Cynthia Louise Palmer

Discovery of (10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo[4,3-h][2,5,11]-benzoxadiazacyclotetradecine-3-carbonitrile (PF-06463922), a macrocyclic inhibitor of anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 (ROS1) with preclinical brain exposure and broad-spectrum potency against ALK-resistant mutations.

Although crizotinib demonstrates robust efficacy in anaplastic lymphoma kinase (ALK)-positive non-small-cell lung carcinoma patients, progression during treatment eventually develops. Resistant patient samples revealed a variety of point mutations in the kinase domain of ALK, including the L1196M gatekeeper mutation. In addition, some patients progress due to cancer metastasis in the brain. Using structure-based drug design, lipophilic efficiency, and physical-property-based optimization, highly potent macrocyclic ALK inhibitors were prepared with good absorption, distribution, metabolism, and excretion (ADME), low propensity for p-glycoprotein 1-mediated efflux, and good passive permeability. These structurally unusual macrocyclic inhibitors were potent against wild-type ALK and clinically reported ALK kinase domain mutations. Significant synthetic challenges were overcome, utilizing novel transformations to enable the use of these macrocycles in drug discovery paradigms. This work led to the discovery of 8k (PF-06463922), combining broad-spectrum potency, central nervous system ADME, and a high degree of kinase selectivity.

Design of Potent and Selective Inhibitors to Overcome Clinical Anaplastic Lymphoma Kinase Mutations Resistant to Crizotinib.

Crizotinib (1), an anaplastic lymphoma kinase (ALK) receptor tyrosine kinase inhibitor approved by the U.S. Food and Drug Administration in 2011, is efficacious in ALK and ROS positive patients. Under pressure of crizotinib treatment, point mutations arise in the kinase domain of ALK, resulting in resistance and progressive disease. The successful application of both structure-based and lipophilic-efficiency-focused drug design resulted in aminopyridine 8e, which was potent across a broad panel of engineered ALK mutant cell lines and showed suitable preclinical pharmacokinetics and robust tumor growth inhibition in a crizotinib-resistant cell line (H3122-L1196M).

Epithelial Tissue Hyperplasia Induced by the RAF Inhibitor PF-04880594 is Attenuated by a Clinically Well-Tolerated Dose of the MEK Inhibitor PD-0325901

Clinical trials of selective RAF inhibitors in patients with melanoma tumors harboring activated BRAFV600E have produced very promising results, and a RAF inhibitor has been approved for treatment of advanced melanoma. However, about a third of patients developed resectable skin tumors during the course of trials. This is likely related to observations that RAF inhibitors activate extracellular signal–regulated kinase (ERK) signaling, stimulate proliferation, and induce epithelial hyperplasia in preclinical models. Because these findings raise safety concerns about RAF inhibitor development, we further investigated the underlying mechanisms. We showed that the RAF inhibitor PF-04880594 induces ERK phosphorylation and RAF dimerization in those epithelial tissues that undergo hyperplasia. Hyperplasia and ERK hyperphosphorylation are prevented by treatment with the mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitor PD-0325901 at exposures that extrapolate to clinically well-tolerated doses. To facilitate mechanistic and toxicologic studies, we developed a three-dimensional cell culture model of epithelial layering that recapitulated the RAF inhibitor–induced hyperplasia and reversal by MEK inhibitor in vitro. We also showed that PF-04880594 stimulates production of the inflammatory cytokine interleukin 8 in HL-60 cells, suggesting a possible mechanism for the skin flushing observed in dogs. The complete inhibition of hyperplasia by MEK inhibitor in epithelial tissues does not seem to reduce RAF inhibitor efficacy and, in fact, allows doubling of the PF-04880594 dose without toxicity usually associated with such doses. These findings indicated that combination treatment with MEK inhibitors might greatly increase the safety and therapeutic index of RAF inhibitors for the treatment of melanoma and other cancers. Mol Cancer Ther; 11(10); 2274–83. ©2012 AACR.

Structural modifications of a 3-methoxy-2-aminopyridine compound to reduce potential for mutagenicity and time-dependent drug–drug interaction

(S)-1-((4-(3-(6-Amino-5-methoxypyridin-3-yl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-yl)amino)propan-2-ol, 1, was recently identified as a potent inhibitor of the oncogenic kinase bRAF. Compounds containing 3-methoxy-2-aminopyridine, as in 1, comprised a promising lead series because of their high ligand efficiency and excellent ADME profile. However, following metabolic oxidation, compounds in this series also demonstrated two significant safety risks: mutagenic potential and time-dependent drug-drug interaction (TDI). Metabolite identification studies revealed formation of a reactive metabolite. We hypothesized that minimizing or blocking the formation of such a metabolite would mitigate the safety liabilities. Our investigation demonstrated that structural modifications which either reduced the electron density of the 3-methoxy-2-aminopyridine ring or blocked the reactive site following metabolic oxidation were successful in reducing TDI and AMES mutagenicity.

Abstract PR10: Is CNS availability for oncology a no-brainer? Discovery of PF-06463922, a novel small molecule inhibitor of ALK/ROS1 with preclinical brain availability and broad spectrum potency against ALK-resistant mutations.

Oncogenic fusions of anaplastic lymphoma kinase (ALK) define a subset of human lung adenocarcinoma. The 1st generation ALK inhibitor crizotinib demonstrated impressive clinical benefit in ALK-fusion positive lung cancers and was approved by the FDA for the treatment of ALK-fusion positive NSCLC in 2011. However, as seen with most kinase inhibitors, patients treated with crizotinib eventually develop resistance to therapy. Acquired ALK kinase domain mutations and disease progression in the central nervous system (CNS) are reported as main contributors to patient relapse after ALK inhibitor therapy. Preclinically, crizotinib lacks significant brain penetration and does not potently inhibit activity of ALK kinase domain mutants, so a drug discovery program was initiated aimed to develop a second generation ALK inhibitor that is more potent than existing ALK inhibitors, capable of inhibiting the resistant ALK mutants and penetrating the blood-brain-barrier. These objectives present a considerable challenge in kinase inhibitor chemical space. Here we report that PF-06463922, a novel small molecule ATP-competitive inhibitor of ALK/ROS1, showed exquisite potencies against non-mutant ALK (Ki 100 fold kinase selectivity against 95% of the kinases tested in a 207 recombinant kinase panel. Specific design considerations were developed leading to novel ATP-competitive kinase inhibitors with desired low efflux in cell lines over-expressing p-glycoprotein and breast cancer resistance protein, providing excellent blood-brain-barrier and cell penetration properties. Efforts to optimize ligand efficiency and lipophilic efficiency leveraging structure based drug design techniques led to ligands with overlapping broad spectrum potency and low efflux. Single and repeat dose preclinical rat in vivo studies of PF-06463922 demonstrated excellent oral bioavailability and CNS availability with free brain exposure approximately 30% of free plasma levels. In addition, CNS-directed safety studies showed no adverse events at predicted efficacious concentrations. It is anticipated that PF-06463922 with its potent activities on non-mutant ALK, ALK kinase domain mutations and CNS metastases would provide great promise for patients with ALK and ROS1 positive cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):PR10. Citation Format: Ted W. Johnson, Simon Bailey, Benjamin J. Burke, Michael R. Collins, J. Jean Cui, Judy Deal, Ya-Li Deng, Martin P. Edwards, Mingying He, Jacqui Hoffman, Robert L. Hoffman, Qinhua Huang, Robert S. Kania, Phuong Le, Michele McTigue, Cynthia L. Palmer, Paul F. Richardson, Neal W. Sach, Graham L. Smith, Lars Engstrom, Wenyue Hu, Hieu Lam, Justine L. Lam, Tod Smeal, Helen Y. Zou. Is CNS availability for oncology a no-brainer? Discovery of PF-06463922, a novel small molecule inhibitor of ALK/ROS1 with preclinical brain availability and broad spectrum potency against ALK-resistant mutations. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PR10.

2-Chloro-4-(1H-pyrazol-1-yl)-5-(trifluoro-meth-yl)pyrimidine.

The reaction of 2,4-dichloro-5-(trifluoromethyl)pyrimidine with 1H-pyrazole gave two structural isomers in a 1:1 ratio that were separable by chromatography. The title compound, C8H4ClF3N4, was the first product to elute and was characterized in the present study to confirm that substitution by the pyrazolyl group had occurred at position 4. The molecule (with the exception of the F atoms) is essentially planar, with a mean deviation of 0.034 Å from the least-squares plane through all non-H and non-F atoms. The bond angles in the pyrimidine ring show a pronounced alternating pattern with three angles, including those at the two N atoms being narrower, and the remaining three wider than 120°.