Nitrons carrying a positive charge and usable in trapping free radicals, especially the superoxide radical

Abstract

The present invention involves new nitrons carrying a positive charge, useful in trapping of free radicals and especially the superoxide radical, of general formula:

Description

BRIEF DESCRIPTION OF THE DRAWINGS

The invention shall now be described in further detail by means of the following figures, in which:

FIG. 1 shows the decay curves obtained for adducts obtained with a molecule according to the invention and a method of the prior art (DEPMPO);

FIG. 2 shows the decay curves obtained for adducts obtained with a molecule according to the invention and a method of the prior art (DEPMPO);

FIG. 3 shows spectra obtained from the trapping of various free radicals with a molecule of the prior art (DEPMPO);

FIG. 4 shows spectra obtained from the trapping of various free radicals with 4HMDEPMPO;

FIG. 5 shows spectra obtained from the trapping of superoxide and hydroxyl free radicals with 4HMDEPMPO;

FIG. 6 shows spectra obtained from the trapping of various free radicals with 4SCMDEPMPO;

FIG. 7 shows spectra obtained from the trapping of various free radicals with 4SCMDEPMPO.

As can be seen in FIG. 1, the decay curve for the Mito-DEPMPO/OOH adducts generated by the enzyme system hypoxanthine/xanthine oxidase at pH 7 reveals that the half-life of these adducts is quite better than that of the DEPMPO/OOH adducts generated by the enzyme system hypoxanthine/xanthine oxidase at pH 7.

Likewise, FIG. 2, decay curve for the Mito-DEPMPO/OH adducts generated by the enzyme system hypoxanthine/xanthine oxidase followed by the addition of SOD and of glutathione and of glutathione peroxidase at pH 7 reveals that the half-life of these adducts is quite better than that of the DEPMPO/OH adducts.

In FIG. 3 are shown spectra obtained by electron paramagnetic resonance (EPR) from the trapping of various free radicals by mito-DEPMPO, as follows:

(a) Signal obtained (Mito-DEPMPO/OOH) after 10 min. of incubation of a mixture containing hypoxanthine (HX) (0.4 mM), xanthine oxidase (XO) (0.04 U/mL), DTPA (1 mM) and Mito-DEPMPO (20 mM) in a phosphate buffer (0.1 M, pH 7.3).(b) Like (a), but after 60 min.(c) Signal obtained after 2 min of incubation of a mixture containing the system KO2/18-c-6 crown ether (10 mM) and Mito-DEPMPO (20 mM) in a phosphate buffer (0.1 M, pH 7.3).(d′) like (a), with 0.2 G of amplitude modulation.(d) Signal obtained 10 min. after reduction of the superoxide adduct obtained in (a) with GPX (10 U/mL)+GSH (1.2 mM) followed by 3 min. of argon bubbling.(e) Signal obtained after 1 min. incubation of a mixture containing Mito-DEPMPO (20 mM), H₂O2 (2 mM), Fe2SO4 (2 mM), DTPA (1 mM) in a phosphate buffer (0.1 M, pH 7.3).(f) like (e), but in presence of 5% of EtOH after 30 min.(g) Like (e), but in presence of 7% of MeOH after 30 min.(h) like (e), but in presence of 10% of DMSO after 30 min. and argon bubbling.(i) like (e) but in presence of 7% of HCOOH.(j) Signal obtained after 10 min. of photolysis of a mixture containing Mito-DEPMPO (20 mM), GSNO (1 mM), DTPA (1 mM) and a phosphate buffer (0.1 M, pH 7.3). Spectrometer adjustment: wave power 10 mW (a-c, e-j), 20 mW (d); amplitude modulation, 0.2 (d′), 0.5 (a, e, f-i), 0.63 (j), 0.7 (b), 0.8 (d-e); time constant, 0.640 ms (a-c, d′, e-f, i), 1.28 ms (d, h, j); gain 10⁵ (a-j); sweep time, 335.54 s (a-c, e-j), 167.77 (d); conversion time 0.163 s (a-c, e-j), 0.082 s (d), 0.327 s (d′).

FIG. 4 shows spectra (EPR) obtained from the trapping of various free radicals with 4-HMDEPMPO, as follows:

(a) signal obtained 34 min. after the production of hydroxyl radicals by the system (H2O2, 2 mM, FeSO4, 2 mM) in a phosphate buffer (0.1 M, pH 7.3) in presence of MeOH (10%), 4HMDEPMPOc (61 mM) and DTPA (1 mM).(b) like (a) after 1 min. incubation in presence of HCOOH (10%).(c) like (a) after 10 min. incubation in presence of DMSO (5%) in an argon purged environment.(d) like (a) after 1 min. incubation in presence of EtOH (10%) and 4HMDEPMPOc (50 mM).(e) like (d) after 1 min. incubation in presence of CH3COH (10%). The signals shown gray represent the spectra simulated by the program ROKI. Registration conditions: power, 10 mW (a-e); amplitude modulation, 0.0991 (b), 0.497 (a, c, e), 0.702 (d); time constant, 1.28 ms (a-e); gain, 10⁵ (a-e); sweep time, 84 s (a-e); conversion time 82 ms (a-e).

FIG. 5 shows spectra (EPR) obtained from the trapping of the superoxide and hydroxyl radicals with 4HM-DEPMPO, as follows:

(a) signal obtained 34 min. after the production of hydroxyl radicals by the system (H2O2, 2 mM, FeSO4, 2 mM) in phosphate buffer (0.1 M, pH 7.3) in presence of MeOH (10%), 4HMDEPMPOc (61 mM) and DTPA (1 mM).(b) like (a) after 1 min. incubation in presence of HCOOH (10%).(c) like (a) after 10 min. incubation in presence of DMSO (5%) in an argon purged environment.(d) like (a) after 1 min. incubation in presence of EtOH (10%) and 4HMDEPMPOc (50 mM).(e) like (d) after 1 min. incubation in presence of CH3COH (10%).

The signals shown gray represent the spectra simulated by the program ROKI. Registration conditions: power, 10 mW (a-e); amplitude modulation, 0.0991 (b), 0.497 (a, c, e), 0.702 (d); time constant, 1.28 ms (a-e); gain, 105 (a-e); sweep time, 84 s (a-e); conversion time 82 ms (a-e).

FIG. 6 shows spectra (EPR) obtained from the trapping of the superoxide and hydroxyl radicals with 4SCMDEPMPO, as follows:

(a) signal 1 min. after the liberation of superoxide radicals by KO2 (5 mM) in a phosphate buffer (0.1 M, pH 7.3) in presence of 4SCMDEPMPOc (50 mM).(b) 2 min. after photolysis of tert-butylhydroperoxide (1.5 M) in presence of 4SCMDEPMPOc (20 mM) in a degassed mixture (9/1) of toluene/CH₂Cl₂.(c) 1 min. after production of methylperoxyl radicals by the Fenton system (H2O2, 2 mM, FeSO4, 2 mM) in a phosphate buffer (0.1 M, pH 7.3) saturated with O₂ in presence of DMSO (5%), 4SCMDEPMPOc (10 mM) and DTPA (1 mM).

The signals shown gray represent the spectra simulated by the program ROKI. Registration conditions: power, 10 mW (a-c); amplitude modulation, 0.056 (b), 0.497 (c), 0.702 (a); time constant, 0.320 ms (b), 1.28 ms (a, c); gain, 3.2×10⁴ (b) 10⁵ (a, c); sweep time, 335.54 s (b), 84 s (a, c); conversion time 327.68 ms (b), 82 ms (a, c).

FIG. 7 shows spectra (EPR) obtained from the trapping of various free radicals with 4SCMDEPMPO, as follows:

(a) signal obtained 1 min. after the production of hydroxyl radicals by the system (H2O2, 2 mM, FeSO4, 2 mM) in a phosphate buffer (0.1 M, pH 7.3) in presence of 4SCMDEPMPOc (20 mM) and DTPA (1 mM).(b) like (a) after 5 min. incubation.(c) like (a) after 1 min. incubation in presence of MeOH (2%) and 4SCMDEPMPOc (20 mM).(d) like (a) after 1 min. incubation in presence of DMSO (5%) in an argon purged environment in presence of 4SCMDEPMPOc (20 mM).(e) like (d) after 1 min. incubation in presence of HCOOH (1%) and 4SCMDEPMPOc (20 mM).(f) like (a) after 1 min. incubation in presence of EtOH (2%) and 4SCMDEPMPOc (20 mM). The signals shown gray represent the spectra simulated by the program ROKI. Registration conditions: power, 10 mW (a-f); amplitude modulation, 0.497 (a-f); time constant, 1.28 ms (a-f); gain, 10⁵ (a-f); sweep time, 84 s (a-f); conversion time 82 ms (a-f).

Claims

1. Compounds derived from cyclical nitrons of general formula

2. Compound according to claim 1 of formula

3. Compound according to claim 2, of formula

4. Precursor compounds, especially for trap compounds of claim 1, of the following formula:

5. Compounds according to claim 4, of formula:

6. Method of preparation of the free radical trap compound of formula