Mauro Mazzei

Synthesis and biological evaluation of novel pyrazole derivatives with anticancer activity.

We synthesized thirty-six novel pyrazole derivatives and studied their antiproliferative activity in human ovarian adenocarcinoma A2780 cells, human lung carcinoma A549 cells, and murine P388 leukemia cells. Four of these substances were selected because of their higher antiproliferative activity and further analyses showed that they were all able to induce apoptosis, although to a different extent. The expression of p53 and p21(waf1), which induce apoptosis and cell cycle arrest, was evaluated by western blot analysis in cells treated with compound 12d. The analysis of the cell cycle showed that all the selected compounds cause a partial G2/M block and the formation of polyploid cells. Furthermore, the four selected compounds were tested for their interaction with the microtubular cytoskeletal system by docking analysis, tubulin polymerization assay and immunofluorescence staining, demonstrating that the compound 12d, unlike the other active derivatives, was able to significantly bind dimers of α- and β-tubulin, probably causing a molecular distortion resulting in the disassembly of microtubules.

Characterization and quantitation of the active polynucleotide fraction (PDRN) from human placenta, a tissue repair stimulating agent

The polydeoxyribonucleotide (PDRN) fraction is an extract which forms the active component in a new formulation of the drug Placentex (a tissue repair stimulating agent), obtained from human placenta through an original proprietory extraction method. From a comparison of the UV, NMR and IR spectra of this fraction (before and after nuclease treatment) with that of a similar standard (Sigma D1501), it was shown that the active substances in the PDRN fraction mainly consist of a mixture of DNA fragments. By gel electrophoresis, the molecular weights of the DNA fragments were shown to range from 50 to 2000 base pairs. Finally, an HPLC method is described, based on an anion-exchange material capable of determining the amount of PDRN in different batches of the extract, which varied from 80 to 90%.

In vitro cell cultures obtained from different explants of Corylus avellana produce Taxol and taxanes

BackgroundTaxol is an effective antineoplastic agent, originally extracted from the bark of Taxus brevifolia with a low yield. Many attempts have been made to produce Taxol by chemical synthesis, semi-synthesis and plant tissue cultures. However, to date, the availability of this compound is not sufficient to satisfy the commercial requirements. The aim of the present work was to produce suspension cell cultures from plants not belonging to Taxus genus and to verify whether they produced Taxol and taxanes. For this purpose different explants of hazel (Corylus avellana species) were used to optimize the protocol for inducing in vitro callus, an undifferentiated tissue from which suspension cell cultures were established.ResultsCalli were successfully induced from stems, leaves and seeds grown in various hormone concentrations and combinations. The most suitable callus to establish suspension cell cultures was obtained from seeds. Media recovered from suspension cell cultures contained taxanes, and showed antiproliferative activity on human tumour cells. Taxol, 10-deacetyltaxol and 10-deacetylbaccatin III were the main taxanes identified. The level of Taxol recovered from the media of hazel cultures was similar to that found in yew cultures. Moreover, the production of taxanes in hazel cell cultures increased when elicitors were used.ConclusionHere we show that hazel cell cultures produce Taxol and taxanes under controlled conditions. This result suggests that hazel possesses the enzymes for Taxol production, which until now was considered to be a pathway particular to Taxus genus. The main benefit of producing taxanes through hazel cell cultures is that hazel is widely available, grows at a much faster rate in vivo, and is easier to cultivate in vitro than yew. In addition, the production of callus directly from hazel seeds shortens the culture time and minimizes the probability of contamination. Therefore, hazel could become a commercial source of Taxol and taxanes, both to be used as new therapeutic agents or as new precursors for Taxol semi-synthesis.

Taxanes from shells and leaves of Corylus avellana

Paclitaxel is an effective antineoplastic agent originally extracted in low yield from the bark of Taxus brevifolia. Although it was generally considered a particular metabolite of Taxus sp., paclitaxel was recently found in hazel cell cultures. The aim of the present work was to verify whether hazel differentiated tissues could be used as a commercial source of paclitaxel and other taxanes. Thus, shells and leaves of hazel plants were analyzed by ELISA and HPLC-MS. Both shell and leaf extracts contained taxanes. Among these, paclitaxel, 10-deacetylbaccatin III, baccatin III, paclitaxel C, and 7-epipaclitaxel were identified and quantified. Hazel extracts also showed biological activity, inhibiting metaphase to anaphase transition in a human tumor cell line. The level of total taxanes in leaves was higher than in shells collected in the same period from the same plants. However, the finding of these compounds in shells, which are considered discarded material and are mass produced by many food industries, is of interest for the future availability of paclitaxel and other antineoplastic compounds.

Antiviral activity of indole derivatives.

Unsymmetrical methylene derivatives 5 were prepared following a known method, by reaction of the Mannich bases of 2-naphthols 4 with indoles. All synthesized compounds were tested against a wide panel of viruses, since previous work showed that Mannich bases on 7-hydroxycoumarin 1 and unsymmetrical methylene derivatives 2 were endowed with some antiviral activities. The symmetrical Mannich bases 4 were completely inactive, whereas the unsymmetrical methylene derivatives 5, although possessing a certain degree of toxicity, showed a significant activity against RSV. Some of compounds 5 showed a moderate antiviral activity against HIV-1, BVDV, YFV and CVB-2. The lack of activity of Mannich bases 4 demonstrates the crucial importance for antiviral activity of coumarin moiety present in Mannich bases 1.

Synthesis and anti-platelet activity of some 2-(dialkylamino)chromones

Substituted 2-(diethylamino) or 2-(ethylamino)chromones 5a—g were obtained from the reaction of suitable phenols with the reagent ethyl N,N-diethyl- or N-ethylmalonamate/POCl3 (2a or 2b). The above compounds, together with other N-substituted 2-aminochromones, either prepared from related chromones by simple reactions (5h—k) or previously obtained by us (5l—w), were tested in vitro for their inhibitory activities against human platelet aggregation induced by collagen, ADP and arachidonic acid. Many compounds showed activity and some were more active than acetylsalicylic acid in the tests with ADP and arachidonic acid. When the 2-amino substituent of tested chromones was a diethylamino group, the highest activity was found. The presence in position 7 of electron releasing substituents (OH, OCH3, CH3) led to an increase of activity, whereas a decrease occurred when an electron withdrawing substituent was present in position 3 (NO2) or 6 (NO2, Cl).

Structure-activity relationship of 1,4-dihydropyridines as potentiators of the cystic fibrosis transmembrane conductance regulator chloride channel

Mutations occurring in the CFTR gene, encoding for the cystic fibrosis transmembrane conductance regulator chloride channel, cause cystic fibrosis (CF). Mutations belonging to class II, such as ΔPhe508, give rise to a protein with both a defective maturation and altered channel gating. Mutations belonging to class III, such as G551D and G1349D, cause only a gating defect. We have previously identified antihypertensive 1,4-dihydropyridines (DHPs), a class of drugs that block voltage-dependent Ca2+ channels, as effective potentiators of CFTR gating, able to correct the defective activity of CFTR mutants (Mol Pharmacol 68:1736-1746, 2005). However, optimization of potency for CFTR versus Ca2+ channels is required to design selective compounds for CFTR pharmacotherapy. In the present study, we have established DHP structure-activity relationship for both CFTR potentiation and Ca2+ channel inhibition using cell-based assays for both types of channels. A panel of 333 felodipine analogs was studied to understand the effect of various substitutions and modifications in the DHP scaffold. Our results show that alkyl substitutions at the para position of the 4-phenyl ring lead to compounds with very low activity on Ca2+ channels and strong effect as potentiators on the ΔPhe508, G551D, and G1349D CFTR mutants.

N,N-dialkylaminosubstituted chromones and isoxazoles as potential anti-inflammatory agents.

The ability of some N,N-dialkylaminosubstituted chromones and isoxazoles to inhibit the protein kinase C (PKC) dependent signal transduction pathway was tested. As a cellular model, human neutrophils stimulated with either phorbol myristate acetate (PMA) or formylmethionine-leucine-phenylalanine (f-MLF) were used. The efficiency of the compounds was established by their capacity to reduce the O2- production by activated human neutrophils. Compounds carrying a 3-bis(2-methoxyethyl)amino group, a substituent found active in previously tested tricyclic compounds, do not show significant anti-PKC activity in this study. On the other hand, substitution with a 1-piperidinyl group leads all tested compounds to a high biological activity against stimulated neutrophils.

Alkaline extraction and reverse-phase high-performance liquid chromatography of adenine and pyridine nucleotides in human platelets

The levels of adenine (ATP, ADP, AMP) and pyridine (NAD, NADH) nucleotides in human platelets have been measured by a simple and reproducible method. A rapid alkaline extraction allows a complete recovery of the compounds concerned. The metabolic ATP and ADP in the cytosolic fraction, the amount released upon thrombin stimulation, and the ADP bound to F-actin have also been evaluated. Analysis was performed by reverse-phase, isocratic high-performance liquid chromatography on a 5-microns Lichrosorb RP-18 column with uv detection at 254 nm.

Synthesis and antiplatelet activity of 2-(diethylamino)-7-ethoxychromone and related compounds

Abstract 2-(Diethylamino)-7-ethoxychromone 3a and its 2-(1-piperidinyl)analogue 3b were synthesized by reaction of 3-ethoxyphenol 1 with 3-(dialkylamino)-3-oxo-propanoic acid ethyl ester 2 in the presence of phosphorus oxychloride. With a view to improve their biological activity the above 7-ethoxychromones 3 were submitted to some structural modifications firstly involving the 4-CO group. The 4H-chromenes 4 and the 4-thiochromones 5 were obtained by action of suitable reagents. The compounds 5 were then easily transformed to 4-(methylthio)chromenylium iodides 6 . Then from the 2-(diethylamino)-7-ethoxychromone 3a were obtained with suitable reactions the 3,6-diamino derivative 8 , the 3- and 6-formyl derivatives 9a,b and the Mannich base 10 . By action of acetic anhydride this latter compound yielded the methylenebis derivative 11 . Most of the above compounds were tested in vitro for their inhibitory activities against human platelet aggregation induced by collagen, ADP and arachidonic acid. Among the tested compounds the 2-(diethylamino)-7-ethoxychromone 3a showed the highest activity.