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  • We have reported previously that


    We have reported previously that β-aminoacrylate derivatives of the general structure () are inhibitors of DHODH. In particular, tricyclic derivatives and displayed sub-micromolar IC values against DHODH, and in the case of there was a 1000-fold greater potency compared to the human enzyme (DHODH). An extensive series of experiments in this previous work with this class of inhibitors showed that compounds and , and related structures, were competitive inhibitors with the enzyme cofactor CoQ and reversible. Compounds such as may in principle exist as a pair of geometric isomers and we considered that different H-bonding groups on either side of the double bond, and their position relative to the aromatic portion of the structure through rotation about the CN bond, would be key in determining their binding to the enzyme. It is perhaps noteworthy to mention that alternative binding modes have been reported with certain DHODH inhibitors, and also differential binding modes for A77 1726, the active metabolite of leflunomide (), when binding to DHODH is compared to its principal target DHODH. We therefore sought to examine whether alterative polar ‘head groups’ to those investigated would lead to improved activity of members in this class of compound, and the results obtained are reported herein. Ten new derivatives (–, ,–) based on the 3-aminocarbazole () and 3-aminodibenzothiophene () core unit were synthesised using the method reported previously (). Derivatives and were prepared by simply heating the aromatic amine with the appropriate commercially available β-ethoxy-α,β-unsaturated carbonyl CGP 57380 and respectively. For and the relevant active methylene compounds were first heated with an excess of triethylorthoformate (or acetate) in toluene, forming the β-ethoxy-α,β-unsaturated carbonyl compounds , followed by addition of either the aromatic amine or and heating for a further short period. After cooling, the products precipitated upon standing, were collected by filtration and then recrystallised. shows the IC values obtained for the compounds discussed in this work. These values were obtained using the screening methodology reported previously. The IC values of derivatives ,, and ,, were above the arbitrary cut-off value of 0.1mM CGP 57380 and thus not further examined. Derivatives and , structures based upon Meldrum’s acid, are conformationally restricted analogues. We assigned their low activity to the restricted freedom imposed by the six-membered ring to position the polar head group for maximal hydrogen bonding to H-bond donors on either side the co-factor channel. The activity of the cyanoacetate (, ) and malonate derivatives (, ) are not too dissimilar, and the IC values are probably subtly altered depending on the influence of the second ethyl ester and/or -ethyl group. The lowest activity is seen with , where both ethyl groups are present, may mean that the molecule is slightly too large to fit well into the ubiquinone binding site. Similarly, derivatives and , which only differ from and by a single methyl group, showed that substitution at this position cannot be tolerated sterically. The lack of activity in and was somewhat surprising, given the similar electron withdrawing effect of the methyl ketone compared to the ethyl ester and cannot so easily be explained. Nitroacrylate derivatives and were as active, if not more so, than cyanoacrylate and malonate derivatives , and ,. As discussed above, we considered that the conformational preferences in the polar ‘head group’ would be important in determining the binding mode of these inhibitors to DHODH, and so it was interesting in particular to compare the isomerism in carbazole derivatives , and , and the corresponding analogues of the dibenzothiophene series , and . In the case of and , evidence of only one isomer is seen in the H NMR. The heavily deshielded NH ( 10.96ppm for and 11.26ppm for ) and large coupling to the CH ( 13.7Hz) indicated the presence of intramolecular hydrogen bonding to the ester carbonyl group, and the antidisplacement of the hydrogen atoms in the ArNC unit showed that the ()-isomer was preferred. In the case of and analysis of the products similarly revealed single isomers [δ 11.56 (s) for , and δ 11.65 (s) for ] in spite of the extra methyl group (). The nitroacrylate derivatives and have the possibility to exhibit intramolecular hydrogen bonding in either isomeric form, and the balance between these isomeric forms was expected to have an important effect on the overall isomeric preferences and therefore binding to the cofactor channel of DHODH. The H NMR of both and showed a 1:1 mixture (in CDCl) however many times it was recrystallised under a range conditions (solvent, rate of cooling etc), indicating no clear preference in solution for one isomer over the other. The C NMR of in CDCl showed doubling of almost all signals. However for in the more polar -DMSO only one set of the aromatic carbon signals and ethyl group were observed, and also the signals for the acrylate unit were completely absent. This was accompanied by changes in the H NMR where in -DMSO the NHCH signals were broadened significantly and almost reduced to baseline level, and the ethyl CH quartet was greatly broadened. This indicates clearly that the geometric forms of are readily interconvertible in a polar solvent on the NMR timescale.