TECHNICAL FIELD

[0001] The present disclosure relates to aromatase inhibitors (Als), and compositions comprising said compounds. In particular, the present disclosure relates to compounds or compositions to be used to ameliorate or treat a condition such as hormonedependent breast cancer or other conditions which are dependent on estrogens for their development, such as uterine cancer and ovarian cancer.

BACKGROUND

[0002] Aromatase is the enzyme responsible for the conversion of androgens into estrogens. The excessive estrogen production can have deleterious effects on the body, including the development or exacerbation of certain hormone-dependent diseases, in particular estrogen-dependent breast cancer, endometriosis, or polycystic ovary syndrome (PCOS).

[0003] Breast cancer, especially metastatic breast cancer, is one of the cancers with highest incidence rate responsible for high mortality and morbidity in women around the world ^[1] .

[0004] Approximately 80% of breast cancers are estrogen-dependent, which means that they need estrogens to develop. Thus, one of the therapeutic strategies to address this disease is the use of drugs that reduce the circulating estrogen levels or block estrogen signalling. These are the principles of endocrine therapy.

[0005] There are two types of drugs for endocrine therapy: the antiestrogens, which block estrogen receptors, preventing natural estrogens from binding and performing their function, and the aromatase inhibitors (Als), which block the conversion of androgens to estrogens by the aromatase enzyme. Als prove to be superior to antiestrogens in monotherapy, but several combinations of the two types of drugs are often used. Further, the combination of Als with cyclin-dependent kinase (CDK) inhibitors, namely CDK4/6 inhibitors, is a major treatment option for first-line therapy of advanced ER-positive/HER2-negative breast cancer in postmenopausal women. Also, the combination of the steroidal Al Exemestane with the mTOR (mammalian target of rapamycin) inhibitor Everolimus has been considered a second-line option to treat metastatic breast cancer ^[2] .

[0006] For all that has been mentioned, Als undoubtedly have a major role in the treatment of breast cancer, in all its stages. However, there are only three Als available for clinical use (Figure 1), the steroidal Exemestane and the non-steroidal Letrozole and anastrozole. The investigation on steroidal Als has been less explored, though steroidal Als are more selective, highly enzyme specific and less toxic due to its structural similarity with the aromatase substrate, androstenedione. In this field, 7|3- substitution in steroidal Als has been much less explored than 7a-substitution, and there are only few examples of 7 ^-substituted Als in the literature ^{[3; 4]} . However, they are not potent Als.

[0007] In any case, Als have several associated side effects, which cannot be overlooked, namely the progressive loss of bone density (osteoporosis) which increases the risk of bone fractures and bone pain, as well as cardiovascular disorders ^[5] . In addition to the side effects, another serious drawback associated with Als is the resistance developed by the tumor cells leading to cancer regrowth and progression ^[6] . For all these reasons, research to discover new Als to increase the therapeutic options for the management of hormone-dependent breast cancer, in particular estrogendependent breast cancer, as well as other diseases dependent on estrogens for their development such as uterine and ovarian cancers, is very relevant.

[0008] Roleira, F. M. F. et al ^[Vi disclosed steroidal Al and its production methods. The effect of the synthetized compounds on aromatase inhibition was tested for 7alpha- methylandrost-4-ene-3, 17-dione, the document being silent on the activity of the compound with Formula I on the aromatase activity.

[0009] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure. GENERAL DESCRIPTION

[0010] The present disclosure relates to the use of a compound of Formula I (7beta- methylandrost-4-ene-3, 17-dione) as a steroidal aromatase inhibitor.

(Formula I)

[0011] In an embodiment, the compound of Formula I is a strong steroidal Al, and it can be more selective, highly enzyme specific and less toxic relatively to the nonsteroidal Als due to its structural similarity with the aromatase substrate, androstenedione.

[0012] The present disclosure relates to a compound of Formula I, or a pharmaceutically acceptable salt, or ester or solvate thereof for use in medicine.

[0013] In an embodiment, the compound is for use in the treatment of any condition susceptible of being improved or prevented by the inhibition of aromatase. Examples of said conditions are hormone-dependent diseases, in particular hormone-dependent breast cancer and further in particular estrogen-dependent breast cancer, endometriosis, or polycystic ovary syndrome (PCOS).

[0014] In an embodiment, the compound is for use in the treatment of hormonedependent cancers.

[0015] In an embodiment, the compound is for use in the treatment of estrogendependent cancer.

[0016] In an embodiment, the compound is for use in the treatment of breast cancer, ovarian cancer, or endometrial cancer.

[0017] In an embodiment, the compound is for use in the treatment of hormonedependent breast cancer. [0018] An aspect the present disclosure relates to a pharmaceutical composition comprising the compound of the present disclosure and a pharmaceutically acceptable carrier, wherein the compound is in a therapeutically effective amount.

[0019] In an embodiment, the pharmaceutical composition is for use in the treatment of hormone-dependent cancers by oral administration or injectable administration.

[0020] In an embodiment, the pharmaceutical composition is administered in a daily dose to a person with a neoplasia condition susceptible of being improved or prevented by the inhibition of aromatase.

[0021] In an embodiment for better results, the dosage amount of the pharmaceutical composition of the present disclosure ranges from 0.5 to 30 mg/day, preferably 1 to 20 mg/day, even more preferably 2-10 mg/day.

[0022] The present disclosure also relates to the use of a compound of Formula I as an in vitro inhibitor of aromatase.

[0023] The present disclosure also relates to the use of a compound of Formula I for the manufacture of a medicament for the treatment of any condition susceptible of being improved or prevented by the inhibition of aromatase.

[0024] In an embodiment, the compound is used for the manufacture of a medicament for the treatment of a hormone-dependent cancer, in particular a hormone dependent breast cancer, further in particular an estrogen-dependent breast cancer.

[0025] The present disclosure also relates to a method for treating or preventing any condition susceptible of being improved or prevented by the inhibition of aromatase in a subject, the method comprising administering compound of Formula I to the subject.

[0026] In an embodiment, the method is for treating a hormone dependent cancer, in particular a hormone dependent breast cancer, further in particular an estrogendependent breast cancer. BRI EF DESCRI PTION OF THE DRAWI NGS

[0027] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0028] Figure 1: The aromatase inhibitors in clinical use (Exemestane, Letrozole and Anastrozole).

[0029] Figure 2: Schematic representation of the synthesis of compound of Formula I (Compound I, 7beta-methylandrost-4-ene-3, 17-dione) and its alpha-isomer, 7alpha- methylandrost-4-ene-3, 17-dione. Reagents and conditions: (i) Chloranil, tert-butanol, reflux; (ii) CuBr, MesAI, trimethylsilyl chloride (TMSCI), anhydrous tetra hydrofuran (THF), room temperature.

[0030] Figure 3: Embodiment of the anti-aromatase activity of the Compound I in MCF-7aro cells. The I Cso (pM) values were determined by the in-cell aromatase assay in MCF-7aro cells incubated with different concentrations (0.001-10 pM) of Als and 50 nM of [l|3- ³ H]-androstenedione. Data are presented as a percentage of the tritiated water control and correspond to three independent experiments carried out in triplicate.

[0031] Figure 4: Effects of Compound I on MCF-7aro cell viability. Cells were treated with different concentrations of Compound I (1- 25 pM), during 3 or 6 days. Cells without compound treatment were considered as control, representing 100% of cell viability. The results are presented as mean ± SEM of at least three independent experiments, performed in triplicate. Statistically significant differences between Compound l-treated cells and control cells are expressed as * (p < 0.05), ** (p < 0.01) and *** (p < 0.001).

[0032] Figure 5: Embodiment of the effects of Compound I on HFF-1 (A) and MCF10A (B) cell viability. Cells were treated with different concentrations of Compound I (1- 25 pM), for 6 days. Cells without compound treatment were considered as control, representing 100% of cell viability. The results are presented as mean ± SEM of at least three independent experiments, performed in triplicate. DETAILED DESCRIPTION

[0033] The present disclosure relates to a compound (7beta-methylandrost-4-ene- 3, 17-dione) for use in medicine, in particular for use in the treatment of any condition susceptible of being improved or prevented by the inhibition of aromatase, such as in the treatment of hormone dependent cancers. A pharmaceutical composition comprising said compound is also described.

[0034] In an embodiment, the compound of Formula I (Compound I) can be prepared as schematic represented in Figure 2 and as previously described ^[7] . Briefly, androstenedione is dehydrogenated with chloranil, in tert-butanol at reflux temperature giving androsta-4,6-diene-3, 17-dione which is than submitted to a 1,6- conjugate addition using trimethylaluminium, CuBr and trimethylsilyl chloride, in tetrahydrofuran, at room temperature. This reaction produced both C-7 methyl isomers 7alpha- and 7beta-methylandrost-4-ene-3, 17-dione, which are separated by column chromatography.

[0035] For the scope and interpretation of the present disclosure it is defined that "room temperature" should be regarded as a temperature between 15-30 °C, preferably between 18-25 °C, more preferably between 20-22 °C.

[0036] In an embodiment, when compared with its alpha-isomer (7alpha- methylandrost-4-ene-3, 17-dione), the compound of Formula I (Compound I) showed a higher aromatase inhibition capacity in human placental microsomes (ICso of 0.270 ± 0.026 pM for 7alpha-isomer vs. ICso of 0.0058 ± 0.00025 pM for 7beta-isomer). In a further embodiment, the compound of Formula I showed higher aromatase inhibition capacity, evaluated by the ICso value, not only than its alpha-isomer, but also than Anastrozole and Exemestane (two of the three aromatase inhibitors currently in clinical use), as well as, of Formestane (the first-generation Al used clinically for the treatment of hormone-dependent breast cancer which was withdrawn from the market in December 2014). These Als and Letrozole were used as controls. [0037] Table 1 - In vitro aromatase inhibition in human placental microsomes and in ER ⁺ breast cancer cells.

“Concentrations of 40 nM [ip- ³ H]-androstenedione, 20 pg protein from human placental microsomes, 150 pM NADPH, 2 pM of the compounds and 15 min incubation were used. ^Concentrations of 100 nM [ip- ³ H]-androstenedione, 20 pg protein from human placental microsomes, 150 pM NADPH, different concentrations (0.01 - 2 pM) of the compounds and 15 min incubation were used. ^c MCF-7aro cells incubated with 50 nM [ip- ³ H]-androstenedione and 10 pM of the compounds during 1 h of aromatase reaction. ^d IC ₅ o value according to ^[8] . ^e IC ₅ o value according to ^[9] . ⁷ IC _S o value according to ^[10] . ^s MCF-7aro cells incubated with 50 nM [ip- ³ H]-androstenedione and 0.001-10 pM of the compounds during 1 h of aromatase reaction. ^h IC ₅ o value according to ^[11] . 'IC ₅ o value according to ^[12] . Anastrozole, Letrozole, Formestane and Exemestane were used as reference Als. All the data are presented as a percentage of the tritiated water to control and are represented as the mean ± SEM of three independent experiments carried out in triplicate.

[0038] In an embodiment, the anti-aromatase activity and the IC50 value were evaluated in human placental microsomes by a radiometric method ^{113, 141} with some modifications ¹⁷¹ in which the tritiated water released during aromatization of [ip- ³ H]- androstenedione, was used as an index of estrogens formation. Briefly, to determine the percentage of aromatase inhibition and for the aromatization reaction it was used 20 pg of microsomes, 150 pM of NADPH, 40 nM of [ip- ³ H]-androstenedione (1 pCi) and 2 pM of Compound I. To evaluate the IC50 value it was used for the aromatization reaction 100 nM (1 pCi) of [ip- ³ H]-androstenedione and different concentrations of the Compound I (0.01 - 2 pM). The aromatase activity was determined as previously described ^[7] . [0039] In a further embodiment, the anti-aromatase activity and the IC50 value were evaluated by performing the same radiometric assay with some modifications ^{115 18]} and using an ER-positive (ER+) aromatase-overexpressing human breast cancer cell line (MCF-7aro), prepared by stable transfection of MCF-7 cells with the human placental aromatase gene and Geneticin selection, as previously described ¹¹⁹ ' ^20] . Briefly, confluent MCF-7aro cells were cultured in serum-free medium, containing the Compound I at different concentrations (0.001-10 pM), 50 nM of the substrate [1|3- ³ H]- androstenedione and 500 nM of progesterone. The aromatase activity was evaluated as previously described ^{[17, 18]} .

[0040] In addition, the in vitro viability effects in MCF-7aro cells were evaluated by MTT assay, as previously described ^{[16 -18]} . In this way, the anti-aromatase activity of Compound I was investigated in an ER ⁺ breast cancer cell model (MCF-7aro cells), which is a more complex model, and since it overexpresses aromatase, it mimics in vitro the tumor microenvironment. Indeed, it is considered the best in vitro cell model to study aromatase inhibitors (Als) and ER ⁺ breast cancer ^{[18; 20-23]} .

[0041] In an embodiment, the percentage of aromatase inhibition (%) for the compound with Formula I (Compound I) was measured by performing a radiometric assay in human placental microsomes using 2 pM of Compound I, and determined the IC ₅₀ value. As reference aromatase inhibitors (Als) it was used the steroidal Als Exemestane and Formestane and the non-steroidal Als Anastrozole and Letrozole, all of them in clinical use. For the Compound I it was obtained an anti-aromatase activity of 97.72 % ± 0.08 and an IC ₅₀ value of 0.0058 pM (Table 1). In addition, in MCF-7aro cells it was also obtained for the Compound I an anti-aromatase activity of 96.77 % ± 0.9 (Table 1), which was similar to the anti-aromatase activity induced by the Al Exemestane (97.42 % ± 0.31) and higher to the one induced by the Al Formestane (94.74% ± 0.92).

[0042] In another embodiment, the IC50 value of the Compound I in breast cancer cells were determined by a radiometric method with some modifications ^[15-18] in which the tritiated water released during aromatization of [l|3- ³ H]-androstenedione, was used as an index of estrogens formation, and by using the ER-positive (ER ⁺ ) aromatase- overexpressing human breast cancer cell line (MCF-7aro), prepared by stable transfection of MCF-7 cells with the human placental aromatase gene and Geneticin selection, as previously described ^{[19, 20]} .

[0043] In an embodiment, it was determined the ICso value of the Compound I in MCF- 7aro cells, and results demonstrate that this compound presents an ICso value of 0.012 pM (Figure 3, Table 1). As reference Als it was used the Als Exemestane (in clinical use) and Formestane (Table 1), which have ICso values almost 10 times higher thanthat of Compound I, data that highlights the potential of this compound.

[0044] In a further embodiment, the effects of the disclosed Compound I on the viability of an ER ⁺ aromatase-overexpressing human breast cancer cell line, MCF-7aro cells, was used to explore the anticancer potential of the Compound I. Said effects were analysed by performing MTT assays after treatment of MCF-7aro cells with different concentrations of Compound I (1- 25 pM), during 3 or 6 days. Cells without compound treatmentwere considered as control, representing 100% of cell viability. The results, presented in Figure 4, showed that Compound I was able to induce a significant (p < 0.05; p < 0.01; p < 0.001) reduction on MCF-7aro cell viability in a dose- and time-dependent manner, being its effects more pronounced after 6 days of treatment.

[0045] In an embodiment, the toxicity of Compound I was ascertained in vitro in two non-tumor cell models, the human foreskin fibroblast cell line (HFF-1) and the human normal breast epithelial cell line (MCF10A), by performing MTT assays, as previously described ^[16-18]

[0046] Figure 5 represents an embodiment of results of the cytotoxicity of the Compound I, on HFF-1 (A) and MCF10A (B) cell viability. Cells were treated with different concentrations of Compound I (1- 25 pM), for 6 days. Cells without compound treatment were considered as control, representing 100% of cell viability. The results are presented as mean ± SEM of at least three independent experiments, performed in triplicate. No significant toxicity was noticed for the tested concentrations in both cell lines, showing the selectivity of Compound I for cancer cells. [0047] In an embodiment, Compound I (7beta-methylandrost-4-ene-3, 17-dione), in addition to being a very potent aromatase inhibitor in microsomes, is also very potent in ER+ breast cancer cells (MCF-7aro), a more complex cell model that mimics the tumour microenvironment. Surprisingly, this compound presents an IC50 value that is almost 10 times lower than the reference steroidal Al used in clinic, Exemestane, and also significantly lower than its alpha-isomer (7alpha-methylandrost-4-ene-3, 17- dione). Thus, Compound I can be effectively used as a treatment of conditions that are susceptible of being improved or prevented by the inhibition of aromatase, such as hormone-dependent breast cancer, in particular estrogen-dependent breast cancer, endometriosis, or polycystic ovary syndrome. Furthermore, it is a safer compound that does not show toxicity in two non-tumoral cell lines.

[0048] In one embodiment, Compound I can be administered to a patient as a standalone therapy. It may be administered orally, intravenously, intratumorally, or via other suitable routes of administration.

[0049] In an embodiment, the daily form consists of a tablet, suppository, ampoule or other device, comprising a definitive amount of the compound of the present disclosure, the whole of which is intended to be administered as a single dose.

[0050] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0051] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above-described embodiments are combinable.

[0052] The following dependent claims further set out particular embodiments of the disclosure.

References

1. Sung, H., et al., Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, 2021. Brufsky, A.M., Long-term management of patients with hormone receptorpositive metastatic breast cancer: Concepts for seguential and combination endocrine-based therapies. Cancer Treat Rev, 2017. 59: p. 22-32. O'Reilly, J.M., et al., Synthesis, structure elucidation, and biochemical evaluation of 7 alpha- and 7 beta-arylaliphatic-substituted androst-4-ene-3, 17-diones as inhibitors of aromatase. J Med Chem, 1995. 38(15): p. 2842-50. Njar, V.C.O., R.W. Hartman, and R.C. H., Synthesis of 6a, 7a- And 66,76- Aziridinoandrost-4-Ene- 3,17-Diones and Related Compounds: Potential Aromatase Inhibitors. J. Chem. Soc., Perkin Trans. 1, 1995. 8: p. 985-991. Zhao, F., et al., Toxicity of extended adjuvant endocrine with aromatase inhibitors in patients with postmenopausal breast cancer: A Systemtic review and Meta-analysis. Crit Rev Oncol Hematol, 2020. 156: p. 103114. Augusto, T., et al., Acguired-resistance to aromatase inhibitors: where we stand! Endocr Relat Cancer, 2018. Roleira, F.M.F., et al., C-6alpha- vs C-7alpha-Substituted Steroidal Aromatase Inhibitors: Which Is Better? Synthesis, Biochemical Evaluation, Docking Studies, and Structure-Activity Relationships. J Med Chem, 2019. 62(7): p. 3636-3657. Augusto, T.V., et al., A novel GC-MS methodology to evaluate aromatase activity in human placental microsomes: a comparative study with the standard radiometric assay. Anal Bioanal Chem, 2019. 411(26): p. 7005-7013. Varela, C., et al., New structure-activity relationships of A- and D-ring modified steroidal aromatase inhibitors: design, synthesis, and biochemical evaluation. J Med Chem, 2012. 55(8): p. 3992-4002. Varela, C.L., et al., Exemestane metabolites: Synthesis, stereochemical elucidation, biochemical activity and anti-proliferative effects in a hormonedependent breast cancer cell line. EurJ Med Chem, 2014. 87: p. 336-45. Cepa, M., et al., Molecular mechanisms of aromatase inhibition by new A, D- ring modified steroids. Biol Chem, 2008. 389(9): p. 1183-91. Amaral, C., et al., Effects of steroidal aromatase inhibitors on sensitive and resistant breast cancer cells: aromatase inhibition and autophagy. J Steroid Biochem Mol Biol, 2013. 135: p. 51-9. Heidrich, D.D., S. Steckelbroeck, and D. Klingmuller, Inhibition of human cytochrome P450 aromatase activity by butyltins. Steroids, 2001. 66(10): p. 763-9. Thompson, E.A., Jr. and P.K. Siiteri, The involvement of human placental microsomal cytochrome P-450 in aromatization. J Biol Chem, 1974. 249(17): p. 5373-8. Varela, C.L., et al., Exemestane metabolites: Synthesis, stereochemical elucidation, biochemical activity and anti-proliferative effects in a hormonedependent breast cancer cell line. EurJ Med Chem, 2014. 87C: p. 336-345. Augusto, T.V., et al., Effects of new C6-substituted steroidal aromatase inhibitors in hormone-sensitive breast cancer cells: Cell death mechanisms and modulation of estrogen and androgen receptors. J Steroid Biochem Mol Biol, 2019. 195: p. 105486. Amaral, C., et al., Anti-tumor efficacy of new 7alpha-substituted androstanes as aromatase inhibitors in hormone-sensitive and resistant breast cancer cells. J Steroid Biochem Mol Biol, 2017. Amaral, C., et al., An Exemestane Derivative, Oxymestane-Dl, as a New MultiTarget Steroidal Aromatase Inhibitor for Estrogen Receptor-Positive (ER(+)) Breast Cancer: Effects on Sensitive and Resistant Cell Lines. Molecules, 2023. 28(2). Sun, X.Z., D. Zhou, and S. Chen, Autocrine and paracrine actions of breast tumor aromatase. A three-dimensional cell culture study involving aromatase transfected MCF-7 and T-47D cells. J Steroid Biochem Mol Biol, 1997. 63(1-3): p. 29-36. Zhou, D.J., D. Pompon, and S.A. Chen, Stable expression of human aromatase complementary DNA in mammalian cells: a useful system for aromatase inhibitor screening. Cancer Res, 1990. 50(21): p. 6949-54. Augusto, T.V., et al., Differential biological effects of aromatase inhibitors: Apoptosis, autophagy, senescence and modulation of the hormonal status in breast cancer cells. Mol Cell Endocrinol, 2021. 537: p. 111426. Amaral, C., et al., Apoptosis and autophagy in breast cancer cells following exemestane treatment. PLoS One, 2012. 7(8): p. e42398. Amaral, C., et al., The potential clinical benefit of targeting androgen receptor (AR) in estrogen-receptor positive breast cancer cells treated with Exemestane. Biochim Biophys Acta Mol Basis Dis, 2020. 1866(5): p. 165661.