Zsuzsanna Riedl

Actions of 3-methyl-N-oleoyldopamine, 4-methyl-N-oleoyldopamine and N-oleoylethanolamide on the rat TRPV1 receptor in vitro and in vivo.

N-oleoyldopamine (OLDA) has been identified as an agonist of the transient receptor potential vanilloid type 1 (TRPV1) receptor. A related fatty acid amide, N-oleoylethanolamide (OEA), was found to excite sensory neurons and produce visceral hyperalgesia via activation of the TRPV1 receptor, however, a recent study described this agent as an antinociceptive one. The aim of the present paper was to characterize two newly synthesized derivatives of N-oleoyldopamine, 3-methyl-N-oleoyldopamine (3-MOLDA) and 4-methyl-N-oleoyldopamine (4-MOLDA) as well as OEA with regard to their effects on the TRPV1 receptor. Radioactive 45Ca2+ uptake was measured in HT5-1 cells transfected with the rat TRPV1 receptor and intracellular Ca2+ concentration was monitored by fura-2 microfluorimetry in cultured trigeminal sensory neurons. Thermonociception was assessed by determining the behavioral noxious heat threshold in rats. 3-MOLDA induced 45Ca2+ uptake in a concentration-dependent manner, whereas 4-MOLDA and OEA were without effect. 4-MOLDA and OEA, however, concentration-dependently reduced the 45Ca2+ uptake-inducing effect of capsaicin. In trigeminal sensory neurons, 3-MOLDA caused an increase in intracellular Ca2+ concentration and this effect exhibited tachyphylaxis upon repeated application. Again, 4-MOLDA and OEA failed to alter intracellular Ca2+ levels. Upon intraplantar injection, 3-MOLDA caused an 8-10 degrees C drop of the noxious heat threshold in rats which was inhibited by the TRPV1 receptor antagonist iodo-resiniferatoxin. 4-MOLDA and OEA failed to alter the heat threshold but inhibited the threshold drop induced by the TRPV1 receptor agonist resiniferatoxin. These data show that 3-MOLDA behaves as an agonist, whereas 4-MOLDA and OEA appear to be antagonists, at the rat TRPV1 receptor.

Structure–activity relationship of antiparasitic and cytotoxic indoloquinoline alkaloids, and their tricyclic and bicyclic analogues

Based on the indoloquinoline alkaloids cryptolepine (1), neocryptolepine (2), isocryptolepine (3) and isoneocryptolepine (4), used as lead compounds for new antimalarial agents, a series of tricyclic and bicyclic analogues, including carbolines, azaindoles, pyrroloquinolines and pyrroloisoquinolines was synthesized and biologically evaluated. None of the bicyclic compounds was significantly active against the chloroquine-resistant strain Plasmodium falciparum K1, in contrast to the tricyclic derivatives. The tricyclic compound 2-methyl-2H-pyrido[3,4-b]indole (9), or 2-methyl-beta-carboline, showed the best in vitro activity, with an IC(50) value of 0.45 microM against P. falciparum K1, without apparent cytotoxicity against L6 cells (SI>1000). However, this compound was not active in the Plasmodium berghei mouse model. Structure-activity relationships are discussed and compared with related naturally occurring compounds.

Direct evidence for activation and desensitization of the capsaicin receptor by N-oleoyldopamine on TRPV1-transfected cell, line in gene deleted mice and in the rat

Effects of the endogenous lipid N-oleoyldopamine (OLDA) were analyzed on the rTRPV1-expressing HT1080 human fibrosarcoma cell line (HT5-1), on cultured rat trigeminal neurons, on the noxious heat threshold of rats and on nocifensive behavior of TRPV1 knockout mice. The EC(50) of capsaicin and OLDA on (45)Ca accumulation of rTRPV1-expressing HT5-1 cells was 36 nM and 1.8 microM, respectively. The efficacy of OLDA was 60% as compared to the maximum response of capsaicin. OLDA (330 nM to 3.3 microM) caused a transient increase in fluorescence of fura-2 loaded cultured small trigeminal neurons of the rat and rTRPV1-transfected HT5-1 cells measured with a ratiometric technique. Repeated application of OLDA and capsaicin caused similar desensitization in the Ca(2+) transients both in cultured neurons and rTRPV1-transfected HT5-1 cells. In the rat intraplantar injection of OLDA (5 nmol) decreased the noxious heat threshold by 6-9 degrees C and this response was strongly inhibited by the TRPV1 antagonist iodoresiniferatoxin (0.05 nmol intraplantarly (i.pl.)). In wild-type mice OLDA (50 nmol i.pl.) evoked paw lifting/licking which was significantly less sustained in TRPV1 knockout mice. It is concluded that on TRPV1 capsaicin receptors OLDA is 50 times less potent than capsaicin and it might serve as an endogenous ligand for TRPV1 in the rat, but more likely in humans.

Synthesis of some diazino-fused tricyclic systems via Suzuki cross-coupling and regioselective nitrene insertion reactions

Suzuki coupling of 5-chloro-2-methyl-6-phenylpyridazin-3(2H)-one, 6-chloro-1,3-dimethyluracil and 2-chloropyrazine with protected aminoaryl boronic acids resulted in the corresponding pivaloylaminophenyl diazines which were transformed to diazino-fused indole and cinnoline derivatives. Suzuki coupling of 5-amino-6-chloro-1,3-dimethyluracil with 2-formylphenyl boronic acid afforded a novel pyrimidoisoquinoline ring system in a one-pot reaction.

Recent advances in ring transformations of five-membered heterocycles and their fused derivatives

Ring transformations of five-membered heterocycles and their benzologues for the recent 10 years are discussed and reviewed. These transformations are classified into four groups: (a) “classical ring transformations”, where the starting and resulting ring system is of the same size, but the heteroatoms and/or their positions have been changed; (b) “degenerate ring transformations”, where during the course of the transformation the starting compound and product have the same ring system, but the reaction proceeds by a ring opening and subsequent ring closure process; (c) “ring contractions” and “ring enlargements”, where the sizes of the product rings are smaller or larger, respectively, than those of the starting compound; and (d) “pseudo ring transformation” or “ring-chain transfer”, where the process is formally a ring transformation, but is realised by a ring closure of a side chain of the starting heterocycle and opening of the original ring. Reaction mechanisms of the most interesting ring transformations are also discussed.

Synthesis of new pyridazino[4,5-c]isoquinolinones by Suzuki cross-coupling reaction

Suzuki cross-coupling reaction of 2-alkyl(methyl and benzyl)-5-chloro-4-methoxy- and 2-alkyl(methyl and benzyl)-4-chloro-5-methoxypyridazin-3(2H)-ones with 2-formylphenylboronic acid afforded the corresponding biaryl products which were cyclized with ammonia to yield hitherto undescribed pyridazino[4,5-c]isoquinolinones. Removal of the N-benzyl protective group in position 2 yielded the unsubstituted tricyclic pyridazinones.

Synthesis of 4-aryl-5-hydroxy- and 5-aryl-4-hydroxypyridazin-3(2H)-ones and their use in the preparation of 4,5-diarylpyridazin-3(2H)-ones and hitherto unknown isochromeno[3,4-d]pyridazinediones

Abstract Easily accessible 2-substituted 4-aryl-5-methoxy- and 2-substituted 5-aryl-4-methoxypyridazin-3(2 H )-ones were transformed into the corresponding aryl-hydroxypyridazin-3(2 H )-ones by alkaline hydrolysis. The use of these compounds in the synthesis of 2-substituted 4,5-diarylpyridazin-3(2 H )-ones with two differently substituted aryl groups was investigated. Two aryl-hydroxypyridazin-3(2 H )-ones, 2-(2-benzyl-5-hydroxy-3-oxo-2,3-dihydropyridazin-4-yl)benzaldehyde and 2-(1-benzyl-5-hydroxy-6-oxo-1,6-dihydropyridazin-4-yl)benzaldehyde, were transformed into 2-benzyl-1 H -isochromeno[3,4- d ]pyridazine-1,6(2 H )-dione and 3-benzyl-3 H -isochromeno[3,4- d ]pyridazine-4,6-dione, respectively, via oxidation of the formyl group with KMnO 4 followed by lactonization.

Designed azolopyridinium salts block protective antigen pores in vitro and protect cells from anthrax toxin.

Background Several intracellular acting bacterial protein toxins of the AB-type, which are known to enter cells by endocytosis, are shown to produce channels. This holds true for protective antigen (PA), the binding component of the tripartite anthrax-toxin of Bacillus anthracis. Evidence has been presented that translocation of the enzymatic components of anthrax-toxin across the endosomal membrane of target cells and channel formation by the heptameric/octameric PA63 binding/translocation component are related phenomena. Chloroquine and some 4-aminoquinolones, known as potent drugs against Plasmodium falciparium infection of humans, block efficiently the PA63-channel in a dose dependent way. Methodology/Principal Findings Here we demonstrate that related positively charged heterocyclic azolopyridinium salts block the PA63-channel in the µM range, when both, inhibitor and PA63 are added to the same side of the membrane, the cis-side, which corresponds to the lumen of acidified endosomal vesicles of target cells. Noise-analysis allowed the study of the kinetics of the plug formation by the heterocycles. In vivo experiments using J774A.1 macrophages demonstrated that the inhibitors of PA63-channel function also efficiently block intoxication of the cells by the combination lethal factor and PA63 in the same concentration range as they block the channels in vitro. Conclusions/Significance These results strongly argue in favor of a transport of lethal factor through the PA63-channel and suggest that the heterocycles used in this study could represent attractive candidates for development of novel therapeutic strategies against anthrax.

Synthesis of the dibenzo[f,h]phthalazine and dibenzo[f,h]cinnoline skeleton via a 'Suzuki-Pd-catalyzed intramolecular arylation' and a 'Suzuki-Pschorr' approach

Abstract Palladium-catalyzed intramolecular arylation of 2-benzyl-5-(2-bromophenyl)-4-phenylpyridazin-3(2 H )-one yielded hitherto unknown 2-benzyldibenzo[f,h]phthalazin-1(2 H )-one. The synthesis of this new tetracyclic pyridazinone from 2-benzyl-5-(2-aminophenyl)-4-phenylpyridazin-3(2 H )-one via a Pschorr type reaction was also investigated. Similarly, the construction of 2-methyldibenzo[f,h]cinnolin-3(2 H )-one from 2-methyl-5-(2-bromophenyl)-6-phenylpyridazin-3(2 H )-one and 2-methyl-5-(2-aminophenyl)-6-phenylpyridazin-3(2 H )-one is also reported. Removal of the N -benzyl protective group of 2-benzyl-dibenzo[f,h]phthalazin-1(2 H )-one with AlCl 3 yielded unsubstituted dibenzo[f,h]phthalazin-1(2 H )-one.

Inhibitions of the translocation pore of Clostridium botulinum C2 toxin by tailored azolopyridinium salts protects human cells from intoxication

C2 toxin from Clostridium botulinum represents the prototype of clostridial binary actin ADP-ribosylating toxins which destroy the actin-cytoskeleton of mammalian cells and cause severe enteric diseases in humans and animals. After receptor-mediated endocytosis of the C2 toxin complex, the binding/translocation component C2IIa forms a heptameric transmembrane pore in membranes of acidified endosomal vesicles. The separate ADP-ribosyltransferase component C2I translocates through this C2IIa-pore from the endosomal lumen into the cytosol. Here we demonstrate that positively charged heterocyclic azolopyridinium salts which were developed as pore blockers for the anthrax toxins, efficiently protect cultured mammalian cells from intoxication with C2 toxin. The inhibitors had no effects on enzyme activity of C2I or receptor binding of C2 toxin but inhibited the pH-dependent membrane translocation of C2I in living cells, most likely by blocking the C2IIa-translocation pores. In vitro, the substances blocked C2IIa-pores in black lipid bilayer membranes when applied to the cis-side of the membrane which corresponds to the endosomal lumen of cells. Thus, heterocyclic azolopyridinium salts could represent lead compounds for development of novel therapeutics against binary clostridial toxins.