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WO2025126129A1 - Composés ayant une activité antitumorale ciblant la voie hippo - Google Patents

Composés ayant une activité antitumorale ciblant la voie hippo Download PDF

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Publication number
WO2025126129A1
WO2025126129A1 PCT/IB2024/062621 IB2024062621W WO2025126129A1 WO 2025126129 A1 WO2025126129 A1 WO 2025126129A1 IB 2024062621 W IB2024062621 W IB 2024062621W WO 2025126129 A1 WO2025126129 A1 WO 2025126129A1
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Prior art keywords
compounds
branched
linear
formula
yap
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Inventor
Maria Paola Costi
Alberto Venturelli
Fabrizio Roncaglia
Glauco Ponterini
Gaetano Marverti
Domenico D'ARCA
Dana ZAPPATERRA
Lorenzo TAGLIAZUCCHI
Daniele AIELLO
Giulia MALPEZZI
Maria Gaetana MOSCHELLA
Matteo SANTUCCI
Marco Mor
Laura SCALVINI
Gian Marco ELISI
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Universita degli Studi di Parma
Universita Degli Studi di Modena e Reggio Emilia
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Universita degli Studi di Parma
Universita Degli Studi di Modena e Reggio Emilia
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • 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.
  • Ovarian cancer affecting 5,200 women each year in Italy
  • CRC colorectal cancer
  • CRC second in Italy and Europe in terms of incidence, with 35,000 cases per year in Italy alone
  • Ovarian cancer affecting 5,200 women each year in Italy
  • CRC colorectal cancer
  • 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 [1 ].
  • 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.
  • TEAD protein function 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 [14].
  • flufenamic and niflumic acid derivatives include flufenamic and niflumic acid derivatives, ionic inhibitors of the TEAD lipoylation pocket, and YAP peptidomimetics [14].
  • 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 [15]. These include MYF-03- 69, K-975, and the scaffold 3-(2-benzylidenehydrazinyl) benzo[d] isothiazole [16].
  • 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.
  • 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 [18].
  • 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 [19].
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • FIG. 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.
  • 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.
  • compositions 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.
  • 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.
  • polymorphic or “polymorphism” refer to the same substance present in different crystalline forms.
  • 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.
  • 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.
  • 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.
  • molecular entities bearing the same atomic connectivity but different spatial orientation, without constituting pairs of non-superimposable mirror structures represent diastereoisomers (or diastereomers).
  • molecular entities characterized by the same molecular formula, but different connectivity are defined as constitutional isomers.
  • 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.
  • 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).
  • terminal groups (-R 2 and -R 4 ), 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
  • 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.
  • 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.
  • the stereogenic carbon center (*) in the cycloalkylamine ring (C), can have a specific (R or S) or nonspecific (partially or completely racemic) configuration.
  • 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.
  • compositions comprising the compounds of formula (I).
  • Such compositions may also comprise one or more carriers, vehicles, excipients or other pharmaceutically acceptable additives.
  • 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.
  • 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.
  • 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.
  • the compounds and/or compositions of the present description are used as inhibitors of the formation of the YAP: TEAD4 transcriptional complex [37].
  • 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.
  • 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.
  • tumors include ovarian cancer, colorectal cancer, mesothelioma, melanoma and blood cancer.
  • 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.
  • 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), m
  • 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:
  • oxygen-phosphorus leaving group (possibly produced in situ), such as that originated during the Mistunobu reaction [38], 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.
  • 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 (R 1 -R 2 -) by the target of Formula I, as shown in Scheme 2.
  • 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 [21] or with a second nitrile group [22], or with a carboxylic acid [23], or with an aldehyde [24].
  • Alternative methods exist that involve the use of aldoximes [25], instead of the aforementioned amidoximes [26].
  • 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.
  • 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.
  • the W substituent can be:
  • alkyloxy linear or branched
  • alkyloxy linear or branched
  • halogen such as: -Cl, -Br or -I
  • nitrogenous heteroaromatic for instance, but not limited to, pyridyl, imidazolyl, or bentrotriazolyl
  • 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 [27] and proceed to decarboxylation [28] 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 [29].
  • 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.
  • 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.
  • the 1 H spectra are acquired at 400.134 MHz and the 13 C 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).
  • 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.
  • 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.
  • Step a (R)-tert-buty ⁇ 3-((methylsulfonyl)oxy)pyrrolidine-1 -carboxylate or (S -terf-butyl 3-((methylsulfonyl)oxy)pyrrolidine-1 -carboxylate (1)
  • Step b S -tert-butyl-3-(4-cyanophenoxy)pyrrolidine-1 -carboxylate or (R)-tert-buty ⁇ -3- (4-cyanophenoxy)pyrrolidine-1 -carboxylate (3)
  • Step c (R)-4-(pyrrolidin-3-yloxy)benzonitrile or (S)- 4-(pyrrolidin-3-yloxy)-benzonitrile (4)
  • 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)
  • 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.
  • 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.
  • Step g (3 -(4- (1 ,2,4-oxadiazol-3-yl)phenoxy)pyrrolidin-1 -y I) (1 -methyl-1 H-indazol-3- yl)methanone (8)
  • 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.
  • 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
  • HPLC High-Performance Liquid Chromatography
  • 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.
  • HPLC applied to chiral molecules 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.
  • a chiral ligand such as a cellulose derivative or a cyclodextrin
  • 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. [33]
  • 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. [33]
  • 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],
  • 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.
  • PPI protein-protein interactions
  • 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.
  • the labeled entity typically ⁇ 5kDa
  • the rotational freedom of the formed complex decreases, increasing the intensity of the emitted light which remains polarized in the direction of excitation.
  • a derivative of the peptide 6 analogous to the Q Loop of YAP was synthesized as per Furet et al, 2019 [34], 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.
  • W(7CI) is 7-chloro tryptophan
  • N(ala) is 1 naphthyl alanine
  • 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).
  • DMEM Dulbecco modified Eagle medium
  • 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.
  • 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.
  • 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.
  • 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.
  • DZ3-(S), DZ5-(S) and DZ5-(R) in all cell lines, sometimes reached ICso values even lower than DZ1 -(S).
  • ICso values 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.
  • 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% CO 2 at 37°C as recommended by the manufacturer.
  • DMEM high-glucose Dulbecco's modified Eagle's medium
  • FBS heat-inactivated foetal bovine serum
  • A2780 ECACC No. 93112519
  • 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% CO 2 at 37°C.
  • 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.
  • RPLPO FW: 5'-CCTTCTCCTTTGGGCTGGTCATCCA-3' - SEQ ID NO 8
  • 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).
  • Relative quantification was performed according to the AACt method [Livak et al] using RPLPO as the reference gene.
  • YAP and TAZ 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.
  • Sd Drosophila Scalloped
  • 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
  • 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.
  • 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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Abstract

La présente invention concerne des composés de formule générale (I) qui agissent sur la voie Hippo et ont une activité antitumorale. L'invention concerne également des procédés de préparation desdits composés ainsi que leurs compositions pharmaceutiques.
PCT/IB2024/062621 2023-12-13 2024-12-13 Composés ayant une activité antitumorale ciblant la voie hippo Pending WO2025126129A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT202300026622 2023-12-13
IT102023000026622 2023-12-13

Publications (1)

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WO2025126129A1 true WO2025126129A1 (fr) 2025-06-19

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003092678A1 (fr) * 2002-04-30 2003-11-13 Schering Aktiengesellschaft Derives de l'imidazole 1-substitues comme inhibiteurs nos
WO2020070610A1 (fr) * 2018-10-01 2020-04-09 Pi Industries Ltd. Nouveaux oxadiazoles
WO2022037568A1 (fr) * 2020-08-17 2022-02-24 Betta Pharmaceuticals Co., Ltd Composés bicycliques, compositions et utilisation de ceux-ci
WO2022232088A1 (fr) * 2021-04-27 2022-11-03 Dana-Farber Cancer Institute, Inc. Inhibiteurs du facteur de transcription à domaine associé transcriptionnel amélioré (tead) et leurs utilisations
WO2023209088A1 (fr) * 2022-04-28 2023-11-02 Astrazeneca Ab Composés hétéroaromatiques bicycliques et leur utilisation dans le traitement du cancer

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003092678A1 (fr) * 2002-04-30 2003-11-13 Schering Aktiengesellschaft Derives de l'imidazole 1-substitues comme inhibiteurs nos
WO2020070610A1 (fr) * 2018-10-01 2020-04-09 Pi Industries Ltd. Nouveaux oxadiazoles
WO2022037568A1 (fr) * 2020-08-17 2022-02-24 Betta Pharmaceuticals Co., Ltd Composés bicycliques, compositions et utilisation de ceux-ci
WO2022232088A1 (fr) * 2021-04-27 2022-11-03 Dana-Farber Cancer Institute, Inc. Inhibiteurs du facteur de transcription à domaine associé transcriptionnel amélioré (tead) et leurs utilisations
WO2023209088A1 (fr) * 2022-04-28 2023-11-02 Astrazeneca Ab Composés hétéroaromatiques bicycliques et leur utilisation dans le traitement du cancer

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