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

Article abstract of DOI:10.1021/jm800295d

Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were prepared and evaluated as inhibitors of PI3 kinase p110α. The synthesis, biological activity, and further profiling of these compounds are described. This work resulted in the discovery of 17, GDC-0941, which is a potent, selective, orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical trials for the treatment of cancer.

Full text of DOI:10.1021/jm800295d

5
522
J. Med. Chem. 2008, 51, 5522–5532
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
,
‡
‡
‡
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‡
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‡
Adrian J. Folkes,* Khatereh Ahmadi, Wendy K. Alderton, Sonia Alix, Stewart J. Baker, Gary Box, Irina S. Chuckowree,
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‡
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§
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‡
Paul A. Clarke, Paul Depledge, Suzanne A. Eccles, Lori S. Friedman, Angela Hayes, Timothy C. Hancox,
Arumugam Kugendradas, Letitia Lensun, Pauline Moore, Alan G. Olivero, Jodie Pang, Sonal Patel,
Giles H. Pergl-Wilson, Florence I. Raynaud, Anthony Robson, Nahid Saghir, Laurent Salphati, Sukhjit Sohal,
Mark H. Ultsch, Melanie Valenti, Heidi J.A. Wallweber, Nan Chi Wan, Christian Wiesmann, Paul Workman,
‡
‡
‡
§
§
‡
‡
|
‡
‡
§
‡
§
|
§
‡
§
|
‡
⊥
‡
Alexander Zhyvoloup, Marketa J. Zvelebil, and Stephen J. Shuttleworth
Piramed Pharma, 957 Buckingham AVenue, Slough, Berks SL1 4NL, United Kingdom, Genentech, Inc., 1 DNA Way, South San Francisco,
California 94080, Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddon Laboratories, Sutton,
Surrey, SN2 5NG, United Kingdom and Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research,
London, SW3 6JB, United Kingdom
ReceiVed March 17, 2008
Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/
Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were
prepared and evaluated as inhibitors of PI3 kinase p110R. The synthesis, biological activity, and further
profiling of these compounds are described. This work resulted in the discovery of 17, GDC-0941, which
is a potent, selective, orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical
trials for the treatment of cancer.
Introduction
a
Phosphatidylinositol-3-kinases (PI3Ks) are lipid kinases that
catalyze the phosphorylation of the 3-hydroxyl position of
1
,2
phosphatidylinositides. To date, a total of eight PI3 kinases
have been identified, which are divided into classes IA, 1B, II,
and III on the basis of sequence homology and substrate
preference. The PI3K superfamily also includes the Class IV
PIK related enzyme family of protein kinases including mTOR,
ATM, ATR, and DNA-PK. The class IA subgroup consists of
p110R, p110ꢀ, and p110δ subtypes, and these primarily generate
phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3), which is
a second messenger that facilitates activation of the downstream
3
kinase Akt. The consequences of biological activation of Akt
include tumor progression, proliferation, survival, growth,
invasion, angiogenesis, and metastasis, and there is significant
evidence suggesting that the PI3K/Akt pathway is deregulated
Figure 1. Structures of PI3K Inhibitors.
3
in many human cancers. Furthermore, the gene encoding the
p110R subunit, PIK3CA, is amplified and overexpressed in
proteins in human malignancy, providing further evidence of
the role of the PI3K pathway in cancer. In addition, persistent
4
several ovarian cancer cell lines and is mutated in a wide variety
8,9
of other cancers including colorectal, glioblastoma, and gastric
signaling through the PI3K/Akt pathway has been shown to be
a major mechanism of resistance to chemotherapeutics as well
as therapeutic agents targeting the epidermal growth factor
4
–7
cancers. PI3K signaling is negatively regulated by the dual
phosphatase PTEN, which is one of the most commonly mutated
1
0
receptor family. Among patients treated with trastuzumab,
activation of the PI3K pathway has been associated with
†
The coordinates of compound 17 in PI3K p110γ have been deposited
1
1,12
(
PDB code 3DBS).
resistance and poor prognosis.
Hence inhibition of PI3K,
*
To whom correspondence should be addressed. Phone: +44 0 1753
and in particular p110R, is a promising target for cancer
2
85823. Fax: +44 0 1753 568367. E-mail: adrian.folkes@piramed.com.
7
,13
‡
treatment.
Several inhibitors of PI3 kinase have been reported in the
Piramed Pharma.
|
Cancer Research UK Centre for Cancer Therapeutics, The Institute of
Cancer Research, Haddon Laboratories.
7,14
15,16
17
literature, including wortmannin
and LY294002 (Figure
§
Genentech, Inc.
⊥
1). Both of these compounds have been widely used to elucidate
Breakthrough Breast Cancer Research Centre, Institute of Cancer
1
4
Research.
the functional role of PI3K. However, their toxicity and lack
of selectivity with respect to targets other than class I PI3K
family members have limited their therapeutic potential. More
recently, Hayakawa et al. have reported the thieno[3,2-d]pyri-
a
Abbreviations: PI3K, phosphatidylinositol-3-kinase; mTOR, mammalian
target of rapamycin; ATM kinase, ataxia telangiectasia mutated kinase; ATR,
ataxia telangiectasia and Rad3-related protein; DNA-PK, DNA protein
kinase; PTEN, phosphatase and tensin homologue; SPA, scintillation
proximity assay.
1
8
midine derivative 1 and the pyrido[3′,2′:4,5]furo-{3,2-
1
0.1021/jm800295d CCC: $40.75

2008 American Chemical Society
Published on Web 08/29/2008

Clinical Candidate of PI3 Kinase
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5523
a
Scheme 1
a
Reagents and conditions: (a) 3-OTBDMS phenyl boronic acid, NaHCO3, H2O, Pd(PPh3)2Cl2, DME, reflux, 51%. (b) (i) nBuLi, THF, -78 °C; (ii) DMF,
9%. (c) (i) RR′NH, 1,2-DCE, Na(OAc)3BH, AcOH; (ii) TBAF, THF. (d) (i) nBuLi, THF, -78 °C; (ii) MeI, 82%. (e) 3-Hydroxyphenyl boronic acid,
9
NaHCO3, H2O, Pd(PPh3)2Cl2, DME, reflux, 10%. (f) (i) nBuLi, THF, -78 °C; (ii) DMF, 77%. (g) RR′NH, 1,2-DCE, Na(OAc)3BH, HC(OMe)3. (h) 25,
MeCN, Na2CO3, H2O, Pd(PPh3)2Cl2, microwave, 130 °C, 20 min.
1
9
d}pyrimidine derivative 2, both potent inhibitors of PI3K
p110R with significant antiproliferative activity in vitro. Whereas
the appropriate amine and sodium triacetoxyborohydride pro-
ceeded smoothly to yield compounds 14-15. Finally, Suzuki
coupling with 4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-
1H-indazole 25 yielded compounds 16-17.
Scheme 2 describes the initial route used for the preparation
of indazole derivatives 20 and 22. Palladium mediated treatment
of 3-bromo-2-methyl-aniline with pinacol borane yielded 2-meth-
yl-3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pheny-
lamine 18. Reaction of 18 with 3 under Suzuki conditions
yielded 19. Cyclisation with isoamyl nitrite resulted in the
desired 4-indazolyl derivative 20. The isomeric 6-indazolyl
derivative 22 was prepared in an analogous manner from
3-amino-4-methylbenzeneboronic acid.
2
demonstrated encouraging activity against several human
2
0
tumor xenografts, the poor pharmacokinetic profile of 1
precluded any further evaluation of this compound, as the half-
life is less than 10 min following intraperitoneal administration
1
8
20,21
in mice. Although 2 has been more extensively profiled,
we viewed 1 as offering greater potential for further optimization
due to the relatively low molecular weight (MW ) 313) and
the opportunity to substitute the 6- and 7- positions of the
thieno[3,2-d]pyrimidine to improve multiple aspects of the
compound, including physicochemical properties, metabolic
stability, and potency. Consequently, 1 was chosen as the
starting point for our medicinal chemistry efforts.
Herein we disclose the synthesis and biological evaluation
of a series of thieno[3,2-d]pyrimidines that demonstrate potent
inhibition of PI3K p110R, culminating in the discovery of 17
4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-inda-
zole 25, which was a more versatile intermediate for the
preparation of indazole derivatives, is prepared in three steps
from 3-bromo-2-methyl aniline as shown in Scheme 3.
(
GDC-0941).
Results and Discussion
Chemistry
18
It has previously been reported that the morpholino group
The general synthetic routes used to prepare thieno[3,2-
d]pyrimidine derivatives are outlined in the following schemes.
In Scheme 1, 2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyri-
midine 3 was treated with 3-(tert-butyldimethylsilyloxy)phenyl
boronic acid under Suzuki conditions to provide 4. Lithiation
of 4, followed by the addition of DMF, gave the aldehyde 5 in
on the related quinazoline template was critical for p110R
inhibitory activity, with all modifications to this position
resulting in large reductions in potency. Studies on analogues
of LY294002 have also shown that the morpholino group is
critical for potency, suggesting that the morpholino groups in
the quinazoline series and LY294002 interact with the PI3K in
a similar manner. As it was believed that the morpholino group
was optimal, this group was retained with all further derivatives
described herein.
Our initial efforts focused on substitutions of the 6- and 7-
positions of the thieno[3,2-d]pyrimidine ring, with the primary aim
of maintaining potency while improving the metabolic stability and
the solubility of the series. Substitution with a methyl group in the
6-position, compound 11, was well tolerated, whereas the 7-Me
derivative 12 showed a slight decrease in potency (Table 1). Taking
into consideration the potential to block any metabolism at the
22
1
7
9
9% yield. Treatment of 5 with the appropriate amine using
standard reductive amination conditions, followed by treatment
with tetrabutyl ammonium fluoride, provided the phenolic
derivatives 6-9.
To prepare the 6-methyl derivative 11, the lithium anion of
3
was quenched with methyl iodide to provide 10. Reaction
with 3-hydroxyphenyl boronic acid under Suzuki conditions
provided the desired compound.
Also in Scheme 1, lithiation of 3 followed by the addition of
DMF yielded the aldehyde 13. Reductive amination of 13 using

5
524 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Folkes et al.
a
Scheme 2
a
Reagents and conditions: (a) pinacol borane, 2-dicyclohexylphosphinobiphenyl, Pd(OAc)2, Et3N, dioxane, 80° C, 85%; (b) DME, Na2CO3, H2O,
Pd(PPh3)2Cl2, reflux, 69%; (c) isoamyl nitrite, CHCl3, AcOH, 20% for 20, 14% for 22; (d) 3-amino-4-methylbenzene boronic acid, DME, Na2CO3, H2O,
Pd(PPh3)2Cl2, reflux, 82%.
a
Scheme 3
a
Reagents and conditions: (a) (i) KOAc, Ac2O; (ii) isoamyl nitrite, 18-crown-6, CHCl3. (b) 6N HCl, MeOH, 86% from 3-bromo-2-methylaniline. (c)
bis(pinacolato)diboron, PdCl2(dppf)2, KOAc, DMSO, 80 °C, 60%.
6
-position of the thieno[3,2-d]pyrimidine ring, and the greater
with recombinant human p110R and 1 µM ATP. Inhibition of
cell proliferation was measured using the Alamar Blue assay
with the PTEN-null U87MG human glioblastoma cell line and
with the PIK3CA mutant, PTEN mutant A2780 human ovarian
synthetic accessibility of this position, we concentrated our efforts
to modifications at the 6- position.
The inhibitory activity of the compounds against p110R was
measured using a radiometric scintillation proximity assay (SPA)
2
0,21
cell line.
Figure 2. (a) ATP-binding site sequence alignment for p110R and p110γ, with key residues highlighted: hinge region Val (red), conserved Tyr
2
4–28
and Asp located at the back end of the pocket (blue and green, respectively), and catalytic Lys (gray).
(b,c) 1 docked into the p110R homology
model reveals key hydrogen bonds between the morpholine oxygen of 1 and Val851 in the hinge and between the exocyclic phenolic hydroxyl and
Asp810, with the 6-position proton on thiophene ring extending out toward solvent.

Clinical Candidate of PI3 Kinase
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5525
Table 1. Inhibition of p110R Activity, Cancer Cell Proliferation, Metabolic Stability, and Oral Bioavailability for Thieno[3,2-d]pyrimidine Derivatives
a
b
The values are averages of at least 2 separate determinations with typical variations of less than (30%. Percent remaining after 30 min incubation of
0 µM compound with 1 mg/mL mouse and human liver microsomes with both NADPH and UDPGA cofactors for glucuronidation. The values are averages
1
c
of at least 2 separate determinations with typical variations of less than (30%. Based on iv and po administration (10 mg/kg). ND: Not determined
compound was not tested).
(
It was found that the 6-position was tolerant to a wide range
of substituents with many analogues of various structural
topologies displaying similar levels of biochemical activity to
1
. Among the more interesting compounds were tertiary amine
derivatives such as compounds 6-9, which offered the potential
for facile salt formation to aid kinetic solubility, dissolution rates,
as well as in vivo absorption and tumor exposure. Compounds
6
-9 showed potent inhibition of p110R and good inhibition of
cancer cell proliferation, and in addition, compounds 7, 8, and
9
demonstrated high solubility (>100 µM) as assessed in a
23
turbidimetric aqueous solubility assay.
Analysis of 1 docked into a homology model of p110R
24
(
Figure 2), which is based upon the crystal structure of p110γ,
suggested that groups located at the 6-position of the thiophene
ring would extend out of the ATP-binding pocket toward
solvent, thereby explaining the tolerance to a large variety of
groups at this position. This model also indicated that the oxygen
atom of the morpholine ring forms a hydrogen bond with the
hinge Val851 (p110R numbering). Crystallographic studies of
Figure 3. Metabolism of 8 (mesylate salt) after ip dosing in female
balb C mice at 50 mg/kg.
conjugates by the phenolic derivatives. In vitro metabolism
studies in microsomes also indicated that glucuronidation of the
parent compound was common to all the phenol derivatives (data
not shown), suggesting that the phenolic group was a metabolic
liability.
2
4
LY294002 with p110γ have shown that the oxygen atom of
the morpholine ring forms a key hydrogen bond with Val882
(
p110γ numbering), supporting the hypothesis that the mor-
pholine groups in both 1 and LY294002 bind in similar manner.
Encouragingly, compounds 8 and 9 also displayed enhanced
metabolic stability in human and mouse liver microsomes
relative to the other members of this series (Table 1). However,
despite the encouraging microsomal stability, pharmacokinetic
studies of several phenolic derivatives, including compounds
To overcome this liability, we explored isosteres for the
phenolic group, which we hoped would provide the required
level of activity without displaying the propensity for glucu-
ronidation shown by the phenolic derivatives. We believed that
a hydrogen bond donor was critical for activity as previous
attempts to replace the 3-hydroxyphenyl group on related
1
8,19
6
, 8, and 9, indicated very low oral bioavailability in mouse
templates had resulted in a significant loss of activity.
and rat, mainly as a result of glucuronidation. Following ip
administration of compound 8 in mice, the circulating concen-
tration of the glucuronide conjugate appeared greater than that
of the parent phenol at all time points after 0.5 h (Figure 3),
demonstrating the propensity for formation of glucuronide
Analysis of the structural model (Figure 2) suggested that the
3-hydroxyl group forms an interaction with Asp810 (p110R
numbering) located at the back end of the ATP binding pocket
of p110R, and consequently, we were particularly interested in
investigating heterocyclic groups that maintained the potential

5
526 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Folkes et al.
Table 4. Inhibition of Classes I, II, III, and IV PI3K Isoforms by 17
Table 2. Inhibition of p110R Activity and Cancer Cell Proliferation for
Indazole Analogues
a
enzyme
p110R (class 1A)
IC50 (µM)
0.003
0.003
0.003
0.033
0.003
0.075
0.670
>10
p110R (class 1A) mutant E545-K
p110R (class 1A) mutant H1047-R
p110ꢀ (class 1A)
p110δ (class 1A)
p110γ (class 1B)
C2ꢀ (class II)
Vps34 (class III)
DNA-PK (class IV)
mTOR (class IV)
1.23
b
0.58
a
The values are averages of at least 2 separate determinations with typical
b
variations of less than ( 30%. Kiapp value.
a
The values are averages of at least 2 separate determinations with typical
variations of less than (30%. ND: Not determined (compound was not
tested).
Table 3. Inhibition of p110R Activity, Cancer Cell Proliferation, and
Metabolic Stability for Indazole Derivatives
Figure 4. Crystal Structure of 17 bound to p110γ (p110γ numbering
displayed).
istrations in the mouse indicated that a glucuronide conjugate
was present but at significantly lower levels than the parent.
Further optimization of 16 followed, in which functional groups
were introduced that had the potential to form additional
hydrogen bonds with residues located near the mouth of the
ATP-binding pocket. Because of the biochemical and cellular
potency of the functionalized piperazines and piperidines for
the phenol-containing series, together with their potential for
protonation to enhance solubility, we focused much of our
attention on groups of this class. This led to the discovery of
the sulfonylpiperazine derivative 17, which showed a marked
improvement in both biochemical and cellular potency compared
to 16 (Table 3).
The crystal structure of 17 bound to p110γ (Figure 4)
confirmed that the binding mode was largely as predicted from
the in silico modeling and clarifies the high potency observed
with compound 17. The morpholine moiety of 17 is in close
proximity to the hinge region of the kinase with the oxygen,
forming a hydrogen bond to the amide of V882 and the carbon
atoms packing against the side chain of I881. The hydrophobic
thienopyrimidine core of the compound packs against the side
chains of M804, W812, and I831 from the N-terminal lobe and
the side chains from M953 and I963 from the C-terminal lobe
of the kinase. The indazole moiety points toward a pocket
formed by residues K833, D836, L838, Y867, and D964 with
the two nitrogen atoms in H-hydrogen bonding distance to the
phenol oxygen of Y867 and the carboxyl group of D841. Finally,
the 4-methanesulfonyl-piperazin-1-ylmethyl group extends to-
ward the solvent with the piperazine ring packing against the
side chain of M804 and the oxygens of the sulfonyl group
positioned in H-bonding distance with the side chain of K802
and the amide nitrogen of A805.
a
The values are averages of at least 2 separate determinations with typical
b
variations of less than (30%. Percent remaining after 30 minutes
incubation of 10 µM compound with 1 mg/mL mouse and human liver
microsomes with both NADPH and UDPGA cofactors for glucuronidation.
The values are averages of at least 2 separate determinations with typical
variations of less than (30%.
to act as a hydrogen bond donor. Homology model analysis
also revealed that the phenol hydroxyl could potentially
participate in hydrogen bonding with Tyr836 (p110R number-
ing), also located at the back end of the pocket. Indazoles were
among the groups explored as replacements for phenols, and
the 4-indazolyl derivative 20 showed encouraging biochemical
activity, albeit 12-fold less potent than the corresponding phenol
derivative 1 (Table 2). In contrast, the 6-indazolyl derivative
2 showed a 300-fold reduction in activity relative to 1.
Introduction of the 4-methylpiperazin-1-ylmethylene group
Table 3), which had provided good solubility and improved
metabolic stability in the phenolic series, resulted in 16. This
compound displayed modest potency and was metabolically
stable in mouse and human microsomes. Encouragingly, com-
pound 16 exhibited improved oral bioavailability in both rat
37%) and mouse (31%). Analysis of the peak areas of the
metabolites of 16 in plasma following both iv and po admin-
2
(
The pharmacokinetic properties of 17 were extensively
studied. Acceptable oral bioavailability was achieved in all
species tested, including mouse (77%), rat (30%), dog (71%),
and monkey (20%). The pharmacokinetic profile in the dog, in
which 17 had moderate clearance, is presented in Figure 5. On
(

Clinical Candidate of PI3 Kinase
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5527
Figure 5. Mean ((SD) plasma concentrations (average of three dogs
dosed) of 17 (dimesylate salt) following a single administration iv (1
mg/kg) or po solution (2 mg/kg) of 17 to male beagle dogs.
Pharmacokinetic parameters: Cl ) 11.9 mL/min/kg; F ) 71%; t1/2
)
Figure 6. Growth inhibitory effect of 17 on established human U87MG
glioblastoma xenografts in female NCr athymic mice (N ) 12 per group,
mean ( SD).
3
.5 h; Vss ) 2.91 L/kg.
Table 5. Inhibition of Cancer Cell Proliferation and Signaling in
Selected Cell Lines by 17
4
73
cell lines with submicromolar IC50s. The antiproliferative effects
of the compound were associated with inhibition of intracellular
PI3K pathway by measuring Akt (Ser473) phosphorylation.
Potent inhibition of Akt (Ser473) phosphorylation was observed
in U87MG, PC3, and MDA-MB-361 cells with IC50s of 46,
molecular
features
proliferation
IC50 (µM)
pAkt
a
cancer cell line
IC50 (µM)
b
U87MG (glioblastoma) PTEN null
PC3 (prostate) PTEN null
MDA-MB-361 (breast) PIK3CA
mutant E545-K
0.95
0.28
0.72
0.046
0.037
0.028
c
c
3
7, and 28 nM, respectively.
a
The values are averages of at least 2 separate determinations with typical
variations of less than ( 30%. Measured using Biosource ELISA.
Measured using MesoScale Discovery electrochemiluminescence ELISA.
Furthermore, 17 shows minimal inhibition of six of the
b
33
principal cytochrome P450 isoforms, and at a concentration
c
of 25 µM, there is negligible induction of CYP1A and
34
CYP3A4. Also, there was no significant blockade of the hERG
the basis of good biochemical and cellular activity and phar-
macokinetic properties, compound 17 was chosen for further
evaluation.
channel for this compound (IC50 ) 64 µM) in the patch clamp
3
5
assay.
1
7 was selected for in vivo efficacy studies in established
The activity of 17 against the PI3K isoform family is
presented in Table 4, which shows that the compound is
equipotent against p110R and 110δ, while displaying modest
levels of selectivity against p110ꢀ (10-fold) and p110γ (25-
fold). However, greater levels of selectivity are observed for
compound 17 when tested against members of PI3K classes II,
III, and IV, including C2ꢀ, Vps34, DNA-PK, and mTOR.
Compound 17 was also tested against two of the p110R mutant
enzymes detected in human cancer. E545-K, which is a hotspot
mutation in the helical domain and H1047-R, a hotspot mutation
located in the C-terminal portion of the kinase domain, have
both been shown to have elevated lipid kinase activity relative
to the wild type and represent two of the three hotspots
U87MG human glioblastoma xenografts grown subcutaneously
in female NCr athymic mice (Figure 6). When dosed orally at
7
5 mg/kg daily, tumor growth inhibition of 83% (based on
weights of excised tumors compared to controls) was observed
after 21 days with no body weight loss. A dose-response
relationship was seen over the range 25-150 mg/kg/day.
Evidence of target inhibition was provided by a decrease in
p-Akt levels as measured in explanted xenograft tumors by
electrochemiluminescence immunoassay. For example p-Akt
levels fell to 5% of control values 30 min following an oral
dose of 75 mg/kg and were undetectable at 1 and 4 h time points.
Further details will be provided in a future publication.
5
,30,31
responsible for 80% of p110R mutations.
Compound 17
Conclusion
was found to be equipotent against both mutants when compared
to the wild type protein (Table 4). Experiments have also
indicated that 17 is a potent, ATP competitive inhibitor of p110R
with a Ki value of 10.2 ( 4.4 nM (data not shown). Compound
7 displayed outstanding selectivity for the PIK family kinases
over a panel of 228 kinases in the KinaseProfiler panel from
Millipore (formerly Upstate Biotechnologies). Of the 228
kinases tested, only two displayed greater than 50% inhibition
by 1 µM 17. The human tyrosine kinase Flt3 displayed 59%
inhibition by the test article, and the human kinase TrkA
displayed 61% inhibition by 1 µM 17. The IC50 value of 17 for
inhibition of TrkA was subsequently determined to be 2.85 µM
A series of thieno[3,2-d]pyrimidine derivatives have been
prepared and evaluated as inhibitors of PI3K. Structural
modifications were targeted primarily at improving the phar-
macokinetic properties of lead compound 1. Replacement of
the 3-hydroxyphenyl group with a 4-indazolyl group reduced
the issue of glucuronidation and resulted in acceptable oral
bioavailability. Further modifications led to the discovery of
17, which has been extensively profiled and shown to be a potent
and highly selective inhibitor of members of Class I PI3K. The
compound shows potent growth inhibitory activity in vitro in a
range of human tumor cell lines and exhibits a strong inhibitory
effect on the growth of human U87MG glioblastoma xenografts
in athymic mice. Compound 17 demonstrates an acceptable
pharmaceutical profile and is currently being evaluated in human
clinical trials for the treatment of cancer.
1
3
2
(
data not shown). Thus, 17 demonstrates at least 300-fold
selectivity for inhibition of p110R /p85R over other assayable
kinases.
Compound 17 was tested using the 96 h Alamar Blue assay
against a wider panel of human tumor cell lines, which included
glioblastoma, breast, and prostate lines that have genetic
aberrations in the PI3K pathway (Table 5). These data demon-
strate that 17 is a potent inhibitor of cell proliferation in these
Experimental Section
Chemistry. All solvents and reagents were used as obtained.
H NMR spectra were recorded with a Bruker Avance DPX400
1

5
528 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Folkes et al.
Spectrometer; 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.
XBridge Phenyl analytical column and H20/MeCN modified with
0.1% formic acid running a linear gradient from 10% MeCN to
100% MeCN monitored by UV wavelength 210 nm and ESI+ TIC
MS showed 95.6% purity.
3-(6-{[(2-Methoxy-ethyl)-methyl-amino]-methyl}-4-morpholin-
4-yl-thieno[3,2-d]pyrimidin-2-yl)-phenol (7). Compound 7 was
prepared from 5 according to the same procedure described for 8,
1
using N-(2-methoxyethyl)methylamine. H NMR (400 MHz,
4
-morpholin-4-yl-2-[3-(1,1,2,2-tetramethyl-propylsilanyloxy)-
3
CDCl ) δ 2.41 (s, 3H), 2.71 (t, J ) 5.6 Hz, 2H), 3.38 (s, 3H), 3.56
22
phenyl]-thieno[3,2-d]pyrimidine (4). A mixture of 3 (2.65 g, 10.37
mmol), 3-(tert-butyldimethylsilyloxy)phenyl boronic acid (3.40 g,
.3 equiv), sodium bicarbonate (1.74 g, 2 equiv), PdCl
mg, 5%), DME (50 mL), and water (15 mL) was heated to reflux.
After 16 h, the reaction mixture was cooled, diluted with ethyl
acetate, washed with brine, dried (MgSO
(t, J ) 5.6 Hz, 2H,), 3.84-3.88 (m, 4H), 3.90 (s, 2H), 4.03-4.07
(m, 4H), 6.92 (dd, 1H), 7.30 (s, 1H), 7.33 (t, J ) 7.9 Hz, 1H), 7.92
+
1
(PPh )
2 3 2
(365
(s, 1H), 7.99 (d, J ) 7.8 Hz, 1H). MS (ESI): m/z (M + H) 415.
Analytical LC-MS using Waters XBridge Phenyl analytical column
and H20/MeCN modified with 0.1% formic acid running a linear
gradient from 10% CH3CN to 100% CH3CN monitored by UV
wavelength 210 nm and ESI+ TIC MS showed 96.1% purity.
3-{6-[4-(2-Hydroxy-ethyl)-piperazin-1-ylmethyl]-4-morpholin-4-
yl-thieno[3,2-d]pyrimidin-2-yl}-phenol (9). Compound 9 was pre-
4
), and the solvent removed
in vacuo. Purification on silica (EtOAC/hexanes, 1:4) yielded the
1
3
desired compound (2.28 g, 51%). H NMR (400 MHz, CDCl ) δ
0
6
1
.25 (s, 6H), 1.03 (s, 9H), 3.85-3.89 (m, 4H), 4.05-4.09 (m, 4H),
.92-6.96 (m, 1H), 7.30-7.33 (m, 1H), 7.53 (d, J ) 5.49 Hz,
H), 7.76 (d, J ) 5.46 Hz, 1H), 7.93 (s, 1H), 8.02-8.05 (m, 1H).
pared from 5 according to the same procedure described for 8 using
1
1-piperazine ethanol. H NMR (400 MHz, CDCl
3
) δ 2.63 (mbr,
4-Morpholin-4-yl-2-[3-(1,1,2,2-tetramethyl-propylsilanyloxy)-
10H), 3.65 (m, 2H), 3.84 (s, 2H), 3.87-3.90 (m, 4H), 4.04-4.06
phenyl]-thieno[3,2-d]pyrimidine-6-carbaldehyde(5). To a solution
of 4 (2.26 g, 5.29 mmol) in dry THF (40 mL) cooled to -78 °C
was added nBuLi (2.5 M solution in hexanes, 2.75 mL). After
stirring for 20 min, dry N,N-dimethylformamide (617 µL, 1.5 equiv)
was added, and the reaction mixture was stirred for 20 min at -78
(m, 4H), 6.93 (d, J ) 10.0 Hz,1H), 7.32-7.36 (m, 2H), 7.91 (s,
+
1H), 8.01 (d, J ) 7.74 Hz, 1H). MS (ESI): m/z (M + H) 456.
Analytical LC-MS using Waters XBridge Phenyl analytical column
and H20/CH3CN modified with 0.1% formic acid running a linear
gradient from 10%CH3CN to 100% CH3CN monitored by UV
wavelength 210 nm and ESI+ TIC MS showed 99% purity.
2-Chloro-6-methyl-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (10).
°
3
C and then warmed slowly to room temperature. After a further
0 min at room temperature, the reaction mixture was quenched
with ice/brine and then extracted exhaustively with chloroform. The
combined organic extracts were dried (MgSO ) and the solvent
removed in vacuo to yield 5 as a yellow solid (2.38 g, 99%). H
NMR (400 MHz, CDCl
2
2
To a suspension of 3 (543 mg, 2.18 mmol) in dry THF cooled at
-78 °C was added a 2.5 M solution of nBuLi in hexanes (1.02
mL, 1.2 equiv). After stirring for 1 h, methyl iodide (158 µL, 1.2
equiv) was added. The reaction mixture was stirred for 1 h at -78
°C and then warmed slowly to room temperature. After stirring
for 2 days, the reaction mixture was poured onto ice/water and
extracted into ethyl acetate. The organic phase was collected, dried
4
1
3
) δ 0.24 (s, 6H), 1.01 (s, 9H), 3.89-3.92
m, 4H), 4.11-4.14 (m, 4H), 6.92-6.96 (m, 1H), 7.32-7.36 (m,
(
1
(
H), 7.92 (s, 1H), 8.02 (d, J ) 7.70 Hz, 1H), 8.13 (s, 1H), 10.19
s, 1H).
3
-[6-(4-Methyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-
(MgSO
4
), and the solvent removed in vacuo to yield 10 as a beige
1
d]pyrimidin-2-yl]-phenol (8). A mixture of 5 (6.67 g, 14.66 mmol),
N-methylpiperazine (2.11 mL, 1.3 equiv), and acetic acid (838 µL,
.0 equiv) was stirred in 1,2-dichloroethane (70 mL) at room
temperature. To this was added sodium triacetoxyborohydride (3.42
g, 1.1 equiv), and the reaction mixture was stirred for 3 days. The
reaction mixture was then quenched with aqueous sodium bicarbon-
solid (470 mg, 82%). H NMR (400 MHz, DMSO-d ) δ 2.62 (s,
6
3H), 3.78-3.81 (m, 4H), 3.86-3.90 (m, 4H), 7.18 (s, 1H).
3-(6-Methyl-4-morpholin-4-yl-thieno[3,2-d]pyrimidin-2-yl)-phe-
nol (11). A mixture of 10 (252 mg, 0.93 mmol), 3-hydroxyphenyl
boronic acid (141 mg, 1.1 equiv), sodium carbonate (235 mg, 3
1
2 3 2
equiv), PdCl (PPh ) (33 mg, 5%), DME (10 mL), and water (2
ate solution, extracted exhaustively with chloroform, dried (MgSO
4
),
mL) was heated to reflux under argon. After 4 h, the reaction
mixture was cooled, diluted with chloroform, washed with water,
and the solvent removed in vacuo to yield a yellow oil. This was
purified using flash chromatography (silica, ethyl acetate to ethyl
acetate/methanol) to yield 6-(4-methyl-piperazin-1-ylmethyl)-4-
morpholin-4-yl-2-[3-(1,1,2,2-tetramethyl-propylsilanyloxy)-phenyl]-
thieno[3,2-d]pyrimidine (6.95 g, 88%). To a solution of 6-(4-methyl-
piperazin-1-ylmethyl)-4-morpholin-4-yl-2-[3-(1,1,2,2-tetramethyl-
propylsilanyloxy)-phenyl]-thieno[3,2-d]pyrimidine (6.95 g, 12.89
mmol) in THF (100 mL) cooled to 0 °C was added a 1.0 M solution
of tetrabutyl ammonium fluoride in THF (14.2 mL, 1.1 equiv). After
4
dried (MgSO ), and the solvent removed in vacuo. Purification using
column chromatography (EtOAc/hexanes to EtOAc) yielded the
1
6
desired compound (30 mg, 10%). H NMR (400 MHz, d -DMSO)
δ 2.65 (s, 3H), 3.80-3.84 (m, 4H), 3.95-3.99 (m, 4H), 6.85-6.88
(m, 1H), 7.25-7.29 (m, 2H), 7.81-7.84 (m, 2H), 9.52 (s, 1H).
+
MS (ESI): m/z (M + H) 328.
2-Chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidine-6-carbalde-
2
2
hyde (13). To a suspension of 3 (1.75 g, 6.85 mmol) in dry
tetrahydrofuran (40 mL) at -78 °C was added a 2.5 M solution of
nBuLi in hexanes (3.3 mL, 1.2 equiv). After stirring for 1 h, dry
N,N-dimethylformamide (796 µL, 1.5 equiv) was added. The
reaction mixture was stirred for 1 h at -78 °C and then warmed
slowly to room temperature. After a further 2 h at room temperature,
the reaction mixture poured onto ice/water, yielding a yellow
3
0 min, the solvent was removed in vacuo and the residue was
purified using flash chromatography (silica, 8% methanol in
dichloromethane) and then triturated using ethyl acetate/methanol
to yield 8 as a white solid (4.62 g, 85%). H NMR (400 MHz,
1
CDCl
3
) δ 2.27 (s, 3H), 2.49 (sbr, 8H), 3.79 (s, 2H), 3.82-3.84 (m,
H), 4.00-4.03 (m, 4H), 6.88-6.91 (m, 1H), 7.24-7.28 (m, 2H),
.69-7.70 (m, 1H), 7.81 (d, J ) 7.8 Hz, 1H). MS (ESI): m/z (M
4
7
+
precipitate. This was collected by filtration and air-dried to yield
+
1
H) 426. Analytical LC-MS using Waters XBridge Phenyl
the title compound (1.50 g, 77%). H NMR (400 MHz, DMSO-d
6
)
analytical column and H20/MeCN modified with 0.1% formic acid
running a linear gradient from 10% MeCN to 100% MeCN
monitored by UV wavelength 210 nm and ESI+ TIC MS showed
δ 3.74-3.78 (m, 4H), 3.94-3.97 (m, 4H), 8.28 (s, 1H), 10.20 (s,
1H).
2-Chloro-6-(4-methyl-piperazin-1-yl methyl)-4-morpholin-4-yl-
thieno[3,2-d]pyrimidine (14). To a mixture of 13 (147 mg, 0.52
mmol), 1-methyl-piperazine (87 µL, 1.5 equiv) and acetic acid (32
µL, 1.05 equiv) in 1,2-dichloroethane (3 mL) was added sodium
triacetoxyborohydride (121 mg, 1.1 equiv) and then stirred at room
temperature overnight. The reaction mixture was diluted with
dichloromethane, washed with a saturated solution of sodium
>
99% purity.
-(6-Dimethylaminomethyl-4-morpholin-4-yl-thieno[3,2-d]pyri-
3
midin-2-yl)-phenol (6). Compound 6 was prepared from 5 according
1
to the same procedure described for 8 using dimethylamine. H
NMR (400 MHz, CDCl
3
) δ 2.39 (s, 6H), 3.74 (s, 2H), 3.82-3.86
(
m, 4H), 4.01-4.04 (m, 4H), 5.70 (s, br, 1H), 6.90-6.92 (m, 1H),
.31-7.35 (m, 2H), 7.89 (d, J ) 2.4 Hz, 1H), 8.00 (d, J ) 7.8 Hz,
7
hydrogen carbonate, brine, separated, and dried (MgSO
4
). The crude
+
1H). MS (ESI): m/z (M + H) 371. Analytical LC-MS using Waters
product was evaporated in vacuo and purified by chromatography

Clinical Candidate of PI3 Kinase
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5529
1
to give 14 as an off-white crystalline solid (51 mg, 45%). H NMR
400 MHz, CDCl ) δ 2.21 (s, 3H), 2.40-2.55 (m, br, 8H), 3.72 (s,
H), 3.75-3.79 (m, 4H), 3.93-3.96 (m, 4H), 7.11 (s, 1H).
-Chloro-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpho-
2-(1H-Indazol-4-yl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (20).
To a solution of 19 (80 mg, 0.25 mmol) in chloroform (8 mL) and
acetic acid (4 mL) was added isoamyl nitrite (36 µL, 1.1 equiv).
The reaction mixture was stirred for 1 day at room temperature.
The mixture was then quenched with sodium bicarbonate solution
and extracted in to chloroform and reduced in vacuo. The residue
was purified using flash chromatography (EtOAC/hexanes to
(
2
3
2
lin-4-yl-thieno[3,2-d]pyrimidine (15). A mixture of 13 (5.56 g, 19.6
mmol), 1-methanesulfonyl-piperazine hydrochloride (4.72 g, 1.2
equiv), and trimethylorthoformate (6.42 mL, 3 equival) was stirred
in 1,2-dichloroethane (100 mL) for 6 h at room temperature. To
this was added sodium triacetoxyborohydride (10.39 g, 2.5 equiv),
and the reaction mixture was stirred for 24 h at room temperature.
The mixture was then quenched with brine, extracted with dichlo-
1
EtOAc) to yield 20 (17 mg, 20%). H NMR (400 MHz, DMSO-
d
6
) δ 3.86-3.91 (m, 4H), 4.05-4.09 (m, 4H), 7.48-7.52 (m, 1H),
7
.63-7.70 (m, 2H), 8.26 (d, 1H), 8.33 (d, 1H), 8.95 (s, 1H), 13.20
+
(
s, br, 1H). MS (ESI): m/z (M + H) 338. Analytical LC-MS using
Waters XBridge Phenyl analytical column and H20/MeCN modified
with 0.1% formic acid running a linear gradient from 10% MeCN
to 100% MeCN monitored by UV wavelength 210 nm and ESI+
TIC MS showed 98.2% purity.
2-(1H-Indazol-6-yl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (22).
A mixture of 3 (806 mg, 3.15 mmol), 3-amino-4-methylbenzenebo-
ronic acid (524 mg, 1.1 equiv), DME (10 mL), water (5 mL),
romethane, dried (MgSO
4
), and the solvent removed in vacuo. The
residue was triturated with hot ethyl acetate to yield 15 as a white
1
solid (1.01 g). NMR H NMR (400 MHz, CDCl
m, 4H), 2.79 (s, 3H), 3.30-3.33 (m, 4H), 3.82-3.86 (m, 6H),
.98-4.01 (m, 4H), 7.18 (s, 1H).
-(1H-Indazol-4-yl)-6-(4-methyl-piperazin-1-ylmethyl)-4-mor-
3
) δ 2.69-2.71
(
3
2
pholin-4-yl-thieno[3,2-d]pyrimidine (16). Compound 16 was pre-
pared from 14 according to the same procedure described for 17.
2 3 2
sodium carbonate (640 mg, 2 equiv), and PdCl (PPh ) (100 mg,
1
5%) was heated to reflux for 16 h. The reaction mixture was then
cooled, diluted with ethyl acetate, and reduced in vacuo. The residue
was purified using flash chromatography to yield 2-methyl-5-(4-
morpholin-4-yl-thieno[3,2-d]pyrimidin-2-yl)-phenylamine (21) (840
mg, 82%). To a solution of 21 (99 mg, 0.30 mmol) in chloroform
H NMR (400 MHz, DMSO-d ) δ 2.18 (s, 3H), 2.30-2.45 (m,
6
4
3
8
H), 2.48-2.55 (m, 4H), 3.82-3.84 (m, 4H), 3.86 (s, 2H),
.98-4.00 (m, 4H), 7.44-7.47 (m, 2H), 7.65 (d, J ) 8.2 Hz, 1H),
.21 (d, J ) 7.2 Hz, 1H), 8.87 (s, 1H), 13.16 (s, br,1H). MS (ESI):
+
m/z (M + H) 450. Analytical LC-MS using Waters XBridge
Phenyl analytical column and H20/MeCN modified with 0.1%
formic acid running a linear gradient from 10% MeCN to 100%
MeCN monitored by UV wavelength 210 nm and ESI+ TIC MS
showed 98.4% purity.
(
1
10 mL) and acetic acid (2 mL) was added isoamyl nitrite (44 µL,
.1 equiv). The reaction mixture was stirred for 2 days at room
temperature. The mixture was then quenched with sodium bicar-
bonate solution and extracted into chloroform and reduced in vacuo.
The residue was purified using flash chromatography (EtOAc) to
2
-(1H-Indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-
-morpholin-4-yl-thieno[3,2-d]pyrimidine (17). A mixture of 15
2.00 g, 4.63 mmol), 25 (2.26 g, 2 equiv), toluene (24 mL), ethanol
12 mL), water (6 mL), sodium carbonate (1.72 g, 3.5 equiv), and
1
yield the title compound (14 mg, 14%). H NMR (400 MHz,
4
DMSO-d ) δ 3.85-3.89 (m, 4H), 4.05-4.09 (m, 4H), 7.58 (d, J )
6
(
5
8
1
.4 Hz, 1H), 7.86 (d, J ) 8.4 Hz, 1H), 8.12 (s, 1H), 8.12 (d, J )
(
.5 Hz, 1H), 8.19 (d, J ) 5.4 Hz, 1H), 8.21 (s, 1H), 13.22 (s, br,
PdCl
microwave for 90 min. The reaction mixture was cooled, diluted
with chloroform, washed with brine, dried (MgSO ), and the solvent
2
(PPh
3
)
2
(325 mg, 0.1 equiv) was heated to 130 °C in the
+
H). MS (ESI): m/z (M + H) 338. Analytical LC-MS using Waters
XBridge Phenyl analytical column and H20/MeCN modified with
4
0
1
.1% formic acid running a linear gradient from 10% MeCN to
00% MeCN monitored by UV wavelength 210 nm and ESI+ TIC
removed in vacuo. The residue was purified using flash chroma-
tography (chloroform to 10% methanol/chloroform) and then
trituration with ether, yielding the desired title compound as a white
MS showed 95.5% purity.
4-Bromo-1H-indazole (24). To a solution of 3-bromo-2-methy-
1
solid (1.42 g, 60%). H NMR (CDCl
3
) δ 2.67-2.71 (m, 4H), 2.81
laniline (5.00 g, 26.9 mmol) in chloroform (50 mL) was added
potassium acetate (2.77 g, 28.2 mmol). Acetic anhydride (5.07 mL,
(
4
s, 3H), 3.29-3.33 (m, 4H), 3.89 (s, 2H), 3.89-3.93 (m, 4H),
.08-4.12 (m, 4H), 7.41 (s, 1H), 7.51 (t, J ) 7.2 Hz, 1H), 7.60 (d,
5
3.7 mmol) was added with concurrent cooling in ice-water. The
J ) 8.3 Hz, 1H), 8.28 (d, J ) 7.5 Hz, 1H), 9.02 (s, 1H), 10.10 (s,
mixture was then stirred at room temperature for 10 min, after which
time a white gelatinous solid formed. 18-crown-6 (1.42 g, 5.37
mmol) was then added, followed by isoamyl nitrite (7.94 mL, 59.1
mmol), and the mixture was heated under reflux for 18 h. The
reaction mixture was allowed to cool and was partitioned between
chloroform and saturated aqueous sodium hydrogen carbonate. The
combined organic extracts were washed with brine, separated, and
+
br,1H). MS (ESI): m/z (M + H) 514. Anal. (C23
27 7 3 2
H N O S ) C,
H, N. Analytical LC-MS using Waters XBridge Phenyl analytical
column and H20/MeCN modified with 0.1% formic acid running
a linear gradient from 10% MeCN to 100% MeCN monitored by
UV wavelength 210 nm and ESI+ TIC MS showed 98% purity.
-Methyl-3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phe-
nylamine (18). To a solution of 3-bromo-2-methylaniline (1.00 g,
.38 mmol) in dioxane (15 mL) was added triethylamine (3.0 mL,
equiv), Pd(OAc) (60 mg, 5%), 2-dicyclohexylphosphinobiphenyl
377 mg, 20%), and pinacol borane (2.34 mL, 3 equiv), and the
reaction mixture was heated to 80 °C for 1 h. The reaction mixture
was then cooled, diluted with ethyl acetate, washed with brine, dried
4
), and the solvent reduced in vacuo. The residue was
purified using flash chromatography (EtOAc/hexane 1:4) to yield
8 as a brown oil (1.07 g, 85%). H NMR (400 MHz, CDCl
.27 (s, 12H), 2.31 (s, 3H), 3.52 (s, 2H), 6.68 (d, J ) 7.8 Hz, 1H),
.92-6.96 (m, 1H), 7.14 (d, J ) 7.4 Hz, 1H)
-Methyl-3-(4-morpholin-4-yl-thieno[3,2-d]pyrimidin-2-yl)-phe-
nylamine (19). A mixture of 3 (169 mg, 0.66 mmol), 18 (155 mg,
.66 mmol), sodium carbonate (140 mg, 2 equiv), DME (10 mL),
water (3 mL), and PdCl (PPh (23 mg, 5%) was heated to reflux.
After 16 h, the reaction mixture was cooled, diluted with ethyl
acetate, washed with brine, dried (MgSO ), and reduced in vacuo.
2
dried (MgSO ). The crude product was evaporated onto silica and
4
5
4
purified by chromatography eluting with 20% to 40% EtOAc-petrol
2
to provide 1-(4-bromo-indazol-1-yl)-ethanone 23 (3.14 g, 49%) and
4-bromo-1H-indazole 24 (2.13 g, 40%). To a solution of 23 (3.09
g, 12.9 mmol) in methanol (50 mL) was added 6M aqueous HCl
(30 mL), and the mixture was stirred at room temperature for 7 h.
The methanol was evaporated and the mixture partitioned between
ethyl acetate and water. The combined organic layers were washed
(
(
MgSO
1
1
1
6
3
) δ
with brine, separated, and dried (MgSO ). The solvent was removed
4
by evaporation under reduced pressure to give 24 (2.36 g, 93%).
1
H NMR (400 MHz, CDCl ) δ 7.25 (t, J ) 7.3 Hz, 1H), 7.33 (d,
3
2
J ) 7.3 Hz, 1H), 7.46 (d, J ) 7.3 Hz, 1H), 8.11 (s, 1H), 10.20 (s,
br,1H).
0
4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indazole (25).
To a solution of 24 (500 mg, 2.54 mmol) and bis(pinacolato)diboron
(967 mg, 3.81 mmol) in DMSO (20 mL) was added potassium
2
3 2
)
4
2 2
acetate (747 mg, 7.61 mmol) and PdCl (dppf) (62 mg, 0.076 mmol,
The residue was purified using flash chromatography (EtOAc/
hexanes 1:1 to EtOAc) to yield 19 (148 mg, 69%). H NMR (400
3 mol%). The mixture was degassed with argon and heated at 80
°C for 40 h. The reaction mixture was allowed to cool and
partitioned between water and ether. The combined organic layers
1
MHz, CDCl
.01-4.06 (m, 4H), 6.73-6.76 (m, 1H), 7.08-7.13 (m, 2H), 7.51
d, J ) 5.5 Hz, 1H), 7.77 (d, J ) 5.5 Hz, 1H).
3
) δ 2.29 (s, 3H), 3.70 (s, br, 2H), 3.85-3.88 (m, 4H),
4
were washed with brine, separated, and dried (MgSO
4
). The crude
(
material was purified by chromatography eluting with 30% to 40%

5
530 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Folkes et al.
EtOAc-petrol to give an inseparable 3:1 mixture of the desired
product 25 (369 mg, 60%) and indazole (60 mg, 20%). H NMR
carried out using either the BioSource p-AKT (Ser473) ELISA
kit (no. KHO0111) and total Akt kit (no. KHO0101) with
absorbance at 450 nm read on a Wallac Victor2 plate reader or
the Meso Scale Discovery (MSD) MS6000 Phospho (Ser473)/
Total Akt Whole Cell Lysate Kit (no. K11100D-1) reading ECL
signal using the MSD SECTOR Imager 6000 reader. Both
assays were carried out according to the manufacturer’s protocol.
The reported IC50 values are means of at least two independent
experiments with variations of less than 20%.
1
(
400 MHz, DMSO-d
Hz, 1H), 7.59 (d, J ) 8.4 Hz, 1H), 7.67 (d, J ) 6.9 Hz, 1H), 8.45
s, 1H), 10.00 (s, br,1H).
6
) δ 1.41 (s, 12H), 7.40 (dd, J ) 8.4 Hz, 6.9
(
Assays
Scintillation Proximity Assay. Recombinant human p110 R,
, and δ were coexpressed in an Sf9 baculovirus system with
ꢀ
the p85R regulatory subunit and purified as GST-fusion proteins
using affinity chromatography on glutathione-sepharose. Re-
combinant human p110γ, Vps34, and C2ꢀ proteins were
expressed as monomeric GST-fusions and purified similarly.
Test compounds were dissolved in DMSO and added to 20 mM
Tris-HCl (pH 7.5) containing 200 µg yttrium silicate (Ysi)
polylysine SPA beads (Amersham), 4 mM MgCl2, 1 mM
Metabolic Stability Studies. Male CD1 mouse liver mi-
crosomes and mixed gender pooled human liver microsomes
were purchased from Tebu-bio (Peterborough, U.K.). Incuba-
tions contained final concentrations of 1 mg/mL microsomal
protein, 10 µmol/L test compound, 3 mmol/L MgCl2, 1 mmol/L
NADPH, 2.5 mmol/L UDP-glucuronic acid, and 10 mmol/L
phosphate buffer (pH 7.4) (all purchased from Sigma Aldrich,
Gillingham, U.K.) in a total volume of 200 µL and were
performed for 0, 15, and 30 min at 37 °C. The reaction was
terminated by the addition of 3 volumes of ice-cold methanol
containing olomoucine (Sigma Aldrich, Gillingham, U.K.) as
an internal standard. Samples were centrifuged at 2800g for 20
min at 4 °C and the supernatants analyzed. Control incubations
were prepared as above but by omission of either cofactors or
microsomes.
3
3
dithiothreitol (DTT), 1 µM ATP, and 0.125 µCi [γ- P]-ATP
Perkin-Elmer)), 4% (v/v) DMSO in a total volume of 50 µL.
(
The recombinant GST-fusion of p110R/p85R (5 ng), p110ꢀ/
p85R (5 ng), p110δ/p85R (5 ng), p110γ (5 ng), p110R/
p85R(H1047-R)(5ng), p110R/p85R(E545-K) (5 ng), Vps 34 (40
ng), or C2ꢀ (5 ng) was added to the assay mixture to initiate
the kinase reaction. After incubation for 1 h at room temperature,
the kinase reaction was terminated with 150 µL PBS. The
mixture was then centrifuged for 2 min at 2000 rpm and read
using a Wallac Microbeta counter (Perkin-Elmer). The reported
IC50 values are means of at least 2 independent experiments
and are calculated using a sigmoidal, dose-response curve fit
Pharmacokinetic Studies. Mouse studies. The mesylate salt
of the appropriate compound was used in the study. Three male
CD1 mice, 25-30 g, were dosed per administration route, per
time point, per compound. The compounds were administered
orally (via gavage) and iv (via the tail vein) at a dose level of
(
4 parameter logistic model [y ) A + ((B - A)/(1 + ((C/x)^D)])
in MDL Assay Explorer.
1
0 mg/kg per compound. Animals were given free access to
mTOR assay. Mammalian target of rapamycin (mTOR) was
assayed by monitoring phosphorylation of GFP-4EBP using a
homogeneous time-resolved fluorescence resonance energy
transfer format and assay reagents from Invitrogen. In the
presence of 8 uM ATP, 50 mM Hepes (pH 7.5), 0.01% (v/v)
Tween 20, 10 mM MnCl2, 1 mM EGTA, and 1 mM DTT, the
mTOR-mediated phosphorylation of 400 nM GFP-4E-BP1 was
measured under initial rate conditions. After incubation at 25
food throughout the studies. At the appropriate times, the
animals were anaesthetized and sacrificed and blood samples
were collected and placed in heparinized tubes. Blood samples
were taken at 0.08, 0.25, 0.5, 1, 2, 4, and 8 h following oral
dosing and at 0.08, 0.25, 0.5, 1, 2, 4, and 8 h following
intravenous dosing. Blood samples were centrifuged to obtain
the plasma, which was aspirated into a separate labeled
container. Aliquots from the three animals at each time point,
for each route, were pooled. Standard curves were prepared in
blank plasma matrices. Standards and samples from plasma were
analyzed using a 3.5 min LC method. Parent compounds were
quantified using specific MRM transition.
°
C for 30 min, the reaction was terminated by addition of 10
mM EDTA, and phosphorylated GFP-4E-BP1 was detected with
nM Tb-anti-p4E-BP1 antibody before reading on a Perkin-
2
Elmer EnVision Fluorescence Reader (exc 340; em 495/520).
Duplicate dose-response curves were fit to an equation of
competitive tight-binding inhibition.
Pharmacokinetic Studies. Dog Study. The dimesylate salt
of 17 was used for the study. Dosing formulations were prepared
in 30% sulfobutylether-ꢀ-cyclodextrin (SBE-ꢀ-CD). Prepared
dosing formulations were stored at 2-8 °C for up to 1 h before
dosing. Three male purebred beagle dogs (Harlan Bioproducts
for Science, Madison, WI) were used in this study. At the
initiation of the study, dogs weighed from 12.8 to 14.1 kg. Only
animals that appeared to be healthy and that were free of obvious
abnormalities were used for the study.
This cross-over study was composed of two phases. In the
first phase, 3 male dogs were given a single iv dose of 1 mg/kg
of 17 via a cephalic vein. In the second phase, the same dogs
were given po doses of 2 mg/kg of 17 in an SBE-ꢀ-CD solution.
The two phases were each separated by a 7-day washout period.
Animals were not fasted before dosing in the first of these
phases, but they were fasted overnight before the second of these
phases until 4 h postdose. Blood samples were collected from
all animals after the first phase at predose and at 0.0033, 0.083,
0.25, 0.5, 1, 2, 4, 8, 12, and 24 h after the intravenous dose.
Blood samples were collected from all animals in the second
phase at predose and at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h
after the oral dose. The samples (approximately 3 mL per
Proliferation Assay. The human tumor cell lines used were
4
obtained from the ATCC. Cells were plated at 4 × 10 cells/
mL and cultured at 37 °C with 5% CO2 in DMEM supplemented
with 10% fetal calf serum, and L-glutamine. Test compound
was added to replicate wells in a volume of 10 µL such that
the final DMSO concentration did not exceed 0.2%. After 4
days of incubation, 10 µL of Alamar Blue reagent was added
and developed for 6 h at 37 °C before measuring the fluores-
cence excitation/emission (wavelength 540/595 nm) using a
Victor plate reader. The reported IC50 values are means of at
least two independent experiments with variations of less than
2
0%.
p-Akt Assay. The human tumor cell lines used were obtained
from the ATCC. Cells were plated in 6-well tissue culture plates
in DMEM/RPMI1640 supplemented with 10% FCS and 2 mM
L-glutamine. Test compound was added to replicate wells in a
volume so that the final DMSO concentration did not exceed
0
.2%. After 2 h incubation at 37 °C with 5% CO2, cells were
lysed at 4 °C and lysates centrifuged for 15 min at 14000 rpm.
Protein concentration was established using the Pierce BCA
protein assay kit (no. 23225). Assessment of p-AKT (S473) was

Clinical Candidate of PI3 Kinase
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5531
sample) were collected from the jugular vein into tubes con-
taining dipotassium ethylenediaminetetraacetic acid (K2EDTA)
as an anticoagulant. Blood samples were kept on ice and
centrifuged for 10-15 min at 2000g and 2-8 °C within 1 h of
collection. Plasma was collected and stored frozen at -60 °C
to -80 °C until ready for analysis. The concentration (expressed
as free base equivalents) of 17 in each plasma sample was
determined by nonvalidated liquid chromatography-tandem mass
spectrometry assays. The assay lower limit of quantitation
Supporting Information Available: Tabulation of compound
purity, and HPLC tracings. This material is available free of charge
via the Internet at http://pubs.acs.org.
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Bowman, Alberto Estevez, Kyle Mortara, and Jiansheng Wu at
Genentech for technical assistance of protein expression and
purification. We thank Delia Bradford for assistance with the
pharmacokinetic studies, and Vickie Tsui for assistance with the
molecular modeling. We thank Alex De Havon Brandon and Alan
Henley for expert technical assistance with in vivo studies, and
Francesco D Stefano and Zahida Ahmad for expert technical
assistance in characterization of cancer cell lines. We thank
Frederique Urban for expert technical assistance with microsomal
incubations. We thank Mohammed Latif for analytical support. We
thank Cristina Lewis and Jim Nonomiya for generating the mTOR
data. We thank the staff at ALS, beamline 5.2. The Advanced Light
Source is supported by the Director, Office of Science, Office of
Basic Energy Sciences, Materials Sciences Division, of the U.S.
Department of Energy under contract no. DE-AC03-76SF00098
at Lawrence Berkeley National Laboratory. The work at The
Institute of Cancer Research was supported by Cancer Research
UK [CUK] Programme Grant numbers C309/A8724 and C2536/
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JM800295D