In silico design, synthesis, and biological evaluation of radioiodinated quinazolinone derivatives for alkaline phosphatase–mediated cancer diagnosis and therapy

Abstract

As part of the development of enzyme-mediated cancer imaging and therapy, a novel technology to entrap waterinsoluble radioactive molecules within solid tumors, we show that a water-soluble, radioactive quinazolinone prodrug, ammonium 2-(2 ¶-phosphoryloxyphenyl)-6-[ 125 I]iodo-4-(3H)-quinazolinone ( 125 IQ 2-P ), is hydrolyzed by alkaline phosphatase to a water-insoluble, radiolabeled drug, 2-(2 ¶-hydroxyphenyl)-6-[ 125 I]iodo-4-(3H)-quinazolinone ( 125 IQ 2-OH ). Biodistribution data suggest the existence of two isoforms of the prodrug (IQ 2-P(I) and IQ 2-P ), and this has been confirmed by their synthesis and characterization. Structural differences of the two isoforms have been examined using in silico molecular modeling techniques and docking methods to describe the interaction/binding between the isoforms and human placental alkaline phosphatase (PLAP), a tumor cell, membrane-associated, hydrolytic enzyme whose structure is known by X-ray crystallographic determination. Docking data show that IQ 2-P , but not IQ 2-P(I) , fits the active binding site of PLAP favorably and interacts with the catalytic amino acid Ser 92 , which plays an important role in the hydrolytic process. The binding free energies (#G binding ) of the isoforms to PLAP predict that IQ 2-P will be the better substrate for PLAP. The in vitro incubation of the isoforms with PLAP leads to the rapid hydrolysis of IQ 2-P only and confirms the in silico expectations. Fluorescence microscopy shows that in vitro incubation of IQ 2-P with mouse and human tumor cells causes the extracellular, alkaline phosphatasemediated hydrolysis of the molecule and precipitation of fluorescent crystals of IQ 2-OH . No hydrolysis is seen in the presence of normal mouse and human cells. Furthermore, the intratumoral injection of 125 IQ 2-P into alkaline phosphatase -expressing solid human tumors grown s.c. in nude rats results in efficient hydrolysis of the compound and retention of f70% of the injected radioactivity, whereas similar injection into normal tissues (e.g., muscle) does not produce any measurable hydrolysis (f1%) or retention of radioactivity at the injected site. These studies support the enzyme-mediated cancer imaging and therapy technology and show the potential of such quinazolinone derivatives in the in vivo radiodetection ( 123 I/ 124 I) and therapy ( 131 I) of solid tumors.

Computational Modeling and Experimental Evaluation of a Novel Prodrug for Targeting the Extracellular Space of Prostate Tumors

“…We then docked IQ 2-P as well as IQ 2-P(I) (Fig. 2), a cyclic analogue that was discovered during the synthesis of IQ 2-P (see Introduction and Results and Discussion) and that had been shown to be a very poor substrate for ALP (3).…”

“…3 The American Cancer Society estimates that 234,300 new cases of invasive prostate cancer will be identified in the United States this year. A substantial proportion of these people will develop metastatic disease at some point, and f30,000 will die.…”

“…Toward these objectives, we present a novel approach, Enzyme-Mediated Cancer Imaging and Therapy (EMCIT; refs. [1][2][3][4][5], which potentially enables the active entrapment of a radioisotopically labeled compound within the extracellular spaces of primary prostate tumors and their metastases (Fig. 1).…”

“…The prototype for this approach was first developed for alkaline phosphatase (ALP; EC 3.1.3.1), a hydrolase with monophosphoesteric activity that is overexpressed on the plasma membranes of many tumor cell types (1,2,6,7). A suitable substrate, the prodrug ammonium 2-(2 ¶-phosphoryloxyphenyl)-6-iodo-4-(3H)-quinazolinone ( 127 IQ 2-P ), and its radioiodinated analogue ( 125 IQ 2-P ) were synthesized, purified, and characterized (1,5), and their ALP-mediated hydrolysis to the water-insoluble, fluorescent 2-(2 ¶-hydroxyphenyl)-6-iodo-4-(3H)-quinazolinone derivatives ( 127 IQ 2-OH and 125 IQ 2-OH ) was shown (1,3,5). These studies also indicated that (a) IQ 2-P is a highly water-soluble molecule (mg/mL) that is stable in human serum and readily dephosphorylated by ALP to the water-insoluble IQ 2-OH derivative; (b) the in vitro incubation of 127 IQ 2-P / 125 IQ 2-P derivatives with several ALP-expressing human and mouse tumor cell lines results in the efficient and rapid formation of the corresponding water-insoluble derivatives 127 IQ 2-OH / 125 IQ 2-OH ; and (c) the intratumoral injection of 125 IQ 2-P into ALP-expressing solid human tumors grown in rats leads to the efficient hydrolysis of the compound and the retention of f70% of the injected radioactivity, whereas similar injection into normal tissues (e.g., muscle) leads to little measurable hydrolysis (f1%) and lack of retention of radioactivity at injected sites.…”

Recent Advances of Quinazolinone Derivatives as Marker for Various Biological Activities

“…Quinazolinone is heterocyclic chemical compound with two conjoined aromatic rings incorporating with two nitrogen atoms and one of the carbons oxidized with keto oxygen It is also called as quinazolindiones, chemically known as Quinazolin-4(3H)-one 17 . There are two structural isomers, 2-quinazolinone (4) and 4-quinazolinone(5), with the 4-isomer being the more common.…”