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    • Having taken a step back from affinity to

    2024-01-15

    Having taken a step back from affinity to gain better exposure and solubility, we started work to replace the unstable oxazolinone, and address the selectivity over the hERG channel. Keeping in mind the binding mode of the series from crystal structures, triazole was identified as another mildly acidic zinc-binder. Compound 9 (Table 4) was prepared analogously to Scheme 3, coupling with 1H-1,2,3-triazole-4-carboxylic acid. Compound 11 was prepared using ‘Click’ chemistry (Scheme 4) which became a robust and efficient method to prepare the relevant triazole carboxylic acids for all subsequent compounds. Although 8c showed good oral exposure, this was dependent on high plasma protein binding, which varied across the series. Compound 9 lost affinity to ATX, but showed good permeability and improved microsomal stability. Both of these compounds exhibited similar inhibition of hERG in the automated patch clamp assay. In an attempt to disrupt this hERG activity, the amide bond was moved closer to the piperidine (Table 4, 11) to redistribute areas of polarity across the molecule. For 11, this approach had no significant effect on hERG activity, but showed a clear improvement in ATX inhibition. Attempts were then made to improve the poor permeability of 11 by lengthening the carbon chain to increase lipophilicity, or by removing a hydrogen bond donor via methylation, as had been shown with 8c. Compounds 12, 13 and 15 were synthesized analogously to 11, using the relevant acetylene acid, and for 13, the relevant 4-aminomethylpiperidine starting material. Compound 14 was formed as shown in Scheme 5. Whilst methylation of the amide nitrogen did indeed improve permeability (13, Table 5), ATX inhibition suffered. Similarly, methylation of the triazole in 14 reduced ATX inhibition considerably, reinforcing that the interaction of the acidic triazole with the catalytic zinc in ATX is key. Extension of the carbon chain by one (12) and two methylenes (15) maintained potency. Improvement of permeability for 15 was marginal, but a similar level of hERG inhibition was seen. For Caspase-1, human recombinant proteinase mg 12, however, the selectivity over hERG inhibition improved. Further exploration of the aryl substitution (Table 6) confirmed what had been found early on in the development of the series – that 3,5 substitution was optimal. No further improvement was seen in permeability, but hERG activity could be reduced to >30 μM by replacement of one of the chlorines with a cyano group (16d). However, this compound demonstrated even further reduced permeability and was subsequently shown to have poor oral exposure. At this point, despite lower permeability in the PAMPA assay, compound 12 was studied further and shown to have good oral exposure in the Sprague Dawley rat. Clearance and volume of distribution were comparable to 8c, leading to good bioavailability (Fig. 5). When tested in an in vitro serum assay, measuring 18:1 LPA levels by LCMS, 12 showed ATX inhibition of IC50 9 nM. The oral exposure levels and activity in the presence of serum led to 12 being studied in a PK/PD model, where inhibition of ATX was measured by LCMS detection of four isoforms of LPA – 16:0, 18:0, 18:1 and 20:4 (Fig. 6). When dosed at 0.3 mg/kg to Sprague Dawley rats, a robust drop in all four LPA levels was seen, the peak reduction of around 70–80% coinciding with the Cmax of 12. This inhibition of LPA production then decayed in line with the clearance of 12, showing the observed PK/PD relationship to be direct. This was simulated, using a turnover model to capture inhibition of the formation of each of the LPA isoforms described above: an IC50 of around 20 nM was determined for 12. In conclusion, a series of potent autotaxin inhibitors has been developed, following a similar binding motif to PF-8380. The benzoxazolone in this molecule was hypothesised to be causing poor solubility through high crystal lattice energy, and was replaced with smaller, monocyclic zinc-binding heterocycles, leading to lower affinity, but more soluble and better orally absorbed compounds. Subsequent optimisation led to compound 12, which shows good oral exposure, and a concentration dependent inhibition of formation of LPA in vivo. This compound has superior solubility and hERG selectivity to PF-8380, and was chosen for further development.