Elsevier

Bioorganic & Medicinal Chemistry

Volume 12, Issue 18, 15 September 2004, Pages 4969-4979
Bioorganic & Medicinal Chemistry

Probing for a hydrophobic a binding register in prostate-specific membrane antigen with phenylalkylphosphonamidates

https://doi.org/10.1016/j.bmc.2004.06.031Get rights and content

Abstract

To explore for the existence of an auxiliary hydrophobic binding register remote from the active site of PSMA a series of phenylalkylphosphonamidate derivatives of glutamic acid were synthesized and evaluated for their inhibitory potencies against PSMA. Both the phenyl- and benzylphosphonamidates (1a and 1b) exhibited only modest inhibitory potency against. The phenethyl analog 1c was intermediate in inhibitory potency while inhibitors possessing a longer alkyl tether from the phenyl ring, resulted in markedly improved Ki values. The greatest inhibitory potency was obtained for the inhibitors in which the phenyl ring was extended furthest from the central phosphorus (1f, n = 5 and 1g, n = 6). The slightly serrated pattern that emerged as the alkyl tether increased from three to six methylene units suggests that inhibitory potency is not simply correlated to increased hydrophobicity imparted by the phenylalkyl chain, but rather that one or more hydrophobic binding registers may exist remote from the substrate recognition architecture in the active site of PSMA.

Graphical abstract

A series of phenylalkylphosphonamidate derivatives of glutamic acid were synthesized and evaluated for their inhibitory potencies against PSMA. The greatest inhibitory potency was observed for the inhibitors 1f (n = 5) and 1g (n = 6) suggesting the presence of a hydrophobic binding register remote from the substrate recognition architecture in the active site of PSMA.

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Introduction

Prostate cancer remains the most common male malignancy and the second most common cause of cancer-related mortality in most Western societies.1 Its incidence is associated with age, family history, and life style factors,2 and varies strikingly among ethnic, racial, and national groups with noteworthy high rates of both incidence and mortality among African Americans.3 As basic research provides an ever-growing understanding of this disease, the strategies for preventing, diagnosing, and treating prostate cancer are being reshaped. One notable discovery has been the identification of an over-expressed membrane-bound cell surface protein on prostate cancer cells, namely, prostate-specific membrane antigen (PSMA). PSMA, also known as folate hydrolase I (FOLH1) and glutamate carboxypeptidase II (GCP II), is a 750-amino acid type II membrane glycoprotein4 and was discovered during the development of the LNCaP cell line; one which retains most of the known features of prostate cancer.5

Although PSMA is primarily expressed in normal human prostate epithelium, the importance of this enzyme lies with the fact that it is upregulated and strongly expressed in prostate cancer cells, including those of the metastatic disease state.6 Recent studies have demonstrated PSMA expression in the endothelium of tumor-associated neovasculature of multiple nonprostatic solid malignancies,7 including metastatic renal carcinoma.8 It is not surprising that PSMA has attracted a great deal of attention as a target for immunotherapy.9 In addition to its immunological importance, PSMA is also reported to possess three, yet poorly understood, enzymatic activities: the hydrolytic cleavage and liberation of glutamic acid from both γ-glutamyl derivatives of folic acid10 and the neuropeptide NAAG11 (N-acetylaspartylglutamate) as shown in Figure 1, and dipeptidyl peptidase IV activity.12 PSMA is highly homologous to NAALADase (N-acetylated alpha-linked L-amino dipeptidase) which is specifically characterized by its ability to hydrolyze the neuropeptide NAAG.13 However, in contrast to NAALADase, which has been extensively studied due to its presumed regulatory role in glutamate neurotransmission, questions of medical interest remain to be answered for PSMA, especially with regard to its role in prostate cancer. It is our hypothesis that these enzymatic activities of PSMA can be exploited for chemotherapeutic strategies such as the selective inhibition by small molecule inhibitors.

There is no crystal structure of PSMA as of yet, and thus the development of specific inhibitors must rely upon rational strategies to identify putative binding sites within or near the architecture of its active site. Although there have been a number of studies aimed at the acquisition of inhibitors of NAALADase,14 there have been far fewer studies on the inhibition of PSMA by rationally designed, synthetic, small-molecule inhibitors.15 The focus of this work was to identify the existence and relative location of hydrophobic binding site remote from the catalytic center of PSMA. To this end, we chose to prepare a series of phenylalkylphosphonamidate derivatives of glutamic acid (1ag) Figure 1. The glutamic acid residue was incorporated to provide a sufficient substrate-recognition element to these inhibitors while the phosphonyl center would bind as a tetrahedral-intermediate analog to the active-site zinc (II) ions. A phenyl ring was selected as a probe for a putative remote hydrophobic binding site and as such, was tethered by an increasing number of (0–6) methylene units from the central phosphorus.

Section snippets

Results and discussion

The series of phenylalkylphosphonamidate inhibitors (1ag) of PSMA were prepared as outlined in Scheme 1. Depending upon the length of the alkyl chain linking the phenyl probe to the central phosphorus, either dibenzyl methylphosphonate (2) or dibenzyl phosphite was selected as the nucleophile for alkylation to provide the phenylalkylphosphonate homologs 3. As such, Michaelis–Becker alkylation of dibenzyl phosphite with the readily available phenylalkyl bromides (Ph(CH2)n Br, n = 1, 3, or 5)

Conclusion

In summary, we have developed a convenient and sensitive HPLC-based assay for monitoring the enzymatic activity of PSMA using a novel synthetic γ-diglutamate derivative bearing a N-acyl chromophore. A comparison of the individual inhibitory potencies of phenylalkylphosphonamidate derivatives of glutamate supports the existence of a hydrophobic binding register remote from the catalytic cleft of PSMA. We expect that further elaboration of both the alkyl tether and the phenyl ring will improve

Synthesis

All solvents used in reactions and diisopropylethylamine (DIPEA) were both anhydrous and obtained as such from commercial sources. The HCl salt of glutamic acid dibenzyl ester [H-Glu(OBn)OBn] was neutralized by extraction with methylene chloride from a saturated aqueous solution of sodium bicarbonate. All other reagents were used as supplied unless otherwise stated. Liquid flash chromatography (silica or C18) was carried out using a Biotage 12i/40i system. 1H, 13C, and 31P NMR spectra were

Acknowledgements

This work was supported in part by grants from the National Institutes of Health, MBRS SCORE Program-NIGMS (Grant No. 2S06-GM052588-09), the National Cancer Institute, U56-Program (Grant No. CA 96217), and the NIH-PREP Scholarship Program (Grant No. 1 R25 GM64078-03). The authors would also like to extend their gratitude to W. Tam and the NMR facility at SFSU for their expert assistance.

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