2007;42(42):365. advantages over ATP-site targeting approaches, including distinct target selectivity and improved therapeutic profiles through inhibition of specific regulatory mechanisms.1C3 Akt (PKB) is usually one example of a kinase that has been targeted by several different small molecule approaches.4C8 Akt is an important regulator of cell growth, cell-cycle progression, transcription and metabolism, and is known to phosphorylate over 20 endogenous substrates.5,9C12 Many of SB 271046 Hydrochloride these substrates are intimately involved the induction of apoptosis and the arrest of cell proliferation, and are inactivated upon phosphorylation by Akt. Overall, enhanced Akt activity through increased expression, upstream amplification of PI3K, or loss of PTEN, its most important negative regulator, is usually observed in over 50% of all human solid tumors.13C17 Akt has thus emerged as a stylish target for the development of novel anticancer therapeutics.4,6,7,18C22 Most small molecules block Akt activity by direct inhibition of the ATP-binding site, interfering with cellular localization (via inhibition of the Pleckstrin Homology domain name), or through allosteric binding. Recently, mimics of the consensus substrate peptide of Akt have also emerged as lead compounds for further development. While achieving ligand complementarity in the relevant protein-protein conversation (PPI) region is usually expected to be more topochemically demanding, such inhibitors may also exhibit better selectivity relative to PH and ATP-binding domain name antagonists. Early work in this area focused polypeptides exhibiting IC50 values in the low to sub-micromolar range (~10C0.1).23C25 A co-crystal structure of Akt1 bound to a substrate peptide in the presence of an ATP-competitive inhibitor revealed that this peptide adopts a highly extended conformation in SB 271046 Hydrochloride the binding cleft.26 Efforts to reduce peptide character while maintaining the bioactive conformation have led to the identification of additional pseudosubstrate Akt1 inhibitors.27C31 Our group recently reported inhibitors of Akt1 based on a consensus sequence incorporating an azabicycloalkane dipeptide surrogate.30 Here, we describe the design and synthesis of a series of imidazopyridine-based peptidomimetics with enhanced potency and proteolytic stability. The undecapeptide Akt substrate GRPRTSSFAEG (Crosstide) was used as a lead structure and the central Thr7-Ser8 dipeptide was identified as a candidate site for conformational constraint (Physique 1). Open in a separate window Physique 1 Design of peptidomimetic Akt inhibitors The general synthesis of Akt substrate mimics is usually depicted in Scheme 1. The imidazo[1,2-a]pyridine (IP)-based dipeptide surrogate32 was prepared by bromination of -ketoester 1 and subsequent condensation with 2,3-diaminopyridine. Amidation of the IP N-terminus with guarded amino acids required stirring in the presence of EDC in DCM for 24C48 hr for optimal yields. The addition of auxiliary base or the use of other common coupling conditions (HBTU/HOBt, HATU, PyBOP, COMU, DEPBT) resulted in significantly lower conversion. The slow rate of amidation also precluded direct coupling to various N-protected arginine derivatives, all of which underwent intramolecular cyclization prior to reacting with the IP amine. In SB 271046 Hydrochloride contrast, 2 was efficiently coupled to Cbz-Orn(Boc)-OH, Cbz-Lys(Boc)-OH, and Cbz-Har(Boc)2-OH without any observable lactam formation. Arginine derivatives were prepared via Boc acidolysis and subsequent guanidinylation using Goodmans reagent to give SB 271046 Hydrochloride guarded tripeptide mimics 3b and 3d. Open in a separate window Scheme 1 Synthesis of imidazo[1,2- em a /em ]pyridine-based inhibitors. Incorporation of various C-terminal fragments was achieved by removal of the allyl ester protecting group and condensation with amino acid and dipeptide derivatives. Notably, the dipeptide amides used in the condensation reaction were efficiently prepared by simple aminolysis of the corresponding Bocprotected dipeptide methyl esters (see Supplementary Data). We found this procedure to be a convenient and racemization-free method to produce a variety of guarded peptide amides. After coupling to the IP-containing fragment, Boc group removal with TFA/DCM was followed by column chromatography to afford inhibitors 4C31. All compounds were assayed in vitro for their ability to inhibit the phosphorylation of Crosstide by Akt1 in the presence of 10 M 33P-labeled ATP (dose-response experiments were repeated 3 times, and IC50 values and 95% confidence intervals were calculated based on a variable slope four parameter model). As shown in Table 1, truncation of SB 271046 Hydrochloride the lead substrate down to tetrapeptide mimics 4C7 afforded compounds with no appreciable Akt1 inhibitory activity at 20 M. Pentapeptide mimic 8, which incorporates the native Ser9-Phe10 motif was also inactive in vitro. Alternative of Ser9 (native phosphorylation site) with the more hydrophobic Leu (9) or Phe (10) residues led to a dramatic increase in potency against Akt1. Optimal potency against Akt1 (IC50 = 0.64 M) was achieved with derivative 11, which incorporates a Val-Phe-NHBn C-terminal dipeptide subunit. Table 1 Structure-activity associations for compounds 4C31. thead th align=”center” colspan=”6″ rowspan=”1″ Open in a separate windows /th th align=”center” colspan=”6″ Rabbit Polyclonal to MUC7 valign=”bottom” rowspan=”1″ hr / /th th align=”left” valign=”middle” rowspan=”1″ colspan=”1″ Compound /th th align=”center” valign=”middle” rowspan=”1″ colspan=”1″ AA3 /th th align=”center” valign=”middle” rowspan=”1″ colspan=”1″ AA2 /th th align=”center” valign=”middle” rowspan=”1″ colspan=”1″ AA1 /th th align=”center” rowspan=”1″ colspan=”1″ Akt1 IC50 br / (M) /th th align=”center” rowspan=”1″ colspan=”1″ 95% CI br / (M) /th /thead 4Cbz-ArgVal-NHBn- 20-5Cbz-ArgVal-OBn- 20-6Cbz-ArgHyp(Bn)-OBn- 20-7Cbz-Argpip( em N /em -Bz)- 20-8Cbz-ArgSerPhe-NHBn 20-9Cbz-ArgLeuPhe-NHBn1.591.43.