Mitoxantrone derivatives as RAS inhibitors

Abstract

The present invention relates to the use of compounds of formula (I) as RAS inhibitors and as medicaments, in particular for the treatment of proliferative disorders, inflammatory diseases and / or genetic disorders. The invention also relates to a pharmaceutical composition comprising the compound of formula (I). Furthermore, the present invention relates to methods of inhibiting the growth, proliferation or metastasis of cancer cells in an individual in need thereof, in particular, which may include a subpopulation of patients defined by RAS oncogene mutation status or patients who may be resistant to care or treatment criteria for RAS mutation-specific inhibitors. The invention also relates to methods of treating genetic disorders such as RAS disease or inflammatory disorders such as adenomyosis by inhibiting RAS molecules, wherein the KRAS gene is mutated and activated. Furthermore, the present invention relates to methods of inhibiting cell population proliferation and / or factor secretion that are sensitive in vitro to inhibition of RAS activation, in particular to inhibition of KRAS, HRAS and NRAS activation in vitro. Furthermore, the present invention relates to a kit comprising a formulation comprising a pharmaceutical composition comprising the compound of formula (I).

Description

[0001] The present invention relates to the use of compounds of formula (I) as RAS inhibitors and as drugs, in particular for the treatment of proliferative disorders, inflammatory diseases and / or genetic disorders. The present invention also relates to pharmaceutical compositions comprising compounds of formula (I). In addition, the present invention relates to methods for inhibiting the growth, proliferation or metastasis of cancer cells in individuals in need, in particular, it may include a patient subgroup defined by the mutation status of the RAS oncogene or a patient who may be resistant to the standard of care or treatment of RAS mutation-specific inhibitors. The present invention also relates to methods for inhibiting RAS molecules to treat genetic disorders such as RAS diseases or inflammatory disorders such as adenomyosis, wherein the KRAS gene is activated by mutation. In addition, the present invention relates to a method for inhibiting the proliferation and / or factor secretion of cell populations that are sensitive to inhibiting RAS activation in vitro, in particular, to inhibiting KRAS, HRAS and NRAS activation in vitro. In addition, the present invention relates to a kit comprising a preparation comprising a pharmaceutical composition containing a compound of formula (I). Background of the Invention

[0003] RAS proteins represent a group of closely related monomeric globular proteins that are associated with the plasma membrane and can bind GDP or GTP. RAS containing bound GDP represents an "inactive" state, while GTP binds to RAS in exchange for GDP and represents an "active" state, enabling the protein to interact with other "effector" proteins of downstream targets. RAS proteins can be regarded as small GTPases that function as molecular switches to control the transmission of extracellular signals from the cell to the nucleus through a variety of effector proteins. Activating RAS mutations have been detected in inflammatory disorders such as adenomyosis / endometriosis, which contribute to the proliferation and invasion of endometrial cells and resistance to progesterone (S. Inoue, Nature Comm. 2019, 10, 5785; PMID: 31857578).

[0004] There are three isoforms of RAS (KRAS, HRAS, and NRAS), and their activation cycle is regulated by GDP or GTP binding, which in turn is controlled by GAPs or GEFs. In their GTP-bound form, they bind to effector proteins and trigger a variety of signaling pathways that control a variety of fundamental cellular processes.

[0005] Typically, mutations in RAS result in defects in GAP-mediated GTP hydrolysis, and thereby lead to accumulation of RAS in a GTP-bound active state. This results in uncontrolled proliferation, a hallmark of cancer cells. Uncontrolled RAS activation has also been detected in inherited disorders such as RASopathy.

[0006] Recent studies have led to the development of mutation-specific KRAS inhibitors targeting the KRAS G12C mutant, which have been approved for clinical use. Additional inhibitors targeting other mutation-specific versions of KRAS are currently under development.

[0007] In addition, it is known that patients often develop resistance to KRAS oncogene inhibitors, such as resistance to KRAS G12C inhibitors (Tanaka et al., Cancer Discov, 2021, PMID 33824136). In addition, patients treated with KRAS G12C inhibitors often develop secondary mutations in other RAS isoforms (Awad MM et al., New England J. Med., 2021 PMID 34161704).

[0008] Mitoxantrone (trade name Novantrone) is an anthracenedione antitumor agent used to treat certain types of cancer, mainly acute myeloid leukemia or to treat multiple sclerosis (MS).

[0009] Wang et al., Investigational New Drugs, 40(2), p.330-339, 2021 relates to the use of mitoxantrone hydrochloride liposome injection in the treatment of breast cancer.

[0010] CN 109 718 228 discloses the anti-tumor lymphatic metastasis function of mitoxantrone. Mitoxantrone is used in local chemotherapy to improve anti-tumor activity, reduce toxic side effects, target the lymphatic system, inhibit tumor lymphatic metastasis, and may reduce tumor volume. When used in neoadjuvant chemotherapy, it can facilitate subsequent surgical resection, kill tumor cells metastasized in the lymphatic vessels, reduce the risk of postoperative tumor recurrence, and improve the quality of life of patients.

[0011] WO2021 / 160115 relates to the use of mitoxantrone hydrochloride liposomes as a pharmaceutical ingredient for the preparation of a drug for treating breast cancer. Clinical trial results show that mitoxantrone hydrochloride liposomes have better efficacy and fewer adverse reactions, and in particular significantly reduce cardiac toxicity compared to commonly used mitoxantrone hydrochloride injections.

[0012] CN 113 209 149 relates to a mixture of mitoxantrone and ginsenoside for treating gastric cancer.

[0013] WO2004 / 041262 relates to mixtures of mitoxantrone and CDK inhibitors for the treatment of cancer and other proliferative disorders.

[0014] None of the documents discloses or suggests that mitoxantrone is suitable for use as an inhibitor of RAS protein activation, in particular, KRAS G12V, NRAS G12V, HRAS G12V, KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12C / Y96C, KRAS G12C / Y96DS, KRAS G13C, KRAS G13D, KRAS G13S, KRAS Q61H, KRAS Q61R or KRAS Q61K.

[0015] To date, it is not known that mitoxantrone inhibits the expression and / or activation of RAS. Activation is defined as the ability of RAS to bind to its effector molecules such as RAF kinase, PI3K kinase in a GTP-dependent manner through the RAS binding domain (RBD) or RAS-associated domain (RA domain) present in effector proteins (such as RASSF). Further targeting of activation rather than stability may be advantageous in certain medical conditions, while targeting both may be used to combat certain subtypes of RAS-mutated cancers.

[0016] Furthermore, the mechanisms driving HRAS, NRAS, and KRAS activation are distinct, and each RAS isoform exhibits distinct functional and biological specificities. Therefore, attempts are underway to target HRAS and NRAS in defined tumor subtypes in which they function as oncogenic drivers.

[0017] Finally, targeting of other RAS isoforms, such as HRAS and NRAS, is needed to combat secondary acquired resistance to standard of care including several cancer treatments.

[0018] Although KRAS G12-specific C inhibitors have been developed, efforts to target HRAS and NRAS in cancers where mutations occur in these isoforms remain a challenge.

[0019] However, effectively targeting and, in particular, inhibiting RAS oncogene activation by small molecules, particularly with limited toxicity, remains a challenge.

[0020] Therefore, the object of the present invention is to provide a pharmaceutically active compound having the ability to inhibit the activation of RAS oncogenes, particularly with high specificity in cells at lower concentrations. This object is achieved by using compounds of formula (I). Surprisingly, it was found that compounds of formula (I), particularly compounds of formula (A) and (B), inhibit the activity of RAS oncogenes, particularly KRAS, HRAS and NRAS, regardless of their mutation status. Without being bound by any theory, it is presumed that the interaction between the mutation-activated RAS and its effector molecules is disrupted. SUMMARY OF THE INVENTION

[0022] The present invention relates to compounds of formula (I)

[0023]

[0024] in

[0025] R 1 is H or C1-C4 alkyl;

[0026] R 2 is H or C1-C4 alkyl;

[0027] R 3 is H or C1-C4 alkyl;

[0028] R 4 is H or C1-C4 alkyl;

[0029] n is 1, 2 or 3;

[0030] or a pharmaceutically acceptable salt thereof, for use as a RAS protein activation inhibitor for the treatment and / or prevention of diseases in which RAS signaling is involved.

[0031] In particular, the present invention also relates to compounds of formula (I)

[0032]

[0033] in

[0034] R 1 is H or C1-C4 alkyl;

[0035] R 2 is H or C1-C4 alkyl;

[0036] R 3 is H or C1-C4 alkyl;

[0037] R 4 is H or C1-C4 alkyl;

[0038] n is 1, 2 or 3;

[0039] or a pharmaceutically acceptable salt thereof, for use as an inhibitor of RAS protein activation for the treatment and / or prevention of proliferative disorders in which RAS signaling is involved.

[0040] In particular, the present invention also relates to compounds of formula (I)

[0041]

[0042] in

[0043] R 1 is H or C1-C4 alkyl;

[0044] R 2 is H or C1-C4 alkyl;

[0045] R 3 is H or C1-C4 alkyl;

[0046] R 4 is H or C1-C4 alkyl;

[0047] n is 1, 2 or 3;

[0048] or a pharmaceutically acceptable salt thereof, for use as a RAS protein activation inhibitor for the treatment and / or prevention of genetic disorders involving RAS signaling, in particular for use as a RAS protein activation inhibitor for the treatment and / or prevention of RAS diseases.

[0049] In particular, the present invention also relates to compounds of formula (I)

[0050]

[0051] in

[0052] R 1 is H or C1-C4 alkyl;

[0053] R 2 is H or C1-C4 alkyl;

[0054] R 3 is H or C1-C4 alkyl;

[0055] R 4 is H or C1-C4 alkyl;

[0056] n is 1, 2 or 3;

[0057] or a pharmaceutically acceptable salt thereof, for use as a RAS protein activation inhibitor for the treatment and / or prevention of inflammatory disorders in which RAS signaling is involved, in particular for use as a RAS protein activation inhibitor for the treatment and / or prevention of endometriosis and / or adenomyosis, more particularly in which KRAS is mutated.

[0058] In particular, the present invention also relates to compounds of formula (I)

[0059]

[0060] in

[0061] R 1 is H or C1-C4 alkyl;

[0062] R 2 is H or C1-C4 alkyl;

[0063] R3 is H or C1-C4 alkyl;

[0064] R 4 is H or C1-C4 alkyl;

[0065] n is 1, 2 or 3;

[0066] or a pharmaceutically acceptable salt thereof, as defined hereinbefore or hereinbefore, for use as an inhibitor of RAS protein activation for the treatment and / or prevention of proliferative disorders and / or inflammatory diseases involving KRAS G12V, NRAS G12V, HRAS G12V, KRASG12A, KRAS G12C, KRAS G12D, KRAS G12C / Y96D, KRAS G12C / Y96C, KRAS G12C / Y96S, KRASG13C, KRAS G13D, KRAS G13S, KRAS Q61H, KRAS Q61R or KRAS Q61K.

[0067] In particular, the present invention also relates to compounds of formula (I)

[0068]

[0069] in

[0070] R 1 is H or C1-C4 alkyl;

[0071] R 2 is H or C1-C4 alkyl;

[0072] R 3 is H or C1-C4 alkyl;

[0073] R 4 is H or C1-C4 alkyl;

[0074] n is 1, 2 or 3;

[0075] or a pharmaceutically acceptable salt thereof, as defined above and below, for use as an inhibitor of RAS protein activation for the treatment and / or prevention of proliferative disorders and / or inflammatory diseases which are resistant to treatment with a RAS mutation-specific inhibitor other than a compound of formula (I), preferably wherein the resistance is due to a secondary mutation, in particular due to a secondary mutation involving KRAS G12C / Y96D, KRAS G12C / Y96C and / or KRAS G12C / Y96S.

[0076] The present invention particularly relates to compounds of formula (I)

[0077]

[0078] in

[0079] R 1 is H or C1-C4 alkyl;

[0080] R 2 is H or C1-C4 alkyl;

[0081] R 3 is H or C1-C4 alkyl;

[0082] R 4 is H or C1-C4 alkyl;

[0083] n is 1, 2 or 3;

[0084] or a pharmaceutically acceptable salt thereof, for use as a RAS protein activation inhibitor.

[0085] The present invention also relates to a composition (1) comprising at least one compound of formula (I) as defined above and below, in particular a compound of formula (A) and (B) as defined above and below, or a pharmaceutically acceptable salt thereof, and a RAS mutation-specific inhibitor. The RAS mutation-specific inhibitor is different from the compound of formula (I).

[0086] The present invention also relates to compounds (I) as defined above and below, in particular compounds of formula (A) and (B) or pharmaceutically acceptable salts thereof, or compositions (1) as defined above and below, or pharmaceutical compositions comprising at least one compound of formula (I) as defined above and below, in particular selected from compounds (A) and (B), or compositions (1) as defined above and below, for use as RAS-RAF disrupting agents.

[0087] The present invention also relates to compounds (I) as defined above and below, in particular compounds of formula (A) and (B) or pharmaceutically acceptable salts thereof, or compositions (1) as defined above and below, or pharmaceutical compositions comprising at least one compound of formula (I) as defined above and below, in particular selected from compounds (A) and (B), or compositions (1) as defined above and below, for use as medicaments.

[0088] The present invention also relates to a compound (I) according to the invention as defined above and below, in particular a compound of formula (A) and / or (B) or a pharmaceutically acceptable salt thereof, or a composition (1) as defined above and below, or a pharmaceutical composition comprising at least one compound of formula (I) as defined above and below, in particular selected from compounds (A) and (B), or a composition (1) as defined above and below, for use in the treatment and / or prevention of a disease.

[0089] The present invention also relates to a compound (I) according to the invention as defined above and below, in particular a compound of formula (A) and / or (B) or a pharmaceutically acceptable salt thereof, or a composition (1) as defined above and below, or a pharmaceutical composition comprising at least one compound of formula (I) as defined above and below, in particular selected from compounds (A) and (B), or a composition (1) as defined above and below, for use in the treatment of proliferative disorders.

[0090] The present invention also relates to a compound (I) according to the invention as defined above and below, in particular a compound of formula (A) and / or (B) or a pharmaceutically acceptable salt thereof, or a composition (1) as defined above and below, or a pharmaceutical composition comprising at least one compound of formula (I) as defined above and below, in particular selected from compounds (A) and (B), or a composition (1) as defined above and below, for use in the treatment of genetic disorders.

[0091] The present invention also relates to a compound (I) according to the invention as defined above and below, in particular a compound of formula (A) and / or (B) or a pharmaceutically acceptable salt thereof, or a composition (1) as defined above and below, or a pharmaceutical composition comprising at least one compound of formula (I) as defined above and below, in particular selected from compounds (A) and (B), or a composition (1) as defined above and below, for use in the treatment of RASopathy.

[0092] The present invention also relates to a compound (I) according to the invention as defined above and below, in particular a compound of formula (A) and / or (B) or a pharmaceutically acceptable salt thereof, or a composition (1) as defined above and below, or a pharmaceutical composition comprising at least one compound of formula (I) as defined above and below, in particular selected from compounds (A) and (B), or a composition (1) as defined above and below, for use in the treatment of inflammatory diseases.

[0093] The present invention also relates to a compound (I) according to the invention as defined above and below, in particular a compound of formula (A) and / or (B) or a pharmaceutically acceptable salt thereof, or a composition (1) as defined above and below, or a pharmaceutical composition comprising at least one compound of formula (I) as defined above and below, in particular selected from compounds (A) and (B), or a composition (1) as defined above and below, for use in the treatment of endometriosis and / or adenomyosis, more particularly wherein KRAS is mutated.

[0094] The present invention also relates to a compound (I) according to the invention as defined above and below, in particular a compound of formula (A) and / or (B) or a pharmaceutically acceptable salt thereof, or a composition (1) as defined above and below, or a pharmaceutical composition comprising at least one compound of formula (I) as defined above and below, in particular selected from compounds (A) and (B), or a composition (1) as defined above and below, for use in the treatment of cancer.

[0095] The present invention also relates to a compound (I) according to the invention as defined above and below, in particular a compound of formula (A) and / or (B) or a pharmaceutically acceptable salt thereof, or a composition (1) as defined above and below, or a pharmaceutical composition comprising at least one compound of formula (I) as defined above and below, in particular selected from compounds (A) and (B), or a composition (1) as defined above and below, for use as an inhibitor of activation of RAS proteins (KRAS, HRAS or NRAS oncogenes).

[0096] The present invention also relates to a compound (I) according to the invention as defined above and below, in particular a compound of formula (A) and / or (B) or a pharmaceutically acceptable salt thereof, or a composition (1) as defined above and below, or a pharmaceutical composition comprising at least one compound of formula (I) as defined above and below, in particular selected from compounds (A) and (B), or a composition (1) as defined above and below, for use in the treatment or prevention of any disease or condition associated with the activity of a RAS protein (RAS oncogene).

[0097] The present invention also relates to compounds (I) according to the invention as defined above and below, in particular compounds of formula (A) and / or (B) or pharmaceutically acceptable salts thereof, or compositions (1) as defined above and below, or pharmaceutical compositions comprising at least one compound of formula (I) as defined above and below, in particular selected from compounds (A) and (B), or compositions (1) as defined above and below, for use in the treatment of proliferative disorders, inflammatory diseases and / or genetic disorders in which RAS signaling is involved, preferably in which KRAS G12V, NRAS G12V, HRAS G12V, KRAS G12A, KRAS G12C, KRAS G12D, KRASG12C / Y96D, KRAS G12C / Y96C, KRAS G12C / Y96S, KRAS G13C, KRAS G13D, KRAS G13S, KRAS Q61H, KRAS Q61R or KRAS Q61K is involved.

[0098] The present invention also relates to a pharmaceutical composition comprising at least one compound (I) according to the invention as defined above and below, in particular a compound of formula (A) and / or (B) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0099] The present invention also relates to a pharmaceutical composition as defined above and below for use as an inhibitor of RAS protein activation for the treatment and / or prevention of proliferative disorders and / or inflammatory diseases which are resistant to treatment with a RAS mutation-specific inhibitor different from the compound of formula (I), preferably, wherein the resistance is caused by a secondary mutation, in particular by a secondary mutation involving KRAS G12C / Y96D, KRAS G12C / Y96C and / or KRAS G12C / Y96S.

[0100] The present invention also relates to a pharmaceutical composition comprising at least one composition (1) as defined above and below and a pharmaceutically acceptable carrier.

[0101] The present invention also relates to a pharmaceutical composition comprising at least one compound (I) of the present invention as defined above and below, in particular a compound of formula (A) and / or (B) or a pharmaceutically acceptable salt thereof, or a composition (1) as defined above and below, wherein the pharmaceutical composition further comprises an additional active substance, preferably selected from chemotherapeutic agents, radiotherapeutic agents, immuno-oncology agents and combinations thereof.

[0102] The present invention also relates to a method for inhibiting the growth, proliferation or metastasis of cancer cells in an individual in need thereof, which method comprises administering to the individual a therapeutically effective amount of a compound (I) of the present invention as defined above and below, in particular a compound of formula (A) and / or (B) or a therapeutically acceptable salt thereof, or a composition (1) as defined above and below, or a pharmaceutical composition comprising at least one compound of formula (I) as defined above and below, in particular selected from compounds (A) and (B), or a composition (1) as defined above and below.

[0103] The present invention also relates to a method for inhibiting the proliferation of a cell population sensitive to inhibition of RAS activation in vitro or ex vivo, which method comprises contacting the cell population with a compound of formula (I) as defined above and below, in particular a compound of formula (A) and / or (B) or a therapeutically acceptable salt thereof, or a composition (1) as defined above and below, or a pharmaceutical composition comprising at least one compound of formula (I) as defined above and below, in particular selected from compounds (A) and (B), or a composition (1) as defined above and below.

[0104] The present invention also relates to a kit comprising a formulation comprising: a) a compound (I) of the present invention as defined above and below, in particular a compound of formula (A) and / or (B), or a composition (1) as defined above and below, or a pharmaceutical composition comprising a compound (I) of the present invention as defined above and below, in particular a compound of formula (A) and / or (B) or a therapeutically acceptable salt thereof, or a composition (1) as defined above and below, and a pharmaceutically acceptable carrier; and b) instructions for administration of the pharmaceutical composition for treating a disorder, wherein inhibition of RAS activation can effectively treat the disorder.

[0105] Description of the Invention

[0106] The present invention has the following advantages:

[0107] - The compounds of the invention show favorable RAS inhibitory properties. In other words, the compounds of the invention qualify as inhibitors of RAS oncogene activation. It is postulated that RAS-effector interactions are disrupted, especially when RAS is activated by oncogenic somatic mutations.

[0108] - These compounds inhibit KRAS at concentrations that are pharmacologically achievable in humans, regardless of mutation. The tolerability of these molecules is well studied, which could help translate these drugs to patients more quickly.

[0109] - These compounds inhibit NRAS and HRAS by functionally uncoupling their binding to their effectors in cells.

[0110] Compound of formula (I)

[0111] Inactivation in the sense of the present invention means the inhibition of the activity of a protein, in particular a RAS protein, in particular a NRAS, KRAS or HRAS protein, based on direct or indirect interaction of at least one compound of formula (I) with the protein, including the inhibition of complexes involving RAS proteins. This interaction is not a translation process and is not part of a translation process.

[0112] Furthermore, activation refers to the ability of RAS to bind its effector molecules such as RAF kinase, PI3K kinase in a GTP-dependent manner through the RAS binding domain (RBD) or RAS-associated domain (RA domain) present in effector proteins (such as RASSF).

[0113] As used herein, the term "synergistic" or "synergistic effect" refers to a therapeutic combination that is more effective than the additive effect of two or more single active agents. This means that the term "synergistic effect" refers to the effect of a given combination of two compounds, wherein the activity of the combination exceeds the sum of the individual activities of the compounds when applied alone. Therefore, based on the individual components, the combination can be used at a lower application rate to achieve a therapeutic effect comparable to the individual components.

[0114] The following formula is used to determine whether a combination of a compound of formula (I) and at least one additional compound different from the compound of formula (I) exhibits a synergistic effect:

[0115] E = X + Y - (X·Y / 100),

[0116] Where X = the effect expressed as a percentage of the compound of formula (I) used at the application rate a;

[0117] Y = effect expressed as a percentage of the compound of formula (II) used at the application rate b;

[0118] E = expected effect (in %) of compound of formula (I) + compound of formula (II) at application rate a+b.

[0119] In this formula, the value E corresponds to the effect that would be expected if the activities of the individual compounds were additive (inhibition). If the observed effect is higher than the value E calculated according to the formula, a synergistic effect is present.

[0120] The term "proliferative disorder" refers to a disorder associated with a certain degree of abnormal cell proliferation. Preferably, the proliferative disorder is cancer. In particular, the cancer is selected from prostate cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, gastric cancer, endometrial cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer (including melanoma and basal carcinoma), mesothelial lining cancer, white blood cell cancer (including lymphoma and leukemia), esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, lung cancer (including small cell lung cancer and non-small cell carcinoma), adrenal cancer, thyroid cancer, kidney cancer or bone cancer; or glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma (including Kaposi's sarcoma), choriocarcinoma, basal cell carcinoma of the skin, hematological malignancies (including blood, bone marrow and lymph nodes) or testicular seminoma.

[0121] Unless otherwise specifically stated herein, the singular number mentioned may also include the plural number. For example, "a" and "an" may refer to one, or one or more.

[0122] In the context of the present invention, the prefix C n -C m Indicates the number of carbon atoms that the molecule or residue specified thereby may contain.

[0123] In the context of the present invention, the expression C1-C4-alkyl refers to an unbranched or branched saturated hydrocarbon radical having 1 to 4 carbon atoms. C1-C4-alkyl is, for example, methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl.

[0124] The compounds of formula (I), (A) and (B) form salts which are also within the scope of the present invention. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, however, other salts are also useful, for example in separation or purification steps which may be used in the preparation process. For example, salts of compounds of formula (I), (A) and (B) may be formed, for example, by reacting the compounds of formula (I), (A) and (B) with at least one acid or base. The amount of acid or base added is suitable for partial or complete neutralization, for example an equivalent amount.

[0125] Unless otherwise indicated, the phrase "pharmaceutically acceptable salt" as used herein includes salts containing a pharmacologically acceptable anion or cation, such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate (hydrogen sulfate), phosphate, hydrogen phosphate, dihydrogen phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, sucrose, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate [i.e., 4,4'-methylene-bis-(3-hydroxy-2-naphthoate)].

[0126] Preferred compounds are those of formula (I) wherein

[0127] R 1 is H or C1-C2 alkyl;

[0128] R 2 is H or C1-C2 alkyl;

[0129] R 3 is H or C1-C2 alkyl;

[0130] R 4 is H or C1-C2 alkyl;

[0131] n is 2;

[0132] or a pharmaceutically acceptable salt thereof, for use as a RAS protein activation inhibitor.

[0133] In particular, R 1 , R 2 , R 3 and R 4 Have the same meaning.

[0134] In particular, R 1 , R 2 , R 3 and R 4 For hydrogen.

[0135] In a first embodiment, the compound of formula (I) is compound A (also known as 1,4-dihydroxy-5,8-bis({2-[(2-hydroxyethyl)amino]ethyl}amino)-9,10-anthraquinone], mitoxantrone, CAS No. 65271-80-9)

[0136]

[0137] or a pharmaceutically acceptable salt thereof, for use as a RAS protein activation inhibitor.

[0138] Preferably, the present invention relates to Compound A or a pharmaceutically acceptable salt thereof for use as an inhibitor of RAS protein activation for the treatment and / or prevention of diseases in which RAS signaling is involved.

[0139] In particular, the present invention relates to Compound A or a pharmaceutically acceptable salt thereof for use as an inhibitor of RAS protein activation for the treatment and / or prevention of proliferative disorders, inflammatory diseases and / or genetic disorders in which RAS signaling is involved.

[0140] In particular, the present invention relates to compound A or a pharmaceutically acceptable salt thereof for use as an inhibitor of RAS protein activation for the treatment and / or prevention of proliferative disorders, inflammatory diseases and / or genetic disorders, wherein KRAS G12V, NRASG12V, HRAS G12V, KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12C / Y96D, KRAS G12C / Y96C, KRAS G12C / Y96S, KRAS G13C, KRAS G13D, KRAS G13S, KRAS Q61H, KRAS Q61R or KRAS Q61K is involved.

[0141] In another preferred embodiment, the invention relates to compound A or a pharmaceutically acceptable salt thereof for use as an inhibitor of RAS protein activation for the treatment and / or prevention of proliferative disorders and / or inflammatory diseases which are resistant to treatment with RAS mutation-specific inhibitors other than the compound of formula (I), preferably, wherein the resistance is caused by secondary mutations, in particular by secondary mutations involving KRAS G12C / Y96D, KRAS G12C / Y96C and / or KRAS G12C / Y96S.

[0142] In a second embodiment, the compound of formula (I) is compound B (also known as 1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10-anthraquinone dihydrochloride], mitoxantrone hydrochloride, CAS No. 70476-82-3)

[0143]

[0144] Acts as an inhibitor of RAS protein activation.

[0145] Preferably, the present invention relates to Compound B or a pharmaceutically acceptable salt thereof for use as an inhibitor of RAS protein activation for the treatment and / or prevention of diseases in which RAS signaling is involved.

[0146] In particular, the present invention relates to Compound B or a pharmaceutically acceptable salt thereof for use as an inhibitor of RAS protein activation for the treatment and / or prevention of proliferative disorders, inflammatory diseases and / or genetic disorders in which RAS signaling is involved.

[0147] In particular, the present invention relates to compound B or a pharmaceutically acceptable salt thereof for use as an inhibitor of RAS protein activation for the treatment and / or prevention of proliferative disorders, inflammatory diseases and / or genetic disorders, wherein KRAS G12V, NRASG12V, HRAS G12V, KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12C / Y96D, KRAS G12C / Y96C, KRAS G12C / Y96S, KRAS G13C, KRAS G13D, KRAS G13S, KRAS Q61H, KRAS Q61R or KRAS Q61K is involved.

[0148] In another preferred embodiment, the present invention relates to compound B or a pharmaceutically acceptable salt thereof for use as an inhibitor of RAS protein activation for the treatment and / or prevention of proliferative disorders and / or inflammatory diseases which are resistant to treatment with RAS mutation-specific inhibitors other than the compound of formula (I), preferably, wherein the resistance is caused by a secondary mutation, in particular by a secondary mutation involving KRAS G12C / Y96D.

[0149] Another aspect of the present invention is a composition (1) comprising at least one compound of formula (I) as defined above and below, or a pharmaceutically acceptable salt thereof, and at least one RAS mutation-specific inhibitor.

[0150] In one embodiment, the composition (1) comprises at least one compound (A) or a pharmaceutically acceptable salt thereof and at least one RAS mutation-specific inhibitor, in particular at least one KRAS mutation-specific inhibitor, in particular sotolacib (also known as AMG510 or (1M)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-[4-methyl-2-(propan-2-yl)pyridin-3-yl]-4-[(2S)-2-methyl-4-(propan-2-yl)pyridin-3-yl]-

[00136] The invention relates to the preparation of pharmaceutical compositions comprising the following: (a) methylpiperazine-1-yl]pyrido[2,3-d]pyrimidin-2(1H)-one) and / or adagracib (also known as MRTX848 or (2S)-4-[7-(8-chloro-1-naphthyl)-5,6,7,8-tetrahydro-2-[[(2"S")-1-methyl-2-pyrrolidinyl]methoxy]pyrido[3,4-"d"]pyrimidin-4-yl]-1-(2-fluoro-1-oxo-2-propen-1-yl)-2-piperazineacetonitrile).

[0151] In a second embodiment, composition (1) comprises at least one compound (B) or a pharmaceutically acceptable salt thereof and at least one RAS mutation-specific inhibitor, in particular at least one KRAS mutation-specific inhibitor, in particular sotolacib and / or adagracib.

[0152] Pharmaceutical composition

[0153] As used herein, the phrase "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0154] The phrase "therapeutically effective" is intended to qualify the amount of each active agent that will achieve the goal of improving the severity and incidence of the disorder while avoiding the adverse side effects typically associated with alternative therapies. For example, an effective anticancer agent may prolong a patient's survival or quality of life, inhibit the growth of rapidly proliferating cells associated with tumor formation, or cause tumor regression.

[0155] As used herein, the terms "treat", "treating" and "treatment" refer to any type of intervention or process performed or administered to an individual with the intent to reverse, alleviate, ameliorate, inhibit or slow down or prevent the progression, development, severity or recurrence of symptoms, complications, symptoms or biochemical indicators associated with a disease. In contrast, "prophylaxis" or "prevention" refers to administration to an individual who does not have a disease to prevent the disease from occurring.

[0156] As used herein, the term "cell" means a cell in vitro, ex vivo or in vivo. In the sense of the present invention, an ex vivo cell can be a part of a tissue sample cut from an organism, such as a mammal. In the sense of the present invention, an in vitro cell can be a cell in a cell culture. In the sense of the present invention, an in vivo cell is a living cell in an organism, such as a mammal.

[0157] The term "patient" includes humans and animals receiving therapeutic or prophylactic treatment.

[0158] The term "subject" includes any human or animal. For example, the methods and compositions disclosed herein can be used to treat a subject having cancer.

[0159] (Non-human) animals include all vertebrates, such as mammals and non-mammals, including cattle, sheep, pigs, goats, horses, poultry, dogs, cats, non-human primates, rodents, etc. In one embodiment, the subject is a human subject.

[0160] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid diluent, solvent, excipient, manufacturing aid (e.g., lubricant), or encapsulating material, which is involved in carrying or transporting the subject compound from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0161] Suitable other ingredients are the above-mentioned carriers and other additives, including adjuvants, preservatives, fillers, flow regulators, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, bittering agents, flavor enhancers, antibacterial agents, antifungal agents, lubricants, dispersants, etc. Suitable additives are selected depending on the mode of administration and the nature of the dosage form; and will not cause harm to the patient.

[0162] The term "pharmaceutical composition" refers to a composition comprising a compound of the present invention in combination with at least one additional compound selected from:

[0163] a) at least one additional pharmaceutically active substance; and

[0164] b) at least one additional pharmaceutically acceptable carrier and or additive.

[0165] The term "RAS inhibitor" refers to an agent that can reduce RAS protein levels, reduce RAS activity levels and / or inhibit RAS expression levels in cells. RAS inhibitors can be reversible or irreversible inhibitors. As used herein, "RAS" protein refers to a protein that is a member of a related protein family expressed in all human and animal cell lineages and organs. All RAS protein family members belong to a class of proteins called small GTPases (also known as small G proteins, a family of hydrolases that can bind and hydrolyze GTP), and are involved in intracellular signaling (cell signal transduction). RAS is a prototype member of the RAS protein superfamily, which is related in three-dimensional structure and regulates different cell behaviors. When RAS is "turned on" by an input signal, it will then turn on other proteins, and eventually turn on genes related to cell growth, differentiation and survival. Mutations in RAS genes can lead to the production of permanently activated RAS proteins, even in the absence of input signals, causing unexpected and overactive signal transduction in cells. Because these signals cause cell growth and division, overactive RAS signal transduction may eventually lead to cancer. Three kinds of RAS genes (HRAS, KRAS and NRAS) of people are the most common oncogenes in human cancer.As mentioned above, the most noteworthy members of RAS subfamily clinically are HRAS, KRAS and NRAS.However, this subfamily also has other members, for example, selected from DIRAS1, DIRAS2, DIRAS3, ERAS, GEM, MRAS, NKIRAS1, NKIRAS2, NRAS, RALA, RALB, RAP1A, RAP1B, RAP2A, RAP2B, RAP2C, RASD1, RASD2, RASL10A, RASL10B, RASL11A, RASL11B, RASL12, REM1, REM2, RERG, RERGL, RRAD, RRAS, RRAS2. In other words, the most common alterations in NRAS were NRAS mutation (2.87%), NRAS exon 3 mutation (1.90%), NRAS exon 3 missense (1.88%), NRAS codon 61 missense (1.72%), and NRAS exon 2 mutation (0.95%). The most common alterations in HRAS were HRAS mutation (0.77%), HRAS missense (0.75%), HRAS exon 2 mutation (0.30%), HRAS codon 61 missense (0.26%), and HRAS Q61R (0.14%). The most common alterations in HRAS were HRAS mutation (0.77%), HRAS missense (0.75%), HRAS exon 2 mutation (0.30%), HRAS codon 61 missense (0.26%), and HRAS Q61R (0.14%) (Source Mycancer genome portal).

[0166] The compounds of formula (I), (A) and (B) as defined above and the composition (1) as defined above and the pharmaceutical compositions comprising at least one compound of formula (I), (A) and (B) as defined above or the composition (1) as defined above can be administered to humans and animals, preferably humans.

[0167] Generally, any administration method can be used to deliver the compounds or pharmaceutical compositions of the present invention to an individual. Suitable administration methods are oral, enteral, parenteral, intravenous, topical, intramuscular, subcutaneous routes.

[0168] The compounds of formula (I), (A) and (B) as defined above can selectively reduce RAS protein levels in cells, reduce RAS activity levels, and particularly reduce HRAS and NRAS, particularly KRAS4A and KRAS4B) activity levels. For example, the compounds of formula (I), (A) and (B) as defined above can be used to selectively reduce RAS activity levels in cells or individuals in which RAS protein levels need to be reduced, by administering an inhibitory amount of the compounds of formula (I), (A) and (B) as defined above or a salt thereof to reduce RAS activity levels.

[0169] In one embodiment, the present invention provides a combination preparation of a compound of formula (I), (A) and (B) as defined above and / or a pharmaceutically acceptable salt thereof and an additional therapeutic agent for simultaneous, separate or sequential use in the treatment and / or prevention of (multiple) diseases, preferably proliferative disorders (e.g. cancer), in particular disorders associated with RAS protein activity.

[0170] The additional therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a radiotherapeutic agent, an immuno-oncology agent, and combinations thereof.

[0171] In one aspect, the compounds of formula (I), (A) and (B) as defined above are administered sequentially prior to administration of the immuno-oncology agent. In another aspect, the compounds of formula (I), (A) and (B) as defined above are administered simultaneously with the immuno-oncology agent. In yet another aspect, the compounds of formula (I), (A) and (B) as defined above are administered sequentially after administration of the immuno-oncology agent.

[0172] In another aspect, the compounds of formula (I), (A) and (B) as defined above may be co-formulated with an immuno-oncology agent.

[0173] Immuno-oncology agents include, for example, small molecule drugs, antibodies or other biological molecules or small molecules. Examples of biological immuno-oncology agents include, but are not limited to, cancer vaccines, antibodies and cytokines. In one aspect, the antibody is a monoclonal antibody. In another aspect, the monoclonal antibody is a humanized or human antibody.

[0174] In one aspect, the immuno-oncology agent is either (i) a stimulatory (including co-stimulatory) receptor agonist or (ii) an inhibitory (including co-inhibitory) signal antagonist on T cells, both of which result in amplification of antigen-specific T cell responses (commonly referred to as immune checkpoint modulators).

[0175] Suitable stimulatory and inhibitory molecules are members of the immunoglobulin superfamily (IgSF). An important family of membrane-bound ligands in conjunction with costimulatory or co-inhibitory receptors is the B7 family, which includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA) and B7-H6. Another family of membrane-bound ligands that bind to co-stimulatory or co-inhibitory receptors is the family of TNF molecules that bind to cognate TNF receptor family members, which include CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR / Fnl4, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTpR, LIGHT, DcR3, HVEM, VEGETL1A, TRAMP / DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin a / TNFp, TNFR2, TNFa, LTpR, lymphotoxin a 1b2, FAS, FASL, RELT, DR6, TROY, NGFR.

[0176] In one aspect, T cell responses may be stimulated using a combination of compounds of formula (I), (A) and (B) as defined above and one or more of the following:

[0177] (i) protein antagonists that inhibit T cell activation (e.g., immune checkpoint inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, galectin-9, CEACAM-1, BTLA, CD69, galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4; and

[0178] (ii) Protein agonists that stimulate T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD 137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3, and CD28H.

[0179] Other active agents that can be combined with the compounds of formula (I), (A) and (B) as defined above to treat cancer include inhibitory receptor antagonists on NK cells or activating receptor agonists on NK cells. For example, the compounds of formula (I), (A) and (B) as defined above can be combined with a KIR antagonist, such as lirelumab.

[0180] Other agents for combination therapy include agents that inhibit or deplete macrophages or monocytes, including but not limited to CSF-1R antagonists, such as CSF-1R antagonist antibodies, including RG7155.

[0181] Combination therapy is intended to include administration of the therapeutic agents in a sequential manner, ie, each therapeutic agent is administered at a different time, as well as administration of the therapeutic agents, or at least two therapeutic agents, in a substantially simultaneous manner.

[0182] For example, by administering a single dosage form of each therapeutic agent with a fixed ratio or a plurality of single dosage forms of each therapeutic agent to an individual, substantially simultaneous administration can be achieved. Each therapeutic agent can be administered sequentially or substantially simultaneously by any appropriate route, including but not limited to oral route, intravenous route, intramuscular route and direct absorption by mucosal tissue. The therapeutic agent can be administered by the same route or different routes. For example, the first therapeutic agent of the selected combination can be administered by intravenous injection, and the other therapeutic agents of the combination can be administered orally. Optionally, for example, all therapeutic agents can be administered orally or all therapeutic agents can be administered by intravenous injection. Combination therapy can also include further combination administration of therapeutic agents as described above with other biologically active ingredients and non-drug therapies (such as surgery or radiotherapy). In the case where combination therapy also includes non-drug therapy, non-drug therapy can be performed at any suitable time, as long as the beneficial effects of the combined action of the therapeutic agent and non-drug therapy combination are achieved. For example, in appropriate cases, when non-drug therapy is temporarily removed from the administration of the therapeutic agent, it may take several days or even weeks to still achieve a beneficial effect.

[0183] The types of cancer that may be treated with the compounds of formula (I), (A) and (B) as defined above include, but are not limited to, prostate cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, stomach cancer, endometrial cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer (including melanoma and basal carcinoma), mesothelial lining cancer, white blood cell cancer (including lymphoma and leukemia), esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, lung cancer (including small cell lung cancer and non-small cell carcinoma), adrenal cancer, thyroid cancer, kidney cancer or bone cancer; or glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma (including Kaposi's sarcoma), choriocarcinoma, basal cell carcinoma of the skin, hematological malignancies (including blood, bone marrow and lymph nodes) or testicular seminoma.

[0184] In one embodiment, the present invention relates to inhibiting HRAS mutations, which are detected in bladder urothelial carcinoma, breast invasive ductal carcinoma, lung adenocarcinoma, prostate cancer and colon adenocarcinoma. This accounts for about 0.94% of all human cancers and about 1.02% of solid tumors. In another embodiment, the present invention relates to inhibiting HRAS mutations, which are detected in other cancers, and the other cancers are selected from chronic myelomonocytic leukemia, non-Hodgkin's lymphoma, thyroid cancer, head and neck squamous cell carcinoma, squamous cell lung cancer, ovarian cancer, poorly differentiated thyroid cancer, squamous cell carcinoma, small cell lung cancer, glioma, low-grade glioma, pancreatic cancer, acute lymphoblastic leukemia, histiocytic and dendritic cell tumors, multiple myeloma, neurofibromatosis type, pancreatic ductal adenocarcinoma, Follicular thyroid carcinoma, embryonal rhabdomyosarcoma, malignant thyroid tumors, undifferentiated (anaplastic) thyroid carcinoma, thymic carcinoma, urothelial carcinoma, papillary thyroid carcinoma, cutaneous melanoma, mucosal melanoma, endometrial carcinoma, malignant peripheral nerve sheath tumor, neuroblastoma, prostate cancer, soft tissue sarcoma, breast cancer, colorectal adenocarcinoma, gastric cancer, diffuse large B-cell lymphoma, diffuse glioma, myelodysplastic syndrome, renal cell carcinoma, astrocytoma, hepatocellular carcinoma and schwannoma.

[0185] Among all human cancers, NRAS mutations are detected in nearly 3.03% of cancers, of which common mutations are in skin melanoma, melanoma, colon adenocarcinoma, aml, thyroid cancer, and lung adenocarcinoma. Therefore, another embodiment of the present invention relates to inhibiting NRAS mutations detected in skin melanoma, melanoma, colon adenocarcinoma, aml, thyroid cancer, and lung adenocarcinoma. This accounts for nearly 2.83% of patients with malignant solid tumors. In another embodiment, the present invention relates to inhibiting NRAS mutations, which are also detected in other cancers, and the cancers are selected from colorectal cancer, non-small cell lung cancer, acute myeloid leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, colorectal adenocarcinoma, multiple myeloma, non-Hodgkin's lymphoma, pancreatic cancer, skin melanoma, ovarian cancer, pancreatic ductal adenocarcinoma, acute lymphocytic leukemia, thyroid cancer, glioma, neurofibroma type 1, poorly differentiated thyroid cancer, secondary acute myeloid leukemia, treatment-related acute myeloid leukemia, Myelodysplastic syndrome with excess blast-2, juvenile myelomonocytic leukemia, histiocytic and dendritic cell tumors, head and neck squamous cell carcinoma, small cell lung cancer, low-grade glioma, squamous cell lung cancer, breast cancer, chronic myelomonocytic leukemia-2, chronic myelomonocytic leukemia, anaplastic thyroid carcinoma, embryonal rhabdomyosarcoma, follicular thyroid carcinoma, T-cell acute lymphoblastic leukemia, mucosal melanoma, chronic myelomonocytic leukemia-1, low-grade ovarian serous adenocarcinoma, papillary thyroid carcinoma, Refractory anemia of myeloid cells, myeloid neoplasms, myelodysplastic / myeloproliferative neoplasms, unclassifiable, rectal cancer, colon cancer, malignant peripheral nerve sheath tumor, bile duct cancer, endometrial cancer, mantle cell lymphoma, secondary myelodysplastic syndrome, therapy-related myelodysplastic syndrome, lymphoma neuronal and mixed neuronal glioma, ganglioglioma, soft tissue sarcoma, bladder cancer, esophageal cancer, sarcoma, thymic carcinoma, lung adenocarcinoma, lung cancer, uveal melanoma, head and neck cancer, diffuse glioma, squamous cell carcinoma, chronic myeloid leukemia, gastric esophageal cancer Tubulotubular junction adenocarcinoma, glioblastoma, neuroblastoma, astrocytoma, hepatocellular carcinoma, pancreatic adenocarcinoma, diffuse large B-cell lymphoma, anaplastic astrocytoma, gastric adenocarcinoma, gastric cancer, prostate cancer, renal cell carcinoma, acute myeloid leukemia due to a previous myelodysplastic syndrome, B-cell acute lymphoblastic leukemia, double hit lymphoma, dysembryoplastic neuroepithelioma, ganglioneuroma, low-grade neuroepithelioma, peripheral T-cell lymphoma, pilocytic astrocytoma, pilocytic myxoid astrocytoma, rhabdoid tumor, and schwannoma.

[0186] One or more additional pharmaceutical agents or treatments, such as antiviral agents, chemotherapeutic agents or other anticancer agents, immunopotentiators, immunosuppressants, radiation, anti-tumor and antiviral vaccines, cytokine therapy (e.g., IL2 and GM-CSF) and / or tyrosine kinase inhibitors may optionally be combined with the compounds of formula (I), (A) and (B) as defined above for the treatment of RAS protein-related diseases, disorders or conditions. These agents may be combined with the compounds of the invention in a single dosage form, or these agents may be administered simultaneously or sequentially as separate dosage forms.

[0187] Suitable chemotherapeutic or other anticancer agents include, for example, alkylating agents (including, but not limited to, nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes), such as uracil mustard, chlormethine, cyclophosphamide, Ifosfamide, melphalan, chlorambucil, piperobroman, triethylenetetracycline, triethylenetetracycline, busulfan, carmustine, lomustine, streptozotocin, dacarbazine, and temozolomide.

[0188] In the treatment of melanoma, suitable active agents for combination with the compounds of formula (I), (A) and (B) as defined above include: dacarbazine (DTIC), optionally with other chemotherapeutic agents such as carmustine (BCNU) and cisplatin; the "Dartmouth regimen", which consists of DTIC, BCNU, cisplatin and tamoxifen; cisplatin, vinblastine and DTIC, temozolomide or YERVOY TM In the treatment of melanoma, the compounds of formula (I), (A) and (B) as defined above may also be combined with immunotherapeutic drugs, including cytokines, such as interferon alpha, interleukin 2 and tumor necrosis factor (TNF). In the treatment of melanoma, the compounds of formula (I), (A) and (B) as defined above may also be used in combination with vaccine treatment. Anti-melanoma vaccines are similar in some respects to antiviral vaccines used to prevent diseases caused by viruses, such as polio, measles and mumps. Weakened melanoma cells or parts of melanoma cells called antigens can be injected into the patient to stimulate the body's immune system to destroy the melanoma cells.

[0189] Melanoma localized to the arm or leg may also be treated using a hyperthermic ex vivo limb perfusion technique with a combination of active agents including one or more compounds of formula (I), (A) and (B) as defined. This treatment regimen temporarily separates the circulation of the affected limb from the rest of the body and injects high doses of the chemotherapy drug into the artery supplying the limb, thereby providing high doses to the tumor area without exposing internal organs to these doses, which may otherwise cause serious side effects. Typically, the fluid is warmed to 38.9°C-40°C. Melphalan is the most commonly used drug in this chemotherapy method. It can be given together with another active agent called tumor necrosis factor (TNF).

[0190] Suitable chemotherapeutic or other anticancer agents include, for example, antimetabolites (including but not limited to folate antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors), such as methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, and gemcitabine.

[0191] Suitable chemotherapeutic or other anticancer agents also include, for example, certain natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines and epipodophyllotoxins), such as vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, ara-C, paclitaxel (taxol), mithramycin, deoxycoformycin, mitomycin-C, L-asparaginase, interferons (particularly IFN-a), etoposide and teniposide.

[0192] Other cytotoxic agents include navelbine, CPT-11, anastrozole, letrozole, capecitabine, raloxifene, and droloxifene.

[0193] Also suitable are cytotoxic agents such as epidophyllotoxin; antitumor enzymes; topoisomerase inhibitors; procarbazine; mitoxantrone; platinum coordination complexes such as cisplatin and carboplatin; biological response modifiers; growth inhibitory agents; antihormonal therapeutics; leucovorin; tegafur; and hematopoietic growth factors.

[0194] Other anticancer agents include antibody therapeutics such as trastuzumab Antibodies against co-stimulatory molecules, such as CTLA-4, 4-1BB, and PD-1, or against cytokines (IL-10 or TGF-b).

[0195] Other anti-cancer agents include those that block the migration of immune cells, such as antagonists to chemokine receptors including CCR2 and CCR4.

[0196] Other anti-cancer agents also include those that enhance the immune system, such as adjuvants or adoptive T cell transfer.

[0197] Anticancer vaccines include dendritic cells, synthetic peptides, DNA vaccines, and recombinant viruses.

[0198] In a particular embodiment of the invention, at least one compound of formula (I), (A) and (B) as defined above and at least one chemotherapeutic agent are administered to the patient simultaneously or sequentially. In other words, at least one compound of formula (I), (A) and (B) as defined above may be administered first, at least one chemotherapeutic agent may be administered first, or at least one compound of formula (I), (A) and (B) as defined above may be administered simultaneously. In addition, when more than one compound of formula (I), (A) and (B) as defined above and / or chemotherapeutic agent is used, the compounds may be administered in any order.

[0199] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of one or more compounds of formula (I), (A) and (B) as defined above, formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents; and optionally one or more additional therapeutic agents as described above.

[0200] The compounds of formula (I), (A) and (B) as defined above may be administered by any suitable route, preferably in the form of a pharmaceutical composition suitable for such route and in a dose effective for the intended treatment. The compounds and compositions of the compounds of formula (I), (A) and (B) as defined above may be administered by any suitable means for any use described herein, for example, orally, such as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders, granules, elixirs, tinctures, suspensions (including nanosuspensions, microsuspensions, spray-dried dispersions), syrups and emulsions; sublingual; buccal; parenteral, such as by subcutaneous, intravenous, intramuscular or intrasternal injection, or infusion techniques (such as sterile injectable aqueous or non-aqueous solutions or suspensions); nasal, including, for example, by inhalation sprays applied to the nasal membrane; topical, such as in the form of creams or ointments; or rectal, such as in the form of suppositories. They may be administered alone, but will typically be administered with a pharmaceutical carrier selected based on the selected route of administration and standard pharmaceutical practice.

[0201] For oral administration, the pharmaceutical composition can be in the form of, for example, tablets, capsules, liquid capsules, suspensions or liquids. The pharmaceutical composition is preferably prepared in the form of a dosage unit, which contains a specific amount of active ingredient. For example, the pharmaceutical composition can be provided as a tablet or capsule, and the amount of the active ingredient contained therein ranges from about 0.1 to 1000 mg, preferably from about 0.25 to 250 mg, and more preferably from about 0.5 to 100 mg. The daily dose suitable for humans or animals can be different in a wide range, depending on the patient's condition and other factors, but can be determined using conventional methods.

[0202] For example, any pharmaceutical composition that this paper pays attention to can be delivered orally, for example, by any acceptable and suitable oral formulation. Exemplary oral formulations include, but are not limited to, for example, tablets, lozenges, lozenges, aqueous and oily suspensions, dispersible powders or granules, emulsions, hard capsules and soft capsules, liquid capsules, syrups and elixirs. The pharmaceutical composition intended for oral administration can be prepared according to any method known in the art for preparing the pharmaceutical composition intended for oral administration. In order to provide pharmaceutically palatable preparations, the pharmaceutical composition of the present invention can include at least one agent selected from sweeteners, flavoring agents, bittering agents, coloring agents, demulcents, antioxidants and preservatives.

[0203] Tablets may be prepared, for example, by mixing at least one compound of formula (I), (A) and (B) as defined above and / or at least one pharmaceutically acceptable salt thereof with at least one non-toxic pharmaceutically acceptable excipient suitable for preparing tablets. Exemplary excipients include, but are not limited to, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate and sodium phosphate; granulating agents and disintegrants such as microcrystalline cellulose, crosslinked sodium carboxymethyl cellulose, corn starch and alginic acid; binders such as starch, gelatin, polyvinyl pyrrolidone and gum arabic; and lubricants such as magnesium stearate, stearic acid and talc. In addition, tablets may be uncoated or coated by known techniques to mask the unpleasant taste of unpleasant tasting drugs or to delay the disintegration and absorption of the active ingredient in the gastrointestinal tract, thereby maintaining the action of the active ingredient over a longer period of time. Exemplary water-soluble taste masking materials include, but are not limited to, hydroxypropyl methylcellulose and hydroxypropyl cellulose. Exemplary time delay materials include, but are not limited to, ethyl cellulose and cellulose acetate butyrate.

[0204] Hard gelatin capsules can be prepared, for example, by mixing at least one compound of formula (I), (A) and (B) as defined above and / or at least one salt thereof with at least one inert solid diluent, such as calcium carbonate, calcium phosphate and kaolin.

[0205] Soft gelatin capsules can be prepared, for example, by mixing at least one compound of formula (I), (A) and (B) as defined above and / or at least one pharmaceutically acceptable salt thereof with at least one water-soluble carrier, such as polyethylene glycol; and at least one oily medium, such as peanut oil, liquid paraffin and olive oil.

[0206] Aqueous suspensions can be prepared, for example, by mixing at least one compound of formula (I), (A) and (B) as defined above and / or at least one pharmaceutically acceptable salt thereof with at least one excipient suitable for preparing aqueous suspensions. Exemplary excipients suitable for preparing aqueous suspensions include, but are not limited to, for example, suspending agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose and hydroxypropyl cellulose, sodium alginate, alginic acid, polyvinyl pyrrolidone, gum tragacanth and gum arabic; dispersants or wetting agents such as naturally occurring phospholipids such as lecithin; condensation products of alkylene oxides with fatty acids such as polyoxyethylene stearate; condensation products of ethylene oxide with long chain fatty alcohols such as heptadecaethyleneoxyhexadecanoate; condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol such as polyoxyethylene sorbitol monooleate; and condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides such as polyethylene sorbitan monooleate. The aqueous suspension may also contain at least one preservative, such as ethyl and n-propyl p-hydroxybenzoate; at least one coloring agent; at least one flavoring agent; and / or at least one sweetening agent, including, but not limited to, for example, sucrose, saccharin, and aspartame.

[0207] Oily suspensions can be prepared, for example, by suspending at least one formula (I), (A) and (B) compound as defined above and / or at least one pharmaceutically acceptable salt thereof in a vegetable oil or in a mineral oil, wherein the vegetable oil is such as peanut oil, olive oil, sesame oil and coconut oil, and the mineral oil is such as liquid paraffin. Oily suspensions can also include at least one thickening agent, such as beeswax, hard paraffin and cetyl alcohol. In order to provide a palatable oily suspension, at least one sweetener and / or at least one flavoring agent described above can be added to the oily suspension. Oily suspensions can also include at least one preservative, including but not limited to, for example, antioxidants, such as butylated hydroxyanisole and alpha-tocopherol.

[0208] Dispersible powders and granules can be prepared, for example, by mixing at least one compound of formula (I), (A) and (B) as defined above and / or at least one pharmaceutically acceptable salt thereof with at least one dispersant and / or wetting agent; at least one suspending agent; and / or at least one preservative. Suitable dispersants, wetting agents and suspending agents are as described above. Exemplary preservatives include, but are not limited to, antioxidants, such as ascorbic acid. In addition, dispersible powders and granules may also contain at least one excipient, including, but not limited to, for example, sweeteners; flavoring agents; and coloring agents.

[0209] For example, an emulsion of at least one compound of formula (I), (A) and (B) as defined above and / or at least one pharmaceutically acceptable salt thereof can be prepared as an oil-in-water emulsion. The oil phase of the emulsion comprising the compound of formula (I), (A) and (B) as defined above can be composed of known ingredients in a known manner. The oil phase can be provided by, but not limited to, for example, vegetable oils, such as olive oil and peanut oil; mineral oils, such as liquid paraffin; and mixtures thereof. Although the phase can contain only an emulsifier, it can contain a mixture of at least one emulsifier with a fat or oil or with both a fat and an oil. Suitable emulsifiers include, but are not limited to, for example, naturally occurring phospholipids, such as soy lecithin; esters or partial esters derived from fatty acids and hexanol anhydride, such as sorbitan monooleate; and condensation products of partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier used as a stabilizer. It is also preferred to include both oil and fat. Emulsifier (whether stable or not) together constitutes so-called emulsifying wax, and wax and oil and fat together constitute so-called emulsifier ointment base, which forms the oily dispersed phase of cream. Emulsion can also include sweetener, flavoring agent, preservative and / or antioxidant. Emulsifier and emulsion stabilizer suitable for preparation of the present invention include Tween 60, Span 80, cetearyl alcohol, myristyl alcohol, glyceryl monostearate, sodium lauryl sulfate, distearic acid glyceryl used alone or with wax, or other materials well known in the art.

[0210] For example, the compounds of formula (I), (A) and (B) as defined above and / or at least one pharmaceutically acceptable salt thereof may also be delivered intravenously, subcutaneously and / or intramuscularly via any pharmaceutically acceptable and suitable injection form. Exemplary injectable forms include, but are not limited to, for example, sterile aqueous solutions containing acceptable vehicles and solvents such as water, Ringer's solution and isotonic sodium chloride solution; sterile oil-in-water microemulsions and aqueous or oily suspensions.

[0211] Preparations for parenteral administration can be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions can be made of sterile powders or granules, using one or more carriers or diluents mentioned for oral administration preparations or by using other suitable dispersants or wetting agents and suspending agents. The compound can be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, tragacanth gum and / or a variety of buffers. Other adjuvants and modes of administration are well known in the pharmaceutical field. The active ingredient can also be used as a composition containing a suitable carrier by injection, and the carrier includes saline, dextrose, water or cyclodextrin solubilization (i.e. Captisol), co-solvent solubilization (i.e. propylene glycol) or micellar solubilization (i.e. Tween 80).

[0212] Sterile injectable preparations can also be sterile injectable solutions or suspensions in nontoxic parenteral acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are generally used as solvents or suspending media. For this reason, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can be used to prepare injectables.

[0213] Sterile injectable oil-in-water microemulsions can be prepared, for example, by the following steps: 1) dissolving at least one compound of formula (I), (A) and (B) as defined above in an oil phase, for example a mixture of soybean oil and lecithin; 2) combining the oil phase containing the compound of formula (I), (A) and (B) as defined above with a mixture of water and glycerol; and 3) processing the combination to form a microemulsion.

[0214] Sterile aqueous or oily suspensions can be prepared according to methods known in the art. For example, sterile aqueous solutions or suspensions can be prepared with nontoxic parenteral acceptable diluents or solvents, such as 1,3-butanediol; and sterile oily suspensions can be prepared with sterile nontoxic acceptable solvents or suspending media, such as sterile fixed oils, such as synthetic monoglycerides or diglycerides; and fatty acids, such as oleic acid.

[0215] Pharmaceutically acceptable carriers are formulated according to many factors within the scope of ordinary technicians in the field. These factors include, but are not limited to: the type and nature of the active agent to be formulated; the individual to whom the active agent-containing composition is administered; the intended route of administration of the composition; and the targeted therapeutic indication. Pharmaceutically acceptable carriers include aqueous and non-aqueous liquid media, as well as a variety of solid and semisolid dosage forms. In addition to the active agent, such carriers may also include many different ingredients and additives, and such additional ingredients are included in the formulation for a variety of reasons, such as the stability of the active agent, adhesive, etc., which is well known to ordinary technicians in the field. Descriptions of suitable pharmaceutically acceptable carriers and factors involved in their selection can be found in a variety of readily available resources, for example, Allen, LVJr. et al., Remington: The Science and Practice of Pharmacy (Volume 2), 22nd Edition (2012), Pharmaceutical Press.

[0216] Pharmaceutically acceptable carriers, adjuvants and vehicles that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-α-tocopheryl polyethylene glycol 1000 succinate, surfactants for pharmaceutical dosage forms such as Tween, polyethoxylated castor oil such as CREMOPHOR surfactant (BASF), or other similar polymer delivery matrices, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin. Cyclodextrins, such as α-, β-, and γ-cyclodextrin, or chemically modified derivatives, such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropylcyclodextrin, or other solubilizing derivatives may also be advantageously used to enhance delivery of compounds of the formulae described herein.

[0217] The pharmaceutically active compounds of the present invention can be processed according to conventional pharmaceutical methods to prepare medicines for administration to patients (including humans and other mammals). Pharmaceutical compositions can be subjected to conventional pharmaceutical operations, such as sterilization and / or can contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc. Tablets and pills can also be prepared with enteric coatings. Such compositions can also contain adjuvants, such as wetting agents, sweeteners, flavoring agents, and flavor enhancers.

[0218] For therapeutic purposes, the active compound of the present invention is usually combined with one or more adjuvants suitable for specifying the route of administration. If administered orally, the compound can be mixed with lactose, sucrose, starch powder, cellulose alkanoate, cellulose alkyl ester, talcum powder, stearic acid, magnesium stearate, magnesium oxide, sodium salt and calcium salt of phosphoric acid and sulfuric acid, gelatin, gum arabic, sodium alginate, polyvinyl pyrrolidone and / or polyvinyl alcohol, then tableted or encapsulated for ease of use. Such capsules or tablets can include controlled release preparations, because the preparation can be provided in the form of a dispersion of the active compound in hydroxypropyl methylcellulose.

[0219] The amount of the compound used and the dosage regimen for treating disease conditions with the compounds of the present invention and / or compositions depend on various factors, including age, body weight, sex, individual medical conditions, disease types, disease severity, route of administration and frequency, and specific compounds used. Therefore, dosage regimens may vary over a wide range, but can be routinely determined using standard methods. The daily dosage of about 0.001-100mg / kg body weight, preferably about 0.0025-about 50mg / kg body weight and most preferably about 0.005-10mg / kg body weight may be suitable. The daily dosage can be administered in 1-4 doses per day. Other dosage regimens include weekly dosages and once-a-week dosages per two days.

[0220] The pharmaceutical composition of the present invention comprises at least one compound of formula (I), (A) and (B) as defined above and / or at least one pharmaceutically acceptable salt thereof or at least one composition (1) as defined above and optionally another agent selected from any pharmaceutically acceptable carrier, adjuvant and vehicle. The optional composition of the present invention comprises a compound of formula (I), (A) and (B) as defined above or a prodrug thereof and a pharmaceutically acceptable carrier, adjuvant or vehicle.

[0221] The present invention also includes a pharmaceutical kit, which can be used, for example, to treat or prevent RAS protein-related diseases. Therefore, the present invention also relates to a kit comprising a preparation, the preparation comprising: a) a pharmaceutical composition comprising a compound of formula (I), (A) and (B) as defined above, or a therapeutically acceptable salt thereof, or at least one composition (1) as defined above, and a pharmaceutically acceptable carrier; and b) instructions for administration of the pharmaceutical composition for treating a disorder, wherein inhibiting RAS activation can effectively treat the disorder.

[0222] If desired, such kits may also include one or more of a variety of conventional pharmaceutical kit components, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, as will be readily understood by those skilled in the art. The kit may also include instructions, whether as an insert or a label, indicating the amounts of the components to be administered, administration instructions, and / or instructions for mixing the components.

[0223] Of course, the dosage regimen of the compounds of the invention will vary depending on known factors, such as the pharmacodynamic characteristics of the specific active agent and its mode and route of administration; the species, age, sex, health condition, medical condition and weight of the recipient; the nature and extent of the symptoms; the type of concurrent treatment; the frequency of treatment; the route of administration, the patient's renal and hepatic function, and the desired effect.

[0224] As a general guideline, when used for indicated effects, the daily oral dosage range for each active ingredient is from about 0.001 to about 5000 mg per day, preferably from about 0.01 to about 1000 mg per day, and most preferably from about 0.1 to about 250 mg per day. The most preferred dosage range for intravenous administration is from about 0.01 to about 10 mg / kg / minute during a constant rate infusion. The compounds of formula (I), (A) and (B) may be administered in a single daily dose, or the total daily dose may be administered in divided doses twice, three times or four times per day.

[0225] These compounds are usually administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as pharmaceutical carriers) appropriately selected according to the intended administration form, such as oral tablets, capsules, elixirs and syrups, and in accordance with conventional pharmaceutical practice.

[0226] Dosage forms (pharmaceutical compositions) suitable for administration may contain from about 1 mg to about 200 mg of active ingredient per dosage unit. In these pharmaceutical compositions, the active ingredient is usually present in an amount of about 0.1-95% by weight based on the total weight of the composition.

[0227] A typical capsule for oral administration comprises at least one compound of formula (I), (A) and (B) (250 mg), lactose (75 mg) and magnesium stearate (15 mg). The mixture is passed through a 60 mesh screen and filled into a size 1 gelatin capsule.

[0228] A typical injectable formulation is prepared by aseptically placing at least one compound of formula (I), (A) and (B) (250 mg) into a vial, aseptically freeze-drying and sealing. For use, the contents of the vial are mixed with 2 mL of saline to prepare an injectable formulation.

[0229] The present invention includes within its scope a pharmaceutical composition comprising as an active ingredient a therapeutically effective amount of at least one compound of formula (I), (A) and (B) as defined above, alone or in combination with a pharmaceutical carrier. Optionally, the compound of formula (I), (A) and (B) as defined above may be used alone, in combination with other compounds of formula (I), (A) and (B) as defined above, or in combination with one or more other therapeutic agents, such as anticancer agents or other pharmaceutically active substances.

[0230] Regardless of the administration route selected, the compounds of formula (I), (A) and (B) as defined above (which may be used in a suitable hydrated form) and / or the pharmaceutical composition of the present invention are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0231] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention or composition (1) may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without causing toxicity to the patient.

[0232] The selected dosage level will depend upon a variety of factors including the activity of the specific compound of Formula (I), (A) and (B) employed, or its ester, salt or amide as defined above, the route of administration, the time of administration, the rate of excretion or metabolism of the specific compound employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and / or materials used in combination with the specific compound employed, the age, sex, weight, condition, general health and prior medical history of the patient to be treated, and other factors well known in the medical art.

[0233] A physician or veterinarian with ordinary skills in the art can easily determine and prescribe the effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian can start with a dose of a compound of formula (I), (A) and (B) as defined above used in a pharmaceutical composition that is lower than the level required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved.

[0234] Typically, a suitable daily dose of a compound of formula (I), (A) and (B) as defined above is the amount of the compound at the lowest dose that effectively produces a therapeutic effect. Such an effective dose generally depends on the above factors. Typically, the oral, intravenous, intracerebroventricular and subcutaneous dose range for a compound of formula (I), (A) and (B) as defined above is about 0.01-about 50 mg / kg body weight / day for a patient.

[0235] If desired, the effective daily dose of active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally in unit dosage form. In certain aspects of the invention, administration is once daily.

[0236] While it is possible for a compound of formula (I), (A) and (B) as defined above to be administered alone, it is preferred to administer the compound as a pharmaceutical formulation (composition).

[0237] When used in combination with a compound of formula (I), (A) and (B) as defined above, the aforementioned other therapeutic agents may be used, for example, in those amounts indicated in the Physicians' Desk Reference (PDR) or otherwise determined by one of ordinary skill in the art. In the methods of the invention, such other therapeutic agents may be administered prior to, concurrently with, or after administration of the compounds of the invention.

[0238] The invention will be further illustrated with reference to the following examples, without limiting the scope to the specific embodiments described.The present invention includes all combinations of described features which are not mutually exclusive, and in particular combinations of preferred features which are not mutually exclusive.

[0239] The invention will be further illustrated with reference to the following examples, without limiting the scope to the specific embodiments described.The present invention includes all combinations of described features which are not mutually exclusive, and in particular combinations of preferred features which are not mutually exclusive.

[0240] Description of the drawings

[0241] Figure 1a) and 1b) :NanoBit Assay for RAS Activation (KRAS G12V, NRAS G12V, HRAS G12V)

[0242] N-terminal LgBiT and C-terminal SmBiT constructs were purchased from Promega, and K, N and HRAS G12V (full length) were cloned into LgBit with Xho I and Bgl II, and CRAF Ras binding domain (1-149) was cloned into SmBiT with EcoRI and Bgl II. For transfection, 1 μg or 2 μg (12 holes / 6 holes) plasmids were transfected into cells with 0.5mM PEI reagent in 100 μL or 200 μL PBS. One day after transfection, cells were harvested and inoculated into 96-well white plates, half area (Greiner). After another day, culture medium was changed into serum-free DMEM, and cells were incubated with inhibitors for 2 hours. NanoGlo assay was performed according to the manufacturer's instructions. Luminescence was measured using Tecan infinite (Tecan).

[0243] Mitoxantrone HCl inhibits K-RAS, N-RAS, and H-RAS.

[0244] Figure 2a and 2b) : MTT assay for cell growth

[0245] Metabolic activity was quantified using Cell Proliferation Kit I (Roche, Basel, Switzerland). Cells were seeded in 96-well cell culture plates, using 5000 cells per well for NCI-H358 cells. After treatment, 10 μL of MTT solution was added and incubated for 2 hours in a CO2 incubator. 100 μL of solubilization buffer was then added to each well and incubated overnight in a CO2 incubator. Cell viability was assessed by the amount of metabolized MTT, which was quantified by measuring the absorbance at 570 nm.

[0246] MTT assay of different tumor cell lines with RAS mutations grown in soft agar containing mitoxantrone hydrochloride. Data from three independent experiments are shown.

[0247] NCl-H358, H358, H2122, ASPC-1, HCT-116, T24 and HT1080 cells were used for soft agar colony formation assay. After treatment with mitoxantrone HCl, colonies were stained with MTT and the values ​​were quantified by measuring the absorbance at 570 nm after solubilization. The results represent the mean ± SEM of 3 independent experiments.

[0248] NCI-H358 NCI-H2122 ASPC1 HCT-116 T24 HT1080 IC50(μM) 1.50 0.25 0.60 0.27 0.43 0.13

[0249] Mitoxantrone HCl blocks the growth of different RAS-isoform addicted cell-lines and stops cancer growth.

[0250] Figure 3 : NanoBit Assay for RAS Activation (G12V, G12C, G12D, G12C / Y96D, G13C, G13D, G13S, Q61K, and Q61R)

[0251] NanoBiT assay of RAS GTP loading was performed in HeLa cells transfected with LgBit KRAS mutants and SmBit-CRAF-RBD. Cells were treated with mitoxantrone HCl in serum-free DMEM for 2 hours. After incubation, the substrate of NanoLuc was added and luminescence was measured in a multiplate reader. The data were calibrated to cells transfected with the indicated mutants and exposed to DMSO for 2 hours. DMSO-treated cells were set to 1. The bar graphs represent the mean ± SEM of 3 independent experiments.

[0252] Mitoxantrone HCl inhibits different RAS-isoforms.

[0253] Figure 4a) and 4b) : MTT assay for cell viability in 96-well cell culture plates

[0254] Cells were treated with mitoxantrone HCl for 48 hours, and cell viability was assessed by MTT assay. DMSO-treated cells were set to 1. Results shown are mean ± SEM of 3 independent experiments.

[0255] Figure 4a ): Mitoxantrone HCl inhibits KRAS-dependent cells regardless of the presence or absence of mutations. G12C / Y96D mutations are commonly found in patients who are resistant to G12C inhibitors. Therefore, patients who are resistant to G12C inhibitors can still be treated with mitoxantrone HCl.

[0256] BaF3 KRAS G12C BaF3 KRAS G12C / Y96D IC50(μM) 24.17 23.99

[0257] Figure 4b ): Single compounds Mitoxantrone HCl and MRTX849 (adagracib) were compared to a mixture of Mitoxantrone HCl (M) MRTX849 (M) in an MTT assay in RAS mutant cells. In addition, the calculated / expected values ​​for the mixture and the measured / true values ​​for the mixture are shown. The expected values ​​were obtained using the following equation:

[0258] Expected value = M + M - (M·M / 100)

[0259] Expected value = [100-[M+M-(M·M / 100)]] / 100 (normalized)

[0260] Figure 5 : Mitoxantrone HCl RASopathy-Model: MTT Assay of Cell Viability in 96-Well Cell Culture Plates

[0261] Cells were treated with mitoxantrone HCl for 48 hours, and cell viability was assessed by MTT assay. DMSO-treated cells were set to 1. Results shown are mean ± SEM of 3 independent experiments.

[0262] sNF96.2 IC50(μM) 3.71

[0263] Mitoxantrone HCl inhibits RAS proteins in RASopathy.

[0264] Cell culture

[0265] HeLa S3 (DSMZ) were authenticated by Eurofin Genomics and cultured in DMEM (10% heat-inactivated FBS). NCI-H358 cells were cultured in RPMI-1640 (10% heat-inactivated FBS). Example

[0266] Mitoxantrone hydrochloride: 1,4-dihydroxy-5,8-bis({2-[(2-hydroxy-ethyl)amino]ethyl}amino)-9,10-anthraquinone dihydrochloride, CAS: 70476-82-3

[0267]

[0268] Commercially available from Sigma-Aldrich / Merck.

Claims

1. Compound of formula (I) in R 1 is H or C1-C4 alkyl; R 2 is H or C1-C4 alkyl; R 3 is H or C1-C4 alkyl; R 4 is H or C1-C4 alkyl; n is 1, 2 or 3; or a pharmaceutically acceptable salt thereof, for use as a RAS protein activation inhibitor for the treatment and / or prevention of diseases in which RAS signaling is involved.

2. The compound of formula (I) according to claim 1, in R 1 is H or C1-C4 alkyl; R 2 is H or C1-C4 alkyl; R 3 is H or C1-C4 alkyl; R 4 is H or C1-C4 alkyl; n is 1, 2 or 3; or a pharmaceutically acceptable salt thereof, for use as a RAS protein activation inhibitor.

3. A compound of formula (I) according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, for use as an inhibitor of RAS protein activation for the treatment and / or prevention of proliferative disorders, inflammatory diseases and / or genetic disorders in which RAS signaling is involved.

4. A compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof for use as an inhibitor of RAS protein activation for the treatment and / or prevention of proliferative disorders, wherein KRAS G12V, NRAS G12V, HRAS G12V, KRAS G12A, KRASG12C, KRAS G12D, KRAS G12C / Y96D, KRAS G12C / Y96C, KRAS G12C / Y96S, KRAS G13C, KRASG13D, KRAS G13S, KRAS Q61H, KRAS Q61R or KRAS Q61K is involved.

5. A compound of formula (I) according to claims 1 to 4 or a pharmaceutically acceptable salt thereof for use as an inhibitor of RAS protein activation for the treatment and / or prevention of proliferative disorders and / or inflammatory diseases which are resistant to treatment with a RAS mutation-specific inhibitor other than the compound of formula (I).

6. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 5, wherein the resistance is caused by a secondary mutation, in particular by a secondary mutation involving KRAS G12C / Y96D, KRAS G12C / Y96C and / or KRAS G12C / Y96S.

7. A compound of formula (I) according to claim 1, wherein R 1 is H or C1-C2 alkyl; R 2 is H or C1-C2 alkyl; R 3 is H or C1-C2 alkyl; R 4 is H or C1-C2 alkyl; n is 2; or a pharmaceutically acceptable salt thereof, for use as a RAS protein activation inhibitor, preferably as a RAS protein activation inhibitor, for treatment and / or prevention, in which RAS signaling is involved, in particular as a RAS protein activation inhibitor, for treatment and / or prevention of proliferative disorders, inflammatory diseases and / or genetic disorders, in which RAS signaling is involved, in particular in which KRASG12V, NRAS G12V, HRAS G12V, KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12C / Y96D, KRASG12C / Y96C, KRAS G12C / Y96S, KRAS G13C, KRAS G13D, KRAS G13S, KRAS Q61H, KRAS Q61R or KRAS Q61K is involved.

8. A compound of formula (I) according to any one of the preceding claims, which is compound A or a pharmaceutically acceptable salt thereof, for use as a RAS protein activation inhibitor.

9. A compound of formula (I) according to any one of claims 1 to 8, which is compound A or a pharmaceutically acceptable salt thereof, for use as an inhibitor of RAS protein activation for the treatment and / or prevention of diseases in which RAS signaling is involved.

10. A compound of formula (I) according to any one of claims 1 to 9, which is compound A or a pharmaceutically acceptable salt thereof, for use as an inhibitor of RAS protein activation for the treatment and / or prevention of proliferative disorders, inflammatory diseases and / or genetic disorders involving RAS signaling, in particular involving KRAS G12V, NRAS G12V, HRAS G12V, KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12C / Y96D, KRAS G12C / Y96C, KRAS G12C / Y96S, KRAS G13C, KRAS G13D, KRAS G13S, KRAS Q61H, KRAS Q61R or KRAS Q61K.

11. A compound of formula (I) according to any one of claims 1 to 9, which is compound A or a pharmaceutically acceptable salt thereof, for use as an inhibitor of RAS protein activation for the treatment and / or prevention of proliferative disorders and / or inflammatory diseases which are resistant to treatment with a RAS mutation-specific inhibitor different from the compound of formula (I), preferably wherein the resistance is caused by a secondary mutation, in particular by a secondary mutation involving KRAS G12C / Y96D, KRAS G12C / Y96C and / or KRAS G12C / Y96S.

12. A compound of formula (I) according to any one of claims 1 to 7, which is compound B Acts as an inhibitor of RAS protein activation.

13. A compound of formula (I) according to any one of claims 1 to 7 or 12, which is compound B or a pharmaceutically acceptable salt thereof, for use as an inhibitor of RAS protein activation for the treatment and / or prevention of diseases in which RAS signalling is involved.

14. A compound of formula (I) according to any one of claims 1 to 7 or 12, which is compound B or a pharmaceutically acceptable salt thereof, for use as an inhibitor of RAS protein activation for the treatment and / or prevention of proliferative disorders, inflammatory diseases and / or genetic disorders involving RAS signaling, in particular, involving KRAS G12V, NRAS G12V, HRAS G12V, KRAS G12A, KRASG12C, KRAS G12D, KRAS G12C / Y96D, KRAS G12C / Y96C, KRAS G12C / Y96S, KRAS G13C, KRASG13D, KRAS G13S, KRAS Q61H, KRAS Q61R or KRAS Q61K.

15. A compound of formula (I) according to any one of claims 1 to 7 or 12, which is compound B or a pharmaceutically acceptable salt thereof, for use as an inhibitor of RAS protein activation for the treatment and / or prevention of proliferative disorders and / or inflammatory diseases which are resistant to treatment with a RAS mutation-specific inhibitor different from the compound of formula (I), preferably wherein the resistance is caused by a secondary mutation, in particular by a secondary mutation involving KRAS G12C / Y96D, KRAS G12C / Y96C and / or KRAS G12C / Y96S.

16. Composition (1) comprising at least one compound of formula (I) as claimed in any one of claims 1 to 15, in particular selected from compound (A) or (B) or a pharmaceutically acceptable salt thereof, and a RAS mutation-specific inhibitor, in particular a KRAS mutation-specific inhibitor, in particular sotolacib and / or adagracib.

17. A compound of formula (I) according to any one of claims 1 to 15, in particular compound (A) or compound (B) or a pharmaceutically acceptable salt thereof, or a composition (1) as claimed in claim 16, for use in the treatment of proliferative disorders, inflammatory diseases and / or genetic disorders in which RAS signalling is involved.

18. A compound of formula (I) according to any one of claims 1 to 15, in particular compound (A) or compound (B) or a pharmaceutically acceptable salt thereof, or a composition (1) as claimed in claim 16, for use in the treatment of proliferative disorders and / or inflammatory diseases involving KRAS G12V, NRAS G12V, HRAS G12V, KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12C / Y96D, KRAS G12C / Y96C, KRAS G12C / Y96S, KRAS G13C, KRAS G13D, KRAS G13S, KRAS Q61H, KRAS Q61R or KRAS Q61K.

19. A compound of formula (I) according to any one of claims 1 to 15, in particular compound (A) or compound (B) or a pharmaceutically acceptable salt thereof, or a composition (1) as claimed in claim 16, for use in the treatment of a proliferative disorder, wherein the proliferative disorder is a cancer, in particular a cancer selected from the group consisting of prostate cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, stomach cancer, endometrial cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer (including melanoma and basal carcinoma), mesothelial lining cancer, white blood cell cancer (including lymphoma and leukemia), esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, lung cancer (including small cell lung cancer and non-small cell carcinoma), adrenal cancer, thyroid cancer, kidney cancer or bone cancer; or glioblastoma, mesothelioma, renal cell carcinoma, stomach cancer, sarcoma (including Kaposi's sarcoma), choriocarcinoma, basal cell carcinoma of the skin, hematological malignancies (including blood, bone marrow and lymph nodes) or testicular seminoma.

20. A pharmaceutical composition comprising at least one compound of formula (I) as claimed in any one of claims 1 to 15, in particular selected from compound (A) or (B) or a pharmaceutically acceptable salt thereof, or composition (1) as claimed in claim 16, for use in the prevention and / or treatment of proliferative disorders, inflammatory diseases and / or genetic disorders in which RAS signaling is involved.

21. A pharmaceutical composition comprising at least one compound of formula (I) as claimed in any one of claims 1 to 15, in particular selected from compound (A) or (B) or a pharmaceutically acceptable salt thereof, or composition (1) as claimed in claim 16, for use in the prevention and / or treatment of proliferative disorders and / or inflammation involving KRAS G12V, NRAS G12V, HRAS G12V, KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12C / Y96D, KRAS G12C / Y96C, KRAS G12C / Y96S, KRAS G13C, KRAS G13D, KRAS G13S, KRAS Q61H, KRAS Q61R or KRAS Q61K.

22. A pharmaceutical composition comprising at least one compound of formula (I) as claimed in any one of claims 1 to 11, in particular selected from compound (A) or a pharmaceutically acceptable salt thereof, or composition (1) as claimed in claim 12, for use as an inhibitor of RAS protein activation for the treatment and / or prevention of proliferative disorders and / or inflammatory diseases which are resistant to treatment with RAS mutation-specific inhibitors other than the compounds of formula (I), preferably wherein the resistance is caused by secondary mutations, in particular by secondary mutations involving KRAS G12C / Y96D, KRAS G12C / Y96C and / or KRAS G12C / Y96S.

23. A pharmaceutical composition according to claims 20-22, comprising at least one compound of formula (I) as claimed in claim 15, in particular selected from compounds (A) and (B) or pharmaceutically acceptable salts thereof, or composition (1) as claimed in claim 15, and a pharmaceutically acceptable carrier.

24. The pharmaceutical composition of claims 20-23, further comprising an additional active substance, preferably selected from chemotherapeutic agents, radiotherapeutic agents, immuno-oncological agents and combinations thereof.

25. A method of inhibiting the growth, proliferation or metastasis of cancer cells in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of at least one compound of formula (I) as defined in any one of claims 1 to 15, in particular selected from compounds (A) and (B) or therapeutically acceptable salts thereof, or a composition (1) as claimed in claim 16.

26. The method of claim 25, wherein the cancer is selected from the group consisting of prostate cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, stomach cancer, endometrial cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer (including melanoma and basal carcinoma), mesothelial lining cancer, white blood cell cancer (including lymphoma and leukemia), esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, lung cancer (including small cell lung cancer and non-small cell carcinoma), adrenal cancer, thyroid cancer, kidney cancer, or bone cancer; or glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma (including Kaposi's sarcoma), choriocarcinoma, basal cell carcinoma of the skin, hematological malignancies (including blood, bone marrow, and lymph nodes), or testicular seminoma.

27. A method for inhibiting the proliferation of a cell population sensitive to inhibition of RAS activation in vitro or ex vivo, which method comprises contacting the cell population with at least one compound of formula (I) as defined in any one of claims 1 to 15, in particular selected from compounds (A) and (B) or therapeutically acceptable salts thereof, or a composition (1) as claimed in claim 16.

28. A kit comprising a formulation comprising: a) at least one compound of formula (I) as defined in any one of claims 1 to 15, in particular selected from compounds (A) and (B), or a composition (1) as claimed in claim 16, or a pharmaceutical composition comprising at least one compound of formula (I) as defined in any one of claims 1 to 15, in particular selected from compounds (A) and (B) or therapeutically acceptable salts thereof, or a composition (1) as claimed in claim 16, and a pharmaceutically acceptable carrier; and b) instructions for administration of the pharmaceutical composition for treating a disorder, wherein inhibition of RAS activation is effective in treating the disorder.

Images