The Pronucleotide Approach. III. Synthesis,Anti-HBV Activity and Stability Studies of the Bis(S-pivaloyl-2-thioethyl) Phosphotriester Derivative of Acyclovir

Abstract

The nucleoside analog Acyclovir (ACV) is used in the treatment of herpes simplex (HSV) and varicella-zoster (VZV) diseases. The possibility to extend the application field of ACV by using the bis[SATE] pronucleotide approach in order to deliver ACVMP inside the cell was investigated. And actually, the title compound has potent anti-hepatitis B activity in cell culture experiments. Here, we also report its synthesis and stability in various media.     

Evaluation of various types of new hypoxic cell sensitizers using the EMT6 single cell-spheroid-solid tumour system

Eleven new hypoxic cell sensitizers representative of those developed in Japan between 1980 and 1985 were evaluated in vitro and in vivo in comparison with misonidazole (MISO), SR-2508, Ro 03-8799, and ANT (2-amino-5-nitrothiazole). The new compounds included 2-nitroimidazole nucleoside analogues, nitrotriazoles and other nitroaromatics, non-nitro compounds, and electron-affinic compounds that readily intercalate DNA. The sensitizing activity in the EMT6 single cells correlated not only with the reduction potential but, for some compounds, also with the reactivity with non-protein sulphydryls. The sensitizers were also tested using the EMT6 spheroids and solid tumours. The patterns of changes in sensitizer enhancement ratios (SERs) for single cells, spheroids, and solid tumours were classified into two types: (1) SERs for the three testing systems were similar; and (2) SERs decreased in the order of: single cells, spheroids, and solid tumours. Only nitroimidazole and nitrotriazole derivatives belonged to the former type. RK-28 and RK-29, 2-nitroimidazoles with sugar analogue components, had in vivo effects almost equal to those of MISO. Also 3- and 4-nitrotriazole derivatives had definite in vivo effects.     

Design and Synthesis of Dually Branched 5′-Norcarbocyclic Adenosine Phosphonodiester Analogue as a New Anti-HIV Prodrug

A novel 3′,4′-dimethyl-5′-norcarbocyclic adenosine phosphonic acid was prepared using acyclic stereoselective route from 4-hydroxybutan-2-one (4). To improve the cellular permeability and enhance the anti-HIV activity of this phosphonic acid, a (bis)SATE phosphonodiester nucleoside prodrug (20) was prepared and its chemical stability was evaluated. The newly synthesized bis(SATE) analogue (20) and its parent nucleoside phosphonic acid (18) were assayed for anti-HIV activity using an in vitro assay system in a CEM cell line.     

SYNTHESIS AND STUDY OF A NEW SERIES OF PHOSPHORAMIDATE DERIVATIVES AS MONONUCLEOTIDE PRODRUGS

The synthesis and the study of new mononucleoside phosphoramidate diesters bearing S-acyl-2-thioethyl (SATE) groups and an alkylamino residue are reported. The studied compounds appear to be able to deliver the corresponding 5′-mononucleotide inside the cells, and could be considered as prototypes for a new kind of mononucleotide prodrugs (pronucleotides).     

Regiospecific Reduction of 4,6-Dinitrobenzimidazoles: Synthesis,Characterization, and Biological Evaluation

The regiospecific reduction of 4,6-dinitrobenzimidazole derivatives leading to the corresponding 4-amino-6-nitrobenzimidazoles was studied. The identification of the formed product structures was accomplished by spectroscopic and X-ray diffraction data. The anticancer and antiparasitic activities of the synthesized compounds were examined, and promising activities against Toxoplasma gondii and Leishmania major parasites were discovered for certain 4,6-dinitrobenzimidazoles in addition to moderate anticancer activities of the 4-amino-6-nitrobenzimidazole derivatives against T. gondii cells. However, the tumor cell experiments revealed a promising sensitivity of p53-negative colon cancer cells to these compounds.     

Neutral glycosphingolipids and gangliosides of human lung and lung tumours.

In order to help determine whether alterations of the profiles of glycosphingolipids occur consistently in human tumours, the neutral glycosphingolipids and gangliosides of nine lung tumours (one adenocarcinoma, four squamous cell, two mixed adeno-squamous cell, one large cell and one oat-cell carcinomata) were analysed. The control tissue consisted of adjacent lung; it contained neutral glycosphingolipids corresponding in properties to glucosyl-, lactosyl-, globotriaosyl- and globotetraosyl-ceramides. All of the tumours also contained these four neutral glycosphingolipids. However, in addition, five of the tumours (two of the squamous, the large cell and the two mixed adeno-squamous cell carcinomata) contained neutral glycosphingolipids corresponding in properties to lactotriaosyl- and neolactotetraosyl-ceramides; these same tumours also exhibited higher amounts of lactosylceramide than the other tumours analysed. Both of the two former neutral glycosphingolipids and very substantial amounts of the latter neutral glycosphingolipid were detected in pneumonic lung and in polymorphonuclear leucocytes; it thus appears possible that these particular compounds were derived from these latter cells rather than from the tumour cells. The ganglioside patterns of the tumours were almost equivalent in complexity to that exhibited by the control lung tissue. This study shows that the profiles of two major classes of glycosphingolipids (neutral glycosphingolipids and gangliosides) occurring in lung tumours are almost as complex as those of the parent tissue, a finding in contrast with the notably simplified patterns of these lipids found in many cancer cells grown in vitro. It also suggests that when lactotriaosyl- and neolactotetraosyl-ceramides and high amounts of lactosylceramide are detected in human tumours, the possibility must be considered that these compounds are derived from polymorphonuclear leucocytes.     

Design and synthesis of dually branched 5'-norcarbocyclic adenosine phosphonodiester analogue as a new anti-HIV prodrug

A novel 3',4'-dimethyl-5'-norcarbocyclic adenosine phosphonic acid was prepared using acyclic stereoselective route from 4-hydroxybutan-2-one (4). To improve the cellular permeability and enhance the anti-HIV activity of this phosphonic acid, a (bis)SATE phosphonodiester nucleoside prodrug (20) was prepared and its chemical stability was evaluated. The newly synthesized bis(SATE) analogue (20) and its parent nucleoside phosphonic acid (18) were assayed for anti-HIV activity using an in vitro assay system in a CEM cell line.     

Induction and repression of enzymes involved in exogenous purine compound utilization in Bacillus cereus

5′-Nucleotidase, adenosine phosphorylase, adenosine deaminase and purine nucleoside phosphorylase, four enzymes involved in the utilization of exogenous purine compounds in Bacillus cereus, were measured in extracts of this organism grown in different conditions. It was found that adenosine deaminase is inducible by addition of adenine derivatives to the growth medium, and purine nucleoside phosphorylase by metabolizable purine and pyrimidine ribonucleosides. Adenosine deaminase is repressed by inosine, while both enzymes are repressed by glucose. Evidence is presented at during growth of B. cereus in the presence of AMP, the concerted action of 5′-nucleotidase and adenosine phosphorylase, two constitutive enzymes, leads to formation of adenine, and thereby to induction of adenosine deaminase. The ionsine formed would then cause induction of the purine nucleoside phosphorylase and repression of the deaminase. Taken together with our previous findings showing that purine nucleoside phosphorylase of B cereus acts as a translocase of the ribose moiety of ionsine inside the cell (Mura, U., Sgarrella, F. and Ipata, P.L. (1978) J. Biol. Chem. 253, 7905–7909), our results provide a clear picture of the molecular events leading to the utilization of the sugar moiety of exogenous AMP, adenosine and inosine as an energy source.     

Inhibition of the synthesis of polyamines and macromolecules by 5′-methylthioadenosine and 5′-alkylthiotubercidins in BHK21 cells

5′-Methylthioadenosine and four 5′-alkylthiotubercidins were tested for their ability to inhibit polyamine synthesis in vitro and to decrease polyamine concentration and prevent growth of baby-hamster-kidney (BHK21) cells. 5′-Methylthioadenosine and 5′-methylthiotubercidin decreased the activity of spermidine synthase from brain to roughly the same extent, whereas brain spermine synthase was much more strongly inhibited by 5′-methylthioadenosine compared with 5′-methylthiotubercidin. These nucleoside derivatives also inhibited the growth of BHK21 cells and increased the concentration of putrescine. 5′-Methylthioadenosine decreased cellular spermine concentration, whereas 5′-methylthiotubercidin lowered the concentration of spermidine. The activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase were enhanced in cells grown in the presence of 5′-methylthiotubercidin. The growth inhibition produced by these nucleoside derivatives was not reversed by exogenous spermidine or spermine. 5′-Ethylthiotubercidin, 5′-propylthiotubercidin and 5′-isopropylthiotubercidin did not appreciably inhibit spermidine or spermine synthase in vitro or decrease the cellular polyamine content, but effectively prevented the growth of BHK21 cells. All nucleoside derivatives at concentrations of 0.2–1 mm caused a rapid inhibition of protein synthesis. It is concluded that the growth inhibition produced by 5′-methylthioadenosine and 5′-alkylthiotubercidins was not primarily due to polyamine depletion but other target sites, for instance the cellular nucleotide pool, cell membranes etc. must be considered.     

Novel semisynthetic derivatives of betulin and betulinic acid with cytotoxic activity

A series of new imidazole carboxylic esters (carbamates) and N-acylimidazole derivatives of betulin and betulinic acid (14–29) have been synthesized. The new compounds were screened for in vitro cytotoxicity activity against human cancer cell lines HepG2, Jurkat and HeLa. A number of compounds have shown IC₅₀ values lower than 2 μM against the cancer cell lines tested and the vast majority has shown a better cytotoxicity profile than betulinic acid, including the betulin derivatives. N-Acylimidazole derivatives 26 and 27 (IC₅₀ 0.8 and 1.7 μM in HepG2 cells) and the C-3 carbamate derivative 16 (IC₅₀ 2.0 μM in HepG2 cells) were the most promising compounds. Based on the observed cytotoxicity, structure–activity relationships have been established.     

Phenyl phosphotriester derivatives of AZT: variations upon the SATE moiety

Synthesis, in vitro anti-HIV activity, stability studies as well as potential for oral absorption of some novel phenyl S-acyl-2-thioethyl (SATE) phosphotriester derivatives of AZT (zidovudine; 3'-azido-2',3'-dideoxythymidine) are described herein. These pronucleotides are characterized by the presence of polar functions on the SATE biolabile phosphate protections. Whereas derivatives incorporating an amino residue in the vicinity of the thioester functionality display low chemical stability, the introduction of one or two hydroxyl groups on the SATE moieties confers high resistance of the resulting prodrugs towards esterase hydrolysis. Thus, one of these pronucleotides, the monohydroxylated SATE derivative of AZT 2, is able to cross a Caco-2 cell monolayer mainly in intact form, probing that further development is warranted as a possible HIV-pronucleotide candidate.     

DNA lesion bypass polymerases and 4’-thio-β-Darabinofuranosylcytosine (T-araC)

The 4’-thio-β-D-arabinofuranosylcytosine (T-araC) is a newly developed nucleoside analog that has shown promising activity against a broad spectrum of human solid tumors in both cellular and xenograft mice models. TaraC shares similar structure with another anticancer deoxycytidine analog, β-D-arabinofuranosylcytosine (araC, cytarabine), which has been used in clinics for the treatment of acute myelogenous leukemia but has a very limited efficacy against solid tumors. T-araC exerts its anticancer activity mainly by inhibiting replicative DNA polymerases from further extension after its incorporation into DNA. DNA lesion bypass polymerases can manage the DNA lesions introduced by therapeutic agents, such as cisplatin and araC, therefore reduce the activity of these compounds. In this study, the potential relationships between the lesion bypass Y-family DNA polymerases η, ι and κ (pol η, pol ι, and pol κ) and T-araC were examined. Biochemical studies indicated that the triphosphate metabolite of T-araC is a less preferred substrate for the Y-family polymerases. In addition, cell viability study indicated that pol η deficient human fibroblast cells were more sensitive to T-araC when compared with the normal human fibroblast cells. Together, these results suggest that bypass polymerases reduced cell sensitivity to T-araC through helping cells to overcome the DNA damages introduced by T-araC.     

Thermophilic Biodesulfurization of Various Heterocyclic Sulfur Compounds and Crude Straight-Run Light Gas Oil Fraction by a Newly Isolated Strain Mycobacterium phlei WU-0103

Various heterocyclic sulfur compounds such as naphtho[2,1-b]thiophene (NTH) and benzo[b]thiophene (BTH) derivatives can be detected in diesel oil, in addition to dibenzothiophene (DBT) derivatives. Mycobacterium phlei WU-0103 was newly isolated as a bacterial strain capable of growing in a medium with NTH as the sulfur source at 50°C. M. phlei WU-0103 could degrade various heterocyclic sulfur compounds, not only NTH and its derivatives but also DBT, BTH, and their derivatives at 45°C. When M. phlei WU-0103 was cultivated with the heterocyclic sulfur compounds such as NTH, NTH 3,3-dioxide, DBT, BTH, and 4,6-dialkylDBTs as sulfur sources, monohydroxy compounds and sulfone compounds corresponding to starting heterocyclic sulfur compounds were detected by gas chromatography–mass spectrometry analysis, suggesting the sulfur-specific desulfurization pathways for heterocyclic sulfur compounds. Moreover, total sulfur content in 12-fold-diluted crude straight-run light gas oil fraction was reduced from 1000 to 475 ppm S, with 52% reduction, by the biodesulfurization treatment at 45°C with growing cells of M. phlei WU-0103. Gas chromatography analysis with a flame photometric detector revealed that most of the resolvable peaks, such as those corresponding to alkylated derivatives of NTH, DBT, and BTH, disappeared after the biodesulfurization treatment. These results indicated that M. phlei WU-0103 may have a good potential as a biocatalyst for practical biodesulfurization of diesel oil.     

bis(S-Acyl-2-thioethyl)esters of 2′,3′-Dideoxyadenosine 5′-Monophosphate Are Potent Anti-HIV Agents in Cell Culture

Abstract

It is shown that ddA bis(SATE)phosphotriesters have potent anti-HIV activity in cell culture. Thus, compared with the parent nucleoside, a decrease of 3 or 4 orders of magnitude was observed in the EC₅₀ values for the bis(S-acetyl-2-thioethyl)phosphotriester derivative, which makes this compound as active as AZT.     

Synthesis of fluorescent nucleoside analogs as probes for 2′-deoxyribonucleoside kinases

We are reporting on the synthesis of fluorescent nucleoside analogs with modified sugar moieties (e.g., sugars other than ribose and 2′-deoxyribose). Four novel derivatives of the fluorescent thymidine analog 6-methyl-3-(β-D-2′-deoxyribofuranosyl) furano-[2,3-d]pyrimidin-2-one were synthesized via Sonogashira reaction and subsequent copper-catalyzed cycloaddition. These compounds represent promising tools for studying nucleoside metabolism inside living cells, as well as for screening directed evolution libraries of 2′-deoxyribonucleoside kinases with new and improved activity for the corresponding nucleoside analogs.     

Synthesis,radiolabeling, and preclinical evaluation of a new octreotide analog for somatostatin receptor‐positive tumor scintigraphy

Radiolabeled somatostatin analogs have become powerful tools in the diagnosis and staging of neuroendocrine tumors, which express somatostatin receptors. The aim of this study was to evaluate a new somatostatin analog, 6‐hydrazinopyridine‐3‐carboxylic acid‐Ser³‐octreotate (HYNIC‐SATE) radiolabeled with ^99mTc, using ethylenediamine‐N,N′‐diacetic acid and tricine as coligands, to be used as a radiopharmaceutical for the in vivo imaging of somatostatin receptor subtype 2 (SSTR2)‐positive tumor. Synthesis of the peptide was carried out on a solid phase using a standard Fmoc strategy. Peptide conjugate affinities for SSTR2 were determined by receptor binding affinity on rat brain cortex and C6 cell membranes. Internalization rate of ^99mTc‐HYNIC‐SATE was studied in SSTR2‐expressing C6 cells that were used for intracranial tumor studies in rat brain. A reproducible in vivo C6 glioma model was developed in Sprague–Dawley rat and confirmed by histopathology and immunohistochemical analysis. Biodistribution and imaging properties of this new radiopeptide were also studied in C6 tumor‐bearing rats. Radiolabeling was performed at high specific activities, with a radiochemical purity of >96%. Peptide conjugate showed high affinity binding for SSTR2 (HYNIC‐SATE IC₅₀ = 1.60 ± 0.05 n m ) and specific internalization into rat C6 cells. After administration of ^99mTc‐HYNIC‐SATE in C6 glioma‐bearing rats, a receptor specific uptake of radioactivity was observed in SSTR‐positive organs and in the implanted intracranial tumor and rapid excretion from nontarget tissues via kidneys. ^99mTc‐HYNIC‐SATE is a new receptor‐specific radiopeptide for targeting SSTR2‐positive brain tumor and might be of great promise in the scintigraphy of SSTR2‐positive tumors. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and Antitumor Activity of Cyclopropane Derivatives of Betulinic and Betulonic Acids

New cyclopropane derivatives of betulin were synthesized by attachment of dichlorocarbenes or dibromocarbenes to the double bond of betulin diacetate, followed by the deprotection of hydroxyl groups. The betulin cyclopropane derivative was obtained from 20,29-dihydro-20,29-dichloromethylenebetulin by treatment with lithium in tert-butanol. These compounds were converted into the corresponding derivatives of betulonic acid by oxidation with chromium trioxide. The reduction of oxo group with sodium borohydride led to the corresponding derivatives of betulinic acid. 20,29-Dihydro-20,29-dichloromethylenebetulinic acid proved to be the most cytotoxic toward human melanoma of the Colo 38 and Bro lines and human ovarian carcinoma of the CaOv line (IC₅₀ 10 μM). 20,29-Dihydro-20,29-dibromomethylenebetulinic acid inhibited the growth of the Bro melanoma cell line and the CaOv carcinoma cell line practically by 50% at a concentration of 10 μM. The other derivatives of betulinic and betulonic acids were active toward all of the three cancer cell lines at concentrations higher than 10 μM.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 320–325.Original Russian Text Copyright © 2005 by Symon, Veselova, Kaplun, Vlasenkova, Fedorova, Lyutik, Gerasimova, Shvets.     

Synthesis and Studies of 3′-C-Trifluoromethyl-β-D-ribonucleosides Bearing the Five Naturally Occurring Nucleic Acid Bases

Abstract

3′-C-Trifluoromethyl-β-D-ribonucleoside derivatives bearing the five naturally occurring nucleic acid bases have been synthesized. All these derivatives were prepared by glycosylation reactions of purine and pyrimidine bases with a suitable peracylated 3-C-trifluoromethyl ribofuranose precursor. After deprotection, the resulting title nucleoside analogues were tested for their inhibitory properties against the replication of HIV, HBV and several RNA viruses. However, none of these compounds showed significant antiviral activity.     

Rapid synthesis of 2′,3′-dideoxy-3′β-fluoro-pyrimidine nucleosides from 2′-deoxypyrimidine nucleosides

A rapid synthesis of 2′,3′-dideoxy-3′-fluoro-β-d-threo-nucleosides bearing the pyrimidine canonical bases of nucleic acids has been developed in order to discover new nucleoside derivatives as potential antiviral drugs. However, when evaluated for their antiviral activity in cell culture experiments, none of these compounds showed any significant antiviral activity.