Regioselective Construction and Screening of 1,3-Disubstituted Tetrahydroindazolones in Enantiomerically Pure Pairs

In this paper, we describe a regioselective synthetic pathway for enantiopure 1,3-disubstituted tetrahydroindazolone derivatives via the condensation of 2-acylcyclohexane-1,3-dione with various alkyl- and arylhydrazines using the steric effects of a Boc-protected pyrrolidine ring. This synthetic method has a broad scope for substrate generality for various hydrazines with excellent regioselectivity. To maximize the molecular diversity, further diversifications of 1,3-disubstituted tetrahydroindazolones were pursued by systematic <i>N</i>-modification of the secondary amine of the pyrrolidine ring using solution-phase parallel synthesis with polymer-supported reagents. A library containing a total of 272 drug-like tetrahydroindazolones, including 85 enantiomeric pairs, was constructed; the average purity, without further purification, was 95%

Synthesis and Biological Evaluation of α‑Galactosylceramide Analogues with Heteroaromatic Rings and Varying Positions of a Phenyl Group in the Sphingosine Backbone

We designed and synthesized seven α-GalCer analogues with a pyrazole moiety and varying positions of a phenyl group in the sphingosine backbone to polarize cytokine secretion. On the basis of in vitro and in vivo biological evaluations, we found that analogue <b>5</b> induced greater polarization toward Th2 and greater secretion of the immunomodulatory cytokine, IL-4, over secretion of pro-inflammatory cytokines, IFN-γ and IL-17. Treatment of a single dose of analogue <b>5</b> markedly ameliorated disease pathogenesis in an animal model of an inflammatory demyelinating disease of the central nervous system, compared to that of KRN7000 (<b>1</b>). Therefore, this new α-GalCer analogue <b>5</b> is a novel iNKT ligand that stimulates the selective secretion of anti-inflammatory cytokines and regulates autoimmune diseases by reducing Th1 and Th17 responses

Inhibitory effects of 4d and 4e on NO and TNF-α production in LTA- or Poly I∶C-stimulated BV2 microglial cells.

<p>Cells were incubated for 16 h with LTA (10 µg/mL) (A) or Poly I∶C (10 µg/mL) (B) in the absence or presence of α-GalCer analogs <b>4d</b> and <b>4e</b> (5 µM), and the amounts of released NO and TNF-α were measured in supernatants. Treatment with α-GalCer analogs alone did not affect NO or TNF-α production. Bars indicate the mean ± S.E.M. of three independent experiments. *<i>P</i><0.05; significantly different from stimulated microglial cells.</p

Synthetic scheme of key intermediates with late-stage diversification of the sphingosine backbone.

<p>Synthetic scheme of key intermediates with late-stage diversification of the sphingosine backbone.</p

The p38 MAPK-specific inhibitor SB203580 inhibited NO and TNF-α production by suppressing NF-κB and AP-1 activities.

<p>(A) BV2 cells were treated with LPS for 16 h in the absence or presence of SB203580, and released NO and TNF-α were measured as previously described. Bars indicate the mean ± S.E.M. of three independent experiments. *<i>P</i><0.05; significantly different from LPS-treated cells. SB indicates SB203580. (B) Effect of SB203580 on LPS-induced NF-κB and AP-1 DNA binding activities.</p

DNA sequences of primers used in real-time PCR reactions, and expected product sizes.

<p>DNA sequences of primers used in real-time PCR reactions, and expected product sizes.</p

Anti-Inflammatory Effects of α-Galactosylceramide Analogs in Activated Microglia: Involvement of the p38 MAPK Signaling Pathway

<div><p>Microglial activation plays a pivotal role in the development and progression of neurodegenerative diseases. Thus, anti-inflammatory agents that control microglial activation can serve as potential therapeutic agents for neurodegenerative diseases. Here, we designed and synthesized α-galactosylceramide (α-GalCer) analogs to exert anti-inflammatory effects in activated microglia. We performed biological evaluations of 25 α-GalCer analogs and observed an interesting preliminary structure-activity relationship in their inhibitory influence on NO release and TNF-α production in LPS-stimulated BV2 microglial cells. After identification of <b>4d</b> and <b>4e</b> as hit compounds, we further investigated the underlying mechanism of their anti-inflammatory effects using RT-PCR analysis. We confirmed that <b>4d</b> and <b>4e</b> regulate the expression of iNOS, COX-2, IL-1β, and IL-6 at the mRNA level and the expression of TNF-α at the post-transcriptional level. In addition, both <b>4d</b> and <b>4e</b> inhibited LPS-induced DNA binding activities of NF-κB and AP-1 and phosphorylation of p38 MAPK without affecting other MAP kinases. When we examined the anti-inflammatory effect of a p38 MAPK-specific inhibitor, SB203580, on microglial activation, we observed an identical inhibitory pattern as that of <b>4d</b> and <b>4e</b>, not only on NO and TNF-α production but also on the DNA binding activities of NF-κB and AP-1. Taken together, these results suggest that p38 MAPK plays an important role in the anti-inflammatory effects of <b>4d</b> and <b>4e</b> via the modulation of NF-κB and AP-1 activities.</p></div

Synthetic scheme of final modifications of the acyl chain resulting in 25 unique α-GalCer analogs.

<p>Synthetic scheme of final modifications of the acyl chain resulting in 25 unique α-GalCer analogs.</p

Effects of 4d and 4e on three types of MAP kinases and NF-κB/AP-1.

<p>(A) Cell extracts were prepared from BV2 microglial cells treated with LPS for 30 min in the absence or presence of <b>4d</b> or <b>4e</b> (5 µM) and then subjected to immunoblot analysis using antibodies against phospho- or total forms of three MAP kinases. Quantification of western blot data (right panel). Levels of the active forms of MAPKs were normalized to total forms and are expressed as fold changes versus untreated control samples, which were arbitrarily set to 1.0. Bars indicate mean ± S.E.M. of three independent experiments. *<i>P</i><0.05; significantly different from LPS-treated cells. (B) EMSA was performed using nuclear extracts isolated from BV2 microglial cells treated with <b>4d</b> or <b>4e</b> in the presence of LPS for 3 h. Images are representative of at least three independent experiments. ‘F’ indicates free probe.</p

Chemical structures of bioactive α-GalCer derivatives and newly designed α-GalCer analogs.

<p>Chemical structures of bioactive α-GalCer derivatives and newly designed α-GalCer analogs.</p