McHardy M. Smith

We report the investigation of sulfonamide-derived Ca_v2.2 inhibitors to address drug-metabolism liabilities with this lead class of analgesics. Modification of the benzamide substituent provided improvements in both potency and selectivity. However, we discovered that formation of the persistent 3-(trifluoromethyl)benzenesulfonamide metabolite was an endemic problem in the sulfonamide series and that the replacement of the center aminopiperidine scaffold failed to prevent this metabolic pathway. This issue was eventually addressed by application of a bioisostere strategy. The new gem-dimethyl sulfone series retained Ca_v2.2 potency without the liability of the circulating sulfonamide metabolite.

The voltage-gated calcium channel Ca_v2.2 (N-type calcium channel) is a critical regulator of synaptic transmission and has emerged as an attractive target for the treatment of chronic pain. We report here the discovery of sulfonamide-derived, state-dependent inhibitors of Ca_v2.2. In particular, 19 is an inhibitor of Ca_v2.2 that is selective over cardiac ion channels, with a good preclinical PK and biodistribution profile. This compound exhibits dose-dependent efficacy in preclinical models of inflammatory hyperalgesia and neuropathic allodynia and is devoid of ancillary cardiovascular or CNS pharmacology at the doses tested. Importantly, 19 exhibited no efficacy in Ca_v2.2 gene-deleted mice. The discovery of metabolite 26 confounds further development of members of this aminopiperidine sulfonamide series. This discovery also suggests specific structural liabilities of this class of compounds that must be addressed.

Biological, genetic, and clinical evidence provide validation for N-type calcium channels (Ca_V2.2) as therapeutic targets for chronic pain. A state-dependent Ca_V2.2 inhibitor may provide an improved therapeutic window over ziconotide, the peptidyl Ca_V2.2 inhibitor used clinically. Supporting this notion, we recently reported that in preclinical models, the state-dependent Ca_V2 inhibitor (3R)-5-(3-chloro-4-fluorophenyl)-3-methyl-3-(pyrimidin-5-ylmethyl)-1-(1H-1,2,4-triazol-3-yl)-1,3-dihydro-2H-indol-2-one (TROX-1) has an improved therapeutic window compared with ziconotide. Here we characterize TROX-1 inhibition of Cav2.2 channels in more detail. When channels are biased toward open/inactivated states by depolarizing the membrane potential under voltage-clamp electrophysiology, TROX-1 inhibits Ca_V2.2 channels with an IC₅₀ of 0.11 μM. The voltage dependence of Ca_V2.2 inhibition was examined using automated electrophysiology. TROX-1 IC₅₀ values were 4.2, 0.90, and 0.36 μM at −110, −90, and −70 mV, respectively. TROX-1 displayed use-dependent inhibition of Ca_V2.2 with a 10-fold IC₅₀ separation between first (27 μM) and last (2.7 μM) pulses in a train. In a fluorescence-based calcium influx assay, TROX-1 inhibited Ca_V2.2 channels with an IC₅₀ of 9.5 μM under hyperpolarized conditions and 0.69 μM under depolarized conditions. Finally, TROX-1 potency was examined across the Ca_V2 subfamily. Depolarized IC₅₀ values were 0.29, 0.19, and 0.28 μM by manual electrophysiology using matched conditions and 1.8, 0.69, and 1.1 μM by calcium influx for Ca_V2.1, Ca_V2.2, and Ca_V2.3, respectively. Together, these in vitro data support the idea that a state-dependent, non–subtype-selective Ca_V2 channel inhibitor can achieve an improved therapeutic window over the relatively state-independent Ca_V2.2-selective inhibitor ziconotide in preclinical models of chronic pain.

Voltage-gated calcium channel (Ca_v)2.2 (N-type calcium channels) are key components in nociceptive transmission pathways. Ziconotide, a state-independent peptide inhibitor of Ca_v2.2 channels, is efficacious in treating refractory pain but exhibits a narrow therapeutic window and must be administered intrathecally. We have discovered an N-triazole oxindole, (3R)-5-(3-chloro-4-fluorophenyl)-3-methyl-3-(pyrimidin-5-ylmethyl)-1-(1H-1,2,4-triazol-3-yl)-1,3-dihydro-2H-indol-2-one (TROX-1), as a small-molecule, state-dependent blocker of Ca_v2 channels, and we investigated the therapeutic advantages of this compound for analgesia. TROX-1 preferentially inhibited potassium-triggered calcium influx through recombinant Ca_v2.2 channels under depolarized conditions (IC₅₀ = 0.27 μM) compared with hyperpolarized conditions (IC₅₀ > 20 μM). In rat dorsal root ganglion (DRG) neurons, TROX-1 inhibited ω-conotoxin GVIA-sensitive calcium currents (Ca_v2.2 channel currents), with greater potency under depolarized conditions (IC₅₀ = 0.4 μM) than under hyperpolarized conditions (IC₅₀ = 2.6 μM), indicating state-dependent Ca_v2.2 channel block of native as well as recombinant channels. TROX-1 fully blocked calcium influx mediated by a mixture of Ca_v2 channels in calcium imaging experiments in rat DRG neurons, indicating additional block of all Ca_v2 family channels. TROX-1 reversed inflammatory-induced hyperalgesia with maximal effects equivalent to nonsteroidal anti-inflammatory drugs, and it reversed nerve injury-induced allodynia to the same extent as pregabalin and duloxetine. In contrast, no significant reversal of hyperalgesia was observed in Ca_v2.2 gene-deleted mice. Mild impairment of motor function in the Rotarod test and cardiovascular functions were observed at 20- to 40-fold higher plasma concentrations than required for analgesic activities. TROX-1 demonstrates that an orally available state-dependent Ca_v2 channel blocker may achieve a therapeutic window suitable for the treatment of chronic pain.