Halogenation of a capsaicin analogue leads to novel vanilloid TRPV1 receptor antagonists

Abstract

1. The C-5 halogenation of the vanillyl moiety of resiniferatoxin, an ultrapotent agonist of vanilloid TRPV1 receptors, results in a potent antagonist for these receptors. Here, we have synthesized a series of halogenated derivatives of 'synthetic capsaicin' (nonanoyl vanillamide=nordihydrocapsaicin) differing for the nature (iodine, bromine-chlorine) and the regiochemistry (C-5, C-6) of the halogenation. 2. The activity of these compounds was investigated on recombinant human TRPV1 receptors overexpressed in HEK-293 cells. None of the six compounds exerted any significant agonist activity, as assessed by measuring their effect on TRPV1-mediated calcium mobilization. Instead, all compounds antagonized, to various extents, the effect of capsaicin in this assay. 3. All 6-halo-nordihydrocapsaicins behaved as competitive antagonists against human TRPV1 according to the corresponding Schild's plots, and were more potent than the corresponding 5-halogenated analogues. The iodo-derivatives were more potent than the bromo- and chloro-derivatives. 4. Using human recombinant TRPV1, 6-iodo-nordihydrocapsaicin (IC(50)=10 nM against 100 nM capsaicin) was about four times more potent than the prototypical TRPV1 antagonist, capsazepine, and was tested against capsaicin also on native TRPV1 in: (i) rat dorsal root ganglion neurons in culture; (ii) guinea-pig urinary bladder; and (iii) guinea-pig bronchi. In all cases, except for the guinea-pig bronchi, the compound was significantly more potent than capsazepine as a TRPV1 antagonist. 5. In conclusion, 6-iodo-nordihydrocapsaicin, a stable and easily prepared compound, is a potent TRPV1 antagonist and a convenient replacement for capsazepine in most of the in vitro preparations currently used to assess the activity of putative vanilloid receptor agonists.

Figure 1

General chemical structure of the compounds synthesized and investigated in this study.

Figure 2

Dose-dependent inhibition by the six novel compounds synthesized here on the effect of 100 nM capsaicin on [Ca²⁺]_i in HEK-293 cells overexpressing the human TRPV1 receptor. The effect was expressed as percent of the maximum possible enhancement of [Ca²⁺]_i induced by 4 μM ionomycin. Data are means of at least n=3 separate experiments. standard error. Bars are not shown for the sake of clarity and were never higher than 10% of the means. CPS=nordihydrocapsaicin.

Figure 3

Antagonistic effect of the 6-halo-nordihydrocapsaicins on the dose-dependent effect of capsaicin on [Ca²⁺]_i in HEK-293 cells overexpressing the human TRPV1 receptor. The effect was expressed as percent of the maximum possible enhancement of [Ca²⁺]_i induced by 4 μM ionomycin. Data are means of at least n=3 separate experiments. standard error. Bars are not shown for the sake of clarity and were never higher than 10% of the means. CPS=nordihydrocapsaicin.

Figure 4

Dose-dependent antagonistic effect of 6-iodo-nordihydrocapsaicin on the effect of 100 nM capsaicin on [Ca²⁺]_i in neonatal rat DRG neurons in culture. The effect was assessed by the calcium-imaging technique described in the Methods section. Data are means±s.e. of n=6 separate determinations.

Figure 5

Effect of two concentrations of 6-iodo-nordihydrocapsaicin on the dose-dependent effects of capsaicin (a) or substance P (b) on guinea-pig urinary bladder contractions. The effect was expressed as percent of the maximal contraction induced by 1 μM CCh. Data are means±s.e. of n⩾4 experiments.

Figure 6

Effect of two concentrations of 6-iodo-nordihydrocapsaicin on the dose-dependent effect of capsaicin on guinea-pig trachea contractions. The effect was expressed as percent of the maximal contraction induced by 1 μM CCh. Data are means±s.e. of n⩾4 experiments.