Plant-derived and dietary phenolic cinnamic acid derivatives: Anti-inflammatory properties.

Cinnamic acids are aromatic acids primarily found in plants and plant-derived food. Phenolic cinnamic acids, with one or more hydroxyl groups in the aromatic ring, often contribute to the biological activities attributed to these compounds. The presence of hydroxyl groups and a carboxyl group makes cinnamic acids very hydrophilic, preventing them from crossing biological membranes and exerting their biological activities. To alleviate this condition, a panel of synthetic modifications have been made leading to a diverse set of phenolic cinnamic structures. In this review, an overview of the natural phenolic cinnamic acid derivatives and their plant sources (more than 200) is described. The synthetic approaches to obtain the referred derivatives (more than 200) namely esters and amides are reviewed. Further, their anti-inflammatory activity (more than 70 compounds) is scrutinized. Finally, future directions will be indicated to translate the research on phenolic cinnamic derivatives into potentially effective anti-inflammatory drugs.

Steroidal epoxides as anticancer agents in lung, prostate and breast cancers: The case of 1,2-epoxysteroids.

Cancer continues to be a serious threat to human health worldwide. Lung, prostate and triple-negative breast cancers are amongst the most incident and deadliest cancers. Steroidal compounds are one of the most diversified therapeutic classes of compounds and they were proven to be efficient against several types of cancer. The epoxide function has been frequently associated with anticancer activity, particularly the 1,2-epoxide function. For this reason, three 1,2-epoxysteroid derivatives previously synthesised (EP1, EP2 and EP3) and one synthesised for the first time (oxysteride) were evaluated against H1299 (lung), PC3 (prostate) and HCC1806 (triple-negative breast) cancer cell lines. A human non-tumour cell line, MRC-5 (normal lung cell line) was also used. EP2 was the most active compound in all cell lines with IC50 values of 2.50, 3.67 and 1.95 µM, followed by EP3 with IC50 values of 12.65, 15.10 and 14.16 µM in H1299, PC3 and HCC1806 cells, respectively. Additional studies demonstrated that EP2 and EP3 induced cell death by apoptosis at lower doses and apoptosis/necrosis at higher doses, proving that their effects were dose-dependent. Both compounds also exerted their cytotoxicity by ROS production and by inducing double-strand breaks. Furthermore, EP2 and EP3 proved to be much less toxic against a normal lung cell line, MRC5, indicating that both compounds might be selective, and they also demonstrated suitable in silico ADME and toxicity parameters. Finally, none of the compounds induced haemoglobin release. Altogether, these results point out the extreme relevance of both compounds, especially EP2, in the potential treatment of these types of cancer.

Design, synthesis, biological activity evaluation and structure-activity relationships of new steroidal aromatase inhibitors. The case of C-ring and 7ß substituted steroids.

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 ß 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 IC50 of 0.011 µM, better than Exemestane, the steroidal AI in clinical use, which presents an IC50 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 7ß-methyl substitution on aromatase inhibition was compared with 7α-methyl substitution, showing that 7ß-methyl is better than 7α-methyl substitution. Molecular modelling studies showed that the 7ß-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 IC50 of 0.0058 µM. Compound 12a showed higher aromatase inhibition capacity than two of the three AIs currently in clinical use.

Design, synthesis, and antitumor activity evaluation of steroidal oximes.

Steroidal compounds were proven to be efficient drugs against several types of cancer. Oximes are also chemical structures frequently associated with anticancer activity. The main goal of this work was to combine the two referred structures by synthesizing steroidal oximes and evaluating them in several cancer cell lines. Compounds (17E)-5α-androst-3-en-17-one oxime (3,4 - OLOX), (17E)-3α,4α-epoxy-5α-androstan-17-one oxime (3,4 - EPOX), (17E)-androst-4-en-17-one oxime (4,5 - OLOX) and (17E)-4α,5α-epoxyandrostan-17-one oxime (4,5 - EPOX) were synthesized and their cytotoxicity evaluated in four human cancer cell lines, namely colorectal adenocarcinoma (WiDr), non-small cell lung cancer (H1299), prostate cancer (PC3) and hepatocellular carcinoma (HepG2). A human non-tumour cell line, CCD841 CoN (normal colon cell line) was also used. MTT assay, flow cytometry, fluorescence and hemocompatibility techniques were performed to further analyse the cytotoxicity of the compounds. 3,4 - OLOX was the most effective compound in decreasing tumour cell proliferation in all cell lines, especially in WiDr (IC50 = 9.1 µM) and PC3 (IC50 = 13.8 µM). 4,5 - OLOX also showed promising results in the same cell lines (IC50 = 16.1 µM in WiDr and IC50 = 14.5 µM in PC3). Further studies also revealed that 3,4 - OLOX and 4,5 - OLOX induced a decrease in cell viability accompanied by an increase in cell death, mainly by apoptosis/necroptosis for 3,4 - OLOX in both cell lines and for 4,5 - OLOX in WiDr cells, and by necrosis for 4,5 - OLOX in PC3 cells. These compounds might also exert their cytotoxicity by ROS production and are not toxic for non-tumour CCD841 CoN cells. Additionally, both compounds did not induce haemoglobin release, proving to be safe for intravenous administration. 3,4 - OLOX and 4,5 - OLOX might be the starting point for an optimization program towards the discover of new steroidal oximes for anticancer treatment.

Effects of new C6-substituted steroidal aromatase inhibitors in hormone-sensitive breast cancer cells: Cell death mechanisms and modulation of estrogen and androgen receptors.

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), 6ß-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.

C-6α- vs C-7α-Substituted Steroidal Aromatase Inhibitors: Which Is Better? Synthesis, Biochemical Evaluation, Docking Studies, and Structure-Activity Relationships.

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 IC50 of 0.06 µM and Ki = 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 IC50 of 0.055 µM and Ki = 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.

Anti-tumor efficacy of new 7α-substituted androstanes as aromatase inhibitors in hormone-sensitive and resistant breast cancer cells.

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-17ß-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.

Exploring new chemical functionalities to improve aromatase inhibition of steroids.

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 5ß-steroids, such as compound 4ß,5ß-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.

Plant derived and dietary phenolic antioxidants: anticancer properties.

In this paper, a review of the literature on the phenolic compounds with anticancer activity published between 2008 and 2012 is presented. In this overview only phenolic antioxidant compounds that display significant anticancer activity have been described. In the first part of this review, the oxidative and nitrosative stress relation with cancer are described. In the second part, the plant-derived food extracts, containing identified phenolic antioxidants, the phenolic antioxidants isolated from plants and plant-derived food or commercially available and the synthetic ones, along with the type of cancer and cells where they exert anticancer activity, are described and summarized in tables. The principal mechanisms for their anti-proliferative effects were also described. Finally, a critical analysis of the studies and directions for future research are included in the conclusion.

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 17ß-hydroxy-6-methylenandrosta-1,4-dien-3-one (17-ßHE) 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 (6ß-spirooxiranandrosta-1,4-diene-3,17-dione (2), 1α,2α-epoxy-6-methylenandrost-4-ene-3,17-dione (3) and 17-ßHE (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-ßHE (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.

Development, optimization and application of an analytical methodology by ultra performance liquid chromatography-tandem mass spectrometry for determination of amanitins in urine and liver samples.

Amanitins, highly toxic cyclopeptides isolated from various Amanita species, are the most potent poisons accounting for the hazardous effects on intestinal epithelium cells and hepatocytes, and probably the sole cause of fatal human poisoning. The present study was focused on the development, optimization and application of an analytical methodology by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), following urine and liver sample preparation by protein precipitation with organic solvents, and solid phase extraction (SPE) procedure, for the determination of the amatoxins, α- and ß-amanitin. Linearity, detection and quantification limits, selectivity, sensitivity, intra and inter-assay precision and recovery were studied, in order to guarantee reliability in the analytical results. The developed method proved to be specific and selective, with LOD (Limit of Detection) values for α- and ß-amanitin of 0.22 and 0.20 ng mL(-1) in urine and 10.9 and 9.7 ng g(-1) in liver, respectively. LOQ (Limit of Quantification) values ranged from 0.46 to 0.57 ng mL(-1) in urine and 12.3-14.7 ng g(-1) in tissue, for both amanitins. Linearity, in the range of 10.0-200.0 ng mL(-1) or ng g(-1), shows that coefficients of correlation were greater than 0.997 for α-amanitin and 0.993 for ß-amanitin. Precision was checked at three levels during three consecutive days with intra-day and inter-day coefficients of variation not greater than 15.2%. The extraction recovery presents good results for the concentrations analyzed, with values ranging from 90.2 to 112.9% for both matrices. Thus, the proposed analytical method is innovative, presents a high potential in the identification, detection and determination of α- and ß-amanitins in urine and tissue samples, as well as in other biological samples, such as kidney and mushrooms.

New steroidal 17ß-carboxy derivatives present anti-5α-reductase activity and anti-proliferative effects in a human androgen-responsive prostate cancer cell line.

The androgens testosterone (T) and dihydrotestosterone (DHT), besides playing an important role in prostate development and growth, are also responsible for the development and progression of benign prostate hyperplasia (BPH) and prostate cancer. Therefore, the actions of these hormones can be antagonized by preventing the irreversible conversion of T into DHT by inhibiting 5α-reductase (5α-R). This has been a useful therapeutic approach for the referred diseases and can be achieved by using 5α-reductase inhibitors (RIs). Steroidal RIs, finasteride and dutasteride, are used in clinic for BPH treatment and were also proposed for chemoprevention of prostate cancer. Nevertheless, due to the increase in bone and muscle loss, impotency and occurrence of high-grade prostate tumours, it is important to seek for other potent and specific molecules with lower side effects. In the present work, we designed and synthesized steroids with the 3-keto-Δ(4) moiety in the A-ring, as in the 5α-R substrate T, and with carboxamide, carboxyester or carboxylic acid functions at the C-17ß position. The inhibitory 5α-R activity, in human prostate microsomes, as well as the anti-proliferative effects of the most potent compounds, in a human androgen-responsive prostate cancer cell line (LNCaP cells), were investigated. Our results showed that steroids 3, 4 and 5 are good RIs, which suggest that C-17ß lipophylic amides favour 5α-R inhibition. Moreover, these steroids induce a decrease in cell viability of stimulated LNCaP cells, in a 5α-R dependent-manner, similarly to finasteride.

Design, synthesis and biochemical studies of new 7α-allylandrostanes as aromatase inhibitors.

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.

Predicting malignant involvement in a thyroid nodule: role of ultrasonography.

OBJECTIVE: To assess how ultrasonography can contribute during the evaluation of a thyroid nodule and whether this technique can have a role in predicting malignant involvement. METHODS: In this retrospective study, data were analyzed on 220 consecutive patients (with 348 thyroid nodules) who underwent thyroidectomy and had previously undergone assessment by high-resolution thyroid ultrasonography. Nodule size, echogenicity, regularity of margins, halo sign, presence or absence of calcifications, and invasion of surrounding tissues were evaluated. The nodules were classified as low, medium, or high risk for malignant involvement on the basis of nodule characteristics found on ultrasonography. All nodules were submitted to cytologic examination by fine-needle aspiration (FNA) before thyroidectomy. Ultrasound, FNA, and pathologic postoperative results were compared. RESULTS: Among the 348 thyroid nodules, 56 were ultrasonographically classified as low risk, 268 as medium risk, and 24 as high risk for malignant potential. Fifty of 56 (89.3%) low-risk nodules and 213 of 268 (79.5%) medium-risk nodules were diagnosed as benign at pathologic postoperative examination. In contrast, however, only 6 of 24 (25%) high-risk nodules were diagnosed as benign. Among the 18 high-risk nodules of 1-cm diameter or larger, FNA showed a 20% false-negative result. CONCLUSION: High-risk classification of a thyroid nodule on ultrasonography had a positive predictive value for malignant involvement of 75%. Nodule characteristics analyzed by ultrasonography should be considered at the time of surgical intervention.

[Slight hormonal alterations in a patient with a large pheochromocytoma]. / Mínimas alterações hormonais em paciente com grande feocromocitoma.

We present a case report that the patient had symptoms suggesting pheochromocytoma, a large tumor (> 50 g) and a single minimally altered laboratorial test, exemplifying a diagnostic pitfall. A 31 y.o. male patient had two acute abdominal events, the last one accompanied by headache, arterial hypertension, facial flushing, perspiration and cutaneous pallor. In another admission, the patient had sustained arterial hypertension and cardiac arrhythmia. From laboratory analysis, the vanililmandelic acid was slightly modified. Scintigraphy disclosed a large adrenal mass suggesting pheochromocytoma. Histopathology confirmed this hypothesis. This report points out that patients with symptoms suggesting pheochromocytoma, even when plasma catecholamines and urinary metanephrines levels are normal, may harbor large tumors with a high catecholamines turnover or that had undergone hemorrhagic necrosis.

Mínimas alterações hormonais em paciente com grande feocromocitoma / Slight hormonal alterations in a patient with a large pheochromocytoma

Relatamos caso clínico no qual o paciente apresentou sintomas sugestivos de feocromocitoma, grande tumor (maior que 50 g) e mínima alteração laboratorial, exemplificando uma armadilha diagnóstica. Um homem de 31 anos apresentou dois episódios de abdômen agudo, sendo o último acompanhado por cefaléia, hipertensão arterial, rubor facial, sudorese e palidez cutânea. Em outra internação, o paciente apresentava hipertensão arterial sustentada e arritmia cardíaca. Em relação aos testes laboratoriais, apenas o ácido vanil-mandélico foi levemente alterado. Cintilografia com MIBG foi realizada e sugeriu a presença de grande massa adrenal compatível com feocromocitoma. Uma amostra histopatológica da peça foi obtida após cirurgia e confirmou esta hipótese. Esse caso sugere que em pacientes que possuem sintomas sugestivos de feocromocitoma, mesmo com valores normais de catecolaminas plasmáticas e metanefrinas urinárias, devemos considerar as possibilidades de um grande tumor metabolizando catecolaminas em seu interior ou que sofreu necrose hemorrágica.

Multimodal molecular encapsulation of nicardipine hydrochloride by beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin and triacetyl-beta-cyclodextrin in solution. Structural studies by 1H NMR and ROESY experiments.

Proton nuclear magnetic resonance spectroscopy (1H NMR), which has become an important tool for in vitro study of cyclodextrin (CD) complexes, was used to study and structurally characterize the inclusion compounds formed in solution between nicardipine hydrochloride (NC) and beta-cyclodextrin (betaCD), hydroxypropyl-beta-cyclodextrin (HPbetaCD) and triacetyl-beta-cyclodextrin (TAbetaCD). The large variation of chemical shifts from protons located around the interior of the hydrophobic cavity (i.e. H-3, H-5 and H-6) coupled with minimal variation of shifts from protons located on the outer sphere of the betaCD (i.e. H-1, H-2 and H-4) provided clear evidence of inclusion complexation. In the presence of the different CDs, the aromatic protons of NC were the most affected, suggesting a strong involvement of the phenyl groups in the inclusion mechanism. The application of continuous variation method indicated the presence of complexes with a 1:1 host/guest stoichiometry for all the studied CDs. Two-dimensional rotating frame nuclear Overhauser effect spectroscopy (ROESY) experiments were carried out to further support the proposed inclusion mode. Inspection of the ROESY spectra allowed the establishment of spatial proximities between several aromatic hydrogens of the guest and the CD protons, indicating that the inclusion occurs by accommodation of the two aromatic groups of NC. All the experimental data were further rationalized to elaborate possible three-dimensional geometric models of inclusion complexes. From the aforementioned observations, we concluded there is no preference for inclusion of a particular aromatic ring. Instead, two types of 1:1 complexes with different inclusion structures may exist simultaneously in solution, being alternatively included through the wider side of the cavity, i.e. the so-called multimodal inclusion occurs in the interaction of NC with the different CDs.