Synthesis and estrogenic activity of BODIPY-labeled estradiol conjugates.

Novel BODIPY-estradiol conjugates have been synthesized by selecting position C-3-O for labeling. The conjugation strategy was based on Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) or etherification. Estradiol derivatives used as azide partners bearing an ω-azidoalkyl function through C4-C8-long linkers have been prepared. CuAAC reactions of estradiol azides with BODIPY alkyne furnished fluorescent 3-O-labeled conjugates bearing the triazole ring as a coupling moiety. Williamson etherifications of 3-O-(ω-bromoalkyl)-17ß-estradiol derivatives with BODIPY-OH resulted in labeled conjugates connected with an ether moiety. Interactions of the conjugates with estrogen receptor (ER) were investigated using molecular docking calculations in comparison with estradiol. The conjugates occupied both the classical and alternative binding sites on human ERα, with slightly lower binding affinity to references estradiol and diethystilbestrol. All compounds have displayed reasonable estrogenic activity. They increased the proliferation of ER-positive breast cancer cell line MCF7 contrary to ER-negative SKBR3 cell line. The most potent compound 13a induced the transcriptional activity of ER in dose-dependent manner in dual luciferase recombinant reporter model and increased progesterone receptor's expression, proving the retained estrogenic activity. The fluorescence of candidate compound 13a co-localised with the ERα. The newly synthesized labeled compounds might serve as good starting point for further development of fluorescent probes for modern biological applications. In addition to studying steroid uptake and transport in cells, e.g. in the processes of biodegradation of estrogen-hormones micropollutants, they could also be utilized in examination of estrogen-binding proteins.

Synthesis of Estrone Heterodimers and Evaluation of Their In Vitro Antiproliferative Activity.

Directed structural modifications of natural products offer excellent opportunities to develop selectively acting drug candidates. Natural product hybrids represent a particular compound group. The components of hybrids constructed from different molecular entities may result in synergic action with diminished side effects. Steroidal homo- or heterodimers deserve special attention owing to their potentially high anticancer effect. Inspired by our recently described antiproliferative core-modified estrone derivatives, here, we combined them into heterodimers via Cu(I)-catalyzed azide-alkyne cycloaddition reactions. The two trans-16-azido-3-(O-benzyl)-17-hydroxy-13α-estrone derivatives were reacted with 3-O-propargyl-D-secoestrone alcohol or oxime. The antiproliferative activities of the four newly synthesized dimers were evaluated against a panel of human adherent gynecological cancer cell lines (cervical: Hela, SiHa, C33A; breast: MCF-7, T47D, MDA-MB-231, MDA-MB-361; ovarian: A2780). One heterodimer (12) exerted substantial antiproliferative activity against all investigated cell lines in the submicromolar or low micromolar range. A pronounced proapoptotic effect was observed by fluorescent double staining and flow cytometry on three cervical cell lines. Additionally, cell cycle blockade in the G2/M phase was detected, which might be a consequence of the effect of the dimer on tubulin polymerization. Computational calculations on the taxoid binding site of tubulin revealed potential binding of both steroidal building blocks, mainly with hydrophobic interactions and water bridges.

Antiproliferative and Antimetastatic Properties of 16-Azidomethyl Substituted 3-O-Benzyl Estrone Analogs.

Four diastereomers of 16-azidomethyl substituted 3-O-benzyl estradiol (1-4) and their two estrone analogs (16AABE and 16BABE) were tested for their antiproliferative properties against human gynecological cancer cell lines. The estrones were selected for additional experiments based on their outstanding cell growth-inhibiting activities. Both compounds increased hypodiploid populations of breast cancer cells, and 16AABE elicited cell cycle disturbance as evidenced by flow cytometry. The two analogs substantially increased the rate of tubulin polymerization in vitro. 16AABE and 16BABE inhibited breast cancer cells' migration and invasive ability, as evidenced by wound healing and Boyden chamber assays. Since both estrone analogs exerted remarkable estrogenic activities, as documented by a luciferase reporter gene assay, they can be considered as promising drug candidates for hormone-independent malignancies.

Synthesis and in vitro photodynamic activity of aza-BODIPY-based photosensitizers.

Aza-BODIPY dyes have recently come to attention owing to their excellent chemical and photophysical properties. In particular, their absorption and emission maxima can efficiently be shifted to the red or even to the NIR spectral region. On this basis, aza-BODIPY derivatives are widely investigated as fluorescent probes or phototherapeutic agents. Here we report the synthesis of a set of novel aza-BODIPY derivatives as potential photosensitizers for use in photodynamic therapy. Triazolyl derivatives were obtained via Cu(I)-catalyzed azide-alkyne cycloaddition as the key step. In vitro photodynamic activities of the newly synthesized compounds were evaluated on the A431 human epidermoid carcinoma cell line. Structural differences influenced the light-induced toxicity of the test compounds markedly. Compared to the initial tetraphenyl aza-BODIPY derivative, the compound bearing two hydrophilic triethylene glycol side chains showed substantial, more than 250-fold, photodynamic activity with no dark toxicity. Our newly synthesized aza-BODIPY derivative, acting in the nanomolar range, might serve as a promising candidate for the design of more active and selective photosensitizers.

Cytotoxic effect of 13α-estrane derivatives on breast, endometrial and ovarian cancer cell lines.

Hormone-dependent cancers such as breast, uterine, and ovarian cancers account for more than 35% of all cancers in women. Worldwide, these cancers occur in more than 2.7 million women/year and account for 22% of cancer-related deaths/year. The generally accepted mechanism for the pathophysiology of estrogen-dependent cancers is estrogen receptor-mediated cell proliferation associated with an increased number of mutations. Therefore, drugs that can interfere with either local estrogen formation or estrogen action via estrogen receptors are needed. Estrane derivatives that have low or minimal estrogenic activity can affect both pathways. In this study, we investigated the effect of 36 different estrane derivatives on the proliferation of eight breast, endometrial, and ovarian cancer cell lines and the corresponding three control cell lines. Estrane derivatives 3 and 4_2Cl showed a stronger effect on the endometrial cancer cell lines KLE and Ishikawa, respectively, compared with the control cell line HIEEC, with IC50 values of 32.6 microM and 17.9 microM, respectively. Estrane derivative 4_2Cl was most active in the ovarian cancer cell line COV362 compared to the control cell line HIO80 with an IC50 value of 3.6 microM. In addition, estrane derivative 2_4I showed a strong antiproliferative effect on endometrial and ovarian cancer cell lines, while the effect on the control cell line was slight or absent. The addition of halogen at carbon 2 and/or 4 in estrane derivatives 1 and 2 increased the selectivity for endometrial cancer cells. Overall, these results suggest that single estrane derivatives are efficient cytotoxic agents for endometrial and ovarian cancer cell lines, and thus potential lead compounds for drug development.

Investigation of the Antineoplastic Effects of 2-(4-Chlorophenyl)-13α-Estrone Sulfamate against the HPV16-Positive Human Invasive Cervical Carcinoma Cell Line SiHa.

Cervical carcinoma is one of the most frequent malignant gynecological cancers in women of reproductive age. Because of the poor tolerability of currently available chemotherapeutic agents, efforts have been focused on developing innovative molecules, including steroids, that exert antineoplastic effects with a better safety profile. In addition to their endocrine properties, certain estrogens exhibit additional biological activities, such as antiangiogenic and anticancer effects. Based on previous studies, the antineoplastic properties of 13α-estrone sulfamate derivatives (13AES1-3) were investigated, and the mechanism of action for the most promising compound 13AES3 was explored. Based on their effects on the viability of different human adherent gynecological cancer cells, the SiHa cervical cell line was used for mechanistic experiments. The most active analog 13AES3 was shown to exert considerable proapoptotic effects, as evidenced by a colorimetric caspase-3 assay and fluorescent double staining. It also elicited antimigratory and anti-invasive effects in a concentration-dependent manner, as evidenced by wound healing and Boyden chamber assays, respectively. Regarding their mechanism of action, 13AES derivatives were shown to inhibit tubulin polymerization, and computer simulations provided a possible explanation for the importance of the presence of the chlorophenyl ring on the estrane skeleton. 13AES3 is considered to be the first 13α-estrone derivative with a significant antineoplastic potency against SiHa cancer cells. Therefore, it might serve as a valuable lead molecule for the design of anticancer agents targeting cervical carcinomas.

Synthesis and Antiproliferative Activity of Steroidal Diaryl Ethers.

Novel 13α-estrone derivatives have been synthesized via direct arylation of the phenolic hydroxy function. Chan-Lam couplings of arylboronic acids with 13α-estrone as a nucleophilic partner were carried out under copper catalysis. The antiproliferative activities of the newly synthesized diaryl ethers against a panel of human cancer cell lines (A2780, MCF-7, MDA-MB 231, HeLa, SiHa) were investigated by means of MTT assays. The quinoline derivative displayed substantial antiproliferative activity against MCF-7 and HeLa cell lines with low micromolar IC50 values. Disturbance of tubulin polymerization has been confirmed by microplate-based photometric assay. Computational calculations reveal significant interactions of the quinoline derivative with the taxoid binding site of tubulin.

Synthesis of hydrocortisone esters targeting androgen and glucocorticoid receptors in prostate cancer in vitro.

Androgen and glucocorticoid receptors have been recently described as key players in processes related to prostate cancer and mainly androgen receptor's inactivation was shown as an effective way for the prostate cancer treatment. Unfortunately, androgen deprivation therapy usually loses its effectivity and the disease frequently progresses into castration-resistant prostate cancer with poor prognosis. The role of the glucocorticoid receptor is associated with the mechanism of resistance; therefore, pharmacological targeting of glucocorticoid receptor in combination with antiandrogen treatment was shown as an alternative approach in the prostate cancer treatment. We introduce here the synthesis of novel 17α- and/or 21-ester or carbamate derivatives of hydrocortisone and evaluation of their biological activity towards androgen and glucocorticoid receptors in different prostate cancer cell lines. A 17α-butyryloxy-21-(alkyl)carbamoyloxy derivative 14 was found to diminish the transcriptional activity of both receptors (in single-digit micromolar range), with comparable potency to enzalutamide towards the androgen receptor, but weaker potency compared to mifepristone towards the glucocorticoid receptor. Lead compound inhibited proliferation and the formation of cell colonies in both androgen and glucocortiocid receptors-positive prostate cancer cell lines in low micromolar concentrations. Candidate compound 14 showed to interact with both receptors in cells and inhibited the translocation of receptors to nucleus and their activation phoshorylation. Moreover, binding to receptor's ligand binding domains was assessed by molecular modelling. Lead compound also induced the accumulation of cells in G1 phase and its combination with enzalutamide was shown to be more effective than enzalutamide alone.

Novel preparation of substituted oxazolines condensed to d-ring of estrane skeleton and characterization of their antiproliferative properties.

A simple and efficient synthesis of novel estrone 16α,17α-oxazoline derivatives substituted at the D ring (compounds 6a-g) is described. The reduction of 16α-azido-3-methoxyestra-1,3,5-trien-17-one (1) in methanol in the presence of CeCl3 under the condition of the Luche reaction produced two epimeric azido alcohol (16α-azido-17α-hydroxy and 16α-azido-17ß-hydroxy) derivatives of estra-1,3,5(10)-triene-3-methyl ether (compounds 2 and 3) in a yield of 90% and 7.6%. The reaction of the sterically unhindered 16α-azido-17α-hydroxy-estra-1,3,5(10)-triene-3-methyl ether (2) with a range of benzaldehydes under the condition of the Schmidt rearrangement yielded d-ring substituted estrone 16α,17α-oxazoline derivatives 6a-g. The in vitro antiproliferative activities of compounds 1, 2, 3, 6a-g were also determined by means of MTT assays on a panel of human cancer cell lines HeLa, SiHa, C-33 A, A2780, MCF-7, MDA-MB-231 and T47D.

Selective antiproliferative effect of C-2 halogenated 13α-estrones on cells expressing Organic anion-transporting polypeptide 2B1 (OATP2B1).

Organic anion-transporting polypeptide 2B1 (OATP2B1) is a multispecific transporter mediating the cellular uptake of steroids and numerous drugs. OATP2B1 is abundantly expressed in the intestine and is also present in various tumors. Increased steroid hormone uptake by OATP2B1 has been suggested to promote progression of hormone dependent tumors. 13α-estrones are effective inhibitors of endogenous estrogen formation and are potential candidates to inhibit proliferation of hormone dependent cancers. Recently, we have identified a variety of 13α/ß-estrone-based inhibitors of OATP2B1. However, the nature of this interaction, whether these inhibitors are potential transported substrates of OATP2B1 and hence may be enriched in OATP2B1-overexpressing cells, has not yet been investigated. In the current study we explored the antiproliferative effect of the most effective OATP2B1 inhibitor 13α/ß-estrones in control and OATP2B1-overexpressing A431 carcinoma cells. We found an increased antiproliferative effect of 3-O-benzyl 13α/ß-estrones in both mock transfected and OATP2B1-overexpressing cells. However, C-2 halogenated 13α-estrones had a selective OATP2B1-mediated cell growth inhibitory effect. In order to demonstrate that increased sensitization can be attributed to OATP2B1-mediated cellular uptake, tritium labeled 2-bromo-13α-estrone was synthesized for direct transport measurements. These experiments revealed increased accumulation of [3H]2-bromo-13α-estrone due to OATP2B1 function. Our results indicate that C-2 halogenated 13α-estrones are good candidates in the design of anti-cancer drugs targeting OATP2B1.

Microwave-assisted Phospha-Michael addition reactions in the 13α-oestrone series and in vitro antiproliferative properties.

Microwave-assisted phospha-Michael addition reactions were carried out in the 13α-oestrone series. The exocyclic 16-methylene-17-ketones as α,ß-unsaturated ketones were reacted with secondary phosphine oxides as nucleophilic partners. The addition reactions furnished the two tertiary phosphine oxide diastereomers in high yields. The main product was the 16α-isomer. The antiproliferative activities of the newly synthesised organophosphorus compounds against a panel of nine human cancer cell lines were investigated by means of MTT assays. The most potent compound, the diphenylphosphine oxide derivative in the 3-O-methyl-13α-oestrone series (9), exerted selective cell growth-inhibitory activity against UPCI-SCC-131 and T47D cell lines with low micromolar IC50 values. Moreover, it displayed good tumour selectivity property determined against non-cancerous mouse fibroblast cells.

Synthesis and evaluation of AKR1C inhibitory properties of A-ring halogenated oestrone derivatives.

Enzymes AKR1C regulate the action of oestrogens, androgens, and progesterone at the pre-receptor level and are also associated with chemo-resistance. The activities of these oestrone halides were investigated on recombinant AKR1C enzymes. The oestrone halides with halogen atoms at both C-2 and C-4 positions (13ß-, 13α-methyl-17-keto halogen derivatives) were the most potent inhibitors of AKR1C1. The lowest IC50 values were for the 13α-epimers 2_2I,4Br and 2_2I,4Cl (IC50, 0.7 µM, 0.8 µM, respectively), both of which selectively inhibited the AKR1C1 isoform. The 13α-methyl-17-keto halogen derivatives 2_2Br and 2_4Cl were the most potent inhibitors of AKR1C2 (IC50, 1.5 µM, 1.8 µM, respectively), with high selectivity for the AKR1C2 isoform. Compound 1_2Cl,4Cl showed the best AKR1C3 inhibition, and it also inhibited AKR1C1 (Ki: AKR1C1, 0.69 µM; AKR1C3, 1.43 µM). These data show that halogenated derivatives of oestrone represent a new class of potent and selective AKR1C inhibitors as lead compounds for further optimisations.

Data-Driven Ensemble Docking to Map Molecular Interactions of Steroid Analogs with Hepatic Organic Anion Transporting Polypeptides.

Hepatic organic anion transporting polypeptides-OATP1B1, OATP1B3, and OATP2B1-are expressed at the basolateral membrane of hepatocytes, being responsible for the uptake of a wide range of natural substrates and structurally unrelated pharmaceuticals. Impaired function of hepatic OATPs has been linked to clinically relevant drug-drug interactions leading to altered pharmacokinetics of administered drugs. Therefore, understanding the commonalities and differences across the three transporters represents useful knowledge to guide the drug discovery process at an early stage. Unfortunately, such efforts remain challenging because of the lack of experimentally resolved protein structures for any member of the OATP family. In this study, we established a rigorous computational protocol to generate and validate structural models for hepatic OATPs. The multistep procedure is based on the systematic exploration of available protein structures with shared protein folding using normal-mode analysis, the calculation of multiple template backbones from elastic network models, the utilization of multiple template conformations to generate OATP structural models with various degrees of conformational flexibility, and the prioritization of models on the basis of enrichment docking. We employed the resulting OATP models of OATP1B1, OATP1B3, and OATP2B1 to elucidate binding modes of steroid analogs in the three transporters. Steroid conjugates have been recognized as endogenous substrates of these transporters. Thus, investigating this data set delivers insights into mechanisms of substrate recognition. In silico predictions were complemented with in vitro studies measuring the bioactivity of a compound set on OATP expressing cell lines. Important structural determinants conferring shared and distinct binding patterns of steroid analogs in the three transporters have been identified. Overall, this comparative study provides novel insights into hepatic OATP-ligand interactions and selectivity. Furthermore, the integrative computational workflow for structure-based modeling can be leveraged for other pharmaceutical targets of interest.

Design, synthesis and biological evaluation of novel estrone phosphonates as high affinity organic anion-transporting polypeptide 2B1 (OATP2B1) inhibitors.

Organic anion-transporting polypeptide 2B1 (OATP2B1) is a multispecific membrane transporter mediating the cellular uptake of various exo- and endobiotics, including drugs and steroid hormones. Increased uptake of steroid hormones by OATP2B1 may increase tumor proliferation. Therefore, understanding OATP2B1's substrate/inhibitor recognition and inhibition of its function, e.g., in hormone-dependent tumors, would be highly desirable. To identify the crucial structural features that correlate with OATP2B1 inhibition, here we designed modifications at four positions of the estrane skeleton. 13α- or 13ß-estrone phosphonates modified at ring A or ring D were synthesized. Hirao and Cu(I)-catalyzed azide-alkyne click reactions served in the syntheses as key steps. 13ß-Derivatives displayed outstanding OATP2B1 inhibitory action with IC50 values in the nanomolar range (41-87 nM). A BODIPY-13α-estrone conjugate was additionally synthesized, modified at C-3-O of the steroid, containing a four-carbon linker between the triazole moiety and the BODIPY core. The fluorescent conjugate displayed efficient, submicromolar OATP2B1 inhibitory potency. The newly identified inhibitors and the structure-activity relationships specified here promote our understanding about drug recognition of OATP2B1.

Transition metal-catalysed A-ring C-H activations and C(sp2)-C(sp2) couplings in the 13α-oestrone series and in vitro evaluation of antiproliferative properties.

Facile syntheses of 3-O-carbamoyl, -sulfamoyl, or -pivaloyl derivatives of 13α-oestrone and its 17-deoxy counterpart have been carried out. Microwave-induced, Ni-catalysed Suzuki-Miyaura couplings of the newly synthesised phenol esters with phenylboronic acid afforded 3-deoxy-3-phenyl-13α-oestrone derivatives. The carbamate and pivalate esters proved to be suitable for regioselective arylations. 2-(4-Substituted) phenyl derivatives were synthesised via Pd-catalysed, microwave-assisted C-H activation reactions. An efficient, one-pot, tandem methodology was elaborated for the introduction of the carbamoyl or pivaloyl group followed by regioselective C-2-arylation and subsequent removal of the directing group. The antiproliferative properties of the novel 13α-oestrone derivatives were evaluated in vitro on five human adherent cancer cell lines of gynaecological origin. 3-Sulfamate derivatives displayed substantial cell growth inhibitory potential against certain cell lines. The newly identified antiproliferative compounds having hormonally inactive core might be promising candidates for the design of more active anticancer agents.

Synthesis and evaluation of anticancer activities of 2- or 4-substituted 3-(N-benzyltriazolylmethyl)-13α-oestrone derivatives.

2- or 4-Substituted 3-N-benzyltriazolylmethyl-13α-oestrone derivatives were synthesised via bromination of ring A and subsequent microwave-assisted, Pd-catalysed C(sp2)-P couplings. The antiproliferative activities of the newly synthesised brominated and phosphonated compounds against a panel of human cancer cell lines (A2780, MCF-7, MDA-MB 231) were investigated by means of MTT assays. The most potent compound, the 3-N-benzyltriazolylmethyl-4-bromo-13α-oestrone derivative exerted substantial selective cell growth-inhibitory activity against A2780 cell line with a submicromolar IC50 value. Computational calculations reveal strong interactions of the 4-bromo derivative with both colchicine and taxoid binding sites of tubulin. Disturbance of tubulin function has been confirmed by photometric polymerisation assay.

Pd-catalyzed Suzuki-Miyaura couplings and evaluation of 13α-estrone derivatives as potential anticancer agents.

13α-Estrones are of great value owing to their potent multiple bioactivity, including anticancer activity. 3-OH or 3-OBn derivatives of 2- or 4-[(subst.) phenyl]-13α-estrone as potential antiproliferative agents have been synthesized via facile, microwave-induced, Pd-catalyzed Suzuki-Miyaura coupling. 2- or 4-Halogenated 13α-estrone derivatives have been reacted with (4-subst.)phenylboronic acids using Pd(PPh3)4 as catalyst. The nature of para substituents at the introduced phenyl group did not influence the outcome of couplings. Certain newly synthesized compounds displayed substantial antiproliferative action against human adherent cancer cell lines of gynecological origin. Important structure-activity relationships were revealed, which might be helpful in the design of potent and selective anticancer derivatives based on the hormonally inactive 13α-estrane core.

Structural dissection of 13-epiestrones based on the interaction with human Organic anion-transporting polypeptide, OATP2B1.

Human OATP2B1 encoded by the SLCO2B1 gene is a multispecific transporter mediating the cellular uptake of large, organic molecules, including hormones, prostaglandins and bile acids. OATP2B1 is ubiquitously expressed in the human body, with highest expression levels in pharmacologically relevant barriers, like enterocytes, hepatocytes and endothelial cells of the blood-brain-barrier. In addition to its endogenous substrates, OATP2B1 also recognizes clinically applied drugs, such as statins, antivirals, antihistamines and chemotherapeutic agents and influences their pharmacokinetics. On the other hand, OATP2B1 is also overexpressed in various tumors. Considering that elevated hormone uptake by OATP2B1 results in increased cell proliferation of hormone dependent tumors (e.g. breast or prostate), inhibition of OATP2B1 can be a good strategy to inhibit the growth of these tumors. 13-epiestrones represent a potential novel strategy in the treatment of hormone dependent cancers by the suppression of local estrogen production due to the inhibition of the key enzyme of estrone metabolism, 17ß-hydroxysteroid-dehydrogenase type 1 (HSD17ß1). Recently, we have demonstrated that various phosphonated 13-epiestrones are dual inhibitors also suppressing OATP2B1 function. In order to gain better insights into the molecular determinants of OATP2B1 13-epiestrone interaction we investigated the effect of C-2 and C-4 halogen or phenylalkynyl modified epiestrones on OATP2B1 transport function. Potent inhibitors (with EC50 values in the low micromolar range) as well as non-inhibitors of OATP2B1 function were identified. Based on the structure-activity relationship (SAR) of the various 13-epiestrone derivatives we could define structural elements important for OATP2B1 inhibition. Our results may help to understand the drug/inhibitor interaction profile of OATP2B1, and also may be a useful strategy to block steroid hormone entry into tumors.

Stereocontrolled synthesis of the four possible 3-methoxy and 3-benzyloxy-16-triazolyl-methyl-estra-17-ol hybrids and their antiproliferative activities.

The four possible isomers of each of 3-methoxy- and 3-benzyloxyestra-1,3,5(10)-trien-17-ols (5-8 and 9-12) were converted through 16-p-tosyloxymethyl- or 16-bromomethyl derivatives into their 3-methoxy- and 3-benzyloxy-16-azidomethylestra(1,3,5(10)-triene derivatives (13-16 and 17-20). The regioselective Cu(I)-catalyzed 1,3-dipolar cycloaddition of these compounds with different terminal alkynes afforded novel 1,4-disubstituted diastereomers (21a-f, 22a-f, 23a-f, 24a-f and 25a-f, 26a-f, 27a-f, 28a-f). The antiproliferative activities of the structurally related triazoles were determined in vitro with the microculture tetrazolium assay on four malignant human cell lines of gynecological origin (Hela, SiHa, MCF-7 and MDA-MB-231).

Synthesis of substituted 15ß-alkoxy estrone derivatives and their cofactor-dependent inhibitory effect on 17ß-HSD1.

17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is a key enzyme in the biosynthesis of 17ß-estradiol. Novel estrone-based compounds bearing various 15ß-oxa-linked substituents and hydroxy, methoxy, benzyloxy, and sulfamate groups in position C3 as potential 17ß-HSD1 inhibitors have been synthesized. In addition, in vitro inhibitory potentials measured in the presence of excess amount of NADPH or NADH were investigated. We observed substantial inhibitory potentials for several derivatives (IC50 < 1 µM) and increased binding affinities compared to unsubstituted core molecules. Binding and inhibition were found to be cofactor-dependent for some of the compounds and we propose structural explanations for this phenomenon. Our results may contribute to the development of new 17ß-HSD1 inhibitors, potential drug candidates for antiestrogen therapy of hormone-dependent gynecological cancers.