Azasteroids from diosgenin: Synthesis and evaluation of their antiproliferative activity.

In this work, we report the synthesis of two new azasteroids through the modification of the A and B rings of diosgenin 1. The 4-azasteroid derivative 12 was prepared in three steps using the α,ß-insaturated-3-keto compound 11 as a precursor, which was first oxidized with KMnO4/KIO4 followed by an oxidative cleavage of ring A, and subsequently cyclized with an ammonium salt, under focused microwave irradiation for a short time of 3 min. A second azasteroid was synthesized, for which the key step was the Beckmann rearrangement of ring B of the oxime 16, affording the lactam-type enamide 17 in good yield. The methodologies developed for the synthesis of the precursors derivatives 10 and 11 contribute to improved yields, compared to those reported in the literature. The biological activity of the azasteroidal compounds 12 and 17 and their precursors has been evaluated in cervical cancer cells (HeLa), colon (HCT-15), and triple negative breast cancer (MDA-MB-231) lines.

Inhibition of Phytosterol Biosynthesis by Azasterols.

Inhibitors of enzymes in essential cellular pathways are potent probes to decipher intricate physiological functions of biomolecules. The analysis of Arabidopsis thaliana sterol profiles upon treatment with a series of azasterols reveals a specific in vivo inhibition of SMT2, a plant sterol-C-methyltransferase acting as a branch point between the campesterol and sitosterol biosynthetic segments in the pathway. Side chain azasteroids that modify sitosterol homeostasis help to refine its particular function in plant development.

Desenvolvimento de uma estratégia para a prospecção de fármacos inibidores da enzima esterol 24-C-metiltransferase (ERG6) de Leishmania spp / Development of a strategy for prospecting drugs inhibiting the enzyme sterol 24-C-methyltransferase (ERG6) from Leishmania spp

A leishmaniose é um grave problema de saúde pública, sendo considerada uma doença extremamente negligenciada, para a qual ainda não existe vacina licenciada para uso em humanos e o tratamento é caro e tóxico. Na busca para o desenvolvimento racional de novos fármacos, a escolha de um alvo seletivo no parasito é essencial. Enquanto os mamíferos produzem colesterol, os tripanossomatídeos produzem esteróis com esqueleto ergostano, como o ergosterol. O passo divergente da via dos parasitos é a transferência de um grupamento metila da S-adenosilmetionina (SAM) para o carbono 24 de esteróis com estrutura colestano, formando uma ramificação no C24 não existente nos esteróis de mamíferos, catalisado pela enzima esterol 24-C-metiltransferase (ERG6). Sendo assim, é proposta do presente trabalho estabelecer uma estratégia para a prospecção de fármacos inibidores da enzima ERG6 de Leishmania spp. Para esse objetivo, preparamos cepas de L. infantum e L. amazonensis que superexpressam a ERG6. A confirmação da expressão gênica aumentada em ambas cepas de Leishmania foi confirmada por PCR quantitativo (qPCR) em diferentes condições experimentais

Em seguida, essas cepas foram utilizadas para realização de triagem de fármacos. Promastigotas e amastigotas de Leishmania spp. superexpressando ou não o alvo estudado foram incubados com os diferentes compostos por 72h. Promastigotas de L. amazonensis superexpressando a ERG6 se tornaram resistentes aos azapterocarpanos LQB 333, 336, 339 e 341, e a cepa recombinante de L. infantum se tornou resistente às substâncias LQB 336, 339, 341 e 343. Em relação aos azasteroides, todos os derivados da série ND apresentaram atividade leishmanicida contra as formas promastigotas de Leishmania spp. Além disso, promastigotas e amastigotas de L. infantum que superexpressam a enzima alvo se tornaram resistentes quando tratadas com as substâncias ND-1 e ND-2. As amastigotas de L. amazonensis que superexpressam a ERG6 também se tornaram resistentes à ND-1 e ND-2. Os resultados apresentados neste trabalho indicam que o mecanismo de ação dos azapterocarpanos e os azasteroides destacados acima pode ser a inibição da ERG6. (AU)

Five-alpha Reductase Inhibitor Influences Expression of Androgen Receptor and HOXB13 in Human Hyperplastic Prostate Tissue

Objectives Five-alpha reductase inhibitors (5ARIs) are known as chemopreventive agents in prostate cancer with a risk of high-grade disease. This study evaluated the effects of 5ARI on androgen receptor (AR) and proteins involved in prostate cell growth such as HOXB13 expression in human prostate tissue and LNCaP prostate cancer cells. Materials and Methods We retrospectively selected 21 patients who underwent TURP between March 2007 and February 2010 for previously confirmed BPH by prostate biopsy. They were grouped into control (group 1, n = 9) and 5ARI treatment (group 2, n = 12) before TURP. AR and HOXB13 expression in prostate tissue was evaluated by immunohistochemical staining. We tested the effect of 5ARI on the expression of AR, prostate specific antigen (PSA) and HOXB13 in LNCaP cells. Cells were assessed by Western blot analysis, MTT in vitro proliferation assay, and ELISA. Results: Group 2 showed stronger reactivity for AR and HOXB13 than those of the group 1. MTT assay showed death of LNCaP cells at 25uM of 5ARI. At the same time, ELISA assay for PSA showed that 5ARI inhibited secretion of PSA in LNCaP cells. Western blot analysis showed that 5ARI did not greatly alter AR expression but it stimulated the expression of HOXB13. Conclusions These results demonstrated that 5ARI influences AR and HOXB13 expression in both LNCaP cells and human prostate tissue. In order to use 5ARI in chemoprevention of prostate cancer, we still need to clarify the influence of 5ARI in ARs and oncogenic proteins and its regulation pathway. .

Five-alpha reductase inhibitor influences expression of androgen receptor and HOXB13 in human hyperplastic prostate tissue.

OBJECTIVES: Five-alpha reductase inhibitors (5ARIs) are known as chemopreventive agents in prostate cancer with a risk of high-grade disease. This study evaluated the effects of 5ARI on androgen receptor (AR) and proteins involved in prostate cell growth such as HOXB13 expression in human prostate tissue and LNCaP prostate cancer cells. MATERIALS AND METHODS: We retrospectively selected 21 patients who underwent TURP between March 2007 and February 2010 for previously confirmed BPH by prostate biopsy. They were grouped into control (group 1, n = 9) and 5ARI treatment (group 2, n = 12) before TURP. AR and HOXB13 expression in prostate tissue was evaluated by immunohistochemical staining. We tested the effect of 5ARI on the expression of AR, prostate specific antigen (PSA) and HOXB13 in LNCaP cells. Cells were assessed by Western blot analysis, MTT in vitro proliferation assay, and ELISA. RESULTS: Group 2 showed stronger reactivity for AR and HOXB13 than those of the group 1. MTT assay showed death of LNCaP cells at 25uM of 5ARI. At the same time, ELISA assay for PSA showed that 5ARI inhibited secretion of PSA in LNCaP cells. Western blot analysis showed that 5ARI did not greatly alter AR expression but it stimulated the expression of HOXB13. CONCLUSIONS: These results demonstrated that 5ARI influences AR and HOXB13 expression in both LNCaP cells and human prostate tissue. In order to use 5ARI in chemoprevention of prostate cancer, we still need to clarify the influence of 5ARI in ARs and oncogenic proteins and its regulation pathway.

Alopecia androgenética femenina: Parte II: tratamiento / Female androgenetic alopecia: Part II: treatment

Como en el varón, el tratamiento tópico de alopecia de patrón femenino (AF) es con minoxidil al 3 por ciento - 5 por ciento dos veces al día. También puede usarse el minoxidil combinado con α-tocoferol o con otros tratamientos tópicos que elevan localmente el factor de crecimiento vascular endotelial. Comentamos nuestra experiencia con esta asociación. Los efectos secundarios más frecuentes en mujeres son la dermatitis de contacto y la hipertricosis de cara y antebrazos. Cuando la alopecia femenina se asocia a elevados niveles de andrógenos hay que utilizar terapéutica antiandrogénica. El síndrome de persistencia de la adrenarquía (SAHA suprarrenal) y alopecia en hiperandrogenismo suprarrenal deben tratarse con supresión suprarrenal y antiandrógenos. La supresión suprarrenal la efectuamos con glucocorticoides como dexametasona, prednisona o deflazacort. La terapia antiandrogénica incluye acetato de ciproterona, drospirenona, espironolactona, flutamida y finasterida. El síndrome por exceso de eliminación de andrógenos ováricos (SAHA ovárico) y alopecia del hiperandrogenismo ovárico pueden tratarse con supresión ovárica y andriandrógenos. La supresión ovárica incluye el uso de anticonceptivos que contengan un estrógeno, etinilestradiol, y un progestágeno. El antiandrógeno acetato de ciproterona, siempre acompañado de un anticonceptivo tricíclico, es la mejor terapéutica de la alopecia femenina. Los antagonistas de las hormonas liberadoras de gonadotropinas (GnRH) como el acetato de leuprolida suprimen la función hipofisaria y gonadal mediante la reducción de los niveles de LH y FSH, y como consecuencia se reducen los niveles de esteroides ováricos, especialmente en el síndrome de los ovarios poliquísticos. El SAHA hiperprolactinémico y alopecia del hiperandrogenismo de procedencia hipofisaria deben tratarse con bromocriptina o cabergolina. Las mujeres con alopecia posmenopáusica y altos niveles séricos de andrógenos en la premenopausia...

Topical treatment of female patgten hair loss (FPHL) is with minoxidil 3 percent-5 percent twice daily. Combination of minoxidil with α-tocopherol or with other topical treatment with possibility to enhance VEGF can be used. Our experience with this association is commented. Side effect of minoxidil is contact dermatitis and hipertricosis on face and forearm. When FPHL is associated with high levels of androgens systemic antiandrogenic therapy must be used. Persistent adrenarche syndrome (adrenal SAHA) and alopecia of adrenal hiperandrogenism must be treated with adrenal suppression and antiandrogens. Adrenal suppression is achieved with glucocorticosteroids such as dexametasona, prednisone ordeflazacort. Antiandrogen therapy includes cyproterone acetate, drospirenone, spironolactone, flutamide and finasteride. Excess release of ovarian androgens (ovarian SAHA) and alopecia of ovarian hiperandrogenism must be treated with ovarian suppression and antiandrogens. Ovarian suppression includes the use of contraceptives containing an estrogen, ethinyl estradiol, and a progestogen. Antiandrogens such as cyproterone acetate, always accompanied by tricyclic contraceptives, are the best antiandrogen to use in FPHL. Gonadotropin-releasing hormone (GnRH) agonists such as leuprolide acetate suppress pituitary and gonadal function through a reduction in LH andFSH levels. Subsequently, ovarian steroids levels will also be reduced, especially in patients with polycystic ovary syndrome. SAHA with hyperprolactinemia and alopecia of hyperprolactinemic hiperandrogenism should be treated with bromocriptineor cabergoline. Postmenopausal alopecia, with previous high levels of androgens or with PSA over 0.02 ng/ml improves with 2.5 mg/day of finasteride or 0,25 mg/day of Dutasteride. Although we do not know the reason, postmenopausal alopecia in normoandrogenic women also improves, probably in relation with the doses of 2.5 mg day that received...

Synthesis and preliminary biological screening of sterol analogues as new antifungal agents against plant pathogens.

In this paper we report the synthesis of a new family of sterol analogues that have two amidic bonds on the side chain. These azasterols were obtained by a straightforward procedure including an Ugi condensation that allows the facile attachment of a polyfunctionalized side chain into the steroidal framework. Some of the new compounds showed an interesting inhibitory effect on the growth of two pathogenic fungi involved in plant diseases.

The effects of combination therapy with dutasteride and tamsulosin on clinical outcomes in men with symptomatic benign prostatic hyperplasia: 4-year results from the CombAT study.

BACKGROUND: Combination therapy with dutasteride and tamsulosin provides significantly greater benefit than either monotherapy for various patient-reported outcomes in men with moderate-to-severe lower urinary tract symptoms (LUTS) due to benign prostatic hyperplasia (BPH) and prostatic enlargement. OBJECTIVE: To investigate whether combination therapy is more effective than either monotherapy in reducing the relative risk for acute urinary retention (AUR), BPH-related surgery, and BPH clinical progression over 4 yr in men at increased risk of progression. DESIGN, SETTING, AND PARTICIPANTS: The Combination of Avodart and Tamsulosin (CombAT) study was a 4-yr, multicenter, randomised, double-blind, parallel-group study in 4844 men > or =50 yr of age with a clinical diagnosis of BPH, International Prostate Symptom Score > or =12, prostate volume > or =30 cm(3), prostate-specific antigen 1.5-10 ng/ml, and maximum urinary flow rate (Q(max)) >5 and < or =15 ml/s with minimum voided volume > or =125 ml. INTERVENTION: Oral daily tamsulosin, 0.4 mg; dutasteride, 0.5 mg; or a combination of both. MEASUREMENTS: The 4-yr primary end point was time to first AUR or BPH-related surgery. Secondary end points included BPH clinical progression, symptoms, Q(max), prostate volume, safety, and tolerability. RESULTS AND LIMITATIONS: Combination therapy was significantly superior to tamsulosin monotherapy but not dutasteride monotherapy at reducing the relative risk of AUR or BPH-related surgery. Combination therapy was also significantly superior to both monotherapies at reducing the relative risk of BPH clinical progression. Combination therapy provided significantly greater symptom benefit than either monotherapy at 4 yr. Safety and tolerability of combination therapy was consistent with previous experience with dutasteride and tamsulosin monotherapies, with the exception of an imbalance in the composite term of cardiac failure among the three study arms. The lack of placebo control is a study limitation. CONCLUSIONS: The 4-yr CombAT data provide support for the long-term use of dutasteride and tamsulosin combination therapy in men with moderate-to-severe LUTS due to BPH and prostatic enlargement. CLINICALTRIALS.GOV IDENTIFIER: NCT00090103 (http://www.clinicaltrials.gov/ct2/show/NCT00090103).

The effects of dutasteride, tamsulosin, and the combination on storage and voiding in men with benign prostatic hyperplasia and prostatic enlargement: 2-year results from the Combination of Avodart and Tamsulosin study.

This article reports the results of a post hoc analysis of the multicenter, randomized, double-blind Combination of Avodart and Tamsulosin (CombAT) study, which aimed to investigate the effects of dutasteride (0.5 mg), tamsulosin (0.4 mg), and their combination on storage and voiding symptoms in 4844 men aged > or =50 years with moderate-to-severe lower urinary tract symptoms (International Prostate Symptom Score > or =12), prostate volume (PV) > or =30 cm(3) and PSA 1.5-10 ng ml(-1). After 24 months, combination treatment achieved significantly greater mean reductions in both voiding and storage symptoms than either monotherapy, in each of the three baseline PV tertiles (30 to <42, 42 to <58, > or =58 cm(3)). Dutasteride was as effective as tamsulosin for control of storage symptoms, but provided significantly greater relief of voiding symptoms than tamsulosin.

Synthesis of 4-azasteroids by an intramolecular Ugi reaction.

In this paper we report the use of an intramolecular Ugi reaction to synthesize new 4-azacholestanes diversely substituted both at N-4 and C-5. Both the scope and the stereochemical outcome of this approach were studied by varying the nature of the components necessary for this multicomponent reaction. In sight of our results we concluded that this methodology can be applied to obtain 4-azasteroids targeted to find new biologically active compounds.

In vivo and in vitro effect of novel 4,16-pregnadiene-6,20-dione derivatives, as 5alpha-reductase inhibitors.

In this study, we report the synthesis and biological evaluation of several new 3-substituted pregna-4,16-diene-6,20-dione derivatives (11a-11d). These compounds were prepared from the commercially available 16-dehydropregnenolone acetate. The biological effect of these steroids was demonstrated in in vivo and in vitro experiments. In the in vivo experiments, we measured the activity of the 11a-11d on the weight of the prostate gland of gonadectomized hamsters treated with testosterone plus finasteride or with the new steroids. For the studies in vitro, we determined the IC50 values by measuring the steroid concentration that inhibits 50% of the activity of 5alpha-reductase present in human prostate. In order to study the mechanism of action of 11a-11d, we also determined the capacity of these steroids to bind to the androgen receptor (AR) present in the rat prostate cytosol using labeled mibolerone as a tracer. The results from this work indicated that compounds 11a-11d significantly decreased the weight of the prostate as compared to testosterone treated animals and this reduction of the weight of the prostate was comparable to that produced by the finasteride. On the other hand 11a-11d exhibited a high inhibitory activity for the human 5alpha-reductase enzyme with IC50 values of 1.4 x 10(-8), 1.8 x 10(-9), 1.0 x 10(-8) and 4 x 10(-5) respectively. However the IC50 value of 11a (1.8 x 10(-9)) was the only one lower than that of finasteride (8.5 x 10(-9)). Nevertheless this compound did not show a higher potency in vivo as compared to that of compounds 11b-11d. The competition analysis for the androgen receptor indicated that the IC50 value of non-labeled mibolerone used in this experiment was 1nM, whereas steroids 10, 11a-11d did not inhibit the labeled mibolerone binding to the androgen receptor. On the other hand, steroid 10 did not show any activities in vitro or in vivo, and for this reason these steroidal derivatives (11a-11d) cannot be considered as prodrugs of compound 10. In conclusion, the compounds containing chlorine 11a, bromine 11b, iodine 11c atoms, and 11d (without any substituent in the ester moiety) at C-3 produce a significant decrease of the prostate weight in castrated animals treated with T and inhibits the activity of the 5alpha-reductase. Apparently the presence of the halogen atoms in compounds 11a-11c enhances the inhibitory activity for the 5alpha-reductase enzyme.

Azasterols impair Giardia lamblia proliferation and induces encystation.

The effects of sterol biosynthesis inhibitors on growth and fine structure of Giardia lamblia P1 strain cultures were analyzed. Azasterols demonstrated high efficacy in killing cells. The IC(50) values for 22,26-azasterol and 24(R,S),25-epiminolanosterol were 7muM and 170nM, respectively. Morphological analysis showed that azasterols induced changes in G. lamblia ultrastructure. The most significant alterations were: (a) considerable increase of the size of the peripheral vesicles, which are part of the parasite endosomal-lysosomal system; (b) appearance of autophagosomal structures; and (c) induction of differentiation, followed by an abnormal enlargement of encystation secretory vesicles. We propose that azasterols are effective chemotherapeutic drugs against Giardia lamblia in vitro and may have another target in cells besides sterol biosynthesis.

Effects of azasteroids on growth and development of the free-living stages of Nippostrongylus brasiliensis and Nematospiroides dubius.

25-Azasteroids were evaluated for their effects on the growth and development of the free-living stages of Nippostrongylus brasiliensis and Nematospiroides dubius. Increasing the concentration of 25-azasteroids in axenic cultures of either species resulted in a decrease in the percentage and mean length of larvae that developed to the third stage. Morphologic abnormalities of inhibited larvae were similar to those shown by larvae cultured in sterol-deficient medium. Addition of cholesterol to the culture medium reversed the inhibitive effects of azasteroid. Azasteroid completely inhibited growth and development of N. brasiliensis when the only sterol present in the culture medium was sitosterol. These results suggest similar pathways of sterol metabolism and similar mechanisms of action by azasteroids in the nematodes and insects that have been studied.