Edson F. da Silva

Antimelanoma activity of 1,3,4-thiadiazolium mesoionics: a structure-activity relationship study.

The effect of a series of 4-phenyl-5-(2′-Y, 4′-X or 4′-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chlorides was evaluated against B16-F10 murine melanoma cells in vitro and against tumors resulting from implanted B16-F10 cells in C57BL/6 mice. These compounds differ from each other only at the cinnamoyl ring substituent (MI-J, X=OH; MI-2,4diF, X=Y=F; MI-4F, X=F and MI-D, X=NO2). The results were compared with those obtained for MI-D, which has already been shown to be a potent and promising drug against melanoma. On exposure of B16-F10 cells to MI-D, MI-2,4diF and MI-4F, all of them at the same micromolar concentration (50 μM) decreased the cell viability to 8, 50 and 22%, respectively, while MI-J did not show any significant effect under the same conditions. However, low doses such as 10 μM MI-D were sufficient to impair cell growth over 72 h, but for MI-2,4diF and MI-4F the effect on B16-F10 proliferation was only observed at a concentration of 25 μM. Furthermore, MI-4F had a slightly better effect than MI-2,4diF in vitro; its effect on tumor growth in vivo was not significant. MI-D inhibited tumor growth by 77%. The greater effectiveness of MI-D compared with MI-2,4diF, MI-4F and MI-J against B16-F10 melanoma cells is probably due to its stronger electron-withdrawing group (NO2), which increases the positive charge on the mesoionic ring and allows extensive conjugation of the side-chain with the exocyclic moiety. This seems to be important for degree of anti-tumor activity of these compounds.

Cytotoxic effect of a new 1,3,4-thiadiazolium mesoionic compound (MI-D) on cell lines of human melanoma.

The structural characteristics of mesoionic compounds, which contain distinct regions of positive and negative charges associated with a poly-heteroatomic system, enable them to cross cellular membranes and interact strongly with biomolecules. Potential biological applications have been described for mesoionic compounds. 1,3,4-Thiadiazolium mesoionic compound (MI-D), a new mesoionic compound, has been demonstrated to be extremely cytotoxic to B16-F10 murine melanoma cells when compared to fotemustine and dacarbazine, drugs of reference in melanoma treatment protocols, describing inhibition of tumours grown in vitro and in vivo. We now evaluate the effects of mesoionic compound MI-D on different human melanoma cell lines. The drug decreased the viability and proliferation of MEL-85, SK-MEL, A2058 and MEWO cell lines in vitro, showing a considerable cytotoxic activity on these human cells. Adhesion of MEL-85 cells was evaluated in the presence of the drug using different extracellular matrix (ECM) constituents. MI-D decreased MEL-85 adhesion to laminin, fibronectin and matrigel. The morphology and actin cytoskeleton organisation of MEL-85 cells were also modified on MI-D treatment. These results on human melanoma cell lines indicate that MI-D is a very encouraging drug against melanoma, a tumour that is extremely resistant to chemotherapy.

The inhibition of lipoperoxidation by mesoionic compound MI-D: A relationship with its uncoupling effect and scavenging activity

Important biological activities have been described for mesoionic compounds. We previously reported that MI-D (4-phenyl-5-(4-nitro-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride) inhibited the respiratory chain, collapsed the transmembrane potential, and stimulated ATPase activity in intact rat liver mitochondria. It is known that drugs that affect mitochondrial membrane potential may facilitate the induction of cell death by apoptosis. Mitochondria have also a central role in the generation of reactive oxygen species, therefore it would be important to investigate how MI-D could affect processes related to oxidative stress. In this work, we evaluated the effects of MI-D on the lipoperoxidation and its ability to scavenge free radicals. Interestingly, it was observed that MI-D promoted a strong inhibition of the lipoperoxidation induced by Fe(3+)-ADP/2-oxoglutarate in isolated mitochondria (95%+/-0.27 at the highest concentration of 80 nmol mg(-1) protein) in a dose-dependent manner. However, at the same concentration its effect was less intense (22%+/-3.46) when the lipoperoxidation was initiated by peroxyl radicals generated from the azocompound AAPH. Lipid peroxidation in both coupled and uncoupled submitochondrial particles initiated with Fe(2+)/NADH was also inhibited by MI-D. The inhibition was about four times greater in coupled particles (approximately 34% at 80 nmol mg(-1) protein) in relation to uncoupled. MI-D inhibited the soybean phosphatidylcholine liposomes lipoperoxidation in a dose-dependent manner (5-80 microM) regardless of the radical being generated in lipid or aqueous phase. The mesoionic compound showed ability of scavenging superoxide radical (7, 11 and 31% for 25, 38 and 80 microM, respectively). Our results strongly suggest that the inhibition of lipoperoxidation promoted by MI-D is due to its scavenger action and to its previously described uncoupling effect.

Antileishmanial Activity of 1,3,4-Thiadiazolium-2-Aminide in Mice Infected with Leishmania amazonensis

ABSTRACT The efficacy of two mesoionic derivatives (MI-H-H and MI-4-OCH3) was evaluated in CBA/J mice infected with Leishmania amazonensis. Treatment with these compounds demonstrated that the MI-4-OCH3 derivative and the reference drug meglumine antimoniate (Glucantime) presented significant activity relative to an untreated control. No apparent hepatic or renal toxicity due to these mesoionic compounds was found.

Effect of mesoionic 4-phenyl-5-(cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivative salts on the activities of the nitric oxide synthase and arginase of Leishmania amazonensis

l-arginine is involved in the production of both nitric oxide (NO), mediated by nitric oxide synthase (NOS) and l-ornithine, by arginase activity. It is generally accepted that NO regulation occurs mainly at the transcriptional level of NOS. In a previous work we purported that there is evidence that Leishmania sp. can produce NO from l-arginine. An arginase activity in its gene sequence has also been reported in Leishmania parasites. In a search for intracellular targets as potential antileishmanicidal agents, such as the l-arginine metabolism, we used 1,3,4-thiadiazolium mesoionic compounds, that have been demonstrated to be cytotoxic to the Leishmania amazonensis, when compared to Pentamidine isethionate as a reference drug. Parasites were assayed in absence/presence of 4′- and 3′- methoxy mesoionic derivatives in order to verify the effect on NO production and arginase activity in L. amazonensis. The results indicated that the drugs reduce from 70 to 90% of the NO production by the parasite and act on a soluble nitric oxide synthase purified from L. amazonensis promastigotes and axenic amastigotes.

Novos derivados do sistema heterocíclico 1H-pirazolo[3,4-b]piridina: síntese e assinalamentos de hidrogênios e carbonos por RMN 1D e 2D

The synthesis and NMR analysis of seven new 4-(aryl)amino-5-carboethoxy-1,3-dimethyl-1H-pyrazolo[3,4- b]pyridines (7-13) are described. The synthetic approach used involved the preparation of intermediates 5-aminopyrazol (4), the enamine derivative (5) and the 4-chloro-1H-pyrazolo[3,4-b]pyridine (6). Compounds (7-13) were obtained by treatment of 6 with the desired aniline. The structures of new heterocyclic compounds and their precursors intermediates were assigned on the basis of spectral analysis including 1D and 2D NMR experiments [1H; 13C{1H} and DEPT; 1H x 1H - COSY; 1H x 13C - COSY, nJCH, n = 1, 2 or 3 (HETECOR and COLOC)].

Rubrofusarina, um policetídeo natural inibidor da topoisomerase II-α humana

This work describes the inhibitory effect of rubrofusarin (5,6-dihydroxy-8-methoxy-2-methylbenzo[g]cromen-4-one, 1) against human DNA topoisomerase II-α. The results for relaxation assays showed total inhibition of topisomerase II-α by rubrofusarin at 120 µM. This result is comparable to the one observed with etoposide as positive control. For this study, rubrofusarin was isolated from Senna macranthera var. nervosa (Voguel) Irwin & Barnebyem and characterized by spectral data, including 2D NMR, as well as its acetylated derivative.

Metabolism of the mesoionic compound (MI-D) by mouse liver microsome, detection of its metabolite in vivo, and acute toxicity in mice.

The mesoionic derivative 4‐phenyl‐5‐[4‐nitrocinnamoyl]‐1,3,4‐thiadiazolyl‐2‐phenylamine chloride (MI‐D) has antitumoral and anti‐inflammatory effects. In this study, we present aspects of its metabolism and toxicity in mice. MI‐D was metabolized in vitro by liver microsome, generating a main product with a much shorter retention time than MI‐D in high‐performance liquid chromatography (HPLC) analysis but with a spectrum similar to that of the original molecule. Mass spectrometry with electrospray ionization in positive mode analysis of the purified compound by HPLC indicated that the product of metabolism has four additional hydroxyl groups (m/z = 465) compared with MI‐D (m/z = 401). The HPLC analyses of plasma and urine samples from mice treated with MI‐D showed the presence of the metabolite product. The kinetic parameters Km (19.5 ± 4.5 μM) and Vmax [1.5 ± 0.4 units of fluorescence/(100 μg of microsomal protein/mL/s)] were estimated, confirming the metabolism of MI‐D and indicating that the reaction follows Michaelis‐Menten kinetics. Acute toxicity was established on the basis of an estimation of mean lethal dose (LD‐50; 181.2 mg/kg) and histopathological analysis of animals that survived the LD‐50 test. Abdominal adhesions, inflammatory foci, and formation of granulomas were observed. Altogether, the results contribute to the advancement of research in support of MI‐D as a future chemotherapeutic drug.