Steroid sulfatase inhibitors and sulfated C19 steroids for proteotoxicity-related diseases: a patent spotlight.

Aging and proteotoxicity go hand in hand. Inhibiting proteotoxicity has been proposed to extend lifespan. This invention describes a new strategy to limit proteotoxicity and to extend the lifespan. Loss of function of sul-2, the Caenorhabditis elegans steroid sulfatase, elevates the pool of sulfated steroid hormones, increases longevity and ameliorates protein aggregation diseases. The present invention provides a group of molecules for use in the prevention of aging-associated proteotoxicity caused by protein aggregation diseases and/or to increase the lifespan of a eukaryotic organism. These molecules are either steroid sulfatase inhibitors or sulfated C19 steroids, both of which reproduce the phenotype of sul-2 mutants. One particular representative example is STX-64. Potential applications of the claims have been demonstrated in animal models of Parkinson's disease, Huntington's disease and Alzheimer's disease.

Aromatase and steroid sulfatase from human placenta.

Cytochrome P450 aromatase (AROM) and steroid (estrone (E1)/dehydroepiandrosterone (DHEA)) sulfatase (STS) are the two key enzymes responsible for the biosynthesis of estrogens in human, and maintenance of the critical balance between androgens and estrogens. Human AROM, an integral membrane protein of the endoplasmic reticulum, is a member of the Fe-heme containing cytochrome P450 superfamily having a cysteine thiolate as the fifth Fe-coordinating ligand. It is the only enzyme known to catalyze the conversion of androgens with non-aromatic A-rings to estrogens characterized by the aromatic A-ring. Human STS, also an integral membrane protein of the endoplasmic reticulum, is a Ca2+-dependent enzyme that catalyzes the hydrolysis of sulfate esters of E1 and DHEA to yield the respective unconjugated steroids, the precursors of the most potent forms of estrogens and androgens, namely, 17ß-estradiol (E2), 16α,17ß-estriol (E3), testosterone (TST) and dihydrotestosterone (DHT). Expression of these steroidogenic enzymes locally within various organs and tissues of the endocrine, reproductive, and central nervous systems is the key for maintaining high levels of the reproductive steroids. Thus, the enzymes have been drug targets for the prevention and treatment of diseases associated with steroid hormone excesses, especially in breast and prostate malignancies and endometriosis. Both AROM and STS have been the subjects of vigorous research for the past six decades. In this article, we review the procedures of their extraction and purification from human term placenta are described in detail, along with the activity assays.

New structural insights provide a different angle on steroid sulfatase action.

A central part of human sulfation pathways is the spatially and temporally controlled desulfation of biologically highly potent steroid hormones. The responsible enzyme - steroid sulfatase (STS) - is highly expressed in placenta and peripheral tissues, such as fat, colon, and the brain. The shape of this enzyme and its mechanism are probably unique in biochemistry. STS was believed to be a transmembrane protein, spanning the Golgi double-membrane by stem region formed by two extended internal alpha-helices. New crystallographic data however challenge this view. STS now is portraited as a trimeric membrane-associated complex. We discuss the impact of these results on STS function and sulfation pathways in general and we hypothesis that this new STS structural understanding suggests product inhibition to be a regulator of STS enzymatic activity.

Dual aromatase-steroid sulfatase inhibitors (DASI's) for the treatment of breast cancer: a structure guided ligand based designing approach.

Dual aromatase-steroid sulfatase inhibitors (DASIs) lead to significant deprivation of estrogen levels as compared to a single target inhibition and thereby exhibited an additive or synergistic effect in the treatment of hormone-dependent breast cancer (HDBC). Triazole-bearing DASI's having structural features of clinically available aromatase inhibitors are identified as lead structures for optimization as DASI's. To identify the spatial fingerprints of target-specific triazole as DASI's, we have performed molecular docking assisted Gaussian field-based comparative 3D-QSAR studies on a dataset with dual aromatase-STS inhibitory activities. Separate contours were generated for both aromatase and steroid sulphates showing respective pharmacophoric structural requirements for optimal activity. These developed 3D-QSAR models also showed good statistical measures with the excellent predictive ability with PLS-generated validation constraints. Comparative steric, electrostatic, hydrophobic, HBA, and HBD features were elucidated using respective contour maps for selective target-specific favourable activity. Furthermore, the molecular docking was used for elucidating the mode of binding as DASI's along with the MD simulation of 100 ns revealed that all the protease-ligand docked complexes are overall stable as compared to reference ligand (inhibitor ASD or Irosustat) complex. Further, the MM-GBSA study revealed that compound 24 binds to aromatase as well as STS active site with relatively lower binding energy than reference complex, respectively. A comparative study of these developed multitargeted QSAR models along with molecular docking and dynamics study can be employed for the optimization of drug candidates as DASI's.Communicated by Ramaswamy H. Sarma.

Is intracrinology of endometriosis relevant in clinical practice? A systematic review on estrogen metabolism.

Endometriosis is a chronic, multifactorial, estrogen-dependent disease. The abnormal endocrine microenvironment of endometriosis lesions is considered a main feature and multiple enzymatic pathways leading to local increased synthesis of estrogens have been identified. However, the relevance of intracrinology in clinical practice is still lacking. Medline, Embase, Scopus database were systematically searched for studies reporting on local estrogens metabolism of endometriotic lesions. The main enzymatic pathways involved in the intracrinology of endometriosis such as aromatase (CYP19A1), 17ß-hydroxysteroid dehydrogenase (HSD17B) type 1, type 2 and type 5, steroid sulfatase (STS), estrogen sulfotransferase (SULT1E1) were assessed with a critical perspective on their role in disease endocrine phenotyping, drug resistance and as therapeutic targets. Overall, studies heterogeneity and missing clinical data affect the interpretation of the clinical role of these enzymes. Although the use of some drugs such as aromatase inhibitors has been proposed in clinical practice for two decades, their potential clinical value is still under investigation as well as their modality of administration. A closer look at new, more realistic drug targets is provided and discussed. Altered expression of these key enzymes in the lesions have far reaching implication in the development of new drugs aimed at decreasing local estrogenic activity with a minimal effect on gonadal function; however, given the complexity of the evaluation of the expression of the enzymes, multiple aspects still remains to be clarified. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022311329, identifier CRD42022311329.

Clinicopathological Significance of Estrogen Receptor ß and Estrogen Synthesizing/Metabolizing Enzymes in Urothelial Carcinoma of Urinary Bladder.

Sex-specific differences in the incidence of urinary bladder carcinomas are well known, and the possible involvement of sex steroids has been proposed. We previously reported the association of the loss of androgen receptors and androgen-producing enzymes with tumor progression of urinary bladder cancer patients. Clinically, the selective estrogen receptor modulators (SERMs) were reported to suppress the progression of these tumors but the status of estrogen receptors (ERs) has not been well studied in patients with bladder urinary cancer. Moreover, not only ERs but also estrogen-related enzymes, such as aromatase, steroid sulfatase (STS), and estrogen sulfotransferase (EST), have been reported in the biological/clinical behavior of various hormone-dependent carcinomas but not studied in urinary bladder carcinoma. Therefore, in this study, we immunolocalized ERs as well as estrogen metabolizing enzymes in urinary bladder carcinoma and performed immunoblotting and cell proliferation assays using the bladder urothelial carcinoma cell line, T24. The results revealed that the loss of STS and aromatase was significantly correlated with advanced stages of the carcinoma. In vitro studies also revealed that T24 cell proliferation rates were significantly ameliorated after treatment with estradiol or diarylpropionitrile (DPN). EST and aromatase were also significantly correlated with the nuclear grade of the carcinoma. The results of our present study, for the first time, demonstrated that biologically active estrogens that bind to ERs could suppress tumor progression and the inactive ones could promote its progression and the potential clinical utility of SERM treatment in selective patients with urinary bladder carcinoma.

Simultaneous expression of steroid sulfatase and androgen receptor reduced overall survival of patients with epithelial ovarian tumors.

BACKGROUND: Ovarian cancer is usually diagnosed at an advanced stage due to its early asymptomatic course and late-stage non-specific symptoms. This highlights the importance of researching the molecular mechanisms involved in ovarian carcinogenesis as well as the discovery of novel prognostic markers that could help improve the survival outcome of patients. The aim of this study was to evaluate the expression of the steroid sulfatase (STS) in 154 samples of primary ovarian tumors. This protein is crucial in the intracellular conversion of sulfated steroid hormones to active steroid hormones. The presence of STS, 3ß-HSD, and 17ß-HSD1 result in the production of testosterone which act through the androgen receptor (AR) in the tumor cell. The presence of STS and AR in epithelial ovarian tumors and their association to the overall survival of patients was evaluated using Kaplan-Meier and Cox regression analyses. RESULTS: Immunoreactivity for STS was detected in 65% of the tumors and no association was observed with histological subtypes and clinical stages of the tumor. The STS expression in the tumors exhibiting immunoreactive AR resulted in a reduced survival (log-rank test, p = 0.032) and a risk factor in univariate and multivariate analysis, HR = 3.46, CI95% 1.00-11.92, p = 0.049 and HR = 5.92, CI95% 1.34-26.09, p = 0.019, respectively. CONCLUSIONS: These findings suggest that the intracellular synthesis of testosterone acting through its receptor can promote tumor growth and progression. Moreover, the simultaneous expression of STS and AR constitutes an independent predictor of poor prognosis in epithelial ovarian tumors.

Steroid Sulphatase and Its Inhibitors: Past, Present, and Future.

Steroid sulphatase (STS), involved in the hydrolysis of steroid sulphates, plays an important role in the formation of both active oestrogens and androgens. Since these steroids significantly impact the proliferation of both oestrogen- and androgen-dependent cancers, many research groups over the past 30 years have designed and developed STS inhibitors. One of the main contributors to this field has been Prof. Barry Potter, previously at the University of Bath and now at the University of Oxford. Upon Prof. Potter's imminent retirement, this review takes a look back at the work on STS inhibitors and their contribution to our understanding of sulphate biology and as potential therapeutic agents in hormone-dependent disease. A number of potent STS inhibitors have now been developed, one of which, Irosustat (STX64, 667Coumate, BN83495), remains the only one to have completed phase I/II clinical trials against numerous indications (breast, prostate, endometrial). These studies have provided new insights into the origins of androgens and oestrogens in women and men. In addition to the therapeutic role of STS inhibition in breast and prostate cancer, there is now good evidence to suggest they may also provide benefits in patients with colorectal and ovarian cancer, and in treating endometriosis. To explore the potential of STS inhibitors further, a number of second- and third-generation inhibitors have been developed, together with single molecules that possess aromatase-STS inhibitory properties. The further development of potent STS inhibitors will allow their potential therapeutic value to be explored in a variety of hormone-dependent cancers and possibly other non-oncological conditions.

Steroid sulfatase inhibitors: the current landscape.

Introduction: Steroid sulfatase (STS) enzyme is responsible for transforming the inactive sulfate metabolites of steroid sex hormones into the active free steroids. Both the deficiency and the over-expression of STS are associated with the pathophysiology of certain diseases. This article provides the readership with a comprehensive review about STS enzyme and its recently reported inhibitors.Areas covered: In the present article, we reviewed the structure, location, and substrates of STS enzyme, physiological functions of STS, and disease states related to over-expression or deficiency of STS enzyme. STS inhibitors reported during the last five years (2016-present) have been reviewed as well.Expert opinion: Irosustat is the most successful STS inhibitor drug candidate so far. It is currently under investigation in clinical trials for treatment of estrogen-dependent breast cancer. Non-steroidal sulfamate is the most favorable scaffold for STS inhibitor design. They can be beneficial for the treatment of hormone-dependent cancers and neurodegenerative disorders without significant estrogenic side effects. Moreover, dual-acting molecules (inhibitors of STS + another synergistic mechanism) can be therapeutically efficient.

Steroid hormones sulfatase inactivation extends lifespan and ameliorates age-related diseases.

Aging and fertility are two interconnected processes. From invertebrates to mammals, absence of the germline increases longevity. Here we show that loss of function of sul-2, the Caenorhabditis elegans steroid sulfatase (STS), raises the pool of sulfated steroid hormones, increases longevity and ameliorates protein aggregation diseases. This increased longevity requires factors involved in germline-mediated longevity (daf-16, daf-12, kri-1, tcer-1 and daf-36 genes) although sul-2 mutations do not affect fertility. Interestingly, sul-2 is only expressed in sensory neurons, suggesting a regulation of sulfated hormones state by environmental cues. Treatment with the specific STS inhibitor STX64, as well as with testosterone-derived sulfated hormones reproduces the longevity phenotype of sul-2 mutants. Remarkably, those treatments ameliorate protein aggregation diseases in C. elegans, and STX64 also Alzheimer's disease in a mammalian model. These results open the possibility of reallocating steroid sulfatase inhibitors or derivates for the treatment of aging and aging related diseases.

Testicular steroid sulfatase overexpression is associated with Leydig cell dysfunction in primary spermatogenic failure.

BACKGROUND: Decreased testosterone (T) to LH ratio and increased 17ß-estradiol (E2) serum concentrations represent a common finding among patients with severe spermatogenic failure, suggesting a concurrent Leydig cell steroidogenic dysfunction. Aromatase overexpression has been associated with increased serum and intratesticular E2 in these patients. However, it is unknown whether the sulfatase pathway contributes to the increased availability of active estrogens in patients with primary spermatogenic failure. OBJECTIVES: To assess estrogen sulfotransferase (SULT1E1) and steroid sulfatase (STS) mRNA abundance in testicular tissue of patients with Sertoli cell-only syndrome (SCOS) and normal tissues, its association with serum and intratesticular hormone levels, and to explore the mRNA and protein testicular localization of both enzymes. MATERIALS AND METHODS: Testicular tissues of 23 subjects with SCOS (cases) and 22 patients with obstructive azoospermia and normal spermatogenesis (controls) were obtained after biopsy. SULT1E1 and STS transcripts accumulation was quantified by RT-qPCR. For mRNA and protein localization, we performed RT-qPCR in Leydig cell clusters and seminiferous tubules isolated by laser-capture microdissection and immunofluorescence in testicular tissues. Serum and intratesticular hormones were measured by immunoradiometric assays. RESULTS: SULT1E1 mRNA accumulation was similar in both groups. The amount of STS mRNA was higher in cases (p = 0.007) and inversely correlated with T/LH ratio (r = -0.402; p = 0.02). Also, a near significant correlation was observed with intratesticular E2 (r = 0.329, p = 0.057), in agreement with higher intratesticular E2 in cases (p < 0.001). Strong STS immunoreaction was localized in the wall of small blood vessels but not in Leydig cells. Both SULT1E1 and STS mRNA abundance was similar in Leydig cell clusters and the tubular compartment, except for lower SUTL1E1 mRNA in the seminiferous tubules of SCOS patients (p = 0.001). CONCLUSIONS: Our results suggest that an unbalance of the STS/SULT1E1 pathway contributes to the testicular hyperestrogenic microenvironment in patients with primary spermatogenic failure and Leydig cell dysfunction.

New potent steroid sulphatase inhibitors based on 6-(1-phenyl-1H-1,2,3-triazol-4-yl)naphthalen-2-yl sulphamate derivatives.

In the present work, we report a new class of potent steroid sulphatase (STS) inhibitors based on 6-(1-phenyl-1H-1,2,3-triazol-4-yl)naphthalen-2-yl sulphamate derivatives. Within the set of new STS inhibitors, 6-(1-(1,2,3-trifluorophenyl)-1H-1,2,3-triazol-4-yl)naphthalen-2-yl sulphamate 3L demonstrated the highest activity in the enzymatic assay inhibiting the STS activity to 7.98% at 0.5 µM concentration. Furthermore, to verify whether the obtained STS inhibitors are able to pass through the cellular membrane effectively, cell line experiments have been carried out. We found that the lowest STS activities were measured in the presence of compound 3L (remaining STS activity of 5.22%, 27.48% and 99.0% at 100, 10 and 1 nM concentrations, respectively). The measured STS activities for Irosustat (used as a reference) were 5.72%, 12.93% and 16.83% in the same concentration range. Moreover, a determined IC50 value of 15.97 nM for 3L showed that this compound is a very promising candidate for further preclinical investigations.

Multimodal Imaging of Pre-Descemet Corneal Dystrophy Associated With X-Linked Ichthyosis and Deletion of the STS Gene.

PURPOSE: To investigate the presence of pre-Descemet corneal dystrophy (PDCD) in association with X-linked ichthyosis (XLI) in an 11-year-old boy using multimodal imaging and genetic analysis. METHODS: Corneal opacities were examined and imaged with slit-lamp biomicroscopy, anterior segment optical coherence tomography, noncontact specular microscopy, and in vivo confocal microscopy. Cytogenomic array analysis was performed using genomic DNA isolated from the patient. RESULTS: Corneal opacities characteristic of PDCD located in the posterior corneal stroma just anterior to Descemet membrane were identified by slit-lamp biomicroscopy. A pre-Descemet hyper-reflective line, consistent with these opacities, was seen with anterior segment optical coherence tomography. Scheimpflug tomography revealed a bimodal peak light scattering. In vivo confocal microscopy findings were unremarkable. Copy number analysis identified a 4389 kbp hemizygous deletion on the X chromosome (chr. X: 6,540,898-8,167,604), resulting in the deletion of 4 genes, including the known locus of XLI, the STS gene. CONCLUSIONS: This report demonstrates that PDCD-associated XLI may present in children and that the diagnosis may be confirmed through multimodal imaging in conjunction with genetic analysis.

Estrogen Formation and Inactivation Following TBI: What we Know and Where we Could go.

Traumatic brain injury (TBI) is responsible for various neuronal and cognitive deficits as well as psychosocial dysfunction. Characterized by damage inducing neuroinflammation, this response can cause an acute secondary injury that leads to widespread neurodegeneration and loss of neurological function. Estrogens decrease injury induced neuroinflammation and increase cell survival and neuroprotection and thus are a potential target for use following TBI. While much is known about the role of estrogens as a neuroprotective agent following TBI, less is known regarding their formation and inactivation following damage to the brain. Specifically, very little is known surrounding the majority of enzymes responsible for the production of estrogens. These estrogen metabolizing enzymes (EME) include aromatase, steroid sulfatase (STS), estrogen sulfotransferase (EST/SULT1E1), and some forms of 17ß-hydroxysteroid dehydrogenase (HSD17B) and are involved in both the initial conversion and interconversion of estrogens from precursors. This article will review and offer new prospective and ideas on the expression of EMEs following TBI.

Recent progress in the development of steroid sulphatase inhibitors - examples of the novel and most promising compounds from the last decade.

The purpose of this review article is to provide an overview of recent achievements in the synthesis of novel steroid sulphatase (STS) inhibitors. STS is a crucial enzyme in the biosynthesis of active hormones (including oestrogens and androgens) and, therefore, represents an extremely attractive molecular target for the development of hormone-dependent cancer therapies. The inhibition of STS may effectively reduce the availability of active hormones for cancer cells, causing a positive therapeutic effect. Herein, we report examples of novel STS inhibitors based on steroidal and nonsteroidal cores that contain various functional groups (e.g. sulphamate and phosphorus moieties) and halogen atoms, which may potentially be used in therapies for hormone-dependent cancers. The presented work also includes examples of multitargeting agents with STS inhibitory activities. Furthermore, the fundamental discoveries in the development of the most promising drug candidates exhibiting STS inhibitory activities are highlighted.

Design and synthesis of 3-benzylaminocoumarin-7-O-sulfamate derivatives as steroid sulfatase inhibitors.

Steroid sulfatase (STS) is a sulfatase enzyme that catalyzes the conversion of sulfated steroid precursors to free steroid. The inhibition of STS could abate estrogenic steroids that stimulate the proliferation and development of breast cancer, and therefore STS is a potential target for adjuvant endocrine therapy. In this study, a series of 3-benzylaminocoumarin-7-O-sulfamate derivatives targeting STS were designed and synthesized. Structure-relationship activities (SAR) analysis revealed that attachment of a benzylamino group at the 3-position of coumarin improved inhibitory activity. Compound 3j was found to have the highest inhibition activity against human placenta isolated STS (IC50  0.13 µM) and MCF-7 cell lines (IC50 1.35 µM). Kinetic studies found compound 3j to be an irreversible inhibitor of STS, with KI and kinact value of 86.9 nM and 158.7 min-1, respectively.

Achieving the ratiometric imaging of steroid sulfatase in living cells and tissues with a two-photon fluorescent probe.

Herein, a novel two-photon ratiometric fluorescence assay was proposed for monitoring endogenous steroid sulfatase (STS) activity, which could be applied for the ratiometric imaging of STS activity in the endoplasmic reticulum of living cells and tissues and also could be used to distinguish estrogen-dependent tumor cells from other types of cells.

Steroid sulfatase inhibiting lanostane triterpenes - Structure activity relationship and in silico insights.

Steroid sulfatase (STS) transforms hormone precursors into active steroids. Thus, it represents a target of intense research regarding hormone-dependent cancers. In this study, three ligand-based pharmacophore models were developed to identify STS inhibitors from natural sources. In a pharmacophore-based virtual screening of a curated molecular TCM database, lanostane-type triterpenes (LTTs) were predicted as STS ligands. Three traditionally used polypores rich in LTTs, i.e., Ganoderma lucidum Karst., Gloeophyllum odoratum Imazeki, and Fomitopsis pinicola Karst., were selected as starting materials. Based on eighteen thereof isolated LTTs a structure activity relationship for this compound class was established with piptolinic acid D (1), pinicolic acid B (2), and ganoderol A (3) being the most pronounced and first natural product STS inhibitors with IC50 values between 10 and 16 µM. Molecular docking studies proposed crucial ligand target interactions and a prediction tool for these natural compounds correlating with experimental findings.

The Phenotype and Secretory Activity of Adipose-Derived Mesenchymal Stem Cells (ASCs) of Patients with Rheumatic Diseases.

Mesenchymal stem/stromal cells (MSCs) have immunosuppressive and regenerative properties. Adipose tissue is an alternative source of MSCs, named adipose-derived mesenchymal stem cells (ASCs). Because the biology of ASCs in rheumatic diseases (RD) is poorly understood, we performed a basic characterization of RD/ASCs. The phenotype and expression of adhesion molecules (intracellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1) on commercially available healthy donors (HD), ASC lines (n = 5) and on ASCs isolated from patients with systemic lupus erythematosus (SLE, n = 16), systemic sclerosis (SSc, n = 17) and ankylosing spondylitis (AS, n = 16) were analyzed by flow cytometry. The secretion of immunomodulatory factors by untreated and cytokine-treated ASCs was measured by ELISA. RD/ASCs have reduced basal levels of CD90 and ICAM-1 expression, correlated with interleukin (IL)-6 and transforming growth factor (TGF)-ß1 release, respectively. Compared with HD/ASCs, untreated and tumour necrosis factor (TNF) + interferon (IFN)-γ (TI)-treated RD/ASCs produced similar amounts of prostaglandin E2 (PGE2), IL-6, leukemia inhibiting factor (LIF), and TGF-ß1, more IL-1Ra, soluble human leukocyte antigen G (sHLA-G) and tumor necrosis factor-inducible gene (TSG)-6, but less kynurenines and galectin-3. Basal secretion of galectin-3 was inversely correlated with the patient's erythrocyte sedimentation rate (ESR) value. IFN-α and IL-23 slightly raised galectin-3 release from SLE/ASCs and AS/ASCs, respectively. TGF-ß1 up-regulated PGE2 secretion by SSc/ASCs. In conclusion, RD/ASCs are characterized by low basal levels of CD90 and ICAM-1 expression, upregulated secretion of IL-1Ra, TSG-6 and sHLA-G, but impaired release of kynurenines and galectin-3. These abnormalities may modify biological activities of RD/ASCs.

Synthesis and in vitro evaluation of piperazinyl-ureido sulfamates as steroid sulfatase inhibitors.

Two new piperazinyl-ureido single ring aryl sulfamate-based inhibitor series were designed against the emerging oncology drug target steroid sulfatase (STS), for which there are existing potent steroidal and non-steroidal agents in clinical trials. 4-(Piperazinocarbonyl)aminosulfamates (5-31) were obtained by reacting 4-hydroxyarylamines with phenylchloroformate, subsequent sulfamoylation of the resulting hydroxyarylcarbamates and coupling of the product with 1-substituted piperazines. Pyrimidinyl-piperazinourea sulfamates (35-42) were synthesized by pyrimidine ring closure of 4-Boc-piperazine-1-carboxamidine with 3-(dimethylamino)propenones, deprotection and coupling with the sulfamoylated building block. Target ureidosulfamates 5-31 and 35-42 were evaluated both as STS inhibitors in vitro using a lysate of JEG-3 human placenta choriocarcinoma cell line and in a whole cell assay. SAR conclusions were drawn from both series. In series 35-42 the best inhibitory activity is related to the presence of a benzofuryl on the pyrimidine ring. In series 5-31 the best inhibitory activity was shown by the ureas bearing 4-chlorophenyl, 3,4-dichlorophenyl groups or aliphatic chains at the piperazino 4-nitrogen displaying IC50 in the 33-94 nM concentration range. Final optimization to the low nanomolar level was achieved through substitution of the arylsulfamate ring with halogens. Four halogenated arylsulfamates of high potency were achieved and two of these 19 and 20 had IC50 values of 5.1 and 8.8 nM respectively and are attractive for potential in vivo evaluation and further development. We demonstrate the optimization of this new series to low nanomolar potency, employing fluorine substitution, providing potent membrane permeant inhibitors with further development potential indicating piperazinyl-ureido aryl sulfamate derivatives as an attractive new class of STS inhibitors.