Compounds with antitumor activity directed at the hippo pathway

Abstract

The present application is directed compounds of general formula (I) which are active on the Hippo pathway and have antitumor activity. The application also discloses processes for the preparation of said compounds as well as their pharmaceutical compositions.

Description

[0001] Compounds with antitumor activity directed to the Hippo pathway

[0002] ★★★★★★

[0003] Field of the invention

[0004] The present invention relates to compounds active on the Hippo pathway with antitumor activity. Said compounds are particularly active against sensitive and resistant tumors.

[0005] The present invention describes phenoxyamine derivatives, pharmaceutical and medicinal compositions that comprise such compounds, methods of production of such compounds and methods of use of such compounds to treat, prevent, alleviate diseases, disorders or conditions associated with neoplastic pathologies, in particular malignant pathologies of the ovary, lung, breast, colorectal, pancreas, liver, skin and neuroglia. The compounds of the invention are also aimed at providing a therapeutic solution to the development of resistance by drugs commonly used in therapy against the same tumors.

[0006] Advantageously, the compounds of the invention can be combined with drugs currently used in antitumor therapy, for instance with inhibitors of important signaling pathways such as EGFR (epithelial growth factor receptor), MEK (mitogen activated kinases), VEGF (Vascular-Endothelial Growth Factor), JAK (Janus kinases), mTOR (mechanistic target of rapamycin), and PI3K (Phosphatidylinositol 3-Kinase). We also recall GR (glucocorticoid-receptor) agonists.

[0007] Background of the invention

[0008] Ovarian cancer (OC, affecting 5,200 women each year in Italy) and colorectal cancer (CRC, second in Italy and Europe in terms of incidence, with 35,000 cases per year in Italy alone) are two neoplastic diseases with a medium-high incidence and a survival rate that is highly dependent on the early diagnosis. Furthermore, despite the numerous treatment regimens available, the onset of drug resistance phenomena remains high in the most advanced stages of the disease, affecting almost two out of three cases of patients affected by OC and CRC and preventing therapeutic success

[0001] . Furthermore, about 70% of all ovarian cancer diagnoses occur in advanced stages, reducing the overall survival rate of patients [2]. The presentation of nonspecific symptoms, combined with limited detection and screening methods, contributes to the high percentage of women diagnosed in advanced stages. Therefore, the development of inhibitors of cell proliferation directed at new molecular targets of these two tumor subtypes represents a priority.

[0009] Among the possible therapeutic targets not explored by the pharmaceutical market is the Hippo pathway and in particular, but not exclusively, one of the proteins belonging to it such as the transcription factor TEAD, in particular its isoform 4 (hereinafter TEAD4), i.e. the isoform most relevant for the oncogenic activity of the aforementioned solid carcinomas as it is mostly overexpressed or mutated. [3,4]

[0010] The transcription factor TEAD4 (Transcriptional Enhancer Associated Domain), associated with its consensus protein YAP (Yes Associated Protein) induces in particular but not exclusively, the pair of anti-apoptotic genes such as CYR61 and CTGF in the carcinoma cell [5]. These genes promote the synthesis of growth factors (GFs) that facilitate cell adhesion, cell cycle progression and the development of tumor metastases. Inhibition of the formation of the YAP:TEAD4 complex results in cytoplasmic retention of the transcription factor itself and proteasomal degradation of YAP, with consequent inhibition of cell growth [6]. This complex represents the final effector of the cellular signaling pathway called Hippo Pathway. It is finely regulated by extra- and intracellular signals, including mechanical stress, cell-cell contact and hormonal signaling, most of which act through the activation of G-protein coupled receptors [7].

[0011] The Hippo Pathway has attracted considerable interest in the fields of regenerative medicine and oncology therapy, due to its central role in the control of cell and organ growth [4]. In colorectal cancer, high expression of YAP and TEAD4 has been shown to be highly associated with increased cell proliferation and poor prognosis [8]. Furthermore, recent studies have shown that the final effectors of the Hippo Pathway could be involved in the initiation, progression and development of resistance of ovarian cancer [9]. At the molecular level, the TEAD4 protein is characterized by a medium molecular weight (48 kDa) and is organized in two main domains, one binding the consensus sequence on DNA (TEA domain, C-terminal), and one interacting with the partner YAP (YBD, Yap Binding Domain, N-terminal). In turn, the YBD domain displays three distinct contact interfaces for the YAP protein

[0010] . The first (Interface 1) consists of 52 residues organized in a strand-strand motif and establishes 7 hydrogen bonds with YAP. Interface 2 consists of an LXXLF motif that organizes itself in an a-helix and contacts YAP through hydrophobic interactions

[0011] . Finally, interface S contacts the corresponding Omega Loop region of YAP through hydrophobic and polar interactions. Among them, the ionic bond between R89 (YAP) and D264 (TEAD) and the hydrogen bond between S94 (YAP) and E255-Y421 (TEAD) are fundamental.

[0012]

[0012] It has been shown that the dissociation of interface 3, as well as the one of interface 2, represents the sufficient condition to separate the partners from the complex. This minimum condition is not satisfied for the contact surface of interface 1, since sitespecific mutations on this contact amino acid sequence do not significantly modify the binding affinity of the complex.

[0013]

[0013] Different classes of inhibitors of TEAD protein function are available, including flufenamic and niflumic acid derivatives, ionic inhibitors of the TEAD lipoylation pocket, and YAP peptidomimetics

[0014] . Furthermore, the palmitoylation site of TEAD protein has recently been validated as a pharmaceutical target, which has led to the development of several covalent compounds that compete with endogenous palmitic / myristic acid for the acylation site of the protein

[0015] . These include MYF-03- 69, K-975, and the scaffold 3-(2-benzylidenehydrazinyl) benzo[d] isothiazole

[0016] . There are also some compounds currently in phase I clinical trials with a competitive action for the palmitoylation site, such as VT3989, a covalent inhibitor of TEAD in NF2- deficient mesothelioma in combination with Osimertinib, and IK-930, a high-potency derivative of flufenamic acid and a reversible inhibitor proposed as monotherapy for the treatment of NF2-deficient mesothelioma and angiosarcoma

[0017] .

[0014] However, the molecules investigated to date have limited action due to their chemical instability, low potency in terms of Ki, or non-optimal pharmacokinetic profiles

[0017] . Added to this is the lack, for the tumors in question, of effective therapeutic alternatives in targeting new biological targets not yet investigated to date, in cases of a recurrent manifestation of drug resistance and in the final phases of therapeutic treatments. All this underlines the current limitation in the number and efficacy of drugs in use.

[0015] Unless specifically excluded in the detailed description that follows, what is described in this chapter is to be considered as an integral part of the detailed description.

[0016] Summary

[0017] The main purpose of the present invention is to describe a new class of molecules of Formula (I) that can overcome the above-mentioned drawbacks and limitations, offering an alternative and improved solution for the treatment, prevention and alleviation of diseases, disorders or conditions associated with the formation and progression of solid tumor subtypes wherein, preferably, the transcription factor TEAD4 is overexpressed, exploiting the latter as a target for pharmacological inhibition.

[0018] Closely connected to the above-mentioned purpose, another purpose of the invention is to provide, through the molecules of Formula (I), a therapeutic alternative for tumor forms that show resistance to the common chemiotherapeutic drugs used, do not respond to common therapeutic treatments and that preferably present an overexpression of TEAD4

[0018] . Clinical studies have shown that TEAD4 expression levels are overexpressed in cancer cells compared to the normal counterpart in several types of tumors, including colorectal and ovarian tumors

[0019] .

[0019] Another purpose of the present invention is to provide different pharmaceutically acceptable forms wherein the above-mentioned molecules of Formula (I) can be present, i.e. in neutral form, salified, as solvates or in the respective polymorphs, to be used as single isomers or as a mixture thereof, individually or in mixture with other drugs.

[0020] A further purpose of the present invention is the description of the synthetic process for the preparation of the compounds of Formula (I) through the coupling of its two precursors of Formula (II) and (III). In particular, for the compounds with a phenoxyamino structure of Formula (I), synthetic routes suitable for industrial scale-up have been designed and then described, which respect to the principles of Green Chemistry and the sustainability criteria in the current state of knowledge, in order to promote their production at a reasonable cost, and therefore their availability in a short time, with obvious ethical implications.

[0021] A further purpose of the present invention is related to the pharmaceutical compositions comprising the compounds of Formula (I) for the treatment, prevention, and alleviation of diseases, disorders or conditions associated with neoplastic pathologies, in particular malignant pathologies of the ovary, lung, breast, colorectal, pancreas, liver, skin and neuroglia.

[0022] These and other purposes are achieved through the invention of a class of molecules of Formula (I) having surprisingly high affinity for the interface 3 of the YBD domain of the TEAD4 protein, which by means of an inhibition mechanism described below, exerts dissociative activity of the YAP:TEAD4 complex. In fact, this class of molecules is characterized by occupying the interface 3 of TEAD4, thus preventing the binding of YAP, with observation of a consequent pharmacological response of the cell.

[0023] The synthetic procedure for obtaining the compounds of Formula (I), the structureactivity correlations, their molecular action starting from modeling studies, their activity on the target by means of fluorescence anisotropy assay, and the in vitro activity are also described.

[0024] These and other purposes, advantages and characteristics of the present invention will be better specified in a detailed description of the preferred embodiments, which follows. Furthermore, the claims describe preferred variants of the invention, forming an integral part of the present description.

[0025] Brief Description of the Figures

[0026] Figure 1 : Results of the crystal violet experiment for preliminary screening on HT29 (CRC, ATCC No. HTB-38) and A2780 (OC, CACC No. 93112519) cell lines at 40 pM concentration. Figure 2. Binding interaction between DZ1 -(S) Fnx1 and the X-ray structure of TEAD, freely available in the PDB with code 5DQ8.

[0027] Figure 3. Examples of the bicyclic system (D).

[0028] Figure 4. Survival of HCT116 (panel A), HT29 (panel B) and A2780 (panel C) and A2780 / CP (panel D) cells after 72 hours of treatment with the compounds of the present invention. 24 h after seeding, cell lines were treated for 72 h with increasing concentrations up to 60 pM) of DZ1 -S (black circles), DZ1 -R (empty circles), DZ3- S) (black triangles up), DZ5- S) (black triangles down), DZ5-R (empty triangles down), DZ7-(S) (black squares), 5-FU (black diamonds), verteporfin (empty hexagons) reference compound, D361 (empty triangles up) as internal reference compound. Then they were fixed and stained with a crystal violet solution. The concentration of the dye extracted and quantified by spectrophotometry was proportional to the number of cells. The percentage of cytotoxicity was obtained by comparing the absorbance of the drug- exposed cultures with that of the unexposed cultures (negative control). The error bars represent the average value ± S.D. of three experiments performed in duplicate.

[0029] Figure 5. Basal expression levels of TEAD protein, in HCT116, HT29 and A2780 cell lines.

[0030] Figure 6. Effects of modification of YAP protein levels and phosphorylation status in tumor cell lines caused by treatments with the compounds (DZ1 -(S), Z5-(S); (A) Western blot, showing YAP and pYAP-Ser397 and pYAP-Ser127 expression levels after 24 h treatment with the selected compounds, in a panel of colorectal cancer cell lines HCT116 and HT29 and ovarian cancer A2780; (B) the corresponding densitometric quantification: YAP (black bar), pYAP-Ser397 (dark grey bar), pYAP- Ser127 (grey bar); (C) quantification of the ratio pYAP-Ser397 / YAP (black bar) and pYAP-Ser127 / YAP (grey bar); Verteporfin was used as reference compound, and D361 was used as internal reference.

[0031] Figure 7. mRNA levels of the RPLPO gene, used as a normalizer gene.

[0032] Figure 8. The mRNA levels of YAP (black bar) and its target genes: CYR61 (dark grey bar) and CTGF (grey bar). The histogram represents the effects of the selected DZ compounds (DZ1 -(S), DZ5-(S)) tested on the colorectal cancer cell lines HCT116 and HT29 and ovarian cancer A2780 for a treatment duration of 48h. Verteporfin (VP) was used as a reference compound.

[0033] Definitions

[0034] In this document, the terms, "comprises", "includes", "has", "having", "bearing", "contains", "containing", "characterized by" or any other variation of these terms, are intended to cover a non-exclusive inclusion, and therefore not subject to any explicitly stated limitation. For instance, a composition, mixture, process or method comprising a list of elements is not necessarily limited only to these elements but may include other elements not expressly listed or inherent in such composition, mixture, process or method.

[0035] The compounds of the present invention may be present either in pure form or as mixtures of different isomeric forms, such as stereoisomers, constitutional isomers or optically active compounds.

[0036] According to the present invention, the definitions "polymorphic" or "polymorphism" refer to the same substance present in different crystalline forms.

[0037] According to the present invention, the definition of “isomer / s” refers to compounds that have the same gross formula, but different structural formula and arrangement of the connectivity of the atoms.

[0038] According to the present invention, a “stereoisomer” is defined as an isomer with identical connectivity but different spatial arrangement. It includes the subcategories of enantiomers (optical or configurational isomers), cis / trans isomers (or geometric isomerism), and diastereoisomers.

[0039] Therefore, a pair of molecular entities of which one is specular and non- superimposable to the other is defined as a pair of enantiomers. An equimolar mixture of two enantiomers is defined as a racemic mixture. Instead, molecular entities bearing the same atomic connectivity but different spatial orientation, without constituting pairs of non-superimposable mirror structures represent diastereoisomers (or diastereomers). Furthermore, molecular entities characterized by the same molecular formula, but different connectivity are defined as constitutional isomers.

[0040] "Alkyl", used alone or in compound words such as "haloalkyl" or "alkylamino" or "alkylcarbonyl", is defined as a molecular moiety comprising alkyl chains.

[0041] "Phenyl", "aryl" or "aromatic", used alone or in compound words such as "heteroaryl" or "heteroaromatic", is defined as a molecular moiety comprising cyclic aromatic structures with 5, 6 or more members, wherein the number of TT electrons follows the Huckel rule. In detail, the term "heteroaromatic" defines cyclic structures wherein one or more carbon atoms are replaced with N, S or O atoms by adjusting the number of hydrogen atoms in relation to the "normal" valence of the heteroatom.

[0042] In the context of the present invention, the term "about" means a numerical value equal to the reported value ± its standard deviation.

[0043] Detailed description.

[0044] The present invention aims to describe a new class of organic compounds of Formula (I) for the treatment, prevention, alleviation of diseases, disorders or conditions associated with malignant neoplastic diseases of the ovary (OC), lung, breast, colorectal (CRC), pancreas, liver, skin, neuroglia and other tumor types wherein the expression of TEAD4 is high. Furthermore, the compounds of Formula (I) may represent, in case of manifestations of drug resistance or lack of therapeutic response, a potential solution to common chemiotherapeutic drugs and / or therapeutic treatments used against the tumors described above.

[0045] In fact, it has been experimentally found that the compounds of Formula (I) exert a completely new mechanism of action in the inhibition / reduction of the concentration of the YAP:TEAD4 complex consisting in the interaction of YAP with interface 3 of the YBD domain of the transcription factor TEAD4. The amino acid sequence (SEQ ID NO:1) of the YBD domain is described below wherein the bolded portions of the sequence represent the direct contact sites between interface 3 and the Q-loop of YAP: AVDIRQIYDKFPEKKGGLKDLFERGPSNAFFLVKFWADLNTNIEDEGSSFYGVSSQY ESPENMIITCSTKVCSFGKQVVEKVETEYARYENGHYSYRIHRSPLCEYMINFIHKLK HLPEKYMMNSVLENFTILQVVTNRDTQETLLCIAYVFEVSASEHGAQH.

[0046] Precisely through this antitumor mechanism of action on patients, different from all the compounds currently studied to inhibit the same complex, to the knowledge of the inventors, and from all the compounds currently in the clinical phase for experimental study on patients, they present a potential advantage on tumor cells resistant to the most common antitumor drugs.

[0047] From a virtual screening study against the TEAD4 protein, 50 compounds of potential interest were identified, deriving from a database of chemical substances with a widely variable structure. The 50 compounds were subjected to in vitro assays to determine their activity against HT29 (colorectal cancer cells, CRC) and A2780 (ovarian cancer cells, OC) cell lines at a concentration of 40 pM (Figure 1).

[0048] Of the 6 most active compounds, a complete inhibition curve was obtained at five different concentrations, also using the HCT116 cell line. These 6 hits were again docked to TEAD4 interface 3 and subjected to a molecular dynamics (MD) simulation, which allowed to identify the first phenoxyamine with high affinity for the binding site. For this molecule (hereinafter referred to as DZ1 -(S) and belonging to the general Formula (I)), the MD simulation shown a fundamental interaction between the indazole bicycle (D) of the molecule with the lipophilic cavity of the protein, mimetic of the central aromatic portion of Peptide-17 of the YAP protein and interacting with the amino acid K289 in an area characterized by the presence of basic residues of the lipophilic cavity. Furthermore, the polar terminus R1-R2- of DZ1 -(S) is stabilized by a hydrogen bridge with S256 of TEAD. Starting from these observations, the authors proceeded to the design of several phenoxyamines, arriving at the definition of the scaffold of Formula (I) shown below.

[0049] Compounds Description The novel phenoxyamine compounds of the present invention are represented by Formula (I) and comprise an oxadiazole ring (A), a six-membered carbocyclic aromatic ring (B), a cycloalkyl amine (C) and a bicyclic system (D).

[0050] The compounds of Formula (I) can be used in their pure isomeric forms or as racemates or as polymorphs and mixtures thereof. They can also be used alone or in mixtures with other ingredients and / or active principles in pharmaceutically acceptable forms (salts, complexes, aggregates, etc.).

[0051] The compounds of Formula (I) contain the substituents specified below which are chosen from:

[0052] • “bridge” groups (-R1- and -R3-), independently equal or different from each other, which can be: o direct bond (no atom between the ring (A) and R2or between the bicycle (D) and R4), o alkylene (linear or branched) C1-4, such as: -CH2-, -C2H4-, -C3H6-, -C4H8- o carbonyl (-(C=O)-), o difluoromethylene (-CF2-);

[0053] • “terminal” groups (-R2and -R4), independently equal or different from each other, which can be: o hydrogen (-H), o alkyl (linear or branched) C1-4, such as: -CH3, -C2H5, -C3H7, -C4H9, o alkyloxy (linear or branched), such as: -OCH3, -OC2H5, -OC3H7, -OC4H9, o alkylthio (linear or branched), such as: -SCH3, -SC2H5, -SC3H7, -SC4H9, o cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, o cyclooxyalkyl (5 or 6-membered), such as: tetrahydrofuryl, tetrahydropyranyl, o phenyl, aryl, possibly substituted, such as: phenyl (-CeHs), tolyl (-C6H4-CH3) o 5- or 6-membered nitrogen- or oxygen-containing heteroaromatic group, such as, but not limited to, pyridyl, pyrimidyl, furyl, pyrrolyl.

[0054] • Y1and Y2, each linked in an available position of the benzene ring (B), independently equal or different from each other, which can be: o hydrogen (-H), o alkyl (linear or branched) C1-4, such as: -CH3, -C2H5, -C3H7, -C4H9, o alkyloxy (linear or branched), such as: -OCH3, -OC2H5, -OC3H7, -OC4H9, o acyloxy (linear or branched), such as: -O(C=O)-CH3, -O(C=O)-C2H5, o -O(C=O)-C3H7, -O(C=O)-C4H9, o carbonyl-alkyl (linear or branched), such as: -(C=O)-CH3, -(C=O)-C2H5, -(C=O)- C3H7, -(C=O)-C4H9, o alkylamino or dialkylamino (linear or branched), such as: -NH2, o -NH(CH3), -NH(C2H5), -NH(C3H7), -NH(C4H9), -N(CH3)2, -N(CH3)C2H5, o -N(CH3)C3H7, -N(CH3)C4H9, -N(C2H5)2, -N(C2H5)C3H7, -N(C2H5)C4H9, o -N(C3H7)2, o cycloalkylamino, such as: -N(CH2)4, -N(CH2)5, o halogen, preferably: -F, -Cl or -Br o halomethyl, preferably: -CF3, -CHF2, CH2F, CH2CI • The moieties (X1, X2and X3) in the bicyclic system (D), independently equal or different from each other, which can be: a C-H group, a nitrogen atom or an N-H group. The variation of these moieties makes the bicyclo (D) an indene, an indole, an isoindole, an indazole, a benzodiazole, a benzimidazole or a benzotriazole. This bicyclic system is linked to the nitrogen atom of the ring (C), through an amide bond. On the side of the bicyclo (D), the bond occurs in one of the available positions on the benzenoid ring or on the five-membered heterocyclic ring (one of the bonds shaded in the structural formulas in figure 3 and reported below), through the substitution of a hydrogen atom.

[0055] The stereogenic carbon center (*) in the cycloalkylamine ring (C), can have a specific (R or S) or nonspecific (partially or completely racemic) configuration.

[0056] Use of compounds of Formula (I) as a drug

[0057] A detailed study of the interaction pocket of the TEAD4 protein has highlighted its predominantly hydrophobic nature. However, amino acid K289 has been identified as a main point of potential interaction. Computational study of the interaction of different small molecules with possible interactions with amino acids of the pocket has allowed the identification of particularly similar functional groups and has identified their optimal relative distance. This study led to the identification of compounds of Formula (I), which were further subjected to computational studies to determine the optimal orientation in the pocket of the biological target (TEAD4).

[0058] For instance, as shown in Figure 2, the indazole portion (bicyclo D) of the compound DZ1 -(S) interacts with amino acid K289, determining an anchoring point of the molecule to the target. The phenyloxadiazole moiety (ring A) interacts via a hydrogen bridge produced by the nitrogen of the condensed heterocycle with a lysine of the binding pocket (K289) and the methylene methoxy substituent (R1= -CH2-; R2= - OCH3) further stabilizes the complex with a second hydrogen bond with S256. The chemical space of this region of the pocket has been explored by defining a series of high-affinity phenoxyamines, some of which are shown in Table 1 .

[0059] Table 1 . Examples of compounds of formula (I).

[0060] The synthesis of the phenoxyamines listed in Table 1 , has allowed to conduct in vitro and in vivo interaction studies, from which a high affinity for the interface 3 of the transcription factor TEAD4 is highlighted. Since, to the best of our knowledge, no substances are currently known that are able to interact with this domain of the TEAD4 protein, the object of the present invention is the use of the compounds of Formula (I) as drugs or medicaments able to inhibit / reduce the concentration of the YAP: TEAD4 complex, in cases where this is over-expressed.

[0061] The present invention also includes compositions comprising the compounds of formula (I). Such compositions may also comprise one or more carriers, vehicles, excipients or other pharmaceutically acceptable additives. Examples of such carriers or additives include an inert organic or inorganic carrier known to those skilled in the art, such as water, saline, glycerol, glucose, natural oils, etc. These compositions may be used as medicaments.

[0062] The present pharmaceutical compositions may be administered orally, for instance in the form of tablets, capsules, pills or the like. They may be administered parenterally, intravenously, intracutaneously and the like. The quantity of the composition to be administered, understood as the dose of the active ingredient of formula (I), will depend on various factors such as, among others, the route of administration, the time of administration, the duration of treatment, the use of any other medicament, the age and sex of the patient, in any case at the discretion of the attending physician.

[0063] The composition may also include other additional components such as flavoring agents, thickeners, stabilizers, and the like. The pharmaceutical form that includes one or more compounds of Formula (I) may also involve prolonged release preparations, retard preparations, liposomes, or other that can be obtained with techniques known and commonly used in the pharmaceutical industry. Preferably the compounds and / or compositions of the present description are used as inhibitors of the formation of the YAP: TEAD4 transcriptional complex

[0037] . The compounds and / or compositions of the present description may also be used as antitumor agents, for instance, for the treatment of tumors wherein TEAD is overexpressed, preferably to treat solid tumors. Examples of such tumors include ovarian cancer, colorectal cancer, mesothelioma, melanoma and blood cancer.

[0064] Furthermore, the compounds and / or compositions of the present description may also be used as antitumor agents, for instance for tumors wherein TEAD4 is overexpressed, or to treat solid tumors resistant to common antitumor drugs. Examples of such tumors include ovarian cancer, colorectal cancer, mesothelioma, melanoma and blood cancer.

[0065] In addition, the compounds of Formula (I) in any of the pharmaceutically acceptable forms may be used in combination with other active substances such as, for instance but not limited to, other antitumor drugs such as inhibitors of important signaling pathways such as EGFR (epithelial growth factor receptor), MEK (mitogen activated kinases), VEGF (Vascular-Endothelial Growth Factor), JAK (Janus kinases), mTOR (mechanistic target of rapamycin), PI3K (Phosphatidylinositol 3-Kinase) and GR (glucocorticoid-receptor) agonists, which act preferably for tumor cell lines with overexpression of TEAD4.

[0066] Preparation of the compounds of Formula (I)

[0067] The present invention also relates to a process for the preparation of the compounds of formula (I) described herein. The compounds can be synthesized by the methods described below or by slight modifications of these methods which are however within the reach of the person skilled in the art. The ways of modifying the methodology include, among others, the temperature, the solvent, the reagents, etc. and other conditions known to those skilled in the art. Further representations are described in detail in the following examples, which are not intended in any way to limit the scope of the claims.

[0068] The compounds of Formula (I) can be prepared by coupling the structure (II) with the structure (III), as shown in Scheme 1 . This method involves the construction of an ether bond (B)-O-(C) between the ring (B) and the ring (C). The ether bond can be built through a nucleophilic substitution, which can be:

[0069] • an aliphatic SN2, wherein Z1= OH and Z2is a suitable leaving group, or

[0070] • an aromatic SN-Ar, wherein Z2= OH and Z1is a suitable leaving group.

[0071] Such leaving groups can be made previously or can be built in situ (even during the coupling reaction itself) starting from a pre-existing group (such as a -OH) through the use of an appropriate activating reagent. This includes the different options known in the art, such as the use of thionyl halides, mesyl halides, tosyl halides, etc. or phosphines (Mitsunobu reaction), etc.

[0072] Consequently, the substituents Z1(formula II) and Z2(formula III), independently equal or different from each other, can be:

[0073] • hydroxyl (-OH),

[0074] • acyloxy (-O(C=O)-H, -O(C=O)-CH3, -O(C=O)-C2H5, -O(C=O)-C3H7, or - O(C=O)-C4H9),

[0075] • halogen, such as: -Cl, -Br, or -I,

[0076] • oxygen-sulphur leaving group, such as, by way of non-limiting example, -OMs (mesylate), -OTs (tosylate), -ONs (nosylate).

[0077] • oxygen-phosphorus leaving group (possibly produced in situ), such as that originated during the Mistunobu reaction

[0038] , by subsequent interaction of triphenylphosphine, diethylazodicarboxylate (DEAD), carboxylate and alcohol or such as those generated in the relevant variants easily identifiable by the expert in the field.

[0078]

[0079] Scheme 1

[0080] The compounds of Formula (II) can be prepared by coupling two carboxylic derivatives, one of which is aromatic (IV) and the other (V) bearing a structure compatible with the required degree of substitution (R1-R2-) by the target of Formula I, as shown in Scheme 2.

[0081] Scheme 2

[0082] Consequently, the substituents K1(formula IV) and K2(formula V), independently equal or different from each other, can be:

[0083] • a carboxylic group, -COOH,

[0084] • a carboxylic ester (linear or branched), such as: -(C=O)OCH3, -(C=O)OC2H5, -(C=O)OC3H7, -(C=O)OC4H9,

[0085] • an acyl halide, -(C=O)CI, -(C=O)Br, -(C=O)I, • a carbonitrile, -C N, • an amidoxime, -(C=N-OH)-NH2 ,

[0086] • an aldehyde or its derivative, such as an oxime, or an imine.

[0087] This method is based on the oxadiazole ring formation strategies, well known in the art. Classic examples include the use of an amidoxime (prepared from a nitrile), and its interaction with an acyl chloride

[0021] or with a second nitrile group

[0022] , or with a carboxylic acid

[0023] , or with an aldehyde

[0024] . Alternative methods exist that involve the use of aldoximes

[0025] , instead of the aforementioned amidoximes

[0026] .

[0088] The use of biobased compounds such as 4-hydroxy-benzoic acid or 4- hydroxybenzaldehyde (or their derivatives) for the preparation of the ring (B) of the compounds of Formula (I) constitutes part of this invention.

[0089] The compounds of Formula (III) can be prepared by coupling a carboxylic derivative of the bicyclic system (D) (formula VI) with a suitable cycloalkylamine (formula VII), as illustrated in scheme 3.

[0090] Scheme 3

[0091] In structure (VI), the W substituent can be:

[0092] • hydroxyl (-OH),

[0093] • alkyloxy (linear or branched), such as: -OCH3, -OC2H5, -OC3H7, -OC4H9,

[0094] • acyloxy, (linear or branched), such as: -O(C=O)H, -O(C=O)CH3, -O(C=O)C2H5, -O(C=O)C3H7, -O(C=O)C4H9

[0095] • halogen, such as: -Cl, -Br or -I, • nitrogenous heteroaromatic (for instance, but not limited to, pyridyl, imidazolyl, or bentrotriazolyl)

[0096] In structure (VII), the Z3substituent can be the same as Z2(structure III) or it can be a precursor of it, capable of being transformed into Z2, after or during the coupling process between VI and VII. Consequently, the Z3substituent (formula VII) can be:

[0097] • equal to the Z2substituent (Formula III)

[0098] • ketone (doubly bonded oxygen)

[0099] • imine (doubly bonded nitrogen)

[0100] • oxime (doubly bonded nitrogen)

[0101] • acetal (between an aldehyde or a ketone and two hydroxyamines). This is a dimeric form, as shown in the dotted box of Scheme 3.

[0102] Although the preferred preparation method for the compounds of Formula (I) is the one shown in Schemes 1 , 2 and 3, it is also possible to prepare the compounds (I) following alternative strategies, which involve a variation on the order of formation of the junctions described previously: formation of the ring (A), bridge (B)-O-(C), amide junction between ring (C) and bicycle (D).

[0103] It is therefore possible to prepare compounds of Formula (I), for instance, through the initial formation of a compound of formula (II) and the subsequent interaction with a compound of formula (VII) - or its derivative - obtaining a condensation compound (VIII) which will then be made to interact with a compound of formula (VI), as shown in scheme 4. Scheme 4

[0104] In some of these preparation methods, it may be appropriate to protect the amino group of the compounds of formula VII, through the use of suitable protecting groups known in the art, such as tert-butoxy carbonyl (Boc), benzyloxycarbonyl (Cbz), fluorenyl methyloxycarbonyl (FMOC) or others having a similar function.

[0105] The compounds of formula VII can be obtained from the bio-oxidation of simpler amines such as proline or pyrrolidine. It is in fact possible to oxidize L-proline into the corresponding hydroxyproline

[0027] and proceed to decarboxylation

[0028] to obtain a compound of formula VII. Alternatively, it is possible to obtain the compounds of formula VII through the oxidation of pyrrolidine or its analogues

[0029] . In consideration of the previous observations, the use of biobased compounds such as proline or pyrrolidine (or their derivatives) for the preparation of the ring (C) of the compounds of Formula (I) constitutes part of this invention.

[0106] With regard to the bicyclic system (D) bearing a carboxylic substituent (formula VI), it could be obtained from simpler precursors. In the case of 1 H-indazole-3-carboxylic acid, its preparation can be conveniently obtained starting from isatin

[0030] (Scheme 5). Therefore, the use of biobased compounds such as isatin (or its derivatives) for the preparation of the ring (D) of the compounds of Formula (I) constitutes a further element of this invention.

[0107] Scheme 5

[0108] The invention will now be illustrated by a series of examples that are to be considered illustrative and not limitative of the scope of the invention itself.

[0109] Examples The present invention is substantiated by the following examples, relating to the synthesis, structural characterization and affinity performance towards the interface 3 of the TEAD protein of some of the phenoxyamines shown in Table 1 . The following examples are to be understood as evidence underlying the claims of the present invention, without limiting to them the generality of the invention.

[0110] Synthesis of compounds of formula fl)

[0111] All commercially available reagents and solvents were used as supplied by the commercial supplier, without further purification, unless otherwise specified. The reactions were monitored by thin layer chromatography (Silicagel 60, F-254, E. Merck) and visualized by UV lamp, iodine vapor, cerium ammonium sulphate solution or alkaline potassium permanganate solution. The following solvents and reagents were abbreviated as follows: tetrahydrofuran (THF), diethyl ether (Et20), dimethyl sulfoxide (DMSO), ethyl acetate (EtOAc), dichloromethane (DCM), dimethyl formamide (DMF), methanol (MeOH), acetonitrile (ACN), triethylamine (TEA). All reactions were carried out by conventional techniques or by microwave irradiation. NMR spectra were acquired on a Bruker spectrometer, mod. Av-Neo 400 or a Bruker spectrometer, mod. Avance III 600 HD. The1H spectra are acquired at 400.134 MHz and the13C spectra at 100.62 MHz. The proton chemical shift is referred to the TMS used as an internal standard. The chemical shifts are reported in parts per million (ppm, 5). The coupling constants are reported in Hertz (Hz). The splitting is described as s (singlet), d (doublet), t (triplet), q (quartet), dd (double doublet), m (multiplet), br (broadcast signal). The mass spectra are acquired by a high-resolution mass spectrometer Orbitrap Q- Exactive Hybrid (Thermo Fisher).

[0112] Determination of the Purity of DZ Compounds

[0113] Each compound reported in this invention was characterized by HPLC-UV / Vis (Agilent Infinity II) to determine its percent purity. The method employed uses a RP Kinetex 2.6 pm Biphenyl 100 A column, operating at a flow rate of 1 mL / min at 30°C. The separation employs a 22-minute gradient with A (0.1% formic acid aq.) and B (0.1% formic acid in ACN), to 5% to t22 95% B. The eluted compounds were monitored at 254nm and 280nm.

[0114] Peak integration was performed using the ChemStation software (Agilent Technologies) using the area method, after subtraction of the blank. All derivatives show a purity > 97% (@250nm).

[0031]

[0115] Determination of chiral descriptors

[0116] The optical activity of the DZ compounds was determined by HPLC-UV coupled to a spectropolarimetric detector (CD, circular dichroism) LC2000 Plus (Jasco). The separation uses an isocratic method 90% ACN, 0.1% FA in 10% aq. for 10 minutes, room temperature, with a directly derivatized RP column Chiralcel-OD analytic 4.6 x 50cm with detection @254nm.

[0117] Example of the synthesis procedure of (3-(4-(1 ,2,4-oxadiazol-3-yl)phenoxy)pyrrolidin-

[0118] 1 -yl)(1 -methyl-1 H-indazol-3-yl)methanones (Scheme 6) a) MeSO2CI, TEA, DCM, from 0°C to RT, 3 hours; b) K2CO3, ACN, 80°C, 6 hours; c) HCI 4M, 1,4-dioxane, from 0°C to RT, 3 hours; d) SOCI2, anhydrous DCM, DMF cat., Ar, from 0°C to reflux, 3 hours; e) TEA, DCM anhydrous, Ar, from 0°C to reflux, 18 hours; f) NH2OH HCI, NaHCO3, MeOH, reflux, 3 hours; g) TEA, DMF, from 0°C to FIT for 30 minutes, reflux 18 hours.

[0119] Step a: (R)-tert-buty\ 3-((methylsulfonyl)oxy)pyrrolidine-1 -carboxylate or (S -terf-butyl 3-((methylsulfonyl)oxy)pyrrolidine-1 -carboxylate (1)

[0120] One enantiomer of tert-butyl-3-hydroxypyrrolidine-1 -carboxylate (1 .0 eq) and TEA (2.0 eq) are dissolved in 10 mL of DCM and the mixture is cooled in an ice bath. Methanesulfonyl chloride (1 .5 eq) is added dropwise, the ice bath is removed, and the mixture is stirred for 3 h at room temperature. After the reaction is complete, the mixture is poured into 10 mL of water and extracted with EtOAc. The organic phase is washed with brine, dried with Na2SO4 and the solvent is removed by evaporation under reduced pressure. The product is obtained as an amber oil (quantitative yield) and is used in the subsequent steps without further purification.

[0121] Step b: S -tert-butyl-3-(4-cyanophenoxy)pyrrolidine-1 -carboxylate or (R)-tert-buty\-3- (4-cyanophenoxy)pyrrolidine-1 -carboxylate (3)

[0122] One of the two enantiomers (1 .0 eq) synthesized in the previous step (a) dissolved in 5 mL of ACN, is added to a suspension of 4-hydroxybenzonitrile 2 (1 .1 eq), K2CO3 (3.0 eq) in ACN. The mixture is stirred at reflux for 18 hours. The mixture is cooled to room temperature, the solvent is removed under reduced pressure and the residue obtained is dissolved with ethyl acetate and water. The aqueous phase is extracted 3 times with ethyl acetate, the combined organic phases are washed with brine, dried with Na2SO4 and the solvent is removed by evaporation under reduced pressure. The crude is purified by automated flash chromatography (Isolera One, Biotage®) with mobile phase 99:1 to 90:10 DCM:Et2O in 15 column volumes. The desired product is obtained as a colourless oil (isolated yield 70%).

[0123] Step c: (R)-4-(pyrrolidin-3-yloxy)benzonitrile or (S)- 4-(pyrrolidin-3-yloxy)-benzonitrile (4)

[0124] An enantiomer of molecule 3 is dissolved in 10 mL of anhydrous DCM and the mixture is cooled in an ice bath. HCI (4 M in 1 ,4-dioxane) is added dropwise, the cold bath is removed, and the reaction is allowed to take place at room temperature for 3 hours. Once the reaction is complete, the solvent is removed by reduced pressure, the residue obtained is dissolved with ethyl acetate. The organic phase is washed with 1 M NaOH, the aqueous phase is extracted 3 times with EtOAc. The combined organic phases are washed with brine, dried with Na2SO4 and the solvent removed by reduced pressure. The product is obtained as an amber oil and is used in the following steps without further purification (quantitative yield).

[0125] Step d: 1 -methyl-1 H-indazole-3-carbonyl chloride (5)

[0126] 1 -methyl-1 H-indazole-3-carboxylic acid (1 .0 eq) is dissolved in 10 mL of DCM, a drop of DMF is added and the mixture is cooled with an ice bath. Thionyl chloride is added dropwise, the ice bath is removed, and when the mixture reaches room temperature it is heated under reflux. At the completion of the reaction the solvent is removed under reduced pressure and the residue is stripped 3 times with Et20. The product is obtained as a white foamy solid (quantitative yield) and is used in the following steps without further purification.

[0127] Step e: (R)-4-((1 -(1 -methyl-1 H-indazole-3-carbonyl)pyrrolidin-3-yl)oxy)benzonitrile or (S)-4-((1 -(1 -methyl-1 H-indazole-3-carbonyl)pyrrolidin-3-yl)oxy)benzonitrile (6)

[0128] One of the two enantiomers obtained by step c (1 .0 eq) and TEA (1 .5 eq), are dissolved in 5 ml of DCM. The mixture is cooled with an ice bath and a solution of acyl chloride obtained in step d is added dropwise in 5 ml of DCM. The mixture is reacted at room temperature for 18 hours. The reaction is then quenched by adding 1 M aq. NaOH, the aqueous phase is extracted 3 times with DCM, the combined organic phases are washed with brine, dried with Na2SO4 and the solvent removed under reduced pressure. The crude is purified by automated flash chromatography (Isolera One, Biotage®) with mobile phase 90:10 to 80:20 DCM:Et2O in 10 column volumes. The desired product is obtained as a white solid (isolated yield = 65%).

[0129] Step f: (R)-N'-hydroxy-4-((1 -(1 -methyl-1 H-indazole-3-carbonyl)pyrrolidin-3-yl)oxy) benzimidamide or (S)-N'-hydroxy-4-((1 -(1 -methyl-1 H-indazole-3-carbonyl)pyrrolidin-3- yl)oxy) benzimidamide (7) One of the two enantiomers obtained by step e (1 eq) is dissolved in 15 ml of EtOH. NH2OH HCI (3 eq) and NaHCOs (4 eq) are added in this order. The mixture is heated under reflux for 18h. At the end, the solvent is removed under reduced pressure, the residue obtained is washed with cold water and dried in air for 1 hour. The desired product is obtained as a white solid (yield 99%) and is used in the subsequent steps without further purification.

[0130] Step g : (3 -(4- (1 ,2,4-oxadiazol-3-yl)phenoxy)pyrrolidin-1 -y I) (1 -methyl-1 H-indazol-3- yl)methanone (8)

[0131] One of the two enantiomers obtained by step f (1 .0 eq) is dissolved in DMF and the mixture is brought to 0°C. TEA is added (1 .1 eq) and the appropriate acyl chloride or acetic anhydride is added drop by drop (1 .1 eq). The cold bath is removed, and it is left to react at room temperature for 30 minutes, then heated at reflux for 18 hours. At the end, the mixture is allowed to cool to room temperature and is diluted with water. The aqueous phase is extracted 3 times with EtOAc, the organic phase is washed with brine, dried with Na2SO4 and the solvent is removed under reduced pressure. The crude is purified by automated flash chromatography ((Isolera One, Biotage®) with mobile phase 90:10 to 70:30 of DCM:Et2O in 10 column volumes. The desired product is obtained as a white solid (isolated yield = 87%).

[0132] EXAMPLE 1 (S)-(3-(4-(5-(methoxymethyl)-1 ,2,4-oxadiazol-3-yl)phenoxy)pyrrolidin-1 - yl)(1 -methyl-1 H-indazol-3-yl)methanone

[0133] The intermediate 7-(S) is reacted with methoxyacetyl chloride (R1= -CH2-; R2= - OMe) according to the procedure described in step g. The product is obtained as a white solid. Yield 36 %.1H NMR (400 MHz, DMSO) 5 8.21 - 8.12 (m, 1 H), 8.02 - 7.91 (m, 2H), 7.81 - 7.68 (m, 1 H), 7.51 - 7.42 (m, 1 H), 7.30 - 7.23 (m, 1 H), 7.23 - 7.12 (m, 2H), 5.26 (d, J = 24.3 Hz, 1 H), 4.81 (d, J = 4.7 Hz, 2H), 4.33 - 4.21 (m, 1 H), 4.12 (d, J = 12.7 Hz, 3H), 4.09 - 3.62 (m, 3H), 3.42 (d, J = 4.1 Hz, 3H), 2.41 - 2.11 (m, 2H).13C NMR (101 MHz, DMSO) 5 176.49, 167.23, 159.50, 140.15, 137.58, 128.88, 126.59, 123.61 , 122.13, 118.54, 116.06, 110.14, 76.95, 74.51 , 64.42, 58.75, 53.80, 52.09, 46.47, 44.62, 36.04, 31 .48, 28.67. C23H24N5O4+[M+H]+Calc= 434,1823; Found= 434,1824. Purity > 97%.

[0134] EXAMPLE 2 (R)-(3-(4-(5-(methoxymethyl)-1 ,2,4-oxadiazol-3-yl)phenoxy)pyrrolidin-1 - yl)(1 -methyl-1 H-indazol-3-yl)methanone

[0135] The intermediate 7- (R) is made to react with methoxy acetylchloride (R1= -CH2-; R2= -OMe) according to the procedure described in the step g. The product is obtained as a white solid. Yield 34%.1H NMR (400 MHz, DMSO) 5 8.21 - 8.12 (m, 1 H), 8.02 - 7.91 (m, 2H), 7.81 - 7.68 (m, 1 H), 7.51 - 7.42 (m, 1 H), 7.30 - 7.23 (m, 1 H), 7.23 -

[0136] 7.12 (m, 2H), 5.26 (d, J = 24.3 Hz, 1 H), 4.81 (d, J = 4.7 Hz, 2H), 4.33 - 4.21 (m, 1 H),

[0137] 4.12 (d, J= 12.7 Hz, 3H), 4.09 - 3.62 (m, 3H), 3.42 (d, J = 4.1 Hz, 3H), 2.41 - 2.11 (m, 2H).13C NMR (101 MHz, DMSO) 5 176.49, 167.23, 159.50, 140.15, 137.58, 128.88, 126.59, 123.61 , 122.13, 118.54, 116.06, 110.14, 76.95, 74.51 , 64.42, 58.75, 53.80, 52.09, 46.47, 44.62, 36.04, 31.48, 28.67. C23H24NsO4+[M+H]+Calc= 434,1823; Found= 434,1824. Purity > 97%.

[0138] EXAMPLE 3 - (S)-(3-(4-(5-methyl-1 ,2,4-oxadiazol-3-yl)phenoxy)pyrrolidin-1 -yl)(1 - methyl-1 H-indazol-3-yl)methanone

[0139] The 7-(S) intermediate is reacted with acetic anhydride according to the procedure described in step g. The product is obtained as a white solid. Yield 28%.1H NMR (400 MHz, DMSO) 5 8.17 (t, J = 1 .1 Hz, 1 H), 7.99 - 7.89 (m, 2H), 7.73 (t, 1 H), 7.52 - 7.43 (m, 1 H), 7.27 (t, J= 0.9 Hz, 1 H), 7.22 - 7.12 (m, 2H), 5.26 (d, J= 24.3 Hz, 1 H), 4.33 - 4.21 (m, 1 ,5H), 4.13 (d, = 12.6 Hz, 3H), 4.09 - 3.99 (m, 0,5H), 3.99 - 3.79 (m, 1 ,5H), 3.75 - 3.64 (m, 0,5H), 2.65 (d, J = 4.5 Hz, 3H), 2.41 - 2.16 (m, 2H).13C NMR (101 MHz, DMSO) 5 177.60, 167.70, 161.80, 161.70, 159.79, 140.63, 140.61 , 138.06, 129.20, 127.07, 124.05, 122.61 , 119.40, 116.53, 116.46, 110.62, 77.40, 74.95, 54.29, 52.57, 46.96, 45.11 , 36.52, 31.96, 29.15, 12.48. C22H22N5O3+[M+H]+Calc= 404,1723; Found= 404,1712. Purity > 97%.

[0140] EXAMPLE 4 - (R)-(3-(4-(5-methyl-1 ,2,4-oxadiazol-3-yl)phenoxy)pyrrolidin-1 -yl)(1 - methyl-1 H-indazol-3-yl)methanone

[0141] The intermediate 7-(R) is reacted with acetic anhydride according to the procedure described in step g. The product is obtained as a white solid. Yield 30%.1H NMR (400 MHz, DMSO) 5 8.17 (t, = 1 .1 Hz, 1 H), 7.99 - 7.89 (m, 2H), 7.73 (t, 1 H), 7.52 - 7.43 (m, 1 H), 7.27 (t, J= 0.9 Hz, 1 H), 7.22 - 7.12 (m, 2H), 5.26 (d, J= 24.3 Hz, 1 H), 4.33 - 4.21 (m, 1 ,5H), 4.13 (d, = 12.6 Hz, 3H), 4.09 - 3.99 (m, 0,5H), 3.99 - 3.79 (m, 1 ,5H), 3.75 - 3.64 (m, 0,5H), 2.65 (d, J = 4.5 Hz, 3H), 2.41 - 2.16 (m, 2H).13C NMR (101 MHz, DMSO) 5 177.60, 167.70, 161.80, 161.70, 159.79, 140.63, 140.61 , 138.06, 129.20, 127.07, 124.05, 122.61 , 119.40, 116.53, 116.46, 110.62, 77.40, 74.95, 54.29, 52.57, 46.96, 45.11 , 36.52, 31.96, 29.15, 12.48. C22H22N5O3+[M+H]+Calc= 404,1723; Found= 404,1712. Purity > 97%.

[0142] EXAMPLE 5 - (S)-(3-(4-(5-butyl-1 ,2,4-oxadiazol-3-yl)phenoxy)pyrrolidin-1 -yl)(1 - methyl-1 H-indazol-3-yl)methanone

[0143] The intermediate 7-(S) is reacted with valerioyl chloride according to the procedure described in step g. The product is obtained as a white solid. Yield 25%.1H NMR (600 MHz, CDCh) 5 8.36 (d, J= 8.2 Hz, 1 H), 8.01 (d, J= 8.5 Hz, 2H), 7.45 - 7.37 (m, 2H), 7.30 - 7.23 (m, 1 H), 6.98 (d, J= 8.4 Hz, 2H), 5.09 (s, 1 H), 4.52 - 3.80 (m, 7H), 2.92 (t, J= 7.6 Hz, 2H), 2.46 - 2.18 (m, 2H), 1 .84 (p, J= 7.6 Hz, 2H), 1 .45 (h, J= 7.4 Hz, 2H), 0.97 (t, J = 7.4 Hz, 3H).13C NMR (151 MHz, CDCh) 5 179.87, 167.83, 162.45, 159.40, 140.42, 138.30, 129.13, 126.76, 124.57, 123.25, 122.37, 119.89, 115.68, 108.82, 65.87, 54.32, 52.53, 46.86, 44.87, 36.10, 33.43, 32.28, 28.70, 26.35, 22.21 , 13.59. C25H28N5O3+[M+H]+Calc: 446,2187 Found= 446,2176. Purity > 97%.

[0144] EXAMPLE 6 - (R)-(3-(4-(5-butyl-1 ,2,4-oxadiazol-3-yl)phenoxy)pyrrolidin-1 -yl)(1 - methyl-1 H-indazol-3-yl)methanone

[0145] The intermediate 7-(R) is reacted with valerioyl chloride according to the procedure described in step g. The product is obtained as a white solid. Yield 32%.1H NMR (600 MHz, CDCh) 5 8.36 (d, J= 8.2 Hz, 1 H), 8.01 (d, J= 8.5 Hz, 2H), 7.45 - 7.37 (m, 2H), 7.30 - 7.23 (m, 1 H), 6.98 (d, J= 8.4 Hz, 2H), 5.09 (s, 1 H), 4.52 - 3.80 (m, 7H), 2.92 (t, J= 7.6 Hz, 2H), 2.46 - 2.18 (m, 2H), 1 .84 (p, J= 7.6 Hz, 2H), 1 .45 (h, J= 7.4 Hz, 2H), 0.97 (t, J = 7.4 Hz, 3H).13C NMR (151 MHz, CDCh) 5 179.87, 167.83, 162.45, 159.40, 140.42, 138.30, 129.13, 126.76, 124.57, 123.25, 122.37, 119.89, 115.68, 108.82, 65.87, 54.32, 52.53, 46.86, 44.87, 36.10, 33.43, 32.28, 28.70, 26.35, 22.21 , 13.59. C25H28N5O3+[M+H]+Calc: 446,2187 Found= 446,2176. Purity > 97%.

[0146] EXAMPLE 7 - (S)-(1 -methyl-1 H-indazol-3-yl)(3-(4-(5-(tetrahydrofuran-3-yl)-1 ,2,4- oxadiazol-3-yl)phenoxy)pyrrolidin-1 -yl)methanone

[0147] The intermediate 7-(S) is reacted with tetrahydrofuran-3-carbonyl chloride according to the procedure described in step g. The product is obtained as a white solid. Yield 34%.1H NMR (400 MHz, CDCh) 5 8.36 (d, J= 8.2 Hz, 1 H), 8.01 (d, 2H), 7.47 - 7.36 (m, 2H), 7.31 - 7.22 (m, 1 H), 6.98 (d, J = 8.5 Hz, 2H), 5.09 (s, 1 H), 4.53 - 3.86 (m, 11 H), 3.74 (q, J = 6.3 Hz, 1 H), 2.50 - 2.15 (m, 4H).13C NMR (101 MHz, CDCh) 5 180.22, 168.08, 162.56, 159.63, 140.53, 138.40, 129.31 , 126.88, 124.67, 123.36, 122.49, 119.72, 115.81 , 108.94, 71.40, 68.37, 54.37, 46.85, 44.99, 37.14, 36.22, 31 .18. C25H26N5O4+[M+H]+Calc= 460,1985; Found= 460,1975. Purity > 97%.

[0148] EXAMPLE 8 - (R)-(1 -methyl-1 H-indazol-3-yl)(3-(4-(5-(tetrahydrofuran-3-yl)-1 ,2,4- oxadiazol-3-yl)phenoxy)pyrrolidin-1 -yl)methanone

[0149] The intermediate 7-(R) is reacted with tetrahydrofuran-3-carbonyl chloride according to the procedure described in step g. The product is obtained as a white solid. Yield 28%.1H NMR (400 MHz, CDCh) 5 8.36 (d, J= 8.2 Hz, 1 H), 8.01 (d, 2H), 7.47 - 7.36 (m, 2H), 7.31 - 7.22 (m, 1 H), 6.98 (d, J = 8.5 Hz, 2H), 5.09 (s, 1 H), 4.53 - 3.86 (m, 11 H), 3.74 (q, J = 6.3 Hz, 1 H), 2.50 - 2.15 (m, 4H).13C NMR (101 MHz, CDCh) 5 180.22, 168.08, 162.56, 159.63, 140.53, 138.40, 129.31 , 126.88, 124.67, 123.36, 122.49, 119.72, 115.81 , 108.94, 71.40, 68.37, 54.37, 46.85, 44.99, 37.14, 36.22, 31 .18. C25H26N5O4+[M+H]+Calc= 460,1985; Found= 460,1975. Purity > 97%.

[0150] EXAMPLE 9 - (S)-(3-(4-(5-(2-cyclohexylethyl)-1 ,2,4-oxadiazol-3- yl)phenoxy)pyrrolidin-1 -yl) (1 -methyl-1 H-indazol-3-yl)methanone

[0151] The intermediate 7-(S) is reacted with 3-cyclohexylpropanoyl chloride according to the procedure described in step g. The product is obtained as a white solid. Yield 27%.1H NMR (400 MHz, CDCh) 5 8.37 (d, J = 8.2 Hz, 1 H), 8.01 (d, 2H), 7.47 - 7.35 (m, 2H), 7.30 - 7.27 (m, 1 H), 5.10 (s, 1 H), 4.54 - 3.88 (m, 7H), 2.93 (t, 2H), 2.50 - 2.14 (m, 2H), 1.91 - 1 .54 (m, 13H), 0.97 (td, J= 12.0, 3.1 Hz, 2H).13C NMR (101 MHz, CDCh) 5 180.31 , 167.96, 162.59, 159.53, 140.56, 138.46, 129.27, 126.90, 124.71 , 123.41 , 122.51 , 120.04, 115.82, 108.94, 77.36, 37.29, 36.24, 34.18, 32.99, 26.60, 26.28, 24.39. C29H34N5O3+[M+H]+Calc= 500,2662; Found= 500,2657. Purity > 97%.

[0152] EXAMPLE 10 - (R)-(3-(4-(5-(2-cyclohexylethyl)-1 ,2,4-oxadiazol-3- yl)phenoxy)pyrrolidin-1 -yl) (1 -methyl-1 H-indazol-3-yl)methanone

[0153] The intermediate 7-(R) is reacted with 3-cyclohexylpropanoyl chloride according to the procedure described in step g. The product is obtained as a white solid. Yield 35%.1H NMR (400 MHz, CDCh) 5 8.37 (d, J = 8.2 Hz, 1 H), 8.01 (d, 2H), 7.47 - 7.35 (m, 2H), 7.30 - 7.27 (m, 1 H), 5.10 (s, 1 H), 4.54 - 3.88 (m, 7H), 2.93 (t, 2H), 2.50 - 2.14 (m, 2H), 1.91 - 1 .54 (m, 13H), 0.97 (td, J= 12.0, 3.1 Hz, 2H).13C NMR (101 MHz, CDCh) 5 180.31 , 167.96, 162.59, 159.53, 140.56, 138.46, 129.27, 126.90, 124.71 , 123.41 , 122.51 , 120.04, 115.82, 108.94, 77.36, 37.29, 36.24, 34.18, 32.99, 26.60, 26.28, 24.39. C29H34N5O3+[M+H]+Calc= 500,2662; Found= 500,2657. Purity > 97%.

[0154] EXAMPLE 11 - (S)-(3-(4-(5-cyclohexyl-1 ,2,4-oxadiazol-3-yl)phenoxy)pyrrolidin-1 - yl)(1 -methyl-1 H-indazol-3-yl)methanone

[0155] The intermediate 7-(S) is reacted with cyclohexancarbonyl chloride according to the procedure described in step g. The product is obtained as a white solid. Yield 22%.1H NMR (400 MHz, CDCh) 58.37 (d, J= 8.2 Hz, 1 H), 8.01 (d, J= 8.6 Hz, 2H), 7.47 - 7.37 (m, 2H), 7.31 - 7.23 (m, 1 H), 6.98 (d, J = 2.0 Hz, 2H), 5.09 (s, 1 H), 4.57 - 3.98 (m, 7H), 2.99 (tt, = 11 .3, 3.7 Hz, 1 H), 2.37 - 2.33 (m, 2H), 2.17 - 2.06 (m, 2H), 1 .92 - 1 .81 (m, 2H), 1 .78 - 1 .62 (m, 2H), 1 .50 - 1 .17 (m, 4H).13C NMR (101 MHz, CDCh) 5 182.94, 167.83, 162.58, 159.48, 140.55, 138.45, 129.28, 126.89, 124.70, 123.40, 122.50, 120.17, 115.79, 108.94, 77.36, 38.29, 36.55, 36.25, 31.38, 30.42, 29.84, 25.68, 25.55. C27H3oN503+[M+H]+Calc= 472,2349; Found= 472,2342. Purity > 97%.

[0156] EXAMPLE 12 - (R)-(3-(4-(5-cyclohexyl-1 ,2,4-oxadiazol-3-yl)phenoxy)pyrrolidin-1 - yl)(1 -methyl-1 H-indazol-3-yl)methanone

[0157] The intermediate 7-(R) is reacted with cyclohexancarbonyl chloride according to the procedure described in step g. The product is obtained as a white solid. Yield 27%.1H NMR (400 MHz, CDCh) 58.37 (d, J= 8.2 Hz, 1 H), 8.01 (d, J= 8.6 Hz, 2H), 7.47 - 7.37 (m, 2H), 7.31 - 7.23 (m, 1 H), 6.98 (d, J = 2.0 Hz, 2H), 5.09 (s, 1 H), 4.57 - 3.98 (m, 7H), 2.99 (tt, J = 11 .3, 3.7 Hz, 1 H), 2.37 - 2.33 (m, 2H), 2.17 - 2.06 (m, 2H), 1 .92 - 1 .81 (m, 2H), 1 .78 - 1 .62 (m, 2H), 1 .50 - 1 .17 (m, 4H).13C NMR (101 MHz, CDCh) 5 182.94, 167.83, 162.58, 159.48, 140.55, 138.45, 129.28, 126.89, 124.70, 123.40, 122.50, 120.17, 115.79, 108.94, 77.36, 38.29, 36.55, 36.25, 31.38, 30.42, 29.84,

[0158] 25.68, 25.55. C27H3oN503+[M+H]+Calc= 472,2349; Found= 472,2342. Purity > 97%.

[0159] EXAMPLE 13 - (S)-1 -(5-(3-(4-(5-methyl-1 ,2,4-oxadiazol-3-yl)phenoxy)pyrrolidin-1 - carbonyl)-1 H-benzo[d] [1 ,2 ,3]triazol-1 -yl)ethane-1 -one The intermediate 7-(S)-4-((1 -(1 H-benzo[d][1 ,2,3]triazol-5-carbonyl)pyrrolidin-3-yl)oxy)-

[0160] N'-hydroxybenzimidammide is reacted with acetic anhydride according to the procedure described in step g. The product is obtained as a nearly white solid. Yield

[0161] 25%.1H NMR (600 MHz, CDCh) 58.39 - 8.23 (m, 1 H), 7.99 (dd, J= 48.5, 8.2 Hz, 1 H),

[0162] 7.93 - 7.82 (m, OH), 7.80 - 7.62 (m, 1 H), 6.96 (dd, J = 67.9, 8.4 Hz, 2H), 5.17 - 4.92 (m, 1 H), 4.10 - 3.52 (m, 4H), 3.02 (d, J = 9.1 Hz, 3H), 2.63 (d, = 15.5 Hz, 3H), 2.51

[0163] - 2.13 (m, 4H). C22H2iN6O4+[M+H]+Calc: 433,1619 Found= 433,1611 . Purity > 97%.

[0164] EXAMPLE 14 - (R)-1 -(5-(3-(4-(5-methyl-1 ,2,4-oxadiazol-3-yl)phenoxy)pyrrolidin-1 - carbonyl)-1 H-benzo[d] [1 ,2 ,3]triazol-1 -yl)ethane-1 -one

[0165]

[0166] (S)-4-((1 -(1 H-benzo[d][1 ,2,3]triazol-5-carbonyl)pyrrolidin-3-yl)oxy)N'- hydroxybenzimidammide is reacted with acetic anhydride according to the procedure described in step g. The product is obtained as a nearly white solid. Yield 21%.1H NMR (600 MHz, CDCh) 5 8.39 - 8.23 (m, 1 H), 7.99 (dd, J= 48.5, 8.2 Hz, 1 H), 7.93 - 7.82 (m, OH), 7.80 - 7.62 (m, 1 H), 6.96 (dd, J= 67.9, 8.4 Hz, 2H), 5.17 - 4.92 (m, 1 H), 4.10 - 3.52 (m, 4H), 3.02 (d, J= 9.1 Hz, 3H), 2.63 (d, J= 15.5 Hz, 3H), 2.51 - 2.13 (m, 4H). C22H2iN6O4+[M+H]+Calc: 433,1619 Found= 433,1611 . Purity > 97%.

[0167] Method for evaluating the enantioselectivity of the synthetic process.

[0168] High-Performance Liquid Chromatography (HPLC), also known as high-performance liquid chromatography, is an analytical technique used to separate, identify, and quantify the components of a complex mixture. In HPLC, a mixture of compounds is passed through a separation column, which may contain a solid or liquid stationary phase. The stationary phase interacts with the components of the mixture based on their chemical properties, such as affinity for certain functional groups or polarity. The separation occurs through the interaction between the stationary phase and the mobile phase, which is a solvent or a mixture of solvents. The mobile phase is pumped through the column at a high pressure, which allows for a rapid and efficient separation of the components of the mixture. As the components of the mixture pass through the column, they separate based on their interaction with the stationary phase. Components that interact more strongly with the stationary phase will take longer to pass through the column than those that interact less strongly. [31 ,32] HPLC applied to chiral molecules, known as chiral HPLC or chiral high-performance liquid chromatography, is an analytical technique used to separate and analyse chiral molecules, that is, molecules that exist in two enantiomeric forms, or stereoisomers, that are mirror images of each other. Chiral HPLC is based on the use of a chiral separation column, which contains a chiral stationary phase. The chiral stationary phase is a material that has selectivity towards one of the two enantiomers of the molecule being analyzed. This selectivity can be achieved through the use of a chiral ligand, such as a cellulose derivative or a cyclodextrin, which interacts specifically with one of the two enantiomers. During the analysis, the mixture containing the two enantiomers is passed through the chiral separation column, using an appropriate mobile phase. Because the chiral stationary phase interacts differently with the two enantiomers, they separate along the column, with one of the enantiomers being retained longer than the other. Once separated, the two enantiomers are detected by a detector, such as a UV / Vis detector or a fluorescence detector, which generates a signal proportional to the concentration of the individual enantiomers. Chiral HPLC finds application in various fields, such as medicinal chemistry and the analysis of natural compounds.

[0033]

[0169] For the determination of enantiomeric purity, a Chiralcel-OD analytic 4.6 x 50 mm column was used. The separation method was isocratic. A mixture of 90% Acetonitrile (ACN) and 10% H2O and 0.1 % Formic Acid was used as the mobile phase. The flow rate was set to 1 mL / min. The wavelength used for UV analysis was 254 nm. The injected volume was 10 pL. The samples were analyzed by UV detector and by circular dichroism (CD) detector. The time of each chromatographic run was 10 minutes.

[0033]

[0170] The compounds used for the analysis were 022-^ and DZ2-(S). To exclude the possibility of racemate formation during the synthetic process, the retention times of the two molecules were evaluated. For this evaluation, two solutions of the two compounds used were prepared at known concentrations: DZ2-(R) [1.26 mM] and DZ2 -(S) [1 .36 mM],

[0171] In vitro assays Measurement of YAP:TEAD4 transcriptional complex dissociation by fluorescence anisotropy

[0172] Fluorescence anisotropy is an effective instrumental method to study protein-protein interactions (PPI) when one of the two entities is sterically smaller and exhibits intrinsic fluorescence or through a conjugated probe. When a peptide / molecule labeled with a fluorescent probe is excited by polarized light, the light emitted by it shows a lower degree of polarization the greater the rotational freedom of the molecule. Fluorescence anisotropy therefore depends on the mass of the molecule, which is inversely proportional to its rotational freedom. For this reason, when the labeled entity (typically < 5kDa) is excited with polarized light, it emits depolarized light. On the contrary, when it interacts with a macromolecule with a higher molecular weight (typically > 10 kDa) the rotational freedom of the formed complex decreases, increasing the intensity of the emitted light which remains polarized in the direction of excitation.

[0173] To perform this experiment, a derivative of the peptide 6 analogous to the Q Loop of YAP was synthesized as per Furet et al, 2019

[0034] , subsequently modified to realize the N-term docking of tetraethyl rhodamine for the detection of fluorescence. The binding domain of the TEAD4 protein (YBD) was obtained by bacterial transformation of E. coli BL21 (DE3) (Agilent Technologies) with a plasmid encoding the His-tagged TEAD4 sequence (pET15b-His6-hTEAD4 YBD, designed by the authors and biosynthesized by Fisher Scientific), and purified by affinity and size exclusion chromatography (Akta Prime, Cytiva). The chemical, physical and structural properties of the obtained protein were evaluated by gel electrophoresis, fluorescence emission, circular dichroism and high-resolution mass spectrometry.

[0174] P-W(7CI)-R-L-R-K-Nle-P-D-S-F-Nala-K-P-P [Peptide 6 from Furet et al] (SEQ ID NO 10)

[0175] Wherein W(7CI) is 7-chloro tryptophan

[0176] - N(ala) is 1 naphthyl alanine

[0177] - N(le) is Norleucine

[0178] Base sequence of hTEAD4-YBD fragment: (SEQ ID NO 11)

[0179] 5’ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCG GCAGCCATATGCGTAGCGTGGCGAGCAGCAAGCTGTGGATGCTGGAGTTCAGC

[0180] GCGTTTCTGGAACAGCAACAGGACCCGGATACCTACAACAAACACCTGTTCGTG

[0181] CACATCGGTCAAAGCAGCCCGAGCTACAGCGACCCGTATCTGGAGGCGGTTGA

[0182] CATCCGTCAGATTTATGATAAGTTCCCGGAGAAGAAAGGTGGCCTGAAAGACCT

[0183] GTTCGAACGTGGCCCGAGCAACGCGTTCTTTCTGGTGAAGTTCTGGGCGGACC TGAACACCAACATCGAGGATGAAGGTAGCAGCTTTTACGGCGTTAGCAGCCAAT

[0184] ATGAGAGCCCGGAAAACATGATCATTACCTGCAGCACCAAAGTGTGCAGCTTCG GCAAGCAGGTGGTTGAGAAAGTTGAGACCGAATACGCGCGTTATGAAAACGGC CACTACAGCTATCGTATTCACCGTAGCCCGCTGTGCGAATACATGATCAACTTC

[0185] ATTCACAAGCTGAAACACCTGCCGGAGAAGTATATGATGAACAGCGTGCTGGAA AACTTTACCATCCTGCAAGTGGTTACCAACCGTGACACCCAGGAGACCCTGCTG TGCATTGCGTACGTGTTTGAAGTTAGCGCGAGCGAACACGGTGCGCAACACCA CATTTATCGTCTGGTTAAAGAATAA- 3’

[0186] To perform the assay, aliquots with increasing concentrations of YAP:TEAD4 dissociative compounds previously solubilized in dimethyl sulfoxide (maximum final concentration 5.9%) are added to a solution of 120 nM recombinant protein and peptide at equal concentration. The cuvette is excited with a polarized 550 nm laser, and the emission is recorded in the range 570-620 nm. The experiment is performed with a FluoroMax-3 spectrofluorimeter (Horiba). The Kd of each tested inhibitor was derived from the previously measured direct Kd of the YAP:TEAD4 complex (250 nM).

[0187] Table 2. Results of anisotropy experiments between the TEAD4 protein: peptide complex 6 and the compounds of Formula (I) of the examples.

[0188] As reported in Table 2, the results of the anisotropy measurements performed demonstrate that the compounds illustrated in the present invention bind with low- micromolar affinity to the YAP-binding domain (YBD) of the TEAD4 protein. Furthermore, the fluorescence anisotropy experiments surprisingly demonstrate that the molecules act in a dissociative manner towards the YAP:TEAD4 transcriptional complex, binding the contact interface 3. In particular, it is verified that the compounds of Formula (I) are able to displace the peptide 6 that binds the interface 3, and this reasonably means that also the compounds of Formula (I) bind to the same interface 3. From the results of the cell survival experiments reported below, it can be deduced that also in tumor cells, the compounds of formula (I) act with this mechanism of action. Cellular assays

[0189] Reagents, Cell Lines and Bacterial Strains

[0190] The HT29 human colorectal carcinoma cell lines HT29 (ATCC No. HTB-38) and HCT116 (ATCC No. CCL-247) were grown in Dulbecco’s modified Eagle medium (DMEM) (Euroclone, Devon, UK) supplemented with 10% heat-inactivated foetal bovine serum and 1% Pen / Strep (Euroclone). The human ovarian carcinoma cell lines A2780 (ECACC No. 93112519) and A2780 / CP (ECACC No. 93112517) were grown in RPM1 1640 medium supplemented with 10% heat-inactivated (30 minutes at 56°C) foetal bovine serum, 1% Pen / Strep (Euroclone). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2.

[0191] Cell growth assays

[0192] 24 hours after seeding cells in 24-well plates, cytotoxicity assays were performed on the cell lines by adding 40 pM of each inhibitor of the formation of the YAP:TEAD4 transcriptional complex. A more in-depth evaluation of the activity of the compounds was developed through cytotoxicity curves at 5, 10, 20 and 40 pM. All experiments were performed in triplicate. 72 hours after treatment, the culture medium was removed, and the cell monolayer was fixed with methanol and stained with a solution of 0.2% Crystal Violet (Sigma Aldrich) in 20% methanol. The incorporated dye was solubilized in acidified isopropanol. The absorbance was determined spectrophoto metrically at 540 nm (TECAN GeniosPro). Percent cytotoxicity was calculated by comparing the absorbance of drug-exposed cultures with negative controls.

[0193] Examples of in vitro biological data against highly and moderately resistant colorectal cancer cell lines (HCT116 and HT29, respectively) and ovarian cancer cell lines sensitive to cisplatin (A2780).

[0194] As reported in the design section of the compounds, the two stereoisomers DZ1 -(S) (dextrorotatory isomer) and DZ1 -(R) (levorotatory isomer) are expected to be able to interact with interface 3 in a stereospecific manner, i.e. the dextrorotatory compound has a different spatial arrangement from the levorotatory one and therefore there may be a more or less favourable interaction. The data reported in Figure 4 highlight the existence of a different specificity.

[0195] The compounds found to be more active in an initial screening at 40 pM against CRC and OC cell lines compared to DZ1 -(S) and DZ1 -(R) (Figure 1) were subjected to a dose-response study at increasing concentrations up to 60 pM and the cytotoxicity expressed as percentage of cell growth inhibition on two colorectal tumor lines and two ovarian tumor lines (Figure 4 and Table 3).

[0196] Among these, the compounds DZ1 -(S), DZ3-(S) and DZ5-(S) showed dose-response cytotoxicity curves such as to be able to determine ICso values higher than 5-FU and verteporfin, reference compounds, but lower than D361 (CAS 1111036-42-0, racemic mixture of DZ1 -(S) and DZ1 -(R)), internal reference compound (Table 3), thus proving more effective. In particular, DZ3-(S), DZ5-(S) and DZ5-(R), in all cell lines, sometimes reached ICso values even lower than DZ1 -(S).

[0197] Table 3. ICso values (pM), the concentration that causes a 50% growth inhibition in treated cells compared to control cells after 72 hours of drug exposure on HT29, HCT116, A2780, A2780 / CP cell lines. Values are average values ± SD from two to four separate experiments performed in duplicate.

[0198] 1. CELL LINES

[0199] This series of experiments was performed on three tumor cell lines: HCT116, HT29, and A2780.

[0200] HCT 116 (ATCC #CCL-247) and HT29 (ATCC #HTB-38) cells are human colon cancer cells and were purchased from the American Type Culture Collection (ATCC) and cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) (#D6429, Sigma Aldrich) supplemented with 10% heat-inactivated foetal bovine serum (FBS) in 5% CO2at 37°C as recommended by the manufacturer. A2780 (ECACC No. 93112519) and A2780 / CP (ECACC No. 93112517) are ovarian tumor cells grown in modified RPMI-1640 medium containing 2 mM L-glutamine, 1 mM sodium pyruvate, 4500 mg / L glucose and foetal bovine serum to a final concentration of 10% in 5% CO2at 37°C.

[0201] 2. TREATMENTS

[0202] The cells under examination were seeded in 6-well plates at 50% confluence, about 500 thousand cells in a final volume of medium corresponding to 3 ml. Once adhered, they were treated by adding the compounds directly into the culture medium: DZ-5(S) 30pM, DZ1 -S 40 pM, and VERTEPORFIN 12 pM. For a treatment duration corresponding to 24h for the study of protein expression levels and 48h for the study of expression levels of the genes under examination.

[0203] 3. STUDY OF PROTEIN EXPRESSION LEVELS AND PHOSPHORYLATION STATUS THEREOF Cells were washed in ice-cold PBS and collected in RIPA buffer supplemented with protease and phosphatase inhibitors (#78442, Thermo scientific). Insoluble debris was removed by centrifugation at 14,000 rpm for 30 minutes at 4°C. After denaturation for 8 minutes at 96°C, samples were subjected to electrophoresis for separation of proteins based on molecular weight using polyacrylamide gel (#4561033, BIO-RAD) and subsequently transferred to nitrocellulose membrane using the Trans-Blot Turbo BIO-RAD instrument. Immunoblot analysis was performed using anti-YAP (cod. sc101199, Santa Cruz Biotechnology, dilution 1 :500), anti-pYAP (s397) (cod. 13619, Cell Signaling, dilution 1 :1000), anti-pYAP (s127) (cod. C66B5, Cell Signaling, 1 :1000 dilution), anti-a tubulin (clone 76199, Sigma Aldrich, dilution 1 :1250). Horseradish peroxidase-conjugated secondary antibodies (Anti-Rabbit A6667, Anti Mouse A5906, Sigma Aldrich) were used to detect the bound primary antibody. Immunocomplexes were visualized by enhanced chemiluminescence (Amersham ECL Prime Western Blotting Reagent) according to the manufacturer's instructions using the Imager instrument (GE AMERSHAM AI680). Band density was calculated using Imaged software.

[0204] 4. STUDY OF YAP AND ITS TARGET GENES EXPRESSION LEVELS

[0205] Total RNA from cultured cells was isolated with Purelink RNA Mini Kit (Life Technologies) according to the manufacturer's instructions and quantified using a NanoDrop ND-1000 instrument (Thermo Scientific). Purity was determined by measuring absorbance at 260 and 280 nm and calculating the A260 / 280 ratio.

[0206] DNase treatment and cDNA synthesis were performed from 1 pg total RNA using Maxima H(-) MasterMix (#M1682, Life Technologies), according to the manufacturer's instructions. Subsequently, quantification of transcript levels (YAP, CYR61 , CTGF and RPLP0, Table 1 ) was achieved by qRT-PCR experiments performed on the CFX96 Connect Real-Time System (Bio-Rad) using SsoAdvanced Universal SYBR® Green Supermix (Bio-Rad) and each sample was analyzed independently in triplicate. Specifically designed primers were used for qRT-PCR analysis, each at a final concentration of 400 nM (oligonucleotides used are listed below). qPCR primers

[0207] CYRS61 : FW: 5’-CCTTGTGGACAGCCAGTGTA-3’ SEQ ID NO 2

[0208] REV: 5’-ACTTGGGCCGGTATTTCTTC-3’ - SEQ ID NO 3

[0209] CTGF: FW: 5’-AGGAGTGGGTGTGTGACGA-3’ - SEQ ID NO 4

[0210] REV: 5’-CCAGGCAGTTGGCTCTAATC-3’ - SEQ ID NO 5

[0211] YAP: FW: 5’-GCACCTCTGTGTTTTAAGGGTCT-3’ - SEQ ID NO 6

[0212] REV: 5’-CAACTTTTGCCCTCCTCCAA-3’ - SEQ ID NO 7

[0213] RPLPO: FW: 5'-CCTTCTCCTTTGGGCTGGTCATCCA-3' - SEQ ID NO 8

[0214] REV: 5'-CAGACACTGGCAACATTGCGGACAC-3' - SEQ ID NO 9

[0215] The amplification reaction (95°C, 2 min; 40 cycles of 95°C, 5 sec and 60°C, 30 sec) was followed by a melting curve generated by increasing the temperature in small increments (from 65°C to 95°C, 0.5°C / s).

[0216] Relative quantification was performed according to the AACt method [Livak et al] using RPLPO as the reference gene.

[0217] RESULTS and DISCUSSION

[0218] YAP levels and phosphorylation changes

[0219] To evaluate the effects of our compounds on the biological responses of tumor cells, we selected two colon cancer cell lines (HCT116 and HT29) and one ovarian cancer cell line (A2780). Since the test compounds were designed on TEAD domain 3, the protein expression levels of TEAD in the cell lines described above were analyzed by immunoblot (Figure 5)

[0220] The cells were treated with the DZ compounds under examination for 24 hours at their ICso values and verteporfin was used as a reference molecule. In Figure 6 A, the expression levels of total Yes-associated protein (YAP) and two specific phosphorylations in position Ser397 and Ser127 are highlighted. We chose to examine the phosphorylation state of YAP because the physiological output of the Hippo Pathway depends on covalent modifications (phosphorylation / dephosphorylation) regulated by a kinase / phosphatase cascade aimed at regulating the activities of YAP and the transcriptional coactivator with PDZ- binding motif (TAZ; two homologs of Drosophila Yorkie [Yki])

[0035] . When YAP and TAZ are active (non-phosphorylated form), they translocate to the nucleus to bind the TEAD family of transcription factors (homologs of Drosophila Scalloped [Sd]) and induce the expression of a wide range of genes involved in cell proliferation, survival and migration, including CYR61 and CTGF.

[0036]

[0221] Figure 6B shows the histogram corresponding to the densitometric analysis with the values normalized with alpha tubulin. In particular, a “cells specific” trend of the response to treatment is observed. Since YAP phosphorylations in the residues Ser127 and Ser397 are considered indicators of the activity of YAP function and its subcellular localization, the ratio between pYAP-Ser397 and total YAP and pYAP- Ser127 and total YAP was also quantified (Figure 6C) and from this ratio it is observed that the compounds under examination determine an increase in both phosphorylations analyzed. gRT-PCR Results

[0222] In the qPCR Primers section, the sequences of the primers that have been specifically designed for the analysis are reported. The RPLPO gene was chosen as a “housekeeping” gene, useful for the normalization of the cDNA levels of all the analyzed samples (see figure 7). qRT-PCR analysis highlighted that the levels of endogenous mRNA of YAP, CTGF and CYR61 , known target genes of the YAP / TEAD complex, were changed after treatment with the selected compounds, compared to the untreated control.

[0223] In particular, as illustrated in Figure 8, the effect of verteporfin is confirmed in all the cell lines examined, with a reduction in the levels of mRNA corresponding to the target genes of YAP (CYR61 , CTGF). Our selected compounds, DZ1 -(S) and DZ5-(S), like the reference compound, cause a reduction in the expression levels of CYR61 and CTGF mRNAs in the colorectal cell lines, HT29 and HCT116, demonstrating that, at least in part, the observed growth-inhibiting effects may depend on the inhibition of the YAP / TEAD complex.

[0224] In A2780 (ovarian cancer) a reduction of CTGF mRNA is observed induced by the activity of DZ1 -(S) and DZ5-(S), but not a reduction in CYR61 expression.

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[0243]

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Claims

CLAIMS1. A compound of general formula (I)a salt or solvate thereof, wherein the General Formula (I) comprises an oxadiazole ring (A), a six-membered carbocyclic aromatic ring (B), a cycloalkyl amine (C) and a bicyclic system (D) and wherein:-R1- and -R3-, independently equal or different from each other, are:• direct bond (no atom between the ring (A) and R2or between the bicyclo (D) and R4);• alkylene, linear or branched, C1-4, such as: -CH2-, -C2H4-, -C3H6-, -C4H8-;• carbonyl (-(C=O)-);• difluoromethylene (-CF2-);-R2and -R4, independently equal or different from each other, are:• hydrogen (-H);• alkyl, linear or branched, C1-4, such as: -CH3, -C2H5, -C3H7, -C4H9;• alkyloxy, linear or branched, such as: -OCH3, -OC2H5, -OC3H7, OC4H9;• alkylthio, linear or branched, such as: -SCH3, -SC2H5, -SC3H7,- SC4H9;• cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl;• cyclooxyalkyl, 5 or 6-membered, such as: tetrahydrofuryl, tetrahydropyranyl;• phenyl, aryl, possibly substituted, such as: phenyl (-CeHs), tolyl (- C6H4-CH3);• heteroaromatic group containing nitrogen or oxygen, 5 or 6 membered, such as: pyridyl, pyrimidyl, furyl, pyrrolyl;Y1and Y2, each bound in an available position of the benzene ring (B), independently equal or different from each other, are:• hydrogen (-H):• alkyl, linear or branched, C1-4, such as: CH3, -C2H5, -C3H7, -C4H9;• alkyloxy, linear or branched, such as: -OCH3, -OC2H5, -OC3H7, OC4H9;• acyloxy, linear or branched, such as: -O(C=O)-CH3, -O(C=O)-C2H5, - O(C=O)-C3H7, -O(C=O)-C4H9;• carbonyl-alkyl, linear or branched, such as: -(C=O)-CH3, -(C=O)- C2H5, -(C=O)-C3H7, -(C=O)-C4H9;• alkylamino or dialkylamino, linear or branched, such as: -NH2, - NH(CH3), -NH(C2H5), -NH(C3H7), -NH(C4H9), -N(CH3)2, -N(CH3)C2H5, -N(CH3)C3H7, -N(CH3)C4H9, -N(C2H5)2, -N(C2H5)C3H7, -N(C2H5)C4H9, -N(C3H7)2;• cycloalkylamino, such as: -N(CH2)4, -N(CH2)s;• halogen, preferably: -F, -Cl, -Br;• halomethyl, preferably: -CF3, -CHF2, -CH2F, -CH2CI;X1, X2and X3in the bicyclical system (D), independently equal or different from each other, are: a C-H group, a nitrogen atom or an N-H group.

2. The compound according to the preceding claim wherein the bicyclo (D) is: an indene, an indole, an isoindole, an indazole, a benzimidazole, a benzodiazole, or a benzotriazole.

3. The compound according to the preceding claim wherein the bicyclo (D) is selected from one of the following structural formulas:

4. The compound according to the preceding claim wherein the bicyclic system (D) is linked to the nitrogen atom of the cycloalkyl amine (C), through an amide bond on one of the available positions of the benzenoid ring of the bicyclic system (D) and on one of the available positions of the cycloalkyl amine (C) through the substitution of a hydrogen atom.

5. The compound according to anyone of the preceding claims, which is selected from the following structural formulas:

6. The compound according to anyone of the preceding claims wherein the stereogenic carbon center in the cycloalkylamine ring (C) may have a specific configuration (R or S) or non-specific, i.e. partially or completely racemic.

7. The compound according to anyone of the preceding claims which is in pure or racemic or polymorphic partially or completely isomeric form and mixtures thereof.

8. The compounds according to anyone of the preceding claims which are stereoisomers, enantiomers or optical isomers, geometrical isomers, diastereoisomers and mixtures thereof.

9. The compositions comprising at least one compound according to anyone of claims 1 -8 and one or more pharmaceutically acceptable components.

10. The compositions according to the preceding claim further comprising one or more antitumor drugs preferably kinase inhibitors.

11. The compositions according to anyone of claims 9-10 in the form of tablets, capsules, pills or injectable formulations, prolonged or delayed release preparations, liposomal formulations.

12. The compounds or compositions according to anyone of the preceding claims for use as a medicament.

13. The compounds or compositions according to anyone of the preceding claims for use in the prevention, treatment and alleviation of symptoms of pathologies, disorders or conditions associated with pathologies wherein the transcription factor TEAD4 is over-expressed compared to a healthy subject, said pathologies being tumor pathologies, preferably malignant neoplastic pathologies of the ovary (OC), lung, breast, colorectal (CRC), pancreas, liver, skin, neuroglia, mesothelioma, melanoma and blood tumors.

14. The compounds or compositions for use according to the preceding claim wherein the administration occurs orally, parenterally, intravenously, intracutaneously.

15. A process for the preparation of a compound of Formula (I) according to anyone of claims 1 -8 which comprises the step of reacting a compound of Formula (II) and a compound of Formula (III):wherein Z1and Z2, equal or different from each other, are:• hydroxyl (-OH);• linear or branched acyloxy, such as: -O(C=O)-CH3, -O(C=O)-C2H5, -O(C=O)-C3H7, -O(C=O)-C4H9;• halogen, such as: -Cl, -Br, -I;• a group containing oxygen-sulphur bonds, such as -OMs, -OTs, -Ons; • a group containing oxygen-phosphorus bonds, optionally produced in situ; and R1, R2, R3, R4, X1, X2, X3, Y1, Y2are defined in claims 1 -8.

16. The process according to the preceding claim wherein the aromatic ring (B) is obtained from 4-hydroxybenzoic acid or 4-hydroxybenzaldehyde, also obtained from natural sources.

17. The process according to the preceding claim wherein the cycloaliphatic ring (C) is obtained from proline, hydroxyproline, pyrrolidine, also obtained from natural sources.

18. The process according to the preceding claim wherein the aromatic ring (D) is obtained from isatin, indole also obtained from natural sources.

Images