Daniel T. Richter

Antitumor Activity and Pharmacology of a Selective Focal Adhesion Kinase Inhibitor, PF-562,271

Cancer cells are characterized by the ability to grow in an anchorage-independent manner. The activity of the nonreceptor tyrosine kinase, focal adhesion kinase (FAK), is thought to contribute to this phenotype. FAK localizes in focal adhesion plaques and has a role as a scaffolding and signaling protein for other adhesion molecules. Recent studies show a strong correlation between increased FAK expression and phosphorylation status and the invasive phenotype of aggressive human tumors. PF-562,271 is a potent, ATP-competitive, reversible inhibitor of FAK and Pyk2 catalytic activity with a IC(50) of 1.5 and 14 nmol/L, respectively. Additionally, PF-562,271 displayed robust inhibition in an inducible cell-based assay measuring phospho-FAK with an IC(50) of 5 nmol/L. PF-562,271 was evaluated against multiple kinases and displays >100x selectivity against a long list of nontarget kinases. PF-562,271 inhibits FAK phosphorylation in vivo in a dose-dependent fashion (calculated EC(50) of 93 ng/mL, total) after p.o. administration to tumor-bearing mice. In vivo inhibition of FAK phosphorylation (>50%) was sustained for >4 hours with a single p.o. dose of 33 mg/kg. Antitumor efficacy and regressions were observed in multiple human s.c. xenograft models. No weight loss, morbidity, or mortality were observed in any in vivo experiment. Tumor growth inhibition was dose and drug exposure dependent. Taken together, these data show that kinase inhibition with an ATP-competitive small molecule inhibitor of FAK decreases the phospho-status in vivo, resulting in robust antitumor activity.

Characterization of PF-4708671, a novel and highly specific inhibitor of p70 ribosomal S6 kinase (S6K1).

S6K1 (p70 ribosomal S6 kinase 1) is activated by insulin and growth factors via the PI3K (phosphoinositide 3-kinase) and mTOR (mammalian target of rapamycin) signalling pathways. S6K1 regulates numerous processes, such as protein synthesis, growth, proliferation and longevity, and its inhibition has been proposed as a strategy for the treatment of cancer and insulin resistance. In the present paper we describe a novel cell-permeable inhibitor of S6K1, PF-4708671, which specifically inhibits the S6K1 isoform with a Ki of 20 nM and IC50 of 160 nM. PF-4708671 prevents the S6K1-mediated phosphorylation of S6 protein in response to IGF-1 (insulin-like growth factor 1), while having no effect upon the PMA-induced phosphorylation of substrates of the highly related RSK (p90 ribosomal S6 kinase) and MSK (mitogen- and stress-activated kinase) kinases. PF-4708671 was also found to induce phosphorylation of the T-loop and hydrophobic motif of S6K1, an effect that is dependent upon mTORC1 (mTOR complex 1). PF-4708671 is the first S6K1-specific inhibitor to be reported and will be a useful tool for delineating S6K1-specific roles downstream of mTOR.

Trifluoromethylpyrimidine-based inhibitors of proline-rich tyrosine kinase 2 (PYK2): structure-activity relationships and strategies for the elimination of reactive metabolite formation.

The synthesis and SAR for a series of diaminopyrimidines as PYK2 inhibitors are described. Using a combination of library and traditional medicinal chemistry techniques, a FAK-selective chemical series was transformed into compounds possessing good PYK2 potency and 10- to 20-fold selectivity against FAK. Subsequent studies found that the majority of the compounds were positive in a reactive metabolite assay, an indicator for potential toxicological liabilities. Based on the proposed mechanism for bioactivation, as well as a combination of structure-based drug design and traditional medicinal chemistry techniques, a follow-up series of PYK2 inhibitors was identified that maintained PYK2 potency, FAK selectivity and HLM stability, yet were negative in the RM assay.

Synthesis of Aryl Ethers via a Sulfonyl Transfer Reaction

A general synthesis of aryl ethers from primary and secondary alcohols and aryl mesylates is presented. The reaction proceeds via a sulfonyl-transfer mechanism. In this paper, we compare the sulfonyl transfer reaction to Mitsunobu ether formation. The reaction can be employed in a multistep synthesis where the aryl mesylate is used as a phenol protecting group and then as an activating group for ether formation. This protecting/activating group strategy is demonstrated using raloxifene as the target.

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.

Achieving selectivity between highly homologous tyrosine kinases: a novel selective erbB2 inhibitor

The discovery of small molecule kinase inhibitors for use as drugs is a promising approach for the treatment of cancer and other diseases, but the discovery of highly specific agents is challenging because over 850 kinases are expressed in mammalian cells. Systematic modification of the 4-anilino functionality of a selective quinazoline inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase can invert selectivity to favor inhibition of the highly homologous erbB2 tyrosine kinase. The selectivity pattern was demonstrated in assays of recombinant kinases and recapitulated in measures of kinase activity in intact cells. The most potent and selective erbB2 inhibitor of the analog series has anti-proliferative activity against an erbB2-overexpressing cell line that was lacking in the original EGFR-selective compound. Subtle changes to the molecular structure of ATP-competitive small molecule inhibitors of tyrosine kinases can yield dramatic changes in potency and selectivity. These results suggest that the discovery of highly selective small molecule inhibitors of very homologous kinases is achievable.

Sustainable chromatography (an oxymoron

Chromatography is routinely used in drug discovery as a means to isolate intermediates and final compounds. From a sustainability perspective, it is one of the largest contributors of solvent waste in the drug discovery process. The medicinal chemistry subgroup within the American Chemical Societys Green Chemistry Institute Pharmaceutical Roundtable (ACS GCI PR) offers a perspective aimed at providing chemists with practical tools and easily implemented techniques to improve the sustainability of drug discovery through reduction of the waste generated during chromatography. This perspective also offers alternatives to traditional, silica gel-based chromatography as well as information on how to avoid chromatography completely through use of crystallization and reaction telescoping.

Abstract A86: Design, synthesis, and SAR of focal adhesion kinase (FAK) inhibitors

Focal adhesion kinase (FAK) is a non‐tyrosine kinase that localizes to focal adhesion plaques. It is activated in response to intergin binding to cellular ligands and when phosphorylated inhibits anoikis allowing for anchorage independent cell growth. Recent studies have shown increased FAK expression and phosphorylation status in many types of invasive and aggressive human tumors strongly suggesting FAK is a possible target for anticancer chemotherapy. Literature, in house HTS and de novo studies identified 2, 4‐diaminopyrimidines as potent FAK inhibitors. Early SAR efforts quickly determined that smaller substituents, particularly CF3, were optimal in the C5 position. Parallel medicinal chemistry strategies were executed for the C2 and C4 positions. These studies suggested that substituted aryl and fused heteroaryl groups at the C2 position in conjunction with substituted phenyl and heterocycles at the C4 position imparted optimum activity and metabolic stability. Inhibitor‐FAK co‐crystal structures were utilized to guide in the SAR strategy around the 2, 4‐diaminopyrimidine template which afforded several lead compounds. The team9s effort culminated in the advancement of PF‐562,271 as a potent and reversible inhibitor of FAK (kinase IC50 of 2 nM and cell IC50 of 5 nM) that is > 100x selective against a long list of non‐target kinases. In summary, detailed SAR studies were executed on the 2, 4‐diaminopyrimidine templates that produced potent inhibitors of FAK with improved ADME properties, and identified a novel and potent series of FAK inhibitors that are selective against most other kinases and have shown activity in clinical trials. This poster will present design, synthesis, challenging chemistry, optimization, and complete inhibitor chemical structures of lead analogs. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A86.

Abstract 753: Novel, potent and selective small molecule inhibitors of 3-phosphoinositide-dependent kinase (PDK1)

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a crucial role in cell growth, proliferation and survival. Genomic aberrations in the PI3K pathway, such as mutational activation of PI3Kα or loss-of-function of the tumor suppressor PTEN, have been closely linked to the development and progression of a wide range of cancers. Inhibition of the key targets in the pathway, PI3K, AKT, mTOR & PDK1, may provide an effective treatment of cancer. In an effort to discover compounds that inhibit PDK1, we have developed a series of 3-Carbonyl-4-Amino-Pyrrolopyrimidine (CAP) compounds that are selective and potent PDK1 inhibitors. Early screening led to a viable starting point, PF-03772304, (4-amino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-(6-methylamino-pyrazin-2-yl)-methanone, which has an IC50 of 94 nM for PDK1 and a ligand efficiency of 0.42. While potent, this lead was not selective against PI3K. Using structure-based drug design, this lead was modified to expand into the selectivity pocket of PDK1 (under the G-Loop), leading to the identification of a potent and pathway-selective compound, PF-05017255 ((4-Amino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-{6-[(3S,4R)-4-(4-fluoro-phenyl)-tetrahydro-furan-3-ylamino]-pyrazin-2-yl}-methanone). PF-05017255 has a Ki of 0.6 nM for PDK1 and is more than 400-fold selective against other PI3K pathway kinases: PI3Kα, AKT, S6K and mTOR. For even greater kinase selectivity, we sought to lower the clogP of our lead (clogP for PF-05017255 is 3.0) to reduce the contribution from the hydrophobic effect. These efforts led to PF-05168899 (1-{(2R,3R)-3-[6-(4-Amino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonyl)-pyrazin-2-ylamino]-2-phenyl-pyrrolidin-1-yl}-ethanone) with a Ki of 0.4 nM for PDK1, a clogP of 2.1, and greater than 1000-fold selectivity against PI3Kα, AKT, S6K, mTOR, CDK2, CHK1 and PAK4. PF-05168899 also showed little inhibitory effect (<50% at 1 uM) against 33 of 35 kinases in a broader panel, demonstrating significant inhibition only against CHK2 (94%) and AuroraB (54%). In addition, the most potent analogs (e.g. PF-05168889) inhibited the phosphorylation of AKT at the residue threonine 308 (IC50 40-200 nM) in a variety of cancer cell lines (e.g. H460, A549). The design, synthesis and SAR of this chemical series will be described. 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 753.

5-Chloro-N-[2-(1H-imidazol-4-yl)eth­yl]-N-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine

The title compound, C12H13ClN6, was prepared by reaction of 4,5-dichloro-7H-pyrrolo[2,3-d]pyrimidine with 2-(1H-imidazol-4-yl)-N-methylethanamine, and the X-ray study confirmed that chloro-substituent in six-membered ring was replaced in the reaction. The exocyclic N atom environment is approximately coplanar with the pyrrolo[2,3-d]pyrimidine [corresponding dihedral angle is 5.5 (1)°], whereas the mean plane of the N—C—C—C link connecting with the imidazolyl ring is almost exactly orthogonal to the plane of the bicyclic system [dihedral angle = 91.6 (2)°]. The imidazolyl plane itself, however, forms a relatively small dihedral angle of 20.8 (1)° with the pyrrolo[2,3-d]pyrimidine plane. There are two independent N—H⋯N hydrogen bonds in the structure, which link molecules into layers parallel to (03).