In this work, we describe the synthesis, bioassay, and docking of ketones 1, Ac-pSerY[C = OCH]-L-pipecolyl (Pip)ryptamine, and rac-2, enantiomeric Ac-pSer-Y[C = OCH]-L-Pipryptamine and Ac-pSerY[C = OCH]-D-Pipryptamine. These inhibitors were designed as electrophilic acceptors of the Pin1 active site Cys113 thiol nucleophile to mimic the enzyme-bound tetrahedral intermediate (Figure 1C). On the other side of the coin, we have described reduced amides designed as twisted-amide transition-state analogues 3 and 4 (Figure 2) [27]. The evidence for a nucleophilic addition mechanism included the proximity of Cys113 to the substrate in the X-ray crystal structure, and the attenuation of activity for Pin1 mutants: 20-fold for C113S and 120-fold for C113A [26]. We anticipated that the ketones would be poor inhibitors, while the reduced amides, as twisted-amide analogues, would fare better. Indeed, the reduced amide 3 is a better Pin1 inhibitor than a similarly substituted substrate analogue (Z)-alkene isostere 5 (Figure 2) [13,27]. Our crystal structure of reduced amide 4 bound to the Pin1 catalytic site adopted a trans-pyrrolidine conformation, supporting the twisted-amide mechanism [27]. Ketones have been widely used as analogues of aldehydes or carboxylic acids to inhibit serine, cysteine [28,29], and aspartyl proteases [30,31]. Substrate-analogue ketones have not yet been developed as inhibitors of Pin1. Juglone is a ketone natural product that was shown to be a non-specific inhibitor of Pin1 through Michael addition to a surface Cys thiol of Pin1, resulting in unfolding [15]. Daum et al developed a series of aryl indanyl ketone inhibitors of Pin1; the best inhibitor had an IC50 value of 0.2 mM [11]. These inhibitors were reversible and cell penetrating, and they showed biological activities against p53 and b-catenin [11]. Daum et al proposed that the aryl indanyl ketones mimic the transition state of the twisted amide, based on the conformation in a crystal structure [11]. Figure 1. Ketone inhibitors were designed to mimic the tetrahedral intermediate of proposed mechanism B. (A) Proposed Pin1 hydrogen-bond assisted twisted amide mechanism [25], (B) Pin1 Cys113 nucleophilic-addition mechanism tetrahedral intermediate proposed by Ranganathan et al [26]. (C) Electrophilic ketone inhibitor designed to mimic the proposed tetrahedral intermediate upon Cys113-S nucleophilic addition. potential transition state analogue inhibitors of Pin1, but their weak inhibition could not be used support either the twisted-amide or the nucleophilic-addition mechanism (Figure 2) [14].
Results Design of Inhibitors
Ketone 1 was designed as a tetrahedral intermediate analogue, incorporating an electrophilic ketone to act as an acceptor for the Pin1 active site Cys113 thiol (Figure 1). Ketone 1 was designed based on substrate and peptide inhibitor specificities [12,32]. The stereoisomer obtained as a side product during synthesis, rac-2, was also tested for Pin1 inhibition because Wildeman et al. found that D-Thr containing peptide inhibitors were more potent than LThr [12]. The carbocyclic analogue of Pip, a cyclohexyl ring, was chosen based on the 100-fold improved inhibition of peptides with a Pip instead of a Pro residue [12,32]. Tryptamine was coupled to the C-terminus, since Pin1 binds large aromatic residues there [3,12,32]. An acetyl was used at the N-terminus because X-ray crystal structures of bound inhibitors showed no electron-density for residues on the N-terminal side of pSer [32,33]. The acetyl group also improved the water solubility of the inhibitors compared with Fmoc analogues for enzyme assays [13].Figure 2. Pin1 inhibitors discussed are cyclohexyl ketones 1 and rac-2 (this work); reduced amides 3 and 4 [27]; (Z)-alkene 5 [13]; and a-ketoamides 6a and 6b [14].
Synthesis
In the synthesis of ketones 1 and rac-2, addition of cyclohexenyl lithium to a Weinreb amide was used to form the ketone functionality (Figure 3). a,b-Unsaturated ketone 7 was obtained by deprotonation of Boc-Ser(Bn)-N(OMe)Me Weinreb amide with i-PrMgCl, followed by addition of cyclohexenyl lithium [34]. The lithium reagent was prepared in situ by treating 1-iodocyclohexene with s-BuLi [34,35]. The Boc group was then removed with TFA, and the amine formed was acetylated with acetic anhydride to give ketone 8 (Figure 3). Michael addition to form orthothioester 9 was accomplished with LiC(SMe)3, similar to a synthesis of (+)methylenolactocin [36]. We first attempted the Michael addition with Boc-protected a, b-unsaturated ketone 7, however a cyclic carbamate was formed as the major product instead of the desired orthothioester. We have used similar cyclic carbamates in stereochemical proofs [34]. The carbamate ring-closure cannot occur with the acetyl amide. After Michael addition, two major diastereomers of the orthothioester were obtained as a mixture; a minor diastereomer was removed during chromatography. Hydrolysis of orthothioester 9 in a mixture of THF and H2O with BF3?Et2O and HgO gave a mixture of diastereomeric carboxylic acids 10 [36]. Without further purification, acids 10 were coupled to tryptamine with EDC to generate the ketone diastereomeric mixture of (1S,3R,4R)-11 and rac-11, which were separated by silica flash chromatography (Figure 3). The two diastereomers were carried on separately to the final compounds 1 (Figure 3), and rac-2. The major diastereomer (1S,3R,4R)-11 was treated with BCl3 to remove the benzyl group and form alcohol (1S,3R,4R)-12 [37,38]. Phosphorylation with dibenzylphosphoramidite gave dibenzyl phosphate (1S,3R,4R)13 [10,39].
