Thiosugars. X. Novel Nucleoside Analogues Derived from 4-Thio-L-lyxofuranose

Abstract

1-O-Acetyl-2,3,5-tri-O-benzyl-4-thio-L-lyxofuranose 1 was transformed into O-benzyl- and O-acetyl-protected 1-(4-thio-L-lyxofuranosyl) nucleoside derivatives by use of the TMSOTf method. Debenzylation with boron tribromide or deacetylation with sodium methoxide yielded the corresponding pyrimidine (7–11, 17, 18, 26 and 27) and purine (29 and 34) nucleoside analogues. The anomeric configurations were determined by NMR spectroscopy and, in the case of the 5-halo- (7–9) and nitrouridine derivative 11 and the 6-methylcytidine derivative 27, by X-ray structural analyses. – The unprotected nucleosides were not antivirically inhibitory at 250 µM.     

Synthesis of imidazo[4,5-e][1,4]diazepine and 3-substituted 3-deazapurine nucleosides from 1-substituted inosines.

New synthetic methods of imidazo[4,5-e][1,4]diazepine nucleosides 8-11 and 3-substituted 3-deazainosines 14 from inosine have been reported. Treatment of N1-substituted inosines 1-3 with aqueous NaOH gave 5-amino-4-(N-substituted carbamoyl)imidazole ribosides 5-7, followed by appropriate manipulations to afford ring-expanded guanosine, inosine, and xanthosine analogues. Additionally, the 5-amino group of 4-N-allylcarbamoyl derivative 12 was converted into corresponding 5-iodo nucleoside 13. We found that 13 was cyclized to form 3-deaza-3-methylinosine (14) in the presence of Pd catalysts.     

Studies on lysine, glutamine and glutamic acid tRNAs from Drosophila.

The minor bases present in the family of Drosophila tRNAs recognising codons of the type NAA or NAG have been studied. Under standard aminoacylating conditions, the acceptor activities of BrCN-treated tRNA-Lys-5 tRNA-Glu-4 and tRNA-G1n-4 were completely eliminated, suggesting the presence of 2-thiouridine derivatives. The two major lysine tRNA species (tRNA-Lys-2 and tRNA-Lys-5) were purified and their nucleoside content determined both directly and by the tritium derivative technique. Both tRNAs contain 1-methyladenosine, N-2-dimethylguanosine, 7-methylguanosine, 5-methylcytidine, pseudouridine and dihydrouridine, and tRNA-Lys-5 contains 1-methylguanosine. Neither species contain ribothymidine, although both may contain 2'-O-methyl ribothymidine. A nucleoside with ultraviolet spectral properties similar to N-4-acetylcytidine was found in tRNA-Lys-5 and a nucleoside with chromatographic properties the same as N-[9-beta-D-ribofuranosyl)-purin-6-yl-carbamoyl] threonine was found in tRNA-Lys-2. A 2-thiouridine derivative was not found in tRNA-Lys-5 using these chromatographic techniques.     

Syntheses of fused heterocyclic compounds and their inhibitory activities for squalene synthase.

A variety of fused heterocyclic compounds (2-11) were synthesized as a modification of the lead compound 1a and evaluated for their inhibition of squalene synthase. 4,1-Benzothiazepine derivative 2, 1,4-benzodiazepine derivative 6, 1,3-benzodiazepine derivative 7, 1-benzazepine derivative 9, and 4,1-benzoxazocine derivative 10 potently inhibited squalene synthase activity, whereas the 4,1-benzoxazepine derivatives 1 was the most potent inhibitor. 4,1-Benzothiazepine S-oxide derivative 4, 1,4-benzodiazepine derivative 5, 1,3,4-benzotriazepine derivative 8, and 1,2,3,4-tetrahydroquinoline derivative 11 were found to be weakly active. Comparison of the X-ray structures of these compounds (1a, 2, 4, 5, 7 and 10) suggests that orientation of the 5- (or 6)-phenyl group is important for activity.     

Solid-phase parallel synthesis of 4-beta-D-ribofuranosylpyrazolo[4,3-d]pyrimidine nucleosides

The synthesis of pyrazolo[4,3-d]pyrimidine nucleoside library using solid-phase parallel synthesis methodology is described. Glycosylation of the trimethylsilyl (TMS) derivative of 1- and 2-(methyl)-1H and 2H-pyrazolo[4,3-d]pyrimidine-5,7-(4H, 6H)-dione (5) with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose in the presence of TMS triflate provided two novel protected nucleosides 6 and 7. The structures of 6 and 7 were assigned by 1H and 2D NMR experiments. Nucleosides 6 and 7 were then transformed to the key intermediates 12 and 15 respectively. Reaction of 12 and 15 with MMTCl resin in the presence of 2,6-lutidine afforded the necessary scaffolds B and C. Different amines (96) were introduced selectively by nucleophilic substitution on scaffolds B and C using solid-phase parallel semi-automated synthesizer. Cleavage of the products from the solid support with 30% HFIP in a parallel fashion yielded nucleoside libraries simultaneously, and they were analyzed and characterized by high-throughput LC-MS.     

Distribution of the modified nucleoside Q and its derivatives in animal and plant transfer RNA''s.

The modified nucleoside, 7-(4,5-cis-dihydroxy-1-cyclopenten-3-yl-aminomethyl)-7-deazaguanosine, designated as Q, and its derivative, Q*, were found in tRNA's from various organisms, including several mammalian tissues, other animals such as starfish, lingula and hagfish, and wheat germ. Q isolated from rat liver tRNA was found to be identical with E. coli Q by mass spectrometry and thin-layer chromatography. Thus the rare modified nucleoside Q originally isolated from E. coli tRNA, is widely distributed in various organisms. Analysis of the mass spectrum of Q* suggested that it has a different side chain from Q.     

Thiosugar nucleosides. Synthesis and biological activity of 1,3,4-thiadiazole, thiazoline and thiourea derivatives of 5-thio-D-glucose

New acylated 5-thio-beta-D-glucopyranosylimino-disusbstituted 1,3,4-thiadiazols 8, and 11 were prepared, via spontaneous rearrangements, by cycloaddition of the glycosyl isothiocyanate 2 with the reactive intermediates 1-aza-2-azoniaallene hexachloroantimonates 4 and 6, respectively. Reaction of 2 with aminoacetone or chloroethylamine afforded the acylated 5-thio-beta-D-glucopyranosyl-4-imidazoline-2-thione nucleoside 16 and glucopyranosylamino-2-thiazoline derivative 18, respectively. Deblocking of 8, 11, 17 and 19 furnished the free nucleoside analogues 9, 12, 18 and 20, respectively. Analogously, treatment of 2 with chloroethylamine in the 1:2 ratio afforded the thioureylendisaccharide 21. No in vitro antiviral activity against HIV-1, HIV-2, human cytomegallovirus (HMCV), has been found for the new synthesized compounds.     

Photolabile and paramagnetic derivatives of the nucleoside X and of Escherichia coli tRNAPhe.

The synthesis of N3-[3-L-(5-azido-2-nitrobenzamido)-3-carboxypropyl]uridine (4b) and N3-[3-carboxy-3-L-(2,2,5,5-tetramethyl-3-pyrroline-3-carbonylamino)propyl]uridine Npyr-oxyl (4c) starting from the nucleoside X (4a) and the appropriate N-hydroxysuccinimide ester 1 or 2 is described. After acylation of tRNAPhe from E. coli (5a) with 1 or 2, the photolabile tRNAPhe derivative 5b and the paramagnetic tRNAPhe derivative 5c could be isolated. The position of modification in the polynucleotide chain was elucidated by comparison of the ribonuclease II/alkaline phosphatase digestion products of the substituted and unsubstituted tRNAPhe samples, and was identified as being exclusively the amino group of the nucleoside X in position 47 of E. coli tRNAPhe.     

Hoogsteen vs. Watson-Crick base pairing: incorporation of 2-substituted adenine- and 7-deazaadenine 2'-deoxy-beta-D-ribonucleosides into oligonucleotides

Various 2-substituted 2'-deoxyadenosines and 7-deazaadenosines have been synthesized. The phosphonate building block 9 of 2-chloro-7-deaza-2'-deoxyadenosine (7-deazacladribine; 2) was prepared by 4,4'-dimethoxytritylation of the parent nucleoside (-->7), followed by protection of the amino function with a formamidine residue (-->8). The latter was reacted with PCl3/N-methylmorpholine/1,2,4-triazole to give compound 9. Moreover, 2-methoxy-2'-deoxyadenosine (2'-deoxyspongosine; 1b) was converted into the fully protected derivative 12, which was then transformed into the 2-cyanoethyl phosphoramidite 14. Also the 2-(trifluoromethyl)-substituted 2'-deoxyadenosines 19-21 were prepared by glycosylation of the chromophore 16 with the halogenose 17, followed by one-pot deprotection and nucleophilic displacement of the 6-Cl substituent. The new DNA building blocks 9 and 14 were used--together with formerly prepared cladribine derivative 4--for solid-phase synthesis of a series of oligodeoxyribonucleotides. These were studied with respect to their thermal stability as well as of the base pairing mode (Watson-Crick vs. Hoogsteen) of modified bases.     

Quinolone Nucleosides: 6,7-Dihalo-N-β- and α-Glycosyl-l 4-dihydro-4-oxo-quinoline-3-carboxylic Acids and Derivatives. Synthesis,Antimicrobial and Antiviral Activity

Abstract

Reaction of the silylated 6,7-dihaloquinoline bases 10–12 with l-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose (13) gave ethyl 7-chloro-6-flouro-l,4-dihydro-4-oxo-1 -(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)quinoline-3-carboxylate (14) and the free acids 15 and 16, respectively, which led on deblocking of the sugar moiety to the free nucleosides 17, 18 and 20, respectively. Treatment of 14 with methanolic ammonia afforded the amide derivative 19. Ribosylation of 11 with l,2-di-O-acetyl-3-azido-3-deoxy-5-p-toluoyl-β-D-ribofuranose (21) afforded the azido nucleoside 22, which was again converted into the free nucleoside 23. Analogously, reaction of 11 with the chloro deoxyribose derivative 24 led to a mixture of α /β (2:1) anomers of 25. Deblocking and recrystallization of the product gave mainly the α-anomer 26. Compounds 17–19, 23 and 26 were evaluated against Escherichia coli and found inactive. Compound 16–18 and 22 were inactive aganist HIV-1 (III B) and HIV-2 (ROD) induced cytopathicity in human MT-4 lymphocyte cells.     

Equilibrative nucleoside transporters: mapping regions of interaction for the substrate analogue nitrobenzylthioinosine (NBMPR) using rat chimeric proteins.

The rat equilibrative nucleoside transporters rENT1 and rENT2 belong to a family of integral membrane proteins with 11 potential transmembrane segments (TMs) and are distinguished functionally by differences in sensitivity to inhibition by nitrobenzylthioinosine (NBMPR). Structurally, the proteins have a large glycosylated extracellular loop between TMs 1 and 2 and a large cytoplasmic loop between TMs 6 and 7. In the present study, we have generated chimeras between NBMPR-sensitive rENT1 and NBMPR-insensitive rENT2, using splice sites at rENT1 residues 99 (end of TM 2), 171 (between TMs 4 and 5), and 231 (end of TM 6) to identify structural domains of rENT1 responsible for transport inhibition by NBMPR. Transplanting the amino-terminal half of rENT2 into rENT1 rendered rENT1 NBMPR-insensitive. Domain swaps within the amino-terminal halves of rENT1 and rENT2 identified two contiguous regions, TMs 3-4 (rENT1 residues 100-171) and TMs 5-6 (rENT1 residues 172-231), as the major sites of NBMPR interaction. Since NBMPR is a nucleoside analogue and functions as a competitive inhibitor of zero-trans nucleoside influx, TMs 3-6 are likely to form parts of the substrate translocation channel.     

Synthesis and rearrangements of D-glucosyl esters of aspartic acid linked through the 1- or 4-carboxyl group.

Catalytic hydrogenation of 2,3,4,6-tetra-O-benzyl-1-O-[1-benzyl N-(benzyloxycarbonyl)-L-aspart-4-oyl]-alpha-D-glucopyranose (1alpha) in acetic acid-2-methoxyethanol gave 1-O-(L-beta-aspartyl)alpha-D-glucopyranose (2alpha) contaminated with 2-O-(L-alpha-aspartyl)-D-glucopyranose (8). Evidence that 8 was formed from the 1-oyl isomer of 1alpha, namely 2,3,4,6-tetra-O-benzyl-1-O-[4-benzyl N-(benzyloxycarbonyl)-L-aspart-1-oyl]-alpha-D-glucopyranose (7alpha), via 1 leads to 2 acyl migration, was obtained by submitting the deprotected D-glucosyl ester to successive N-acetylation, esterification, and O-acetylation; the final product was identified as a approximately 4:1 mixture of 2,3,4,6-tetra-O-acetyl-1-O-[1-methyl N-(acetyl)-L-aspart-4-oyl]-alpha-D-glucopyranose (4alpha) and 1,3,4,6-tetra-O-acetyl-2-O-[4-methyl N-(acetyl)-L-aspart-1-oyl]-D-glucopyranose (6) which were also prepared by definitive methods. On the other hand, deprotection of 1beta gave isomerically pure 2beta which was converted into the peracetylated ester derivative 4beta; an explanation for the differences in aglycon isomeric purity of 2alpha and 2beta is given. Hydrogenolysis of 7beta under the above conditions led to intermolecular transesterification with scission of the C-1 ester bond to give 1-(2-methoxyethyl) L-aspartic acid and D-glucose. Catalytic hydrogenation of 7alpha and 7beta, performed in the presence of trifluoroacetic acid, afforded 1-O-(L-alpha-aspartyl)-alpha- and -beta-D-glucopyranoside trifluoroacetate salts (11alpha and 11beta), respectively. The structure of 11beta was established by successive conversion into 2,3,4,6-tetra-O-acetyl-1-O-[4-methyl N-(acetyl)-L-aspart-1-oyl]-beta-D-glucopyranose (5beta) which was also prepared by definitive methods. Analogous treatment of 11alpha gave the N-acetyl derivative 12 which underwent 1 leads to 2 acyl migration during esterification with diazomethane to give the N-acetyl methyl ester derivative 10; acetylation of 10 afforded 6.     

Terpenoid and phenolic metabolites from the fungus xylaria sp. associated with termite nests

Three new sesquiterpene acids, xylaric acids A-C (1-3, resp.), and a new tetralone (=3,4-dihydronaphthalen-1(2H)-one) derivative, 4, along with nine known compounds, xylaric acid D (5), hydroheptelidic acid (6), gliocladic acid (7), chlorine heptelidic acid (8), trichoderonic acid A (9), 16-(α-D-mannopyranosyloxy)isopimar-7-en-19-oic acid (10), 16-(α-D-glucopyranosyloxy)isopimar-7-en-19-oic acid (11), 5-carboxymellein (12), and naphthalen-1,8-diol 1-O-α-D-glucopyranoside (13) have been isolated from the solid culture of the ascomycete fungus Xylaria sp. associated with termite nest. The structures of these compounds were elucidated primarily by NMR experiments. The absolute configurations of compounds 1-3 and 5-9 were determined by combination of X-ray data and CD spectral analysis. The absolute configuration of 4 was assigned by Snatzke's method. Compounds 8 and 11 showed slight cytotoxicities against two cell lines A549 and SGC7901.     

Iridoids from Kigelia pinnata DC. fruits

From the fruits of Kigelia pinnata DC., a new furanone derivative formulated as 3-(2'-hydroxyethyl)-5-(2"-hydroxypropyl)-dihydrofuran-2(3H)-one (1), and four new iridoids named; 7-hydroxy viteoid II (2), 7-hydroxy eucommic acid (3), 7-hydroxy-10-deoxyeucommiol (4) and 10-deoxyeucommiol (5) have been isolated together with seven known iridoids, jiofuran (6), jioglutolide (7), 1-dehydroxy-3,4-dihydroaucubigenin (8), des-p-hydroxybenzoyl kisasagenol B (9), ajugol (10), verminoside (11) and 6-trans-caffeoyl ajugol (12). The structures of the isolated compounds were characterized by different spectroscopic methods.     

5''-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5''-thioadenosine (SAENTA), a novel ligand with high affinity for polypeptides associated with nucleoside transport. Partial purification of the nitrobenzylthioinosine-binding protein of pig erythrocytes by affinity chromatography.

Derivatives of N6-(4-aminobenzyl)adenosine (substituted at the aminobenzyl group) and 5'-linked derivatives of N6-(4-nitrobenzyl)adenosine (NBAdo) were evaluated as inhibitors of site-specific binding of [3H]nitrobenzylthioinosine (NBMPR) to pig erythrocyte membranes. Potent inhibitors were SAENTA [5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine] and acetyl-SAENTA (the 2-acetamidoethyl derivative of SAENTA). SAENTA was coupled to derivatized agarose-gel beads (Affi-Gel 10) to form an affinity matrix for chromatographic purification of NBMPR-binding polypeptides, which in pig erythrocytes are part of, or are associated with, the equilibrative nucleoside transporter. When pig erythrocyte membranes were solubilized with octyl glucoside (n-octyl beta-D-glucopyranoside) and applied to SAENTA-Affi-Gel 10 (SAENTA-AG10), polypeptides that migrated as a broad band on SDS/PAGE with an apparent molecular mass of 58-60 kDa were selectively retained by the affinity gel. These polypeptides were identified as components of the nucleoside transporter of pig erythrocytes by reactivity with a monoclonal antibody (mAb 11C4) that recognizes the NBMPR-binding protein of pig erythrocytes. Retention of the immunoreactive polypeptides by SAENTA-AG10 was blocked by NBAdo. The immunoreactive polypeptides were released from SAENTA-AG10 by elution under denaturing conditions with 1% SDS or by elution with detergent solutions containing competitive ligands (NBAdo or NBMPR). A 72-fold enrichment of the immunoreactive polypeptides was achieved by a single passage of solubilized, protein-depleted membranes through a column of SAENTA-AG10, followed by elution with detergent solutions containing NBAdo. These results demonstrate that polypeptide components of NBMPR-sensitive nucleoside-transport systems may be partly purified by affinity chromatography using gel media bearing SAENTA groups.     

Synthesis and characterization of nucleoside derivatives,n-(benzoyl)-n-(deoxyguanosin-8-yl)4-aminobiphenyl and n-(2'-deoxyguanosin-8-yl)4-aminobiphenyl via alpha-phenyl-n-(4-biphenyl)nitrone

Lead tetraacetate (LTA) oxidation of alpha-Phenyl-N-(4-bipheny])nitrone (8) to give a new ultimate carcinogen, N-acetoxy-N-benzoyl-4-aminobiphenyl (9) which was reacted with deoxyguanosine (dG) at pH 6.9 to give nucleoside derivative, N-(benzoyl)-N-(deoxyguanosin-8-yl)-4-aminobiphenyl (10). Following debenzoylation with sodium carbonate-methanol leads to N-(2'-deoxyguanosin-8-yl)-4-aminobiphenyl (11).     

Increase of the adenallene anti-HIV activity in cell culture using its bis(tBuSATE) phosphotriester derivative.

The bis(S-pivaloyl-2-thioethyl) phosphotriester derivative of 9-(4'-hydroxy-1',2'-butadienyl)adenine (adenallene) was synthesized. This mononucleotide prodrug proved to be more effective than the parent nucleoside in inhibiting HIV-1 replication in several human T4 lymphoblastoid cell lines.     

Introduction of antigenic determining 2,4-dinitrophenyl residues into 4-thiouridine, N3-(3-L-amino-3-carboxypropyl) uridine and tRNA-Phe from E. coli.

The introduction of antigenic determining 2,4-dinitrophenyl residues into the rare ribonucleosides 4-thiouridine (1a), and N3-(3-L-amino-3-carboxypropyl) uridine (2) as well as into tRNA-Phe from E. coli has been investigated. Alkylation of 1a with omega-bromo-2,4-dinitroacetophenone (3b) gives S-(2,4-dinitrophenacyl)-4-thiouridine (5A). Applying the reaction to the 5'-monophosphate of 1a, 5b is formed, but this product decomposes at pH 7. However, acylation of 2 with 2,4-dinitrobenzoic acid N-hydroxysuccinimide ester (4b) leads to N3-[3-carboxy-3-L-(2,4-dinitrobenzamido)propyl]uridine (6) which is stable in aqueous solution. The latter reaction was used for the introduction of an antigenic determining 2,4-dinitrophenyl residue into tRNA-Phe from E. coli. The modified tRNA-Phe was isolated and by degradation of the molecule with RNase T2 and alkaline phosphatase the nucleoside derivative 6 was obtained and found to be identical with the synthetic product.     

Synthesis and anti-CVB3 activity of 4-amino acid derivative substituted pyrimidine nucleoside analogues

Seven novel 4-amino acid derivative substituted pyrimidine nucleoside analogues were designed, synthesized, and tested for their anti-CVB3 activity. Initial biological studies indicated that among these 4-amino acid derivative substituted pyrimidine nucleoside analogues, 4-N-(2′-amino-glutaric acid-1′-methylester)-1-(2′- deoxy-2′-β-fluoro-4′-azido)-furanosyl-cytosine 2 exhibited the most potent anti-CVB activity (IC₅₀ = 9.3 μM). The cytotoxicity of these compounds has also been assessed. The toxicity of compound 2 was similar to that of ribavirin.     

Antagonistic properties are shifted back to agonistic properties by further N-terminal shortening of pituitary adenylate-cyclase-activating peptides in human neuroblastoma NB-OK-1 cell membranes.

N-terminally shortened analogs of the 27-amino-acid and 38-amino-acid forms of the pituitary-adenylate-cyclase-activating neuropeptide, PACAP(1-27) and PACAP(1-38), were synthesized by a solid-phase method. Systematic deletion of the first 13 amino acids of both PACAP was tested by evaluating their ability to occupy the specific and selective PACAP receptor of human neuroblastoma NB-OK-1 cell membranes and to stimulate adenylate cyclase or, when inactive per se, to inhibit PACAP-stimulated adenylate cyclase activity. For each peptide, the Kact (concentration required for half-maximal adenylate cyclase activation) or Ki [concentration required to shift the dose/response curve of PACAP(1-27) twofold to the right] was in good agreement with the corresponding IC50 [concentration inhibiting 50% of 125I-[AcHis1]PACAP(1-27) binding to membranes], suggesting interaction with the same homogeneous class of adenylate cyclase-coupled receptors. The deletion of the two first amino acids (His1 and Ser2) sufficed to decrease the affinity for receptors and to suppress the capacity to activate adenylate cyclase. The shorter fragments 3-27 and 3-38, 4-27 and 4-38, 5-27 and 5-38, 6-27 and 6-38, 7-27 and 7-38, 8-27 and 8-38, and 9-27 and 9-38 were all competitive antagonists of PACAP(1-27)-stimulated activity with the N-terminally shortened PACAP(1-38) derivatives being 4-30-fold more potent than the equivalent PACAP(1-27) derivatives. In this group PACAP(6-38) was the most potent antagonist (Ki 1.5 nM). Surprisingly, the N-terminally shorter fragments 10-27 and 10-38, 11-27 and 11-38, 12-27 and 12-38, 13-27 and 13-38, and 14-27 and 14-38 were again able to stimulate adenylate cyclase, the smallest fragments, PACAP(14-27) and PACAP(14-38), being the most potent and efficient (Kact 2 microM and 0.1 microM, respectively). In this group of agonists, PACAP(1-38) derivatives deleted at the N-terminus were also more potent than the equivalent PACAP(1-27) derivatives.