Image contrast agents activated by prostate specific antigen (PSA) Graham B. Jones, a, * Longfei Xie, a Ahmed El-Shafey, a Curtis F. Crasto, a Glenn J. Bubley b and Anthony V. D’Amico c a Bioorganic and Medicinal Chemistry Laboratories, Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA b Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA c Dana Farber Cancer Institute and Brigham and Womens Hospital, Harvard Medical School, Boston, MA 02215, USA Received 4 March 2004; accepted 10 April 2004 Abstract—A family of image contrast agent conjugates designed to undergo enzymatic activation has been synthesized. The agents underwent activation both with enzymatically active prostate specific antigen and a-chymotrypsin, releasing free fluorophore via cleavage of a three-component system. Ó 2004 Elsevier Ltd. All rights reserved. Despite improvements in local therapy and increased awareness, prostate cancer continues to be second only to lung cancer as a cause for cancer deaths in men. 1 Prior investigations show that the presence of prosta- tectomy Gleason grade P4 in the radical prostatec- tomy specimen is the most important predictor of progression following surgery. 2 Unfortunately, the transrectal ultrasound guided sextant sampling of the prostate is subject to sampling error, and therefore biopsy Gleason grade will underestimate prostatectomy Gleason grade 4 or 5 disease in as many as 40% of men with clinically localized disease. 3 Therefore, an imaging method capable of identifying Gleason grade P4 dis- ease within the prostate gland could provide the basis for patient selection for more aggressive initial thera- peutic approaches. 4 A number of image contrast enhancing agents have been studied for use in conjunc- tion with ultrasound methods of detection. 5 However, immunohistochemical studies have also shown that Gleason grade bears an inverse correlation with the concentration of enzymatically active prostate specific antigen (PSA). 6 PSA is a serine protease; however, PSA in serum (but not in the prostatic microenvironment) is rapidly inactivated by binding to serum proteins. 7 An attractive possibility, therefore, would be the design of an imaging system, which exploits the enzymatic effi- ciency of PSA in the prostatic microenvironment. Our strategywastoconjugateaproteinogenicPSAsubstrate to a masked fluorophore via an inert spacer/linker group, such that the free fluorescent molecule is liber- ated on proteolysis (Scheme 1). 8 Our preferred choice for the inert linker is the p-amino- benzyl alcohol pioneered by Katzenellenbogen, 9 having previously employed this method for enzyme mediated cytotoxin release. 10 Though a number of high-affinity peptide substrates for PSA have been identified, we initially wished to provide proof-of-principle with a minimal substrate and selected tyrosine conjugates for examination of appropriate fluorophores (Scheme 2). 11 *Corresponding author. Tel.: +1-617-373-8619; fax: +1-617-373-8795; e-mail: gr.jones@neu.edu fluorophore fluorophore fluorophore LINKER PSA Cleavage Site LINKER spontaneous release LINKER peptide sequence (recognized by PSA) Scheme 1. Three-component system for PSA activated image contrast agent. 0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2004.04.041 Bioorganic & Medicinal Chemistry Letters 14 (2004) 3081–3084