EP3652170A1 - 5-{4-allyl-5-[2-(4-alkoxyphenyl)quinolin-4-yl]-4h-1,2,4-triazol-3-ylsulphanylmethyl}furan-2-carboxylic acids in the treatment of cancer - Google Patents

Abstract

The present invention relates to a compound of formula (I) below: formula (I) or a pharmaceutically acceptable salt thereof, in which R is an alkyl group comprising 1 to 10 carbon atoms. The invention also relates to the use thereof as a medicament, in particular in the treatment of cancer. The invention also relates to pharmaceutical compositions containing such compounds and to the process for preparing same.

Description

 5-F4-allyl-5-r2- (4-alkoxyphenyl) quinolin-4-yn-4H-1,2,4-triazol-3-ylsulphanylmethyl] furan-2-carboxylic acids in the treatment of cancer

TECHNICAL AREA

 The present invention relates to furfuryl derivatives of 5- [2- (4'-alkoxyphenyl) quinolin-4-yl] -4H-1,2,4-triazol-3-thiol, pharmaceutical compositions comprising them, a method of preparation of these, as well as their use in the treatment of cancer.

STATE OF THE PRIOR ART

 Targeting the Epidermal Growth Factor Receptor (EGFR) receptor binding site by tyrosine kinase inhibitors (TKIs) makes it possible to interrupt aberrant signaling cascades in cancer cells.

EGFR belongs to a family of ErbB transmembrane proteins that control the signaling pathways and expression of genes involved in cell proliferation, survival, angiogenesis, adhesion and motility. A protein ligand, such as EGF, binding to the extracellular region of EGFR leads to tyrosine kinase activation and tyrosine autophosphorylation at multiple sites in the receptor's cytoplasmic region (Schlessinger, 2014). Mutations leading to overexpression or deregulation of EGFR have been shown to be associated with the progression of different types of cancer. Therefore, targeting of this protein has been an important goal in medicinal chemistry over the last two decades (Mendelsohn and Baselga, 2006). Competitive tyrosine kinase inhibitors (TKIs) of ΑΤΡ, including the FDA-approved Gefitinib (ZD1839), have been developed to inhibit the catalytic site of EGFR (Herbst et al., 2004). TKIs with alternative modes of action, such as Carnetinib (CI-1033), have also been designed to bind nucleophilic cysteine (Cys797) near the catalytic site of EGFR (Allen et al., 2003). ). The reversible and irreversible covalent inhibitors effectively block the catalytic site in EGFR and prolong attenuation of the downstream signaling cascade compared to competitive inhibitors (Kawakita et al., 2013, Jia et al., 2016, Smail et al. 2016, Cheng et al., 2016). However, a limitation of anti-EGFR TKI is the acquisition of somatic mutations in the catalytic pocket or other receptor sites leading to Antitumor drug resistance in clinical trials (Campbell et al., 2008, Hao et al., 2016, Wang et al., 2016).

 The inactivation of tyrosine kinase in EGFR by small molecules is based on the observation that the driving force to activate the binding site at ΑΤΡ is the action of hydrogen peroxide (H2O2) generated during binding of the parent ligand to receptor (Bae et al., 1997; Paulsen et al., 2012). It has also been shown that the action of small molecules, such as 4-nitro-benzoxadiazole derivatives, depends on the generation of H2O2 by cytoplasmic superoxide dismutase in cancer cells (Sakanyan et al., 2016).

 These results suggest that highly reactive hydrogen peroxide generated by a variety of metabolic reactions could increase phosphorylation in EGFR-triggered pathways and quench the effects of TKIs in cancer cells and, consequently, decrease therapeutic expectations. . Also, the development of anti-EGFR compounds leading to a reduction in the level of the receptor could be an alternative strategy to disrupt aberrant signaling in cancer cells.

 Endocytosis of EGFR is an autophagic process known as micro-autophagy that governs the fate of the receptor in cells (Klionsky et al., 201). The EGFR-ligated EGFR undergoes clathrin-dependent and non-clathrin-dependent endocytosis, followed by receptor recycling and / or degradation with proteolytic enzymes in endosome-fused lysosomes (Goh and Sorkin, 2013). Low doses of EGF activate clathrin-dependent endocytosis, whereas high doses of the ligand favor clathrin-dependent or non-cladrin-dependent endocytosis (Sigismund et al., 2005, Sigismund et al., 2008). Endocytic degradation of the EGFR-ligated EGFR is performed more rapidly than for the unbound receptor (Stoscheck and Carpenter, 1984). It has been shown that Gefitinib suppresses EGF-stimulated endocytosis (Nisimura et al., 2007) and induces endocytic degradation of the receptor as a cytoprotective response in lung cancer cells (Han et al., 2011). .

In addition, EGFR is a client protein for Hsp90a, which, in cooperation with Hsp70, controls the proper folding and maturation of nascent polypeptides by a super-chaperone complex in normal and cancer cells (Taipale et al., 2010). . The Hsp70 chaperone initially recognizes an incorrectly folded client protein, then translocates the Hsp90a-related protein, and the latter supplements the processing of the client protein (Li et al., 2012). About 700 proteins have been identified as interaction partners with Hsp90a (Echeverria et al., 2011), and a 20 amino acid sequence, called aC64 in EGFR, is conserved in Hsp90a-recognized protein kinases (Citri et al., 2006). The Hsp90a chaperone is highly expressed in cancer cells (Pick et al., 2007) and the inhibition of Hsp90 / Hsp70 leads to the degradation of misfolded client proteins by the cellular proteasome (Miyata et al., 2013).

 The phosphorylation state of EGFR determines the functions of integrins, transmembrane receptors that allow cells to adhere to the extracellular matrix (ECM) (Vlahakis and Debnath, 2017). The binding of EGF to EGFR triggers, by phosphorylation of the MAPK / ERK pathway, the activation of Bim and Beclin-1 proteins which bind to microtubules and actin which, in conjunction with other proteins, provide the attachment of α / β-integrins to ECM. Nutrient deprivation adversely affects the EGFR signaling cascade, resulting in cell detachment and eventually a programmed death, called anoikosis (Frish and Francis, 1994). Anoikosis is preceded by autophagic degradation of damaged proteins and sequestration of the cytoskeleton (Fung et al., 2008). In comparison to healthy cells, cancer cells have a higher tolerance to anoïkose, and this feature appears to be important for metastatic progression of inflammatory tumors (Buchheit et al., 2015).

 Thus, there is a possible pharmacological window for attenuating aberrant tumor signaling by targeting the degradation of EGFR by small molecules to overcome the limitations of current therapies due to the heterogeneity of somatic mutations acquired in the cells. the same tumor that represents a major obstacle in the fight against the progression of cancer.

SUMMARY OF THE INVENTION

It has thus been demonstrated in the context of the present invention that furfuryl derivatives of 5- [2- (4'-alkoxyphenyl) quinolin-4-yl] -4H-1,2,4-triazol-3-thiol make it possible to reduce the abundance of EGFR by activating authentic degradation pathways of the receptor. Indeed, these derivatives bind to the EGFR and decrease the concentration of the receptor in breast cancer cells. The active compounds briefly inhibit the phosphorylation of EGFR and rapidly induce degradation of EGFR and concomitantly Hsp90a chaperone by endocytosis. The decrease in the receptor and chaperone proteins degrades cytoskeletal proteins leading to detachment of cancer cells and finally to their death (anoïkose). The compounds of the invention thus inhibit tumor growth, which has been demonstrated in mice, and represent a promising tool for attenuating the progression of metastatic and drug-resistant forms of cancer.

The present invention therefore relates to a compound of formula (I) below:

or a pharmaceutically acceptable salt thereof,

wherein R represents an alkyl group having 1 to 10 carbon atoms.

The results obtained with the compounds according to the invention open up promising possibilities for improving the targeted anti-cancer therapy with chemical agents, by reinforcing the degradation of the EGFR which leads to the death of the cancer cells. To date, no anti-EGFR molecule has been described with an action associated with the detachment of cancer cells from the ECM. The novel furfuryl derivatives of 5- [2- (4'-alkoxyphenyl) quinolin-4-yl] -4H-1,2,4-triazole-3-thiol have a low ability to inhibit EGFR tyrosine kinase, but increased to degrade proteins.

The ability of similar molecules to degrade EGFR (in order of efficiency 26>25>24> 23) correlates with the length of the alkyl ether chain in their structures, CH3 (CH2) 4, CH3 (CH2) 3, CH3 (CH2) 2, and CH3CH2, respectively. Therefore, the local hydrophobicity in the catalytic pocket caused by a longer alkyl ether substituent (CH2) _n in compounds could enhance the degradation of the EGFR / Hsp90a complex by proteases.

The suppression of phosphorylation flux in the MAPK / ERK pathway has been considered as a promising way to reduce the metastatic dissemination of cancer cells (Akiyama et al., 2009). According to the invention, a weak inhibition of catalytic site in EGFR may be offset by increased degradation of the receptor and associated Hsp90a chaperone during endocytosis. The degradation of the EGFR seems to be advantageous compared to a strong inhibition of the signaling pathways by the known TKIs molecules, since it more specifically provides the interruption of the phosphorylation of the Bim protein and the sequestration of the cytoskeleton proteins.

 The apoptotic death of cancer cells with chemical intervention due to increased protein degradation attracts special attention in the therapy of tumor-resistant forms. Indeed, the complete interruption of phosphorylation pathways with TKIs creates selective conditions favoring the survival of mutant forms of EGFR through alternative mechanisms of receptor activation with H2O2 generated in the branched web of protein interactions. relationship with the metabolism and the tumor microenvironment. In contrast, direct and dual protein degradation by simultaneously reducing the amount of EGFR and Hsp90a decreases the development of such selective conditions and, therefore, may be less predisposing to the emergence of EGFR-resistant mutants.

 Thus, the targeting of EGFR degradation represents a fundamentally different alternative and alternative to tyrosine kinase inhibition, which is attractive for mitigating metastatic progression and decreasing drug resistance of mutated cancer cells and tumors. malignant.

DEFINITIONS

 In the present invention, the term "pharmaceutically acceptable" is intended to mean that which is useful in the preparation of a pharmaceutical composition which is generally safe, non-toxic and neither biologically nor otherwise undesirable and which is acceptable for veterinary use as well as human pharmaceutical.

 The pharmaceutically acceptable salts comprise in particular:

(1) pharmaceutically acceptable acid addition salts formed with pharmaceutically acceptable inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or formed with pharmaceutically acceptable organic acids such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, acid mandelic acid, methanesulfonic acid, muconic acid, 2-naphthalenesulfonic acid, propionic acid, salicylic acid, succinic acid, dibenzoyl-L-tartaric acid, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid, trifluoroacetic acid and the like, and

 (2) pharmaceutically acceptable base addition salts formed when an acidic proton present in the parent compound is either replaced by a metal ion, for example an alkali metal ion, an alkaline earth metal ion or a aluminum; is coordinated with a pharmaceutically acceptable organic base such as diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine and the like; or with a pharmaceutically acceptable inorganic base such as aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide and the like.

 For the purposes of the present invention, the term "halogen atom" means the fluorine, chlorine, bromine and iodine atoms.

 By "alkyl" is meant, in the sense of the present invention, a saturated hydrocarbon chain, linear or branched. It may be in particular an n-propyl, n-butyl or n-pentyl group.

 By "(d-C6) alkyl" is meant, in the sense of the present invention, a saturated hydrocarbon chain, linear or branched, having 1 to 6, preferably 1 to 4, carbon atoms. By way of example, mention may be made of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl groups.

 For the purposes of the present invention, the term "aryl" means an aromatic hydrocarbon group, preferably comprising from 6 to 10 carbon atoms, and comprising one or more contiguous rings, for example a phenyl or naphthyl group. Advantageously, it is phenyl.

By "aryl- (d-C6) alkyl" is meant, in the sense of the present invention, an aryl group as defined above, linked to the rest of the molecule via a chain (d-C6 ) alkyl as defined above. By way of example, mention may be made of the benzyl group. For the purposes of the present invention, the term "(C 6 -C 6) alkyl-aryl" means a (C 1 -C 6) alkyl group as defined above, linked to the remainder of the molecule via a group aryl as defined above. By way of example, mention may be made of the tolyl group (ChhPh).

 By "leaving group" is meant, in the sense of the present invention, a chemical group that can be easily moved by a nucleophile during a nucleophilic substitution reaction, the nucleophile being for example a thiol. Such a leaving group may be more particularly a halogen atom such as a chlorine or bromine atom or a sulfonate. The sulfonate may in particular be a group -OSO 2 -OR with Ro representing a (C 1 -C 6) alkyl, aryl, aryl- (C 1 -C 6) alkyl or (C 1 -C 6) alkyl-aryl group, said group being optionally substituted by one or more halogen atoms such as fluorine atoms. The sulphonate may be in particular a mesylate (-OS (O 2) -CH 3), a triflate (-OS (O) 2-CF 3) or a tosylate (-OS (O) 2- (p-Me-C 6 H 4)) . Preferably the leaving group will be a halogen atom, such as Cl or Br, in particular Cl.

DETAILED DESCRIPTION

 The present invention therefore firstly relates to a compound of formula (I) above or a pharmaceutically acceptable salt thereof.

 The group R will preferably represent a linear alkyl group. This alkyl group comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, in particular 3 to 10 carbon atoms, in particular 3 to 6 carbon atoms, advantageously 3, 4 or 5 carbon atoms. carbon atoms.

The compound according to the invention may be more particularly chosen from:

and their pharmaceutically acceptable salts.

The present invention relates as a second object to a pharmaceutical composition comprising at least one compound according to the present invention.

 Advantageously, the pharmaceutical composition will also comprise at least one pharmaceutically acceptable excipient.

 The pharmaceutical compositions according to the invention may be intended for enteral (for example oral) or parenteral (for example intravenous) administration, preferably orally or intravenously. The active ingredient can be administered in unit forms for administration, mixed with conventional pharmaceutical carriers, to animals, preferably mammals, including humans.

 For oral administration, the pharmaceutical composition may be in solid or liquid form (solution or suspension).

A solid composition may be in the form of tablets, capsules, powders, granules and the like. In tablets, the active ingredient can be mixed with one or more pharmaceutical carriers such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic and the like, before being compressed. The tablets may further be coated, especially with sucrose or other suitable materials, or they may be treated in such a way that they have prolonged or delayed activity. In powders or granules, the active ingredient may be mixed or granulated with dispersants, wetting agents or suspending agents and with flavor correctors or sweeteners. In the capsules, the active ingredient can be introduced into soft or hard capsules in the form of a powder or granules as mentioned above or in the form of a liquid composition as mentioned below.

 A liquid composition may contain the active ingredient with a sweetener, flavor enhancer or a suitable colorant in a solvent such as water. The liquid composition can also be obtained by suspending or dissolving a powder or granules, as mentioned above, in a liquid such as water, juice, milk, etc. It may be for example a syrup or an elixir.

 For parenteral administration, the composition may be in the form of an aqueous suspension or a solution which may contain suspending agents and / or wetting agents. The composition is advantageously sterile. It can be in the form of an isotonic solution (especially with respect to blood).

 The compounds according to the invention can be used in a pharmaceutical composition at a dose ranging from 0.01 mg to 1000 mg per day, administered in a single dose once a day or in several doses during the day, for example twice. per day at equal doses. The dose administered daily is advantageously between 5 mg and 500 mg, and more preferably between 10 mg and 200 mg. However, it may be necessary to use doses outside these ranges, which can be appreciated by those skilled in the art.

The third subject of the present invention is the compounds or pharmaceutical compositions according to the invention for their use as medicaments, especially for the prevention or treatment of cancer, including in particular the prevention of metastases.

The invention also relates to the use of a compound or a pharmaceutical composition according to the invention in the prevention or treatment of cancer, including in particular the prevention of metastases. The invention also relates to the use of a compound or a pharmaceutical composition according to the invention for the manufacture of a medicament for the prevention or treatment of cancer, including in particular the prevention of metastases.

 The invention also relates to a method for the prevention or treatment of cancer, including in particular the prevention of metastases, comprising the administration to a patient in need of an effective amount of a compound or a pharmaceutical composition according to the invention .

 The cancer that can be treated with the compounds and pharmaceutical compositions according to the invention may be any cancer of the epithelial cells, and more particularly breast cancer, non-small cell lung cancer, prostate cancer, pancreatic cancer. , or colorectal cancer.

 The compounds and pharmaceutical compositions according to the invention can also be used in the treatment of other pathologies related to the overexpression or deregulation of EGFR and corresponding signaling pathways, such as cardiovascular diseases such as stroke (stroke). , ischemia, neurodegenerative diseases such as Alzheimer 's disease or glaucoma, or to promote the regeneration of injured optical nerve fibers.

The fourth subject of the present invention is a process for the preparation of a compound according to the invention comprising the hydrolysis of the ester function of a compound of formula (I I) below:

in which R is as defined above and R 1 represents an alkyl group comprising 1 to 6 carbon atoms, in particular a methyl or ethyl group. Such a hydrolysis reaction can be carried out in an acidic or basic medium, in particular basic, according to methods well known to those skilled in the art. The reaction can thus be carried out in the presence of a base such as KOH or NaOH. The reaction may be carried out in a solvent such as methanol, water or a mixture thereof.

 This hydrolysis step may be followed by a salification step in the presence of a pharmaceutically acceptable acid or base to give the corresponding salt.

 The compound thus obtained may be separated from the reaction medium by methods well known to those skilled in the art, such as, for example, by extraction, evaporation of the solvent or by precipitation and filtration.

 The compound may be further purified if necessary by techniques well known to those skilled in the art, such as by recrystallization if the compound is crystalline, by distillation, by column chromatography on silica gel or by high performance liquid chromatography (HPLC ).

The compounds of formula (II) may be prepared from compounds of formula (III) below:

in which R is as defined above,

by substitution of the thiol function with a compound of formula (IV) below:

wherein R 1 is as defined above and GP is a leaving group such as a halogen atom, for example Cl.

Such a reaction is advantageously carried out in the presence of a base such as KOH or NaOH. This reaction can be carried out in a solvent such as methanol, for example at room temperature, that is to say a temperature between 15 and 30 ° C, especially between 20 and 25 ° C.

The compounds of formula (III) exist under the two forms tautomers (III) and (ΙΙ) below:

 (III) (NI ')

The compounds of formula (III) may be prepared from the compounds of formula (V) below:

in which R is as defined above,

by cyclization in basic medium.

 Such a reaction is advantageously carried out in the presence of a base such as KOH or NaOH. The reaction can be carried out in a solvent such as water, especially at the reflux temperature.

The compounds of formula (V) may be prepared from compounds of formula (VI) below:

in which R is as defined above,

by reaction with aylisothiocyan below

 Such a reaction can be carried out in a solvent such as ethanol.

 The compounds of formula (VI) can be prepared according to a protocol described in Avetyan et al. , 1973.

The present invention is illustrated by the non-limiting examples and figures detailed below.

FIGURES

 Figure 1. Western blotting of tyrosine phosphorylation of EGFR and protein expression in breast cancer cells treated with various compounds.

 (A) Serum-deprived MDA MB468 cells were treated with compounds according to the invention (100 μl) or Cl-1033 (1 μl) for 90 min, washed with PBS, and then incubated without or with 200 ng / ml. EGF for 15 min. Total tyrosine phosphorylation of EGFR (pTyr) was detected with an anti-pTyr-100 antibody that recognizes a pan-epitope of tyrosine phosphorylated in proteins (www.cellsignal.com).

 (B) Compounds 25 and 26 exhibit a dose-dependent effect on phosphorylation and expression of EGFR. The cells were treated with different concentrations of compounds for 120 min and then stimulated with EGF as in

(AT).

 Figure 2. Immunoprecipitation test revealing a combination of EGFR with Hsp90a in cancer cells treated with compounds according to the invention.

Figure 3. Effects of compounds according to the invention on the expression of proteins in cancer cells transfected with EGFR siRNA. (A) Western blot of proteins extracted from MDA MB468 cells after transfection with siRNA EGFR or siRNA control and exposure to compounds 25 and 26.

 (B) Relative amounts of protein were estimated as EGFR / 6-actin, Hsp90a / 6-actin or LC36 / 6-actin and adjusted to the corresponding values with a referenced vehicle (0.1% DMSO) as 100%.

Figure 4. Proof of concept of detachment of cancer cells after 18 h of growth in serum-free medium and treatment with compounds according to the invention for 6 h.

 Figure 5. Proof of concept of detachment of cancer cells with compounds according to the invention after 48 hours in private cancer cells and not deprived of serum.

 Figure 6. Proof of concept of the weak and reversible inhibition of autophosphorylation of EGFR in cancer cells with compound 26.

EXAMPLES

I - Synthesis of the compounds according to the invention

Nuclear magnetic resonance spectra (NMR) ¹ H were recorded in DMSO-d at ambient temperature using a Varian Mercury VX-300 NMR spectrometer and chemical shifts were measured in ppm using TMS as a reference. Electrospray ionization (ESI) and high resolution (HRMS) mass spectrometry analyzes were obtained using a Thermo Finnigan LCQ Advantage spectrometer or a Thermo Scientific Exactive Orbitrap spectrometer, respectively. The melting points (mp) are defined in ° C and were measured on a Boecius micro-heating table. Thin layer chromatography (TLC) was performed on Silufol UV-254 plates in the chloroform-methanol solvent system, 10: 0.5 for compounds 6-10; in the solvent system ethyl acetate-benzene 1: 3 for compounds 1 1 -15; in the 2: 1 ethyl acetate-benzene solvent system for compounds 17-21; and in the 10: 2: 1 ethyl acetate-methanol-water solvent system for compounds 22-26. The plates were exposed by UV (254 nm).

The compounds according to the invention were synthesized according to the reaction scheme below:

with R = CH ₃ , C ₂ H ₅ , C ₃ H ₇ , C ₄ H ₉ and C ₅ Hn.

Hydrazides of 2- (4-alkoxyphenyl) quinoline-4-carboxylic acids 1-5:

 Starting materials 1-5 were synthesized according to the protocol described in Avetyan et al., 1973.

N ⁴ -allylthiosemicarbazides of 2- (4-alkoxyphenyl) quinoline-4-carboxylic acids 6-10:

 Compounds 6-10 were synthesized by reaction of compounds 1-5 with ethylsothiocyanate in ethanol. A mixture of 0.3 g (0.003 mol) of allylisothiocyanate, 0.003 mol of hydrazide (compounds 1 -5) (Avetyan et al., 1973) and 12 ml of ethanol was refluxed for 3-4 h. and left overnight at 18 ° C. The precipitate was filtered and washed on a filter with ether.

4-Allyl-5- [2- (4-alkoxyphenyl) quinolin-4-yl] -4H-1,2,4-triazol-3-thiol 1-15:

The compounds 6-10 were cyclized in the presence of KOH to give the compounds 1-15. A mixture of 0.01 mole of N4-allylthiosemicarbazide 2- (4-alkoxyphenyl) -quinolin-4-carboxylic acid (compounds 6-10), 0.84 g (0.015 mol) of KOH and 35 ml of water was refluxed for 2-3 h, and the solution was filtered and acidified with acetic acid. The precipitate was filtered and recrystallized from absolute ethanol. Triazole-3-thiols may exist in tautomeric forms of thiolthione type (Cretu et al., 2010). S-substituted derivatives 17-21:

 Compounds 17-21 were synthesized by reaction of compounds 11-15 with 5-chloromethylfuran-2-carboxylic acid methyl ester (prepared according to Mndzhoyan and Grigoryan, 1956) in the presence of an equimolar amount of KOH. Dissolve 0.56 g (0.01 mol) of KOH in 25 ml of methanol, and then dissolve 0.01 mol of the corresponding triazole 11-15 under heating. After cooling the solution to ambient temperature, 1.76 g (0.01 mol) of 5-chloromethylfuran-2-carboxylic acid methyl ester (16) dissolved in 6 ml of ethanol and left for one hour were added. night. The mixture was refluxed for 4 h, the methanol was distilled off and the precipitate was filtered and recrystallized from methanol.

5- {4-Allyl-5- [2- (4-alkoxyphenyl) quinolin-4-yl] -4H-1,2,4-triazol-3-ylsulphanylmethyl} furan-2-carboxylic acids (22-26).

 0.001 mol of the corresponding 17-21 ester and 0.11 g (0.002 mol) of KOH were diluted in 16 ml of 50% by volume methanol in water. The mixture was refluxed for 3-4 h. The resulting solution was acidified with acetic acid at 18 ° C and the resulting precipitate was filtered and recrystallized from methanol. The products below have been obtained.

5- {4-Allyl-5- [2- (4-methoxyphenyl) quinolin-4-yl] -4H-1,2,4-triazol-3-ylsulphanylmethyl} -furan-2-carboxylic acid (22). 0.51 g (0.001 mole) of 5- {4-allyl-5- [2- (4-methoxyphenyl) quinolin-4-yl] -4H-1,2,4-triazol-3-ylsulfanylmethyl} -2- Furoate and 0.11 g (0.002 mol) of KOH were diluted in 16 ml of 50/50 mixture of water and methanol. The mixture was refluxed for 3-4 h. The solution was acidified with acetic acid (pH 4) and the precipitate was filtered and recrystallized from ethanol. Yield 0.4 g (82%), m.p. 150-152 ° C. Rf 0.65. IR-spectra, γ, cm ¹ : 1602, 1519, 837, 770 (CH = CH, aromatic), 1711 (C = O), 2500-3100 (OH). ¹ H NMR,? (Ppm): 3.88 (s, 3H, O (s, 2H, SCH _2), 4.80 (dq, 1H, Hz, J ₂ = 1.5 Hz, 6.51 (d, 1H, J = 3.3 Hz, H-fur), 7.00-7.06 (m, 3H, C ₆ H ₄ OCH ₃ ), 7.05 (d, 1H, J). = 3.3 Hz, H-fur.), 7.51-7.57 (m, 1H, C ₆ H ₄ ), 7.72-7.79 (m, 2H, C ₆ H ₄ ), 8, 09 (s, 1H, = CH, pyr), 8.11 (dd, 1H, Hz, J ₂ = 1.0 Hz, C ₆ H ₄ ), 8.21-8.26 (m, 2H, C ₆ H ₄ OCH ₃ ), 12.50 (br, 1H, COOH). HRMS [ESI ⁺ , MeOH]: calcd for C ₂₇ H ₂₃ O ₄ N ₄ S [M + H] ⁺ 499.1435, found 499.1441. Anal. el. calcd for C ₂₇ H ₂₂ N ₄ O ₄ S: C 65.04; H, 4.45; N, 11.24; S, 6.43; found: C, 65.17; H, 4.39; N, 11.13; S, 6.27. 5- {4-Allyl-5- [2- (4-ethoxyphenyl) quinolin-4-yl] -4H-1,2,4-triazol-3-ylsulphanylmethyl} -furan-2-carboxylic acid (23) - compound reference. 0.51 g (0.001 mol) of 5- {4-allyl-5- [2- (4-ethoxyphenyl) quinolin-4-yl] -4H-1,2,4-triazol-3-ylsulfanylmethyl} -2 Furate and 0.11 g (0.002 mol) of KOH were diluted in 16 ml of 50/50 mixture of water and methanol. The mixture was refluxed for 3-4 h. The solution was acidified with acetic acid (pH 4) and the precipitate was filtered and recrystallized from ethanol. Yield 0.41 g (80%), mp 156-157 ° C. Rf 0.68. IR-spectra, γ, cm ¹ : 1599, 1455, 847, 766 (CH = CH, aromatic), 1693 (C = O), 2500-3100 (OH). ¹ H NMR, δ (ppm): 1.45 (t, 3H, J = 7.0 Hz, CH ₃ ), 4.13 (q, 2H, J = 7.0 Hz, OCH ₂ ), 4.48 (dt, 2H, 4.60 (s, 2H, SCH ₂ ), 4.80 (dq, 1H,

5.72 (ddt, 1H, 6.51 (d, 1H, J = 3.4 Hz, H-fur), 6.98-7.03 (m, 2H, C ₆ H ₄ OC ₂ H ₅ ), 7.05 (d, 1H). , J = 3.4 Hz, H-fur.), 7.50-7.57 (m, 1H, C ₆ H ₄ ), 7.72-7.79 (m, 2H, C ₆ H ₄ ), 8.08 (s, 1H, = CH, pyr), 8.11 (dd, 1H, Hz, J ₂ = 1.0 Hz, C ₆ H ₄ ), 8.19-8.25 (m, 2H, C ₆ H ₄ OC ₂ H ₅ ), 12.50 (br, 1H, COOH). HRMS [ESI ⁺ , MeOH]: Calculated for C ₂₈ H ₂₅ O ₄ N ₄ S [M + H] ⁺ 513.1591, found 513.1590. Anal. el. calcd for C ₂₈ H ₂₄ N ₄ O ₄ S: C 65.61; H, 4.72; N, 10.93; S, 6.26; found: C 65.54; H, 4.66; N, 10.85; S, 6.07.

5- {4-Allyl-5- [2- (4-propoxyphenyl) quinolin-4-yl] -4H-1,2,4-triazol-3-ylsulphanylmethyl} -furan-2-carboxylic acid (24). 0.53 g (0.001 mol) of 5- {4-allyl-5- [2- (4-propoxyphenyl) quinolin-4-yl] -4H-1,2,4-triazol-3-ylsulfanylmethyl} -2 Furate and 0.11 g (0.002 mol) of KOH were diluted in 16 ml of 50/50 mixture of water and methanol. The mixture was refluxed for 3-4 h. The solution was acidified with acetic acid (pH 4) and the precipitate was filtered and recrystallized from ethanol. Yield 0.45 g (86%), mp 149-150 ° C. Rf 0.62. IR-spectra, γ, cm ¹ : 1598, 1456, 840, 766 (CH = CH, aromatic), 1694 (C = O), 2500-3100 (OH). ¹ H NMR,? (Ppm): 1.09 (t, 3H, J = 7.4 Hz, CH _3), 1, 78-1, 90 (m, 2H, CJH ₂ CH _3), 4.02 ( t, 2H, J = 6.4 Hz, OCH ₂ ), 4.48 (br, d, 2H, J = 5.0 Hz, CH ₂ CH = CH ₂ ), 4.60 (s, 2H, SCH ₂ ), 4.80 (br, d, 1H, J = 17.2 Hz, CH ₂ CH = CH ₂ ), 5.08 (br, d, 1H, J = 10.4 Hz,

Hz, CH ₂ CJH = CH _2), 6.51 (d, 1H, J = 3.4 Hz, H-fur.), 6.98 to 7.03 (m, 2H, C ₆ H ₄ OC H ₇ ), 7.04 (d, 1H, J = 3.4 Hz, H-fur), 7.53 (ddd, 1H, Hz, C ₆ H ₄ ), 7.73-7.79 (m, 2H, C ₆ H ₄ ), 8.08 (s, 1H, = CH, pyr), 8.11 (br, d, 1H). , J = 8.3 Hz, C ₆ H ₄ ), 8.19-8.24 (m, 2H, C ₆ H ₄ OC H ₇ ), 12.50 (br, 1H, COOH). HRMS [ESI ⁺ , MeOH] calcd for C ₂₉ H ₂₇ O ₄ N ₄ S [M + H] ⁺ 527.1748, found 527.1753. Anal. el. calcd for C ₂₉ H ₂₆ N ₄ O ₄ S: C, 66.14; H, 4.98; N, 10.64; S, 6.09; found: C, 66.03; H, 4.75; N, 10.90; S 6.17. 5- {4-Allyl-5- [2- (4-butoxyphenyl) quinolin-4-yl] -4H-1,2,4-triazol-3-ylsulphanylmethyl} -furan-2-carboxylic acid (25). 54 g (0.001 mol) of 5- {4-allyl-5- [2- (4-butoxyphenyl) quinolin-4-yl] -4H-1,2,4-triazol-3-ylsulfanylmethyl} -2- Furoate and 0.11 g (0.002 mol) of KOH were diluted in 16 ml of 50/50 mixture of water and methanol. The mixture was refluxed for 3-4 h. The solution was acidified with acetic acid (pH 4) and the precipitate was filtered and recrystallized from ethanol. Yield 0.44 g (81%), mp 158-159 ° C. Rf 0.70. IR-spectra, γ, cm ¹ : 1599, 1455, 840, 766 (CH = CH, aromatic), 1693 (C = O), 2500-3100 (OH). ¹ H NMR, δ (ppm): 1.02 (t, 3H, J = 7.3 Hz, CH ₃ ), 1.48-1.61 (m, 2H, CH ₂ CH ₃ ), 1.75-1.85 (m, 2H, CH ₂ CH ₂ O), 4.05 (t, 2H, J = 6.4 Hz, OCH ₂ ), 4.48 (dt, 2H, J = 1.7 Hz, aH ₂ CH = CH ₂ ), 4.60 (s, 2H, SCH ₂ ), 4.80 (dq,

, J ₂ = 1.7 Hz, 6.52 (d, 1H, J = 3.4 Hz, H-fur), 6.97-7.03 (m, 2H, C6H4OC4H9), 7.05 (d, 1H, J = 3.4 Hz). , H-fur.), 7.53 (ddd, 1H, Hz, C ₆ H ₄ ), 7.73-7.79 (m, 2H, C ₆ H ₄ ), 8.08 (s, 1H, = CH, pyr), 8.11 (dd, 1H, Hz, J ₂ = 1.2 Hz, C ₆ H ₄ ), 8.19-8.24 (m, 2H, C ₆ H ₄ OC ₄ H 9), 12.70 (br, 1H, COOH). HRMS [ESI ⁺ , MeOH]: calcd for C30H29O4N4S [M + H] ⁺ 541.1904, found 541.1915. Anal. el. calcd for C30H28N4O4S: C, 66.65; H, 5.22; N, 10.36; S, 5.93; found: C, 66.52; H, 5.10; N, 10.23; S, 6.07.

5- {4-Allyl-5- [2- (4-pentyloxyphenyl) quinolin-4-yl] -4H-1,2,4-triazol-3-ylsulphanylmethyl} -furan-2-carboxylic acid (26). 0.55 g (0.001 mol) of 5- {4-allyl-5- [2- (4-pentyloxyphenyl) quinolin-4-yl] -4H-1,2,4-triazol-3-ylsulfanylmethyl} -2 furoate and 0.11 g (0.002 mol) of KOH were diluted in 16 ml of 50/50 mixture of water and methanol. The mixture was refluxed for 3-4 h. The solution was acidified with acetic acid (pH 4) and the precipitate was filtered and recrystallized from ethanol. Yield 0.47 g (85%), mp 165-166 ° C. Rf 0.69. IR-spectra, γ, cm ^"1: 1600, 1455, 841, 766 (CH = CH, aromatic), 1693 (C = 0), 2500-3100 (OH) ¹ H NMR? (Ppm):. 0, 97 (t, 3H, J = 7.1Hz, CH ₃ ), 1.36-1.55 (m, 4H, CH ₂ CH ₂ ), 1.76-1.86 (m, 2H, OCH ₂ CH ₂ ), 4.04 (t, 2H, J = 6.5 Hz, OCH ₂ ), 4.48 (dt, 2H,

4.60 (s, 2H, SCH ₂ ), 4.80 (dq, 1H, 5.08 (dq, 1H, Hz, J ₂ = 10.4 Hz, J ₃ = 5.1 Hz, CH ₂ CH = CH ₂ ), 6.52 (d, 1H, J = 3.4 Hz, H-fur), 6.97. -7.03 (m, 2H, C 6 H 4 OC 5 H 11), 7.05 (d, 1H, J = 3.4 Hz, H-fur), 7.53 (ddd, 1H, Hz, C ₆ H ₄ ), 7.73-7.79 (m, 2H, C ₆ H ₄ ), 8.08 (s, 1H, = CH, pyr), 8.11 (dd, 1H, Hz, J ₂ = 1.2 Hz, C ₆ H ₄ ), 8.19-8.24 (m, 2H, C ₆ H ₄ OC 5 H 11), 12.66 (br, 1H, COOH). HRMS [ESI ⁺ , MeOH]: calculated for C31H31O4N4S [M + H] ⁺ 555.2061, found 555.2057. Anal. el. calcd for C31H30N4O4S: C, 67.13; H, 5.45; N, 10.10; S, 5.78; found: C, 66.95; H, 5.37; N, 10.18; S, 5.66.

Il - Biological results

Material and method :

 Chemical products and reagents. Compounds Carnetinib (CI-1033) and Gefitinib (ZD1839) were purchased respectively from Sigma-Aldrich and Santa Cruz Biotechnology. 25mM stock solutions of the synthesized and commercial chemical compounds were prepared in DMSO (99.9% purity, OriGen Biomedical) and aliquots were stored at -80 ° C. Pierce® IP and RIPA lysis buffers , protease inhibitor / phosphatase cocktail tablets, human anti-EGFR, anti-ErbB2 and anti-6-actin primary antibodies are from Thermo Fisher. The primary human antibodies anti-phospho-EGFR (Tyr1068), anti-Hsp90a, anti-HDAC2 and rabbit and mouse secondary antibodies are from R & D Systems. The human monoclonal antibodies against EGFR (A-10), LC3a / 6 and Hsp70 bovine are from Santa Cruz Biotechnology. The anti-pTyr mouse monoclonal antibody (pTyr-100) conjugated to biotin is from Cell Signaling Technology. Protein G is from Bio-Rad. The WesternSure chemiluminescent substrate was provided courtesy of Li-Cor.

Cell culture. MDA MB468 triple negative breast cancer cells were cultured in DMEM medium (Dulbecco's modified Eagle's medium) supplemented with 10% fetal calf serum (FCS), penicillin (100 units / ml), and streptomycin (100 mcg / ml). To evaluate the action of the compounds, the cells were washed with PBS and deprived of nutrients in DMEM media free of FBS for 18 hours. To stimulate phosphorylation of EGFR, cells treated or not treated with the compounds were washed with TBS and incubated in FCS-free medium containing EGF natural ligand (200 ng / ml). Cells were washed twice with cold TBS and then lysed in IP or RIPA lysis buffers in the presence of protease and phosphatase cocktail inhibitors, scraped off, transferred to tubes and centrifuged at 14,000 g. 10 minutes. Supernatant fractions of cell lysates were stored at -80 ° C. Relative protein levels were measured as a ratio of individual β-actin proteins in the same sample and normalized with corresponding values of untreated samples (vehicle) being considered 100%. Unless otherwise indicated, concentrations of 25 μΜ and 50 μΜ of compounds were used in the DMEM medium free from FCS or in DMEM supplemented with 10% (v / v) FCS.

 Cell viability test. Stock solutions of compounds were diluted to the appropriate concentrations in fresh media prior to each run. The final concentration of DMSO was less than 0.1%. MDA MB468 breast cancer cells were cultured in DMEM medium not supplemented or supplemented with FCS in the presence of the compounds for 72 h. Cell viability was determined by a colorimetric method (Vindelov, 1977). The tests were performed three times and the calculated values of ICso represent an average.

 Cell detachment test. Culture medium and a suspension washed once with PBS of MB468 MDA cells grown after treatments at the indicated times were collected by pipetting and combined in a tube (called detached cells). The detached cells collected were collected by centrifugation at 14,000 g for 10 min, and after having removed the supernatant, the pellet was incubated in 100 μl or 40 μl of RIPA lysis solution. The rest of the cells attached to a surface were lysed in 400 μl of RIPA lysis solution and collected by scraping. The efficiency of cell detachment by the compounds was estimated as the ratio of protein levels in the detached cell fraction to attached cells using Western Blot analysis. This approach has made it possible to simplify the evaluation of cell detachment efficiency and cell death, anoïkose. Immunoprecipitation. The supernatant fractions of extracts obtained by lysis of cells in a moderate strength IP lysis buffer were incubated with an anti-EGFR monoclonal antibody (A-10) for 2 h, washed with PBS and the immunoprecipitated proteins were collected. on G protein conjugated to magnetic beads according to the Bio-Rad protocol.

RNA interference for knockdown of EGFR. MDA MB468 cells were incubated in DMEM medium supplemented with 10% FCS for 18 h. Transfection of the siRNAs was performed with 60 pmol of siRNA duplex specific for human EGFR or siRNA control according to the recommendations of the manufacturer Santa Cruz Biotechnology. The transfected cells were allowed to recover in DMEM supplemented with FCS for 48 h and then deprived of nutrients in DMEM without FCS for 18 h before exposure to 25 or 26 for 2 h. Receptor knockdown was approximately 40% with EGFR siRNA compared to siRNA-transfected cells in two experiments independent. A moderate decrease effect can be explained by overexpression of EGFR in MDA MB468 cells.

 Western blot. Equal amounts of samples were used for protein electrophoresis by SDS-PAGE and then transferred to nitrocellulose membrane with a Trans-Blot Turbo system (Bio-Rad). Chemiluminescent detection of the transferred proteins and quantification were performed with the C-Digit scanner (Li-COR). The level of a charged control protein, 6-actin, was generally unchanged in cells treated with different compounds at the concentrations used. However, it was noticed that compounds at concentrations greater than 25 μΜ and longer exposure could lead to detachment of cells in the plate. In order to exclude the interference of cell detachment on the decrease of the amount of protein to be tested, the ratio of the signal intensity of each individual protein to β-actin was estimated in the same treated sample and normalized values. were obtained with the ratio of the same protein to β-actin in the samples incubated with a vehicle considered as 100%.

Results:

 Evaluation of the decrease of EGFR level by compounds in cancer cells.

To evaluate the ability of compounds 22 to 26 to modulate EGFR tyrosine phosphorylation, an MDA MB468 negative triple breast cancer cell line, in which the receptor is overexpressed compared to the low expression of its ErbB2 homologue (Filmus et al. ., 1985), was used. The cells were cultured in serum-free DMEM for 18 h, and the cells were treated with 100 μl of the compounds for 90 min, followed by induction of EGFR with an EGF parent ligand. Western blot analysis revealed that compounds 25 and 26 remarkably suppress tyrosine phosphorylation and decrease the level of expression of the receptor (Figure 1A). On the other hand, the cells are incubated with compound 25 or 26 without subsequent addition of EGF, the level of expression of EGFR decreases again relative to cells incubated with a vehicle (DMSO). A covalent EGFR inhibitor, namely CI-1033 used as a control, suppresses tyrosine phosphorylation without reducing the level of expression of the receptor. A comparative evaluation of the action of the compounds showed that Tyr1068 phosphorylation and EGFR expression decreased dose-dependently in MB468 MDA cells deprived of serum and treated with compounds 25 and 26 for 2 h then incubated with EGF (Figure 1B). The cells showed a progressive suppression of tyrosine phosphorylation of other protein kinases detected by the anti-pTyr antibody. These data show that the compounds of the invention are capable of attenuating signal transduction in signaling pathways downstream of EGFR.

 The decrease in the level of EGFR is accompanied by a decrease in the amount of Hsp90a, which is clearly visible at the concentration of 25 μΜ of compounds. In addition, a decrease in functionally independent protein levels, such as ErbB2, HDAC2 and β-actin, has also been detected in cells with higher concentrations of compounds.

 The ability of compounds 25 and 26 to suppress tyrosine phosphorylation and to reduce the level of EGFR, resulting in decreased levels of Hsp90a and other functionally independent proteins, shows that the compounds of the invention induce degradation of the proteins in breast cancer cells.

 The compounds according to the invention lead to the simultaneous degradation of EGFR and Hsp90a.

The action of the compounds on the interactions of EGFR with Hsp90a was studied using immunoprecipitation tests. MDA MB468 cells cultured in serum-free DMEM were treated with compounds 23, 25 and 26 and then lysed in IP moderately strong buffer. The putative complexes formed between EGFR and other proteins were captured with anti-EGFR antibody from extracts and characterized by Western blotting. A small amount of EGFR was detected in the immunoprecipitated extract from cells exposed to compound 26 which is consistent with the low level of receptor detected in in-put control samples (Figure 2). Analysis revealed a 90 kDa diffuse band corresponding to Hsp90a co-immunoprecipitated with EGFR from extracts of cells treated with the compounds, with the highest yield of chaperone in the sample for treatment with compound 23 and lowest with compound 26 (Figure 2A). No band corresponding to Hsp70 was detected in immunoprecipitated extracts, whereas this 70 kDa chaperone was detected with in-put samples and at lower levels in cells exposed to compound 25 or 26 (Figure 2B).

 Thus, compounds 25 and 26 do not interfere with protein interactions between EGFR and Hsp90a. Co-immunoprecipitation of Hsp90a with EGFR, even from extracts with low abundance of the receptor, demonstrates the degradation of an EGFR / Hsp90a complex rather by compound-induced endocytosis in MDA MB468 cells.

 Protein profiles were also evaluated in cells in which the level of EGFR expression was reduced by specific siRNA knockdown. MB468 MDA cells transfected with EGFR-specific siRNA were cultured in serum-free DMEM medium and then treated with the compounds. The cells displayed a remarkable decrease in EGFR and Hsp90a protein levels when exposed to compound 26 (and less visibly to compound 25) relative to the vehicle (Figure 3). The effect was lower in cells transfected with scrambled siRNA. Thus, decreasing the amount of EGFR simultaneously reduces the amounts of the Hsp90a receptor and chaperone thereby highlighting the role of EGFR related to compounds 25 and 26 in the eventual loss of proteins.

 LC3a / LC36 autophagic biomarkers were also evaluated in siRNA-transfected cells, taking into account that LC36 accumulation is correlated with increased autophagosome numbers, resulting in degradation of protein deprived of nutrients ( Kabeya et al., 2000). A 16 kDa band corresponding to LC36 was detected in cells transfected with EGFR-specific siRNA or scrambled siRNA as an indication of the autophagic degradation of the proteins during growth in serum-free medium. However, quantification of the LC36 ratio to a charged control β-actin revealed that the relative level of this autophagic biomarker is about 50% higher after exposure to compounds 26 and 25 relative to the vehicle. Therefore, compounds 26 and 25 improve the degradation of nutrition-promoting proteins in cancer cells.

Two different methods demonstrated that compounds 25 and 26 result in a decrease in EGFR that is associated with the reduction of the amount of Hsp90a in the serum-deprived cells for two hours. It can be concluded that the compounds according to the invention, bound to the receptor protein, induce endocytosis of the EGFR / Hsp90a complex, which undergoes an increased degradation with lysosomal proteases. By therefore, this leads to the depletion of Hsp90a and thus to the failure of the functional capacity of the chaperone machinery to correct the misfoldings in the client proteins, which would affect the viability of the cancer cells. The compounds according to the invention cause the detachment of cancerous cells leading to an anokosis.

 Fetal calf serum (FCS) provides a wide range of growth factors, including EGFs required for growth of human cells in culture medium (Brunner et al., 2010). In the absence of growth factors, healthy cells undergo ECM detachment followed by anoïkose, while cancer cells appear to be tolerant to detachment during nutrient deprivation (Bucheit et al., 2015). It was noted that incubation of MDA MB468 cells with compounds 25 and 26 in serum-free medium for more than one hour resulted in a decrease in the level of β-actin, used as a loaded control during SDS-PAGE. Since β-actin is one of the key cytoskeletal proteins, it was hypothesized that binding of the compound to EGFR affected attachment of cells to the surface of plaques, mimicking ECM, resulting in the loss of cancer cells that are typically the adherent cells.

To test this hypothesis, MDA MB468 cells were cultured in serum-free DMEM for 18 h and then treated with compounds 23, 25 and 26 for 6 hours. The culture medium on the cells attached to the surface of the plate was carefully collected and transferred to Falcon tubes. The wells were gently washed with hot PBS, and the washings were transferred to the tubes. The collected samples were centrifuged and the pellet containing the probably detached cells was treated with RIPA lysis solution. The cells attached to the plate surface were also collected and lysed with RIPA. Proteins in the detached attached and putative cell lysates were separated by electrophoresis on the same gel and analyzed by Western Blot.

This methodological approach revealed the presence of high amounts of cytoskeletal β-actin and α-tubulin proteins, as well as Hsp90a, but only traces of EGFR in the culture medium collected on the attached cells, which proves the detachment of cells treated with compounds 25 and 26 (Figure 4A). Although cell detachment was more pronounced with compound 26 than with compound 25, the relative levels of proteins in detached cells were close to both molecules, suggesting that proteins degrade by the same degradation pathways (Figure 4B).

Then, the ability of all the compounds according to the invention to detach cancer cells at the concentration of 25 μΜ after a treatment of 48 h in serum-free medium was evaluated. It has been found that a longer incubation with the vehicle results in cellular detachment as demonstrated by detection of β-actin, α-tubulin and Hsp90a, but not EGFR (Figure 5A). Long incubation of MB468 MDA cells with vehicle or with compounds 22 and 23 in serum-free medium resulted in the accumulation of nutritionally favorable proteins leading to a low rate of detachment of cancer cells. On the contrary, important differences were observed in the cells treated with compounds 24, 25 and 26. The detached cells displayed a lower level of 6-actin compared to the vehicle and no longer had Hsp90a or a-tubulin. . Similarly, cells attached after treatment with compounds 25 and 26 exhibited a lower level of β-actin and traces of α-tubulin relative to the vehicle. In addition, the cells do not contain EGFR and Hsp90a. Thus, active compounds 26, 25 and 24 not only increased protein degradation in cancerous cells deprived of nutrients, but probably altered signal transduction to the cytoskeleton leading to death by cellular detachment, anokosis. In parallel assays, protein profiles were evaluated in MB468 MDA cells treated with compounds at 50 μΜ after 48 h growth in DMEM supplemented with FCS. If no significant difference was detected in the protein profiles in the attached cells after treatment with the vehicle or compounds 22 and 23, low levels of 6-actin and α-tubulin were detected in detached cells (FIG. 5C). This observation suggests that, during a long culture, cells consume growth factors, including EGF from a medium supplemented with FCS, and cell growth becomes sensitive to some extent to nutrient deprivation leading to detachment of the cells. cells. Indeed, the other three active compounds 26, 25 and 24 showed a strong ability to degrade proteins, as demonstrated by the lower levels of EGFR and Hsp90a in attached cells and higher amounts of cytoskeleton proteins in detached cells. . As in the case of serum-deprived cells, the action of compound 24 was more visible in detached cells. To estimate the efficiency of the cell detachment induced by the compounds according to the invention, the ratio of β-actin was used because this protein was detected in all the samples. The efficiency of cell detachment was nearly 4% and 11% of cells after exposure of the cells to the vehicle and compound 26 in the medium with FCS, respectively, and it increased by almost 21% and 29% after exposure. vehicle and compound 25 in the serum-deprived medium, respectively (Figures 5B and 5D).

 Thus, the compounds of the invention 24, 25 and 26 have been found to be active in inducing degradation of EGFR and other proteins leading to detachment of cancer cells deprived of nutrients and not deprived of nutrients.

Inhibition of reversible and weak EGFR

 Since compound 26 causes visible degradation at 60 minutes of treatment, the question was whether this compound could inhibit phosphorylation of EGFR during a shorter exposure time. To exclude degradation of the recipient protein, MDA MB468 cells were incubated with 26 or Gefitinib (as a reference) for 10 and 25 minutes, then EGFR was induced in cells with EGF for 5 min. Compared to the strong inhibition of EGFR tyrosine phosphorylation with FDA-approved Gefitinib at both treatment times, the decrease in receptor band intensity with compound 26 was only detected at one time. 10 min exposure, while the receptor was perfectly phosphorylated at longer exposure (Figure 6). The ability of compound 26 to inhibit EGF-induced tyrosine phosphorylation has demonstrated its ability to inhibit in a very short period of time.

Inhibition of tumor growth in mice.

 Compounds 22 to 26 showed moderate to low toxicity in MDA MB468 cells, with an IC50 as follows:> 120 μΜ for compound 22, 116 μΜ for 23, 114 μΜ for 24, 31, 6 μΜ for 25 and 35 , 7 μΜ for 26.

The level of expression of EGFR is high in many types of epithelial cancer, including S180 mouse sarcoma (Sun et al., 2011). Thus, S180 sarcoma cells were implanted in mice to examine the antitumor ability of the compounds according to the invention. The animals were treated with compounds 22 to 26 at doses of 150-200 mg / kg for 6 days and tumor growth inhibition was determined on a group of 7-8 animals for each test compound. after implantation of sarcoma 180. All compounds inhibited tumor growth with an efficiency as follows: 43.3% for Compound 22, 24.4% for 23, 25.0% for 24, 39.0% for 25 and 36.7% for 26. The relative difference in mouse tumor inhibition and degradation efficiency between the compounds tested suggests that the metabolism of the compounds in the animal body is different from that in the cultured cells. Nevertheless, these data are consistent with protein degradation leading to anokosis of cancer cells through a cascade of events induced by EGFR - targeting compounds.

BIBLIOGRAPHIC REFERENCES

 Akiyama T., Dass C.R., Choong P.F. Bim-targeted cancer therapy: a link between drug action and the underlying molecular changes. Mol. Cancer Ther. 8; 3173-3180. doi: 10.1 158 / 1535-7163. MCT-09-0685 (2009).

 Allen L.F., Eiseman I.A., Fry D.W., LenehanPF.CI-1033, irreversible pan-erbB receptor inhibitor and its potential application for the treatment of breast cancer. Semin. Oncol. Suppl 16, 65-78 (2003).

 Avetyan S.A., Azaryan A.S., Aroyan A.A. Arm. Chem. J 1973, 26 (9), 763-767.

Bae, Y. S. et al. Epidermal growth factor (EGF) -induced generation of hydrogen peroxide. Role in EGF receptor-mediated tyrosine phosphorylation. J. Biol. Chem. 272, 217-221 (1997).

 Brunner D., Frank J., Appl H., Schofff H., Pfaller W., Gstraunthaler G. Serum-free cell culture: the serum-free interactive media online database. ALTEX 27, 53-62 (2010). Buchheit C.L., Angarola B.L., Steiner A., Weigel K.J., Schafer Z. Anoikis T. Escape in inflammatory breast cancer cells is mediated by Bim-EL sequestration. Cell Death Differ. 22.1275-86. doi: 10.1038 / cdd.2014.209 (2015).

 Campbell PJ, Pleasance E, Stephens PJ, et al. Subclonal phylogenetic structures in cancer revealed by ultra-deep sequencing. Proc Natl Acad Sci U.S.A. 105, 13081-13086 (2008).

 Cheng H., Nair S.K., Murray B.W. Recent progress on third generation covalent EGFR inhibitors. Bioorç. Med. Chem. Lett. 26, 1861-1868; doi: 10.1016 / j.bmcl.2016.02.067 (2016).

Citri A., Harari D., Shohat G., Ramakrishnan P., Gan J., Lavi S., Eisenstein M., Kimchi A., Wallach D., Pietrokovski S., Yarden Y. Hsp90 A common surface on customer kinases. J. Biol. Chem. 1281 (20), 14361-14369 (2006). Cretu, D. D., Barbuceanu, SF, Saramet, G., Draghici, C. Synthesis and characterization of some 1, 2,4-triazole-3 (4h) -thiones obtained from intramolecular cyclization of new 1 - (4- (4-x-phenylsulfonyl) benzoyl) -4- (4-iodophenyl) thiosemicarbazides. J. Serb. Chem. Soc.75, 1463-1471 (2010).

Echeverria P.C., Bernthaler A., Dupuis P., Mayer B., Picard D. An interaction network predicted from a data discovery tool: application to the Hsp90 molecular chaperone machine. PloS One 6 (10): e26044 (2011).

 Filmus, J., Pollak, MN, Cailleau, R., Buick, RN MDA-468, a human breast cancer cell receptor (EGF) receptors, has an amplified EGF receptor gene and is growth inhibited by EGF. Biochem Biophys Res Commun 128, 898-905 (1985).

 Frish S. M., and Francis H. Disruption of epithelial cell-matrix interactions induces apoptosis. J. Cell. Biol. 124, 619-626 (1994).

 Fung C, Lock R., Gao S., Salas E., Debnath J. Induction of autophagy during extracellular matrix detachment promotes cell survival. Mol. Biol. Cell 19, 797-806 (2008).

 Goh, L. K., Sorkin, A. Endocytosis of receptor tyrosine kinases. Cold Spring Harb Perspect Biol 5, a017459 (2013).

 Hao, JJ., DC Lin, Dinh HQ, Mayakonda A, Jiang YY, Chang C, Jiang Y, Lu CC, Shi ZZ, Xu X, Zhang Y, Y Cai, Wang JW, QM Zhan, Wei WQ, Berman BP, Wang MR, Koeffler, HP Spatial intratumoral heterogeneity and temporal clonal evolution in esophageal squamous cell carcinoma. Nature Genetics 48, 1500-1507, doi: 10.1038 / ng.3683 (2016).

 Han, W., Pan, H., Chen, Y., Sun, J., Wang, Y., Li, J., Ge, W., Feng, L., Lin, X., Wang, X., et al. EGFR tyrosine kinase inhibitors activate autophagy as a cytoprotective response in human lung cancer cells. PLoS ONE 6, e18691 (2011).

Herbst RS, Fukuoka M and Baselga J: Gefitinib - a novel targeted approach to treating cancer. Nat. Rev. Cancer 4, 956-965 (2004).

Jia, Y., Yun, CH, Park, E., Ercan, D., Manuia M., Juarez J., Xu C, Rhee K., Chen T., Zhang H., Palakurthi S., Jang J., Lelais G., DiDonato M., Bursulaya B., Michellys PY, Epple R., Marsilje TH, McNeill M., Lu W., Harris J., Bender S., Wong KK, Janne PA, Eck MJ Overcoming EGFR (T790M ) and EGFR (C797S) resistance with mutant-selective allosteric inhibitors. Nature https: //www.ncbi. nlm.nih.gov/pubmed/27251290534(7605), 129-32. doi: 10.1038 / nature17960 (2016). Kabeya Y., Mizushima N., Ueno T., Yamamoto A., Kirisako T., et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBOJ.19, 5720-5728 (2000).

 Kawakita Y., Seto M., Ohashi T., Tamura T., Yusa T., Miki H., Iwata H., Kamiguchi H., Tanaka T., Sogabe S., Ohta Y., Ishikawa T. Design and synthesis of novel pyrimido [4,5-b] azepine derivatives as HER2 / EGFR dual inhibitors. Bioorg. Med. Chem.21 (8), 2250-2221; doi: 10.1016 / j.bmc.2013.02.014 (2013).

 Klonsky, D.J., Abdelmohsen, K., Abe, A., Avedin, M.J., Abeliovich, H., Arozena, A.A. et al. Guidelines for the use and interpretation of assays for autophagy monitoring (3rd edition). Autophagy 12 (2), 443 (2016).

 Li S., Guo C, Sun X., Li Y., Zhao H., Zhan D., Lan M., Tang Y. Synthesis and biological evaluation of quinazoline and quinoline bearing 2,2,6,6-tetramethylpiperidine-N Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors and EPR bio-probe agents, fur. J. Med. Chem.49, 271-278. doi: 10.1016 / j.ejmech.2012.01.021. (2012).

 Mendelsohn, J., and Baselga, J. Epidermal growth factor receptor targeting in cancer. Semin. Oncol.33, 369-385 (2006).

 Miyata Y., Nakamoto H., Neckers L. The therapeutic target Hsp90 and cancer hallmarks. Current Pharma.19, 347-365 (2013).

 Mndzhoyan A. L., Grigoryan. M. T. In: The synthesis of heterocyclic compounds, Yerevan, 1, 36-38 (1956).

 Nisimura, Y., Bereczky, B., Ono, M. The EGFR inhibitor gefitinib suppresses ligand-stimulated endocytosis of EGFR via the early / late endocytic pathway in non-small cell lung cancer cells. Histochem. Cell. Biol. 127 (5), 541-553 (2007).

Paulsen, CE., Truong, T., Garcia, F., Homann, A., Gupta, V., Leonard, S. and Carroll, K. Peroxide-dependent sulfenylation of the EGFR catalytic site enhances kinase activity. Nat. Chem. Biol.8,57-64 (2012).

 Pick E., Kluger Y., Giltnane J.M., Moeder C, Camp R.L., D.L.M.M., Kluger H.M.HSP90 High expression is associated with decreased survival in breast cancer. Cancer Res.67, 2932-2937 (2007).

Sakanyan, V., Hulin, P., Sousa, R., Silva, V., Hambardsumyan, A., Nedellec, S., Tomasoni, C, Lodge, C, Pineau, C., Roussakis, C. et al. Activation of EGFR by small compounds through coupling the generation of hydrogen peroxide to stable dimerization of Cu / Zn SOD1. Sci. Rep.6 (2016). Schlessinger, J. Receptor Tyrosine Kinases: Legacy of the first two decades. Cold Spring Harb Perspect. Biol. 6, a008912 (201).

 Sigismund S., Woelk T., Puri C, Maspero E., Tacchetti C, Transidico P., Di Fiore P. P., Polo S. Clathrin-independent endocytosis of ubiquitinated cargos. Proc Natl Acad Sci 102, 2760-2765 (2005).

 Sigismund S, Argenzio E, Tosoni D, Cavallaro E, Polo S, Di Fiore PP. Clathrin-mediated internalization is essential for sustained EGFR signaling but dispensable for degradation. Dev Cell 15, 209-219, (2008).

 Smail JB, AJ Gonzales, Spicer JA, Lee H, Reed JE, Sexton K, Althaus IW, Zhu T, Black SL, Blaser A, Denny WA, Ellis PA, Fakhoury S, Harvey PJ, K Hook, McCarthy FO, Palmer BD , Rivault F, Schlosser K, Ellis T, Thompson AM, Trachet E, Winters RT, Tecle H, A. Tyrosine Kinase Inhibitors: 20. Optimization of substituted quinazoline and pyrido [3,4-d] pyrimidine derivatives as orally active, irreversible inhibitors of the Epidermal Growth Factor Receptor family. J. Med. Chem. 59 (17), 8103-8124. doi: 10.1021 / acs. jmedchem.6b00883, (2016).

 Stoscheck, C., Carpenter G. Characterization of the metabolic turnover of epidermal growth factor receptor protein in A-431 cells. J. Cell. Physiol. 120, 296-302 (1984). Sun, J.; Zhao, L.; Teng, L.; Lin, F.; Zhang, H.; Li, Z .; Gao, Q. Solid tumor-targeted infiltrating cytotoxic T lymphocytes retained by a superantigen fusion protein. PLoS One 6, e16642 (201 1).

 Taipale, M., Jarosz, D.F., Lindquist, S.HSP90 at the hub of protein homeostasis: emerging mechanistic insights. Nat. Rev. Mol. Cell. Biol. 1, 51-5-528 (2010).

Vindelov, L. L. Flow microfluorometric analysis of nuclear DNA in cells from solid tumors and cell suspensions. A new method for rapid isolation and straining of nuclei. Virchows Arch. B Cell. Pathol. 24, 227-242 (1977).

 Vlahakis A., Debnath J. The interconnections between autophagy and integrin-mediated cell adhesion. J. Mol. Biol. 429, 51-5-530 (2017).

 Wang, M., Shen, A., Zhang, C., Song, Z., Ai, J., Liu, H., Sun, L., Ding, J., Geng, M., Zhang, A. Development of Heat Shock Protein (Hsp90) inhibitors to fight resistance to tyrosine kinase inhibitors through Hsp90-kinase interactions. J. Med. Chem. 59, 5563-5586. doi: 10.1021 /acs.jmedchem.5b01 106 (2016).

Claims

Compound of formula (below:

or a pharmaceutically acceptable salt thereof,

wherein R represents an alkyl group having 1 to 10 carbon atoms.

2. Compound according to claim 1, characterized in that R is a linear alkyl group.

3. Compound according to claim 1 or 2, characterized in that R has 3 to 6 carbon atoms, in particular 3, 4 or 5 carbon atoms.

4. Compound according to any one of claims 1 to 3, characterized in that it is chosen from:

and their pharmaceutically acceptable salts.

5. A compound according to any one of claims 1 to 4 for use as a medicament.

6. Compound according to any one of claims 1 to 4 for its use in the prevention or treatment of cancer, including in particular the prevention of metastasis, the treatment of cardiovascular diseases such as stroke, stroke and stroke. ischemia, neurodegenerative diseases such as Alzheimer 's disease or glaucoma, or the regeneration of injured optical nerve fibers.

Pharmaceutical composition comprising at least one compound according to any one of claims 1 to 4.

8. Pharmaceutical composition according to claim 7, characterized in that it further comprises at least one pharmaceutically acceptable excipient.

9. Pharmaceutical composition according to claim 7 or 8 for its use in the prevention or treatment of cancer, including in particular the prevention of metastases, the treatment of cardiovascular diseases such as stroke, ischemia, neurodegenerative diseases such as Alzheimer 's disease or glaucoma, or the regeneration of injured optical nerve fibers.

10. Process for the preparation of a compound according to any one of claims 1 to 4 comprising the hydrolysis of the ester function of a compound of formula (I I) below:

in which R is as defined in claim 1 and R 1 represents an alkyl group comprising 1 to 6 carbon atoms, in particular a methyl or ethyl group, to give a compound of formula (I) according to claim 1,

optionally followed by a salification step in the presence of a pharmaceutically acceptable acid or base to give a pharmaceutically acceptable salt of a compound of formula (I) according to claim 1.