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Sex steroid hormones such as estrogen estradiol (E2) and androgen dihydrotestosterone (DHT) are involved in the development of hormone-dependent cancers. Blockade of 17β-hydroxysteroid dehydrogenase type 7 (17β-HSD7), a member of the short chain dehydrogenase/reductase superfamily, is thought to decrease E2 levels while increasing those of DHT. Therefore, its unique double action makes this enzyme as an interesting drug target for treatment of breast cancer. The chemical synthesis, molecular characterization, and preliminary biological evaluation as 17β-HSD7 inhibitors of novel carbamate derivatives 3 and 4 are described. Like previous 17β-HSD7 inhibitors 1 and 2, compounds 3 and 4 bear a hydrophobic nonyl side chain at the C-17β position of a 4-aza-5α-androstane nucleus, but compound 3 has an oxygen atom replacing the CH 2 in the steroid A-ring C-2 position, while compound 4 has a C17-spiranic E-ring containing a carbamate function. They both inhibited the in vitro transformation of estrone (E1) into E2 by 17β-HSD7, but the introduction of a (17 R)-spirocarbamate is preferable to replacing C-2 methylene with an oxygen atom since compound 4 (IC 50 = 63 nM) is an inhibitor 14 times more powerful than compound 3 (IC 50 = 900 nM). Furthermore, when compared to the reference inhibitor 1 (IC 50 = 111 nM), the use of a C17-spiranic E-ring made it possible to introduce differently the hydrophobic nonyl side chain, without reducing the inhibitory activity., Competing Interests: Declaration of Competing Interests MZ declares no competing interests, while JYS, RM, SXL, and DP are owners of a patent on 17β-HSD type 7 inhibitors., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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.

Microwave-assisted phospha-Michael addition reactions were carried out in the 13α-oestrone series. The exocyclic 16-methylene-17-ketones as α,β-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 IC 50 values. Moreover, it displayed good tumour selectivity property determined against non-cancerous mouse fibroblast cells.

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 13β-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. 13β-Derivatives displayed outstanding OATP2B1 inhibitory action with IC 50 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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The interest in the introduction of the oxime group in molecules aiming to improve their biological effects is increasing. This work aimed to develop new steroidal oximes of the estrane series with potential antitumor interest. For this, several oximes were synthesized by reaction of hydroxylamine with the 17-ketone of estrone derivatives. Then, their cytotoxicity was evaluated in six cell lines. An estrogenicity assay, a cell cycle distribution analysis and a fluorescence microscopy study with Hoechst 3358 staining were performed with the most promising compound. In addition, molecular docking studies against estrogen receptor α, steroid sulfatase, 17β-hydroxysteroid dehydrogenase type 1 and β-tubulin were also accomplished. The 2-nitroestrone oxime showed higher cytotoxicity than the parent compound on MCF-7 cancer cells. Furthermore, the oximes bearing halogen groups in A-ring evidenced selectivity for HepaRG cells. Remarkably, the Δ 9,11 -estrone oxime was the most cytotoxic and arrested LNCaP cells in the G 2 /M phase. Fluorescence microscopy studies showed the presence of condensed DNA typical of prophase and condensed and fragmented nuclei characteristic of apoptosis. However, this oxime promoted the proliferation of T47-D cells. Interestingly, molecular docking studies estimated a strong interaction between Δ 9,11 -estrone oxime and estrogen receptor α and β-tubulin, which may account for the described effects.

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) is a key enzyme in the biosynthesis of 17β-estradiol. Novel estrone-based compounds bearing various 15β-oxa-linked substituents and hydroxy, methoxy, benzyloxy, and sulfamate groups in position C3 as potential 17β-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 (IC 50  < 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 17β-HSD1 inhibitors, potential drug candidates for antiestrogen therapy of hormone-dependent gynecological cancers.

Fluorination of 13-epimeric estrones and their 17-deoxy counterparts was performed with Selectfluor as the reagent. In acetonitrile or trifluoroacetic acid (TFA), 10β-fluoroestra-1,4-dien-3-ones were formed exclusively. Mechanistic investigations suggest that fluorinations occurred via SET in acetonitrile, but another mechanism was operative in TFA. Simultaneous application of N-chlorosuccinimide (NCS) and Selectfluor in TFA led to a 1.3:1 mixture of 10β-fluoroestra-1,4-dien-3-one and 10β-chloroestra-1,4-dien-3-one as the main products. The potential inhibitory action of the 10-fluoro- or 10-chloroestra-1,4-dien-3-one products on human aromatase was investigated via in vitro radiosubstrate incubation. The classical estrane conformation with trans ring anellations and a 13β-methyl group seems to be crucial for the inhibition of the enzyme, while test compounds bearing the 13β-methyl group exclusively displayed potent inhibitory action with submicromolar or micromolar IC 50 values. Concerning molecular level explanation of biological activity or inactivity, computational simulations were performed. Docking studies reinforced that besides the well-known Met374 H-bond connection, the stereocenter in the 13 position has an important role in the binding affinity. The configuration inversion at C-13 results in weaker binding of 13α-estrone derivatives to the aromatase enzyme.

Development of hybrid drug candidates is well known strategy for designing antitumor agents. Herein, a novel class of nitric oxide donating cucurbitacin inspired estrone analogs (NO-CIEAs) were designed and synthesized as multitarget agents. Synthesized analogs were initially evaluated for their anti-hepatocellular carcinoma activities. Among the tested analogs, NO-CIEAs 17 and 20a exhibited more potent activity against HepG2 cells (IC 50  = 4.69 and 12.5 µM, respectively) than the reference drug Erlotinib (IC 50  = 25 µM). Interestingly, NO-CIEA 17 exerted also a high potent activity against Erlotinib-resistant HepG2 cell line (HepG2-R) (IC 50  = 8.21 µM) giving insight about its importance in drug resistance therapy. Intracellular measurements of NO revealed that NO-CIEAs 17 and 20a showed a significant increase in NO production in tumor cells after 1 h of incubation comparable to the reference prodrug JS-K. Flow cytometric analysis showed that both NO-CIEAs 17 and 20a mainly arrested the HepG2 cells in the G0/G1 phase. Also, In-Cell Based ELISA screening showed that NO-CIEA 17 resulted in a potential inhibitory activity towards the EGFR and MAPK (25% and 29% inhibition compared to untreated control cells, respectively). This data suggests the binding ability of NO-CIEA 17 to the EGFR and ERK to be well correlated along with the docking and cellular studies. Also, treatment of HepG2-R cells with NO-CIEA 17 showed a potential reduction of MRP2 expression in a dose dependent manner providing a significant impact on the chemotherapeutic resistance. Overall, the current study provides a potential new approach for the discovery of a novel antitumor agent against HCC., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Heterocyclization of steroids were reported to give biologically active products where ring D modification occured. Estrone (1) was used as a template to develop new heterocyclic compounds. Ring D modification of 1 through its reaction with cyanoacetylhydrazine and elemental sulfur gave the thiophene derivative 3. The latter compound reacted with acetophenone derivatives 4a-c to give the hydrazide-hydrazone derivatives 5a-c, respectively. In addition, compound 3 formed thiazole derivatives through its first reaction with phenylisothiocyanate to give the thiourea derivative 9 followed by the reaction of the later with α-halocarbonyl compounds. In the present work a series of novel estrone derivatives were designed, synthesized and evaluated for their in vitro biological activities against c-Met kinase, and six typical cancer cell lines (A549, H460, HT-29, MKN-45, U87MG and SMMC-7721). The most promising compounds 5b, 5c, 11a, 13c, 15b, 15c, 15d, 17a and 17b were further investigated against the five tyrosine kinases c-Kit, Flt-3, VEGFR-2, EGFR, and PDGFR. Compounds 5b, 15d, 17a and 17b were selected to examine their Pim-1 kinase inhibition activity where compounds 15d and 17b showed high activities. Molecular docking of some of the most potent compounds was demonstrated., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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.

A series of new estrone derivatives were designed and synthesized, and their structures were confirmed by spectroscopic methods. All new estrone derivatives were investigated for their in vitro cytotoxic efficacies against a panel of three human prostate cancer cell lines (PC-3, LNCaP, and DU145). The derivatives 6, 7, 10, 15, 16, 20, 21, 22, 24 and 26 showed important cytotoxic actions against individual carcinoma cell line collections. Moreover, antagonistic activities of compounds (7, 15, 16 and 21) towards a 1 -ARs (α 1A , α 1B , and α 1D ) were further evaluated using dual-luciferase reporter assays, and the compounds 16 and 21 exhibited better a 1 -ARs subtype selectivity. The structure-activity relationship (SAR) suggested that the substitute's type and position on the phenyl group leads to the interesting variations within pharmacological effects of resultant molecular systems., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A novel series of 3-ketolithocholic acid derivatives as well as estrone derivatives bearing a small ring for the conformational fixation of the side chain were synthesized by using a catalytic [2+2] cycloaddition and a ring-contraction rearrangement. The steroidal derivatives were evaluated for transcriptional activation of vitamin D receptor by luciferase reporter assays. Among them, two estrone derivatives showed a higher efficacy of the transactivation of vitamin D receptor than 3-ketolithocholic acid, and the small ring moieties were found to be important for the efficacy., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The syntheses of monosaccharide-d-secoestrone conjugates are reported. They were prepared from 3-(prop-2-inyloxy)-d-secoestrone alcohol or oxime and monosaccharide azides via Cu(I)-catalyzed azide-alkyne cycloaddition reactions (CuAAC). The antiproliferative activities of the conjugates were investigated in vitro against a panel of human adherent cancer cell lines (HeLa, A2780 and MCF-7) by means of MTT assays. The protected d-glucose-containing d-secoestrone oxime bioconjugate (24b) proved to be the most effective with an IC 50 value in the low micromolar range against A2780 cell line., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Various novel arylated estrone derivatives, such as 2-aryl-, 4-aryl- and 2,4-diaryl-estrones, by Suzuki-Miyaura reactions. While the synthesis of 4-arylestrones could be carried out under standard conditions, the synthesis of 2-arylestrones and 2,4-diarylestrones required a thorough optimization of the conditions and it proved to be important to use sterically encumbered biaryl ligands. The best results were obtained by the use of RuPhos. Combination of developed Suzuki coupling reactions with subsequent cyclization reactions afforded more complex hybrid structures, containing dibenzofuran, benzocoumarin and steroid moieties. These derivatives were tested as pancreatic lipase inhibitors and it was found that most of the compounds exhibited inhibition of pancreatic lipase but the maximum inhibitory potential was shown by 4-arylestrones. All of the synthesized derivatives showed inhibitory values in the range of 0.82±0.01-59.7±3.12μM. The biological activity was also rationalized on the bases of docking studies., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

13α-Estrone derivatives containing various substituents on C-3 and C-17 were synthesized, and evaluated by means of MTT assays for in vitro antiproliferative activity against a panel of human adherent cancer cell lines (HeLa, MCF-7, A2780 and A431). Compounds with N-benzyltriazolylmethoxy moieties on C-3 proved to be more potent than their 3-hydroxy or 3-ether counterparts. Some triazoles exerted substantial cytostatic effects against particular tumor cell lines, with submicromolar IC50 values., (Copyright © 2016 Elsevier Inc. All rights reserved.)

An efficient synthesis of several N-[(1-benzyl-1,2,3-triazol-4-yl)methyl]carboxamides in the 13β- and 13α-d-secoestrone series is reported. Novel triazoles were synthesized via the Cu(I)-catalyzed azide-alkyne cycloaddition of steroidal alkynyl carboxamides and p-substituted benzyl azides. Each of the products was evaluated in vitro by means of MTT assays for antiproliferative activity against a panel of human adherent cancer cell lines (HeLa, MCF-7, A431 and A2780). Some of them exhibited activities similar to those of the reference agent cisplatin. On change of the substitution pattern of the benzyl group of the azide, great differences in the cell growth-inhibitory properties were observed. The p-alkylbenzyl-substituted triazoles selectively exerted high cytostatic action against A2780 cells, with IC50 values of 1 µM. We investigated the potential inhibitory action exerted on the human 17β-HSD1 activity of the new secosteroids. Three triazoles effectively suppressed the estrone to 17β-estradiol conversion with IC50 values in low micromolar range.

A two-step synthesis of gold mesoionic carbene complexes containing estrone moieties has been developed. The method uses the methylation of the triazole nucleus, followed by the treatment of the triazolium salt with Ag2O and transmetalation with [AuCl(SMe2)]. Mono-, bi-, tri-, and tetrametallic gold complexes can be obtained depending on the structure of the starting triazolium salts. Tetrametallic gold carbene embedded in a macrocylic stereoidal cavity containing four estrone nuclei has been also prepared. Additionally, the mono- and bimetallic silver carbene complexes containing triazole-steroid ligands have been isolated and characterized. These complexes resulted to be stable and have been characterized by spectroscopic and HRMS techniques. The gold and silver complexes having triazole-steroid ligands are unprecedented in the literature and the method reported here to access to these compounds is easy and efficient. Preliminary results regarding the catalytic activity of some of the gold-carbenes prepared in the insertion of diazoalkanes into alcohols are presented.

13α-Estrone and its 3-methyl or benzyl ether were halogenated in ring A with N-bromo- or N-iodosuccinimide or 1,3-dibromo-5,5-dimethylhydantoin as electrophile triggers. The chemo- and regioselectivities of the reactions depended greatly on the nature of the substituent on C-3. Bromination of the ethers led to 2- and 4-regioisomers. Bis-halogenation occurred only in the case of the phenolic derivative. Iodination and bromination resulted in similar products, except that the 3-benzyl ether could not be iodinated under the applied conditions. The potential inhibitory action of the new halogenated 13α-estrones on human 17β-hydroxysteroid dehydrogenase 1 activity was investigated via in vitro radiosubstrate incubation. Some compounds proved to be effective inhibitors, with IC50 values in the submicromolar range., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Novel 16-triazoles in the 13α-estrone series were synthesized via Cu(I)-catalyzed azide-alkyne cycloaddition of the two diastereomeric (on C-16 and on C-17) 16-azido-13α-estra-1,3,5(10)-trien-17-ol 3-benzyl ethers with substituted phenylacetylenes. The new heterocyclic derivatives were evaluated in vitro by means of MTT assays for antiproliferative activity against a panel of human adherent cancer cell lines (HeLa, MCF-7, A431, A2780, T47D, MDA-MB-231 and MDA-MB-361). The inversion of the configurations at C-16 and C-17 selectively affected the growth-inhibitory properties of the tested compounds. The 16β,17α isomers generally proved to be potent on all cell lines, with IC50 values comparable to those of the reference agent cisplatin. Change of the substitution pattern of the phenyl group of the acetylene led to great differences in antiproliferative properties. Exclusively the p-phenyl-substituted triazoles exerted high cytostatic effects. One of the most potent compounds activated caspase-3 and caspase-9 without influencing caspase-8, confirming the induction of apoptosis via the intrinsic pathway., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Introduction: Organic Anion Transporting Polypeptides (OATP) are a family of membrane associated transporters that facilitate estrone-3-sulphate (E3S) uptake by hormone dependent, post-menopausal breast cancers. We have established E3S as a potential ligand for targeting hormone dependent breast cancer cells, and in this study sought to prepare and investigate radioiodinated E3S as a tool to study the OATP system., Methods: 2- and 4-Iodoestrone-3-sulfates were prepared from estrone via aromatic iodination followed by a rapid and high yielding sulfation procedure. The resulting isomers were separated by preparative HPLC and verified by (1)H NMR and analytical HPLC. Transport studies of 2- and 4-[(125)I]-E3S were conducted in hormone dependent (i.e. MCF-7) and hormone independent (i.e. MDA-MB-231) breast cancer cells in the presence or absence of the specific transport inhibitor, bromosulfophthalein (BSP). Cellular localization of OATP1A2, OATP2B1, OATP3A1 and OATP4A1 were determined by immunofluorescence analysis using anti-Na(+)/K(+) ATPase-α (1:100 dilution) and DAPI as plasma membrane and nuclear markers, respectively., Results: Significantly (p<0.01) higher total accumulation of 2-[(125)I]-E3S was observed in hormone dependent MCF-7 as compared to hormone independent MDA-MB-231 breast cancer cells. In contrast 4-[(125)I]-E3S did not show cellular accumulation in either case. The efficiency of 2-[(125)I]-E3S transport (expressed as a ratio of Vmax/Km) was 2.4 times greater in the MCF-7 as compared to the MDA-MB-231 breast cancer cells. OATP1A2, OATP3A1 and OATP4A1 expression was localized in plasma membranes of MCF-7 and MDA-MB-231 cells confirming the functional role of these transporters in radioiodinated E3S cellular uptake., Conclusion: An efficient method for the preparation of 2- and 4-[(125)I]-E3S was developed and where the former demonstrated potential as an in vitro probe for the OATP system. The new E3S probe can be used to study the OATP system and as a platform to create radiopharmaceuticals for imaging breast cancer., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A simple and efficient synthesis of novel, D-ring substituted estrone derivatives containing a 16α-triazolyl moiety is described. Two epimeric azido alcohols (16α-azido-17α-hydroxy and 16α-azido-17β-hydroxy) of estra-1,3,5(10)-triene-3-methyl ether were prepared, followed by copper(I)-catalyzed azide-alkyne cycloaddition with various terminal alkynes. The steroidal triazoles were obtained in high yields and their activities against three human cancer cell lines (HeLa, MCF7 and A431) were screened. The most effective analogs were submitted to additional experiments in order to characterize their antiproliferative properties. As evidenced by flow cytometry, the selected steroids induced a disturbance in the HeLa cell cycle in a concentration- and exposure-dependent manner, through an increase of the hypodiploid population (subG1) and a cell cycle arrest in the G2/M phase. A noncancerous human fibroblast cell line (MRC5) was used to determine the selectivities of these compounds. Fluorescent microscopy after Hoechst 33258 - propidium iodide (HOPI) double staining revealed nuclear condensation and disturbed cell membrane integrity. The enhanced activities of caspase-3 and caspase-9 without activation of caspase-8 in the treated cells indicated the activation of the intrinsic pathway of apoptosis. The levels of cell cycle regulators (CDK1, cyclin B1/B2 and cdc25B) were decreased and the ratio Bax/Bcl-2 was increased 24 h after the treatment of HeLa cells (determined at an mRNA level by means of an RT-PCR technique). Under the same conditions, two agents elicited substantially increased degrees of phosphorylation of stathmin, as evidenced by Western blotting. The presented results demonstrate that these steroids can be regarded as appropriate structural scaffolds for the design and synthesis of further steroid analogs as innovative drug candidates with good efficacy.

d-Secooximes were synthesized from the d-secoaldehydes in the 13β- and 13α-estrone series. The oximes were modified at three sites in the molecule: the oxime function was transformed into an oxime ether, oxime ester or nitrile group, the propenyl side-chain was saturated and the 3-benzyl ether was removed in order to obtain a phenolic hydroxy function. Triazoles were formed via Cu(I)-catalysed azide-alkyne cycloaddition (CuAAC) from 3-(prop-2-yniloxy)-d-secooximes and benzyl azides. All the products were evaluated in vitro by means of MTT assays for antiproliferative activity against a panel of human adherent cell lines (HeLa, MCF-7, A2780 and A431). Some of them exhibited activities with submicromolar IC50 values, better than that of the reference agent cisplatin. The structural modifications led to significant differences in the cytostatic properties. Flow cytometry indicated that one of the most potent agents resulted in a cell cycle blockade., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Substituted and/or heterocyclic d-homoestrone derivatives were synthetized via the intramolecular cyclization of a δ-alkenyl-d-secoaldehyde, -d-secoalcohol or -d-secocarboxylic acid of estrone 3-benzyl ether. The d-secoalcohol was modified at three sites in the molecule. The in vitro antiproliferative activities of the new d-homo- and d-secoestrone derivatives were determined on HeLa, MCF-7, A431 and A2780 cells through use of MTT assay. d-Homoalcohols 3 and 5 displayed cell line-selective cytostatic effects against ovarian and cervical cell lines, respectively. Two d-secoestrones (6 and 12c) proved to be effective, with IC50 values comparable with those of the reference agent cisplatin. A selected compound (6) was tested by tubulin polymerization assay and its cancer specificity was additionally determined by using noncancerous human fibroblast cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

An asymmetric Torgov cyclization, catalyzed by a novel, highly Brønsted acidic dinitro-substituted disulfonimide, is described. The reaction delivers the Torgov diene and various analogues with excellent yields and enantioselectivity. This method was applied in a very short synthesis of (+)-estrone., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The one pot reaction of estrone with the aromatic aldehydes 2a-c and either of malononitrile or ethyl cyanoacetate afforded the fused pyran derivatives 4a-f. On the other hand, carrying the same reaction using thiourea instead of the cyanomethylene reagent gave the fused pyrimidine derivatives 6a-c. The latter compounds reacted with phenacyl bromide to give the thiazolo[3,2-a]pyrimidine derivatives 8a-c. The reaction of the title compound with bromine gave the monobromo derivative 13 which in turn reacted with either thiourea or cyanothioacetamide to give the thiazole derivatives 14 and 16, respectively. The cytotoxicity of the newly synthesized products was evaluated against six human cancer and normal cell lines where the results showed that compounds 4c, 4f, 6b, 8b, 8c, 10, 13, 16, 18c and 19c exhibited optimal cytotoxic effect against the cancer cell lines, with IC50's in the nM range., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Functionalized estrogen analogs have received interest due to their unique and differing biological activity compared to their parent compounds. The synthesis of a new class of 3-methoxyestrone analogs functionalized at the C17 position possessing both alkyl and aryl substituted α,β-unsaturated ketones is described, along with their thiophenol conjugate addition products., (Copyright © 2013. Published by Elsevier Inc.)

Cyclic nitrones of estrone 3-methyl or 3-benzyl ether were reacted with phenyl isocyanate or nonsubstituted phenyl isocyanates as reactive CN dipolarophiles, yielding condensed homosteroidal oxadiazolidinones. These dipolar cycloadditions were carried out under conventional heating or microwave irradiation. The chemo- and stereoselectivities of the reactions and the effects of the aromatic substituents on the reaction rates and yields were investigated and compared. The structures of the new products were determined by NMR (one- and two-dimensional) and MALDI-MS techniques, with C70 fullerenes as matrix in the latter case. The antiproliferative properties of the synthetized compounds were determined on a panel of human adherent cell lines (HeLa, MCF7, A2780 and A431) by means of MTT assays. Some of them exhibited activities comparable to that of the reference agent cisplatin. Flow cytometry indicated that one of the most potent agents (11a) resulted in a cell cycle blockade., (Copyright © 2013 Elsevier Inc. All rights reserved.)

To generate the first series of prodigiosene conjugates, the tripyrrolic skeleton was appended to estrone, tamoxifen and porphyrin frameworks by way of ester linkers and various hydrocarbon chain lengths. The ability of the conjugates to inhibit various types of cancer cells was evaluated in vitro. The porphyrin conjugates did not exhibit significant activity. The estrone conjugates exhibited modest activity, for the most part. However, significantly greater growth inhibition activity against certain breast, colon, lung, leukemia, melanoma and prostate cell lines was noted. This unusual effect for this first generation model class of compound warrants further investigation and comparison to cases where estrogens are linked to prodigiosenes via connection points that do not feature in estrogen receptor binding. The 4-hydroxytamoxifen conjugates exhibit nanomolar range activity against the MCF-7 breast cancer cell line, paving the way to expand the scope and connectivity of prodigiosene-tamoxifen conjugates., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

An expanding body of evidence indicates the possible role of estrane derivatives as useful anticancer agents. The aim of this study was to describe the cytotoxic effects of 63 newly synthetized estrone-16-oxime ethers on human cancer cell lines (cervix carcinoma HeLa, breast carcinoma MCF7 and skin epidermoid carcinoma A431), studied by means of the MTT assay. Four of the most promising compounds were selected for participation in additional experiments in order to characterize the mechanism of action, including cell cycle analysis, morphological study and the 5-bromo-2'-deoxyuridine incorporation assay. The cancer selectivity was tested on a noncancerous fibroblast cell line (MRC-5). Since apoptosis and cell cycle disturbance were observed, caspase-3 activities were further assayed for the two most effective agents. These estrone-16-oxime analogs activated caspase-3 and changed the mRNA level expression of endogenous factors regulating the G1-S phase transition (retinoblastoma protein, CDK4 and p16). The repression of retinoblastoma protein was reinforced at a protein level too. These experimental data lead to the conclusion that estrone-16-oxime ethers may be regarded as potential starting structures for the design of novel anticancer agents., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Sixteen platinum(II) complexes of estrone and estradiol were synthesized in this work to evaluate their cytotoxic activity against several cancer cell lines including estrogen dependent and independent ones. The synthesis of all the complexes was done in three steps. The reaction of steroids with dibromoalkanes was followed by a reaction of the bromoalkyl steroids with 2-(aminomethyl)pyridine or 2-(2-aminoethyl)pyridine. The last step was a reaction of steroidal diamino ligands with potassium tetrachloroplatinate to obtain the desired platinum(II) complexes. Cytotoxicity assays showed that most of the complexes prepared are active against the cancer cell lines used-CEM, U-2 OS, MCF7, MCF7 AL, MDA-MB-468, BT-474, BT-549, and BJ fibroblasts. The six-membered platinum complexes are more active than five-membered ones., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Regioselective 1,3-dipolar cycloadditions of different aryl nitrile oxides to mestranol were carried out to furnish novel steroidal 17α-isoxazoles in good to excellent yields. Copper(I) was found to be an efficient catalyst, accelerating the intermolecular ring-closures and leading exclusively to 3,5-disubstituted isoxazoles. The yields of the cycloadducts, however, were influenced by the substituents on the aromatic moiety of the 1,3-dipoles. Moreover, dehydration of the primary products resulted in the corresponding Δ(16,17)exo-heterocyclic derivatives. All the synthesized compounds were subjected to in vitro pharmacological studies of their antiproliferative effects relative to three human malignant cell lines (HeLa, MCF7 and A2780)., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Protein disulfide isomerase (PDI) is a promiscuous protein with multifunctional properties. PDI mediates proper protein folding by oxidation or isomerization and disrupts disulfide bonds by reduction. The entry of HIV-1 into cells is facilitated by the PDI-catalyzed reductive cleavage of disulfide bonds in gp120. PDI is regarded as a potential drug target because of its reduction activity. We screened a chemical library of natural products for PDI-specific inhibitors in a high-throughput fashion and identified the natural compound juniferdin as the most potent inhibitor of PDI. Derivatives of juniferdin were synthesized, with compound 13 showing inhibitory activities comparable to those of juniferdin but reduced cytotoxicity. Both juniferdin and compound 13 inhibited PDI reductase activity in a dose-dependent manner, with IC(50) values of 156 and 167 nM, respectively. Our results also indicated that juniferdin and compound 13 exert their inhibitory activities specifically on PDI but do not significantly inhibit homologues of this protein family. Moreover, we found that both compounds can inhibit PDI-mediated reduction of HIV-1 envelope glycoprotein gp120.

We present in this report the development and realization of a novel formal total synthesis of estrone (1) via the Torgov diene (24) by the furano diene approach, first attempted by Woodward in 1937. The core ring structure 16 was established by an acid-mediated regioselective and stereospecific cyclization of the endo-oxabicyclo[2.2.1]heptene derivative 14, which is readily available from the AlCl(3)-catalyzed Diels-Alder cycloaddition of 2-(3-methoxyphenethyl)furan (4) and dimethyl maleate. The mechanistic pathway of this S(N)' type cyclization is discussed, and the earlier perspectives in our preliminary report (Org. Lett. 2004, 6, 1333) are corrected.

We report here dicationic ruthenium PNNP complexes that promote the enantioselective Diels-Alder reaction of alpha-methylene beta-ketoesters with various dienes. Complex [Ru(OEt2)2(PNNP)](PF6)2, formed in situ from [RuCl2,(PNNP)] and (Et3O)PF6 (2 equiv.), catalyzes the Diels-Alder reaction of such unsaturated beta-ketoesters to give novel alkoxycarbonyltetrahydro-1-indanone derivatives (nine examples) with up to 93% ee. The crystal structure of the substrate-catalyst adduct shows that the lower face of the substrate is shielded by a phenyl ring of the PNNP ligand, which accounts for the high enantioselectivity. The attack of the diene from the open re enantioface of the unsaturated beta-ketoester is consistent with the absolute configuration of the product. A useful application of this method is the reaction with Dane's diene to give estrone derivatives with up to 99% ee and an ester-exo:endo ratio of up to 145:1 (after recrystallization). Besides the enantioselective formation of all-carbon quaternary centers, this methodology is notable because unsaturated beta-ketoesters have been rarely used in Diels-Alder reactions. Furthermore, enantiomerically pure estrone derivatives are interesting in view of their potential applications, including the treatment of breast cancer.

Starting from Dane's diene and methylcyclopentenedione, (+)-estrone is synthesized along the Quinkert-Dane route in 24% total yield. The key step is an enantioselective Diels-Alder reaction promoted by an amidinium catalyst as efficiently as by a traditional Ti-TADDOLate Lewis acid.

A sensitive and selective assay method for labile estrogen o-quinones, estrone (E(1))-2,3-quinone (Q), E(1)-3,4-Q, estradiol (E(2))-2,3-Q and E(2)-3,4-Q, based on the use of phenazine (Phz) derivatization with o-phenylenediamine and high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) was described. The Phz derivatives of four estrogen o-quinones were purified by solid phase extraction and analyzed by HPLC-ESI-MS/MS. The protonated molecule was observed as a base peak for all Phz derivatives in their ESI-mass spectra (positive mode). In multiple reaction monitoring, the transition from [M+H]+ to m/z 231 was chosen for quantification. Calibration curves for the o-quinones were obtained using standard catechol estrogens after sodium metaperiodate treatment and Phz derivatization. Using this method, these four estrogen o-quinones were analyzed with the limit of quantification of 5 ng/ml in acetonitrile (MeCN)-blank matrix (1:4, v/v), respectively, on a basis of the weight of catechol estrogens. Assay accuracy and precision for four estrogen o-quinones were 89.6-113.0% and 3.1-12.6% (5, 125 and 2000 ng/ml in MeCN-blank matrix). Applications of this method enabled to determine the catalytic activities on hydroxylation and subsequent oxidation of E(1) and E(2) of Mushroom tyrosinase and rat liver microsomal fraction. It was confirmed by this method that tyrosinase exhibited 2- and 4-hydroxylation and further oxidation activities for catechols in the ring-A of estrogens. Whereas rat liver microsomal fraction possessed only 2- and 4-hydroxylation activities, and further oxidation activity for catechol estrogens was low., (2010 Elsevier Ltd. All rights reserved.)

A general steroid synthesis is presented that relies on prior formation of three stereogenic centers (C8, C13, and C14) on a D ring template, followed by C- and B-ring cyclizations. The assembly of the key D ring template, achieved by a 3-component conjugate addition/alkylation process, allows introduction of structural variety as required. The method is illustrated by the total synthesis of estrone via a C-ring closing metathesis and a B-ring Heck cyclization.

In search for specific drugs against steroid-dependent cancers we have developed a novel set of potent inhibitors of steroidogenic human 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD 1). The X-ray structure of 17beta-HSD 1 in complex with estradiol served as basis for the design of the inhibitors. 2-Substituted estrone and D-homo-estrone derivatives were synthesized and tested for 17beta-HSD 1 inhibition. The best 17beta-HSD 1 inhibitor, 2-phenethyl-D-homo-estrone, revealed an IC(50) of 15 nM in vitro. The inhibitory potency of compounds is comparable or better to that of previously described inhibitors. An interaction within the cofactor binding site is not necessary to obtain this high binding affinity for substances developed.

A novel series of 17-modified and 2,17-modified analogs of 2-methoxyestradiol (2ME2) were synthesized and characterized. These analogs were designed to retain or potentiate the biological activities of 2ME2 and have diminished metabolic liability. The analogs were evaluated for antiproliferative activity against MDA-MB-231 breast tumor cells, antiangiogenic activity in HUVEC, and estrogenic activity on MCF-7 cell proliferation. Several analogs were evaluated for metabolic stability in human liver microsomes and in vivo in a rat cassette dosing model. This study lead to several 17-modified analogs of 2ME2 that have similar or improved antiproliferative and antiangiogenic activity, lack estrogenic properties and have improved metabolic stability compared to 2ME2.

The synthesis and biological evaluation of structurally simplified, metabolically stable cyclopamine-like Sonic Hedgehog (SHH) signaling inhibitors, i.e., 5, is described in four chemical steps from commercially available steroidal precursors. Biological evaluation of this cyclopamine analogue in two different systems establishes the high potency of 5 as a SHH signaling inhibitor. This approach provides important new lead structures for the development of new cancer chemotherapeutic agents based on the inhibition on SHH signaling.

Aromatase, which is responsible for the conversion of androgens to estrogens, is a potential therapeutic target for the selective lowering estrogen level in patients with estrogen-dependent breast cancer. We prepared and tested series of the pyridine- and other heterocyclic ring-containing derivatives of 2- and 4-aminoestrones, estrone, and estradiol, compounds 5, 10, 12 and 15. The isonicotinyl derivatives of 2- and 4-aminoestrone, compounds 5c and 10c, were fairly potent competitive inhibitors of aromatase (K(i), 2.1+/-0.14 and 1.53+/-0.08 microM for 5c and 10c, respectively) and other compounds did not show, to a significant extent, the aromatase inhibitory activity. This result suggests that the isonicotinyl-substituted derivatives 5c and 10c would be accessible to the active site of aromatase.

A straightforward approach to macrocycles having four estrone-derived nuclei by the sequential Cu-catalyzed Huisgen azide-alkyne cycloaddition-Glaser-Eglington Cu homocoupling has been developed. Due to its efficiency and simplicity, this sequence is useful for application to different natural product scaffolds.

A new and diastereoselective method for the synthesis of the estrone skeleton from a substituted styrene based on sequential 3-fold use of Cp 2ZrBu 2 (oxidative addition-alkylation and two cyclization-alkylation sequences) and a ruthenium complex catalyzed RC-metathesis of a sterically hindered diene was developed. The prepared estratetraene was obtained in 7 steps from a commercially available starting material and thus the overall synthesis of estrone could be accomplished in 9 steps. Moreover, we have also found that the course of the reaction of substrates bearing the 2-halo-1,7-diene moiety with Cp 2ZrBu 2, i.e., cyclization or oxidative addition to the C-X bond, could be controlled by the nature of the halogen leaving group.

17-Alkoxycarbonyl- and 17-carboxamido-13alpha-estra-1,3,5(10),16-tetraenes were synthesized from the 17-iodo-13alpha-estra-1,3,5(10),16-tetraene derivative in palladium-catalyzed alkoxycarbonylation and aminocarbonylation reactions, respectively. The synthesis of the 17-iodo-16-ene derivative, used as substrate, is based on the transformation of the 17-keto derivative (epiestrone methyl ether) to hydrazone, which was treated with iodine in the presence of a base (1,1,3,3-tetramethyl guanidine). 17-Carboxamides were obtained in good yields (up to 88%) not only with simple alkyl/aryl amines but also with amino acid methyl esters as N-nucleophiles. The use of alcohols as O-nucleophiles in alkoxycarbonylation resulted in the corresponding 17-esters; however, yields of synthetic interest were obtained only with methanol.

Hydrolysis of 3-methoxy-16alpha-nitro-14,17-ethenoestra-1,3,5(10)-trien-17beta-yl acetate under weakly basic conditions leads to formation of 3-methoxy-2'-oxopyrrolidino-[4',5':14beta,15beta]-estra-1,3,5 (10)-trien-17-one, the structure of which has been confirmed by X-ray analysis and some chemical transformations. The reactivity of 3-methoxy-16alpha-nitro-14,17-ethanoestra-1,3,5(10)-trien-17beta-yl acetate under various conditions of basic hydrolysis has been investigated. The derived compounds have been identified by means of NMR spectroscopy and X-ray analysis.

A library of 17 novel estrogen analogues 3 and 4 containing different substituents at rings A and D (steroid nomenclature) was prepared in a five- to seven-step synthesis. The key transformation is a Sonogashira-coupling of cyclic vinyl iodides of type 7 or 8 with phenylacetylenes of type 9. Reduction of the keto function in 3 led to the estradiol analogue 5.

4-Formyl estrone was synthesized in overall good yield in three steps starting from estrone. This was achieved by conducting an electrophilic aromatic substitution reaction using formaldehyde, triethylamine, and MgCl2 on 2-tert-butyl estrone, which was readily prepared in 96% yield from estrone using tert-butyl alcohol and BF3OEt2. The tert-butyl group acted as a positional protecting group to prevent reaction at the 2-position. The tert-butyl group was readily removed in good yield using AlCl3 in dichloromethane/CH3NO2. To our knowledge, this represents the first use of a positional protecting group for the synthesis of a C-4-modified estrogen. 4-Formyl estrone was used as a common precursor to obtain a variety of other C-4 modified estrogens in very high yields such as 4-methylestrone and 4-hydroxymethylestrone as well as the novel estrogen 4-carboxyestrone. The syntheses of 4-formyl, -methyl-, and -hydroxymethyl estrone represent dramatic improvements over previously reported syntheses of these compounds.

16Beta-aminomethyl-17beta-hydroxyestrone 3-methyl ether 6 and its N-propyl (17), N-benzyl (18) and N-arylmethyl derivatives (19-22) were subjected to ring closure reactions with phenylphosphonic dichloride in order to synthetize P-epimeric oxazaphosphorinanes 23a, 24-29 in which the hetero ring is condensed to ring D of the sterane skeleton. The stereostructures of the products were evaluated by 1H, 13C and 31P NMR spectroscopy. The geometry was optimized by utilizing the B3LYP DFT method. The NMR spectral data and the results of the ab initio calculations demonstrated that the stereostructure of the hetero ring was strongly affected by the rigid sterane framework condensed to it, and the phosphoramidate ring proved to adopt predominantly a distorted-boat conformation, regardless of the P-configuration.

D-ring-fused dioxaphosphorinanes (4-6) in the estrone series were synthetized as epimeric pairs and investigated by NMR and computational methods in order to determine their stereostructures and predominant conformations. The study was performed to evaluate the influence of the rigid sterane framework on the geometry of the condensed hetero ring, with regard to the possible steric effect of the angular methyl group at position 13. Additionally, the steric and electronic effects of the P-substituents on the conformational equilibrium were examined. The distorted-boat conformation of the hetero ring of dioxaphosphorinoestrone 3-methyl ether 4a was confirmed by single-crystal X-ray analysis. This is in good agreement with the observation in solution that, in the case of the boat conformation, the anisotropic shielding effect of the phenyl group of cyclic phosphonate 4a generates an upfield shift for 17-H, as compared with the corresponding chemical shift for epimer 4b. A similar boat conformation was substantiated for derivatives 4b, 5a, 5b and 6b on the basis of the J(H, H) and J(H, P) coupling constants and also ab initio calculations, regardless of the P-configuration. At the same time, the hetero ring of 6a seems to tilt towards a chair-like conformation due to the strong equatorial preference of the N-bis(2-chloroethyl) group.