a subject; or the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for increasing immune cell activation and / or treating cancer in a subject.

[0023] A second aspect provides a method for increasing immune cell activation and / or treating cancer in a subject, which comprises administering an effective amount of a compound of formula (II) or a pharmaceutically acceptable salt thereof:

[0024]

[0025] Formula (II).

[0026] An alternative second aspect provides a compound of formula (II) or a pharmaceutically acceptable salt thereof for increasing immune cell activation and / or treating cancer in a subject; or the use of a compound of formula (II) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for increasing immune cell activation and / or treating cancer in a subject.

[0027] A third aspect provides a method for reducing ERK phosphorylation and / or increasing JUN expression in tumor cells of a subject, and / or reducing PD-1 expression and / or increasing JUN expression and / or increasing JNK phosphorylation in T cells of a subject, which comprises administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0028] An alternative third aspect provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for reducing ERK phosphorylation and / or increasing JUN expression in tumor cells of a subject, and / or for reducing PD-1 expression and / or increasing JUN expression and / or increasing JNK phosphorylation in T cells of a subject; or the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for reducing ERK phosphorylation and / or increasing JUN expression in tumor cells of a subject, and / or for reducing PD-1 expression and / or increasing JUN expression and / or increasing JNK phosphorylation in T cells of a subject.

[0029] A fourth aspect provides a method for reducing ERK phosphorylation and / or increasing JUN expression in tumor cells of a subject, and / or for reducing PD-1 expression and / or increasing JUN expression and / or increasing JNK phosphorylation in T cells of a subject, which comprises administering an effective amount of a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0030] An alternative fourth aspect provides a compound of formula (II) or a pharmaceutically acceptable salt thereof for reducing ERK phosphorylation and / or increasing JUN expression in tumor cells of a subject, and / or for reducing PD-1 expression and / or increasing JUN expression and / or increasing JNK phosphorylation in T cells of a subject; or the use of a compound of formula (II) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for reducing ERK phosphorylation and / or increasing JUN expression in tumor cells of a subject, and / or for reducing PD-1 expression and / or increasing JUN expression and / or increasing JNK phosphorylation in T cells of a subject.

[0031] A fifth aspect provides a method for reducing ERK phosphorylation in tumor cells of a subject and for reducing PD-1 expression in T cells of a subject, which comprises administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0032] An alternative fifth aspect provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for reducing ERK phosphorylation in tumor cells of a subject and for reducing PD-1 expression in T cells of a subject; or the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for reducing ERK phosphorylation in tumor cells of a subject and for reducing PD-1 expression in T cells of a subject.

[0033] A sixth aspect provides a method for reducing ERK phosphorylation in cancer cells of a subject and for reducing PD-1 expression in T cells of a subject, which comprises administering an effective amount of a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0034] An alternative sixth aspect provides a compound of formula (II) or a pharmaceutically acceptable salt thereof for reducing ERK phosphorylation in tumor cells of a subject and reducing PD-1 expression in T cells of the subject; or the use of a compound of formula (II) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for reducing ERK phosphorylation in tumor cells of a subject and reducing PD-1 expression in T cells of the subject.

[0035] A seventh aspect provides a method of treating a disease or disorder associated with PD-1 expression in T cells of a subject, which comprises administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0036] An alternative seventh aspect provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for treating a disease or disorder associated with PD-1 expression in T cells of a subject; or the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a disease or disorder associated with PD-1 expression in T cells of a subject.

[0037] An eighth aspect provides a method of treating a disease or disorder associated with PD-1 expression in T cells of a subject, which comprises administering an effective amount of a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0038] An alternative eighth aspect provides a compound of formula (II) or a pharmaceutically acceptable salt thereof for treating a disease or disorder associated with PD-1 expression in T cells of a subject; or the use of a compound of formula (II) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a disease or disorder associated with PD-1 expression in T cells of a subject.

[0039] A ninth aspect provides a method of increasing immune cell activation and / or reducing tumor growth rate in a subject suffering from cancer, which comprises administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0040] An alternative ninth aspect provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for increasing immune cell activation and / or reducing tumor growth rate in a subject suffering from cancer; or the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for increasing immune cell activation and / or reducing tumor growth rate in a subject suffering from cancer.

[0041] A tenth aspect provides a method of increasing immune cell activation and / or reducing tumor growth rate in a subject suffering from cancer, which comprises administering an effective amount of a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0042] An alternative tenth aspect provides a compound of formula (II) or a pharmaceutically acceptable salt thereof for increasing immune cell activation and / or reducing tumor growth rate in a subject having cancer; or the use of a compound of formula (II) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for increasing immune cell activation and / or reducing tumor growth rate in a subject having cancer.

[0043] The eleventh aspect provides a method for reducing PD-1 expression in T cells of a subject, which comprises administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0044] An alternative eleventh aspect provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for reducing PD-1 expression in T cells of a subject; or the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for reducing PD-1 expression in T cells of a subject.

[0045] The twelfth aspect provides a method for reducing PD-1 expression in T cells of a subject, which comprises administering an effective amount of a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0046] An alternative twelfth aspect provides a compound of formula (II) or a pharmaceutically acceptable salt thereof for reducing PD-1 expression in T cells of a subject; or the use of a compound of formula (II) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for reducing PD-1 expression in T cells of a subject.

[0047] The thirteenth aspect provides a method for reducing ERK phosphorylation and / or increasing JUN expression in tumor cells, and / or reducing PD-1 expression and / or increasing JUN expression and / or increasing JNK phosphorylation in T cells, which comprises contacting the tumor cells or T cells with an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0048] An alternative thirteenth aspect provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for reducing ERK phosphorylation and / or increasing JUN expression in tumor cells, and / or reducing PD-1 expression and / or increasing JUN expression and / or increasing JNK phosphorylation in T cells; or the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for reducing ERK phosphorylation and / or increasing JUN expression in tumor cells, and / or reducing PD-1 expression and / or increasing JUN expression and / or increasing JNK phosphorylation in T cells.

[0049] The fourteenth aspect provides a method for reducing ERK phosphorylation and / or increasing JUN expression in tumor cells, and / or reducing PD-1 expression and / or increasing JUN expression and / or increasing JNK phosphorylation in T cells, which comprises contacting the tumor cells or T cells with an effective amount of a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0050] An alternative fourteenth aspect provides a compound of formula (II) or a pharmaceutically acceptable salt thereof for reducing ERK phosphorylation and / or increasing JUN expression in tumor cells, and / or reducing PD-1 expression and / or increasing JUN expression and / or increasing JNK phosphorylation in T cells; or the use of a compound of formula (II) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for reducing ERK phosphorylation and / or increasing JUN expression in tumor cells, and / or reducing PD-1 expression and / or increasing JUN expression and / or increasing JNK phosphorylation in T cells.

[0051] The fifteenth aspect provides a method for reducing the expression of the genes listed in Table 2 and / or increasing the expression of the genes listed in Table 3 in T cells, which comprises contacting the T cells with an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0052] An alternative fifteenth aspect provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for reducing the expression of the genes listed in Table 2 and / or increasing the expression of the genes listed in Table 3 in T cells; or the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for reducing the expression of the genes listed in Table 2 and / or increasing the expression of the genes listed in Table 3 in T cells.

[0053] The sixteenth aspect provides a method for reducing the expression of the genes listed in Table 2 and / or increasing the expression of the genes listed in Table 3 in T cells, which comprises contacting the T cells with an effective amount of a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0054] An alternative sixteenth aspect provides a compound of formula (II) or a pharmaceutically acceptable salt thereof for reducing the expression of the genes listed in Table 2 and / or increasing the expression of the genes listed in Table 3 in T cells; or the use of a compound of formula (II) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for reducing the expression of the genes listed in Table 2 and / or increasing the expression of the genes listed in Table 3 in T cells.

[0055] The seventeenth aspect provides a kit for reducing ERK phosphorylation and / or increasing JUN expression in tumor cells, and / or reducing PD-1 expression and / or increasing JUN expression and / or increasing JNK phosphorylation in T cells, the kit comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0056] The eighteenth aspect provides a kit for reducing ERK phosphorylation and / or increasing JUN expression in tumor cells, and / or reducing PD-1 expression and / or increasing JUN expression and / or increasing JNK phosphorylation in T cells, the kit comprising a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0057] The nineteenth aspect provides a kit for reducing inflammation in a tumor and / or reducing PD-1 expression and / or increasing JUN expression and / or increasing JNK phosphorylation in T cells and / or increasing JUN expression in the tumor cells of a subject, the kit comprising a compound of formula (II) or a pharmaceutically acceptable salt thereof. Description of the Drawings

[0058] Preferred embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0059] Figure 1 Images (A, C) of Western blots performed with extracts from A375 (A, B) and MeWo (C, D) melanoma cells are shown. The melanoma cells were incubated with 10 or 100 nM BT2 (Table 1) or SCH772984 for 24 hours, incubated with an antibody against p-ERK or total ERK, and then incubated with a secondary antibody. The approximate positions of the molecular weight markers are shown. SCH represents SCH772984. The graphs in (B, D) show the band intensities of the Western blots quantified using Image J. The data plotted represent the mean ± SEM. The data represent 2 biologically independent experiments.

[0060] Figure 2 Shows the quantification of migration into the cell-free area (A, C) and invasion into the cell-free area (B, D) of A375 cells (A, B) and MeWo cells (C, D) after incubation with 1 μM BT2 or SCH772984 for 24 hours and 48 hours, respectively. The data plotted represent the mean ± SEM of the means of 3 - 4 biologically independent experiments. Statistical significance was assessed by one-way ANOVA or Kruskal Wallis test as appropriate. Are morphological images of A375 and MeWo cells after exposure to 1 μM vehicle (Veh), BT2, or SCH772984 for 24 hours.

[0061] Figure 3 Are morphological images of A375 and MeWo cells after exposure to 1 μM vehicle (Veh), BT2, or SCH772984 for 24 hours.

[0062] Figure 4Is a graph showing the results of apoptosis assays performed using (A) A375 cells and (B) MeWo cells, which were exposed to 1 μM BT2 or SCH772984 for 24 hours. The data plotted (Annexin V-FITC + PI - ) represent the mean ± SEM of the means of 3 biologically independent experiments. Statistical significance was evaluated by one-way ANOVA.

[0063] Figure 5 Shows the results of treatment of C.B.17 SCID mice bearing subcutaneous melanoma (MDA-MB-435) with BT2 (20 mg / kg intratumorally) or vehicle, with a treatment schedule of once daily, 5 days on / 2 days off. Treatment began on day 16. (A) is a schematic diagram showing the treatment schedule. (B) is a graph showing tumor growth (volume), and (C) is a graph showing body weight data from an animal study with the same vehicle group as described in Li et al. (Li et al., 2019). Data are represented as mean ± SEM. n = 10 mice / group. Statistical significance was evaluated by t-test.

[0064] Figure 6 Is a graph showing the quantification of immunohistochemical analysis of MDA-MB-435 tumors from mice treated with BT2 or vehicle using antibodies against (A, B) p-ERK or (C, D) total ERK. Integrated optical density (IOD) and tissue area were quantified using Image-Pro Plus. Data are represented as mean ± SEM of n = 9 - 10 mice per group. Statistical significance was evaluated by Mann-Whitney test.

[0065] Figure 7 Shows the results of the effect of treatment of C.B.17 SCID mice bearing subcutaneous MDA-MB-435 tumors with BT2 200 mg / kg or vehicle intraperitoneally (i.p.) on tumor volume and body weight, with a treatment schedule of once daily, 5 days on / 2 days off. Treatment began on day 16. (A) is a schematic diagram of the treatment schedule. (B) is a graph showing tumor growth in animals treated with BT2 and vehicle, and (C) is a graph showing body weight in animals treated with vehicle and BT2. Body weight data are from an animal study described in Li et al. (Li et al., 2019) with the same vehicle group. Data are represented as mean ± SEM. n = 10 mice / group.

[0066] Figure 8Shows the effect of twice-weekly treatment with anti-mouse PD-1 monoclonal antibody or control IgG (100 μg, intraperitoneally) on tumor volume and body weight in C57BL / 6J mice bearing subcutaneous B16F10 melanoma. Treatment started on day 7. (A) is a schematic of the treatment protocol. (B) is a graph showing the change in tumor volume over time in animals treated with BT2 or vehicle. (C) shows the measured body weight over time. Data are represented as mean ± SEM. n = 6 mice / group. Statistical significance was evaluated by Mann-Whitney or t-test as appropriate.

[0067] Figure 9 Shows the effect of once-daily treatment with BT2 or vehicle (200 mg / kg or 20 ml / kg, respectively, intraperitoneally) on tumor volume and body weight in C57BL / 6J mice bearing subcutaneous B16F10 tumors on a schedule of 5 days of dosing / 2 days of rest. Treatment started on day 5. (A) is a schematic of the treatment protocol. (B) is a graph showing the change in tumor volume over time in animals treated with BT2 or vehicle. (C) also shows the measured body weight over time. Data are represented as mean ± SEM. n = 8 mice / group. Statistical significance was evaluated by Mann-Whitney or t-test as appropriate.

[0068] Figure 10 Are graphs showing (A) tumor size and (B) isolated tumor weight on day 17 from mice treated with BT2 or vehicle, respectively. Data are represented as mean ± SEM. n = 8 mice / group. Statistical significance was evaluated by t-test.

[0069] Figure 11 Is a graph showing the Kaplan-Meier analysis of survival conducted at a tumor size limit of 500 mm as described previously (Haynes et al., 2018), n = 8 mice / group. Statistical significance was evaluated by log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test. 3

[0070] Figure 12 Is a graph showing the quantification of immunohistochemical analysis of B16F10 tumors (day 17) from mice treated with BT2 or vehicle using antibodies against (A) p-ERK or (B) total ERK. IOD and tissue area were quantified using Image-Pro Plus. Data are represented as mean ± SEM for n = 8 mice per group. Statistical significance was evaluated by Mann-Whitney test.

[0071] Figure 13It is a graph showing the quantification of immunohistochemical analysis of B16F10 tumors (day 17) from BT2- or vehicle-treated mice using an antibody against CD68. IOD and tissue area were evaluated using Image-Pro Plus, and IOD / μm was determined. 2 Data are presented as mean ± SEM of mean / animal. n = 8 per group. Statistical significance was evaluated by t-test.

[0072] Figure 14 It is a graph showing the quantification of immunohistochemical analysis of B16F10 tumors (day 17) from BT2- or vehicle-treated mice using an antibody against CD3. (A) IOD and tissue area were evaluated using Image-Pro Plus, and IOD / μm was determined. 2 (B) The number of CD3-positive cells and total cells was quantified using Image-Pro Plus, and CD3 + cell % was determined. Data are presented as mean ± SEM of mean / animal. n = 7 - 8 per group. Statistical significance was evaluated by Mann-Whitney test.

[0073] Figure 15 It is a graph showing serum IFN-γ levels in vehicle- or BT2-treated mice determined by ELISA. n = 8 per group. Statistical significance was evaluated by t-test. Data are presented as mean ± SEM.

[0074] Figure 16 It is a graph showing the quantification of immunohistochemical analysis of B16F10 tumors (day 17) from BT2- or vehicle-treated mice using antibodies against (A) PD-1 and (B) PD-L1. IOD and tissue area were evaluated using Image-Pro Plus, and IOD / μm was determined. 2 Data are presented as mean ± SEM of mean / animal. n = 7 - 8 per group. Statistical significance was evaluated by Mann-Whitney test.

[0075] Figure 17 It is a graph showing the results of flow cytometry with: (A) a PD-1 antibody (or IgG) and Jurkat T cells treated with the indicated concentration of BT2 or vehicle for 48 h; (B) a PD-1 antibody (or IgG) and Jurkat T cells treated with vehicle or the indicated concentration of BT3 for 48 h; and (C) a PD-1 antibody (or IgG) and Jurkat T cells treated with 3 μM BT2 or vehicle for the indicated time. Statistical significance was evaluated by one-way ANOVA. Data are presented as mean ± SEM of the mean of 3 biologically independent experiments.

[0076] Figure 18 Shows the images of Western blots performed using extracts of Jurkat T cells treated with BT2 or SCH772984 at various concentrations for 24 hours. The membranes were incubated with PD-1 or β-actin antibodies and then with secondary antibodies. (B) is a graph showing the band intensities of the Western blots in (A) quantified using Image J, and the data plotted represent the mean ± SEM. The data represent 2 biologically independent experiments. The approximate positions of the molecular weight markers are shown.

[0077] Figure 19 Shows the images of Western blots performed using extracts of Jurkat T cells treated with BT2 or PD98059 at various concentrations for 24 hours. The membranes were incubated with PD-1 or β-actin antibodies and then with secondary antibodies. (B) is a graph showing the band intensities of the Western blots quantified using Image J, and the data plotted represent the mean ± SEM. The data represent 2 biologically independent experiments. The approximate positions of the molecular weight markers are shown.

[0078] Figure 20 Shows the images of Western blots performed using extracts of Jurkat T cells incubated with BT2 or PD98059 for 24 hours. The membranes were incubated with DUSP8 antibody and then with secondary antibodies. (B) is a graph showing the band intensities of the Western blots quantified using Image J, and the data plotted represent the mean ± SEM. The data represent 2 biologically independent experiments. The approximate positions of the molecular weight markers are shown.

[0079] Figure 21 Shows the images of Western blots performed using extracts of Jurkat T cells incubated with BT2 or PD98059 for 24 hours. The membranes were incubated with c-MAF antibody and then with secondary antibodies. (B) is a graph showing the band intensities of the Western blots quantified using Image J, and the data plotted represent the mean ± SEM. The data represent 2 biologically independent experiments. The approximate positions of the molecular weight markers are shown.

[0080] Figure 22 Shows the images of Western blots performed using extracts of Jurkat T cells incubated with BT2 for different times. The membranes were incubated with the indicated antibodies and then with secondary antibodies. (B) is a graph showing the band intensities of the Western blots quantified using Image J, and the data plotted represent the mean ± SEM. The data represent 2 biologically independent experiments. The approximate positions of the molecular weight markers are shown.

[0081] Figure 23Shows images of Western blots performed with extracts of (A) A375, (B) MDA-MB-435, and (C) MeWo cells incubated with the indicated amount of BT2 or SCH772984 for 24 hours. The membranes were incubated with JUN or β-actin antibodies and then with secondary antibodies. Below each Western blot is a graph showing the intensity of the Western blot bands quantified using Image J, and the data plotted represent the mean ± SEM. The data represent 2 biologically independent experiments. The approximate positions of the molecular weight markers are shown.

[0082] Figure 24 Is a sensorgram showing the binding of BT2 to CD28 in the concentration range of 0.156 - 15 μM of BT2. Measurements were performed on a Biacore T200 at 12 °C in 20 mM HEPES, 150 mM NaCl, 5% DMSO (pH 7.5).

[0083] Figure 25 Shows images of (A) Western blots in which vehicle, BT2 (3 nmol), or a mixture of BT2 (3 nmol) and CD28 (3 nmol) or EGF (3 nmol) were pre-incubated in growth medium at 37 °C for 30 minutes and then added to Jurkat T cells in a 12-well plate (final concentration of BT2, CD28, or EGF was 3 μM). After 24 hours, total cell lysates were prepared in RIPA buffer. The membranes were incubated with the indicated primary antibodies and then with secondary antibodies. (B) is a graph showing the intensity of the Western blot bands quantified using Image J, and the data plotted represent the mean ± SEM. The data represent 2 biologically independent experiments. The approximate positions of the molecular weight markers are shown. Detailed Description

[0084] Compound BT2 is a dibenzoxapinone that has previously been shown to inhibit endothelial cell proliferation and migration, angiogenesis, and wound repair. BT2 has been shown to inhibit ERK phosphorylation in endothelial cells as well as the expression of FosB / ΔFosB and VCAM-1 and VEGF, among others.

[0085] As described in the Examples, the inventors have now found that BT2 (Compound of Formula (II)):

[0086] (a) Reduces ERK phosphorylation in cancer cells;

[0087] (b) Reduces tumor cell migration;

[0088] (c) Reduces tumor cell invasion;

[0089] (d) Increases apoptosis of cancer cells;

[0090] (e) Reducing the tumor growth rate;

[0091] (f) Reducing the tumor volume;

[0092] (g) Binding to CD28 of T cells;

[0093] (h) Reducing the expression of PD-1 in T cells;

[0094] (i) Increasing the expression of JUN in T cells;

[0095] (j) Increasing the expression of JUN in tumor cells;

[0096] (k) Increasing the phosphorylation of JNK in T cells;

[0097] (l) Inhibiting tumor inflammation;

[0098] (m) Increasing tumor immunity.

[0099] Accordingly, the inventors contemplate that the compounds of formulas (I) and (II) will effectively increase immune cell activation, reduce tumor inflammation, and treat cancer.

[0100] One aspect provides a method for increasing immune cell activation and / or treating cancer in a subject, which comprises administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0101]

[0102] Wherein:

[0103] R 1 is a straight-chain or branched C1-C6 alkyl; and

[0104] R 2 is a straight-chain or branched C1-C6 alkyl,

[0105] or R 2 is

[0106]

[0107] where q is 1, 2, 3 or 4; and R 3 is a straight-chain or branched C1-C6 alkyl.

[0108] In one embodiment, the compound of formula (I) has the structure of formula (II):

[0109]

[0110] One aspect provides a method for increasing immune cell activation and treating cancer in a subject, which comprises administering an effective amount of a compound of formula (I), generally formula (II), or a pharmaceutically acceptable salt thereof.

[0111] Another aspect provides a method for reducing ERK phosphorylation in tumor cells of a subject and / or reducing PD-1 expression in T cells of a subject, which comprises administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof:

[0112]

[0113] Wherein:

[0114] R 1 is a straight-chain or branched C1-C6 alkyl; and

[0115] R 2 is a straight-chain or branched C1-C6 alkyl,

[0116] or R 2 is

[0117]

[0118] where q is 1, 2, 3 or 4; and R 3 is a straight-chain or branched C1-C6 alkyl.

[0119] In one embodiment, the compound of formula (I) has the structure of formula (II):

[0120]

[0121] In one embodiment, the method increases JUN expression and JNK phosphorylation in T cells of the subject.

[0122] In one embodiment, the method increases JUN expression in tumor cells of the subject.

[0123] In one embodiment, the method reduces ERK phosphorylation and increases JUN expression in tumor cells of the subject, and reduces PD-1 expression in T cells of the subject.

[0124] In one embodiment, the method reduces ERK phosphorylation and increases JUN expression in tumor cells of the subject, and reduces PD-1 expression in T cells of the subject.

[0125] In one embodiment, the method reduces ERK phosphorylation and increases JUN expression in tumor cells of the subject, and increases JUN expression, increases JNK phosphorylation and reduces PD-1 expression in T cells of the subject.

[0126] In one embodiment, the method increases DUSP8 expression in T cells of a subject.

[0127] In one embodiment, the method decreases MAF expression in T cells of a subject.

[0128] In one embodiment, the method reduces tumor cell migration and invasion in a subject.

[0129] In one embodiment, the method increases tumor cell apoptosis in a subject.

[0130] In one embodiment, the method decreases the tumor growth rate in a subject.

[0131] In one embodiment, the method reduces tumor cell migration, reduces tumor cell invasion, increases tumor cell apoptosis, and decreases the tumor growth rate in a subject.

[0132] A compound of formula (I) is:

[0133]

[0134] Wherein:

[0135] R 1 is a straight-chain or branched C1-C6 alkyl; and

[0136] R 2 is a straight-chain or branched C1-C6 alkyl,

[0137] or R 2 is

[0138]

[0139] where q is 1, 2, 3 or 4; and R 3 is a straight-chain or branched C1-C6 alkyl.

[0140] In some embodiments of formula (I), R 1 is a straight-chain C1-C6 alkyl or a branched C1-C6 alkyl. In some embodiments of formula (I), R 1 is -CH2CH3 or -CH2CH(CH3)2.

[0141] In some embodiments of formula (I), R 2 is a straight-chain C1-C6 alkyl or a branched C1-C6 alkyl. In some embodiments of formula (I), R 2 is -CH2CH3 or -CH2CH(CH3)2.

[0142] In some embodiments of formula (I), R2 is wherein q is 1, 2, 3 or 4; and R 3 is a straight-chain C1-C6 alkyl group or a branched-chain C1-C6 alkyl group. In some embodiments of formula (I), q is 2. In some embodiments of formula (I), R 3 is -CH3. In some embodiments of formula (I), q is 2 and R 3 is -CH3.

[0143] In some embodiments, the compound of formula (I) can be a compound of formula (I-1):

[0144]

[0145] wherein:

[0146] R 2 is a straight-chain or branched-chain C1-C6 alkyl group;

[0147] or R 2 is:

[0148]

[0149] wherein q is 1, 2, 3 or 4; and R 3 is a straight-chain or branched-chain C1-C6 alkyl group.

[0150] In one embodiment, the compound of formula (I) is a compound of formula (II).

[0151] The compound of formula (II) is:

[0152]

[0153] (also referred to herein as BT2)

[0154] A compound that increases immune cell activation refers to a compound that can induce or cause or promote an increase in the biological function or activity of immune cells (usually T cells) relative to the biological function or activity of immune cells that have not been exposed to the compound after contact with the compound. Examples of increased immune cell activation include increased responsiveness of T cells to antigens, increased proliferation, increased IFN-γ secretion by T cells, increased expression of JUN in T cells, decreased expression of PD-1 in T cells, increased expression of DUSP8 in T cells, and increased phosphorylation of JNK in T cells.

[0155] A compound that reduces PD-1 expression is a compound that reduces the amount of PD-1 protein produced by a cell or tissue after contact with the compound or agent, relative to the amount of PD-1 protein produced by a cell or tissue that has not been contacted with the compound.

[0156] A compound that increases JNK phosphorylation is a compound that increases the degree of JNK phosphorylation in a cell or tissue after contact with the compound, as compared to the degree of JNK phosphorylation in a cell or tissue not contacted with the compound.

[0157] A compound that increases JUN expression is a compound that increases the amount of JUN protein produced by a cell or tissue after contact with the compound, as compared to the amount of JUN protein produced by a cell or tissue not contacted with the compound.

[0158] A compound that reduces ERK phosphorylation is a compound that reduces the degree of ERK phosphorylation in a cell or tissue after contact with the compound, as compared to the degree of ERK phosphorylation in a cell or tissue not contacted with the compound.

[0159] A compound that increases DUSP8 expression is a compound that increases the amount of DUSP8 protein produced by a cell or tissue after contact with the compound, as compared to the amount of DUSP8 protein produced by a cell or tissue not contacted with the compound.

[0160] A compound that reduces MAF expression is a compound that reduces the amount of MAF protein produced by a cell or tissue after contact with the compound or agent, as compared to the amount of MAF protein produced by a cell or tissue not contacted with the compound.

[0161] In one embodiment, the compound increases JUN expression in T cells.

[0162] In one embodiment, the compound increases JUN expression in tumor cells.

[0163] In one embodiment, the compound reduces PD-1 expression in T cells.

[0164] In one embodiment, the compound increases DUSP8 expression in T cells.

[0165] In one embodiment, the compound reduces MAF expression in T cells.

[0166] In one embodiment, the compound increases circulating IFN-γ.

[0167] In one embodiment, the compound increases PD-L1 expression in tumor cells.

[0168] In one embodiment, the compound increases netrin-1 expression in T cells.

[0169] In one embodiment, the compound binds to CD28.

[0170] In one embodiment, the compound reduces tumor inflammation and / or tumor size, and / or increases tumor immunity.

[0171] In some embodiments, the compound is a pharmaceutically acceptable salt of a compound of formula (I) or formula (II). Examples of pharmaceutically acceptable salts include salts of pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium, and alkylammonium salts; acid addition salts of pharmaceutically acceptable inorganic acids such as hydrochloric acid, orthophosphoric acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, boric acid, sulfamic acid, and hydrobromic acid; or salts of pharmaceutically acceptable organic acids such as acetic acid, propionic acid, butyric acid, tartaric acid, maleic acid, hydroxymaleic acid, fumaric acid, citric acid, lactic acid, mucic acid, gluconic acid, benzoic acid, succinic acid, oxalic acid, phenylacetic acid, trihaloacetic acid (e.g., trifluoroacetic acid), methanesulfonic acid, trihalomethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, salicylic acid, p-aminobenzenesulfonic acid, aspartic acid, glutamic acid, ethylenediaminetetraacetic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid, and valeric acid.

[0172] In one embodiment, the compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof is deuterated.

[0173] In one embodiment, the compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof is the E isomer.

[0174] In one embodiment, the compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof is the Z isomer.

[0175] In one embodiment, the compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof is a mixture of the E isomer and the Z isomer.

[0176] A pharmaceutical composition is described herein that comprises a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof.

[0177] The pharmaceutical composition of the present invention can be used in the methods of the present invention described herein.

[0178] The pharmaceutical composition generally comprises a pharmaceutically acceptable carrier.

[0179] The compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof can be used to treat any disease or disorder associated with PD-1 expression in T cells or associated with ERK phosphorylation. If the development and / or maintenance of a disease or disorder requires the activity of a protein or phosphoprotein, the disease or disorder is associated with the protein or phosphoprotein.

[0180] In one embodiment, the disease or disorder is cancer. In one embodiment, a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof can be used to treat any cancer in which PD-1 / PD-L1 inhibition with an anti-tumor immune response is present.

[0181] Examples of cancers that can be treated with a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof include melanoma, squamous cell carcinoma, basal cell carcinoma, dermatofibrosarcoma, Merkel cell carcinoma, head and neck cancer, non-small cell lung cancer, urothelial carcinoma, thyroid cancer, renal cell carcinoma, breast cancer, and hepatocellular carcinoma.

[0182] In one embodiment, the cancer is a cancer of a subject in which the subject's T cells express PD-1.

[0183] In one embodiment, the cancer is a cancer in which one or more tumors of the cancer contain T cells that express PD-1.

[0184] The cancer treated by the methods described herein may contain T cells that express PD-1. However, as described in the Examples, the compounds described herein not only inhibit PD-1 expression, but also bind to CD28 on T cells and stimulate T cell activity even in tumors that are unresponsive to anti-PD-1 antibodies.

[0185] Thus, in some embodiments, the cancer can be a cancer that is resistant to treatment with anti-PD-1 antibody therapy.

[0186] As described in the Examples, the inventors further found that the compound of formula (II) is effective against BRAF mutant cell lines and tumors formed from BRAF mutant cell lines. Advantageously, the compound of formula (II) thus appears to be able to treat cancer regardless of the BRAF mutation status of the tumor.

[0187] In some embodiments, the cancer includes cells that are resistant to treatment with dabrafenib and trametinib.

[0188] In some embodiments, the cancer includes BRAF mutant cells.

[0189] The methods described herein may involve administering a pharmaceutical composition comprising a compound described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0190] A pharmaceutical composition is described herein that comprises a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0191] In some embodiments, the carrier is a non-naturally occurring carrier.

[0192] In some embodiments, the compounds of formula (I) or (II) or pharmaceutically acceptable salts thereof can be used in combination with one or more other agents.

[0193] It is to be understood that the combined administration of the compounds of formula (I) or (II) or pharmaceutically acceptable salts thereof with one or more other agents can be simultaneous, sequential or separate administration.

[0194] The term "composition" encompasses formulations containing the active ingredient with conventional carriers and excipients, as well as formulations having an encapsulating material as a carrier to provide a capsule in which the active ingredient (with or without other carriers) is surrounded by the encapsulating carrier. In a pharmaceutical composition, the carrier is "pharmaceutically acceptable", which means that it is compatible with the other ingredients of the composition and is not harmful to the subject. The pharmaceutical compositions of the present invention can contain other agents or other active agents as described above and can be formulated, for example, by using conventional solid or liquid solvents or diluents and types of pharmaceutical additives suitable for the desired mode of administration (e.g., excipients, binders, preservatives, stabilizers, flavoring agents, etc.) according to techniques such as those known in the field of pharmaceutical formulations (see, e.g., Remington: The Science and Practice of Pharmacy, 21st Edition, 2005, Lippincott Williams & Wilkins).

[0195] The pharmaceutical compositions can be suitable for intravitreal, oral, rectal, nasal, topical (including dermal, buccal and sublingual), vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation.

[0196] In some embodiments, the compounds described herein can be formulated for administration, for example, in nanoparticles or liposomes or polymer formulations. Methods for preparing formulations containing liposomes, lipid nanoparticles and polymer formulations are known in the art and are described, for example, in Neervannan, 2006; Zhang et al., 2022. Liposomes, nanoparticles or polymer formulations can contain cationic lipids such as DOTAP, DOPE, DC-Chol / DOPE, DOTMA and DOTMA / DOPE, polymers such as hydroxypropyl methylcellulose (HPMC), polyethylene glycol (PEG), poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), poly(glycolic acid) (PGA).

[0197] Accordingly, the compounds described herein or pharmaceutically acceptable salts thereof can be formulated in pharmaceutical compositions and unit dosage forms thereof together with a pharmaceutically acceptable carrier. The pharmaceutical compositions can be in solid form, such as tablets or filled capsules, or in liquid form, such as solutions, suspensions, emulsions, elixirs, or capsules filled with them, for oral administration. The pharmaceutical compositions can be in liquid form, such as solutions, suspensions, or emulsions, for intravitreal administration. The pharmaceutical compositions can also be in the form of suppositories for rectal administration or in the form of sterile injectable solutions for parenteral (including subcutaneous) use.

[0198] Such pharmaceutical compositions and unit dosage forms thereof may contain conventional proportions of conventional ingredients, with or without additional active compounds or ingredients, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be used.

[0199] For the preparation of pharmaceutical compositions from the compounds described herein, the pharmaceutically acceptable carrier can be solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, lozenges (solid or chewable), suppositories, and dispersible granules. The solid carrier can be one or more substances which may also serve as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or encapsulating materials.

[0200] Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting wax, cocoa butter, and the like. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.

[0201] Liquid form preparations include solutions, suspensions, and emulsions, such as water or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in polyethylene glycol aqueous solutions.

[0202] Compositions in sterile liquid form include sterile solutions, suspensions, emulsions, syrups, and elixirs. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable carrier, such as sterile water, sterile organic solvents, or mixtures of both.

[0203] Accordingly, the pharmaceutical compositions according to the present invention can be formulated for parenteral administration (e.g., by injection, such as bolus injection or continuous infusion), and can be present in unit dosage form in ampoules, pre-filled syringes, small volume infusions or multi-dose containers which are added with preservatives. The pharmaceutical compositions can take the form of, for example, suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulating agents, such as suspending agents, stabilizers and / or dispersing agents. Alternatively, the active ingredient can be in powder form, obtained by aseptic isolation of a sterile solid or by lyophilization from a solution, for constitution with a suitable vehicle (e.g., sterile, pyrogen-free water) before use.

[0204] Pharmaceutical forms suitable for injectable use include sterile injectable solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions. They should be stable under the conditions of manufacture and storage, and can be preserved against the contaminating action of microorganisms such as oxidation and bacteria or fungi.

[0205] The solvent or dispersion medium for injectable solutions or dispersions can contain any conventional solvent or carrier system for injectable solutions or dispersions, and can contain, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and vegetable oils.

[0206] Pharmaceutical forms suitable for injectable use can be delivered by any suitable route, including intravenous, intramuscular, intracerebral, intrathecal, epidural injection or infusion.

[0207] Sterile injectable solutions are prepared by incorporating the required amount of the active ingredient, as required, with various other ingredients (such as those listed above) into a suitable solvent, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is to vacuum dry or lyophilize a previously sterile filtered solution of the active ingredient plus any additional desired ingredients. The formulations can also be sterilized by heat treatment (e.g., boiling) or autoclaving.

[0208] The compounds described herein can be formulated into compositions suitable for oral administration, such as with an assimilable edible carrier, or encapsulated in hard or soft shell gelatin capsules, or compressed into tablets, or directly incorporated into the food in the diet. For oral therapeutic administration, the active compounds can be incorporated with excipients and used in ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc.

[0209] As described in the examples, the compounds of formula (II) are bioavailable upon oral and intraperitoneal administration.

[0210] One aspect provides a pharmaceutical composition comprising a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof, a surfactant, and a solvent or polymer.

[0211] In one embodiment, the surfactant is polysorbate. In one embodiment, the polysorbate is Tween 80.

[0212] In one embodiment, the solvent is a polar aprotic solvent. In one embodiment, the polar aprotic solvent is dimethyl sulfoxide (DMSO).

[0213] In one embodiment, the polymer is hydroxypropyl methylcellulose (HPMC).

[0214] The amount of the active compound in the therapeutically useful composition should be sufficient to obtain a suitable dosage.

[0215] Tablets, lozenges, pills, capsules, troches, implants, etc. may also contain the following components: binders such as gums, acacia, corn starch or gelatin; excipients such as dibasic calcium phosphate; disintegrants such as corn starch, potato starch, alginic acid, etc.; lubricants such as magnesium stearate; and sweetening agents such as sucrose, lactose or saccharin or flavoring agents such as peppermint, wintergreen oil or cherry flavoring may be added. When the dosage unit form is a capsule, it may also contain a liquid carrier in addition to the above types of materials.

[0216] Various other materials may be present as coatings or otherwise modify the physical form of the dosage unit. For example, tablets, pills or capsules may be coated with shellac, sugar or both. Syrups or elixirs may contain the active compound, sucrose as a sweetening agent, methylparaben and propylparaben as preservatives, dyes and flavoring agents such as cherry or orange flavoring. Of course, any material used in the preparation of any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active ingredient may be incorporated into sustained release preparations and formulations, including those that permit specific delivery of the active ingredient to a particular region of the intestine.

[0217] An aqueous solution suitable for oral use can be prepared by dissolving the active component in water and adding, as required, suitable coloring agents, flavoring agents, stabilizers and thickening agents. An aqueous suspension suitable for oral use can be prepared by dispersing the finely divided active component in water with a viscous material such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose or other well-known suspending agents.

[0218] Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.

[0219] Also included are solid form preparations that are intended to be converted shortly before use to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. In addition to the active ingredient, these preparations may also contain coloring agents, flavoring agents, stabilizers, buffers, artificial and natural sweeteners, dispersing agents, thickening agents, solubilizing agents, and the like.

[0220] For topical application, the compounds described herein can be formulated in an aqueous or oily base and admixed with suitable thickening and / or gelling agents. Lotions can be formulated in an aqueous or oily base and generally also contain one or more emulsifying agents, stabilizers, dispersing agents, suspending agents, thickening agents, or coloring agents.

[0221] Preparations suitable for topical application in the mouth include lozenges that contain the active agent in a flavored base, usually sucrose and gum arabic or tragacanth; pastilles that contain the active ingredient in an inert base such as gelatin and glycerin or sucrose and gum arabic; and mouthwashes that contain the active ingredient in a suitable liquid carrier.

[0222] Solutions or suspensions for nasal administration can be administered directly to the nasal cavity by conventional means, such as with a dropper, pipette, or spray. The preparations can be provided in single-dose or multi-dose form. In the case of a dropper or pipette, this can be accomplished by the patient administering an appropriate predetermined volume of the solution or suspension. In the case of a spray, this can be accomplished, for example, with the aid of a metered atomizing spray pump. To improve nasal delivery and retention, the compounds of the present invention can be encapsulated with cyclodextrin or formulated with other agents expected to enhance delivery and retention in the nasal mucosa.

[0223] Administration to the respiratory tract can also be achieved by aerosol formulations, wherein the active ingredient is provided in a pressurized package together with a suitable propellant such as a chlorofluorocarbon (CFC) (e.g., dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane), carbon dioxide, or other suitable gas.

[0224] The aerosol can also conveniently contain a surfactant such as lecithin. The dosage of the active ingredient can be controlled by providing a metering valve.

[0225] Alternatively, the active ingredient may be provided in dry powder form, e.g., a powder mixture of the compound in a suitable powder matrix such as lactose, starch, starch derivatives such as hydroxypropylmethylcellulose, and polyvinylpyrrolidone (PVP). Conveniently, the powder carrier will form a gel in the nasal cavity. The powder composition may be presented in unit dose form, e.g., in a capsule or cartridge such as gelatin, or in a blister pack from which the powder can be administered via an inhaler.

[0226] In formulations intended for administration to the respiratory tract, including intranasal formulations, the active ingredient typically has a small particle size, e.g., on the order of 5 to 10 microns or smaller. Such particle sizes can be obtained by methods known in the art, e.g., by micronization.

[0227] When desired, formulations suitable for sustained release of the active ingredient may be used.

[0228] The pharmaceutical formulation is preferably in unit dosage form. In this form, the formulation is subdivided into unit doses containing an appropriate amount of the active ingredient. The unit dosage form may be a packaged formulation, the package containing discrete quantities of the formulation, e.g., packaged tablets, capsules, and powders in vials or ampoules. Additionally, the unit dosage form itself may be a capsule, tablet, cachet, or lozenge, or it may be any of these dosage forms in a suitable number taken in packaged form.

[0229] Particularly advantageous is the formulation of parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. The parenteral composition may be in the form of physically discrete units suitable as a unit dose for the subject to be treated, each unit containing a predetermined quantity of the active ingredient calculated to produce the desired therapeutic effect in association with the pharmaceutical carrier.

[0230] The compound may also be administered in the absence of a carrier, where the compound is in unit dosage form.

[0231] The term "effective amount" refers to the amount of the compound effective to achieve the desired response.

[0232] The effective amount of the compound or its pharmaceutically acceptable salt described herein can be determined by those skilled in the art according to the particular compound.

[0233] It should be understood that the specific dosage levels and dosage frequencies for any particular subject may vary and will depend on a variety of factors, including the activity of the specific compound employed, the metabolic stability and duration of action of that compound, the age, body weight, general health, sex, and diet of the subject, the mode and time of administration, the rate of excretion, drug combination, and the severity of the particular disorder.

[0234] The appropriate dosage of the compounds described herein or other active agents administered in combination with the compounds described herein can be readily determined by those skilled in the art based on the particular compound or other active agent selected in accordance with the present invention.

[0235] It should also be understood that when the compounds described herein are administered in combination with one or more pharmaceutical agents or other active agents, the dosage form and dosage level can be formulated for simultaneous, sequential, or separate administration or combinations thereof.

[0236] The methods of the present invention are intended for use in any subject who can experience the benefits of the methods of the present invention. Thus, the term "subject" includes humans as well as non-human mammals. A subject can be, for example, a domestic animal, a zoo animal, or livestock.

[0237] Unless otherwise defined herein, the following terms will be understood to have the following general meanings. Unless otherwise indicated, the terms mentioned below have the general meanings that follow when the term is used alone and when the term is used in combination with other terms. Thus, for example, the definition of "alkyl" applies to the "alkyl" portion of "alkyl" as well as "haloalkyl", "heteroalkyl", "arylalkyl", etc.

[0238] The term "alkyl" refers to a straight-chain or branched-chain saturated hydrocarbon group. Unless otherwise indicated, preferably C 1-6 alkyl and C 1-4 The term "C x-y alkyl", where x and y are integers, refers to an alkyl group having from x to y carbon atoms. For example, the term "C 1-6 alkyl" refers to an alkyl group having from 1 to 6 carbon atoms. Examples of C 1-6 alkyl include methyl (Me), ethyl (Et), propyl (Pr), isopropyl (i-Pr), butyl (Bu), isobutyl (i-Bu), sec-butyl (s-Bu), tert-butyl (t-Bu), pentyl, neopentyl, hexyl, and the like. Unless the context otherwise requires, the term "alkyl" also encompasses an alkyl group having one less hydrogen atom such that the group is attached via two positions, i.e., divalent.

[0239] As used herein, "treatment" refers to affecting a subject, tissue, or cell to obtain a desired pharmacological and / or physiological effect and includes inhibiting a disorder, i.e., preventing its development; or alleviating or ameliorating the effects of a disorder, i.e., causing a reversal or regression of the effects of the disorder. As used herein, "prevention" refers to preventing the occurrence of a disorder in a cell or subject that is at risk of developing the disorder, but does not necessarily mean that the disorder will not ultimately develop, or that the subject will not ultimately develop the disorder. Prevention includes delaying the onset of a disorder in a cell or subject.

[0240] The term "effective amount" refers to the amount of a compound that elicits a biological or medical response in a tissue, system, animal, or human being that a researcher, veterinarian, physician, or other clinician is seeking.

[0241] Table 1. Compounds

[0242]

[0243]

[0244]

[0245] The compounds described herein can be synthesized by methods known in the art. The compound referred to herein as BT2 is commercially available. For example, BT2 can be purchased from Aurora Building Blocks in the United States or Life Chemicals HTS Compounds in Canada.

[0246] All publications mentioned in this specification are incorporated herein by reference. Those skilled in the art will understand that various changes and / or modifications can be made to the invention shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. Accordingly, the embodiments of the invention are to be considered in all respects as illustrative and not restrictive.

[0247] The present invention is further described below by reference to the following non-limiting examples.

[0248] Embodiment

[0249] Here, we report that BT2, a dibenzoxazepinone (Li et al., 2020), acts as a novel pharmaceutical immunotherapeutic agent and inhibits tumor growth through a dual mechanism. In tumor cells, BT2 acts as an inhibitor of ERK phosphorylation and stimulates JUN expression, while in T cells, BT2 binds to CD28, activates JNK phosphorylation, stimulates JUN expression, and inhibits PD-1 expression.

[0250] Materials and Methods

[0251] Compound synthesis and purification. Compounds BT2, BT3, BT2-MeOA, BT2-EOMe, BT2-Pr, BT2-IC, BT2-IMO, BT2-MO, and BT2-deut were synthesized and purified (>95%) as described in WO 2021 / 184059.

[0252] Cell culture. Human A375 and MeWo melanoma cells were donated by Dr. Helen Rizos (Department of Biomedical Sciences, Macquarie University, Sydney). A375 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) pH 7.4 containing 10% fetal bovine serum (FBS) in a humidified incubator at 37 °C and 5% CO2, and were routinely passaged after trypsinization of adherent cells. MeWo cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium (pH 7.4) containing 10% FBS. Mouse B16F10 and human MDA-MB-435 (American Type Culture Collection, USA) cells were grown in DMEM at pH 7.4 containing 10% FBS. Jurkat T cells (ATCC) were grown in RPMI-1640 medium at pH 7.4 containing 10% FBS.

[0253] Western blot. Cells were treated with compounds for a specified time and total cell lysates were prepared in RIPA buffer. Lysates (10 μg) were resolved by SDS-PAGE and transferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore, USA).

[0254] Membranes were blocked with 5% non-fat milk and incubated with rabbit monoclonal anti-c-Jun (Abcam, catalog number ab32137; reactive with mouse, rat, and human) antibody, rabbit monoclonal anti-PD-1 (Abcam, catalog number ab214421) antibody, mouse monoclonal β-actin antibody (Sigma-Aldrich, catalog number A5316), anti-phospho-SAPK / JNK (Thr 183 / Tyr 185 )(98F2)(CST, catalog number 4671) antibody, anti-phospho-p44 / 42 MAPK (ERK1 / 2) (Thr 202 / Tyr 204 )(D13.14.4E)(CST, catalog number 4370) antibody, anti-phospho-p38 MAPK (Thr 180 / Tyr 182(D3F9) (CST, catalog number 4511), anti-SAPK / JNK antibody (CST, catalog number 9252), anti-p44 / 42 MAPK (ERK1 / 2) (137F5) antibody (CST, catalog number 4695), anti-p38 MAPK antibody (CST, catalog number 9212), anti-DUSP8 antibody (Abcam, catalog number ab198175), or anti-c-MAF (BLR045F) antibody (Abcam, catalog number ab243901). Subsequently, incubated with horseradish peroxidase-conjugated secondary goat anti-rabbit (DAKO, catalog number P0448) or goat anti-mouse (DAKO, catalog number P0447) antibody. Chemiluminescence was detected using the Western Lightning chemiluminescence system (Thermo Scientific, USA) and the ImageQuant TM LAS 4000 biomolecular imager (GE Healthcare Life Sciences, USA). The band intensities in the images generated by the LAS4000 were quantified using NIH Image J.

[0255] Migration assay. Cells were grown in medium containing 10% FBS in 6-well plates. The next day, the cells were scratched with a sterile toothpick, washed with PBS, and incubated in medium containing 10% FBS and 1 μM vehicle, BT2, or SCH772984. At 0, 24, and 48 hours, the cells were photographed using an Olympus CKX41 microscope at a 4× objective.

[0256] Invasion assay. Cells were grown in medium containing 10% FBS in 24-well plates. The next day, the cells were scratched with a sterile toothpick, washed with PBS, and overlaid with 90% Matrigel (catalog number 354230, Corning) (200 μl / well) containing 1 μM vehicle, BT2, or SCH772984. The cells were immediately photographed using an Olympus CKX41 microscope at a 10× objective, incubated at 37 °C for 1 hour, and medium containing 10% FBS and 1 μM vehicle, BT2, or SCH772984 (800 μl / well) was added. After 24 or 48 hours, the cells were photographed again using the same microscope at a 10× objective.

[0257] Cell morphology study. Cells were grown in a medium containing 10% FBS in 4-well chamber slides. The next day, the cells were incubated in a medium containing 10% FBS and 1 μM of vehicle, BT2, or SCH772984 for 24 hours, and then fixed with 4% paraformaldehyde solution for 15 minutes. After a brief wash in PBS, the cells were stained with hematoxylin and eosin. The slides were scanned using an Aperio ScanScope XT slide scanner (Leica Biosystems, Mt Waverley, Vic, Australia), and images were captured using ImageScope software (Leica Biosystems).

[0258] Flow cytometry using Annexin V-FITC. Cells were seeded into a 6-well plate in normal medium containing 10% FBS. After overnight culture, the cells were incubated and allowed to stand for 24 hours in a complete medium containing 1 μM of vehicle, BT2, or SCH772984. The medium was removed and the cells were washed with PBS. The cells were detached using Accutase (Stem Cell Technologies, catalog number 07920). The cells were then washed and centrifuged at 300 g for 5 minutes, and resuspended at 1 × 10 6 cells / ml in 500 μl of 1× binding buffer (Annexin V-FITC Apoptosis Staining / Detection Kit, Abcam, catalog number ab14085). The cells were transferred to a 12 × 75 mm tube, Annexin V-FITC / propidium iodide (PI) was added, and the cells were incubated for 5 minutes at 22 °C in the dark. The stained cell suspension was analyzed by flow cytometry using a BD LSRFortessa X20.

[0259] Flow cytometry using anti-PD-1 antibody. Jurkat T cells were grown in a 6-well plate containing RPMI 1640 and 10% FBS. After 24 hours, the cells were treated with different concentrations of BT2 or BT3 for 48 hours. Alternatively, the cells were incubated with 3 μM BT2 for different times. After treatment, the cells were washed with PBS, centrifuged at 300 × g for 5 minutes, and resuspended at 5 × 10 6 cells / ml. The cells were then incubated with BV421-conjugated mouse anti-human CD279 (PD-1) (BD, catalog number 562516) or BV421-conjugated mouse IgG1 (Bd, catalog number 562438) for 45 minutes at 22 °C. The cells were then washed twice with 1 ml of staining buffer, centrifuged, and the pellet was resuspended in 0.5 ml of staining buffer. The stained cell suspension was analyzed by flow cytometry using a BD FACSCanto (II).

[0260] CD28 Blocking Assay. BT2 (3 nmol), vehicle, or a mixture of BT2 (3 nmol) and recombinant CD28 (3 nmol, Sino biological, catalog number 90182-C08H) or EGF (3 nmol, Sigma, catalog number E9644) in 150 μl of growth medium (10% FBS / RPMI 1640 medium) was pre-incubated at 37 °C for 30 min. The mixture was added to a 12-well plate containing 850 μl of 10% FBS / RPMI 1640 medium and 0.5×10 6 Jurkat T cells / well. After 24 h, total cell lysates were prepared in RIPA buffer. Lysates (10 μg) were resolved by SDS-PAGE and transferred to Immobilon-P polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membrane was blocked with 5% non-fat milk and incubated with rabbit monoclonal anti-JUN (Abcam, catalog number ab32137) antibody, mouse monoclonal β-actin antibody (Sigma-Aldrich, catalog number A5316). Subsequently, it was incubated with horseradish peroxidase-conjugated secondary goat anti-rabbit (DAKO, catalog number P0448) or goat anti-mouse (DAKO, catalog number P0447) antibody. Chemiluminescence was detected using the Western Lightning chemiluminescence system (ThermoScientific, USA) and ImageQuant TM LAS 4000 biomolecular imager (GE Healthcare LifeSciences, USA). Band intensities in the images generated by LAS 4000 were quantified using NIH Image J.

[0261] Melanoma Growth Study in Mice. C57BL / 6J mice (6 - 8 weeks old, obtained from the Australian Resource Centre, Perth) were subcutaneously (s.c.) inoculated with B16F10 cells (1×10 5 cells / mouse, in 100 μl of DMEM containing 10% FBS and 50% Matrigel). InVivoPlus anti-mouse PD-1 (CD279) (BioXCell, BP0033-2) or InVivoPlus polyclonal Armenian hamster IgG (Bio X Cell, BP0091) (100 μg) was administered intraperitoneally (i.p.) twice a week. Treatment started on day 7.

[0262] Alternatively, BT2 was suspended at 10 mg / ml in vehicle (saline containing 0.5% (v / v) Tween 80 and 0.01% (v / v) DMSO) and sonicated before administration. The vehicle or BT2 (20 ml / kg and 200 mg / kg, respectively) was administered intraperitoneally once daily on a schedule of 5 days on / 2 days off. Treatment was initiated when tumors were palpable on day 5.

[0263] In experiments using SCID mice, on day 0, human MDA-MB-435 cells (2.5 × 10 6 cells / animal in 100 μl PBS) were subcutaneously inoculated into the third mammary fat pad of 5-week-old CB17 / Icr-Prkdcscid / IcrIcoCrl mice (from Charles River Laboratories, USA). These cells were also from ATCC. BT2 was suspended at 9.6 mg / ml in vehicle (saline containing 0.5% (v / v) Tween 80 and 0.01% (v / v) DMSO) and sonicated. The vehicle or BT2 was administered intraperitoneally or intratumorally (i.t.) at 20 mg / kg (i.t.) or 200 mg / kg (intraperitoneally) once daily on a schedule of 5 days on / 2 days off.

[0264] Treatment was initiated on day 16 and ended on day 43. Tumor growth and body weight data were sourced from the animal study described in Li et al. (Li et al., 2019) using the same vehicle group.

[0265] For MDA-MB-435 in SCID mice, the length and width of tumors were measured twice weekly in mm, and for B16F10 in C57BL / 6J mice, the length, width, and height of tumors were measured daily in mm. Tumor volume was calculated using the formula V = (L x W x H x π) / 6 (B16F10) or V = L x W x W / 2 (MDA-MB-435). If a second tumor was detected in an animal, the volumes of both tumors were measured and combined. The animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of ExploraBiolabs and the UNSW Animal Care and Ethics Committee.

[0266] Mass spectrometry. Proteins in serum (50 μl) were precipitated by dilution with acetone (150 μl). The mixture was left at 4 °C for 14 h, and the proteins were precipitated by centrifugation (10 min at 14,000 g) and the supernatant was removed. The solution (10 μl) was diluted to 40 μl in buffer A (H2O (0.1% formic acid)) for MS analysis. The BT2 standard was prepared by diluting the BT2 stock suspension (10 μg, 30 mg / ml) in buffer A (990 μl), then taking 10 μl and diluting it in 990 μl, and then diluting it 1 / 100 further. The concentration of the standard BT2 for LC-MS was 92 fmol / μl. LC-MS was performed using a Thermo QExactive HF, Gold C 18 column (50 × 2.1 mm), where solvent A (H2O (0.1% formic acid)) and solvent B (H2O:CH3CN 20:80 (0.1% formic acid)) were used, with a gradient of T = 0, 1% B, T = 26 min, 100% B, T = 27 min, 100%, T = 27.1 min, 1% B, T = 30 min, 1% B; column temperature 45 °C. MS1 scan, m / z 140 - 800, 3 × 10 6 ions, maximum IT 25 ms, resolution 120,000 and the top 5 MS2 (2 × 10 5 ions, maximum IT 50 ms, resolution 30,000 and HCD 20, 30 50 V). Extracted ion chromatograms (m / z 327.134 ± 5 ppm) of the blank, standard, and sample were obtained, and the integrated peak areas were calculated and compared).

[0267] RNA-seq and bioinformatics analysis. Jurkat T cells were grown to confluence in complete medium in 100 mm plates and incubated with 10 μM BT2 or vehicle for 4 h. Total RNA was extracted using a modified RNeasy Mini kit (Qiagen, catalog number 74004). Briefly, cells were washed twice with pre-cooled 1×PBS and TRIzol reagent (ThermoFisher Scientific, catalog number 15596026) was added to lyse the cells. Chloroform was added to the mixture and then centrifuged at 13,000 rpm in a microcentrifuge at 4 °C for 15 min. The upper aqueous layer containing total RNA was transferred to a fresh microtube, isopropanol was added and loaded onto an RNeasy column. The column was washed with buffer RW1 and RPE. Total RNA was eluted from the column using ribonuclease-free water. Samples were submitted to the UNSW Ramaciotti Centre for Genomics for TruSeq Stranded mRNA-seq preparation and sequencing via a One NextSeq 500 1×75bp high-output flow cell, with data output up to 400M reads. The quality control of the samples was set to be >80% higher than Q30 at 1×75bp.

[0268] First, the quality of RNA-seq reads was evaluated using the tool FastQC (v0.11.8, www.bioinformatics.babraham.ac.uk / projects / fastqc / ). The tool Salmon was used to quantify the transcript abundances of RNA-seq reads (Patro et al., 2017). Then, the R package DESeq2, which includes methods for differential analysis of count data, was used to identify differentially expressed genes across specific comparisons (Love et al., 2014). This package was also used to perform principal component analysis. Significantly differentially expressed genes were defined as having an adjusted p-value < 0.05 and a log2 fold change > 1 (absolute fold change > 2). The ggplot2 R package was used to prepare bar plots, scatter plots, and density plots. The pheatmap R package was used to generate a distance matrix and a clustered heatmap. The DESeq2 output was used to generate a ranked list for input into GSEA preranked (v6.0.12, Broad Institute) (Subramanian et al., 2005) for pathway analysis against curated (CanonicalPathway), Gene Ontology, and Hallmarks gene collections from MSigDb (Liberzon et al., 2015; Liberzon et al., 2011). The rank score was calculated by multiplying the sign of the log2 fold change by the log10-transformed adjusted p-value. Gene sets were shown only if the false discovery rate of the gene set was the lowest < 0.25, and a maximum of 60 gene sets were shown in the figure. All immune response-related gene sets were selected for display. No other preselection of the shown gene sets was performed.

[0269] Immunohistochemical staining and analysis. Rabbit monoclonal anti-CD3 (catalog number ab16669; reactive with mouse, rat, and human), rabbit monoclonal anti-PD-1 (catalog number ab214421), and rabbit monoclonal anti-CD68 (catalog number ab125212) antibodies were obtained from Abcam. Rabbit monoclonal anti-phosphorylated ERK (Thr 202 / Tyr 204 )(catalog number CST4370) and rabbit monoclonal total ERK (catalog number CST4695) antibodies were obtained from Cell Signaling Technology. Rabbit polyclonal anti-PD-L1 (catalog number PA5-20343) antibody was obtained from ThermoFisher.

[0270] Prepare formalin-fixed, paraffin-embedded sections from the tumor. Perform heat-induced epitope retrieval for 5 minutes at 110 °C on all deparaffinized sections (4-μm sections on Superfrost slides) using citrate buffer at pH 6.0. Block the sections with DualEndogenous Enzyme Block (DAKO, S2003) for 10 minutes and then with 2% skim milk for 20 minutes. Incubate the slides with the primary antibody at room temperature for 60 minutes or overnight at 4 °C, and then incubate with the probe of MACH3 Rabbit AP-Polymer Detection Solution (Biocare Medical, M3R533 G,H,L) for 10 minutes. After rinsing with buffer, incubate the slides with the polymer of MACH3 Rabbit AP-Polymer Detection Solution (Biocare Medical, M3R533 G,H,L) for another 10 minutes. Incubate the slides with the red chromagen (Warp Red TM Chromagen Kit) for 5 minutes and counterstain with hematoxylin and Scott blue. Dry the slides with filter paper and dehydrate in xylene, and then cover with coverslips. Scan the immunostained slides using an Aperio ScanScope XT slide scanner (Leica Biosystems, Mt Waverley, Vic, Australia) and capture images using ImageScope software (Leica Biosystems). Evaluate the integrated optical density (IOD) or the positive staining area (red chromagen) using Image-Pro Plus software (Cybernetics, Bethesda, MD, USA). Positive staining is the average quantitative value in 4-8 fields of view under a 20× or 40× objective lens.

[0271] Mouse serum IFN-γ levels. Collect whole blood from day 17 mice and centrifuge at 1500 g for 10 minutes at 4 °C. Extract the serum from the top layer and store at -80 °C. Measure IFN-γ levels using a Mouse IFN-γ (modified) ELISA kit (Invitrogen, catalog number KMC4021) according to the manufacturer's protocol, and read the absorbance at 450 nm. Generate a standard curve using GraphPad Prism 9 software. Measure the IFN-γ levels in mouse serum using GraphPad Prism 9 alongside the standard curve.

[0272] Surface Plasmon Resonance (SPR). Using a Biacore T200 (Cytiva), at 25 °C and a flow rate of 10 μl / min, CD28 (Sino Biological) was immobilized onto a Series S sensor chip CM5 (GE) on flow cells 2 and 4 by amine coupling to a level of approximately 3000 RU. 0.2 M EDC + 0.05 M NHS was injected for 420 s, then CD28 (37.5 μg / ml dissolved in 10 mM acetate pH 5) was injected for 450 s with the flow rate reduced to 2 μl / min. Unreacted NHS was blocked by injecting 1 M ethanolamine-HCl pH 8.5 for 420 s. Flow cells 1 and 3 were used as reference cells and were activated and blocked as described above. The immobilization running buffer was 20 mM HEPES, 150 mM NaCl, pH 7.5. The SPR runs were performed at 12 °C using 20 mM HEPES, 150 mM NaCl, 5% DMSO, pH 7.5 as the running buffer. BT2 was dissolved in DMSO to a concentration of 50 mM and then diluted to a final concentration of 20 μM in the running buffer. The concentration was confirmed using 1 H1D NMR. An 8-point dilution series with a maximum concentration of 20 μM was performed. Samples were injected at a flow rate of 40 μl / min for 60 s and the dissociation time was 60 s. Solvent correction was performed to correct for the excluded volume effect. The integrity of the coupled CD28 was verified by injecting the known binding partner CD80 (Sino Biological) (Waite et al., 2020) (5-point dilution series, maximum concentration 2 μM).

[0273] Systemic pharmacokinetics after oral (PO) and intraperitoneal (IP) administration. Male CD-1 mice were injected with BT2 in formulation 1, formulation 2, or formulation 3 (Table 4), and blood was collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h. BT2 was administered by PO or IP injection.

[0274] Table 4 - Formulation components

[0275]

[0276] Approximately 110 μl of blood samples (n = 3 / time point, 3 mice / group) were collected into tubes containing EDTA-K2 anticoagulant. All blood samples were placed on wet ice prior to centrifugation (6000 g, 4 °C, 5 min) to obtain plasma, which was stored at -70 °C or on dry ice until analysis.

[0277] Plasma samples were analyzed by LC-MS / MS (SCIEX ExionLC)-MS / MS (Triple Quad 6500+ With Analyst 1.7.1 AB Sciex). HPLC was performed using a Waters Acquity UPLC HSS T3 1.8 μm, 2.1×50 mm column, with mobile phase A: H2O-0.025% FA+1 mM NH4OAc, and mobile phase B: MeOH-0.025% FA+1 mM NH4OAc, and an injection volume of 3 μl.

[0278] Statistics. Statistical analysis was performed using Graphpad PRISM v9, noting that PRISM does not draw error bars when the error bars are shorter than the height of the symbols. If the distribution is not normal, Mann-Whitney or Kruskal-Wallis is performed as appropriate. Normally distributed data are analyzed by t-test or one-way ANOVA as appropriate. The data plotted represent the mean ± SEM. n represents biological triplicates, not technical triplicates. Differences were considered significant when p≤0.05. As indicated, *p≤0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0279] Results

[0280] BT2 inhibits ERK phosphorylation, inhibits the migration and invasion of human melanoma cells and stimulates apoptosis

[0281] Given that MAP kinase activation is crucial for melanoma progression and the lack of clinically approved ERK inhibitors, the effect of BT2 on ERK phosphorylation (p-ERK) was evaluated in human melanoma cells. The study by Rossi et al. compared the invasiveness (a combination of growth, invasion and migration rates) of 10 human melanoma cell lines and found that A375 cells (mutant BRAF V600E ) had the most invasive phenotype (Rossi et al., 2018). MeWo cells (wild-type BRAF) are melanoma cells that exhibit the least invasive phenotype studied by Rossi et al. Western blotting was performed using extracts from A375 and MeWo cells incubated with 10 or 100 nM BT2 or SCH772984 for 24 hours. The membrane was incubated with antibodies against p-ERK (Thr 202 / Tyr 204 ) or total ERK, and then with a secondary antibody. The results are shown in Figure 1 A and 1C. Band intensities were quantified using Image J, and the data plotted represent the mean ± SEM ( Figure 1B and 1D). Data represent 2 biologically independent experiments. Results showed that BT2 inhibited ERK phosphorylation, as did SCH772984, an ERK inhibitor that overcomes resistance to BRAF and MEK inhibitors and is also the precursor of MK-8353 in clinical trials for advanced malignancies.

[0282] To evaluate the effect of BT2 on melanoma cell migration, A375 and MeWo cells were incubated with 1 μM BT2 or SCH772984, and migration of A375 and MeWo into cell-free areas was quantified after 24 or 48 hours, respectively. Results are shown in Figure 2 A to 2D. It can be seen that in A375 and MeWo cells, BT2 inhibited melanoma migration ( Figure 2 A and 2C) and invasion ( Figure 2 B and 2D) with greater potency than SCH772984.

[0283] The morphology of A375 and MeWo cells exposed to 1 μM BT2 or SCH772984 for 24 hours was also evaluated. Results are shown in Figure 3 . Different from SCH772984, BT2 had a significant morphological effect ( Figure 3 ), resulting in loss of spindle morphology and cell rounding in both cell lines. Flow cytometry confirmed that BT2 increased apoptosis in both cell lines, while SCH772984 had no or only a weak pro-apoptotic effect ( Figure 4 ).

[0284] BT2 inhibits melanoma growth after local but not systemic delivery in immunodeficient mice

[0285] The ability of BT2 to inhibit melanoma growth was investigated in a murine xenograft model. Immunocompromised SCID mice (C.B.17 / lcr-Prkdcscid / IcrIcoCrl) bearing human melanoma (MDA-MB-435 cells, also mutant BRAF V600E ) were treated intratumorally (i.t.) with 20 mg / kg BT2 on a 5-day-on / 2-day-off schedule ( Figure 5 A). BT2 caused growth inhibition ( Figure 5 B), without an adverse effect on body weight ( Figure 5 C).

[0286] Immunohistochemical staining using an alkaline phosphatase-conjugated secondary antibody rather than 3,3'-diaminobenzidine to avoid interference with brown pigment (melanin) confirmed that BT2 inhibited ERK phosphorylation in tumors ( Figure 6 A and 6B), without affecting total ERK levels in this local delivery model ( Figure 6C and 6D).

[0287] To evaluate the effect of BT2 when delivered systemically (as opposed to locally) in the same immunodeficient mouse model, C.B.17 SCID mice bearing subcutaneous MDA-MB-435 tumors were given BT2 at 200 mg / kg or vehicle intraperitoneally once daily on a 5-day dosing / 2-day off schedule. Treatment began on day 16 ( Figure 7 A). The effects on tumor volume and body weight are shown in Figure 7 B and 7C. Surprisingly, when BT2 was delivered systemically in this model, no inhibition was observed even at 200 mg / kg in the 5-day dosing / 2-day off regimen ( Figure 7 B).

[0288] In a murine immunocompetent allograft model resistant to tumor suppression by anti-PD-1 antibody, systemically delivered BT2 inhibits melanoma growth and ERK phosphorylation

[0289] Previous xenograft studies have shown antitumor effects of BT2 in immunocompromised mice when delivered intratumorally (i.t.) rather than systemically (i.p.), leading the inventors to hypothesize that BT2 may rely on the active immune system to achieve tumor growth inhibition. Accordingly, the effect of BT2 was tested in an immunocompetent mouse model of melanoma growth using B16F10 melanoma. B16F10 grown in C57BL / 6 mice provides a highly invasive, poorly immunogenic, ERK-dependent syngeneic melanoma model. This model is resistant to inhibition by anti-PD-1 antibody (Kleffel et al., 2015). Anti-mouse PD-1 monoclonal antibody or control IgG (100 μg, intraperitoneally) was administered twice weekly to C57BL / 6J mice bearing subcutaneous B16F10 tumors. Treatment began on day 7 ( Figure 8 A). Tumor volume and body weight were evaluated over time, and the results are shown in Figure 8 B and 8C. These experiments showed that the anti-PD-1 antibody showed a transient but non-sustained inhibition of B16F10 growth (i.e., on days 12 and 13) ( Figure 8 B), as observed by Kleffel et al. (Kleffel et al., 2015).

[0290] To evaluate the effect of BT2 in the same model, BT2 or vehicle (200 mg / kg or 20 ml / kg, respectively, intraperitoneally) was administered once daily to C57BL / 6J mice bearing subcutaneous B16F10 tumors on a 5-day dosing / 2-day off schedule. Treatment began on day 5 ( Figure 9 A). The evaluated results of tumor volume and body weight are shown in Figure 9In B and 9C, the evaluation of tumor size and the weight of the isolated tumors is shown in Figure 10 In A and 10B, and the evaluation of survival rate is shown in Figure 11 . Different from anti-PD-1 treatment, systemically delivered BT2 caused significant B16F10 growth inhibition from day 9, which persisted during the study duration ( Figure 9 B), and had no adverse effect on body weight during this period ( Figure 9 C). The tumor size measured by calipers on day 17 ( Figure 10 A) was correlated with the isolated weighed tumors ( Figure 10 B). Kaplan-Meier analysis showed that on day 17, the tumor size in 75% of the animals treated with vehicle exceeded 500 mm 3 (Haynes et al., 2018), while on day 17, none of the tumors in the BT2-treated mice exceeded this size ( Figure 10 A and 11). LC-MS confirmed the bioavailability of BT2, with a serum concentration of 4 μg / ml or 12.3 μM on day 17 (data not shown). Immunohistochemical staining of tumors on day 17 showed that BT2 inhibited ERK phosphorylation ( Figure 12 A), without affecting the total ERK level ( Figure 12 B). In summary, these findings demonstrate that BT2 inhibits ERK phosphorylation and B16F10 growth in a xenograft model resistant to tumor suppression using anti-PD-1 antibody.

[0291] BT2 inhibits inflammation and increases anti-tumor immunity in immunocompetent mice

[0292] The inventors' recent studies have shown that BT2 has anti-inflammatory properties in arthritic mice (Yeh et al., 2021). The inventors hypothesized that this agent might have similar activity within tumors. Staining of the pan-macrophage marker CD68 in tumor cell nests of melanoma is associated with tumor recurrence and low survival rate. Immunohistochemical analysis of B16F10 tumors (day 17) from mice treated with BT2 or vehicle was performed using an antibody against CD68. IOD and tissue area were evaluated using Image-Pro Plus, and IOD / μm 2 was determined. The results are shown in Figure 13 . It was found that BT2 reduced the level of CD68 in tumors ( Figure 13 ).

[0293] Further immunohistochemical analysis of B16F10 tumors (day 17) from mice treated with BT2 or vehicle was performed using an antibody against CD3. The results are as shown in Figure 14 . This revealed that BT2 stimulated CD3 in the periphery of the tumor (i.e., within 250 μm) +Staining( Figure 14 A and 14B). The B16F10 tumor is an immunologically "cold tumor", meaning that tumor-infiltrating T cells are generally lacking (Bonaventura et al., 2019). Fu et al. found that CD3 + Tumor-infiltrating lymphocytes are prognostic markers for overall survival in melanoma (Fu et al., 2019). The inventors found increased CD3 + staining around the tumor, indicating that the immune system is involved in the anti-tumor activity of BT2.

[0294] IFN-γ is a biomarker for cellular and anti-tumor immune responses. Elevated IFN-γ is associated with systemic immune responses in cancer patients and tumor-bearing mice after immune checkpoint therapy. Since IFN-γ is produced by T cells, the levels of IFN-γ in the sera of tumor-bearing mice treated with vehicle or BT2 were measured. The results are presented in Figure 15 . The results showed that circulating IFN-γ levels were elevated in BT2-treated mice compared to vehicle-treated mice ( Figure 15 ).

[0295] BT2 inhibits the expression of PD-1 in tumors and human T cells

[0296] The increase in serum IFN-γ levels in BT2-treated mice prompted the inventors to determine the effect of the agent on intratumoral PD-1 levels. Immunohistochemical analysis of B16F10 tumors (day 17) from BT2- or vehicle-treated mice was also performed using antibodies against PD-1 and PD-L1. The results are shown in Figure 16 A and 16B. Immunohistochemical staining showed that PD-1 levels were reduced in B16F10 tumors treated with BT2 compared to those treated with vehicle ( Figure 16 A). Interestingly, it was found that PD-L1 levels also increased with BT2 treatment ( Figure 16 B). These findings have clinical relevance. Studies by Gettinger et al. showed that when patients' tumors express PD-L1, the overall response rate to anti-PD-1 therapy is higher (and there is a trend towards greater responses). Similarly, Vilain et al. found that tumor PD-L1 expression is a determinant of patients' responses to pembrolizumab / nivolumab. Inspired by these findings, the inventors investigated the effect of BT2 on PD-1 expression on cultured T cells.

[0297] Jurkat T cells express PD-1 (Yi et al., 2023) and are widely used as a model human T cell (e.g., Repas et al., 2022). Flow cytometry showed that 0.1 μM BT2 reduced PD-1 expression in Jurkat T cells by 50%, and 3 μM reduced PD-1 expression by 80% ( Figure 17A), these concentrations are much lower than those in tumor-bearing mice treated with BT2 in the above-mentioned study. The structural analogue of BT2, BT3 (Li et al., 2020), showed no inhibitory effect in the same concentration range ( Figure 17 B). The effect of BT2 is not only dose-dependent but also time-dependent. BT2 (3 μM) inhibited PD-1 levels by 50% after 24 hours and by 80% after 48 hours ( Figure 17 C).

[0298] Western blotting confirmed the flow cytometry data by showing that PD-1 expression in Jurkat T cells was inhibited by BT2 in a dose-dependent manner ( Figure 18 A and 18B). SCH772984 also inhibited PD-1 expression, but with >10-fold lower potency than BT2 ( Figure 18 A and 18B). The MEK1 / 2 inhibitor PD98059 had no effect on PD-1 expression ( Figure 19 A and 19B).

[0299] BT2 stimulated p-JNK / JUN and affected the enrichment of genes mediating immune responses and T cell activation

[0300] To gain insight into the mode of action of BT2, we performed next-generation RNA sequencing (RNA-seq) on extracts of Jurkat T cells exposed to 10 μM BT2 for 4 hours. Principal component analysis (PCA) and clustering analysis showed a clear separation between the treatment groups and a close association between biological replicates. RNA-seq showed that, based on a pool of 19,071 gene IDs, 856 genes were inhibited and 1,320 genes were induced by BT2, with an adjusted p-value < 0.05, of which 14 genes (Table 2) and 30 genes (Table 3) were differentially expressed by ≥2-fold (absolute fold change), respectively.

[0301] Table 2 - Gene expression inhibited by BT2 by at least 2-fold in Jurkat T cells

[0302]

[0303]

[0304] Table 3 - Gene expression increased by BT2 by at least 2-fold in Jurkat T cells

[0305]

[0306]

[0307]

[0308] Since BT2 was identified from an AP-1-dependent firefly luciferase screen (Li et al., 2020), it was surprisingly found that the most significantly induced genes (logFC≥2) by BT2 in these cells were the proto-oncogene JUN (Table 3). JUN was induced 5.9-fold by BT2 within 4 hours (Table 3). However, BT2 did not induce the expression of all AP-1 family members. For example, while BT2 increased the level of JUND by 2.4-fold, its effects on FOSB and FOS like 2 were negligible, and FOS was excluded from the analysis because it did not pass the low read filter cut-off.

[0309] BT2 altered the expression of multiple other genes. For example, BT2 increased the mRNA expression of dual specificity phosphatase 8 (DUSP8) by 14.1-fold (Table 3), and inhibited the expression of the transcription factor MAF by 2-fold (Table 2). These RNA data were confirmed by western blotting with extracts from Jurkat T cells incubated with BT2. The blots were incubated with anti-DUSP8 or anti-c-MAF antibodies and then with secondary antibodies. The results are shown in Figure 20 A and 20B as well as 21A and 21B. In Jurkat cells treated with BT2, the protein levels of DUSP8 ( Figure 20 ) and c-MAF ( Figure 21 ) were elevated. Gene set enrichment analysis (GSEA) showed that BT2 affected changes in the expression of different gene ontologies. Notably, genes mediating immune response and T cell activation were significantly enriched, including JUN, and multiple other members of the DUSP family.

[0310] Western blotting was performed with extracts from Jurkat T cells incubated with BT2 for different times, and the membranes were incubated with JUN, p-JNK, p-ERK and p38 antibodies and then with secondary antibodies. This confirmed that BT2 induced JUN ( Figure 22 A and 22B) at the protein level within 2 - 4 hours. BT2 stimulated JNK phosphorylation within 1 hour, but had no effect on the levels of ERK phosphorylation or p38 phosphorylation ( Figure 22 A and 22B).

[0311] BT2 induces JUN in tumor cells independent of BRAF mutation status

[0312] Western blotting was performed to determine the effect of BT2 on JUN expression in multiple melanoma cell lines. Before preparing the extracts and western blotting, A375, MDA-MB-435, MeWo were incubated with different amounts of BT2 or SCH772984 for 24 hours. Figure 23The results shown in A, 23B, and 23C demonstrated that in all three melanoma cell lines, BT2 increased JUN expression in a dose-dependent manner. The results also showed that JUN was induced in tumor cells regardless of the BRAF mutation status.

[0313] BT2 induces JUN in T cells through CD28 binding

[0314] In T cells, the c-jun promoter was activated by anti-CD28 binding. CD28 is a homodimeric cell surface glycoprotein and a co-stimulatory signal required for T cell activation. The inventors investigated whether BT2 might interact with CD28. In this regard, surface plasmon resonance (SPR) was measured using a Biacore T200 at 12 °C in 20 mM HEPES, 150 mM NaCl, 5% DMSO (pH 7.5) in the concentration range of 0.156 to 15 mM of BT2. The resulting sensorgrams are shown in Figure 24 . BT2 bound to CD28 in a dose-dependent manner.

[0315] To further evaluate the interaction between BT2 and CD28, the vehicle, BT2 (3 nmol), or a mixture of BT2 (3 nmol) and soluble recombinant CD28 (3 nmol) or recombinant epidermal growth factor (3 nmol) were pre-incubated in growth medium at 37 °C for 30 minutes and then added to Jurkat T cells in a 12-well plate (the final concentration of BT2, CD28, or EGF was 3 μM). After 24 hours, total cell lysates were prepared in RIPA buffer and subjected to Western blotting. The results are shown in Figure 25 A and 25B. Pre-incubation of BT2 with soluble recombinant CD28 inhibited BT2 from inducing JUN in Jurkat T cells, while BT2 still induced JUN after pre-incubation with EGF ( Figure 25 A and 25B).

[0316] After oral or intraperitoneal administration, BT2 was bioavailable in the circulating blood

[0317] BT2 in the plasma of mice administered BT2 by oral gavage (PO) or intraperitoneal (IP) injection was detected by LC-MS / MS. Plasma samples were processed (using protein precipitation) and injected into LC-MS / MS (SCIEX ExionLC coupled with TripleQuad 6500+).

[0318] The highest PO BT2 exposure was obtained with formulation 3 (G5) (AUClast = 340 h*ng / mL = 1.04 h*nM, Cmax at 1 hour (Tmax) = 53.7 ng / ml = 0.165 μM, and relative bioavailability was 28.8%). The second highest PO BT2 exposure was obtained with formulation 1 (G1) (AUClast = 189 h*ng / mL = 0.579 h*nM, Cmax at 15 minutes (Tmax) = 57.9 ng / mL = 0.177 μM, and relative bioavailability was 13.1%).

[0319] The highest IP BT2 exposure was obtained with formulation 1 IP (G2) (AUClast = 1440 h*ng / mL = 4.41 h*nM, Cmax at 15 minutes (Tmax) = 1073 ng / ml = 3.29 μM, and T1 / 2 = 8.72 hours). The second highest IP BT2 exposure was obtained with formulation 3 (G6) (AUClast = 1182 h*ng / ml = 3.62 h*nM, Cmax at 30 minutes (Tmax) = 846 ng / ml = 2.59 μM, and T1 / 2 = 8.42 hours).

[0320] Conclusion

[0321] An immunocompetent mouse model resistant to sustained inhibition of the PD-1 antibody (Kleffel et al., 2015) was used to test the ability of dibenzooxazepinone BT2 to inhibit PD-1 expression and tumor growth. In tumor cells, BT2 inhibits ERK phosphorylation and increases JUN expression. In T cells, BT2 increases JUN expression and decreases PD-1 expression; it also interacts with CD28 and increases JNK phosphorylation. BT2 inhibits tumor growth in a syngeneic mouse model resistant to PD-1 antibody growth inhibition. Although B16F10 (BRAF WT) is a "cold tumor" (Okada et al., 2020), representing resistance of melanoma patients to immune checkpoint inhibitors (Bonaventura et al., 2019), Kleffel et al. found that forced overexpression of pdcd1, which encodes PD-1, enhanced the growth of B16F10 tumors in C57BL / 6 mice, while shRNA knockdown of pdcd1 decreased the growth of B16F10 (Kleffel et al., 2015). This study shows that BT2 stimulates JUN in T cells and melanoma cells, and BT2 negatively regulates PD-1 in T cells. BT2 may be useful in small molecule strategies to increase JUN in T cells to help prevent T cell exhaustion and maintain effective and sustained tumor cell killing.

[0322] BT2 binds to CD28 and activates downstream signaling, particularly JUN, which negatively regulates PD-1 expression and increases T cell activity (Lynn et al., 2019). CD28 is a T cell co-stimulator, and its binding promotes naive T cell priming. Binding of CD28 can trigger JUN induction. Anti-CD28 monoclonal antibodies strongly promote T cell activity. CD80- and CD86-Ig RFP (recombinant fusion protein) targeting CD28 improves T cell responses and inhibits tumor growth. Different from anti-PD-1 antibodies, BT2 not only inhibits PD-1 expression but also binds to CD28, indicating that T cell activity is enhanced by BT2 and PD-1 antibody resistance is overcome. In addition, this article demonstrates that BT2 inhibits ERK activation (Thr 202 / Tyr 204 phosphorylation) in melanoma cells and can stimulate JUN expression in tumor cells, regardless of the BRAF mutation status (A375 (BRAF V600E ), MeWo (BRAF WT), MDA-MB-435 (BRAF V600E )) or the strength of tumor cell invasiveness. BT2 inhibits a series of cellular processes including migration and invasion more effectively than the ERK inhibitor SCH772984 and stimulates tumor cell apoptosis.

[0323] Tumor-associated macrophages (TAMs) are clinically associated with melanoma recurrence and low survival rates. The density of TAMs in invasive melanoma is higher compared to benign melanocytic lesions. In addition, TAMs promote cancer development and progression to malignancy. TAMs create a pro-inflammatory microenvironment and produce IL-1β, which is involved in BRAF inhibitor-induced tolerance in melanoma. Many chemotherapeutic drugs induce the processing and production of IL-1β. For example, the BRAF inhibitors dabrafenib and vemurafenib increase IL-1β gene expression and inflammasome activation in dendritic cells and macrophages. BT2 reduces the accumulation of CD68 + macrophages in melanoma.

[0324] In addition to stimulating JUN, BT2 also increased the levels of DUSP8 and other members of this family. DUSPs are a group of phosphatases that negatively regulate MAP kinases including JNK, p38, and ERK1 / 2, and inhibit the production of IL-1β, IL-6, and other key pro-inflammatory cytokines including TNF-α. The induction of DUSP by BT2 suggests possible autoregulation, including increased JNK phosphorylation and JUN expression. This has therapeutic implications. For example, DUSP4 regulates responsiveness to MEK inhibition in BRAF wild-type tumors, and depletion of DUSP4 induces resistance to MEK inhibitors. DUSP is upregulated in T cells stimulated with anti-CD28 and anti-CD3. Recent studies have shown an inverse relationship between DUSP8 and T cell exhaustion. DUSP8 also negatively regulates ERK phosphorylation. BT2 decreased the level of c-MAF, a transcription factor and key regulator of T cells, which is also induced by anti-CD28 and anti-CD3. Increased c-MAF expression in T cells is associated with tumor metastasis, and its induction requires IL-6 and TGF-β. c-MAF is a driver of T cell exhaustion, in which c-MAF overexpression inhibits the production of IFN-γ and IL-2 by T cells and enhances PD-1 expression, which is associated with T cell dysfunction and exhaustion. Giordano et al. knocked out c-MAF, resulting in increased IFN-γ production in tumor-infiltrating lymphocytes (TILs), and after adoptive transfer, reduced tumor growth and increased the survival rate of tumor-bearing mice. Chihara et al. reported that c-MAF deficiency inhibits PD-1 expression in T cells. c-MAF functionally interacts with other T cell function regulators such as PRDM1 that regulate PD-1 expression on T cells. Recently, ChIP-seq of human T cells immunoprecipitated with c-MAF revealed that approximately 70% of the induced gene loci were associated with c-MAF, and lentiviral c-MAF overexpression in T cells increased PD-1 expression. By regulating c-MAF and DUSP expression, BT2 can control PD-1 and prevent T cell exhaustion. At the same time, the data in this article show that BT2 promotes intratumoral T cell infiltration. For example, BT2 increased netrin-1 (NTN1) by 90.6-fold, which is associated with increased CD4 + T cell chemotaxis and inflammatory cell infiltration.

[0325] As a small molecule inhibitor of PD-1, BT2 offers many potential advantages over antibody or macromolecular products, including lower production costs, the ability to penetrate cell membranes, and a tendency for oral administration. Moreover, all currently approved PD-L1 or PD-1 inhibitors are antibodies that require intravenous infusion. There are no small molecule drugs clinically available that can simultaneously inhibit ERK and PD-1. Given the high cost of immunotherapy, an effective small molecule may reduce the cost to the health system and eliminate the reliance on clinical drug management. Compared with "bispecific" antibodies, the production of the bifunctional small molecule BT2 is simpler and less costly. BT2 inhibits melanoma growth in models resistant to PD-1 antibody inhibition, indicating its potential use in patients in whom tumors are unresponsive to immune checkpoint immunotherapy. As a potential anti-cancer drug that simultaneously inhibits the ERK and PD-1 / PD-L1 systems, BT2 can be used to treat tumors other than melanoma, including skin tumors such as, but not limited to, squamous cell carcinoma, basal cell carcinoma, skin sarcoma, and Merkel cell carcinoma, as well as other tumor types such as head and neck cancer, non-small cell lung cancer, urothelial cancer, thyroid cancer, renal cell carcinoma, breast cancer, and hepatocellular carcinoma, particularly where there is evidence that PD-1 / PD-L1 inhibition anti-tumor immune responses.

[0326] The ability of BT2 to act as both an ERK and a PD-1 inhibitor may reduce the risk of treatment-related toxicities, thereby interfering with current clinical strategies, as one drug rather than multiple drugs will be administered. Additionally, a drug that can inhibit ERK activation and PD-1 function would be a potentially invaluable tool, particularly in cases of PD-1 antibody resistance.

[0327] In the following claims and the prior description of the present invention, unless the context otherwise requires by reason of express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" are used in an inclusive sense, i.e., to specify the presence of the stated features, but not to exclude the presence or addition of other features in the various embodiments of the present invention.

[0328] References

[0329] Bonaventura, P. et al., 2019. Front Immunol 10, 168.

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[0334] Giordano, M. et al., 2015. EMBO J 34, 2042 - 2058.

[0335] Kleffel, S. et al., 2015. Cell 162, 1242 - 1256.

[0336] Li, Y. et al., 2019. Cancer Lett 459, 268 - 276.

[0337] Li, Y. et al., 2020. Science Advances 6, eaaz7815.

[0338] Liberzon, A. et al., 2011. Bioinformatics 27, 1739 - 1740.

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Claims

1. A method for increasing immune cell activation and / or treating cancer in a subject, which comprises administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof: Wherein: R 1 is a straight-chain or branched C1-C6 alkyl group; and R 2 is a straight-chain or branched C1-C6 alkyl group, or R 2 is where q is 1, 2, 3 or 4; and R 3 is a straight-chain or branched C1-C6 alkyl group.

2. The method according to claim 1, wherein R 1 is a straight-chain C1-C6 alkyl group or a branched C1-C6 alkyl group.

3. The method according to claim 1, wherein R 1 is -CH2CH3 or -CH2CH(CH3)2.

4. The method according to claim 1, wherein R 2 is a straight-chain C1-C6 alkyl group or a branched C1-C6 alkyl group.

5. The method according to claim 1, wherein R 2 is -CH2CH3 or -CH2CH(CH3)2.

6. The method according to claim 1, wherein the compound of formula (I) is a compound of formula (I-1): Wherein: R 2 is a straight-chain or branched C1-C6 alkyl group; or R 2 is: where q is 1, 2, 3 or 4; and R 3 is a straight-chain or branched C1-C6 alkyl group.

7. The method according to claim 1, wherein the compound of formula (I) is a compound of formula (II):

8. The method according to claim 1, wherein the expression of PD-1 in the T cells of the subject is reduced.

9. The method according to any one of claims 1 to 8, wherein the cancer is resistant to the treatment with dabrafenib and trametinib, or comprises cells resistant to the treatment with dabrafenib and trametinib.

10. The method according to claim 9, wherein the cancer comprises BRAF mutant cells or wild-type cells.

11. The method according to any one of claims 1 to 10, wherein the compound increases the expression of JUN in the tumor cells of the cancer.

12. The method according to any one of claims 1 to 8, wherein the cancer is resistant to PD-1 antibody treatment.

13. A method for reducing the expression of PD-1 in the T cells of a subject, which comprises administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

14. The method according to claim 13, wherein the compound of formula (I) is:

15. A method for reducing ERK phosphorylation and increasing JUN expression in the tumor cells of a subject, and / or reducing PD-1 expression and / or increasing JUN expression and / or increasing JNK phosphorylation in the T cells of a subject, which comprises administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

16. The method according to claim 15, wherein the compound of formula (I) is a compound of formula (II):

17. The method according to any one of claims 1 to 16, wherein the expression of JUN in the T cells of the subject is increased.

18. The method according to any one of claims 1 to 17, wherein the phosphorylation of JNK in the T cells of the subject is increased.

19. The method according to any one of claims 1 to 18, wherein the expression of DUSP8 in the T cells of the subject is increased.

20. The method according to any one of claims 1 to 19, wherein the expression of MAF in the T cells of the subject is reduced.

21. The method according to any one of claims 15 to 20, wherein the tumor cells are resistant to the treatment with dabrafenib and trametinib.

22. The method according to any one of claims 15 to 21, wherein the tumor cells are BRAF mutant cells or wild-type cells.

23. A kit for increasing immune cell activation and / or treating cancer, which comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof.

24. A kit for reducing ERK phosphorylation in tumor cells and / or reducing PD-1 expression in T cells and / or increasing JUN expression and / or increasing JNK phosphorylation, the kit comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof.

25. The medicine box according to claim 23 or 24, wherein the compound of formula (I) is: or a pharmaceutically acceptable salt thereof.

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