New precursors of probes for oxidative stress

Abstract

Novel Probe Precursors for Oxidative Stress The present invention relates to a compound of formula (I): wherein n = 1 or 2, A and A' are independently selected from the group consisting of the following substituents (II-a) and (II-b): and , R and R' being independently selected from H and linear or C1-C3 branched alkyls, Z being selected from a linear or C1-C4 branched alkyl group and an aryl group optionally substituted by a linear or C1-C3 branched alkyl group and / or by a hydroxyl group, and the motif of formula (N) is selected from the following motifs (III-a), (III-b), (III-c), (III-d) and (III-e): , , , and , Ra, Rb, Rc and Rd being, for each motif (III-a), (III-b), (III-c), (III-d) and (III-e), independently selected among the alkyl groups, linear or branched, in the C1-C4 range. Figure for the abstract: None

Description

Title of the invention: Novel precursors of probes for oxidative stress

[0001] The present invention relates to novel precursors of probes for oxidative stress, and their various uses.

[0002] One of the greatest societal challenges is to promote active and healthy aging of the population. To achieve this, it is essential to discover new diagnostic methods or effective therapies against cardiovascular and neurological pathologies or cancer. It is increasingly clear that oxidative stress has a major impact on the evolution of all these diseases. A method for non-invasive detection and mapping of oxidative stress in vivo would therefore provide valuable information on the development of these pathologies and would help in the design of diagnostic and treatment tools.

[0003] Protein and lipid oxidation biomarkers in biological fluids are interesting tools but they do not allow localization of the pathology. Therefore, structural and / or functional imaging methods based on positron emission tomography (PET), magnetic susceptibility imaging (SWI) and magnetic resonance imaging (MRI) are commonly used. However, they have certain limitations. PET requires expensive and short-lived radioactive tracers as well as access to a local cyclotron for their production, and is not compatible with certain pathologies. Magnetic susceptibility imaging (SWI) evaluates the tissue content of metals (Cu, non-heme iron) as potential sources of reactive oxygen species (ROS) by Fenton reaction, but many regions accumulate iron (in ferritin for example) without generating overproduction of radicals.More specific, magnetic resonance spectroscopy is capable of measuring part of the antioxidant defenses (ascorbate, glutathione) but ignores the essential enzymes for detoxifying ROS.

[0004] Minimally invasive methods using electron paramagnetic resonance (EPR) and exogenous molecular probes have already demonstrated their usefulness in assessing and mapping the "redox status" in the brain under conditions of oxidative stress, for example in rat models suffering from hypertension, stroke or epilepsy and in rodents exposed to ischemia-reperfusion injury. The American Heart Association even considers EPR combined with the use of molecular probes as the gold standard in redox physiology (KK Griendling, RM Touyz, JL Zweier, S. Dikalov, W. Chilian, Y.-R. Chen, DG Harrison, A. Bhatnagar, Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signaling in the Cardiovascular System: A Scientific Statement From the American Heart Association, Wax. Res. 119 (2016) e39-e75. doi: 10.1161 / RES.0000000000000110).

[0005] Among the molecular probes already known, we distinguish the probes of the hydroxylamine family, which are oxidized in cells into nitroxide which is detectable by EPR.

[0006] It is particularly known to use precursors of these probes, such as the following compound l-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP): / x H3C CH3

[0007] This compound comprises a protective ester function of the hydroxylamine group. The objective of these precursors is to deliver the probe to the interior of the cells with the least possible degradation. Masking the hydroxylamine function limits the spontaneous oxidation of hydroxylamines before internalization into the cells, which facilitates manipulation by non-chemist experimenters. Once internalized, the ester functions are hydrolyzed by the cell's esterases, thus releasing the hydroxylamine unit, which can be oxidized to nitroxide and become detectable in EPR.

[0008] However, these probes have a number of limitations, such as their low biological stability, particularly in vivo, their lack of specificity and / or sensitivity and the low number of commercially available probes.

[0009] There is therefore a need for new, more efficient molecular probes. This need is all the more acute as new technological advances in EPR spectrometers have recently been commercialized, and the limitations of the probes are a barrier to the development of redox EPR applications.

[0010] The aim of the invention is therefore to propose new molecular probes for oxidative stress, exhibiting improved performance compared to already existing probes.

[0011] In particular, an aim of the invention is to propose new molecular probes for oxidative stress, having improved stability, and / or better sensitivity and / or easier handling.

[0012] For this purpose, the invention relates to a compound of formula (I):

[0013]

[0014]

[0015] in which n = 1 or 2, preferably n = 1, A and A' are independently selected from the group consisting of the following substituents (II-a) and (II-b): R' (Ha) Z R and R' being independently selected from H and alkyls

[0016] linear or branched C1-C4, Z being chosen from a linear or branched C1-C4 alkyl group and an aryl group optionally substituted by a linear or branched C1-C4 alkyl group and / or by a methoxyl group (such as 4-methoxyphenyl), and the pattern of formula (N) Z''"'"' > is chosen from the patterns (Ill-a), (Ill-b), ( N~O-|- (N) (III-c), (Ill-d) and (Ill-e) following: Ra Rb ' Rd Rc (Hl-a)

[0017]

[0018]

[0019] Rd Rd (Okay <i) Ra Rb Rd Rc (IH-e) Ra, Rb, Rc and Rd being, for each unit (Ill-a), (Ill-b), (III-c), (Ill-d) and (Ill-e), independently chosen from linear or branched C1-C4 alkyls. By linear or branched C1-C4 alkyl is meant a hydrocarbon chain comprising n carbon atoms, n being equal to 1, 2, 3 or 4, and comprising 2n+2 hydrogen atoms. This is for example a methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert-butyl group. Indeed, the inventors have surprisingly discovered that the compounds of formula (I) according to the invention are precursors of probes for oxidative stress, detectable in particular by EPR spectroscopy, exhibiting significantly improved performance compared to existing compounds, particularly in terms of in vivo stability and sensitivity. Without wishing to be bound by any theory, the inventors believe that the Ra, Rb, Rc and Rd groups of the unit of formula (N) in the pre Prior art probe sliders induce steric hindrance that prevents cell esterases from hydrolyzing the ester protecting the hydroxylamine function. Indeed, since the deprotection kinetics are low and the reaction incomplete, a very small amount of probe is actually released into the cells, which is difficult to detect in medical imaging or by EPR spectroscopy. In the compounds of formula (I) according to the invention, the presence of a spacer group between the nitrogen of the unit of formula (N) and the carboxyl targeted by the esterase in the A' group allows rapid and complete deprotection of the hydroxylamine function. For example, when A' is the (II-a) substituent with R=R'=CH3, 100% of hydroxylamine is released in 10 min at 37°C vs. deprotection of the commercial ACP probe (l-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine) limited to 27% under the same conditions.The compounds of formula (I) according to the invention thus allow easier handling (suitable for non-chemist experimenters for applications in biology), more efficient release of the probe, which results in a gain in sensitivity.

[0020] In formula (I), and except in the case where the unit of formula (N) is the unit of formula (IILe), a person skilled in the art will understand that each group o JL? CT A can be linked to any substitutable carbon atom (bearing at least one hydrogen atom) constituting the ring(s) of the unit of formula (N) of formula (in-a), (mb), (III-c) and (in-d).

[0021] The presence of the group A in the compound of formula (I) advantageously makes it possible to improve the accumulation of the probe in the cells, therefore the quantity of probe detectable in the cells after oxidation, and therefore the quality of the image or signal obtained. The groups A allow the compounds to penetrate passively into the cells and are sensitive to hydrolysis by intracellular esterases, which leads to the release of a negatively charged carboxylate ion which does not diffuse freely through the membranes.

[0022] It is understood that each atom of the compound of formula (I) may be present in the form of any of its natural isotopes.

[0023] According to a particular embodiment, the nitrogen atom of the unit of formula N is a nitrogen 15.

[0024] Alternatively, or in combination, the hydrogen atoms of the unit of formula (N), and in particular of the groups Ra, Rb, Rc and Rd, are preferably hydrogen 2 (more commonly called deuterium).

[0025] The isotopic composition of the compound of formula (I), and in particular its nitrogen 15 and / or hydrogen 2 content, is determined according to techniques known to man of the profession. For example, high-resolution mass spectrometry (HRMS) can determine the average concentration of the isotope studied across all sites of the compound of formula (I). Quantitative nuclear magnetic resonance (NMR) of deuterium and nitrogen-15 can also be used, and can study the specific isotopic distribution at each position of the compound of formula (I). Also, after hydrolysis and oxidation (by potassium ferricyanide, for example) of a compound of formula (I), the electron paramagnetic resonance (EPR) spectrum is different for a molecule containing nitrogen-15 (2 lines) and one containing nitrogen-14 (3 lines).

[0026] Preferably, in the substituent (ILb), Z is chosen from methyl, ethyl, propyl, isopropyl, tert-butyl, phenyl and 4-methoxyphenyl. Advantageously, for the remainder of the description, Z = Me.

[0027] Preferably, A is selected from the group consisting of the substituent (Il-b) in which Z = Me and the substituent (Il-a) in which R = H, and A' is selected from the group consisting of the substituent (ILb) in which Z = Me and the substituent (Il-a) in which R = Me.

[0028] Preferably, R' is methyl or ethyl, preferably methyl.

[0029] Preferably, A and A' are identical.

[0030] According to certain embodiments, A = A' = (Il-a).

[0031] According to other embodiments, A = A' = (ILb).

[0032] The use of the (ILb) groups advantageously makes it possible to avoid the release of an equivalent of acetaldehyde and an equivalent of formaldehyde after the action of the esterase, which could induce a potential problem of toxicity at very high doses as well as a potential cellular stress which could interfere with the measurement of oxidative stress. Indeed, the hydrolysis by esterases of the (ILb) group only results in the formation of 3-hydroxybutan-2-one and 2,3-butanediol, which are non-toxic.

[0033] Preferably, the unit of formula (N) is chosen from the units (IILb), (IILc), (IILd) and (IILe), preferentially from the units (IILb), (IILc) and (IILd), more preferentially (IILb) and (IILd), or alternatively (IILc).

[0034] Preferably, the compound of formula (I) is chosen from the following compounds (La), (Lb), (Lb”), (Le), (Le”), (Ld), (Ld'), (Ld”), (Ld'”) and (Le): (l>0 Pïd O—N / °4 .VO qy o-< yo— / .y O—•< / y Q- y L --\^\XOxv Il O (..ol) .4 loy T^Z 0 O—N | f V"K / O, 4 qy F v (3-1) Pi! O““N | Kry% 0 qy b ÿ (>.qi) UQ—N^ ] \ Vv% 0 J™ fv (qi) PiJ p—r / J ^0 q / èy )T" 'V 0 (ie)

[0035] A, A', Ra, Rb, Rc and Rd being, for each compound (Ia), (Ib), (Ib”), (Ic), (Ic”), (Id), (I-d'), (Id”), (I-d'”) and (Ie), each independently as defined above.

[0036] Preferably, the compound of formula (I) is chosen from compounds (Ia), (Ib), (Ib”), (Ic), (Id) and (Ie), preferentially from compounds (Ia), (Ib), (Ib”), (Ic) and (Id), more preferentially from compounds (Ib), (Ic) and (Id), even more preferentially (Ib) and (Id), or alternatively (Ic).

[0037] More preferably, the compound of formula (I) is chosen from the compounds

[0038]

[0039]

[0040] (IV), (V), (VI) and (VII) following, / CH? h3c (IV) H H3C CH 3 O A' AV \\ / ■' v-\ | n—d bw3 (V) CH<î o / :ch. Il V f OAN / i N—O \~ \ / CH> HgC' A' d' (V!) And çh3 / d~i-^ AO . / / A' of ,A( A—ô" \ / \ / Sc==< N--0 \ / \_____7 / \ H3ç—' ) h3c (VH) A and A' being as defined above. Preferably, the compound of formula (I) is chosen from compounds (IV), (V) or alternatively from compounds (VI). Preferably, Ra, Rb, Rc and Rd are independently selected from methyl, ethyl and isopropyl, preferably from methyl and ethyl.

[0041]

[0042] preferably, Ra, Rb, Rc and Rd are the same, and are preferably selected from methyl and ethyl. The Ra, Rb, Rc and Rd groups advantageously allow for better image or signal quality. Without being bound by any theory, the inventors believe that the bulk induced by the four Ra, Rb, Rc and Rd groups allows for stabilization of the nitroxide radical, and also allows for reduction of the superoxide dismutase mimic activity of the nitroxide produced, which improves the response of the hydroxylamine probe to the production of superoxide anion. According to a particular embodiment, the compound of formula (I) is chosen from:

[0043] According to one embodiment, the compound of formula (I) is the compound

[0044] According to another embodiment, the compound of formula (I) is the compound

[0045] According to an alternative embodiment, the compound of formula (I) is different from the following compound:

[0046]

[0047] The present invention also relates to a method for preparing compounds of formula (I) according to the invention. This method is in particular as described in the examples. A typical method for preparing compounds of formula (I) according to the invention comprises at least one step (SI) of reduction to hydroxylamine of the compound of formula (X) (X) for example in the presence of dihydrogen, to obtain a compound of formula (X')

[0048] (X*) then a step (SI') of nucleophilic addition of the compound (X') to a compound of formula (XI), GOLD

[0049] or of formula (XI')

[0050] to give a compound of formula (XII) (XH)

[0051] or of formula (XII'), respectively (xsn

[0052] A, A', n, R, and the unit of formula (N) being as defined above for the compounds of formula (I).

[0053] Preferably, step (S1) is carried out in the presence of palladium on carbon and dihydrogen. The dihydrogen is then removed by purging with argon before carrying out step (S1').

[0054] Preferably, step (SI') of adding the compound of formula (XI) to the compound of formula (X') is carried out between -5°C and 8°C, then the reaction mixture is stirred for 12 to 20 h at room temperature (20-28°C).

[0055] This method may further comprise a step (S2) of nucleophilic substitution between the compound of formula (XII) and a compound of formula (XIII) R'-C(O)-O (XIII), R' being as defined above for the compounds of formula (I), to give a compound of formula (I) according to the present invention.

[0056] Preferably, step (S2) is carried out in the presence of an acetate salt, preferably mercury acetate. Preferably, the reaction mixture is stirred for 12 to 20 h at room temperature (20-28°C).

[0057] The method may further comprise a step (S0) of nucleophilic substitution between a compound of formula (XIV) 0 HOX YN""0' rr (XIV)

[0058] and a compound of formula (XV) A- X ' (XV)

[0059] to obtain a compound of formula (X),

[0060] A, n, and the unit of formula (N) being as defined above for the compounds of formula (I) and X = Cl or Br, preferably A = (Il-a).

[0061] Preferably, step (S0) is carried out in the presence of an inorganic base, preferably in the presence of potassium carbonate. Preferably, the reaction mixture is stirred for 1 to 5 hours at room temperature (20-28°C).

[0062] Alternatively, the method may further comprise a step (S0') of activating the compound of formula (XIV) into an acyl chloride of formula (XIV') Q (XIV)

[0063] followed by a step (S0”) of coupling between the compound (XIV') and a compound (XV') A—OH (XV)

[0064] to obtain a compound of formula (X), A, n and the unit of formula (N) being as defined above for the compounds of formula (I), preferably A = (II-b).

[0065] The activation step (S0') is preferably carried out in the presence of an acyl dichloride, for example oxalyl chloride and dimethylformamide.

[0066] Step (S0”) is preferably carried out in the presence of bases, for example triethylamine and 4-dimethylaminopyridine. Preferably, the reaction mixture is stirred for 10 to 48 h at room temperature (20-28°C).

[0067] Any alternative method well known in the prior art may be used.

[0068] The present invention further relates to a pharmaceutical composition, comprising in a physiologically acceptable medium, at least the compound of formula (I) according to the invention.

[0069] Preferably, the pharmaceutical composition according to the invention is in the form of a nanoemulsion, preferably an oil-in-water nanoemulsion.

[0070] Preferably, the nanoemulsion comprises an aqueous phase and an oily phase, the oily phase comprising the compound of formula (I). Preferably, at least 90% by mass, preferably from 95 to 99.99% by mass, of the total mass quantity of the compound of formula (I) of the nanoemulsion is included in the oily phase.

[0071] Preferably, the oily phase further comprises at least one oily compound comprising a triglyceride, preferably a C4-C24 triglyceride, preferentially a C8-C18 triglyceride. The triglycerides are generally triesters of fatty acids and glycerol, the fatty acids of which may have chain lengths varying from C4 to C24, the latter being able to be linear or branched, saturated or unsaturated.

[0072] Preferably, the oily compound is chosen from vegetable oils and medium chain triglycerides in C6-C12, preferably in C8-C10.

[0073] The triglycerides according to the invention are in particular wheat germ, sunflower, grape seed, sesame, corn, apricot, castor, shea, avocado, olive, soybean oils, almond oil and in particular sweet almond, palm, rapeseed, cotton, hazelnut, macadamia, jojoba, alfalfa, poppy, pumpkin, sesame, squash, rapeseed, blackcurrant, evening primrose, millet, barley, quinoa, rye, safflower, candlenut, passionflower, musk rose, coconut oil; or the triglycerides of caprylic / capric acids.

[0074] Advantageously, the oily compound of the oily phase is chosen from olive oil and lipophilic Labrafac® WL 1349.

[0075] Preferably, the aqueous phase comprises water, and optionally NaOH, NaCl, glycerol, sorbitol, dextrose or xylitol. Preferably, the aqueous phase comprises NaOH in order to adjust the pH of the nanoemulsion between 6.8 and 7.2, preferably around 7, and optionally glycerol, in an amount preferably ranging between 0.5 and 5% by mass relative to the total mass of the emulsion.

[0076] Preferably, the composition, preferably the nanoemulsion, further comprises at least one surfactant, preferably a non-ionic surfactant, preferably chosen from organosilicon surfactants, polysorbate, cetostearyl alcohol, cetyl alcohol, oleyl alcohol, stearyl alcohol, cocamide DEA, cocamide MEA, polyalkylglucoside, decyl glucoside, lauryl glucoside, octyl glucoside, monolaurin, poloxamer, sorbitan monostearate, sorbitan tristearate, or any combination thereof, preferably from poloxamers and sorbitan monostearate.

[0077] Preferably, the composition, preferably the nanoemulsion, comprises a first surfactant as defined above, and a second surfactant different from the first surfactant and as defined above.

[0078] Advantageously, the first surfactant is sorbitan monostearate and the second surfactant is chosen from poloxamers, preferably poloxamer 188.

[0079] An example of sorbitan monostearate is the commercial product Span® 80. An example of poloxamer is the commercial product Kolliphor® P 188.

[0080] Preferably, the total mass content of surfactant ranges from 4% to 12% relative to the total mass of the composition, preferably of the nanoemulsion, preferably ranges from 5% to 10%.

[0081] Preferably, the mass content of first surfactant ranges from 3% to 7% relative to the total mass of the composition, preferably of the nanoemulsion, preferably ranges from 4% to 6%.

[0082] Preferably, the mass content of second surfactant ranges from 1% to 5% relative to the total mass of the composition, preferably of the nanoemulsion, preferably ranges from 1% to 4%.

[0083] Preferably, in the composition, preferably in the nanoemulsion, the mass ratio between the first surfactant and the second surfactant ranges from 1 to 6, preferably from 1.2 to 4.5, advantageously from 1.5 to 4.2.

[0084] Preferably, in the composition, preferably in the nanoemulsion, the mass ratio between the aqueous phase and the oily phase ranges from 1 to 8, preferably from 2 to 6, preferably from 3 to 5, advantageously from 3.5 to 4.5.

[0085] Preferably, in the composition, preferably in the nanoemulsion, the mass ratio between the oily compound and all of the surfactants ranges from 1 to 5, preferably from 1.5 to 4, preferably from 2 to 3.5, advantageously from 2.5 to 3.5.

[0086] Preferably, the nanoemulsion has an average droplet size ranging from 50 to 500 nm, preferably ranging from 80 to 300 nm, advantageously ranging from 100 to 200 nm.

[0087] Preferably, the nanoemulsion has a droplet size distribution ranging from 0.05 to 0.20, preferably ranging from 0.07 to 0.15.

[0088] The average droplet size is determined by photon correlation spectroscopy using a Zetasizer® Nano ZS (Malvem Panalytical, Worcestershire, UK). 10 qL of nanoemulsion is diluted one hundred times with ultrapure water. All measurements are recorded at 25°C with a scattering angle of 173°. Curve fitting of the correlation functions is performed using an exponential fit (cumulative approach) for the evaluation of the hydrodynamic diameter and PDI. All measurements are performed in triplicate to determine the average droplet size expressed as Z-average diameter (Z-Ave) and PDI.

[0089] Preferably, in the nanoemulsion, the mass ratio between the compound of formula (I) and the oily phase ranges from 0.1 to 0.5, preferably from 0.15 to 0.45, preferentially from 0.2 to 0.4, more preferentially from 0.25 to 0.35, advantageously from 0.30 to 0.35.

[0090] Preferably, the molar concentration of the compound of formula (I) in the oily phase ranges from 0.01 mol / L to 0.5 mol / L, preferably from 0.05 mol / L to 0.25 mol / L, advantageously from 0.08 mol / L to 0.12 mol / L.

[0091] Preferably, the molar concentration of the compound of formula (I) in the nanoemulsion ranges from 1 mmol / L to 500 mmol / L, preferably from 5 mmol / L to 250 mmol / L, advantageously from 10 mmol / L to 50 mmol / L.

[0092] The present invention further relates to the use of the compound of formula (I) or of the composition according to the invention, as a precursor of compounds for electron paramagnetic resonance spectroscopy or for imaging, preferably medical or pre-clinical imaging, in particular for electron paramagnetic resonance spectroscopy, for electron paramagnetic resonance imaging or for magnetic resonance imaging.

[0093] According to one embodiment, the compound of formula (I) or the composition, preferably the nanoemulsion, according to the invention are used as radical probe precursors for electron paramagnetic resonance spectroscopy or for electron paramagnetic resonance imaging.

[0094] According to another embodiment, the compound of formula (I) or the composition, preferably of the nanoemulsion, according to the invention are used as precursors of contrast agent for magnetic resonance imaging.

[0095] As mentioned above, the compounds of formula (I) have the ability to penetrate inside cells, to be hydrolyzed by the esterases of said cells in order to generate a probe comprising a hydroxylamine unit.

[0096] This hydroxylamine unit can be oxidized under the action of oxidative stress of the cell, becoming a nitroxide radical, directly detectable in spectroscopy or electron paramagnetic resonance imaging. The paramagnetic nitroxide radical can also cause a modification of the longitudinal (TJ) and / or transverse (T2) magnetic relaxation time of the protons of nearby water, or even of the aliphatic protons of the tissues, leading to a variation of the contrast in magnetic resonance imaging.

[0097] Preferably, the use of the compound of formula (I) or of the composition according to the invention is in vivo or in vitro.

[0098] According to one embodiment, the use of the compound of formula (I) or the composition according to the invention is in vitro.

[0099] The present invention also relates to a method for in vitro detection of oxidative stress, comprising the following steps:

[0100] - bringing into contact the compound of formula (I) or the composition according to the invention, with a medium comprising cells, a fluid or a biological tissue, and

[0101] - record the signal from the medium by electro-paramagnetic resonance spectroscopy electronics, or capture one or more images of the environment by an imaging technique, preferably medical or pre-clinical, preferably by electron paramagnetic resonance imaging or by magnetic resonance imaging.

[0102] The present invention also relates to a method for in vivo detection of oxidative stress in a subject, comprising:

[0103] - either a step of capturing one or more images of a subject or at least one part of a subject previously treated with the compound of formula (I) according to the invention, or with the composition according to the invention, by an imaging technique, preferably by a medical or pre-clinical imaging technique, preferably by electronic paramagnetic resonance imaging or by magnetic resonance imaging.

[0104] - either a step of recording the signal of a subject or at least part of a subject previously treated with the compound of formula (I) according to the invention, or with the composition according to the invention, by electronic paramagnetic resonance spectroscopy.

[0105] Preferably, the subject is treated with an amount detectable by said imaging technique or by said electron paramagnetic resonance spectroscopy.

[0106] By "detectable quantity" is meant according to the invention a quantity of compound of formula (I) or of the composition sufficient to obtain, by the imaging technique considered, an interpretable image or to obtain, by said electronic paramagnetic resonance spectroscopy, an interpretable spectrum.

[0107] Preferably, the method for in vivo detection of oxidative stress in a subject comprises the following steps:

[0108] - administering to the subject an amount of compound of formula (I) or of the composition according to the invention detectable by said imaging technique,

[0109] - capturing the image(s) of the subject or at least part of the subject treated by said imaging technique, preferably by electron paramagnetic resonance imaging or magnetic resonance imaging or recording the signal of at least part of the subject by electron paramagnetic resonance spectroscopy.

[0110] According to one embodiment, the method for in vivo detection of oxidative stress according to the invention comprises capturing the image(s) of the subject or at least part of the subject treated by electron paramagnetic resonance imaging.

[0111] According to another embodiment, the method for in vivo detection of oxidative stress according to the invention comprises capturing the image(s) of the subject or at least part of the subject treated by magnetic resonance imaging.

[0112] According to yet another embodiment, the method for in vivo detection of oxidative stress according to the invention comprises recording the signal from at least part of the subject by electronic paramagnetic resonance spectroscopy.

[0113] The invention also relates to the compound of formula (I) or the composition according to the invention, for their use as a diagnostic tool, preferably in combination with an electron paramagnetic resonance spectroscopy technique, an electron paramagnetic resonance imaging technique or a magnetic resonance imaging technique.

[0114] Preferably, the compound of formula (I) or the composition according to the invention are used as a diagnostic tool in an in vivo diagnostic method, preferably comprising an electron paramagnetic resonance spectroscopy technique, an electron paramagnetic resonance imaging technique or a magnetic resonance imaging technique.

[0115] Preferably, the in vivo diagnostic method comprises:

[0116] - either a step of capturing images by an imaging technique of a subject or of at least a portion of a subject previously treated with an amount of compound of formula (I) or composition according to the invention detectable by said imaging technique, the imaging technique preferably being chosen from an electron paramagnetic resonance imaging technique and a magnetic resonance imaging technique,

[0117] - either a recording step by paramagnetic resonance spectroscopy electronic signal of a subject or at least part of a subject previously treated with an amount of compound of formula (I) or composition according to the invention detectable by said electronic paramagnetic resonance spectroscopy.

[0118] The present invention also relates to the compound of formula (I) or the composition according to the invention, for their use as a radical probe precursor in an in vivo diagnostic method, said method preferably comprising an electron paramagnetic resonance imaging technique.

[0119] The present invention also relates to the compound of formula (I) or the composition according to the invention, for their use as a contrast agent precursor in an in vivo diagnostic method, said method preferably comprising a magnetic resonance imaging technique.

[0120] The present invention also relates to the compound of formula (I) or the composition according to the invention, for their use as a precursor of contrast agent in an in vivo diagnostic method, said method preferably comprising an electron paramagnetic resonance spectroscopy technique.

[0121] The present invention also relates to the compound of formula (I) or the composition according to the invention, for their use in a method of detecting a disease of a diagnosed subject, comprising the following steps:

[0122] - capturing one or more images by an imaging technique, preferably medical, of a healthy subject previously treated with a quantity of compound of formula (I) or composition according to the invention detectable by said technique imaging,

[0123] - capturing one or more images by the imaging technique of the diagnosed subject previously treated with an amount of compound of formula (I) or composition according to the invention detectable by said imaging technique,

[0124] - determine the difference between the healthy subject and the diagnosed subject, and

[0125] - compare this deviation with reference values ​​in order to determine the presence or the absence of the disease in the diagnosed subject.

[0126] The present invention also relates to the compound of formula (I) or the composition according to the invention, for their use in a method of detecting a disease of a diagnosed subject, comprising the following steps:

[0127] - record a signal by electron paramagnetic resonance spectroscopy of a healthy subject previously treated with a quantity of compound of formula (I) or of composition according to the invention detectable by said electronic paramagnetic resonance spectroscopy,

[0128] - record a signal by electron paramagnetic resonance spectroscopy of the diagnosed subject previously treated with a quantity of compound of formula (I) or of composition according to the invention detectable by said electronic paramagnetic resonance spectroscopy,

[0129] - determine the difference between the healthy subject and the diagnosed subject, and

[0130] - compare this deviation with reference values ​​in order to determine the presence or the absence of the disease in the diagnosed subject.

[0131] The present invention also relates to the compound of formula (I) or the composition according to the invention, for its use in a method of monitoring a disease of a subject, comprising the following steps:

[0132] - at a time t0, capture one or more images by an imaging technique of the subject previously treated with a quantity of compound of formula (I) or composition according to the invention detectable by said imaging technique,

[0133] - at a time tl after time t0, capture one or more images by said technique for imaging the subject previously treated with a quantity of compound of formula (I) or composition according to the invention detectable by said imaging technique,

[0134] - determine the difference between the image(s) captured at time t0 and the image(s) captured at time tl,

[0135] - determine the progression of the disease.

[0136] The present invention also relates to the compound of formula (I) or the composition according to the invention, for its use in a method of monitoring a disease of a subject, comprising the following steps:

[0137] - at a time t0, record at least one signal by para resonance spectroscopy magnetic electron of the subject previously treated with a quantity of compound of formula (I) or of composition according to the invention detectable by said electronic paramagnetic resonance spectroscopy,

[0138] - at a time tl after time t0, record at least one signal per spec electron paramagnetic resonance spectroscopy of the subject previously treated with a quantity of compound of formula (I) or composition according to the invention detectable by said electron paramagnetic resonance spectroscopy,

[0139] - determine the difference between the signal recorded at time t0 and the signal recorded at the instant tl,

[0140] - determine the progression of the disease.

[0141] The present invention also relates to the compound of formula (I) or the composition according to the invention, for its use in a method for determining the effectiveness of a treatment of a disease of a sick subject, comprising the following steps:

[0142] - at a time t0, capture one or more images by an imaging technique of the sick subject previously treated with a quantity of compound of formula (I) or composition according to the invention detectable by said imaging technique, and optionally also treated with an effective quantity of said treatment for a disease,

[0143] - at a time tl after time t0, capture one or more images by said imaging technique of the sick subject previously treated with a quantity of compound of formula (I) or of composition according to the invention detectable by said imaging technique, and optionally also treated with an effective quantity of said treatment of a disease,

[0144] - determine the difference between the image(s) captured at time t0 and the image(s) captured at time tl,

[0145] - determine the effectiveness of the treatment.

[0146] These steps can be repeated several times for monitoring of the treatment over time.

[0147] The present invention also relates to the compound of formula (I) or the composition according to the invention, for its use in a method for determining the effectiveness of a treatment of a disease of a sick subject, comprising the following steps:

[0148] - at a time t0, record at least one signal by para resonance spectroscopy magnetic resonance spectroscopy of the sick subject previously treated with a quantity of compound of formula (I) or of composition according to the invention detectable by said electronic paramagnetic resonance spectroscopy,

[0149] - at a time tl after time t0, record at least one signal per spec electron paramagnetic resonance troscopy of the previously ill subject treated with an amount of compound of formula (I) or composition according to the invention detectable by said electron paramagnetic resonance spectroscopy,

[0150] - determine the difference between the signal(s) captured at time t0 and the signal(s) captured at time tl,

[0151] - determine the effectiveness of the treatment. FIGURES

[0152] [Fig.l] [Fig.l] is an EPR spectrum obtained for the incubation of compound 106 (100 pM) with 5 million U87 cells in serum-free DMEM medium showing a majority component (stars), characteristic of a nitroxide in an isotropic regime, and a minority (arrows), characteristic of a nitroxide in an intermediate regime.

[0153] [Fig.2] [Fig.2] is an EPR spectrum of 5 million U87 cells in suspension incubated with compound 106 (100 pM) with (dotted line) and without Tempone-15N (solid line).

[0154] [Fig.3] [Fig.3] is a representative EPR spectrum obtained in X-band from blood and brain homogenate from a mouse injected with compound 106 (0.5 pmol / g body weight) formulated in an oil-in-water nanoemulsion (106 NE-00 100 mM emulsion) before (solid lines) and after the addition of potassium ferricyanide (dotted lines).

[0155] The present invention will now be described with the aid of the following non-limiting examples. EXAMPLES Example 1: Synthesis of compounds of formula (I)

[0156] Materials and methods

[0157] All reagents were purchased from commercial suppliers and used without purification. Thin layer chromatography (TLC) was performed using silica gel 60 F254 TLC plates and preparative column chromatography was performed using silica gel (230-400 mesh). Dry oxygen-free THF was prepared by a Pure SolvTM micro-solvent purification system (SigmaAldrich). 1H and 13C NMR spectra were recorded on Bruker Biospin Advance II 500 MHz or Bruker ARX 250 250 MHz spectrometers and referenced to the corresponding solvent peak (CDCl3; δH =7.26 ppm, δc =77.0 ppm). Fourier transform infrared (IR) spectra were recorded on a Perkin Elmer FT-IR spectrometer equipped with an ATR objective. High-resolution mass spectra were recorded on a Thermo Exactive_HCD spectrometer (Thermo Scientific, Les Ulis, France).Electron paramagnetic resonance (EPR) spectra were obtained at 21°C on an Elexsys E500 spectrometer (Bruker, Wissembourg, France) operating in X-band (9.8 GHz) and equipped with a . high sensitivity cavity SHQ. The nitrogen coupling constant AN and the half-maximum linewidth AB are given in gauss (G) for a 100 pM nitroxide radical solution in 0.1 M potassium phosphate buffer, pH 7.4, containing 1 mM diethylenetriaminepentaacetic acid (DTPA).

[0158] Synthesis of compound 106 1. 5-Acetoxymethoxycarbonyl-1,1,3,3-tetraethylisoindolin-2-yloxyl radical 91

[0159] Compound 83 was obtained from 4-methylphthalic anhydride following the procedure published by Fairfull-Smith et al (KE Fairfull-Smith, F. Brackmann, SE Bottle, The Synthesis of Novel Isoindoline Nitroxides Bearing Water-Solubilizing Functionality, Eur. J. Org. Chem. (2009) 1902-1915).

[0160] To a solution of compound 83 (250 mg, 0.861 mmol, 1.00 eq.) in dry DMSO (1.60 mL) was added anhydrous potassium carbonate (236 mg, 1.71 mmol, 2.00 eq.). This mixture was stirred at 22 °C for 5 min, after which bromomethyl acetate (149 mg, 95.6 μL, 0.975 mmol, 1.13 eq.) was added. The reaction was stirred at 22 °C for 2 h. A mixture of ice and water was then added and the solution was extracted with dichloromethane. The organic solution was dried over anhydrous sodium sulfate, and evaporated to dryness. The crude product was purified by silica gel column chromatography (cyclohexane / ethyl acetate, 8:1) to give nitroxide 91 as a thick yellow oil (309 mg, 87%).Rf = 0.47 (cyclohexane / ethyl acetate, 4:1), IR vmax (ATR, ZnSe, pure, cm1): 2973, 2940, 2881, 1767, 1738, 1617, 1457, 1418, 1370, 1284, 1259, 1231, 1203, 1159, 1101, 1032, 983, 909, 885, 846, 769, 743; HRMS-ESI: m / z (C20H28NO5) calculated for [M+H]+ 363.2040, found 363.2027, calculated for [M+2H]+ 364.2118, found 364.2110; EPR (100 pM solution in potassium phosphate buffer (0.1 M, pH 7.4, 1 mM DTPA)): An = 15.4 G, AB = 1.7 G.

[0161] 2) 2-(((l-chloroethoxy)carbonyl)oxy)-l,l,3,3-tetraethylisoindoline-5-carboxylate acetoxymethyl 105

[0162] In a septum-sealed flask, compound 91 (259 mg, 0.715 mmol, 1 eq.) and palladium (30 mg, 10% on carbon) were placed and the atmosphere was exchanged with argon. Dry oxygen-free THF (7.0 mL) was added and hydrogen was bubbled through the stirred mixture for 30 minutes and then argon for 5 minutes to remove the hydrogen. After cooling to 0 °C, triethylamine (149.6 μL, 108.5 mg, 1.07 mmol, 1.5 eq.) was added, followed by

[0163] 1-Chloroethyl chloroformate (106 μL, 120 mg, 0.979 mmol, 1.37 eq.) and the resulting mixture was stirred for 1 h at 22 °C. The reaction mixture was filtered through cotton in a Pasteur pipette and concentrated in vacuo. The crude product was purified by silica gel chromatography (dichloromethane / cyclohexane, 2:1) to give compound 105 as a colorless oil (227 mg, 68%). Rf = 0.56 (dichloromethane); *H NMR (CDC13, 500 MHz): ô = 0.77-0.82 (m, 6 H, (Hll)), 0.94-1.01 (m, 6 H, (H11)), 1.73-1.84 (m, 4 H, (H10)), 1.87 (d, J = 5.8 Hz, 3 H, (H26)), 1.91-2.02 (m, 2 H, (H10)), 2.02-2.13 (m, 2 H, (H10)), 2.14 (s, 3 H, (H20)), 6.00 (s, 2 H, (H16)), 6.49 (q, J = 5.8 Hz, 1 H, (H24)), 7.15 (br. d, J = 8.0 Hz, 1 H, (H3)), 7.77 (br.s, 1 H, (H6)), 8.00 (dd, J = 8.0, 1.6 Hz, 1 H, (H2)) ppm; 13C NMR (CDC13, 125 MHz): ô = 169.8 (C18), 165.3 (C13), 154.1 (C21), 147.6 (C5), 142.1 (C4), 129.1 (C2), 128.1 (Cl), 125.4 (C6), 123.9 (C3), 85.0 (C24), 79.8 (C16), 74.7 (C7), 74.5 (C9), 30.2 (CIO), 29.13 (CIO), 29.06 (CIO), 29.00 (CIO), 25.3 (C26), 20.9 (C20), 9.4 (Cil), 8.7 (Cil), 8.6 (Cil) ppm; HRMS-ESI: m / z (C23H32NO7C1) calculated for [M+H]+ 470.1940, found 470.1930, calculated for [M+Na]+ 492.1760, found 492.1748, calculated for [M+K]+ 508.1499, found 508.1486. ​​. 3) 2-(((l-acetoxyethoxy)carbonyl)oxy)-l,l,3,3-tetraethylisoindoline-5-acetoxymethyl carboxylate 106

[0164] Mercury(II) acetate (158 mg, 0.496 mmol, 1.22 eq.) was added to a solution of compound 105 (192 mg, 0.408 mmol, 1.00 eq.) in ethanoic acid (0.73 mL), and the mixture was stirred at 22 °C overnight during which time a fine white precipitate of mercury(II) chloride appeared. Most of the ethanoic acid was removed under reduced pressure and the residue was suspended in diethyl ether and filtered. The ether solution was washed with water, saturated aqueous sodium hydrogen carbonate solution and then water again, dried over anhydrous sodium sulfate, and the solvent was removed in vacuo. The crude product was purified by silica gel chromatography (dichloromethane) to give product 106 a thick colorless oil (164 mg, 82%).Rf = 0,47 (dichlorométhane) ; >H NMR (CDC13, 500 MHz) : ô = 0,75-0,81 (m, 6 H, (H14)), 0,92-1,00 (m, 6 H, (H14)), 1,56 (d, J = 5,5 Hz, 3 H, (H32)), 1,72-1,85 (m, 4 H, (H13)), 1,90-2,02 (m, 2 H, (H13)), 2,03-2,12 (m, 2 H, (H13)), 2,10 (s, 3 H, (H35)), 2,14 (s, 3 H, (H26)), 6,00 (s, 2 H, (H22)), 6,77 (q, J = 5.5 Hz, 1 H, (H30)), 7,15 (d, J = 8,0 Hz, 1 H, (H3)), 7,76 (s, 1 H, (H6)), 7,99 (dd, J = 8,0, 1,6 Hz, 1 H, (H2)) ppm ; 13C NMR . (CDC13, 125 MHz): ô = 169.8 (C24), 169.0 (C33), 165.4 (C19), 154.1 (C27), 147.8 (C5), 142.2 (C4), 129.0 (C2), 128.0 (Cl), 125.4 (C6), 123.9 (C3), 92.2 (C30), 79.8 (C22), 74.6 (C7), 74.4 (C9), 30.1 (C13), 30.2 (C13), 29.0 (C13), 29.1 (C13), 20.9 (C26, C35), 19.6 (C32), 9.4 (C14), 8.6 (C14) ppm; IR vmax (ATR, ZnSe, pure, cm >): 2968, 2942, 1790, 1765, 1740, 1450, 1373, 1283, 1245, 1204, 1159, 1103, 1079, 1032, 1010, 983, 944, 864, 768; HRMS-ESI: m / z (C25H35NO9) calcd for [M+H]+ 494.2385, found 494.2370, calcd for [M+Na]+ 516.2204, found 516.2189, calcd for [M+K]+ 532.1943, found 532.1926.

[0165] Synthesis of compound 108

[0166] 1) l-(((l-chloroethoxy)carbonyl)oxy)-2,2,5,5-tetramethylpyrrolidine-3-carboxylate acetoxymethyl 110

[0167] The radical 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethylpyrrolidin-l-yloxyl 109 (500 mg, 1.94 mmol), synthesized according to published procedures (Hatano, H. Araya, Y. Yoshimura, H. Sato, T. Ito, T. Ogata, T. Kijima, Facile Synthesis of 3-Methoxycarbonyl-2,2,5,5-tetra-methylpyrrolidine-l-oxyl and Derivatives, Hete-rocycles, 81 (2010) 349-356, and JPY Kao, GM Rosen, Esterase-assisted accumulation of 3-carboxy-2,2,5,5-tetramethyll-pyrrolidinyloxyl into lymphocytes, Org. Biomol. Chem. 2 (2004) 99-102), was dissolved in Dry THF (10 mL), treated with palladium on carbon (10%, 50 mg, 47 pmol, 2.5 mol-%), and stirred under a dihydrogen atmosphere (balloon) for 1.5 h at 25°C. The reaction mixture was cooled to 0°C, then triethylamine (540 pL, 3.9 mmol, 2 eq.) and 1-chloroethyl chloroformate (520 pL, 4.8 mmol, 2.5 eq.) were added dropwise while continuing stirring at 0°C for 30 min.The cooling bath was removed and the mixture was stirred overnight at 25°C. The catalyst was removed by filtration through Celite and the solvent was evaporated in vacuo. The residual oil was diluted with water (25 mL) and extracted with ethyl acetate (3 x 25 mL). The combined organic extracts were dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The product was isolated as a colorless oil (640 mg, 91%) after column chromatography on silica gel (cyclohexane / ethyl acetate = 4:1). Rf = 0.40 (cyclohexane / ethyl acetate = 4:1; phosphomo- acid staining. lybdique), 'H NMR (CDC13, 500 MHz, diastéréo-isomère principal ~ 90%) : ô = 1,00-1,15 (s large, 3 H, CH3), 1,15-1,25 (m large, 6 H, 2 x CH3), 1,30-1,35 (s, 3 H, CH 3), 1,76-1,88 (m, 4 H, CHH + C / ÇCHCI), 2,08 (s, 3 H, CHCO). 2,13-2,35 (m large, 1 H, CHH), 2,80-3,05 (m large, 1 H, CH), 5,70 (d, J = 5,2 Hz, 1 H, 0CH / / 0), 5,78 (d, J = 5,2 Hz, 1 H, OCHHO), 6,43 (q, J = 5,7 Hz, 1 H, CfLŒCI) ppm, 13C NMR (CDC13, 125 MHz, l'inversion relativement lente de l'azote divise et élargit certains des signaux) : ô = 17,2 (large, CH3), 20,8 (CH3CO), 22,1 (large, CH3), 25,3 (CHCH3), 25,7 (large, CH3), 28,0 (large, CH3), 31,6 (large, CH3), 37,1 (large, CH2), 38,2 (large, CH2), 47,9 (large, CH), 50,7 (large, CH), 59,7 (large, Cquat), 62,8 (large, Cquat), 64,7 (large, Cquat), 67,3 (large, Cquat), 79,6 (OCH2O), 85,0 (CHCH3), 85,1 (CHCH3), 154,1 (O(C=O)O), 154,2 (O(C=O)O), 169,6 (C=O), 170,9 (C=O) ppm.HRMS-ESI: m / z (C15H24C1NO7) calculated for [M+H]+ 366.1314, found 366.1306, calculated for [M+Na]+ 388.1134, found 388.1124, calculated for [M+K]+ 404.0873, found 404.0863.

[0168] 2) l-(((l-acetoxyethoxy)carbonyl)oxy)-2,2,5,5-tetramethylpyrrolidine-3-carboxylate acetoxymethyl 108 108

[0169] Compound 110 (0.3 g, 0.8 mmol) was dissolved in acetic acid (1.2 mL) and mercury(II) acetate (0.25 mg, 0.8 mmol) was added. The resulting mixture was stirred overnight at 25°C. A fine white precipitate of mercury(II) chloride formed. The reaction mixture was concentrated in vacuo and the residue was suspended in diethyl ether and filtered. The ether solution was washed with water, saturated aqueous sodium hydrogen carbonate solution, and water again, then dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The resulting oil was purified by silica gel column chromatography (cyclohexane / ethyl acetate = 4:1) and the desired product was isolated as a colorless oil (302 mg, 94%).Rf = 0.21 (cyclohexane / ethyl acetate = 4:1; phosphomolybdic acid staining), 1H NMR (CD3OD, 500 MHz, major diastereomer ~ 90%): δ = 1.00-1.36 (m, 12 H, 4 x CH3), 1.51 (m, 3 H, CHCHj), 1.86 (m broad, 1 H, ŒH), 2.07 (s, 6 H, 2 x CH3CO), 2.10-2.35 (m broad, 1 H, CHH), 2.80-3.15 (m broad, 1 H, CH), 5.70 (m, 1 H, OCHHO), 5.82 (m, 1 H, OCH / / O), 6.71 (m, 1 H, C / / CH3) ppm, 13C NMR (CD3OD, 125 MHz, slow nitrogen reversal divides. and broadens some of the signals): ô = 17.5 (broad, CH3), 19.7 (CHCH3), 20.6 (CH3CO), 20.7 (CH3CO), 22.2 (broad, CH3), 26.2 (broad, CH3), 26.4 (broad, CH3), 26.8 (broad, CH3), 28.2 (broad, CH3), 28.9 (broad, CH3), 31.7 (broad, CH3), 37.7 (broad, CH2), 39.1 (broad, CH2), 48.3 (broad, CH), 51.6 (broad, CH), 63.7 (broad, Cquat), 65.5 (broad, Cquat), 67.3 (broad, Cquat), 68.5 (broad, Cquat), 80.8 (OCH2O), 93.3 (CHCH3), 156.2 (O(C=O)O), 170.7 (C=O), 171.2 (C=O), 172.1 (C=O) ppm. HRMS-ESI: m / z (C17H27NO9) calcd for [M+H]+ 390.1759, found 390.1751, calcd for [M+Na]+ 412.1578, found 412.1570, calcd for [M+K]+ 428.1317, found 428.1308.

[0170] Synthesis of compound 112

[0171] 1) Radical 5-(((5-methyl-2-oxo-l,3-dioxol-4-yl)methoxy)carbonyl)-l,l,3,3-tetraethylisoindolin-2-yloxyl 112 DMF. DCM

[0172] The 5-carboxy-1,1,3,3-tetraethylisoindol-2-yloxyl radical 83 (103 mg, 0.35 mmol) was dissolved in dry dichloromethane under an argon atmosphere. Oxalyl chloride (152 μL, 1.75 mmol, 5 eq.) was added dropwise, followed by a 0.15 M solution of anhydrous dimethylformamide in dry dichloromethane (118 μL, 0.02 mmol, 0.05 eq.). The reaction mixture was allowed to react for two hours under argon at 20 °C, and then the mixture was evaporated in vacuo. In a second flask under argon, 4-hydroxymethyl-5-methyl-1,3-dioxol-2-one 111 (33 μL, 0.35 mmol, 1 eq.) was dissolved in dry dichloromethane (1.3 mL). Triethylamine (59 μL, 0.42 mmol, 1.2 eq.) and 4-dimethylaminopyridine (2.2 mg, 0.02 mmol, 0.05 eq.) were added with vigorous stirring. This mixture was transferred to the flask containing the previously synthesized acyl chloride dissolved in dry dichloromethane (1 mL), and the reaction mixture was stirred for 24 h under argon at 20 °C.It was washed with saturated aqueous sodium hydrogen carbonate solution. The organic phase was washed with brine and dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude product. The latter was purified on a flash column with silica gel as the stationary phase and a mixture of cyclohexane and ethyl acetate (4:1) as the elution system. Compound 112 was obtained as a yellow oil in 66% yield over both steps (91 mg). Rf = 0.22 (cyclohexane:ethyl acetate = 4:1); HRMS-ESI m / z (C22H28NO6) calcd for [M+] 402.1911 Da, found 402.1916 Da, calcd for [(M+H)+] 403.1995 . Da, found 403.1957 Da, calculated for [(M+2H)+] 404.2073 Da, found 404.2050 Da; IR vmax (ATR, ZnSe, pure, cm1): 1740 (C=O ester), 1830 (C=O carbonate).

[0173] Example 2: Stability and reactivity of compounds of formula (I) with respect to esterases and comparison with a radical probe precursor of the prior art

[0174] To confirm that the deprotection of the compounds of formula (I) is esterase-dependent, assays in the presence of modified esterase and lipase were performed. The cleavage of the protection was tested by incubating the compounds of formula (I) with esterase or lipase, followed by oxidation of rhydroxylamine released by potassium iron ricyanide and measurement of the EPR signal of the nitroxide produced. Potassium iron ricyanide is a mild oxidant that reacts rapidly with hydroxylamines by accepting an electron. The commercial acetyl-protected, tetramethylated PCR probe was used as a control.

[0175] Preparation of Compound 106 Solution: Two volumes of Compound 106 stock solution in DMSO (50 or 500 mM) were thoroughly mixed with one volume of 20% w / v Pluronic F-127 (Sigma-Aldrich) solution in DMSO. The resulting mixture was diluted in the desired aqueous medium and mixed vigorously by vortexing.

[0176] Incubation with enzymes: 100 μM of compound 106 solution was incubated for 10 minutes at 37°C with porcine liver carboxylesterase (1 U ml1; Sigma E3019; one unit hydrolyzes 1.0 micromole of ethyl butyrate to butyric acid and ethanol per minute at pH 8.0 at 25°C), or with lipase (0.51 mg mL 1; Sigma L3126; 1 mg of enzyme hydrolyzes 25.6 nmol of 4-nitrophenol acetate per minute at pH 7.4 at 25°C in potassium phosphate buffer (0.1 M, pH 7.4, 1 mM DTPA) and then the released hydroxylamine was mixed with 1 μL of 100 mM potassium ferricyanide (1 mM final). The mixture was transferred in a 50 pL glass capillary (Hirschmann) and the nitroxide EPR signal was recorded with a Bruker Elexsys E500 X-band EPR spectrometer (9.86 GHz) at 21 °C, equipped with a high-sensitivity SHQ cavity.Typical parameters used were: microwave power, 10 mW; modulation frequency, 100 kHz; modulation amplitude, 0.14 mT; receiver gain, 60 dB; time constant, 40.96 ms; conversion time, 40.96 ms; 1024 points; scan width, 5 mT; scan time, 41.94 s.

[0177] It was observed that lipase exhibited low activity (as expected with short-chain esters), whereas porcine liver carboxylesterase (EC 3.1.1.1) was more effective.

[0178] Incubation of compound 106 with 1 U mL 1 of porcine liver esterase for 10 minutes at 37 °C, followed by reaction with ferricyanide (1 mM), resulted in the recovery complete nitroxide operation compared to an independently prepared 100 pM solution of compound 83 (authentic isoindoline nitroxide).

[0179] When the ACP compound (comparative probe precursor known from the prior art, acetylated and substituted by four methyl groups) was subjected to the same conditions, only 27% of the corresponding nitroxide was recovered.

[0180] Stability of compound 106: To test the stability of compound 106 in aqueous solution, a 100 μM solution of compound 106 was incubated in potassium phosphate buffer (0.1 M, pH 7.4, 1 mM DTPA) for 24 hours at 21 °C. After addition of potassium ferricyanide, no nitroxide signal was detected, confirming that the protection of tetraethylated hydroxylamine by an (acyloxy)alkyl carbonate is stable against spontaneous hydrolysis at neutral pH under these conditions.

[0181] Example 3: Behavior of compounds of formula (I) on cells

[0182] After the synthesis and initial characterization of the compounds of formula (I), several in vitro tests were carried out in order to answer the following questions:

[0183] 3.1. Are the compounds of formula (I) toxic?

[0184] 3.2. Do compounds of formula (I) accumulate inside the cell?

[0185] 3.3. Is the deprotection of compounds of formula (I) a limiting step towards the oxidation of hydroxylamine?

[0186] 3.4. Do the compounds of formula (I) account for oxidative stress?

[0187] 3.5. Is it possible to detect oxidative stress under more physiological conditions? logical?

[0188] The in vitro model on which these tests were mainly carried out is the U-87 MG cell line.

[0189] Protocol for cell culture of cancer cell lines

[0190] Human brain cells (glioblastoma astrocytoma) U87-MG were cultured in Dulbecco's minimal essential medium (DMEM), 10% fetal calf serum (FCS), 200 U / mL penicillin and 50 mg / mL streptomycin (Invitrogen, Cergy-Pontoise, France) in a humidified atmosphere of 5% CO2. Human breast cancer cell line MCF-7 (ATCC® HTB-22™) cells were cultured at 37°C in DMEM supplemented with non-essential amino acids and supplemented with 10 pg / mL human recombinant insulin, 10% FCS, 200 U / mL penicillin, 50 pg / mL streptomycin (Invitrogen, Cergy-Pontoise, France) and 0.5 pg / mL fungizon in a humidified 5% CO2 atmosphere.

[0191] 3.1. Are the compounds of formula (I) toxic?

[0192] Protocols:

[0193] Treatment of cells for measuring the toxicity of compounds of formula (I)

[0194] U87 cells were seeded in a 96-well plate (10,000 per well) day before the experiment). The culture medium was removed, the cells were washed with PBS (Dulbecco's phosphate-buffered saline pH 7.1-7.5) and then incubated with a series of concentrations of compound 106 solubilized using Pluronic F-127 in DMSO according to the method described previously (from 1 to 500 pM in DMEM without FCS, 100 pL per well) for 6 hours.

[0195] Cell toxicity test

[0196] For fluorescence imaging analysis, the incubation medium was removed and the cells washed once with PBS and then incubated for 30 minutes with FCS-free medium containing Hoechst dye (0.1 pg / mL) and propidium iodide (0.1 mg / mL). Fluorescence was read and analyzed using an ImageXpress® Pico instrument (Molecular Devices).

[0197] Results:

[0198] The following table shows the results of the counting of dead cells after 6 hours of incubation measured by fluorescence imaging with propidium iodide labeling compared to the total number of cells in the wells (Hoechst labeling).

[0199] [Tableauxl] Fluorescence imaging count (propidium iodide labeling) Sample Dead cells (%) Culture medium 16.7 + 5.9 Solubilizing vehicle 15.3 + 2.3 5 pM 13.2 + 0.8 50 pM 12.7 + 3.7 100 pM 13.6+1.6 250 pM 13.7 + 4.9 500 pM 12.8+1.4

[0200] The number of dead cells does not vary significantly, regardless of the concentration of compound 106 of formula (I). These results show that compound 106 is not toxic to U87 cells, even at the highest concentration tested of 500 pM for 6 hours. This is consistent with the incubation conditions commonly used for hydroxylamine probe on cultured cells (50 - 500 pM for 15-60 min).

[0201] 3.2. Do compounds of formula (I) accumulate inside the cell?

[0202] Membrane permeation and cellular accumulation of compound 106 was tested on U87 cells in suspension. Trypsinized cells were incubated with the compound for 30 minutes at 37°C in serum-free DMEM under a 5% CO2 atmosphere (in a water-jacketed incubator). After centrifugation, the cell pellet was thoroughly washed three times with medium, and resuspended in 50 μL of fresh serum-free medium before EPR recording.

[0203] RPE measurement protocol on cells in suspension

[0204] After trypsinization, a suspension of U87 cells was prepared in serum-free medium (10 million cells per mL). In a typical experiment, compound 106 (emulsified in a mixture of Pluronic F-127 and DMSO as in Example 2) was diluted in 500 μL of the cell suspension (5 million cells). The final concentration of compound 106 was typically 100 μM. After incubation (30 min, 37 °C, 5% CO2, with occasional mixing to resuspend the cells), the cells were separated by centrifugation (2000 rpm for 3 min on a miniSpin microcentrifuge, Eppendorf), the incubation medium was removed, and the cells were washed 3 times by resuspension in 500 μL of serum-free medium followed by centrifugation.Finally, the cell pellet was resuspended in 50 μL of serum-free medium optionally containing 15 N-TEMPONE (15-nitrogen-labeled 4-oxo-2,2,6,6-tetramethylpiperidine-l-oxyl radical, 100 μM), placed in a gas-permeable PTFE tube (Extruded Sub-LiteWall®, inner diameter: 0.635 mm, wall thickness: 0.051 mm, Zeus Industrial Products Ltd, Ireland), folded into a W shape, inserted into a 4 mm quartz tube and the EPR spectrum recorded as before (see Example 2). Results :

[0205] Cells incubated with compound 106 showed strong nitroxide EPR signals after washing. The esterase-sensitive acetoxymethyl-masked carboxylic group of compound 106 allows it to cross the membrane and, once inside the cell, to be deprotected by the cell's esterases. Deprotection results in a charged product that can no longer easily cross the membrane and, therefore, accumulates inside the cell. Deprotection of hydroxylamine also takes place, with oxidation of the released hydroxylamine to nitroxide. The experimental EPR spectrum results from the superposition of two components, one major (marked by stars in [Fig.l]), characteristic of a nitroxide in the isotropic regime and compatible with localization in the cytosol, and the other minor (marked by arrows), characteristic of a nitroxide in the intermediate and compatible with a location in the cell membrane.

[0206] 3.3. Is the deprotection of compounds of formula (I) a limiting step? towards the oxidation of hydroxylamine?

[0207] The rate of deprotection of a hydroxylamine probe precursor protected against oxidative stress must be faster than the rate of oxidation.

[0208] The presence of unoxidized hydroxylamine in cells can be tested using 15N-TEMPONE. This method relies on the ability of TEMPONE to freely cross the cell membrane and on the rapid transfer of hydrogen between nitroxides and hydroxylamines. Isotopically labeled 15N-TEMPONE is used because its spectrum has only two lines that do not completely overlap with the signal of the 14N-nitroxide derived from compound 106 (which has three lines), allowing simultaneous measurement of both species.

[0209] For this, 5 million U87 cells in suspension were incubated with compound 106 (100 pM in serum-free DMEM, 30 min at 37 °C, 5% CO2). After the final wash, one part was suspended in serum-free DMEM, while the other part was suspended in serum-free DMEM containing 100 pM 15N-TEMPONE.

[0210] The EPR spectra were acquired according to the protocol described in section 3.2 above. Results:

[0211] The sample of cells treated with 15N-TEMPONE showed a significantly higher signal of 14N-nitroxide derived from compound 106 (4 times more) compared to the untreated sample ([Fig.2]). This experiment showed that a significant amount of free hydroxylamine is present in the cells, indicating that deprotection is not a limiting reaction in the transformation of compound 106 into nitroxide in the cell.

[0212] 3.4. Do the compounds of formula (I) account for oxidative stress?

[0213] To test the compounds of formula (I) under oxidative stress conditions, a cellular model of oxidative stress induction by juglone was chosen. Juglone (5-hydroxy-1,4-naphthalenedione) is a quinone-type natural product that can produce superoxide by futile redox cycling in the cell. Principle:

[0214] Confluent U87 cells were incubated on coverslips with compound 106 (250 pM in serum-free DMEM, 30 min at 37 °C, 5% CO2) with or without juglone (2.5 and 5 pM) before EPR recording. In some cases, cells were preincubated with the potential antioxidants in DMEM containing 10% FCS: SOD-PEG (superoxide dismutase-polyethylene glycol, 100 U mL 1 for 2 h), Cat-PEG (catalase-polyethylene glycol, 100 U mL 1 for 2 h), chelators (sodium diethyldithiocarbamate, DETC, 5 pM and deferoxamine, DFO, 25 pM for 1 h), NAC (N-acetylcysteine, 5 mM for 1 h). The EPR spectrum signal corresponds to the nitroxide generated by incubation of cells with juglone. The signal intensity was normalized to the average protein content of each day's samples. All experiments were performed with DMEM medium containing phenol red, which does not interfere with the measurements.

[0215] Protocol for EPR measurement using hydroxylamine probe precursors on cells grown on glass slides

[0216] U87-MG cells were cultured on round microscope slides (diameter 12 mm, thickness 0.13-0.16 mm, Thermo Scientific Menzel, Braunschweig, Germany) inserted into each well of a 24-well plate. In general, high confluence (cells almost completely covering the surface of the glass on which they grow) was required to obtain sufficient EPR signal intensity. Cells were seeded at least 24 hours before EPR experiments to avoid interference due to cell passage stress. Typically, 56 x 103 U87 cells per well were seeded three days before EPR experiments. (As for MCF-7 cells, they were seeded at a concentration of 4 x 105 cells per well two days before the experiment.) For each RPE measurement, two coverslips were transferred to clean wells of a 24-well plate, each coverslip was washed with 500 μL of FCS-free medium and incubated for 30 minutes (37 °C, 5% CO2) with 300 μL of a 250 μM solution / emulsion of compound 106 or ACP in FCS-free medium containing different concentrations of juglone (prepared from a 100 mM juglone stock in DMSO; aliquots of juglone stock in DMSO can be stored for weeks at -80 °C, but once thawed, they should be used promptly before they turn dark brown; the intermediate solution of juglone in culture medium was prepared freshly for each sample and used within minutes). Since protected hydroxylamine 106 is not soluble in water, a suspension was prepared using the surfactant Pluronic F-127.The stock solution of 106 (10 μL of a 500 mM solution in DMSO) was mixed with a solution of Pluronic F-127 (5 μL of a 20% w / v solution in DMSO). The mixture was dispersed in 85 μL of serum-free medium by vortex mixing, yielding a 50 mM white emulsion that was used for further dilutions during the day. The total maximum final concentration of DMSO in the cell experiments was 0.08% (including the dilution of the juglone stock). When indicated, preincubation with antioxidants (2 h in the case of SOD-PEG (100 U / mL), Cat-PEG (100 U / mL); 1 h for NAC (5 mM), DETC (5 μM), and DFO (25 μM)) was performed in medium with 10% FCS. The coverslips were removed from the wells using a bent-tip needle and tweezers, and excess liquid was carefully removed. with a paper towel and both coverslips were placed on the flat side of a suprasil quartz flat cell (WG-806-AQ, Wilmad) with the cell layer facing towards it (to prevent the sample from drying out). The coverslips were attached to the tissue cell only by capillary forces (caution: if excess liquid is left on the coverslips, they tend to slide down the smooth surface of the EPR cell). The EPR cell was fixed inside the cylindrical mode EPR resonator (EM4103TM, Bruker) with the sample facing towards the experimenter, and EPR recording began immediately.The parameters used were: microwave frequency, 9.81 GHz; microwave power, 10 mW; modulation frequency, 100 kHz; modulation amplitude, 0.14 mT; receiver gain, 60 dB; time constant, 40.96 ms; conversion time, 41.04 ms; 512 points; center field, 346 mT; scan width, 6 mT; scan time, 20.97 s. EPR spectra were recorded sequentially at 21°C for 5 min. Data acquisition and processing were performed using Bruker Xepr software and MathWorks MATLAB software with the Easyspin toolbox. After acquisition, each coverslip was placed in a clean well of a 24-well plate and cells were lysed and collected on the coverslips by RIPA lysis buffer (Thermo Scientific) (50 pL / coverslip) for protein titration using the BCA assay.A paired-observation equality of expectation test was applied on a set of 3 to 5 independent experiments with a critical value p < 0.05 (*) and p < 0.001 (**) compared to the no-juglone condition. Results :

[0217] The results obtained are presented in the following table:

[0218] [Tables2] Juglone concentration (pM) Normalized RPE signal intensity# (AU.mg prot ') Without antioxidant With SOD-PEG With Cat-PEG With chelators With NAC 0 6.6+1.1 5.8 + 0.9 7.2 + 0.7 7.2 + 0.5 6.7 + 1.0 2.5 8.3 + 1.3 * - - - - 5 10.4+1.3 ** 10.8 + 2.2 10.9+1.3 * 12.6 + 2.9 * 12.9 + 2.6 *

[0219] # Mean ± standard deviation obtained for 3 to 5 independent experiments.

[0220] Two concentrations of juglone were tested (2.5 and 5 pM), and an increase dose-dependent increase in the RPE signal was observed. The maximum increase in signal was obtained with 5 pM (~50% compared to untreated cells). These results demonstrate show that compound 106 is indeed a reporter of intracellular oxidative stress. Basal oxidation of compound 106 in untreated cell samples was relatively high, which may suggest that compound 106 is sensitive to intracellular oxidation processes even under basal conditions in cancer cells.

[0221] DETC, a universal "mild" metal ion chelator, and DFO, an iron chelator, are commonly added to incubations with unprotected hydroxylamine probes (such as MHC, 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine) to prevent autoxidation in the buffer. To test whether metal ion chelation is necessary when using compounds of formula (I), cells were preincubated with DFO and DETC. We confirm here that they are not necessary with compound 106 since these compounds do not alter the EPR response. This makes compound 106 simpler to use than unprotected hydroxylamine probes because it is less prone to interference.

[0222] No antioxidant (SOD-PEG, Cat-PEG or NAC) reduced basal oxidation, nor juglone-induced oxidation, which does not allow us to conclude precisely on the chemical species responsible for oxidation in the cells. But interestingly, visual inspection of the cells showed that juglone-induced cell contraction is reversed by NAC treatment. It is known that the antioxidant effect of NAC is mediated by an increase in intracellular glutathione concentration. It is therefore likely that compound 106 is sensitive to oxidative stress processes upstream of glutathione.

[0223] In parallel, the commercially available ACP probe was tested according to the same protocol. Under the conditions tested, no nitroxide signal could be detected with ACP, regardless of juglone treatment. This demonstrates that compound 106 is a better reporter of juglone-induced oxidative stress in cells than ACP under the conditions tested.

[0224] In addition, another cell line (MCF-7 cells) was tested, and gave similar results to those obtained with U87 cells. The dose-dependent effect of juglone was clearly visible, with a maximum signal increase of ~60% with 5 pM juglone.

[0225] 3.5. Is it possible to detect oxidative stress under more physiological conditions? logical?

[0226] It was important to test compound 106 on normal cells, as well as to have a more physiological model of oxidative stress to test the detection threshold. A good model was that of rabbit articular chondrocytes (also adherent cells) induced to express an osteoarthritis-like inflammatory phenotype by the addition interleukin 1 beta (IL-1). IL-1 is a family of cytokines—small proteins important in cell signaling—that play a role in regulating the inflammatory response. Rabbit articular chondrocytes were cultured on microscope slides (see protocol below).

[0227] Chondrocyte harvesting and culture protocol

[0228] All experiments were performed in accordance with ethical guidelines. A 5-week-old New Zealand White rabbit was euthanized by vertebral dislocation followed by strangulation. Articular cartilage was harvested from the femoral and humeral heads, tibial plateau, and femoral condyles. Cartilage was removed from the articular surfaces tangentially and cut into small pieces in a 0.5 mg / mL hyaluronidase solution. The samples were then washed with PBS and placed in a digestion chamber for trypsinization and subsequent collagenase digestion. Chondrocytes were isolated from the collagenase solution by centrifugation.The resulting cells were then seeded at 60,000 cells / cm2 in 2 T75 flasks and cultured in proliferation medium (Ham's Nutrient Mixture F12 medium supplemented with 10% FCS, 1% L-glutamine, and 100 U / mL penicillin and 100 U / mL streptomycin) at 8% CO2 for five days. The medium was then changed to DMEM with 1 g / L D-glucose, 4 mM L-glutamine, and 10% FCS. After two days, cells were passaged, seeded at 250,000 cells / well in 24-well plates on glass coverslips (previously coated with FCS and dried; diameter 12 mm, thickness 0.13-0.16 mm, Thermo Scientific Menzel, Braunschweig, Germany) with 2 mL of complete DMEM and cultured for 3 days at 37 °C under 5% CO2 before the experiment. A secondary culture assay was also performed, for which 80,000 cells per well were seeded on coverslips and cultured for 3 days before the experiment.

[0229] RPE measurement protocol

[0230] RPE recording was performed, as indicated in section 3.4, 1 h, 24 h or 48 h after stimulation with 1TL-1 (1 ng / mL in DMEM without FCS) or medium change to serum-free DMEM. Cells were incubated with compound 106 (250 pM or 500 pM in serum-free DMEM at 37 °C, 5% CO2) for 1 h or 30 min before RPE recording. For 1 h incubation, IL-1 was added together with compound 106, while in the cases of 24 h and 48 h, cells were preincubated with IL-1. Given the small amount of cells available, each condition was tested only once unless otherwise indicated. Results :

[0231] The results obtained are presented in the tables below.

[0232] Primary culture

[0233] [Tables3] Conditions RPE signal intensity (AU) Cell incubation time with compound 106 before recording the RPE spectrum Concentration of compound 106 solution Time between the start of IL-1 stimulation and recording of the RPE signal (if protein present) 0 ng / mL IL-1 protein 1 ng / mL IL-1 protein (variation compared to the control without IL-1) 1h 250 pM 1h 15.8 15.1 (-4%) 30 min 250 pM 24h 6.3 6.6 (+5%) 30 min 250 pM 48h 4.8 1.9 (+65%) 30 min 500 pM 48h 4.5 7.2 (+60%)

[0234] Secondary culture

[0235] [Tables4] Conditions EPR signal intensity (AU) Cell incubation time with compound 106 before recording EPR spectrum Concentration of compound 106 solution Time between start of IL-1 stimulation and recording of EPR signal (if protein present) 0 ng / mL IL-1 protein 1 ng / mL IL-1 protein 1h 250 pM 1h 10.7 10.0 (-7%) 30 min 250 pM 24h 8.4 10.8 (+29%) 30 min 250 pM 48h 6.1 ± 1.1 (n=2) 10.4 ± 0.4 (n=2) (+70%)

[0236] Treatment of chondrocytes with IL-1 rapidly induces the expression of pro-inflammatory proteins through various signaling pathways. A significant amount of nitrite can be detected in the culture medium 12 to 24 hours after IL-1 treatment, indicating an overexpression of iNOS, whereas it is absent under basal conditions. Prolonged incubation in DMEM without FCS likely induces the death of a portion of the cells over time, which explains why the signal obtained with compound 106 without IL-1 stimulation decreases over time, but the RPE signal was not normalized to the amount of protein.Using compound 106, the difference between treated and untreated cells could only be detected at 48 hours on primary cultured cells (Table 3), and from 24 hours, with a more pronounced effect at 48 hours, on secondary cultured cells (Table 4), suggesting that the compound is only sensitive to downstream events and not to the initial stimulus. Although preliminary, these results show that compound 106 can detect part of the response of rabbit chondrocytes to IL-1.

[0237] Example 4: Preparation and characterization of emulsions according to the invention

[0238] Nanoemulsions comprising compound 106 obtained according to Example 1 were prepared as follows: the aqueous phase (composed of ultrapure water with Kolliphor® 188) and the oil phase (Labrafac® or olive oil with Span® 80) were separately heated to 40°C, and then the aqueous phase was added dropwise to the oil phase maintained at 40°C under constant stirring. A 0.1 M aqueous sodium hydroxide solution was used to adjust the pH to 7.0. Isotonicity was adjusted by adding glycerol to a final concentration of 2.5% (w / w). The resulting coarse emulsion was directly introduced into the microfluidizer (LV1, Microfluidics) without homogenization. Microfluidization was carried out at 20 kpsi for 10 cycles. A concentration of 0.1 M and 0.5 M of compound 106 in the oil phase was used to prepare the loaded nanoemulsions, resulting in final concentrations of 20 mM and 100 mM in the nanoemulsions, respectively. Table 5 below summarizes the different emulsions prepared.

[0239] [Tables5] Oil Emulsion Oil phase / aqueous phase mass ratio Oil / surfactant mass ratio Mass ratio Span80:K188 NE—Ll* Labrafac® 20:80 74:26 75:25 NE—L2* Labrafac® 20:80 74:26 63:37 NE—OO* Olive oil 20:80 75:25 80:20 106 NE—Ll Labrafac® 20:80 74:26 75:25 106 NE—L2 Labrafac® 20:80 74:26 63:37 106 NE—OO Olive oil 20:80 75:25 80:20

[0240] Determination of droplet size and size distribution

[0241] The hydrodynamic diameter and polydispersity index (PDI) of the nanoemulsion droplets were determined by photon correlation spectroscopy using a Zetasizer® Nano ZS (Malvem Panalytical, Worcestershire, UK). Approximately 10 qL of each nanoemulsion sample was diluted 100 times with ultrapure water for measurements. All measurements were recorded at 25°C with a scattering angle of 173°. Curve fitting of the correlation functions was performed using an exponential fit (cumulative approach) for the evaluation of the hydrodynamic diameter and PDI. All measurements were performed in triplicate to determine the average droplet size expressed as Z-mean diameter (Z-Ave) and in PDI.

[0242] Determination of zeta potential

[0243] The Zeta potential (ZP) of the nanoemulsions was measured by laser Doppler velocimetry using the Zetasizer® Nano ZS (Malvem Panalytical, Worcestershire, UK). The Smoluchowski approximation was used to determine the electrophoretic mobility required for ZP determination. All measurements were performed in triplicate at 25 °C by diluting 10 μl of each sample to 1000 μl with 20 mM NaCl.

[0244] Table 6 below presents a summary of the hydrodynamics, polydispersity and Zeta potential of the droplets for the blank nanoemulsions (blank NE) and the nanoemulsions loaded with compound 106 at the final concentration of 20 or 100 mM (NE comprising compound 106).

[0245] [Tableauxô] Formulation Span 80: K188 Droplet size (nm) PDI ZP (mV) NE—L1 75:25 127.8 ±1.4 0.107 +0.00 -4.95 + 0.23 NE—L2 63:37 112.3+1.3 0.105 +0.00 -3.37 + 0.26 NE—OO 80:20 166.9+1.0 0.118+0.01 -5.91 + 0.81 106 NE—L1 (20 mM) 75:25 136.9+1.9 0.073 +0.00 -4.17 + 0.55 106 NE—L2 (20 mM) 63:37 126.3 +0.6 0.110+0.02 -3.78 + 0.39 106 NE—OO (20 mM) 80:20 158.6+1.6 0.115+0.01 -5.45 + 0.62 106 NE—OO (100 mM) 80:20 172.6+1.6 0.127 +0.00 -

[0246] Encapsulation efficiency of emulsions

[0247] The encapsulation efficiency of compound 106 in the nanoemulsion was determined by separating the aqueous phase from the formulation by ultrafiltration / centrifugation, using Amicon ultra-0.5 mL 30KD centrifugal filters (Merck Millipore, France). The nanoemulsion was centrifuged at 14,000 g for 30 minutes in a Minispin plus microcentrifuge (Eppendorf, Switzerland). The aqueous phase was then analyzed for compound 106 content using the SHIMADZU LC-20 HPLC system with a Nucleosil 100 Â C18 column (25 cm x 4.6 mm; 5 μm), maintained at 40°C. Mobile phase composed of 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). A gradient from 40%B to 90%B in 30 minutes was used with an injection volume of 50 μl and a flow rate of 1.0 mL / min. UV detection of compound 106 was performed at 254 nm. Compound 106 was diluted in the mobile phase to concentrations ranging from 10 μM to 50 μM and run through the HPLC-UV system to obtain the calibration curve of formula y (AU) = 2474.4*x(pM concentration) + 5717.1 (R2 = 0.9986).

[0248] Using this calibration curve, the concentration of compound 106 in the aqueous phase was estimated and the encapsulation efficiency (EE) determined as shown below:

[0249] [Math.l] EE (%) Total compound 106 in nanoemulsion — compound 106 in aqueous phase Total compound 106 in nanoemulsion X 100

[0250] The probe precursor loading capacity (drug loading = DL) of the nanoemulsion encapsulating the highest concentration of compound 106 (0.5 M in the oil phase) in this study was estimated as follows:

[0251] [Math.2] Mass of compound 106 in the oil phase DL W =----r.---- --------x 100 Total mass of the oil phase

[0252] In all cases, compound 106 was not detected in the aqueous phase (<0.01%), indicating almost complete encapsulation of compound 106 in the oil phase. Neither the type of oil nor the Span 80:Kolliphor 188 ratio had any effect on the retention of compound 106 in the oil phase. The probe precursor loading capacity for the olive oil or Labrafac®-based nanoemulsions encapsulating the highest amount of compound 106 (final concentration 100 mM) in this study was estimated to be approximately 33.33%.

[0253] Stability under storage conditions

[0254] The stability of the nanoemulsions was studied by photon correlation spectroscopy as previously described for the determination of droplet size. The analysis of droplet size and size distribution was carried out at different times (D1, D7, D14, D28, D50) up to 50 days. The samples were stored at 25°C throughout this period. The studies were carried out on two independent samples for each formulation.

[0255] No visible phase separation was observed for any formulation throughout the study period. All formulations demonstrated good stability.

[0256] Example 5: Evaluation of the cytotoxicity of nanoemulsions comprising compound 106

[0257] A dye exclusion assay was used to study the cytotoxicity of compound 106 nanoemulsions on brain endothelial cells (b.End 3). Initially, the cells were cultured in an appropriate medium composed of DMEM medium, 10% FCS, penicillin (100 IU / mL), and streptomycin (100 pg / mL). Incubation was carried out in a humidified environment at 37 °C, with 5% CO2. Subculture to approximately 90% confluence was carried out weekly by 1:10 dilution after detaching the cells with trypsin.

[0258] Then, these cells were seeded into 24-well plates (2.5 x 105 cells / well) and incubated for 24 hours before being treated with the nanoemulsion samples and incubated for another 4 hours. Each well was treated with either nanoemulsions loaded with compound 106 (range 0.1 mM - 5 mM), blank nanoemulsions, or the culture medium as controls. All formulations were diluted in the cell culture medium to prepare the required concentrations. After 4 hours, the media containing the nanoemulsions were removed and the cells were washed with 100 μL of trypsin. Subsequently, they were incubated with 200 μL of 0.25% trypsin / EDTA solution. Finally, harvested cells (20 pL) were mixed with 0.4% trypan blue dye (20 pL) after neutralization of enzymes with complete DMEM.The number of live and dead cells was counted using a hemocytometer under an inverted microscope (Primo Vert, Zeiss). Cells treated with cell culture medium (control) were considered to have 100% viability. Results: .

[0259] [Tables7] Emulsion 106 NE-00 Emulsion 106 NE-L1 Emulsion 106 NE-L2 Medium control 100 + 5.7% 100 + 4.3% 100 + 5.3% Virgin NE 96.4 + 7.5% 96.5 + 4.0% 94.0 + 6.0% Concentration of compound 106 in the nanoemulsion 0.1 mM 93.0 + 7.0% 93.0 + 6.2% 85.5 + 4.7% 0.25 mM 91.1 + 8.3% 83.3 + 5.5% 78.3 + 4.7% 0.5 mM 94.8 + 7.7% - 73.7 + 6.2% 1 mM 89.2+1.6% 84.6 + 6.0% 71.7 + 6.2% 2.5 mM 78.3 + 7.0% 79.3 + 6.3% 69.6 + 5.4% 5 mM 36.2 + 3.4% 50.9 + 6.8% 65.8 + 6.1%

[0260] The evaluation of the cytotoxicity of the nanoemulsions 106 NE-00 and 106 NE-L1 revealed cell viability greater than 80% at concentrations ranging from 0.1 mM to 2.5 mM. For 106 NE-L2 nanoemulsion, cell viability >80% was observed at 0.25 mM and below, but a slightly more significant decrease in cell viability was observed from 0.5 mM. However, a higher percentage of viability was observed at a concentration of 5 mM (69.8%) for this formulation, compared to 106 NE-00 (36.17%) and 106 NE-L1 (50.91%) nanoemulsions.

[0261] Example 6: EPR studies of nanoemulsions comprising compound 106 Protocols:

[0262] All animal experimentation procedures were reviewed by the local Animal Ethics Committee (CEEA 34), in accordance with French regulations and the Council of the European Communities Directive on the protection of animals used for scientific purposes. The project was authorized by the French Ministry of Higher Education and Research under reference number APAFIS#4594-2015092117546157 v9. The experiments were performed on healthy male BALB / c Ola Hsd mice (15-21 g, 4 weeks old, Envigo) previously adapted to the laboratory for seven days with free access to water and laboratory food.

[0263] In vivo EPR studies of nanoemulsions comprising compound 106

[0264] Compound 106-loaded nanoemulsions were injected into the tail vein of BALB / c mice using a 26 G needle (0.45x13 mm). The injection volume was 5 pL / g body mass, in accordance with Good Animal Practice guidelines. The injected dose was 0.1 - 0.5 pmol of compound per g body mass of the mice, with two mice injected per group. The nanoemulsions were prepared with final concentrations of compound 106 of 20 and 100 mM. After injection, the mice were anesthetized by isoflurane inhalation. Then, the heads were placed in the EPR spectrometer cavity using a modified mouse holder. During recording, the mice were under anesthesia (2-3% isoflurane) through a face mask. The body temperature of the mice was maintained between 32 and 37 °C. Respiratory rate was monitored during the experiment.RPE signal acquisition began approximately 4 minutes after injection.

[0265] EPR measurements were performed using a Bruker Elexsys 540 EPR spectrometer (Bruker, Wissembourg, France) operating in L-band (1.2 GHz) with a BLGR_23 cavity. The parameters used for EPR spectroscopy were: microwave power, 10 mW; modulation frequency, 100 kHz; modulation amplitude, 0.17 mT; receiver gain, 60 dB; time constant, 81.92 ms; conversion time, 81.92 ms; 512 points; center of field, 39.52 mT; scan width, 19 mT; scan time, 41.98 s. The spectra were recorded sequentially as a function of time for approximately 100 minutes. Bruker Xepr software was used for data acquisition and processing.

[0266] Ex vivo EPR studies of nanoemulsions comprising compound 106

[0267] An ex vivo study was conducted to evaluate the NE distribution of compound 106 and confirm the intracellular cleavage of compound 106 by esterases in vivo. Mice injected with the nanoemulsions were euthanized approximately 100 min after intravenous injection. Deep anesthesia was performed with ketamine (100 mg / kg) / xylazine (10 mg / kg) injected into the peritoneum. Whole blood samples were collected from the left ventricle, stored in heparinized tubes, and frozen in liquid nitrogen. Blood replacement by cardiac perfusion of the mice was performed with 10 to 15 mL of saline containing 25 U / mL of heparin. The major organs, including the brain, heart, liver, spleen, lungs, and kidneys, were then removed, weighed, and frozen in liquid nitrogen at -80°C for ex vivo EPR analysis.

[0268] Homogenates of each organ were prepared by mixing them with saline (0.1 g wet tissue / 0.1 mL saline) and disintegrating them using a potter (Dounce homogenizer). After vortexing, the samples (30 pL) were transferred to a 5 cm long piece of gas-permeable PTFE tubing (Extruded Sub-Lite-Wall, inner diameter 0.635 mm, wall thickness 0.051 mm; Zeus Industrial Products Ltd., Ireland) which was folded (V-shaped) into a 4 mm quartz tube and then placed in the EPR cavity. The blood was not diluted before recording. Spectra were recorded with an Elexsys E500 X-band (9.8 GHz) EPR spectrometer at 21 °C and equipped with a high-sensitivity SHQ cavity.The parameters used were: microwave power, 1 mW; modulation frequency, 100 kHz; modulation amplitude, 0.1 mT; receiver gain, 60 dB; time constant, 40.96 ms; conversion time, 40.96 ms; 1024 points; magnetic field center, 350 mT; scan width, 8 mT; scan time, 41.94 s.

[0269] 100 mM potassium ferricyanide was added gradually (0.2-1 μL) to the samples for subsequent EPR analysis. Potassium ferricyanide is a mild oxidant whose addition to the compound should oxidize the hydroxylamine released by compound 106 to the nitroxide visible by EPR. Results :

[0270] No acute toxicity was observed during in vivo experiments.

[0271] As mentioned previously, the probe from compound 106 is an EPR-invisible probe that is cleaved intracellularly by esterase to free hydroxylamine. The resulting hydroxylamine reacts under oxidative stress conditions to produce an EPR-visible nitroxide radical. No signal is expected. in a healthy system since the oxidant level is very low and there will be little or no reaction with free hydroxylamine. As expected, no EPR signal was observed in the head of mice after injection of compound 106 NEs for the entire recording time, i.e., 100 min. Neither the concentration of compound 106 in the oil phase nor the type of oil used to formulate the nanoemulsions affected their in vivo stability in healthy mice.

[0272] The in vivo results suggest that the probe from compound 106 was not significantly oxidized to the nitroxide form in healthy mice. However, the sensitivity of the L-band EPR spectrometer is lower than the sensitivity of the X-band spectrometer, meaning that X-band analysis of ex vivo samples allows quantification of the amount of probe that reaches the different tissues and is cleaved and / or oxidized. First, an EPR acquisition of the organ homogenates was performed. Then, potassium ferricyanide [K3(FeCN6)] was added gradually to recover the total amount of cleaved probe 106 precursor distributed to each organ. Potassium ferricyanide oxidizes the free hydroxylamine product of probe 106 to a nitroxide visible by EPR.For each organ, the addition of the oxidant led to an increase in the intensity of the EPR signal, thus indicating the sensitivity of the formulated probes to oxidizing species.

[0273] The results obtained (mean ± standard deviation) are shown in the following Table 8:

[0274] [Tables8] Concentration (pM) Emulsion 106 NE-00 (20 mM) (0.1 pmol / g body weight) Emulsion 106 NE-00 (100 mM) (0.5 pmol / g body weight) Emulsion 106 NE-L1 (100 mM) (0.5 pmol / g body weight) As isolated After oxidation As isolated After oxidation As isolated After oxidation Blood 0.47 + 0.47 0.99 + 0.16 7.44 + 3.55 12.4 + 0.34 1.47 + 0.06 1.82 + 0.12 Heart 0.60 + 0.60 0.60 + 0.60 7.54 + 2.40 8.38 + 3.12 1.94 + 0.66 2.53 + 0.73 Liver 3.87 + 0.65 9.05 + 0.65 27.9 + 4.3 70.1 + 10.4 16.5 + 9.3 28.3 + 11.4 Spleen 0.60 + 0.60 0.88 + 0.88 6.60 + 0.90 7.90 + 1.22 2.65 + 0.07 3.45 + 0.53 Kidneys 1.36 + 1.36 1.51 + 1.51 19.9 + 4.9 26.8 + 7.1 4.17 + 1.15 5.47 + 1.09 Lungs 0 + 0 0.83 + 0.83 9.84 + 1.06 11.1 + 2.05 14.8 + 11.8 27.7 + 24.2 Brain 0.82 + 0.82 0.96 + 0.96 4.53 + 0.75 6.89 + 2.73 1.95 + 0.33 2.30 + 0.60

[0275] An EPR signal was observed in different organs, indicating the successful uptake of compound 106. As expected, an increase in intensity was noticed when the dose of the probe precursor increased from 0.1 to 0.5 pmol / g body weight using olive oil-based formulations. In addition, the EPR evaluation revealed a higher accumulation of compound 106 in the liver compared to other organs. The preferential accumulation of the probe precursor in the liver was observed for both concentrations tested.

[0276] At the highest dose of compound 106 (0.5 pmol / g body weight) in an olive oil-based nanoemulsion with Span8O:K188 = 80:20 (emulsion 106 NE-OO), the EPR spectrum of the blood sample indicates the presence of a free nitroxide in an aqueous environment characterized by a nitrogen coupling constant of 1.56 mT (stars, [Fig. 3]) and a nitroxide in a more lipophilic environment such as cell membranes (arrows, [Fig. 3]). Traces of protein-bound nitroxide were detected after the addition of ferricyanide (triangle, [Fig. 3]). A species unidentified six-line species (AN = 1.58 mT, AH = 2.36 mT), appeared upon oxidation in blood (diamonds, [Fig. 3]). The unidentified six-line species was not observed in the EPR spectra of other organs. The distribution of nitroxide in organs before and after ferricyanide treatment is shown in Table 7. A significant proportion of free hydroxylamine is observed only in the liver, whereas most of the detected probe precursor is oxidized to nitroxide in other organs.

Claims

Claims

1. Compound of formula (I): in which n = 1 or 2, A and A' are independently selected from the group consisting of the following substituents (II-a) and (II-b): R and q, R and R' being independently K / :lat° R' Z (H_a) Ohb) chosen from H and linear or branched C1-C4 alkyls, Z being chosen from a linear or branched C1-C4 alkyl group and an aryl group optionally substituted by a linear or branched C1-C4 alkyl group and / or by a methoxyl group, and the unit of formula (N) X > is chosen from the units < N—OJ- (N) (Ill-a), (Ill-b), (III-c), (Ill-d) and (Ill-e) following: , Ra, Rb, Rc and Rd

2.

3.

4. being, for each unit (III-a), (III-b), (III-c), (III-d) and (III-e), independently chosen from linear or branched C1-C4 alkyls. Compound according to claim 1, in which A is selected from the group consisting of the substituent (II-b) in which Z = Me and the substituent (II-a) in which R = H, and A' is selected from the group consisting of the substituent (II-b) in which Z = Me and the substituent (II-a) in which R = Me. A compound according to claim 1 or 2, wherein R' is methyl or ethyl, preferably methyl. A compound according to any one of claims 1 to 3, wherein the compound of formula (I) is selected from the following compounds (Ia), (Ib), (Ib”), (Ic), (Id) and (Ie): (THE)

5. A compound according to any one of claims 1 to 4, wherein the unit of formula (N) is selected from units (III-b), (III-c), (III-d) and (III-e).

6. A compound according to any one of claims 1 to 5, wherein Ra, Rb, Rc and Rd are the same, and selected from methyl and ethyl.

7. A compound according to any one of claims 1 to 6, selected from: o and CH, CH, H3C CH, H ? / r “ow AA^.A vh,coo ?r \ / N—° / ^ch3 h3c o h3c \^ch3 Il || H3C CH3 0 y—0 H,C^ 'A 0 N—0 ch3

8. Pharmaceutical composition, comprising in a physiologically acceptable medium, at least the compound of formula (I) according to any one of claims 1 to 7.

9. Pharmaceutical composition according to claim 8, characterized in that it is in the form of a nanoemulsion, preferably an oil-in-water nanoemulsion.

10. Pharmaceutical composition according to claim 9, wherein the nanoemulsion comprises an aqueous phase and an oily phase, the oily phase comprising the compound of formula (I) and further comprising at least one oily compound comprising a triglyceride, preferably a C4-C24 triglyceride, preferentially C8-C18.

11. Pharmaceutical composition according to any one of claims 8 to 10, further comprising at least one non-ionic surfactant, preferably chosen from organosilicon surfactants, polysorbate, cetostearyl alcohol, cetyl alcohol, oleyl alcohol, stearyl alcohol, cocamide DEA, cocamide MEA, polyalkylglucoside, decyl glucoside, lauryl glucoside, octyl glucoside, monolaurin, poloxamer, sorbitan monostearate, sorbitan tristearate, or any combination thereof, preferably among poloxamers and sorbitan monostearate.

12. Use of the compound of formula (I) according to any one of claims 1 to 7 or of the pharmaceutical composition according to any one of claims 8 to 11, as a precursor of compounds for electron paramagnetic resonance spectroscopy or for imaging, in particular for electron paramagnetic resonance spectroscopy, for electron paramagnetic resonance imaging or for magnetic resonance imaging.

13. A method for in vitro detection of oxidative stress, comprising the following steps: - contacting the compound of formula (I) according to any one of claims 1 to 7, or the composition according to any one of claims 8 to 11, with a medium comprising cells or biological tissue, and - recording the signal of the medium by electron paramagnetic resonance spectroscopy, or capturing one or more images of the medium by an imaging technique, preferably by electron paramagnetic resonance imaging or by magnetic resonance imaging.

14. Method for in vivo detection of oxidative stress in a subject, comprising: - either a step of capturing one or more images of a subject or at least part of a subject previously treated with the compound of formula (I) according to any one of claims 1 to 7, or with the composition according to any one of claims 8 to 11, by an imaging technique, preferably by electron paramagnetic resonance imaging or by magnetic resonance imaging, - or a step of recording the signal of a subject or at least part of a subject previously treated with the compound of formula (I) according to any one of claims 1 to 7, or with the composition according to any one of claims 8 to 11, by electron paramagnetic resonance spectroscopy.

15. A compound of formula (I) as defined in any one of claims 1 to 7 or a composition according to any one of claims 8 to 11, for use as a diagnostic tool.

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