Replacement of the ribofuranose oxygen of 2-5A derivatives by methylene: synthesis of an aristeromycin analogue of 2-5A core 5'-monophosphate (5'-O-phosphoryladenylyl)(2'----5')adenyl(2'----5')adenosine

The ribofuranose oxygens of the three adenosine residues of the 5'-monophosphate of the 2-5A core [adenylyl(2'----5')adenylyl(2'----5')adenosine] were replaced by methylenes through the synthesis of an aristeromycin [9-[(1R,2S,3R,4R)-2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl]adenosine] analogue. In the synthetic approach, the chlorophosphite triester procedure was employed together with the use of dimethoxytrityl and tert-butyldimethylsilyl protecting groups. The final product 14 was bound to the 2-5A-dependent endonuclease of mouse, rabbit, or human cells 100-300 times less effectively than parent p5'A2'p5'A2'p5'A. In extracts of human Daudi cells where the monophosphate p5'A2'p5'A2'p5'A was able to effect ribosomal RNA cleavage at 2 X 10(-7) M, 14 required a concentration of 2 X 10(-5) M to bring about discernible rRNA cleavage.     

Synthesis and biological activity of uronic acid analogues of 2-5A[5'-O-triphosphoryladenylyl(2----5')adenylyl-(2'----5')adenosine]

The oligonucleotide ppp5'A2'p5'A2'p5'A, known as 2-5A, is a potent translational inhibitor involved in some aspects of interferon action. To explore the specific function of the charged 5'-triphosphate moiety, we prepared a series of congeners in which the 5' region was hypermodified. Thus, uronic acid derivatives were substituted for the 5' terminal adenosine residue of 2-5A. Compounds 9, 10, 11 and 12 carried adenosine 5'-uronic acid, ethyl adenosine 5'-uronate, adenosine 5'-uronamide, and adenosine 5'-(N-ethyl)uronamide, respectively, in place of the 5' terminal adenosine triphosphate moiety of 2-5A. While all the analogues showed some weak interaction with the 2-5A-dependent endonuclease (RNase L), compound 9 showed the strongest binding ability, and while unable to activate the mouse RNase L, could activate human RNase at a concentration 100-fold greater than that required for the parent 2-5A. This result suggests that the function of the 5'(poly)phosphate moiety of 2-5A may be fulfilled by some other anionic moiety.     

A new and potent 2-5A analogue which does not require a 5'-polyphosphate to activate mouse L-cell RNase L.

In order to explore the possibility of supplanting the requirement of a 5'-triphosphate moiety for the activation of the 2-5A-dependent endonuclease (RNase L) of mouse L-cells, two new tetrameric analogues of 2-5A were synthesized. The first tetramer, obtained by both a modified prebiotic synthetic approach as well as a phosphite triester solid phase oligonucleotide synthesis method, was p5'A2'p5'A2'p5'(br8A)2'p5'(br8A). The second oligonucleotide was derived from the former by a sequence involving periodate oxidation, reaction with n-hexylamine, and cyanoborohydride reduction, resulting in conversion of the 2'-terminal adenosine residue to 9-(3'-aza-4'-hexyl-1',2',3',4'-tetradeoxyhexopyranos-1(1)-yl)-8-++ +bromoadenine. Both of these oligomers, bearing only 5'-monophosphate groups, were found to be as potent as 2-5A itself as activators of the RNase L of mouse L-cells.     

Synthesis and biological activities of oligo(8-bromoadenylates) as analogues of 5'-O-triphosphoadenylyl(2'----5')adenylyl(2'----5')adenosine

The 8-bromoadenosine analogue of 5'-O-triphosphoadenylyl(2'----5')adenylyl(2'----5')adenosine (2-5A) and its derivatives were synthesized, and their biological activity was evaluated in mouse L cell extracts. All compounds, except 5'-dephosphorylated "cores" bound to the 2-5A-dependent endonuclease with a relative activity, depending on the derivative, of 1 to 0.035 of that of 2-5A trimer. 8-Bromoadenylate trimer 5'-mono-, -di, and -triphosphates inhibited protein synthesis with a relative activity of 0.0023, 0.050, and 0.015 compared to 2-5A. Tetramer 5'-monophosphate also inhibited protein synthesis (relative activity 0.0033). The corresponding pentamer 5'-monophosphate did not; however, the pentamer 5'-diphosphate was able to inhibit translation (relative activity 0.0092). All compounds that possessed inhibitory activity in the protein synthesis inhibition assay gave ribosomal RNA cleavage patterns characteristic of the action of 2-5A-dependent endonuclease. Thus, 8-bromination of the all of the adenine rings of 2-5A leads to 20- to 70-fold reduction in the biological activity of the corresponding 5'-di- and -triphosphate, respectively; however, this same alteration of the three adenine moieties gives rise to a 5'-monophosphate with much enhanced translational inhibitory activity compared to the parent 2-5A trimer 5'-monophosphate.     

Inhibition of the RNA polymerase of vesicular stomatitis virus by ppp5'A2'p5'A and related compounds

The diadenylate triphosphates ppp5'A2'p5'A and ppp5'A3'p5'A were found to inhibit the purified RNA polymerase ('nucleocapsid') complex from vesicular stomatitis virus (VSV). The corresponding diadenylate monophosphate p5'A2'p5'A did not inhibit, nor did the triadenylate triphosphate ppp5'A2'p5'A2'p5'A; the diadenylate diphosphate pp5'A2'p5'A had intermediate inhibitory activity. Increasing the concentration of ATP, GTP or CTP in the reaction mixture decreased inhibition by ppp5'A2'p5'A, while UTP had minimal or no protective effect. ppp5'A2'p5'A did not protect the RNA polymerase from inactivation by N-ethylmaleimide. This suggests that the action of ppp5'A2'p5'A occurs at a site on the enzyme that is distinct from the N-ethylmaleimide-protecting, ATP-binding site characterized previously.     

Hydrolysis and solvent-dependent 2'----5'and 3'----5' acyl migration in prodrugs of 9-beta-D-arabinofuranosyladenine

As a prerequisite to quantitative in vivo studies to further explore the promising topical activity of the 2',3'-di-O-acetyl derivative of 9-beta-D-arabinofuranosyladenine (ara-A) against herpes virus infections, the kinetics of solution degradation of the 2',3'-di-O-acetyl derivative and the 2'-,3'-, and 5'-monoacetates were investigated. The rates of aqueous solution hydrolysis were found to be consistent with rank order predictions based on a consideration of substituent effects. Preliminary in vivo hydrolysis data, however, do not correlate with such predictions, indicating a need for more systemic studies of the effect of molecular structure on enzyme-catalyzed hydrolysis. An important reaction of the 2'-3'-diester and the 3'-monoester in aqueous solution, in addition to ester hydrolysis, is 3'----5' acyl migration. 2'----5' Acyl migration does not occur in water but is the predominant migration pathway in organic solvents, as verified by studies in acetonitrile. 1H NMR spectroscopy was employed to study the dependence of the conformation of the sugar ring on the solvent environment. Although a change in the equilibrium between the C(2')endo and C(3')endo conformational states does occur, it is not a dramatic change and cannot explain the solvent selectivity observed in the acyl migration kinetics.     

Synthesis, characterization, and biological properties of 8-azido- and 8-amino-substituted 2',5'-oligoadenylates

A series of 8-azido- and 8-amino-substituted 2',5'-oligoadenylatyes was prepared by a uranyl-ion catalyzed polymerization of the corresponding 8-substituted adenosine phosphorimidazolide. Subsequent 5'-dephosphorylation of the resulting 5'-phosphoryl 2',5'-linked oligomers with alkaline phosphatase gave the corresponding core oligomers. The CD spectra indicated that the 8-aminoadenosine analogue of the 2',5'-linked trimer has an anti-orientation as in naturally occurring 2',5'-oligoadenylates, while 8-azido-substituted 2',5'-oligoadenylates have a syn-orientation. The 8-substituted oligomers showed enhanced resistance against digestion by snake venom phosphodiesterase. The 2',5'-linked 8-azidoadenylate trimer and tetramer displayed strong RNase L binding and activating ability, although the corresponding dimer is devoid of such activities. In contrast, very low or no RNase L binding and activating ability were observed in the 8-aminoadenosine analogue of 2',5'-oligoadenylates. Results indicate that the bulkiness and ionic character of the 8-substituting group have significant effects on the ability of these analogues to bind and activate RNase L. Furthermore, the orientation of the glycosidic base in the 2-5A analogues may change from syn to anti during binding to RNase L. The 8-azidoadenosine analogues of 2-5A will be useful tools in the photoaffinity labeling of RNase L, due to their strong RNase L binding ability. In addition, these 8-azidoadenosine compounds may be considered as candidates for experimental therapeutic agents because they have enhanced stability to enzyme degradation while retaining the ability to activate RNase L.     

Synthesis and characterization of oligonucleotides containing 5',8-cyclopurine 2'-deoxyribonucleosides: (5'R)-5',8-cyclo-2'-deoxyadenosine, (5'S)-5',8-cyclo-2'-deoxyguanosine, and (5'R)-5',8-cyclo-2'-deoxyguanosine.

Radiation-induced degradation of purine and pyrimidine nucleosides gave rise to carbon-bridged cyclocompounds. Such cyclonucleosides represent a class of tandem lesions in which modification of both the base and 2-deoxyribose has occurred. A solid-phase synthetic method was designed for the incorporation of both 5'R and 5'S diastereoisomers of 5',8-cyclopurine 2'-deoxyribonucleosides into oligodeoxynucleotides to facilitate the assessment of the biochemical and biophysical features of such lesions. We report the preparation of the phosphoramidite synthons of (5'R)-5', 8-cyclo-2'-deoxyadenosine (2), (5'S)-5',8-cyclo-2'-deoxyguanosine (3), and (5'R)-5',8-cyclo-2'-deoxyguanosine (4). Fully protected compounds 10, 18, and 25 were then inserted into several oligonucleotides by automated procedures. Analysis of modified DNA oligomers 26-31 by electrospray mass spectrometry and enzymatic digestions with exo- and endonucleases confirmed the base compositions and the integrity of free radical-induced tandem lesions 2-4 that were chemically inserted.     

A new one-step method for the preparation of 3',5'-bisphosphates of acid-labile deoxynucleosides

A method is described for preparing 3',5'-bisphosphates of labile deoxynucleosides. Under strictly anhydrous conditions, pyrophosphoryl tetrakistriazole apparently forms a ring structure bridging the 3'- and 5'-hydroxyl groups of deoxynucleosides, since upon the addition of water the ring opens and the 3',5'-bisphosphate is formed. Due to the presence of triethylamine no acid is generated at any time so that the entire procedure is in neutral solution. The bisphosphates of N2,3-ethenodeoxyguanosine, O2-ethyldeoxythymidine, and O4-methyldeoxythymidine, all of which are acid-labile, were prepared in good yield without degradation. Other modified bisphosphates prepared include O6-benzyldeoxyguanosine and 1,N6-ethenodeoxyadenosine, as well as those of unmodified deoxyguanosine and thymidine. Characterization was by 31P NMR and UV spectroscopy. Both 5'p(dT)p3' and 5'p(dG)p3' were substrates for RNA ligase, further proving the structure of the phosphorylated compounds.     

Binding sites for bilobalide, diltiazem, ginkgolide, and picrotoxinin at the 5-HT3 receptor

Bilobalide (BB), ginkgolide B (GB), diltiazem (DTZ), and picrotoxinin (PXN) are 5-hydroxytryptamine type 3 (5-HT(3)) receptor antagonists in which the principal sites of action are in the channel. To probe their exact binding locations, 5-HT(3) receptors with substitutions in their pore lining residues were constructed (N-4'Q, E-1'D, S2'A, T6'S, L7'T, L9'V, S12'A, I16'V, D20'E), expressed in Xenopus laevis oocytes, and the effects of the compounds on 5-HT-induced currents were examined. EC(50) values at mutant receptors were less than 6-fold different from those of wild type, indicating that the mutations were well tolerated. BB, GB, DTZ, and PXN had pIC(50) values of 3.33, 3.14, 4.67, and 4.97, respectively. Inhibition by BB and GB was abolished in mutant receptors containing T6'S and S12'A substitutions, but their potencies were enhanced (42- and 125-fold, respectively) in S2'A mutant receptors. S2'A substitution also caused GB ligand trap. PXN potency was modestly enhanced (5-fold) in S2'A, abolished in T6'S, and reduced in L9'V (40-fold) and S12'A (7-fold) receptors. DTZ potency was reduced in L7'T and S12'A receptors (5-fold), and DTZ also displaced [(3)H]granisetron binding, indicating mixed competitive/noncompetitive inhibition. We conclude that regions close to the hydrophobic gate of M2 are important for the inhibitory effects of BB, GB, DTZ, and PXN at the 5-HT(3) receptor; for BB, GB, and PXN, the data show that the 6' channel lining residue is their major site of action, with minor roles for 2', 9', and 12' residues, whereas for DTZ, the 7' and 12' sites are important.     

Synthesis and complement inhibitory activity of B/C/D-ring analogues of the fungal metabolite 6,7-diformyl-3',4',4a',5',6',7',8',8a'-octahydro-4,6',7'-trihydroxy- 2',5',5',8a'-tetramethylspiro[1'(2'H)-naphthalene-2(3H)-benzofuran

This paper reports the synthesis and the bioassay of 4-methoxy- and 4-hydroxyspiro[benzofuran-2(3H)-cyclohexane] partial analogues (5) of the complement inhibitory sesquiterpene fungal metabolite 6,7-diformyl-3',4',4a',5',6',7',8',8a'-octahydro-4,6',7'-trihydroxy-2',5',5',8a'-tetramethylspiro[1'(2'H)-naphthalene-2(3H)-benzofuran] (1a, K-76) and its silver oxide oxidized product (1b, K-76COOH). The described target compounds represent spirobenzofuran B/C/D-ring analogues lacking the A-ring component of the prototype structure. The target compounds were evaluated by the inhibition of total hemolytic complement activity in human serum. It was observed that the structurally simplified analogue 4-methoxyspiro[benzofuran-2(3H)-cyclohexane]-6-carboxylic acid (5a) exhibited an IC(50) = 0.53 mM similar to the IC(50) = 0.57 mM that was observed for the natural product derivative 1b. Exhibiting an IC(50) = 0.16 mM, the three-ringed partial structure 6-carboxy-7-formyl-4-methoxyspiro[benzofuran-2(3H)-cyclohexane] (5k)was found to be the most potent target compound. Like the natural product, 5k appears to inhibit primarily at the C5 activation step and inhibits both the classical and alternative human complement pathways. Several other analogues inhibited complement activation in vitro at concentrations similar to those required for inhibition by the natural product 1b.     

A new flavone glycoside, 5-hydroxy 7,3',4',5'-tetra-methoxyflavone 5-O-beta-D-xylopyranosyl-(1-->2)-alpha-L-rhamnopyranoside from Bauhinia variegata Linn.

A new flavone glycoside m.f. C(30)H(36)O(15) m.p. 252-253 degrees C, [M]+ 636 (EIMS) was isolated from the acetone soluble fraction of the concentrated 95% ethanolic extract of the seeds of Bauhinia variegata (Linn). It was identified as 5-hydroxy7,3',4',5'-tetra-methoxyflavone 5-O-beta-D-xylopyranosyl-(1-->2)-alpha-L-rhamnopyranoside (1) by various colour reactions, chemical degradations and spectral techniques.     

Synthesis and biological evaluation of 2-amino-3-(3',4',5'-trimethoxybenzoyl)-5-aryl thiophenes as a new class of potent antitubulin agents

A new series of compounds in which the 2-amino-5-chlorophenyl ring of phenstatin analogue 7 was replaced with a 2-amino-5-aryl thiophene was synthesized and evaluated for antiproliferative activity and for inhibition of tubulin polymerization and colchicine binding to tubulin. 2-Amino-3-(3',4',5'-trimethoxybenzoyl)-5-phenyl thiophene (9f) as well as the p-fluoro-, p-methyl-, and p-methoxyphenyl substituted analogues (9i, j, and l, respectively) displayed high antiproliferative activities with IC50 values from 2.5 to 6.5 nM against the L1210 and K562 cell lines. Compounds 9i and j were more active than combretastatin A-4 as inhibitors of tubulin polymerization. Molecular docking simulations to the colchicine site of tubulin were performed to determine the possible binding mode of 9i. The results obtained demonstrated that antiproliferative activity correlated well with the inhibition of tubulin polymerization and the lengthening of the G2/M phase of the cell cycle. Moreover, a good correlation was found between these inhibitory effects and the induction of apoptosis in cells treated with the compounds.     

Synthesis and biological evaluation of dinucleoside methylphosphonates of 3'-azido-3'-deoxythymidine and 2', 3'-dideoxycytidine

The 5'----5' dinucleoside methylphosphonates of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (DDC) were prepared and evaluated for their inhibitory properties against different viruses, including human immunodeficiency virus (HIV). The synthesis of the compounds was achieved by reaction of AZT or N4-(4-monomethoxytrityl)-2',3'-dideoxycytidine with in situ prepared methylphosphonic bis (triazolide), followed in the latter case by an acidic treatment. The two title compounds showed in vitro anti-HIV activity, that was 200- to 450-fold less pronounced that that shown by the corresponding monomeric nucleosides AZT and DDC. The decreased antiviral activity may be ascribed to nuclease resistance of the methylphosphonate linkage.     

Metabolism and disposition of 2',3'-di-O-nitro-adenosine-5'-(N-ethyl-carboxamide) in dogs

2',3'-Di-O-nitro-[8-3H]-adenosine-5'-(N-ethyl-carboxamide) (20 micrograms/kg) was denitrated completely within 1-3 hr in perorally and intravenously dosed dogs. Extremely rapid disappearance of the unchanged drug in serum was parallelled by the instantaneous appearance of mononitrates with 3'-mononitrate levels exceeding those of 2'-mononitrate three-fold. The mononitrates were eliminated with a half-life of 30-70 min, giving rise to the completely denitrated product, adenosine-5'-(N-ethyl-carboxamide) (NECA). The latter product was not further metabolized and was eliminated with a half-life of about 4 hr. Urinary excretion averaged 50% of the administered dose within 4 days and was represented essentially by the completely denitrated drug. Volatile 3H-label of the drug was found in serum and urine during in vivo experiments. Oral bioavailability of the drug was about 90%. In vitro studies indicated that thiols are involved in denitration and reactions are catalysed by glutathione S-transferases, which were partially purified from dog liver. Nitrate ester cleavage was more easily accomplished at the 2'-position than at the 3'-position of the drug and resulted in the liberation of inorganic nitrite. Comparison of in vitro denitration rates gave the following ranking order; 2',3'-di-O-nitro-NECA greater than isosorbide-2,5-dinitrate greater than 2'-nitro-NECA greater than 3'-nitro-NECA greater than isosorbide-2-mononitrate, while nitrate ester cleavage of isosorbide-5-mononitrate was not detectable.     

Muscarinic receptor subtype specificity of 5'-(isobutylthio)-adenosine (SIBA) and its analogs

1. 5'(Isobutylthio)-adenosine (SIBA) and its analogs, at 100 microM, inhibited [3H]N-methyl-scopolamine binding to homogenates of whole brain and cortex (mainly M1 subtype receptors) by 11-30% and to cerebellum (mainly M2 subtype receptors) by 20-39%. 2. At 0.01-1.0 microM, stimulation of [3H]QNB and NMS-inaccessible [3H]QNB binding was observed, with the most induced by 1 microM 3-deaza-SIBA. 3. In contrast, [3H]pirenzepine ([3H]PZ) binding to whole brain and cortex was inhibited in a dose-dependent manner with Ki values in the microM range. 4. As antagonists of acetylcholine-induced contraction of guinea pig ileum (mainly M2 subtype receptors), the analogs were slightly more potent than pirenzepine, but several orders of magnitude less than atropine; the order of potency was opposite that determined for the binding of [3H]PZ to cortex. 5. Thus, SIBA and its analogs may have differential effects on muscarinic receptor subtypes and show some specificity for the M1 receptor subtype.     

TSAO analogues. Stereospecific synthesis and anti-HIV-1 activity of 1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro -5'- (4'-amino-1',2'-oxathiole 2',2'-dioxide) pyrimidine and pyrimidine-modified nucleosides.

Several analogues of a new lead for anti-HIV-1 agents [1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-thymine] -3'-spiro-5'-(4'-amino-1',2'-oxathiole 2',2'-dioxide) (TSAO) modified at positions N-3, O-4 and C-5 of the thymine moiety, have been prepared and evaluated as inhibitors of HIV-1 replication. A new stereoselective synthetic procedure is described. Reaction of 1,2-di-O-acetyl-5-O-benzoyl-3-C-cyano-3-O-mesyl-D-ribofuranose with pyrimidine bases, followed by treatment with Cs2CO3 afforded stereoselectively, beta-D-ribofuranosyl-3'-spiro nucleosides. 2',5'-O-Deacylation and subsequent treatment with tert-butyldimethylsilyl chloride gave the TSAO derivatives. Only those analogues having a tBDMSi group at both the C-5' and C-2' positions of the ribose moiety showed potent anti-HIV-1 activity. The activity ranged from 0.060 microM to 1.0 microM. Introduction of an alkyl or alkenyl function at N-3 of the thymine ring markedly decreased cytotoxicity without affecting the antiviral activity. While markedly active against HIV-1, the TSAO derivatives had no activity against HIV-2 or SIV. They represent the first example of nucleoside analogues with an intact ribose moiety that discriminate between HIV-1 and other retroviruses.     

Mutations of the 2' proline in the M2 domain of the human GABAC rho1 subunit alter agonist responses

Mutations of the proline residue at the 2' position (P2') within the second transmembrane (M2) domain of the gamma-aminobutyric acid(C) (GABA(C)) rho1 subunit are known to produce receptors with altered pharmacology. In the present study, P2' was mutated to alanine (rho1P2'A), phenylalanine (rho1P2'F), glycine (rho1P2'G) and serine (rho1P2'S). Mutant receptors were characterized using a range of agonists, partial agonists and antagonists. rho1P2'A, rho1P2'G and rho1P2'S receptors were less susceptible than wild-type receptors to agonist activation. Most notably, the partial agonists, (+/-)-trans-2-(aminomethyl)cyclopropanoic acid ((+/-)-TAMP) and imidazole-4-acetic acid (I4AA) were converted to antagonists at rho1P2'G and rho1P2'S receptors and the partial agonist CACA acted as an antagonist at rho1P2'S receptors. In contrast, rho1P2'F receptors were more prone to activation by agonists. A correlation was observed between the pharmacological properties of the mutant receptors and the hydrophobicity of each residue. Unlike the agonists or partial agonists, the affinity of competitive antagonists, (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA) and 4,5,6,7-tetrahydroisoxazole[4,5-c]pyridine-3-ol (THIP), did not change significantly between wild-type and mutant receptors. Thus, the results suggest that the agonist/competitive antagonist binding site(s) were not significantly affected by the mutations, but that receptor activation properties altered such that the more hydrophobic the residue at the 2' position, the more prone the receptor is to agonist activation.