A unique ring-expanded acyclic nucleoside analogue that inhibits both adenosine deaminase (ADA) and guanine deaminase (GDA; guanase): synthesis and enzyme inhibition studies of 4,6-diamino-8H-1-hydroxyethoxymethyl-8-iminoimidazo[4,5-e][1,3]diazepine.

The synthesis and enzyme inhibition studies of a novel ring-expanded acyclic nucleoside analogue are reported. Compound has been found to be a competitive inhibitor of both adenosine deaminase (ADA) and guanine deaminase (GDA; guanase) with K(i)'s equal to 1.52+/-0.34 x 10(-4) M and 2.97+/-0.25 x 10(-5) M, respectively. Inhibition of two enzymes of purine metabolism may bear beneficial implications in antiviral therapy.

Acyclic nucleoside/nucleotide analogues with an imidazole ring skeleton.

Syntheses of a few acyclic nucleoside and acyclic nucleoside phosphonate analogues containing an imidazole ring have been reported. These analogues include methyl 1-(2-hydroxyethoxymethyl)imidazole-4, 5-dicarbo-xylate (1), 4,5-dicarbamoyl-1-(2-hydroxyethoxymethyl)imidazole (2), 4,5-dicyano-1-(2-hydroxyethoxymethyl)imidazole (4), Methyl 1-(2-bromoethoxymethyl)imidazole-4,5-dicarboxylate (7), 4,5-dicyano-(2-bromoethoxymethyl)imidazole (8), and Methyl 1-(2-phosphonomethoxyethyl)imidazole (10). Also reported are a few potential prodrugs of the above compounds, including the acetyl derivatives 5 and 6 (of 1 and 4, respectively), and the diethyl phosphonate ester 9 (of 10). In addition, the corresponding benzyl-protected precursors 11 and 12 (of 1 and 4, respectively), along with their common hydrolysis product, 1-(2-benzyloxy-ethoxymethyl)-4,5-imidazoledicarboxylic acid (3), are reported. Another potential prodrug included in the list is 1-(2-acetoxyethyl)-4,5-dicyanoimidazole (15). The compounds were screened for in vitro antiviral activity against a wide variety of herpes and respiratory viruses. The most active compound was the phosphonate analogue 9 which exhibited an anti-measles virus activity with an EC50 of <2.5 microg/mL and an SI value of > 176.

Potent in vitro anticancer activities of ring-expanded ("fat") nucleosides containing the imidazo[4,5-e][1,3]diazepine ring system.

The ring-expanded ("fat") nucleoside, 4,8-diamino-6-imino-6H-1-beta-D-ribofuranosylimidazo[4,5-e][1,3]diazepine (1) and its 2',3',5'-tri-O-benzoyl derivative (2) exhibited potent broad spectrum anticancer activities in vitro against a wide variety of human tumor cell lines. The tribenzoyl derivative 2 was found to be considerably more active than the parent nucleoside 1. Further studies using human prostate cancer cells PC-3 and DU-145 suggest that the treatment of exponentially growing culture cells with 1 and 2 leads to marked loss of cell viability in a dose-dependent manner.

Investigations into biochemical mode of inhibition of guanase by azepinomycin: synthesis and biochemical screening of several analogues of azepinomycin.

In an effort to biochemical mode of guanase inhibition as well as the structure-activity relationships of azepinomycin, five analogues (I-V) of azepinomycin were synthesized and screened against guanase from rabbit liver. Our results suggest that while the 6-hydroxy group of azepinomycin is crucial for activity, its putative transition state mode of inhibition of guanase is questionable. The additional H-bonding sites at position 5, and hydrophobic groups in and around position 3 of azepinomycin appear to be tolerated, and may in fact enhance the potency of inhibition.

Potent anti-hepatitis B viral activity and inhibition of bacteriophage T7 RNA polymerase by a "fat" nucleoside and its 5'-triphosphate derivative: synthetic, biochemical, and biological studies of 4,8-diamino-6-imino-6H-1-beta-D-ribofuranosyl-imidazo[4,5-E] [1,3]diazepine-5'-triphosphate.

The title nucleoside, 4,8-diamino-6-imino-6H-1-beta-d-ribofuranosylimidazo[4,5-e][1,3]-d iazepine, exhibited potent anti-hepatitis B viral activity with minimum toxicity in vitro, and its 5'-triphosphate derivative strongly inhibited the bacteriophage T7 RNA polymerase.

An efficient, short synthesis and potent anti-hepatitis B viral activity of a novel ring-expanded purine nucleoside analogue containing a 5:7-fused, planar, aromatic, imidazo[4,5-e][1,3]diazepine ring system.

An efficient, short synthesis of a ring-expanded nucleoside analogue containing a novel 5:7-fused, planar, and potentially aromatic imidazo[4,5-e][1,3]diazepine heterocyclic ring system is reported. The target compound, 6-amino-8-hydroxy-4H-1-beta-D- ribofuranosylimidazo[4,5-e][1,3]diazepin-4-one (2) was synthesized in a single step in > or = 90% yield by condensation of guanidine with either methyl 1-beta-D-ribofuranosylimidazole-4,5- dicarboxylate(1a) or its 2',3',5'-tri-O-benzoyl derivative (1b). Compound 2 showed potent anti-hepatitis B virus (anti-HBV) activity with an EC50 value of 0.17 microM in the transfected hepatoma cell line 2.2.15, and a low cellular toxicity with a CC50 value of 2.4 mM (TI > 14,000).

Affinity reagents for cross-linking hemoglobin: bis(phenoxycarbonylethyl)phosphinic acid (BPCEP) and bis(3-nitrophenoxycarbonylethyl)phosphinic acid (BNCEP).

Synthesis and properties of two new macrobiomolecular cross-linking reagents, bis(phenoxycarbonylethyl) phosphinic acid (BPCEP) and bis(3-nitrophenoxycarbonylethyl)phosphinic acid (BNCEP), have been reported. The reagents were successfully employed to cross-link human hemoglobin under oxygenated conditions. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high performance liquid chromatography (HPLC), and fast protein liquid chromatography (FPLC) analyses of the reaction products indicated that the cross-link was intramolecular in nature, and that it was between the two beta subunits of hemoglobin in each case. The products were purified by DEAE-cellulose chromatography, and the purified material was employed for oxygen-binding assessments. The oxygen equilibrium curve of the cross-linked material, in each case, was right-shifted toward lower oxygen affinity as desired. The sigmoidal shapes of oxygen curves, in each case, suggested retainment of oxygen-binding cooperativity, although considerably lower than that of the native hemoglobin

Substrate/inhibition studies of bacteriophage T7 RNA polymerase with the 5'-triphosphate derivative of a ring-expanded ('fat') nucleoside possessing potent antiviral and anticancer activities.

As part of an effort to explore the mechanism of potent, broad spectrum antiviral and anticancer activities of a number of ring-expanded ('fat') nucleosides that we recently reported, a representative 'fat' nucleoside 4,6-diamino-8-imino-8H-1-beta-D-ribofuranosylimidazo[4,5-e][1,3]di azepine (1) was converted to its 5'-triphosphate derivative (2), and biochemically screened for possible inhibition of nucleic acid polymerase activity, employing synthetic DNA templates and the bacteriophage T7 RNA polymerase as a representative polymerase. Our results suggest that 2 is a moderate inhibitor of T7 RNA polymerase, and that the 5'-triphosphate moiety of 2 appears to be essential for inhibition as nucleoside 1 alone failed to inhibit the polymerase reaction.

Inhibitors of glycogen phosphorylase b: synthesis, biochemical screening, and molecular modeling studies of novel analogues of hydantocidin.

The synthesis and biochemical screening of four novel spironucleosides 1-4 against rabbit liver glycogen phosphorylase b (Gpb), along with molecular modeling studies on compound 2 and its 4-hydroxy analogue VII, have been presented. Gpb is a key enzyme of glycogen metabolism, and is known to be involved in the control of diabetes mellitus. The general strategy for synthesis involved base-catalyzed condensation of diethyl 2,4-dioxoimidazolidine-5-phosphonate (5) with either 2-deoxy-D-ribose or D-ribose, followed by sequential reactions involving ring-closure with phenylselenenyl chloride and reduction with tri-n-butyltin hydride catalyzed by azobisisobutyronitrile. Compounds 2 and 4 were found to be weak competitive inhibitors of Gpb, whereas 1 and 3 were inactive.

Analogues of azepinomycin as inhibitors of guanase.

Synthesis and biochemical screening against guanase of analogues of the naturally occurring guanase inhibitor azepinomycin (2) are reported. Compound 6-amino-5,6,7,8,-tetrahydro-4H-imidazo[4,5-e][1,4]diazepine-5,8-dione (3) was synthesized in six steps commencing with 1-benzyl-5-nitroimidazole-4-carboxylic acid (5). Compound 3 and its synthetic precursor 3-benzyl-6-(N-benzyloxycarbonyl)amino-5,6,7,8-tetrahydro-4H-imidazo[4,5- e] [1,4]diazepine-5,8-dione (12) were screened against rabbit liver guanase. Both were found to be moderate inhibitors of the enzyme with K's in the range of 10(-4) M.

Unification of some literature models of aromaticity: calculational and conceptual study of a set of one-ring species.

Numerous models have been suggested for the important concept of aromaticity. In the current study, a set of recently suggested models of aromaticity/homoaromaticity/anti-aromaticity for one-ring species [e.g. pyridazine, oxazole, tropilidene (cycloheptatriene), 1,4-dithiin, [8]-annulene (cyclooctatetraene)] is shown to have a common mathematical framework from which a new, unifying quantitative equation has been derived. Calculational and conceptual application is made to a well defined set of one-ring carbocycles.

Bis[2-(4-carboxyphenoxy)carbonylethyl]phosphinic acid (BCCEP): a novel affinity reagent for the beta-cleft modification of human hemoglobin.

The design, synthesis, and hemoglobin cross-linking studies of a novel organic reagent, bis[2-(4-carboxyphenoxy)carbonylethyl]phosphinic acid (BCCEP, 1) have been reported. The reagent was designed with the aid of molecular modeling, employing crystal coordinates of human hemoglobin A0. It was synthesized in three steps commencing from 4-t-butoxycarbonylphenol. The tri-sodium salt of 1 was employed to cross-link human oxyHb. While SDS-PAGE analyses of the modified hemoglobin product pointed to the molecular mass range of 32 kDa, the HPLC analyse suggested that the cross-link had formed between the beta 1-beta 2 subunits. The oxygen equilibrium measurements of the modified hemoglobin at 37 degrees C showed significantly reduced oxygen affinity (P50 = 31.3 Torr) as compared with that of cell-free hemoglobin (P50 = 6.6 Torr). The sigmoidal shape of O2 curves of the modified Hb pointed to reasonable retainment of oxygen-binding cooperativity after the cross-link formation. Molecular dynamics simulation studies on the reagent-HbA0 complex suggested that the most likely amino acid residues involved in the cross-linking are N-terminus Val-1 or Lys-82 on one of the-chains, and Lys-144 on the other. These predictions were consistent with the results of MALDI-MS analyses of the peptide fragments obtained from tryptic digestion of the cross-linked product.

Synthesis and guanase inhibition studies of a novel ring-expanded purine analogue containing a 5:7-fused, planar, aromatic heterocyclic ring system.

The synthesis of a novel planar, potentially aromatic, ring-expanded xanthine analogue (1), containing the 5:7-fused imidazo[4,5-e][1,4]diazepine ring system, along with guanase inhibition studies are reported. The compound was synthesized in six steps, starting from 1-benzyl-5-nitroimidazole-4-carboxylic acid (2), and was biochemically screened against rabbit liver guanase. Compound 1 is a moderate competitive inhibitor of the enzyme with a Ki of 2.27 +/- 0.66 x 10(-4) M.

Bis[2-(4-carboxyphenoxy)carbonylethyl]phosphinic acid (BCCEP): a new reagent for hemoglobin modification.

The synthesis and hemoglobin cross-linking studies of a novel organic reagent, bis[2-(4-carboxyphenoxy)carbonylethyl]phosphinic acid (BCCEP; 2) has been reported. The reagent was synthesized in four steps from hydroxybenzoic acid. The tri-sodium salt of BCCEP was employed to cross-link oxyHb, and the product was purified by DEAE-cellulose chromatography. The purified material was analyzed by SDS-PAGE, IEF, and HPLC analyses, which clearly showed the formation of covalent, intramolecular cross-links. While SDS-PAGE analyses of individual bands pointed to the molecular weight range of 32 kDa, the HPLC analyses suggested that the cross-links had formed between beta 1-beta 2 subunits. The oxygen equilibrium measurements and the Hill plots were performed on the purified bands to assess oxygen affinity as well as cooperativity of oxygen binding of the modified hemoglobins. All bands corresponding to modified hemoglobins showed significantly reduced oxygen affinity as compared with that of cell-free hemoglobin, as desired. The modified hemoglobins, however, exhibited somewhat reduced oxygen-binding cooperativity as contrasted with human stroma-free hemoglobin. Molecular dynamics simulation studies (Insight II/Discover/Biosym) on the Reagent-HbA0 complex suggested that the most likely amino acid residues involved in the cross-linking are Lys82 or N-terminal Val1 on one of the beta chains, and Lys144 on the other.

How important is the N-3 sugar moiety in the tight-binding interaction of coformycin with adenosine deaminase?

Preliminary findings on the possible important role of the N-3 sugar moiety of coformycin in its tight-binding interaction with adenosine deaminase (ADA) are reported. The compound 3-beta-D-Ribofuranosyl-5,6,7,8-tetrahydro-4H-imidazo[4,5-d][1,3]diaze pin-5-one-8-ol (1), its 3-benzyl analogue (6), and the aglycon (7) served as probes. The first two were both found to be competitive inhibitors of ADA with Ki's in the range of 10(-5) M, while the last one was inactive.

The use of fluorophore-containing spin traps as potential probes to localize free radicals in cells with fluorescence imaging methods.

Central to the study of free radical processes is the ability to identify and localize their cellular site of formation. Under the best of experimental conditions, spin trapping/ESR spectroscopy can only characterize intracellular production of specific free radicals and confocal microscopy can only localize the site of their formation. In this article, we report on the development of a fluorophore-containing nitrone, alpha-[4-[5-((2-carboxy)phenyl)-5-hydroxy-4-oxo-3-phenyl)-2-pyrrolin+ -1-yl]phenyl]-N-(tert-butyl)nitrone sodium salt (4). This nitrone (4) reacts with alpha-hydroxyethyl radical with a second order rate constant of 1.7 x 10(5) M-1 s-1 to give a characteristic ESR spectrum. However, we were unable to decrease the fluorescence emission, due in part to the small concentration of nitroxide generated from the reaction of alpha-hydroxyethyl radical with nitrone (4). Using the fluorophore-containing nitroxide (7) as a model, we found that only 12% of the nitroxide needs to be reduced to give an almost 400% increase in the fluorescent emission of (7). Our findings suggest new approaches to the development of various fluorophore-containing nitrones that can both characterize specific free radicals and localize their site of intracellular formation.

"Slim" nucleosides and nucleotides. I. Synthetic, structural and biophysical investigations of showdomycins.

A new, convenient, and short synthesis of 2'-deoxyshowdomycin, along with an improved procedure for the preparation of showdomycin, have been presented. A single-crystal X-ray structure of 1-benzyl-2'-deoxyshowdomycin (9) has been reported. Conformational studies using C.D. indicated that showdomycin exists predominantly in an anti conformation in aqueous solution. Molecular mechanics calculations using AMBER point to comparable binding energy of showdomycin-adenosine pair with the natural uridine-adenosine pair, but with a significant base-ribose conformational deviation from the natural array in the former. Implications of such a conformational deviation on tumor and viral replications have been discussed. Base-pairing studies employing high resolution NMR spectroscopy indicate that both showdomycin and epishowdomycin base-pair with adenosine-5'-monophosphate (AMP); however, while showdomycin also shows evidence of stacking, that was absent in epishowdomycin. Molecular modeling studies using QUANTA/CHARMm show that showdomycin is capable of forming a homopolymer duplex by base-pairing with poly(A), but with a considerably broader and deeper major groove. A heteropolymer duplex with a single insert of showdomycin exhibits tighter coiling at the point of insertion. A ten-picosecond dynamics simulation of the above heteroduplex revealed relaxation of the helix with disruption of H-bonding for two base pairs on either side of the insertion point, forming a large central cavity.

A fluorophore-containing nitroxide as a probe to detect superoxide and hydroxyl radical generated by stimulated neutrophils.

Toward the development of a fluorescence assay in combination with confocal microscopy to image free radicals generated by cells, we synthesized a fluorophore-nitroxide, 5-((2-carboxy)phenyl)-5-hydroxy-1-((2,2,5,5-tetramethyl-1-oxypyrrolid in-3- yl)methyl)-3-phenyl-2-pyrrolin-4-one sodium salt, and tested the applicability of this probe to detect oxygen-centered free radicals. The reaction of the fluorophore-nitroxide with superoxide (10 microM/min) generated either by the reaction of xanthine oxidase on xanthine or by PMA-activated neutrophils in the presence of cysteine (200 microM) resulted in a loss of electron spin resonance (ESR) signal intensity concurrent with an increase in fluorescence emission. The decrease in ESR signal and the augmentation in fluorescence emission were inhibited by the addition of superoxide dismutase. This fluorophore-nitroxide also reacted with methyl radical generated by the reaction of hydroxyl radical with DMSO (0.14 M). In this case a loss in ESR signal intensity concomitant with an increase in fluorescence emission which were inhibited by catalase (300 U/ml), was recorded. These results clearly demonstrated the feasibility of using fluorescence methodology in conjunction with a fluorophore-nitroxide to detect oxygen-centered free radicals in biological systems.

A versatile, highly reactive, cross-linking reagent: 2,2'-sulfonylbis[3-methoxy-(E,E)-2-propenenitrile].

Adequate aqueous stability and cross-linking ability of the novel title reagent, recently discovered in this laboratory, have been demonstrated by comparison of its rate of hydrolysis with the rate of reaction with an amine nucleophile and by cross-linking deoxy- and oxyhemoglobins, as an example.

Conformational studies of two isomeric ring-expanded purine nucleosides and their 5'-mono- and -diphosphate derivatives.

The nucleosides Ia and IIa exist in syn and anti conformations, respectively, both in solid state and solution. Compound Ia undergoes significant conformational change, accompanied by increased population of the anti conformer, upon conversion to the corresponding 5'-mono- and- diphosphate derivatives, whereas conformation of IIa remains reasonably constant between nucleoside and nucleotides. While Ia possessed the C2'-endo-C3'-exo geometry, IIa had the opposite C2'-exo-C3'-endo conformation. The C5' of the two nucleosides bore axial and equatorial conformations, respectively.