Metabolism and hydrophilicity of the polarised ‘Janus face’ all-cis tetrafluorocyclohexyl ring, a candidate motif for drug discovery

The metabolism and polarity of the all-cis tetra-fluorocyclohexane motif is explored in the context of its potential as a motif for inclusion in drug discovery programmes.


Introduction
Approximately one third of drugs on the market or in development contain at least one uorine atom 1 and around a third of herbicides historically, 2 and half the commercial pesticides introduced in the last period (2010-2016) 3 contain uorine atoms. The element is also important in organic materials with applications in next generation displays 4 and high value materials. 5 The investigation of new products bearing uorinated moieties is an ever expanding eld, given the particular properties that uorine bestows on organic compounds. 6 We have recently synthesised all-cis 1,2,4,5-tetrauorocyclohexane ring systems such as 1 and 2 as a novel motif in organic chemistry. 7 This tetrauorocyclohexane isomer displays a particular polar property across the cyclohexane, largely because all of the uorines are on one face of the ring and there are two 1,3-diaxial C-F bonds, with dipoles orientated parallel to each other.
As an extreme example, we extended this concept to the preparation and analysis of all-cis hexauorocyclohexane 3 which is even more polar due to a cyclohexane ring accommodating three axial C-F bonds. 8a This cyclohexane has been referred to as a 'Janus'-like molecule, 8b because of its very well differentiated faces, and recent experimental and theoretical studies have indicated that these rings will coordinate cations to the uorine face and anions to the hydrogen face, consistent with the electrostatic polarity of the ring system. 9 The conformational and polar properties of these multi-vicinal uorinated aliphatics is beginning to attract the attention of the synthesis community and new methods are emerging for their preparation, for example, from the Gilmour, 9 Jacobsen 10 and Carreira 11 laboratories. For the cyclohexanes, a recent report from Glorius's laboratory 12 has demonstrated the direct catalytic hydrogenation of uorinated aromatics to generate all-cis uorinated cyclohexanes in a single step, and this methodology promises to make compounds such as cyclohexanes 1 and 3 much more accessible to the organic chemistry community. With these developments in synthesis methods, we believe the cyclohexane motif merits exploration as a candidate substituent for agrochemicals or pharmaceutical drug discovery programmes.
Immediate questions which arise are how will these selectively uorinated cyclohexane rings be metabolised and how lipophilic are these ring systems. It is commonly understood that increasing the level of uorination of an organic motif will generally result in increasing its resistance to metabolism at certain sites. 13 Also, the prevailing dogma is that increased levels of uorination render a motif more lipophilic and, thus, its introduction would have a tendency to raise log P values in a manner detrimental to judicious selection in medicinal chemistry. However, it is more complex than that, and Müller and Carreira have exemplied this extensively in recent contributions e.g. mapping log Ps of RCH 3 compounds through progressive uorination to RCF 3 , where intermediate uorinations (RCH 2 F & RCF 2 H) decrease lipophilicity. 14 It is a feature of these ring systems, 15 where the uorines have a relative stereochemistry such that they are all on one face of the cyclohexane, that the rings become polar, and thus increasing uorination could reasonably increase hydrophilicity. Thus we set out to explore the nature of these ring systems in the context of their properties and potential as a novel motif for inclusion in bioactive research programmes. To that end we focus on phenylcyclohexane 2, because it is readily prepared 7c and has been shown to be amenable to a range of synthetic transformations and diversication. 16,7b The study compared the metabolism of 2 to close analogues 4-7 by incubation with the human metabolism model organism Cunninghamella elegans. 17 Lipophilicity trends (log P) were also explored comparing cyclohexanes with four, three, two and no uorine atoms. Lastly, a molecular dynamics simulation study was carried out to elucidate the structural basis of the observed lipophilicity trends.

Biotransformations with Cunninghamella elegans
The fungus Cunninghamella elegans represents a wellestablished model for drug metabolism in mammals due to its ability to biotransform and degrade a wide range of xenobiotics. 18 The organism contains a range of cytochrome P 450 enzymes and this gives an oxidative metabolic prole which mimics phase-I oxidative metabolism. In order to investigate how the tetrauorocyclohexyl motif may be metabolised, we have explored the incubation of phenyl tetrauorocyclohexanes and also compounds with three and two uorine atoms. Five compounds were investigated in total, three of which were the phenyl derivatives 2, 4 and 5, and two were the benzoic acid derivatives 6 and 7. 7d Incubations with C. elegans were carried out in triplicate in submerged liquid cultures. In each case, the incubations were worked up aer three days and products were extracted and analysed.
Phenylcyclohexane 2 gave rise to only one obvious metabolite in a conversion of around 30%. This product arose by direct hydroxylation at the benzylic position of 2 to give benzyl alcohol 8. Only one product as a single isomer could be detected, with the hydroxyl group congured anti to the adjacent uorine atoms of the cyclohexane ring. The identity of 8 and its stereochemistry was conrmed by X-ray structure analysis.
Phenyl triuorocyclohexane 4 was similarly incubated with the fungus and it too gave rise to the analogous benzyl hydroxylated product 9. The extent of microbial conversion was approximately 50% aer the three day incubation. The residual 4 was assayed for enantiomeric purity by chiral HPLC, and it was shown to be almost racemic, thus there is no indication that the microbial hydroxylation was signicantly enantioselective. Finally in this series, diuorocyclohexane 5 was subject to a similar incubation with C. elegans. This compound was completely and extensively metabolised, and it generated a much greater product prole of which compounds 10-13 were isolated. Compounds 11-13 were characterised by X-ray crystallography as illustrated in Fig. 1. Monohydroxylated products Fig. 1 Biotransformations of selectively fluorinated phenyl fluorocyclohexanes 2 and 4-7 by C. elegans. Some of the products were crystalline and amenable to X-ray structure analysis.
10-12, can be rationalised by direct methylene P 450 type hydroxylations, however the monouorinated cyclohexanol 13 is less easily rationalised and presumably arises from a series of biotransformations involving uoride elimination. More generally, it is clear that removal of two of the ring uorines from positions 2 and 3 of the phenyl all-cis tetracyclohexyl ring system has rendered the aliphatic ring much more susceptible to metabolism.
The benzoic acids 6 and 7 were also incubated with C. elegans. The tetrauorocyclohexyl benzoic acid 6 was poorly biotransformed and only a very low conversion to alcohol 14 was obvious aer the three day incubation. Triuorocyclohexyl benzoic acid 7 was more readily transformed, but only to benzylalcohol 15 ($50% conversion). This product was isolated and crystallised and X-ray analysis conrmed its structure. Again, in order to explore any enantioselectivity for this biotransformation, the methyl ester of the residual carboxylic acid 7 was analysed by chiral HPLC and this indicated a very low enantioselectivity, thus in a similar outcome to substrate 4, there was no obvious selectivity for 7 by the hydroxylation enzyme involved.

Lipophilicity study of selectively uorinated phenyl cyclohexanes
An important measure of the druggability of a substituent is its lipophilicity, 19 and given the polarity of the phenyl all-cis tetra-uorocyclohexyl moiety it was of interest to explore the relative log Ps of various analogous compounds. log P's were measured by reverse phase HPLC (AcCN 60%: water 40%, with TFA 0.05%), as previously described. 20 The measured log P's of a series of phenyl uorocyclohexane derivatives are summarised in Fig. 2 and against a series of compounds, of known log P values, which were re-measured for comparison, including biphenyl 16 and phenylcyclohexane 17.
It is clear that there is a signicant reduction in log P with increasing uorination. Phenyldiuorocyclohexane 5 (log P 3.30) is signicantly more polar than the phenylcyclohexane (log P 4.99), and then both the tri-and tetra-uoro cyclohexanes progressively increase in polarity (log Ps of 2.64 and 2.58 respectively) with additional uorine atoms. An interesting comparison on log Ps can be made with the two triuorinated stereoisomers 4 and 18. Compound 4 is more polar, and this presumably arises as it has a preferred diaxial arrangement of the C2 and C6 C-F bonds. 7c This parallel alignment can be expected to increase the molecular dipole relative to isomer 18 which has one of these uorines lying in an equatorial orientation.
The study extended to substituted aryls of the benzoic acids 6 and 7 and the anilines 21 and 22(ref. 7b) as illustrated in Fig. 3. In each case both the triuoro-and tetrauoro-cyclohexanes are around two log P units more lipophilic than the non-uorinated cyclohexanes 20 and 24, whereas the phenyl derivatives 19 and 23 lie in between. There is a clear trend that selective uorinations around the ring increases the polarity of the cyclohexane.

Computational analysis of lipophilicity trends
Molecular dynamics (MD) simulations were carried out for phenylcyclohexanes 2, 4, 5, 16 and 17 to clarify the mechanisms by which progressive uorination decreases lipophilicity. log Ps were predicted by computing absolute solvation free energies in aqueous and organic phases using explicit solvent molecular dynamics simulations. 21 Fig. 4 shows a comparison of calculated (log P pred ) and measured (log P exp ) log P values, as well as calculated solvation free energies in aqueous (DG aq ) and cyclohexane (DG org ) phases. Overall, the log P calculations are in good agreement with the experimental data (Kendall tau 0.5 AE 0.1 and mean unsigned error 0.77 AE 0.07 log P units). Inspection of the solvation free energies shows that the trend for decreased log P upon increased uorination is due to a more rapid decrease in solvation free energies in the aqueous phase (from ca. À2.5 to À5.2 kcal mol À1 for 16 and 2 respectively) vs. the cyclohexane phase (ca. À7.5 kcal mol À1 for all compounds).
Further insights were investigated to help rationalise the calculated differences in hydration free energies by grid-cell theory (GCT) analyses of the MD simulation trajectories. 22  GCT is a MD trajectory post-processing method that spatially resolves the water contribution to enthalpies, entropies and free energies of the hydration for small molecules, host/guests and protein-ligands complexes. 23 Fig. 5 depicts spatially resolved hydration thermodynamics around the non-uorinated cyclohexane 17 and the tetra-uorinated cyclohexane 2. Comparison of water density contours show water structuring above and below the p-cloud of the phenyl ring due to the expected weak hydrogen bonding interactions in this region. In addition the four uorine atoms in 2 induce further structuring of water around the cyclohexyl moiety, with a more pronounced effect around the hydrogen face of the cyclohexane (panels A and B). Owing to the different polarities of the cyclohexyl ring in 2, water near the uorine-face preferentially orients hydrogen atoms towards the ring, whereas water near the hydrogen-face preferentially orients oxygen atoms towards the ring. Water near the hydrogen-face is more enthalpically stabilised and entropically destabilised with respect to bulk, whereas the energetics are not signicantly different from the bulk in the vicinity of the uorine face (panels C and D and E and F). Overall favourable enthalpic contributions offset unfavourable entropic contributions for water near the hydrogen face and water in this region makes additional favourable contributions to the hydration free energy (panels G and H). Therefore the decreased lipophilicity of 2 with respect to 17 is attributed to enhanced hydrogen bonding interactions between water and the hydrogen face of the all-cis tetrauorocyclohexane ring.

Conclusions
The all-cis tetrauorocyclohexane motif has been recognised to have particularly polar properties and the ease of synthesis of the phenyl derivative 2 has prompted us to investigate it properties further as it emerges as a building block for the introduction of this new motif into medicinal chemistry and other bioactives discovery programmes. The metabolism of the phenyl cyclohexane derivatives 2, 4-7 with varying levels of uorination was explored in incubations with Cunninghamella elegans. This fungus has been used as a microbial model for mammalian metabolism. In the present study we observed that increasing the degree of uorination of cyclohexyl ring leads to a more stable xenobiotic. The phenyl all-cis tetra-uorocyclohexane 2 was signicantly less metabolised than the triuoro-4 and then diuoro-5, the latter of which was extensively metabolised. In the case of 2, 4, 6 and 7 metabolism is conned to benzylic hydroxylation.
A systematic log P evaluation of these ring systems shows an increase in hydrophilicity with increasing uorination, and for the phenyl all-cis tetrauorocyclohexanes (including anilines and benzoic acids) there is a maximal effect. These ring systems are at least two full log P units (100 fold) more hydrophilic than their non-uorinated cyclohexane counterparts.
Molecular dynamics simulations reproduce the experimental trends and suggest that the decreased lipophilicity of 2 is due to enhanced hydrogen bonding interactions of water molecules with the hydrogen face of the cyclohexane ring with respect to bulk water. The orientation of the water near this face of the ring was consistent with the hydrogen bonding donor ability of the polarised hydrogens of the ring.
This contrasts with the energetics of water near the uorine face of the ring which are comparable to bulk water. Altogether these studies indicate that metabolism of the all-cis tetra-uorocyclohexyl motif is slow, and that the ring system is signicantly hydrophilic for an aliphatic motif. These factors Fig. 4 The positive y-axis depicts a comparison between calculated (cyan) and measured (blue) log P values for compounds 2, 4, 5, 16 and 17. The negative y-axis depicts calculated solvation free energy in cyclohexane, DG org (red), and aqueous, DG aq (yellow), phases. . Panels A and B show isocontours for density (red: r wat > 2.33 bulk density, blue: r wat < 0.5 bulk density). Panels C and D show isocontours for regions where water is enthalpically stabilised with respect to bulk water (red: DH w < À0.0055 kcal mol À1 A À3 ). Panels E and F show isocontours for regions where water is entropically destabilised with respect to bulk water (blue: ÀTDS w > 0.0033 kcal mol À1 A À3 ). Panels G and H show isocontours for regions where water is more stable than bulk water (red: DG w < À0.0055 kcal mol À1 A À3 ).
add to the unique facially polarised aspect of this motif and make it an attractive option for inclusion in medicinal chemistry or crop protection studies.

Conflicts of interest
There are no conicts of interest.