Non-vanillyl resiniferatoxin analogues as potent and metabolically stable transient receptor potential vanilloid 1 agonists

Abstract

A series of non-vanillyl resiniferatoxin analogues, having 4-methylsulfonylaminophenyl and fluorophenyl moieties as vanillyl surrogates, have been investigated as ligands for rat TRPV1 heterologously expressed in Chinese hamster ovary cells. Although lacking the metabolically problematic 4-hydroxy substituent on the A-region phenyl ring, the compounds retained substantial agonist potency. Indeed, the 3-methoxy-4-methylsulfonylaminophenyl analog (1) was modestly (2.5-fold) more potent than RTX, with an EC(50)=0.106 nM. Further, it resembled RTX in its kinetics and pattern of stimulation of the levels of intracellular calcium in individual cells, as revealed by imaging. Compound 1 displayed modestly enhanced in vitro stability in rat liver microsomes and in plasma, suggesting that it might be a pharmacokinetically more favorable surrogate of resiniferatoxin. Molecular modeling analyses with selected analogues provide evidence that the conformational differences could affect their binding affinities, especially for the ester versus amide at the B-region.

Figure 1

Modulation of intracellular calcium levels in response to vanilloid treatment. Chinese hamster ovary cells heterologously expressing rat TRPV1 were preloaded with Fura-2 and treated with capsaicin, RTX, or compound 1 as indicated. The cells were imaged and the 340/380 fluorescence ratio of the Fura-2 was monitored to detect changes in intracellular calcium levels as described. Ligand concentrations, chosen to be approximately at their EC₅₀’s for TRPV1, were: capsaicin, 30 nM; RTX, 75 pM; compound 1, 100 pM. (a) In each panel, 20 cells responding to the stimuli are shown; each line represents the response of an individual cell. Data are from a single experiment representative of three independent experiments for capsaicin and RTX and of four experiments for compound 1. (b) The average percent of responding cells as a function of time. Values represent the mean data for 3 (capsaicin and RTX) or 4 (compound 1) experiments. Bars, SEM. The data for capsaicin and RTX are from experiments reported previously; the experiments with compound 1 were done in parallel.

Figure 2

(a) The comparison of in vitro metabolic stability for RTX derivatives in the presence of rat liver microsomes. The initial concentration was fixed at 10 μg/mL for the three derivatives. The vertical axis indicates the percent of the initial concentration remaining after incubation for 60 min with the microsomes. RTX, resiniferatoxin. Values represent the mean ± SEM of three replicates. **p < 0.01, from RTX value by the unpaired t-test. (b) Comparative metabolic stability of RTX derivatives in the presence of rat plasma. The initial concentration was fixed at 10 μg/mL for the three derivatives. The vertical axis indicates the percent of the initial concentration remaining after the 240 min of incubation. RTX, resiniferatoxin. Values represent the mean ± SEM of three replicates. **p < 0.01, from RTX value by the unpaired t-test.

Figure 3

The representative low energy conformers of RTX and its analogue 1. (a) The low energy conformers of RTX. Carbon atoms are shown in green colors. (b) Two major low energy conformers of compound 1, a sulfonamide analogue of RTX. The lowest energy conformer shows a bent conformation with a distinctive internal H-bonding. Carbon atoms are shown in magenta or purple. Non-polar hydrogens are undisplayed for clarity.

Figure 4

Low energy conformers of sulfonamide analogues of RTX, 3, and 14. (a) Lowest energy conformers, showing bent conformation due to internal H-bonding, (b) Extended conformers as alternative low energy conformations. Carbon atoms are shown in sky-blue and white for compounds 3 and 14, respectively. Non-polar hydrogens are undisplayed for clarity.

Figure 5

Low energy conformers of RTX and RTX-amide. The representative low energy conformers of RTX-amide analogue are converged on one bent conformer, resulting in the global minimum. Carbon atoms are displayed in green and brown for RTX and RTX-amide, respectively. Non-polar hydrogens are undisplayed for clarity.

Scheme 1

Synthesis of RTX analogues.

Scheme 2

Synthesis of RTX-amide analogue.