Compound was synthesized by the directed
Compound () was synthesized by the directed -metallation procedure (DOM) at the stage of the amide using butyl lithium and dry ice as CO source for the introduction of the carboxylic group. Thiophene amides were prepared from commercial 3-thiophenecarboxylic body language by conversion into the acid chloride and reaction with the corresponding 4-phenylaniline, which in turn were obtained by the Suzuki cross-coupling method as described earlier. Compounds with a second carboxylic group, like in or , were either built up by 2-fold DoM/CO-quenching or from 3,4-thiophene dicarboxylate upon its conversion into the anhydride with acetic anhydride and nucleophilic anhydride opening with the respective 4-phenylaniline to give compounds –. Regiochemistry for the carboxylate introduction in 3-thiophenamides was in analogy to a literature protocol and was finally deduced from NMR analysis to be in the 2-position. Introduction of a third carboxylic group into yielded a major and a minor component, which were separated by HPLC. Major product possessed a completely identical NMR spectrum as the major product from carboxylation of 3-thiophene-2-carboxylate by the DoM protocol. Therefore, the structure has to be as depicted in . Enzyme inhibition was measured in an in vitro enzyme assay. For the assay N-terminally truncated recombinant human DHODH was used and the data are presented in . Direct comparison of the previously described compounds – with their thiophene analogs , , and showed a clear trend toward an increased inhibitory activity of the thiophene analogs. As with the cyclopentene series, the activity increases with fluoro constituents in the aromatic ring adjacent to the amide bond and with a methoxy group in the terminal aromatic, but the trend is more pronounced in the thiophene series. A X-ray structure investigation of the cyclopentene series revealed a binding mode with the possibility of the carboxylic acid either having an ion bridge toward Arg 136 of the enzyme or pointing into the opposite direction toward Tyr 365 and 147 upon turning by 180°. To test the possibility that both binding modes could be obtained within one molecule, compounds with two free carboxylic acids (compounds –) were synthesized. These compounds, however, were inactive, which was explained by the fact that for the alternative binding mode, a conformational change of some residues in the active site of the enzyme was required. There was not much difference between the regioisomers and in their inhibitory activity, indicating the relatively diminished importance of the sulfur position. If one compares the X-ray structures of the cyclopentene analog versus those of the thiophene derivative , it is of significance that the cyclopentene analog displayed a single binding mode which we termed ‘non-brequinar’ like. In contrast, the thiophene analog showed a dual binding mode which was brequinar-like and non-brequinar-like, which can be deduced from the electron density pattern in the crystal structure. We have previously shown that the brequinar-like binding mode leads to a better inhibitory activity, reflecting a high affinity binding mode. Thus, the higher activity of the compounds of the thiophene series as compared to that of the respective representatives of the cyclopentene series can easily be explained by taking into account their possibility for binding in this high affinity mode. Compound represents an exception within the cyclopentene class, as it likewise binds in both modes, resulting in a comparable excellent activity (cf. compound vs ). Furthermore, an increased number of fluoro substituents in the aromatic ring adjacent to the amide bond correlate with an increased tendency toward a ‘brequinar-like’ binding mode, thus with a better inhibitory activity. As can be seen from the furan analogs and , a replacement of sulfur by oxygen in the pentacyclic ring is well tolerated. From the few furan analogs prepared, it can be deduced that this series will have a similar SAR compared with that of the thiophene compounds. A somewhat lower activity can be expected from higher hydrophilicity which results in a hindered diffusion into the hydrophobic environment of the active site, a phenomenon we have encountered with all analogs.