Coumarin derivatives for use in the treatment of a synucleinopathy

Abstract

The present invention relates to coumarin derivatives for use in the treatment of a synucleinopathy

Description

[0001] DESCRIPTION

[0002] TITLE OF THE INVENTION: COMPOUNDS FOR TREATING SYNUCLEOPATHY

[0003] The present invention relates to compounds for use in the treatment of synucleinopathy.

[0004] Synucleinopathies are a group of neurodegenerative proteinopathies characterized by pathological lesions composed of alpha-synuclein (or α-syn) aggregates. These lesions can be located in various parts of the brain and affect neurons or glial cells. The main synucleinopathies are Parkinson's disease, Lewy body dementia, and multiple system atrophy. Although environmental factors have been identified through epidemiological studies, the causes of these diseases remain poorly understood. Despite significant research efforts, these diseases remain incurable.

[0005] Parkinson's disease is one of the few degenerative disorders of the central nervous system that can be treated with medication. These treatments are purely symptomatic, meaning they can alleviate or even eliminate the manifestations of the disease, but they do not affect its progression. In the long term, however, these treatments become less and less effective and can lead to motor complications.

[0006] For over a decade now, the involvement of serine proteases in the functioning of the central nervous system (CNS) has been continually highlighted. Among serine proteases, tissue kallikreins form a family of proteases present in at least six mammalian orders. They are involved in functions as diverse as the regulation of synaptic plasticity, neuronal survival, memory acquisition, and vascular function. Among the tissue kallikreins, KLK6 (kallikrein-6 or hK6) is the most abundant in the adult CNS. It is produced by neurons and oligodendrocytes and has tryptic proteolytic activity. KLK6 thus plays a central role in the brain and spinal cord. Various studies converge on the hypothesis that KLK6 is involved in numerous neurodegenerative diseases, including synucleinopathies.The involvement of KLK6 in α-syn degradation was reported as early as 2003. These initial observations demonstrated the in vivo colocalization of KLK6 with α-syn within Lewy bodies found in Parkinson's disease. It has also been shown that in vitro, KLK6 prevents α-syn polymerization by reducing the amount of monomers available for polymerization and by generating α-syn fragments that, in turn, inhibit polymerization. Finally, peripheral administration of a lentivirus expressing KLK6 in murine models of synucleinopathies led to the accumulation of this protease in the brain, associated with a decrease in α-syn deposition in oligodendrocytes and astrocytes, as well as an improvement in parkinsonian behavior.Thus, these studies suggest that strong KLK6 activity, including under unfavorable pH conditions, could limit or stop the spread of a-syn in the brain and consequently prevent and / or treat alpha-synucleinopathies.

[0007] Biochemical studies on KLK6 had reported that certain high molecular weight polymers belonging to the glycosaminoglycan and heparin families could act as KLK6 activators. However, these compounds exhibit low activating efficacy and are unlikely to cross the blood-brain barrier.

[0008] The identification of small molecules capable of activating KLK6 and likely to distribute in the CNS is therefore a real challenge in the context of the treatment of synucleinopathies.

[0009] In a particularly unique development, the inventors discovered that the compounds according to the invention are effective and highly selective activators of KLK6. These compounds also have the advantage of not being cytotoxic, particularly neurotoxic, for example, to primary murine neurons. They are therefore of significant therapeutic interest in the context of synucleinopathies.

[0010] Furthermore, these compounds are small molecules that are easy to synthesize, making their production quick and economical.

[0011] Thus, the present invention relates to a compound for use in the treatment of a disease linked to a deficiency in the expression and / or activity of KLK6, said compound having the following formula (I):

[0012] (I), in which:

[0013] Ri represents:

[0014] H, a linear or branched alkyl group of 1 to 6 carbon atoms, a phenyl group possibly substituted by a group R' in particular chosen from OR”, with R” represents a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me, or a group of the following formula (A): in which:

[0015] X is chosen from -C(=O)-O-, -C(=O)-S-, -OC(=O)-, and -SC(=O)-, Ra represents a heterocycle chosen from:

[0016] , these heterocycles may optionally be substituted by at least one group chosen from linear or branched alkyl groups of 1 to 6 carbon atoms, in particular Me, and halogens, in particular Cl or in which:

[0017] Xi, X2 and X3 independently represent CH or N, one representing N, the other two CH.

[0018] X4 and X5 independently represent CH or N, one representing N, the other CH.

[0019] R2 represents: H, a linear or branched alkyl group of 1 to 6 carbon atoms, or a group of the following formula (B):

[0020] (B), in which:

[0021] Y is chosen from -OC(=O)-, -CH2-C(=O)-, -SC(=O)-, -C(=O)-O-, -C(=O)-S-, -OH- (Rb being absent), -NH-, -CONH-, -C(=O)-NRb'-,

[0022] Rb represents a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me, or Rb forms with Ri, especially when Y represents -OC(=O)-, a bicycle of the following formula: , which is possibly substituted, being in particular , with

[0023] Rd representing a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me,

[0024] Rb' represents a linear or branched alkyl group of 1 to 6 carbon atoms, in particular Me, Ri representing H, a linear or branched alkyl group of 1 to 6 carbon atoms, or a phenyl group possibly substituted by a group R' in particular chosen from OR”, where R” represents a linear or branched alkyl group of 1 to 6 carbon atoms, in particular Me, and R2 being of formula (B), or R2 representing H or a linear or branched alkyl group of 1 to 6 carbon atoms, and Ri being of formula (A),

[0025] R3, R4, Rs and RÔ represent, independently of each other, H, OH, ORc or OC(=O)Rc, Rc representing a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me, or R4 and R5 together form a possibly substituted heterocycle, for example a heterocycle of the following formula: or , with e and e representing n pen amment a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me.

[0026] The present invention also relates to a compound for use in the treatment of a disease being a synucleinopathy, said compound having the following formula (I):

[0027] (I), in which:

[0028] Ri represents:

[0029] H, a linear or branched alkyl group of 1 to 6 carbon atoms, a phenyl group possibly substituted by a group R' in particular chosen from OR”, with R” represents a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me, or a group of the following formula (A): in which:

[0030] X is chosen from -C(=O)-O-, -C(=O)-S-, -OC(=O)-, and -SC(=O)-, Ra represents a heterocycle chosen from: , these heterocycles may optionally be substituted by at least one group chosen from linear or branched alkyl groups of 1 to 6 carbon atoms, in particular Me, and halogens, in particular Cl or Br, in which:

[0031] Xi, X2 and X3 independently represent CH or N, one representing N, the other two CH.

[0032] X4 and X5 independently represent CH or N, one representing N, the other CH.

[0033] R2 represents:

[0034] H, a linear or branched alkyl group of 1 to 6 carbon atoms, or a group of the following formula (B):

[0035] (B), in which:

[0036] Y is chosen from -OC(=O)-, -CH2-C(=O)-, -SC(=O)-, -C(=O)-O-, -C(=O)-S-, -OH- (Rb being absent), -NH-, -C(=O)-NH-, -C(=O)-NRb'-,

[0037] Rb represents a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me, or Rb forms with Ri, especially when Y represents -OC(=O)-, a bicycle of the following formula:

[0038] , which is possibly substituted, being in particular , with

[0039] Rd representing a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me,

[0040] Rb' represents a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me,

[0041] Ri representing H, a linear or branched alkyl group of 1 to 6 carbon atoms, or a phenyl group possibly substituted by a group R' in particular chosen from OR”, where R” represents a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me, and R2 being of formula (B), or R2 representing H or a linear or branched alkyl group of 1 to 6 carbon atoms, and Ri being of formula (A),

[0042] R3, R4, Rs and RÔ represent, independently of each other, H, OH, ORc or OC(=O)Rc, Rc representing a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me, or R4 and R5 together form a possibly substituted heterocycle, for example a heterocycle of the following formula: , mment a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me.

[0043] According to a particular embodiment, R3, R4, Rs and Re represent H.

[0044] According to a particular embodiment, R3, R4, and Re represent H, R5 represents -

[0045] C(=O)Rc, with in particular Ri being H.

[0046] According to a particular embodiment, Ri does not represent H.

[0047] According to a particular embodiment, Y is chosen from -OC(=O)- and -CH2-C(=O)-, with in particular Ri being H.

[0048] According to a particular embodiment, Rb forms with Ri, especially when Y represents -OC(=O)-, a bicycle with the following formula:

[0049] which is possibly substituted, being in particular with Rd representing a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me, with in particular R4 being -OH and / or R3, R5 and Re representing H.

[0050] According to a particular embodiment, R4 and R5 together form a possibly substituted heterocycle, for example a heterocycle with the following formula: , ment a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me, notably with:

[0051] R3 and RO representing H, or R3 representing ORc and RO representing H; and / or

[0052] Y chooses between -OC(=O)- and -OH- (Rb being absent); and / or

[0053] - Ri representing a phenyl group possibly substituted by a group R' in particular chosen from OR”, with R” represents a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me.

[0054] The present invention also relates to a compound for use in the treatment of a disease being a synucleinopathy, said compound having the following formula (I): in which:

[0055] Ri represents:

[0056] H, a linear or branched alkyl group of 1 to 6 carbon atoms, or a group of the following formula (A): in which:

[0057] X is chosen from -C(=O)-O-, -C(=O)-S-, -OC(=O)-, -SC(=O)-, Ra represents a heterocycle chosen from: These heterocycles may optionally be substituted by at least one group chosen from linear or branched alkyl groups of 1 to 6 carbon atoms, in particular Me, and halogens, in particular Cl or

[0058] Br, in which:

[0059] Xi, X2 and X3 independently represent CH or N, one representing N, the other two CH.

[0060] X4 and X5 independently represent CH or N, one representing N, the other CH.

[0061] R2 represents:

[0062] H, a linear or branched alkyl group of 1 to 6 carbon atoms, or a group of the following formula (B):

[0063] (B), in which:

[0064] Y is chosen from -OC(=O)-, -SC(=O)-, -C(=O)-O-, -C(=O)-S-,

[0065] Rb represents a linear or branched alkyl group of 1 to 6 carbon atoms, in particular Me,

[0066] Ri representing H, a linear or branched alkyl group of 1 to 6 carbon atoms, or a phenyl group possibly substituted by a group R' in particular chosen from OR”, where R” represents a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me, and R2 being of formula (B), or R2 representing H or a linear or branched alkyl group of 1 to 6 carbon atoms, and Ri being of formula (A),

[0067] R3, R4, Rs and RÔ represent H.

[0068] According to a particular embodiment, said compound has the following formula (Ii):

[0069] (II).

[0070] According to a particular embodiment, said compound has the following formula (III): in which R2 represents: H, or a linear or branched alkyl group of 1 to 6 carbon atoms.

[0071] According to a particular embodiment, said compound has the following formula (II2):

[0072] (II2).

[0073] According to a particular embodiment, said compound has the following formula (II3):

[0074] According to a particular embodiment, said compound has the following formula (II4):

[0075] According to a particular embodiment, Ra has the following formula: in which Xi, X2 and X3 independently represent CH or N, one representing N, the other two CH. In particular, Xi represents N, and X2 and X3 represent CH.

[0076] According to a particular embodiment, said compound has the following formula (III): in which Ri represents H, a phenyl group possibly substituted by a group R' in particular chosen from OR”, where R” represents a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me, or a linear or branched alkyl group of 1 to 6 carbon atoms, in particular in which Ri represents H, or a linear or branched alkyl group of 1 to 6 carbon atoms.

[0077] According to a particular embodiment, said compound has the following formula (III2):

[0078] (III2). In a particular realization, said compound has the following formula (III3):

[0079] (III3).

[0080] According to a particular embodiment, Rb is Me. According to a particular embodiment, Ri is Me, Et, nPr or nBu.

[0081] According to a particular embodiment, said compound is chosen from: , 0402 (or 402),

[0082]

[0083] According to a particular embodiment, synucleinopathy is an alpha-synucleinopathy of the group comprising Parkinson's disease (or PD for "Parkinson's Disease"), Lewy body dementia (or DLB for "Dementia with Lewy Bodies"), multiple system atrophy (or MSA for "Multiple System Atrophy"), amyotrophic lateral sclerosis, pure autonomic failure (or PAF for "Pure Autonomy Failure") and REM sleep behavior disorder (or RBD for "REM sleep Behavior Disorder").

[0084] According to one particular embodiment, synucleinopathy is Parkinson's disease, Lewy body dementia, multiple system atrophy, or amyotrophic lateral sclerosis.

[0085] In one particular embodiment, synucleinopathy is Parkinson's disease. In another particular embodiment, synucleinopathy is dementia with a specific underlying condition.

[0086] Lewy.

[0087] DEFINITIONS

[0088] As used here, the term "alkyl" refers to a linear or branched alkyl group having the number of carbon atoms indicated before the term, specifically 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl, neopentyl, 1-ethylpropyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, hexyl, etc. Thus, an expression such as "C1-C4 alkyl" refers to an alkyl radical containing 1 to 4 carbon atoms.

[0089] FIGURES

[0090] [Figure 1]: Activation reaction scheme for the activator CFL33. E = enzyme, S = substrate, KM = Michaelis constant, A = activator, KA = activation constant, a = association factor, P = catalytic factor.

[0091] [Figure 2]: AC50 measurements to evaluate the activating effect of the hit compounds. AC50 curves of the four hit compounds 402 (A), 410 (B), 2718 (C), and CFL33 (D) at three different pH values ​​where activation occurs: pH 7 (circles), pH 8 (squares), and pH 9 (diamonds) for 402, 410, and 2718, and at pH 9 for CFL33. (E) AC50 values ​​of the compounds. ND: not determined. The compounds at different concentrations (from 0.19 to 50 pM for CFL33 and from 3.12 to 100 pM for the others) were tested on KLK6 (0.5 nM) with the Boc-QAR-AMC substrate (100 pM) in 50 mM Tris buffer, IM citrate, and 0.05% Brij-35 at 37°C. The tests were carried out in duplicate.

[0092] [Figure 3]: Activation mechanisms of molecule 402 with respect to KLK6. A - Lineweaver-Burk (LB) plot for KLK6 and 402 (from 0 to 100 pM). B - Primary plot of the specific velocity VO / VA as a function of (0 to 100 pM), in the absence (dashed blue line) or in the presence of 402 (1.2, 3.7, 11.1, 33.3, 100 pM, vertical lines from top to bottom). C - Secondary plot representing the variation of the values ​​of a / (al) (red line) and b / (ba) (pink line), extracted from the primary curve, as a function of l /

[402] to determine a, P, and KA. D - Values ​​of the kinetic parameters KA, a, and P of compounds 402, 410, and 2718 and their activation mode. The activities of KLK6 (0.5nM) under these conditions were measured for the hydrolysis of Boc-QAR-AMC (100pM), in duplicates in Tris 50mM buffer, IM Citrate, Brij-35 0.05%; pH7 at 37°C.

[0093] [Figure 4]: Selectivity profile of hit compounds towards KLK6. The activities of KLK1, KLK5, KLK6, KLK13, KLK14, plasmin, and the trypsin-like (TL) and caspase-like (CL) activities of the constitutive proteasome (C) were measured, in the absence of molecules (green) or in the presence of pM molecules (blue) 402 (A), 410 (B), 2718 (C), or CFL33 (D), for the hydrolysis of Boc-QAR-AMC 100 pM in 50 mM Tris buffer, IM citrate, Brij-35 0.05%; pH 7 at 37°C (pH 9 for CFL33). The tests were performed in duplicate.

[0094] [Figure 5]: Inhibition of prion-like propagation of α-synuclein aggregates by compounds of the invention, as shown in a histogram:

[0095] • First column (control): PB S (without PFF).

[0096] • Second column (control): DMSO + PFF (in PBS).

[0097] • Other columns: composed of CFL33, 402, 2718 or 410 (in DMSO) + PFF (in PBS).

[0098] Thus, in the first line of the legend, "-" signifies the absence of PFF and DMSO, and "+" signifies the presence of PFF (in PBS) and DMSO. The second line illustrates the presence or absence of a fixed-concentration activator.

[0099] EXAMPLES

[0100] Example 1: Enzymatic kinetics and mechanistic studies

[0101] 1.1 Materials and methods

[0102] 1.1.1 Experimental Procedure

[0103] A miniaturized assay was developed for each protease studied. The conditions used for screening molecules in 96-well plates allow for good reproducibility of activity measurements on very small volumes using 50 mM Tris-HCl buffer, 1 M sodium citrate, 0.05% (v / v) Brij-35, and the foliogenic peptide substrates Boc-VPR-AMC, Boc-QAR-AMC, Suc-RPY-AMC, or MeOSuc-AAPV-AMC at 37 °C and pH 7.0. In a typical assay, 100 pL of reaction medium contains the buffer, 1 pL of recombinant enzyme, and 1 pL of compound previously dissolved in DMSO at the appropriate concentration (in the control, 1 pL DMSO). The release of the fluorescent group (X) ex= 360 nm, em = 460 nm) following enzyme-catalyzed substrate hydrolysis is monitored for 15 to 120 min using a BMG-FLUOStar instrument. A fluorogenic FRET substrate mimicking the TV-terminal portion of the PAR-2 ​​receptor (Protease-Activated Receptor 2 or protease-activated G protein-coupled receptor) synthesized by GL-Biochem was also used for KLK6 under the same experimental conditions but at wavelengths: Ux = 320 nm, em = 405 nm.

[0104] Table 1: Summary of experimental conditions used for the enzyme assays for each enzyme. All enzymes used are of human origin except for pancreatic elastase (of porcine origin). Abbreviations: Abz: aminobenzoyl; AMC: 7-amino-4-methyl coumarin; EDDnp: ethylenediamine 2,4-dinitrophenyl.

[0105] The fluorogenic substrates Boc-QAR-AMC and Boc-VPR-AMC were purchased from Bachem®. The FRET substrates MBP1 (Abz-RPSQR HATQ-EDDnp), MBP2 (Abz-HPAR TAHQ-EDDnp), MBP3 (Abz-YGGR ASDQ-EDDnp), and PAR2 (Abz-SSKGRJ.SLIGQ-EDDnp) were purchased from GL Biochem®. The sequences of these peptide substrates replicate the cleavage sites of the biological substrates of KLK6 (MBP, PAR2) and plasmin (PAR4) and were custom-synthesized. All substrates were prepared at concentrations of 10 mM or 20 mM in 100% DMSO (Sigma-Aldrich®) and stored at room temperature, protected from light.

[0106] The ACso of each of compounds 402, 410, 2718, and CFL33 was measured to evaluate their activating power against KLK6 and to rank them. ACso curves were plotted at pH 7, 8, and 9 to assess the stability of the activating effect (Figure 2). The ACso and A maThe percentage of maximum activation (x) of each molecule at each pH was extracted (Table 1). Compounds 402, 410, and 2718 exhibited conservation of their activating effect with varying pH. However, compound CFL33 exhibited a pH-dependent effect, with activation occurring only at pH 9. This compound showed excellent values ​​(ACso = 9.5 pM; A ma x = 540.8% at pH 9). Among the other compounds, 402 shows high activation even at pH 7 (A max : 255.5 ± 6.6) with an excellent ACso (20 pM), stable with pH variation.

[0107] According to the taxonomic model of A. Baici (2015), with parameters KA = 18.5 pM, a = 0.29, and P = 1.3, determined graphically (Figure 3.B, C), molecule 402 acts via a "mixed hyperbolic activation with predominantly catalytic" mechanism. The activation mode and parameters of the other molecules were also determined (Table 2). Mixed activation means that the molecule acts on both the enzyme's affinity for its substrate (KM) and its catalytic capacity (V). max ). This is confirmed by the Lineweaver-Burk curve (Figure 3.A), representing the variation of the inverse of the initial velocity (1 / Vo) as a function of the inverse of the substrate concentration (1 / [S]) in the presence of increasing concentrations of activators (0 to 100 pM)).

[0108] The selectivity of action of the compounds of the invention towards KLK6 was evaluated on a broad panel of kallikreins and proteases abundant in the CNS. The enzymatic activities of KLK1, KLK5, KLK6, KLK13, KLK14, plasmin, and the trypsin-like (TL) and caspase-like (CL) activities of the constitutive proteasome (clearance system also established) were then measured in the absence and presence of the four molecules at 100pM (Figure 4). The relative activities (%) compared to the initial activities of these enzymes suggest that molecule 402 exhibits an excellent selectivity profile at physiological pH among the four hit compounds. Indeed, the strong increase in relative activity (300%) induced by 402 occurs only with KLK6. Activation by molecule 2718 is also highly selective towards KLK6.

[0109] CFL33 directly and selectively increases KLK6 activity with an ACso of 1.6 pM at basic pH. Its action is characterized by a completely original mechanism that can significantly amplify KLK6 activity, up to 7 times greater than basal activity. The observed effect has the following characteristics:

[0110] - it becomes more pronounced at a basic pH;

[0111] - it is stable over time for 24 hours;

[0112] - It is significant (increasing basal activity by up to 7 times at the considered pH). This effect is very important given the catalytic nature of the target (KLK6) and its involvement in various enzymatic cascades. Indeed, an activation of only 10% (VA / VO > 1.1) is sufficient to trigger an in vivo response (where Vo represents the basal activity of an enzyme; VA represents the activity in the presence of the activator).

[0113] Thus, the activating compound maintains significant catalytic activity at basic pH. In the context of neurodegenerative diseases such as Parkinson's disease, pH dysregulation is observed, and alkaline and acidic microenvironments can emerge (Obara et al., Neurochem Int 2008, 52 (6), 905-19). This activator therefore possesses mechanistic and pharmacological characteristics (sustained action) compatible with the development of new KLK6-specific activity probes in pathological microenvironments where local pH fluctuations are observed. These highly specific properties pave the way for the design of molecules of therapeutic or diagnostic interest for Parkinson's disease and synucleinopathies.

[0114] Table 2 Summary of the main pharmacological properties of the four hit compounds.

[0115] The ADME-Tox (Absorption, Distribution, Metabolism, Elimination, and Toxicity) properties of the compounds of the invention were evaluated. Only the main findings are given in Table 2. Among these properties, the permeability of a molecule can be strongly influenced by its lipophilicity and therefore its oral absorption and even its passage across the blood-brain barrier. The latter is measured using the logD parameter, which represents the octanol / water partition coefficient of the molecule. The solubility / stability of the molecules in PBS and simulated gastric flow was tested and compared to reference drugs such as ketoconazole and rifampicin. The four compounds have solubility and logD values ​​that are higher than or equivalent to those of the reference compounds and consistent with oral absorption.Permeability properties were evaluated using intestinal and renal cell models (Caco2 and hMDRI-MDCKII cMDRI KO), which mimic a functional intestinal barrier and measure an active substance's ability to cross the intestinal wall and distribute into the bloodstream. The permeability of the four compounds was, on average, greater than or equivalent to that of the reference compounds. The Safety47 panel (SAFETYscan E / ICso ELECT) is an essential selection of 78 targets identified by the pharmaceutical industry for drug development. These targets include those of interest in the central nervous system (CNS), receptor tyrosine kinases (RTKs), cyclooxygenases, monoamine oxidases, acetylcholinesterase, ion channels, dopamine receptors, and more. A key aspect of this Safety47® test is its prediction of cardiotoxicity.Importantly, none of the compounds present a risk of cardiotoxicity. Indeed, there is no significant inhibition of ion channels, particularly the hERG potassium channel, which is essential for heart function and therefore cannot predict potential toxic effects. Furthermore, and interestingly, the CFL33 molecule inhibits the ROCK1 (Rho-associated protein kinase 7) kinase with an IC50 of 1.26 pM, a target of interest in Parkinson's disease. In vitro studies using human cells and in vivo studies have shown that ROCK1 inhibition protects dopaminergic cells from apoptosis and improves the symptoms of Parkinson's disease.

[0116] Example 2: Biological study of compounds.

[0117] 3.1 Materials and methods

[0118] Analysis of cytotoxicity on primary culture of murine cortical neurons

[0119] Cortical neurons were harvested from E1 6 stage embryos of pregnant Swiss females (Janvier, Le Genest Saint Isle, France). All dissection steps were performed in Gey's balanced saline solution (GBSS, Life Technologies, Inc., Gaithersburg, MD, USA) supplemented with 0.1% D-(+)-glucose (Sigma, St. Louis, MO, USA). The dissected structures were chemically dissociated using papain (45 U / mL, Sigma) resuspended in Dulbecco's Modified Eagle Medium + GlutaMAX-I (DMEM, Life Technologies, Inc.) containing penicillin and streptomycin (100 U / mL and 100 pg / mL, respectively, Life Technologies, Inc.), at 37°C. The cells were cultured for 48 h in DMEM medium supplemented with 10% fetal bovine serum, penicillin (100 pg / mL) and streptomycin (100 pg / mL), at 37°C under a humid atmosphere with 5% CO2. The cells were then incubated with the compound of interest CFL33 (100; 25; 6.25; 1.56 pM) for 48 h.The medium is then replaced with 100 pL of XTT (0.3 mg / mL in DMEM F 12 medium (without phenol red); 8.3 pM PMS, Sigma-Aldrich). The cells are incubated for 3 h at 37°C, and the absorbance is measured at 485 nm. XTT is a tetrazolium salt derivative, the reduction of which by functional mitochondrial dehydrogenases produces an orange-yellow color. The mitochondrial activity of the cells is determined by measuring the absorbance at 485 nm. This mitochondrial activity assay allows for the evaluation of cell viability. In control experiments, the cells are treated with the vehicle molecule (DMSO), used at the same concentration. Differentiation of SH-SY5Y cells into dopan-nergic-like neurons.

[0120] Human SH-SY5Y neuroblastoma cells (ATCC, Manassas, VA, USA) were maintained and cultured to 80% confluence in their DMEM culture medium "Dulbecco's Modified Eagle's media" supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin and 100 pg / ml streptomycin (Gibco Grand Island, NY, USA), and 2 mM L-Glutamine, at 37°C in a humidified atmosphere with 5% CO2. The cells were then cultured on 96-well plates (Falcon®) at a density of 5000 cells / well. 24h later, on day 1 of the differentiation protocol with retinoic acid, the medium was replaced with the first differentiation medium (DMEM, 2.5% FBS, 100 U / ml penicillin, 100 pg / ml streptomycin, L-Glutamine 2Mm, retinoic acid 1OpM), on day 3, the medium is removed and replaced with the second differentiation medium (DMEM, 1% FBS, 100 U / ml penicillin, 100 pg / ml streptomycin, L-Glutamine 2mM, retinoic acid 1OpM).This step was repeated during days 3, 5, 7, 9 and 11 of the differentiation protocol.

[0121] Analysis of the cytotoxicity and neuro-cytoprotection of the compounds of the invention on rotenone-treated differentiated SH-SY5Y cells

[0122] SH-SY5Y cells differentiated in 96-well plates (5000 cells) were incubated with (or without) 50 pM of hits molecules or DMSO control and then incubated for 27 h at 37°C in a humidified atmosphere with 5% CO2. The cells were then treated with rotenone (1 pM) or DMSO 3 h later and incubated for 24 h at 37°C in a humidified atmosphere with 5% CO2. The medium was then replaced with 100 pM of phenol red-free detection medium containing XTT (0.3 mg / ml) and PMS (8.3 pM) for 3 h at 37°C. Absorbance was measured at 485 nm to quantify cell viability. XTT is a tetrazolium salt derivative that turns yellow-orange after its reduction by functional mitochondrial dehydrogenases in the cells.

[0123] Quantitative analysis of the neuro-cytoprotection of the compounds of the invention on SH-SY5Y cells treated with 6-hydroxydopamine

[0124] Another test was performed in a 6-hydroxydopamine (6OHDA)-induced toxicity model in SH-SY5Y cells to determine the molecules' ability to inhibit cell death. The known PHD2 inhibitor, Adaptaquin, was used as a control.

[0125] The compounds were prepared in 10 mM DMSO, and other dilutions were made in the assay buffer (serum-free medium for 6OHDA). The day after plate inoculation, the medium was replaced with serum-free medium. The compound dilutions were added to the plates, which were incubated for 30 minutes before the addition of 60HDA to a final concentration of 75 pM (compounded in ice-cold, deoxygenated saline; 0.001% ascorbic acid). After 30 minutes, the media were removed and replaced with fresh serum-free medium containing 1:1000 of CellTox™ Green. The compound dilutions were added back to the plates, which were incubated for 24 hours at 37°C and then read on a plate reader (Ex 490, Em 535).

[0126] The compounds were tested with and without 60HDA in triplicate for their ability to protect cells against 60HDA-induced cell death. The control compound (PHD2 inhibitor, Adaptaquin) was tested at the same concentration range. The percentage recovery from 60HDA-induced cell death was calculated by comparing the fluorescence of the control and treated cells. Percentage recovery values ​​were calculated for each concentration of the tested compound and for the controls.

[0127] % recovery = 100 - [(combined RFU — untreated control RFU) / (RFU 10 M toxin alone - untreated control RFU) *100]

[0128] 3.2 Results

[0129] Analysis of cytotoxicity on primary culture of murine cortical neurons

[0130] According to the cell survival assay using the XTT, compounds CFL33 and 402 were found to be weakly cytotoxic to a primary culture of murine cortical neurons exposed for 48 hours to concentrations of 100, 25, 6.25, and 1.56 pM (1% DMSO). Cell survival was greater than 65% for each concentration of CFL33 and greater than 80% for 402.

[0131] Analysis of the cytotoxicity and neuro-cytoprotection of the compounds of the invention on differentiated SH-SY5Y

[0132] To mimic the microenvironment of Parkinson's disease (PD) in cells, differentiated SH-SY5Y cells were exposed for 24 hours to a neurotoxic agent: rotenone. The cells were pretreated with 100pM of compounds 402 and 410 to determine whether they protect dopaminergic-like neurons against rotenone-induced apoptosis. Rotenone reduces cell viability by 20%, and treatment with the compounds of the invention restores the initial viability percentage.

[0133] Quantitative analysis of the neuro-cytoprotection of the compounds of the invention in a 6-hydroxydopamine-induced toxicity model in SH-SY5Y

[0134] The effective concentration values ​​EC50 or the maximum recovery values ​​in % obtained are reported in Table 3.

[0135] Table 3: Quantitative analysis of the neuro-cytoprotection of the compounds of the invention in a 6-hydroxydopamine-induced toxicity model on SH-SY5Y cells

[0136] Example 3: Inhibition of prion-like propagation of α-synuclein aggregates by the compounds of the invention.

[0137] WT primary hippocampal neurons were treated with 70 nM of unphosphorylated α-synuclein or PFF (pre-formed fibrils) for 21 days before being fixed, immunolabeled for pS129 α-synuclein (antibody 81A), and imaged (INCell 2200) (Fares et al. PNAS 2016, 113(7):E912-21). Automated quantification of inclusions (cellProfiler) showed that co-treatment with the compounds of the invention (10 pM) from day 10 to day 21 reduced the formation of pS129 α-synuclein inclusions (newly formed endogenous α-synuclein aggregates) compared to the DMSO control (Figure 5).

Claims

DEMANDS 1. Compound for use in the treatment of a disease being a synucleinopathy, said compound having the following formula (I): in which: Ri represents: H, a linear or branched alkyl group of 1 to 6 carbon atoms, a phenyl group possibly substituted by a group R' in particular chosen from OR”, with R” represents a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me, or a group of the following formula (A): in which: X is chosen from -C(=O)-O-, -C(=O)-S-, -OC(=O)-, and -SC(=O)-, Ra represents a heterocycle chosen from: These heterocycles may optionally be substituted by at least one group chosen from linear or branched alkyl groups of 1 to 6 carbon atoms, in particular Me, and halogens, in particular Cl or Br, in which: Xi, X2 and X3 independently represent CH or N, one representing N, the other two CH. X4 and X5 independently represent CH or N, one representing N, the other CH. R2 represents: H, a linear or branched alkyl group of 1 to 6 carbon atoms, or a group of the following formula (B): (B), in which: Y is chosen from -OC(=O)-, -CH2-C(=O)-, -SC(=O)-, -C(=O)-O-, -C(=O)-S-, -OH- (Rb being absent), -NH-, -CONH-, -C(=O)-NRb'-, Rb represents a linear or branched alkyl group of 1 to 6 carbon atoms, in particular Me, Or Rb forms with Ri, particularly when Y represents -OC(=O)-, a bicycle with the following formula: , which is possibly substituted, being in particular , with Rd representing a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me, Rb' represents a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me, Ri representing H, a linear or branched alkyl group of 1 to 6 carbon atoms, or a phenyl group possibly substituted by a group R' in particular chosen from OR”, where R” represents a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me, and R2 being of formula (B), or R2 representing H or a linear or branched alkyl group of 1 to 6 carbon atoms, and Ri being of formula (A), R3, R4, Rs and RÔ represent, independently of each other, H, OH, ORc or OC(=O)Rc, Rc representing a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me, or R4 and R5 together form a possibly substituted heterocycle, for example a heterocycle of the following formula: , pp damment a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me.

2. Compound for its use according to claim 1, said compound having the following formula (Ii): (II).

3. Compound for its use according to claim 1, said compound having the following formula (III): in which R2 represents: H, or a linear or branched alkyl group of 1 to 6 carbon atoms, said compound having in particular the following formula (II2): (II2), said compound being more particularly of the following formula (II3):

4. Compound for use according to any one of the preceding claims, said compound having the following formula (II4): (I - 5. Compound for use according to any one of the preceding claims, wherein Ra has the following formula: in which Xi, X2 and X3 independently represent CH or N, one representing N, the other two CH.

6. Compound for its use according to claim 1, said compound having the following formula (IIIi): (nii), in which Ri represents H, a phenyl group possibly substituted by a group R' in particular chosen from OR”, with R” represents a linear or branched alkyl of 1 to 6 carbon atoms, in particular Me, or a linear or branched alkyl group of 1 to 6 carbon atoms 5. The said compound being in particular of the following formula (III2): (IH2), said compound being more particularly of the following formula (III3): (III3).

7. Compound for its use according to claim 6, wherein Rb is Me.

8. Compound for its use according to any one of claims 6 to 7, wherein Ri is Me, Et, nPr or nBu.

9. Compound for use according to any one of the preceding claims, which is selected from:

10. Compound for use according to any one of the preceding claims, wherein synucleinopathy is an alpha-synucleinopathy of the group comprising Parkinson's disease, Lewy body dementia, multiple system atrophy, amyotrophic lateral sclerosis, pure autonomic insufficiency (and REM sleep behavior disorder).

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