The synthesis of 5-fluorokynurenine and 6-fluorokynurenic acid as metabolic probes

(2S)-5-Fluorokynurenine 9 and 6-fluorokynurenic acid 14 were synthesised as fluorinated probes to enable the metabolism of kynurenine to be monitored in vivo by 19 F NMR spectroscopy. The (2S)-5-fluorokynurenine 9 was prepared using a Friedel-Crafts acylation of N -( t butoxycarbonyl)- 4-fluoroaniline with a chiral oxazolidine derivative 6 , derived from 2s-aspartic acid. This represents a novel, and simple, method for the synthesis of kynurenine derivatives. The 6-fluorokynurenic acid 14 was synthesised using a Conrad-Limpach type synthesis. Preliminary biological studies are described.


Introduction
NMR spectroscopy offers the possibility of non-invasive measurement of metabolism in vivo1 and 19F-NMR spectroscopy has particular advantages.The sensitivity of 19F-NMR spectroscopy is high, the chemical shift range is wider than 1H-NMR spectroscopy and there are few background signals as there is little or no dietary source of fluorine.The aim of this work was to synthesise a fluorinated derivative of 2S-kynurenine 1 so that its in vivo conversion into kynurenic acid 2 (Scheme 1), as catalysed by the PLP (pyridoxal 5'-phosphate) dependent enzyme kynurenine aminotransferase, 2 could be monitored.This reaction lies on the kynurenine pathway of tryptophan metabolism 3 which produces a number of important neuroactive metabolites including quinolinic acid and kynurenic acid.Recently it has been postulated by Schwarcz et al. that kynurenic acid is an important marker for excitatory brain damage.4 They showed that after a certain level of kynurenic acid had been released into the brain the damage became irreversible.The eventual aim is to use the fluorinated derivatives to produce a noninvasive assay to monitor brain damage using this hypothesis.

Results and Discussion
The original and simplest synthesis5,6of kynurenine involves the ozonolysis of N-protected tryptophan derivatives and has been successfully employed in our laboratory to produce 2S-[2-2H]-kynurenine.7 It was thus decided to prepare 5-fluorokynurenine from commercially available (2S)-5-fluorotryptophan, which was protected as the ethyl ester and N-carbobenzyloxy (CBz) derivative 3.However, ozonolysis of this compound, using previously established conditions7 did not produce the expected product. 19F NMR spectroscopy showed the loss of the fluorinecontaining ring and the only isolated product was an aspartic acid derivative 4. It appeared that ozonolysis had taken place but then excess ozone brought about a Baeyer-Villiger type insertion reaction followed by hydrolysis during work-up (Scheme 2).The Baeyer-Villiger reaction appeared to be faster than oxidative cleavage as it was not possible to stop reaction after the first step by using shorter reaction times.As this was not observed in the ozonolysis of other tryptophans, it is likely that reaction takes place because of the increased reactivity of the ketone 5 formed by ozonolysis, as a result of the electron withdrawing effect of the fluorine.Alternatives to ozonolysis were investigated including oxidative cleavage using 4-tbutyliodoxybenzene8 but none were successful.An alternative synthesis was thus sought.A number of synthetic routes to kynurenine derivatives have employed a protected oxazolidine derivative 6 prepared from 2S-aspartic acid, which is then coupled to an aromatic fragment using palladium (II) chemistry. 9,10In this case it was decided to investigate a simple Friedel Crafts type acylation reaction using a protected fluoroaniline derivative (Scheme 3).Thus 4-fluoroaniline was protected as the Nt butoxycarbonyl (Boc) derivative 7 and then reacted with oxazolidine 6, prepared via standard literature methodology, 11,12 using boron trifluoride as the Lewis acid catalyst.This gave the protected (2S)-5-fluorokynurenine 8 in 63% yield.The t butoxycarbonyl protection was found to be cleaved from the aniline during reaction.Further deprotection was then carried out in 6N HCl under reflux to give the final product, which was isolated as the free amino acid in 33% yield following neutralisation using propylene oxide.The Mosher's amide 13,14 was prepared from the (2S)-5-fluorokynurenine 9 to ensure that no racemisation had taken place during the synthesis.A single peak was observed in the 19 F NMR spectrum at -78.1 ppm for the amide implying that only one diastereomer was present and that there had indeed been no racemisation.The fluorine at the 5-position of the product gave a peak at -113.6 ppm under these conditions.
The synthesis of kynurenic acid analogues is well documented.There are established routes such as the Conrad-Limpach synthesis and related procedures.15,16The desired fluorinated derivative was thus synthesised from 4-fluoroaniline 10 and diethyl acetylenedicarboxylate 11 as shown in Scheme 4. The two starting materials were heated under reflux in methanol to give the butenedioate 12 in 91% yield.However, as this compound was difficult to purify further it was carried straight through into the second step.Cyclisation was achieved by heating to reflux in diphenyl ether (250 ˚C) giving the product 13 in 72% yield. 1 H NMR spectroscopy confirmed the structure, including a characteristic signal at 6.97 ppm for the hydrogen at the 3-position.The mass spectrum gave a strong signal at the correct M + of 235 and microanalysis established the purity.
In the final step the ethyl ester was hydrolysed under basic conditions to yield the 6fluorokynurenic acid 14 in 84% yields.The 1 H NMR spectrum confirmed the structure and the 19 F NMR spectrum showed a single peak -117.4 ppm.
It can thus be seen that using the methods described the two target compounds were successfully synthesised.The (2S)-5-fluorokynurenine 9 was prepared using a novel, but simple, modification of previous procedures.This Friedel-Crafts route may prove useful for other kynurenine derivatives and its applications are being further explored.In order to check that the compounds could be used for metabolic studies the 19 F NMR spectrum of a solution containing both compounds in aqueous was measured and two singlets were readily observed with a separation of 1.6 ppm.The metabolism of the (2S)-5-fluorokynurenine 9 by a related enzyme, kynureninase, was also examined.Kynureninase is also a PLP-dependent enzyme and catalyses the hydrolytic β,γ-cleavage of kynurenine to give anthranilic acid and alanine (Scheme 5). 17When (2S)-5-fluorokynurenine 9 was incubated with bacterial kynureninase (isolated from Pseudomonas fluorescens 18 ) in 10 mM potassium phosphate buffer (pH 7.4) and the reaction followed by 19 F NMR spectroscopy, a clean reaction was observed showing the decrease in the signal due to (2S)-5-fluorokynurenine 9 at -125.15 ppm and the appearance of a new signal at 128.0 ppm.This was shown to be due to the expected product, 5-fluoroanthranilic acid 15, by comparison with the 19 F NMR spectrum of a commercial sample under identical conditions.It can thus be seen that the (2S)-5-fluorokynurenine 9 is a substrate for kynureninase.Bearing in mind the mechanistic similarities between the two PLP-dependent enzymes it is likely that the compound will also be a substrate for kynurenine aminotransferase.These results are in good agreement with some previous studies by Harada, who examined the metabolism of 5fluorotryptophan in rat kidney and observed its transformation to 5-fluorokynurenine by 19 F NMR spectroscopy. 19Future studies will assess the utility of (2S)-5-fluorokynurenine 9 for monitoring the kynurenine aminotransferase reaction in vivo.

Experimental Section
General procedures.Analytical TLC was performed using aluminium plates precoated with silica gel 60 F 254 (Merck) and visualized using UV light.Flash chromatography was carried out on silica gel 60 (35-70 µ, Fluorochem).Melting points (uncorrected) were determined on a Gallenkamp Melting Point apparatus.All NMR spectra were recorded on a Varian Gemini f.t.spectrometer (1H, 300 MHz; 19F, 298 MHz; 13C, 74.76 MHz) or a Varian Gemini f.t.spectrometer (1H, 200 MHz; 13C, 50.31MHz). 1 H-and 13 C-NMR spectra were referenced on chloroform, TMS, methanol or DMSO.19F NMR spectra were referenced on fluorotrichloromethane.Coupling constants are given in Hz and without sign.The IR-spectra were recorded as nujol mulls on a Perkin-Elmer series 1420 IR instrument.Optical rotations were measured at room temperature using an Optical Activity Ltd.AA 1000 polarimeter with 10 cm path-length cells.The measurements are given in 10-1 ˚ cm2 g-1.Mass spectra were recorded on an A. E. I./Kratos MS-50 instrument or a Micromass TofSpec 2E for MALDI-TOF spectra.HPLC purifications were carried out on a Cecil CE1200 series chromatograph using Luna 5u C-18 silica-gel on a 150 x 4.60 mm 5 micron sized column.Ozonolysis was carried out using a Fischer Ozon Ozon-generator 500.
Materials.Unless otherwise stated, these were commercial samples.Solvents were dried and purified according to the methods of Perrin and Armarego.20Solvent mixtures are defined by volume ratios (v/v).