Piperidine, pyrrolidine and isoindole precursors for probes for oxidative stress

Abstract

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

Description

[0001] The present invention relates to new precursors for oxidative stress probes and various uses thereof.

[0002] One of the biggest challenges facing society is to promote the positive healthy aging of the population. To achieve this, new diagnostic methods or effective therapies for cardiovascular and nervous system diseases or cancer must be discovered. It is increasingly evident that oxidative stress has a major impact on the progression of all these diseases. Therefore, non-invasive methods for detecting and mapping oxidative stress in vivo would provide valuable information about the development of these diseases and help in the design of diagnostic and therapeutic tools.

[0003] Protein and lipid oxidation biomarkers in biological fluids are interesting tools, but they do not allow the localization of the lesion. This is why structural and / or functional imaging methods based on positron emission tomography (PET), susceptibility-weighted imaging (SWI) and magnetic resonance imaging (MRI) are usually used. However, these methods have certain limitations. PET requires expensive, short-lived radioactive tracers and needs to be produced in a local cyclotron and is not compatible with certain lesions. Susceptibility imaging (SWI) evaluates the metal content of tissues (Cu, non-haem iron) as a potential source of reactive oxygen species (ROS) through the Fenton reaction, but many regions accumulate iron (for example in ferritins) without causing the excessive production of free radicals. More particularly, magnetic resonance spectroscopy is able to measure some antioxidant defense substances (ascorbate, glutathione), but ignores the essential enzymes involved in ROS detoxification.

[0004] Methods using Electron Paramagnetic Resonance (EPR) and exogenous molecular probes have proven that they can be used to assess and map the "redox state" in the brain under oxidative stress conditions, for example in rat models suffering from hypertension, stroke or epilepsy, and in rodents exposed to ischemia-reperfusion injury. The American Heart Association even considers EPR associated with the use of molecular probes as the preferred method for measuring redox physiology (K.K. Griendling, R.M. Touyz, J.L. Zweier, S. Dikalov, W. Chilian, Y.-R. Chen, D.G. Harrison, A. Bhatnagar, Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signalling in the Cardiovascular System: A Scientific Statement From the American Heart Association, Circ. Res. 119 (2016) e39-e75 (doi: 10.1161 / RES.0000000000000110)).

[0005] Among the known molecular probes, we can distinguish the probes from the hydroxylamine family, which are oxidized in the cell into nitroxides detectable by EPR.

[0006] In particular, precursors of these probes such as the following compound 1-acetyloxy-3- aminocarbonyl-2,2,5,5-tetramethylpyrrolidine (ACP) are known to be used:

[0007]

[0008] This compound comprises an ester function protecting the hydroxylamine group. The purpose of these precursors is to transport the probe into the cell while minimizing degradation. Masking the hydroxylamine function limits the spontaneous oxidation of the hydroxylamine before its internalization into the cell, which facilitates handling by non-chemists. Once internalized, the ester function is hydrolyzed by the esterases of the cell, releasing the hydroxylamine moiety which can be oxidized into a nitroxide and become detectable by EPR.

[0009] However, these probes have many limitations, such as their low biological stability, in particular in vivo, their lack of specificity and / or sensitivity, and the small number of commercially available probes.

[0010] There is therefore a need for new, more efficient molecular probes. This need is even more acute in view of the recent commercialization of new technical progress in EPR spectrometers and the limitations of probes that are hindering the development of redox EPR applications.

[0011] It is therefore an object of the present invention to propose new molecular probes for oxidative stress that offer improved performances compared to existing probes.

[0012] In particular, it is an object of the present invention to propose new molecular probes for oxidative stress that offer improved stability and / or better sensitivity and / or simpler handling.

[0013] To this end, the present invention relates to a compound of formula (I):

[0014]

[0015] wherein

[0016] n = 1 or 2, preferably n = 1,

[0017] A and A’ are independently selected from the group consisting of the following substituents (II-a) and (II-b):

[0018] and R and R’ are independently selected from H and linear or branched C1-C4 alkyl, Z is selected from linear or branched C1-C4 alkyl groups and aryl groups optionally substituted by linear or branched C1-C4 alkyl groups and / or by methoxy groups (such as 4-methoxyphenyl), and

[0019] the structural unit of formula (N) is selected from the following structural units (III-a), (III-b), (III-c), (III-d) and (III-e):

[0020] , , , , and ,

[0021] For each structural unit (III-a), (III-b), (III-c), (III-d) and (III-e), Ra, Rb, Rcand Rdare independently selected from linear or branched C1-C4 alkyl.

[0022] Linear or branched C1-C4 alkyl refers to a hydrocarbon chain comprising n carbon atoms, with n equal to 1, 2, 3 or 4, and comprising 2n+2 hydrogen atoms. Examples include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl and t-butyl groups.

[0023] Indeed, the inventors have surprisingly found that the compounds of formula (I) according to the application are probe precursors for oxidative stress, particularly detectable by EPR spectroscopy, with significantly improved performances compared to prior art compounds, in particular in terms of in vivo stability and sensitivity. Without wishing to be bound by any theory, the inventors believe that the Ra, Rb, Rcand Rdgroups of the structural unit of formula (N) induce a steric hindrance in the prior art probe precursors, which prevents the hydrolysis of the ester protecting the hydroxylamine function by the esterases of the cell. Indeed, as the kinetics of deprotection is slow and the reaction is incomplete, very small amounts of probe are really released into the cell, making it difficult to detect by medical imaging or EPR spectroscopy. In the compounds of formula (I) according to the application, the presence of a spacer group between the nitrogen of the structural unit of formula (N) and the carboxyl targeted by the esterase in the group A’ allows a fast and complete deprotection of the hydroxylamine function. For example, when A’ is a substituent (II-a) with R = R’ = CH3, 100% of the hydroxylamine is released in 10 minutes at 37°C, whereas under the same conditions, the deprotection of the commercial ACP probe (1-acetyloxy-3- aminocarbonyl-2,2,5,5-tetramethylpyrrolidine) is limited to 27%. Thus, the compounds of formula (I) according to the application allow a more convenient handling (adapted to biological applications by non-chemists) and a more efficient release of the probe, resulting in an increased sensitivity.

[0024] In formula (I), and except in the case where the structural unit of formula (N) is a structural unit of formula (III-e), the skilled person will understand that each of the groups can be bonded to any substitutable carbon atom (bearing at least one hydrogen atom) of one or more rings of the structural unit of formula (N) constituting formulae (III-a), (III-b), (III-c) and (III-d). The groups can be bonded to any substitutable carbon atom (bearing at least one hydrogen atom) of one or more rings of the structural unit of formula (N) constituting formulae (III-a), (III-b), (III-c) and (III-d).

[0025] The presence of the group A in the compounds of formula (I) advantageously improves the accumulation of the probe in the cell and thus increases the amount of probe detectable in the cell after oxidation and thus improves the quality of the image or signal obtained. The group A allows the compound to passively penetrate the cell and to be sensitive to the hydrolysis by esterases in the cell, which leads to the release of a negatively charged carboxylate ion that does not freely diffuse through the membrane.

[0026] It will be understood that each atom of the compounds of formula (I) can be present in the form of any of its natural isotopes.

[0027] In one particular embodiment, the nitrogen atom of the structural unit of formula N is nitrogen-15.

[0028] Alternatively or in combination, the hydrogen atoms of the structural unit of formula (N), in particular the hydrogen atoms of the Ra, Rb, Rcand Rdgroups, are preferably hydrogen-2 (more commonly referred to as deuterium).

[0029] The isotopic composition of the compounds of formula (I), in particular their nitrogen-15 and / or hydrogen-2 content, is determined using techniques known to the person skilled in the art. For example, high resolution mass spectrometry (HRMS) can be used to determine the average concentration of the isotopes of interest at all sites of the compounds of formula (I). Quantitative nuclear magnetic resonance (NMR) of deuterium and nitrogen-15 can also be used, this technique allowing to study the specific isotopic distribution at each position of the compounds of formula (I). Similarly, after hydrolysis and oxidation of the compounds of formula (I), for example by potassium ferricyanide, the electron paramagnetic resonance (EPR) spectrum is different for molecules containing nitrogen-15 (2 lines) and nitrogen-14 (3 lines).

[0030] Preferably, in the substituent (II-b), Z is selected from the group consisting of methyl, ethyl, propyl, isopropyl, tert-butyl, phenyl and 4-methoxyphenyl. Advantageously, for the remainder of the present specification, Z = Me.

[0031] Preferably, A is selected from the group consisting of the substituent (II-b) wherein Z = Me and the substituent (II-a) wherein R = H, and A' is selected from the group consisting of the substituent (II-b) wherein Z = Me and the substituent (II-a) wherein R = Me.

[0032] Preferably, R' is methyl or ethyl, preferably methyl.

[0033] Preferably, A and A' are identical.

[0034] According to certain embodiments, A = A' = (II-a).

[0035] According to other embodiments, A = A' = (II-b).

[0036] The use of the group (II-b) advantageously avoids the release of one equivalent of acetaldehyde and one equivalent of formaldehyde upon esterase action, which can cause potential toxicity issues at very high doses and potential cellular stress that can interfere with oxidative stress measurements. Indeed, the hydrolysis of the group (II-b) by esterases only leads to the formation of the non-toxic 3-hydroxybutan-2-one and 2,3-butanediol.

[0037] Preferably, the structural unit of formula (N) is selected from the group consisting of the structural units (III-b), (III-c), (III-d) and (III-e), preferably from the group consisting of the structural units (III-b), (III-c) and (III-d), more preferably (III-b) and (III-d), or alternatively (III-c).

[0038] Preferably, the compound of formula (I) is selected from the following compounds (I-a), (I-b), (I-b”), (I-c), (I-c”), (I-d), (I-d’), (I-d”), (I-d”’) and (I-e):

[0039] , , , , , , , , and ,

[0040] For each compound (I-a), (I-b), (I-b”), (I-c), (I-c”), (I-d), (I-d’), (I-d”), (I-d”’) and (I-e), A, A’, Ra, Rb, Rcand Rdare each independently as defined above.

[0041] Preferably, the compound of formula (I) is selected from the compounds (I-a), (I-b), (I-b”), (I-c), (I-d) and (I-e), preferably from the compounds (I-a), (I-b), (I-b”), (I-c) and (I-d), more preferably from the compounds (I-b), (I-c) and (I-d), even more preferably (I-b) and (I-d), or alternatively (I-c).

[0042] More preferably, the compound of formula (I) is selected from the following compounds (IV), (V), (VI) and (VII),

[0043] , , and ,

[0044] A and A’ are as defined above.

[0045] Preferably, the compound of formula (I) is selected from the compounds (IV), (V) or alternatively from the compound (VI).

[0046] Preferably, Ra, Rb, Rcand Rdare independently selected from methyl, ethyl and isopropyl, preferably from methyl and ethyl. Preferably, Ra, Rb, Rcand Rdare identical and preferably selected from methyl and ethyl.

[0047] The Ra, Rb, Rcand Rdgroups advantageously enable better image or signal quality to be obtained. Without being bound by any theory, the inventors believe that the steric hindrance induced by the four groups Ra, Rb, Rcand Rdstabilizes the nitroxide radical and also reduces the superoxide dismutase mimetic activity of the generated nitroxide, which improves the response of the hydroxylamine probe to superoxide anion production.

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

[0049] and .

[0050] According to one embodiment, the compound of formula (I) is the compound

[0051] .

[0052] According to another embodiment, the compound of formula (I) is the compound

[0053] .

[0054] According to one alternative embodiment, the compound of formula (I) is different from the following compounds:

[0055] .

[0056] The present application also relates to a process for preparing a compound of formula (I) according to the present application. This process is in particular as described in the examples.

[0057] A typical process for preparing a compound of formula (I) according to the present application comprises at least one step (S1 ) of reducing a compound of formula (X)

[0058] to obtain a compound of formula (X'),

[0059]

[0060] followed by a step (S1 ') of nucleophilic addition of compound (X') to a compound of formula (XI)

[0061]

[0062] or of formula (XI'),

[0063]

[0064] to obtain respectively a compound of formula (XII)

[0065]

[0066] Or compounds of formula (XII'),

[0067]

[0068] The structural units A, A', n, R and formula (N) are as defined above for compounds of formula (I).

[0069] Preferably, step (S1) is carried out in the presence of carbon-supported palladium and dihydrogen. The dihydrogen is then removed by purging with argon gas before step (S1').

[0070] Preferably, the step (S1') of adding the compound of formula (XI) to the compound of formula (X') is carried out between -5°C and 8°C, and then the reaction mixture is stirred at room temperature (20°C-28°C) for 12 to 20 hours.

[0071] The method may further include step (S2): reacting the compound of formula (XII) with the compound of formula (XIII) R'-C(O)-O - Nucleophilic substitution is performed between (XIII) (R' as defined above for compounds of formula (I)) to obtain compounds of formula (I) according to the invention.

[0072] Preferably, step (S2) is carried out in the presence of acetate, preferably mercuric acetate. Preferably, the reaction mixture is stirred at room temperature (20°C-28°C) for 12 to 20 hours.

[0073] The method may also include step (S0): in equation (XIV)

[0074]

[0075] The compound and formula (XV)

[0076]

[0077] Nucleophilic substitution occurs between compounds.

[0078] To obtain a compound of formula (X),

[0079] The structural units of A, n and formula (N) are as defined above for compounds of formula (I), and X = Cl or Br, preferably A = (II-a).

[0080] Preferably, step (S0) is carried out in the presence of an inorganic base, more preferably in the presence of potassium carbonate. Preferably, the reaction mixture is stirred at room temperature (20°C-28°C) for 1 to 5 hours.

[0081] Alternatively, the method can also comprise a step (S0') of activating the compound of formula (XIV) to an acyl chloride of formula (XIV')

[0082]

[0083] followed by a step (S0'') of coupling between the compound (XIV') and the compound (XV)

[0084]

[0085] to obtain a compound of formula (X), A, n and the structural unit of formula (N) being as defined above for the compound of formula (I), preferably A = (II-b).

[0086] The activation step (S0') is preferably carried out in the presence of an acyl dichloride, such as oxalyl chloride and dimethylformamide.

[0087] The step (S0'') is preferably carried out in the presence of a base, such as triethylamine and 4-dimethylaminopyridine. Preferably, the reaction mixture is stirred at room temperature (20-28°C) for 10 hours to 48 hours.

[0088] Any alternative method well known in the state of the art can be used.

[0089] The present application also relates to a pharmaceutical composition comprising at least a compound of formula (I) according to the present application in a physiologically acceptable medium.

[0090] Preferably, the pharmaceutical composition according to the present application is in the form of a nanoemulsion, preferably in the form of an oil-in-water nanoemulsion.

[0091] Preferably, the nanoemulsion comprises an aqueous phase and an oily phase comprising the compound of formula (I). Preferably, at least 90% by weight, preferably 95% to 99.99% by weight of the total mass of the compound of formula (I) in the nanoemulsion is comprised in the oily phase.

[0092] Preferably, the oily phase further comprises at least one oily compound, the at least one oily compound comprising a triglyceride, preferably a C4-C24 triglyceride, more preferably a C8-C18 triglyceride. Triglycerides are generally triesters of fatty acids and glycerol, the fatty acids of which can have a chain length of C4 to C24, these fatty acids being linear or branched, saturated or unsaturated.

[0093] Preferably, the oily compound is selected from the group consisting of a plant oil and a medium-chain triglyceride having a C6-C12 chain, preferably a C8-C10 chain.

[0094] ​The triglyceride according to the application is in particular wheat germ oil, sunflower oil, grape seed oil, sesame oil, corn oil, apricot oil, castor oil, shea oil, avocado oil, olive oil, soybean oil, almond oil (in particular sweet almond oil), palm oil, rapeseed oil, cottonseed oil, hazelnut oil, macadamia oil, jojoba oil, alfalfa oil, poppy oil, pumpkin oil, sesame oil, zucchini oil, rapeseed oil, black currant oil, evening primrose oil, millet oil, barley oil, quinoa oil, rye oil, safflower oil, candlenut oil, passionflower oil, muscadine oil and coconut oil; or a triglyceride of caprylic / capric acid.

[0095] Advantageously, the oily compound of the oily phase is selected from olive oil and Labrafac ® The lipophilic WL 1349.

[0096] Preferably, the aqueous phase comprises water and optionally NaOH, NaCI, glycerol, sorbitol, dextrose or xylitol. Preferably, the aqueous phase comprises NaOH so as to adjust the pH of the nanoemulsion to between 6.8 and 7.2, preferably about 7, and optionally glycerol in an amount preferably ranging from 0.5% to 5% by weight relative to the total weight of the emulsion.

[0097] Preferably, the composition, preferably the nanoemulsion, further comprises at least one surfactant, preferably a non-ionic surfactant, the at least one surfactant being preferably selected from silicone surfactants, polysorbates, cetostearyl alcohol, cetyl alcohol, oleyl alcohol, stearyl alcohol, cocamide DEA, cocamide MEA, polyalkyl glucosides, decyl glucoside, lauryl glucoside, octyl glucoside, monolaurin, poloxamer, sorbitan monostearate, sorbitan tristearate or any combination thereof, preferably from poloxamer and sorbitan monostearate.

[0098] 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.

[0099] Advantageously, the first surfactant is sorbitan monostearate and the second surfactant is selected from poloxamer, preferably poloxamer 188.

[0100] An example of sorbitan monostearate is the commercial product Span ® 80. An example of poloxamer is the commercial product Kolliphor ® P 188.

[0101] Preferably, the total mass content of surfactants ranges from 4% to 12%, preferably from 5% to 10%, relative to the total mass of the composition, preferably the nanoemulsion.

[0102] Preferably, the mass content of the first surfactant is in the range of 3% to 7%, preferably in the range of 4% to 6%, relative to the total mass of the composition, preferably of the nanoemulsion.

[0103] Preferably, the mass content of the second surfactant is in the range of 1% to 5%, preferably in the range of 1% to 4%, relative to the total mass of the composition, preferably of the nanoemulsion.

[0104] Preferably, in the composition, preferably in the nanoemulsion, the mass ratio between the first surfactant and the second surfactant is in the range of 1 to 6, preferably of 1.2 to 4.5, advantageously of 1.5 to 4.2.

[0105] Preferably, in the composition, preferably in the nanoemulsion, the mass ratio between the aqueous phase and the oily phase is in the range of 1 to 8, preferably of 2 to 6, preferably of 3 to 5, advantageously of 3.5 to 4.5.

[0106] Preferably, in the composition, preferably in the nanoemulsion, the mass ratio between the oily compound and the total surfactant is in the range of 1 to 5, preferably of 1.5 to 4, preferably of 2 to 3.5, advantageously of 2.5 to 3.5.

[0107] Preferably, the nanoemulsion has an average droplet size in the range of 50 nm to 500 nm, preferably in the range of 80 nm to 300 nm, advantageously in the range of 100 nm to 200 nm.

[0108] Preferably, the nanoemulsion has a droplet size distribution in the range of 0.05 to 0.20, preferably in the range of 0.07 to 0.15.

[0109] The average droplet size is determined by photon correlation spectroscopy using a Zetasizer Nano ZS (Malvern Panalytical, Worcestershire, UK) at 25 °C. 10 pL of the nanoemulsion are diluted 100-fold with ultrapure water. All measurements are recorded at a scattering angle of 173°. The correlation function curves are fitted using an exponential fit law (cumulant method) to evaluate the hydrodynamic diameter and the PDI. All measurements are performed in triplicate to determine the average droplet size expressed as the average Z diameter (Z-Ave) and the PDI. ® Nano ZS (Malvern Panalytical, Worcestershire, UK) at 25 °C. 10 pL of the nanoemulsion are diluted 100-fold with ultrapure water. All measurements are recorded at a scattering angle of 173°. The correlation function curves are fitted using an exponential fit law (cumulant method) to evaluate the hydrodynamic diameter and the PDI. All measurements are performed in triplicate to determine the average droplet size expressed as the average Z diameter (Z-Ave) and the PDI.

[0110] Preferably, in the nanoemulsion, the mass ratio between the compound of formula (I) and the oily phase is in the range of 0.1 to 0.5, preferably of 0.15 to 0.45, more preferably of 0.2 to 0.4, most preferably of 0.25 to 0.35, advantageously of 0.30 to 0.35.

[0111] Preferably, the molar concentration of the compound of formula (I) in the oil phase is in the range of 0.01 mol / L to 0.5 mol / L, preferably 0.05 mol / L to 0.25 mol / L, advantageously 0.08 mol / L to 0.12 mol / L.

[0112] Preferably, the molar concentration of the compound of formula (I) in the nanoemulsion is in the range of 1 mmol / L to 500 mmol / L, preferably 5 mmol / L to 250 mmol / L, advantageously 10 mmol / L to 50 mmol / L.

[0113] The present application also relates to the use of a compound of formula (I) or a composition according to the present application as a precursor of a compound for electron paramagnetic resonance spectroscopy or for imaging, preferably medical or preclinical imaging, in particular for electron paramagnetic resonance spectroscopy, for electron paramagnetic resonance imaging or for magnetic resonance imaging.

[0114] According to one embodiment, a compound of formula (I) or a composition according to the present application, preferably a nanoemulsion, is used as a radical probe precursor for electron paramagnetic resonance spectroscopy or for electron paramagnetic resonance imaging.

[0115] According to another embodiment, a compound of formula (I) or a composition according to the present application, preferably a nanoemulsion, is used as a contrast agent precursor for magnetic resonance imaging.

[0116] As mentioned above, the compound of formula (I) has the ability to penetrate into a cell and to be hydrolyzed by an esterase in said cell to generate a probe comprising a hydroxylamine moiety.

[0117] This hydroxylamine building block can be oxidized under the action of oxidative stress in the cell to become a nitroxide radical which can be directly detected by spectroscopy or electron paramagnetic resonance imaging. Paramagnetic nitroxide radicals can also induce changes in the longitudinal (T1) and / or transverse (T2) magnetic relaxation times of nearby water protons or even aliphatic protons in the tissue, leading to changes in contrast in magnetic resonance imaging.

[0118] Preferably, the compound of formula (I) or the composition according to the present application is used in vivo or in vitro.

[0119] According to one embodiment, the compound of formula (I) or the composition according to the present application is used in vitro.

[0120] The present application also relates to a method for detecting oxidative stress in vitro, the method comprising the steps of:

[0121] - contacting a compound of formula (I) or a composition according to the present application with a medium comprising a cell, a fluid or a biological tissue, and

[0122] - recording a signal from the medium by electron paramagnetic resonance spectroscopy, or capturing one or more images of the medium by an imaging technique, preferably a medical or preclinical imaging technique, preferably by electron paramagnetic resonance imaging or magnetic resonance imaging.

[0123] The present application also relates to a method for detecting oxidative stress in a subject in vivo, the method comprising:

[0124] - a step of capturing one or more images of the subject or of at least one portion of the subject that has been previously treated with a compound of formula (I) according to the application or with a composition according to the application, using an imaging technique, preferably a medical or preclinical imaging technique, preferably electron paramagnetic resonance imaging or magnetic resonance imaging.

[0125] - or a step of recording a signal from the subject or from at least one portion of the subject that has been previously treated with a compound of formula (I) according to the application or with a composition according to the application, by electron paramagnetic resonance spectroscopy.

[0126] Preferably, the subject is treated with an amount detectable by said imaging technique or by said electron paramagnetic resonance spectroscopy.

[0127] According to the application, by "detectable amount" is meant an amount of compound (I) or of composition sufficient to obtain an interpretable image by the imaging technique considered or an interpretable spectrum by said electron paramagnetic resonance spectroscopy.

[0128] Preferably, the method for detecting oxidative stress in a subject in vivo comprises the following steps:

[0129] - administering to the subject an amount of a compound of formula (I) according to the application or of a composition detectable by said imaging technique,

[0130] - capturing an image of the treated subject or of at least one portion of the subject by said imaging technique, preferably by electron paramagnetic resonance imaging or magnetic resonance imaging, or recording a signal from at least one portion of the subject by electron paramagnetic resonance spectroscopy.

[0131] According to one embodiment, the method for detecting oxidative stress in vivo according to the application comprises capturing an image of the treated subject or of at least one portion of the subject by electron paramagnetic resonance imaging.

[0132] According to another embodiment, the method for detecting oxidative stress in vivo according to the application comprises capturing an image of the treated subject or of at least one portion of the subject by magnetic resonance imaging.

[0133] According to yet another embodiment, the method for detecting oxidative stress in vivo according to the application comprises recording the signal of at least one part of the subject by electron paramagnetic resonance spectroscopy.

[0134] The application also relates to a compound of formula (I) or a composition according to the application for 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.

[0135] Preferably, the compound of formula (I) or the composition according to the application is for use as a diagnostic tool in an in vivo diagnostic method, the diagnostic method preferably comprising an electron paramagnetic resonance spectroscopy technique, an electron paramagnetic resonance imaging technique or a magnetic resonance imaging technique.

[0136] Preferably, the in vivo diagnostic method comprises:

[0137] - a step of image capture of a subject or of at least one part of a subject who has previously been treated with a compound of formula (I) or a composition according to the application in an amount detectable by said imaging technique, the imaging technique preferably being selected from the group consisting of an electron paramagnetic resonance imaging technique and a magnetic resonance imaging technique,

[0138] - or a step of electron paramagnetic resonance spectroscopy recording of a signal from a subject or from at least one part of a subject who has previously been treated with a compound of formula (I) or a composition according to the application in an amount detectable by said electron paramagnetic resonance spectroscopy.

[0139] The application also relates to a compound of formula (I) or a composition according to the application for use as a free radical probe precursor in an in vivo diagnostic method, the method preferably comprising an electron paramagnetic resonance imaging technique.

[0140] The application also relates to a compound of formula (I) or a composition according to the application for use as a contrast agent precursor in an in vivo diagnostic method, the method preferably comprising a magnetic resonance imaging technique.

[0141] The application also relates to a compound of formula (I) or a composition according to the application for use as a contrast agent precursor in an in vivo diagnostic method, the method preferably comprising an electron paramagnetic resonance spectroscopy technique.

[0142] The application also relates to a compound of formula (I) or a composition according to the application for use in a method for detecting a disease in a subject, the method comprising the steps of:

[0143] - capturing one or more images of a healthy subject who has previously been treated with a compound of formula (I) or a composition according to the application in an amount detectable by said imaging technique by an imaging technique, preferably medical imaging,

[0144] - capturing one or more images of a diagnosed subject that has been previously treated with an amount of a compound or composition of formula (I) according to the application detectable by said imaging technique,

[0145] - determining the difference between the healthy subject and the diagnosed subject, and

[0146] - comparing the difference to a reference value in order to determine the presence or absence of the disease in the diagnosed subject.

[0147] The application also relates to a compound or composition of formula (I) according to the application for use in a method of detecting a disease in a diagnosed subject, the method comprising the steps of:

[0148] - recording a signal from a healthy subject that has been previously treated with an amount of a compound or composition of formula (I) according to the application detectable by said electron paramagnetic resonance spectroscopy,

[0149] - recording a signal from a diagnosed subject that has been previously treated with an amount of a compound or composition of formula (I) according to the application detectable by said electron paramagnetic resonance spectroscopy,

[0150] - determining the difference between the healthy subject and the diagnosed subject, and

[0151] - comparing the difference to a reference value in order to determine the presence or absence of the disease in the diagnosed subject.

[0152] The application also relates to a compound or composition of formula (I) according to the application for use in a method of monitoring a disease in a subject, the method comprising the steps of:

[0153] - capturing one or more images of the subject that has been previously treated with an amount of a compound or composition of formula (I) according to the application detectable by said imaging technique at a time t0,

[0154] - capturing one or more images of the subject that has been previously treated with an amount of a compound or composition of formula (I) according to the application detectable by said imaging technique at a time t1 after the time t0,

[0155] - determining the difference between the images captured at the time t0 and the images captured at the time t1,

[0156] - determining the change in the disease.

[0157] The application also relates to a compound or composition of formula (I) according to the application for use in a method of monitoring a disease in a subject, the method comprising the steps of:

[0158] - at a time t0, recording at least one signal from a subject who has been previously treated with an amount of a compound or composition of formula (I) according to the application detectable by said electron paramagnetic resonance spectroscopy,

[0159] - at a time t1, after time t0, recording at least one signal from a subject who has been previously treated with an amount of a compound or composition of formula (I) according to the application detectable by said electron paramagnetic resonance spectroscopy,

[0160] - determining the difference between the signal recorded at time t0 and the signal recorded at time t1,

[0161] - determining the change in the disease.

[0162] The application also relates to a compound or composition of formula (I) according to the application for use in a method of determining the efficacy of a therapy of a disease in a subject suffering from the disease, the method comprising the steps of:

[0163] - at a time t0, capturing one or more images of a subject suffering from a disease who has been previously treated with an amount of a compound or composition of formula (I) according to the application detectable by said imaging technique and optionally also with an effective amount of said therapy of the disease,

[0164] - at a time t1, after time t0, capturing one or more images of a subject suffering from a disease who has been previously treated with an amount of a compound or composition of formula (I) according to the application detectable by said imaging technique and optionally also with an effective amount of said therapy of the disease,

[0165] - determining the difference between the image captured at time t0 and the image captured at time t1,

[0166] - determining the efficacy of said therapy.

[0167] These steps can be repeated several times to monitor the therapy over time.

[0168] The application also relates to a compound or composition of formula (I) according to the application for use in a method of determining the efficacy of a therapy of a disease in a subject suffering from the disease, the method comprising the steps of:

[0169] - at a time t0, recording at least one signal from a subject who has been previously treated with an amount of a compound or composition of formula (I) according to the application detectable by said electron paramagnetic resonance spectroscopy,

[0170] - at a time tl, after the time to, recording at least one signal from the diseased subject previously treated with a compound or a composition of formula (I) according to the application in an amount detectable by said electron paramagnetic resonance spectroscopy,

[0171] - determining the difference between the signal captured at time to and the signal captured at time tl,

[0172] - determining the efficacy of said treatment. BRIEF DESCRIPTION OF DRAWINGS

[0173] [ Figure 1 ] Figure 1 is an EPR spectrum obtained with 5 million U87 cells incubated with compound 1 (100 µM) in serum-free DMEM medium, showing the main component (asterisk) characteristic of nitroxide in an isotropic state and the minor component (arrow) characteristic of nitroxide in an intermediate state.

[0174] [ Figure 2 ] Figure 2 is an EPR spectrum obtained with 5 million U87 cells in suspension incubated with compound 1 (100 µM) in the presence (dashed line) and in the absence (solid line) of Tempone- 15

[0175] [ Figure 3 ] Figure 3 are representative EPR spectra obtained in the X-band from blood and brain homogenate of mice injected with compound 1 (0.5 µmol / g body weight) formulated in an oil-in-water nanoemulsion (100 mM NE-OO emulsion 1) before (solid line) and after (dashed line) addition of potassium ferricyanide.

[0176] The application will now be described with reference to the following non-limiting examples.

[0177] Examples

[0178] Example 1 : Synthesis of compounds of formula (I)

[0179] Materials and methods

[0180] ​All reagents were purchased from commercial suppliers and used as received without purification. Thin layer chromatography (TLC) was performed using silica gel 60 F254 TLC plates and preparative column chromatography was performed using silica gel (230-400 mesh). Dry oxygen-free THF was prepared using a Pure Solv™ microsolvation purification system (Sigma Aldrich). NMR spectra were recorded on a Bruker Biospin Advance II 500 MHz or a Bruker ARX 250 250 MHz spectrometer 1 H and 13 C spectra and referenced to the peak of the corresponding solvent (CDC13; δ H = 7,26 ppm, δ C = 77,0 ppm). Fourier transform infrared (IR) spectra were recorded on a Perkin Elmer FT-IR spectrometer equipped with an ATR objective. High resolution mass spectra were recorded on a Thermo Exactive_HCD spectrometer (Thermo Scientific, Les Ulis, France). Electron paramagnetic resonance (EPR) spectra were obtained at 21 °C by an Elexsys E500 spectrometer (Bruker, Wissembourg, France) running at the X band (9.8 GHz) and equipped with a high sensitivity SHQ cavity. Nitrogen coupling constants A N and half-width linewidths AB are given in millitesla (mT). Melting temperatures of solid products were measured using a Stuart ® SMP20 (Cole-Palmer ® ) apparatus.

[0181] General procedure A

[0182] Anhydrous potassium carbonate (2 eq.) was added to a solution of the carboxylic acid derivative (1 eq.) in anhydrous DMSO ([C] = 0.4 M). The mixture was stirred at 22 °C for 5 min, then bromomethyl acetate (1.2 eq. - 1.5 eq.) was added. The reaction was kept under stirring at 22 °C for 2 h. Then a mixture of ice and water was 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 column chromatography on silica gel under the elution conditions indicated for each case to obtain the desired compound.

[0183] General procedure B

[0184] Nitroxide derivative (1 equiv.) and palladium (10%, on carbon) were placed in a flask closed with a septum and purged with 3 vacuum / argon cycles. Dry oxygen-free THF ([C]=0.1M) was introduced under an argon atmosphere and dihydrogen was bubbled into the mixture under stirring for 30 minutes. To remove dihydrogen, argon was bubbled for 5 minutes. After cooling to 0°C, triethylamine (1.5 equiv. - 2.5 equiv.) was added, followed by the chloroformate derivative (1.5 equiv. - 2 equiv.) and the resulting mixture was stirred at 22°C for 3 hours. The reaction mixture was filtered through celite and concentrated under vacuum. The crude product was purified by silica gel column chromatography under the elution conditions indicated for each case to obtain the desired compound.

[0185] General procedure C

[0186] A solution of the chlorinated derivative (1 equiv.) in acetic acid ([C]=0.55M) was added to mercury(II) acetate (1.5 equiv.) in acetic acid ([C]=0.55M) and the mixture was stirred at 22°C overnight, during which 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 bicarbonate solution and then again with water, dried over anhydrous magnesium sulfate and the solvent was removed under vacuum. The crude product was purified by silica gel column chromatography under the elution conditions indicated for each case to obtain the desired compound. In some cases, no purification was required.

[0187] General procedure D

[0188] To a solution of the carboxylic acid derivative (1 equiv.) in dry dichloromethane under argon was added oxalyl chloride (5 equiv.) dropwise, followed by a 0.15M solution of dry dimethylformamide (5 mol%) in dry dichloromethane. The reaction mixture was stirred at 20°C under argon for 2 hours, then the mixture was evaporated under vacuum. In a second flask, 4-hydroxymethyl-5-methyl-1,3-dioxol-2-one (1 equiv.) was dissolved in dry dichloromethane ([C]=0.03M) under argon. Triethylamine (1.2 equiv.) and 4-dimethylaminopyridine (5 mol%) were added under vigorous stirring. The mixture was transferred to the flask containing the previously synthesized acid chloride dissolved in dry dichloromethane and the reaction mixture was stirred at 20°C under argon for 24 hours. After the addition of saturated aqueous sodium bicarbonate solution, the aqueous phase was removed and the organic phase was washed with saturated aqueous sodium chloride solution, 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 under the elution conditions indicated for each case to obtain the desired compound.

[0189] Synthesis of probe precursors incorporating isoindoline building blocks

[0190] Compounds 1 Synthesis of

[0191] 1) 5-acetyloxymethoxycarbonyl-l, 1,3,3-tetraethylisoindolin-2-yloxy radical (S2)

[0192] 5-Carboxy-1,1,3,3-Tetraethylisoindoline-2-yloxy radical S1 was obtained from 4-methylphthalic anhydride according to the procedure published by Fairfull-Smith et al. (KE Fairfull-Smith, F. Brackmann, SEBottle, The Synthesis of Novel Isoindoline Nitroxides Bearing Water-Solubilising Functionality, Eur. J. Org. Chem. (2009) 1902-1915).

[0193] Following general procedure A, S1 (250 mg, 0.861 mmol, 1 equivalent), K2CO3 (236 mg, 1.71 mmol, 2 equivalents), and ethyl bromoacetate (96 µL, 0.975 mmol, 1.2 equivalents) were used. The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 90 / 10) to give the desired compound as a yellow oil (309 mg, 87%).

[0194] Rf = 0.47 (cyclohexane / ethyl acetate, 4:1).

[0195] IR ν max (ATR, ZnSe, pure, cm) -1 ): 2973, 2940, 2881, 1767, 1738, 1617, 1457, 1418, 1370, 1284, 1259, 1231, 1203, 1159, 1101, 1032, 983.

[0196] HRMS (ESI-orbital trap): for C 20 H 29 NO5 calculation m / z [M+H]+: 363.2040; Measured value: 363.2027; For C 20 H 30Calculated [M+2H]+for NO5: 364.2118; found 364.2110.

[0197] EPR (100 µM solution in 0.1 M potassium phosphate buffer at pH = 7.4): A N = 1.54 mT, ΔΒ = 0.17 mT.

[0198] 2) 2-(((l-chloroethoxy)carbonyl)oxy)-l, 1,3,3-tetraethylisoindoline-5-carboxylate acetyloxy methyl ester (S3)

[0199] According to the general procedure B using S2 (262 mg, 0.723 mmol, 1 eq), Pd / C (30 mg), Et3N (151 µL, 1.084 mmol, 1.5 eq) and 1-chloroethyl chloroformate (117 µL, 1.084 mmol, 1.5 eq). The crude was purified by silica gel column chromatography (cyclohexane / AcOEt, 90 / 10) to give the desired compound as a yellowish oil (298 mg, 88%).

[0200] Rf = 0.56 (dichloromethane)

[0201] NMR 1 H (500 MHz, CDCl3) δ (ppm) 8.00 (d, J = 7.9, 1H), 7.77 (s, 1H),7.15 (d, J = 7.9 Hz, 1H), 6.49 (q, J = 5.8 Hz, 1H), 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).

[0202] NMR 13 C (126 MHz, CDCl3) δ (ppm) 169.8; 165.3; 154.0; 147.7; 142.0; 129.1; 128.1; 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; 8.60.

[0203] IR v max (ATR, ZnSe, neat, cm -1 ): 2968, 2942, 2863, 1790, 1690, 1450, 1373, 1282, 1204, 1159, 1080, 1010, 983.

[0204] HRMS (ESI-Orbitrap): [M+H]+calcd for C 23 H 33 ClNO7, 470.1940; found 470.1930. m / z [M+H]+: 470.1940; found 470.1930; for C 23 H 32 ClNNaO7, [M+Na]+492.1760; found 492.1748.

[0205] 3) 2-(((l-acetyloxyethoxy)carbonyl)oxy)-l, 1,3,3-tetraethylisoindoline-5-carboxylate acetyloxy methyl ester (compound 1)

[0206] According to the general procedure C, using 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 was purified by silica gel column chromatography (cyclohexane / AcOEt, 80 / 20) to give the desired compound as a yellowish oil (254 mg, 84%).

[0207] Rf= 0.47 (dichloromethane)

[0208] NMR 1 H (500 MHz, CDCl3) δ (ppm) 7.99 (d, J = 7.9 Hz, 1H), 7.76 (s, 1H), 7.15 (d, J = 7.9 Hz, 1H), 6.77 (q, J = 5.4 Hz, 1H), 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).

[0209] NMR13 C (CDCl3, 125MHz) δ (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.

[0210] IR v max (ATR, ZnSe, pure, cm -1 ): 2968, 2942, 1790, 1765, 1740, 1450, 1373, 1283, 1245, 1204, 1159, 1079, 1032, 1010, 983.

[0211] HRMS (ESI-Orbitrap): [M+H]+calcd for C 25 H 36 NNaO9 516.2204; found 516.2189. m / z [M+H]+: 494.2385; found 494.2370; [M+Na]+: 516.2204; found 516.2189. 25 H 35 NNaO9 516.2204; found 516.2189.

[0212] Compounds 2 Synthesis of

[0213] 1) radical 5-(((5-methyl-2-oxo-l,3-dioxol-4-yl)methoxy)carbonyl)-l, 1,3,3-tetraethylisoindolin-2-yloxy (S4) m / z

[0214] According to the general procedure D, using 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) and DMAP (8.1 mg, 0.066 mmol, 5 mol%). The crude was purified by silica gel column chromatography (cyclohexane / AcOEt, 80 / 20) to yield the desired compound as a yellow oil (180 mg, 34%). 3) N (219 µL, 1.57 mmol, 1.2 equiv) and DMAP (8.1 mg, 0.066 mmol, 5 mol%). The crude was purified by silica gel column chromatography (cyclohexane / AcOEt, 80 / 20) to yield the desired compound as a yellow oil (180 mg, 34%).

[0215] Rf= 0.22 (cyclohexane: ethyl acetate = 4:1)

[0216] IR ν max (ATR, ZnSe, pure, cm) -1 ): 2984, 2942, 2908, 1739, 1447, 1373, 1238, 1098, 1046, 938.

[0217] HRMS (ESI-orbital trap): for C 22 H 28 NO6 calculation methyl-2-(((l-chloroethoxy)carbonyl)oxy)-l, 1,3,3-tetraethylisoindoline-5-carboxylate of (5-methyl-2-oxo-l,3-dioxol-4-yl) (S5) [M]+: 402.1911; Measured value: 402.1916; For C 22 H 29 NO6 calculated [M+H]+: 403.1995; measured value: 403.1957; for C 22 H 30 The calculated value of [M+2H]+ for NO6 is 404.2073; the measured value is 404.2050.

[0218] EPR (100µM solution, dissolved in 0.1M potassium phosphate buffer at pH 7.4, containing 1mM DTPA): A N =1.54mT, ΔB=0.17mT.

[0219] 2) m / z methyl-2-(((l-acetyloxyethoxy)carbonyl)oxy)-l, 1,3,3-tetraethylisoindoline-5-carboxylate of (5-methyl-2-oxo-l,3-dioxol-4-yl) (compound 2)

[0220] Following general procedure B, S4 (65 mg, 0.162 mmol, 1 equivalent), Pd / C (7 mg), Et3N (34 µL, 0.243 mmol, 1.5 equivalent), and 1-chloroethyl chloroformate (26 µL, 0.243 mmol, 1.5 equivalent) were used. The crude product was purified by column chromatography on silica gel (cyclohexane / AcOEt, 90 / 10) to give the desired compound as a pale yellow oil (21 mg, 26%).

[0221] NMR 1 H (500MHz, CDCl3) δ (ppm) 7.99 (d, J = 7.0Hz, 1H), 7.75 (s, 1H), 7.18 (d, J= 7.0Hz, 1H), 6.51 (q, J = 5.6Hz, 1H), 5.11 (s, 2H), 2.27 (s, 3H),2.15 - 2.06 (m, 2H), 2.05 - 1.95 (m, 2H), 1.89 (d, J = 5.6Hz, 3H), 1.85-1.74(m, 4H), 1.06-0.95 (m, 6H), 0.90-0.77 (m, 6H).

[0222] NMR 13 C (126MHz, CDCl3) δ (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.

[0223] IR ν max (ATR, ZnSe, pure, cm -1 ): 2985, 2864, 1710, 1670, 1578, 1414, 1298, 1259, 1121, 1097, 1056, 985, 913.

[0224] HRMS (ESI-Orbitrap): [M+H]+calcd for C 25 H 33 ClNO8 510.1895; found 510.1864. m / z [M+H]+: 510.1895; found 510.1864.

[0225] 3) 3-acetyloxymethoxycarbonyl-2,2,5,5-tetraethyl-2,5-dihydro-lH-pyrrole-N-oxyl radical Org. Lett.

[0226] According to the general procedure C, using 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 yellowish oil (16 mg, 77%).

[0227] NMR 1 H (500MHz, CDCl3) δ (ppm) 7.97 (dd, J= 8.0, 1.4Hz, 1H), 7.73 (s,1H), 7.16 (dd, J = 8.0, 2.1Hz, 1H), 6.78 (q, J = 5.4Hz, 1H), 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 = 5.4Hz, 3H), 1.01-0.75 (m, 12H).

[0228] NMR 13 C (126MHz, CDCl3) δ (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.

[0229] IR ν max (ATR, ZnSe, pure, cm) -1 ): 2986, 2864, 1710, 1698, 1670, 1414, 1382, 1121, 1110, 1097, 985.

[0230] HRMS (ESI-orbital trap): for C 27 H 36 NO 10 Calculated Tetrahedron Lett. [M+H]+: 534.2339; Measured value: 534.2311; For C 27 H 35 NNaO 10 Calculated [M+Na]+: 556.2159; Measured value: 556.2134.

[0231] Synthesis of probe precursors incorporating pyrroline structural units: Compounds 3

[0232] 1) m / z （S7）

[0233] 3-carboxy-2,2,5,5-tetraethyl-1-pyrroline-N-oxyl radical S6 was prepared according to the procedure 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, l-(((l-chloroethoxy)carbonyl)oxy)-2,2,5,5-tetraethyl-2,5-dihydro-lH-pyrrole-3-carboxylate acetyloxy methyl ester (S8) 16 (2014) 5298-5300) by the route 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-labelled 4-oxo-2,2,6,6-tetraethylpiperidine nitroxide for EPR brain imaging, m / z 55(2014), 2146-2149) was synthesized 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

[0234] According to the general procedure A, using S6 (100 mg, 0.416 mmol, 1 equiv), K2CO3 (115 mg, 0.832 mmol, 2 equiv) and ethyl bromoacetate (61 μL, 0.624 mmol, 1.5 equiv). The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 90 / 10) to give the desired compound as a yellow oil (129 mg, 99%).

[0235] IR v max (ATR, ZnSe, neat, cm -1 ): 2978, 2927, 2881, 2856, 1767, 1734, 1458, 1371, 1274, 1248, 1217, 1197, 1161, 1014.

[0236] HRMS (ESI-Orbitrap): calculated for C 16 H 26 NNaO5 [M+Na]+: 335.1709; found 335.1696. l-(((l-acetyloxyethoxy)carbonyl)oxy)-2,2,5,5-tetraethyl-2,5-dihydro-lH-pyrrole-3-carboxylate acetyloxy methyl ester (compound 3) [M+Na]+: 335.1709; found 335.1696.

[0237] EPR (100 µM solution in 0.1 M potassium phosphate buffer, pH = 7.4): A N = 1.54 mT, ΔΒ = 0.18 mT.

[0238] 2) m / z 3-acetyloxymethoxycarbonyl-2,2,5,5-tetraethylpyrrolidine-N-oxyl radical (S10)

[0239] According to the general procedure B using 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 was purified by silica gel column chromatography (cyclohexane / AcOEt, 95 / 05) to yield the desired compound as a yellow oil (70 mg, 40%).

[0240] NMR 1 H (500 MHz, CDCl3) δ (ppm) (slow tumbling of the nitrogen in the ring causes some signals to be split) 6.80-6.79 (m, 1H), 6.44 (q, J = 5.6 Hz, 1H), 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, 1H), 1.73-1.55 (m, 3H), 0.97-0.86 (m, 12H).

[0241] NMR 13 C (126 MHz, CDCl3) δ (ppm) (slow tumbling of the nitrogen in the ring causes some signals to be split) 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.

[0242] IR v max (ATR, ZnSe, neat, cm -1) : 2978, 2941, 2885, 1789, 1768, 1734, 1462, 1454, 1371, 1276, 1219, 1193, 1157, 1097, 1008.

[0243] HRMS (ESI-Orbitrap): [M+H]+calcd for C 19 H 31 ClNO7calcd Org. Lett. [M+H]+: 420.1789; found 420.1762.

[0244] 3) m / z l-(((l-chloroethoxy)carbonyl)oxy)-2,2,5,5-tetraethylpyrrolidine-3-carboxylate acetyloxy methyl ester (S11)

[0245] According to general procedure C using 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 yellowish oil (45 mg, 70%).

[0246] NMR 1 H (500MHz, CDCl3) δ (ppm) (slow nitrogen inversion in the ring causes some signals to be split) 6.80-6.78 (m, 1H), 6.72 (q, J = 5.4Hz, 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 = 5.4Hz, 3H), 0.97-0.84 (m, 12H).

[0247] NMR 13 C (126MHz, CDCl3) δ (ppm) (slow nitrogen inversion in the ring causes some signals to be split) 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.

[0248] IR vmax (ATR, ZnSe, pure, cm) -1 ): 2978, 2941, 2884, 1791, 1766, 1735, 1452, 1373, 1249, 1213, 1197, 1082, 1014, 910.

[0249] HRMS (ESI-orbital trap): for C 21 H 33 Calculated by NNaO9 m / z [M+Na]+: 466.2053; measured value: 466.2044.

[0250] Synthesis of probe precursors incorporating pyrrolidine structural units: Compounds 4 Synthesis of

[0251] 1) l-(((l-acetyloxyethoxy)carbonyl)oxy)-2,2,5,5-tetraethylpyrrolidine-3-carboxylate acetyloxy methyl ester (compound 4)

[0252] 3-Carboxy-2,2,5,5-Tetraethyl-1-pyrrolidine-N-oxy radical S9, according to JT Paletta et al. (JT Paletta, M. Pink, B. Foley, S. Rajca, A. Rajca, Synthesis and Reduction Kinetics of Sterically Shielded Pyrrolidine Nitroxides, m / z The synthesis scheme described in 14 (2012) 5322-5325).

[0253] Following general procedure A, S9 (57 mg, 0.235 mmol, 1 equivalent), K2CO3 (65 mg, 0.470 mmol, 2 equivalents), and ethyl bromoacetate (34 µL, 0.352 mmol, 1.5 equivalents) were used. The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 80 / 20) to give the desired compound as a yellow oil (48 mg, 64%).

[0254] IR ν max (ATR, ZnSe, pure, cm) -1 ): 2978, 2824, 2884, 2854, 1766, 1456, 1417, 1369, 1219, 1201, 1159, 1139, 1066, 983.

[0255] HRMS (ESI-orbital trap): for C 16 H 28 Calculated by NNaO5 l-(((l-chloroethoxy)carbonyl)oxy)-2,2,5,5-tetramethylpyrrolidine-3-carboxylate acetyloxy methyl ester (S13) [M+Na]+: 337.1865; measured value: 337.1859.

[0256] EPR (100µM solution, dissolved in 0.1M potassium phosphate buffer, pH=7.4): A N =1.53mT, ΔB=0.30mT.

[0257] 2) Heterocycles, Org. Biomol. Chem.

[0258] According to standard procedure B, S10 (40 mg, 0.127 mmol, 1 equivalent), Pd / C (6 mg), and Et ( 3)N (35 µL, 0.254 mmol, 2 equivalents) and 1-chloroethyl chloroformate (42 µL, 0.382 mmol, 3 equivalents). The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 95 / 05) to give the desired compound as a pale yellow oil (31 mg, 58%).

[0259] NMR 1 H (500MHz, CDCl3) δ (ppm) (a mixture of two diastereomers, with slow flipping of nitrogen in the ring causing some signal splitting and broadening) 6.41-6.33 (m, 1H), 5.76-5.65 (m, 2H), 2.89 - 2.82 (m, 0.5H), 2.24 (broad t, J = 12.4Hz, 0.5H), 2.03 (s, 3H), 1.94 - 1.80 (m, 2H), 1.78 - 1.76 (m, 3H), 1.72 - 1.50 (m, 6H), 1.48 - 1.32 (m, 2H), 0.95 -0.73 (m, 12H).

[0260] NMR 13C (126 MHz, CDCl3) δ (ppm) (mixture of two diastereoisomers, slow nitrogen flipping in the ring 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.

[0261] IR v max (ATR, ZnSe, pure, cm -1 ): 2984, 2932, 2834, 1690, 1670, 1608, 1482, 1356, 1280, 1111.

[0262] HRMS (ESI-Orbitrap): [M+Na]+: 444.1765; found 444.1759. 19 H 32 calcd for C m / z [M+Na]+: 444.1765; found 444.1759.

[0263] 3) l-(((l-acetyloxyethoxy)carbonyl)oxy)-2,2,5,5-tetramethylpyrrolidine-3-carboxylate acetyloxy methyl ester (compound 5) m / z

[0264] According to the general procedure C, using 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 was purified by silica gel column chromatography (cyclohexane / AcOEt, 95 / 05) to afford the desired compound as a colorless oil (15 mg, 60%).

[0265] NMR 1H (500 MHz, CDCl3) δ (ppm) (mixture of two diastereoisomers, slow nitrogen ring flipping of some signals split and broadened) 6.75-6.69 (m, 1H), 5.84-5.69 (m, 2H), 2.93 - 2.88 (m, 0.5H), 2.29 (broad t, J = 12.8Hz, 0.5H), 2.09 (s, 3H), 2.07 (s, 3H), 2.01-1.83 (m, 2H), 1.79 - 1.57 (m, 6H), 1.53 - 1.50 (m, 3H), 1.48 - 1.31 (m, 2H), 1.10 - 0.79 (m, 12H).

[0266] NMR 13 C (126 MHz, CDCl3) δ (ppm) (mixture of two diastereoisomers, slow nitrogen ring flipping of some signals split and broadened) 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.

[0267] IR ν max (ATR, ZnSe, pure, cm -1 ): 2984, 2868, 1706, 1690, 1672, 1608, 1514, 1482, 1356, 1280, 1111, 985.

[0268] HRMS (ESI-Orbitrap): for C 21 H 35 calcd for C 4-acetyloxymethoxycarbonyl-2,2,6,6-tetraethylpiperidine-N-oxyl radical (S15) [M+Na]+: 468.2209; found 468.2203.

[0269] Compounds 5 Synthesis of

[0270] 1) ​ ​

[0271] The free radical 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyloxyS12, according to the published procedure (Hatano, H. Araya, Y. Yoshimura, H. Sato, T. Ito, T. Ogata, T. Kijima, Facile Synthesis of 3-Methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyloxy and Derivatives), ​ 81 (2010) 349-356, and JPY Kao, GM Rosen, Esterase-assisted accumulation of 3-carboxy-2,2,5,5-tetramethyl1-pyrrolidinyloxyl into lymphocytes, ​ 2 (2004) 99-102) Synthesis.

[0272] According to standard procedure B, S12 (500 mg, 1.94 mmol, 1 equivalent), Pd / C (50 mg), and Et ( 3)N (540 µL, 3.90 mmol, 2 equivalents) and 1-chloroethyl chloroformate (520 µL, 4.80 mmol, 2.5 equivalents). The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 8 / 2) to give the desired compound as a colorless oil (640 mg, 91%).

[0273] Rf=0.40 (cyclohexane / ethyl acetate = 4:1; phosphomolybdic acid staining).

[0274] NMR 1 H (500MHz, CDCl3) δ (ppm) (major diastereomer ≈ 90%) 6.43 (q, J =5.7Hz, 1H), 5.78 (d, J = 5.2Hz, 1H), 5.70 (d, J = 5.2Hz, 1H), 3.05-2.80 (width m, 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).

[0275] NMR 13C (126 MHz, CDCl3) δ (ppm) (slow inversion of nitrogen causes some signals to be split and broadened) 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.

[0276] HRMS (ESI-Orbitrap): [M+H]+calcd for C 15 H 25 CINaO7 calculated ​ [M+H]+: 366.1314; found 366.1306; [M+Na]+: 388.1134; found 388.1124. 15 H 24 CINaO7 calculated

[0277] 2) ​ ​

[0278] According to general procedure C, using 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 was purified by silica gel column chromatography (cyclohexane / AcOEt, 80 / 20) to give the desired compound as a colorless oil (302 mg, 94%).

[0279] Rf= 0.21 (cyclohexane / ethyl acetate = 4:1; molybdenum blue staining).

[0280] NMR 1 H (500MHz, CD3 OD) δ (ppm) (main diastereoisomer ~90%) 6.71 (m, 1H), 5.82 (m, 1H), 5.70 (m, 1H), 3.15 - 2.80 (broad m, 1H), 2.35-2.10 (broad m, 1H), 2.07 (s, 6H), 1.86 (m, 1H), 1.51 (m, 3H), 1.00-1.36 (m, 12H).

[0281] NMR 13C (126 MHz, CD3OD) δ (ppm) (relatively slow tumbling of the nitrogen causes some signals to be split and broadened) 172.1; 171.2; 170.7; 156.2; 93.3; 80.8; 68.5; 67.3; 65.5; 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; 19.7; 17.5.

[0282] HRMS (ESI-Orbitrap): [M+H]+calcd for C 17 H 28 NO9calcd for C ​ [M+H]+: 390.1759; found 390.1751; [M+Na]+: 412.1578; found 412.1570. 17 H 27 ClNNaO7calcd for C

[0283] Synthesis of probe precursor incorporating a piperidine structural unit: Compounds 6 Synthesis of

[0284] 1) ​

[0285] 4-carboxy-2,2,6,6-tetraethyl(piperidin-l-yloxy) radical S14 was prepared according to the protocol published by N. Babi et al. (N. Babi , F. Peyrot, New synthetic route to 2,2,6,6-tetraethylpiperidin-4-one, a key-intermediate towards tetraethyl nitroxides, Tetrahedron Lett. 60 (2019) 15207).

[0286] According to the general procedure A, using S14 (200 mg, 0.780 mmol, 1 equiv), K2CO3 (215 mg, 1.56 mmol, 2 equiv) and ethyl bromoacetate (92 µL, 0.936 mmol, 1.2 equiv). The crude was purified by silica gel column chromatography (cyclohexane / AcOEt, 80 / 20) to give the desired compound as a yellow oil (156 mg, 61%).

[0287] IR v max (ATR, ZnSe, neat, cm -1 ): 2978, 2881, 1763, 1724, 1463, 1369, 1319, 1301, 1224, 1151, 1136, 1020.

[0288] HRMS (ESI-Orbitrap): calculated for C 17 H 30 NNaO5: 351.2016; found 351.2003. m / z [M+Na]+: 351.2016; found 351.2003.

[0289] EPR (100 µM solution in 0.1 M potassium phosphate buffer, pH = 7.4): A N = 1.61 mT, AB = 0.30 mT.

[0290] 2) 1-(((1 -chloroethoxy)carbonyl)oxy)-2,2,6,6-tetraethylpiperidine-4-carboxylate acetyloxymethyl ester （S16）

[0291] According to the general procedure B, using 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 was purified by silica gel column chromatography (cyclohexane / AcOEt, 95 / 05) to afford the desired compound as a yellowish oil (44 mg, 21%).

[0292] NMR 1 H (500 MHz, CDCl3) δ (ppm) 6.42 (q, J = 5.7 Hz, 1H), 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).

[0293] NMR 13C (126 MHz, CDCl3) δ (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.

[0294] IR v max (ATR, ZnSe, pure, cm -1 ): 2986, 2864, 1778, 1768, 1698, 1465, 1256, 1193, 1136, 1078, 1011, 1006, 987.

[0295] HRMS (ESI-Orbitrap): [M+H]+: 436.2102; found 436.2087; for C 20 H 35 ClNO7 calculated m / z [M+H]+: 436.2102; found 436.2087; for C 20 H 34 ClNNaO7 calculated [M+Na]+: 458.1933; found 458.1887.

[0296] 3) 1-(((1 -acetyloxyethoxy)carbonyl)oxy)-2,2,6,6-tetraethylpiperidine-4-carboxylate acetyloxy methyl ester (compound 6) m / z

[0297] According to the general procedure C, using S16 (40 mg, 0.092 mmol, 1 eq), AcOH (164 µL) and Hg(OAc)2(44 mg, 0.138 mmol, 1.5 eq). The pure product was obtained without purification as a yellowish oil (40 mg, 95%).

[0298] NMR 1 H (500MHz, CDCl3) δ (ppm) 6.70 (q, J = 5.3Hz, 1H), 5.75 (s, 2H),2.65 (t, J= 12.6Hz, 1H), 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 = 5.3Hz, 3H), 1.44-1.33 (m, 2H), 1.01-0.86 (m, 12H).

[0299] NMR 13 C (126MHz, CDCl3) δ (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.

[0300] IR ν max (ATR, ZnSe, pure, cm -1 ): 2970, 2945, 2881, 1788, 1764, 1467, 1454, 1373, 1247, 1211, 1193, 1136, 1078, 1020, 1006, 985.

[0301] HRMS (ESI-Orbitrap): [M+H]+calcd for C 22 H 38 NO9 460.2547; found 460.2520. 4-(2-ethoxy-2-oxoethylidene)-2,2,6,6-tetraethylpiperidin-1 -yloxy radical (S18) [M+H]+: 460.2547; found 460.2520; [M+Na]+: 482.2366; found 482.2339. 22 H 37 ClNNaO9 482.2366; found 482.2339.

[0302] Compounds 7 Synthesis of

[0303] 1) Tetrahedron Lett.

[0304] The free radical 2,2,6,6-tetraethyl-4-oxo(piperidin-1-yloxy) or TEEPONE S17 is based on the synthesis of free radicals 2,2,6,6-tetraethyl-4-oxo(piperidin-1-yloxy) or TEEPONE S17 according to ... 15 N-labelled 4-oxo-2,2,6,6-tetraethylpiperidine nitroxide for EPR brain imaging, m / z The procedure described in 55(2014), 2146-2149) is used to obtain the results.

[0305] Triethyl phosphonoacetate (197 µL, 0.994 mmol, 1.5 equivalents) was added to an anhydrous THF (8 mL) solution of TEEPONE S17 (150 mg, 0.663 mmol, 1 equivalent) at 0 °C, and sodium hydride (32 mg, 1.326 mmol, 2 equivalents) was added to the solution. The mixture was then stirred at room temperature for 4 hours. Excess sodium hydride was then neutralized by adding a saturated ammonium chloride solution. The aqueous phase was extracted with ethyl acetate (×3), and the combined organic phases were washed with a saturated sodium chloride aqueous solution 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 give the desired product (154 mg, 78%) as a yellow oil.

[0306] IR ν max (ATR, ZnSe, pure, cm) -1 ): 2978, 2935, 2881, 1741, 1712, 1456, 1390, 1273, 1155.

[0307] HRMS (ESI-orbital trap): for C 17 H 30 NO3 calculation 4-(carboxymethylene)-2,2,6,6-tetraethylpiperidin-1 -yloxy radical (S19) [M]+: 296.2226; Measured value: 296.2221; For C 17 H 32 NO3 calculated [M+2H]+: 298.2382; measured value: 298.2375; for C 17 H 30 [M+Na]+ calculated from NNaO3: 319.2123; measured value: 319.2118.

[0308] EPR (100 µM solution in 0.1 M potassium phosphate buffer at pH = 7.4): A N = 1.59 mT, AB = 0.22 mT.

[0309] 2) m / z

[0310] A solution of compound S18 (150 mg, 0.506 mmol, 1 eq) in methanol (5 mL) was added to a solution of sodium hydroxide (61 mg, 1.52 mmol, 3 eq) in water (5 mL). The solution was then stirred at 40 °C for 1 hour. Water and ethyl acetate were then added. The organic phase was removed and the aqueous phase was acidified by adding a hydrochloric solution (1 M) until the pH reached 2. Ethyl acetate was then 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 as a yellow solid without purification (120 mg, 89%).

[0311] IR v max (ATR, ZnSe, neat, cm -1 ): 2972, 2935, 2881, 1715, 1641, 1458, 1419, 1382, 1276, 1176, 941.

[0312] HRMS (ESI-Orbitrap): calculated for C 15 H 25 NO3 4-(2-(acetyloxymethoxy)-2-oxoethylidene)-2,2,6,6-tetraethylpiperidin-1 -yloxy radical (S20) [M-H : 267.1834; found 267.1835.

[0313] Melting point: 136-138 °C.

[0314] EPR (100 µM solution in 0.1 M potassium phosphate buffer, pH = 7.4): A N = 1.60 mT, AB = 0.23 mT.

[0315] 3) m / z 2-(1 -(((1 -chloroethoxy)carbonyl)oxy)-2,2,6,6-tetraethylpiperidin-4- ylidene)acetic acid acetyloxymethyl ester (S21 )

[0316] Following general procedure A, S19 (200 mg, 0.745 mmol, 1 equivalent), K2CO3 (206 mg, 1.49 mmol, 2 equivalents), and ethyl bromoacetate (109 µL, 1.118 mmol, 1.5 equivalents) were used. The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 90 / 10) to give the desired compound as a yellow oil (80 mg, 32%).

[0317] IR ν max (ATR, ZnSe, pure, cm) -1 ): 2980, 2931, 2885, 2856, 1768, 1745, 1371, 1226, 1207, 1132, 1010, 987.

[0318] HRMS (ESI-orbital trap): for C 18 H 30 Calculated by NNaO5 m / z [M+Na]+: 363.2022; measured value 363.2008.

[0319] EPR (100µM solution, dissolved in 0.1M potassium phosphate buffer, pH=7.4): A N =1.59mT, ΔB=0.22mT.

[0320] 4) 2-(1 -(((1 -acetyloxyethoxy)carbonyl)oxy)-2,2,6,6-tetraethylpiperidin-4- ylidene)acetic acid acetyloxymethyl ester (compound 7) m / z

[0321] Following general procedure B, S20 (47 mg, 0.138 mmol, 1 equivalent), Pd / C (6 mg), Et3N (48 µL, 0.345 mmol, 2.5 equivalents), and 1-chloroethyl chloroformate (30 µL, 0.276 mmol, 2 equivalents) were used. The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 95 / 05) to give the desired compound as a pale yellow oil (33 mg, 54%).

[0322] NMR (1) H(500MHz, CDCl3)δ 6.36 (q, J = 5.7Hz, 1H), 5.72 (s, 2H), 5.68(s, 1H), 3.55 (d, J = 13.2Hz, 1H), 3.04 - 2.95 (m, 1H), 2.33 (d, J = 13.2Hz, 1H), 2.21 (d, J = 15.0Hz, 1H), 2.05 (s, 3H), 1.88 - 1.79 (m, 2H), 1.77 (d, J= 5.7Hz, 3H), 1.58 - 1.46 (m, 4H), 1.45 - 1.30 (m, 2H), 0.94 - 0.78 (m, 12H).

[0323] NMR 13 C (126MHz, CDCl3) δ 169.9; 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.

[0324] IR ν max (ATR, ZnSe, pure, cm) -1 ): 2978, 2924, 2885, 2854, 1791, 1764, 1456, 1215, 1193, 1136, 1099, 1022, 1001, 987.

[0325] HRMS (ESI-orbital trap): for C 21 H 35 ClNO7 calculation Example 2: Stability and reactivity of compounds of formula (I) with respect to esterases and comparison with prior art radical probe precursors [M+H]+: 448.2102; Measured value: 448.2093.

[0326] 5) Example 3: Behaviour of compounds of formula (I) on cells Cell culture protocol for cancer cell lines

[0327] Following standard procedure C, S21 (15 mg, 0.033 mmol, 1 equivalent), AcOH (5 µL), and Hg(OAc)2 (16 mg, 0.050 mmol, 1.5 equivalent) were used. The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 95 / 05) to give the desired compound as a pale yellow oil (7 mg, 45%).

[0328] NMR 1 H (500MHz, CDCl3) δ 6.71 (q, 5.3Hz, 1H), 5.78 (s, 2H), 5.74 (s,1H), 3.59 (d, J 1.64 - 1.54 (m,4H), 1.52 (d, J = 5.3Hz, 3H), 1.49 - 1.41 (m, 2H), 1.05 - 0.82 (m, 12H).

[0329] NMR 13 C (126MHz, CDCl3) δ 169.9; 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.

[0330] IR ν max (ATR, ZnSe, pure, cm) -1 ): 2980, 2878, 2864, 1791, 1764, 1698, 1670, 1456, 1213, 1111, 1099, 1027, 1001, 987.

[0331] HRMS (ESI-orbital trap): for C 23 H 37 Calculated by NNaO9 Toxicity of compounds of formula (I) [M+Na]+: 494.2366; Measured value: 494.2360.

[0332] Protocol Cytotoxicity test

[0333] To confirm that the deprotection of the compound of formula (I) is esterase-dependent, tests were performed in the presence of modified esterases and lipases. Cleavage of the protecting group was tested by incubating the compound of formula (I) with either an esterase or a lipase, followed by oxidation of the released hydroxylamine with potassium ferricyanide and measurement of the EPR signal of the resulting nitrogen oxides. Potassium ferricyanide is a mild oxidant that reacts rapidly with hydroxylamine by accepting electrons. A tetramethylated and acetyl-protected commercial ACP probe was used as a control.

[0334] Preparation of Compound 1 solution: Two volumes of DMSO stock solution of Compound 1 (50 mM or 500 mM) were thoroughly mixed with one volume of 20% m / v Pluronic F-127 (Sigma-Aldrich) DMSO solution. The resulting mixture was diluted in the desired aqueous medium and vigorously mixed using a vortex mixer.

[0335] Incubation with enzyme: Incubate a 100 µM solution of compound 1 with the following enzyme at 37 °C for 10 minutes: porcine liver carboxylesterase (1 U / ml) -1 ; Sigma E3019; One unit of enzyme hydrolyzes 1.0 μmol of ethyl butyrate to butyrate and ethanol per minute at 25°C and pH 8.0, or lipase (0.51 mg / mL). -1 Sigma L3126; 1 mg of enzyme was hydrolyzed at 25 °C and pH 7.4 in potassium phosphate buffer (0.1 M, pH 7.4, DTPA 1 mM) at a rate of 25.6 nmol / min, and the released hydroxylamine was mixed with 1 µL of 100 mM potassium ferricyanide (final concentration 1 mM). This mixture was transferred to a 50 µL glass capillary (Hirschmann), and the nitrogen oxide EPR signal was recorded in the X-band (9.86 GHz) at 21 °C using a Bruker Elexsys E500 EPR spectrometer equipped with a high-sensitivity SHQ chamber. The typical parameters used are as follows: microwave power, 10mW; modulation frequency, 100kHz; modulation amplitude, 0.14mT; receiver gain, 60dB; time constant, 40.96ms; conversion time, 40.96ms; 1024 points; scan width, 5mT; scan time, 41.94s.

[0336] Lipases were observed to have low activity (as expected for short-chain esters), while porcine liver carboxylesterase (EC 3.1.1.1) was more effective.

[0337] Compared to a solution of compound 83 (true isoindoline nitride) prepared independently at 100 µM, the concentration of compound 1 with 1 U mL... -1The porcine liver esterase was incubated at 37°C for 10 minutes, followed by a reaction with ferricyanide (1 mM), allowing for complete recovery of nitrogen oxides.

[0338] When compound ACP (a known comparative probe precursor in the art, acetylated and substituted with four methyl groups) was subjected to the same conditions, only 27% of the corresponding nitrogen oxides were recovered.

[0339] Stability of Compound 1: To test the stability of Compound 1 in aqueous solution, a 100 µM solution of Compound 1 was incubated at 21 °C in potassium phosphate buffer (0.1 M, pH 7.4, 1 mM DTPA) for 24 hours. No nitrogen oxide signal was detected after the addition of potassium ferricyanide, confirming that the protection of tetraethylenediamine by (acyloxy)alkyl carbonates under these conditions is stable against spontaneous hydrolysis at neutral pH.

[0340] Results

[0341] Following the synthesis and initial characterization of the compound of formula (I), several in vitro tests were performed to answer the following questions: 3.1. Are the compounds of formula (I) toxic? 3.2. Do compounds of formula (I) accumulate in cells? 3.3. Is the deprotection of the compound of formula (I) the rate-limiting step in the oxidation of hydroxylamine? 3.4. Does the compound of formula (I) cause oxidative stress? 3.5. Is it possible to detect oxidative stress under conditions closer to physiological conditions? The in vitro model used for most of these tests was the U-87 MG cell line.

[0342] [Table 1]

[0343] Human brain cells (glioblastoma-astrocytoma) U87-MG were cultured in Duchenne minimum essential medium (DMEM), 10% fetal bovine serum (FCS), 200 U / mL penicillin, and 50 mg / mL streptomycin (Invitrogen, Sergiy-Pontoise, France) under a humid atmosphere containing 5% CO2. Human breast cancer cell line MCF-7 (ATCC) was cultured at 37°C under a humid atmosphere containing 5% CO2 in DMEM supplemented with non-essential amino acids, 10 µg / mL recombinant human insulin, 10% FCS, 200 U / mL penicillin, 50 µg / mL streptomycin (Invitrogen, Sergiy-Pontoise, France), and 0.5 µg / mL amphotericin B. ® HTB-22 ™ ) cells.

[0344] 3.1. Accumulation of compounds of formula (I) in cells ?

[0345] EPR : Cell processing for measuring toxicity of compounds of Formula (I) ​ U87 cells were seeded in 96-well plates (10,000 cells per well, one day before the experiment). The culture medium was removed, and the cells were washed with PBS (Dupuy phosphate-buffered saline, pH 7.1–7.5) and then incubated for 6 hours with a series of concentrations of Compound 1 (1 µM to 500 µM, dissolved in FCS-free DMEM, 100 µL / well) using Pluronic F-127 dissolved in DMSO.

[0346] Results

[0347] For fluorescence imaging analysis, the incubation medium was removed, and the cells were washed once with PBS, then incubated for 30 minutes in FCS-free medium containing Hoechst dye (0.1 µg / mL) and propidium iodide (0.1 mg / mL). ImageXpress was used. ® Pico (Molecular Devices) reads and analyzes fluorescence.

[0348] Figure 1 : The table below shows the results of the dead cell count compared to the total number of cells in the well after 6 hours of incubation, measured by fluorescence imaging with propidium iodide staining (Hoechst staining).

[0349] Deprotection of compounds of formula (I) is it a limiting step for oxidation of hydroxylamine?

[0350] Regardless of the concentration of compound 1 of formula (I), the number of dead cells did not change significantly. These results indicate that compound 1 is non-toxic to U87 cells even at the highest concentration of 500 µM for 6 hours. This is consistent with the incubation conditions (50 µM-500 µM for 15-60 min) commonly used for hydroxylamine probes on cultured cells.

[0351] 3.2. Results ?

[0352] Membrane permeability and cell accumulation of compounds 1, 2, 3, and 7 were tested on U87 cells in suspension. Trypsin-treated cells were incubated with each compound at 37°C for 30 min in serum-free DMEM (in a water-jacketed incubator) under a 5% CO2 atmosphere. After centrifugation, the cell pellet was thoroughly washed three times with culture medium and resuspended in 50 µL of fresh serum-free medium before EPR recording.

[0353] For cells in suspension Figure 2 Measurement protocol

[0354] Following trypsin treatment, a suspension of U87 cells (10 million cells / mL) was prepared in serum-free medium. In a typical experiment, compounds 1, 2, 3, or 7 (emulsified in a mixture of Pluronic F-127 and DMSO as described in Example 2) were diluted in 500 µL of cell suspension (5 million cells). The final concentration of compounds 1, 2, 3, or 7 was typically 100 µM. After incubation (30 min, 37 °C, 5% CO2, with occasional mixing to resuspend cells), 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 three times by resuspending in 500 µL of serum-free medium, followed by centrifugation. Finally, the cell pellet was resuspended in 50 µL of serum-free medium containing... 15 N-TEMPONE (4-oxo-2,2,6,6-tetramethylpiperidine-1-oxy radical labeled with nitrogen-15, 100 µM) was placed in serum-free medium in gas-permeable PTFE tubes (extruded Sub-LiteWall). ® The inner diameter is 0.635 mm and the wall thickness is 0.051 mm. It is folded into a W shape and inserted into a 4 mm quartz tube. The EPR spectrum is recorded as described above (see Example 2).

[0355] Oxidative stress induced by compounds of formula (I) : Cells incubated with compound 1 showed a strong nitride EPR signal after washing. The carboxyl group in compound 1, masked by the esterase-sensitive acetoxymethyl group, allows it to cross the membrane and, once inside the cell, is deprotected by the cell's esterases. Deprotection produces charged products that may no longer easily cross the membrane and thus accumulate inside the cell. During the deprotection of hydroxylamine, the released hydroxylamine is also oxidized to nitrides. The experimental EPR spectrum was produced by the superposition of two components: a major component (in...) PrincipleCompounds 1 (marked with an asterisk) exhibit characteristics of nitrogen oxides in an isotropic state and are compatible with the position in the cytosol; and a minor component (marked with an arrow) exhibits characteristics of nitrogen oxides in an intermediate state and is compatible with the position in the cell membrane. Similar results were obtained with compounds 2, 3, and 7 (strong EPR signal of nitrogen oxides in incubated cells after washing), indicating that these compounds accumulate in cells.

[0356] 3.3. EPR ?

[0357] The deprotection rate of hydroxylamine probe precursors protected from oxidative stress must be faster than the oxidation rate.

[0358] It can be used 15 N-TEMPONE assays detect the presence of unoxidized hydroxylamine in cells. This method relies on the ability of TEMPONE to freely cross the cell membrane and the rapid transfer of hydrogen between nitrogen oxides and hydroxylamine. Isotope labeling is used. 15 N-TEMPONE, because its spectrum has only two spectral lines, these two spectral lines do not correspond to those derived from compounds 1, 2, 3, or 7. 14 The signals of N-nitrogen oxides (which have three spectral lines) are completely overlapping, thus allowing for the simultaneous measurement of two substances.

[0359] For this purpose, 5 million U87 cells in suspension were incubated with compound 1 or with compounds 2, 3, or 7 (100 µM, in serum-free DMEM, at 37°C, 5% CO2 for 30 min). After the final wash, a portion was resuspended in serum-free DMEM, while the other portion was resuspended in a solution containing 100 µM... 15 N-TEMPONE in serum-free DMEM.

[0360] EPR spectra were acquired according to the scheme described in Section 3.2 above.

[0361] Results : Compared with untreated samples, using 15 N-TEMPONE-treated cell samples showed a derivative of compound 1. 14 The signal of N-nitrogen oxides was significantly higher (4 times higher). [Table 2] In the case of compound 2, compared with the untreated sample, using 15 N-TEMPONE processing enables the corresponding 14The signal for N-nitrosamines increased threefold. For compound 3, the signal increased fourfold, and for compound 7, the signal increased sixfold. This experiment showed that significant amounts of free hydroxylamine were present in the cells, suggesting that deprotection in the cells is not the limiting reaction for the conversion of compounds 1, 2, 3, and 7 to N-nitrosamines.

[0362] 3.4. Is it possible to detect oxidative stress under more physiological conditions? ?

[0363] To test the compound of formula (I) under oxidative stress conditions, a cell model of oxidative stress induced by juglone was selected. Juglone (5-hydroxy-1,4-naphthyldione) is a natural quinone product that can generate superoxide through ineffective redox cycles in cells.

[0364] Protocol for harvesting and culturing chondrocytes : Prior to EPR recording, confluent U87 cells were incubated on coverslips with compound 1 (250 µM, in serum-free DMEM, at 37°C, 5% CO2 for 30 min) with or without juglone (2.5 µM and 5 µM). In some cases, cells were pre-incubated with a potential antioxidant in DMEM containing 10% FBS: SOD-PEG (superoxide dismutase-polyethylene glycol, 100 U / mL). -1 (lasts for 2 hours), Cat-PEG (catalase-polyethylene glycol, 100 U / mL) -1 The incubation process involved chelating agents (sodium diethyldithiocarbamate, DETC, 5 µM and deferoxamine, DFO, 25 µM, for 1 hour) and NAC (N-acetylcysteine, 5 mM, for 1 hour). EPR spectral signals corresponded to nitrogen oxides produced by incubating cells with juglone. Signal intensity was normalized based on the average protein content of samples taken daily. All experiments were performed in DMEM medium containing phenol red, which did not interfere with the measurements.

[0365] Cells cultured on glass coverslips were probed with hydroxylamine probe precursor EPR Protocol for measurements

[0366] U87-MG cells were cultured on circular microscope coverslips (12 mm in diameter, 0.13 mm–0.16 mm thick, Thermo Scientific Menzel, Braunschweig, Germany) inserted into each well of a 24-well plate. Generally, high confluence (cells almost completely covering the glass surface on which they grow) is required to obtain sufficient EPR signal intensity. Cells were seeded at least 24 hours before the EPR experiment to avoid interference due to cell passage stress. Typically, 56 × 10⁶ cells per well were seeded three days before the EPR experiment. 3 10 U87 cells. (Two days before the experiment, at a rate of 4 × 10⁶ cells / year) 5MCF-7 cells were seeded at a concentration of cells / well. For each EPR 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 with Compound 1 or ACP for 30 minutes in 300 µL of 250 µM solution / emulsion in FCS-free medium containing different concentrations of juglone (prepared from a DMSO stock solution of 100 mM juglone; aliquots of the DMSO stock solution of juglone can be stored at -80°C for several weeks, but once thawed, they must be used as soon as possible before turning dark brown; for each sample, an intermediate solution of juglone in medium was freshly prepared and used within minutes). Since protected hydroxylamine 1 is insoluble in water, a suspension was prepared using the surfactant Pluronic F-127. The stock solution of 1 (10 µL of 500 mM DMSO solution) was mixed with the solution of Pluronic F-127 (5 µL of 20% m / v DMSO solution). The mixture was dispersed by vortexing in 85 µL of serum-free medium to produce a white 50 mM emulsion, which was used for further dilution on the same day. The maximum total final concentration of DMSO in cell experiments was 0.08% (including dilutions of the juglone stock solution). At indication, pre-incubation with antioxidants (2 h for 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 containing 10% FCS. Using a bent-tipped needle and tweezers, remove the coverslips from the well. Carefully remove any excess liquid with a paper towel and place both coverslips on the flat side of a flat quartz suprasil cell (WG-806-AQ, Wilmad), with the cell surface facing that side (to prevent the sample from drying out). The coverslips adhere to the tissue cells only by capillary action (note: if excess liquid remains on the coverslips, they tend to slip off the smooth surface of the EPR cells). Secure the EPR cell within a cylindrical-mode EPR resonator (EM4103TM, Bruker), with the sample facing the experimenter, and begin EPR recording immediately. The parameters used are as follows: microwave power, 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; sweep width, 6 mT; sweep time, 20.97 s. EPR spectra were continuously recorded at 21 °C for 5 min. Data acquisition and processing were performed using Bruker Xepr software and MathWorks MATLAB software with the Easyspin toolkit.After collection, each coverslip was placed in a clean well of a 24-well plate, and cells were lysed using RIPA lysis buffer (Thermo Scientific) (50 µL / coverslip) and collected on the coverslip for protein titration using the BCA assay. Paired-sample t-tests were applied to a set of 3 to 5 independent experiments with a critical value of p < 0.05. ) and p<0.001 ( (and compared with conditions without juglone).

[0367] Results : The results are presented in the table below: Primary culture

[0368] # The mean ± standard deviation of 3 to 5 independent experiments.

[0369] Two concentrations of juglone (2.5 µM and 5 µM) were tested, and a dose-dependent increase in the EPR signal was observed. The maximum signal amplification was obtained with 5 µM (approximately 50% compared to untreated cells). These results demonstrate that compound 1 is indeed a reporter molecule for intracellular oxidative stress. The relatively high basal oxidation of compound 1 in untreated cell samples may indicate that compound 1 is sensitive to intracellular oxidative processes even under basal conditions in cancer cells.

[0370] Typically, DETC (a general-purpose "soft" metal ion chelator) and DFO (an iron chelator) are added and incubated with unprotected hydroxylamine probes (such as CMH, 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine) to prevent auto-oxidation in the buffer. To verify whether metal ion chelation is required when using compounds of formula (I), cells were pre-incubated with DFO and DETC. We here confirm that DFO and DETC are not required for compound 1, as these compounds do not alter the EPR response. This makes compound 1 easier to use than unprotected hydroxylamine probes, as compound 1 is less susceptible to interference.

[0371] No antioxidants (SOD-PEG, Cat-PEG, or NAC) reduced basal oxidation or juglone-induced oxidation, preventing us from drawing any precise conclusions about the chemicals responsible for oxidation in cells. Interestingly, however, visual examination of cells showed that juglone-induced cell contraction was reversed by NAC treatment. The antioxidant effect of NAC is known to be mediated by an increase in intracellular glutathione concentration. Therefore, compound 1 may be sensitive to oxidative stress processes upstream of glutathione.

[0372] In parallel, commercially available ACP probes were tested using the same protocol. Under the tested conditions, no nitrogen oxide signal was detected with ACP regardless of juglone treatment. This indicates that, under the tested conditions, compound 1 is a better reporter molecule than ACP for juglone-induced cellular oxidative stress.

[0373] In addition, another cell line (MCF-7 cells) was tested, and the results were similar to those obtained with U87 cells. A clear dose-dependent effect of juglone was observed, with a maximum signal amplification of approximately 60% at 5 µM juglone.

[0374] 3.5. [Table 3] ?

[0375] It is important to test compound 1 on normal cells, and to use a physiological oxidative stress model to test the detection threshold. A good model is rabbit articular chondrocytes (also adherent cells) induced to express an osteoarthritis-like inflammatory phenotype by the addition of interleukin-1β (IL-1). IL-1 is a family of cytokines (small proteins important in cell signaling) that play a role in regulating inflammatory responses. Rabbit articular chondrocytes were cultured on microscope slides (see protocol below).

[0376] Sub-cultures

[0377] All experiments were conducted in accordance with ethical guidelines. Five-week-old New Zealand white rabbits were euthanized by vertebral dislocation followed by strangulation. Articular cartilage was harvested from the femoral and humeral heads, tibial plateau, and femoral condyles. The cartilage was harvested tangentially from the articular surface 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 trypsin treatment, followed by collagenase digestion. Chondrocytes were isolated from the collagenase solution by centrifugation. The obtained cells were then seeded at 60,000 cells / cm² in two T75 flasks and cultured for five days in proliferation medium (supplemented with Ham nutrient mixture F12 containing 10% FCS, 1% L-glutamine, and 100 U / mL penicillin and 100 U / mL streptomycin) at 8% CO2. The medium was then replaced with DMEM containing 1 g / L D-glucose, 4 mM L-glutamine, and 10% FCS. Two days later, cells were passaged and seeded at 250,000 cells / well in 24-well plates on glass coverslips containing 2 mL of complete DMEM (pre-coated and dried with FCS; 12 mm in diameter, 0.13 mm–0.16 mm thick, Thermo Scientific Menzel, Braunschweig, Germany), and cultured for 3 days at 37°C and 5% CO2 prior to the experiment. Subculture assays were also performed, for which 80,000 cells / well were seeded on coverslips and cultured for 3 days prior to the experiment.

[0378] [Table 4] Measurement scheme

[0379] As described in Section 3.4, EPR recordings were performed 1 hour, 24 hours, or 48 hours after stimulation with IL-1 (1 ng / mL, in FCS-free DMEM) or after the culture medium was changed to serum-free DMEM. Prior to EPR recording, cells were incubated with Compound 1 (250 µM or 500 µM, in serum-free DMEM, at 37°C, 5% CO2) for 1 hour or 30 minutes. For the 1-hour incubation, IL-1 was added simultaneously with Compound 1, while for the 24-hour and 48-hour incubations, cells were pre-incubated with IL-1. Given the limited number of available cells, each condition was tested only once unless otherwise specified.

[0380] Example 4: Preparation and characterization of emulsions according to the application : The results are presented in the table below.

[0381] [Table 5]

[0382] Determination of droplet size and size distribution

[0383] zeta

[0384] [Table 6]

[0385] Treatment of chondrocytes with IL-1 rapidly induced the expression of pro-inflammatory proteins through various signaling pathways. Significant amounts of nitrite were detected in the culture medium 12 to 24 hours after IL-1 treatment, indicating overexpression of iNOS, which was absent under basal conditions. Prolonged incubation in FCS-free DMEM may have induced some cell death over time, explaining why the signal obtained with compound 1 decreased over time in the absence of IL-1 stimulation, although the EPR signal was not normalized relative to protein levels. When using compound 1, the difference between treated and untreated cells was only detectable in primary cultured cells at 48 hours (Table 3), and the effect was more pronounced in subcultured cells starting at 24 hours (Table 4), indicating that the compound is sensitive only to downstream events and not to the initial stimulus. Although these results are preliminary, they demonstrate that compound 1 can detect a partial response of rabbit chondrocytes to IL-1.

[0386] Emulsion encapsulation efficiency

[0387] The nanoemulsion comprising compound 1 obtained according to Example 1 was prepared as follows: Aqueous phase (composed of ultrapure water and Kolliphora) was separately prepared by mixing the aqueous phase (composed of ultrapure water and Kolliphora) with ... ® 188 composition) and oil phase (Labrafac) ® Or olive oil and Span ® 80) The mixture was heated to 40°C, and then the aqueous phase was added dropwise to the oil phase, which was maintained at 40°C, with continuous stirring. The pH was adjusted to 7.0 using a 0.1M sodium hydroxide aqueous solution. Isotonicity was adjusted by adding glycerol to a final concentration of 2.5% (m / m). The resulting crude emulsion was introduced directly into a microfluidic bed (LV1) without homogenization. Microfluidization was performed for 10 cycles at 20 kpsi. Supported nanoemulsions were prepared using compound 1 in the oil phase at concentrations of 0.1M and 0.5M, i.e., final concentrations of 20 mM and 100 mM, respectively. Table 5 below summarizes the different emulsions prepared.

[0388] [Mathematical formula 1]

[0389] [Mathematical formula 2]

[0390] Hydrodynamic diameter and polydispersity index (PDI) of nanoemulsion droplets were determined by photon correlation spectroscopy using Zetasizer. ® Nano ZS (Malvern Panalytical, Worcestershire, UK) measurements were performed. Approximately 10 µL of each nanoemulsion sample was diluted 100-fold with ultrapure water for measurement. All measurements were recorded at 25 °C and a scattering angle of 173°. An exponential fitting method (cumulative method) was used to fit correlation function curves to evaluate the hydrodynamic diameter and PDI. All measurements were performed in triplicate to determine the average droplet size and PDI, expressed as the average Z-Ave diameter.

[0391] Stability under storage conditions Measurement of potential

[0392] The zeta potential (ZP) of the nanoemulsion was determined using laser Doppler velocimetry with Zetasizer. ® Nano ZS (Malvern Panalytical, Worcestershire, UK) measurements were performed. 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.

[0393] Table 6 below summarizes the hydrodynamics, polydispersity, and zeta potential of droplets of the original nanoemulsion (original NE) and nanoemulsions of compound 1 loaded at a final concentration of 20 mM or 100 mM (NE containing compound 1).

[0394] Example 5: Evaluation of cytotoxicity of nanoemulsions comprising compound 1

[0395] Results

[0396] The encapsulation efficiency of compound 1 in the nanoemulsion was determined by separating the aqueous phase of the formulation using an Amicon ultra-0.5 mL 30KD centrifugal filter (Merck Millipore, France). The nanoemulsion was centrifuged at 14,000 g for 30 min in a Minispin Plus microcentrifuge (Eppendorf, Switzerland). The aqueous phase was then analyzed using a SHIMADZU LC-20 HPLC system equipped with a Nucleosil 100 Å C18 column (25 cm × 4.6 mm; 5 µm) maintained at 40 °C to determine the content of compound 1. The mobile phase consisted of an aqueous solution of 0.1% formic acid (mobile phase A) and an acetonitrile solution of 0.1% formic acid (mobile phase B). A gradient of 40% B to 90% B was used over 30 min, 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 µM to 50 µM and passed through an HPLC-UV system to obtain the formula y(UA) = 2474.4. The calibration curve is calculated as x (concentration in µM) + 5717.1 (R² = 0.9986).

[0397] Using this calibration curve, the concentration of compound 1 in the aqueous phase was estimated, and the encapsulation efficiency (EE) was determined as follows: [Table 7]

[0398] In this study, the probe precursor loading capacity (drug loading = DL) of the nanoemulsion encapsulating the highest concentration of compound 1 (0.5 M in the oil phase) was estimated as follows: Example 6: EPR study of nanoemulsions comprising compound 1

[0399] In all cases, compound 1 was not detected in the aqueous phase (<0.01%), indicating that compound 1 was almost completely encapsulated in the oil phase. The type of oil and the Span 80:Kolliphor 188 ratio had no effect on the retention of compound 1 in the oil phase. In this study, the highest amount of compound 1 encapsulated (final concentration 100 mM) was found in oil-based or Labrafac-based oils. ® The probe precursor loading capacity of the nanoemulsion is estimated to be approximately 33.33%.

[0400] Protocol

[0401] The stability of the nanoemulsions was investigated using photon correlation spectroscopy as previously described for droplet size determination. Droplet size and size distribution were analyzed at different time points (D1, D7, D14, D28, D50) for a maximum of 50 days. Samples were stored at 25°C during this period. Two independent samples of each formulation were studied.

[0402] No visible phase separation was observed in the formulations throughout the study period. All formulations exhibited good stability.

[0403] EPR

[0404] The cytotoxicity of Compound 1 nanoemulsion to brain endothelial cells (b.End 3) was investigated using a dye exclusion assay. Initially, cells were cultured in a suitable medium consisting of DMEM, 10% FCS, penicillin (100 IU / mL), and streptomycin (100 µg / mL). Culture was carried out under humid conditions at 37°C and 5% CO2. After trypsin-dissociated cell division, cells were passaged weekly at approximately 90% confluence using a 1:10 dilution.

[0405] These cells were then seeded into 24-well plates (2.5 × 10⁻⁶). 5 Cells were incubated with nanoemulsion (0.1 mM–5 mM) for 24 hours, then treated with the nanoemulsion sample and incubated for another 4 hours. Each well was treated with nanoemulsion loaded with Compound 1 (range 0.1 mM–5 mM), blank nanoemulsion, or culture medium as a control. All formulations were diluted in cell culture medium to prepare the desired concentration. After 4 hours, the culture medium containing the nanoemulsion was removed, and the cells were washed with 100 µL of trypsin. They were then incubated with 200 µL of 0.25% trypsin / EDTA solution. Finally, after neutralizing the enzyme with complete DMEM, the harvested cells (20 µL) were mixed with 0.4% trypan blue dye (20 µL). 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.

[0406] EPR : Results

[0407] Cytotoxicity assessments of nanoemulsions 1 NE-OO and 1 NE-L1 showed cell viability greater than 80% at concentrations ranging from 0.1 mM to 2.5 mM. For nanoemulsion 1 NE-L2, cell viability >80% was observed at 0.25 mM and below, but a slightly more significant decrease in cell viability was observed at concentrations above 0.5 mM. However, a higher percentage of viability was observed at a concentration of 5 mM (69.8%) compared to nanoemulsions 1 NE-OO (36.17%) and 1 NE-L1 (50.91%).

[0408] [Table 8]

[0409] Figure 3 : All animal experimental procedures were reviewed by the local animal ethics committee (CEEA 34) in accordance with French regulations on the protection of animals used for scientific purposes and directives from the European Commission. The project was authorized by the French Ministry of Higher Education and Research with authorization number APAFIS#4594-2015092117546157 v9. Experiments were conducted on healthy male BALB / c OlaHsd mice (15g-21g, 4 weeks old, Envigo) that had been acclimatized to the laboratory for seven days and had free access to water and laboratory food.

[0410] Nanemulsion containing compound 1 in vivo Figure 3 studies

[0411] The nanoemulsion loaded with compound 1 was injected into the tail vein of BALB / c mice using a 26 G (0.45 mm × 13 mm) needle. Following good animal practice guidelines, the injection volume was 5 µL / g body weight. The injected dose was 0.1 µmol–0.5 µmol of compound per g of mouse body weight, with two mice injected per group. Nanoemulsions of compound 1 were prepared at final concentrations of 20 mM and 100 mM. Following injection, mice were anesthetized by inhalation of isoflurane. The mice were then placed headfirst into the cavity of the EPR spectrometer using a modified mouse cage. During recording, the mice were anesthetized with a mask (2%–3% isoflurane). The mice's body temperature was maintained between 32°C and 37°C. Body temperature and respiratory rate were monitored during the experiment. EPR signal acquisition began approximately 4 minutes after injection.

[0412] EPR measurements were performed using a Bruker Elexsys 540 EPR spectrometer (Bruker, Vissambau, France) equipped with a BLGR_23 cavity, operating in the L-band (1.2 GHz). The parameters used for EPR spectroscopy were as follows: 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; field center, 39.52 mT; scan width, 19 mT; scan time, 41.98 s. Spectral changes over time were continuously recorded for approximately 100 minutes. Data acquisition and processing were performed using Bruker Xepr software.

[0413] Nanemulsions comprising compounds 1 of the invention Figure 3 in vitro

[0414] In vitro studies were conducted to evaluate the distribution of compound 1 (NE) and to confirm the intracellular cleavage of compound 1 by esterases in vivo. Mice injected with the nanoemulsion were euthanized approximately 100 min after intravenous injection. Deep anesthesia was achieved by intraperitoneal injection of ketamine (100 mg / kg) / toluidine (10 mg / kg). Whole blood samples were collected from the left ventricle, stored in heparinized tubes, and frozen in liquid nitrogen. Blood replacement of the mice's hearts was performed using 10 to 15 mL of saline solution containing 25 U / mL heparin. Major organs (including brain, heart, liver, spleen, lungs, and kidneys) were then removed, weighed, and frozen in liquid nitrogen at -80°C for in vitro EPR analysis.

[0415] Homogenizes were prepared from each organ by mixing it with physiological saline (0.1 g wet tissue / 0.1 mL physiological saline) and depolymerizing it using a potter (Dounce homogenizer). After vortexing, the sample (30 µL) was transferred to a 5 cm long piece of breathable PTFE tubing (extruded sub-Lite-Wall, 0.635 mm inner diameter, 0.051 mm wall thickness; Zeus Industrial Products Ltd., Ireland), folded (V-shaped) into a 4 mm quartz tube, and then placed in the EPR chamber. Blood was not diluted before recording. Spectroscopy was performed using an Elexsys E500 EPR spectrometer operating at X-band (9.8 GHz), 21 °C, and equipped with a high-sensitivity SHQ chamber. The parameters used are as follows: 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; center, 350 mT; sweep width, 8 mT; sweep time, 41.94 s.

[0416] 100 mM potassium ferricyanide was gradually added to the sample (0.2 µL–1 µL) for subsequent EPR analysis. Potassium ferricyanide is a mild oxidizing agent, and its addition to the compound should oxidize the hydroxylamine released from compound 1 to nitrogen oxides visible via EPR.

[0417] Figure 3 : No acute toxicity was observed during in vivo experiments.

[0418] As described above, the probe derived from compound 1 is an EPR-invisible probe, which is cleaved intracellularly by esterases into free hydroxylamine. The resulting hydroxylamine reacts under oxidative stress conditions to generate nitric oxide radicals visible via EPR. No signal is expected in healthy systems because the levels of oxidants are very low and react little or not at all with free hydroxylamine. As expected, no EPR signal was observed in the heads of mice following injection of compound 1 NE throughout the entire recording period (i.e., 100 min). The concentration of compound 1 in the oil phase and the type of oil used to formulate the nanoemulsion did not affect its in vivo stability in healthy mice.

[0419] In vivo results showed that the probe derived from compound 1 was not significantly oxidized to nitrogen oxides in healthy mice. However, the sensitivity of the L-band EPR spectrometer was lower than that of the X-band spectrometer, meaning that X-band analysis of ex vivo samples allowed for the quantification of the amount of probe that reached different tissues and was lysed and / or oxidized. First, EPR acquisition was performed on organ homogenates. Then, potassium ferricyanide [K3(FeCN6)] was gradually added to recover the total amount of lysed probe precursor 1 distributed to each organ. Potassium ferricyanide oxidized the free hydroxylamine product of probe 1 to nitrogen oxides visible via EPR. For each organ, the addition of the oxidant resulted in an increase in the EPR signal intensity, thus indicating the sensitivity of the formulated probe to oxidants.

[0420] The results obtained (mean ± standard deviation) are shown in Table 8 below: Example 7: NMR study of nitroxide radicals derived from compounds of formula (I)

[0421] EPR signals were observed in different organs, indicating successful absorption of compound 1. As expected, an increase in potency was noted when the dose of the probe prodrug was increased from 0.1 µmol / g body weight to 0.5 µmol / g body weight using an olive oil-based formulation. Furthermore, EPR evaluation showed greater accumulation of compound 1 in the liver compared to other organs. Preferential accumulation of the probe prodrug in the liver was observed for both concentrations tested.

[0422] At the highest dose of compound 1 (0.5 µmol / g body weight) in an olive oil-based nanoemulsion (emulsion 1 NE-OO) with a Span80:K188 = 80:20, EPR spectra of blood samples indicated the presence of free nitrogen oxides (asterisks, asterisks) in an aqueous environment characterized by a nitrogen coupling constant of 1.56 mT. MRI Furthermore, nitrogen oxides are present in more lipophilic environments such as cell membranes (arrows). Results Trace amounts of protein-bound nitrogen oxides (triangular) were detected after the addition of ferricyanide. [Table 9] Unidentified six-line substances (A) appeared during oxidation in the blood. (N) =1.58mT, A (H) =2.36mT)(rhombus, ​ No unidentified six-line substances were observed in the EPR spectra of other organs. The distribution of nitrogen oxides in organs before and after ferricyanide treatment is shown in Table 7. A significant proportion of free hydroxylamine was observed only in the liver, while in other organs, most of the detected probe precursors were oxidized to nitrogen oxides.

[0423] ​

[0424] As described above, the compound of formula (I) has the ability to penetrate cells and be hydrolyzed by esterases in these cells into a probe containing a hydroxylamine moiety. Under oxidative stress, this hydroxylamine moiety is oxidized into nitric oxide radicals, which can shorten the relaxation time T of water protons. (1) and T (2) And used as MRI contrast agents. For modeling purposes, we measured the effect of nitride radicals of various compounds derived from formula (I) on relaxation time in a mixture of 90 vol% 0.1 M potassium phosphate buffer (pH 7.4) and 10% DMSO to demonstrate their ability to act as MRI contrast agents.

[0425] ​ Measurement scheme

[0426] MRI images were acquired on test subjects consisting of samples containing one of the nitrogen oxides of interest at concentrations of 0 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, and 15 mM. Each nitrogen oxide was dissolved in DMSO and then diluted in 0.1 M potassium phosphate buffer at pH 7.4 to obtain a final DMSO proportion of 10% by volume in the final mixture, regardless of the nitrogen oxide concentration. Samples were prepared in tubes with an inner diameter of 5 mm containing 0.6 mL of solution. The tubes were placed horizontally and parallel to each other for image acquisition. Measurements were performed using five tubes at a time and repeated to analyze samples at all concentrations.

[0427] MRI studies were performed using a Bruker BioSpec7 T system connected to an Advance III spectrometer (Bruker BioSpin MRI GmbH) at approximately 21°C (room temperature). T-wavelengths were obtained using saturation-recovery pulse sequences. (1) (T(1) map) (Echo time (TE) = 5.9ms, number of acquisitions (NA) = 1, number of repetitions (NR) = 1, slice thickness = 3mm, field of view (FOV) = 25×25mm) 2 The matrix was 125×125, with 15 recovery time (TR) values: 63.148ms - 6500ms - 100ms - 5000ms - 200ms - 4000ms - 400ms - 3000ms - 600ms - 2500ms - 800ms - 2000ms - 1000ms - 1600ms - 1300ms. T2 spectra were recorded using a multi-spin multi-echo (MSME) sequence (32 TE values ​​increasing in 25ms increments between 25ms and 800ms, TR=3500ms, NA=1, NR=1, slice thickness=3mm, FOV=25×25mm). 2 (Matrix = 125×125). MRI images were processed using ITK-SNAP 4.2.0 software (Free Software Foundation, Inc.).

[0428] ​ : MRI results showed increased contrast in different samples containing varying concentrations of nitrogen oxides. The relaxation times T1 and T2 of water protons decreased with increasing nitrogen oxide concentration. The longitudinal relaxation rate 1 / T1 and the transverse relaxation rate 1 / T2 increased linearly with nitrogen oxide concentration. The slopes of the corresponding lines give the values ​​of the longitudinal relaxation rate r1 and the transverse relaxation rate r2, which are grouped together in the table below. These values ​​are consistent with those reported in the literature for organic single radicals of the nitrogen oxide family.

[0429] ​ The relatively low values ​​are due to the incomplete dissolution of the compound in the solvent mixture (slight turbidity was observed).

Claims

1. A compound of formula (I): wherein n = 1 or 2, A and A’ are independently selected from the group consisting of the following substituents (II-a) and (II-b):

2. The compound according to claim 1, wherein and wherein R and R' are independently selected from H and linear or branched C1-C4 alkyl groups, Z is selected from linear or branched C1-C4 alkyl groups and aryl groups optionally substituted by linear or branched C1-C4 alkyl groups and / or by methoxy groups, and Formula (N) The structural unit of formula (III) is selected from the following structural units (III-a), (III-b), (III-c), (III-d) and (III-e): , , , and for each structural unit (III-a), (III-b), (III-c), (III-d), and (III-e), Ra, Rb, Rc, and Rdare independently selected from a linear or branched C1-C4alkyl group. A is selected from the group consisting of substituent (II-b) wherein Z = Me and substituent (II-a) wherein R = H, and A’ is selected from the group consisting of substituent (II-b) wherein Z = Me and substituent (II-a) wherein R = Me.

3. The compound according to claim 1 or 2, wherein R’ is methyl or ethyl, preferably methyl.

4. The compound according to any one of claims 1 to 3, wherein the compound of formula (I) is selected from the following compounds (I-a), (I-b), (I-b”), (I-c), (I-d) and (I-e):

5. The compound according to any one of claims 1 to 4, wherein the structural unit of formula (N) is selected from the structural units (III-b), (III-c), (III-d) and (III-e). 、 、 、 、 、 。 6. The compound according to any one of claims 1 to 5, wherein Ra, Rb, Rc and Rd are identical and selected from methyl and ethyl.

7. The compound according to any one of claims 1 to 6, selected from:

8. A pharmaceutical composition comprising at least a compound of formula (I) according to any one of claims 1 to 7 in a physiologically acceptable medium. and . The pharmaceutical composition is in the form of a nanoemulsion, preferably in the form of an oil-in-water nanoemulsion.

9. The pharmaceutical composition of claim 8, wherein, 10. The 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, the at least one oily compound comprising a triglyceride, preferably a C4-C24 triglyceride, more preferably a C8-C18 triglyceride.

11. The pharmaceutical composition according to any one of claims 8 to 10, further comprising at least one non-ionic surfactant, the at least one non-ionic surfactant being preferably selected from a silicone surfactant, a polysorbate, a cetearyl alcohol, a cetyl alcohol, an oleyl alcohol, a stearyl alcohol, a cocamide DEA, a cocamide MEA, a polyalkyl glucoside, a decyl glucoside, a lauryl glucoside, an octyl glucoside, a monolaurin, a poloxamer, a sorbitan monostearate, a sorbitan tristearate or any combination thereof, preferably from a poloxamer and a sorbitan monostearate.

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

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

14. A method for detecting oxidative stress in a subject in vivo, said method comprising: - a step of capturing one or more images of a subject or of at least one portion of a subject that has been previously treated with a compound of formula (I) according to any one of claims 1 to 7 or by a composition according to any one of claims 8 to 11 using an imaging technique, preferably electron paramagnetic resonance imaging or magnetic resonance imaging, - or a step of recording a signal from a subject or from at least one portion of a subject that has been previously treated with a compound of formula (I) according to any one of claims 1 to 7 or with a composition according to any one of claims 8 to 11 by electron paramagnetic resonance spectroscopy.

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

Images