Conclusions and Future Work 6.1. Conclusions
6.1.1. Activity-Directed Synthesis exploiting intramolecular reactions
The Activity-Directed Synthesis of sub-micromolar Androgen Receptor modulators with chemotypes with no previously annotated activity has been successfully developed and demonstrated. By exploiting promiscuous metal-carbenoid reactions with multiple possible outcomes, generating mixtures of products allowed the rapid and facile identification of small biologically active molecules, in a more efficient manner compared to conventional approaches. A key aspect of Activity-Directed Synthesis was the utilisation of evolutionary feedback, through the analysis of the information from the screening of one reaction array to design a subsequent reaction array. The development of the approach was retrospectively analysed and rationalised in the case of intramolecular reactions, demonstrating that Activity-Directed Synthesis is able to focus on active molecules rather than treating molecules in large collections with the same significance. The approach was demonstrated to facilitate the optimisation of both the structure of the active component as well as a route for its synthesis simultaneously.
6.1.2. Activity-Directed Synthesis exploiting intermolecular reactions
A limiting factor when exploiting intramolecular reactions was the necessity to synthetically incorporate the various reactive groups on the same substrate, increasing the synthetic effort required for their preparation and diminishing the number of possible combinations used. These issues were addressed by exploiting intermolecular reactions, as demonstrated by the greater diversity of structures and chemotypes of the major products isolated from the scale-up of selected intermolecular reactions. It was also shown that exhaustive combinations of reaction arrays were not needed, as
sparse combinations were enough to provide sufficient information for the design of a subsequent reaction array. In addition, it was demonstrated that structural leaps through chemical space provide an additional mechanism for the evolution Activity-Directed Syntheis.
6.2. Future work
6.2.1. Retrospective exploration of the intermolecular reaction arrays
In order to fully rationalise and understand the development of Activity-Directed Synthesis using intermolecular reactions a retrospective exploration, similar to the case of intramolecular reactions, is needed.
Initially, reactions which were not prioritised from each reaction array should be scaled-up and the products isolated, characterised and evaluated biologically, in order to investigate whether products of different structural scaffolds were formed.
In the case of diazo-substrate 18k, with co-substrate 36g’ (Scheme 6.1), two significantly different activities were observed with the use of the two enantiomers of the Rh2(DOSP)4 catalyst. These results suggested that that the reaction was enantioselective and the product was chiral. As the major product formed, 37i is indeed chiral, it is possible that the catalyst is kinetically resolving the two enantiomers of the racemic co-substrate used, resulting in an enantiomerically enriched bioactive product. Performing the same reaction using the S enantiomer instead of the R, and isolating, characterising and evaluating the isolated products will aid to the verification of this hypothesis. The enantioselectivity can be determined by determining the enantiomeric excess using chiral HPLC analysis. This analysis will also help verify the above hypothesis, as well as add to the overall understanding of the methodology.
N
Scheme 6.1: Scaled reaction of diazo-substrate 18k with co-substrate 36g’
with Rh2(R-DOSP)4 in CH2Cl2.
6.2.2. Crystallographic Studies
There is currently no evidence for the structural basis of the novel Androgen Receptor modulators discovered through this project. Although it is hypothesised that these molecules bind in the same cavity of the AR LBD as other flutamide or bicalutamide analogues,56 it would not be surprising if the binding conformation or orientation is different. It would also be possible that these molecules act through covalent binding, as these has been previously reported for other β-lactam based molecules.57 A crystallographic study to obtain an X-Ray crystal structure with the isolated molecules co-crystallised with the AR LBD would provide additional information and elucidate the binding site and orientation, as well as provide evidence of potentially new interactions which could be used to further optimise the activity of these molecules.
6.2.3. Further Future Work
The general applicability of Activity-Directed Synthesis as an efficient approach for the discovery of novel small bioactive molecules could be effectively demonstrated by applying this methodology on different biological targets exploiting different chemical toolbox.
The TR-FRET assay has proven to be an effective, high throughput and reliable screening method once it was established. Similar assays are commercially available for different biological targets such as PDK1 or VEGFR-2 or other kinases.58 As they as very well studied and understood, with limited novel inhibitors reported, kinases provide an attractive target to demonstrate the applicability of Activity-Directed Synthesis.
When choosing a different chemical toolbox the same considerations should be taken into account. The chemistry needs to be equally versatile and tunable, with a demonstrated record of applicability. In this regards, both Au catalysis and N-heterocyclic carbene (NHC) catalysis are very attractive.
Au-catalysed chemistry can be considered complementary to carbenoid chemistry as shown in Scheme 6.2A. Both intra- and intermolecular applications of this chemistry have been reported, leading to the formation of diverse molecular scaffolds with the reaction outcome critically depended on conditions used (Scheme 6.2B).59 Similarly, NHC-catalysed cascades have been reported, where the use of either NHC-I or NHC-II type catalysed may result in different products with the use of the same starting materials (Scheme 6.2C).60
Finally, however, in order to demonstrate the versatility, efficiency and greater potential of Activity-Directed Synthesis, it would be ideal to turn to unbiased libraries. Such an application would allow the same library of starting materials and its respective chemical toolbox to be exploited against different biological targets and ultimately turn Activity-Directed synthesis into a general methodology. This application would be far more efficient, as any synthetic preparations would need to be undertaken to a minimal extend whilst the starting materials could be used for more than one assays.
Furthermore, it would be possible to extract multivariant information which would in turn facilitate SAR deduction and add to the subsequent optimisation of small molecules discovered by such an application of Activity-Directed Synthesis.
Scheme 6.2: Representative examples of the potentially attractive chemical transformations to be exploited for Activity-Directed Synthesis. A: Au-catalysed chemistry can be considered complementary to metal-carbenoid chemistry considering the reactive species. B: Representative examples of Au-catalysed transformations to prepare molecules with diverse scaffolds through either intra- or intermolecular reactions. C: Representative example of NHC-catalysed transformation where different molecular scaffolds are obtained by treating the same starting materials with different types of NHC catalysts.