Design and synthesis of biotinylated cardiac glycosides for probing Nur77 protein inducting pathway.

The orphan nuclear receptor Nur77 (also known as TR3 or nerve growth factor-induced clone B NGFI-B) functions as a nuclear transcription factor in the regulation of target gene expression and plays a critical role in the regulation of differentiation, proliferation, apoptosis, and survival of many different cell types. Recent studies demonstrate that Nur77 also involves many important physiological and pathological processes including cancer, inflammation and immunity, cardiovascular diseases, and bone diseases. Our previous studies showed that cardiac glycosides could induce the expression of Nur77 protein and its translocation from the nucleus to the cytoplasm and subsequent targeting to mitochondria, leading to apoptosis of cancer cells. In order to probe the Nur77 protein inducting pathway, we designed and synthesized a series of novel biotinylated cardiac glycosides from ß-Antiarin and α-Antiarin, two typical cardiac glycosides from the plant of Antiaris toxicaria. The induction of Nur77 protein expression of these biotinylated cardiac glycosides and their inhibitory effects on NIH-H460 cancer cell proliferation were evaluated. Results displayed that some biotinylated cardiac glycosides could significantly induce the expression of Nur77 protein comparable with their parent compounds ß-Antiarin and α-Antiarin. Also, their streptavidin binding activities were evaluated. Among them, biotinylated cardiac glycosides P4b and P5a exhibited significant effect on the induction of Nur77 expression along with high binding capacity with streptavidin, suggesting that they can be used as probes for probing Nur77 protein inducting pathway.

Spin-labeled derivatives of cardiotonic steroids as tools for characterization of the extracellular entrance to the binding site on Na+ ,K+ -ATPase.

The information obtained from crystallized complexes of the Na+ ,K+ -ATPase with cardiotonic steroids (CTS) is not sufficient to explain differences in the inhibitory properties of CTS such as stereoselectivity of CTS binding or effect of glycosylation on the preference to enzyme isoforms. The uncertainty is related to the spatial organization of the hydrophilic cavity at the entrance of the CTS-binding site. Therefore, there is a need to supplement the crystallographic description with data obtained in aqueous solution, where molecules have significant degree of flexibility. This work addresses the applicability of the electron paramagnetic resonance (EPR) method for the purpose. We have designed and synthesized spin-labeled compounds based on the cinobufagin steroid core. The length of the spacer arms between the steroid core and the nitroxide group determines the position of the reporting group (N-O) confined to the binding site. High affinity to Na+ ,K+ -ATPase is inferred from their ability to inhibit enzymatic activity. The differences between the EPR spectra in the absence and presence of high ouabain concentrations identify the signature peaks originating from the fraction of the spin labels bound within the ouabain site. The degree of perturbations of the EPR spectra depends on the length of the spacer arm. Docking of the compounds into the CTS site suggests which elements of the protein structure might be responsible for interference with the spin label (e.g., steric clashes or immobilization). Thus, the method is suitable for gathering information on the cavity leading to the CTS-binding site in Na+ ,K+ -ATPase in all conformations with high affinity to CTS.

Production of the Cytotoxic Cardenolide Glucoevatromonoside by Semisynthesis and Biotransformation of Evatromonoside by a Digitalis lanata Cell Culture.

Recent studies demonstrate that cardiac glycosides, known to inhibit Na+/K+-ATPase in humans, have increased susceptibility to cancer cells that can be used in tumor therapy. One of the most promising candidates identified so far is glucoevatromonoside, which can be isolated from the endangered species Digitalis mariana ssp. heywoodii. Due to its complex structure, glucoevatromonoside cannot be obtained economically by total chemical synthesis. Here we describe two methods for glucoevatromonoside production, both using evatromonoside obtained by chemical degradation of digitoxin as the precursor. 1) Catalyst-controlled, regioselective glycosylation of evatromonoside to glucoevatromonoside using 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide as the sugar donor and 2-aminoethyldiphenylborinate as the catalyst resulted in an overall 30 % yield. 2) Biotransformation of evatromonoside using Digitalis lanata plant cell suspension cultures was less efficient and resulted only in overall 18 % pure product. Structural proof of products has been provided by extensive NMR data. Glucoevatromonoside and its non-natural 1-3 linked isomer neo-glucoevatromonoside obtained by semisynthesis were evaluated against renal cell carcinoma and prostate cancer cell lines.

Concise Enantioselective Total Synthesis of Cardiotonic Steroids 19-Hydroxysarmentogenin and Trewianin Aglycone.

The expedient and scalable approach to cardiotonic steroids carrying oxygenation at the C11- and C19-positions has been developed and applied to the total asymmetric synthesis of steroids 19-hydroxysarmentogenin and trewianin aglycone as well as to the assembly of the panogenin core. This new approach features enantioselective organocatalytic oxidation of an aldehyde, diastereoselective Cu(OTf)2-catalyzed Michael reaction/tandem aldol cyclizations, and one-pot reduction/transposition reactions allowing a rapid (7 linear steps) assembly of a functionalized cardenolide skeleton. The ability to quickly set this steroidal core with preinstalled functional handles and diversity elements eliminates the need for difficult downstream functionalizations and substantially improves the accessibility to the entire class of cardenolides and their derivatives for biological evaluation.

High Throughput Screening for Colorectal Cancer Specific Compounds.

The development of new anti-cancer therapeutic agents is necessary to improve antitumor efficacy and reduce toxicities. Here we report using a systematic anticancer drug screening approach we developed previously, to concurrently screen colon and glioma cancer cell lines for 2000 compounds with known bioactivity and 1920 compounds with unknown activity. The hits specific to each tumor cell line were then selected, and further tested with the same cells transfected with EGFP (Enhanced Green Fluorescent Protein) alone. By comparing the percentage of signal reduction from the same cells transfected with the sensor-conjugated reporter system; hits preferably causing apoptosis were identified. Among the known lead compounds, many cardiac glycosides used as cardiotonic drugs were found to effectively and specifically kill colon cancer cells, while statins (hypolipidemic agents) used as cholesterol lowering drugs were relatively more effective in killing glioma cells.

Synthesis of cardiac glycoside analogs by catalyst-controlled, regioselective glycosylation of digitoxin.

The cardiac glycoside natural product digitoxin was selectively glycosylated at one of its five hydroxyl groups using a borinic acid derived catalyst. This method provided access to the glycosylation pattern characteristic of a subclass of natural products from Digitalis purpurea. Variation of the glycosyl donor was tolerated, enabling the synthesis of novel cardiac glycoside analogs from readily available materials.

Chemical synthesis of the cardiotonic steroid glycosides and related natural products.

The active components from the extracts of Digitalis, cardiotonic steroid glycosides, have been ingested by humans for more than 200 years as a medicinal therapy for heart failure and abnormal heart rhythms. The positive inotropic activity of the cardiotonic steroids that mediates clinically useful physiological effects in patients has been attributed largely to a high affinity inhibitory interaction with the extracellular surface of the membrane-bound sodium pump (Na(+)/K(+)-ATPase). However, previously unrecognized intracellular signaling pathways continue to be uncovered. This Review examines both partial and de novo synthetic approaches to the medicinally important and structurally captivating cardenolide and bufadienolide steroid families, with an emphasis on the stereocontrolled construction of the pharmacophoric aglycone (genin) framework.

The cephalostatins. 21. Synthesis of bis-steroidal pyrazine rhamnosides (1).

The synthesis of bis-steroidal pyrazines derived from 3-oxo-11,21-dihydroxypregna-4,17(20)-diene (4) and glycosylation of a D-ring side chain with α-L-rhamnose have been summarized. Rearrangement of steroidal pyrazine 10 to 14 was found to occur with boron triflouride etherate. Glycosylation of pyrazine 10 using 2,3,4-tri-O-acetyl-α-L-rhamnose iodide led to 1,2-orthoester-α-L-rhamnose pyrazine 17b. By use of a persilylated α-L-rhamnose iodide as donor, formation of the orthoester was avoided. Bis-steroidal pyrazine 10 and rhamnosides 17b and 21c were found to significantly inhibit cancer cell growth in a murine and human cancer cell line panel. Pyrazine 9 inhibited growth of the nosocomial pathogen Enterococcus faecalis.

First total synthesis of rhodexin A.

An efficient total synthesis of rhodexin A (1) is reported. An initial inverse-electron-demand Diels-Alder reaction of the acyldiene 6 with the silyl enol ether 7 gave the cycloadduct 8 with the required 4 contiguous stereocenters in a single step. This compound was then transformed into the tetracyclic enone 16, which was converted to rhodexin A (1).

Synthesis of several cleistrioside and cleistetroside natural products via a divergent de novo asymmetric approach.

The de novo asymmetric syntheses of several partially acylated dodecanyl tri- and tetra-rhamnoside natural products (cleistriosides-5 and 6 and cleistetrosides-2 to 7) have been achieved (19-24 steps). The divergent route requires the use of three or less protecting groups. The asymmetry was derived via Noyori reduction of an acylfuran. The rhamno-stereochemistry was installed by a diastereoselective palladium-catalyzed glycosylation, ketone reduction and dihydroxylation.

Enhancing the anticancer properties of cardiac glycosides by neoglycorandomization.

Glycosylated natural products are reliable platforms for the development of many front-line drugs, yet our understanding of the relationship between attached sugars and biological activity is limited by the availability of convenient glycosylation methods. When a universal chemical glycosylation method that employs reducing sugars and requires no protection or activation is used, the glycorandomization of digitoxin leads to analogs that display significantly enhanced potency and tumor specificity and suggests a divergent mechanistic relationship between cardiac glycoside-induced cytotoxicity and Na+/K+-ATPase inhibition. This report highlights the remarkable advantages of glycorandomization as a powerful tool in glycobiology and drug discovery.

Oxidative fragmentation of pregna-14,16-dien-20-ones to 14 beta-hydroxyandrost-15-en-17-ones.

Two methods have been developed for efficient conversion of pregna-14,16-dien-20-ones into 14 beta-hydroxyandrost-15-en-17-ones. One procedure consists of treatment of the ring-D dienone successively with sodium borohydride and singlet oxygen. The reaction is illustrated by the conversion of pregna-14,16-dien-20-one 1 into 14 beta-hydroxyandrost-15-en-17-one 3, via the corresponding allylic alcohol 2. Although this two-step procedure is simple, it provides 3 in relatively low yield, accompanied by a smaller amount of the isomeric 14 alpha-hydroxyandrost-15-en-17-one 6. An alternative one-step conversion is achieved by treatment of dienone 1 with a peroxyacid in the presence of a strong protic acid. This process is illustrated by the two-step conversion of dienone 1 into hydroxy ketone 11 in 51% overall yield (Scheme 5) and by the analogous conversion of dienone 13 into hydroxy ketone 24 in 61% overall yield (Scheme 11).

Stereoselective synthesis of 2-S-phenyl-2-deoxy-beta-glycosides using phenyl 2,3-O-thionocarbonyl-1-thioglycoside donors via 1,2-Migration and concurrent glycosidation.

[figure: see text] 1,2-Migration and concurrent glycosidation of phenyl 2,3-O-thionocarbonyl-1-thlo-alpha-L-rhamnopyranosides under the action of methyl trifluoromethanesulfonate (MeOTf) afforded in high yields the 3-O-(methylthio)carbonyl-2-S-phenyl-2,6-dideoxy-beta-L-glucopyranosides, ready precursors to the corresponding 2-deoxy-beta-glycosides.

Triagem fitoquímica preliminar de tetrapterys multiglandulosa A. Juss. (Malpighiaceae) / Preliminary phytochemical screening of tetrapterys multiglandulosa A. Juss. (Malpighiaceae)

Este trabalho foi desenvolvido com o objetivo de identificar os princípios tóxicos da tetrapterys multigalndulosa A. Juss. Realizou-se triagem fitoquímica nas folhas (brotos e maduras) frescas, através de marcha analítica clássica e Cromatografia em Camada Delgada (CCD) pesquisando-se os seguintes princípios ativos: heterosídeos antrasênicos, saponínicos, flavônicos, cardiotônicos, taninos (hidrolisáveis e condensados), alcalóides (terciários e quaternários), composto esteróides e cumarínicos. Os resultados obtidos mostraram na folha jovem e madura, presença de heterosídeos flavônicos e esteróides. Taninos condensados e alcalóides quaternários foram encontrados somente na folha madura.

[Synthesis of lactone ring-containing bioactive natural products. Cardiotonic steroids, naphthoquinone antibiotics, and tetronic acid ionophore antibiotics].

Achievements in the synthestic study on cardiotonic steroids, pyranonaphthoquinone antibiotics, and tetronic acid ionophore antibiotics from author's laboratory are reviewed.

Structure-activity relationships of synthetic cardiac glycosides.

The antiviral, cytotoxic and anti-ATPase activities of 14 synthetic bufalyl glycosides were compared with each other. Among these glycosides, the activities of the gentiobioside and the melibioside were much weaker than those of the others. On the other hand, these three activities were found to be highly correlated with each other. These were parallel to the case of the digitoxigenyl glycosides in our previous paper.

[Studies on cardiac ingredients of plants. X. Preparation of nitrates of tetrahydroproscillaridin and their pharmacological activities].

To reduce the vascular contracting effect of the hydrogenated cardiac glycosides, 20-(R)- and 20-(S)-tetrahydroproscillaridins (THPs, 1a, 1b), and to extend the concentration-dependent range, mono- and dinitrates of THPs were prepared. The pharmacological activities of the nitrates of THP were evaluated by use of isolated guinea-pig papillary muscle preparations and Na+,K(+)-adenosine triphosphatase preparations from dog kidney. Furthermore, the effect for smooth muscle was examined using the helical strips isolated from 13-week-old spontaneously hypertensive rat. The positive inotropic effects of mononitrates (11a, 11b, 2a, 2b, 8a, and 8b) were more potent than those of THPs. Nitration of the sugar moiety in THPs resulted in a vascular relaxing effect unobserved in the case of THPs.

[A new class of cardiotonic steroids]. / Novyi klass kardiotonicheskikh steroidov.

A series of 23-oxosteroid derivatives have been synthesized and tested for their inhibiting Na+, K(+)-dependent ATPase from rat brain in the 1 x 10(-6)-1 x 10(-4) M concentrations. Natural 23-oxogenins from sea star Asterias amurensis and synthetic monoesters showed the inhibiting activity upto 50-55%. These compounds caused heart contraction in frogs at the level of the known cardiotonic strophanthin G, and inotropic activity on isolated heart of mollusk Spisula sachalinensis.

Synthesis of 17 beta-(3'-thiophenyl)-5 beta-androstane-3 beta,14 beta-diol 3-D-glucopyranoside, an anti-inotropic cardiac glucoside.

In a continuing effort to synthesize cardiac glucoside analogs with modified 17 beta-functional groups for pharmacologic testing, we used 3 beta-benzyloxy-5 beta-androst-15-en-17-one as an efficient intermediate. This report describes a preparation of 17 beta-(3'-thiophenyl)-5 beta-androstane-3 beta,14 beta-diol 3-D-glucopyranoside.