A forward chemical screen using zebrafish embryos with novel 2-substituted 2H-chromene derivatives

Abstract

We synthesized 2-substituted 2H-chromene derivatives from salicylaldehyde using potassium vinylic borates in the presence of secondary amines. Our goal was to generate novel compounds that might modulate transforming growth factor-beta signaling, based on limited rational design. Potassium vinyl trifluoroborates react with salicylaldehydes at 80 degrees C in the presence of a secondary amine and produce 2-substituted 2H-chromene derivatives with a 70-90% yield. A small library of these compounds, predicted to potentially interact with transforming growth factor-beta receptors, was screened for bioactivity in living zebrafish embryos. We found that the related compounds differentially affect development, and demonstrate one compound that produces severe body axis alterations in early embryogenesis and at lower doses affects specifically cardiovascular development. This compound modulates specifically a Smad-independent transforming growth factor-beta-regulated mitogen-activated protein kinase pathway, namely p-SAPK/JNK. These compounds, as suggested by our biological assays, may prove useful to manipulate developmental programs and develop therapeutic tools.

Figure 1

Zebrafish can be used to screen bioactivity of the novel 2-chromene compounds. Zebrafish embryos were treated with compounds, as indicated, at 100 µm. Compounds were added at 5 hpf and embryos were cultured until 24 hpf. Shown are representative embryos from batches in which all embryos looked similar (n = at least 10). Embryos treated with compounds 10 (A) or 6 (B) (or 8, not shown) are essentially normal, compared to embryos treated with vehicle only (not shown). Embryos treated with compounds 1 (C) or 2 (D) (similar to 4 or 9, not shown) develop significant defects in the hindbrain (arrow in C), eye (arrowhead in D), and a shortened tail (indicated by the double-headed arrows). Embryos treated with compound 7 (E), 3 or 5 (not shown) are disrupted entirely for normal body axis development, and do not survive to 48 hpf. Views are lateral, anterior to the left.

Figure 2

Compound 7 affects early embryonic processes including epiboly. Shown are representative embryos (n = 10) that were treated with vehicle (A, control) or compound 7 at 50 µm, added at 3 hpf (B), and cultured until 8 hpf. Note that epiboly in control embryos has reached by this time approximately 80% completion, while this is significantly delayed in 84-treated embryos. Views are lateral.

Figure 3

Compound 7 affects both early and subsequent developmental processes. Shown are representative embryos (n = 25) treated with 50 µm compound 7, which was added at 3, 5, or 8 hpf, compared with embryos treated with vehicle alone (D). All embryos were cultured until 24 hpf. As indicated by the double-headed arrows, the tail effects are progressively less severe in later embryos, but still significant when added at 8 hpf compared to control. Views are lateral, anterior to the left.

Figure 4

Compound 7 alters hematopoietic and cardiac development. Shown are representative embryos (n = 25) treated with vehicle alone (A, control) 10 µm compound 7 added at 3 hpf (B), or 10 µm compound 7 added at 30 hpf (C). Embryos were cultured until 55 hpf (A, B) or 5 dpf (C). Note the relative lack of circulating erythroid cells in B, compared to A (white arrows), and the defective and linear heart (black arrow) and edema in C.

Figure 5

Compound 7 modulates specifically levels of phospho-SAPK/JNK. Shown are representative results from western blotting experiments. Fifty embryos at 24 hpf were cultured for 5 h either in vehicle (DMSO, indicated by lanes marked D) or in 100 µm compound 7 (indicated by lanes marked 7). Embryos were then deyolked and dissociated directly into SDS sample buffer (4 µL per embryo). Each lane contains a lysate representing 5 embryo equivalents. Samples were electrophoresed in a 10% SDS-PAGE-Tris gel (Invitrogen, Carlsbad, CA, USA), transferred to nylon and probed using primary antibodies (Cell Signaling Technology, Danvers, MA, USA) specific to the phosphorylated (active) isoforms for each protein. After incubation with appropriate secondary antibodies, filters were developed using ECL (Santa Cruz, Santa Cruz, CA, USA) and exposed to film. An equivalent set of samples was stained directly from the gel using Coomasie Blue (CB), demonstrating equal protein loading. Antibodies used are indicated next to each panel. Note that only p-SAPK/JNK levels are changed, being increased substantially by exposure of embryos to compound 7. Equivalent results were reproduced with independent experiments.

Scheme 1

An efficient procedure for generating chromene derivatives (see ref. 18).

Scheme 2

A small library of chromene derivatives was synthesized and used to screen bioactivity with zebrafish embryos. Compounds tested are shown and labelled 1–10.