Discovery of (thienopyrimidin-2-yl)aminopyrimidines as potent, selective, and orally available Pan-PI3-kinase and dual Pan-PI3-kinase/mTOR inhibitors for the treatment of cancer

Article abstract of DOI:10.1021/jm901284w

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 in

Full text of DOI:10.1021/jm901284w

1086 J. Med. Chem. 2010, 53, 1086–1097
DOI: 10.1021/jm901284w
Discovery of (Thienopyrimidin-2-yl)aminopyrimidines as Potent, Selective, and Orally Available
Pan-PI3-Kinase and Dual Pan-PI3-Kinase/mTOR Inhibitors for the Treatment of Cancer
Daniel P. Sutherlin,*,† Deepak Sampath,† Megan Berry,† Georgette Castanedo,† Zhigang Chang,† Irina Chuckowree,‡
Jenna Dotson,† Adrian Folkes,‡ Lori Friedman,† Richard Goldsmith,† Tim Heffron,† Leslie Lee,† John Lesnick,†
Cristina Lewis,† Simon Mathieu,† Jim Nonomiya,† Alan Olivero,† Jodie Pang,† Wei Wei Prior,† Laurent Salphati,†
Steve Sideris,† Qingping Tian,† Vickie Tsui,† Nan Chi Wan,‡ Shumei Wang,† Christian Wiesmann,† Susan Wong,† and
Bing-Yan Zhu†
†Genentech, Inc., 1 DNA Way, South San Francisco, California 94080 and ‡Piramed Pharma, 957 Buckingham Avenue, Slough, Berks SL1 4NL,
United Kingdom
Received August 27, 2009
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 discove-
red as potent inhibitors of PI3K or both PI3K and mTOR. Structural information derived from
PI3Kγ-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 PI3KR mutated
MCF7.1 xenograft model and were found to have equivalent efficacy when normalized for exposure.
Introduction
4-phosphate and are generally resistant to class I inhibitors.
Class III PI3Ks phosphorylate only phosphatidylinositol to
generate phosphatidylinositol 3-phosphate. The sole member
of this class, known as Vps34, functions in protein trafficking
and autophagy.2 A fourth class of PI3K-related enzymes
(PIKK), which contains a catalytic core similar to the PI3Ks,
is involved in tumor growth and DNA-damage response as
mediated by mTOR and DNA-dependent protein kinase,
respectively.3
Upon formation of PIP3 by class I PI3Ks, PDK1 is
recruited to the plasma membrane and in turn phosphorylates
Akt, a serine/threonine kinase that integrates multiple signal-
ing pathways involved in growth and metabolism regulation.4
The activation of Akt results in phosphorylation of several
proximal substrates, including mTOR and PRAS40, which
triggers a cascade of signaling events that culminates in the
activation of mTOR, a serine/threonine kinase that regulates
cellular growth, nutrient metabolism, and protein translation.
Class IA PI3Ks, and PI3KR in particular, are promising
therapeutic targets for cancer based on the identification of
activating mutations in the PI3KR catalytic subunit and loss
of function of its negative regulator PTEN in a notable
percentage of solid human tumors representing, breast, pros-
tate, ovarian, colorectal cancer, and glioblastoma.5 In addi-
tion, PI3Ks are activated upstream by known oncogenes such
as HER2, EGFR, and Ras, all of which lead to aberrant
activation of the pathway. Accordingly, pharmaceutical and
academic laboratories are actively pursuing inhibitors of the
central nodes of the PI3K/Akt/mTOR pathway, and PI3KR
inhibitors in particular, for the treatment of human cancers.6
Deregulation of the mTOR pathway has also been strongly
The PI3K/Akt/mTORa pathway plays a central role in cell
proliferation, migration, survival, and angiogenesis upon acti-
vation by growth factor or integrin receptors. PI3Ks are lipid
kinases, which can be divided into three classes: Class I, II, and
III. Class I PI3Ks are activated by growth factor receptors or
G-protein coupled receptors that are subdivided into Class IA
and Class IB enzymes, respectively.1 Class IA PI3Ks exist as
heterodimers that contain one of three PI3K catalytic subunits
(PI3KR, PI3Kβ, and PI3Kδ) and a p85 regulatory subunits,
whereas Class IB PI3Ks consist of a PI3Kγ catalytic sub-
unit and a p101 regulatory subunit.1 Class I PI3Ks convert
the membrane bound substrate PIP2 (4,5-phosphatidyl-
inositolbisphosphate) to PIP3 (3,4,5-phosphatidylinositoltris-
phosphate), which provides lipid docking sites for the pleckstrin
homology domains of the effector kinases PDK1 and Akt.1
PTEN and other phosphatases negatively regulate PI3K by
dephosphorylation of PIP3 to PIP2, attenuating further
downstream signaling.1 Class II PI3Ks display the ability to
phosphorylate phosphatidylinositol and phosphatidylinositol
*To whom correspondence should be addressed. E-mail: sutherlin.dan@
gene.com; phone: 650-225-2061; fax: 650-225-3171.
a Abbreviations: Akt, protein kinase B; DMF, dimethylformamide;
DMSO, dimethylsulfoxide; EGFR, epidermal growth factor receptor 1;
HER2, epidermal growth factor receptor 2; PEG, polyethylene glycol;
PRAS40, proline-rich Akt substrate of 40 kDa; PI3K, phosphoinositide
3-kinase; PDK1, pyruvate dehydrogenase kinase isoform 1; PTEN,
phosphatase and tensin homologue; pS6 RP, phosphorylated S 6
ribosomal protein; mTOR, mammalian target of rapamycin; TFA,
trifluoroacetic acid; THF, tetrahydrofuran; DCE, dichloroethane;
SAR, structure activity relationships.
r
pubs.acs.org/jmc
Published on Web 01/05/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1087
Figure 1. Discovery of compounds 5 and 21, starting from phenols 1 and 2 (GDC-941).
implicated in cancer and inhibitors of this pathway are
currently under clinical development.3b In addition, mTOR
is a validated target in humans as demonstrated by the clinical
use of Torisel (temsirolimus), approved for the treatment of
advanced renal cell. Because both PI3K and mTOR are
known to activate Akt independently, developing dual inhibi-
tors of both PI3KR and mTOR is a potential strategy for
generating more effective therapeutics for the treatment of
human cancer.7
The discovery of the pan isoform (R, β, δ, γ) PI3K inhibitor,
2, starting from the simple morpholino thienopyrimidine1 has
previously been described.8 Key discoveries leading to the
clinical compound 2 were the identification of an indazole as a
phenolic isostere which significantly improved the oral bio-
availability of the class and the addition of a piperazino-
methane sulfonamide substituent to the C-6 position of the
thienopyrimidine core that improved potency and metabolic
stability relative to compound 1.
Herein we describe our efforts to identify novel PI3K
inhibitors that are structurally diverse from 2 and that have
varying activity toward mTOR. In order to do this, we
examined nonisostere replacements of the phenol resulting
in new interactions within the same binding site, highlighted
by the aminopyrimidine 4, a compound that possessed in-
creased mTORpotency relativeto2. We also strove to remove
potential sites of metabolism and reduce the molecular weight
of the lead, resulting in tertiary alcohol 3. These two directions
of research were continued, culminating in the discovery of 5,
a low molecular weight, potent dual inhibitor of pan-PI3
kinases and mTOR. Following this, we discovered 21, a
structurally similar compound to 5, which lacked mTOR
but maintained pan-PI3K inhibitory activity.
following similar procedures previously described in this
journal, where DMF was used as an electrophile to prepare
aldehyde 7 followed by reductive amination and Suzuki
coupling.8 Aldehyde 7 was also used to prepare compounds
17 and 18 by either reduction with sodium borohydride or
addition of methyl Grignard to give the primary and second-
ary alcohols, respectively. The alcohol intermediate 9 could be
converted to the acylated amine 19 through a series of steps
that include bromination, transformation to an amine, acyla-
tion, and coupling. Alternatively, the anion of 6 could be
trapped with acetone to give a tertiary alcohol which was
subsequently used in a cross coupling reactions to generate
compounds 3 and 5.
Results and Discussion
The potency of each compound for inhibition of purified,
recombinant PI3K isoforms R, β, γ, δ was determined using a
competitive displacement fluorescence polarization assay
monitoring formation of the PIP3 product (Table 1).
Despite some minor differences in potency for the isoforms,
the compounds in Table 1-3 were generally pan-inhibitors of
Synthesis
Compounds were prepared generally in a two-step process
from common intermediate 6 (Scheme 1). In all cases, the C-6
position of the thienopyrimidine was functionalized prior to
performing a Suzuki coupling with the C-2 aryl chloride and
the corresponding boronic ester or acid to yield the 2-aryl
thienopyrimidines. Compounds 4 and 10-16 were prepared
Figure 2. Crystal Structure of 2 in PI3Kγ.

1088 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Scheme 1. Synthesis of compounds found in Tables 1-3a
Sutherlin et al.
a Reagents and conditions: (a) nBuLi, THF, -78 °C, DMF; (b) N-sulphonylpiperazine, 1,2-DCE, HC(OCH3)3, Na(OAc)3BH; (c) boronic acid/ester,
Pd(PPh3)2Cl2, NaHCO3, H2O, CH3CN, microwave, 150-140 °C, 15-30 min; (d) NaBH4, MeOH; (e) PBr3, benzene, 80 °C, 1 h; (f) phthalimide, K2CO3,
DMF, 20 h; (g) N2H4, MeOH, 65 °C, 1 h; (h) AcCl, CH2Cl2, rt, 16 h; (i) nBuLi, THF, -78 °C, acetone; (j) MeMgBr, THF, 0 °C.
Table 1. Inhibition of PI3KR, β, γ, δ, and Cancer Cell Proliferation for
Phenol Replacements for Compounds 2, 4, and 10-16a
the class I PI3-Kinases, with the greatest fold-selectivity for
PI3KR of approximately 20-fold versus other PI3K isoforms.
The antiproliferative activity was compared in the PTEN-null
human tumor prostate cancer cell line PC3 and the PI3KR
mutant breast cancer cell line MCF7.1. The clinical lead 2 has
biochemical potencies of 3 nM, 32 nM, 3 nM, and 66 nM,
versus PI3K R, β, δ, and γ, respectively, and cellular antipro-
liferative potencies in the PC3 and MCF7.1 cell lines of
0.39 μM, and 0.34 μM, respectively.
In order to design phenol replacements, we relied on X-ray
crystal structures of 1 and 2 bound to PI3Kγ to observe the
interactions that were contributing to the potency of these
compounds as well as to evaluate the potential for other
beneficial interactions. All four Class I PI3K isoforms are
highly homologous within the active site and all residues
discussed herein are conserved. As previously described, the
morpholine in both molecules makes a key contact with
the backbone NH of the hinge and is critical for potency.9
The thienopyrimidine core can direct substituents toward
solvent-exposed regions of the binding site from the C-6
position of the heterocycle as seen in 2, and can position a
C-2 aryl group into an affinity pocket, (exemplified by the
indazole of 2, Figure 2). From the X-ray cocrystal structure of
PI3Kγ and 2 it was observed that the indazole 2-nitrogen
makes a key hydrogen bond with the OH of Tyr 867 and the
indazole NH forms an interaction with Asp 841. We recogni-
zed that there were a number of potential hydrogen bonding
interactions that were not utilized by the indazole of 2 or the
phenol of compound 1. We theorized that these residues, Asp
836 as an example(PI3Kγ numbering), could form productive
binding partners with a variety of structures that were neither
phenols nor phenol isosteres.
Synthetically, the C-2 aryl group was installed at the last
step of the synthesis and could easily be modified with a large
number of boronic acids or esters. As a comparison to 2,
compound 10 was very potent (Table 1), but as previously
described,8 lacked oral bioavailability. In contrast to 10, the
a ND = not determined.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1089
Figure 3. Crystal structures of 14 (panel A) and 16 (panel B) bound to PI3Kγ.
Table 2. Inhibition of PI3KR and PC3 Cancer Cell Proliferation; Hepatic Clearance Calculated from in Vitro Rat Liver Microsome Incubations; and
Pharmacokinetic Results for Compounds Intravenously and Orally Dosed in Rats for Alcohol Analoguesa
a Male Sprague-Dawley rats were dosed with mono TFA salts of each compound intravenously at 1 mg/kg and orally at 2 mg/kg as a solution in
5% DMSO/76% PEG400. Hepatic clearance was predicted from liver microsome incubations using the “in vitro t1/2 method”.11
Table 3. IV and PO PK Data for 5 in Mouse, Rat, Cynomolgus Monkey, and Doga
IV (1 mg/kg)
PO
species
in vitro Cl (mL/min/kg)
in vitro Cl (mL/min/kg)
Vss (L/kg)
dose (mg/kg)
C
max (μM)
AUC (μM*h)
F%
PPB%
mouse
rat
45
23
17
9
2.8
2.8
3.0
1.6
5
5
2
2
2.1
2.2
1.1
4.4
14
21
3.8
63
100
83
64
49
79
73
cyno
dog
5.4
1.8
15
1.6
62
100
a Female nu/nu mice were dosed orally with compounds as a solution in 60% PEG.
unsubstituted phenyl derivative 13 was over 400-fold less
potent, highlighting the critical interaction that the phenol
has with Asp 841. Potency was regained by 3-hydroxymethyl
phenyl 14 and demonstrated that activity could be attained

1090 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Sutherlin et al.
without directly mimicking the phenol. 3-Substituted pyridyl
11 was also capable of recovering activity whereas the
4-pyridyl 12 could not. On the basis of this SAR, it was
thought that 3-pyridyl 11 could be making contacts to Tyr867
and Asp841 through a water-mediated H-bond. Interestingly,
the crystal structure of 14 shows a clear interaction with
the benzylic alcohol and Tyr 867 and Asp 841, anticipating
the position of a potential water molecule bound to a pyridyl
ring and this same tyrosine, strengthening this hypothesis
(Figure 3A).
that were both small and would maintain some degree of
solubility. Each had similar potencies in the biochemical
assays but had in vitro and in vivo clearance that improved
with increased steric bulk around the hydroxyl, presumably
decreasing the likelihood of oxidation or conjugation of the
alcohol (Table2). Comparablepotency andmicrosomalstabi-
lities could also be obtained with the secondary amide 19,
but the clearance in the rat remained high in contrast to that of
the tertiary alcohol, 3.
The potency seen previously with the aminopyrimidine 4
along with the favorable pharmacokinetic properties of 3
prompted us to make 5. This compound had improved
potency and overall pharmacokinetic parameters relative to 3.
The increase in potency gained in going from an indazole to the
aminopyrimidine was consistent with most compounds that had
matched R1 substituents other than the optimized piperazine-
sulfonamide found in 2. The promising PK in rat translated well
across species resulting in low to moderate clearances and good
oral bioavailability in mice, dogs, and Cynomolgus monkeys
(Table 3).
Aminopyridine 16 and aminopyrimidine 4 had excellent
potencies, and analysis of crystal structures revealed potential
H-bonding interactions with Asp 841 and Asp 836 (H-bond-
˚
ing distances are 3.2 and 3.6 A, respectively for 16, Figure 3B).
These two compounds maintain the heterocyclic ring nitrogen/
s similar to 3-pyridyl 11 while also increasing the acidity of the
hydrogen on the para amino group relative to aniline 15, a
molecule that was 100-fold less potent than aminopyrimidine
4 (pKa’s for the -NH2 of aniline, 2-aminopyridine, and
2-aminopyrimidine are 30.6, 27.7, and 25.3, respectively).10
The substitutions of the phenol with either the aminopyridine
or aminopyrimidine, in 16 and 4, also greatly improved the
PK profile in rodents, leading to good exposure, in contrast to
the primary alcohol 14 which had 0% oral bioavailability.
As an example, when dosed as a solution at 5 mg/kg in the
rat, aminopyrimidine 4 had a moderate clearance of 24 mL/
min/kg, a volume of distribution of 5.6 L/kg, and 75% oral
bioavailability. However, both 4 and 16 had moderate to high
clearance in male beagle dogs (22 and 16 mL/min/kg, res-
pectively) that were not predicted by in vitro microsomal
stability data (predicted hepatic clearance of 4.8 and 0.5,
respectively) nor could they be explained via phase II
metabolism. Thus, metabolic stability was an area for further
optimization.
Male Sprague-Dawley rats were dosed orally with com-
pounds as a solution in 5% DMSO/76% PEG400. Male
Cynomolgus monkeysandmale beagle dogswere dosed orally
with the HCl salt of the compounds as a suspension in 0.5%
methylcellulose/0.2% Tween-80.
mTOR Activity. In addition to potency against PI3K, we
discovered that several compounds had increased potency
for mTOR relative to 2 (Table 4). For the indazoles, 2 and 3,
reduction in the size of the left-hand portion of the molecule
from the sulfonyl piperazine to the tertiary alcohol increased
mTOR potency slightly while reducing PI3KR potency. On
the other hand, 4 and 5 showed similar and increased mTOR
inhibition regardless of the left-hand side group, suggesting
that for these compounds the aminopyrimidine is driving
most of the potency for both PI3K and mTOR inhibition.
Further evaluation of the R2 position led to the 6-methyl
substituted 20 and 21 which had >200 fold selectivity for
mTOR while maintaining identical potency for PI3KR
relative to their unsubstituted precursors. Overall, potency
in the PC3 cell line appeared to correlate well with the PI3KR
In parallel, we were also interested in modifying the physio-
chemical and phamacodynamic (PD) properties of the mole-
cule by modifying the 6 position of the thienopyrimidine, a
region that was both exposed to solvent and had the greatest
degree of flexibility in terms of SAR. Primary, secondary, and
tertiary alcohols 17, 18, and 3 were targeted as substitutions
Table 4. Inhibition of PI3K Isoforms, mTOR, and Cancer Cell Proliferation for Selected Compounds

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1091
biochemical potency and was not dramatically influenced by
mTOR potency.
To rationalize the dramatic shifts in selectivity derived
from the addition of a single methyl group onto the amino-
pyrimidine, we examined a crystal structure of 20 with
PI3Kγ (Figure 4). The 4-methyl group, as expected, twists
the amino pyrimidine ring out of the plane of the thienopyr-
imidine in contrast to the unsubstituted aminopyrimidine
analogues (not shown). Furthermore, steric clashes with
Tyr867 cause the 4-methyl to point away from that residue
and toward the upper surface of the ATP binding pocket
where differences in mTOR and PI3K exist, presumably
resulting in the observed selectivity.
Kinase Selectivity. Compound 2, the pan-PI3K inhibitor
currently in clinical oncology trials, displays an excellent
selectivity profile relative to other Ser/Thr and Tyr kinases.8
The highly optimized 5 and 21, which each display approxi-
mately equipotent inhibition of Class I PI3K isoforms, were
submitted for screening in a 142 kinase panel provided by
Invitrogen’s SelectScreen service. Of these kinases, only three
were subject to greater than 50% inhibition by 5, and none
were inhibited greater than 80% when tested at 1 μM.
Subsequently measured IC50s demonstrated that 5 is more
than 100-fold selective for PI3KR over these three unrelated
kinases (Aurora A IC50 > 10 μM, MLK1 IC50 = 591 nM
and SYK IC50 = 371 nM). In the same panel, all kinases
displayed less than 50% inhibition at 1 μM (or IC50’s >
1 μM) by 21. Several members of the related PIK family
kinases were also screened at Invitrogen: 5 displayed rela-
tively weak inhibition of PIK3 C2 alpha and PIK3 C2 beta
(65 and 78% inhibition at 1 μM, respectively), but no
significant inhibition of hVPS34 or PI4K beta was seen.
Similarly, 21 caused no significant inhibition of PIK3 C2
alpha, hVPS34, or PI4K beta, and 81% inhibition of PIK3
C2 beta at 1 μM (data not shown). Thus, both of these
molecules were deemed sufficiently selective for PI3KR or
PI3KR and mTOR to warrant further evaluation in human
tumor xenograft efficacy studies.
To better understand the potency and selectivity of our
compounds, we examined the residue differences between
mTOR and PI3K isoforms in the vicinity of the inhibitors.
All of the PI3Kγ residues discussed throughout are identical
to those found in PI3KR. Our analysis suggests that mTOR
has identical residues corresponding to Tyr867 and Asp841
and a homologous Glu in the place of Asp836 (Figure 4). The
binding site of the two enzymes also differ in the upper
portion of the ATP binding pocket where Met804 and Ile831
of PI3Kγ align to Ile2163 and Leu2186 in mTOR, respec-
tively, potentially altering the hydrophobic surface in this
region.
The increased potency for mTOR in compounds with an
aminopyrimidine relative to those with an indazole is possi-
bly because this portion of the molecule is adjacent to
Asp836(PI3Kγ)/Glu2190(mTOR). This residue resides on
a loop which is one amino acid longer in mTOR than in
PI3Kγ. mTOR’s Glu2190 likely adopts different conforma-
tions and experiences different flexibility compared to
PI3Kγ’s Asp836, such that its favorable interactions with
the aminopyridine are disrupted by the indazole, giving rise
to the observed SAR.
Pharmacokinetic Data
Two representative compounds, 5 and 21, were chosen as
examples of a dual pan-PI3K þ mTOR inhibitor and a pan-
PI3K inhibitor (Table 5). Each of these compounds displayed
promising PK parameters in mice, suggesting that both mole-
cules could achieve sufficient exposure to warrant testing their
antitumor efficacy in human tumor xenograft models. Com-
pound 5 achieved an almost 2.5-fold greater exposure in vivo
Figure 4. Structure of 20 bound to PI3Kγ. Residues labeled in blue
are identical in mTOR, while residues in black align to different
amino acids in mTOR.
Table 5. Potency and Mouse PK Data for 5 and 21a
IC50 (nM)
mouse PK (1 mg/kg iv/5 mg/kg po)
compd PI3KR PI3Kβ/δ/γ mTOR PC3 MCF7.1 Cl (mL/min/kg) Vss (L/kg) AUC (μM*h, po) T 1/2 (h, po) F% PPB% mouse
5
21
3.4
3.5
12/16/16
25/5.2/15
32
740
330
490
180
280
17
51
2.8
3.0
13.7
4.8
3.6
4.6
100
100
64
73
a Female nu/nu mice were dosed orally with the HCl salt of each compound as a solution intraveneously in 5% DMSO/5% cremophor. Compound 5
was dosed orally as a solution in 60% PEG and 21 was dosed orally in 0.5% methylcellulose/0.2% Tween-80. AUCs for 5 when dosed in MCT were
equivalent to the AUC reported in PEG.

1092 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Sutherlin et al.
Figure 5. In vivo efficacy of 5 versus PC3 prostate cancer xenografts and compounds 5 and 21, versus MCF7.1 breast cancer xenografts. (A)
Nu/nu (nude) female mice bearing PC3 tumors established from human prostate cancer cells implanted subcutaneously were dosed orally either
with 5 at the indicated concentrations as a solution/suspension in vehicle (0.5% methylcellulose/0.2% Tween-80) or with vehicle daily (QD) for
14 days. (B) Nu/nu (nude) female mice bearing tumors established from MCF7.1 breast cancer cells implanted subcutaneously were dosed
orally with vehicle, or as a solution/suspension of 5 or 21 at the indicated concentrations in the aforementioned vehicle daily (QD) for 21 days.
Dunnett t test was used to calculate p-values comparing the drug-treated groups with vehicle control group at the end of dosing either on day
14 or 21.
than 21 when dosed at 5 mg/kg, exposure differences which
seemed to be maintained at higher doses. Additionally, 21
showed 2 and 0.5 times less potency on PC3 and MCF7.1 cells
lines, respectively, than 5. These important differences in
cellular potency and in vivo exposure were taken into con-
sideration as we designed and interpreted results from in vivo
efficacy studies.
Anti-Tumor In Vivo Efficacy. The favorable in vitro
potency and PK profiles of 5 and 21 (Table 5) prompted us
to test these compounds in vivo against the same human
cancer cell lines used in the in vitro cellular assays, but grown
as subcutaneous xenografts in nude mice. For PC3, a human
prostate cancer cell line that is PTEN -/-, once daily oral
dosing with 10 mg/kg of 5 for 14 continuous days resulted
significant in tumor regressions, with 6 partial regressions
(50-99% tumor inhibition) and 1 complete regression
(100% tumor inhibition) out of 10 animals (Figure 5A).
The mean reduction in tumor volume at day 14 compared to
vehicle-treated animals was 89%. At a 10-fold lower dose of
1 mg/kg, 5 showed no statistically significant efficacy in the
PC3 prostate cancer xenograft model. All doses tested were
tolerated and did not cause significant body weight loss (data
not shown).
To confirm and compare in vivo efficacy, 5 and 21 were
examined in the human MCF7.1 breast cancer xenograft
model that harbors a PI3KR activating mutation. Mice
bearing xenografts were dosed orally once daily with
10 mg/kg of 5 or escalating doses of 21 from 6.25 mg/kg to
50 mg/kg for 21 continuous days. Similar to observations
made in the PC3 prostate cancer xenograft model, 10 mg/kg
of 5 resulted in 73% tumor growth inhibition at day 21 when
compared to vehicle control animals (Figure 5B). Com-
pound 21 also displayed 88% tumor growth inhibition at
50 mg/kg and doses as low as 6.25 mg/kg were also effica-
cious (28% tumor growth inhibition; p < 0.05) (Figure 5B).
Comparable efficacy was observed with 10 mg/kg of 5 and
25 mg/kg of 21 and was attributed to both compounds
showing similar drug exposures at these doses (data not
shown). All doses of 21 and 5 tested were tolerated with
insignificant body weight loss observed (data not shown).
The similar efficacy profiles of 21 and 5 suggested that the
effect on PI3K/Akt pathway markers might be similar in
vivo. Upon completion of continuous dosing (up to day 21),
MCF7.1 tumors were collected and analyzed to assess the
PD regulation of the primary PI3K/Akt/mTOR path-
way markers, phospho-Akt (pAkt), phospho-PRAS40
(pPRAS40) and phospho-S6RP, (pS6RP), relative to total
proteinmarker levels, respectively. Dosesaslow as6.25 mg/kg
of 21 caused significant suppression of all three pathway
markers within 1 h of treatment (Figure 6A-C) and was
comparable to 10 mg/kg of 5. However, at 4 h, maximum
suppression of all three markers was observed only at
25 mg/kg of 21 and at 10 mg/kg of 5. These same doses
showed equivalent antitumor responses for both compounds
versus the MCF7.1 xenograft, suggesting that in this model
the PI3K/Akt pathway must be suppressed for at least 4 h
in order to achieve maximum efficacy. Furthermore, as
observed in the efficacy study, the PD response to 21 and 5
correlated well with plasma drug levels (data not shown).
Thus, when achieving comparable levels of drug exposure, 5
and 21 showed a similar suppression of the PI3K pathway
and consequently, a similar efficacy profile against MCF7.1
breast tumors.
Conclusion
Two potent, selective, and efficacious pan-PI3K inhibitors
have been discovered. Using 1 as a template, SAR was
developed that led to the replacement of a phenol with an
aminopyrimidine resulted in improved exposure in rodents.
Additionally, the piperazine-sulfonamide of 4 was replaced
with a tertiary alcohol to give 5, reducing the molecular weight
and increasing the metabolic stabilityrelative to4. Compound
5 has excellent pharmacokinetic properties in all species tested

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1093
Figure 6. Effect of 5 and 21 on PD markers in the MCF7.1 breast cancer xenograft model after 21 days of continuous dosing. Tumors were
excised from animals 1 and 4 h after the last administered dose on day 21 and processed for analysis of PI3K/Akt pathway markers as described
in Methods. (A) Levels of pAkt (Ser473) and total Akt were measured by electrochemiluminescence using Meso Scale Discovery according to
manufacturer’s instructions (Gaithersburg, MD). (B) Levels of pPras40 (Thr246) and total Pras40 were measured by ELISA at an absorbance of
450 nm as described in Methods. (C) pS6RP(Ser235/236) and total S6RP were assessed using assays measuring electrochemiluminescence from
Meso Scale Discovery (Gaithersburg, MD) according to manufacturer’s instructions. (%) = % of phospho-protein suppression compared to
vehicle control groups (100%); p-values determined by Student’s t test.
and is remarkably selective against >130 kinases. Evaluation
of structure-activity relationships around this series revealed
a method for modulating potency against mTOR, offering
a significant opportunity to expand upon the therapeutic
potential of this chemical series. Specifically, the addition of
a 4-methyl group on the pyrimidine modulated the mTOR
potency, resulting in 21. Compound 5 was profiled in tumor
models in which the PI3K pathway is activated such as PC3
prostate cancer and MCF7.1 breast cancer xenograft models
and was highly efficacious in vivo at 10 mg/kg when adminis-
tered orally on a daily schedule. The mTOR selective 21 was
also profiled in the MCF7.1 tumor model and was found
to have equivalent efficacy and PD responses to the dual
PI3K/mTOR inhibitor 5 when differences in exposure were
taken into account. The fact that MCF7.1 cells possess an
activating mutation of PI3KR and are likely more dependent
on this pathway may be one explanation for the apparent
equivalency for these two compounds in this particular xeno-
graft model, regardless of differences in mTOR potency. Both
21 (GNE-490) and 5 (GNE-493) are excellent tools to further
evaluate the therapeutic potential of PI3K and dual PI3K/
mTOR inhibitors in the treatment of cancer.
Experimental Procedures
Chemistry. All solvents and reagents were used as obtained.
1H NMR spectra were recorded with a Bruker Avance DPX400

1094 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Sutherlin et al.
Spectrometer 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 spectrometer using an ESI source
coupled to a Waters 600MS HPLC system operating in reverse
mode with an X-bridge Phenyl column of dimensions 150 mm by
4.6 mm, with 5 μm sized particles.
added sodium triacetoxyborohydride (0.074 g, 1.6 equiv), and the
reaction mixture was stirred for 1 day. The reaction mixture was
then quenched with aqueous sodium bicarbonate solution, ex-
tracted exhaustively with chloroform, dried (MgSO4), and the
solvent was removed in vacuo to yield an oil. This was purified
using flash chromatography (silica, ethyl acetate/hexane to ethyl
acetate to yield 2-[3-(tert-butyl-dimethyl-silanyloxy)-phenyl]-6-(4-
methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno-
[3,2-d]pyrimidine. To a solution of 2-[3-(tert-butyl-dimethyl-
silanyloxy)-phenyl]-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-
4-morpholin-4-yl-thieno[3,2-d]pyrimidine in THF (10 mL) cooled
to 0 °C was added a 1.0 M solution of tetrabutyl ammonium
fluoride in THF (84 μL). After 30 min, the solvent was removed in
vacuo and the residue was purified using flash chromatography
(silica, 2% methanol in dichloromethane) and then triturated using
dichloromethane/hexane to yield the title compound as a white
solid (21 mg, 21%). 1H NMR (400 MHz, CDCl3) δ 2.59 (m, 4H),
2.72 (s, 3H), 3.20 (m, 4H), 3.80 (m, 6H), 3.94-3.97 (m, 4H), 6.87
(d, J = 7.8 Hz 1H), 7.19-7.27 (m, 2H), 7.88(m, 2H). MS(ESI): m/
z (M þ H)þ 490. Analytical LC-MS using Waters XBridge Phenyl
analytical column and H2O/MeCN modified with 0.1% formic
acid running a linear gradient from 10% MeCN to 100% MeCN
monitored by UV wavelength 210 nm and ESIþ TIC MS showed
100% purity.
Compounds 2, 6-8, and intermediates in route to compounds
10-16 have been previously described in this journal.7
2-(2-Chloro-4-morpholinothieno[3,2-d]pyrimidin-6-yl)propan-2-ol.
4-(2-Chlorothieno[3,2-d]pyrimidin-4-yl)morpholine (20.0 g, mmol)
was combined with THF (300 mL) and cooled to -78 °C prior to
slow addition of 2.5 M n-BuLi in hexane (40.6 mL) via an addition
funnel to maintain a temperature below -70 °C. The reaction was
brought to -60 °C and allowed to stir for 1 h. The reaction mixture
was recooled to -78 °C and acetone was added slowly via an
addition funnel to maintain the temperature below -70 °C. After
stirring for 2 h, the reaction was quenched into a mixture of 1 N HCl
(100 mL), water (600 g), and ice (600 g). The slurry was filtered and
washed with water (150 mL) and dried in a vacuum oven overnight
at 50 °C to yield 22.3 g of 2-(2-chloro-4-morpholinothieno[3,2-
d]pyrimidin-6-yl)propan-2-ol (83.5% yield).
2-(2-(2-Aminopyrimidin-5-yl)-4-morpholinothieno[3,2-d]pyrimidin-
6-yl)propan-2-ol (5).2-(2-Chloro-4-morpholinothieno[3,2-d]pyrimidin-
6-yl)propan-2-ol (26.0 g), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)pyrimidin-2-amine (23 g), Na2CO3 (17.6 g), dioxane (390 mL),
and water (156 mL) were combined to give a light yellow suspension.
The reaction mixture was degassed through several vacuum and
purge cycles with N2. PdCl2(PPh3)2 (1.116 g) was added fol-
lowed by additional vacuum and purge cycles with N2. The
reaction was then heated for 12 h at an internal temperature of
90 °C and then cooled to room temperature. Water (400 mL)
was added and the mixture was allowed to stir for 1 h prior to
filtration and an additional water wash (100 mL). The resultant
gray solid was combined with THF (1 L), water (100 mL), and
finally Florisil (52 g, 60-100 mesh) and stirred for 15 min prior
to filtration. The Florisil was washed with two portions of THF
(300 and 200 mL) and the filtrate was evaporated, suspended in
EtOAc, heated to 80 C for 1 h, and cooled to room temp. The
resultant solid was filtered to give 25.8 g of 5 (85.5% yield). 1H
NMR (400 MHz, DMSO) δ 9.28 (s, 2H), 7.78 (bs, 1H), 7.52
(s, 1H), 4.11 (m, 4H), 3.80 (m, 4H), 1.60 (s, 6H). MS (ESI): m/z
(M þ H)þ 373. Analytical LC-MS on an Agilent (1100/MSD)
using a 3 mm ꢀ 100 mm SD-C18 analytical column and H2O/
MeCN modified with 0.05% trifluoroacetic acid running a
linear gradient from 5% MeCN to 95% MeCN monitored by
UV wavelength 254 nm and ESIþ TIC MS showed 99% purity.
2-(2-(2-Aminopyrimidin-5-yl)-4-morpholinothieno[3,2-d]pyrimidin-
6-yl)propan-2-ol hydrochoride (5). 2-(2-(2-Aminopyrimidin-
5-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-yl)propan-2-ol (15.8 g)
was suspended in 160 mL of methanol and combined with a
solution of HCl in ethanol (1.25 M, 36 mL), stirred for 2 h,
cooled to 0 °C, and filtered to give 14.8 g of the HCl salt of 5
(85.5% yield). 1H NMR (400 MHz, DMSO) δ 9.28 (s, 2H), 7.78
(bs, 1H), 7.52 (s, 1H), 4.11 (m, 4H), 3.80 (m, 4H), 1.60 (s, 6H).
MS (ESI): m/z (M þ H)þ 373. Analytical LC-MS on an Agilent
(1100/MSD) using a 3 mm ꢀ 100 mm SD-C18 analytical column
and H2O/MeCN modified with 0.05% trifluoroacetic acid run-
ning a linear gradient from 5% MeCN to 95% MeCN moni-
tored by UV wavelength 254 nm and ESIþ TIC MS showed
99% purity. Anal. (C17H21ClN6O2S) C, H, N, Cl.
6-(4-Methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-2-
pyridin-3-yl-thieno[3,2-d]pyrimidine (11). A mixture of 2-chloro-
6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno-
[3,2-d]pyrimidine8 (100 mg, 0.23 mmol), pyridine-3-boronic acid
(57 mg, 2 equiv), sodium carbonate (95 mg, 3 equiv), water (1 mL),
acetonitrile (3 mL), and PdCl2(PPh3)2 (0.1 equiv) was heated to
140 °C for 30 min in the microwave. The reaction mixture was
cooled, diluted with dichloromethane, washed with brine, dried
(MgSO4), and the solvent was removed in vacuo. The residue was
purified using flash chromatography (3% methanol in ethyl
acetate) and then trituration with ether yielded the desired title
1
compound as a white solid (84 mg, 77%). H NMR (400 MHz,
CDCl3) δ 2.68-2.72 (m, 4H), 2.82 (s, 3H), 3.29-3.33 (m, 4H),
3.90 (s, 2H), 3.90-3.94 (m, 4H), 4.05-4.10 (m, 4H),7.33 (s, 1H),
7.34-7.38 (m, 1H), 8.68 (d, J = 5.6 Hz, 1H), 9.64 (s, 1H). MS
(ESI): m/z (M þ H)þ 475. Analytical LC-MS using Waters
XBridge Phenyl analytical column and H2O/MeCN modified with
0.1% formic acid running a linear gradient from 10% MeCN to
100% MeCN monitored by UV wavelength 210 nm and ESIþ TIC
MS showed 100% purity.
6-(4-Methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-2-
pyridin-4-yl-thieno[3,2-d]pyrimidine (12). Compound 12 was
prepared from 2-chloro-6-(4-methanesulfonyl-piperazin-1-yl-
methyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine8 according to
a similar Suzuki coupling procedure as described for com-
pound 11, using 4-pyridyl boronic acid. Yield = 71%. 1H NMR
(400 MHz, CDCl3) δ 2.68-2.70 (m, 4H), 2.81 (s, 3H), 3.29-3.32
(m, 4H), 3.89-3.91 (m, 6H), 4.06-4.08 (m, 4H), 7.35 (s, H), 8.26
(dd, J = 4.5, 3.0 Hz, 2H), 8.72 (dd, J = 4.66, 3 Hz, 2H). MS
(ESI): m/z (M þ H)þ 475. Analytical LC-MS using Waters
XBridge Phenyl analytical column and H2O/MeCN modified
with 0.1% formic acid running a linear gradient from 10%
MeCN to 100% MeCN monitored by UV wavelength 210 nm
and ESIþ TIC MS showed 100% purity.
(4-(6-((4-(Methylsulfonyl)piperazin-1-yl)methyl)-2-phenylthieno-
[3,2-d]pyrimidin-4-yl)morpholine (13). Compound 13 was pre-
pared from 2-chloro-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-
4-morpholin-4-yl-thieno[3,2-d]pyrimidine8 according to a similar
Suzuki coupling procedure as described for compound 11, using
phenyl boronic acid and purified on reversed phase HPLC. Yield =
59%. 1H NMR (400 MHz, CDCl3) δ 8.33 (d, J = 7.2 Hz, 2H),
7.98 (s, 1H), 7.64 (t, J = 7.3 Hz, 1H), 7.57 (t, J = 7.4, 2H),
4.30-4.22 (m, 4H), 4.17(s, 2H), 3.99-3.92 (m, 4H), 3.43 (br s,
4H), 2.96-2.89 (m, 4H), 2.85 (s, 3H). MS (ESI): m/z (MþH)þ
474.2. Analytical LC-MS on an Agilent (1100/MSD) using a
3 mm ꢀ 100 mm SD-C18 analytical column and H2O/MeCN
3-[6-(4-Methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-
4-yl-thieno[3,2-d]pyrimidin-2-yl]-phenol (10). A mixture of 4-
morpholin-4-yl-2-[3-(1,1,2,2-tetramethyl-propylsilanyloxy)-phenyl]-
thieno[3,2-d]pyrimidine-6-carbaldehyde8 (0.101 g, 0.221 mmol),
1-methanesulphonyl-piperazine hydrochloride (0.06 g, 1.3 equiv),
acetic acid (13 μL, 1.0 equiv), and triethylamine (42 μL) was stirred
in 1,2-dichloroethane (6 mL) at room temperature. To this was

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1095
modified with 0.05% trifluoroacetic acid running a linear gradi-
ent from 5% MeCN to 95% MeCN monitored by UV wave-
length 254 nm and ESIþ TIC MS showed 97% purity.
157.25, 156.88, 150.01, 123.06, 120.04, 111.55, 65.97, 56.09,
51.78, 45.80, 45.34, 33.76. MS (ESI): m/z (M þ H)þ 491.
Analytical LC-MS on an Agilent (1100/MSD) using a 3 mm ꢀ
100 mm SD-C18 analytical column and H2O/MeCN modified
with 0.05% trifluoroacetic acid running a linear gradient from
5% MeCN to 95% MeCN monitored by UV wavelength 254 nm
and ESIþ TIC MS showed 100% purity.
(3-(6-((4-(Methylsulfonyl)piperazin-1-yl)methyl)-4-morpholino-
thieno[3,2-d]pyrimidin-2-yl)phenyl)methanol (14). Compound 14
was prepared from 2-chloro-6-(4-methanesulfonyl-piperazin-
1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine8 accord-
ing to a similar Suzuki coupling procedure as described for
compound 11, using 3-(hydroxymethyl)phenylboronic acid
and purified on reversed phase HPLC. Yield = 62%. 1H NMR
(400 MHz, DMSO-d6) δ 8.35 (s,1H), 8.26 (t, 1H), 7.59 (s, 1H),
7.47 (m, 1H), 4.61 (s, 2H), 4.04 (d, 2H), 3.83 (d, 2H), 3.31 (d, 2H),
2.96 (d, 2H), 2.96 ppm (s, 2H). MS (ESI): m/z (M þ H)þ 504.2.
Analytical LC-MS on an Agilent (1100/MSD) using a 3 mm ꢀ
100 mm SD-C18 analytical column and H2O/MeCN modified
with 0.05% trifluoroacetic acid running a linear gradient from
5% MeCN to 95% MeCN monitored by UV wavelength 254 nm
and ESIþ TIC MS showed 98% purity.
(2-Chloro-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methanol (9).
A solution of 4-morpholin-4-yl-2-[3-(1,1,2,2-tetramethyl-pro-
pylsilanyloxy)-phenyl]-thieno[3,2-d]pyrimidine-6-carbaldehyde
78 (0.2 g, 0.7 mmol) in MeOH (10 mL) at 0 °C was treated with
sodium borohydride (0.1 g, 6 mmol). The solution was allowed
to warm to room temperature and stirred for 15 min. The
reaction mixture was quenched with water and extracted with
ethyl acetate. The organic layer was dried with magnesium
sulfate, filtered via Buchner funnel, and concentrated in vacuo
to dryness. Crude yield = 100%. 1H NMR (500 MHz, DMSO)
δ 7.23 (s, 1H), 5.94 (t, J = 5.6 Hz, 1H), 4.82 (d, J = 4.9 Hz, 2H),
4.01-3.83 (m, 4H), 3.83-3.60 (m, 4H). MS (ESI): m/z (M þ H)þ
286.1. Analytical LC-MS on an Agilent (1100/MSD) using a
3 mm x 100 mm SD-C18 analytical column and H20/MeCN
modified with 0.05% trifluoroacetic acid running a linear
gradient from 5% MeCN to 95% MeCN monitored by UV
wavelength 254 nm and ESIþ TIC MS showed 100% purity.
1-(2-(1H-Indazol-4-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-
yl)ethanol (18). Compound 18 was prepared from 1-(2-chloro-4-
morpholinothieno[3,2-d]pyrimidin-6-yl)ethanol (167 mg, 5.6 mmol)
according to a similar Suzuki coupling procedure as described
for compound 11, using 4-(4,4,5,5-tetramethyl-1,3,2-dioxabor-
olan-2-yl)-1H indazole and purified via reverse phase HPLC.
Yield = 20%. 1H NMR (500 MHz, DMSO) δ 13.17 (s, 1H), 8.90
(s, 1H), 8.24 (dd, J = 7.2 Hz, 0.6, 1H), 7.67 (d, J = 8.2 Hz, 1H),
7.53-7.46 (m, 1H), 7.43 (t, J = 3.4 Hz, 1H), 5.99 (d, J = 4.9 Hz,
1H), 5.15 (p, J = 5.5 Hz, 1H), 4.10-3.97 (m, 4H), 3.94 - 3.73
(m, 4H), 1.55 (d, J = 6.4 Hz, 3H). MS (ESI): m/z (M þ H)þ
382.1. Analytical LC-MS using Waters XBridge Phenyl analy-
tical column and H2O/MeCN modified with 0.1% formic acid
running a linear gradient from 5% MeCN to 95% MeCN
monitored by UV wavelength 210 nm and ESIþ TIC MS
showed 100% purity.
4-[6-(4-Methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-
thieno[3,2-d]pyrimidin-2-yl]-phenylamine (15). Compound 15
was prepared from 2-chloro-6-(4-methanesulfonyl-piperazin-
1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine8 accord-
ing to a similar Suzuki coupling procedure for compound
11, using 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline.
1
Yield = 83% H NMR (400 MHz, CDCl3) δ 2.67-2.71 (m,
4H), 2.81 (s, 3H), 3.29-3.33 (m, 4H), 3.89 (s, 2H), 3.89-3.93 (m,
4H), 4.08-4.12 (m, 4H), 6.75 (d, J= 8.6 Hz, 2H), 7.30 (s, 1H), 8.29
(d, J = 8.6 Hz, 2H). MS (ESI): m/z (M þ H)þ 489 Analytical
LC-MS using Waters XBridge Phenyl analytical column and H2O/
MeCN modified with 0.1% formic acid running a linear gradient
from 10% MeCN to 100% MeCN monitored by UV wavelength
210 nm and ESIþ TIC MS showed 100% purity.
5-[6-(4-Methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-
thieno[3,2-d]pyrimidin-2-yl]-pyridin-2-ylamine (16). Compound
16 was prepared from 2-chloro-6-(4-methanesulfonyl-piper-
azin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine8 accord-
ing a similar Suzuki coupling procedure for compound 11, using
2-amino-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine.
1
Yield = 39%. H NMR (400 MHz, CDCl3) δ 2.67-2.71 (m,
4H), 2.81 (s, 3H), 3.29-3.33 (m, 4H), 3.89 (s, 2H), 3.89-3.93 (m,
4H), 4.08-4.12 (m, 4H), 4.60-4.65 (br s, 2H), 6.57 (d, J =
8.6 Hz,1H), 7.40 (s, 1H), 8.45 (dd, J = 8.6, 2.2 Hz, 1H), 9.17 (d,
J = 2.2 Hz, 1H). MS (ESI): m/z (M þ H)þ 490. Analytical LC-
MS using Waters XBridge Phenyl analytical column and H2O/
MeCN modified with 0.1% formic acid running a linear gra-
dient from 10% MeCN to 100% MeCN monitored by UV
wavelength 210 nm and ESIþ TIC MS showed 99% purity.
2-(2-(1H-indazol-4-yl)-4-morpholinothieno[3,2-d]pyrimidin-6-
yl)propan-2-ol (3). Compound 3 was prepared from 2-chloro-
6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-
thieno[3,2-d]pyrimidine8 according a similar Suzuki coupling
procedure for compound 11. Mesylate salt 1H NMR (400 MHz,
DMSO) δ 8.66 (s, 1H), 8.30 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.8
Hz, 1H), 7.58 (dd, J = 8.8, 8.0 Hz, 1H), 7.42 (s, 1H), 4.14 (m,
4H), 3.86 (m, 4H), 2.33 (s, 3H), 1.63 (s, 6H). MS (ESI): m/z (M þ
H)þ 396. Analytical LC-MS on an Agilent (1100/MSD) using
a 3 mm ꢀ 100 mm SD-C18 analytical column and H2O/MeCN
modified with 0.05% trifluoroacetic acid running a linear
gradient from 5% MeCN to 95% MeCN monitored by UV
wavelength 254 nm and ESIþ TIC MS showed 99% purity.
5-(6-((4-(Methylsulfonyl)piperazin-1-yl)methyl)-4-morpholino-
thieno[3,2-d]pyrimidin-2-yl)pyrimidin-2-amine (4). Compound 4
was prepared from 2-chloro-6-(4-methanesulfonyl-piperazin-
1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine8 accord-
ing a similar Suzuki coupling procedure for compound 11, using
5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-amine.
(41 mg, 57%). 1H NMR (500 MHz, DMSO) δ 9.11 (s, 2H), 7.34
(s, 1H), 7.10 (s, 2H), 3.95-3.91 (m, 4H), 3.90 (s, 2H), 3.84-3.73
(m, 4H), 3.20-3.07 (m, 4H), 2.91 (s, 3H), 2.63-2.54 (m, 4H). 13C
NMR (126 MHz, DMSO) δ 164.04, 162.99, 161.92, 157.77,
2-[2-(2-Amino-4-methyl-pyrimidin-5-yl)-4-morpholin-4-yl-thieno-
[3,2-d]pyrimidin-6-yl]-propan-2-ol (19). Compound 19 was pre-
pared from 1-(2-chloro-4-morpholinothieno[3,2-d]pyrimidin-
6-yl)ethanol (70 mg, 0.2 mmol) according to a similar Suzuki
coupling procedure as described for compound 11, using 4-methyl-
5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyrimidin-2-yl-
amine and purified via reverse phase HPLC. (15 mg, 20%) MS:
387.2 (M þ H); 1H NMR (400 MHz, DMSO) δ 8.84 (s, 1H), 7.42
(s, 1H), 4.08 (m, 4H), 3.81 (m, 4H), 1.61(m, 9H). MS (ESI): m/z
(M þ H)þ 387. Analytical LC-MS on an Agilent (1100/MSD)
using a 3 mm x 100 mm SD-C18 analytical column and H2O/
MeCN modified with 0.05% trifluoroacetic acid running a
linear gradient from 5% MeCN to 95% MeCN monitored
by UV wavelength 254 nm and ESIþ TIC MS showed 100%
purity.
5-[6-(4-Methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-
thieno[3,2-d]pyrimidin-2-yl]-4-methyl-pyrimidin-2-ylamine (20).
2-Chloro-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-
4-yl-thieno[3,2-d]pyrimidine (80 mg, 0.2 mmol), 4-methyl-5-(4,4,
5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyrimidin-2-ylamine
(65 mg, 0.28 mmol), and bis(triphenylphosphine)palladium(II)
dichloride (9.0 mg, 0.01mmol) in 1 M aqueous sodiumcarbonate
(0.8 mL) and acetonitrile (0.8 mL) were heated to 130 °C in a
sealed microwave reactor for 15 min. Upon completion, the
organic layer was separated and the aqueous layer was extracted
with dichloromethane. The combined organic layers were con-
centrated in vacuo. The product was purified by reverse phase
HPLC to yield 5-[6-(4-methanesulfonyl-piperazin-1-ylmethyl)-
4-morpholin-4-yl-thieno[3,2-d]pyrimidin-2-yl]-4-methyl-pyrimi-
1
din-2-ylamine (10 mg, 10%). MS: 505.2 (M þ H); H NMR

1096 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Sutherlin et al.
(400 MHz, DMSO) δ 8.83 (s, 1H), 7.48 (s, 1H), 3.98 (m, 4H), 3.80
(m, 4H), 3.24 (m, 4H), 2.93 (m, 2H), 2.82 (m, 4H), 2.66 (m, 6H).
MS (ESI): m/z (M þ H)þ 505. Analytical LC-MS on an Agilent
(1100/MSD) using a 3 mm ꢀ 100 mm SD-C18 analytical column
and H2O/MeCN modified with 0.05% trifluoroacetic acid run-
ning a linear gradient from 5% MeCN to 95% MeCN monitored
by UV wavelength 254 nm and ESIþ TIC MS showed 100%
purity.
(500 MHz, DMSO): δ 8.77 (s, 1H), 7.38 (br s, 2H), 7.29 (s, 1H),
4.01 (app t, 4H), 3.80 (app t, 4H), 2.64 (s, 3H), 1.60 (s, 6H). MS
(ESI): m/z 387.2 (M þ H)þ.
Characterization of Biochemical and Cellular Activity In Vitro.
Enzymatic activity of the Class I PI3K isoforms was measured
using a fluorescence polarization assay that monitors formation
of the product 3,4,5-inositoltriphosphate molecule as it com-
petes with fluorescently labeled PIP3 for binding to the GRP-1
pleckstrin homology domain protein. An increase in phospha-
tidyl inositide-3-phosphate product results in a decrease in
fluorescence polarization signal as the labeled fluorophore is
displaced from the GRP-1 protein binding site. Class I PI3K
isoforms were purchased from Perkin-Elmer or were expressed
and purified as heterodimeric recombinant proteins (ref original
JACS PI3K paper). Tetramethylrhodamine-labeled PIP3
(TAMRA-PIP3), di-C8-PIP2, and PIP3 detection reagents were
purchased from Echelon Biosciences. PI3K isoforms were as-
sayed under initial rate conditions in the presence of 10 mM Tris
(pH 7.5), 25 μM ATP, 9.75 μM PIP2, 5% glycerol, 4 mM MgCl2,
50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 2% (v/v)
DMSO at the following concentrations for each isoform:
PI3KR,β at 60 ng/mL; PI3Kγ at 8 ng/mL; PI3Kδ at 45 ng/
mL. After assay for 30 min at 25 °C, reactions were terminated
with a final concentration of 9 mM EDTA, 4.5 nM TAMRA-
PIP3, and 4.2 μg/mL GRP-1 detector protein before reading
fluorescence polarization on an Envision plate reader. IC50s
were calculated from the fit of the dose-response curves to a
4-parameter equation.
Synthesis of N-[2-(1H-Indazol-4-yl)-4-morpholin-4-yl-thieno-
[3,2-d]pyrimidin-6-ylmethyl]-acetamide (19). 6-(Bromomethyl)-
2-chloro-4-morpholinothieno[3,2-d]pyrimidine. To a solution of
(2-chloro-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methanol
(100 mg, 0.4 mmol) in benzene (3.0 mL) at 0 °C was added PBr3
(30 μL, 0.4 mmol). The reaction was heated at reflux for 1 h.
After cooling to room temperature, the reaction was quenched
by the addition of water. The aqueous layer was extracted with
EtOAc. The combined organics were dried over Na2SO4 and
concentrated in vacuo. The crude material was not purified
1
further (115 mg). H NMR (400 MHz, CDCl3) δ 7.32 (s,1H),
4.71 (s, 2H), 3.99-3.97 (m, 4H), 3.85-3.83 (m, 4H). MS (ESI):
m/z (M þ H)þ 347.9
(2-Chloro-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methanamine.
To a solution of 6-(bromomethyl)-2-chloro-4-morpholinothieno-
[3,2-d]pyrimidine (0.3 g, 0.9 mmol) in DMF (10 mL) was added
K2CO3 (0.2 g, 1.3 mmol), and phthalimide (0.1 g, 0.9 mmol).
The resulting solution was stirred 20 h at room temperature.
The reaction was concentrated in vacuo and diluted with water
(10 mL). The heterogeneous mixture was filtered and the solid
intermediate was carried onto the next step without purification.
MS (ESI): m/z (M þ H)þ 415. To the solid phtalimide inter-
mediate (100 mg, 0.24 mmol) in MeOH (7 mL) was added
Human recombinant mTOR(1360-2549) was expressed and
purified from insect cells and assayed using a Lanthascreen
fluorescence resonance energy transfer format from Invitrogen
in which phosphorylation of recombinant green fluorescent
protein (GFP)-4-EBP1 is detected using a terbium-labeled anti-
body to phospho-threonine 37/46 of 4-EBP1. Reactions were
initiated with ATP and conducted in the presence of 50 mM
Hepes (pH 7.5), 0.25 nM mTOR, 400 nM GFP-4E-BP1, 8 μM
ATP, 0.01% (v/v) Tween 20, 10 mM MnCl2, 1 mM EGTA,
1 mM dithiothreitol, and 1% (v/v) DMSO. Assays were con-
ducted under initial rate conditions at room temperature for
30 min before terminating the reaction and detecting product in
the presence of 2 nM Tb-anti-p4E-BP1 antibody and 10 mM
EDTA. Dose-response curves were fit to an equation for
competitive tight-binding inhibition and apparent Ki’s were
calculated using the determined Km for ATP of 6.1 μM.
Antiproliferative cellular assays were conducted 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). Cell lines were cultured in RPMI supplemented
with 10% fetal bovine serum, 100 units/mL penicillin, and
100 μg/mL streptomycin, 10 mM HEPES, and 2 mM glutamine
at 37 °C under 5% CO2. MCF7.1 cells or PC3 cells were seeded
in 384-well plates in media at 1000 cells/well or 3000 cells/well,
respectively, and incubated overnight prior to the addition of
compounds to a final DMSO concentration of 0.5% v/v.
MCF7.1 cells and PC3 cells were incubated for 3 and 4 days,
respectively, prior to the addition of CellTiter-Glo reagent
(Promega) and reading of luminescence using an Analyst plate
reader. For antiproliferative assays, a cytostatic agent such
as aphidicolin and a cytotoxic agent such as staurosporine
were included as controls. Dose-response curves were fit to a
4-parameter equation and relative IC50s were calculated using
Assay Explorer software.
H2NNH2 H2O (24 μL, 0.48 mmol). The reaction was heated
3
1 h at reflux. After cooling to room temperature, the reaction
was quenched with water (10 mL) and extracted with EtOAc.
The combined organics were dried over Na2SO4 and concen-
trated in vacuo to afford (2-chloro-4-morpholinothieno[3,2-d]-
pyrimidin-6-yl)methanamine (0.05 g). 1H NMR (400 MHz, CDCl3)
δ 7.15 (s,1H), 4.20 (s, 2H), 4.00-3.96 (m, 4H), 3.84-3.82 (m, 4H).
MS (ESI): m/z (M þ H)þ 285.1
N-((2-Chloro-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)-
acetamide. To a solution of 4-morpholinothieno[3,2-d]pyrimidin-
6-yl)methanamine (50 mg, 0.2 mmol) in CH2Cl2 (4 mL) was added
Et3N(84μL, 0.6 mmol) and acetyl chloride (0.3 mmol). The reaction
was stirred overnight at room temperature before being quenched
with water. The aqueous layer was extracted with EtOAc. The
combined organics were dried over Na2SO4 and concentrated in
vacuo and used in the next step without purification. 1H NMR (400
MHz, CDCl3) δ7.17 (s,1H), 4.68 (d, J= 6.1 Hz, 2H), 3.98-3.95(m,
4H), 3.84-3.81 (m, 4H), 2.07(s, 3H). MS(ESI):m/z (M þ H)þ 327.
N-[2-(1H-Indazol-4-yl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidin-
6-ylmethyl]-acetamide (19). Compound 19 was prepared from
N-((2-chloro-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)-
acetamide according a similar Suzuki coupling procedure as
described above for 16, using 4-(4,4,5,5-tetramethyl-1,3,2-diox-
aborolan-2-yl)-1H-indazole. (41 mg from 50 mg of 4-mor-
pholinothieno[3,2-d]pyrimidin-6-yl)methanamine). 1H NMR
(500 MHz, CDCl3) δ 9.00 (s, 1H), 8.27 (d, J = 7.1 Hz, 1H),
7.60 (d, J = 8.3 Hz, 1H), 7.53-7.47 (m, 2H), 4.76 (d, J = 5.9 Hz,
2H), 4.09-4.07 (m, 4H), 3.92-3.90 (m, 4H), 2.62 (s, 1H), 2.10
(s, 3H), 3.90 (s, 2H), 3.84-3.73 (m, 4H), 3.20-3.07 (m, 4H), 2.91
(s, 3H), 2.63-2.54 (m, 4H). MS (ESI): m/z (M þ H)þ 409.1.
2-(2-(2-Amino-4-methylpyrimidin-5-yl)-4-morpholinothieno-
[3,2-d]pyrimidin-6-yl)propan-2-ol (20). To a solution of 2-(2-chloro-
4-morpholinothieno[3,2-d]pyrimidin-6-yl)propan-2-ol (1.0 g) in
acetonitrile (6 mL) and 1 M aq Na2CO3 (6 mL) was added
Pd(PPh3)4 (180 mg) and the reaction mixture was heated 15 min
in a microwave reactor (300 W) at 140 °C. The reaction mixture
was concentrated in vacuo and the desired product was purified
by silica gel chromatography (2-8% MeOH in DCM) followed
by recrystallization from EtOAc to afford 20 (650 mg). 1H NMR
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 ꢀ
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 150-200 mm3 prior to the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1097
References
initiation of dosing. MCF7.1 cells resuspended in a 1:1 mix-
ture of Hank’s Buffered Salt Solution and Matrigel Basement
Membrane Matrix (No. 356237, BD Biosciences; Santa Cruz,
CA), were 5 ꢀ 106 subcutaneously implanted into the right hind
flank of athymic nu/nu (nude) mice. Prior to cell inoculation,
17 β-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 250-350 mm3 prior to
initiating dosing. Compound 5 and 21 were dissolved in 0.5%
methylcellulose with 0.2% Tween-80 (MCT). Female nude
(nu/nu) mice that were 6-8 weeks old and weighed 20-30 g
were obtained from Charles River Laboratories (Hollister, CA).
Tumor bearing mice were dosed daily for 14 to 21 days depend-
ing 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 54-10-111; 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 Corporation;
Pine Brook, NJ). Percent weight change = [1 - (new weight /
initial weight)] ꢀ 100. Tumor sizes were recorded twice weekly
over the course of the study (14-21 days). Mouse body weights
were also recorded twice weekly and the mice were observed
daily. Mice with tumor volumes g 2000 mm3 or with losses in
body weight g 20% from their initial body weight were
promptly euthanized per IACUC guidelines. Mean tumor vo-
lume and 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 - 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).
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Acknowledgment. The authors wish to thank Mengling
Wong, Martin Struble, 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 purification, and Jeremy Murray for depositing
structures to the PDB. We thank Emil Plise for plasma protein
binding data. We are grateful to Michelle Nannini, Janeko
Bower, Alex Vanderbilt, Alfonso Arrazate, Judi-Anne Ramiscal,
Vincent Javinal, and Kenton Wong for coordinating and dosing
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