Anthracycline Analogues Research Articles

Synthesis of (±)-7-(3,4,5-trimethoxyphenyl)-7-deoxyidarubicinone. A new family of anthracycline analogues

The synthesis of the first representative compound of 7-aryl anthracycline analogues, is described. 1-Alkyl-3-trialkylsiloxydienes, prepared from readily available materials, are transformed through a Diels-Alder cycloaddition into a tetracyclic ketone, that is converted into (±)-7-(3,4,5-trimethoxyphenyl)-7-deoxyidarubicinone. The conformations of the target compound and intermediate products are studied.

Additive effect of concomitant multiple low-dose doxorubicin and thoracic irradiation on ex vivo cardiac performance of the rat heart.

The effects of doxorubicin alone or combined with local heart irradiation on ex vivo cardiac performance were studied in Sprague-Dawley rats. Rats were treated with doxorubicin either administered as a single bolus injection or administered weekly during a period of 10 weeks. In "combined" experiments, local heart irradiation with a single dose of 15 Gy was given prior to drug administration. Evaluation of cardiac performance was performed 14 weeks after initiation of treatment. At drug doses that were tolerated by the rat, single injections with doxorubicin (sd-DXR; up to a dose of 5 mg/kg) did not lead to a change in cardiac performance whereas multiple injections with low-dose doxorubicin (mld-DXR; up to a cumulative dose of 20 mg/kg) led to a dose-dependent decrease in cardiac function. Extracardial toxicity as a result of mld-DXR (cumulative dose < or =15 mg/kg) was mild when compared to the toxicities observed after sd-DXR (5.0 and 7.5 mg/kg). When administration of mld-doxorubicin was preceded by 15 Gy, cardiac performance further decreased. The present data indicate that the interaction between doxorubicin and local heart irradiation with a dose of 15 Gy is additive, when the treatments are given concomitantly. Irradiation did not lead to an increase of DXR-mediated extracardial toxicities. The isolated working rat heart preparation offers a reliable method to evaluate the effects of doxorubicin and new anthracycline analogue on the heart.

Induction of DNA damage, inhibition of DNA synthesis and suppression of c-myc expression by the anthracycline analog, idarubicin (4-demethoxy-daunorubicin) in the MCF-7 breast tumor cell line.

Studies were designed to elucidate the basis for the antiproliferative activity of the anthracycline antibiotic, idarubicin (4-demethoxy-daunorubicin) in MCF-7 breast tumor cells. Growth inhibition was evaluated using the MTT tetrazolium dye assay, induction of DNA strand breaks was determined by alkaline elution, inhibition of DNA synthesis was assessed by measuring the incorporation of labelled thymidine into DNA, modulation of the expression of the c-myc oncogene was determined by Northern blotting and the induction of apoptosis was evaluated by alkaline unwinding, static field gel electrophoresis, terminal end labelling and assessment of cell morphology. MCF-7 cells were relatively sensitive to idarubicin, with an IC50 value for growth inhibition of approximately 0.01 microM. While DNA strand breakage was not evident below a concentration of 0.1 microM idarubicin, where growth inhibition exceeded 70%, both the inhibition of DNA synthesis and suppression of c-myc expression closely paralleled the profile of antiproliferative activity for idarubicin. Finally, while exposure to idarubicin resulted in a substantial loss of viable cells within 48-72 h, there was no morphological evidence of apoptotic body formation. The absence of apoptosis in cells exposed to idarubicin was supported by studies demonstrating the absence of DNA fragmentation using gel electrophoresis, alkaline elution and in situ DNA end-labelling assays. The results of these studies extend previous results from this laboratory indicating an association between suppression of c-myc expression, inhibition of DNA synthesis and growth arrest by topoisomerase II inhibitors, as well as the lack of induction of apoptotic cell death by topoisomerase II inhibitors in MCF-7 breast tumor cells.

The prevention of anthracycline cardiomyopathy

The use of anthracyclines, a family of chemotherapeutic agents with efficacy against many solid tumors and leukemias has been limited by their cardiotoxicity. This cardiotoxicity manifests as a reversible acute myopericarditis, an irreversible subacute cardiomyopathy and a progressive late, chronic form. It is particularly important in children who can be expected to survive decades after cure of their malignancy. The incidence of the subacute and late forms have been shown to increase with increasing peak and total cumulative anthracycline dose. Prevention of cardiotoxicity has been attempted by: (1) arbitrary limitation of total cumulative dose; (2) dose limitation guided by monitoring; (3) variation of administrative methods to decrease peak dose by the use of weekly divided boluses or continuous infusion; (4) liposomal encapsulation; (5) use of anthracycline analogues; and (6) concomitant administration of cardioprotective agents. The most promising cardioprotective agent, to date is an intracellular iron chelator which interferes with the formation of free radicals by the anthracyclines, thus limiting cardiac damage from lipid peroxidation. None of these methods has provided full cardiac protection, hence further study of the pathophysiology of anthracycline cardiotoxicity and its prevention is warranted as is long term monitoring of the cancer survivors who have received anthracyclines.

Equilibrium binding of anthracycline cytostatics to serum albumin and small unilamellar phospholipid vesicles as measured by gel filtration

A Sephadex G'200 gel filtration method was used to measure directly the equilibrium binding of five importam anthracycline analogs to serum albumin. The order of the overall binding constant ( K) in a 150 mM NaCl, 20 mM Hepes buffer (pH 7.45) was doxorubicin < daunorubicin < 4-demethoxydaunorubicin ≈ 13-dihydro-4′-deoxy-4′-iododoxorubicin < 4′-deoxy-4′-iododoxorubicin for human serum albumin ( K = 2.67 ± 0.07 mM −1 to 24-5 ± 3.1 mM −1) and bovine serum albumin ( K = 1.36 ± 0.25 mM −1 to 48.4 ± 5.2 mM −1). Data were given on the pH-dependence of K. The anthracycline-albumin association reaction was compared with measurements of drug partitioning into unilamellar phospholipid membranes and octanol. The results provide important new data required for a systematic kinetic analysis of anthracycline transport in tumor cells under serum conditions in a biological system.

New 7-aryl analogues of anthracyclines: Synthesis and cytotoxic activity of (±)-7-(3,4,5-trimethoxyphenyl)-7-deoxyidarubicinone

Abstract The synthetic methodology for the preparation of 7-aryl-7-deoxyanthracyclinones, a new class of anthracycline analogues, is described. Readily available materials are easily transformed into the key intermediate ketone, that is converted into bent and planar tetracyclic compounds. Cytotoxic studies of the final and intermediate products reveal a moderate activity of bent products and a lack of activity of planar products, thus supporting a non-intercalating mechanism for their cytotoxic activity.

The synthesis of anthraquinone derivatives as potential anticancer agents

The simple anthracycline analogues 3a-c were synthesized as potential anticancer agents. Anodic oxidation of the phenol derivative 4, phthalide anion annelation of p-quinone monoacetals 5 and selective attachment of the side chain were the three key steps towards these target compounds.

Mechanisms for high methoxymorpholino doxorubicin cytotoxicity in doxorubicin‐resistant tumor cell lines

Methoxymorpholino doxorubicin (MMRDX) is an anthracycline analogue that is able to overcome tumor cell resistance to classical anthracyclines. Mechanisms for increased MMRDX cytotoxicity were analyzed in a small cell lung carcinoma cell line (GLC4), its 300-fold doxorubicin-resistant and multidrug resistance-associated protein (MRP)–over-expressing subline (GLC4/ADR), an ovarian carcinoma cell line (A2780) and its 100-fold doxorubicin resistant and P-glycoprotein (P-gp)-over-expressing subline A2780AD. Cross-resistance, measured with the MTT assay at MMRDX concentration resulting in 50% growth inhibition, was 1.8-fold in GLC4/ADR and 4.5-fold in A2780AD compared to their respective parental cell lines. Cellular MMRDX accumulation was equal in GLC4 and GLC4/ADR and 2-fold lower in A2780AD compared to A2780. Doxorubicin fluorescence was analyzed with confocal laser scan microscopy. Fluorescence was nuclear in sensitive, and cytoplasmic in resistant, cell lines, while MMRDX fluorescence was found in the nucleus in all cell lines. Pre-incubation with the MRP blocker MK 571 restored in GLC4/ADR cells the nuclear doxorubicin fluorescence pattern, as observed in GLC4 cells. MMRDX, thus, can largely overcome cross-resistance in these P-gp- and MRP-overexpressing doxorubicin-resistant cell lines. Our results suggest that MMRDX is not a substrate for MRP-mediated resistance. Int. J. Cancer 73:362–366, 1997. © 1997 Wiley-Liss, Inc.

Mechanisms for high methoxymorpholino doxorubicin cytotoxicity in doxorubicin-resistant tumor cell lines.

Methoxymorpholino doxorubicin (MMRDX) is an anthracycline analogue that is able to overcome tumor cell resistance to classical anthracyclines. Mechanisms for increased MMRDX cytotoxicity were analyzed in a small cell lung carcinoma cell line (GLC4), its 300-fold doxorubicin-resistant and multidrug resistance-associated protein (MRP)-over-expressing subline (GLC4/ADR), an ovarian carcinoma cell line (A2780) and its 100-fold doxorubicin resistant and P-glycoprotein (P-gp)-overexpressing subline A2780AD. Cross-resistance, measured with the MTT assay at MMRDX concentration resulting in 50% growth inhibition, was 1.8-fold in GLC4/ADR and 4.5-fold in A2780AD compared to their respective parental cell lines. Cellular MMRDX accumulation was equal in GLC4 and GLC4/ADR and 2-fold lower in A2780AD compared to A2780. Doxorubicin fluorescence was analyzed with confocal laser scan microscopy. Fluorescence was nuclear in sensitive, and cytoplasmic in resistant, cell lines, while MMRDX fluorescence was found in the nucleus in all cell lines. Pre-incubation with the MRP blocker MK 571 restored in GLC4/ADR cells the nuclear doxorubicin fluorescence pattern, as observed in GLC4 cells. MMRDX, thus, can largely overcome cross-resistance in these P-gp- and MRP-overexpressing doxorubicin-resistant cell lines. Our results suggest that MMRDX is not a substrate for MRP-mediated resistance.

Diels-alder reactivity of 1-alkoxyphenyl-3-trialkylsiloxy-1,3-dienes

The synthesis of 1-(3,4,5-trimethoxyphenyl)-3-trialkylsiloxy-1,3-butadienes and their Diels-Alder reaction with selected dienophiles at room temperature is described. These aryldienes are useful building blocks for the synthesis of natural and pharmacological active products, as has been shown by the preparation of a tetracyclic ketone needed for the synthesis of new anthracycline analogues.

Cellular uptake and antitumor activity of the new anthracycline analog DA-125 in human cancer cell lines.

To predict the clinical usefulness of DA-125, a newly developed doxorubicin analog, we compared its antitumor activity against 20 different human cancer cell lines with that of doxorubicin using the MTT in vitro chemosensitivity test. We also measured and compared the cellular uptake of this drug and doxorubicin in two cancer cell lines and their doxorubicin-resistant sublines. In the MTT test, DA-125 showed lower IC50 values than doxorubicin for 14 of 20 cell lines. DA-125 was more potent than doxorubicin for hepatocellular cancer cells with high mdr 1 expression. Among cancer cells from the stomach and colon, DA-125 was more potent than doxorubicin in 12 of 14 cell lines. We also investigated the cross-resistance of this drug with doxorubicin using four doxorubicin-resistant cancer cell sublines. Except in one cell line, there was very low cross-resistance. Cellular drug-uptake experiments were performed for two gastric cancer cell lines and their doxorubicin-resistant sublines. In this experiment, DA-125 was found to be very rapidly and completely converted to its active metabolite, M1, in the culture media. After this conversion, M1 was incorporated into these cancer cells more rapidly and reached higher intracellular concentrations than doxorubicin, suggesting that DA-125 (as M1) could achieve earlier and higher levels of intracellular accumulation than doxorubicin in its target tissues from the bloodstream. As a possible alternative antineoplastic agent to doxorubicin, DA-125 awaits further evaluation for its antitumor activity and toxicity.

Activation of erythroid-specific promoters during anthracycline-induced differentiation of K562 cells

Anthracycline antitumor drugs such as aclacinomycin (ACM) and doxorubicin (DOX) used in subtoxic concentrations induce erythroid differentiation of the erythroleukemic cell line K562. To elucidate the possible role of erythroid genes of the erythropoietin receptor (EpoR) and the transcription factor GATA-1 in this effect, the regulatory regions of the above genes and human epsilon- and gamma-globin and porphobilinogen deaminase (PBGD) genes were fused to the firefly luciferase gene. The resulting reporter constructs were tested in a transfection assay of the erythroleukemic cell line K562 stimulated to differentiate by treatment with the anthracycline drugs ACM and DOX or hemin (HEM). The results showed activation of the tested promoters after cell treatment with ACM, but not with DOX or HEM. In contrast to the mouse EpoR gene promoter, the activity of the human EpoR gene promoter (-659/-60) in the reporter construct was not modified by addition of the first intron sequence. In ACM-treated K562 cells, EpoR gene promoter activity completely correlated with EpoR and GATA-1 mRNA levels and the degree of erythroid maturation. In addition, ACM strongly activated the erythroid gene promoters that contain GATA binding sites. Nevertheless, less activation was also observed for the GATA-1 gene promoter (-312/-31) lacking any known GATA binding sites. Insertion of the GATA-1 gene enhancer with two canonic GATA binding sites, stimulated the ACM activation effect for EpoR and GATA-1 promoter-containing constructs. Mutation of the enhancer GATA binding sites abolished this effect. All the regulatory regions tested (except gamma-globin promoter) were completely inactive in nonerythroid COS7 cells. These data indicate that (1) two structurally different anthracycline analogues, DOX and ACM, differ in their differentiation mechanisms, and (2) ACM switches on the erythroid program of K562 cells, at least in part because of interaction with a factor(s) that recognizes the GATA binding sites in the promoter region of erythroid genes leading to their activation.

Pharmacology of N,N-di(n-butyl)adriamycin-14-valerate in the rat.

Lipophilic N-alkylanthracyclines such as AD 198 (N-benzyladriamycin-14-valerate) or AD 201 [N,N-di(n-propyl)adriamycin-14-valerate], which exert their cytotoxicity through mechanisms which are not yet fully defined, possess inherent abilities to circumvent multidrug resistance in vitro and in vivo, possibly though alterations in normal intracellular drug trafficking. As part of structure-activity studies with this class of agent, we have now examined the pharmacology of AD 202 [N,N-di(n-butyl)adriamycin-14-valerate], another analog possessing superior antitumor activity to doxorubicin in vivo and an ability to circumvent multidrug resistance in vitro. Following the administration of AD 202 (20 mg/kg, i.v.) to anesthetized rats, rapid drug distribution (T1/2 5 min) was followed by more gradual elimination (T1/2 3.6h). Plasma clearance of AD 202 (224 +/- 63.6 ml/min per kg) and steady state volume of distribution (25.7 +/- 11.1 l/kg) were indicative of extensive tissue sequestration and/or a large degree of extra-hepatic metabolism. The parent drug predominated in plasma until 20 min, thereafter N,N-di(n-butyl)adriamycin became the principal circulating anthracycline. The systemic exposure to this biotransformation product (area under the plasma concentration-time curve from time zero to 480 min AUC(0-480) 28 1672 ng.min/ml) was > tenfold higher than for the other detected plasma products (N-butyladriamycin-14-valerate, N-butyladriamycin, and three unidentified fluorescent signals; P1-3). Total urinary elimination over 8h was limited (1.9% of dose), occurring predominantly as N,N-di(n-butyl)adriamycin (1.2% of dose), N-butyladriamycin (0.4% of dose), and their corresponding 13-carbinol metabolites (<0.1% of dose each). Low levels of adriamycin (ADR), aglycones and two unidentified products were also seen. Parental AD 202 was found in urine only up to 1h. By contrast, hepatic elimination of parent drug was seen, albeit at low levels, through 8h. Excretion by this route (22% of dose) occurred principally as N-butyl-adriamycin (8% of dose), N-butyladraimycinol (2.1% of dose) with lower levels of N,N-di(n-butyl)adriamycin (1.6% of dose), N,N-di(n-butyl)adriamycin (0.8% of dose), and aglycones (4.3% of dose, combined). Other products included ADR (1.1% of dose) and two unidentified signals (3.4% of dose, combined). The relatively poor mass balance in these studies is attributed to prolonged intracellular retention (elimination T1/2 24.2h) of N,N-di(n-butyl)adriamycin. Thus, in common with other N-alkylanthracyclines, the pharmacology of AD 202 is complex but its therapeutic properties clearly are not derived from an ADR prodrug effect. Significant differences continue to be noted as to the metabolic fate of congeners of this class of anthracycline analogs.

Efficacy of, and toxicoses associated with, oral idarubicin administration in cats with neoplasia

Summary Idarubicin, a new synthetic anthracycline analogue, was administered orally to 34 cats with spontaneous tumors. The maximum tolerated dosage was determined to be 2 mg/cat/d given for 3 consecutive days every 3 weeks. Anorexia and leukopenia were found to be dose limiting in cats receiving the drug at a higher dosage. The most common toxicoses seen at the maximum tolerated dosage were leukopenia, anorexia, and vomiting; however, development of toxicoses was not found to be associated with sex, FeLV test result, tumor type, dosage, age, or weight. Idarubicin (2 mg/cat/d for 3 days, q 3 wks) was used to treat 18 cats with lymphoma in which complete remission had been achieved by administration of other chemotherapeutic agents. Median remission duration for these cats was comparable to that reported for cats treated with other protocols. We concluded that orally administered idarubicin would be useful in the treatment of cats with lymphoma.

Growth-inhibitory properties of novel anthracyclines in human leukemic cell lines expressing either Pgp-MDR or at-MDR.

The objective of the experiments reported in this paper was the identification of promising anthracycline analogs on the basis of lack of cross-resistance against tumor cells presenting either P-glycoprotein multidrug resistance (Pgp-MDR) or the altered topoisomerase multidrug resistant (at-MDR) phenotype. Differently modified anthracycline analogs known to be active against MDR cells were assayed in vitro against CEM human leukemic cells, and the sublines CEM/VLB100 and CEM/VM-1 exhibiting respectively the Pgp-MDR and the at-MDR phenotype. Two classes of molecules, in which the -NH2 group in C-3' position is substituted with a morpholino, methoxymorpholino (morpholinyl-anthracycline), or an alkylating moiety, present equivalent efficacy in the drug-sensitive and the two drug-resistant sublines. These results indicate that such molecules may exert their cytotoxic effect through a mode of action different from that of "classical" anthracyclines and is not mediated through topoisomerase II inhibition. Both molecules represent novel concepts in the field of new anthracyclines derivatives.

Potential Role of Oral Anthracyclines in Older Patients with Cancer

Two anthracycline analogues, idarubicin and menogaril, have acceptable bioavailability via the oral route of administration. Encouraging antitumour activity of oral idarubicin has been reported in breast cancer, non-lymphocytic leukaemia, non-Hodgkin's lymphoma and myeloma. The outlook for menogaril is less clear, given the modest antitumour activity reported so far. Although the oral formulations of idarubicin and menogaril remain investigational, they represent a step forward in the direction of developing new active anticancer drugs with oral bio-availability. Further prospective studies of the orally-active anthracyclines in elderly patients with cancer are justified. These studies should address specific issues such as optimal dosage regimens as a function of 'physiological age', and quality of life.

The efflux of anthracyclines in multidrug-resistant cell lines

In order to address the association of enhanced drug efflux with the multidrug-resistant (MDR) phenotype, we have studied the cellular pharmacokinetics of anthracyclines in the P-glycoprotein (Pgp)-positive MDR cell lines H69/LX4 (human small cell lung cancer) and EMT6/AR1.0 (mouse mammary tumour). Both doxorubicin (DOX) and daunorubicin (DNR) were accumulated to a lesser extent and effluxed at a higher rate by MDR cells than by their drug-sensitive counterparts. In contrast, the 9-alkyl substituted compound, aclacinomycin A (ACL), was accumulated and effluxed from parent and MDR cells at an identical rate. In experiments designed to examine energy-dependent efflux, DOX and DNR were shown to be efficiently effluxed against the concentration gradient in the presence of glucose. However, in the same experiments the analogues ACL and Ro 31-3294 (9-alkyl and morpholinyl substituted), which have previously been shown to retain activity against MDR cell lines, were accumulated and effluxed at identical rates in parent and MDR EMT6 cells. Hence, 9-alkyl and morpholinyl substituted compounds appear to behave less favourably as substrates for energy-driven drug efflux by Pgp-positive MDR cells than do DOX or DNR. Resistance modifiers verapamil and cyclosporin A appeared to abolish energy-dependent efflux for DOX and DNR in both the EMT6 and H69 MDR lines whereas they had no effect on the cellular efflux of ACL. The altered cellular pharmacology in MDR cell lines may provide a rational basis for the use of modified anthracycline analogues (e.g. 9-alkyl and morpholinyl substituted) and resistance of modifying agents in the treatment of tumours expressing a Pgp-mediated phenotype.

Chemotherapy for Soft-Tissue Sarcomas

Single-agent and combination chemotherapy have greater than 20% single-agent activity: doxorubicin and ifosfamide. Certain other anthracycline analogues possess similar activity. It remains a question whether combination therapy is any better than single-agent doxorubicin. Randomized trials have demonstrated enhanced response rates and increased toxicity for the combinations, but survival, with limited follow-up data, is not improved. The role of adjuvant chemotherapy also is not yet established. Two of 12 trials demonstrate survival benefits; however, most show a delay in relapse with about a 10% risk of irreversible toxicity. New agents, targets, and methods to circumvent resistance mechanisms developed in the laboratory are necessary to advance the field.

THE DEVELOPMENT OF ANTHRACYCLINE ANALOGS (REVIEW)

The anthracycline class of antitumour agents have the widest spectrum of activity of all drugs used to treat malignant disease. Consequently they are ubiquitous in their use worldwide, but are not without problems. The abrogation of cardiac toxicity and the development of drug resistance has been attempted by modifying the molecule chemically, producing over 1000 structural analogues since the discovery of Daunorubicin in the late 1950's. This review charts this developmental process and points to the possible future of this important class of drugs.

Treatment of myelodysplastic syndromes with daily oral idarubicin. A phase I–II study

Idarubicin, a new anthracycline analogue, is available in an oral preparation, and responses have been observed using relatively aggressive therapy in patients with myelodysplastic syndromes (MDS). The authors studied whether a chronic low-dose schedule would be effective but less myelotoxic. Forty-two patients with MDS received daily low-dose oral idarubicin in 5-week courses that included 3 weeks of treatment, followed by a 2-week rest period. Doses were escalated when possible after the second course, and each patient was to receive six courses. Only one partial response was observed. Although no patient had fatal bone marrow toxicity, only eight patients received the full six courses, primarily because of myelosuppression. This schedule of oral idarubicin is relatively safe but produces fewer responses than are reported with the high-dose pulse regimens.