Fragments vs JAK1, a sequel from LEO

Four years ago we highlighted a paper from LEO Pharma describing inhibitors of the kinase JAK1, which is implicated in a host of inflammatory conditions. Although they developed low nanomolar inhibitors, these showed phototoxicity, which was unacceptable for the topical applications the researchers had in mind. The molecules were also not selective against closely related JAK2, whose inhibition can cause neutropenia and anemia. A recent paper in J. Med. Chem from Andrea Ritźen and collaborators at LEO and GVK Biosciences describes a more selective series.

As mentioned previously, hits came from about 500 fragments screened against JAK2 using SPR and validated in a biochemical assay against JAK1. Most fragments had similar activities against both proteins, but compound 1 was moderately selective for the latter. Initial SAR around the fragment revealed that the methyl group was essential to activity and that methylating the pyrazole nitrogen atoms also obliterated binding. The molecule looks like a hinge-binder, but because it can assume four different tautomers docking was difficult. Fortunately, replacing the difluoromethyl substituent with a phenyl ring in compound 6 improved affinity and led to a crystal structure, which showed the methyl group making lipophilic interactions with the protein.

The crystal structure also revealed that the phenyl ring didn’t quite fill the lipophilic ribose-binding pocket, so the researchers replaced this with the more three-dimensional cyclohexyl substituent in compound 7, which yielded a ten-fold improvement in biochemical potency as well as the first cellular activity. The philosophy behind further optimization is described eloquently: “in the spirit of fragment-based drug design – start small and make every added atom count – small substituents with balanced polarity were added to the cyclohexyl analogue 7.” Unfortunately, although several polar substituents introduced onto the cyclohexyl ring improved biochemical potency, they did not do much for cell activity.

Replacing the cyclohexane moiety of compound 7 with a norbornane led to compound 11, which was not only more potent against JAK1 but also less lipophilic and more soluble. The researchers then borrowed a nitrile from an approved pan-JAK inhibitor, leading to compound 40. This molecule has low nanomolar activity against JAK1 and is somewhat selective against closely related JAK2, JAK3, and TYK2. It is quite selective against a panel of 50 other kinases and does not inhibit several cytochrome P450 enzymes or bind to hERG. Oral bioavailability in rats is fairly low, but this should not be a problem for topical indications.

As noted in the paper, the researchers were able to benefit from published work from multiple other companies that has led to five approved JAK inhibitors plus several more in clinical development. While another JAK inhibitor may not be the most pressing medical need, this paper is still a nice example of structure-based design that illustrates several points. First, ligand efficiency was improved during the optimization process, in contrast to common perceptions. Second, fragment selectivity was also improved during optimization. And finally, although this sounds banal, it matters what you have in your fragment library: had the des-methyl version of compound 1 been the only representative of this core in the LEO library the researchers would not have discovered it. In other words, while simplifying your fragments will decrease molecular complexity, sometimes a single methyl group can make all the difference.