Synthesis of 4′-Thio-β-D-arabinofuranosylcytosine (4′-Thio-ara-C) and Comparison of Its Anticancer Activity with That of Ara-C

Abstract

4′-thio-β-D-arabinofuranosylcytosine was synthesized by a facile route in high yields. It was evaluated for antitumor activity against a panel of human tumors, both in vitro and in vivo.     

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.     

The Synthesis of Some 5-Alkyl (Cycloalkyl)-Substituted 2′ -Deoxy-4′-Thiouridines

Abstract

The silylated pyrimidine bases IIa-d were condensed with the benzyl 3,5-di-O-benzyl-2-deoxy-1,4-dithio-d-erythro-pentofuranoside III in acetonitrile under activation by N-iodosuccinimide, giving ca 1.5: 1/α: β anomeric mixtures of the blocked nucleosides IVa-d and Va-d. in yields of 55–58%. After the separation on a silica column the pure anomers were deprotected by BCI₃ or TiCI₄, providing the free nucleosides VIa-d and VIIa,c,d in moderate to good overall yields. The β- or α-anomeric configuration, anti-glycosidic conformation and prevailing C2′endo(S) thiosugar pucker in the synthesized compounds were established by the combined use of the ¹H, ¹³C NMR and X-ray crystallography.

     

Anti-AIDS agents 84. Synthesis and anti-human immunodeficiency virus (HIV) activity of 2′-monomethyl-4-methyl- and 1′-thia-4-methyl-(3′R,4′R)-3′,4′-di-O-(S)-camphanoyl-(+)-cis-khellactone (DCK) analogs

In a continuing investigation into the pharmacophores and structure–activity relationship (SAR) of (3′R,4′R)-3′,4′-di-O-(S)-camphanoyl-(+)-cis-khellactone (DCK) as a potent anti-HIV agent, 2′-monomethyl substituted 1′-oxa, 1′-thia, 1′-sulfoxide, and 1′-sulfone analogs were synthesized and evaluated for inhibition of HIV-1 replication in H9 lymphocytes. Among them, 2′S-monomethyl-4-methyl DCK (5a)³ and 2′S-monomethyl-1′-thia-4-methyl DCK (7a) exhibited potent anti-HIV activity with EC₅₀ values of 40.2 and 39.1 nM and remarkable therapeutic indexes of 705 and 1000, respectively, which were better than those of the lead compound DCK in the same assay. In contrast, the corresponding isomeric 2′R-monomethyl-4-methyl DCK (6) and 2′R-monomethyl-1′-thia-4-methyl DCK (8) showed much weaker inhibitory activity against HIV-1 replication. Therefore, the bioassay results suggest that the spatial orientation of the 2′-methyl group in DCK analogs can have important effects on anti-HIV activity of this compound class.     

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.     

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.     

Mechanism of killing of HeLa cells by the antitumor sulfonylurea,N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY 181984)

Summary The antitumor sulfonylureas appear to inhibit both mitochondrial activity in susceptible human colon lines and to inhibit the oxidation of NADH by isolated plasma membrane vesicles from HeLa cells. The results reported here describe the morphological appearance of HeLa cells treated with the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N-(4-chlorophenyl)urea (LY181984). The cells remain viable for several days although the rate of increase in cell number is slowed especially at high concentrations of the drug. Cells become smaller with normal nuclei or maintain a normal size but contain multiple or enlarged nuclei. The morphological observations suggest that the drug may somehow interfere with the ability of the cells to enlarge following cytokinesis. Between 72 and 96 h, the cells begin to die. Cell death is accompanied by a condensed and fragmented appearance of the nuclear DNA as revealed by fluorescence microscopy with 4,6-diamidino-2-phenylindole suggestive of apoptosis. Early transients in loss of pH control (4 min after sulfonylurea addition) and an increase in cytoplasmic calcium (4 h after sulfonylurea addition) were observed but were small and perhaps secondary to the mechanism responsible for the failure of the cells to grow and ensuing cell death.     

Synthesis of 4′-(Hydroxymethyl)Guanosine and a Phosphonate Analogue of Guanylic Acid

Abstract

The synthesis of 4′-(hydroxymethyl)guanosine (7) and the phosphonate analogue 8 of guanylic acid proceed from a common intermediate, 2′, 3′-O-isopropylidene-N ²-(monomethoxytrityl)-guanosine-5′-aldehyde (13).     

The oxidation of N,N′-bis(4-aminophenyl)-N,N′-dimethylethylenediamine (BED) by rat liver

We have examined the oxidation of N,N′-bis(4-aminophenyl)-N,N′-dimethylethylenediamine (BED) by tissue homogenates and fractions of liver homogenates. We find that this agent both gives osmiophilic deposits in tissue blocks and readily increases the uptake of oxygen by hepatic homogenates. The highest activity was in the mitochondrial and, next, in the microsomal fractions. Kinetic evidence indicates that the former represents two enzymatic activities while the latter is only a single site. The activity was greatest in the outer membrane of the mitochondria, in agreement with electron micrographic studies and in the rough microsomal fraction. Further, it was very sensitive to both formaldehyde and detergents. The activity was not well associated with either monamine oxidase (benzylamine substrate) or xanthine oxidase activities. Activity was observed in a large number of tissues.     

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.     

Chemoenzymatic Synthesis of 3′-Deoxy-3′-(4-Substituted-Triazol-1-YL)-5-Methyluridine

An efficient protocol has been developed for the synthesis of a small library of 3′-deoxy-3′-(4-substituted-triazol-1-yl)-5-methyluridine using Cu(I)-catalyzed Huisgen–Sharpless–Meldal 1,3-dipolar cycloaddition reaction of 3′-azido-3′-deoxy-5-methyluridine with different alkynes under optimized condition in an overall yields of 76%–92%. Here, the azido precursor compound, i.e., 3′-azido-3′-deoxy-5-methyluridine was chemoenzymatically synthesized from D-xylose in good yield. Some of the alkynes used in cycloaddition reaction were synthesized by the reaction of hydroxycoumarins or naphthols with propargyl bromide in acetone using K₂CO₃in excellent yields. All synthesized compounds were unambiguously identified on the basis of their spectral (IR, ¹H-, ¹³C NMR spectra, and high-resolution mass spectra) data analysis.     

Syntheses,properties, and use of fluorescent N-(5′-phospho-4′-pyridoxyl)amines in assay of pyridoxamine (pyridoxine) 5′-phosphate oxidase

A sensitive and simple fluorometric assay has been developed for detection of pyridoxamine (pyridoxine) 5′-phosphate oxidase. This technique utilizes fluorescent N-(5′-phospho-4′-pyridoxyl)amines as substrates that, upon incubation with the oxidase, release the free fluorescent amine. The substrates were prepared by condensation of pyridoxal 5′-phosphate with fluorescent amines and subsequent hydrogenation of the Schiff bases. Since N-(1-naphthyl)ethylenediamine is 15 times less fluorescent in the intramolecularly quenched substrate than the product amine, the direct increase of fluorescence, as well as selective extraction of more fluorescent product, can be utilized for assay. The apparent K_m value for this substrate is 8 μm, which is slightly less than that of pyridoxamine 5′-phosphate; V is larger than the natural substrate value. The greater sensitivity gained by this fluorimetric method allows detection of the oxidase in smaller quantities than can be determined by the conventional colorimetric assay.     

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%.     

DFT-based molecular modeling and vibrational study of the La(III) complex of 3,3′-(benzylidene)bis(4-hydroxycoumarin)

Molecular modeling of the La(III) complex of 3,3′-(benzylidene)bis(4-hydroxycoumarin) (PhDC) was performed using density functional theory (DFT) methods at B3LYP/6-31G(d) and BP86/TZP levels. Both Stuttgart-Dresden effective core potential and ZORA approximation were applied to the La(III) center. The electron density distribution and the nucleophilic centers of the deprotonated ligand PhDC^2- in a solvent environment were estimated on the basis of Hirshfeld atomic charges, electrostatic potential values at the nuclei, and Nalewajski-Mrozek bond orders. In accordance with the empirical formula La(PhDC)(OH)(H₂O), a chain structure of the complex was simulated by means of two types of molecular fragment: (1) two La(III) cations bound to one PhDC^2- ligand, and (2) two PhDC^2- ligands bound to one La(III) cation. Different orientations of PhDC^2-, OH^- and H₂O ligands in the La(III) complexes were investigated using 20 possible [La(PhDC^2-)₂(OH)(H₂O)]^2- fragments. Energy calculations predicted that the prism-like structure based on “tail-head” cis-LML2 type binding and stabilized via HO...HOH intramolecular hydrogen bonds is the most probable structure for the La(III) complex. The calculated vibrational spectrum of the lowest energy La(III) model fragment is in very good agreement with the experimental IR spectrum of the complex, supporting the suggested ligand binding mode to La(III) in a chain structure, namely, every PhDC^2- interacts with two La(III) cations through both carbonylic and both hydroxylic oxygens, and every La(III) cation binds four oxygen atoms of two different PhDC^2-. Figure Low energy prism-like model fragment, [La(PhDC^2-)₂(OH)(H₂O)]^2-, optimized at BP86/TZP level of theory     

Synthesis,reactivity and biological activity of N(4)-boronated derivatives of 2′-deoxycytidine

By seeking new stable boron-containing nucleoside derivatives, potential BNCT boron delivery agents, a novel synthetic approach was tested, aimed at a boron attachment via a single bond to an aliphatic carbon of sp³ hybridization. The latter allowed successful modification of deoxycytidine in the reaction with 2-(iodomethyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane of the deoxynucleoside amino group. For new compounds, detailed NMR, LDI HRMS (Laser Desorption/Ionization High-Resolution Mass Spectrometry) analyses along with in vivo phosphorylation studies, toxicity assays and DFT modelling are presented.     

Electrochemical Analysis of 3′-Azidothymidine (AZT)

Abstract

3′-Azido-3′-deoxythymidine (AZT) exhibits a two-electron diffusion—controlled polarographic reduction wave, with conversion to 3^′?amino-3′-deoxythymidine. The mechanism of reduction, analytical and clinical applications, and its use for one-step synthesis of amino from azido nucleosides, are described.     

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.

     

The antihypertensive effect of U-0521 (3′,4′-dihydroxy-2-methylpropiophenone)

The synthetic catechol, U-0521, (3′,4′-dihydroxy-2-methylpropiophenone) is a competitive inhibitor of both tyrosine hydroxylase and catechol-O-methyltransferase. Continuous subcutaneous administration of 10 μmoles per day of U-0521 via Alzet osmotic minipumps to adult male Spontaneously Hypertensive Rats (SHR) reduced blood pressure from 160 mmHg to 125 mmHg. This effect occurred within two days, persisted for the two weeks that the pumps were in place, and reversed gradually upon cessation of U-0521 administration. Similar treatment of U-0521 to normotensive Wistar Kyoto rats (WKY) did not result in a similar hypotentensive effect. Subcutaneous administration of the same dose to juvenile SHRs led to a blockade in the expression of hypertension. After the five week treatment period, the blood pressure of the U-0521 treated animals escalated rapidly to match the saline treated controls. The antihypertensive effect of U-0521 on SHRs also occured when the compound was delivered by the oral route at the rate of 50 mg/kg/day.