Synthesis of new tetralone derivatives as modulators of the GPER pathway

Abstract

The present invention relates to novel tetralone derivatives as modulators of the GPER pathway, and their application in particular to the control of nociception. Abstract figure: Figure 13

Description

Title of the invention: Synthesis of novel tetralone derivatives as modulators of the GPER pathway Technical field of the invention

[0001] The present invention relates to novel tetralone derivatives as modulators of the GPER (Gprotein-coupled estrogen receptor) pathway.

[0002] The present invention finds particular application in the control of nociception, especially in the prevention or treatment of cancer-related pain (cancer, metastatic, chemotherapy-induced, and inflammatory pain), particularly in the context of triple-negative breast cancer (TNBC). The novel derivatives also find application as anticancer agents, particularly in the context of triple-negative breast cancer (TNBC), but also in the prevention of chemotherapy-induced peripheral neuropathies as well as other neuropathies (diabetes) and neurodegenerative diseases.

[0003] In the description below, references in brackets ([ ]) refer to the list of references presented at the end of the text. Prior art

[0004] Since 2020, pain has been defined by the IASP (International Association for the Pain is defined as "an unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage" (Raja et al., 2020) [1]. In terms of temporal framework, pain can evolve in two ways: 1) acute, constituting an alarm signal for the body, and 2) chronic (lasting more than at least three months), thus indicating a true pathology whose prevalence in the general population is far from trivial (International Classification of Diseases ICD-11, 2019).

[0005] Currently, and according to the 1TASP terminology, three types of chronic pain are identified based on the underlying neurobiological mechanisms that cause them: 1) nociceptive pain (corresponding to local tissue damage, mechanical or inflammatory: inflammatory pain, osteoarthritis, etc.), 2) neuropathic pain (related to a lesion of the central or peripheral nervous system), and 3) nociplastic pain, also called psychogenic pain (due to a disruption of pain modulation at the cerebral and / or peripheral level and, until 2017, referred to as dysfunctional pain such as fibromyalgia, irritable bowel syndrome, and complex regional pain syndrome type 1, etc.). Cancer pain is a special case because it frequently combines nociceptive and neuropathic pain.

[0006] The prevalence of chronic pain is significant, estimated at 31.7% in France. This pain represents a major factor in impaired quality of life and can be associated with comorbidities such as depression, anxiety, insomnia, etc. It results in a significant economic and social burden. People with chronic pain are generally more prone to absenteeism than presenteeism at work, and the medical expenses incurred for these patients are generally higher.

[0007] Many patients report that the pain they experience is not under control. Indeed, the various treatments indicated for chronic pain conditions have limited effectiveness or significant side effects.

[0008] Faced with this observation, the management of chronic pain is not always optimal due to an aging pharmacopoeia and an often unsatisfactory benefit-risk ratio.

[0009] It is therefore important to have access to treatment solutions for this pain with an improved benefit-risk ratio, or alternative solutions for pain management. This major public health problem calls for significant innovation.

[0010] Since 2004, cancer has been the leading cause of premature death in France, surpassing cardiovascular diseases. Thus, cancer is the leading cause of death in men and the second leading cause in women. It is estimated that 3.8 million people are currently living in France with a cancer diagnosis. Globally, according to the latest estimates from the International Agency for Research on Cancer (IARC), 19.3 million new cases of cancer were diagnosed worldwide in 2020, with 9.9 million deaths, particularly from triple-negative breast cancer (TNBC).

[0011] Cancer treatment often relies on chemotherapy, which can cause chemotherapy-induced neuropathies. Chemotherapy-induced neuropathies are particularly common, debilitating, and difficult to treat. Moreover, they are dose-dependent. Peripheral nerve damage accounts for the majority of neurological impairments related to chemotherapy toxicity. It results from direct toxic damage to the axon or its demyelination and represents the most frequent limiting factor after hematological toxicity.

[0012] Thus, in the face of the appearance of chemo-induced neuropathies, the doses of chemotherapy are reduced, or even the treatment stopped, which constitutes a real loss of opportunity for the patient.

[0013] This is how neuropathies have been observed following treatment with alkaloids (vincristine, vinblastine, vinorelbine) with damage to small fibers, as, for example, with platinum derivatives (oxaliplatin, cisplatin, carboplatin), Anti-topoisomerase (VP16), proteasome inhibitors (bortezomib, carfilzomib), thalidomide derivatives such as lenalidomide, or taxanes such as Taxol or Taxotere, which primarily target large nerve fibers, are all potential treatments. Neuropathies can also occur following immunotherapy, such as with anti-CD20, anti-CD30, or anti-CD38 agents.

[0014] In patients with cancer, in addition to these peripheral neuropathic pains, bone metastatic pains may be added, at least in cases of advanced CSTN.

[0015] TNBC is an aggressive form of breast cancer that is heterogeneous and presents a variety of clinical signs and morphological and molecular characteristics (Won and Spruck, 2020) [2]. TNBCs represent approximately 15 to 20% of breast cancers (Yin et al., 2020; Almansour, 2022) [3,4]. Their immunohistochemical profile reveals an absence of estradiol receptor (ER) and progesterone receptor (PR) expression, and overexpression of HER2 (human epidermal growth factor receptor 2) receptors (Yin et al., 2020) [3]. TNBCs often occur in premenopausal women under 40 years of age (Yin et al., 2020) [3]. The average survival rate for CSTN is approximately 10.2 months with current treatments and rises to approximately 65% ​​at 5 years in cases of local tumors and 11% in cases of metastatic tumors (Won and Spruck, 2020) [2].The clinical profile of TNBC reveals a particularly aggressive type of cancer with a high risk of local and distant recurrence, as half of patients with early-stage TNBC experience a recurrence within 3 to 5 years of diagnosis (Li et al., 2022) [5]. Approximately 45% of patients with TNBC develop metastases (Okorafor et al., 2024) [6], particularly bone metastases. Breast cancer management relies on a multidisciplinary approach involving medical oncology, surgical oncology, radiation oncology, and drug therapy. The absence of hormone receptors or HER2 overexpression limits treatment options despite the recent development of personalized therapies such as PARP (poly(ADP-ribose) polymerase) inhibitors and immunotherapy. Consequently, intensive chemotherapy remains the standard pharmacological treatment for patients with TNBC.These include alkylating agents (e.g., cyclophosphamide), anthracyclines (e.g., doxorubicin, a topoisomerase blocker, and DNA intercalating agents), antimetabolites (e.g., fluorouracil), and microtubule-stabilizing agents (e.g., taxane) such as paclitaxel (Almansour, 2022) [4]. Used as first-line therapy for various cancers (Alves et al., 2018) [7], paclitaxel causes chemotherapy-induced neuropathic pain (CINP) in 80% of patients with TNBC (Molassiotis et al., 2019) [8], which is managed by [methods]. analgesics, antidepressants, or anticonvulsants (Staff et al., 2017; Moisset et al., 2020) [9,10]. However, there is no effective medication to prevent their development, which may be accompanied by bone metastatic pain (Zajqczkowska et al., 2019)

[11] for which no treatment is available.

[0016] The G protein-coupled estrogen receptor (GPER) is a seven-transmembrane-domain protein identified as binding λ7[3-estradiol and structurally distinct from the classical estrogen receptors α and [3] (ROα and RO[3]) (Wnuk et al., 2023)

[12] . GPER regulates numerous physiological functions such as metabolism, cell proliferation, and pain perception, and is implicated in multiple pathologies such as cancer, autoimmune diseases, and visceral hypersensitivity (Prossnitz and Barton, 2023)

[13] . Studies concerning GPER have notably highlighted an important role of the latter in pain processes (Deliu et al., 2012; An et al., 2014; Zielihska et al., 2017; Xu et al., 2021; Jiao et al., 2023) [14-18], tumor growth and metastasis formation (Wang et al., 2010; Lappano et al., 2014; Jacenik et al., 2016; Xu et al., 2019; Tirado-Garibay et al., 2024) [19-23].The Inventors highlighted that the GPER membrane protein could be used as an innovative therapeutic target in the control of chronic and cancer pain (Mallet, 2021)

[28] . In the context of TNBC, modulation of GPER, which exhibits higher expression levels than in other breast cancer subtypes (Steiman et al., 2013)

[25] , particularly by an inverse agonist (e.g., peptide ERal7p, ROa residues 295-311; PLMI motif "Pro Leu Met Ile" (SEQ ID NO: 1), ROa residues 295-298), induces both anti-tumor (Lappano et al., 2019; Kampa et al., 2023) [26,27] and analgesic (Mallet et al., 2021; Jouffre et al., 2023) [28,29] effects, even at the same low doses. However, this peptide has several limitations: it is not orally bioavailable, it forms aggregates at high concentrations, and it has a short half-life. Description of the invention

[0017] The present invention provides a solution to all or part of the problems of the prior art. The inventors have thus developed new tetralone derivatives targeting the GPER pathway. These new derivatives were tested on an animal model (mouse) of chronic neuropathic pain induced by chronic paclitaxel injections and on tumor growth (cell assay). The anti-allodynic effects were evaluated by measuring the 50% paw withdrawal threshold and using an adaptation of Dixon's up-down method by applying von Frey filaments. These original tetralone derivatives targeting GPER have shown their usefulness as antitumor and analgesic agents, particularly in the treatment of triple negative breast cancer (TNBC) and associated chronic pain (e.g., bone metastatic pain).

[0018] The present invention therefore relates to a tetralone derivative of general formula (I) following:

[0019] [Chem.l] (I) where Ri is H or CH3; R2 is H, OH or OCH3; R3 is H or F, OCH3, CH3 or Br; R4 and R5 are H, OCH3 or together form a 1,3-dioxolane; R6 is H or Br.

[0020] According to a particular embodiment of the present invention, said derivative has a formula (I) where Rh R2, R3 and R6 are as defined in claim 1 and where R4 and R5 are H.

[0021] According to a particular embodiment of the present invention, said derivative has a formula (I) where Rh R2, R3 and R6 are as defined in claim 1 and where R4 and R5 together form a 1,3-dioxolane.

[0022] According to a particular embodiment of the present invention, said derivative has one of the following formulas:

[0023] [Chem.2] E 1

[0024] [Chem.3] E2

[0025] [Chem.4]

[0026] [Chem.5] E4

[0027] The present invention also relates to a pharmaceutical composition comprising at least one derivative according to the invention, and a pharmaceutically acceptable excipient.

[0028] According to a particular embodiment of the present invention, the pharmaceutical composition further comprises at least one anti-tumor agent and / or one pain-relieving (analgesic) agent, different from the derivatives according to the invention.

[0029] According to a particular embodiment of the present invention, said at least one anti-tumor agent is paclitaxel, oxaliplatin or bortezomib and / or said at least one analgesic is a morphine-based pain reliever, for example an agent selected from codeine, tramadol, morphine, oxycodone, or a non-morphine-based pain reliever, in particular a non-steroidal anti-inflammatory agent (NSAID), a pain reliever (e.g. paracetamol) or a local anesthetic agent.

[0030] The present invention also relates to a derivative or composition according to the invention, for use as a medicinal product.

[0031] According to a particular embodiment of the present invention, said drug is an analgesic and / or an anti-tumor agent. In particular, the drug is intended for the control of nociception.

[0032] The present invention also relates to a derivative or composition according to the invention, for use in the prevention or treatment of pain, for example, acute or chronic pain, skin pain, muscle pain, joint pain, osteoarticular pain, visceral pain, tendon pain, postoperative pain, dental pain, cancer pain (e.g., metastatic bone pain), or post-traumatic pain. This may include pain associated with arthritis, osteoarthritis, inflammatory bowel disease, irritable bowel syndrome, migraine, headache, myalgia, tendinitis, back pain, or lower back pain. In particular, it relates to neuropathic or arthritic pain. More specifically, pain is associated with cancer or its treatments, particularly the treatment of triple-negative breast cancer (TNBC).

[0033] The present invention also relates to a derivative or composition according to the invention, for use in the prevention or treatment of cancer, in particular triple negative breast cancer (TNBC). Brief description of the figures: Antiproliferative effect

[0034] [Figure 1] represents the percentage of cell viability in the presence of the different compounds according to the invention (AC: E 1-E 3) and comparative (DK: EC 1-EC 6, EC 8, EC 9), at different concentrations (i.e. 0.1 pM, 1 pM, 5 pM or 10 pM) on human triple negative breast cancer cells (MDA-MB-231).

[0035] [Fig.2] represents the inhibition of cell growth of cancer cells Triple-negative breast cancer (TNBC) 4T1-Luc2 cells were treated with compound E2 and paclitaxel. Cell viability and IC50 were assessed by measuring the bioluminescence of 4T1-Luc2 cells treated with the indicated concentrations of paclitaxel (0.01 to 1000 nM) and E2 (0.5 to 100 pM) for 24 hours. Data are expressed as mean ± SEM (n = 6) in quadruplicate. *p < 0.05, **p < 0.01, ***p < 0.001, compared to the vehicle dose by one-way ANOVA followed by Dunnett's test for the dose-response of E2 and Kruskal-Wallis followed by Dunn's test for paclitaxel.

[0036] [Fig.3] represents the synergistic antiproliferative effect of compound E 2 and paclitaxel on 4T1-Luc2 triple-negative breast cancer (TNBC) cells. (A) Dose-effect curve of the combination of E2 and paclitaxel. (B) Dose reduction index (DRI) of the molecules. Favorable dose reduction when DRI > 1. The dotted lines show the DRI of paclitaxel (8.6) and E2 (2.39) when 50% of viable cells are inhibited. (C) Combination index showing that the interaction between E2 and paclitaxel is synergistic because IndC < 1. Isobologram of the IC25 (D) and IC50 (E) of the combination. Data are expressed as mean ± IC95 (n = 6) in quadruplicate.

[0037] [Fig.4] represents the apoptotic effect of the combination of compound E 2 and the Paclitaxel in 4T1-Luc2 triple-negative breast cancer (TNBC) cells. (A) Representative flow cytometry data of Annexin V / IP dual staining after 24 h of treatment with E2 (15.13 µM), paclitaxel (117.5 nM), or the combination in the murine 4T1-Luc2 TNBC cell line. (B) Frequency of apoptotic and (C) necrotic cells. Data are expressed as mean ± SEM (n = 4) in quadruplicate. **p < 0.01, compared to the control group by Kruskal-Wallis test followed by Dunn's test. Analgesic effect (neuropathic pain)

[0038] [Fig. 5] represents the evaluation of the analgesic effect of compound E2 (administered by Intraperitoneal (IP) administration, as shown by the von Frey test in a paclitaxel-induced murine model of neuropathic pain. Top: effect kinetics. Bottom: area under the curve.

[0039] [Fig.6] represents the evaluation of the analgesic effect of E 2 (administered by intravenous route os) by the von Frey test in a mouse model of paclitaxel-induced neuropathic pain. Left: effect kinetics. Right: area under the curve.

[0040] [Fig.7] represents the evaluation of the GPER-dependent analgesic effect of the compound E 2 (15 mg / kg, ip) by von Frey test in a murine model of paclitaxel-induced neuropathic pain following administration of vehicle or G-15 (GPER-specific antagonist ip).

[0041] [Fig.8] represents the effect of GPER deletion specifically in fibers primary afferents on paclitaxel-induced neuropathic pain.

[0042] [Fig.9] represents the evaluation of the peripheral analgesic effect of compound E 2 by von Frey's test in a mouse model of paclitaxel-induced neuropathic pain. (A) E2 injected intraplantarly (IPL) into the animal's paw. (B) Vehicle or G-15 injection (IPL) 10 min before vehicle or E2 injection (IPL). (C) Vehicle or G-15 injection (IPL) 10 min before vehicle or E2 injected intraperitoneally (IPL). Left: effect kinetics. Right: area under the curve.

[0043] [Fig. 10] represents the effect of E2 and PLMI(ip) on a paclitaxel-induced neuropathic pain model after specific deletion of GPER in primary afferent fibers. Top: effect kinetics. Bottom: area under the curve.

[0044] [Fig. 11] represents the effect of GPER deletion specifically in the spinal cord on paclitaxel-induced neuropathic pain.

[0045] [Fig. 12] shows the evaluation of the spinal analgesic effect of compound E2 by the von Frey test in a murine model of paclitaxel-induced neuropathic pain. (A) E2 injected intrathecally (it). (B) Vehicle or G-15 injection (it) 10 min before vehicle or E2 (it). (C) Vehicle or G-15 injection (it) 10 min before vehicle or E2 intraperitoneally (ip). Left: effect kinetics. Right: area under the curve.

[0046] [Fig. 13] shows the kinetics of E2 analogs in female mice where pain was assessed before pain induction (baseline), and then after receiving four intraperitoneal (IP) injections of paclitaxel (4 mg / kg) at least 10 days before the first kinetic study. All molecules were dissolved in dimethyl sulfoxide (DMSO) stock solution, then diluted in DMSO, Tween 80, and saline to a final solution of 1 mg / ml in 5% DMSO, 2% Tween, and saline. The administered doses were 10 mg / kg IP for each group and each molecule. The kinetics studies were performed in randomized block and in blinded, each mouse being reused four times (with a wash period of more than 48 hours). Each group consisted of eight mice. The statistical tests used were ANOVA for repeated measures followed by Tukey's post-hoc test for kinetics, and the Kruskal-Wallis test (the 4k, 4h, and 4g mice did not pass the normality tests) followed by Dunn's post-hoc test. * vs vehicle, # vs E 2.

[0047] [Fig. 14] represents the evaluation of the effect of repeated treatment with compound E 2 or PLMI (three ip per day from J1 to J10) on the paclitaxel-induced neuropathic pain model.

[0048] [Fig. 15] illustrates the effect of combining compound E2 and paclitaxel in vivo on reducing paclitaxel-induced neuropathic pain and tumor growth. (A) Study overview. Nineteen days after tumor implantation, mice received compound E2 at a dose of 10 mg / kg three times daily, followed by paclitaxel injections of 4 mg / kg every other day starting on day 20. A von Frey test was performed before each paclitaxel injection and on day 30 at the end of the experiment. Tumor growth was measured with calipers three times weekly. (B) Monitoring tumor growth over time using calipers. (C) Tumor mass of mice collected on day 31 that received vehicle, paclitaxel 4 mg / kg, compound E 2 (10 mg / kg) or co-administration, (D) Evaluation of mechanical allodynia according to the different treatment groups over ten days. Data are expressed as mean ± SEM (n = 8 per group).*p < 0.05, **p < 0.01, ***p < 0.001 compared to the vehicle group and ##p < 0.01 compared to the paclitaxel 4 mg / kg group. Kruskal-Wallis tests followed by Dunn's test (tumor weight measurement), 2-way ANOVA followed by Dunnett's test (analysis of 50% of the withdrawal thresholds obtained by von Frey, tumor volume).

[0049] [Fig. 16] shows the evaluation of the analgesic effect of compound E 2, administered intraperitoneally, by the von Frey test in a murine model of neuropathic pain induced by oxaliplatin (top, 3 mg / kg, twice weekly for four weeks) and by bortezomib (bottom, 0.4 mg / kg, three times weekly for four weeks). Left: effect kinetics. Right: area under the curve. Analgesic effect (osteoarthritic pain)

[0050] [Fig. 17] represents the analgesic effect of compound E 2 or PLMI in a murine model of osteoarthritic pain induced by an intra-articular injection of monoiodoacetate (MIA). Top: kinetics of the effect. Bottom: area under the curve.

[0051] [Fig. 18] represents the effect of GPER deletion specifically in primary afferent fibers on pain in a murine model of osteoarthritic pain induced by destabilization of the medial meniscus.

[0052] [Fig. 19] represents the effect of compound E 2 (ip) after specific deletion of the GPER of primary afferent fibers in a murine model of osteoarthritic pain induced by Destabilization of the medial meniscus. Top: kinetics of the effect. Bottom: area under the curve.

[0053] [Fig.20] represents the effect of viral deletion of GPER specifically in the spinal cord pain in a murine model of osteoarthritic pain induced by destabilization of the medial meniscus.

[0054] [Fig.21] represents the effect of compound E 2 (ip) on a pain model Osteoarthritic pain after specific deletion of GPER in the spinal cord in a murine model of osteoarthritic pain induced by destabilization of the medial meniscus. Top: effect kinetics. Bottom: area under the curve. EXAMPLES: MATERIALS AND METHODS

[0055] The reactions leading to the desired molecules were carried out in an organic medium (with variable reaction times) using reflux setups. After returning to room temperature, the synthesized molecules were purified by silica gel chromatography, precipitation, crystallization, and / or recrystallization. The physicochemical characteristics of the desired molecules were determined by thin-layer chromatography (TLC, measurement of Rf values) and by measuring their melting points on a Kofler hot stage microscope (melting point, mp, °C). Their color and appearance (amorphous or crystalline powder, oil, turbidity) were also recorded.Once purified, the molecules were characterized by high-resolution mass spectrometry (HRMS) and their structure (including their stereochemistry) elucidated by Fourier transform infrared spectroscopy (FTIR), 1D and 2D nuclear magnetic resonance (*H, 13C-NMR, NOESY, COSY, HMBC, HSQC, DEPT135) and X-ray diffraction. A- Chemistry

[0056] 1- Purification by silica gel column chromatography

[0057] The crude reaction products were purified, where necessary, by silica gel column chromatography (40-63 pm). The eluents are, as appropriate, composed of the solvent pairs: cyclohexane (CHX) / ethyl acetate (AE) or dichloromethane (CH2Cl2) / MeOH, in varying proportions. 2- Thin-layer chromatography (TLC)

[0058] TLC was performed on Kieselgel 60 F254 silica gel aluminum plates (0.20 mm thick, Macherey-Nagel, Düren, Germany) and developed under a 254 nm UV lamp. The Rf values ​​were calculated using the equation Rf = h / H, where h and H correspond respectively to the distance between the application line and the center of the spot of interest (h) and the distance between the application line and the solvent front. Ideally, 0.25 < Rf < 0.80. 3- Melting points (Tf)

[0059] The melting points were measured after calibration on a Kofler bench (REICHERT-JUNG, 220 V, measuring range: 50-260°C, accuracy: ±1 °C). The values ​​obtained were not corrected.

[0060] 4- Fourier Transform Infrared (FTIR) Spectroscopy

[0061] The FTIR analyses were carried out on a PERKIN ELMER Spectrum 65FT-IR Fourier transform instrument (measurement range: 650 - 4000 cm1) equipped with Spectrometer 10.02 software. Wavenumbers (abscissa) are expressed in cm' and transmittances (ordinate) as a percentage (%).

[0062] 5- Nuclear magnetic resonance (NMR) spectroscopy

[0063] 1D (¹H, ¹³C, DEPT) and 2D (COSY, HSQC, HMBC, NOESY, ROESY) NMR spectra were recorded on a Bruker 300 MHz spectrometer equipped with a QNP direct probe (¹H, ¹³C, ¹⁵N, ¹⁹F, ³¹P) and the Avance I console, or on a Bruker 400 MHz spectrometer equipped with a BBFO probe (¹H, ¹³C, ¹⁵N, ¹⁹F, ³¹P) and the Avance III console, or on a Bruker 600 MHz spectrometer equipped with a BBI inverse probe (¹H, ¹³C, ¹⁵N, ³¹P) and the Avance I console. The samples were diluted in 0.5 ml of the appropriate deuterated solvent (CDC13 or DMSO-d6) and then introduced into a Norell® NMR tube (length: 7 in (17.78 cm) ; DE x DI: 5 mm x 4.2 mm; Landisville, USA). Chemical shifts (φ) are expressed in ppm (parts per million) relative to the residual solvent peak (βH CDC13: 7.26 ppm; βH DMSO-d6: 2.50 ppm; βC CDC13: 77.16 ppm; βC DMSO-d6: 39.52 ppm). Multiplicities are expressed as follows: s (singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet).Coupling constants (J) are expressed in Hertz (Hz). Spectral data were acquired and processed using TopSpin Bruker software (Billerica, USA).

[0064] 6- High-resolution mass spectrometry (HRMS)

[0065] The samples to be analyzed were resuspended in a suitable solvent at a concentration of approximately 1 mg / ml and then diluted five hundredfold (final concentration ~2 ng / pL). The analyses were performed on a Bruker maXis Q-TOF instrument coupled to a Dionex Ultimate 3000 RSLC system. The analysis of the compounds of interest was carried out by flow injection analysis (FIA) without a column. The solvent used was an acetonitrile (ACN) / water (H2O) mixture: 65 / 35 at a flow rate of 200 pL / min. The injection volume was 0.2 pL. The analyses were performed in positive or negative mode by electrospray ionization (ESI). The positive mode analyses were performed in the presence of 0.1% formic acid (HCOOH). In the case of negative mode analyses, the samples were free of HCOOH.

[0066] 7- Synthesis of compounds E according to the invention and comparative compounds EC

[0067] [Chem.6] S- R;

[0068] [Tables 1] Compound RI R2 R3 R4 R5 R6 R7 R8 El H och3 HHHHHH E2 HHH 1,3-dioxolane Br HH E3 ch3 HH 1,3-dioxolane Br HH E4 HHF HH och3 1,3-dioxolane Br HH EC 4 HHH 1,3-dioxolane Br och3 H EC 5 HH ch3 1,3-dioxolane Br ch3 H EC 6 HHH 1,3-dioxolane Br OH H EC 7 H och3 och3 1,3-dioxolane Br HOH HEC 1,3-dioxolane Br HH EC 9 HH Br 1,3-dioxolane Br HH

[0069] Compounds E 1-E 4 and EC 1-EC 9 correspond to the compounds according to the invention and the comparative compounds, respectively.

[0070] In a 100 mL round-bottom single-neck flask, 1.2 mmol of substituted tetralone and 1 eq. Simple benzaldehyde or 6-bromobenzo[( / ][l,3]dioxole-5-carbaldehyde were dissolved in 10 mL of ethanol. 10 mL of a 40% NaOH ethanolic solution were added dropwise and the mixture was stirred vigorously for 15 h at room temperature. The reaction mixture was then neutralized with 6N HCl aqueous solution. The precipitate formed was filtered and rinsed with cold ethanol. The purification of molecules E1-E4 and EC1-EC9 was carried out either by recrystallization in ethanol (compounds E1 and EC1-EC2, E2, E3, and EC3-EC6) or by silica gel chromatography (eluent CHX / AE: 80 / 20, compounds E4 and EC7-EC9).

[0071] - (E)-2-benzylidenyl-6-methoxy-3,4-dihydronaphthalenyl-1(2H)-one (E Ij. Physicochemical characteristics: Ci8Hi6O2 (MW = 264.32 g / mol); yield = 79 %. Poudre cristalline jaune. T°f = 97 °C ; CCM : Rf = 0,50 (CHX / AE : 60 / 40). Caractéristiques spectrales : FTIR (cm1) : 3013, 2938, 2906, 2843, 1657, 1601, 1583, 1490, 1440, 1138. RMN *H (400 MHz, CDC13) ô : 2,91 (2H, m, H-4) ; 3,11 (2H, m, H-3) ; 3,87 (3H, s, OCH3) ; 6,71 (1H, d, 7= 2,4 Hz, H-5) ; 6,88 (1H, dd, 7=8,7 Hz,4 J= 2,5 Hz, H-7) ; 7,30 - 7,46 (5H, m, H-Ar) ; 7,84 (1H, s, H-9) ; 8,12 (1H, d, 7=8,7 Hz, H-8). RMN 13C (400 MHz, CDC13) ô : 27,2 ; 29,3 ; 55,4 ; 112,2 ; 113,3 ; 127,0 ; 128,3 ; 128,4 (2C) ; 129,8 (2C) ; 130,7 ; 135,6 ; 135,9 ; 136,0 ; 145,7 ; 163,5 ; 186,7. HR-MS (m / z) : 265 [M-H]+

[0072] - ( E )-2-[6'-bromobenzo[ d ][l',3']dioxolo-5'-methylenyl]-3,4-dihydronaphtaleny 1-1(2 H)-one (E 2). Caractéristiques physicochimiques : Ci8HnO3Br (MW = 357,20 g / mol) ; rdt = 65 %. Poudre cristalline jaune. T°f= 162 °C ; CCM : Rf = 0,68 (CHX / AE : 70 / 30). Caractéristiques spectrales : FTIRn (cm1) : 3024, 2908, 1654, 1588, 1496, 1239. RMN *H (300 MHz, CDC13) ô (ppm) : 2,97 (4H, s, H-3 et H-4) ; 6,03 (2H, s, H-2’) ; 6,80 (1H, s, H-4’) ; 7,12 (1H, s, H-7’) ; 7,25 (1H, d, 7=7,5 Hz, H-5) ; 7,37 (1H, m, 7=7,5 Hz, 7=0,9 Hz, H-7) ; 7,50 (1H, m, 7=7,5 Hz, 7=1,3 Hz, H-6) ; 7,78 (1H, s, H-9) ; 8,15 (1H, dd, 7=7,9 Hz, 7=1,3 Hz, H-8). RMN 13C (400 MHz, CDC13 ) ô (ppm) : 26,3 ; 27,9 ; 101,0 ; 108,8 ; 112,1 ; 112,2 ; 115,6 ; 126,0 ; 127,2 ; 128,1 ; 132,3 ; 132,4 ; 134,3 ; 135,0 ; 142,3 ; 142,9 ; 147,5 ; 186,5. HR-MS (m / z) : 357 [M]+.

[0073] - ( E )-2-[6'-bromobenzo[ d ][l',3']dioxolo-5'-methylenyl]-4-methyl-3,4- dihydronaphtalenyl-l(2H)-one (E 3). Caractéristiques physicochimiques : Ci9Hi5O3 Br (MW = 371,23 g / mol) ; rdt = 75 % ; poudre cristalline orange. T°f = 162°C ; CCM : Rf = 0,67 (CHX / AE : 70 / 30). Caractéristiques spectrales : FTIR n (cm1) : 2965, 2918, 2875, 1665, 1600, 1586, 1498, 1479, 1240. RMN ‘H (400 MHz, CDC13) ô (ppm) : 2,61 (3H, d, 7=7,0 Hz, CH3) ; 2,81 (1H, m, H-3a) ; 3,00 (1H, m, H-3b) ; 3,12 (1H, m, 7=5,6 Hz, H-4) ; 6,03 (2H, s, H-2’) ; 6,77 (1H, s, H-4’) ; 7,11 (1H, s, H-7’) ; 7,29 (1H, d, 7=7,66 Hz, H-5) ; 7,37 (1H, m, 7=7,7 Hz, 7=1,0 Hz, H-7) ; 7,53 (1H, m,3 7=7,7 Hz, 7=1,4 Hz, H-6) ; 7,83 (1H, s, H-9) ; 8,15 (1H, dd, 7=7,9 Hz, 7=1,3 Hz, H-8). RMN 13C (400 MHz, CDC13) ô (ppm) : 21,8 ; 33,3 ; 34,5 ; 102,0 ; 109,8 ; 113,1 ; 116,5 ; 127,0 ; 127,1 ; 128,3 ; 129,2 ; 132,3 ; 133,6 ; 134,6 ; 137,1 ; 147,0 ; 148,3 ; 148,5 ; 187,5. HR-MS (m / z) : 371 [M]+

[0074] ( E )-2-[6'-bromobenzo[ d ][l',3']dioxolo-5'-methylenyl]-7-fluoro-3,4- dihydronaphthalenyl-l(2H)-one (E 4). Physicochemical characteristics: C[8H12BrFO3(MW = 375.19 g / mol); yield = 16%; yellow crystalline powder. Temperature = 208 °C; TLC: Rf = 0.53 (CHX / AE: 70 / 30). Spectral characteristics: FTIR n (cm1): 2913, 2841, 1656, 1582, 1493, 1418, 1243. ¹H NMR (400 MHz, CDC13) δ (ppm): 2.86 3.00 (4H, m, H-3 and H-4); 6.02 (2H, s, H-2'); 6.79 (1H, s, H-4'); 7.11 (1H, s, H-7'); 7.15 - 7.26 (2H, m, H-5 and H-6); 7.75 - 7.82 (2H, m, H-8 and H-9). 13C NMR (400 MHz, CDC13) δ (ppm): 27.5; 28.2; 102.1; 109.8; 113.1; 114.0; 116.8; 120.5; 128.9; 130.1; 134.8; 135.2; 136.5; 139.0; 147.0; 148.8; 163.0; 186.6. HR-MS (m / z): 375 [M]+.

[0075] - (E)-2-[2'-methoxybenzylidenyl]-6-methoxy-3,4-dihydronaphthalenyl-1(2H)- one (EC 1). Physicochemical characteristics: Ci9Hi8O3 (MW = 294.35 g / mol); yield = 83%. Yellow crystalline powder. Temperature = 142°C; TLC: Rf = 0.56 (CHX / AE: 60 / 40). Spectral characteristics: FTIR (cm1): 3063, 3019, 2968, 2945, 2838, 1661, 1590, 1507, 1440, 1137. ¹H NMR (400 MHz, CDC13): 2.92 (2H, m,H⁻⁴); 3.13 (2H, m,H⁻³); 3.85 (3H, s, OCH₃); 3.87 (3H, s, OCH₃); 6.70 (1H, d, 4J=2.5 Hz, H-5); 6.87 (1H, dd, 3J=8.8 Hz, 4 / = 2.6 Hz, H-7); 6.92 - 6.97 (2H, m, H-Ar); 7.38 - 7.44 (2H, m, H-Ar); 7.80 (1H, s, H-9); 8.10 (1H, d, 3J=8.8 Hz, H-8). 13C NMR (400 MHz, CDC13) δ: 26.2; 28.2; 54.3; 54.4; 111.2; 112.2; 112.8 (2C); 126.1; 127.5; 129.6; 130.6 (2C); 132.7; 134.9; 144.5; 158.7; 162.4; 185.7. HR-MS (m / z): 295 [MH]+.

[0076] - (E)-2-[2'-chlorobenzylidenyl]-7-methoxy-3,4-dihydronaphthalenyl-1(2H)-one (EC 2). Physicochemical characteristics: Ci8Hi5O2Cl (MW = 298.77 g / mol); yield = 75%; yellow crystalline powder. Temperature = 141 °C; TLC: Rf = 0.60 (CHX / AE: 60 / 40). Spectral characteristics: FTIR (cm1): 3061, 2955, 2937, 2835, 1666, 1601, 1572, 1493, 1461, 1237. ¹H NMR (CDC13, 400 MHz): 2.87–2.92 (2H, m, H⁻⁴); 2.92–2.98 (2H, m, H⁻³); 3.88 (3H, s, OCH₃); 7.08 (1H, dd, 3J=8.4 Hz, 4J=2.8 Hz, H-6); 7.17 (1H, d, V=8.4 Hz, H-5); 7.27 - 7.49 (4H, m, H-Ar); 7.65 (1H, d, 4J=2.8 Hz, H-8); 7.89 (1H, s, H-9); 13C NMR (CDC13, 400 MHz) δ: 26.5; 27.1; 54.5; 109.2; 120.7; 125.3; 128.5; 128.6; 128.7; 129.3; 132.4; 133.0; 133.5; 133.7; 135.1; 136.1; 157.6; 186.4. HR-MS (m / z): 299 [MH]+.

[0077] - (E)-2-[6'-bromobenzo[d][l',3']dioxolo-5'-methyleneyl]-7-methoxy-3,4- dihydronaphtalenyl-l(2H)-one (EC 3). Caractéristiques physicochimiques : C19H15 O4Br(MW = 387,23 g / mol) ; rdt = 43 % ; poudre amorphe jaune. T°f = 140 °C ; CCM : Rf = 0,35 (CHX / AE : 80 / 20 AE). Caractéristiques spectrales : FTIR n (cm-1) : 2912, 2838, 1660, 1582, 1493, 1460, 1250. RMN ‘H (CDC13, 400 MHz) ô (ppm) : 2,86 -3,00 (4H, m, H-3 et H-4) ; 3,87 (3H, s, OCH3) ; 6,02 (2H, s, H-2’) ; 6,79 (1H, s, H-4’) ; 7,07 (1H, dd, V=8,4 Hz, 4J=2,8 Hz, H-6) ; 7,10 (1H, s, H-7’) ; 7,15 (1H, d, 3J=8,4 Hz, H-5) ; 7,63 (1H, d, 4J=2,8 Hz, H-8) ; 7,74 (1H, s, H-9). RMN 13C (CDC13, 400 MHz) ô (ppm) : 27,5 ; 28,1 ; 55,5 ; 102,0 ; 109,8 ; 110,2 ; 113,1 ; 116,6 ; 121,7 ; 129,2 ; 129,5 ; 134,1 ; 135,8 ; 136,0 ; 136,1 ; 147,0 ; 148,5 ; 158,7 ; 187,5. HR-MS (m / z) : 387 [M]+.

[0078] - ( E )-2-[6'-bromobenzo[ d ][l',3']dioxolo-5'-methylenyl]-5-methoxy-3,4- dihydronaphtalenyl-l(2.H)-one (EC 4). Caractéristiques physicochimiques : C19H i5O4Br (MW = 387,23 g / mol) ; rdt = 70 % ; poudre cristalline jaune. T°f = 195 °C ; CCM : Rf = 0,61 (CHX / AE : 70 / 30). Caractéristiques spectrales : FTIR n (cm1) : 2970, 2945, 2914, 2839, 1656, 1580, 1505, 1472, 1240. RMN ‘H (400 MHz, CDC13) ô (ppm) : 2,92 (4H, s, H-3 et H-4) ; 3,87 (3H, s, OCH3) ; 6,03 (2H, s, H-2’) ; 6,79 (1H, s, H-4’) ; 7,04 (1H, dd, 7=8,1 Hz, 7=0,6 Hz, H-6) ; 7,11 (1H, s, H-7’) ; 7,32 (1H, m,3 J =7,9 Hz, H-7) ; 7,72 (1H, s, H-9) ; 7,78 (1H, dd, 7=7,9 Hz, 7=0,9 Hz, H-8). RMN 13C (400 MHz, CDC13) ô (ppm) : 20,7 ; 25,6 ; 54,7 ; 100,9 ; 108,8 ; 112,0 ; 113,3 ; 115,6 ; 118,8 ; 126,2 ; 128,2 ; 131,4 ; 133,2 ; 134,4 ; 135,1 ; 145,9 ; 147,4 ; 155,3 ; 186,8. HR-MS (m / z) : 387 [M]+.

[0079] - ( E )-2-[6'-bromobenzo[ d ][l',3']dioxolo-5'-methylenyl]-5,7-dimethyl-3,4- dihydronaphtalenyl-l(2H)-one (EC 5). Caractéristiques physicochimiques : C2o Hi7O3Br (MW = 385,26 g / mol) ; rdt = 78 % ; poudre amorphe jaune. T°f = 198 °C ; CCM : Rf = 0,74 (CHX / AE : 70 / 30). Caractéristiques spectrales : FTIR n (cm1) : 2901, 1660, 1591, 1499, 1472, 1246. RMN ‘H (400 MHz, CDC13) ô (ppm) : 2,28 (3H, s, CH3 ) ; 2,36 (3H, s, CH3) ; 2,83 (2H, t, 7=6,4 Hz, H-4) ; 2,94 (2H, m, 7=6,4 Hz, 7=1,4 Hz, H-3) ; 6,02 (2H, s, H-2’) ; 6,79 (1H, s, H-4’) ; 7,11 (1H, s, H-7’) ; 7,20 (1H, s, H-6) ; 7,70 (1H, s, H-9) ; 7,85 (1H, s, H-8). RMN 13C (400 MHz, CDC13) ô (ppm) : 19,3 ; 20,9 ; 25,2 ; 26,8 ; 102,0 ; 109,9 ; 113,1 ; 116,5 ; 126,2 ; 129,3 ; 133,3 ; 135,0 ; 135,6 ; 135,9 ; 136,1 ; 136,2 ; 138,9 ; 147,0 ; 148,4 ; 188,2. HR-MS (m / z) : 385 [M]+

[0080] - ( E )-2-[6'-bromobenzo[ d ][l',3']dioxolo-5'-methylenyl]-5-hydroxy-3,4- dihydronaphtalenyl-l(2H)-one (EC 6). Caractéristiques physicochimiques : Ci8H i3O4Br (MW = 373,20 g / mol) ; rdt = 47 % ; poudre cristalline jaune. T°f = 228 °C ; CCM : Rf = 0,38 (CHX / AE : 70 / 30). Caractéristiques spectrales : FTIR n (cm1) : 3266, 2968, 2917, 2834, 1656, 1603, 1587, 1560, 1500, 1466, 1243. RMN ‘H (300 MHz, DMSO-d6) ô (ppm) : 2,76 - 3,00 (4H, m, H-3 et H-4) ; 6,15 (2H, s, H-2’) ; 7,08 (1H, s, H-4’) ; 7,08 (1H, dd, 7=7,9 Hz, 7=1,2 Hz, H-Ar) ; 7,25 (1H, m, 7=7,9 Hz, H-7) ; 7,37 (1H, s, H-7’) ; 7,47 (1H, dd, 7=7,9 Hz, 7=1,2 Hz, H-Ar) ; 7,55 (1H, s, H-9) ; 9,86 (1H, s, OH). RMN 13C (300 MHz, DMSO-d6,) ô (ppm) : 21,7 ; 26,5 ; 102,8 ; 110,4 ; 113,1 ; 116,4 ; 118,4 ; 119,8 ; 127,5 ; 128,7 ; 130,7 ; 134,3 ; 134,5 ; 136,6 ; 147,5 ; 149,1 ; 154,8 ; 187,3. HR-MS (m / z) : 373 [M]+.

[0081] - {E )-2-[6'-bromobenzo[ d ][l',3']dioxolo-5'-methylenyl] - 6,7-dimethoxy-3,4- dihydronaphthalenyl-l(2H)-one (EC 7). Physicochemical characteristics: C2O Hi7BrO5 (MW = 417.26 g / mol); yield = 3%; orange crystalline powder. Melting temperature > 260 °C; TLC: Rf = 0.37 (CHX / AE: 70 / 30). Spectral characteristics: FTIR n (cm1): 2937, 1645, 1599, 1571, 1425, 1265. 'H NMR (400 MHz, CDC13) δ (ppm): 2.86 - 3.03 (4H, m, H-3 and H-4); 3.86 (6H, s, OCH3-6 and OCH3-7); 6.03 (2H, s, H-2'); 6.69 (1H, s, H-4'); 6.84 (1H, s, H-5); 7.11 (1H, s, H-7'); 7.65 (1H, s, H-8); 7.72 (1H, s, H-9). 13C NMR (400 MHz, CDC13) δ (ppm): 27.6; 28.7; 56.1 (2C); 102.0; 109.5; 109.8; 109.9; 113.0; 116.5; 126.5; 129.4; 135.1; 136.0; 138.3; 146.9; 148.3; 148.4; 153.6; 186.4. HR-MS (m / z): 417 [M]+.

[0082] - (E)-2-[6'-bromobenzo[d][l',3']dioxolo-5'-methyleneyl]-6-hydroxy-3,4- dihydronaphtalenyl-l(2H)-one (EC 8). Caractéristiques physicochimiques : C[8 Hi3O4Br (MW = 373,20 g / mol) ; rdt = 13 % ; poudre amorphe jaune. T°f = 250 °C ; CCM : Rf = 0,50 (CHX / AE : 60 / 40). Caractéristiques spectrales : FTIR n (cm1) : 3060, 2844, 1651, 1594, 1562, 1496, 1472, 1240. RMN ‘H (400 MHz, DMSO-d6) ô (ppm) : 2,80 - 2,93 (4H, m, H-3 et H-4) ; 6,14 (2H, s, H-2’) ; 6,69 (1H, dd, 7=8,0 Hz, 7=2,4 Hz, H-5) ; 6,78 (1H, dd, 7=8,6 Hz, 7=2,4 Hz, H-7) ; 7, 07 (1H, s, H-4’) ; 7,36 (1H, s, H-7’) ; 7,51 (1H, s, H-9) ; 7,86 (1H, d, 7=8,6 Hz, H-8) ; 10,44 (1H, s, OH). RMN 13 C (400 MHz, DMSO-d6) ô (ppm) : 26,1 ; 27,6 ; 101,7 ; 109,4 ; 112,1 ; 113,4 ; 114,2 ; 115,3 ; 124,3 ; 127,8 ; 129,7 ; 132,8 ; 135,8 ; 145,6 ; 146,4 ; 147,9 ; 161,8 ; 184,4. HR-MS (m / z) : 373 [M]+.

[0083] ( E )-2-[6'-bromobenzo[ d ][l',3']dioxolo-5'-methylenyl]-7-bromo-3,4- dihydronaphtalenyl-l(2H)-one (EC 9). Caractéristiques physicochimiques : C[8H i2Br2O3(MW = 436,10 g / mol) ; rdt = 13 % ; poudre cristalline jaune. T°f = 186 °C ; CCM : Rf = 0,50 (CHX / AE : 90 / 10). Caractéristiques spectrales : FTIR n (cm1) : 2920, 1653, 1578, 1498, 1473, 1217. RMN ‘H (400 MHz, CDC13) ô (ppm) : 2,85 - 3,00 (4H, m, H-3 et H-4) ; 6,02 (2H, s, H-2’) ; 6,77 (1H, s, H-4’) ; 7,10 (1H, s, H-7’) ; 7,14 (1H, d, 7=8,1 Hz, H-5), 7,59 (1H, dd, 3 7=8,1 Hz, 7=2,1 Hz, H-6) ; 7,77 (1H, s, H-9) ; 8,24 (1H, s, 7=2,1 Hz, H-8). RMN 13C (400 MHz, CDC13) ô (ppm) : 27,0 ; 28,4 ; 102,1 ; 109,8 ; 113,2 ; 116,8 ; 121,0 ; 128,8 ; 130,1 ; 130,9 ; 134,8 ; 135,2 ; 136,1 ; 136,7 ; 141,9 ; 147,0 ; 148,7 ; 186,3. HR-MS (m / z) : 435 [M]+. B- Pharmacologie in vitro 1- Réactifs

[0084] Paclitaxel was obtained from LeanCare (Greenfield, UK) and compounds E 1-E 4 (compounds according to the invention) and EC 1-EC 9 (comparative compounds) were synthesized as described above. DMSO was obtained from Sigma Aldrich (France). 2- Cell culture

[0085] MDA-MB-231 cells (human triple-negative breast cancer cell line) were obtained from ATCC (Manassas, VA, USA) and maintained in medium DMEM / F12 culture medium (Dulbecco's modified Eagle's medium) containing phenol red and supplemented with 5% fetal bovine serum (FBS) and 1% of a penicillin / streptomycin mixture (Thermo Fisher Scientific, Monza, Italy) was used. Cells were cultured at 37°C in an atmosphere composed of 95% air and 5% CO2.

[0086] 4T1-Luc2 cells (a murine triple-negative breast cancer cell line) were obtained from ATCC (Manassas, VA, USA) and maintained in Roswell Park Memorial Institute (RPMI) 1640 culture medium in the presence of GlutaMAX™ supplemented with 10% FBS, 8 pg / mL blasticidin, and 2 mM penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). The cells were cultured at 37°C in an atmosphere composed of 95% air and 5% CO2. 3- Cell viability of MDA-MB-231

[0087] The viability of MDA-MB-231 cells was assessed using an automated cell counter (Life Technologies, Milan, Italy) according to the manufacturer's recommendations. MDA-MB-231 cells were seeded onto 24-well plates containing standard growth medium. The plates were incubated in 2.5% FBS before being cultured with increasing concentrations of the compounds of interest for 72 hours. The treatment was repeated daily, and the cells were counted on day 4. The number of live cells is expressed as a percentage relative to the number of cells that received serum alone (100%, reference). Each point represents the mean of three experiments (n=3) ± standard deviation, with the experiments performed in triplicate (N=3). The x-axis represents the concentrations of the different products (in pM), and the y-axis represents cell viability (%). 4- Cell viability of 4T1-Luc2

[0088] The viability of 4T1-Luc2 cells was measured by bioluminescence using 4T1-Luc2 cells expressing luciferase under the control of the elongation factor promoter EF-la. 4T1-Luc2 cells were seeded onto 96-well plates (6 x 10³ / well) containing 200 pL of standard growth medium. The 96-well plates were incubated for 24 h, and then the cells were exposed to increasing concentrations of compound E2 (0.5, 1, 5, 7.5, 10, 25, 50, 75, 100 pM) or paclitaxel (0.01, 0.1, 1, 5, 10, 50, 100, 1000 nM) for 24 h. A concentration of 0.1% DMSO was used as a vehicle.

[0089] After 24 hours of incubation with the different treatment concentrations (E 2 or paclitaxel), luciferin (Promega) was added to each well (20 pL per well for a final concentration of 0.15 mg / mL). After 16 minutes of incubation in the dark, the bioluminescence (in RLU, Relative Light Unit) of each well was read by the Flexstation for 1 second per well, in quadruplicate (n=4) per group. The The number of living cells is expressed as a percentage relative to the number of cells that received the vehicle compound (0.1% DMSO). Cell viability in the presence of the vehicle alone is considered to be 100%. Cell viability was calculated using the following formula: % cell viability = (RLU treated cells / RLU vehicle cells) x 100.

[0090] Dose-response curves and their nonlinear regression curve were plotted using GraphPad Prism software (version 9.0) to determine the IC50 values ​​and the slope of the Hill curve. We then defined the affected fraction (Fa) and its counterpart, the unaffected fraction (Fu). The percentage of viable cells is defined by Fa, while 100 - Fa determines Fu. These data allowed the calculation of concentrations leading to the inhibition percentages of 6.25, 12.5, 18.75, 25, 31.25, 37.5, and 50.

[0091] [Math.l] pa ï / hill [molecules] = jÿ ^CI5Q

[0092] Compound E2 was then combined with paclitaxel at E2 and paclitaxel concentrations resulting in the same percentage of inhibition. The concentration of the single molecule that provides the same percentage of viability as that obtained with the combination (FaCombined) was then calculated. The concentrations of the single molecules, inducing the same effect as the combination, and the concentration of the molecules used in the combination were used to calculate the dose reduction index (DRI) and the combination index (IndC).

[0093] [Math.2]

[0094] । [Paclitaxel£^^ [MJ0!032æJ [Paclitaxel^

[0095] The DRI values ​​were used to assess whether the combination reduces the molecule dose required to achieve an equivalent effect when DRI > 1. The combination index was used to determine the type of interaction between two molecules, such as antagonistic (IndC > 1), additive (IndC = 1), or synergistic (IndC < 1), using the Chou-Talalay method (Chou, 2010)

[30] . The values ​​for the isobologram are presented as mean ± 95% CI. 5- Flow cytometry

[0096] The apoptosis assay was performed using the F1TC Annexin V Apoptosis Detection with PI kit (BioLegend), according to the manufacturer's instructions. 4T1-Luc2 cells were seeded onto a 6-well plate (250 x 10³ cells / well) and allowed to attach for 24 h. The cells were then treated with a concentration of E 2, corresponding to its IC50 (15.17 µM), and a paclitaxel concentration corresponding to its IC50 (117.5 nM), or a combination thereof, were incubated for 24 h. The cells were then collected by trypsinization and labeled for 15 minutes in the dark with Annexin V conjugated to FITC and propidium iodide, prior to flow cytometry analysis. Data were acquired using a flow cytometer (BD LSR II) and analyzed using BD FACSDiva 9.0.1 software. Cells were classified according to physical parameters to select cells of interest (FSC / SSC, Forward SCatterlSide SCatterf) and cell clusters (width / area). C- In vivo pharmacology 1- Reagents

[0097] Paclitaxel was obtained from LeanCare (Greenfield, UK), and compounds E1-E4 and EC1-EC9 were synthesized as previously described. For in vivo testing, paclitaxel was dissolved in a mixture of 10% 96% ethanol, 10% Cremophor (Sigma Aldrich, France), and 80% physiological saline. The reference compound E2 was dissolved in 5% DMSO, 2% Tween80 (Sigma Aldrich, France), and physiological saline. 2- Animals

[0098] Eight-week-old female C57BL6 / J and Balb / cJRj mice from Janvier (Le Genest-St-Isle, France) were housed with ad libitum access to food and water in an environment free of specific pathogens, at 22 ± 2°C and 20% humidity with a 12h / 12h day / night cycle. All experiments were conducted according to IASP recommendations and were approved by the Animal Experimentation Ethics Committee (CEMEA) of the Auvergne Region, in accordance with Directive 2010 / 63 / EU.

[0099] 3- Model of paclitaxel-induced peripheral neuropathies

[0100] The paclitaxel-induced peripheral neuropathy model was generated by four intraperitoneal injections, one every two days, of paclitaxel (LeanCare, Greenfield, United Kingdom) dissolved in 10% 96% ethanol, 10% Cremophor EL (Sigma-Aldrich, Saint-Quentin-Fallavier, France), and 80% physiological saline. Neuropathies were considered chronic fourteen days after model initiation. The animals were divided into different groups of eight mice per group by equal block randomization. 4- Orthotopic breast tumor model

[0101] Cultured 4Tl-luc2 cells were harvested and resuspended in phosphate saline buffer (PBS) at different densities (250,000, 500,000, 1,000,000 cells / injection site) or in a mixture of PBS and Matrigel® 60% (Corning). Mice were anesthetized by isoflurane inhalation, and then cells were implanted into the 4th left mammary fat pad by subcutaneously injecting 50 pL of cell suspension using a Hamilton syringe and a 26G needle. Tumor volume was calculated using the formula (length x (width)² / 2) from caliper measurements of tumor length and width.

[0102] To evaluate the optimal density of cells to be implanted, the tumor volume was assessed three times a week for thirty-five days.

[0103] To evaluate the effect of compound E 2 in combination with or alone from paclitaxel, tumor volume and pain sensitivity were measured three times a week after treatment initiation. When the tumors began their second growth phase (day 18), the mice received the different treatments.

[0104] 5- Murine model of mono-iodoacetate-induced osteoarthritis (MIA)

[0105] Mice are anesthetized by isoflurane gas anesthesia (Iso-Vet®, 5% induction, 6% maintenance). An intra-articular injection of monoiodoacetate (MIA, 5 mg / 10 pL) is performed at the left knee of the mice.

[0106] 6- Murine model of osteoarthritis induced by destabilization of the median meniscus

[0107] Osteoarthritis is induced in mice by destabilization of the medial meniscus (MMD, (Glasson, 2007)

[31] . Mice are anesthetized by intraperitoneal (IP) injection of 10 mL / kg of a mixture of ketamine (hnalgene® 500, 100 mg / kg) and xylazine (Rompun®, 10 mg / kg) diluted in physiological saline (0.9% NaCl). The surgical area is disinfected with 90% alcohol and then with Betadine® (povidone-iodine solution). A longitudinal incision of approximately 5 mm is made, from the bottom of the tibial plateau to the top of the patella. The patella is then dislocated, the synovial capsule opened with a scalpel, and the infrapatellar fat pad divided to expose the medial meniscotibial ligament, which is then divided. The The kneecap is then put back in place, and the skin is closed with Monocryl® 5.0. Control mice (Sham) undergo the same operation until the ligament is exposed, but keep it intact.

[0108] 7- Evaluation of mechanical allodynia: the von Frey test

[0109] Mechanical pain sensitivity was assessed using the von Frey filament test. Nylon filaments (Bioseb) of varying diameters were applied to the animal's paw. Withdrawal or licking of the paw was considered a positive response. The measurement was then repeated with a finer or larger diameter filament if the animal did not react. The paw withdrawal threshold of 50% (PWT, in grams) was determined using the Dixon up-down method. 8- Treatments

[0110] G-15 (a specific GPER antagonist, Tocris Bio-Techne, Noyal-Châtillon-sur-Seiche, France), at a dose of 0.3 mg / kg, dissolved in 5% DMSO (SigmaAldrich, Saint-Quentin-Fallavier, France), 5% Tween80 (Sigma-Aldrich, Saint-Quentin-Fallavier, France), and 90% physiological saline, and E 2, dissolved in 5% DMSO and 95% physiological saline, were administered at a dose of 10 mL / kg intraperitoneally or orally. G-15 was injected 15 minutes before E 2. Monoiodoacetate (MIA) is supplied by Sigma-Aldrich, Saint-Quentin-Fallavier, France. EXAMPLE 2: RESULTS A- Antiproliferative effect

[0111] 1- In vitro evaluation of the antiproliferative effect of compounds according to the invention (E) and comparatives (EC)

[0112] Cell viability studies performed on MDA-MB-231 TNBC cells show in all cases a concentration-dependent antiproliferative action (control: vehicle alone, 100%) ([Figure 1]).

[0113] Regarding compound E 1, the antiproliferative action is visible at concentrations above 1 pM. It is visible from 5 pM for compound E 2. As for compound E3, an antiproliferative action is observed at concentrations above 0.1 pM.

[0114] Regarding the derivatives of the EC series, an antiproliferative action is observed at concentrations above 0.1 pM for derivatives EC 3, EC 5, EC 6 and EC 8. This activity is observed at concentrations above 1 pM for derivatives EC 1, EC 4 and EC 9. For compound EC 2, this action is observed from 5 pM.

[0115] 2- Compound E 2 induces a cytotoxic effect on murine TNSC cells

[0116] The antiproliferative effects of compound E2 and paclitaxel (positive control) were evaluated on murine TNBC cells. 4T1-Luc2 cells were treated with increasing concentrations of compound E2 (0.5 to 100 pM) or paclitaxel (0.01 to 1000 nM) for 24 h, and cell viability was measured by bioluminescence. As shown in [Fig. 2], compound E2 and paclitaxel reduced cell viability with an inhibitory concentration 50 (IC50) of 15.1 pM (95% confidence interval [CI] 12.2 to 18.7 pM) and 117.5 nM (95% CI 63.3 to 242.9 nM), respectively. These results show that compound E 2 and paclitaxel have an antiproliferative effect on murine TNBC cells.

[0117] 3- Compound E 2 and paclitaxel exhibit a synergistic cytotoxic effect in vitro

[0118] The effect of compound E 2 was then evaluated by bioluminescence on the antiproliferative response of paclitaxel on 4T1-Luc2 cells. Co-treatment with compound E 2 and paclitaxel, at concentrations corresponding to their percentages Inhibition concentrations of 6.25, 12.5, 18.75, 25, 31.25, 37.5, and 50 induced an antiproliferative effect (Figure 3A). Indeed, the use of these two compounds at an IC50 of 36.4% (95% CI 33.6–39.9%) reduced cell viability by 50%. These data allowed us to determine the dose reduction index of each molecule by calculating the ratio of the concentration of the single molecule, which produces the same effect as the combination, to the concentration of the molecule in the combination. The use of the combination allows the dose of paclitaxel used to be reduced by a factor of 8.60 (95% CI 4.84 to 13.80) and by a factor of 2.39 (95% CI 1.89 to 2.91) for compound E 2 to reduce cell viability by 50% (Figure 3B).

[0119] Since the combination index (IndC) of compound E2 and paclitaxel is less than 1, it is possible to conclude that these two molecules exhibit synergy at the different doses tested, although this index increases in a dose-dependent manner (IndC of 0.32 ± 0.05 for 75% viability, 0.46 ± 0.06 for 55% viability, and 0.87 ± 0.31 for 39% viability) (Figure 3C). These results therefore indicate a synergistic inhibitory effect of the paclitaxel-E2 combination at the tested doses on the growth of 4T1-Luc2 cells, as shown by the isobologram analysis at IC25 (Figure 3D) and IC50 (Figure 3E).

[0120] 4- The combination of compound E 2 and paclitaxel induces apoptosis cellular

[0121] To determine whether the cytotoxic effect of compound E 2 and paclitaxel is due to the induction of cell death by apoptosis and / or necrosis, a double-label analysis with Annexin V and propidium iodide on 4T1-Luc2 cells was performed (Figure 4A). Flow cytometry analysis showed that compound E 2 (at its IC50 of 15.13 pM) alone and paclitaxel (at its LC50 of 117.5 nM) alone induced apoptosis of 4T1-Luc2 cells in a non-significant manner (Figure 4B). Furthermore, the combination of the two compounds significantly increased induced apoptosis compared to cells treated with the vehicle (28.55 ± 1.45% apoptosis for the combination versus 12.05 ± 0.51% for the vehicle, p = 0.001). The different treatments did not induce necrosis (Figure 4C). B- Analgesic effect: neuropathic pain

[0122] 1- Compound E 2 induces a dose-dependent analgesic effect on a model murine paclitaxel-induced neuropathy

[0123] Repeated administration of paclitaxel induces neuropathic pain, observed by a decrease in thresholds (50% of the withdrawal threshold) between baseline (before paclitaxel administration) and time 0 (after paclitaxel treatment) ([Fig. 5]). Compound E2 administered intraperitoneally increases pain thresholds in animals in a dose-dependent manner. The positive control is duloxetine. A At 20 mg / kg, E2 has an effect comparable to duloxetine at 30 mg / kg. At 10 mg / kg, E2 has an effect comparable to PLMI at the same dose. Similarly, a dose-dependent analgesic effect is observed when E2 is administered orally, highlighting its oral bioavailability ([Fig. 6]).

[0124] 2- Compound E 2 induces a GPER-dependent analgesic effect on a model murine paclitaxel-induced neuropathy

[0125] The analgesic effect of compound E 2 observed on a murine model of paclitaxel-induced neuropathic pain is suppressed by co-administration of G-15 ([Fig.7]).

[0126] 3- Compound E 2 induces an analgesic effect on a murine model of Paclitaxel-induced neuropathy via a GPER pool, the functional localization of which is partly peripheral

[0127] Genetic deletion of the GPER of primary afferent fibers (GPERSNS KO mice) reduces paclitaxel-induced neuropathic pain ([Fig.8]) and shows that this receptor, when localized peripherally, is involved in neuropathic pain.

[0128] When administered peripherally (subcutaneous injection into the animal's paw), compound E 2 induces an analgesic effect (positive control: lidocaine) (Figure 9A). The analgesic effect of peripherally administered compound E 2 is suppressed by peripheral co-administration of G-15 (Figure 9B). The analgesic effect of systemic administration of compound E 2 (ip) is partially reduced by intraplantar co-administration of G-15 (Figure 9C).

[0129] The analgesic effect of compound E 2 or PLMI in a mouse model of paclitaxel-induced neuropathic pain is reduced in mice with GPER specifically deleted in primary afferent fibers ([Fig. 10]).

[0130] All these results mean that the peripherally localized GPER is at least partly responsible for the analgesic effect of compound E 2.

[0131] 4- Compound E 2 induces an analgesic effect on a murine model of Paclitaxel-induced neuropathy dependent on GPER, the functional localization of which is partly spinal.

[0132] Viral deletion of GPER specifically in the spinal cord (GPERDH KO mice) reduces paclitaxel-induced neuropathic pain ([Fig. 11]) and shows that this receptor, when localized at the spinal level, is involved in neuropathic pain.

[0133] When administered into the spinal cord (intrathecal injection), compound E 2 induces an analgesic effect (positive control: morphine) (Figure 12A). The analgesic effect of compound E 2 administered intrathecally is suppressed by intrathecal co-administration of G-15 (Figure 12B). The analgesic effect of systemic administration of compound E 2 (ip) is partially reduced by co-administration of G-15 via the it route (Figure 12C).

[0134] Taken together, these results mean that the GPER localized at the spinal level is at least partly responsible for the analgesic effect of compound E 2.

[0135] 5- Evaluation of the analgesic effect of compounds E on a murine model of paclitaxel-induced neuropathy

[0136] Repeated administration of paclitaxel induces neuropathic pain, as observed by the decrease in thresholds (50% of the withdrawal threshold) between baseline (before paclitaxel administration) and time 0 (after paclitaxel treatment) ([Fig. 13]). The effect of the E compounds at a dose of 10 mg / kg (ip) is shown. The E compounds are more effective than the EC compounds.

[0137] 6- Repeated injection of compound E 2, in conjunction with paclitaxel, reduces the induced neuropathy pain

[0138] Chronic administration of compound E2 or PLMI (10 mg / kg, intraperitoneal) three times daily for 11 days (days 0 to 10) concomitantly with paclitaxel (PIPN design, 4 injections on days 1, 3, 5, and 7) resulted in a significant reduction in tactile allodynia as assessed by the von Frey test on day 10 (top of Fig. 14). This represents a residual analgesic effect. Daily pain thresholds were measured 6 hours after treatment with E2 or PLMI, a time point exceeding the duration of the acute effect of the compounds. Notably, the acute effect is still maintained after injection of E2 or PLMI, as shown by the kinetics performed after the last injection of E2 or PLMI (bottom of Fig. 14).

[0139] It should be noted that acute or chronic administration of E2 in animals does not induce weight loss or stereotypical behaviors that could be considered adverse effects of the molecule.

[0140] 7- The combination of compound E 2 and paclitaxel in vivo reduces pain neuropathic effects of paclitaxel and tumor growth

[0141] The antinociceptive and antitumor effect of compound E 2 was evaluated on the murine model of primary TNBC tumor (implantation at Jo) treated or not with paclitaxel. After tumor establishment for 19 days, mice were treated with compound E 2 (10 mg / kg, administered ip 3 times daily from Day 9 to Day 30) or its vehicle (physiological saline, 10 mL / kg) and with paclitaxel (4 mg / kg administered ip on Days 20, 22, 24 and 26) or its vehicle (physiological saline, 10 mL / kg) (Figure 15A).

[0142] After 30 days, tumor growth in animals treated with compound E 2 is 35% lower than in animals treated with the vehicle (376.71+75.93 mm3 for E 2-treated mice vs 577.98+54.317 mm3 for the vehicle group, p = 0.12) and 23% lower in paclitaxel-treated mice compared to vehicle mice (443.92 ± 38.73 mm³) (Figure 15B). Furthermore, the paclitaxel-compound E2 combination resulted in the greatest inhibition of tumor growth, with a significant 42% reduction compared to the vehicle group (339.5 ± 55.1 mm³, p = 0.02). These results are similar to those obtained when measuring tumor mass at the end of treatment, where a significant decrease in tumor mass was observed from the paclitaxel and combination treated groups compared to the vehicle group (363.4+59.43 mg for the paclitaxel group vs 561.740.41 mg for the vehicle group, p = 0.0488, and 31855.37 mg for the combination treated group, p = 0.0328 compared to the vehicle group) (Figure 15C).Paclitaxel-induced mechanical allodynia (0.13+0.05 g for paclitaxel vs 0.52+0.04 g for the vehicle group at day 24, p = 0.0004, and 0.058+0.007 g for paclitaxel at day 30 vs 0.41+0.05 g for the vehicle group, p = 0.002) is significantly reduced by co-administration of compound E 2 (0.29+0.07 g for the combination group vs 0.058+0.007 g for the paclitaxel alone group at day 30, p = 0.002) (Figure 15D).

[0143] These results show that co-administration of compound E 2 and paclitaxel does not alter the antiproliferative effect of paclitaxel, and that it allows for a significant reduction in paclitaxel-induced mechanical allodynia.

[0144] 8- Compound E 2 induces an analgesic effect in pain models oxaliplatin- and bortezomib-induced neuropathic pain

[0145] Compound E 2 and PLMI (administered intraperitoneally) exhibit an analgesic effect in the von Frey test in a murine model of neuropathic pain induced by oxaliplatin (top, 3 mg / kg, twice weekly for 4 weeks) and by bortezomib (bottom, 0.4 mg / kg, three times weekly for 4 weeks). C- Analgesic effect: arthritic pain

[0146] 1- Compound E 2 induces an analgesic effect on a murine model of pain osteoarthritis induced by an intra-articular injection of monoiodoacetate (MIA)

[0147] The onset of osteoarthritic pain is objectively demonstrated by the decrease in thresholds (50% of the withdrawal threshold) between baseline (before MIA injection) and time 0 (18 days after MIA injection) ([Fig. 17]). Compound E2, administered intraperitoneally, increases the pain thresholds in animals. The effect of PLMI is also shown.

[0148] 2- Compound E 2 induces an analgesic effect on a murine model of pain GPER-dependent arthritic osteoarthritis, the functional location of which is partly peripheral

[0149] Genetic deletion of the GPER of primary afferent fibers (GPERSNS KO mice) reduces surgery-induced osteoarthritic pain ([Fig. 18]) and shows that this receptor, when located peripherally, is involved in arthritic pain.

[0150] The analgesic effect of compound E 2 in a murine model of osteoarthritic pain is reduced in mice whose GPER is specifically deleted in primary afferent fibers ([Fig. 19], control: morphine 1 mg / kg, ip).

[0151] All these results mean that GPER, when localized in the periphery, is at least partly responsible for the analgesic effect of compound E 2.

[0152] 3- Compound E 2 induces an analgesic effect on a murine model of pain osteoarthritic dependent on the GPER and whose functional localization is partly spinal

[0153] Viral deletion of GPER specifically in the spinal cord (GPERDH KO mice) reduces osteoarthritic pain ([Fig.20]) and shows that this receptor, when localized at the spinal level, is involved in osteoarthritic pain.

[0154] The analgesic effect of compound E 2 in a murine model of osteoarthritic pain is reduced in GPERDH KO mice (positive control: morphine 1 mg / kg, ip) ([Fig.21]).

[0155] Taken together, these results mean that GPER, when localized to the spinal cord, is at least partly responsible for the analgesic effect of compound E 2. List of references

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Claims

1. Demands Derivative of tetralone with the following general formula (I): [Chem.7] U

2.

3.

4. (I) where Ri is H or CH3; R2 is H, OH or OCH3; R3 is H or F, OCH3, CH3 or Br; R4 and R5 are H, OCH3 or together form a 1,3-dioxolane; R6 is H or Br. Derived according to claim 1, where Ri, R2, R3 and R6 are as defined in claim 1; and R4 and R5 are H. Derived according to claim 1, where Ri, R2, R3 and R6 are as defined in claim 1; and R4 and R5 together form a 1,3-dioxolane. Derived according to any one of claims 1 to 3; of the following formula: [Chem. 8] 0

5. E3 E4 Pharmaceutical composition comprising at least one derivative according to any one of claims 1 to 4 and a pharmaceutically acceptable excipient.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15. A pharmaceutical composition according to claim 5, further comprising at least one antitumor agent and / or analgesic agent. A pharmaceutical composition according to claim 6, wherein said at least one antitumor agent is paclitaxel, oxaliplatin, or bortezomib. Derived according to any one of claims 1 to 4 or composition according to any one of claims 5 to 7, for use as a medicinal product. Pharmaceutical derivative or composition for use according to claim 8, wherein said medicinal product is an analgesic and / or an anti-tumor agent. Pharmaceutical derivative or composition for use according to claim 8 or 9, wherein the drug is intended for the control of nociception. A derivative according to any one of claims 1 to 4 or a composition according to any one of claims 5 to 7, for use in the prevention or treatment of pain. A pharmaceutical derivative or composition for use according to claim 11, where the pain is neuropathic or arthritic. A pharmaceutical derivative or composition for use according to claim 12, where the pain is associated with cancer treatment. A pharmaceutical derivative or composition for use according to claim 13, where the cancer is triple-negative breast cancer (TNBC). Derived according to any one of claims 1 to 4 or composition according to any one of claims 5 to 7, for use in the prevention or treatment of cancer, in particular triple negative breast cancer (TNBC).

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