Studies designed to increase the stability and antiviral activity (HCMV) of the active benzimidazole nucleoside, TCRB.

The potent activity of 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) against Human Cytomegalovirus with the concomitant low cellular toxicity at concentrations that inhibit viral growth prompted considerable interest in this research area. This interest was moderated by the pharmacokinetic studies of TCRB in rats and monkeys that revealed the instability of TCRB in vivo. These studies suggested that the instability was due to a cleavage of the glycosidic bond in vivo which released the heterocycle (2,5,6-trichlorobenzimidazole) into the bloodstream. This prompted us to initiate synthetic studies designed to increase the stability of the glycosidic bond of TCRB and BDCRB. Several synthetic approaches to address this and other problems are presented.     

Design,Synthesis, and Antiviral Evaluation of Some Polyhalogenated Indole C-nucleosides

2,5,6-Trichloro-1-(β-D-ribofuranosyl)benzimidazole (TCRB), 2-bromo-5,6-dichloro-1-(β-D-ribofuranosyl)benzimidazole (BDCRB) and 2-benzylthio-5,6-dichloro-1-(β-D-ribofuranosyl)benzimidazole (BTDCRB) are benzimidazole nucleosides that exhibit strong and selective anti-HCMV activity. Polyhalogenated indole C-nucleosides were prepared as 1-deaza analogs of the benzimidazole nucleosides TCRB and BDCRB. A mild Knoevenagel coupling reaction between an indol-2-thione and a ribofuranose derivative was developed for the synthesis of 2-benzylthio-5,6-dichloro-3-(β-D-ribofuranosyl)indole (12). 3-(β-D-ribofuranosyl)-2,5,6-trichloroindole (16) was prepared from 12 in 4 steps. A Lewis acid-mediated glycosylation method was then developed to prepare the targeted 2-haloindole C-nucleoside 16 stereoselectively in four steps from the corresponding 2-haloindole aglycons. Only 12 was active against HCMV but it also was somewhat cytotoxic.     

Design,Synthesis, and Antiviral Evaluation of 2-Deoxy-D-Ribosides of Substituted Benzimidazoles as Potential Agents for Human Cytomegalovirus Infections

Abstract

Stereoselective glycosylation of 2,5,6-trichlorobenzimidazole (1b), 2-bromo-5,6-dichlorobenzimidazole (1c), 5,6-dichlorobenzimidazole (1d), 5,6-dichlorobenzimidazole-2-thione (1e), 5,6-dichloro-2-(methylthio)benzimidazole (1f), 2-(benzylthio)-5,6-dichlorobenzimidazole (1g), and 2-chloro-5,6-dimethylbenzimidazole (1h) with 2-deoxy-3,5-di-O-p-toluoyl-α-D-erythro-pentofuranosyl chloride was achieved to give the desired β nucleosides 2b-h. Subsequent deprotection afforded the corresponding free β-D-2-deoxyribosides 3b-h. The 2-methoxy derivative 3i was synthesized by the treatment of 2b with methanolic sodium methoxide. Displacement of the 2-chloro group of 2b with lithium azide followed by a removal of the protective groups gave the 2-azido-5,6-dichlorobenzimidazole derivative (5). The 2-amino derivative (6) was obtained by hydrogenolysis of 5 over Raney nickel. 5,6-Dichloro-2-isopropylamino-1-(2-deoxy-β-D-erythro-pentofuranosyl)benzimidazole (10) was prepared using 2′-deoxyuridine (7), N-deoxyribofuranosyl transferase and 1d followed by functionalization of the C2 position. Antiviral evaluation of target compounds established that compounds 3b and 3c were active against human cytomegalovirus (HCMV) at non-cytotoxic concentrations. The activity of these 2-deoxy ribosides, however, was less than the activity of the parent riboside, 2,5,6-trichloro-1-β-D-ribofuranosylbenzimidazole (TCRB). Compared to TCRB, 3b and 3c were somewhat more cytotoxic and active against herpes simplex virus type 1. Compounds 3d-i with other substituents in the 2-position were inactive against both viruses and non-cytotoxic. In contrast, compounds with amine substituents in the 2-position (5, 6, 10) were active against HCMV albeit less so than TCRB. These results establish that 2-deoxy-D-ribosyl benzimidazoles are less active against the DNA virus HCMV than are the corresponding D-ribosides.     

Benzimidazole 2′-Isonucleosides: Design,Synthesis, and Antiviral Activity of 2-Substituted-5,6-Dichlorobenzimidazole 2′-Isonucleosides

Abstract

2,5,6-Trihalogenated benzimidazole-β-D-ribofuranosyl nucleosides and 2-substituted amino-5,6-dichlorobenzimidazole-β-L-ribofuranosyl nucleosides are potent and selective inhibitors of human cytomegalovirus (HCMV). The D-ribofuranosyl analogs are metabolized rapidly in vivo rendering them unsuitable as drug candidates. The primary source of instability is thought to be the anomeric bond. The synthesis of a series of chemically stable benzimidazole-2′-isonucleosides is presented. The synthetic schemes employed are based on nucleophilic displacements of a 2′-tosylate from carbohydrate intermediates with 2-bromo-5,6-dichlorobenzidazole. 2-Bromo and 2-isopropyl amino analogs with 3′- and 5′-oxo and deoxy substitutions were prepared. The benzimidazole-2′-isonucleosides presented here demonstrated reduced activity against HCMV when compared to other D-ribofuranosyl benzimidazole analogs. In addition, they were not found to be inhibitors of HIV.     

Insect Transgenesis: Mechanisms,Applications, and Ecological Safety

Abstract

Glycosylation is considered to be an important reaction for the chemical modification of compounds with useful biological activities. Glycoside hydrolases are biotechnologically attractive enzymes which can be used in synthetic reactions for assembling glycosidic linkages with absolute stereoselectivity at an anomeric centre. Most of these enzymes are commercially available but there is great interest in the search for new biocatalysts with original catalytic characteristics. The marine environment has shown to be a very interesting source for new glycosyl hydrolases for both hydrolytic and synthetic aspects. In particular, Aplysia fasciata a marine herbivorous mollusc has been shown to be a potent producer of a library of glycoside hydrolases applied to the synthesis of glycosidic bonds. The impressive assortment of glycosidases in marine organisms clearly indicates that the potential biodiversity of these enzymes is still largely unexplored and that potential applications of biocatalysts from the sea will increase in the near future.     

Effective Anomerisation of 2′‐Deoxyadenosine Derivatives During Disaccharide Nucleoside Synthesis

The formation of a disaccharide nucleoside (11) by O3′‐glycosylation of 5′‐O‐protected 2′‐deoxyadenosine or its N ⁶‐benzoylated derivative has been observed to be accompanied by anomerisation to the corresponding α‐anomeric product (12). The latter reaction can be explained by instability of the N‐glycosidic bond of purine 2′‐deoxynucleosides in the presence of Lewis acids. An independent study on the anomerisation of partly blocked 2′‐deoxyadenosine has been carried out. Additionally, transglycosylation has been utilized in the synthesis of 3′‐O‐β‐d‐ribofuranosyl‐2′‐deoxyadenosines and its α‐anomer.     

Sarcomelicope alkaloids as leads for the discovery of new antitumor acronycine derivatives

The acridone alkaloid acronycine first isolated from Acronychia baueri Schott (Rutaceae) in 1948, was later shown to exhibit a promising activity against a broad spectrum of solid tumors. Nevertheless, clinical trials only gave poor results, probably due to the moderate potency and low water solubility of this alkaloid. Early studies on structure-activity relationships in the series concluded that the 1,2-double bond was an essential structural requirement to observe cytotoxic activity. It is the reason why the isolation in our laboratory of the unstable acronycine epoxide from several New-Caledonian Sarcomelicope species led to the hypothesis of bioactivation of acronycine by transformation of the 1,2-double bond into the corresponding oxirane in vivo. Consequently, there was interest in the search for acronycine derivatives modified in the pyran ring and having improved stability, but a similar reactivity toward nucleophilic agents as acronycine epoxide. Accordingly, we synthesized a series of cis- and trans-1,2-dihydroxy-1,2-dihydroacronycine diesters which exhibited interesting antitumor properties with a broadened spectrum of activity and increased potency when compared with acronycine. (±)-Cis-1,2-diacetoxy-1,2-dihydroacronycine was of particular interest, due to its marked activity in vivo against the resistant solid tumor C 38 colon carcinoma. The demonstration that acronycine should interact with DNA by some noncovalent process able to stabilize the double helix against thermal denaturation prompted us to develop benzo[b]acronycine analogues possessing an additional aromatic ring linearly fused on the natural alkaloid basic skeleton. When tested against a panel of cancer cell lines in vitro, cis-1,2-dihydroxy-1,2-dihydrobenzo[b]acronycine diesters exhibited cytotoxic activities within the same range of potency as the most active drugs currently used in cancer chemotherapy. In vivo, cis-1,2-diacetoxy-1,2-dihydrobenzo[b]acronycine (S23906-1), selected for further preclinical development, demonstated a marked antitumor activity in human orthotopic models of lung, ovarian and colon cancers xenografted in nude mice. This revised version was published online in June 2006 with corrections to the Cover Date.     

Twisting of glycosidic bonds by hydrolases

Patterns of scissile bond twisting have been found in crystal structures of glycoside hydrolases (GHs) that are complexed with substrates and inhibitors. To estimate the increased potential energy in the substrates that results from this twisting, we have plotted torsion angles for the scissile bonds on hybrid Quantum Mechanics::Molecular Mechanics energy surfaces. Eight such maps were constructed, including one for α-maltose and three for different forms of methyl α-acarviosinide to provide energies for twisting of α-(1,4) glycosidic bonds. Maps were also made for β-thiocellobiose and for three β-cellobiose conformers having different glycon ring shapes to model distortions of β-(1,4) glycosidic bonds. Different GH families twist scissile glycosidic bonds differently, increasing their potential energies from 0.5 to 9.5 kcal/mol. In general, the direction of twisting of the glycosidic bond away from the conformation of lowest intramolecular energy correlates with the position (syn or anti) of the proton donor with respect to the glycon’s ring oxygen atom. This correlation suggests that glycosidic bond distortion is important for the optimal orientation of one of the glycosidic oxygen lone pairs toward the enzyme’s proton donor.     

Beta Receptor Sensitivity Testing in Humans

Abstract

Understanding of beta receptor function in vitro and at the molecular level has advanced enormously in the last five years. With that improved understanding has come the recognition that some of the changes seen in certain pathophysiological states may be related to altered function of the beta receptor in vivo. For example changes in beta receptor function have been described following catecholamine administration (1), with altered sodium intake (2), pseudohypoparthyroidism (3), propranolol withdrawal (2, 4), corticosteroid administration (1), cardiac failure (5), hypertension (6), exercise (7), aging (9, 11) and recently endogenous depression (8). However prior to extrapolating the findings from ex vivo studies of receptor function to the in vivo setting it is necessary to correlate the ex vivo changes to in vivo in sensitivity. Unfortunately in contrast to the plethora of studies reporting changes in receptor function ex vivo the number of studies which have demonstrated a correlation between ex vivo change in beta receptor function and some in vivo change in sensitivity are relatively few. To some extent this reflects the difficulty in defining beta receptor sensitivity in vivo in patients or normal volunteers. The purpose of this paper is to briefly review the available techniques for assessing beta receptor sensitivity in man and suggest a potential improvement.

When assessing beta receptor sensitivity the first issue to be determined is the tissue of interest. Traditionally the response which has been evaluated is the chronotropic response of the heart (10) to a beta agonist such as isoproterenol. However, the response of other tissues including the lungs and vasculature have also been used.     

Influence ofL-Cysteine on the Oxidation Chemistry of (-)-Epinephrine: Formation of Cysteinyl Conjugates and Novel Dihydrobenzothiazines

Oxidation of epinephrine (EPI) in aqueous solution at pH 7.4 generates anortho-quinone(1)that normally deprotonates and undergoes a rapid intramolecular cyclization and secondary reactions, ultimately leading to an indolic melanin polymer. In this investigation, it is demonstrated thatL-cysteine (CySH) can intervene in this reaction by scavengingo-quinone1to give 5-S-cysteinylepinephrine (5-S-CyS-EPI) and 2-S-cysteinylepinephrine (2-S-CyS-EPI). Subsequent oxidation (2e, 2H⁺) of the latter cysteinyl conjugates giveso-quinones that can either react further with free CySH to give the 2,5-bi-S- and 2,5,6-tri-S-cysteinyl conjugates of EPI or cyclize to give 7-[(2-methylamino)ethyl]-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-E1) and 8-[(2-methylamino)ethyl]-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-E2), respectively, Oxidations of 2,5-bi-S-CyS-EPI and 2,5,6-tris-S-CyS-EPI and of DHBT-E1 and DHBT-E2 in the presence of CySH provide routes to a number of other dihydrobenzothiazines (DHBTs). Four new cysteinyl conjugates of EPI and seven DHBTs have been isolated and their structures elucidated by spectroscopic methods. Based upon a number of lines of converging evidence, it is suggested that these compounds might include unusual metabolites of EPI formed in adrenergic neurons under certain pathological brain conditions.     

Synthesis of an Epoxide Derivative of Thymidine as a Potential Enzyme Inhibitor

Abstract

Attempts to prepare I -[7,8-anhydro-2,5,6-trideoxy-α-L-lyxo-(and β-n-ribo)-octofuranosyl]thymine (10) by treatment of halohydrins 6–9 with sodium hydride in DMF or sodium methoxide in methanol gave mixtures of the epoxides 10 or 11 and the 3′,8′-anhydronucleoside 12. The structure of 12 was confirmed by oxidation to the cyclic ketone 14. The successful synthesis of 10 involved a Wittig reaction on the thymidine-5′-aldehyde 16 to give the unsaturated ketoacetate 18 which was reduced in two steps to the diacetate 20. The 7′-O-tosyl derivative 21 upon treatment with sodium methoxide in chloroform gave the pure epoxide 10 which was marginally toxic to L1210 cells in culture (I₅₀=25 μM) and demonstrated borderline in vivo activity (24% ILS) against P388 murine leukemia.     

Synthetic Studies on Guanofosfocin: Glycosylation of 8-Oxo-purine Nucleosides via Mitsunobu Reaction

Abstract

In model studies directed to the total synthesis of guanofosfocins, a unique glycosidic bond formation between the 8-oxo-purine nucleosides and mannopyranose derivatives under Mistunobu conditions is described.     

Solution NMR Conformational Analysis of the Potent Equilibrative Sensitive (ES) Nucleoside Transporter Inhibitor,S 6-(4-Nitrobenzyl)mercaptopurine Riboside (NBMPR)

Abstract

High resolution NMR analysis involving one-dimensional (1-D) ¹H and nuclear Overhauser (NOE) difference spectroscopy was applied to solutions of NBMPR in DMSO-d ₆. Coupling constants were obtained at different temperatures between 285 and 353 K, and used to analyze the rotamer preferences about the C-4′-C-5′ bond. The results revealed a rotamer distribution about the χ tortion angle that favors the high-anti range, a preponderance of the γ⁺ rotamer (at ～64 %) with respect to the γ torsion angle, and a higher population of the south (S) conformer, which was favored by as little as the 4 % to as much as 31 % over the north (N) conformer as calculated by the program PSEUROT 6.2. The high-anti glycosidic torsion orientation appears to be the major conformational difference between the solution structure of NBMPR determined in this study and the structure previously observed in the solid state.     

Comparison of Acid- and Enzyme-Catalyzed Cleavage of the Glycosidic Bond of N(7)-Substituted Guanosines

Abstract

Kinetic parametrs for enzymatic cleavage of the glycosidic bond (phosphorolysis) of ten N(7)-substituted guanosines were determined, and used to establish a structure-activity relation for the Michaelis constants. Results were compared with those for acid-catalyzed cleavage of the glycosidic bonds, and are consistent with a mechanism for phosphorolysis via protonation of the purine ring N(7).     

Hypoglycemic activity of curcumin synthetic analogues in alloxan-induced diabetic rats

The currently available therapies for type 2 diabetes have been unable to achieve normoglycemic status in the majority of patients. The reason may be attributed to the limitations of the drug itself or its side effects. In an effort to develop potent and safe oral antidiabetic agents, we evaluated the in vitro and in vivo hypoglycemic effects of 10 synthetic polyphenolic curcumin analogues on alloxan-induced male diabetic albino rats. In vitro studies showed 7-bis(3,4-dimethoxyphenyl)hepta-1,6-diene-3,5-dione (4) to be the most potential hypoglycemic agent followed by 1,5-bis(4-hydroxy-3-methoxyphenyl)penta-1,4-dien-3-one (10). Structure activity relationship (SAR) of the tested compounds was elucidated and the results were interpreted in terms of in vitro hypoglycemic activities. Furthermore, oral glucose tolerance test (OGTT) with compounds 4, 10 and reference hypoglycemic drug glipizide showed that compound 4 and glipizide had relatively similar effects on the reduction of blood glucose levels within 2?h. Thus, compound 4 might be regarded as a potential hypoglycemic agent being able to reduce glucose concentration both in vitro and in vivo.     

Synthesis and/or accumulation of bioactive molecules in the in vivo and in vitro root

Abstract

Roots of many species are studied because of the presence of high-value bioactive molecules, yet few studies have attempted to determine the biosynthetic pathways of these compounds or the way in which synthesis is regulated. The presence of secondary metabolites in the root does not necessarily mean that this organ is also the site of synthesis. Thus the identification of organ-specific intermediate precursors and key enzymes is important for understanding the biosynthetic pathway and the regulation of bioactive molecules. This knowledge could allow researchers to predict the suitability of in vitro systems, such as regenerated roots and hairy roots, for producing the molecules of interest. In the present review, the production of bioactive molecules in in vivo roots is compared to that in in vitro untransformed and transformed roots, concentrating on recent developments in the study of the biosynthesis of the anti-cancer alkaloid camptothecin in Camptotheca acuminata Decne. The results of a recent study performed in our laboratory on the production of camptothecin and other secondary metabolites in in vivo and in vitro C. acuminata roots are also presented.     

Synthesis and biological activity of progesterone derivatives as 5α-reductase inhibitors,and their effect on hamster prostate weight

In this study, we report the synthesis and biological evaluation of four 6- and 17-substituted progesterone derivatives (7–10). These compounds were prepared from the commercially available 17α-acetoxyprogesterone. The biological effect of these steroids was demonstrated in in vivo as well as in vitro experiments. In the in vivo experiments, we measured the activity of 6–10 on the weight of the prostate glands of gonadectomized hamsters treated with testosterone (T). For the studies in vitro, we determined the IC₅₀ value by measuring the concentration of steroidal derivative that inhibited 50% of the activity of 5α-reductase present in the human prostate. The results from this work indicated that compounds 6–9 significantly decreased the weight of the prostate as compared to testosterone-treated animals and this reduction of prostate weight was comparable to that produced by finasteride. Steroid 8 was the most effective of the tested compounds. However, compound 10 did not exhibit this capacity. On the other hand, 6–9 exhibited a high inhibitory activity for the human 5α-reductase enzyme with IC₅₀ values of 10, 70, 22, and 19?nM, respectively. However, 10 was not effective for the inhibition of 5α-reductase activity. In conclusion, the compounds that contained the acetate ester moiety in the molecule (6, 7, 8, and 9) inhibited the activity of 5α-reductase and decreased the weight of the prostate. Nevertheless, the double bond in ring B seems to diminish the inhibitory potency (7 and 9), since 6, which does not possess a double bond at C-6, had the highest inhibitory activity (the lowest IC₅₀ value).     

Formation of an Inclusion Complex of Anthocyanin with Cyclodextrin

A new gallotannin named kurigalin was isolated from the bark of chestnut tree. The chemical structure of kurigalin was established as 2,5,6-tri-O-galloyl-α,β-d-hamamelose (I) on the basis of enzymatic hydrolyses and spectral analyses.     

Nucleophilic N1 → N3 Rearrangement of 5′-O-Trityl-O2,3′-Cycloanhydrothymidine

Abstract

5′-O-Trityl-O²,3′-cycloanhydrothymidine (1) heated at 150°C in the presence of O,O-diethyl phosphate or O,O-diethyl phosphorothioate anions undergoes rearrangement into N³-isomer (2); its structure was established by both advanced NMR methods and X-ray crystallographic studies. The most probable mechanism of 1→2 rearrangement relies upon reversibility of glycosidic bond cleavage process.     

Preferential radioprotection to DNA of normal tissues by ferulic acid under ex vivo and in vivo conditions in tumor bearing mice

Our previous study showed that ferulic acid (FA) offered good radioprotection under in vitro and in vivo conditions to DNA and enhanced the DNA repair process in the peripheral blood leucocytes of mice in vivo. This study concerns radioprotection of normal versus tumor cells. Administration of FA (50 mg/kg body weight) to mice bearing fibrosarcoma tumor, 1 h prior to/ or immediately after radiation exposure (4 Gy) showed preferential radioprotection to normal cells i.e. peripheral blood leucocytes and bone marrow cells in comparison to tumor cells. This preferential protection under in vivo conditions could be attributed to poor vasculature in the tumor or peculiar characteristics of the tumor cells either to restrict its entry inside the cells or metabolize or inactivate the drug. To resolve these ex vivo study was carried out using bone marrow and tumor cells. It was found that under ex vivo condition also only bone marrow cells were protected by FA. Thus the studies revealed that FA showed preferential protection to normal cells under both in vivo and ex vivo conditions. (Mol Cell Biochem xxx: 1–10, 2005)