Development of (p-O-sulfamoyl)-N-alkanoyl-phenylalkyl amines as non-steroidal estrone sulfatase inhibitors

Estrogen levels in breast tumors of postmenopausal women are as much as 10 times higher than estrogen levels in plasma, presumably due to in situ formation of estrogen. The major source of estrogen in breast cancer cells may be conversion of estrone sulfate to estrone by the enzyme estrone sulfatase. Thus, inhibitors of estrone sulfatase are potential agents for treatment of estrogen-dependent breast cancer. Several steroidal compounds have been developed that are potent estrone sulfatase inhibitors, most notably estrone-3-O-sulfamate. However, these compounds and their metabolites may have undesired effects, including estrogenicity. To avoid the problems associated with a potentially active steroid nucleus, we designed and synthesized a series of nonsteroidal estrone sulfatase inhibitors, the (p-O-sulfamoyl)-N-alkanoyl phenylalkyl amines. The compounds synthesized vary in the length of their alkanoyl chain and in the number of carbons separating the phenyl ring and the carbonyl carbon. The ability of these compounds to inhibit estrone sulfatase activity was tested using human placental microsomes and intact cultured human breast cancer cells. Estrogenicity was also evaluated, using growth of estrogen-dependent human breast cancer cells. All of the test compounds inhibited estrone sulfatase activity of human placental microsomes to some extent, with the most effective compound having an IC50 value of 72 nM. In general, compounds with longer alkanoyl chains (12-14 carbons) were more effective than those with shorter chains. The test compounds also inhibited estrone sulfatase activity in intact cultures of MDA-MB-231 human breast cancer cells. Again, the longer chain compounds were more effective. In both the placental and breast cancer cell sulfatase assays, the optimal distance between the phenyl ring and the carbonyl carbon was 1-2 carbons. The MCF-7 cell proliferation assay revealed that estrone and estrone-3-O-sulfamate were both estrogenic, but the (p-O-sulfamoyl)-N-alkanoyl phenylalkyl amines were not. Our data indicate the utility of (p-O-sulfamoyl)-N-alkanoyl phenyl alkylamines for inhibition of estrone sulfatase activity. Furthermore, our data support the concept that nonsteroidal estrone sulfatase inhibitors may be useful as therapeutic agents for estrogen-dependent breast cancers.     

3Beta-sulfamate derivatives of C19 and C21 steroids bearing a t-butylbenzyl or a benzyl group: synthesis and evaluation as non-estrogenic and non-androgenic steroid sulfatase inhibitors

A series of C19 and C21 steroids bearing one or two inhibiting groups (3beta-sulfamate and 17alpha- or 20(S)-t-butylbenzyl or benzyl) were synthesized and tested for inhibition of steroid sulfatase activity. When only a sulfamate group was added to dehydroepiandrosterone, androst-5-ene-3beta,17beta-diol, pregnenolone and 20-hydroxy-pregnenolone, no significant inhibition of steroid sulfatase occurred at concentrations of 0.3 and 3 microM. With only a t-butylbenzyl or a benzyl group, a stronger steroid sulfatase inhibition was obtained in the androst-5-ene than in the pregn-5-ene series. Comparative results from the screening tests and the IC50 values have shown that the effect of a sulfamate moiety as a second inhibiting group can be combined to the t-butylbenzyl or benzyl effect in the C19 and C21 steroid series. The 3beta-sulfamoyloxy-17alpha-t-butylbenzyl-5-androsten-17beta-ol (10) was thus found to be the most active compound with IC50 values of 46 +/- 8 and 14 +/- 1 nM, respectively for the transformations of E1S to E1 and DHEAS to DHEA. The IC50 values of compound 10 are similar to that of 17alpha-t-butylbenzyl-estradiol, which was previously reported by our group as a good steroid sulfatase reversible inhibitor, but remains higher than that of the potent inactivators estrone-3-O-sulfamate (EMATE) and 17alpha-t-butylbenzyl-EMATE. However, contrary to these two latter inhibitors, compound 10 did not induce any proliferative effect on estrogen-sensitive ZR-75-1 cells nor on androgen-sensitive Shionogi cells at concentrations tested, suggesting that this steroid sulfatase inhibitor is non estrogenic and non androgenic.     

The difluoromethylene group as a replacement for the labile oxygen in steroid sulfates: a new approach to steroid sulfatase inhibitors

Several estrone sulfate and estradiol sulfate analogues, in which the sulfate group was replaced with an alpha,alpha-difluoromethylenesulfonate group or an alpha,alpha-difluoromethylenetetrazole group, were examined as inhibitors of steroid sulfatase (STS). These compounds were 4.5-10.5 times more potent than their non-fluorinated analogues. Moreover, the presence of the fluorines changed the mode of inhibition from mixed to competitive. The inhibitor bearing the alpha,alpha-difluoromethylenetetrazole group exhibited an affinity for STS approaching that of the natural STS substrate, estrone sulfate. Possible reasons for the enhanced affinity of the fluorinated compounds compared to their non-fluorinated counterparts are discussed.     

3β-Sulfamate Derivatives of C19 and C21 Steroids Bearing a t -Butylbenzyl or a Benzyl Group: Synthesis and Evaluation as Non-estrogenic and Non-androgenic Steroid Sulfatase Inhibitors

A series of C19 and C21 steroids bearing one or two inhibiting groups (3 β -sulfamate and 17 α - or 20(S)- t -butylbenzyl or benzyl) were synthesized and tested for inhibition of steroid sulfatase activity. When only a sulfamate group was added to dehydroepiandrosterone, androst-5-ene-3 β,17 β -diol, pregnenolone and 20-hydroxy-pregnenolone, no significant inhibition of steroid sulfatase occurred at concentrations of 0.3 and 3 μM. With only a t -butylbenzyl or a benzyl group, a stronger steroid sulfatase inhibition was obtained in the androst-5-ene than in the pregn-5-ene series. Comparative results from the screening tests and the IC 50 values have shown that the effect of a sulfamate moiety as a second inhibiting group can be combined to the t -butylbenzyl or benzyl effect in the C19 and C21 steroid series. The 3 β -sulfamoyloxy-17 α - t -butylbenzyl-5-androsten-17 β -ol (10) was thus found to be the most active compound with IC 50 values of 46 ± 8 and 14 ± 1 nM, respectively for the transformations of E 1 S to E 1 and DHEAS to DHEA. The IC 50 values of compound 10 are similar to that of 17 α - t -butylbenzyl-estradiol, which was previously reported by our group as a good steroid sulfatase reversible inhibitor, but remains higher than that of the potent inactivators estrone-3- O -sulfamate (EMATE) and 17 α - t -butylbenzyl-EMATE. However, contrary to these two latter inhibitors, compound 10 did not induce any proliferative effect on estrogen-sensitive ZR-75-1 cells nor on androgen-sensitive Shionogi cells at concentrations tested, suggesting that this steroid sulfatase inhibitor is non estrogenic and non androgenic.     

17Alpha-alkan (or alkyn) amide derivatives of estradiol as inhibitors of steroid-sulfatase activity

To develop inhibitors of steroid sulfatase without residual estrogenic activity, we have designed a series of estradiol (E2) derivatives bearing an alkan (or alkyn) amide side chain at position 17alpha. A hydrophobic alkyl group was selected from our previous study where 17alpha-octyl-E2 was found to inhibit strongly the steroid-sulfatase activity. Furthermore, it is known that an alkylamide side chain blocks the estrogen-receptor activation. Starting from ethynylestradiol, the chemical synthesis of target compounds was short and efficient with overall yields of 22-42% (3 or 4 steps). Among these compounds, N-octyl,N-methyl-3-(3',17'beta-dihydroxy-1',3',5'(10')-estratrien- 17'alpha-yl)-propanamide (15) was the most potent inhibitor, with an IC50 value of 0.08 microM for the transformation of estrone sulfate (E1S) to estrone (E1) by homogenated JEG-3 cells. N-butyl, N-hexyl, and N,N-dioctyl propanamide derivatives of E2 (IC50 values of 6.4, 2.8, and >20 microM, respectively) were less potent inhibitors than N-octyl analog 15. Furthermore, the unsaturated propynamide analog of 15 gave lower inhibition (four times) than the saturated compound. Compound 15 is also about 100-fold more effective in interacting with the enzyme than substrate E1S itself. The ability of target compounds to bind the estrogen receptor, to stimulate the proliferation of estrogen-sensitive ZR-75-1 cells, or to inhibit the E2-stimulation of ZR-75-1 cells was also evaluated. Although a mixed estrogenic/anti-estrogenic activity was obtained for tested compounds at 1 microM, no estrogenic activity was observed at 0.03 microM for 15. In conclusion, a promising inhibitor of steroid-sulfatase activity was obtained by introducing a hydrophobic octyl group in a 17alpha-propanamide side chain of E2, but further structure-activity relationships (SAR) studies are necessary to minimize the residual estrogenic activity.     

Interaction between rat prostatic steroid 5 alpha-reductase and 3-carboxy-17 beta-substituted steroids: novel mechanism of enzyme inhibition

Efforts to identify novel compounds capable of blocking the steroid 5 alpha-reductase (SR) catalyzed conversion of testosterone (T) to 5 alpha-dihydrotestosterone have resulted in the development of 17 beta-substituted-3-androstene-3-carboxylic acids as potent inhibitors of the rat prostatic enzyme. The dead-end inhibition patterns of one of these steroidal acrylates, 17 beta-N-(2-methyl-2-propyl)-carbamoyl-androst-3,5-diene-3-carboxylic acid were best evaluated with a linear uncompetitive kinetic model vs both T (Kii = 11 +/- 1 nM) and NADPH (Kii = 22 +/- 2 nM). To interpret these observations, the kinetic mechanism of the rat prostatic SR was shown to involve the binding of NADPH prior to that of T through a series of dead-end and product inhibition experiments. Within the context of this preferentially ordered kinetic mechanism, it is proposed that the uncompetitive inhibition patterns result from the association of the steroidal acrylate to an enzyme complex containing NADP+ in formation of a dead-end ternary complex of enzyme, NADP+, and inhibitor.     

Inhibition of hepatic 17 beta- and 3 alpha-hydroxysteroid dehydrogenases by antiinflammatory drugs and nonsteroidal estrogens

Antiinflammatory agents and estrogens have been tested as inhibitors of two isozymes of guinea pig liver testosterone 17 beta-dehydrogenase (NADP) 1.1.1.64) and rat liver 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50). Antiinflammatory steroids and estradiols were highly inhibitory to 3 alpha-hydroxysteroid dehydrogenase and one isozyme of testosterone 17 beta-dehydrogenase, respectively, but nonsteroidal antiinflammatory agents and nonsteroidal estrogens such as hexestrol, dienstrol, diethylstilbestrol and zearalenone showed potent inhibitions on all the enzymes. Although the inhibitory potency of indomethacin for one isozymes of testosterone 17 beta-dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase decreased with changing pH from 9.7 to 7.0, that of the nonsteroidal estrogens for all the enzymes was little affected by pH. No additive effect in double inhibitor experiments with indomethacin and the nonsteroidal estrogens was observed, and the compounds were all competitive inhibitors with respect to steroidal substrate. The results suggest that there is a very similar region in substrate binding sites of the enzymes.     

D-ring modified estrone derivatives as novel potent inhibitors of steroid sulfatase

A series of novel D-ring modified derivatives of estrone was synthesized and tested as inhibitors of steroid sulfatase (STS). The steroidal D-ring was cleaved via an iodoform reaction and thermal condensation of the resulting marrianolic acid derivative gave 16,17-seco-estra-1,3,5(10)-triene-16,17-imide derivatives, where a piperidinedione moiety is in place of the D-ring. This synthetic approach was found to give a higher overall yield than the literature method of Beckmann rearrangement. A range of alkyl side chains have been introduced on the nitrogen atom of the imido-ring and the corresponding 3-O-sulfamates synthesized. The new D-ring modified estrone derivatives bearing a propyl (39) and a 1-pyridin-3-ylmethyl (46) moiety had IC(50) values of 1 nM when tested in placental microsomes for the inhibition of STS. These compounds are therefore up to 18-fold more potent than EMATE, the very first highly potent irreversible steroidal STS inhibitor.     

Synthesis and biochemical evaluation of novel inhibitors of aromatase (AR) using an enhanced representation of the active site of AR derived from the consideration of the reaction mechanism

A novel molecular modeling study, involving inhibitors bound to the iron of cytochrome P450 heme, is described for nonsteroidal inhibitors of aromatase (AR). Study of compounds such as aminoglutethimide (AG) suggests that it utilizes hydrogen bonding group(s) at the active site which would usually H-bond to the steroid C(17) carbonyl group. Interaction between AG's carbonyl groups and the area of the active site corresponding to the substrate C(3)==O group is not possible due to steric interaction. Possible reasons for the difference in activity of enantiomers of alternative inhibitors is also suggested, as well as the mode of action of the new AR inhibitor, Arimidex-whose inhibitory activity previously has not been rationalized. The present study proposes that it is able to use hydrogen bonding groups at the active site corresponding to the steroid C(17)==O and C(3)==O area, contradicting a previous study where it is postulated that azole-type compounds only use polar groups at the active site corresponding to the steroid D ring. Using the hypotheses of the modeling study, we designed and synthesized a number of novel (enantiomerically pure) inhibitors, which upon biochemical evaluation were found to be good inhibitors; the N-nonyl derivative of the S-enantiomer was found to possess 39% inhibition at 100 microM inhibitor concentration (using androstenedione as the substrate), under similar conditions, and AG possessed 20% inhibition.     

The design,synthesis, and biochemical evaluation of derivatives of biphenyl sulfamate-based compounds as novel inhibitors of estrone sulfatase

We report the initial results of our study into the use of a potential transition state (TS) of the reaction catalyzed by the enzyme estrone sulfatase (ES) in the design of a series of simple 4'-O-sulfamoyl-4-biphenyl-based compounds as novel inhibitors of ES. The results of the study show that these compounds are: potent inhibitors, possessing greater inhibitory activity than 4-methylcoumarin-7-O-sulfamate (COUMATE); weaker inhibitors than the tricyclic derivative of COUMATE, namely 667-COUMATE and the steroidal inhibitor estrone-3-O-sulfamate (EMATE), and irreversible inhibitors of ES.     

Thio- and aminocaffeine analogues as inhibitors of human monoamine oxidase

In a recent study it was shown that 8-benzyloxycaffeine analogues act as potent reversible inhibitors of human monoamine oxidase (MAO) A and B. Although the benzyloxy side chain appears to be particularly favorable for enhancing the MAO inhibition potency of caffeine, a variety of other C8 oxy substituents of caffeine also lead to potent MAO inhibition. In an attempt to discover additional C8 substituents of caffeine that lead to potent MAO inhibition and to explore the importance of the ether oxygen for the MAO inhibition properties of C8 oxy-substituted caffeines, a series of 8-sulfanyl- and 8-aminocaffeine analogues were synthesized and their human MAO-A and -B inhibition potencies were compared to those of the 8-oxycaffeines. The results document that the sulfanylcaffeine analogues are reversible competitive MAO-B inhibitors with potencies comparable to those of the oxycaffeines. The most potent inhibitor, 8-{[(4-bromophenyl)methyl]sulfanyl}caffeine, exhibited an IC₅₀ value of 0.167 μM towards MAO-B. While the sulfanylcaffeine analogues also exhibit affinities for MAO-A, they display in general a high degree of MAO-B selectivity. The aminocaffeine analogues, in contrast, proved to be weak MAO inhibitors with a number of analogues exhibiting no binding to the MAO-A and -B isozymes. The results of this study are discussed with reference to possible binding orientations of selected caffeine analogues within the active site cavities of MAO-A and -B. MAO-B selective sulfanylcaffeine derived inhibitors may act as lead compounds for the design of antiparkinsonian therapies.     

The design,synthesis, and in vitro biochemical evaluation of a series of esters of 4-[(aminosulfonyl)oxy]benzoate as novel and highly potent inhibitors of estrone sulfatase

We report the initial results of our study into the use of a potential transition-state (TS) of the reaction catalysed by the enzyme estrone sulfatase (ES) in the design of a series of cyclic esters of 4-[(aminosulfonyl)oxy]benzoate as novel inhibitors of ES. The results of the study show that these compounds are some of the most potent inhibitors known todate, possessing greater inhibitory activity than the three standard compounds: 4-methylcoumarin-7-O-sulfamate (COUMATE); the tricyclic derivative of COUMATE, namely 667-COUMATE (which is in Phase I of clinical trials) and; the steroidal inhibitor estrone-3-O-sulfamate (EMATE).     

Inhibition of 3(17)beta-hydroxysteroid dehydrogenase from Pseudomonas testosteroni by steroidal A ring fused pyrazoles

Several 2,3- and 3,4-steroidal fused pyrazoles have been investigated as potential inhibitors of NAD(P)H-dependent steroid oxidoreductases. These compounds are proven to be potent, specific inhibitors for 3(17) beta-hydroxysteroid dehydrogenase from Pseudomonas testosteroni with Ki values of 6-100 nM. In contrast, the activities of 3 alpha,20 beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans, steroid 5 alpha-reductase from rat prostate, and 3 alpha-hydroxysteroid dehydrogenase from rat liver were unaffected by micromolar concentrations of these compounds. Product and dead-end inhibition studies indicate an ordered association to the beta-dehydrogenase with the cofactor binding prior to substrate or inhibitor. From the results of double inhibition experiments, it is proposed that inhibition occurs through formation of an enzyme-NAD+-inhibitor ternate. On the basis of pH profiles of Vm/Km, Vm, and 1/Ki and of absorbance difference spectra, a hypothetical mechanism of inhibition by the steroidal pyrazoles, drawn by analogy from the inhibition of liver alcohol dehydrogenase by alkylpyrazoles [Theorell, H., & Yonetani, T. (1963) Biochem. Z. 338, 537-553; Andersson, P., Kvassman, J. K., Lindstr?m, A., Oldén, B., & Pettersson, G. (1981) Eur. J. Biochem. 113, 549-554], is reconsidered. The pH studies and enzyme modification experiments by diethyl pyrocarbonate suggest the involvement of histidine in binding of the inhibitor. A modified proposal for the structure of the enzyme-NAD+-steroidal pyrazole complex is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human placental sterylsulfatase. Interaction of the isolated enzyme with substrates, products, transition-state analogues, amino-acid modifiers and anion transport inhibitors.

The enzymatic properties of a homogeneous sterylsulfatase preparation isolated from human term placenta were studied. The enzyme exhibited both arylsulfatase and sterylsulfatase activity: it catalysed the hydrolysis of sulfuric acid esters of (in the order of decreasing specific activity) non-steroidal phenols, of a phenolic steroid, and of neutral 3 beta-, 21- and (though at a very low rate) 17 beta-hydroxysteroids. However, among all the substrates tested only the 3-sulfates of phenolic and neutral steroids exhibited high affinity towards the sulfatase. Vitamin D3 sulfate was not hydrolysed by the sterylsulfatase but strongly inhibited its activity. The products of the catalytic reaction, free steroids or phenols as well as the sulfate anion or analogues thereof, likewise interfered with the enzyme's activity. Ki values of unconjugated steroids were ten- to hundredfold higher than Km values of the respective sulfoconjugates. Inorganic sulfate only slightly inhibited the sulfatase activity; its inhibitory potency, however, increased in a time-dependent manner when it was preincubated with the enzyme prior to assay. In contrast to sulfate, the hypothetical transition-state analogues sulfite and vanadate acted as strong inhibitors of the sulfatase activity. According to the results of an analysis of the effect of pH on sterylsulfatase kinetics, enzyme constituents with pK values of approximately 5.8 and 8.0 are involved in a general acid-base catalysed reaction. Treatment of the sulfatase with amino-acid side chain modifying reagents directed against arginine, cysteine, cystine, serine or tyrosine residues did not result in significant alteration of its activity. Diethyl-pyrocarbonate known to react primarily with histidyl groups, however, rapidly inactivated the enzyme; this inactivation reaction was markedly retarded in the presence of substrate. Histidine thus appears to be essential for the catalytic activity of the sulfatase. Taken together, the present results reveal a considerable similarity between the catalytic mechanism of human placental sterylsulfatase and the ones already proposed for the lysosomal arylsulfatases A and B. Taurocholate, salicylate, ouabain, and 4,4'-substituted stilbene-2,2'-disulfonates are well known inhibitors of carrier-mediated transport of anions across cellular membranes. With the exception of ouabain, these compounds likewise turned out to inhibit the enzymatic hydrolysis of steryl sulfates; the pattern of dose dependences of their interference with the sulfatase activity resembles the one reported for inhibition of anion transport. Since the sterylsulfatase in vivo strongly is associated with cellular membranes including the plasma membrane of the syncytiotrophoblast, this finding supports the speculation that similar molecular structures may be involved in both placental transport and hydrolysis of anionic steryl sulfates.     

Boronic acids as inhibitors of steroid sulfatase

Steroid sulfatase (STS) catalyzes the hydrolysis of steroidal sulfates such as estrone sulfate (ES1) to the corresponding steroids and inorganic sulfate. STS is considered to be a potential target for the development of therapeutics for the treatment of steroid-dependent cancers. Two steroidal and two coumarin- and chromenone-based boronic acids were synthesized and examined as inhibitors of purified STS. The boronic acid analog of estrone sulfate bearing a boronic acid moiety at the 3-position in place of the sulfate group was a good competitive STS inhibitor with a K_i of 2.8 μM at pH 7.0 and 6.8 μM at pH 8.8. The inhibition was reversible and kinetic properties corresponding to the mechanism for slow-binding inhibitors were not observed. An estradiol derivative bearing a boronic acid group at the 3-position and a benzyl group at the 17-position was a potent reversible, non-competitive STS inhibitor with a K_i of 250 nM. However, its 3-OH analog, a known STS inhibitor, exhibited an almost identical affinity for STS and also bound in a non-competitive manner. It is suggested that these compounds prefer to bind in a hydrophobic tunnel close to the entrance to the active site. The coumarin and chromenone boronic acids were modest inhibitors of STS with IC₅₀s of 86 and 171 μM, respectively. Surprisingly, replacing the boronic acid group of the chromenone derivative with an OH group yielded a good reversible, mixed type inhibitor with a K_i of 4.6 μM. Overall, these results suggest that the boronic acid moiety must be attached to a platform very closely resembling a natural substrate in order for it to impart a beneficial effect on binding affinity compared to its phenolic analog.     

Synthesis and structure–activity relationships of 2- and/or 4-halogenated 13β- and 13α-estrone derivatives as enzyme inhibitors of estrogen biosynthesis

Ring A halogenated 13α-, 13β-, and 17-deoxy-13α-estrone derivatives were synthesised with N-halosuccinimides as electrophile triggers. Substitutions occurred at positions C-2 and/or C-4. The potential inhibitory action of the halogenated estrones on human aromatase, steroid sulfatase, or 17β-hydroxysteroid dehydrogenase 1 activity was investigated via in vitro radiosubstrate incubation. Potent submicromolar or low micromolar inhibitors were identified with occasional dual or multiple inhibitory properties. Valuable structure–activity relationships were established from the comparison of the inhibitory data obtained. Kinetic experiments performed with selected compounds revealed competitive reversible inhibition mechanisms against 17β-hydroxysteroid dehydrogenase 1 and competitive irreversible manner in the inhibition of the steroid sulfatase enzyme.     

4,4'-Benzophenone-O,O'-disulfamate: a potent inhibitor of steroid sulfatase

We investigated whether the benzophenone moiety can be used as core element of steroid sulfatase (STS) inhibitors. While 4- and 3-benzophenone-O-sulfamates inhibit STS with IC(50) values between 5 and 7 microM irrespective of additional hydroxy and methoxy substituents at the second phenyl ring, benzophenone-O,O'-disulfamates show increased activity. With an IC(50) value of 190 nM the 4,4'-derivative is the first small monocyclic STS inhibitor coming close to the potency of the steroidal standard estrone sulfamate.     

Studies on the cholinesterases inhibiting compounds from the Cassiopea andromeda venom

Cholinesterase inhibitors find application in the combat and care of several diseases, especially AD. Jellyfish venoms are the most promising sources of potent cholinesterase inhibitors. Therefore, it is of interest to study cholinesterases inhibiting compounds from the Cassiopea andromeda venom. We report bioactive compounds using the GC-MC method followed by molecular modeling and docking data analysis. The GC-MS analysis of the crude venom led to the identification of seven bioactive compounds (C1-C7), comprising the steroidal alkaloids, phenolic and carotenoid derivatives. The venom exhibited inhibitory activities against the cholinesterase enzymes. The compound C2, a Dioxolane steroid, displayed the strongest inhibition on both AChE and BChE activities for further consideration.     

Retinoid-mediated stimulation of steroid sulfatase activity in myeloid leukemic cell lines requires RARalpha and RXR and involves the phosphoinositide 3-kinase and ERK-MAP kinase pathways

All-trans retinoic acid and 9-cis-retinoic acid stimulate the activity of steroid sulfatase in HL60 acute myeloid leukemia cells in a concentration- and time-dependent manner. Neither of these 'natural retinoids' augmented steroid sulfatase activity in a HL60 sub-line that expresses a dominant-negative retinoic acid receptor alpha (RARalpha). Experiments with synthetic RAR and RXR agonists and antagonists suggest that RARalpha/RXR heterodimers play a role in the retinoid-stimulated increase in steroid sulfatase activity. The retinoid-driven increase in steroid sulfatase activity was attenuated by inhibition of phospholipase D (PLD), but not by inhibitors of phospholipase C. Experiments with inhibitors of protein kinase C (PKC) show that PKCalpha and PKCdelta play an important role in modulating the retinoid-stimulation of steroid sulfatase activity in HL60 cells. Furthermore, we show that pharmacological inhibition of the RAF-1 and ERK MAP kinases blocked the retinoid-stimulated increase in steroid sulfatase activity in HL60 cells and, by contrast, inhibition of the p38-MAP kinase or JNK-MAP kinase had no effect. Pharmacological inhibitors of the phosphatidylinositol 3-kinase, Akt, and PDK-1 also abrogated the retinoid-stimulated increase in steroid sulfatase activity in HL60 cells. These results show that crosstalk between the retinoid-stimulated genomic and non-genomic pathways is necessary to increase steroid sulfatase activity in HL60 cells.     

Synthesis, in vitro and in vivo activity of benzophenone-based inhibitors of steroid sulfatase

Steroid sulfatase (STS) is an important new therapeutic target in oncology. Attempts to design nonsteroidal STS inhibitors, because of the oestrogenicity of the original lead oestrone 3-O-sulfamate in rodents, have led to the discovery of benzophenone-4,4'-O,O-bis-sulfamate (BENZOMATE, 3). The nonfused bicyclic BENZOMATE is a highly potent STS inhibitor in vitro, inhibiting STS activity in intact MCF-7 breast cancer cells by > 70% at 0.1 microM and in placental microsomes by > 98% at 10 microM. When MCF-7 cells were pre-treated with 3 at 1 microM and then washed to remove unbound inhibitor, the initial 94% inhibition was reduced to 89% suggesting that 3, like other sulfamate-based STS inhibitors, inhibits the enzyme irreversibly. This agent also inhibits rat liver STS activity by 84% and 93% respectively 24 h after a single dose of 1 or 10 mg/kg, demonstrating that BENZOMATE possesses similar in vivo potency to the established potent nonsteroidal inhibitor 667COUMATE. Several modifications were made to BENZOMATE structurally and effects on in vitro activity were examined. These structure-activity relationship studies show that its carbonyl and bis-sulfamate groups are pivotal for activity, although conformational flexibility is not required. Two rigid anthraquinone-based sulfamate derivatives however showed inhibitory activity significantly better than BENZOMATE in the MCF-7 cell assay. BENZOMATE and related analogues therefore represent an important class of non-steroidal STS inhibitor and lead compounds for future drug design.