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Here we report mild, environmentally-friendly reaction conditions which enable the addition–elimination S N Ar reaction between weakly reactive substrates––an aminopyrrolopyrazole template and several substituted pyrimidines. The method was developed during our efforts to synthesize a series of novel P21-activated kinase (PAK) inhibitors.

Pin1 is a member of the cis-trans peptidyl-prolyl isomerase family with potential anti-cancer therapeutic value. Here we report structure-based de novo design and optimization of novel Pin1 inhibitors. Without a viable lead from internal screenings, we designed a series of novel Pin1 inhibitors by interrogating and exploring a protein crystal structure of Pin1. The ligand efficiency of the initial concept molecule was optimized with integrated SBDD and parallel chemistry approaches, resulting in a more attractive lead series.

Novel tricyclic benzimidazole carboxamide poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors have been synthesized. Several compounds were found to be powerful chemopotentiators of temozolomide and topotecan in both A549 and LoVo cell lines. In vitro inhibition of PARP-1 was confirmed by direct measurement of NAD+ depletion and ADP-ribose polymer formation caused by chemically induced DNA damage.

The design of potent Pin1 inhibitors has been challenging because its active site specifically recognizes a phospho-protein epitope. The de novo design of phosphate-based Pin1 inhibitors focusing on the phosphate recognition pocket and the successful replacement of the phosphate group with a carboxylate have been previously reported. The potency of the carboxylate series is now further improved through structure-based optimization of ligand–protein interactions in the proline binding site which exploits the H-bond interactions necessary for Pin1 catalytic function. Further optimization using a focused library approach led to the discovery of low nanomolar non-phosphate small molecular Pin1 inhibitors. Structural modifications designed to improve cell permeability resulted in Pin1 inhibitors with low micromolar anti-proliferative activities against cancer cells.

The P21-activated kinases (PAK) are emerging antitumor therapeutic targets. In this paper, we describe the discovery of potent PAK inhibitors guided by structure-based drug design. In addition, the efflux of the pyrrolopyrazole series was effectively reduced by applying multiple medicinal chemistry strategies, leading to a series of PAK inhibitors that are orally active in inhibiting tumor growth in vivo.

Despite abundant evidence that aberrant Rho-family GTPase activation contributes to most steps of cancer initiation and progression, there is a dearth of inhibitors of their effectors (e.g., p21-activated kinases). Through high-throughput screening and structure-based design, we identify PF-3758309, a potent (K d = 2.7 nM), ATP-competitive, pyrrolopyrazole inhibitor of PAK4. In cells, PF-3758309 inhibits phosphorylation of the PAK4 substrate GEF-H1 (IC 50 = 1.3 nM) and anchorage-independent growth of a panel of tumor cell lines (IC 50 = 4.7 ± 3 nM). The molecular underpinnings of PF-3758309 biological effects were characterized using an integration of traditional and emerging technologies. Crystallographic characterization of the PF-3758309/PAK4 complex defined determinants of potency and kinase selectivity. Global high-content cellular analysis confirms that PF-3758309 modulates known PAK4-dependent signaling nodes and identifies unexpected links to additional pathways (e.g., p53). In tumor models, PF-3758309 inhibits PAK4-dependent pathways in proteomic studies and regulates functional activities related to cell proliferation and survival. PF-3758309 blocks the growth of multiple human tumor xenografts, with a plasma EC 50 value of 0.4 nM in the most sensitive model. This study defines PAK4-related pathways, provides additional support for PAK4 as a therapeutic target with a unique combination of functions (apoptotic, cytoskeletal, cell-cycle), and identifies a potent, orally available small-molecule PAK inhibitor with significant promise for the treatment of human cancers.

The p21-activated kinase (PAK) family members are key effectors of Rho family GTPases, which act as regulatory switches that control such cellular processes as motility, proliferation, and cell survival. Some members of this family (such as Cdc42) have been shown to be required for Ras driven tumorigenesis. PAK4 is a key effector for Cdc42 and mediates downstream signals that control cell motility, proliferation and cell survival. The PAK family consists of PAK1,2,3 (‘group 1’) and PAK4,5,6 (‘group 2’). Both PAK4 and PAK1 have been shown to be required for Ras driven transformation. PAK4 has been shown to be oncogenic and able to drive anchorage independent growth when activated. PAK4 expression and activity is broadly up-regulated in solid tumors such as colon, ovarian and pancreatic cancers. Pfizer's PAK4 inhibitor program started with HTS of kinase focus library compounds. Multiple series were identified from the screening effort. Initial optimization mainly focused on kinase selectivity, ADME properties and ability to modulate target in vivo, leading to selection of pyrrolopyrazoles as the lead series. By hybridizing the pyrrolopyrazole core with aminopyrimidine series, we discovered potent PAK4 inhibitors with low to sub nM cellular activity. For the new hybrid series, the challenge was to attain good oral bioavailability. The observed poor absorption was likely due to high efflux nature of the template. Medicinal chemistry strategies, such as reducing molecular charge, lowering polar surface area and improving ligand efficiency, were applied to reduce efflux. As a result, two sub-series achieved excellent in vivo tumor growth inhibition when dosed orally. PF-03758309 has demonstrated excellent profile, leading to its selection as a clinical development candidate. PF-3758309 is a potent ATP-competitive inhibitor of PAK4 kinase domain (Kd = 4.5 nM). In engineered cell assays, PF-3758309 inhibited PAK4 dependent phosphorylation of its substrate GEF-H1 (IC50 = 1 nM). It potently inhibits the anchorage independent growth of HCT116 cells (IC50 = 0.24 nM). PF-3758309 exhibited broad anti-proliferative activity across a panel of 67 cell lines (CRC/pancreatic/NSCLC): 66% IC50 < 100 nM and 36% IC50 < 10 nM. In a human xenograft tumor model, PF-3758309 demonstrated a dose-dependent inhibition of both GEF-H1 and PAK4 phosphorylation. PF-3758309 is highly efficacious in human xenograft tumor models. Five of seven models tested showed robust tumor growth inhibition (>70%TGI at 15–20 mg/kg PO) by PF-3758309: HCT116, A549, MDAMB231, M24met, and Colo205. Broad kinase screening has demonstrated that this is a selective pan-PAK inhibitor with potential additional activities (e.g. AMPK). The pharmacodynamic and antitumor effects of PF-3758309 support its evaluation as an anticancer agent. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):PR-2.