WO2023030687A1

WO2023030687A1 - Cyclopenta[4,5]furo[3,2-c]pyridine derivatives as ras inhibitors for use in the treatment of hyperproliferative diseases or genetic disorders

Info

Publication number: WO2023030687A1
Application number: PCT/EP2022/025399
Authority: WO
Inventors: Krishnaraj Rajalingam
Original assignee: KHR Biotec GmbH
Current assignee: KHR Biotec GmbH
Priority date: 2021-09-01
Filing date: 2022-08-30
Publication date: 2023-03-09
Anticipated expiration: 2024-03-01
Also published as: EP4395775A1

Abstract

The present invention relates to the use of CYCLOPENTA[4,5]FURO[3,2-C]PYRIDINE derivatives of formula (I) as RAS inhibitors and as a medicament, in particular for use in treating proliferative disorders. The present invention relates further to a pharmaceutical composition comprising the compounds of formula (I). Moreover, the present invention relates to a method of inhibiting growth, proliferation or metastasis of cancer cells in a subject in need thereof. In addition, the present invention relates to a method of inhibiting proliferation of a cell population sensitive towards inhibiting RAS activation in vitro, in particular sensitive towards inhibiting KRAS,HRAS and NRAS activation in vitro. Furthermore, the present invention relates to a kit containing a formulation comprising a pharmaceutical composition comprising CYCLOPENTA[4,5]FURO[3,2-C]PYRIDINE derivatives of formula (I).

Description

CYCLOPENTA[4,5]FURO[3,2-C]PYRIDINE DERIVATIVES AS RAS INHIBITORS FOR USE IN THE TREATMENT OF

HYPERPROLIFERATIVE DISEASES OR GENETIC DISORDERS

Novel RAS inhibitors

The present invention relates to the use of compounds of formula (I) as RAS inhibitors and as a medicament, in particular for use in treating proliferative disorders. The present invention relates further to a pharmaceutical composition comprising the compounds of formula (I). Moreover, the present invention relates to a method of inhibiting growth, proliferation or metastasis of cancer cells in a subject in need thereof, in particular which may encompass subsets of patients defined by their mutational status of the RAS oncogene or patients who might have developed resistance to the standard of care or treatment with RAS inhibitors. In addition, the present invention relates to a method of inhibiting proliferation of a cell population sensitive towards inhibiting RAS activation in vitro, in particular sensitive towards inhibiting KRAS, HRAS and NRAS activation in vitro. Furthermore, the present invention relates to a kit containing a formulation comprising a pharmaceutical composition comprising a compound of formula (I).

BACKGROUND OF THE INVENTION

RAS proteins represent a group of closely related monomeric globular proteins which are associated with the plasma membrane and are able to bind either GDP or GTP. RAS that contains bound GDP represents the "inactive" state, whereas the binding of GTP to RAS in exchange to a GDP represents the "active" state, such that the protein is able to interact with other proteins of downstream targets. RAS proteins can be regarded as small GTPases that function as molecular switches controlling the transmission of extracellular signals from outside of the cell to the nucleus by various effector proteins.

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

Usually, mutations of RAS lead to defects in GAP-mediated GTP hydrolysis and thus result in the accumulation of RAS in the GTP-bound active state.

It is known that some flavaglines like rocaglamide, a class of natural anti-tumour drugs and chemical ligands of prohibitins, inhibit the interaction between RASGTP and the putative effector molecules indirectly thus inhibiting oncogene activation in cells.

It is also known that various flavagline derivatives exhibit cytotoxic properties and have been described as potent anti-cancer and cytoprotective agents. However, it is also known that some flavagline derivatives do not show any inhibition properties. Yurugi et al., (J. Cell Sience, 133 ,2020) 1 , relates to flavagline derivates e.g. FL1 , FL6, FL26, FL30 which despite carrying the core cyclo benzofuran ring failed to inhibit RAS.

Furthermore, it is known that patients frequently develop resistance to KRAS oncogene inhibitors, e.g. to KRAS G12 C inhibitors (Tanaka et al., Cancer Discov, 2021 , PMID 33824136 ). In addition, the patients treated with KRAS G12 C inhibitors often develop secondary mutations in other RAS isoforms (Awad MM et al. New England J. Med., 2021 PMID34161704).

WO 2017091585 describes compounds having activity as inhibitors of elF4A.

Eukaryotic initiation factor 4A (elF4A) is a DEAD-box protein containing ATPase and ATP- dependent RNA helicase required to melt local secondary structure and facilitate access of the ribosome to the mRNA template. The factor regulates the cap-dependent protein synthesis.

In mammals, there are three isoforms of elF4A (elF4AI, II and III), where elF4AII and el F4AI 11 share -90% and -65% identity, respectively, with the most abundant cellular factor elF4AI. All isoforms are DEAD-box RNA helicase family members but only the paralogs elF4AI and el F4AI I are found in the elF4F complex and participate in translation initiation.

In contrast to the inhibition of RAS oncogenes, which takes place at the plasma membrane level, the inhibition of the eukaryotic initiation factor 4A occurs in the cytosol. Therefore, in the inhibition of RAS oncogenes and the inhibition of the eukaryotic initiation factor 4A two different pathways leads to a profound inhibition of the tumour cell growth and survival.

One of the most popular elF4A inhibitor is Zotatifin (4-{(5aR,6S,7S,8R,8aS)-7- [(dimethylamino)methyl]-8,8a-dihydroxy-1 ,3-dimethoxy-6-phenyl-6,7,8,8a-tetrahydro-5aH- cyclopenta[4,5]furo[3,2-c]pyridin- 5a-yl}benzonitrile, CAS: 2098191 -53-6). Zotatifin (eFT226) is a potent, selective, and well-tolerated elF4A inhibitor. Zotatifin promotes elF4A binding to specific mRNA sequences with recognition motifs in the 5’-UTRs and interferes with the assembly of the elF4F initiation complex.

In the article announcing that eFFECTOR Therapeutics initiates Phase 1/2 Safety and Efficacy Study of Zotatifin (eFT226) in Patients with Advanced Solid Tumor Malignancies San Diego, November 5, 2019 it is described that Zotatifin inhibits the translation of mRNA encoding several important oncogenes and survival factors, including several RTKS, KRAS, Cyclin D, MCL1 and BCL-2. In other words Zotatifin inhibits the expression and not the activation of KRAS. Activation is defined as ability of RAS to bind to its effector molecules like RAF kinases, PI3K kinases through the RAS binding domain (RBD) or RAS -Associated domain (RA domain) present in the effector proteins (Like RASSF) in a GTP dependent manner. Further targeting the activation but not the stability can be advantageous in defined medical conditions while targeting both could be useful in combating certain subtypes of RAS mutated cancers.

The mechanisms driving the activation of the three isoforms HRAS, NRAS, KRAS are different and each RAS isoform exhibit distinct functions and biological specificites. Thus efforts are being made to target HRAS and NRAS in defined tumour subtypes where they function as an oncogenic driver.

Finally, targeting of other RAS isoforms like HRAS and NRAS is required to combat secondary, acquired resistance to the standard of care like RAS inhibitors among other cancer therapeutics.

While KRASG12 specific C inhibitors have been developed, efforts to target HRAS and NRAS in cancers where these isoforms are mutated remains a challenge.

However, effective targeting, in particular inhibition of RAS oncogene activation of this RAS oncogenes with small molecules is still a challenge.

C. R. Stumpf et al., Experimental and Molecular Therapeutics, Abstract 1955, AACR Meeting April and June 2020, discusses the ability of eFT226 to block tumor cell growth, that means eFT226 inhibits the expression and not the activation of KRAS.

Anonymous: History of changes for study NCT04092673 study of eFT226 with subjects with selected advanced solid tumor malignancies, clinical trials. Gov. 2021 , relates to a clinlical trial, phase 1 -2, for evaluating the safety, pharmacokinetics, pharmacodynamics and antitumor actifity of Zotatifin in a subject with selected advanced solid tumor malignancies. It is stated that eFT226 is a selective inhibitor of elF4A1 -mediated translation and selectively regulates the translation of a subset of mRNAs based on sequence specific recognition motifs in their 5’-UTR. elF4A1 inhibition by eFT226 downregulates the expression of receptor tyrosine kinase and KRAS. However, there is no disclosure for the abilitiy of eFT226 to inhibit the activity of other RAS oncogenes. HRAS, NRAS though they belong to the same RAS family are activated by different mechansims and thus exhibit functional differences and drive different types of cancers. D. Uprety et al. relates to an overview on the role of K-RAS in tumorgenesis, past approaches to inhibiting K-RAS, including SHP2, SOS1 and elF4 inhibition. Zotatifin is merely mentioned as an inhibitor of elF4A1 .

J. T. Ernst et al. J. Med. Chem., 63, 2020, 5879, investigate the properties of Zotatifin as antitumor agent. However, Zotatifin is merely mentioned as an inhibitor of elF4A1.

WO2018/218072 relates to inhibitors of elF4A. there is no disclosure for the abilitiy of eFT226 to inhibit the activity of RAS oncogenes.

W02020/078975 relates to flavagline derivatives for the inhibition of KRAS oncogene activation. The flavagline derivatives are structurally different compared to Zotatifin.

WO2021/195128 relates to a composition comprising a compounds for inhibition of elF4A an a cyclin-depent kinase (CDK) inhibitor.

To sum up, prior art documents, if at all, describe that Zotatifin inhibits the expression of KRAS oncocene. However, activation of a protein is fundamentally different from its expression which has different functional consequences in both physiology and pathophysiology. For instance, RAS gene expression and tightly regulated activation/inactivation by GEFs and GAPs is not pathological while mutation at specific residues leads to persistent activation of RAS which leads to cancer.

Prohibitins are evolutionarily conserved proteins and recent studies revealed a critical role for prohibitins in the activation of RAS by enabling RAS-effector interaction in the plane of the plasma membrane. Polier et al, Chemistry and Biology, 19, 2012, 1093, showed that rocaglamides target this interaction (PHB1 -CRAF) to inhibit RAS-CRAF interaction. These are several follow up studies confirming these effects.

It is therefore the object of the present invention to provide pharmaceutically active compounds that have the capability to inhibit the activation of RAS oncogenes, in particular in cells at nanomolar concentrations with high specificity. This object is achieved by the use of compounds of formula (I). It was suprisingly found that the compounds of formula (I), in particular of formulae (A) and (B), especially of formulae (A), (B), (C), (D) and (E), or an enantiomeric mixture thereof, inhibit the activity of RAS oncogenes, in particular KRAS, HRAS and NRAS irrespective of their mutational status. SUMMARY OF THE INVENTION

The invention relates to compounds of formula (I)

wherin

R¹ is selected from halogen, CN and OH;

R² is C1-C4 alkyl, wherein alkyl is unstubstituted or substituted by 1 , 2 or 3 substituents selected from OH, C1-C3 alkoxy and NR^aR^b;

R³ is selected from C1-C4 alkyl;

R⁴ is selected from C1-C4 alkyl;

R^a and R^b independently from each other are selected from hydrogen, C1-C4 alkyl and C3- C7 cycloalkyl; or a pharmaceutically acceptable salt thereof, for use as inhibitor of RAS proteine activation.

The invention further relates to compounds (I), in particular to compounds of formulae (A) (B), (C), (D), (E) or an enantiomeric mixture thereof, as defined above and below, or a pharmaceutically acceptable salt thereof, or pharmaceutical compositions comprising at least one compound of formula (I), in particular selected from compounds (A), (B) (C), (D) and (E) as defined above and below, for use as a medicament.

The invention further relates to compounds (I), in particular to compounds of formulae (A) and (B) as defined above and below, or a pharmaceutically acceptable salt thereof, or pharmaceutical compositions comprising at least one compound of formula (I), in particular selected from compounds (A) and (B) as defined above and below, for use as a medicament. The invention further relates to compounds (I) according to the invention, in particular to compounds of formulae (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above and below, or a pharmaceutically acceptable salt thereof, or pharmaceutical compositions comprising at least one compound of formula (I), in particular selected from compounds (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above and below, for the treatment and/or prophylaxis of diseases.

The invention further relates to compounds (I) according to the invention, in particular to compounds of formulae (A) and/or (B) as defined above and below, or a pharmaceutically acceptable salt thereof, or pharmaceutical compositions comprising at least one compound of formula (I), in particular selected from compounds (A) and (B) as defined above and below, for the treatment and/or prophylaxis of diseases.

The invention further relates to compounds (I) according to the invention, in particular to compounds of formulae (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above and below, or a pharmaceutically acceptable salt thereof, or pharmaceutical compositions comprising at least one compound of formula (I), in particular selected from compounds (A), (B) (C), (D), (E) or an enantiomeric mixture thereo fas defined above and below, for use in treating proliferative disorders.

The invention further relates to compounds (I) according to the invention, in particular to compounds of formulae (A) and/or (B) as defined above and below, or a pharmaceutically acceptable salt thereof, or pharmaceutical compositions comprising at least one compound of formula (I), in particular selected from compounds (A) and (B) as defined above and below, for use in treating proliferative disorders.

The invention further relates to compounda (I) according to the invention, in particular to compounds of formulae (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above and below or a pharmaceutically acceptable salt thereof, or pharmaceutical compositions comprising at least one compound of formula (I), in particular selected from compounds (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above and below, for use in treating cancer.

The invention further relates to compounda (I) according to the invention, in particular to compounds of formulae (A) and/or (B) as defined above and below or a pharmaceutically acceptable salt thereof, or pharmaceutical compositions comprising at least one compound of formula (I), in particular selected from compounds (A) and (B) as defined above and below, for use in treating cancer. The invention further relates to compounds (I) according to the invention, in particular to compounds of formulae (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above and below or a pharmaceutically acceptable salt thereof, or pharmaceutical compositions comprising at least one compound of formula (I), in particular selected from compounds (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above or below for use as inhibitor of RAS protein (KRAS, HRAS or NRAS oncogenes) activation.

The invention further relates to compounds (I) according to the invention, in particular to compounds of formulae (A) and/or (B) as defined above and below or a pharmaceutically acceptable salt thereof, or pharmaceutical compositions comprising at least one compound of formula (I), in particular selected from compounds (A) and (B) as defined above or below for use as inhibitor of RAS protein (KRAS, HRAS or NRAS oncogenes) activation.

The invention further relates to compounds (I) according to the invention, in particular to compounds of formulae (A), (B) (C), (D) and(E) as defined above and below or a pharmaceutically acceptable salt thereof, or pharmaceutical compositions comprising at least one compound of formula (I), in particular selected from compounds (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above or below for treating or preventing any diseases or conditions that are associated with the activity of RAS proteine (RAS oncogene).

The invention further relates to compounds (I) according to the invention, in particular to compounds of formulae (A) and/or (B) as defined above and below or a pharmaceutically acceptable salt thereof, or pharmaceutical compositions comprising at least one compound of formula (I), in particular selected from compounds (A) and (B) as defined above or below for treating or preventing any diseases or conditions that are associated with the activity of RAS proteine (RAS oncogene).

The invention further relates to compounds (I) according to the invention, in particular to compounds of formulae (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above and below or a pharmaceutically acceptable salt thereof, or pharmaceutical compositions comprising at least one compound of formula (I), in particular selected from compounds (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above for use in treating proliferative disorders, wherein RAS-signalling is involved, preferably wherein KRAS G12V, NRAS G12V, HRAS G12V, KRAS G12C, KRAS G12D, KRAS G12C/Y96D, KRAS G13C, KRAS G13D, KRASG13S, KRAS Q61 H, KRAS Q61 R or KRAS Q61 K is involved or or wherein any activating mutation in KRAS, HRAS and NRAS is involved or wherein any mutation that conferes resistance to other RAS inhibitors is involved.

The invention further relates to compounds (I) according to the invention, in particular to compounds of formulae (A) and/or (B) as defined above and below or a pharmaceutically acceptable salt thereof, or pharmaceutical compositions comprising at least one compound of formula (I), in particular selected from compounds (A) and (B) as defined above for use in treating proliferative disorders, wherein RAS-signalling is involved, preferably wherein KRAS G12V, NRAS G12V, HRAS G12V, KRAS G12C, KRAS G12D, KRAS G12C/Y96D, KRAS G13C, KRAS G13D, KRASG13S, KRAS Q61 H, KRAS Q61 R or KRAS Q61 K is involved or wherein any activating mutation in KRAS, HRAS and NRAS is involved or wherein any mutation that conferes resistance to other RAS inhibitors is involved.

The invention further relates to compounds of formula (I) in particular compound (A), (B),

(C), (D) or (E) according to any of claims 1 to 7 or a pharmaceutically acceptable salt thereof, for use as a ligand of prohibitins (PHB1/2 complex) in the plasma membrane.

The invention further relates to a pharmaceutical composition comprising at least one compound (I) according to the invention, in particular compounds of formulae (A), (B) (C),

(D), (E) or an enantiomeric mixture thereof as defined above and below, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

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

The invention further relates to a pharmaceutical composition comprising at least one compound (I) according to the invention, in particular compound of formulae (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above and below, or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition additionally comprises a further active substance, preferably selected from chemotherapeutic agents, radiotherapeutic agents, immuno-oncology agents and combinations thereof.

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

The invention further relates to a pharmaceutical composition comprising at least one compound of formula (I), in particular selected from compounds (A), (B), (C), (D) or (E) or a pharmaceutically acceptable salt for use in the prophylaxis and/or treatment of genetic disorders where RAS signaling is involved, in particular including RASopathies, craniofacial syndrome and Neurofibromatosis type I.

The invention further relates to a method of inhibiting growth, proliferation, or metastasis of cancer cells in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound (I) according to the invention, in particular a compound of formulae (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above and below, or a therapeutically acceptable salt thereof, or a pharmaceutical composition comprising at least one compound of formula (I), in particular selected from compounds (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above.

The invention further relates to a method of inhibiting growth, proliferation, or metastasis of cancer cells in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound (I) according to the invention, in particular a compound of formulae (A) and/or (B) as defined above and below, or a therapeutically acceptable salt thereof, or a pharmaceutical composition comprising at least one compound of formula (I), in particular selected from compounds (A) and (B) as defined above.

The invention further relates to a method of inhibiting proliferation of a cell population sensitive towards inhibiting RAS activation in vitro or ex vivo, the method comprising contacting the cell population with a compound (I) according to the invention, in particular a compound of formulae (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above and below, or a therapeutically acceptable salt thereof or a pharmaceutical compositions comprising at least one compound of formulae (I), in particular selected from compounds (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above.

The invention further relates to a method of inhibiting proliferation of a cell population sensitive towards inhibiting RAS activation in vitro or ex vivo, the method comprising contacting the cell population with a compound (I) according to the invention, in particular a compound of formulae (A) and/or (B) as defined above and below, or a therapeutically acceptable salt thereof or a pharmaceutical compositions comprising at least one compound of formulae (I), in particular selected from compounds (A) and (B) as defined above.

The invention further relates to a kit containing a formulation comprising: a) a compound (I) according to the invention, in particular a compound of formulae (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above and below, or a pharmaceutical composition comprising a compound (I) according to the invention, in particular a compound of formulae (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above or below, or a therapeutically acceptable salt thereof and a pharmaceutically acceptable carrier; and b) instructions for dosing of the pharmaceutical composition for the treatment of a disorder in which inhibition of RAS activation is effective in treating the disorder.

The invention further relates to the compound of formula (I), as defined above and below, an enantiomeric mixture thereof, or a pharmaceutically acceptable salt thereof, for use of treating patients having developed resistance to RAS inhibitors, different from compounds of formula (I).

The invention further relates in particular selected from compounds (A), (B), (C), (D) and (E), as defined above and below, an enantiomeric mixture thereof, or a pharmaceutically acceptable salt thereof, for use of treating patients having developed resistance to RAS inhibitors, different from compounds of formula (I), in particular KRAS G12C inhibitors.

The invention further relates in particular selected from compounds (A), (B), (C), (D) and (E), as defined above and below, an enantiomeric mixture thereof, or a pharmaceutically acceptable salt thereof, for use of treating patients having developed resistance to KRAS G12C inhibitors different from compounds of formula (I).

The invention further relates to a kit containing a formulation comprising: a) a compound (I) according to the invention, in particular a compound of formulae (A) and/or (B) as defined above and below, or a pharmaceutical composition comprising a compound (I) according to the invention, in particular a compound of formulae (A) and/or (B) as defined above or below, or a therapeutically acceptable salt thereof and a pharmaceutically acceptable carrier; and b) instructions for dosing of the pharmaceutical composition for the treatment of a disorder in which inhibition of RAS activation is effective in treating the disorder.

DESCRIPTION OF THE INVENTION The invention has the following advantages:

The compounds according to the invention exhibit advantageous RAS inhibition properties. In other words the compounds according to the invention qualify as inhibitors of RAS oncogene activation by inhibiting the prohibitin pathway, in particular inhibiting EGF-induced RAS-GTP loading in cells.

The compounds according to the invention prevent the activation of RAS, in particular KRAS, as the interaction between RAS, in particular KRAS, and its effectors is uncoupled possibly due to defects in nanoclustering of RAS, in particular KRAS, in the plane of the plasma membrane.

The compounds inhibit KRAS irrespective of the mutations at nanomolar range.

The compounds inhibit NRAS and HRAS by functionally uncoupling their binding to their effectors in the plane of the plasma membrane.

Patients frequently develop resistance to several anti tumour drugs. The activation of RAS or its downstream signalling machinery in many cases is the mechanism driving the resistance to the standard of care. Under these settings where patients develop resistance to the drugs, these compounds could still be employed as a RAS inhibitor for better management of disease progression and metastasis.

It is possible to employ compounds of formula (I) against cancers driven by multiple KRAS mutations. This allows defining subsets of patients within different cancer cohorts which can be identified by employing companion diagnostics.

- The effects of compounds of formula (I) on the activation of three different oncogenes e.g. KRAS, HRAS and NRAS has been shown. Though they belong to the same family, their mechanism of activation and functions are different. They drive different set of cancers and thus it is possible to employ compounds (I) as a therapy with patients having cancers driven by HRAS and NRAS mutations.

It has been shown that compounds of formula (I), in particular Zotatifin can be employed in patients who have developed resistance to KRAS G12C inhibitors.

Compounds of formula (I)

Inactivation in the sense of the invention means inhibiting the activity of a protein, in particular RAS protein, especially NRAS, KRAS or HRAS protein, based on direct interaction of at least one of the compound of formula (I) and the proteins including prohibitins (PHB1/2) involved in complex with RAS proteins. This interaction is not a translation process or part of a translation process. Further, activation means promoting the activity or physiological effect of a protein based on direct interactions of two or more molecules, whereby this interaction is not a translation process or part thereof. In particular, activations means the ability of RAS to bind to its effector molecules like RAF kinases, PI3K kinases through the RAS binding domain (RBD) or RAS -Associated domain (RA domain) present in the effector proteins (Like RASSF) in a GTP dependent manner.

The inhibition of RAS protein activation refers to the delay or slowing down of the ability of RAS to bind to its effector molecule.

Expression in the sense of the invention means gene expression, which is the formation of a gene product encoded by a gene, especially proteins or RNA molecules. Gene expression consists of several individual processes. These include transcription, splicing, translation, and post-translational modification, as well as their regulatory mechanisms. Overexpression is the expression of a particular gene in a cell that is significantly above the norm. This usually results in increased synthesis of the protein for which the gene codes.

The inhibition of RAS protein expression refers to the prevention of the process by which information from a gene is used in the synthesis of a functional gene product.

Unless specifically stated otherwise herein, references made in the singular may also include the plural. For example, “a” and “an” may refer to either one, or one or more.

In the context of the invention, the prefix C_n-C_m indicates the number of carbon atoms that a molecule or residue designated thereby may contain.

In the context of the invention, the expression Ci-C4-alkyl refers to unbranched or branched saturated hydrocarbon groups having 1 to 4 carbon atoms. Ci-C4-alkyl are e.g. methyl, ethyl, propyl, 1 -methylethyl, butyl, 1 -methylpropyl, 2-methylpropyl, 1 ,1 -dimethylethyl.

In the context of the invention the expression Ci-C4-alkoxy refers to an unbranched or branched saturated Ci-C4-alkyl group as defined above, which is bound via an oxygen atom. Alkoxy radicals with 1 or 2 carbon atoms are preferred. Ci-C2-alkoxy is methoxy or ethoxy. Ci-C4-alkoxy is e.g. methoxy, ethoxy, n-propoxy, 1 -methylethoxy (isopropoxy), butoxy, 1 - methylpropoxy (sec-butoxy), 2-methylpropoxy (isobutoxy) or 1 ,1 -dimethylethoxy (tertbutoxy).

In the context of the invention the expressions haloalkyl and haloalkoxy refer to partially or fully halogenated alkyl or alkoxy. In other words, one or more hydrogen atoms, for example 1 , 2, 3, 4 or 5 hydrogen atoms bonded to one or more carbon atoms of alkyl or alkoxy are replaced by a halogen atom, in particular by fluorine or chlorine.

In the context of the invention the expression "Cs-Cy-cycloalkyl" refers to monocyclic cycloaliphatic radicals having from 3 to 7 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl, preferably cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

The expression "halogen" denotes in each case fluorine, chlorine, bromine or iodine.

The compounds of formulae (I), (A) and (B) form salts which are also within the scope of this invention. Pharmaceutically acceptable (i.e. non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful, e.g., in isolation or purification steps which may be employed during preparation. Salts of the compounds of formulae (I), (A) and (B) may be formed, for example, by reacting a compound of formulae (I), (A) and (B) with at least one acid or base. The acid or base is added in an amount suitable for partial or complete neutralization e.g. an equivalent amount.

The phrase "pharmaceutically acceptable salt(s)" as used herein, unless otherwise indicated, includes salts containing pharmacologically acceptable anions or cations, such as the chloride, bromide, iodide, 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, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate [i.e. 4,4'-methylene-bis-(3-hydroxy-2-naphthoate)] salts.

In the context of the present invention, the chemical structure that does not explicitly show a specific stereochemical orientation usually means all possible stereoisomers and mixtures thereof, unless indicated otherwise. For example, the compounds of formula (I)

in which * designates the asymmetry centers, represent the isomers of formulae (1.1 ), (1.2), (I.3), (I.4), (I.5), (I.6), (I.7), (I.8), (I.9), (1.10), (1.1 1 ), (1.12), (1.13), (1.14), (1.15), (1.16), (1.17), (1.18(, (1.19), (I.20), (1.21 ), (I.22), (I.23), (I.24), (I.25), (I.26), (I.27), (I.28), (I.29), (I.30), (1.31 ) ad (1.32):

wherein, R¹, R², R³ and R⁴ has one of the meanings as defined above or below. “Chiral compounds” in the sense of the invention are compounds that contain no improper axis of rotation (S_n). In the context of the present invention, they are in particular compounds with at least four chirality centers and without S_n-symmetry.

“Stereoisomers” in the context of the invention are compounds of identical constitution but different atomic arrangement in the three-dimensional space.

“Enantiomers” are stereoisomers which behave like image to mirror image to one another. The “enantiomeric excess” (ee) achieved during asymmetric synthesis is given here by the following formula: ee [%]=(R-S)/(R+S)*100. R and S are the descriptors of the CIP system for the two enantiomers and describe the absolute configuration on the asymmetric atom. The enantiomerically pure compound (ee=100%) is also referred to as “homochiral compound”.

“Diastereomers” are stereoisomers which are not enantiomeric to one another.

The compound of the invention can exist in various isomeric forms, as well as in one or more tautomeric forms, including both single tautomers and mixtures of tautomers. The term “isomer” is intended to encompass all isomeric forms of a compound of this invention, including tautomeric forms of the compound.

Some compounds described herein can have asymmetric centers and therefore exist in different enantiomeric and diastereomeric forms. A compound of the invention can be in the form of an optical isomer or a diastereomer. Accordingly, the invention encompasses compounds of the invention and their uses as described herein in the form of their optical isomers, diastereoisomers and mixtures thereof, including a racemic mixture. Optical isomers of the compounds of the invention can be obtained by known techniques such as asymmetric synthesis, chiral chromatography, or via chemical separation of stereoisomers through the employment of optically active resolving agents.

Unless otherwise indicated, “stereoisomer” means one stereoisomer of a compound that is substantially free of other stereoisomers of that compound. Thus, a stereomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound. A typical stereomerically pure compound comprises greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, for example greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, or greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound.

A “stereoisomer” refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. The present invention contemplates various stereoisomers and mixtures thereof and includes “enantiomers”, which refers to two stereoisomers whose molecules are nonsuperimposable mirror images of one another.

Compounds of the invention or their pharmaceutically acceptable salts may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- or as (D)- or (L)- for amino acids. The present invention is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography.

Relative configuration in stereochemistry (relative stereochemistry) is the arrangement of atoms or groups of atoms that is described relative to other atoms or groups of atoms in the molecule. In other words, this term describes the position of atoms or groups of atoms in space in relation to other atoms or groups of atoms that are located elsewhere in the molecule.

Absolute configuration in stereochemistry (absolute stereochemistry) is the arrangement of atoms or group of atoms that is described independently of any other atom or group of atoms in the molecule. This type of configuration is defined for chiral molecular entities and their stereochemical descriptions (e.g. R or S).

Racemic mixture or racemate is defined as a mixture of compounds consisting of two molecules structured like image and mirror image (= enantiomers) and which are present in an equimolar mixture, i.e. in the ratio 1 :1 (50:50). Preferred are compounds of formula (I), wherein

R¹ is selected from F, Cl and CN;

R² is C1-C4 alkyl, wherein alkyl is unstubstituted or substituted by 1 , 2 or 3 substituents NR^aR^b;

R³ is selected from C1-C2 alkyl;

R⁴ is selected from C1-C2 alkyl;

R^a and R^b independently from each other are selected from hydrogen and C1-C4.

In a first embodiment the compound of formula (I) is the compound A

In a second embodiment the compound of formula (I) is the compound B (also II.3, Zotatifin CAS 2098191 -53-6) or a mixture of B with its enatiomer B1 , preferably with enantiomer excess (ee) of the enantiomer of formula B of at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99%

B B1 , or a pharmaceutically acceptable salt thereof, for use as inhibitor of RAS proteine activation. Preferred is a compound of formula (B) or an enantiomeric mixture comprising the compounds of formula (B) and its enantiomer (B1 ), in particular wherein the enantiomer excess (ee) of the enantiomer of formula (B) is at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99% for use as inhibitor of RAS proteine activation.

Especially preferred is the compound B.

In a third embodiment the compound of formula (I) is the compound C (also 11.1 ) or a mixture of C with its enatiomer C1 , preferably with enantiomer excess (ee) of the enantiomer of formula C of at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99%

or a pharmaceutically acceptable salt thereof, for use as inhibitor of RAS proteine activation.

Preferred is a compound of formula (C) or an enantiomeric mixture comprising the compounds of formula (C) and its enantiomer (C1 ), in particular wherein the enantiomer excess (ee) of the enantiomer of formula (C) is at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99% for use as inhibitor of RAS proteine activation.

In a fourth embodiment the compound of formula (I) is the compound D (also 11.2) or a mixture of D with its enatiomer D1 , preferably with enantiomer excess (ee) of the enantiomer of formulaD of at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99%

D D1 or a pharmaceutically acceptable salt thereof, for use as inhibitor of RAS proteine activation.

Preferred is a compound of formula (D) or an enantiomeric mixture comprising the compounds of formula (D) and its enantiomer (D1 ), in particular wherein the enantiomer excess (ee) of the enantiomer of formula (D) is at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99% for use as inhibitor of RAS proteine activation.

In a fifth embodiment the compound of formula (I) is the compound E (also II.4) or a mixture of E with its enatiomer E1 , preferably with enantiomer excess (ee) of the enantiomer of formula E of at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99%

E E1 or a pharmaceutically acceptable salt thereof, for use as inhibitor of RAS proteine activation.

Preferred is a compound of formula (E) or an enantiomeric mixture comprising the compounds of formula (E) and its enantiomer (E1 ), in particular wherein the enantiomer excess (ee) of the enantiomer of formula (E) is at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99% for use as inhibitor of RAS proteine activation. In a further embodiment the compound of formula (I) is the compound (11.1 ) depicted below.

In a further embodiment the compound of formula (I) is the compound (II.2) depicted below.

In a further embodiment the compound of formula (I) is the compound (II.4) depicted below.

In a further embodiment the compound of formula (I) is the compound (II.5) depicted below.

In a further embodiment the compound of formula (I) is the compound (II.6) depicted below.

In a further embodiment the compound of formula (I) is the compound (II.7) depicted below.

In a further embodiment the compound of formula (I) is the compound (II.8) depicted below.

In a further embodiment the compound of formula (I) is the compound (II.9) depicted below.

In a further embodiment the compound of formula (I) is the compound (11.10) depicted below.

In a further embodiment the compound of formula (I) is the compound (11.1 1 ) depicted below.

In a further embodiment the compound of formula (I) is the compound (11.12) depicted below.

In a further embodiment the compound of formula (I) is the compound (11.13) depicted below.

In a further embodiment the compound of formula (I) is the compound (11.14) depicted below.

In a further embodiment the compound of formula (I) is the compound (11.15) depicted below.

In a further embodiment the compound of formula (I) is the compound (11.16) depicted below.

In a further embodiment the compound of formula (I) is the compound (11.17) depicted below.

In a further embodiment the compound of formula (I) is the compound (11.18) depicted below.

In a further embodiment the compound of formula (I) is the compound (11.19) depicted below.

In a further embodiment the compound of formula (I) is the compound (11.20) depicted below. In a further embodiment the compound of formula (I) is the compound (11.21 ) depicted below.

In a further embodiment the compound of formula (I) is the compound (11.22) depicted below.

In a further embodiment the compound of formula (I) is the compound (II.23) depicted below.

In a further embodiment the compound of formula (I) is the compound (II.24) depicted below.

In a further embodiment the compound of formula (I) is the compound (II.25) depicted below.

In a further embodiment the compound of formula (I) is the compound (II.26) depicted below.

In a further embodiment the compound of formula (I) is the compound (II.27) depicted below.

In a further embodiment the compound of formula (I) is the compound (II.28) depicted below.

In a further embodiment the compound of formula (I) is the compound (II.29) depicted below.

In a further embodiment the compound of formula (I) is the compound (II.30) depicted below.

In a further embodiment the compound of formula (I) is the compound (11.31 ) depicted below.

In a further embodiment the compound of formula (I) is the compound (II.32) depicted below.

Preferred is a compound of formula (B) or an enantiomeric mixture comprising the compounds of formula (B) and its enantiomer (B1 ), in particular wherein the enantiomer excess (ee) of the enantiomer of formula (B) is at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99% for use in treating proliferative disorders, wherein RAS-signaling is involved, preferably wherein KRAS G12V, NRAS G12V, HRAS G12V, KRAS G12C, KRAS G12D, KRAS G12C/Y96D, KRAS G13C, KRAS G13D, KRASG13S, KRAS Q61 H, KRAS Q61 R or KRAS Q61 K is involved, or wherein any activating mutation in KRAS, HRAS and NRAS is involved.

Preferred is a compound of formula (C) or an enantiomeric mixture comprising the compounds of formula (C) and its enantiomer (C1), in particular wherein the enantiomer excess (ee) of the enantiomer of formula (C) is at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99% for use in treating proliferative disorders, wherein RAS-signaling is involved, preferably wherein KRAS G12V, NRAS G12V, HRAS G12V, KRAS G12C, KRAS G12D, KRAS G12C/Y96D, KRAS G13C, KRAS G13D, KRASG13S, KRAS Q61 H, KRAS Q61 R or KRAS Q61 K is involved, or wherein any activating mutation in KRAS, HRAS and NRAS is involved.

Preferred is a compound of formula (D) or an enantiomeric mixture comprising the compounds of formula (D) and its enantiomer (D1 ), in particular wherein the enantiomer excess (ee) of the enantiomer of formula (D) is at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99% for use in treating proliferative disorders, wherein RAS-signaling is involved, preferably wherein KRAS G12V, NRAS G12V, HRAS G12V, KRAS G12C, KRAS G12D, KRAS G12C/Y96D, KRAS G13C, KRAS G13D, KRASG13S, KRAS Q61 H, KRAS Q61 R or KRAS Q61 K is involved, or wherein any activating mutation in KRAS, HRAS and NRAS is involved.

Preferred is a compound of formula (E) or an enantiomeric mixture comprising the compounds of formula (E) and its enantiomer (E1 ), in particular wherein the enantiomer excess (ee) of the enantiomer of formula (E) is at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99% for use in treating proliferative disorders, wherein RAS-signaling is involved, preferably wherein KRAS G12V, NRAS G12V, HRAS G12V, KRAS G12C, KRAS G12D, KRAS G12C/Y96D, KRAS G13C, KRAS G13D, KRASG13S, KRAS Q61 H, KRAS Q61 R or KRAS Q61 K is involved, or wherein any activating mutation in KRAS, HRAS and NRAS is involved.

Preferred is a compound of formula (B) or an enantiomeric mixture comprising the compounds of formula (B) and its enantiomer (B1 ), in particular wherein the enantiomer excess (ee) of the enantiomer of formula (B) is at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99% for use as a ligand of prohibitins (PHB1/2 complex) in the plasma membrane.

Preferred is a compound of formula (C) or an enantiomeric mixture comprising the compounds of formula (C) and its enantiomer (C1), in particular wherein the enantiomer excess (ee) of the enantiomer of formula (C) is at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99% for use as a ligand of prohibitins (PHB1/2 complex) in the plasma membrane. Preferred is a compound of formula (D) or an enantiomeric mixture comprising the compounds of formula (D) and its enantiomer (D1 ), in particular wherein the enantiomer excess (ee) of the enantiomer of formula (D) is at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99% for use as a ligand of prohibitins (PHB1/2 complex) in the plasma membrane.

Preferred is a compound of formula (E) or an enantiomeric mixture comprising the compounds of formula (E) and its enantiomer (E1), in particular wherein the enantiomer excess (ee) of the enantiomer of formula (E) is at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99% for use as a ligand of prohibitins (PHB1/2 complex) in the plasma membrane.

Preferred is a compound of formula (B) or an enantiomeric mixture comprising the compounds of formula (B) and its enantiomer (B1 ), in particular wherein the enantiomer excess (ee) of the enantiomer of formula (B) is at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99% for use in the prophylaxis and/or treatment of genetic disorders where RAS signaling is involved, in particular including RASopathies, craniofacial syndrome and Neurofibromatosis type I.

Preferred is a compound of formula (C) or an enantiomeric mixture comprising the compounds of formula (C) and its enantiomer (C1 ), in particular wherein the enantiomer excess (ee) of the enantiomer of formula (C) is at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99% for use in the prophylaxis and/or treatment of genetic disorders where RAS signaling is involved, in particular including RASopathies, craniofacial syndrome and Neurofibromatosis type I.

Preferred is a compound of formula (D) or an enantiomeric mixture comprising the compounds of formula (D) and its enantiomer (D1 ), in particular wherein the enantiomer excess (ee) of the enantiomer of formula (D) is at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99% for use in the prophylaxis and/or treatment of genetic disorders where RAS signaling is involved, in particular including RASopathies, craniofacial syndrome and Neurofibromatosis type I.

Preferred is a compound of formula (E) or an enantiomeric mixture comprising the compounds of formula (E) and its enantiomer (E1 ), in particular wherein the enantiomer excess (ee) of the enantiomer of formula (E) is at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99% for use in the prophylaxis and/or treatment of genetic disorders where RAS signaling is involved, in particular including RASopathies, craniofacial syndrome and Neurofibromatosis type I.

Preferred is a compound of formula (B) or an enantiomeric mixture comprising the compounds of formula (B) and its enantiomer (B1 ), in particular wherein the enantiomer excess (ee) of the enantiomer of formula (B) is at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99% for use of treating patients having developed resistance to RAS inhibitors, different from compounds of formula (I), in particular KRAS G12 C inhibitors.

Preferred is a compound of formula (C) or an enantiomeric mixture comprising the compounds of formula (C) and its enantiomer (C1 ), in particular wherein the enantiomer excess (ee) of the enantiomer of formula (C) is at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99% for use of treating patients having developed resistance to RAS inhibitors, different from compounds of formula (I), in particular KRAS G12 C inhibitors.

Preferred is a compound of formula (D) or an enantiomeric mixture comprising the compounds of formula (D) and its enantiomer (D1 ), in particular wherein the enantiomer excess (ee) of the enantiomer of formula (D) is at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99% for use of treating patients having developed resistance to RAS inhibitors, different from compounds of formula (I), in particular KRAS G12 C inhibitors.

Preferred is a compound of formula (E) or an enantiomeric mixture comprising the compounds of formula (E) and its enantiomer (E1 ), in particular wherein the enantiomer excess (ee) of the enantiomer of formula (E) is at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99% for use of treating patients having developed resistance to RAS inhibitors, different from compounds of formula (I), in particular KRAS G12 C inhibitors.

Pharmaceutical composition

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which 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 problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase "therapeutically effective" is intended to qualify the amount of each agent, which will achieve the goal of improvement in disorder severity and the frequency of incidence, while avoiding adverse side-effects typically associated with alternative therapies. For example, effective anticancer agents prolong the survivability of the patient or his/her life quality, inhibit the rapidly proliferating cell growth associated with the neoplasm, or effect a regression of the neoplasm.

The terms “treat,” “treating,” and “treatment,” as used herein, refer to any type of intervention or process performed on, or administering an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting or slowing down or preventing the progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease. By contrast, “prophylaxis” or “prevention” refers to administration to a subject who does not have a disease to prevent the disease from occurring.

As used herein, the term "cell" is meant to refer to a cell that is in vitro, ex vivo or in vivo. In the sense of the invention, an ex vivo cell can be part of a tissue sample excised from an organism such as a mammal. In the sense of the invention, an in vitro cell can be a cell in a cell culture. In the sense of the invention, an in vivo cell is a cell living in an organism such as a mammal.

The term “patient” includes humans and animals that receive either therapeutic or prophylactic treatment.

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

A (non-human) animal includes all vertebrates, e.g. mammals and non-mammals, including cows, sheep, pigs, goats, horses, poultry, dogs, cats, non-human primates, rodents etc. In one embodiment, the subject is a human subject.

The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid diluent, solvent, excipient, manufacturing aid (e.g. lubricant) or encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable other ingredients are the afore-mentioned carrier and further additives, including adjuvants, preserving agents, fillers, flow regulating agents, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, bittering agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents, dispensing agents, etc.. Suitable additives are selected depending on the nature of the mode of administration and dosage forms; and not injurious to the patient.

The term "pharmaceutical composition" means a composition comprising a compound of the invention in combination with at least one further compound selected from a) at least one further pharmaceutically active substance and b) at least one additional pharmaceutically acceptable carrier and or additive.

The term "RAS inhibitor" refers to an agent capable of decreasing RAS protein levels, decreasing RAS activity levels and/or inhibiting RAS expression levels in the cells, in particular refers to an agent capable of decreasing RAS activity levels in the cells. The RAS inhibitor may be a reversible or irreversible inhibitor. As used herein, “RAS” protein refers to a protein that is a member of a family of related proteins that are expressed in all human and animal cell lineages and organs. All RAS protein family members belong to a class of proteins called small GTPase (also known as small G proteins, a family of hydrolase enzymes that can bind and hydrolyse GTP), and are involved in transmitting signals within cells (cellular signal transduction). RAS is the prototypical member of the RAS superfamily of proteins, which are all related in three-dimensional structure and regulate diverse cell behaviours. When RAS is 'switched on' by incoming signals, it subsequently switches on other proteins, which ultimately turn on genes involved in cell growth, differentiation, and survival. Mutations in RAS genes can lead to the production of permanently activated RAS proteins, which can cause unintended and overactive signaling inside the cell, even in the absence of incoming signals. Because these signals result in cell growth and division, overactive RAS signaling can ultimately lead to cancer. The three RAS genes in humans (HRAS, KRAS, and NRAS) are the most common oncogenes in human cancer. As mentioned, the clinically most notable members of the RAS subfamily are HRAS, KRAS and NRAS. However, there are other members of this subfamily, which are e.g. selected from DIRAS1 , DIRAS2, DIRAS3, ERAS, GEM, MRAS, NKIRAS1 , NKIRAS2, NRAS, RALA, RALB, RAP1 A, RAP1 B, RAP2A, RAP2B, RAP2C, RASD1 , RASD2, RASL10A, RASL10B, RASL1 1A, RASL11 B, RASL12, REM1 , REM2, RERG, RERGL, RRAD, RRAS, RRAS2. In other words, the most common alterations in NRAS are 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 are HRAS Mutation (0.77%), HRAS Missense (0.75%), HRAS Exon 2 Mutation (0.30%), HRAS Codon 61 Missense (0.26%), and HRAS Q61 R (0.14%) The most common alterations in HRAS are HRAS Mutation (0.77%), HRAS Missense (0.75%), HRAS Exon 2 Mutation (0.30%), HRAS Codon 61 Missense (0.26%), and HRAS Q61 R (0.14%) (Source Mycancer genome portal).

The compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above and a pharmaceutical composition comprising at least one compound of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above may be administered to humans and animals, preferably humans.

In principle any method of administration may be used to deliver the compound or pharmaceutical composition according to the invention to a subject. Suitable methods of administration are orally, enterally, parenterally, intravenously, topically, intramuscular, subcutaneous routes.

The compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above can selectively decrease RAS protein levels, decrease RAS activity levels, in particular decrease the activity levels of HRAS and NRAS, especially KRAS4A,and KRAS4B) in the cells. For example, the compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above can be used to selectively decrease RAS activity levels in cells or in an individual in need of a decrease in RAS protein levels, decrease in RAS activity levels by administering an inhibiting amount of compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above or a salt thereof.

In one embodiment, the present invention provides a combined preparation of a compound or compounds of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above, and/or a pharmaceutically acceptable salt thereof, and (an) additional therapeutic agent(s) for simultaneous, separate or sequential use in the treatment and/or prophylaxis of (multiple) diseases, preferably of proliferative disorders (e.g. cancer), in particular disorders associated with the activity of RAS protein.

Additional therapeutic agent(s) are selected from chemotherapeutic agents, radiotherapeutic agents, immuno-oncology agents, and combinations thereof. In one aspect, the compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above are sequentially administered prior to administration of the immuno-oncology agent. In another aspect, compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above are administered concurrently with the immuno-oncology agent. In yet another aspect, compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above are sequentially administered after administration of the immuno-oncology agent.

In another aspect, compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above may be co-formulated with an immuno-oncology agent.

Immuno-oncology agents include, for example, a small molecule drug, antibody or other biologic or small molecule. Examples of biologic 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 humanized or human.

In one aspect, the immuno-oncology agent is (i) an agonist of a stimulatory (including a costimulatory) receptor or (ii) an antagonist of an inhibitory (including a co-inhibitory) signal on T cells, both of which result in amplifying antigen-specific T cell responses (often referred to as immune checkpoint regulators).

Suitable of the stimulatory and inhibitory molecules are members of the immunoglobulin super family (IgSF). One important family of membrane-bound ligands that bind to 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 TNF family of molecules that bind to cognate TNF receptor family members, which includes CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1 BBL, CD137 (4-1 BB), 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 1 b2, FAS, FASL, RELT, DR6, TROY, NGFR.

In one aspect, T cell responses can be stimulated by a combination of compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above and one or more of: (i) an antagonist of a protein that inhibits 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, PD1 H, LAIR1 , TIM-1 , and TIM-4, and

(ii) an agonist of a protein that stimulates T cell activation such as B7-1 , B7-2, CD28, 4-1 BB (CD 137), 4-1 BBL, ICOS, ICOS-L, 0X40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3 and CD28H.

Other agents that can be combined with compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above for the treatment of cancer include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above can be combined with antagonists of KIR, such as Lirilumab.

Yet other agents for combination therapies include agents that inhibit or deplete macrophages or monocytes, including but not limited to CSF-1 R antagonists such as CSF- 1 R antagonist antibodies including RG7155.

The combination therapy is intended to embrace administration of these therapeutic agents in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner.

Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single dosage form having a fixed ratio of each therapeutic agent or in multiple, single dosage forms for each of the therapeutic agents. Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination selected may be administered by intravenous injection while the other therapeutic agents of the combination may be administered orally. Alternatively, for example, all therapeutic agents may be administered orally or all therapeutic agents may be administered by intravenous injection. Combination therapy can also embrace the administration of the therapeutic agents as described above in further combination with other biologically active ingredients and non-drug therapies (e.g surgery or radiation treatment). Where the combination therapy further comprises a non-drug treatment, the non-drug treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and nondrug treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the non-drug treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.

Types of cancers that may be treated with the compounds of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above include, but are not limited to, prostate, colon, rectum, pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary, testis, head, neck, skin (including melanoma and basal carcinoma), mesothelial lining, white blood cell (including lymphoma and leukemia), esophagus, breast, muscle, connective tissue, lung (including small cell lung carcinoma and non-small-cell carcinoma), adrenal gland, thyroid, kidney, or bone; or glioblastoma, mesothelioma, renal cell carcinoma, gastric carcinoma, sarcoma (including Kaposi's sarcoma), choriocarcinoma, cutaneous basocellular carcinoma, haematological malignancies (including blood, bone marrow and lymph nodes) or testicular seminoma.

In one embodiment the invention relats to the inhibition of HRAS mutations, which are detected in bladder urothelial carcinoma, breast invasive ductal carcinoma, lung adenocarcinoma, prostatecarcinoma and colon adenocarcinoma. This accounts to nearly 0.94% of all human cancers and nearly 1 .02% of solid tumours. In another embodiment the invention relats to the inhibition of HRAS mutations, which are also detected in other cancers selected from chronic myelomonocytic leukemia, non-nodgkin lymphoma, thyroid gland carcinoma, head and neck squamous cell carcinoma, squamous cell lung carcinoma, ovarian carcinoma, poorly differentiated thyroid gland carcinoma, squamous cell carcinoma, small cell lung carcinoma, glioma, low grade glioma, pancreatic carcinoma, acute lymphoblastic leukemia, histiocytic and dendritic cell neoplasm, multiple myeloma, neurofibromatosis type, pancreatic ductal adenocarcinoma, thyroid gland follicular carcinoma, embryonal rhabdomyosarcoma, malignant thyroid gland neoplasm, thyroid gland undifferentiated (anaplastic) carcinoma, thymic carcinoma, urothelial carcinoma, thyroid gland papillary carcinoma cutaneous melanoma, mucosal melanoma, endometrial carcinoma, malignant peripheral nerve sheath tumor, neuroblastoma, prostate carcinoma, soft tissue sarcoma, breast carcinoma, colorectal adenocarcinoma, gastric carcinoma, diffuse, large b-cell lymphoma, diffuse gliom, myeloid dysplastic syndrome, renal cell carcinoma, astrocytic tumor, hepatocellular carcinoma and shwannoma.

NRAS mutations are detected in nearly 3.03% of all human cancers with frequent mutations cutaneous melanoma, melanoma, colonadenocarcinoma, ami, thyroid carcinoma and lung adenocarcinoma. Therefore another embodiment of the invention relats to the inhibition of NRAS mutations detected in cutaneous melanoma, melanoma, colonadenocarcinoma, ami, thyroid carcinoma and lung adenocarcinoma. This accounts for nearly 2.83% of malignant solid tumour patients. In another embodiment the invention relats to the inhibition of NRAS mutations, which are also detected in other cancers selected from colorectal carcinoma, non-small cell lung carcinoma, acute myeloid leukemia, myelodysplastic syndromes, chronic myelomonocytic leukemia, colorectal adenocarcinoma, multiple myeloma, non- hodgkin lymphoma, pancreatic carcinoma, cutaneous melanoma, ovarian carcinoma, pancreatic ductal adenocarcinoma, acute lymphoblastic leukemia, thyroid gland carcinoma, glioma, neurofibromatosis type 1 , poorly differentiated thyroid gland carcinoma secondary acute myeloid leukemia, therapy-related acute myeloid leukemia, myelodysplastic syndrome with excess blasts-2, juvenile myelomonocytic leukemia, histiocytic and dendritic cell neoplasm, head and neck squamous cell carcinoma, small cell lung carcinoma, low grade glioma, squamous cell lung carcinoma, breast carcinoma, chronic myelomonocytic leukemia-2, chronic myelomonocytic leukemia, thyroid gland undifferentiated (anaplastic) carcinoma, embryonal rhabdomyosarcoma, thyroid gland follicular carcinoma, t-cell acute lymphoblastic leukemia, mucosal melanoma, chronic myelomonocytic leukemia-1 ,low grade ovarian serous adenocarcinoma, thyroid gland papillary carcinoma, refractory anemia with excess blasts, myeloid neoplasm myelodysplastic/myeloproliferative neoplasm, unclassifiable, rectal carcinoma, colon carcinoma, malignant peripheral nerve sheath tumor, cholangiocarcinoma, endometrial carcinoma mantle cell lymphoma, secondary myelodysplastic syndrome, therapy-related myelodysplastic syndrome, lymphoma neuronal and mixed neuronal-glial tumors, ganglioglioma, soft tissue sarcoma, bladder carcinoma, esophageal carcinom, sarcoma, thymic carcinoma, lung adenocarcinoma, lung carcinoma, uveal melanoma head and neck carcinoma, diffuse glioma, squamous cell carcinoma, chronic myeloid leukemia, adenocarcinoma of the gastroesophageal junction, glioblastoma neuroblastoma, astrocytic tumo, hepatocellular carcinoma, pancreatic adenocarcinom, diffuse large b-cell lymphoma, anaplastic astrocytoma, gastric adenocarcinoma, gastric carcinoma, prostate carcinoma, renal cell carcinoma, acute myeloid leukemia arising from previous myelodysplastic syndrome, b-cell acute lymphoblastic leukemia, double-hit lymphoma, dysembryoplastic neuroepithelial tumor, gangliocytoma, low-grade neuroepithelial tumor, peripheral t-cell lymphoma, mpilocytic astrocytoma, pilomyxoid astrocytoma, rhabdoid tumor and schwannoma.

One or more additional pharmaceutical agents or treatment methods such as, for example, anti-viral agents, chemotherapeutics or other anti-cancer agents, immune enhancers, immunosuppressants, radiation, anti-tumor and anti-viral vaccines, cytokine therapy (e.g ., IL2 and GM-CSF), and/or tyrosine kinase inhibitors can be optionally used in combination with the compound(s) of formulae (I), (A), (B), (C), (D) and (E), as defined above, for treatment of RAS protein associated diseases, disorders or conditions. The agents can be combined with the present compounds in a single dosage form, or the agents can be administered simultaneously or sequentially as separate dosage forms.

Suitable chemotherapeutic or other anti-cancer agents include, for example, alkylating agents (including, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes) such as uracil mustard, chlormethine, cyclophosphamide (CYTOXAN®), ifosfamide, melphalan, chlorambucil, pipobroman, triethylene-melamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, and temozolomide.

In the treatment of melanoma, suitable agents for use in combination with the compound(s) of formulae (I), (A), (B), (C), (D) and (E), as defined above, include: dacarbazine (DTIC), optionally, along with other chemotherapy drugs such as carmustine (BCNU) and cisplatin; the "Dartmouth regimen", which consists of DTIC, BCNU, cisplatin and tamoxifen; a combination of cisplatin, vinblastine, and DTIC, temozolomide orYERVOY™. Compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above may also be combined with immunotherapy drugs, including cytokines such as interferon alpha, interleukin 2, and tumor necrosis factor (TNF) in the treatment of melanoma. Compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above may also be used in combination with vaccine therapy in the treatment of melanoma. Antimelanoma vaccines are, in some ways, similar to the anti-virus vaccines which are used to prevent diseases caused by viruses such as polio, measles, and mumps. Weakened melanoma cells or parts of melanoma cells called antigens may be injected into a patient to stimulate the body's immune system to destroy melanoma cells.

Melanomas that are confined to the arms or legs may also be treated with a combination of agents including one or more compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined, using a hyperthermic isolated limb perfusion technique. This treatment protocol temporarily separates the circulation of the involved limb from the rest of the body and injects high doses of chemotherapy into the artery feeding the limb, thus providing high doses to the area of the tumor without exposing internal organs to these doses that might otherwise cause severe side effects. Usually the fluid is warmed to 38.9 °C to 40 °C. Melphalan is the drug most often used in this chemotherapy procedure. This can be given with another agent called tumor necrosis factor (TNF). Suitable chemotherapeutic or other anti-cancer agents include, for example, antimetabolites (including, without limitation, folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors) such as methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatine, and gemcitabine.

Suitable chemotherapeutic or other anti-cancer agents further include, for example, certain natural products and their derivatives (for example, vinca alkaloids, antitumor antibiotics, enzymes, lymphokines and epipodophyllotoxins) such as vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, ara-C, paclitaxel (Taxol), mithramycin, deoxyco-formycin, mitomycin-C, L-asparaginase, interferons (especially IFN-a), etoposide, and teniposide.

Other cytotoxic agents include navelbene, CPT-1 1 , anastrazole, letrazole, capecitabine, reloxafme, and droloxafme.

Also suitable are cytotoxic agents such as epidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor; procarbazine; mitoxantrone; platinum coordination complexes such as cisplatin and carboplatin; biological response modifiers; growth inhibitors; antihormonal therapeutic agents; leucovorin; tegafur; and haematopoietic growth factors.

Other anti-cancer agent(s) include antibody therapeutics such as trastuzumab (HERCEPTIN®), antibodies to costimulatory molecules such as CTLA-4, 4-1 BB and PD-1 , or antibodies to cytokines (IL-IO or TGF-b).

Other anti-cancer agents also include those that block immune cell migration such as antagonists to chemokine receptors, including CCR2 and CCR4.

Other anti-cancer agents also include those that augment the immune system such as adjuvants or adoptive T cell transfer.

Anti-cancer vaccines include dendritic cells, synthetic peptides, DNA vaccines and recombinant viruses.

In a specific embodiment of the present invention, at least one compound of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above and at least one chemotherapeutic agent are administered to the patient concurrently or sequentially. In other words, at least one compound of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above may be administered first, at least one chemotherapeutic agent may be administered first, or at least one compound of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above may be administered at the same time. Additionally, when more than one compound of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above and/or chemotherapeutic agent is used, the compounds may be administered in any order.

The invention also provides pharmaceutically compositions which comprise a therapeutically effective amount of one or more of the compound(s) of formulae(l), (A), (B), (C), (D) and (E), 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.

The compound(s) of formulae (I), (A), (B), (C), (D) and (E), as defined above, may be administered by any suitable route, preferably in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the treatment intended. The compound(s) and compositions of the compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above, can be administered for any of the uses described herein by any suitable means, 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; sublingually; bucally; parenterally, such as by subcutaneous, intravenous, intramuscular, or intrasternal injection, or infusion techniques ( e.g. as sterile injectable aqueous or non-aqueous solutions or suspensions); nasally, including administration to the nasal membranes, such as by inhalation spray; topically, such as in the form of a cream or ointment; or rectally, such as in the form of suppositories. They can be administered alone, but generally will be administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

For oral administration, the pharmaceutical composition may be in the form of, for example, a tablet, capsule, liquid capsule, suspension, or liquid. The pharmaceutical composition is preferably made in the form of a dosage unit containing a particular amount of the active ingredient. For example, the pharmaceutical composition may be provided as a tablet or capsule comprising an amount of active ingredient in the range of 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. A suitable daily dose for a human or animal may vary widely depending on the condition of the patient and other factors, but, can be determined using routine methods. Any pharmaceutical composition contemplated herein can, for example, be delivered orally via any acceptable and suitable oral preparation. Exemplary oral preparations, include, but are not limited to, for example, tablets, troches, lozenges, aqueous and oily suspensions, dispersible powders or granules, emulsions, hard and soft capsules, liquid capsules, syrups, and elixirs. Pharmaceutical compositions intended for oral administration can be prepared according to any methods known in the art for manufacturing pharmaceutical compositions intended for oral administration. In order to provide pharmaceutically palatable preparations, a pharmaceutical composition in accordance with the invention can contain at least one agent selected from sweetening agents, flavoring agents, bittering agents, coloring agents, demulcents, antioxidants, and preserving agents.

A tablet can, for example, be prepared by admixing at least one compound of formulae (I), (A), (B), (C), (D) and (E), as defined above, and/or at least one pharmaceutically acceptable salt thereof with at least one non-toxic pharmaceutically acceptable excipient suitable for the manufacture of tablets. Exemplary excipients include, but are not limited to, for example, inert diluents, such as, for example, calcium carbonate, sodium carbonate, lactose, calcium phosphate, and sodium phosphate; granulating and disintegrating agents, such as, for example, microcrystalline cellulose, sodium crosscarmellose, corn starch, and alginic acid; binding agents, such as, for example, starch, gelatin, polyvinyl-pyrrolidone, and acacia; and lubricating agents, such as, for example, magnesium stearate, stearic acid, and talc. Additionally, a tablet can either be uncoated, or coated by known techniques to either mask the bad taste of an unpleasantly tasting drug, or delay disintegration and absorption of the active ingredient in the gastrointestinal tract thereby sustaining the effects of the active ingredient for a longer period. 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.

Hard gelatin capsules can, for example, be prepared by mixing at least one compound of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above, and/or at least one salt thereof with at least one inert solid diluent, such as, for example, calcium carbonate; calcium phosphate; and kaolin.

Soft gelatin capsules can, for example, be prepared by mixing at least one compound of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above, and/or at least one pharmaceutically acceptable salt thereof with at least one water soluble carrier, such as, for example, polyethylene glycol; and at least one oil medium, such as, for example, peanut oil, liquid paraffin, and olive oil.

An aqueous suspension can be prepared, for example, by admixing at least one compound of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above and/or at least one pharmaceutically acceptable salt thereof with at least one excipient suitable for the manufacture of an aqueous suspension. Exemplary excipients suitable for the manufacture of an aqueous suspension, include, but are not limited to, for example, suspending agents, such as, for example, sodium carboxymethylcellulose, hydroxypropylmethylcellulose and hydroxypropyl- cellulose, sodium alginate, alginic acid, polyvinylpyrrolidone, gum tragacanth, and gum acacia; dispersing or wetting agents, such as, for example, a naturally-occurring phosphatide, e.g., lecithin; condensation products of alkylene oxide with fatty acids, such as, for example, polyoxyethylene stearate; condensation products of ethylene oxide with long chain aliphatic alcohols, such as, for example heptadecaethylene-oxycetanol; condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol, such as, for example, polyoxyethylene sorbitol monooleate; and condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, such as, for example, polyethylene sorbitan monooleate. An aqueous suspension can also contain at least one preservative, such as, for example, 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.

Oily suspensions can, for example, be prepared by suspending at least one compound of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above and/or at least one pharmaceutically acceptable salt thereof in either a vegetable oil, such as, for example, arachis oil, olive oil, sesame oil and coconut oil or in mineral oil, such as, for example, liquid paraffin. An oily suspension can also contain at least one thickening agent, such as, for example, beeswax, hard paraffin and cetyl alcohol. In order to provide a palatable oily suspension, at least one of the sweetening agents already described hereinabove, and/or at least one flavoring agent can be added to the oily suspension. An oily suspension can further contain at least one preservative, including, but not limited to, for example, an anti-oxidant, such as, for example, butylated hydroxyanisol, and alphatocopherol.

Dispersible powders and granules can, for example, be prepared by admixing at least one compound of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above and/or at least one pharmaceutically acceptable salt thereof with at least one dispersing and/or wetting agent; at least one suspending agent; and/or at least one preservative. Suitable dispersing agents, wetting agents, and suspending agents are as already described above. Exemplary preservatives include, but are not limited to, for example, anti-oxidants, e.g., ascorbic acid. In addition, dispersible powders and granules can also contain at least one excipient, including, but not limited to, for example, sweetening agents; flavoring agents; and coloring agents.

An emulsion of at least one compound of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above and/or at least one pharmaceutically acceptable salt thereof can, for example, be prepared as an oil-in-water emulsion. The oily phase of the emulsions comprising compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above may be constituted from known ingredients in a known manner. The oil phase can be provided by, but is not limited to, for example, a vegetable oil, such as, for example, olive oil and arachis oil; a mineral oil, such as, for example, liquid paraffin; and mixtures thereof. While the phase may comprise merely an emulsifier, it may comprise a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Suitable emulsifying agents include, but are not limited to, for example, naturally-occurring phosphatides, e.g., soy bean lecithin; esters or partial esters derived from fatty acids and hexitol anhydrides, such as, for example, sorbitan monooleate; and condensation products of partial esters with ethylene oxide, such as, for example, polyoxyethylene sorbitan monooleate. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabilize) make-up the so- called emulsifying wax, and the wax together with the oil and fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations. An emulsion can also contain a sweetening agent, a flavoring agent, a preservative, and/or an antioxidant. Emulsifiers and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate, sodium lauryl sulfate, glyceryl distearate alone or with a wax, or other materials well known in the art.

The compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above and/or at least one pharmaceutically acceptable salt thereof can, for example, also be delivered intravenously, subcutaneously, and/or intramuscularly via any pharmaceutically acceptable and suitable injectable form. Exemplary injectable forms include, but are not limited to, for example, sterile aqueous solutions comprising acceptable vehicles and solvents, such as, for example, water, Ringer’s solution, and isotonic sodium chloride solution; sterile oil-in-water microemulsions and aqueous or oleaginous suspensions.

Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions may be prepared from sterile powders or granules using one or more of the carriers or diluents mentioned for use in the formulations for oral administration or by using other suitable dispersing or wetting agents and suspending agents. The compounds may 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 various buffers. Other adjuvants and modes of administration are well and widely known in the pharmaceutical art. The active ingredient may also be administered by injection as a composition with suitable carriers, including saline, dextrose, water or with cyclodextrin solubilization (i.e. Captisol), cosolvent solubilization (i.e. propylene glycol) or micellar solubilization (i.e. Tween 80).

The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1 ,3- butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

A sterile injectable oil-in-water microemulsion can, for example, be prepared by 1 ) dissolving at least one compound of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above in an oily phase, such as, for example, a mixture of soybean oil and lecithin; 2) combining the compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above containing oil phase with a water and glycerol mixture; and 3) processing the combination to form a microemulsion.

A sterile aqueous or oleaginous suspension can be prepared in accordance with methods already known in the art. For example, a sterile aqueous solution or suspension can be prepared with a non-toxic parenterally-acceptable diluent or solvent, such as, for example, 1 ,3-butane diol; and a sterile oleaginous suspension can be prepared with a sterile non- toxic acceptable solvent or suspending medium, such as, for example, sterile fixed oils, e.g., synthetic mono- or diglycerides; and fatty acids, such as, for example, oleic acid. Pharmaceutically acceptable carriers are formulated according to a number of factors well within the purview of those of ordinary skill in the art. These include, without limitation: the type and nature of the active agent being formulated; the subject to which the agentcontaining composition is to be administered; the intended route of administration of the composition; and the therapeutic indication being targeted. Pharmaceutically acceptable carriers include both aqueous and non-aqueous liquid media, as well as a variety of solid and semi-solid dosage forms. Such carriers can include a number of different ingredients and additives in addition to the active agent, such additional ingredients being included in the formulation for a variety of reasons, e.g., stabilization of the active agent, binders, etc., well known to those of ordinary skill in the art. Descriptions of suitable pharmaceutically acceptable carriers, and factors involved in their selection, are found in a variety of readily available sources such as, for example, Allen, L. V. Jr. et al. Remington: The Science and Practice of Pharmacy (2 Volumes), 22nd Edition (2012), Pharmaceutical Press.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-alpha-tocopherol poly ethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens, polyethoxylated castor oil such as CREMOPHOR surfactant (BASF), or other similar polymeric 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 silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat. Cyclodextrins such as alpha-, beta-, and gamma-cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-cyclodextrins, or other solubilized derivatives may also be advantageously used to enhance delivery of compounds of the formulae described herein.

The pharmaceutically active compounds of this invention can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, including humans and other mammals. The pharmaceutical compositions may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc. Tablets and pills can additionally be prepared with enteric coatings. Such compositions may also comprise adjuvants, such as wetting, sweetening, flavoring, and perfuming agents.

For therapeutic purposes, the active compounds of this invention are ordinarily combined with one or more adjuvants appropriate to the indicated route of administration. If administered orally, the compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted or encapsulated for convenient administration. Such capsules or tablets may contain a controlled-release formulation as may be provided in a dispersion of active compound in hydroxypropylmethyl cellulose.

The amounts of compounds that are administered and the dosage regimen for treating a disease condition with the compounds and/or compositions of this invention depend on a variety of factors, including the age, weight, sex, the medical condition of the subject, the type of disease, the severity of the disease, the route and frequency of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods. A daily dose of about 0.001 to 100 mg/kg body weight, preferably between about 0.0025 and about 50 mg/kg body weight and most preferably between about 0.005 to 10 mg/kg body weight, may be appropriate. The daily dose can be administered in one to four doses per day. Other dosing schedules include one dose per week and one dose per two day cycle.

Pharmaceutical compositions of this invention comprise at least one compound of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above and/or at least one pharmaceutically acceptable salt thereof, and optionally an additional agent selected from any pharmaceutically acceptable carrier, adjuvant, and vehicle. Alternate compositions of this invention comprise a compound of the formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above, or a prodrug thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

The present invention also includes pharmaceutical kits useful, for example, in the treatment or prevention of RAS protein-associated diseases. Thus, the present invention also relates to a kit containing a formulation comprising: a) a pharmaceutical composition comprising a compound of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above, or a therapeutically acceptable salt thereof and a pharmaceutically acceptable carrier; and b) instructions for dosing of the pharmaceutical composition for the treatment of a disorder in which inhibition of RAS activation is effective in treating the disorder.

Such kits can further include, if desired, one or more of various conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, as will be readily apparent to those skilled in the art. Instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.

The dosage regimen for the compounds of the present invention will, of course, vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient, and the effect desired.

By way of general guidance, the daily oral dosage of each active ingredient, when used for the indicated effects, will range between about 0.001 to about 5000 mg per day, preferably between about 0.01 to about 1000 mg per day, and most preferably between about 0.1 to about 250 mg per day. Intravenously, the most preferred doses will range from about 0.01 to about 10 mg/kg/minute during a constant rate infusion. Compound(s) of the formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily.

The compounds are typically administered in admixture with suitable pharmaceutical diluents, excipients, or carriers (collectively referred to herein as pharmaceutical carriers) suitably selected with respect to the intended form of administration, e.g. oral tablets, capsules, elixirs, and syrups, and consistent with conventional pharmaceutical practices.

Dosage forms (pharmaceutical compositions) suitable for administration may contain from about 1 milligram to about 200 milligrams of active ingredient per dosage unit. In these pharmaceutical compositions the active ingredient will ordinarily be present in an amount of about 0.1 -95 % by weight based on the total weight of the composition.

A typical capsule for oral administration contains at least one of the compound of the formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof (250 mg), lactose (75 mg), and magnesium stearate (15 mg). The mixture is passed through a 60 mesh sieve and packed into a no. 1 gelatin capsule.

A typical injectable preparation is produced by aseptically placing at least one of the compound of the formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof (250 mg) into a vial, aseptically freeze-drying and sealing. For use, the contents of the vial are mixed with 2 mL of physiological saline, to produce an injectable preparation.

The present invention includes within its scope pharmaceutical compositions comprising, as an active ingredient, a therapeutically effective amount of at least one of the compound of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above, alone or in combination with a pharmaceutical carrier. Optionally, compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above can be used alone, in combination with other compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above, or in combination with one or more other therapeutic agent(s), e.g. an anticancer agent or other pharmaceutically active material.

Regardless of the route of administration selected, the compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factors including the activity of the particular compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start with doses of compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, oral, intravenous, intracerebroventricular and subcutaneous doses of the compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above for a patient will range from about 0.01 to about 50 mg per kilogram of body weight per day.

If desired, the effective daily dose of the 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 forms. In certain aspects of the invention, dosing is one administration per day.

While it is possible for compound(s) of formulae (I), (A), (B) (C), (D), (E) or an enantiomeric mixture thereof as defined above to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).

The above other therapeutic agents, when employed in combination with the compound(s) of formulae (I), (A) and (B) as defined above, may be used, for example, in those amounts indicated in the Physicians’ Desk Reference (PDR) or as otherwise determined by one of ordinary skill in the art. In the methods of the present invention, such other therapeutic agent(s) may be administered prior to, simultaneously with, or following the administration of the inventive compounds.

The invention will be illustrated further with reference to the examples that follow, without restricting the scope to the specific embodiments described. The invention includes all combinations of described and especially of preferred features that do not exclude each other.

DESCRIPTION OF THE DRAWINGS Figurel : NanoBit assay for RAS activation (KRAS G12, KRAS G13, KRAS Q61 )

NanoBiT assay for the RAS GTP-loading was performed in HeLa cells transfected with LgBit-KRAS mutants and and HRAS G12V and SmBit-CRAF-RBD. Cells were treated with compounds according to the invention (compound B/Zotatifin) (250 nM) for 2 h in serum- free DMEM. After incubation, the substrate for NanoLuc was added, and the luminescence was measured in a multiplate reader. Data were normalized to cells transfected with the indicated mutant and exposed to DMSO for 2 h. DMSO-treated cells were set as 1. The bars represent mean ± SEM from 3 independent experiments.

Figure 2: NanoBiT assay for RAS activation (KRAS G12V, NRAS G12V and HRAS G12V).

NanoBiT assay for the RAS GTP-loading was performed in HeLa cells transfected with LgBit-KRAS mutants and SmBit-CRAF-RBD. Cells were treated with compounds according to the invention (250 nM) for 2 h in serum-free DMEM. After incubation, the substrate for NanoLuc was added, and the luminescence was measured in a multiplate reader. Data were normalized to cells transfected with the indicated mutant and exposed to DMSO for 2 h. DMSO-treated cells were set as 1 . The bars represent mean ± SEM from 3 independent experiments.

Figure 3: MTT assay for cell viability in 96 well cell culture plate

Cells with the KRAS mutations were treated with Zotatifin for 48h and cell viability was evaluated by MTT assay. The bars represent mean ± SEM from 3 independent experiments. Each dot indicates the value from each experiment.

It is shown that compound B (Zotatifin) can inhibit the growth of different tumour cells with defined RAS mutations (MDAMB231 with KRASG13D mutation, HCT-116 with KRASG13D mutation, NCI-H2122 with KRASG12C mutation, NCI-358 with KRASG12C, Aspcl with KRASG12D,HT-1080with NRASQ61 K mutation, T24 with HRASG12V and NCI-358 with KRASG12C mutations.

Figure 4A 4B 4C: The effect of Zotatifin treatment on EGF mediated RAS-MAPK activation

Figure 4A: HeLa cells were cultured in a serum-free medium with Zotatifin (200 nM) for 4 h. After incubation, cells were stimulated with EGF (final concentration 100 ng/ml) and the cells were collected after 30 min of stimulation. Immunoblot analyses were performed with the cell lysates to detect the activation of MEK1/2.

It is demonstrated that Zotatifin treatment inhibits the binding of endogenous RAS proteins activated by the growth factor EGF to the RAS-binding domain of CRAF kinase.

Figure 4B and C: The level of pMEK1/2 and Ras activation was quantified by Imaged software. The bars represent mean ± SD from 3 independent experiments. The values of DMSO-treated cells were taken as 1 .

Figure 5A und 5B: The effect of Zotatifin treatment on mutation dependent RAS-MAPK activation.

Figure 5A: Mutated Ras oncogene was transfected to HeLa cells and the cells were cultured in the medium with Zotatifin (200 nM) for 24 h. After incubation, cells were harvested and subjected to SDS-PAGE and westernblotting analyses.

It is shown that the action of Zotatifin as a mere elF4A inhibitor can be delineated. For instance, Zotatifin inhibits the action of exogenously expressed RAS proteins in cells with defined mutations without compromising the expression of the total proteins.

Figure 5B: The level of pMEK1/2 was quantified using Imaged software. The bars represent mean ± SD from 3 independent experiments. DMSO-treated cells were set as 1 .

Figure 6: MTT assay for cell viability in 96 well cell culture plate

Cells with the KRAS, NRAS, and HRAS mutations were treated with Zotatifin for 48h and cell viability was evaluated by MTT assay. The bars represent mean ± SEM from 3 independent experiments. Each dot indicates the value from each experiment.

It is shown that Zotatifin can inhibit the growth of different tumour cells with defined RAS mutations (MDAMB231 with KRASG13D mutation, HCT-1 16 with KRASG13D mutation, NCI-H2122 with KRASG12C mutation, NCI-358 with KRASG12C, Aspcl with KRASG12D,HT-1080with NRASQ61 K mutation, T24 with HRASG12V and NCI-358 with KRASG12C mutations.

Figure 7: IC 50 values of Zotatifin for KRAS inhibition

Figure 7A: KRAS G12V NanoBiT NanoBiT assay for the RAS GTP-loading was performed in HeLa cells transfected with LgBit-KRAS G12V and SmBit-CRAF-RBD. Cells were treated with Zotatifin (0-400 nM) for 2 h in serum-free DMEM. After incubation, the substrate of NanoLuc was added and the luminescence was measured using a multiplate reader. Data were normalized to cells transfected with the indicated mutant and treated with DMSO for 2 h. DMSO-treated cells were set as 1 . IC50 is 31 nM.

Figure 7B: MTT assay for cell viability in 96 well cell culture plate

Cells with the KRAS mutations were treated with Zotatifin for 48h and cell viability was evaluated by MTT assay. IC50 was obtained using Prism 9.

Cell culture

Calu-1 cells were obtained from Sigma-Aldrich and cultured in Mccoy’s 5A medium (10 % heat inactivated FBS). HeLa (DSMZ), MDA-MB-231 , and HCT-1 16 were authenticated by Eurofin genomics and cultured in DMEM (10 % heat inactivated FBS). ASPC-1 cells were purchased from DSMZ and cultured in RPMI-1640 (10 % heat inactivated FBS). NCI-H358 and NCI-H2122 were purchased from ATCC and cultured in RPMI-1640 (10 % heat inactivated FBS). T24 and HT-1080 were cultured in Mccoy’s 5A medium (10% heat inactivated FBS) and EMEM (10 % heat inactivated FBS + 1 mM Na Pyruvate) respectively. HeLa cells were starved in the serum free medium with Zotatifin for 4 hours and stimulated with EGF (100 ng/ml) for 30min.

DNA Transfection

HeLa cells were harvested with 0.05% Trypsin/0.02% EDTA in PBS and seeded in 6 well or 12 well cell culture plates at the concentration of 5x104 cells in complete DMEM (2 ml for 6 well plate and 1 ml for 12 well plate). After 1 day from seeding, the DNA were transfected with Flag tagged Ras or NanoBiT plasmid using PEI/PBS solution.

NanoBiT assay

The constructs were transfected to HeLa cells and the cells were harvested after 1 day of transfection. The cells were seeded to 96 well white plate. Next day, the medium was changed to serum free DMEM for 2h with Zotatifin. After incubation, Nano Gio assay was performed. The luminescence was measured using Tecan SPARKS (Tecan).

Cell viability assay (MTT)

Cancer cells were seeded in 96 well plates (5x103 cells/well) in 80 pl in growth medium and cultured for 1 day in the incubator. Next day, 20 pl of compound B (Zotatifin) containing growth medium was added to each well and the cells were cultured for 48h. After incubation with Zotatifin, 10 pl of MTT solution was added to the wells and incubated for 2-4 hours. After incubation with MTT, solubilization buffer was added and incubated over night. MTT was measured at O.D. 570 nm with a plate reader (Tecan).

Soft agar colony formation assay

Agarose solution of 1 .5% was mixed with equal volume of 2x growth medium supplemented with 20% FBS. The resulting agarose/medium solution was dispensed in a 96-well plate, using 50 pl per well, and incubated at room temperature for 10 min to allow bottom layer to solidify. To each well a 75 pl upper layer was added containing 5000 cells per well in 0.5% agarose/1x complete medium. After solidification of the upper layer, 125 pl of 2x compound dilution was added to each well. The cells seeded in soft agar were cultured for 5 to 10 days to allow formation of colonies.

Cell viability was then assessed using Cell Proliferation Kit I (Roche, Basel, Switzerland), by adding 25 pl MTT solution and incubating for 4 h in CO2 incubator. Then medium was removed and 175 pl of solubilization buffer was added to each well and incubated 1 h at 70 °C. Once agar was melted, cell viability, assessed by the amount of metabolized MTT, was quantified by measuring absorbance at 570 nm.

Active Ras pull down assay

After the stimulation with EGF, active Ras pull down buffer (25 mM T ris-HCI pH7.2, 150 mM NaCI, 5 mM MgCI2, 1% NP-40, 5% Glycerol with protease inhibitor cocktail) was added to the cells and the cells were sonicated for 3 seconds. The cell lysates were centrifuged for 15 min at 4C, 13000 rpm. Protein concentration was adjusted by 660 nm protein assay reagent (Thermoscientific) and 20% of the lysate was collected for the total cell lysate control. 10 pl of CRAF-RBD protein coated agarose beads were added to the rest of lysates and rotated at 4°C for 1 h. After incubation, the beads were washed with binding buffer twice and 50 pl of SDS-PAGE sample buffer (125mM Tris-HCI pH 6.8, 4% SDS, 10% Glycerol, BPB) was added.

SDS-PAGE and Western blotting

The samples were subjected to 12% SDS-PAGE followed by western blotting. The membrane was incubated with 3% BSA/TBST (20mM T ris-HCI, pH7.5, 150mM NaCI, 0.05% Tween-20) for 1 h at room temperature. After blocking, the membrane was incubated with primary antibody diluted in 1% BSA/TBST and incubated over night at 4C. After the over night incubation, the membrane was washed with TBST (5minx5) and incubated with HRP conjugated secondary antibody in TBST for 1 h at RT. After the secondary antibody treatment, the membrane was washed and the signal was detected using chemiluminescence substrate (Millipore) and Chemidoctouch (Bio-Rad).

Antibodies Anti-phospho CRAF antibody S338 (#9427), Anti-phospho MEK1/2 (#9154), Anti-MEK1 antibody (#2352) were purchased from Cell Signalling Technology. Anti-CRAF antibody (sc- 133) and Anti-pan Ras antibody (SC-166691 ) were purchased Santa Cruz. Anti-Vinculin antibody (SAB4200080) was purchased from Sigma-Aldrich. HRP conjugated antibodies for mouse and rabbit IgG were obtained from Novex (A16066 and A16096, respectively.)

EXAMPLES

Zotatifin: 4-{(5aR,6S,7S,8R,8aS)-7-[(dimethylamino)methyl]-8,8a-dihydroxy-1 ,3- dimethoxy-6-phenyl-6,7,8,8a-tetrahydro-5aH-cyclopenta[4,5]furo[3,2-c]pyridin-5a- yljbenzonitrile, CAS: 2098191 -53-6

can be prepared analogous to WO2017/091585.

Claims

56

Claims A compound of the formula (I)

wherein

R¹ is selected from halogen, CN and OH;

R² is C1-C4 alkyl, wherein alkyl is unsubstituted or substituted by 1 , 2 or 3 substituents selected from OH, C1-C3 alkoxy and NR^aR^b;

R³ is selected from C1-C4 alkyl;

R⁴ is selected from C1-C4 alkyl;

R^a and R^b independently from each other are selected from hydrogen, C1-C4 alkyl and C3-C7 cycloalkyl; or a pharmaceutically acceptable salt thereof, for use as inhibitor of RAS proteine activation. The compound formula (I) according to claim 1 , wherein

R¹ is selected from F, Cl and CN;

R³ is selected from C1-C2 alkyl;

R⁴ is selected from C1-C2 alkyl;

R^a and R^b independently from each other are selected from hydrogen and C1-C4; or a pharmaceutically acceptable salt thereof, for use as inhibitor of RAS proteine activation. 57 The compound of formula (I) according to any of the proceeding claims, which is a compound A

A or a pharmaceutically acceptable salt thereof, for use as inhibitor of RAS proteine activation. The compound of formula (I) according to any of the proceeding claims, which is a compound B or a mixture of B with its enatiomer B1 , preferably with enantiomer excess (ee) of the enantiomer of formula B of at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99%

or a pharmaceutically acceptable salt thereof, for use as inhibitor of RAS protein activation. The compound of formula (I) according to any of the proceeding claims, which is a compound C or a mixture of C with its enatiomer C1 , preferably with enantiomer excess (ee) of the enantiomer of formula C of at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99%

or a pharmaceutically acceptable salt thereof, for use as inhibitor of RAS protein activation. The compound of formula (I) according to any of the proceeding claims, which is a compound D or a mixture of D with its enatiomer D1 , preferably with enantiomer excess (ee) of the enantiomer of formula D of at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99%

D D1 or a pharmaceutically acceptable salt thereof, for use as inhibitor of RAS protein activation. The compound of formula (I) according to any of the proceeding claims, which is a compound E or a mixture of E with its enatiomer E1 , preferably with enantiomer excess (ee) of the enantiomer of formula E of at least 20%, preferably at least 50%, in particular at least 80%, especially at least 99%

59 or a pharmaceutically acceptable salt thereof, for use as inhibitor of RAS protein activation. The compound of formula (I) in particular compounds (A), (B), (C), (D) or (E) according to any of claims 1 to 7, an enantiomeric mixture thereof, or a pharmaceutically acceptable salt thereof, for use in treating proliferative disorders, wherein RAS-signaling is involved, preferably wherein KRAS G12V, NRAS G12V, HRAS G12V, KRAS G12C, KRAS G12D, KRAS G12C/Y96D, KRAS G13C, KRAS G13D, KRASG13S, KRAS Q61 H, KRAS Q61 R or KRAS Q61 K is involved, or wherein any activating mutation in KRAS, HRAS and NRAS is involved or wherein any mutation that conferes resistance to other RAS inhibitors is involved. The compound of formula (I) in particular compound (A), (B), (C), (D) or (E) according to any of claims 1 to 7, an enantiomeric mixture thereof, or a pharmaceutically acceptable salt thereof, for use as a ligand of prohibitins (PHB1/2 complex) in the plasma membrane. A pharmaceutical composition, comprising at least one compound of formula (I), in particular selected from compounds (A), (B), (C), (D) or (E), as claimed in any one of claims 1 to 7, an enantiomeric mixture thereof, or a pharmaceutically acceptable salt thereof for the prophylaxis and/or treatment of proliferative disorders, wherein RAS-signaling is involved, preferably wherein KRAS G12V, NRAS G12V, HRAS G12V, KRAS G12C, KRAS G12D, KRAS G12C/Y96D, KRAS G13C, KRAS G13D, KRASG13S, KRAS Q61 H, KRAS Q61 R or KRAS Q61 K is involved, or wherein any activating mutation in KRAS, HRAS and NRAS is involved. The pharmaceutical composition according to claim 10, comprising at least one compound of formula (I), in particular selected from compounds (A), (B), (C), (D) or (E), an enantiomeric mixture thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 10 or 1 1 , comprising additionally a further active substance, preferably selected from chemotherapeutic agents, radiotherapeutic agents, immuno-oncology agents and combinations thereof. The pharmaceutical composition as claimed in any of claims 10 to 12 for use in the prophylaxis and/or treatment of genetic disorders where RAS signaling is involved, in particular including RASopathies, craniofacial syndrome and Neurofibromatosis type I. 60 A method of inhibiting growth, proliferation, or metastasis of cancer cells in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of at least one compound of formula (I), in particular selected from compounds (A), (B), (C), (D) or (E), as defined in any one of claims 1 to 7, an enantiomeric mixture thereof, or a therapeutically acceptable salt thereof. The method of claim 14, wherein the cancer is selected from prostate, colon, rectum, pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary, testis, head, neck, skin (including melanoma and basal carcinoma), mesothelial lining, white blood cell (including lymphoma and leukemia), esophagus, breast, muscle, connective tissue, lung (including small cell lung carcinoma and non-small-cell carcinoma), adrenal gland, thyroid, kidney, or bone; or glioblastoma, mesothelioma, renal cell carcinoma, gastric carcinoma, sarcoma (including Kaposi's sarcoma), choriocarcinoma, cutaneous basocellular carcinoma, Haematological malignancies (including blood, bone marrow and lymph nodes) or testicular seminoma. A method of inhibiting proliferation of a cell population sensitive towards inhibiting RAS activation in vitro or ex vivo, the method comprising contacting the cell population with at least one compound of the formula (I), in particular selected from compounds (A), (B), (C), (D) and (E), as defined in any one of claims 1 to 7, an enantiomeric mixture thereof, or a therapeutically acceptable salt thereof. A kit containing a formulation comprising: a) at least one compound of the formula (I), in particular selected from compounds (A), (B), (C), (D) and (E), as defined in any one of claims 1 to 7, an enantiomeric mixture thereof, or a pharmaceutical composition comprising at least one compound of the formula (I), in particular selected from compounds (A), (B), (C), (D) and (E), as defined in any one of claims 1 to 7, or a therapeutically acceptable salt thereof and a pharmaceutically acceptable carrier; and b) instructions for dosing of the pharmaceutical composition for the treatment of a disorder in which inhibition of RAS activation is effective in treating the disorder. The compound of formula (I), in particular selected from compounds (A), (B), (C), (D) and (E), as defined in any one of claims 1 to 7, an enantiomeric mixture thereof, or a pharmaceutically acceptable salt thereof, for use of treating patients having developed resistance to RAS inhibitors, different from compounds of formula (I), in particular KRAS G12 C inhibitors.