Academic literature on the topic 'Digitoxina'

We previously showed that digitoxin prolongs the survival of rats with heart failure due to myocardial infarction (MI). In this study, we evaluated the effect of digitoxin on myocardial structure, ventricular function, and proteins involved in calcium kinetics. Seventy-two rats with MI >35% of the left ventricle were randomly assigned to 4 treatment groups: sham (n = 15), digitoxin (n = 11), infarction (n = 20), and infarction + digitoxin (n = 26). The rats were assessed 120 days after surgery by echocardiogram, hemodynamics, papillary muscle mechanics, collagen content, cardiomyocyte nuclear volume, and Western blot analysis of proteins involved in calcium kinetics. Digitoxin was administered via the rat chow. Two-way analysis of variance was used for comparisons. Myocardial infarction caused inotropic impairment, pulmonary congestion, increase of nuclear volume, myocardial collagen, and Na+/Ca2+ exchanger levels, and decreased SERCA2 and phosphorylated phospholamban levels. Treatment with digitoxin showed improvements in cardiac remodeling, inotropism, ventricular performance, pulmonary congestion, collagen accumulation, nuclear volume, and proteins involved in calcium kinetics. In rats with heart failure due to MI, long-term treatment with digitoxin attenuates congestive heart failure, mitigates myocardial remodeling and contractile impairment, and preserves myocardial levels of proteins involved in calcium kinetics.

The 1H and 13C NMR spectra of the cardenolides digitoxigenin, digoxigenin, digitoxin, and mono- and bis-digitoxigenin digitoxosides have been completely assigned by two-dimensional NMR spectroscopy. The techniques used include phase-sensitive COSY, multiple relay COSY, and carbon–proton correlation (HETCOR and HMQC) spectra. Various aspects of the solution conformation of the steroid moiety of digitoxin and digoxigenin could be determined from coupling constants and NOE difference experiments and they are indicative of an all-chair conformation. The carbohydrate rings in digitoxin and the mono- and bis-digitoxigenin digitoxosides are also in the chair conformation. Keywords: cardenolides, digitoxigenin, digitoxin, 2-dimensional NMR, conformational analysis.

ABSTRACT Host-directed therapeutics for human cytomegalovirus (HCMV) requires elucidation of cellular mechanisms that inhibit HCMV. We report a novel pathway used by cardiac glycosides to inhibit HCMV replication: induction of AMP-activated protein kinase (AMPK) activity and autophagy flux through the Na+,K+/ATPase α1 subunit. Our data illustrate an intricate balance between the autophagy regulators AMPK, mammalian target of rapamycin (mTOR), and ULK1 during infection and treatment with the cardiac glycoside digitoxin. Both infection and digitoxin induced AMPK phosphorylation, but ULK1 was differentially phosphorylated at unique sites leading to opposing effects on autophagy. Suppression of autophagy during infection occurred via ULK1 phosphorylation at Ser757 by enhanced mTOR activity. Digitoxin continuously phosphorylated AMPK, leading to ULK1 phosphorylation at Ser317, and suppressed mTOR, resulting in increased autophagy flux and HCMV inhibition. In ATG5-deficient human fibroblasts, digitoxin did not inhibit HCMV, supporting autophagy induction as a mechanism for virus inhibition. Drug combination studies with digitoxin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) further confirmed the role of autophagy activation in HCMV inhibition. Individually, each compound phosphorylated AMPK, but their combination reduced autophagy rather than inducing it and was antagonistic against HCMV, resulting in virus replication. The initial ULK1 activation by digitoxin was counteracted by AICAR, which prevented the downstream interaction of Beclin1 and phosphatidylinositol 3-kinase class III (PI3K-CIII), further supporting digitoxin-mediated HCMV inhibition through autophagy. Finally, the α1 subunit was required for autophagy induction, since in α1-deficient cells neither AMPK nor autophagy was activated and HCMV was not inhibited by digitoxin. In summary, induction of a novel pathway (α1-AMPK-ULK1) induces autophagy as a host-directed strategy for HCMV inhibition. IMPORTANCE Infection with human cytomegalovirus (HCMV) creates therapeutic challenges in congenitally infected children and transplant recipients. Side effects and selection of resistant mutants with the limited drugs available prompted evaluation of host-directed therapeutics. We report a novel mechanism of HCMV inhibition by the cardiac glycoside digitoxin. At low concentrations that inhibit HCMV, digitoxin induced signaling through the α1 subunit of the Na+,K+/ATPase pump and the cellular kinase AMPK, resulting in binding and phosphorylation of ULK1 (Ser317) and autophagy activation. HCMV suppressed autophagy through ULK1 phosphorylation (Ser757) by activating the mTOR kinase. The pump-autophagy pathway was required for HCMV inhibition, since in α1- or ATG5-deficient cells the virus was not inhibited. Furthermore, the AMPK activator AICAR antagonized digitoxin activity against HCMV, a phenomenon resulting from opposing effects downstream in the autophagy pathway, at the Beclin1 stage. In summary, autophagy may provide a strategy for harnessing HCMV replication.

Binding of warfarin, digitoxin, diazepam, salicylate and Phenol Red, individually or in different pair combinations, to defatted human serum albumin at ligand/protein molar ratios less than 1:1 was studied at pH 7.0. The binding was determined by ultrafiltration. Some of the experiments were repeated with the use of equilibrium dialysis in order to strengthen the results. Irrespective of the method used, all ligands bind to one high-affinity binding site with an association constant in the range 10(4)-10(6) M-1. High-affinity binding of the following pair of ligands took place independently: warfarin-Phenol Red, warfarin-diazepam, warfarin-digitoxin and digitoxin-diazepam. Simultaneous binding of warfarin and salicylate led to a mutual decrease in binding of one another, as did simultaneous binding of digitoxin and Phenol Red. Both effects could be accounted for by a coupling constant. The coupling constant is the factor by which the primary association constants are affected; in these examples of anti-co-operativity the factor has a value between 0 and 1. In the first example it was calculated to be 0.8 and in the latter 0.5. Finally, digitoxin and salicylate were found to compete for a common high-affinity binding site. The present findings support the proposal of four separate primary binding sites for warfarin, digitoxin (and salicylate), diazepam and Phenol Red. An attempt to correlate this partial binding model for serum albumin with other models in the literature is made.

The cytochrome P-450-dependent monooxygenase digitoxin 12β-hydroxylase from cell cultures of Digitalis lanata needs NADPH and molecular oxygen and hydroxylates cardiac glycosides with the aglycon of digitoxigenin to the corresponding derivatives of the C-series. Other electron donors cannot replace NADPH. The apparent Km-values are 26 μᴍ for NADPH, 7.1 μᴍ for β-methyldigitoxin and 10 μᴍ for digitoxin. The reaction is inhibited by NADP+ and cytochrome c in a competitive mode. The optimum temperature was at 20 °C. Low concentrations of Mn2+, Mg2+, and EDTA were slightly stimulatory, but there was no strict dependence on divalent cations. Digitoxin 12β-hydroxylase is very stable at room temperature and the reaction proceeds for more than 20 h. After the addition of 15% glycerol, 70% of the original activity can be retained subsequent to freezing at -18 °C. By means of linear sucrose gradient fractionation of cellular membranes the digitoxin 12β-hydroxylase was found to be located in the endoplasmic reticulum.

The effects of chronic treatment with digitoxin on arterial baroreceptor sensitivity for heart rate (HR) and renal sympathetic nerve activity (rSNA) control, cardiopulmonary reflex, and autonomic HR control in an animal model of heart failure (HF) were evaluated. Wistar rats were treated with digitoxin, which was administered in their daily feed (1 mg/kg per day) for 60 days. The following 3 experimental groups were evaluated: sham, HF, and HF treated with digitoxin (HF + DIG). We observed an increase in rSNA in the HF group (190 ± 29 pps, n = 5) compared with the sham group (98 ± 14 pps, n = 5). Digitoxin treatment prevented an increase in rSNA (98 ± 14 pps, n = 7). Therefore, arterial baroreceptor sensitivity was decreased in the HF group (−1.24 ± 0.07 bpm/mm Hg, n = 8) compared with the sham group (−2.27 ± 0.23 bpm/mm Hg, n = 6). Digitoxin did not alter arterial baroreceptor sensitivity in the HF + DIG group. Finally, the HF group showed an increased low frequency band (LFb: 23 ± 5 ms2, n = 8) and a decreased high frequency band (HFb: 77 ± 5 ms2, n = 8) compared with the sham group (LFb: 14 ± 3 ms2; HFb: 86 ± 3 ms2, n = 9); the HF+DIG group exhibited normalized parameters (LFb: 15 ± 3 ms2; HFb: 85 ± 3 ms2, n = 9). In conclusion, the benefits of decreasing rSNA are not directly related to improvements in peripheral cardiovascular reflexes; such occurrences are due in part to changes in the central nuclei of the brain responsible for autonomic cardiovascular control.

Callus derived from Digitalis purpurea hypocotils were grown during a 6-week period on solid Murashige–Skoog medium supplemented with 1 mg/L 6-benzylaminopurine, 0.01 mg/L gibberellic acid and 0.1 mg/L indole-3-acetic acid or α-naphthaleneacetic acid, with or without phenobarbital (40 mg/L). The presence of phenobarbital in the culture medium caused a reduction of the vacuole/cytoplasm ratio. At the same time, the chloroplastic volume fraction decreased in callus tissue cells grown in media supplemented with phenobarbital, while the mitochondrial volume ratio increased. Digitoxin content was enhanced in callus tissues, especially in those grown on indole-3-acetic acid medium supplemented with phenobarbital. The relationship between ultrastructure of D. purpurea callus and digitoxin content is discussed. Keywords: Digitalis purpurea tissue cultures, digitoxin, phenobarbital, mitochondria, chloroplast.

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.

Despite a gradual decline in the clinical use of digitalis glycosides, digitalis toxicity continues to be responsible for substantial morbidity and mortality, particularly among the elderly. Digitalis poisoning may occur acutely, after intentional overdose, but is more often seen as the result of chronic intoxication among patients receiving digitalis therapy. Clinical findings in chronic digitalis poisoning are often subtle. The astute clinician will enquire about digitalis use in older patients with vague complaints and will not be dissuaded from considering digitalis toxicity in the face of a ‘therapeutic’ digitalis blood concentration. Two digitalis preparations continue to be used with frequency, depending on geography. Digoxin is the digitalis glycoside of choice in the USA, while digitoxin prevails in some parts of Europe. While the methods and half-lives of elimination differ markedly for these two substances, the approach to poisoning by either is similar. Advanced age, underlying cardiovascular disease, and severe hyperkalaemia represent poor prognostic factors in digitalis poisoning. Early administration of digitalis Fab fragments should be undertaken when life-threatening symptoms are present. Prophylactic therapy with reduced doses of Fab fragments should be strongly considered for less serious toxicity.