Systematic Exploration of the Potential of HPQ as the Core Scaffold of Probes for in Vivo Imaging: A Case Study of FAP-Responsive Probes

The limited tissue penetration depth of optical probes based on 2-(2'-hydroxyphenyl)-4(3H)-quinazolinone (HPQ) hindered dynamic monitoring of the actual distribution and metabolism of them in deep tissues. Herein, using fibroblast activating protein (FAP)-activatable dual-modal probes as a prototype, we explored the potential of HPQ as a core scaffold for in vivo imaging probes via stepwise biological characterization. The tumor-to-muscle uptake ratios (T/M) of <b>LWF-1</b>, <b>LWF-2</b>, and <b>LWF-3</b> were consistently above 4.05 during in vivo fluorescence imaging with sustained tumor visualization exceeding 48 h. All ⁶⁸Ga-radiolabeled probes exhibited a maximum tumor uptake of up to 12.83 ± 9.37%ID/g in model mice, with the T/M of <b>[</b>^<b>68</b><b>Ga]LWF-3</b> remaining above 4.89 throughout the PET scanning. The ex vivo biodistribution study revealed that <b>[</b>^<b>177</b><b>Lu]LWF-3</b> reached the peak uptake in the tumor at 4 h postinjection (10.41 ± 0.68%ID/g). Accordingly, structural design and optimization governing their in vivo metabolism are critical for acquisition of ideal HPQ-based probes.