Amalia Cipollone

Configurational requirements of the sugar moiety for the pharmacological activity of anthracycline disaccharides.

The amino sugar is recognized to be a critical determinant of the activity of anthracycline monosaccharides related to doxorubicin and daunorubicin. In an attempt to improve the pharmacological properties of such agents, novel anthracycline disaccharides have been designed in which the amino sugar, daunosamine, is separated from the aglycone by another carbohydrate moiety. In the present study, we examined the influence of the orientation of the second sugar residue on drug biochemical and biological properties in a series of closely related analogs. This structure-activity relationship study showed that the substitution of the daunosamine for the disaccharide moiety dramatically reduced the cytotoxic potency of the drug in the 4-methoxy series (daunorubicin analogs). In contrast, in the 4-demethoxy series (idarubicin analogs), the C-4 axial, but not the equatorial, configuration conferred a cytotoxic potency and antitumor activity comparable to that of doxorubicin. The configuration also influenced the drugs ability to stimulate topoisomerase II alpha-mediated DNA cleavage. Indeed, the glycosides with the equatorial orientation were ineffective as topoisomerase II poisons, whereas the compounds with axial orientation were active, although the daunorubicin analog exhibited a lower activity than the idarubicin analog. It is conceivable that the axial orientation allows an optimal interaction of the drug with the DNA-enzyme complex only in the absence of the methoxy group. Our results are consistent with a critical role of the sugar moiety in drug interaction with the target enzyme in the ternary complex.

Impairment of myocardial contractility by anticancer anthracyclines: role of secondary alcohol metabolites and evidence of reduced toxicity by a novel disaccharide analogue

The anticancer anthracycline doxorubicin (DOX) causes cardiotoxicity. Enzymatic reduction of a side chain carbonyl group converts DOX to a secondary alcohol metabolite that has been implicated in cardiotoxicity. We therefore monitored negative inotropism, assessed as inhibition of post‐rest contractions, in rat right ventricle strips exposed to DOX or to analogues forming fewer amounts of their alcohol metabolites (epirubicin, EPI, and the novel disaccharide anthracycline MEN 10755). Thirty μM EPI exhibited higher uptake than equimolar DOX, but formed comparable amounts of alcohol metabolite due to its resistance to carbonyl reduction. MEN 10755 exhibited also an impaired uptake, and consequently formed the lowest levels of alcohol metabolite. Accordingly, DOX and EPI inhibited post‐rest contractions by ∼40 – 50%, whereas MEN 10755 inhibited by ∼6%. One hundred μM EPI exhibited the same uptake as equimolar DOX, but formed ∼50% less alcohol metabolite. One hundred μM MEN 10755 still exhibited the lowest uptake, forming ∼60% less alcohol metabolite than EPI. Under these conditions DOX inhibited post‐rest contractions by 88%. EPI and MEN 10755 were ∼18% (P<0.05) or ∼80% (P<0.001) less inhibitory than DOX, respectively. The negative inotropism of 30 – 100 μM DOX, EPI, or MEN 10755 correlated with cellular levels of both alcohol metabolites (r=0.88, P<0.0001) and carbonyl anthracyclines (r=0.79, P<0.0001). Nonetheless, multiple comparisons showed that alcohol metabolites were ∼20 – 40 times more effective than carbonyl anthracyclines in inhibiting contractility. The negative inotropism of MEN 10755 was therefore increased by chemical procedures, like side chain valeryl esterification, that facilitated its uptake and conversion to alcohol metabolite but not its retention in a carbonyl form. These results demonstrate that secondary alcohol metabolites are important mediators of cardiotoxicity. A combination of reduced uptake and limited conversion to alcohol metabolite formation might therefore render MEN 10755 more cardiac tolerable than DOX and EPI.

New anthracycline disaccharides. Synthesis of L-daunosaminyl-α(1 →4)-2-deoxy-L-rhamnosyl and of L-daunosaminyl-α(1 →4)-2-deoxy-L-fucosyl daunorubicin analogues

The synthesis of the new disaccharide anthracyclines 20, 21, 24 and 25, where the daunosamine moiety is separated from the aglycone by either a rhamnose or a fucose residue, performed following a convergent procedure, gives insight into the configurational requirement of the first sugar residue and opens the way to a new class of antitumour anthracyclines.

Synthesis, biological evaluation, and molecular modeling studies of rebeccamycin analogues modified in the carbohydrate moiety.

A new series of indolocarbazole glycosides containing disaccharides were synthesized and their in vitro antiproliferative activity was evaluated against three human cancer cell lines (A2780, H460, and GLC4). Cytotoxicity appeared to be remarkably affected by the regio‐ and stereochemical features of the disaccharide moiety. In vivo antitumor activity of the compounds studied, two of which having IC50<100 nm, was determined using ovarian cancer cell line A2780 xenografted on nude mice. One compound showed an efficacy similar to that of the reference compound edotecarin, though with a lower long‐lasting activity. The topoisomerase I inhibitory properties of some compounds were also examined. Molecular dynamics simulations of the ternary topoisomerase I–DNA–ligand complexes were performed to analyze the structural features of topoisomerase I poisoning with this class of indolocarbazoles. A plausible explanation of their biological behavior was provided. These theoretical results were compared with the recently published crystal structure of an indolocarbazole monosaccharide bound to the covalent human topoisomerase I–DNA complex.

Synthesis and biological evaluation of rebeccamycin analogues modified at the imide moiety.

Glycosylated indolocarbazoles related to the antibiotic rebeccamycin represent an important class of antitumour drugs. In the course of our structure-activity relationship studies, new rebeccamycin analogues modified at the imide moiety were synthesised. The antiproliferative activity of the compounds was evaluated on three human cancer cell lines, A2780 (ovarian cancer), H460 (lung cancer), and GLC4 (small-cell lung cancer). The in vitro cytotoxicity of compounds 2 and 4, characterised respectively by a 1,3-dioxolan and (1,3-dioxolan-4-yl)methylene groups linked to the imide moiety, was higher than the reference compound, edotecarin. The effect of compound 2 in inducing tumour regression in the A2780 xenograft model was also investigated.

Benzofused hydroxamic acids: Useful fragments for the preparation of histone deacetylase inhibitors. Part 2: 7-Fluorobenzothiophenes and benzofurans

In the search for a new class of histone deacetylase inhibitors, we prepared a series of very simple benzofused hydroxamic acids to find an anchoring fragment of minimal molecular weight: they showed very good ligand efficiencies. Following these findings, classical fragment growing work was performed to increase binding energy and selective cytotoxicity. In the second phase of the work, information from the SARs of the benzothiophene series and data available in literature, we explored the in vitro pharmacological properties of the 6-substituted-7-fluoro-benzothiophene hydroxamates and the 5-susbtituted-benzofuran hydroxamates.

NMR-Spectroscopic, computational and mass-spectrometric investigations on the cis/trans analogues of 2,3,4-trihydronaphthalene-1-one

Abstract NMR-Spectroscopic, computational and mass-spectrometric studies of the cis/trans isomers of N-[8-(acetylamino)-4-(2,2-dimethyl-1,1-diphenyl-silapropoxy)-6-fluoro-5-methyl-1-one-2,3,4-trihydronaphthyl]acetamide ( 1a and 1b ), obtained as intermediates in the synthesis of an important class of alkaloid molecules, are reported. 1H and 13C NMR analyses show an unusual axial preference of the TBDPSi– (tert-butyldiphenylsilyl) group in position 4 in both the isomers. Mass spectrometric evidence demonstrates that trans isomer has a higher affinity for ammonium ions than the cis isomer and that only the ammonium adduct [ 1b +NH4]+ and the protonated molecule [ 1b +H]+ show the fragmentation in which loss of benzene is observed. Moreover, molecular mechanics and semi-empirical calculations indicate that a group of trans conformers tend to place one of the phenyl rings of the TBDPSiO– group in a offset π-stacked geometry with the compounds aromatic ring. The combination and the detailed analyses of these experimental and theoretical results could support the π–π interaction obtained as a conformational preference in the trans isomer.