Madecine derivative compounds, compositions and use thereof for treating cancer

Abstract

The present invention relates to the field of medicine, in particular cancer treatment. More specifically, the present invention relates to new compounds that are generally used as drugs. In particular, the present invention relates to the use of these new compounds for improving the presentation, generally the production and presentation, of antigens derived from prior translation products (PTPs) by cells, in particular cancer cells, or for changing the immunopeptidase group in a subject and inducing or stimulating an immune response in the subject. The present disclosure also relates to the use of such compounds, in particular for the preparation of pharmaceutical compositions and / or for allowing or improving the efficiency of therapy in subjects in need. The present invention also discloses methods for treating diseases, in particular cancer, preventing or treating cancer metastasis and / or cancer recurrence in a subject. In addition, the present invention also provides a kit suitable for preparing a composition according to the present invention and / or for performing the methods described herein.

Description

Technical Field

[0001] The present disclosure relates generally to the field of medicine, in particular to the field of cancer treatment. More specifically, the present invention relates to novel compounds that are derivatives of Madrasin and are each generally useful as a medicament. In particular, the present invention relates to the use of these novel compounds to enhance the presentation, generally the production and presentation, of (antigenic) peptides, preferably antigens derived from prior translation products (PTPs), by cells, particularly cancer cells, or to alter the immunopeptide group in a subject and to induce or stimulate an immune response in the subject. The immune response is generally directed against a tumor antigen, more generally against a cancerous tumor that the subject is suffering from.

[0002] The present disclosure also relates to the use of such compounds, in particular for the preparation of pharmaceutical compositions and / or to allow or improve the efficiency of therapy, in particular cancer therapy, in a subject in need thereof. In fact, each of the compounds of the present invention can be advantageously combined with at least one different anticancer agent, generally a chemotherapeutic agent and / or with 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 treating a disease, particularly cancer, in a subject and for preventing or treating cancer metastasis and / or cancer recurrence in a subject. In addition, the present invention also provides a kit suitable for preparing a composition according to the present invention and / or for performing the method described herein. Background Art

[0004] All nucleated cells present antigenic peptides (APs) on their surface via the class I major histocompatibility complex (MHC-I) pathway. APs are 8 to 10 amino acids long and reflect intrinsic cellular activity (Caron et al.). Due to their presentation, they direct immune cells, primarily cytotoxic CD8 + T cells (CTL) and CD4 +T helper cells monitor potentially dangerous elements, so APs are the targets of therapeutic anti-cancer vaccines currently under development. Despite the optimistic outlook, the results of clinical trials using therapeutic vaccines targeting tumor-associated antigens (TAAs) have not met expectations. The main failures are related to immunosuppressive mechanisms and suboptimal selection of antigens (Mellman et al.; Burg et al.). One of the important events that drives tumor immune selection and is associated with poor prognosis is the loss or downregulation of class I MHC antigen presentation by tumor cells (Watson et al.; Liu et al.). Due to defects in components of the class I MHC pathway, the tumor cells can avoid recognition by CTLs and natural killer cells (Leone et al.). With the overall decline in class I MHC antigen presentation, the nature of the antigen presented on the cell surface, the class I MHC immune peptide group (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 by immunotherapy, the loss of expression of this TAA on the surface of tumor cells can lead to immune escape (Lee et al.; Kmieciak et al.). To counteract this, current strategies aim to expand the range of cancer peptides that are targeted and restore MHC antigen presentation.

[0005] In order to understand the dynamics of MIP, one can focus on the source of AP for class I MHC presentation pathway. Initially, endogenous AP was thought to be strictly derived from the degradation of senescent proteins. However, models proposing alternative sources challenged this concept. In 1996, the research group of J. Yewdell introduced the concept of defective ribosomal products (DRIP) (Yewdell et al.), which were initially described as products that were rapidly degraded due to their unstable conformation. More recently, the inventors have explored this concept from a different perspective, showing that the main source of AP is derived from a pre-translation event, which occurs before the intron is spliced ​​off and is independent of the translation event of the full-length protein (Apcher et al.). Therefore, the non-canonical peptides produced can be derived from intron sequences, 3' or 5' UTR regions, and alternative reading frames. These polypeptides are called pre-translation products (PTPs). The discovery of PTPs indicates the existence of a complex nuclear translation mechanism that is partly intended to shape MIPs by producing relevant and suitable polypeptides for the class I MHC pathway. In addition, PTPs appear to play a role in the dynamics of cancer development. When inoculated in mice, it has been shown that cancer cells presenting PTP-derived antigens on their surface can be recognized by specific T cells, resulting in reduced tumor growth. In addition, purified PTPs containing model epitopes are highly effective in promoting anti-cancer immune responses when injected into mice as peptide vaccines (Duvallet et al.).

[0006] In the nucleus, precursor-mRNA (pre-mRNA) splicing is catalyzed by the spliceosome, a conserved and dynamic multiprotein complex composed of five small nuclear RNAs (snRNAs) U1, U2, U4, U5 and U6 complexed with more than 200 proteins. An increasing number of studies have reported that in many cancers, dysregulation of the spliceosome complex is bound to lead to abnormal splicing patterns, resulting in 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, the first international conference at Paris-Saclay University, April 2018) taught that the spliceosome is a druggable target for epitope-based immunotherapy. WO2017 / 165495 describes the combination of PIM kinase inhibitors with 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. In recent years, several microbial natural products that inhibit the spliceosome and their synthetic analogs have been reported, including pladienolide B and D, spliceostatin A, FR901464, E7107, isoginkgetin and madexin. The inventors tested some compounds in each class for their potential effects as regulators of antigen presentation.

[0008] In this article, they show that Madecine and some of their derivatives can be used as positive immunomodulators against cancer. They studied the antigen presentation of PTP-derived antigen models expressed in mouse cancer cell lines, and observed that in vitro treatment with Madecine and different derivatives described herein increased the presentation of the antigen. In addition, they also showed that in vivo treatment of mice with sarcomas with Madecine hydrochloride (also referred to herein as "Madra.HCl", EYP34 or compounds of Formula I) dissolved in water slowed tumor growth in an immune-dependent manner. In order to improve its effect, they tested different Madecine derivatives that were soluble or insoluble in water, and observed that some derivatives were more effective inhibitors of mouse sarcoma growth compared to Madecine itself. Since the natural products and derivatives had no effect on tumor growth in immunodeficient Nu / Nu mice, they concluded that their effects depended on the immune response. These results show that antigen presentation from PTP sources can be regulated by treatment, and inhibition of splicing can enhance anticancer responses. The inventors describe herein another mechanism of action of splicing inhibitors, which can be used as positive immunomodulators to enhance anti-tumor immune responses.

[0009] The inventors now describe herein novel compounds which are useful for treating a disease, in particular treating cancer, preventing cancer metastasis and / or preventing cancer recurrence in a subject. Summary of the invention

[0010] The present inventors have produced the following specific compounds derived from Madecine and described their structural formulas for the first time herein:

[0011] -Madr.HCl (also referred to herein as "Madr.HCl", "EYP34" or "compound of formula I")

[0012]

[0013] -5,6-dimethyl-2-((4-methyl-7-(2-morpholinoethoxy)quinazolin-2-yl)amino)pyrimidin-4(3H)-one (also referred to herein as "EYP201", "Compound 6" or "Compound of Formula II")

[0014]

[0015] -Sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazolin-7-yl phosphate (also referred to herein as "EYP59," "Compound 7," or "Compound of Formula III")

[0016] Wherein "Na" represents "sodium", and the radical of formula -ONa represents "sodium hydroxide",

[0017] -N-(2-methoxypyrimidin-5-yl)quinazolin-2-amine (also referred to herein as "EYP281," "Compound 32," or "Compound of Formula IV")

[0018]

[0019] -N-(1H-indol-3-yl)-7-methoxy-4-methylquinazolin-2-amine (also referred to herein as "EYP165", "Compound 57" or "Compound of Formula V")

[0020]

[0021] -((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 referred to herein as "EYP86", "Compound 10" or "Compound of Formula VI")

[0022] The atomic group of the formula -OAc represents an "acetoxy group" having a structure of CH3-C(=O)-O-,

[0023] -7-methoxy-4-methyl-N-(3,4,5-trimethoxyphenyl)quinazolin-2-amine (also referred to herein as "EYP188," "Compound 42," or "Compound of Formula VII")

[0024]

[0025] -7-methoxy-N-(6-methoxypyridin-3-yl)-4-methylquinazolin-2-amine (also referred to herein as "EYP174," "Compound 41," or "Compound of Formula VIII")

[0026]

[0027] -N2-(7-methoxy-4-methylquinazolin-2-yl)-N4-methylpyrimidine-2,4-diamine (also referred to herein as "EYP181", "Compound 49" or "Compound of Formula IX")

[0028]

[0029] -7-methoxy-N-(4-methoxypyrimidin-2-yl)-4-methylquinazolin-2-amine (also referred to herein as "EYP179," "Compound 50," or "Compound of Formula X")

[0030]

[0031] -7-methoxy-N-(2-methoxypyrimidin-4-yl)-4-methylquinazolin-2-amine (also referred to herein as "EYP190," "Compound 54," or "Compound of Formula XI")

[0032]

[0033] Each of these compounds and any combination thereof is described herein for use as a medicament.

[0034] Any of these compounds and their pharmaceutically acceptable salts can be advantageously used as a medicament.

[0035] In the preferred cases described herein, any one of the compounds of formula (I) to (XI) described above or a pharmaceutically acceptable salt thereof or any combination thereof is used for treating a disease in a subject, in particular for treating cancer, for preventing cancer metastasis and / or for preventing cancer recurrence.

[0036] Also described are the in vivo, in vitro or ex vivo uses of compounds having a structural formula selected from formula (I) to (XI) for inducing or increasing the presentation, typically the production and presentation, of (antigenic) peptides, preferably antigens derived from prior translation products (PTPs), by cells, particularly cancer cells, in a subject, or for altering the immunopeptidome.

[0037] A compound having a structural formula selected from formula (I) to (XI) allows a physician to prevent or control, preferably reduce, cancer cell proliferation by stimulating the subject's immune system. In addition, in favorable cases, it can also improve the effectiveness of other cancer treatments. The inventors herein have demonstrated that such a compound can additionally reduce the risk of cancer metastasis and / or cancer recurrence.

[0038] Also described herein is a composition comprising such a compound having a structural formula selected from formula (I) to (XI) and a pharmaceutically acceptable carrier, preferably used simultaneously, separately or sequentially with at least one different anticancer agent. Such a compound is generally used to treat a disease, particularly cancer, prevent cancer metastasis and / or prevent cancer recurrence in a subject.

[0039] Also described herein is a method of treating a disease, particularly cancer, in a subject, comprising the step of administering to the subject a compound described herein, typically a compound or composition having a structural formula selected from Formula (I) to (XI).

[0040] Also described herein is a kit comprising a compound having a structural formula selected from formula (I) to (XI) and preferably at least one different anticancer agent in different containers, and the use of the kit, in particular for preparing the composition described herein. DETAILED DESCRIPTION OF THE INVENTION

[0042] The inventors have produced the madecine derivatives which are described for the first time in the context of the present invention and which are more precisely identified as compounds of the following formulae (I) to (XI):

[0043] -Madr.HCl (also referred to herein as "Madr.HCl", "EYP34" or "Compound of Formula I")

[0044]

[0045] -5,6-dimethyl-2-((4-methyl-7-(2-morpholinoethoxy)quinazolin-2-yl)amino)pyrimidin-4(3H)-one (also referred to herein as "EYP201", "Compound 6" or "Compound of Formula II")

[0046]

[0047] -Sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazolin-7-yl phosphate (also referred to herein as "EYP59," "Compound 7," or "Compound of Formula III")

[0048] Wherein "Na" represents "sodium", and the radical of formula -ONa represents "sodium hydroxide",

[0049] -N-(2-methoxypyrimidin-5-yl)quinazolin-2-amine (also referred to herein as "EYP281," "Compound 32," or "Compound of Formula IV")

[0050]

[0051] -N-(1H-indol-3-yl)-7-methoxy-4-methylquinazolin-2-amine (also referred to herein as "EYP165", "Compound 57" or "Compound of Formula V")

[0052]

[0053] -((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 referred to herein as "EYP86", "Compound 10" or "Compound of Formula VI")

[0054] The atomic group of the formula -OAc represents an "acetoxy group" having a structure of CH3-C(=O)-O-,

[0055] -7-methoxy-4-methyl-N-(3,4,5-trimethoxyphenyl)quinazolin-2-amine (also referred to herein as "EYP188," "Compound 42," or "Compound of Formula VII")

[0056]

[0057] -7-methoxy-N-(6-methoxypyridin-3-yl)-4-methylquinazolin-2-amine (also referred to herein as "EYP174," "Compound 41," or "Compound of Formula VIII")

[0058]

[0059] -N2-(7-methoxy-4-methylquinazolin-2-yl)-N4-methylpyrimidine-2,4-diamine (also referred to herein as "EYP181", "Compound 49" or "Compound of Formula IX")

[0060]

[0061] -7-methoxy-N-(4-methoxypyrimidin-2-yl)-4-methylquinazolin-2-amine (also referred to herein as "EYP179," "Compound 50," or "Compound of Formula X")

[0062]

[0063] -7-methoxy-N-(2-methoxypyrimidin-4-yl)-4-methylquinazolin-2-amine (also referred to herein as "EYP190," "Compound 54," or "Compound of Formula XI")

[0064]

[0065] In a specific case, the inventors describe herein a compound of formula (A):

[0066]

[0067] in:

[0068] R 1 and R 2 independently selected from H, CH4 (methyl) and Cl (chloro),

[0069] R 3 is H, CH4 (methyl), C2H6 (ethyl), n-propyl, N(CH2)4 or N(CH2)5, Z is CH2 or C=O,

[0070] n is 1, 2, or 3, and

[0071] X is O, N or CH.

[0072] In a specific and preferred case, the compound of formula (A) is a compound of formula II (also referred to as "EYP201" or "Compound 6").

[0073] In another specific aspect, the inventors describe herein a compound of formula (B):

[0074]

[0075] in:

[0076] R 1 and R 2 are independently selected from H, CH4 (methyl), Cl (chloro), and thiosaccharides, and

[0077] R 3 is P(O)(ONa)2 or

[0078] in:

[0079] R 4 is H, CH4(methyl), C2H6(ethyl), n-propyl, N(CH2)4 or N(CH2)5,

[0080] Z is CH2 or C=O,

[0081] n is 1, 2, or 3, and

[0082] X is O, N or CH.

[0083] The aryl group may be, for example, 3,4,5-trimethoxyphenyl, 2-chloro-3,4,5-trimethoxyphenyl, 3,5-trimethoxyphenyl or 2-chloro-3,5-trimethoxyphenyl.

[0084] The heteroaryl group may 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.

[0085] In certain cases, 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).

[0086] Any of the compounds of formulae (A) and (B) or (I) to (XI) described above, and pharmaceutically acceptable salts thereof, can be advantageously used as medicaments.

[0087] As used herein, the term "pharmaceutically acceptable" refers to compositions, compounds, salts, etc., which are suitable for contact with the tissues of a subject within the scope of sound medical judgment, or can be administered to a subject without excessive toxicity or other complications, with a reasonable benefit / risk ratio. For example, pharmaceutically acceptable salts include sodium salts, potassium salts, chlorides, ammonium salts, acetates, etc.

[0088] The inventors have demonstrated herein that compounds of formula (A), (B) and (I) to (XI) can be advantageously used as positive immunomodulators, in particular against cancer.

[0089] The inventors have studied the antigen presentation of a model PTP-derived antigen expressed in a mouse cancer cell line and observed that their in vitro treatment with madecine, more preferably a compound having a structural formula selected from formula (A) and (B) or (I) to (XI), increased the presentation of this antigen. Furthermore, they showed that in vivo treatment of mice bearing sarcomas with madecine dissolved in DMSO slowed tumor growth in an immune-dependent manner. In order to improve their effect, they tested water-soluble madecine derivatives described herein and surprisingly observed that they were much more effective inhibitors of cancer growth than madecine itself. Since the natural product and derivatives had no effect on tumor growth in immunodeficient Nu / Nu mice, they concluded that their effect was dependent on the immune response. These results confirm that PTP-derived antigen presentation can be modulated and that the inventors provide new promising molecules that can be used for market development, which, in contrast to other derivatives of madecine, can be used to enhance, in particular, anti-cancer responses and treat cancer.

[0090] In the preferred cases described herein, the compound having a structural formula selected from formula (A) and (B) or (I) to (XI), in particular a compound of formula (I), (II), (III), (V), (V), (VI), (VII), (VIII), (IX), (X) or (XI) (also referred to herein as "compound of interest"), is used for treating a disease, in particular cancer, in a subject, for preventing cancer metastasis and / or for preventing cancer recurrence.

[0091] In another preferred aspect described herein, the compound having a structural formula selected from formula (A) and (B) or (I) to (XI) is used to stimulate an anti-cancer immune response in a subject in need thereof.

[0092] In another preferred embodiment, the compound having a structural formula selected from formula (A) and (B) or (I) to (XI) is used to induce or increase the presentation, generally the production and presentation, of a prior translation product (PTP)-derived antigen by cancer cells in a subject, or to alter the immune peptide group.

[0093] The compounds of the present invention can be obtained by methods known to the skilled person, such as semi-synthesis or total synthesis. Examples of methods for producing compounds of interest are described herein in the experimental section and in Fig. 9 and 10 The compounds of formulae (A), (B) and (I) to (XI) are artificial products which are not found in nature per se.

[0094] Compounds of formula (A), (I) to (III) and (VI) can generally be prepared from madecins which have been described as permeabilizing inhibitors of pre-mRNA splicing. Compounds of formula (IV), (V), (VII) to (XI) can be prepared by chemical synthesis using conventional chemical reactions.

[0095] Another object of the present invention is the use of a compound having a structural formula selected from formula (A) and (B) or (I) to (XI) (or a pharmaceutically acceptable salt thereof) for reducing the resistance of cancer or a subject suffering from cancer to different anticancer agents, usually different chemotherapeutic agents.

[0096] Also described herein are compounds having a structural formula selected from formula (A) and (B) or (I) to (XI) (or a pharmaceutically acceptable salt thereof) according to the present invention, or compositions comprising such compounds and a pharmaceutically acceptable carrier, in combination with at least one different anticancer agent, typically a different chemotherapeutic drug and / or with radiotherapy, for use in treating a disease, in particular treating cancer, preventing cancer metastasis and / or preventing cancer recurrence in a subject.

[0097] The term "subject" refers to any object, preferably a mammal.

[0098] Examples of mammals include humans and non-human animals, such as, but not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), non-human primates (e.g., monkeys), rabbits, and rodents (e.g., mice and rats). The treatment is preferably for humans in need, regardless of age or sex.

[0099] The term "subject" generally refers to a patient, particularly a patient having a tumor. Unless otherwise indicated in the present disclosure, a tumor is a cancerous or malignant tumor. In certain instances, the subject is a subject undergoing cancer treatment, such as chemotherapy and / or radiotherapy, or a subject who has or is suspected of being at risk of developing cancer.

[0100] The subject is, for example, a human who has cancer and is resistant to cancer treatment, typically chemotherapy.

[0101] The subject may have been exposed to a portion of a complete conventional treatment regimen, such as to at least one cycle of the complete treatment regimen, such as to two cycles of the complete treatment regimen.

[0102] The cancer or tumor may be any type of cancer or neoplasm. The tumor is typically a solid tumor, in particular a solid tumor of epithelial, neuroectodermal or mesenchymal origin. The cancer is typically also selected from epithelial cancer, sarcoma, lymphoma, germ cell tumor, blastoma, leukemia and multiple myeloma, preferably selected from epithelial cancer, sarcoma, blastoma, lymphoma, leukemia and multiple myeloma. The cancer may or may not be a metastatic cancer.

[0103] The cancer may be, for example, selected from, but 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, stomach cancer, bladder cancer, malignant hepatoma, breast cancer, colon cancer, head and neck cancer, gastric cancer, germ cell tumors, pediatric sarcoma, 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.

[0104] In a preferred embodiment, the cancer is selected from the group consisting of lung cancer, breast cancer, genitourinary tract 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).

[0105] In certain embodiments, the cancer is a sarcoma.

[0106] 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.

[0107] Even more preferably, the cancer is melanoma or lung cancer.

[0108] In one instance, the cancer is lung cancer, typically small cell lung cancer or non-small cell lung cancer.

[0109] In another instance, the cancer is a leukemia, typically acute myeloid leukemia (AML) or chronic lymphocytic leukemia.

[0110] In another embodiment, the cancer is colon cancer, typically colon epithelial cancer. The cancer can also be colorectal cancer.

[0111] In another instance, the cancer is a pediatric cancer, typically a pediatric sarcoma, lymphoma, leukemia, neuroblastoma, brain cancer, or a central nervous system cancer.

[0112] In the specific cases described herein, the anti-cancer agent is selected from a chemotherapeutic agent, an immune checkpoint blocker, and an anti-cancer vaccine (also referred to herein as a "cancer vaccine"). These agents are generally considered to be "conventional" agents for treating cancer.

[0113] The chemotherapeutic agent is typically an agent selected from, for example, anti-tumor / cytotoxic antibiotics, alkylating agents, antimetabolites, topoisomerase inhibitors, mitotic inhibitors, platinum-based compositions, specific kinase inhibitors, hormones, cytokines, anti-angiogenic agents, antibodies, DNA methyltransferase inhibitors, and vascular disrupting agents.

[0114] The anti-tumor agent or cytotoxic antibiotic may, for example, be selected from anthracyclines (eg, doxorubicin, daunomycin, adriamycin, idarubicin, epirubicin, mitoxantrone, valrubicin), actinomycin, bleomycin, mitomycin C, plicamycin and hydroxyurea.

[0115] The alkylating agent can be, for example, selected from nitrogen mustard, cyclophosphamide, melphalan, chlorambucil, ifosfamide, temozolomide busulfan, N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, streptozotocin, dacarbazine, mitozolamide, thiotepa, tadalafil, azepine (AZQ), procarbazine, hexamethylmelamine and uramustine.

[0116] The antimetabolite can be, for example, selected from pyrimidine analogs (e.g., fluoropyrimidine analogs, 5-fluorouracil (5-FU), floxuridine (FUDR), cytosine arabinoside (cytarabine), gemcitabine capecitabine), purine analogs (e.g., azathioprine, mercaptopurine, thioguanine, fludarabine, pentostatin, cladribine, clofarabine), folic acid analogs (e.g., methotrexate, folic acid, pemetrexed, aminopterin, raltitrexed, trimethoprim, pyrimethamine).

[0117] The topoisomerase inhibitor may, for example, be selected from camptothecin, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate and teniposide.

[0118] The mitotic inhibitor may be selected, for example, from the group consisting of taxanes [paclitaxel (PG-paclitaxel and DHA-paclitaxel) Docetaxel larotaxel, cabazitaxel, ortataxel, tesetaxel, or taxoprexin], vinca alkaloids or vincristine (e.g., vincristine, vinblastine, vinorelbine, vindesine, or vinflunine), mebendazole, and colchicine.

[0119] The platinum-based component may, for example, be selected from platinum, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin and triplatinium tetranitrate.

[0120] The specific kinase inhibitor may, for example, be selected from BRAF kinase inhibitors such as vemurafenib, MAPK inhibitors (eg dabrafenib), MEK inhibitors (eg trametinib) and tyrosine kinase inhibitors such as imatinib, gefitinib, erlotinib, sunitinib or cabozantinib.

[0121] Tamoxifen, an anti-aromatase or anti-estrogen drug, is also commonly used in the context of hormone therapy.

[0122] Cytokines that can be used in the context of immunotherapy can, for example, be selected from IL-2 (interleukin-2), IL-11 (interleukin-11), IFN (interferon) alpha (IFNa) and granulocyte-macrophage colony stimulating factor (GM-CSF).

[0123] The anti-angiogenic agent may, for example, be selected from bevacizumab, sorafenib, sunitinib, pazopanib and everolimus.

[0124] The antibody, in particular a monoclonal antibody (mAb), can be selected from an anti-CD20 antibody (anti-all B-cell antigen antibody), an anti-Her2 / Neu (human epidermal growth factor receptor-2 / NEU) antibody, antibodies targeting the surface of cancer cells (e.g., rituximab and alemtuzumab), antibodies targeting growth factors (e.g., bevacizumab, cetuximab, panitumumab and trastuzumab), agonistic antibodies (e.g., anti-ICOS mAb, anti-OX40 mAb, anti-41BB mAb) and immunoconjugates (e.g., 90Y-ibritumomab tiuxetan, 131I-tositumomab or trastuzumab emtansine).

[0125] The DNA methyltransferase inhibitor may, for example, be selected from 2'-deoxy-5-azacytidine (DAC), 5-azacytidine, 5-aza-2'-deoxycytidine, 1-[β]-D-arabinofuranosyl-5-azacytosine and dihydro-5-azacytidine.

[0126] The vascular disrupting agent may, for example, be selected from the group consisting of flavone acetic acid derivatives, 5,6-dimethylxanthone-4-acetic acid (DMXAA) and flavone acetic acid (FAA).

[0127] Other chemotherapeutic drugs include proteasome inhibitors (eg, bortezomib), DNA strand-breaking compounds (eg, tirapazamine), inhibitors of both thioredoxin reductase and ribonucleotide reductase (eg, xcytrin), and enhancers of the Th1 immune response (eg, thymalfasin).

[0128] In a preferred embodiment, the chemotherapeutic drug or agent is selected from anti-tumor / cytotoxic antibiotics, alkylating agents, antimetabolites, topoisomerase inhibitors, mitotic inhibitors, platinum-based compositions, specific kinase inhibitors, anti-angiogenic agents, antibodies and DNA methyltransferase inhibitors.

[0129] Immune checkpoint blockers are usually antibodies that target immune checkpoints. Such immune checkpoint blockers can be selected from anti-CTLA4 antibodies (ipilimumab and tremelimumab), anti-PD-1 antibodies (nivolumab and pembrolizumab), anti-PD-L1 antibodies (atezolizumab, duruvalumab and avelumab), anti-PD-L2 antibodies and anti-Tim3 antibodies in favorable situations.

[0130] The cancer vaccine may for example be selected from a vaccine composition comprising (antigenic) peptides, in particular PTPs, a human papillomavirus (HPV) vaccine (e.g. and ), a vaccine that stimulates an immune response to prostatic acid phosphatase (PAP) Oncolytic viruses, and talimogene laherparepvec (T-VEC or ).

[0131] In another specific aspect, the ("conventional") cancer treatment is radiation (also referred to herein as "radiotherapy"). The radiotherapy typically involves radiation selected from X-rays ("XR"), gamma rays and / or UVC rays.

[0132] The treatment, which may include several anti-cancer agents, is selected by an oncologist depending on the specific cancer to be prevented or treated.

[0133] Specific melanomas are melanomas conventionally treated with ipilimumab, nivolumab, pembrolizumab, IFNα, dacarbazine, BRAF inhibitors, dabrafenib, trametinib, sorafenib, temozolomide, electrochemotherapy, TNFα, and / or fotemustine.

[0134] In a specific embodiment, the melanoma is a melanoma that is resistant to the above-mentioned cytotoxic conventional therapies.

[0135] Particular breast cancer is breast cancer that has been routinely treated with anthracyclines, taxanes, trastuzumab, anti-PARP (poly ADP-ribose polymerase) antibodies, anti-PI3K (phosphoinositide 3-kinase) antibodies, mTOR (mammalian target of rapamycin) inhibitors, vinorelbine, gemcitabine, anti-estrogen and / or anti-aromatase agents, either before or after a surgical procedure to remove a breast tumor, preferably before such a surgical procedure.

[0136] In a specific embodiment, the breast cancer is a breast cancer that is resistant to the above conventional therapies.

[0137] A specific lung cancer is one that is conventionally treated with XR and either platinum or pemetrexed.

[0138] Specific early stage NSCLC is NSCLC that is conventionally treated with paclitaxel, docetaxel, gemcitabine, vinorelbine, etoposide, taxanes, Avastin [anti-VEGF (vascular endothelial growth factor) antibody], erlotinib and / or gefitinib. In a specific embodiment, the lung cancer is resistant to conventional therapy.

[0139] Certain colon cancers, also known as colorectal or intestinal cancers, are routinely treated with antimetabolites (5-FU, raltitrexed), platinum-based compositions (oxaliplatin), topoisomerase inhibitors (irinotecan), antibodies targeting growth factors (bevacizumab, cetuximab, panitumumab) following surgical procedures to remove breast tumors.

[0140] The present disclosure also relates to the use of a compound having a structural formula selected from formula (A) and (B) or (I) to (XI) (or a pharmaceutically acceptable salt thereof) according to the present invention for preparing a pharmaceutical composition or a drug, wherein the composition is capable of treating a disease, in particular cancer, in a subject in need thereof by stimulating the subject's immune system, or improving the efficiency of the therapy.

[0141] In certain instances, the compound is selected from compounds of Formula (A), (B), II, III, IV, V, VI and VII, and the cancer is a sarcoma.

[0142] In another specific instance, the compound is selected from compounds of Formula II, III, IV, V, VIII, IX, X and XI, and the epithelial cancer is melanoma.

[0143] In particular, the compounds of the present invention can be advantageously combined with at least one different anticancer agent or any other therapeutically active compound described previously and / or with radiotherapy for treating a disease, in particular treating cancer, preventing cancer metastasis and / or preventing cancer recurrence in a subject.

[0144] Therefore, also described herein is a composition comprising a compound having a structural formula selected from formula (A) and (B) or (I) to (XI) and a pharmaceutically acceptable carrier, typically as a combined preparation, and preferably together with at least one different therapeutic agent, especially an anticancer agent, for simultaneous, separate or sequential use in the treatment of a disease, especially cancer.

[0145] Also described herein are (i) a method of treating a disease, particularly cancer, (ii) a method of increasing the sensitivity of a disease, particularly cancer, to a therapeutic / anti-cancer agent, and (iii) a method of reducing the resistance of a disease / cancer to a therapeutic / anti-cancer agent, each of which comprises administering to a subject in need thereof an effective amount, typically a therapeutically effective amount, of at least one compound or pharmaceutical composition having a structural formula selected from formula (A) and (B) or (I) to (XI) as defined above, preferably together with another therapeutic / anti-cancer agent conventionally used to treat the disease / cancer as described herein (as a combined preparation).

[0146] In another specific aspect, the method further comprises administering an effective amount of another therapeutically active compound for treating the disease / cancer or for preventing or treating a side effect of the disease / cancer treatment.

[0147] As used herein, "treatment" refers to therapeutic intervention that attempts to alter the natural course of the subject being treated, and is generally performed for the purpose of cure. Desirable therapeutic effects include, but are not limited to, prevention of recurrence of the disease, alleviation of symptoms and alleviation of any direct or indirect pathological consequences of the disease, reduction in the rate of progression of the disease, alleviation or alleviation of the disease state, and alleviation or improvement of prognosis. In preferred embodiments, the compositions and methods of the present invention are used to delay the onset of cancer or slow the progression of cancer, typically tumor growth.

[0148] Typically, the treatment will induce a therapeutic response in the subject's immune system, typically CD4 + and / or CD8 + T cell response.

[0149] Inducing T cell response generally means inducing T cell response to a certain antigen in this article.Before the induction, the T cell response does not exist or is below the detection level or does not work.Enhancing T cell response means in this article that the overall effect of the T cell for a certain antigen is higher and / or more effective than the overall effect of the T cell before the enhancement.For example, after the enhancement, more T cells for the antigen can be produced.As a result, the effect of the T cell produced additionally increases the overall effect for the antigen.Alternatively, the enhancement can include an increase in the effect of the T cell for the antigen.The T cell may, for example, react more strongly and / or faster with the antigen.Certainly, the result of the enhancement can be the generation of additional T cells and the increase of the T cell effect.Alternatively, the enhancement can only include the generation of additional T cells or only include the increase of T cell effect.

[0150] 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 cancer / tumor cell or tissue, comprising injecting into the subject, typically an effective amount of a compound according to the invention having a structural formula selected from formula (A) and (B) or (I) to (XI) or a composition according to the invention comprising such a compound.

[0151] Detection of a therapeutic immune response can be readily determined by one skilled in the art using techniques such as ELISA, ELISPOT, delayed-type hypersensitivity, intracellular cytokine staining, and / or extracellular cytokine staining.

[0152] As used herein, "effective amount or dose" or "therapeutically effective amount or dose" refers to the amount of the compound of the present invention that removes, slows down a disease, particularly cancer, or alleviates or delays one or more symptoms or disorders caused by or associated with the disease, or induces a measurable immune response in a subject, preferably a human. The effective amount of the compound of the present invention and its pharmaceutical composition, more generally the dosage regimen, can be determined and varied by those skilled in the art. The effective dose can be determined using conventional techniques and by observing the results obtained under similar circumstances. The therapeutically effective dose of the compound of the present invention varies with the disease to be treated, its severity, the route of administration, any combined therapy involved, the patient's age, weight, overall medical condition, medical history, etc.

[0153] Typically, for human patients, the amount of the compound to be administered to the patient can be in the range of about 0.01 mg / kg to 500 mg / kg body weight. In a specific embodiment, the pharmaceutical composition according to the present invention comprises 0.1 mg / kg to 100 mg / kg, for example 0.5 mg / kg to 10 mg / kg of the compound of the present invention.

[0154] In certain cases, the compound of interest of the present invention can be administered to the object by parenteral route, oral route or intravenous (IV), intratumoral (IT) or intraperitoneal (IP) injection. The compound of the present invention can be administered to the object every day (once a day) for consecutive days, such as consecutive 2 to 10 days, preferably consecutive 3 to 6 days. The treatment can be repeated during 1, 2, 3, 4, 5, 6 or 7 weeks or every 2 or 3 weeks or every 1, 2 or 3 months. Alternatively, several treatment cycles such as 1, 2, 3, 4 or 5 weeks can be performed, optionally with an interruption period between two treatment cycles. Any of the compounds of the present invention described herein can be administered, for example, as a single agent once a week, once every two weeks or once a month. The treatment can be repeated once or several times a year.

[0155] The medicament can be administered at appropriate intervals determined by a skilled person. The amount selected depends on a variety of factors, including the route of administration, duration of administration, time of administration, elimination rate of the selected compound having a structural formula selected from formula (A) and (B) or (I) to (XI) or various products used in combination with the compound, the age, weight and physical condition of the patient and his or her medical history, and any other information known in medicine.

[0156] The administration route can be carried out by various different routes. For example, it can be oral or parenteral. It is usually carried out by systemic injection such as intravenous, intramuscular, intraperitoneal, intratumoral, subcutaneous injection, etc. The pharmaceutical composition is modified to adapt to one or more of the above-mentioned routes. The pharmaceutical composition is preferably administered by injection or intravenous infusion of a suitable sterile solution or in the form of a liquid or solid dosage form through the digestive tract.

[0157] The pharmaceutical composition may be formulated in a manner known in the art, possibly by providing a sustained and / or delayed release dosage form or device, as a solution in a pharmaceutically compatible solvent or medium, or as a pill, tablet, capsule, powder, suppository, etc. containing a solid medium. For such dosage forms, agents such as celluloses, lipids, carbonates or starches may be advantageously used.

[0158] The agents or media that can be used for the dosage forms (liquid and / or injectable and / or solid) are excipients or inert media, ie, media that are inactive and non-toxic.

[0159] Mention may be made, for example, of saline, physiological, isotonic and / or buffered solutions compatible with pharmaceutical use and known to the person skilled in the art. The composition may contain one or more agents or media selected from dispersants, solubilizers, stabilizers, preservatives and the like.

[0160] Dosage forms of the invention suitable for oral administration may take the form of discrete units such as capsules, sachets, tablets or lozenges, each containing a predetermined amount of the active ingredient; may take the form of a powder or granules; may take the form of a solution or suspension in an aqueous liquid or a non-aqueous liquid; or may take the form of an oil-in-water emulsion or a water-in-oil emulsion.

[0161] Dosage forms 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. Each such dosage form may also contain other pharmaceutically compatible and non-toxic adjuvants, such as stabilizers, antioxidants, binders, dyes, emulsifiers or flavoring substances.

[0162] The dosage form of the present invention comprises an active ingredient, i.e. a compound (or a pharmaceutically acceptable salt thereof) having a structural formula selected from formula (A) and (B) or (I) to (XI) according to the present invention, in combination with a pharmaceutically acceptable carrier and optionally with other active or therapeutic ingredients. The carrier must be "acceptable" in the sense that it is compatible with the other ingredients in the dosage form and harmless to its recipient. Methods for safe and effective administration of most of these anticancer agents are known to those skilled in the art. In addition, their administration is also described in standard literature.

[0163] Another object of the present invention is a kit comprising, in different containers, at least one compound according to the present invention having a structural formula selected from formula (A) and (B) or (I) to (XI) (or a pharmaceutically acceptable salt thereof), and preferably at least one different therapeutic agent, in particular an anticancer agent, typically a chemotherapeutic agent. The kit may also comprise instructions for preparing a composition according to the present invention, for performing any of the methods described herein, for example for treating a disease, in particular cancer, in a subject, for preventing or treating cancer metastasis and / or preventing or treating cancer recurrence.

[0164] In a particular embodiment, the invention relates to the use of a kit according to the invention for the preparation of a composition as described herein.

[0165] In another specific embodiment, the kit is suitable for performing any of the methods described herein, in particular for treating cancer, preventing cancer metastasis and / or preventing cancer recurrence in a subject.

[0166] Other aspects and advantages of the invention will be disclosed in the following experimental section and figures which should be regarded as merely illustrative. Sequence Listing <110> INSTITUT GUSTAVE ROUSSY, France <120> Madecine derivative compounds, compositions and use thereof for treating cancer <130> B2887PC00 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Epitope <400> 1 Ser Ile Ile Asn Phe Glu Lys Leu 1 5 BRIEF DESCRIPTION OF THE DRAWINGS

[0167] Figure 1 A and 1B: Madecine and Madra.HCl treatment increases intron-derived antigen presentation in cancer cells.

[0168] B3Z-specific T-cell activation in MCA205 sarcoma cells and B16F10 melanoma cells expressing the intron-derived SL8 antigen after treatment with 5 μM or 10 μM (A) Madercin and (B) Madra.HCl. Free SL8 peptide was added in each condition to ensure that the T-cell assay was performed under non-saturating conditions and that the expression of MHC-I molecules was taken into account in the results. Each figure represents at least three independent experiments. Data are given as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired student t test).

[0169] Figure 1 C-1F: Madra.HCl slows the growth of tumors bearing the intron-derived SL8 epitope

[0170] Wild-type MCA205 sarcoma cells or MCA205 sarcoma cells expressing globin-SL8-intron constructs (MCA205 globin-SL8-intron) were subcutaneously inoculated into the flanks of immunocompetent C57BL / 6 mice, followed by intraperitoneal injections of 20 mg / kg or 40 mg / kg of Madra.HCl on days 4, 7, 10, and 14 after tumor inoculation. Tumor size was assessed every 3 to 4 days until the established ethical endpoint was reached. Figure C represents the growth curve of MCA205 sarcoma cells expressing globin-SL8-intron constructs (MCA205 globin-SL8-intron), and Figure D represents the tumor size on day 25. Figure E represents the growth curve of wild-type MCA205 sarcoma cells (MCA205 WT), and Figure F represents the tumor size on day 21. Data are given as mean ± SEM. *p<0.05, **p<0.01 (ANOVA and Tukey's multiple comparison test comparing all groups).

[0171] Figure 1 G: Madra.HCl prolongs overall survival of mice bearing tumors expressing intron-derived SL8

[0172] Kaplan-Meier survival curves of mice injected subcutaneously with MCA205 globin-SL8-intron and subsequently treated intraperitoneally with 20 mg / kg or 40 mg / kg of Madra.HCl at days 4, 7, 10 and 14 after tumor inoculation.

[0173] Figure 1 H: Madra.HCl induces a long-lasting and specific antitumor response

[0174] Growth curves of MCA205 globin-SL8-intron cells and B16F10 WT cells inoculated into the right and left flanks, respectively, of C57BL / 6 mice at day 100 that underwent complete tumor regression after treatment with Madra.HCl.

[0175] Figure 2 : MTT test for each madecine derivative

[0176] MTT assay was performed on MCA205 (A) or B16F10 (B) cells treated with increasing doses of different Madecine derivatives. Data are expressed as half maximum inhibitory concentration (IC50) of the different derivatives. The IC50 represents the concentration of the test compound required to inhibit cell viability by 50% compared to the control condition. The threshold value was specified to be 60 μM. It is the highest dose that did not show any cell death for all compounds.

[0177] Figure 3: Madecine derivatives that increase antigen presentation in two murine cell lines

[0178] Activation of B3Z-specific T-cells in MCA205 and B16F10 expressing intron-derived SL8 antigen after treatment with 60 μM (A) EYP59 (Compound 7), 20 μM (B) EYP201 (Compound 6), 60 μM (C) EYP165 (Compound 57) or 50 μM (D) 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 the expression of MHC-I molecules was taken into account in the results. Each figure represents at least three independent experiments. Data are given as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired student t-test).

[0179] Figure 4 : Madecine derivatives that increase antigen presentation only in the MCA205 sarcoma cell line

[0180] B3Z-specific T-cell activation in MCA205 expressing intron-derived SL8 antigen after treatment with 60 μM (A) EYP188 (Compound 42), 50 μM (B) EYP86 (Compound 10). Free SL8 peptide was added in each condition to ensure that the T-cell assay was performed under non-saturating conditions and that the expression of MHC-I molecules was taken into account in the results. Each figure represents at least three independent experiments. Data are given as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired student t test).

[0181] Figure 5 : Madecine derivatives that increase antigen presentation only in the B16F10 melanoma cell line

[0182] B3Z-specific T-cell activation in MCA205 expressing intron-derived SL8 antigen after treatment with 20 μM (A) EYP174 (Compound 41), 20 μM (B) EYP179 (Compound 50), 60 μM (C) EYP190 (Compound 54) and 10 μM (D) 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 the expression of MHC-I molecules was taken into account in the results. Each figure represents at least three independent experiments. Data are given as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired student t test).

[0183] Figure 6: A madecine derivative that does not increase antigen presentation in two tumor cell lines

[0184] B3Z-specific T-cell activation in MCA205 expressing intron-derived SL8 antigen after treatment with 60 μM (A) EYP177 (Compound 46), 60 μM (B) EYP156 (Compound 43), 10 μM (C) EYP113 (Compound 11) and 20 μM (D) 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 the expression of MHC-I molecules was taken into account in the results. Each figure represents at least three independent experiments. Data are given as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired student t test).

[0185] Figure 7 : EYP59 (Compound 7) slows the growth of MCA-WT sarcomas and MCA-Intron-SL8

[0186] Wild-type MCA205 sarcoma cells or MCA205 sarcoma cells expressing globin-SL8-intron constructs (MCA205 globin-SL8-intron) were subcutaneously inoculated into the flanks of immunocompetent C57BL / 6 mice, followed by intraperitoneal injections of 20 mg / kg of EYP59 on days 4, 7, 10, and 14 after tumor inoculation. Tumor size was assessed every 3 to 4 days until the established ethical endpoint (A) was reached. The upper B represents the growth curve of MCA205 sarcoma cells expressing globin-SL8-intron constructs (MCA205 globin-SL8-intron), and the lower B represents the tumor size on day 23. The upper C represents the growth curve of wild-type MCA205 sarcoma cells (MCA205 WT), and the lower C represents the tumor size on day 19. Panel D shows the growth curve of wild-type MCA205 sarcoma cells (MCA205WT) in immunodeficient nu / nu mice with the same setup as described above for immunocompetent mice. Data are given as mean ± SEM. *p<0.05, **p<0.01 (ANOVA and Tukey's multiple comparison test comparing all groups).

[0187] Figure 8 Madecine derivative EYP59 (Compound 7) effectively reduces tumor growth in vivo when injected intratumorally or intravenously

[0188] 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 of EYP59 on days 4, 7, 10 and 14 after tumor inoculation or intravenous injection of 5 mg / kg of EYP59 on days 4, 7, 10 and 14 after tumor inoculation. The upper panel A represents the growth curve of wild-type MCA205 sarcoma cells, and the lower panel A represents the tumor size on day 21. The upper panel B represents the growth curve of wild-type MCA205 sarcoma cells, and the lower panel B represents the tumor size on day 25. Data are given as mean ± SEM. *p<0.05, **p<0.01 (ANOVA and Tukey's multiple comparison test comparing all groups).

[0189] Fig. 9 :Synthesis of Madecine and Madecine Hydrochloride (Madra.HCl)

[0190] Fig.10 :Drug preparation of madecine and synthesis of madecine derivatives Example

[0191] Materials and methods

[0192] Cell culture

[0193] The MCA205 mouse sarcoma cell line was cultured in RPMI 1640 medium (Life Technologies) at 37°C and 5% CO2 in the presence of 1% glutamine, 1% sodium pyruvate, 1% non-essential amino acids, 1% penicillin / streptomycin, and 10% FBS (Life Technologies). The B16F10 mouse melanoma cell line was cultured in DMEM medium (Life Technologies) containing 1% glutamine, 1% penicillin / streptomycin, and 10% FCS at 37°C and 5% CO2 under standard conditions. The stable MCA205-globin-SL8-intron cell line was cultured under the same conditions as the MCA205 cell line and 500 μg / ml G418 (Life Technologies) was added for selection. The stable B16F10-globin-SL8-intron cell line was cultured under the same conditions as the B16F10 cell line and 500 μg / ml G418 (Life Technologies) was added for selection. SL8 / Kb-specific (B3Z) T-cell reporter hybridomas were cultured in RPMI 1640 medium (Life Technologies) at 37°C and 5% CO2 in the presence of 1% glutamine, 1% penicillin / streptomycin, 50 μM β-mercaptoethanol and 10% FCS.

[0194] Synthetic route of Madecine and all its derivatives

[0195] 1) Synthesis of Madecine and Madecine Hydrochloride (Madra.HCL)

[0196]

[0197] a.7-Methoxy-2,2,4-trimethyl-1,2-dihydroquinoline (Compound 1) (reference Org.Lett.2015,17,4125)

[0198]

[0199]

[0200] Under argon, m-anisidine (2.3 mL, 20.3 mmol) and InCl3 (232 mg, 1.03 mmol) in acetone (30 mL) were heated at 50 °C for 14 h. The solvent was removed and the crude product was partitioned between DCM and a saturated aqueous solution of Na2CO3. The organic layer was dried over MgSO4, filtered and concentrated. Purification by column chromatography (cyclohexane / EtOAc 100:0->99:1) gave the desired compound as a slightly yellow solid. The spectral data were consistent with the literature (Tamariz, J. et al., J. Org. Chem. 2013, 78, 9614-9626). Yield: 61% (2.51 g, 12.3 mmol). Mp 68.8 °C. TLC Rf: 0.5 (cyclohexane / EtOAc 9:1). 1 H NMR (300 MHz, CDCl3) δ 6.97 (d, J = 8.4 Hz, 1H), 6.20 (dd, J = 8.4, 2.5 Hz, 1H), 6.01 (d, J = 2.5 Hz, 1H), 5.19 (s, 1H), 3.75 (s, 3H), 1.96 (d, J = 1.5 Hz, 3H), 1.26 (s, 6H). 13 H 18 NO, HRMS (ESI) (M+H) + The calculated value of m / z is 204.1388 and the measured value is 204.1385.

[0201] b. 1-(7-methoxy-4-methylquinazolin-2-yl)guanidine (Compound 2)

[0202]

[0203] 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, in dihydroquinoline) were added. The mixture was stirred at room temperature for 30 min in 2% ethanol (1% ethanol, 0.76 g). H2O (95 mL) and dicyandiamide (831 mg, 0.98 mmol) were then added and the mixture was refluxed for 48 h. After cooling at 60 °C, the oil was filtered and the pH was adjusted to 11 with a saturated aqueous solution of NaHCO3. The precipitate formed was filtered and dried in vacuo. Compound 2 was isolated as an off-white powder. Yield: 78% (1.76 g, 0.76 mmol). Mp 235-236 °C. 1 H NMR (300 MHz, DMSO-d6) δ7.95 (d, J = 9.1 Hz, 1H), 7.82 (s, 2H), 7.11 (d, J = 2.5 Hz, 1H), 7.00 (dd, J = 9.0, 2.5 Hz, 1H), 3.90 (s, 3H), 2.70 (s, 3H). 11 H 14 N5O, HRMS (ESI) (M+H) + The calculated value of m / z is 232.1198 and the measured value is 232.1193.

[0204] c. 2-((7-methoxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one (madecine, compound 3)

[0205]

[0206] 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), NaHCO3 (1.43 g, 17 mmol) in DMSO (22 mL) was heated at 110°C for 48 h. After cooling at room temperature, cold water was added. The formed precipitate was filtered off and purified by column chromatography (DCM / MeOH 100:0->98:2) to give Madecine 3 as a light brown powder. Yield: 83% (3.58 g, 11.5 mmol). Mp 216.4-217.5°C. TLC Rf: 0.26 (DCM / MeOH 98:2). 1H NMR (300 MHz, CDCl3) δ13.25 (s, 1H), 8.54 (s, 1H), 7.86 (d, J = 9.1 Hz, 1H), 7.17 (d, J = 2.5 Hz, 1H), 7.07 (dd, J = 9.1, 2.5 Hz, 1H), 3.98 (s, 3H), 2.80 (s, 3H), 2.27 (s, 3H), 2.06 (s, 3H). 16 H 18 N5O2, HRMS (ESI) (M+H) + The calculated value of m / z is 312.1462 and the measured value is 312.1458. d. Madr.HCl (EYP34)

[0207]

[0208] Madecine (Compound 3) (600 mg, 1.98 mmol) was dissolved in anhydrous distilled water. To the solution in oxane (10 mL) was added HCl (0.5 mL, 2 mmol, 4 M in dioxane). The mixture was allowed to stir at room temperature for 10 min. The formed precipitate was filtered off and dried in vacuo to give madecine hydrochloride (565 mg, 1.68 mmol, 85%) as a transparent green powder. 1 H NMR (300MHz, deuterium oxide) δ7.76 (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). Mp>330℃ (decomposition). For C 16 H 18 N5O2, HRMS (ESI) (M+H) + The calculated value of m / z is 312.1462 and the measured value is 312.1460. 16 H 18 Calculated values ​​for ClN5O2.2 / 3H2O, Anal.: C, 53.41; H, 5.42. Found values: C, 53.40; H, 5.47.

[0209] 2) Madexin’s drug preparation

[0210] Part 1:

[0211]

[0212] a. 2-((7-Hydroxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one (Compound 4, EYP107)

[0213]

[0214] To a solution of Madecine (compound 3) (250 mg, 0.8 mmol) in DCE (15 mL) was added BBr3 (12 mL, 12 mmol) dropwise under argon and -78 °C. The solution was allowed to warm to room temperature and heated at 60 °C for 20 h. After quenching, the solvent was removed under reduced pressure. The crude product was collected with EtOAc and washed with a saturated aqueous solution of 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). Mp 348.8-349.9 °C. TLC Rf: 0.18 (DCM / MeOH 97: 3). 1 HNMR (300 MHz, DMSO) δ 13.52 (s, 1H), 10.93 (s, 2H), 8.05 (d, J = 9.0 Hz, 1H), 7.03 (dd, J = 9.8, 1.1 Hz, 1H), 6.94 (d, J = 2.2 Hz, 1H), 2.77 (s, 3H), 2.18 (s, 3H), 1.90 (s, 3H). 15 H 16 N5O2, HRMS (ESI) (M+H) + The calculated value of m / z is 298.1304 and the found value is 298.1312.

[0215] b. 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazoline-7-ol sodium (Compound 5, EYP112)

[0216]

[0217] 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, 1 M in H2O). Stirring was continued for 5 minutes until a clear solution was obtained. After evaporation to dryness, a yellow solid was obtained. Yield: >99% (15 mg, 0.05 mmol). 1 H NMR (300 MHz, D2O) δ7.51 (d, J = 8.8 Hz, 1H), 6.67 (d, J = 8.8 Hz, 1H), 6.40 (s, 1H), 2.49 (s, 3H), 2.03 (s, 3H), 1.71 (s, 3H). 15H 16 N5O2, HRMS (ESI) (M+H) + The calculated value of m / z is 298.1304 and the found value is 298.1259.

[0218] c. 5,6-Dimethyl-2-((4-methyl-7-(2-morpholinoethoxy)quinazolin-2-yl)amino)pyrimidin-4(3H)-one (Compound 6, EYP201)

[0219]

[0220] Under an inert atmosphere, compound 4 (35 mg, 0.118 mmol) was suspended in DMF (1 mL), and KOH (13 mg, 0.23 mmol) was added. The mixture was stirred at room temperature for 1 h until it became transparent. 2-Chloro-N-ethylmorpholine (22 mg, 0.12 mmol) was then added, and the mixture was stirred at room temperature for 14 h. The solvent was removed in vacuo. Compound 6 was obtained as an off-white powder after column chromatography (DCM / MeOH 100:0->97:3). Yield: 19% (9.1 mg, 0.022 mmol). TLC Rf: 0.2 (DCM / MeOH 96:4). 1 H NMR (300MHz, CDCl3) δ13.26 (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). For C 21 H 27 N6O3, HRMS (ESI) (M+H) + The calculated value of m / z is 411.2145 and the found value is 411.2152.

[0221] d. Sodium 2-((4,5-dimethyl-6-oxo-1,6-dihydropyrimidin-2-yl)amino)-4-methylquinazolin-7-yl phosphate (Compound 7, EYP59)

[0222]

[0223] A suspension of compound 4 (100.4 mg, 0.33 mmol) and KOH (25 mg, 0.44 mmol) in H2O (5 mL) was stirred at room temperature for 30 min. DCM (5 mL) was then 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 in vacuo. The intermediate diethyl phosphate derivative was obtained after column chromatography (DCM / MeOH 100:0->97:3). Yield: 38% (55 mg, 0.12 mmol). 1 H NMR (300MHz, CDCl3) δ13.16(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).

[0224] The purified diethyl phosphate (55 mg, 0.12 mmol) compound was then 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 product was ground with DCM, filtered, and then suspended in H2O. An aqueous solution of NaOH (0.250 mL, 0.254 mmol) was slowly added and the mixture became clear after stirring for 30 min. The solvent was removed under vacuum to obtain Madecine-phosphate (Compound 7). Yield: 93% (50 mg, 0.11 mmol). 1 H NMR (300MHz, D2O) δ7.97 (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 (81MHz, D2O) δ-174.17. 15 H 16 N5NaO5P, HRMS (ESI) (M+H) + The calculated value of m / z is 400.0787 and the found value is 400.0792.

[0225] Part 2:

[0226]

[0227] a. 2-((8-iodo-7-methoxy-4-methylquinazolin-2-yl)amino)-5,6-dimethylpyrimidin-4(3H)-one (Compound 8)

[0228]

[0229] To a cooled solution of Madecine (Compound 3) (360 mg, 1.16 mmol) in concentrated H2SO4 (1.2 mL) was added (in the dark) NIS (248 mg, 1.10 mmol). The solution was stirred at room temperature for 5 h. The mixture was poured into cold 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->98:2) gave the desired compound 8. Yield: 25% (127 mg, 0.29 mmol). TLC Rf: 0.22 (DCM / MeOH 98:2). 1 H NMR (300 MHz, CDCl3) δ 13.50 (s, 1H), 8.88 (s, 1H), 7.96 (d, J = 9.1 Hz, 1H), 7.09 (d, J = 9.1 Hz, 1H), 4.08 (s, 3H), 2.83 (s, 3H), 2.28 (s, 3H), 2.07 (s, 3H). 16 H 17 N5O2, HRMS (ESI) (M+H) + The calculated value of m / z is 438.0427 and the measured value is 438.0427.

[0230] 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)

[0231]

[0232]

[0233] Under inert atmosphere, 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), PdG3Xantphos (5 mg, 0.005 mmol) and THF (0.1 mL) were added in a sealable vial. 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->70:30) to give the desired compound 9. A second purification by preparative TLC (DCM / MeOH 75:25) was necessary to provide 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 (300 MHz, MeOD) δ 8.11 (d, J = 9.1 Hz, 1H), 7.32 (d, J = 9.0 Hz, 1H), 4.08 (s, 3H), 3.70-3.55 (m, 2H), 2.80 (s, 3H), 2.70 (sl, 1H), 2.23-1.98 (m, 6H), 1.73 (s, 3H). 21 H 25 N6O5S, HRMS (ESI) (M+H) + The calculated value of m / z is 473.1607 and the measured value is 473.1608.

[0234] 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]oct-2-yl)methyl acetate (Compound 10, EYP86)

[0235]

[0236] Under inert atmosphere, 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 glucosinolate (22 mg, 0.06 mmol) in THF (0.5 mL) was purged with argon. 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 in vacuo and purified by column chromatography (DCM / MeOH 100:0->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 (300 MHz, MeOD) δ 8.18 (d, J = 9.1 Hz, 1H), 7.34 (d, J = 9.5 Hz, 1H), 5.17 (t, J = 9.3 Hz, 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 = 9.4 Hz, 6H), 1.66 (s, 3H). 30 H 35 N5O 11 NaS, HRMS (ESI) (M+Na) + The calculated value of m / z is 696.1651 and the measured value is 696.1657.

[0237] 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)

[0238]

[0239] A solution of compound 10 in MeONa (0.19 mL, 0.038 mmol, 0.2 M in MeOH) was stirred at room temperature for 1 h. After evaporating the solvent in vacuo, compound 11 was obtained as a yellow powder. Yield: >99% (7 mg, 0.014 mmol). 1HNMR (300 MHz, MeOD) δ7.88 (d, J = 9.0 Hz, 1H), 7.02 (d, J = 9.1 Hz, 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). 22 H 28 N5O7S, HRMS (ESI) (M+H) + The calculated value of m / z is 506.1709 and the found value is 506.1729.

[0240] 3) General Procedure A:

[0241]

[0242] 2-aminoquinazoline derivative (1 equivalent) (compound 12), aryl halide (1.5 equivalents; unless otherwise stated), Pd2dba3 (7.5mol% or 10mol%), Xantphos (15mol% or 20mol%) and Cs2CO3 (2 equivalents) were added under an inert atmosphere. The mixture was purged with argon. THF was added. The vial was sealed and the reaction mixture was stirred at 80°C for 14h. After cooling at room temperature, the solvent was removed under vacuum and the crude product was purified by column chromatography to obtain the desired compound.

[0243] N-(2-methoxypyrimidin-5-yl)quinazolin-2-amine (Compound 32, EYP281, C 13 H 11 N5O, 253, 2650 g / mol): Following general procedure A, starting from 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). Light brown powder. Mp 210.9-211.7 °C. TLC Rf: 0.2 (DCM / MeOH 98:2). 1 HNMR (300MHz, CDCl3) δ9.11 (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).

[0244] 4) Another series from the methodology section

[0245]

[0246] a. 7-methoxy-N-(6-methoxypyridin-3-yl)-4-methylquinazolin-2-amine (Compound 41, EYP174)

[0247]

[0248] Following general procedure A (except that only 1.1 equivalents of heteroaryl bromide were used), starting from 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. Mp 172.1-173.0°C. TLC Rf: 0.6 (DCM / MeOH 98:2). 1 H NMR (300 MHz, CDCl3) δ8.59 (d, J = 2.8 Hz, 1H), 8.04 (dd, J = 8.8, 2.8 Hz, 1H), 7.75 (d, J = 9.1 Hz, 1H), 7.36 (s, 1H), 6.98 (d, J = 2.5 Hz, 1H), 6.89 (dd, J = 9.0, 2.5 Hz, 1H), 6.76 (d, J = 8.8 Hz, 1H), 3.93 (s, 3H), 3.89 (s, 3H), 2.72 (s, 3H). 16 H 17 N4O2, HRMS (ESI) (M+H) + The calculated value of m / z is 297.1352 and the found value is 297.1328.

[0249] b. 7-Methoxy-4-methyl-N-(3,4,5-trimethoxyphenyl)quinazolin-2-amine (Compound 42, EYP188)

[0250]

[0251] Following general procedure 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 (300 MHz, CDCl3) δ7.78 (d, J = 9.0 Hz, 1H), 7.39 (s, 1H), 7.11 (s, 2H), 6.97 (d, J = 2.6 Hz, 1H), 6.92 (dt, J = 9.0, 2.4 Hz, 1H), 3.91 (s, 3H), 3.89 (s, 6H), 3.83 (s, 3H), 2.75 (s, 3H). 19 H 22 N3O4, HRMS (ESI) (M+H) + The calculated value of m / z is 356.1610 and the found value is 356.1546.

[0252] c. 7-Methoxy-4-methyl-N-(p-tolyl)quinazolin-2-amine (Compound 43, EYP156)

[0253]

[0254]

[0255] Following general procedure 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. Mp 142.8-143.3 °C. TLC Rf: 0.7 (DCM 100%). 1H NMR (300 MHz, CDCl3) δ7.77 (d, J = 9.0 Hz, 1H), 7.68 (d, J = 8.2 Hz, 2H), 7.24–7.12 (m, 3H), 7.05 (d, J = 2.5 Hz, 1H), 6.90 (dd, J = 9.1, 2.5 Hz, 1H), 3.93 (s, 3H), 2.75 (s, 3H), 2.34 (s, 3H). 17 H 18 N3O, HRMS (ESI) (M+H) + The calculated value of m / z is 280.1450 and the found value is 280.1444.

[0256] d. tert-Butyl 3-((7-methoxy-4-methylquinazolin-2-yl)amino)-1H-indole-1-carboxylate (Compound 46, EYP177)

[0257]

[0258] Following general procedure A (except that only 1.1 equivalents of heteroaryl bromide were used), starting from 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% (16 mg, 0.04 mmol). Orange powder. Mp 114-116 ° C. TLC Rf: 0.55 (DCM / MeOH 98:2). 1 H NMR (300MHz, CDCl3) δ8.57(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). For C 23 H 25 N4O3, HRMS (ESI) (M+H) + The calculated value of m / z is 405.1927 and the measured value is 405.1932.

[0259] E 2 -(7-methoxy-4-methylquinazolin-2-yl)-N 4-Methylpyrimidine-2,4-diamine (Compound 49, EYP181)

[0260]

[0261] Following general procedure A (except that only 1.1 equivalents of heteroaryl chloride were used), starting from 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. Mp 247.4-249.1° C. TLC Rf: 0.45 (DCM / MeOH 9:1). 1 H NMR (300 MHz, MeOD) δ 8.02 (d, J = 9.2 Hz, 2H), 7.49 (s, 1H), 7.13 (d, J = 9.2 Hz, 1H), 6.39 (d, J = 6.5 Hz, 1H), 4.00 (s, 3H), 3.07 (s, 3H), 2.85 (s, 3H). 15 H 17 N6O, HRMS (ESI) (M+H) + The calculated value of m / z is 297.1464 and the found value is 297.1473.

[0262] f. 7-Methoxy-N-(4-methoxypyrimidin-2-yl)-4-methylquinazolin-2-amine (Compound 50, EYP179)

[0263]

[0264] Following general procedure A (except that only 1.1 equivalents of heteroaryl chloride were used), starting from 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% (23 mg, 0.08 mmol). Yellow powder. Mp 160.8-161.5° C. TLC Rf: 0.3 (DCM / MeOH 98:2). 1H NMR (300 MHz, CDCl3) δ8.42–8.33 (m, 1H), 8.28–8.16 (m, 1H), 7.84 (d, J = 9.0 Hz, 1H), 7.23 (s, 1H), 7.01 (dd, J = 9.0, 2.3 Hz, 1H), 6.35 (d, J = 5.6 Hz, 1H), 3.97 (s, 3H), 3.92 (s, 3H), 2.81 (s, 3H). 15 H 16 N5O2, HRMS (ESI) (M+H) + The calculated value of m / z is 298.1304 and the measured value is 298.1302.

[0265] g. 7-methoxy-N-(2-methoxypyrimidin-4-yl)-4-methylquinazolin-2-amine (Compound 54, EYP190)

[0266]

[0267] 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 in vacuo and purified by column chromatography (DCM / MeOH 100:0->99:1) to afford the desired compound 54 as a white solid. Yield: 61% (6 mg, 0.02 mmol). Mp 173.9-174.3 °C. TLC Rf: 0.27 (DCM / MeOH 98:2). 1 HNMR (300MHz, CDCl3) δ8.41 (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). 15 H 16 N5O2, HRMS (ESI) (M+H) + The calculated value of m / z is 298.1304 and the found value is 298.1309.

[0268] hN-(1H-Indol-3-yl)-7-methoxy-4-methylquinazolin-2-amine (Compound 57, EYP165)

[0269]

[0270] 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 a saturated aqueous solution of 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->200:1) gave 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) δ10.69(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). For C 18 H 17 N4O, HRMS (ESI) (M+H) + The calculated value of m / z is 305.1402 and the found value is 305.1408.

[0271] T-cell assay

[0272] MCA205 and B16F10 mouse cell lines were transfected with plasmid YFP-globin-SL8-intron or PCDNA3 empty plasmid (negative control) using transfection reagent jetPRIME (Ozyme) or GeneJuice (Millipore) according to the respective manufacturers' protocols. 24 hours after transfection, cells were treated with different doses of Maderxin (Sigma SML 1409), MaderHCl (3.HCl) or different compounds cited in this article. Cells were then washed three times with PBS 1X and 5x10 4 Tumor cells and 1x10 5B3Z hybridomas were co-cultured in a 96-well plate. In the positive control well, 4 μg / ml of the synthetic peptide SL8 was added. The cells were then incubated overnight at 37°C and 5% CO2. The plate was centrifuged at 1200 rpm for 5 min, the cells were washed twice with PBS 1X, and lysed in 0.2% TritonX-100, 0.2% DTT, 0.5M K2HPO4, 0.5M KH2PO4, with shaking at 4°C for 5 min. The lysate was centrifuged at 3000 rpm for 10 min, and the supernatant was transferred to a 96-well optiplate (OptiPlaque-96, PerkinElmer). PBS containing 10 mM MgCl2, 11.2 mM β-mercaptoethanol, 0.0015% CA-630 and 40 μM 4-methylumbelliferyl β-D-pyranogalactoside (MUG) developing buffer, and the plate was incubated at room temperature for 3 hours. Finally, β-galactosidase activity was measured using FLUOstar OPTIMA (BMG LABTECH Gmbh, 30 Offenburg, Germany). Results are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired student t test).

[0273] Tumor stimulation and treatment

[0274] C57Bl / 6J female mice were obtained from Harlan Laboratories Ltd, Switzerland. Seven-week-old mice were treated with 1×10 5 MCA205 sarcoma or B16F10 melanoma cells or 1.5x10 5 MCA205 globin-SL8-intron or B16F10 globin-SL8-intron cells were injected subcutaneously into the right flank. Sarcoma cells were injected in 100 μL sterile PBS, while melanoma cells were injected in 50:50 PBS: Matrigel to prevent tumor cell dissemination. Madra.HCl in 100 μL H2O+30% (2-hydroxypropyl)-β-cyclodextrin (w / v) was injected 4, 7, 10 and 14 days after tumor inoculation. In the same way, EYP59 (Compound 7) in 100 μL H2O was injected 4, 7, 10 and 14 days after tumor inoculation. The area of ​​the tumor was recorded every 3 to 4 days until the tumor reached the ethical endpoint. All animal experiments were performed in accordance with French and European laws and regulations. The results are expressed as mean ± SEM. *p<0.05, **P<0.01, ***P<0.001 (ANOVA and Tukey's multiple comparison test comparing all groups).

[0275] result

[0276] Madecine treatment increases antigen presentation of PTP-derived antigens in cancer cells

[0277] In recent studies, the inventors have shown that PTPs are the major source of peptides for the endogenous class I MHC pathway in vitro. To modulate the presentation of PTP-derived antigens at the surface of cancer cells, the inventors tested the effects of madecine treatment on mouse tumor cell lines, a melanoma (B16F10) and a sarcoma (MCA205) cell line. Both murine cell lines transiently expressed the PTP-SL8 epitope derived from an intron in the β-globin gene construct or remained untransfected. Madecine triggered an increase in PTP-dependent antigen presentation in both mouse cell lines with a dose-dependent effect ( Figure 1 A). These results show that the production and presentation of PTP-derived antigens can be upregulated in cancer cell lines after madecine treatment. They support the hypothesis that this molecule can be used as a positive immunomodulator to enhance specific antitumor immune responses that are dependent on PTP production and presentation.

[0278] Treatment with synthetic madera hydrochloride (Madra.HCl) increases antigen presentation of PTP-derived antigens in vitro and slows tumor growth in vivo

[0279] The above results confirmed that madecine can increase the production and presentation of PTP-dependent antigens encoded by intronic sequences on the cell surface of treated tumor cell lines in vitro. The next question was to investigate whether madecine, which can only be dissolved in DMSO, can have an effect on tumor growth and CD8 + T cell proliferation has the same effect. Unfortunately, the inventors were unable to perform in vivo experiments with madecine due to its low concentration in DMSO. Since this first result was encouraging, they decided to produce derivatives of madecine in order to improve the cancer immune response. In fact, madecine is insoluble in water and can only be dissolved in DMSO solvent, making its pharmacokinetic efficiency in mice low.

[0280] In order to make madecine available for more extensive in vitro and in vivo validation without the use of toxic carriers or cosolvents (DMSO), the present inventors considered it necessary to find a strategy to synthesize madecine and certain derivatives to enhance their solubility and immunomodulator activity.

[0281] To this end, for example, phosphate prodrugs can generally exhibit excellent water solubility, chemical stability, and the ability to be rapidly bioconverted back to the parent drug by phosphatases. The formation of phosphate prodrugs has been used to improve the water solubility of a variety of different molecules including antitumor phenolic natural products and their derivatives such as Compretin A-4.

[0282] In order to test the new compounds as positive immunomodulators against tumor cell lines, the inventors first decided to test them in an in vitro assay. As expected, the new compound referred to herein as "Madra.HCl" was able to dissolve in water. After treating both murine cell lines MCA205 or B16F10 transiently expressing the PTP-SL8 epitope derived from an intron in the β-globin gene (globin-intron-SL8) with 5 μM or 10 μM Madra.HCl, the inventors observed that PTP-dependent antigen presentation increased in a dose-dependent manner ( Figure 1 B) 1x10 5 MCA205 sarcoma cells stably expressing the SIINFEKL (SL8) epitope from an intron in the β-globin gene (globin-intron-SL8) were subcutaneously inoculated into mice. Four days after this inoculation, the mice were vaccinated intraperitoneally with a defined dose of Madra.HCl. Then 20 or 40 mg / kg of Madra.HCl were injected every 3 days. During this period, tumor growth was monitored every 2 to 3 days ( Figure 1 C). The inventors observed a significant reduction of 50% in tumor growth at day 27 after challenge in mice treated with 20 mg / kg Madra.HCl ( Figure 1 D), and in mice treated with 40 mg / kg Madra.HCl, tumor growth was reduced by 60 to 70% on day 27 after challenge ( Figure 1 D) The same experiment was performed on B16F10 tumor cells expressing the globin-intron-SL8 construct.

[0283] In the same way, 1x10 5 Untransfected MCA sarcoma cells were subcutaneously inoculated into mice. Four days after this inoculation, the mice were vaccinated intraperitoneally with a determined dose of Madra.HCl. Then, the same dose was injected again every 3 days. During this period, tumor growth was monitored every 2 to 3 days ( Figure 1 E). The inventors observed a significant reduction of 40% in tumor growth at day 21 after challenge in mice treated with 20 mg / kg or 40 mg / kg of Madra.HCl ( Figure 1 F).

[0284] Furthermore, it has been shown that Madra.HCl treatment prolonged the survival of mice, with approximately 25% survival at 120 days post tumor inoculation when mice were treated with 40 mg / kg Madra.HCl and approximately 15% survival at 120 days post tumor inoculation when mice were treated with 20 mg / kg Madra.HCl ( Figure 1 G).

[0285] Finally, mice inoculated with MCA205 tumor cells expressing the globin-intron-SL8 construct as described above and that experienced complete tumor regression after treatment with Madra.HCl were re-challenged 100 days later with MCA205 tumor cells expressing the globin-intron-SL8 construct on the right flank and with B16F10 tumor cells on the left flank. While B16F10 tumors grew over time, MCA205 tumors did not grow in the mice ( Figure 1 H). These results demonstrate that following Madra.HCl treatment, mice develop a long-term anti-tumor response specific to MCA205 tumors.

[0286] Madecine derivatives effectively increase class I MHC presentation of intron-derived antigens in vitro

[0287] In order to improve the immunomodulatory activity of Madra.HCl for more extensive in vitro and in vivo validation without the use of toxic carriers or cosolvents (DMSO), the inventors considered it necessary to find a strategy to modify its structure while retaining its activity. As described above, the following compounds of the invention were prepared.

[0288] To test the new compounds as positive immunomodulators against tumor cell lines, the inventors first decided to test them in an in vitro assay. As expected, several derivatives of Madra.HCl were soluble in water, while some others were not. Using the MTT assay, they identified the IC25 and IC50 (0.05) of each compound in MCA sarcoma and B16F10 melanoma cell lines. Figure 2 ). For the rest of the experiments, they decided to treat two cell lines with the IC50 of each compound, either soluble or insoluble in water. For this purpose, two murine tumor cell lines transiently expressing the PTP-SL8 epitope derived from an intron in the β-globin gene (globin-intron-SL8) were treated overnight with each compound. Four different observations were made on the immunomodulatory activity of each compound:

[0289] The first observation was that they noted an increase in PTP-dependent antigen presentation in both murine tumor cell lines when certain compounds were used ( Figure 3 After treating murine cell lines with EYP59 (compound 7) (A), EYP201 (compound 6) (B), EYP165 (compound 57) (C), and EYP281 (compound 32) (D), they could see an increase in PTP-SL8 presentation.

[0290] The second observation was that some compounds could positively increase the presentation of their PTP-derived antigen only in MCA cell lines transiently expressing the PTP-SL8 epitope derived from an intron in the β-globin gene ( Figure 4 They saw an increase in PTP-SL8 presentation after treating murine sarcoma cell lines with EYP188 (compound 42) (A) and EYP86 (compound 10) (B), respectively.

[0291] Furthermore, a third observation was that some compounds positively increased the presentation of our PTP-derived antigen only in the B16F10 cell line transiently expressing the PTP-SL8 epitope derived from an intron in the β-globin gene ( Figure 5 The inventors were able to see an increase in PTP-SL8 presentation after treatment of murine melanoma cell lines with EYP174 (compound 41) (A), EYP179 (compound 50) (B), EYP190 (compound 54) (C), and EYP181 (compound 49) (D), respectively.

[0292] Finally, a fourth observation was that some compounds failed to positively increase the presentation of our PTP-derived antigen in both murine cell lines tested ( Figure 6 The inventors failed to see any increase in PTP-SL8 presentation after treatment of both murine cell lines with EYP177 (compound 46) (A), EYP156 (compound 43) (B), EYP113 (compound 11) (C), and EYP102 (compound 9) (D), respectively.

[0293] The madecine derivative EYP59 (Compound 7) effectively reduces tumor growth in an immune-dependent manner in vivo

[0294] 1x10 5 MCA205 sarcoma cells stably expressing the SIINFEKL (SL8) epitope from an intron in the β-globin gene (globin-intron-SL8) were subcutaneously inoculated into mice. Four days after this inoculation, the mice were vaccinated intraperitoneally with a defined dose of EYP59. Then, 20 mg / kg was injected every 3 days. Tumor growth was monitored every 2 to 3 days during this period ( Figure 7 A). The inventors observed a significant reduction of 85% in tumor growth at day 23 after challenge in mice treated with 20 mg / kg of EYP59 ( Figure 7 B) In the same way, 1x10 5 Untransfected MCA205 sarcoma cells were subcutaneously inoculated into mice. Four days after this inoculation, the mice were vaccinated intraperitoneally with a determined dose of EYP59. Then, the same dose was injected again every 3 days. Tumor growth was monitored every 2 to 3 days during this period. The inventors observed a significant reduction in tumor growth of 70% (19 days after challenge) in mice treated with 20 mg / kg of EYP59. Figure 7 C).

[0295] To assess the requirement for an immune response for this effect, the inventors tested the effect of 20 mg / kg EYP59 in immunodeficient nu / nu mice with the same setup as above and observed that it had no effect on tumor growth ( Figure 7 D) These results show that the reduction in tumor size after EYP59 treatment requires the presence of an active immune response in vivo.

[0296] The madecine derivative EYP59 (Compound 7) effectively reduces tumor growth in vivo when injected intratumorally or intravenously

[0297] 1x10 5 Wild-type MCA205 sarcoma cells were subcutaneously inoculated into mice. Four days after this inoculation, the mice were injected with a determined dose of EYP59 intratumorally (2.5 mg / kg) or intravenously (5 mg / kg). Then, the same amount of product was injected every 3 days in the corresponding dosing mode. During this period, tumor growth was monitored every 2 to 3 days. The inventors observed that in mice treated with 2.5 mg / kg of EYP59 intratumorally, tumor growth was significantly reduced by 75% (2.5 mg / kg) on ​​day 21 after challenge. Figure 8 A). In addition, the inventors observed that in mice treated with 5 mg / kg of EYP59 intravenously, tumor growth was significantly reduced by 80% on day 25 after challenge ( Figure 8 B).

[0298] discuss

[0299] The present invention reveals that specific spliceosome inhibitors have a positive effect on anti-tumor immune response and therefore have a positive effect on tumor growth. Splicing abnormalities appear as specific characteristics of cancer and are studied as predictive markers for patient survival and targets for treating cancer using splicing inhibitors, some of which are currently being developed in acute myeloid leukemia. In the present invention, the inventors confirmed that certain specific derivatives of Madecine are effective stimulators of in vitro anti-tumor immune responses in sarcoma and melanoma tumor cell lines. They also confirmed that certain Madecine derivatives specifically increase PTP-dependent antigen presentation in sarcoma cancer models, and that certain other derivatives, unlike the first group, can also increase PTP-dependent antigen presentation in melanoma cancer models. By emphasizing the original biomolecular spectrum, they pave the way for new applications within the framework of targeted molecular therapy.

[0300] The PTP model describes that pre-spliced ​​mRNAs become templates for PTPs through alternative translation events that occur in the nucleus. In studies describing this alternative translation, the inventors demonstrated that forced nuclear retention of mRNA encoding the intronic SL8 peptide resulted in increased SL8 antigen presentation. In addition, pladienolide and spliceostatin A (SSA) have been shown to inhibit splicing by targeting SF3b, a subcomplex of the U2 intranuclear small nuclear ribonucleoprotein (snRNP) in the spliceosome, and have been described to promote the accumulation of pre-RNA in the nucleus. Madecine inhibits splicing in a different way by preventing the recruitment of stable U4 / U6 / U5 tri-snRNPs immediately after U2 snRNP fixation. However, it is possible that it also induces the accumulation of pre-RNA in the nucleus, leading to increased antigen presentation. The connection between pre-mRNA nuclear accumulation and increased antigen presentation is unknown. It is tempting to assume that the accumulation of pre-mRNA in the nucleus provides more templates for the production of PTPs, leading to the enrichment of PTPs containing SL8, which are used as the main source for direct presentation of SL8.

[0301] In addition, pre-mRNA splicing is an essential mechanism required for the normal function of all mammalian cells. In recent years, several studies have reported the presence of mutations and overexpression of major spliceosome factors associated with aberrant splicing activity in various different cancers. A few years ago, the inventors also provided some evidence that inhibition of the spliceosome increases class I MHC PTP-dependent antigen presentation. These findings focus on the spliceosome as a potential target for anticancer therapy.

[0302] As already mentioned, several small molecules have been reported to inhibit the spliceosome, and in particular to inhibit the function of the spliceosomal factor SF3B1. Although the exact mechanism of these small molecules is not fully understood, they have been reported to be effective in cancer therapy by reducing tumor size by 40% to 80% depending on the compound used. The only compound that has been tested in humans so far is E7107. Its use has been discontinued due to toxicity issues. This compound is known to inhibit the spliceosome by interacting with SF3B1.

[0303] Furthermore, the failure of cytotoxic T lymphocytes to reject tumors could be explained in part by the initial inappropriate activation of CTLs by pAPCs. It has been described that a subset of dendritic cells (DCs) identified in the tumor microenvironment (TME) are able to migrate to tumor-draining lymph nodes, deliver intact antigens encountered in the TME, and directly or indirectly sensitize naive CD8 + In addition, it has been proposed that some DCs can directly sensitize naive CD8 T cells in the TME. The present inventors recently demonstrated that tumor-associated PTPs are mediated by cross-priming of CD8 T cells by DCs. +The results suggest that PTPs for endogenous and cross-presentation are generated by the same translational event and that the two pathways are then rapidly separated. PTPs are rare products and the efficiency of PTP vaccines or exosome-containing PTP vaccines has been shown to rely on the enrichment of PTPs by previous PTP proteasome inhibitors. The inventors believe that the splicing inhibitor Madecine and its different derivatives, in addition to providing more source material for direct antigen presentation, also enrich the pool of SL8-containing PTPs that serve as source material for DC uptake and cross-presentation within the tumor, inducing enhanced SL8-specific CD8 + T cell proliferation.

[0304] In this specification, the inventors provide both in vitro and in vivo evidence that by modulating spliceosome activity using specific derivative compounds from madecin, specific anti-tumor immune responses can be induced against different cancer models. Madecin has been reported to interfere with early steps of spliceosome assembly. In fact, it has been shown that madecin inhibits the formation of the pre-spliceosome A complex to form the larger catalytic pre-spliceosome B complex. We have demonstrated that by inhibiting the formation of the spliceosome as early as possible in vitro and in vivo using madecin derivatives, specific induction of CD8 + T cells proliferated and anti-tumor antigen presentation was significantly increased. They reported that these derivatives could be used as chemotherapeutic agents against melanoma and sarcoma.

[0305] References

[0306] -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).

[0307] -Burg, SH, et al., Vaccines for established cancer: overcoming the challenges posed by immune evasion, Nat. Publ. Gr. 16, 219–233 (2016).

[0308] -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).

[0309] -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).

[0310] -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).

[0311] -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).

[0312] -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).

[0313] -Liu, Y. et al., Expression of antigen processing and presenting molecules in brain metastasis of breast cancer, Cancer Immunol. Immunother. 61, 789–801 (2012).

[0314] -Mellman, I. et al., Cancer immunotherapy comes of age, Nature 480, 480–489 ​​(2014).

[0315] - Watson, NFS, 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).

[0316] - Yewdell, JW. et al., Defective ribosomal products (DRiPs): major source of antigenic peptides for MHC class I molecules?, J Immunol 157(5): 1823-6 (1996).

Claims

1. A compound of formula (II), which is: 5,6-Dimethyl-2-((4-methyl-7-(2-morpholinoethoxy)quinazolin-2-yl)amino)pyrimidin-4(3H)-one 2. Use of a compound according to claim 1 in the preparation of a medicament for treating cancer in a subject, wherein the cancer is a sarcoma or a melanoma.

3. Use according to claim 2, wherein the compound is combined with at least one different anticancer agent and / or with radiotherapy.

4. The use according to claim 3, wherein the at least one different anti-cancer agent is selected from a chemotherapeutic agent, an immune checkpoint blocker and an anti-cancer vaccine.

5. Use of a compound according to claim 1 in the preparation of a medicament for stimulating an anti-cancer immune response in a subject in need thereof, wherein the cancer is a sarcoma or a melanoma.

6. Use of the compound according to claim 1 in the preparation of a medicament for inducing or enhancing the production and presentation of a prior translation product (PTP)-derived antigen by cancer cells or altering the immune peptide group in a subject, wherein the cancer cells are sarcoma or melanoma cells.

7. The use according to any one of claims 2 to 6, wherein the subject is a mammal.

8. The use according to claim 7, wherein the subject is a human.

9. A composition comprising the compound according to claim 1 and a pharmaceutically acceptable carrier.

10. The composition according to claim 9, wherein the composition further comprises at least one different anticancer agent for simultaneous, separate or sequential use.

11. The composition of claim 9 or 10 for use in treating cancer in a subject, wherein the cancer is a sarcoma or a melanoma.

12. The composition of claim 11, wherein the subject is a mammal.

13. The composition of claim 12, wherein the subject is a human.

14. A kit comprising, in separate containers, a compound according to claim 1 and at least one different anticancer agent.

15. Use of the kit according to claim 14 for preparing the composition according to claim 10.

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