Diversity through phosphine catalysis identifies octahydro-1,6-naphthyridin-4-ones as activators of endothelium-driven immunity

Abstract

The endothelium plays a critical role in promoting inflammation in cardiovascular disease and other chronic inflammatory conditions, and many small-molecule screens have sought to identify agents that prevent endothelial cell activation. Conversely, an augmented immune response can be protective against microbial pathogens and in cancer immunotherapy. Yet, small-molecule screens to identify agents that induce endothelial cell activation have not been reported. In this regard, a bioassay was developed that identifies activated endothelium by its capacity to trigger macrophage inflammatory protein 1 beta from primary monocytes. Subsequently, a 642-compound library of 39 distinctive scaffolds generated by a diversity-oriented synthesis based on the nucleophilic phosphine catalysis was screened for small molecules that activated the endothelium. Among the active compounds identified, the major classes were synthesized through the sequence of phosphine-catalyzed annulation, Tebbe reaction, Diels-Alder reaction, and in some cases, hydrolysis. Ninety-six analogs of one particular class of compounds, octahydro-1,6-naphthyridin-4-ones, were efficiently prepared by a solid-phase split-and-pool technique and by solution phase analog synthesis. Structure-function analysis combined with transcriptional profiling of active and inactive octahydro-1,6-naphthyridin-4-one analogs identified inflammatory gene networks induced exclusively by the active compound. The identification of a family of chemical probes that augment innate immunity through endothelial cell activation provides a framework for understanding gene networks involved in endothelial inflammation as well as the development of novel endothelium-driven immunotherapeutic agents.

Scheme 1.

Diversity-oriented synthesis based on nucleophilic phosphine catalysis. For clarity, detailed designation of stereochemistry and substituents are removed. For the detailed structural information, see the following references: For scaffold s1, refs.  and ; s2, refs.  and ; s4, ref. ; s5, ref. ; s6, refs.  and ; s7, ref. ; s8, ref. ; s9–s12, ref. ; s13–s20, ref. ; s21–s23, ref. ; s24–s39, ref. .

Fig. 1.

Identification of small molecules that activate human endothelial cells. (A) IFNγ (10 ng/mL), DMSO controls (n = 60 total replicates), and 642 compounds (10 μM) were tested for their ability to promote MIP1β production. The library contained 37 compounds capable of inducing MIP1β at least to the level of IFNγ (red dashed line). * represents five distinct scaffolds with activity, whereas ** represents the two naphtyridine families. (B) Structures of five octahydronaphthyridinones selected for further study. (C) Confirmation of a subset of active compounds: Coculture is required for MIP1β production. Data represents mean ± SEM of triplicate wells from one of two comparable experiments. # p values for MIP1β: (a) 105A3 = 0.008, (b) A8 = 0.003, (c) A10 = 0.026, (d) A11 = 0.007, and (e) B2 = 0.046. ## p values for MIP1α: (a) 105A3 = 0.042, (b) A8 = 0.016, (c) A10 = 0.009, (d) A11 = 0.041, and (e) B2 = 0.13. (D) Activated endothelium triggers MIP1β production from CD14+ monocytes. Percent positive MIP1β cells and mean fluorescence intensity (MFI) of MIP1β⁺ cells are shown.

Scheme 2.

Solid-phase synthesis of octahydro-1,6-naphthyridin-4-ones.

Scheme 3.

Solution-phase synthesis of octahydro-1,6-naphthyridin-4-ones.

Fig. 2.

EC-activating effects of 96 naphthyridinones. (A) Data represent percent induction relative to IFNγ for each of the 96 analogs analyzed over two experiments. (B) Structures of selected active (H6, 2B6, 2B7, 105A3, D6, A10, D10, D11, E3, C2, C8, and C9) and inactive octahydro-1,6-naphthyridin-4-one analogs (H11, 2B2, D5, D9, E2, E8, F7, F10, and G9). (C) Validation of seven (active and inactive) analogs. Data represent the mean ± SEM from two independent experiments performed in triplicate wells (n = 6 wells per condition), * p value < 0.05, ** p value < 0.03, *** p value < 0.003.

Fig. 3.

SAR analysis of octahydro-1,6-naphthyridin-4-ones.

Fig. 4.

Gene expression profiling of endothelial cells treated with active and inactive analogs. (A) Microarray analysis comparing gene expression induction in EC treated with active analog (D10, 10 μM) and inactive analog (E2, 10 μM). (B) Subset of genes from A with known involvement in inflammation: relative expression by active and inactive analogs. (C) E-selectin induction by naphthyridinone analogs. Percentages for intercellular adhesion molecule (ICAM) and ICAM/E-selectin expressing cells are displayed.