CDC42 / rhogdi inhibitors for use in the treatment of ras-related cancer and metastasizing osteosarcoma

Abstract

The present invention relates to a compound for use in a method of treatment of RAS-related cancer and metastasizing osteosarcoma.

Description

CDC42 / RH0GDI INHIBITORS FOR USE IN THE TREATMENT OF RAS-RELATED CANCER AND METASTASIZING OSTEOSARCOMA

[0001] This application claims priority to European Application No. EP24191998 filed 31 July 2024, which is incorporated herein by reference in its entirety.TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates to a compound for use in a method of treatment of RAS- related cancer and metastasizing osteosarcoma.BACKGROUND ART

[0003] Osteosarcoma is the most common malignant bone tumor and is characterized by aggressive growth and early micrometastasis.

[0004] As a highly malignant type of tumor with aggressive growth and early metastasis, osteosarcoma is a great burden, especially for young patients. Osteosarcomas are the most common malignant bone tumors in children and adolescents.1Here, the relative 10-year survival rate is about 68% and decreases significantly again in middle and older adulthood.2

[0005] The treatment of osteosarcoma usually consists of chemotherapy, surgery and radiotherapy. Since many patients already have metastases at the time of diagnosis, chemotherapy is first used before surgical removal in order to reduce the size of the tumor and eliminate any existing metastases in the blood and other organs. In more than 80% of cases, surgery can be performed to preserve the arm and leg, and the resulting bone defects can be treated with tumor endoprostheses and the body's own bone tissue. Radiotherapy, on the other hand, is used if the osteosarcoma is inoperable in the present case, as this type of tumor has only low radiation sensitivity.34

[0006] In the presence of localized osteosarcoma, the average 5-year survival rate is about 80%. However, early micrometastasis leads to a very poor prognosis, with metastases often settling in the lungs.5Thus, there is need for furher therapies for the treatment of osteosarcoma in particular therapies which reduce or prevent metastasizing of osteosarcoma.SUMMARY OF THE INVENTION

[0007] A compound for use in a method of treatment of RAS-related cancer and metastasizing osteosarcoma, wherein the compound has a structure according to formula (I)or an enantiomer and / or a pharmaceutically acceptable salt thereof; wherein Y is selected from the group consisting of -OR6, -NR7R8;R6is selected from the group consisting of H, -(Ci-C6)alkyl, -O(Ci-Ce)alkyl, -(CH2)n(C3- C7)cycloalkyl, -(C2-C6)alkenyl, -(Ci-Ce)alkylOH, phenyl,-(Ci-Ce)alkyl, -O(Ci-Ce)alkyl, -(CH2)n(C3-C7)cycloalkyl, -(C2-C6)alkenyl, -(Ci-Ce)alkylOH, phenyl each are optionally substituted with one or more substituents each independently selected from the group consisting of halogen, -CN, -OH, -O(Ci-C6)alkyl, -(Cs-Cio)heteroaryl; n is 1 to 5;R7and R8are independently selected from the group consisting of H, (Ci-C6)alkyl, (C3- C7)cycloalkyl and phenyl, wherein optionally (Ci-C6)alkyl, (C3-C7)cycloalkyl and phenyl are substituted with one or more substituents each independently selected from the group consisting of halogen, -CN, -NO2, -OH, -(Ci-C6)alkyl, -O(Ci-C6)alkyl, orR7and R8are optionally taken together with the nitrogen to which they are attached to form indolinyl, pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl, each optionally substituted with one or more substituents independently selected from the group consisting of halogen, -CN, -NO2, - OH, -(Ci-Ce)alkyl, -O(Ci-Ce)alkyl, or R7and R1come together to be (Ci-C3)alkyl linking together as a ring;R1is selected from the group consisting of H, (Ci-Ce)alkyl, (C3-C7)cycloalkyl and phenyl, wherein optionally (Ci-Ce)alkyl, (C3-C7)cycloalkyl and phenyl are substituted with one or more substituents each independently selected from the group consisting of halogen, -CN, -NO2, -OH, -(Ci-Ce)alkyl, -O(Ci-Ce)alkyl, or R7and R1come together to be (Ci-C3)alkyl linking together as a ring;R2, R3, R4, R5are independently selected from the group consisting ofH, halogen, -CN, -NO2, -OH, -(Ci-C6)alkyl, -O(Ci-C6)alkyl, -(CH2)u(C3-C7)cycloalkyl, -O(CH2)u(C3- C7)cycloalkyl, -(C2-Ce)alkenyl, -(Ci-Ce)alkylOH, phenyl, wherein optionally (Ci-Ce)alkyl, -O(Ci- Ce)alkyl, -(CH2)u(C3-C7)cycloalkyl, -O(CH2)u(C3-C7)cycloalkyl, -(C2-Ce)alkenyl, -(Ci-Ce)alkylOH, phenyl is substituted with one or more substituents independently selected from the groupconsisting of halogen -CN, -NO2, -OH, -(Ci-C6)alkyl, -(CH2)u(C3-C7)cycloalkyl, -(C2-Ce)alkenyl, - (Ci-Ce)alkylOH, phenyl; u is 1 to 5.

[0008] The invention is further directed to a pharmaceutical composition for use in the treatment of RAS-related cancer and metastasizing osteosarcoma comprising the compound as defined above and a pharmaceutically acceptable carrier.

[0009] As a GTPase protein, CDC42 (cell division control protein 42) belongs to the Rho subfamily of the RAS superfamily of GTPases and alternates between an active, GTP-bound state and an inactive, GDP-bound state. The Rho family of GTPases regulates many aspects of actin cytoskeleton dynamics6, including the formation of stress fibers, contractile bundles of actin filaments. It has been shown that in the osteosarcoma cell line 143B the actin-binding tumor suppressor NF2 is deregulated compared to non-metastatic human cell lines, as well as an overactivation of CDC42 and reduced formation of F-actin fibers (see Figure 2). Treatment with a compound according to the present invention, such as CASIN, stimulates the expression of NF2 and repairs the actin cytoskeleton in metastatic cancer cells (see Figure 3). Furthermore, the inventors were able to show that a compound according to the present invention, such as CASIN inhibits the proliferation and migration of metastatic cells and at the same time promotes their adhesion (see Figures 4, 5, 6).BRIEF DESCRIPTION OF THE FIGURES

[0010] Fig. 1 : Transformation of non-tumourigenic and non-metastatic HOS (human osteosarcoma) cell line into high tumourigenic and / or non-metastatic variants.

[0011] Fig. 2: Human cell line: HOS: non-tumourigenic and non-metastatic. HOS-143B: tumourigenic and metastatic cell line and mouse metastatic cell line (NF2 Knockout). Jn the human metastatic cell line, Figure A, and in the mouse metastatic cell line (NF2 Knockdown) a downregulation of tumorsuppressor NF2, a CDC42 overactivation, and a de-structured F-actin is observed.

[0012] Fig. 3: CASIN stimulates the expression of the tumor suppressor NF2 and repairs actin cytoskeletal fibers in metastatic cancer cells.

[0013] Fig. 4: CASIN inhibits the proliferation of metastatic cancer cells (HOS 143B OS cell line).

[0014] Fig. 5: CASIN increases the adhesion of metastatic cancer cells (HOS 143B OS cell line).

[0015] Fig. 6: CASIN reduces migration und invasion behaviour of metastatic cancer cells (HOS 143B OS cell line).DETAILED DESCRIPTION OF THE INVENTION

[0016] Definitions

[0017] The term "alkyl" refers to a monoradical of a saturated straight or branched hydrocarbon. Preferably, the alkyl group comprises from 1 to 6 carbon atoms, i.e., 1 , 2, 3, 4, 5, 6, carbon atoms (such as 1 , 2, 3, 4, 5, 6, carbon atoms), more preferably 1 1 to 4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1 ,2-dimethyl-propyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, and the like.

[0018] The term "cycloalkyl" or "cycloaliphatic" represents cyclic non-aromatic versions of "alkyl" and "alkenyl" with preferably 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 3 to 8 carbon atoms, even more preferably 3 to 7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cylcodecyl, cylcodecenyl, and adamantyl. The term "cycloalkyl" is also meant to include bicyclic and tricyclic versions thereof. If bicyclic rings are formed it is preferred that the respective rings are connected to each other at two adjacent carbon atoms, however, alternatively the two rings are connected via the same carbon atom, i.e., they form a spiro ring system or they form "bridged" ring systems. Preferred examples of cycloalkyl include Ca-Cs- cycloalkyl, in particular cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, spiro[3,3]heptyl, spiro[3,4]octyl, spiro[4,3]octyl, bicyclo[4.1.0]heptyl, bicyclo[3.2.0]heptyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[5.1.0]octyl, and bicyclo[4.2.0]octyl.

[0019] The term "alkenyl" refers to a monoradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximal number of carbon-carbon double bonds in the alkenyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenyl group by 2 and, if the number of carbon atoms in the alkenyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 4, i.e., 1 , 2, 3, or 4, carbon-carbon double bonds. Preferably, the alkenyl group comprises from 2 to 10 carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenyl group comprises from 2 to 10 carbon atoms and 1 , 2, 3, 4, or 5 carbon-carbon double bonds, more preferably it comprises 2 to8 carbon atoms and 1 , 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1 , 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenyl groups include vinyl, 1-propenyl, 2-propenyl (i.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1- pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1 -hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5- hexenyl, 1 -heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1 -octenyl, 2- octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4- nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1 -decenyl, 2-decenyl, 3-decenyl, 4- decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, and the like. If an alkenyl group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom.

[0020] The term "halogen" means fluoro, chloro, bromo, or iodo, preferably fluoro, or chloro.

[0021] Some of the inventive compounds may exist as single stereoisomers (i.e., essentially free of other stereoisomers), racemates, and / or mixtures of enantiomers and / or diastereomers. All such single stereoisomers, racemates and mixtures thereof are intended to be within the scope of the present invention. Preferably, the inventive compounds that are optically active are used in optically pure form.

[0022] “A pharmaceutically acceptable salt” is intended to mean a salt that retains the biological effectiveness of the free acids and bases of the specified compound and that is not biologically or otherwise undesirable. A compound of the invention may possess a sufficiently acidic, a sufficiently basic, or both functional groups, and accordingly react with any of a number of inorganic or organic bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt. Exemplary pharmaceutically acceptable salts include those salts prepared by reaction of the compounds of the present invention with a mineral or organic acid or an inorganic base, such as salts including sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1 ,4-dioates, hexyne-1 ,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, y-hydroxy butyrates, glycolates, tartrates, methane-sulfonates, propanesulfonates, naphthalene-1 -sulfonates, naphthalene-2-sulfonates, and mandelates.

[0023] It is noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[0024] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0025] The term "and / or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0026] The term “less than” or in turn “more than” does not include the concrete number.

[0027] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”. When used herein “consisting of" excludes any element, step, or ingredient not specified.

[0028] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0029] The content of all documents and patent documents cited herein is incorporated by reference in their entirety.A better understanding of the present invention and of its advantages will be had from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way.Compounds

[0030] The invention comprises a compound for use in a method of treatment of RAS-related cancer and metastasizing osteosarcoma, wherein the compound has a structure according to formula (I)or an enantiomer and / or a pharmaceutically acceptable salt thereof.

[0031] Wherein Y is selected from the group consisting of -OR6, -NR7R8.

[0032] R6is selected from the group consisting of H, -(Ci-C6)alkyl, -O(Ci-Ce)alkyl, -(CH2)n(C3- C7)cycloalkyl, -(C2-C6)alkenyl, -(Ci-Ce)alkylOH, phenyl,-(Ci-Ce)alkyl, -O(Ci-Ce)alkyl, -(CH2)n(C3-C7)cycloalkyl, -(C2-C6)alkenyl, -(Ci-Ce)alkylOH, phenyl each are optionally substituted with one or more substituents each independently selected from the group consisting of halogen, -CN, -OH, -O(Ci-C6)alkyl, -(Cs-Cio)heteroaryl.

[0033] n is 1 to 5.

[0034] R7and R8are independently selected from the group consisting of H, (Ci-C6)alkyl, (C3- C7)cycloalkyl and phenyl, wherein optionally (Ci-C6)alkyl, (C3-C7)cycloalkyl and phenyl are substituted with one or more substituents each independently selected from the group consisting of halogen, -CN, -NO2, -OH, -(Ci-C6)alkyl, -O(Ci-C6)alkyl, orR7and R8are optionally taken together with the nitrogen to which they are attached to form indolinyl, pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl, each optionally substituted with one or more substituents independently selected from the group consisting of halogen, -CN, -NO2, - OH, -(Ci-Ce)alkyl, -O(Ci-Ce)alkyl, or R7and R1come together to be (Ci-C3)alkyl linking together as a ring.

[0035] R1is selected from the group consisting of H, (Ci-Ce)alkyl, (C3-C7)cycloalkyl and phenyl, wherein optionally (Ci-Ce)alkyl, (C3-C7)cycloalkyl and phenyl are substituted with one or more substituents each independently selected from the group consisting of halogen, -CN, -NO2, -OH, -(Ci-Ce)alkyl, -O(Ci-Ce)alkyl, or R7and R1come together to be (Ci-C3)alkyl linking together as a ring.

[0036] R2, R3, R4, R5are independently selected from the group consisting ofH, halogen, -CN, -NO2, -OH, -(Ci-C6)alkyl, -O(Ci-C6)alkyl, -(CH2)U(C3-C7)cycloalkyl, -O(CH2)U(C3- C7)cycloalkyl, -(C2-C6)alkenyl, -(Ci-Ce)alkylOH, phenyl, wherein optionally (Ci-Ce)alkyl, -0(C C6)alkyl, -(CH2)U(C3-C7)cycloalkyl, -O(CH2)U(C3-C7)cycloalkyl, -(C2-C6)alkenyl, -(Ci-C6)alkylOH, phenyl is substituted with one or more substituents independently selected from the group consisting of halogen -CN, -NO2, -OH, -(Ci-Ce)alkyl, -(CH2)U(C3-C7)cycloalkyl, -(C2-C6)alkenyl, - (Ci-CejalkylOH, phenyl. u is 1 to 5.or an enantiomer and / or a pharmaceutically acceptable salt thereof.Medical applications

[0038] The compound according to formula (I) is for use in a method of treatment of RAS-related cancer and metastasizing osteosarcoma.

[0039] The compound according to formula (I) is preferably an inhibitor of CDC42 activity.

[0040] Preferably, the method comprises: a) measuring the level of CDC42-GTP, preferably measured by immunofluorescence ( for example by Anti-Cdc42-GTP Monoclonal Antibody; New East Biosciences) and b) based on the level of CDC42-GTP in the subject, determining whether to administer to the subject the compound of Formula (I).Preferably, the compound according to formula (I) is administered when the level of CDC42-GTP is elevated. Preferably, an elevated level of CDC42 activity is a level of CDC42 activity that is higer than that found in a non-cancerous cell of the same cell type, preferably measured by immunofluorescence.

[0041] Preferably, the elevated level of CDC42 activity is a level of CDC42 activity that is 1.5 to 5 times higher than that found in a non-cancerous cell.

[0042] Preferably, the RAS-related cancer is selected from the group consisting of breast, colon, lung, liver, and pancreatic cancer.

[0043] |ln general, Cheng et al.13shows that a RAS mutant binds to and activates CDC42.|[Further, Stengel et al14demonstrate that |CDC42 activation is required for RAS-induced transformation. Wang et al15show that CDC42 and RAS cooperate to transform cells. CDC42 deregulation and / or upregulation plays an important role in the progression of breast cancer7, colon cancer8, lung cancer9 17, [pancreatic cancer10, liver

[0044] Preferably, metastasizing comprises at least one of the steps:(i) local infiltration of tumor cells into the adjacent tissue(ii) transendothelial migration of cancer cells into vessels known as intravasation(iii) survival in the circulatory system(iv) extravasation and(v) subsequent proliferation in competent organs leading to colonization.

[0045] Preferably, the adjacent tissue in step (i) is selected from the group consisting of connective, fat, muscle and nerve tissue.

[0046] Preferably, the compound inhibits at least one of the steps (i), (ii), and / or (iii), more preferably steps (i) and (ii) in the metastasizing process.

[0047] Preferably, the compound is an inhibitor16of the interaction between CDC42, RhoJ,RhoQ, and RhoGDI, more preferably between CDC42, and RhoGDI..

[0048] Preferably, metastasizing osteosarcoma NF2 is deregulated.

[0049] In one embodiment, the compound is for use in the treatment as described above, wherein the patients are selected from the group consisting of children and adolescents, preferably patients within the age range of 8 to 18, and / or 19 to 22, more preferably 10 to 18 and / or 19 to 20.Formulation

[0050] The invention is further directed to a pharmaceutical composition for use in the treatment of RAS-related cancer and metastasizing osteosarcoma comprising the compound as described above and at least one pharmaceutically acceptable excipient.

[0051] The pharmaceutical composition may be administered to an individual by any route, such as enterally or parenterally. In one embodiment the pharmaceutical composition is administered by intraperitoneal injection or intravenous injection.

[0052] The expressions "enteral administration" and "administered enterally" as used herein mean that the drug administered is taken up by the stomach and / or the intestine. Examples of enteral administration include oral and rectal administration. The expressions "parenteral administration" and "administered parenterally" as used herein mean modes of administration other than enteral administration, usually by injection or topical application, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraosseous, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, intracerebral, intracerebroventricular, subarachnoid, intraspinal, epidural and intrasternal administration (such as by injection and / or infusion) as well as topical administration (e.g., epicutaneous, inhalational, or through mucous membranes (such as buccal, sublingual or vaginal)).

[0053] The compounds used in the present invention are generally applied in "pharmaceutically acceptable amounts" and in "pharmaceutically acceptable preparations". Such compositions may contain salts, buffers, preserving agents, carriers and optionally other therapeutic agents.

[0054] The term "excipient" when used herein is intended to indicate all substances in a pharmaceutical composition which are not active ingredients (e.g., which are therapeutically inactive ingredients that do not exhibit any therapeutic effect in the amount / concentration used), such as, e.g., carriers, binders, lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffers, flavoring agents, colorants, or antioxidants.

[0055] The compositions described in the present invention may comprise a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like that are physiologically compatible. The "pharmaceutically acceptable carrier" may be in the form of a solid, semisolid, liquid, or combinations thereof. Preferably, the carrier is suitable for enteral (suchas oral) or parenteral administration (such as intravenous, intramuscular, subcutaneous, spinal or epidermal administration (e.g., by injection or infusion)). Depending on the route of administration, the active compound, i.e., the compound used in the present invention, either alone or in combination with one or more additional active compounds, may be coated in a material to protect the active compound(s) from the action of acids and other natural conditions that may inactivate the active compound.

[0056] Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions used according to the present invention include water (e.g., water for injection), ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), aqueous solutions of a salt, carbohydrate, sugar alcohol, or an amino acid (such as saline or an aqueous amino acid solution), and suitable mixtures and / or buffered forms thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0057] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active compounds is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions used according to the present invention is contemplated.

[0058] A better understanding of the present invention and of its advantages will be had from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way.EXAMPLES OF THE INVENTION1. Methods and Materials

[0059] Osteosarcoma cell lines:The HOS (human osteosarcoma) cell line was derived from a tumour biopsy from a 13-year-old female osteosarcoma patient in 1971 and was called HOS (HOS-TE85), clone F5. The patient was amputated and treated with radiation therapy and chemotherapy but nevertheless died with extensive pulmonary metastases Derived from the TE85 another subclone was generated by using the chemical agent N-methyl-N'-nitro-N-nitroguanidine (MNNG), named MNNG (HOS MNNG).12An additional derivate was introduced, called 143B (HOS -143B). The 143B cell line iwas generated by transforming the TE85 cells with Ki-RAS. This cell line was described to be a 5-bromodeoxyuridine (BrdU)-resistant and thymidine kinase-negative mutant.See Figure 1.

[0060] Cell culture and treatment:Cells were provided by a partner institute of TUM, University Medical Center of the Johannes Gutenberg University in Mainz or derived from ATCC. HOS, HOS-MNNG, FBMD and NF22A / +(Ma et al., Molecular Cancer Biology, 2020) were grown in alphaMEM with 10 % fetal calf serum and 1 % PenStrep (P / S) and HOS-143B in DMEM with 10% fetal calf serum, 1 % PenStrep (P / S) and 0,1 % BrdU under standard conditions. The CDC42 acitivity inhibitor CASIN was incubated for 1 h every day for one to two weeks. The CASIN (5pM) containing medium was prepared freshly before adding it to the cells. After the treatment, the cells were washed with PBS to remove the drug properly and new culture medium was added.

[0061] Immunofluorescence staining and microscopy:The cells (10 000 cells) grow on slides with 200-250 pl medium surrounded by a liquid blocking line (IHC PAP Pen). After overnight growth, cells can be fixated with 4 % paraformaldehyde (PFA) for 5 - 10 min. Unspecific binding sites are blocked by addition of blocking buffer, containing 30 ml PBS, 1 ml FCS and 100 pl Triton. After 30 minutes the buffer is tapped off and the cells are stained according to specific experiments. Mainly two different stains where used (AlexaFluor- 488 and AlexaFluor-546 coupled antibodies). All antibodies were diluted in the predefined ratio with blocking buffer and added with a volume of 50 pl to the spots. The primary antibodies incubate overnight and after washing with blocking buffer for 5 min, secondary antibody is incubated for 1 h. Images were taken by fluorescence microscope and analysis were performed with Imaged by measuring the pixel intensity of the certain stain in order to calculate mean values and standard deviation.

[0062] Proliferation:The growth rate of each cell line and each condition was determined throughout 5 passages. In the format of 6-well plates, 1.0 x 105cells per well were seeded and cultured until 80-90 % confluence. For every passage a defined number (1.0 x 105cells) of cells was cultured in a new well again to monitor the growth, calculate the growth rate and summarize the total cell amount in the end.

[0063] Adhesion:This assay starts with pre-drawn spots by the IHC PAP pen (liquid blocker) on poly-lysine coated slides where 5000 / 2500 cells were put in 100-200 pl medium. During 2 hours of incubation the cells can adhere to the coated glass at 37°C. After two gentle washing steps with PBS and fixation with 4 % PFA for 10 min the cells were stained with DAPI and counted under the fluorescent microscope.

[0064] Migration:The cells face pores (6.5 pm pore size) in a transwell chamber. The migration is triggered by a serum gradient as the upper chamber is serum-free. The 50 000 cells migrate towards the bottom chamber containing 600 pl medium with 10 % FCS overnight. Subsequently, the bottom chamber was analysed by flow cytometry and the migrated cells were counted.

[0065] Invasion:The difference to previously described migration experiment is the additional Matrigel layer on the filter. Matrigel needs to be handled with pre-cooled tips and media to prevent coagulation. In advance, Matrigel is diluted 1 :4 in serum-free medium and 100 pl is added on the transwell. The coating takes 30-60 min at 37°C. The bottom chamber is filled with 10 % FCS medium as described for the Migration Assay. In the upper chamber 50 000 cells are added with serum-free medium and incubated overnight at 37°C. Afterwards, the uninvaded cells are removed with a cotton swab. The transwell filter is washed with 70 % ethanol for 10 min and dried with a cotton swab. For fixation the filter incubates in 4 % PFA for 10 min and is washed with PBS before it is removed with a scalpel and put on a slide.The bottom chamber can be counted via FACS optionally, but the main observation is counting the cells on the filter by DAPI stain under the fluorescent microscope.

[0066] The results are displayed in Fig. 2 to Fig. 6.REFERENCES1. https: / / www.kinderkrebsstiftung.de / krebs-bei-kindern / krankheitsbilderbehandlung / osteosarkom / .2. https: / / www.onkopedia.eom / de / onkopedia / guidelines / osteosarkome / @@guideline / html / index. html3. https: / / www.krebsgesellschaft.de / onko-internetportal / basis- informationenkrebs / krebsarten / andere-krebsarten / knochenkrebs / therapie.html.4. https: / / www.klinikum.uni-heidelberg.de / erkrankungen / osteosarkom-201718 / .5. https: / / onlinelibrary.wiley.com / doi / 10.1002 / ijc.24320.6. N. P. Murphy et al., Biochemical Society Transactions (2021), 49, 1425-1442.7. Zhang et al. Cells 2019, 8(2), 146.8. Wang D-N, Ni J-J, Li J-H, Gao Y-Q, Ni F-J, Zhang Z-Z, et al. (2023) Bacterial infection promotes tumorigenesis of colorectal cancer via regulating CDC42 acetylation. PLoS Pathog 19(2): e1011189. https: / / doi.org / 10.1371 / journal.ppat.1011189.9. Yao et al., Cancer Letters, Volume 502, 1 April 2021 , Pages 1-8.10. Pistoni et al. Carcinogenesis . 2021 Aug 19;42(8): 1037-1045.11. Zhang et al. Journal of Experimental & Clinical Cancer Research volume 41 , Article number: 230 (2022).12. Luu, H. H., Kang, Q., Park, J. K., Si, W., Luo, Q., Jiang, W., Yin, H., Montag, A. G., Simon, M. A., Peabody, T. D., et al. An orthotopic model of human osteosarcoma growth and spontaneous pulmonary metastasis. Clinical & experimental metastasis 22 (2005), 319-329.13. Cheng CM, Li H, Gasman S, Huang J, Schiff R, Chang EC. Compartmentalized RAS proteins transform NIH 3T3 cells with different efficiencies. Mol Cell Biol. 2011 Mar;31 (5):983-97. doi: 10.1128 / MCB.00137-10.14. Stengel KR, Zheng Y. Essential role of Cdc42 in RAS-induced transformation revealed by gene targeting. PLoS One. 2012;7(6):e37317. doi: 10.1371 / journal. pone.0037317.15. Wang JB, Wu WJ, Cerione RA. Cdc42 and RAS cooperate to mediate cellular transformation by intersectin-L. J Biol Chem. 2005 Jun 17;280(24):22883-91. doi: 10.1074 / jbc.M414375200.16. Petersen et al., Chem Biol. DOI: 10.1016 / j.chembiol.2006.02.009.

Claims

Claims1. A compound for use in a method of treatment of RAS-related cancer and metastasizing osteosarcoma, wherein the compound has a structure according to formula (I)or an enantiomer and / or a pharmaceutically acceptable salt thereof; whereinY is selected from the group consisting of -OR6, -NR7R8;R6is selected from the group consisting of H, -(Ci-C6)alkyl, -O(Ci-Ce)alkyl, -(CH2)n(C3- C7)cycloalkyl, -(C2-C6)alkenyl, -(Ci-Ce)alkylOH, phenyl,-(Ci-Ce)alkyl, -O(Ci-Ce)alkyl, -(CH2)n(C3-C7)cycloalkyl, -(C2-C6)alkenyl, -(Ci-Ce)alkylOH, phenyl each are optionally substituted with one or more substituents each independently selected from the group consisting of halogen, -CN, -OH, -O(Ci-C6)alkyl, -(Cs-Cio)heteroaryl; n is 1 to 5;R7and R8are independently selected from the group consisting of H, (Ci-Ce)alkyl, (C3- C7)cycloalkyl and phenyl, wherein optionally (Ci-C6)alkyl, (C3-C7)cycloalkyl and phenyl are substituted with one or more substituents each independently selected from the group consisting of halogen, -CN, -NO2, -OH, -(Ci-C6)alkyl, -O(Ci-C6)alkyl, orR7and R8are optionally taken together with the nitrogen to which they are attached to form indolinyl, pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl, each optionally substituted with one or more substituents independently selected from the group consisting of halogen, -CN, -NO2, - OH, -(Ci-Ce)alkyl, -O(Ci-Ce)alkyl, or R7and R1come together to be (Ci-C3)alkyl linking together as a ring;R1is selected from the group consisting of H, (Ci-Ce)alkyl, (C3-C7)cycloalkyl and phenyl, wherein optionally (Ci-C6)alkyl, (C3-C7)cycloalkyl and phenyl are substituted with one or more substituents each independently selected from the group consisting of halogen, -CN, -NO2, -OH, -(Ci-Ce)alkyl, -O(Ci-Ce)alkyl, or R7and R1come together to be (Ci-C3)alkyl linking together as a ring;R2, R3, R4, R5are independently selected from the group consisting ofH, halogen, -CN, -NO2, -OH, -(Ci-C6)alkyl, -O(Ci-C6)alkyl, -(CH2)u(C3-C7)cycloalkyl, -O(CH2)u(C3- C7)cycloalkyl, -(C2-Ce)alkenyl, -(Ci-Ce)alkylOH, phenyl, wherein optionally (Ci-Ce)alkyl, -O(Ci- Ce)alkyl, -(CH2)u(C3-C7)cycloalkyl, -O(CH2)u(C3-C7)cycloalkyl, -(C2-Ce)alkenyl, -(Ci-Ce)alkylOH, phenyl is substituted with one or more substituents independently selected from the groupconsisting of halogen -CN, -NO2, -OH, -(Ci-Ce)alkyl, -(CH2)u(C3-C7)cycloalkyl, -(C2-C6)alkenyl, - (Ci-Ce)alkylOH, phenyl; u is 1 to 5.

2. The compound for use of claim 1 , wherein the compound has the structureor an enantiomer and / or a pharmaceutically acceptable salt thereof.

3. The compound for use of claims 1 or 2, wherein the method of treatment of metastasizing osteosarcoma comprises prevention or reducing the metastasizing.

4. The compound for use of claim 3, wherein metastasizing comprises at least one of the steps:(i) local infiltration of tumor cells into the adjacent tissue(ii) transendothelial migration of cancer cells into vessels known as intravasation(iii) survival in the circulatory system(iv) extravasation and(v) subsequent proliferation in competent organs leading to colonization5. The compound for use of claim 4, wherein the compound inhibits at least one of the steps (i), (ii), and / or (iii), preferably steps (i) and (ii) in the metastasizing process.

6. The compound for use of claim 4 or 5, wherein the adjacent tissue is selected from the group consisting of connective, fat, muscle and nerve tissue.

7. The compound for use of any of claims 1 to 6, wherein the compound is an inhibitor of the interaction between CDC42, RhoJ, RhoQ, and RhoGDI, more preferably between CDC42, and RhoGDI.

8. The compound for use of any of claims 1 to 7, wherein the compound stimulates the expression of NF2.

9. The compound for use of any of claims 1 to 8, wherein in metastasizing osteosarcoma NF2 is deregulated.

10. The compound for use of any of claims 1 to 9, wherein the RAS-related cancer is selected from the group consisting of breast, colon, lung, and pancreatic cancer.

11. The compound for use of claims 1 to 10, wherein the method comprises: a) measuring the level of CDC42-GTP; and b) based on the level of CDC42-GTP in the subject, determining whether to administer to the subject the compound of Formula (I).

12. The compound for use of 1 to 11 , wherein the patients are selected from the group consisting of children and adolescents, preferably patients within the age range of of 8 to 18, and / or 19 to 22, more preferably 10 to 18 and / or 19 to 20.

13. A pharmaceutical composition for use in the treatment of RAS-related cancer and metastasizing osteosarcoma comprising the compound of any one claims 1 to 12 and a pharmaceutically acceptable carrier.

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