Piperidine, pyrrolidine, and isoindole precursors as probes for oxidative stress

Abstract

The present invention relates to a compound of formula (I), TIFF2026525175000071.tif28170 wherein, n is 1 or 2, A and A' are independently selected from the group consisting of the following substituents (II-a) and (II-b), TIFF2026525175000072.tif30170 R and R' are independently selected from H and linear or branched C1-C3 alkyl, and 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 a hydroxyl group, formula (N) TIFF2026525175000073.tif23170 is selected from the following motifs (III-a), (III-b), (III-c), (III-d), and (III-e), TIFF2026525175000074.tif29170 Ra, Rb, Rc, and Rd are independently selected from linear or branched C1-C4 alkyl groups for each of the motifs (III-a), (III-b), (III-c), (III-d), and (III-e).

Description

[Technical Field]

[0001] This invention relates to novel precursors for oxidative stress probes and various uses thereof. [Background technology]

[0002] One of the greatest social challenges is promoting active and healthy aging among the population. To achieve this, it is essential to discover new diagnostic methods or effective therapies for cardiovascular and neurological diseases or cancer. It is becoming increasingly clear that oxidative stress has a significant 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 onset of these diseases and would be useful in designing diagnostic and treatment tools.

[0003] While oxidative biomarkers of proteins and lipids in biological fluids are interesting tools, they cannot pinpoint the location of pathology. 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 commonly used. However, they have certain limitations. PET requires expensive, short-lived radiotraces, necessitates access to a local cyclotron for their manufacture, and is not suitable for certain medical conditions. Susceptibility imaging (SWI) assesses the metal content of tissues (Cu, non-heme iron) as a potential source of reactive oxygen species (ROS) via the Fenton reaction, but many areas accumulate iron (e.g., in ferritin) without causing excessive radical production. More specifically, magnetic resonance spectroscopy can measure some antioxidant defenses (ascorbic acid, glutathione), but misses essential enzymes involved in ROS detoxification.

[0004] Methods using electron paramagnetic resonance (EPR) and exogenous molecular probes have already demonstrated their usefulness in evaluating and mapping the "redox state" in the brain under oxidative stress conditions, for example, in rat models with 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, to be a preferred method for measuring redox physiology (KKGriendling, RMTouyz, JLZweier, S.Dikalov, W.Chilian, Y.Chen, DGHarrison, 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 molecular probes already known, we can distinguish probes from the hydroxylamine family, which are oxidized to nitroxides in cells and detectable by EPR.

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

[0007] [ka]

[0008] This compound contains an ester functional group that protects the hydroxylamine group. The purpose of these precursors is to transport the probe into the cell with minimal degradation. Masking the hydroxylamine functional group limits the spontaneous oxidation of hydroxylamine before internalization into the cell, making it easier for non-chemist experimenters to handle. Once internalized, the ester functional group is hydrolyzed by cellular esterases, releasing the hydroxylamine moiety, which is oxidized to a nitroxide and becomes detectable by EPR.

[0009] However, these probes have several limitations, including poor biological stability, particularly in vivo, lack of specificity and / or sensitivity, and a limited number of commercially available probes.

[0010] Therefore, there is a need for novel and more effective molecular probes. This need is becoming increasingly urgent, given the recent commercialization of new technological advancements in EPR spectrometers and the limitations of probes hindering the development of redox EPR applications.

[0011] Therefore, the object of the present invention is to propose a novel molecular probe for oxidative stress that provides improved performance compared to existing probes.

[0012] In particular, one objective of the present invention is to propose a novel molecular probe for oxidative stress that provides improved stability and / or better sensitivity and / or easier handling. [Overview of the project]

[0013] For this purpose, the present invention relates to a compound of formula (I),

[0014] [ka] (In the formula, n=1 or 2, preferably n=1. A and A' are independently selected from the group consisting of the following substituents (II-a) and (II-b):

[0015] [ka] R and R' are independently selected from H and linear or branched C1-C4 alkyl groups, and Z is selected from linear or branched C1-C4 alkyl groups, and aryl groups optionally substituted with linear or branched C1-C4 alkyl groups and / or methoxyl groups (such as 4-methoxyphenyl). Motif of formula (N)

[0016] [ka] The following motifs are selected: (III-a), (III-b), (III-c), (III-d), and (III-e).

[0017] [ka] Ra, Rb, Rc, and Rd are independently selected from linear or branched C1-C4 alkyl groups for each motif (III-a), (III-b), (III-c), (III-d), and (III-e).

[0018] A linear or branched C1-C4 alkyl group consists of n carbon atoms (where n is equal to 1, 2, 3, or 4). This refers to a hydrocarbon chain containing 2n+2 hydrogen atoms. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl groups.

[0019] In fact, the inventors have surprisingly discovered that the compound of formula (I) according to the present invention is a probe precursor for oxidative stress, particularly detectable by EPR spectroscopy, exhibiting significantly improved performance compared to existing compounds, especially in terms of in vivo stability and sensitivity. While not wishing to be bound by any theory, the inventors believe that the Ra, Rb, Rc, and Rd groups of the formula (N) motif in prior art probe precursors induce steric hindrance, preventing cellular esterases from hydrolyzing the ester protecting the hydroxylamine functional group. Indeed, the deprotection reaction is slow and incomplete, resulting in the release of very small amounts of probe into the cell, making detection by medical imaging or EPR spectroscopy difficult. In the compound of formula (I) according to the present invention, the presence of a spacer group between the nitrogen of the formula (N) motif and the carboxyl targeted by the esterase in group A' enables rapid and complete deprotection of the hydroxylamine functional group. For example, when A' is a substituent (II-a) where R=R'=CH3, 100% of the hydroxylamine is released in 10 minutes at 37°C, compared to the deprotection of a commercially available ACP probe (1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine) which is limited to 27% under the same conditions. Therefore, the compounds of formula (I) according to the present invention enable easier handling (suitable for non-chemist experiments for biological applications) and more efficient release of the probe, resulting in improved sensitivity.

[0020] In formula (I), except when the motif of formula (N) is the motif of formula (III-e), a person skilled in the art can determine each

[0021] [ka] It will be understood that the group can be bonded to any substituteable carbon atom (having at least one hydrogen atom) that constitutes the ring(s) of the motif of formula (N) in formulas (III-a), (III-b), (III-c), and (III-d).

[0022] The presence of group A in the compound of formula (I) favorably improves the accumulation of the probe in cells, and therefore the amount of the probe detectable in the post-oxidation cell, and consequently the quality of the resulting image or signal. Group A allows the compound to pass through cells passively and is sensitive to hydrolysis by intracellular esterases, which results in the release of negatively charged carboxylate ions that do not freely diffuse across the membrane.

[0023] It is understood that each atom of the compound of formula (I) can exist in any of its natural isotopes.

[0024] In certain embodiments, the nitrogen atom of the motif of formula N is nitrogen-15.

[0025] Alternatively, or in combination, the hydrogen atom of the (N) motif, particularly the hydrogen atoms of the Ra, Rb, Rc, and Rd groups, is preferably hydrogen-2 (more commonly known as deuterium).

[0026] The isotopic composition of the compound of formula (I), particularly its nitrogen-15 and / or hydrogen-2 content, can be determined using techniques known to those skilled in the art. For example, high-resolution mass spectrometry (HRMS) can be used to determine the average concentration of isotopes during testing across all sites of the compound of formula (I). Quantitative nuclear magnetic resonance (NMR) of deuterium and nitrogen-15 can also be used to test specific isotopic distributions at each site of the compound of formula (I). Similarly, after hydrolysis and oxidation of the compound of formula (I) (e.g., by potassium ferricyanide), the electron paramagnetic resonance (EPR) spectra differ for molecules containing nitrogen-15 (two lines) and molecules containing nitrogen-14 (three lines).

[0027] Preferably, in substituent (II-b), Z is selected from methyl, ethyl, propyl, isopropyl, tert-butyl, phenyl, and 4-methoxyphenyl. Advantageously, for the remainder of the description, Z = Me.

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

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

[0030] Preferably, A and A' are the same.

[0031] According to a particular embodiment, A = A' = (II - a).

[0032] According to another embodiment, A=A'=(II-b).

[0033] The use of group (II-b) favorably avoids the release of one equivalent of acetaldehyde and one equivalent of formaldehyde after esterase action, which could potentially induce cellular stress that could interfere with the measurement of oxidative stress, as well as potential toxicity issues at very high doses. In fact, hydrolysis of group (II-b) by esterase results only in the formation of the non-toxic 3-hydroxybutan-2-one and 2,3-butanediol.

[0034] Preferably, the motif of formula (N) is selected from motifs (III-b), (III-c), (III-d), and (III-e), preferably from motifs (III-b), (III-c), and (III-d), more preferably from (III-b) and (III-d), or from (III-c).

[0035] Preferably, the compound of formula (I) is selected from the following compounds (Ia), (Ib), (I-b''), (Ic), (I-c''), (Id), (I-d'), (I-d''), (I-d''''), and (Ie):

[0036] [ka] A, A', Ra, Rb, Rc, and Rd are the respective compounds (Ia), (Ib), (I-b''), (Ic), (I-c''), (Id), (I-d'), (I-d''), (I-d''') and (Ie) are defined independently as described above.

[0037] Preferably, the compound of formula (I) is selected from compounds (Ia), (Ib), (I-b''), (Ic), (Id), and (Ie), more preferably from compounds (Ia), (Ib), (I-b''), (Ic), and (Id), more preferably from compounds (Ib), (Ic), and (Id), even more preferably from (Ib) and (Id), or alternatively from (Ic).

[0038] More preferably, the compound of formula (I) is selected from the following compounds (IV), (V), (VI), and (VII):

[0039] [ka] A and A' are defined as above.

[0040] Preferably, the compound of formula (I) is selected from compounds (IV) and (V), and alternatively from compound (VI).

[0041] Preferably, Ra, Rb, Rc, and Rd are independently selected from methyl, ethyl, and isopropyl, preferably from methyl and ethyl. Preferably, Ra, Rb, Rc, and Rd are the same, preferably selected from methyl and ethyl.

[0042] The Ra, Rb, Rc, and Rd groups are advantageous in enabling better image or signal quality. Without being bound by any theory, the inventors believe that the steric hindrance induced by the four Ra, Rb, Rc, and Rd groups stabilizes the nitroxide radical and reduces the superoxide dismutase-mimicking activity of the resulting nitroxide, which improves the response of the hydroxylamine probe to the generation of the superoxide anion.

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

[0044] [ka] It is selected from the group that includes it.

[0045] According to one embodiment, the compound of formula (I) is as follows:

[0046] [ka]

[0047] According to another embodiment, the compound of formula (I) is as follows:

[0048] [ka]

[0049] According to the alternative embodiment, the compound of formula (I) is different from the following compounds.

[0050] [ka]

[0051] The present invention also relates to a method for preparing a compound of formula (I) according to the present invention. This method is described in particular in the examples.

[0052] A typical method for preparing the compound of formula (I) according to the present invention involves using a hydroxylamine to prepare the compound of formula (X).

[0053] [ka] For example, at least one step (S1) to obtain a compound of formula (X') by reduction in the presence of dihydrogen,

[0054] [ka] Next, compound (X') is the compound of formula (XI).

[0055] [ka] Alternatively, by nucleophilic addition to the compound of formula (XI'),

[0056] [ka] Compound of formula (XII)

[0057] [ka] or includes a step (S1') that yields a compound of formula (XII').

[0058] [ka]

[0059] The motifs A, A', n, R, and formula (N) are as defined above for the compound of formula (I).

[0060] Preferably, step (S1) is carried out in the presence of palladium carbon and dihydrogen. Then, before carrying out step (S1'), dihydrogen is removed by purging with argon.

[0061] Preferably, the step of adding the compound of formula (XI) to the compound of formula (X') (S1') is carried out at -5°C to 8°C, and the reaction mixture is then left at room temperature (20 to 28°C) for 12 to 20 hours. It is stirred.

[0062] This method involves the compound of formula (XII) and the compound of formula (XIII)R'-C(O)-O - The process may further include a nucleophilic substitution step (S2) with the compound of (XIII), where R' is as defined above for the compound of formula (I), resulting in the compound of formula (I) according to the present invention.

[0063] Preferably, step (S2) is carried out in the presence of an acetate, preferably mercury acetate. Preferably, the reaction mixture is stirred at room temperature (20-28°C) for 12-20 hours.

[0064] This method involves the compound of formula (XIV) and

[0065] [ka] Compound of formula (XV)

[0066] [ka] The process may further include step (S0) of nucleophilic substitution to obtain a compound of formula (X). The motifs of A, n, and formula (N) are as defined above for the compound of formula (I), where X = Cl or Br, preferably A = (II-a).

[0067] Preferably, step (S0) is carried out in the presence of an inorganic base, preferably potassium carbonate. Preferably, the reaction mixture is stirred at room temperature (20-28°C) for 1-5 hours.

[0068] Alternatively, this method involves converting the compound of formula (XIV) to the acyl chloride of formula (XIV').

[0069] [ka] The process involves activation (S0'), followed by coupling compound (XV') and compound (XIV'),

[0070] [ka] The process may further include a step (S0'') to obtain a compound of formula (X), where A, n and the motif of formula (N) are as defined above for the compound of formula (I), and preferably A=(II-b).

[0071] The activation step (S0') is preferably carried out in the presence of acyl dichloride, such as oxalyl chloride and dimethylformamide.

[0072] 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 10-48 hours.

[0073] Any alternative method known in the prior art may be used.

[0074] The present invention also relates to a pharmaceutical composition comprising at least one compound of formula (I) according to the present invention in a physiologically acceptable medium.

[0075] Preferably, the pharmaceutical composition according to the present invention is in the form of a nanoemulsion, preferably an oil-in-water nanoemulsion.

[0076] Preferably, the nanoemulsion comprises an aqueous phase and an oil phase, the oil phase comprising the compound of formula (I). Preferably, at least 90% by weight, preferably 95-99.99% by weight, of the total mass of the compound of formula (I) in the nanoemulsion is contained in the oil phase.

[0077] Preferably, the oil phase further comprises at least one oil compound containing triglycerides, preferably C4-C24 triglycerides, more preferably C8-C18 triglycerides. Triglycerides are generally triesters of fatty acids and glycerol, the fatty acids may have chain lengths in the range of C4-C24, and the latter may be linear or branched, saturated or unsaturated.

[0078] Preferably, the oily compound is selected from vegetable oils and medium-chain triglycerides having C6-C12 chains, preferably C8-C10 chains.

[0079] The triglycerides according to the present invention are, in particular, triglycerides of 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, especially sweet almond oil, coconut oil, rapeseed oil, cottonseed oil, hazelnut, macadamia, jojoba, alfalfa, poppy, pumpkin, sesame, pumpkin, rapeseed, blackcurrant, evening primrose, millet, barley, quinoa, rye, safflower, kukui tree, passionflower, muscat rose, and coconut, or caprylic / capric acid triglycerides.

[0080] Advantageously, the oily compound of the oil phase is selected from olive oil and Labrafac® lipophile WL 1349.

[0081] Preferably, the aqueous phase contains water and optionally NaOH, NaCl, glycerol, sorbitol, dextrose, or xylitol. Preferably, the aqueous phase contains nanoemulsified NaOH is included to adjust the pH of the emulsion to 6.8-7.2, preferably about 7, and optionally, glycerol is included in an amount preferably in the range of 0.5-5% by weight relative to the total weight of the emulsion.

[0082] Preferably, the composition, preferably the nanoemulsion, further comprises at least one surfactant, preferably a nonionic surfactant, preferably an organosilicon surfactant, polysorbate, cetostearyl alcohol, cetyl alcohol, oleyl alcohol, stearyl alcohol, cocamide DEA, cocamide MEA, polyalkyl glucoside, decyl glucoside, lauryl glucoside, octyl glucoside, monolaurin, poloxamer, sorbitan monostearate, sorbitan tristearate, or any combination thereof, preferably selected from poloxamer and sorbitan monostearate.

[0083] Preferably, the composition, preferably a nanoemulsion, comprises a first surfactant as defined above and a second surfactant as defined above, which is different from the first surfactant.

[0084] Advantageously, the first surfactant is sorbitan monostearate, and the second surfactant is selected from poloxamers, preferably poloxamer 188.

[0085] An example of sorbitan monostearate is the commercially available product Span® 80. An example of poloxamer is the commercially available product Kolliphor® P188.

[0086] Preferably, the total mass content of the surfactant is in the range of 4% to 12%, preferably 5% to 10%, relative to the total mass of the composition, preferably the nanoemulsion.

[0087] Preferably, the mass content of the first surfactant is in the range of 3% to 7%, preferably 4% to 6%, relative to the total mass of the composition, preferably the nanoemulsion.

[0088] Preferably, the mass content of the second surfactant is in the range of 1% to 5%, preferably 1% to 4%, relative to the total mass of the composition, preferably the nanoemulsion.

[0089] Preferably, in the composition, preferably in the nanoemulsion, the mass ratio of the first surfactant to the second surfactant is in the range of 1 to 6, preferably 1.2 to 4.5, and advantageously 1.5 to 4.2.

[0090] Preferably, in the composition, preferably in the nanoemulsion, the mass ratio of the aqueous phase to the oil phase is in the range of 1 to 8, preferably 2 to 6, preferably 3 to 5, and advantageously 3.5 to 4.5.

[0091] Preferably, in the composition, preferably in the nanoemulsion, the mass ratio of the oily compound to all surfactants is in the range of 1 to 5, preferably 1.5 to 4, preferably 2 to 3.5, and advantageously 2.5 to 3.5.

[0092] Preferably, the nanoemulsion has an average droplet size in the range of 50 to 500 nm, preferably in the range of 80 to 300 nm, and advantageously in the range of 100 to 200 nm.

[0093] 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.

[0094] The average droplet size is Zetasizer® Nano ZS (Malvern The results are determined by photon correlation spectroscopy using Panalytical (Worcestershire, UK). 10 μL of nanoemulsion is diluted 100-fold with ultrapure water. All measurements are performed. The scattering angle is recorded at 173° at 25°C. The correlation function curve is fitted using an exponential fit (cumulative approach) to evaluate the hydrodynamic diameter and PDI. All measurements are performed triple to determine the average droplet size and PDI, expressed as the average Z diameter (Z-Ave).

[0095] Preferably, in the nanoemulsion, the mass ratio of the compound of formula (I) to the oil phase is in the range of 0.1 to 0.5, preferably 0.15 to 0.45, more preferably 0.2 to 0.4, most preferably 0.25 to 0.35, and advantageously 0.30 to 0.35.

[0096] 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, and advantageously 0.08 mol / L to 0.12 mol / L.

[0097] 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, and advantageously 10 mmol / L to 50 mmol / L.

[0098] The present invention further relates to the use of compounds or compositions of formula (I) according to the present invention as precursors of compounds for electron paramagnetic resonance spectroscopy, or imaging, preferably for medical or preclinical imaging, particularly for electron paramagnetic resonance spectroscopy, for electron paramagnetic resonance imaging, or for magnetic resonance imaging.

[0099] According to one embodiment, a compound or composition of formula (I) according to the present invention, preferably a nanoemulsion, is used as a radical probe precursor for electron paramagnetic resonance spectroscopy or electron paramagnetic resonance imaging.

[0100] According to another embodiment, the compound or composition of formula (I) according to the present invention, preferably a nanoemulsion, is used as a contrast agent precursor for magnetic resonance imaging.

[0101] As described above, the compound of formula (I) has the ability to penetrate into cells and be hydrolyzed by esterases within those cells to produce probes containing a hydroxylamine moiety.

[0102] This hydroxylamine motif can be oxidized under intracellular oxidative stress to form a nitroxide radical, which can be directly detected by spectroscopy or electron paramagnetic resonance imaging. Paramagnetic nitroxide radicals can also cause changes in the longitudinal magnetic relaxation time (T1) and / or transverse magnetic relaxation time (T2) of nearby water protons or aliphatic protons in tissue, resulting in changes in contrast in magnetic resonance imaging.

[0103] Preferably, the compound or composition of formula (I) according to the present invention is used in vivo or in vitro.

[0104] According to one embodiment, the compound or composition of formula (I) according to the present invention is used in vitro.

[0105] The present invention also relates to a method for in vitro detection of oxidative stress, - A step of contacting a compound or composition of formula (I) according to the present invention with a culture medium containing cells, body fluids, or biological tissue, - Record the signal from the culture medium by electron paramagnetic resonance spectroscopy, or by imaging techniques, preferably medical or preclinical imaging techniques, preferably electron paramagnetic resonance imaging or magnetic resonance imaging. The present invention relates to a method comprising the step of capturing one or more images of a culture medium by imaging.

[0106] The present invention also provides a method for in vivo detection of oxidative stress in a subject, - A step of capturing one or more images of an object or at least a portion of an object previously treated with a compound of formula (I) according to the present invention, or with a composition according to the present invention, using imaging techniques, preferably medical or preclinical imaging techniques, preferably electron paramagnetic resonance imaging or magnetic resonance imaging, The present invention relates to a method comprising the step of recording a signal from an object or at least a portion of an object previously treated with a compound of formula (I) or a composition according to the present invention by electron paramagnetic resonance spectroscopy.

[0107] Preferably, the target is treated in an amount detectable by the imaging technique or the electron paramagnetic resonance spectroscopy.

[0108] According to the present invention, "detectable amount" means an amount of compound (I) or composition sufficient to obtain an image interpretable by the imaging technique or a spectrum interpretable by the electron paramagnetic resonance spectroscopy.

[0109] Preferably, a method for detecting oxidative stress in vivo in a subject is: - A step of administering a detectable amount of the compound or composition of formula (I) according to the present invention to the subject using the imaging technique, - The imaging technique includes capturing an image of an object or at least a portion of an object that has been treated, preferably by electron paramagnetic resonance imaging or magnetic resonance imaging, or recording a signal of at least a portion of an object by electron paramagnetic resonance spectroscopy.

[0110] According to one embodiment, the method for in vivo detection of oxidative stress according to the present invention includes capturing images of an object or at least a portion of an object being treated by electron paramagnetic resonance imaging.

[0111] According to another embodiment, the method for in vivo detection of oxidative stress according to the present invention includes capturing images of an object or at least a portion of an object to be treated by magnetic resonance imaging.

[0112] According to yet another embodiment, the method for in vivo detection of oxidative stress according to the present invention includes recording the signal of at least a portion of the subject by electron paramagnetic resonance spectroscopy.

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

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

[0115] Preferably, the in vivo diagnostic method is: - An image acquisition step using imaging technology on an object or at least a portion of an object previously treated with a detectable amount of compound (I) of the present invention, wherein the imaging technology is preferably selected from electron paramagnetic resonance imaging technology and magnetic resonance imaging technology. -or an amount of the compound of formula (I) according to the present invention that can be detected by the said electron paramagnetic resonance spectroscopy. The process includes any step of recording a signal from an object or at least a portion of an object that has been previously treated with a material or composition using electron paramagnetic resonance spectroscopy.

[0116] The present invention also relates to a compound or composition of formula (I) according to the present invention for use as a radical probe precursor in an in vivo diagnostic method, the method preferably comprising electron paramagnetic resonance imaging technique.

[0117] The present invention also relates to a compound or composition of formula (I) according to the present invention for use as a contrast agent precursor in an in vivo diagnostic method, the method preferably including magnetic resonance imaging technology.

[0118] The present invention also relates to a compound or composition of formula (I) according to the present invention for use as a contrast agent precursor in an in vivo diagnostic method, the method preferably comprising electron paramagnetic resonance spectroscopy techniques.

[0119] The present invention also provides a compound of formula (I) or a composition according to the present invention for use in a method for detecting a disease in a subject to be diagnosed, the method being - A step of capturing images of one or more healthy subjects previously treated with a compound or composition of formula (I) according to the present invention in an amount detectable by said imaging technique, preferably medical imaging, - A step of capturing by imaging technology one or more images of a diagnostic target previously treated with a compound or composition of formula (I) according to the present invention in an amount detectable by the imaging technology, - A process to determine the difference between a healthy subject and a subject to be diagnosed, - This includes the step of comparing this difference with a reference value to determine whether or not a disease is present in the subject of diagnosis.

[0120] The present invention also relates to a compound or composition of formula (I) according to the present invention for use in a method for detecting a disease in a subject to be diagnosed, wherein the method is - A step of recording a signal from a healthy subject previously treated with a detectable amount of compound (I) of the present invention by electron paramagnetic resonance spectroscopy, - A step of recording, by electron paramagnetic resonance spectroscopy, a signal from a diagnostic target previously treated with a compound or composition of formula (I) according to the present invention in an amount detectable by said electron paramagnetic resonance spectroscopy, - A process to determine the difference between a healthy subject and a subject to be diagnosed, - This includes the step of comparing this difference with a reference value to determine whether or not a disease is present in the subject of diagnosis.

[0121] The present invention also relates to a compound or composition of formula (I) according to the present invention for use in a method for monitoring a disease in a subject, wherein the method - A step of capturing one or more images of an object previously treated with a detectable amount of the compound (I) of the present invention at time t0 using the imaging technique, - A step of capturing one or more images of an object previously treated with a detectable amount of compound (I) of the present invention at time t1 after time t0 using imaging technology, - A step of determining the difference between the image captured at time t0 and the image captured at time t1, - Includes a step of determining changes in the disease.

[0122] The present invention also relates to a compound or composition of formula (I) according to the present invention for use in a method for monitoring a disease in a subject, wherein the method - A step of recording at least one signal from an object previously treated with a detectable amount of the compound or composition of formula (I) according to the present invention at time t0 by electron paramagnetic resonance spectroscopy, - A step of recording, at time t1 after time t0, at least one signal from an object previously treated with a detectable amount of the compound or composition of formula (I) according to the present invention by electron paramagnetic resonance spectroscopy, - A step of determining the difference between the signal recorded at time t0 and the signal recorded at time t1, - Includes a step of determining changes in the disease.

[0123] The present invention also relates to a compound of formula (I) or a composition according to the present invention for use in a method for determining the effectiveness of a treatment for a disease in a subject of disease, the method is - A step of capturing images by imaging technology of one or more diseased subjects that have been previously treated with a detectable amount of the compound of formula (I) or a composition according to the present invention at time t0, and optionally also treated with an effective amount of said treatment for the disease, - A step of capturing one or more images of a patient who, at time t1 after time t0, has been previously treated with a detectable amount of compound (I) of the present invention, and optionally also treated with an effective amount of the treatment for a disease, using the imaging technique, - A step of determining the difference between the image captured at time t0 and the image captured at time t1, -Includes a step of determining the effectiveness of the treatment.

[0124] These steps may be repeated several times to monitor the treatment over time.

[0125] The present invention also relates to a compound of formula (I) or a composition according to the present invention for use in a method for determining the effectiveness of a treatment for a disease in a subject of disease, the method is - A step of recording, by electron paramagnetic resonance spectroscopy, at time t0, at least one signal from a diseased subject previously treated with a detectable amount of the compound or composition of formula (I) according to the present invention, - A step of recording, at time t1 after time t0, at least one signal from a diseased subject previously treated with a detectable amount of the compound or composition of formula (I) according to the present invention by electron paramagnetic resonance spectroscopy, - A step of determining the difference between the signal captured at time t0 and the signal captured at time t1, -Includes a step of determining the effectiveness of the treatment. [Brief explanation of the drawing]

[0126] [Figure 1] This is the EPR spectrum obtained when compound 1 (100 μM) was incubated with 5 million U87 cells in serum-free DMEM medium. It shows a large number of components characteristic of nitroxides in the isotropic region (stars) and a small number of components characteristic of nitroxides in the intermediate region (arrows). [Figure 2] These are the EPR spectra of 5 million U87 cells in a suspension incubated with compound 1 (100 μM), with and without Tempone-15N (dotted line). [Figure 3] These are representative EPR spectra obtained in the X-band from the blood and brain homogenates 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 (dotted line) the addition of potassium ferricyanide. [Modes for carrying out the invention]

[0127] The present invention will be described with reference to the following non-limiting embodiments. [Examples]

[0128] Example 1: Synthesis of the compound of formula (I) Materials and methods All reagents were purchased from commercial suppliers and used without purification. Thin-layer chromatography (TLC) was performed using silica gel 60 F254 TLC plates, and preparative column chromatography was performed using silica gel (230-400 mesh). Dry, oxygen-free THF was prepared using the Pure Solv™ microsolvent purification system (Sigma-Aldrich). NMR spectra were obtained. 1 H and 13 The 1C spectrum was recorded using a Bruker Biospin Advance II 500MHz or Bruker ARX 250 250MHz spectrometer, and the corresponding solvent peak was referenced (CDCl3;δ H = 7.26 ppm, δ C (=77.0 ppm). Fourier transform infrared (IR) spectra were recorded with a Perkin Elmer FT-IR spectrometer equipped with an ATR objective lens. High-resolution mass spectra were recorded with a Thermo Exactive_HCD spectrometer (Thermo Scientific, Les Ulis, France). Electron paramagnetic resonance (EPR) spectra were manipulated in the X band (9.8 GHz) and obtained at 21°C with an Elexsys E500 spectrometer (Bruker, France, Weissenburg) equipped with a high-sensitivity SHQ cavity. Nitrogen bond constant A N The full width at half maximum (FWHM) ΔB is given in millitesla (mT) for a 100 μM nitroxide radical solution in 0.1 M potassium phosphate buffer at pH 7.4, with or without 1 mM diethylenetriaminepentaacetic acid (DTPA). The melting temperature of the solid product was measured using a Stuart® SMP20 (Cole-Palmer®) instrument.

[0129] General Procedure A Anhydrous potassium carbonate (2 equivalents) was added to a dry DMSO solution ([C]=0.4M) of a carboxylic acid derivative (1 equivalent). This mixture was stirred at 22°C for 5 minutes, and then bromomethyl acetate (1.2-1.5 equivalents) was added. The reaction was maintained at 22°C with stirring for 2 hours. Next, 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 silica gel column chromatography under the elution conditions shown for each case to obtain the desired compound.

[0130] General Procedure B A nitroxide derivative (1 equivalent) and palladium (10% on carbon) were placed in a membrane-closed flask and purged with a three-phase vacuum / argon atmosphere. Under an argon atmosphere, dry oxygen-free THF ([C]=0.1M) was introduced, and dihydrogen was bubbled into the mixture for 30 minutes with stirring. To remove the dihydrogen, argon was bubbled for 5 minutes. After cooling to 0°C, triethylamine (1.5-2.5 equivalents) was added, followed by the addition of a chloroformate derivative (1.5-2 equivalents), 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.

[0131] General Procedure C A solution of a chlorinated derivative (1 equivalent) in acetic acid ([C]=0.55M) was added to mercury(II) acetate (1.5 equivalents) which had been added to a solution of the chlorinated derivative (1 equivalent) in acetic acid ([C]=0.55M). The mixture was stirred overnight at 22°C, during which time a fine white precipitate of mercury(II) chloride appeared. Most of the acetic acid was removed under reduced pressure, and the residue was suspended in diethyl ether and filtered. The ether solution was washed with water, a saturated aqueous solution of sodium bicarbonate, and then again with water. The product was 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 shown for each case to obtain the desired compound. In some cases, purification was not necessary.

[0132] General Procedure D To a solution of a carboxylic acid derivative (1 equivalent) in dry dichloromethane, oxalyl chloride (5 equivalents) was added dropwise under argon, followed by the addition of a 0.15 M solution of anhydrous dimethylformamide (5 mol%) in dry dichloromethane. The reaction mixture was stirred under argon at 20°C for 2 hours, and then evaporated under vacuum. In a second flask under argon, 4-hydroxymethyl-5-methyl-1,3-dioxol-2-one (1 equivalent) was dissolved in dry dichloromethane ([C]=0.03M). Triethylamine (1.2 equivalents) and 4-dimethylaminopyridine (5 mol%) were added with vigorous stirring. This mixture was transferred to a flask containing pre-synthesized acyl chloride dissolved in dry dichloromethane, and the reaction mixture was stirred under argon at 20°C for 24 hours. After adding saturated sodium bicarbonate aqueous solution, the aqueous phase was removed, the organic phase was washed with saturated sodium chloride aqueous solution, and then dried over anhydrous magnesium sulfate. After filtration and evaporation of the solvent under reduced pressure, the crude product was purified by silica gel column chromatography under the elution conditions shown for each case to obtain the desired compound.

[0133] Synthesis of probe precursors incorporating isoindoline motifs [ka]

[0134] Synthesis of Compound 1 1) 5-acetoxymethoxycarbonyl-1,1,3,3-tetraethylisoindoline-2-yloxyl radical (S2)

[0135] [ka]

[0136] 5-Carboxy-1,1,3,3-tetraethylisoindol-2-yloxyl radical Cal S1 was obtained from 4-methylphthalic anhydride according to the procedure published by Fairfull-Smith et al. (K.E. Fairfull-Smith, F. Brackmann, S.E. Bottle, The Synthesis of Novel Isoindoline Nitroxide Bearing Water-Solubilising Functionality, Eur. J. Org.. Chem. (2009) 1902-1915).

[0137] 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 and the general procedure A was followed. 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%).

[0138] Rf = 0.47 (cyclohexane / ethyl acetate, 4:1).

[0139] IR v max (ATR, ZnSe, pure, cm[[ID=2S]] -1 ): 2973, 2940, 2881, 1767, 1738, 1617, 1457, 1418, 1370, 1284, 1259, 1231, 1203, 1159, 1101, 1032, 983.

[0140] HRMS (ESI-Orbitrap) m / z [M+H] + for C 20 H 29 NO5 calculated value 363.2040; measured value 363.2027, [M+2H] + for C I 20 H 30 NO5 calculated value 364.2118; measured value 364.2110.

[0141] EPR (100 μM in solution in 0.1 M potassium phosphate buffer at pH=7.4): A N =1.54mT, ΔB=0.17mT.

[0142] 2) 2-(((1-chloroethoxy)carbonyl)oxy)-1,1,3,3-tetraethylisoindoline-5-carboxylate acetoxymethyl ester (S3)

[0143] [ka]

[0144] S2 (262 mg, 0.723 mmol, 1 equivalent), Pd / C (30 mg), Et3N (151 μL, 1.084 mmol, 1.5 equivalents), and 1-chloroethyl chloroformate (117 μL, 1.084 mmol, 1.5 equivalents) were used, and the general procedure B was followed. The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 90 / 10) to obtain the desired compound as a pale yellow oil (298 mg, 88%).

[0145] Rf = 0.56 (dichloromethane) NMR 1 H(500MHz,CDCl3)δ(ppm)8.00(d,J=7.9,1H),7.77(s,1H),7.15(d,J=7.9Hz,1H),6.49(q,J= 5.8Hz,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=5.8Hz ,3H),1.84-1.73(m,4H),1.03-0.92(m,6H),0.84-0.75(m,6H).

[0146] NMR 13C(126MHz, 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.

[0147] IRv max (ATR, ZnSe, pure, cm -1 ): 2968, 2942, 2863, 1790, 1690, 1450, 1373, 1282, 1204, 1159, 1080, 1010, 983.

[0148] HRMS(ESI-Orbitrap)m / z[M+H] + C 23 H 33 Calculated value for ClNO7: 470.1940; measured value: 470.1930; [M+Na] + C 23 H 32 The calculated value for ClNNaO7 is 492.1760; the measured value is 492.1748.

[0149] 3) 2-(((1-acetoxyethoxy)carbonyl)oxy)-1,1,3,3-tetraethylisoindoline-5-carboxylate acetoxymethyl ester (compound 1)

[0150] [ka]

[0151] S3 (290 mg, 0.617 mmol, 1 equivalent), AcOH (1.1 mL), and Hg(OAc)2 (294 mg, 0.925 mmol, 1.5 equivalents) were used, and the general procedure C was followed. The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 80 / 20) to obtain the desired compound as a pale yellow oil (254 mg, 84%).

[0152] Rf = 0.47 (dichloromethane) NMR1 H(500MHz,CDCl3)δ(ppm)7.99(d,J=7.9Hz,1H),7.76(s,1H),7.15(d,J=7.9Hz,1H),6.77(q,J=5.4Hz,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=5.4Hz,3H),1.03-0.90(m,6H),0.86-0.72(m,6H).

[0153] NMR 13 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.

[0154] IRv max (ATR, ZnSe, pure, cm -1 ): 2968, 2942, 1790, 1765, 1740, 1450, 1373, 1283, 1245, 1204, 1159 , 1079, 1032, 1010, 983.

[0155] HRMS(ESI-Orbitrap)m / z[M+H] + C 25 H 36 Calculated value for NO9: 494.2385; measured value: 494.2370, [M+Na] + C 25 H 35 Calculated value for NNaO9: 516.2204; measured value: 516.2189.

[0156] Synthesis of Compound 2 1) Radical 5-((5-methyl-2-oxo-1,3-dioxol-4-yl)methoxy)carbonyl)-1,1,3,3-tetraethylisoindoline-2-yloxyl(S4)

[0157] [Chemical formula]

[0158] S1 (380 mg, 1.31 mmol, 1 equivalent), oxalyl chloride (561 μL, 6.54 mmol, 5 equivalents), DMF (5 μL, 0.066 mmol, 5 mol%), 4-hydroxymethyl-5-methyl-1,3-dioxol-2-one (170 mg, 1.31 mmol, 1 equivalent), and Et3N (219 μL, 1.57 mmol, 1.2 equivalents) and DMAP (8.1 mg, 0.066 mmol, 5 mol%) were used and the general procedure D was followed. The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 80 / 20) to obtain the desired compound as a yellow oil (180 mg, 34%).

[0159] Rf = 0.22 (cyclohexane:ethyl acetate = 4:1) IR v max (ATR, ZnSe, pure, cm -1 ): 2984, 2942, 2908, 1739, 1447, 1373, 1238, 1098, 1046, 938.

[0160] HRMS (ESI-Orbitrap) m / z [M] + for C 22 H 28 NO6 calculated value: 402.1911; measured value: 402.1916, [M + H] + for C 22 H 29 NO6 calculated value: 403.1995; measured value: 403.1957, [M + 2H] + for C 22 H 30 NO6 calculated value: 404.2073; measured value: 404.2050.

[0161] EPR (in a solution of 100 μM in 0.1 M potassium phosphate buffer at pH = 7.4 containing 1 mM DTPA): A N = 1.54 mT, ΔB = 0.17 mT.

[0162] 2) Methyl - 2 - (((1 - chloroethoxy)carbonyl)oxy)-1,1,3,3 - tetraethylisoindoline - 5 - carboxylate of (5 - methyl - 2 - oxo - 1,3 - dioxol - 4 - yl) (S5)

[0163]

Chemical formula

[0164] S4 (65 mg, 0.162 mmol, 1 equivalent), Pd / C (7 mg), Et3N (34 μL, 0.243 mmol, 1.5 equivalents), and chloroformic acid 1 - chloroethyl (26 μL, 0.243 mmol, 1.5 equivalents) were used and the general procedure B was followed. The crude product was purified by column chromatography on silica gel (cyclohexane / AcOEt, 90 / 10) to obtain the desired compound as a pale yellow oil (21 mg, 26%).

[0165] NMR 1 H (500 MHz, CDCl3) δ (ppm) 7.99 (d, J = 7.0 Hz, 1H), 7.75 (s, 1H), 7.18 (d, J = 7.0 Hz, 1H), 6.51 (q, J = 5.6 Hz, 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 = 5.6 Hz, 3H), 1.85 - 1.74 (m, 4H), 1.06 - 0.95 (m, 6H), 0.90 - 0.77 (m, 6H).

[0166] [[ID=2​​​​​​​​​): 2985, 2864, 1710, 1670, 1578, 1414, 1298, 1259, 1121, 1097, 1056, 985, 913.

[0168] HRMS(ESI-Orbitrap)m / z[M+H] + C 25 H 33 Calculated value for ClNO8: 510.1895; measured value: 510.1864.

[0169] 3) Methyl-2-(((1-acetoxyethoxy)carbonyl)oxy)-1,1,3,3-tetraethylisoindoline-5-carboxylate (compound 2)

[0170] [ka]

[0171] S5 (20 mg, 0.039 mmol, 1 equivalent), AcOH (69 μL), and Hg ( OAc)2 (18.7 mg, 0.059 mmol, 1.5 equivalents) was used, and the general procedure C was followed. The pure product was obtained as a pale yellow oil (16 mg, 77%) without purification.

[0172] 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).

[0173] NMR 13C (126 MHz, 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。

[0174] IRv max (ATR, ZnSe, pure, cm -1 ): 2986, 2864, 1710, 1698, 1670, 1414, 1382, 1121, 1110, 1097, 985。

[0175] HRMS (ESI - Orbitrap) m / z [M + H] + of C 27 H 36 NO 10 Calculated value for 534.2339; measured value 534.2311, [M + Na] + of C 27 H 35 NNaO 10 Calculated value for 556.2159; measured value 556.2134。

[0176] Synthesis of probe precursor incorporating pyrroline motif: Compound 3

[0177]

Chem.

[0178] 1) 3 - acetoxymethoxycarbonyl - 2,2,5,5 - tetraethyl - 2,5 - dihydro - 1H - pyrrole - N - oxyl radical (S7)

[0179]

Chem.

[0180] The 3-carboxy-2,2,5,5-tetraethyl-1-pyrroline-N-oxyl radical S6 was prepared from 2,2,6,6-tetraethylpiperidine-4-one prepared according to the procedure of X. Wang et al., via the route described by Y. Wang et al. (Wang, JTPaletta, K. Berg, E. Reinhart, S. Rajca, A. Rajca, Synthesis of Unnatural Amino Acids Functionalized with Sterically Shielded Pyrroline Nitroxides, Org. Lett. 16 (2014) 5298-5300) (X. Wang, M. Emoto, A. Sugimoto, Y. Miyake, K. Itto, M. Amasaka, S. Xu, H. Hirata, H. Fujii, H. Arimoto, Synthesis of ( 15 )N-labeled 4-oxo-2,2,6,6-tetraethylpiperidine nitroxide for EPR brain imaging, Tetrahedron Lett.55(2014),2146-2149).

[0181] S6 (100 mg, 0.416 mmol, 1 equivalent), K2CO3 (115 mg, 0.832 mmol, 2 equivalents), and ethyl bromo (61 μL, 0.624 mmol, 1.5 equivalents) were used, and the general procedure A was followed. The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 90 / 10) to obtain the desired compound as a yellow oil (129 mg, 99%).

[0182] IRv max (ATR, ZnSe, pure, cm -1 ): 2978, 2927, 2881, 2856, 1767, 1734, 1458, 1371, 1274, 1248, 1217, 1197, 1161, 1014.

[0183] HRMS(ESI-Orbitrap)m / z[M+Na] + C 16 H26 Calculated value for NNaO5: 335.1709; measured value: 335.1696.

[0184] EPR (100 μM solution in 0.1 M potassium phosphate buffer, pH=7.4): A N =1.54mT, ΔB=0.18mT.

[0185] 2) 1-((1-chloroethoxy)carbonyl)oxy)-2,2,5,5-tetraethyl-2,5-dihydro-1H-pyrrole-3-carboxylate acetoxymethyl ester (S8)

[0186] [ka]

[0187] S7 (129 mg, 0.416 mmol, 1 equivalent), Pd / C (18 mg), Et3N (144 μL, 1.040 mmol, 2.5 equivalents), and 1-chloroethyl chloroformate (89 μL, 0.832 mmol, 2 equivalents) were used, and the general procedure B was followed. The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 95 / 05) to obtain the desired compound as a yellow oil (70 mg, 40%).

[0188] NMR 1 H(500MHz,CDCl3)δ(ppm) (slow inversion of nitrogen in the ring separates several signals): 6.80-6.79(m,1H), 6.44(q,J=5.6Hz,1H), 5.82-5.79(m,2H), 2.12(s,3H), 2.01-1.86(m,4H), 1.84(d,J=5.6Hz,3H), 1.82-1.76(m,1H), 1.73-1.55(m,3H), 0.97-0.86(m,12H).

[0189] NMR 13(C126MHz,CDCl3)δ(ppm) (Slow nitrogen reversal during the cycle separates several signals) 169.7;161.6;161.6;153.9;153.8;147.7;147.5;133.7;133.5;84.8;84.8;79.0;77.6;77.6;75.2;75.1;29.7;29.1;29.1;28.5;28.4;28.2;28.1;25.1;20.7;10.1;10.1;9.4;9.0;9.0;8.7;8.7.

[0190] IRv max (ATR, ZnSe, pure, cm -1 ): 2978, 2941, 2885, 1789, 1768, 1734, 1462, 1454, 1371, 1276, 1219, 1193, 1157, 1097, 1008.

[0191] HRMS(ESI-Orbitrap)m / z[M+H] + C 19 H 31 Calculated value for ClNO7: 420.1789; measured value: 420.1762.

[0192] 3) 1-(((1-acetoxyethoxy)carbonyl)oxy)-2,2,5,5-tetraethyl-2,5-dihydro-1H-pyrrole-3-carboxylate acetoxymethyl ester (compound 3)

[0193] [ka]

[0194] S8 (61 mg, 0.145 mmol, 1 equivalent), AcOH (258 μL), and Hg(OAc)2 (69 mg, 0.218 mmol, 1.5 equivalents) were used, and the general procedure C was followed. A pure product was obtained as a pale yellow oil without purification (45 mg, 70%).

[0195] NMR 1H(500MHz,CDCl3)δ(ppm) (slow inversion of nitrogen in the ring separates several signals) 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).

[0196] NMR 13 (C126MHz,CDCl3)δ(ppm) (Slow nitrogen reversal during the cycle separates several signals) 169.7;168.9;161.6;154.0;147.8;147.7;133.7;133.6;91.7;79.0;77.6;77.5;75.1;75.0;29.7;29.7;29.1;28.4;28.4;28.2;28.1;20.8;20.8;20.77;19.4;10.1;10.1;9.5;9.5;9.0;8.7;8.7.

[0197] IRv max (ATR, ZnSe, pure, cm -1 ): 2978, 2941, 2884, 1791, 1766, 1735, 1452, 1373, 1249, 1213, 1197, 1082, 1014, 910.

[0198] HRMS(ESI-Orbitrap)m / z[M+Na] + C 21 H 33 The calculated value for NNaO9 is 466.2053; the measured value is 466.2044.

[0199] Synthesis of probe precursors incorporating pyrrolidine motifs: Synthesis of Compound 4

[0200] [ka]

[0201] 1) 3-Acetoxymethoxycarbonyl-2,2,5,5-tetraethylpyrrolidine-N-oxyl radical (S10)

[0202]

Chem.

[0203] 3-Carboxy-2,2,5,5-tetraethyl-1-pyrrolidine-N-oxyl radical S9 was synthesized according to the protocol described by J.T. Paletta et al. (J.T. Paletta, M. Pink, B. Foley, S. Rajca, A. Rajca, Synthesis and Reduction Kinetics of Sterically Shielded Pyrrolidine Nitroxides, Org. Lett. 14(2012)5322 - 5325).

[0204] 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 and the general procedure A was followed. The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 80 / 20) to obtain the desired compound as a yellow oil (48 mg, 64%).

[0205] IRv max (ATR, ZnSe, pure, cm -1 ): 2978, 2824, 2884, 2854, 1766, 1456, 1417, 1369, 1219, 1201, 1159, 1139, 1066, 983.

[0206] HRMS(ESI - Orbitrap) m / z [M + Na] + of C 16 H 28 Calculated value for C H NNaO5: 337.1865; Measured value: 337.1859.<​​EPR (100 μM solution in 0.1 M potassium phosphate buffer, pH=7.4): A N =1.53mT, ΔB=0.30mT.

[0208] 2) 1-((1-chloroethoxy)carbonyl)oxy)-2,2,5,5-tetraethylpyrrolidine-3-carboxylate acetoxymethyl ester (S11)

[0209] [ka]

[0210] S10 (40 mg, 0.127 mmol, 1 equivalent), Pd / C (6 mg), Et3N (35 μL, 0.254 mmol, 2 equivalents), and 1-chloroethyl chloroformate (42 μL, 0.382 mmol, 3 equivalents) were used, and the general procedure B was followed. The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 95 / 05). The desired compound was obtained as a pale yellow oil (31 mg, 58%).

[0211] NMR 1 H(500MHz,CDCl3)δ(ppm) (a mixture of two diastereoisomers; slow inversion of nitrogen in the ring splits and broadens the specific signal) 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).

[0212] NMR 13C (126 MHz, CDCl3) δ (ppm) (mixture of two diastereoisomers, slow inversion of the nitrogen in the ring splits and broadens specific 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.

[0213] IRv max (ATR, ZnSe, pure, cm -1 ): 2984, 2932, 2834, 1690, 1670, 1608, 1482, 1356, 1280, 1111.

[0214] HRMS (ESI - Orbitrap) m / z [M+Na] + of C 19 H 32 Calculated for C H ClNNaO7 444.1765; found 444.1759.

[0215] 3) 1 - ((((1 - acetoxyethoxy) carbonyl) oxy) - 2,2,5,5 - tetraethylpyrrolidine - 3 - carboxylate acetoxymethyl ester (Compound 4)

[0216]

Chem.

[0217] S11 (24 mg, 0.057 mmol, 1 eq), AcOH (9 μL), and Hg(OAc)2 (27 mg, 0.085 mmol, 1.5 eq) were used and the general procedure C was followed. The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 95 / 05) to afford the desired compound as a colorless oil (15 mg, 60%).

[0218] NMR 1H(500MHz,CDCl3)δ(ppm) (a mixture of two diastereoisomers; slow inversion of nitrogen in the ring splits and broadens a specific signal) 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).

[0219] NMR 13 C(126MHz,CDCl3)δ(ppm) (a mixture of two diastereoisomers; slow inversion of nitrogen in the ring splits and broadens a specific signal) 169.8;1 69.0;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.

[0220] IRv max (ATR, ZnSe, pure, cm -1 ): 2984, 2868, 1706, 1690, 1672, 1608, 1514, 1482, 1356, 1280, 1111, 985.

[0221] HRMS(ESI-Orbitrap)m / z[M+Na] + C 21 H 35 The calculated value for NNaO9 is 468.2209; the measured value is 468.2203.

[0222] Synthesis of Compound 5

[0223] [ka]

[0224] 1) 1-(((1-chloroethoxy)carbonyl)oxy)-2,2,5,5-tetramethylpyrrolidine-3-carboxylate acetoxymethyl ester (S13)

[0225] [ka]

[0226] 3-Acetoxymethoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-yloxyl S12 was synthesized 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-oxyl a nd Derivatives.

[0227] S12 (500 mg, 1.94 mmol, 1 equivalent), Pd / C (50 mg), Et3N (540 μL, 3.90 mmol, 2 equivalents), and 1-chloroethyl chloroformate (520 μL, 4.80 mmol, 2.5 equivalents) were used, and the general procedure B was followed. The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 8 / 2) to obtain the desired compound as a colorless oil (640 mg, 91%).

[0228] Rf = 0.40 (cyclohexane / ethyl acetate = 4:1; phosphomolybdate staining).

[0229] NMR 1H (500 MHz, CDCl3) δ (ppm) Main diastereoisomers

[0230]

number

[0231] NMR 13 (C126MHz, CDCl3)δ(ppm) (Relatively slow nitrogen inversion splits and spreads part of the signal) 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.

[0232] HRMS(ESI-Orbitrap)m / z[M+H] + C 15 H 25 Calculated value for ClNO7: 366.1314; measured value: 366.1306; [M+Na] + C 15 H 24 The calculated value for ClNNaO7 is 388.1134; the measured value is 388.1124.

[0233] 2) 1-(((1-acetoxyethoxy)carbonyl)oxy)-2,2,5,5-tetramethylpyrrolidine-3-carboxylate acetoxymethyl ester (compound 5)

[0234] [ka]

[0235] Using S13 (300 mg, 0.80 mmol, 1 equivalent), AcOH (1.2 mL), and Hg(OAc)2 (250 mg, 0.80 mmol, 1 equivalent), follow general procedure C. The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 80 / 20) to obtain the desired compound as a colorless oil (302 mg, 94%).

[0236] Rf = 0.21 (cyclohexane / ethyl acetate = 4:1; phosphomolybdate staining).

[0237] NMR 1 H(500MHz,CD3OD)δ(ppm)(Main diastereoisomers)

[0238]

number

[0239] NMR 13 C(126MHz, CD3OD)δ(ppm) (relatively slow nitrogen inversion splits and spreads part of the signal) 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.

[0240] HRMS(ESI-Orbitrap)m / z[M+H] + C 17 H 28 Calculated value for NO9: 390.1759; measured value: 390.1751; [M+Na] + C 17 H 27The calculated value for ClNNaO7 is 412.1578; the measured value is 412.1570.

[0241] Synthesis of probe precursors incorporating piperidine motifs: Synthesis of Compound 6

[0242] [ka]

[0243] 1) 4-Acetoxymethoxycarbonyl-2,2,6,6-tetraethylpiperidine-N-oxyl radical (S15)

[0244] [ka]

[0245] The 4-carboxy-2,2,6,6-tetraethyl(piperidine-1-yloxy) radical S14 was prepared according to the protocol published by N. Babic et al. (N. Babic, F. Peyrot, New synthetic route) to 2,2,6,6-tetraethylpiperidin-4-one,a key-intermediate towards tetraethyl nitroxides,Tetrahedron Lett.60(2019)151207).

[0246] S14 (200 mg, 0.780 mmol, 1 equivalent), K2CO3 (215 mg, 1.56 mmol, 2 equivalents), and ethyl bromo (92 μL, 0.936 mmol, 1.2 equivalents) were used, and the general procedure A was followed. The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 80 / 20) to obtain the desired compound as a yellow oil (156 mg, 61%).

[0247] IRv max (ATR, ZnSe, pure, cm -1): 2978, 2881, 1763, 1724, 1463, 1369, 1319, 1301, 1224, 1151, 1136, 1020.

[0248] HRMS(ESI-Orbitrap)m / z[M+Na] + C 17 H 30 Calculated value for NNaO5: 351.2016; measured value: 351.2003.

[0249] EPR (100 μM solution in 0.1 M potassium phosphate buffer, pH=7.4): A N =1.61mT, ΔB=0.30mT.

[0250] 2) 1-((1-chloroethoxy)carbonyl)oxy)-2,2,6,6-tetraethylpiperidine-4-carboxylate acetoxymethyl ester (S16)

[0251] [ka]

[0252] S15 (156 mg, 0.475 mmol, 1 equivalent), Pd / C (21 mg), Et3 N (99 μL, 0.712 mmol, 1.5 equivalents) and 1-chloroethyl chloroformate (77 μL, 0.712 mmol, 1.5 equivalents) were used, and the general procedure B was followed. The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 95 / 05) to obtain the desired compound as a pale yellow oil (44 mg, 21%).

[0253] NMR 1H(500MHz,CDCl3)δ(ppm)6.42(q,J=5.7Hz,1H),5.75(s,2H),2.66(t,J=12.4Hz,1H),2.13(s,3H),2.03-1.92(m,2H),1 .83(d,J=5.7Hz,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).

[0254] NMR 13 C(126MHz, 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.

[0255] IRv max (ATR, ZnSe, pure, cm -1 ): 2986, 2864, 1778, 1768, 1698, 1465, 1256, 1193, 1136, 1078, 1011, 1006, 987.

[0256] HRMS(ESI-Orbitrap)m / z[M+H] + C 20 H 35 Calculated value for ClNO7: 436.2102; measured value: 436.2087; [M+Na] + C 20 H 34 Calculated value for ClNNaO7: 458.1933; measured value: 458.1887.

[0257] 3) 1-(((1-acetoxyethoxy)carbonyl)oxy)-2,2,6,6-tetraethylpiperidine-4-carboxylate acetoxymethyl ester (compound 6)

[0258] [ka]

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

[0260] 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).

[0261] 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.

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

[0263] HRMS(ESIC22H38NO9 + )m / z[M+H] + C 22 H 38 Calculated value for NO9: 460.2547; measured value: 460.2520; [M+Na] + C 22 H 37 The calculated value for ClNNaO9 was 482.2366; the measured value was 482.2339.

[0264] Synthesis of Compound 7

[0265] [ka]

[0266] 1) 4-(2-ethoxy-2-oxoethylidene)-2,2,6,6-tetraethylpiperidine-1-oxyl radical (S18)

[0267] [ka]

[0268] The radical 2,2,6,6-tetraethyl-4-oxo-(piperidine-1-yloxy) or TEEPONE S17 was obtained by following the procedure described by X. Wang et al. (X. Wang, M. Emoto, A. Sugimoto, Y. Miyake, K. Itto, M. Amasaka, S. Xu, H. Hirata, H. Fujii, H. Arimoto, Synthesis of 15 N-labeled 4-oxo-2,2,6,6-tetraethylpiperidine nitroxide for EPR brain imaging, Tetrahedron Lett. 55 (2014), 2146-2149).

[0269] To a solution of TEEPONE S17 (150 mg, 0.663 mmol, 1 equivalent) in dry THF (8 mL), triethyl phosphonoacetate (197 μL, 0.994 mmol, 1.5 equivalents) was added, and sodium hydride (32 mg, 1.326 mmol, 2 equivalents) was added to the solution at 0°C. The mixture was then stirred at room temperature for 4 hours. The 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 phase was washed with a saturated aqueous sodium chloride solution and dried over anhydrous magnesium sulfate. After evaporating the solvent under reduced pressure, the crude product was purified by chromatography on a silica gel column (cyclohexane / AcOEt, 8 / 2) to obtain the desired product in the form of a yellow oil (154 mg, 78%).

[0270] IRv max (ATR, ZnSe, pure, cm -1 ): 2978, 2935, 2881, 1741, 1712, 1456, 1390, 1273, 1155.

[0271] HRMS(ESI-Orbitrap)m / z[M] + C 17 H 30 Calculated value for NO3: 296.2226; measured value: 296.2221, [M+2H] + C 17 H 32 Calculated value for NO3: 298.2382; measured value: 298.2375; [M+Na] + C 17 H 30 Calculated value for NNaO3: 319.2123; measured value: 319.2118.

[0272] EPR (100 μM in solution in 0.1 M potassium phosphate buffer at pH=7.4): A N =1.59mT, ΔB=0.22mT.

[0273] 2) 4-(carboxymethylene)-2,2,6,6-tetraethylpiperidine-1-oxyl radical (S19)

[0274] [ka]

[0275] Compound S18 (150 mg, 0.506 mmol, 1 equivalent methanol solution (5 mL)) was added to an aqueous solution (5 mL) of sodium hydroxide solution (61 mg, 1.52 mmol, 3 equivalents). This 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 hydrochloric acid solution (1 M) until a pH of 2 was obtained. Ethyl acetate was then added, and the aqueous phase was separated. The organic phase was anhydrous The product was dried over magnesium sulfate, and the solvent was removed under reduced pressure. The pure product was then obtained in the form of a yellow solid without further purification (120 mg, 89%).

[0276] IRv max (ATR, ZnSe, pure, cm -1 ): 2972, 2935, 2881, 1715, 1641, 1458, 1419, 1382, 1276, 1176, 941.

[0277] HRMS(ESI-Orbitrap)m / z[MH] - C 15 H 25 Calculated value for NO3: 267.1834; measured value: 267.1835.

[0278] Melting point: 136-138°C.

[0279] EPR (100 μM in solution in 0.1 M potassium phosphate buffer, pH=7.4): A N =1.60mT, ΔB=0.23mT.

[0280] 3) 4-2-(acetoxymethoxy)-2-oxoethylidene)-2,2,6,6-tetraethylpiperidine-1-oxyl radical (S2O)

[0281] [ka]

[0282] S19 (200 mg, 0.745 mmol, 1 equivalent), K2CO3 (206 mg, 1.49 mmol, 2 equivalents), and ethyl bromo (109 μL, 1.118 mmol, 1.5 equivalents) were used, and the general procedure A was followed. The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 90 / 10) to obtain the desired compound as a yellow oil (80 mg, 32%).

[0283] IRv max (ATR, ZnSe, pure, cm -1 ): 2980, 2931, 2885, 2856, 1768, 1745, 1371, 1226, 1207, 1132, 1010, 987.

[0284] HRMS(ESI-Orbitrap)m / z[M+Na] + C 18 H 30 Calculated value for NNaO5: 363.2022; measured value: 363.2008.

[0285] EPR (100 μM solution in 0.1 M potassium phosphate buffer, pH=7.4): A N =1.59mT, ΔB=0.22mT.

[0286] 4) Acetoxymethyl 2-(1-(((1-chloroethoxy)carbonyl)oxy)-2,2,6,6-tetraethylpiperidine-4-ylidene)acetate (S21)

[0287] [ka]

[0288] 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, and the general procedure B was followed. The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 95 / 05) to obtain the desired compound as a pale yellow oil (33 mg, 54%).

[0289] NMR 1 H(500MHz,CDCl3)δ6.36(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).

[0290] NMR 13 C(126MHz, CDCl3)δ169.9;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.

[0291] IRv max (ATR, ZnSe, pure, cm -1 ): 2978, 2924, 2885, 2854, 1791, 1764, 1456, 1215, 1193, 1136, 1099, 1022, 1001, 987.

[0292] HRMS(ESI-Orbitrap)m / z[M+H] + C 21 H 35 Calculated value for ClNO7: 448.2102; measured value: 448.2093.

[0293] 5) Acetoxymethyl 2-(1-(((1-acetoxyethoxy)carbonyl)oxy)-2,2,6,6-tetraethylpiperidine-4-ylidene)acetate (Compound 7)

[0294] [ka]

[0295] S21 (15 mg, 0.033 mmol, 1 equivalent), AcOH (5 μL), and Hg(OAc)2 (16 mg, 0.050 mmol, 1.5 equivalents) were used, and the general procedure C was followed. The crude product was purified by silica gel column chromatography (cyclohexane / AcOEt, 95 / 05) to obtain the desired compound as a pale yellow oil (7 mg, 45%).

[0296] NMR 1 H(500MHz,CDCl3)δ6.71(q,5.3Hz,1H),5.78(s,2H),5.74(s,1H),3.59(d,J=12.8Hz,1H),3.10-3.01(m,1H),2.43-2.35(m,1H),2.32-2. 23(m,1H),2.11(s,3H),2.07(s,3H),1.93-1.83(m,2H),1.64-1.54(m,4H),1.52(d,J=5.3Hz,3H),1.49-1.41(m,2H),1.05-0.82(m,12H).

[0297] NMR 13 C(126MHz, CDCl3)δ169.9;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.

[0298] IRv max (ATR, ZnSe, pure, cm -1 ): 2980, 2878, 2864, 1791, 1764, 1698, 1670, 1456, 1213, 1111, 1099, 1027, 1001, 987.

[0299] HRMS(ESI-Orbitrap)m / z[M+Na] + C 23 H 37 The calculated value for NNaO9 is 494.2366; the measured value is 494.2360.

[0300] Example 2: Stability and reactivity of the compound of formula (I) with respect to esterase, and comparison with radical probe precursors from the prior art. 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. The cleavage of the protecting group was tested by incubating the compound of formula (I) with esterase or lipase, followed by oxidation of the hydroxylamine released by potassium ferricyanide, and measuring the EPR signal of the resulting nitroxide. Potassium ferricyanide is a mild oxidizing agent that reacts rapidly with hydroxylamine by accepting electrons. A commercially available tetramethylated and acetyl-protected ACP probe was used as a control.

[0301] Preparation of Compound 1 Solution: A stock solution of Compound 1 (50 or 500 mM) in 2 volumes of DMSO was thoroughly mixed with a 20% m / v Pluronic F-127 (Sigma-Aldrich) solution in 1 volume of DMSO. The resulting mixture was diluted in the desired aqueous medium and vigorously mixed using a vortex mixer.

[0302] Incubation with enzyme: Incubation of 100 μM compound 1 solution with porcine liver carboxylesterase (1 U mL) for 10 minutes at 37°C. -1 Sigma E3019; 1 unit hydrolyzes 1.0 micromolar of ethyl butyrate to butyric acid and ethanol per minute at pH 8.0 and 25°C), or lipase (0.51 mg / mL) -1Sigma L3126; 1 mg of the enzyme was incubated with potassium phosphate buffer (0.1 M, pH 7.4, DTPA 1 mM) at pH 7.4 and 25°C (hydrolyzing 25.6 nmol of 4-nitrophenol acetate per minute), and the released hydroxylamine was mixed with 1 μL of 100 mM potassium ferricyanide (final concentration 1 mM). The mixture was transferred to a 50 μL glass capillary (Hirschmann), and the nitroxide EPR signal was recorded in the X-band (9.86 GHz) at 21°C using a Bruker Elexsys E500 EPR spectrometer with a high-sensitivity SHQ cavity. Typical parameters used were as follows: Microwave output, 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, 41.94 seconds.

[0303] Lipases were observed to have low activity (as expected for short-chain esters), but porcine liver carboxylesterase (EC3.1.1.1) was more effective.

[0304] Compound 1, 1U mL -1 Incubation with porcine liver esterase at 37°C for 10 minutes, followed by reaction with ferricyanide (1 mM), allowed for the complete recovery of the nitroxide compared to a solution of compound 83 (true isoindoline nitroxide) prepared independently at 100 μM.

[0305] When compound ACP (a comparative probe precursor known from prior art, acetylated and substituted with four methyl groups) was subjected to the same conditions, only 27% of the corresponding nitroxide was recovered.

[0306] Stability of Compound 1: To test the stability of Compound 1 in aqueous solution, a 100 μM solution of Compound 1 was incubated in potassium phosphate buffer (0.1 M, pH 7.4, 1 mM DTPA) at 21°C for 24 hours. After the addition of potassium ferricyanide, no nitroxide signal was detected, confirming that the protection of tetraethylenediamine by (acyloxy)alkyl carbonate is stable against spontaneous hydrolysis at neutral pH under these conditions.

[0307] Example 3: Behavior of the compound of formula (I) on cells After the synthesis and initial characterization of the compound of formula (I), several in vitro tests were conducted, and the following questions were answered.

[0308] 3.1. Is the compound in formula (I) toxic? 3.2. Does the compound in formula (I) accumulate in cells? 3.3. Is the deprotection of the compound in formula (I) the rate-determining step for the oxidation of hydroxylamine? 3.4. Does the compound in equation (I) explain oxidative stress? 3.5. Is it possible to detect oxidative stress under more physiological conditions? The in vitro model used in most of these trials was the U-87mg cell line.

[0309] Cell culture protocols for cancer cell lines Human brain cells (glioblastoma astrocytoma) U87-MG were mixed with Dulbecco's minimal essential medium (DMEM), 10% fetal bovine serum (FCS), 200 U / mL penicillin, and 50 mg / m². Cells were cultured in L-streptomycin (Invitrogen, Cergy-Pontoise, France) in a humidified atmosphere containing 5% CO2. Human breast cancer cell line MCF-7-derived cells (ATCC® HTB-22™) were cultured at 37°C in a humidified 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, Cergy-Pontoise, France), and 0.5 μg / mL fungizone.

[0310] 3.1. Is the compound in formula (I) toxic? protocol: Treatment of cells for measuring the toxicity of the compound of formula (I) U87 cells were seeded in 96-well plates (10,000 cells / well the day before the experiment). The culture medium was removed, the cells were washed with PBS (Dulbeccio phosphate-buffered saline, pH 7.1-7.5), and then incubated for 6 hours with a series of concentrations of compound 1 (1-500 μM in FCS-free DMEM, 100 μL per well) solubilized using Pluronic F-127 in DMSO according to the method described above.

[0311] Cytotoxicity test For fluorescence imaging analysis, the incubation medium was removed, the cells were washed once with PBS, and then incubated for 30 minutes with FCS-free medium containing Hoechst dye (0.1 μg / mL) and propidium iodide (0.1 mg / mL). Fluorescence was read and analyzed using ImageXpress® Pico (Molecular Devices).

[0312] result: The table below shows the dead cell count after 6 hours of incubation, measured by fluorescence imaging using propidium iodide staining, compared to the total number of cells in the wells (Hoechst staining).

[0313] [Table 1]

[0314] The number of dead cells did not change significantly regardless of the concentration of compound 1 in formula (I). These results indicate that compound 1 is not toxic to U87 cells, even at the highest concentration tested over 6 hours at 500 μM. This is consistent with incubation conditions commonly used for hydroxylamine probes on cultured cells (15–60 minutes at 50–500 μM).

[0315] 3.2. Does the compound in formula (I) accumulate in cells? The membrane permeability and cellular accumulation of compounds 1, 2, 3, and 7 were tested in U87 cells in suspension. Trypsin-treated cells were incubated with each compound in serum-free DMEM at 37°C for 30 minutes under a 5% CO2 atmosphere (in a water-jacketed incubator). 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.

[0316] EPR measurement protocol for cells in suspension After trypsin treatment, a suspension of U87 cells was prepared in serum-free medium (10 million cells per mL). In a typical experiment, compound 1, 2, 3, or 7 (emulsified in a mixture of Pluronic F-127 and DMSO as in Example 2) was diluted in 500 μL of cell suspension (5 million cells). The final concentration of compound 1, 2, 3, or 7 was generally 100 μM. After culturing (30 minutes, 37°C, 5% CO2, stirring occasionally to resuspend cells), cells were separated by centrifugation (miniSpin microcentrifuge, Eppendorf, 2000 rpm, 3 minutes), culture medium was removed, and the cells were washed three times by resuspending them in 500 μL of serum-free medium, followed by centrifugation. Finally, the cell pellet was... 15The mixture was resuspended in 50 μL of serum-free medium containing 100 μM of 4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl radical labeled with N-TEMPONE nitrogen-15, placed in a gas-permeable PTFE tube (Extruded Sub-LiteWall®, inner diameter: 0.635 mm, wall thickness: 0.051 mm, Zeus Industrial Products Ltd, Ireland), bent into a W-shape, inserted into a 4 mm quartz tube, and the EPR spectrum was recorded as described above (see Example 2).

[0317] result: Cells incubated with compound 1 showed a strong nitroxide 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, it is deprotected by the cell's esterase. Deprotection leads to a charged product that can no longer easily cross the membrane and therefore accumulates inside the cell. Deprotection of hydroxylamine also occurs, and the released hydroxylamine is oxidized to nitroxide. The experimental EPR spectrum arises from the superposition of two components: one major component (marked with a star in Figure 1) that characterizes nitroxide under isotropic conditions, representing its position and compatibility in the cytosol, and a minor component (marked with an arrow) that characterizes nitroxide under intermediate conditions, representing its position and compatibility in the cell membrane. Similar results were obtained with compounds 2, 3, and 7 (strong EPR signals of nitroxide present in cells incubated after washing), indicating that this compound accumulates inside the cell.

[0318] 3.3. Is the deprotection of the compound of formula (I) a limiting step against the oxidation of hydroxylamine? The deprotection rate of a hydroxylamine probe precursor protected against oxidative stress must be faster than the oxidation rate.

[0319] The presence of non-oxidized hydroxylamine in cells indicates that 15This can be tested using N-TEMPONE. This method relies on TEMPONE's ability to freely pass through the cell membrane and the rapid transfer of hydrogen between the nitroxide and hydroxylamine. Isotope-labeled 15 N-TEMPONE's spectrum was derived from compounds 1, 2, 3, or 7 (which have three lines). 14 It is used because it has only two lines that do not completely overlap with the signal from N-nitroxide, allowing for simultaneous measurement of both species.

[0320] To do this, 5 million U87 cells in suspension were incubated with compound 1, or with compound 2, 3, or 7 (100 μM in serum-free DMEM, 37°C, 5% CO2 for 30 minutes). After final washing, one group was suspended in serum-free DMEM and the other in 100 μM. 15 It was suspended in serum-free DMEM containing N-TEMPONE.

[0321] EPR spectra were obtained according to the protocol described in Section 3.2 above.

[0322] result: 15 Cell samples treated with N-TEMPONE showed differences from untreated samples compared to those derived from compound 1. 14 The N-nitroxide showed a significantly higher signal (4 times higher) (Figure 2). In the case of compound 2, 15 Treatment with N-TEMPONE, compared to untreated samples, 14 The N-nitroxide signal was tripled. For compound 3, the signal was quadrupled, and for compound 7, it was six-folded. This experiment demonstrated the presence of significant amounts of free hydroxylamine in the cells and showed that deprotection is not a limiting reaction in the conversion of compounds 1, 2, 3, and 7 to nitroxides in the cells.

[0323] 3.4. Does the compound in equation (I) explain oxidative stress? To test the compound of formula (I) under oxidative stress conditions, a cell model of oxidative stress induction by jugron was selected. Jugron (5-hydroxy-1,4-naphthalenedione) is a native quinone-type product that can produce superoxide via a futile redox cycle within cells.

[0324] principle: Confluent U87 cells were incubated on coverslips with compound 1 (250 μM in serum-free DMEM, 37°C, 5% CO2 for 30 minutes) containing or not containing jugron (2.5 and 5 μM) prior to EPR recording. In some cases, cells were incubated with 10% FBS:SOD-PEG (superoxide dismutase-polyethylene glycol, 100 μM). -1 (2 hours), Cat-PEG (catalase-polyethylene glycol, 100 mL) -1 Cells were pre-incubated with available antioxidants in DMEM containing chelating agents (sodium diethyldithiocarbamate, DETC, 5 μM and deferoxamine, DFO, 25 μM for 1 hour) and NAC (N-acetylcysteine, 5 mM for 1 hour). The EPR spectral signal corresponds to the nitroxide produced by incubation with juglon. Signal intensity was normalized according to the average protein content of the sample each day. All experiments were performed using DMEM medium containing phenol red that does not interfere with the measurements.

[0325] Protocol for EPR measurement using a hydroxylamine probe precursor on cells cultured on glass coverslips U87-MG cells were cultured on circular microscope coverslips (12 mm in diameter, 0.13-0.16 mm thick, Thermo Scientific Menzel, Braunschweig, Germany) inserted into each well of a 24-well plate. Generally, to obtain sufficient EPR signal intensity, high confluence (cells growing through glass) is required. It was necessary to cover the surface almost completely. To avoid interference from cell passage stress, cells were seeded at least 24 hours before the EPR experiment. Generally, 56 × 10⁶ cells per well were seeded 3 days before the EPR experiment. 3 We seeded 4 x 10 U87 cells per well. (MCF-7 cells were seeded 2 days before the experiment.) 5Cells were seeded at the specified concentration. 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 for 30 minutes (37°C, 5% CO2) with 300 μL of a 250 μM solution / emulsion of compound 1 or ACP in FCS-free medium containing different concentrations of juglon (juglon was prepared from 100 mM juglon stock in DMSO. Aliquots of juglon stock in DMSO can be stored at -80°C for several weeks, but once thawed, they must be used quickly before turning dark brown. A fresh intermediate solution of juglon in medium was prepared for each sample and used within minutes). Since protected hydroxylamine 1 is insoluble in water, a suspension was prepared using the surfactant Pluronic F-127. A stock solution of 1 (10 μL of 500 mM solution in DMSO) was mixed with a solution of Pluronic F-127 (5 μL of 20% m / v solution in DMSO). The mixture was dispersed by vortexing in 85 μL of serum-free medium to obtain a white 50 mM emulsion, which was used for further dilution throughout the day. The maximum total final concentration of DMSO in cell experiments was 0.08% (including dilution of the Juglon stock). Where indicated, pre-incubation with antioxidants (2 hours for SOD-PEG (100 U / mL) and Cat-PEG (100 U / mL); 1 hour for NAC (5 mM), DETC (5 μM), and DFO (25 μM)) was performed in medium containing 10% FCS. The coverslips were removed from the wells using a bent-tip needle and tweezers, and any excess liquid was carefully removed with a paper towel. Both coverslips were then placed on the flat side of a flat quartz suprasil cell (WG-806-AQ, Wilmad) with the cell layer facing it (to prevent the sample from drying out). The coverslips were adhered to the tissue cells solely by capillary force (Note: If excess liquid remains on the coverslips, it tends to slide off the smooth surface of the EPR cell). The EPR cell was then fixed inside a cylindrical mode EPR resonator (EM4103TM, Bruker) with the sample facing the experimenter, and EPR recording was immediately started. The parameters used were as follows:Microwave frequency, 9.81 GHz; microwave output, 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; central magnetic field, 346 mT; sweep width, 6 mT; sweep time, 20.97 seconds. EPR spectra were recorded continuously for 5 minutes at 21°C. Data acquisition and processing were performed using Bruker Xepr software and MathWorks MATLAB software with the Easyspin toolkit. After acquisition, each coverslip was placed in a clean well of a 24-well plate and RIPA lysis buffer (Thermo) was used for protein titration using the BCA assay. Cells were lysed and collected on coverslips using Scientific (50 μL / coverslip). Paired sample t-tests were performed compared to the condition without juglon, with p<0.05. * ) and p<0.001( ** This was applied to a set of 3-5 independent experiments with critical values ​​of ).

[0326] result: The results are shown in the table below.

[0327] [Table 2] # Mean ± standard deviation obtained from 3 to 5 independent experiments.

[0328] Two concentrations of jugron were tested (2.5 and 5 μM), and a dose-dependent increase in EPR signaling was observed. The maximum increase in signaling was obtained at 5 μM (approximately 50% compared to untreated cells). These results demonstrate that compound 1 is indeed a reporter of intracellular oxidative stress. Basal oxidation of compound 1 in untreated cell samples is relatively high, which may suggest that compound 1 is sensitive to intracellular oxidative processes even under basal conditions in cancer cells.

[0329] DETC, a common "soft" metal ion chelator, and DFO, an iron chelator, are generally added to incubation along with unprotected hydroxylamine probes (such as CMH, 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine) to prevent auto-oxidation in the buffer. To determine whether metal ion chelation is necessary in the use of the compound of formula (I), cells were pre-incubated with DFO and DETC. We hereby confirm that these compounds do not alter the EPR response and are therefore not necessary for compound 1. This makes compound 1 less susceptible to interference and thus easier to use than unprotected hydroxylamine probes.

[0330] Antioxidants (SOD-PEG, Cat-PEG, or NAC) did not reduce basal oxidation or jugron-induced oxidation, making it impossible to draw precise conclusions about the chemical species involved in oxidation in cells. Interestingly, however, visual examination of cells showed that jugron-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 is likely sensitive to oxidative stress processes upstream of glutathione.

[0331] In parallel, commercially available ACP probes were tested according to the same protocol. Under the tested conditions, the nitroxide signal could not be detected with ACP, regardless of juglon treatment. This is because compound 1, under the tested conditions, was more effective in juglon than ACP in cells. This demonstrates that it is a better reporter for ron-induced oxidative stress.

[0332] Furthermore, another cell line (MCF-7 cells) was tested, yielding results similar to those obtained with U87 cells. The dose-dependent effect of juglon was clearly visible, with a maximum signal increase of approximately 60% at 5 μM juglon.

[0333] 3.5. Is it possible to detect oxidative stress under more physiological conditions? It was important to test compound 1 against normal cells and to have a more physiological model of oxidative stress to test the detection threshold. A good model was rabbit articular chondrocytes (which are also adherent cells) induced to express an osteoarthritis-like inflammatory phenotype upon addition of interleukin-1 beta (IL-1). IL-1 is a family of cytokine small proteins that play an important role in cellular signaling, regulating inflammatory responses. Rabbit articular chondrocytes were cultured on microscope slides (see protocol below).

[0334] Protocol for collecting and culturing cartilage cells 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 collected 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, placed in a digestion chamber for trypsin treatment, and subsequently digested with collagenase. Chondrocytes were isolated from the collagenase solution by centrifugation. The resulting cells were stored in two T75 flasks at a rate of 60,000 cells / cm³. 2 Seeds were sown in this manner, and growth medium (Ham's) supplemented with 10% FCS, 1% L-glutamine, 100 U / mL penicillin, and 100 U / mL streptomycin. Cells were cultured for 5 days in Nutrient Mixture F12 under 8% CO2. The medium was then replaced with DMEM containing 1 g / L D-glucose, 4 mM L-glutamine, and 10% FCS. After 2 days, the cells were subcultured and seeded at 250,000 cells / well in 24-well plates on glass coverslips (pre-coated with FCS and dried: 12 mm diameter, 0.13-0.16 mm thickness, Thermo Scientific Menzel, Braunschweig, Germany) with 2 mL of complete DMEM. The cells were cultured for 3 days at 37°C under 5% CO2 before the experiment. A secondary culture assay was also performed, in which 80,000 cells per well were seeded on coverslips and cultured for 3 days before the experiment.

[0335] EPR measurement protocol EPR recording was performed 1 hour, 24 hours, or 48 hours after stimulation with IL-1 (1 ng / mL in FCS-free DMEM) or medium change to serum-free DMEM, as described in Section 3.4. 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 prior to EPR recording. For the 1-hour incubation, IL-1 was added concurrently with compound 1, while for the 24-hour and 48-hour incubations, cells were pre-incubated with IL-1. Given the limited number of cells available, each condition was tested only once unless otherwise specified.

[0336] result: The results obtained are shown in the table below.

[0337] primary culture

[0338] [Table 3]

[0339] Secondary culture

[0340] [Table 4]

[0341] Treatment of chondrocytes with IL-1 rapidly induces the expression of pro-inflammatory proteins via various signaling pathways. Significant amounts of nitrite can be detected in the culture medium 12–24 hours after IL-1 treatment, indicating overexpression of iNOS, which is not present under basal conditions. Long-term incubation in DMEM without FCS may induce some cell death over time, as observed with compound 1 without IL-1 stimulation. The decrease in signal over time explains why the EPR signal was not normalized with respect to protein levels. Using compound 1, differences between treated and untreated cells could only be detected at 48 hours in primary cultured cells (Table 3), while in secondary cultured cells the effect was more pronounced at 48 hours, starting from 24 hours (Table 4), suggesting that the compound is sensitive only to downstream events and not to the initial stimulus. Although preliminary, these results suggest that compound 1 may be able to detect part of the rabbit chondrocyte response to IL-1.

[0342] Example 4: Preparation and characterization of emulsion according to the present invention A nanoemulsion containing compound 1 obtained according to Example 1 was prepared as follows: The aqueous phase (consisting of ultrapure water containing Kolliphor® 188) and the oil phase (olive oil containing Labrafac® or Span® 80) were heated separately to 40°C, and then the aqueous phase was added dropwise to the oil phase, which was maintained at 40°C under constant stirring. The pH was adjusted to 7.0 using a 0.1 M aqueous sodium hydroxide solution. Isotonicity was adjusted by adding glycerol to a final concentration of 2.5% (m / m). The resulting crude emulsion was directly introduced into a microfluidizer (LV1, Microfluidics) without homogenization. Microfluidization was performed for 10 cycles at 20 kpsi. Packed nanoemulsions were prepared using compound 1 at concentrations of 0.1 M and 0.5 M in the oil phase (i.e., the final concentrations in the nanoemulsions were 20 mM and 100 mM, respectively). Table 5 below summarizes the various emulsions that were prepared.

[0343] [Table 5]

[0344] Determination of droplet size and size distribution The hydrodynamic diameter and polydispersity index (PDI) of nanoemulsion droplets were determined by photon correlation spectroscopy using Zetasizer® Nano ZS (Malvern Panalytical, Worcestershire, UK). Each nanoemulsion sample (approximately 10 μL) was diluted 100-fold with ultrapure water before measurement. All measurements were recorded at 25°C with a scattering angle of 173°. The correlation function curve was fitted using an exponential fit (cumulative approach) to evaluate the hydrodynamic diameter and PDI. All measurements were performed triple to determine the average droplet size (expressed as the average Z diameter, Z-Ave) and PDI.

[0345] Zeta potential determination The zeta potential (ZP) of nanoemulsions is measured by Zetasizer® Nano Measurements were performed using laser Doppler kinetics with ZS (Malvern Panalytical, Worcestershire, UK). The electrophoretic mobility necessary for ZP determination was determined using the Smoluchowski approximation. All measurements were performed three times at 25°C by diluting 10 μl of each sample with 20 mM NaCl to 1000 μl.

[0346] Table 6 below summarizes the droplet hydrodynamics, polydispersity, and zeta potential of virgin nanoemulsions (virgin NE) and nanoemulsions filled with compound 1 at a final concentration of 20 or 100 mM (NE containing compound 1).

[0347] [Table 6]

[0348] Emulsion encapsulation efficiency The encapsulation efficiency of compound 1 in nanoemulsion is Amicon ultra-0.5mL The composition was determined by separating the aqueous phase of the formulation by ultrafiltration / centrifugation using a 30KD centrifugation filter (Merck Millipore, France). The nanoemulsion was centrifuged at 14,000 g for 30 minutes in a Minispin Plus microcentrifuge (Eppendorf, Switzerland). The aqueous phase was then analyzed using a SHIMADZU 100ÅC18 column (25 cm × 4.6 mm; 5 μm). Compound 1 was identified using an LC-20 HPLC system and maintained at 40°C. The mobile phases were 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The gradient from 40%B to 90%B was performed over 30 minutes using 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 to concentrations ranging from 10 μM to 50 μM and passed through an HPLC-UV system, yielding the equation y(UA) = 2474.4 * x (concentration in μM units) + 5717.1 (R 2A calibration curve of (=0.9986) was obtained.

[0349] Using this calibration curve, the concentration of compound 1 in the aqueous phase was estimated, and the encapsulation efficiency (EE) was determined as shown below.

[0350]

number

[0351] The probe precursor packing volume (drug packing = DL) of the nanoemulsion containing the highest concentration of compound 1 (0.5 M in the oil phase) in this test was estimated as follows.

[0352]

number

[0353] In all cases, compound 1 was not detected in the aqueous phase (<0.01%), indicating almost complete encapsulation of compound 1 in the oil phase. Neither the type of oil nor the Span 80 to Kolliphor 188 ratio had any effect on the retention of compound 1 in the oil phase. The probe precursor packing capacity of olive oil-based or Labrafac®-based nanoemulsions encapsulating the highest amount of compound 1 (final concentration 100 mM) in this test was estimated to be approximately 33.33%.

[0354] Stability under storage conditions The stability of the nanoemulsions was tested by photon correlation spectroscopy, as previously described for determining droplet size. Droplet size and size distribution were analyzed at different time points (D1, D7, D14, D28, D50) up to 50 days. Throughout this period, the samples were stored at 25°C. Two independent samples were tested for each formulation.

[0355] Throughout the testing period, no visible phase separation was observed in the formulations. All formulations demonstrated good stability.

[0356] Example 5: Evaluation of cytotoxicity of nanoemulsion containing compound 1 The cytotoxicity of compound 1 nanoemulsion to brain endothelial cells was tested using a dye exclusion test (brain endothelial cells 3). First, cells were cultured in a suitable medium consisting of DMEM medium, 10% FCS, penicillin (100 IU / mL), and streptomycin (100 μg / mL). Incubation was performed at 37°C, 5%, and humidified CO2. Subculture at approximately 90% confluence was performed weekly at a 1:10 dilution after detaching cells with trypsin.

[0357] Next, these cells were seeded into a 24-well plate (2.5 × 10⁻⁶). 5 Cells / wells) After incubation for 24 hours, the cells were treated with the nanoemulsion sample and incubated for a further 4 hours. Each well was treated with either a nanoemulsion packed with compound 1 (ranging from 0.1 mM to 5 mM), a blank nanoemulsion, or a control culture medium. All formulations were diluted in cell culture medium to prepare the required concentrations. After 4 hours, the 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 viable 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 a 100% viability rate.

[0358] result:

[0359] [Table 7]

[0360] Cytotoxicity evaluation of nanoemulsions 1 NE-OO and 1 NE-L1 revealed cell viability exceeding 80% at concentrations ranging from 0.1 mM to 2.5 mM. For 1 NE-L2, a cell viability rate exceeding 80% was observed at concentrations below 0.25 mM, but a slightly more significant decrease in cell viability was observed at concentrations above 0.5 mM. However, this formulation showed a higher viability rate (69.8%) at a concentration of 5 mM compared to nanoemulsions 1 NE-OO (36.17%) and 1 NE-L1 (50.91%).

[0361] Example 6: EPR test of nanoemulsion containing compound 1 protocol: All animal experiment procedures were reviewed by the local animal ethics committee (CEEA34) in accordance with the European Council Directive on Regulation and Protection of Animals Used for Scientific Purposes in France. This project was authorized by the French Ministry of Higher Education and Research under reference number APAFIS#4594-2015092117546157 v9. The experiments were conducted with healthy male BALB / c Ola Hsd mice (15-21g, 4 weeks old, Envigo) that had been acclimatized to the laboratory for 7 days with free access to water and laboratory feed.

[0362] In vivo EPR test of nanoemulsion containing compound 1 Nanoemulsions filled with compound 1 were injected into the tail vein of BALB / c mice using a 26G (0.45 × 13 mm) needle. The injection volume was 5 μL / g body weight, in accordance with good animal experiment guidelines. The injected dose was 0.1–0.5 μmol of compound per g of mouse body weight, and two mice were injected per group. Nanoemulsions were prepared at final concentrations of compound 1 of 20 and 100 mM. After injection, the mice were anesthetized by inhalation of isoflurane. Next, the heads of the mice were placed in the cavity of an EPR spectrometer using a modified mouse holder. During recording, the mice were anesthetized using a face mask (2–3% isoflurane). The body temperature of the mice was maintained at 32–37°C. Body temperature and respiratory rate were monitored during the experiment. Acquisition of the EPR signal began approximately 4 minutes after injection.

[0363] EPR measurements were performed using a Bruker Elexsys 540 EPR spectrometer (Bruker, Weissenburg, France) operating in the L-band (1.2 GHz) using the BLGR_23 cavity. 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; central magnetic field, 39.52 mT; scan width, 19 mT; scan time, 41.98 seconds. Spectra were recorded continuously as a function of time for approximately 100 minutes. Bruker Xepr software was used for data acquisition and processing.

[0364] Ex vivo EPR test of nanoemulsion containing compound 1 Ex vivo studies were performed to evaluate the distribution of NE of compound 1, and intracellular cleavage of compound 1 by esterases was confirmed in vivo. Mice injected with the nanoemulsion were euthanized approximately 100 minutes after intravenous injection. Deep anesthesia was achieved with ketamine (100 mg / kg) / xylazine (10 mg / kg) injected intraperitoneally. Whole blood samples were collected from the left ventricle, stored in heparinized tubes, and frozen in liquid nitrogen. Blood replacement by cardiac perfusion of mice was performed with 10-15 mL of saline containing 25 U / mL heparin. Subsequently, major organs, including the brain, heart, liver, spleen, lungs, and kidneys, were removed, weighed, and frozen in liquid nitrogen at -80°C for ex vivo EPR analysis.

[0365] Homogenates from each organ were prepared by mixing them with physiological saline (0.1 g moist tissue / 0.1 mL physiological saline) and deaggregating them using a Potter (Dounce homogenizer). After vortexing, the sample (30 μL) was placed in a 5 cm long gas-permeable PTFE tube (Extruded Sub-Lite-Wall, inner diameter 0.635 mm, wall thickness 0.051 mm; Zeus Industrial Products Ltd). d. The blood was transferred to Ireland, folded (V-shaped) into a 4mm quartz tube, and then placed in the EPR cavity. The blood was not diluted before recording. The spectrum was recorded at 21°C in the X-band (9.8 GHz) using an Elexsys E500 EPR spectrometer with a high-sensitivity SHQ cavity. The parameters used were as follows: microwave power, 1mW; modulation frequency, 100kHz; modulation amplitude, 0.1mT; receiver gain, 60dB; time constant, 40.96ms; conversion time, 40.96ms; 1024 points; center -350mT; sweep width, 8mT; sweep time, 41.94 seconds.

[0366] 100 mM potassium ferricyanide was gradually added to the sample (0.2–1 μL) for subsequent EPR analysis. Potassium ferricyanide is a mild oxidizing agent, and its addition to the compound should oxidize the hydroxylamine released by compound 1 to a visible nitroxide via EPR.

[0367] result: No acute toxicity was observed during in vivo experiments.

[0368] As described above, the probe derived from compound 1 is an EPR-invisible probe that is cleaved intracellularly into free hydroxylamine by esterase. The resulting hydroxylamine reacts under oxidative stress conditions to produce a nitroxide radical visible by EPR. Since the level of the oxidizing agent is very low and there is little to no reaction with free hydroxylamine, no signal is expected in a healthy system. As expected, no EPR signal was observed in the mouse heads after injection of compound 1 NE throughout the recording period, i.e., 100 minutes. Neither the concentration of compound 1 in the oil phase nor the type of oil used to formulate the nanoemulsion affected their in vivo stability in healthy mice.

[0369] In vivo results suggest that the probe derived from compound 1 was not significantly oxidized to nitroxide 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 would allow for the quantification of the amount of probe that reaches different tissues and is cleaved and / or oxidized. First, EPR acquisition of organ homogenates was performed. Next, potassium ferricyanide [K3(FeCN6)] was gradually added to recover the total amount of cleaved probe precursor 1 distributed to each organ. Potassium ferricyanide oxidizes the free hydroxylamine product of probe 1 to visible nitroxide by EPR. For each organ, the addition of the oxidizing agent resulted in an increase in the intensity of the EPR signal, thus demonstrating the sensitivity of the formulated probe to the oxidized species.

[0370] The results obtained (mean ± standard deviation) are shown in Table 8 below.

[0371] [Table 8]

[0372] EPR signals were observed in various organs, indicating successful absorption of compound 1. As expected, an increase in intensity was observed when the probe precursor dose was increased from 0.1 to 0.5 μmol / g body weight using an olive oil-based formulation. Furthermore, EPR evaluation revealed greater accumulation of compound 1 in the liver compared to other organs. Preferential accumulation of the probe precursor in the liver was observed for both concentrations tested.

[0373] At the highest dose (0.5 μmol / g body weight) of compound 1 in an olive oil-based nanoemulsion with Span80:K188=80:20 (Emulsion 1 NE-OO), the EPR spectrum of blood samples showed the presence of free nitroxides in an aqueous environment (star, Figure 3) and nitroxides in more lipophilic environments such as cell membranes (arrow, Figure 3), characterized by a nitrogen binding constant of 1.56 mT. Trace amounts of protein-bound nitroxides were detected after the addition of ferricyanide (triangle, Figure 3). During oxidation in blood, six unidentified line species (A) were detected. N =1.58mT,A H A 2.36mT (rhomboid, Figure 3) was observed. The six unidentified line species were not observed in the EPR spectra of other organs. Table 7 shows the distribution of nitroxides in organs before and after ferricyanide treatment. A significant proportion of free hydroxylamine was observed only in the liver, while most of the detected probe precursors were oxidized to nitroxides in other organs.

[0374] Example 7: NMR analysis of nitroxide radicals derived from the compound of formula (I). As described above, the compound of formula (I) has the ability to penetrate cells and be hydrolyzed by esterases in those cells to a probe containing a hydroxylamine moiety. Under the action of oxidative stress, this hydroxylamine moiety undergoes the relaxation times T1 and T2 of water protons. By shortening the time, it is oxidized to a nitroxide radical that can function as an MRI contrast agent. For modeling purposes, the inventors measured the effect of nitroxide radicals derived from various compounds of formula (I) on relaxation time in a mixture of 90 vol% 0.1 M potassium phosphate buffer at pH 7.4 and 10% DMSO to demonstrate their ability to act as MRI contrast agents.

[0375] MRI measurement protocol MRI images were acquired for a test subject consisting of samples containing one of the target nitroxides at concentrations of 0, 0.5, 1, 2, 5, 10, and 15 mM. Each nitroxide was dissolved in DMSO and then diluted in 0.1 M potassium phosphate buffer at pH 7.4, so that the final DMSO ratio in the final mixture was 10% by volume, regardless of the nitroxide concentration. Samples were prepared in 5 mm inner diameter tubes containing 0.6 mL of solution. For image acquisition, the tubes were placed horizontally and parallel to each other. Measurements were performed using five tubes at a time and repeated to analyze the samples at all concentrations.

[0376] The MRI examination was performed at approximately 21°C (room temperature) using a Bruker BioSpec 7 T system interfaced with an Advance III spectrometer (Bruker BioSpin MRI GmbH). T1 mapping was performed with a saturation-recovery pulse sequence echo time (TE) = 5.9 ms, number of acquisitions (NA) = 1, number of repetitions (NR) = 1, slice thickness = 3 mm, field of view (FOV) = 25 × 25 mm. 2 The T2 mapping was obtained using a matrix of 125 × 125 pixels and 15 recovery time (TR) values ​​(63.148-6500-100-5000-200-4000-400-3000-600-2500-800-2000-1000-1600-1300 ms). T2 mapping was recorded using a multi-spin multi-echo MSME sequence (32 TE values ​​from 25 ms to 800 ms in 25 ms increments, TR=3500 ms, NA=1, NR=1, slice thickness=3 mm, FOV=25 × 25 mm). 2 (Matrix = 125 × 125). MRI images were processed using ITK-SNAP 4.2.0 software (Free Software Foundation, Inc.).

[0377] result: MRI results showed increased contrast in different samples containing varying concentrations of nitroxide. The relaxation times T1 and T2 of the water proton decreased with increasing nitroxide concentration. The longitudinal relaxation rate 1 / T1 and transverse relaxation rate 1 / T2 increased linearly with nitroxide concentration. The slope of the corresponding line can be determined using the values ​​of longitudinal relaxation degree r1 and transverse relaxation degree r2 summarized in the table below. These values ​​are consistent with those reported in the literature for organic monoradicals of the nitroxide family.

[0378] [Table 9] * The relatively low values ​​are due to the incomplete solubility of the compound in the solvent mixture (slight cloudiness was observed).

Claims

1. Compound of formula (I): 【Chemistry 1】 During the ceremony, n is either 1 or 2. A and A' are independently selected from the group consisting of the following substituents (II-a) and (II-b): 【Chemistry 2】 In the formula, R and R' are independently selected from H and linear or branched C1-C4 alkyl groups, and Z is selected from linear or branched C1-C4 alkyl groups and aryl groups optionally substituted with linear or branched C1-C4 alkyl groups and / or methoxyl groups. Motif of formula (N) 【Transformation 3】 The following motifs are selected: (III-a), (III-b), (III-c), (III-d), and (III-e). 【Chemistry 4】 Ra, Rb, Rc, and Rd are compounds independently selected from linear or branched C1-C4 alkyl groups for each motif (III-a), (III-b), (III-c), (III-d), and (III-e).

2. A is selected from the group consisting of substituents (II-b) where Z = Me and substituents (II-a) where R = H. The compound according to claim 1, wherein A' is selected from the group consisting of substituents (II-b) where Z = Me and substituents (II-a) where R = Me.

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

4. The compound of formula (I) is selected from the following compounds (I-a), (I-b), (I-b''), (I-c), (I-d), and (I-e), according to any one of claims 1 to 3: 【Transformation 5】

5. The compound according to any one of claims 1 to 4, wherein the motif of formula (N) is selected from motifs (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 the same and selected from methyl and ethyl. 【Request Item 7】 【Transformation 6】 A compound according to any one of claims 1 to 6, selected from the group including the compound.

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

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

10. The pharmaceutical composition according to claim 9, wherein the nanoemulsion comprises an aqueous phase and an oil phase, the oil phase comprising the compound of formula (I), and further comprising at least one oil 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, preferably further comprising an organosilicon surfactant, polysorbate, cetostearyl alcohol, cetyl alcohol, oleyl alcohol, stearyl alcohol, cocamide DEA, cocamide MEA, polyalkyl glucoside, decyl glucoside, lauryl glucoside, octyl glucoside, monolaurin, poloxamer, sorbitan monostearate, sorbitan tristearate, or any combination thereof, preferably at least one nonionic surfactant selected from poloxamer and sorbitan monostearate.

12. Use of a compound of formula (I) according to any one of claims 1 to 7 or 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, particularly electron paramagnetic resonance spectroscopy, electron paramagnetic resonance imaging or magnetic resonance imaging.

13. A method for in vitro detection of oxidative stress, A step of 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 culture medium containing cells or biological tissue, A method comprising the steps of recording a signal from the culture medium by electron paramagnetic resonance spectroscopy, or capturing one or more images of the culture medium by imaging technology, preferably by electron paramagnetic resonance imaging or magnetic resonance imaging.

14. A method for detecting oxidative stress in vivo in a subject, A step of capturing one or more images of an object or at least a portion of an object previously treated with 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, using imaging techniques, preferably electron paramagnetic resonance imaging or magnetic resonance imaging; A method comprising the step of recording the signal of an object or at least a portion of an object previously treated with 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 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.

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