Synthesis of Deprenyl-like nitroxide free radicals and their diamagnetic derivatives

Synthesis of paramagnetically modified deprenyl and oxotremorine is reported. Starting from 5-and 6-membered 2,5-disubstituted nitrones 1, 6 or 4-phenyl-2,5,5-trimethyl-1 H -pyrroline 1-oxide 11 deprenyl or oxotremorine like nitroxides were synthesized via Grignard reactions. The corresponding pre-nitroxides with propargylamine structure were achieved by reduction of nitroxides followed by methylation.


Introduction
Parkinson's disease is an age-related disorder that afflicts as many as 2% of all individuals. 1The biochemical basis for the motor symptoms of Parkinson's disease is a loss of dopamine. 2,3herefore Parkinson's disease can be relieved by treating patients with L-3,4dihydroxyphenylalanine (L-DOPA) and an inhibitor of peripheral L-DOPA decarboxylase.In order to preserve brain dopamine, it is also common to treat patients with a monoamine oxidase B (MAO-B) inhibitor. 4Selegiline (L-Deprenyl) and Rasagiline (Figure 1) [5][6][7][8][9][10] are selective inhibitors of MAO-B and Selegiline is currently used for the treatment of Parkinson's and Alzheimer's diseases.This compound was reported to have a neuroprotective activity due to the prevention of apoptosis. 11The propargylamine pharmacophore of Selegiline and Rasagiline appears to be responsible for neuroprotective activity.Crystallographic analysis revealed that rasagiline covalently binds with its propargyl group to flavine enzyme to form an iminopropene chain. 12amiecinski et al. found that nitroxides with propargylamine chain such as JSAK-648 (Figure 1) can cross the blood-brain barrier and have been shown to have antioxidant properties, cell protection against oxidative stress and Reactive Oxygen Species (ROS) cytotoxicity. 13,14It is important to have a radical scavenger in statu nascendi to prevent damages caused by ROS, because the MAO-B mediated metabolism of dopamine and its autooxidation generate O2 .-,H2O2 and the highly toxic .OH in the presence of trace levels of free iron ions.Continuing our research in the synthesis of experimental drugs with dual activity containing nitroxide or its precursor, [15][16][17][18][19] we wish to extend this idea for neuroprotective drugs such as Deprenyl and Oxotremorine.Oxotremorine (Figure 1) is known as a muscarinic agonist and is also used in the Alzheimer's therapy.][22] The metabolic oxidation of 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to the oxidation product MPDP + is also catalyzed by MAO-B.Following a second two-electron oxidation, the ultimate neurotoxic metabolite, 1-methyl-4-phenylpyridinium (MPP + ) is generated. 23,24MPP + is a mitochondrial toxin, which selectively damages nigrostriatal neurons and induces Parkinsonian syndrome in humans. 25Several other allylamines act as good MAO-B substrates, such as the 5-membered ring analogue of MPTP. 23(Figure 2) Motivated by these findings the synthesis of Deprenyl and Oxotremorine derivatives with 5-or 6-membered nitroxide rings was planned.We desired to investigate the oxidation of their sterically hindered secondary or tertiary amine precursors and a six membered model compound.We hope that these compounds will act as MAO-B antagonists or muscarinic agonists and as antioxidant compounds in the central nervous system (CNS).

Results and Discussion
An evident approach for the combination of Deprenyl and Oxotremorine structure with nitroxides was the reaction of five-or six-memberd nitrones and properly chosen Grignardreagents, supporting the introduction of the required aryl or alkynyl group easily (Figure 3).

Figure 3
Treatment of 2,5-dimethyl-3,4-dihydro-2H-pyrrole 1-oxide 1 or 2,6-dimethyl-2,3,4,5tetrahydro-pyridine 1-oxide 6 with benzylmagnesium chloride in dry Et2O followed by the oxidation of N-hydroxy compound with activated MnO2 afforded the appropriate nitrones 2, 7.The reaction of ethynylmagnesium bromide with 2-benzyl substituted nitrones 2 and 7 in THF yielded the stereoisomers of nitroxide 3 and 8, respectively.The ratio of trans/cis isomers was approximately 5:1.For further reactions the trans isomers were used.Complete assignments of compound 3 could be obtained with conventional 1D and 2D NMR spectroscopy methods after the treatment of the free radical compound with diphenylhydrazine in CDCl3.
The Grignard reaction of nitrone 11 with ethynylmagnesium bromide yielded 3-phenyl-5ethynyl substituted nitroxide 12.In the latter case the spacer between the nitrogen and aryl group is more rigid, being a part of a five-membered ring.To get more Deprenyl-like nitroxides 3, 8 and 12 were reduced to secondary amines by treating them with 5 equiv Fe powder in glacial acetic acid at 60 °C producing amines 4, 9 and 13.These secondary amines were alkylated by refluxing them with methyl iodide excess in THF affording tertiary amines 5, 10 and 14 (Scheme 1).The advantages of synthesized secondary and tertiary amines beyond the structural fidelity are their better water solubility and oxygen scavenging ability such detoxyfying ROS during non-toxic, stable nitroxide free radical formation.
The oxidation of both 5-and 6-membered sterically hindered tertiary amines was investigated in MeOH with H2O2.However, upon oxidation in case of compound 14 or model compound 15 26 upon oxidation nitroxides 12 and 16 27 were formed and because of tetramethyl group no MPP + -like product is possible even at harsh oxidation conditions.
For Oxotremorine analogue synthesis we prepared compound 17 from nitrone 11 and a Grignard-reagent generated from propargylic alcohol with 2 equiv.EtMgBr in situ.After the oxidation of hydroxylamine with catalytic amount MnO2 17 propargylic alcohol was converted to 18 bromo compound by treating mesylate with LiBr in acetone.This bromo compound was used to alkylate pyrrolidine or 2-pyrrolidinone to yield spin labelled tremorine 19 or oxotremorine 20 derivatives.Nitroxides 19 and 20 were reduced to sterically hindered secondary amines 21, 22 with Fe powder in AcOH as mentioned above.
To avoid the quaternary salt formation of tertiary amine of compounds 21 and 22 the Nmethylation of secondary amines by refluxing with formaldehyde in the presence of formic acid, (Eschweiler-Clark conditions) 21 furnished the tertiary pyrrolidine derivatives 23 and 24 (Scheme 2).

Conclusions
In conclusion, starting from disubstituted or trisubstituted five-or six-membered nitrones paramagnetic Deprenyl and Oxotremorine analogues were synthesized by means of Grignard reactions.Reduction of nitroxides and N-methylation offered a closer analogue of Deprenyl and Oxotremorine neuroprotective drugs with ROS scavenging structural elements, such as sterically hindered amine (pre nitroxide) compounds.The biological study of these compounds is in progress and will be reported in due course.

Experimental Section
General.Melting points were determined with a Boetius micro melting point apparatus and are uncorrected.Elemental analyses (C, H, N, S) were performed on Fisons EA 1110 CHNS elemental analyzer.The IR (Specord 85) spectra were in each case consistent with the assigned structure.Mass spectra were recorded on a Thermoquest Automass Multi and VG TRIO-2 instruments in the EI mode.To ethynylmagnesium bromide (0.5 M sol. in THF, 50 mL) a solution of nitrone (2) (4.06 g, 0.02 mol) in dry THF (30 mL) was added dropwise at 0 °C.The mixture was stirred at r.t. for 2 h then aq.sat.NH4Cl (40 mL) was added.The organic layer was separated and the aqueous layer was extracted with CHCl3 (3 x 20 mL).The combined organic layer was dried (MgSO4) and evaporated.The residue was dissolved in CHCl3 (50 mL), activated MnO2 (cat., 100 mg) was added and the mixture was bubbled with O2 for 30 min at r.t.The reaction mixture was then filtered, evaporated and purified by flash column chromatography with hexane/Et2O to give the cis and trans stereoisomers of nitroxide.

Oxidation of tertiary amines (14, 15) to nitroxides (12, 16)
To a solution of tertiary amine 14 (227 mg, 1 mmol) or 15 (229 mg, 1 mmol) in MeOH (5 mL) H2O2 (30 % sol. in water, 0.5 mL, 5 mmol) and Na2WO4 (cat, 10 mg) was added at r.t.The reaction mixture was allowed to stand for 24 h at this temperature.The solvent was evaporated, brine (5 mL) was added and the aqueous phase was extracted with CHCl3 (3 x 5 mL).The organic phase was dried (MgSO4), evaporated and purified with flash column chromatography to yield nitroxides 12 (96 mg, 42 %) and 16 (80 mg, 35 %).The physical and spectroscopic data of these compounds are identical described above.A mixture of the free base of 21 (296 mg, 1 mmol) or 22 (310 mg, 1 mmol) and 10-fold excess each of 37 % formalin and 88 % formic acid was heated at reflux for 6 h.After cooling to room temperature, water (5 mL) was added and the aqueous phase was basified with solid K2CO3 and extracted with CHCl 3 (3 x 10 mL).The organic phase was dried (MgSO 4 ) and concentrated under vacuum to give N-methyl derivatives.

Synthesis
Figure 1
CHCl3solution and all monoradicals gave triplett spectrum aN = 14.7-16.5G. Flash column chromatography was performed on Merck Kieselgel 60 (0.040-0.063 mm).Qualitative TLC was carried out on commercially available plates (20 x 20 x 0.02 cm) coated with Merck Kieselgel GF254.°C.The reaction mixture was stirred at r.t. for 3 h, then aq.sat.NH4Cl (80 mL) was added.The organic layer was separated and the aqueous layer was extracted with CHCl 3 (3 x 30 mL).The combined organic layer was dried (MgSO 4 ) and evaporated.The residue was dissolved in CHCl3 (50 mL), activated MnO2 (cat., 100 mg) was added and the mixture was bubbled with O2 for 1 h at r.t.The reaction mixture was then filtered, evaporated and purified by flash column chromatography with CHCl3/MeOH to give 2-benzyl-nitrone.