Madracine derivative compounds, compositions and their uses for treating cancer - Patents.com

Abstract

The present invention relates to the field of medicine, particularly cancer treatment. More specifically, the present invention relates to novel compounds, typically for use as pharmaceuticals. In particular, the present invention relates to the use of these novel compounds for increasing the presentation, typically production and presentation, of Pioneer Translation Products (PTP)-derived antigens by cells, particularly cancer cells, in a subject, or for altering the immunopeptidome, and for eliciting or stimulating an immune response in the subject. The present invention also relates to the use of such compounds, particularly for preparing pharmaceutical compositions and / or for enhancing or improving the efficacy of therapeutic methods, particularly cancer therapy, in a subject in need of such treatment. The present invention also discloses methods for treating diseases, particularly cancer, and for preventing or treating cancer metastasis and / or cancer recurrence in a subject. The present invention further provides kits suitable for preparing the compositions according to the present invention and / or for carrying out the methods described herein.

Description

[Technical field]

[0001] The present invention relates generally to the field of medicine, in particular to cancer treatment. In particular, the present invention relates to novel compounds that are madrasin derivatives, each of which is typically used as a medicine. In particular, the present invention relates to the use of these novel compounds for increasing the presentation, typically the production and presentation, or altering the immune peptidome, of (antigenic) peptides, preferably Pioneer Translation Products (PTP) derived antigens, by cells, particularly cancer cells, in a subject, and for eliciting or stimulating an immune response in the subject. The immune response is typically directed against tumor antigens, more commonly against a cancerous tumor borne by the subject.

[0002] The present invention also relates to the use of such compounds, in particular for the manufacture of pharmaceutical compositions and / or for enabling or improving the efficiency of treatment, in particular cancer treatment, in subjects in need of such treatment.Each of the compounds of the present invention may in fact be advantageously used in combination with at least one different anti-cancer agent, typically a chemotherapeutic agent and / or radiotherapy, for the treatment of cancer, the prevention or treatment of cancer metastasis and / or the prevention of cancer recurrence in a subject.

[0003] The present invention also discloses a method for the treatment of a disease, in particular cancer, the treatment or prevention of cancer metastasis and / or the prevention or treatment of cancer recurrence in a subject. The present invention further provides a kit suitable for the preparation of the composition according to the invention and / or for carrying out the methods described herein. [Background technology]

[0004] All nucleated cells present antigenic peptides (APs) on their surface via the class I major histocompatibility complex (MHC-I) pathway. APs are 8-10 amino acids long and reflect intrinsic cellular activities (Caron et al.). Their presentation is primarily mediated by cytotoxic CD8 + T cells (CTL) and CD4 +APs are targets of therapeutic anticancer vaccines currently being developed, since they induce the surveillance of potentially dangerous elements by immune cells, namely T helper cells. Despite the promise, the results of clinical trials with therapeutic vaccines targeting tumor-associated antigens (TAA) have not met expectations. The main failures were related to immunosuppressive mechanisms and suboptimal selection of antigens (Mellman et al.; Burg et al.). One of the key events driving tumor immune selection and correlating with poor prognosis is the reduction or downregulation of MHC class I antigen presentation by tumor cells (Watson et al.; Liu et al.). The last of these could be an escape from recognition by CTLs and natural killer cells, due to a deficiency in the organization of the MHC class I pathway (Leone et al.). Alongside the overall reduction of MHC class I antigen presentation, the nature of antigens presented on the cell surface, referred to as the MHC class I immunopeptidome (MIP), is crucial for immune recognition. In cancers where specific TAAs, such as Her / neu in breast cancer or CEA in colon cancer, have been identified and targeted with immunotherapy, reduction of this TAA expression on the tumor cell surface results in immune evasion (Lee et al.; Kmieciak et al.). To combat this, current strategies aim to broaden the scope of targeted cancer peptides and restore antigen presentation.

[0005] To understand the dynamics of MIPs, the source of APs for the MHC class I presentation pathway may be noted. Endogenous APs were initially thought to be derived entirely from the degradation of aged proteins. However, models suggesting alternative sources have challenged this concept. In 1996, J. Yewdell's group introduced the concept of defective ribosomal products (DRIPs), which were initially described as products that degrade rapidly due to their unstable conformation (Yewdell et al.). More recently, we have investigated the concept from a different perspective, showing that the main source of APs originates from precursor translation events that occur before introns are excised and are independent of the translation events of full-length proteins (Apcher et al.). Thus, the non-standard peptides produced may originate from intron sequences, 3' or 5' UTR regions, and alternative reading frames. These polypeptides are described as Pioneer Translation Products (PTPs). The discovery of PTPs suggests the existence of a complex translational nuclear mechanism that is related to the MHC class I pathway and is aimed in part at forming MIPs by producing appropriate polypeptides. Furthermore, PTPs are believed to play a role in the rate of cancer progression. When inoculated into mice, cancer cells presenting PTP-derived antigens on their cell surface can be recognized by specific T cells, resulting in reduced tumor growth. Furthermore, purified PTPs containing model epitopes efficiently promote anti-cancer immune responses when injected into mice as peptide vaccines (Duvallet et al.).

[0006] Precursor mRNA (pre-mRNA) splicing is catalyzed in the nucleus by the spliceosome, a conserved and dynamic multiprotein complex consisting of five small nuclear RNAs (snRNAs), U1, U2, U4, U5 and U6, in complex with more than 200 proteins. An increasing number of studies report that deregulated spliceosome complexes entail abnormal splicing patterns in many cancers that contribute to abnormal tumor cell proliferation and progression. Since 2011, recurrent spliceosome mutations have been reported in several cancers, including myelomonocytic leukemia, myeloid leukemia, chronic lymphocytic leukemia, breast cancer or multiple myeloma.

[0007] Darrigrand et al. (Drug Discovery and new Therapeutics, First international symposium at Paris-Saclay University, April 2018) teach that it is a druggable target for epitope-based immunotherapy. WO 2017 / 165495 describes the combination of PIM kinase inhibitors and RNA splicing regulators / inhibitors for the treatment of cancer. Salton et al. disclose the use of small molecule pre-mRNA splicing regulators in cancer therapy. Recently, several microbial natural products and their synthetic analogs have been reported to inhibit the spliceosome, including pladienolides B and D, splicostatin A, FR901464, E7107, Isoginkgetin and madrasin. We have tested the potential effect of each of several as regulators of antigen presentation.

[0008] We show here that madracin and some of the derivatives produced can be used as positive immune modulators against cancer. We observed antigen presentation of a PTP-derived antigen model expressed in a mouse cancer cell line, and observed that in vitro treatment with madracin and various derivatives described herein increased antigen presentation. Furthermore, we showed that in vivo treatment of sarcoma-bearing mice with madracin hydrochloride (also identified herein as "Madra.HCl", EYP34 or compound of formula I) dissolved in water slowed down tumor growth in an immune-dependent manner. To improve the effect, we tested various madracin derivatives that are soluble or insoluble in water, and observed that some derivatives were more potent inhibitors of mouse sarcoma growth than madracin itself. Since the natural product and its derivatives have no effect on tumor growth in immunodeficient Nu / Nu mice, we conclude that their effect depends on the immune response. These results indicate that PTP-derived antigen presentation can be modulated by treatment, and splicing inhibition can boost anti-cancer responses. Herein, we describe an alternative mechanism of action of splicing inhibitors that can be used as positive immunomodulators to enhance anti-tumor immune responses.

[0009] We now describe novel compounds for use in the treatment of disease in a subject, in particular in the treatment of cancer, in the prevention of cancer metastasis and / or in the prevention of cancer recurrence. Summary of the Invention

[0010] The present inventors have prepared for the first time and herein describe the following specific compounds derived from madrasine: - Madracine hydrochloride (also identified herein as "Madr.HCl", "EYP34" or "compound of formula I") [ka] - 5,6-dimethyl-2-((4-methyl-7-(2-morpholinoethoxy)quinazolin-2-yl)amino)pyrimidin-4(3H)-one (also identified herein as "EYP201", "compound 6" or "compound of formula II"); [ka] sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazolin-7-yl phosphate (also identified herein as "EYP59", "compound 7" or "compound of formula III"); [ka] [During the ceremony, "Na" stands for "sodium" The radical of the formula -ONa represents "sodium hydroxide". - N-(2-methoxypyrimidin-5-yl)quinazolin-2-amine (also identified herein as "EYP281", "compound 32" or "compound of formula IV"), [ka] - N-(1H-indol-3-yl)-7-methoxy-4-methylquinazolin-2-amine (also identified herein as "EYP165", "compound 57" or "compound of formula V"); [ka] - ((1S,2S,4R,5S,6S)-5-acetoxy-7,8-diacetyl-4-((2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-7-methoxy-4-methylquinazolin-8-yl)thio)-3,7λ 3 ,8λ 3 -trioxabicyclo[4.2.0]octan-2-yl)methyl acetate (also identified herein as "EYP86," "compound 10" or "compound of formula VI"); [ka] (wherein the radical of the formula -OAc denotes an "acetoxy group" of the structure CH3-C(=O)-O-). - 7-methoxy-4-methyl-N-(3,4,5-trimethoxyphenyl)quinazolin-2-amine (also identified herein as "EYP188", "compound 42" or "compound I of formula VI"); [ka] - 7-methoxy-N-(6-methoxypyridin-3-yl)-4-methylquinazolin-2-amine (also identified herein as "EYP174", "compound 41" or "compound of formula VIII"); [ka] - N2-(7-methoxy-4-methylquinazolin-2-yl)-N4-methylpyrimidine-2,4-diamine (also identified herein as "EYP181", "compound 49" or "compound of formula IX"), [ka] - 7-methoxy-N-(4-methoxypyrimidin-2-yl)-4-methylquinazolin-2-amine (also identified herein as "EYP179", "compound 50" or "compound of formula X"); [ka] - 7-Methoxy-N-(2-methoxypyrimidin-4-yl)-4-methylquinazolin-2-amine (also identified herein as "EYP190", "compound 54" or "compound of formula XI") [ka]

[0011] Each of these compounds, and any combination thereof, is described herein for use as a pharmaceutical.

[0012] All of these compounds and their pharma- ceutically acceptable salts are advantageously used as medicines.

[0013] In a preferred embodiment described herein, any of the above compounds of formula (I)-(XI) or a pharma- ceutically acceptable salt thereof or any combination thereof is for use in the treatment of disease in a subject, in particular for use in the treatment of cancer, for use in the prevention of cancer metastasis and / or for use in the prevention of cancer recurrence.

[0014] Further described is the in vivo, in vitro or ex vivo use of a compound of a formula selected from formulae (I)-(XI) for inducing or increasing the presentation, typically the production and presentation, of (antigenic) peptides, preferably Pioneer Translation Products (PTP)-derived antigens, by cells, in particular cancer cells, or for altering the immunopeptidome in a subject.

[0015] The compounds of the formulae selected from formulae (I) to (XI) allow the physician to prevent or control, preferably reduce, cancer cell proliferation by stimulating the immune system of the subject. In addition, it is advantageously possible to increase the effectiveness of other cancer treatments. The inventors now show that the compounds can further reduce the risk of metastasis and / or cancer recurrence.

[0016] Also described herein are compositions comprising such a compound of a formula selected from Formulae (I)-(XI) and a pharma- ceutically acceptable carrier, preferably together with at least one different anti-cancer agent used simultaneously, separately or sequentially; such compositions are typically used for the treatment of disease, in particular the treatment of cancer, the prevention of cancer metastasis and / or the prevention of cancer recurrence in a subject.

[0017] Also described herein are methods of treating a disease, particularly cancer, in a subject, comprising administering to the subject a compound described herein, typically a compound or composition of a formula selected from Formulas (I)-(XI).

[0018] Also described herein are kits comprising a compound of a formula selected from formulae (I)-(XI) and preferably at least one different anti-cancer agent in different containers and their uses, in particular for the manufacture of the compositions described herein.

[0019] Detailed Description of the Invention The present inventors have synthesised madrasine derivatives which are described for the first time herein and more specifically identified below as compounds of formulae (I)-(XI): - madrasine hydrochloride (also identified herein as "Madr.HCl", "EYP34" or "compound of formula I"), [ka] - 5,6-dimethyl-2-((4-methyl-7-(2-morpholinoethoxy)quinazolin-2-yl)amino)pyrimidin-4(3H)-one (also identified herein as "EYP201", "compound 6" or "compound of formula II"); [ka] sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazolin-7-yl phosphate (also identified herein as "EYP59", "compound 7" or "compound of formula III"); [ka] [During the ceremony, "Na" stands for "sodium" The radical of the formula -ONa represents "sodium hydroxide". - N-(2-methoxypyrimidin-5-yl)quinazolin-2-amine (also identified herein as "EYP281", "compound 32" or "compound of formula IV"), [ka] - N-(1H-indol-3-yl)-7-methoxy-4-methylquinazolin-2-amine (also identified herein as "EYP165", "compound 57" or "compound of formula V"); [ka] - ((1S,2S,4R,5S,6S)-5-acetoxy-7,8-diacetyl-4-((2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-7-methoxy-4-methylquinazolin-8-yl)thio)-3,7λ3,8λ3-trioxabicyclo[4.2.0]octan-2-yl)methyl acetate (also identified herein as "EYP86", "compound 10" or "compound of formula VI"); [ka] (wherein the radical of the formula -OAc denotes an "acetoxy group" of the structure CH3-C(=O)-O-). - 7-methoxy-4-methyl-N-(3,4,5-trimethoxyphenyl)quinazolin-2-amine (also identified herein as "EYP188", "compound 42" or "compound I of formula VI"); [ka] - 7-methoxy-N-(6-methoxypyridin-3-yl)-4-methylquinazolin-2-amine (also identified herein as "EYP174", "compound 41" or "compound of formula VIII"); [ka] - N2-(7-methoxy-4-methylquinazolin-2-yl)-N4-methylpyrimidine-2,4-diamine (also identified herein as "EYP181", "compound 49" or "compound of formula IX"), [ka] - 7-methoxy-N-(4-methoxypyrimidin-2-yl)-4-methylquinazolin-2-amine (also identified herein as "EYP179", "compound 50" or "compound of formula X"); [ka] - 7-Methoxy-N-(2-methoxypyrimidin-4-yl)-4-methylquinazolin-2-amine (also identified herein as "EYP190", "compound 54" or "compound of formula XI") [ka]

[0020] In certain embodiments, we have provided a compound of formula (A): [ka] [During the ceremony, R 1 and R 2 is independently selected from H, CH4 (methyl) and Cl (chlorine); R 3 is H, CH4 (methyl), C2H6 (ethyl), n-propyl, N(CH2)4 or N(CH2)5, Z is CH2 or C=O; n is 1, 2 or 3; X is O, N or CH. The compounds of the formula are described herein.

[0021] In specific and preferred embodiments, the compound of formula (A) is a compound of formula II (also identified as "EYP201" or "compound 6").

[0022] In another particular embodiment, we have found a compound of formula (B): [ka] [During the ceremony, R 1 and R 2 is independently selected from H, CH4 (methyl), Cl (chlorine) and thiosugar; R 3 is P(O)(ONa)2 or [ka] (In the formula, R 4 is H, CH4 (methyl), C2H6 (ethyl), n-propyl, N(CH2)4 or N(CH2)5, Z is CH2 or C=O; n is 1, 2 or 3; X is O, N or CH is The compounds of the formula are described herein.

[0023] The aryl group can be, for example, 3,4,5-trimethoxyphenyl; 2-chloro-3,4,5-trimethoxyphenyl; 3,5-trimethoxyphenyl; or 2-chloro-3,5-trimethoxyphenyl.

[0024] The heteroaryl group can be, for example, 3-indolyl; 2-indolyl; 2-imidazole; 2-benzimidazole; carbazole; N-methylpyrimidine; methoxypyrimidin-2-yl; methoxypyrimidin-4-yl; methoxypyridin-2-yl or methoxypyridin-4-yl.

[0025] In certain embodiments, the compound of formula (B) is selected from a compound of formula (III), a compound of formula (V), a compound of formula (VI), a compound of formula (VII), a compound of formula (VIII), a compound of formula (IX), a compound of formula (X), and a compound of formula (XI).

[0026] The compounds of any of formula (A) and formula (B) or formula (I) to (XI) herein above and their pharma- ceutically acceptable salts may be advantageously used as medicines.

[0027] As used herein, the term "pharmaceutical acceptable" refers to a composition, compound, salt, etc. that may be suitable for contact with the tissue of a subject within the scope of sound medical judgment or may be administered to a subject without excessive toxicity or other complications commensurate with a reasonable benefit / risk ratio. For example, pharmaceutical acceptable salts include sodium salts, potassium salts, chloride salts, ammonium salts, acetate salts, and the like.

[0028] We now show that compounds of formulae (A), (B) and (I)-(XI) may be advantageously used as positive immunomodulators, particularly against cancer.

[0029] The inventors observed antigen presentation of a PTP-derived antigen model expressed in a mouse cancer cell line, and observed that their in vitro treatment with madracin and, preferably, a compound of formula (A) and (B) or formula selected from (I)-(XI) increases the presentation of this antigen. Furthermore, in vivo treatment of sarcoma-bearing mice with madracin dissolved in DMSO was shown to slow down tumor growth in an immune-dependent manner. To improve the effect, the water-soluble madracin derivatives described herein were tested, and surprisingly, they were observed to be much more potent cancer inhibitors than madracin itself. In immunodeficient Nu / Nu mice, the natural product and derivatives had no effect on tumor growth, and therefore, it was concluded that their effect was dependent on the immune response. The results showed that PTP-derived antigen presentation can be modulated, and the inventors provide a new promising molecule for market development that, in contrast to other madracin derivatives, can be used to boost anticancer responses and treat cancer, among others.

[0030] In a preferred embodiment described herein, the compounds of formulae (A) and (B) or of a formula selected from (I)-(XI), in particular the compounds of formula (I), formula (II), (III), formula (V), formula (VI), formula (VII), formula (VIII), formula (IX), formula (X) or (XI) (also identified herein as "compounds of interest") are for use in the treatment of a disease, in particular cancer, in the prevention of cancer metastasis and / or in the prevention of cancer recurrence in a subject.

[0031] In another preferred embodiment described herein, the compound of Formula (A) and (B) or a formula selected from (I)-(XI) is for use in stimulating an anti-cancer immune response in a patient in need of treatment.

[0032] In a further preferred embodiment, the compound of formula (A) and (B) or a formula selected from (I)-(XI) is used to induce, increase the presentation, typically the production and presentation, of Pioneer Translation Products (PTP)-derived antigens by cancer cells in a subject or to alter the immunopeptidome.

[0033] The compounds of the present invention can be obtained by methods known to those skilled in the art, such as semi-synthesis or total synthesis. Examples of methods for preparing the compounds of interest are described herein in the experimental section and further illustrated in Figures 9 and 10. The compounds of formulae (A), (B) and (I)-(XI) are man-made products that cannot be found in nature.

[0034] The compounds of formulae (A), (I)-(III) and (VI) can typically be prepared from madrasin, which has been described as a permeant inhibitor of pre-mRNA splicing. The compounds of formulae (IV), (V), (VII)-(XI) can be prepared by chemical synthesis using conventional chemical reactions.

[0035] A further object of the present invention is the use of a compound of formula (A) and (B) or a formula selected from (I)-(XI) (or a pharma- ceutically acceptable salt thereof) for reducing the resistance of a cancer or a subject suffering from a cancer to different anti-cancer agents, typically different chemotherapeutic agents.

[0036] Also described herein is a composition comprising a compound of formula (A) and (B) or a formula selected from (I)-(XI) according to the present invention or a pharma- ceutically acceptable salt thereof) or such a salt and a pharma- ceutically acceptable carrier for use in combination with at least one different anti-cancer agent, typically a chemotherapeutic agent and / or radiation therapy, for the treatment of a disease in a subject, in particular for the treatment of cancer, for the prevention of cancer metastasis and / or for the prevention of cancer recurrence.

[0037] The term "subject" refers to any subject, preferably a mammal.

[0038] Examples of mammals include humans and non-human animals, including, but not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), non-human primates (e.g., monkeys), rabbits, and rodents (e.g., mice and rats). Treatment is preferably directed to humans of any age and sex in need of treatment.

[0039] The term "subject" typically refers to a patient, particularly a patient with tumor.Unless otherwise specified, in the present invention, tumor is a cancerous tumor or a malignant tumor.In certain embodiments, the subject is a subject undergoing cancer treatment, such as chemotherapy and / or radiation therapy, or is a subject at risk of developing cancer, or is a subject suspected of being at risk of developing cancer.

[0040] The subject is, for example, a human who has cancer and is resistant to cancer treatment, typically chemotherapy.

[0041] The subject may have been exposed to the entire conventional treatment protocol, for example, at least one cycle of the entire treatment protocol, for example, part of two cycles of the entire treatment protocol.

[0042] The cancer or tumor can be any kind of cancer or neoplasm. The tumor is typically a solid tumor, particularly a solid tumor of epithelial, neuroectodermal or mesenchymal origin. The cancer is also typically selected from carcinoma, sarcoma, lymphoma, germ cell tumor, blastoma, leukemia and multiple myeloma, preferably selected from carcinoma, sarcoma, blastoma, lymphoma, leukemia and multiple myeloma. The cancer may or may not be metastatic cancer.

[0043] Cancers include, but are not limited to, chronic myeloid leukemia, acute lymphoblastic leukemia, Philadelphia chromosome positive acute lymphoblastic leukemia (Ph + ALL), Hodgkin's disease, Hodgkin or non-Hodgkin's lymphoma, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, kidney cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, brain cancer, central nervous system cancer, pancreatic cancer, glioblastoma multiforme, cervical cancer, gastric cancer, bladder cancer, malignant hepatocellular carcinoma, breast cancer, colon cancer, head and neck cancer, gastric cancer, germ cell tumors, childhood cancer, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, soft tissue sarcoma, sinonasal NK / T cell lymphoma, myeloma, melanoma, multiple myeloma, acute myeloid leukemia (AML) and chronic lymphocytic leukemia.

[0044] In preferred embodiments, the cancer is selected from the group consisting of lung cancer, breast cancer, genitourinary cancer (e.g., prostate cancer, bladder cancer, testicular cancer, cervical cancer or ovarian cancer) and sarcoma (e.g., osteosarcoma or soft tissue sarcoma, including pediatric soft tissue sarcoma, neuroblastoma, myeloma and melanoma).

[0045] In certain embodiments, the cancer is a sarcoma.

[0046] More preferably, the cancer is selected from melanoma, lung cancer (including non-small cell lung cancer (or NSCLC) and small cell lung cancer (or SCLC)) and breast cancer.

[0047] Even more preferably, the cancer is melanoma or lung cancer.

[0048] In some embodiments, the cancer is lung cancer, typically small cell lung cancer or non-small cell lung cancer.

[0049] In another embodiment, the cancer is leukemia, typically acute myeloid leukemia (AML) or chronic lymphocytic leukemia.

[0050] In a further embodiment, the cancer is colon cancer, typically colon cancer. The cancer may also be colorectal cancer.

[0051] In a further embodiment, the cancer is a childhood cancer, typically a pediatric sarcoma, lymphoma, leukemia, neuroblastoma, brain cancer or central nervous system cancer.

[0052] In certain embodiments described herein, the anti-cancer agent is selected from chemotherapeutic agents, immune checkpoint inhibitors, and anti-cancer vaccines (also identified herein as "cancer vaccines"). These agents are typically considered "conventional" agents for treating cancer.

[0053] The chemotherapeutic agent is typically an agent selected from, for example, antitumor / cytotoxic antibiotics, alkylating agents, antimetabolites, topoisomerase inhibitors, mitotic inhibitors, platinum-based moieties, specific kinase inhibitors, hormones, cytokines, antiangiogenic agents, antibodies, DNA methyltransferase inhibitors and vascular disrupting agents.

[0054] The antitumor agent or cytotoxic antibiotic may be selected, for example, from anthracyclines (eg, doxorubicin, daunorubicin, adriamycin, idarubicin, epirubicin, mitoxantrone, valrubicin), actinomycin, bleomycin, mitomycin C, plicamycin, and hydroxyurea.

[0055] The alkylating agent may be selected from, for example, mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide, temozolomide, busulfan, N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, streptozotocin, dacarbazine, temozolomide, thiotepa, mitomycin, diaziquone (AZQ), procarbazine, hexamethylmelamine, and uramustine.

[0056] The antimetabolite may be selected, for example, from pyrimidine analogs (e.g., fluoropyrimidine analogs, 5-fluorouracil (5-FU), floxuridine (FUDR), cytosine arabinoside (cytarabine), gemcitabine (Gemzar®), capecitabine); purine analogs (e.g., azathioprine, mercaptopurine, thioguanine, fludarabine, pentostatin, cladribine, clofarabine); folate analogs (e.g., methotrexate, folic acid, pemetrexed, aminopterin, raltitrexed, trimethoprim, pyrimethamine).

[0057] The topoisomerase inhibitor may be selected, for example, from camptothecin, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, and teniposide.

[0058] The mitotic inhibitor may be selected, for example, from a taxane [paclitaxel (PG-paclitaxel and DHA-paclitaxel) (Taxol®), docetaxel (Taxotere®), larotaxel, cabazitaxel, ortataxel, tesetaxel or taxoplexin]; a spindle inhibitor or vinca alkaloid (e.g., vincristine, vinblastine, vinorelbine, vindesine or vinflunine); mebendazole; and colchicine.

[0059] The platinum-based component may be selected from, for example, platinum, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, and triplatin tetranitrate.

[0060] Specific kinase inhibitors may be selected from, for example, BRAF kinase inhibitors such as vemurafenib; MAPK inhibitors (e.g., dabrafenib); MEK inhibitors (e.g., trametinib); and tyrosine kinase inhibitors such as imatinib, gefitinib, erlotinib, sunitinib, or carbozantinib.

[0061] Tamoxifen, anti-aromatase or anti-estrogen drugs may also typically be used in hormone therapy.

[0062] Cytokines that can be used in immunotherapy can be selected from, for example, IL-2 (interleukin-2), IL-11 (interleukin-11), IFN (interferon) alpha (IFNα), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0063] The anti-angiogenic agent may be selected from, for example, bevacizumab, sorafenib, sunitinib, pazopanib, and everolimus.

[0064] Antibodies, particularly monoclonal antibodies (mAbs), may be selected from anti-CD20 antibodies (anti-pan B-cell antigen), anti-Her2 / Neu (human epidermal growth factor receptor-2 / NEU) antibodies; cancer cell surface targeting antibodies (e.g., rituximab and alemtuzumab); growth factor targeting antibodies (e.g., bevacizumab, cetuximab, panitumumab and trastuzumab); agonist antibodies (e.g., anti-ICOS mAb, anti-OX40 mAb, anti-41BB mAb); and immunoconjugates (e.g., 90Y-ibritumomab tiuxetan, 131I-tositumomab or ado-trastuzumab emtansine).

[0065] The DNA methyltransferase inhibitor may be selected from, for example, 2'-deoxy-5-azacytidine (DAC), 5-azacytidine, 5-aza-2'-deoxycytidine, 1-[β]-D-arabinofuranosyl-5-azacytosine and dihydro-5-azacytidine.

[0066] The vascular disrupting agent may be selected, for example, from the flavone acetic acid derivatives, 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and flavone acetic acid (FAA).

[0067] Other chemotherapeutic agents include proteasome inhibitors (e.g., bortezomib), DNA strand-breaking compounds (e.g., tirapazamine), inhibitors of both thioredoxin reductase and ribonucleotide reductase (e.g., xytrin) and Thl immune response enhancers (e.g., thymalfasin).

[0068] In preferred embodiments, the chemotherapeutic drug or agent is selected from antitumor / cytotoxic antibiotics, alkylating agents, antimetabolites, topoisomerase inhibitors, mitotic inhibitors, platinum-based moieties, specific kinase inhibitors, antiangiogenic agents, antibodies and DNA methyltransferase inhibitors.

[0069] The immune checkpoint inhibitor is typically an immune checkpoint targeting antibody. Such immune checkpoint inhibitor is advantageously selected from anti-CTLA4 (ipilimumab and tremelimumab), anti-PD-1 (nivolumab and pembrolizumab), anti-PD-L1 (atezolizumab, durvalumab and avelumab), anti-PD-L2 and anti-Tim3.

[0070] Cancer vaccines may, for example, be selected from vaccine compositions comprising (antigenic) peptides, in particular PTPs; human papillomavirus (HPV) vaccines (e.g., Gardasil®, Gardasil 9® and Cervarix®); vaccines stimulating an immune response against prostatic acid phosphatase (PAP) sipuleucel T (Provenge®); oncolytic viruses; and talimogene laherparepvec (T-VEC or Imlygic®).

[0071] In another specific embodiment, the ("conventional") cancer treatment is radiation (also identified herein as "radiotherapy"). Radiation therapy typically involves light rays selected from X-rays ("XR"), gamma rays, and / or UVC rays.

[0072] Treatments, which may include several anti-cancer agents, are selected by an oncologist depending on the particular cancer being prevented or treated.

[0073] Particular melanomas are those that are conventionally treated with ipilimumab, nivolumab, pembrolizumab, IFNα, dacarbazine, BRAF inhibitors, dabrafenib, trametinib, sorafenib, temozolomide, electrochemotherapy, TNFα and / or fotemustine. In certain embodiments, the melanoma is resistant to said cytotoxic conventional therapies.

[0074] Certain breast cancers are conventionally treated with anthracyclines, taxanes, trastuzumab, anti-PARP (poly(ADP-ribose)polymerase), anti-PI3K (phosphoinositide 3-kinase), mTOR (mammalian target of rapamycin) inhibitors, vinorelbine, gemcitabine, anti-estrogens and / or anti-aromatase, either before or after surgery to remove the breast tumor, preferably after surgery. In certain embodiments, the breast cancer is a breast cancer that is resistant to conventional therapies as described above.

[0075] Certain lung cancers are traditionally treated with XR and platin or permetrexed.

[0076] Particular early stage NSCLC is NSCLC that is conventionally treated with paclitaxel, docetaxel, gemcitabine, vinorelbine, etoposide, taxane, Avastin [anti-VEGF (vascular endothelial growth factor) antibody], erlotinib and / or gefitinib.In certain embodiments, lung cancer is resistant to conventional therapy.

[0077] Certain colon cancers, also known as colorectal or intestinal cancer, are traditionally treated with antimetabolites (5-FU, raltitrexed), platinum-based components (oxaliplatin), topoisomerase inhibitors (irinotecan), and growth factor-targeting antibodies (bevacizumab, cetuximab, panitumumab) after surgery to remove breast tumors.

[0078] The present invention further relates to the use of a compound of formula (A) and formula (B) or a formula selected from formulas (I) to (XI) according to the present invention (or a pharma- ceutically acceptable salt thereof) for the manufacture of a pharmaceutical composition or a medicament, wherein said composition is capable of treating a disease, in particular cancer, in a subject or improving the efficacy of treatment by stimulating the immune system of the subject in need of treatment.

[0079] In certain embodiments, the compound is selected from compounds of formula (A), formula (B), formula II, formula III, formula IV, formula V, formula VI, and formula VII, and the cancer is a sarcoma.

[0080] In another particular embodiment, the compound is selected from a compound of formula II, a compound of formula III, formula IV, formula V, formula VIII, formula IX, formula X, and formula XI, and the cancer is melanoma.

[0081] The compounds of the present invention may be advantageously used in combination with at least one different said anti-cancer agent or any other therapeutically active compound and / or radiation therapy to treat a disease in a subject, in particular to treat cancer, to prevent cancer metastasis and / or to prevent cancer recurrence.

[0082] Also described herein are compositions comprising a compound of Formula (A) and (B) or a formula selected from (I)-(XI) and a pharma- ceutically acceptable carrier, preferably together with at least one different therapeutic agent, particularly an anti-cancer agent, typically as a combined preparation, for simultaneous, separate or sequential use in the treatment of disease, particularly the treatment of cancer.

[0083] Also described herein are (i) methods of treating a disease, particularly cancer, (ii) methods of increasing the sensitivity of a disease, particularly cancer, to a therapeutic / anticancer agent, and (iii) methods of decreasing the resistance of a disease / cancer to a therapeutic / anticancer agent, each of said methods comprising administering to a subject in need of treatment an effective amount, typically a therapeutically effective amount, of at least one compound of formula (A) and formula (B) or a formula selected from formulas (I)-(XI) or a pharmaceutical composition as described above, preferably together with another therapeutic / anticancer agent classically used in the treatment of the disease / cancer described herein (as a combined preparation).

[0084] In another particular embodiment, the method further comprises administering an effective amount of another therapeutically active compound to treat the disease / cancer or to prevent or treat a disease / cancer treatment side effect.

[0085] As used herein, "treatment" or "treating" refers to therapeutic intervention to change the natural course of the subject being treated, and is typically performed for curative purposes. The desired effects of treatment include, but are not limited to, prevention of disease recurrence, alleviation of symptoms, and reduction of any direct or indirect pathological consequences of disease, reduction of disease progression rate, improvement or alleviation of disease state, and remission or improvement of prognosis.

[0086] In a preferred embodiment, the compositions and methods herein are used to slow the progression of cancer or to slow the progression of cancer, typically tumor growth.

[0087] Typically, the treatment involves the induction of a therapeutic response of the subject's immune system, typically CD4 + and / or CD8 + Induce a T cell response.

[0088] Induction of a T cell response typically means that a T cell response directed to a particular antigen is induced. Prior to the induction, the T cell response was absent or below detectable levels or was not functional. Enhancement of a T cell response means that the overall action of T cells directed to a particular antigen is enhanced and / or more efficient compared to the overall action of the T cells before the enhancement. For example, after the enhancement, more T cells directed to the antigen may be produced. As a result, the action of the further produced T cells increases the overall action against the antigen. Alternatively, the enhancement may include an increase in the action of T cells directed to the antigen. For example, the T cells may react more strongly and / or faster with the antigen. Of course, the result of the enhancement may be the production of additional T cells with an increase in the action of the T cells. Alternatively, the enhancement may include only one of the production of additional T cells or the increase in the action of T cells.

[0089] Another object described herein relates to a method for generating an immune response in a subject, typically against a specific target, preferably a tumor antigen or a cancer / tumor cell or tissue, which comprises injecting said subject with a compound of formula (A) and formula (B) or of a formula selected from formulas (I) to (XI) according to the invention, typically in an effective amount.

[0090] Detection of a therapeutic immune response can be readily determined by one of skill in the art by techniques such as ELISA, ELISPOT, delayed type hypersensitivity, intracellular cytokine staining and / or extracellular cytokine staining.

[0091] As used herein, "effective amount or dose" or "therapeutically effective amount or dose" refers to an amount of the compound of the present invention that eliminates, slows down, or reduces or delays one or more symptoms or disorders caused by or associated with a disease in a subject, particularly cancer, or induces a measurable immune response in a subject, preferably a human. The effective amount, and more generally the administration regimen, of the compounds of the present invention and their pharmaceutical compositions can be determined and adapted by those skilled in the art. Effective doses can be determined by the use of conventional techniques and by observing the results obtained under analogous circumstances. The therapeutically effective dose of the compounds of the present invention varies depending on the disease to be treated, its severity, the route of administration, any associated concomitant therapy, the age, weight, general medical condition, medical history, etc. of the patient.

[0092] Typically, the amount of the compound administered to a patient may range from about 0.01 mg / kg to 500 mg / kg body weight for a human patient. In certain embodiments, the pharmaceutical composition according to the invention comprises 0.1 mg / kg to 100 mg / kg, e.g., 0.5 mg / kg to 10 mg / kg, of a compound of the invention.

[0093] In certain embodiments, the compounds of interest of the present invention may be administered to a subject via parenteral routes, oral routes, or intravenous (IV), intratumoral (IT) or intraperitoneal (IP) injection. The compounds of the present invention may be administered to a subject daily (once a day) for several consecutive days, for example, for 2-10 consecutive days, preferably for 3-6 consecutive days. The treatment may be repeated for a period of 1, 2, 3, 4, 5, 6 or 7 weeks, or every two weeks, every three weeks, or every month, every two months or every three months. Alternatively, multiple treatment cycles may be performed, optionally with a rest period of, for example, 1, 2, 3, 4 or 5 weeks between two treatment cycles. Any one of the compounds of the present invention described herein may be administered as a single dose, for example, once a week, once every two weeks or once a month. The treatment may be repeated one or more times per year.

[0094] Doses are administered at appropriate intervals that can be determined by one skilled in the art. The amount selected depends on several factors, including the route of administration, duration of administration, time of administration, excretion rate of the selected compound having formula (A) and formula (B) or a formula selected from formulas (I)-(XI) or various products used in combination with said compound, age, weight and health condition of the patient and the medical history of the patient and any other information known in medicine.

[0095] The route of administration can be carried out by a variety of routes, for example, the route of administration can be oral or parenteral.

[0096] It is typically performed by systemic injection, for example, intravenously, intramuscularly, intraperitoneally, intratumorally, subcutaneously, etc. The pharmaceutical composition is adapted to one or more of the above routes. The pharmaceutical composition is preferably administered by injection or intravenous infusion of a suitable sterile solution, or via the digestive tract in the form of a liquid or solid dose.

[0097] The pharmaceutical compositions may be formulated as solutions in pharma- ceutically compatible solvents or vehicles, or as pills, tablets, capsules, powders, suppositories, etc., containing solid vehicles, optionally through dosage forms or devices providing sustained and / or delayed release, in a manner known in the art. For this type of formulation, substances such as celluloses, lipids, carbonates or starches are advantageously used.

[0098] The substances or vehicles (liquids and / or injectable solutions and / or solids) that can be used in the formulation are excipients or inert vehicles, ie, pharma- ceutically inert and non-toxic vehicles.

[0099] For example, saline, physiological, isotonic and / or buffer solutions compatible with pharmaceutical use and known to those skilled in the art may be mentioned. The composition may include one or more substances or vehicles selected from dispersants, solubilizers, stabilizers, preservatives, etc.

[0100] Formulations of the present invention suitable for oral administration may be present in the form of discrete units such as capsules, sachets, tablets or lozenges, each containing a predetermined amount of the active ingredient in powder or granular form; in the form of a solution or suspension in an aqueous liquid or a non-aqueous liquid; or in the form of an oil-in-water or water-in-oil emulsion.

[0101] Formulations suitable for parenteral administration conveniently comprise a sterile oily or aqueous preparation of the active ingredient, which is preferably isotonic with the blood of the recipient. All such preparations may also contain other pharma- ceutically compatible and non-toxic auxiliary substances, such as stabilizers, antioxidants, binders, dyes, emulsifiers, or flavoring agents.

[0102] The formulations of the present invention contain the active ingredient, a compound having formula (A) and formula (B) or a formula selected from formulas (I) to (XI) according to the present invention (or a pharma- ceutically acceptable salt thereof), together with a pharma- ceutically acceptable carrier and, optionally, other active or therapeutic ingredients. The carrier must be "acceptable" in the sense of being compatible with the other formulation ingredients and not deleterious to the recipient thereof. Safe and effective administration of many of these anticancer agents is known to those skilled in the art. Moreover, these administrations are described in the standard literature.

[0103] Another object of the present invention is a kit comprising a compound having formula (A) and formula (B) or a formula selected from formulas (I) to (XI) according to the present invention (or a pharma- ceutically acceptable salt thereof) and preferably at least one different therapeutic agent, in particular an anti-cancer agent, typically a chemotherapeutic agent, in different containers. The kit further comprises instructions for carrying out a method as described herein for a third party, e.g. a method for treating a disease, in particular cancer, for preventing or treating cancer metastasis and / or for treating cancer recurrence in a subject, for manufacturing a composition according to the present invention.

[0104] In another particular embodiment, the present invention relates to the use of a kit according to the present invention for producing a composition as described herein.

[0105] In another particular embodiment, a third party is suitable to perform the methods described herein, in particular the methods for treating cancer, preventing or treating cancer metastasis and / or treating cancer recurrence in a subject.

[0106] Further aspects and advantages of the present invention are disclosed in the following experimental section and drawings which are to be considered as illustrative only. [Brief description of the drawings]

[0107] [Figure 1A-1B] 1A and 1B: Madrasin and Madra.HCl treatment increases intron-derived antigen presentation in cancer cells. B3Z-specific T cell activation in MCA205 sarcoma cells and B16F10 melanoma expressing intron-derived SL8 antigen after treatment with 5 μM or 10 μM (A) Madrasin or (B) Madra.HCl. Free SL8 peptide was added in each condition to ensure that T cell assays were performed under non-saturating conditions and that expression of MHC-I molecules was factored into the results. Each graph is representative of one of at least three independent experiments. Data are given as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (unpaired Student's t-test).

[0108] [Fig. 1C-1F]Madra.HCl slows the growth of tumors bearing intron-derived SL8 epitopes. Wild-type MCA205 sarcoma cells or MCA205 sarcoma cells expressing a globulin-SL8-intron construct (MCA205 globulin-SL8-intron) were inoculated subcutaneously into the flank of immunocompetent C57BL / 6 mice, followed by intraperitoneal injection of 20 mg / kg or 40 mg / kg Madra.HCl on days 4, 7, 10 and 14 after tumor inoculation. Tumor size was assessed every 3-4 days until the established ethical endpoint was reached. Panel C represents the growth curve of MCA205 sarcoma cells expressing a globulin-SL8-intron construct (MCA205 globulin-SL8-intron) and panel D represents tumor size at day 25. Panel E shows the growth curve of wild type MCA205 sarcoma cells (MCA205 WT), and panel F shows the tumor size at day 21. Data are shown as mean ± SEM. *p<0.05, **p<0.01 (ANOVA with Tukey's multiple comparison test comparing all groups).

[0109] [Figure 1G] Madra.HCl prolongs overall survival of intron-derived SL8-expressing tumor-bearing mice. Kaplan-Meier survival curves for mice subcutaneously injected with MCA205 globulin-SL8-intron followed by intraperitoneal treatment with 20 mg / kg or 40 mg / kg Madra.HCl on days 4, 7, 10, and 14 after tumor inoculation.

[0110] [Figure 1H] Madra.HCl induces a sustained and specific anti-tumor response. Growth curves of MCA205 globulin-SL8-Intron and B16F10WT cells inoculated into the right and left flanks on day 100 of C57BL / 6 mice that underwent complete tumor regression after treatment with Madra.HCl, respectively.

[0111] [Diagram 2]MTT test of each madracin derivative. MTT assay performed on MCA205 cells (A) or B16F10 cells (B) treated with increasing doses of various madracin derivatives. Data are expressed as half-maximal inhibitory concentration (IC50) of various derivatives. IC50 represents the concentration of test compound required for 50% inhibition of cell viability compared to control conditions. A threshold of 60 μM is indicated. It is the maximum dose used for all compounds that does not show any cell death.

[0112] [Diagram 3] Madrasin derivatives increase antigen presentation in both mouse cell lines. B3Z-specific T cell activation in MCA205 and B16F10 expressing intron-derived SL8 antigen after treatment with (A) 60 μM EYP59 (compound 7), (B) 20 μM EYP201 (compound 6), (C) 60 μM EYP165 (compound 57), or (D) 50 μM EYP281 (compound 32). Free SL8 peptide was added in each condition to ensure that the T cell assay was performed under non-saturating conditions and that expression of MHC-I molecules was factored into the results. Each graph is representative of one of at least three independent experiments. Data are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (unpaired Student's t-test).

[0113] [Figure 4] Madrasin derivatives increase antigen presentation only in MCA205 sarcoma cell line. B3Z-specific T cell activation in MCA205 expressing intron-derived SL8 antigen after treatment with (A) 60 μM EYP188 (compound 42) and (B) 50 μM EYP86 (compound 10). Free SL8 peptide was added in each condition to ensure that T cell assays were performed under non-saturating conditions and that expression of MHC-I molecules was factored into the results. Each graph is representative of one of at least three independent experiments. Data are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (unpaired Student's t-test).

[0114] [Diagram 5] Madrasin derivatives increase antigen presentation only in B16F10 melanoma cell line. B3Z-specific T cell activation in MCA205 expressing intron-derived SL8 antigen after treatment with (A) 20 μM EYP174 (compound 41), (B) 20 μM EYP179 (compound 50), (C) 60 μM EYP190 (compound 54), and (D) 10 μM EYP181 (compound 49). Free SL8 peptide was added in each condition to ensure that the T cell assay was performed under non-saturating conditions and that expression of MHC-I molecules was factored into the results. Each graph is representative of one of at least three independent experiments. Data are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (unpaired Student's t-test).

[0115] [Figure 6] Madrasin derivatives do not increase antigen presentation in both tumor cell lines. B3Z-specific T cell activation in MCA205 expressing intron-derived SL8 antigen after treatment with (A) 60 μM EYP177 (compound 46), (B) 60 μM EYP156 (compound 43), (C) 10 μM EYP113 (compound 11) and (D) 20 μM EYP102 (compound 9). Free SL8 peptide was added in each condition to ensure that the T cell assay was performed under non-saturating conditions and that expression of MHC-I molecules was factored into the results. Each graph is representative of one of at least three independent experiments. Data are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (unpaired Student's t-test).

[0116] [Figure 7]EYP59 (compound 7) slows down the growth of MCA-WT sarcoma and MCA-intron-SL8. Wild-type MCA205 sarcoma cells or MCA205 sarcoma cells expressing the globulin-SL8-intron construct (MCA205 globulin-SL8-intron) were inoculated subcutaneously into the flank of immunocompetent C57BL / 6 mice, followed by intraperitoneal injection of 20 mg / kg EYP59 on days 4, 7, 10 and 14 after tumor inoculation. Tumor size was assessed every 3-4 days until the established ethical endpoint was reached (A). The top of panel B represents the growth curve of MCA205 sarcoma cells expressing the globulin-SL8-intron construct (MCA205 globulin-SL8-intron), and the bottom of panel B represents tumor size at day 23. The top of panel C represents the growth curve of wild type MCA205 sarcoma cells (MCA205 WT), and the bottom of panel C represents the tumor size on day 19. Panel D represents the growth curve of wild type MCA205 sarcoma cells (MCA205 WT) in immunodeficient nu / nu mice with a similar setup as previously described for immunocompetent mice. Data are shown as mean ± SEM. *p<0.05, **p<0.01 (ANOVA with Tukey's multiple comparison test comparing all groups).

[0117] [Figure 8] Madracin derivative EYP59 (compound 7) efficiently reduces tumor growth in vivo when injected intratumorally or intravenously. Wild-type MCA205 sarcoma cells were inoculated subcutaneously into the flank of immunocompetent C57BL / 6 mice, followed by intratumoral injection of 2.5 mg / kg EYP59 on days 4, 7, 10, and 14 after tumor inoculation, or intravenous injection of 5 mg / kg EYP59 on days 4, 7, 10, and 14 after tumor inoculation. The top of panel A represents the growth curve of wild-type MCA205 sarcoma cells, and the bottom of panel A represents the tumor size at day 21. The top of panel B represents the growth curve of wild-type MCA205 sarcoma cells, and the bottom of panel B represents the tumor size at day 25. Data are shown as mean ± SEM. *p<0.05, **p<0.01 (ANOVA with Tukey's multiple comparison test comparing all groups).

[0118] [Figure 9] Synthesis of Madracine and Madracine Hydrochloride (Madra.HCl).

[0119] [Figure 10] Pharmacomodulation of madracine and synthesis of madracine derivatives. EXAMPLES

[0120] material and method cell culture The MCA205 mouse sarcoma cell line is cultured in RPMI 1640 medium (Life Technologies) in the presence of 1% glutamine, 1% sodium pyruvate, 1% unnatural amino acids, 1% penicillin / streptomycin and 10% FBS (Life Technologies) at 37° C. under 5% CO2. The B16F10 mouse melanoma cell line is cultured under standard conditions in DMEM medium (Life Technologies) containing 1% glutamine, 1% penicillin / streptomycin and 10% FCS at 37° C. under 5% CO2. For selection, the stable MCA205-globulin-SL8-intron cell line is cultured under the same conditions as the MCA205 cell line, with the additional addition of 500 μg / ml G418 (Life Technologies). For selection, the stable B16F10-globulin-SL8-intron cell line is cultured under the same conditions as the B16F10 cell line with the addition of 500 μg / ml G418 (Life Technologies). The SL8 / Kb-specific (B3Z) T cell reporter hybridoma is cultured at 37°C under 5% CO2 in RPMI1640 medium (Life Technologies) in the presence of 1% L-glutamine, 1% penicillin / streptomycin, 50 μM β-mercaptoethanol and 10% FCS.

[0121] Synthetic scheme of madrasine and all derived compounds 1) Synthesis of Madracine and Madracine Hydrochloride (Madra.HCL) [ka]

[0122] a. 7-Methoxy-2,2,4-trimethyl-1,2-dihydroquinoline (compound 1) (see Org. Lett. 2015, 17, 4125) [ka] A solution of m-anisidine (2.3 mL, 20.3 mmol) and InCl3 (232 mg, 1.03 mmol) in acetone (30 mL) was heated at 50 °C under argon for 14 h. The solvent was removed and the crude was partitioned between DCM and saturated aqueous Na2CO3. The organic layer was dried over MgSO4, filtered and concentrated. Purification by column chromatography (cyclohexane / EtOAc 100:0 to 99:1) afforded the desired compound as a yellow solid. Spectral data are consistent with those in the literature (Tamariz, J. et al. J. Org. Chem. 2013, 78, 9614-9626). Yield: 61% (2.51 g, 12.3 mmol). Melting point 68.8 °C. TLC Rf: 0.5 (cyclo / EtOAc 9:1). 1 H NMR (300MHz, CDCl3) δ 6.97(d, J=8.4Hz, 1H), 6.20(dd, J=8.4, 2.5Hz, 1H), 6.01(d, J=2.5Hz, 1H), 5.19(s, 1H), 3.75(s, 3H), 1.96(d, J=1.5Hz, 3H), 1.26(s, 6H). HRMS(ESI)(M+H) + m / z C 13 H 18 Calculated value for NO: 204.1388; measured value: 204.1385.

[0123] b. 1-(7-Methoxy-4-methylquinazolin-2-yl)guanidine (Compound 2) [ka] 7-Methoxy-2,2,4-trimethyl-1,2-dihydroquinoline (compound 1) (2.0 g, 0.98 mmol) and HCl (0.25 mL, 0.98 mmol, 4 M dioxane solution) were stirred at room temperature for 30 min. Then, HO (95 mL) and dicyandiamide (831 mg, 0.98 mmol) were added and the mixture was refluxed for 48 h. After cooling to 60 °C, the oil was filtered and the pH was adjusted to 11 using saturated aqueous NaHCO3 solution. The precipitate formed was filtered and dried under reduced pressure. Compound 2 was isolated as an off-white powder. Yield: 78% (1.76 g, 0.76 mmol). Melting point 235-236 °C. 1 H NMR (300MHz, DMSO-d6) δ 7.95(d, J=9.1Hz, 1H), 7.82(s, 2H), 7.11(d, J=2.5Hz, 1H), 7.00(dd, J=9.0, 2.5Hz, 1H), 3.90(s, 3H), 2.70(s, 3H). HRMS(ESI)(M+H) + m / z C 11 H 14 Calculated value for N5O: 232.1198; measured value: 232.1193.

[0124] c. 2-((7-Methoxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one (Madrasine, compound 3) [ka] A solution of 1-(7-methoxy-4-methylquinazolin-2-yl)guanidine (compound 2) (3.2 g, 13.8 mmol), ethyl 2-methyl-3-oxobutanoate (2.3 mL, 16.2 mmol), and NaHCO3 (1.43 g, 17 mmol) in DMSO (22 mL) was heated at 110 °C for 48 h. After cooling to room temperature, water was added. The formed precipitate was filtered and purified by column chromatography (DCM / MeOH 100: to 98:2) to give madrasine 3 as a beige powder. Yield: 83% (3.58 g, 11.5 mmol). Melting point 216.4-217.5 °C. TLC Rf: 0.26 (DCM / MeOH 98:2). 1H NMR (300MHz, CDCl3) δ 13.25(s, 1H), 8.54(s, 1H), 7.86(d, J=9.1Hz, 1H), 7.17(d, J=2.5Hz, 1H), 7.0 7(dd, J=9.1, 2.5Hz, 1H), 3.98(s, 3H), 2.80(s, 3H), 2.27(s, 3H), 2.06(s, 3H). HRMS(ESI)(M+H) + m / z C 16 H 18 Calculated value for N5O2: 312.1462, measured value: 312.1458.

[0125] d. Madr.HCl (EYP34) [ka] To a solution of madracine (compound 3) (600 mg, 1.98 mmol) in dry dioxane (10 mL) was added HCl (0.5 mL, 2 mmol, 4 M in dioxane). The mixture was stirred at room temperature for 10 min. The precipitate that formed was filtered and dried under reduced pressure to give madracine hydrochloride as a clear green powder (565 mg, 1.68 mmol, 85%). 1 H NMR (300MHz, heavy water) δ 7.76(s, 1H), 6.88(s, 1H), 6.80(s, 1H), 3.87(s, 3H), 2.71(s, 3H), 2.11(s, 3H), 1.74(s, 3H). Melting point >330℃ (decomposition). HRMS(ESI)(M+H) + m / z C 16 H 18 Calculated value for N5O2: 312.1462, measured value: 312.1460. 16 H 18 Analytical calculated for ClN5O2.2 / 3H2O: C, 53.41 H, 5.42. Found: C, 53.40; H, 5.47.

[0126] 2) Pharmacomodulation of madrasin Part 1: [ka] a. 2-((7-hydroxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one (compound 4, EYP107) [ka] To a solution of madracine (compound 3) (250 mg, 0.8 mmol) in DCE (15 mL) was added BBr3 (12 mL, 12 mmol) dropwise under argon at -78 °C. The solution was allowed to warm to room temperature and heated at 60 °C for 20 h. After quenching, the solvent was concentrated under reduced pressure. The crude was taken up in EtOAc and washed with saturated aqueous NaHCO3. The aqueous layer was extracted 10 times with EtOAc. The combined organic layers were dried over MgSO4, filtered and concentrated. The desired compound 4 was precipitated in DCM, filtered and washed several times with DCM. Yield: 42% (1.0 g, 0.34 mmol). Melting point 348.8-349.9 °C. TLC Rf: 0.18 (DCM / MeOH 97:3). 1 H NMR (300MHz, DMSO) δ 13.52(s, 1H), 10.93(s, 2H), 8.05(d, J=9.0Hz, 1H), 7.03(dd, J=9.8, 1.1Hz, 1H), 6.94(d, J=2.2Hz, 1H), 2.77(s, 3H), 2.18(s, 3H), 1.90(s, 3H). HRMS(ESI)(M+H) + m / z C 15 H 16 Calculated value for N5O2: 298.1304, measured value: 298.1312.

[0127] b. Sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazolin-7-olate (Compound 5, EYP112) [ka] To a suspension of 2-((7-hydroxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one (compound 4) (15 mg, 0.05 mmol) in water (1.5 mL) was added NaOH (50 μL, 0.05 mmol, 1M in HO). Stirring was continued for 5 min until a clear solution was obtained. Evaporation to dryness gave a yellow solid. Yield: >99% (15 mg, 0.05 mmol). 1 H NMR (300MHz, D2O) δ 7.51(d, J=8.8Hz, 1H), 6.67(d, J=8.8Hz, 1H), 6.40(s, 1H), 2.49(s, 3H), 2.03(s, 3H), 1.71(s, 3H). HRMS(ESI)(M+H) + m / z C 15 H 16 Calculated value for N5O2: 298.1304, measured value: 298.1259.

[0128] c. 5,6-Dimethyl-2-((4-methyl-7-(2-morpholinoethoxy)quinazolin-2-yl)amino)pyrimidin-4(3H)-one (compound 6, EYP201) [ka] Compound 4 (35 mg, 0.118 mmol) was suspended in DMF (1 mL) under inert atmosphere and KOH (13 mg, 0.23 mmol) was added. The mixture was stirred at room temperature for 1 h until clear. Then 2-chloro-N-ethylmorpholine (22 mg, 0.12 mmol) was added and the mixture was stirred at room temperature for 14 h. The solvent was removed under reduced pressure. After column chromatography (DCM / MeOH 100:0 to 97:3), compound 6 was obtained as an off-white powder. Yield: 19% (9.1 mg, 0.022 mmol). TLC Rf: 0.2 (DCM / MeOH 96:4). 1H NMR (300MHz, CDCl3) δ 13.26(s, 1H), 7.85(d, J=9.0Hz, 1H), 7.19(s, 1H), 7.08(d, J=9.0Hz, 1H), 4.29(t, J=5.6Hz, 2H), 3.76( t, J=4.7Hz, 4H), 2.89(t, J=5.7Hz, 2H), 2.80(s, 3H), 2.62(t, J=4.6Hz, 4H), 2.26(s, 3H), 2.05(s, 3H). HRMS(ESI)(M+H) + m / z C 21 H 27 Calculated value for N6O3: 411.2145, measured value: 411.2152.

[0129] d. Sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazolin-7-yl phosphate (Compound 7, EYP59) [ka] A suspension of compound 4 (100.4 mg, 0.33 mmol) and KOH (25 mg, 0.44 mmol) in HO (5 mL) was stirred at room temperature for 30 min. Then DCM (5 mL) was added, followed by (EtO)2P(O)Cl (0.064 mL, 0.44 mmol) and TBAB (141 mg, 0.44 mmol). The mixture was stirred at room temperature for 2 h. The organic layer was separated and the aqueous layer was extracted with DCM. The combined organic layers were dried over MgSO4, filtered and concentrated under reduced pressure. After column chromatography (DCM / MeOH 100:0 to 97:3), the intermediate diethyl phosphate derivative was obtained. Yield: 38% (55 mg, 0.12 mmol). 1 H NMR (300MHz, CDCl3) δ 13.16(s, 1H), 7.98(d, J=9.0Hz, 1H), 7.66(d, J=2.3Hz, 1H), 7.42(dd, J=9.0, 2.4Hz, 1H ), 4.28(p, J=7.4Hz, 4H), 2.84(s, 3H), 2.27(s, 3H), 2.05(s, 3H), 1.39(t, J=7.0Hz, 6H).

[0130] The purified diethyl phosphate compound (55 mg, 0.12 mmol) was dissolved in DCM (2.5 mL); TMSI was added and the reaction was stirred at room temperature for 3 h. The solvent was evaporated and the crude was triturated with DCM, filtered, and then suspended in H2O. Aqueous NaOH (0.250 mL, 0.254 mmol) was added slowly and after stirring for 30 min the mixture became clear. The solvent was removed under vacuum to give madrasine-phosphate (compound 7). Yield: 93% (50 mg, 0.11 mmol). 1 H NMR (300MHz, D2O) δ 7.97 (d, J=9.1Hz, 1H), 7.47-7.38 (m, 2H), 2.73 (s, 3H), 2.26 (s, 3H), 1.87 (s, 3H). 31 P NMR (81 MHz, D2O) δ -174.17. HRMS(ESI)(M+H) + m / z C 15 H 16 Calculated value for N5NaO5P: 400.0787; found value: 400.0792.

[0131] Part 2 [ka] a. 2-((8-iodo-7-methoxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one (compound 8) [ka] To a cooled solution of madrasin (compound 3) (360 mg, 1.16 mmol) in concentrated H2SO4 (1.2 mL) was added NIS (248 mg, 1.10 mmol) (in the dark). The solution was stirred at room temperature for 5 min. The mixture was poured into ice water and the pH was adjusted to 5. After extraction with DCM, the combined organic layers were dried over MgSO4, filtered and concentrated. Purification by column chromatography (DCM / MeOH 100:0 to 98:2) gave the desired compound 8. Yield: 25% (127 mg, 0.29 mmol). TLC Rf: 0.22 (DCM / MeOH 98:2). 1H NMR (300MHz, CDCl3) δ 13.50(s, 1H), 8.88(s, 1H), 7.96(d, J=9.1Hz, 1H), 7.09(d, J=9.1Hz, 1H), 4.08(s, 3H), 2.83(s, 3H), 2.28(s, 3H), 2.07(s, 3H). HRMS(ESI)(M+H) + m / z C 16 H 17 Calculated value for N5O2: 438.0427, measured value: 438.0427.

[0132] bN-Acetyl-S-(2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-7-methoxy-4-methylquinazolin-8-yl)cysteine ​​(compound 9, EYP102) [ka] In a sealable vial under inert atmosphere was added 2-((8-iodo-7-methoxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one compound 8 (10.6 mg, 0.025 mmol), cysteine ​​NHAc (7 mg, 0.04 mmol), PdG3 xantphos (5 mg, 0.005 mmol) and THF (0.1 mL). The mixture was purged with argon and NEt3 (10 μL, 0.07 mmol) was added. The mixture was stirred at 50 °C for 18 h and at 70 °C for 6 h. The solvent was removed and purified by column chromatography (DCM / MeOH 100:0 to 70:30) to give the desired compound 9. A second purification by preparative TLC (DCM / MeOH 75:25) was required to obtain pure compound 9 as a white powder. Yield: 48% (5.7 mg, 0.012 mmol). TLC Rf: 0.1 (DCM / MeOH 8:2). 1 H NMR (300MHz, MeOD) δ 8.11 (d. HRMS(ESI)(M+H) +m / z C 21 H 25 Calculated value for N6O5S: 473.1607; measured value: 473.1608.

[0133] c.((1S,2S,4R,5S,6S)-5-acetoxy-7,8-diacetyl-4-((2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-7-methoxy-4-methylquinazolin-8-yl)thio)-3,7λ 3 ,8λ 3 -Trioxabicyclo[4.2.0]octan-2-yl)methyl acetate (compound 10, EYP86) [ka] A solution of 2-((8-iodo-7-methoxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one compound 8 (25 mg, 0.055 mmol) and thioglucose (22 mg, 0.06 mmol) in THF (0.5 mL) was purged with argon under inert atmosphere. Catalyst PdG3Xantphos (10 mg, 0.01 mmol) and Et3N (20 μL, 0.14 mmol) were added and the reaction mixture was heated at 50 °C for 3 h. The solvent was removed under reduced pressure and purified by column chromatography (DCM / MeOH 100:0 to 97:3) to give the desired compound 10 as an orange solid. Yield: 41%. (15.4 mg, 0.022 mmol). TLC Rf: 0.3 (DCM / MeOH 96:4). 1 H NMR (300MHz, MeOD) δ 8.18(d, J=9.1Hz, 1H), 7.34(d, J=9.5Hz, 1H), 5.17(t, J=9.3Hz, 1H), 4.75-4.66(m, 1H), 4.07(s, 3H), 3.99- 3.89(m, 1H), 3.62-3.52(m, 2H), 2.83(s, 3H), 2.62(s, 2H), 2.10(s, 6H), 1.94(d, J=9.4Hz, 6H), 1.66(s, 3H). HRMS(ESI)(M+Na) + m / z C 30 H 35 N5O 11Calculated value for NaS: 696.1651; measured value: 696.1657.

[0134] d. 2-((7-Methoxy-4-methyl-8-(((2R,3S,4R,5R,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)thio)quinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one (compound 11, EYP113) [ka] A solution of compound 10 in MeONa (0.19 mL, 0.038 mmol, 0.2 M MeOH solution) was stirred at room temperature for 1 h. After evaporation of the solvent under reduced pressure, compound 11 was obtained as a yellow powder. Yield: >99% (7 mg, 0.014 mmol). 1 H NMR (300MHz, MeOD) δ 7.88(d, J=9.0Hz, 1H), 7.02(d, J=9.1Hz, 1H), 3.87(s, 3H), 3.53-3.28 (m, 5H), 2.98-2.91(m, 2H), 2.58(s, 2H), 2.10(s, 3H), 1.80(s, 3H). HRMS(ESI)(M+H) + m / z C 22 H 28 Calculated value for N5O7S: 506.1709; measured value: 506.1729.

[0135] 3) General method A: [ka] Under an inert atmosphere, 2-aminoquinazoline derivative (1 eq.) (compound 12), aryl halide (1.5 eq. unless otherwise stated), Pd2dba3 (7.5 mol% or 10 mol%), Xantphos (15 mol% or 20 mol%) and Cs2CO3 (2 eq.) were added. The mixture was purged with argon. THF was added. The vial was sealed and the reaction mixture was stirred at 80° C. for 14 h. After cooling to room temperature, the solvent was removed under vacuum and the crude was purified by column chromatography to give the desired compound.

[0136] N-(2-methoxypyrimidin-5-yl)quinazolin-2-amine (compound 32, EYP281, C 13 H 11 NO, 253,2650 g / mol): Following general method A, starting with 2-aminoquinazoline (21 mg, 0.13 mmol), 5-bromo-2-methoxypyrimidine (26 mg, 0.19 mmol), Pd2dba3 (9 mg, 0.01 mmol), Xantphos (12 mg, 0.02 mmol), Cs2CO3 (84 mg, 0.26 mmol), THF (0.65 mL). Yield: 42% (13 mg, 0.05 mmol). Beige powder. Melting point 210.9-211.7 °C. TLC Rf: 0.2 (DCM / MeOH 98:2). 1 H NMR (300MHz, CDCl3) δ 9.11(s, 1H), 9.03(s, 2H), 7.77(d, J=7.4Hz, 3H), 7.38(t, J=7.3Hz, 1H), 7.21(s, 1H), 4.04(s, 3H).

[0137] 4) Another method from the methodology section [ka] a. 7-Methoxy-N-(6-methoxypyridin-3-yl)-4-methylquinazolin-2-amine (compound 41, EYP174) [ka] Following general procedure A, except only using 1.1 equiv of heteroaryl bromide, starting with 2-amino-4-methyl-7-methoxyquinazoline (compound 39) (25 mg, 0.13 mmol), 2-methoxy-5-bromopyridine (0.017 mL, 0.14 mmol), Pd2dba3 (12 mg, 0.013 mmol), Xantphos (15 mg, 0.026 mmol), Cs2CO3 (84 mg, 0.26 mmol), THF (0.65 mL). Yield: 76% (29 mg, 0.10 mmol). Orange powder. Melting point 172.1-173.0 °C. TLC Rf: 0.6 (DCM / MeOH 98:2). 1H NMR (300MHz, CDCl3) δ 8.59(d, J=2.8Hz, 1H), 8.04(dd, J=8.8, 2.8Hz, 1H), 7.75(d, J=9.1Hz, 1H), 7.36(s, 1H), 6.98(d, J=2. 5Hz, 1H), 6.89(dd, J=9.0, 2.5Hz, 1H), 6.76(d, J=8.8Hz, 1H), 3.93(s, 3H), 3.89(s, 3H), 2.72(s, 3H). HRMS(ESI)(M+H) + m / z C 16 H 17 Calculated value for N4O2: 297.1352, measured value: 297.1328.

[0138] b. 7-Methoxy-4-methyl-N-(3,4,5-trimethoxyphenyl)quinazolin-2-amine (compound 42, EYP188) [ka] According to general method A, starting from 2-amino-4-methyl-7-methoxyquinazoline (compound 39) (25 mg, 0.13 mmol), 1-bromo-3,4,5-trimethoxybenzene (47 mg, 0.19 mmol), Pd2dba3 (12 mg, 0.013 mmol), Xantphos (15 mg, 0.026 mmol), Cs2CO3 (84 mg, 0.26 mmol), THF (0.65 mL). Yield: 58% (26 mg, 0.07 mmol). Light yellow powder; Mp 159.5-160.1 °C. TLC Rf: 0.8 (DCM / MeOH 98:2). 1 H NMR (300MHz, CDCl3) δ 7.78(d, J=9.0Hz, 1H), 7.39(s, 1H), 7.11(s, 2H), 6.97(d, J=2.6Hz, 1H), 6.92 (dt, J=9.0, 2.4Hz, 1H), 3.91(s, 3H), 3.89(s, 6H), 3.83(s, 3H), 2.75(s, 3H). HRMS(ESI)(M+H) + m / z C 19 H 22 Calculated value for N3O4: 356.1610, measured value: 356.1546.

[0139] c. 7-Methoxy-4-methyl-N-(p-tolyl)quinazolin-2-amine (compound 43, EYP156) [ka] According to general method A, starting from 2-amino-4-methyl-7-methoxyquinazoline (compound 39) (25 mg, 0.13 mmol), 4-bromotoluene (32 mg, 0.19 mmol), Pd2dba3 (12 mg, 0.013 mmol), Xantphos (15 mg, 0.026 mmol), Cs2CO3 (84 mg, 0.26 mmol), THF (0.65 mL). Yield: 39% (14.0 mg, 0.05 mmol). Off-white solid. Melting point 142.8-143.3 °C. TLC Rf: 0.7 (DCM 100%). 1 H NMR (300MHz, CDCl3) δ 7.77(d, J=9.0Hz, 1H), 7.68(d, J=8.2Hz, 2H), 7.24-7.12(m, 3H), 7.05(d, J=2. 5Hz, 1H), 6.90(dd, J=9.1, 2.5Hz, 1H), 3.93(s, 3H), 2.75(s, 3H), 2.34(s, 3H). HRMS(ESI)(M+H) + m / z C 17 H 18 Calculated value for N3O: 280.1450; measured value: 280.1444.

[0140] dt-Butyl 3-((7-methoxy-4-methylquinazolin-2-yl)amino)-1H-indole-1-carboxylate (compound 46, EYP177) [ka] According to general procedure A, except that only 1.1 equivalents of heteroaryl bromide were used, 2-amino-4-methyl-7-methoxyquinazoline (compound 39) (25 mg, 0.13 mmol), 3-bromoindole derivative (41 mg, 0.14 mmol), Pd2dba3 (12 mg, 0.013 mmol), Xantphos (15 mg, 0.026 mmol), Cs2CO3 (84 mg, 0.26 mmol), THF (0.65 mL). Yield: 31% starting from (16 mg, 0.04 mmol). Orange powder. Melting point 114-116 °C. TLC Rf: 0.55 (DCM / MeOH 98:2). 1 H NMR (300MHz, CDCl3) δ 8.57(s, 1H), 8.17(s, 1H), 7.82(d, J=9.0Hz, 1H), 7.58(d, J=7.7Hz, 1H), 7.40-7.27(m, 4H), 7.09(d, J=2.5Hz, 1H), 6.94(dd, J=9.1, 2.5Hz, 1H), 3.95(s, 3H), 2.80(s, 3H), 1.73(s, 9H). HRMS(ESI)(M+H) + m / z C 23 H 25 Calculated value for N4O3: 405.1927, measured value: 405.1932.

[0141] eN 2 -(7-Methoxy-4-methylquinazolin-2-yl)-N 4 -Methylpyrimidine-2,4-diamine (compound 49, EYP181) [ka] Following general procedure A, but using only 1.1 equivalents of heteroaryl chloride, starting with 2-amino-4-methyl-7-methoxyquinazoline (compound 39) (25 mg, 0.13 mmol), 2-chloro-N-methylpyrimidin-4-amine (20 mg, 0.14 mmol), Pd2dba3 (12 mg, 0.013 mmol), Xantphos (15 mg, 0.026 mmol), Cs2CO3 (84 mg, 0.26 mmol), THF (0.65 mL). Yield: 31% (11 mg, 0.04 mmol). Grey powder. Melting point 247.4-249.1 °C. TLC Rf: 0.45 (DCM / MeOH 9:1). 1 H NMR (300MHz, MeOD) δ 8.02(d, J=9.2Hz, 2H), 7.49(s, 1H), 7.13(d, J=9.2Hz, 1H), 6.39(d, J=6.5Hz, 1H), 4.00(s, 3H), 3.07(s, 3H), 2.85(s, 3H). HRMS(ESI)(M+H) + m / z C 15 H 17 Calculated value for N6O: 297.1464; measured value: 297.1473.

[0142] f. 7-Methoxy-N-(4-methoxypyrimidin-2-yl)-4-methylquinazolin-2-amine (compound 50, EYP179) [ka] According to general procedure A, except that only 1.1 equivalents of heteroaryl chloride were used, 2-amino-4-methyl-7-methoxyquinazoline (compound 39) (25 mg, 0.13 mmol), 2-chloro-4-methoxypyrimidine (20 mg, 0.14 mmol), Pd2dba3 (12 mg, 0.013 mmol), Xantphos (15 mg, 0.026 mmol), Cs2CO3 (84 mg, 0.26 mmol), THF (0.65 mL). Yield: 62% starting from (23 mg, 0.08 mmol). Yellow powder. Melting point 160.8-161.5 °C. TLC Rf: 0.3 (DCM / MeOH 98:2). 1H NMR (300MHz, CDCl3) δ 8.42-8.33(m, 1H), 8.28-8.16(m, 1H), 7.84(d, J=9.0Hz, 1H), 7.23(s, 1H), 7.01(dd , J=9.0, 2.3Hz, 1H), 6.35(d, J=5.6Hz, 1H), 3.97(s, 3H), 3.92(s, 3H), 2.81(s, 3H). HRMS(ESI)(M+H) + m / z C 15 H 16 Calculated value for N5O2: 298.1304; measured value: 298.1302.

[0143] g. 7-Methoxy-N-(2-methoxypyrimidin-4-yl)-4-methylquinazolin-2-amine (compound 54, EYP190) [ka] To a solution of compound 53 (EYP193) (10 mg, 0.033 mmol) in MeOH (0.5 mL) was added NaOMe (0.15 mL, 0.075 mmol). The mixture was heated at 80 °C for 6 h. The solvent was then removed under reduced pressure and purified by column chromatography (DCM / MeOH 100:0 to 99:1) to give the desired compound 54 as a white solid. Yield: 61% (6 mg, 0.02 mmol). Melting point: 173.9-174.3 °C. TLC Rf: 0.27 (DCM / MeOH 98:2). 1 H NMR (300MHz, CDCl3) δ 8.41(d, J=5.7Hz, 1H), 8.35(d, J=5.8Hz, 1H), 7.96(s, 1H), 7.86(d, J=9.0Hz, 1H), 7.12 (d, J=2.6Hz, 1H), 7.05(dd, J=9.1, 2.5Hz, 1H), 3.98(s, 3H), 3.97(s, 3H), 2.81(s, 3H). HRMS(ESI)(M+H) + m / z C 15 H 16 Calculated value for N5O2: 298.1304; measured value: 298.1309.

[0144] hN-(1H-indol-3-yl)-7-methoxy-4-methylquinazolin-2-amine (compound 57, EYP165) [ka] To a solution of compound 46 (EYP177) (35 mg, 0.08 mmol) in DCM (2 mL) was added TFA (0.1 mL, 1.3 mmol). The solution was stirred at room temperature for 2 h. After addition of saturated aqueous NaHCO3 and extraction with DCM, the organic layer was dried over MgSO4, filtered and concentrated to dryness. Purification by column chromatography (DCM / MeOH 100:1 to 200:1) afforded the desired compound 57 as a white solid. Yield: 48% (11 mg, 0.04 mmol). White powder. TLC Rf: 0.6 (DCM / MeOH 98:2). 1 H NMR (300MHz, DMSO-d6) δ 10.69(s, 1H), 9.51(s, 1H), 8.17(s, 1H), 8.01(d, J=8.0Hz, 1H), 7.91(d, J=8.9Hz, 1H), 7.34(d, J=8.1Hz, 1H), 7.09(t , J=7.5Hz, 1H), 7.01(d, J=2.4Hz, 1H), 6.96(t, J=7.5Hz, 1H), 6.88(dd, J=9.0, 2.5Hz, 1H), 3.91(s, 3H), 2.73(s, 3H). HRMS(ESI)(M+H) + m / z C 18 H 17 Calculated value for N4O: 305.1402; measured value: 305.1408.

[0145] T cell assay MCA205 and B16F10 mouse cell lines are transfected with the plasmid YFP-globulin-SL8-intron or PCDNA3 deletion plasmid (negative control) using the transfection reagent jetPRIME (Ozyme) or GeneJuice (Millipore), respectively, according to the respective manufacturer's protocol. 24 hours after transfection, cells are treated with various doses of Madrasin (Sigma SML 1409), Madra.HCl (3.HCl) or various compounds cited herein. Cells are washed three times with PBS 1× and incubated at 5.10°C for 24 hours. 4 1.10 tumor cells 5 The cells are co-cultured with 100 B3Z hybridomas in a 96-well plate. In the positive control wells, 4 μg / ml of synthetic peptide SL8 is added. The cells are then incubated overnight at 37° C., 5% CO2. The plates are centrifuged at 1200 rpm for 5 min, the cells are washed twice with PBS 1× and lysed in 0.2% TritonX-100, 0.2% DTT, 0.5 M K2HPO4, 0.5 M KH2PO4 for 5 min at 4° C. with shaking. The lysates are centrifuged at 3000 rpm for 10 min and the supernatants are transferred to 96-well optiplates (OptiPlaque-96, PerkinElmer). Expression buffer containing 10 mM MgCl2, 11.2 mM β-mercaptoethanol, 0.0015% IGEPAL® CA-630 and 40 μM 4-methylumbelliferyl β-D-galactopyranoside (MUG) in PBS is added and the plates are incubated for 3 h at room temperature. Finally, β-galactosidase activity is measured using a FLUOstar OPTIMA (BMG LABTECH Gmbh, Offenburg 30, Germany). Results are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (unpaired Student's t-test).

[0146] Tumor burden and treatment C57Bl / 6J female mice are obtained from Harlan Laboratories Ltd (Switzerland). 5MCA205 sarcoma or B16F10 melanoma cells or 1,5.10 5 MCA205 globulin-SL8-intron or B16F10 globulin-SL8-intron cells are injected subcutaneously into the right flank of 7-week-old mice. Sarcoma cells are injected in 100 μL sterile PBS solution and melanoma cells in 50:50 PBS:Matrigel solution to prevent tumor cell spreading. 4, 7, 10 and 14 days after tumor inoculation, 100 μL Madra.HCl in HO + 30% (2-hydroxypropyl)-β-cyclodextrin (w / v) solution is injected. In a similar manner, EYP59 (compound 7) is injected in 100 μL HO solution 4, 7, 10 and 14 days after tumor inoculation. Tumor area is recorded every 3-4 days until the tumor reaches the ethical endpoint. All animal experiments were performed in accordance with French and European laws and regulations. Results are expressed as mean ± SEM. *p<0.05, **P<0.01, ***P<0.001 (ANOVA with Tukey's multiple comparison test comparing all groups).

[0147] result Madrasin treatment increases antigen presentation of PTP-derived antigens in cancer cells. In a recent study, we showed that PTPs are the major source of peptides for the endogenous MHC class I pathway in vitro. To modulate PTP-derived antigen presentation on the cancer cell surface, we tested the effect of madrasin treatment on mouse tumor cell lines, one melanoma (B16F10) and the other sarcoma (MCA205). Both mouse cell lines transiently expressed the PTP-SL8 epitope derived from an intron in a β-globulin gene construct or remained untransfected. Madrasin induces an increase in PTP-dependent antigen presentation with dose-dependent efficacy in both mouse cell lines (Figure 1A). These results indicate that the production and presentation of PTP-derived antigens can be positively regulated in cancer cell lines after madrasin treatment. They support the hypothesis that this molecule can be used as a positive immunomodulator to enhance specific antitumor immune responses that depend on PTP production and presentation.

[0148] Treatment with the synthetic madrasine hydrochloride (Madra.HCl) increases antigen presentation of PTP-derived antigens in vitro and slows tumor growth in vivo. The above results indicate that madrasin can increase the in vitro production and presentation of PTP-dependent antigens encoded by intronic sequences on the cell surface of treated tumor cell lines. The next question is whether madrasin, which is soluble only in DMSO, can increase tumor growth and CD8 in vivo. + The question was whether madracine would have a similar effect on T cell proliferation. Unfortunately, we could not carry out in vivo experiments with madracine due to its dilute concentration in DMSO. As this first result was promising, we decided to produce a madracine derivative to increase the cancer immune response. In fact, madracine is insoluble in water and can only be dissolved in DMSO solvent, which reduces the efficiency of its pharmacokinetics in mice.

[0149] It was deemed necessary to find these synthetic strategies to enhance the solubility and immunomodulatory activity of madracine and its derivatives in order to make it available for broader in vitro and in vivo validation without the use of toxic carriers or cosolvents (DMSO).

[0150] To achieve this goal, for example, phosphate prodrugs can typically exhibit excellent aqueous solubility, chemical stability, and rapid bioconversion to the parent drug by phosphatases. Phosphate prodrug formation has been applied to increase the aqueous solubility of a variety of molecules, including antineoplastic phenolic natural products and their derivatives, exemplified by combretastatin A-4.

[0151] With a view to testing the novel compounds as positive immunomodulators against tumor cell lines, we decided to first test them in an in vitro assay. As expected, the novel compound, identified herein as "Madra.HCl", could be dissolved in water. After treating both mouse cell lines MCA205 or B16F10, which transiently express the PTP-SL8 epitope from an intron of the β-globulin gene (globulin-intron-SL8), with 5 μM or 10 μM Madra.HCl, we observed an increase in PTP-dependent antigen presentation in a dose-dependent manner (Figure 1B). 1.10 cells, which stably express the SIINFEKL(SL8) epitope from an intron of the β-globulin gene (globulin-intron-SL8), showed a significant increase in PTP-dependent antigen presentation. 5 Mice were subcutaneously inoculated with 1.10 MCA205 sarcoma cells. Four days after this inoculation, the mice were intraperitoneally inoculated with the determined dose of Madra.HCl. Thereafter, 20 mg / kg or 40 mg / kg Madra.HCl was injected every 3 days. During that period, tumor growth was monitored every 2-3 days (Figure 1C). We observed a significant 50% reduction in tumor growth 27 days after inoculation in mice treated with 20 mg / kg Madra.HCl (Figure 1D), and further a 60-70% reduction in tumor growth 27 days after inoculation in mice treated with 40 mg / kg Madra.HCl (Figure 1D). In a similar manner, 1.10 5 Mice were inoculated subcutaneously with non-transfected MCA205 sarcoma cells (Figure 1D). Similar experiments are performed in B16F10 tumor cells expressing the globulin-intron-SL8 construct.

[0152] In a similar way, 1.10 5 Mice were inoculated subcutaneously with non-transfected MCA sarcoma cells. Four days after this inoculation, the mice were inoculated intraperitoneally with the determined dose of Madra.HCl. The same dose was then injected every 3 days. During that period, tumor growth was monitored every 2-3 days (Figure 1E). We observed a significant 40% reduction in tumor growth 21 days after inoculation in mice treated with 20 mg / kg or 40 mg / kg Madra.HCl (Figure 1F).

[0153] Furthermore, treatment with Madra.HCl extended mouse survival; when mice were treated with 40 mg / kg Madra.HCl, approximately 25% survived 120 days after tumor inoculation, and when mice were treated with 20 mg / kg Madra.HCl, approximately 15% survived 120 days after tumor inoculation (Figure 1G).

[0154] Finally, mice inoculated with MCA205 tumor cells expressing the globulin-intron-SL8 construct and that experienced tumor regression following treatment with Madra.HCl as described above were inoculated with MCA205 tumor cells expressing the globulin-intron-SL8 construct in the right flank and B16F10 tumor cells in the left flank 100 days later. B16F10 tumors grew over time in the mice, whereas MCA205 tumor cells did not (Figure 1H). These results indicate that the mice acquired a long-lasting antitumor response specific for MCA205 tumors.

[0155] Madracine derivatives efficiently enhance MHC class I presentation of intron-derived antigens in vitro. In order to improve the immunomodulatory activity of Madra.HCl for broader in vitro and in vivo validation of Madracin without using toxic carriers or cosolvents (DMSO), it was deemed necessary to find a strategy of structural transformation to maintain its activity. The following compounds of the present invention were prepared as described above.

[0156] To test the new compounds as positive immunomodulators against tumor cell lines, we decided to first test them in an in vitro assay. As expected, some Madra.HCl derivatives were soluble in water, while others were insoluble in water. Using the MTT test, the IC 25 and I.C. 50 For the remaining experiments, the IC of each water-soluble or water-insoluble compound was determined (Figure 2). 50We decided to treat both cell lines with β-globulin introns. To this end, both mouse cell lines transiently expressing the PTP-SL8 epitope derived from an intron of the β-globulin gene (globulin-intron-SL8) were treated overnight with each compound. Four distinct observations were made regarding the immunomodulatory activity of each compound.

[0157] First, we found that several compounds showed an increase in PTP-dependent antigen presentation in both mouse tumor cell lines (Figure 3). Increased PTP-SL8 presentation was observed after treatment of mouse cell lines with EYP59 (compound 7) (A), EYP201 (compound 6) (B), EYP165 (compound 57) (C) and EYP281 (compound 32) (D), respectively.

[0158] The second observation was that some compounds were able to positively increase the presentation of PTP-SL8-derived antigens only in MCA cell lines that transiently express the PTP-SL8 epitope derived from an intron in the β-globin gene (Figure 4). Increased PTP-SL8 presentation was observed after treatment of mouse sarcoma cell lines with EYP188 (compound 42) (A) and EYP86 (compound 10) (B), respectively.

[0159] Furthermore, a third observation was that some compounds were able to positively increase the presentation of PTP-derived antigens only in the B16F10 cell line, which transiently expresses the PTP-SL8 epitope derived from an intron in the β-globulin gene (Figure 5). We were able to find an increase in PTP-SL8 presentation after treating mouse melanoma cell lines with EYP174 (compound 41) (A), EYP179 (compound 50) (B), EYP190 (compound 54) (C) and EYP181 (compound 49) (D), respectively.

[0160] And finally, the fourth observation was the failure of several compounds to positively increase the presentation of PTP-derived antigens in both mouse cell lines tested (Figure 6). We were unable to see an increase in PTP-SL8 presentation after treating both mouse cell lines with EYP177 (compound 46) (A), EYP156 (compound 43) (B), EYP113 (compound 11) (C), and EYP102 (compound 9) (D), respectively.

[0161] The madrasine derivative EYP59 (compound 7) efficiently reduces tumor growth in vivo in an immune-dependent manner. 1.10 stably expressing the SIINFEKL (SL8) epitope from an intron of the β-globulin gene (globulin-intron-SL8) 5 Mice were subcutaneously inoculated with MCA205 sarcoma cells. Four days after this inoculation, the mice were intraperitoneally inoculated with the determined dose of EYP59. Then, 20 mg / kg was injected every 3 days. During that period, tumor growth was monitored every 2-3 days (Figure 7A). We observed a significant 85% reduction in tumor growth 23 days after inoculation in mice treated with 20 mg / kg EYP59 (Figure 7B). In a similar manner, 1.10 5 Mice were subcutaneously inoculated with non-transfected MCA205 sarcoma cells. Four days after this inoculation, the mice were intraperitoneally inoculated with the determined dose of EYP59. The same dose was then injected every 3 days. During that period, tumor growth was monitored every 2-3 days. We observed a significant 70% reduction in tumor growth 19 days after inoculation in mice treated with 20 mg / kg EYP59 (Figure 7C).

[0162] To assess the requirement of an immune response for these effects, we tested the effect of 20 mg / kg EYP59 treatment in immunodeficient nu / nu mice in a similar setting as described above and observed no effect on tumor growth (Figure 7D). These results indicate that tumor size reduction by EYP59 treatment requires the presence of an active immune response in vivo.

[0163] The madrasine derivative EYP59 (compound 7) efficiently reduces tumor growth in vivo when injected in an intratumoral or intravenous manner. 1.10 5 Mice were subcutaneously inoculated with wild-type MCA205 sarcoma cells. Four days after this inoculation, a determined dose of EYP59 was injected intratumorally (2.5 mg / kg) or intravenously (5 mg / kg). The same dose was then injected every 3 days using each administration method. During that period, tumor growth was monitored every 2-3 days. We observed a significant 80% reduction in tumor growth 25 days after inoculation in mice treated intravenously with 5 mg / kg EYP59 (Figure 8B).

[0164] Consideration The present invention reveals that certain spliceosome inhibitors have a positive effect on antitumor immune response and therefore on tumor growth. Splicing abnormalities appear as specific features of cancer and are investigated as predictive markers for patient survival and targets for cancer treatment with splicing inhibitors, some of which are currently under development in acute myeloid leukemia. In this invention, we show that some specific madrasin derivatives are potent stimulators of antitumor immune response in vitro in sarcoma and melanoma tumor cell lines. We also show that some madrasin derivatives specifically increase PTP-dependent antigen presentation in sarcoma cancer models, and some other derivatives that are different from them can specifically increase PTP-dependent antigen presentation in melanoma cancer models. By focusing on traditional biomolecular profiles, we disclose a new application method within the framework of targeted molecular therapy.

[0165] The PTP model describes pre-spliced ​​mRNA as a template for PTP by alternative translation events occurring in the nucleus. In a study describing this alternative translation, we showed that forced nuclear retention of mRNA encoding the intronic SL8 peptide leads to an increase in SL8 antigen presentation. Furthermore, pladienolide and splicostatin A (SSA) inhibit splicing by targeting SF3b, a subcomplex of the U2 small nuclear ribonucleoprotein (snRNP) in the spliceosome. Madrasin has been shown to differentially inhibit splicing by inhibiting stable U4 / U6 / U5 tri-snRNP recruitment immediately after U2 snRNP fixation. However, it is also thought to induce pre-mRNA accumulation in the nucleus, leading to increased antigen presentation. The association between pre-mRNA nuclear accumulation and increased antigen presentation is not known. It is hypothesized that pre-mRNA accumulation in the nucleus provides more templates for PTP production, leading to enrichment of SL8-containing PTPs, which are used as the main basis for SL8-directed presentation.

[0166] Moreover, pre-mRNA splicing is an essential mechanism required for the normal function of all mammalian cells. In the last few years, several studies have reported the presence of mutations and overexpression of key spliceosomal factors associated with abnormal splicing activity in various cancers. A few years ago, we also provided some evidence that inhibition of the spliceosome increases MHC class I PTP-dependent antigen presentation. These findings focus on the spliceosome as a potential target in anti-cancer treatment.

[0167] As already mentioned, several small molecules have already been reported that inhibit the spliceosome and specifically inhibit the function of the spliceosomal factor SF3B1. Although the detailed mechanisms of these small molecules are not yet fully understood, they have been reported to be effective in cancer treatment by reducing tumor size by 40-80% depending on the compound used. To date, the only small molecule tested in humans is E7107. It was discontinued due to toxicity issues. This compound is known to inhibit the spliceosome by interacting with SF3B1.

[0168] Moreover, the failure of cytotoxic T lymphocytes to reject tumors may be explained in part by early inappropriate CTL activation by pAPCs. Defined subsets of dendritic cells (DCs) migrate to tumor-draining lymph nodes in the tumor microenvironment (TME), delivering intact antigens encountered in the TME and targeting naive CD8 + It has been described that it can directly or indirectly stimulate T cells. Furthermore, it has been reported that it can stimulate naive CD8 + It has been suggested that tumor-associated PTPs can directly stimulate CD8 T cells. + We showed that SL8-containing PTPs, which are the basis for T cell cross-stimulation, can be mainly transported from tumor cells to DCs by PTP-carrying exosomes. Furthermore, we provided a suggestion that PTPs for endogenous and cross-presentation are produced by the same translation event, and that the two pathways then rapidly diverge. It was shown that PTPs are rare products, and the efficacy of PTP vaccines or exosome-containing PTP vaccines depends on prior PTP enrichment with PTP proteasome inhibitors. We showed that the splicing inhibitor madrasin and its various derivatives enrich the pool of SL8-containing PTPs that serve as the basis for intratumoral DC uptake and cross-presentation, in addition to providing more basis for direct antigen presentation, and that SL8-specific CD8 + It is believed to induce enhanced T cell proliferation.

[0169] Herein, we provide evidence that modulating spliceosome activity with specific madrasin derivative compounds may elicit specific antitumor immune responses against various cancer models. Indeed, madrasin inhibits the formation of the large precatalytic spliceosome B complex from the pre-spliceosomal A complex. We show that by inhibiting spliceosomal formation as early as possible in vitro and in vivo with madrasin derivatives, antitumor antigen presentation is significantly increased and CD8 expression is specifically directed against PTP-dependent epitopes. + It has been shown that these derivatives induced T cell proliferation and the inventors report that these derivatives may be used as chemotherapeutic agents against melanoma and sarcoma.

[0170] References - Apcher, S. et al. Major source of antigenic peptides for the MHC class I pathway is produced during the pioneer round of mRNA translation. Proc. Natl. Acad. Sci. USA 108, 11572-7 (2011) - Burg, SH et al. Vaccines for established cancer: overcoming the challenges posed by immune evasion. Nat. Publ. Gr. 16, 219-233 (2016) - Caron, E. et al. The MHC I immunopeptidome conveys to the cell surface an integrative view of cellular regulation. Mol Syst Biol. 7, 533 (2011) - Duvallet, E., et al. Exosome-driven transfer of tumor-associated Pioneer Translation Products (TA-PTPs) for the MHC class I cross-presentation pathway. Oncoimmunology 5, e1198865 (2016) - Kmieciak, M.,et al. HER-2 / neu antigen loss and relapse of mammary carcinoma are actively induced by T cell-mediated anti-tumor immune responses. Eur. J. Immunol. 37, 675-685 (2007) - Lee, S. & Sin, J. MC32 tumor cells acquire Ag-specific CTL resistance through the loss of CEA in a colon cancer model. Hum Vaccin Immunother 11, 2012-2020 (2015) - Leone, P. et al. MHC class I antigen processing and presenting machinery: organization, function, and defects in tumor cells. J. Natl. Cancer Inst. 105, 1172-87 (2013) - Liu, Y. et al. Expression of antigen processing and presenting molecules in brain metastasis of breast cancer. Cancer Immunol. Immunother. 61, 789-801 (2012) - Mellman, I.,et al. Cancer immunotherapy comes of age. Nature 480, 480-489 (2014) - Watson, N. F. S. et al. Immunosurveillance is active in colorectal cancer as downregulation but not complete loss of MHC class I expression correlates with a poor prognosis. Int. J. Cancer 118, 6-10 (2006) - Yewdell, JW., et al. Defective ribosomal products (DRiPs): a major source of antigenic peptides for MHC class I molecules? J Immunol 157(5):1823-6 (1996)

Claims

1. Formula II, Formula III, Formula IV, Formula V, Formula VI and Formula VII: 5,6-Dimethyl-2-((4-methyl-7-(2-morpholinoethoxy)quinazolin-2-yl)amino)pyrimidin-4(3H)-one 【Chemistry 1】 Sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazolin-7-yl phosphate 【Chemistry 2】 N-(2-Methoxypyrimidin-5-yl)quinazolin-2-amine 【Chemistry 3】 N-(1H-indol-3-yl)-7-methoxy-4-methylquinazolin-2-amine 【Chemistry 4】 ((1S,2S,4R,5S,6S)-5-acetoxy-7,8-diacetyl-4-((2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-7-methoxy-4-methylquinazolin-8-yl)thio)-3,7λ3,8λ3-trioxabicyclo[4.2.0]octan-2-yl)methyl acetate 【Chemistry 5】 7-Methoxy-4-methyl-N-(3,4,5-trimethoxyphenyl)quinazolin-2-amine 【Chemistry 6】 1. A pharmaceutical composition for use in treating cancer, preventing cancer metastasis and / or preventing cancer recurrence in a subject, comprising a compound selected from the group consisting of:

2. Formula II, Formula III, Formula IV, Formula V, Formula VII, Formula VIII, Formula IX, Formula X and Formula XI: 5,6-Dimethyl-2-((4-methyl-7-(2-morpholinoethoxy)quinazolin-2-yl)amino)pyrimidin-4(3H)-one 【Chemistry 7】 Sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazolin-7-yl phosphate 【Chemistry 8】 N-(2-Methoxypyrimidin-5-yl)quinazolin-2-amine 【Chemistry 9】 N-(1H-indol-3-yl)-7-methoxy-4-methylquinazolin-2-amine 【Chemistry 10】 7-Methoxy-4-methyl-N-(3,4,5-trimethoxyphenyl)quinazolin-2-amine 【Chemistry 11】 7-Methoxy-N-(6-methoxypyridin-3-yl)-4-methylquinazolin-2-amine 【Chemistry 12】 N2-(7-methoxy-4-methylquinazolin-2-yl)-N4-methylpyrimidine-2,4-diamine 【Chemistry 13】 7-Methoxy-N-(4-methoxypyrimidin-2-yl)-4-methylquinazolin-2-amine 【Chemistry 14】 7-Methoxy-N-(2-methoxypyrimidin-4-yl)-4-methylquinazolin-2-amine 【Chemistry 15】 1. A pharmaceutical composition for use in treating cancer, preventing cancer metastasis and / or preventing cancer recurrence in a subject, comprising a compound selected from the group consisting of:

3. Formula III: 【Chemistry 16】 13. A pharmaceutical composition comprising a compound of formula (I) for use in treating cancer, preventing cancer metastasis and / or preventing cancer recurrence in a subject, wherein said cancer is blastoma.

4. A pharmaceutical composition according to any one of claims 1 to 3 for use in the treatment of cancer in combination with at least one different anti-cancer agent and / or radiation therapy.

5. 5. The pharmaceutical composition of claim 4, wherein the at least one different anti-cancer agent is selected from a chemotherapeutic agent, an immune checkpoint inhibitor, and an anti-cancer vaccine.

6. Sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazolin-7-yl phosphate (EYP59), ((1S,2S,4R,5S,6S)-5-acetoxy-7,8-diacetyl-4-((2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-7-methoxy-4-methylquinazolin-8-yl)thio)-3,7λ3,8λ3-trioxabicyclo[4.2.0]octan-2-yl)methyl acetate (EYP86), N-(1H-indol-3-yl)-7-methoxy-4-methylquinazolin-2-amine (EYP165), 7-methoxy-N-(6-methoxypyridin-3-yl)-4-methylquinazolin-2-amine (EYP174), 7-methoxy-N-(4-methoxypyrimidin-2-yl)-4-methylquinazolin-2-amine (EYP179), N2-(7-methoxy-4-methylquinazolin-2-yl)-N4-methylpyrimidine-2,4-diamine (EYP181), 7-methoxy 20. A pharmaceutical composition for use in stimulating an anti-cancer immune response in a subject in need of treatment comprising a compound selected from 4-methyl-N-(3,4,5-trimethoxyphenyl)quinazolin-2-amine (EYP188), 5,6-dimethyl-2-((4-methyl-7-(2-morpholinoethoxy)quinazolin-2-yl)amino)pyrimidin-4(3H)-one (EYP201) and N-(2-methoxypyrimidin-5-yl)quinazolin-2-amine (EYP281), wherein the cancer is a sarcoma.

7. Sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazolin-7-yl phosphate (EYP59), N-(1H-indol-3-yl)-7-methoxy-4-methylquinazolin-2-amine (EYP165), 7-methoxy-N-(6-methoxypyridin-3-yl)-4-methylquinazolin-2-amine (EYP174), 7-methoxy-N-(4-methoxypyrimidin-2-yl)-4-methylquinazolin-2-amine (EYP179), N2-(7-methoxy-4-methylquinazolin-2-yl)-N4-methylpyrimidine-2,4-diamine (EYP181), 7-methoxy-4-methyl-N-(3,4,5-trimethoxyphenyl 1. A pharmaceutical composition for use in stimulating an anti-cancer immune response in a subject in need of treatment comprising a compound selected from N-(2-methoxypyrimidin-4-yl)-quinazolin-2-amine (EYP188), 7-methoxy-N-(2-methoxypyrimidin-4-yl)-4-methylquinazolin-2-amine (EYP190), 5,6-dimethyl-2-((4-methyl-7-(2-morpholinoethoxy)quinazolin-2-yl)amino)pyrimidin-4(3H)-one (EYP201) and N-(2-methoxypyrimidin-5-yl)quinazolin-2-amine (EYP281), wherein the cancer is melanoma.

8. A pharmaceutical composition comprising sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazolin-7-yl phosphate (EYP59) for use in stimulating an anti-cancer immune response in a subject in need of treatment, wherein the cancer is blastoma.

9. 4. The pharmaceutical composition according to claim 1, 2 or 3, comprising a pharma- ceutically acceptable carrier together with at least one different anti-cancer agent, used simultaneously, separately or sequentially.

10. Sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazolin-7-yl phosphate (EYP59), ((1S,2S,4R,5S,6S)-5-acetoxy-7,8-diacetyl-4-((2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-7-methoxy-4-methylquinazolin-8-yl)thio)-3,7λ3,8λ3-trioxabicyclo[4.2.0]octan-2-yl)methyl acetate (EYP86), N-(1H-indol-3-yl)-7-methoxy-4-methylquinazolin-2-amine (EYP165), 7-methoxy-N-(6-methoxypyridin-3-yl)-4-methylquinazolin-2-amine (EYP174), 7-methoxy-N-(4-methoxypyrimidin-2-yl)-4-methylquinazolin-2-amine (EYP179), N2-(7-methoxy-4-methylquinazolin-2-yl)-N4-methylpyrimidine-2,4-diamine (EYP181), 7-methoxy-4-methyl-N-(3,4,5-trimethoxyphenyl)quinazolin-2-amine 1. A pharmaceutical composition comprising a compound selected from: 5,6-dimethyl-2-((4-methyl-7-(2-morpholinoethoxy)quinazolin-2-yl)amino)pyrimidin-4(3H)-one (EYP188), 5,6-dimethyl-2-((4-methyl-7-(2-morpholinoethoxy)quinazolin-2-yl)amino)pyrimidin-4(3H)-one (EYP201) and N-(2-methoxypyrimidin-5-yl)quinazolin-2-amine (EYP281), for use in treating cancer, preventing cancer metastasis and / or preventing cancer recurrence in a subject, wherein the cancer is a sarcoma, and the pharmaceutical composition comprises a pharma- ceutical carrier together with at least one different anti-cancer agent used simultaneously, separately or sequentially.

11. Sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazolin-7-yl Phosphate (EYP59), N-(1H-indol-3-yl)-7-methoxy-4-methylquinazolin-2-amine (EYP165), 7-methoxy-N-(6-methoxypyridin-3-yl)-4-methylquinazolin-2-amine (EYP174), 7-methoxy-N-(4-methoxypyrimidin-2-yl)-4-methylquinazolin-2-amine (EYP179), N2-(7-methoxy-4-methylquinazolin-2-yl)-N4-methylpyrimidine-2,4-diamine (EYP181), 7-methoxy-4-methyl-N-(3,4,5-trimethoxyphenyl)quinazolin-2-amine (EYP188), 7-methoxy-N-(2-methoxy 2. A pharmaceutical composition comprising a compound selected from N-(2-methoxypyrimidin-5-yl)quinazolin-2-amine (EYP281), 5,6-dimethyl-2-((4-methyl-7-(2-morpholinoethoxy)quinazolin-2-yl)amino)pyrimidin-4(3H)-one (EYP201) and N-(2-methoxypyrimidin-5-yl)quinazolin-2-amine (EYP281), for use in treating cancer, preventing cancer metastasis and / or preventing cancer recurrence in a subject, wherein the cancer is melanoma, and the pharmaceutical composition comprises a pharma- ceutical acceptable carrier together with at least one different anti-cancer agent used simultaneously, separately or sequentially.

12. 1. A pharmaceutical composition comprising sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazolin-7-yl phosphate (EYP59) for use in treating cancer, preventing cancer metastasis and / or preventing cancer recurrence in a subject, wherein said cancer is blastoma, and said pharmaceutical composition comprises a pharmacologic acceptable carrier together with at least one different anti-cancer agent used simultaneously, separately or sequentially.

13. Sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazolin-7-yl phosphate (EYP59), ((1S,2S,4R,5S,6S)-5-acetoxy-7,8-diacetyl-4-((2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-7-methoxy-4-methylquinazolin-8-yl)thio)-3,7λ3,8λ3-trioxabicyclo[4.2.0]octan-2-yl)methyl acetate (EYP86), N-(1H-indol-3-yl)-7-methoxy-4-methylquinazolin-2-amine (EYP165), 7-methoxy-N-(6-methoxypyridin-3-yl)-4-methylquinazolin-2-amine (EYP174), 7-methoxy-N-(4-methoxypyrimidin-2-yl)-4-methylquinazolin-2-amine (EYP179), N2-(7-methoxy-4-methylquinazolin-2-yl)-N4-methylpyrimidin-2,4- 20. A pharmaceutical composition for use in inducing or increasing presentation of antigens derived from Pioneer Translation Product (PTP) by cancer cells or altering the immune peptidome in a subject comprising a compound selected from N-(2-methoxypyrimidin-5-yl)quinazolin-2-amine (EYP281), 7-methoxy-4-methyl-N-(3,4,5-trimethoxyphenyl)quinazolin-2-amine (EYP188), 5,6-dimethyl-2-((4-methyl-7-(2-morpholinoethoxy)quinazolin-2-yl)amino)pyrimidin-4(3H)-one (EYP201) and N-(2-methoxypyrimidin-5-yl)quinazolin-2-amine (EYP281), wherein the cancer is a sarcoma.

14. Sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazolin-7-yl phosphate (EYP59), N-(1H-indol-3-yl)-7-methoxy-4-methylquinazolin-2-amine (EYP165), 7-methoxy-N-(6-methoxypyridin-3-yl)-4-methylquinazolin-2-amine (EYP174), 7-methoxy-N-(4-methoxypyrimidin-2-yl)-4-methylquinazolin-2-amine (EYP179), N2-(7-methoxy-4-methylquinazolin-2-yl)-N4-methylpyrimidine-2,4-diamine (EYP181), 7-methoxy-4-methyl- 20. A pharmaceutical composition for use in inducing or increasing presentation of antigens derived from Pioneer Translation Product (PTP) by cancer cells or altering the immune peptidome in a subject comprising a compound selected from N-(3,4,5-trimethoxyphenyl)quinazolin-2-amine (EYP188), 7-methoxy-N-(2-methoxypyrimidin-4-yl)-4-methylquinazolin-2-amine (EYP190), 5,6-dimethyl-2-((4-methyl-7-(2-morpholinoethoxy)quinazolin-2-yl)amino)pyrimidin-4(3H)-one (EYP201) and N-(2-methoxypyrimidin-5-yl)quinazolin-2-amine (EYP281), wherein the cancer is melanoma.

15. A pharmaceutical composition comprising sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazolin-7-yl phosphate (EYP59) for use in inducing or increasing presentation of antigens derived from Pioneer Translation Product (PTP) by cancer cells or altering the immunopeptidome in a subject, wherein the cancer is blastoma.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the subject is a mammal, preferably a human.

17. A kit for use in treating sarcoma, comprising the pharmaceutical composition of claim 1, 9 or 10 and at least one different anti-cancer agent in different containers; a kit for use in treating melanoma, comprising the pharmaceutical composition of claim 2, 9 or 11 and at least one different anti-cancer agent in different containers; or a kit for use in treating blastoma, comprising the pharmaceutical composition of claim 3, 9 or 12 and at least one different anti-cancer agent in different containers.

Images