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.

A framework for evaluating commercial fragment libraries

The easiest way to build a fragment library is to purchase one. Quite a few vendors sell fragments, and as our poll from a few years ago demonstrated, most buyers are quite happy with them. But what exactly do they offer? This is the subject of a new paper by Gilles Marcou, Esther Kellenberger, and colleagues at CNRS Université de Strasbourg in RSC Med. Chem.

 

The researchers analyzed 86 different libraries from 14 vendors that were available in February of 2021. These were classified into ten categories, such as “general,” “3D-shaped,” “metal chelating,” “diverse,” “covalent,” etc. Individual library sizes ranged considerably: 41 had ≤ 2000 compounds, 31 had 2000-10,000, and 14 libraries had > 10,000 molecules. The total number of fragments came to 754,646, of which 512,284 were unique, indicating some redundancy between libraries. Laudably, the structures and several analyses are all provided as downloadable files here.

 

Most of the fragments are 200-300 Da, with only 13% less than 200 Da. This skew towards larger molecules is common but may not be desirable, as researchers at Astex demonstrated back in 2014. On the other hand, people do seem to be paying attention to lipophilicity: nearly half the fragments have AlogP < 1. Interestingly, less than a quarter of fragments strictly fulfill the rule of three, though the majority of violations are for more than 3 hydrogen bond acceptors, which is probably not as important as the other criteria, according to analyses of approved drugs.

 

Two different methods were used to assess diversity. These were applied to 433,433 compounds from fifty libraries; specialized libraries such as fluorine-rich and covalent libraries were excluded. The first analysis deconstructed fragments into 59,270 component scaffolds. Not surprisingly, benzene was the most common, present in nearly 5% of all fragments. Quinoline, indole, pyridine, and benzimidazole all were present in at least 1% of compounds. At the other end of the spectrum, 36,555 scaffolds occurred only once. Not surprisingly, these tended to be more complex.

 

In addition to assessing scaffolds, the researchers developed a “Generative Topographical Map (GTM) model to represent the chemical space in a landscape.” The resulting figures do indeed look like topographical maps, with darker regions corresponding to more populated areas. For example, since substituted benzimidazoles are common and similar to one another, they form a dark cluster. Not unexpectedly, the landscape for the set of 433,433 compounds is heterogenous, with denser regions separated by sparsely-populated regions.

 

A nice feature of the GTM model is that it allows easy, intuitive comparisons. For example, some of the “diverse” libraries are more diverse than others, or emphasize different regions of chemical space, and potential customers may want to take these into account.

 

Fragment shapeliness was assessed using plane of best fit (PBF), where lower values correspond to “flatter” molecules, such as benzene, with PBF = 0. The libraries varied considerably in their average PBF, though reassuringly the “3D-shaped” libraries did have higher values. Interestingly, GTM models showed both flat (PBF < 0.1) and non-planar (PBF ≥ 0.1) fragments had similar distributions across fragment space.

 

Overall this is a valuable snapshot of the current state of commercial libraries, and makes a useful complement to the ongoing analysis Chris Swain does at Cambridge MedChem Consulting. Of course, the devil is in the details; PAINS still sometimes show up in commercial libraries, and quality control can vary. In the end you’ll want to do your own vetting, but this is a good place to start.

Fragments vs AXL, with help from an ELF

The receptor tyrosine kinase AXL has been implicated in multiple cancers, and more recently as a possible target for COVID-19. The protein has been pursued by many groups, mostly starting from high-throughput screens or existing inhibitors. In a recent Bioorg. Med. Chem. paper Pearly Ng, Alvin Hung, and collaborators at Experimental Drug Development Centre Singapore, University of Sussex, and LigatureTX describe a fragment-based approach.

 

The researchers started with a biochemical screen of 1700 fragments at 500 µM. Subsequent dose-response experiments yielded 16 fragment hits with IC₅₀ < 400 µM, all of which are shown in the paper. Four of these were based on 7-azaindole, a kinase hinge-binding motif that has previously been used in AXL inhibitors.

 

Seeking novelty, the researchers focused on the remaining 12 fragments and turned to their “expanded library of fragments (ELF),” which they have been building for several years. Though not described in depth, this collection was designed such that near-neighbors of their primary library could be rapidly screened to establish SAR and identify productive growth vectors. Compound 11, an indazole, led to several analogs from the ELF such as compound 24. This was docked into a publicly available crystal structure of AXL, and structure-based design led to compound 32. Further optimization ultimately led to molecules such as compound 52, the most potent in the paper.

 

Although compounds in this series were potent in the biochemical assay, they were typically 100-1000 less potent in a cell-based NanoBRET target-engagement assay, an annoying but not uncommon phenomenon. Several of the compounds showed high nanomolar activity in an ovarian cancer cell line with AXL overexpression. Interestingly, two compounds showed considerably more potent antiproliferative activity than target-engagement, and the researchers speculate they may be hitting other kinases. In support of this hypothesis, the researchers found that one of their compounds inhibited 12 of 97 kinases by >90% at 1 µM. This is not surprising given that indazoles are privileged fragments for kinases. Mouse pharmacokinetic studies revealed poor oral exposure for most of the compounds, though this could be improved by decreasing the basicity of the molecules.

 

A nice aspect of this paper is that most of the work was done without the benefit of structural information. Crystallography attempts with AXL and any of the ligands were unsuccessful, though the researchers were eventually able to obtain a structure of one of their more potent molecules bound to a related kinase. And just in time for the holidays, the paper also illustrates the utility of having an ELF on your shelf.

Poll results: synthetic challenges are pervasive in FBLD

Last month we wrote about “sociable” vs “unsociable” fragments: the former are amenable to straightforward chemistry for fragment growing, merging, or linking, while the latter are not. This categorization prompted a poll on how often synthetic challenges have kept you from pursuing a particular fragment or impeded a fragment-to-lead project. The results are now in.

 

(Methods note: the poll was run on Crowdsignal from 14 June through 16 July. There were 42 responses to the first question and 44 to the second. Respondents came from a dozen countries with the most from the US, UK, Germany, Denmark, Netherlands, Australia, France, and Italy.)

 

 

Responses were similar for both questions: a fifth to a quarter of respondents often found that synthetic challenges kept them from pursuing a particular fragment or impeded work on a fragment-to-lead project, nearly half sometimes found this to be the case, while a fifth to a third rarely experienced this. Only a handful of people never found synthetic challenges limiting.

 

It is heartening that around a third of respondents are rarely if ever impeded by chemistry, perhaps because they have intentionally constructed their libraries with sociable fragments. Still, the fact that nearly two thirds of researchers sometimes or often run into problems suggests a continuing need for synthetically enabling fragment chemistry.

 

For academic chemists looking to make a practical impact, this could be a fertile area. And fragment library vendors looking to differentiate themselves may want to consider providing robust synthetic routes and methodologies for their available compounds.