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- Development of novel 4-azasteroid derivatives as potential 5a-reductase inhibitors Synthesis, biological and computational evaluationPublication . Brito, Vanessa Sofia Figueiredo de; Silvestre, Samuel MartinsThe enzyme steroid 5a-reductase (5AR) is responsible for the NADPH-dependent reduction of testosterone to the androgen 5a-dihydrotestosterone (DHT). The abnormally high 5AR activity in humans results in excessively high DHT levels in peripheral tissues, which has been mainly implicated in the pathogenesis of prostate cancer (PCa) and benign prostatic hyperplasia (BHP). Therefore, the role of DHT had contributed to the interest in finding 5AR inhibitors. Accordingly, several steroidal and non-steroidal inhibitors have been synthesized and tested against 5AR enzymes. Finasteride is a semi-synthetic 4-azasteroid and it was the first 5AR type II inhibitor approved for the treatment of BHP. In addition, it was also demonstrated that dutasteride acts as a 5AR type I and type II inhibitor and turosteride is selective for the type II isoform of this enzyme. Since these compounds have low potency and produce side effects, it becomes necessary to develop more potent and safe compounds. In the present work, several novel 4-azasteroid derivatives were synthesised from testosterone and progesterone, considering the pharmacophore which finasteride, dutasteride and turosteride have in common. Additionally, to introduce a bulky group and improve the affinity to the enzyme, through an aldol condensation reaction, an aldehyde was added in 16-C and 21-C positions of testosterone and progesterone, respectively. The cell proliferation effects of these compounds was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay in NHDF, LNCaP and T47D cell lines, In addiction, the cell viability effects was also analyzed using propidium iodide staning in LNCaP cells. In order to understand the affinity of the new compounds to the 5AR enzyme, molecular docking studies were performed. However, as the 3D structural coordinates of 5AR are not available, the docking studies have to be performed with 5ß-reductase (5BR) enzyme. Docking simulations with androgen receptor, estrogen receptor and CYP17A1 were also accomplished. To evaluate the toxicity of compounds, in silico toxicity studies were realized. In conclusion, the new compounds were successfully obtained in reasonable global yields. Molecular docking results support the information relative to the 5AR inhibition and show that the majority of the novel compounds have a high affinity to 5BR, while a less number of compounds presented low binding energies for the other studied receptors. From in silico toxicity studies, the data showed that compounds with furan substituent, after metabolized, can cause tumorigenic and mutagenic effects. Compound 16-[(4-methylphenyl)methylideny)-4-azaandrost-5-ene-3,17-dione, 6b, is the most promising compound, having a behaviour in LNCaP cell line very similar to which was observed with finasteride and show a lower IC50 than this approved drug in these experimental conditions.
- Antimicrobial and antitumor activity of S-methyl dithiocarbazate Schiff base zinc(II) complexesPublication . Gomes, Filipa Ramilo; Addis, Yemataw; Tekamo, Israel; Cavaco, Isabel; Campos, Débora L.; Pavan, Fernando R.; Gomes, Clara S.B.; Brito, Vanessa; Santos, Adriana O.; Domingues, F.C.; Luís, Ângelo; Marques, M. Matilde; Pessoa, João Costa; Ferreira, Susana; Silvestre, Samuel; Correia, IsabelSchiff bases (SB) obtained from S-methyl dithiocarbazate and aromatic aldehydes: salicylaldehyde (H2L1), o-vanillin (H2L2), pyridoxal (H2L3) and 2,6-diformyl-4-methylphenol (H3L4), and their corresponding Zn(II)-complexes (1-4), are synthesized. All compounds are characterized by elemental analyses, infrared, UV-Vis, nuclear magnetic resonance spectroscopy and mass spectrometry. The structures of H2L2 and [Zn2(L1)2(H2O)(DMF)] (1a) (DMF = dimethylformamide) are solved by single crystal X-ray diffraction. The SB coordinates the metal center through the Ophenolate, Nimine and Sthiolate atoms. The radical scavenging activity is tested using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, with all ligand precursors showing IC50 values ~40 μM. Cytotoxicity studies with several tumor cell lines (PC-3, MCF-7 and Caco-2) as well as a non-tumoral cell line (NHDF) are reported. Interestingly, 1 has relevant and selective antiproliferative effect against Caco-2 cells (IC50 = 9.1 μM). Their antimicrobial activity is evaluated in five bacterial strains (Klebsiella pneumoniae, Acinetobacter baumannii, Listeria monocytogenes, Pseudomonas aeruginosa and Staphylococcus aureus) and two yeast strains (Candida albicans and Candida tropicalis) with some compounds showing bacteriostatic and fungicidal activity. The minimal inhibitory concentration (MIC90) of HnL against Mycobacterium tuberculosis is also reported, with H2L2 and H3L4 showing very high activity (MIC90 < 0.6 μg/mL). The ability of the compounds to bind bovine serum albumin (BSA) and DNA is evaluated for H3L4 and [Zn2(L4)(CH3COO)] (4), both showing high binding constants to BSA (ca. 106 M-1) and ability to bind DNA. Overall, the reported compounds show relevant antitumor and antimicrobial properties, our data indicating they may be promising compounds in several fields of medicinal chemistry.
- New Steroidal Arylidene Derivatives Potentially Useful in the Treatment of Prostatic DiseasesPublication . Brito, Vanessa Sofia Figueiredo de; Almeida, Paulo Jorge da Silva; Silvestre, Samuel Martins; Alves, Gilberto LourençoThe prostate is a male-specific hormone-responsive gland with a crucial role in the male reproductive system and requires androgenic hormones and an androgen receptor (AR) for proper growth and development. Generally, this gland is mainly affected by three pathologies, namely prostatitis, benign prostatic hyperplasia (BPH), and malignant prostate cancer (PCa). These disorders constitute a source of significant morbidity and mortality for men worldwide. Thus, pharmacological therapy's relevance in managing these prostatic disorders is unquestionable. Currently, it is well established that the importance of steroidal hormones in the pathophysiology of BPH and PCa. In this context, it is known that the abnormally high 5α-reductase (5AR) activity in humans results in excessively high 5α-dihydrotestosterone (DHT) levels in peripheral tissues, which have been mainly implicated in the pathogenesis of BPH and PCa. Therefore, the role of DHT had contributed to the interest in finding 5AR inhibitors (5ARIs), comprising an important and commonly applied strategy principally in the treatment of BPH. Considering the unmet medical need, it is recognized that the 5ARIs clinically used, such as finasteride, exhibit low potency and several adverse side effects that significantly reduce the quality of life of patients. Furthermore, the effect of these drugs in the PCa treatment is controversial, although it seems a valid and logical approach to explore. In this context, arylidenosteroids, a class of modified steroids, have been reported with important antiproliferative activity and also with potential 5AR inhibitory activity. In the present work, the preparation of novel steroidal arylidene derivatives potentially useful in prostatic diseases, displaying inhibitory capacity against 5AR and/or antiproliferative properties against tumoral cells was intended. Essentially, this thesis aimed the discover of hit compounds for further development in the context of BPH and PCa. To achieve this goal, different synthetic strategies were exploited and new series of steroidal arylidene derivatives were obtained, and their antitumoral and 5AR inhibitory activities were evaluated. Moreover, molecular docking simulations against important targets of steroidal molecules, 5AR type 2, estrogen receptor α (ERα), AR, steroid 17α-hydroxylase/17,20 lyase (CYP17A1), and aromatase, were performed to understand the possible affinity and interactions. This thesis also contemplates the conclusion of the biological evaluation of arylidene-4-azasteroids (VB 4a-g and VB 7a-g) synthesized at the Master’s project and a comprehensive three-dimensional quantitative structure-activity relationship (3D-QSAR study considering the antiproliferative activity in prostate cells, since this work fits into the context of this thesis. In relation to this last part, robust 3D-QSAR models generated to elucidate the relation of the proliferation of LNCaP (androgen-dependent) and PC-3 (non-androgen dependent) cells and the structure of tested compounds showed the importance of the presence of bulky groups and electronegative atoms. Concerning the doctoral project, after several failed attempts to prepare new 3-,4- and 6-azasteroids modified at D-ring with fused/attached heterocycles, a new synthetic strategy was studied and applied and three distinct series of novel arylidene oxidized steroids were successfully obtained from dehydroepiandrosterone. The chemical structures and high purity of the new 16E-arylidene-5α,6α-epoxyepiandrosterone, 16E-arylidene-3β,5α,6β-trihydroxyandrosten-17-one, and 16E-arylidene-androst-4-ene-3,6,17-trione derivatives were corroborated by melting point determination, infrared spectroscopy, nuclear magnetic resonance spectroscopy, and high-resolution mass spectroscopy. Then, to determine their antitumoral potential, the activity of all synthesized analogs over the viability of tumoral cells was evaluated against several human tumoral and non-tumoral cell lines were assessed by (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effect of compounds on cell proliferation was evaluated against prostate cancer androgen-dependent cells (LNCaP), prostate cancer non-androgen dependent cells (PC-3), breast cancer cell line (MCF-7), normal prostate epithelium cells (PNT1A), and normal human dermal fibroblasts (NHDF). Furthermore, preliminary studies of the mechanism of action of the most promising derivatives were performed in MCF-7, LNCaP, and PC-3 cell lines using techniques, such as immunocytochemistry, fluorescence microscopy, and caspase activity assessment. Lastly, the potential 5AR inhibitory activity of all compounds, including the arylidene-4-azasteroids synthesized during the Master’s project, were determined through testosterone quantification in mice liver microsomes by high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD). This method was previously adapted, partially developed and validated, also in the context of the present project. Moreover, from the results of this assay, the main interactions of the best compounds against 5AR type 2 was also evaluated in the attempt of understanding the structure-activity relationship. Several of these new steroidal derivatives exhibited an interesting growth-inhibition effect on human tumoral cells, and generally, these tested compounds revealed to be less cytotoxic to non-tumoral cells producing higher IC50 values. The most relevant reduction of cell proliferation was observed with compounds VB 9e in MCF-7 cells (IC50= 3.47μM), VB 10e in PC-3 cells (IC50= 6.96 μM), and VB 11c in LNCaP cells (IC50= 6.48 μM). Moreover, the determined IC50 values of these compounds are very similar to the positive control, 5-fluouracil (5-FU). Additional biological studies showed that steroidal derivatives VB 9e and VB 11c seemed to trigger apoptosis in MCF-7 and LNCaP cells, respectively. VB 9e caused significant alterations in Ki67 (↓) and propidium iodide (↑) staining and modifications in nuclear morphology and cell distribution analysis in MCF-7 cells. On the other hand, VB 11c caused the same morphological and cell distribution alterations, and also seemed to increase the activity of caspase-3/7 in the LNCaP cell line. Molecular docking studies showed that these new derivatives presented stronger affinity to 5AR type 2 and CYP17A1, and poor affinity to ERα and AR. Relative to aromatase, it was verified higher affinity for steroidal 4-ene-3,6,17-triones. To finish, the effect of the steroidal arylidene derivatives on 5AR activity and the main results revealed that compound VB 11c seemed to be the most potent inhibitor (IC50= 6.12 nM). In addition, the in silico interactome study was performed and it was observed that this steroid displayed a higher number of interactions in common with finasteride, the reference, compared with other tested compounds. Interestingly, in this case, there is a correlation between in silico and experimental data. Taken together, the results showed that steroid VB 11c seemed to be the most promising molecule to be considered as a hit compound, presenting selectivity to tumoral prostate cells, potentially triggering the apoptotic cell death mechanism in the LNCaP cell line, and being the most potent against 5AR. In conclusion, the research work constitutes an important contribution to the knowledge concerning steroidal arylidene derivatives as agents with antiproliferative and 5AR inhibitory properties. Therefore, new paths were opened, showing the possibility of successfully developing potential new inhibitors based on steroid molecules for further development in the context of BPH and PCa.