XX TrxR Thioredoxin reductase
I. Introduction
7. Conclusion
“Enjoy the journey of life and not just the endgame.”
Benedict Cumberbatch The search for effective non-steroidal anti-inflammatory compounds without serious side effects is an ongoing one, and with the failure of High-Throughput Screening (HTS) in providing new drug-like compounds, many researchers are returning to the world of natural products and their derivatives in order to identify new lead compounds. One compound which has been the focus of much investigation is curcumin, the diferuloyl methane compound found in turmeric. This compound was chosen as a model for compounds which would selectively target the inducible form of cyclooxygenase (COX), the enzyme responsible for the first step in the conversion of arachidonic acid into the various prostaglandins and thromboxanes in the inflammatory process. An in silico investigation into the binding of curcumin and celecoxib, a known COX-2 selective analgesic compound, was conducted and important ligand-protein interactions were identified which would need to be mimicked by a novel COX-2 selective compound. However, as the docking and binding scores for celecoxib when docked into COX-2 are similar to the results obtained on docking into COX-1, computational results cannot be the sole criterion used when identifying a potential lead compound, as literature reports celecoxib showing 10-20-fold selectivity for COX-2 over COX-1 in vivo.
Based on these results, two potential COX-2 selective compounds were designed using moieties found in curcumin as well as moieties common to celecoxib and other known COX-2 selective compounds. Initial docking results showed that both compounds interact with the secondary pocket present in COX-2 as desired, and a number of ligand-protein interactions are made that mimic those seen between celecoxib and COX-2, while also identifying the potential for improvement, both in docking and binding scores as well as with interactions between the protein and the ligands. Therefore, a range of modifications were made to these two parent compounds in order to explore the impact of the various substitutions on the docking and binding scores and on the protein-ligand interactions. In all cases, the modifications resulted in an increase in the COX-2 binding scores when the scores were compared to that calculated for the parent compound, and in a few cases, reductions in the COX-1 binding scores were observed.
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Thirty of the 166 compounds designed were selected for synthesis and biological screening as these compounds exemplified the range of changes observed in the full complement of compounds.
Retrosynthetic analysis identified two potential routes involving simple chemistry, which would allow for the formation of the correct Z-isomer about the double bond. The initial synthetic route, which involved the formation of the double bond through a base-catalysed condensation of an acetophenone with a benzaldehyde prior to the formation of the ether bond proved unsuccessful as the base-catalyzed intramolecular reaction of the chalcones yielded a flavanone rather than the desired benzyl ether, and as such this pathway was abandoned in favour of the alternate pathway. Early investigations into this second synthetic pathway, which involved the formation of the ether bond prior to the base-catalyzed condensation, yielded the correct benzylated compound in high yields, however the addition of the sulfonyl chloride moiety proved unsuccessful, with low yields and addition to other positions within the molecule observed. At the outset, use of a benzenesulfonyl chloride compound in order to circumvent the need for chlorosufonic acid resulted in the formation of a tosyl derivative, rather than the desired ether. Conversion of the sulfonyl chloride into a sulfonate or sulfonamide prior to the ether formation yielded “protected” species, however the protected sulfonamide proved too insoluble to be of use.
High yields were obtained when an ethyl-protected benzenesulfonate was combined with three 4-substituted 2’-hydroxyacetophenone, however, the corresponding 5-substituted 2’-hydroxyacetophenone proved too unstable and rapid decomposition was observed even with the use of milder reaction conditions. Removal of the ethyl protecting group occurs during the ether formation reaction; nevertheless this does not affect the subsequent condensation. The current understanding of this reaction is that the ethyl-protected sulfonate species is the reactive species, with the ethyl group removed after ether formation. This hypothesis is supported by the lack of intermolecular addition between two benzenesulfonate molecules, as would be expected if the ethyl group is removed prior to ether formation. With suitable reactions and conditions identified, the synthesis of the thirty compounds previously identified was perfomed, using three 2’-hydroxyacetophenones and twelve benzaldehydes, in moderate to good yields. These products were isolated cleanly with only simple recrystallization techniques required to remove unwanted starting materials and side products.
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Complete spectroscopic analysis of all synthesized compounds was carried out in order to definitively determine the identity of all the compounds. While appearing daunting, the NMR spectroscopic analysis of the final benzenesulfonate compounds was simplified through the identification and analysis of simpler compounds which are components of the final compound. During the course of analysis, broadening of the line widths of methylene peak in the 1H NMR spectrum was observed on formation of the ether bond, indicating a change in the solution mobility of the compound. DOSY experiments were carried out on two compounds, and the diffusion coefficients and hydrodynamic radii were determined for both compounds. The size of the hydration sphere was shown to be solvent-dependent, and a need for the determination of the viscosity of the solution used for the DOSY experiments was identified, as effective radius calculations make use of literature viscosity values, rather than taking into account the viscosity of the solution under investigation.
In order to identify the conformations present in solution, NMR Analysis of Molecular Flexibility In Solution (NAMFIS) made use of NOE correlations to identify six conformers as existing in solution, from a pool of 3630 potential conformations. Of these six conformers, four poses comprise >95% of the solution population, with one pose comprising almost 50%. While none of the poses are exact matches when compared to the pose generated for 6 in either COX-1 or COX-2, all but one pose show RMSD values of less than 2 Å, which indicates that any of these five conformers could bind into the proteins.
Initial inhibition screening results of the unsubstituted parent benzenesulfonate compound appeared to show three-fold selectivity of COX-2 over COX-1 at 100 nM. Testing of the substituted compounds revealed that these compounds are not COX-2 selective as desired, rather a number show promise as COX-1 selective compounds, with inhibition scores of over 40%, and several other compounds show potential as non-selective COX inhibitors. There appears to be no correlation between the inhibition results and either the Glide XP docking scores or the Prime binding scores, and as such, additional computational analysis as well as experimental testing is required to identify a correlation between the theoretical results and the experimental data. It is possible that these compounds could behave differently in vivo than is observed in vitro, as is the case for lumiracoxib, and it is also possible that the mode of inhibition is allosteric in nature, rather than the competitive inhibition model used here. Should these compounds not prove suitable as analgesic compounds, a number of alternative uses exist for
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COX-isoform selective and non-selective compounds, ranging from cancer treatments to the reduction of neuroinflammation seen in diseases such as Alzheimer’s disease and Parkinson’s disease.
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