Synthesis and Biological Evaluation of Substrate-Based Imaging Agents for the Prostate-Specific Membrane Antigen

Abstract

Prostate-specific membrane antigen (PSMA) is an attractive target for the imaging of prostate cancer (PCa) due to the elevated expression on the surface of prostate tumor cells. Most PSMA-targeted low molecular weight imaging agents are inhibitors of PSMA. We have synthesized a series of substrate-based PSMA-targeted imaging agents by mimicking poly-γ-glutamyl folic acid, an endogenous substrate of PSMA. In vitro the γ-linked polyglutamate conjugates proved to be better substrates than the corresponding α-linked glutamates. However, in vivo imaging studies of γ-ray-emitting and γ-linked glutamates did not demonstrate selective uptake in PSMA-pos-itive over PSMA-negative tumors. Subsequent chromatographic studies and in silico molecular dynamics simulations indicated that hydrolysis of the substrates is slow and access to the enzymatic active site is limited. These results inform the design of future substrate-based imaging agents for PSMA.

Keywords: PSMA; SPECT; molecular imaging; prodrug; prostate cancer.

Figure 1

Design of aminostrylpyridium (ASP)-polyglutamate conjugates.

Figure 2

Glutamate production of 11b, 11c, 13b, and NAAG.

Figure 3

SPECT-CT images of 15a at 6 days (A) and of 15c at 24 h post-injection (B). PIP=PSMA⁺ PC3 tumors; flu=PSMA⁻ PC3 tumors.

Figure 4

HPLC trace of 11c.

Figure 5

Binding mode of 11c in the active site of PSMA.

Scheme I

Synthetic route to aminostrylpyridine moieties. Reagents and conditions: (a) K₂CO₃, DMF, 1-iodohexane (2a) or 1-iodo-dodecane (2b), (b) POCl₃, DMF, (c) 3-bromo-4-methylpyridine, LDA, Et₂O, (d) 3N-HCl, dioxane, 60 °C, (e) ethylacrylate, PPh₃, Pd(OAc)₂, TEA, DMF, 130 °C, (f) NaOH, H₂O/EtOH/i-PrOH.

Scheme II

Synthetic route for resin-bound polyglutamates. Reagents and conditions: (a) i: 20% piperidine in DMF, rt, 60 min, ii: Fmoc-Glu-OtBu, HATU, HOBt, DIPEA, DMF, (b) HATU, HOBt, DIPEA, DMF, 7a (for 10a), 7b (for 10b), or 7c (for 10c), (c) i: 4-iodobenzyl chloride, CH₃CN, ii: TFA/CH₂Cl₂, (d) 20% piperidine in DMF, rt, 60 min, ii: Fmoc-Glu(OtBu)-OH, HATU, HOBt, DIPEA, DMF.

Scheme III

Synthetic route to [¹²⁵I]15a and [¹²⁵I]15c. Reagents and conditions: (a) i: 4-((bromomethyl)phenyl)tributylstannane, CH₃CN, reflux, 24 h, (b) i: Na[¹²⁵I]I, N-chlorosuccinimide, methanol (0.1% acetic acid), rt, 2 h, ii: TFA/CH₂Cl₂ (1:1), rt, 90 min.