SYNTHESIS AND ANTI-HIV ACTIVITY OF [D4U]-[TROVIRDINE ANALOGUE] AND [D4T]-[TROVIRDINE ANALOGUE] HETERODIMERS AS INHIBITORS OF HIV-1 REVERSE TRANSCRIPTASE

ABSTRACT

A series of eleven heterodimers containing both a nucleoside analogue (d4U, d4T) and a non-nucleoside type inhibitor (Trovirdine analogue) were synthesized and evaluated for their ability to inhibit HIV replication. Unfortunately, the (N-3)d4U-Trovirdine conjugates (9a–e) and (N-3)d4T-Trovirdine conjugates (10a–f) were found to be inactive suggesting that the two individual inhibitor compounds do not bind simultaneously in their respective sites.     

Conjugates of Fluorescein and Saenta (5′-S-(2-Aminoethyl)-N 6-(4-nitrobenzyl)-5′-thioadenosine): Flow Cytometry Probes for the ES Nucleoside Transporter Elements of the Plasma Membrane

Abstract

SAENTA was linked to the C-5 or C-6 positions of fluorescein through several structures to form conjugates that were bound tightly to plasma membrane sites associated with es nucleoside transport activity. The conjugates imparted fluorescence to cells that expressed es nucleoside transport activity and served as es-selective plasma membrane stains suitable for flow cytometry. Prior treatment of es-expressing cells with nitrobenzylthioinosine prevented fluorescent staining with the conjugates. Seven SAENTA-fluorescein conjugates served as flow cytometric stains with high affinities for es sites, despite substantial differences in the SAENTA-fluorescein linkage structures.     

Ethyl[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-α- and β-D-erythro-Pentofuranosyl] Acetates as Versatile Intermediates in Nucleic Acid Chemistry

Abstract

The title compounds (1a,b) were synthesized in three steps from 2-deoxy-D-ribose, and used in the preparation of oligonucleotide conjugates, branched oligonucleotides as well as homo-N-nucleosides.     

Systematic considerations for a multicomponent pharmacokinetic study of Epimedii wushanensis herba: From method establishment to pharmacokinetic marker selection

BackgroundPrenylflavonoids are major active components of Epimedii wushanensis herba (EWH). The global pharmacokinetics of prenylflavonoids are unclear, as these compounds yield multiple, often unidentified metabolites.PurposeThis study successfully elucidated the pharmacokinetic profiles of EWH extract and five EWH-derived prenylflavonoid monomers in rats.Study designThe study was a comprehensive analysis of metabolic pathways and pharmacokinetic markers.MethodsMajor plasma compounds identified after oral administration of EWH-derived prototypes or extract included: (1) prenylflavonoid prototypes, (2) deglycosylated products, and (3) glucuronide conjugates. To select appropriate EWH-derived pharmacokinetic markers, a high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was established to simultaneously monitor 14 major compounds in unhydrolyzed plasma and 10 potential pharmacokinetic markers in hydrolyzed plasma.ResultsThe pharmacokinetic profiles indicated that the glucuronide conjugates of icaritin were the principle circulating metabolites and that total icaritin accounted for ∼99% of prenylflavonoid exposure after administration of EWH-derived materials to rats. To further investigate icaritin as a prospective pharmacokinetic marker, correlation analysis was performed between total icaritin and its glucuronide conjugates, and a strong correlation (r > 0.5) was found, indicating that total icaritin content accurately reflected changes in the exposure levels of the glucuronide conjugates over time. Therefore, icaritin is a sufficient pharmacokinetic marker for evaluating dynamic prenylflavonoid exposure levels. Next, a mathematical model was developed based on the prenylflavonoid content of EWH and the exposure levels in rats, using icaritin as the pharmacokinetic marker. This model accurately predicted exposure levels in vivo, with similar predicted vs. experimental area under the curve (AUC)_0–96 h values for total icaritin (24.1 vs. 32.0 mg/L h).ConclusionIcaritin in hydrolyzed plasma can be used as a pharmacokinetic marker to reflect prenylflavonoid exposure levels, as well as the changes over time of its glucuronide conjugates.     

New Polyazamacrocycle-Nucleoside Conjugates: Synthesis,Anti-HIV Evaluation and Interaction with CXCR-4 Coreceptor

Abstract

We report the synthesis of new conjugates that incorporate in their structure bis-tetraazamacrocycle coupled with AZT via enzymolabile bond.

Two series of bis-polyazamacrocycles-AZT conjugates were designed, synthesized and evaluated for their antiviral effect in vitro as well as their capability to bind to CXCR-4 coreceptor.     

Tri-N-Boc-Tetraazamacrocycle-Nucleoside Conjugates: Synthesis and anti-HIV activities

Abstract

As far as linear N-Boc-polyamines conjugates elicited remarkable anti-HIV activity, the synthesis and anti-HIV properties of cyclic N-Boc-polyamines conjugates such as tetraazamacrocycle-nucleoside were studied. These new conjugates include an ester linkage between the two moieties. They were synthesized using Benzotriazol-l-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate coupling reagent, in the case of N-alkyl polyazamacrocycle derivatives, or through direct condensation of the acyl chloride derivative with nucleoside in the case of N-acyl polyazamacrocycle compounds. None of the new conjugates presented anti-HIV activity greater than that of the corresponding parent nucleosides.     

Synthesis and Analytical Characterization of RNA-Polyethylene Glycol Conjugates

Abstract

Polyethylene glycols with degrees of polymerization from 5 to more than 100 were incorporated into synthetic oligoribonucleotides by automated solid phase synthesis at 3′-terminal, 5′-terminal and internal positions. The conjugates were characterized by chromatographic, electrophoretic and mass-spectrometric methods. The influence of coupling site, polymer size and number of coupled polymers per oligonucleotide on the molecular properties of the conjugates is investigated.     

Studies Towards the Synthesis of Peptide-Oligonucleotide Conjugates

Abstract

We describe a peptide fragment, solid-phase coupling strategy for synthesis of peptide-oligonucleotide conjugates. Model conjugates contained a hydrophobic tetrapeptide, a hydrophobic influenza virus fusion nonapeptide, or a basic octapeptide of the HIV-1 Tat protein coupled to either dT₁₂ or a 16-mer anti-Tat oligodeoxyribonucleotide. Conjugation yields were improved by removal of internucleotide 2-cyanoethyl groups prior to peptide coupling and by use of a C12 spacer between peptide and oligonucleotide.     

Enhanced Fluorescence in the Binding of Oligonucleotides With a Pyrene Group in the Sugar Fragment to Complementary Polynucleotides

Abstract

The fluorescence intensity and lifetime of oligonucleotides with a pyrenylmethyl group at the specific sugar residue were increased upon binding to their complementary polynucleotide in aqueous solution. The present oligonucleotide-pyrene conjugates provide new fluorescent probes for detection of specific nucleic acids.     

Oligonucleotide Conjugates Designed for Discriminative Hybridization at Physiological Temperature

Abstract

We report the synthesis of oligonucleotide conjugates engineered to allow discriminative hybridization at temperatures around physiological. Two types of structural modifications were introduced: 1) internal oligomethylene and oligoethylene glycol spacers, and 2) terminal phenazinium residues. The thermal denaturation behaviour of the complexes formed by these oligonucleotide conjugates with a target sequence is compared to that of natural duplexes. We observed a lowering of the T_m of the duplexes formed by the internal modified oligonucleotides, whilst the terminal phenazinium residues enhance their stability. The effect of the spacers is modulated by their length and hydrophobic or hydrophilic nature. Alkylating substituents, which modify the target DNA strand on hybridization, were introduced on all conjugates, and the target cleavage obtained after piperidine treatment used as a further indicator of hybridization.     

“Mixed Inhibitors” of HIV-Reverse Transcriptase: Synthesis and Antiviral Activity

Abstract

Some “AZT-HEPT” and “ddC-HEPT” conjugates were designed, synthesized and evaluated for their anti-HIV activity.     

Selective Attachment of Oligonucleotides to Interleukin-1β and Targeted Delivery to Cells

Abstract

Conjugates between oligodeoxyribonucleotides and an interleukin-1β mutant protein have been constructed using a heterobifunctional cross-linker. These protein-DNA conjugates had conserved binding activity to the interleukin-1 receptor. The oligonucleotide hybridization properties were unchanged.     

Synthesis of DNA interactive C3-trans-cinnamide linked β-carboline conjugates as potential cytotoxic and DNA topoisomerase I inhibitors

A series of new C3-trans-cinnamide linked β-carboline conjugates has been synthesized by coupling between various β-carboline amines and substituted cinnamic acids. Evaluation of their anti-proliferative activity against a panel of selected human cancer cell lines such as A549 (lung cancer), MCF-7 (breast cancer), B16 (melanoma), HeLa (cervical cancer) and a normal cell line NIH3T3 (mouse embryonic fibroblast cell line), suggested that the newly designed conjugates are considerably active against all the tested cancer cell lines with IC₅₀ values 13–45?nM. Moreover, the conjugates 8v and 8x were the most active against MCF-7 cells (14.05?nM and 13.84?nM respectively) and also even potent on other cell lines tested. Further, detailed investigations such as cell cycle analysis, apoptosis induction study, topoisomerase I inhibition assay, DNA binding affinity and docking studies revealed that these new conjugates are DNA interactive topoisomerase I inhibitors.     

The Cytotoxicity of Anti-PAI-I Oligonucleotides and Their Conjugates

Abstract

The cytotoxicity of anti-PAI-5 hexadecanucleotides (phosphodiesters and phosphorothioates) and their conjugates with lipophilic alcohols was tested in EA.hy 926 hybrid endothelial cells. Some cytotoxicity was found for cholesteryl and bornyl conjugates at concentrations higher than those used for antisense inhibition experiments.     

Use of Oligonucleotide Conjugates in Creating Self-Assembling Supramolecular Systems

Abstract

The chemistry of two types of oligonucleotide conjugates containing novel chromophores are described. One, containing a stilbenedicarboxamide bridge, generates unusually stable hairpin structures that are useful in assessing rates of electron transfer through the π system of a DNA double helix. The other, containing gold nanoparticle conjugates, provides a highly selective system for detecting nucleotide sequences in oligonucleotides.     

G-specific RNA-cleaving Conjugates of Short Peptides and Oligodeoxyribonucleotides

Abstract

Artificial ribonucleases, conjugates of short oligodeoxyribonucleotides and peptides built of arginine, leucine, proline, and serine, were synthesized and assessed in terms of ribonuclease activity and specificity of RNA cleavage. A specific group of the conjugates was identified that display T1-ribonuclease-like activity and cleave RNA predominantly at G-X sequences. Circular dichroism study of the structures of the most active conjugates, free peptide (LR)4G, and oligonucleotides revealed that conjugation of oligonucleotide to the peptide results in a specific peptide folding that possibly provides ribonuclease activity to the conjugate.     

Stabilization of DNA Double and Triple Helices by Conjugation of Minor Groove Binders to Oligonucleotides

Abstract

New conjugates containing two parallel or antiparallel carboxamide minor groove binders (MGB) attached to the same terminal phosphate of one oligonucleotide strand were synthesized. The conjugates interact with their target DNA stronger than the individual components. Effect of conjugated MGB on DNA duplex and triplex stability and their sequence specificity was demonstrated on the short oligonucleotide duplexes and on the triplex formed by model 16-mer oligonucleotide with HIV polypurine tract.     

Cleavage of Leishmania Mini-exon Sequence by Oligonucleotides Conjugated to a Dimidazole Construction

Abstract

RNA sequences derived from the Leishmania amazonensis mini-exon and pre-mini-exon sequences have been targeted with complementary oligonucleotides bearing a diimidazole construction mimicking active center of ribonuclease A. The conjugates were shown to cleave the target RNAs at specific positions.     

A new bisphosphonate-containing <Superscript>99m</Superscript>Tc(I) tricarbonyl complex potentially useful as bone-seeking agent: synthesis and biological evaluation

Aiming to develop new bone-seeking radiotracers based on the organometallic core fac-[^99mTc(CO)₃]⁺ with improved radiochemical and biological properties, we have prepared new conjugates with phosphonate pendant groups. The conjugates comprise a chelating unit for metal coordination, which corresponds to a pyrazolyl-containing backbone (pz) with a N,N,N donor-atom set, and a pendant diethyl phosphonate (pz-MPOEt), phosphonic acid (pz-MPOH) or a bisphosphonic acid (pz-BPOH) group for bone targeting. Reactions of the conjugates with the precursor [^99mTc(H₂O)₃(CO)₃]⁺ yielded (mote than 95%) the single and well-defined radioactive species [^99mTc(CO)₃(κ³-pz-MPOEt)]⁺ (1a), [^99mTc(CO)₃(κ³-pz-MPOH]⁺ (2a) and [^99mTc(CO)₃(κ³-pz-BPOH)]⁺ (3a), which were characterized by reversed-phase high-performance liquid chromatography . The corresponding Re surrogates (1–3), characterized by the usual analytical techniques, including X-ray diffraction analysis in the case of 1, allowed for macroscopic identification of the radioactive conjugates. These radioactive complexes revealed high stability both in vitro (phosphate-buffered saline solution and human plasma) and in vivo, without any measurable decomposition. Biodistribution studies of the complexes in mice indicated a fast rate of blood clearance and high rate of total radioactivity excretion, occurring primarily through the renal–urinary pathway in the case of complex 3a. Despite presenting moderate bone uptake (3.04 ± 0.47% injected dose per gram of organ, 4 h after injection), the high stability presented by 3a and its adequate in vivo pharmacokinetics encourages the search for new ligands with the same chelating unit and different bisphosphonic acid pendant arms.     

2′- and 3′- Cholesterol-Conjugated Adenosine and Cytosine Nucleoside Building Blocks: Synthesis of Lipidic Nucleic Acids

Abstract

Recently our laboratory introduced¹ chemistries to synthesize 2′- and 3′- cholesteroluridine conjugates which were incorporated into several antisense oligonucleotides. We have now extended this chemistry to other nucleosides (adenosine and cytosine) and synthesized antisense oligonucleotide conjugates for several disease targets. Synthesis of these cholesterol nucleosides was carried out hy condensing choleskrol chloroformate with 2′-O-alkylamine or 3′-O-alkylamine of the appropriate nucleoside. The 2′-O-alkylamines were deiived from direct alkylation procedure.