Beware of fused tetrahydroquinolines

Practical Fragments has written frequently about pan-assay interference compounds, or PAINS. These molecules contain substructures that frequently show up in hits that tend not to be advanceable, often wasting considerable effort. One criticism of the PAINS concept is that the original definitions were based on a limited number of screens in one assay format. In a new (open-access) J. Med. Chem. paper, Alison Axtman and collaborators at University of North Carolina Chapel Hill, Emory University, and Oxford University characterize one class of PAINS in more detail.

 

The researchers focused on fused tetrahydroquinolines, or THQs. Of the 51 molecules containing this substructure in the original 2010 PAINS paper, 34 hit in at least one of the assays, and one hit all six. At the time Jonathan Baell and Georgina Holloway noted that “it is not clear for some PAINS, such as the fused tetrahydroquinolines, what the relevant mechanisms of interference may be.” 

 

 

The new paper notes that fused THQs are common in screening libraries, with more than 15,000 commercially available. They also frequently show up as hits: the researchers summarize more than two dozen examples against a wide variety of targets including phosphatases, kinases, protein-protein-interactions, and more. In most cases the hits are modestly active, with low to mid micromolar IC₅₀ values, though a few are high nanomolar. 

 

Promiscuity per se is not necessarily bad. Just last week we noted that the 7-azaindole fragment was the starting point for three approved drugs. However, despite showing up as hits in so many screens, only one peer-reviewed paper reports a crystal structure of a fused THQ bound to a protein, and the researchers note that “no optimized chemical probes or approved drugs contain the chemotype.”

 

Importantly, fused THQs hit in a variety of different assay formats, including spectrophotometric, chemiluminescent, SPR, and radiochemical. Thus, these are not merely problematic in the AlphaScreen format studied in the original PAINS paper.

 

So what’s going on? The researchers found that, while molecules containing the fused THQ core were initially colorless, they darkened when dissolved in DMSO or chloroform, turning purple within 72 hours. Interestingly, the reaction seems to be light-dependent: solutions stored in the dark remained colorless. Thin layer chromatography and NMR showed new species forming, and mass spectrometry revealed oxidation with loss of two or four hydrogen atoms. The isolated double bond in the cyclopentene ring seemed to be the culprit, as the saturated analog was stable. Indeed, all of the hits shown in the paper contain the double bond, so fused THQs that lack this feature may be fine – if they ever show up in your assay.

 

It is still not clear exactly how the decomposition products light up so many assays, but in general it’s a good idea to steer clear of molecules that fall apart in ambient light, unless you’re trying to make a photosensitizer.

 

The researchers conclude that “it is tragic to continue to watch groups invest time and resources in dead-end hit-to-lead campaigns, and the medicinal chemistry community will benefit everyone if the cycle stops.”

 

This concludes our public service announcement.