Design and synthesis of new cross-linking agents

We have previously synthesized 6-vinylated guanosine derivatives by new Pd(0)-catalyzed cross-coupling reaction using guanosine 6-O-tosylate and vinyltributyl-stannane. Its potentials as a cross-linking agent have been demonstrated by adduct formation with guanosine and cytidine. But these 6-vinylated derivatives could not be incorporated to oligonucleotide because of chemical instability. In this paper, we report new deoxyguanosine derivatives with substituted olefin (2 and 3), which were designed to control the reactivity of vinyl functional group. Although either 2 or 3 did not formed the adducts with nucleobases, these compounds reacted with hydroxylamine hydrochloride in the presence of acid catalyst. The reactivity was in the order of 1 > 2 > 3. These results suggest that new guaosine derivatives (2 and 3) have moderate reactivity toward nucleophiles and are expected to be stable in incorporating into oligonucleotides as a cross-linking agent.     

Biochemistry of 1,3-butadiene metabolism and its relevance to 1,3-butadiene-induced carcinogenicity

Recently, the roles of specific P450 isoforms, myeloperoxidase (MPO), GSH-S-transferase and epoxide hydrolase in the metabolism of 1,3-butadiene, and its major oxidative metabolite, butadiene monoxide (BM), were investigated. The results provided evidence for P450s 2A6 and 2E1 being major catalysts of 1,3-butadiene oxidation in human liver microsomes. cDNA-expressed human P450s 2E1, 2A6, and 2C9 catalyzed BM oxidation to meso- and (+/-)-diepoxybutane (DEB), but the rates of BM oxidation in mouse, rat, or human liver microsomes were much lower than the rates of 1,3-butadiene oxidation in these tissues. Human MPO catalyzed 1,3-butadiene oxidation to BM, but MPO incubations with BM did not yield DEB. Rates of BM formation in mouse and human liver microsomes were similar and were nearly 3.4-fold higher than that obtained with rat liver microsomes. However, rat liver epoxide hydrolase activity was nearly 2-fold higher than that of mouse liver microsomes. Rat and mouse liver GSH-S-transferases exhibited similar BM conjugation kinetics, but rats excreted more BM-mercapturic acids compared to mice given low equimolar doses of BM. BM reacted with guanosine and adenosine to yield N7-, N2-, and N1-guanosinyl and N6-adenosinyl adducts, respectively. These results may contribute to a better understanding of the biochemical basis of 1,3-butadiene-induced carcinogenicity.     

Chlorothioketene, the ultimate reactive intermediate formed by cysteine conjugate beta-lyase-mediated cleavage of the trichloroethene metabolite S-(1,2-Dichlorovinyl)-L-cysteine, forms cytosine adducts in organic solvents, but not in aqueous solution

Chlorothioketene has been suggested as a reactive intermediate formed by the cysteine conjugate beta-lyase-mediated cleavage of S-(1,2-dichlorovinyl)-L-cysteine, a minor metabolite of trichloroethene. Halothioketenes are highly reactive, and their intermediate formation may be confirmed by reactions such as cycloadditions and thioacylations of nucleophiles. A precursor of chlorothioketene, S-(1,2-dichlorovinyl)thioacetate, is readly accessible by the reaction of dichloroethyne with thioacetic acid. In presence of base, S-(1,2-dichlorovinyl)thioacetate is cleaved to chlorothioketene. Chlorothioketene is not stable at room temperature and was characterized after transformation to stable products by reaction with compounds such as cyclopentadiene, N,N-diethylamine, and ethanol. In organic solvents, the cleavage of S-(1, 2-dichlorovinyl)thioacetate in the presence of cytosine results in N4-acetylcytosine, N4-(chlorothioacetyl)cytosine, and small amounts of 3-(N4-thioacetyl)cytosine. No reaction products were seen with guanosine, adenosine, and thymidine under identical conditions. When cytosine was reacted with S-(1,2-dichlorovinyl)thioacetate in aqueous solutions, only N4-acetylcytosine was formed. N4-(Chlorothioacetyl)cytosine and 3-(N4-thioacetyl)cytosine were not detected even when using a very sensitive method, derivatization with pentafluorobenzyl bromide and electron capture mass spectrometry with a detection limit of 50 fmol/microliter of injection volume. Aqueous solutions of DNA cleave S-(1, 2-dichlorovinyl)thioacetate to give N4-acetyldeoxycytidine in DNA, but chlorothioketene adducts of deoxynucleosides were also not detected in these experiments. These results confirm the electrophilic reactivity of chlorothioketene toward nucleophilic groups of DNA constituents in inert solvents but also demonstrate that the formation of DNA adducts under physiological conditions likely is not efficient. Therefore, DNA adducts may not represent useful biomarkers of exposure and biochemical effects for trichloroethene.     

Therapeutic window of serum haloperidol concentration in acute schizophrenia and schizoaffective disorder

Starting from 3-(3-chloro-1H-pyrazol-5-yl)-1H-quinoxalin-2-one (2) a series of substituted [1,2,4]triazolo[4,3-a]quinoxalines (3a-f) was prepared via a multistep reaction sequence. Affinities of the novel derivatives 3a-f for benzodiazepine as well as for adenosine A1- and A2A-receptors of rat brain were determined by radioligand binding assays. 1-Methyl-4-(3-chloro-1H-pyrazol-5-yl) derivative 3a exhibited submicromolar affinity for the benzodiazepine binding site of GABAA receptors (Ki = 340 nM) and was less potent at A1-(Ki = 7.85 microM) and A2A-(Ki = 1.43 microM) adenosine receptors (AR). Derivatives with larger substituents in the 1-position showed reduced binding to benzodiazepine and A2A-AR, but increased A1-AR affinity, the 2-thienylmethyl derivative 3f being the most potent and selective A1-AR ligand of the present series (Ki = 200 nM).     

Location of the carbohydrate groups of ovomucoid

The aims of this investigation were to establish the optimum reaction conditions for silylation of nucleotides with bis(trimethylsilyl)trifluoroacetamide (BSTFA) and to investigate the chromatographic properties of the following nucleosides: adenosine, guanosine, cytidine, thymine, inosine, xanthosine, and uridine. Closed tube silylations were performed with a 1000 molar excess of BSTFA at 25, 75, 120, 150, and 175 degrees for 15, 30, 60, 120 and 240 min. The optimal time and temperature for derivatization were found to be 150 degrees and 15 min. Using these reaction conditions, samples were then silylated with 50, 100, 200, 500, and 1000 molar excess of BSTFA; a molar excess of 225 was best. The stability of the nucleoside derivatives on standing at room temperature for 1-7 days was investigated. Quantitative gas-liquid chromatography of trimethylsilyl (TMS) nucleosides can be performed if samples are analyzed within 48 h, after which time the relative weight response for the nucleosides decreased somewhat. Chromatographic column studies were made using various liquid phases and supports. With methylsiloxanes as the liquid phase, Supelcoport as support was found to be superior. Resolution of TMS guanosine from TMS cytidine was attempted at different column lengths using 3% (w/w) SE-30 on Supelcoport, different loadings (%, w/w) of SE-30 on Supelcoport, and different polarity liquid phases, 4% (w/w) OV-11 or 3% (w/w) OV-17 or 4% (w/w) Dexsil-300 on Supelcoport. A complete separation of the six ribonucleosides including guanosine and cytidine was obtained with 1 m x 4 mm I.D. glass columns of 4% (w/w) OV-11 and 3% (w/w) OV-17 on 100-120 mesh Supelcoport. It was observed that with 2'-deoxycytidine, one obtains the TMS cytosine peak (retention temperature 150 degrees) plus another peak with a retention temperature of 120 degrees, under all derivatization conditions with BSTFA and bis(trimethylsilyl)-acetamide. Similar formation of bases from other 2'-deoxyribonucleosides occurred when the molar excess of BSTFA was greater than 500. The minimal detectable amounts obtained for all the nucleosides ranged from 5 to 10 ng injected with a signal-to-noise ratio of 3. The relative standard deviations for all nucleosides and deoxynucleosides ranged from 1.2 to 4.8% on different methylsiloxanes on Supelcoport columns.     

N2,7-bis(1-hydroxy-2-oxopropyl)-2'-deoxyguanosine: identical noncyclic adducts with 1,3-dichloropropene epoxides and methylglyoxal

cis- and trans-1,3-dichloropropene epoxides (1,3-D-epoxides) are proposed to be the penultimate or ultimate genotoxic metabolites of the major soil fumigant nematicide 1,3-dichloropropene. The 1, 3-D-epoxide isomers and the potential aldehydes from their degradation readily form adducts with 2'-deoxyguanosine (dGuo) but not with 2'-deoxyadenosine or 2'-deoxycytidine. The reaction of dGuo with the 1,3-D-epoxides (1:20 molar equiv) in phosphate buffer at pH 7.4 for 24 h at 37 degreesC results in complete conversion to four adducts that can be separated by HPLC with the same UV spectra and electrospray (ES)/MS molecular ion and fragmentation patterns. These adducts contain no chlorine and are identical to those obtained more rapidly with methylglyoxal in place of the 1,3-D-epoxides. The four isomeric methylglyoxal adducts with dGuo were proposed originally by others to be the cyclic adducts 1,N2-(1, 2-dihydroxy-2-methyl)ethano-dGuo, but they are reassigned here as the four diastereomers of the noncyclic bis adducts N2, 7-bis(1-hydroxy-2-oxopropyl)-dGuo. The assignments are based on HPLC/UV and HPLC/ES/MS experiments and 1H NMR spectral analysis of the first of the four adducts eluted with HPLC. Acid-catalyzed depurination converts the four dGuo derivatives to the two corresponding isomers of N2,7-bis(1-hydroxy-2-oxopropyl)guanine, assigned by ES/MS and 1H and 13C NMR. Although identical adducts are formed from dGuo with the 1,3-D-epoxides or methylglyoxal, the latter alpha,beta-dicarbonyl compound is not an intermediate in the reaction; instead, the 1,3-D-epoxides hydrolyze to 3-chloro-2-hydroxypropanal which adds to dGuo at N2 and N7. The adducts dehydrochlorinate, in a rate-limiting reaction, thereby giving the same end products obtained on direct reaction with methylglyoxal. Thus, 3-chloro-2-hydroxypropanal (not the 1, 3-D-epoxides or methylglyoxal) is the derivatizing agent for dGuo and therefore probably the mutagenic agent on 1,3-D bioactivation. On the basis of the dGuo model studied here, the DNA adducts of 1, 3-D and its epoxides may be the same as those with methylglyoxal [Vaca, C. E., Fang, J.-L., Conradi, M., and Hou, S.-M. (1994) Carcinogenesis 15, 1887-1894].     

Reactions of OH and eaq- adducts of cytosine and its nucleosides or nucleotides with Cu(II) ions in dilute aqueous solutions: a steady-state and pulse radiolysis study

The reactions of OH and eaq- adducts of cytosine, cytidine and deoxycytidine in the presence of Cu(II) ions have been studied by product analysis and pulse radiolysis. The product analysis studies show that the degradation of the base is enhanced in N2O-saturated conditions in the presence of Cu(II) ions and the major radiolytic products are Cu(I), cytosine glycols and 5(6)-hydroxycytosine. It is also interesting to note that the yields of Cu(I) are equivalent to cytosine degradation yields, which suggests that the interaction of the OH adducts with Cu(II) ions restricts the radical recombination reactions (known to be the major physicochemical repair process) which partly regenerate the parent cytosine. The rate constants of the reactions of cytosine OH adducts with Cu(II) ions determined by pulse radiolysis lie between 10(7) and 10(8) dm3 mol-1 s-1. The growth in the transient absorption spectra of cytosine OH adducts in the range 330-400 nm, observed in the presence of copper(II) ions in free and complexed state, suggests formation of copper radical adduct which decays by water insertion at the copper-carbon bond to give glycol as the major product. Such copper radical adduct formation was also observed in the case of cytidine and deoxycytidine. The protonated electron adducts (at the hetero atoms) of cytosine, cytidine and deoxycytidine transfer electrons to the Cu(II) ions with rate constants of 10(8) and 10(9) dm3 mol-1 s-1. Here no adduct formation is observed. The steady-state results show that such electron transfer reactions regenerate the parent molecules themselves. Hence such electron transfer reactions do not contribute to enhanced base degradation in the presence of copper ions.     

Derivatives of the triazoloquinazoline adenosine antagonist (CGS 15943) having high potency at the human A2B and A3 receptor subtypes

The adenosine antagonist 9-chloro-2-(2-furanyl)[1,2,4]triazolo[1, 5-c]quinazolin-5-amine (CGS 15943) binds nonselectively to human A1, A2A, and A3 receptors with high affinity. Acylated derivatives and one alkyl derivative of the 5-amino group and other modifications were prepared in an effort to enhance A2B or A3 subtype potency. In general, distal modifications of the N5-substituent were highly modulatory to potency and selectivity at adenosine receptors, as determined in radioligand binding assays at rat brain A1 and A2A receptors and at recombinant human A3 receptors. In Chinese hamster ovary cells stably transfected with human A2B receptor cDNA, inhibition of agonist-induced cyclic AMP production was measured. An N5-(2-iodophenyl)acetyl derivative was highly selective for A2A receptors. An (R)-N5-alpha-methyl(phenylacetyl) derivative was the most potent derivative at A3 receptors, with a Ki value of 0.36 nM. A bulky N5-diphenylacetyl derivative, 13, displayed a Ki value of 0. 59 nM at human A3 receptors and was moderately selective for that subtype. Thus, a large, nondiscriminating hydrophobic region occurs in the A3 receptor in proximity to the N5-substituent. A series of straight-chain N5-aminoalkylacyl derivatives demonstrated that for A2B receptors the optimal chain length occurs with three methylene groups, i.e., the N5-gamma-aminobutyryl derivative 27 which had a pA2 value of 8.0 but was not selective for A2B receptors. At A1, A2A, and A3 receptors however the optimum occurs with four methylene groups. An N5-pivaloyl derivative, which was less potent than 27 at A1, A2A, and A3 receptors, retained moderate potency at A2B receptors. A molecular model of the 27-A2B receptor complex based on the structure of rhodopsin utilizing a "cross-docking" procedure was developed in order to visualize the environment of the ligand binding site.     

Pyrazolopyridine derivatives act as competitive antagonists of brain adenosine A1 receptors: [35S]GTPgammaS binding studies

The effects of adenosine receptor ligands and three novel pyrazolopyridine derivatives on guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding to rat cerebral cortical membranes were examined. [35S]GTPgammaS binding was stimulated in a concentration dependent manner by several adenosine receptor agonists. The adenosine A2a receptor selective agonist, 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680), was ineffective confirming specificity for adenosine A1 receptor activation. 2-Chloro-N6-cyclopentyladenosine (CCPA; 10(-7) M)-stimulated [35S]GTPgammaS binding was inhibited by xanthine and pyrazolopyridine based adenosine receptor antagonists. The concentration-response curve for CCPA-stimulated [35S]GTPgammaS binding was shifted to the right with increasing concentrations of antagonist without significant changes in maximal response. Schild analyses determined pK(B) values of 8.97, 8.88, 8.21, 8.16, 7.79 and 7.65 for 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), (R)-1-[(E)-3-(2-phenylpyrazolo[1,5a]pyridin-3-yl) acryloyl]-2-piperidine ethanol (FK453), 6-oxo-3-(2-phenylpyrazolo[1,5a]pyridin-3-yl)-1(6H)-pyridazinebutyric+ ++ acid (FK838), 9-chloro-2-(2-furyl)[1,2,4]triazolo-[1,5c]quinazolin-5-amine (CGS 15943), 8-cyclopentyl-1,3-methylxanthine (CPT) and (R)-1-[(E)-3-(2-phenylpyrazolo[1,5a]pyridin-3-yl) acryloyl]-piperidin-2-yl acetic acid (FK352), respectively. Schild slopes were close to unity, confirming that these novel pyrazolopyridine derivatives act as competitive antagonists at rat brain adenosine A1 receptors.     

Capillary isotachophoresis with fiber-optic Raman spectroscopic detection. Performance and application to ribonucleotides

A fiber-optic Raman probe fitted with a microscope objective was used to obtain on-line normal Raman spectra of adenosine 5'-monophosphate, cytidine 5'-monophosphate, guanosine 5'-monophosphate and uridine 5'-monophosphate separated by capillary isotachophoresis. With multimode optical fiber, the system interrograted a 40-micron length of capillary. Fiber-optic coupling facilitated use of an unmodified spectrograph and conventional capillary mounting systems. Raman spectra were excited with a 2W 532 nm NdYVO4, laser as the excitation source, with collection of 1 spectrum per second. Even at 2.10(-5) M initial concentration, Raman spectra were obtained at a good signal-to-noise ratio.     

Molecular simulation of 8-styrylxanthines

Regarding theophylline as representative xanthine derivative, it was shown that the net charges of various semiempirical quantum chemistry methods are transferable by scaling. PM3 should be preferred for calculation of xanthine derivatives. Molecular modelling indicated that there is a conformational similarity of the lead structure of xanthine and adenosine derivatives. The substituents bound to the C8 of xanthine and to the C2 of adenosine derivatives are involved in the discrimination into adenosine A2 antagonists and agonists. The A2 affinity of xanthines is mainly determined by the type of N7 substitution (hydrogen/methyl), the lipophilic substituent constant related to the C8 substituents, and the dipole moment of the molecules. To simulate chemically the A1 affinity, a further term (lowest unoccupied molecular orbital energy) must be added. In addition, hydrogen-bonding forces were hypothesized using a newly synthesized 3,6-diaminocarbazole derivative as synthetic adenosine pseudoreceptor.     

Characterization of four N-3-thymidine adducts formed in vitro by the reaction of thymidine and butadiene monoxide

Four products were characterized from the reaction of thymidine with butadiene monoxide (BM), a known human mutagen and possible human carcinogen. These products were purified by HPLC and characterized as diastereomeric pairs of N-3-(1-hydroxy-3-buten-2-yl)thymidine and N-3-(2-hydroxy-3-buten-1-yl)thymidine based upon their UV spectra, 1H NMR and fast atom bombardment mass spectra. Incubation of thymidine with an excess of BM at pH 7.4 and 37 degrees C allowed calculation of the pseudo-first-order kinetic rate constants for the adduct formation, but when these rate constants were compared with the rates we previously determined with guanosine, adenosine and deoxycytidine, the results suggested a lower reactivity with thymidine in comparison with the other nucleosides. When incubations were carried out at lower BM concentrations, the formation of adducts appeared to be linearly dependent on BM concentration. The four thymidine adducts were completely stable for 1 week when incubated at 37 degrees C in pH 7.4 phosphate buffer. These results suggest that the interactions of BM with thymidine may play a role in the molecular mechanisms of mutagenesis and carcinogenesis of BM.     

Synthesis and evaluation of 4-amino-substituted 7-methoxy (and 7-hydroxy)- 11-methyl (and 5,11-dimethyl)-10H-pyrido [2,3-b] carbazoles and 4-amino- substituted 11-methyl (and 5,11-dimethyl)-10H-pyrido[3',4':4,5] pyrrolo [3,2-g] quinolines, two new series related to antitumor ellipticine derivatives

2-Acetyl-4-chloro-3-lithiopyridine ethylene glycol ketal (6b) was reacted with 3-formyl-5-methoxy-1-methyl-indole (9) and 3-formyl-1-methyl-1H-pyrrolo [3,2-c] pyridine (12), giving the corresponding expected alcohols. Reduction of these intermediates with triethylsilane trifluoroacetic acid and subsequent cyclodehydration then led to 4-chloro-7-methoxy-10,11-dimethyl-10H-pyrido [2,3-b] carbazole (8a) and the corresponding 7-aza-analog (8b). The synthesis of 4-chloro-11-methyl (and 5,11-dimethyl)-10-unsubstituted derivatives of these two series was performed through an independent pathway, involving condensation of conveniently substituted 2-amino carbazoles (17) and 7-amino-5H-pyrido [4,3-b] indoles (18) with 5-(ethoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione, thermal cyclization of the resulting compounds with concomitant decarboxylation to the corresponding tetracyclic fused-4-quinolone systems and final chlorination with phosphorus oxychloride. Nucleophilic substitution of various 4-chloro derivatives was then easily performed in an excess of the required dialkylamino alkylamines at reflux and 4-amino substituted-7-hydroxy-10H- pyrido [2,3-b] carbazoles (25d-e) were obtained from 7-methoxy precursors (25a-b), by demethylation with boron tribromide in methylene chloride at -65 degrees C or with boiling 47% hydrobromic acid. Cytotoxicity determination of all new aminosubstituted derivatives and in vivo antitumor evaluation of the most active compounds clearly show that these two series of ellipticine analogs closely related to highly active products are devoid of antitumor properties in two experimental models shown to be sensitive to ellipticines. The place of the pyridinic nitrogen atom in these series has thus been demonstrated to play a crucial role in antitumor activity.     

Synthesis and structure-activity relationships of adenosine analogs as inhibitors of trypanosomal glyceraldehyde-3-phosphate dehydrogenase. Modifications at positions 5' and 8

A number of 5', N6- and C8, N6-disubstituted adenosine analogs was synthesized and tested for inhibition of trypanosomal glyceraldehyde 3-phosphate dehydrogenase. The most active compound, N6-(3-methyl-2-butenyl)-8-(2-thienyl)adenosine, had Kl of 9 microM and was marginally selective for the parasite enzyme.     

DNA vaccines for bacterial infections

The uptake and transportation of purine and pyrimidine based nucleosides by trophozoites of axenically grown Entamoeba histolytica (HMI-IMSS) were studied. The trophozoites transported adenosine and its analog tubercidin (1 microM) at a significant rate but poor transportation was observed in case of uridine (about 10% relative rate), inosine (3%), thymidine (2%) and formycin B (1%). The Km for adenosine was 160 +/- 42 microM. Unlabeled nucleosides (100 microM) inhibited adenosine and tubercidin transport. Adenosine related compounds 5'-deoxyadenosine and nebularin inhibited adenosine and tubercidine transport by 50% or more. However, inosine related compounds guanosine, 3'-deoxyinosine and formycin B were less inhibitory. The pyrimidine nucleosides uridine, thymidine and cytidine were poorly inhibitory. 6-[(4 nitrobenzyl)-mercapto] purine ribonucleoside, an inhibitor of mammalian nucleoside transporter, inhibited adenosine or tubercidin transport in E. histolytica variably between 0-30% at 10 microM, but dilazep, a known inhibitor, was inactive upto 10 microM. Increase in temperature from 22 degrees C to 33 degrees C enhanced the rate of transport of adenosine 4.5 fold, tubercidin 7.3 fold and of inosine 4 fold. These findings along with the structure activity figures suggested that transport was mediated and not passive.     

Oxidation and nitration of catecholamines by nitrogen oxides derived from nitric oxide

The reactivity of catecholamines with nitrogen oxides formed from NO in aerated solutions, nitrite, and peroxynitrite was evaluated. Dopamine and norepinephrine in aerobic buffer (pH 7.4) were almost completely converted to their 6-nitro-derivatives by nitric oxide (NO) at room temperature, while epinephrine was nitrated and above all oxidized. The products obtained from each catecholamine treated with sodium nitrite at pH 4-7 were compared to those produced by NO at pH 7.4. Peroxynitrite, which can nitrate tyrosinyl residues, did not produce nitro-derivatives, only oxidized ones. The physiological relevance, particularly for the vascular and nervous system, is discussed. Catecholamine oxidation reactions could be relevant to physiological conditions and also explain neurotoxicity in Parkinson's disease and aging. Nitration reactions, requiring such high NO concentrations, do not seem possible to occur directly under normal physiological conditions, but could take place in acidic vesicules where nitrite, catecholamines, and their nitrated products could accumulate. Finally, the ability of dopamine to increase 2',5'-cyclic adenosine monophosphate (cAMP) formation in cultured striatal neurons was blocked by its nitration by NO or its nitrogen oxide derivatives.     

Processing of adenosine receptor agonists in rat and human whole blood

A stability study of adenosine receptor agonists in rat and human whole blood was performed. The compounds were incubated at 37 degrees in fresh blood, and aliquots of the incubation mixture were hemolyzed at regular time intervals and analyzed with HPLC. N6-cyclopentyladenosine (CPA) and N6-cyclobutyladenosine (CBA) were degraded, whereas N6-cyclohexyladenosine, N6-cycloheptyladenosine and N6-sulfophenyladenosine were not. 2-Chloroadenosine had a half-life very similar to that of CPA. However, the 2'-, 3'-, and 5'-deoxyribose derivatives of CPA remained intact. The nucleoside transport inhibitor nitrobenzylthioinosine attenuated CBA and CPA metabolism in rat blood as did the inhibitor of adenosine deaminase erythro-9-(2-hydroxy-3-nonyl)adenine, albeit at relatively high concentrations. Complete blockade of CBA and CPA degradation was achieved by a preincubation of rat and human blood with the adenosine kinase (AK) inhibitor 5'-amino-5'-deoxyadenosine. We conclude that the two adenosine analogues are metabolized by AK both in rat and in human whole blood.     

Structure-activity relationships of 4-(phenylethynyl)-6-phenyl-1,4-dihydropyridines as highly selective A3 adenosine receptor antagonists

4-(Phenylethynyl)-6-phenyl-1,4-dihydropyridine derivatives are selective antagonists at human A3 adenosine receptors, with Ki values in a radioligand binding assay vs [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5'-(N-methylcarbamoyl)adenosine) in the submicromolar range. In this study, structure-activity relationships at various positions of the dihydropyridine ring (the 3- and 5-acyl substituents, the 4-aryl substituent, and 1-methyl group) were probed synthetically. Using the combined protection of the 1-ethoxymethyl and the 5-[2-(trimethylsilyl)ethyl] ester groups, a free carboxylic acid was formed at the 5-position allowing various substitutions. Selectivity of the new analogues for cloned human A3 adenosine receptors was determined vs radioligand binding at rat brain A1 and A2A receptors. Structure-activity analysis at adenosine receptors indicated that pyridyl, furyl, benzofuryl, and thienyl groups at the 4-position resulted in, at most, only moderate selectivity for A3 adenosine receptors. Ring substitution (e.g., 4-nitro) of the 4-phenylethylnyl group did not provide enhanced selectivity, as it did for the 4-styryl-substituted dihydropyridines. At the 3-position of the dihydropyridine ring, esters were much more selective for A3 receptors than closely related thioester, amide, and ketone derivatives. A cyclic 3-keto derivative was 5-fold more potent at A3 receptors than a related open-ring analogue. At the 5-position, a homologous series of phenylalkyl esters and a series of substituted benzyl esters were prepared and tested. (Trifluoromethyl)-, nitro-, and other benzyl esters substituted with electron-withdrawing groups were specific for A3 receptors with nanomolar Ki values and selectivity as high as 37000-fold. A functionalized congener bearing an [(aminoethyl)amino]carbonyl group was also prepared as an intermediate in the synthesis of biologically active conjugates.     

Angiographic progression in patients with angina pectoris and normal or near normal coronary angiograms who are restudied due to unstable symptoms

In this work we studied the mechanism of nitric oxide (NO) release underlying the vasorelaxant and antiaggregant effect of 3,4-dihydrodiazete 1,2-dioxides (DD). Six derivatives were included in the investigations, namely, 3-bromo- and 3-chloro-3,4,4-trimethyl-DD (1a,b), 3-bromo- and 3-chloro-4-methyl-3,4-hexamethylene-DD (2a,b), 3,3,4,4-tetramethyl-DD (3), and 3-methyl-3,4-hexamethylene-DD (4), and their reactivity toward thiols was analyzed. The 3-bromo- and 3-chloro-DD derivatives were found to react with thiols; this reaction can lead to NO formation, DD 2a being the most reactive compound. 2-(Hydroxyamino)-2-methylbutan-3-one oxime (5a) and 2-hydroxy-2-methylbutan-3-one oxime (6) were the main products isolated from the reaction of 1a with cysteine. Reaction rates of DD with thiols were dependent upon pH and concentration of the reagents. Maximum rates of NO release corresponded to thiol concentrations in the range of 1 mM. Consistent with reaction kinetics data and products isolated, a reaction mechanism was proposed. Addition of 2a to bovine aortic endothelial cells led to strong NO release indicating a reaction with endogenous thiols. In rat mesenterial arteries, the vasorelaxant action of 2a was only slightly influenced by addition of thiol to the incubation medium. For the most reactive DD derivatives, cytotoxic effects were observed at concentrations roughly 2 orders of magnitude higher than those inducing vasorelaxation.     

Reactivity of haloketenes and halothioketenes with nucleobases: chemical characterization of reaction products

Halothioketenes and haloketenes are postulated as intermediates in haloolefin bioactivation. Little is known about the interactions of these reactive intermediates with macromolecules such as DNA. DNA binding, however, may be relevant in the toxicity of the parent olefins since they or their proximate metabolites are genotoxic. This prompted us to elucidate the structures and properties of potential DNA adducts formed. Adenine, cytosine, guanine, and thymine were reacted with chloro- and dichlorothioketene, chloro- and dichloroketene, and chloro- and dichloroacyl chloride. While thymine did not react, adenine and cytosine formed stable DNA base adducts with all reaction partners as demonstrated by HPLC analysis. Guanine yielded only products with chloroketene and chloroacetyl chloride. The pH-dependent UV spectra, 1H and 13C NMR, FT-IR, and elemental analysis showed (i) nucleophilic attack of the exocyclic amino groups of the DNA bases yielded haloacyl (thio)amides with all reactants as clearly demonstrated by the FT-IR spectra; (ii) the sulfur in the initial thioamides seems to be rapidly exchanged with oxygen; (iii) the acyl chlorides form identical products but in lower yields as compared to the haloketenes. Reactions of the nucleosides with haloketenes showed the formation of similar nucleoside adducts upon HPLC and MS analysis. Beside the modification of the base moieties, additional peaks in the reaction mixtures analyzed suggested acylation of the deoxyribose hydroxyl groups. In aqueous solutions at pH 7 N6-(chloroacetyl)adenine, N4-(chloroacetyl)cytosine, and N2-(chloroacetyl)guanine are not stable and cleaved to the original base or form 1,N6-acetyladenine, 3,N4-acetylcytosine, 1,N2-acetylguanine, and N2,3-acetylguanine. Under the same conditions, N6-(dichloroacetyl)adenine and N4-(dichloroacetyl)cytosine were completely hydrolyzed to adenine and cytosine, respectively. All haloacyl DNA base adducts proved to be stable at pH 5 but were rapidly degraded at neutral or alkaline pH. The compounds with an additional five-membered ring remained unchanged after 1 week at room temperature. All synthesized DNA base adducts except N2-(chloroacetyl)guanine and 1,N2-acetylguanine were fluorescent. The characterized compounds, especially the etheno (epsilon) base adduct-related derivatives, may represent potential DNA adducts formed as a consequence of haloolefin bioactivation.