Discovery of 1-(4-(4-Propionylpiperazin-1-yl)-3-(trifluoromethyl)phenyl)-9- (quinolin-3-yl)benzo[h][1,6]naphthyridin-2(1 H)-one as a highly potent, selective mammalian target of rapamycin (mTOR) inhibitor for the treatment of cancer

Article abstract of DOI:10.1021/jm101144f

The mTOR protein is a master regulator of cell growth and proliferation, and inhibitors of its kinase activity have the potential to become new class of anticancer drugs. Starting from quinoline 1, which was identified in a biochemical mTOR assay, we developed a tricyclic benzonaphthyridinone inhibitor 37 (Torin1), which inhibited phosphorylation of mTORC1 and mTORC2 substrates in cells at concentrations of 2 and 10 nM, respectively. Moreover, Torin1 exhibits 1000-fold selectivity for mTOR over PI3K (EC₅₀ = 1800 nM) and exhibits 100-fold binding selectivity relative to 450 other protein kinases. Torin1 was efficacious at a dose of 20 mg/kg in a U87MG xenograft model and demonstrated good pharmacodynamic inhibition of downstream effectors of mTOR in tumor and peripheral tissues. These results demonstrate that Torin1 is a useful probe of mTOR-dependent phenomena and that benzonaphthridinones represent a promising scaffold for the further development of mTOR-specific inhibitors with the potential for clinical utility.

Full text of DOI:10.1021/jm101144f

7146 J. Med. Chem. 2010, 53, 7146–7155
DOI: 10.1021/jm101144f
Discovery of 1-(4-(4-Propionylpiperazin-1-yl)-3-(trifluoromethyl)phenyl)-9-
(quinolin-3-yl)benzo[h][1,6]naphthyridin-2(1H)-one as a Highly Potent,
Selective Mammalian Target of Rapamycin (mTOR) Inhibitor for the Treatment of Cancer
Qingsong Liu,†,‡ Jae Won Chang,†,‡ Jinhua Wang,†,‡ Seong A. Kang,§ Carson C. Thoreen,†,‡
Andrew Markhard,§ Wooyoung Hur,†,‡ Jianming Zhang,†,‡ Taebo Sim,†,‡ David M. Sabatini,§, ,^ and
Nathanael S. Gray*,†,‡
†Department of Cancer Biology, Dana Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115, ‡Department of Biological
Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, Massachusetts 02115,
§Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, Howard Hughes Medical Institute,
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and
^Koch Center for Integrative Cancer Research at MIT, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139
Received June 27, 2010
The mTOR protein is a master regulator of cell growth and proliferation, and inhibitors of its kinase
activity have the potential to become new class of anticancer drugs. Starting from quinoline 1, which was
identified in a biochemical mTOR assay, we developed a tricyclic benzonaphthyridinone inhibitor 37
(Torin1), which inhibited phosphorylation of mTORC1 and mTORC2 substrates in cells at concentra-
tions of 2 and 10 nM, respectively. Moreover, Torin1 exhibits 1000-fold selectivity for mTOR over PI3K
(EC50 = 1800 nM) and exhibits 100-fold binding selectivity relative to 450 other protein kinases. Torin1
was efficacious at a dose of 20 mg/kg in a U87MG xenograft model and demonstrated good
pharmacodynamic inhibition of downstream effectors of mTOR in tumor and peripheral tissues. These
results demonstrate that Torin1 is a useful probe of mTOR-dependent phenomena and that benzo-
naphthridinones represent a promising scaffold for the further development of mTOR-specific
inhibitors with the potential for clinical utility.
Introduction
mTOR signaling pathway is often implicated in cellular growth
deregulation in cancer, and therefore there has been a substan-
tial effort to develop mTOR inhibitors for potential clinical
application.3,4
The mammalian target of rapamycin (mTORa) is a key node
in the PI3K/Akt/mTOR signaling pathway that is often deregu-
lated in human cancer.1 In conjunction with PI3K and Akt/
PKB, mTOR integrates signals derived from extracellular cues,
such as growth factors, energy, stress, and nutrients, and
regulates growth-related cellular processes, including mRNA
translation, ribosome biogenesis, autophagy, and metabolism.
The mTOR serine/threonine protein kinase shares a structurally
similar catalytic domain with other PIKK family members,
including PI3Ks, DNA-PK, ATM, ATR, and SMG-1.2 mTOR
exists in at least two distinct protein complexes in cells: mTOR
complex 1 (mTORC1) and mTOR complex 2 (mTORC2).
mTORC1 regulates mRNA translation through the phosphor-
ylation of S6K1 and 4EBP1, while mTORC2 regulates cell
survival through the phosphorylation of Akt/PKB and other
AGC-family kinases, such as SGK. Hyperactivation of the
The development of ATP-competitive mTOR inhibitors was
not viewed as a priority until recently because of rapamycin
and its analogues (known as rapalogues), which were widely
considered highly potent and selective allosteric inhibitors of
mTORC1. In some cell types, rapamycin can also inhibit
mTORC2.5 Unfortunately, clinical success of these compounds
has been limited to a small number of relatively rare cancers,
including mantle cell lymphoma, renal cell carcinoma, and
endometrial cancer.6 Several explanations for this limited effi-
cacy have been proposed: (1) rapamycin may only be effective in
tumors where it is also capable of inhibiting mTORC2, (2) the
induction by rapamycin of a feedback signal that leads to the
hyperactivation of PI3K signaling might undermine the anti-
proliferative effect of mTORC1 inhibition, or (3) in many cell
types, rapamycin fails to significantly inhibit mTORC1 kinase
activity toward substrates, such as 4E-BP1, that are major
regulators of proliferation.7,8 It is currently unknown whether
any or all of these explanations account for the apparently
limited clinical efficacy of rapamycin and its derivatives. None-
theless, these potential deficiencies in rapamycin-based thera-
pies have spurred the rapid development of ATP-competitive
mTOR inhibitors that target both mTOR complexes. Recently,
a number of selective ATP-competitive mTOR inhibitors
*To whom correspondence should be addressed. Phone: 1-617-582-
8590. Fax: 1-617-582-8615. E-mail: Nathanael_gray@dfci.harvard.edu
a Abbreviations: mTOR, mammalian target of rapamycin; Akt, v-akt
murine thymoma viral oncogene homologue 1; ATP, adenosine tripho-
sphate; PIKKs, PI3K related kinases; DNA-PK, DNA activated protein
kinase; ATM, ataxia telangiectasia mutated kinase; ATR, ataxia telan-
giectasia and Rad-3-related kinase; SMG-1, serine/threonine protine
kinase-1; 4EBP1, eukaryotic translation initiation factor 4E-binding
protein 1; PK, pharmacokinetics; PD, pharmacodynamics; ADME,
absorption, distribution, metabolism, excretion; MEFs, mouse embryo
fibroblasts.
r
pubs.acs.org/jmc
Published on Web 09/22/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7147
Figure 1. Chemical structures of reported ATP-competitive mTOR inhibitors.
have been reported, including 37 (Torin1),7 1 (PP242),8 2
(KU-0063794),9 3 (WYE-354),10 4 (OSI-027),11 5 (AZD-8055),12
6(KU-BMCL-200908069-1),13 and 7(Wyeth-BMCL-200910096-
27),14 as well as a number of compounds that are capable of
inhibiting both PI3K and mTOR and other PIKK-family kinases
such as 8 (PI-103),15 9 (NVP-BEZ235),16 10 (GNE-493),17 and 11
(GSK2126458)18 (Figure 1). Here we describe our screening and
medicinal chemistry efforts that resulted in the identification of
benzonapthridinones exemplified by 37 as highly potent and
selective mTOR inhibitors.
Results and Discussion
To identify compounds capable of inhibiting mTOR kinase
activity, we established a medium-throughput biochemical
assay utilizing purified mTORC1 from mammalian cells.19
We screened a compound library consisting of both known
kinase inhibitors as well as heterocycles that would be antici-
pated to have the propensity to bind to the ATP binding pocket
of kinases.20 This screening effort resulted in the identification
of several classes of compounds; however, based on the criteria
of the scaffold selectivity, we focused our attention on quino-
Figure 2. Screening flowchart for development of mTOR inhibitors.
line hit compound 12. Although compound 12 exhibited only
moderate biochemical inhibitory activity (mTORC1 IC50 = 5
μM) and was unable to inhibit phosphorylation of the cellular
mTORC1 substrate S6K at a concentration of 10 μM, it did
exhibit selectivity for PIKK-family kinases when screened in
binding assays against a panel of approximately 400 kinases at
a concentration of 10 μM (KinomeScan). In addition, the
quinoline and isosteric quinazoline scaffolds are “privileged”
ATP-site binders and several highly selective kinase inhibitors
such as HKI-27221 and lapatinib22 are built from these hetero-
cyclic cores.
To optimize the cellular potency and selectivity of “hit”
compound 12, we established a streamlined flowchart of
biochemical and cellular assays to explore the structure-
activity relationship (Figure 2). Newly synthesized compounds
were initially screened for their ability to inhibit mTORC1
activity by monitoring the inhibition of phosphorylation of

7148 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Liu et al.
Scheme 1a
a Reagents and conditions: (1) appropriate aniline, 1,4-dioxane, 100 ꢀC, 4-12 h; (2) appropriate boronic acid, PdCl2(Ph3P)2, t-Bu-Xphos, Na2CO3,
dioxane, 100 ꢀC, 6 h.
Table 1. Enzymatic and Cellular Activities Profile of Acyclic Quinoline
A substructure search of compound 12 identified a broad
Seriesa
class of tricyclic quinoline-derived dual PI3K/mTOR inhibi-
tors exemplified by 9 as containing a similar substructure24,25
(Figure 1). Tricylic core structure have been exploited in the
development of numerous other kinase inhibitors targeting
EGFR,26a PDGFR,26b Aurora,26c CK2,26d and IKK.26e 9 was
predicted tobind toPI3Kusing the quinoline nitrogen toform
a key hydrogen bond to the kinase “hinge region”.25 The cyclic
urea moiety was predicted to be important for constraining
the pendant phenyl ring to the presumed bioactive conforma-
tion. We decided to introduce a similar constraint by introdu-
cing a six-membered lactam that would result in a tricyclic
scaffold which would facilitate additional diversification
points as well as introduce the potentially important carbonyl
oxygen for the potency.
The introduction of this additional ring constraint resulted
in benzonaphthridinone compound 18, which exhibited a
dramatic 1000-fold improved mTOR cellular potency com-
pared tothatof the screening hit compound 12. Compound 18
possessed a biochemical IC50 of 5.4 nM against mTORC1
enzyme and an IC50 of 3 nM for inhibition of S6K phosphor-
ylation in cells without any detectable inhibitory activity on
PI3K up to a concentration of 300 nM (Table 2).
a The IC50 determinations are the mean of two of independent
measurement with standard error of <20%. For cellular assay, 300 nM
was the highest concentration tested
T389 of S6K1 by immunoblot analyses in mouse embryonic
fibroblasts (MEFs). The cellular assay was primarily used to
prioritize compounds and, in parallel, compounds were also
tested using an in vitro mTORC1 kinase assay. Compounds
that exhibited promising cellular mTORC1 activity were as-
sessed for selectivity versus inhibition of cellular PI3K activity
by monitoring inhibition of Akt T308 in PC-3 cells that stably
express the phosphomimetic S473D mutant of Akt.7 The
S473D mutant of Akt was used to achieve a more pure
PI3K-dependent phosphorylation read-out by eliminating the
effect of mTORC2 phosphorylation at that site, which nor-
mally facilitates phosphorylation at T308 by PDK1.23 The
most potent and selective candidates to emerge from this
reiterative process between the chemical modification and
biological evaluation were subject to broad kinase selectivity
profiling using the Ambit KinomeScan and LifeTechnologies
LanthaScreen platforms. Compounds that passed our criteria
for triage were further profiled for their in vitro and in vivo drug
metabolism and pharmacokinetic properties (DMPK) and
finally tested in a U87MG tumor xenograft model.
To begin the SAR exploration of compound 12, we gener-
ated a number of analogues by varying the side chains at
4- and 6-positions of the quinoline (Scheme 1). A few represen-
tative compounds that emerged from this effort are illustrated
by compounds 15, 16, and 17 (Table 1). Unfortunately,
these substitutions generally resulted in very minor changes in
biochemical IC50 values and none of the compounds inhibited
mTOR or PI3K activity in cellular assays at submicromolar
concentrations (Table 1).
To rationalize the potency of 18 in a structural context, we
docked compound 18 into the ATP-binding site of an mTOR
homology model built using the related Pi3Kγ crystal struc-
ture (PDB code: 3DBS) (Figure 3). Similar to the published
homology model of 9 with Pi3Kγ,27 a hydrogen bond was pre-
dicted to form between the quinolione nitrogen of compound 18
and Val2240 within the kinase hinge region.16 A comparison to
the proposed binding mode of morpholine-derived mTOR
inhibitors such as 8 suggests that the morpholine oxygen makes
a similar hydrogen bond. The quinoline moiety of compound 18
was predicted to reside within the inner hydrophobic pocket
formed by Glu2190, Leu2192, Asp2195, Tyr2225, Asp2357,
Phe2358, Gly2359, and Asp2360. The phenyl piperazine side
chain was oriented below a loop defined by residues from
Ile2183 to Gln2187, which occupy a similar region of the
ATP-binding site as the “P-loop” of protein kinases. Using this
homology model as a guide, we embarked on a further elabora-
tion of the benzonaphthridione scaffold at the quinoline (R1)
and lactam phenyl (R2) positions. Previous efforts to optimize
PI3K inhibitors have suggested that modification at either of
these positions could modulate potency, while the R1 position
may provide a stronger contribution to specificity versus other
PIKK-family kinases.27 In addition, to improve the extremely
poor water-solubility of this compound class, we continued our
SAR exploration with R2 fixed as a phenyl piperazine, which
introduces a water solubility enhancing basic secondary nitro-
gen versus the acyl piperazine present in our library compounds
(Figure 4). The target compounds were synthesized using a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7149
Figure 3. Model demonstration of compound 18 binding with mTOR.
Scheme 2a
a Reagents and conditions: (1) aniline (R2NH2), 1,4-dioxane, 100 ꢀC, 4 h; (2) LAH, THF, 0 ꢀC-rt, 4 h; (3) (a) MnO2, CH2Cl2, rt, 6 h, (b) triethyl
phosphonoacetate, K2CO3, EtOH, 100 ꢀC, 12 h; (4) R1B(OH)2, PdCl2(Ph3P)2, t-Bu-Xphos, Na2CO3, 1,4-dioxane, 100 ꢀC, 12 h.
Table 2. Enzymatic and Cellular Activities Profile of Compound 18
four-step sequence starting from dichloroquinoline scaffold S1
(Scheme 2). Introduction of substituted anilines in the first step
was followed by construction of the six-membered lactam using
a Horner-Wadsworth-Emmons reaction. The R1 side chain
was then introduced via palladium-mediated coupling with the
desired boronic acid. The biological activities of the compounds
that resulted from this effort are illustrated in Table 3 and 4.
Removal of the acyl group of compound 18 to yield
compound 19 resulted in a 2-10-fold reduction in potency
Figure 4. Representative structures of variations of R1.
in both biochemical and cellular assays. On the basis of the
homology model, this could be rationalized by the potential
loss of a hydrogen bond between the amide carbonyl oxygen
and epsilon amine of Lys2186. Removal of the quinoline
nitrogen of compound 19 to yield compound 25 resulted in
more than a 10-fold loss in cellular mTOR potency, which
may be attributable to the loss of a hydrogen bond between
the quinoline N and the phenol of Tyr2225. Introduction of
the tricyclic thianthrene side chain (22) significantly reduced
the cellular mTOR potency, providing a potential upper limit
on the size of the functionality that can be accommodated
within the inner hydrophobic pocket. Most of the bicyclic side
chains such as benzothiophene (21), isoquinoline (23),
naphthalene (25), benzodioxane (26), and benzodioxole (27)
maintained reasonable enzymatic activities, but only those
with appropriately placed hydrogen-bond accepting potential
(26, 27) displayed good cellular activity. Monocyclic R1
groups with substitution at the para-position (20) possessed
activity in the same range as the bicyclic ring systems. The

7150 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Liu et al.
Table 3. Enzymatic and Cellular Activities Profile of Compounds
Varying R1a
Table 4. Enzymatic and Cellular Activities Profile of Compounds
Varying R2a
a IC50 determinations are the mean of two of independent measurement
with standard error of <20%. None of the tested compounds exhibited
IC50 for inhibition of cellular PI3K activity of less than 300 nM.
dramatic loss of activity observed between compound 24 and
20 may be attributed to the lack of tolerance for R-substitu-
tion, which is consistent with the observations made for the
compound 10 series.17 Cumulatively, these data suggest that a
combination of hydrophobic and hydrogen bonding interac-
tions are critical for achieving potent cellular inhibition of
mTOR and that the quinoline moiety was the preferred core
for this pharmacophore. None of the compounds in this series
displayed cellular activity against PI3K up to a concentration
of 300 nM. A number of discordances were observed between
the biochemical and cellular IC50 measurements that may be
attributed to differential cellular permeability and/or the
inability of the biochemical assay to accurately reflect the
functionally relevant intracellular mTOR. On the basis of this
observation, the cell based IC50 was used as a guide for the
chemical optimization process.
Following the initial optimization steps, we introduced a
variety of side chains to the R2 position while fixing the R1
side chain as a quinoline (Figure 5 and Table 4). Introducing
sulfonyl (31) or methylene spacer (33) before the piperazine
functionality resulted in a loss in mTOR potency. Introducing
an ortho-methoxy substitution (32 versus 19) resulted in a
5-foldloss in cellular mTORinhibition. Replacement of anR2
phenyl moiety with small aliphatic side chains (represented by
38) resulted in inactive compounds. Most compounds that
a IC50 determinations are the mean of two of independent measurement
with standard error of <20%. None of the tested compounds exhibited an
IC50 for inhibition of cellular PI3K activity of less than 300 nM.
retained the meta-CF3 substituted aniline and also those
derivatized at the para-position with piperazinyl, phenyl,
and piperidinyl groups retained similar or superior potency
relative to compound 18. Introducing a carbonyl moietyin the
form of an acetyl group to the piperazine nitrogen (18 versus
19) resulted in a 13-fold improvement in cellular potency,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7151
Table 5. Enzymatic and Cellular Activities Profile of Fixed Conforma-
tion Compoundsa
Figure 5. Representative structures of variations of R2.
potentially due to the introduction of a hydrogen bond with
Lys2186. (Figure 3) To avoid an 1,4-dianiline moiety that
could potentially represent a metabolic liability in compound
18, several piperazine replacements were explored such as
reversed piperidine (41), unsaturated piperidine (40), or phenyl
(39) linkage; however, they all resulted in weaker biochemical
and cellular mTOR inhibition. Further exploration of the
piperazine acyl moiety, which the modeling predicted to be
directed toward the solvent area, resulted in the identification
of the propyl amide (37, named Torin1) as conferring the
greatest potency and selectivity. Compound 37 is a picomolar
inhibitor of mTORC1 enzymatic activity and single digit
nanomolar inhibitor of cellular mTOR activity. Although
most compounds in this series have over 100-fold selectivity
relative to inhibition of cellular PI3K activity, a more detailed
study of 37 revealed that it has more than 800-fold selectivity
between mTOR and PI3K.7
We next investigated the SAR of the lactam ring using
Torin1 as a staring point. The resultant SAR indicated that
the rigid six-membered lactam was preferred, as urea, carba-
mate, and pyrimidinedione analogues lost more than 500-fold
activity against the mTORC1 complex (Table 5). Introduc-
tion of a methyl group to either the R (42) or β position (43) of
the lactam ring significantly diminished potency in both the
biochemical and cellular assays, suggesting these positions
make a close contact to the ATP-binding site.
Kinase Selectivity
Biochemical characterization of 37 at varying ATP con-
centrations demonstrated that it is an ATP competitive
inhibitor of mTOR. The selectivity of 37 was assessed using
the KinomeScan methodology across a panel of 442 human
kinases at a concentration of 10 μM. Only three kinases
displayed tight binding to 37 (Ambit scores of less than 2),
and dissociation constants were determined for mTOR,
PI3KR, and PI3KR (I800L) (Table 6). This analysis revealed
that 37 is extremely selective for PIKK family kinases relative
to all other serine/threonine and tyrosine kinases. To further
investigate these observed binding events, we performed en-
zymatic assays across most of the knownPIKK family kinases
using the LanthaScreen technology. This analysis revealed
that 37 is very selective relative to other PIKK family kinases
with the exception of DNA-PK (Table 7). The activity against
DNA-PK was further characterized using a radiometric
kinase assay where the compound exhibited an IC50 of
approximately 1 μM.7 The origin of the 150-fold discrepancy
between the LanthaScreen IC50 for DNA-PK versus the
radiometric assay is currently unknown.
a IC50 determinations are the mean of two of independent measure-
ments with standard error of <20%. 37 has a cellular IC50 of 1800 nM
for PI3K. None of other compounds exhibited a cellular IC50 for PI3K
of less than 300 nM.
human liver microsome metabolism observed in the presence
and absence of NADPH indicates a NADPH dependent
metabolism mechanism. The in vivo pharmacokinetic para-
meters of 37 in mice were determined following the intrave-
nous administration of 1 mg/kg, oral administration of 10 mg/
kg, or intraperitoneal administration of 10 mg/kg of 37 (Table
9). As expected from the in vitro studies, 37 exhibited a short
T1/2 of 0.5 h that may partially due to the rapid first-pass
metabolism. 37 also exhibited low exposure and limited bio-
availability following the oral administration, possibly sug-
gestive of low absorption or high first pass metabolism. The
better exposure and much longer half-life achieved following
intraperitoneal administration further confirmed the instabil-
ity of 37 to the liver metabolism.
Pharmacokinetic Data
Pharmacodynamic Data
The pharmacokinetic properties of 37 were next evaluated
both in vitro and in vivo. In both human and mouse liver
microsome stability studies, 37 was rapidly consumed with a
half-life of 4 min (Table 8). The significant difference in
To assess the pharmacodynamic effects of 37, mice were
treated with a single intraperitoneal dose of 20 mg/kg 37, and
the phosphorylation of S6 at S235/236 and of Akt1 at S473
were measured by Western blot in lysates derived from liver

7152 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Liu et al.
and lung. 37 suppressed Akt1 phosphorylation at S473, the
phosphorylation site of mTORC2, for approximately 2-3 h,
after which the phosphorylation gradually returned to base-
line levels. Inhibition of S6 at S235/236, an indirect substrate
of mTORC1, was more markedly suppressed with complete
inhibition of phosphorylation observed up to 6 h in the lung
and to greater than 10 h in the liver (Figure 6). These phar-
macodynamic studies indicate that despite the poor pharma-
cological properties of 37, suppression of both mTORC1 and
mTORC2 dependent outputs can be achieved in mice.
decided to perform once daily IP dosing of 20 mg/kg of 37.
Continuous dosing for 10 days resulted in greater than 99%
inhibition of tumor growth as assessed by caliper measure-
ments. (Figure 7) Upon cessation of drug treatment, the tumors
continued to grow, suggesting that 37 treatment is primarily
cytostatic and that a significant number of tumor cells remain
viable during treatment (data not shown).
Conclusion
Starting from a quinoline screening “hit” compound 12
discovered using a medium-throughput biochemical mTORC1
assay, a focused medicinal chemistry effort facilitated by mo-
lecular modeling and other known PIKK-family inhibitors
Antitumor in Vivo Efficacy
The results from the pharmacodynamic studies encouraged
us to investigate whether 37 would inhibit growth in a U87MG
xenograft model. U87MG is a PTENnull glioblastoma cell line
that exhibits strong activation of the PI3K-mTOR-Akt path-
way and has been commonly used for xenograft experiments to
evaluate the efficacy of PI3K and mTOR kinase inhibitors.11,28
Because of the relatively poor pharmacokinetic properties, we
Table 6. KinomeScan Profile of 37a
%
Kd
control (nM)
0.42 PIK3CA(E545K) 0.6
%
Kd
kinase
mTOR
PIK3CA(C420R) 0.7
PIK3CA 1.3
kinase
control (nM)
0
ND
14
Figure 6. Pharmacodynamic inhibition of Akt S473 and S6 S235/
236 phosphorylation in lung and liver of mice treated with a single
intraperitoneal dose of 20 mg/kg 37. Six-week-old male C57BL/6
mice were fasted overnight prior to treatment with 37 as a suspen-
sion in 20% N-methyl-2-pyrrolidone/40% PEG400/40% water or
with vehicle by IP injection. Tissues were collected and analyzed at
2, 6, and 10 h after dosing.
ND PIK3CA(I800L)
23 MRCKA
0
0.65
10000
a Compound 37 was profiled at a concentration of 10 μM against a
diverse panel of 442 kinases by Ambit Biosciences.
Table 7. LanthaScreen Profile of 37 in Invitrogen Lipid Kinase Panela
kinase
PI4K R
IC50 (nM)
kinase
IC50 (nM)
>10000
6680
176
P110 R/p85R
P110δ/P85R
P110γ
DNA-PK
mTOR
250
564
171
6.34
4.32
PI4K β
PI3K-C2 R
PI3K-C2β
hVPS34
549
533
a Kinase targets were tested with biochemical enzymatic kinase assays
using the SelectScreen Kinase Profiling Service (Life Technologies
Corporation, Madison, WI) to determine IC50 values as shown in
Table 7. The compounds were assayed at 10 concentrations (3-fold
serial dilutions starting from 1 μM) at an ATP concentration equal to the
ATP Km,app for the assay following the detailed procedures described in
the SelectScreen Customer Protocol and Assay Conditions documents
located at www.invitrogen.com/kinaseprofiling.
Table 8. Human and Mouse Liver Microsome Stability Data of 37a
Figure 7. In vivo efficacy of 37 in U87MG xenografts. Mice model
was established with six-week-old immunodeficient mice by sub-
cutaneous injection of U87MG cells. 37 as a solution/suspension
in vehicle (20% N-methyl-2-pyrrolidone, 40% PEG400, and
40% water) or vehicle was delivered by intraperitoneal injection
once daily after the tumor reached 1 cm3 in size and continued for
10 days.
MR
(nmol/min/mg)
T1/2
(min)
%R at
60 min
%R at 60 min
(-NADPH)
species
human
mouse
1.30
1.53
4
4
1
0.2
69
1
a Human liver microsome (7 males þ 2 females, cat. 452165) and male
mouse liver microsomes (cat. 452220) from BD Gentest.
Table 9. Mouse PK Data of 37a
route
Cmax (ng/mL)
Tmax (h)
AUC (h ng/mL)
T1/2 (h)
MRT (h)
CL (mL/min/kg)
Vss (L/kg)
F (%)
3
720
IV
PO
IP
2757
223
ND
0.25
0.08
0.5
0.43
1.51
ND
23.0
ND
ND
0.59
ND
ND
ND
5.49
ND
396
5718
0.79
4.52
5121
a C57BL/6 male mouse was dosed at 1 mg/kg for the intravenous (IV), 10 mg/kg for the oral (PO), and 10 mg for the intraperitoneal (IP: Swiss albino
mice) studies. IV formulation used 10% N-methyl pyrrolidone (NMP) and 50% polyethylene glycol (PEG-200) in water. PO formulation used 0.5% w/v
NaCMC with 0.1% w/v Tween-80. IP administration formulation was 10% v/v dimethylacetamide (DMA), 20% v/v polyethylene glycol (PEG-200),
20% v/v PG in water.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7153
resulted in the identification of a novel benzonaphthridinone
scaffold exemplified by 37. Compound 37 inhibits phosphor-
ylation of both mTORC1 and mTORC2 at single-digit nano-
molar concentrations while exhibiting 800-fold selectivity
relative to cellular PI3K inhibitory activity. Compound 37
exhibited a short in vivo half-life and low oral bioavailability
but displayed pharmacodynamic inhibition of both mTORC1
and mTORC2 outputs in lung and liver. Compound 37 dosed
once a day at 20 mg/kg for 10 days demonstrated efficacy in a
U87MG xenograft mouse model. Compound 37 is a valuable
tool for investigating mTOR mediated signal pathways and
provides a valuable starting point for identification of com-
pounds with improved “drug-like” properties.
room temperature and stirred for 1-4 h before carefully
quenching with methanol and water. Subsequent filtration
through Celite furnished crude S3, which was used in the next
step without further purification.
To a solution of compound S3 in CH2Cl2 (1 equiv) at room
temperature was added MnO2 (10 equiv mass). After 4 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 (triethyl
2-phosphonopropionate for compound (42) were added. The
resultant mixture was heated to 100 ꢀC for 12 h before cooling to
room temperature. Upon removal of the solvents under vac-
uum, the residue was diluted with water following by extraction
with ethyl acetate. Purification of the residue by column chro-
matography provided compound S4.
To a solution of compound S4 in 1,4-dioxane at room
temperature was added subsequently PdCl2(Ph3P)2 (0.1 equiv),
t-Bu-Xphos (0.1 equiv), Na2CO3 (3 equiv, 1 N), and boronic
acids or boronate pinacol esters. After degassing, the resultant
mixture was heated to 100 ꢀC for 6 h before cooling to room
temperature and filtration through Celite. Upon removal of the
solvents, the residue was subjected to column purification to
furnish the desired compounds (18-42).
Experimental Procedures
Chemistry. All solvents and reagents were used as obtained.
1HNMR spectra were recorded with a Varian Inova 600 NMR
spectrometer and referenced to dimethyl sulfoxide. Chemical
shifts are expressed 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 Micro-
mass ZQ using an ESI source coupled to a Waters 2525 HPLC
system operating in reverse mode with an 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-
mentioned 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 15-17.
To a solution of compound 14 (4,6-dichloroquinoline, 1 equiv)
in 1,4-dioxane at room temperature in a sealed tube was added
aniline (1 equiv). The resultant solution was heated to 100-
120 ꢀC for 4-12 h. After cooling to room temperature, a
solution of NaOH (1 N) was added to neutralize the solution
followed by dilution with water and extraction with ethyl
acetate. After drying with Na2SO4, the solvents were removed
and the residue was purified by ISCO to furnish compound 13.
To a solution of compound 13 (1 equiv) in 1,4-dioxane at room
temperature was added PdCl2(Ph3P)2 (0.1 equiv), t-Bu-Xphos
(0.1 equiv), Na2CO3 (3 equiv, 1 N), and quinoline-3-boronic acid.
After degassing, the resultant mixture was heated to 100 ꢀC for 6 h
before cooling down to room temperature and filtration through
Celite. Upon removal of the solvents, the residue was purified by
column chromatography to furnish compounds 15-17.
1-(4-(4-Acetylpiperazin-1-yl)-3-(trifluoromethyl)phenyl)-9-(quino-
lin-3-yl)benzo[h][1,6]naphthyridin-2(1H)-one (18). Yield 15%
overall from S1. 1H NMR (600 MHz, DMSO-d6) δ 9.20 (s,
1H), 8.58 (d, J = 1.8 Hz, 1H), 8.34 (d, J = 9.6 Hz, 1H), 8.27 (s,
1H), 8.20 (d, J = 8.4 Hz, 1H), 8.16 (d, J = 7.2 Hz, 1H), 8.04 (d,
J = 8.4 Hz, 1H), 7.97-7.98 (m, 2H), 7.79-7.82 (m, 2H),
7.67-7.71 (m, 2H), 7.11 (s, 1H), 6.95 (d, J = 9.0 Hz, 1H),
3.28 (m, 4H), 2.65-2.70 (m, 2H), 2.59 (m, 2H), 1.98 (s, 3H). MS
(ESI): m/z (M þ H)þ 594.81.
1-(4-(4-Propionylpiperazin-1-yl)-3-(trifluoromethyl)phenyl)-
9-(quinolin-3-yl)benzo[h][1,6]naphthyridin-2(1H)-one (37). Over-
all yield 7% from S1. 1H NMR (600 MHz, DMSO-d6) δ 9.19 (s,
1H), 8.58 (d, J = 2.4 Hz, 1H), 8.34 (d, J = 9.6 Hz, 1H), 8.26 (d,
J = 1.8 Hz, 1H), 8.19 (d, J = 9.0 Hz, 1H), 8.14 (dd, J = 9.0, 1.8
Hz, 1H), 8.03 (d, J = 9.0 Hz, 1H), 7.96-7.98 (m, 2H), 7.77-7.82
(m, 2H), 7.66-7.70 (m, 2H), 7.09 (d, J = 1.8 Hz, 1H), 6.94 (d,
J = 9.6 Hz, 1H), 3.40-3.42 (m, 4H), 2.60-2.65 (m, 4H), 2.28
(q, J = 7.8 Hz, 2H), 0.98 (t, J = 7.8 Hz, 3H). MS (ESI): m/z
(M þ H)þ 608.23.
General Procedure for the Preparation of Compounds 43-47.
Compound S2 was subjected to Pd mediated coupling with
quinoline 3-boronic acid under the conditions mentioned above
to furnish compound S2a (ethyl 4-(3-(trifluoromethyl)-4-(4-
propionylpiperazin-1-yl)phenylamino)-6-(quinolin-3-yl)quino-
line-3-carboxylate). Compound S2a was dissolved in a mixture
of MeOH/H2O (1:1) and treated with NaOH (1 N, 5 equiv) at
room temperature. The reaction mixture was then stirred over-
night. The pH of the solution was adjusted to 4, followed by
extraction with ethyl acetate to afford the crude carboxylic acid
S2b. Acid S2b (1 equiv) was dissolved in THF at room tem-
perature and treated with triphosgene (0.4 equiv). After 2 h,
methylamine (2 equiv) was added and the resulting mixture was
heated at 40 ꢀC for 1 h before quenching with saturated Na2CO3.
Extraction with ethyl acetate followed by column chromatog-
raphy afforded compound 44.
1-(4-(4-([3,60-Biquinolin]-40-ylamino)-2-(trifluoromethyl)phenyl)-
piperazin-1-yl)ethanone (15). Yield 20% overall from 14. 1H
NMR (600 MHz, DMSO-d6) δ 10.12 (s, 1H), 9.49 (d, J = 2.4 Hz,
1H), 9.05 (d, J = 1.8 Hz, 1H), 8.84 (d, J = 2.4 Hz, 1H), 8.67 (s,
1H), 8.42 (dd, J = 8.4, 2.4 Hz, 1H), 8.26 (dd, J = 8.4, 2.4 Hz,
1H), 8.19 (d, J = 2.4 Hz, 1H), 8.10-8.12 (m, 2H), 7.96 (d, J =
8.4 Hz, 1H), 7.82 (ddd, J = 7.2, 6.6, 1.8 Hz, 1H), 7.69 (ddd, J =
8.4, 7.8, 1.2 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 3.54-3.56 (m,
4H), 2.87 (t, J = 4.8 Hz, 2H), 2.81 (t, J = 4.8 Hz, 2H), 2.04 (s,
3H). MS (ESI): m/z (M þ H)þ 542.87.
To a solution of compound S2b in THF was added methyl
methoxyamine (2 equiv), Et3N (3 equiv), and HATU (2 equiv).
The resultant solution was stirred at room temperature for 4 h
before extraction with ethyl acetate. Column purification afforded
Weinreb amide S2c, which was then dissolved in THF and treated
with methyl magnesium bromide (3 equiv) at room temperature.
The resulting solution was stirred for 4 h before quenching with
satd Na2CO3. Column chromatography afforded methyl ketone
S2d, which was further elaborated to compound 43 in the same
fashion as the synthesis of compound 18.
General Procedure for Compounds 18-42. To a solution
of compound S1 (ethyl 4,6-dichloroquinoline-3-carboxylate,
1 equiv) in 1,4-dioxane at room temperature was added aniline
(1 equiv) at room temperature. The reaction mixture was heated
to 100 ꢀC for 4 h, followed by cooling to room temperature and
the addition of NaOH (1 N) to neutralize the solution. The
resultant solution was diluted with water and extracted with
ethyl acetate. After removal of the solvents, the residue was
purified by flash chromatography to afford compound S2.
To a solution of compound S2 (1 equiv) in THF at 0 ꢀC was
added LAH (3 equiv). After 15 min, the solution was warmed to
To a solution of compound S3 (1 equiv) in THF at room
temperature was added triphosgene (0.4 equiv). The resultant

7154 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Liu et al.
solution was stirred at room temperature for 2 h before it was
quenched with satd Na2CO3. Column chromatography af-
forded compound 47.
using formulation as 10% v/v dimethylacetamide(DMA), 20%
v/v polyethylene glycol(PEG-200), 20% v/v PG in water. The
blood samples were collected from sets of three mice at each time
point in labeled microcentrifuge tubes containing K2EDTA as
an anticoagulant. Plasma samples were separated by centrifuga-
tion and stored below -70 ꢀC until bioanalysis. All samples were
processed for analysis by precipitation using acetonitrile and
analyzed with a partially validated LC/MS/MS method (LLOQ,
1.138 ng/mL). Pharmacokinetic parameters were calculated
using the noncompartmental analysis tool of WinNonlin En-
terprise software (version 5.2).
Antitumor (U87MG model) Efficacy Studies. Immunodefi-
cient mice (NCR nude, nu/nu; Taconic Laboratories) were
maintained in a pathogen-free facility and were given auto-
claved food and water ad libitum. U87-MG glioblastoma cells
were xenografted into six-week-old immunodeficient mice.
Briefly, 2 ꢀ 106 U87MG cells were resuspended in 100 μL of
media that had been premixed with Matrigel and was injected
subcutaneously in the upper flank region of mice that had been
anaesthetized with isoflurane. Tumors were allowed to grow to 1
cm3 in size, and the animals were randomized into two treatment
groups: vehicle and 37. For 37 injections, 37 powder was first
dissolved at 25 mg/mL in 100% N-methyl-2-pyrrolidone and
subsequently diluted 1:4 with sterile 50% PEG400 to a final
concentration of 5 mg/mL 37. Vehicle or 37 was delivered by
IP injection at the indicated dosage once daily. Tumors were
measured with calipers in two dimensions every other day. Tumor
volumes were estimated with the formula: volume = (2a ꢀ b)/2,
where a = short and b = long tumor axes, respectively, in
millimeters. All animal studies were performed according to the
official guidelines from the MIT Committee on Animal Care and
the American Association of Laboratory Animal Care.
To a solution of compound S3 in CH2Cl2 (1 equiv) at room
temperature was added MnO2 (10 equiv mass). The mixture was
stirred for 4 h before being filtered through Celite. The filtrate
was concentrated, dissolved in THF, and treated with methyl
amine (2 equiv for compound 46) or 2,4-dimethoxylbenzyla-
mine (2 equiv for compound 45), NaBH(OAc)3 (5 equiv), and
AcOH (1 drop) at room temperature. The reaction mixture was
stirred for 4 h before it was quenched with satd Na2CO3. After
compound purification, the corresponding product was sub-
jected to the same procedure as in the preparation of compound
43 to afford compounds 45 and 46.
Characterization of in Vitro Biochemical Activity with the
mTORC1 Complex. Human mTORC1 complex was obtained
as previously described.7,29 In vitro mTORC1 activity was
assayed using the Lanthascreen time-resolved FRET assay
(Invitrogen). Briefly, mTORC1 (0.1 μg each) was incubated
with serially diluted inhibitors (3-fold, 11 points) for 30 min in
5 μL of kinase buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2,
4 mM MnCl2, 50 mM KCl) in a 384-well low-volume plate
(Corning). The kinase reaction was initiated by the addition of
an equal volume of kinase buffer containing 0.8 μM GFP-
labeled 4E-BP1 and 100 μM ATP at room temperature and,
after 1 h, the reaction was stopped with 10 μL of solution
containing 20 mM EDTA and 4 nM Tb-labeled antiphospho
4E-BP1 (T46) antibody. After incubation for 30 min, the FRET
signal between Tb and GFP within the immune complex was
read using an Envision plate reader (PerkinElmer). Each data
point was duplicated and IC50 values were calculated using
Prism4 software (GraphPad).
In Vivo Pharmacodynamic Studies. For pharmacodynamic
experiments, 37 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 37 (20 mg/kg for 2, 6, or 10 h) by IP
injection and then re-fed 1 h prior to sacrifice (CO2 asphyxiation).
Tissues 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 pyropho-
sphate, 10 mM sodium β-glycerophosphate, 0.1% SDS, 1.0%
sodium deoxycholate, and 1.0% Triton, supplemented with pro-
tease inhibitor cocktail 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.
mTOR and PI3K Cellular Assays. Cellular IC50 values for
mTOR were determined using p53-/- MEFs. Cells were treated
with vehicle or increasing concentrations of compound for 1 h
and then lysed. Phosphorylation of S6K1 Thr-389 was mon-
itored by immunoblotting using a phospho-specific antibody.
Meanwhile, cellular IC50 values for PI3Ka were determined
based on phosphorylation of Akt Thr-308 in p53-/-/mLST8-/-
MEFs or human PC3 cells expressing the S473D mutant of Akt1
as previously described.7
Ambit in Vitro KinomeScan Kinase Selectivity Profile. 37 was
profiled at a concentration of 10 μM against a diverse panel of
442 kinases by Ambit Biosciences. Scores for primary screen hits
are reported as a percent of the DMSO control (% control). For
kinases where no score is shown, no measurable binding was
detected. The lower the score, the lower the Kd is likely to be,
such that scores of zero represent strong hits. Scores are related
to the probability of a hit but are not strictly an affinity
measurement. At a screening concentration of 10 μM, a score
of less than 10% implies that the false positive probability is less
than 20% and the Kd is most likely less than 1 μM. A score
between 1% and 10% implies that the false positive probability
is less than 10%, although it is difficult to assign a quantitative
affinity from a single-point primary screen. A score of less than
1% implies that the false positive probability is less than 5% and
the Kd is most likely less than 1 μM.
In Vivo Pharmacokinetic Studies. The study was performed at
the Sai Advantium Pharma Limited Company (India) with male
Swiss albino mice following single intravenous bolus and oral
administration. A group of 18 male mice were divided into two
groups (group 1, IV; group 2, PO), with each group comprised of
nine mice. Animals in group 1 were dosed intravenously via the
tail vein at 1 mg/kg of 37 solution (10% v/v N-methyl pyrro-
lidone and 50% v/v polyethylene glycol-200 in normal saline).
Group 2 animals were dosed orally at 10 mg/kg with a suspen-
sion formulation (0.5% w/v Na CMC with 0.1% v/v Tween-80
in water) of 37. Blood samples were collected at 0, 0.08 (for IV
only), 0.25, 0.5, 1, 2, 4, 6 (For PO only), 8, 12, and 24 h for the IV
and PO groups. IP experiment was one with Swiss albino mice
Molecular Modeling. An mTOR homology model was built
with Modeler(9v6) based on a published PI3Kγ crystal structure
complexed with GDC-0941 (PDB: 3DBS). 37 was docked into
the model with Pair fitting, followed by energy minimization
with Discovery Studio II.
Acknowledgment. We thank Life Technologies Corporation,
SelectScreen Kinase Profiling Service for performing enzymatic
biochemical kinase profiling. We also thank Ambit Bioscience for
performing KinomeScan profiling and Dr. David Waller (Dana
Farber Cancer Institute) for the useful proofreading.
Supporting Information Available: Spectral data of 16-17,
19-36, 38-47. This material is available free of charge via the
Internet at http://pubs.acs.org.
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