Discovery of 9-(6-aminopyridin-3-yl)-1-(3-(trifluoromethyl)phenyl)benzo[ h ][1,6]naphthyridin-2(1 H)-one (torin2) as a potent, selective, and orally available mammalian target of rapamycin (mTOR) inhibitor for treatment of cancer

Article abstract of DOI:10.1021/jm101520v

The mTOR mediated PI3K/AKT/mTOR signal transduction pathway has been demonstrated to play a key role in a broad spectrum of cancers. Starting from the mTOR selective inhibitor 1 (Torin1), a focused medicinal chemistry effort led to the discovery of an improved mTOR inhibitor 3 (Torin2), which possesses an EC₅₀ of 0.25 nM for inhibiting cellular mTOR activity. Compound 3 exhibited 800-fold selectivity over PI3K (EC₅₀: 200 nM) and over 100-fold binding selectivity relative to 440 other protein kinases. Compound 3 has significantly improved bioavailability (54%), metabolic stability, and plasma exposure relative to compound 1.

Full text of DOI:10.1021/jm101520v

ARTICLE
pubs.acs.org/jmc
Discovery of 9-(6-Aminopyridin-3-yl)-1-(3-(trifluoromethyl)-
phenyl)benzo[h][1,6]naphthyridin-2(1H)-one (Torin2) as a
Potent, Selective, and Orally Available Mammalian Target
of Rapamycin (mTOR) Inhibitor for Treatment of Cancer
Qingsong Liu,†,‡ Jinhua Wang,†,‡ Seong A. Kang,§ Carson C. Thoreen,†,‡ Wooyoung Hur,†,‡ Tausif Ahmed,||
David M. Sabatini,§,^,# and Nathanael S. Gray*,†,‡
†Department of Cancer Biology, Dana Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115, United States
‡Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston,
Massachusetts 02115, United States
§Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, United States
Sai Advantium Pharma Ltd., 8-2-120/86/9/B “Luxor Park”, Road No. 2, Banjara Hills, Hyderabad 500033, India
^Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139,
United States
#Koch Center for Integrative Cancer Research at MIT, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States
S Supporting Information
b
ABSTRACT: The mTOR mediated PI3K/AKT/mTOR signal transduction pathway has been
demonstrated to play a key role in a broad spectrum of cancers. Starting from the mTOR
selective inhibitor 1 (Torin1), a focused medicinal chemistry effort led to the discovery of an
improved mTOR inhibitor 3 (Torin2), which possesses an EC50 of 0.25 nM for inhibiting
cellular mTOR activity. Compound 3 exhibited 800-fold selectivity over PI3K (EC50: 200 nM)
and over 100-fold binding selectivity relative to 440 other protein kinases. Compound 3 has
significantly improved bioavailability (54%), metabolic stability, and plasma exposure relative to
compound 1.
’ INTRODUCTION
The PI3K/Akt/mTOR signal transduction pathway is frequently
deregulated in human cancers and thereby has attracted con-
siderable attention as an oncology drug discovery target.6-9
Successful development of rapalogues (rapamycin and related
analogues) as treatments of specific cancers has provided clinical
validation of mTOR as an anticancer drug discovery target.10
However, existing rapalogues do not fully inhibit mTORC1 and
are unable to acutely inhibit mTORC2.11,12 In addition, a negative
feedback loop exists through S6K/IRS1/PI3K that results in
pathway activation upon inhibition of mTORC1 by rapamycin
which may also serve to limit the efficacy of the rapalogues.13
There is currently great interest in clinically testing the hypoth-
esis that ATP-competitive mTOR inhibitors will exhibit more
broad-based and profound antitumor activity relative to the
rapalogues. In the recent years there has been a significant invest-
ment in developing potent and selective mTOR inhibitors which
The mammalian target of rapamycin (mTOR) is a highly con-
served serine/threonine protein kinase and is a key element in
the PI3K/AKT/mTOR signal pathway.1 Upon activation by
extracellular inputs such as insulin, energy stress, growth factors,
and nutrients, mTOR regulates a variety of fundamental cellular
processes such as proliferation, growth, and metabolism.2,3 mTOR
is a member of PIKK kinase family, which also includes PI3K, ATR,
ATM, DNA-PK, and SMG-1. Structurally their catalytic domains
all share high sequence identity to the PI3K kinase but differ
significantly in sequence and secondary structure from the protein
kinases.4 In cells mTOR exists in at least two functionally distinct
protein complexes, mTORC1 and mTORC2. The mTORC1
complex is composed of Raptor, LST8, PRAS40, and Deptor and
is responsible for regulated protein synthesis through phosphor-
ylation of S6K1 and 4E-BP1. The mTORC2 complex consists
of Rictor, LST8, SIN1, Deptor, and Protor and regulates cell
proliferation and survival through phosphorylation of Akt/PKB.5
Received: November 26, 2010
Published: February 15, 2011
r
2011 American Chemical Society
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Scheme 1. Discovery of 3 Starting from 1
Table 1. Pharmacological Characterization of Compounds
1 and 2a
mTORC1
IC50(nM)
mTOR
PI3K
mouse microsome
compd
EC50(nM)
EC50(nM)
stability, T1/2(min)
1
2
0.29
37.1
2
1800
4
25
>1250
17.7
a Determinations are the mean of two of independent measurements
with standard error of <20%.
Figure 1. Representative structure of the library.
reasoned that if we could reduce the molecular weight of 1,
we might remove sites of metabolism and also obtain a more
water-soluble compound. Replacement of the propionylpi-
perazine moiety of 1 with a fluorine yielded compound 2
has resulted in compounds such as 2-(4-amino-1-isopropyl-1H-
pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-5-ol(PP242),14 (5-(2-((2R,
6S)-2,6-dimethylmorpholino)-4-morpholinopyrido[2,3-d]pyri-
midin-7-yl)-2-methoxyphenyl)methanol(KU63794),15 (5-(2,4-bis-
((S)-3-methylmorpholino)pyrido[2,3-d]pyrimidin-7-yl)-2-meth-
oxyphenyl)methanol (AZD8055),16 methyl 4-(1-(1-(methoxy-
carbonyl)piperidin-4-yl)-4-morpholino-1H-pyrazolo[3,4-d]pyri-
midin-6-yl)phenylcarbamate (WYE-354),17 (1s,4s)-4-(4-amino-
5-(7-methoxy-1H-indol-2-yl)imidazo[1,5-f][1,2,4]triazin-7-yl)cyclo-
hexanecarboxylic acid (OSI-027),18 1-(4-(8-oxa-3-azabicyclo-
[3.2.1]octan-3-yl)-1-(1,4-dioxaspiro[4.5]decan-8-yl)-1H-py-
razolo[3,4-d]pyrimidin-6-yl)-3-methylurea (WYE-125132),19
(E)-4-((2-(4,6-dimorpholino-1,3,5-triazin-2-yl)hydrazono)-
methyl)-2,6-dimethoxyphenol (KU-BMCL-200908069-1),20
1-(4-(4-(8-oxa-3-aza-bicyclo[3.2.1]octan-3-yl)-1-(2,2,2-trifluoro-
ethyl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)phenyl)-3-(4-(4-methyl-
piperazin-1-yl)phenyl)urea (Wyeth-BMCL-200910096-27),21
and compound 1.22
Compound 1 is a highly potent and selective mTOR
inhibitor that exhibits the ability to inhibit mTOR in vivo.
However, the low yielding synthetic route, poor water
solubility, short half-life (T1/2), and low oral bioavailability
limits the use of 1 as a pharmacological tool in vivo. In order
to address some of these deficiencies, we initiated a medicinal
chemistry campaign that resulted in the identification of a
highly potent and selective mTOR inhibitor 3 (Torin2),
which is easier to produce on scale and exhibits improved
pharmacokinetic properties that should enable its use
in vivo.
(Scheme 1) which maintained potent cellular mTOR (EC50
25 nM) inhibitory activity and selectivity versus PI3K (EC50
=
>
1250 nM). The isolated mouse microsome stability of com-
pound was significantly improved compared to 1 (T1/2 of 17.7
min) (Table 1). This result encouraged us to prepare a
focused series of analogues with replacements to the quino-
line moiety which ultimately resulted in the identification of 3
(Figure 1).
The targeted library was generated using a four-step synthetic
sequence that commenced from 4-chloro-6-bromoquinoline
compound 4 (Scheme 2). A nucleophilic substitution with
appropriate anilines provided general intermediate 5, which was
subsequently subjected to NaBH4 reduction to afford the alcohol
6. Benzylic alcohol oxidation with MnO2 followed by Horner-
Wadsworth-Emmons olefination furnished the tricyclic core
scaffold 7. Finally variation of the Ar2 position was achieved using
palladium mediated coupling reactions to afford the target
compounds 8.
By variation the substitutions of the Ar1 and Ar2 groups, a
focused library was generated (Figure 1) and the biological
characterizations are summarized in Table 2.
Substitution of the acylpiperazine ring with fluoro at the R1
position (compound 2) resulted in a 10-fold loss of potency in
cellular mTOR assays. Replacement of the quinoline side chain
at the Ar2 position with pyrazole (9) and methylpyrazole (10)
resulted in 5- to 25-fold reduction in activity. Replacement of
the quinoline of 2 with aminopyridine (11), aminopyrimidine
(12), and 7-azaindole (14) groups resulted in compounds with
potent mTOR inhibitory activity. However, compounds 11 and
related analogues demonstrated higher PI3K inhibitory activity.
Indazole replacement (13) resulted in 10-fold loss of potency
relative to 14, indicating that a β nitrogen was required for the
activity, potentially suggesting a hydrogen-bond accepting
moiety is required at this position. A similar activity trend
was observed when CF3 was substituted with CH3 (15-21) at
the R2 position. 7-azaindole analogue (21) possessed the most
’ RESULTS AND DISCUSSION
Compound 1 exhibits very rapid metabolism upon incuba-
tion with mouse and human hepatocytes (T1/2 of 4 min),
which suggests that the compound may be subject to rapid
first-pass metabolism in the liver. Consistent with this finding,
the observed half-life following intravenous delivery of 1 to
mice was 30 min. Oral administration of 1 revealed that the
compound possessed very limited oral bioavailability. We
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Scheme 2. Chemical Synthesis of a Focused Librarya
a Reagents and conditions: (1) appropriate aniline, 1,4-dioxane, 90 ꢀC, 4-12 h; (2) NaBH4, EtOH, room temp, 2-4 h; (3) (a) MnO2, CH2Cl2, room
temp, 2-6 h; (b) triethyl phosphonoacetate, K2CO3, EtOH, 100 ꢀC, 12 h; (4) Ar2B(OH)2, PdCl2(Ph3P)2, Na2CO3, 1,4-dioxane, 80 ꢀC, 4-12 h.
potent mTOR inhibitory activity but also exhibited significant
activity against PI3K. Replacement of CF3 with smaller groups
such as Cl (22-26) and F (27, 28) resulted in compounds with
similar potency against mTOR and PI3K. 7-Azaindole analogue
(24) and aminopyrimidine analogue (25) exhibited the best
potency as inhibitors of mTOR while still possessing more than
500-fold selectivity relative to PI3K. Introduction of the CF3
group at R3 position (29-31) resulted in a 20- to 100-fold loss
of activity against mTOR, suggesting either steric impediments
to substitution at this position or unfavorable effects on the
conformation of the compound. Removal of F from the R1
position while maintaining the aminopyridine side chain (3)
resulted in the best balance of highly potent inhibition of
mTOR (EC50 = 0.25 nM) while maintaining 800-fold selec-
tivity relative to PI3K (EC50 = 200 nM).
We attempted to rationalize the observed structure-activity
relationship (SAR) by docking compound 3 into a homology
model of mTOR built using a PI3Kγ crystal structure (PDB code
3DBS) (Figure 2A). Similar to 1,22 compound 3 is predicted to
engage in a “hinge” hydrogen bond with V2240 using the
quinoline nitrogen as well as engage a second hydrogen bond
between Y2225 and pyridine nitrogen inside inner hydrophobic
pocket. In addition, two hydrogen bonds are predicted to exist
between the aniline amino group of 3 with D2195 and D2357 in
the inner hydrophobic pocket, and this may provide an explana-
tion for the approximate 10-fold potency increase in mTOR
inhibition compared to 1. The same binding mode was observed
in PI3Kγ modeling study with compound 3. V882 served as a
hinge binding point, and in the inner hydrophobic pocket Y867,
D841 and D964 provided three more hydrogen bonds with
aminopyridine side chain analogous to Y2225, D2195, and
D2357 of mTOR. This may provide a rationale for why
compound 3 begins to inhibit PI3K (Figure 2B)
80-95%, “þþ” indicates 60-80%, and “þ” indicates 40-60%
inhibition of pS6K and pAkt compared to vehicle). The results
are summarized in Table 3. Compounds 3, 9, 14, 16, and 17
exhibited similar or better pharmacodynamic effects relative to 1,
while compounds 2, 15, 23, and 24 were less effective than 1.
Compounds 19, 21, 25, 26, 27, and 28 did not exhibit inhibition
of the S6K or Akt phosphorylation after 6 h. The liver microsome
stability results varied significantly among the compounds.
Compounds 2, 3, 19, 25, 26, and 27 all exhibited more than a
10 min half-life, but this did not predict which compounds would
exhibit a robust pharmacodynamic effect. Several compounds
such as 19, 25, 26, and 27 exhibited poor pharmacodynamics
despite having superior microsome stability relative to 1, while
other very short-lived compounds such as 9, 14, 16, and 17
exhibited more sustained pharmacodynamics. The basis for this
discrepancy is currently unknown but may result from a sus-
tained mTOR inhibition due to a slow off-rate from the kinase for
some compounds. The oral PD studies employed a different
formulation (10% NMP, 40% PEG in water) relative to the oral
PK studies (0.5% w/v Na cmc with 0.1% w/v Tween-80), so the
degree of drug exposure in the PD studies cannot be directly
extrapolated from the PK studies.
Compounds that exhibited the best pharmacodynamic prop-
erties were subject to pharmacokinetic evaluation in mice
following intravenous and oral administration (Table 4). As
has been predicted by the mouse liver microsome stability study,
all compounds exhibited a half-life of less than 2 h when dosed to
male Swiss albino mice. Compound 3 exhibited the best bioavail-
ability (51%) and overall exposure despite possessing a short
half-life and was selected for further evaluation. Compared to
compound 1, the bioavailability of 3 was improved by approxi-
mately 10-fold.22 Most of the compounds exhibited rapid
absorption following oral administration to mice. All the com-
pounds studied had low to moderate clearance and exhibited
volumes of distribution close to the total body water. The
possible reasons for the low bioavailability for the majority of
the compounds could be poor absorption that may be due to the
limited water solubility of the compounds.
Pharmacodynamics and Pharmacokinetics. In order to
determine which compounds might be optimal for use in murine
tumor studies, we selected a set of compounds based on the
mTOR activity, selectivity over PI3K, synthetic feasibility, and
structural diversity. First the compounds were evaluated for their
stability when incubated in the presence of isolated mouse liver
microsomes. A subset of compounds were then advanced into
pharmacodynamic (PD) studies monitoring the phosphoryla-
tion status of S6K T389 and Akt T308 in the lung and liver by
Western blot 6 h after oral administration. The pharmacody-
namic response was approximately scored with 1 being assigned a
“þþþ” score (“þþþþ” indicates over 95%, “þþþ” indicates
Kinase Selectivity Profiling. To characterize the kinase
selectivity of 3, we profiled its ability to bind to a panel of 440
kinases using the Ambit KinomeScan technology23 (Table 5).
Compound 3 exhibited excellent overall selectivity (S(5) score
(1 μM) of 0.02)24 and has strong binding to mTOR (4.6),
CSNK1E (2.7), several PI3Ks (0-3.5), CSF1R (2.4), and MKNK2
(0.9). Compound 3 was subject to further profiling against a
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Table 2. Enzymatic and Cellular Activities of Compounds 3 and 9-31a
a IC50 determinations are the mean of two of independent measurement with standard error of <20%.
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Figure 2. Model of 3 (green sticks) docked into a homology model of mTOR (gray ribbons) (A) and PI3Kγ (PDB code 3DBS) (gray ribbons) (B).
Potential hydrogen bonds are shown with hatched red lines.
panel of lipid kinases using the Invitrogen SelectScreen technol-
ogy which exhibited potent biochemical inhibition of PI3Ks and
DNA-PK in addition to mTOR (Table 6). From our cellular
PI3K assays we know there is approximately a 40-fold dis-
crepancy between the biochemical PI3K IC50 and the cellular
EC50. It remains to be determined whether DNA-PK is
inhibited by 3 in cellular assays. It is interesting to note that
the enzymatic selectivity ratio for inhibition of mTor versus
PI3K for both 3 (enzyme ratio of 2) and Torin1 (enzyme ratio
of 60)22 substantially underestimates the observed ratio for
cellular inhibition of these targets: 3 (cell ratio of 800) and
Torin1 (cell ratio of 900).
to 90 ꢀC for 4-12 h. After the mixture was cooled to room tem-
perature, a solution of NaOH (1 N) was added to neutralize the
solution followed by dilution with water and extraction with ethyl
acetate. After the mixture was dried with Na2SO4, the solvents were
removed and the residue was purified by ISCO (hexanes/EtOAc) to
furnish compound 5.
To a solution of compound 5 (1 equiv) in EtOH at room tempera-
ture was added NaBH4 (10 equiv). The resultant solution was stirred at
room temperature for 2-4 h before being diluted with EtOAc and
filtered through Celite. The filtrate was concentrated under vacuum,
and the residue was dissolved in EtOAc and brine. After separation, the
organic layer was dried over MgSO4 and concentrated under vacuum.
The residue was purified by ISCO (hexanes/EtOAc) to afford com-
pound 6.
To a solution of compound 6 in CH2Cl2 (1 equiv) at room tempera-
ture was added MnO2 (5 equiv mass). After 2-6 h, the reaction mixture
was filtered through Celite. The filtrate was concentrated in a sealed tube
and dissolved in dry EtOH, after which K2CO3 (3 equiv) and triethyl
phosphonoacetate were added. The resulting mixture was heated to
100 ꢀC for 12 h before cooling to room temperature. Upon removal of
the solvents under vacuum, the residue was diluted with water followed
by extraction with EtOAc. Purification of the residue by ISCO (hexanes/
EtOAc) provided compound 7.
To a solution of compound 7 in 1,4-dioxane at room temperature was
subsequently added PdCl2(Ph3P)2 (0.1 equiv), Na2CO3(3 equiv, 1 N),
and boronic acids or pinacol boronate esters. After degassing, the
resulting mixture was heated to 80 ꢀC for 4-12 h before cooling to
room temperature and filtering through Celite. Upon removal of the
solvents, the residue was subjected to column purification (CH2Cl2/
MeOH) to furnish the desired compounds 8.
’ CONCLUSION
Starting from the selective mTOR inhibitor 1, we used a
focused medicinal chemistry approach guided by cellular assays
and pharmacokinetic and pharmacodynamic assays to develop
3 which possesses properties that make it more suitable for use
in murine tumor studies. Compound 3 possesses a 250 pM
EC50 for inhibiting mTOR in cells while maintaining 800-fold
cellular selectivity relative to inhibition of PI3K and most other
protein kinases. Compound 3 exhibits good bioavailability and
exposure and can maintain strong inhibition of mTOR activity
in lung and liver to at least 6 h after a single dose of 20 mg/kg.
Compound 3 is a useful pharmacological tool for further explor-
ing the therapeutic potential of mTOR inhibitors for the
treatment of cancer.
Compound 3 was prepared following the above procedures (15%
overall yield). LC-MS: (M þ H) 433.13. 1H NMR (600 MHz, DMSO-
d6) δ 9.09 (s, 1H), 8.31 (d, J = 9.6 Hz, 1H), 8.11 (s, 1H), 8.03 (d, J = 9.0
Hz, 1H), 8.00 (d, J = 7.8 Hz, 1H), 7.92 (dd, J = 8.4, 1.8 Hz, 1H), 7.86 (dd,
J = 8.4, 7.8 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.72 (d, J = 2.4 Hz, 1H),
7.04 (dd, J = 8.4, 2.4 Hz, 1H), 6.91-6.93 (m, 2H), 6.38 (d, J = 8.4 Hz,
1H), 6.18 (s, 2H).
For characterization of in vitro biochemical activity with the
mTORC1 complex, mTOR and PI3K cellular assays, Ambit in vitro
KinomeScan kinase selectivity profile, and in vivo pharmacokinetic
studies, see ref 22.
In Vivo Pharmacodynamic Studies. Compound 3 powder was
first dissolved at 25 mg/mL in 100% N-methyl-2-pyrrolidone and then
diluted 1:4 with sterile 50% PEG400 prior to injection. Six-week-old
male C57BL/6 mice were fasted overnight prior to drug treatment. The
mice were treated with vehicle (for 10 h) or 3 (20 mg/kg for 6 h) via oral
gavage and then refed 1 h prior to sacrifice (CO2 asphyxiation). Liver
and lung were collected and frozen on dry ice. The frozen tissue was
thawed on ice and lysed by sonication in tissue lysis buffer (50 mM
HEPES, pH 7.4, 40 mM NaCl, 2 mM EDTA, 1.5 mM sodium
orthovanadate, 50 mM sodium fluoride, 10 mM sodium pyrophosphate,
’ EXPERIMENTAL PROCEDURES
1
Chemistry. All solvents and reagents were used as obtained. H
NMR spectra were recorded with a Varian Inova 600 NMR spectro-
meter and referenced to dimethylsulfoxide. Chemical shifts are ex-
pressed in ppm. In the NMR tabulation, s indicates singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; and br, broad peak. Mass
spectra were measured with Waters Micromass ZQ using an ESI source
coupled to a Waters 2525 HPLC system operating in reverse mode with
a Waters Sunfire C18 5 μm, 4.6 mm ꢀ 50 mm column. Purification of
compounds was performed with either a Teledyne ISCO CombiFlash Rf
system or a Waters Micromass ZQ preparative system. The purity of all
compounds was g95%. The purity was analyzed on an above-men-
tioned Waters LC-MS Symmetry (C18 column,4.6 mm ꢀ 50 mm,
5 μM) using a gradient of 5-95% acetonitrile in water containing
0.05% trifluoacetic acid (TFA) over 8 min (10 min run time) at a flow
rate of 2 mL/min.
General Procedure for the Preparation of Compounds.
To a solution of compound 4 (ethyl 6-bromo-4-chloroquinoline-3-
carboxylate, 1 equiv) in 1,4-dioxane at room temperature in a sealed
tube was added aniline (1 equiv). The resultant solution was heated
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Table 3. Pharmacodynamics and Liver Microsome Stability of Selected Compounds
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Table 4. PK Properties of Selected Compoundsa
compd
Cmax (ng/mL) iv/po
Tmax (h) po
AUC (h ng/mL) iv/po
T1/2 (h) iv
CL ((mL/min)/kg) iv
Vss (L/kg) iv
F (%) po
3
3
1540/3968
426/430
1639/98
1035/853
-/1039
0.25
0.5
1.0
0.5
0.5
850/4311
391/755
0.72
19.6
42.6
20.8
15.0
22.9
1.0
51
9
0.63
2.42
1.30
1.16
1.08
19.2
4.2
21.6
28
14
16
17
803/352
1.09
1110/2410
1.46
728/2068
0.99/1.31
a C57BL/6 male mouse was dosed at 1 mg/kg for the intravenous (iv) and at 10 mg/kg for the oral (po).
Table 5. Kinome Scan Profile of 3a
’ ACKNOWLEDGMENT
We thank Life Technologies Corporation, SelectScreen
Kinase Profiling Service for performing enzymatic biochemical
kinase profiling, and Ambit Bioscience for performing KinomeS-
can profiling. We also want to thank Sai Advantium Pharma Ltd.
(India) for the pharmacokinetic studies and Michael Cameron
for performing in vitro microsome stability studies. This work
was partially supported by NIH Grant GM079575-03.
kinase
% control
kinase
% control
mTOR
4.6
2.7
3.5
0.1
0.25
1.4
1
CSF1R
2.4
0.9
0.55
0.25
0
CSNK1E
MKNK2
PIK3C2G
PIK3CA
PIK3CA(C420R)
PIK3CA(E545K)
PIK3CA(H1047Y)
PIK3CA(M1043I)
PIK3CG
PIK3CA(E542K)
PIK3CA(H1047L)
PIK3CA(I800L)
PIK3CA(Q546K)
PIK4CB
0
0.35
1.7
’ ABBREVIATIONS USED
0.2
mTOR, mammalian target of rapamycin; Akt, v-akt murine
thymoma viral oncogene homologue 1; ATP, adenosine tripho-
sphate; PIKK, PI3K related kinases; DNA-PK, DNA activated
protein kinase; ATM, ataxia telangiectasia mutated kinase; ATR,
ataxia telangiectasia and Rad-3-related kinase; SMG-1, serine/
threonine protine kinase -1; 4E-BP1, eukaryotic translation in-
itiation factor 4E-binding protein 1; PK, pharmacokinetics
a Compound 3 was profiled at 1 μM against a diverse panel of 442
kinases by Ambit Biosciences.
Table 6. LanthaScreen Profile of 3 Using the Invitrogen Lipid
Kinase Panela
kinase
IC50 (nM)
kinase
IC50 (nM)
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PI4K R
>10000
18.3
P110 R/p85R
P110δ/P85R
P110γ
4.68
17.5
5.67
0.5
PI4K β
PI3K-C2R
PI3K-C2β
hVPS34
28.1
24.5
DNA-PK
8.58
mTOR
2.81
a Kinase targets were tested with biochemical enzymatic kinase assays
using the SelectScreen Kinase Profiling Service (Life Technologies
Corporation, Madison, WI).
10 mM sodium β-glycerophosphate, 0.1% SDS, 1.0% sodium deox-
ycholate, and 1.0% Triton, supplemented with protease inhibitor cock-
tail tablets (Roche)). The concentration of clear lysate was measured
using the Bradford assay, and samples were subsequently normalized by
protein content and analyzed by SDS-PAGE and immunoblotting.
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’ ASSOCIATED CONTENT
S
Supporting Information. Spectral data of 2 and 9-31.
b
This material is available free of charge via the Internet at http://
pubs.acs.org.
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’ AUTHOR INFORMATION
Corresponding Author
*Phone: 1-617-582-8590. Fax: 1-617-582-8615. E-mail: Nathanael_
gray@dfci.harvard.edu.
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