Everybody working within drug discovery is acutely aware of the high attrition rate of small molecule drug candidates throughout the drug discovery process – this becomes especially troublesome during late stage development as the time and money already invested to progress these candidates is significant.
As a synthetic medicinal chemist, I strive to develop metabolically stable molecules that show a desired effect against a particular target – avoiding known problematic functional groups, substitution patterns and alteration of the electronic properties of compounds can reduce potential liabilities that plague late stage candidate progression. One (of many) increasingly recognised problematic areas is that of aldehyde oxidase (AO) metabolism of heteroaromatic compounds; current predictive tools have proven difficult to refine, and so medicinal chemists are required to submit individual compounds for biotransformation testing, which is both costly and time consuming. Therefore, a quick and robust chemical test indicating the propensity for a compound to undergo AO metabolism could prove to be an early warning sign for medicinal chemists.
Figure 1: Aldehyde oxidase and proposed litmus test
The mechanism by which AO is proposed to operate is via nucleophilic attack of the carbon adjacent to the heteroaromatic nitrogen by a molybdenum bound oxygen (Figures 1 and 2A). This reactivity is similar to the Authors developed method for direct C-H functionalisation of heteroarenes using alkylsulfinate radicals – that being nucleophilic attack of the aromatic carbon and subsequent C-H cleavage. Due to this similar reactivity, the Authors decided to apply their chemistry to create a Litmus Test for AO metabolism. In order to make this Litmus Test easily accessible to medicinal chemists, the following conditions needed to be met:
- Must use readily available reagents that are not moisture or air sensitive
- Easy to handle and analyse
- The conditions must tolerate a plethora of commonly encountered functional groups
- Not overly sensitive to stoichiometry of substrates/reagents (tip of a spatula accuracy)
Figure 2: A) AO and Litmus Test mechanism of action; B) Indication of substrate susceptibility to AO
After extensive optimisation (as highlighted by the Baran Groups blog) using Bis(((difluoromethyl)sulfinyl)oxy)zinc (DFMS) as the radical source, the addition of a new LCMS peak with a mass of substrate +50 would indicate a positive result, and therefore that compound is potentially an AO sensitive substrate (Figure 2B).
In order to validate their Litmus Test, the Authors initially subjected known AO substrates to their newly developed conditions (Table 1) – it is clear to see that these five compounds appear positive in their chemical test.
Table 1: Optimised Litmus Test with five known AO substrates
As AO is an enzyme, subtle structural changes of a molecule can alter substrate susceptibility, and as it is difficult for chemical reactions to mimic such a sensitive environment (and so the potential for false positives), the Authors took structurally related substrates to compound 5 – some of which are known to be AO resistant, and investigated their reactivity (Table 2). On the whole, the Litmus Test proved predictive of AO susceptibility, with only one false positive being encountered amongst the compounds described (compound 13, a ketolide antibiotic).
Table 2: Predictive accuracy of Litmus Test
This is by no means a fall-proof method to gauge AO substrate susceptibility, neither is it designed to replace biotransformation tests as false positives are possible due to the non-enzymatic nature of the chemical reaction. However, when used with caution, it is both a quick and cheap early warning sign that trouble may lie ahead with your otherwise promising compound.
Blog written by Mark Honey