Incorporation of shikimate and 4-(2′-carboxyphenyl)-4-oxobutyrate into phylloquinone

The patterns of incorporation of d-[G-¹⁴C]shikimate and variously labelled ¹⁴C-4-(2′-carboxy-phenyl)-4-oxobutyrate into the naphthoquinone nucleus of phylloquinone by maize shoots have been investigated. The results show that (a) the alicyclic ring and C-7 of shikimate give rise to Ring A and either C-1 or C-4, and (b) the phenyl ring, 2′-carboxy and C-4, and C-2 and -3 of 4-(2′-carboxyphenyl)-4-oxobutyrate give rise to Ring A, C-1 and -4 and C-2 and -3. Radioactivity from α-[1-¹⁴C]naphthol, 1,4-[1,4-¹⁴C]naphthoquinone and [Me-¹⁴C]menadione is not incorporated into phylloquinone to any significant extent.     

Synthesis of (±)-4′-Ethynyl and 4′-Cyano Carbocyclic Analogues of Stavudine (d4T)

The synthesis of (±)-4′-ethynyl (8) and 4′-cyano (9) carbocyclic analogues of the anti-HIV agent stavudine (5, d4T) is reported. The carbocyclic unit (16) was constructed from readily available β-keto ester 10. The ethynyl or cyano group of 8 and 9 were prepared, after the introduction of thymine base to 16, by manipulation of the ester function. Evaluation of the anti-HIV activity of 8 and 9 was also carried out.     

4-(2′-Carboxyphenyl)-4-oxobutyrate: An obligatory intermediate in lawsone biosynthesis

4-(2′-Carboxyphenyl)-4-oxobutyric acid (6) has been detected in cuttings of Impatiens balsamina. It is labelled under conditions where activity from U-¹⁴C-glutamate is incorporated effectively into lawsone (1). 3-(2′-Carboxyphenyl)-3-oxopropionic acid (7) has also been encountered in the cuttings.     

2′-(E)-O-p-coumaroylgalactaric acid and 2′-(E)-O-feruloylgalactaric acid in citrus

2′-(E)-O-p-Coumaroyl- and 2′-(E)-O-feruloylgalactaric acids, hitherto unknown in nature, have been isolated and identified from orange peel.     

Resistance Profiles of (+) 2′-Deoxy-3′-oxa-4′-thiocytidine and (-) 2′-Deoxy-3′-oxa-4′-thio-5-fluorocytidine

Abstract

Resistant variants were selected in vitro against two novel nucleoside analogues, (+) dOTC and (-) dOTFC using the HIV-1 molecular clone HXB2D. The variants obtained displayed 6.5-fold and 10-fold resistance to these compounds, respectively. Cloning and sequencing of the RT genes of the selected viruses identified two mutations, M184I for (+) dOTC and M184V for (-) dOTFC. Results with mutated recombinant clones of HXB2D confirmed the importance of these mutations in MT-4 cells. The resistance profiles of clinical samples with wild-type or 3TC-resistant phenotypes were also studied; low to moderate levels of cross-resistance were observed against the novel compounds.     

Mechanism of Action of the Fungicide Thiabendazole, 2-(4′-Thiazolyl) Benzimidazole

Thiabendazole, 2-(4'-thiazolyl) benzimidazole (TBZ) inhibited the growth of Penicillium atrovenetum at 8 to 10 mug/ml. Oxygen consumption with exogenous glucose was inhibited at 20 mug/ml, but endogenous respiration required more than 100 mug/ml. TBZ inhibited completely the following systems of isolated heart or fungus mitochondria: reduced nicotinamide adenine dinucleotide oxidase, succinic oxidase, reduced nicotinamide adenine dinucleotide-cytochrome c reductase, and succinic-cytochrome c reductase at concentrations of 10, 167, 10, and 0.5 mug/ml, respectively. Cytochrome c oxidase was not inhibited. Antimycin A and sodium azide caused the usual inhibition patterns for both fungus and heart terminal electron transport systems. In the presence of antimycin, the fungicide inhibited completely succinate-dichloro-phenolindophenol reductase and succinate-2, 2-di-p-nitrophenyl-(3, 3-dimethoxy-4, 4-biphenylene-5, 5-diphenylditetrazolium)-reductase at 2 and 4 mug of TBZ per ml, respectively. Coenzyme Q reductase required 15 mug/ml. TBZ reduced the uptake by P. atrovenetum of glucose and amino acids and decreased the synthesis of various cell components. At 120 mug/ml, the incorporation of labeled carbon from amino acids-U-(14)C was decreased: lipid, 73%; nucleic acids, 80%; protein, 80%; and a residual fraction, 89%. TBZ did not inhibit peptide synthesis in a cell-free protein-synthesizing system from Rhizoctonia solani. Probably the primary site of inhibition is the terminal electron transport system and other effects are secondary.     

Synthesis of (±)-Methyl 5-(1′,2′-Epoxy-2′,6′-dimethylcyclohexyl)-3-methyl-(2Z,4E)-2,4-pentadienoate

The effect of tripropyltin chloride (TPT) on transport systems in E. coli was investigated. The inhibition on uptakes of ¹⁴C-l-leucine, l-proline, adenine and methyl-(α-d-gluco)pyrano-side (α-methylglucoside) by TPT was examined. The active uptake of l-leucine which utilized ATP molecule as an energy source was 100% inhibited at the concentration of 10 µg/ml TPT. On the other hand, the uptake of l-proline which was generated by an “energied” membrane state of the cells was inhibited only 40% at the same concentration of TPT. α-Methylglucoside uptake was scarcely inhibited. Adenine uptake was intensely inhibited at 20 µg/ml TPT. The effect of the delayed addition of TPT on transport systems was also examined. l-Leucine incorporated into cells was completely released from cells by TPT. Leucine binding protein (LBP) was prepared from E. coli cells and the effect of TPT on LBP activity was examined. TPT scarcely inhibited LBP activity.     

Synthesis of 2-(3-Deoxy-β-D-erythropentofuranosyl)-thiazole-4-carboxamide (3′-Deoxytiazofurin)

Abstract

2-(3-Deoxy-β-D-erythropentofuranosyl)-thiazole-4-carboxamide was synthesized in four steps from its β-D-ribofuranosyl nucleoside precursor.     

Selective synthesis of cerebrosides: (2S, 3R, 4E)-1-O-β-d-galactopyranosyl-N-(2′R and 2′S)-2′-hydroxytetracosanoyl-sphingenine

The cerebrosides were first isolated by Thudicum in 1874 and the structures were established by Carteret al. in 1950 (for review, see [2]). In 1961 Shapiro and Flowers [3] reported the first total synthesis of a cerebroside1 (Fig. 1) which was identified with the natural sample, only through comparison of their i.r. data. In order to confirm the absolute configuration at C-2 of natural cerebroside1, we describe here an unambiguous synthesis of two stereoisomeric cerebrosides1 and2, and found that the¹H-NMR spectra of the synthetic1 (Fig. 2) was completely identical with that of the natural cerebroside reported recently by Dabrowskiet al. [4].In planning the synthetic route, the target structures1 and2 were disconnected at the dotted lines to give three key synthetic intermediates3, 4 and5 or6 (Fig. 1).Abbreviations Bu butyl - Ph phenyl - t-BuPh₂SiCl t-butyldiphenylsilyl chloride - MTPA -methoxy--trifluoromethylphenylacetic acid - THF tetrahydrofuran Part 36 in the series Synthetic Studies on Cell-surface Glycans, for part 35, see [1]     

Total synthesis of cerebrosides: (2S, 3R, 4E)-1-O-β-d-galactopyranosyl-N-(2′R and 2′S)-2′-hydroxytetracosanoylsphingenine

Total syntheses of both (2S, 3R, 4E)-1-O-β-d-galactopyranosyl-N-(2′R)-2′-hydroxytetracosanoylsphingenine 23 and the (2′S) stereoisomer were performed in an unambiguous way by employing either (2S, 3R, 4E)-N-(2′R)-2′-(tert-butyl-diphenylsilyloxy)tetracosanoylsphingenine or its (2′S) stereoisomer as the key glycosyl acceptors. The synthetic cerebroside 23 was shown to be identical with the natural product through comparison of their 400-MHz, ¹H-n.m.r. spectra, thus providing synthetic evidence for the 2′R configuration of the natural cerebroside.     

Highly Diastereoselective Synthesis of (2′S)-[2′-2H]-2′-Deoxyribonucleosides from the Corresponding Ribonucleosides

Abstract

The four (2′S)-[2′-²H]-2′-deoxynucleosides (>90 atom % ²H), were synthesized from the corresponding ribonucleosides involving six steps of reactions, i.e., oxidation of their 2′-hydroxyl group, stereoselective reductive deuteration of the resulting 2′-ketonucleoside intermediates with NaB²H₄ in EtOH-H₂O or EtOH, triflation, bromination with LiBr, highly stereoselective Bu₃SnH-Et₃B reduction of the resulting bromide, and, finally, unmasking.     

The metabolism of 2-chloro-1-(2′,4′-dichlorophenyl)vinyl diethyl phosphate (Chlorfenvinphos) in the dog and rat

1. A single oral dose of [(14)C]Chlorfenvinphos to rats is quantitatively eliminated in 4 days. Rats do not show a sex difference in the elimination pattern and show only a small degree of biological variation in the total excretion data. Of the label 87.2% is excreted in the urine (67.5% in the first day after dosage), 11.2% in the faeces and 1.4% in the expired gases; less than 0.9% of (14)C is present in the gut and contents after 4 days. 2. After oral administration of [(14)C]Chlorfenvinphos to dogs, 94.0% (91.8-97.6%) of the (14)C is excreted in the urine and faeces during 4 days. Dogs do not show a sex difference in the pattern of elimination, and excretion of radioactivity in the urine is very rapid: 86.0% of (14)C during 0-24hr. 3. Chlorfenvinphos is completely metabolized in rats and dogs: unchanged Chlorfenvinphos is absent from the urine and from the carcass, when elimination is complete. In rats, 2-chloro-1-(2',4'-dichlorophenyl)vinyl ethyl hydrogen phosphate accounts for 32.3% of a dose of Chlorfenvinphos, [1-(2',4'-dichlorophenyl)ethyl beta-d-glucopyranosid]uronic acid for 41.0%, 2,4-dichloromandelic acid for 7.0%, 2,4-dichlorophenylethanediol glucuronide for 2.6% and 2,4-dichlorohippuric acid for 4.3%; in dogs, 2-chloro-1-(2',4'-dichlorophenyl)vinyl ethyl hydrogen phosphate accounts for 69.6%, [1-(2',4'-dichlorophenyl)ethyl beta-d-glucopyranosid] uronic acid for 3.6%, 2,4-dichloromandelic acid for 13.4% and 2,4-dichlorophenylethanediol glucuronide for 2.7%. 4. Dogs and rats show a species difference in the rate of excretion of (14)C in the urine, and in the proportions of the metabolites, with the exception of 2,4-dichlorophenylethanediol glucuronide, that are excreted in the urine. Alternative explanations for the latter species difference are suggested. 5. 2-Chloro-1-(2',4'-dichlorophenyl)vinyl ethyl hydrogen phosphate and 2,4-dichlorophenacyl chloride probably lie on the main metabolic pathway of Chlorfenvinphos, since, in common with that insecticide, they give rise to [1-(2',4'-dichlorophenyl)ethyl beta-d-glucopyranosid]uronic acid and 2,4-dichloromandelic acid as major metabolites in the urine. 6. The proposed scheme for the metabolism of Chlorfenvinphos represents a detoxication mechanism.     

Discovery of N-(4′-(indol-2-yl)phenyl)sulfonamides as novel inhibitors of HCV replication

A series of novel 2-phenylindole analogs were synthesized and evaluated for activity in subgenomic HCV replicon inhibition assays. Several compounds containing small alkyl sulfonamides on the phenyl ring exhibiting submicromolar EC₅₀ values against the genotype 1b replicon were identified. Among these, compound 25d potently inhibited the 1b replicon (EC₅₀ = 0.17 μM) with 147-fold selectivity with respect to cytotoxicity. Compound 25d was stable in the presence of human liver microsomes and had a good pharmacokinetic profile in rats with an IV half-life of 4.3 h and oral bioavailability (F) of 58%.     

Asymmetric incorporation of 4-(2′-carboxyphenyl)-4-oxobutyrate into phylloquinone by Zea mays

Radioactivity from 4-(2′-carboxyphenyl)-4-oxobutyrate-[2-¹⁴C] and 4-(2′-carboxyphenyl)- 4-oxobutyrate-[3-¹⁴C] was incorporated into C-3 and C-2 respectively of phylloquinone in maize shoots. These results show that this substrate is incorporated in the same asymmetric manner into phylloquinone as it is into the bacterial menaquinones.     

Microbiological synthesis of 5-ethyl- and (E)-5-(2-bromovinyl)-2′-deoxyuridine

Summary The title compounds were prepared by an enzymatic transdeoxyribosylation from 2 dGuo or 2 dThd to the respective heterocyclic bases, 5-ethyluracil and (E)-5-(2-bromovinyl)uracil, using the whole bacterial cells ofEscherichia coli as a biocatalyst.     

Synthesis of (E)-5-(2-cIOdovinyl)-3′-0-(1-Methyl-1,4-Dihydropyridyl-3 -Carbonyl)-2′-Fluoro-2′-Deoxyuridine (Ivfru-Cds) for Brain Targetted Delivery of Ivfru,an Antiviral Nucleoside

Abstract

(E)-5-(2-lodovinyl)-2′-fluoro-3′-0-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-2′-deoxyuridine (11) was synthesized for future evaluation as a lipophilic, brain-selective, pyrimidine phosphorylase-resistant, antiviral agent for the treatment of Herpes simplex encephalitis (HSE). Treatment of (E)-5-(2-iodovinyl)-2′-fluoro-2′-deoxyuridine (6) with TBDMSCI in the presence of imidazole in DMF yielded the protected 5′-O-t-butyldimethylsilyl derivative (7). Subsequent reaction with nicotinoyl chloride hydrochloride in pyridine afforded (E)-5-(-2-iodovinyl)-2′-fluoro-3′-O-(3-pyridylcarbonyl)-5′-O-t-butyldimethylsily-2′-deoxyuridine (8). Deprotection of the silyl ether moiety of 8 with n-Bu₄N⁺F^? and quaternization of the resulting 3′-O-(3-pyridylcarbonyl) derivative 9 using iodomethane afforded the corresponding 1-methylpyridinium salt 10. The latter was reduced with sodium dithionite to yield (E)-5-(2-iodovinyl)-2′-fluoro-3′-O-(1-methyl-1,4-dihydropyridyl-3-carbonyl)-2′-deoxyuridine (11).     

Metabolism of O,O-Dipropyl S-[2-(2′-Methyl-1′-piperidinyl)-2-oxo-ethyl] Phosphorodithioate (C 19 490) in Paddy Rice

The fate of O,O-dipropyl S-[2-(2′-methyl-1′-piperidinyl)-2-oxo-ethyl] phosphorodithioate (C 19 490, piperophos) was followed in greenhouse-grown rice after application of 1.6 kg/ha ¹⁴C-labeled C 19 490 into the paddy water. At maturity the shoots contained 2.5 ppm and the hulled grains 0.16 ppm ¹⁴C-C 19 490 equivalents. Small amounts of unchanged C 19 490 were found in the shoots but none in the grains.

The main metabolites found were:- 2-(2′-methyl-1′-piperidiny])-2-oxo-ethane sulfonic acid, 2-(2?-methyl-1?-piperidinyl)-2-oxo-ethanoic acid and a fraction of polar unknown substances. O,O-Dipropyl S-[2-(2?-methyl-1′ -piperidinyl)-2-oxo-ethyl] phosphorothioate, 2-(2?-methyl-1?-piperidinyl)- 1-methyl-sulfinyl-2-oxo-ethane, 2-(2?-methyl-1?-piperidinyl)-1-methyl-sulfonyl-2-oxo- ethane, 2-methyl-piperidine and CO₂ were found in smaller amounts.     

Deuteration and Tritiation of 2′-Deoxyribonucleosides in the 5′ and 4′ Positions

Abstract

The deuterations of 2′-deoxyguanosine in the 4′ and 5′ positions have been described elsewhere (1). The starting material is the 5′-aldehyde formed by mild oxidation with N,N-dicyclohexyl carbodiimide in dimethyl sulphoxide of the fully protected nucleoside with free 5′-alcoholic function. The 5′4euteration was achieved by reduction with deuterated sodium borohydride. Incorporation of deuterium in the 4′-position was achieved v i a an enhanced keto-enol tautomerim by heating the aldehyde in 50/50 D20/pyridine, with subsequent reduction of the aldehyde with NaBH4. The 6-furanoid form was isolated from the I-lyxo by-product by reverse phase HPLC. Applied to pyrimidine 2′-deoxyribonucleosides, this method was shown to give deuterated 2′-deoxycytidine and thymidine in good yield.     

Synthesis of 2′-Deoxynucleoside 5′-Methylenebis-(phosphonate)s Using 2-(4-Nitrophenyl)ethyl Methylenebis(phosphonate) as the Phosphonylating Agent.

Abstract

2-(4-Nitrophenylethyl) methylenebis(phosphonate) (1) has been prepared by reaction of 2-(4-nitrophenyl)ethyl alcohol with methylenebis(phosphonyl) tetrachloride. Compound 1 was treated with diisopropylcarbodiimide (DIC) to give bicyclic intermediate 2, which in reaction with suitably protected 2′-deoxynucleosides 3 gave P¹,P²-disubstituted methylenebis(phosphonate)s 4. Removal of the nitrophenylethyl group by β-elimination with DBU afforded the corresponding 2′-deoxynucleoside 5′-methylenebis(phosphonate) analogues 5.

     

Role of oxidative stress in the manganese and 1-methyl-4-(2′-ethylphenyl)-1,2,3,6-tetrahydropyridine-induced apoptosis in PC12 cells

Oxidative stress is thought to play a key role in the apoptotic death of several cellular systems, including neurons. Oxidative stress is proposed also as a mechanism of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- and manganese (Mn)-induced neuronal death. We have recently shown that Mn and the MPTP analogue 1-methyl-4-(2′-ethylphenyl)-1,2,3,6-tetrahydropyridine (2′Et-MPTP), which is metabolized by MAO-A to 1-methyl-4-(2′-ethylphenyl)-pyridinium ion, induce apoptosis in PC12 cells. In the present study, we evaluated the effects of deprenyl and the antioxidant drugs N-acetylcysteine (NAC) and ascorbic acid (AA) on Mn- and 2′Et-MPTP-induced apoptosis in PC12 cells. Apoptosis was tested by terminal deoxynucleotidyl transferase-mediated 2′-deoxy-uridine-5′-triphosphate nick end labelling (TUNEL) technique, flow cytometry and fluorescence microscopy. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Mn-induced apoptosis and decrease in cell viability was inhibited by the antioxidants NAC and AA. Deprenyl failed to inhibit the above Mn effects. Neither NAC, AA nor deprenyl were able to inhibit both 2′Et-MPTP-induced apoptosis and decrease in cell viability. These results confirm that apoptosis may be an important mechanism of cell death in MPTP- and Mn-induced parkinsonism. However, an oxidative stress mechanism may be recognized, at least in vitro, only in the Mn-induced apoptosis.