Clicking 3′-Azidothymidine into Novel Potent Inhibitors of Human Immunodeficiency Virus

3′-Azidothymidine (AZT) was the first approved antiviral for the treatment of human immunodeficiency virus (HIV). Reported efforts in clicking the 3′-azido group of AZT have not yielded 1,2,3-triazoles active against HIV or any other viruses. We report herein the first AZT-derived 1,2,3-triazoles with submicromolar potencies against HIV-1. The observed antiviral activities from the cytopathic effect (CPE) based assay were confirmed through a single replication cycle assay. Structure–activity-relationship (SAR) studies revealed two structural features key to antiviral activity: a bulky aromatic ring and the 1,5-substitution pattern on the triazole. Biochemical analysis of the corresponding triphosphates showed lower ATP-mediated nucleotide excision efficiency compared to AZT, which along with molecular modeling suggests a mechanism of preferred translocation of triazoles into the P-site of HIV reverse transcriptase (RT). This mechanism is corroborated with the observed reduction of fold resistance of the triazole analogue to an AZT-resistant HIV variant (9-fold compared to 56-fold with AZT)

7-Deazapurine and 8-Aza-7-deazapurine Nucleoside and Oligonucleotide Pyrene “Click” Conjugates: Synthesis, Nucleobase Controlled Fluorescence Quenching, and Duplex Stability

7-Deazapurine and 8-aza-7-deazapurine nucleosides related to dA and dG bearing 7-octadiynyl or 7-tripropargylamine side chains as well as corresponding oligonucleotides were synthesized. “Click” conjugation with 1-azidomethyl pyrene (<b>10</b>) resulted in fluorescent derivatives. Octadiynyl conjugates show only monomer fluorescence, while the proximal alignment of pyrene residues in the tripropargylamine derivatives causes excimer emission. 8-Aza-7-deazapurine pyrene “click” conjugates exhibit fluorescence emission much higher than that of 7-deazapurine derivatives. They are quenched by intramolecular charge transfer between the nucleobase and the dye. Oligonucleotide single strands decorated with two “double clicked” pyrenes show weak or no excimer fluorescence. However, when duplexes carry proximal pyrenes in complementary strands, strong excimer fluorescence is observed. A single replacement of a canonical nucleoside by a pyrene conjugate stabilizes the duplex substantially, most likely by stacking interactions: 6–12 °C for duplexes with a modified “adenine” base and 2–6 °C for a modified “guanine” base. The favorable photophysical properties of 8-aza-7-deazapurine pyrene conjugates improve the utility of pyrene fluorescence reporters in oligonucleotide sensing as these nucleoside conjugates are not affected by nucleobase induced quenching

Effect of MPX-004 on isolated NMDA receptor-mediated fEPSPs in rat hippocampal CA1 stratum radiatum in response to stimulation of Schaffer collateral input.

<p>Slices were exposed to MPX-004 from 100 nM to 30 μM in half log concentration increments as well as to 50 μM. Right panel- Time course for inhibition of fEPSPs after application of different concentrations of MPX-004. Left panel- Percent inhibition at 40 min after application for each MPX-004 concentration. Maximum inhibition was approximately 60% of the fEPSPs at 30 or 50 μM. The IC₅₀ of MPX-004 for inhibition of fEPSPs was 3.4 μM. Curves in the right panel and data point in the left panel are a mean (± SEM) of 4, 5, 6, 8, 6, 5 and 2 slices obtained from 2, 2, 3, 3, 2, 2 and 1 rats exposed to 0.1, 0.3, 1, 3, 10, 30 and 50 μM MPX-004, respectively. In separate experiments, a highly selective GluN2B NAM inhibited approximately 40% of the fEPSP (data not shown).</p

Effect of MPX-004 and MPX-007 on glutamate/glycine-induced currents in <i>Xenopus</i> oocytes expressing GluN1 and GluN2A, B, C, or D.

<p>Oocytes were exposed to MPX-004 or MPX-007 at concentrations from 10 nM to 10 μM as indicated. Inhibition curves were generated using GraphPad Prism and IC₅₀ values were generated using CCD Vault. The IC₅₀s for inhibition of GluN2A-mediated currents were 198 ± 17 and 143 ± 10 nM for MPX-004 and MPX-007, respectively. Each data point is a mean (± standard deviation) of data from 4–12 oocytes.</p

Effects of MPX-004 and MPX-007 on NMDA/glycine-induced currents of rat cortical neurons in primary culture.

<p>Cortical neurons were maintained in primary culture for 13–15 days and then examined using whole-cell manual patch clamp for current evoked by NMDA (100 μM) + glycine (10 μM) applied for 4 seconds during 10 second pulses to +20 or +40 mV from a holding potential of -70 mV. Inhibition of NMDA-activated currents was quantified during exposure to 10 μM MPX-007, MPX-004, Ro 25–6981, or a combination of Ro 25–6981 + MPX-004. Currents were blocked ~25–30% by either MPX-007 or MPX-004 alone, ~70% by Ro 25–6981 alone, and ~85% by Ro 25–6981 plus MPX-004.</p

Synthetic schemes for compounds 2–10.

<p>Synthetic schemes for compounds 2–10.</p

Concentration-response of TCN-201, MPX-004, and MPX-007 inhibition of Ca²⁺ responses mediated by GluN2A expressed in HEK cells.

<p>Cells were stimulated with glutamate and glycine (3 μM each) in the presence of compounds at a range of concentrations. Curves for inhibition of the Ca²⁺ response in GluN2A-expressing cells were derived from fits to the Hill equation using GraphPad Prism (v6.00 for Mac, GraphPad Software, La Jolla California USA). Whereas MPX-004 and MPX-007 achieve full inhibition of the GluN2A Ca²⁺ response by ~ 3 μM, TCN-201 never inhibits more than ~40% of the response. Each data point is a mean (± standard deviation) of data from 20–86 experiments).</p

The structures of TCN-201, MPX-004 and MPX-007.

<p>The values are IC₅₀s for inhibition of Ca²⁺ responses mediated by GluN2A receptors expressed in HEK cells.</p