Doxorubicin disaccharide analogue: apoptosis-related improvement of efficacy in vivo.

BACKGROUND: Although doxorubicin remains one of the most effective agents for the treatment of solid tumors, there is an intensive effort to synthesize doxorubicin analogues (compounds with similar chemical structures) that may have improved antitumor properties. We have synthesized a novel doxorubicin disaccharide analogue (MEN 10755) and have characterized some of its relevant biochemical, biologic, and pharmacologic properties. METHODS: The antitumor activity of this compound (MEN 10755) was studied in a panel of human tumor xenografts, including xenografts of A2780 ovarian tumor cells, MX-1 breast carcinoma cells, and POVD small-cell lung cancer cells. MEN 10755 was compared with doxorubicin according to the optimal dose and schedule for each drug. The drug's cytotoxic effects, induction of DNA damage, and intracellular accumulation were studied in A2780 cells. DNA cleavage mediated by the enzyme topoisomerase II was investigated in vitro by incubating fragments of simian virus 40 DNA with the purified enzyme at various drug concentrations and analyzing the DNA cleavage-intensity patterns. Drug-induced apoptosis (programmed cell death) in tumors was determined with the use of MX-1 and POVD tumor-bearing athymic Swiss nude mice. RESULTS: MEN 10755 was more effective than doxorubicin as a topoisomerase II poison and stimulated DNA fragmentation at lower intracellular concentrations. In addition, MEN 10755 exhibited striking antitumor activity in the treatment of human tumor xenografts, including those of the doxorubicin-resistant breast carcinoma cell line MX-1. CONCLUSIONS: The high antitumor activity of MEN 10755 in human tumor xenografts, including doxorubicin-resistant xenografts, and its unique pharmacologic and biologic properties make this disaccharide analogue a promising candidate for clinical evaluation.

New developments in antitumor anthracyclines.

Doxorubicin is a major anticancer agent introduced to extended clinical use in the early 1970s. The fulfillment of a wide program of analogue synthesis led to the development of the better tolerated epirubicin and of a highly potent antileukemic drug, idarubicin. In recent years, on the basis of the available information on the molecular requirements for action, a new synthetic program, coupled with target-oriented pharmacological experiments, was carried out. Various interesting derivatives, namely, the 8- and 10-fluoro compounds and the disaccharides, were obtained. The latter compounds exhibited a strong dependence of biological activity on the orientation (axial vs. equatorial) of the second sugar moiety, daunosamine. A member of this group, namely, 7-O-(4'-O-alpha-L-daunosaminyl-2'-deoxy-alpha-L-fucosyl)-4-demetho xy-adriamycinone, is presently undergoing clinical trials as a third generation antitumor anthracycline.

Anthracyclines: selected new developments.

Anthracycline antibiotics play an important role in cancer chemotherapy. The need for an improvement of their therapeutic index has stimulated an ongoing search for anthracycline analogues with improved properties. Analogue development was originally limited by a lack of information on the cellular drug target, nevertheless almost 20 years ago the mechanism of action of doxorubicin and daunorubicin was revealed and DNA topoisomerase II was recognised to be their main cellular target. Several anthracyclines interfere with topoisomerase II functions by stabilizing a reaction intermediate in which DNA strands are cut and covalently linked to tyrosine residues of the enzyme. Investigations on the sequence specificity of doxorubicin in vitro and in nuclear chromatin of living cell have led to a molecular model of drug receptor on the topoisomerase II-DNA complex. Anthracyclines are likely placed at the interface between the DNA cleavage site and the active site of the enzyme, forming a DNA-drug-enzyme ternary complex. Moreover, a quite detailed structure-function relationship has been established for anthracyclines. First, drug intercalation is necessary but not sufficient for topoisomerase II poisoning; second, the removal of the 4-methoxy and 3'-amino substituents greatly increases the drug activity and third, the 3' substituent of the sugar moiety markedly influences the sequence selectivity of anthracycline-stimulated DNA cleavage. These relationships have been exploited during the last decade by several groups, including ours, in the search for new anthracycline drugs with lower side effects and higher activity against resistant cancer cells. This review will focus on areas of the anthracycline field including synthesis of new analogues, new strategies of synthesis and recent developments in the area of drug delivery.

Synthesis, DNA-binding properties, and antitumor activity of novel distamycin derivatives.

A group of potential alkylating agents have been synthesized that are structurally related to the oligopeptide antiviral antibiotic distamycin. All derivatives form complexes with native calf-thymus DNA but compounds 2, 3, and 6 give rise to covalent adducts. Cytostatic activity against both human and murine tumor cell lines in vitro is displayed by the new compounds. Compounds 3 and 4 are active on melphalan-resistant L1210 leukemia in mice.

Synthesis of two distamycin analogues and their binding mode to d(CGCAAATTTGCG)2 in the 2:1 solution complexes as determined by two-dimensional 1H-NMR.

In the course of a study aimed at the synthesis of pyrrole amidine carboxamide DNA-binding agents as novel pharmacological agents, a series of carbamoyl analogues of distamycin, containing an increasing number of pyrrole units, have been obtained by total synthesis. The interaction of the tetrapyrrole carbamoyl 4 with the dodecamer d(CGCAAATTTGCG)2 in comparison with that of the corresponding formylamino analogue 3 has been examined by high-resolution 1H-NMR and molecular modeling. Either ligand binds to DNA in one-drug and symmetric two-drug modes at low drug:DNA ratios, while at high ratios only the two-drug complex was observed. In this article, the structure of 2:1 drugs DNA complexes has been studied by NMR and molecular modeling, which indicate that the two analogues bind the DNA in a similar fashion, in the minor groove of the 5'-AATTT region. In both complexes the two drugs are symmetrically placed along the complementary strands of DNA with the pyrrole ring of one molecule in close contact with those of the other one. Although another region of five consecutive A-T base pairs is available, no evidence of sliding of drug molecules between different binding sites, as in the case of the 2:1 complex of distamycin with the same dodecamer, is observed, thus indicating that increasing the number of N-methylpyrrolecarboxamide units from three to four cases a lengthening of the recognition sequence.

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

1. 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). 2. Thirty microM 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 approximately 40-50%, whereas MEN 10755 inhibited by approximately 6%. 3. One hundred microM EPI exhibited the same uptake as equimolar DOX, but formed approximately 50% less alcohol metabolite. One hundred microM MEN 10755 still exhibited the lowest uptake, forming approximately 60% less alcohol metabolite than EPI. Under these conditions DOX inhibited post-rest contractions by 88%. EPI and MEN 10755 were approximately 18% (P<0.05) or approximately 80% (P<0.001) less inhibitory than DOX, respectively. 4. The negative inotropism of 30-100 microM 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 approximately 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. 5. 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.

Distamycin analogues with improved sequence-specific DNA binding activities.

In the present study we have investigated the effect of unprecedented chemical modifications introduced in the distamycin molecule, with the aim of assessing their ability to interfere with sequence-specific DNA-protein interactions in vitro. By using an electrophoretic mobility shift assay, we have been able to identify novel distamycin analogues with improved displacing abilities on the binding of octamer nuclear factors to their target DNA sequence. While variations in the number of pyrrole rings and/or reversion of an internal amide bond result in distamycin-like compounds with identical or very similar properties, the reversion of the formamido into a carboxyamido group or its replacement with the charged formimidoyl moiety significantly improves the ability of the resulting novel distamycin derivatives to compete with OCT-1 (octamer 1 nuclear factor) for its target DNA sequence. Tissue-specific octamer-dependent in vitro transcription is similarly affected by these chemical modifications, suggesting that the ability of distamycins to bind octamer sequences has a direct influence on the functional state of octamer-containing promoters. These data represent an initial, successful attempt to rationalize the design of DNA binding drugs, using distamycins as a model.

A comparative study of cellular and molecular pharmacology of doxorubicin and MEN 10755, a disaccharide analogue.

MEN 10755 is a disaccharide anthracycline endowed with a broader spectrum of antitumour activity than doxorubicin (DOX). To investigate the cellular and molecular basis of its action, cytotoxic activity, drug uptake, subcellular localisation, induction of DNA damage, and apoptosis were assessed in the human A2780 ovarian carcinoma cell line. Experiments with radiolabelled anthracyclines indicated that MEN 10755 exhibited reduced cellular accumulation and a different subcellular distribution (higher cytoplasmic/nuclear ratio) than DOX. In spite of the lower nuclear concentration, MEN 10755 was as potent as DOX in eliciting DNA single- and double-strand breaks, G2/M cell arrest, and apoptosis. Sequencing of drug-induced topoisomerase II cleavage sites showed a common DNA cleavage pattern for MEN 10755 and DOX. Cleavage sites were always characterised by the presence of adenine in -1 position. However, the extent of DNA cleavage stimulation induced by MEN 10755 was greater than that produced by DOX. Reversibility studies showed that MEN 10755-stimulated DNA cleavage sites were more persistent than those induced by DOX, thus suggesting a more stable interaction of the drug in the ternary complex. As a whole, the study indicated that the cellular pharmacokinetics of MEN 10755 substantially differs from that of DOX, showing a lower uptake and a different subcellular disposition. In spite of the apparently unfavourable cellular pharmacokinetics, MEN 10755 was still as potent as DOX in inducing topoisomerase-mediated DNA damage. Although the extent and persistence of protein-associated DNA breaks may contribute to the cytotoxic effects, the drug's efficacy as apoptosis inducer and antitumour agent could not be adequately explained on the basis of DNA damage mediated by the known target (i.e. topoisomerase II), thus supporting additional cellular effects that may be relevant in cellular response.

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 drug's 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.

Binding of Epstein-Barr virus nuclear antigen 1 to DNA: inhibition by distamycin and two novel distamycin analogues.

Modulation of the interaction between cellular or viral transcription factors and target DNA sequences may represent a potential experimental strategy to control proliferation of neoplastic cells as well as virus DNA replication. Distamycin represents a likely candidate to mediate such modulation by pharmacological means. In order to obtain more detailed information on structure-activity relationships of these compounds, we have analysed the effects of distamycin and two distamycin analogues on the binding of a recombinant protein, the Epstein-Barr virus nuclear antigen 1 (EBNA-1) to its target sequence of Epstein-Barr virus, containing the 12 bp palindromic consensus TAGCATATGCTA. The sequence selectivity in the binding of distamycin to DNA was evaluated by footprinting experiments, while the effects of distamycins on DNA-protein interactions was analysed by means of electrophoretic mobility shift assay. The data presented in this paper suggest that distamycin and its analogues differentially inhibit the interaction between DNA-binding proteins and target DNA sequences.

Synthesis and conformational preference of novel 8-fluoroanthracyclines.

Analogues of daunorubicin possessing a fluorine atom at position C(8) of ring A have been synthesized with the aim of comparing their DNA-drug interaction and antitumour properties with those of the clinically useful anthracyclines doxorubicin and idarubicin. The synthesis of (8S)-8-fluoro-4-demethoxydaunorubicin, 1, is reported and molecular mechanics and NMR studies which guided towards the synthesis of the epimeric (8R)-8-fluoro-4-demethoxydaunorubicin, 2, are discussed. Both compounds were prepared by divergent routes starting from the common intermediate, 6, obtained via the Diels-Alder cyclisation between quinizarin diquinone, 3, and 2-(1-hydroxyethyl)-1,3-butadiene, 4. The synthesis of the (8S)-fluoroepimer proceeded via epoxidation of the C(8)-C(9) olefinic bond of 6, oxidation, oxirane cleavage by BF3.Et2O to give the fluorohydroxyketone, 9, followed by the introduction of the hydroxyl moiety at C(7) and glycosylation. Conversely, the synthesis of the (8R)-fluoroepimer involved the fluorobromination of the C(8)-C(9) olefinic bond of 6, formation of the C(9)-C(13) epoxide, 20 which, after regioselective hydrolysis and oxidation of the resulting fluorodiol to the epimeric fluorohydroxyketone, 21, similarly gave the desired fluoroaglycone, 25 and, hence, the corresponding glycoside, 2. The cytotoxic properties of the two 8-fluoroanthracycline analogues, 1 and 2, were markedly affected by the stereochemistry of the fluorine substituent.

Croton macrostachys, a plant used in traditional medicine: purgative and inflammatory activity.

Croton macrostachys seeds are widely used in Somalia as a purgative. In the present study, pharmacologic and chemical investigations confirm the laxative effect of the seeds and indicate the presence of phorbolesters. There appears to be no direct correlation between phorbolester content and laxative effect.

Solution chemistry and DNA binding properties of MEN 10755, a novel disaccharide analogue of doxorubicin.

The behavior under physiological conditions of MEN 10755, a novel disaccharide analogue of doxorubicin, was investigated in detail by a variety of spectroscopic techniques including spectrophotometry, fluorescence, and (1)H NMR. The pH dependent properties of MEN 10755 were also analysed by spectrophotometry and potentiometry within the pH range 5--11. It is found that MEN 10755 behaves very similarly to doxorubicin and reproduces closely its pH dependent pattern. Like doxorubicin, MEN 10755 undergoes dimerization with a significantly smaller association constant. The interaction of MEN 10755 with calf thymus DNA was studied in detail. Spectrophotometric and fluorescence titrations of MEN 10755 with calf thymus DNA show spectral patterns almost identical to those obtained with doxorubicin implying that the binding mechanism and the stability of the resulting adducts are very similar. An apparent affinity constant of 1.2 x 10(6) was determined for the interaction of MEN 10755 with calf thymus DNA to be compared with the value of 3.3 x 10(6) measured for doxorubicin, under the same conditions. The effects of both anthracyclines on the thermal denaturation profiles of calf thymus DNA were also analyzed; both compounds turned out to stabilize to a similar extent the DNA double helix and to give rise to a characteristic two-step melting profile. The implications of the present results for the pharmacological activity and the mechanism of action of this novel and promising antitumor compound are discussed.

Cytotoxic and antitumor activity of MEN 10710, a novel alkylating derivative of distamycin.

MEN 10710 is a new synthetic distamycin derivative possessing four pyrrole rings and a bis-(2-chloroethyl)aminophenyl moiety linked to the oligopyrrole backbone by a flexible butanamido chain. Its biological properties have been investigated in comparison with the structurally related compound, tallimustine (FCE24517), and the classical alkylating agent, melphalan (L-PAM). Cytotoxic potency of MEN 10710 was increased from 10- to 100-fold, as compared to tallimustine or L-PAM in murine L1210, human LoVo and MCF7 tumor cell lines. MEN 10710 was still active against L1210/L-PAM leukemic cells, while a partial cross-resistance was observed in LoVo/DX and in MCF7/DX cells selected for resistance to doxorubicin and expressing a MDR phenotype. Treatment with verapamil (VRP) reduced the resistance to tallimustine, but not to MEN 10710, in MCF7/DX cells. The cytotoxic effects reflect in vivo antitumor potency and toxicity in the treatment of human tumor xenografts. MEN 10710 was more effective in A2780/DDP, an ovarian carcinoma selected for resistance to cisplatin. On the other hand, the IC30 for inhibiting murine granulocyte/macrophage colony formation was 50 times higher for MEN 10710 than for tallimustine, suggesting a lower myelotoxic potential. In conclusion, the particular biological profile of MEN 10710 characterized by a marked cytotoxic potency, an interesting antitumor efficacy and a reduced in vitro myelosuppressive action may represent a further improvement in the rational design of a novel distamycin-related alkylating compound.

Anthracycline metabolism and toxicity in human myocardium: comparisons between doxorubicin, epirubicin, and a novel disaccharide analogue with a reduced level of formation and [4Fe-4S] reactivity of its secondary alcohol metabolite.

Secondary alcohol metabolites have been proposed to mediate chronic cardiotoxicity induced by doxorubicin (DOX) and other anticancer anthracyclines. In this study, NADPH-supplemented human cardiac cytosol was found to reduce the carbonyl group in the side chain of the tetracyclic ring of DOX, producing the secondary alcohol metabolite doxorubicinol (DOXol). A decrease in the level of alcohol metabolite formation was observed by replacing DOX with epirubicin (EPI), a less cardiotoxic analogue characterized by an axial-to-equatorial epimerization of the hydroxyl group at C-4 in the amino sugar bound to the tetracyclic ring (daunosamine). A similar decrease was observed by replacing DOX with MEN 10755, a novel anthracycline with preclinical evidence of reduced cardiotoxicity. MEN 10755 is characterized by the lack of a methoxy group at C-4 in the tetracyclic ring and by intercalation of 2, 6-dideoxy-L-fucose between daunosamine and the aglycone. Multiple comparisons with methoxy- or 4-demethoxyaglycones, and a number of mono- or disaccharide 4-demethoxyanthracyclines, showed that both the lack of the methoxy group and the presence of a disaccharide moiety limited alcohol metabolite formation by MEN 10755. Studies with enzymatically generated or purified anthracycline secondary alcohols also showed that the presence of a disaccharide moiety, but not the lack of a methoxy group, made the metabolite of MEN 10755 less reactive with the [4Fe-4S] cluster of cytoplasmic aconitase, as evidenced by its limited reoxidation to the parent carbonyl anthracycline and by a reduced level of delocalization of Fe(II) from the cluster. Collectively, these studies (i) characterize the different influence of methoxy and sugar substituents on the formation and [4Fe-4S] reactivity of anthracycline secondary alcohols, (ii) lend support to the role of alcohol metabolites in anthracycline-induced cardiotoxicity, as they demonstrate that the less cardiotoxic EPI and MEN 10755 share a reduction in the level of formation of such metabolites, and (iii) suggest that the cardiotoxicity of MEN 10755 might be further decreased by the reduced [4Fe-4S] reactivity of its alcohol metabolite.

Biochemical and pharmacological activity of novel 8-fluoroanthracyclines: influence of stereochemistry and conformation.

In an attempt to better understand the role of the cyclohexene ring (ring A) in the biochemical and pharmacological properties of anthracyclines related to doxorubicin and daunorubicin, we investigated the effects of introduction of a fluorine atom at position 8 of idarubicin (4-demethoxydaunorubicin) on drug molecular conformation and biochemical and pharmacological activities. The study showed that the stereochemistry of the substituent at position 8 influenced the "half-chair" conformation, so that in the (8R)-fluoroepimer the A ring retained the alpha half-chair conformation, which is the most stable for natural compounds (i.e., daunorubicin and doxorubicin), and the (8S)-fluoroepimers preferred the beta half-chair conformation. The (8R)-fluoroepimer was more effective than the (8S)-fluoroepimer and idarubicin in stimulating topoisomerase II-mediated DNA cleavage. Similarly, the epimer with the alpha conformation was markedly more potent than the (8S)-epimer as a cytotoxic agent in a variety of human tumor cell lines and was more effective as an antitumor agent in the treatment of an ovarian carcinoma xenograft. In addition, 8-fluoro derivatives were able to overcome the resistance to doxorubicin in a number of human tumor cell lines expressing different mechanisms of resistance. In conclusion, these findings provide evidence that drug interactions involving the external (nonintercalating) moiety of the anthracycline chromophore play an important role in determining pharmacological properties, including drug ability to induce DNA cleavage, and therefore their antitumor efficacy.

Novel anthracycline oligosaccharides: influence of chemical modifications of the carbohydrate moiety on biological activity.

Several observations highlight the importance of the carbohydrate moiety for the biological activity of antitumoural anthracyclines. Here is reported the synthesis, cytotoxicity and topoisomerase II-mediated DNA cleavage intensity of the new oligosaccharide anthracyclines 1--4 modified in the sugar residue. Evaluation of cytotoxic potency on different cell lines, resulted in quite similar values among the different analogues. On the other hand, topoisomerase II-mediated DNA breaks level was different for the various compounds, and was not related to cytotoxicity, thus supporting previous observations reported for some monosaccharide anthracyclines modified in the carbohydrate portion.

Topoisomerase poisoning activity of novel disaccharide anthracyclines.

Doxorubicin and idarubicin are very effective anticancer drugs in the treatment of human hematological malignancies and solid tumors. These agents are well known topoisomerase II poisons; however, some anthracycline analogs recently have been shown to poison topoisomerase I. In the present work, we assayed novel disaccharide analogs and the parent drug, idarubicin, for their poisoning effects of human topoisomerase I and topoisomerases IIalpha and IIbeta. Drugs were evaluated with a DNA cleavage assay in vitro and with a yeast system to test whether the agents were able to poison the enzymes in vivo. We have found that the test agents are potent poisons of both topoisomerases IIalpha and IIbeta. The axial orientation of the second sugar relative to the first one of the novel disaccharide analogs was shown to be required for poisoning activity and cytotoxicity. Interestingly, idarubicin and the new analogs stimulated topoisomerase I-mediated DNA cleavage at low levels in vitro. As expected, the cytotoxic level of the drug was highly affected by the content of topoisomerase II; nevertheless, the test agents had a yeast cell-killing activity that also was weakly dependent on cellular topoisomerase I content. The results are relevant for the full understanding of the molecular mechanism of topoisomerase poisoning by anticancer drugs, and they define structural determinants of anthracyclines that may help in the rational design of new compounds directed against topoisomerase I.