C-H Functionalization-Enabled 11-Step Semisynthesis of (-)-Veragranine A and Characterization of Synthetic Analogs in Osteoarthritis-related Pain Treatment.

We report an efficient semisynthesis of the cholestane steroidal alkaloid (-)-veragranine A with a 6/6/6/5/6/6 hexacyclic ring system, eight stereocenters, and a unique C12-C23 linkage. Our synthesis features a Schönecker-Baran C-H oxidation at C12, a Suzuki-Miyaura cross-coupling to form the C12-C23 bond, and a hydrogen atom transfer (HAT)-initiated Minisci C-H cyclization to forge the C20-C22 bond with desired stereochemistry at C20. These enabling transformations significantly enhanced the overall synthetic efficiency and delivered (-)-veragranine A in 11 steps and over 200 mg from cheap and readily available dehydroepiandrosterone. In addition, this approach allowed flexible syntheses of novel synthetic analogs for biological evaluations in sensory neurons in vitro and in an in vivo model of arthritic pain, from which two novel lead compounds were identified for further development.

Betulinic acid analogs inhibit N- and T-type voltage-gated calcium channels to attenuate nerve-injury associated neuropathic and formalin models of pain.

Over the past three decades, there has been a significant growth in the use of natural products, with approximately 80% of individuals using them for some aspect of primary healthcare. Our laboratories have identified and studied natural compounds with analgesic effects from dry land plants or their associated fungus during the past ten years. Here, we isolated and characterized thirteen betulin analogs and fifteen betulinic acid analogs for their capacity to prevent calcium influx brought on by depolarization in sensory neurons. The in vitro inhibition of voltage-gated calcium channels by the top drugs was then assessed using whole cell patch clamp electrophysiology. In vivo experiments, conducted at two sites, evaluated the best compound in acute and tonic, neuropathic, inflammatory, post-operative and visceral models of pain. We found that the betulinic acid analog 8 inhibited calcium influx in rat dorsal root ganglion neurons by inhibiting N- (CaV2.2) and T- (CaV3) type voltage-gated calcium channels. Moreover, intrathecal delivery of analog 8 had analgesic activity in both spared nerve injury model of neuropathic pain and acute and tonic pain induced by formalin. The results presented herein highlight the potential antinociceptive properties of betulinic acid analog 8 and set the stage for the development of novel non-opioid pain therapeutics based on the triterpenoid scaffold of betulinic acid.

The natural product argentatin C attenuates postoperative pain via inhibition of voltage-gated sodium and T-type voltage-gated calcium channels.

BACKGROUND AND PURPOSE: Postoperative pain occurs in as many as 70% of surgeries performed worldwide. Postoperative pain management still relies on opioids despite their negative consequences, resulting in a public health crisis. Therefore, it is important to develop alternative therapies to treat chronic pain. Natural products derived from medicinal plants are potential sources of novel biologically active compounds for development of safe analgesics. In this study, we screened a library of natural products to identify small molecules that target the activity of voltage-gated sodium and calcium channels that have important roles in nociceptive sensory processing. EXPERIMENTAL APPROACH: Fractions derived from the Native American medicinal plant, Parthenium incanum, were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion (DRG) neurons. Further separation of these fractions yielded a cycloartane-type triterpene identified as argentatin C, which was additionally evaluated using whole-cell voltage and current-clamp electrophysiology, and behavioural analysis in a mouse model of postsurgical pain. KEY RESULTS: Argentatin C blocked the activity of both voltage-gated sodium and low-voltage-activated (LVA) calcium channels in calcium imaging assays. Docking analysis predicted that argentatin C may bind to NaV 1.7-1.9 and CaV 3.1-3.3 channels. Furthermore, argentatin C decreased Na+ and T-type Ca2+ currents as well as excitability in rat and macaque DRG neurons, and reversed mechanical allodynia in a mouse model of postsurgical pain. CONCLUSION AND IMPLICATIONS: These results suggest that the dual effect of argentatin C on voltage-gated sodium and calcium channels supports its potential as a novel treatment for painful conditions.

Stereospecific Effects of Benzimidazolonepiperidine Compounds on T-Type Ca2+ Channels and Pain.

T-type calcium channels activate in response to subthreshold membrane depolarizations and represent an important source of Ca2+ influx near the resting membrane potential. These channels regulate neuronal excitability and have been linked to pain. For this reason, T-type calcium channels are suitable molecular targets for the development of new non-opioid analgesics. Our previous work identified an analogue of benzimidazolonepiperidine, 5bk, that preferentially inhibited CaV3.2 channels and reversed mechanical allodynia. In this study, we synthesized and screened a small library of 47 compounds derived from 5bk. We found several compounds that inhibited the Ca2+ influx in DRG neurons of all sizes. After separating the enantiomers of each active compound, we found two compounds, 3-25-R and 3-14-3-S, that potently inhibited the Ca2+ influx. Whole-cell patch clamp recordings from small- to medium-sized DRG neurons revealed that both compounds decreased total Ca2+. Application of 3-14-3-S (but not 3-25-R) blocked transiently expressed CaV3.1-3.3 channels with a similar IC50 value. 3-14-3-S decreased T-type, but not N-type, Ca2+ currents in DRG neurons. Furthermore, intrathecal delivery of 3-14-3-S relieved tonic, neuropathic, and inflammatory pain in preclinical models. 3-14-3-S did not exhibit any activity against G protein-coupled opioid receptors. Preliminary docking studies also suggest that 3-14-3-S can bind to the central pore domain of T-type channels. Together, our chemical characterization and functional and behavioral data identify a novel T-type calcium channel blocker with in vivo efficacy in experimental models of tonic, neuropathic, and inflammatory pain.