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[Structure: see text] The asymmetric synthesis of a highly functionalized tetrahydroisoquinoline relevant to the total synthesis of Et-743 is described. The key step involves a highly diastereoselective radical cyclization on a glyoxalimine derivative.
[formula: see text] A short synthesis of saframycin A is described which begins with a readily available intermediate previously utilized for the total synthesis of ecteinascidin 743. A key step in this synthesis is the use of 1-fluoro-3,5-dichloropyridinium triflate to oxidize a phenolic ring to a 1,4-benzoquinone unit while simultaneously cleaving a methoxymethyl ether of a different phenolic ring to the corresponding phenol (4-->5). The common intermediate (2) for the synthesis of saframycin A (1) and ecteinascidin 743 also allowed the synthesis of two hybrids of these structures (6 and 7). Whole cell bioassays for antitumor activity using lung, colon, melanoma, and prostate-derived tumor cell lines allowed a clear correlation of structure with biological activity in this series.
BACKGROUND: Ecteinascidin-743 (ET-743), a member of the ecteinascidin family selected for clinical development, is a tetrahydroisoquinolone alkaloid isolated from the marine ascidian, Ecteinascidia turbinata. This novel compound is a minor groove binding, guanine-specific alkylating agent which also interacts with the microtubule network and blocks cell cycle progression at late S/G2. MATERIALS AND METHODS: A soft agar cloning assay was used to determine the in vitro effects of ET-743 against primary human tumor specimens taken directly from patients. A total of 93 evaluable specimens were exposed to ET-743 for one-hour (n = 25) and/or 14-day continuous exposure (n = 92) at concentrations ranging from 0.1 nM to 1 microM. In vitro responses were defined as an inhibition > or = 50% of human tumor colony forming units at a given concentration. RESULTS: One-hour exposure to ET-743 at concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM and 1 microM induced in vitro responses in 0% (0/17), 6% (1/17), 16% (4/25), 13% (1/8), and 25% (2/8) of specimens, respectively. Continuous exposure to ET-743 at concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM and 1 microM, inhibited 0% (0/16), 13% (2/16), 49% (44/90), 62% (47/76), and 77% (58/75) of tumor specimens, respectively. Tumor-specific responses and concentration-dependent relationships were observed with a continuous exposure to ET-743. At 100 nM, the compound inhibited 79% (11/14) breast, 69% (9/13) non-small-cell lung, 58% (7/12) ovary, and 88% (7/8) melanoma specimens. At 1 microM, ET-743 inhibited 100% (14/14) breast specimens, 85% (11/13) non-small-cell lung, 67% (8/12) ovary and 86% (6/7) melanoma specimens. Activity of ET-743 at and above 10 nM was also observed against sarcoma and kidney tumors. At 10 nM concentration and continuous exposure ET-743 demonstrated incomplete cross-resistance with paclitaxel, alkylating agents, doxorubicin and cisplatin. CONCLUSIONS: Our data from the cloning assay indicate that the duration of exposure to ET-743 is an important factor in human tumors. Therefore, long-term exposure to ET-743 may be preferred in future clinical trials. The activity of ET-743 in breast, non-small-cell lung, and ovarian cancers as well as in melanoma may deserve further clinical evaluations. The potential of ET-743 in sarcoma and renal tumors might also be considered. In addition, our data indicate that a plasma concentration of 100 nM of ET-743 must be considered as a target during the clinical development of the compound; also the concept of continuous/protracted exposure in clinical trials with ET-743 has to be taken into account.
BACKGROUND: Ecteinascidin-743 (ET743) is a novel antitumour agent originating from the Caribbean tunicate Ecteinascidia turbinata. It has potent cytotoxic and antitumour activity and a potential new mechanism of action. The aim of the present study was to further explore the antitumour activity of ET743 in human tumour xenografts from melanoma, non-small-cell lung and ovarian cancer. DESIGN: As the antitumour profile of ET743 was largely unknown a chemo-sensitive and a marginal chemo-resistant human tumour xenograft were selected for each tumour type. ET743 was administered intravenously using two administration schedules (days 0, 4, 8 and 0-2, 13-15). RESULTS: ET743 was very active at the maximum tolerated dose (MTD) in the chemo-sensitive xenograft melanoma MEXF 989, non-small-cell lung cancer LXFL 529, and ovarian cancers HOC22 and (marginally resistant to cisplatin) HOC18. Activity was also seen at 1/2 MTD. Apart from HOC18, ET743 caused complete remissions in the responding xenografts. The compound was inactive in the chemo-resistant xenograft melanoma MEXF 514 and non-small-cell lung cancer LXFA 629. In terms of antitumour activity the days 0, 4, 8 schedule had advantages over the days 0-2, 13-15 schedule. CONCLUSIONS: ET743 is a very effective agent in chemo-sensitive and marginal chemo-resistant xenografts, but inactive in chemo-resistant tumour xenografts. The activity of ET743 in the marginally cisplatin-resistant ovarian cancer HOC18 might indicate absence or incomplete cross-resistance against cisplatin. It is recommended to include melanoma, non-small-cell lung cancer, and ovarian cancer in phase II clinical trials and to use an intermittent schedule.
BACKGROUND: Ecteinascidin (ET-743) is a marine derived compound with an interesting preclinical profile currently completing phase I clinical trials. The present study was undertaken to compare the toxicity of different schedules of ET-743 against human hemopoietic progenitors and tumour cell lines. MATERIALS AND METHODS: Human hemopoietic progenitors and solid tumour cell lines were incubated with ET-743 for one hour, 24 hours and one hour daily for five consecutive days to define by comparison an 'in vitro therapeutic index'. Additional experiments were set up to assess whether incubation for 24 hours or five days could change either the sensitivity of cells or the activity of ET-743. RESULTS: Prolonged or repeated exposures were more toxic than a single one hour exposure (P < 0.001), but due to the higher sensitivity to prolonged exposure of several tumor cell lines, prolonged treatment yielded a more favorable in vitro therapeutic index. After incubation for 24 hours, ET-743 showed a significantly (P < 0.01) lower inhibiting capacity. Incubation before treatment rendered progenitors more resistant, but incubation after treatment increased their sensitivity, so that overall the toxicity of ET-743 on hemopoietic cells appears to be close to AUC dependency. CONCLUSIONS: Despite the possible effect of some experimental artefacts, prolonged exposure could represent the best schedule of administration of ET-743.
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Cytochrome P450 3A4 is an important mediator of drug catabolism that can be regulated by the steroid and xenobiotic receptor (SXR). We show here that SXR also regulates drug efflux by activating expression of the gene MDR1, which encodes the protein P-glycoprotein (ABCB1). Paclitaxel (Taxol), a commonly used chemotherapeutic agent, activated SXR and enhanced P-glycoprotein-mediated drug clearance. In contrast, docetaxel (Taxotere), a closely related antineoplastic agent, did not activate SXR and displayed superior pharmacokinetic properties. Docetaxel's silent properties reflect its inability to displace transcriptional corepressors from SXR. We also found that ET-743, a potent antineoplastic agent, suppressed MDR1 transcription by acting as an inhibitor of SXR. These findings demonstrate how the molecular activities of SXR can be manipulated to control drug clearance.
While investigating the novel anticancer drug ecteinascidin 743 (Et743), a natural marine product isolated from the Caribbean sea squirt, we discovered a new cell-killing mechanism mediated by DNA nucleotide excision repair (NER). A cancer cell line selected for resistance to Et743 had chromosome alterations in a region that included the gene implicated in the hereditary disease xeroderma pigmentosum (XPG, also known as Ercc5). Complementation with wild-type XPG restored the drug sensitivity. Xeroderma pigmentosum cells deficient in the NER genes XPG, XPA, XPD or XPF were resistant to Et743, and sensitivity was restored by complementation with wild-type genes. Moreover, studies of cells deficient in XPC or in the genes implicated in Cockayne syndrome (CSA and CSB) indicated that the drug sensitivity is specifically dependent on the transcription-coupled pathway of NER. We found that Et743 interacts with the transcription-coupled NER machinery to induce lethal DNA strand breaks.
The effect of selected marine compounds with anti-tumoral activity on the cell microtubule network was tested by immunofluorescence analyses, or by other in vitro analyses involving competition with colchicine or with GTP for tubulin binding and tubulin polymerisation, studies that were carried out in parallel with other microtubule poisons used as controls. Three compounds were found to disorganise the microtubule network: isohomohalichondrin B, LL-15 and ecsteinascidin 743. The first two compounds prevent microtubule assembly and GTP binding to tubulin. Ecteinascidin 743 disorganises the microtubule network but it does not seem to interact directly with tubulin.
Ecteinascidin 743 (ET-743) is a new marine-derived agent with promising activity against a number of solid tumours. In four human tumour cell lines, the interaction between ET-743 and radiation was investigated in relation to the effects of ET-743 on the cell cycle, in vitro. Cell survival was measured based on quantitative staining of cellular protein by sulforhodamine B. A 24 h treatment with ET-743 before radiation resulted in a moderate increase in radiosensitivity in three out of four cell lines. Dose enhancement factors > or =1.8 were observed for concentrations resulting in 52, 46 and 30% cell kill in ECV304, H292 and CAL-27, respectively, whereas in A549 no radiosensitisation was observed (no significant increase in radiosensitivity). According to the combination index analysis, synergism was observed only in ECV304 and CAL-27 cells. A 24 h incubation with ET-743 resulted in a concentration-dependent G2/M block, which might explain the moderate radiosensitising effects in ECV304 and H292. The lack of radiosensitisation in A549 might be due to the S phase delay preceding the G2/M block at the moment of radiation, which only occurred in this cell line. In conclusion, ET-743 has moderate cell line-dependent radiosensitising properties; however, only when cytotoxic concentrations of ET-743 are used. In one of the four cell lines tested, no radiosensitisation was observed.
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By exposing Igrov-1 human ovarian cancer cells to increasing concentrations of Ecteinascidin-743 (ET-743), either for a short or prolonged time, we obtained sublines resistant to ET-743 which overexpress Pgp. The most resistant clone (Igrov-1/25 ET) was evaluated for biological and pharmacological characterizations. The increased Pgp levels of Igrov-1/25 ET were not due to amplification of the mdr-1 gene but to increased mRNA levels. No increase in other multidrug resistance-related proteins such as MRP or LRP was observed in Igrov-1/25 ET. The IC50 values of ET-743 against Igrov-1/25 ET was approximately 50 times higher than the parental cell line. Resistance was not reversed while maintaining the cell line in drug-free medium for at least 24 months. Igrov-1/25 ET was cross-resistant to Doxorubicin and VP16 while it was equally sensitive to L-PAM, MNNG, CPT and only marginally less sensitive to Cis-DDP and Oxaliplatin compared to the parental cell line. Igrov-1/25 ET exposed to Doxorubicin retained this drug much less, mainly because of a more efficient drug efflux. The cyclosporine analogue SDZ PSC-833 reversed the resistance of Igrov-1/25 ET to ET-743, without any enhancement of the drug activity against the parental Igrov-1 cell line. Igrov-1/25 ET exhibits typical features of cell lines overexpressing the mdr-1 gene and can be a potentially useful tool in selecting ET-743 non-cross-resistant analogues as well as to investigate methods to counteract resistance to this drug.
Cockayne syndrome (CS) is an inherited neurodevelopmental disorder with progeroid features. Although the genes responsible for CS have been implicated in a variety of DNA repair- and transcription-related pathways, the nature of the molecular defect in CS remains mysterious. Using expression microarrays and a unique method for comparative expression analysis called L2L, we sought to define this defect in cells lacking a functional CS group B (CSB) protein, the SWI/SNF-like ATPase responsible for most cases of CS. Remarkably, many of the genes regulated by CSB are also affected by inhibitors of histone deacetylase and DNA methylation, as well as by defects in poly(ADP-ribose)-polymerase function and RNA polymerase II elongation. Moreover, consistent with these microarray expression data, CSB-null cells are sensitive to inhibitors of histone deacetylase or poly(ADP-ribose)-polymerase. Our data indicate a general role for CSB protein in maintenance and remodeling of chromatin structure and suggest that CS is a disease of transcriptional deregulation caused by misexpression of growth-suppressive, inflammatory, and proapoptotic pathways.
Ecteinascidin 743 (Et743, National Service Center 648766) is a potent antitumor agent from the Caribbean tunicate Ecteinascidia turbinata. Although Et743 is presently in clinical trials for human cancers, the mechanisms of antitumor activity of Et743 have not been elucidated. Et743 can alkylate selectively guanine N2 from the DNA minor groove, and this alkylation is reversed by DNA denaturation. Thus, Et743 differs from other DNA alkylating agents presently in the clinic (by both its biochemical activities and its profile of antitumor activity in preclinical models). In this study, we investigated cellular proteins that can bind to DNA alkylated by Et743. By using an oligonucleotide containing high-affinity Et743 binding sites and nuclear extracts from human leukemia CEM cells, we purified a 100-kDa protein as a cellular target of Et743 and identified it as topoisomerase I (top1). Purified top1 was then tested and found to produce cleavage complexes in the presence of Et743, whereas topoisomerase II had no effect. DNA alkylation was essential for the formation of top1-mediated cleavage complexes by Et743, and the distribution of the drug-induced top1 sites was different for Et743 and camptothecin. top1-DNA complexes were also detected in Et743-treated CEM cells by using cesium chloride gradient centrifugation followed by top1 immunoblotting. These data indicate that DNA minor groove alkylation by Et743 induces top1-mediated protein-linked DNA breaks and that top1 is a target for Et743 in vitro and in vivo.
A series of totally synthetic molecules that are structurally related to the marine natural product ecteinascidin 743 (Et 743) has been prepared and evaluated as antitumor agents. The most active of these, phthalascidin, is very similar to Et 743 with regard to in vitro potency and mode of action across a variety of cell types. The antiproliferative activity of phthalascidin (IC50 = 0.1-1 nM) is greater than that of the agents Taxol, camptothecin, adriamycin, mitomycin C, cisplatin, bleomycin, and etoposide by 1-3 orders of magnitude, and the mechanism of action is clearly different from these currently used drugs. Phthalascidin and Et 743 induce DNA-protein cross-linking and, although they seem to interact with topoisomerase (topo) I (but not topo II), topo I may not be the primary protein target of these agents. Phthalascidin and Et 743 show undiminished potency in camptothecin- and etoposide-resistant cells. Phthalascidin is more readily synthesized and more stable than Et 743, which is currently undergoing clinical trials. The relationship of chemical structure and antitumor activity for this class of molecules has been clarified by this study.
Ecteinascidin 743 (ET-743), a highly promising marine-based antitumor agent presently in phase II clinical trials, has been shown to interfere with the binding of minor-groove-interacting transcription factors, particularly NF-Y, with their cognate promoter elements in vitro. We have shown that NF-Y is a central mediator of activation of transcription of the human P glycoprotein gene (MDR1) by a variety of inducers and that NF-Y functions by recruiting the histone acetyltransferase PCAF to the MDR1 promoter. In the present study, we tested whether ET-743 could block activation of the MDR1 promoter by agents that mediate their effect through the NF-Y/PCAF complex. We report that physiologically relevant concentrations of ET-743 abrogate transcriptional activation of both the endogenous MDR1 gene and MDR1 reporter constructs by the histone deacetylase inhibitors as well as by UV light, with minimal effect on constitutive MDR1 transcription. Notably, this inhibition does not alter the promoter-associated histone hyperacetylation induced by histone deacetylase inhibitors, suggesting an in vivo molecular target downstream of NF-Y/PCAF binding. ET-743 is therefore the prototype for a distinct class of transcription-targeted chemotherapeutic agents and may be an efficacious adjuvant to the treatment of multidrug-resistant tumors.
Ecteinascidin-743 (ET-743) is a tetrahydroisoquinoline alkaloid isolated from the tunicate Ecteinascidia turbinata currently under phase II clinical trials for its potent anticancer activity. ET-743 binds DNA in the minor groove and forms covalent adducts with some sequence specificity. It selectively inhibits in vitro binding of the CCAAT box factor NF-Y. In this study, we assayed ET-743 function in vivo on the HSP70 promoter. On heat induction, the drug blocks transcription rapidly at pharmacological concentrations and in a CCAAT-dependent manner, whereas the activity of the CCAAT-less simian virus 40 promoter is not affected. The effect is exerted at the mRNA level. The distamycin-like alkylating tallimustine is inactive in these assays. Binding of NF-Y and of the heat-shock factor is normal in ET-743-treated cells. Run-on analysis of several endogenous genes further proves that the drug has rapid, profound, and selective negative effects on transcription. Thus, this marine-derived compound is a promoter-specific, transcription-interfering agent.