This application claims the right of priority of European Patent Application EP22182501.1 filed 01 July 2022, which is incorporated by reference herein.

Field

The present invention relates to small-molecule inhibitors of the FRS2-FGFR interaction. The present invention relates the small-molecule inhibitors for use as a medicament and for use in cancer treatment or prevention.

Background of the Invention

Metastasis, the dissemination and growth of neoplastic cells in an organ distant from that in which they originated, causes as much as 90% of cancer-associated mortality. Effective cancer therapy is largely dependent on the capability to prevent metastasis specifically and less toxic, targeted anti-metastatic therapies are urgently needed. An important and fundamental cause of metastasis in most of all solid tumours is the deregulated motile behaviour of the cancer cells. The microenvironment shapes cell behaviour and determines metastatic outcomes of tumours. Kumar et al. (Cell Reports, 2018, vol. 23, issue 13, P3798-3812) addressed how microenvironmental cues control tumour cell invasion in pediatric brain tumour, medulloblastoma (MB). They show that bFGF promotes MB tumour cell invasion through FGF receptor (FGFR) in vitro and that blockade of FGFR represses brain tissue infiltration in vivo. TGF-p regulates pro-migratory bFGF function in a context-dependent manner. Under low bFGF, the non-canonical TGF-p pathway causes ROCK activation and cortical translocation of ERK1/2, which antagonizes FGFR signaling by inactivating FGFR substrate 2 (FRS2), and promotes a contractile, non-motile phenotype. Under high bFGF, negative-feedback regulation of FRS2 by bFGF-induced ERK1/2 causes repression of the FGFR pathway. Under these conditions, TGF-p counters inactivation of FRS2 and restores pro-migratory signalling. These findings pinpoint coincidence detection of bFGF and TGF-p signalling by FRS2 as a mechanism that controls tumour cell invasion. Thus, targeting FRS2 represents an emerging strategy to abrogate aberrant FGFR signalling.

Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to provide small-molecule inhibitors of the FRS2-FGFR interaction. This objective is attained by the subject-matter of the independent claims of the present specification. Summary of the Invention

A first aspect of the invention relates to a compound of the general formula (100) wherein - Y is O or S;

- X is N or CH;

L is a linker selected from

, a C1-C4 alkyl, a C1-C3 amine, a C1-C3 amide, and a C1-C3 amide-ether, wherein Q is N or CH, particularly Q is N, and R ^L is selected from H and a C1-C3 alkyl, particularly L is a linker selected from

, a C1-C3 amide, and a C1-C3 amide-ether; and wherein L is unsubstituted or substituted with Ci-Ce alkyl, -(Ci-C4)-NR ^N1 R ^N2 ,

OH, CN, halogen, NR ^N1 R ^N2 , and/or SO2R ^s with R ^N1 , R ^N2 , and R ^s being independently selected from H, and C1-C3 alkyl, n is 0, 1 , 2, 3 or 4, particularly n is 1 or 2, more particularly n is 2; each R ¹ is independently selected from an unsubstituted or substituted Ci-Ce alkyl,

OH, OR°, COOR ^A , CN, NO ₂ , halogen, NR ^N1 R ^N2 , SO ₂ R ^s , with R ^N1 , R ^N2 , R ^s , R ^A , and R°being independently selected from H, and Ci-Ce alkyl; particularly each R ¹ is independently selected from OR°, COOR ^A , COOH and OH; or two R ¹ form a C4-C8 cyclo-alkyl or a 4- to 8-membered heterocycle, particularly two R ¹ form a Cs-Ce cyclo-alkyl or a 5- to 6-membered heterocycle, wherein the cyclo-alkyl or the heterocycle is unsubstituted or substituted with C1-C3 alkyl, NO ₂ , COOR ^A , OR° CN, halogen, NR ^N1 R ^N2 , SO ₂ R ^S with R ^N1 , R ^N2 , R°, R ^A , and R ^s being independently selected from H, and C1-C3 alkyl; m is 0, 1 , or 2; - each R ² is independently selected from C1-C3 alkyl, OH, NH ₂ , CN, and halogen; with the proviso that the compound is not characterized by the formula (001), (002), or (003)

(003). In another embodiment, the present invention relates a pharmaceutical composition comprising at least one of the compounds of the present invention or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent or excipient.

A second aspect relates to a compound as described in the first aspect for use as a medicament.

A third aspect of the invention relates to a compound as described in the first aspect for use in treatment or prevention of cancer. In certain embodiments, said cancer is selected from ependymoma, prostate cancer, esophageal cancer, thyroid cancer, hepatocellular carcinoma, testicular cancer, pediatric brain tumour, medulloblastoma, rhabdomyosarcoma, gastric cancer, pulmonary pleomorphic carcinoma, breast cancer, non-small cell lung cancer, liposarcoma, cervical cancer, colorectal cancer, melanoma, multiple myeloma, endometrial cancer, bladder cancer, glioblastoma, squamous cell carcinoma of the lung, ovarian cancer, head and neck cancer, and pancreatic cancer, sarcoma. In certain embodiments, said cancer is selected from bladder cancer, multiple myeloma, gastric cancer, pediatric brain tumour, medulloblastoma, glioblastoma, ependymoma, and sarcoma. In certain embodiments, said cancer is selected from bladder cancer, colorectal cancer, pediatric brain tumour, medulloblastoma, multiple myeloma, and gastric cancer.

A fourth aspect of the invention relates to a compound as described in the first aspect use in treatment or prevention of metastasis. In certain embodiments, said metastasis arises from a cancer selected from bladder cancer, colorectal cancer, pediatric brain tumour, medulloblastoma, multiple myeloma, and gastric cancer.

A fifth aspect of the invention relates to a compound as described in the first aspect for use as an angiogenesis antagonist.

The compound - contrary to the first aspect - characterized by the formula (001), (002) or (003) is included in the scope of protection of the second to the fifth aspect of the invention.

The transmission of signals from activated fibroblast growth factor receptor (FGFR) tyrosine kinases promotes oncogenic functions in tumor cells, including proliferation, survival and cell migration and invasion. The interruption of signal transmission from activated FGFRs to downstream signal transduction cascades by kinase inhibitors designed against FGFRs is an established means of attenuating these oncogenic functions. In addition to aberrant activation of FGFRs in numerous malignancies, FGFR activation is also observed to act as an evasion mechanism in cancers of patients subjected to targeted therapies with kinase inhibitors, which results in tumor re-growth and progression. The small molecule compounds described in this application will prevent signal transmission from activated FGFRs to downstream effector molecules, specifically to the mitogen activated protein kinases (MAPKs), a key driver of tumorigenesis. The compounds bind to FRS2. FGFR substrate 2 (FRS2) is a key adaptor protein that is largely specific to FGF signalling pathway. It is an exclusive downstream effector of FGFRs. FRS2 interacts with the FGFRs via the c-terminal phospho-tyrosine binding (PTB) domain and serves as a molecular hub by assembling both positive and negative signalling proteins to mediate important FGF-induced cellular functions. It transmits the signal from the FGFRs (outside of the cell) to the inside of the cell. Hence, targeting FRS2, which is very upstream of the FGF signalling pathway, effectively shuts down the downstream effectors, especially MAPKs of FGFR signaling.

The compounds specifically bind to the phosphotyrosine binding (PTB) domain of the FRS2 protein (Fig. 10). Compound binding induces a conformational shift in the PTB domain that prevents FGFR-induced signal transmission through FRS2. Two potential binding sites were initially selected: Binding site 1 is not involved in FGFR binding and located below the interaction site of FGFR’s N-terminus with FRS2. Binding site 2 is the extended surface area interacting with FGFR’s C-terminal end.

The mechanism of compound-target interaction, conformational change in the target domain and transmission blockade is unique and does not depend on receptor tyrosine kinase inhibition. In addition, unlike FGFRs, FRS2 does not have any shared protein domains with other adapter proteins. Thus, compared to the existing kinase inhibitors, much less off-target activity is expected. In contrast to existing FGFR targeting strategies, the compounds also interfere specifically with those FGFR functions that are particularly relevant for tumorigenesis and tumor progression.

In contrast to existing FGFR targeting strategies, the compounds also interfere specifically with those FGFR functions that are particularly relevant for tumorigenesis and tumor progression, such as proliferation, migration and invasion and angiogenesis. There is evidence of the FRS2- FGFR interaction being altered in many types of cancer, for example in prostate cancer (Yang, F. et al. Cancer Res 73, 3716-3724, 2013, Liu J et al. Oncogene. 2016 Apr 7;35(14):1750-9), esophageal cancer (Nemoto, T., Ohashi, K., Akashi, T., Johnson, J. D. & Hirokawa, K. Pathobiology 65, 195-203, 1997), thyroid cancer (St Bernard, R. et al. Endocrinology 146, 1145-1153, 2005), hepatocellular carcinoma (Zheng, N., Wei, W. Y. & Wang, Z. W. Transl Cancer Res 5, 1-6, 2016, Matsuki M et al. Cancer Med. 2018 Jun;7(6):2641-2653), testicular cancer (Jiang, X. et al. J Diabetes Res, 2013), medulloblastoma (Santhana Kumar, K. et al. Cell Rep 23, 3798-3812 e3798, 2018), rhabdomyosarcoma (Goldstein, M., Meller, I. & Orr- Urtreger, A. Gene Chromosome Cane 46, 1028-1038, 2007), gastric cancer (Kunii, K. et al. Cancer Res 68, 3549-3549, 2008), pulmonary pleomorphic carcinoma (Lee, S. et al. J Cancer Res Clin 137, 1203-1211 , 2011), breast cancer (Penaultllorca, F. et al. Int J Cancer 61 , 170- 176, 1995), non-small cell lung cancer (Dutt, A. et al. Pios One 6, 2011), Liposarcoma (Zhang, K. Q. et al. Cancer Res 73, 1298-1307, 2013), cervical cancer (Jang, J. H., Shin, K. H. & Park, J. G. Cancer Res 61 , 3541-3543, 2001), colorectal cancer (Sato, T. et al. Oncol Rep 21 , 211- 216, 2009), melanoma (Becker, D., Lee, P. L., Rodeck, II. & Herlyn, M. Oncogene 7, 2303- 2313, 1992), multiple myeloma (Kalff, A. & Spencer, A. Blood Cancer J, 2, 2012), endometrial cancer (Konecny, G. E. et al. Mol Cancer Ther 12, 632-642, 2013), bladder cancer (Cappellen, D. et al. Nat Genet 23, 18-20, 1999, Wu S et al. Nat Commun. 2019 Feb 12; 10(1):720), glioblastoma (Morrison, R. S. et al. Cancer Res 54, 2794-2799, 1994), squamous cell carcinoma of the lung (Weiss, J. et al. Sci Transl Med 4, 2012), ovarian cancer (Cole, C. et al. Cancer Biol Ther 10, 2010), head and neck cancer (Koole, K. et al. Virchows Arch 469, S31- S31 , 2016), and pancreatic cancer (Ishiwata, T. et al. Am J Pathol 180, 1928-1941 , 2012).

Detailed Description of the Invention

Terms and definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc.) and chemical methods.

A Cv-Ce alkyl in the context of the present specification signifies a saturated linear or branched hydrocarbon having 1 , 2, 3, 4, 5 or 6 carbon atoms, wherein one carbon-carbon bond may be unsaturated and/or one CH2 moiety may be exchanged for oxygen (ether bridge) or nitrogen (NH, or NR with R being methyl, ethyl, or propyl; amino bridge). Non-limiting examples for a Ci-Ce alkyl include the examples given for C1-C4 alkyl above, and additionally 3-methylbut-2- enyl, 2-methylbut-3-enyl, 3-methylbut-3-enyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 1 ,1- dimethylpropyl, 1 ,2-dimethylpropyl, 1 ,2-dimethylpropyl, pent-4-inyl, 3-methyl-2-pentyl, and 4- methyl-2-pentyl. In certain embodiments, a C5 alkyl is a pentyl or cyclopentyl moiety and a Ce alkyl is a hexyl or cyclohexyl moiety.

The term C3-C7 cycloalkyl in the context of the present specification relates to a saturated hydrocarbon ring having 3, 4, 5, 6 or 7 carbon atoms, wherein in certain embodiments, one carbon-carbon bond may be unsaturated. Non-limiting examples of a C3-C7 cycloalkyl moiety include cyclopropanyl (-C3H5), cyclobutanyl (-C4H7), cyclopentenyl (C5H9), and cyclohexenyl (CeHn) moieties. In certain embodiments, a cycloalkyl is substituted by one Ci to C4 unsubstituted alkyl moiety. In certain embodiments, a cycloalkyl is substituted by more than one Ci to C4 unsubstituted alkyl moieties. The term carbocycle in the context of the present specification relates to a cyclic moiety composed of carbon and hydrogen atoms only. An aromatic carbocycle is also named aryl. A non-aromatic carbocycle is also named cycloalkyl.

The term heterocycle in the context of the present specification relates to a cyclic moiety, wherein at least one ring atom is replaced or several ring atoms are replaced by a nitrogen, oxygen and/or sulphur atom. An aromatic heterocycle is also named heteroaryl. A non- aromatic heterocycle is a cycloalkyl, wherein at least one ring atom is replaced or several ring atoms are replaced by a nitrogen, oxygen and/or sulphur atom. A 6-membered heterocycle may be, for example, tetrahydropyran or dioxane.

The term heterobicycle in the context of the present specification relates to two directly connected cycles, wherein at least one ring atom is replaced or several ring atoms are replaced by a nitrogen, oxygen and/or sulphur atom. A heterobicycle is composed of two heterocycles or of one heterocycle and one carbocycle.

The term unsubstituted C _n alkyl when used herein in the narrowest sense relates to the moiety -C _n H2n- if used as a bridge between moieties of the molecule, or -C _n H2n+i if used in the context of a terminal moiety.

The terms unsubstituted C _n alkyl and substituted C _n alkyl include a linear alkyl comprising or being linked to a cyclical structure, for example a cyclopropane, cyclobutane, cyclopentane or cyclohexane moiety, unsubstituted or substituted depending on the annotation or the context of mention, having linear alkyl substitutions. The total number of carbon and -where appropriate- N, O or other hetero atom in the linear chain or cyclical structure adds up to n.

Where used in the context of chemical formulae, the following abbreviations may be used: Me is methyl CH3, Et is ethyl -CH2CH3, Prop is propyl -(CH ₂ ) ₂ CH3 (n-propyl, n-pr) or -CH(CH ₃ ) ₂ (iso-propyl, i-pr), but is butyl -C ₄ H ₉ , -(CH ₂ ) ₃ CH3, -CHCH3CH2CH3, -CH ₂ CH(CH ₃ )2 or -C(CH ₃ ) ₃ .

The term substituted alkyl in its broadest sense refers to an alkyl as defined above in the broadest sense, which is covalently linked to an atom that is not carbon or hydrogen, particularly to an atom selected from N, O, F, B, Si, P, S, Cl, Br and I, which itself may be -if applicable- linked to one or several other atoms of this group, or to hydrogen, or to an unsaturated or saturated hydrocarbon (alkyl or aryl in their broadest sense). In a narrower sense, substituted alkyl refers to an alkyl as defined above in the broadest sense that is substituted in one or several carbon atoms by groups selected from amine NH2, alkylamine NHR, imide NH, alkylimide NR, amino(carboxyalkyl) NHCOR or NRCOR, hydroxyl OH, oxyalkyl OR, oxy(carboxyalkyl) OCOR, carbonyl O and its ketal or acetal (OR)2, nitril CN, isonitril NC, cyanate CNO, isocyanate NCO, thiocyanate CNS, isothiocyanate NCS, fluoride F, choride Cl, bromide Br, iodide I, phosphonate PO3H2, PO3R2, phosphate OPO3H2 and OPO3R2, sulfhydryl SH, suflalkyl SR, sulfoxide SOR, sulfonyl SO2R, sulfanylamide SO2NHR, sulfate SO3H and sulfate ester SO3R, wherein the R substituent as used in the current paragraph, different from other uses assigned to R in the body of the specification, is itself an unsubstituted or substituted Ci to C12 alkyl in its broadest sense, and in a narrower sense, R is methyl, ethyl or propyl unless otherwise specified.

The term hydroxyl substituted group refers to a group that is modified by one or several hydroxyl groups OH.

The term amino substituted group refers to a group that is modified by one or several amino groups NH2.

The term carboxyl substituted group refers to a group that is modified by one or several carboxyl groups COOH.

Non-limiting examples of amino-substituted alkyl include -CH2NH2, -CH2NHMe, -CH2NHEt,

-CH2CH2NH2, -CH ₂ CH ₂ NHMe, -CH ₂ CH ₂ NHEt, -(CH ₂ ) ₃ NH2, -(CH ₂ ) ₃ NHMe, -(CH ₂ ) ₃ NHEt,

-CH ₂ CH(NH ₂ )CH ₃ , -CH ₂ CH(NHMe)CH ₃ , -CH ₂ CH(NHEt)CH ₃ , -(CH ₂ )3CH ₂ NH ₂ ,

(CH ₂ ) ₃ CH ₂ NHMe, -(CH ₂ )3CH ₂ NHEt, -CH(CH ₂ NH2)CH ₂ CH3, -CH(CH ₂ NHMe)CH ₂ CH ₃ ,

CH(CH ₂ NHEt)CH ₂ CH ₃ , -CH ₂ CH(CH ₂ NH2)CH3, -CH ₂ CH(CH ₂ NHMe)CH ₃ ,

CH ₂ CH(CH ₂ NHEt)CH ₃ , -CH(NH ₂ )(CH ₂ )2NH ₂ , -CH(NHMe)(CH ₂ ) ₂ NHMe,

CH(NHEt)(CH ₂ ) ₂ NHEt, -CH ₂ CH(NH2)CH ₂ NH2, -CH ₂ CH(NHMe)CH ₂ NHMe,

CH ₂ CH(NHEt)CH ₂ NHEt, -CH ₂ CH(NH2)(CH ₂ )2NH2, -CH ₂ CH(NHMe)(CH ₂ )2NHMe,

CH ₂ CH(NHEt)(CH ₂ ) ₂ NHEt, -CH ₂ CH(CH ₂ NH ₂ )2, -CH ₂ CH(CH ₂ NHMe) ₂ and

CH ₂ CH(CH ₂ NHEt) ₂ for terminal moieties and -CH2CHNH2-, -CH ₂ CHNHMe-, -CH ₂ CHNHEt- for an amino substituted alkyl moiety bridging two other moieties.

Non-limiting examples of hydroxy-substituted alkyl include -CH2OH, -(CH ₂ )2OH, -(CH2)3OH, -CH ₂ CH(OH)CH ₃ , -(CH ₂ ) ₄ OH, -CH(CH ₂ OH)CH ₂ CH ₃ , -CH ₂ CH(CH ₂ OH)CH ₃ , -CH(OH)(CH ₂ ) ₂ OH, -CH ₂ CH(OH)CH ₂ OH, -CH ₂ CH(OH)(CH ₂ )2OH and -CH ₂ CH(CH ₂ OH) ₂ for terminal moieties and -CHOH-, -CH2CHOH-, -CH ₂ CH(OH)CH ₂ -, -(CH ₂ ) ₂ CHOHCH2-, - CH(CH ₂ OH)CH ₂ CH ₂ -, -CH ₂ CH(CH ₂ OH)CH ₂ -, -CH(OH)(CH ₂ CHOH-, -CH ₂ CH(OH)CH ₂ OH, - CH2CH(OH)(CH2)2OH and -CH2CHCH2OHCHOH- for a hydroxyl substituted alkyl moiety bridging two other moieties.

The term sulfoxyl substituted group refers to a group that is modified by one or several sulfoxyl groups -SO2R, or derivatives thereof, with R having the meaning as laid out in the preceding paragraph and different from other meanings assigned to R in the body of this specification.

The term amine substituted group refers to a group that is modified by one or several amine groups -NHR or -NR2, or derivatives thereof, with R having the meaning as laid out in the preceding paragraph and different from other meanings assigned to R in the body of this specification.

The term carbonyl substituted group refers to a group that is modified by one or several carbonyl groups -COR, or derivatives thereof, with R having the meaning as laid out in the preceding paragraph and different from other meanings assigned to R in the body of this specification.

An ester refers to a group that is modified by one or several ester groups -CO2R, with R being defined further in the description.

An amide refers to a group that is modified by one or several amide groups -CONHR, with R being defined further in the description.

The term halogen-substituted group refers to a group that is modified by one or several halogen atoms selected (independently) from F, Cl, Br, I.

The term fluoro substituted alkyl refers to an alkyl according to the above definition that is modified by one or several fluoride groups F. Non-limiting examples of fluoro-substituted alkyl include -CH ₂ F, -CHF ₂ , -CF ₃ , -(CH ₂ ) ₂ F, -(CHF) ₂ H, -(CHF) ₂ F, -C ₂ F ₅ , -(CH ₂ ) ₃ F, -(CHF) ₃ H, - (CHF) ₃ F, -C ₃ F ₇ , -(CH ₂ ) ₄ F, -(CHF) ₄ H, -(CHF) ₄ F and -C ₄ F ₉ .

Non-limiting examples of hydroxyl- and fluoro-substituted alkyl include -CHFCH ₂ OH, - CF ₂ CH ₂ OH, -(CHF) ₂ CH ₂ OH, -(CF ₂ ) ₂ CH ₂ OH, -(CHF) ₃ CH ₂ OH, -(CF ₂ ) ₃ CH ₂ OH, -(CH ₂ ) ₃ OH, -CF ₂ CH(OH)CH ₃ , -CF ₂ CH(OH)CF ₃ , -CF(CH ₂ OH)CHFCH ₃ , and -CF(CH ₂ OH)CHFCF ₃ .

The term aryl in the context of the present specification signifies a cyclic aromatic C5-C10 hydrocarbon. Examples of aryl include, without being restricted to, phenyl and naphthyl.

A heteroaryl is an aryl that comprises one or several nitrogen, oxygen and/or sulphur atoms. Examples for heteroaryl include, without being restricted to, pyrrole, thiophene, furan, imidazole, pyrazole, thiazole, oxazole, pyridine, pyrimidine, thiazin, quinoline, benzofuran and indole. An aryl or a heteroaryl in the context of the specification additionally may be substituted by one or more alkyl groups.

The term amide-ether refers to an amide-group (-NH-(C=O)-) coupled to an ether group (-O-). The C _x designates the number of C atoms between the amide group and the ether group. Thus, a Ci amide-ether is a group of the following formula: -NH-(C=O)-CH ₂ -O-.

As used herein, the term pharmaceutical composition refers to a compound of the invention, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the invention is provided in a form suitable for topical, parenteral or injectable administration. As used herein, the term pharmaceutically acceptable earner includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (for example, antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington: the Science and Practice of Pharmacy, ISBN 0857110624).

As used herein, the term treating or treatment of any disease or disorder (e.g. cancer) refers in one embodiment, to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and/or prevention of disease.

A first aspect of the invention relates to a compound of the general formula (100) wherein

- Y is O or S;

- X is N or CH;

L is a linker selected from

, a C1-C4 alkyl, a C1-C3 amine, a C1-C3 amide, and a C1-C3 amide-ether, wherein Q is N or CH, particularly Q is N, and R ^L is selected from H and a C1-C3 alkyl, particularly L is a linker selected from

, a C1-C3 amide, and a C1-C3 amide-ether; and wherein L is unsubstituted or substituted with Ci-Ce alkyl, -(Ci-C4)-NR ^N1 R ^N2 , OH, CN, halogen, NR ^N1 R ^N2 , and/or SO2R ^s with R ^N1 , R ^N2 , and R ^s being independently selected from H, and C1-C3 alkyl, n is 0, 1 , 2, 3 or 4, particularly n is 1 or 2, more particularly n is 2; each R ¹ is independently selected from an unsubstituted or substituted Ci-Ce alkyl, OH, OR°, COOR ^A , CN, NO ₂ , halogen, NR ^N1 R ^N2 , SO ₂ R ^s , with R ^N1 , R ^N2 , R ^s , R ^A , and R°being independently selected from H, and Ci-Ce alkyl; particularly each R ¹ is independently selected from OR°, COOR ^A , COOH and OH; or two R ¹ form (with the atoms they are attached to) a C4-C8 cyclo-alkyl or a 4- to 8- membered heterocycle, particularly two R ¹ form (with the atoms they are attached to) a Cs-Ce cyclo-alkyl or a 5- to 6-membered heterocycle, wherein the cyclo-alkyl or the heterocycle is unsubstituted or substituted with C1-C3 alkyl, NO ₂ , COOR ^A , OR° CN, halogen, NR ^N1 R ^N2 , SO ₂ R ^S with R ^N1 , R ^N2 , R°, R ^A , and R ^s being independently selected from H, and C1-C3 alkyl; m is 0, 1 , or 2; each R ² is independently selected from C1-C3 alkyl, OH, NH2, CN, and halogen; with the proviso that the compound is not characterized by the formula (001), (002), or (003)

(003).

An amine function in the linker L is beneficial, as it increases the solubility of the compound.

In certain embodiments, the compound is of the general formula (201) or (202) wherein

X, L, R ² , and m have the same meanings as defined above; k is 0, 1 , or 2; each R ³ is independently selected from Ci- Cs-alkyl, OR°, COOR ^A , CN, NO2, halogen, NR ^N1 R ^N2 , with R ^N1 , R ^N2 , R ^A , and R° being independently selected from H, and C1-C3 alkyl;

V ¹ and V ² are independently selected from CH2, CHR ³ , O, S, NH, and NR ³ ; with the proviso that at least one of R ³ , V ¹ and V ² comprises a heteroatom;

- W is selected from -CH ₂ -, -CHR ³ - , -CH2-CH2-, -CHR ³ -CH ₂ -, -CH2-CHR ³ , and - CHR ³ -CHR ³ -.

The heteroatom in at least one of R ³ , V ¹ and V ² is important to stabilize the interaction of this moiety with the target protein.

In certain embodiments, the compound is of the general formula (300) wherein

- X, L, R ¹ , and n have the same meanings as defined above;

R ^2A and R ^2B are selected from H and C1-C3 alkyl, particularly R ^2A and R ^2B are selected from H and methyl.

In certain embodiments, L is according to formula (401), (402), or (403), (40T), (402’), or (403’) particularly L is according to formula (401), (402), or (403), wherein

Q is N or CH, particularly Q is N;

R ^L is selected from H and a C1-C3 alkyl, particularly R ^L is methyl;

R ^N is selected from H, C1-C3 alkyl; -(Ci-C4)-NR ^N1 R ^N2 , with R ^N1 and R ^N2 being independently selected from H, and C1-C3 alkyl.

In certain embodiments, is selected from

wherein

V ¹ and V ² have the same meanings as defined above, particularly at least one of V ¹ and V ² is independently selected from O, S, and NH, particularly at least one of V ¹ and V ² is O,

R ^3A and R ^3B are independently selected from H, Ci- Cs-alkyl, OR°, COOR ^A , CN, NO2, halogen, NR ^N1 R ^N2 , with R ^N1 , R ^N2 , R ^A , and R° being independently selected from H, and C1-C3 alkyl, particularly R ^3A and R ^3B are independently selected from H and Ci- Cs-alkyl.

In certain embodiments, Y is S.

In certain embodiments, X is N.

A second aspect of the invention relates to a compound as described in the first aspect for use as a medicament, with the proviso that the compound includes the compounds characterized by the formula (001), (002) or (003).

A third aspect of the invention relates to a compound as described in the first aspect for use in treatment or prevention of cancer, with the proviso that the compound includes the compounds characterized by the formula (001), (002) or (003). In certain embodiments, said cancer is selected from ependymoma, prostate cancer, esophageal cancer, thyroid cancer, hepatocellular carcinoma, testicular cancer, pediatric brain tumour, medulloblastoma, rhabdomyosarcoma, gastric cancer, pulmonary pleomorphic carcinoma, breast cancer, nonsmall cell lung cancer, liposarcoma, cervical cancer, colorectal cancer, melanoma, multiple myeloma, endometrial cancer, bladder cancer, glioblastoma, squamous cell carcinoma of the lung, ovarian cancer, head and neck cancer, and pancreatic cancer, sarcoma. In certain embodiments, said cancer is selected from bladder cancer, multiple myeloma, gastric cancer, pediatric brain tumour, medulloblastoma, glioblastoma, ependymoma, and sarcoma. In certain embodiments, said cancer is selected from bladder cancer, colorectal cancer, pediatric brain tumour, medulloblastoma, multiple myeloma, and gastric cancer.

A fourth aspect of the invention relates to a compound as described in the first aspect use in treatment or prevention of metastasis, with the proviso that the compound includes the compounds characterized by the formula (001), (002) or (003). In certain embodiments, said metastasis arises from a cancer selected from bladder cancer, colorectal cancer, pediatric brain tumour, medulloblastoma, multiple myeloma, and gastric cancer.

A fifth aspect of the invention relates to a compound as described in the first aspect for use as an angiogenesis antagonist, with the proviso that the compound includes the compounds characterized by the formula (001), (002) or (003).

Similarly, a dosage form for the prevention or treatment of cancer is provided, comprising a non-agonist ligand or antisense molecule according to any of the above aspects or embodiments of the invention.

The skilled person is aware that any specifically mentioned drug may be present as a pharmaceutically acceptable salt of said drug. Pharmaceutically acceptable salts comprise the ionized drug and an oppositely charged counterion. Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetate, benzoate, besylate, bitatrate, bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide and valerate. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminium, benzathine, calcium, ethylene diamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine and zinc.

Dosage forms may be for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or as an inhalation form or suppository. Alternatively, parenteral administration may be used, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms. Optionally, a pharmaceutically acceptable carrier and/or excipient may be present.

Topical administration is also within the scope of the advantageous uses of the invention. The skilled artisan is aware of a broad range of possible recipes for providing topical formulations, as exemplified by the content of Benson and Watkinson (Eds.), Topical and Transdermal Drug Delivery: Principles and Practice (1st Edition, Wiley 2011 , ISBN-13: 978-0470450291); and Guy and Handcraft: Transdermal Drug Delivery Systems: Revised and Expanded (2 ^nd Ed., CRC Press 2002, ISBN-13: 978-0824708610); Osborne and Amann (Eds.): Topical Drug Delivery Formulations (1 ^st Ed. CRC Press 1989; ISBN-13: 978-0824781835).

Pharmaceutical Composition and Administration

Another aspect of the invention relates to a pharmaceutical composition comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In further embodiments, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.

In certain embodiments of the invention, the compound of the present invention is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient an elegant and easily handleable product.

In embodiments of the invention relating to topical uses of the compounds of the invention, the pharmaceutical composition is formulated in a way that is suitable for topical administration such as aqueous solutions, suspensions, ointments, creams, gels or sprayable formulations, e.g., for delivery by aerosol or the like, comprising the active ingredient together with one or more of solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives that are known to those skilled in the art.

The pharmaceutical composition can be formulated for oral administration, parenteral administration, or rectal administration. In addition, the pharmaceutical compositions of the present invention can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including without limitation solutions, suspensions or emulsions).

The dosage regimen for the compounds of the present invention will 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. In certain embodiments, the compounds of the invention 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.

In certain embodiments, the pharmaceutical composition or combination of the present invention can be in unit dosage of about 1-1000 mg of active ingredient(s) for a subject of about 50-70 kg. The therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations thereof, is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease.

The pharmaceutical compositions of the present invention can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc. They may be produced by standard processes, for instance by conventional mixing, granulating, dissolving or lyophilizing processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see for example L. Lachman et al. The Theory and Practice of Industrial Pharmacy, 4th Ed, 2013 (ISBN 8123922892).

Wherever alternatives for single separable features such as, for example, a ligand type or medical indication are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein. Thus, any of the alternative embodiments for a ligand type may be combined with any medical indication mentioned herein.

The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

Description of the Figures

Fig. 1 Efficacy of the compound - ability to inhibit cancer cell invasion. The graph represents the efficacy of E12, E15 and E15 at 3 different concentrations - 1 .M , 5 .M and 10 .M.

Fig. 2 Efficacy of the compound - ability to inhibit cancer cell invasion. The graph represents the efficacy of E12, E15 and E25 at 10 .M.

Fig. 3 Binding E12, E15 and E25 to the target protein - binding affinities. Nano diffraction scanning fluorimetry (nanoDSF) and Microscale thermophoresis (MST) are biophysical assays used to assess the binding of the compounds to the target protein. Any temperature shift above 1.5 degree Celsius is considered as indication for significant binding.

Fig. 4 Effective inhibitory concentration of E12 - EC50 ( .M).

Fig. 5 Effective inhibitory concentration of E15 - EC50 ( .M).

Fig. 6 Effective inhibitory concentration of E25 - EC50 ( .M). Fig. 7 Overview of effective inhibitory concentration of E12, E15, E25 - EC50 (|iM).

Fig. 8 Biochemical specificity of E12, E15 and E25 - Ability of the compounds to inhibit

FGF signalling pathway without affecting other signalling pathways. 1. Control - DAOY l-A-EGFP cells unstimulated, serum starved overnight and then lysed. 2. bFGF (100ng/ml) - Overnight serum starved DAOY LA-EGFP cells stimulated with bFGF for 10 minutes and then lysed. 3. E12 (10pM) - Overnight serum starved DAOY l_A-EGFP cells treated with E12 for four hours, cells stimulated with bFGF for 10 minutes and then lysed. 4. E15 (10pM) - Overnight serum starved DAOY l_A-EGFP cells treated with E15 for four hours, cells stimulated with bFGF for 10 minutes and then lysed. 5. E25 (10pM) - Overnight serum starved DAOY l_A-EGFP cells treated with E25 for four hours, cells stimulated with bFGF for 10 minutes and then lysed.

Fig. 9 shows the structures of the compounds E12, E15, and E25.

Fig. 10 A) Binding site 1 is not involved in FGFR binding and located below the interaction site of FGFR’s N-terminus with FRS2. B) Binding site 2 is the extended surface area interacting with FGFR’s C-terminal end.

Fig. 11 shows spheroid invasion assay using DAOY cells stimulated with bFGF +/- BGJ398 or E12 or E25 to determine the EC50 of E12 and E25.

Fig. 12 shows cell titer gio assay performed with DAOY cells treated with BGJ398, E12 or E25.

Fig. 13 shows cell titer gio assay performed with AGS cells treated with BGJ398, E12 or E25.

Fig. 14 shows cell titer gio assay performed with M059K cells treated with BGJ398 or E12.

Fig. 15 shows cell titer gio assay performed with RT 112 cells treated with BGJ398, E12 or E25.

Fig. 16 shows cell titer gio assay performed with DMS114 cells treated with BGJ398, E12 or E25.

Fig. 17 shows cell titer gio assay performed with HCT116 cells treated with BGJ398, E12 or E25.

Fig. 18 shows cell titer gio assay performed with SKOV3 cells treated with BGJ398, E12 or E25. Fig. 19 shows cell titer gio assay performed with SNLI16 cells treated with BGJ398, E12 or E25.

Fig. 20 shows pediatric primary brain tumor cells viability after 72 h tratment in 3D culture.

Fig. 21 shows E12 concentration in serum.

Fig. 22 shows immunoblots using various FGFR-driven cell lines treated with BGJ398 or E12 showing the effect of the treatment on the downstream effectors of FGF signaling.

Methods and instruments:

Spheroid invasion assay (SIA) and automated cell dissemination counter (aCDc)

1000 cells/100 mL per well were seeded in cell-repellent 96 well microplate (650790, Greiner Bio-one). The cells were incubated at 37°C overnight to form spheroids. 70 ml of the medium were removed from each well and remaining medium with spheroid overlaid with 2.5% bovine collagen 1. Following the polymerization of collagen, fresh medium was added to the cells and treated with bFGF and/or with compounds. The cells were allowed to invade the collagen matrix for 24 h, after which they were fixed with 4% PFA and stained with Hoechst. Images were acquired on an Axio Observer 2 mot plus fluorescence microscope (Zeiss, Munich, Germany) using a 5x objective. Cell invasion is determined as the average of the distance invaded by the cells from the center of the spheroid as determined using automated cell dissemination counter (aCDc) with our cell dissemination counter software aSDIcs (Kumar et al., Sci Rep 5, 15338 (2015)) (Fig. 11).

Nano differential scanning fluorimetry (nanoDSF)

Purified FRS2 protein tagged with 6X Histidine residues and Guanine nucleotide-binding protein subunit beta (GB1) was diluted in the protein buffer (100mM sodium phosphate, 50mM NaCI, 0.5mM EDTA, 50mM arginine, 1mM TCEP, pH 7.0) to final concentration of 30 .M. The compounds were dissolved in 100% at 50 or 100mM and further diluted to 1 mM with a final concentration of 100% DMSO. Compound and protein were mixed at 1 :1 ration yielding final concentrations of 15 .M and 500 .M for the compounds. The mixture was incubated at room temperature for 15 minutes before measurement. The measurement was performed on a Prometheus system in high sensitivity capillaries. Samples were subjected to a temperature gradient of 20 to 95°C with 1°C/min intervals. Microscale thermophoresis (MST)

Purified FRS2 protein tagged with 6X Histidine residues and Guanine nucleotide-binding protein subunit beta (GB1) was labelled with 2 ^nd generation BLUE-NHS dye. The protein was labelled at a final concentration of 20 .M with 60 .M dye. The labelling was performed in the protein buffer without arginine supplementation. Arginine was re-buffered to protein’s buffer post-labelling. The compounds were dissolved in 100% at 50 or 100mM and further diluted to 1mM with a final concentration of 100% DMSO. The compounds were then diluted. In a 1 :1 serial dilution from 1mM to 61.04nM in protein buffer supplemented with 10% DMSO. 10 J of 50nM labelled protein was added to 10 l of each compound dilution for a final labelled protein concentration of 25nM and DMSO-concentration of 5%. The samples were incubated at room temperature for 15 minutes. The experiments were performed in premium-coated capillaries. Excitation power was set at 20%, MST power to 40% (4 Kelvin temperature gradient) with a laser-on time of 20 seconds and a laser-off time of 3 seconds. Temperature was set to 25°C. Each measurement was repeated twice. The interaction was measured in two independent duplicates.

Immunoblotting (IB)

Cancer cells were treated with bFGF (100ng/ml) and/or with compounds and lysed using Radioimmunoprecipitation assay (RIPA) buffer. RIPA buffer lysates were resolved by SDS- PAGE and transferred to a nitrocellulose membrane using a transfer apparatus according to the manufacturer’s instructions (Bio-Rad). Membranes were probed with primary antibodies against phospho-FRS2, FRS2, ERK1/2, phospho-ERK1/2, AKT, phosphor-AKT, phospho- PKC and tubulin. HRP-linked secondary antibodies (1 :5000) were used to detect the primary antibodies. Chemiluminescence detection was performed using ChemiDoc Touch Gel and Western Blot imaging system (BioRad).

Availability of Compounds

All compounds were purchased and have the following CAS numbers:

E12: CAS number: 385424-29-3

E15: CAS number: 1215614-53-1

E25: CAS number: 924454-71-7

Cell titer gio assay

The metabolic activity and the proliferation of the cells were determined using the Cell Titer gio assay from Promega according to the manufacturer’s instructions. In brief, 250 cells/1 OOpl/per well (for up to 72 h incubation) were seeded in Greiner Bio-One p-clear 384 well plates (655090, Greiner Bio-One) and incubated overnight at 37°C. The old media was then replaced with fresh serum-free media and the cells were treated with BGJ398, E12 or E25 till the desired time point. Following appropriate incubation for each timepoint, 10 pl of the Cell titer gio reagent was added to each well (final concentration of cell titer gio reagent per well is 1 :10) and incubated at 37° C for 30 minutes. The luminescence was then measured with a signal integration time of 0.5 to 1 second per well (Fig. 12-19).

In vitro ADME:

Table. 1 shows the in vitro absorption, distribution, metabolism, elimination and toxicity (ADMET) properties of E12 and E25. Efflux ration represents the permeability of E12 and E25, Semi-thermodynamic solubility shows the solubility of E12 and E25 in aqueous solutions.

Intrinsic clearance and t1/2 shows the metabolic stability of E12 and E25, MTT shows the toxicity of E12 and E25 and potency shows the efficacy of E12 and E25 (Fig. 21).

In vitro absorption, distribution, metabolism, elimination and toxicity (ADMET) properties of E12 and E25.

In vivo pharmacokinetics

3 Healthy non-SCID mice were per oral gavage (10mg/Kg) treated with E12. Additional 3 healthy non-SCID mice were treated with E12 intravenously (1mg/kg). Blood samples were collected at 2, 4, 6, 8 and 24 hours after treatment. Serum from the collected blood samples were isolated and the concentration of E12 in the serum was measured to determine the intrinsic clearance of E12 (Fig. 21).

Table 1: In vivo pharmacokinetic (PK) properties of E12. Pathway analysis

RIPA buffer FGFR-driven cell lysates were resolved by SDS-PAGE and transferred to a nitrocellulose membrane using a transfer apparatus according to the manufacturer’s instructions (Bio-Rad). Membranes were probed with primary antibodies against phospho- FRS2, FRS2, ERK1/2, phospho-ERK1/2, AKT, phospho-AKT, phospho-PKC and tubulin. HRP-linked secondary antibodies (1:5000) were used to detect the primary antibodies.

Chemiluminescence detection was performed using ChemiDoc Touch Gel and Western Blot imaging system (BioRad) (Fig. 22). NMR data - structural binding details of E12

Purified PTB domain of FRS2 is combined with E12 in equimolar concentrations. This complex in incubated at room temperature for 45 minutes. The interactions between the PTB domain of FRS2 and E12 is monitored and recorded using 600nm nuclear magnetic resonance spectrometer.

A chemical shift occurs for amino acids T159, E224, R173, A198, S179, I220, H168, R100, D138, and 1196, which all are shifted in the PTB domain of FRS2 when combined with E12.