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In this work, new steroidal aromatase inhibitors (AIs) were designed, synthesized, and tested. In one approach, C-ring substituted steroids namely those functionalized at C-11 position with an α or β hydroxyl group or with a carbonyl group as well as C-9/C-11 steroidal olefins and epoxides were studied. It was found that the carbonyl group at C-11 is more beneficial for aromatase inhibition than the hydroxyl group, and that the C-ring epoxides were more potent than the C-ring olefins, leading to the discovery of a very strong AI, compound 7, with an IC 50 of 0.011 μM, better than Exemestane, the steroidal AI in clinical use, which presents an IC 50 of 0.050 μM. In another approach, we explored the biological activity of A-ring C-1/C-2 steroidal olefins and epoxides in relation to aromatase inhibition and compared it with the biological activity of C-ring C-9/C-11 steroidal olefins and epoxides. On the contrary to what was observed for the C-ring olefins and epoxides, the A-ring epoxides were less potent than A-ring olefins. Finally, the effect of 7β-methyl substitution on aromatase inhibition was compared with 7α-methyl substitution, showing that 7β-methyl is better than 7α-methyl substitution. Molecular modelling studies showed that the 7β-methyl on C-7 seems to protrude into the opening to the access channel of aromatase in comparison to the 7α-methyl. This comparison led to find the best steroidal AI (12a) of this work with IC 50 of 0.0058 μM. Compound 12a showed higher aromatase inhibition capacity than two of the three AIs currently in clinical use., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Estrogen receptor-positive (ER + ) breast cancers require estrogens for their growth. Aromatase inhibitors (AIs) are considered the first-line therapy for this type of tumours. Despite the well-established clinical benefit of this therapy, the search for novel potent AIs that present higher efficacy and fewer side effects is still demanded. Thus, taking into account the known interactions of the natural substrate, androstenedione, within the aromatase active-site, a range of new steroidal compounds have been designed, synthesized and studied by our group. In this work, it was evaluated in MCF-7aro, an ER + breast cancer cell line that overexpress aromatase, the anti-aromatase efficacy and the biological effects of eight new AIs: 6α-methyl-5α-androst-3-en-17-one (1a), 6α-methyl-3α,4α-epoxy-5α-androstan-17-one (3a), 6α-methylandrost-4-ene-3,17-dione (9), 6α-allylandrosta-1,4-diene-3,17-dione (13), 6α-allylandrost-4-ene-3,17-dione (15), 6α-allylandrost-4-en-17-one (17), 6β-hydroxyandrost-4-ene-3,17-dione (19) and 6α-hydroxyandrost-4-ene-3,17-dione (20). Their anti-cancer properties were elucidated, as well as, the dependence of their mechanism of action on aromatase inhibition and/or on steroid receptors modulation, such as estrogen and androgen receptors, which are key targets for this type of cancer. Results demonstrate that the studied AIs present high anti-aromatase activity, disrupt MCF-7aro cell cycle progression and induce apoptosis, through the mitochondrial pathway. Compounds 1a, 3a, 9, 13, 15 and 17 exhibited an aromatase-dependent effect on cells and, interestingly, steroids 9 and 13 displayed the ability to decrease aromatase protein levels without affecting CYP19A1 mRNA levels. Furthermore, the effects of compounds 1a, 3a and 15 were dependent on ER and on AR modulation, whereas compounds 9 and 19 were only dependent on AR modulation. From a clinical point of view, these actions can be considered as a therapeutic advantage for this type of tumours. Thus, new promising AIs that impair ER + breast cancer cell growth, by acting on aromatase, and even, on ER and AR were discovered. Furthermore, new insights on the most favourable structural modifications in the steroidal core structure were provided, helping to a more rational drug design of new and potent AIs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

C-6α and C-7α androstanes were studied to disclose which position among them is more convenient to functionalize to reach superior aromatase inhibition. In the first series, the study of C-6 versus C-7 methyl derivatives led to the very active compound 9 with IC 50 of 0.06 μM and K i = 0.025 μM (competitive inhibition). In the second series, the study of C-6 versus C-7 allyl derivatives led to the best aromatase inhibitor 13 of this work with IC 50 of 0.055 μM and K i = 0.0225 μM (irreversible inhibition). Beyond these findings, it was concluded that position C-6α is better to functionalize than C-7α, except when there is a C-4 substituent simultaneously. In addition, the methyl group was the best substituent, followed by the allyl group and next by the hydroxyl group. To rationalize the structure-activity relationship of the best inhibitor 13, molecular modeling studies were carried out.

The majority of breast cancer cases are estrogen receptor positive (ER + ). Although, third-generation aromatase inhibitors (AIs) are used as first-line treatment in post-menopausal women, they cause endocrine resistance and bone loss, which limits their success. Therefore, there is a demand to discover new potent molecules, with less toxicity that can circumvent these drawbacks. Our group has previously demonstrated that new 7α-substituted steroidal molecules, 7α-(2ξ,3ξ-epoxypropyl)androsta-1,4-diene-3,17-dione (3), 7α-allylandrost-4-ene-3,17-dione (6), 7α-allylandrost-4-en-17-one (9), 7α-allyl-3-oxoandrosta-1,4-dien-17β-ol (10) and 7α-allylandrosta-1,4-diene-3,17-dione (12) are potent AIs in placental microsomes. In this work, it was investigated their anti-aromatase activity and in vitro effects in sensitive and resistant breast cancer cells. All the steroids efficiently inhibit aromatase in breast cancer cells, allowing to establish new structure-activity relationships for this class of compounds. Moreover, the new AIs can inhibit breast cancer cell growth, by causing cell cycle arrest and apoptosis. The effects of AIs 3 and 12 on sensitive cells were dependent on aromatase inhibition and androgen receptor (AR), while for AI 9 and AI 10 were AR- and ER-dependent, respectively. In addition, it was shown that all the AIs can sensitize resistant cancer cells being their behavior similar to the sensitive cells. In summary, this study contributes to the understanding of the structural modifications in steroidal scaffold that are translated into better aromatase inhibition and anti-tumor properties, providing important information for the rational design/synthesis of more effective AIs. In addition, allowed the discovery of new potent 7α-substituted androstane molecules to inhibit tumor growth and prevent endocrine resistance., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In this work, new potent steroidal aromatase inhibitors both in microsomes and in breast cancer cells have been found. The synthesis of the 3,4-(ethylenedioxy)androsta-3,5-dien-17-one (12), a new steroid containing a heterocycle dioxene fused in the A-ring, led to the discovery of a new reaction for which a mechanism is proposed. New structure-activity relationships were established. Some 5β-steroids, such as compound 4β,5β-epoxyandrostan-17-one (9), showed aromatase inhibitory activity, because they adopt a similar A-ring conformation as those of androstenedione, the natural substrate of aromatase. Moreover, new chemical features to increase planarity were disclosed, specifically the 3α,4α-cyclopropane ring, as in 3α,4α-methylen-5α-androstan-17-one (5) (IC50=0.11μM), and the Δ(9-11) double bond in the C-ring, as in androsta-4,9(11)-diene-3,17-dione (13) (IC50=0.25μM). In addition, induced-fit docking (IFD) simulations and site of metabolism (SoM) predictions helped to explain the recognition of new potent steroidal aromatase inhibitors within the enzyme. These insights can be valuable tools for the understanding of the molecular recognition process by the aromatase and for the future design of new steroidal inhibitors., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Different hormonal therapies are used for estrogen receptor positive (ER(+)) breast cancers, being the third-generation of aromatase inhibitors (AIs), an effective alternative to the classical tamoxifen. AIs inhibit the enzyme aromatase, which is responsible for catalyzing the conversion of androgens to estrogens. In this study, it was evaluated the effects of several steroidal AIs, namely 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), on cell proliferation, cell cycle progression and cell death in an ER(+) aromatase-overexpressing human breast cancer cell line (MCF-7aro). All AIs induced a decrease in cell proliferation and these anti-proliferative effects were due to a disruption in cell cycle progression and cell death, by apoptosis. AIs 1 and 16 caused cell cycle arrest in G0/G1, while AIs 12 and 13a induced an arrest in G2/M. Moreover, it was observed that these AIs induced apoptosis by different pathways, since AIs 1, 12 and 13a activated the apoptotic mitochondrial pathway, while AI 16 induced apoptosis through activation of caspase-8. These results are important for the elucidation of the cellular effects of steroidal AIs on breast cancer cells and will also highlight the importance of AIs as inducers of apoptosis in hormone-dependent breast cancers.

Two series of derivatives of 7α-allylandrostenedione, namely its 3-deoxo and 1-ene analogs, were designed and synthesised and their biochemical activity towards aromatase evaluated. In each of these series, the C-17 carbonyl group was further replaced by the hydroxyl and acetoxyl groups. The attained data pointed out that the absence of the C-3 carbonyl group led to a slightly decrease in the inhibitory activity and the introduction of an additional double bond in C-1 revealed to be a very beneficial structural change in the studied compounds (compound 12, IC₅₀ = 0.47 μM, K(i) = 45.00 nM). Furthermore, the relevance of the C-17 carbonyl group in the D-ring as a structural feature required to achieve maximum aromatase inhibitory activity is also observed for this set of derivatives., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.

Simple Summary: The occurrence of resistance to the standard treatment for estrogen receptor-positive (ER+) (Luminal A) breast cancer emphasizes the search for new molecules. In recent years, the interest in multi-target drugs has been growing, and they have even been identified as future therapeutic strategies. In line with this, this study aims to understand the anti-cancer action and multi-target potential of three new steroidal aromatase inhibitors (AIs): 7α-methylandrost-4-en-17-one (6), 7α-methylandrost-4-ene-3,17-dione (10a) and androsta-4,9(11)-diene-3,17-dione (13). We report new compounds that are able to impair the growth of sensitive and resistant ER+ breast cancer cells by acting as AIs, ER modulators and AR agonists, which is a therapeutic advantage over the current drugs applied in clinic. Therefore, this study is a breakthrough on drug discovery as it allows the discovery of new multi-target drugs for the treatment of ER+ breast cancer. Background/Objectives: Endocrine therapies that comprise anti-estrogens and aromatase inhibitors (AIs) are the standard treatment for estrogen receptor-positive (ER+) (Luminal A) breast cancer—the most prevalent subtype. However, the emergence of resistance restricts their success by causing tumor relapse and re-growth, which demands a switch towards other therapeutic approaches in order to minimize or overcome resistance. Indeed, this clinical limitation highlights the search for new molecules to improve cancer treatment. Recently, strategies that address multiple targets have been emerging, and multi-target drugs have the potential to become the future anti-cancer molecules. Our group has been searching for new multi-target compounds, and as part of this, our study aims to understand the anti-cancer and multi-target potential of three new steroidal aromatase inhibitors (AIs): 7α-methylandrost-4-en-17-one (6), 7α-methylandrost-4-ene-3,17-dione (10a) and androsta-4,9(11)-diene-3,17-dione (13). Methods: Their in vitro actions and molecular mechanisms were elucidated in a sensitive ER+ aromatase-overexpressing breast cancer cell line, MCF-7aro cells, as well as in an AI-resistant ER+ breast cancer cell line, LTEDaro cells. Results: All the new AIs (10 µM) prevented the proliferation of MCF-7aro cells by arresting cell cycle progression. Interestingly, all AIs (10 µM) act as androgen receptor (AR) agonists and modulate ER levels, synthesis and signaling to induce the apoptosis of ER+ breast cancer cells. Additionally, these new AIs (10 µM) also re-sensitize resistant cells by promoting apoptosis, offering a therapeutic benefit. Conclusions: Overall, new steroidal polypharmacological compounds have been discovered that, by acting as AIs, ER modulators and AR agonists, impair ER+ breast cancer cell growth. Overall, this study is a breakthrough on drug discovery as it presents new molecules with appealing anti-cancer properties and multi-target action for the treatment of ER+ breast cancer. [ABSTRACT FROM AUTHOR]

Around 70–85% of all breast cancer (BC) cases are estrogen receptor-positive (ER+). The third generation of aromatase inhibitors (AIs) is the first-line treatment option for these tumors. Despite their therapeutic success, they induce several side effects and resistance, which limits their efficacy. Thus, it is crucial to search for novel, safe and more effective anti-cancer molecules. Currently, multi-target drugs are emerging, as they present higher efficacy and lower toxicity in comparison to standard options. Considering this, this work aimed to investigate the anti-cancer properties and the multi-target potential of the compound 1α,2α-epoxy-6-methylenandrost-4-ene-3,17-dione (Oxy), also designated by Oxymestane-D1, a derivative of Exemestane, which we previously synthesized and demonstrated to be a potent AI. For this purpose, it was studied for its effects on the ER+ BC cell line that overexpresses aromatase, MCF-7aro cells, as well as on the AIs-resistant BC cell line, LTEDaro cells. Oxy reduces cell viability, impairs DNA synthesis and induces apoptosis in MCF-7aro cells. Moreover, its growth-inhibitory properties are inhibited in the presence of ERα, ERβ and AR antagonists, suggesting a mechanism of action dependent on these receptors. In fact, Oxy decreased ERα expression and activation and induced AR overexpression with a pro-death effect. Complementary transactivation assays demonstrated that Oxy presents ER antagonist and AR agonist activities. In addition, Oxy also decreased the viability and caused apoptosis of LTEDaro cells. Therefore, this work highlights the discovery of a new and promising multi-target drug that, besides acting as an AI, appears to also act as an ERα antagonist and AR agonist. Thus, the multi-target action of Oxy may be a therapeutic advantage over the three AIs applied in clinic. Furthermore, this new multi-target compound has the ability to sensitize the AI-resistant BC cells, which represents another advantage over the endocrine therapy used in the clinic, since resistance is a major drawback in the clinic. [ABSTRACT FROM AUTHOR]

The classical synthesis, followed by purification of the steroidal A-ring Δ1-olefin, 5 α-androst-1-en-17-one ( 5), from the Δ1-3-keto enone, (5 α,17 β)-3-oxo-5-androst-1-en-17-yl acetate ( 1), through a strategy involving the reaction of Δ1-3-hydroxy allylic alcohol, 3 β-hydroxy-5 α-androst-1-en-17 β-yl acetate ( 2), with SOCl2, was revisited in order to prepare and biologically evaluate 5 as aromatase inhibitor for breast cancer treatment. Surprisingly, the followed strategy also afforded the isomeric Δ2-olefin 6 as a by-product, which could only be detected on the basis of NMR analysis. Optimization of the purification and detection procedures allowed us to reach 96% purity required for biological assays of compound 5. The same synthetic strategy was applied, using the Δ4-3-keto enone, 3-oxoandrost-4-en-17 β-yl acetate ( 8), as starting material, to prepare the potent aromatase inhibitor Δ4-olefin, androst-4-en-17-one ( 15). Unexpectedly, a different aromatase inhibitor, the Δ3,5-diene, androst-3,5-dien-17-one ( 12), was formed. To overcome this drawback, another strategy was developed for the preparation of 15 from 8. The data now presented show the unequal reactivity of the two steroidal A-ring Δ1- and Δ4-3-hydroxy allylic alcohol intermediates, 3 β-hydroxy-5 α-androst-1-en-17 β-yl acetate ( 2) and 3 β-hydroxyandrost-4-en-17 β-yl acetate ( 9), towards SOCl2, and provides a new strategy for the preparation of the aromatase inhibitor 12. Additionally, a new pathway to prepare compound 15 was achieved, which avoids the formation of undesirable by-products. [ABSTRACT FROM AUTHOR]

Around 60–80% of all breast tumors are estrogen receptor-positive. One of the several therapeutic approaches used for this type of cancers is the use of aromatase inhibitors. Exemestane is a third-generation steroidal aromatase inhibitor that undergoes a complex and extensive metabolism, being catalytically converted into chemically active metabolites. Recently, our group showed that the major exemestane metabolites, 17β-hydroxy-6-methylenandrosta-1,4-dien-3-one and 6-(hydroxymethyl)androsta-1,4,6-triene-3,17-dione, as well as, the intermediary metabolite 6β-Spirooxiranandrosta-1,4-diene-3,17-dione, are potent aromatase inhibitors in breast cancer cells. In this work, in order to better understand the biological mechanisms of exemestane in breast cancer and the effectiveness of its metabolites, it was investigated their effects in sensitive and acquired-resistant estrogen receptor-positive breast cancer cells. Our results indicate that metabolites induced, in sensitive breast cancer cells, cell cycle arrest and apoptosis via mitochondrial pathway, involving caspase-8 activation. Moreover, metabolites also induced autophagy as a promoter mechanism of apoptosis. In addition, it was demonstrated that metabolites can sensitize aromatase inhibitors-resistant cancer cells, by inducing apoptosis. Therefore, this study indicates that exemestane after metabolization originates active metabolites that suppress the growth of sensitive and resistant breast cancer cells. It was also concluded that, in both cell lines, the biological effects of metabolites are different from the ones of exemestane, which suggests that exemestane efficacy in breast cancer treatment may also be dependent on its metabolites. [ABSTRACT FROM AUTHOR]