Discovery of a potent, selective, and orally available class i phosphatidylinositol 3-kinase (PI3K)/Mammalian target of rapamycin (mTOR) kinase inhibitor (GDC-0980) for the treatment of cancer

Article abstract of DOI:10.1021/jm2009327

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 IC₅₀s 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.

Full text of DOI:10.1021/jm2009327

ARTICLE
pubs.acs.org/jmc
Discovery of a Potent, Selective, and Orally Available Class I
Phosphatidylinositol 3-Kinase (PI3K)/Mammalian Target of
Rapamycin (mTOR) Kinase Inhibitor (GDC-0980) for the Treatment of
Cancer
Daniel P. Sutherlin,*,† Linda Bao,† Megan Berry,† Georgette Castanedo,† Irina Chuckowree,‡ Jenna Dotson,†
Adrian Folks,‡ Lori Friedman,† Richard Goldsmith,† Janet Gunzner,† Timothy Heffron,† John Lesnick,†
Cristina Lewis,† Simon Mathieu,† Jeremy Murray,† Jim Nonomiya,† Jodie Pang,† Niel Pegg,‡ Wei Wei Prior,†
Lionel Rouge,† Laurent Salphati,† Deepak Sampath,† Qingping Tian,† Vickie Tsui,† Nan Chi Wan,‡
Shumei Wang,† BinQing Wei,† Christian Wiesmann,† Ping Wu,† Bing-Yan Zhu,† and Alan Olivero†
†Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States
‡Piramed Pharma, 957 Buckingham Avenue, Slough, Berks SL1 4NL, United Kingdom
ABSTRACT: The discovery of 2 (GDC-0980), a class I PI3K
and mTOR kinase inhibitor for oncology indications, is de-
scribed. mTOR inhibition was added to the class I PI3K
inhibitor 1 (GDC-0941) scaffold primarily through the sub-
stitution 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 com-
parison 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 IC50s of 5, 27, 7, and 14 nM for PI3Kα, β, δ, and γ, respectively, inhibits mTOR with a Ki 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.
’ INTRODUCTION
Both PI3K and mTOR have been identified as promising
kinase 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.1À5 An interest in targeting two important
points along this critical signaling pathway, along with the fact
that PI3Ks and mTOR have a high degree of active site similarity,
has spurred the development of PI3K/mTOR inhibitors for
oncology indications.6À8
PI3Kα, a memberofthe classI PI3Kinases (α, β, δ, γ), has been
linked to cancer through the identification of activating muta-
tions in the kinase domain,9À12 loss of function mutations in the
PI3K negative regulator PTEN,13,14 through its key role in trans-
mitting signals to or from known oncogenes (such as EGFR,
HER2, RAS, and AKT)1,2 and as a recognized mechanism for
resistance to known therapies.15 Functionally, this lipid kinase is
responsible for the phosphorylation of phosphatidylinositol
(4,5) bis-phosphate to phosphatidylinositol (3,4,5) tris-phos-
phate, which then recruits additional kinases to the membrane,
activating the oncogene AKT. AKT, in turn is then responsible
for a broad number of downstream signaling events that includes
mTOR activation. Several class I PI3K inhibitors, including 1
Figure 1. Structures of morpholino thienopyrimidine inhibitors of class
I PI3Ks, 1, and class I PI3Ks and mTOR Kinase, 2.
(GDC-0941, Figure 1),16,17 have entered clinical trials. Addi-
tionally, isoform selective compounds have also entered clinical
development for various oncology indications.18
The kinase mTOR, a member of the PIKK (phosphatidylinositol
like kinase) family, is activated downstream of AKT and leads to
increased protein synthesis and growth.5 Rapamycin analogues
such as temsirolimus, which inhibits mTOR when complexed in
Received: July 13, 2011
Published: October 07, 2011
r
2011 American Chemical Society
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Table 1. Relevant Potency and PK Data for Previously Described Molecules 1, 3, and 4a
a Male SpragueÀDawley rats and male beagle dogs were dosed intravenously at 1 mg/kg for rat and dog (1 rat, dimesylate salt in 5% DMSO, 5%
cremaphore (5 mg/kg); dog, dimesylate salt in 30% SBE-β-CD. 3 rat, TFA salt in 5% DMSO, 5% cremaphore; dog, HCl salt in 20% HP-β-CD. 4 rat,
TFA salt in 5% DMSO, 5% cremaphore; dog, HCl salt in 10% HP-β-CD). *Ki app is reported for mTOR, all others are IC50s.16,31,32
part to rictor (mTORC1 complex), have been approved for the
treatment of advanced renal cell carcinoma and mantel cell
lymphoma, validating this target in humans.19 One potential
limitation of exclusive mTORC1 inhibition by rapamycin ana-
logues are that mTOR kinase also participates in the mTORC2
protein complex that can activate the oncogene AKT, through
phosphorylation of AKT Ser473, and promote cell survival
through other signaling mechanisms.20 Experimentally, it has
been shown that inhibition of mTORC1 by rapamycin analo-
gues fails to repress a negative feedback loop that results in the
phosphorylation and activation of AKT.21À24 To avoid this
undesired feedback mechanism and potential to reactivate the
pathway and cause resistance, ATP competitive mTOR kinase
inhibitors that can inhibit both mTORC1 and mTORC2 have
been pursued as alternatives to the rapamycin analogues.25
Given the quantity of evidence implicating both PI3K and
mTOR in cancer, we and others have developed compounds that
inhibit both kinases. Several of these inhibitors have begun early
clinical development.26À29 The discovery of 2 (GDC-0980), also
in the clinic, is described herein. By inhibiting two nodes in this
critical pathway, this compound may provide a therapeutic ad-
vantage relative to inhibitors that target PI3K or mTOR alone.
the scaffold while also maintaining or improving potency and
solubility.
Recent communications from our lab have described the
discovery of the 2-aminopyrimidyl-thienopyrimidine 3 and the
7-methyl analogue 4 (Table 1).31À33 These compounds are po-
tent inhibitors of mTOR and the class I PI3Ks and confirm the
potential to inhibit both kinases in this structural class. In addi-
tion to biochemical assays, these compounds are also potent in
cell-based proliferation assays where human tumor cell growth is
driven by loss of the negative regulator PTEN (PC3 line) or
activating mutations in PI3Kα (MCF7-neo/HER2 line). Prolif-
eration IC50s correlated well with cellular pAKT IC50s measured
in PC3 cells, a downstream readout of pathway inhibition (corre-
lation coefficients of 0.78 and 0.74 for the PC3 and MCF7-neo/
HER2 lines, respectively, when looking across >200 compounds
from multiple scaffolds). The replacement of the indazole with a
2-aminopyrimidine, found in 1 and 3, respectively, was a key
factor in increasing potency for mTOR by 10-fold on average
across a number of diverse morpholino thienopyrimidines. When
this change was applied to the lead compound 1 to generate 3,
the result was a 20-fold improvement in mTOR inhibition.
Proliferation IC50s for potent PI3Kα inhibitors were not drama-
tically affected by additional mTOR inhibition in these two
specific cell lines. This observation was not surprising in that
these two lines used in the discovery phase of the program
depended exclusively on PI3Kα activity for growth.
’ RESULTS AND DISCUSSION
The class I PI3K inhibitor 1 served as a useful template to
explore the potential addition of mTOR potency, as this com-
pound possessed good potency, was selective against a large
panel of kinases, and was efficacious in mice xenograft studies
when dosed orally.16 At the outset of our work, it was known that
the tool compound PI-103, also possessing a morpholino pyrim-
idine core, did inhibit mTOR and PI3Ks, and it was conceivable
that mTOR potency could be acquired through modification
of 1.30 Secondary to this effort, we aimed to improve clearance of
In addition to the increased mTOR inhibition brought about
through the aminopyrimidine substitution, the cLogP of the
scaffold was lowered by 1.9 units via this change and conse-
quently increased the free fraction and the in vitro stability of the
molecules across species (measured logDs for 1 and 3 were 2.4
and 1.3, respectively). To systematically observe these effects, we
examined matched pairs of compounds where the core and
pendant R groups are held constant and the data is compared
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Figure 2. Comparisons of rat liver microsome stability, plasma protein binding, and unbound clearance in rat for matched pairs of compounds having
either an indazole or a 2-aminopyrimidine. Points on each graph represents two compounds where R = indazole (data on the y axis) or R =
2-aminopyrimidine (x axis). Blue points have undisclosed X groups, and the red point indicates the data for compounds 1 and 3 specifically. The black
lines represent the line of unity, and the blue line in (A) represents a 2-fold change from the line of unity. Hepatic clearance in (A) was predicted from
liver microsome incubations using the “in vitro t1/2 method”.35
Scheme 1. Synthesis of Compounds Found in Table 2,a
Where 3 displayed good clearance in rats, methyl substitution
of the core found in 4 was required to give low clearances in dogs
as well. While this change improved dog clearances, compounds
such as 4 suffered from relatively low solubility and was con-
sidered to increase the compounds potential risk of solubility
limited exposure and PK variability in humans. Thus, the focus of
further work was to determine whether analogues could be
generated with better solubility while maintaining the biological
and pharmacokinetic properties of 4. With this goal in mind, we
considered solubility improvements to be best accessed through
modification of the solvent exposed sulfonamide found on the
piperazine ring. This was thought to be possible due to the added
potency afforded by the 2-aminopyrimidine that allowed greater
flexibility in the SAR surrounding the solvent exposed region of
the core.31
Chemistry. Piperazine amides 2 and 8À13 (Scheme 1 and
Table 2) were pursued to maintain the neutral charge of the distal
nitrogen of the piperazine ring to provide a balance of polarity
without added molecular weight and to utilize readily available
amino acid starting materials. We explored both amino and
hydroxy acids to improve solubility and with and without sub-
stitution at the α carbon to modulate metabolic stabilities. Synth-
etically, the amides were installed in the last step due to instability
of this group to the high temperature Suzuki reaction used
to install the aminopyrimidine. Unsubstituted piperazine 7 was
generated from the previously described aldehyde 5 after re-
ductive amination with Boc-piperazine, Suzuki coupling with
2-aminopyrimidine-4-boronic acid, and Boc deprotection of 6.
The final products were made with Boc protected amino acids
which were deprotected following coupling or with the unpro-
tected hydroxy acids to give 2 and 8À13.
a Reagents and conditions: (a) Boc-piperazine, 1,2-DCE, HC(OCH3)3,
Na(OAc)3BH, 96%; (b) 2-aminopyrimidine-5-boronic acid pinacol
ester, Pd(PPh3)2Cl2, 1 M Na2CO3, CH3CN, microwave, 140 °C, 15 min,
82%; (c) 4N HCl in dioxane, DCM, 25 °C, 3 h, 100%; (d) RCOOH,
DIPEA, HATU, DMF, 25 °C, 30 min, 42À56%.
exclusively between the indazole and the aminopyrimidine
(Figure 2AÀC). Figure 2A shows that when 46 indazole/
pyrimidine matched pairs are compared, 21 of the pyrimidines
were >2 fold more stable than the corresponding indazole-
containing analogue in rat liver microsomes. The remaining 30
compounds are within an accepted 2-fold margin of error for this
assay. Additionally, for the comparisons that can be made, the
PPB in rat is also lowered significantly for all compounds
(Figure 2B). These findings were not restricted to rat and could
be seen across species, including human (data not shown). The
increased microsomal stability correlated well with in vivo data
and contributed to equivalent or significantly lower unbound
clearances for all molecules tested in rat (Figure 2C).
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Table 2. Biochemical and Cell-Based Potency, Predicted and in Vivo Hepatic Clearances, and Thermodynamic Solubility for
Compounds 2 and 8À13a
a Male SpragueÀDawley rats were dosed with TFA salts of each compound intravenously at 1 mg/kg as a solution in 5% DMSO/5% cremophor and
dosed orally in 80% PEG400 (or 0.5% methylcellulose)/0.2% Tween-80 for compound 2). *Ki app is reported for mTOR and PI3Kα for compound 11,
all others are IC50s. Hepatic clearance was predicted from liver microsome incubations using the “in vitro t1/2 method”.35
A subset of structurally related analogues are shown in Table 2.
The amine-based glycine, alanine, and dimethyl gylcine deriva-
tives of 4 all had respectable potencies in biochemical and cell-
based assays along with the desired significant improvements in
thermodynamic solubility at pH 6.5. However, in vivo clearance
for the two analogues tested, 8 and 10, were at or above liver
blood flow34 and the compounds had little or no oral bioavail-
ability. The alcohol-based analogues had much better PK proper-
ties as exemplified by 2, 12, and 13, which had increased
solubility, low to moderate clearance, and good oral bioavail-
ability. Compound 11 was not advanced further as the cellular
potency in the PC3 line was above 400 nM. Lactic amide 12 had
biochemical potencies and human microsomal stabilities that
were less preferred relative to it is enantiomer 2, even though
rodent PK and cellular potencies were roughly similar. Lactic
amide 2 was chosen for further study primarily due to its low
predicted clearance in human as measured in vitro with human
liver microsomes, its low in vivo clearance in rat, and maximized
oral bioavailability relative to tertiary alcohol 13. In addition to
measuring cellular proliferation in the PC3 line, direct inhibition
of PI3K could be monitored in these same cells by monitoring
the phosphorylation of Ser473 on AKT. In this assay, 2 had an
IC50 of 36 nM. Additionally, to confirm the binding mode of 2,
we obtained a cocrystal structure of this compound bound to
PI3Kγ (Figure 3). The structure bound in accordance with a
similar previously disclosed amino-pyridine.31
Kinase Selectivity. Previous PI3K inhibitors from this and
other chemical classes have maintained a high degree of selec-
tivity over off-target kinases even while maintaining dual inhibi-
tion for mTOR and the PI3K isoforms. To confirm this, 2 was
submitted for screening in a panel of kinases provided by
Invitrogen’s SelectScreen service. Of the 240 total kinases in
the SelectScreen panel, only 5 kinases other than the PI3K
isoforms and mTOR consistently displayed greater than 60%
inhibition when treated with 1 μM of 2: Fgr, Mlk1, PAK4, Syk,
and Yes1. Measurement of IC50s for the most potently inhibited
kinases in the panel revealed significantly weaker values for the
off-target kinases compared to PI3K/mTOR; IC50s for Fgr,
Mlk1, and Syk were 697, 232, and 134 nM, respectively.
Compound 2 was also remarkably selective for several other
members of the closely related PIKK family kinases: C2alpha
IC50 = 1300 nM;C2beta IC50 = 7 94 nM;VPS34 IC50 = 2000 nM;
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Table 3. Pharmacokinetic and Plasma Protein Binding Data
for Compound 2 in Mousea
IV (1 mg/kg)
Clp Vss
PO
predicted Clh
dose
Cmax AUC F PPB
(mL/min/kg) (mL/min/kg) (L/kg) (mg/kg) (μM) (μM h) %
%
3
33
9.2
1.7
5
2.0
24
10 56 71
121 66
50
a Female nu/nu mice were dosed with the HCl salt, either intraveneously
as a solution in 5% DMSO/5% cremophor, orally at 5 mg/kg in 80%
PEG400, and orally at 50 mg/kg as a crystalline suspension in 0.5%
methylcellulose/0.2% Tween-80. Hepatic clearance was predicted from
mouse liver microsome incubations using the “in vitro t1/2 method”.35
Figure 3. Crystal structure (2.8 Å) of 2 bound to PI3Kγ. The backbone
atoms of PI3Kγ are shown in a cartoon representation with carbon
atoms colored gray, 2 is shown in ball and stick representation with
carbon atoms colored cyan. Critical contacts include the morpholine
oxygen with the hinge residue Val882 and key contacts from the amino-
pyrimidine NH2 to the carbonyl oxygens of Asp841 and Asp964.
Crystals were grown at 18 °C from 20% PEG 3350, 0.2 M NH4SO4
by hanging drop vapor diffusion.16
vehicle treated group based on Dunnett’s t test on day 14 (for PC3)
and day 22 (for MCF-7 neo/HER2)]. The significant antitumor
activity of compound 2 at low doses is likely due to high cellular
potency, low plasma clearance, and relatively high free fraction of
the drug in vivo.
’ CONCLUSION
PI4Kalpha >10 μM; PI4Kbeta >10 μM; DNA-PK Kiapp = 623 nM,
respectively. Thus, the biochemical data illustrate that 2 had the
desired selectivity profile, with potent inhibition of all class I PI3K
isoforms and mTOR and selectivity over the other profiled PIKK
family enzymes and a large, diverse panel of kinases.
Pharmacokinetics. On the basis of this promising profile, 2
was further characterized through PK studies conducted in
several preclinical species. PK parameters obtained in mice after
intravenous administration at 1 mg/kg and when dosed orally at
5 and 50 mg/kg are highlighted in Table 3 and are relevant to
xenograft studies discussed herein. Clearance and PPB were low,
and 2 showed dose-proportional exposure from 5 mg/kg dosed
in PEG to 50 mg/kg dosed in suspension in MCT, a finding
attributed partially to the compound’s good solubility. PK param-
eters for 2 were also favorable in the other species tested, in-
cluding dog (data not shown, manuscript in preparation). Plasma
protein binding was similar across species.
A potent and selective inhibitor of class I PI3 kinases and
mTOR kinase was developed using lead compound 1 as a struc-
tural template. The replacement of an indazole with a 2-amino-
pyrimidine was responsible for increased potency for mTOR,
lower PPB, and improved predicted and measured total and
unbound clearances in rats and mice. A methyl group was added
to the thienopyrimidine core to lower the in vivo clearances in
rats and dogs further. Finally, a lactic amide was used to replace a
sulfonamide in order to increase the thermodynamic solubility at
a neutral pH, resulting in the discovery of 2. On the basis of the
potency for PI3K and mTOR, selectivity against other kinases,
pharmacokinetics, solubility, and efficacy in PI3K driven animal
models of cancer, this compound was advanced into develop-
ment where it is currently in phase I clinical trials.
’ EXPERIMENTAL SECTION
In Vivo Efficacy. We evaluated the efficacy of compound 2
in vivo using xenografts derived from the same two tumor cell
lines that were used to screen for cellular potency: PC-3 and
MCF-7 neo/HER2 (Figure 4). The compound was dosed orally
as a crystalline suspension in 0.5% methylcellulose/0.2% Tween-
80 (MCT) once a day for either 14 or 22 days, and the tumor
volume was measured relative to control animals treated with
MCT vehicle only. In both PC-3 and MCF-7 neo/HER2 xeno-
graft models, treatment with 1 mg/kg of compound 2 resulted
in statistically significant antitumor activity, characterized as tu-
mor growth delay [p < .001 vs vehicle treated group based on
Dunnett’s t test on day 14 (for PC3) and day 22 (for MCF-7 neo/
HER2)]. At the maximum tolerated dose of 7.5 mg/kg, com-
pound 2, tumor stasis or regressions were observed in the PC-3 and
MCF-7 neo/HER2 xenograft models, respectively [p < .0001 vs
Chemistry. All solvents and reagents were used as obtained.
1H NMR spectra were recorded with a Bruker Avance DPX400 spectrom-
eter or a Varian Inova 400 NMR spectrometer and referenced to
tetramethyl silane. Chemical shifts are expressed as δ units using
tetramethylsilane as the external standard (in NMR description, s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and br, broad peak).
Mass spectra were measured with a Finnigan SSQ710C spectrom-
eter using an ESI source coupled to a Waters 600MS HPLC system
operating in reverse mode with an X-bridge Phenyl column of dimen-
sions 150 mm by 4.6 mm, with 5 μm sized particles. All compounds were
purified to g95% purity using reversed phase HPLC.
2-Chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidine-
6-carbaldehyde (5). To 4-(2-Chloro-7-methylthieno[3,2-d]pyrimidin-
4-yl)morpholine (3.18 g, 11.8 mmol) in THF (65 mL) at À78 °C
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Figure 4. In vivo efficacy for 2 in PC3 and MCF-7 neo/HER2 xenograft tumor models when dosed orally at 1 and 7.5 mg/kg on a once daily schedule.
was added a 2.5 M solution of n-butyllithium in hexane (10.3 mL). The
resulting solution was warmed to À40 °C and stirred 2 h. DMF (4.2 mL,
54.2 mmol) wasadded, andthereactionmixture stirred2 hatÀ40 °C. The
reactionwasquenchedby pouring slowly onto 0.25 M HClwith ice. After 1
h, the solid was filtered and lyophilized to yield 3.44 g of (5). 1H NMR
(400 MHz, CDCl3) δ 10.32 (s, 1H), 4.02 (m, 4H), 3.79 (m, 4H), 2.68 (s,
3H). MS: (ESI+) 298.1.
of 4 M HCl in 1,4-dioxane and stirred for 3 h. The solution was evapo-
rated and dried overnight under vacuum to afford 7 in 100% yield. NMR
(400 MHz, DMSO) δ 9.15 (s, 2H), 7.06 (s, 2H), 4.05À3.86 (m, 4H),
3.85À3.69 (m, 6H), 2.70 (brs, 4H), 2.42 (brs, 4H), 2.34 (s, 3H). MS:
(ESI+) 427.2.
2-Amino-1-(4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-
morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-
1-yl)ethanone (8). N,N-Diisopropylethylamine (150 μL, 0.87 mmol)
was added to a solution of 5-(7-methyl-4-morpholino-6-(piperazin-
1-ylmethyl)thieno[3,2-d]pyrimidin-2-yl)pyrimidin-2-amine 7 (74 mg,
0.17 mmol) in DMF (1 mL). HATU (97 mg, 0.26 mmol) and Boc-
glycine (60 mg, 0.3 mmol) was added to the reaction mixture, which was
stirred for 30 min at room temperature. The reaction mixture was diluted
with ethyl acetate (10 mL) and extracted with a saturated sodium bicar-
bonate solution (10 mL). The organic layer was dried with magnesium
sulfate, filtered, and then concentrated to afford the penultimate pro-
duct. This intermediate was then subjected to a Boc-deprotection with
TFA before final reverse-phase HPLC purification to give 75.3 mg
(56%) of 8 (2Â TFA salt) as a white solid. 1H NMR (400 MHz, DMSO)
δ 9.18 (s, 2H), 8.03 t, J = 5.2 Hz, 3H), 7.15 (brs, 2H), 4.00À3.94 (m,
4H), 3.89 (q, J = 5.2 Hz, J = 10.8 Hz, 2H), 3.82À3.76 (m, 4H), 3.51 (br
m, 4H), 2.89 (br m, 4H), 2.42 (s, 3H). MS: (ESI+) 484.1.
tert-Butyl
4-((2-chloro-7-methyl-4-morpholinothieno-
[3,2-d]pyrimidin-6-yl)methyl)piperazine-1-carboxylate. 2-
Chloro-7-methyl-4-morpholinothieno [3, 2-d] pyrimidine-6-carbalde-
hyde (0.58 g) and tert-butyl piperazine-1-carboxylate (0.52 g) were
dissolved in 1,2-dichloroethane (10 mL). Acetic acid (1.22 mL) was
added and the reaction stirred 15 min before the addition of sodium
triacetoxyborohydride (0.55 g). The resulting solution was stirred 24 h
at room temperature. The reaction mixture was quenched with saturated
aqueous NaHCO3, extracted with CH2Cl2, dried over Na2SO4, filtered,
and concentrated in vacuo. The crude material was purified by flash
chromatography (EtOAc/hexanes) to provide 880 mg of tert-butyl
4-((2-chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-
1
yl)methyl)piperazine-1-carboxylate. H NMR (400 MHz, DMSO) δ
3.88 (m, 4H), 3.82 (s, 2H), 3.74 (m, 4H), 3.34 (m, 4H), 2.43 (m, 4H),
2.23 (s, 3H), 1.39 (s, 9H). MS: (ESI+) 468.3.
(S)-2-Amino-1-(4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-
morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-
propan-1-one (9). Compound 9 was prepared from intermediate
7 and Boc-L-alanine by the procedure for compound 8 in 42% yield.
1H NMR (400 MHz, DMSO) δ 9.18 (s, 2H), 8.09 (d, J = 3.8 Hz, 3H),
7.10 (br s, 2H), 4.43À4.32 (m, 1H), 4.25 (br s, 2H), 4.01À3.93 (m, 4H),
3.83À3.75 (m, 5H), 3.62 (br m, 4H), 2.89 (br m, 4H), 2.42 (s, 3H), 1.31
(d, J = 6.8 Hz, 3H). MS: (ESI+) 498.1.
2-Amino-1-(4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-mor-
pholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-
2-methylpropan-1-one (10). Compound 10 was prepared from
intermediate 7 and N-Boc-aminoisobutyric acid by the procedure for
compound 8 in 56% yield. 1H NMR (400 MHz, DMSO) δ 9.18 (s, 2H),
8.12 (s, 3H), 7.09 (br s, 2H), 4.32 (br s, 2H), 4.03À3.91 (m, 4H),
3.90À3.53 (m, 4H), 2.93 (br m, 4H), 2.43 (s, 3H), 1.55 (s, 6H). MS:
(ESI+) 512.1.
tert-Butyl 4-((2-(2-Aminopyrimidin-5-yl)-7-methyl-4-mor-
pholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazine-1-
carboxylate (6). tert-Butyl 4-((2-chloro-7-methyl-4-morpholinothieno-
[3,2-d]pyrimidin-6-yl)methyl)piperazine-1-carboxylate (2.74 g, 5.8 mmol)
was combined with bis(triphenylphosphine)palladium(II) chloride (0.21 g)
and 2-aminoprimidine-5-boronic acid pinacol ester (1.68 g) in 12 mL of
acetonitrile and 12 mL of 1 M sodium carbonate in water. The reaction
mixture was heated in a 50 mL microwave vessel and microwaved at
300 W to 140 °C for 12 min. After cooling to room temperature, the
resultant gray solid was washed with water and acetonitrile before
solubilizing in a mixture of DMF, methanol, and DCM and stirred with
charcoalovernightprior tofiltering. This filtrate was treatedwith florisil for
2 h, filtered, and evaporated to give 6 as an off-white solid (2.52 g). 1H
NMR (400 MHz, DMSO) δ 9.15 (s, 2H), 7.06 (s, 2H), 3.97À3.91 (m,
4H), 3.83 (s, 2H), 3.80À3.74 (m, 4H), 3.34 (brs, 4H), 2.48À2.41 (m,
4H), 2.35 (s, 3H), 1.40 (s, 9H). MS: (ESI+) 527.2.
(7-Methyl-4-morpholino-6-(piperazin-1-ylmethyl)thieno-
[3,2-d]pyrimidin-2-yl)pyrimidin-2-amine (7). tert-Butyl 4-((2-
(2-aminopyrimidin-5-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-
6-yl)methyl)piperazine-1-carboxylate 6 (9.0 g) was dissolved in 43 mL
1-(4-((2-(2-Aminopyrimidin-5-yl)-7-methyl-4-morpholino-
thieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2-hydro-
xyethanone (11). Compound 11 was prepared from intermediate 7
and 2-hydroxyacetic acid by the procedure for compound 8 in 42% yield
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but did not necessitate final Boc-deprotection prior to reverse phase
HPLC purification. 1H NMR (400 MHz, DMSO) δ 9.15 (s, 2H), 7.04
(s, 2H), 4.51 (t, J = 5.5 Hz, 1H), 4.07 (d, J = 5.5 Hz, 2H), 3.97À3.91 (m,
4H), 3.85 (s, 2H), 3.81À3.75 (m, 4H), 3.54À3.45 (m, 2H), 3.42À3.32
(m, 2H), 2.35 (s, 3H). MS: (ESI+) 485.3.
All IC50 or Kiapp values for both biochemical and cellular assays represent
geometric means of multiple replicates or of duplicates. Biochemical
assays typically showed <30% difference between duplicates or repli-
cates and cellular assays were typically within 2-fold agreement between
replicates.
In Vivo Xenograft Studies. Human prostate cancer PC3 cells
obtained from the National Cancer Institute (Frederick, MD) were
resuspended in Hank’s Balanced Salt Solution and 3 Â 106 cells
implanted subcutaneously into the right hind flank of athymic nu/nu
(nude) mice. Tumors were monitored until they reached a mean tumor
volume of 150À200 mm3 prior to the initiation of dosing. MCF7.1 cells
resuspended in a 1:1 mixture of Hank’s Buffered Salt Solution and
Matrigel Basement Membrane Matrix (no. 356237, BD Biosciences;
Santa Cruz, CA) were 5 Â 106 subcutaneously implanted into the right
hind flank of athymic nu/nu (nude) mice. Prior to cell inoculation, 17β-
estradiol (0.36 mg/pellet, 60-day release, no. SE-121) obtained from
Innovative Research of America (Sarasota, FL) were implanted into the
dorsal shoulder blade area of each nude mouse. After implantation of
cells, tumors were monitored until they reached a mean tumor volume of
250À350 mm3 prior to initiating dosing. Compound 2 was dissolved in
0.5% methylcellulose with 0.2% Tween-80 (MCT). Female nude (nu/
nu) mice that were 6À8 weeks old and weighed 20À30 g were obtained
from Charles River Laboratories (Hollister, CA). Tumor bearing mice
were dosed daily for 14À21 days depending on the xenograft model with
100 μL of vehicle (MCT) or test agent orally.
Tumor volume was measured in two dimensions (length and width)
using Ultra Cal-IV calipers (model 54À10À111; Fred V. Fowler Company;
Newton, MA) and was analyzed using Excel version 11.2 (Microsoft
Corporation; Redmond, WA). The tumor volume (mm3) = (longer
measurement  shorter measurement2)  0.5. Animal body weights
were measured using an Adventurer Pro AV812 scale (Ohaus Corpora-
tion; Pine Brook, NJ). Percent weight change = [1 À (new weight/initial
weight)] Â 100. Tumor sizes were recorded twice weekly over the
course of the study (14À21 days). Mouse body weights were also
recorded twice weekly, and the mice were observed daily. Mice with
tumor volumes g2000 mm3 or with losses in body weight g20% from
their initial body weight were promptly euthanized per IACUC guide-
lines. Meantumor volumeand SEM values (n = 10) were calculated using
JMP statistical software, version 5.1.2 at end of treatment. % Tumor
inhibition = 100(mean volume of tumors in vehicle treated animals À
mean volume of tumors in test article treated animals given the test
article)/mean volume of tumors in vehicle treated animals. Data were
analyzed and p-values were determined using the Dunnett t test with JMP
statistical software, version 5.1.2 (SAS Institute; Cary, NC).
(S)-1-(4-((2-(2-Aminopyrimidin-5-yl)-7-methyl-4-morpho-
linothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2-
hydroxypropan-1-one (2). Compound 2 was prepared from inter-
mediate 7 and L-lactic acid by the procedure for compound 11 in 87%
yield. Chiral HPLC analysis for 2 was performed with a Chiral Techno-
logies Chiralcel OJ-RH (4.6 mm  150 mm, 5 μm) column and found
to be >99% enantiomerically pure (isocratic elution with 0.1% DEA in
methanol (v/v), run time = 20 min, flow rate of 1.0 mL/min, detection =
270 nm, injection volume = 5 μL, 2 tR = 5.8 min, 12 tR = 7.6 min). 1H
NMR (400 MHz, DMSO-d6) δ 1.18 (d, J = 6.53 Hz, 3H), 2.34 (s, 3H),
2.38À2.60 (br, 4H), 3.36À3.64 (br, 4H), 3.67À3.78 (m, 4H), 3.84
(s, 2H), 3.89À4.00 (m, 4H), 4.35À4.48 (m, 1H), 4.84 (d, J = 6.98 Hz,
1H), 7.05 (s, 2H), 9.15 (s, 2H). MS: (ESI+) 499.1.
(R)-1-(4-((2-(2-Aminopyrimidin-5-yl)-7-methyl-4-morpho-
linothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2-
hydroxypropan-1-one (12). Compound 12 was prepared from
intermediate 7 and L-lactic acid by the procedure for compound 11 in
61% yield. 1H NMR (400 MHz, DMSO) δ 9.15 (s, 2H), 7.02 (s, 2H),
4.81 (d, J= 6.9 Hz, 1H), 4.41 (dq, J= 6.6 Hz, 1H), 3.97À3.91 (m, 4H), 3.84
(s, 2H), 3.81À3.74 (m, 4H), 3.55À3.41 (br m, 4H), 2.55À2.41 (br m,
4H), 2.35 (s, 3H), 2.07 (s, 2H), 1.18 (d, J = 6.5 Hz, 3H). MS: (ESI+) 499.1.
1-(4-((2-(2-Aminopyrimidin-5-yl)-7-methyl-4-morpholino-
thieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2-hydroxy-
2-methylpropan-1-one (13). Compound 13 was prepared from
intermediate 7 and 2-hydroxy-2-methylpropanoic acid by the procedure
1
for compound 11 in 54% yield. H NMR (400 MHz, DMSO) δ 9.16
(s, 2H), 7.04 (s, 2H), 5.35 (s, 1H), 3.98À3.91 (m, 5H), 3.85À3.81 (m,
2H), 3.81À3.75 (m, 5H), 2.51À2.47 (m, 4H), 2.35 (s, 3H), 1.31 (s, 6H).
MS: (ESI+) 513.3.
Characterization of Biochemical and Cellular Activity in
Vitro. Enzymatic activity of the class I PI3K isoforms and human
recombinant mTOR were measured using a fluorescence polarization
assay as described previously.31 VPS34 and C2b were assayed using a
scintillation proximity assay as described previously.16 DNA-PK was
assayed using a capillary electrophoresis assay that monitors production
of phosphorylated peptide as well as loss of unphosphorylated sub-
strate. In a total volume of 5 μL, DNA-PK (2 units/uL; Promega) was in-
cubated with 1.5 μM peptide substrate (5-FAM-AEPPLSQEAFADL-
WKK-NH2.) in the presence of 10 mM Hepes (pH 7.2), 10 mM MgCl2,
10 μM ATP, 10 μg/mL calf thymus DNA, 0.015% (v/v) Brij-35, 1 mM
dithiothreitol, and 2% dimethylsulfoxide.36 After incubation for 60 min
at 25 °C the reaction was terminated with a final concentration of 25 mM
Hepes (pH 7.2), 0.0037% Brij-35, 37.5 mM EDTA. DoseÀresponse
curves were fit to an equation for tight-binding inhibition using the
measured Km for ATP for DNA-PK of 9.3 μM.
Accession Codes
The coordinates and structure factors for compound 2 has been
deposited (PDB code 3TL5).
’ AUTHOR INFORMATION
Kinase selectivity was measured using the SelectScreen service at In-
vitrogen (LifeTechnologies), profiling 145 or 240 kinases for % inhibi-
tion at 1 μM compound using the Z-lyte assay format or the Adapta assay
format as specified by Lifetechnologies.
Corresponding Author
*Phone: 650-225-3171. Fax: 650-225-2061. E-mail: sutherlin.dan@
gene.com.
IC50s were calculated from the fit of the doseÀresponse curves to a
four-parameter equation and were measured at the experimental Km for
ATP for the kinase of interest. Alternatively, apparent Kis were calculated
by fitting the data to an equation for tight-binding inhibition as previ-
ously described.31
’ ACKNOWLEDGMENT
We thank Mengling Wong, Martin Struble, Yanzhou Liu,
and Wen Chiu for compound purification and determination
of purity by HPLC, mass spectroscopy, and 1H NMR. We thank
Krista K. Bowman, Alberto Estevez, Kyle Mortara, and Jiansheng
Wu for technical assistance of protein expression and purifica-
tion. We thank Emil Plise for plasma protein binding data. We are
grateful to Michelle Nannini, Janeko Bower, and Alex Vanderbilt
Antiproliferative cellular assays were conducted as described31 using
PC3 and MCF7.1 human tumor cell lines provided by the ATCC or
Genentech Research laboratories, respectively. MCF7.1 is an in vivo
selected line developed at Genentech and originally derived from the
parental human MCF7 breast cancer cell line (ATCC, Manassas, VA).
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Journal of Medicinal Chemistry
ARTICLE
for coordinating and dosing animals for the reported in vivo
efficacy studies.
Moore, P.; Olivero, A. G.; Pang, J.; Patel, S.; Pergl-Wilson, G. H.;
Raynaud, F. I.; Robson, A.; Saghir, N.; Salphati, L.; Sohal, S.; Ultsch,
M. H.; Valenti, M.; Wallweber, H. J. A.; Wan, N. C.; Wiesmann, C.;
Workman, P.; Zhyvoloup, P.; Zvelebil, M. J.; Shuttleworth, S. J. The
Identification of 2-(1H-Indazol-4-yl)-6-(4-methanesulfonyl-piperazin-
1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941)
as a Potent, Selective, Orally Bioavailable Inhibitor of Class I PI3 Kinase
for the Treatment of Cancer. J. Med. Chem. 2008, 51, 5522–5532.
(17) Salphati, L.;Wong, H.;Belvin, M.;Bradford, D.;Edgar, K. A.;Prior,
W. W.; Sampath, D.; Wallin, J. J. PharmacokineticÀpharmacodynamic
modeling of tumor growth inhibition and biomarker modulation by the
novel phosphatidylinositol 3-kinase inhibitor GDC-0941. Drug. Metab.
Dispos. 2010, 38, 1436–1442.
(18) Lannutti, B. J.; Meadows, S. A.; Herman, S. E. M.; Kashishian,
A.; Steiner, B.; Johnson, A. J.; Byrd, J. C.; Tyner, J. W.; Loriaux, M. M.;
Deininger, M.; Druker, B. J.; Puri, K. J.; Ulrich, R. G.; Giese, N. A. CAL-
101, a p110delta selective phosphatidylinositol-3-kinase inhibitor for the
treatment of B-cell malignancies, inhibits PI3K signaling and cellular
viability. Blood 2011, 117, 591–594.
’ ABBREVIATIONS USED
AKT, protein kinase B; DCE, dichloroethane; DMF, dimethyl-
formamide; DMSO, dimethylsulfoxide; EGFR, epidermal growth
factor receptor 1; HATU, (2-(7-aza-1H-benzotriazole-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate); HER2, epi-
dermal growth factor receptor 2; PEG, polyethylene glycol;
PI3K, phosphoinositide 3-kinase; PPB, plasma protein binding;
PTEN, phosphatase and tensin homologue; mTOR, mammalian
target of rapamycin; TFA, trifluoroacetic acid; THF, tetrahydrofuran
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(36) PK/PD studies were performed in tumor bearing animals at
doses similar to those used in the PC3 xenograft efficacy study and
showed evidence of pharmacologic inhibition of the pathway. At a 5 and
10 mg/kg doses, pAKT was reduced to 385% of vehicle control at all time
points up to 8 h and 63% and 78% of vehicle control at 24 h with
unbound plasma concentrations of 0.05 mM and 0.45 mM, respectively.
A more detailed account of PK/PD and efficacy of this molecule is
forthcoming.
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