Syntheses of Ribonucleoside 5′-S-Methyl Phosphorothiolates and Ribonucleoside 3′: 5′-Cyclic Phosphates from Nucleosides Applying a New Phosphorylating Agent,MTBO

When adenosine (Ia) was allowed to react with 2-methylthio-4H-l,3,2-benzodioxaphosphorin-2-oxide (MTBO) (II), a new phosphorylating agent, in the presence of cyclohexylamine, adenosine 5′-S-methyl phosphorothiolate (IVa) and adenosine 2′: 3′-cyclic phosphate (Va) were obtained.

Ribonucleoside 5′-S-methyl phosphorothiolates (IV) were selectively synthesized directly from borate complexes of ribonucleosides (VI) and MTBO in the presence of cyclohexylamine, followed by removal of metaboric acid by co-distillation with methanol, in 68~86% yields.

Under anhydrous conditions, the phosphorothiolates (IV) were cyclized to give ribonucleoside 3′: 5′-cyclic phosphates (X) by iodine oxidation. Five 3′: 5′-cyclic nucleotides including cyclic AMP were synthesized by this procedure in 38~64% yields after purification.     

Adenosine A 2B receptors modulate cAMP levels and induce CREB but not ERK1/2 and p38 phosphorylation in rat skeletal muscle cells

The present study examined the existence of the adenosine A(1),A(2A), and A(2B) receptors and the effect of receptor activation on cAMP accumulation and protein phosphorylation in primary rat skeletal muscle cells. Presence of mRNA and protein for all three receptors was demonstrated in both cultured and adult rat skeletal muscle. NECA (10(-9)-10(-4)M) increased the cAMP concentration in cultured muscle cells with an EC(50) of (95% confidence interval)=15 (5.9-25.1) micro M, whereas CGS 21680 (10(-9)-10(-4)M) had no effect on cAMP accumulation. Concentrations of [R]-PIA below 10(-6)M had no effect on cAMP accumulation induced by either isoproterenol or forskolin. NECA resulted in phosphorylation of CREB with an EC(50) of (95% confidence interval)=1.7 (0.40-7.02) micro M, whereas ERK1/2 and p38 phosphorylation was unchanged. The results show that, although the A(1),A(2A), and A(2B) receptors are all present in skeletal muscle cells, the effect of adenosine on adenylyl cyclase activation and phosphorylation of CREB is mainly mediated via the adenosine A(2B) receptor.     

A new method for the synthesis of 2'-O-benzyladenosine using Mitsunobu reaction

A new method to introduce a benzyl group onto the 2'-OH of purine ribonucleoside is described. Thus, 6-chloropurine 3'-O-benzoylriboside and its 5'-O-trityl congener were condensed with benzyl alcohol using the Mitsunobu reaction to give the 2'-O-benzyl derivative. The yields were varied from 4.6 to 62.9% depending on the solvent used. The product was converted to adenosine, indicating that the stereochemistry at C-2' is retained.     

Contribution of a single hydroxyl group to transition-state discrimination by adenosine deaminase: evidence for an "entropy trap" mechanism

Adenosine deaminase was found to bind 6-hydroxy-1,6-dihydropurine ribonucleoside (II), formed by reversible addition of water to purine ribonucleoside (I) in a reaction analogous to formation of a tetrahedral intermediate in substrate deamination, with an apparent Ki value of 3 x 10(-13) M at 20 degrees C. 1,6-Dihydropurine ribonucleoside (IV), synthesized by photolysis of purine ribonucleoside in the presence of NaBH4, exhibited a Ki value of 5.4 x 10-6 M. After correction for differences between the relative free energies of solvation of II and IV, the 6-hydroxyl group of II was estimated to contribute more than 16 kcal to the free energy of binding, approaching the enthalapy of formation of a single hydrogen bond to charged group in the vapor phase. The relatively weak binding of IV and of substrate water suggests that entropic effects, arising from the cooperative action of binding determinants contained within these separate molecules, contribute more than 10 kcal/mol to the free energy of binding of II in which these binding determinants are contained within a single molecule. In free solution, the entropy of reversible hydration of I was evaluated by measuring the temperature dependence of equilibria of protonation of I and of pseudobase formation from I-methylpurinium ribonucleoside as -35 eu, comparable with the entropy of activation for the uncatalyzed hydrolysis of adenosine. In the active site of adenosine deaminase, this thermodynamic obstacle is evidently climbed spontaneously as a result of attractive interactions between the active site and the critical hydroxyl group at the 6-position.(ABSTRACT TRUNCATED AT 250 WORDS)     

A transition state in pieces: major contributions of entropic effects to ligand binding by adenosine deaminase.

Nebularine undergoes hydration at the active site of adenosine deaminase, in a reaction analogous to a partial reaction in the displacement of ammonia from adenosine by water, to generate an inhibitory complex that captures much of the binding affinity expected of an ideal transition-state analogue. Enzyme affinities of several compounds related to nebularine 1,6-hydrate, and to its stable analog 2'-deoxycoformycin, were compared in an effort to identify the structural origins of strong binding. Binding of the stable transition-state analog inhibitor 2'-deoxycoformycin was rendered 9.8 kcal/mol less favorable by removal of substituent ribose, 9.7 kcal/mol less favorable by inversion of the 8-hydroxyl substituent of the diazepine ring, and 10.0 kcal/mol less favorable by removal of atoms 4-6 of the diazepine ring. Binding of the unstable transition-state analog nebularine hydrate was rendered at least 9.9 kcal/mol less favorable by removal of the 6-hydroxyl group and 10.2 kcal/mol less favorable by removal of atoms 1-3 of the pyrimidine ring. In each case, the enzyme exhibited only modest affinity (Kd greater than or equal to 10(-2) M) for the "missing piece", indicating that incorporation of 2 binding determinants within a single molecule permits an additional 7-12 kcal/mol of intrinsic binding energy to be manifested as observed binding energy. These results are consistent with earlier indications that adenosine deaminase may use 10.5 kcal/mol of the intrinsic free energy of binding of the two substrates to place them in positions appropriate for reaction at the active site, overcoming the unfavorable entropy change of -35 eu for the equilibrium of 1,6-hydration of purine ribonucleoside and reducing the equilibrium constant for attainment of the transition state in deamination of adenosine. Thus, adenosine deaminase may achieve up to 8 orders of magnitude of its catalytic power by converting the nonenzymatic, bimolecular, hydration reaction to a monomolecular reaction at its active site. Several new 6-substituted 1,6-dihydropurine ribonucleosides, prepared by photoaddition of formate and by low-temperature addition of organolithium reagents to a derivative of purine ribonucleoside, exhibited Ki values of 9-1400 microM against adenosine deaminase, in accord with the active site's considerable tolerance of bulky leaving groups in substrates. Inhibition by one diastereomer of 6-carboxy-1,6-dihydropurine ribonucleoside was found to be time-dependent, progressing from a weakly bound to a more strongly bound complex.     

Formaldehyde-induced mutagenesis: a novel mechanism for its action

A novel and unique mechanism for formaldehyde-induced mutagenesis is described which is mediated by the formation of an N6-substituted adenine ribonucleoside analogue, N6-hydroxymethyl adenosine, after an in vitro reaction of formaldehyde with adenosine. This type of ribonucleoside analogue (the deoxyribose derivative is ineffective) exhibits a powerful and remarkable germ-cell-stage-specific mutagenic effect in male Drosophila larvae, apparently by interfering with DNA repair. Circumstantial evidence is presented which indicates that the analogue most probably acts by its utilisation in the synthesis of diadenosine tetraphosphate (Ap4A) to form an antimetabolite(s) of Ap4A which subsequently interferes with Ap4A-mediated intracellular events, amongst which an effect on DNA repair would appear to be its mutagenic mechanism of action.     

Structural basis for the specificity, catalysis, and regulation of human uridine-cytidine kinase

Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine and cytidine and activates pharmacological ribonucleoside analogs. Here we present the crystal structures of human UCK alone and in complexes with a substrate, cytidine, a feedback inhibitor, CTP or UTP, and with phosphorylation products, CMP and ADP, respectively. Free UCK takes an alpha/beta mononucleotide binding fold and exists as a homotetramer with 222 symmetry. Upon inhibitor binding, one loop region was loosened, causing the UCK tetramer to be distorted. Upon cytidine binding, a large induced fit was observed at the uridine/cytidine binding site, which endows UCK with a strict specificity for pyrimidine ribonucleosides. The first UCK structure provided the structural basis for the specificity, catalysis, and regulation of human uridine-cytidine kinase, which give clues for the design of novel antitumor and antiviral ribonucleoside analogs that inhibit RNA synthesis.     

Sodium-dependent and inhibitor-insensitive uptake of adenosine by mouse peritoneal exudate cells

[8-3H]Adenosine uptake in mouse peritoneal exudate cells, harvested following i.p. challenge with Complete Freund's Adjuvant from BALB/c mice, was found to be insensitive to common nucleoside transport inhibitors such as dilazep or 6-[(4-nitrobenzyl)mercapto]purine ribonucleoside and to require sodium ion, being inactive when sodium was replaced by lithium or potassium. These findings also applied to the adherent (macrophages) and nonadherent (polymorphonuclear cells) cell fractions prepared from the peritoneal cell mixture. Uptake was inhibited by several nucleosides including deoxyadenosine, inosine, uridine, thymidine and, to a lesser extent, by the adenosine analog tubercidin, while adenine, fructose, glucose and ribose were without effect. Uptake [8-3H]adenosine was fully matched by rapid intracellular phosphorylation to AMP, ADP and ATP. Inosine was a substrate for the transporter, but tubercidin was not. The system clearly is distinct from carrier-mediated, nonconcentrative transport and has similarities to concentrative, sodium-dependent nucleoside transporters described in other cell types.     

Prostaglandin E2 up-regulates macrophage-derived chemokine production but suppresses IFN-inducible protein-10 production by APC

PGE(2) has been known to suppress Th1 responses. We studied the role of PGE(2) in two representative chemokines, macrophage-derived chemokine (MDC) and IFN-inducible protein-10, production by LPS- or CD40-stimulated spleen cells. The production of MDC, one of the ligands for CCR4 preferentially expressed on Th2, was enhanced in nonstimulated, LPS-, CD40-, or CD3-stimulated spleen cells by the pretreatment with PGE(2), while the production of IFN-inducible protein-10, a representative ligand for CXC chemokine receptor 3 expressed on Th1, was suppressed. MDC production was also enhanced by IL-4, IL-5, and intracellular cAMP-elevating agents such as dibutyryl cAMP and 3-isobutyl-1-methylxanthine, and the effect of IL-4, IL-5, and PGE(2) was additive. However, the pretreatment with IL-6, IL-10, or TGF-beta, or the neutralization of IFN-gamma or IL-12 had no effect on MDC production. B cells, macrophages, and dendritic cells were main producers of MDC, while T cells produced only a small amount of MDC. MDC production by B cells was equally stimulated by LPS and anti-CD40 Ab, while that by macrophages and dendritic cells was more markedly stimulated by anti-CD40 Ab, and PGE(2) further enhanced MDC production by these stimulated cells. These results indicate that PGE(2) regulates Th1/Th2-related chemokine production by B cells, macrophages, and dendritic cells, and that this is a new function of PGE(2) for the regulation of Th2 immune responses at the induction and activation stages.     

Introduction of a benzyl group onto the 2'-OH of 6-chloropurine 3'-O-benzoylriboside

A new method to introduce a benzyl group onto the 2'-OH of purine ribonucleoside is described. Thus, 6-chloropurine 3'-O-benzoylriboside and its 5'-O-trityl congener were condensed with benzyl alcohol using the Mitsunobu reaction to give the 2'-O-benzyl derivative. The yields were varied from 4.6 to 62.9% depending on the solvent. The product was converted to adenosine, indicating that the stereochemistry at C-2' is retained.     

Introduction of a Benzyl Group onto the 2′-OH of 6-Chloropurine 3′-O-Benzoylriboside

Abstract

A new method to introduce a benzyl group onto the 2′-OH of purine ribonucleoside is described. Thus, 6-chloropurine 3′-O-benzoylriboside and its 5′-O-trityl congener were condensed with benzyl alcohol using the Mitsunobu reaction to give the 2′-O-benzyl derivative. The yields were varied from 4.6 to 62.9% depending on the solvent. The product was converted to adenosine, indicating that the stereochemistry at C-2′ is retained.     

Inhibition of the Equilibrative Nucleoside Transporter 1 and Activation of A2A Adenosine Receptors by 8-(4-Chlorophenylthio)-modified cAMP Analogs and Their Hydrolytic Products

Cyclic AMP analogs containing hydrophobic modification of C⁸ at the adenine ring such as 8-(4-chlorophenylthio)-cAMP (8-pCPT-cAMP) and 8-(4-chlorophenylthio)-2′-O-methyl-cAMP (8-pCPT-2′-O-methyl-cAMP) can penetrate membranes due to their high lipophilicity and directly activate intracellular cAMP effectors. Therefore, these cAMP analogs have been used in numerous studies, assuming that their effects reflect the consequences of direct activation of cAMP effectors. The present study provides evidence that 8-pCPT-modified cAMP analogs and their corresponding putative hydrolysis products (8-(4-chlorophenylthio)-adenosine (8-pCPT-ado) and 8-(4-chlorophenylthio)-2′-O-methyl-adenosine (8-pCPT-2′-O-methyl-ado)) inhibit the equilibrative nucleoside transporter 1 (ENT1). In PC12 cells, in which nucleoside transport strongly depended on ENT1, 8-pCPT-ado, 8-pCPT-2′-O-methyl-ado, and, to a smaller extent, 8-pCPT-2′-O-methyl-cAMP caused an increase of protein kinase A substrate motif phosphorylation and anti-apoptotic effect by an A_2A adenosine receptor (A_2AR)-dependent mechanism. In contrast, the effects of 8-pCPT-cAMP were mainly A_2AR-independent. In HEK 293 showing little endogenous ENT1-dependent nucleoside transport, transfection of ENT1 conferred A_2AR-dependent increase in protein kinase A substrate motif phosphorylation. Together, the data of the present study indicate that inhibition of ENT1 and activation of adenosine receptors have to be considered when interpreting the effects of 8-pCPT-substituted cAMP/adenosine analogs.     

Functional analysis of the PP2A subfamily of protein phosphatases in regulating Drosophila S6 kinase

Phosphorylation and activation of ribosomal S6 protein kinase is an important link in the regulation of cell size by the target of rapamycin (TOR) protein kinase. A combination of selective inhibition and RNA interference were used to test the roles of members of the PP2A subfamily of protein phosphatases in dephosphorylation of Drosophila S6 kinase (dS6K). Treatment of Drosophila Schneider 2 cells with calyculin A, a selective inhibitor of PP2A-like phosphatases, resulted in a 7-fold increase in the basal level of dS6K phosphorylation at the TOR phosphorylation site (Thr398) and blocked dephosphorylation following inactivation of TOR by amino acid starvation or rapamycin treatment. Knockdown of the PP2A catalytic subunit increased basal dS6K phosphorylation and inhibited dephosphorylation induced by amino acid withdrawal. In contrast, depletion of the catalytic subunits of the other two members of the subfamily did not enhance dS6K phosphorylation. Knockdown of PP4 caused a 20% decrease in dS6K phosphorylation and knockdown of PP6 had no effect. Knockdown of the Drosophila B56-2 subunit resulted in enhanced dephosphorylation of dS6K following removal of amino acids. In contrast, knockdown of the homologs of the other PP2A regulatory subunits had no effects. Knockdown of the Drosophila homolog of the PP2A/PP4/PP6 interaction protein alpha4/Tap42 did not affect S6K phosphorylation, but did induce apoptosis. These results indicate that PP2A, but not other members of this subfamily, is likely to be a major S6K phosphatase in intact cells and is consistent with an important role for this phosphatase in the TOR pathway.     

Adenosine deaminase from bovine brain: purification and partial characterization.

Bovine brain adenosine deaminase cytoplasmatic form was purified about 450 fold by salt fractionation, column chromatography on DEAE-cellulose, octyl-sepharose 4B and affinity chromatography on CH-sepharose 4B 9-(p-aminobenzyl)adenine. The purified enzyme was homogeneous on disc gel electrophoresis; the enzyme had a molecular mass of about 65 kDa with an isoelectric point at pH 4.87. The Km values for adenosine and 2'-deoxyadenosine were 4 x 10(-5) and 5.2 x 10(-5) M, respectively. The enzyme showed a great stability to temperature with a half life of 15 hours at 53 degrees C significantly different compared to that known for other mammalian forms of this enzyme. Aza and deaza analogs of adenosine and erythro-9-(2-hydroxy-3-nonyl) adenine were good inhibitors of the bovine brain enzyme with little difference with respect to those reported for the adenosine deaminases purified from other sources. Kinetic constants for the association and dissociation of coformycin and 2'-deoxycoformycin with the bovine brain adenosine deaminase are reported.     

Characterization of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC)-induced protein phosphorylation in rabbit platelets: inhibitory effects of AGEPC analogs

1-O-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) induced phosphorylation of two proteins having molecular masses of approximately 20- and 40-kDa in washed rabbit platelets in a concentration- and time-dependent manner. Sequential stimulation with AGEPC did not induce additional protein phosphorylation, supporting the concept of desensitization of the AGEPC receptors responsible for biological activity. AGEPC analogs 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphoric acid-6'-trimethylammonium hexyl ester and 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphoric acid-10'-trimethylammonium decyl ester (U66985 and U66982), containing polar head groups with methylene chain lengths of C6 and C10, did not cause protein phosphorylation, but they did inhibit the AGEPC-induced events. Thus protein phosphorylation is closely associated with the receptor-mediated stimulation of platelets and is a useful indicator of the signaling process initiated through the receptors. Other synthetic analogs of AGEPC such as rac-3-(N-n-octadecylcarbamoyloxy)-2-methoxypropyl 2-thiazolioethyl phosphate and 1-(N-n-pentadecylcarbamoyloxy)-2-methoxy-rac-glycero-3-phosphochol ine (CV3988 and U68043) were also shown to be inhibitors of the AGEPC-induced protein phosphorylation. Inhibition by these analogs was specific for AGEPC since there was no observed effect of thrombin, ADP, 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and arachidonic acid-induced changes. The extent of inhibition was dependent on the concentration of AGEPC and its analogs and did not change with time after the addition of AGEPC. In platelets incubated with AGEPC analogs before and simultaneously with the addition of AGEPC, protein phosphorylation was prevented; however, addition of AGEPC to platelets shortly before the addition of these analogs showed a high response. In experiments where platelets were previously incubated with AGEPC analogs and washed with buffer containing 0.5% bovine serum albumin, AGEPC-induced protein phosphorylation was recovered to a level of 80%. These observations support the conclusion that AGEPC stimulates platelets through its specific receptor, and that the AGEPC analogs bind to the AGEPC receptor and block that pathway sensitive to AGEPC stimulation but not because of the desensitization of its receptor. On the other hand, in platelets where phosphorylation of the 40-kDa protein was induced by a 2-min preincubation with 3 X 10(-10) M TPA, 5 X 10(-10) M AGEPC-induced serotonin release decreased by 51% compared to a control value.(ABSTRACT TRUNCATED AT 400 WORDS)     

Adenosine receptors in smooth muscle: structure-activity studies and the question of adenylate cyclase involvement in control of relaxation

Structure-activity studies with a number of adenosine derivatives and analogs, measuring their relaxant effects in a variety of smooth muscle systems, were conducted in the hope of obtaining indications of the possible involvement of adenylate cyclase in their mechanism of action. While it was confirmed that a C6 aminofunction is of importance for agonist activity, several compounds, in particular the relatively potent N6-hydroxylaminopurine ribonucleoside, were not antagonized by 8-p-sulfophenyltheophylline, indicating that some nucleosides cause smooth muscle relaxation by a mechanism other than adenosine receptor stimulation. Nucleosides not bearing a C6 aminofunction were essentially inactive in rabbit intestine but showed weak relaxant effects in bovine coronary artery; this may indicate a difference between the adenosine receptor systems in these tissues and the intracellular mechanisms of relaxation. Comparing the relative potencies of compounds such as adenosine, 2-chloroadenosine, 5'-(N-ethylcarboxamido)adenosine, and (-)N6-(R-phenylisopropyl)adenosine, which have been used widely to classify adenylate cyclase-coupled adenosine receptors, no uniform pattern became apparent among different smooth muscle systems used in this study and reported in the recent literature. Thus, we conclude that a classification of smooth muscle adenosine receptors according to criteria established for cyclase-coupled receptors may be inappropriate or misleading, particularly with respect to implications of adenylate cyclase involvement in the relaxant effects of adenosine and related nucleosides.     

Activation of Na+/Ca2+ exchange by adenosine in ewe heart sarcolemma is mediated by a pertussis toxin-sensitive G protein

We studied the effect of adenosine on Na+/Ca2+ exchange activity in ewe heart ventricular sarcolemmal vesicles. Adenosine was found to stimulate Na+/Ca2+ exchange activity in a dose-dependent manner from 0.1 nM to 10 microM, with maximal stimulation (40%) at 0.1 microM adenosine. The Vmax of Na+/Ca2+ exchange was increased, but the Km for Ca2+ was not altered. The effect of adenosine was specific since 1 microM adenine, inosine, and guanosine led to less than 15% stimulation, and adenosine diphosphate had no effect. Caffeine antagonized the activation of Na+/Ca2+ exchange by adenosine, and the order of potency of adenosine analogs was N6-(L-2-phenylisopropyl)adenosine = N6-cyclohexyladenosine = 5'-(N- ethylcarboxamido)adenosine much greater than N6-(D-2-phenylisopropyl)adenosine, indicating the involvement of A1 subclass receptors. The effect of adenosine was mimicked by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and blocked by pertussis toxin treatment. Taken together, these results suggest that A1 subclass receptors coupled to a pertussis toxin-sensitive G protein mediate the activation of Na+/Ca2+ exchange activity by adenosine. We conclude that the negative inotropic effect of adenosine in ventricular muscle, antagonistic toward cyclic AMP, may involve activation of Na+/Ca2+ exchange.     

Pharmacological preconditioning with resveratrol: role of CREB-dependent Bcl-2 signaling via adenosine A3 receptor activation

Recent studies demonstrated that resveratrol, a grape-derived polyphenolic phytoalexin, provides pharmacological preconditioning (PC) of the heart through a NO-dependent mechanism. Because adenosine receptors play a role in PC, we examined whether they play any role in resveratrol PC. Rats were randomly assigned to groups perfused for 15 min with 1) Krebs-Henseleit bicarbonate buffer (KHB) only; 2) KHB containing 10 microM resveratrol; 3) 10 microM resveratrol + 1 microM 8-cyclopentyl-1,3-dimethylxanthine (CPT; adenosine A(1) receptor blocker); 4) 10 microM resveratrol + 1 microM 8-(3-chlorostyryl)caffeine (CSC; adenosine A(2a) receptor blocker); 5) 10 microM resveratrol + 1 microM 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS-1191; adenosine A(3) receptor blocker); or 6) 10 microM resveratrol + 3 microM 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride [LY-294002, phosphatidylinositol (PI)3-kinase inhibitor], and groups perfused with adenosine receptor blockers alone. Hearts were then subjected to 30-min ischemia followed by 2-h reperfusion. The results demonstrated significant cardioprotection with resveratrol evidenced by improved ventricular recovery and reduced infarct size and cardiomyocyte apoptosis. CPT and MRS 1191, but not CSC, abrogated the cardioprotective abilities of resveratrol, suggesting a role of adenosine A(1) and A(3) receptors in resveratrol PC. Resveratrol induced expression of Bcl-2 and caused its phosphorylation along with phosphorylation of cAMP response element-binding protein (CREB), Akt, and Bad. CPT blocked phosphorylation of Akt and Bad without affecting CREB, whereas MRS 1191 blocked phosphorylation of all compounds, including CREB. LY-294002 partially blocked the cardioprotective abilities of resveratrol. The results indicate that resveratrol preconditions the heart through activation of adenosine A(1) and A(3) receptors, the former transmitting a survival signal through PI3-kinase-Akt-Bcl-2 signaling pathway and the latter protecting the heart through a CREB-dependent Bcl-2 pathway in addition to an Akt-Bcl-2 pathway.     

Facile preparation of new 4-phenylamino-3-quinolinecarbonitrile Src kinase inhibitors via 7-fluoro intermediates: identification of potent 7-amino analogs

A more efficient preparation of 4-[(2,4-dichloro-5-methoxyphenyl)amino]-7-fluoro-6-methoxy-3-quinolinecarbonitrile (2), the penultimate intermediate in the synthesis of bosutinib (1a), was developed. New 7-alkoxy-4-phenylamino-3-quinolinecarbonitrile Src inhibitors were prepared from 5 and 9, the 6-ethoxy and 6-hydrogen analogs of 2. In addition, the fluoro group of 2 was readily displaced by primary and secondary amines to give 7-amino analogs. Two of these 7-amino analogs, 15 and 18, were potent Src inhibitors with in vivo activity.     

Adenosine Inhibition of Calmodulin-Sensitive Adenylate Cyclase from Bovine Cerebral Cortex

Calmodulin (CaM)-sensitive adenylate cyclase has recently been purified extensively from bovine brain. In this study, the sensitivity of the CaM-sensitive adenylate cyclase to adenosine and adenosine analogs was examined. The highly purified enzyme preparation retained sensitivity to inhibition by adenosine and adenosine analogs with ribose ring modifications, but not to those with purine ring modifications. Adenosine inhibition of this enzyme was not dependent on GTP and was noncompetitive with respect to ATP. Enzyme that had been dissociated from functional guanine nucleotide binding protein interactions by gel filtration in the presence of the zwitterionic detergent 3-[3-(cholamidopropyl)-dimethylammonio]-propanesulfonate and Mn2+ retained sensitivity to adenosine inhibition. The Ki for adenosine inhibition of the CaM-sensitive adenylate cyclase was approximately 2.6 X 10(-4) M. 5'-Guanylylimidodiphosphate and CaM did not affect the Ki of 3'-deoxyadenosine for the enzyme, but the presence of Ca2+ in the millimolar range raised the Ki by a factor of 5. These results show that the CaM-sensitive form of adenylate cyclase from bovine brain is subject to adenosine inhibition, and strongly suggest that this inhibition is due to interaction of ligands with a purine-specific ("P") site located on the catalytic subunit of the enzyme.