Discovery of GSK2126458, a highly potent inhibitor of PI3K and the mammalian target of rapamycin

Article abstract of DOI:10.1021/ml900028r

Phosphoinositide 3-kinase α (PI3Kα) is a critical regulator of cell growth and transformation, and its signaling pathway is the most commonly mutated pathway in human cancers. The mammalian target of rapamycin (mTOR), a class IV PI3K protein kinase, is also a central regulator of cell growth, and mTOR inhibitors are believed to augment the antiproliferative efficacy of PI3K/AKT pathway inhibition. 2,4-Difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)- 6-quinolinyl]-3-pyridinyl}benzenesulfonamide (GSK2126458, 1) has been identified as a highly potent, orally bioavailable inhibitor of PI3Kα and mTOR with in vivo activity in both pharmacodynamic and tumor growth efficacy models. Compound 1 is currently being evaluated in human clinical trials for the treatment of cancer.

Full text of DOI:10.1021/ml900028r

pubs.acs.org/acsmedchemlett
Discovery of GSK2126458, a Highly Potent Inhibitor of
PI3K and the Mammalian Target of Rapamycin
Steven D. Knight,* Nicholas D. Adams, Joelle L. Burgess, Amita M. Chaudhari,
Michael G. Darcy, Carla A. Donatelli, Juan I. Luengo, Ken A. Newlander, Cynthia A. Parrish,
Lance H. Ridgers, Martha A. Sarpong, Stanley J. Schmidt, Glenn S. Van Aller, Jeffrey D. Carson,
Melody A. Diamond, Patricia A. Elkins, Christine M. Gardiner, Eric Garver, Seth A. Gilbert,
Richard R. Gontarek, Jeffrey R. Jackson, Kevin L. Kershner, Lusong Luo, Kaushik Raha,
Christian S. Sherk, Chiu-Mei Sung, David Sutton, Peter J. Tummino, Ronald J. Wegrzyn,
Kurt R. Auger, and Dashyant Dhanak
GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426
ABSTRACT Phosphoinositide 3-kinase R (PI3KR) is a critical regulator of cell
growth and transformation, and its signaling pathway is the most commonly
mutated pathway in human cancers. The mammalian target of rapamycin (mTOR),
a class IV PI3K protein kinase, is also a central regulator of cell growth, and mTOR
inhibitors are believed to augment the antiproliferative efficacy of PI3K/AKT
pathway inhibition. 2,4-Difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinoli-
nyl]-3-pyridinyl}benzenesulfonamide (GSK2126458, 1) has been identified as a
highly potent, orally bioavailable inhibitor of PI3KR and mTOR with in vivo activity
in both pharmacodynamic and tumor growth efficacy models. Compound 1 is
currently being evaluated in human clinical trials for the treatment of cancer.
KEYWORDS GSK2126458, phosphoinositide 3-kinase R, mammalian target of
rapamycin, PI3K/AKT pathway
he phosphoinositide 3-kinase (PI3K) signaling path-
way is activated in a broad spectrum of human
cancers.1 Activation of this pathway often occurs
evaluated in a phase I, open-label, dose-escalation study in
subjects with solid tumors or lymphoma.
T
GSK1059615 (2),13 our first PI3K clinical compound,
recently entered a dose-escalation study in patients with
refractory malignancies. In follow-up studies, we sought to
identify a second inhibitor with improved potency, selectiv-
ity, and pharmacokinetic (PK) profiles. Key to our approach
for achieving the desired levels of PI3K activity was to pursue
structure-based design utilizing crystallography of the more
amenable PI3Kγ as a surrogate protein.14-17 The inhibitor-
bound crystal structure of 2 in PI3Kγ indicated that the
thiazolidinedione (TZD) ring formed an interaction with
the catalytic lysine (Lys833) within the ATP-binding pocket.
However, the structure also showed that larger groups could
potentially be accommodated. We reasoned that filling the
empty space in the enzyme pocket could lead to inhibitors
with improved potency and potentially selectivity, and this
was the basis for the initial strategy to identify alternates to
the TZD moiety.
indirectly by the activation of receptor tyrosine kinases or
the inactivation of the phosphotase and tensin homologue
(PTEN) tumor suppressor.2,3 Direct activation of PI3K has
been demonstrated with the discovery of several activating
mutations in the PIK3CA gene itself, the gene that encodes
the p110R catalytic subunit of PI3KR.4 Several of the muta-
tions found in PIK3CA have been shown to increase the lipid
kinase activity of PI3KR, induce activation of signaling path-
ways, and promote transformation of cells both in vitro and
in vivo.5-7 Furthermore, the PI3K pathway is the most
commonly mutated signaling pathway in human cancers.8,9
The PI3K family of enzymes is comprised of 15 lipid
kinases with distinct substrate specificities, expression pat-
terns, and modes of regulation.10 In particular, PI3KR has
emerged as an attractive target for cancer therapeutics,
and several PI3K inhibitors are currently under evaluation
in human clinical trials, including BEZ235 (Novartis), GDC-
0941 (Genentech), PX-866 (ProlX), and XL765 (Exelixis).11, 1 2
We describe herein the discovery of 2,4-difluoro-
N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridi-
nyl}benzenesulfonamide (GSK2126458, 1, Figure 1), a highly
potent and selective inhibitor of class I PI3Ks and the mam-
malian target of rapamycin (mTOR). Compound 1 is being
Synthesis of these derivatives began with conversion of
6-bromo-4-chloroquinoline (3) to the corresponding 4-iodo
Received Date: December 23, 2009
Accepted Date: January 4, 2010
Published on Web Date: January 19, 2010
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Figure 1. GlaxoSmithKline PI3K clinical compounds.
Table 1. TZD Replacement SAR
Figure 2. X-ray cocrystal structure of p110γ with 6d. The protein
is shown in the solid ribbon. Selected residues are shown in gray.
Figures 2 and 3 were prepared using PyMOL.21
Table 2. Selected Pyridylsulfonamide Analog SARa
Scheme 1. Synthesis of TZD Replacement Analogsa
a Units: Dose-normalized area under the curve (DNAUC) (ng h mL-1
mg-1 kg-1); NQ, not quantifiable.
a Conditions: (a) 2 M HCl, ether; then NaI, EtCN, reflux. (b) 4-Pyridyl-
boronic acid, Pd(PPh3)4, 2 M aqueous K2CO3, dioxane, 100 °C.
(c) Pinacolato(diboron), PdCl2(dppf)-CH2Cl2, KOAc, dioxane, 100 °C;
then ArBr, 2 M aqueous Na2CO3, 110 °C.
pyridine 6b and indazole 6c, although neither showed
much improvement in the cellular assay relative to 6a.
Molecular modeling overlays indicated that the pyridyl and
indazolyl nitrogens of 6b and 6c, respectively, were most
likely making distinct interactions with the enzyme. We
therefore merged the two heterocycles to form azaindazole
6d, which provided a substantial boost in biochemical and
cellular potency. Cocrystallization of 6d with PI3Kγ con-
firmed that the nitrogen at the 2-position of the indazole
forms a hydrogen bond with Lys833 and that the pyridyl
nitrogen interacts with a conserved active site water mole-
cule (Figure 2).
Although 6d exhibited a promising activity profile, the
compound displayed very low aqueous solubility and a poor
PK profile, characterized by high clearance, a short half-life,
and a lack of oral bioavailability. Opening of the pyrazole
portion of the azaindazole to give 2,3,5-trisubstituted
pyridine analogs led to the identification of pyridylsulfona-
mide 6e, which retained the gains made in potency and
intermediate 4, followed by installation of the 4-pyridyl group
under standard palladium-catalyzed cross-coupling conditions
to provide quinoline 5 (Scheme 1). Various aryl (Ar) groups
were then attached to the 6-positionofthe quinoline core using
an in situ borylation and palladium-catalyzed cross-coupling to
furnish the desired analogs 6.18,19
Inhibition of PI3KR was measured using a continuous
read time-resolved fluorescence resonance energy transfer
displacement assay.20 The analogs were also evaluated in a
PI3KR-driven mechanistic cellular assay, which measured
the ability of the compounds to decrease intracellular
phosphorylation of AKTat S473 (pAKT-S473) in T47D and
BT474 cancer cells. Replacement of the TZD with a simple
phenyl group (6a) resulted in a dramatic loss in potency
(Tabl e 1 ). The activity was much less attenuated for both
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Table 3. Biochemical Activity of 1
p110 isoform app Ki (nM)
Table 4. mTOR Complex Activity of GSK2126458 (1)
mTORC1 app Ki (nM)
mTORC2 app Ki (nM)
p110R mutant app Ki (nM)
0.18
0.3
R
β
δ
γ
E542K
0.008
E545K
0.008
H1047R
0.009
0.019
0.13
0.024
0.06
Table 5. Mechanistic and Functional Cellular Activity of 1
pAKT-S473 IC50 (nM) growth IC50 (nM)
T47D
0.41
BT474
0.18
T47D
BT474
2.4
3
inhibitors, 1 is also active against the other class I PI3K
isoforms (β, γ, and δ).
Acocrystal structure of PI3Kγ in complex with 1 shows the
inhibitor bound in the ATP-binding site of the enzyme
(Figure 3). The structure was determined to 2.7 Å resolution
and shows, like 6e, that the pyridyl nitrogen forms a key
hydrogen bond with the conserved water molecule. The
sulfonamide interacts with Lys833, making a strong charged
interaction. On the basis of the pKa of the sulfonamide-NH
(6.56),24 ∼87% of the moiety exists in its deprotonated form
at physiological pH. This charged interaction may help to
explain the superior potency of 1 as compared to the other
reported PI3K inhibitors. In addition, the difluorophenyl
group fills a hydrophobic region in the back pocket of the
enzyme, while the quinoline nitrogen forms an interaction
with the hinge (Val882).
Compound 1 shows excellent selectivity over protein
kinases (>10,000-fold vs >240 kinases evaluated) with
the notable exception of the class IV PI3K family. mTOR, a
class IV PI3K protein kinase, is a central regulator of cell
growth and exists in two functional complexes, mTORC1
and mTORC2.25 mTORC2 is proposed to regulate AKT S473
phosphorylation, and its inhibition is believed to augment
the antiproliferative efficacy of a PI3K inhibitor by dual
inhibition of the PI3K/AKT pathway.26 The kinase domain
of mTOR is homologous to the p110R catalytic subunit of the
class I PI3Ks,27 and 1 is a potent inhibitor of both mTOR
complexes with subnanomolar activity (Table 4). Compound
1 is also a potent inhibitor of the class IV PI3 kinase, DNA-PK
(IC50 = 0.28 nM).
In mechanistic cellular assays, 1 caused a significant
reduction in the levels of pAKT-S473 with remarkable po-
tency (Table 5). Consistent with its activity against both
PI3KR and mTOR, 1 also inhibits phosphorylation of AKT-
T308 and p70S6K at low nanomolar concentrations (data not
shown). Compound 1 induces a G1 cell cycle arrest and
inhibits cell proliferation in a large panel of cell lines, includ-
ing T47D and BT474 breast cancer lines.
The PK profile of 1 was studied in four preclinical species
(mouse, rat, dog, and monkey). The compound showed low
blood clearance and good oral bioavailability (Table 6). In
addition, 1 had minimal potential to inhibit the human
cytochrome P450 isoforms (IC50 > 25 μM vs CYPs 3A4,
1A2, 2C9, 2C19, and 2D6).
Figure 3. X-ray cocrystal structure of p110γ with GSK2126458 (1).
showed increased solubility. However, no improvement in
the PK profile was noted.
We next examined the structure-activity relationship
(SAR) of the sulfonamide moiety and found that arylsulfo-
namides (e.g., 6f) exhibited oral exposure in rats with no loss
of inhibitory activity (Table 2). Removal of the 2-amino group
to yield 6g further increased exposure ∼3-fold. Reversal of
the sulfonamide connectivity (6h) led to an increase in
biochemical and cellular potency, while maintaining the
improved oral exposure.
Reintroduction of a small substituent at the 2-position of
the pyridine (e.g., methoxy, methyl, halogen, etc.) resulted
in a significant boost in both enzyme and cellular potency
(data not shown). We reasoned that this was due to the
substituent filling unoccupied space deep within the enzyme
pocket, as well as a potential effect on the orientation of the
neighboring sulfonamide moiety. Further efforts showed
that the incorporation of a pyridazine at the 4-position of
the quinoline resulted in a moderate improvement in the
CYP inhibition profile as compared to the pyridine (data not
shown).
These studies eventually led to the identification of 1, an
extraordinarily potent inhibitor of PI3KR (p110R/p85R) with
low picomolar activity (PI3KR IC50 = 0.04 nM). In biochem-
ical assays, compound 1 is significantly more potent than 2
(PI3KR IC50 = 2 nM) and is the most potent PI3KR inhibitor
reported to date. In comparison with other clinical PI3K
inhibitors, 1 is ∼100-fold more potent than BEZ235 (IC50
=
6 nM)22 and GDC-0941 (IC50 = 9 nM)23 and ∼1000-fold
more active than XL-765 (IC50 = 39 nM).11,12 Importantly, 1
is also a low picomolar inhibitor of the common activating
mutants of p110R (E542K, E545K, and H1047R) found in
human cancer (Table 3). Similar to the other reported PI3K
In an in vivo setting, 1 exhibited a dose-dependent reduc-
tion in pAKT-S473 levels in human BT474 tumors implanted
in mice. In the study, designed to measure the magnitude
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Table 6. Preclinical PK Profile of 1a
iv dosing
oral dosing
DNAUC
species
Clb
10
Vdss
T1/2
% F
mouse
rat
1.0
1.1
0.7
0.8
2.1
6.2
1.3
3.5
1100
6100
2400
2300
100
81
2.3
5.8
3.6
dog
80
monkey
49
a Units: Clb, mL min-1 kg-1; Vdss, L kg-1; T1/2, h; and DNAUC, ng h
mL-1 mg-1 kg-1
.
Figure 5. Tumor growth efficacy of 1 in BT474 human tumor
xenografts as measured by median tumor volume (cu mm). Mice
were dosed once daily for 5 days/week (M-F) for 3 weeks (days
17-21, 24-28, and 31-35). Error bars denote the standard error
of the mean (SEM).
SUPPORTING INFORMATION AVAILABLE Biological as-
says, biological data, and experimental procedures. This material
is available free of charge via the Internet at http://pubs.acs.org.
Accession Codes: The coordinates for 1 and 6d have been
deposited with the RCSB Protein Data Bank under the accession
codes 3L08 (1) and 3L54 (6d).
Figure 4. PD effect of 1 in BT474 human tumor xenografts
following a single 300 μg kg-1 oral dose. The ratio of pAKT/total
AKT was measured and compared to control. [Blood], concentra-
tion of drug in the blood in ng mL-1; NQ, not quantifiable.
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should be
and duration of the pharmacodynamic (PD) response, mice
were treated orally with drug, and pAKT levels were deter-
mined over the course of 24 h. Following a single 300 μg/kg
dose, 1 showed a profound and sustained PD response over
the 10 h observation period with pAKT levels returning to
those of control by 24 h (Figure 4). Remarkably, the sus-
tained PD response was achieved with very low circulating
levels of drug, consistent with the high in vitro potency of 1.
Compound 1 was also evaluated in a BT474 human tumor
xenograft growth efficacy model where mice were adminis-
tered a single oral dose five times per week for 3 weeks.
Consistent with inhibition of the PI3K/AKT/mTOR pathway,
the drug exhibited dose-dependent tumor growth inhibition
(Figure 5). The top dose (3 mg kg-1) was well-tolerated in the
study. As reported previously,13 compound 2 exhibited
efficacy in BT474 xenografts following twice daily dosing
at 25 mg kg-1. In comparison, compound 1 exhibited
similar efficacy at a much lower dose and less frequent
administration.28
In conclusion, we report the discovery of 1, a structurally
novel inhibitor of the PI3K/AKT/mTOR signaling pathway
with picomolar activity against PI3KR and mTOR. Com-
pound 1 displays remarkable potency in both mechani-
stic and antiproliferative cellular assays. Compound 1 also
exhibits excellent in vivo activity, highlighted by a sustained
PD effect at very low circulating drug levels. Inhibition of the
PI3K/AKT/mTOR pathway is expected to have a beneficial
effect on cancer therapy, and 1 has been advanced into a
phase I, open-label, dose-escalation study in subjects with
solid tumors or lymphoma.
addressed. E-mail: steven.d.knight@gsk.com.
ABBREVIATIONS PI3K, phosphoinositide 3-kinase; mTOR,
mammalian target of rapamycin; PTEN, phosphotase and
tensin homologue; DNAUC, dose-normalized area under the
curve.
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