Developing piperine towards TRPV1 and GABAA receptor ligands--synthesis of piperine analogs via Heck-coupling of conjugated dienes.

Piperine, the pungent alkaloid of black pepper, and several of its derivatives are modulators of γ-amino butyric acid type A (GABAA) receptors. Concomitantly, this natural product has also been reported to activate transient receptor potential vanilloid type 1 (TRPV1) receptors. We have developed a Heck cross-coupling reaction of conjugated dienamides enabling the rapid assembly of piperine derivatives containing a modified aromatic core. Upon assessment of a focussed compound library, key aromatic substituents were identified selectively affecting either the GABAA or the TRPV1 receptor.

Identification of dihydrostilbenes in Pholidota chinensis as a new scaffold for GABAA receptor modulators.

A dichloromethane extract of stems and roots of Pholidota chinensis (Orchidaceae) enhanced GABA-induced chloride currents (I(GABA)) by 132.75 ± 36.69% when tested at 100 µg/mL in a two-microelectrode voltage clamp assay, on Xenopus laevis oocytes expressing recombinant α1ß2γ2S GABA(A) receptors. By means of an HPLC-based activity profiling approach, the three structurally related stilbenoids coelonin (1), batatasin III (2), and pholidotol D (3) were identified in the active fractions of the extract. Dihydrostilbene 2 enhanced I(GABA) by 1512.19 ± 176.47% at 300 µM, with an EC50 of 52.51 ± 16.96 µM, while compounds 1 and 3 showed much lower activity. The relevance of conformational flexibility for receptor modulation by stilbenoids was confirmed with a series of 13 commercially available stilbenes and their corresponding semisynthetic dihydro derivatives. Dihydrostilbenes showed higher activity in the oocyte assay than their corresponding stilbenes. The dihydro derivatives of tetramethoxy-piceatannol (12) and pterostilbene (20) were the most active among these derivatives, but they showed lower efficiencies than compound 2. Batatasin III (2) showed high efficiency but no significant subunit specificity when tested on the receptor subtypes α1ß2γ2s, α2ß2γ2s, α3ß2γ2s, α4ß2γ2s, α5ß2γ2s, α1ß1γ2s, and α1ß3γ2s. Dihydrostilbenes represent a new scaffold for GABA(A) receptor modulators.

Identification of dehydroabietc acid from Boswellia thurifera resin as a positive GABAA receptor modulator.

In a two-microelectrode voltage clamp assay with Xenopus laevis oocytes, a petroleum ether extract (100 µg/mL) of the resin of Boswellia thurifera (Burseraceae) potentiated GABA-induced chloride currents (IGABA) through receptors of the subtype α1ß2γ2s by 319.8% ± 79.8%. With the aid of HPLC-based activity profiling, three known terpenoids, dehydroabietic acid (1), incensole (2), and AKBA (3), were identified in the active fractions of the extract. Structure elucidation was achieved by means of HR-MS and microprobe 1D/2D NMR spectroscopy. Compound 1 induced significant receptor modulation in the oocyte assay, with a maximal potentiation of IGABA of 397.5% ± 34.0%, and EC50 of 8.7 µM ± 1.3 µM. This is the first report of dehydroabietic acid as a positive GABAA receptor modulator.

Efficient modulation of γ-aminobutyric acid type A receptors by piperine derivatives.

Piperine activates TRPV1 (transient receptor potential vanilloid type 1 receptor) receptors and modulates γ-aminobutyric acid type A receptors (GABAAR). We have synthesized a library of 76 piperine analogues and analyzed their effects on GABAAR by means of a two-microelectrode voltage-clamp technique. GABAAR were expressed in Xenopus laevis oocytes. Structure-activity relationships (SARs) were established to identify structural elements essential for efficiency and potency. Efficiency of piperine derivatives was significantly increased by exchanging the piperidine moiety with either N,N-dipropyl, N,N-diisopropyl, N,N-dibutyl, p-methylpiperidine, or N,N-bis(trifluoroethyl) groups. Potency was enhanced by replacing the piperidine moiety by N,N-dibutyl, N,N-diisobutyl, or N,N-bistrifluoroethyl groups. Linker modifications did not substantially enhance the effect on GABAAR. Compound 23 [(2E,4E)-5-(1,3-benzodioxol-5-yl)-N,N-dipropyl-2,4-pentadienamide] induced the strongest modulation of GABAA (maximal GABA-induced chloride current modulation (IGABA-max = 1673% ± 146%, EC50 = 51.7 ± 9.5 µM), while 25 [(2E,4E)-5-(1,3-benzodioxol-5-yl)-N,N-dibutyl-2,4-pentadienamide] displayed the highest potency (EC50 = 13.8 ± 1.8 µM, IGABA-max = 760% ± 47%). Compound 23 induced significantly stronger anxiolysis in mice than piperine and thus may serve as a starting point for developing novel GABAAR modulators.

GABAA receptor modulation by piperine and a non-TRPV1 activating derivative.

The action of piperine (the pungent component of pepper) and its derivative SCT-66 ((2E,4E)-5-(1,3-benzodioxol-5-yl))-N,N-diisobutyl-2,4-pentadienamide) on different gamma-aminobutyric acid (GABA) type A (GABA(A)) receptors, transient-receptor-potential-vanilloid-1 (TRPV1) receptors and behavioural effects were investigated. GABA(A) receptor subtypes and TRPV1 receptors were expressed in Xenopus laevis oocytes. Modulation of GABA-induced chloride currents (I(GABA)) by piperine and SCT-66 and activation of TRPV1 was studied using the two-microelectrode-voltage-clamp technique and fast perfusion. Their effects on explorative behaviour, thermoregulation and seizure threshold were analysed in mice. Piperine acted with similar potency on all GABA(A) receptor subtypes (EC50 range: 42.8±7.6 µM (α2ß2)-59.6±12.3 µM (α3ß2). I(GABA) modulation by piperine did not require the presence of a γ(2S)-subunit, suggesting a binding site involving only α and ß subunits. I(GABA) activation was slightly more efficacious on receptors formed from ß(2/3) subunits (maximal I(GABA) stimulation through α1ß3 receptors: 332±64% and α1ß2: 271±36% vs. α1ß1: 171±22%, p<0.05) and α3-subunits (α3ß2: 375±51% vs. α5ß2:136±22%, p<0.05). Replacing the piperidine ring by a N,N-diisobutyl residue (SCT-66) prevents interactions with TRPV1 and simultaneously increases the potency and efficiency of GABA(A) receptor modulation. SCT-66 displayed greater efficacy on GABA(A) receptors than piperine, with different subunit-dependence. Both compounds induced anxiolytic, anticonvulsant effects and reduced locomotor activity; however, SCT-66 induced stronger anxiolysis without decreasing body temperature and without the proconvulsive effects of TRPV1 activation and thus may serve as a scaffold for the development of novel GABA(A) receptor modulators.