Methods of Increasing Immune Cell Activation and / or Treating Cancer Using Dibenzoxazepinones

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) or a pharmaceutically acceptable salt thereof. TIFF2025535140000031.tif23161

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to methods and pharmaceutical compositions for increasing immune cell activation and methods for treating cancer. [Background technology]

[0002] background Cancer is one of the leading causes of death worldwide. Despite progress in the development of treatments for cancer, there remains a high need for improved therapies, for example, to treat cancers where tumor resistance to existing therapies is an issue or tumors that are unresponsive to existing therapies.

[0003] One approach that has shown promise in the treatment of cancer is cancer immunotherapy. Cancer immunotherapy is a type of treatment that involves modulating a patient's immune system to treat cancer. Antibodies targeting programmed cell death protein-1 (PD-1) have shown particular promise in cancer immunotherapy. PD-1 and its ligand, PD-L1, are checkpoint regulators that suppress the body's immune response to 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 expressed primarily by T cells, but also 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 cell lysis 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 against PD-1 and PD-L1 have been developed and have shown promise in the treatment of multiple malignancies. For example, antibodies approved for use in the treatment of various cancers and / or in clinical trials include the anti-PD-1 antibodies nivolumab (for metastatic melanoma) and pembrolizumab (for the treatment of metastatic melanoma, lymphoma, mesothelioma, and non-small cell lung cancer); and anti-PD-L1 antibodies, including avelumab (for urothelial carcinoma, Merkel cell carcinoma, and 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] Antibodies against PD-1 have shown promising results in cancer treatment, but the use of antibody-based therapies has limitations. For example, 40% of melanoma patients who initially respond to PD-1 inhibitors experience progression of existing or new lesions within 3 years. In metastatic melanoma, long-term survival (>3 years) with anti-CTLA-4 therapy is only approximately 20% and approximately 30–50% when combined with a PD-1 antibody. KEYNOTE-001 demonstrated that the overall 5-year survival rate for patients with advanced melanoma treated with anti-PD-1 (pembrolizumab) was 34% for all patients and 41% for treatment-naive patients, with treatment-related adverse events recorded in 86% of patients. The combination of anti-PD-1 (nivolumab) and anti-CTLA-4 (ipilimumab) increased the response rate to 53%, but treatment-related severe adverse events (grade ≥3) were recorded in 53% of patients, leading to treatment discontinuation in 21%. Of concern, a recent study identified venous thromboembolism in 24% of cancer patients receiving immunotherapy, resulting in reduced overall survival.

[0006] There is a need for safer and more effective immune checkpoint therapies. Small molecules offer potential advantages over antibodies, such as favorable pharmacokinetics and druggability, and are amenable to oral formulation and outpatient administration. This potentially avoids intravenous (IV) administration and associated risks, and offers greater patient convenience, particularly among frail patients. Small molecules are typically cheaper to produce and more stable than antibodies. Currently, there are no clinically approved small molecule inhibitors of the PD-1 / PD-L1 system, despite the system being a primary target in immunotherapy.

[0007] Inhibitors of BRAF and MEK have also shown promise in cancer treatment. For example, first-line treatment of patients with metastatic melanoma with dabrafenib and trametinib inhibits BRAF. V600E or V600KOne of three melanoma patients with the mutation achieved 5-year survival. However, 30% of patients developed higher-grade 3 / 4 toxicity, which often led to dose reductions and treatment delays. Importantly, resistance can develop after 9–12 months, likely due to activation of other signaling pathways or modulation of the immune system. Increased downstream ERK signaling is a mechanism of resistance to BRAF / MEK inhibition, leading to various preclinical and clinical initiatives targeting ERK. Furthermore, recent studies in mice bearing non-small cell lung cancer have shown that an ERK inhibitor (PD0325901) can enhance the efficacy of anti-PD-1 antibodies. ASN007 is another ERK1 / 2 kinase inhibitor that has demonstrated efficacy in resistant melanoma PDX models. However, of the 62 FDA-approved small molecule therapeutics targeting over 20 different protein kinases, eight were approved in 2020, and although several are in clinical investigation, none are ERK inhibitors (e.g., ulixertinib NCT03698994 National Cancer Institute; LY3214996 NCT02857270 Eli Lilly; BVD-523 NCT03417739 BioMed Valley Discoveries; LTT462 NCT02711345 Novartis; MK-8353 NCT02972034 Merck Sharp & Dohme).

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

[0009] What is needed are non-antibody alternatives for the treatment of cancer that target immune checkpoint inhibition and ERK signaling.

[0010] overview The present inventors have found that compound BT2 (compound of formula (II)) functions as both an immune checkpoint inhibitor and a targeted inhibitor of ERK signaling. In this regard, the present inventors have found that BT2: (i) inhibiting ERK phosphorylation and increasing JUN (also known as c-Jun) expression in tumor cells; and (ii) In T cells, it reduces PD-1 expression, increases JUN expression, increases JNK phosphorylation, and interacts with CD28. We found that...

[0011] Therefore, the inventors reasoned that BT2 may be beneficial in increasing immune cell activation and in the treatment of cancer through its combined activities as an immune checkpoint inhibitor, an inhibitor of ERK phosphorylation, and a promoter of JUN expression.

[0012] The first aspect is a compound of formula (I): [ka] [In formula: R 1 is a straight or branched C1-C6 alkyl; R 2 is a straight or branched C1-C6 alkyl; Or, R 2 teeth, [ka] (wherein q is 1, 2, 3, or 4; R 3 is a straight-chain or branched C1-C6 alkyl is] The present invention provides a method for increasing immune cell activation and / or treating cancer in a subject, comprising administering a compound of the formula:

[0013] An alternative first aspect provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in increasing immune cell activation and / or treating cancer in a subject; or 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.

[0014] A second aspect is a compound of formula (II): [ka] The present invention provides a method for increasing immune cell activation and / or treating cancer in a subject, comprising administering a compound of the formula:

[0015] An alternative second aspect provides a compound of formula (II) or a pharmaceutically acceptable salt thereof for use in increasing immune cell activation and / or treating cancer in a subject; or 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.

[0016] 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, comprising administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0017] An alternative third aspect provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in 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; or 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 reducing PD-1 expression and / or increasing JUN expression and / or increasing JNK phosphorylation in T cells of a subject.

[0018] A fourth 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, comprising administering an effective amount of a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0019] An alternative fourth aspect provides a compound of formula (II) or a pharmaceutically acceptable salt thereof for use in 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; or 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 reducing PD-1 expression and / or increasing JUN expression and / or increasing JNK phosphorylation in T cells of a subject.

[0020] A fifth aspect provides a method of reducing ERK phosphorylation in tumor cells of a subject and reducing PD-1 expression in T cells of the subject, comprising administering an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

[0021] An alternative fifth aspect provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in reducing ERK phosphorylation in tumor cells of a subject and reducing PD-1 expression in T cells of the subject; or 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 reducing PD-1 expression in T cells of a subject.

[0022] A sixth aspect provides a method of reducing ERK phosphorylation in cancer cells of a subject and reducing PD-1 expression in T cells of the subject, comprising administering an effective amount of a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0023] An alternative sixth aspect provides a compound of formula (II) or a pharmaceutically acceptable salt thereof for use in reducing ERK phosphorylation in tumor cells of a subject and reducing PD-1 expression in T cells of the subject; or 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 a subject.

[0024] A seventh aspect provides a method of treating a disease or condition associated with PD-1 expression in T cells in a subject, comprising administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0025] An alternative seventh aspect provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in treating a disease or condition associated with PD-1 expression on T cells 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 treating a disease or condition associated with PD-1 expression on T cells in a subject.

[0026] An eighth aspect provides a method of treating a disease or condition associated with PD-1 expression in T cells in a subject, comprising administering an effective amount of a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0027] An alternative eighth aspect provides a compound of formula (II), or a pharmaceutically acceptable salt thereof, for use in treating a disease or condition associated with PD-1 expression on T cells 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 treating a disease or condition associated with PD-1 expression on T cells in a subject.

[0028] A ninth aspect provides a method of increasing immune cell activation and / or reducing the rate of tumor growth in a subject suffering from cancer, comprising administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0029] An alternative ninth aspect provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in increasing immune cell activation and / or reducing the rate of tumor growth in a subject suffering from cancer; or 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 the rate of tumor growth in a subject suffering from cancer.

[0030] A tenth aspect provides a method of increasing immune cell activation and / or reducing the rate of tumor growth in a subject suffering from cancer, comprising administering an effective amount of a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0031] An alternative tenth aspect provides a compound of formula (II) or a pharmaceutically acceptable salt thereof for use in increasing immune cell activation and / or reducing the rate of tumor growth in a subject suffering from cancer; or 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 the rate of tumor growth in a subject suffering from cancer.

[0032] An eleventh aspect provides a method of reducing PD-1 expression in T cells of a subject, comprising administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0033] An alternative eleventh aspect provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in reducing PD-1 expression in T cells of a subject; or 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.

[0034] A twelfth aspect provides a method of reducing PD-1 expression in T cells in a subject, comprising administering an effective amount of a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0035] An alternative twelfth aspect provides a compound of formula (II) or a pharmaceutically acceptable salt thereof for use in reducing PD-1 expression in T cells of a subject; or 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.

[0036] A thirteenth aspect provides a method of 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, comprising contacting tumor cells or T cells with an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0037] An alternative thirteenth aspect provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in 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 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.

[0038] A fourteenth aspect provides a method of 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, comprising contacting tumor cells or T cells with an effective amount of a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0039] An alternative fourteenth aspect provides a compound of formula (II) or a pharmaceutically acceptable salt thereof for use in 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 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.

[0040] A fifteenth aspect provides a method of decreasing expression of a gene listed in Table 2 and / or increasing expression of a gene listed in Table 3 in a T cell, comprising contacting the T cell with an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0041] An alternative fifteenth aspect provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in decreasing expression of a gene listed in Table 3 and / or increasing expression of a gene listed in Table 3 in a T cell; or use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for decreasing expression of a gene listed in Table 2 and / or increasing expression of a gene listed in Table 3 in a T cell.

[0042] A sixteenth aspect provides a method of decreasing expression of a gene listed in Table 2 and / or increasing expression of a gene listed in Table 3 in a T cell, comprising contacting the T cell with an effective amount of a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0043] An alternative sixteenth aspect provides a compound of formula (II) or a pharmaceutically acceptable salt thereof for use in decreasing the expression of genes listed in Table 2 and / or increasing the expression of genes listed in Table 3 in a T cell; or use of a compound of formula (II) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for decreasing the expression of genes listed in Table 2 and / or increasing the expression of genes listed in Table 3 in a T cell.

[0044] A 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.

[0045] An 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.

[0046] A nineteenth aspect provides a kit for reducing inflammation in a tumor, and / or reducing PD-1 expression in T cells, and / or increasing JUN expression, and / or increasing JNK phosphorylation, and / or increasing JUN expression in tumor cells of a subject, the kit comprising a compound of formula (II) or a pharmaceutically acceptable salt thereof. [Brief explanation of the drawings]

[0047] Preferred embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings in which: [Figure 1]Figure 1 shows (A, C) images of Western blots performed on extracts from A375 (A, B) and MeWo (C, D) melanoma cells incubated with either 10 or 100 nM BT2 (Table 1) or SCH772984 for 24 hours, followed by incubation with antibodies against p-ERK or total ERK, followed by secondary antibodies. The approximate positions of molecular weight markers are indicated. SCH refers to SCH772984. Graphs in (B, D) show Western blot band intensities quantified using Image J. Plotted data represent mean ± SEM. Data represent two biologically independent experiments. [Figure 2] Figure 2 shows quantification of migration into (A, C) the denuded zone of Matrigel® and invasion into (B, D) the denuded zone of A375 cells (A, B) and MeWo cells (C, D) after 24 and 48 hours of incubation with 1 μM BT2 or SCH772984, respectively. Data plotted represent the mean ± SEM of the mean of three to four biologically independent experiments. Statistical significance was assessed by one-way analysis of variance or Kruskal-Wallis test, as appropriate. [Figure 3] FIG. 3 shows images of the morphology of A375 and MeWo cells exposed to 1 μM vehicle (Veh), BT2, or SCH772984 for 24 hours. [Figure 4] Figure 4 is a graph showing the results of an apoptosis assay performed using (A) A375 cells and (B) MeWo cells exposed to 1 μM of either BT2 or SCH772984 for 24 hours. Plotted data (Annexin V-FITC + PI-) represent the mean ± SEM of the mean of three biologically independent experiments. Statistical significance was assessed by one-way analysis of variance. [Figure 5]Figure 5 shows the results of intratumoral (it) treatment of CB17 SCID mice bearing subcutaneous (sc) melanoma (MDA-MB-435) with 20 mg / kg BT2 or vehicle once daily on a 5-day on / 2-day off schedule. Treatment began on day 16. (A) is a schematic diagram illustrating the treatment regimen. (B) is a graph showing tumor growth (volume), and (C) is a graph showing body weight data provided by the animal study described in Li et al. (Li et al., 2019) using the same vehicle group. Data represent mean ± SEM. n = 10 mice per group. Statistical significance was assessed by t-test. [Figure 6] Figure 6 shows the quantification of immunohistochemical analysis performed with antibodies against (A, B) p-ERK or (C, D) total ERK in MDA-MB-435 tumors from mice treated with BT2 or vehicle. IOD and tissue area were quantified using Image-Pro Plus. Data represent the mean ± SEM for n = 9–10 mice per group. Statistical significance was assessed by the Mann-Whitney test. [Figure 7] Figure 7 shows the effect of daily intraperitoneal (ip) treatment of CB17 SCID mice bearing subcutaneous MDA-MB-435 tumors with 200 mg / kg BT2 or vehicle on a 5-day on / 2-day off schedule on tumor volume and body weight. Treatment began on day 16. (A) is a schematic diagram of the treatment regimen. (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 provided by the animal study described in Li et al. (Li et al., 2019) using the same vehicle group. Data represent mean ± SEM. n = 10 mice per group. [Figure 8]Figure 8 shows the effect of twice-weekly treatment of C57BL / 6J mice bearing subcutaneous melanoma (B16F10) with anti-mouse PD-1 monoclonal antibody or control IgG (100 μg, i.p.) on tumor volume and body weight. Treatment began on day 7. (A) Schematic representation of the treatment regimen. (B) Graph showing the change in tumor volume over time for animals treated with BT2 or vehicle. (C) Measurements of body weight over time. Data represent mean ± SEM. n = 6 mice per group. Statistical significance was assessed by Mann-Whitney or t-test, as appropriate. [Figure 9] Figure 9 shows the effect on tumor volume and body weight of once-daily treatment of C57BL / 6J mice bearing subcutaneous B16F10 tumors with BT2 or vehicle (200 mg / kg or 20 ml / kg, i.p., respectively) on a 5-day on / 2-day off schedule. Treatment began on day 5. (A) is a schematic diagram of the treatment regimen. (B) is a graph showing the change in tumor volume over time for animals treated with BT2 or vehicle. (C) also shows body weight measurements over time. Data represent mean ± SEM. n = 8 mice per group. Statistical significance was assessed by Mann-Whitney or t-test, as appropriate. [Figure 10] Figure 10 is a graph showing (A) tumor size and (B) isolated tumor weight at individual days 17 from mice treated with BT2 or vehicle. Data represent mean ± SEM. n = 8 mice / group. Statistical significance was assessed by t-test. [Figure 11] Figure 11 shows a Kaplan-Meier analysis of viability performed with a tumor size limit of 500 mm3 as previously described (Haynes et al., 2018), n = 8 mice / group. Statistical significance was assessed by the log-rank (Mantel-Cox) test and the Gehan-Breslow-Wilcoxon test. [Figure 12]Figure 12 is a graph showing quantification of immunohistochemistry performed with antibodies against (A) p-ERK or (B) total ERK on B16F10 tumors (day 17) from mice treated with BT2 or vehicle. IOD and tissue area were quantified using Image-Pro Plus. Data represent mean ± SEM for n = 8 mice / group. Statistical significance was assessed by the Mann-Whitney test. [Figure 13] Figure 13 is a graph showing quantification of immunohistochemistry analysis performed with an antibody against CD68 on B16F10 tumors (day 17) from mice treated with BT2 or vehicle. IOD and tissue area were assessed using Image-Pro Plus, and IOD / μm2 was determined. Data represent mean ± SEM of the mean / animal. n=8 animals / group. Statistical significance was assessed by t-test. [Figure 14] Figure 14 is a graph showing quantification of immunohistochemistry performed using an antibody against CD3 on B16F10 tumors (day 17) from mice treated with BT2 or vehicle. (A) IOD and tissue area were assessed using Image-Pro Plus, and IOD / µm2 was determined. (B) CD3-positive cell counts and total cell counts were quantified using Image-Pro Plus, and the % of CD3+ cells was determined. Data represent the mean ± SEM of the mean / animal. n = 7–8 animals / group. Statistical significance was assessed by the Mann-Whitney test. [Figure 15] Figure 15 is a graph showing serum IFN-γ levels, as determined by ELISA, in mice treated with vehicle or BT2. n=8 per group. Statistical significance was assessed by t-test. Data represent mean ± SEM. [Figure 16]Figure 16 shows the quantification of immunohistochemistry performed with antibodies against (A) PD-1 and (B) PD-L1 on B16F10 tumors (day 17) from mice treated with BT2 or vehicle. IOD and tissue area were assessed using Image-Pro Plus, and IOD / µm2 was determined. Data represent mean ± SEM of the mean / animal. n = 7–8 animals / group. Statistical significance was assessed by the Mann-Whitney test. [Figure 17] Figure 17 is a graph showing the results of flow cytometry performed using (A) Jurkat T cells treated with PD-1 antibody (or IgG) and the indicated concentrations of BT2 or vehicle for 48 hours; (B) Jurkat T cells treated with PD-1 antibody (or IgG) and vehicle or the indicated concentrations of BT3 for 48 hours; and (C) Jurkat T cells treated with PD-1 antibody (or IgG) and 3 μM BT2 or vehicle for the indicated times. Statistical significance was assessed by one-way analysis of variance. Data represent the mean ± SEM of the average of three biologically independent experiments. [Figure 18] Figure 18 shows (A) images of Western blots performed on extracts of Jurkat T cells treated with various concentrations of BT2 or SCH772984 for 24 hours. Membranes were incubated with PD-1 or β-actin antibodies, followed by secondary antibodies. (B) is a graph showing band intensities from the Western blot in (A) quantified using Image J; plotted data represent mean ± SEM. Data represent two biologically independent experiments. The approximate locations of molecular weight markers are indicated. [Figure 19]Figure 19 shows (A) images of Western blots performed on extracts of Jurkat T cells treated with various concentrations of BT2 or PD98059 for 24 hours. Membranes were incubated with PD-1 or β-actin antibodies, followed by secondary antibodies. (B) is a graph showing band intensities from the Western blots quantified using Image J; plotted data represent mean ± SEM. Data represent two biologically independent experiments. The approximate locations of molecular weight markers are indicated. [Figure 20] Figure 20 shows (A) an image of a Western blot performed on extracts of Jurkat T cells incubated with BT2 or PD98059 for 24 hours. The membrane was incubated with DUSP8 antibody followed by a secondary antibody. (B) is a graph showing band intensities from the Western blot quantified using Image J; plotted data represent mean ± SEM. Data represent two biologically independent experiments. The approximate positions of molecular weight markers are indicated. [Figure 21] Figure 21 shows (A) an image of a Western blot performed on extracts of Jurkat T cells incubated with BT2 or PD98059 for 24 hours. The membrane was incubated with c-MAF antibody followed by a secondary antibody. (B) is a graph showing band intensities from the Western blot quantified using Image J; plotted data represent mean ± SEM. Data represent two biologically independent experiments. The approximate positions of molecular weight markers are indicated. [Figure 22-1] Figure 22 shows (A) images of Western blots performed on extracts from Jurkat T cells incubated with BT2 for various times. Membranes were incubated with the indicated antibodies followed by secondary antibodies. [Figure 22-2] (B) Graph showing band intensities from Western blots quantified using Image J. Plotted data represent mean ± SEM. Data are representative of two biologically independent experiments. Approximate positions of molecular weight markers are indicated. [Figure 23] Figure 23 shows images of Western blots performed on extracts from (A) A375, (B) MDA-MB-435, and (C) MeWo cells incubated with the indicated amounts of BT2 or SCH772984 for 24 hours. Membranes were incubated with JUN or β-actin antibodies, followed by secondary antibodies. Beneath each Western blot is a graph showing Western blot band intensities quantified using Image J; plotted data represent mean ± SEM. Data represent two biologically independent experiments. The approximate locations of molecular weight markers are indicated. [Figure 24] Figure 24 is a sensorgram showing BT2 binding to CD28 over a concentration range of 0.156 to 15 μM BT2. Measurements were performed on a Biacore T200 at 12°C in 20 mM HEPES, 150 mM NaCl, 5% DMSO (pH 7.5). [Figure 25] Figure 25 shows (A) images of Western blots in which vehicle, BT2 (3 nmol), or a mixture of BT2 (3 nmol) and CD28 (3 nmol) or EGF (3 nmol) was preincubated in growth medium for 30 minutes at 37°C and then added to Jurkat T cells in 12-well plates (final concentrations of BT2, CD28, or EGF were 3 μM). After 24 hours, whole-cell lysates were prepared in RIPA buffer. Membranes were incubated with the indicated primary antibodies followed by secondary antibodies. (B) is a graph showing band intensities from the Western blots quantified using Image J; plotted data represent mean ± SEM. Data represent two biologically independent experiments. The approximate locations of molecular weight markers are indicated.

[0048] Detailed Description The compound BT2 is a dibenzoxapinone that has previously been shown to inhibit endothelial cell proliferation and migration, angiogenesis, and wound healing. BT2 has been shown to inhibit ERK phosphorylation and expression of FosB / ΔFosB and VCAM-1, among others, VEGF, in endothelial cells.

[0049] As described in the Examples, the present inventors have now discovered that BT2 (a compound of formula (II)) is (a) Reduce ERK phosphorylation in cancer cells; (b) reducing tumor cell migration; (c) reducing tumor cell invasion; (d) increasing apoptosis in cancer cells; (e) reducing the rate of tumor growth; (f) reducing tumor volume; (g) associates with CD28 on T cells; (h) reducing PD-1 expression on T cells; (i) Increase JUN expression in T cells; (j) increasing JUN expression in tumor cells; (k) increasing JNK phosphorylation in T cells; (l) inhibiting tumor inflammation; (m) Increase tumor immunity We found that...

[0050] Therefore, the inventors expect that compounds of formula (I) and (II) will be effective in increasing immune cell activation, reducing tumor inflammation, and treating cancer.

[0051] One aspect is a method of increasing immune cell activation and / or treating cancer in a subject, comprising administering an effective amount of a compound of Formula (I): [ka] [In formula: R 1is a straight or branched C1-C6 alkyl; R 2 is a straight or branched C1-C6 alkyl, or R 2 is the group: [ka] (wherein q is 1, 2, 3, or 4; R 3 is a straight-chain or branched C1-C6 alkyl is] or a pharmaceutically acceptable salt thereof.

[0052] In one embodiment, the compound of formula (I) has formula (II): [ka] It has the structure shown below.

[0053] One aspect provides a method of increasing immune cell activation and treating cancer in a subject, comprising administering an effective amount of a compound of Formula (I), typically Formula (II), or a pharmaceutically acceptable salt thereof.

[0054] Another aspect is a method of reducing ERK phosphorylation in tumor cells of a subject and / or reducing PD-1 expression in T cells of a subject, comprising administering to the subject an effective amount of a compound of Formula (I): [ka] [In formula: R 1 is a straight or branched C1-C6 alkyl; R 2 is a straight or branched C1-C6 alkyl, or R 2 teeth, [ka] (wherein q is 1, 2, 3, or 4; R 3 is a straight-chain or branched C1-C6 alkyl is] or a pharmaceutically acceptable salt thereof.

[0055] In one embodiment, the compound of formula (I) has formula (II): [ka] It has the structure shown below.

[0056] In one embodiment, the method increases JUN expression and JNK phosphorylation in T cells of the subject.

[0057] In one embodiment, the method increases JUN expression in tumor cells of the subject.

[0058] In one embodiment, the method reduces ERK phosphorylation, increases JUN expression, and reduces PD-1 expression in tumor cells of the subject.

[0059] In one embodiment, the method reduces ERK phosphorylation, increases JUN expression, and reduces PD-1 expression in tumor cells of the subject.

[0060] 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.

[0061] In one embodiment, the method increases DUSP8 expression in T cells of the subject.

[0062] In one embodiment, the method reduces MAF expression in T cells of the subject.

[0063] In one embodiment, the method reduces tumor cell migration and invasion in the subject.

[0064] In one embodiment, the method increases apoptosis of tumor cells in a subject.

[0065] In one embodiment, the method reduces the rate of tumor growth in a subject.

[0066] In one embodiment, the method reduces tumor cell migration, reduces tumor cell invasion, increases tumor cell apoptosis, or reduces tumor growth rate in a subject.

[0067] The compound of formula (I) is: [ka] [In formula: R 1 is a straight or branched C1-C6 alkyl; R 2 is a straight or branched C1-C6 alkyl, or R 2 teeth, [ka] (wherein q is 1, 2, 3, or 4; R 3 is a straight-chain or branched C1-C6 alkyl is] is.

[0068] 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.

[0069] In some embodiments of Formula (I), R 2is 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.

[0070] In some embodiments of Formula (I), R 2 is the following: [ka] (wherein q is 1, 2, 3, or 4; R 3 is a straight chain C1-C6 alkyl or a branched C1-C6 alkyl) In some embodiments of Formula (I), q is 2. In some embodiments of Formula (I), R 3 In some embodiments of Formula (I), q is 2 and R 3 is -CH3.

[0071] In some embodiments, the compound of formula (I) has the formula (I-1): [ka] [In formula: R 2 is a straight chain or branched C1-C6 alkyl; or R 2 teeth, [ka] (wherein q is 1, 2, 3, or 4; R 3 is a straight-chain or branched C1-C6 alkyl is] The compound may be a compound represented by the formula:

[0072] In one embodiment, the compound of formula (I) is a compound of formula (II).

[0073] The compound of formula (II) is: [ka] (also referred to herein as BT2).

[0074] A compound that increases immune cell activation refers to a compound that induces, induces, or promotes immune cells, typically T cells, to have an increased biological function or activity after contact with the compound compared to the biological function or activity of immune cells not contacted with the compound. Examples of increased immune cell activation include an increased T cell response to an antigen, increased proliferation, increased IFN-γ secretion from T cells, increased JUN expression in T cells, decreased PD-1 expression in T cells, increased DUSP8 expression in T cells, and increased JNK phosphorylation in T cells.

[0075] 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 compared to the amount of PD-1 protein produced by the cell or tissue not contacted with the compound.

[0076] 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 compared to the degree of JNK phosphorylation in a cell or tissue that has not been contacted with the compound.

[0077] 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 compared to the amount of JUN protein produced by a cell or tissue that has not been contacted with the compound.

[0078] 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 compared to the degree of ERK phosphorylation in a cell or tissue that has not been contacted with the compound.

[0079] 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 compared to the amount of DUSP8 protein produced by a cell or tissue that has not been contacted with the compound.

[0080] 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 compared to the amount of MAF protein produced by the cell or tissue that has not been contacted with the compound.

[0081] In one embodiment, the compound increases JUN expression in T cells.

[0082] In one embodiment, the compound increases JUN expression in tumor cells.

[0083] In one embodiment, the compound reduces PD-1 expression on T cells.

[0084] In one embodiment, the compound increases DUSP8 expression in T cells.

[0085] In one embodiment, the compound reduces MAF expression in T cells.

[0086] In one embodiment, the compound increases circulating IFN-γ.

[0087] In one embodiment, the compound increases PD-L1 expression in tumor cells.

[0088] In one embodiment, the compound increases netrin-1 expression in T cells.

[0089] In one embodiment, the compound associates with CD28.

[0090] In one embodiment, the compound reduces tumor inflammation and / or tumor size and / or increases tumor immunity.

[0091] 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; 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, sulfanilic acid, aspartic acid, glutamic acid, edetic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid, and valeric acid.

[0092] In one embodiment, the compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof is deuterated.

[0093] In one embodiment, the compounds of Formula (I) or (II) or pharmaceutically acceptable salts thereof are E isomers.

[0094] In one embodiment, the compounds of formula (I) or (II) or pharmaceutically acceptable salts thereof are Z isomers.

[0095] In one embodiment, the compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof is a mixture of the E and Z isomers.

[0096] Described herein are pharmaceutical compositions comprising a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof.

[0097] The pharmaceutical compositions of the present invention can be used in the methods of the present invention described herein.

[0098] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier.

[0099] The compounds of Formula (I) or (II) or pharmaceutically acceptable salts thereof can be used to treat any disease or condition associated with PD-1 expression in T cells or associated with ERK phosphorylation. The disease or condition is associated with a protein or phosphoprotein when the activity of the protein or phosphoprotein is required for the development and / or maintenance of the disease or condition.

[0100] In one embodiment, the disease or condition is cancer. In one embodiment, the compounds of Formula (I) or (II) or pharmaceutically acceptable salts thereof can be used to treat any cancer in which there is PD-1 / PD-L1 inhibition of an anti-tumor immune response.

[0101] Examples of cancers that may be treated with a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof include melanoma, squamous cell carcinoma, basal cell carcinoma, sarcoma of the skin, Merkel cell carcinoma, head and neck cancer, non-small cell lung cancer, urothelial carcinoma, thyroid cancer, renal cell carcinoma, breast cancer, and hepatocellular carcinoma.

[0102] In one embodiment, the cancer is a cancer in a subject in which T cells of the subject express PD-1.

[0103] In one embodiment, the cancer is a cancer in which one or more tumors of the cancer contain T cells that express PD-1.

[0104] Cancers that can be treated using the methods described herein may involve T cells that express PD-1. However, as described in the Examples, the compounds described herein not only inhibit PD-1 expression, but also associate with CD28 on T cells and stimulate T cell activity, even in tumors that do not respond to anti-PD-1 antibodies.

[0105] Thus, in some embodiments, the cancer may be a cancer that is resistant to treatment with anti-PD-1 antibody therapy.

[0106] As described in the Examples, the inventors have further found that the compound of formula (II) is effective against BRAF-mutated cell lines and tumors arising from BRAF-mutated cell lines. Advantageously, the compound of formula (II) is therefore believed to be capable of treating cancer regardless of the BRAF mutation status of the tumor.

[0107] In some embodiments, the cancer comprises cells that are resistant to treatment with dabrafenib and trametinib.

[0108] In some embodiments, the cancer comprises BRAF-mutated cells.

[0109] The methods described herein can include administering a pharmaceutical composition comprising a compound described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0110] Described herein are pharmaceutical compositions comprising a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0111] In some embodiments, the carrier is a non-naturally occurring carrier.

[0112] In some embodiments, a compound of Formula (I) or (II) or a pharmaceutically acceptable salt thereof may be used in combination with one or more other drugs.

[0113] It will be appreciated that the administration of a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof in combination with one or more other pharmaceutical agents may be simultaneous, sequential or separate administration.

[0114] The term "composition" encompasses formulations containing active ingredients together with conventional carriers and excipients, as well as formulations that include an encapsulating material as a carrier, thereby providing a capsule in which the active ingredient (with or without other carriers) is surrounded by the encapsulating carrier. In pharmaceutical compositions, the carrier is "pharmaceutically acceptable," meaning that the carrier is compatible with the other components of the composition and is not harmful to the subject. The pharmaceutical compositions of the present invention may contain other medicinal or additional active agents as described above, and can be formulated, for example, by using conventional solid or liquid vehicles or diluents, as well as pharmaceutical additives (e.g., excipients, binders, preservatives, stabilizers, flavoring agents, etc.) of a type appropriate for the desired mode of administration, according to techniques known in the art of pharmaceutical formulation (see, for example, Remington: The Science and Practice of Pharmacy, 21st Ed., 2005, Lippincott Williams & Wilkins).

[0115] Pharmaceutical compositions may be in a form suitable for intravitreal, oral, rectal, nasal, topical (including cutaneous, buccal and sublingual), vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration, or for administration by inhalation or insufflation.

[0116] In some embodiments, the compounds described herein can be formulated for administration, for example, in nanoparticles or liposomes, or polymeric formulations. Methods for making formulations, including liposomes, lipid nanoparticles, and polymeric formulations, are known in the art and are described, for example, in Neervannan, 2006; Zhang et al., 2022. The liposome, nanoparticle, or polymeric formulation can include cationic lipids such as DOTAP, DOPE, DC-Chol / DOPE, DOTMA, and DOTMA / DOPE, and polymers such as hydroxypropylmethylcellulose (HPMC), polyethylene glycol (PEG), poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), and poly(glycolic acid) (PGA).

[0117] Therefore, the compound described herein or its pharmaceutically acceptable salt can be made into the form of pharmaceutical compositions and unit dosage forms thereof together with pharmaceutically acceptable carriers.The pharmaceutical composition can be a solid, such as a tablet or a filled capsule, for oral administration, or a liquid, such as a solution, suspension, emulsion, elixir, or a capsule filled with it.The pharmaceutical composition can be a liquid, such as a solution, suspension, or emulsion, for intravitreal administration.The pharmaceutical composition can also be in the form of a suppository for rectal administration or in the form of a sterile injection solution for parenteral (including subcutaneous) use.

[0118] Such pharmaceutical compositions and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or ingredients, and such unit dosage forms may contain any suitable effective amount of the active ingredient consistent with the intended daily dosage range employed.

[0119] For preparing pharmaceutical compositions from the compounds described herein, pharmaceutically acceptable carriers can be either solid or liquid.Solid form preparations include powder, tablets, pills, capsules, cachets, lozenges (solid or chewable), suppositories and dispensable granules.Solid carriers can be one or more substances that can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents or encapsulating materials.

[0120] Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, etc. Tablets, powders, capsules, pills, cachets and lozenges can be used as solid forms suitable for oral administration.

[0121] Liquid form preparations include solutions, suspensions and emulsions, for example, water or water-propylene glycol solutions.For example, parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution.

[0122] Sterile liquid form compositions 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 solvent, or a mixture of both.

[0123] Therefore, the pharmaceutical composition of the present invention may be formulated for parenteral administration (e.g., by injection, e.g., bolus injection or continuous infusion), and may be provided in unit dosage form in ampoules, pre-filled syringes, small-volume drip infusions, or multi-dose containers with added preservatives.The pharmaceutical composition may take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and may contain formulation agents such as suspending agents, stabilizers, and / or dispersing agents.Alternatively, the active ingredient may 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.

[0124] 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 oxidation and the contaminating action of microorganisms such as bacteria or fungi.

[0125] The solvent or dispersion medium for the injectable solution or dispersion may contain any conventional solvent or carrier system for injectable solutions or dispersions, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.

[0126] Pharmaceutical forms suitable for injectable use may be delivered by any suitable route, including intravenous, intramuscular, intracerebral, intrathecal, epidural injection or infusion.

[0127] Sterile injectable solution is prepared by incorporating the required amount of active ingredient into a suitable solvent with various other ingredients, such as those listed above, as needed, followed by filtration sterilization.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and the other necessary ingredients listed above.For sterile powders for preparing sterile injectable solution, the preferred preparation method is vacuum drying or freeze-drying the solution of active ingredient and any additional desired ingredients that has been previously sterilized and filtered.Preparation can also be sterilized by heat treatment (for example, boiling) or autoclave.

[0128] The compounds described herein can be formulated in compositions suitable for oral administration, for example, with an assimilable edible carrier, or enclosed in hard or soft shell gelatin capsules, or compressed into tablets, or incorporated directly with dietary food. For oral therapeutic administration, the active compounds can be incorporated with excipients, which can be used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

[0129] As described in the Examples, the compound of formula (II) was bioavailable when administered orally and intraperitoneally.

[0130] 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.

[0131] In one embodiment, the surfactant is a polysorbate. In one embodiment, the polysorbate is Tween 80.

[0132] In one embodiment, the solvent is a polar aprotic solvent. In one embodiment, the polar aprotic solvent is dimethyl sulfoxide (DMSO).

[0133] In one embodiment, the polymer is hydroxypropyl methylcellulose (HPMC).

[0134] The amount of active compound in therapeutically useful compositions should be sufficient that a suitable dosage will be obtained.

[0135] Tablets, troches, pills, capsules, lozenges, implants and the like may also contain ingredients such as those listed below: binders such as gums, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; disintegrating agents such as corn starch, potato starch, alginic acid and the like; lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose or saccharin, or flavoring agents such as peppermint, oil of wintergreen or cherry flavor. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier.

[0136] Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. A syrup or elixir may contain the active compound, sucrose as a sweetener, methyl and propylparabens as preservatives, a dye, and a flavoring such as cherry or tangerine flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts used. In addition, the active ingredient(s) may be incorporated into sustained-release preparations and formulations, including those that allow specific delivery of the active ingredient to a specific region of the gastrointestinal tract.

[0137] Aqueous solutions suitable for oral use can be prepared by dissolving the active ingredient in water and, if desired, adding suitable colorants, flavorings, stabilizers and thickeners.Aqueous suspensions suitable for oral use can be prepared by dispersing the finely divided active ingredient in water together with a viscous material, such as natural or synthetic gum, resin, methylcellulose, sodium carboxymethylcellulose or other well-known suspending agent.

[0138] Pharmaceutically acceptable carriers include any and all pharmaceutically acceptable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.

[0139] Also comprise solid form preparations that are intended to be converted into liquid form preparations for oral administration immediately before use.Such liquid forms include solution, suspension and emulsion.These preparations may contain, in addition to active ingredients, coloring agents, flavoring agents, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizers etc.

[0140] For topical administration, the compounds described herein may be formulated in an aqueous or oily base with the addition of suitable thickening and / or gelling agents.Lotions may be formulated in an aqueous or oily base and generally also contain one or more emulsifiers, stabilizers, dispersants, suspending agents, thickening agents or coloring agents.

[0141] Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0142] The solution or suspension for nasal administration can be directly applied to the nasal cavity by conventional means, for example, using a nasal dropper, pipette or spray.The formulation can be provided in a single or multi-dose form.In the case of a nasal dropper or pipette, this can be achieved by the patient administering an appropriate predetermined volume of the solution or suspension.In the case of a spray, this can be achieved, for example, by using a metered atomizing spray pump.In order to improve nasal delivery and retention, the compound of the present invention can be encapsulated with cyclodextrin or formulated with other agents that are expected to enhance delivery and retention to the nasal mucosa.

[0143] Administration to the respiratory tract can also be achieved by means of an aerosol formulation in which the active ingredient is provided in a pressurized pack with a suitable propellant, such as a chlorofluorocarbon (CFC), for example, dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.

[0144] The aerosol may conveniently also contain a surfactant such as lecithin.The dose of active ingredient may be controlled by providing a metered valve.

[0145] Alternatively, the active ingredient can be provided in the form of dry powder, for example, the powder mixture of the compound in a suitable powder base, for example, lactose, starch, starch derivatives such as hydroxypropylmethylcellulose and polyvinylpyrrolidone (PVP).Advantageously, the powder carrier forms a gel in the nasal cavity.The powder composition can be provided in a unit dosage form, for example, in capsules or cartridges (for example, made of gelatin), or in blister packs, which can be used to administer the powder by inhaler.

[0146] In formulations intended for administration to the respiratory tract, including intranasal formulations, the active ingredient generally has a small particle size, for example, of the order of 5 to 10 microns or smaller. Such a particle size may be obtained by means known in the art, for example, by micronization.

[0147] When desired, formulations adapted to give sustained release of the active ingredient may be employed.

[0148] Pharmaceutical preparation is preferably in unit dosage form.In this form, preparation is divided into unit doses that contain appropriate amounts of active ingredients.Unit dosage form can be a packaged preparation, and this package contains individual amounts of preparation, such as packaged tablets, capsules, and powders in vials or ampoules.Also, unit dosage form can be capsules, tablets, cachets or lozenges themselves, or any of these can be packaged in appropriate numbers.

[0149] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. Parenteral compositions may be in the form of physically discrete units adapted as unitary dosages for the treated subject, each unit containing a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in association with the pharmaceutical carrier.

[0150] The compound may also be administered in the absence of a carrier, where the compound is in unit dosage form.

[0151] The term "effective amount" refers to an amount of a compound effective to achieve a desired response.

[0152] The effective amount of the compounds described herein or a pharmaceutically acceptable salt thereof can be determined by one skilled in the art in view of the individual compound.

[0153] It will be understood that the specific dose level and frequency of administration 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 length of action of the compound, the subject's age, weight, general health, sex and diet, mode and time of administration, rate of excretion, drug combinations and the severity of the particular condition.

[0154] Appropriate dosages of the compounds described herein or additional active agents administered in combination with the compounds described herein can be readily determined by one of ordinary skill in the art in view of the particular compound of the invention or selected additional active agent.

[0155] Furthermore, it will be understood that when the compounds described herein are to be administered in combination with one or more drugs or other active agents, the dosage forms and levels may be formulated for either simultaneous, sequential or separate administration or any combination thereof.

[0156] The methods of the invention are intended for use with any subject that may benefit from the methods of the invention. Thus, the term "subject" includes both human and non-human mammals. The subject may be, for example, a domestic animal, a zoo animal, or a farm animal.

[0157] Unless otherwise defined herein, the following terms shall be understood to have the following general meanings: The terms referred to below have the following general meanings when used by themselves and when used in combination with other terms, unless otherwise indicated. Thus, for example, the definition of "alkyl" applies to "alkyl" as well as the "alkyl" portions of "haloalkyl," "heteroalkyl," "arylalkyl," etc.

[0158] The term "alkyl" refers to a straight or branched chain saturated hydrocarbyl group. Unless otherwise indicated, preferred are C 1-6 Alkyl and C 1-4 It is an alkyl group. x-y The term "alkyl" (where x and y are integers) refers to an alkyl group having x to y carbon atoms. For example, "C 1-6 The term "alkyl" refers to an alkyl group having 1 to 6 carbon atoms. 1-6Examples of 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, etc. Unless the context dictates otherwise, the term "alkyl" also encompasses alkyl groups containing one less hydrogen atom, where the alkyl group is attached through two positions, i.e., is divalent.

[0159] As used herein, "treating" means affecting a subject, tissue, or cell to achieve a desired pharmacological and / or physiological effect, including inhibiting a disease state, i.e., halting its occurrence; or reducing or ameliorating the effects of a disease state, i.e., causing a reversal or regression of the effects of a disease state. As used herein, "preventing" means preventing the onset of a disease state in a cell or subject that may be at risk of having the disease state, but does not necessarily mean that the disease state will not eventually occur or that the subject will not eventually develop the disease state. Preventing includes delaying the onset of a disease state in a cell or subject.

[0160] The term "effective amount" refers to the amount of a compound that elicits the biological or medical response of a tissue, system, animal or human that is desired by a researcher, veterinarian, physician or other clinician.

[0161] [Table 1] TIFF2025535140000018.tif66161

[0162] 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, USA, or Life Chemicals HTS Compounds, Canada.

[0163] All publications mentioned herein are incorporated herein by reference. Those skilled in the art will recognize that numerous variations and / or modifications as shown in the particular embodiments may be made to the present invention without departing from the spirit or scope of the invention as broadly described. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive.

[0164] The invention will now be further described by reference to the following non-limiting examples. [Example]

[0165] Here, we report that BT2, a dibenzoxazepinone (Li et al., 2020), functions as a novel pharmacological-immunotherapeutic agent and inhibits tumor growth through a dual mechanism: in tumor cells, BT2 functions as an inhibitor of ERK phosphorylation and stimulates JUN expression, whereas in T cells, BT2 associates with CD28, activates JNK phosphorylation, stimulates JUN expression, and suppresses PD-1 expression.

[0166] material and method 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.

[0167] Cell Culture. Human A375 and MeWo melanoma cells were kindly provided 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 with 5% CO2. Adherent cells were routinely passaged after trypsinization. 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 (pH 7.4) containing 10% FBS. Jurkat T cells (ATCC) were grown in RPMI-1640 medium (pH 7.4) containing 10% FBS.

[0168] Western blotting. Cells were treated with compounds for the indicated times, and whole-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).

[0169] The membrane was blocked with 5% skim milk and then incubated with rabbit monoclonal anti-c-Jun (Abcam, cat. no. ab32137; mouse, rat, and human reactive) antibody, rabbit monoclonal anti-PD-1 (Abcam, cat. no. ab214421) antibody, mouse monoclonal β-actin antibody (Sigma-Aldrich, cat. no. A5316), anti-phospho-SAPK / JNK (Thr 183 / Tyr 185 )(98F2)(CST, cat. no. 4671) antibody, anti-phospho-p44 / 42 MAPK(ERK1 / 2)(Thr 202 / Tyr 204)(D13.14.4E)(CST, cat. no. 4370) antibody, anti-phospho-p38 MAPK(Thr 180 / Tyr 182 The sections were incubated with anti-SAPK / JNK (CST, cat. no. 9252), anti-p44 / 42 MAPK (ERK1 / 2) (137F5) (CST, cat. no. 4695), anti-p38 MAPK (CST, cat. no. 9212), anti-DUSP8 (Abcam, cat. no. ab198175), or anti-c-MAF (BLR045F) (Abcam, cat. no. ab243901), followed by incubation with horseradish peroxidase-conjugated secondary goat anti-rabbit (DAKO, cat. no. P0448) or goat anti-mouse (DAKO, cat. no. P0447) antibodies. Chemiluminescence was detected using a Western Lightning Chemiluminescence system (Thermo Scientific, USA) and an ImageQuant™ LAS 4000 biomolecular imager (GE Healthcare Life Sciences, USA). Band intensities in images generated by the LAS 4000 were quantified using NIH Image J.

[0170] Migration assay. Cells were grown in 6-well plates in medium containing 10% FBS. The next day, cells were scraped with a sterile toothpick, washed with PBS, and incubated in medium containing 10% FBS and 1 μM vehicle, BT2, or SCH772984. Cells were photographed under a 4x objective using an Olympus CKX41 microscope at 0, 24, and 48 hours.

[0171] Invasion assay. Cells were grown in 24-well plates in medium containing 10% FBS. The next day, cells were scraped with a sterile toothpick, washed with PBS, and overlaid with 90% Matrigel (cat. no. 354230, Corning) (200 μl / well) containing 1 μM vehicle, BT2, or SCH772984. Cells were immediately photographed under a 10× objective using an Olympus CKX41 microscope, incubated at 37°C for 1 hour, and then medium containing 1 μM vehicle, BT2, or SCH772984 and 10% FBS (800 μl / well) was added. After 24 or 48 hours, cells were photographed again under a 10× objective using the same microscope.

[0172] Cell morphology studies. Cells were grown in 4-well chamber slides in medium containing 10% FBS. The following day, cells were incubated for 24 hours in medium containing 10% FBS and 1 μM vehicle, BT2, or SCH772984, then fixed with 4% paraformaldehyde solution for 15 minutes. After a brief wash in PBS, cells were stained with hemotoxylin and eosin. Slides were scanned using an Aperio ScanScope XT slide scanner (Leica Biosystems, Mt. Waverley, Vic, Australia), and images were acquired using ImageScope software (Leica Biosystems).

[0173] Flow cytometry using Annexin V-FITC. Cells were seeded in 6-well plates in regular culture medium containing 10% FBS. After overnight culture, cells were incubated in complete medium containing 1 μM vehicle, BT2, or SCH772984 for 24 hours. The culture medium was removed, and cells were washed with PBS. Accutase (Stem Cell Technologies, cat. no. 07920) was used to detach cells. Cells were then washed, centrifuged at 300 g for 5 minutes, and 1 × 10 cells were collected in 500 μl of 1× binding buffer (Annexin V-FITC Apoptosis Staining / Detection Kit, Abcam, cat. no. ab14085). 6 Cells were resuspended at 1000 cells / ml. Cells were transferred to a 12 x 75 mm tube, and Annexin V-FITC / propidium iodide (PI) was added and incubated for 5 minutes at 22°C in the dark. The stained cell suspension was analyzed by flow cytometry using a BD LSRFortessa X20.

[0174] Flow cytometry using anti-PD-1 antibody. Jurkat T cells were grown in 6-well plates containing RPMI 1640 and 10% FBS. After 24 hours, cells were treated with various concentrations of BT2 or BT3 for 48 hours. Alternatively, cells were incubated with 3 μM BT2 for various times. After treatment, cells were washed with PBS and centrifuged at 300 × g for 5 minutes. 5 × 10 cells were then collected. 6 The cells were then resuspended at 0.5 ml / ml. The cells were then incubated with BV421-conjugated mouse anti-human CD279 (PD-1) (BD, cat. no. 562516) or BV421-conjugated mouse IgG1 (BD, cat. no. 562438) for 45 minutes at 22°C. The cells were then washed twice with 1 ml of Stain Buffer, centrifuged, and the pellet was resuspended in 0.5 ml of Stain Buffer. The stained cell suspension was analyzed by flow cytometry using a BD FACSCanto (II).

[0175] CD28 blocking experiments. BT2 (3 nmol), vehicle, or a mixture of BT2 (3 nmol) and recombinant CD28 (3 nmol, Sino biological cat. no. 90182-C08H) or EGF (3 nmol, Sigma cat. no. E9644) in 150 μl of growth medium (10% FBS / RPMI 1640 medium) was preincubated at 37°C for 30 min. The mixture was then diluted with 850 μl of 10% FBS / RPMI 1640 medium and 0.5 × 10 6 Jurkat T cells were added to 12-well plates containing 1000 cells / well. After 24 hours, whole-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). The membranes were blocked with 5% skim milk and incubated with rabbit monoclonal anti-JUN (Abcam, cat. no. ab32137) or mouse monoclonal β-actin (Sigma-Aldrich, cat. no. A5316) antibodies. This was followed by incubation with horseradish peroxidase-conjugated secondary goat anti-rabbit (DAKO, cat. no. P0448) or goat anti-mouse (DAKO, cat. no. P0447) antibodies. Chemiluminescence was detected using a Western Lightning Chemiluminescence system (Thermo Scientific, USA) and an ImageQuant™ LAS 4000 biomolecular imager (GE Healthcare Life Sciences, USA). Band intensities in images generated by the LAS 4000 were quantified using NIH Image J.

[0176] Melanoma growth studies in mice. Six- to eight-week-old C57BL / 6J mice (provided by the Australian Resource Centre, Perth) were cultured with B16F10 cells (1 × 10 cells in 100 μl of DMEM containing 50% Matrigel and 10% FBS). 5Mice were inoculated subcutaneously (sc) with 100 μg of InVivoPlus anti-mouse PD-1 (CD279) (BioX Cell, BP0033-2) or InVivoPlus polyclonal Armenian hamster IgG (Bio X Cell, BP0091) intraperitoneally (ip) twice weekly. Treatment began on day 7.

[0177] 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 prior to administration. Vehicle or BT2 was administered ip once daily on a 5-day on / 2-day off schedule (20 ml / kg and 200 mg / kg, respectively). Treatment began on day 5 when tumors were palpable.

[0178] For experiments using SCID mice, human MDA-MB-435 cells (2.5 × 10 cells in 100 μl of PBS) were injected into the third mammary fat pad of 5-week-old CB17 / Icr-Prkdcscid / IcrIcoCrl mice (provided by Charles River Laboratories, USA) on day 0. 6 Cells (cells / animal) were inoculated sc. These cells were also provided by 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. Vehicle or BT2 was administered intratumorally or intratumorally (it) at 20 mg / kg (it) or 200 mg / kg (ip) once daily on a 5-day on / 2-day off schedule. Treatment began on day 16 and ended on day 43. Tumor growth and body weight data are provided by the animal study described in Li et al. (Li et al., 2019) using the same vehicle group.

[0179] Tumors were measured twice weekly by length and width (in mm) in MDA-MB-435 SCID mice and once daily by length, width, and height (in mm) in B16F10 C57BL / 6J mice. Tumor volume was calculated using the formula V = (L × W × H × π) / 6 (B16F10) or V = L × W × W / 2 (MDA-MB-435). If a second tumor was detected in an animal, both tumor volumes were measured and their volumes were summed. The animal protocol was approved by the Explora Biolabs Institutional Animal Care and Use Committee (IACUC) and the UNSW Animal Care and Ethics Committee.

[0180] Mass spectrometry. Proteins in serum (50 μl) were precipitated by dilution with acetone (150 μl). The mixture was left at 4°C for 14 hours, centrifuged (14,000 g for 10 minutes) to pellet the proteins, and the supernatant was removed. For MS analysis, 10 μl of the solution was diluted in 40 μl of Buffer A (HO (0.1% formic acid)). BT2 standards were prepared by diluting a stock suspension of BT2 (10 μg, 30 mg / ml) in Buffer A (990 μl), then diluting 10 μl of this into 990 μl, followed by a further 1:100 dilution. The standard BT2 used for LC-MS was at a concentration of 92 fmol / μl. LC-MS was performed on a Thermo QExactive HF, Gold C 18 A 50 x 2.1 mm column was used to run the elution using solvent A (HO (0.1% formic acid)) and solvent B (HO:CHCN 20:80 (0.1% formic acid)) with a gradient of T = 0, 1% B, T = 26 min, 100% B, T = 27 min, 100%, T = 27.1, 1% B, and T = 30 min, 1% B; column temperature 45 °C. MS1 scan, m / z 140-800, 3 x 10 6 ions, max IT 25 msec, resolution 120,000 and top 5 MS2 (2 x 10 5 The analysis was performed with a maximum IT of 50 msec, a resolution of 30,000, and HCD at 20, 30, and 50 V. Extracted ion chromatograms (m / z 327.134 ± 5 ​​ppm) were obtained for the blank, standard, and sample, and the peak area integrals were calculated and compared.

[0181] 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 hours. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, cat. no. 74004) with modifications. Briefly, cells were washed twice with pre-chilled 1x PBS and lysed with TRIzol Reagent (Thermo Fisher Scientific cat. no. 15596026). Chloroform was added to the mixture, followed by microfuge centrifugation at 13,000 rpm for 15 minutes at 4°C. The upper aqueous layer containing total RNA was transferred to a new microfuge tube, isopropanol was added, and the mixture was 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 sent to the UNSW Ramaciotti Centre for Genomics for TruSeq Stranded mRNA-seq preparation and sequencing on a One NextSeq 500 (1 × 75 bp) high-output flow cell with a data output of up to 400M reads. Sample quality control was set at >80% Q30 for 1 × 75 bp.

[0182] First, RNA-seq reads were assessed for quality using the FastQC tool (v0.11.8) (www.bioinformatics.babraham.ac.Uk / projects / fastqc / ). To quantify transcript abundance from RNA-seq reads, we used the Salmon tool (Patro et al., 2017). We then used the R package DESeq2, which incorporates a method for differential analysis of count data, to identify differentially expressed genes between specific comparisons (Love et al., 2014). We also performed principal component analysis using this package. Significantly differentially expressed genes were defined as those with an adjusted p-value <0.05 and a log2 fold change >1 (absolute fold change >2). Bar plots, scatter plots, and density plots were created using the ggplot2 R package. Distance matrices and clustering heatmaps were generated using the pheatmap R package. For pathway analysis against the Curated (Canonical Pathway), Gene Ontology, and Hallmarks gene collections from MSigDb (Liberzon et al., 2015; Liberzon et al., 2011), DESeq2 output was used to generate rankings for input into GSEA Pre-ranked (v6.0.12, Broad Institute) (Subramanian et al., 2005). Ranking scores were calculated by multiplying the sign of the log2 fold change by the log10-transformed adjusted p-value. Gene sets were displayed only if they had at least a false discovery rate <0.25, and a maximum of 60 gene sets were displayed in the figure. All immune response-related gene sets were selected for display. No other preselection was performed on the displayed gene sets.

[0183] Immunohistochemical staining and analysis. Rabbit monoclonal anti-CD3 (cat. no. ab16669; mouse, rat, and human reactive), rabbit monoclonal anti-PD-1 (cat. no. ab214421), and rabbit monoclonal anti-CD68 (cat. no. ab125212) antibodies were obtained from Abcam, and rabbit monoclonal anti-phospho-ERK (Thr) 202 / Tyr 204 ) (cat. no. CST4370), rabbit monoclonal pan-ERK (cat. no. CST4695) antibody was obtained from Cell Signaling Technology. Rabbit polyclonal anti-PD-L1 (cat. no. PA5-20343) antibody was obtained from ThermoFisher.

[0184] Formalin-fixed, paraffin-embedded sections were prepared from tumors. All deparaffinized sections (4 μm sections on Superfrost slides) underwent heat-induced antigen retrieval using citrate buffer (pH 6.0) at 110°C for 5 minutes. Sections were blocked with Dual Endogenous Enzyme Block (DAKO, S2003) for 10 minutes, followed by 2% skim milk for 20 minutes. Slides were incubated with primary antibodies for 60 minutes at room temperature or overnight at 4°C, followed by 10 minutes with the probe in MACH3 Rabbit AP-Polymer Detection solution (Biocare Medical, M3R533 G, H, L). After rinsing with buffer, slides were incubated with the polymer in MACH3 Rabbit AP-Polymer Detection solution (Biocare Medical, M3R533 G, H, L) for an additional 10 minutes. Slides were incubated with red chromagen (Warp Red™ Chromagen Kit) for 5 minutes and counterstained with hematoxylin and Scott Blue. Slides were dried on filter paper, dehydrated in xylene, and then coverslipped. Immunostained slides were scanned using an Aperio ScanScope XT slide scanner (Leica Biosystems, Mt. Waverley, Vic, Australia), and images were acquired using ImageScope software (Leica Biosystems). The integrated optical density (IOD) or area of ​​positive staining (red chromagen) was assessed using Image-Pro Plus software (Cybernetics, Bethesda, MD, USA). Positive staining was the average of quantification of 4–8 fields under a 20× or 40× objective.

[0185] Mouse serum IFN-γ levels. Whole blood was collected from mice on day 17 and spun at 1500 g for 10 minutes at 4°C. Serum was extracted from the top layer and stored at -80°C. IFN-γ levels were measured using a mouse IFN-γ (Improved) ELISA kit (Invitrogen, cat. no. KMC4021) according to the manufacturer's protocol, and absorbance was read at 450 nm. A standard curve was generated using GraphPad Prism 9 software. Mouse serum IFN-γ levels were measured in parallel with the standard curve using GraphPad Prism 9.

[0186] Surface plasmon resonance (SPR). CD28 (Sino Biological) was immobilized on a Series S sensor chip CM5 (GE) by amine coupling to a level of approximately 3000 RU at a flow rate of 10 μl / min at 25°C using a Biacore T200 (Cytiva) on flow cells 2 and 4. 0.2 M EDC + 0.05 M NHS was injected for 420 s, followed by CD28 (37.5 μg / ml in 10 mM acetate, pH 5) for 450 s, with the flow rate reduced to 2 μl / min. Unreacted NHS was blocked by a 420 s injection of 1 M ethanolamine-HCl (pH 8.5). Flow cells 1 and 3 served as reference cells, and activation and blocking were performed as described above. The immobilization running buffer was 20 mM HEPES, 150 mM NaCl (pH 7.5). 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 solubilized in DMSO to a concentration of 50 mM and then diluted in running buffer to a final concentration of 20 μM. 1This was confirmed using H 1D NMR. An 8-point dilution series was performed with a maximum concentration of 20 μM. Samples were injected for 60 seconds at a flow rate of 40 μl / min with a dissociation time of 60 seconds. Solvent correction was performed to correct for excluded volume effects. The integrity of the coupled CD28 was verified by injection of its known binding partner, CD80 (Sino Biological) (Waite et al., 2020) (a 5-point dilution series with a maximum concentration of 2 μM).

[0187] Systemic pharmacokinetics after oral (PO) and intraperitoneal (IP) administration. Male CD-1 mice were injected with BT2 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 hours. BT2 was given by PO or IP injection.

[0188] [Table 2]

[0189] Approximately 110 μl of blood samples were collected into tubes containing EDTA-K2 anticoagulant (n=3 / time point, 3 mice / group). All blood samples were placed on wet ice and then centrifuged (6000 g, 4°C for 5 minutes) to obtain plasma, which was stored at -70°C or on dry ice until analysis.

[0190] 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: HO-0.025% FA + 1 mM NH4OAc and mobile phase B: MeOH-0.025% FA + 1 mM NH4OAc, with an injection volume of 3 μl.

[0191] Statistics. Statistical analysis was performed using Graphpad PRISM v9. Note that PRISM does not draw error bars if they are shorter than the symbol height. If the distribution was not normal, a Mann-Whitney or Kruskal-Wallis was performed, as appropriate. Normally distributed data were analyzed by t-test or one-way ANOVA, as appropriate. Plotted data represent mean ± SEM. n indicates biological triplicates, not technical triplicates. Differences were considered significant when p ≤ 0.05. Where indicated, * p ≤ 0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0192] result BT2 inhibits ERK phosphorylation, suppresses migration and invasion, and stimulates apoptosis by human melanoma cells Given that MAP kinase activation is key to 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. A study by Rossi et al. comparing malignant potential (a combination of growth, invasion, and migration rates) among 10 human melanoma cell lines showed that A375 cells (mutated BRAF V600E ) had the most aggressive phenotype (Rossi et al., 2018). MeWo cells (wild-type BRAF) are the melanoma cells that exhibited the least aggressive phenotype among those studied by Rossi et al. Western blotting was performed on extracts from A375 and MeWo cells incubated with either 10 or 100 nM BT2 or SCH772984 for 24 hours. Membranes were analyzed for p-ERK (Thr 202 / Tyr 204) or total ERK, followed by incubation with a secondary antibody. The results are shown in Figures 1A and 1C. Band intensities were quantified using Image J, and plotted data represent the mean ± SEM (Figures 1B and 1D). Data are representative of two biologically independent experiments. The results showed that BT2 suppressed ERK phosphorylation, similar to SCH772984 (an ERK inhibitor that overcomes resistance to BRAF and MEK inhibitors and is the precursor to MK-8353, which is currently in clinical trials for advanced malignancies).

[0193] To evaluate the effect of BT2 on melanoma cell migration, A375 and MeWo cells were incubated with 1 µM BT2 or SCH772984, and migration into the denuded zone of A375 and MeWo cells was quantified after 24 or 48 hours, respectively. The results are shown in Figure 2A-2D. As can be seen, BT2 more potently inhibited melanoma migration (Figure 2A and 2C) and invasion (Figure 2B and 2D) than SCH772984 in both A375 and MeWo cells.

[0194] We also evaluated the morphology of A375 and MeWo cells exposed to 1 μM BT2 or SCH772984 for 24 hours. The results are shown in Figure 3. Unlike SCH772984, BT2 had a dramatic morphological effect (Figure 3), causing 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 a less potent pro-apoptotic effect (Figure 4).

[0195] BT2 inhibits melanoma growth after local but not systemic delivery in immunodeficient mice The ability of BT2 to inhibit melanoma growth was investigated in a mouse xenograft model. Human melanoma (MDA-MB-435 cells) and mutant BRAF V600EImmune-deficient SCID mice bearing the CB17 / lcr-Prkdcscid / IcrIcoCrl gene were treated intratumorally (it) with 20 mg / kg BT2 on a 5-day on / 2-day off regimen (Figure 5A). BT2 caused growth inhibition (Figure 5B) but had no adverse effect on body weight (Figure 5C).

[0196] Immunohistochemical staining, using alkaline phosphatase-conjugated secondary antibodies rather than 3,3'-diaminobenzidine to avoid interference from brown (black) dye, confirmed BT2 inhibition of ERK phosphorylation in tumors (Figures 6A and 6B) and no effect on total ERK levels (Figures 6C and 6D) in this local delivery model.

[0197] To assess the efficacy of BT2 when delivered systemically (as opposed to locally) in the same immunodeficient mouse model, CB17 SCID mice bearing subcutaneous MDA-MB-435 tumors were given 200 mg / kg BT2 or vehicle by i.p. once daily on a 5-day on / 2-day off schedule. Treatment began on day 16 (Figure 7A). The effects on tumor volume and body weight are shown in Figures 7B and 7C. Surprisingly, no inhibition was observed when BT2 was delivered systemically in this model, even at 200 mg / kg on a 5-day on / 2-day off regimen (Figure 7B).

[0198] Systemically delivered BT2 inhibits melanoma growth and ERK phosphorylation in an immunocompetent mouse allograft model resistant to tumor inhibition using anti-PD-1 antibodies Previous xenograft studies demonstrating antitumor efficacy in immune-deficient mice when BT2 was delivered intratumorally (it) rather than systemically (ip), led us to hypothesize that BT2 might rely on an active immune system to achieve tumor growth inhibition. Therefore, we tested the effects of BT2 in an immunocompetent mouse model of melanoma growth using B16F10 melanoma. B16F10, grown in C57BL / 6 mice, provides a highly aggressive, low-immunogenic, syngeneic ERK-dependent melanoma model. This model is resistant to PD-1 antibody inhibition (Kleffel et al., 2015). C57BL / 6J mice bearing subcutaneous B16F10 tumors were administered anti-mouse PD-1 monoclonal antibody or control IgG (100 μg, i.p.) twice weekly. Treatment began on day 7 (Figure 8A). Tumor volume and body weight were assessed over time, and the results are shown in Figures 8B and 8C. These experiments demonstrated that the PD-1 antibody exhibited transient and non-sustained B16F10 growth inhibition (i.e., days 12 and 13) (Figure 8B), as observed by Kleffel et al. (Kleffel et al., 2015).

[0199] To evaluate the efficacy of BT2 in this model, C57BL / 6J mice bearing subcutaneous B16F10 tumors were administered BT2 or vehicle (200 mg / kg or 20 ml / kg, i.p., respectively) once daily on a 5-day on / 2-day off schedule. Treatment began on day 5 (Figure 9A). Tumor volume and body weight assessments are shown in Figures 9B and 9C, tumor size and isolated tumor weight assessments are shown in Figures 10A and 10B, and viability assessments are shown in Figure 11. Unlike anti-PD-1 treatment, systemically delivered BT2 caused significant B16F10 growth inhibition beginning on day 9, which persisted for the duration of the study (Figure 9B), with no adverse effect on body weight during this time course (Figure 9C). Tumor size measured using calipers on day 17 (Figure 10A) correlated with the isolated tumor weights (Figure 10B). Kaplan-Meier analysis showed that on day 17, tumor size was 500 mm in 75% of vehicle-treated animals. 3(Haynes et al., 2018), whereas none of the tumors in BT2-treated mice exceeded this size by day 17 (Figures 10A and 11). LC-MS confirmed the bioavailability of BT2 at serum concentrations of 4 μg / ml or 12.3 μM on day 17 (data not shown). Immunohistochemical staining of tumors on day 17 revealed BT2-mediated suppression of ERK phosphorylation (Figure 12A) without affecting total ERK levels (Figure 12B). Collectively, these findings demonstrate that BT2 inhibits ERK phosphorylation and B16F10 growth in an allograft model resistant to tumor inhibition using anti-PD-1 antibodies.

[0200] BT2 inhibits inflammation and enhances antitumor immunity in immunocompetent mice Recent studies by the present inventors have shown that BT2 has anti-inflammatory properties in arthritic mice (Yeh et al., 2021). We hypothesized that this agent may have similar activity within tumors. CD68, a pan-macrophage marker that stains in tumor cell nests in melanoma, is associated with tumor recurrence and poor survival. Immunohistochemistry analysis was performed using an antibody against CD68 on B16F10 tumors (day 17) from mice treated with BT2 or vehicle. IOD and tissue area were assessed using Image-Pro Plus and expressed as IOD / μm 2 The results are shown in Figure 13. It was found that BT2 reduced the level of CD68 in the tumor (Figure 13).

[0201] Further immunohistochemical analysis was performed on B16F10 tumors (day 17) from mice treated with BT2 or vehicle using an antibody against CD3. The results are shown in Figure 14. This demonstrated that BT2 inhibited CD3 in the tumor periphery (i.e., within 250 μm). +The results showed that B16F10 tumors stimulated CD3 T cell staining (Figures 14A and 14B). B16F10 tumors are immunologically "cold tumors," meaning a general lack of tumor-infiltrating T cells (Bonaventura et al., 2019). Fu et al. + Tumor-infiltrating lymphocytes were found to be a prognostic marker for overall survival in melanoma (Fu et al., 2019). + The finding of increased staining suggests that the antitumor activity of BT2 involves the immune system.

[0202] IFN-γ is a biomarker of cellular immunity and anti-tumor immune responses. Elevated IFN-γ is associated with systemic immune responses after immune checkpoint therapy in cancer patients and tumor-bearing mice. Because IFN-γ is produced by T cells, we determined the levels of IFN-γ in the serum of tumor-bearing mice treated with vehicle or BT2. The results are shown in Figure 15. The results show that circulating IFN-γ levels were elevated in mice treated with BT2 compared to mice treated with vehicle (Figure 15).

[0203] BT2 suppresses PD-1 expression in tumors and human T cells Encouraged by the increased serum IFN-γ levels in BT2-treated mice, we decided to determine the effect of this agent on intratumoral PD-1 levels. We also performed immunohistochemical analysis using antibodies against PD-1 and PD-L1 on B16F10 tumors (day 17) from mice treated with BT2 or vehicle. The results are shown in Figures 16A and 16B. Immunohistochemical staining revealed that PD-1 levels were reduced in BT2-treated B16F10 tumors compared with those treated with vehicle (Figure 16A). Interestingly, PD-L1 levels were also found to increase with BT2 treatment (Figure 16B). These findings have clinical relevance. A study by Gettinger et al. revealed a higher overall response rate (and a trend toward greater response) to anti-PD-1 therapy when patients' tumors expressed PD-L1. Similarly, Vilain et al. found that tumor PD-L1 expression was a determinant of patient response to pembrolizumab / nivolumab. These findings prompted us to investigate the effect of BT2 on PD-1 expression in cultured T cells.

[0204] 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 PD-1 expression in Jurkat T cells was reduced by 50% at 0.1 μM BT2 and 80% at 3 μM, concentrations well below those observed in tumor-bearing mice treated with BT2 in the above study (Figure 17A). BT3, a structural analog of BT2 (Li et al., 2020), showed no inhibitory effect over the same concentration range (Figure 17B). The effect of BT2 was not only dose-dependent but also time-dependent. BT2 (3 μM) inhibited PD-1 levels by 50% at 24 hours and by 80% at 48 hours (Figure 17C).

[0205] Western blotting confirmed the flow cytometry data by demonstrating that BT2 suppressed PD-1 expression in Jurkat T cells in a dose-dependent manner (Figures 18A and 18B). SCH772984 also suppressed PD-1 expression, but with >10-fold less potency than BT2 (Figures 18A and 18B). The MEK1 / 2 inhibitor PD98059 had no effect on PD-1 expression (Figures 19A and 19B).

[0206] BT2 stimulates p-JNK / JUN, leading to enrichment of genes that mediate immune responses and T cell activation To gain insight into the mechanism of BT2 action, we performed next-generation RNA sequencing (RNA-seq) on extracts from Jurkat T cells exposed to 10 μM BT2 for 4 hours. Principal component analysis (PCA) and cluster analysis demonstrated clear distinctions between treatment groups and close associations between biological replicates. From a population of 19,071 gene IDs, RNA-seq revealed 856 genes repressed and 1,320 genes induced by BT2 with adjusted p-values ​​<0.05, including 14 genes (Table 2) and 30 genes (Table 3) that were expressed with a ≥2-fold change (absolute fold change).

[0207] [Table 3]

[0208] [Table 4]

[0209] Because BT2 was identified from an AP-1-dependent firefly luciferase screen (Li et al., 2020), it was surprising to discover that the most significantly induced gene (logFC ≥ 2) by BT2 in these cells was the proto-oncogene JUN (Table 3). JUN was induced 5.9-fold by BT2 within 4 h (Table 3). However, BT2 did not induce the expression of all AP-1 family members. For example, BT2 increased the levels of JUND by 2.4-fold but had little effect on FOSB and FOS-like 2, whereas FOS did not pass the low read filter cutoff and was therefore excluded from the analysis.

[0210] BT2 altered the expression of several 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 using extracts from Jurkat T cells incubated with BT2. Blots were incubated with anti-DUSP8 or anti-c-MAF antibodies, followed by secondary antibodies. The results are shown in Figures 20A and 20B, and 21A and 21B. DUSP8 (Figure 20) and c-MAF (Figure 21) protein levels are elevated in Jurkat cells treated with BT2. Gene set enrichment analysis (GSEA) revealed that BT2 altered the expression of distinct gene ontologies. Notably, there was significant enrichment for genes mediating immune responses and T cell activation, including JUN and several other members of the DUSP family.

[0211] Western blotting was performed on extracts from Jurkat T cells incubated with BT2 for various times. Membranes were incubated with JUN, p-JNK, p-ERK, and p38 antibodies, followed by secondary antibodies. This confirmed the induction of JUN at the protein level by BT2 within 2–4 h (Figures 22A and 22B). BT2 stimulated JNK phosphorylation within 1 h but had no effect on the levels of ERK or p38 phosphorylation (Figures 22A and 22B).

[0212] BT2 induces JUN in tumor cells regardless of BRAF mutation status Western blotting was performed to determine the effect of BT2 on JUN expression in multiple melanoma cell lines. A375, MDA-MB-435, and MeWo were incubated with various amounts of BT2 or SCH772984 for 24 hours prior to extract preparation and Western blotting. The results, shown in Figures 23A, 23B, and 23C, demonstrate that BT2 increased JUN expression in a dose-dependent manner in all three melanoma lines. The results also demonstrate that JUN was induced in tumor cells regardless of BRAF mutation status.

[0213] JUN is induced by BT2 through CD28 engagement in T cells In T cells, anti-CD28 binding activates the c-jun promoter. CD28, a homodimeric cell surface glycoprotein, is a costimulatory signal required for T cell activation. We investigated whether BT2 interacts with CD28. To this end, we measured surface plasmon resonance (SPR) measurements over a BT2 concentration range of 0.156 to 15 mM using a Biacore T200 in 20 mM HEPES, 150 mM NaCl, and 5% DMSO (pH 7.5) at 12°C. The resulting sensorgrams are shown in Figure 24. BT2 bound to CD28 in a dose-dependent manner.

[0214] To further evaluate the interaction between BT2 and CD28, vehicle, BT2 (3 nmol), or a mixture of BT2 (3 nmol) and soluble recombinant CD28 (3 nmol) or recombinant epidermal growth factor (3 nmol) was preincubated in growth medium at 37°C for 30 minutes and then added to Jurkat T cells in 12-well plates (the final concentration of BT2, CD28, or EGF was 3 μM). After 24 hours, whole-cell lysates were prepared in RIPA buffer and subjected to Western blotting. The results are shown in Figures 25A and 25B. Preincubation of BT2 with soluble recombinant CD28 inhibited the induction of JUN by BT2 in Jurkat T cells, whereas BT2 still induced JUN after preincubation with EGF (Figures 25A and 25B).

[0215] BT2 is bioavailable in the circulation after oral or intraperitoneal administration LC-MS / MS detection of BT2 in the plasma of mice given BT2 either by oral gavage (PO) or intraperitoneal (IP) injection was performed. Plasma samples were processed (using a protein precipitation method) and injected into an LC-MS / MS (SCIEX ExionLC connected to a Triple Quad 6500+).

[0216] Highest PO BT2 exposure (AUClast = 340 h * ng / mL=1.04 hours * The next highest PO BT2 exposure (AUClast = 189 h) was achieved with formulation 3 (G5). * ng / mL=0.579 hours * nM, Cmax = 57.9 ng / mL = 0.177 μM at 15 min (Tmax), relative bioavailability of 13.1%) was obtained with formulation 1 (G1).

[0217] Highest IP BT2 exposure (AUClast = 1440 h) * ng / mL=4.41 hours *nM, Cmax = 1073 ng / ml = 3.29 μM (Tmax) at 15 minutes, T = 8.72 hours) was obtained with Formulation 1 IP (G2). The next highest IP BT2 exposure (AUClast = 1182 hours) * ng / ml=3.62 hours * nM, Cmax = 846 ng / ml = 2.59 μM at 30 min (Tmax, T1 / 2 = 8.42 h) was obtained with formulation 3 (G6).

[0218] conclusion Using an immunocompetent mouse model resistant to sustained PD-1 antibody blockade (Kleffel et al., 2015), we tested the ability of the dibenzoxazepinone BT2 to inhibit PD-1 expression and suppress 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 inhibited tumor growth in a syngeneic mouse model resistant to PD-1 antibody blockade. B16F10 (BRAF WT) is a typical "cold tumor" (Okada et al., 2020) that exhibits resistance to immune checkpoint inhibitors in melanoma patients (Bonaventura et al., 2019). However, Kleffel et al. found that forced overexpression of pdcd1 (encoding PD-1) enhanced B16F10 tumor growth in C57BL / 6 mice, whereas shRNA knockdown of pdcd1 reduced B16F10 growth (Kleffel et al., 2015). This study revealed that BT2 stimulates JUN in both T cells and melanoma cells and negatively regulates PD-1 in T cells. BT2 helps prevent T cell exhaustion by increasing JUN in T cells, potentially making it useful in small molecule strategies to maintain effective and sustained tumor cell killing.

[0219] BT2 associates with CD28 and activates downstream signaling, particularly JUN, which negatively regulates PD-1 expression and enhances T cell activity (Lynn et al., 2019). CD28 is a T cell costimulator, and its association promotes priming of naive T cells. CD28 engagement can induce JUN induction. Anti-CD28 monoclonal antibodies strongly promote T cell activity. CD80- and CD86-Ig RFP (recombinant fusion protein), which targets CD28, improves T cell responses and inhibits tumor growth. Unlike anti-PD-1 antibodies, BT2 not only inhibits PD-1 expression but also associates with CD28, suggesting that BT2 enhances T cell activity and overcomes PD-1 antibody resistance. Additionally, BT2 inhibits ERK activation (Thr) in melanoma cells. 202 / Tyr 204 phosphorylation) and inhibits BRAF mutation status (A375(BRAF V600E ), MeWo(BRAF WT), MDA-MB-435(BRAF V600E It was demonstrated herein that BT2 can stimulate JUN expression regardless of tumor cell grade or relative tumor cell malignancy. BT2 suppressed a wide range of cellular processes, including migration and invasion, and stimulated tumor cell apoptosis more effectively than the ERK inhibitor SCH772984.

[0220] Tumor-associated macrophages (TAMs) are clinically associated with melanoma recurrence and poor survival. TAM density is higher in invasive melanoma compared with benign melanocytic lesions. Furthermore, TAMs promote cancer initiation and progression to malignancy. TAMs create a proinflammatory microenvironment and produce IL-1β, which is involved in BRAF inhibitor-induced resistance in melanoma. Many chemotherapeutic agents 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 upregulates CD68 in melanoma. + It reduced macrophage accumulation.

[0221] In addition to stimulating JUN, BT2 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 important proinflammatory cytokines, including IL-1β, IL-6, and TNF-α. BT2 induction of DUSPs suggests a possible autoregulatory mechanism involving increased JNK phosphorylation and JUN expression. This has therapeutic implications. For example, DUSP4 regulates responsiveness to MEK inhibition in BRAF wild-type tumors, and its depletion induces resistance to MEK inhibitors. DUSPs are upregulated in T cells stimulated with anti-CD28 and anti-CD3. Recent studies have demonstrated an inverse relationship between DUSP8 and T cell exhaustion. DUSP8 also negatively regulates ERK phosphorylation. BT2 reduced the levels of c-MAF, a transcription factor and key regulator of T cells that 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, and c-MAF overexpression inhibits IFN-γ and IL-2 production by T cells and enhances PD-1 expression, which is associated with T cell dysfunction and exhaustion. Giordano et al. knocked out c-MAF and increased IFN-γ production in tumor-infiltrating lymphocytes (TILs), resulting in reduced tumor growth and increased survival in tumor-bearing mice after adoptive transfer. Chihara et al. reported that c-MAF deletion inhibited PD-1 expression by T cells. c-MAF functionally interacts with other regulators of T cell function, such as PRDM1, which regulates PD-1 expression on T cells. Recently, ChIP-seq of human T cells immunoprecipitated with c-MAF revealed that approximately 70% of the induced loci were c-MAF-associated, and lentiviral c-MAF overexpression in T cells increased PD-1 expression. By modulating c-MAF and DUSP expression, BT2 may regulate PD-1 and prevent T cell exhaustion.At the same time, the data herein suggest that BT2 promotes intratumoral T cell infiltration. For example, BT2 enhances CD4+ expression. + It increased netrin-1 (NTN1), which is associated with T cell chemokinesis and inflammatory cell infiltration, by 90.6-fold.

[0222] As a small molecule inhibitor of PD-1, BT2 offers many potential advantages over antibody or macromolecule products, including lower production costs, the ability to penetrate cell membranes, and oral administration. Furthermore, all currently approved PD-L1 or PD-1 inhibitors are antibodies that require intravenous infusion. There are no clinically available small molecule drugs capable of inhibiting both ERK and PD-1. Given the high cost of immunotherapy, an effective small molecule could potentially reduce the financial burden on the healthcare system and eliminate reliance on in-hospital drug administration. Compared to "bispecific" antibodies, the bifunctional small molecule BT2 is simpler and less expensive to produce. Inhibition of melanoma growth by BT2 in models resistant to PD-1 antibody blockade suggests its potential use in patients whose tumors are unresponsive to immune checkpoint immunotherapy. As a promising anti-cancer drug that inhibits both the ERK and PD-1 / PD-L1 systems, BT2 may be applicable to the treatment of melanoma as well as other tumor types, including, but not limited to, skin tumors such as squamous cell carcinoma, basal cell carcinoma, cutaneous sarcoma, and Merkel cell carcinoma, as well as head and neck cancer, non-small cell lung cancer, urothelial carcinoma, thyroid cancer, renal cell carcinoma, breast cancer, and hepatocellular carcinoma, especially those with evidence of PD-1 / PD-L1 inhibition of anti-tumor immune responses.

[0223] The ability of BT2 to function as both an ERK and PD-1 inhibitor may reduce the risk of treatment-related toxicities that complicate current strategies, as a single drug is administered instead of multiple. Furthermore, a drug that can inhibit ERK activation and PD-1 function would be a potentially valuable tool, especially in the setting of PD-1 antibody resistance.

[0224] In the following claims and the foregoing description of the invention, unless the context dictates otherwise by reason of the express language or necessary inference, the words "comprise" or variations such as "comprises" or "comprising" are used in the inclusive sense, i.e., to specify the presence of mentioned features but not to exclude the presence or addition of further features in various embodiments of the invention.

[0225] References [Table 5]

Claims

1. 1. A method of increasing immune cell activation and / or treating cancer in a subject, comprising: A subject is administered an effective amount of a compound of formula (I): 【Chemistry 16】 [In the formula: R １ is a linear or branched C １ -C ６ is alkyl; R ２ is a linear or branched C １ -C ６ alkyl, or R ２ is the following group: 【Chemistry 17】 wherein q is 1, 2, 3, or 4; R ３ is a linear or branched C １ -C ６ alkyl) is] or a pharmaceutically acceptable salt thereof.

2. R １ But linear C １ -C ６ Alkyl or branched C １ -C ６ The method of claim 1 , wherein the alkyl is alkyl.

3. R １ But -CH ２ CH ３ or -CH ２ CH (CH ３ ) ２ The method of claim 1, wherein

4. R ２ But linear C １ -C ６ Alkyl or branched C １ -C ６ The method of claim 1 , wherein the alkyl is alkyl.

5. R ２ But -CH ２ CH ３ or -CH ２ CH (CH ３ ) ２ The method of claim 1, wherein

6. The compound of formula (I) is represented by formula (I-1): 【Chemistry 18】 [In the formula: R ２ is a linear or branched C １ -C ６ is alkyl; or R ２ is the following group: 【Chemistry 19】 wherein q is 1, 2, 3, or 4; R ３ is a linear or branched C １ -C ６ alkyl) is] The method according to claim 1, wherein the compound is

7. The compound of formula (I) may be a compound of formula (II): 【Chemistry 20】 The method according to claim 1, wherein the compound is

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

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

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

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

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

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

14. The compound of formula (I) is 【Chemical 21】 The method of claim 13, wherein

15. A method for reducing ERK phosphorylation and 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 the subject, comprising administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

16. The compound of formula (I) may be a compound of formula (II): 【Chemical 22】 The method according to claim 15, wherein the compound is

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

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

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

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

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

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

23. A kit for increasing immune cell activation and / or treating cancer comprising 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 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.

25. The compound of formula (I) is 【Chemical 23】 or a pharmaceutically acceptable salt thereof.

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