Matthew A. Marx

Discovery of the highly potent PI3K/ mTOR dual inhibitor PF-04691502 through structure based drug design

The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays crucial roles in cell growth, proliferation and survival. Genomic aberrations in the PI3K pathway, such as mutational activation of PI3Kα or loss of function of tumor suppressor PTEN, have been closely linked to the development and progression of a wide range of cancers. Hence, inhibition of the key targets in the pathway, e.g. PI3K, AKT, mTOR, offers great potential for the treatment of cancer. Lead optimization through integration of structure based drug design (SBDD) and physical properties-based optimization (PPBO) led to the discovery of 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxypyridin-3-yl)-4-methylpyrido[2,3-d]pyrimidin-7(8H)-one (PF-04691502, 1) that demonstrated potent in vitro inhibitory activity against both PI3K and mTOR, excellent kinase selectivity, good ADMET, and robust in vivo efficacy in a mouse xenograft tumor growth model. Compound 1 is currently being evaluated in human clinical trials for the treatment of cancer.

Design of selective, ATP-competitive inhibitors of Akt.

This paper describes the design and synthesis of novel, ATP-competitive Akt inhibitors from an elaborated 3-aminopyrrolidine scaffold. Key findings include the discovery of an initial lead that was modestly selective and medicinal chemistry optimization of that lead to provide more selective analogues. Analysis of the data suggested that highly lipophilic analogues would likely suffer from poor overall properties. Central to the discussion is the concept of optimization of lipophilic efficiency and the ability to balance overall druglike propeties with the careful control of lipophilicity in the lead series. Discovery of the nonracemic amide series and subsequent modification produced an advanced analogue that performed well in advanced preclinical assays, including xenograft tumor growth inhibition studies, and this analogue was nominated for clinical development.

Discovery of the Highly Potent PI3K/mTOR Dual Inhibitor PF-04979064 through Structure-Based Drug Design.

PI3K, AKT, and mTOR are key kinases from PI3K signaling pathway being extensively pursued to treat a variety of cancers in oncology. To search for a structurally differentiated back-up candidate to PF-04691502, which is currently in phase I/II clinical trials for treating solid tumors, a lead optimization effort was carried out with a tricyclic imidazo[1,5]naphthyridine series. Integration of structure-based drug design and physical properties-based optimization yielded a potent and selective PI3K/mTOR dual kinase inhibitor PF-04979064. This manuscript discusses the lead optimization for the tricyclic series, which both improved the in vitro potency and addressed a number of ADMET issues including high metabolic clearance mediated by both P450 and aldehyde oxidase (AO), poor permeability, and poor solubility. An empirical scaling tool was developed to predict human clearance from in vitro human liver S9 assay data for tricyclic derivatives that were AO substrates.

4-methylpteridinones as orally active and selective PI3K/mTOR dual inhibitors.

Pteridinones were designed based on a non-selective kinase template. Because of the uniqueness of the PI3K and mTOR binding pockets, a methyl group was introduced to C-4 position of the peteridinone core to give compounds with excellent selectivity for PI3K and mTOR. This series of compounds were further optimized to improve their potency against PI3Kα and mTOR. Finally, orally active compounds with improved solubility and robust in vivo efficacy in tumor growth inhibition were identified as well.

Discovery of Novel, Potent, and Selective Inhibitors of 3-Phosphoinositide-Dependent Kinase (PDK1)

Analogues substituted with various amines at the 6-position of the pyrazine ring on (4-amino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)pyrazin-2-ylmethanone were discovered as potent and selective inhibitors of PDK1 with potential as anticancer agents. An early lead with 2-pyridine-3-ylethylamine as the pyrazine substituent showed moderate potency and selectivity. Structure-based drug design led to improved potency and selectivity against PI3Kα through a combination of cyclizing the ethylene spacer into a saturated, five-membered ring and substituting on the 4-position of the aryl ring with a fluorine. ADME properties were improved by lowering the lipophilicity with heteroatom replacements in the saturated, five-membered ring. The optimized analogues have a PDK1 Ki of 1 nM and >100-fold selectivity against PI3K/AKT-pathway kinases. The cellular potency of these analogues was assessed by the inhibition of AKT phosphorylation (T308) and by their antiproliferation activity against a number of tumor cell lines.

Highly Selective and Potent Thiophenes as PI3K Inhibitors with Oral Antitumor Activity

Highly selective PI3K inhibitors with subnanomolar PI3Kα potency and greater than 7000-fold selectivity against mTOR kinase were discovered through structure-based drug design (SBDD). These tetra-substituted thiophenes were also demonstrated to have good in vitro cellular potency and good in vivo oral antitumor activity in a mouse PI3K driven NCI-H1975 xenograft tumor model. Compounds with the desired human PK predictions and good in vitro ADMET properties were also identified. In this communication, we describe the rationale behind the installation of a critical triazole moiety to maintain the intricate H-bonding network within the PI3K receptor leading to both better potency and selectivity. Furthermore, optimization of the C-4 phenyl group was exploited to maximize the compounds mTOR selectivity.

Mitotic Checkpoint Kinase Mps1 Has a Role in Normal Physiology which Impacts Clinical Utility

Cell cycle checkpoint intervention is an effective therapeutic strategy for cancer when applied to patients predisposed to respond and the treatment is well-tolerated. A critical cell cycle process that could be targeted is the mitotic checkpoint (spindle assembly checkpoint) which governs the metaphase-to-anaphase transition and insures proper chromosomal segregation. The mitotic checkpoint kinase Mps1 was selected to explore whether enhancement in genomic instability is a viable therapeutic strategy. The basal-a subset of triple-negative breast cancer was chosen as a model system because it has a higher incidence of chromosomal instability and Mps1 expression is up-regulated. Depletion of Mps1 reduces tumor cell viability relative to normal cells. Highly selective, extremely potent Mps1 kinase inhibitors were created to investigate the roles of Mps1 catalytic activity in tumor cells and normal physiology (PF-7006, PF-3837; K i<0.5 nM; cellular IC50 2–6 nM). Treatment of tumor cells in vitro with PF-7006 modulates expected Mps1-dependent biology as demonstrated by molecular and phenotypic measures (reduced pHH3-Ser10 levels, shorter duration of mitosis, micro-nucleation, and apoptosis). Tumor-bearing mice treated with PF-7006 exhibit tumor growth inhibition concomitant with pharmacodynamic modulation of a downstream biomarker (pHH3-Ser10). Unfortunately, efficacy only occurs at drug exposures that cause dose-limiting body weight loss, gastrointestinal toxicities, and neutropenia. Mps1 inhibitor toxicities may be mitigated by inducing G1 cell cycle arrest in Rb1-competent cells with the cyclin-dependent kinase-4/6 inhibitor palbociclib. Using an isogenic cellular model system, PF-7006 is shown to be selectively cytotoxic to Rb1-deficient cells relative to Rb1-competent cells (also a measure of kinase selectivity). Human bone marrow cells pretreated with palbociclib have decreased PF-7006-dependent apoptosis relative to cells without palbociclib pretreatment. Collectively, this study raises a concern that single agent therapies inhibiting Mps1 will not be well-tolerated clinically but may be when combined with a selective CDK4/6 drug.

Discovery and synthesis of novel 4-aminopyrrolopyrimidine Tie-2 kinase inhibitors for the treatment of solid tumors.

The synthesis and biological evaluation of novel Tie-2 kinase inhibitors are presented. Based on the pyrrolopyrimidine chemotype, several new series are described, including the benzimidazole series by linking a benzimidazole to the C5-position of the 4-amino-pyrrolopyrimidine core and the ketophenyl series synthesized by incorporating a ketophenyl group to the C5-position. Medicinal chemistry efforts led to potent Tie-2 inhibitors. Compound 15, a ketophenyl pyrrolopyrimidine urea analog with improved physicochemical properties, demonstrated favorable in vitro attributes as well as dose responsive and robust oral tumor growth inhibition in animal models.

2-Morpholino-4-oxo-4,5-dihydro-thio-phene-3-carbonitrile.

The title compound, C9H10N2O2S, was obtained from the treatment of ethyl 4-cyano-3-hydroxy-5-morpholinothiophene-2-carboxylate with concentrated HCl. The mean plane of the essentially planar dihydrothiophene ring is almost orthogonal to the mirror plane of the N-morpholine substituent, making a dihedral angle of 87.2 (2)°.

Abstract 3484: In vivo activity of combined PI3k/mTOR and MEK-inhibition in a K-RASG12D; PTEN deletion mouse model of ovarian cancer

The PI3K/Akt pathway is commonly deregulated in human cancer, making it an attractive target for novel anti-cancer therapeutics. We have utilized a mouse model of ovarian cancer generated by K-RAS G12D activation and PTEN deletion in the ovarian surface epithelium (Dinulescu, et al, 2005) for the pre-clinical assessment of a novel PI3K/mTOR inhibitor (PF-04691502, Pfizer). To enable high throughput studies, we have transplanted primary tumours from these mice orthotopically into the ovarian bursa. Tumour growth rates were monitored by small animal-Ultrasound, and when tumours reached an average of 200mm 3 , mice received vehicle or 10mg/kg PF-04691502 daily for 7 days. Ultrasound and FDG-PET scans were performed on day 0, day 2 and day 7 of therapy. PF-04691502 inhibited tumour growth at 7 days by 71.64%±8.93. Tumour weights measured post-mortem following 7 days treatment also showed a significant decrease in tumour mass in treated mice (0.78±0.13g in vehicle treated versus 0.38±0.06g in drug treated mice; p PF-04691502 was sufficient to inhibit PI3K/mTOR resulting in inhibition of tumour growth; however, we did not observe tumour regression in this model. Notably, tumours from PF-04691502 treated mice displayed activation of pERK, suggesting that activation of the MAPK pathway is a mechanism by which KRAS-mutant tumours can limit the efficacy of PI3K/mTOR inhibitors alone. To overcome the effects of RAS/MAPK signalling, we have performed studies evaluating PF-04691502 (7.5mg/kg, daily) in combination with an inhibitor of MEK (PD-0325901, Pfizer, 10mg/kg, daily). Ultrasound monitoring of tumour volume shows that over a 7 day treatment period, PF-04691502 led to tumour growth inhibition of 55%±10.27, PD-0325901 alone led to tumour regression (36.3%±20.45) and combined PD-0325901 and PF-04691502 led to a striking tumour regression of 80.7%±4.97. These data show that combined inhibition of PI3K/mTOR and MEK converts tumor growth delay with PI3K-inhibition alone to tumour regression in this KRAS and PTEN mutant mouse model of ovarian cancer. Therefore inhibition of MEK may enhance activity of PI3K/mTOR-inhibitors in tumours showing activation of the PI3K pathway by genomic changes such as mutation in KRAS combined with loss of PTEN. Dinulescu D. M., et al. (2005) Nat. Med. 11:63-70. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3484.