A new approach to phosphorylation of nucleosides using oxyonium phosphobetaines as intermediates.

Oxyonium phosphobetaines are recently discovered molecules with a unique -O-P-O-N+ bond system, which makes them useful and versatile intermediates for the synthesis of phosphates and their derivatives. In this paper, the preliminary data on the application of these compounds in nucleoside phosphorylation were presented.

Azomethine Ylides-Versatile Synthons for Pyrrolidinyl-Heterocyclic Compounds.

Azomethine ylides are nitrogen-based three-atom components commonly used in [3+2]-cycloaddition reactions with various unsaturated 2π-electron components. These reactions are highly regio- and stereoselective and have attracted the attention of organic chemists with respect to the construction of diverse heterocycles potentially bearing four new contiguous stereogenic centers. This review article complies the most important [3+2]-cycloaddition reactions of azomethine ylides with various olefinic, unsaturated 2π-electron components (acyclic, alicyclic, heterocyclic, and exocyclic ones) reported over the past two decades.

H-Phosphonate Chemistry in the Synthesis of Electrically Neutral and Charged Antiviral and Anticancer Pronucleotides.

In this review a short account of our work on the synthesis and biological activity of electrically neutral and charged anti-HIV and anticancer pronucleotides, presented on the background of the contemporary research in this area, is given.

Reaction of Boranephosphonate Diesters with Pyridines or Tertiary Amines in the Presence of Iodine: Synthetic and Mechanistic Studies.

Boranephosphonate diesters react with heteroaromatic and certain tertiary amines in the presence of an oxidant (I2) to afford the boron-modified phosphodiester analogues containing a P-B-N structural motif. Our multinuclear 31P and 11B NMR spectroscopy studies lend support for a two-step mechanism involving generation of a λ3-boranephosphonate intermediate that immediately coordinates an amine in the solvent cage, leading to B-pyridinium or B-ammonium boranephosphonate betaine derivatives. We found that the type of the solvent used (e.g., dichloromethane vs acetonitrile) significantly affected the course of the reaction, resulting in either formation of boron-modified derivatives or loss of the boron group with a subsequent oxidation of the phosphorus atom. In aprotic, electron-donating, polar solvents., e.g., acetonitrile (ACN) and tetrahydrofuran (THF), a λ3-boranephosphonate intermediate can also coordinate solvent molecules forming P-B-ACN or P-B-THF complexes that may influence the type of the products formed.

Aryl H-phosphonates. 19. New anti-HIV pronucleotide phosphoramidate diesters containing amino- and hydroxypyridine auxiliaries.

We have designed a new type of AZT and ddU phosphoramidate diesters containing various combinations of 2-, 3-, 4-aminopyridine and 2-, 3-, 4-hydroxypyridine moieties attached to the phosphorus center, as potential anti-HIV pronucleotides. Depending on the pKa values of the aminopyridines and the hydroxypyridines used, alternative synthetic strategies based on H-phosphonate chemistry were developed for their preparation. Synthetic aspects of these transformations and the biological activity of the synthesized compounds are discussed.

Reaction of Boranephosphonate Diesters with Amines in the Presence of Iodine: The Case for the Intermediacy of H-Phosphonate Derivatives.

Mechanistic and stereochemical aspects of the reaction of boranephosphonate diesters with amines promoted by iodine were investigated. This is a complex, multistep reaction that ultimately produces the corresponding phosphoramidate diesters via a formal replacement of the borane group by an amine moiety. We found by a stereochemical correlation analysis that, contrary to a literature report, the whole transformation proceeded with total inversion of the configuration at the phosphorus center. Our study also showed that instead of the postulated nucleophilic substitution by iodide at the phosphorus center of the initially formed intermediate, the corresponding iodoboranephosphonate, the crucial step of the reaction involved intermediacy of H-phosphonate derivatives that reacted with iodine to afford ultimately phosphoramidate diesters. The reaction of the iodoboranephosphonate with the amine to produce an aminoboranephosphonate diester that rapidly dissociated into the corresponding H-phosphonate and the borane parts was apparently instrumental to the formation of an H-phosphonate diester intermediate.

New antiglioma zwitterionic pronucleotides with an FdUMP framework.

We have designed and synthesized new 5-fluoro-2'-deoxyuridine 5'-phosphate pronucleotides which can function as potential agents against the glioblastoma multiforme tumor. Their anti-malignant potency has been tested against T98G, U-118 MG, U-87 MG gliomas, HeLa, and Caco-2 cancer cell lines, using MRC-5 healthy cells as a reference. Five of the sixteen compounds (4c, 4f-i) exhibited significant anticancer potency and high selectivity indices (SI 12-66). It is likely that these zwitterionic pronucleotides may function in a similar manner to zwitterionic phospholipids, by inducing cell membrane charge disorder, making the cell permeable to bioactive agents. The most promising therapeutic pronucleotides 4c, 4f-h, have high intestinal-blood uptake potency (Caco-2 cell line), and may be considered as potential, orally administrated, anticancer drugs.

Searching for anti-glioma activity. Ribonucleoside analogues with modifications in nucleobase and sugar moieties.

Several ribonucleoside analogues with modifications in the nucleobase and sugar moiety have been screened for anti-glioma activity in the T98G glioma cell line using cervical (HeLa) cell line as reference human malignant cells, and lung fibroblast (MCR-5) cell line as non-cancerous reference cells. Among the investigated compounds, ribonucleosides containing 6-chloropurine (3), 7-guanine (5) and a pyrrolopyrimidine (18) as nucleobases, show promising anti-glioma activity with good selectivity indices, and can be considered as lead structures for further anti-cancer studies.

New 3'-O-aromatic acyl-5-fluoro-2'-deoxyuridine derivatives as potential anticancer agents.

New aromatic and aliphatic 3'-O-acyl-5-fluoro-2'-deoxyuridine derivatives were synthesized and evaluated as candidates for prodrugs against various cancer cell lines. As the most promising candidate for antimalignant therapeutics was found a dual-acting acyl derivative 7h, which apparently released not only the known anticancer nucleoside, 5-fluoro-2'-deoxyuridine (FdU), but also an additional active metabolite, acetylsalicylic acid, reinforcing thus therapeutic effect of FdU. Promising therapeutic indices showed also some aromatic dicarboxylic acids derivatives decorated with FdU esters (11 and 12).

Aryl H-Phosphonates 18. Synthesis, properties, and biological activity of 2',3'-dideoxynucleoside (N-heteroaryl)phosphoramidates of increased lipophilicity.

Recently, AZT (N-pyridyl)phosphoramidates were reported as a new type of potential anti-HIV therapeutics. In continuation of that work, here we present new (N-heteroaryl)phosphoramidate derivatives of antiviral 2',3'-dideoxynucleosides containing other types of N-heteroaryl moieties, particularly those with higher lipophilicity. The present studies comprise mechanistic investigations using (31)P NMR correlation analysis, which permitted improvements in the synthetic procedures. The obtained compounds were tested in biological systems to establish their cytotoxicity and anti-HIV activity. The results were analyzed with respect to possible correlations between biological and physico-chemical properties of the phosphoramidates studied, to get some insight into their antiviral mode of action.

Novel reactivity of Fhit proteins: catalysts for fluorolysis of nucleoside 5'-phosphoramidates and nucleoside 5'-phosphosulfates to generate nucleoside 5'-phosphorofluoridates.

Fragile histidine triad (HIT) proteins (Fhits) occur in all eukaryotes but their function is largely unknown. Human Fhit is presumed to function as a tumour suppressor. Previously, we demonstrated that Fhits catalyse hydrolysis of not only dinucleoside triphosphates but also natural adenosine 5'-phosphoramidate (NH2-pA) and adenosine 5'-phosphosulfate (SO4-pA) as well as synthetic adenosine 5'-phosphorofluoridate (F-pA). In the present study, we describe an Fhit-catalysed displacement of the amino group of nucleoside 5'-phosphoramidates (NH2-pNs) or the sulfate moiety of nucleoside 5'-phosphosulfates (SO4-pNs) by fluoride anion. This results in transient accumulation of the corresponding nucleoside 5'-phosphorofluoridates (F-pNs). Substrate specificity and kinetic characterization of the fluorolytic reactions catalysed by the human Fhit and other examples of involvement of fluoride in the biochemistry of nucleotides are described. Among other HIT proteins, human histidine triad nucleotide-binding protein (Hint1) catalysed fluorolysis of NH2-pA 20 times and human Hint2 40 times more slowly than human Fhit.

Recent advances in H-phosphonate chemistry. Part 2. Synthesis of C-phosphonate derivatives.

This chapter provides an overview of recent advances in the development of new methods and protocols for the formation of the P-C bond using H-phosphonate diesters as starting materials. Various chemical and stereochemical aspects of the transition metal-catalyzed cross-coupling and organocatalyst-promoted reactions which are relevant to the synthesis of structurally diverse C-phosphonate derivatives are surveyed.

Recent advances in H-phosphonate chemistry. Part 1. H-phosphonate esters: synthesis and basic reactions.

This review covers recent progress in the preparation of H-phosphonate mono- and diesters, basic studies on mechanistic and stereochemical aspects of this class of phosphorus compounds, and their fundamental chemistry in terms of transformation of P-H bonds into P-heteroatom bonds. Selected recent applications of H-phosphonate derivatives in basic organic phosphorus chemistry and in the synthesis of biologically important phosphorus compounds are also discussed.

The case of triethylammonium cation loss during purification of certain nucleotide analogues: a cautionary note.

Nucleotides, their analogues, and other phosphate esters and phosphoramidates often contain the triethylammonium cation as a counterion. We found that this may be lost during chromatographic purification or concentration of solutions, yielding products in acidic forms or containing sub-stoichiometric amounts of the counterion. This in turn may be detrimental, e.g., due to possible decomposition of a compound or inaccurate sample preparation. Correlations between the structure of studied compounds and their susceptibility for cation loss were analyzed. Modifications in preparative techniques were developed to obtain the studied compounds with stoichiometric anion to cation ratios.

Design, synthesis and QSAR studies of dispiroindole derivatives as new antiproliferative agents.

A variety of 4'-aryl-3-(arylmethylidene)-1″-[(cyclic-amino)methylene]-1'-methyl-dispiro[cyclohexane-1,3'-pyrrolidine-2',3″-[3H]indole]-2,2″(1″H)-diones 4a-u were prepared via reaction of 2E,6E-bis(arylidene)-1-cyclohexanones 1a-i with azomethine ylides, generated in situ via a decarboxylative condensation of isatins 2a-c and sarcosine (3). Single crystal X-ray study of 4a, revealed structural and stereochemical features of these derivatives. While most of the synthesized compounds exhibit mild antitumor properties when tested against various human tumor cell lines (HEPG2 "liver", HELA "cervical" and PC3 "prostate" cancers), three of them, 4d and 4p (active against HEPG2), and compound 4g (active against HELA), demonstrated higher activities, that were close or even higher than that of the reference standard Doxorubicin. QSAR studies revealed good predictive and statistically significant 3 descriptor models (r2=0.903-0.812, r2adjusted=0.855-0.672, r2prediction=0.773-0.605).

Nucleoside 3',5'-cyclic H-phosphonates, new useful precursors for the synthesis of nucleoside 3',5'-cyclic phosphates and their analogues.

Nucleoside H-phosphonates activated with a condensing agent spontaneously formed nucleoside 3',5'-cyclic H-phosphonates. The cyclization was stereoselective and produced one of the P-diastereomers in preponderance (de ca. 80%). Nucleoside 3',5'-cyclic H-phosphonates were stereochemically unstable and underwent epimerization affording the thermodynamically more stable diastereomer as a major product (de ca. 80%). They were susceptible to hydrolysis, transesterification, and oxidation and by changing oxidation protocols nucleoside 3',5'-cyclic phosphate analogues, e.g. phosphodiesters, phosphorothioate diesters, and phosphotriesters, were obtained.

Computational study of the mechanism and selectivity of palladium-catalyzed propargylic substitution with phosphorus nucleophiles.

The mechanism and sources of selectivity in the palladium-catalyzed propargylic substitution reaction that involves phosphorus nucleophiles, and which yields predominantly allenylphosphonates and related compounds, have been studied computationally by means of density functional theory. Full free-energy profiles are computed for both H-phosphonate and H-phosphonothioate substrates. The calculations show that the special behavior of H-phosphonates among other heteroatom nucleophiles is indeed reflected in higher energy barriers for the attack on the central carbon atom of the allenyl/propargyl ligand relative to the ligand-exchange pathway, which leads to the experimentally observed products. It is argued that, to explain the preference of allenyl- versus propargyl-phosphonate/phosphonothioate formation in reactions that involve H-phosphonates and H-phosphonothioates, analysis of the complete free-energy surfaces is necessary, because the product ratio is determined by different transition states in the respective branches of the catalytic cycle. In addition, these transition states change in going from a H-phosphonate to a H-phosphonothioate nucleophile.

Synthesis and QSAR study of novel cytotoxic spiro[3H-indole-3,2'(1'H)-pyrrolo[3,4-c]pyrrole]-2,3',5'(1H,2'aH,4'H)-triones.

1,3-Dipolar cycloaddition reaction of 1-aryl-1H-pyrrole-2,5-diones 1a-e with non-stabilized azomethine ylides, generated in situ via decarboxylative condensation of isatins 2a-c and sarcosine (3) in refluxing ethanol, afforded 4'-aryl-5'a,6'-dihydro-1'-methyl-spiro[3H-indole-3,2'(1'H)-pyrrolo[3,4-c]pyrrole]-2,3',5'(1H,2'aH,4'H)-triones 4a-o in good yields. Compound 4l exhibited high anti-tumor activity against HEPG2 (liver cancer) cell line (IC(50) = 12.16 µM) compared to that of Doxorubicin (IC(50) = 7.36 µM), and the other synthesized compounds revealed moderate anti-tumor properties against HCT116 (colon), MCF7 (breast) and HEPG2 (liver) human tumor cell lines. 3D-Pharmacophore modeling and quantitative structure-activity relationship (QSAR) analysis were combined to explore the structural requirements controlling the observed anti-tumor properties. It was found that the major structural factors affecting potency of these compounds were related to their basic skeleton.

31P NMR and computational studies on stereochemistry of conversion of phosphoramidate diesters into the corresponding phosphotriesters.

31P NMR spectroscopy was used to investigate a stereochemical course of a nitrite-promoted conversion of phosphoramidate diesters into the corresponding phosphotriesters. It was found that this reaction occurred with almost complete epimerization at the phosphorus center and at the C1 atom in the amine moiety. On the basis of the 31P NMR data, a plausible mechanism for the reaction was proposed. The density functional theory calculation of the key step of the reaction, i.e., breaking of the P-N bond and formation of the P-O bond, suggested a one-step S(N)2(P) process with retention of configuration at the phosphorus center.