Inhibition of phosphoinositide 3-kinase (PI3K) signaling is an appealing approach to treat brain tumors, especially glioblastoma multiforme (GBM). We previously disclosed our successful approach to prospectively design potent and blood-brain barrier (BBB) penetrating PI3K inhibitors. The previously disclosed molecules were ultimately deemed not suitable for clinical development due to projected poor metabolic stability in humans. We, therefore, extended our studies to identify a BBB penetrating inhibitor of PI3K that was also projected to be metabolically stable in human. These efforts required identification of a distinct scaffold for PI3K inhibitors relative to our previous efforts and ultimately resulted in the identification of GDC-0084 (16). The discovery and preclinical characterization of this molecule are described within.

Inhibitors of the class I phosphoinositide 3-kinase (PI3K) isoform PI3Kα have received substantial attention for their potential use in cancer therapy. Despite the particular attraction of targeting PI3Kα, achieving selectivity for the inhibition of this isoform has proved challenging. Herein we report the discovery of inhibitors of PI3Kα that have selectivity over the other class I isoforms and all other kinases tested. In GDC-0032 (3, taselisib), we previously minimized inhibition of PI3Kβ relative to the other class I insoforms. Subsequently, we extended our efforts to identify PI3Kα-specific inhibitors using PI3Kα crystal structures to inform the design of benzoxazepin inhibitors with selectivity for PI3Kα through interactions with a nonconserved residue. Several molecules selective for PI3Kα relative to the other class I isoforms, as well as other kinases, were identified. Optimization of properties related to drug metabolism then culminated in the identification of the clinical candidate GDC-0326 (4).

Abstract Modifications of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway are frequent in cancer due to multiple mechanisms, including activating mutations of the alpha isoform of PI3K. The dysregulation of this pathway has been implicated in many processes involved in oncogenesis. Thus, PI3K is a promising therapeutic target for cancer. Previously we have disclosed GDC-0941, a class 1 selective PI3K inhibitor and our class 1 PI3K/mTOR kinase inhibitor, GDC-0980. In this presentation we describe the design and discovery of a new class of PI3K inhibitors, which selectively inhibit the activated PI3Kα isoform relative to the PI3Kβ isoform. A lead was identified from a high throughput screen (HTS) that resulted in a novel chemical series of kinase inhibitors. Through a structure-based approach, this lead was optimized to provide very potent inhibitors of PI3K. In addition, this chemical series allowed for designing molecules that have different selectivity patterns with respect to the class 1 PI3K isoforms. In particular, a series of inhibitors were designed that could preferentially inhibit PI3Kα relative to PI3Kβ (“beta-sparing”). Further modification of the physicochemical properties led to the discovery of GDC-0032. GDC-0032 is a potent inhibitor of PI3Kα (PIK3CA) isoform with a Ki =0.2 nM, and with reduced inhibitory activity against PI3Kβ. This selectivity profile allowed for greater efficacy in vivo at the maximum tolerated dose relative to a pan inhibitor in representative PI3Kα (PIK3CA) mutant xenografts. It is notable that GDC-0032 preferentially inhibited PI3Kα (PIK3CA) mutant cells relative to cells with wild-type PI3K. Taken together, GDC-0032 is a potent and effective beta-sparing PI3K inhibitor, which currently is in clinical trials. Citation Format: Alan G. Olivero, Timothy P. Heffron, Matthew Baumgardner, Marcia Belvin, Leanne Berry Ross, Nicole Blaquiere, Erin Bradley, Georgette Castanedo, Mika Derynck, Steven Do, Jennafer Dotson, Danette Dudley, Kyle Edgar, Adrian Folkes, Ross Francis, Tony Gianetti, Richard Goldsmith, Paul Goldsmith, Jane Guan, Trevor Harrison, Robert Heald, Jerry Hsu, Phillip Jackson, Graham Jones, Amy Kim, Aleks Kolesnikov, Mark Lackner, Leslie Lee, John Lesnick, Cristina Lewis, Michael Mamounas, Neville McLean, Jeremy Murray, Chudi Ndubaku, Jim Nonomiya, Jodie Pang, Neil Pegg, Wei Wei Prior, Laurent Salphati, Deepack Sampath, Stephen Sideris, Michael Siu, Steven Staben, Daniel Sutherlin, Mark Ultsch, Jeff Wallin, Lan Wang, Christian Wiesmann, Xiaolin Zhang, Lori S. Friedman. Discovery of GDC-0032: A beta-sparing PI3K inhibitor active against PIK3CA mutant tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr DDT02-01. doi:10.1158/1538-7445.AM2013-DDT02-01

Aberrant activation of the PI3K-Akt-mTOR signaling pathway has been observed in human tumors and tumor cell lines, indicating that these protein kinases may be attractive therapeutic targets for treating cancer. Optimization of advanced lead 1 culminated in the discovery of clinical development candidate 8h, GDC-0349, a potent and selective ATP-competitive inhibitor of mTOR. GDC-0349 demonstrates pathway modulation and dose-dependent efficacy in mouse xenograft cancer models.

A series of suitable five-membered heterocyclic alternatives to thiophenes within a thienobenzoxepin class of PI3-kinase (PI3K) inhibitors was discovered. Specific thiazolobenzoxepin 8-substitution was identified that increased selectivity over PI3Kβ. PI3Kβ-sparing compound 27 (PI3Kβ Ki,app/PI3Kα Ki,app=57) demonstrated dose-dependent knockdown of pAKT, pPRAS40 and pS6RP in vivo as well as differential effects in an in vitro proliferation cell line screen compared to pan PI3K inhibitor GDC-0941. A new structure-based hypothesis for reducing inhibition of the PI3K β isoform while maintaining activity against α, δ and γ isoforms is presented.

Substructural class effects surrounding replacement of a 'cis' N-methyl aniline amide within potent and selective thienobenzoxepin PI3-kinase inhibitors are disclosed. While a simple aryl to alkyl switch was not tolerated due to differences in preferred amide conformation, heterocyclic amide isosteres with maintained aryl substitution improved potency and metabolic stability at the cost of physical properties. These gains in potency allowed lipophilic deconstruction of the arene to simple branched alkyl substituents. As such, overall lipophilicity-neutral, MW decreases were realized relative to the aniline amide series. The improved properties for lead compound 21 resulted in high permeability, solubility and bioavailability.

A potent inhibitor of PI3Kδ that is ⩾200 fold selective for the remaining three Class I PI3K isoforms and additional kinases is described. The hypothesis for selectivity is illustrated through structure activity relationships and crystal structures of compounds bound to a K802T mutant of PI3Kγ. Pharmacokinetic data in rats and mice support the use of 3 as a useful tool compound to use for in vivo studies.

PI3Kδ is a lipid kinase and a member of a larger family of enzymes, PI3K class IA(α, β, δ) and IB (γ), which catalyze the phosphorylation of PIP2 to PIP3. PI3Kδ is mainly expressed in leukocytes, where it plays a critical, nonredundant role in B cell receptor mediated signaling and provides an attractive opportunity to treat diseases where B cell activity is essential, e.g., rheumatoid arthritis. We report the discovery of novel, potent, and selective PI3Kδ inhibitors and describe a structural hypothesis for isoform (α, β, γ) selectivity gained from interactions in the affinity pocket. The critical component of our initial pharmacophore for isoform selectivity was strongly associated with CYP3A4 time-dependent inhibition (TDI). We describe a variety of strategies and methods for monitoring and attenuating TDI. Ultimately, a structure-based design approach was employed to identify a suitable structural replacement for further optimization.

Inhibition of phosphoinositide 3-kinase (PI3K) signaling through PI3Kα has received significant attention for its potential in cancer therapy. While the PI3K pathway is a well-established and widely pursued target for the treatment of many cancer types due to the high frequency of abnormal PI3K signaling, glioblastoma multiforme (GBM) is particularly relevant because the pathway is implicated in more than 80% of GBM cases. Herein, we report the identification of PI3K inhibitors designed to cross the blood-brain barrier (BBB) to engage their target where GBM tumors reside. We leveraged our historical experience with PI3K inhibitors to identify correlations between physicochemical properties and transporter efflux as well as metabolic stability to focus the selection of molecules for further study.

Of the four class I phosphoinositide 3-kinase (PI3K) isoforms, PI3Kα has justly received the most attention for its potential in cancer therapy. Herein we report our successful approaches to achieve PI3Kα vs PI3Kβ selectivity for two chemical series. In the thienopyrimidine series of inhibitors, we propose that select ligands achieve selectivity derived from a hydrogen bonding interaction with Arg770 of PI3Kα that is not attained with the corresponding Lys777 of PI3Kβ. In the benzoxepin series of inhibitors, the selectivity observed can be rationalized by the difference in electrostatic potential between the two isoforms in a given region rather than any specific interaction.

Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutation of the PIK3CA gene, loss or mutation of phosphatase and tensin homolog(PTEN), and deregulation of mammaliantargetof rapamycin (mTOR) complexes. The dysregulation of this pathway has been implicated in tumor initiation, cell growth and survival, invasion and angiogenesis, thus, PI3K and mTOR are promising therapeutic targets for cancer. We discovered GDC-0980, a selective, potent, orally bioavailable inhibitor of Class I PI3 kinase and mTOR kinase (TORC1/2) with excellent pharmacokinetic and pharmaceuticalproperties. GDC-0980potently inhibitssignaltransductiondownstream of both PI3K and mTOR, as measured by pharmacodynamic (PD) biomarkers, thereby acting upon two key pathway nodes to produce the strongest attainable inhibition of signaling in the pathway. Correspondingly, GDC-0980waspotentacrossabroadpanelofcancercelllines,withthegreatestpotencyinbreast,prostate,and lung cancers and less activity in melanoma and pancreatic cancers, consistent with KRAS and BRAF acting as resistancemarkers.TreatmentofcancercelllineswithGDC-0980resultedinG1cell-cyclearrest,andincontrast

Abstract PI3 Kinase and mTOR have been identified as promising targets for the treatment of cancer. These enzymes participate in related, but not redundant, signaling networks to transmit cellular growth and survival signals, which are hallmarks of tumor growth. An interest in targeting both of these two important points along this critical signaling pathway, and the ability to leverage the high degree of structural similarity in the active sites of PI3K and mTOR kinase, has resulted in the discovery of GDC-0980 as a Class I PI3K and mTOR kinase inhibitor for oncology indications. The structure, efficacy, and medicinal chemistry behind the discovery of this compound is described. Beginning with the morpholin-4-yl-thieno[3,2-d]pyrimidine core of the Class I PI3K inhibitor GDC-0941, structural substitutions were made external to the core that added mTOR potency, improved the metabolic stability in vitro and in vivo, and lowered the plasma protein binding of the scaffold. Homology models of mTOR using PI3Kγ structures with bound inhibitors provided hypotheses for increasing mTOR potency relative to previous compounds. The solubility of the modified compounds was improved through the addition of polar functionality in the solvent exposed region of the scaffold, resulting in GDC-0980. GDC-0980 is potent across Class I isoforms with IC50's of 5, 27, 7, and 14 nM for PI3Kα, β, Δ, and γ, and inhibits mTOR with a Ki of 17 nM. The compound is highly selective versus a large panel of kinases including others in the PIK family. Based on the excellent PK profile, linear increase in exposure, strong potency in a broad range of cancer cells, and high free fraction, GDC-0980 is efficacious in animal models of cancer when dosed orally at low doses. Furthermore, this compound is efficacious when dosed intermittently as well as on a daily schedule. These preclinical data provide compelling support for GDC-0980 as a clinical candidate, and early stage clinical trials are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2787. doi:10.1158/1538-7445.AM2011-2787

Abstract Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutation of the PIK3CA gene, loss or mutation of phosphatase and tensin homolog (PTEN), and deregulation of mammalian target of rapamycin (mTOR) complexes. The dysregulation of this pathway has been implicated in tumor initiation, cell growth and survival, invasion and angiogenesis, thus, PI3K and mTOR are promising therapeutic targets for cancer. We discovered GDC-0980, a selective, potent, orally bioavailable inhibitor of Class I PI3 kinase and mTOR kinase (TORC1/2) with excellent pharmacokinetic and pharmaceutical properties. GDC-0980 potently inhibits signal transduction downstream of both PI3K and mTOR, as measured by pharmacodynamic (PD) biomarkers, thereby acting upon two key pathway nodes to produce the strongest attainable inhibition of signaling in the pathway. Correspondingly, GDC-0980 was potent across a broad panel of cancer cell lines, with the greatest potency in breast, prostate, and lung cancers and less activity in melanoma and pancreatic cancers, consistent with KRAS and BRAF acting as resistance markers. Treatment of cancer cell lines with GDC-0980 resulted in G1 cell-cycle arrest, and in contrast to mTOR inhibitors, GDC-0980 induced apoptosis in certain cancer cell lines, including those with direct pathway activation via PI3K and PTEN. Low doses of GDC-0980 potently inhibited tumor growth in xenograft models including those with activated PI3K, loss of LKB1 or PTEN, and elicited an exposure-related decrease in PD biomarkers. These preclinical data show that GDC-0980 is a potent and effective dual PI3K/mTOR inhibitor with promise for the clinic. Mol Cancer Ther; 10(12); 2426–36. ©2011 AACR.

Starting from thienobenzopyran HTS hit 1, co-crystallization, molecular modeling and metabolic analysis were used to design potent and metabolically stable inhibitors of PI3-kinase. Compound 15 demonstrated PI3K pathway suppression in a mouse MCF7 xenograft model.

The discovery of 2 (GDC-0980), a class I PI3K and mTOR kinase inhibitor for oncology indications, is described. mTOR inhibition was added to the class I PI3K inhibitor 1 (GDC-0941) scaffold primarily through the substitution of the indazole in 1 for a 2-aminopyrimidine. This substitution also increased the microsomal stability and the free fraction of compounds as evidenced through a pairwise comparison of molecules that were otherwise identical. Highlighted in detail are analogues of an advanced compound 4 that were designed to improve solubility, resulting in 2. This compound, is potent across PI3K class I isoforms with IC(50)s of 5, 27, 7, and 14 nM for PI3Kα, β, δ, and γ, respectively, inhibits mTOR with a K(i) of 17 nM yet is highly selective versus a large panel of kinases including others in the PIKK family. On the basis of the cell potency, low clearance in mouse, and high free fraction, 2 demonstrated significant efficacy in mouse xenografts when dosed as low as 1 mg/kg orally and is currently in phase I clinical trials for cancer.

The PI3K/AKT/mTOR pathway has been shown to play an important role in cancer. Starting with compounds 1 and 2 (GDC-0941) as templates, (thienopyrimidin-2-yl)aminopyrimidines were discovered as potent inhibitors of PI3K or both PI3K and mTOR. Structural information derived from PI3K gamma-ligand cocrystal structures of 1 and 2 were used to design inhibitors that maintained potency for PI3K yet improved metabolic stability and oral bioavailability relative to 1. The addition of a single methyl group to the optimized 5 resulted in 21, which had significantly reduced potency for mTOR. The lead compounds 5 (GNE-493) and 21 (GNE-490) have good pharmacokinetic (PK) parameters, are highly selective, demonstrate knock down of pathway markers in vivo, and are efficacious in xenograft models where the PI3K pathway is deregulated. Both compounds were compared in a PI3K alpha mutated MCF7.1 xenograft model and were found to have equivalent efficacy when normalized for exposure.