The Discovery and Process Chemistry Development of GDC-0084, a

Chapter 6

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The Discovery and Process Chemistry Development of GDC-0084, a Brain Penetrating Inhibitor of PI3K and mTOR Timothy P. Heffron,1 Andrew McClory,2 and Andreas Stumpf*,2 1Department

of Discovery Chemistry, Genentech, Inc. 1 DNA Way, South San Francisco, California 94080, United States 2Small Molecule Process Chemistry, Genentech, Inc. 1 DNA Way, South San Francisco, California 94080, United States *E-mail: [email protected].

Aberrant signaling of the PI3K pathway has been implicated in the majority of cases of glioblastoma multiforme (GBM), a malignant brain tumor with an associated poor prognosis. In order for a PI3K/mTOR inhibitor to inhibit PI3K pathway signaling where GBM tumors reside, such molecules must be capable of penetrating the blood-brain barrier (BBB). This chapter describes the medicinal chemistry efforts that led to the discovery of GDC-0084, a BBB penetrating inhibitor of PI3K and mTOR, followed by the process chemistry development that enabled its advancement to clinical studies.

The Discovery of GDC-0084 Glioblastoma multiforme (GBM) is an aggressive form of primary brain tumor with more than 20,000 new diagnoses each year (1). Unfortunately, there is significant room for improvement upon the limited chemotherapeutic treatment options for GBM as the two-year survival rate ranges from 4-29% (2). Inhibition of PI3Kα is a compelling potential approach to treating GBM as aberrant PI3K signaling is implicated in more than 80% of cases (3, 4). With the apparent medical need and biological rationale in mind, we sought to realize a blood-brain barrier (BBB) penetrating PI3Kα inhibitor to reach the © 2016 American Chemical Society Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


brain where GBM tumors reside. At the onset of this program, we had extensive experience in the discovery of PI3K inhibitors as we had recently concluded a multi-year effort pursuing inhibitors of PI3Kα for the treatment of peripheral disease (5–14). However, there was still a need for a PI3K inhibitor that achieved brain penetration as, at the time, we were not aware of any PI3K inhibitors capable of penetrating the BBB. To achieve free BBB penetration, we anticipated that we would need to minimize efflux transport mediated by P-gp and Bcrp, two transporters highly expressed at the BBB which actively limit brain penetration of small molecules (15). Achieving brain penetration with a PI3K inhibitor, then, presented a significant challenge due to the need to avoid the P-gp and Bcrp mediated efflux normally experienced by kinase inhibitors. Indeed, when we studied the extent to which our previous clinical PI3Kα inhibitor GDC-0941 (5) and clinical PI3K/mTOR inhibitor GDC-0980 (10) were capable of penetrating the BBB in mice, we found that essentially no penetration was achieved (Table 1). While the [brain]/[plasma] ratios were low, the more meaningful measure of free BBB penetration, [brain]u/[plasma]u also indicated that no free penetration was achieved. Furthermore, the lack of BBB penetration was consistent with in vitro permeability assay results using Madin-Darby canine kidney (MDCK) cells transfected to highly express P-gp or Bcrp (Table 1). In these assays, both GDC-0941 and GDC-0980 experience high B-A/A-B efflux ratios, demonstrating that they are substrates of both P-gp and Bcrp.

Table 1. In Vitro Permeability Efflux Ratios from MDR1 and Bcrp1 Transfected MDCK Cell Line Assays and in Vivo Brain-to-Plasma Ratios for Compounds GDC-0941 and GDC-0980

148 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.


That GDC-0941 and GDC-0980 were each substrates of P-gp and Bcrp was not unexpected given their physicochemical properties are inconsistent with the median values of 119 marketed CNS drugs (Table 2), for which low transporter mediated efflux is expected (16). Particularly noteworthy, the molecular weights of GDC-0941 and GDC-0980 are significantly greater than those of marketed CNS drugs. We thought that attempts to harmonize the physical properties of our PI3K inhibitors with the median value of marketed CNS drugs might increase the probability that the PI3K inhibitors would lack efflux mediated by P-gp or Bcrp.

Table 2. Comparison of Calculated Physicochemical Properties of Compounds GDC-0941 and GDC-0980 with Marketed CNS Targeting Drugsa

In an effort to reduce the MW, to realize PI3K inhibitors with physical properties more consistent with marketed CNS drugs, we evaluated 1 (Table 3) in which the piperazine sulfonamide moiety of GDC-0941 is absent. With the reduction of MW from 513 to 337 when compared to GDC-0941, we were pleased to see a dramatic reduction in P-gp and Bcrp mediated efflux (Table 3). Unfortunately, 1 also had much weaker potency than GDC-0941 and accordingly was deemed inadequate. Encouragingly however, 2, an analog of GDC-0980 in which the piperazine amide has been eliminated, retained most of the potency of GDC-0980, but with substantially reduced MW. This encouraging result demonstrated that adequate PI3Kα potency could be achieved in a comparatively low MW molecule that, most excitingly, was not a substrate of either P-gp or Bcrp (Table 3). Despite this important discovery, however, 2 was not by itself adequate as it was rapidly metabolized in in vitro microsomal incubations. Nevertheless, this initial result gave us the encouragement to pursue further morpholinopyrimidine-based PI3K inhibitors. 149

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Table 3. A Comparison of the Potency, Permeability Efflux Ratios, and in Vitro Metabolic Stability of GDC-0941 and GDC-0980 with Analogs That Do Not Have Substitution in the Solvent Exposed Region (1 and 2)

From our previous studies, we knew that substitution of the thiophene of the thienopyrimidine at the 6-position (numbering in Table 3) had allowed for us to achieve molecules with reasonable in vitro and in vivo ADME properties (e.g. GDC-0980) and so we began to design and synthesize analogs of 2 that were substituted at the 6-position. A substantial number of analogs were generated in this effort that were evaluated in permeability assays to determine if they were P-gp and/or Bcrp substrates. From this effort we rapidly identified that hydrogen bond donor (HBD) count had a dramatic effect on transporter-mediated efflux. For example, the pairs of molecules 36 with 4 and 5 with 6 (Table 4) differ in each case by the presence of a hydroxyl group or a methyl ether. In each case, elimination of the hydrogen bond by alkylation to the methyl ether results in a molecule that is a much weaker efflux transporter substrate. In the end, 4 and 6 also had reasonable exposure in mice (Table 5) with evident free BBB penetration (Table 5) (17, 18). With these molecules in hand we were prepared for in vivo studies to verify that they could achive potent inhibition of PI3K/mTOR signalling behind the BBB as intended. In order to demonstrate inhibition of our kinase target by 4 and 6 in the intended compartment we conducted experiments to evaluate pharmacodynamic response in the brain. Oral doses of 4 or 6 to healthy mice inhibited pAKT, a downstream marker of PI3K signaling, in normal brain tissue (i.e. behind a fully intact BBB, Figure 1). Additionally, 4 and 6 were shown to inhibit PI3K signaling (pAKT, pS6) in a U87 (human primary glioblastoma tumor cell line) tumor model in mice (17) that led to a corresponding inhibition of tumor growth (Figure 2). Furthermore, 6 also demonstrated inhibition of growth of tumors implanted in mouse brains, providing even further evidence of target inhibition in the brain that suggests such a molecule might provide benefit to patients with glioblastoma (18).



Table 4. Efflux Ratios for Select Thienopyrimidines

Table 5. Potency, Efflux Ratios, Mouse PK and Brain Exposure for 4 and 6



Figure 1. Inhibition of pAKT in female CD-1 mice brain tissue after oral administration of 4 and 6 as MCT suspension. Normalized pAKT was measured 1 h and 6 h post dose and are indicated by the bars in the chart and error bars indicate ± standard error of the mean. The percentage reduction in pAKT compared to the untreated group is indicated where applicable. The mean pAKT for the untreated groups are based on samplings of brain tissue from 3 animals per time point. Sampling of 1 animal per time point was analyzed for compound 4. This Figure is reproduced from Reference (17), copyright 2012, American Chemical Society.

Figure 2. In vivo efficacy of 4 and 6 versus U87 MG/M human glioblastoma xenografts. Female NCr nude mice bearing subcutaneous tumors were administered escalating doses of 4 or 6 orally as a suspension in vehicle (0.5% methylcellulose/0.2% Tween-80) or vehicle once daily (QD) for 24 days. Changes in tumor volumes over time by dose for each compound are depicted as cubic spline fits generated via Linear Mixed Effects analysis of log-transformed volumes. This Figure is reproduced from Reference (17), copyright 2012, American Chemical Society. 152 Abdel-Magid et al.; Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage ... ACS Symposium Series; American Chemical Society: Washington, DC, 2016.

While excellent preclinical tool compounds, 4 and 6 were determined to be not suitable for advancement to human clinical study as human liver microsomal incubations indicated that they were likely to have rapid clearance in humans (Table 6). We, therefore, extended our studies from 4 and 6 in the hopes of improving upon human metabolic stability while also maintaining potency and continuing to limit transporter efflux to maintain CNS penetration.


Table 6. MDR1 and Bcrp1 Transfected MDCK Cell Permeability Efflux Ratios and Human Liver Microsomal Stability for Select Compounds

Many efforts to modify the substitution at C-6 of the thiophene within 4 and 6 (numbering in Table 3) were attempted to achieve improved human metabolic stability. Unfortunately, those efforts were not successful or, when human metabolic stability improved, either reduced CNS penetration or potency was also encountered. During these efforts we also returned to alternative morpholinopyrimidine cores that we had evaluated during studies that led to the discovery of GDC-0980. One such core was a purine, instead of thienopyrimidine, and 7 was a molecule we had identified that had excellent PI3K potency and metabolic stability. We now hoped to modify 7 so that it could also achieve BBB penetration (Table 6). We had previously demonstrated the importance of minimizing HBD count to minimize efflux and so from 7, which contains four HBD, we recognized we would need a molecule with reduced HBD count. Previously we alkylated hydroxyl groups to their corresponding methyl ethers to reduce HBD count (e.g. 3 to 4, 5 to 6). In this case, with two hydroxyl groups, we decided to hydrolyze one hydroxyl group in the course of a cyclization to realize a new tricyclic core (19). Compound 8 (Table 7), containing this new tricyclic core, 153

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was found to have low transporter mediated efflux, suggesting it should penetrate the BBB, and exhibits good metabolic stability. However, its cellular potency was modest. To improve the potency of 8, we used the same aminopyridine group found in GDC-0980, known to improve mTOR potency relative to the aminopyridine in 8 (6). Gratifyingly, the resultant molecule, GDC-0084 (Table 7), is a potent PI3K/mTOR inhibitor with excellent human metabolic stability and low transporter efflux in vitro (19).


Table 7. Potency, Efflux Ratios and Human Metabolic Stability of Tricyclic Purine-Based PI3K Inhibitors 8 and GDC-0084

Figure 3. Inhibition of p-AKT by GDC-0084 in normal mouse brain tissue along with corresponding brain and unbound brain concentrations. *Significantly different from untreated control. p

The Discovery and Process Chemistry Development of GDC-0084, a

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