Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
  • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
  • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
  • C07C323/52—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
  • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
  • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
  • C07C323/60—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton with the carbon atom of at least one of the carboxyl groups bound to nitrogen atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C327/00—Thiocarboxylic acids
  • C07C327/20—Esters of monothiocarboxylic acids
  • C07C327/30—Esters of monothiocarboxylic acids having sulfur atoms of esterified thiocarboxyl groups bound to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms, not being part of nitro or nitroso groups

Definitions

• the present invention relates to new acylated aminopropanediols and their nitrogen and sulfur analogues, pharmaceutical compositions comprising them, their therapeutic applications, in particular for the treatment of cerebral ischemia. It also relates to a process for the preparation of these derivatives.
• the compounds of the invention have advantageous pharmacological, antioxidant and anti-inflammatory properties.
• the invention also describes the methods of therapeutic treatment using these compounds and pharmaceutical compositions containing them.
• the compounds of the invention can be used in particular for preventing or treating cerebrovascular accidents.
• cerebrovascular pathology (150,000 new cases per year) represents the third cause of death and the first cause of disability in adults.
• Ischemic and hemorrhagic accidents respectively concern 80% and 20% of this pathology.
• Ischemic strokes are an important therapeutic issue to reduce the morbidity and mortality of this condition. Advances have been made not only in the treatment of the acute phase of ischemia but also in its prevention. It is also important to note that the identification and management of risk factors are essential to the treatment of this pathology.
• Drug treatments for ischemic strokes are based on different strategies.
• a first strategy is to prevent the occurrence of ischemic strokes by preventing risk factors (high blood pressure, high cholesterol, diabetes, atrial fibrillation, etc.) or by preventing thrombosis, in particular using anti - platelet aggregators or anticoagulants (Adams 2002) and (Gorelick 2002).
• a second strategy is to treat the acute phase of ischemia in order to reduce the long-term consequences (Lutsep and Clark 2001).
• the pathophysiology of cerebral ischemia can be described as follows: the penumbra zone, the intermediate zone between the heart of the ischemia - where the neurons are necrotized - and the intact nervous tissue, is the site of a pathophysiological cascade which leads to neuronal death within a few days if reperfusion is not ensured or if neuroprotection is not effective enough.
• the first event which occurs within the first few hours, is a massive release of glutamate which results in neuronal depolarization as well as cellular edema.
• the entry of calcium into the cell induces mitochondrial damage promoting the release of free radicals as well as the induction of enzymes which cause the membrane degradation of neurons.
• NF-B transcription factors
• This activation induces inflammatory processes such as the induction of adhesion proteins at the endothelial level, the infiltration of the ischemic focus by neutrophils, microglial activation, induction of enzymes such as nitric oxide (NO) synthase type II or cyclooxygenase type II.
• NO nitric oxide
• cyclooxygenase type II enzymes
• NO or prostanoids which are toxic to the cell. All of these processes result in a phenomenon of apoptosis causing irreversible lesions (Dirnagl, ladecola et al. 1999).
• prophylactic neuroprotection is based on experimental bases demonstrating resistance to ischemia in animal models. Indeed, various procedures applied prior to the realization of an experimental cerebral ischemia make it possible to make it less severe. Different stimuli make it possible to induce resistance to cerebral ischemia: preconditioning (brief ischemia preceding prolonged ischemia); thermal stress; administration of a low dose of bacterial lipopolysaccharide (Bordet, Deplanque et al. 2000). These stimuli induce resistance mechanisms which activate signals triggering the protective mechanisms. Different triggering mechanisms have been highlighted: cytokines, pathways of inflammation, free radicals, NO, ATP-dependent potassium channels, adenosine.
• PPARs ( ⁇ , ⁇ , ⁇ ) belong to the family of hormone activated nuclear receptors. When activated by an association with their ligand, they heterodimerize with the Retinoid-X-Re ⁇ ptor (RXR) and then bind to a Peroxisome Proliferator Response Elements ”(PPREs) which are located in the promoter sequence of the target genes. The binding of PPAR to PPRE thus induces the expression of the target gene (Fruchart, Staels et al. 2001).
• PPARs are distributed in a wide variety of organs, but with a certain specificity for each of them, with the exception of PPAR ⁇ , the expression of which seems ubiquitous.
• the expression of PPAR ⁇ is particularly important in the liver and along the intestinal wall whereas PPAR ⁇ is mainly expressed in adipose tissue and the spleen.
• PPAR ⁇ is mainly expressed in adipose tissue and the spleen.
• ⁇ , ⁇ , ⁇ the three subtypes ( ⁇ , ⁇ , ⁇ ) are expressed!
• Cells such as oligodendrocytes as well as astrocytes express more particularly the PPAR ⁇ subtype (Kainu, Wikstrom et al. 1994).
• the target genes of PPARs control the metabolism of lipids and carbohydrates.
• PPARs participate in other biological processes.
• PPARs The activation of PPARs by their ligands induces a change in the transcriptional activity of genes which modulate the inflammatory process, antioxidant enzymes, angiogenesis, cell proliferation and differentiation, apoptosis, the activities of iNOS, MMPases and TIMPs. (Smith, Dipreta et al. 2001) and (Clark 2002).
• Free radicals are involved in a very broad spectrum of pathologies such as allergies, cancer initiation and promotion, cardiovascular pathologies (atherosclerosis, ischemia), genetic and metabolic disorders (diabetes), infectious and degenerative diseases (Prion, etc. .) as well as ophthalmic problems (Mates, Perez-Gomez et al. 1999).
• ROS Reactive oxygen species
• the management of ROS is done via an antioxidant system which includes an enzymatic and non-enzymatic component.
• the enzymatic system consists of several enzymes whose characteristics are as follows: - Superoxide dismutase (SOD) destroys the superoxide radical by converting to peroxide. The latter is itself supported by a other enzyme system.
• SOD Superoxide dismutase
• a low level of SOD is constantly generated by aerobic respiration.
• Three classes of SOD have been identified in humans, each containing Cu, Zn, Fe, Mri, or Ni as a cofactor.
• the three forms of human SOD are distributed as follows: Cu-Zn SOD at the cytosolic level, Mn-SOD at the mitochondrial level and an extracellular SOD.
• Catalase is very effective in converting hydrogen peroxide (H 2 O 2 ) into water and oxygen. Hydrogen peroxide is catabolized enzymatically in aerobic organisms. Catalase also catalyzes the reduction of a variety of hydroperoxides (ROOH).
• ROOH hydroperoxides
• Glutathione peroxidase contains selenium as a cofactor and catalyzes the reduction of hydroperoxides (ROOH and H 2 O 2 ) using glutathione, and thus protects cells against oxidative damage.
• Non-enzymatic antioxidant cell defenses are made up of molecules that are synthesized or provided by food.
• antioxidant molecules present in different cellular compartments.
• Detoxifying enzymes are for example responsible for eliminating free radicals and are essential for the life of the cell.
• the three most important types of antioxidant compounds are carotenoids, vitamin C and vitamin E (Gilgun-Sherki, Melamed et al. 2001).
• the inventors have developed new compounds capable of preventing the appearance of the risk factors described above and capable of exerting a prophylactic activity in term of neuroprotection, but also to ensure active neuroprotection in the acute phase of ischemic strokes.
• the compounds according to the invention have at the same time properties of PPAR activators, antioxidants and anti-inflammatory drugs and, as such, the compounds have a high therapeutic or prophylactic potential for ischemic strokes.
• the present invention thus provides a new family of compounds having advantageous pharmacological properties and usable for the curative or preventive treatment of cerebral ischemia. It also relates to a process for the preparation of these derivatives.
• G2 and G3 independently represent an oxygen atom, a sulfur atom or an N-R4 group, G2 and G3 cannot simultaneously represent an N-R4 group,
• R and R4 independently represent a hydrogen atom or a linear or branched alkyl group, saturated or not, optionally substituted, containing from 1 to 5 carbon atoms,
• R1, R2 and R3, identical or different, represent a hydrogen atom, a CO-R5 group or a group of formula CO- (CH 2 ) 2n + rX-R6, at least one of the groups R1, R2 or R3 being a group of formula CO- (CH 2 ) 2n + rX-R6,
• R5 is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, optionally comprising a cyclic group, the main chain of which contains from 1 to 25 carbon atoms,
• X is a sulfur atom, a selenium atom, an SO group or an SO 2 group
• n is an integer between 0 and 11
• R6 is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, optionally comprising a cyclic group, the main chain of which contains from 3 to 23 carbon atoms, preferably 10 to 23 carbon atoms and optionally one or more heterogroups chosen from an oxygen atom, a sulfur atom, a selenium atom, an SO group and an S0 group 2)
• the group or groups R5, identical or different preferably represent a linear or branched, saturated or unsaturated, substituted or unsubstituted alkyl group, the main chain of which comprises from 1 to 20 carbon atoms, even more preferably 7 to 17 carbon atoms, even more preferably 14 to 17.
• the group or groups R5, identical or different may also represent a group lower alkyl containing 1 to 6 carbon atoms, such as in particular the methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl or hexyl radical.
• the group or groups R6, which are identical or different, preferably represent a linear or branched, saturated or unsaturated, substituted or unsubstituted alkyl group, the main chain of which comprises from 3 to 23 carbon atoms, preferably 13 to 20 carbon atoms, even more preferably 14 to 17 carbon atoms, and even more preferably 14 carbon atoms.
• saturated long chain alkyl groups for R5 or R6 are in particular the groups C7H15, C10H21, C11H23, C13H27, C14H29, C15H31, C 16 H 33 , C 17 H 35 .
• unsaturated long chain alkyl groups for R5 or R6 are in particular the groups C14H27, C14H25, C15H29,
• Examples of branched long chain alkyl groups are in particular the groups (CH 2 ) n-CH (CH3) C2H 5 , or (CH2) 2 + ⁇ -C (CH 3 ) 2 - (CH2) n "-CH3 (x being an integer equal to or between 1 and 11, n 'being an integer equal to or between 1 and 22, n "being an integer equal to or between 1 and 5, n '" being an integer equal to or between 0 and 22, and (2x + n'") being less than or equal to 22, preferably less than or equal to 20).
• alkyl groups R5 or R6 may optionally include a cyclic group.
• cyclic groups are especially cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
• the alkyl groups R5 or R6 may be optionally substituted by one or more substituents, identical or different.
• This invention also relates to the optical and geometric isomers of these compounds, their racemates, their salts, their hydrates and their mixtures.
• the compounds of formula (la) are the compounds of formula (I) according to the invention in which only one of the groups R1, R2 or R3 represents a hydrogen atom.
• the compounds of formula (Ib) are the compounds of formula (I) according to the invention in which two of the groups R1, R2 or R3 represent a hydrogen atom.
• the present invention also includes the prodrugs of the compounds of formula (I), which, after administration in a subject, will transform into compounds of formula (I) and or the metabolites of the compounds of formula (I) which exhibit therapeutic activities, especially for the treatment of cerebral ischemia, comparable to the compounds of formula (I).
• X most preferably represents a sulfur or selenium atom and advantageously a sulfur atom.
• n is preferably between 0 and 3, more specifically between 0 and 2 and is in particular equal to 0.
• R6 can comprise one or more heterogroups, preferably 0, 1 or 2, more preferably 0 or 1, chosen from an oxygen atom, a sulfur atom, a selenium atom, an SO group and an SO 2 group.
• a specific example of a CO- (CH 2 ) 2n + rX-R6 group according to the invention is the group CO-CH2-S-C14H 2 9.
• Preferred compounds within the meaning of the invention are therefore compounds of general formula (I) above in which at least one of the groups R1, R2 and R3 represents a group CO- (CH 2 ) 2n + ⁇ -X-R6 in which X represents a sulfur or selenium atom and preferably a sulfur atom and / or R6 is a saturated and linear alkyl group comprising from 3 to 23 carbon atoms, preferably 13 to 20 carbon atoms, preferably 14 to 17, more preferably 14 to 16, and even more preferably 14 carbon atoms.
• R1, R2 and R3 are groups CO- (GH2) 2n + - 6, identical or different, in which X represents a sulfur or selenium atom and preferably a sulfur atom.
• G2 represents an oxygen or sulfur atom, and preferably an oxygen atom.
• R2 advantageously represents a group of formula CO- (CH2) 2n + -R6 î®l ue defined above.
• Particularly preferred compounds are the compounds of general formula (I) above in which: * G3 is an N-R4 group in which R4 is a hydrogen atom or a methyl group, and G2 is an oxygen atom; and or
• R2 represents a group CO- (CH 2 ) 2n + ⁇ -X-R6 as defined above.
• FIG. 1 Examples of preferred compounds according to the invention are represented in FIG. 1.
• the present invention more particularly relates to compounds chosen from:
• the subject of the present invention is also a pharmaceutical composition
• a pharmaceutical composition comprising, in a pharmaceutically acceptable carrier, at least one compound of general formula (I) as described above, including the compounds of formula (I) in which the G2R2 and G3R3 groups simultaneously represent hydroxyl groups, optionally in association with another therapeutic active.
• This composition is in particular intended to treat a cerebrovascular pathology, such as cerebral ischemia or a hemorrhagic stroke.
• Another object of the present invention thus relates to any pharmaceutical composition
• any pharmaceutical composition comprising in a support acceptable on the plan pharmaceutical at least one compound of formula (I) as described above, including the compounds of formula (I) in which the groups G2R2 and G3R3 simultaneously represent hydroxyl groups.
• the compounds of formula (I), including the compounds of formula (I) in which the groups G2R2 and G3R3 simultaneously represent hydroxyl groups have both PPAR activator properties , antioxidants and anti-inflammatories and have a prophylactic and curative neuroprotective activity for cerebral ischemia.
• the invention also relates to the use of a compound as defined above for the preparation of a pharmaceutical composition intended for the implementation of a method of treatment or prophylaxis in humans or in animals .
• the invention also relates to a method of treatment of cerebrovascular pathologies and more particularly of cerebral ischemia, comprising the administration to a subject, in particular human, of an effective dose of a compound of formula (I) or of a pharmaceutical composition as defined above, including the compounds of general formula (I) in which the groups G2R2 and G3R3 simultaneously represent hydroxyl groups.
• the compounds of formula (I) used are as defined above and also include 3- (tetradecylthioacetylamino) propane-1,2-diol.
• compositions according to the invention advantageously comprise one or more excipients or vehicles, acceptable on the pharmaceutical plan. Mention may be made, for example, of saline, physiological, isotonic, buffered solutions, etc., compatible with pharmaceutical use and known to those skilled in the art.
• the compositions may contain one or more agents or vehicles chosen from dispersants, solubilizers, stabilizers, surfactants, preservatives, etc.
• Agents or vehicles usable in formulations are in particular methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, lactose, vegetable oils, acacia, etc.
• compositions may be formulated in the form of an injectable suspension, gels, oils, tablets, suppositories, powders, capsules, capsules, etc., optionally by means of dosage forms or devices ensuring sustained and / or delayed release.
• an agent such as cellulose, carbonates or starches is advantageously used.
• the compounds or compositions according to the invention can be administered in different ways and in different forms.
• they can for example be administered systemically, orally, parenterally, by inhalation or by injection, such as for example by intravenous, intramuscular, subcutaneous, trans-dermal, intra-arterial, etc.
• the compounds are generally packaged in the form of liquid suspensions, which can be injected using syringes or infusions, for example.
• the compounds are generally dissolved in saline, physiological, isotonic, buffered solutions, etc., compatible with pharmaceutical use and known to those skilled in the art.
• compositions can contain one or more agents or vehicles chosen from dispersants, solubilizers, emulsifiers, stabilizers, surfactants, preservatives, buffers, etc.
• Agents or vehicles that can be used in liquid and / or injectable formulations are in particular methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, lactose, vegetable oils, acacia, liposomes, etc. .
• the compounds can thus be administered in the form of gels, oils, tablets, suppositories, powders, capsules, capsules, aerosols, etc., optionally by means of dosage forms or devices ensuring sustained and / or delayed release.
• an agent such as cellulose, carbonates or starches is advantageously used.
• the compounds can be administered orally in which case the agents or vehicles used are preferably chosen from water, gelatin, gums, lactose, starch, magnesium stearate, talc, an oil, polyalkylene glycol, etc. .
• the compounds are preferably administered in the form of solutions, suspensions or emulsions with in particular water, oil or polyalkylene glycols to which it is possible to add, in addition to preservatives, stabilizers, emulsifiers , etc., salts to adjust the osmotic pressure, buffers, etc.
• the flow rate and / or the dose injected can be adapted by a person skilled in the art depending on the patient, the pathology concerned, the mode of administration, etc.
• the compounds are administered in doses which can vary between 1 ⁇ g and 2 g per administration, preferably from 0.1 mg to 1 g per administration.
• the administrations can be daily or repeated several times a day, if necessary.
• the compositions according to the invention can also comprise other agents or active principles.
• the invention also relates to processes for the preparation of the compounds as defined above.
• the compounds of the invention can be prepared from commercial products, using a combination of chemical reactions known to those skilled in the art.
• G3 are oxygen or sulfur atoms or an NH group
• R is a hydrogen atom and
• the compounds of formula (I) according to the invention in which (i) G2 and G3 are oxygen atoms or an NH group, (ii) R is a hydrogen atom and (iii) R1, R2 and R3 are hydrogen atoms or represent a CO-R5 or CO- (CH2) 2n + rX-R6 group can be obtained according to different methods which allow the synthesis of compounds in which the groups carried by the same heteroatom (nitrogen or oxygen) have the same meaning.
• a first mode a molecule of 1-aminoglycerol, 1, 3-diaminoglycerol or 1,2-diaminoglycerol (obtained by adapting the protocol described by (Morris, Atassi et al.
• A1 is a reactive group chosen for example from OH, Cl and OR7, R7 being an alkyl group
• a ° is the group R5 or the group (CH 2 ) 2n + ⁇ -X- R6 optionally in the presence of coupling agents or activators known to those skilled in the art.
• This reaction makes it possible to obtain respective particular forms of compounds of formula (I), called compounds (lla-c), and can be implemented by adapting the protocols described (Urakami and Kakeda 1953), (Shealy, Frye et al . 1984), (Marx, Piantadosi et al. 1988) and (Rahman, Ziering et al. 1988) or (Nazih, Cordier et al. 1999).
• the groups carried by the same heteroatom, respectively, (R1 and R3) and (R1 and R2) have the same meaning.
• the compounds of formula (I) according to the invention in which (i) G2 and G3 are oxygen atoms or a NH group, (ii) R is a hydrogen atom and (iii) R1, R2 and R3, identical or different, represent a group CO-R5 or CO- (CH 2 ) 2n + - 6, can be obtained from a compound of formula (Ia-c) and a compound of formula A ° -CO- A2 in which A2 is a reactive group chosen for example between OH and Cl, and A ° is the group R5 or the group (CH 2 ) 2 n + ⁇ -X-R6, optionally in the presence of coupling agents or activators known to those skilled in the art.
• R is a hydrogen atom and (iii) R1, R2 and R3, identical or different, represent a group CO-R5 or CO- (CH 2 ) 2 n + X-R5, can be obtained according to following steps :
• reaction of 1-aminoglycerol, 1, 3-diaminoglycerol or 1,2-diaminoglycerol with a compound (PG) 2 O in which PG is a protective group to give a compound of general formula (IIIa-c).
• the reaction can advantageously be carried out by adapting the protocols described by (Nazih, Cordier et al. 2000) and (Kotsovolou, Chiou et al. 2001) in which (PG) 2 ⁇ represents di-tert-butyl dicarbonate;
• a compound of formula (I) according to the invention is reacted, in which (i) G2 and G3 are oxygen atoms, (ii) R and R2 are hydrogen atoms and (iii ) R1, R3, identical or different, represent a group CO-R5 or CO- (CH 2 ) 2n + rX-R6, with a compound of formula A ° -CO-A2 in which A2 is a reactive group chosen for example between OH and Cl, and A ° is the group R5 or the group (CH 2 ) 2n + rX-R6, optionally in the presence of coupling agents or activators known to those skilled in the art.
• the compounds of formula (I) in which (i) G2 and G3 are oxygen atoms, (ii) R and R2 are hydrogen atoms and (iii) R1 and R3, identical or different, represent a group CO-R5 or CO- (GHa iX-R ⁇ ) can be obtained from a compound of formula (IIa) as defined above with a compound of formula A ° -CO-A2 in which A2 is a reactive group chosen, for example, from OH and CI, and A ° is the group R5 or the group (CH2) 2n + - 6, optionally in the presence of coupling agents or activators known to those skilled in the art.
• the compounds of formula (I) in which (i) G2 and G3 are oxygen atoms, (ii) R is a hydrogen atom and (iii) R1, R2 and R3, identical or different, represent a group CO-R5 or CO- (CH 2 ) 2n + rX-R6, can be obtained from a compound of formula (I) according to the invention in which (i) G2 and G3 are oxygen atoms, (ii) R, R2 and R3 represent a hydrogen atom and (iii) R1 is a CO-R5 or CO- (CH 2 ) 2n + ⁇ -X-R6 group (composed of formula (IIa)) according to the following steps (diagram 2): a) reaction of the compound of formula (IIa) with a compound PG-E in which PG is a protective group and E is a reactive group chosen, for example, from OH or a halogen, to give a compound of general formula (V) in which
• reaction can advantageously be carried out by adapting the protocols described by (Marx, Piantadosi et al. 1988) and (Gaffney and Reese 1997) in which PG-E can represent triphenylmethyl chloride or 9-phenylxanthene-9-ol or also 9-chloro-9-phenylxanthene;
• a ° is the group R5 or the group (CH 2 ) 2n + rX-R6, optionally in the presence of coupling agents or activators known to those skilled in the art.
• the reaction may advantageously be implemented by adapting the protocols described by (Nazih, Cordier et al., 2000) and (Kotsovolou, Chiou et al., 2001) wherein (PG) 2 0 represents the di-tert-butyl dicarbonate;
• A2 is a reactive group chosen, for example, between OH and Cl
• a ° is the group R5 or the group (CH2 1-X-R6, optionally in the presence of coupling agents or known activators of those skilled in the art to obtain a compound of formula (XI) in which R and R4 represent different linear or branched alkyl groups, saturated or unsaturated, optionally substituted, containing from 1 to 5 carbon atoms, R3 represents the group R5 or the group (CH 2 ) 2n + rX-R6 and PG is a protective group; f) deprotection of the compound (XI) according to conditions known to those skilled in the art.
• the reaction can advantageously be carried out by adapting the protocols described by (Marx, Piantadosi et al. 1988) and (Gaffney and Reese 1997) in which PG'-E can represent triphenylmethyl chloride or 9-phenylxanthene-9- ol or also 9-chloro-9-phenylxanthene;
• LG in which LG a reactive group chosen, for example, from iodine, bromine, etc., in the presence of possible activators known to a person skilled in the art to give a compound of general formula (XVId-f);
• This reaction scheme allows the synthesis of compounds of general formula (I) in which the groups carried by the same heteroatom (nitrogen or sulfur) respectively (R2 and R3), (R1 and R3) and (R1 and R2) have the same meaning .
• the compounds of formula (I) according to the invention in which (i) G2 and G3 are sulfur atoms or an NH group, (ii) R is a d atom hydrogen and (iii) R1, R2 and R3 are hydrogen atoms or represent a CO-R5 or CO- (CH2) 2n + rX-R6 group can be prepared from the compounds of formula (IIIa-c) by a process comprising:
• This reaction scheme allows the synthesis of compounds of general formula (I) in which the groups carried by the same heteroatom (nitrogen or sulfur) respectively (R2 and R3), (R1 and R3) and (R1 and R2) have the same meaning .
• the compounds of general formula (I) in which (i) the group G2 and G3 represent sulfur atoms or an N-R4 group, (ii) R and R4 independently represent groups as defined above, (iii) R1 is a hydrogen atom and (iv) R2 and R3, identical or different, represent a CO-R5 group or a GO- (CH 2 ) 2 n + -R6 group, can be obtained according to the following methods:
• R and R4 independently represent different linear or branched alkyl groups, saturated or unsaturated, optionally substituted, having from 1 to 5 carbon atoms, (iv) R1 is a hydrogen atom and (v) R2 and R3, identical or different, represent a CO-R5 group or a CO- (CH) 2n + ⁇ -X-R6 group are obtained in the following manner (diagram 7):
• the compounds of formula (I) according to the invention in which (i) G2 represents an N-R4 group, (ii) G3 is a sulfur atom, (iii) R and R4 represent independently of the different linear or branched alkyl groups, saturated or unsaturated, optionally substituted, comprising from 1 to 5 carbon atoms, (iv) R1 is a hydrogen atom and (v) R2 and R3, identical or different, represent a group CO-R5 or a CO- (GH2) 2n + -R6 group are obtained in the following manner:
• LG-E in which E represents a halogen and LG a reactive group chosen for example from mesyl, tosyle, etc., to give a compound of general formula (XXVII) in which PG represents a protective group; c) reaction of the compound (XXVII) with a compound of formula R4-NH 2 in which R4 represents a linear or branched alkyl group, saturated or unsaturated, optionally substituted, containing from 1 to 5 carbon atoms and NH 2 represents the amino function , according to the method described by (Ramalingan, Raju et al. 1995), to obtain a compound of formula (XXVIII) in which R and R4 independently represent different linear or branched alkyl groups, saturated or unsaturated, optionally substituted, comprising 1 to 5 carbon atoms;
• the compounds of formula (I) according to the invention in which (i) G2 is a sulfur atom, (ii) G3 is an oxygen atom, (iii) R is an atom hydrogen, (iv) R1 and R2 represent a group CO-R5 or GO- (GH2) 2n + ⁇ - -R @ e (v) R3 is a hydrogen atom or represents a group CO-R5 or GO- (GH2 ) 2n + - 6, can be prepared from the compounds of formula (V) by the following process (scheme 9B):
• the reaction can advantageously be carried out by adapting the protocols described by (Marx, Piantadosi et al. 1988) and (Gaffney and Reese 1997) in which PG-E can represent triphenylmethyl chloride or 9-phenylxanthene-
• reaction can advantageously be carried out works by adapting the protocols described by (Nazih, Cordier et al. 2000) and (Kotsovolou, Chiou et al. 2001) in which (PG) 2 ⁇ represents di-tert-butyl dicarbonate;
• the reaction can advantageously be carried out by adapting the protocols described by (Marx, Piantadosi et al. 1988) and (Gaffney and Reese 1997), in which PG-E can represent triphenylmethyl chloride or 9-phenylxanthene-9- ol or also 9-chloro-9-phenylxanthene;
• R6 optionally in the presence of coupling agents or activators known to those skilled in the art for obtaining a compound of general formula (XLVIII) in which R1 and R3, identical or different, represent a group CO-R5 or CO - (CH 2 ) 2n + rX-R6;
• the reaction can advantageously be carried out by adapting the protocols described by (Marx, Piantadosi et al. 1988) and (Gaffney and Reese 1997) in which PG'-E can represent triphenylmethyl chloride or 9-phenylxanthene-9- ol or also 9-chloro-9-phenylxanthene;
• the reaction can advantageously be carried out by adapting the protocols described by (Nazih, Cordier et al. 2000) and (Kofsovolou, Ghiou et al. 2001) in which (P) 2 ⁇ represents di-tert-butyl dicarbonate;
• R2 and R3, identical, are hydrogen atoms or represent a group CO-R5 or CO- (CH 2 ) 2n + ⁇ -X-R6 and (v) R1 represents a group CO-R5 or CO- (CH 2 ) 2n + rX-R6, can be prepared from the compounds of formula (IIIa) according to the following process (diagram 13):
• FIG. 1 Structure of particular compounds according to the invention, the preparation of which is described in Examples 2, 4, 5, 6, 8, 10 to 14, 16, 18, 19, 21 and 23 noted in FIG. 1A.2 1A.4, 1A.5, 1A.6, 1A.8, 1A.10, 1A.11, 1A.12, 1A.13, 1A.14, 1A.16, 1A.18, 1A.19, 1A. 21 and 1A.23.
• Figure 2 Evaluation of the antioxidant properties of compounds according to the invention on the oxidation of LDL by copper (Cu).
• Figure 3 Evaluation of the properties of PPAR ⁇ agonists of compounds according to the invention with the Gal4 / PPAR ⁇ transactivation system.
• Example 2 the compounds according to the invention used in the examples for measuring or evaluating activity will be abbreviated as "Ex 2" to denote the compound according to the invention, the preparation of which is described in example 2.
• GGM Thin layer chromatographies
• the column chromatographies were carried out on silica gel 60 with a particle size 40-63 ⁇ m (reference 9385-5000 MERCK).
• the melting points (PF) were measured using a BUCHI B 540 device by the capillary method.
• IR infrared
• NMR nuclear magnetic resonance
• Mass spectra were performed on a PERKIN-ELMER SCIEX API 1 spectrometer (ESI-MS for Electrospray lonization Mass Spectrometry) or on an APPLIED BIOSYSTEMS Voyager DE-STR spectrometer of MALDI-TOF type (Matrix-Assisted Laser Desorption / lonization - Time Of Flight).
• the product is obtained in the form of a colorless oil which crystallizes slowly.
• 1,3- (di-fert-butoxycarbonylamino) -propan-2-ql (1 g; 3.45 mmol), tetradecylthioacetic acid (example 1) (0.991 g; 3.45 mmol) and dimethylaminopyridine ( 0.042 g; 0.34 mmol) are dissolved in dichloromethane (40 ml) at 0 ° C before adding dropwise dicyclohexylcarbodiimide (0.709 g; 3.45 mmol), diluted in dichloromethane ,. The reaction medium is left stirring at 0 ° C for 30 min and then brought to room temperature.
• the 1,3-diamino-2-tetradecylthioacetyloxypropane dihydrochloride (example 10) (0.400 g; 0.92 mmol) and the tetradecylthioacetic acid (example 1) (0.532 g; 1.84 mmol) are dissolved in dichloromethane (50 ml) at 0 ° C before adding triethylamine (0.3 ml; 2.1 mmol), dicyclohexylcarbodiimide (0.571 g; 2.77 mmol) and f hydroxybenzotriazole (HOBt) (0.249 g; 1.84 mmol).
• the 2,3-diaminopropionic acid hydrochloride (1 g; 7 mmol) is dissolved in methanol (40 ml). The medium is cooled by an ice bath before adding dropwise the thionyl chloride (2.08 ml; 28 mmol). The medium is brought to room temperature and then brought to reflux for 20 hours. The solvent is evaporated and the residue is triturated in heptane. The resulting precipitate is filtered, rinsed and dried to give a white-yellow solid.
• Methyl 2,3-diaminopropanoate dihydrochloride (example 13a) (0.500 g; 2.62 mmol) and tetradecylthioacetic acid (example 1) (1.51 g; 5.23 mmol) are dissolved in dichloromethane (80 ml) at 0 ° C before adding triethylamine (0.79 ml), dicyclohexylcarbodiimide (1.62 g; 7.85 mmol) and
• EXAMPLE 14 Preparation of 2.3-ditetradecylthioacetylamino-1-tetradecylthioacetyloxypropane 2,3-ditetradecylthioacetylaminopropan-1-ol (example 13) (0.200 g; 0.32 mmol) is dissolved in tetrahydrofuran (40 ml) before adding dicycloheimidecarbyl mg; 0.32 mmol), dimethylaminopyridine (39 mg; 0.32 mmol) and tetradecylthioacetic acid (Example 1) (91 mg; 0.32 mmol). The mixture is left stirring at room temperature for 20 hours.
• the product obtained (0.48 g) is purified by chromatography on silica gel (eluent dichloromethane-ethyl acetate 98-2) and allows the desired product to be obtained in the form of an ocher solid. Efficiency: 84%
• CHSH-CH 2 -SH CHSH-CH 2 -SH); 3.26-3.35 (m, 2H, BOGNH-CH 2 -GHSH-CH 2 -SH); 3.43-3.52 (m, 2H, BOGNH-GH 2 -GH-CH 2 -SH); 4.91 (m, 2H, SH); 5.08 (s, 1H, -NHBOC).
• Example 17 This compound is obtained according to the procedure described above (Example 15) from 1- (tert-butyloxycarbonylamino) -2,3-dissetradécylthioacétyl-thiopropane
• 3-tetradecylthioacetylamino-1-triphenylmethyloxypropan-2-ol (2 g; 3.31 mmol) is dissolved in toluene (100 ml) before adding imidazole (0.564 g; 8.28 mmol), triphenylphosphine ( 2.171 g; 8.28 mmol) and iodine (1.681 g; 6.62 mmol) in this order.
• the reaction medium is left under stirring at room temperature for 20 hours.
• a saturated solution of sodium bisulfite is added until complete discoloration of the reaction medium.
• Hydrated sodium hydrogen sulfate 38 mg; 0.68 mmol is suspended in ethanol (20 ml) before adding 2-iodo-3-tetradecylthioacetylamino-1-triphenylmethyloxypropane (example 20b) (200 mg; 0.28 mmol).
• the reaction medium is heated to 70 ° G. 238 mg of hydrated sodium hydrogen sulfate are added over several days. After 6.5 days, the solvent is evaporated, the residue taken up in dichloromethane and washed with water. The aqueous phase is reextracted and the combined organic phases are washed with a 0.5N hydrochloric acid solution and then with a saturated solution of sodium chloride, dried over magnesium sulfate.
• reaction medium is left under stirring at room temperature for 17 hours and 0.5 equivalents of imidazole, triphenylphosphine and iodine are added. After 21 hours of reaction, a saturated solution of sodium sulfite is added until the reaction medium is completely discolored.
• the phases are decanted and the aqueous phase is extracted twice with toluene.
• the combined organic phases are washed with a saturated aqueous sodium chloride solution, dried over magnesium sulfate, filtered and the solvent evaporated.
• the residue obtained (11.02 g) is purified by chromatography on silica gel (eluent dichloromethane-ethyl acetate 95-5) to give the desired compound in the form of a yellow paste which is rapidly reacted.
• the compounds according to the invention were prepared in the form of an emulsion as described below.
• the emulsion comprising a compound according to the invention and phosphatidylcholine (PC) is prepared according to the protocol of Spooner et al. (Spooner, Clark et al. 1988).
• the compound according to the invention is mixed with the PC in a 4: 1 ratio (w / w) in chloroform, the mixture is dried under nitrogen, then evaporated overnight under vacuum, the resulting powder is taken up by 0 , 16 M of potassium chloride containing 0.01 M of EDTA then the lipid particles are dispersed by ultrasound for 30 minutes at 37 ° C.
• the liposomes formed are then separated by ultracentrifugation (ultracentrifuge XL 80, Beckman Coulter, Villepinte, France) at 25,000 rpm for 45 minutes to recover liposomes whose size is greater than 100 nm and approaches that of chylomicrons.
• Liposomes consisting solely of PC are prepared in parallel to serve as a negative control.
• the composition of the liposomes in compounds according to the invention is estimated using the enzymocolorimetric assay kit for triglycerides. The assay is performed against a standard range, prepared using the CFAS lipid calibrator (Ref. No. 759350, Boehringer Mannheim GmbH, Germany). The standard range was built from 16 to 500 ⁇ g / ml.
• liposomes containing the compounds according to the invention, thus prepared are used in the in vitro experiments described in Examples 26, 27 and 28.
• the LDLs are prepared according to the method described by Lebeau et al. (The beautiful,
• test compounds are prepared at 10 "2 M in ethanol and diluted in PBS to have final concentrations ranging from 0.1 to 100 ⁇ M for a total ethanol concentration of 1% (v / v ).
• EDTA is removed from the LDL preparation by dialysis.
• the oxidation then takes place at 30 ° C. by adding 100 ⁇ l of a solution with 16.6 ⁇ M of copper sulphate to 800 ⁇ L of LDL (125 ⁇ g of proteins per ml) and 100 ⁇ L of a solution of the compound to test.
• the formation of dienes, the species to be observed, is measured by optical density at 234 nm in the samples treated with the compounds in the presence or absence of copper.
• the measurement of the optical density at 234 nm is carried out every 10 minutes for 8 hours using a thermostated spectrophotometer (Kontron Uvikon 930). The analyzes are carried out in triplicate.
• FIG. 2 shows that the compounds according to the invention Ex 2, 4, 5, 6 and 11 have intrinsic antioxidant properties.
• FIG. 2a shows that the compounds according to the invention induce a lag lag phase of more than 13% for the compound Ex 2 up to 34.3% for the compound Ex 4.
• the compounds according to the invention do not seem modify the oxidation rate (see Figure 2b) or the quantity of dienes formed (see Figure 2c).
• the compounds according to the invention, tested are the compounds whose preparation is described in Examples 2 to 23.
• the measurement of the oxidation of LDL is carried out by the method of TBARS (Thiobarbituric Acid Reactive Substances).
• the LDLs are oxidized with copper sulphate and the lipid peroxidation is determined as follows:
• the TBARS are measured using a spectrophotometric method, the lipid hydroperoxidation is measured in using peroxide-lipid oxidation dependent on iodide iodine.
• the results are expressed in nmol of malonodialdehyde (MDA) or in nmol of hydroperoxide per mg of proteins.
• MDA malonodialdehyde
• the results obtained previously, by measuring the inhibition of the formation of conjugated dienes, are confirmed by the experiments of measurement of lipid peroxidation of LDL.
• the compounds according to the invention therefore also effectively protect LDL against lipid peroxidation induced by copper (oxidizing agent).
• Example 26 Measurement of the antioxidant properties of the compounds according to the invention on cell cultures
• the cell lines used for this type of experiment are of the neuronal, neuroblastoma (human) and PC12 (rat) cells type.
• PC12 cells were prepared from a rat pheochromocytoma and are characterized by Greene and Tischler (Greene and Tischler 1976). These cells are commonly used for studies of neuronal differentiation, signal transduction and neuronal death.
• the PC12 cells are cultured as previously described (Farinelli, Park et al. 1996), in complete RPMI medium (Invitrogen) supplemented with 10% horse serum and 5% fetal calf serum.
• the mRNAs are extracted from the cells in culture treated or not with the compounds according to the invention. The extraction is carried out using reagents from the Absolutely RNA RT-PCR miniprep Kit (Stratagene, France) according to the supplier's instructions. The mRNAs are then assayed by spectrometry and quantified by quantitative RT-PCR using the Light Cycler Fast start DNA Master Sybr Green I kit (Roche) on a Light Cycler System device (Roche, France). Pairs of primers specific for Super Oxide Dismutase (SOD), Catalase and Glutathione Peroxidase (GPx) genes, antioxidant enzymes, are used as probes.
• SOD Super Oxide Dismutase
• GPx Glutathione Peroxidase
• Pairs of primers specific for the ⁇ -actin and cyclophilin genes are used as control probes.
• the increase in the expression of mRNAs, measured by quantitative RT-PCR, of the genes of the antioxidant enzymes is demonstrated in the different cell types used, when the cells are treated with the compounds according to the invention.
• the antioxidant properties of the compounds are also evaluated using a fluorescent indicator, the oxidation of which is followed by the appearance of a fluorescent signal.
• the reduction in intensity of the fluorescent signal emitted is measured in the cells treated with the compounds as follows: the PC12 cells cultured as previously described (black plate 96 wells transparent background, Falcon) are incubated with increasing doses of peroxide hydrogen (0.25 mM - 1 mM) in serum-free medium for 2 and 24 hours.
• DCFDA dichlorodihydrofluorescein diacetate
• the different cell lines (cell models mentioned above) as well as the cells in primary culture are treated as above.
• the cell supernatant is recovered after the treatment and the cells are lysed and recovered for the determination of the protein concentration.
• the detection of lipid peroxidation is determined as follows: lipid peroxidation is measured using thiobarbituric acid (TBA) which reacts with lipoperoxidation of aldehydes such as malonodialdehyde (MDA).
• TSA thiobarbituric acid
• MDA malonodialdehyde
• the supernatant from cells is collected (900 ⁇ l) and 90 ⁇ l of butylated hydroxytoluene are added thereto (Moriiere, Moysan et al. 1991).
• the compounds according to the invention advantageously have intrinsic antioxidant properties which make it possible to slow down and / or inhibit the effects of oxidative stress.
• the inventors also show that the compounds according to the invention are capable of inducing the expression of the genes of antioxidant enzymes. These particular characteristics of the compounds according to the invention allow the cells to fight more effectively against oxidative stress and therefore to be protected from damage caused by free radicals.
• Example 27 Evaluation of the activation of PPARs in vitro by the compounds according to the invention
• the nuclear receptors belonging to the PPAR subfamily which are activated by two major classes of pharmaceutical compounds, fibrates and glitazones, which are widely used in human clinics for the treatment of dyslipidemias and diabetes, play an important role in homeostasis lipid and carbohydrate.
• the following experimental data show that the compounds according to the invention activate PPAR ⁇ in vitro.
• PPARs The activation of PPARs is evaluated in vitro in lines of fibroblastic type RK13 or in a hepatocyte line HepG2, by measuring the transcriptional activity of chimeras consisting of the DNA binding domain of the yeast transcription factor Gal4 and of the ligand binding domain of the different PPARs.
• the example presented below is given for HepG2 cells.
• the HepG2 cells come from EGAGG (Porton Down, UK) and are cultured in DMEM medium supplemented with 10% vol / vol fetal calf serum, 100 U / ml penicillin (Gibco, Paisley, UK) and 2 mM L- Glutamine (Gibco,
• the culture medium is changed every two days.
• the cells are stored at 37 ° C. in a humid atmosphere containing 5% carbonic acid and 95% air.
• the plasmids pG5TkpGL3, pRL-CMV, pGal4-hPPAR ⁇ , pGal4-hPPAR ⁇ and pGal4-f have been described by Rascourt et al. (Raspe, Madsen et al. 1999).
• the constructs pGal4-mPPAR ⁇ and pGal4-hPPAR ⁇ were obtained by cloning into the vector pGal4-f DNA fragments amplified by PCR corresponding to the DEF domains of the mouse PPAR ⁇ and human PPAR ⁇ nuclear receptors respectively.
• the HepG2 cells are seeded in 24-well culture dishes at the rate of 5 ⁇ 10 4 cells / well, are transfected for 2 hours with the reporter plasmid pG5TkpGL3 (50 ng / well), the expression vectors pGal4-f, pGal4- mPPAR ⁇ , pGal4-hPPAR ⁇ , pGal4-hPPAR ⁇ or pGal4-hPPAR ⁇ (100 ng / well) and the vector for controlling the transfection efficiency pRL-CMV (1 ng / well) according to the protocol described above (Raspe, Madsen et al . 1999) and incubated for 36 hours with the test compounds.
• the cells are lysed (Gibco, Paisley, UK) and the luciferase activities are determined using the Dual-Luciferase TM Reporter Assay System assay kit (Promega, Madison, Wl, USA) according to the supplier's instructions.
• the protein content of the cell extracts is then evaluated using the Bio-Rad Protein Assay assay kit (Bio-Rad, M ⁇ nchen, Germany) according to the supplier's instructions.
• the inventors demonstrate an increase in luciferase activity in cells treated with the compounds according to the invention and transfected with the plasmid pGal4-hPPAR ⁇ . This induction of luciferase activity indicates that the compounds according to the invention are activators of PPAR ⁇ .
• An example of results obtained with compounds according to the invention is presented in FIG. 3.
• FIG. 3 HepG2 cells, transfected with the plasmids of the Gal4 / PPAR ⁇ system, are incubated with different concentrations (5, 15, 50 and 100 ⁇ M) of the compounds according to the invention (Ex 2, Ex 4, Ex 5, Ex 6 , Ex 11) for 24 h and with different vehicle concentrations (PC) noted 1, 2, 3, 4 as controls respectively for the concentrations 5, 15, 50 and 100 ⁇ M of the compounds according to the invention (according to 4: 1 ratio w / w described in Example 24 (Method for preparing the compounds of formula (I) according to the invention)).
• the results are represented by the induction factor (luminescent signal of the treated cells divided by the luminescent signal of the untreated cells) according to the different treatments.
• the compound according to the invention Ex 5 also induces an increase in the induction factor with a dose effect of 10.5 to 100 ⁇ M, 7 to 50 ⁇ M, 2.5 to 15 ⁇ M and 1.2 to 5 ⁇ M.
• the compound according to the invention Ex 6 also induces an increase in the luminescent signal, revealing an activity on the nuclear receptor PPAR ⁇ .
• the induction factors for compound Ex 6 are 14.5 to 100 ⁇ M, 9.6 to 50 ⁇ M, 2.2 to 15 ⁇ M and 1.1 to 5 ⁇ M.
• the vehicle PC liposome
• cytokines and free radicals The inflammatory response appears in many neurological disorders, such as cerebral ischemia, and inflammation is one of the important factors in neurodegeneration.
• One of the first reactions of glia cells to stroke is to release cytokines and free radicals.
• cytokines and free radicals The consequence of this release of cytokines and free radicals is an inflammatory response in the brain which can lead to the death of neurons (Rothwell 1997).
• Cell lines and primary cells are grown as described above.
• the lipopolysaccharide (LPS), bacterial endotoxin (Escherichia coli 0111: B4) (Sigma, France) is reconstituted in distilled water and stored at 4 ° C.
• the cells are treated with an LPS concentration of 1 ⁇ g / ml for 24 hours.
• the cell culture medium is completely changed.
• TNF- ⁇ is an important factor in the inflammatory response to stress (oxidant for example).
• the culture medium of the stimulated cells is removed and the amount of TNF- ⁇ is evaluated with an ELISA-TNF- ⁇ kit (Immunotech, France ).
• the samples are diluted 50 times in order to be in line with the standard range (Chang, Hudson et al. 2000).
• the anti-inflammatory property of the compounds according to the invention is characterized in the following manner: the culture medium of the cells is completely changed and the cells are incubated with the compounds to be tested for 2 hours. After this incubation, LPS is added to the culture medium at a final concentration of 1 ⁇ g / ml. After 24 hours of incubation, the cell supernatant is recovered and stored at -80 ° C when it is not treated directly. The cells are lysed and the amount of protein is measured, using the Bio-Rad Protein Assay assay kit (Bio-Rad, Kunststoff, Germany) according to the supplier's instructions.
• the measurement of the decrease in TNF- ⁇ secretion favored by the treatment with the test compounds is expressed in pg / ml / ⁇ g of protein and reported as a percentage relative to the control. This shows that the compounds according to the invention have anti-inflammatory properties.
• A- / Prophylactic model 1 / Treatment of animals
• Wistar rats weighing 200 to 350 g were used for this experiment.
• the animals are kept under a 12 hr light / dark cycle at a temperature of 20 ⁇ 3 ° C. Animals have free access to water and food. Food gain and weight gain are recorded.
• the animals are treated by gavage with the compounds according to the invention.
• CMC carboxymethylcellulose
• Tween 0.16% a sodium salt of carboxymethylcellulose of medium viscosity (Ref. C4888, Sigma-aldrich, France).
• the Tween used is Polyoxyethylenesorbitan Monooleate (Tween 80, Ref. P8074, Sigma-aldrich, France).
• Brains are quickly frozen and sectioned.
• the sections are colored in Cresyl purple.
• the non-colored areas of the brain sections were considered to be injured by the infarction.
• Analysis of the brain sections of animals treated with the compounds according to the invention reveals a marked reduction in the volume of the infarction compared to the untreated animals.
• the compounds according to the invention are administered to animals before ischemia (prophylactic effect), they are capable of inducing neuroprotection.
• the brains of rats are frozen, crushed and reduced to powder and then resuspended in saline.
• the different enzymatic activities are then measured as described by the following authors: superoxide dismutase (Flohe and Otting 1984); glutathione peroxidase (Paglia and Valentine 1967); glutathione reductase (Spooner, Delides et al. 1981); glutathione-S-transferase (Habig and Jakoby 1981); catalase (Aebi 1984).
• superoxide dismutase Flohe and Otting 1984
• glutathione peroxidase Paglia and Valentine 1967
• glutathione reductase Spooner, Delides et al. 1981
• glutathione-S-transferase Habig and Jakoby 1981
• catalase Aebi 1984.
• the various enzymatic activities mentioned above are increased in the preparations of brains of animals treated with the compounds
• Animals as described above are used for this experiment.
• the animals are anesthetized using an intraperitoneal injection of 300 mg / kg of chloral hydrate.
• a rectal probe is placed and the body temperature is maintained at 37 ⁇ 0.5 ° C. Blood pressure is measured during the whole experiment.
• the right carotid is updated using a medial cervical incision.
• the pterygopalatine artery was ligated at its origin and an arteriotomy is performed in the external carotid artery in order to slip a nylon monofilament into it.
• This filament is then gently advanced into the common carotid artery and then into the internal carotid artery in order to close off the origin of the middle cerebral artery. After 1 hour, the filament is removed to allow reperfusion. 2 / Treatment of animals:
• Animals having undergone prior ischemia-reperfusion are treated with the compounds according to the invention by the oral route (as already described in a CMC + Tween vehicle) one or more times after the reperfusion (600 mg / kg / d or 2 administrations of 300mg / kg / d).
• the animals previously treated or not treated with the compounds according to the invention are killed by an overdose of pentobarbital.
• Brains are quickly frozen and sectioned.
• the sections are colored in Cresyl purple.
• the non-colored areas of the brain sections are considered to be injured by the infarction.
• a curative treatment treatment of the acute phase
• the animals treated with the compounds according to the invention have reduced damage to the brain level compared to the untreated animals.
• the volume of the infarction is reduced when the compounds according to the invention are administered for 24, 48 or 72 hours after ischemia-reperfusion.
• the compounds according to the invention therefore have neuroprotective activity during treatment subsequent to acute ischemia.
• the use of the compounds according to the invention shows that these new compounds have an intrinsic antioxidant activity, capable of delaying and reducing the effects of oxidative stress.
• they induce the expression of the genes of antioxidant enzymes, which combined with their antioxidant nature makes it possible to strengthen anti-radical protections.
• the compounds according to the invention have an anti-inflammatory power and the property of activating the nuclear receptor PPAR ⁇ .

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