Structure-Based Design and Synthesis of Potent and Selective KRAS G12D Inhibitors.

KRAS G12D mutation has been found in approximately 45% of pancreatic ductal adenocarcinoma (PDAC) cases, making it an attractive therapeutic target. Through structure-based drug design, a series of potent and selective KRAS G12D inhibitors were designed. The lead compound, ERAS-5024, inhibited ERK1/2 phosphorylation and cell proliferation in three-dimensional Cell-Titer Glo assays in AsPC-1 PDAC cells with single-digit nanomolar potency and caused tumor regression in the in vivo efficacy studies. We describe here the details of the design and synthesis program that led to the discovery of ERAS-5024.

The pharmacologic and toxicologic characterization of the potent and selective KRAS G12D inhibitors ERAS-4693 and ERAS-5024.

Two potent and selective KRASG12D inhibitors, ERAS-4693 and ERAS-5024, were generated as possible clinical candidates to treat patients harboring G12D mutations in solid tumors. Both molecules exhibited strong anti-tumor activity in the KRASG12D mutant PDAC xenograft mouse models while ERAS-5024 also showed tumor growth inhibition when administered on an intermittent dosing regimen. Acute dose-limiting toxicity consistent with an allergic reaction was observed for both molecules shortly after administration at doses just above those which demonstrated anti-tumor activity, indicative of a narrow therapeutic index. A series of studies were subsequently conducted to identify a common underlying mechanism for the observed toxicity, including CETSA® (Cellular Thermal Shift Assay) as well as several functional off-target screens. Both ERAS-4693 and ERAS-5024 were identified to agonize MRGPRX2 which has been linked to pseudo-allergic reactions. In vivo toxicologic characterization of both molecules included repeat-dose studies in the rat and dog. Dose-limiting toxicities were observed in both species with ERAS-4693 and ERAS-5024 and plasma exposure levels at the maximum tolerated doses were generally below that which caused strong anti-tumor activity, supporting the initial observation of a narrow therapeutic index. Additional overlapping toxicities included a reduction in reticulocytes and clinical pathological changes suggestive of an inflammatory response. Furthermore, increases in plasma histamine were observed in dogs administered ERAS-5024, supporting the hypothesis that MRGPRX2 agonism may be the cause of the pseudo-allergic reaction. This work highlights the importance of balancing both the safety and efficacy of KRASG12D inhibitors as this class of molecules begins to enter clinical development.

Structure-Based Drug Design and Synthesis of PI3Kα-Selective Inhibitor (PF-06843195).

The phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway is a frequently dysregulated pathway in human cancer, and PI3Kα is one of the most frequently mutated kinases in human cancer. A PI3Kα-selective inhibitor may provide the opportunity to spare patients the side effects associated with broader inhibition of the class I PI3K family. Here, we describe our efforts to discover a PI3Kα-selective inhibitor by applying structure-based drug design (SBDD) and computational analysis. A novel series of compounds, exemplified by 2,2-difluoroethyl (3S)-3-{[2'-amino-5-fluoro-2-(morpholin-4-yl)-4,5'-bipyrimidin-6-yl]amino}-3-(hydroxymethyl)pyrrolidine-1-carboxylate (1) (PF-06843195), with high PI3Kα potency and unique PI3K isoform and mTOR selectivity were discovered. We describe here the details of the design and synthesis program that lead to the discovery of 1.

Precedence and Promise of Covalent Inhibitors of EGFR and KRAS for Patients with Non-Small-Cell Lung Cancer.

Epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog (KRAS) oncogenic mutations are leading causes for lung cancer. Extensive drug discovery efforts targeting EGFR have led to the discovery and FDA approval of both reversible and covalent inhibitors. Second and third generation covalent inhibitors for EGFR have also been described, with the latter targeting specific emerging mutations. After decades of extensive effort, KRAS is widely regarded as an intractable therapeutic target; however, recent publications suggest covalent inhibition is a promising strategy to deliver inhibitors of the KRASG12C mutation.

Discovery of N-((3R,4R)-4-Fluoro-1-(6-((3-methoxy-1-methyl-1H-pyrazol-4-yl)amino)-9-methyl-9H-purin-2-yl)pyrrolidine-3-yl)acrylamide (PF-06747775) through Structure-Based Drug Design: A High Affinity Irreversible Inhibitor Targeting Oncogenic EGFR Mutants with Selectivity over Wild-Type EGFR.

Mutant epidermal growth factor receptor (EGFR) is a major driver of non-small-cell lung cancer (NSCLC). Marketed first generation inhibitors, such as erlotinib, effect a transient beneficial response in EGFR mutant NSCLC patients before resistance mechanisms render these inhibitors ineffective. Secondary oncogenic EGFR mutations account for approximately 50% of relapses, the most common being the gatekeeper T790M substitution that renders existing therapies ineffective. The discovery of PF-06459988 (1), an irreversible pyrrolopyrimidine inhibitor of EGFR T790M mutants, was recently disclosed.1 Herein, we describe our continued efforts to achieve potency across EGFR oncogenic mutations and improved kinome selectivity, resulting in the discovery of clinical candidate PF-06747775 (21), which provides potent EGFR activity against the four common mutants (exon 19 deletion (Del), L858R, and double mutants T790M/L858R and T790M/Del), selectivity over wild-type EGFR, and desirable ADME properties. Compound 21 is currently being evaluated in phase-I clinical trials of mutant EGFR driven NSCLC.

Recent progress on third generation covalent EGFR inhibitors.

First generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (gefitinib and erlotinib) demonstrate excellent clinical efficacy for NSCLC patients carrying EGFR oncogenic mutations (L858R, del exon 19 deletions between amino acids 746 and 750). Invariable, drug resistance occurs with around 60% of it driven by the EGFR-T790M gatekeeper mutation. To counter the T790M-dependent resistance, third generation covalent EGFR inhibitors have been developed with high potency toward T790M containing mutants and selectivity over WT EGFR. This review provides an overview of the third generation drugs currently in clinical trials and also encompasses novel methodologies developed to discover third generation covalent EGFR drugs.

Discovery of 1-{(3R,4R)-3-[({5-Chloro-2-[(1-methyl-1H-pyrazol-4-yl)amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl}prop-2-en-1-one (PF-06459988), a Potent, WT Sparing, Irreversible Inhibitor of T790M-Containing EGFR Mutants.

First generation EGFR TKIs (gefitinib, erlotinib) provide significant clinical benefit for NSCLC cancer patients with oncogenic EGFR mutations. Ultimately, these patients' disease progresses, often driven by a second-site mutation in the EGFR kinase domain (T790M). Another liability of the first generation drugs is severe adverse events driven by inhibition of WT EGFR. As such, our goal was to develop a highly potent irreversible inhibitor with the largest selectivity ratio between the drug-resistant double mutants (L858R/T790M, Del/T790M) and WT EGFR. A unique approach to develop covalent inhibitors, optimization of reversible binding affinity, served as a cornerstone of this effort. PF-06459988 was discovered as a novel, third generation irreversible inhibitor, which demonstrates (i) high potency and specificity to the T790M-containing double mutant EGFRs, (ii) minimal intrinsic chemical reactivity of the electrophilic warhead, (iii) greatly reduced proteome reactivity relative to earlier irreversible EGFR inhibitors, and (iv) minimal activity against WT EGFR.

Structure-based design, SAR analysis and antitumor activity of PI3K/mTOR dual inhibitors from 4-methylpyridopyrimidinone series.

PI3K, AKT and mTOR, key kinases from a frequently dysregulated PI3K signaling pathway, have been extensively pursued to treat a variety of cancers in oncology. Clinical trials of PF-04691502, a highly potent and selective ATP competitive kinase inhibitor of class 1 PI3Ks and mTOR, from 4-methylpyridopyrimidinone series, led to the discovery of a metabolite with a terminal carboxylic acid, PF-06465603. This paper discusses structure-based drug design, SAR and antitumor activity of the MPP derivatives with a terminal alcohol, a carboxylic acid or a carboxyl amide.

N-(Pyridin-2-yl) arylsulfonamide inhibitors of 11ß-hydroxysteroid dehydrogenase type 1: strategies to eliminate reactive metabolites.

N-(Pyridin-2-yl) arylsulfonamides 1 and 2 (PF-915275) were identified as potent inhibitors of 11ß-hydroxysteroid dehydrogenase type 1. A screen for bioactivation revealed that these compounds formed glutathione conjugates. This communication presents the results of a risk benefit analysis carried out to progress 2 (PF-915275) to a clinical study and the strategies used to eliminate reactive metabolites in this series of inhibitors. Based on the proposed mechanism of bioactivation and structure-activity relationships, design efforts led to N-(pyridin-2-yl) arylsulfonamides such as 18 and 20 that maintained potent 11ß-hydroxysteroid dehydrogenase type 1 activity, showed exquisite pharmacokinetic profiles, and were negative in the reactive metabolite assay.

Targeting the mTOR pathway in tumor malignancy.

The mammalian target of rapamycin (mTOR) plays a critical role in the regulation of cell growth, proliferation,and metabolism by integrating growth factor stimulation and energy/nutrient input through a complex signaling network.The mTOR kinase is a part of two structurally and functionally distinct multiple protein complexes, mTORC1 and mTORC2. The mammalian target of rapamycin complex 1 (mTORC1) is rapamycin-sensitive and mediates temporal control of cell growth by regulating several cellular processes, such as translation, transcription, and nutrient transport while the mammalian target of rapamycin complex 2 (mTORC2) is in sensitive to rapamycin and is involved in spatial control of cell growth via cytoskeleton regulation. Here we discuss the mechanism of mTOR regulation in tumor malignancy through a variety of signaling pathways and the potential of mTOR inhibitors for the treatment of cancer.

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.

Discovery of a novel class of exquisitely selective mesenchymal-epithelial transition factor (c-MET) protein kinase inhibitors and identification of the clinical candidate 2-(4-(1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)ethanol (PF-04217903) for the treatment of cancer.

The c-MET receptor tyrosine kinase is an attractive oncology target because of its critical role in human oncogenesis and tumor progression. An oxindole hydrazide hit 6 was identified during a c-MET HTS campaign and subsequently demonstrated to have an unusual degree of selectivity against a broad array of other kinases. The cocrystal structure of the related oxindole hydrazide c-MET inhibitor 10 with a nonphosphorylated c-MET kinase domain revealed a unique binding mode associated with the exquisite selectivity profile. The chemically labile oxindole hydrazide scaffold was replaced with a chemically and metabolically stable triazolopyrazine scaffold using structure based drug design. Medicinal chemistry lead optimization produced 2-(4-(1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)ethanol (2, PF-04217903), an extremely potent and exquisitely selective c-MET inhibitor. 2 demonstrated effective tumor growth inhibition in c-MET dependent tumor models with good oral PK properties and an acceptable safety profile in preclinical studies. 2 progressed to clinical evaluation in a Phase I oncology setting.

Design and synthesis of a novel pyrrolidinyl pyrido pyrimidinone derivative as a potent inhibitor of PI3Kα and mTOR.

Lead optimization efforts that employed structure base drug design and physicochemical property based optimization leading to the discovery of a novel series of 4-methylpyrido pyrimidinone (MPP) are discussed. Synthesis and profile of 1, a PI3Kα/mTOR dual inhibitor, is highlighted.

PF-04691502, a potent and selective oral inhibitor of PI3K and mTOR kinases with antitumor activity.

Deregulation of the phosphoinositide 3-kinase (PI3K) signaling pathway such as by PTEN loss or PIK3CA mutation occurs frequently in human cancer and contributes to resistance to antitumor therapies. Inhibition of key signaling proteins in the pathway therefore represents a valuable targeting strategy for diverse cancers. PF-04691502 is an ATP-competitive PI3K/mTOR dual inhibitor, which potently inhibited recombinant class I PI3K and mTOR in biochemical assays and suppressed transformation of avian fibroblasts mediated by wild-type PI3K γ, δ, or mutant PI3Kα. In PIK3CA-mutant and PTEN-deleted cancer cell lines, PF-04691502 reduced phosphorylation of AKT T308 and AKT S473 (IC(50) of 7.5-47 nmol/L and 3.8-20 nmol/L, respectively) and inhibited cell proliferation (IC(50) of 179-313 nmol/L). PF-04691502 inhibited mTORC1 activity in cells as measured by PI3K-independent nutrient stimulated assay, with an IC(50) of 32 nmol/L and inhibited the activation of PI3K and mTOR downstream effectors including AKT, FKHRL1, PRAS40, p70S6K, 4EBP1, and S6RP. Short-term exposure to PF-04691502 predominantly inhibited PI3K, whereas mTOR inhibition persisted for 24 to 48 hours. PF-04691502 induced cell cycle G(1) arrest, concomitant with upregulation of p27 Kip1 and reduction of Rb. Antitumor activity was observed in U87 (PTEN null), SKOV3 (PIK3CA mutation), and gefitinib- and erlotinib-resistant non-small cell lung carcinoma xenografts. In summary, PF-04691502 is a potent dual PI3K/mTOR inhibitor with broad antitumor activity. PF-04691502 has entered phase I clinical trials.

Quinazolines with intra-molecular hydrogen bonding scaffold (iMHBS) as PI3K/mTOR dual inhibitors.

Intra-molecular hydrogen bonding was introduced to the quinazoline motif to form a pseudo ring (intra-molecular H-bond scaffold, iMHBS) to mimic our previous published core structures, pyrido[2.3-D]pyrimidin-7-one and pteridinone, as PI3K/mTOR dual inhibitors. This design results in potent PI3K/mTOR dual inhibitors and the purposed intra-molecular hydrogen bonding structure is well supported by co-crystal structure in PI3Kγ enzyme. In addition, a novel synthetic route was developed for these analogs.

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.

The development and SAR of pyrrolidine carboxamide 11beta-HSD1 inhibitors.

The design and development of a series of highly selective pyrrolidine carboxamide 11beta-HSD1 inhibitors are described. These compounds including PF-877423 demonstrated potent in vitro activity against both human and mouse 11beta-HSD1 enzymes. In an in vivo assay, PF-877423 inhibited the conversion of cortisone to cortisol. Structure guided optimization effort yielded potent and stable 11beta-HSD1 selective inhibitor 42.

In vivo evaluation of 11beta-hydroxysteroid dehydrogenase activity in the rabbit eye.

PURPOSE: Steroids are used in a diverse range of conditions in clinical ophthalmology and one of the most significant complications is corticosteroid-induced glaucoma, which is characterized by an increase in intraocular pressure (IOP). 11beta-Hydroxysteroid dehydrogenase-1 (11beta-HSD1) is known to catalyze the interconversion of hormonally inactive cortisone to hormonally active cortisol and is widely expressed in the eye, particularly ciliary epithelium. Carbenoxolone (CBX), an 11beta-HSD1 inhibitor, has been shown to reduce IOP in healthy volunteers and patients with ocular hypertension (OHT). The purpose of this study was to: (1) develop an in vivo model for the assessment of cortisone to cortisol conversion in the eye, that is, 11beta-HSD1 activity and (2) assess the pharmacokinetic/pharmacodynamic relationship following topical treatment with 11beta-HSD1 inhibitors using an in vivo rabbit model. METHODS: Potent and selective 11beta-HSD1 inhibitors were topically administered to the rabbit eye and exogenous cortisone to endogenous cortisol conversion in the eye was assessed in rabbits. Tissues were then evaluated for cortisone, cortisol, and 11beta-HSD1 inhibitor levels by LC/MS/MS. Concomitantly cortisol activity in ocular tissue samples was determined using a secondary mechanistic pLuc-GRE assay. RESULTS: Topical treatment with potent and selective 11beta-HSD1 inhibitors resulted in complete inhibition in the conversion of cortisone to cortisol in the rabbit eye as well as decreased pLuc-GRE luciferase activity. The reduction of cortisone conversion was time- and dose-dependent as well as dependent on dosing volume (suggestive of increased spillover and washout with greater dosing volume). CONCLUSIONS: In conclusion, topical delivery of 11beta-HSD1 inhibitors can reduce or inhibit the conversion of cortisone to cortisol in the eye, indicating that the rabbit eye possesses an active enzyme for glucocorticoid synthesis. Dosing concentration and volume play an important role in the pharmacokinetic and pharmacodynamic effects of topically delivering an 11beta-HSD1 inhibitor. The rabbit model is useful for mechanistically assessing the conversion of cortisone to cortisol in the eye.

Comparative structure-activity relationship studies of 1-(5-methylsulfonylpyrid-2-yl)-5-alkyl and (hetero)aryl triazoles and pyrazoles in canine COX inhibition.

Structure-activity relationship (SAR) studies of novel 5-alkyl and 5-aryl/heteroaryl substituted 1,2,4-triazoles are described. The in vitro activity is compared to the pyrazole class of compounds with analogous side chains to delineate the contribution of the triazole ring nitrogen in binding to the active site. Both series are quite potent and selective in the canine whole blood (CWB) COX-2 assay, suggesting the increased binding contribution of the hydrophobic side chains.

5-Heteroatom substituted pyrazoles as canine COX-2 inhibitors. Part III: molecular modeling studies on binding contribution of 1-(5-methylsulfonyl)pyrid-2-yl and 4-nitrile.

The structure-activity relationship toward canine COX-1 and COX-2 in vitro whole blood activity of 4-hydrogen versus 4-cyano substituted 5-aryl or 5-heteroatom substituted N-phenyl versus N-2-pyridyl sulfone pyrazoles is discussed. The differences between the pairs of compounds with the 4-nitrile pyrazole derivatives having substantially improved in vitro activity are highlighted for both COX-2 and COX-1. This difference in activity may be due to the contribution of the hydrogen bond of the 4-cyano group with Ser 530 as shown by our molecular modeling studies. In addition, our model suggests a potential contribution from hydrogen bonding of the pyridyl nitrogen to Tyr 355 for the increased activity over the phenyl sulfone analogs.