Enantiomers of C(5)-chiral 1-acetyl-3,5-diphenyl-4,5-dihydro-(1H)-pyrazole derivatives: Analytical and semipreparative HPLC separation, chiroptical properties, absolute configuration, and inhibitory activity against monoamine oxidase

The HPLC enantiomer separation of a novel series of C(5)-chiral 1-acetyl-3-(4-hydroxy- and 2,4-dihydroxyphenyl)-5-phenyl-4,5-dihydro-(1H)-pyrazole derivatives, with inhibitory activity against monoamine oxidases (MAO) type A and B, was accomplished using polysaccharide-based chiral stationary phases (CSPs: Chiralpak AD, Chiralcel OD, and Chiralcel OJ). Pure alcohols, such as ethanol and 2-propanol, and typical normal-phase binary mixtures, such as n-hexane and alcohol modifier, were used as mobile phases. Single enantiomers of several analytes examined were isolated on a semipreparative scale, and their chiroptical properties were measured. The assignment of the absolute configuration was established for one compound by single-crystal X-ray diffraction method and for the other three by CD spectroscopy. The inhibitory activity against MAO of racemic samples and single enantiomers were evaluated in vitro.     

Enantioselective chromatography and absolute configuration of N,N-dimethyl-3-(naphthalen-2-yl)-butan-1-amines: potential sigma1 ligands

We describe the preparation of racemic N,N-dimethyl-3-(naphthalen-2-yl)-butan-1-amines, potential sigma1 ligands, and their resolution via chiral HPLC. In order to obtain enantiopure compounds, direct chromatographic methods of separation using chiral stationary phases were investigated. Different methods suitable for both analytical and semipreparative purposes are proposed. The best resolutions were achieved using cellulose tris (3,5-dimethylphenyl carbamate) (Chiralcel OD and OD-H) and amylose tris (3,5-dimethylphenyl carbamate) (Chiralpak AD). On the basis of the preliminary chromatographic results, the resolution of compound 1 was transferred onto a Chiralcel OD semipreparative column. The enantiomers were obtained in high enantiomeric excess. The configurational assignment was performed by circular dichroism. Computational analysis was used to explore the enantioselective recognition process of compound 1 with the Chiralcel OD stationary phase.     

Solid-phase synthesis of chiral stationary phases based on 2,4,5,6-tetrachloro-1,3-dicyanobenzene derivatives spaced from N-3,5-dinitrobenzoyl alpha-amino acids: comparative study of their resolution efficacy

Two new chiral stationary phases, 3-[5-chloro-1,3-dicyano-2,4-[2'-(N'-1,3-dinitrobenzoyl-D-phenylglycinyl) aminoethyl]aminophen-1-yl] aminopropyl silica (CSP-1) and 3-[5-chloro-1,3-dicyano-2,4-[2'-(N'-1,3-dinitrobenzoyl-L-leucinyl) aminoethyl] aminophen-1-yl] aminopropyl silica (CSP-2), were prepared by solid-phase synthesis. They comprise chiral unit, 3,5-dinitrobenzoyl derivative of the amino acid, D-PhGly or L-Leu, bound via spacer 1,2-diaminoethane to 2,4-positions of the persubstituted benzene ring, derived from compound 1, and possess pseudo-C2 symmetry. Preparation of model compounds 6 and 7 confirmed the structure of chiral selectors, which comprise pi-donor persubsituted aromatic ring and two strong pi-acceptor 3,5-dinitrobenzoyl amido units. CD spectra of model selectors 6 and 7, run in DMSO above 250 nm, exhibit negative exciton coupling (EC) between pi-acceptor and pi-donor chromophores, C(1) symmetric model compound 8 exhibited much weaker EC and 9, devoid of pi-donor unit, does not exhibit any significant CD. Combined pi-donor and pi-acceptor properties enable the new CSPs to separate a broad range of racemates. The columns with CSP-1 and CSP-2 were tested for the separation of 22 racemates by HPLC with two different mobile phase systems and the results are compared with those obtained by using a structurally related commercial column.     

An exploration of the binding site of aldolase using alkyl glycolamido phosphoric esters and alkyl monoglycolate phosphoric esters.

Alkyl glycolamido phosphoric esters (P-O-CH2-CO-NH-(CH2)n-CH3) and alkyl monoglycolate phosphoric esters (P-O-CH2-CO-O-(CH2)n-CH3), which are analogs of the aldolase substrate fructose-1-phosphate, were synthesized and use for probing the active site of rabbit muscle aldolase. The inhibition constants (Ki) were affected by the length of the alkyl groups of these compounds and a maximum value of Ki was observed between the number of methylene groups 2 and 4, depending on the type of compound. In the previous investigation, N-(omega-hydroxyalkyl)-glycolamido bisphosphoric esters (P-O-CH2-CO-NH-(CH2)n-O-P) and alkanediol monoglyclolate bisphosphoric esters (P-O-CH2-CO-O-(CH2)n-O-P) have a minimum Ki value between the number of methylene groups 1 and 4. The difference spectra of aldolase caused by binding of alkyl glycoamido phosphoric esters or alkyl monophosphates resembled that of their analogous bisphosphoric esters, but the intensity of absorbance was smaller than that of the bisphosphoric ester analogs. These results suggest that rabbit muscle aldolase has two binding sites for the phosphate groups on the entrance end of the active site cavity, the singly wound beta-barrel of the parallel alpha/beta class structure. The distance between the phosphate binding site Lys-107 in the beta-sheet structure (c) and Arg-148 in the beta-sheet structure (d) may possibly be expanded or contracted by the forms of the bending structure of the biphosphate compounds. Also, the change of distance between the beta-sheet structure (c) and (d) containing Trp-147, may have an effect on the environment of the tryptophan and cause a change of the absorbance of aldolase especially at 295-299 nm. On the other hand, the synthetic monophosphate compounds bind at only one of the two phosphate binding sites and have very little effect on the absorbance of Trp-147, in a similar manner as orthophosphate. The alkyl groups of monophosphate may be repelled by the ionic amino acid side chains, Asp-33, Lys-146, Glu-187 and/or Lys-229 in the middle of the active site cavity. However, the end of the long alkyl group of some monophosphates may possibly contact the hydrophobic bottom of the active site cavity without effect on the environment of Trp-147.     

The relationship between the structure of plastoquinone derivatives and their biological activity in Photosystem II of spinach chloroplasts

The relationship between the structure of reconstituted plastoquinone derivatives and their ability to recover the Hill reaction was investigated by extraction and reconstitution of lyophilized chloroplasts from spinach, followed by monitoring DCIP photoreduction at 600 nm. The results show that: It is not essential that the plastoquinone side chain be an isoprenoid or a phytol; the activity increases with increasing length of the side chain up to 13–15 carbon atoms; for chains longer than 15 carbon atoms, the activity is practically constant. Lipophilic groups (such as -Br) in the side chain increased the activity, hydrophilic groups (such as -OH) decreased the activity. Conjugated double bonds in the side chain decreased the activity greatly, but non-conjugated double bonds had almost no effect on the activity, indicating a requirement of flexibility of the side chain. The activity is decreased in the order of PQ, UbiQ and MQ, showing a large effect of the ring structure.Abbreviations DCIP 2,6-dichlorophenolindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Q_A primary electron acceptor in PS II reaction centers - Q_B secondary electron acceptor in PS II reaction centers - PQ _n plastoquinones with an isoprenoid side chain (n, number of the isoprenoid units in the side chain) - PQ-n synthetic plastoquinones with alkyl side chain (n, number of the carbon atoms in the alkyl side chain) - PQ-n synthetic plastoquinones with a double bond in the alkyl side chain - UQ _n ubiquinones with an isoprenoid side chain (n, number of the isoprenoid units in the side chain) - UQ-n synthetic ubiquinones with alkyl side chain (n, number of the carbon atoms in the akyl side chain) - MQ-n 2-alkyl-1,4-naphthoquinone (n, number of the carbon atoms in the alkyl side chain)     

Deoxamuscaroneoxime derivatives as useful muscarinic agonists to explore the muscarinic subsite: demox, a modulator of orthosteric and allosteric sites at cardiac muscarinic M2 receptors

A series of muscarinic agonists, straight chained, branched, cyclic alkyl and aromatic derivatives of the oxime 1 (demox) was designed with the aim of investigating their activity on muscarinic receptor subtypes. Effects on M1 receptor were assessed functionally by a microphysiometer apparatus, while M2, M3, and M4 receptor potency and affinity were studied on isolated preparations of guinea pig heart, ileum, and lung, respectively. The results suggest that the substitution of a hydrogen with a long side-chain or bulky group generally induces a decrease in potency at M1 and M3 subtypes, while a general increase in this parameter is obtained at M2 subtype. Among the agonists 2-18, compound 4 behaves as a full agonist with a preference for M3 subtype. Moreover, compound 12 is inactive at M1 and M4 receptors while it displays a full agonist activity at M2 and M3 subtypes. Since demox displays a variable response on cardiac M2 receptors regulating heart force, an in-depth inquiry of the functional behaviour of this compound was carried out at M2 receptors. In presence of 10(-11) and 10(-10) M demox, the binding of [3H]-NMS was increased by approximately 30% as a consequence of an increase of the association of [3H]-NMS to membranes; this effect was not observed in presence of a higher concentration of [3H]-NMS. Higher concentrations of demox decreased the binding of [3H]-NMS to heart atrial membranes but significantly retarded the dissociation of this radioligand. Our results suggest that demox may interact with orthosteric and allosteric sites of atrial M2 muscarinic receptor.     

Synthesis and evaluation of 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexanecarboxamides as Rho kinase inhibitors and neurite outgrowth promoters

The influence of stereochemistry and alkyl side chain length on the bioactivity of the Rho kinase inhibitor Y-27632 [(+)-1, R=Me] was examined by the synthesis of (+)- and (-)-1, and two alkyl chain analogs (+)- and (-)-2 (R=n-propyl) and (-)-3 (R=n-octyl) as well as their evaluation in enzymatic and neurite outgrowth assays.     

Pharmacophore models based studies on the affinity and selectivity toward 5-HT1A with reference to α1-adrenergic receptors among arylpiperazine derivatives of phenytoin

The study is focused on (2-alkoxy)phenylpiperazine derivatives of 1-(2-hydroxy-3-(4-arylpiperazin-1-yl)propyl)-5,5-diphenylimidazolidine-2,4-dione with alkyl or ester substituents at N3 of hydantoin ring, as well as a new designed and synthesized series of compounds with a free N3H group or N3-acetic acid terminal fragment. The compounds were assessed on their affinity for 5-HT_1A and ??₁-adrenoceptors and evaluated in functional bioassays for antagonistic properties. Classical molecular mechanics (MMFFs force field, MCMM, MacroModel) and DFT methods (B3LYP functional, Gaussian 0.3) were used to investigate 3D structure of the compounds. SAR analysis was based on two pharmacophore models, the one described by Barbaro et al. for ??₁-adenoceptor antagonist and the model of Lepailleur et al. for 5-HT_1A receptor ligands. All compounds exhibited significant to moderate affinities for 5-HT_1A receptors in nanomolar range (7-610 nM). The highest activity (7 nM) and selectivity (17.38) for 5-HT_1A was observed for 1-(3-(4-(2-ethoxyphenyl)piperazin-1-yl)-2-hydroxypropyl)-3-methyl-5,5-diphenylimidazolidine-2,4-dione (13a). Among new synthesized compounds 1-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-5,5-diphenylimidazolidine-2,4-dione hydrochloride (20a) displayed the highest affinity (16.6 nM) and selectivity (5.72) for ??₁-AR.     

Benzimidazole derivatives endowed with potent antileishmanial activity

Two sets of benzimidazole derivatives were synthesised and tested in vitro for activity against promastigotes of Leishmania tropica and L. infantum. Most of the tested compounds resulted active against both Leishmania species, with IC₅₀ values in the low micromolar/sub-micromolar range. Among the set of 2-(long chain)alkyl benzimidazoles, whose heterocyclic head was quaternised, compound 8 resulted about 100-/200-fold more potent than miltefosine, even if the selectivity index (SI) versus HMEC-1 cells was only moderately improved. In the set of 2-benzyl and 2-phenyl benzimidazoles, bearing a basic side chain in position 1, compound 28 (2-(4-chlorobenzyl)-1-lupinyl-5-trifluoromethylbenzimidazole) was 12-/7-fold more potent than miltefosine, but exhibited a further improved SI. Therefore, compounds 8 and 28 represent interesting hit compounds, susceptible of structural modification to improve their safety profiles.     

Enantiomers of 2-[(Acylamino)ethyl]-1,4-benzodiazepines, potent ligands of kappa-opioid receptor: chiral chromatographic resolution, configurational assignment and biological activity

Compounds 2a and 3a-e are racemic 2-[(acylamino)ethyl]-1,4-benzodiazepines, tifluadom analogs, with high affinity and selectivity towards the kappa-opioid receptor. We describe the enantiomeric separation of all compounds through liquid chromatography with chiral stationary phases, as well as the resolution of the enantiomers of the most interesting compounds, 2a and 3a, by the semipreparative column Chiralpak AD. The configuration of the resolved enantiomers was investigated: the comparative study of CD and (1)H NMR spectra shows that compounds (-)-2a and (-)-3a have the same absolute configuration of (+)-(S)-tifluadom. A study on the stereoselective interaction with opiate receptors is reported.     

An isocratic separation of underivatized gentamicin components, 1H NMR assignment and protonation pattern

A simple method for the separation of the major components of commercial gentamicin sulfate (C-1, C-1a, C-2, C-2a) by high-performance liquid chromatography (HPLC) on an analytical and a semipreparative scale was developed. The method utilized ion-pair reversed-phase chromatography, isocratic elution with an aqueous solution containing 9% trifluoroacetic acid and 2.5% acetonitrile as the mobile phase at a flow rate of 2 and 9 mL/min for analytical and semipreparative columns, respectively. Detection was carried out at 213 nm without derivatization. The protonation pattern of the separated gentamicins was determined by potentiometry and 15N and 1H NMR. The full proton NMR assignment for gentamicin C-1 was obtained through the use of 1H 1D and 2D 1H-1H COSY measurements.     

Synthesis and SAR studies of a novel class of S1P1 receptor antagonists

A series of Sodium 4-[(4-butoxyphenyl)thio]-2'-substituted-1,1'-biphenyl-3- sulfonates were identified as functional sphingosine-1-phosphate (S1P) antagonists with selectivity for the S1P(1) receptor subtype starting from chemical lead 2, which was found while screening our in-house compound library. We performed chemical modifications on each regional structure of compound 2, for example, on the three ring compartments, the benzyl substituents, and the long alkyl chain part. The introduction of a biphenyl skeletal structure and the installation of a hydroxyl group onto the terminal carbon in the side-chain region resulted in the potent derivative 35c, which showed >500-fold more potent S1P(1) inhibitory activity than lead compound 2. We report herein the synthesis and structure-activity relationships of structurally novel S1P(1) receptor antagonists.     

New reagents and methods for the synthesis of internal and 3'-labeled DNA

The syntheses of two new nucleoside phosphoramidites containing a hydroxyl functionality masked by a levulinate protecting group are presented; N(4)-(2-(ethylene glycol-2-levulinate)ethyl)-5-methyl-5'-(4,4'-dimethoxytrityl)-3'-O-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxycytidine 1 and 5-(N-(6-O-levulinoyl-1-aminohexyl)-3(E)-acrylamido)-5'-(4,4'-dimethoxytrityl)-3'-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxyuridine 3. Optimization of solid-phase-supported synthetic parameters for incorporation of these into DNA, removal of the levulinate group by exposure to dilute hydrazine, and subsequent attachment of dye labels is described. Synthesis of the known compound 5-(N-(6-trifluoroacetylaminohexyl)-3(E)-acrylamido)-5'-(4,4'-dimethoxytrityl)-3'-(2-cyanoethyldiisopropylphosphoramidite)-2'-deoxyuridine 2 (1), containing a masked amine at the end of an alkyl chain attached at the 5 position, was also revisited using new techniques developed for 3.     

2- and 8-alkynyl-9-ethyladenines: Synthesis and biological activity at human and rat adenosine receptors

The synthesis of a series of 9-ethyladenine derivatives bearing alkynyl chains in 2- or 8-position was undertaken, based on the observation that replacement of the sugar moiety in adenosine derivatives with alkyl groups led to adenosine receptor antagonists. All the synthesized compounds were tested for their affinity at human and rat A₁, A_2A, and A₃ adenosine receptors in binding assays; the activity at the human A_2B receptor was determined in adenylyl cyclase experiments. Biological data showed that the 2-alkynyl derivatives possess good affinity and are slightly selective for the human A_2A receptor. The same compounds tested on the rat A₁ and A_2A subtypes showed in general lower affinity for both receptors. On the other hand, the affinity of the 8-alkynyl derivatives at the human A₁, A_2A, and A_2B receptors proved to be lower than that of the corresponding 2-alkynyl derivatives. On the contrary, the affinity of the same compounds for the human A₃ receptor was improved, resulting in A₃ selectivity. As in the case of the 2-alkynyl-substituted compounds, the 8-alkynyl derivatives showed decreased affinity for rat receptors. However, it is worthwhile to note that the 8-phenylethynyl-9-ethyladenine was the most active compound of the two series (K_i in the nanomolar range) at both the human and rat A₃ subtype. Docking experiments of the 2- and 8-phenylethynyl-9-ethyladenines, at a rhodopsin-based homology model, gave a rational explanation of the preference of the human A₃ receptor for the 8-substituted compound.     

N-alkylated chitosan as a potential nonviral vector for gene transfection

Alkylated chitosans (ACSs) were prepared by modifying chitosan (CS) with alkyl bromide. The self-aggregation of ACSs in acetic acid solution was characterized by fluorescence spectroscopy and dynamic light scattering method. The results indicate that introducing alkyl side chains leads to the self-aggregation of ACSs, and CS with a 99% deacetylation degree shows no aggregation due to the electrostatic repulsion. The electrophoresis experiment demonstrates that the complex between CS and DNA was formed at a charge ratio (+/-) of 1/1; ACS/DNA complexes were formed at a lower charge ratio (+/-) of 1/4. A small amount of alkylated chitosans play the same shielding role as chitosan in protecting DNA from DNase hydrolysis. Differential scanning calorimetry (DSC) and atomic force microscopy (AFM) were employed separately to investigate the thermodynamic behavior of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/CS and DPPC/ACS mixtures and the variation in topological structure of DPPC membrane induced by CS and ACS. It is shown that CS and ACS can cause the fusion of DPPC multilamellar vesicles as well as membrane destabilization. In contrast, the perturbation effect induced by ACS is more evident due to the hydrophobic interaction. CS and ACS were used to transfer plasmid-encoding CAT into C(2)C(12) cell lines. Upon elongating the alkyl side chain, the transfection efficiency is increased and levels off after the number of carbons in the side chain exceeds 8. It is proposed that the higher transfection efficiency of ACS is attributed to the increasing entry into cells facilitated by hydrophobic interactions and easier unpacking of DNA from ACS carriers due to the weakening of electrostatic attractions between DNA and ACS.     

Synthesis and biochemical evaluation of benzyl propargyl ethers as inhibitors of 5-lipoxygenase

A series of benzyl propargyl ethers were synthesized and tested as inhibitors of 5-lipoxygenase, the key enzyme involved in leukotriene biosynthesis. Among these, optimum activity was displayed by 1-(2-heptynyloxymethyl) benzene 12 (IC50 1.2 microM). Addition of carboxyl group at the end of the alkyl side chain attached to the acetylenic group abolished the inhibition. Selective reduction of the acetylenic group to cis or trans double bond reduced the inhibitory potential, the cis isomer 24 showing more than 20-fold higher inhibition than the trans isomer 25. Introduction of sulphur in place of oxygen in the alkyl side chain attached to the (carboxyalkyl) benzyl group also reduced the inhibition. The IC50 value of 12, towards rabbit reticulocyte 15-LOX is > 50 fold higher than that of 5-LOX. These results indicate that compound 12 is a specific inhibitor of 5-LOX.     

Structure of the Ring Cleavage Product of 1-Hydroxy-2-Naphthoate, an Intermediate of the Phenanthrene-Degradative Pathway of Nocardioides sp. Strain KP7

1-Hydroxy-2-naphthoate (compound I) is a metabolite of the phenanthrene-degradative pathway in Nocardioides sp. strain KP7. This singly hydroxylated aromatic compound is cleaved by 1-hydroxy-2-naphthoate dioxygenase. In this study, the structure of the ring cleavage product generated by the action of homogeneous 1-hydroxy-2-naphthoate dioxygenase was determined upon separation by high-performance liquid chromatography at pH 2.5 by using nuclear magnetic resonance (NMR) and mass spectroscopic techniques. The ring cleavage product at this pH existed in equilibrium between two forms, 2-oxo-3-(3-oxo-1,3-dihydro-1-isobenzofuranyl)propanoate (compound III) and 2,2-dihydroxy-3-(3-oxo-1,3-dihydro-1-isobenzofuranyl)propanoate (compound IV). After the pH of the solution was raised to 7.5, the structure of the major species became (E)-4-(2-carboxylatophenyl)-2-oxo-3-butenoate (compound II; common name, trans-2′-carboxybenzalpyruvate), which was in equilibrium with compound III. Direct monitoring of the enzymatic formation of the ring cleavage product by ¹H-NMR in a deuterated potassium phosphate buffer (pH 7.5) detected only compound II as a product, and the proton on carbon 3 of compound II was not exchanged with deuterium. Thus, compound II is likely to be the first stable product of dioxygenation of 1-hydroxy-2-naphthoate.     

Effect of ginger constituents and synthetic analogues on cyclooxygenase-2 enzyme in intact cells.

Seventeen pungent oleoresin principles of ginger (Zingiber officinale, Roscoe) and synthetic analogues were evaluated for inhibition of cyclooxygenase-2 (COX-2) enzyme activity in the intact cell. These compounds exhibited a concentration and structure dependent inhibition of the enzyme, with IC(50) values in the range of 1-25 microM. Ginger constituents, [8]-paradol and [8]-shogaol, as well as two synthetic analogues, 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decane and 5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecane, showed strong inhibitory effects on COX-2 enzyme activity. The SAR analysis of these phenolic compounds revealed three important structural features that affect COX-2 inhibition: (i) lipophilicity of the alkyl side chain, (ii) substitution pattern of hydroxy and carbonyl groups on the side chain, and (iii) substitution pattern of hydroxy and methoxy groups on the aromatic moiety.     

The crystal structure of isopropyl 1-thio-beta-D-galactopyranoside monohydrate at 123 K

The crystal structure of isopropyl 1-thio-beta-D-galactopyranoside monohydrate is orthorhombic, P2(1)2(1)2(1), Z = 4, with cell dimensions at 123 K [293 K] of a = 7.983(1) [8.037(1)], b = 24.574(5) [24.709(4)], c = 6.329(1) [6.3736(8)] A, V = 1241.84 [1265.71] A3. The calculated and measured density is Dx = 1.371 [1.345] g cm-3, Dm = [1.340] g cm-3. Diffraction data were obtained with CuK alpha radiation and a Nonius CAD-4 diffractometer. The structure was solved by using MULTAN, and refined to R(F2) = 0.051, RW(F2) = 0.078, R(F) = 0.029, S = 1.16 for 1502 reflections. The molecule has the 4C1(D) conformation. The orientation of the primary alcohol group is gauche/trans, and that about the glycosidic C-S bond is (-)synclinal relative to the ring C-O bond. Although this compound does not form thermotropic liquid crystals, it has two crystal-to-crystal phase-transitions, at 70 and 104 degrees, prior to melting at 126 degrees. The crystal structure has a characteristic, amphiphilic, head-to-head bilayer molecular packing, with intercalated alkyl groups. The water molecule is included in the hydrogen-bond structure that links the galactoside moieties.     

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.