Mathew P. Leese

Inhibition of in vitro angiogenesis by 2-methoxy- and 2-ethyl-estrogen sulfamates

Sulfamoylation of 2‐methoxyestrone (2‐MeOE1) was shown previously to enhance its potency as an anti‐proliferative agent against breast cancer cells. We have examined the ability of a series of 2‐methoxyestradiol (2‐MeOE2) and 2‐ethylestradiol (2‐EtE2) sulfamates to inhibit angiogenesis in vitro. 2‐MeOE2 bis‐sulfamate and 2‐EtE2 sulfamate were potent inhibitors of human umbilical vein endothelial cell (HUVEC) proliferation with IC50 values of 0.05 μM and 0.01 μM, respectively. A novel co‐culture system, in which endothelial cells were cultured in a matrix of human dermal fibroblasts, was also used to assess the anti‐angiogenic potential of these drugs. In this system endothelial cells proliferate and migrate through the culture matrix to form tubule structures. Whereas 2‐MeOE2 (1.0 μM) caused a small reduction in tubule formation, both 2‐MeOE2 bis‐sulfamate (0.1 μM) and 2‐EtE2 sulfamate (0.1 μM) almost completely abolished tubule formation. 2‐MeOE2 bis‐sulfamate and 2‐EtE2 sulfamate both induced BCL‐2 phosphorylation, p53 protein expression and apoptosis in HUVECs. Microarray analysis of a limited number of genes known to be involved in the angiogenic process did not show any gross changes in cells treated with the 2‐substituted estrogens. The sulfamoylated derivatives of 2‐MeOE2 and 2‐EtE2 are potent inhibitors of in vitro angiogenesis and both compounds should have therapeutic potential.

Inhibition of MCF-7 breast cancer cell proliferation and in vivo steroid sulphatase activity by 2-methoxyoestradiol-bis-sulphamate.

The endogenous oestrogen metabolite, 2-methoxyoestradiol (2-MeOE2) inhibits the growth of breast cancer cells and is also a potent anti-angiogenic agent. We have previously shown that the 3-sulphamoylated derivatives of 2-methoxyoestrogens are more potent than the non-sulphamoylated compounds. In this study, we have compared the abilities of 2-methoxyoestradiol-bis-sulphamate (2-MeOE2bisMATE) and 2-MeOE2 to inhibit the growth of MCF-7 breast cancer cells. Both compounds inhibited cell growth with the IC(50) for 2-MeOE2bisMATE (0.4 microM) being six-fold lower than that for 2-MeOE2 (2.5 microM). Oestrogen sulphamates are potent inhibitors of steroid sulphatase (STS) activity. 2-MeOE2bisMATE was found to retain its STS inhibitory activity and in a placental microsome assay system it was equipotent with oestrone-3-O-sulphamate (EMATE). An in vivo study was also carried out to compare the potency of 2-MeOE2bisMATE with that of EMATE and the non-steroidal STS inhibitor, 667 coumarin sulphamate (667 COUMATE). After a single oral dose (10mg/kg) some recovery of STS activity was detected by day 3 (10%) with activity partially restored (55%) by day 7 after administration of 667 COUMATE. For the other two steroidal compounds, STS activity remained almost completely inactivated for up to 5 days with complete restoration of activity occurring by day 15. The anti-proliferative and STS inhibitory properties of 2-MeOE2bisMATE suggest that it has considerable potential for development as a novel anti-cancer drug.

The role of 17β-hydroxysteroid dehydrogenases in modulating the activity of 2-methoxyestradiol in breast cancer cells

The bis-sulfamoylated derivative of 2-methoxyestradiol (2-MeOE2), 2-methoxyestradiol-3,17-O,O-bis-sulfamate (2-MeOE2bisMATE), has shown potent antiproliferative and antiangiogenic activity in vitro and inhibits tumor growth in vivo. 2-MeOE2bisMATE is bioavailable, in contrast to 2-MeOE2 that has poor bioavailability. In this study, we have examined the role of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type 2 in the metabolism of 2-MeOE2. In MDA-MB-231 cells, which express high levels of 17beta-HSD type 2, and in MCF-7 cells transfected with 17beta-HSD type 2, high-performance liquid chromatography analysis showed that a significant proportion of 2-MeOE2 was metabolized to inactive 2-methoxyestrone. Furthermore, MCF-7 cells transfected with 17beta-HSD type 2 were protected from the cytotoxic effects of 2-MeOE2. In contrast, no significant metabolism of 2-MeOE2bisMATE was detected in transfected cells and 17beta-HSD type 2 transfection did not offer protection against 2-MeOE2bisMATE cytotoxicity. This study may go some way to explaining the poor bioavailability of 2-MeOE2, as the gastrointestinal mucosa expresses high levels of 17beta-HSD type 2. In addition, this study shows the value of synthesizing sulfamoylated derivatives of 2-MeOE2 with C17-position modifications as these compounds have improved bioavailability and potency both in vitro and in vivo.

Structure-activity relationships of C-17 cyano-substituted estratrienes as anticancer agents

The synthesis, SAR, and preclinical evaluation of 17-cyanated 2-substituted estra-1,3,5(10)-trienes as anticancer agents are discussed. 2-Methoxy-17beta-cyanomethylestra-1,3,5(10)-trien-3-ol ( 14), but not the related 2-ethyl derivative 7, and the related 3- O-sulfamates 8 and 15 display potent antiproliferative effects (MCF-7 GI 50 300, 60 and 70 nM, respectively) against human cancer cells in vitro. Investigation of the SAR reveals that a sterically unhindered hydrogen bond acceptor attached to C-17 is most likely key to the enhanced activity. Compound 8 displayed significant in vitro antiangiogenic activity, and its ability to act as a microtubule disruptor was confirmed. Inhibitory activity of the sulfamate derivatives against steroid sulfatase and carbonic anhydrase II (hCAII) was also observed, and the interaction between 15 and hCAII was investigated by protein crystallography. The potential of these multimechanism anticancer agents was confirmed in vivo, with promising activity observed for both 14 and 15 in an athymic nude mouse MDA-MB-231 human breast cancer xenograft model.

Anti-cancer activities of novel D-ring modified 2-substituted estrogen-3-O-sulfamates

Sulfamoylated derivatives of the endogenous estrogen metabolite 2-methoxyestradiol (2-MeOE2 (7)), such as 2-methoxy-3-O-sulfamoyl estrone (2-MeOEMATE (1)), display greatly enhanced activity against the proliferation of human cancer cells and inhibit steroid sulphatase (STS), another current oncology target. We explore here the effects of steroidal D-ring modification on the activity of such 2-substituted estrogen-3-O-sulfamates in respect of inhibition of tumour cell proliferation and steroid sulphatase. The novel 17-deoxy analogues of 2-MeOEMATE and the related 2-ethyl and 2-methylsulfanyl compounds showed greatly reduced inhibition of MCF-7 proliferation. Introduction of a 17alpha-benzyl substituent to such 2-substituted estrogen sulfamates also proved deleterious to anti-proliferative activity but could, in one case, enhance STS inhibition with respect to the parent substituted estrone sulfamate. In contrast, selected 17-oxime derivatives of 2-MeOEMATE displayed an enhanced anti-proliferative activity. These results illustrate that enhanced in vitro anti-cancer activity can be achieved in the 2-substituted estrogen sulfamate series and highlight, in particular, the importance of potential hydrogen bonding effects around the steroidal D-ring in the activity of these molecules. The SAR parameters established herein will assist the future design of anti-proliferative and anti-endocrine agents as potential therapeutics for both hormone dependent and independent cancers.

STX140 Is Efficacious In vitro and In vivo in Taxane-Resistant Breast Carcinoma Cells

Purpose: The aim of these studies was to characterize the action of STX140 in a P-glycoprotein–overexpressing tumor cell line both in vitro and in vivo. In addition, its efficacy was determined against xenografts derived from patients who failed docetaxel therapy. Experimental Design: The effects of STX140, Taxol, and 2-methoxyestradiol (2-MeOE2) on cell proliferation, cell cycle, and apoptosis were assessed in vitro in drug-resistant cells (MCF-7DOX) and the parental cell line (MCF-7WT). Mice bearing an MCF-7DOX tumor on one flank and an MCF-7WT tumor on the other flank were used to assess the in vivo efficacy. Furthermore, the responses to STX140 of three xenografts, derived from drug-resistant patients, were assessed. Results: In this study, STX140 caused cell cycle arrest, cyclin B1 induction, and subsequent apoptosis of both MCF-7DOX and MCF-7WT cells. Taxol and 2-MeOE2 were only active in the MCF-7WT parental cell line. Although both STX140 and Taxol inhibited the growth of xenografts derived from MCF-7WT cells, only STX140 inhibited the growth of tumors derived from MCF-7DOX cells. 2-MeOE2 was ineffective at the dose tested against both tumor types. Two out of the three newly derived docetaxel-resistant xenografts, including a metastatic triple-negative tumor, responded to STX140 but not to docetaxel treatment. Conclusions: STX140 shows excellent efficacy in both MCF-7WT and MCF-7DOX breast cancer xenograft models, in contrast to Taxol and 2-MeOE2. The clinical potential of STX140 was further highlighted by the efficacy seen in xenografts recently derived from patients who had failed on taxane therapy.

Synthesis, Antitubulin, and Antiproliferative SAR of Analogues of 2-Methoxyestradiol-3,17-O,O-bis-sulfamate

The synthesis and antiproliferative activity of analogues of estradiol 3,17-O,O-bis-sulfamates (E2bisMATEs) are discussed. Modifications of the C-17 substituent reveal that an H-bond acceptor is essential for high antiproliferative activity. The local environment in which this H-bond acceptor lies can be varied to an extent. The C-17-oxygen linker can be deleted or substituted with an electronically neutral methylene group, and replacement of the terminal NH(2) with a methyl group is also acceptable. Mesylates 10 and 14 prove equipotent to the E2bisMATEs 2 and 3, while sulfones 20 and 35 display enhanced in vitro antiproliferative activity. In addition, the SAR of 2-substituted estradiol-3-O-sulfamate derivatives as inhibitors of tubulin polymerization has been established for the first time. These agents inhibit the binding of radiolabeled colchicine to tubulin.

Inhibition of MDA‐MB‐231 cell cycle progression and cell proliferation by C‐2‐substituted oestradiol mono‐ and bis‐3‐O‐sulphamates

A natural metabolite of oestradiol (E2), 2‐methoxyoestradiol (2‐MeOE2), exerts both antitumour and antiangiogenic effects. 2‐MeOE2 is currently in clinical trials for the treatment of a variety of cancers. We have previously shown that a number of sulphamoylated analogues of 2‐MeOE2 possess enhanced potency and bioavailability with respect to 2‐MeOE2. In our study, the effects of C‐2‐substituted E2 derivatives, with sulphamoylation at the C‐3 and/or C‐17 position, on ERα −ve MDA‐MB‐231 breast cancer cells were evaluated. Sulphamoylated derivatives were potent inhibitors of cell proliferation, and these effects were irreversible when compared to growth inhibitory effects induced by 2‐MeOE2. Cell cycle analysis suggested that these derivatives caused cells to arrest at the G2‐M phase of the cell cycle. Sulphamoylated analogues suppressed the clonogenic potential of MDA‐MB‐231 cells and also their growth on Matrigel® culture substratum. Immunofluorescence studies showed fragmented nuclear bodies and an abnormal microtubule cytoskeleton in cells exposed to one of the potent compounds, 2‐MeOE2‐bis‐sulphamate. In addition, these analogues induced phosphorylation of BCL‐2, a protein considered to be the guardian of microtubule integrity. In each of the assays, the sulphamoylated derivatives were at least 10‐fold more potent than the parent compound 2‐MeOE2. In view of the enhanced potencies associated with sulphamoylated E2 derivatives in ERα −ve cells, these analogues should hold considerable therapeutic potential for the treatment of hormone‐independent breast cancers.

Effects of C-17 heterocyclic substituents on the anticancer activity of 2-ethylestra-1,3,5(10)-triene-3-O-sulfamates: synthesis, in vitro evaluation and computational modelling.

The potent activity of 2-substituted estra-1,3,5(10)-triene-3-O-sulfamates against the proliferation of cancer cells in vitro and tumours in vivo highlights the therapeutic potential of such compounds. Optimal activity is derived from a combination of a 2-XMe group (where X = CH(2), O or S), a 3-O-sulfamate group in the steroidal A-ring and a H-bond acceptor around C-17 of the D-ring. Herein, we describe the synthesis and anti-proliferative activities of a series of novel 2-substituted estra-1,3,5(10)-triene-3-O-sulfamates bearing heterocyclic substituents (oxazole, tetrazole, triazole) tethered to C-17. In vitro evaluation of these molecules revealed that high anti-proliferative activity in breast and prostate cancer cells lines (GI(50) of 340-850 nM) could be retained when the heterocyclic substituent possesses H-bond acceptor properties. A good correlation between the calculated electron density of the heterocyclic ring and anti-proliferative activity was observed. Docking of the most active compounds into their putative site of action, the colchicine binding site of tubulin, suggests that they bind through a different mode to the previously described bis-sulfamate derivatives and 1 and 2, which possess similar in vitro activity.

Chimeric microtubule disruptors

A chimeric approach is used to discover microtubule disruptors with excellent in vitro activity and oral bioavailability; a ligand-protein interaction with carbonic anhydrase that enhances bioavailability is characterised by protein X-ray crystallography. Dosing of a representative chimera in a tumour xenograft model confirms the excellent therapeutic potential of the class.