Emission tuning of fluorescent kinase inhibitors: conjugation length and substituent effects

Abstract

Fluorescent N-phenyl-4-aminoquinazoline probes targeting the ATP-binding pocket of the ERBB family of receptor tyrosine kinases are reported. Extension of the aromatic quinazoline core with fluorophore "arms" through substitution at the 6- position of the quinazoline core with phenyl, styryl, and phenylbutadienyl moieties was predicted by means of TD-DFT calculations to produce probes with tunable photoexcitation energies and excited states possessing charge-transfer character. Optical spectroscopy identified several synthesized probes that are nonemissive in aqueous solutions and exhibit emission enhancements in solvents of low polarity, suggesting good performance as turn-on fluorophores. Ligand-induced ERBB2 phosphorylation assays demonstrate that despite chemical modification to the quinazoline core these probes still function as ERBB2 inhibitors in MCF7 cells. Two probes were found to exhibit ERBB2-induced fluorescence, demonstrating the utility of these probes as turn-on, fluoroescent kinase inhibitors.

Figure 1

Crystal structures of the EGFR ATP-binding pocket with (A) erlotinib (PDB ID: 1M17) and (B) lapatinib (PDB ID: 1XKK) reveal the inhibitor binding modes. The arms at the 6-position of the quinazoline core (in blue; C, D) may be replaced by fluorophore arms without disturbing the key binding contacts. (E) General structure and substituent key for the synthesized fluorescent quinazolines.

Scheme 1. Synthesis Routes to Fluorescent Quinazolines 1a–3e

Representative structures 1a and 3d are shown.

Figure 2

Frontier molecular orbitals of 2a–2e calculated at the CAM-B3LYP/6-31G* level: the polarization of the HOMO and LUMO shifts across the series (compare 2a and 2e); strong CT character is expected for both 2a and 2e, although the localization of charge should be reversed.

Figure 3

Chloroform solutions of selected probes (5 μM) under UV illumination (354 nm); aqueous solutions showed weak or no emission (vials not shown).

Figure 4

Absorption (solid lines) and emission (dashed lines) spectra of (A) 1a–1e, (B) 2a–2e, and (C) 3a–3e in CHCl₃. Emission intensities are given relative to 3d, which has the highest quantum yield (see Table 2).

Figure 5

Emission intensities obtained in octanol and water reveal that several probes are highly responsive to changes in their chemical microenvironment and possess high ON/OFF ratios.

Figure 6

(A) Comparative inhibitory properties of selected probes: the inhibition of NRGβ1-induced ERBB2 tyrosine phosphorylation in MCF7 cells was evaluated at 0.1 and 10 μM concentrations of the indicated compounds. For comparison, lapatinib was used at its clinically used serum concentration of 3 μM. (B) Compounds 3d and 1a show comparable affinity in inhibition studies but a difference in the maximum achievable inhibition.

Figure 7

(A) 1a and (B) 2a exhibit turn-on emission upon binding to soluble ERBB2 kinase domain. In PBS alone, emission (in black) is largely quenched, whereas in the presence of ERBB2 kinase, emission (in green) is significantly enhanced; conditions: [1a] = [2a] = 1 μM; [ERBB2] = 100 nM, λ_ex = 370 nm.

Figure 8

R₁, R₂, and R₃ are possible sites for chemical modification of 1a and 2a to enhance aqueous solubility.