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.

Interaction between double helix DNA fragments and the new antitumor agent sabarubicin, Men10755.

Among the disaccharide derivatives of the antitumor anthracycline doxorubicin, sabarubicin (Men10755) is more active and less cytotoxic than doxorubicin. It showed a strong in vivo antitumor activity in all preclinical models examined, in conjunction with a better tolerability, and is now in phase II clinical trials. The interaction of sabarubicin and Men10749 (a similar disaccharide with a different configuration at C-4' of the proximal sugar) with the hexanucleotides d(CGTACG)(2) and d(CGATCG)(2) was studied by a combined use of 2D-(1)H and (31)P NMR techniques. Both (1)H and (31)P chemical shifts of imino protons and phosphates allowed to established the intercalation sites between the CG base pairs, as it occurs for other anthracyclines of the series. The dissociation rate constants (k(off)) of the slow step of the intercalation process were measured for Men10755 and Men10749, by NMR NOE-exchange experiments. The increase of k(off) , with respect of doxorubicin, showed that the intercalation process is significantly faster for both drugs, leading to an average residence time for sabarubicin into d(CGTACG)(2) sixfold shorter than for doxorubicin. This could give account of both higher cytoplasmic/nuclear ratio and lower cellular uptake of sabarubicin in comparison with doxorubicin and accordingly of the lower cytotoxicity of these disaccharide analogues. A relevant number of NOE interactions allowed the structure of the complexes in solution to be derived through restrained MD calculations. NMR-DOSY experiments were performed with several drug/oligonucleotide mixtures in order to determine the structure and the dimension of the aggregates.

Antitumor activity of delimotecan against human metastatic melanoma: pharmacokinetics and molecular determinants.

Delimotecan (MEN 4901/T-0128) is a new cytotoxic prodrug constituted by a camptothecin analog (T-2513) bound to carboxymethyl dextran through a triglycine linker. A significant antitumor activity of delimotecan against human metastatic melanoma xenograft model Me15392 is reported. Dacarbazine, the drug approved for the treatment of metastatic melanoma, was ineffective in this melanoma model. Pharmacokinetic studies, together with the expression analysis of mRNA for enzymes involved in delimotecan metabolism, showed that T-2513 and other cytotoxic metabolites of delimotecan (SN 38 and T-0055) are generated in greater quantities in the tumor tissue than in toxicity target tissues, such as liver, thus accounting for the antitumoral activity. Moreover, we demonstrated that human metastatic melanoma cells are able to phagocytose delimotecan and cleave it to release the cytotoxic moieties T-2513 in the tumoral environment. Further flow cytometric analysis showed a higher recruitment of macrophages in xenografted human metastatic melanoma, when compared with other human tumors. Thus, the antitumoral activity of delimotecan exerted on metastatic melanoma is due to several factors: (i) the ability of melanoma cells to phagocytose and metabolise delimotecan; (ii) the accumulation of delimotecan in tumoral mass; (iii) the recruitment of macrophage cells to the melanoma nodule and (iv) the expression in melanoma cells of a pattern of enzymes that converts delimotecan into cytotoxic metabolites. Based on these results, delimotecan might be exploited as a new anticancer agent for the therapy of metastatic melanoma because of its high efficacy and good selectivity, and therefore clinical trials for this indication are warranted.

Clinical and pharmacologic study of the novel prodrug delimotecan (MEN 4901/T-0128) in patients with solid tumors.

PURPOSE: To investigate i.v. administration of delimotecan (MEN 4901/T-0128), a carboxymethyldextran polymer prodrug of the active camptothecin derivative T-2513, and to assess the maximum tolerated dose, safety profile, clinical pharmacology, and antitumor activity of delimotecan and metabolites. EXPERIMENTAL DESIGN: Patients with solid tumors refractory to standard therapy received i.v. delimotecan as 3-hour infusion once every 6 weeks. The starting dose was 150 mg/m2, followed by an accelerated dose escalation with at least one patient per dose level. The pharmacokinetics of delimotecan, T-2513, and its metabolites, SN-38, SN-38G, T-1335, T-0055, and T-3921, were assessed in plasma and urine, and their pharmacodynamics were determined by measuring the effect of the treatment on hematologic and nonhematologic toxicity. RESULTS: Twenty-two patients received 35 courses. Dose-limiting toxicities were observed at 5,400 mg/m2 (n = 1), 3,600 mg/m2 (n = 1), and 2,400 mg/m2 (n = 2). The dose level of 1,800 mg/m2 was determined as maximum tolerated dose. Two partial responses were observed in patients with anal cancer (1800 mg/m2) and head and neck cancer (2400 mg/m2). Delimotecan had a long terminal half-life of 109 h, and relatively high exposures to T-2513 and SN-38 were obtained. The percentage decrease in WBC and absolute neutrophil count significantly correlated with the dose of delimotecan. CONCLUSIONS: Based on its preliminary antitumor activity, safety profile, and pharmacokinetic profile, we recommend to evaluate delimotecan given as 3-hour infusion once every 6 weeks at a dose level of 1,800 mg/m2 in a phase II study.

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 IC(50)<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.

Oxidative degradation of cardiotoxic anticancer anthracyclines to phthalic acids. Novel function or ferrylmyoglobin.

We show that the pseudoperoxidase activity of ferrylmyoglobin (MbIV) promotes oxidative degradation of doxorubicin (DOX), an anticancer anthracycline known to induce severe cardiotoxicity. MbIV, formed in vitro by reacting horse heart MbIII with H2O2, caused disappearance of the spectrum of DOX at 477 nm and appearance of UV-absorbing chromophores that indicated opening and degradation of its tetracyclic ring. Electron spray ionization mass spectrometry analyses of DOX/MbIV ultrafiltrates showed that DOX degradation resulted in formation of 3-methoxyphthalic acid, the product of oxidative modifications of its methoxy-substituted ring D. Other methoxy-substituted anthracyclines similarly released 3-methoxyphthalic acid after oxidation by MbIV, whereas demethoxy analogs released simple phthalic acid. Kinetic and stoichiometric analyses of reactions between DOX and MbIII/H2O2 or hemin/H2O2 showed that the porphyrin radical of MbIV-compound I and the iron-oxo moiety of MbIV-compound II were sequentially involved in oxidizing DOX; however, oxidation by compound I formed more 3-methoxyphthalic acid than oxidation by compound II. Sizeable amounts of 3-methoxyphthalic acid were formed in the heart of mice treated with DOX, in human myocardial biopsies exposed to DOX in vitro, and in human cardiac cytosol that oxidized DOX after activation of its endogenous myoglobin by H2O2. Importantly, H9c2 cardiomyocytes were damaged by low concentrations of DOX but could tolerate concentrations of 3-methoxyphthalic acid higher than those measured in murine or human myocardium. These results unravel a novel function for MbIV in the oxidative degradation of anthracyclines to phthalic acids and suggest that this may serve a salvage pathway against cardiotoxicity.

Chronic cardiotoxicity of anticancer anthracyclines in the rat: role of secondary metabolites and reduced toxicity by a novel anthracycline with impaired metabolite formation and reactivity.

(1) The anticancer anthracycline doxorubicin (DOX) causes cardiomyopathy upon chronic administration. There is controversy about whether DOX acts directly or after conversion to its secondary alcohol metabolite DOXol. Here, the role of secondary alcohol metabolites was evaluated by treating rats with cumulative doses of DOX or analogues--like epirubicin (EPI) and the novel disaccharide anthracycline MEN 10755--which were previously shown to form less alcohol metabolites than DOX when assessed in vitro. (2) DOX induced electrocardiographic and haemodynamic alterations, like elongation of QalphaT or SalphaT intervals and suppression of isoprenaline-induced dP/dt increases, which developed in a time-dependent manner and were accompanied by cardiomegaly, histologic lesions and mortality. EPI caused less progressive or severe effects, whereas MEN 10755 caused essentially no effect. (3) DOX and EPI exhibited comparable levels of cardiac uptake, but EPI formed approximately 60% lower amounts of its alcohol metabolite EPIol at 4 and 13 weeks after treatment suspension (P<0.001 vs DOX). MEN 10755 exhibited the lowest levels of cardiac uptake; hence, it converted to its alcohol metabolite MEN 10755ol approximately 40% less efficiently than did EPI to EPIol at either 4 or 13 weeks. Cardiotoxicity did not correlate with myocardial levels of DOX or EPI or MEN 10755, but correlated with those of DOXol or EPIol or MEN 10755ol (P=0.008, 0.029 and 0.017, respectively). (4) DOX and EPI inactivated cytoplasmic aconitase, an enzyme containing an Fe-S cluster liable to disassembly induced by anthracycline secondary alcohol metabolites. DOX caused greater inactivation of aconitase than EPI, a finding consistent with the higher formation of DOXol vs EPIol. MEN 10755 did not inactivate aconitase, which was because of both reduced formation and impaired reactivity of MEN 10755ol toward the Fe-S cluster. Aconitase inactivation correlated (P<0.01) with the different levels of cardiotoxicity induced by DOX or EPI or MEN 10755. (5) These results show that (i) secondary alcohol metabolites are important determinants of anthracycline-induced cardiotoxicity, and (ii) MEN 10755 is less cardiotoxic than DOX or EPI, a behaviour attributable to impaired formation and reactivity of its alcohol metabolite.

The crystal structure of the complex between a disaccharide anthracycline and the DNA hexamer d(CGATCG) reveals two different binding sites involving two DNA duplexes.

The crystal structure of the complex formed between the anthracycline antibiotic 3'-deamino-3'- hydroxy-4'-(O-L-daunosaminyl)-4-demethoxydoxo rubicin (MEN 10755), an active disaccharide analogue of doxorubicin, and the DNA hexamer d(CGATCG) has been solved to a resolution of 2.1 A. MEN 10755 exhibits a broad spectrum of antitumor activities, comparable with that of the parent compound, but there are differences in the mechanism of action as it is active in doxorubicin-resistant tumors and is more effective in stimulating topoisomerase DNA cleavage. The structure is similar to previously crystallised anthracycline- DNA complexes. However, two different binding sites arise from drug intercalation so that the two halves of the self-complementary duplex are no longer equivalent. In one site both sugar rings lie in the minor groove. In the other site the second sugar protrudes out from the DNA helix and is linked, through hydrogen bonds, to guanine of a symmetry-related DNA molecule. This is the first structure of an anthracycline-DNA complex where an interaction of the drug with a second DNA helix is observed. We discuss the present findings with respect to the relevance of the amino group for DNA binding and to the potential role played by the second sugar in the interactions with topoisomerases or other cellular targets.

An unusual rearrangement of Zofenopril, a new ACE inhibitor drug: mass spectrometric and conformational studies.

Zofenopril (1) is a new ACE inhibitor, used in therapy for hypertension and post-myocardial infarction. The protonated quasi-molecular ion (m/z 430) of 1, obtained under positive electrospray ionization conditions, loses a benzoic acid molecule (m/z 308), which in turn decomposes via loss of CO (m/z 280) when low-energy collisional-induced dissociation (CID) and in-source experiments are performed. This rearrangement is the main fragmentation process and can be observed both in-source and in the product ion tandem mass spectra, using either an ion trap or a triple quadrupole instrument. Other known diastereoisomers of 1, an impurity with an acetyl in the place of the benzoyl group (2) and an impurity with two propanoyl chains in series (3), give the same rearrangement. On the other hand, the mass spectra of the methyl ester (4) and an impurity with two proline moieties (5) do not show this unusual fragmentation. Time-resolved CID spectra of 1 show that the rearrangement occurs after about 2 ms, a time scale comparable to those of the other non-rearrangement cleavages. These experiments suggest a conformation in the gas phase for 1 in which the benzoyl group is close to the hydroxyl of the carboxylic acid group, from which the rearrangement could readily occur. Since compounds 4 and 5 do not show the same behaviour, the presence of a carboxylic acid in the proline ring seems to play a crucial role in the rearrangement, probably due to an intramolecular hydrogen bond. To confirm this hypothesis, deuterium exchanges in mass spectrometric experiments and a conformational analysis via computational methods were performed.