A Quantitative Systems Pharmacology Platform to Investigate the Impact of Alirocumab and Cholesterol-Lowering Therapies on Lipid Profiles and Plaque Characteristics.

Reduction in low-density lipoprotein cholesterol (LDL-C) is associated with decreased risk for cardiovascular disease. Alirocumab, an antibody to proprotein convertase subtilisin/kexin type 9 (PCSK9), significantly reduces LDL-C. Here, we report development of a quantitative systems pharmacology (QSP) model integrating peripheral and liver cholesterol metabolism, as well as PCSK9 function, to examine the mechanisms of action of alirocumab and other lipid-lowering therapies, including statins. The model predicts changes in LDL-C and other lipids that are consistent with effects observed in clinical trials of single or combined treatments of alirocumab and other treatments. An exploratory model to examine the effects of lipid levels on plaque dynamics was also developed. The QSP platform, on further development and qualification, may support dose optimization and clinical trial design for PCSK9 inhibitors and lipid-modulating drugs. It may also improve our understanding of factors affecting therapeutic responses in different phenotypes of dyslipidemia and cardiovascular disease.

Correlating thyroid cytology and histopathology: Implications for molecular testing.

BACKGROUND: A gene expression classifier (GEC) has been advocated in management of some indeterminate nodules without surgery. We assessed the potential negative predictive value (NPV) of the GEC at our academic center. METHODS: Retrospectively, all cytologically indeterminate fine-needle aspirates (FNAs) diagnosed by University of Iowa cytopathologists over a 3-year period were identified. Histopathologic findings were recorded. Using published sensitivity and specificity, NPVs were calculated. RESULTS: Of 178 nodules (17, 135, and 26 in classes III, IV, and V, respectively), 71 (40%) were malignant. Prevalence of malignancy was 41%, 29%, and 96% for classes III, IV, and V, respectively. Using sensitivities and specificities for the GEC, NPVs were 91% for the cohort: 88%, 92%, and 26% for classes III, IV, and V, respectively. CONCLUSION: Molecular testing should be associated with an NPV no lower than that from clinical criteria alone. With the prevalences reported, GEC use may result in more missed cancer diagnoses. © 2016 Wiley Periodicals, Inc. Head Neck 38: 1104-1106, 2016.

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.

STX140, but not paclitaxel, inhibits mammary tumour initiation and progression in C3(1)/SV40 T/t-antigen transgenic mice.

Despite paclitxael's clinical success, treating hormone-refractory breast cancer remains challenging. Paclitaxel has a poor pharmacological profile, characterized by a low therapeutic index (TIX) caused by severe dose limiting toxicities, such as neutropenia and peripheral neuropathy. Consequently, new drugs are urgently required. STX140, a compound previously shown to have excellent efficacy against many tumors, is here compared to paclitaxel in three translational in vivo breast cancer models, a rat model of peripheral neuropathy, and through pharmacological testing. Three different in vivo mouse models of breast cancer were used; the metastatic 4T1 orthotopic model, the C3(1)/SV40 T-Ag model, and the MDA-MB-231 xenograft model. To determine TIX and pharmacological profile of STX140, a comprehensive dosing regime was performed in mice bearing MDA-MD-231 xenografts. Finally, peripheral neuropathy was examined using a rat plantar thermal hyperalgesia model. In the 4T1 metastatic model, STX140 and paclitaxel significantly inhibited primary tumor growth and lung metastases. All C3(1)/SV40 T-Ag mice in the control and paclitaxel treated groups developed palpable mammary cancer. STX140 blocked 47% of tumors developing and significantly inhibited growth of tumors that did develop. STX140 treatment caused a significant (P<0.001) survival advantage for animals in early and late intervention groups. Conversely, in C3(1)/SV40 T-Ag mice, paclitaxel failed to inhibit tumor growth and did not increase survival time. Furthermore, paclitaxel, but not STX140, induced significant peripheral neuropathy and neutropenia. These results show that STX140 has a greater anti-cancer efficacy, TIX, and reduced neurotoxicity compared to paclitaxel in C3(1)/SV40 T-Ag mice and therefore may be of significant benefit to patients with breast cancer.

STX2171, a 17ß-hydroxysteroid dehydrogenase type 3 inhibitor, is efficacious in vivo in a novel hormone-dependent prostate cancer model.

17ß-Hydroxysteroid dehydrogenases (17ß-HSDs) catalyse the 17-position reduction/oxidation of steroids. 17ß-HSD type 3 (17ß-HSD3) catalyses the reduction of the weakly androgenic androstenedione (adione) to testosterone, suggesting that specific inhibitors of 17ß-HSD3 may have a role in the treatment of hormone-dependent prostate cancer and benign prostate hyperplasia. STX2171 is a novel selective non-steroidal 17ß-HSD3 inhibitor with an IC(50) of ∼200 nM in a whole-cell assay. It inhibits adione-stimulated proliferation of 17ß-HSD3-expressing androgen receptor-positive LNCaP(HSD3) prostate cancer cells in vitro. An androgen-stimulated LNCaP(HSD3) xenograft proof-of-concept model was developed to study the efficacies of STX2171 and a more established 17ß-HSD3 inhibitor, STX1383 (SCH-451659, Schering-Plough), in vivo. Castrated male MF-1 mice were inoculated s.c. with 1×10(7) cells 24 h after an initial daily dose of testosterone propionate (TP) or vehicle. After 4 weeks, tumours had not developed in vehicle-dosed mice, but were present in 50% of those mice given TP. One week after switching the stimulus to adione, mice were dosed additionally with the vehicle or inhibitor for a further 4 weeks. Both TP and adione efficiently stimulated tumour growth and increased plasma testosterone levels; however, in the presence of either 17ß-HSD3 inhibitor, adione-dependent tumour growth was significantly inhibited and plasma testosterone levels reduced. Mouse body weights were unaffected. Both inhibitors also significantly lowered plasma testosterone levels in intact mice. In conclusion, STX2171 and STX1383 significantly lower plasma testosterone levels and inhibit androgen-dependent tumour growth in vivo, indicating that 17ß-HSD3 inhibitors may have application in the treatment of hormone-dependent prostate cancer.

The role of women in dental education: monitoring the pipeline to leadership.

The purpose of this study was to analyze data collected by the American Dental Association and the American Dental Education Association over the past two decades relating to changes in the number of women active in dental education and dental practice. The concept of a pipeline of women in dentistry was explored by analyzing predoctoral, postdoctoral, dental practice, and dental education domains for the inclusion of women. Statistical analyses show that there has been a consistent and progressive increase in the number of women in all stages of the pipeline. Over the past two decades, the number of female students attending and graduating from dental school has steadily increased. In 1984-85, 23.7 percent of all predoctoral students were women; in 2009-10, 45.1 percent were women. Similarly, in 1999, the graduating class was 35.3 percent women; in 2009, it was 46.1 percent women. In the postdoctoral domain, in 1996, 29.9 percent of all residents were women; in 2010, this had increased to 39.0 percent. In dental practice, the number of actively licensed women dentists in 1999 was 15.3 percent of the workforce; in 2010, this percentage had grown to 24.0 percent. In dental education, the number of women clinical faculty members has gradually increased from 669 in 1997-98 to 902 in 2007-08. Until 2000, there had been only two women deans and very few associate/assistant deans, with only sixteen in 1990. In 2000, major changes began with three women deans and seventy-two women associate/assistant deans. In 2009-10, there were 111 associate/assistant women deans and twelve women deans. These data show a progressive increase in the presence of women in all domains of dentistry, especially in leadership positions in dental education.

Bicyclic derivatives of the potent dual aromatase-steroid sulfatase inhibitor 2-bromo-4-{[(4-cyanophenyl)(4h-1,2,4-triazol-4-yl)amino]methyl}phenylsulfamate: synthesis, SAR, crystal structure, and in vitro and in vivo activities.

The design and synthesis of a series of bicyclic ring containing dual aromatase-sulfatase inhibitors (DASIs) based on the aromatase inhibitor (AI) 4-[(4-bromobenzyl)(4H-1,2,4-triazol-4-yl)amino]benzonitrile are reported. Biological evaluation with JEG-3 cells revealed structure-activity relationships. The X-ray crystal structure of sulfamate 23 was determined, and selected compounds were docked into the aromatase and steroid sulfatase (STS) crystal structures. In the sulfamate-containing series, compounds containing a naphthalene ring are both the most potent AI (39, IC(50 AROM)=0.25 nM) and the best STS inhibitor (31, IC(50 STS)=26 nM). The most promising DASI is 39 (IC(50 AROM)=0.25 nM, IC(50 STS)=205 nM), and this was evaluated orally in vivo at 10 mg kg(-1), showing potent inhibition of aromatase (93 %) and STS (93 %) after 3 h. Potent aromatase and STS inhibition can thus be achieved with a DASI containing a bicyclic ring system; development of such a DASI could provide an attractive new option for the treatment of hormone-dependent breast cancer.

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.

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.

Highly potent first examples of dual aromatase-steroid sulfatase inhibitors based on a biphenyl template.

Single agents against multiple drug targets are of increasing interest. Hormone-dependent breast cancer (HDBC) may be more effectively treated by dual inhibition of aromatase and steroid sulfatase (STS). The aromatase inhibitory pharmacophore was thus introduced into a known biphenyl STS inhibitor to give a series of novel dual aromatase-sulfatase inhibitors (DASIs). Several compounds are good aromatase or STS inhibitors and DASI 20 (IC(50): aromatase, 2.0 nM; STS, 35 nM) and its chlorinated congener 23 (IC(50): aromatase, 0.5 nM; STS, 5.5 nM) are examples that show exceptional dual potency in JEG-3 cells. Both biphenyls share a para-sulfamate-containing ring B and a ring A, which contains a triazol-1-ylmethyl meta to the biphenyl bridge and para to a nitrile. At 1 mg/kg po, 20 and 23 reduced plasma estradiol levels strongly and inhibited liver STS activity potently in vivo. 23 is nonestrogenic and potently inhibits carbonic anhydrase II (IC(50) 86 nM). A complex was crystallized and its structure was solved by X-ray crystallography. This class of DASI should encourage further development toward multitargeted therapeutic intervention in HDBC.

Developing dental faculty for the future: ADEA/AAL Institute for Teaching and Learning, 2006-09.

This report summarizes the history and curriculum of the American Dental Education Association/Academy for Academic Leadership Institute for Teaching and Learning (ADEA/AAL ITL) Program for Dental School Faculty, describes participant feedback, and reviews how the program serves the faculty development initiatives of the American Dental Education Association. The fifty-hour program (6.5 days), conducted in two phases at collaborating dental schools, enhances core academic competencies of new and transitional faculty, including faculty members whose responsibilities include predoctoral, allied, and postdoctoral dental education. The program's mission is to prepare participants to become more effective teachers and develop other skills that will facilitate confidence, job satisfaction, and professional growth in the academic environment. From 2005 to 2009, 174 individuals graduated from the program, representing forty-three schools of dentistry in the United States and Canada and twenty-nine private practices. A total of forty scholarships have been awarded to participants by the American Academy of Periodontology Foundation, the American Academy of Pediatric Dentistry, and the American Association of Orthodontists. In an online survey completed by 75 percent of ADEA/AAL ITL participants, 99 percent indicated they were positive or highly positive about their learning experience in this faculty development program. Ninety-six percent stated that the program had been important or very important in their effectiveness as a teacher. In 2010, the program will be held at the University of North Carolina at Chapel Hill School of Dentistry, with phase I occurring on August 19-22, 2010, and phase II on October 22-24, 2010. In summary, the ADEA/AAL ITL is addressing an unmet need through a formal professional development program designed to help new and potential faculty members thrive as educators and become future leaders in academic health care.

STX140 and STX641 cause apoptosis via the intrinsic mitochondrial pathway and down-regulate survivin and XIAP expression in ovarian and prostate cancer cells.

Many anticancer drugs target microtubules and induce apoptosis. However, improved microtubule-targeting drugs, such as STX140 and STX641, are being developed. These compounds induce cell cycle arrest and apoptosis in a variety of tumour cells. The mechanisms that induce apoptosis and the key mediators involved are elucidated in this study. Results demonstrate that STX140 and STX641 depolarise mitochondrial bioenergetics and activate caspase 3/7 in A2780, LNCaP and MCF-7 cancer cells. Furthermore, both compounds cause a significant reduction in the expression of survivin and XIAP. This work details the temporal organisation of apoptosis induced by two microtubule disruptors and highlights the role that the down-regulation of survivin and XIAP may play in this process.

The development of steroid sulfatase inhibitors for hormone-dependent cancer therapy.

Steroid sulfatase (STS) regulates the hydrolysis of steroid sulfates to their unconjugated forms. Estrone sulfate and dehydroepiandrosterone sulfate can be hydrolyzed by STS to estrone and dehydroepiandrosterone, respectively, with these steroids being the precursors for the synthesis of more biologically active estrogens or androgens. A number of potent STS inhibitors have now been developed including STX64, which entered a phase I trial for the treatment of postmenopausal women with advanced metastatic hormone-dependent breast cancer. The results from this phase I trial were encouraging, suggesting that STS inhibitors may also have a role in the treatment of other hormone-dependent cancers including those of the endometrium, ovary, and prostate. In this paper the potential use of STS inhibitors to treat these hormone-dependent cancers is reviewed. In addition, results from in vitro studies show that Ishikawa endometrial cancer cells, OVCAR-3 ovarian cancer cells, and LNCaP prostate cancer cells all possess significant STS activity. Furthermore, STS activity in these cells can be almost completely inhibited by STX64 or the second-generation STS inhibitor, STX213. Results from these investigations therefore suggest that STS inhibitors could have therapeutic potential for the treatment of a range of hormone-dependent cancers.

The design of novel 17beta-hydroxysteroid dehydrogenase type 3 inhibitors.

17beta-Hydroxysteroid dehydrogenase type 3 (17beta-HSD3) is expressed at high levels in the testes and seminal vesicles but has also been shown to be present in prostate tissue, suggesting its potential involvement in both gonadal and non-gonadal testosterone biosynthesis. The role of 17beta-HSD3 in testosterone biosynthesis makes this enzyme an attractive molecular target for small molecule inhibitors for the treatment of prostate cancer. Here we report the design of selective inhibitors of 17beta-HSD3 as potential anti-cancer agents. Due to 17beta-HSD3 being a membrane-bound protein a crystal structure is not yet available. A homology model of 17beta-HSD3 has been built to aid structure-based drug design. This model has been used with docking studies to identify a series of lead compounds that may give an insight as to how inhibitors interact with the active site. Compound 1 was identified as a potent selective inhibitor of 17beta-HSD3 with an IC(50)=700nM resulting in the discovery of a novel lead series for further optimisation. Using our homology model as a tool for inhibitor design compound 5 was discovered as a novel potent and selective inhibitor of 17beta-HSD3 with an IC(50) approximately 200nM.

Development of hormone-dependent prostate cancer models for the evaluation of inhibitors of 17beta-hydroxysteroid dehydrogenase type 3.

17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) are responsible for the pre-receptor reduction/oxidation of steroids at the 17-position into active/inactive hormones, and the 15 known enzymes vary in their substrate specificity, localisation, and directional activity. 17beta-HSD Type 3 (17beta-HSD3) has been seen to be over-expressed in prostate cancer, and catalyses the reduction of androstenedione (Adione) to testosterone (T), which stimulates prostate tumour growth. Specific inhibitors of 17beta-HSD3 may have a role in the treatment of hormone-dependent prostate cancer and benign prostate hyperplasia, and also have potential as male anti-fertility agents. A 293-EBNA-based cell line with stable expression of transfected human 17beta-HSD3 was created and used to develop a whole cell radiometric TLC-based assay to assess the 17beta-HSD3 inhibitory potency of a series of compounds. STX2171 and STX2624 (IC(50) values in the 200-450nM range) were two of several active inhibitors identified. In similar TLC-based assays these compounds were found to be inactive against 17beta-HSD1 and 17beta-HSD2, indicating selectivity. A novel proof of concept model was developed to study the efficacy of the compounds in vitro using the androgen receptor positive hormone-dependent prostate cancer cell line, LNCaPwt, and its derivative, LNCaP[17beta-HSD3], transfected and selected for stable expression of 17beta-HSD3. The proliferation of the parental cell line was most efficiently stimulated by 5alpha-dihydrotestosterone (DHT), but the LNCaP[17beta-HSD3] cells were equally stimulated by Adione, indicating that 17beta-HSD3 efficiently converts Adione to T in this model. Adione-stimulated proliferation of LNCaP[17beta-HSD3] cells was inhibited in the presence of either STX2171 or STX2624. The compounds alone neither stimulated proliferation of the cells nor caused significant cell death, indicating that they are non-androgenic with low cytotoxicity. STX2171 inhibited Adione-stimulated growth of xenografts established from LNCaPwt cells in castrated mice in vivo. In conclusion, a primary screening assay and proof of concept model have been developed to study the efficacy of 17beta-HSD3 inhibitory compounds, which may have a role in the treatment of hormone-dependent cancer. Active compounds are selective for 17beta-HSD3 over 17beta-HSD1 and 17beta-HSD2, non-androgenic with low toxicity, and efficacious in both an in vitro proof of concept model and in an in vivo tumour model.

Recent developments of steroid sulfatase inhibitors as anti-cancer agents.

The steroid sulfatase (STS) enzyme plays a pivotal role in the formation of biologically active steroid hormones. Its involvement in the hydrolysis of estrone sulfate and dehydroepiandrosterone sulfate to estrone and dehydroepiandrosterone, respectively, is an important step in the formation of estradiol and androstenediol, both of which are estrogenic steroids that can stimulate tumor growth. Consequently, as STS is widely distributed throughout the entire body, it has a substantial influence on hormone-dependent cancer mitogenesis. It is a useful prognostic marker of disease as a significant majority of breast tumors over-express the enzyme and there are indications of STS having a role in prostate cancer. This knowledge has led to the development of potent STS inhibitors for use as anti-cancer agents. There are now several steroidal and non-steroidal STS inhibitors available. New in vivo models, using ovariectomized female nude mice, have been developed to pre-clinically test these inhibitors. These studies have demonstrated the excellent efficacy and effect of STS inhibitors on breast carcinoma development. Recently, 667 COUMATE, an irreversible type of inhibitor which utilizes a phenol sulfamate ester as its active pharmacophore, has completed a Phase I clinical trial in postmenopausal women with breast cancer. These studies have indicated the potential clinical benefit for the use of STS inhibitors. Most pre-clinical and clinical studies have focused on breast cancer as the target for STS inhibition. However, there are other hormone-dependent malignancies, such as endometrial and prostate cancer, that could in the future be treated with these new potent STS inhibitors.

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 new therapeutic strategy against hormone-dependent breast cancer: the preclinical development of a dual aromatase and sulfatase inhibitor.

PURPOSE: The production of E2 is paramount for the growth of estrogen receptor-positive breast cancer. Various strategies have been used, including the use of enzyme inhibitors against either aromatase (AROM) or steroid sulfatase (STS), in an attempt to ablate E2 levels. Both these enzymes play a critical role in the formation of estrogenic steroids and their inhibitors are now showing success in the clinic. EXPERIMENTAL DESIGN: We show here, in a xenograft nude mouse model, that the inhibition of both enzymes using STX681, a dual AROM and STS inhibitor (DASI), is a potential new therapeutic strategy against HDBC. MCF-7 cells stably expressing either AROM cDNA (MCF-7(AROM)) or STS cDNA (MCF-7(STS)) were generated. Ovariectomized MF-1 female nude mice receiving s.c. injections of either androstenedione (A(4)) or E2 sulfate and bearing either MCF-7(AROM) or MCF-7(STS) tumors were orally treated with STX64, letrozole, or STX681. Treatment was administered for 28 days. Mice were weighed and tumor measurements were taken weekly. RESULTS: STX64, a potent STS inhibitor, completely blocked MCF-7(STS) tumor growth but failed to attenuate MCF-7(AROM) tumor growth. In contrast, letrozole inhibited MCF-7(AROM) tumors but had no effect on MCF-7(STS) tumors. STX681 completely inhibited the growth of both tumors. AROM and STS activity was also completely inhibited by STX681, which was accompanied by a significant reduction in plasma E2 levels. CONCLUSIONS: This study indicates that targeting both the AROM and the STS enzyme with a DASI inhibits HDBC growth and is therefore a potentially novel treatment for this malignancy.

Synthesis of aromatase inhibitors and dual aromatase steroid sulfatase inhibitors by linking an arylsulfamate motif to 4-(4H-1,2,4-triazol-4-ylamino)benzonitrile: SAR, crystal structures, in vitro and in vivo activities.

4-(((4-Cyanophenyl)(4H-1,2,4-triazol-4-yl)amino)methyl)phenyl sulfamate (6 a) was the first dual aromatase-sulfatase inhibitor (DASI) reported. Several series of its derivatives with various linker systems between the steroid sulfatase (STS) and the aromatase inhibitory pharmacophores were synthesised and evaluated in JEG-3 cells. The X-ray crystal structures of the aromatase inhibitors, DASI precursors 42 d and 60, and DASI 43 h were determined. Nearly all derivatives show improved in vitro aromatase inhibition over 6 a but decreased STS inhibition. The best aromatase inhibitor is 42 e (IC(50)=0.26 nM) and the best DASI is 43 e (IC(50 aromatase)=0.45 nM, IC(50 STS)=1200 nM). SAR for aromatase inhibition shows that compounds containing an alkylene- and thioether-based linker system are more potent than those that are ether-, sulfone-, or sulfonamide-based, and that the length of the linker has a limited effect on aromatase inhibition beyond two methylene units. Compounds 43 d-f were studied in vivo (10 mg kg(-1), single, p.o.). The most potent DASI is 43 e, which inhibited PMSG-induced plasma estradiol levels by 92 % and liver STS activity by 98 % 3 h after dosing. These results further strengthen the concept of designing and developing DASIs for potential treatment of hormone-related cancers.

Anticancer steroid sulfatase inhibitors: synthesis of a potent fluorinated second-generation agent, in vitro and in vivo activities, molecular modeling, and protein crystallography.

An improved steroid sulfatase inhibitor was prepared by replacing the N-propyl group of the second-generation steroid-like inhibitor (2) with a N-3,3,3-trifluoropropyl group to give (10). This compound is 5-fold more potent in vitro, completely inhibits rat liver steroid sulfatase activity after a single oral dose of 0.5 mg/kg, and exhibits a significantly longer duration of inhibition over (2). These biological properties are attributed to the increased lipophilicity and metabolic stability of (10) rendered by its trifluoropropyl group and also the potential H-bonding between its fluorine atom(s) and Arg(98) in the active site of human steroid sulfatase. Like other sulfamates, (10) is expected to be sequestered, and transported by, erythrocytes in vivo because it inhibits human carbonic anhydrase II (hCAII) potently (IC(50), 3 nmol/L). A congener (4), which possesses a N-(pyridin-3-ylmethyl) substituent, is even more active (IC(50), 0.1 nmol/L). To rationalize this, the hCAII-(4) adduct, obtained by cocrystallization, reveals not only the sulfamate group and the backbone of (4) interacting with the catalytic site and the associated hydrophobic pocket, respectively, but also the potential H-bonding between the N-(pyridin-3-ylmethyl) group and Nepsilon(2) of Gln(136). Like (2), both (10) and its phenolic precursor (9) are non-estrogenic using a uterine weight gain assay. In summary, a highly potent, long-acting, and nonestrogenic steroid sulfatase inhibitor was designed with hCAII inhibitory properties that should positively influence in vivo behavior. Compound (10) and other related inhibitors of this structural class further expand the armory of steroid sulfatase inhibitors against hormone-dependent breast cancer.