Accessing simply-substituted 4-hydroxytetrahydroisoquinolines via Pomeranz-Fritsch-Bobbitt reaction with non-activated and moderately-activated systems.

Background: 1,2,3,4-Tetrahydroisoquinolines (THIQs) are common motifs in alkaloids and in medicinal chemistry. Synthetic access to THIQs via the Pomeranz-Fritsch-Bobbit (PFB) methodology using mineral acids for deactivated, electron-poor aromatic systems, is scarcely represented in the literature. Here, the factors controlling the regiochemical outcome of cyclization are evaluated. Results: A double reductive alkylation was telescoped into a one-pot reaction delivering good to excellent yields of desired aminoacetals for cyclization. Cyclization of activated systems proceeded smoothly under standard PFB conditions, but for non-activated systems the use of HClO4 alone was effective. When cyclization was possible in both para- and ortho-positions to the substituent, 7-substituted derivatives were formed with significant amounts of 5-substituted byproduct. The formation of the 4-hydroxy-THIQs vs the 4-methoxy-THIQ products could be controlled through modification of the reaction concentration. In addition, while a highly-activated system exclusively cyclized to the indole, this seems generally highly disfavored. When competition between 6- and 7-ring formation was investigated in non-activated systems, 5,7,8,13-tetrahydro-6,13-methanodibenzo[c,f]azonine was exclusively obtained. Furthermore, selective ring closure in the para-position could be achieved under standard PFB conditions, while a double ring closure could be obtained utilizing HClO4. Conclusion: Reactivity differences in aminoacetal precursors can be employed to control cyclization using the PFB methodology. It is now possible to select confidently the right conditions for the synthesis of N-aryl-4-hydroxy-1,2,3,4-tetrahydroisoquinolines.

Targeted NF1 cancer therapeutics with multiple modes of action: small molecule hormone-like agents resembling the natural anticancer metabolite, 2-methoxyoestradiol.

BACKGROUND: Both the number and size of tumours in NF1 patients increase in response to the rise in steroid hormones seen at puberty and during pregnancy. The size of tumours decreases after delivery, suggesting that hormone-targeting therapy might provide a viable new NF1 treatment approach. Our earlier studies demonstrated that human NF1 tumour cell lines either went through apoptosis or ceased growth in the presence of 2-methoxyoestradiol (2ME2), a naturally occurring anticancer metabolite of 17-ß estradiol. Previous reports of treatment with sulfamoylated steroidal and non-steroidal derivatives of 2ME2 showed promising reductions in tumour burden in hormone-responsive cancers other than NF1. Here we present the first studies indicating that 2ME2 derivatives could also provide an avenue for treating NF1, for which few treatment options are available. METHODS: STX3451, (2-(3-Bromo-4,5-dimethoxybenzyl)-7-methoxy-6-sulfamoyloxy-1,2,3,4-tetrahydroisoquinoline), a non-steroidal sulphamate analogue of 2ME2, was tested in dose-dependent studies of malignant and benign NF1 human tumour cell lines and cell lines with variable controlled neurofibromin expression. The mechanisms of action of STX3451 were also analysed. RESULTS: We found that STX3451-induced apoptosis in human malignant peripheral nerve sheath tumour (MPNST) cell lines, even in the presence of elevated oestrogen and progesterone. It inhibits both PI3 kinase and mTOR signalling pathways. It disrupts actin- and microtubule-based cytoskeletal structures in cell lines derived from human MPNSTs and in cells derived from benign plexiform neurofibromas. STX3451 selectively kills MPNST-derived cells, but also halts growth of other tumour-derived NF1 cell lines. CONCLUSION: STX3451 provides a new approach for inducing cell death and lowering tumour burden in NF1 and other hormone-responsive cancers with limited treatment options.

N-Phenyl-1,2,3,4-tetrahydroisoquinoline: An Alternative Scaffold for the Design of 17ß-Hydroxysteroid Dehydrogenase 1 Inhibitors.

17ß-Hydroxysteroid dehydrogenases catalyse interconversion at the C17 position between oxidized and reduced forms of steroidal nuclear receptor ligands. The type 1 enzyme, expressed in malignant cells, catalyses reduction of the less-active estrone to estradiol, and inhibitors have therapeutic potential in estrogen-dependent diseases such as breast and ovarian cancers and in endometriosis. Synthetic decoration of the nonsteroidal N-phenyl-1,2,3,4-tetrahydroisoquinoline (THIQ) template was pursued by using Pomeranz-Fritsch-Bobbitt, Pictet-Spengler and Bischler-Napieralski approaches to explore the viability of this scaffold as a steroid mimic. Derivatives were evaluated biologically in vitro as type 1 enzyme inhibitors in a bacterial cell homogenate as source of recombinant protein. Structure-activity relationships are discussed. THIQs possessing a 6-hydroxy group, lipophilic substitutions at the 1- or 4-positions in combination with N-4'-chlorophenyl substitution were most favourable for activity. Of these, one compound had an IC50 of ca. 350 nM as a racemate, testifying to the applicability of this novel approach.

Structures of human carbonic anhydrase II/inhibitor complexes reveal a second binding site for steroidal and nonsteroidal inhibitors.

Carbonic anhydrase (CA) catalyzes the reversible hydration of carbon dioxide to hydrogen carbonate, and its role in maintaining pH balance has made it an attractive drug target. Steroidal sulfamate esters, inhibitors of the cancer drug target steroid sulfatase (STS), are sequestered in vivo by CA II in red blood cells, which may be the origin of their excellent drug properties. Understanding the structural basis of this is important for drug design. Structures of CA II complexed with 2-methoxyestradiol 3-O-sulfamate (3), 2-ethylestradiol 3,17-O,O-bis(sulfamate) (4), and 2-methoxyestradiol 17-O-sulfamate (5) are reported to 2.10, 1.85, and 1.64 A, respectively. Inhibitor 3 interacts with the active site Zn(II) ion through the 3-O-sulfamate, while inhibitors 4 and 5 bind through their 17-O-sulfamate. Comparison of the IC(50) values for CA II inhibition gave respective values of 56, 662, 2113, 169, 770, and 86 nM for estrone 3-O-sulfamate (1), 2-methoxyestradiol 3,17-O,O-bis(sulfamate) (2), 3, 4, 5, and 5'-((4H-1,2,4-triazol-4-yl)methyl)-3-chloro-2'-cyanobiphenyl-4-yl sulfamate (6), a nonsteroidal dual aromatase-sulfatase inhibitor. Inhibitors 2, 5, and 6 showed binding to a second adjacent site that is capable of binding both steroidal and nonsteroidal ligands. Examination of both IC(50) values and crystal structures suggests that 2-substituents on the steroid nucleus hinder binding via a 3-O-sulfamate, leading to coordination through a 17-O-sulfamate if present. These results underline the influence of small structural changes on affinity and mode of binding, the degree of flexibility in the design of sulfamate-based inhibitors, and suggest a strategy for inhibitors which interact with both the active site and the second adjacent binding site simultaneously that could be both potent and selective.

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-7(DOX)) and the parental cell line (MCF-7(WT)). Mice bearing an MCF-7(DOX) tumor on one flank and an MCF-7(WT) 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-7(DOX) and MCF-7(WT) cells. Taxol and 2-MeOE2 were only active in the MCF-7(WT) parental cell line. Although both STX140 and Taxol inhibited the growth of xenografts derived from MCF-7(WT) cells, only STX140 inhibited the growth of tumors derived from MCF-7(DOX) 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-7(WT) and MCF-7(DOX) 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.

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.

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.

A comparison of two orally bioavailable anti-cancer agents, IRC-110160 and STX140.

UNLABELLED: This study characterises two recently developed anticancer agents in vitro and in vivo, 2-methoxyoestra-1,3,5(10), 16-tetraene-3-carboxamide (IRC-110160) and STX140. MATERIALS AND METHODS: Hormone-dependent (MCF-7), hormone-independent (MDA-MB-231) and P-glycoprotein overexpressing (MCF-7Dox) cells were used for proliferation experiments. For the tumour efficacy studies, female nude mice were inoculated with MDA-MB-231 cells. RESULTS: IRC-110160 is a potent inhibitor of both MCF-7 and MDA-MB-231 cell proliferation. Furthermore, the potency of IRC-110160 was unaffected by the over-expression of the P-glycoprotein drug efflux pump. IRC-110160 and 2-methoxyoestradiol-3,17-O,O-bis-sulfamate (STX140) induced apoptosis in a similar timeframe in the MDA-MB-231 cell line, but only STX140 caused G2/M arrest in these cells. In the MDA-MB-231 xenograft model 300 mg/kg p.o. (daily) of IRC-110160 and 20 mg/kg p.o. STX140 (daily) both completely inhibited tumour growth; however some toxicity was observed with IRC-110160. After 28 days of daily dosing STX140 (20 mg/kg p.o.) had minimal effect on the white blood population of mice with tumours. The masking of STX140 from white blood cells may be due to its interaction with carbonic anhydrase II (CAII) in the red blood cells. In contrast to STX140, IRC-110160 does not inhibit CAII. These studies highlight the activity of two orally bioavailable anti-cancer agents one of which, STX140, may offer a significant clinical advantage over existing drugs as a common dose limiting factor, haemotoxicity, may be minimised.

A new micronized formulation of 2-methoxyestradiol-bis-sulfamate (STX140) is therapeutically potent against breast cancer.

UNLABELLED: There is a continued need for orally bioavailable anticancer compounds that exhibit good efficacy against breast cancer. STX140, a derivative of 2-methoxyestradiol (2-MeOE2), has been shown to have excellent oral bioavailability and significantly reduces tumor growth. A new micronized formulation of STX140 has now been developed and its pharmacokinetics (PK) in rats and effect on MDA-MB-231 breast cancer growth in nude mice was investigated. MATERIALS AND METHODS: For the PK studies, female Wistar rats were treated orally with STX140 in two separate vehicles (10% tetrahydrofuran (THF) in propylene glycol (PG) or 0.5% methyl cellulose (MC) in saline) and plasma samples taken for high performance liquid chromatography analysis over 48 h. For the tumor efficacy studies, female nude mice were inoculated with MDA-MB-231 breast cancer cells and then treated orally with a range of doses of STX140. RESULTS: The PK studies demonstrated that the THF/PG vehicle resulted in a greater oral bioavailability of STX140 compared to the 0.5% MC vehicle. However, this was not translated to the tumor efficacy studies where STX140 at 20 mg/kg in either vehicle caused a significant reduction in tumor volume. CONCLUSION: The new micronized formulation of STX140 is orally bioavailable and efficacious at inhibiting MDA-MB-231 breast tumor growth.

The role of 17beta-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.

Quinazolinone-Based Anticancer Agents: Synthesis, Antiproliferative SAR, Antitubulin Activity, and Tubulin Co-crystal Structure.

Quinazolinone-based anticancer agents were designed, decorated with functional groups from a 2-methoxyestradiol-based microtubule disruptor series, incorporating the aryl sulfamate motif of steroid sulfatase (STS) inhibitors. The steroidal AB-ring system was mimicked, favoring conformations with an N-2 substituent occupying D-ring space. Evaluation against breast and prostate tumor cell lines identified 7b with DU-145 antiproliferative activity (GI50 300 nM). A preliminary structure-activity relationship afforded compounds (e.g., 7j GI50 50 nM) with activity exceeding that of the parent. Both 7b and 7j inhibit tubulin assembly in vitro and colchicine binding, and 7j was successfully co-crystallized with the αß-tubulin heterodimer as the first of its class, its sulfamate group interacting positively at the colchicine binding site. Microtubule destabilization by 7j is likely achieved by preventing the curved-to-straight conformational transition in αß-tubulin. Quinazolinone sulfamates surprisingly showed weak STS inhibition. Preliminary in vivo studies in a multiple myeloma xenograft model for 7b showed oral activity, confirming the promise of this template.

3,17-disubstituted 2-alkylestra-1,3,5(10)-trien-3-ol derivatives: synthesis, in vitro and in vivo anticancer activity.

Estradiol-3,17-O,O-bis-sulfamates inhibit steroid sulfatase (STS), carbonic anhydrase (CA), and, when substituted at C-2, cancer cell proliferation and angiogenesis. C-2 Substitution and 17-sulfamate replacement of the estradiol-3,17-O,O-bis-sulfamates were explored with efficient and practical syntheses developed. Evaluation against human cancer cell lines revealed the 2-methyl derivative 27 (DU145 GI(50) = 0.38 microM) as the most active novel bis-sulfamate, while 2-ethyl-17-carbamate derivative 52 (GI(50) = 0.22 microM) proved most active of its series (cf. 2-ethylestradiol-3,17-O,O-bis-sulfamate 4 GI(50) = 0.21 microM). Larger C-2 substituents were deleterious to activity. 2-Methoxy-17-carbamate 50 was studied by X-ray crystallography and was surprisingly 13-fold weaker as an STS inhibitor compared to parent bis-sulfamate 3. The potential of 4 as an orally dosed anti-tumor agent is confirmed using breast and prostate cancer xenografts. In the MDA-MB-231 model, dramatic reduction in tumor growth or regression was observed, with effects sustained after cessation of treatment. 3-O-Sulfamoylated 2-alkylestradiol-17-O-carbamates and sulfamates have considerable potential as anticancer agents.

2-substituted estradiol bis-sulfamates, multitargeted antitumor agents: synthesis, in vitro SAR, protein crystallography, and in vivo activity.

The anticancer activities and SARs of estradiol-17-O-sulfamates and estradiol 3,17-O,O-bis-sulfamates (E2bisMATEs) as steroid sulfatase (STS) inhibitors and antiproliferative agents are discussed. Estradiol 3,17-O,O-bis-sulfamates 20 and 21, in contrast to the 17-O-monosulfamate 11, proved to be excellent STS inhibitors. 2-Substituted E2bisMATEs 21 and 23 additionally exhibited potent antiproliferative activity with mean graph midpoint values of 18-87 nM in the NCI 60-cell-line panel. 21 Exhibited antiangiogenic in vitro and in vivo activity in an early-stage Lewis lung model, and 23 dosed p.o. caused marked growth inhibition in a nude mouse xenograft tumor model. Modeling studies suggest that the E2bisMATEs and 2-MeOE2 share a common mode of binding to tubulin, though COMPARE analysis of activity profiles was negative. 21 was cocrystallized with carbonic anhydrase II, and X-ray crystallography revealed unexpected coordination of the 17-O-sulfamate of 21 to the active site zinc and a probable additional lower affinity binding site. 2-Substituted E2bisMATEs are attractive candidates for further development as multitargeted anticancer agents.

A-ring-substituted estrogen-3-O-sulfamates: potent multitargeted anticancer agents.

Efficient and flexible syntheses of 2-substituted estrone, estradiol and their 3-O-sulfamate (EMATE) derivatives have been developed using directed ortho-lithiation methodology. 2-Substituted EMATEs display a similar antiproliferative activity profile to the corresponding estradiols against a range of human cancer cell lines. 2-Methoxy (3, 4), 2-methylsulfanyl (20, 21) and 2-ethyl EMATEs (32, 33) proved the most active compounds with 2-ethylestradiol-3-O-sulfamate (33), displaying a mean activity over the NCI 55 cell line panel 80-fold greater than the established anticancer agent 2-methoxyestradiol (2). 2-Ethylestradiol-3-O-sulfamate (33) was also an effective inhibitor of angiogenesis using three in vitro markers, and various 2-substituted EMATEs also proved to be inhibitors of steroid sulfatase (STS), a therapeutic target for the treatment of hormone-dependent breast cancer. The potential of this novel class of multimechanism anticancer agents was confirmed in vivo with good activity observed in the NCI hollow fiber assay and in a MDA-MB-435 xenograft mouse 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.

The In Vitro and In Vivo Activity of the Microtubule Disruptor STX140 Is Mediated by Hif-1 Alpha and CAIX Expression.

Tumor neo-angiogenesis is regulated, in part, by the hypoxia-inducible gene HIF1. Evidence suggests HIF1 associates with polymerized microtubules and traffics to the nucleus. This study investigated the role of HIF1 in mediating the antitumor activity of two steroid-based sulfamate ester microtubule disruptors, STX140 and STX243, in vitro and in vivo. The effects of STX140, STX243 and the parental compound 2-methoxyestradiol (STX66) on HIF1α and HIF2α protein expression were assessed in vitro in MCF-7 and MDA-MB-231 cells cultured under hypoxia. More pertinently, their effects were examined on HIF1-regulated genes in vivo in mice bearing MCF-7 or MDA-MB-231 tumors. The level of mRNA expression of vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUTI), phosphoglycerate kinase (PGK), ATP-binding cassette sub-family B member 1 (ABCB1) and carbonic anhydrase IX (CAIX) was quantified by Real-time Polymerase Chain Reaction (RT-PCR). Despite inhibiting nuclear HIF1α protein accumulation under hypoxia in vitro, STX140 and STX243 did not significantly regulate the expression of four out of five HIF1α-regulated genes in vitro and in vivo. Only CAIX mRNA expression was down-regulated both in vitro and in vivo. Immunoblot analysis showed that STX140 and STX243 reduced CAIX protein expression in vitro. These compounds had no effect on HIF2α translocation. The potential for inhibition of CAIX by STX140 and STX243 was examined by docking the ligands to the active site in comparison with a known sulfamate-based inhibitor. Microtubule disruption and antitumor activity of STX140 and STX243 is most likely HIF1-independent and may, at least in part, be mediated by inhibition of CAIX expression and activity.

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.

Tetrahydroisoquinolinone-based steroidomimetic and chimeric microtubule disruptors.

A structure-activity relationship (SAR) translation strategy was used for the discovery of tetrahydroisoquinoline (THIQ)-based steroidomimetic and chimeric microtubule disruptors based upon a steroidal starting point. A steroid A,B-ring-mimicking THIQ core was connected to methoxyaryl D-ring ring mimics through methylene, carbonyl and sulfonyl linkers to afford a number of steroidomimetic hits (e.g., 7-methoxy-2-(3- methoxybenzyl)-6-sulfamoyloxy-1,2,3,4-tetrahydroisoquinoline (20 c) GI50=2.1 µM). Optimisation and control experiments demonstrate the complementary SAR of this series and the steroid derivatives that inspired its design. Linkage of the THIQ-based A,B-mimic with the trimethoxyaryl motif prevalent in colchicine site binding microtubule disruptors delivered a series of chimeric molecules whose activity (GI50=40 nM) surpasses that of the parent steroid derivatives. Validation of this strategy was obtained from the excellent oral activity of 7-methoxy-6-sulfamoyloxy-2-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroisoquinoline relative to a benchmark steroidal bis- sulfamate in an in vivo model of multiple myeloma.

Optimisation of tetrahydroisoquinoline-based chimeric microtubule disruptors.

Tetrahydroisoquinoline (THIQ)-based "chimeric" microtubule disruptors were optimised through modification of the N-benzyl motif, in concert with changes at C3 and C7, resulting in the identification of compounds with improved in vitro antiproliferative activities (e.g. 15: GI50 20 nM in DU-145). The broad anticancer activity of these novel structures was confirmed in the NCI 60-cell line assay, with 12 e,f displaying MGM values in the 40 nM region. In addition, their profiles as inhibitors of tubulin polymerisation and colchicine binding to tubulin were confirmed. Compound 15, for example, inhibited tubulin polymerisation with an IC50 of 1.8 µM, close to that of the clinical drug combretastatin A-4, and also proved effective at blocking colchicine binding. Additionally, compound 20 b was identified as the only phenol in the series to date showing both better in vitro antiproliferative properties than its corresponding sulfamate and excellent antitubulin data (IC50=.6 µM). Compound 12 f was selected for in vivo evaluation at the NCI in the hollow fibre assay and showed very good activity and wide tissue distribution, illustrating the value of this template for further development.

Synthesis, anti-tubulin and antiproliferative SAR of steroidomimetic dihydroisoquinolinones.

A SAR translation strategy adopted for the discovery of tetrahydroisoquinolinone (THIQ)-based steroidomimetic microtubule disruptors has been extended to dihydroisoquinolinone (DHIQ)-based compounds. A steroid A,B-ring-mimicking DHIQ core was connected to methoxyaryl D-ring mimics through methylene, carbonyl, and sulfonyl linkers, and the resulting compounds were evaluated against two cancer cell lines. The carbonyl-linked DHIQs in particular exhibit significant in vitro antiproliferative activities (e.g., 6-hydroxy-7-methoxy-2-(3,4,5-trimethoxybenzoyl)-3,4-dihydroisoquinolin-1(2H)-one (16 g): GI50 51 nM in DU-145 cells). The broad anticancer activity of DHIQ 16 g was confirmed in the NCI 60-cell line assay giving a mean activity of 33 nM. Furthermore, 6-hydroxy-2-(3,5-dimethoxybenzoyl)-7-methoxy-3,4-dihydroisoquinolin-1(2H)-one (16 f) and 16 g and their sulfamate derivatives 17 f and 17 g (2-(3,5-dimethoxybenzoyl)-7-methoxy-6-sulfamoyloxy-3,4-dihydroisoquinolin-1(2H)-one and 7-methoxy-2-(3,4,5-trimethoxybenzoyl)-6-sulfamoyloxy-3,4-dihydroisoquinolin-1(2H)-one, respectively) show excellent activity against the polymerization of tubulin, close to that of the clinical combretastatin A-4, and bind competitively at the colchicine binding site of tubulin. Compounds 16 f and 17 f were also shown to demonstrate in vitro anti-angiogenic activity. Additionally, X-ray and computational analyses of 17 f reveal that electrostatic repulsion between the two adjacent carbonyl groups, through conformational biasing, dictates the adoption of a "steroid-like" conformation that may partially explain the excellent in vitro activities.