Tesi sul tema "Digitoxina"

Estudos recentes sobre disfunção ventricular demonstram o potencial terapêutico da modulação da matriz extracelular. Isso se dá pela influência que a referida matriz tem sobre a função sistólica e a diastólica do coração. Outros estudos demonstram a influência do digital sobre os sistemas neurohormonais desbalanceados no cenário de disfunção ventricular levantando uma questão acerca do potencial do digital como modulador da deposição do colágeno intersticial e perivascular miocárdico. Sabendo-se da importância prognóstica que a concentração do colágeno no referido cenário tem, a literatura apresenta uma lacuna de conhecimento. Objetivo: Avaliar o papel do digital no remodelamento miocárdico em um modelo experimental. Material e Métodos: 60 ratos Wistar foram separados em 3 grupos de 20. Um grupo controle (GC); outro grupo submetido ao modelo experimental de uninefrectomia, administração de água de beber com 1% de NaCl e de aldosterona subcutânea (GA); e outro grupo submetido ao mesmo modelo experimental, mas também recebendo digitoxina na ração de comer na dose de 100 g/Kg/dia (GAD). Resultados: A fração de volume de colágeno intersticial e perivascular mostrou-se maior no GA comparado aos outros dois grupos (GC e GAD). O índice de desempenho miocárdico mostrou diferença estatisticamente significativa entre o GA (0,49 ± 0,08) e o GC (0,32 ± 0,06) e o GAD (0,4 ± 0,13) (p=0,001). Os níveis séricos de BNP mostraram diferença estatisticamente significativa entre o GA (1,07 ± 0,32 ng/ml) e o GC (0,75 ± 0,19 ng/ml) e o GAD (0,84 ± 0,21 ng/ml) (p=0,01). Os níveis de metaloproteinases não diferiram entre os grupos. Houve uma correlação positiva entre uma maior fração de encurtamento e menores níveis séricos de BNP no GAD. Conclusões: Esses dados demonstram que a digitoxina teve efeito reduzindo a deposição de colágeno intersticial e perivascular e melhorando a função cardíaca avaliada pelo BNP e IDM nesse modelo experimental
Recent studies on myocardial dysfunction are highlighting the therapeutic potential of the myocardial extracellular matrix management. Its interesting to highlight the importance of the dynamics of the cardiac extracellular matrix, because even the systolic and diastolic functions are implicated on it. Other studies showed that digital compounds may regulates the neuroendocrin misbalance due to myocardial impairment and influencing these systems the digital compounds may regulates the interstitial collagen deposition. Objective: To evaluate the role of the digital on a myocardial fibrosis in an experimental model, examining if the digital is able to prevent the collagen deposition. Methods: The sample was divided in 20 rats from the control group (CG); 20 rats submitted to a fibrosis experimental model in which the rats are uninefrectomized, drink water with 1% NaCl during the protocol and receive aldosterone through an osmotic minipump (AG); and 20 rats submitted to the same experimental model treated with digitoxin in a daily dose of 100 g/Kg (DAG). Results: The interstitial and perivascular collagen volume fraction showed a significant difference between the AG and the other 2 groups (CG and DAG). The myocardial performance index showed a significant difference between the AG (0.49 ± 0.08) and the CG (0.32 ± 0.06) and the DAG (0.40 ± 0.13) (p=0.001). The BNP levels showed a significant difference between the AG (1.07 ± 0.32 ng/ml) and the CG (0.75 ± 0.19 ng/ml) and the DAG (0.84 ± 0.21 ng/ml) (p=0.01). The metalloproteinases levels did not differ among the groups and there was a positive correlation between the shortening fraction and the BNP levels among the GAD animals. Conclusion: These data demonstrate the digitoxin positive effect on the myocardial collagen deposition in this experimental model of interstitial fibrosis and could have a new therapeutic target previously unexplored

Made available in DSpace on 2015-12-06T23:47:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2008
Objetivo Insuficiência cardíaca congestiva (ICC) é o maior preditor de mortalidade entre as doenças cardíacas. Este trabalho foi conduzido para analisar, pela primeira vez, se a digitoxina afeta a sobrevida de ratos com ICC que se segue ao Infarto do Miocárdio. Material e métodos e Resultados Durante período de observação de 280 dias, foi avaliada a influência da administração de digitoxina (0,1 mg/100 g/dia, via oral) na sobrevida de ratas fêmeas (n = 170) infartadas e randomizadas em dois grupos: Controle (C: n = 85) ou Digitoxina (D: n = 85). A sobrevida média foi de 235 ± 7 dias (95% IC: 220 to 249) em C e 255 ± 5 dias (95% IC: 244 to 265) em D; o teste logrank aproximou-se dos limites da significância (p = 0,0602). A digitoxina não afetou a sobrevida de ratos com ICC dependente de Infarto do Miocárdio (IM) < 40% do ventrículo esquerdo, mas prolongou a sobrevida de ratos com IM ≥ 40%. Morte foi observada em 56% de C ≥ 40% e em 34% de D ≥ 40%. O teste logrank caracterizou maior mortalidade em C (p = 0,0161), com razão de risco igual a 2,03. ICC foi identificada em todas as ratas que faleceram. O teor de água do pulmão e a mecânica miocárdica – analisada em músculo papilar – foram analisadas em C (n = 7) e D (n = 14). Diferenças significantes foram observadas (x±epm) no teor de água do pulmão (C: 82 ± 0,4; D: 80 ± 0,3%; p = 0,0014), tensão desenvolvida (C: 2,7 ± 0,3; D: 3,8 ± 0,3 g/mm2 ; p = 0,0286) e em sua primeira derivada temporal (C: 24 ± 3; D: 39±4 mg/mm2 /s; p = 0,0109). A depressão da contração pós-pausa foi atenuada em D. Conclusões: A administração por período prolongado de digitoxina reduziu marcadamente o comprometimento miocárdico após Infarto do Miocárdio, atenuou a disfunção miocárdica, reduziu a congestão pulmonar, fornecendo a primeira evidência da eficiência da digitoxina em prolongar a sobrevida na Insuficiência Cardíaca experimental.
Congestive heart failure (CHF) is a major predictor of death. We design this protocol aiming to analyze, for the first time, whether digitoxin affect survival of rats with CHF due to Myocardial Infarction. Methods and Results – The influence of digitoxin administration (0.1 mg/100 g/day, orally) on the survival of decompensate infarcted female rats (n=170) randomized as Control (C: n=85) or Digitoxin (D: n=85) was evaluated during a 280-day observational period. Mean survival was 235±7 days (95%CI: 220; 249) for C and 255±5 days (95%CI: 244; 265) for D; the logrank test revealed a borderline significant difference (p=0.06). Digitoxin did not affect (p=0.5595) survival in rats presenting CHF due to myocardial infarction (MI) <40% of the left ventricle; however, prolonged survival in rats presenting MI ≥40%. Indeed, death was reported for 56% in C≥40% and 34% rats in D≥40%; the logrank test defined significantly (p=0.0161) higher mortality in C, with a hazard ratio of 2.03 (95%CI: 1.13; 3.55). CHF was present in all deceased rats. Pulmonary water content (PWC) and papillary muscle mechanics were analyzed in C (n=7) and D (n=14) survivors. Significant differences were observed (x±epm) regarding PWC (C: 82±0.4; D: 80±0.3%; p=0.0014), developed tension (C: 2.7±0.3; D: 3.8±0.3 g/mm2 ; p=0.0286) and +dT/dt (C: 24±3; D: 39±4 mg/mm2 /s; p=0.0109). Post-rest contraction was more depressed in C than in D. Conclusion – long-term digitoxin administration markedly reduced cardiac impairment after myocardium infarction, attenuated myocardial dysfunction, reduced pulmonary congestion, and provided the first evidence regarding the efficiency of digitalis in prolonging survival in experimental cardiac failure.
BV UNIFESP: Teses e dissertações

"Stereoselective Synthesis of 2-Deoxyglycosides. Approach to the synthesis of Digitoxine"

2-Deoxy and 2,6-dideoxy-glycosides are important structural units in many natural products including antitumor drugs (anthracyclines, aureolic acids, calicheamicin, esperamicin), antibiotics active against Gram-positive bacteria (erythromycins, orthosomycins), antibiotics inhibiting platelet aggregation (angucyclines), drugs used in the treatment of cardiac insufficiency (cardiac glycosides), antiparasitic agents (avermectins).
The stereocontrolled formation of the glycosidic linkage in 2-deoxy-oligosaccharides has been found to be one of the most challenging tasks in glycosylation reactions. This could be solved using C-2 substituents (I, SeR, SR) in order to aid stereocontrol in the glycosylation step. These groups can be then easily removed under mild conditions after stereocontrol has been achieved. On the other hand, one of the most important intermediates in the synthesis of 2 deoxycarbohydrates are 1,2-unsaturated glycosides, commonly called glycals.
The aim of the study reported in this thesis was to develop a new method for the stereoselective synthesis of 2-deoxyoligosaccharides and precursors such as glycals. Moreover, an approach to the synthesis of digitoxine was also reported. Thus, in a preliminary introduction, previous reported methods for the synthesis of deoxysugars were discussed. In chapter 1, we developed a new procedure for synthesizing phenyl 2-deoxy-2-iodo-1-thio-glycosides and their use as glycosyl donors for the stereocontrolled synthesis of 2-deoxy-2-iodo-oligosaccarides. The key step for the synthesis of 2-deoxy-2-iodo-1-thio-glycosides is a regio- and stereoselective 6-endo cyclization of alkenols induced by iodine electrophiles. In chapter 2, Deoxy-2-iodopyranosides were synthesized from sulfanyl alkenes using a "one pot" consecutive cyclization-glycosylation process. The "one pot" procedure was also applied to the synthesis of a 2,6-dideoxy-2-iodo-glycoside, which was successfully deiodinated to afford the 2,6-dideoxyglycoside. In chapter 3, it is showed that pyranoid glycals of all configurations can be obtained from pentoses through an olefination-cyclization-elimination sequence. This method provides access to non conventional glycals having C3-alkoxy substituent in axial configuration, such as D-allal and D gullal, which are difficult to synthesize by usual methods. Furthermore, other functionalized glycals such as 2 phenylselenenyl or 2 iodoglycals can be synthesized starting from enolthioethers by direct selenium-mediated elimination or through dehydrative reaction of 2-iodolactols, respectively. Finally, Starting from a common precursor such as D-ribonolactone, we have explored a new approach to the synthesis of digitoxine and other cardiac glycosides as application of the previous methodology developed.



Títol: "Stereoselective Synthesis of 2-Deoxyglycosides. Approach to the synthesis of Digitoxine"

Paraules Clau: Síntesi estereoselectiva, 2-desoxi i 2,6-didesoxiglicòsids, glicósids cardiacs, glicals i iodoglicals, síntesi "one-pot", ciclació (electròfila), digitoxina.
Informe Preceptiu sobre la Tesi doctoral "Estereoselective Synthesis of 2 Deoxyglycosides. Approach to the síntesis of Digitoxine" presentada per Miguel Àngel Rodríguez Gómez.


La tesi s'emmarca dins el camp de la síntesis de carbohidrats i glicoconjugats amb estructures que presenten posicions on una o més de les funcions hidroxil característiques del sacàrids no hi estan presents. El treball realitzat ha tingut com objectiu final la síntesis estereoselectiva de 2-desoxi i 2,6-didesoxiglicòsids ja que són part constituent de moltes substàncies biològicament actives i/o productes naturals com antitumorals, antibiòtics, agents antiparasitaris, cardiotònics...i a més a més són difícils d'obtenir a partir de carbohidrats naturals.
D'aquesta forma en aquesta tesi s'aborda la síntesis de 2-desoxi-2-iodo-1-tiopiranósids com a nous dadors de glicosil i la seva aplicació en la síntesis estereoselectiva d'oligosacàrids i glicòsids. Aquest dadors de glicosil es caracteritzen per la presència d'un grup fenilsulfanil com a grup sortint en la posició anomèrica (C1) i un grup iodo en el C2 que actua com element de control en la reacció de glicosilació. Aquests dos grups funcionals donen, a més a més, moltes possibilitats de derivatització i una variada reactivitat.

La memòria s'ha organitzat en una introducció general sobre la biologia i la química dels 2-desoxi i 2,6-didesoxiglicòsids, un objectius, quatre capítols on es s'exposen i discuteixen els resultats obtinguts amb les seves corresponents conclusions i un annex amb els espectres dels productes seleccionats.

La introducció tracta sobre la importància i el variat paper biològic dels 2-desoxi i 2,6-didesoxicarbohidrats a la vegada que parla de la dificultat i els especials problemes que comporta la síntesis química d'aquests tipus de compostos. D'aquesta forma es fa una revisió dels mètodes desenvolupats fins avui per la síntesis d'aquest glicòsids. Lligat amb aquests mètodes anteriors, en els objectius es posa de manifest la necessitat d'arribar a un nou mètode de síntesis de 2-desoxicarbohidrats que permeti assolir totes les configuracions de piranòsids possibles.

En el primer capítol es desenvolupa el nou mètode d'obtenció de 2-desoxi-2 iodo-1-tioglicòsids que després es faran servir com a dadors de glicosil. D'aquesta forma, partint de pentoses de totes les configuracions i diferentment protegides es van olefinar per diversos mètodes, obtenint polihidroxihexenilsulfurs. El mètode més convenient per aquesta reacció en termes de rendiment i estereoselectivitat fou la olefinació amb oxid de fosfina (Wittig-Horner-WH). Aquests alquenols es van ciclar amb electrofils de iode, conduint de forma regioselectiva als 2-desoxi-2-iodo-1 tiopiranòsids. Aquest dadors de glicosil es van fer reaccionar amb colesterol com a model d'aglicona de diferents compostos bioactius i amb un glucosid com a model de síntesi d'oligosacàrid.
En el segon capítol s'aborda la síntesis dels mateixos compostos del capítol primer aprofitant la semblança de les condicions de ciclació i de glicosilació, que permet en una sola etapa la glicosilació de diversos compostos partint de l'alquenol, un precursor acíclic molt més estable i fàcil de sintetizar que el 2-desoxi-2-iodo-tioglicòsid corresponent. Aquest procediment "one-pot" es mostra igual en diasteroselectivitat i superior en termes de rendiment i de facilitat de manipulació que la síntesi per passos del capítol 1. A més es va sintetizar un 2,6-didesoxicarbohidrat model del supressor de l'apetit P57AS3.

En el tercer capítol s'exposa la síntesi de glicals a partir del 1-tio-2-desoxi-2 iodo-piranosids. El glicals són compostos molt versàtils i útils en la síntesis de carbohidrats i amb el procediment desenvolupat en aquest capítol s'arribà a obtenir glicals de configuracions difícils d'obtenir per altres mètodes, com el D-allal i el D gullal. A més, en una segona part del capítol tercer, aplicant un procediment de glicosilació estàndar per a com el de Gin ("dehydrative glycosylation") s'obtenen a partir de 2 iodolactols diversos compostos com a 2 iodoglicals, glicals o 1,1'-disacàrids.

En el quart capítol tots els anteriors procediments s'apliquen en la aproximació a la síntesis d'un glicósid cardíac, la digitoxina, molt utilitzat en el tractament de la insuficiència cardíaca. D'aquesta forma es realitzà la síntesi dels alquenols precursors dels 2,6-dideoxiglicòsids que formen part de l'estructura d'aquest fàrmac (unitats de digitoxosa) i es va unir a la aglicona obtenint el monosacàrid de la digitoxigenina. A més a més es van obtenir altres intermedis valuosos, tals com el corresponents 2 iodolactols o els trichloroacetimidats, en el camí cap a la síntesis de la digitoxina i/o anàlegs.

Amb el treball d'aquesta tesi els objectius inicialment proposats de desenvolupament d'un nou mètode de glicosilació per la síntesi de 2-desoxiglicòsids i han estat ampliament assolits i a més a més, s'han desenvolupat vies alternatives (glicals, lactols, 2-iodoglicals,...) que amplien els procediments sintètics inicials, ampliant les vies de síntesis dels 2-desoxiglicòsids.

Prostate cancer is the most occurring form of cancer in men in Sweden and new candidates for treatment towards advanced phases of prostate cancer needs to be investigated. One suggested treatment is vitamin D which will mediate effect via VDR and PDIA3 and cause cell cycle arrest.  Another treatment is digitoxin which will cause accumulation of intracellular Ca2+ leading to apoptosis. The aim of the project of was to investigate potential synergistic effects of 25-hydroxyvitamin D3 and digitoxin in prostate cancer cell lines and find out effects mediated by PDIA3. DU145 and LNCaP cells were seeded and treated with 25-hydroxyvitamin D3 (10-10, 10-9, 10-7 M), digitoxin (25 ng/ml and 50 ng/ml) and four combinations of 25-hydroxyvitamin D3 and digitoxin. Cell viability assay was performed for determining the number of viable cells. Treatment with 25-hydroxyvitamin D3 10-7M + digitoxin 50ng/ml (68%), 25-hydroxyvitamin D3 10-9M + digitoxin 25ng/ml (39%), 25-hydroxyvitamin D3 10-9M + digitoxin 50ng/ml (69%), digitoxin 25ng/ml (26%) and digitoxin 50 ng/ml (44%) was statistically significant with increased cell viability compared to untreated control in DU145 after 48h of treatment. Treatment with 25-hydroxyvitamin D3 10-7M (12%) and 25-hydroxyvitamin D3 10-9M (12%) was statistically significant with increased cell viability compared to untreated control in LNCaP after 24h of treatment. The conclusion based on results from this study is that a combination of digitoxin and 25-hydroxyvitamin D3 does not inhibit cell viability in DU145 or LNCaP cancer cell lines.

In the present work, a complete study for the synthesis of 2-deoxy-glycosides is described, applying
a strategy previously developed in our group for the preparation of 2-deoxy-2-iodo-pyranoses. This
strategy, that involves Wittig¨CHorner olefination from fully protected furanoses to give alkenyl sulfides,
electrophilic¨Cinduced cyclization to furnish 2-deoxy-2-iodo-pyranosyl thioglycosides, gives access to a
new type of glycosyl donor that can be used in glycosylation reactions of the desired glycosyl acceptors to
give 2-deoxy-2-iodo-glycosides.
This method is based, on one hand, in the availability of sulfanylmethylphosphine oxides to perform
the olefination reaction over the furanoses. The usual access to these reagents is the Arbuzov reaction,
that requires chloro derivatives as starting materials that are not easy to prepare and in many cases are
unstable. Furthermore, the efficiency of the cyclization is limited by the obtaintion of E/Z alkene mixtures
in the olefination step, because Z alkenes were proved to be reluctant to cyclization.
To increase the efficiency of the whole process, two implementations were studied in this work.
First, a new approach for the preparation of sulfanylmethylphosphine oxides was investigated starting
from (tosyloxymethyl)phosphine oxide. The method was also extended to heteroatomic substituted
methylphosphine oxides (X, Se, Te, NR2, etc).
Application of these novel sulfanylmethylphosphine oxides in the olefination of ribo- and
arabinofuranoses resulted in the formation of the corresponding sulfanyl alkenes with increased E/Z
stereoselectivity.
The sulfanyl ribo and arabino alkenes were investigated in the iodonium¨Cinduced cyclization
reaction. The effect of the bulkiness of the substituent at sulfur was studied and the results of cyclization
compared to that of phenyl at the phenylsulfanyl parent compound. Cyclization of the arabino derivatives
led to 6-endo cyclization products in lower yields whereas the t-butylsulfanyl arabino-1-hex-enitol
proceeded in higher yield. No cyclization took place from 2,6-dimethylphenyl arabino-1-hex-enitol. The
yields in al cases were higher from ribo-hex-enitols than from the corresponding arabino-hex-enitol.
Glycosylation of some of the allopyranoside thioglycosides synthesized were explored and
compared with those obtained from phenylsulfanyl parent thioglycoside. t-Butyl thioglycoside was
reacted with cholesterol to render alloglycoside product as an anomeric mixture in higher yield without
almost affecting the stereoselectivity whereas with 2,6-dimethylphenyl thioglycoside the stereoselectivity
increased but the yield was lower.
The synthesis of septanosides was studied starting from pyranosides with the strategy of Wittig¨C
Horner olefination and subsequent electrophile¨Cinduced cyclization reaction, but the desired 7-endo
cyclization did no work with secondary alcohols. To overcome this problem, starting from
conformationally¨Crestricted 2,3-O-isopropylidenefuranosides, hex-1-enitols with a free primary hydroxyl
function were prepared, from which 7-endo cyclization reaction took place to furnish the desired
oxepanes with moderate yields.
The total syntheses of 2,6-dideoxyoligosaccharides digitoxin and appetite suppressant P57, with
common 2,6-dideoxypyranose units, were explored, applying the three-step (olefination¨Ccyclization¨C
glycosylation) methodology. For the synthesis of common intermediate C, two different permanent
protecting groups for free hydtoxyl group at C-3 were used: benzyl ethers for digitoxin and methyl ethers
for P57. Different silyl groups (TBS, TES and TBDPS) were used for hydroxyl at C-4 that required
temporary protection. Olefination of the different 6-deoxyribofuranoses rendered the corresponding 5-Osilyl
hex-1-enitols (167, 169, and 173) as a consequence of silyl migration from hydroxyl at C-4 to C-5,
altogether with the expected 4-O-silyl hex-1-enitols (164, 168, and 172). These products were analyzed
by 1D and 2D NMR techniques.
5-O-TES, 5-O-TBS or 5-O-TBDPS protected hex-1-enitols were submitted to iodonium¨Cinduced
cyclization reactions to afford exclusively 5-endo cyclization products. Furthermore, 5-endo cyclization
product 2-iodofuranose 189 was formed as a major product by cyclization from the C-4 unprotected enitol
176.
Digitoxin and P57 synthesis will be reconsidered in a near future using other protecting groups that
do not migrate under the basic conditions of the olefination.
En este trabajo se describe un estudio completo para la s¨ªntesis de 2-desoxyglicosidos, aplicando la
estrategia desarrollada anteriormente en nuestro grupo de investigaci¨®n para la preparaci¨®n de 2-desoxi-2-
yodo-piranosidos. Este estrategia incluye la olefinaci¨®n de Wittig¨CHorner de furanosas protegidas
completamente para obtener alquenil sulfidos, las ciclaciones inducidas electrofilicamente para conseguir
2-desoxi-2-yodo-piranisil tioglicosidos y permite el acceso a nuevo tipos de glicosil donores que pueden
ser utilizados en glicosilaci¨®n reacciones con aceptores deseados para suministrar 2-desoxi-2-yodoglicosidos.
Esto m¨¦todo se basa, por un lado, en la disponibilidad de sulfanilmetil oxido fosfinas para llevar a
cabo la reacci¨®n de olefinaci¨®n empezando por furanosas. La preparaci¨®n corriente de estos reactivos es a
trav¨¦s de la reacci¨®n de Arbuzov, que requiere derivados cloratos para materiales de partida cuya
obtenci¨®n, en muchas ocasiones, es complicada o no son estables en condiciones est¨¢ndar. Adem¨¢s, la
eficacia de la ciclaci¨®n est¨¢ limitada a la obtenci¨®n de una mezcla de alquenes en una mezcla de E/Z en la
etapa de olefinaci¨®n, porque los Z alquenos resultan de ciclar m¨¢s lento o no ciclan.
Para mejorar la eficacia de todo el proceso se ha investigado el desarrollo para la obtenci¨®n de
sulfanilmetil oxido fosfinas a partir de tosiloximetil oxido fosfina. Este m¨¦todo se podr¨ªa ampliar para la
s¨ªntesis de otras metil oxido fosfinas ¦Á-sustituidas por heteroatomo (X, Se, Te, NR2, etc).
De la aplicaci¨®n de estas nuevas sulfanilmetil oxido fosfinas en reacciones de olefinaci¨®n con riboy
arabinofuranosas result¨® la formaci¨®n de las sulfanil alquenos correspondientes de estereoselectividad
mejorada con una relaci¨®n de E/Z m¨¢s alta.
Los sulfanil alquenos con configuraciones ribo y arabino se investigaron en reacciones de ciclaci¨®n
inducida electrof¨ªlicamente. Se estudi¨® el efecto de voluminosidad del sustituyente en el azufre y los
resultados de las ciclaciones se compararon con el resultado obtenido del fenil sustituyente en el fenil
sulfanil an¨¢logo. La ciclaci¨®n de alqueno con la configuraci¨®n arabino condujo al producto 6-endo
ciclado con menor rendimiento, mientras que con el t-butilsulfanil arabino-1-hex-enitol se consigui¨®
mejorar el rendimiento. El derivado 2,6-dimetilfenil arabino-1-hex-enitol no particip¨® en ciclaci¨®n.
Se ha explorado la glicosilaci¨®n de unos de los tioglicosidos sintetizados y se ha comprobado con lo
obtenido de la tioglicosido piloto con grupo fenil. El tioglicosido con grupo t-butil ha reaccionado con
colesterol para dar el producto glicosilado con mayor rendimiento y selectividad casi inalterada, mientras
con 2,6-dimethilphenil tioglicosido la estereoselectividad ha aumentado pero con menor rendimiento.
Se ha estudiado la s¨ªntesis de los septanosidos empezando por los piranosidos y furanosidos con la
estrategia de olefinaci¨®n de Wittig¨CHorner y posteriormente ciclaci¨®n inducida electrofilicamente pero el
deseado 7-endo ciclaci¨®n no se di¨® con alcoholes secundarios. Para solucionar el problema se decidi¨®
aplicar 2,3-O-isopropilidenefuranosidos con conformaci¨®n restringida como material de partida,
obteniendo hex-1-enitols con alcoholes primarios libres, que se utiliz¨® en 7-endo ciclaci¨®n para dar los
oxepanes deseados con rendimientos moderados.
Se han explorado las s¨ªntesis totales de 2,6-didesoxioligosaccaridos, de la digitoxina y el supresor
del apetito P57, con 2,6-didesoxipiranosa como unidad estructural com¨²n aplicando la metodolog¨ªa de
tres etapas (olefinaci¨®n¨Cciclaci¨®n¨Cglicosilaci¨®n). Para la s¨ªntesis del intermediario com¨²n C, se utilizaron
dos grupos protectores permanentes diferentes en el grupo hidroxil en la posici¨®n C-3: bencil eteres para
la s¨ªntesis de la digitoxina y metil eteres para la de P57. Se estudi¨® la aplicaci¨®n de diversos grupos sililes
(TBS, TES y TBDPS) para la protecci¨®n del grupo hidroxil en la posici¨®n C-4, que requiere un grupo
protector temporal. La olefinaci¨®n de los diferentes 6-desoxiribofuranosas ha dado los 5-O-silil hex-1-
enitoles correspondientes (167, 169, y 173) como consecuencia de la migraci¨®n del grupo silil, desde el
grupo hidroxil C-4 hasta el C-5, conjuntamente con el deseado 4-O-silil hex-1-enitoles (164, 168, y 172).
Se ha analizado y se ha confirmado la estructura de todos estos productos con t¨¦cnicas de 1D y 2D NMR.
Se ha estudiado la ciclaci¨®n de hex-1-enitoles con grupos protectores como 5-O-TES, 5-O-TBS o 5-
O-TBDPS y de estas reacciones inducidas con yodo se ha obtenido exclusivamente productos de 5-endo
ciclos. Adem¨¢s, se ha obtenido como producto mayoritario el producto de 5-endo ciclo 189 con la
ciclaci¨®n de enitol no protegido en posici¨®n C-4 176.
Se reconsidera la s¨ªntesis total de la digitoxina y la P57 en un futuro cercano aplicando otros grupos
protectores que no participan en reacciones de migraci¨®n bajo las condiciones b¨¢sicas de la olefinaci¨®n.

Pancreatic ductal adenocarcinoma (PDAC) yet remains one of the top 10 most prevalent and top five most common cause of cancer-related deaths solely in the United States. Its metastatic deviation from most cancer types rends it one of the most subtle yet perturbating disease. Hope lies within the traces of reproducibly mutated genes which are said to be found in nearly 95% of all PDAC. This study aimed to examine how digitoxin affects glycolysis and gene expression in Panc-1 cell lines. Panc-1 cells were cultured and treated with various concentrations of digitoxin for 24h-48h depending on the analysis and cell viability was then examined via the MTS assay. Lactate assay was conducted in order to investigate the degree to which panc-1 production of lactate was affected by digitoxin. Specific genes of interest (LDHA, C-Myc, PFKM, PDP1, SLC2A and PMM1) that take part in the glycolytic segment of the panc-1 metabolism were analyzed with qPCR in order to study their expression. Cell cycle assay was conducted in order to examine whether digitoxin also affected the panc-1 cell cycle and to what degree. Results from the MTS assay indicated that cell viability was indeed affected with increasing concentrations of digitoxin whereas; the lactate assay indicated that panc-1 cells were sensitive to 25nM digitoxin/higher due to how the decrease in lactate production became evident at that point. The gene expression of PDP1 was significantly increased with high concentrations of digitoxin compared to other genes. The cell cycle assay indicated that most treatment groups did not make it to the G2 and M phase due to apoptosis. All in all, digitoxin showed to have apoptotic effects on panc-1 cells line although this effect was not evident in certain assays.

This project had a focus on the effect of digitoxin on inflammation in cancer Panc-1 cell line compared to BxPC-3 cell line. Pancreatic cancer, especially the form pancreatic adenocarcinoma is the fourth cause for cancer deaths, seen in the Western world. The progression and the initiation of the disease is a consequence of an interplay between genetic and inflammation events that are linked. Digitoxin is a cardiac glycoside with effect on various cancer types. Panc-1 cells and BxPC-3 cells were treated with different concentrations of digitoxin for 48 hours. Cell viability was measured, ELISA to measure expression of pro-IL-1β and IL-1β, since they are involved in the pathway and qPCR to measure gene expression. Gene expression was done to see if digitoxin affected the genes in the inflammasome pathway. The cell viability decreased with increasing concentration of digitoxin for both cell lines. TNFα was mostly downregulated in both Panc-1 and BxPC-3. NF-κB was upregulated in both cell lines. IL-1β was upregulated in BxPC-3 while gene expression was not seen in Panc-1. Lastly, IL-18 was downregulated in Panc-1, but upregulated in BxPC-3. A downregulation of IL-1β protein expression was seen in Panc-1 and in BxPC-3, a downregulation of pro IL-1β in Panc-1 and a downregulation of pro IL-1β in BxPC-3 was seen in ELISA. This study might provide an insight that is valuable for the researchers in the future to treat cancer from the perspective of an immune disorder.

碩士
高雄醫學院
藥學研究所
85
A simple and sensitive high performance liquid chromatographic method has been established for the trace determination of digoxin and digitoxin by derivatization. The method is based on the derivatization of digoxin and digitoxin with derivatizing agent, 1-naphthoyl chloride in pyridine at 50 ℃. Under the mild condition, the digoxin and digitoxin were derivatized as their highlysesitive derivatives. After the derivatization reaction, a dimethylamine aqueous solution was added to the reacted solution to avoid the interference of digoxin and digitoxin determination from the excess derivatizing reagent. The derivatives obtained were performed on a reversed-phase C8 column with 88 % acetonitrile as mobile phase and 7-methoxy-4-(docosanoyloxy methyl) coumarin as the internal standard. Several parameters affecting the derivatization of digoxin and digitoxin, including reaction temperature, reaction time and the amount of derivatizing agent, were investigated by high performance liquid chromatography with ultraviolet detection at 295 nm. The linear ranges of the method for the determination of digoxin and digitoxin were over 1-50 nmol/mL and 5-50 nmol/mL; the detection limit (signal-to-noise ratio = 5; injection volume, 20 μL) were 0.25 nmol/mL and 0.30 nmol/mL, respectively. Partial application of the method for analysis of digoxin in commercial tablets proved saticfactory. The HPLC conditions showed good selectivity for digoxin and digitoxin determination. Because the digoxin and digitoxin derivatives are highly responsive to a fluorimetric detection; an improved LC with fluorimetric detection is developed for trace determination of digoxin and digitoxin. The effects of several parameters on the derivatives formation were evaluated. The linear ranges for the quantitation of digoxin and digitoxin were over 30-300 pmol/mL and 50-300 pmol/mL; the detection limit (singnal-to-noise ratio = 5, injection volume, 20 μL) were 2.5 pmol/mL and 3.0 pmol/mL, respectively. Further application of the proposed method for trace analysis of digoxin and digitoxin in plasma by fluorimetric detection is in progress.

博士
國立臺灣大學
醫學檢驗暨生物技術學研究所
99
Human herpes virus type 1 (HSV-1) is known to have a tightly-regulated cascade expression of its immediate early (IE), early (E), and late (L) genes. Previously, digitoxin, which was widely-used as cardiac drug, was shown to inhibit in vitro replication of HSV-1. In this study, we found that digitoxin can repress HSV-2 and acyclovir-resistant HSV-1, and several structural analogues of digitoxin such as digoxin, ouabain octahydrate and G-strophanthin also exhibited anti-HSV activity. The inhibitory effects of digitoxin are likely to be introduced at the early stage of HSV-1 replication. In HSV-infected Vero cells, the protein levels of IE gene ICP4 and ICP27 as well as the ratio of ICP4-positive cells decreased in the presence of digitoxin. The decrease of ICP4 protein level by digitoxin was most effective when digitoxin was added at 3-4 h post-infection, indicating that the inhibitory effect was less likely attributed to blocking of virus entry or post-entry events, such as uncoating and nuclear localization. Moreover, this reduction was not due to degradation of ICP4 and ICP27 protein, and the steady mRNA level suggests that the ICP4 and ICP27 protein translation was interrupted by digitoxin. At present, the involvement of HSV-encoded small RNA, miR-H6, and some eukaryotic translation initiation factors, including eIF3m, eIF4G and eIF4E in digitoxin-induced interruption of viral protein translation was excluded, and the specific interaction between digitoxin and Na+/K+-ATPase (NKA) pump was proved to be essential for such inhibition. Our results demonstrated that digitoxin is a specific inhibitor of HSV IE protein translation and could be a candidate of anti-HSV agents.

碩士
國立陽明大學
生理學研究所
102
The cardiac glycosides are a group of compounds isolated from plants and some animals, and have been used in therapy for heart failure clinically since many years ago. Digoxin and digitoxin are the two major cardiac glycosides extracted from the foxglove plant, and the first traditional Chinese medicine extracts introduced to western medicine. Recently, some reports have shown that cardiac glycosides affect growth or survival in many cancer cell lines, including prostate cancer, lung cancer and human hepatoma cells. However, there are rare findings between cardiac glycosides and ovarian cancer cells. It has been well known that SKOV-3 is a human epithelium ovarian cancer cell line from sixty-years-old female Caucasian. Based on MTT assay, both digoxin and digitoxin have been shown to inhibit the proliferation of SKOV-3 in a dose-dependent manner for 24 and 48 hours. Moreover, there were no formation of colonies during digoxin (IC50, 10-6M) and digitoxin (IC50, 10-6M) treatments for 10 days. To examine the cytotoxicity of digoxin and digitoxin, LDH assay was employed. Digoxin (10-4M) and digitoxin (10-5M) treatments for 24 hours leading to a 10% elevated cytotoxicity. The continuous cell cycle is one of the key points when normal cells become cancer cells. In our results, we found that digoxin and digitoxin lead the cell cycle of SKOV-3 cells to arrest in G0/G1 phase measured by DNA content analysis, and increase the hypodiploid cells ratio for 72 hours . These data suggest that digoxin and digitoxin inhibit the growth of human epithelium ovarian cancer cell line, SKOV-3, through cell cycle arrest and cytotoxic effect. We expect that the present results will provide a new strategy for the therapy of ovarian cancer.

碩士
高雄醫學大學
藥學研究所
91
The purpose of this work is directed to the development of simple and sensitivity methods for the different medicines by micellar electrokinetic hromatography. The main study included simultaneous determination of cardiac glycosides and trace analysis of cefepime in human cerebral spinal fluid and plasma by micellar electrokinetic chromatography. The results are summarized as follow： 1. Simultaneous Determination of Digoxin and Digitoxin by Micellar Electrokinetic Chromatography A simple micellar electrokinetic chromatography is described for simultaneous determination of digoxin and digitoxin. The simultaneous analysis of digoxin and digitoxin was performed in Tris buffer (10 mM; pH 9) with 90 mM sodium dodecyl sulfate and 10% isopropyl alcohol as an anionic surfactant and organic modifier, respectively. Several parameters affecting the separation of the drugs were studied, including the pH and concentrations of the Tris buffer and sodium dodecyl sulfate. Partial application of the proposed method to the determination of digoxin in commercial tablets and in injections proved to be feasible. 2. Quantitative Determination of Cefepime in Human Cerebral Spinal Fluid and Plasma by Micellar Electrokinetic Chromatography A simple micellar electrokinetic chromatography is described for determination of cefepime in human cerebral spinal fluid and plasma. The analysis of cefepime in human cerebral spinal fluid was performed in Tris buffer (20 mM; pH 9) with 200 mM sodium dodecyl sulfate as an anionic surfactant. The analysis of cefepime in in human plasma was performed in Tris buffer (10 mM; pH 8) with 150 mM sodium dodecyl sulfate as an anionic surfactant. Several parameters affecting the separation of the drugs were studied, including the pH and concentrations of the Tris buffer and sodium dodecyl sulfate. Application of the method to the determination of cefepime in human cerebral spinal fluid and plasma proved to be feasible.