Function-Oriented and Modular (+/-)-cis-Pseudoguaianolide Synthesis: Discovery of New Nrf2 Activators and NF-κB Inhibitors

Abstract

Described herein is a function-oriented synthesis route and biological evaluation of pseudoguaianolide analogues. The 10-step synthetic route developed retains the topological complexity of the natural product, installs functional handles for late-stage diversification, and forges the key bioactive Michael acceptors early in the synthesis. The analogues were found to be low-micromolar Nrf2 activators and micromolar NF-κB inhibitors and dependent on the local environment of the Michael acceptor moieties.

Keywords: NF-κB inhibitors; Nrf2 activators; enyne metathesis; modular synthesis; pseudoguaianolides.

Figure 1.

A) Representative covalent modifier drugs. B–C) Covalent-modifier natural products and analogues.

Figure 2.

A) Pseudoguaianolide natural products. B) Summary of this work.

Figure 3.

Pseudoguaianolide analogues and intermediates examined in the below outlined biological studies.

Figure 4.

(A) NF-κB luciferase reporter assay and Alamar Blue cytotoxicity assay in A549 cells and (B) NF-κB Secreted Embryonic Alkaline Phosphatase (SEAP) reporter assay and Alamar Blue cytotoxicity assay in HEK293 cells. The NF-κB pathway was induced by addition of TNF-α (15 ng mL⁻¹ for A; 22.5 ng mL⁻¹ for B) in experiments except for the non-induced controls. Experimental values in both assays were normalized to the induced (no compound) control. Relative NF-κB activity is represented by the dark colored bars superimposed over the relative cell viability represented by the lighter color bars. Data shown are the mean ±S.D. of N≥2 biological replicates performed in technical triplicate (individual biological replicates shown in the SI). Cell viability columns occluded by activity columns were all above 85% viability and marked NT (non-toxic). 12 and 13 were tested at the highest possible soluble concentration. Parthenolide (PTL) was used as a positive control.

Figure 5.

Nrf2/ARE activity of pseudoguainolide analogues in HEK293 cells. (A,B) Pseudoguainolide analogues activated the ARE-luc reporter (24 h) in a dose-dependent manner. Data represents average of three replicates with ±SD. (C) Western blot analysis of the Nrf2 target HO-1 after 24 h treatment with 11b. *indicates cytotoxicity.

Figure 6.

Anti-inflammatory activity of pseudoguainolide analogues in RAW 264.7 (A,B) and MEF cells (C,D). (A,B) RAW264.7 were pretreated with epi-11b and 11b (1 h) and then stimulated with LPS (1 μg mL⁻¹). (A) Nqo1 was measured by TaqMan analysis, using actin as endogenous control. (B) NO production was measured after 24 h using Griess reagent (n = 3 with ±SD). (C,D) Wild-type MEFs (C) or Nrf2^−/− MEFs (D) were pretreated with compounds for 1 h and then challenged with IFN-γ/TNF-α (10 ng mL⁻¹). NO levels were measured 24 h later using Griess reagent.

Scheme 1.

Proposed four-step route to pseudoguaianolide scaffolds. Pg = Protecting group, E = electron-withdrawing group

Scheme 2.

Scope of pseudoguaianolide scaffold synthesis. Conditions: i) 1.4 equiv. methyl cyanoacetate, 1.4 equiv. ammonium acetate, PhH:AcOH 4:1, reflux, Dean-Stark, ii) 1.5 equiv. allyl bromide (4-bromobut-2-yn-1-yl acetate for 7I), 2 equiv. potassium carbonate, DMF, 0 °C. iii) 1 mol% Grubbs II, toluene (0.05 M), 80 °C. iv) 1 mol% Grubbs II, 1 atm ethylene, toluene (0.05 M), 80 °C. ^a in all cases, the major and minor diastereomers from the deonjugative alkylation step are seperable upon ring-closing enyne metathesis.

Scheme 3.

Knoevenagel condensation of α-quaternary cyclopentan-1,3-diones.

Scheme 4.

A) Synthesis of isomeric scaffolds. B) The targeted scaffolds can also be prepared by diene metathesis. i. – iii. same general conditions as Scheme 2

Scheme 5.

A potential model for predicting the diastereoselectivity of alkylation.

Scheme 6.

Functional group interconversion to pseudoguaianolide analogues. ^a an X-Ray crystal structure of the major epimer was obtained on an analog of this scaffold, confirming the stereochemistry. See SI for details.

Scheme 7.

A) A unique stereospecific decarboxylative protonation. B) proposed mechanism for stereospecificity.