A facile synthesis and anti-avian influenza virus (H5N1) screening of some novel pyrazolopyrimidine nucleoside derivatives

Treatment of 5-amino-1-(9-methyl-5,6-dihydronaphtho[1',2':4,5]thieno[2,3-d]pyrimidin-11-yl)-1H-pyrazole-4-carbonitrile (1) with formic acid afforded pyrazolo[3,4-d]pyrimidin-4-one derivative 2. The sodium salt of the latter compound (generated in situ) was treated with some alkyl halides to afford the corresponding N-substituted compounds 3-7. The siloxy derivative 8 (generated also in situ from 2) was ribosylated and glycosylated to yield compounds 9 and 11, respectively. Deprotection of compounds 9 and 11 in methanolic ammonia produced the free nucleosides 10 and 12, respectively. Moreover, the prepared compounds were tested for antiviral activity against H5N1 virus [A/chicken/Egypt/1/2006] and some of them revealed moderate results compared with the other tested compounds.     

From mutagenic to non-mutagenic nitroarenes: effect of bulky alkyl substituents on the mutagenic activity of 4-nitrobiphenyl in Salmonella typhimurium. Part I. Substituents ortho to the nitro group and in 2'-position

Eleven alkyl substituted derivatives of 4-nitrobiphenyl (4NBp) and two corresponding nitroso compounds were synthesised and tested for mutagenic potency in strains TA98 and TA100 of Salmonella typhimurium. The mutagenicity of compounds substituted ortho to the nitro group (3-methyl-, 3-ethyl-, 3-isopropyl-, 3-tertbutyl-, 3, 5-diethyl-, 3,5-diisopropyl-, and 3,5-ditertbutyl-4NBp) decreased with growing steric demand of the alkyl substituents in both tester strains. The most sterically hindered compounds were non-mutagenic even at highest concentrations. This reduction of mutagenicity is correlated with deviations from the coplanar orientation of the nitro group relative to the aromatic plane. Since a comparable decrease of mutagenicity for the nitroso compounds (4NOBp and 3-tertbutyl-4NOBp) was not observed, the first reduction step of non-planar nitro groups must be inhibited.Alkyl groups in the 2'-position of 4NBp (2'-methyl-, 2'-ethyl-, 2'-isopropyl-, and 2',4', 6'-trimethyl-4NBp) also reduced mutagenic activity with increasing size and removed it completely for the most sterically hindered species (2'-isopropyl-, 2',4',6'-trimethyl-4NBp). In this case, the co-planarity of the nitro group is not affected but the twisting of the two aromatic rings, which is associated with a less effective charge delocalisation of the nitrenium ion.The experimental mutagenicities of all nitro compounds were compared to predicted values, that are based on recently developed empirical equations. While there was reasonable correspondence for the parent compound (4NBp), its ortho methylated derivative (3-methyl-4NBp) and two highly hydrophobic dialkylated species (3,5-diisopropyl- and 3, 5-ditertbutyl-4NBp), predictions for all other alkyl substituted compounds were too high. Thus, steric parameters should be included to improve the general validity of predictions by means of quantitative structure-activity relationships (QSAR).     

3'-Fluoro-3'-deoxy-5'-noraristeromycin derivatives: synthesis and antiviral analysis

The promising antiviral properties of 3'-fluoro-3'-deoxyadenosine and 4',4'-difluoro-4'-deoxy-5'-noraristeromycin prompted the synthesis of the corresponding 3'-fluoro derivatives of 5'-noraristeromycin. These target compounds, which were prepared from the same readily accessible cyclopentenol, were found to be inactive when subjected to 31 viral assays. The results have some implications on the mechanism of antiviral action of 5'-noraristeromycin and provide guidance for future geminal-difluoro analog design.     

Synthesis and anti-inflammatory activity of N-phthalimidomethyl 2,3-dideoxy- and 2,3-unsaturated glycosides

3,4,6-Tri-O-acetyl-D-galactal, 3,4,6-tri-O-acetyl-D-glucal and 3,6,2',3',4'6'-hexa-O-acetyl-D-lactal were reacted with N-hydroxymethylphthalimide and boron trifluoride etherate to produce the corresponding phthalimidomethyl unsaturated glycosides via Ferrier rearrangement. When the galactal derivative was used, a non-Ferrier rearrangement product was also isolated as a minor product under classical Ferrier conditions. Phthalimidomethyl deoxy glycosides were readily prepared by hydrogenation of the unsaturated glycosides. Following deacetylation, the anti-inflammatory activities of these compounds were tested on mice and three were found to possess potent activity compared to hydrocortisone sodium succinate (HSS).     

Synthesis and biological evaluation of some novel triazol-3-ones as antimicrobial agents

A new series of triazol-3-one derivatives bearing 4-methyl-4H-thieno[3',2': 5,6]thiopyrano[4,3-d][1,3]thiazolyl or 4-(thiophene-3-yl) thiazolyl moiety at 4-position and alkyl substitution at 2-position are synthesized. All the synthesized compounds are characterized by elemental analysis, IR, (1)H NMR, (13)C NMR, and mass spectral data. The newly synthesized compounds are screened for antifungal and antibacterial activities.     

Metabolism of Lignin Model Compounds of the Arylglycerol-beta-Aryl Ether Type by Pseudomonas acidovorans D(3)

A natural bacterial isolate that we have classified as Pseudomonas acidovorans grows on the lignin model compounds 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (compound 1) and 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (compound 1'), as well as on the corresponding 1-oxo compounds (2 and 2') as sole sources of carbon and energy. Metabolic intermediates present in cultures growing on compound 1 included compound 2, 2-methoxyphenol (guaiacol [compound 3]), beta-hydroxypro-pioveratrone (compound 4), acetoveratrone (compound 5), and veratric acid (compound 6). Also identified were compounds 1', 2', beta-hydroxypropiovanillone (compound 4'), and acetovanillone (compound 5'), indicating that 4-O demethylation also occurs. The phenolic intermediates were the same as those found in cultures growing on compound 1'. Compounds 2 and 2' were in part also reduced to compounds 1 and 1', respectively. Compound 3 was shown to be derived from the 2-methoxyphenoxy moiety. A suggested degradation scheme is as follows: compound 1-->2-->(3 + 4)-->5-->6 (and similarly for 1'). In this scheme, the key reaction is cleavage of the ether linkage between C-2 (C(beta)) of the phenylpropane moiety and the 2-methoxyphenoxy moiety in compounds 2 and 2' (i.e., beta-aryl ether cleavage). On the basis of compounds identified, viz., 3 and 4 (4'), cleavage appears formally to be reductive. Because this is unlikely, the initial cleavage products probably were not detected. The implications of these results for the enzyme(s) responsible are discussed.     

Protease inhibitors: Synthesis of L-alanine hydroxamate sulfonylated derivatives as inhibitors of clostridium histolyticum collagenase

L-alanine hydroxamate derivatives were obtained by reaction of alkyl/arylsulfonyl halides with L-alanine, followed by treatment with benzyl chloride, and conversion of the COOH moiety to the CONHOH group with hydroxylamine in the presence of carbodiimides. Other derivatives were obtained by reaction of N-benzyl-alanine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate, followed by a similar conversion of the COOH to the CONHOH moiety. The obtained compounds were assayed as inhibitors of Clostridium histolyticum collagenase, ChC (EC 3.4.24.3), a zinc enzyme which degrades triple helical collagen. The hydroxamate derivatives were generally 100-500 times more active than the corresponding carboxylates. In the series of synthesized derivatives, substitution patterns leading to the most potent ChC inhibitors were those involving perfluoroalkylsulfonyl- and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl, 3- and 4-protected-aminophenylsulfonyl-, 3- and 4-carboxy-phenylsulfonyl-, 3-trifluoromethyl-phenylsulfonyl-, or 1- and 2-naphthylsulfonyl among others. Similarly to the matrix metalloproteinase (MMP) hydroxamate inhibitors, ChC inhibitors of the type reported here must incorporate hydrophobic moieties at the P(2') and P(3') sites, in order to achieve tight binding to the enzyme.     

Synthesis of glyceroyl beta-N-acetyllactosaminide and its derivatives through a condensation reaction by cellulase

A condensation reaction between N-acetyllactosamine and glycerol was directly catalyzed by using a commercially available cellulase preparation from Trichoderma reesei. 1-O-beta-N-Acetyllactosaminyl-(R, S)-glycerols (1) were readily synthesized in a 5% yield based on the N-acetyllactosamine added and conveniently isolated by two-step column chromatographies. The use of a partially purified enzyme increased 2.3-fold the yield of 1, compared to that of the crude enzyme containing beta-D-galactosidase activity. When various alkanols (n:2-4) were used in the condensation reaction, the corresponding alkyl beta-N-acetyllactosaminides were obtained in yields of 0.3-1.1% of the desired compounds.     

Chemoenzymatic synthesis and antiviral evaluation of conformationally constrained and 3'-methyl-branched carbanucleosides using both enantiomers of the same building block

Starting from both enantiomers of a readily available building block, a straightforward enantioselective approach to constrained 3'-methyl-2',3'-alpha-oxirane-fused and 3'-methyl-3',4'-alpha-oxirane-fused carbanucleosides bearing different purine base analogues is described. The title compounds were evaluated as potential antiviral agents against important viruses. None of the new compounds had significant antiviral activity at a concentration of 100 microg/mL, which was the highest concentration tested.     

Protease inhibitors. Part 8: synthesis of potent Clostridium histolyticum collagenase inhibitors incorporating sulfonylated L-alanine hydroxamate moieties

A series of hydroxamates was prepared by reaction of alkyl/arylsulfonyl halides with N-2-chlorobenzyl-L-alanine, followed by conversion of the COOH moiety to the CONHOH group, with hydroxylamine in the presence of carbodiimides. Other structurally related compounds were obtained by reaction of N-2-chlorobenzyl-L-alanine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate, followed by the similar conversion of the COOH into the CONHOH moiety. The new compounds were assayed as inhibitors of the Clostridium histolyticum collagenase, ChC (EC 3.4.24.3), a bacterial zinc metallo-peptidase which degrades triple helical collagen as well as a large number of synthetic peptides. The prepared hydroxamate derivatives proved to be 100-500 times more active collagenase inhibitors than the corresponding carboxylates. Substitution patterns leading to best ChC inhibitors (both for carboxylates as well as for the hydroxamates) were those involving perfluoroalkylsulfonyl- and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl; 3- and 4-protected-aminophenylsulfonyl-; 3- and 4-carboxyphenylsulfonyl-; 3-trifluoromethyl-phenylsulfonyl; as well as 1- and 2-naphthyl-, quinoline-8-yl- or substituted-arylsulfonylamidocarboxyl moieties among others. Similarly to the matrix metalloproteinase (MMP) hydroxamate inhibitors, ChC inhibitors of the type reported here must incorporate hydrophobic moieties at the P2' and P3' sites, in order to achieve tight binding to the enzyme. This study also proves that the 2-chlorobenzyl moiety, investigated here for the first time, is an efficient P2' anchoring moiety for obtaining potent ChC inhibitors.     

Facile reductive cleavage of purine nucleosides to acyclonucleosides using diisobutylaluminium hydride (DIBAL): a new synthetic method for the preparation of 9-ribitylpurine derivatives.

Reaction of purine nucleosides, such as 2',3'-isopropylideneinosine (1a) and 2',3'-isopropylideneadenosine (1c), with diisobutylaluminum hydride (DIBAL) in dry tetrahydrofurane resulted in the formation of the corresponding 9-(2',3'-isopropylideneribity)purines (2) in good yields. Oxidation of the ribityl derivatives (2) with NalO4 and subsequent reduction with NaBH4 gave the corresponding 9-(2',3',4'-trihydroxybutyl)-purine derivatives (4) in high yields. Deprotection of compounds 2 and 4 in 80% acetic acid gave the corresponding 9-(2',3',4'-trihydroxybutyl)purines (5) and 9-ribitylpurines (6), respectively.     

Synthesis and cytotoxicity of epoxide and pyrazole analogs of the combretastatins

Twenty-six epoxide and corresponding pyrazole derivatives, of the structurally related chalcones and combretastatin A-4 (CA-4), were synthesized and tested for in vitro cytotoxicity. These molecules were synthesized by epoxidation of the relevant chalcones, followed by reaction with hydrazine. The structures of epoxides 3 and 7, and pyrazole 17, were confirmed by X-ray diffraction studies. The relatively coplanar conformation of a 3',3',4',4',5',5'-hexamethoxypyrazole 17 was in good agreement with the shape for 3',3',4',4',5'-pentamethoxypyrazole 16, which was determined from molecular mechanics optimization. In vitro cytotoxicity of each class of compounds was obtained using a 72 h continuous exposure MTT assay against two murine cancer cell lines; B16 melanoma and L1210 leukemia. The effect of substitution in the A-ring is addressed: three methoxy groups versus two, generally increased cytotoxicity across both cell lines. In the majority of cases, the pyrazoles are generally more active than the epoxides, with the most active, 5-(3'-amino-4'-methoxyphenyl)-3-(3',4',5'-trimethoxyphenyl)pyrazole 21, possessing an IC(50) value of 5 and 2.4 microM (B16 and L1210, respectively). Due to their planar conformations, the pyrazoles are typically less active than the corresponding chalcones, which adopt angular conformations similar to CA-4. B-ring modifications confirmed that in general the amino compounds are more active than the corresponding nitro compounds. Varying the number and orientation of methoxy groups on the A-ring did not produce any significant differences in toxicity in the cell lines studied.     

Synthesis and reactivities of 2,2'-anhydro-1-(3'-deoxy-3'-iodo-5'-O-trityl- beta-D-arabinofuranosyl)thymine.

2,2'-Anhydro-1-(3'-deoxy-3'-iodo-5'-O-trityl-beta-D-arabinofuranosyl) thymine (2) was synthesized from 2',3'-didehydro-3'-deoxythymidine (DHT). Compound 2 was readily converted into the 2',3'-anhydrolyxofuranosyl derivatives 4-6. Treatment of 4a with some nucleophiles (N3-, OMe-, Cl-) gave the corresponding 3'-substituted arabinosyl nucleosides (7a,c,e) together with the minor xylosyl isomers (8a,c,d). 7a,c,e were deprotected to 7b,d,f, respectively.     

Structure-activity studies of 3'-4'-dichloro-meperidine analogues at dopamine and serotonin transporters

The structure-activity relationships of 3',4'-dichloro-meperidine were investigated at dopamine (DAT) and serotonin transporters (SERT). Large ester substituents and lipophilic groups at the 4-position favored molecular recognition at the SERT. The benzyl ester of 3',4'-dichloro-meperidine exhibited high potency and high selectivity for the SERT (DAT/SERT=760). Chemical modification of the ester group and N-substitution generally led to compounds with decreased DAT affinity. Only small esters and alkyl groups were tolerated at the 4-position of the meperidine ring system by the DAT. Overall, the meperidine analogues were generally more selective for the SERT than for the DAT.     

Anti-AIDS agents. Part 62: anti-HIV activity of 2'-substituted 4-methyl-3',4'-di-O-(-)-camphanoyl-(+)-cis-khellactone (4-methyl DCK) analogs

Four 4-methyl-3',4'-di-O-(-)-camphanoyl-(+)-cis-khellactone (4-methyl DCK) analogs (7a-d) with different alkyl substituents at the 2'-position were synthesized and evaluated for inhibition of HIV-1 replication in H9 lymphocytes. 2'-Methyl-2'-ethyl-4-methyl DCK (7b) was more potent (EC(50)=0.22 microM, TI>175) than the other three compounds (7a, 7c, and 7d), but significantly less potent than 4-methyl DCK (2, EC(50)=0.0059 microM, TI>6600). The bioassay results indicated that the 2'-substituents had a strong effect on the anti-HIV activity, and gem-dimethyl substitution at the 2'-position was greatly preferable to larger alkyl substituents or hydrogen atoms.     

Substrate specificity and enantioselectivity of 4-hydroxyacetophenone monooxygenase

The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of carbonylic compounds that are structurally more or less similar to 4-hydroxyacetophenone are also substrates for this flavin-containing monooxygenase. On the other hand, several carbonyl compounds that are substrates for other Baeyer-Villiger monooxygenases (BVMOs) are not converted by HAPMO. In addition to performing Baeyer-Villiger reactions with aromatic ketones and aldehydes, the enzyme was also able to catalyze sulfoxidation reactions by using aromatic sulfides. Furthermore, several heterocyclic and aliphatic carbonyl compounds were also readily converted by this BVMO. To probe the enantioselectivity of HAPMO, the conversion of bicyclohept-2-en-6-one and two aryl alkyl sulfides was studied. The monooxygenase preferably converted (1R,5S)-bicyclohept-2-en-6-one, with an enantiomeric ratio (E) of 20, thus enabling kinetic resolution to obtain the (1S,5R) enantiomer. Complete conversion of both enantiomers resulted in the accumulation of two regioisomeric lactones with moderate enantiomeric excess (ee) for the two lactones obtained [77% ee for (1S,5R)-2 and 34% ee for (1R,5S)-3]. Using methyl 4-tolyl sulfide and methylphenyl sulfide, we found that HAPMO is efficient and highly selective in the asymmetric formation of the corresponding (S)-sulfoxides (ee > 99%). The biocatalytic properties of HAPMO described here show the potential of this enzyme for biotechnological applications.     

Novel bicyclic sugar modified nucleosides: synthesis, conformational analysis and antiviral evaluation

Methodology previously described by us was applied to the formation of novel conformationally restrained bicyclic sugar modified nucleosides, with introduction of an oxazole and a thiocarbamate ring at the 2('),3(')-positions of the ribonucleosides. Two novel alkyl derivatives of 2('),3(')-dideoxy-2('),3(')-oxazole-beta-d-uridine and a novel uridine 2('),3(')-thiocarbamate were successfully synthesised. Conformational evaluation of all the synthesised compounds was conducted using the theoretical potential energy calculation via the macromodel v.6.0 molecular modelling programme. The conformationally restrained nucleosides described were evaluated against a wide range of DNA and RNA viruses. None of the compounds showed specific antiviral effects at subtoxic concentrations.     

Convenient synthesis and evaluation of glycosidase inhibitory activity of alpha- and beta-galactose-type valienamines, and some N-alkyl derivatives

Valienamine analogues having alpha- and beta-galactose-type structures were synthesized by racemic modification from (1SR,2RS,3SR)-6-methylenecyclohex-4-ene-1,2,3-triol. Four N-alkyl derivatives of the beta-anomer were readily prepared selectively by treatment of key intermediate 2,6-di-O-acetyl-3,4-O-isopropylidene-5a-carba-alpha- and beta-l-arabino-hex-5(5a)-enopyranosyl bromides with alkyl amines. All compounds were assayed for inhibitory activity against six glycosidases, and the N-dodecyl derivative was shown to be a very strong inhibitor of beta-galactosidase (IC(50) 0.01 microM, bovine liver).     

Chemical synthesis of 5''-pyrophosphate and triphosphate derivatives of 3''-5'' ApA, ApG, GpA and GpG. CD study of the effect of 5''-phosphate groups on the conformation of 3''-5'' GpG.

A simple, two-step method is described for the synthesis of the 5'-pyro- and triphosphate derivatives of 3'-5' ApA, ApG, GpA and GpG. The readily accessible 2'(3')-5' ApA, ApG, GpA and GpG were converted in one step to the corresponding 5'-phosphoramidate derivatives which were then transformed to the 5'-pyro- and triphosphates. CD spectra of 3'-5' pn GpG (n = 0,1,2 or 3) derivatives, measured at pH 1, indicated stabilization of the (syn) G+p (anti)G conformation by the 5'-phosphate groups.     

Acyclic analogs of ribavirin. Synthesis and antiviral activity

Activity of several ribavirin analogues, viz.1-(2-hydroxyethoxymethyl)-, 1-(3-hydroxypropoxymethyl)-, 1-(4-hydroxybutoxymethyl)- and 1-(2,3-dihydroxypropyl)-1,2,4-triazole 5- and 3-carboxamides, against human adenovirus type 2 in the Hep-2 cell culture has been studied. The ether oxygen atom imitating the ribose O4' was shown to be essential for the antiviral activity. 1-(2-Hydroxyethoxymethyl)-1,2,4-triazole 3-carboxamide, a structural analogue of ribavirin in which the hydroxyl group is apparently equivalent to the ribose 5'-OH, possesses the highest activity among the compounds studied. Lengthening of the alkyl side chain reduces essentially the antiviral activity.