Rupintriq, icatibant and related compounds for use in the treatment of skin cancer

Abstract

The present invention relates to the therapeutic treatment of skin cancer, in particular melanoma, by using rubitecan, PM14 (ecubectedin) and compound IA.

Description

Technical Field

[0001] This invention relates to the therapeutic treatment of skin cancer, particularly melanoma, using rubitidine, PM14 (ecubectedin), and compound IA. Background Technology

[0002] Skin cancer is one of the most common cancers in Western countries. Melanoma is the most aggressive form of skin cancer, accounting for only 1% of all skin cancer cases, but it accounts for more than 80% of skin cancer-related deaths due to its high metastatic tendency.

[0003] The mitogen-activated protein kinase (MAPK) pathway is a key oncogenic signaling system of a series of kinases that ultimately lead to cell proliferation, differentiation, and survival. The BRAF gene, part of the MAPK pathway, is crucial for the regulation of cell growth, proliferation, and survival. Mutations in the BRAF gene occur in approximately 50% of melanomas.

[0004] BRAF V600E It is the most common BRAF mutation in melanoma, accounting for 80% of all BRAF mutations, and leads to constitutive activation of BRAF and downstream activation of MEK and ERK, which promote uncontrolled growth. In addition to BRAF mutations, NRAS and NF1 mutations are found in the majority of the remaining patients (40%). These genomic alterations are considered driver mutations in melanoma development.

[0005] Immunotherapy represents the first-line treatment for metastatic or unresectable melanoma using immune checkpoint inhibitors (nivolumab, pembrolizumab, etc.). However, patients carrying BRAF mutations can benefit from BRAF or MEK-targeted therapies.

[0006] Current BRAF inhibitors (i.e., vemurafenib, dabrafenib, and cannefenib) and MEK (downstream protein in the BRAF signaling cascade) inhibitors (i.e., trametinib and cobimetinib) have been approved for targeted therapy of unresectable or metastatic BRAF-mutant melanoma because they have shown improved efficacy.

[0007] Despite these recent advances, the most critical phenomenon hindering the clinical success of such therapies remains intrinsic or acquired resistance to targeted therapies.

[0008] In fact, the difficulty in eradicating melanoma lies in the intratumoral heterogeneity driven by cellular phenotypic plasticity. Melanoma comprises subpopulations of cancer cells with different phenotypes, different transcriptional and epigenetic driving characteristics, and different sensitivities to actual treatments. Following environmental stress or drug therapy, melanoma cells can interconvert between at least two major phenotypes: a proliferative / differentiation high MITF phenotype (also known as a "melanocyte-like" state) and an invasive / undifferentiated low MITF phenotype (known as a "mesenchymal-like" state).

[0009] Furthermore, it has been demonstrated that BRAF mutations in melanoma cells are not associated with phenotype-specific gene expression.

[0010] Therefore, this phenotypic shift represents one of the major obstacles to clinical success and underscores the need for alternative compounds that consistently target different tumor phenotypes controlled by different transcriptional programs, such as transcription factors like MITF, SOX10, SOX9, or c-JUN. Summary of the Invention

[0011] The inventors have surprisingly determined that compounds IA, PM14, and rubitidine are effective in treating skin cancer, particularly proliferative / differentiated and / or invasive / undifferentiated melanoma, as well as melanoma resistant to prior treatment with inhibitors of the MAP kinase signaling pathway.

[0012] Therefore, a first aspect of the present invention provides compound IA or a pharmaceutically acceptable salt or ester thereof:

[0013] Compound IA,

[0014] It is used to treat skin cancer.

[0015] In one specific embodiment, skin cancer is melanoma.

[0016] In another specific embodiment, the melanoma is aggressive. Melanomas exhibiting an aggressive phenotype were previously difficult to treat, and current therapies are ineffective against them.

[0017] In another specific embodiment, the melanoma is proliferative / differentiated.

[0018] In another specific embodiment, melanoma is a mixture of proliferative / differentiated and invasive / undifferentiated phenotypes. The present invention is effective in treating both phenotypes, which current therapies are ineffective against.

[0019] In another aspect, a method for treating skin cancer is provided, comprising administering to a patient in need a therapeutically effective amount of compound IA or a pharmaceutically acceptable salt or ester thereof.

[0020] In another aspect, the use of compound IA or a pharmaceutically acceptable salt or ester thereof in the preparation of a medicament for treating skin cancer is provided, wherein said treatment comprises administering a therapeutically effective amount of compound IA or a pharmaceutically acceptable salt or ester thereof to a patient in need.

[0021] In another aspect of the invention, a pharmaceutical composition or dosage form is provided comprising compound IA according to the invention or a pharmaceutically acceptable salt or ester thereof for the treatment of skin cancer.

[0022] In another aspect of the invention, the use of a pharmaceutical composition or dosage form comprising a compound IA according to the invention or a pharmaceutically acceptable salt or ester thereof in the preparation of a medicament for treating skin cancer is provided.

[0023] In another aspect, a drug package is provided that contains compound IA or a pharmaceutically acceptable salt or ester thereof, along with instructions for use in the treatment of skin cancer.

[0024] In another aspect, a method for inhibiting the growth of cancer cells is provided, the method comprising contacting cancer cells with compound IA or a pharmaceutically acceptable salt or ester thereof, wherein the cancer cells are skin cancer cells.

[0025] In another aspect, the present invention provides rubitidine or a pharmaceutically acceptable salt or ester thereof:

[0026] ,

[0027] It is used to treat aggressive / undifferentiated and / or proliferative / differentiated melanoma.

[0028] In this embodiment, the melanoma is aggressive. Melanomas exhibiting an aggressive phenotype were previously difficult to treat, and current therapies are ineffective against them.

[0029] In a further embodiment, the melanoma is proliferative / differentiated.

[0030] In a further embodiment, melanoma is a mixture of proliferative / differentiated and invasive / undifferentiated phenotypes. The present invention is effective in treating both phenotypes, which current therapies are ineffective against.

[0031] In another aspect, a method for treating aggressive / undifferentiated and / or proliferative / differentiated melanoma is provided, the method comprising administering to a patient in need a therapeutically effective amount of rubitidine or a pharmaceutically acceptable salt or ester thereof.

[0032] In another aspect, the use of rubitidine or a pharmaceutically acceptable salt or ester thereof in the preparation of a medicament for treating aggressive / undifferentiated and / or proliferative / differentiated melanoma is provided, wherein said treatment comprises administering a therapeutically effective amount of rubitidine or a pharmaceutically acceptable salt or ester thereof to a patient in need.

[0033] In another aspect of the invention, a pharmaceutical composition or dosage form is provided comprising rubitidine according to the invention or a pharmaceutically acceptable salt or ester thereof for the treatment of aggressive / undifferentiated and / or proliferative / differentiated melanoma.

[0034] In another aspect of the invention, the use of a pharmaceutical composition or dosage form comprising rubitidine according to the invention or a pharmaceutically acceptable salt or ester thereof in the preparation of a medicament for treating aggressive / undifferentiated and / or proliferative / differentiated melanoma is provided.

[0035] In another aspect, a drug package is provided comprising rubitidine or a pharmaceutically acceptable salt or ester thereof, along with instructions for use for the treatment of aggressive / undifferentiated and / or proliferative / differentiated melanoma.

[0036] In another aspect, a method for inhibiting cancer cell growth is provided, the method comprising contacting cancer cells with rubitidine or a pharmaceutically acceptable salt or ester thereof, wherein the cancer cells are invasive / undifferentiated and / or proliferative / differentiated melanoma cells.

[0037] In another aspect, the present invention provides PM14 or a pharmaceutically acceptable salt or ester thereof:

[0038] ,

[0039] It is used to treat aggressive / undifferentiated and / or proliferative / differentiated melanoma.

[0040] In this embodiment, the melanoma is aggressive. Melanomas exhibiting an aggressive / undifferentiated phenotype were previously difficult to treat, and current therapies are ineffective against them.

[0041] In a further embodiment, the melanoma is proliferative / differentiated.

[0042] In a further embodiment, melanoma is a mixture of proliferative / differentiated and invasive / undifferentiated phenotypes. The present invention is effective in treating both phenotypes, which current therapies are ineffective against.

[0043] In another aspect, a method for treating aggressive / undifferentiated and / or proliferative / differentiated melanoma is provided, the method comprising administering a therapeutically effective amount of PM14 or a pharmaceutically acceptable salt or ester thereof to a patient in need.

[0044] In another aspect, the use of PM14 or a pharmaceutically acceptable salt or ester thereof in the preparation of a medicament for treating aggressive / undifferentiated and / or proliferative / differentiated melanoma is provided, wherein said treatment comprises administering a therapeutically effective amount of PM14 or a pharmaceutically acceptable salt or ester thereof to a patient in need.

[0045] In another aspect of the invention, a pharmaceutical composition or dosage form is provided comprising PM14 according to the invention or a pharmaceutically acceptable salt or ester thereof for the treatment of aggressive / undifferentiated and / or proliferative / differentiated melanoma.

[0046] In another aspect of the invention, the use of a pharmaceutical composition or dosage form comprising PM14 according to the invention or a pharmaceutically acceptable salt or ester thereof in the preparation of a medicament for treating aggressive / undifferentiated and / or proliferative / differentiated melanoma is provided.

[0047] In another aspect, a drug package is provided comprising PM14 or a pharmaceutically acceptable salt or ester thereof, along with instructions for use for the treatment of aggressive / undifferentiated and / or proliferative / differentiated melanoma.

[0048] In another aspect, a method for inhibiting cancer cell growth is provided, the method comprising contacting cancer cells with PM14 or a pharmaceutically acceptable salt or ester thereof, wherein the cancer cells are invasive / undifferentiated and / or proliferative / differentiated melanoma cells.

[0049] The following embodiments apply to all aspects of the present invention:

[0050] In the embodiments, melanoma may include an aggressive / undifferentiated phenotype. Melanoma may include at least about 10% aggressive / undifferentiated phenotype, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% aggressive / undifferentiated phenotype.

[0051] In the embodiments, melanoma may contain a proliferative / differentiated phenotype. Melanoma may contain at least about 10% proliferative / differentiated phenotype, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% proliferative / differentiated phenotype.

[0052] In an embodiment, melanoma may comprise a mixture of proliferative / differentiated and invasive / undifferentiated phenotypes.

[0053] In this embodiment, the melanoma is an unresectable or metastatic BRAF.

[0054] In the embodiments, melanoma is RAS (including NRAS, HRAS and KRAS).

[0055] In this embodiment, the melanoma is a triple WT mutant melanoma.

[0056] In the embodiments, the melanoma is selected from cutaneous melanoma, mucosal melanoma, or acral melanoma.

[0057] In this embodiment, the melanoma is resistant to previous treatment with inhibitors of the MAP kinase signaling pathway.

[0058] In this embodiment, the inhibitor of the MAP kinase signaling pathway is selected from BRAF or MEK kinase inhibitors or combinations thereof.

[0059] In the examples, the BRAF or MEK kinase inhibitor is selected from vemurafenib, dabrafenib, cannefenib, trametinib, cobimetinib, or bimetinib, or combinations thereof. Attached Figure Description

[0060] Figure 1 Protein lysates from proliferative / differentiated melanoma cells 501mel, MM011, MM074, MM117, IGR37, and SKMel-28, or invasive / undifferentiated melanoma cells MM029, MM047, MM099, and IGR39, are shown, after Western blotting. The molecular weight (kDa) of the proteins is indicated.

[0061] Figure 2 It shows the use of their respective ICs 50 Melanoma cells were treated with concentrations of DMSO, rubitidine, PM14, or compound IA for 48 hours, and then allowed to regrow for 10 days in the absence of the drug. Results are shown as mean colony number + / - SD of three biological replicates. P-values ​​are shown (ordinary one-way ANOVA, using Dunnett's multiple comparison test).

[0062] Figure 3 The results showed incubation with CellTrace followed by incubation with DMSO, rubitidine, PM14, or compound IA at their respective IC50 values. 50 Melanoma cells treated with the concentration for 72 h. Quantification of the population with high CellTrace signal in cells treated with DMSO or the drug is shown as the mean + / - SD of three biological replicates. Proliferating cells showed low CellTrace signal, while non-proliferating cells showed high CellTrace signal. P-values ​​are shown (ordinary one-way ANOVA, using Dunnett's multiple comparison test).

[0063] Figure 4The results showed the use of DMSO, rubitidine, PM14, or compound IA at their respective IC50 values. 50 Melanoma cells were treated with a concentration of 501 mel for 72 h. Cell cycle was studied by flow cytometry, and the results are shown as the mean of three biological replicates with + / - SD.

[0064] Figure 5 It shows the use of their respective ICs 50 Melanoma cells were treated for 72 h with concentrations of DMSO, rubitidine, PM14, or compound IA. Apoptosis was studied by flow cytometry using annexin V-APC staining. Results are shown as the mean of three biological replicates + / - SD. P-values ​​are shown (plain one-way ANOVA, using Dunnett's multiple comparison test).

[0065] Figure 6 Displayed from 5xIC 50 Protein lysates from 501 melon of proliferative / differentiated melanoma cells treated for 24 h with concentrations of rubitidine, PM14, or compound IA were subjected to Western blotting. The molecular weight (kDa) of the proteins was indicated.

[0066] Figure 7 It shows the use of their respective ICs 50 MM029 and MM099 melanoma cells were treated for 48 h with concentrations of DMSO, rubitidine, PM14, or compound IA. Invasion was determined using the Boyden chamber assay. Results are shown as the mean + / - SD of the coverage index of three biological replicates. P-values ​​are shown (plain one-way ANOVA, using Dunnett's multiple comparison test).

[0067] Figure 8 The image shows a confluent monolayer of 501 melatonnes of scratch-treated melanoma cells, supplemented with reduced FCS % and DMSO, rubitidine, PM14, or compound IA (IC). 50 Fresh culture medium (concentration). Wound size was measured at indicated time points, and results are shown as the mean + / - SD of the fold change in wound area compared to DMSO treatment in three biological replicates. P-values ​​are shown (ordinary one-way ANOVA, using Dunnett's multiple comparison test).

[0068] Figure 9A and 9BThe results show that MM074 melanoma globules were treated with trametinib, vemurafenib, or dabrafenib (A) and the compound of the present invention (B) for 72 h, and cell viability was measured using the CellTiter-Glo assay. Results are shown as the mean + / - SD of viability compared to DMSO from three biological replicates. P-values ​​are shown (ordinary one-way ANOVA, using Dunnett's multiple comparison test).

[0069] Figure 10 MM074 melanoma spheres treated with DMSO, rubitidine, PM14, compound IA, or vemurafenib for 72 h are shown. Apoptosis was investigated by flow cytometry using annexin V-APC and propidium iodide staining. Results are shown as mean + / - SD of three biological replicates.

[0070] Figure 11A The qRT-PCR analysis was shown, which demonstrated the use of solvent (DMSO), rubitidine, PM14, or compound IA at 5x IC50. 50 Mean 18S normalized expression of MITF in differentiated / proliferating MM074, 501mel, and IGR37 cells treated with the concentration for 12 h. Error bars indicate the mean +SD of three biological replicates. P-values ​​are shown (ordinary one-way ANOVA, using Dunnett's multiple comparison test).

[0071] Figure 11B The qRT-PCR analysis was shown, which demonstrated the use of solvent (DMSO), rubitidine, PM14, or compound IA at 5x IC50. 50 Mean 18S normalized expression of AXL in undifferentiated / invasive MM029, MM099, and IGR39 cells treated with the concentration for 12 h. Error bars indicate the mean + / - SD of the three biological replicates. P-values ​​are shown (ordinary one-way ANOVA, using Dunnett's multiple comparison test).

[0072] Figure 12A and 12B Differentiated / proliferative MM074 (A) or undifferentiated / invasive MM099 (B) cells were shown using solvent (DMSO), rubitidine, PM14, or compound IA at 5x IC50. 50 Concentration treatment for 24 h. Western blotting of the protein lysates was performed as instructed. The molecular weight (kDa) of the protein was indicated.

[0073] Figure 13 The qRT-PCR analysis was shown, which demonstrated the use of solvent (DMSO), rubitidine, PM14, or compound IA at 5x IC50. 50Mean 18S normalized expression of RPL13A, TBP, MITF, and SOX10 in MM074 melanoma spheres treated with concentrations for 24 h. Results are shown as mean + / - SD of three biological replicates. P-values ​​are shown (ordinary one-way ANOVA, using Dunnett's multiple comparison test).

[0074] Figure 14 The qRT-PCR analysis was shown, which demonstrated the use of solvent (DMSO), rubitidine, PM14, or compound IA at 5x IC50. 50 Mean 18S normalized expression of RPL13A, TBP, and AXL in MM029 melanoma spheres treated with concentrations for 24 h. Results are shown as mean + / - SD of three biological replicates. P-values ​​are shown (ordinary one-way ANOVA, using Dunnett's multiple comparison test).

[0075] Figure 15A and 15B The qRT-PCR analysis was shown, which demonstrated the use of solvent (DMSO), rubitidine, PM14, or compound IA at 5x IC50. 50 Mean 18S normalized expression of SOX10 in differentiated / proliferating cells 501mel, MM074, and IGR37 (A) treated for 12 h, and mean 18S normalized expression of EGFR in undifferentiated / invasive cells MM029, MM099, and IGR39 (B). Error bars indicate the mean +SD of three biological replicates. P-values ​​are shown (ordinary one-way ANOVA, using Dunnett's multiple comparison test).

[0076] Figure 16A and 16B The results showed that when using rubitidine, PM14, or compound IA at 10x IC50... 50 Venn diagram of significantly downregulated (left) and upregulated (right) genes identified by RNA-seq in MM074(A) and MM029 (B) cells after 8 h of concentration treatment.

[0077] Figure 17 The diagram shows the Venn diagram between genes identified by RNA-seq as being universally downregulated (left) or upregulated (right) by three compounds in both MM074 and MM029 cells. The representation factors and hypergeometric p-values ​​are shown.

[0078] Figure 18 Gene ontology analysis, as shown in (b), reveals 757 genes (left) that were significantly downregulated by the three compounds and 110 genes that were significantly upregulated in MM074 and MM029 cells. Histograms show the top-ranked dysregulated biological pathways according to FDR and enrichment fold.

[0079] Figure 19A and 19B A heatmap was displayed, depicting the heat from Figure 17 All dysregulated genes from rubitidine, PM14, or compound IA treatment in MM074 cells (A) or MM029 cells (B) as described in RNA-Seq in A and 17B. RPKM values ​​are expressed as z-scores.

[0080] Figure 20 The study showed that after 24 hours of treatment with a single dose of placebo, PM14, or compound IA at 1.2 mg / kg, 501 mel and 501 mel were... Vemur Quantification of the mitotic index (% of pHH3 positive cells / tumor slice) for the SKMel28 CDX model, where results are shown as the mean + / - SD of tumor slices from three tumors for each condition. P-values ​​are shown (ordinary one-way ANOVA, using Dunnett's multiple comparison test).

[0081] Figure 21 The results showed that after 24 hours of treatment with a single dose of placebo, PM14, or compound IA at 1.2 mg / kg, 501 mel and 501 mel VemuR Quantification of the apoptosis index (% of lysed caspase-3 positive cells / tumor slice) in the SKMel28 CDX model, where results are shown as the mean + / - SD of tumor slices from three tumors for each condition. P-values ​​are shown (ordinary one-way ANOVA, using Dunnett's multiple comparison test).

[0082] Figure 22A , 22B 22C showed that 501mel(A), 501mel, were treated weekly with 1.2 mg / kg of placebo, PM14, or compound IA. Vemur CDX models (N=8 / condition) for (B) and SKmel-28 (C) were used to assess survival. P-values ​​(log-rank (Mantel-Cox) test) are shown.

[0083] Figure 23A and 23B The results showed that 501 melanomas treated intravenously with compounds IA and PM14 at 1.2 mg / kg Q7 dx3, respectively, were effective against melanomas. Vemur Tumor volume assessment in xenograft models of cell lines (resistant to vemurafenib).

[0084] Figure 24A and 24BTumor volume assessment was shown in a xenograft model of 501mel melanoma cell line treated intravenously with compounds IA and PM14 at 1.2 mg / kg Q7 dx3, respectively.

[0085] Figure 25A and 25B The tumor volume assessment of a xenograft model of the melanoma cell line IGR-37 treated intravenously with compounds IA and PM14 at 1.2 mg / kg Q7 dx3, respectively, is shown.

[0086] Figure 26A and 26B Tumor volume assessment is shown in a xenograft model of the melanoma cell line Skmel-28 treated intravenously with compounds IA and PM14 at 1.2 mg / kg Q7 dx3, respectively.

[0087] Figure 27A and 27B The tumor volume assessment of a xenograft model of melanoma cell line WM-2664 treated intravenously with compounds IA and PM14 at 1.2 mg / kg Q7 dx3, respectively, is shown.

[0088] Figure 28A and 28B Tumor volume assessment is shown in a xenograft model of the LOX-IMVI melanoma cell line treated intravenously with compounds IA and PM14 at 1.2 mg / kg Q7 dx3, respectively. Detailed Implementation

[0089] Many common terms and phrases are used in this application, which should be interpreted as follows.

[0090] Unless otherwise stated, as used herein, the term "treating" means reversing, reducing, alleviating, or inhibiting the progression of a disease or condition to which the term applies, or one or more symptoms of that disease or condition. As used herein, unless otherwise stated, the term "treatment" refers to the therapeutic act immediately following the definition of "treatment" above.

[0091] "Patients" include humans, non-human mammals (e.g., dogs, cats, rabbits, cattle, horses, sheep, goats, pigs, deer, etc.) and non-mammals (e.g., birds, etc.), preferably humans.

[0092] Ecteinascidia turbinata is a highly effective antitumor agent isolated from the marine tunicate mangrove tunicate.

[0093] WO2018 / 197663 describes synthesized seaweed extract compounds, including compound 39-S having the following formula:

[0094]

[0095] This compound has demonstrated in vitro activity against non-small cell lung cancer (NSCLC), colorectal adenocarcinoma, breast adenocarcinoma, pancreatic adenocarcinoma, prostate adenocarcinoma, and prostate cancer cell lines, as well as in vivo activity in xenograft models of fibrosarcoma, breast adenocarcinoma, NSCLC, ovarian cancer, gastric cancer, small cell lung cancer (SCLC), and prostate cancer. This compound can be synthesized using the synthetic route disclosed in WO2018 / 197663.

[0096] WO2018 / 197663 also describes synthesized tunicate compounds, including “ecubetidine” called PM14, which is described as compound 4-S having the following formula:

[0097]

[0098] This compound has demonstrated in vitro activity against non-small cell lung cancer (NSCLC), colorectal adenocarcinoma, breast adenocarcinoma, pancreatic adenocarcinoma, prostate adenocarcinoma, melanoma, and prostate cancer cell lines, as well as in vivo activity in fibrosarcoma, breast adenocarcinoma, NSCLC, ovarian cancer, gastric cancer, small cell lung cancer (SCLC), prostate adenocarcinoma, and prostate cancer xenograft models. This compound can be synthesized using the synthetic route disclosed in WO2018 / 197663.

[0099] Rubitidine, also known as PM01183, originally called tryptamicidin, is a synthetic alkaloid with antitumor activity and is the subject of WO 03 / 014127. Rubitidine is a selective inhibitor of oncogenic transcription, induces DNA double-strand breaks leading to apoptosis, and modulates the tumor microenvironment. For example, by inhibiting active transcription in tumor-associated macrophages, rubitidine downregulates IL-6, IL-8, CCL2, and VEGF. Rubitidine can be synthesized using the synthetic route disclosed in WO 03 / 014127.

[0100] The chemical structure of rubitidine is represented as follows:

[0101]

[0102] It is a selective inhibitor of oncogenic transcriptional programs that many tumors are particularly dependent on. In addition to its effects on cancer cells, rubitidine also inhibits oncogenic transcription in tumor-associated macrophages and downregulates the production of cytokines essential for tumor growth. Transcriptional addiction is a recognized target in these diseases, many of which lack other actionable targets.

[0103] Rubitridine was approved and marketed in the United States in 2020 for the treatment of adult patients with metastatic small cell lung cancer (SCLC) whose disease has progressed during or after platinum-based chemotherapy. The recommended dose is 3.2 mg / m2 administered intravenously every 21 days. In 2021, rubitridine also received marketing authorization in the United Arab Emirates, Canada, Australia, and Singapore.

[0104] In the context of this invention, "skin cancer" should be understood as cancer that forms in skin tissue. There are several types of skin cancer: melanoma, which forms in cells called melanocytes in the skin; basal cell carcinoma, which forms in the lower part of the epidermis (outer layer of skin); squamous cell carcinoma, which forms in squamous cells (flat cells that form the surface of the skin); and neuroendocrine carcinoma of the skin, which forms in neuroendocrine cells (cells that release hormones in response to signals from the nervous system).

[0105] Melanoma is a type of skin cancer that occurs when melanocytes begin to grow uncontrollably. Melanoma is much less common than some other types of skin cancer. However, it is more dangerous because it is more likely to spread to other parts of the body if not detected and treated early. The number of people diagnosed with melanoma has increased over the past few decades.

[0106] Melanoma can occur anywhere on the skin, but it is more likely to begin on the male torso (chest and back) and female legs. The neck and face are other common sites. Melanoma can also form in other parts of the body, such as the eyes, mouth, genitals, and anus, but these sites are much less common than cutaneous melanoma. Regarding this invention, the compound treats melanoma on the skin as well as on any non-cutaneous site.

[0107] According to the National Cancer Institute (NCI), 52% of cutaneous melanomas have a BRAF-mutated genomic subtype, 28% have a RAS-mutated genomic subtype (NRAS, HRAS, and KRAS), 14% have an NF1-mutated subtype, and 14.5% are triple WT, meaning they are heterogeneous subgroups lacking BRAF, NRAS, HRAS, KRAS, and NF1 mutations.

[0108] In the context of this invention, "inhibitor of the MAP kinase signaling pathway" should be understood as a compound that targets components of the MAPK pathway.

[0109] MAPK, or mitogen-activated protein kinase (MAPK), is a component of many signal transduction pathways and is activated by protein kinase cascades. The MAPK pathway relays, amplifies, and integrates signals from a variety of stimuli, triggering appropriate physiological responses, including cell proliferation, differentiation, development, inflammation, and apoptosis in mammalian cells. In this invention, the MAPK pathway is represented by the Raf-MEK-ERK kinase cascade, which generates proliferation, differentiation, and development as cellular responses.

[0110] BRAF inhibitors are drugs that can reduce or slow the growth of melanoma in individuals whose tumors have BRAF mutations. MEK inhibitors are drugs that target the kinases MEK1 and / or MEK2. The MEK gene is closely related to the BRAF gene, therefore drugs that target MEK can also help treat melanomas with BRAF mutations.

[0111] Current BRAF inhibitors include vemurafenib, dabrafenib, and cannefenib, while MEK inhibitors include trametinib, cobimetinib, and bimetinib.

[0112] Furthermore, the positive results led to the FDA approval of three BRAFi / MEKi combinations, including:

[0113] • Dabrafenib plus trametinib (approved for metastatic and resected stage III melanoma, NSCLC and ATC, and low-grade glioma)

[0114] • Vemofinib plus cobimetinib (approved for metastatic melanoma)

[0115] • Cannefenib (approved for metastatic melanoma)

[0116] In embodiments of the present invention, compound IA is used to treat melanoma that has previously been resistant to MAP kinase signaling pathway inhibitors.

[0117] In an embodiment of the invention, PM14 is used to treat melanoma that has previously been resistant to MAP kinase signaling pathway inhibitors.

[0118] In embodiments of the present invention, rubitidine is used for melanoma that is previously resistant to MAP kinase signaling pathway inhibitors.

[0119] In the embodiments, compound IA is used in a method of treating patients who have progressed from treatment with an inhibitor of the MAP kinase signaling pathway (or who have otherwise been identified as resistant to that inhibitor).

[0120] In another embodiment, PM14 is used in a method of treating patients who have progressed from treatment with an inhibitor of the MAP kinase signaling pathway (or who have otherwise been identified as resistant to that inhibitor).

[0121] In a further embodiment, rubitidine is used in a method of treating patients who have progressed from treatment with an inhibitor of the MAP kinase signaling pathway (or who have otherwise been identified as resistant to that inhibitor).

[0122] Inhibitors of the MAP kinase signaling pathway can be BRAF inhibitors.

[0123] BRAF inhibitors can include vemurafenib.

[0124] BRAF inhibitors can include dabrafenib.

[0125] BRAF inhibitors can include cannefenib.

[0126] Inhibitors of the MAP kinase signaling pathway can be MEK inhibitors.

[0127] MEK inhibitors can include trametinib.

[0128] MEK inhibitors can include cobimetinib.

[0129] MEK inhibitors can include bimetinib.

[0130] Inhibitors of the MAP kinase signaling pathway can be either BRAF inhibitors or MEK inhibitors.

[0131] BRAF / MEK inhibitors can be dabrafenib plus trametinib.

[0132] BRAF / MEK inhibitors can be vemofinib plus cobimetinib.

[0133] BRAF / MEK inhibitors can include caninefenib plus memetinib.

[0134] Inhibitors of the MAP kinase signaling pathway can be MEK inhibitors.

[0135] Transcription in melanoma is dysregulated. One example of a melanoma-driving transcription factor is MITF, which was identified as a lineage-specific oncogene using an integration approach that combines single nucleotide polymorphism (SNP) array data with gene expression analysis of the NCI-60 cell line.

[0136] Furthermore, transcription factors represent a highly dysregulated class of cellular effectors in melanoma cells carrying BRAF V600E that are resistant to BRAF inhibition.

[0137] Compounds IA, PM14, or rubitidine can balance the dysregulation caused by transcription factors. Compounds IA, PM14, or rubitidine induce a damaging response and significant degradation of the largest subunit of RNAPII, which leads to the suppression of the expression of essential genes in melanoma cells, such as MITF or SOX10.

[0138] Examples of administration methods include, but are not limited to, oral, topical, parenteral, sublingual, rectal, vaginal, ocular, and intranasal administration. Parenteral administration includes subcutaneous injection, intravenous, intramuscular, intrasternal injection, or infusion techniques. Preferably, the composition is administered parenterally. The pharmaceutical compositions of the present invention can be formulated using methods well known in the pharmaceutical industry to make the compounds according to the invention bioavailable when administered to animals, preferably humans. The compositions can be in the form of one or more dose units, wherein, for example, tablets can be single dose units, and containers for the compounds according to the invention can contain the compound in liquid or aerosol form and can hold single or multiple dose units. Compositions intended for parenteral administration can be prepared by combining the compounds of the invention with water or other physiologically suitable diluents to form a solution or suspension. Example

[0139] Example 1: Human melanoma cell models with different genotypes and phenotypes are highly sensitive to rubitidine / PM14 / compound IA

[0140] To investigate the response of human melanoma cells to rubitidine, PM14, or compound IA, cells representing the two major phenotypes (invasive or proliferative) and the most common driver mutations in melanoma, as well as human melanocytes (produced in vitro in the laboratory, Hermes 3A cell line) were examined (Table 1).

[0141] Table 1. Melanoma cell lines used to test the cytotoxicity of the compounds of the present invention. The phenotype and genotype of these cells are indicated.

[0142]

[0143] (*)Note: Triple negative refers to BRAF, NRAS, and NF1 genes.

[0144] On the one hand, the melanocyte-type culture MM011 (NRAS) derived from differentiated / proliferative patients was evaluated. Q61K ), MM074 (BRAF) V600E ), MM117 (triple WT) and melanoma cell line 501mel (BRAF) V600E ), IGR37 (BRAF) V600E ) and SKMel-28 (BRAF) V600EOn the other hand, patient-derived undifferentiated / invasive mesenchymal-like melanoma cell cultures MM029 (BRAF) were examined. V600K ), MM047 (NRAS) Q61R ), MM099 (BRAF) V600E ) and melanoma cell line IGR39 (BRAF) V600E ).

[0145] Cells were grown at 37°C and 5% CO2 (10% for Hermes 3A) with regular checks for mycoplasma contamination. Short-term melanoma cultures derived from MM patients (MM011, MM074, MM117, MM029, MM047, MM099) were grown in HAM-F10 (Gibco, Invitrogen) supplemented with 10% fetal bovine serum (FCS), 25 mM HEPES, 5.2 mM GLUTAMAX, and penicillin-streptomycin. Melanoma cell lines 501mel and SKmel28 were grown in HEPES-free (Gibco, Invitrogen) supplemented with 10% FCS and gentamicin. Vemurafenib-resistant cells (501melVemuR and MM074VemuR) were additionally supplemented with 1.5 µM vemurafenib. Melanoma IGR cell lines (IGR37 and IGR39) were grown in RPMI without HEPES (Gibco, Invitrogen) supplemented with 15% FCS and gentamicin.

[0146] Proliferating / differentiated cells exhibited moderate to high expression of lineage-specific transcription factors MITF and SOX10, and low to undetectable levels of metastatic EGFR and AXL (Table 2 and ). Figure 1 ).

[0147] Invasive / undifferentiated cells showed low to undetectable levels of MITF and SOX10, and high levels of EGFR and / or AXL.

[0148] To determine the IC50 of these cells against various inhibitors 50 (Half-maximal inhibitory concentration) was used to determine cell viability.

[0149] After treatment with different concentrations of the compound for 72 hours, cells were treated with PrestoBlue reagent according to the manufacturer's instructions. The absorbance of each well was measured using a Cellinsight CX5 microplate reader. Data were analyzed using Prism9 statistical software. The IC50 values ​​obtained for each cell line were... 50 The values ​​are shown in Table 2.

[0150] Table 2. IC50 values ​​obtained in each cell line using the compounds of the present invention, compared to currently used inhibitors vemurafenib (Vemu), dabrafenib (Dabra), and trametinib (Trame). 50 .

[0151]

[0152] Patient-derived cell cultures and melanoma cell lines exhibit varying sensitivities to commonly used targeted therapies in the clinical management of melanoma, such as BRAF inhibitors vemurafenib and dabrafenib (Table 2), and the MEK inhibitor trametinib (Table 2) . Differentiated / proliferative BRAF V600E Melanoma cells (such as MM074 or IGR37) are most sensitive to these drugs, while undifferentiated / invasive melanoma cultures and cell lines show high resistance (in the micromolar range).

[0153] In contrast, all melanoma cells were observed to exhibit high sensitivity to rubitidine, PM14, and compound IA, with an IC50 value of [missing value]. 50 The values ​​are in the low nanomolar range, spanning from 0.3 to 4.73 nM (Table 2).

[0154] In addition, vemurafenib-resistant cells, i.e., 501 metronidazole cells, were generated by exposing cells to increased drug concentrations in vitro. Vemur and MM074 Vemur (Table 2). These vemurafenib-resistant cells exhibited cross-resistance to dabrafenib (in the case of MM074) and trametinib (Table 2), but maintained high sensitivity to rubitidine, PM14, and compound IA (Table 2). When these compounds were tested on non-cancerous Hermes3A immortalized melanocytes, this cell line was observed to be less sensitive to rubitidine, PM14, or compound IA than to melanoma cells.

[0155] In summary, melanoma cells have been shown to exhibit high sensitivity to rubitidine, PM14, and compound IA, with an IC50 concentration of 100%. 50 The values ​​are in the nanomolar range and are independent of cell phenotype or driver mutations.

[0156] Example 2: Rubitidine / PM14 / Compound IA inhibits proliferation, blocks cell cycle progression, causes DNA damage, and induces apoptotic death in melanoma cells.

[0157] Based on the cell viability assays described above, the efficacy of rubitidine, PM14, or compound IA on melanoma cell proliferation was investigated. Initially, a colony formation assay was performed. Cells were analyzed at an IC50 concentration over 48 hours. 50 The cells were treated with a drug at a concentration of 1x10. 3Or 2x10 3 Cells were seeded into drug-free 6-well plates and allowed to grow for 10 days to allow for colony formation. The cells were then fixed with 4% formaldehyde solution for 10 min, washed once with PBS, and stained with 0.2% crystal violet solution for 15 min. Finally, the wells were washed twice with deionized water, air-dried, scanned, and analyzed using Fiji software to count the number of colonies.

[0158] The results demonstrated that rubitididine, PM14, and compound IA had significant effects on all melanoma cell cultures or cell lines tested. Figure 2 ).

[0159] Subsequently, additional experiments were conducted to elucidate the affected cellular responses in melanoma cells after drug treatment, and flow cytometry was used to analyze proliferation and apoptosis induction.

[0160] Cells (2x10) 6 Inoculate into 6-well plates and incubate with 1 μM CellTrace Violet reagent (ThermoFisher) according to the manufacturer's instructions after 24 hours, then immediately incubate at IC50. 50 Cells were washed and treated with the drug at the specified concentrations. After 48 hours of incubation, cells were washed and incubated with annexin V-APC (BD Biosciences). Cell proliferation and apoptosis were detected using a BDLSLRFortessa™ flow cytometer. Data were analyzed using FlowJo software. To define slowly proliferating or apoptotic cells: slowly proliferating cells were considered to represent the 30% of cells in the DMSO control with the highest concentration of CellTrace Violet signal. The percentage of cells treated with the drug and having a signal greater than or equal to that value was then calculated. For apoptotic cells, the 20% of cells in the DMSO control with the highest annexin V-APC signal were considered.

[0161] For cell cycle analysis, use 2x10 6 Cells were seeded in 6-well plates. In IC50... 50 After treatment with the drug at the specified concentration, the cells were pelleted and fixed with 70% ethanol at 4°C for 1 h. After washing twice with cold PBS, the cells were incubated with RNase A and PI in the dark for 1 h, and then analyzed on a BD LSRFortessa™ flow cytometer. Data were analyzed using FlowJo software.

[0162] Compared with DMSO, exposure to rubitidine, PM14, or compound IA significantly inhibited melanoma cell proliferation. Figure 3 Meanwhile, treatment with rubitidine, PM14, or compound IA significantly inhibited cell cycle progression. Figure 4 And apoptosis was significantly induced. Figure 5 ).

[0163] To assess potential DNA damage, an alternative biomarker was used: detection of γH2AX (gH2AX), which signals double-strand breaks. At 5x IC50 for each drug... 50 After 24 hours of treatment, distinct gH2AX activation was observed in the nuclei of differentiated / proliferative 501mel melanoma cells or undifferentiated / invasive MM029 cell cultures. This was confirmed by the different activation rates observed in differentiated / proliferative 501mel and MM074 cells. Figure 6 Immunoblotting in undifferentiated / invasive MM029 cells was confirmed.

[0164] Phosphorylation of ATM, a core protein involved in the DNA damage response, was observed in 501 mel concurrently with the accumulation of gH2AX. Figure 6 Interestingly, RPB1 (RNA pol II elongation subunit) degradation was minimal in the presence of rubitidine, but significant in the presence of PM14 and compound IA. Figure 6 ).

[0165] The results showed that rubitidine, PM14, and compound IA exerted strong cell-inhibiting and cytotoxic effects on both proliferating / differentiated and invasive / undifferentiated melanoma cells, characterized by the generation of DNA breaks and degradation of RNAPII, which were particularly significant for PM14 and compound IA, forcing cells to undergo apoptosis and cell death.

[0166] Example 3: Effects of rubitididine / PM14 / compound IA on the migration and invasion of undifferentiated / invasive melanoma cell cultures

[0167] The undifferentiated / invasive melanoma cell cultures MM029 and MM099 were studied. Figure 7 and 8 Invasion and migration due to the presence of rubitidine, PM14 or compound IA.

[0168] Boyden chamber invasion and wound healing assays were performed.

[0169] Use 2x10 6For Boyden chamber invasion assays, cells were seeded in Boyden chamber inserts (Fisher Scientific) containing 4% Matrigel (Corning) and covered with serum-free medium. The inserts were placed in 24-well plates filled with complete medium. After 24 hours, the inserts were fixed with 4% formaldehyde solution for 10 min, washed once with PBS, and stained with 0.2% crystal violet solution for 15 min. Finally, the wells were washed twice with deionized water, air-dried, and images were collected using an EVOS xlCore microscope. These images were analyzed using Fiji to assess cell occupancy.

[0170] Wound healing migration was assessed in 6-well plates using confluent melanoma cell monolayers, achieved by scraping with the tip of a 20 µL pipette to create homogeneous, cell-free wounds. Fresh culture medium with or without drugs and a low FCS % (to mitigate proliferation) was added. Micrographs of the wounds were taken under an inverted microscope at 0, 24, and 48 hours. Wound area was then quantified using ImageJ software.

[0171] Boyden chamber and trauma assays clearly demonstrate that all three drugs of this invention significantly affect the invasion and migration of undifferentiated / invasive melanoma cell cultures. Figures 7 to 8 ).

[0172] Example 4: Rubitidine / PM14 / Compound IA exhibits strong cytotoxic activity against BRAF mutant 3D melanoma spheres.

[0173] The effects of rubitidine, PM14, or compound IA on three-dimensional (3D) melanoma cultures were investigated using melanoma sphere culture assays. Initially, the response of 3D melanoma spheres to BRAF (vemurafenib and dabrafenib) and MEK (trametinib) inhibitors was examined.

[0174] MM074 proliferating / differentiating cell lines (5x10) 4 Cells were seeded in 96-well plates coated with an ultra-low adhesion hydrogel in KO DMEM medium supplemented with 20% KSR, AANE, 2 mM Glutamax, penicillin / streptomycin, and 100 μM β-mercaptoethanol. To induce melanoma sphere formation, cells were allowed to grow for 4 days prior to drug treatment. They were then seeded at [IC50]... 50 ]x1 or [IC 50 x5 was exposed to the compound of the present invention for 72 hours and reacted with [IC] 50 ]x1 or [IC 50 The treatment with vemurafenib, dabrafenib, and trametinib for 72 hours was compared.

[0175] To analyze the viability of melanoma spheres after drug treatment, cells were treated with CellTiterGlo reagent (Promega) according to the manufacturer's instructions. The luminescence signal was measured using a Centro XS LB 960 microplate reader (Berthold).

[0176] Interestingly, in stark contrast to the response observed in 2D cultures, even at the equivalent of 5x IC50... 50 At the doses, vemurafenib, dabrafenib, and trametinib also failed to reduce cell viability in 3D cultures. Figure 9A ).

[0177] Conversely, rubitidine, PM14, and compound IA are equivalent to 5x IC 50 At the specified dose, it exhibited significant cytotoxicity against MM074 melanoma globules. Figure 9B ).

[0178] For apoptosis assays of 3D-growing melanoma cells, melanoma spheres were dissociated using TrypLe Select 10x reagent (Gibco) to obtain single-cell suspensions. These cells were then incubated with annexin V-APC (Biolegend) and propidium iodide (PI, Biolegend). Bivariate dot plots were used to distinguish between viable cells (annexin V- / PI-), early apoptotic cells (annexin V+ / PI-), late apoptotic cells (annexin V+ / PI+), and necrotic cells (annexin V- / PI+).

[0179] Melanoma spheres treated with rubitididine, PM14, and compound IA showed a large population of late apoptotic cells positive for annexin V and propidium iodide (in contrast to vemurafenib), indicating abundant DNA breaks compared to the control sample. Figure 10 ).

[0180] These findings highlight the potent cytotoxic activity of rubitidine, PM14, and compound IA against mutant BRAF melanoma globules, in contrast to the limited efficacy of currently used targeted therapies based on BRAF and MEK inhibition.

[0181] Example 5: Rubitidine, PM14, and compound IA affect key melanoma genes

[0182] To investigate the potential mechanisms underlying the sensitivity of melanoma cells to rubitidine, PM14, and compound IA, 5x IC50 was used. 502D cell cultures of differentiated / proliferative melanoma cells (501mel, MM074, and IGR37) and undifferentiated / invasive melanoma cells (MM029, MM099, and IGR39) were treated with doses of rubitidine, PM14, and compound IA for 12 h.

[0183] The aim was to evaluate whether rubitidine, PM14, and compound IA dysregulate key genes in melanoma, such as lineage-specific transcription factors MITF and SOX10 expressed in proliferative / differentiated melanoma cells, and pro-invasive / migration transmembrane protein AXL and pro-transfer protein EGFR expressed in invasive / differentiated melanoma cells.

[0184] RT-qPCR analysis was performed. Total RNA was isolated using the NucleoSpin RNA Plus kit (Macherey-Nagel) according to the manufacturing protocol. RNA was reverse transcribed using Superscript IV (Invitrogen), and qPCR was performed using SYBR Green (Roche) on a LightCycler 480 (Roche). Target gene expression was normalized using the 18S reference gene. Primers for RT-qPCR were designed using Primer-BLAST. The following primers were used:

[0185]

[0186] RT-qPCR analysis revealed that, compared with the housekeeping gene β-actin, which showed stable expression after treatment, the expression of MITF and SOX10 in differentiated cells and the expression of AXL and EGFR in undifferentiated cells were significantly affected by the compounds of this invention. Figures 11A to 11B and Figures 15A to 15B In addition, immunoblotting was performed 24 hours after treatment using antibodies that specifically recognize each protein.

[0187] Cells were washed once with cold PBS, rinsed with a cell scraper, and the pellet was precipitated and resuspended in LSDB 0.5M buffer (0.5M KCl, 50mM Tris HCl pH 7.9, 20% glycerol, 1% NP40, 1mM DTT, PIC). Cells were then completely disrupted by heat shock in liquid nitrogen and a 37°C water bath for three cycles, followed by centrifugation at 14,000 rpm for 15 min to precipitate cell debris. Ten micrograms of extract were loaded onto an SDS-PAGE plate and then transferred to a nitrocellulose membrane for Western blotting.

[0188] Immunoblotting further confirmed that the protein levels of these genes were significantly reduced. Figures 12A to 12B ).

[0189] In addition, the expression of MITF and SOX10 in MM074 melanoma spheres and the expression of AXL in MM029 melanoma spheres were evaluated in 3D melanoma cultures to assess the effectiveness of treatment with 5x IC50. 50 After 24 hours of treatment with the specified dosage, whether the RPL13A or TBP gene was significantly affected by the compound of the present invention compared to the control gene (RPL13A or TBP) was significantly affected. Figure 13 and Figure 14 ).

[0190] In summary, these findings suggest that short-term treatment with rubitidine, PM14, or compound IA affects key melanoma master regulatory genes (MRGs) in 2D and 3D melanoma cell cultures, leading to apoptosis.

[0191] Example 6: Differential set of rubitidin, PM14 and compound IA inhibiting melanoma genes

[0192] To investigate the effects of rubitidine, PM14, or compound IA on the transcriptome of melanoma cells, gene expression profiling was performed in 2D cultures of representative melanoma cells MM074 and MM029, representing differentiated / proliferative and undifferentiated / invasive phenotypes, respectively.

[0193] Use 10xIC 50 Cells were treated with the drug for 8 hours, and total RNA was extracted using the QIAquick RNA Purification Kit (Qiagen). The SuperScript II RT reverse RT Reverse Transcription Kit (Thermo Scientific) was used according to the manufacturer's recommendations. Following the manufacturer's instructions, the purified RNA was used for library preparation and high-throughput sequencing on Illumina NovaSeq at 50-base reads. Sequencing samples were analyzed using the nf-core RNAseq pipeline v1.3 for quality control, trimming, and alignment to a reference human genome using HISAT2. Transcripts were identified and quantified using feature counts from the subread package. Differentially expressed genes (DEG) analysis was performed using DeSeq2.

[0194] Treatment with the compounds of this invention resulted in a significant downregulation of genes expressed in MM074 and MM029 cells, and a smaller number of genes upregulation.

[0195] It is noteworthy that the compounds of this invention typically downregulate 1,365 genes in differentiated MM074 cells and 1,104 genes in undifferentiated MM029 cells. Figures 16A to 16BOf these genes, 757 were consistently downregulated by all three drugs in MM074 and MM029 cells. Figure 17 ).

[0196] Gene Ontology (GO; https: / / geneontology.org / ) analysis revealed that many of these 757 genes are involved in transcription processes, indicating that a large proportion of the genes sensitive to rubitidine and its derivatives are related to transcription factor function. Figure 18 ).

[0197] Furthermore, gene set enrichment analysis (GSEA; https: / / www.gsea-msigdb.org / gsea / index.jsp) showed that genes involved in the G2M checkpoint and WNT β-catenin signaling pathways in both MM074 and MM029 were particularly affected by the three drugs.

[0198] The transcriptional effects of compound IA were compared with those of rubitidine in differentiated / proliferative MM074 and undifferentiated / invasive MM029 melanoma cell cultures. Compound IA induced different transcriptional effects compared to rubitidine, resulting in different and fewer gene dysregulations in both differentiated / proliferative and undifferentiated / invasive melanoma cells. Figures 19A to 19B ).

[0199] In summary, this indicates that rubitidine, PM14, and compound IA have a significant impact on the transcriptional program of melanoma cells, with compound IA exerting the least effect on transcriptional activity compared to rubitidine and PM14, while exhibiting equally high cytotoxic activity.

[0200] GSEA and GO analyses further revealed that compound IA affects more genes involved in transcriptional regulation, while rubitidine mainly downregulates genes related to cell cycle pathways.

[0201] Example 7: Antiproliferative and proapoptotic effects of PM14 and compound IA in a xenograft mouse melanoma model

[0202] To investigate the effects of the aforementioned test drugs in living organisms, it was decided to examine the effects of PM14 and compound IA on a melanoma cell-derived xenograft (CDX) mouse model. Human 501mel and 501mel... VemuR Alternatively, SKMEL28 melanoma cells were subcutaneously implanted into the right abdomen of NSG mice.

[0203] Once the tumor reaches 150 mm 3The size of the sample was determined by administering a single intravenous (IV) dose of PM14 or compound IA at a concentration of 1.2 mg / kg to animals (N=3 / group).

[0204] Large 8-bit digital scan images (Hamamatsu, NanozoomerHT 2.0) of tumors stained with DAPI (10,000 to 30,000 nuclei per slice) and pHH3 or caspase 3 were processed using an internal Python (v3.8) algorithm to quantify positive cells. Essentially, the blue channel was fed into a Cellpose2 model (a deep learning model with a PyTorch process backbone) to segment the nuclei. Nuclei-specific signals were then analyzed. For pHH3, a nucleus was considered positive if the total number of pixels with an intensity value greater than 50 exceeded 20% of the nucleus surface (values ​​ranged from 0 [no signal] to 255 in the 8-bit image). The same procedure was applied to caspase 3, with pixel values ​​set to 50 and a minimum coverage area set to 30%. For each image, the ratio of positive cells to total nuclei was returned as an experimental variable. Statistical data were generated using Python's pingouin library (v0.5.3) and built-in bidirectional ANOVA and post-hoc test functions.

[0205] Twenty-four hours later, the level of phosphohistone H3 (pHH3) was assessed to measure the mitotic index (% of pHH3-positive cells / tumor section). Based on the results, compared with xenografts not treated with the drug or DMSO, at 24 hours after drug treatment, 501 mil and 501 mil were significantly higher. VemuR The proportion of pHH3-positive cells in xenografts derived from SKMEL28 was reduced fourfold. Figure 20 ).

[0206] In addition, after drug treatment, 501mel and 501mel were evaluated using the IF (intracellular fibrosis) caspase-3 cleavage assay. VemuR Apoptotic cell density (% of lysed caspase-3 positive cells / tumor section) in xenografts derived from SKMEL28 at 24 hours. A significantly increased proportion of apoptotic cells was observed in all three tumors treated with PM14 or compound IA compared to untreated or DMSO-treated xenografts. Figure 21 ).

[0207] Tumor volume was monitored after weekly IV treatment with PM14 at a concentration of 1.2 mg / kg or compound IA. Tumors in 4- to 6-week-old female NSG mice (N=8 / group) reached 150 mm. 3Treatment began on day 0. When comparing the group receiving weekly treatment to the placebo group, we observed antitumor activity of PM14 and compound IA (including activity against 501 mel). VemuR Statistically significant differences were observed in the origin of the tumor. Furthermore, a significant increase in overall survival was noted in all three CDX models with both drugs (PM14 and compound IA). Figures 22A to 22C ), including those carrying 501mel VemuR The survival rate of mice with tumor-derived tumors was significantly affected. Figure 22B These findings indicate that PM14 and compound IA possess potent antitumor activity in living organisms and have a significant impact on animal survival rates.

[0208] Example 8: Reduction of in vivo tumor volume in a xenograft model

[0209] Female athymic nu / nu mice (Harlan Laboratories Models, SL Barcelona, ​​Spain, or Envigo, Spain) were used for all experiments. Animals were housed in individual, ventilated cages (Sealsafe® Plus, Techmplast SPA) at 21–23°C and 40–60% humidity, with a maximum of ten mice per cage, under a 12-hour light-dark cycle. Mice were allowed free access to an irradiated standard rodent diet (Tecklad 2914C) and sterile water. Animals were acclimatized for at least 5 days before tumor cell suspension implantation.

[0210] The cell line used is:

[0211] •501mel

[0212] •IGR-37

[0213] •SK-Mel-28

[0214] •WM-266-4

[0215] •LOX-IMVI

[0216] •501mel Vemur

[0217] In xenotransplantation studies of cell lines:

[0218] -Use equation (ab) 2The tumor volume was calculated as a / 2, where a is the length (longest diameter) and b is the width (shortest diameter), measured in mm using digital calipers (Fowler Sylvac, S235PAT). Tumor size and weight were recorded 2-3 times per week, starting from the first day of treatment.

[0219] -When the tumor reaches approximately 150-250 mm 3 Based on body weight and tumor measurements, tumor-bearing animals (N=8-10 / group) were randomly assigned to treatment groups using NewLabOncology Software (version 2.25.06.00).

[0220] - The antitumor efficacy was assessed by comparing the median tumor volume between the treatment group and the placebo group.

[0221] - When the animal's tumor reaches approximately 1,500 mm 3 Euthanasia may be performed on animals when severe necrosis is observed.

[0222] Treatments that produce a mortality rate >20% and / or a net weight loss of 20% are considered toxic.

[0223] The placebo was provided in the form of a lyophilized cake containing 200 mg sucrose + 13.6 mg potassium dihydrogen phosphate + qs phosphate pH 3.8-4.1 + qs potassium hydroxide pH 3.8-4.1, which was reconstituted with water for infusion.

[0224] In these experiments, PM14 and compound IA, along with a placebo, were administered intravenously once a week for three weeks, on days 0, 7, and 14, whenever possible.

[0225] Table 3 reports the number of melanomas in mice treated with placebo and intravenously with the present invention compounds PM14 and IA (at a dose of 1.2 mg / kg). Vemur Median tumor volume assessment of the cell line. Results also showed... Figures 23A to 23B middle.

[0226] Table 3. Tumor volume assessment in the 501 melVemur cell line using the present invention's compounds PM14 and IA, as well as placebo.

[0227]

[0228] Compared to controls, the compounds of the present invention resulted in a reduction in tumor volume in various xenograft models with vemurafenib-resistant cell lines. Administration of the compounds of the present invention led to a delay in disease progression.

[0229] These results are also shown in Tables 4 to 8 and Figures 24-28 AB (501mel, IGR-37, SK-Mel-28, WM-266-4, LOX-IMVI, respectively).

[0230] Table 4. Tumor volume of 501 mel melanoma xenografts evaluated using PM14, compound IA, and placebo.

[0231]

[0232] Table 5. Tumor volume of IGR-37 melanoma xenografts evaluated using PM14, compound IA, and placebo.

[0233]

[0234] Table 6. Tumor volume of Sk-mel-28 melanoma xenografts evaluated using PM14, compound IA, and placebo.

[0235]

[0236] Table 7. Tumor volume of WM-266-4 melanoma xenografts evaluated with PM14, compound IA, and placebo.

[0237]

[0238] Table 8. Evaluation of tumor volume of LOX-IMVI melanoma xenografts using PM14, compound IA, and placebo.

[0239]

[0240] Compared to placebo, PM14 and compound IA produced tumor volume reduction in various xenografts with different melanoma cell lines. The compounds of this invention achieved a delay in disease progression in all types of melanoma.

[0241] in conclusion:

[0242] Low nanomolar doses of rubitidine, PM14, and compound IA all reduced the proliferation and invasion of melanoma cells, while inducing apoptosis and S-phase cell cycle arrest. Similarly, the viability of 3D-cultured melanoma spheres was severely affected by treatment with the three drugs. Surprisingly, however, even high doses of MAPKi did not affect BRAF-containing cells. V600EThe viability of melanoma spheres in mutant melanocyte MM074 cells, which showed high sensitivity to BRAFi and MEKi in a 2D environment, was demonstrated. These results highlight the importance of assessing novel drugs in different environments, as factors such as hypoxic signaling or drug penetration can severely limit their therapeutic efficacy. However, these factors did not appear to limit the efficacy of rubitidine, PM14, or compound IA in a 3D model. Furthermore, all three compounds rapidly induced gH2AX in melanoma cells, a phenomenon not observed with MAPKi, and significant differences were observed between rubitidine and treatment with PM14 or compound IA in some cell models. For example, in 501mel cells, RNAPII degradation and gH2AX were more pronounced when treated with PM14 or compound IA, suggesting potential differences in efficacy and intracellular pharmacodynamics.

[0243] The results also highlighted the dual mechanism of action of rubitidine and PM14, as well as compound IA, in exerting their cytotoxic effects. Simultaneously, drug treatment also led to significant disruption of oncogene expression, as emphasized by the induction of DNA breaks, such as the appearance of gH2AX. Importantly, the transcriptional effects of the compounds appeared to exhibit high specificity for significantly overexpressed oncogenes, depending on the melanoma cell state. Thus, while the expression of housekeeping genes (such as ACTb, TBP, or RPL13a) was unaffected by short-term drug treatment under 2D or 3D conditions, oncogenic overexpression and MRGs (such as MITF or SOX10) were specifically and severely suppressed in melanocytes. However, in mesenchymal-like cells, different genes were affected, such as overexpressed RTKAXL or EGFR.

[0244] Through their unique mechanisms of action, they selectively inhibit different transcriptional programs relied upon by a given subset of cancer cells. Because non-cancer cells exhibit fewer open chromatin conformations and are less "transcriptionally addicted," they should experience less cytotoxicity than cancer cells.

[0245] Furthermore, GSEA and GO analyses showed that drug treatment triggered a strong stress response, activating AP-1 and EMT factors while disrupting β-catenin signaling, representing events associated with melanoma phenotypic transformation.

[0246] The compounds of this invention overcome the negative effects of phenotypic conversion induction because even mesenchymal-like cells show sensitivity to these drugs.

[0247] In vitro and in vivo data indicate that PM14 and compound IA significantly affect melanocyte-like melanoma cells, even those cells that have acquired resistance to MAPKi.

[0248] This invention has identified rubitidine, PM14, and compound IA as effective in treating skin cancer, particularly melanoma. These compounds are particularly effective in treating mesenchymal-like melanoma (aggressive / undifferentiated phenotype). They are also particularly effective in treating melanocytic-like melanoma, even those melanomas that have acquired resistance to MAPKi. By being effective against these phenotypes, the compounds of this invention can mitigate or overcome the negative effects of phenotype conversion induction. By overcoming this resistance mechanism used by melanoma to overcome prior therapies, the compounds of this invention are particularly effective in the treatment of melanoma.

[0249] The compounds of this invention can be used in melanoma patients who have developed resistance to prior treatment with inhibitors of the MAP kinase signaling pathway. The patients may have primary resistance. The patients may have progressed from previous treatment with inhibitors of the MAP kinase signaling pathway. The inhibitor of the MAP kinase signaling pathway may be a BRAF inhibitor. The inhibitor of the MAP kinase signaling pathway may be a MEK kinase inhibitor. The inhibitor of the MAP kinase signaling pathway may be a combination of a BRAF inhibitor and a MEK kinase inhibitor.

[0250] Overall, the compounds of the present invention are effective in treating skin cancer. In particular, the compounds of the present invention are effective in treating melanoma.

[0251] References

[0252] Tsao H et al Management of cutaneous melanoma. N Engl J Med 2004;351:998-1012

[0253] Zhang et al., Cell Research, Vol. 12, pp. 9-18, 2002

[0254] Flaherty KT et al. Combined BRAF and MEK inhibition in melanoma withBRAF V600 mutations. N Engl J Med 2012;367:1694-703

[0255] Nazarian, R. et al. 2010. Melanomas acquire resistance toB-RAF(V600E)inhibition by RTK or N-RAS upregulation. Nature 468, 973-U377.

[0256] WO2018 / 197663

[0257] WO 2022243482

[0258] Santamaria Nunez, G, et al., Lurbinectedin Specifically Triggers the Degradation of Phosphorylated RNA Polymerase II and the Formation of DNA Breaks in Cancer Cells, Molecular Cancer Therapeutics (2016), 15(10), 2399-2412

[0259] Verfaillie, A. et al., 2015. Decoding the regulatory landscape of melanoma reveals TEADS as regulators of the invasive cell state. Nature Communications 6

[0260] Garnett, M.J., Marais, R., 2004. Guilty as charged: B-RAF is a human oncogene. Cancer Cell 6, 313–9

[0261] Ribas, A. et al., J., 2013. Phase I trial evaluating concurrent Vemurafenib and Ipilimumab in patients with advanced BRAF-mutant melanoma. European Journal of Cancer 49, S867–S867

[0262] Siegel, R.L. et al., 2018. Cancer Statistics, 2018. Cancer Journal for Clinicians 68, 7–30

[0263] Casper et al., Use of anti-phosphohistone H3 immunohistochemistry to determine mitotic rate in thin melanoma, Am J Dermatopathol, 32 (2010), pp. 650-654

[0264] Melanoma treatment (PDQ)-Health Professional Version-NCI (cancer.gov)

[0265] NCCN Guidelines cutaneous melanoma version 2 / 2023

[0266] Decoding the regulatory landscape of melanoma reveals TEADS as regulators of the invasive cell state Nature Communications Vol. 6, Article number: 6683 (2015).

[0267] Systematic classification of melanoma cells by phenotype‐specific gene expression mapping - Widmer - 2012 - Pigment Cell & Melanoma Research - Wiley Online Library

[0268] Najem A et al., Understanding Molecular Mechanisms of Phenotype Switching and Crosstalk with TME to Reveal New Vulnerabilities of Melanoma.Cells. March 29, 2022;11(7):1157.

[0269] Garraway LA, et al. Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma. Nature. 2005;436:117–22。

[0270] NCI-SEER-Database, 2023

[0271] Akbani, R.., 2015. Genomic Classification of Cutaneous Melanoma. Cell 161, 1681–1696。

[0272] Anders, S., Huber, W., 2010. Differential expression analysis for sequence count data. Genome Biol.11, 1–12。

[0273] Arozarena, I., Wellbrock, C., 2019. Phenotype plasticity as enabler of melanoma progression and therapy resistance. Nat. Rev. Cancer 19, 377–391。

[0274] Atkins, M.B., 2021. The State of Melanoma: Emergent Challenges and Opportunities. Clin.Cancer Res.27, 2678–2697。

[0275] Benboubker, V., Boivin, F., Dalle, S., Caramel, J., 2022. Cancer Cell Phenotype Plasticity as a Driver of Immune Escape in Melanoma. Front.Immunol.13, 873116。

[0276] Benjamini, Y., Hochberg, Y., 1995. Controlling the False DiscoveryRate: A Practical and Powerful Approach to Multiple Testing. J. R. Stat.Soc.Ser. B Methodol. 57, 289–300。

[0277] Berico, P., Cigrang, 2021. CDK7 and MITF repress a transcriptionprogram involved in survival and drug tolerance in melanoma. EMBO Rep。

[0278] Bradner, J.E., Hnisz, D., Young, R.A., 2017. TranscriptionalAddiction in Cancer. Cell 168, 629–643。

[0279] Bushweller, J.H., 2019. Targeting transcription factors in cancer —from undruggable to reality. Nat. Rev. Cancer 19, 611–624。

[0280] Carlino, M.S., Larkin, J., Long, G.V., 2021. Immune checkpointinhibitors in melanoma. The Lancet 398, 1002–1014。

[0281] Chauhan, J.S., 2022. The MITF regulatory network in melanoma. PigmentCell Melanoma Res. 35, 517–533。

[0282] Comandante-Lou, N., 2022. AP-1 transcription factor network explainsdiverse patterns of cellular plasticity in melanoma cells. Cell Rep. 40,111147。

[0283] Costanzo, F., 2022. Promoters of ASCL1‐ and NEUROD1‐dependent genesare specific targets of lurbinectedin in SCLC cells. EMBO Mol. Med。

[0284] Curti, B.D., Faries, M.B., 2021. Recent Advances in the Treatment ofMelanoma. N. Engl. J. Med.384, 2229–2240。

[0285] De Velasco, G., 2017. Comprehensive Meta-analysis of Key Immune-Related Adverse Events from CTLA-4 and PD-1 / PD-L1 Inhibitors in CancerPatients. Cancer Immunol. Res. 5, 312–318。

[0286] Delyon, J., Lebbe, C., Dumaz, N., 2020. Targeted therapies inmelanoma beyond BRAF: targeting NRAS-mutated and KIT-mutated melanoma.Curr.Opin.Oncol.32, 79–84。

[0287] Dickson, P.V., Gershenwald, J.E., 2011. Staging and Prognosis ofCutaneous Melanoma. Surg.Oncol.Clin.N. Am. 20, 1–17。

[0288] Donati, B., Lorenzini, E., Ciarrocchi, A., 2018. BRD4 and Cancer:going beyond transcriptional regulation. Mol.Cancer 17, 164。

[0289] Fontanals-Cirera, B., 2017. Harnessing BET Inhibitor SensitivityReveals AMIGO2 as a Melanoma Survival Gene. Mol.Cell 68, 731-744.e9。

[0290] Guo, W., Wang, H., Li, C., 2021. Signal pathways of melanoma andtargeted therapy. Signal Transduct. Target. Ther.6, 424。

[0291] Hoek, K.S., Goding, C.R., 2010. Cancer stem cells versus phenotype-switching in melanoma. Pigment Cell Melanoma Res 23, 746–59。

[0292] Hogg, S.J., Beavis, P.A., Dawson, M.A., Johnstone, R.W., 2020.Targeting the epigenetic regulation of antitumour immunity. Nat. Rev. DrugDiscov.19, 776–800。

[0293] Huang, A.C., Zappasodi, R., 2022. A decade of checkpoint blockadeimmunotherapy in melanoma: understanding the molecular basis for immunesensitivity and resistance. Nat. Immunol. 23, 660–670。

[0294] Jenkins, R.W., Fisher, D.E., 2021. Treatment of Advanced Melanoma in2020 and Beyond. J. Invest.Dermatol.141, 23–31。

[0295] Jerby-Amon, L., 2018. A Cancer Cell Program Promotes T Cell Exclusionand Resistance to Checkpoint Blockade. Cell 175, 984-+。

[0296] Karras, P., 2022. A cellular hierarchy in melanoma uncouples growthand metastasis. Nature 610, 190–198。

[0297] Kozar, I., Margue, C., Rothengatter, S., Haan, C., Kreis, S., 2019.Many ways to resistance: How melanoma cells evade targeted therapies.Biochim.Biophys.Acta BBA - Rev. Cancer 1871, 313–322。

[0298] Kwiatkowski, N., 2014. Targeting transcription regulation in cancerwith a covalent CDK7 inhibitor. Nature 511, 616–20。

[0299] Laham-Karam, N., 2018. Cutaneous melanoma: From pathogenesis totherapy (Review). Int. J. Oncol。

[0300] Love, M.I., Huber, W., Anders, S., 2014. Moderated estimation of foldchange and dispersion for RNA-seq data with DESeq2.Genome Biol.15, 1–21。

[0301] Luke, J.J., Flaherty, K.T., Ribas, A., Long, G.V., 2017. Targetedagents and immunotherapies: optimizing outcomes in melanoma. Nat. Rev. Clin.Oncol.14, 463–482。

[0302] Luskin, M.R., Murakami, M.A., Manalis, S.R., Weinstock, D.M., 2018.Targeting minimal residual disease: a path to cure? Nat. Rev. Cancer 18, 255–263。

[0303] Marin-Bejar, O., 2021. Evolutionary predictability of genetic versusnongenetic resistance to anticancer drugs in melanoma. Cancer Cell 39, 1135-1149.e8。

[0304] Mehta, A., 2018. Immunotherapy Resistance by Inflammation-InducedDedifferentiation. Cancer Discov.8, 935–943。

[0305] Newell, F., 2022. Comparative Genomics Provides Etiologic andBiological Insight into Melanoma Subtypes. Cancer Discov.12, 2856–2879。

[0306] Radke, J., 2022. Decoding molecular programs in melanoma brainmetastases. Nat. Commun. 13, 7304。

[0307] Rambow, F., Marine, J.C., Goding, C.R., 2019. Melanoma plasticity andphenotypic diversity: therapeutic barriers and opportunities. Genes Dev 33,1295–1318。

[0308] Rambow, F., 2018. Toward Minimal Residual Disease-Directed Therapy inMelanoma. Cell 174, 843。

[0309] Rebecca, V.W., Herlyn, M., 2020. Nongenetic Mechanisms of DrugResistance in Melanoma. Annu. Rev. Cancer Biol. 4, 315–330。

[0310] Ribas, A., 2019. Five-Year Outcomes with Dabrafenib plus Trametinibin Metastatic Melanoma. N. Engl. J. Med.381, 626–636。

[0311] Rubanov, A., Berico, P., Hernando, E., 2022. Epigenetic MechanismsUnderlying Melanoma Resistance to Immune and Targeted Therapies. Cancers 14,5858。

[0312] Saginala, K., 2021. Epidemiology of Melanoma. Med. Sci. 9, 63。

[0313] Schadendorf, D., 2018. Melanoma. The Lancet 392, 971–984。

[0314] Sengupta, S., George, R.E., 2017. Super-Enhancer-DrivenTranscriptional Dependencies in Cancer. Trends Cancer 3, 269–281。

[0315] Shain, A.H., Bastian, B.C., 2016. From melanocytes to melanomas. NatRev Cancer 16, 345–58。

[0316] Sung, H., Ferlay, 2021. Global Cancer Statistics 2020: GLOBOCANEstimates of Incidence and Mortality Worldwide for 36 Cancers in 185Countries. CA. Cancer J. Clin. 71, 209–249。

[0317] Switzer, B., 2022. Managing Metastatic Melanoma in 2022: A ClinicalReview. JCO Oncol. Pract. 18, 335–351。

[0318] Tirosh, I., 2016. Dissecting the multicellular ecosystem ofmetastatic melanoma by single-cell RNA-seq. Science 352, 189–96。

[0319] Tsoi, J., 2018. Multi-stage Differentiation Defines Melanoma Subtypeswith Differential Vulnerability to Drug-Induced Iron-Dependent OxidativeStress. Cancer Cell 33, 890-904.e5。

[0320] Vervoort, SJ, 2022. Targeting transcription cycles in cancer. Nat. Rev. Cancer 22, 5–24.

[0321] Widmer, DS, 2012. Systematic classification of melanoma cells by phenotype-specific gene expression mapping. Pigment Cell Melanoma Res 25,343–53.

[0322] Wouters, J., 2020. Robust gene expression programs underlie recurrent cell states and phenotype switching in melanoma. Nat Cell Biol 22, 986–998.

[0323] Zanconato, F., 2018. Transcriptional addiction in cancer cells ismediated by YAP / TAZ through BRD4. Nat. Med. 24, 1599–161

[0324] Terms:

[0325] 1. Compound IA

[0326] Or its pharmaceutically acceptable salts or esters, which are used to treat skin cancer.

[0327] 2. The compound used according to Clause 1, wherein the skin cancer is melanoma.

[0328] 3. The compound used according to Clause 2, wherein the melanoma is aggressive / undifferentiated.

[0329] 4. The compound used according to Clause 2, wherein the melanoma is proliferative / differentiated.

[0330] 5. The compound used according to Clause 2, wherein the melanoma is invasive / undifferentiated and proliferative / differentiated.

[0331] 6. The compound for use according to any of the preceding clauses, wherein the melanoma is an unresectable or metastatic BRAF or RAS, or a triple WT mutant melanoma.

[0332] 7. The compound for use according to any of the preceding clauses, wherein the melanoma is selected from cutaneous melanoma, mucosal melanoma or acral melanoma.

[0333] 8. The compound for use according to any of the preceding clauses, wherein the melanoma is resistant to inhibitors of the MAP kinase signaling pathway.

[0334] 9. The compound used according to Clause 8, wherein the inhibitor of the MAP kinase signaling pathway is selected from BRAF or MEK kinase inhibitors or combinations thereof.

[0335] 10. The compound for use as described in Clause 9, wherein the BRAF or MEK kinase inhibitor is selected from vemurafenib, dabrafenib, cannefenib, trametinib, cobimetinib, or bimetinib, or combinations thereof.

[0336] 11. The compound used according to Clause 10, wherein the BRAF or MEK kinase inhibitor is vemurafenib, dabrafenib, or trametinib, or a combination thereof.

[0337] 12. The compound for use as described in Clause 9, wherein the melanoma is resistant to BRAF and MEK kinase inhibitors, including dabrafenib in trametinib, vemofenib in cobimetinib, and caninefenib in bimetinib.

[0338] 13. The compound for use as described in Clauses 8 to 12, wherein the melanoma is resistant to vemurafenib.

[0339] 14. A pharmaceutical composition or dosage form comprising a compound as defined in Clause 1 for the treatment of skin cancer as defined in any of the preceding clauses.

[0340] 15. A pharmaceutical package comprising a compound as defined in Clause 1, and instructions for use in treating skin cancer as defined in any of the preceding clauses.

[0341] 16. A method for inhibiting the growth of cancer cells, the method comprising contacting cancer cells with a therapeutically effective amount of a compound as defined in Clause 1, wherein the cancer cells are skin cancer cells.

[0342] 17. PM14 compounds

[0343]

[0344] Or a pharmaceutically acceptable salt or ester thereof, for the treatment of aggressive / undifferentiated and / or proliferative / differentiated melanoma.

[0345] 18. The compound used according to Clause 17, wherein the melanoma is aggressive / undifferentiated.

[0346] 19. The compound used according to Clause 17, wherein the melanoma is proliferative / differentiated.

[0347] 20. The compound used according to Clause 17, wherein the melanoma is invasive / undifferentiated and proliferative / differentiated.

[0348] 21. The compound for use according to any one of clauses 17 to 20, wherein the melanoma is an unresectable or metastatic BRAF or RAS, or a triple WT mutant melanoma.

[0349] 22. The compound for any of the uses described in any of clauses 17 to 21, wherein the melanoma is selected from cutaneous melanoma, mucosal melanoma or acral melanoma.

[0350] 23. The compound for any of the uses described in any of clauses 17 to 22, wherein the melanoma is resistant to inhibitors of the MAP kinase signaling pathway.

[0351] 24. The compound used according to Clause 23, wherein the inhibitor of the MAP kinase signaling pathway is selected from BRAF or MEK kinase inhibitors or combinations thereof.

[0352] 25. The compound used according to Clause 24, wherein the BRAF or MEK kinase inhibitor is selected from vemurafenib, dabrafenib, cannefenib, trametinib, cobimetinib, or bimetinib, or combinations thereof.

[0353] 26. The compound used according to Clause 25, wherein the BRAF or MEK kinase inhibitor is vemurafenib, dabrafenib, or trametinib, or a combination thereof.

[0354] 27. The compound for use as described in Clause 24, wherein the melanoma is resistant to BRAF and MEK kinase inhibitors, including dabrafenib in trametinib, vemofenib in cobimetinib, and caninefenib in bimetinib.

[0355] 28. A compound for any of the uses described in any of clauses 17 to 27, wherein the melanoma is resistant to vemurafenib.

[0356] 29. A pharmaceutical composition or dosage form comprising a compound as defined in Clause 17 for the treatment of skin cancer as defined in any one of Clauses 17 to 28.

[0357] 30. A pharmaceutical package containing a compound as defined in Clause 17, and instructions for use in treating skin cancer as defined in any one of Clauses 17 to 28.

[0358] 31. A method for inhibiting the growth of cancer cells, the method comprising contacting cancer cells with a therapeutically effective amount of a compound as defined in Clause 17, wherein the cancer cells are melanoma cancer cells.

[0359] 32. Rubitil

[0360]

[0361] Or a pharmaceutically acceptable salt or ester thereof, for the treatment of aggressive / undifferentiated and / or proliferative / differentiated melanoma.

[0362] 33. The compound used according to Clause 32, wherein the melanoma is aggressive / undifferentiated.

[0363] 34. The compound used according to Clause 32, wherein the melanoma is proliferative / differentiated.

[0364] 35. The compound used according to Clause 32, wherein the melanoma is invasive / undifferentiated and proliferative / differentiated.

[0365] 36. The compound for any of the uses described in any of clauses 32 to 35, wherein the melanoma is an unresectable or metastatic BRAF or RAS, or a triple WT mutant melanoma.

[0366] 37. The compound for any of the uses described in any of clauses 32 to 36, wherein the melanoma is selected from cutaneous melanoma, mucosal melanoma or acral melanoma.

[0367] 38. A compound for any of the uses described in any of clauses 32 to 37, wherein the melanoma is resistant to inhibitors of the MAP kinase signaling pathway.

[0368] 39. The compound used according to Clause 38, wherein the inhibitor of the MAP kinase signaling pathway is selected from BRAF or MEK kinase inhibitors or combinations thereof.

[0369] 40. The compound for use as described in Clause 39, wherein the BRAF or MEK kinase inhibitor is selected from vemurafenib, dabrafenib, cannefenib, trametinib, cobimetinib, or bimetinib, or combinations thereof.

[0370] 41. The compound used according to Clause 40, wherein the BRAF or MEK kinase inhibitor is vemurafenib, dabrafenib, or trametinib, or a combination thereof.

[0371] 42. The compound used according to Clause 39, wherein the melanoma is resistant to BRAF and MEK kinase inhibitors, including dabrafenib in trametinib, vemofenib in cobimetinib, and caninefenib in bimetinib.

[0372] 43. The compound for any of the uses described in any of clauses 32 to 42, wherein the melanoma is resistant to vemurafenib.

[0373] 44. A pharmaceutical composition or dosage form comprising a compound as defined in Clause 32 for the treatment of skin cancer as defined in any one of Clauses 32 to 43.

[0374] 45. A pharmaceutical package comprising a compound as defined in Clause 32, and instructions for use in treating skin cancer as defined in any one of Clauses 32 to 43.

[0375] 46. ​​A method for inhibiting the growth of cancer cells, the method comprising contacting cancer cells with a therapeutically effective amount of a compound as defined in Clause 32, wherein the cancer cells are melanoma cancer cells.

Claims

1. A compound selected from compounds IA, PM14, and rubitidine. Or a pharmaceutically acceptable salt or ester thereof, which is used to treat cancer, wherein the cancer is aggressive / undifferentiated and / or proliferative / differentiated melanoma.

2. A compound, which is compound IA. Or a pharmaceutically acceptable salt or ester thereof, which is used to treat cancer, wherein said cancer is skin cancer.

3. The compound for the use of claim 2, wherein the skin cancer is melanoma.

4. The compound for use according to any of the preceding claims, wherein the cancer is invasive / undifferentiated melanoma.

5. The compound for use according to any of the preceding claims, wherein the cancer is proliferative / differentiated melanoma.

6. A compound for use according to any of the preceding claims, wherein the cancer includes aggressive / undifferentiated and proliferative / differentiated melanoma.

7. The compound for use according to any of the preceding claims, wherein the cancer is unresectable or metastatic BRAF or RAS, or triple WT mutant melanoma.

8. A compound for use according to any of the preceding claims, wherein the cancer is selected from cutaneous melanoma, mucosal melanoma, or acral melanoma.

9. A compound for use according to any of the preceding claims, wherein the cancer is melanoma and is resistant to inhibitors of the MAP kinase signaling pathway.

10. The compound for use according to claim 9, wherein the inhibitor of the MAP kinase signaling pathway is selected from BRAF or MEK kinase inhibitors or combinations thereof.

11. The compound for the use of claim 10, wherein the BRAF or MEK kinase inhibitor is selected from vemurafenib, dabrafenib, cannefenib, trametinib, cobimetinib, or bimetinib, or combinations thereof.

12. The compound for use according to claim 11, wherein the BRAF or MEK kinase inhibitor is vemurafenib, dabrafenib, or trametinib, or a combination thereof.

13. The compound for use according to claim 10, wherein the cancer is melanoma resistant to BRAF and MEK kinase inhibitors.

14. The compound for use according to claim 13, wherein the cancer is melanoma resistant to dabrafenib plus trametinib and / or vemofenib plus cobimetinib and / or caninefenib plus bimetinib.

15. The compound for use according to claims 9 to 14, wherein the cancer is vemurafenib-resistant melanoma.

Images