Piperidine, pyrrolidine and isoindole precursors of probes for oxidative stress

Abstract

The present invention relates to a compound of formula (I), wherein n = 1 or 2, and A and A' are independently chosen from among the group consisting of the following substituents (ll-a) and (ll-b), R and R' being independently chosen from among H and C1-C3 linear or branched alkyls, Z being chosen from among a C1-C4 linear or branched alkyl group and an aryl group optionally substituted with a C1-C3 linear or branched alkyl group or / and with a hydroxyl group, and the unit of formula (N) is chosen from among the following units (III-a), (III-b), (III-c), (III-d) and (III-e), Ra, Rb, Rc and Rd being, for each unit (lll-a), (lll-b), (lll-c), (lll-d) and (lll-e), independently chosen from among C1-C4 linear or branched alkyls.

Description

[0001] DESCRIPTION

[0002] PIPERIDINE, PYRROLIDINE, AND ISOINDOLE PRECURSORS OF PROBES FOR OXIDATIVE STRESS

[0003] The present invention relates to novel precursors of probes for oxidative stress, and their various uses.

[0004] 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 for cardiovascular and neurological pathologies or cancer. It is increasingly clear that oxidative stress has a major impact on the progression of all these diseases. A non-invasive method for detecting and mapping oxidative stress in vivo would therefore provide valuable information on the development of these pathologies and would aid in the design of diagnostic and treatment tools.

[0005] Protein and lipid oxidation biomarkers in biological fluids are valuable tools but do not allow pathology to be localized. 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 some limitations. PET requires expensive and short-lived radioactive tracers and access to a local cyclotron for their production, and is not compatible with some pathologies. Magnetic susceptibility imaging (SWI) assesses tissue metal content (Cu, non-heme iron) as potential sources of reactive oxygen species (ROS) by Fenton reaction, but many regions accumulate iron (e.g., in ferritin) without causing 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.

[0006] Minimally invasive methods using electron paramagnetic resonance (EPR) and exogenous molecular probes have already demonstrated their utility in assessing and mapping the "redox status" in the brain under conditions of oxidative stress, for example in rat models of 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, Circ. Res. 119 (2016) e39-e75. doi: 10.1161 / RES.0000000000000110).

[0007] Among the already known molecular probes, we distinguish probes of the hydroxylamine family, which are oxidized in cells to nitroxide which is detectable by EPR.

[0008] In particular, it is known to use precursors of these probes, such as the following compound 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP):

[0009] This compound includes a protective ester function for the hydroxylamine group. The objective of these precursors is to deliver the probe into cells with the least possible degradation. Masking the hydroxylamine function limits spontaneous oxidation of hydroxylamines before internalization into cells, which facilitates manipulation by non-chemist experimenters. Once internalized, the ester functions are hydrolyzed by the cell's esterases, releasing the hydroxylamine moiety, which can be oxidized to nitroxide and become detectable in EPR.

[0010] 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.

[0011] 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 hampering the development of redox EPR applications.

[0012] The aim of the invention is therefore to propose new molecular probes for oxidative stress, presenting improved performances compared to already existing probes.

[0013] 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. To this end, the invention relates to a compound of formula (I): in which n = 1 or 2, preferably n = 1,

[0014] A and A' are independently selected from the group consisting of the following substituents (ll-a) and (ll-b): , R and R' being independently chosen from

[0015] 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 (such as 4-methoxyphenyl), and the motif of formula (N) is chosen from the motifs (lll-a), (lll-b), (lll-c),

[0016] (lll-d) and (lll-e) following:

[0017]

[0018] (lll-e)

[0019] Ra, Rb, Rc and Rd being, for each unit (lll-a), (lll-b), (lll-c), (lll-d) and (lll-e), independently chosen from linear or branched C1-C4 alkyls.

[0020] 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.

[0021] Indeed, the inventors have surprisingly discovered that the compounds of formula (I) according to the invention are probe precursors 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 probe precursors of the prior art induce steric hindrance which prevents the cell's esterases from hydrolyzing the ester protecting the hydroxylamine function. Indeed, since the deprotection kinetics are low and the reaction is 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 substituent (ll-a) with R=R'=CH3, 100% of hydroxylamine is released in 10 min at 37°C vs. a deprotection of the commercial probe ACP (1-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), a more efficient release of the probe, which leads to a gain in sensitivity.

[0022] In formula (I), and except in the case where the motif of formula (N) is the motif of formula (lll-e), a person skilled in the art will understand that each group can be linked to any substitutable carbon atom (carrying at least one hydrogen atom) constituting the ring(s) of the unit of formula (N) of formula (lll-a), (lll-b), (lll-c) and (lll-d).

[0023] The presence of the A group in the compound of formula (I) advantageously improves 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 A groups 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.

[0024] It is understood that each atom of the compound of formula (I) may be present in the form of any of its naturally occurring isotopes.

[0025] According to a particular embodiment, the nitrogen atom of the unit of formula N is a nitrogen 15.

[0026] 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).

[0027] The isotopic composition of the compound of formula (I), and in particular its nitrogen-15 and / or hydrogen-2 content, is determined using techniques known to those skilled in the art. For example, high-resolution mass spectrometry (HRMS) makes it possible to determine the average concentration of the isotope studied over all the sites of the compound of formula (I). Quantitative nuclear magnetic resonance (NMR) of deuterium and nitrogen-15 can also be used, and makes it possible to 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).Preferably, in substituent (ll-b), Z is selected from methyl, ethyl, propyl, isopropyl, tert-butyl, phenyl and 4-methoxyphenyl. Advantageously, for the remainder of the description, Z = Me.

[0028] Preferably, A is selected from the group consisting of substituent (ll-b) wherein Z = Me and substituent (ll-a) wherein R = H, and A' is selected from the group consisting of substituent (ll-b) wherein Z = Me and substituent (ll-a) wherein R = Me.

[0029] Preferably, R' is methyl or ethyl, preferably methyl.

[0030] Preferably, A and A' are identical.

[0031] According to some embodiments, A = A' = (ll-a).

[0032] According to other embodiments, A = A' = (ll-b).

[0033] The use of (ll-b) groups advantageously avoids the release of an equivalent of acetaldehyde and an equivalent of formaldehyde after the action of the esterase, which could induce a potential toxicity problem 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 (ll-b) group only results in the formation of 3-hydroxybutan-2-one and 2,3-butanediol, which are non-toxic.

[0034] Preferably, the unit of formula (N) is chosen from the units (lll-b), (lll-c), (lll-d) and (lll-e), preferentially from the units (lll-b), (lll-c) and (lll-d), more preferentially (lll-b) and (lll-d), or alternatively (lll-c).

[0035] Preferably, the compound of formula (I) is chosen from the following compounds (la), (lb), (lb”), (lc), (lc”), (ld), (l-d'), (ld”), (l-d'”) and (le):

[0036]

[0037]

[0038] A, A', Ra, Rb, Rc and Rd being, for each compound (la), (lb), (lb”), (lc), (lc”), (ld), (I- d'), (ld”), (l-d'”) and (le), each independently as defined above.

[0039] Preferably, the compound of formula (I) is chosen from compounds (Ia), (Ib), (Ib”), (Ic), (Id) and (Ic), preferentially from compounds (Ia), (Ib), (Ib”), (Ic) and (I-d), more preferentially from compounds (Ib), (Ic) and (Id), even more preferentially (Ib) and (Id), or alternatively (Ic). More preferentially, the compound of formula (I) is chosen from compounds (IV),

[0040] (V), (VI) and (VII) following,

[0041]

[0042] A and A' being as defined above.

[0043] Preferably, the compound of formula (I) is chosen from compounds (IV), (V) or alternatively from compounds (VI).

[0044] Preferably, Ra, Rb, Rc and Rd are independently selected from methyl, ethyl and isopropyl, preferably from methyl and ethyl. Preferably, Ra, Rb, Rc and Rd are the same, and are preferably selected from methyl and ethyl.

[0045] 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.

[0046] According to a particular embodiment, the compound of formula (I) is chosen from:

[0047]

[0048] According to one embodiment, the compound of formula (I) is the compound

[0049] According to another embodiment, the compound of formula (I) is the compound

[0050] According to an alternative embodiment, the compound of formula (I) is different from the following compound:

[0051] 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.

[0052] A typical method for preparing compounds of formula (I) according to the invention comprises at least one step (S1) of reducing the compound of formula (X) to hydroxylamine. for example in the presence of dihydrogen, to obtain a compound of formula (X') then a step (ST) of nucleophilic addition of the compound (X') to a compound of formula (XI), or of formula (XI') to give a compound of formula (XII) or of formula (XII'), respectively

[0053] A, A', n, R, and the unit of formula (N) being as defined above for the compounds of formula (I).

[0054] 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 (ST).

[0055] Preferably, step (ST) 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).

[0056] 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.

[0057] 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).

[0058] The method may further comprise a step (S0) of nucleophilic substitution between a compound of formula (XIV) (XIV) and a compound of formula (XV) to obtain a compound of formula (X),

[0059] 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 = (ll-a).

[0060] Preferably, step (SO) 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 h at room temperature (20-28°C).

[0061] Alternatively, the method may further comprise a step (SO') of activating the compound of formula (XIV) into an acyl chloride of formula (XIV') followed by a coupling step (SO”) between compound (XIV') and a compound (XV')

[0062] A— OH

[0063] (XV) 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 = (ll-b).

[0064] The activation step (SO') is preferably carried out in the presence of an acyl dichloride, for example oxalyl chloride and dimethylformamide.

[0065] Step (SO”) 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).

[0066] Any alternative method well known in the prior art may be used.

[0067] 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.

[0068] Preferably, the pharmaceutical composition according to the invention is in the form of a nanoemulsion, preferably an oil-in-water nanoemulsion.

[0069] 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 comprised in the oily phase.

[0070] Preferably, the oily phase further comprises at least one oily compound comprising a triglyceride, preferably a C4-C24 triglyceride, preferably a C8-C18 triglyceride. 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.

[0071] Preferably, the oily compound is chosen from vegetable oils and medium chain triglycerides in C6-C12, preferably in C8-C10.

[0072] 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.

[0073] Advantageously, the oily compound of the oily phase is chosen from olive oil and lipophilic Labrafac® WL 1349.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] Advantageously, the first surfactant is sorbitan monostearate and the second surfactant is chosen from poloxamers, preferably poloxamer 188. An example of sorbitan monostearate is the commercial product Span® 80. An example of poloxamer is the commercial product Kolliphor® P 188.

[0078] 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%.

[0079] 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%.

[0080] 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%.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] Preferably, the nanoemulsion has a droplet size distribution ranging from 0.05 to 0.20, preferably ranging from 0.07 to 0.15.

[0086] The average droplet size was determined by photon correlation spectroscopy using a Zetasizer® Nano ZS (Malvern Panalytical, Worcestershire, UK). 10 pL of nanoemulsion was diluted 100 times with ultrapure water. 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-average diameter (Z-Ave) and PDI.

[0087] 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. 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] According to another embodiment, the compound of formula (I) or the composition, preferably the nanoemulsion, according to the invention are used as precursors of contrast agent for magnetic resonance imaging.

[0092] 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 motif.

[0093] This hydroxylamine unit can be oxidized under the action of oxidative stress of the cell, becoming a nitroxide radical, directly detectable by spectroscopy or electron paramagnetic resonance imaging. The paramagnetic nitroxide radical can also cause a modification of the longitudinal (Ti) and / or transverse (T2) magnetic relaxation time of nearby water protons, or even of aliphatic protons in tissues, leading to a variation in contrast in magnetic resonance imaging.

[0094] Preferably, the use of the compound of formula (I) or of the composition according to the invention is in vivo or in vitro.

[0095] According to one embodiment, the use of the compound of formula (I) or the composition according to the invention is in vitro.

[0096] The present invention also relates to a method for in vitro detection of oxidative stress, comprising the following steps: - bringing the compound of formula (I) or the composition according to the invention into contact with a medium comprising cells, a fluid or a biological tissue, and

[0097] - record the signal of the medium by electron paramagnetic resonance spectroscopy, or capture one or more images of the medium by an imaging technique, preferably medical or pre-clinical, preferably by electron paramagnetic resonance imaging or by magnetic resonance imaging.

[0098] The present invention also relates to a method for in vivo detection of oxidative stress in a subject, comprising:

[0099] - 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 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.

[0100] - 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.

[0101] Preferably, the subject is treated with an amount detectable by said imaging technique or by said electron paramagnetic resonance spectroscopy.

[0102] 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.

[0103] Preferably, the method for in vivo detection of oxidative stress in a subject comprises the following steps:

[0104] - administering to the subject a quantity of compound of formula (I) or of the composition according to the invention detectable by said imaging technique,

[0105] - 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 by magnetic resonance imaging or recording the signal of at least part of the subject by electron paramagnetic resonance spectroscopy. 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] Preferably, the in vivo diagnostic method comprises:

[0111] - either a step of capturing images by an imaging technique of a subject or at least part of a subject previously treated with a quantity of compound of formula (I) or of 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,

[0112] - either a step of recording by electronic paramagnetic resonance spectroscopy the signal of a subject or at least part of a 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.

[0113] 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. 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.

[0114] 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.

[0115] 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:

[0116] - 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 imaging technique,

[0117] - 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,

[0118] - determine the gap between the healthy subject and the diagnosed subject, and

[0119] - compare this deviation with reference values ​​in order to determine the presence or absence of the disease in the diagnosed subject.

[0120] 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:

[0121] - recording a signal by electronic paramagnetic resonance spectroscopy of a healthy subject previously treated with a quantity of compound of formula (I) or composition according to the invention detectable by said electronic paramagnetic resonance spectroscopy,

[0122] - recording a signal by electron paramagnetic resonance spectroscopy of the diagnosed subject previously treated with a quantity of compound of formula (I) or composition according to the invention detectable by said electron paramagnetic resonance spectroscopy,

[0123] - determine the gap between the healthy subject and the diagnosed subject, and

[0124] - comparing this deviation with reference values ​​in order to determine the presence or absence of the disease in the diagnosed subject. 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 monitoring a disease of a subject, comprising the following steps:

[0125] - at a time t0, capturing one or more images by an imaging technique of the subject previously treated with a quantity of compound of formula (I) or of composition according to the invention detectable by said imaging technique,

[0126] - at a time t1 after time t0, capturing one or more images by said 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,

[0127] - determine the difference between the image(s) captured at time t0 and the image(s) captured at time t1,

[0128] - determine the progression of the disease.

[0129] 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:

[0130] - at a time t0, record at least one signal by electronic paramagnetic resonance spectroscopy 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,

[0131] - at a time t1 after time t0, record at least one signal by electronic paramagnetic resonance spectroscopy 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,

[0132] - determine the difference between the signal recorded at time t0 and the signal recorded at time t1,

[0133] - determine the progression of the disease.

[0134] 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:

[0135] - at a time t0, capturing 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 of a disease,

[0136] - at a time t1 after time t0, capturing one or more images by said 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,

[0137] - determine the difference between the image(s) captured at time t0 and the image(s) captured at time t1,

[0138] - determine the effectiveness of the treatment.

[0139] These steps can be repeated several times for monitoring of the treatment over time.

[0140] 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:

[0141] - at a time t0, record at least one signal by electronic paramagnetic 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,

[0142] - at a time t1 after time t0, record at least one signal by electronic paramagnetic 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,

[0143] - determine the difference between the signal(s) captured at time t0 and the signal(s) captured at time t1,

[0144] - determine the effectiveness of the treatment.

[0145] Brief description of the figures

[0146] [Fig 1] Figure 1 is an EPR spectrum obtained for the incubation of compound 1 (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. [Fig 2] Figure 2 is an EPR spectrum of 5 million U87 cells in suspension incubated with compound 1 (100 pM) with (dotted lines) and without Tempone- 15 N (solid line).

[0147] [Fig 3] Figure 3 is a representative X-band EPR spectrum obtained from blood and brain homogenate of a mouse injected with compound 1 (0.5 pmol / g body weight) formulated in an oil-in-water nanoemulsion (100 mM NE-00 emulsion 1) before (solid lines) and after the addition of potassium ferricyanide (dotted lines).

[0148] The present invention will now be described with the aid of the following non-limiting examples.

[0149] EXAMPLES

[0150] Example 1: Synthesis of compounds of formula (I)

[0151] Materials and methods

[0152] 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). Oxygen-free dry THF was prepared by a Pure SolvTM micro-solvent purification system (SigmaAldrich). NMR spectra 1 H and 13C spectrometers were recorded on Bruker Biospin Advance II 500 MHz or Bruker ARX 250 250 MHz spectrometers and referenced to the corresponding solvent peak (CDCh; 5H = 7.26 ppm, 5c = 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) spectrometers were obtained at 21°C on an Elexsys E500 spectrometer (Bruker, Wissembourg, France) operating at X-band (9.8 GHz) and equipped with a high-sensitivity SHQ cavity. The nitrogen coupling constant AN and the half-maximum linewidth AB are given in millitesla (mT) for a 100 pM nitroxide radical solution in 0.1 M potassium phosphate buffer, pH 7.4, containing or not 1 mM diethylenetriaminepentaacetic acid (DTPA).The melting temperature of the solid products was measured with a Stuart® SMP 20 apparatus (Cole-Palmer®).

[0153] General Procedure A

[0154] To a solution of the carboxylic acid derivative (1 equiv.) in dry DMSO ([C]=0.4 M) was added anhydrous potassium carbonate (2 equiv.). This mixture was stirred at 22 °C for 5 min, after which bromomethyl acetate (1.2 - 1.5 equiv.) was added. The reaction was kept stirring at 22 °C for 2 h. A mixture of ice and water was then added and the solution was extracted with dichloromethane. The organic phase was dried over anhydrous magnesium sulfate and evaporated to dryness. The crude product was purified by silica gel column chromatography with the elution conditions indicated for each case, to yield the desired compound.

[0155] General Procedure B

[0156] In a septum-sealed flask, the nitroxide derivative (1 equiv.) and palladium (10% on carbon) were placed and the atmosphere was purged by 3 vacuum / argon phases. Under an argon atmosphere, dry oxygen-free THF ([C]=0.1 M) was introduced and dihydrogen was bubbled into the mixture while stirring for 30 minutes. To evacuate the dihydrogen, argon was bubbled for 5 minutes. After cooling to 0 °C, triethylamine (1.5-2.5 equiv.) was added, followed by the chloroformate derivative (1.5-2 equiv.) and the resulting mixture was stirred for 3 h at 22 °C. The reaction mixture was filtered through Celite and concentrated in vacuo. The crude product was purified by silica gel column chromatography with the elution conditions indicated for each case, in order to yield the desired compound.

[0157] General Procedure C

[0158] To a solution of the chlorinated derivative (1 equiv.) in acetic acid ([C]=0.55 M) was added mercury(II) acetate (1.5 equiv.) and the mixture was stirred at 22°C overnight during which time a fine white precipitate of mercury(II) chloride appeared. Most of the acetic 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 again with water, dried over anhydrous magnesium sulfate, and the solvent was removed in vacuo. The crude product was purified by silica gel column chromatography with the elution conditions indicated for each case, to yield the desired compound. In some cases, no purification was necessary.

[0159] General procedure D a solution of the carboxylic acid derivative (1 equiv.) in dry dichloromethane, was added under argon oxalyl chloride (5 equiv.) dropwise, followed by a 0.15 M solution of anhydrous dimethylformamide (5 mol%) in dry dichloromethane. The reaction mixture was stirred for 2 h under argon at 20 °C, then the mixture was evaporated in vacuo. In a second flask under argon, 4-hydroxymethyl-5-methyl-1,3-dioxol-2-one (1 equiv.) was dissolved in dry dichloromethane ([C] = 0.03 M). Triethylamine (1.2 equiv.) and 4-dimethylaminopyridine (5 mol% were added under vigorous stirring). This mixture was transferred into the flask containing the previously synthesized acyl chloride dissolved in dry dichloromethane and the reaction mixture was stirred for 24 h under argon at 20 °C.After adding saturated aqueous sodium hydrogen carbonate solution, the aqueous phase was removed and the organic phase was washed with saturated aqueous sodium chloride solution and then dried over anhydrous magnesium sulfate. After filtration and evaporation of the solvent under reduced pressure, the crude product was purified by silica gel column chromatography with the elution conditions indicated for each case, to yield the desired compound.

[0160] Synthesis of probe precursors incorporating an isoindoline motif

[0161]

[0162] Synthesis of compound 1

[0163] 1) 5-acetoxymethoxycarbonyl-1,1,3,3-tetraethylisoindolin-2-yloxyl radical (S2) The 5-carboxy-1,1,3,3-tetraethylisoindol-2-yloxyl radical S1 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).

[0164] According to general procedure A, employing S1 (250 mg, 0.861 mmol, 1 equiv.), K2CO3 (236 mg, 1.71 mmol, 2 equiv.) and bromoethyl acetate (96 μL, 0.975 mmol, 1.2 equiv.). The crude product was purified by silica gel column chromatography (Cyclohexane / AcOEt, 90 / 10) to afford the desired compound as a yellow oil (309 mg, 87%).

[0165] Rf = 0.47 (cyclohexane / ethyl acetate, 4:1).

[0166] IR vmax (ATR, ZnSe, pure, cm' 1 ) : 2973, 2940, 2881, 1767, 1738, 1617, 1457, 1418, 1370, 1284, 1259, 1231, 1203, 1159, 1101, 1032, 983.

[0167] HRMS (ESI-Orbitrap) m / z [M+H] + Calculated for C20H29NO5 363.2040; Found 363.2027, [M+2H] + Calculated for C20H30NO5 364.2118; Found 364.2110.

[0168] RPE (100 pM dissolved in 0.1 M potassium phosphate buffer and pH = 7.4): AN = 1.54 mT, AB = 0.17 mT.

[0169] 2) 2-(((1-chloroethoxy)carbonyl)oxy)-1,1,3,3-tetraethylisoindoline-5-carboxylate d'acetoxyméthyle (S3)

[0170] According to general procedure B, employing S2 (262 mg, 0.723 mmol, 1 equiv.), Pd / C (30 mg), Et3N (151 pL, 1.084 mmol, 1.5 equiv.) and 1-chloroethyl chloroformate (117 pL, 1.084 mmol, 1.5 equiv.). The crude product was purified by silica gel column chromatography (Cyclohexane / AcOEt, 90 / 10) to afford the desired compound as a pale yellow oil (298 mg, 88%).

[0171] Rf = 0.56 (dichloromethane)

[0172] NMR 1H (500 MHz, CDCI3) 5 (ppm) 8,00 (d, J = 7,9, 1H), 7,77 (s, 1H), 7,15 (d, J = 7,9 Hz, 1 H), 6.49 (q, J = 5,8 Hz, 1 H), 6,00 (s, 2H), 2,14 (s, 3H), 2,12 - 2,03 (m, 2H), 2,02 - 1,92 (m, 2H), 1 ,87 (d, J = 5,8 Hz, 3H), 1 ,84 - 1 ,73 (m, 4H), 1 ,03 - 0,92 (m, 6H), 0,84 - 0,75 (m, 6H). RMN 13 C (126 MHz, CDCh) 5 (ppm) 169,8 ; 165,3 ; 154,0 ; 147,7 ; 142,0 ; 129,1 ; 128,1 ;

[0173] 125,4 ; 124,0 ; 85,0 ; 79,8 ; 74,8 ; 74,5 ; 30,2 ; 29,1 ; 29,0 ; 29,0 ; 25,3 ; 20,9 ; 9,4 ; 8,7 ;

[0174] 8,60.

[0175] IR v max (ATR, ZnSe, pur, cm’ 1 ) : 2968, 2942, 2863, 1790, 1690, 1450, 1373, 1282, 1204, 1159, 1080, 1010, 983.

[0176] HRMS (ESI-Orbitrap) m / z [M+H] + Calculé pour C23H33CINO7 470,1940 ; Trouvé 470,1930, [M+Na] + Calculé pour C23H32CINNaO7492,1760 ; Trouvé 492,1748.

[0177] 3) 2-(((1-acetoxyethoxy)carbonyl)oxy)-1,1,3,3-tetraethylisoindoline-5-acetoxymethylcarboxylate (Compound 1)

[0178] According to general procedure C, employing S3 (290 mg, 0.617 mmol, 1 equiv.), AcOH (1.1 mL) and Hg(OAc)2 (294 mg, 0.925 mmol, 1.5 equiv.). The crude product was purified by silica gel column chromatography (Cyclohexane / AcOEt, 80 / 20) to afford the desired compound as a pale yellow oil (254 mg, 84%). Rf = 0.47 (dichloromethane)

[0179] NMR 1 H (500 MHz, CDCI3) 5 (ppm) 7.99 (d, J = 7.9 Hz, 1 H), 7.76 (s, 1 H), 7.15 (d, J = 7.9 Hz, 1 H), 6.77 (q, J = 5.4 Hz, 1 H), 5.99 (s, 2H), 2.14 (s, 3H), 2.10 (s, 3H), 2.11 - 2.04 (m, 2H), 2.00 - 1.89 (m, 2H), 1.85 - 1.72 (m, 4H), 1.56 (d, J = 5.4 Hz, 3H), 1.03 - 0.90 (m, 6H), 0.86 - 0.72 (m, 6H).

[0180] NMR 13C (CDCI3, 125 MHz) 5 (ppm) 169.8; 169.0; 165.4; 154.1; 147.8; 142.2; 129.0; 128.0; 125.4; 123.9; 92.2; 79.8; 74.6; 74.4; 30.2; 30.1; 29.1; 29.0; 20.9; 20.8; 19.6; 9.4; 8.6.

[0181] IR v max (ATR, ZnSe, pure, cm' 1 ): 2968, 2942, 1790, 1765, 1740, 1450, 1373, 1283, 1245, 1204, 1159, 1079, 1032, 1010, 983.

[0182] HRMS (ESI-Orbitrap) m / z [M+ H] + Calculated for C25H36NO9 494.2385; Found 494.2370, [M+Na] + Calculated for C25H35NNaOg 516.2204; Found 516.2189.

[0183] Synthesis of compound 2 1) Radical 5-(((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)carbonyl)-1,1,3,3-tetraethylisoindolin-2-yloxyl (S4)

[0184] According to general procedure D, employing S1 (380 mg, 1.31 mmol, 1 equiv.), oxalyl chloride (561 μL, 6.54 mmol, 5 equiv.), DMF (5 μL, 0.066 mmol, 5 mol%), 4-hydroxymethyl-5-methyl-1,3-dioxol-2-one (170 mg, 1.31 mmol, 1 equiv.), Et3N (219 μL, 1.57 mmol, 1.2 equiv.) and DMAP (8.1 mg, 0.066 mmol, 5 mol%). The crude product was purified by silica gel column chromatography (Cyclohexane / AcOEt, 80 / 20) to afford the desired compound as a yellow oil (180 mg, 34%).

[0185] Rf = 0.22 (cyclohexane:ethyl acetate = 4:1)

[0186] IR v max (ATR, ZnSe, pure, cm' 1 ): 2984, 2942, 2908, 1739, 1447, 1373, 1238, 1098, 1046, 938.

[0187] HRMS (ESI-Orbitrap) m / z [M] + Calculated for C22H28NO6402.1911; Found 402.1916, [M+H] + Calculated for C22H29NO6 403.1995; Found 403.1957, [M+2H] + Calculated for C22H30NO6 404.2073; Found 404.2050.

[0188] RPE (100 pM dissolved in 0.1 M potassium phosphate buffer and pH = 7.4 containing 1 mM DTPA): AN = 1.54 mT, AB = 0.17 mT.

[0189] 2) Methyl-2-(((1-chloroethoxy)carbonyl)oxy)-1,1,3,3-tetraethylisoindoline-5-carboxylate de (5-méthyl-2-oxo-1,3-dioxol-4-yl) (S5)

[0190] According to general procedure B, employing S4 (65 mg, 0.162 mmol, 1 equiv.), Pd / C (7 mg), Et3N (34 μL, 0.243 mmol, 1.5 equiv.) and 1-chloroethyl chloroformate (26 μL, 0.243 mmol, 1.5 equiv.). The crude product was purified by silica gel column chromatography (Cyclohexane / AcOEt, 90 / 10) to afford the desired compound as a pale yellow oil (21 mg, 26%).

[0191] NMR 1H (500 MHz, CDCI3) 5 (ppm) 7.99 (d, J = 7.0 Hz, 1 H), 7.75 (s, 1 H), 7.18 (d, J = 7.0 Hz, 1 H), 6.51 (q, J = 5.6 Hz, 1 H), 5.11 (s, 2H), 2.27 (s, 3H), 2.15 - 2.06 (m, 2H), 2.05 - 1.95 (m, 2H), 1.89 (d, J = 5.6 Hz, 3H), 1.85 - 1.74 (m, 4H), 1.06 - 0.95 (m, 6H), 0.90 - 0.77 (m, 6H).

[0192] NMR 13 C (126 MHz, CDCI3) 5 (ppm) 165.6; 153.5; 151.8; 147.0; 141.6; 140.0; 133.2; 128.4; 127.6; 124.7; 123.5; 84.5; 74.2; 74.0; 54.0; 29.7; 29.3; 28.6; 28.5; 28.4; 24.8; 9.2; 9.0; 8.2; 8.1.

[0193] IR vmax (ATR, ZnSe, pure, cm' 1 ) : 2985, 2864, 1710, 1670, 1578, 1414, 1298, 1259, 1121, 1097, 1056, 985, 913.

[0194] HRMS (ESI-Orbitrap) m / z [M+H] + Calculated for C25H33CINO8 510.1895; Found 510.1864.

[0195] 3) Methyl-2-(((1-acetoxyethoxy)carbonyl)oxy)-1,1,3,3-tetraethylisoindoline-5-carboxylate de (5-methyl-2-oxo-1,3-dioxol-4-yl) (Compound 2)

[0196] According to general procedure C, employing S5 (20 mg, 0.039 mmol, 1 equiv.), AcOH (69 μL) and Hg(OAc)2 (18.7 mg, 0.059 mmol, 1.5 equiv.). The pure product was obtained without purification as a pale yellow oil (16 mg, 77%).

[0197] NMR 1 H (500 MHz, CDCI3) 5 (ppm) 7.97 (dd, J = 8.0, 1.4 Hz, 1 H), 7.73 (s, 1 H), 7.16 (dd, J = 8.0, 2.1 Hz, 1 H), 6.78 (q, J = 5.4 Hz, 1 H), 5.09 (s, 2H), 2.26 (s, 3H), 2.11 (s, 3H), 2.13 - 2.04 (m, 2H), 2.02 - 1.90 (m, 2H), 1.85 - 1.72 (m, 4H), 1.56 (t, J = 5.4 Hz, 3H), 1.01 - 0.75 (m, 12 p.m.).

[0198] NMR 13 C (126 MHz, CDCh) 5 (ppm) 168.5; 165.5; 153.5; 151.7; 147.1; 141.6; 139.9; 133.1; 128.2; 127.5; 124.6; 123.4; 91.4; 73.9; 73.7; 53.9; 29.9; 29.3; 28.4; 28.3; 22.3; 20.4; 19.1; 9.2; 8.9; 8.1; 8.0.

[0199] IR vmax (ATR, ZnSe, pure, cm' 1): 2986, 2864, 1710, 1698, 1670, 1414, 1382, 1121, 1110, 1097, 985. HRMS (ESI-Orbitrap) m / z [M+H] + Calculated for C27H36NO10 534.2339; Found 534.2311, [M+Na] + Calculated for C27H3sNNaOio 556.2159; Found 556.2134.

[0200] Synthesis of the probe precursor incorporating a pyrroline motif: compound 3

[0201] 1) 3-acetoxymethoxycarbonyl-2,2,5,5-tetraethyl-2,5-dihydro-1 H-pyrrole- / V-oxyl radical (S7) The 3-carboxy-2,2,5,5-tetraethyl-1-pyrrolin- / V-oxyl radical S6 was synthesized by the route described by Y. Wang et al. (Y. Wang, J.T. Paletta, K. Berg, E. Reinhart, S. Rajca, A. Rajca, Synthesis of Unnatural Amino Acids Functionalized with Sterically Shielded Pyrroline Nitroxides, Org. Lett. 16 (2014) 5298-5300) from 2,2,6,6-tetraethylpiperidin-4-one prepared according to the procedure of X. Wang et al. (X. Wang, M. Emoto, A. Sugimoto, Y. Miyake, K. Itto, M. Amasaka, S. Xu, H. Hirata, H. Fujii, H. Arimoto, Synthesis of 15 / V-labeled 4-oxo-2,2,6,6-tetraethylpiperidine nitroxide for EPR brain imaging, Tetrahedron Lett. 55 (2014), 2146-2149). According to general procedure A, employing S6 (100 mg, 0.416 mmol, 1 equiv.), K2CO3 (115 mg, 0.832 mmol, 2 equiv.) and bromoethyl acetate (61 μL, 0.624 mmol, 1.5 equiv.). The crude product was purified by silica gel column chromatography (Cyclohexane / AcOEt, 90 / 10) to afford the desired compound as a yellow oil (129 mg, 99%).

[0202] IR v max (ATR, ZnSe, pure, cm' 1 ) : 2978, 2927, 2881 , 2856, 1767, 1734, 1458, 1371 , 1274, 1248, 1217, 1197, 1161 , 1014.

[0203] HRMS (ESI-Orbitrap) m / z [M+Na] + Calculated for CieHLeNNaOs 335.1709; Found 335.1696. RPE (100 pM in solution in 0.1 M potassium phosphate buffer and pH = 7.4): AN = 1.54 mT, AB = 0.18 mT.

[0204] 2) 1-(((1-chloroethoxy)carbonyl)oxy)-2,2,5,5-tetraethyl-2,5-dihydro-1 H-pyrrole-3-acetoxymethyl carboxylate (S8)

[0205] According to general procedure B, employing S7 (129 mg, 0.416 mmol, 1 equiv.), Pd / C (18 mg), Et3N (144 μL, 1.040 mmol, 2.5 equiv.) and 1-chloroethyl chloroformate (89 μL, 0.832 mmol, 2 equiv.). The crude product was purified by silica gel column chromatography (Cyclohexane / AcOEt, 95 / 05) to afford the desired compound as a yellow oil (70 mg, 40%).

[0206] NMR 1 H (500 MHz, CDCh) 5 (ppm) (slow cycle nitrogen reversal splits some signals) 6.80 - 6.79 (m, 1 H), 6.44 (q, J = 5.6 Hz, 1 H), 5.82 - 5.79 (m, 2H), 2.12 (s, 3H), 2.01 - 1.86 (m, 4H), 1.84 (d, J = 5.6 Hz, 3H), 1.82 - 1.76 (m, 1 H), 1.73 - 1.55 (m, 3H), 0.97 - 0.86 (m, 12H).

[0207] NMR 13C (126 MHz, CDCh) 5 (ppm) (slow cycle nitrogen reversal splits some signals) 169.7; 161.6; 161.6; 153.9; 153.8; 147.7; 147.5; 133.7; 133.5; 84.8; 84.8; 79.0; 77.6; 77.6; 75.2; 75.1; 29.7; 29.1; 29.1; 28.5; 28.4; 28.2; 28.1; 25.1; 20.7; 10.1; 10.1; 9.4; 9.0; 9.0; 8.7; 8.7.

[0208] IR v max (ATR, ZnSe, pure, cm' 1 ) : 2978, 2941, 2885, 1789, 1768, 1734, 1462, 1454, 1371, 1276, 1219, 1193, 1157, 1097, 1008.

[0209] HRMS (ESI-Orbitrap) m / z [M+H] + Calculated for C19H31CINO7 420.1789; Found 420.1762. 3) Acetoxymethyl 1-(((1-acetoxyethoxy)carbonyl)oxy)-2,2,5,5-tetraethyl-2,5-dihydro-1 H-pyrrole-3-carboxylate (Compound 3)

[0210] According to general procedure C, employing S8 (61 mg, 0.145 mmol, 1 equiv.), AcOH (258 μL) and Hg(OAc)2 (69 mg, 0.218 mmol, 1.5 equiv.). The pure product was obtained without purification as a pale yellow oil (45 mg, 70%).

[0211] NMR 1 H (500 MHz, CDCh) 5 (ppm) (slow cycle nitrogen reversal splits some signals) 6.80 - 6.78 (m, 1 H), 6.72 (q, J = 5.4 Hz, 1H), 5.81 - 5.78 (m, 2H), 2.11 (s, 3H), 2.07 (s, 3H), 1 .99 - 1.83 (m, 4H), 1.81 - 1.56 (m, 4H), 1.52 (d, J = 5.4 Hz, 3H), 0.97 - 0.84 (m, 12H).

[0212] NMR 13 C (126 MHz, CDCh) 5 (ppm) (slow cycle nitrogen reversal splits some signals) 169.7; 168.9; 161.6; 154.0; 147.8; 147.7; 133.7; 133.6; 91.7; 79.0; 77.6; 77.5; 75.1; 75.0; 29.7; 29.7; 29.1; 28.4; 28.4; 28.2; 28.1; 20.8; 20.8; 20.77; 19.4; 10.1; 10.1; 9.5; 9.5; 9.0; 8.7; 8.7.

[0213] IR vmax (ATR, ZnSe, pure, cm' 1): 2978, 2941, 2884, 1791, 1766, 1735, 1452, 1373, 1249, 1213, 1197, 1082, 1014, 910.

[0214] HRMS (ESI-Orbitrap) m / z [M+Na] + Calculated for C2iH 33 NNaO9466.2053; Found 466.2044.

[0215] Synthesis of probe precursors incorporating a pyrrolidine motif:

[0216] Synthesis of compound 4

[0217]

[0218] S11 Compound 4

[0219] 1) 3-acetoxymethoxycarbonyl-2,2,5,5-tetraethylpyrrolidin- / \ / -oxyl radical (S10) The 3-carboxy-2,2,5,5-tetraethyl-1-pyrrolidin- / \ / -oxyl radical S9 was synthesized following a protocol described by JT Paletta et al. (JT Paletta, M. Pink, B. Foley, S. Rajca, A. Rajca, Synthesis and Reduction Kinetics of Sterically Shielded Pyrrolidine Nitroxides, Org. Lett. 14 (2012) 5322-5325).

[0220] According to general procedure A, employing S9 (57 mg, 0.235 mmol, 1 equiv.), K2CO3 (65 mg, 0.470 mmol, 2 equiv.) and bromoethyl acetate (34 μL, 0.352 mmol, 1.5 equiv.). The crude product was purified by silica gel column chromatography (Cyclohexane / AcOEt, 80 / 20) to afford the desired compound as a yellow oil (48 mg, 64%).

[0221] IR v max (ATR, ZnSe, pure, cm' 1 ) : 2978, 2824, 2884, 2854, 1766, 1456, 1417, 1369, 1219, 1201 , 1159, 1139, 1066, 983.

[0222] HRMS (ESI-Orbitrap) m / z [M+Na] + Calculated for Ciet^sNNaOs 337.1865; Found 337.1859. RPE (100 pM in solution in 0.1 M potassium phosphate buffer and pH = 7.4): AN = 1.53 mT, AB = 0.30 mT. 2) Acetoxymethyl 1-(((1-Chloroethoxy)carbonyl)oxy)-2,2,5,5-tetraethylpyrrolidine-3-carboxylate (S11)

[0223] According to general procedure B, employing S10 (40 mg, 0.127 mmol, 1 equiv.), Pd / C (6 mg), Et3N (35 μL, 0.254 mmol, 2 equiv.) and 1-chloroethyl chloroformate (42 μL, 0.382 mmol, 3 equiv.). The crude product was purified by silica gel column chromatography (Cyclohexane / AcOEt, 95 / 05) to afford the desired compound as a pale yellow oil (31 mg, 58%).

[0224] NMR 1 H (500 MHz, CDCh) 5 (ppm) (mixture of two diastereomers, slow inversion of ring nitrogen splits and broadens some signals) 6.41 - 6.33 (m, 1 H), 5.76 - 5.65 (m, 2 H), 2.89 - 2.82 (m, 0.5 H), 2.24 (broad t, J = 12.4 Hz, 0.5 H), 2.03 (s, 3 H), 1.94 - 1.80 (m, 2 H), 1.78 - 1.76 (m, 3 H), 1.72 - 1.50 (m, 6 H), 1.48 - 1.32 (m, 2 H), 0.95 - 0.73 (m, 12 H). NMR 13C (126 MHz, CDCh) 5 (ppm) (mixture of two diastereomers, slow inversion of ring nitrogen splits and broadens some signals) 171.4; 169.8; 153.8; 84.9; 79.4; 75.3; 72.4; 42.4; 32.9; 31.1; 29.8; 29.3; 28.5; 25.3; 20.8; 10.2; 9.4; 8.9; 8.2.

[0225] IR v max (ATR, ZnSe, pure, cm' 1 ): 2984, 2932, 2834, 1690, 1670, 1608, 1482, 1356, 1280, 1111.

[0226] HRMS (ESI-Orbitrap) m / z [M+Na] + Calculated for Ci9H32CINNaO? 444.1765; Found 444.1759.

[0227] 3) Acetoxymethyl 1-(((1-acetoxyethoxy)carbonyl)oxy)-2,2,5,5-tetraethylpyrrolidine-3-carboxylate (Compound 4) According to general procedure C, employing S11 (24 mg, 0.057 mmol, 1 equiv.), AcOH (9 μL) and Hg(OAc)2 (27 mg, 0.085 mmol, 1.5 equiv.). The crude product was purified by silica gel column chromatography (Cyclohexane / AcOEt, 95 / 05) to afford the desired compound as a colorless oil (15 mg, 60%).

[0228] NMR 1 H (500 MHz, CDCh) 5 (ppm) (mixture of two diastereoisomers, slow inversion of ring nitrogen splits and broadens some signals) 6.75 - 6.69 (m, 1 H), 5.84 - 5.69 (m, 2 H), 2.93 - 2.88 (m, 0.5 H), 2.29 (t broad, J= 12.8 Hz, 0.5 H), 2.09 (s, 3 H), 2.07 (s, 3 H), 2.01 - 1 .83 (m, 2 H), 1 .79 - 1 .57 (m, 6 H), 1 .53 - 1 .50 (m, 3 H), 1 .48 - 1 .31 (m, 2 H), 1 .10 - 0.79 (m, 12H).

[0229] NMR 13C (126 MHz, CDCh) 5 (ppm) (mixture of two diastereomers, slow inversion of ring nitrogen splits and broadens some signals) 169.8; 169.0; 161.8; 153.8; 91.8; 79.1; 72.3; 67.4; 42.5; 32.9; 30.4; 28.3; 26.0; 24.3; 20.9; 20.8; 19.6; 9.6; 9.4; 8.8; 8.2.

[0230] IR vmax (ATR, ZnSe, pure, cm' 1 ) : 2984, 2868, 1706, 1690, 1672, 1608, 1514, 1482, 1356, 1280, 1111, 985.

[0231] HRMS (ESI-Orbitrap) m / z [M+Na] + Calculated for C2iH 35 NNaO9468.2209; Found 468.2203.

[0232] Synthesis of compound 5

[0233] 94%

[0234] Compound 5

[0235] 1) 1-(((1-chloroethoxy)carbonyl)oxy)-2,2,5,5-tetramethylpyrrolidine-3-carboxylate acetoxymethyl (S13)

[0236] The 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethylpyrrolidin-1-yloxyl radical S12 was 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-1-oxyl and Derivatives, Heterocycles, 81 (2010) 349-356, and J. P. Y. Kao, G. M. Rosen, Esterase-assisted accumulation of 3-carboxy-2,2,5,5-tetramethyl1-pyrrolidinyloxyl into lymphocytes, Org. Biomol. Chem. 2 (2004) 99-102).

[0237] According to general procedure B, employing S12 (500 mg, 1.94 mmol, 1 equiv.), Pd / C (50 mg), Et3N (540 μL, 3.90 mmol, 2 equiv.) and 1-chloroethyl chloroform iate (520 μL, 4.80 mmol, 2.5 equiv.). The crude product was purified by silica gel column chromatography (Cyclohexane / AcOEt, 8 / 2) to afford the desired compound as a colorless oil (640 mg, 91%).

[0238] Rf = 0.40 (cyclohexane / ethyl acetate = 4:1; phosphomolybdic acid staining). NMR 1 H (500 MHz, CDCh) 5 (ppm) (main diastereoisomer “90%) 6.43 (q, J= 5.7 Hz, 1 H), 5.78 (d, J= 5.2 Hz, 1H), 5.70 (d, J= 5.2 Hz, 1H), 3.05 - 2.80 (m large, 1H), 2.35 - 2.13 (m, 1H), 2.08 (s, 3H), 1.88 - 1.76 (m, 4H), 1.35 - 1.30 (s, 3H), 1.25 - 1.15 (m, 6H), 1.15 - 1.00 (s, 3H).

[0239] NMR 13 C (126 MHz, CDCh) 5 (ppm) (relatively slow nitrogen inversion splits and broadens some of the signals) 170.9; 169.6; 154.2; 154.1; 85.1; 85.0; 79.6; 67.3; 64.7; 62.8; 59.7; 50.7; 47.9; 38.2; 37.1; 31.6; 28.0; 25.7; 25.3; 22.1; 20.8; 17.2.

[0240] HRMS (ESI-Orbitrap) m / z [M+H] + Calculated for C15H25CINO7 366.1314; Found 366.1306, [M+Na] + Calculated for Ci5H24CINNaO7388.1134; Found 388.1124.

[0241] 2) 1-(((1-acetoxyethoxy)carbonyl)oxy)-2,2,5,5-tetramethylpyrrolidine-3-acetoxymethyl carboxylate (Compound 5) According to general procedure C, employing S13 (300 mg, 0.80 mmol, 1 equiv.), AcOH (1.2 mL) and Hg(OAc)2 (250 mg, 0.80 mmol, 1 equiv.). The crude product was purified by silica gel column chromatography (Cyclohexane / AcOEt, 80 / 20) to afford the desired compound as a colorless oil (302 mg, 94%). Rf = 0.21 (cyclohexane / ethyl acetate = 4:1; phosphomolybdic acid staining). NMR 1 H (500 MHz, CD3OD) 5 (ppm) (main diastereoisomer “90%) 6.71 (m, 1 H), 5.82 (m, 1 H), 5.70 (m, 1 H), 3.15 - 2.80 (m wide, 1 H), 2.35 - 2.10 (m wide, 1 H), 2.07 (s, 6H), 1.86 (m, 1H), 1.51 (m, 3H), 1.00 - 1.36 (m, 12H).

[0242] NMR 13C (126 MHz, CD3OD) 5 (ppm) (relatively slow nitrogen inversion splits and broadens some of the signals) 172.1; 171.2; 170.7; 156.2; 93.3; 80.8; 68.5; 67.3; 65.5;

[0243] 63.7; 51.6; 48.3; 39.1; 37.7; 31.7; 28.9; 28.2; 26.8; 26.4; 26.2; 22.2; 20.7; 20.6;

[0244] 19.7; 17.5.

[0245] HRMS (ESI-Orbitrap) m / z [M+H] + Calculated for C17H28NO9 390.1759; Found 390.1751, [M+Na] + Calculated for Ci7H27CINNaO7412.1578; Found 412.1570.

[0246] Synthesis of probe precursors incorporating a piperidine motif:

[0247] Synthesis of compound 6

[0248] 1) 4-acetoxymethoxycarbonyl-2,2,6,6-tetraethylpiperidine- / \ / -oxyl radical (S15)

[0249] The 4-carboxy-2,2,6,6-tetraethyl(piperidin-1-yloxyl) radical S14 was prepared following the protocol published by N. Babic et al. (N. Babic, F. Peyrot, New synthetic route to 2,2,6,6-tetraethylpiperidin-4-one, a key-intermediate towards tetraethyl nitroxides, Tetrahedron Lett. 60 (2019) 151207).

[0250] According to general procedure A, employing S14 (200 mg, 0.780 mmol, 1 equiv.), K2CO3 (215 mg, 1.56 mmol, 2 equiv.) and bromoethyl acetate (92 μL, 0.936 mmol, 1.2 equiv.). The crude product was purified by silica gel column chromatography (Cyclohexane / AcOEt, 80 / 20) to afford the desired compound as a yellow oil (156 mg, 61%).

[0251] IR vmax (ATR, ZnSe, pure, cm' 1 ): 2978, 2881, 1763, 1724, 1463, 1369, 1319, 1301, 1224, 1151, 1136, 1020.

[0252] HRMS (ESI-Orbitrap) m / z [M+Na] +Calculated for Ci7H3oNNa05351,2016; Found 351,2003. RPE (100 pM in solution in 0.1 M potassium phosphate buffer and pH = 7.4): AN = 1.61 mT, AB = 0.30 mT.

[0253] 2) Acetoxymethyl 1-(((1-chloroethoxy)carbonyl)oxy)-2,2,6,6-tetraethylpiperidine-4-carboxylate (S16)

[0254] According to general procedure B, employing S15 (156 mg, 0.475 mmol, 1 equiv.), Pd / C (21 mg), Et3N (99 μL, 0.712 mmol, 1.5 equiv.) and 1-chloroethyl chloroformate (77 μL, 0.712 mmol, 1.5 equiv.). The crude product was purified by silica gel column chromatography (Cyclohexane / AcOEt, 95 / 05) to afford the desired compound as a pale yellow oil (44 mg, 21%). NMR 1H (500 MHz, CDCI3) 5 (ppm) 6.42 (q, J = 5.7 Hz, 1 H), 5.75 (s, 2H), 2.66 (t, J = 12.4 Hz, 1H), 2.13 (s, 3H), 2.03 - 1.92 (m, 2H), 1.83 (d, J = 5.7 Hz, 3H), 1.87 - 1.74 (m, 4H), 1.69 - 1.66 (m, 2H), 1.61 - 1.48 (m, 2H), 1.44 - 1.33 (m, 2H), 1.01 - 0.84 (m, 12H).

[0255] NMR 13 C (126 MHz, CDCI3) 5 (ppm) 173.4; 169.0; 153.0; 84.2; 79.0; 65.5; 65.4; 33.4; 32.6; 29.2; 29.1; 25.9; 24.6; 20.2; 9.5; 7.4; 7.4.

[0256] IR vmax (ATR, ZnSe, pure, cm' 1 ) : 2986, 2864, 1778, 1768, 1698, 1465, 1256, 1193, 1136, 1078, 1011, 1006, 987.

[0257] HRMS (ESI-Orbitrap) m / z [M+H] + Calculated for C20H35CINO7436.2102; Found 436.2087, [M+Na] + Calculated for C2oH34CINNa07458.1933; Found 458.1887.

[0258] 3) Acetoxymethyl 1-(((1-acetoxyethoxy)carbonyl)oxy)-2,2,6,6-tetraethylpiperidine-4-carboxylate (Compound 6)

[0259] According to general procedure C, employing S16 (40 mg, 0.092 mmol, 1 equiv.), AcOH (164 μL) and Hg(OAc)2 (44 mg, 0.138 mmol, 1.5 equiv.). The pure product was obtained without purification as a pale yellow oil (40 mg, 95%).

[0260] NMR 1 H (500 MHz, CDCI3) 5 (ppm) 6.70 (q, J = 5.3 Hz, 1H), 5.75 (s, 2H), 2.65 (t, J = 12.6 Hz, 1 H), 2.13 (s, 3H), 2.07 (s, 3H), 2.02 - 1.91 (m, 2H), 1.85 - 1.72 (m, 4H), 1.68 - 1.63 (m, 2H), 1.58 - 1.47 (m, 2H), 1.51 (d, J = 5.3 Hz, 3H), 1.44 - 1.33 (m, 2H), 1.01 - 0.86 (m, 12H).

[0261] NMR 13 C (126 MHz, CDCI3) 5 (ppm) 173.7; 169.2; 168.4; 153.4; 91.1; 79.2; 65.5; 65.4; 33.6; 32.9; 29.4; 29.3; 26.1; 20.4; 19.1; 9.7; 7.6.

[0262] IR vmax (ATR, ZnSe, pure, cm' 1 ) : 2970, 2945, 2881, 1788, 1764, 1467, 1454, 1373, 1247, 1211, 1193, 1136, 1078, 1020, 1006, 985.

[0263] HRMS (ESI-Orbitrap) m / z [M+ H] +Calculated for C22H38NO9460.2547; Found 460.2520, [M+Na] + Calculated for C 22 H37CINNaO9482.2366; Found 482.2339.

[0264] Synthesis of compound 7

[0265] 1) 4-(2-ethoxy-2-oxoethylidene)-2,2,6,6-tetraethylpiperidin-1-oxyl radical (S18) The radical 2,2,6,6-tetraethyl-4-oxo(piperidin-1-yloxyl) or TEEPONE S17 was obtained according to the procedure described by X. Wang et al. (X. Wang, M. Emoto, A. Sugimoto, Y. Miyake, K. Itto, M. Amasaka, S. Xu, H. Hirata, H. Fujii, H. Arimoto, Synthesis of 15 N- labeled 4-oxo-2,2,6,6-tetraethylpiperidine nitroxide for EPR brain imaging, Tetrahedron Lett. 55 (2014), 2146-2149). To a solution of TEEPONE S17 (150 mg, 0.663 mmol, 1 equiv.) in dry THF

[0266] (8 mL) were added triethyl phosphonoacetate (197 μL, 0.994 mmol, 1.5 equiv.) and sodium hydride (32 mg, 1.326 mmol, 2 equiv.) at 0 °C. The mixture was then stirred for 4 hours at room temperature. Then the excess sodium hydride was neutralized by adding a saturated solution of ammonium chloride. The aqueous phase was extracted with ethyl acetate (x3), the combined organic phases were then washed with a saturated aqueous solution of sodium chloride and dried over anhydrous magnesium sulfate. After evaporation of the solvent under reduced pressure, the crude product was purified by chromatography on a silica gel column (Cyclohexane / AcOEt, 8 / 2) to obtain the desired product in the form of a yellow oil (154 mg, 78%).

[0267] IR vmax (ATR, ZnSe, pure, cm' 1 ) : 2978, 2935, 2881, 1741, 1712, 1456, 1390, 1273, 1155.

[0268] HRMS (ESI-Orbitrap) m / z [M] +Calculated for C17H30NO3 296.2226; Found 296.2221, [M+2H] + Calculated for C17H32NO3 298.2382; Found 298.2375, [M+Na] + Calculated for Ci7H3oNNa03319.2123; Found 319.2118.

[0269] RPE (100 pM dissolved in 0.1 M potassium phosphate buffer and pH = 7.4): AN = 1.59 mT, AB = 0.22 mT.

[0270] 2) 4-(carboxymethylene)-2,2,6,6-tetraethylpiperidin-1-oxyl radical (S19)

[0271] To a solution of compound S18 (150 mg, 0.506 mmol, 1 equiv.) in methanol (5 mL) was added a solution of sodium hydroxide (61 mg, 1.52 mmol, 3 equiv.) in water (5 mL). This solution was then stirred for 1 hour at 40 °C. Then water and ethyl acetate were added. The organic phase was removed and the aqueous phase was acidified by adding a hydrochloric acid solution (1 M) to a pH of 2. Then ethyl acetate was added and the aqueous phase was separated. The organic phase was dried over anhydrous magnesium sulfate and the solvent was removed under reduced pressure. The pure product was then obtained without purification in the form of a yellow solid (120 mg, 89%).

[0272] IR vmax (ATR, ZnSe, pure, cm' 1 ) : 2972, 2935, 2881 , 1715, 1641 , 1458, 1419, 1382, 1276, 1176, 941.

[0273] HRMS (ESI-Orbitrap) m / z [MH]' Calculated for C15H25NO3 267.1834; Found 267.1835. Melting point: 136-138 °C.

[0274] RPE (100 pM dissolved in 0.1 M potassium phosphate buffer and pH = 7.4): A N = 1.60 mT, AB = 0.23 mT. 3) Radical 4-(2-(acetoxymethoxy)-2-oxoethylidene)-2,2,6,6-tetraethylpiperidin-1-oxyl (S20)

[0275] According to general procedure A, employing S19 (200 mg, 0.745 mmol, 1 equiv.), K2CO3 (206 mg, 1.49 mmol, 2 equiv.) and bromoethyl acetate (109 μL, 1.118 mmol, 1.5 equiv.). The crude product was purified by silica gel column chromatography (Cyclohexane / AcOEt, 90 / 10) to afford the desired compound as a yellow oil (80 mg, 32%).

[0276] IR v max (ATR, ZnSe, pure, cm' 1 ) : 2980, 2931, 2885, 2856, 1768, 1745, 1371, 1226, 1207, 1132, 1010, 987.

[0277] HRMS (ESI-Orbitrap) m / z [M+Na] +Calculated for CisHsoNNaOs 363.2022; Found 363.2008. RPE (100 pM in solution in 0.1 M potassium phosphate buffer and pH = 7.4): AN = 1.59 mT, AB = 0.22 mT.

[0278] 4) 2-(1-(((1-chloroethoxy)carbonyl)oxy)-2,2,6,6-tetraethylpiperidin-4- ylidene)acetoxymethyl acetate (S21)

[0279] According to general procedure B, employing S2O (47 mg, 0.138 mmol, 1 equiv.), Pd / C (6 mg), Et3N (48 μL, 0.345 mmol, 2.5 equiv.) and 1-chloroethyl chloroformate (30 μL, 0.276 mmol, 2 equiv.). The crude product was purified by silica gel column chromatography (Cyclohexane / AcOEt, 95 / 05) to afford the desired compound as a pale yellow oil (33 mg, 54%). NMR 1H (500 MHz, CDCI3) 5 6.36 (q, J = 5.7 Hz, 1 H), 5.72 (s, 2H), 5.68 (s, 1 H), 3.55 (d, J = 13.2 Hz, 1 H), 3.04 - 2.95 (m, 1H), 2.33 (d, J = 13.2 Hz, 1 H), 2.21 (d, J = 15.0 Hz, 1 H), 2.05 (s, 3H), 1.88 - 1.79 (m, 2H), 1.77 (d, J = 5.7 Hz, 3H), 1.58 - 1.46 (m, 4H), 1.45 - 1.30 (m, 2H), 0.94 - 0.78 (m, 12H).

[0280] NMR 13 C (126 MHz, CDCI3) 5169.9; 164.4; 159.3; 153.6; 116.0; 84.8; 69.1; 66.9; 42.7; 36.9; 35.3; 30.3; 29.7; 29.1; 28.1; 25.2; 20.9; 9.9; 9.7; 8.8; 8.1.

[0281] IR v max (ATR, ZnSe, pure, cm' 1 ) : 2978, 2924, 2885, 2854, 1791, 1764, 1456, 1215, 1193, 1136, 1099, 1022, 1001 , 987.

[0282] HRMS (ESI-Orbitrap) m / z [M+H] + Calculated for C21H35CINO7448.2102; Found 448.2093.

[0283] 5) 2-(1-(((1-acetoxyethoxy)carbonyl)oxy)-2,2,6,6-tetraethylpiperidin-4- ylidene)acetoxymethyl acetate (Compound 7)

[0284] According to general procedure C, employing S21 (15 mg, 0.033 mmol, 1 equiv.), AcOH (5 μL) and Hg(OAc)2 (16 mg, 0.050 mmol, 1.5 equiv.). The crude product was purified by silica gel column chromatography (Cyclohexane / AcOEt, 95 / 05) to afford the desired compound as a pale yellow oil (7 mg, 45%).

[0285] NMR 1 H (500 MHz, CDCI3) 5 6.71 (q, 5.3 Hz, 1H), 5.78 (s, 2H), 5.74 (s, 1 H), 3.59 (d, J = 12.8 Hz, 1 H), 3.10 - 3.01 (m, 1 H), 2.43 - 2.35 (m, 1H), 2.32 - 2.23 (m, 1H), 2.11 (s, 3H), 2.07 (s, 3H), 1.93 - 1.83 (m, 2H), 1.64 - 1.54 (m, 4H), 1.52 (d, J = 5.3 Hz, 3H), 1.49 - 1.41 (m, 2H), 1.05 - 0.82 (m, 12H).

[0286] NMR 13 C (126 MHz, CDCh) 5169.9; 168.9; 164.5; 159.6; 153.9; 116.0; 91.7; 79.0; 69.2; 68.6; 42.4; 35.5; 29.4; 28.1; 27.0; 25.2; 20.9; 20.9; 19.6; 9.9; 9.8; 8.3; 8.2.

[0287] IR v max (ATR, ZnSe, pure, cm' 1) : 2980, 2878, 2864, 1791, 1764, 1698, 1670, 1456, 1213, 1111, 1099, 1027, 1001 , 987.

[0288] HRMS (ESI-Orbitrap) m / z [M+Na] + Calculated for C23H37NNaO9494.2366; Found 494.2360.

[0289] Example 2: Stability and reactivity of compounds of formula (I) towards esterases and comparison with a prior art radical probe precursor To confirm that the deprotection of compounds of formula (I) is esterase-dependent, assays in the presence of modified esterase and lipase were performed. Protection cleavage was tested by incubating compounds of formula (I) with esterase or lipase, followed by oxidation of the released hydroxylamine by potassium ferricyanide and measurement of the EPR signal of the produced nitroxide. Potassium ferricyanide is a mild oxidant that reacts rapidly with hydroxylamines by accepting an electron. The commercial acetyl-protected, tetramethylated ACP probe was used as a control.

[0290] Preparation of Compound 1 Solution: Two volumes of Compound 1 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.

[0291] Incubation with enzymes: 100 μM solution of compound 1 was incubated for 10 minutes at 37 °C with pig liver carboxylesterase (1 U ml 1 ; 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 to a 50 μL glass capillary (Hirschmann) and the nitroxide EPR signal was recorded with a Bruker Elexsys E500 X-band (9.86 GHz) EPR spectrometer 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.

[0292] Lipase was observed to exhibit low activity (as expected with short-chain esters), whereas pig liver carboxylesterase (EC 3.1.1.1) was more effective.

[0293] Incubation of compound 1 with 1 U mL' 1 of pig liver esterase for 10 minutes at 37 °C, followed by reaction with ferricyanide (1 mM), resulted in complete recovery of the nitroxide compared to an independently prepared 100 pM solution of compound 83 (authentic isoindoline nitroxide). When compound ACP (a comparative probe precursor known from the prior art, acetylated and substituted with four methyl groups) was subjected to the same conditions, only 27% of the corresponding nitroxide was recovered.

[0294] Stability of compound 1: To test the stability of compound 1 in aqueous solution, a 100 μM solution of compound 1 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.

[0295] Example 3: Behavior of compounds of formula (I) on cells

[0296] Following 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:

[0297] 3.1. Are compounds of formula (I) toxic?

[0298] 3.2. Do compounds of formula (I) accumulate inside the cell?

[0299] 3.3. Is deprotection of compounds of formula (I) a rate-limiting step towards the oxidation of hydroxylamine?

[0300] 3.4. Do compounds of formula (I) account for oxidative stress?

[0301] 3.5. Is it possible to detect oxidative stress under more physiological conditions?

[0302] The in vitro model on which these tests were mainly carried out is the U-87 MG cell line.

[0303] Cell culture protocol for cancer cell lines

[0304] 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 5% CO2 atmosphere. 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 recombinant human 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. 3.1. Are the compounds of formula (I) toxic?

[0305] Protocols:

[0306] Treatment of cells for the measurement of toxicity of compounds of formula (I)

[0307] U87 cells were seeded in a 96-well plate (10,000 per well one 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 1 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.

[0308] Cell toxicity test

[0309] For fluorescence imaging analysis, the incubation medium was removed and the cells were 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).

[0310] Results :

[0311] The following table shows the results of the dead cell count 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).

[0312] [Table 1]

[0313] The number of dead cells does not vary significantly, regardless of the concentration of compound 1 of formula (I). These results show that compound 1 is not toxic to U87 cells, even at the highest concentration tested of 500 pM for 6 hours. This is consistent with commonly used incubation conditions for a hydroxylamine probe on cultured cells (50 - 500 pM for 15 - 60 min).

[0314] 3.2. Do compounds of formula (I) accumulate inside the cell?

[0315] Membrane permeation and cell accumulation of compounds 1, 2, 3, and 7 were tested on U87 cells in suspension. Trypsinized cells were incubated with each 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.

[0316] RPE measurement protocol on suspended cells

[0317] After trypsinization, a suspension of U87 cells was prepared in serum-free medium (10 million cells per mL). In a typical experiment, compound 1, 2, 3, or 7 (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 1, 2, 3, or 7 was typically 100 μM. After incubation (30 min, 37 °C, 5% CO2, with occasional mixing to resuspend 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 pL of serum-free medium possibly containing 15N-TEMPONE (nitrogen-15-labeled 4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl radical, 100 pM), 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).

[0318] Results: Cells incubated with compound 1 showed strong nitroxide EPR signals after washing. The esterase-sensitive acetoxymethyl-masked carboxylic group of compound 1 allows it to cross the membrane and, once inside the cell, 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 occurs, with oxidation of the released hydroxylamine to nitroxide.The experimental EPR spectrum results from the superposition of two components, one main (marked by stars in Figure 1), characteristic of a nitroxide in the isotropic regime and compatible with a localization in the cytosol, and the other minor (marked by arrows), characteristic of a nitroxide in the intermediate regime and compatible with a localization in the cell membrane. A similar result was obtained with compounds 2, 3 and 7 (intense EPR signals of nitroxide present in the cells incubated after washing) indicating that this compound accumulates in the cells.

[0319] 3.3. Is deprotection of compounds of formula (I) a rate-limiting step towards the oxidation of hydroxylamine?

[0320] The deprotection rate of a hydroxylamine probe precursor protected from oxidative stress must be faster than the oxidation rate.

[0321] The presence of unoxidized hydroxylamine in cells can be tested using the 15 N-TEMPONE. This method is based on the ability of TEMPONE to freely cross the cell membrane and on the rapid transfer of hydrogen between nitroxides and hydroxylamines. 15 Isotopically labeled N-TEMPONE is used because its spectrum has only two lines that do not completely overlap with the signal of the 14 N-nitroxide derived from compound 1, 2, 3 or 7 (which exhibits three lines), which allows simultaneous measurement of both species.

[0322] For this, 5 million U87 cells in suspension were incubated with compound 1 or with compound 2, 3 or 7 (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 of 15 N-TEMPONE.

[0323] EPR spectra were acquired according to the protocol described in section 3.2 above.

[0324] Results: The sample of cells treated with the 15 N-TEMPONE showed a significantly higher signal of the 14 N-nitroxide derived from compound 1 (4 times more) compared to the untreated sample (Figure 2). In the case of compound 2, treatment with the 15 N- TEMPONE multiplies by three the signal of the 14 Corresponding N-nitroxide compared to the untreated sample. For compound 3, the signal is increased fourfold and for compound 7 by sixfold. 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 compounds 1, 2, 3 and 7 into nitroxides in the cell.

[0325] 3.4. Do compounds of formula (I) account for oxidative stress?

[0326] 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-like natural product that can produce superoxide by futile redox cycling in the cell.

[0327] Principle:

[0328] Confluent U87 cells were incubated on coverslips with compound 1 (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 -1for 2 h), chelators (sodium diethyldithiocarbamate, DETC, 5 μM and deferoxamine, DFO, 25 μM 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.

[0329] Protocol for EPR measurement using hydroxylamine probe precursors on cells grown on glass coverslips

[0330] 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 confluency (cells almost completely covering the surface of the glass on which they are growing) 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 10 3 1187 cells per well were seeded three days before the RPE experiments. (MCF-7 cells were seeded at a concentration of 4 x 10 5cells 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 1 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 1 is not soluble in water, a suspension was prepared using the surfactant Pluronic F-127.The stock solution of 1 (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, 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 toward 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 toward 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.

[0331] Results :

[0332] The results obtained are presented in the following table:

[0333] [Table 2]

[0334] # Mean ± standard deviation obtained for 3 to 5 independent experiments.

[0335] Two concentrations of juglone were tested (2.5 and 5 pM), and a dose-dependent increase in the EPR signal was observed. The maximum signal increase was obtained with 5 pM (~50% compared to untreated cells). These results demonstrate that compound 1 is indeed a reporter of intracellular oxidative stress. The basal oxidation of compound 1 in untreated cell samples was relatively high, which may suggest that compound 1 is sensitive to intracellular oxidation processes even under basal conditions in cancer cells.

[0336] 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 1 since these compounds do not alter the EPR response. This makes compound 1 simpler to use than unprotected hydroxylamine probes because it is less prone to interference. No antioxidant (SOD-PEG, Cat-PEG, or NAC) reduced basal oxidation or juglone-induced oxidation, which does not allow us to conclude precisely on the chemical species responsible for oxidation in cells.Interestingly, however, visual inspection of the cells showed that juglone-induced cell contraction was reversed by NAC treatment. It is known that the antioxidant effect of NAC is mediated by an increase in intracellular glutathione concentration. Therefore, it is likely that compound 1 is sensitive to oxidative stress processes upstream of glutathione.

[0337] In parallel, the commercially available ACP probe was tested using the same protocol. Under the tested conditions, no nitroxide signal could be detected with ACP, regardless of juglone treatment. This demonstrates that compound 1 is a better reporter of juglone-induced oxidative stress in cells than ACP under the tested conditions.

[0338] Additionally, another cell line (MCF-7 cells) was tested, and yielded 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.

[0339] 3.5. Is it possible to detect oxidative stress under more physiological conditions?

[0340] It was important to test compound 1 on normal cells, as well as to have a more physiological model of oxidative stress to test the detection threshold. A good model was rabbit articular chondrocytes (also adherent cells) induced to express an osteoarthritis-like inflammatory phenotype by the addition of 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).

[0341] Chondrocyte harvesting and culture protocol

[0342] 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 / cm 2in 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 DM EM 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.

[0343] RPE measurement protocol

[0344] RPE recording was performed, as described in section 3.4, 1 h, 24 h, or 48 h after IL-1 stimulation (1 ng / mL in FCS-free DMEM) or medium change to serum-free DMEM. Cells were incubated with compound 1 (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 1, while for 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 stated.

[0345] Results :

[0346] The results obtained are presented in the tables below.

[0347] Primary culture

[0348] [Table 3]

[0349] Secondary culture

[0350] [Table 4] 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 some cells over time, which explains why the signal obtained with compound 1 without IL-1 stimulation decreases over time, but the RPE signal was not normalized to the amount of protein.Using compound 1, 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 1 can detect part of the response of rabbit chondrocytes to IL-1.

[0351] Example 4: Preparation and characterization of emulsions according to the invention

[0352] Nanoemulsions comprising compound 1 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 heated separately to 40°C, 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 obtained 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 1 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.

[0353] [Table 5]

[0354] Droplet Size Determination and Size Distribution The hydrodynamic diameter and polydispersity index (PDI) of the nanoemulsion droplets were determined by photon correlation spectroscopy using a Zetasizer® Nano ZS (Malvern Panalytical, Worcestershire, UK). Approximately 10 pL 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-average diameter (Z-Ave) and PDI.

[0355] Determination of zeta potential

[0356] The Zeta potential (ZP) of the nanoemulsions was measured by laser Doppler velocimetry using the Zetasizer® Nano ZS (Malvern 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.

[0357] 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 1 at the final concentration of 20 or 100 mM (NE including compound 1).

[0358] [Table 6]

[0359] Encapsulation efficiency of emulsions

[0360] The encapsulation efficiency of compound 1 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 1 content using the SHIMADZU LC-20 HPLC system with a Nucleosil 100 Å 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 1 was performed at 254 nm.Compound 1 was diluted in the mobile phase at concentrations ranging from 10 pM to 50 pM and run through the HPLC-UV system to obtain the calibration curve of formula y (UA) = 2474.4*x(pM concentration) + 5717.1 (R. 2 = 0.9986).

[0361] Using this calibration curve, the concentration of compound 1 in the aqueous phase was estimated and the encapsulation efficiency (EE) determined as shown below:

[0362] [Math 1]

[0363] Total compound 1 in nanoemulsion — compound 1 in aqueous phase

[0364] EE(%) = - - - — - - — - x 100

[0365] Total in composition 1

[0366] The drug loading capacity (DL) of the nanoemulsion encapsulating the highest concentration of compound 1 (0.5 M in the oil phase) in this study was estimated as follows:

[0367] [Math 2]

[0368] Mass of compound 1 in the oil phase DL(%) = - 77 - - - , , - x 100

[0369] Total mass in the oil phase

[0370] In all cases, compound 1 was not detected in the aqueous phase (<0.01%), indicating almost complete encapsulation of compound 1 in the oil phase. Neither the oil type nor the Span 80:Kolliphor 188 ratio had any effect on the retention of compound 1 in the oil phase. The probe precursor loading capacity for olive oil-based or Labrafac®-based nanoemulsions encapsulating the highest amount of compound 1 (final concentration 100 mM) in this study was estimated to be approximately 33.33%.

[0371] Stability under storage conditions

[0372] The stability of the nanoemulsions was studied by photon correlation spectroscopy as previously described for droplet size determination. The analysis of droplet size and size distribution was performed 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 performed on two independent samples for each formulation. No visible phase separation was observed for the formulations throughout the study period. All formulations demonstrated good stability.

[0373] Example 5: Evaluation of the cytotoxicity of nanoemulsions comprising compound 1

[0374] A dye exclusion assay was used to study the cytotoxicity of compound 1 nanoemulsions on brain endothelial cells (b. End 3). Initially, cells were cultured in an appropriate medium composed of DMEM medium, 10% FCS, penicillin (100 IU / mL), and streptomycin (100 pg / mL). Incubation was performed in a humidified environment at 37°C, with 5% CO2. Subculture to approximately 90% confluence was performed weekly by 1:10 dilution after detaching the cells with trypsin.

[0375] Then, these cells were seeded in 24-well plates (2.5 x 10 5cells / well) and incubated for 24 hours before being treated with the nanoemulsion samples and incubated for an additional 4 hours. Each well was treated with either nanoemulsions loaded with compound 1 (range 0.1 mM - 5 mM), blank nanoemulsions, or culture medium as controls. All formulations were diluted in 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, the harvested cells (20 μL) were mixed with 0.4% trypan blue dye (20 μL) after neutralizing the 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.

[0376] Results :

[0377] [Table 7]

[0378] Cytotoxicity assessment of 1 NE-00 and 1 NE-L1 nanoemulsions revealed cell viability above 80% at concentrations ranging from 0.1 mM to 2.5 mM. For 1 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 1 NE-00 (36.17%) and 1 NE-L1 (50.91%) nanoemulsions.

[0379] Example 6: EPR studies of nanoemulsions comprising compound 1

[0380] Protocols:

[0381] All animal testing procedures were reviewed by the local Animal Ethics Committee (CEEA 34), in accordance with French regulations and the European Communities Council 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. 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 chow.

[0382] In vivo EPR studies of nanoemulsions comprising compound 1

[0383] Nanoemulsions loaded with compound 1 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. Nanoemulsions were prepared with final concentrations of compound 1 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.

[0384] EPR measurements were performed using a Bruker Elexsys 540 EPR spectrometer (Bruker, Wissembourg, France) operating at 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. Spectra were recorded sequentially as a function of time for approximately 100 minutes. Bruker Xepr software was used for data acquisition and processing.

[0385] Ex vivo EPR studies of nanoemulsions comprising compound 1

[0386] An ex vivo study was conducted to evaluate the NE distribution of compound 1 and confirm the intracellular cleavage of compound 1 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 10–15 mL of saline containing 25 U / mL heparin. 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.

[0387] Homogenates of each organ were prepared by mixing with saline (0.1 g wet tissue / 0.1 mL saline) and disintegrating 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) that was folded (V-shaped) into a 4 mm quartz tube and then placed in the EPR cavity. 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.

[0388] 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 1 to the nitroxide visible by EPR.

[0389] Results :

[0390] No acute toxicity was observed during in vivo experiments.

[0391] As mentioned earlier, the probe from compound 1 is an EPR-invisible probe that is cleaved intracellularly by esterase to free hydroxylamine. The resulting hydroxylamine reacts under oxidative stress conditions to produce a nitroxide radical that is visible by EPR. 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 1 NEs for the entire recording time, i.e., 100 min. Neither the concentration of compound 1 in the oil phase nor the type of oil used to formulate the nanoemulsions affected their in vivo stability in healthy mice.

[0392] In vivo results suggest that the probe from compound 1 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 reaching different tissues and being cleaved and / or oxidized. First, EPR acquisition of organ homogenates was performed. Then, potassium ferricyanide [K3(FeCNe)] was added gradually to recover the total amount of cleaved probe 1 precursor distributed to each organ. Potassium ferricyanide oxidizes the free hydroxylamine product of probe 1 to a nitroxide visible by EPR. For each organ, the addition of the oxidant led to an increase in the EPR signal intensity, thus indicating the sensitivity of the formulated probes to oxidizing species.

[0393] The results obtained (mean ± standard deviation) are shown in the following Table 8: [Table 8]

[0394] An EPR signal was observed in different organs, indicating the successful absorption of compound 1. 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. Furthermore, EPR evaluation revealed a higher accumulation of compound 1 in the liver compared to other organs. The preferential accumulation of the probe precursor in the liver was observed for both tested concentrations.

[0395] At the highest dose of compound 1 (0.5 pmol / g body weight) in an olive oil-based nanoemulsion with Span80:K188 = 80:20 (emulsion 1 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, Figure 3) and a nitroxide in a more lipophilic environment such as cell membranes (arrows, Figure 3). Traces of protein-bound nitroxide were detected after the addition of ferricyanide (triangle, Figure 3). An unidentified six-line species (AN = 1.58 mT, AH = 2.36 mT), appeared upon oxidation in blood (diamonds, Figure 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. Example 7: MRI study of nitroxide radicals derived from compounds of formula.

[0396] As mentioned previously, the compounds of formula (I) have the ability to penetrate inside cells and be hydrolyzed by the esterases of said cells into a probe comprising a hydroxylamine unit. Under the action of oxidative stress, this hydroxylamine unit is oxidized into a nitroxide radical which can serve as a contrast agent in MRI by shortening the T1 and T2 relaxation times of water protons. For model reasons, we measured the effect of nitroxide radicals derived from the different compounds of formula (I) on the relaxation times in a mixture of 90% by volume of 0.1 M potassium phosphate buffer and pH 7.4 and 10% DMSO in order to demonstrate their ability to act as an MRI contrast agent.

[0397] MRI measurement protocol

[0398] MRI image acquisitions were performed on test objects consisting of samples containing one of the nitroxides of interest at concentrations of 0; 0.5; 1; 2; 5; 10 and 15 mM. Each nitroxide was dissolved in DMSO and then diluted in 0.1 M potassium phosphate buffer and pH 7.4, so as to obtain a final proportion of DMSO of 10% by volume in the final mixture regardless of the nitroxide concentration. Samples were prepared in 5 mm internal diameter tubes with 0.6 mL of solution. The tubes were placed in a horizontal and parallel position for image acquisition. Measurements were performed using 5 tubes at a time and repeated to analyze samples at all concentrations.

[0399] MRI studies were performed at approximately 21 °C (room temperature) with a Bruker BioSpec 7 T system interfaced with an Advance III spectrometer (Bruker BioSpin MRI GmbH). T1 mapping was obtained by a saturation-recovery pulse sequence (echo time (TE) = 5.9 ms, number of acquisitions (NA) = 1, number of repetitions (NR) = 1, slice thickness = 3 mm, field of view (FOV) = 25 x 25 mm 2 , matrix = 125 x 125, 15 recovery time (TR) values ​​= 63, 148 - 6500 - 100 - 5000 - 200 - 4000 - 400 - 3000 - 600 - 2500 - 800 - 2000 - 1000 - 1600 - 1300 ms). T2 mappings were recorded using a multi-spin multi-echo (MSME) sequence (32 TE values ​​ranging from 25 ms to 800 ms with 25 ms increments, TR = 3500 ms, NA = 1, NR = 1, slice thickness = 3 mm, FOV = 25 x 25 mm 2, matrix = 125 x 125). MRI image processing was performed with ITK-SNAP 4.2.0 software (Free Software Foundation, Inc).

[0400] Results: The results of the MRI studies showed an increase in contrast in the different samples containing varying concentrations of nitroxides. The relaxation times Ti and T2 of water protons decrease under the effect of the increasing nitroxide concentration. The longitudinal 1 / Ti and transverse I / T2 relaxation rates increase linearly with the nitroxide concentration. The slopes of the corresponding lines give access respectively to the longitudinal n and transverse r2 relaxivity values ​​which are grouped in the following table. These values ​​are in agreement with those of the literature for organic monoradicals of the nitroxide family.

[0401] [Table 9]

[0402]

[0403] The relatively low values ​​are due to the imperfect solubility of the compound in the solvent mixture (slight cloudiness observed).

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): being independently 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) is chosen from the following units (lll-a), (I ll-b), (II lc), (lll-d) and (lll-e): and (lll-e) R a Rb, Rc and Rd being, for each pattern (lll-a), (lll-b), (lll-c), (lll-d) and (lll-e), independently selected from linear or branched C1-C4 alkyls.

2. Compound according to claim 1, wherein A is selected from the group consisting of the substituent (ll-b) in which Z = Me and the substituent (ll-a) in which R = H, and A' is selected from the group consisting of the substituent (ll-b) in which Z = Me and the substituent (ll-a) in which R = Me.

3. Compound according to claim 1 or 2, wherein R' is methyl or ethyl, preferably methyl.

4. 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 (Ic):

5. Compound according to any one of claims 1 to 4, in which the unit of formula (N) is chosen from the units (lll-b), (lll-c), (lll-d) and (lll-e).

6. Compound according to any one of claims 1 to 5, in which Ra, Rb, Rc and Rd are identical, and chosen from methyl and ethyl.

7. Compound according to any one of claims 1 to 6, chosen from:

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 from 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. 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, - 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 any one of claims 1 to 7, or with the composition according to any one of claims 8 to 11, by electronic paramagnetic resonance spectroscopy.

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

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