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In order to explore the metabolic fate of these ether derivatives, three candidates were selected, based on their availability and low volatility. Accordingly, the selected

compounds were (1,1-difluoroethyl)(4-methoxyphenyl)sulfane (104), (1,1-

difluoroethyl) (naphthalene-2-yl)sulfane (105) and 1-(1,1-difluoroethoxy)-4- methoxybenzene (106). These compounds, shown in Fig. 24, were prepared in the University of St Andrews by Dr Tomita (104 and 105) and Dr Al-Maharik (106). Incubation, isolation and purification procedures followed those previously described.

Fig. 24 Selected molecules for the study on metabolic fate in C. elegans

2.2.4.1 (1,1-Difluoroethyl)(4-methoxyphenyl)sulfane (104)

Incubation of thioether 104 in C. elegans led to the isolation of three new fluorometabolites, products of oxidation from the fungal CYP enzymes.

Analysis of the extracts by 19F{1H}NMR spectroscopy showed a clear feature. While the starting material appears as a singlet peak for the fluorines, two of the fluorometabolites displayed a doublet of doublets, indicating an AB system and a structural change in the close environment of the fluorine atoms.

Isolation of each peak by reverse-phase HPLC, and subsequent NMR spectroscopy and high-resolution mass spectrometry, revealed sulfoxides 112 and 113 and sulfone 114.

S O F F S F F O O F F 104 105 106 O S F F C. elegans 72 h, 28 ºC 180 rpm O S F F O HO S F F O HO S F F O O + +

sulfoxide 112. The sulfoxides are chiral on the sulfur atom, resulting in non- equivalence of the fluorine atoms, and hence, the observed AB system. The lower level of sulfone 114 could be an indication that it is the last product to be generated in the metabolic pathway.

Additional to sulfur oxidation, C. elegans is also able to de-methylate the para

methoxyl group, generating a phenol. The fact that there is no oxidation of the aromatic ring, nor elimination of the fluorines, seems to point towards a reasonably stable motif overall.

In an effort to establish the sequence of events through which these metabolites are generated, a series of control experiments was carried out. It was hypothesised that sulfoxide 112 would be generated first. A demethylation would then act to generate

113, which would finally be oxidised to sulfone 114 (Scheme 20a). Alternatively, the final steps could possibly invert in order, and generate sulfone 115 prior to demethylation (Scheme 20b).

Scheme 20. Hypothetical pathways for (1,1-difluoroethyl)(4-methoxyphenyl)sulfane (100) metabolism

S F F O CYP S F F O O S F F HO O CYP S F F HO O O A B S F F O CYP S F F O O CYP _S F _F O O O Demethylating enzyme Demethylating enzyme S F F HO O 104 112 113 114 113 104 112 115 CYP X X

In order to establish if any of these routes is valid, each of the fluorometabolites was separately incubated, with different rates of success. It proved difficult to establish outcomes by 19F{1H} NMR spectroscopy from the crude extracts, so it was necessary to use HPLC. Comparison of retention times and NMR analyses of the collected peaks proved more successful and allowed characterisation.

Incubation of sulfoxide 112 (tr = 24 min) led to the formation of phenol 113 and

sulfone 114, both being securely identified against previous data. No starting material (112) was observed either by NMR spectroscopy or HPLC, indicating full conversion. These results prove that oxidation of the sulfide happens first in the metabolism.

An incubation of sulfoxide 113 (tr = 16 min) with C. elegans did not result in any

conversion and only the starting material 113 was recovered.

Finally, given the very low amount that was recovered from the original culture, an experiment with sulfone 114 did not show any new products, indicating that the metabolite is either exuded and not taken up by the fungus, or possibly fully metabolised.

These experiments established the first step of the metabolism, and also allowed route

A of Scheme 21 to be discarded. In an attempt to establish whether route B is valid,

the sulfone 115 was synthesised for an incubation study (Scheme 21). Given that the synthesis was carried out on analytical amounts of 104 (5 mg), a complete conversion was estimated by NMR spectroscopy only, but the yield could not be established.

Scheme 21. Synthesis of 1-((1,1-difluoroethyl)sulfonyl)-4-methoxybenzene (sulfone 115)

S F F O mCPBA CH₂Cl₂ 16 h, r.t. 100% conversion S F F O O O 104 115

2.2.4.2 (1,1-Difluoroethyl)(naphthalene-2-yl) sulfane (105)

In order to study the influence of the p-methoxy substituent on the metabolism of the thioether motif, a derivative of difluorinated 104 without the –OMe group was explored. (1,1-Difluoroethylphenyl)sulfane would have been the most obvious choice, but this compound is too volatile for the complete cycle of incubation, extraction and purification. Instead, the metabolic fate of (1,1-difluoroethyl) (naphthalene-2-yl) sulfane (105) was investigated.

After incubation with C. elegans, 19_F{1_{H} NMR analysis of the extract showed three}

sets of signals, indicating the formation of two sulfoxide metabolites and one sulfone

(Scheme 22) – reproducing the observed products in the methoxy derivative 104.

Scheme 22. C. elegans incubation results for 105

Reverse-phase HPLC allowed the separation of these metabolites. NMR analysis, followed by high-resolution mass spectrometry enabled a preliminary identification of each of the structures. However, the recovery of fluorometabolites from the fungal culture was low, making full characterisation challenging.

The major product (tr = 37 min) was positively identified as sulfoxide 116. The second

most abundant fluorometabolite (tr = 13 min) is sulfoxide 117, as determined by 1H

and 19F{1H} NMR spectroscopy, as well as high-resolution mass spectrometry. Unfortunately, the amount of material recovered was too low to be able to carry out

13_{C NMR analysis. The} 1_{H NMR spectrum is however quite distinctive, and points}

towards this structure. In particular, three signals in the aromatic region, between 7.27 and 7.75 ppm, match the substitution pattern for the aromatic ring adjacent to the sulfur atom; while two doublets at 6.5 and 6.1 ppm correspond to protons vicinal to the hydroxyl groups. S F F S F F O + S F F OH OH + S F F O (HO)2 O O C. elegans 72 h, 28 ºC 180 rpm 105 116 117 118

Finally, a third fluorometabolite of general sulfone structure 118 (tr = 17 min),

according to mass spectrometry, was recovered by HPLC. This is a very minor metabolite and little information was obtained for its unambiguous identification.

2.2.4.3 1-(1,1-Difluoroethoxy)-4-methoxybenzene (106)

Finally, in order to extend the study to the oxy ether series, difluoroethoxy ether 106 (5 mg) was fed to cultures of C. elegans, employing the standard conditions.

Examination of the 19F NMR spectrum of the extracts showed only residual starting material, although only at low levels. Isolation of the major components by HPLC and their subsequent analysis proved that there was a non-fluorinated metabolite present. This was determined to be 4-acetoxyphenol 119 (tr = 25 min), the identity of which

was confirmed by comparison of literature NMR data. 49

Scheme 23. Incubation of 106 in C. elegans, gave 4-acetoxyphenol 119

These results were reproduced in separate experiments (4 times). However, on one occassion, the 19F NMR spectrum showed the formation of a new fluorine peak. HPLC purification showed that the main metabolite was still 4-acetoxyphenol 119, but also allowed the isolation of this new fluorinated compound (tr = 19 min). Unfortunately,

the recovery from the fungal culture was once again very low, and taking into account that this was a very minor metabolite, only 1H and 19F NMR spectra were obtained. These analyses were enough to observe that the OCF2CH3 motif was intact, and that

demethylation had occurred. A residual para substitution pattern was obvious in the 1H NMR spectrum, suggesting 4-(1,1-difluoro)ethoxy phenol 120.

O O F F _{C. elegans} 72 h, 28 ºC 180 rpm HO O O 106 119

In order to further establish what happened in this incubation, scale-up (3 × 25 mg) batches of 106 were added to three different cultures of C. elegans. The outcome of each was exactly the same as that for the first assays, and showed the formation of 4- acetoxyphenol 119 along with some residual starting material, and some minor traces of decomposition, but no phenol 120.

Therefore, it was determined that 1-(1,1-difluoroethoxy)-4-methoxybenzene (106) suffers from a rapid hydrolytic metabolism, as well as demethylation, and seems to be a vulnerable motif for bioactive discovery programmes, given its very low metabolic stability.