Conformational Restriction of Flexible Ligands Guided by the Transferred NOE Experiment:  Potent Macrocyclic Inhibitors of Farnesyltransferase

Abstract

The optimization of enzyme inhibitor potency and specificity is an important goal of drug design since both properties contribute to clinical efficacy and safety. Restricting an inhibitor's conformation to one recognized by the enzyme increases potency by lowering the entropic barrier to complex formation, and could potentially enhance specificity by limiting its interactions with other macromolecules. 1 In lieu of detailed structural characterization of enzyme-inhibitor interactions, the transferred nuclear Overhauser effect (trNOE) NMR method has proven valuable in defining conformations of ligands weakly associated with macromolecules. 2 However, despite the considerable implications of the trNOE technique for drug design, there are few instances of the method playing an influential role in inhibitor optimization, 3 and none is directed at designing specific conformational constraints. This report describes the design of a highly potent macrocyclic enzyme inhibitor based on the trNOE structure of a conformationally flexible analogue. Farnesyltransferase (FTase) is an important posttranslational processing enzyme that prenylates proteins using farnesylpyrophosphate (FPP) and enables the participation by some in signal transduction during cell proliferation. 4 Inhibitors of this enzyme (FTIs) are promising antitumor agents, and several are currently being evaluated in human clinical trials. 5 In our investigations of structure-activity relationships of the clinical candidate 1, 6 we found that the related FTI 2, with diminished inhibitory activity (FTase IC 50 475 nM vs 2 nM), was an appropriate ligand for the trNOE experiment.In the absence of added enzyme, NMR spectroscopic evaluation of 2 reveals no defined solution conformation. NMR-derived intramolecular distance constraint data was generated in the presence of the putative FTase‚FPP complex. Ligand-competition experiments with a potent peptidomimetic FTI served to disqualify non-active-site bound contributions. The calculated lowest-energy structures depict folded conformations with the cyanophenyl group flanking the piperazinone ring (Figure 1). 7a Stabilization of this orientation by covalent linkage of the cyanophenyl and piperazinone N-aryl substituent in a macrocycle appeared to be an attractive approach to optimize the properties of 1.The synthesis of a macrocyclic version of 1 is described in Scheme 1. The piperazinone 8, prepared by a Mitsunobu cyclodehydration reaction, 8 was reductively coupled with aldehyde 5 to give 9. Compound 9 was subjected to a tandem basepromoted arylmethanesulfonate deprotection and S N Ar cyclization 9 to give the cyclophane 10 in good yield. Interestingly, 10 exhibits planar chirality, and its enantiomeric conformers are readily resolved by chiral HPLC, due to a sufficient activation energy for their interconversion. 10 The calculated lowest-energy structure of (+)-10 7c (Figure 2, gray) bears close resemblance to available FTase-bound conformations of 2 (Figure 1), especially with regard to the relative positions of ...

2D Autocorrelation modeling of the activity of trihalobenzocycloheptapyridine analogues as farnesyl protein transferase inhibitors

“…However, the peptidic nature or the presence of a free thiol group in these FPT inhibitors may have disadvantages in the development of such compounds as therapeutic agents. In recent years the number of non-peptidic, non-thiolcontaining selective FPT inhibitors is increased [7][8][9][10].…”

Farnesyltransferase inhibitors: recent advances

“…The macrocyclic series was designed based on transferred nuclear Overhauser effect nuclear magnetic resonance (NMR) studies of L-778123 analogues, which indicated that the cyanobenzyl group on the imidazole and the N-aryl moiety are in close proximity [54]. This macrocyclic compound 23 exhibited an enhanced FT IC 50 value of 0.1 nM.…”

“…Moreover, the conformational constraint altered the GGT-I mechanism of inhibition of these compounds. L-778123 is competitive with geranylgeranyl pyrophosphate, whereas compound 23 is competitive with the K-Ras-derived peptide substrate [54]. In another series, the macrocyclic ring was formed between the benzyl imidazole and a substitutent on the C-3 carbon of the piperazinone core [150], with exemplified compound 24 showing FT activity of < 10 µM.…”

Inhibition of farnesyltransferase: A rational approach to treat cancer?

“…This was designed to simultaneously ligate the active site zinc ion and occupy a nearby hydrophobic binding site, which resulted in a potent peptidomimetic FTI like 7 (FTase IC 50 ¼ 0.15 nM) [45]. Fusion of the non-peptide 8 with the thiol replacement in 7 and further optimization of the piperazine template led to piperazinone FTIs, which displayed significantly improved cell based activity compared with the earlier peptidomimetic FTIs [46] [47]. Members of this class of FTIs exhibited the highest cell potency yet described for inhibitors of FTase.…”