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Ilustraciones y fotografías With the objective to contribute the discovering of drugs to treat the disorders endogen androgen-dependent like prostate cancer (CP) and prostatic benign hyperplasia, which are public health problem worldwide and in Colombia because their high morbidity and mortality in mature men, in this investigation the in vivo activity of the lichen origin compounds sphaerophorin (1) and atraric acid (2) was evaluated. These compounds have been active in vitro on targets involved in the biosynthesis and action of endogen androgens. The compounds 1 y 2 were obtained from the lichens Bunodophoron melanocarpum and Stereocaulon strictum using phytochemical techniques. Their identity was confirmed by their spectroscopic RMN 1H and 13C data that was compared with the literature. The in vivo activity on the biosynthesis and action of endogen androgens was determined by Hershberger bioassay using male Wistar rats. The animals were castrated under anesthesia (Ketamine 75 mg/kg and xilacine 10 mg/kg) intraperitoneal ten days before the experiment. Then, the rats were distributed randomly in 5 groups of 7 individuals each one. Three groups were the controls: vegetal oil (AV) (200 µL, vehicle); testosterone propionate (TP) (0.4 mg/kg dissolved in the vehicle); and TP (0.4 mg/kg) + finasteride (F) (positive control) (1 mg/kg dissolved in 200 µL of vehicle) subcutaneously. The fourth and fifth groups had the test compounds (both 2 mg/kg dissolved in 200 µL of vehicle) + TP (0.4 mg/kg) that were administrated once daily for 10 days subcutaneously. The day after the last administration, the animals were sacrificed and their organs androgen dependents (prostate, seminal vesicles, bulbocavernosus muscle, Cowper glands and glans) and no androgen dependents (adrenal glands, kidneys, and liver) were obtained and weight. This data was expressed as mg of organ per 100 grams of corporal weight. Additionally, the prostates were analyzed semiquantitatively using histoscore to observe some characteristics that could be a possible antiandrogenic effect in that organ. Considering that 1 and 2 did not decrease significatively (p

El cáncer de próstata (CP) y la hiperplasia prostática benigna (HPB) son las principales patologías que afectan a la próstata cuya ocurrencia en la población masculina mayor de 45 años ha aumentado en la última década. Al ser enfermedades de una alta incidencia y mortalidad, cada día se destinan más esfuerzos para estudiar sus tratamientos, con el fin de mejorar la calidad de vida de los afectados. La HPB es la más común entre las dos enfermedades. En la actualidad su tratamiento se lleva a cabo teniendo en cuenta las guías de la Asociación Americana de Urología, por medio de su algoritmo que clasifica a la enfermedad y ayuda a la elección de su tratamiento ya sea farmacológico o quirúrgico. Los principales andrógenos endógenos son la testosterona (T) y la 5α-dihidrotestosterona (DHT) que tienen actividades sexuales y anabólicas, además de controlar el desarrollo y mantenimiento de órganos como las vesículas seminales, el pene y la próstata, entre otros. En la próstata, la T es reducida enzimáticamente a DHT por la 5α-reductasa tipo II (5αR-II), siendo este metabolito el responsable de su agrandamiento. Por esta razón, la inhibición de la 5αR-II constituye un blanco de acción clave para el tratamiento de la HPB. Dentro de los fármacos para tratar a la HPB se encuentran los inhibidores de la 5αR como la finasterida y la dutasterida, que aunque son muy efectivos se les ha asociado una serie de eventos adversos relacionados con la función sexual masculina. Por lo tanto, la búsqueda y diseño de inhibidores de 5αR-II novedosos potencialmente con menores efectos adversos se encuentra justificada. En este sentido, el diseño de fármacos asistido por computadores o in silico constituye una herramienta valiosa para encontrar compuestos potencialmente bioactivos. A través de la estrategia de docking o acoplamiento molecular se pueden encontrar compuestos que se adapten tanto geométrica como químicamente a una determinada cavidad (sitio activo) de una diana biológica siendo esto representativo de su potencial actividad por la misma. Sin embargo, para llevar a cabo esta estrategia, es necesario conocer la estructura tridimensional (3D) de la diana de interés. Cabe mencionar que a la fecha, no se conoce la estructura 3D de la 5αR-II humana, por lo tanto, el presente trabajo tuvo como objetivo su modelado in silico partiendo de su secuencia de aminoácidos conocida, empleando técnicas de modelado por homología y por plegamiento (threading). A partir del alineamiento de la secuencia de aminoácidos conocida de la 5αR con las proteínas de la base de datos del Protein Data Bank (PDB) en el programa BLAST, se determinaron las plantillas proteicas (1MG7, 1J0A, 1UTH, 3BUR, 3BUV, 4QUV) para el modelado, mismo que se llevó a cabo en el programa computacional MODELLER y las plataformas informáticas I-TASSER y PHYRE2. Se construyeron un total de doce modelos que fueron validados mediante las plataformas QMEAN, ProQ y el gráfico de Ramachandran. A estos modelos se les determinó el sitio catalítico, representativo de las regiones de mayor accesibilidad y probabilidad de unión a la T, mediante una estrategia de acoplamiento molecular amplio (blind docking) con AutoDock 4.6 y una estrategia de acoplamiento molecular fino (en el programa SiteMap) entre la T o los inhibidores y los doce modelos 3D construidos. Se determinó que el sitio catalítico se encuentra en la región comprendida entre los aminoácidos histidina 230 a 232 de la estructura proteica concordando con el sitio catalítico previamente establecido por mutagénesis experimental. El modelo ITA3BUR, construido por la estrategia de plegamiento (threading) en el programa I-TASSER, empleando como plantilla proteica a la enzima 5β-reductasa (3BUR) de origen humano resultó un modelo válido de la estructura 3D de la 5αR. Adicionalmente, se determinó que el modelo ITA3BUR se une de una manera energéticamente más favorable tanto al sustrato endógeno como a los inhibidores adicionando elementos para considerarlo en una aproximación teórica válida a la estructura tridimensional de la 5αR-II humana. Abstract Prostate cancer (PC) and benign prostatic hyperplasia (BPH) are the main diseases that affect the prostate, whose occurrence on male population over 45 years has increased in the last decade. Since they are diseases with a high incidence, every day many efforts are oriented to study their treatments, in order to improve the quality of life of those affected. BPH is the most common of the two diseases. Nowadays, its treatment is performed according to the American Urological Association guidelines using its algorithm that classifies the disease and helps to choice of drug therapy or surgery. The main endogenous androgens are testosterone (T) and 5α-dihydrotestosterone (DHT) both have sexual and anabolic activities as well as control the development and maintenance of organs such as seminal vesicles, penis and prostate. In the prostate, T is reduced to 5α-dihydrotestosterone (DHT), catalyzed by type II 5α-reductase (5αR-II) which in turn is the responsible of its enlargement. For this reason, the inhibition of 5αR-II is a key target for the treatment of BPH. Dutasteride and finasteride are 5αR inhibitors, which are the choice drugs for BPH treatment. Although they are effective, a series of side effects related to male sexual function have been attributed to them. Therefore, the discovery and design of novel 5αR-II inhibitors with potentially less side effects is justified. In this sense, the in silico or computer assisted drug design is a valuable tool for finding potentially bioactive compounds. By molecular docking can be found compounds that geometrically or chemically suit to a biological target, which in turn represents its activity on it. However, in order to do this, it is necessary to know the three dimensions (3D) structure of the biological target. Due to nowadays the 3D structure for human 5αR-II it is not known the objective of this study was to model it in silico starting from its known amino acid sequence using homology and threading modeling techniques. For this purpose, different protein templates (1MG7, 1J0A, 1UTH, 3BUR, 3BUV, 4QUV) and software (MODELLER, I-TASSER y PHYRE2) were used. A total of twelve models were constructed, which were validated by QMEAN, ProQ and Ramachandran plot. Their catalytic site, representing the more accessible and binding probability to T regions were established by blind docking by AutoDock 4.6 and SiteMap software. The catalytic site was determined in the region between amino acids 230 to 232, which was also found by mutagenesis in previous studies. ITA3BUR model constructed by threading with the software I-TASSER using human 5β-reductase (3BUR) as protein template was a valid model. By molecular docking between each model and the endogenous substrate T or its inhibitors dutasteride and finasteride, In addition, it was determined that ITA3BUR model binds in a way energetically more propitious to these molecules, which helped to consider ITA3BUR model as a valid theoretical approach to 3D structure of human 5αR-II. Maestría

Con la finalidad de contribuir al descubrimiento y desarrollo de moléculas novedosas para el tratamiento de desórdenes androgénicos de alta incidencia en la población masculina, como el cáncer de próstata e hiperplasia prostática benigna, se llevó a cabo la síntesis y evaluación de la actividad disruptora endocrina de la N-ciclohexil-3β-hexanoiloxiandrostan-4,16-dien-6-oxo-17-carboxamida, como un análogo novedoso del androstano con características estructurales para inhibir a la enzima 5alfa-reductasa. Para la síntesis de dicho compuesto, se diseñó e implementó una ruta sintética a partir del acetato de 16-dehidropregnenolona. La identidad tanto del análogo novedoso como de los intermediarios de síntesis fue comprobada mediante análisis de sus datos espectrométricos (EM-ESI) y espectroscópicos (RMN, IR). Como medida de la posible biodisponibilidad oral del análogo novedoso, se llevó a cabo la determinación de su coeficiente de reparto n-octanol/agua y la verificación del cumplimiento del criterio de biodisponibilidad oral de Lipinski. Además, la actividad antiandrogénica de dicho análogo fue determinada in vivo mediante el ensayo de Hershberger en ratas Wistar macho adultas. Se encontró que a pesar de que la N-ciclohexil-3β-hexanoiloxiandrostan-4,16-dien-6-oxo-17-carboxamida posee características estructurales para inhibir a la enzima 5alfa-reductasa, carece de efecto antiandrogénico in vivo concluyente sobre la próstata, presentando dicho efecto sobre las vesículas seminales. Este hallazgo convierte al análogo novedoso en un candidato apto para la investigación de la afección de desórdenes androgénicos sobre vesículas seminales y como posible prototipo de uso como anticonceptivo. Por otro lado, se encontró que el compuesto no presenta toxicidad aparente a la dosis evaluada (2 mg/kg, rata) a corto plazo sobre hígado, riñones ni glándulas suprarrenales debido a que no se apreciaron daños a nivel macroscópico sobre los mismos. Finalmente, se encontró que el análogo novedoso final N-ciclohexil-3β-hexanoiloxiandrostan-4,16-dien-6-oxo-17-carboxamida presenta posible biodisponibilidad oral según criterios de comparación basados en el cumplimiento de las reglas de Lipinski. Abstract. In the pursuit of discovering and developing novel molecules for the treatment of high impact androgenic disorders, such as prostate cancer and benign prostatic hyperplasia, the synthesis and endocrine disrupting activity evaluation of N-cyclohexyl-3β-hexanoyloxyandrostan-4,16-dien-6-oxo-17-carboxamide was carried out. This is a novel androstane analog with structural features to inhibit the 5alpha-reductase enzyme. A synthetic route starting from 16-dehydropregnenolone acetate was designed and implemented in order to obtain the titled compound. In addition, the identity of both, the novel analog and synthetic intermediates was determined by analysis of their spectrometric (ESI-MS) and spectroscopic (NMR, IR) data. As a measurement of a possible oral bioavailability of the novel analog, the partition coefficient n-octanol/water and the compliance of Lipinski criteria for oral bioavailability was determined, as well as its in vivo antiandrogenic activity by the Hershberger assay in adult male Wistar rats. Although the novel analog N-cyclohexyl-3β-hexanoyloxyandrostan-4,16-dien-6-oxo-17-carboxamide had structural features to inhibit the 5alpha-reductase enzyme, no conclusive in vivo antiandrogenic effect on prostate was shown, however it showed antiandrogenic effect on seminal vesicles. This finding makes the novel analogue in a suitable candidate for further investigation on androgenic disorders on seminal vesicles and also as a possible leads for its use as a male contraceptive. On the other hand, it was shown that the novel analog had no apparent short-term toxicity at evaluated dose (2 mg/kg, rat) on liver, adrenals and kidneys due to their macroscopically examination did not show appreciable changes. Finally, it was found that the final novel analog, N-cyclohexyl-3β-hexanoyloxyandrostan-4,16-dien-6-oxo-17-carboxamide has possible oral bioavailability according to comparison criteria based on compliance with the Lipinski rules. Maestría