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European Journal of Medicinal Chemistry 87 (2014) 336e345

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Short communication

Exemestane metabolites: Synthesis, stereochemical elucidation,biochemical activity and anti-proliferative effects in a hormone-dependent breast cancer cell line

Carla L. Varela a, 1, Cristina Amaral b, c, 1, Elisi�ario Tavares da Silva a, Andreia Lopes b, c,Georgina Correia-da-Silva b, c, Rui A. Carvalho d, e, Saul C.P. Costa a, Fernanda M.F. Roleira a,Nat�ercia Teixeira b, c, *

a CEF, Center for Pharmaceutical Studies & Pharmaceutical Chemistry Group, Faculty of Pharmacy, University of Coimbra, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugalb Laboratory of Biochemistry, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, n� 228, 4050-313Porto, Portugalc Institute for Molecular and Cell Biology (IBMC), University of Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugald Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, 3001-401, Portugale Center for Neuroscience and Cell Biology (CNC), University of Coimbra, 3001-401, Portugal

a r t i c l e i n f o

Article history:Received 14 May 2014Received in revised form22 September 2014Accepted 23 September 2014Available online

Keywords:Hormone-dependent breast cancerAromataseExemestaneExemestane metabolitesAromatase inhibitorsStructureeactivity relationships (SAR)studies

* Corresponding author. Laboratory of BiochemistSciences, Faculty of Pharmacy, University of Porto, R228, 4050-313 Porto, Portugal.

E-mail address: natercia@ff.up.pt (N. Teixeira).1 Both authors contributed equally to this work.

http://dx.doi.org/10.1016/j.ejmech.2014.09.0740223-5234/© 2014 Elsevier Masson SAS. All rights re

a b s t r a c t

Exemestane is a third-generation steroidal aromatase inhibitor that has been used in clinic for hormone-dependent breast cancer treatment in post-menopausal women. It is known that exemestane undergoesa complex metabolization, giving rise to some already identified metabolites, the 17b-hydroxy-6-methylenandrosta-1,4-dien-3-one (17-bHE) and the 6-(hydroxymethyl)androsta-1,4,6-triene-3,17-dione(6-HME). In this study, four metabolites of exemestane have been analyzed, three of them were syn-thesized (6b-spirooxiranandrosta-1,4-diene-3,17-dione (2), 1a,2a-epoxy-6-methylenandrost-4-ene-3,17-dione (3) and 17-bHE (4)) while one was acquired, the 6-HME (6). The stereochemistry of the epoxidegroup of 2 and 3 has been unequivocally elucidated for the first time on the basis of NOESY experiments.New structureeactivity relationships (SAR) have been established through the observation that thesubstitution of the double bonds by epoxide groups led to less potent derivatives in microsomes.However, the reduction of the C-17 carbonyl group to a hydroxyl group originating 17-bHE (4) resulted ina significant increase in activity in MCF-7aro cells when compared to exemestane (IC50 0.25 mM vs0.90 mM, respectively). All the studied metabolites reduced MCF-7aro cells viability in a dose and time-dependent manner, and metabolite 3 was the most potent one. Altogether our results showed that notonly exemestane but also its main metabolites are potent aromatase inhibitors and reduce breast cancercells viability. This suggests that exemestane efficacy may also be due to the active metabolites that resultfrom its metabolic transformation. Our results emphasize the importance of performing further studiesto expand our understanding of exemestane actions in breast cancer cells.

© 2014 Elsevier Masson SAS. All rights reserved.

1. Introduction

Breast cancer is the most commonly diagnosed cancer and theleading cause of cancer death in women worldwide [1]. About 70%

ry, Department of Biologicalua Jorge Viterbo Ferreira, n�

served.

of breast tumors are dependent on estrogens for their developmentand growth [2]. Estrogens are obtained from androgens in the lastreaction of steroid biosynthesis, which is catalyzed by the enzymearomatase. Therefore, aromatase inhibitors (AIs) greatly reduce theproduction of estrogens hence offering a valuable therapeuticapproach to treat estrogen-dependent breast tumors [3].

There are only three AIs available on the market, one steroidal(exemestane (1)) and two non-steroidal compounds (letrozole andanastrozole). The steroidal AI exemestane (1) (Fig. 1) is orally active,long-lasting and safe for the treatment of hormone-responsivebreast cancer in postmenopausal women [4]. It binds irreversibly

C.L. Varela et al. / European Journal of Medicinal Chemistry 87 (2014) 336e345 337

to aromatase inactivating and producing suicide aromatase inhi-bition [5e7].

Exemestane (1) undergoes a complex metabolization process,and the first step is a reduction of the 17-keto group, giving theprimary metabolite to be identified in plasma, the 17b-hydroxylderivative (17b-hydroxy-6-methylenandrosta-1,4-dien-3-one (17-bHE)) (4) (Fig. 1), which is followed by the P450-catalyzed oxidationof the 6-methylene group with formation of many secondary me-tabolites. These metabolites were found to be either inactive or lesspotent than exemestane [4,8e11]. However, the 17b-hydroxyl de-rivative was further studied by Goss and collaborators [12], whichfound that it produced similar effects to exemestane. Buzzetti et al.have also synthesized some potent potential metabolites ofexemestane through oxidation of the 6-methylene group [13]. Dueto the huge clinical interest of exemestane and since it is exten-sively metabolized giving, in some cases, active compounds, wewere interested in synthesizing epoxide derivatives of exemestane,since these compounds have never been identified or isolated,though they are proposed as potential metabolites [4,8,9]. Inaddition, they may exhibit strong aromatase inhibition as thesubstitution of double bonds by epoxide functions, which havesimilar bond geometries, allow the molecule to maintain planarity,an important chemical feature for anti-aromatase activity [14e17].Therefore, this prompted us to synthesize epoxide derivatives(compounds 6b-spirooxiranandrosta-1,4-diene-3,17-dione (2) and1a,2a-epoxy-6-methylenandrost-4-ene-3,17-dione (3)) (Scheme 1)as potential metabolites of exemestane (1). The stereochemistry ofthe epoxide group of compounds 2 and 3 was also unequivocallyelucidated on the basis of NMR two-dimensional NOESY experi-ments, with the stereochemistry of the C-6 spiro epoxide group ofcompound 2 described for the first time. Beyond that, we were alsointerested in further study the well-established metabolites ofexemestane, 17-bHE (4) and 6-(hydroxymethyl)androsta-1,4,6-triene-3,17-dione (6-HME (6)) (Fig. 1 and Scheme 1). For this, 17-bHE (4) was synthesized, with compound (5) as a by-product, while6-HME (6) was commercially acquired. For all compounds, theinhibitory activity against aromatase was evaluated in microsomesand in a hormone-dependent (ERþ) breast cancer cell line thatoverexpress aromatase, MCF-7aro cells. Their biological effects inMCF-7aro cells were also investigated.

2. Materials and methods

2.1. Chemistry

Reactions were controlled by thin layer chromatography (TLC)using silica gel 60 F254 plates. Column chromatography was per-formed using silica gel 60 (0.063e0.200 mm). Melting points (MPs)were determined on a Reichert Thermopan hot block apparatus andwere not corrected. IR spectra were recorded on a Jasco 420 FT/IR

Fig. 1. Chemical structures of exemestane (1) and their

spectrometer. The 1H NMR and 13C NMR spectra were recorded at600 MHz and 150 MHz, respectively on a Varian Unity 600.Chemical shifts were recorded in d values in parts permillion (ppm)downfield from tetramethylsilane as an internal standard. All Jvalues are given in Hz. Mass spectra ESI and LC-MS were obtainedwith a mass spectrometer QIT-MS Thermo Finningan, model LCQAdvantage MAX coupled to a Liquid Chromatograph of High Per-formance Thermo Finningan. Exemestane (1) was purchased fromSequoia Research Products (Pangbourne, United Kingdom) and 6-HME (6) from Carbosynth Limited (Berkshire, United Kingdom).Reagents and solvents were used as obtained from supplierswithout further purification, with exception to dichloromethane,which was dried through reflux and distilled from CaH2 [18].

All compounds possess a purity superior to 98%. The purity waschecked by HPLC with a C18-reversed phase column and water/acetonitrile 40:60 as solvent. The purity of individual compoundswas determined from the area of the peaks in the chromatogram ofthe sample solution.

2.1.1. 6b-Spirooxiranandrosta-1,4-diene-3,17-dione (2)To a solution of exemestane (1) (250.4 mg, 0.84 mmol) in

dichloromethane (7 mL), a solution of performic acid, generated insitu by addition of HCOOH 98e100% (0.12 mL) to H2O2 35%(0.31 mL), was added and the reaction was stirred at room tem-perature for 29 h, time after which an equivalent amount of per-formic acid solution was added and let to react for more 67 h. Thereaction was worked up by addition of dichloromethane (200 mL)and the organic layer was washed with 10% aqueous NaHCO3(2� 100mL) followed by water (4� 100mL), dried over anhydrousMgSO4, filtered and concentrated to dryness, giving a white solidresidue. Purification of this residue by column chromatography(petroleum ether 40e60 �C/ethyl acetate) afforded the pure com-pound 2 (55.7 mg, 21%) as a white crystalline solid. A secondfraction gave 76.1 mg of a mixture of 2 and its 6a-isomer 2a (65:35,respectively, by NMR).

6b-Spirooxiranandrosta-1,4-diene-3,17-dione (2): Mp (petroleum

ether 40e60 �C/ethyl acetate) 222e224 �C. IR (NaCl plates, CHCl3) nmaxcm�1: 3045 (HeC]), 1722 (C]O), 1662 (C]O), 1638 (C]C), 1053(CeO). 1H NMR (600 MHz, CDCl3) d: 0.96 (3H, s, 18-H3), 1.37 (3H, s,19-H3), 2.79 (1H, d, J60a-60b ¼ 4.2, 60-Ha), 3.13 (1H, d, J60b-60a ¼ 4.2, 60-Hb), 6.15 (1H, d, J4-2 ¼ 1.8, 4-H), 6.26 (1H, dd, J2e1 ¼10.2, J2e4 ¼ 1.8,2-H), 7.08 (1H, d, J1e2¼10.2, 1-H). 13C NMR (150MHz, CDCl3) d: 13.8(C-18), 18.5 (C-19), 21.7, 21.9, 31.1, 32.9, 35.5, 38.9, 44.8, 47.6, 50.3(eCH2eOe), 50.4, 51.6, 59.6 (C-6), 124.9 (C-2), 127.4 (C-1), 155.7 (C-4), 161.4 (C-5), 185.9 (C-3), 219.4 (C-17). ESI: 313.2 for C20H25O3([M þ H]þ, 100%).

6a-Spirooxiranandrosta-1,4-diene-3,17-dione (2a) (identified inthe 6b:6amixture): 1H NMR (600MHz, CDCl3) d: 0.95 (3H, s,18-H3),1.27 (3H, s, 19-H3), 2.69 (1H, d, J60ae60b ¼ 5.7, 60-Ha), 3.01 (1H, dd,

identified metabolites 17-bHE (4) and 6-HME (6).

Scheme 1. Reagents and conditions: (i) H2O2, HCOOH, dichloromethane, rt, 96 h; (ii) H2O2, NaOH, methanol, 0 �C, 30 min and then rt, 24 h; (iii) CF3COOH, CH3COOH, CH3CN, NaBH4,anhydrous dichloromethane, rt, 11 h.

C.L. Varela et al. / European Journal of Medicinal Chemistry 87 (2014) 336e345338

J60be60a ¼ 5.7, 60-Hb), 6.26 (1H, dd, J2e1 ¼ 10.1, J2e4 ¼ 1.8, 2-H), 6.32(1H, d, J4e2 ¼ 1.8, 4-H), 7.04 (1H, d, J1e2 ¼ 10.1, 1-H).

2.1.2. 1a,2a-epoxy-6-methylenandrost-4-ene-3,17-dione (3)To an ice cold solution of exemestane (1) (250.4 mg, 0.84 mmol)

in methanol (10 mL), it was added dropwise a cooled solution of35% H2O2 (0.81 mL, 34.83 mmol) followed by a cooled solution of4 N aqueous NaOH (0.61 mL, 2.44 mmol). The reaction was stirredat 0 �C over 30 min, and then at room temperature for 24 h. Thereaction was neutralized by addition of 0.5 N aqueous HCl. Afterthat, water (150mL)was added and the resulting solution extractedwith dichloromethane (3 � 100 mL). The organic layer was washedsuccessively with 10% aqueous NaHCO3 (2 � 100 mL) and water(3 � 100 mL), dried over anhydrous MgSO4, filtered and concen-trated to dryness giving a white solid residue. This residue waspurified by silica gel 60 column chromatography (petroleum ether40e60 �C/ethyl acetate) affording the pure compound 3 (28.7 mg,11%) as a white solid. Mp (petroleum ether 40e60 �C/ethyl acetate)201e203 �C. IR (NaCl plates, CHCl3) nmax cm�1: 3029 (]CH2), 1722(C]O), 1675 (C]O), 1664 (C]C), 1052 (CeO). 1H NMR (600 MHz,CDCl3) d: 0.93 (3H, s, 18-H3), 1.18 (3H, s, 19-H3), 3.46 (1H, dd,J2be1b ¼ 3.9, J2b-4 ¼ 1.9, 2b-H), 3.58 (1H, d, J1be2b ¼ 3.9, 1b-H), 4.98(1H, dd, J60ae60b ¼ 1.75, J60ae7b ¼ 1.75, 60-Ha), 5.08 (1H, dd,J60be60a ¼ 1.75, J60be7b ¼ 1.75, 60-Hb), 5.88 (1H, d, J4-2b ¼ 1.9, 4-H). 13CNMR (150 MHz, CDCl3) d: 13.7 (C-18), 18.9 (C-19), 21.3, 21.7, 31.0,34.9, 35.6, 38.6, 41.3, 47.3, 47.5, 50.7, 55.1 (C-2), 60.6 (C-1), 114.6 (C-60), 118.6 (C-4),145.1 (C-6),163.9 (C-5),194.3 (C-3), 219.7 (C-17). ESI:313.4 for C20H25O3 ([M þ H]þ, 100%).

2.1.3. 17b-Hydroxy-6-methylenandrosta-1,4-dien-3-one (17-bHE(4))

Sodium borohydride (63.2 mg, 1.66 mmol) was added in smallportions to a stirred and cooled mixture of trifluoroacetic acid(0.4 mL), glacial acetic acid (0.4 mL) and acetonitrile (0.4 mL). Asolution of exemestane (1) (100.0 mg, 0.34 mmol) in drydichloromethane (8 mL) was then added to the former mixture.After this, the reaction was stirred at room temperature under ni-trogen, until all the starting material had been consumed (11 h,TLC). The reaction mixture was then neutralized with a solution of10% aqueous NaHCO3 and extracted with dichloromethane(3 � 100 mL). The organic layer was washed with water(3 � 100 mL), dried over anhydrous MgSO4, filtered and concen-trated to dryness giving a white solid residue (102.5 mg). Thisresidue was purified by silica gel column chromatography

(petroleum ether 40e60 �C/ethyl acetate) affording 39.1 mg of 17-bHE (4) (39%) and 11.3 mg of a mixture mainly composed by 6-methylandrosta-1,4,6-trien-17b-ol 5.

17b-Hydroxy-6-methylenandrosta-1,4-dien-3-one (17-bHE (4)):Mp (petroleum ether 40e60 �C/ethyl acetate) 87e90 �C. IR (NaCl plates,CHCl3) nmax cm�1: 3421 (OeH), 3037 (HeC]), 1735 (C]O), 1656(C]O), 1644 (C]C), 1057 (CeO). 1H NMR (600 MHz, CDCl3) d: 0.81(3H, s, 18-H3), 1.14 (3H, s, 19-H3), 3.66 (1H, dd,J17ae16a ¼ J17ae16b ¼ 8.6, 17a-H), 4.94 (1H, t, J ¼ 2.0, 60-Ha), 5.01 (1H,t, J¼ 2.0, 60-Hb), 6.14 (1H, t, J4e2¼ 1.8, 4-H), 6.23 (1H, dd, J2e1 ¼10.2,J2e4 ¼ 1.8, 2-H), 7.08 (1H, d, J1e2 ¼ 10.2, 1-H). 13C NMR (150 MHz,CDCl3) d: 11.1 (C-18), 19.7 (C-19), 22.4, 23.4, 30.3, 35.8, 36.2, 40.0,43.0, 43.8, 50.0, 50.5, 81.4 (C-17), 111.9 (C-60), 122.5 (C-4), 127.6 (C-2), 145.8 (C-6), 154.6 (C-1), 167.9 (C-5), 186.6 (C-3). ESI: 299.3 forC20H27O2 ([M þ H]þ, 100%).

6-Methylandrosta-1,4,6-trien-17b-ol (5) (identified in mixture):1H NMR (600 MHz, CDCl3) d: 0.81 (3H, s, 18-H3), 0.99 (3H, s, 19-H3),1.81 (3H, s, 6-H3), 2.77 (2H, m, 3-H), 3.65 (1H, dd,J17ae16a ¼ J17ae16b ¼ 8.4, 17a-H), 5.36 (1H, s, 7-H), 5.66 (1H, t,J4e2a ¼ J4-2b ¼ 4.3, 4-H), 5.70 (1H, m, 2-H), 5.92 (1H, dt, J1e2 ¼ 10.0,J1-3a ¼ J1-3b ¼ 2.0, 1-H). 13C NMR (150 MHz, CDCl3) d: 11.1 (C-18),20.0 (C-19), 20.6, 21.2 (C-20), 23.2, 27.1, 30.5, 36.5, 36.7, 37.1, 43.6,49.1, 49.5, 81.6 (C-17), 117.8 (C-4), 122.6 (C-2), 126.1 (C-1), 131.4 (C-5), 133.3 (C-7), 142.4 (C-6).

2.2. Biochemistryearomatase activity in human placentalmicrosomes

2.2.1. Preparation of placental microsomesThe human placental microsomes were obtained as described

previously [14,15]. Human placentas, obtained after delivery from alocal hospital were placed in cold 67 mM potassium phosphatebuffer (pH 7.4) containing 1% KCl. The cotyledon tissue was sepa-rated and homogenized in a Polytron homogenizer with 67 mMpotassium phosphate buffer (pH 7.4) containing 0.25M sucrose and0.5 mM dithiothreitol (DTT, 1:1, w/v). The homogenate wascentrifuged at 5000 � g for 30 min and the supernatant wascentrifuged at 20,000 � g for 30 min and after at 54,000 � g for45 min to yield the microsomal pellet. After ultra-centrifugation,the microsomes were washed and resuspended in 67 mM potas-sium phosphate buffer (pH 7.4) containing 0.25 M sucrose, 20%glycerol, and 0.5 mM DTT and stored at �80 �C. All the procedureswere carried out at 4 �C. Protein content was estimated by the Bio-

C.L. Varela et al. / European Journal of Medicinal Chemistry 87 (2014) 336e345 339

Rad protein assay (Bio-Rad Labs, Munich, Germany) using bovineserum albumin (BSA) as a standard.

2.2.2. Aromatase assay procedureAromatase activity was measured according to Thompson and

Siiteri [19] and Heidrich et al. [20] method with modifications [17],by measuring the tritiated H2O released from [1b-3H] androstene-dione (PerkineElmer Life Sciences, Boston, MA, USA), during thearomatization process. The steroidal compounds studied weredissolved in DMSO (SigmaeAldrich Co., Saint Louis, USA) anddiluted in 67 mM potassium phosphate buffer (pH 7.4), prior toassays. In order to determine the percentage of aromatase inhibi-tion it was performed a screening assay, where it was used for thereaction mixture (1 mL) 20 mg of microsomal protein, 40 nM of[1b-3H] androstenedione (1 mCi) and 2 mM of each steroidal com-pound tested. To determine the IC50 it was used 100 nM (1 mCi) of[1b-3H] androstenedione and different concentrations of thecompounds (0.01e5 mM). The aromatase-catalyzed reaction wasinitiated by the addition of reduced nicotinamide adenine dinu-cleotide phosphate (NADPH, 150 mM) (SigmaeAldrich Co., SaintLouis, USA), and incubations were performed in a shaking waterbath at 37 �C for 15 min. The aromatase reactions were terminatedby addition of 250 mL of 20% trichloroacetic acid (TCA) and themixture was transferred to microcentrifuge tubes containing acharcoaledextran pellet, vortexed and incubated for 1 h. Aftercentrifugation at 14,000 � g for 10 min, the supernatants weretransferred to new charcoaledextran pellets and incubated for10 min. The supernatant containing the tritiated water was mixedwith a liquid scintillation cocktail (ICN Radiochemicals, Irvine, CA,USA) and counted in a liquid scintillation counter (LS-6500, Beck-man Coulter, Inc., Fullerton, CA). All the experiments were carriedout in triplicate. As a reference AI it was used exemestane (1)(Sequoia Research Products Ltd., Pangbourne, UK) at 1 mM.

2.3. Biologyearomatase activity and viability effects in humanbreast cancer cells

2.3.1. Cell cultureThe ER-positive (ERþ) aromatase-overexpressing human breast

cancer cell line, MCF-7aro, prepared by stable transfection of MCF-7cells with the human placental aromatase gene and Geneticin se-lection [21,22], was kindly provided by Dr. Shiuan Chen (BeckmanResearch Institute, City of Hope, Duarte, CA, U.S.A.). The cells weremaintained with Eagles's minimum essential medium (MEM)supplemented with 1 mmol/L sodium pyruvate, 1% penicillin-streptomycin-amphotericin B, 100 mg/mL G418 and 10% heat-inactivated fetal bovine serum (FBS) (Gibco Invitrogen Co.,Paisley, Scotland, UK) in 5% CO2 atmosphere at 37 �C. The mediumwas changed every three days.

In order to avoid the interference of steroids present in FBS andof the estrogenic effects of phenol-red [23], three days beforestarting the experiments, cells were cultured in an E2-free MEMmediumwithout phenol-red, containing 5% of pre-treated charcoalheat-inactivated fetal bovine serum (CFBS), 1 mmol/L sodium py-ruvate, 2 mmol/L glutamine and 1% penicillin-streptomycin-amphotericin B. All the biological experiments were performedaccording to these conditions.

The stock solution of each steroidal compound was prepared in100% DMSO and stored at �20 �C. The stock solutions of testos-terone (T) and estradiol (E2) (SigmaeAldrich Co., Saint Louis, USA)were prepared in absolute ethanol and stored at �20 �C. Appro-priate dilutions were freshly prepared with medium, just prior theassays and the final concentration of DMSO and ethanol in culturemedium was less than 0.05% and 0.01%, respectively.

2.3.2. In cell aromatase assayAromatase activity and IC50 of each steroidal compound in MCF-

7aro cells were determined according to Thompson and Siiteri [24]and Zhou et al. [21] methods with modifications [25]. Briefly,confluent MCF-7aro cells plated in a 24-well plate, were cultured inserum-free medium containing the inhibitors at 10 mM, for aro-matase activity screening, or at various concentrations(0.01e10 mM), for IC50 determination, with 50 nM of [1b-3H] an-drostenedione as substrate and also 500 nM of progesterone (thatwas used to suppress the 5a-reductase activity, which also use theandrogen as substrate) and incubated at 37 �C during 1 h. Thearomatase reaction was finished by addition of 100 mL of 20% TCA.The aromatase activity was evaluated as previously described[25,26]. All experiments were carried out in triplicate in three in-dependent experiments. Exemestane (1) at 10 mM was used asreference AI.

2.3.3. Cell viabilityCell viability of each steroidal compound in MCF-7aro cells was

assessed by tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-difenyltetrazolium (MTT) assay and by measuring the lactate de-hydrogenase (LDH) release. Cells were cultured in 96-well plates ata cellular density of 2.5 � 104 cells/ml (for 2 and 3 days) and1 � 104 cells/mL (for 6 days), with different concentrations of eachcompound (0.01e15 mM). MCF-7aro cells cultured in MEM withoutphenol-red containing CFBS were treated with each compound andwith 1 nM of testosterone (T), the aromatase substrate, or with1 nM of estradiol (E2), the product of aromatase reaction. Themedium and drugs were refreshed every 3 days. Cells incubatedonly with 1 nM of T or E2 were used as control.

After each incubation time, MTT (0.5 mg/mL) (SigmaeAldrichCo., Saint Louis, USA) was added to each well and cells were incu-bated for 2 h and 30 min at 37 �C in 5% CO2. The formazan wasquantified spectrophotometrically by addition of DMSO:isopropa-nol mixture (3:1). LDH release was measured using CytoTox 96nonradioactive cytotoxity assay kit (Promega Corporation, Madi-son, USA) according to the manufacturer's protocol.

All the assays were performed in triplicate in three independentexperiments and results are expressed as a percentage of the un-treated control cells.

2.4. Statistical analysis

Statistical analysis of data was performed using analysis ofvariance (ANOVA) followed by Bonferroni test for multiple com-parisons and values of p < 0.05 were considered as statisticallysignificant. The data presented are expressed as the mean ± SEM.

3. Results

3.1. Chemistry

Two different reaction conditions for the epoxidation reactionwere explored in order to obtain distinct epoxide derivatives ofexemestane, depending on whether the double bond belongs to ana,b-unsaturated ketone, or not (Scheme 1). In fact, the epoxidationof an olefin can be achieved by the conventional oxidation withperacids however, this method of epoxidation is not applicable toa,b-unsaturated ketones. This is because the reaction of such olefinswith peracids occurs very slowly, due to the deactivation of thedouble bond by the carbonyl group [27]. The synthesis of com-pound 2 (Scheme 1) was made by treating exemestane (1) withperformic acid in dichloromethane, at room temperature, for 96 h[17,28]. The resulting crude mixture was then purified by columnchromatography affording the desired 6b-epoxide 2, in 21% yield. In

α α,

Fig. 3. Compound 3 showing NOESY correlations (full line e strong correlation).

C.L. Varela et al. / European Journal of Medicinal Chemistry 87 (2014) 336e345340

other fraction, a mixture of 2with its 6a-isomer 2awas obtained ina 65:35 proportion (NMR analysis), respectively. Efforts have beendone to isolate its 6a-isomer (compound 2a) nevertheless, all at-tempts resulted unfruitful being 2a always obtained in mixture.Although compound 2 has already been described [13], the ste-reochemistry of its C-6 epoxide group had not been unequivocallyestablished. Therefore, two-dimensional NOESY experiments wereused to assign the C-6 epoxide stereochemistry of compound 2. Themost significant correlation to be studied was between the H-atoms at C-60 and at C-19 (Fig. 2). For the 6a-isomer 2a it wasobserved a strong correlation between the above mentioned H-atoms, which implies that these atoms are spatially close enough tocorrelate. Hence the H-atoms at C-60 will be pointing towards the b-face of the molecule, the oxygen atom towards the a-face andconsequently the epoxide functional group was assigned with a-stereochemistry. For the 6b-isomer 2, it was not observed anycorrelation between the referred H-atoms. This observationtogether with the study of the chemical shifts for the H-atoms at C-19, which are shifted downfield, when compared with those of the6a-isomer 2a, allows one to conclude that the more abundant andisolated compound is the 6b-isomer 2. In fact, the H-atoms at C-19of the 6b-isomer 2, due to closeness to the epoxide oxygen atomexperience higher influence of its electronegativity.

The synthesis of epoxide 3 (Scheme 1) was performed bytreatment of a methanol solution of exemestane (1) with an alka-line oxidizing solution of 35% hydrogen peroxide in a 4 N sodiumhydroxide solution [29]. The reaction was carried out at 0 �C for30min and then at room temperature during 24 h and the resultingcrude product was purified by column chromatography affordingthe desired epoxide 3 in 11% yield. Despite the reaction has notbeen complete, only compound 3 was detected. NOESY experi-ments were also used to unequivocally identify compound 3(Fig. 3). The most significant signal analyzed was the H-atom at C-1(3.6 ppm). It was observed a strong correlation between this H-atom and the H-atoms of C-19, which reveals that the H-atom at C-1 must be assuming the b-configuration allowing an enhancedspatial proximity towards the C-19 angular methyl group (Fig. 3).Hence, the isolated isomer was the 1a,2a-epoxide compound 3.

The reaction of exemestane (1) (Scheme 1) with a mixture ofsodium borohydride in trifluoroacetic acid, glacial acetic acid andacetonitrilewas performed in a nitrogen atmosphere [30]. It led to amixture of several compounds, being 17-bHE (4) and compound 5(Scheme 1) the two main products, which were isolated throughcolumn chromatography. 17-bHE (4) was isolated in higher yield(39%) as a pure white solid residue and compound 5was identifiedin a mixture as the major component.

β α

Fig. 2. Compound 2 (6b-isomer) and compound 2a (6a-isomer) showing NOESY cor-relations (dashed line e absence of correlation; full line e strong correlation).

3.2. Biochemistry

3.2.1. Aromatase inhibition in human placental microsomesThe anti-aromatase activity of the exemestane metabolites was

evaluated in human placental microsomes using a radiometricassay in which tritiated water, released from [1b-3H] androstene-dione into the incubationmedium,was used as an index of estrogenformation [19]. By a screening assay, it was determined the per-centage of aromatase inhibition (%) for all steroidal compounds at2 mM (Fig. 4), relative to an assay carried out in the absence of theinhibitor. The percentage of aromatase inhibition obtained forsteroids 2, 3, 17-bHE (4) and 6-HME (6) was 91.7 ± 2.0%, 86.8 ± 1.0%,97.7 ± 0.2% and 81.0 ± 0.9%, respectively (Table 1). The 17-bHE (4) isthe most potent one with an aromatase inhibition similar toexemestane (1) (98.3 ± 0.6%). The IC50, in human placental micro-somes, for all compounds was also determined, being 0.62 mM forcompound 2, 0.81 mM for compound 3, 0.10 mM for 17-bHE (4) and0.67 mM for 6-HME (6) (Table 1 and Fig. 4A). Among metabolites,the lowest IC50 was obtained for 17-bHE (4). For exemestane (1) itwas obtained an IC50 of 0.05 mM (Table 1 and Fig. 4A).

3.3. Biology

3.3.1. Aromatase inhibition in MCF-7aro cellsIn this study, it was also evaluated for all metabolites, the anti-

aromatase activity and the IC50 values in MCF-7aro cells by aradiometric assay, using [1b-3H] androstenedione as substrate. Thesteroids 2, 3, 17-bHE (4) and 6-HME (6) induced an aromatase in-hibition of 95.3 ± 0.4%, 88.6 ± 3.0%, 98.1 ± 0.3% and 84.0 ± 1.4%,respectively (Table 1). The exemestane (1) that was used as refer-ence AI, presented an aromatase inhibition of 95.17 ± 0.80%. Inwhich concerns IC50, themetabolites 2, 3,17-bHE (4) and 6-HME (6),presented an IC50 of 0.73 mM, 1.18 mM, 0.25 mM and 0.98 mM,respectively (Table 1 and Fig. 4B). Our results demonstrate thatmetabolite 17-bHE (4) was the most potent inhibitor in MCF-7arocells, while metabolite 3 was the less effective. It was alsoobserved that exemestane (1), which presented an IC50 of 0.9 mM, asalready described by our group [25], presented an IC50 higher thanthe metabolite 17-bHE (4) (Table 1 and Fig. 4B). Furthermore, theresults obtained in MCF-7aro cells are in accordance with thoseobtained in human placental microsomes, being the 17-bHE (4) themost potent metabolite.

3.3.2. Cell viability in T-treated and E2-treated MCF-7aro cellsThe effects of the exemestanemetabolites 2, 3,17-bHE (4) and 6-

HME (6) (0.01e15 mM), after 2, 3 and 6 days of treatment, in MCF-7aro cells viability and cytotoxicity were studied by MTT and LDHrelease assays. Cells treated only with T or E2 were considered ascontrols.

As presented in Fig. 5, the steroids 2, 3,17-bHE (4) and 6-HME (6)induced a decrease in cell viability in a dose- and time-dependent

Fig. 4. Graphical representation of the IC50 (mM) values in human placental microsomes (A) and in MCF-7aro cells (B) of the exemestane metabolites 2, 3, 17-bHE (4) and 6-HME (6).Exemestane (1) was used as reference AI. Data are presented as a percentage of the tritiated water control and correspond to three independent experiments carried out intriplicate. * Previously published data in Amaral C. et al., 2013 [25].

Table 1Aromatase inhibition (%) and IC50 (mM) values of the exemestane metabolites 2, 3,17-bHE (4) and 6-HME (6), as well as exemestane (1), in human placental micro-somes and in MCF-7aro cells, a breast cancer cell line over-expressing aromatase.

Compounds Human placental microsomes MCF-7aro cells

Aromatase inhibitiona

(%) ± SEMIC50

b

(mM)Aromatase inhibitionc

(%) ± SEMIC50

d

(mM)

2 91.7 ± 2.0 0.62 95.3 ± 0.4 0.733 86.8 ± 1.0 0.81 88.6 ± 3.0% 1.1817-bHE (4) 97.7 ± 0.2 0.10 98.1 ± 0.3 0.256-HME (6) 81.0 ± 0.9 0.67 84.0 ± 1.4 0.98Exemestane

(1)98.3 ± 0.6 0.05 95.2 ± 0.8 0.90

[25]

a Concentrations of 40 nM [1b-3H] androstenedione, 20 mg protein from humanplacental microsomes, 2 mM of the steroids and 15 min incubation were used.

b Concentrations of 100 nM [1b-3H] androstenedione, 20 mg protein from humanplacental microsomes, different concentrations of the steroids and 15 min incuba-tion were used.

c Concentrations of 50 nM [1b-3H] androstenedione, confluent MCF-7aro cells,10 mM of the steroids and 1 h of incubation were used.

d Concentrations of 50 nM [1b-3H] androstenedione, confluent MCF-7aro cells,different concentrations of the steroids and 1 h of incubation were used. The resultsrepresent the mean ± S.E.M. of three different experiments performed in triplicate.

C.L. Varela et al. / European Journal of Medicinal Chemistry 87 (2014) 336e345 341

manner. Steroids 2, 17-bHE (4) and 6-HME (6) induced a significant(p < 0.05; p < 0.01; p < 0.001) decrease in cell viability for all theconcentrations and incubation times, except for the lower con-centrations (1 and 2.5 mM) and after 2 days of treatment (Fig. 5).Metabolite 3 induced a more pronounced and statistically signifi-cant (p < 0.01; p < 0.001) reduction in cell viability (Fig. 5B).Moreover, this steroid in the same concentrations used for theother metabolites (5e15 mM) induced a drastic decrease in cellviability, lower than 25% after all the times of treatment (Fig. 5B).

To evaluate the cytotoxicity of exemestane metabolites, it wasalso analyzed the LDH release for all compounds. Contrary to theother studied metabolites that did not induce LDH release in anyconcentration, compound 3 induced a significant (p < 0.001) in-crease in LDH release (Fig. 6B) between 5 and 15 mM after 2 and 3days of treatment, suggesting, for these concentrations, a disrup-tion of the MCF-7aro cell membrane integrity.

In order to understand if the reduction in MCF-7aro cellsviability was due to aromatase inhibition, it was evaluated andcompared the effects of exemestanemetabolites in E2-treatedMCF-7aro cells with T-treated cells. It was observed that all metabolitesinduced a slight decrease in viability of E2-treated MCF-7aro cells,being the behavior similar after 3 and 6 days of treatment. More-over, for steroids 2, 3 and 6-HME (6) the effects on viability of T-

treated cells were more pronounced than in E2-treated cells,causing significant differences (p < 0.05; p < 0.01; p < 0.001) be-tween T- versus E2-treated cells (Fig. 7). On the contrary, for themetabolite 17-bHE (4) the effects on viability of E2-treated cellswere more pronounced than in T-treated cells. Even though, thismetabolite induced significant differences (p < 0.01; p < 0.001)between T- versus E2-treated cells (Fig. 7C). Comparing the effectsof exemestane (1) on T- versus E2-treated cells, it was also notedthat the effects on viability of T-treated cells were more pro-nounced than in E2-treated cells, for 10 and 15 mM, though withonly significant (p < 0.05) differences for 15 mM (Fig. 7E). Thus, aswell as exemestane (1), the metabolites 2, 3 and 6-HME (6) induce adecrease in viability of MCF-7aro cells in an aromatase-dependentmanner, while for metabolite 17-bHE (4) the reduction in cellviability seems to be also due to other mechanisms than aromataseinhibition.

4. Discussion

In this work, some metabolites were synthesized by trans-forming one double bond of A- or B-rings of exemestane (1) in anepoxide (compounds 2 and 3) and by reducing its C-17 carbonylgroup to a hydroxyl group (17-bHE (4)). Additionally, the well-established metabolite 6-HME (6) was acquired for furtherstudies. Subsequently, the anti-aromatase activity of the referredexemestane metabolites, in human placental microsomes and in anERþ aromatase-overexpressing human breast cancer cell line, MCF-7aro cells, was investigated. This cell line was stably transfectedwith aromatase gene, corresponding to a good model to study AIsin breast cancer [31]. It was also explored the biological effects ofthese metabolites in MCF-7aro cells viability and investigated ifthese effects were dependent on aromatase inhibition.

By the evaluation of the anti-aromatase activity of exemestanemetabolites in human placental microsomes, using a radiometricassay, it was observed that all the steroidal compounds have apercentage of aromatase inhibition (%) higher than 80% (Table 1),showing that they are also potent AIs. Looking at compounds 2 and3, we observed that the substitution of the C-6 exocyclic and the C-1 double bonds, respectively, by epoxide groups led to derivativesless potent in microsomes but, in the case of 2, more potent in MCF-7aro cells than exemestane (1) (Table 1), being the exocyclic sub-stitution slightly more favorable (compound 2 with an IC50 of0.62 mM, in microsomes, and 0.73 mM in MCF-7aro cells), than thesubstitution at C-1 (compound 3, with an IC50 of 0.81 mM and1.18 mM, in microsomes and MCF-7aro cells, respectively) (Table 1).

Fig. 5. Effects of exemestane metabolites 2 (A), 3 (B), 17-bHE (4) (C) and 6-HME (6) (D) on cell viability of MCF-7aro cells treated with testosterone (T). MCF-7aro cells were culturedwith different concentrations of each AI (0.01e15 mM) and T at 1 nM during 2, 3 and 6 days. Cells cultured with T represent the maximum of cell viability and were considered aspositive control. Results are the mean ± SEM of three independent experiments, performed in triplicate. Significant differences between the control and cells treated with each AIare denoted by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001).

Fig. 6. Effects of exemestane metabolites 2 (A), 3 (B), 17-bHE (4) (C) and 6-HME (6) (D) on MCF-7aro cells cytotoxicity, evaluated by LDH release assay. MCF-7aro cells were culturedwith different concentrations of each AI (0.1e15 mM) and T at 1 nM during 2 and 3 days. Cells cultured with T were considered as positive control. Results are the mean ± SEM ofthree independent experiments, performed in triplicate. Significant differences between the control and cells treated with each AI are denoted by *** (p < 0.001).

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In 17-bHE (4), the C-17 carbonyl group was transformed into ahydroxyl group affording the well-established metabolite ofexemestane with a significant increase in the anti-aromatase ac-tivity (IC50 of 0.25 mM) in MCF-7aro cells when comparing withexemestane (1) (IC50 of 0.9 mM [25]). 17-bHE (4) was actually themost potent aromatase inhibiting metabolite. In 6-HME (6), thepresence of a C-6 hydroxymethyl group instead of the exocyclicdouble bond revealed to be less favorable leading to a decrease inthe anti-aromatase activity in microsomes. Some of these metab-olites, 2,17-bHE (4) and 6-HME (6) were already studied in placental

microsomes [13], revealing slightly lower IC50 values (0.21 mM,0.07 mM and 0.56 mM, respectively). For the reference AI exemes-tane (1) it was obtained an IC50 of 0.051 mM in human placentalmicrosomes, which is in accordance to the values alreadydescribed, 0.043 mM [32] or 0.050 mM [7].

To evaluate the biological effects of the exemestane metabolites,2, 3, 17-bHE (4) and 6-HME (6), it was also studied their effects inMCF-7aro cells viability. Our results demonstrate that all metabo-lites induced a significant decrease in the viability of T-treatedMCF-7aro cells, in a dose- and time-dependent manner, as described for

Fig. 7. Comparison of the biological effects of metabolites 2 (A), 3 (B), 17-bHE (4) (C) and 6-HME (6) (D) and exemestane (1)a,b on viability of T-treated MCF-7aro cells versus E2-treated MCF-7aro cells, after 6 days of treatment. Results are the mean ± SEM of three independent experiments, performed in triplicate. Significant differences between the controland cells treated with each AI are denoted by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001) and between the T-treated MCF-7aro cells versus E2-treated MCF-7aro cells are denoted by# (p < 0.05), ## (p < 0.01) and ### (p < 0.001). aPreviously published data in Amaral C. et al., 2012 [33]. bPreviously published data in Amaral C. et al., 2013 [25].

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exemestane [33] and for other steroidal AIs synthesized by ourgroup [25,26], which induced also apoptosis and autophagy in thereferred cells [34,35]. Moreover, steroid 3 revealed to be the mostpotent in decreasing MCF-7aro cells viability. Though, contrary tothe other metabolites, for higher concentrations, metabolite 3 in-duces loss of plasma membrane integrity. Nevertheless, it must bestressed that it causes a dramatic reduction in MCF-7aro cellviability for concentrations 10 to 100 times lower than the othermetabolites, indicating that this steroid should be further studied.As described for exemestane (1) [33,36], it was also referred that17-bHE (4), at low concentrations (growth EC50 of 2.7 mM) inducedproliferation of breast cancer cells [9], although, in our study, thiseffect was not observed. Further, it was noted that all exemestanemetabolites induced a more marked reduction of cell viability thanexemestane (1) [33], which suggests that the studied metabolitesare even more potent in reducing viability of breast cancer cellsthan exemestane.

In order to understand if the observed biological effects weredependent on aromatase inhibition, it was studied the effects ofeach metabolite in MCF-7aro cells stimulated with estradiol (E2),the product of the aromatization reaction of testosterone by theenzyme aromatase. Comparing T-treated MCF-7aro cells versus E2-treated cells, our results suggest that, except for the case of 17-bHE(4), the metabolites 2, 3 and 6-HME (6), as well as exemestane (1),induced a decrease in cell viability mainly in an aromatase-

dependent manner. Although, we cannot exclude the hypothesisthat other mechanisms, independent of aromatase inhibition, mayalso be involved. As exemestane (1) and its metabolites, othersteroidal AIs described by our group, also induced a reduction inMCF-7aro cell viability in an aromatase-dependent manner [25].

5. Conclusion

In summary, four metabolites of exemestane (1) have beenstudied being three of them synthesized (2, 3 and 17-bHE (4)) usingconvenient synthetic methodologies. The stereochemistry of theepoxide group of 2 and 3 has been unequivocally established, forthe first time, on the basis of NOESY experiments. In terms ofstructureeactivity relationships (SAR) it was found that the sub-stitution of the double bonds by epoxide groups, led to derivativesless potent in microsomes but, in the case of 2, more potent in MCF-7aro cells than exemestane (1), being the exocyclic substitution inC-6 slightly more favorable, than the substitution at C-1/C-2. Thesubstitution of the C-17 carbonyl group by a hydroxyl group,affording 17-bHE (4), resulted in significant increase in anti-aromatase activity in MCF-7aro cells, being this molecule morepotent than exemestane (1) and the most potent studied metabo-lite in inhibiting aromatase. The substitution of the C-6 methylenegroup by a hydroxymethyl group as in 6-HME (6), resulted in adecrease in the anti-aromatase activity. In terms of the biological

C.L. Varela et al. / European Journal of Medicinal Chemistry 87 (2014) 336e345344

effects, all the studied metabolites reduced cell viability of MCF-7aro cells in a more pronounced way than exemestane (1). Inaddition, metabolite 3, in much lower concentrations than theother metabolites and even exemestane (1), dramatically decreaseMCF-7aro cells viability. Therefore, our data suggest for the firsttime that exemestane originates active metabolites after metabolictransformation, which are also able to inhibit aromatase and reducehormone-dependent breast cancer cells viability. Since, exemes-tane efficacy may also be due to its active metabolites, it will beimportant to further study its metabolites in order to understandthe underlying mechanisms to improve exemestane effectiveness.

Acknowledgments

The authors are grateful to Fundaç~ao para a Ciencia e Tecnologia(FCT) for the PhD grants attributed to Cristina Amaral and CarlaVarela (SFRH/BD/48190/2008 and SFRH/BD/44872/2008, respec-tively) and also for the strategic project Pest-OE/SAU/UI0177/2011.This work was funded by FEDER Funds through the OperationalCompetitiveness Program- COMPETE and by National Fundsthrough FCT under the project FCOMP-01-0124-FEDER-020970(PTDC/QUI-BIQ/120319/2010). We also thank Dr. Shiuan Chen(Department of Cancer Biology, Beckman Research Institute of theCity of Hope, Duarte, CA, USA) for kindly supplying MCF-7aro cells.

Abbreviations

AIs aromatase inhibitorsBSA bovine serum albuminCFBS charcoal fetal bovine serumDTT dithiothreitolE2 estradiolER estrogen receptorERþ estrogen receptor positiveHPLC High-performance liquid chromatographyMCF-7aro cells ER-positive aromatase-overexpressing human

breast cancer cell lineFBS fetal bovine serumLDH lactate dehydrogenaseMEM Eagles's minimum essential mediumMPs Melting pointsMTT tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-

difenyltetrazoliumNADPH nicotinamide adenine dinucleotide phosphateNMR nuclear magnetic resonanceNOESY nuclear overhauser effect spectroscopyppm parts per millionSAR structureeactivity relationshipsSEM standard error of the meanT testosteroneTCA trichloroacetic acidTLC thin layer chromatography1 exemestane17-bHE or 4 17b-hydroxy-6-methylenandrosta-1,4-dien-3-one6-HME or 6 6-(hydroxymethyl)androsta-1,4,6-triene-3,17-dione2 6b-spirooxiranandrosta-1,4-diene-3,17-dione3 1a,2a-epoxy-6-methylenandrost-4-ene-3,17-dione.

References

[1] A. Jemal, M.M. Center, C. DeSantis, E.M. Ward, Global patterns of cancerincidence and mortality rates and trends, Cancer Epidemiol. Biomarkers Prev.Publ. Am. Assoc. Cancer Res. Cospons. Am. Soc. Prev. Oncol. 19 (2010)1893e1907.

[2] W.R. Miller, Aromatase inhibitorsewhere are we now? Br. J. Cancer 73 (1996)415e417.

[3] V.C. Jordan, A.M. Brodie, Development and evolution of therapies targeted tothe estrogen receptor for the treatment and prevention of breast cancer,Steroids 72 (2007) 7e25.

[4] P. Lombardi, Exemestane, a new steroidal aromatase inhibitor of clinicalrelevance, Biochim. Biophys. Acta 1587 (2002) 326e337.

[5] Y. Hong, S. Chen, Aromatase inhibitors: structural features and biochemicalcharacterization, Ann. N. Y. Acad. Sci. 1089 (2006) 237e251.

[6] X. Wang, S. Chen, Aromatase destabilizer: novel action of exemestane, a foodand drug administration-approved aromatase inhibitor, Cancer Res. 66 (2006)10281e10286.

[7] D. Ghosh, J. Lo, D. Morton, D. Valette, J. Xi, J. Griswold, S. Hubbell, C. Egbuta,W. Jiang, J. An, H.M. Davies, Novel aromatase inhibitors by structure-guideddesign, J. Med. Chem. 55 (2012) 8464e8476.

[8] A. Cavalcanti Gde, B.C. Garrido, F.D. Leal, M.C. Padilha, X. de la Torre, F.R. deAquino Neto, Detection of new urinary exemestane metabolites by gaschromatography coupled to mass spectrometry, Steroids 76 (2011)1010e1015.

[9] E.A. Ariazi, A. Leitao, T.I. Oprea, B. Chen, T. Louis, A.M. Bertucci, C.G. Sharma,S.D. Gill, H.R. Kim, H.A. Shupp, J.R. Pyle, A. Madrack, A.L. Donato, D. Cheng,J.R. Paige, V.C. Jordan, Exemestane's 17-hydroxylated metabolite exerts bio-logical effects as an androgen, Mol. Cancer Ther. 6 (2007) 2817e2827.

[10] L.K. Kamdem, D.A. Flockhart, Z. Desta, In vitro cytochrome P450-mediatedmetabolism of exemestane, Drug Metab. Dispos. 39 (2011) 98e105.

[11] E.D. Deeks, L.J. Scott, Exemestane: a review of its use in postmenopausalwomen with breast cancer, Drugs 69 (2009) 889e918.

[12] P.E. Goss, S. Qi, A.M. Cheung, H. Hu, M. Mendes, K.P. Pritzker, Effects of thesteroidal aromatase inhibitor exemestane and the nonsteroidal aromataseinhibitor letrozole on bone and lipid metabolism in ovariectomized rats,clinical cancer research, Off. J. Am. Assoc. Cancer Res. 10 (2004) 5717e5723.

[13] F. Buzzetti, E. Di Salle, A. Longo, G. Briatico, Synthesis and aromatase inhibitionby potential metabolites of exemestane (6-methylenandrosta-1,4-diene-3,17-dione), Steroids 58 (1993) 527e532.

[14] C. Varela, E.J. Tavares da Silva, C. Amaral, G. Correia da Silva, T. Baptista,S. Alcaro, G. Costa, R.A. Carvalho, N.A. Teixeira, F.M. Roleira, New structure-activity relationships of A- and D-ring modified steroidal aromatase in-hibitors: design, synthesis, and biochemical evaluation, J. Med. Chem. 55(2012) 3992e4002.

[15] C.L. Varela, C. Amaral, G. Correia-da-Silva, R.A. Carvalho, N.A. Teixeira,S.C. Costa, F.M. Roleira, E.J. Tavares-da-Silva, Design, synthesis and biochem-ical studies of new 7alpha-allylandrostanes as aromatase inhibitors, Steroids78 (2013) 662e669.

[16] M. Numazawa, K. Yamada, S. Nitta, C. Sasaki, K. Kidokoro, Role of hydrophilicinteraction in binding of hydroxylated 3-deoxy C(19) steroids to the activesite of aromatase, J. Med. Chem. 44 (2001) 4277e4283.

[17] M.M. Cepa, E.J. Tavares da Silva, G. Correia-da-Silva, F.M. Roleira, N.A. Teixeira,Structure-activity relationships of new A,D-ring modified steroids as aroma-tase inhibitors: design, synthesis, and biological activity evaluation, J. Med.Chem. 48 (2005) 6379e6385.

[18] D.D. Perrin, W.L.F. Armarego, Purification of organic chemicals. Purification ofLaboratory Chemicals, third ed., Pergamon, Oxford, 1988.

[19] E.A. Thompson Jr., P.K. Siiteri, The involvement of human placental micro-somal cytochrome P-450 in aromatization, J. Biol. Chem. 249 (1974)5373e5378.

[20] D.D. Heidrich, S. Steckelbroeck, D. Klingmuller, Inhibition of human cyto-chrome P450 aromatase activity by butyltins, Steroids 66 (2001) 763e769.

[21] D.J. Zhou, D. Pompon, S.A. Chen, Stable expression of human aromatasecomplementary DNA in mammalian cells: a useful system for aromatase in-hibitor screening, Cancer Res. 50 (1990) 6949e6954.

[22] X.Z. Sun, D. Zhou, S. Chen, Autocrine and paracrine actions of breast tumoraromatase. A three-dimensional cell culture study involving aromatasetransfected MCF-7 and T-47D cells, J. Steroid Biochem. Mol. Biol. 63 (1997)29e36.

[23] Y. Berthois, J.A. Katzenellenbogen, B.S. Katzenellenbogen, Phenol red in tissueculture media is a weak estrogen: implications concerning the study ofestrogen-responsive cells in culture, Proc. Natl. Acad. Sci. U. S. A. 83 (1986)2496e2500.

[24] E.A. Thompson Jr., P.K. Siiteri, Utilization of oxygen and reduced nicotinamideadenine dinucleotide phosphate by human placental microsomes duringaromatization of androstenedione, J. Biol. Chem. 249 (1974) 5364e5372.

[25] C. Amaral, C. Varela, M. Azevedo, E.T. da Silva, F.M. Roleira, S. Chen, G. Correia-da-Silva, N. Teixeira, Effects of steroidal aromatase inhibitors on sensitive andresistant breast cancer cells: aromatase inhibition and autophagy, J. SteroidBiochem. Mol. Biol. 135 (2013) 51e59.

[26] M. Cepa, G. Correia-da-Silva, E.J. Tavares da Silva, F.M. Roleira, Y. Hong,S. Chen, N.A. Teixeira, Molecular mechanisms of aromatase inhibition by newA, D-ring modified steroids, Biol. Chem. 389 (2008) 1183e1191.

[27] H.O. House, R.S. Ro, The stereochemistry of base-catalyzed epoxidation, J. Am.Chem. Soc. 80 (1958) 2428e2433.

[28] A. Roebuck, H. Adkins, Trans-1,2-cyclohexanediol - 1,2-cyclohexanediol,Trans. Org. Synth. 28 (1948) 35e37.

[29] F. Buzzetti, A. Longo, E. Di Salle, Fluorinated 6-methylenandrosta-1,4-dien-3-one derivatives and process for their preparation, in WO 95/01366, 1995.

[30] J.R. Hanson, P.B. Hitchcock, M.D. Liman, S. Nagaratnam, Facial selectivity in thehydroboration of androst-4-enes, J. Chem. Soc. Perk. Trans. 1 (1995)2183e2187.

C.L. Varela et al. / European Journal of Medicinal Chemistry 87 (2014) 336e345 345

[31] T. Itoh, K. Karlsberg, I. Kijima, Y.C. Yuan, D. Smith, J. Ye, S. Chen, Letrozole-,anastrozole-, and tamoxifen-responsive genes in MCF-7aro cells: a microarrayapproach, Mol. Cancer Res. 3 (2005) 203e218.

[32] D. Giudici, G. Ornati, G. Briatico, F. Buzzetti, P. Lombardi, E. di Salle, 6-methylenandrosta-1,4-diene-3,17-dione (FCE 24304): a new irreversiblearomatase inhibitor, J. Steroid Biochem. 30 (1988) 391e394.

[33] C. Amaral, M. Borges, S. Melo, E.T. da Silva, G. Correia-da-Silva, N. Teixeira,Apoptosis and autophagy in breast cancer cells following exemestane treat-ment, PLoS One 7 (2012) e42398.

[34] M. Cepa, G. Correia-da-Silva, E.J. da Silva, F.M. Roleira, M. Borges, N.A. Teixeira,New steroidal aromatase inhibitors: suppression of estrogen-dependent

breast cancer cell proliferation and induction of cell death, BMC Cell. Biol. 9(2008) 41.

[35] C. Amaral, C. Varela, M. Borges, E. Tavares da Silva, F.M. Roleira, G. Correia-da-Silva, N. Teixeira, Steroidal aromatase inhibitors inhibit growth of hormone-dependent breast cancer cells by inducing cell cycle arrest and apoptosis,Apoptosis 18 (2013) 1426e1436.

[36] S. Masri, K. Lui, S. Phung, J. Ye, D. Zhou, X. Wang, S. Chen, Characterization ofthe weak estrogen receptor alpha agonistic activity of exemestane, BreastCancer Res. Treat. 116 (2009) 461e470.