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WO2025125248A1 - Composés destinés à être utilisés dans le traitement de cancers qui surexpriment tspan1 - Google Patents

Composés destinés à être utilisés dans le traitement de cancers qui surexpriment tspan1 Download PDF

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Publication number
WO2025125248A1
WO2025125248A1 PCT/EP2024/085543 EP2024085543W WO2025125248A1 WO 2025125248 A1 WO2025125248 A1 WO 2025125248A1 EP 2024085543 W EP2024085543 W EP 2024085543W WO 2025125248 A1 WO2025125248 A1 WO 2025125248A1
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compound
phenyl
acetamide
pyridin
ethylthio
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Inventor
Matilde Esther LLEONART PAJARÍN
Gerard Pujadas Anguiano
Santiago GARCÍA VALLVÉ
Yoelsis GARCÍA MAYEA
Almudena SÁNCHEZ GARCÍA
Aleix GIMENO VIVES
Júlia MESTRES TRUYOL
Amadeo Llebaria Soldevilla
Maria Del Carmen Serra Comas
Juan Lorenzo Catena Ruiz
Laia JOSA CULLERÉ
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Consejo Superior de Investigaciones Cientificas CSIC
Universitat Rovira i Virgili URV
Fundacio Institut de Recerca Hospital Universitari Vall dHebron
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Consejo Superior de Investigaciones Cientificas CSIC
Universitat Rovira i Virgili URV
Fundacio Institut de Recerca Hospital Universitari Vall dHebron
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
    • C07D263/52—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings condensed with carbocyclic rings or ring systems
    • C07D263/54—Benzoxazoles; Hydrogenated benzoxazoles
    • C07D263/56—Benzoxazoles; Hydrogenated benzoxazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
    • C07D263/57—Aryl or substituted aryl radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/04—Ortho-condensed systems

Definitions

  • the invention refers to compounds for use in the treatment of cancers that overexpress TSPAN1 protein. It also relates to pharmaceutical compositions comprising these compounds, and to their therapeutical indication in the treatment of different types of cancer, in particular those resistant to conventional methods of treatment.
  • Cancer is an heterogeneous disease characterized by the accumulation of tumor cells, which can result in death in both animals and humans.
  • Conventional methods of cancer treatment include surgical treatments, radiotherapy, administration of chemotherapeutic agents, and more recently immune-based treatments. However, so far, such treatments have had limited success in certain types of cancer.
  • Chemotherapy despite all its limitations, is still today one of the most widespread methods for the treatment of different types of cancer.
  • the inability of chemical agents to distinguish between normal, rapidly dividing cells and tumor cells can lead to the depression of the patient's immune system. This has been considered one of the main problems associated with chemotherapy, as well as the resistance mechanisms developed by cancer cells.
  • Drug discovery for use in cancer treatment is of vital importance for several reasons, in particular, to overcome the growing resistance of cancer cells to currently available treatments.
  • One of the possible approaches is to combine new drugs with conventional treatments, thus enhancing effecti vity by synergy or by targeting different characteristics of tumor cells, especially in heterogenous tumors. This approach would also help reducing adverse effects associated to conventional treatments, such as chemotherapy and radiotherapy, given that doses would be reduced, improving tolerance and adherence to treatment.
  • the development of new drugs may also enable a more selective and targeted approach, by focusing on specific signaling pathways or genetic mutations present in resistant cancers.
  • Tetraspanin-1 is a member of the family of proteins tetraspanins (TSPAN), whose principal characteristic is the capacity of forming aggregates between them or with several other transmembrane receptors, to become TSPAN-enriched microdomains.
  • TSPANTs are essential for basic biological activities, such as cell adhesion, proliferation, and cell motility.
  • TSPANTs overexpression has been associated to different types of cancers and to poor prognosis.
  • TSPANTs oncogenic role in cancer has been evidenced, mainly in digestives malignancies as pancreatic, gastric, colon cancer, and esophageal cancers, but also in hepatocellular carcinoma (Wang, G. L., et al., Oncol Rep 27, (2012), 1944-1952), skin squamous cell carcinoma, prostate cancer, osteosarcoma (Duan, J., et al., (2017), 5568- 5574) and non-small cell lung cancer (Wang, L., et al., Cell Death Dis 8, (2017), e2746).
  • TSPAN1 overexpression of TSPAN1 at mRNA and/or protein level in human tumor samples versus adjacent noncancerous tissues has been widely documented in cholangiocarcinoma, skin squamous cell carcinoma, esophageal carcinoma (Gu, T., et al., Oncology letters 14, (2017), 6815-6822), ovarian carcinomas (Scholz, C.J., et al., Cancer Lett 275 (2009), 198-203), prostate cancer (Munkley, J., et al., Sci Rep 7, (2017), 5249), pancreatic cancer and gastric carcinoma.
  • TSPAN1 is overexpressed (at mRNA and protein level) in colon cancer tissues. Its expression in tumor tissue is significantly associated with histological grade, proliferating cell nuclear antigen (PCNA), lymph node metastasis, and distant tumor node metastasis (TNM staging), according to Chen, L., et al., in “TSPAN1 protein expression: a significant prognostic indicator for patients with colorectal adenocarcinoma”, World J. Gastroenterol. 15 (2009), 2270. High TSPAN 1 expression is linked to poor survival and serves as an independent prognostic factor for colon cancer.
  • PCNA proliferating cell nuclear antigen
  • TAM staging distant tumor node metastasis
  • TSPAN 1 pancreatic cancer, known for its poor prognosis, identifies TSPAN 1 as an independent poor prognostic factor.
  • TSPAN 1 has also been included in a diagnostic model for predicting pancreatic carcinoma. In head and neck squamous cell carcinoma (HNSCC), high TSPAN1 expression is related to poorly differentiated tumors.
  • HNSCC head and neck squamous cell carcinoma
  • TSPAN1 promotes invasiveness of cervical cancer cells
  • AMPK AMP-activated protein kinase
  • TSPAN1 promotes epithelial-mesenchymal transition (EMT) and metastasis, acting through the p- ERK and p-AKT pathways, thus having a direct effect on the progression of the cancer.
  • TSPAN1 accelerates the progression, especially, of digestive malignancies, such as hepatocellular carcinoma (HCC), pancreatic, gastric, colorectal, and esophageal cancers, and some non-digestive cancers such as osteosarcoma and cervical cancer.
  • digestive malignancies such as hepatocellular carcinoma (HCC), pancreatic, gastric, colorectal, and esophageal cancers
  • non-digestive cancers such as osteosarcoma and cervical cancer.
  • TSPAN1 A Novel Protein Involved in Head and Neck Squamous Cell Carcinoma Chemoresistance
  • Cancers 2020, vol. 12, Article number 3269 describe that an increase in TSPAN1 expression, when compared to healthy tissue, can be observed in cancer cells resistant to cisplatin (CDDP-R), in cancer stem cells (CSC) and in biopsies obtained from patients with HNSCC.
  • CDDP-R cancer cells resistant to cisplatin
  • CSC cancer stem cells
  • biopsies obtained from patients with HNSCC biopsies obtained from patients with HNSCC.
  • TSPAN1 has been identified as an oncogenic protein that contributes to the acquisition of chemoresistance of cancerous cells through the activation of its main target, p-Src.
  • TSPAN1 emerges as a promising target for HNSCC therapy, given that its inhibition decreases size and proliferation of parental and resistant tumors, reduces metastasis, induces apoptosis, and sensitizes tumor cells to chemotherapeutic agents.
  • HNSCC is a form of cancer that affects the squamous cells lining the inner surfaces of the head and neck. This type of cancer can affect areas such as the oral cavity, pharynx, larynx, tonsils and sinuses. HNSCC accounts for approximately 90% of all head and neck cancers. It is a prevalent form of cancer with a significant burden of disease and mortality worldwide, mainly due to the lack of effective treatments and the resistance to conventional treatments, which in most cases leads to metastasis and relapse in patients, the latter occurring in more than 50% of HNSCC patients in the first 3 years after treatment.
  • TSPAN1 tetraspanin 1
  • the compounds of the invention inhibit TSPAN1 expression and, as a result, not only do they halt tumor proliferation, but moreover block activation of chemotherapy resistance and metastasis signaling pathways.
  • the compounds of the invention have effect against cancer cells but are not toxic for non-cancerous cells. Therefore, these compounds are advantageous because they represent a new treatment opportunity, in particular for cancers involving metastasis or chemotherapy-resistant cells, and provide more effective and targeted treatments for these challenging cancers.
  • a first aspect of the present invention relates to a compound of formula (I) or its pharmaceutically acceptable salts, for use in the treatment of a cancer expressing
  • TSPAN1 in a mammal, including a human,
  • Ri is a radical selected from (Ci-C4)-alkyl, (C3-C6)-cycloalkyl, phenyl, and chlorophenyl;
  • A is a biradical selected from S, N and C;
  • Cy is a biradical selected from: on a single or double bond, m is an integer from 0 to 2; n is an integer from 0 to 2; o is an integer from 0 to 1 ; p is an integer from 0 to 1 ; q is an integer from 0 to 1 ;
  • X is selected from O, S, N, and NH;
  • Y is selected from C and N;
  • Z is selected from C and N;
  • R2, R3, R4, and Rs are selected from H, halogen, OH, (Ci-Ce)-alkyl, and O-(Ci-Ce)-alkyl;
  • Re is selected from H and (Ci-C4)-alkyl-phenyl;
  • the compounds of formula (I) as defined above in combination with cisplatin have synergistic effect, thus in a particular aspect of the invention, the combination of compounds of formula (I) and CDDP is for use in the treatment of cancers overexpressing TSPAN1.
  • a second aspect of the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof of formula (I),
  • R1 is a radical selected from (Ci-C4)-alkyl, and (C3-C6)-cycloalkyl;
  • A is S; Cy is a biradical selected from: a single or double bond; m is 1 ; n is an integer from 0 to 2; o is an integer from 0 to 1 ; p is an integer from 0 to 1 ; q is an integer from 0 to 1 ;
  • X is selected from O, S, N; Y is selected from CH and N; Z is selected from C and N; R2 is selected from H, and halogen; Ra is selected from H, halogen, and OH; R4 is H; Rs is selected from H, halogen, and OH; Rs is selected from H and (Ci-C4)-alkyl-phenyl; and R7-R20 is selected from H, halogen, OH; with the proviso that: when (a) is a double bond then (b) is a single bond, and when
  • (b) is a single bond, p is 1 ; when Y is N, q is 0; when Z is N, o is 0; and when Z is C, o is 1 ; and with the proviso that: when Cy is phenyl and n is 0, R1 is other than methyl; when Cy is phenyl, n is 0 and X is O, R1 is other than ethyl or cyclopentyl; when Cy is phenyl, n is 0 and X is NH, R1 is other than isopropyl or 2-methylpropyl; and when Cy is phenyl, n is 0 and X is S, R1 is other than tert-butyl or cyclopentyl;
  • a third aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein: R1 is a radical selected from (Ci-C4)-alkyl, and (Ca-Ce)- cycloalkyl; A is S; Cy has the same meaning as in formula (I), being a biradical selected from: formula (i), formula (ii), formula (iii), formula (iv), formula (v), and formula (vi); is a single or double bond; m is 1 ; n is an integer from 0 to 2; o is an integer from 0 to 1 ; p is an integer from 0 to 1 ; q is an integer from 0 to 1 ; X is selected from O, S, N; Y is selected from CH and N; Z is selected from C and N; R2 is selected from H, and halogen; Ra is selected from H, halogen, and OH; R4
  • (b) is a single bond, p is 1 ; when Y is N, q is 0; when Z is N, o is 0; and when Z is C, o is
  • FIG. 1 shows expression of TSPAN1 in 4 cell line models of HNSCC (JHLI029, HTB-43, CCL-138 and SCC-25) by Western Blot (WB) (FIG. 1A) and the overexpression of TSPAN1 in CDDP-resistant cells (JHLI029, HTB-43 and CCL-138), as well as in CSC (generation 2 and 3 of HTB-43).
  • WB Western Blot
  • FIG. 1 B shows expression of TSPAN1 in 4 cell line models of HNSCC (JHLI029, HTB-43, CCL-138 and SCC-25) by Western Blot (WB)
  • FIG. 1A shows expression of TSPAN1 in CDDP-resistant cells (JHLI029, HTB-43 and CCL-138), as well as in CSC (generation 2 and 3 of HTB-43).
  • p-actin and Vinculin were used as loading controls (FIG. 1 B).
  • FIG. 2 shows that depletion of TSPAN1 affects different signal cascades with p-Src as a central node as it is a commonly de-regulated protein.
  • FIG. 3 shows results of an in vivo study in mice, comparing growth of tumors formed by cells transfected with siRNA against TSPAN1 (siTSPANI) versus control cells, until day 41.
  • FIG. 4 shows reduction in tumor size, both in JHLI029 and CDDP resistant (R) cells, under TSPAN1 depletion (NC, negative control).
  • FIG. 5 shows a graphical representation (logarithmic scale) of a cytotoxic assay to calculate the respective IC50 values of each compound (If) for the lines JHLI029 and JHU029-R.
  • FIG. 6 shows the signaling pathway modulated for compound (If) (WB image) on HNSCC cell lines JHLI029, HTB-43 and CCL-138, both Parental and CDDP-resistant cells, at 8h and 24h after exposure to compound (If).
  • FIG. 7 shows morphological and proliferation changes on JHU029-R cells after 24hours of exposure to compound (If) at different concentrations.
  • FIG. 8 shows IC50 results of compound (If) on fibroblasts IMR90 and a cell line from HNSCC that doesn’t express TSPAN1 (line RPMI).
  • FIG. 9 shows a Volume vs Time graph comparing tumor growth of control group to that of the experimental group, treated with compound (If).
  • FIG. 10 shows tumor size comparison (at end point) between tumor samples of control group and tumor samples of experimental group.
  • FIG. 11 shows a graph representation of the mean tumor weight of the control group and that of the experimental group.
  • FIG. 12 shows anti-tumoral effect on JHLI029 cell line of CDDP, compound (If) and the combination of compound (If) and CDDP.
  • FIG. 13 shows a WB image of the effects of compound (If), CDDP and the combination of both on cell lines JHLI029 and JHU029-R, at 24h of exposition.
  • FIG. 14 shows microphotographies of the effect on the proliferation of JHU029-R cells of different treatment options, each option at two different concentrations.
  • FIG.15 shows a WB image of the effects of compounds (If), (Ir) and (Is) on cells lines JHU029-R and CCL-138-R at 24h and 48h.
  • FIG. 16 shows in vitro effectivity studies of compound (Is) in a graphic representation of tumor volume (mm 3 ) vs time (days).
  • FIG. 17 shows the proliferation effect in number of cells at different times in JHLI029 and JHU029-R with compound (Is) versus control.
  • FIG. 18 shows differences in tumor weight (mean of each group) between control group and group treated with compound (Is) by the end of the treatment.
  • FIG. 19 shows the differences in tumor size by the end of the treatment with compound (Is).
  • FIG. 20 shows the expression in the proliferation marker Ki67 and the hematoxilin and eosin (H&E) staining in treated tumors with compound (If) and (Is).
  • FIG. 21 shows the cytotoxic effect of 11 compounds of the invention, at 100pM, in the HNSCC cell lines JHU029 and JHU029-R.
  • FIG. 22 shows IC50 values (mean ⁇ SD) of compound (Is) at 72 hours when tested against cancer cells lines of different types of cancers (Hep27 for hepatocellular carcinoma, MDA-MD-23 for breast cancer, MNK-45 for gastric adenocarcinoma, SK-OV-3 for ovarian adenocarcinoma, and LI-2-OS and MG63 for osteosarcoma), where (%) is the survival rate of the given cells.
  • FIG. 23 shows WB test for TSPAN1 downstream signaling pathways of cancer cell lines of different types of cancers when treated for 24 hours with the corresponding IC50 concentration of compound (Is) obtained in FIG. 22, where NC DMSO is the “control” cell line, that is, without treatment, and asterisks (*) below the cell line indicate differences with respect to the control.
  • treatment is meant to include alleviating or eradicating a disorder, disease, or condition, or one or more of the symptoms associated with the disorder, disease or condition, or alleviating or eradicating the cause(s) of the disorder, disease, or condition itself.
  • halogen is meant to include the chemically related elements fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).
  • an aspect of the present invention relates to compounds of formula (I) as defined above, or their pharmaceutically acceptable salts, for use in the treatment of a cancer expressing TSPAN1 , in a mammal, including a human, where: Ri is a radical selected from (Ci-C4)-alkyl, (C3-C6)-cycloalkyl, phenyl, and chlorophenyl; A is a biradical selected from S, N and C; Cy is a biradical selected from: formula (i).
  • formula (ii), formula (iii), formula (iv), formula (v), and formula (vi) as defined above; is a single or double bond, m is an integer from 0 to 2; n is an integer from 0 to 2; o is an integer from 0 to 1 ; p is an integer from 0 to 1 ; q is an integer from 0 to 1 ;
  • X is selected from O, S, N, and NH;
  • Y is selected from C and N;
  • Z is selected from C and N;
  • R2, R3, R4, and Rs are selected from H, halogen, OH, (Ci-Cs)-alkyl, and O-(Ci-Cs)-alkyl; Rs is selected from H and (C1-C4)- alkyl-phenyl;
  • R7-R20 is selected from H, halogen, OH, (Ci-Cs)-alkyl, and O-(Ci-Cs)-alkyl; with the proviso
  • the invention also relates to a method of treatment of a mammal, including a human, suffering from or being susceptible of suffering from cancer expressing TSPAN1 , in particular, to one of the cancers mentioned above, said method comprising the administration to said patient of a therapeutically effective amount of a compound of formula (I) as defined above, together with pharmaceutically acceptable excipients or carriers.
  • the compounds of formula (I) as defined above, or their pharmaceutically acceptable salts, for use as defined above are those where: A is a biradical selected from S and C, and m is an integer from 1 to 2.
  • the compounds of formula (I) as defined above, or their pharmaceutically acceptable salts, for use as defined above are those where: A is a biradical selected from S and C, m is an integer from 1 to 2, Cy is a biradical selected from formulas (i), (ii), (iii), (iv) and (v), and Re is selected from H and benzyl.
  • the compounds of formula (I) or their salts can exist in solvated, as well as unsolvated forms, including hydrated forms. Thus, they can contain in its structure stoichiometric amounts of solvent in the case of solvates, or of water in the case of hydrates. It is to be understood that the invention encompasses all such solvated, as well as unsolvated forms.
  • the pharmaceutically acceptable salts of the compounds of formula (I) include acid addition salts such as the hydrochloride, but also any other pharmaceutically acceptable salts of other acids such as hydrobromic, hydrofluoric, sulphuric, phosphoric, acetic, citric, fumaric, gluconic, lactic, maleic, succinic or tartaric acid.
  • the compounds of the present invention may be prepared by the process illustrated in Schemes I, II, III, IV and V. All radicals in the formulas of Schemes I, II, III, IV and V have the same meaning as the corresponding radicals in formula (I), as defined above. Cy in Schemes I, II, III, IV and V has the same meaning as the corresponding Cy in formula (I), as defined above. A, o, p, q, m, and n in Schemes I, III, IV and V have the same meaning as the corresponding A, o, p, q, m, and n in formula (I), as defined above. X in Schemes I, III, IV and V has the same meaning as the corresponding X in formula (I), and is thus selected from O, S, and NH, as defined above, with the proviso that in Scheme I X is selected from S or O.
  • Compound (IVi) can be prepared by reacting (VI) with (VII) ((Re)NH2) in the presence of a base, such as CS2CO3, for 2 hours, obtaining intermediated Vlh, followed by a hydrogenation reaction, using, for example, H2 and a palladium catalyst, for 3 hours. Both reactions may be carried out at room temperature and in an organic solvent, such as dioxane, and methanol.
  • Compound (VIII) can be prepared through a cyclation reaction mediated by a coupling reagent, such as CDI, to obtain intermediate VII h, followed by a chlorination reaction, using, for example, a mixture of POCI3 and PCI5. Both reactions may be carried out in an organic solvent, such as THF.
  • the cyclation reaction may be carried out at room temperature for 12 hours and the chlorination reaction may be carried out at a temperature comprised in a range from 90 to 110°C for 6 hours.
  • Compound of (II) can be prepared by reacting compound of (VIII) with compound of (III) boronic acid in the presence of a base, such as CS2CO3. Suzuki coupling reaction may be carried with a palladium catalyst, such as Pd(PPhs)4, in an organic solvent, such as DME, and with a reflux system.
  • a base such as CS2CO3.
  • Suzuki coupling reaction may be carried with a palladium catalyst, such as Pd(PPhs)4, in an organic solvent, such as DME, and with a reflux system.
  • Compounds of formula (I) can be prepared by reacting (II) with (V) in the presence of a solvent, such as DMF, coupling reagents, such as EDC.HOBt, and HATLI, and an organic base, such as DI PEA, and triethylamine.
  • a solvent such as DMF
  • coupling reagents such as EDC.HOBt, and HATLI
  • organic base such as DI PEA
  • triethylamine organic base
  • the reaction may be stirred at room temperature, diluted with a solvent, such as EtOAc, extracted with a solvent, such as EtOAc, washed, dried over an anhydrous reagent, such as Na2SC>4, filtered, and concentrated under pressure.
  • the product may be later purified to give (I).
  • Compounds of formula (V) can be prepared by using, for instance, mercaptoacetic acid (thioglycolic acid) and a halogenated compound, such as methyl iodide or propylbromide; or, for instance, by using mercaptoacetic ester instead of thioglycolic acid, and then a saponification process.
  • mercaptoacetic acid thioglycolic acid
  • a halogenated compound such as methyl iodide or propylbromide
  • Compounds of formula (I) can be prepared by reacting (II) with (V3) in the presence of a solvent, such as toluene. The reaction may be left stirring overnight at a temperature comprised in a range from 65 to 110°C to give the urea compounds of formula (I).
  • compositions of formula (I) can be carried out by methods known in the art. For instance, they can be prepared from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts are, for example, prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate pharmaceutically acceptable base in water or in an organic solvent or in a mixture of them.
  • TSPAN1 is frequently overexpressed in several cancers and is correlated with advanced tumor stage and poor prognosis.
  • the inventors have found, surprisingly, that the compounds of the present invention efficiently inhibit TSPAN1 overexpression, having a direct impact on proliferation and metastasis signaling pathways, as well as considerably reducing tumor size and weight, as can be seen in the examples and figures herein disclosed. This represents a major advance in cancer therapy, given that the compounds of the invention target a protein that is associated to several cancers, and have proved to be efficient against it.
  • the compounds of the present invention for use as defined above are those where the cancer expressing TSPAN1 is selected from the group consisting of pancreatic cancer, colon cancer, gastric cancer, colorectal cancer, esophageal cancer, ovarian cancers, endometrial cancer, hepatocellular carcinoma, prostate cancer, osteosarcoma, non-small cell lung cancer, skin squamous cell carcinoma, cholangiocarcinoma, cervical intraepithelial neoplasia, and head and neck squamous cell carcinoma.
  • the cancer expressing TSPAN1 is selected from the group consisting of pancreatic cancer, colon cancer, gastric cancer, colorectal cancer, esophageal cancer, ovarian cancers, endometrial cancer, hepatocellular carcinoma, prostate cancer, osteosarcoma, non-small cell lung cancer, skin squamous cell carcinoma, cholangiocarcinoma, cervical intraepithelial neoplasia, and head and neck squam
  • the compounds of the present invention for use as defined above are those where the cancer expressing TSPAN1 is selected from head and neck squamous cell carcinoma, hepatocellular carcinoma, breast cancer, gastric and ovarian adenocarcinoma, and osteosarcoma. In a more particular embodiment, the compounds of the present invention for use as defined above are those where the cancer expressing TSPAN1 is selected from HNSCC.
  • TSPAN1 activates several pathways involved in proliferation and apoptosis evasion, and even in metastasis. By inhibiting the overexpression of this protein, the compounds of the present invention halt these processes, therefore, ameliorating the patient’s prognosis, as well as their life expectancy and quality.
  • TSPAN1 is also involved in the activation of tumor cell mechanisms to develop resistance against currently approved cancer treatments, such as chemotherapy.
  • the inventors have found that, unexpectedly, the compounds of the present invention are able to sensitize tumor cells to currently used chemotherapeutic agents, overcoming thus one of the major challenges in cancer therapy. Therefore, in a particular embodiment, the compounds of the present invention for use as defined above are those where the cancer is resistant to chemotherapeutic agents. In a more particular embodiment, the compounds of the present invention for use as defined above are those where the chemotherapeutic agent to which tumor cells are resistant is cisplatin (CDDP).
  • CDDP cisplatin
  • TSPAN1 is overexpressed in cancer stem cells (CSCs) and resistant HNSCC cells and in their CDDP-resistant derivatives JHU029-R, HTB-43-R and CCL-138-R.
  • CSCs cancer stem cells
  • the compounds of the present invention for use as defined above show antitumoral activity in a cancer cell line selected from JHLI029, JHU029-R, HTB-43, HTB-43-R, CCL-138, and CCL-138-R.
  • the compounds of the present invention show antitumoral activity in the cancer cell line JHU029-R.
  • the compounds of formula (I) for use as defined above are those where each one of the radicals R4, Rs, and R7-20 is H.
  • the compounds of formula (I) for use as defined above are those where each one of the radicals R2 and R3 is selected from H and halogen.
  • the compounds of formula (I) for use as defined above are those where the biradical A is S.
  • compounds of formula (I) are those R1 is a radical selected from (Ci-C4)-alkyl, (Cs-Csj-cycloalkyl, phenyl, and chlorophenyl.
  • compounds of formula (I) are those where R1 is a radical selected from (Ci-C4)-alkyl and (C3-C6)-cycloalkyl.
  • compounds of formula (I) for use as defined above are those where m is 1.
  • compounds of formula (I) for use as defined above are those where X is O.
  • compounds of formula (I) for use as defined above are those where Y is N.
  • compounds of formula (I) for use as defined above are those where Z is C.
  • compounds of formula (I) for use as defined above are those where the biradical Cy is a phenyl.
  • the compounds of formula (I) for use as defined above are selected from the following list: a) /V-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)-2-(phenylthio)acetamide (la) b) /V-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)butyramide (lb) c) /V-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)hexanamide (Ic) d) 1-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)-3-propylurea (Id) e) / ⁇ /-(3-(benzo[c(]oxazol-2-yl)phenyl)-2-(ethylthio)acetamide (le)
  • the compound of formula (I) for use as defined above is that where: f) when Ri is a chlorophenyl radical; n is 0; m is 1 ; Cy is a phenyl biradical; (a) is a single bond and (b) is a double bond; p is 0; o is 0; q is 0; A is S; Z is C; X is O; Y is N; and each one of R2 and R4 is H, its IIIPAC name is 2-((4-chlorophenyl)thio)-/V-(3- (oxazolo[4,5-b]pyridin-2-yl)phenyl)acetamide having formula (If) below,
  • the compounds of formula (I) for use as defined above are selected from the following list: g) N-(5-(benzo[d]oxazol-2-yl)-2-chlorophenyl)-2-(ethylthio)acetamide (lea), h) N-(2-chloro-5-(oxazolo[4,5-b]pyridin-2-yl)phenyl)-2-(ethylthio)acetamide (Iva), i) 2-(ethylthio)-N-(3-(7-hydroxybenzo[d]oxazol-2-yl)phenyl)acetamide, (leb), j) 2-(ethylthio)-N-(3-(4-hydroxybenzo[d]oxazol-2-yl)phenyl)acetamide (lec), and k) N-(3-(benzo[d]oxazol-2-yl)-4-hydroxyphenyl)-2-(e
  • the compounds of the present invention can be used in the same manner as other known chemotherapeutic agents, i.e., in combination with other treatments, either simultaneously or sequentially, depending on the condition to be treated.
  • the compounds of formula (I) for use in the treatment of cancers overexpressing TSPAN1 as defined above are administered in combination with a chemotherapeutic agent.
  • the chemotherapeutic agent is cisplatin.
  • heterogenous cancers more than a single population of cancer cells, i.e., bulk of cancer cells, CSCs, resistant cells [non-CSCs]
  • CSCs cancer cells
  • non-CSCs resistant cells
  • the combination of both therapies have resulted in a synergistic effect, achieving a reduction of the tumor with a smaller dose and hence reduced side effects related to these treatments.
  • the compounds of the invention are administered simultaneously with the chemotherapeutic agent.
  • the compounds of the invention and the chemotherapeutic agent are administered separately, in any order, within a therapeutically effective interval.
  • the compounds of the invention and the chemotherapeutic agent are administered simultaneously or sequentially.
  • the compounds of the invention are administered in combination with a chemotherapeutic agent at 50 to 800pM and 0,5 to 30pM, respectively.
  • compositions of the present invention may be administered in parenteral form suitable for injection such as intravenous bolus injections, intravenous infusion, implantation into the body, oral, intratecal, or intranasal.
  • parenteral form suitable for injection such as intravenous bolus injections, intravenous infusion, implantation into the body, oral, intratecal, or intranasal.
  • Intratumoral administration may also be suitable for HNSCC tumors, due to their accessibility.
  • the compounds for use in the treatment of a cancer expressing TSPAN1 , in a mammal, including a human, according to the present invention are compounds of formula (11), or a pharmaceutically acceptable salt thereof, as described above.
  • Ri is a radical selected from (Ci-C4)-alkyl, and (Cs-Cej-cycloalkyl;
  • A is S; Cy is a biradical selected from: a single or double bond; m is 1 ; n is an integer from 0 to 2; o is an integer from 0 to 1 ; p is an integer from 0 to 1 ; q is an integer from 0 to 1 ;
  • X is selected from O, S, N; Y is selected from CH and N; Z is selected from C and N; R2 is selected from H, and halogen; R 3 is selected from H, halogen, and OH; R4 is H; Rs is selected from H, halogen, and OH; Rs is selected from H and (Ci-C4)-alkyl-phenyl; and R7-R20 is selected from H, halogen, OH; with the proviso that: when (a) is a double bond then (b) is a single bond, and when
  • (b) is a single bond, p is 1 ; when Y is N, q is 0; when Z is N, o is 0; and when Z is C, o is 1 , and with the proviso that: when Cy is phenyl and n is 0, R 1 is other than methyl; when Cy is phenyl, n is 0 and X is O, R1 is other than ethyl or cyclopentyl; when Cy is phenyl, n is 0 and X is NH, R1 is other than isopropyl or 2-methylpropyl; and when Cy is phenyl, n is 0 and X is S, R1 is other than tert-butyl or cyclopentyl, are also part of the invention.
  • the previous compounds of formula (I) are those where q is 1 and Rs is OH.
  • the previous compounds of formula (I) are those where R 3 is OH.
  • the previous compounds of formula (I) are those where R9 is Cl.
  • the previous compounds of formula (I) are those where R 7 is OH.
  • the compound of formula (I) is a compoundof formula (h), or a pharmaceutically acceptable salt thereof,
  • Ri is a radical selected from (Ci-C4)-alkyl and (C3-C6)-cycloalkyl; Cy has the same meaning as in formula (I), being a biradical selected from: formula (i), formula (ii), formula (iii), formula (iv), formula (v), and formula (vi); a single or double bond; n is an integer from 0 to 2; o is an integer from 0 to 1 ; p is an integer from 0 to 1 ; X is selected from O, S and N; Y is selected from CH and N; Z is selected from C and N; R2 and R 3 are selected from H and halogen; Re is selected from H and (Ci-C4)-alkyl-phenyl; R7-R20 are H; with the proviso that: when (a) is a double bond then (b) is a single bond, and when (a) is a single bond then (b) is a double bond; when (a) is a double bond,
  • compounds of formula (h) are those where, when Cy is phenyl and n is 0, R1 is other than methyl; when Cy is phenyl, n is 0 and X is O, R1 is other than ethyl or cyclopentyl; when Cy is phenyl, n is 0 and X is NH, R1 is other than isopropyl or 2-methylpropyl; and when Cy is phenyl, n is 0 and X is S, R1 is other than tert-butyl or cyclopentyl.
  • the compounds of formula (h) are those where n is an integer from 0 to 2; o is 1 ; p is 0; (a) is a single bond; (b) is a double bond; X is O; Y is N; Z is C; Cy is phenyl; Ri is a radical selected from (Ci-C4)-alkyl and (C3-C6)-cycloalkyl; R2 and R 3 are selected from H and halogen.
  • the compounds of formula (h) are those where n is an integer from 0 to 2; o is 0; p is 0; (a) is a single bond; (b) is a double bond; X is O; Y is N; Z is C; Cy is phenyl; R1 is a radical selected from (Ci-C4)-alkyl and (C3-C6)-cycloalkyl; R2 is H.
  • the compounds of formula (h) as defined above are those selected from the following list: a) /V-(3-(3-benzyl-3/7-imidazo[4,5-b]pyridin-2-yl)phenyl)-2-(ethylthio)acetamide (Ig); b) 2-(ethylthio)-/V-(3-(oxazolo[5,4-c]pyridin-2-yl)phenyl)acetamide (Ih); c) 2-(ethylthio)-/V-(5-(oxazolo[4,5-b]pyridin-2-yl)oxazol-2-yl)acetamide (li); d) 2-(ethylthio)-/V-(5-(oxazolo[4,5-b]pyridin-2-yl)thiazol-2-yl)acetamide (Ij); e) /V-(5-(benzo[d]thiazol
  • the compounds of formula (h) as defined above are selected from the following list: a) /V-(3-(7-bromooxazolo[4,5-b]pyridin-2-yl)phenyl)-2-(ethylthio)acetamide (Ip); b) /V-(3-(6-chlorooxazolo[4,5-b]pyridin-2-yl)phenyl)-2-(ethylthio)acetamide (Iq); c) 2-(isopropylthio)-/V-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)acetamide (Ir); d) 2-(ethylthio)-/V-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)acetamide (Is); e) 2-(cyclopropylthio)-/V-(3-(7-bromoox
  • the compounds of formula (h) as defined above are selected from the following list: a) 2-(isopropylthio)-/V-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)acetamide (Ir); b) 2-(ethylthio)-/V-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)acetamide (Is); c) 2-(cyclopropylthio)-/V-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)acetamide (It); d) 2-(cyclopentylthio)-/V-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)acetamide (lu).
  • the compound of formula (I) is selected from the following list:
  • a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein: Ri is a radical selected from (C1-C4)- alkyl, and (C3-C6)-cycloalkyl; A is S; Cy has the same meaning as in formula (I), being a biradical selected from: formula (i), formula (ii), formula (iii), formula (iv), formula (v), and formula (vi); is a single or double bond; m is 1 ; n is an integer from 0 to 2; o is an integer from 0 to 1 ; p is an integer from 0 to 1 ; q is an integer from 0 to 1 ; X is selected from O, S, N; Y is selected from CH and N; Z is selected from C and N; R2 is selected from H, and halogen; Ra is selected from H, halogen, and OH; R4 is H; Rs is selected from H,
  • the pharmaceutical composition according to the invention comprises a therapeutically effective amount of the compound of formula (h), or a pharmaceutically acceptable salt thereof as defined above, including the provisos mentioned above, together with appropriate amounts of one or more pharmaceutical excipients or carriers.
  • the pharmaceutical composition according to the invention comprises a therapeutically effective amount of a compound of formula (I) which is selected from the group consisting of:
  • terapéuticaally effective amount refers to the amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disease to be treated.
  • the particular dose of compound administered according to this invention will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and the similar considerations.
  • pharmaceutical composition refers to a mixture of a compound disclosed herein with other chemical components, such as diluents or carriers.
  • the pharmaceutical composition facilitates administration of the compound to an organism.
  • pharmaceutically acceptable excipients or carriers refer to pharmaceutically acceptable material, composition or vehicle, such as liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • Stepl Compound (Vh g ), with IIIPAC name /V-benzyl-3-nitropyridin-2-amine, was prepared from 2-chloro-3-nitropyridine (3.2 g, 20.2 mmol) and phenylmethanamine (2.2 mL, 20 mmol). Yield 81% (4.34 g, 16.3 mmol), yellow solid.
  • Step2 compound (I Vi g ), with IIIPAC name /V-benzylpyridine-2,3-diamine, was prepared from compound (Vh g ) (4.34 g, 16.3 mmol). Yield (3.92 g, 86%) as a brown solid.
  • Step3 Compound (Vllli g ), with IIIPAC name 3-benzyl-1/7-imidazo[4,5-b]pyridin-2(3/7)-one was prepared from compound (I Vi g ) (3.92 g, 16.3 mmol). Yield (3.52 g, 94%) as a brown semi-solid.
  • Step4 Compound (VII Ig), with IIIPAC name 3 imidazopyridine 3-benzyl-2-chloro-3/7- imidazo[4,5-b]pyridine was prepared from compound (Vllli g ) (1.03 g, 4.57 mmol). Yield (1.11 mg, 0.76 mmol, 99%) as a brown oil.
  • Step5 Compound (I Ig), with IIIPAC name 3-(3-benzyl-3/7-imidazo[4,5-b]pyridin-2- yl)aniline was prepared from compound (VI I Ig) (800 mg, 3.28 mmol) and 3- aminophenylboronic acid (517mg, 3.78 mmol) and purified by column cromatography (DCM:MeOH 2%). Yield (390 mg, 40%) as a solid.
  • Example 19 Preparation process of compound (Vd)
  • Compound (Vd), with IIIPAC name 2-(isopropylthio)acetic acid was prepared from 2- mercaptoacetic acid (500 pL, 7.16 mmol) and 2-bromopropane (710 pL, 7.52 mmol) following General Method F at room temperature for 2 h; yield 92% (881 mg, 6.56 mmol), light yellow oil.
  • Stepl To a solution of ethyl 2-mercaptoacetate (0.91 ml, 8.32 mmol) in DMSO (8.32 ml) were added potassium tert-butoxide (1.17 g, 10.40 mmol), and bromocyclopropane (0.67 ml, 8.32 mmol). The resulting mixture were heated at 80 °C overnight. The resulting mixture was cooled at room temperature, and added saturated NaHCO3, extracted with EtOAc, three times, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give compound (Vei) with IIIPAC name ethyl 2- (cyclopropylthio)acetate, as a light yellow oil (721 mg, 4.50 mmol, 54%).
  • Step2 Ethyl 2-(cyclopropylthio)acetate (520 mg, 3.25 mmol) was dissolved in a 1 :1 mixture of THF/MeOH (32.5 mL) and then aqueous sodium hydroxide (2.5 M, 6.5 ml, 16 mmol) was added and reacted at room temperature overnight. The mixture was neutralised with 1 M hydrochloric acid until acidic pH and extracted with EtOAc three times. The organic layers were washed with brine, dried, and concentrated in vacuo to compound (Ve) as a yellow oil (480 mg, 3.63 mmol, quant.).
  • 1 H NMR 400 MHz, CDCI3 6 3.33 (s, 2H), 2.12-2.03 (m, 1H), 0.98-0.82 (m, 2H), 0.70-0.57 (m, 2H).
  • Example 22 Preparation process of compound (Vea) Compound (Vea), with IIIPAC name 2-(ethylthio)acetyl chloride, was prepared from compound Vc (2.00 g, 16.6 mmol) following General Method H; yield: quantitative. (2.31 g, 16.6 mmol), light brown oil.
  • Example 40 Preparation process of compound (Iw) Compound (Iw), with IIIPAC name /V-(3-(6-chlorooxazolo[4,5-b]pyridin-2-yl)phenyl)-2- (cyclopropylthio)acetamide, was prepared from compound (Ilf) (65 mg, 0.26 mmol) and compound (Ve) (52 mg, 0.40 mmol) following General Method C; yield 10% (9 mg, 0.03 mmol), beige solid.
  • Step 1 Compound (lin) (70 mg, 0.31 mmol) and compound (Vc) (64 mg, 0.46 mmol) following General Method C; yield 35% (46 mg, 0.11 mmol) of 2-(3-(2- (ethylthio)acetamido)phenyl)benzo[d]oxazol-7-yl 2-(ethylthio)acetate.
  • Step 2 To a solution of 2-(3-(2-(ethylthio)acetamido)phenyl)benzo[d]oxazol-7-yl 2- (ethylthio)acetate (40 mg, 0.09 mmol) in THF (1 mL), LiOH 1 N (186 .L, 0.18 mmol) was added and stirred at r.t. for 6 h. Then THF was evaporated and the aqueous solution was purified by biotage, reverse phase, yield 15% (5 mg, 0.14 mmol)
  • Example 52 TSPAN1 inhibition sensitize chemotherapy resistant cancers and blocks tumoriqenic signaling pathways
  • TSPAN1 is most expressed in the CCL-138 cell line (FIG. 1A). Furthermore, the overexpression of TSPAN1 can be corroborated in the resistant lines as well as in CSC (generation 2 and 3 of HTB-43) (FIG.1 B).
  • p-Src Phosphorylation of Src kinase
  • AKT protein kinase B
  • ERK mitogen-activated protein kinase
  • FIG. 2 shows these results, hence confirming that inhibition of TSPAN1 affects different signal cascades with Src as a central node.
  • Inhibition of chemoresistance pathways in cancerous cells represents an important advantage and progress in the management of cancer, given that this is one of the main obstacles in cancer treatment and would ameliorate prognosis, life expectancy and life quality of patients.
  • Tumor size was also compared between groups at the end point, showing that in both cases (parental (P) cells and CDDP-R cells) tumor size was reduced when TSPAN1 was inhibited (FIG. 4). Phenotypic characteristics and proliferative potential of the tumors formed in mice were also observed, showing similarities to human HNSCC. Hence, the correlation between TSPAN1 inhibition and halt of tumor progression, which is part of the state of the art, is confirmed with these in vitro results obtained by the inventors, meaning that TSPAN1 is a valuable target for drug development in cancer treatment, in particular HNSCC.
  • HNSCC cell lines JHLI029, HTB-43 and CCL-138) were used, including both parental cells and CDDP-resistant cells (R), to determine the IC50 of compound (If) with the MTS method in p96 plates as described in Riss TL, et al. (in “Cell Viability Assays”, 2013 [Updated 2016 Jul 1], in: Markossian S., et al., “Assay Guidance Manual” [Internet], Bethesda (MD), Eli Lilly & Company and the National Center for Advancing Translational Sciences, 2004), (see Table 1).
  • Example 55 Western blot (WB) assays under compound (If) effect in HNSCC cell lines at three different times
  • FIG. 5 is a graphical representation of IC50 results of compound (If) against TSPAN1 for the cell lines JHLI029 and JHU029-R at 72h by MTS assay, as described in Riss TL, et al. (in “Cell Viability Assays”, 2013 [Updated 2016 Jul 1], in: Markossian S., et al., “Assay Guidance Manual” [Internet], Bethesda (MD), Eli Lilly & Company and the National Center for Advancing Translational Sciences, 2004).
  • TSPAN1 inhibition as well as inhibition of active form of Src and ERK (p-Src y p-ERK), is achieved with compound (If) on HNSCC cell lines JHU029, HTB-43 and CCL-138, both parental and resistant to CDDP (R), at 8h and 24h after exposure to compound (If) at 2 times the IC50.
  • apoptosis marker PARP1 is also cleaved at 8 and 24 h in HTB- 43-P cells (FIG. 6).
  • Example 56 In vitro toxicological studies on non-tumorous and other HNSCC cell lines
  • mice In vivo toxicological studies with the compounds of the invention were conducted on mice, at different concentrations of drug (20, 40 and 80mg/Kg), based on IC50 results obtained.
  • the vehicle used in all treatments was PBS + 12,5% chromophore + 5% DMSO and the treatment were administrated via intraperitoneal injection, 3 times a week for a total of 4 weeks.
  • Possible toxicological effects were evaluated by means of visual examination of pain and weight loss in the mice. Samples of serum, spleen, liver, lungs, and kidneys were extracted from the dead animals (sacrificed under an overdose of 5% deep anesthesia) to evaluate possible toxicity by means of histological technigues and molecular biology.
  • Example 58 In vivo experiments to show antitumoral efficacy on JHU029-R cells
  • an intermediate concentration of compound (If) was used.
  • 1x10 6 cancerous CDDP resistant cells (JHU029-R) that overexpress TSPAN1 were injected subcutaneously on the side of the mouse, allowing tumor growth for approximately 7-9 days before starting the treatment (40mg/kg).
  • Necropsies of the animals were performed once treatment had finished, extracting samples of the spleen, kidneys, liver, lungs, and serum, as well as extracting the tumor.
  • FIG. 9 shows that tumor volume was significantly reduced in the experimental group after 2-3 weeks of treatment, when compared to the control group.
  • total size (FIG. 10) and weight (FIG. 11) of the tumor were also significantly reduced in the experimental group, in comparison to the control group.
  • Example 59 Drug levels in tumor, liver and serum detected by HPLC technique
  • HPLC technique was used to detect drug levels at end point in tumor, liver, and serum in 3 mice of each group (control and experimental). A final dose of treatment was injected in these animals 2h before euthanasia was conducted. The presence of compound (If) in serum and tumor indicates that it is distributed correctly and that it effectively reaches the tumor. In this way we can conclude that the reduction in size and weight is due to the presence of the drug and not to indirect/unspecific effects of the compound (If), mentioned in Example 47, in the experimental group. Drug levels were also reported in liver tissue, as expected, given that the drug is injected in peritoneal zone and, furthermore, the liver has metabolic functions. The variability is due to the heterogeneity of the in vivo model and the quantity is not as important as the mere presence of the compound in the tumor and serum samples.
  • Tables 3 and 4 show Cl results for non-constant ratio combinations of compound (If) at fixed IC50 concentration and CDDP at different concentrations, and vice versa, respectively in JHU029-R.
  • synergy (Cl ⁇ 1) of the combination of drugs was confirmed by the Cl results, meaning that treatment could be improved by such combination.
  • FIG. 12 shows a graphic representation of the effects of CDDP (0,5
  • a WB assay was performed with the obtained ratio (1 ,8:444 pM CDDP: compound (If)) and the most adequate concentration (one time the ratio and two times), in order to evaluate the expression of aforementioned markers (p-ERK, p-Src and TSPAN1), as well as other markers that will indicate the effect of CDDP on cells.
  • FIG. 12 shows a graphic representation of the effects of CDDP (0,5
  • FIG. 13 shows the corresponding WB assay, where inhibition of TSPAN1 , p-Src and p-ERK both in cell lines JHLI029 and JHU029-R (resistant to CDDP) is increased by the combination of compound (If) and CDDP when compared to CDDP alone.
  • CD44 a classic CSC marker in HNSCC and other types of cancers
  • CD44 also significantly reduced its expression with both compound (If) concentrations and with their respective combinations with CDDP.
  • FIG. 14 shows microphotographies that represent the effect of different treatment conditions: control (DMSO), CDDP alone, compound (If) alone and combination of compound (If) and CDDP, on the proliferation of JHU029-R cells. All three treatment options were evaluated at two different concentrations and, as can be seen in the microphotologies, proliferation is proportionally reduced with increasing concentrations, but most importantly, it is significantly reduced with compound (If) in comparison to CDDP (probably due to this cell line’s resistance to CDDP) and even more reduced with the combination of both drugs, as suggested by the Cl synergistic results from Tables 3 and 4.
  • the fact that both drugs are used in combination represents an advantage, since when using each drug individually the concentrations are higher (than when using them in combination with each other) and therefore the side effects associated with each drug individually are reduced (this is very significant in the case of CDDP).
  • Example 61 In vitro tests of compounds (Ir) and (Is) of the invention
  • Example 62 In vivo toxicological and effectivity studies of compounds (Ir) and (Is) of the invention
  • Figure 21 shows two graphs, in which 11 compounds of the invention (namely (lb), (Ic), (Id), (le), (Ig), (Ih), (Ik), (lu), (lx), (Iz), and (Icc)) were tested against HNSCC cell lines JHLI029 and JHU029-R.
  • the survival effect was measured at 72h after treatment withlOOpM of each compound by MTS assay, as described in Riss TL, et al.
  • Example 64 In vitro toxicological studies and WB tests performed in cancer cell lines of hepatocellular carcinoma, breast cancer, gastric and ovarian adenocarcinoma, and osteosarcoma.
  • the cell lines were cultured in DM EM F-12 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) with 10% FBS (Biowest, Nuaille-France), 1% L- glutamine (200 mM, Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and 1% penicillin/streptomycin (Pen 20 U/mL and Strep 20 pg/mL, Gibco, Thermo Fisher Scientific, Waltham, MA, USA).
  • TSPAN1 protein expression a significant prognostic indicator for patients with colorectal adenocarcinoma”, World J. Gastroenterol, 15 (2009), 2270.

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Abstract

L'invention concerne des composés de formule (I) destinés à être utilisés dans le traitement de cancers qui surexpriment la protéine TSPAN1. L'invention concerne également des compositions pharmaceutiques comprenant ces composés, et leur indication thérapeutique dans le traitement de différents types de cancer, en particulier ceux résistants aux méthodes classiques de traitement.
PCT/EP2024/085543 2023-12-11 2024-12-10 Composés destinés à être utilisés dans le traitement de cancers qui surexpriment tspan1 Pending WO2025125248A1 (fr)

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