Structural optimization of thiol-based inhibitors of glutamate carboxypeptidase II by modification of the P1' side chain.

A series of thiol-based inhibitors containing a benzyl moiety at the P1' position have been synthesized and tested for their abilities to inhibit glutamate carboxypeptidase II (GCP II). 3-(2-Carboxy-5-mercaptopentyl)benzoic acid 6c was found to be the most potent inhibitor with an IC(50) value of 15 nM, 6-fold more potent than 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA), a previously discovered, orally active GCP II inhibitor. Subsequent SAR studies have revealed that the phenoxy and phenylsulfanyl analogues of 6c, 3-(1-carboxy-4-mercaptobutoxy)benzoic acid 26a and 3-[(1-carboxy-4-mercaptobutyl)thio]benzoic acid 26b, also possess potent inhibitory activities toward GCP II. In the rat chronic constriction injury (CCI) model of neuropathic pain, compounds 6c and 26a significantly reduced hyperalgesia following oral administration (1.0 mg/kg/day).

High-affinity near-infrared fluorescent small-molecule contrast agents for in vivo imaging of prostate-specific membrane antigen.

Surgical resection remains a definitive treatment for prostate cancer. Yet, prostate cancer surgery is performed without image guidance for tumor margin, extension beyond the capsule and lymph node positivity, and without verification of other occult metastases in the surgical field. Recently, several imaging systems have been described that exploit near-infrared (NIR) fluorescent light for sensitive, real-time detection of disease pathology intraoperatively. In this study, we describe a high-affinity (9 nM), single nucleophile-containing, small molecule specific for the active site of the enzyme PSMA. We demonstrate production of a tetra-sulfonated heptamethine indocyanine NIR fluorescent derivative of this molecule using a high-yield LC/MS purification strategy. Interestingly, NIR fluorophore conjugation improves affinity over 20-fold, and we provide mechanistic insight into this observation. We describe the preparative production of enzymatically active PSMA using a baculovirus expression system and an adenovirus that co-expresses PSMA and GFP. We demonstrate sensitive and specific in vitro imaging of endogenous and ectopically expressed PSMA in human cells and in vivo imaging of xenograft tumors. We also discuss chemical strategies for improving performance even further. Taken together, this study describes nearly complete preclinical development of an optically based small-molecule contrast agent for image-guided surgery.

Enantiospecificity of glutamate carboxypeptidase II inhibition.

Two representative glutamate carboxypeptidase II (GCP II) inhibitors, 2-(hydroxypentafluorophenylmethyl-phosphinoylmethyl)pentanedioic acid 2 and 2-(3-mercaptopropyl)pentanedioic acid 3, were synthesized in high optical purities (>97%ee). The two enantiomers of 2 were prepared from previously reported chiral intermediates, (R)- and (S)-2-(hydroxyphosphinoylmethyl)pentanedioic acid benzyl esters 8. The synthesis of (R)- and (S)-3 involves the hydrolysis of (R)- and (S)-3-(2-oxo-tetrahydro-thiopyran-3-yl)propionic acids, (R)- and (S)-11, the corresponding optically pure thiolactones delivered by chiral chromatographic separation of the racemic 11. GCP II inhibitory assay revealed that (S)-2 is 40-fold more potent than (R)-2. In contrast, both enantiomers of 3 inhibited GCP II with nearly equal potency. The efficacy observed in subsequent animal studies with these enantiomers correlated well with the inhibitory potency in a GCP II assay.

Facile synthesis of alpha-substituted acrylate esters.

Treatment of 5-monosubstituted Meldrum's acids with dimethylmethyleneimmonium iodide (Eschenmoser's iodide salt) in methanol gives alpha-substituted acrylate methyl esters in good yields. Easy access to 5-monosubstituted Meldrum's acids allowed us to synthesize a wide variety of alpha-substituted acrylate methyl esters. The reaction conditions are mild and tolerate many functional groups commonly used in organic synthesis; thus, this new method has potential as an alternative to conventional preparative methods for alpha-substituted acrylate esters.