Synthesis, physicochemical characterization and neuroprotective evaluation of novel 1-hydroxypyrazin-2(1H)-one iron chelators in an in vitro cell model of Parkinson's disease

Iron dysregulation, dopamine depletion, cellular oxidative stress and α-synuclein protein mis-folding are key neuronal pathological features seen in the progression of Parkinson's disease. Iron chelators endowed with one or more therapeutic modes of action have long been suggested as disease modifying therapies for its treatment. In this study, novel 1-hydroxypyrazin-2(1H)-one iron chelators were synthesized and their physicochemical properties, iron chelation abilities, antioxidant capacities and neuroprotective effects in a cell culture model of Parkinson's disease were evaluated. Physicochemical properties (log β, log D7.4, pL0.5) suggest that these ligands have a poorer ability to penetrate cell membranes and form weaker iron complexes than the closely related 1-hydroxypyridin-2(1H)-ones. Despite this, we show that levels of neuroprotection provided by these ligands against the catecholaminergic neurotoxin 6-hydroxydopamine in vitro were comparable to those seen previously with the 1-hydroxypyridin-2(1H)-ones and the clinically used iron chelator Deferiprone, with two of the ligands restoring cell viability to ≥89% compared to controls. Two of the ligands were endowed with additional phenol moieties in an attempt to derive multifunctional chelators with dual iron chelation/antioxidant activity. However, levels of neuroprotection with these ligands were no greater than ligands lacking this moiety, suggesting the neuroprotective properties of these ligands are due primarily to chelation and passivation of intracellular labile iron, preventing the generation of free radicals and reactive oxygen species that otherwise lead to the neuronal cell death seen in Parkinson's disease.


Organic Synthesis Discussion
The target 1-hydroxypyrazin-2(1H)-ones 6 were synthesized in two steps from amino acid ethyl esters following the literature procedures as shown below in Scheme S1. 1 Initially, reaction of glycine ethyl ester hydrochloride 4a with hydroxylamine hydrochloride in alkaline water afforded the known glycine hydroxamic acid 5a in 64 % yield. 1a,2 Condensation reaction of 5a with 2,3-butanedione afforded the known 1-hydroxypyrazin-2(1H)-one 6a 1b, [3][4][5] in 24 % yield (Scheme S1). Unfortunately, application of this two-step procedure to the synthesis of 6b from alanine ethyl ester 4b failed to give the desired product, due to the high solubility of the hydroxamic acid 5b in water. We subsequently modified this procedure by using methanol as the solvent and we were able to obtain 5a from 4a in 56 % yield (Scheme S1). However, application of this modified procedure to the synthesis of alanine hydroxamic acid 5b 2b,2c,6 from alanine ethyl ester 4b gave a mixture of 5b and another compound (presumed to be the corresponding diketopiperazine) in low yield as judged by 1 H NMR spectroscopy.
Reaction of this mixture with 2,3-butanedione gave an intractable mixture of products from which the novel 1-hydroxypyrazin-2(1H)-one 6b could not be isolated by chromatography. However, 1hydroxypyrazin-2(1H)-ones 6c and 6d were successfully obtained by this modified procedure from the known hydroxamic acids 5c 1b,2b,7 and 5d, 8 albeit in only 13 % and 14 % overall yields from 4c and 4d, respectively (Scheme S1). There are some reports of multifunctional hydroxypyridinone metal chelators containing phenolic antioxidant moieties that show promising efficacy against neurodegenerative diseases by acting as radical traps as well as metal chelators. 9 Accordingly, we synthesized 1-hydroxypyrazin-2(1H)-one 6d that contains a phenol moiety which could provide a beneficial antioxidant mode of action in addition to iron chelation. Unfortunately, all our attempts to isolate hydroxamic acids 5e-5g from amino esters 4e-4g met with no success.
Due to the low yields obtained above and the failure to synthesize certain 1-hydroxypyrazin-2(1H)ones 6 by the procedure shown in Scheme S1, we sought a more general synthetic method which could be applied to the synthesis of a broader range of these compounds. The synthesis of 1-hydroxypyrazin-2(1H)-ones 6 in 4 steps from N-Boc amino acids via their protected hydroxamic acid benzyl esters was previously reported. [3][4][5]10 Inspired by this approach, we explored a new synthesis of 1-hydroxypyrazin-2(1H)-ones 6 from activated N-Boc amino acid N-hydroxysuccinimide esters 7 as shown below in Scheme S2.
We also explored the reactions of glycine hydroxamic acid 5a with both aromatic and aliphatic αketoaldehydes (glyoxals) as shown below in Scheme S3. Reaction of 5a with phenylglyoxal in ethanol/water at reflux afforded the novel 1-hydroxypyrazin-2(1H)-one 10a in 30 % yield as a single regioisomer. Similarly, reaction of 5a with 4-methoxyphenylglyoxal and 4-fluorophenylglyoxal gave 10b and 10c as single regioisomers in 27 % and 24 % yields, respectively. As with 1-hydroxypyrazin-2(1H)-one 6d, we sought to convert 10b into a 1-hydroxypyrazin-2(1H)-one bearing a phenol moiety with potential antioxidant activity. Accordingly, deprotection of the methoxy group of 10b with boron tribromide in DCM afforded the novel 1-hydroxypyrazin-2(1H)-one 10d in 21 % yield. Reaction of 5a with pyruvaldehyde gave the novel 1-hydroxypyrazin-2(1H)-ones 11a and 12a as a 12:1 mixture of regioisomers, as judged by 1 H NMR spectroscopy (Scheme S3). The major regioisomer was tentatively assigned as 11a on the basis that the free primary amino group of 5a would preferentially react with the aldehyde carbonyl group of the glyoxal, rather than the less electrophilic ketone carbonyl group. These regioisomers proved inseparable by recrystallisation or chromatography, and were studied without further purification.

Synthesis of N-Boc hydroxamic acid benzyl esters 8a-8f: General procedure
To a solution of the appropriate N-Boc amino acid N-hydroxysuccinimide (OSu) ester 7 (1.47 mmol) in DCM (20 mL) at room temperature was added O-benzylhydroxylamine (1.47 mmol, 1 eq). The solution was allowed to stir at room temperature for 24 hours. The solvent was evaporated to afford the crude N-Boc hydroxamic acid benzyl ester 8 as an oil that crystallised contaminated with Nhydroxysuccinimide. This mixture was used in the next step without further purification. [3][4][5]11 δH (

Synthesis of hydroxamic acid benzyl ester TFA salts 9b-9f: General procedure
The appropriate crude N-Boc hydroxamic acid benzyl ester 8 (1.47 mmol) was dissolved in DCM (10 mL) and trifluoroacetic acid (10 mL) was added. The solution was allowed to stir at room temperature for 24 hours. The solvents were evaporated to afford the crude TFA salt 9 as a clear oil. The oil was triturated with diethyl ether (10 mL) and the resulting white solid was filtered and washed with diethyl ether (10 mL) and allowed to dry in air to afford the pure TFA salt 9 as a white solid.