US20090281330A1 - Nitrons Carrying a Positive Charge and Usable in Trapping Free Radicals, Especially the Superoxide Radical - Google Patents
Nitrons Carrying a Positive Charge and Usable in Trapping Free Radicals, Especially the Superoxide Radical Download PDFInfo
- Publication number
- US20090281330A1 US20090281330A1 US12/307,709 US30770907A US2009281330A1 US 20090281330 A1 US20090281330 A1 US 20090281330A1 US 30770907 A US30770907 A US 30770907A US 2009281330 A1 US2009281330 A1 US 2009281330A1
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- US
- United States
- Prior art keywords
- atom
- aryl
- alkyl
- group
- hydrogen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 title abstract description 18
- 150000003254 radicals Chemical class 0.000 claims description 59
- 125000004429 atom Chemical group 0.000 claims description 58
- 125000003118 aryl group Chemical group 0.000 claims description 44
- 229910052739 hydrogen Inorganic materials 0.000 claims description 34
- 239000001257 hydrogen Substances 0.000 claims description 34
- 125000006702 (C1-C18) alkyl group Chemical group 0.000 claims description 33
- 150000002431 hydrogen Chemical class 0.000 claims description 33
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 26
- 229910052760 oxygen Inorganic materials 0.000 claims description 26
- 239000001301 oxygen Substances 0.000 claims description 26
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 24
- 150000001875 compounds Chemical class 0.000 claims description 20
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 18
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 16
- YZCKVEUIGOORGS-OUBTZVSYSA-N Deuterium Chemical compound [2H] YZCKVEUIGOORGS-OUBTZVSYSA-N 0.000 claims description 16
- 229910052805 deuterium Inorganic materials 0.000 claims description 16
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 claims description 16
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 13
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 12
- 239000011593 sulfur Substances 0.000 claims description 12
- 229910052717 sulfur Inorganic materials 0.000 claims description 12
- 230000015572 biosynthetic process Effects 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- 125000000217 alkyl group Chemical group 0.000 claims description 9
- 238000003786 synthesis reaction Methods 0.000 claims description 9
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- 125000004430 oxygen atom Chemical group O* 0.000 claims description 8
- 125000005704 oxymethylene group Chemical group [H]C([H])([*:2])O[*:1] 0.000 claims description 8
- 125000004437 phosphorous atom Chemical group 0.000 claims description 8
- 229910052698 phosphorus Inorganic materials 0.000 claims description 8
- CWGBFIRHYJNILV-UHFFFAOYSA-N (1,4-diphenyl-1,2,4-triazol-4-ium-3-yl)-phenylazanide Chemical compound C=1C=CC=CC=1[N-]C1=NN(C=2C=CC=CC=2)C=[N+]1C1=CC=CC=C1 CWGBFIRHYJNILV-UHFFFAOYSA-N 0.000 claims description 7
- 229920006395 saturated elastomer Polymers 0.000 claims description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- ZRALSGWEFCBTJO-UHFFFAOYSA-O guanidinium Chemical compound NC(N)=[NH2+] ZRALSGWEFCBTJO-UHFFFAOYSA-O 0.000 claims description 4
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 4
- XYFCBTPGUUZFHI-UHFFFAOYSA-O phosphonium Chemical compound [PH4+] XYFCBTPGUUZFHI-UHFFFAOYSA-O 0.000 claims description 4
- RWSOTUBLDIXVET-UHFFFAOYSA-O sulfonium Chemical compound [SH3+] RWSOTUBLDIXVET-UHFFFAOYSA-O 0.000 claims description 4
- DZZPYICWHXVKEZ-UHFFFAOYSA-N (2-diethoxyphosphoryl-2-methyl-1-oxido-3,4-dihydropyrrol-1-ium-3-yl)methanol Chemical compound CCOP(=O)(OCC)C1(C)C(CO)CC=[N+]1[O-] DZZPYICWHXVKEZ-UHFFFAOYSA-N 0.000 claims description 3
- IEDLFWPVJJOQLZ-UHFFFAOYSA-M 2-aminoethyl(triphenyl)phosphanium;bromide Chemical compound [Br-].C=1C=CC=CC=1[P+](C=1C=CC=CC=1)(CCN)C1=CC=CC=C1 IEDLFWPVJJOQLZ-UHFFFAOYSA-M 0.000 claims description 3
- RSOCSVWZTHPBBQ-UHFFFAOYSA-N carbonic acid;pyrrolidine-2,5-dione Chemical compound OC(O)=O.O=C1CCC(=O)N1 RSOCSVWZTHPBBQ-UHFFFAOYSA-N 0.000 claims description 2
- 230000037361 pathway Effects 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 claims 1
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 22
- 0 C.C.[1*]C1([2*])N(O)=C([7*])C([5*])([6*])C1([3*])[4*] Chemical compound C.C.[1*]C1([2*])N(O)=C([7*])C([5*])([6*])C1([3*])[4*] 0.000 description 19
- 239000000203 mixture Substances 0.000 description 17
- 238000011534 incubation Methods 0.000 description 16
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 15
- -1 nitroxides Chemical class 0.000 description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 14
- 238000001228 spectrum Methods 0.000 description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 238000004435 EPR spectroscopy Methods 0.000 description 11
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 10
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 10
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- 108010093894 Xanthine oxidase Proteins 0.000 description 8
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- 238000006243 chemical reaction Methods 0.000 description 7
- OKCDBZSDRSXFIB-UHFFFAOYSA-N 2-diethoxyphosphoryl-2-methyl-1-oxido-3,4-dihydropyrrol-1-ium Chemical compound CCOP(=O)(OCC)C1(C)CCC=[N+]1[O-] OKCDBZSDRSXFIB-UHFFFAOYSA-N 0.000 description 6
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 description 6
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- CZDYPVPMEAXLPK-UHFFFAOYSA-N tetramethylsilane Chemical compound C[Si](C)(C)C CZDYPVPMEAXLPK-UHFFFAOYSA-N 0.000 description 6
- VCUVETGKTILCLC-UHFFFAOYSA-N 5,5-dimethyl-1-pyrroline N-oxide Chemical compound CC1(C)CCC=[N+]1[O-] VCUVETGKTILCLC-UHFFFAOYSA-N 0.000 description 5
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 5
- 102100033220 Xanthine oxidase Human genes 0.000 description 5
- 229910052786 argon Inorganic materials 0.000 description 5
- CBOIHMRHGLHBPB-UHFFFAOYSA-N hydroxymethyl Chemical compound O[CH2] CBOIHMRHGLHBPB-UHFFFAOYSA-N 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000000741 silica gel Substances 0.000 description 5
- 229910002027 silica gel Inorganic materials 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 241000894007 species Species 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N Formic acid Chemical compound OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 239000007832 Na2SO4 Substances 0.000 description 4
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 4
- 239000012043 crude product Substances 0.000 description 4
- 239000003480 eluent Substances 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 238000003818 flash chromatography Methods 0.000 description 4
- 235000019253 formic acid Nutrition 0.000 description 4
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 4
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 4
- 150000002832 nitroso derivatives Chemical class 0.000 description 4
- 230000002085 persistent effect Effects 0.000 description 4
- 239000011541 reaction mixture Substances 0.000 description 4
- 229910052938 sodium sulfate Inorganic materials 0.000 description 4
- WPFFFZGOXPERKA-UHFFFAOYSA-N 4-(1-diethoxyphosphoryl-1-nitroethyl)oxolan-2-one Chemical compound CCOP(=O)(OCC)C(C)([N+]([O-])=O)C1COC(=O)C1 WPFFFZGOXPERKA-UHFFFAOYSA-N 0.000 description 3
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- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 238000000921 elemental analysis Methods 0.000 description 3
- CCGKOQOJPYTBIH-UHFFFAOYSA-N ethenone Chemical compound C=C=O CCGKOQOJPYTBIH-UHFFFAOYSA-N 0.000 description 3
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- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
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- 230000003244 pro-oxidative effect Effects 0.000 description 3
- 230000003595 spectral effect Effects 0.000 description 3
- VOLGAXAGEUPBDM-UHFFFAOYSA-N $l^{1}-oxidanylethane Chemical compound CC[O] VOLGAXAGEUPBDM-UHFFFAOYSA-N 0.000 description 2
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
- C07F9/65515—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a five-membered ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
- C07F9/572—Five-membered rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
- C07F9/65583—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system each of the hetero rings containing nitrogen as ring hetero atom
Definitions
- the present invention concerns molecules, in particular, new nitrons carrying a positive charge, for the trapping of free radicals, and especially for the trapping of the superoxide anion radical, as well as a method for preparing them.
- oxygen brought into the organism by respiration can be reduced to form various reactive oxygenated species, prooxidants such as the superoxide radical (O2* ⁇ ), the hydroxyl radical (HO*), and the perhydroxyl radical (HOO*).
- prooxidants such as the superoxide radical (O2* ⁇ ), the hydroxyl radical (HO*), and the perhydroxyl radical (HOO*).
- these species are eliminated by reaction with enzymatic or nonenzymatic antioxidizing substances, such as catalase, superoxide dismutase, glutathione peroxidase, glutathione, vitamin E or ascorbate, so that the concentration of reactive oxygenated species remains low in biological environments.
- enzymatic or nonenzymatic antioxidizing substances such as catalase, superoxide dismutase, glutathione peroxidase, glutathione, vitamin E or ascorbate, so that the concentration of reactive oxygenated species remains low in biological environments.
- prooxidants being highly unstable and present in excess amount, can then react nonselectively with a large number of compounds present in the cellular environment, such as amino acids, proteins, carbohydrates, the bases of DNA, as well as organic acids, especially lipids, which are unsaturated fatty acids. This may result in the inactivation of numerous enzymes, as well as modifications in regard to the permeability and fluidity of the cell membranes.
- free radicals and especially the superoxide anion radical are implicated in many physiological and pathological situations in animals and man, such as coronary ischemia, arteriosclerosis, infarction, neurodegenerative diseases, numerous inflammatory, infectious, and aging processes.
- the superoxide radical is the primary radical in the radical chain reaction in the organism. Its detection, its quantification and the determination of its production site are consequently important factors in the developing of therapeutic treatments.
- the oxygenated radicals are species of very short lifetime. They cannot be isolated and detected directly.
- One of the strategies used at present to study these radicals is to combine the radical trapping technique with paramagnetic electron resonance (EPR).
- EPR paramagnetic electron resonance
- the principle of this radical trapping is to trap the radical on a diamagnetic molecule to yield a paramagnetic species, one which is persistent and can be detected by EPR.
- the trap needs to be stable and nontoxic under the conditions of the experiment, it should interact selectively with the target free radicals, and their values of trapping speed should be elevated.
- the radical adducts resulting from the trapping should be sufficiently persistent to be observed by EPR and should possess EPR parameters characteristic of the trapped radical.
- the radicals having at least these properties and thus being used most often belong to two families of compounds:
- the trap molecules most often used to trap radicals resulting from oxygen are the nitrons PBN (a-phenyl-N-tert-butyl nitron) and DMPO (5,5-dimethyl-1-pyrroline-N-oxide).
- the adduct DMPO-OOH obtained with the superoxide radical has a half-life of only 60 seconds at physiological pH.
- the adduct DMPO-OOH breaks down partially into the adduct DMPO-OH, thus causing difficulties in the analysis of the mechanisms involved in the formation of the trapped radicals.
- the superoxide radical has a very short lifetime in biological environments, which means that the existing rates of molecule trapping are too low to enable a detection of the molecules.
- one of the goals of the invention is to propose a trap molecule for free radicals and in particular for the superoxide radical that has an improved trapping rate, especially for the superoxide radical, and a greater stability for the adduct formed with said superoxide radical.
- the object of the invention is compounds derived from cyclical nitrons of general formula
- R1 represents a phenyl, a (C6-C12) aryl or a (C1-C18) alkyl
- A1 is a single bond, a methylene group, or an oxymethylene group (where the oxygen atom is bound to the phosphorus atom of the radical Z1)
- X represents an atom of oxygen or of sulfur
- Y represents an atom of oxygen and R represents an atom of hydrogen, a (C1-C18) alkyl group or a (C6-C18) aryl group or Y represents a methylene group and R represents an atom of hydrogen, a (C1-C17) alkyl or a (C6-C18) aryl
- X represents an atom of oxygen or of sulfur
- Y represents an atom of oxygen and R represents an atom of hydrogen, a (C1-C18) alkyl group or a (C6-C18) aryl group
- Y represents a methylene group and R represents an atom of hydrogen, a (C1-C17) alkyl or a (C6-C18) aryl
- R represents an atom of hydrogen, a (C1-C18) alkyl or a (C6-C18) aryl group
- X is a (C1-C18) alkyl or (C6-C18) aryl
- A2 is a single bond, a methylene group, or an oxymethylene group (where the oxygen atom is bound to the phosphorus atom of the radical Z2)
- X represents an atom of oxygen or of sulfur
- Y represents an atom of oxygen and R represents an atom of hydrogen, a (C1-C18) alkyl or a (C6-C18) aryl
- Y represents a methylene group and R represents an atom of hydrogen, a (C1-C17) alkyl or (C6-C18) aryl
- the molecules according to the invention possess superior performance at trapping of free radicals and especially the superoxide radical, due to a long life for the adducts obtained and a trapping rate, especially for the superoxide radical, which is higher than that of the existing molecules, such as DMPO and DEPMPO, in particular thanks to the presence of the positive charge on the molecule of the invention, which attracts by electrostatic interaction the radicals being trapped, and carrying a negative charge, to the nitron radical.
- the compounds according to the invention correspond to the following general formula
- X is a (C1-C18) alkyl or (C6-C18) aryl
- the compound is the Mito-DEPMPO compound of formula:
- the present invention also deals with precursor compounds, especially for trap compounds of the invention, of the following formula:
- R1 represents a phenyl, a (C6-C18) aryl or a (C1-C18) alkyl
- A1 is a single bond, a methylene group, or an oxymethylene group (where the oxygen atom is bound to the phosphorus atom of the radical Z1)
- X represents an atom of oxygen or of sulfur
- Y represents an atom of oxygen and R represents an atom of hydrogen, a (C1-C18) alkyl or a (C6-C18) aryl or Y represents a methylene group and R represents an atom of hydrogen, a (C1-C17) alkyl or (C6-C18) aryl
- A2 is a single bond, a methylene group, or an oxymethylene group (where the oxygen atom is bound to the phosphorus atom of the radical Z2)
- X represents an atom of oxygen or of sulfur
- Y represents an atom of oxygen and R represents an atom of hydrogen, a (C1-C18) alkyl or a (C6-C18) aryl
- Y represents a methylene group and R represents an atom of hydrogen, a (C1-C17) alkyl or (C6-C18) aryl
- the precursor compound is:
- Such a free radical trap molecule carries a freely activated group which makes it possible to prepare new trap molecules by functionalization with ligands providing appropriate properties and thus leading to the preparation of bifunctional traps having superior trapping performance, such as when a cation group is introduced.
- the present invention also deals with a method for preparation of the free radical trap compound that is preferable according to the invention, involving the synthesis of 4HMDEPMPO by the following synthesis pathway:
- the crude product is purified by flash chromatography on silica gel with the eluent being a mixture of CH 2 Cl 2 /EtOH (80/20) to give 3 fractions with a yield of 64%.
- the eluent being a mixture of CH 2 Cl 2 /EtOH (80/20)
- 3 fractions with a yield of 64%.
- one being a hygroscopic red solid (1.22 g, 0.006 mol, 38% yield) corresponded to 4HMDEPMPOc (4S, 5R or 4R, 5S).
- Another one, being a red oil (630 mg, 0.0024, 19% yield) was composed entirely of 4HMDEPMPOt (4R, 5R or 4S, 5S).
- the stereochemistry of the two nitrons was confirmed by NOESY NMR.
- the crude product is purified by flash chromatography on silica gel with the eluent being a mixture of CH 2 Cl 2 /EtOH (90/10) to give 4SCMDEPMPOc (170 mg, 0.419 mmol) in the form of a white powder with a yield of 55%.
- 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.
- 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.
- 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.
- FIG. 3 spectra obtained by electron paramagnetic resonance (EPR) from the trapping of various free radicals by mito-DEPMPO, as follows:
- FIG. 4 shows spectra (EPR) obtained from the trapping of various free radicals with 4-HMDEPMPO, as follows:
- FIG. 5 shows spectra (EPR) obtained from the trapping of the superoxide and hydroxyl radicals with 4HM-DEPMPO, as follows:
- 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, 10 5 (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:
- 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 ⁇ 10 4 (b) 10 5 (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:
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Abstract
Description
- This application is a National Phase patent application of PCT/FR2007/001157, filed Jul. 6, 2007, which claims priority to French Patent Application 0606237, filed Jul. 7, 2006, and a continuing application of U.S. Ser. No. 11/585,731, filed Oct. 24, 2006 and claims priorities to the foregoing applications in their entireties herein by reference thereto.
- 1. Field of the Invention
- The present invention concerns molecules, in particular, new nitrons carrying a positive charge, for the trapping of free radicals, and especially for the trapping of the superoxide anion radical, as well as a method for preparing them.
- Under the action of many intracellular processes, oxygen brought into the organism by respiration can be reduced to form various reactive oxygenated species, prooxidants such as the superoxide radical (O2*−), the hydroxyl radical (HO*), and the perhydroxyl radical (HOO*). In a normal situation, these species are eliminated by reaction with enzymatic or nonenzymatic antioxidizing substances, such as catalase, superoxide dismutase, glutathione peroxidase, glutathione, vitamin E or ascorbate, so that the concentration of reactive oxygenated species remains low in biological environments. *the nitroso compounds*the nitrons
- However, a disruption may occur in the equilibrium between the prooxidants and the antioxidants, leading to a situation of oxidative stress. These prooxidants, being highly unstable and present in excess amount, can then react nonselectively with a large number of compounds present in the cellular environment, such as amino acids, proteins, carbohydrates, the bases of DNA, as well as organic acids, especially lipids, which are unsaturated fatty acids. This may result in the inactivation of numerous enzymes, as well as modifications in regard to the permeability and fluidity of the cell membranes.
- Thus, free radicals and especially the superoxide anion radical are implicated in many physiological and pathological situations in animals and man, such as coronary ischemia, arteriosclerosis, infarction, neurodegenerative diseases, numerous inflammatory, infectious, and aging processes. Very often the superoxide radical is the primary radical in the radical chain reaction in the organism. Its detection, its quantification and the determination of its production site are consequently important factors in the developing of therapeutic treatments.
- It is thus important to be able to propose molecules acting as effective free radical traps, that is, ones which have a sufficiently considerable reactivity to compete with die other cell components, especially the superoxide radical.
- The oxygenated radicals are species of very short lifetime. They cannot be isolated and detected directly. One of the strategies used at present to study these radicals is to combine the radical trapping technique with paramagnetic electron resonance (EPR). The principle of this radical trapping is to trap the radical on a diamagnetic molecule to yield a paramagnetic species, one which is persistent and can be detected by EPR.
- The trap needs to be stable and nontoxic under the conditions of the experiment, it should interact selectively with the target free radicals, and their values of trapping speed should be elevated. The radical adducts resulting from the trapping should be sufficiently persistent to be observed by EPR and should possess EPR parameters characteristic of the trapped radical. The radicals having at least these properties and thus being used most often belong to two families of compounds:
-
R1N═O - With these two families of molecules, the capture of a free radical results in an adduct of nitroxide type, which in the great majority of cases is persistent and observable by EPR. However, the nitroso compounds have major intrinsic difficulties, which limit their uses in aqueous environments. They are toxic, not very soluble in water, and have poor thermal or photochemical stability. What is more, the trapping of oxygenated radicals by nitroso derivatives results in adducts of very low persistence, quickly leading to products of molecular rearrangement. Consequently, the EPR spectra obtained during an experiment with radical trapping in presence of a nitroso derivative often correspond to the superpositioning of the signals of several nitroxides and are difficult to interpret.
- The trap molecules most often used to trap radicals resulting from oxygen are the nitrons PBN (a-phenyl-N-tert-butyl nitron) and DMPO (5,5-dimethyl-1-pyrroline-N-oxide).
- Recent works have shown that PBN and certain of its derivatives have a protector effect with regard to neuron death resulting from ischemia-reperfusion in the cerebral region. This protector therapeutic effect is attributed to a good cellular penetration of PBN, which can thus compete with the other cell components to react with the oxygenated radicals. Unfortunately, PBN does not constitute a trap of interest to the radical trapping technique, because of the very low persistence of its oxygenated adducts and their not very characteristic EPR parameters. Although its intracellular distribution is less than that of PBN, DMPO performs better in this regard, since it forms more persistent adducts with oxygen-centered radicals, which are more easy to detect and characterize. Even so, the use of DMPO to detect and characterize oxygen-centered radicals also has serious limits. For example, the adduct DMPO-OOH obtained with the superoxide radical has a half-life of only 60 seconds at physiological pH. On the other hand, the adduct DMPO-OOH breaks down partially into the adduct DMPO-OH, thus causing difficulties in the analysis of the mechanisms involved in the formation of the trapped radicals.
- Other compounds are also known, such as 1′ α-(4-pyridyl-1-oxide-N-tertbutyl nitron (POBN) and 2-methyl-2-nitrosopropane (MNP).
- 2. Background and Discussion of the Prior Art
- From
patent EP 0 660 841 one is also aware of cyclical nitrons comprising, in particular, a phosphorylated compound, such as 2-diethylphosphono-2-methylpyrroline-N-oxide (DEPMO), resulting in trapping adducts relatively more stable than those obtained with the already known nitron derivatives. - However, in view of the stability of the adducts obtained with such compounds, a need still exists to propose molecules having superior performance to that of these already existing compounds, in particular, ones having an even more improved stability of the resulting adducts.
- In particular, the superoxide radical has a very short lifetime in biological environments, which means that the existing rates of molecule trapping are too low to enable a detection of the molecules. Thus, one of the goals of the invention is to propose a trap molecule for free radicals and in particular for the superoxide radical that has an improved trapping rate, especially for the superoxide radical, and a greater stability for the adduct formed with said superoxide radical.
- Toward this end, the object of the invention is compounds derived from cyclical nitrons of general formula
- in which R1 represents a phenyl, a (C6-C12) aryl or a (C1-C18) alkyl,
- R2 represents an atom of hydrogen, of deuterium, a phenyl group, a (C6-C18) aryl group, a (C1-C18) alkyl group, or a radical Z1 of formula
- in which A1 is a single bond, a methylene group, or an oxymethylene group (where the oxygen atom is bound to the phosphorus atom of the radical Z1), X represents an atom of oxygen or of sulfur, Y represents an atom of oxygen and R represents an atom of hydrogen, a (C1-C18) alkyl group or a (C6-C18) aryl group or Y represents a methylene group and R represents an atom of hydrogen, a (C1-C17) alkyl or a (C6-C18) aryl,
- or a radical Z3 of formula
- in which R represents an atom of hydrogen, a (C1-C18) alkyl or a (C6-C18) aryl group,
- R3 and R5 represent, independently, an atom of hydrogen, of deuterium, a phenyl, (C6-C18) aryl, or (C1-C18) alkyl group,
- R4 represents a radical of formula
- in which X is a (C1-C18) alkyl or (C6-C18) aryl,
- A2 is an atom of oxygen, of nitrogen, or of carbon and Z is a positively charged group, such as a triaryl phosphonium —P+(Ar)3, Ar being a (C6-C18) aryl group, guanidinium —NH—C(=NH)NH3 +, ammonium —N+(R)3, sulfonium S+(R)2, in which R is a group chosen from among the (C1-C18) alkyl and (C6-C18) aryl groups, substituted or not, saturated or not, possibly containing at least one atom of oxygen, nitrogen, and sulfur,
- R6 is an atom of hydrogen, of deuterium, a phenyl, (C6-C18) aryl, (C1-C18) alkyl group, or a radical Z2 of formula
- in which A2 is a single bond, a methylene group, or an oxymethylene group (where the oxygen atom is bound to the phosphorus atom of the radical Z2), X represents an atom of oxygen or of sulfur, Y represents an atom of oxygen and R represents an atom of hydrogen, a (C1-C18) alkyl or a (C6-C18) aryl, or Y represents a methylene group and R represents an atom of hydrogen, a (C1-C17) alkyl or (C6-C18) aryl, and
- R7 represents an atom of hydrogen, of deuterium, or a methyl group.
- In very advantageous manner, it has been observed that the molecules according to the invention possess superior performance at trapping of free radicals and especially the superoxide radical, due to a long life for the adducts obtained and a trapping rate, especially for the superoxide radical, which is higher than that of the existing molecules, such as DMPO and DEPMPO, in particular thanks to the presence of the positive charge on the molecule of the invention, which attracts by electrostatic interaction the radicals being trapped, and carrying a negative charge, to the nitron radical.
- Preferably, the compounds according to the invention correspond to the following general formula;
- in which X is a (C1-C18) alkyl or (C6-C18) aryl,
- A2 is an atom of oxygen, of nitrogen or of carbon and Z is a positively charged group, such as triaryl phosphonium —P+(Ar)3, guanidinium —NH—C(=NH)NH3 +, ammonium —N+(R)3, sulfonium S+(R)2, in which R is a group chosen from among the (C1-C18) alkyl and (C6-C18) aryl groups, substituted or not, saturated or not, possibly containing at least one atom of oxygen, nitrogen, and sulfur.
- According to one preferred embodiment of the invention, the compound is the Mito-DEPMPO compound of formula:
- The present invention also deals with precursor compounds, especially for trap compounds of the invention, of the following formula:
- in which R1 represents a phenyl, a (C6-C18) aryl or a (C1-C18) alkyl,
- R2 represents an atom of hydrogen, of deuterium, a phenyl group, a (C6-C18) aryl, (C1-C18) alkyl, or a radical Z1 of formula
- in which A1 is a single bond, a methylene group, or an oxymethylene group (where the oxygen atom is bound to the phosphorus atom of the radical Z1), X represents an atom of oxygen or of sulfur, Y represents an atom of oxygen and R represents an atom of hydrogen, a (C1-C18) alkyl or a (C6-C18) aryl or Y represents a methylene group and R represents an atom of hydrogen, a (C1-C17) alkyl or (C6-C18) aryl,
- R3 and R5 represent, independently, an atom of hydrogen, of deuterium, a phenyl, (C6-C18) aryl, or (C1-C18) alkyl group,
- R4 represents a radical of formula
- R6 is an atom of hydrogen, of deuterium, a phenyl, (C6-C18) aryl, (C1-C18) alkyl group, or a radical Z2 of formula
- in which A2 is a single bond, a methylene group, or an oxymethylene group (where the oxygen atom is bound to the phosphorus atom of the radical Z2), X represents an atom of oxygen or of sulfur, Y represents an atom of oxygen and R represents an atom of hydrogen, a (C1-C18) alkyl or a (C6-C18) aryl, or Y represents a methylene group and R represents an atom of hydrogen, a (C1-C17) alkyl or (C6-C18) aryl, and
- R7 represents an atom of hydrogen, of deuterium, or a methyl group.
- According to one preferred embodiment of the invention, the precursor compound is:
- Such a free radical trap molecule carries a freely activated group which makes it possible to prepare new trap molecules by functionalization with ligands providing appropriate properties and thus leading to the preparation of bifunctional traps having superior trapping performance, such as when a cation group is introduced.
- The present invention also deals with a method for preparation of the free radical trap compound that is preferable according to the invention, involving the synthesis of 4HMDEPMPO by the following synthesis pathway:
- Then the carbonation of the nitron 5-diethoxyphosphoryl-5-methyl-4-hydroxymethyl-1-pyrroline N-oxide (4HMDEPMPO) thus obtained in solution in acetonitrile with succinimide carbonate and triethylamine
- The nitron thus formed is then caused to react with 1 equivalent of (2-aminoethyl) triphenyl phosphonium bromide in presence of triethylamine in dichlormethane, thereby producing the Mito-DEPMPO compound
- We shall now describe the following preparation examples which illustrate without limiting the invention.
- Example of synthesis of 4-(1-diethoxyphosphoryl-1-nitroethyl)-tetrahydrofuran-2-one
- 2.16 g of tributylphosphane (0.01 mol) are added drop by drop to a mixture of nitrophosphate (12.58 g, 0.059 mol) and furanone (5 g, 0.059 mol) in 70 ml of cyclohexane. Methylene chloride (7 ml) is added to fully solubilize the reaction mixture, which is agitated for 24 h at ambient temperature. The solvent is evaporated under reduced pressure. The crude product is purified by flash chromatography on silica gel with the eluent being a mixture of Et2O/pentane (9/1). The nitrofuranone, in diastereoisomeric mixture (55/45), is obtained in the form of a pale yellow oil (14.01 g, 0.047 mol) with a yield of 80%.
dP (121.49 MHz; CDCl3; H3PO4) 13.70 (55%), 13.79 (45%).
Remark: the 2 diastereoisomers have signals which are superposed or nonattributable in NMR 1H.
dH [see original] -
multiplicity of the carbons d (in ppm) of the d (in ppm) of form of the signal diastereoisomer of the diastereoisomer of (attribution) d31P = 13.70 ppm d31P = 13.70 ppm 2 × 1C, s, (CO) 174.48 174.63 2 × 1C, d, CIVNO2 90.09 (JCP = 147.1) 90.42 (JCP = 148.2) 2 × 2C, d, CH 2 OC(O) 68.27 (JCP = 2.8) 68.14 (JCP = 9.3) 2 × 1C, d, 2CH2OP 64.80 (JCP = 7.7) 65.05 (JCP = 7.2) 2 × 1C, d, CHCIV 40.20 39.75 2 × 1C, s, CH2CO 29.85 (JCP = 7.7) 30.19 (JCP = 3.3) 2 × 1C, d, CH3CIV 16.37 (JCP = 9.4) 16.36 (JCP = 7.7) 2 × 2C, d, CH 3 CH2O 16.25 (JCP = 5.2) 16.28 (JCP = 5.2) - Elemental analysis for C10H18NO7P (molecular weight=295.23): Calculated: C, 40.68; H, 6.15; N, 4.74%. Found: C, 40.67; H, 6.07; N, 4.61%.
- Example of synthesis of 2-hydroxy-4-(1-diethoxyphosphoryl-1-nitroethyl)-tetrahydrofurane:
- To a solution of 1.3 g (4.4 mmol) of 4-(1-diethoxyphosphoryl-1-nitroethyl)-tetrahydrofuran-2-one in 35 ml of CH2Cl2 at −78° C. are added drop by drop 11.45 ml of a solution of DIBAL-H 1M in hexane. The reaction mixture is agitated for 3 h and then the mixture is hydrolyzed. The mixture is quickly filtered on silica gel, then dried on Na2SO4. The solvent is evaporated under reduced pressure. The crude product (1.4 g) is purified by flash chromatography on silica gel with the eluent being a mixture of CH2Cl2/Et2O. The 4 diastereoisomers of the hemiacetal are obtained in mixture (650 mg, 2.19 mmol) in the form of a pale yellow oil with a yield of 50%.
- dP (121.49 MHz; CDCl3; H3PO4) 15.15 (50%), 15.28 (25%), 15.48 (25%).
Remark: the 4 diastereoisomers have signals which are superposed or nonattributable in NMR 1H.
dH [see original] [key: respectivement=respectively] - Elemental analysis for C10H20NO7P (297.24): Calculated: C, 40.41; H, 6.78; N, 4.71%. Found: C, 40.42; H, 6.65; N, 4.49%.
- Example of synthesis of 5-diethoxyphosphoryl-5-methyl-4-hydroxymethyl-1-pyrroline N-oxide or 4HMDEPMPO:
- To a solution of 3.8 g (0.128 mol) of hemiacetal 28 in a mixture of THF/H2O (1/1) is added NH4Cl (1.71 g, 0.032 mol) and then finely powdered zinc (2.00 g, 0.032 mol) is added. The reaction mixture is left under agitation for 6 h at ambient temperature. The precipitated ZnCl2 is then eliminated by filtration and rinsed with CH2Cl2 (3×40 ml). The filtrate is then concentrated under reduced pressure and the residue is dissolved in 40 ml of CH2Cl2. The combined organic phases are dried on Na2SO4 and the solvent is evaporated under reduced pressure to yield 3 g of a yellow oil. The crude product is purified by flash chromatography on silica gel with the eluent being a mixture of CH2Cl2/EtOH (80/20) to give 3 fractions with a yield of 64%. Of the 3 fractions obtained, one being a hygroscopic red solid (1.22 g, 0.006 mol, 38% yield) corresponded to 4HMDEPMPOc (4S, 5R or 4R, 5S). Another one, being a red oil (630 mg, 0.0024, 19% yield), was composed entirely of 4HMDEPMPOt (4R, 5R or 4S, 5S). The stereochemistry of the two nitrons was confirmed by NOESY NMR.
- Spectral characteristics of (4S*, 5R*)-4HMDEPMPOc: dP (121.49 MHz; CDCl3, H3PO4) 20.85. dH (300.13 MHz; CDCl3; Me4Si) 6.92 (1H, q, JHH=JHP=2.7, HC═N), 4.39-4.14 (4H, m, CH3CH2OP), 3.90 (2H, d, JHH=6.1, CH2OH), 2.78-2.39 (4H, m, CHCH2OH, CH2C═NO, OH), 1.76 (3H, d, JHP=14.5, CH3CIVNO), 1.38 (3H, t, JHH=7.0, CH3CH2OP), 1.34 (3H, t, JHH=7.0, CH3CH2OP). dC (75.47 MHz; CDCl3; Me4Si) 134.52 (1C, d, JCP=8.8, HC═N), 76.93 (1CIV, d, JCP=149.8, CH3CIV), 63.86 (1C, d, JCP=7.1, CH3CH2OP), 63.81 (1C, d, JCP=7.7, CH3CH2OP), 62.23 (1C, d, JCP=5.0, CH2OH), 49.32 (1C, d, JCP=2.8, CH2C=), 29.16 (1C, s, CHCH2OH), 21.31 (1C, d, JCP=1.6, CH3CIV), 16.40 (1C, d, JCP=5.5, CH3CH2OP), 16.25 (1C, d, JCP=6.0 CH3CH2OP). Pf. 48° C. (décomposition).
- Spectral characteristics of (4R*, 5R*)-4HMDEPMPOt: dP (121.49 MHz; CDCl3; H3PO4)22.0. dH (300.13 MHz; CDCl3; Me4Si) 6.85 (Hv, q, JHH=J HP=2.5, HC═N), 4.40-4.11 (4H, m, CH3CH2O), 3.82 (Ha, ABd, JHaHb=11.0 & JHaHc=7.0, CH2OH), 3.66 (Hb, ABd, JHbHa=11.0 & JHbHc=5.9, CH2OH); 3.25-3.05 (Hc, m, CHCH2OH), 2.87 (Hd, ABdt, JHdHe=18.3 & JHdHe=6.6 & JHdHv=JHdP=2.5, CHC═NO), 2.48 (1H & He, ABtd, JHcHd=18.3, JHcHc=6.6, JHeP=6.6, JHeHv=2.5, CHC═NO, OH), 1.67 (3H, d, JHP=16.0, CH3CIVNO), 1.34 (3H, t, JHH=6.9, CH3CH2OP), 1.33 (3H, t, JHH=6.9, CH3CH2OP). dC (75.47 MHz; CDCl3; Me4Si) 134.87 (1C, d, JCP=8.8, HC═N), 76.74 (1CIV, d, JCP=158.1, CH3CIV), 64.08 (1C, d, JCP=6.6, CH3CH2OP), 63.25 (1C, d, JCP=7.3, CH2OH), 61.26 (1C, d, JCP=6.6, CH2OP), 40.85 (1C, s, CHCH2OH), 30.14 (1C, d, JCP=4.8, CH2C═N) 16.24 (2C, d, JCP=5.9 CH3CH2OP), 14.01 (1C, s, CH3CIV), Elemental analysis for C10H20NO7P+1/2 of H2O (274.25) of the two diastereoisomers: Calculated: C, 43.80; H, 7.72; N, 5.11%. Found: C, 44.06; H, 7.60; N, 5.33%.
- ESI-MS/MS [see original, data]
Example of synthesis of (4R*, 5S*)-5-diethoxyphosphoryl-4-(succinimidyloxycarbonyloxymethyl)-5-methyl-1-pyrroline-N-oxide: - To a solution of 0.234 g of disuccinimide carbonate (0.91 mmol) in acetonitrile (5 ml) is added 0.1 ml of triethylamine (0.76 mmol) and 0.202 g of nitron (0.76 mmol). The reaction mixture is left under agitation for 16 h. It is then concentrated under reduced pressure. It is dissolved in dichlormethane and then washed with a saturated solution of NaHCO3 until pH=8, then with a saturated solution of NaCl. The organic phase is dried on Na2SO4 and the solvent is evaporated to yield 220 mg of a yellow oil. The crude product is purified by flash chromatography on silica gel with the eluent being a mixture of CH2Cl2 /EtOH (90/10) to give 4SCMDEPMPOc (170 mg, 0.419 mmol) in the form of a white powder with a yield of 55%.
- Spectral characteristics of the nitron (4S*, 5R*): dP (81.01 MHz; CDCl3; H3PO4) 18.0, dH (200.13 MHz, CDCl3; Me4Si) 6.97 (Hv, q, JHH=JHP=2.9, HC═N), 4.76 (1H, ABd, JHAHB=10.8 & JHAH=5.9, CH2OCO), 4.57 (1H, ABd, JHBHA=10.8 & JHBH=7.8, CH2OCO)), 4.36-4.08 (4H, m, CH3CH2O), 2.83 (4H, s, CH2CO), 2.8-2.57 (3H, m, CHCH2O, CH2C═NO), 1.71 (3H, d, JHP=13.9, CH3CIVNO), 1.31 (3H, t, JHH=7.0, CH3CH2OP), 1.33 (3H, t, JHH=7.0, CH3CH2OP). dC (50.32 MHz; CDCl3; Me4Si) 168.44 (2C, s, CON), 151.10 (1C, COO), 133.73 (1C, d, JCP=7.7, HC═N), 75.86 (1CIV, d, JCP=148.7, CH3CIV), 70.00 (1C, d, JCP=3.0, CH2OCO), 64.51 (1C, d, JCP=6.5, CH2OP), 62.80 (1C, d, JCP=7.2, CH2OP), 45.45 (1C, d, JCP=2.3, CHCH2O), 29.68 (1C, d, JCP=0.8, CH2C═N), 25.28 (2C, s, CH2CO), 20.1 (1C, s, CH3CIV), 16.13 (2C, d, JCP=5.7, CH3CH2OP). Pf. 81.4° C., décomposition. Exact mass for C15H23N2O9P, [C15H23N2O9P+Na]+. Calculated: 429.1039. Found: 429.1062.
- Example of synthesis of the trap molecule according to the invention.
- To a mixture of 4-NHSDEPMPO (0.5 g, 1.23 mmol) and (2-aminoethyl) triphenylphosphonium bromide (0.48 g, 1.24 mmol) in solution in CH2Cl2 (30 ml) is added triethylamine (0.23 mL, 1.61 mmol) at ambient temperature. The mixture is agitated for 2 hours, then washed with 15 ml of water. The organic phase is dried on Na2SO4 and the solvent is eliminated under reduced pressure. The product is purified on a silica column (CH2Cl2/EtOH 70:30) to give a white solid (0.57 g, 69%) corresponding to 4-MitoDEPMPO.
- 31P NMR (121.49 MHz) d 20.13, 21.52. 1H NMR (300.13 MHz) d 7.89 (1H, t, J=6.1, 7.76-7.60 (15H, m), 6.88 (1H, q, J=2.4), 4.34-4.01 (6H, m), 3.92-3.76 (2H, m), 3.64-3.52 (2H, m), 2.65-2.45 (3H, m), 1.60 (3H, d, J=14.1), 1.28 (3H, t, J=7.0), 1.24 (3H, t, J=6.9). 13C NMR (75.47 MHz) d 156.4 (1CIV, s), 135.1 (3C, d, J=2.9), 134.8 (1C, d, J=8.0), 133.5 (6C, d, J=10.3), 130.4 (6C, d, J=13.2), 122.7 (3C, d, J=86.1), 75.8 (1C, d, J=149.2), 64.1 (2C, d, J=6.3), 62.5 (1C, d, J=7.5), 46.3 (1C, d, J=2.3), 35.0 (1C, s), 30.5 (1C, s), 23.0 (1C, d, J= 48.7), 20.1 (1C, s), 16.3 (1C, d, J=5.7), 16.2 (1C, d, J=5.7). ESI-MS/MS (60 eV) m/z 597.0 [M+H]+ (100), 378.3 (20.1), 350.0 (9.3), 332.2 (22.6), 306.3 (1.0), 288.9 (7.9), 277.2 (0.6), 275.3 (0.4), 262.9 (0.4), 262.0 (0.6), 248.0 (0.6), 220.0 (2.5), 174.0 (2.7), 155.7 (0.6), 115.9 (0.2), 110.2 (0.4), 107.9 ) (0.2), 94.0 (0.7), 88.0 (0.7). HRMS calculated for [C31H39N2O6P2]+, Br−; [C31H39N2O6P2]+597.2253, found: 597.2283.
- 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), H202 (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 a phosphate buffer (0.1 M, pH 7.3) in presence of MeOH (10%), 4HMDEPMPOc (61 mM) and DTP A (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 (6)
1. A method for preparing a compound derived from cyclical nitrons of the general formula
in which R1 represents a phenyl, a (C1-C18) aryl or a (C1-C18) alkyl,
R2 represents an atom of hydrogen, of deuterium, a phenyl group, a (C1-C18) aryl, (C1-C18) alkyl, or a radical Z1 of formula
in which A1 is a single bond, a methylene group, or an oxymethylene group (where the oxygen atom is bound to the phosphorus atom of the radical Z1), X represents an atom of oxygen or of sulfur, Y represents an atom of oxygen and R represents an atom of hydrogen, a (C1-C18) alkyl or a (C6-C18) aryl or Y represents a methylene group and R represents an atom of hydrogen, a (C1-C17) alkyl or (C6-C18) aryl,
or a radical Z3 of formula
in which R represents an atom of hydrogen, a (C1-C17) alkyl or a (C6-C18) aryl group
R3 and R5 represent, independently, an atom of hydrogen, of deuterium, a phenyl, (C1-C18) aryl, or (C1-C18) alkyl group,
R4 represents a radical of formula
in which X is a (C1-C18) alkyl or (C6-C18) aryl,
A2 is an atom of oxygen, of nitrogen, or of carbon and Z is a positively charged group, such as triaryl phosphonium —P+(Ar)3, Ar being a (C6-C18) aryl group, guanidinium —NH—C(=NH)NH3 +, ammonium —N+(R)3, sulfonium S+(R)2, in which R is a group chosen from among the (C1-C18) alkyl and (C6-C18) aryl groups, substituted or not, saturated or not, possibly containing at least one atom of oxygen, nitrogen, and sulfur,
R6 is an atom of-hydrogen, of deuterium, a phenyl, (C1-C18) aryl, (C1-C18) alkyl group, or a radical Z2 of formula
in which A2 is a single bond, a methylene group, or an oxymethylene group (where the oxygen atom is bound to the phosphorus atom of the radical Z2), X represents an atom of oxygen or of sulfur, Y represents an atom of oxygen and R represents an atom of hydrogen, a (C1-C18) alkyl or a (C6-C18) aryl, or Y represents a methylene group and R represents an atom of hydrogen, a (C1-C17) alkyl or (C6-C18) aryl, and
R7 represents an atom of hydrogen, of deuterium, or a methyl group, said method comprises synthesizing 4HMDEPMPO.
2. The method of claim 1 , said compound further comprises;
in which X is a (C1-C18) alkyl or (C6-C18) aryl,
A2 is an atom of oxygen, of nitrogen or of carbon and Z is a positively charged group, such as triaryl phosphonium —P+(Ar)3, guanidinium —NH—C(=NH)NH3 +, ammonium —N+(R)3, sulfonium S+(R)2, in which R is a group chosen from among the (C1-C18) alkyl and (C6-C18) aryl groups, substituted or not, saturated or not, possibly containing at least one atom of oxygen, nitrogen, and sulfur.
4. A method for preparing a trap compound of the following formula:
in which R1 represents a phenyl, a (C1-C18) aryl or a (C1-C18) alkyl,
R2 represents an atom of hydrogen, of deuterium, a phenyl group, a (C1-C18) aryl, (C1-C18) alkyl, or a radical Z1 of formula
in which A1 is a single bond, a methylene group, or an oxymethylene group (where the oxygen atom is bound to the phosphorus atom of the radical Z1), X represents an atom of oxygen or of sulfur, Y represents an atom of oxygen and R represents an atom of hydrogen, a (C1-C18) alkyl or a (C6-C18) aryl or Y represents a methylene group and R represents an atom of hydrogen, a (C1-C17) alkyl or (C6-C18) aryl,
R3 and R5 represent, independently, an atom of hydrogen, of deuterium, a phenyl, (C1-C18) aryl, or (C1-C18) alkyl group,
R4 represents a radical of formula
R6 is an atom of hydrogen, of deuterium, a phenyl, (C1-C18) aryl, (C1-C18) alkyl group, or a radical Z2 of formula
in which A2 is a single bond, a methylene group, or an oxymethylene group (where the oxygen atom is bound to the phosphorus atom of the radical Z2), X represents an atom of oxygen or of sulfur, Y represents an atom of oxygen and R represents an atom of hydrogen, a (C1-C18) alkyl or a (C6-C18) aryl, or Y represents a methylene group and R represents an atom of hydrogen, a (C1-C17) alkyl or (C6-C18) aryl, and
R7 represents an atom of hydrogen, of deuterium, or a methyl group.
6. A method for preparing the free radical trap compound of the formula
followed by the carbonation of the nitron 5-diethoxyphosphoryl-5-methyl-4-hydroxymethyl-1-pyrroline N-oxide (4HMDEPMPO) thus obtained in solution in acetonitrile with succinimide carbonate and triethylamine
the nitron thus formed (SCMDEPMPO) being then caused to react with 1 equivalent of (2-aminoethyl) triphenyl phosphonium bromide in presence of triethylamine in dichlormethane, thereby producing the Mito-DEPMPO compound
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR0606237 | 2006-07-07 | ||
FR0606237A FR2903407B1 (en) | 2006-07-07 | 2006-07-07 | NOVEL NITRONES CARRYING A POSITIVE LOAD USED FOR TRAPPING FREE RADICALS, ESPECIALLY RADICAL SUPEROXIDE |
PCT/FR2007/001157 WO2008003871A1 (en) | 2006-07-07 | 2007-07-06 | New nitrones bearing a positive charge and suitable for use for the trapping of free radicals, and more particularly the peroxide radical |
Publications (1)
Publication Number | Publication Date |
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US20090281330A1 true US20090281330A1 (en) | 2009-11-12 |
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ID=37651177
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
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US11/585,731 Expired - Fee Related US7589210B2 (en) | 2006-07-07 | 2006-10-24 | Nitrons carrying a positive charge and usable in trapping free radicals, especially the superoxide radical |
US12/307,709 Abandoned US20090281330A1 (en) | 2006-07-07 | 2007-07-06 | Nitrons Carrying a Positive Charge and Usable in Trapping Free Radicals, Especially the Superoxide Radical |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
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US11/585,731 Expired - Fee Related US7589210B2 (en) | 2006-07-07 | 2006-10-24 | Nitrons carrying a positive charge and usable in trapping free radicals, especially the superoxide radical |
Country Status (4)
Country | Link |
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US (2) | US7589210B2 (en) |
EP (1) | EP2044015A1 (en) |
FR (1) | FR2903407B1 (en) |
WO (1) | WO2008003871A1 (en) |
Families Citing this family (2)
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US8143420B2 (en) * | 2007-12-31 | 2012-03-27 | Medical Collage of Wisconsin, Inc. | Bifunctional and trifunctional nitrone spin trapping compounds and uses thereof |
JP5482196B2 (en) * | 2009-12-25 | 2014-04-23 | 東京エレクトロン株式会社 | Film forming apparatus, film forming method, and storage medium |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5354871A (en) * | 1992-11-27 | 1994-10-11 | National Research Council Of Canada | Pyrroline N-oxide derivatives |
US5750710A (en) * | 1993-07-20 | 1998-05-12 | Centre National De La Recherche Scientifique (C.N.R.S.) | Nitrones which are usable for the scavenging of free radicals |
-
2006
- 2006-07-07 FR FR0606237A patent/FR2903407B1/en not_active Expired - Fee Related
- 2006-10-24 US US11/585,731 patent/US7589210B2/en not_active Expired - Fee Related
-
2007
- 2007-07-06 EP EP07803855A patent/EP2044015A1/en not_active Withdrawn
- 2007-07-06 US US12/307,709 patent/US20090281330A1/en not_active Abandoned
- 2007-07-06 WO PCT/FR2007/001157 patent/WO2008003871A1/en active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5354871A (en) * | 1992-11-27 | 1994-10-11 | National Research Council Of Canada | Pyrroline N-oxide derivatives |
US5750710A (en) * | 1993-07-20 | 1998-05-12 | Centre National De La Recherche Scientifique (C.N.R.S.) | Nitrones which are usable for the scavenging of free radicals |
Also Published As
Publication number | Publication date |
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EP2044015A1 (en) | 2009-04-08 |
FR2903407A1 (en) | 2008-01-11 |
US7589210B2 (en) | 2009-09-15 |
FR2903407B1 (en) | 2008-10-03 |
WO2008003871A1 (en) | 2008-01-10 |
US20080009636A1 (en) | 2008-01-10 |
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