The present invention relates to modified fumarate molecules capable of inducing HO-1 protein expression, their synthesis and their use in therapeutic applications, in particular their use in the treatment of inflammatory and cardiovascular diseases.

BACKGROUND ART:

The nuclear factor erythroid derived 2-related factor 2 (Nrf2) and the antioxidant protein heme oxygenase-1 (HO-1 ) are crucial components of the cellular stress response. These two systems work together to combat oxidative stress and inflammation and are attractive drug targets for counteracting different pathologies, including cardiovascular diseases and inflammatory diseases such as neuroinflammation. Fumaric acid esters, including dimethyl fumarate (DMF) and its primary in vivo metabolite monomethyl fumarate (MMF), are a class of compounds that have anti-inflammatory and immune-modulating effects in vitro and in vivo.

DMF have been shown to induces the expression of NF E2-related factor 2 (Nrf2)-driven antioxidant response genes, including heme oxygenase-1 (HO-1 ) and NAD(P)H quinone oxidoreductase 1 (NQ01 ).

A formulation of DMF and other fumaric acid esters have been used in Europe since 1995 as effective treatments for psoriasis. Furthermore, DMF has been approved by the FDA and is sold as Tecfidera by Biogen for use in multiple sclerosis as it demonstrated a significant benefit in suppressing relapses, disease progression, and brain lesion inflammation.

It has been shown that MMF, the primary in vivo metabolite of DMF, has the same cytotoxic profile as DMF and that it inhibit NF- Β signaling, suppress the production of inflammatory mediators, and induce an antioxidant response in a variety of cell types (Cross et al;, J Immunol. 2011 Nov 15; 187(10): 5015-5025).

However, MMF has been shown to have a more than 4 fold decrease on activity compared to DMF. Hence, derivatives of DMF can exhibit a biological activity, in particular HO-1 activation, but this activity is usually lower than the DMF. There is thus a need for new molecules related to DMF that will exhibit a similar activity at least on HO-1 activation and a low cytotoxicity.

BRIEF SUMMARY OF THE INVENTION

Aiming to identify effective HO-1 activators that modulate the inflammatory response in microglia cells. Inventors of the present invention have discovered a new family molecules, based on a fumarate structure, capable of activating the HO-1 pathway to the same extent or even better than DMF.

Moreover, those molecules can further be advantageously coupled with CO-releasing molecules (CO-RMs) resulting in a dual activation of the inflammatory defenses in cells and an improved therapeutic efficacy compared to several other compounds.

The present invention therefore concerns compounds, their pharmaceutically acceptable salts, hydrates and solvates, of formula (I):

or a pharmaceutically acceptable salt and/or solvate thereof, wherein:

R1 represents -(Ci-C ₆ )alkyl, preferably -CH ₃

X represents O, N or S

Y represents O, N or S

A represents -(CrC ₆ )alkyl-, -(C ₂ -C ₆ )alkenyl-, -aryl-, -heteroaryl-, - (C ₂ -C ₆ )alkynyl-, -(C ₃ -C ₈ )heterocyclyl-, -(C ₃ -C ₁₄ )cycloalkyl, -(CrCeJalkyl-Rz-iC C ₆ )alkyl-, -(C ₂ -C ₆ )alkenyl-R ₂ -(Co-C ₆ )alkyl-, -(C-i-CeJalkyl-Rz-iCz-CeJalkenyl-, -(C^ C6)alkenyl-R ₂ -(C ₂ -C ₆ )alkenyl-, -(Ci-C ₆ )alkyl-R ₂ -(C ₂ -C ₆ )alkynyl-, -(C ₂ -C ₆ )alkenyl-R ₂ -(C ₂ - C ₆ )alkynyl-, -(C ₂ -C ₆ )alkynyl-R2-(C ₂ -C ₆ )alkynyl-, -(C2-C ₆ )alkynyl-R ₂ -(C _r C ₆ )alkyl-, -(C2- C ₆ )alkynyl-R ₂ -(C ₂ -C ₆ )alkenyl-, -(Ci-C ₆ )alkyl-OCO-, -CH ₂ -(CHOR ₂ )CH ₂ 0-(Ci-C ₆ )alkyl-, -(C ₀ -C ₆ )alkyl-CO-R ₂ -, or -(C ₀ -C ₆ )alkenyl-CO-R ₂ -,

where R ₂ represents -(C ₀ -C ₆ )alkyl-aryl, -(C ₀ -C ₆ )alkenyl-aryl, heteroaryl, (C ₃ -

C ₈ )heterocyclyl, or (C ₃ -Ci ₄ )cycloalkyl,

A is optionally substituted by a CO-releasing molecule. In particular, X represent N

In particular, Y represent O

In particular, A represent -(Ci-C ₆ )alkyl-, -aryl-, -(CrC ₆ )alkyl-aryl-, -heteroaryl-,

CO-R2-, -(C ₀ -C ₆ )alkenyl-CO-R ₂ -,

where R ₂ represents -(C ₀ -C ₆ )alkyl-aryl-, or -(C ₀ -C ₆ )alkenyl-aryl-

Preferably, A represent -(C _r C ₆ )alkyl-, -aryl-, -(C CeJalkyl-aryl-, -heteroaryl-,

CO-R2-, -(C ₀ -C ₆ )alkenyl-CO-R ₂ -,

where R ₂ represents -aryl-, or -(C ₀ -C ₆ )alkenyl-aryl-

In particular, compounds of the invention can be chosen from among:

Preferably, A is substituted by a CO-releasing molecule chosen from among:

W-(' Ru(CO) ₃ L , , -|-=|= and -|-≡|≡

S I CO ₂ (CO)B CO^CO)! o

M(CO) _N ^{2 M}

where W represents O or NR ₃ , where R3 represents -(C ₁ -C ₆ )alkyl-,

L represents an ionic ligand such as halogen, BF ₄ , PF ₆

M represents Rh, Co, Ru, Mn, Mo, V or Fe, preferably Co, Ru or Mn, and

n is an integer chosen so that the metal M has no free valency In particular, the CO-releasing molecule is chosen from among:

Preferably, the CO-releasing molecule is chosen from among:

where W represents O or NR ₃ , where R ₃ represents -(Ci-C ₆ )alkyi

In particular, compound of the invention can be chosen from among:

In another aspect, compounds of the invention are for use as a drug, preferably for use in the treatment of cardiovascular or inflammatory diseases.

In another aspect, the invention encompasses a pharmaceutical composition comprising compound of the invention

DETAILED DESCRIPTION

Definitions:

The present invention encompasses stable isomers with a geometry of the carbon- carbon double bound which is not restricted to E isomers and also include Z isomers.

Within the groups, radicals or fragments defined in the description and the claims, the number of carbon atoms is specified inside the brackets. For example, (CrC ₆ )alkyl designates an alkyl group or radical having 1 to 6 carbon atoms. When C ₀ is mentioned, it means that there is no atoms and hence the group encompass a single bond.

In the formulas, ^ indicates the bond linked to the rest of the molecule.

In the formulas, i indicates a single or double bond depending on the valence of linked atoms.

For the groups comprising two or more subgroups, the attachment is indicated with "- ". For example, "-(C ₁ -C ₆ )alkyl-aryl-(C ₁ -C ₆ )alkenyl-" indicates a radical alkyl bound to a radical aryl itself bound to an alkenyl wherein the alkyl and alkenyl groups are bound to the rest of the molecule.

In the sense of the present invention, the expression "-(d-CeJalkyl" designates an acyclic, saturated, linear or branched hydrocarbon chain comprising 1 to 6 carbon atoms. Examples of -(Ci-C ₆ )alkyl groups include methyl, ethyl, propyl, butyl, pentyl or hexyl. Unless explicitly stated, the definitions propyl, butyl, pentyl and hexyl include all possible isomers, in particular structural isomers. For example, butyl comprises n-butyl, / ^' so-butyl, sec-butyl and ierf-butyl. The alkyl group may be substituted, preferably with one or more groups independently selected from the group consisting of alkyl, alkoxy, halogen, hydroxyl, amino, nitro, cyano, trifluoro, carboxylic acid, CHO, amide, cetone thiol, thioester, thioalkyl, urea, sulfoxyde sulfone or carboxylic ester.

In the sense of the present invention, the expression "-(C2-C ₆ )alkenyl" designates an acyclic, saturated, linear or branched hydrocarbon chain comprising 2 to 6 carbon atoms, at least two of which are linked via a double bond. Examples of "-(C ₂ -C ₆ )alkenyl" include ethenyl or vinyl, propenyl, butenyl, pentenyl or hexenyl. Unless explicitly stated, the definitions of propenyl, butenyl, pentenyl and hexenyl include all possible isomers, in particular structural and/or position isomers.

In the sense of the present invention, the expression "-(C2-C ₆ )alkynyl" designates an acyclic, saturated, linear or branched hydrocarbon chain comprising 2 to 6 carbon atoms, at least two of which are linked via a triple bond. Examples of "-(C2-C6)alkynyl" include ethynyl, propynyl, butynyl, pentynyl or hexynyl. Unless explicitly stated, the definitions of propynyl, butynyl, pentynyl and hexynyl include all possible isomers, in particular structural and/or position isomers.

The term "substituted" as used herein means that any of the hydrogen atoms can be replaced by a substituent, such as fluorine.

In the sense of the present invention, the expression "-(C ₃ -C ₁₄ )cycloalkyl" designates a saturated or partially saturated mono-, di- or tri-cyclic structure comprising from 3 to 14 carbon atoms. Examples of "-(C ₃ -C ₄ )cycloalkyl" include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl and cyclohexenyl.

Examples of "-(C ₃ -C ₈ )cycloalkyl" include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl. Unless explicitly stated, the cycloalkyls can be substituted by one or more groups such as methyl, ethyl, isopropyl, hydroxy, fluoro, chloro, bromo and iodo.

In the sense of the present invention, the expression "-(C ₃ -C ₈ )heterocyclyl" designates saturated heterocycles having 3, 4, 5, 6, 7 or 8 atoms in the ring where 1 , 2 or 3 heteroatoms chosen from among N, O and S replace the corresponding number of carbon atoms. Examples of "-(C ₃ -C ₈ )heterocyclyl" include aziridinyl, oxyranyl, pyrrolidinyl, tetrahydrofuranyl, oxazolyl, piperidinyl, piperazinyl and morpholinyl.

The term "aryl" designates an aromatic, monocyclic ring that may be fused with a second saturated, unsaturated or aromatic ring. The term "aryl" include, without restriction to the following examples, phenyl, indanyl, indenyl, naphtyl, anthracenyl, phenanthrenyl, tetrahydronaphtyl and dihydronaphtyl. The most preferred aryl are those comprising one six- membered aromatic ring. The aryl group may be substituted, preferably with one or more groups independently selected from the group consisting of alkyl, alkoxy, halogen, hydroxyl, amino, nitro, cyano, trifluoro, CHO, amide, cetone thiol, thioester, thioalkyl, urea, sulfoxyde sulfone, carboxylic acid or carboxylic ester.

The term "heteroaryl" designates a mono- or polycyclic aryl as defined above where one or more carbon atoms have been replaced with one or more heteroatoms chosen from among N, O and S. Unless explicitly stated, the term "heteroaryl" includes all possible isomers, in particular position isomers.

Examples of heteroaryl groups include furyl, thienyl, imidazolyl, pyridyl, pyrrolyl, N- alkyl pyrrolyl, pyrimidinyl, pyrazinyl, tetrazolyl, triazolyl and triazinyl. The heteroaryl group may be substituted, preferably with one or more groups independently selected from the group consisting of alkyl, alkoxy, halogen, hydroxyl, amino, nitro, cyano, trifluoro, carboxylic acid or carboxylic ester. Preferred heteroaryls are those having 5 or 6 atoms in the ring, such as indolyl, pyrrolyl, pyridinyl, pyrrazolyl, triazolyl, furanyl or thienyl.

As used, herein, the term "halogen" designates a fluorine, chlorine, bromine or iodine atom.

For the purpose of the invention, the term "pharmaceutically acceptable" is intended to mean what is useful to the preparation of a pharmaceutical composition, and what is generally safe and non-toxic, for a pharmaceutical use.

The term "pharmaceutically acceptable salt, hydrate of solvate" is intended to mean, in the framework of the present invention, a salt of a compound which is pharmaceutically acceptable, as defined above, and which possesses the pharmacological activity of the corresponding compound. Such salts comprise:

(1) hydrates and solvates,

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

(3) salts formed when an acid proton present in the compound is either replaced by a metal ion, such as an alkali metal ion, an alkaline-earth metal ion, or an aluminium ion; or coordinated with an organic or inorganic base. Acceptable organic bases comprise diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine and the like. Acceptable inorganic bases comprise aluminium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and sodium hydroxide.

Fumarate derivative

In a first aspect, the invention concerns a molecule of formula (I):

(I) or a pharmaceutically acceptable salt and/or solvate thereof, wherein:

R1 represents -(Ci-CeJalkyl, preferably -CH ₃

X represents O, N or S

Y represents O, N or S

A represents -(CrC ₆ )alkyl-, -(C ₂ -C ₆ )alkenyl-, -aryl-, -(Ci-C ₆ )alkyl-aryl-, -heteroaryl-, - (C ₂ -C ₆ )alkynyl-, -(C ₃ -C ₈ )heterocyclyl-, -(C ₃ -C ₁₄ )cycloalkyl, -(Ci-C ₆ )alkyl-R ₂ -(Ci- C ₆ )alkyl-, -(C ₂ -C ₆ )alkenyl-R ₂ -(C ₀ -C ₆ )alkyl-, -(C -C ₆ )alkyl-R ₂ -(C ₂ -C ₆ )alkenyl-, -(<¾- C ₆ )alkenyl-R ₂ -(C ₂ -C ₆ )alkenyl-, -(Ci-C ₆ )alkyl-R ₂ -(C ₂ -C ₆ )alkynyl-, -(p _z -C _e )alkenyl-R ₂ -(C ₂ - C ₆ )alkynyl-, -(C ₂ -C ₆ )alkynyl-R ₂ -(C ₂ -C ₆ )alkynyl-, -(C ₂ -C ₆ )alkynyl-R ₂ -(C C ₆ )alkyl-, -(C _r

C ₆ )alkynyl-R ₂ -(C ₂ -C ₆ )alkenyl-, -iC _r Ce)alky1-OCO-, -CH ₂ -(CHOR ₂ )CH ₂ 0-(C ₁ -C ₆ )alkyl-, -(C ₀ -C ₆ )alkyl-CO-R ₂ -, or -(C ₀ -C ₆ )alkenyl-CO-R ₂ -,

where R ₂ represents -(C ₀ -C ₆ )alkyl-aryl, -(C ₀ -C ₆ )alkenyl-aryl, heteroaryl, (C ₃ - C ₈ )heterocyclyl, or (C ₃ -C ₁₄ )cycloalkyl,

A is optionally substituted by a CO-releasing molecule.

As shown in the examples, those compounds, when incubated with cells, induce an activation of HO-1 which is comparable or even more potent than the one obtained with DMF. Furthermore, as shown in the example, those compounds do not induce cytotoxicity at their active concentrations. Moreover, it has been found by the inventors that the presence of a nitrogen on position 3 of the heterocycle (which could be oxadiazole or thiadiazole) is of utmost importance since the compound 1 which is missing such a nitrogen on position 3 of the heterocycle show a low HO-1 activation.

The inventors found that the presence of an aryl linked, directly or not, to the heterocycle induce a better HO-1 activation.

Hence, preferably A represent -(C _r C _B )a\ky\-, -aryl-, -(d-C ₆ )alkyl-aryl-, -heteroaryl-, -(C ₀ - C ₆ )alkyl-CO-R ₂ -, -(C ₀ -C ₆ )alkenyl-CO-R2-, where R ₂ represents -aryl-, or -(C ₀ -C ₆ )alkenyl-aryl-

Particularly, X represent N.

Particularly, Y represent O. example, the compounds of the invention can be chosen from among

Moreover, inventors found that those new structures are advantageous for the integration of CO-releasing molecules.

Hence, the invention particularly refer to compounds wherein A is substituted by a CO- releasing molecule chosen from among:

where W represents O or NR^, where f¾ represents -(CrC^alkyl-,

L represents an ionic ligand such as halogen, BF ₄ , PF ₆

M represents Rh, Co, Ru, Mn, Mo, V or Fe, preferably Co, Ru or Mn, and

n is an integer chosen so that the metal M has no free valency

Preferably, the CO-releasing molecule is chosen from among:

Among those CORM, inventors have identified more potent CORM that show faster release of CO. Hence more preferably, the CO-releasing molecule is chosen from among: where W represents O or NR ₄ , where R4 represents -(Ci-C ₆ )alkyl-,

For example, the compounds of the invention can be chosen from among:

Pharmaceutical compositions, methods and uses

The invention also concerns a pharmaceutical composition comprising at least one compound of the invention, a pharmaceutically acceptable salt, solvate or hydrate thereof, as defined previously and at least one pharmaceutically acceptable excipient.

The pharmaceutical compositions of the invention are advantageously suitable for administration via oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical or rectal route. The pharmaceutical compositions of the invention may also be administered by inhalation, for example by means of an aerosol. The active ingredient can be administered in unit forms for administration, mixed with conventional pharmaceutical carriers, to animals or to humans.

When a solid composition is prepared in the form of tablets, the main active ingredient is mixed with a pharmaceutical vehicle and other conventional excipients known to those skilled in the art.

The compounds of the invention can be used in a pharmaceutical composition at a dose ranging from 0.01 mg to 1000 mg a day, administered in only one dose once a day or in several doses along the day, for example twice a day. The daily administered dose is advantageously comprises between 5 mg and 500 mg, and more advantageously between 10 mg and 200 mg. However, it can be necessary to use doses out of these ranges, which could be noticed by the person skilled in the art.

The present invention further concerns a compound of the invention, a pharmaceutically acceptable salt, solvate or hydrate thereof, or a pharmaceutical composition comprising at least one compound of the invention, a salt, solvate or hydrate thereof, for use as a drug.

The present invention further concerns at least one compound of the invention, a pharmaceutically acceptable salt, solvate or hydrate thereof, or a pharmaceutical composition comprising at least one compound of the invention, a salt, solvate or hydrate thereof, for use in the treatment of cardiovascular or inflammatory diseases.

The present invention further concerns the use of at least one compound of the invention, a pharmaceutically acceptable salt, solvate or hydrate thereof, for the manufacture of a medicament for use in the treatment of cardiovascular or inflammatory diseases.

The present invention further concerns a method for treating cardiovascular or inflammatory diseases, comprising the administration of at least one compound of the invention, a pharmaceutically acceptable salt, solvate or hydrate thereof, or of a pharmaceutical composition comprising at least one compound of the invention, a pharmaceutically acceptable salt, solvate or hydrate thereof, to a person in need thereof.

Inflammatory and cardiovascular diseases according to the present invention include for example myocardial ischemia and heart diseases, rheumatoid arthritis, acute and chronic skin wound (wound healing), inflammatory bowel disease, post-operative ileus, brain ischemia, psoriasis, diabetes, diabetic nephropathy, metabolic syndrome, sickle-cell disease, neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease, neuropathic pain, hypertension, pulmonary arterial hypertension, septicemia, septic or endotoxic shock, hemorrhagic shock, multiple sclerosis, cancer and chronic obstructive pulmonary disease. Preferred inflammatory and cardiovascular diseases according to the present invention are skin wound (wound healing), brain and cardiac ischemia, psoriasis, diabetes, multiple sclerosis, cancer and chronic obstructive pulmonary disease.

The present invention further concerns a process for preparing the compounds of the invention, their salts, hydrates or solvates.

Synthesis methods

The compounds of formula (I) can be obtained according to following methods.

Step 1 :

Preparation of (I) as 1 ,2.4-oxadiazole derivative:

The preparation follows the general procedure described in the following scheme:

A-CN

The conversion of a benzonitrile derivative into the corresponding 1 ,2,4-oxadiazole proceeds according reported procedures by treatment with hydroxylamine and subsequent reaction with an activated carboxylic acid.

Preparation of (I) as 1 ,3,4-oxadiazole derivative:

The preparation follows the general procedure described in the following scheme:

The formation of 1 ,3,4 oxadiazole follows reported procedures (e.g. Tetrahedron Letters, 2014, 3252-3254). It proceeds by cyclisation of the corresponding A/./V-diacylhydrazine in the presence of phosphorus oxychloride.

Preparation of (I) as a-keto-1 ,3,4-oxadiazole derivative:

The preparation follows the general procedure described in the following scheme

a-keto-1 ,3,4-oxadiazoles are prepared by reacting successively the benzoyl chloride derivative and the carboxylic acid with (A/-isocyanimine) triphenylphosphorane (Tetrahedron Letters, 2011 , 5530-5533).

Step 2:

Preparation of non-hydrolysable hybrids with Co-based CORM:

_R - ^OH BrCH ₂ CCH _R .0^^ Co ₂ (CO) _{8 R} -°^^Co ₂ (CO) _& R = formula (I) K ₂ C0 ₃

Preparation of non-hydrolysable hybrids with Mn/Ru-based CORM:

_^ NHMe 1) NaH ^M®

R *■ R ^r S

^{2) CS2} S-[M] [M] = Mn(CO) ₄ ou Ru(CO) ₃ CI

3) [M]CI

R = formula (I)

Description of the figures:

Figure 1 represents the cytotoxicity as measured in BV2 microglia by LDH release after 24h treatment with increasing concentrations of compound A (figure 1A), compound B (figure 1 B), compound C (figure 1C), and comparative compound 1 (figure 1 D).

Figure 2 represents HO-1 protein expression measured in BV2 cells 6 h after treatment with 10 μΜ of compounds A, B, C compared to 10 μ of DMF. The lower panels in (A) represent the densitometric analysis of Western blots.

The present invention is illustrated by the non-limiting following examples.

Example 1 : Synthesis of fumarate derivatives according to the invention: Compound A:

Step 1 :

KM-153

MW: 74.08

C ₂ H ₆ N ₂ 0

To a solution of 0.57 g (14.2 mmol, 1 eq) of NaOH in 5 ml of water, 1.00 g (14.2 mmol, 1 eq) of hydroxylamine was added. 15 mL of acetonitrile were added dropwise, the mixture was stirred at room temperature overnight. Acetonitril and water were removed under vacuum and ethanol was added to the crude product. The product was filtered off and washed with ethanol giving 600 mg of the expected product (60%).

H NMR 400MHz fCDC ) δ: 1.86 (s, 3H) Step 2: Compound A

200 mg (0.83 mmol, 1 eq) of 2,5-dioxopyrrolidin-1-yl ethyl fumarate (Kidd, et al., Angewandte Chemie International Edition, 2003, 42, 3379-3383) were dissolved in 10 ml of dioxane. After cooling at 0 °C, 60 mg (0.83 mmol, 1 eq) of KM-153 and 120 mg (0.83 mmol, 1 eq) of potassium carbonate were added. The mixture was then stirred overnight at room temperature. The crude product was purified by chromatography on silica gel with ethyl acetate/cyclohexane (30/70). 42 mg (28%) of the expected product were obtained.

H NMR 400MHz (CDCI- δ: 7.46 (d, J = 16.2 Hz, 1 H); 7.00 (d, J = 16.2 Hz, 1 H); 4.31 (q, J = 7.3 Hz); 4.25 (s, 3H); 1.35 (t, J = 7.3 Hz, 3H)

3C NMR 100MHz fCDCI δ: 172.8. 168.2, 164.7, 131 .4, 125.1 , 61.7, 14.2, 1 1.7 Compound B:

MW: 272.26

144 mg (1 mmol, 1 eq) of mono ethyl ester fumaric acid were dissolved in 10 ml of dichloromethane. 302 mg (1 mmol, 1 eq) W-lsocyaniminotriphenylphosphorane were added portionwise. The solution was stirred for 2 h at room temperature. 135 μΙ (1 mmol, 1 eq) of trimethylamine and 117 μΙ (1 mmol, 1 eq) of benzoyl chloride were added to the solution. The mixture was stirred at room temperature overnight. The solvent was then removed under vacuum. The crude product was purified by chromatography on silica gel with ethyl acetate/cyclohexane (15/85). 30 mg (1 1 %) of the expected product were obtained.

1H NMR 400MHz (CDC ) δ: 8.53 (m, 2H); 7.72 (m, 1 H); 7.63 (d, J = 15.6 Hz, 1 H); 7.57 (m, 2H); 7.06 (d, J = 15.6 Hz, 1 H); 4.42 (q, J = 7.2 Hz, 2H); 1 .36 (t, J = 7.2 Hz, 3H).

3C NMR 100MHz fCDCU) δ: 177.4, 164.5, 163.6, 160.6, 135.3, 134.1 , 131.1 , 129.1 , 124.0, 61 .8, 31.0, 14.2

Compound C:

Step 1 :

100 mg (0.41 mmol, 1 eq) of 2,5-dioxopyrrolidin-1 -yl ethyl fumarate ¹ were dissolved in 10 ml of dichloromethane. After cooling at 0 °C, 20 μΙ (0.41 mmol, 1 eq) of hydrazine monohydrate were added. The solution was stirred for 2 h at 0 °C. 50 μΙ (0.41 mmol, 1 eq) of benzoyl chloride and 1 15 mg (0.82 mmol, 2 eq) of potassium carbonate were then added to the solution. The mixture was stirred at 0 "C overnight. The crude product was purified by chromatography on silica gel with ethyl acetate/cyclohexane (30/70). 65 mg (60%) of the expected product were obtained.

H NMR 400MHz fCDCI. 5: 7.87 (m, 2H); 7.58 (m, 1 H); 7.47 (m, 2H); 7.19 (d, J = 15.7 Hz, 1 H); 6.96 (d, J = 15.7 Hz, 1 H); 4.27 (q, J = 7.3 Hz, 2H); 1.31 (t, J = 7.3 Hz, 3H)

1 ³ C NMR 100MHz fCDCU) δ: 165.0, 132.8, 132.5, 132.2, 130.8, 129.0, 127.3, 100.6, 61.4, 14.2

Step 2: compound C

65 mg (0.248 mmol) of KM-178 were dissolved in 10 ml of POCI ₃ , the mixture was stirred at reflux during 3 h. The crude mixture was then quenched with cooled water. The aqueous phase was extracted with dichloromethane. After chromatography on silica gel with ethyl acetate/cyclohexane (15/85), 40 mg of a solid were recovered.

Example 2: Assessment of the cytotoxicity of compounds of the invention

Assessment of the cytotoxicity of compound A. B and C on BV2 microglia

Cells were grown in an atmosphere of 5% C0 ₂ at 37°C in either 75 cm ² flasks, 100 mm diameter Petri dishes, 6- or 24-well plates containing medium supplemented with 10% fetal bovine serum (Lonza) and penicillin (100 U.ml_ ^"1 )/streptomycin (100 mg.mL ^"1 ; Life Technologies). BV2 mouse microglial cells were grown in RPMI-1640 containing 2 g.L ¹ glucose and supplemented with 0.3 g.L ^"1 l-glutamine.

Cytotoxicity was evaluated in BV2 microglia cells 24 h after incubation with increasing concentrations of hybrids using a Cytotoxicity Detection Kit (LDH) (Roche Applied Science) to measure lactate dehydrogenase released from damaged cells. X-100 Triton solution (2 %) prepared in medium was used as a positive control (100 % cytotoxicity). The assay was performed according to the manufacturer's instructions. Briefly, at the end of the incubation, cell plates were centrifuged at 300 x g for 5 min and 100 μΙ cell-free supernatant transferred to a 96-well plate. A reaction mixture was added to the supernatant and the plate was incubated in the dark at room temperature with gentle shaking for 10 min. Absorbance was measured at 485 nm.

Data are expressed as percentage of LDH released by treating cells with 2% triton (100% toxicity).

The results are presented in figure 1.

Increasing concentrations of compound 1 , B, C of the invention from 1 to 20 μΜ caused no or a low release of lactate dehydrogenase (LDH), an index of cell injury, from BV2 cells exposed to the compounds for 24 h (Figure 1A, B, C).

Example 3: In vitro assessment of the biological activity of compounds of the invention: Assessment of HO-1 activation after exposure of BV2 microglia cells to compounds A, B and C

To determine HO-1 protein expression, BV2 microglia cells were incubated with 10 μ of compounds A, B and C for 6 h. At the end of the incubation cells were washed with ice cold DPBS (-Ca,-Mg; Gibco ^® Cell Culture, Life Technologies) and lysed during 30 min incubation at 4 °C in cell lysis buffer (50 mM HEPES, 150 mM NaCI, 50 mM NaF, 50 μΜ Na ₃ V0 ₄ , 1 % v/v Triton X-100 and 1 % mammalian protease inhibitor). Lysates were centrifuged for 10 min at 15,000 x g and 4 °C; supernatants were collected and stored at - 80 °C. Protein concentrations were measured using a Pierce BCA Protein Assay kit (Thermo Scientific).

Whole cell lysates (20 μg protein/sample) were resolved on 12% acrylamide gels, respectively, and proteins transferred to polyvinylidene difluroride membranes (Millipore, Brussels, Belgium). Membranes were blocked for 1 h at room temperature in 1 x Tris- buffered saline (pH 7.5) containing 0.1 % v/v TWEEN 20 and 5 % w/v non-fat dry milk and incubated overnight at 4 "C with the following primary antibodies: HO-1 (rabbit polyclonal, Enzo Life Sciences) and β-actin (clone 8H10D10, mouse monoclonal, Cell Signaling Technology) as a loading control. Membranes were then incubated with secondary antibodies coupled to horseradish peroxidase (goat anti-mouse or anti-rabbit, Cell Signaling Technology or donkey anti-goat, Jackson ImmunoResearch) for 1 h at room temperature. Bands were detected with chemiluminescent substrates (Pierce ECL®, Thermo Scientific or RevelBIOt® Intense, Ozyme) and images captured using a G:Box F3 Imagery Station and GeneSys Software (Syngene, Cambridge, UK).

The results are presented in figure 2A-D. Exposure of BV2 microglia cells to 10 μΜ of compounds A, B and C and compound B for 6 h strongly promoted HO-1 protein expression.

In comparison, compounds 1 was less effective as an HO-1 inducing agent.

Compared to a twofold concentration of DMF, compounds B and C show a better activation of HO-1 expression (more than 4 fold increase) whereas compound A show an almost 3 fold increase compared to control.