Elisiário J. Tavares da Silva

Lipophilic phenolic antioxidants: Correlation between antioxidant profile, partition coefficients and redox properties

Lipophilic compounds structurally based on caffeic, hydrocaffeic, ferulic and hydroferulic acids were synthesized. Subsequently, their antioxidant activity was evaluated as well as their partition coefficients and redox potentials. The structure-property-activity relationship (SPAR) results revealed the existence of a clear correlation between the redox potentials and the antioxidant activity. In addition, some compounds showed a proper lipophilicity to cross the blood-brain barrier. Their predicted ADME properties are also in accordance with the general requirements for potential CNS drugs. Accordingly, one can propose these phenolic compounds as potential antioxidants for tackling the oxidative status linked to the neurodegenerative processes.

New structure-activity relationships of A- and D-ring modified steroidal aromatase inhibitors: design, synthesis, and biochemical evaluation.

A- and D-ring androstenedione derivatives were synthesized and tested for their abilities to inhibit aromatase. In one series, C-3 hydroxyl derivatives were studied leading to a very active compound, when the C-3 hydroxyl group assumes 3β stereochemistry (1, IC(50) = 0.18 μM). In a second series, the influence of double bonds or epoxide functions in different positions along the A-ring was studied. Among epoxides, the 3,4-epoxide 15 showed the best activity (IC(50) = 0.145 μM) revealing the possibility of the 3,4-oxiran oxygen resembling the C-3 carbonyl group of androstenedione. Among olefins, the 4,5-olefin 12 (IC(50) = 0.135 μM) revealed the best activity, pointing out the importance of planarity in the A,B-ring junction near C-5. C-4 acetoxy and acetylsalicyloxy derivatives were also studied showing that bulky substituents in C-4 diminish the activity. In addition, IFD simulations helped to explain the recognition of the C-3 hydroxyl derivatives (1 and 2) as well as 15 within the enzyme.

Apoptosis and Autophagy in Breast Cancer Cells following Exemestane Treatment

Aromatase inhibitors (AIs), which block the conversion of androgens to estrogens, are used for hormone-dependent breast cancer treatment. Exemestane, a steroidal that belongs to the third-generation of AIs, is a mechanism-based inhibitor that binds covalently and irreversibly, inactivating and destabilizing aromatase. Since the biological effects of exemestane in breast cancer cells are not totally understood, its effects on cell viability, cell proliferation and mechanisms of cell death were studied in an ER-positive aromatase-overexpressing breast cancer cell line (MCF-7aro). The effects of 3-methyladenine (3-MA), an inhibitor of autophagy and of ZVAD-FMK, an apoptotic inhibitor, in exemestane treated cells were also investigated. Our results indicate that exemestane induces a strong inhibition in MCF-7aro cell proliferation in a dose- and time-dependent manner, promoting a significant cell cycle arrest in G0/G1 or in G2/M phases after 3 and 6 days of treatment, respectively. This was accompanied by a decrease in cell viability due to activation of cell death by apoptosis, via mitochondrial pathway and the occurrence of autophagy. Inhibition of autophagy by the autophagic inhibitor, 3-MA, resulted in a reduction of cell viability and activation of caspases. All together the results obtained suggest that exemestane induced mitochondrial-mediated apoptosis and autophagy, which act as a pro-survival process regulating breast cancer cell apoptosis.

New steroidal aromatase inhibitors: suppression of estrogen-dependent breast cancer cell proliferation and induction of cell death.

BackgroundAromatase, the cytochrome P-450 enzyme (CYP19) responsible for estrogen biosynthesis, is an important target for the treatment of estrogen-dependent breast cancer. In fact, the use of synthetic aromatase inhibitors (AI), which induce suppression of estrogen synthesis, has shown to be an effective alternative to the classical tamoxifen for the treatment of postmenopausal patients with ER-positive breast cancer. New AIs obtained, in our laboratory, by modification of the A and D-rings of the natural substrate of aromatase, compounds 3a and 4a, showed previously to efficiently suppress aromatase activity in placental microsomes. In the present study we have investigated the effects of these compounds on cell proliferation, cell cycle progression and induction of cell death using the estrogen-dependent human breast cancer cell line stably transfected with the aromatase gene, MCF-7 aro cells.ResultsThe new steroids inhibit hormone-dependent proliferation of MCF-7aro cells in a time and dose-dependent manner, causing cell cycle arrest in G0/G1 phase and inducing cell death with features of apoptosis and autophagic cell death.ConclusionOur in vitro studies showed that the two steroidal AIs, 3a and 4a, are potent inhibitors of breast cancer cell proliferation. Moreover, it was also shown that the antiproliferative effects of these two steroids on MCF-7aro cells are mediated by disrupting cell cycle progression, through cell cycle arrest in G0/G1 phase and induction of cell death, being the dominant mechanism autophagic cell death. Our results are important for the elucidation of the cellular effects of steroidal AIs on breast cancer.

IMPROVED SYNTHESES OF AROMATASE INHIBITORS AND NEUROACTIVE STEROIDS EFFICIENT OXIDATIONS AND REDUCTIONS AT KEY POSITIONS FOR BIOACTIVITY

An Henbest reduction, followed by the preparation of a silyl enol ether and oxidation in situ with m-CPBA has led to the neurosteroids 3α-hydroxy- and 3α,21-dihydroxy-5α-pregnanolones. Using testosterone as starting material, a new short synthesis of an aromatase inhibitor, 4-OHA, has been achieved through hydroboration/oxidation followed by a Swern type oxidation and epimerization. Another aromatase inhibitor, androst-4-ene-3,6-17-trione, has been efficiently prepared using PCC on montmorillonite K10, under ultrasonic irradiation.

Synthesis and biochemical studies of 17-substituted androst-3-enes and 3,4-epoxyandrostanes as aromatase inhibitors

A series of 5alpha-androst-3-enes and 3alpha,4alpha-epoxy-5alpha-androstanes were synthesized and tested for their abilities to inhibit aromatase in human placental microsomes. In these series the original C-17 carbonyl group was replaced by hydroxyl, acetyl and hydroxyimine groups. Inhibition kinetic analysis on the most potent steroid of these series revealed that it inhibits the enzyme in a competitive manner (IC(50)=6.5 microM). The achieved data pointed out the importance of the C-17 carbonyl group in the D-ring of the studied steroids as a structural feature required to reach maximum aromatase inhibitory activity. Further, at least one carbonyl group (C-3 or C-17) seems to be essential to effective aromatase inhibition.

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

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 undergoes a complex metabolization, giving rise to some already identified metabolites, the 17β-hydroxy-6-methylenandrosta-1,4-dien-3-one (17-βHE) 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 synthesized (6β-spirooxiranandrosta-1,4-diene-3,17-dione (2), 1α,2α-epoxy-6-methylenandrost-4-ene-3,17-dione (3) and 17-βHE (4)) while one was acquired, the 6-HME (6). The stereochemistry of the epoxide group of 2 and 3 has been unequivocally elucidated for the first time on the basis of NOESY experiments. New structure-activity relationships (SAR) have been established through the observation that the substitution 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-βHE (4) resulted in a significant increase in activity in MCF-7aro cells when compared to exemestane (IC50 0.25 μM vs 0.90 μM, 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 not only exemestane but also its main metabolites are potent aromatase inhibitors and reduce breast cancer cells viability. This suggests that exemestane efficacy may also be due to the active metabolites that result from its metabolic transformation. Our results emphasize the importance of performing further studies to expand our understanding of exemestane actions in breast cancer cells.

Effects of steroidal aromatase inhibitors on sensitive and resistant breast cancer cells: Aromatase inhibition and autophagy

Several therapeutic approaches are used in estrogen receptor positive (ER(+)) breast cancers, being one of them the use of aromatase inhibitors (AIs). Although AIs demonstrate higher efficacy than tamoxifen, they can also exhibit de novo or acquired resistance after prolonged treatment. Recently, we have described the synthesis and biochemical evaluation of four steroidal AIs, 3β-hydroxyandrost-4-en-17-one (1), androst-4-en-17-one (12), 4α,5α-epoxyandrostan-17-one (13a) and 5α-androst-2-en-17-one (16), obtained from modifications in the A-ring of the aromatase substrate, androstenedione. In this study, it was investigated the biological effects of these AIs in different breast cancer cell lines, an ER(+) aromatase-overexpressing human breast cancer cell line (MCF-7aro cells), an estrogen-receptor negative (ER(-)) human breast cancer cell line (SK-BR-3 cells), and a late stage of acquired resistance cell line (LTEDaro cells). The effects of an autophagic inhibitor (3-methyladenine) plus AIs 1, 12, 13a or exemestane in LTEDaro cells were also studied to understand the involvement of autophagy in AI acquired resistance. Our results showed that these steroids inhibit aromatase of MCF-7aro cells and decrease cell viability in a dose- and time-dependent manner. The new AI 1 is the most potent inhibitor, although the AI 12 demonstrates to be the most effective in decreasing cell viability. Besides, and in advantage over exemestane, AIs 12 and 13a also reduced LTEDaro cells viability. The use of the autophagic inhibitor allowed AIs to diminish viability of LTEDaro cells, presenting a similar behavior to the sensitive cells. Thus, inhibition of autophagy may sensitize hormone-resistant cancer cells to anti-estrogen therapies.

Development of a new gas chromatography–mass spectrometry (GC–MS) methodology for the evaluation of 5α-reductase activity

The androgens testosterone (T) and dihydrotestosterone (DHT) play a key role in the function and integrity of prostate tissue, but are also implicated in prostate cancer and benign prostatic hyperplasia (BPH). The reduction of androgen levels can be achieved by the inhibition of 3-oxo-5α-steroid-4-dehydrogenase (5α-reductase), which is responsible for the irreversible conversion of T into its more active metabolite DHT. In fact, the use of 5α-reductase inhibitors (RIs), like finasteride, can be a valuable strategy for the treatment of BPH and in chemoprevention of prostate tumors. In this work a new method based on a dispersive liquid-liquid microextraction (DLLME) procedure, followed by gas chromatography-mass spectrometry (GC-MS), to evaluate the 5α-reductase activity, by measuring the conversion percentage of T into DHT was optimized and validated. Enzymatic assays were carried out in human prostate microsomes, using T as substrate. T and DHT were extracted by the developed DLLME technique and quantified, after silylation, by GC-MS. Variables affecting the extraction efficiency and derivatization of T and DHT were evaluated. The optimized method showed good linearity (with correlation coefficients over 0.9994 for T and 0.9995 for DHT), good recoveries (higher than 80%), and good intra- and inter-day precision (below 13%, 3 levels, n=6). The detection limits for T and DHT were 0.5 nM and the limits of quantification were 5 nM. The new GC-MS method is a good alternative to the already described methods, to evaluate 5α-reductase activity, since it avoids the use of radioactive compounds and corresponds to a fast and sensitive methodology with a good extraction efficiency, accuracy and high recovery. As this method allows the evaluation of 5α-reductase activity, also permits the study of inhibitory efficacy of new molecules as potential RIs.

X-ray and deuterium labeling studies on the abnormal ring cleavages of a 5β-epoxide precursor of formestane

A new convergent synthesis of the antitumor steroid formestane (4-OHA) 5 has been performed from the easily available epimeric mixture of 5 alpha- and 5 beta-androst-3-en-17-one 1a and 1b in order to attempt a yield improvement. A two-step oxidative route followed by base-catalyzed isomerization was applied to the 5 alpha- and 5 beta-epimers 1a and 1b, either as a mixture or separately, leading to the title compound 5. From epimer 1a an efficient process was attained to prepare the desired aromatase inhibitor formestane. Epimer 1b led to the formation of the same compound 5. Additionally, 1b have also been converted in 5 beta-hydroxyandrostane-3,17-dione 12 and androst-4-ene-3,17-dione 13, revealing an unexpected reactivity of the 3 beta,4 beta-epoxy-5 beta-androstan-17-one intermediate 6 formed from 1b during the first oxidative step with performic acid. Cleavage of the epoxide 6 led to the trans-diaxial and the trans-diequatorial vic-diols 7 and 8 and to the 1,3-diol 9. The formation of the abnormal products 8 and 9 were investigated through X-ray and deuterium labeling studies. Diol 8 was formed through a trans-diequatorial epoxide ring opening and the 1,3-diol 9 was formed through an intramolecular rearrangement involving a 1,2-hydride shift. All the vic-diols 3, 7 and 8 formed, proved to be good precursors for the synthesis of the target compound 5.