Synthesis of 3,4-dihydro-2 H -pyrrole 1-oxide based aldonitrones as potential spin trapping agents

Different synthetic approaches to fluorescent 3,4-dihydro-2 H -pyrrole 1-oxide aldonitrones that are of potential interest as spin traps are discussed


Introduction
Aldonitrones based on pyrroline N-oxide derivatives (type A, scheme 1) are widely used as spin traps of short-living radicals in biological systems 1,2,3 that permits the determination of both the concentration and a structure of these radicals by the analysis of ESR of spin adducts of type B formed (scheme 1).This method possesses some obvious limitations.Different types of radicals react with nitrones with different rates, 4 and spin adducts formed have different lifetime and the main route of their transformation is bio-reduction to the hydroxylamines (type C, scheme 1).Thereby, the concentration of the spin adduct may be insufficient for the ESR spectrum registration and its interpretation.Furthermore, signals in spectra can broaden due to high solution viscosity or presence of dissolved oxygen, etc., hindering the interpretation of the spectra observed.

Scheme 1. The essence of spin trapping.
It is well known that the presence of both fluorescent fragment and nitroxide group in one molecule leads to significant decrease of quantum yield of fluorescence.This phenomenon was proposed to use in the systems where transformation "nitroxidehydroxylamine" takes place (as a result of bio-reduction) for biochemical studies. 5This phenomenon probably may be used for the detecting of short-living radicals in "inversed manner".In the case of conversion of diamagnetic spin trap into spin adductnitroxide the initial fluoresce quenching should be observe, that could provide the possibility of measurement of the radical concentration based on fluorescent quantum yield decrease. 6,7This approach doesn't allow the determination of the nature of free radical, but seems to be prospective to increase the sensitivity in comparison to ESR method.
Thus, the aim of this work is the synthesis of aldonitrones (ketonitrones possess lower rates of free radicals capture 8 ) based on 3,4-dihydro-2H-pyrrole 1-oxides containing fluorescent substituent.Noteworthy, that the presence of hydrophilic group is highly desirable in order to increase the solubility of spin trap in water phase that is very important particularly in biochemical investigations.

Results and Discussion
Different synthetic approaches were used to achieve the posed goal.They can be conditionally divided into two types.The first is the insertion of fluorescent moiety at the stage of heterocycle ring construction and the second is based on modifications of suitable heterocycle using reactivity of nitrone or other groups in a molecule.
The general method to synthesize the derivatives of 3,4-dihydro-2H-pyrrole 1-oxide is based on the condensation reaction of aliphatic nitro compounds with α,β-unsaturated carbonyl compounds followed by reductive cyclization of initially formed 1,4-nitroaldehydes or 1,4nitroketones. 9,10tarting 2b, containing naphthalene-1-yl group as fluorescent moiety and ester group for further transformation into hydrophilic carboxyl group could be synthesized by reaction of ethyl 2-bromo-2-(naphthalene-1-yl)acetate with sodium nitrite in DMF in the presence of phloroglucinol similarly to that, described for ethyl 2-bromophenylacetate. 11 However in the case of 1 the main, if not the sole product of the reaction was ethyl 2-hydroxy-2-(naphthalene-1yl)acetate 2a, probably due to significant steric hindrance (Scheme 2).Scheme 2. Reaction of bromide 1 with sodium nitrite.
An alternative approach for fluorescent nitrones synthesis could be the condensations of acinitro compounds salts bearing fluorescent fragment and cyano group as a precursor of hydrophilic moiety, with acrolein.Sodium salts 4a,b, were synthesized by condensation of nitriles 3a,b with methyl nitrate in the presence of sodium methylate (Scheme 3).Reaction of compounds 4a,b with acrolein in the presence of double excess of acetic acid leads to compounds 5a,b, but complete consumption of reagents wasn't observe.Increasing of the reaction time from 24 to 48 hours under similar conditions leads to products yield decrease (from 65% to 48% for 5a) (Scheme 3).The structure of 5a was proved by X-ray analysis (Figure 1).Thus, we failed to insert the fluorescent fragment into a molecule that may be a precursor of desired heterocycle and the next step of our work was the attempts of introducing of fluorescent moiety using reactivity of functional groups already presented in suitable heterocycle.
We succeed to insert fluorescent moiety into the aldonitrone molecule with the use of 2-(ethoxycarbonyl)-2-methyl-3,4-dihydro-2H-pyrrole 1-oxide (EMPO) 1 8 as starting material.Commercially available compound 8 nowadays is used as a spin trap that readily reacts with superoxide radical. 12We found that reaction of 8 with excess of ethylenediamine (Scheme 5) gives aminoamide 9 and further reaction of 9 with 2,4-dinitrofluorobenzene or 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride) in the presence of triethylamine yields 10a and 10b respectively.Nitrone 8 could be very promising precursor for fluorescent spin traps, since variation of fluorescent moiety on the last step of synthesis allows to produce spin traps with different physical and chemical properties.Limitations of this approach are high commercial value of 8 and its limited availability.
Another way of introduction of the fluorescent moiety is the synthetic sequence including reaction of nitrones with appropriate organometallic compounds followed by oxidation of hydroxylamines formed into nitrones.This method was previously widely used in the synthesis of pyrrolidine (proxyl) nitroxides. 13,14eaction of benzylmagnesium chloride as a model reagent with nitrone 11 leads to hydroxylamine 12a, which was oxidized without purification by manganese dioxide into nitrone 13a (Scheme 6).Similarly, nitrone 13b was obtained by reaction of (naphthalen-1ylmethyl)magnesium chloride with nitrone 11.Addition of naphthalen-1-ylmagnesium bromide to nitrone 11 did not proceed probably due to steric hindrance.In this case cross-metallation of methyl group in the position 5 of heterocycle seems to proceed with further self-condensation products formation. 15heme 6. Synthesis of nitrones 13a-b.