Bioactive Natural Product and Superacid Chemistry for Lead Compound Identification: A Case Study of Selective hCA III and L-Type Ca2+ Current Inhibitors for Hypotensive Agent Discovery.

Dodoneine (Ddn) is one of the active compounds identified from Agelanthusdodoneifolius, which is a medicinal plant used in African pharmacopeia and traditional medicine for the treatment of hypertension. In the context of a scientific program aiming at discovering new hypotensive agents through the original combination of natural product discovery and superacid chemistry diversification, and after evidencing dodoneine's vasorelaxant effect on rat aorta, superacid modifications allowed us to generate original analogues which showed selective human carbonic anhydrase III (hCA III) and L-type Ca2+ current inhibition. These derivatives can now be considered as new lead compounds for vasorelaxant therapeutics targeting these two proteins.

Superacid-Catalyzed Trifluoromethylthiolation of Aromatic Amines.

Upon activation under superacid conditions, functionalized tailor-made N-SCF3 sulfenamides served as reagents for the trifluoromethylthiolation of aromatic amines. This method has a broad substrate scope and can be used for the late-stage functionalization of complex molecules such as alkaloids or steroids. Mechanistic studies based on in situ low-temperature NMR spectroscopy revealed the involvement of dicationic superelectrophilic intermediates.

Vasorelaxation induced by dodoneine is mediated by calcium channels blockade and carbonic anhydrase inhibition on vascular smooth muscle cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Dodoneine (Ddn) is one of the active compounds identified from Agelanthus dodoneifolius (DC.) Polhill and Wiens, a medicinal plant used in traditional medicine for the treatment of hypertension. This dihydropyranone exerts hypotensive and vasorelaxant effects on rats, and two molecular targets have been characterized: the carbonic anhydrase and the L-type calcium channel in cardiomyocytes with biochemical and electrophysiological techniques, respectively. To further evaluate the involvement of these two molecular targets in vasorelaxation, the effect of Ddn on rat vascular smooth muscle was investigated. MATERIAL AND METHODS: The effects of Ddn on L-type calcium current and on resting membrane potential were characterized in A7r5 cell line using the whole-cell patch-clamp configuration. The molecular identities of carbonic anhydrase isozymes in smooth muscle cells were examined with RT-PCR. Vascular response was measured on rat aortic rings in an organ bath apparatus and the effect of Ddn on intracellular pH was determined by flow cytometry using the pH-sensitive fluorescent probe BCECF-AM [2,7-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester]. RESULTS: 100µM Ddn reduced calcium current density of about 30%. In addition, carbonic anhydrase II, III, XIII and XIV were shown to be expressed in rat aorta and inhibited in smooth muscle cells by Ddn. This inhibition resulted in a rise in pHi of about 0.31, leading to KCa channel activation, thereby inducing membrane hyperpolarization and vasorelaxation. The results of vascular reactivity experiments obtained with pharmacological tools acting on the L-type calcium current and carbonic anhydrase suggest that Ddn produces its vasorelaxant effect via the inhibition of these two molecular targets. CONCLUSION: This study demonstrates that Ddn induced vasorelaxation by targeting two proteins involved in the modulation of excitation-contraction coupling: L-type calcium channels and carbonic anhydrase.

The hypotensive agent dodoneine inhibits L-type Ca2+ current with negative inotropic effect on rat heart.

Agelanthus dodoneifolius is one of the medicinal plants used in African pharmacopeia and traditional medicine for the treatment of cardiovascular diseases. A chemical analysis has identified one of the active principles: Dodoneine (Ddn). It is a new dihydropyranone which exerts hypotensive and vasorelaxant effects on rat. Since the mechanism of the hypotensive effect is unknown, we performed a variety of preclinical and mechanistic studies to characterize the specific cardiac effect of Ddn at tissue (ex-vivo) and cellular levels (in-vitro) in order to determine a molecular target. Ddn effects were evaluated in an isolated rat heart preparation using Langendorff retrograde perfusion and then, the effects of Ddn were characterized in freshly dissociated cardiac ventricular myocytes using the whole-cell patch-clamp configuration. Ex-vivo, Ddn produced a dose-dependent negative inotropic effect with an IC50 value of 10 µM without changed heart rate. 100 µM Ddn decreased left ventricular developed pressure of about 40%. In isolated cardiac myocytes, Ddn reduced I(Ca),L density of about 30% with an IC50 value estimated at 3 µM. Ddn did not change current-voltage relation but it shifted the inactivation curve toward negative potentials and modified the half inactivation potentials. Furthermore, Ddn induced a phasic-dependent blocking on ICa,L. This study demonstrates that the hypotensive property of dodoneine is likely associated with a negative inotropic effect and the blockade of the L-type calcium channels.

Natural product hybrid and its superacid synthesized analogues: dodoneine and its derivatives show selective inhibition of carbonic anhydrase isoforms I, III, XIII and XIV.

The natural product dodoneine (a dihydropyranone phenolic compound), extracted from African mistletoe Agelanthus dodoneifolius, has been investigated as inhibitor of several human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms. By using superacid chemistry, analogues of the lactone phenolic hybrid lead compound have been synthesized and tested as CA inhibitors. Small chemical modifications of the basic scaffold revealed strong changes in the selectivity profile against different CA isoforms. These new compounds selectively inhibited isoforms CA I (K(I)s in the range of 0.13-0.76 µM), III (K(I)s in the range of 5.13-10.80 µM), XIII (K(I)s in the range of 0.34-0.96 µM) and XIV (K(I)s in the range of 2.44-7.24 µM), and can be considered as new leads, probably acting as non-zinc-binders, similar to other phenols/lactones investigated earlier.

On the elucidation of the mechanism of Vinca alkaloid fluorination in superacidic medium.

Detailed investigations on one of the key steps of the superacidic fluorination of Vinca alkaloids that is the origin of C20' activation are reported. While two different pathways can be envisioned for the emergence of the transient secondary carbocationic intermediate, isotopic labeling experiments unambiguously revealed the involvement of a 1,2-hydride shift mechanism.

Gem-difluorination of aminoalkynes via highly reactive dicationic species in superacid HF-SbF5: application to the efficient synthesis of difluorinated cinchona alkaloid derivatives.

A variety of alkynylated amines, amides, and imides are reacted in the superacid system HF-SbF5 to give regioselectively new beta-gem-difluoroamines. The reaction, which is not observed in pure HF, is consistent with the formation of a dicationic intermediate (i.e., both vinylic and adjacent protonated N-ammonium cations). Application to the regioselective and efficient synthesis of difluorinated cinchona alkaloid derivatives is described.

Structure elucidation of a dihydropyranone from Tapinanthus dodoneifolius.

A new dihydropyranone, ( R)-6-[( S)-2-hydroxy-4-(4-hydroxyphenyl)butyl]-5,6-dihydropyran-2-one ( 1), was isolated from Tapinanthus dodoneifolius. The structure was determined from spectroscopic and X-ray crystallographic analysis. Compound 1 (named dodoneine) showed a relaxing effect on preconstricted rat aortic rings (IC 50 of 81.4 +/- 0.9 microM).