Structure-activity relationships of 2-, 4-, or 6-substituted estrogens as aromatase inhibitors

Aromatase, which is responsible for the conversion of androgens to estrogens, is a potential therapeutic target for the selective lowering of estrogen levels in patients with estrogen-dependent breast cancer. To develop a novel class of aromatase inhibitors, we tested series of 2- and 4-substituted (halogeno, methyl, formyl, methoxy, nitro, and amino) estrones (7 and 9), as well as series of 6alpha- and 6beta-substituted (alkyl, phenalkyl, and alkoxy) estrones (13 and 14), and their estradiol analogs (8, 10, 11, and 12) as aromatase inhibitors. All of the inhibitors examined blocked the androstenedione aromatization in a competitive manner. Introduction of halogeno and methyl functions at C-2 of estrone as well as that of a phenalkyl or methyl function at the C-6alpha or C-6beta position markedly increased affinity to aromatase (apparent K(i) value=0.10-0.66 microM for the inhibitors versus 2.5 microM for estrone). 6alpha-Phenylestrone (13c) was the most powerful inhibitor among the estrogens studied, and its affinity was comparable to that of the androgen substrate androstenedione. Estradiol analogs were much weaker inhibitors than the corresponding estrone compounds in each series, indicating that the 17-carbonyl group plays a critical role in the formation of a thermodynamically stable enzyme-inhibitor complex.     

Inhibition of human placental aromatase in a perfusion model. Comparison with kinetic, cell-free experiments

In vitro perfusion of human placenta was evaluated for characterization of aromatase inhibitors. The results were compared with those in kinetic experiments in cell-free system. Inhibition constants (Ki) were determined by measuring the release of tritiated water during coincubation of human placenta microsomes with varying amounts of [1 beta,2 beta 3H]androstenedione and inhibitor in the presence of NADPH-generating system. Irreversible inactivation constants (Kinact) were determined in a similar manner following preincubation of the microsomes with different amounts of inhibitor for varying times. Lineweaver-Burk plots indicated a competitive type of inhibition with Ki values of 37 nM for 4-hydroxy-androstenedione, 3,700 nM for testolactone, 15 nM for 1-methyl-androsta-1,4-diene-3,17-dione, and 7.5 nM for 19-azido-androstenedione. Additionally, irreversible enzyme inactivation by all four substances could be demonstrated with Kinact values of 3.64 x 10(-3), 0.57 x 10(-3), 0.34 x 10(-3), and 0.69 x 10(-3)sec-1, respectively. Perfusion of a single cotyledon of human term placenta was performed by infusing medium through catheters placed in a fetal artery and in the maternal intervillous space. Perfused medium was collected from a cannulated fetal vein and from the maternal basal plate. The medium was supplemented with [3H]androstenedione (4.2 nM) and inhibitor. The perfusates were analyzed for their [3H]estrone and estradiol content following phenolic partition and Sephadex-LH 20 chromatography. The main results were, (1) the recovery of labelled steroids increased rapidly after perfusion started and reached a plateau within 60 min, when 55 and 30% (mean values) of the infused radioactivity were recovered in the fetal and maternal perfusates, respectively, (2) similar amounts of estrone and estradiol were found in both effluates, whereas androgens (mainly androstenedione and lower amounts of 5 alpha-androstane-3,17-dione) were found nearly exclusively in the fetal perfusate, (3) formation of estrogens (estrone + estradiol) reached a plateau within 20 min of perfusion. (4) The percentage of estrogens formed was not changed by increasing androstenedione concentration in the perfusion medium unless this concentration exceeded 3.5 microM indicating limited capacity of aromatase. (5) The four aromatase inhibitors reduced estrogen formation by 50% at concentrations about 100-fold of their Ki determined in the cell-free system, (6) irreversible aromatase inhibition could not be demonstrated in the perfusion model. It was concluded that the human placenta perfusion model can be successfully used to evaluate aromatase inhibitors.     

Sulfamoyloxy-substituted 2-phenylindoles: antiestrogen-based inhibitors of the steroid sulfatase in human breast cancer cells

Estrone sulfate (E1S) is an endogenous prodrug that delivers estrone and, subsequently, estradiol to the target cells following the hydrolysis by the enzyme estrone sulfatase which is active in various tissues including hormone dependent breast cancer cells. Blockade of this enzyme should reduce the estrogen level in breast cancer cells and prevent hormonal growth stimulation. Sulfamates of a variety of phenolic compounds have been shown to be inhibitors of estrone sulfatase. Our rational is based on findings that these inhibitors can undergo hydrolysis and the pharmacological effects of the free hydroxy compounds contribute to the bioactivity of the sulfamates. A desirable action of the metabolites would be an estrogen antagonism to block stimulatory effects of residual amounts of estrogens. Thus, we synthesized a number of sulfamoyloxy-substituted 2-phenylindoles with side chains at the indole nitrogen that guarantee antiestrogenic activity. All of the new sulfamates were studied for their inhibitory effects on the enzyme estrone sulfatase from human breast cancer cells and their (anti)hormonal activities in stably transfected human MCF-7/2a mammary carcinoma cells. The hormonal profile of the sulfamates was partly reflected by the properties of the corresponding hydroxy precursors. Some of the sulfamoylated antiestrogens strongly inhibited estrone sulfatase activity with IC₅₀ values in the submicromolar range. They were devoid of agonist activity and suppressed estrone sulfate-stimulated gene expression mainly by blocking the enzyme. Examples are the disulfamates of the indoles ZK 119, 010 and ZK 164, 015. Their IC₅₀s for sulfatase inhibition were 0.3 and 0.2 μM, respectively, and 50 and 80 nM, respectively, for the inhibition of E1S-stimulated luciferase expression in transfected MCF-7 cells. With some of the new sulfamates an additional direct antiestrogenic effect was noticed which might be due to a partial hydrolysis during incubation and would improve the growth inhibitory effect on estrogen-sensitive breast cancer cells.     

Carbonic anhydrase inhibitors: inhibition of the human isozymes I, II, VA, and IX with a library of substituted difluoromethanesulfonamides

An inhibition study of the human cytosolic isozymes I, and II, the mitochondrial isoform VA, and the tumor-associated, transmembrane isozyme IX of carbonic anhydrase (CA, EC 4.2.1.1) with a library of aromatic/heteroaromatic/polycyclic difluoromethanesulfonamides is reported. Most of the inhibitors were derivatives of benzenedifluoromethanesulfonamide incorporating substituted-phenyl moieties, or were methylsulfonamide and difluoromethyl-sulfonamide derivatives of the sulfamates COUMATE and EMATE, respectively. Except for the methylsulfonamide-COUMATE derivative which behaved as a potent CA II inhibitor (K(I) of 32nM), these sulfonamides were moderate inhibitors of all isozymes, with inhibition constants in the range of 96-5200nM against hCA I, of 80-670nM against hCA II, and of 195-9280nM against hCA IX, respectively. Remarkably, some derivatives, such as 3-bromophenyl-difluoromethanesulfonamide, showed a trend to selectively inhibit the mitochondrial isoform CA VA, showing selectivity ratios for inhibiting CA VA over CA II of 3.53; over CA I of 6.84 and over CA IX of 9.34, respectively, although it is a moderate inhibitor (K(I) of 160nM). Some of these derivatives may be considered as leads for the design of isozyme selective CA inhibitors targeting the mitochondrial isozyme CA VA, with potential use as anti-obesity agents.     

Synthesis and evaluation of analogues of estrone-3-O-sulfamate as potent steroid sulfatase inhibitors

Estrone sulfamate (EMATE) is a potent irreversible inhibitor of steroid sulfatase (STS). In order to further expand SAR, the compound was substituted at the 2- and/or 4-positions and its 17-carbonyl group was also removed. The following general order of potency against STS in two in vitro systems is observed for the derivatives: The 4-NO(2) > 2-halogens, 2-cyano > EMATE (unsubstituted)>17-deoxyEMATE > 2-NO(2) > 4-bromo>2-(2-propenyl), 2-n-propyl > 4-(2-propenyl), 4-n-propyl > 2,4-(2-propenyl)= 2,4-di-n-propyl. There is a clear advantage in potency to place an electron-withdrawing substituent on the A-ring with halogens preferred at the 2-position, but nitro at the 4-position. Substitution with 2-propenyl or n-propyl at the 2- and/or 4-position of EMATE, and also removal of the 17-carbonyl group are detrimental to potency. Three cyclic sulfamates designed are not STS inhibitors. This further confirms that a free or N-unsubstituted sulfamate group (H(2)NSO(2)O-) is a prerequisite for potent and irreversible inhibition of STS as shown by inhibitors like EMATE and Irosustat. The most potent derivative synthesized is 4-nitroEMATE (2), whose IC(50)s in placental microsomes and MCF-7 cells are respectively 0.8 nM and 0.01 nM.     

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.     

The mechanism of the irreversible inhibition of estrone sulfatase (ES) through the consideration of a range of methane- and amino-sulfonate-based compounds

We report the results of our study into a series of simple phenyl and alkyl sulfamates and alkyl methanesulfonates as potential inhibitors of the enzyme estrone sulfatase (ES). The results of the study show that the substituted phenyl sulfamates are good irreversible inhibitors; the alkyl sulfamate compounds were found to lack inhibitory activity; whilst the large alkyl chain containing methanesulfonate-based compounds were found to possess weak reversible inhibitory activity. Using the results of the inhibition study, we postulate the probable mechanism for ES and suggest that an attack by the gem-diol is a major requirement prior to the hydrolysis of the sulfamate group, following which, attack on the active site C=O occurs and which therefore leads to the production of an imine type functionality, resulting in irreversible inhibition.     

Probing the binding pocket of the active site of aromatase with 6-ether or 6-ester substituted androst-4-ene-3,17-diones and their diene and triene analogs

A series of 6-ester- (3 and 4) and 6-ether- (7 and 8) substituted androst-4-ene-3,17-diones (androstenediones) and their 1,4-diene analogs (5 and 6, and 9 and 10) as well as C6-substituted 4,6-diene and 1,4,6-triene steroids 11 and 12 were synthesized as aromatase inhibitors to gain insight into the structure-activity relationship between various substituents and inhibitory activity. All of the inhibitors synthesized blocked aromatase in a competitive manner. The inhibitory activities of all of the steroids, except for the 6beta-benzoates 4g and 6h and the 6beta-acetate 6a, were fairly effective to very powerful (K(i): 7.0-320 nM). The 6alpha-n-hexanoyloxy- and 6alpha-benzyloxyandrostenediones (3e and 7e) were the most potent inhibitors (K(i): 7.0 nM each). In the series of 4-ene and 1,4-diene steroids, the 6alpha-substituted steroids had higher affinity for the enzyme than the corresponding 6beta-isomers. In the 1,4-diene steroid series, 6beta-substituted steroids 6a, e, g, and 10a, b, e caused a time-dependent inactivation of aromatase, whereas their 6alpha-isomers 5 and 9 essentially did not. The ether-substituted 1,4,6-trienes 12 inactivated the enzyme in a time-dependent manner; in contrast, their 4,6-diene analogs 11 did not. The substrate androstenedione blocked the inactivation, but no significant effect of L-cysteine was observed. Based on molecular modeling with the PM3 method, along with the present inhibition and inactivation results, it is thought that both the steric effects of the 6-substituents as well as the electronic effects of the C-6 oxygen functions play a critical role in the binding of inhibitors to the active site of aromatase.     

Inhibition studies of the β-carbonic anhydrases from the bacterial pathogen Salmonella enterica serovar Typhimurium with sulfonamides and sulfamates

The two β-carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Salmonella enterica serovar Typhimurium, stCA 1 and stCA 2, were investigated for their inhibition with a large panel of sulfonamides and sulfamates. Unlike inorganic anions, which are weak, millimolar inhibitors of the two enzymes [Vullo et al., Bioorg. Med. Chem. Lett.2011, 21, 3591], sulfonamides and sulfamates are effective micro-to nanomolar inhibitors of the two enzymes. Various types of inhibitors have been detected among the 38 investigated sulfonamides/sulfamates, with K(I)s in the range of 31 nM-5.87 μM. The best stCA 1 inhibitors were acetazolamide and benzolamide-based compounds, whereas the best stCA 2 inhibitors were sulfonylated benzenesulfonamides and amino-benzolamide derivatives (K(I)s in the range of 31-90 nM). 3-Fluoro-5-chloro-4-aminobenzolamide showed an inhibition constant of 51 nM against stCA 1 and of 38 nM against stCA 2, being the best inhibitor detected so far for these enzymes. As many strains of S. enterica show extensive resistance to classical antibiotics, inhibition of the β-CAs investigated here may be useful for developing novel antibacterials, targeting β-CAs which may be involved in pathogenicity and invasion of some bacteria.     

Biological characterization of A-ring steroids

Hydroxylated 2,19-methylene-bridged androstenediones were designed as potential mimics of enzyme oxidized intermediates of androstenedione. These compounds exhibited competitive inhibition with low micromolar affinities for aromatase. These inhibitory constants (K_i values) were 10 times greater than the 2,19-methylene-bridged androstenedione constant (K_i = 35–70 nM). However, expansion of the 2,19-carbon bridge to ethylene increased aromatase affinity by 10-fold (K_i = 2 nM). Substitution pf a methylene group with oxygen and sulfur in this expanded bridge resulted in K_i values of 7 and 20 nM, respectively. When the substituent was an NH group, the apparent inhibitory kinetics changed from competitive to uncompetitive. All of these analogs exhibited time-dependent inhibition of aromatase activity following preincubation of the inhibitor with human placental microsomes prior to measuring residual enzyme activity. Part of this inhibition was NADPH cofactor-dependent for the 2,19-methyleneoxy- but not for the 2,19-ethylene-bridged androstenedione. The time-dependent inhibition for these four analogs was very rapid since they exhibited τ₅₀ values, the t_1/2 for enzyme inhibition at infinite inhibitor concentration, of 1 to 3 min. These A-ring-bridged androstenedione analogs represent a novel series of potent steroidal aromatase inhibitors. The restrained A-ring bridge containing CH₂, O, S, or NH could effectively coordinate with the heme of the P450 aromatase to allow the tight-binding affinities reflected by their nanomolar K_i values.     

Biochemistry and pharmacology of 7α-substituted androstenediones as aromatase inhibitors

The inhibition of aromatase, the enzyme responsible for converting androgens to estrogens, is therapeutically useful for the endocrine treatment of hormone-dependent breast cancer. Research by our laboratory has focused on developing competitive and irreversible steroidal aromatase inhibitors, with an emphasis on synthesis and biochemistry of 7α-substituted androstenediones. Numerous 7α-thiosubstituted androst-4-ene-3,17-diones are potent competitive inhibitors, and several 1,4-diene analogs, such as 7α-(4′-aminophenylthio)-androsta-1,4-diene-3,17-dione (7α-APTADD), have demonstrated effective enzyme-activated irreversible inhibition of aromatase in microsomal enzyme assays. One focus of current research is to examine the effectiveness and biochemical pharmacology of 7α-APTADD in vivo. In the hormone-dependent 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinoma model system, 7α-APTADD at a 50 mg/kg/day dose caused an initial decrease in mean tumor volume during the first week, and tumor volume remained unchanged throughout the remaining 5-week treatment period. This agent lowers serum estradiol levels and inhibits ovarian aromatase activity. A second research area has focused on the synthesis of more metabolically stable inhibitors by replacing the thioether linkage at the 7α position with a carbon-carbon linkage. Several 7α-arylaliphatic androst-4-ene-3,17-diones were synthesized by 1,6-conjugate additions of appropriate organocuprates to a protected androst-4,6-diene or by 1,4-conjugate additions to a seco-A-ring steroid intermediate. These compounds were all potent inhibitors of aromatase with apparent K_is ranging between 13 and 19 nM. Extension of the research on these 7α-arylaliphatic androgens includes the introduction of a C₁---C₂ double bond in the A-ring to provide enzyme-activated irreversible inhibitors. The desired 7α-arylaliphatic androsta-1,4-diene-3,17-diones were obtained from their corresponding 7α-arylaliphatic androst-4-ene-3,17-diones by oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). These inhibitors demonstrated enzyme-mediated inactivation of aromatase with apparent k_inacts ranging from 4.4 × 10⁻⁴ to 1.90 x 10⁻³ s⁻¹. The best inactivator of the series was 7α-phenpropylandrosta-1,4-diene-3,17-dione, which exhibited a T_1/2 of 6.08 min. Aromatase inhibition was also observed in MCF-7 human mammary carcinoma cell cultures and in JAr human choriocarcinoma cell cultures, exhibiting IC₅₀ values of 64-328 nM. The 7α-arylaliphatic androgens thus demonstrate potent inhibition of aromatase in both microsomal incubations and in choriocarcinoma cell lines expressing aromatase enzymatic activity. Additionally, the results from these studies provide further evidence for the presence of a hydrophobic binding pocket existing near the 7α-position of the steroid in the active site of aromatase. The size of the 7α-substituent influences optimal binding of steroidal inhibitors to the active site and affects the extent of enzyme-mediated inactivation observed with androsta-1,4-diene-3,17-dione analogs.     

2-phenylindole sulfamates: inhibitors of steroid sulfatase with antiproliferative activity in MCF-7 breast cancer cells

A number of 2-phenylindole sulfamates with lipophilic side chains in 1- or 5-position of the indole were synthesized and evaluated as steroid sulfatase (estrone sulfatase) inhibitors. Most of the new sulfamates inhibited the enzymatic hydrolysis of estrone sulfate in MDA-MB 231 breast cancer cells with IC₅₀ values between 2 nM and 1 μM. A favorable position for a long side chain is the nitrogen of a carbamoyl group at C-5 of the indole when the phenyl ring carries the sulfamate function. These derivatives inhibit gene activation in estrogen receptor (ER)-positive MCF-7 breast cancer cells in submicromolar concentrations and reduce cell proliferation with IC₅₀ values of ca. 1 μM. All of the potent inhibitors were devoid of estrogenic activity and have the potential for in vivo application as steroid sulfatase inhibitors.     

Competitive product inhibition of aromatase by natural estrogens

In order to better understand the function of aromatase, we carried out kinetic analyses to asses the ability of natural estrogens, estrone (E₁), estradiol (E₂), 16-OHE₁, and estriol (E₃), to inhibit aromatization. Human placental microsomes (50 μg protein) were incubated for 5 min at 37°C with [1β-³H]testosterone (1.24 × 10³ dpm ³H/ng, 35–150 nM) or [1β-³H,4-¹⁴C]androstenedione (3.05 × 10³ dpm ³H/ng, ³H/¹⁴C = 19.3, 7–65 nM) as substrate in the presence of NADPH, with and without natural estrogens as putative inhibitors. Aromatase activity was assessed by tritium released to water from the 1β-position of the substrates. Natural estrogens showed competitive product inhibition against androgen aromatization. The K_i of E₁, E₂, 16-OHE₁, and E₃ for testosterone aromatization was 1.5, 2.2, 95, and 162 μM, respectively, where the K_m of aromatase was 61.8 ± 2.0 nM (n = 5) for testosterone. The K_i of E₁, E₂, 16-OHE₁, and E₃ for androstenedione aromatization was 10.6, 5.5, 252, and 1182 μM, respectively, where the K_m of aromatase was 35.4 ± 4.1 nM (n = 4) for androstenedione. These results show that estrogens inhibit the process of andrigen aromatization and indicate that natural estrogens regulate their own synthesis by the product inhibition mechanism in vivo. Since natural estrogens bind to the active site of human placental aromatase P-450 complex as competitive inhibitors, natural estrogens might be further metabolized by aromatase. This suggests that human placental estrogen 2-hydroxylase activity is catalyzed by the active site of aromatase cytochrome P-450 and also agrees with the fact that the level of catecholestrogens in maternal plasma increases during pregnancy. The relative affinities and concentration of androgens and estrogens would control estrogen and catecholestrogen biosynthesis by aromatase.     

Novel silylated steroids as aromatase inhibitors

Androst-4-en-3-one analogs incorporating a trimethylsilyl or a trimethylsilylmethyl group at C-1, C-2 or C-19 were prepared and evaluated as inhibitors of aromatase. Only 10-[1-hydroxy-2-(trimethylsilyl)ethyl]estr-4-ene-3,17-dione inhibited human placental aromatase. Enzyme kinetic analysis revealed competitive inhibition [apparent dissociation constant (Ki) of 562 +/- 12 nM] associated with marginal time-dependent inhibition.     

Carbonic anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, IX, and XII with N-hydroxysulfamides--a new zinc-binding function in the design of inhibitors

A small library of N-hydroxysulfamides was synthesized by an original approach in order to investigate whether this zinc-binding function is efficient for the design of inhibitors targeting the cytosolic (hCA I and II) and transmembrane, tumor-associated (hCA IX and XII) isozymes of carbonic anhydrase (CA, EC 4.2.1.1). The parent derivative, N-hydroxysulfamide was a more potent inhibitor as compared to sulfamide or sulfamic acid against all isozymes, with inhibition constants in the range of 473 nM-4.05 microM. Its substituted n-decyl-, n-dodecyl-, benzyl-, and biphenylmethyl-derivatives were less inhibitory against hCA I (K(I)s in the range of 5.8-8.2 microM) but more inhibitory against hCA II (K(I)s in the range of 50.5-473 nM). The same situation was true for the tumor-associated isozymes, with K(I)s in the range of 353-790 nM against hCA IX and 372-874 nM against hCA XII. Some sulfamides/sulfamates possessing similar substitution patterns have also been investigated for the inhibition of these isozymes, being shown that in some particular cases sulfamides are more efficient inhibitors as compared to the corresponding sulfamates. Potent CA inhibitors targeting the cytosolic or tumor-associated CA isozymes can thus be designed from various classes of sulfonamides, sulfamides, or sulfamates and their derivatives, considering the extensive interactions in which the inhibitor and the enzyme active site are engaged, based on recent X-ray crystallographic data.     

Carbonic anhydrase inhibitors: X-ray crystallographic structure of the adduct of human isozyme II with EMATE, a dual inhibitor of carbonic anhydrases and steroid sulfatase

The X-ray crystal structure for the adduct of human carbonic anhydrase II (hCA II) with estrone-3-O-sulfamate (EMATE), an antiendocrine agent showing both CA and estrone sulfatase inhibitory properties, has been resolved at a resolution of 1.5A. Its binding to the enzyme is similar to that of other sulfamates/sulfonamides, considering the interactions of the zinc anchoring group, but differs considerably when the steroidal scaffold of the inhibitor is analyzed. This part of the inhibitor interacts only within the hydrophobic half of the CA active site, interacting with residues Val 121, Phe 131, Val 135 and Pro 202, and leaving the hydrophilic half able to accommodate several water molecules not present in the uncomplexed enzyme. In addition, a very short bond of 1.78A between the zinc ion and the coordinated nitrogen atom of the sulfamate moiety is observed, which may explain the high affinity of this inhibitor for hCA II (K(i) of 10nM).     

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).     

Hydrophobicity, a physicochemical factor in the inhibition of the enzyme estrone sulfatase (ES).

We report the initial structure-activity relationship study (SAR) (in particular logP) of a series of compounds based upon 4-sulfamated phenyl ketones as potent inhibitors of the enzyme estrone sulfatase (ES). The results of the study show that these compounds are irreversible inhibitors of ES and that they are more potent than COUMATE, but weaker than EMATE. Analysis of the SAR data shows a strong correlation between IC(50) and logP but also supports our previous study, which suggests a very strong relationship between pK(a) and IC(50).     

Rainbow trout, Oncorhynchus mykiss, as a model for aromatase inhibition.

The feasibility of utilizing rainbow trout, Oncorhynchus mykiss, as an alternative model for studying the inhibition of aromatase (CYP 19) was investigated. The suppression of estrogen-dependent tumors by aromatase inhibitors has been important in the treatment of breast cancer. Estrogens, estrogen precursors and xenoestrogens have been found to promote liver cancer in the trout model. A steroid, 4-hydroxy-4-androstene-3,17-dione (4-OHA), and non-steroids, aminoglutethimide (AG) and Letrozole (CGS 20267), all of which are known aromatase inhibitors in rats and humans, were examined in vitro for activity in trout ovarian microsomes. Aromatase activity was quantified as the release of 3H2O from the conversion of [3H]-4-androstene-3,17-dione to 17beta-estradiol and estrone. Trout ovarian microsomes exhibited activity between 39-60 fmol mg(-1) min(-1) with a calculated Vmax of 71.1 fmol mg(-1) min(-1) when incubated at 25 degrees C with 200 nM 4-androstene-3,17-dione (K(M) = 435 nM). Significant inhibition by 4-OHA up to 80% was seen at 1.5 microM. At 2000 microM, AG decreased aromatase activity by up to 82%. Letrozole reduced aromatase activity a maximum of 90% in a dose-dependent manner, but the Ki (2.3 microM) was 1000-fold higher than reported in human trials. Indole-3-carbinol and some of its derivatives, two DDE isomers and four flavones (except alpha-naphthoflavone) at 1000 microM did not significantly inhibit aromatase in vitro. Letrozole and clotrimazole, fed to juvenile rainbow trout at doses up to 1000 ppm for 2 weeks, were not effective in suppressing dehydroepiandrosterone (DHEA) induced increases in vitellogenin and 17beta-estradiol levels. These results document that trout aromatase is sensitive to inhibition in vitro by known inhibitors of the mammalian enzyme. The mechanism(s) for lack of inhibition in vivo is currently unknown and must be further investigated in order to develop a trout model for studying the role of aromatase in carcinogenesis.     

Design, synthesis and biochemical evaluation of AC ring mimics as novel inhibitors of the enzyme estrone sulfatase (ES)

We report the initial results of our study into a series of simple 4'-O-sulfamoyl-4-biphenyl based compounds as novel inhibitors of the enzyme estrone sulfatase (ES). The results of the study show that these compounds are potent inhibitors, possessing greater inhibitory activity than COUMATE, but weaker inhibitory activity than EMATE or the tricyclic derivative of COUMATE, namely 667-COUMATE. Furthermore, the compounds are observed to be irreversible inhibitors.