Design, synthesis, and structure-activity relationships of 3-ethynyl-1 H-indazoles as inhibitors of the phosphatidylinositol 3-kinase signaling pathway

Article abstract of DOI:10.1021/jm100825h

A new series of 3-ethynyl-1H-indazoles has been synthesized and evaluated in both biochemical and cell-based assays as potential kinase inhibitors. Interestingly, a selected group of compounds identified from this series exhibited low micromolar inhibitio

Full text of DOI:10.1021/jm100825h

8368 J. Med. Chem. 2010, 53, 8368–8375
DOI: 10.1021/jm100825h
Design, Synthesis, and Structure-Activity Relationships of 3-Ethynyl-1H-indazoles as Inhibitors
of the Phosphatidylinositol 3-Kinase Signaling Pathway
Elisa Barile,†,§ Surya K. De,†,§ Coby B. Carlson,‡ Vida Chen,† Christine Knutzen,† Megan Riel-Mehan,† Li Yang,†
Russell Dahl,† Gary Chiang,† and Maurizio Pellecchia*,†
†Sanford-Burnham Medical Research Institute, La Jolla, California 92037, United States, and ‡Invitrogen Discovery Assays and Services
(Now a Part of Life Technologies), 501 Charmany Drive, Madison, Wisconsin 53719, United States. §These authors contributed equally to this work
Received July 2, 2010
A new series of 3-ethynyl-1H-indazoles has been synthesized and evaluated in both biochemical and cell-
based assays as potential kinase inhibitors. Interestingly, a selected group of compounds identified from
this series exhibited low micromolar inhibition against critical components of the PI3K pathway,
targeting PI3K, PDK1, and mTOR kinases. A combination of computational modeling and structure-
activity relationship studies reveals a possible novel mode for PI3K inhibition, resulting in a PI3KR
isoform-specific compound. Hence, by targeting the most oncogenic mutant isoform of PI3K, the
compound displays antiproliferative activity both in monolayer human cancer cell cultures and in three-
dimensional tumor models. Because of its favorable physicochemical, in vitro ADME and drug-like
properties, we propose that this novel ATP mimetic scaffold could prove useful in deriving novel
selecting and multikinase inhibitors for clinical use.
Introduction
Because of its involvement in a variety of cellular events, such
as survival, proliferation, cell motility, and invasion,4 hyper-
activation of the PI3K signaling pathway is one of the most
common molecular events in nearly every form of human
cancer.5-8 Indeed, aberrant activation of the PI3K cascade,
including PTEN inactivation and PI3K activating mutations, is
present in about 32% of colon cancers, 30% of breast cancers,
30% of melanomas,9 27% of brain cancers, and 25% of
stomach cancers.10,11 It is for these reasons that considerable
drug discovery efforts are ongoing, targeting several compo-
nents of this signaling cascade using either single and multiple
target strategies.12-16 Interestingly, clinical data suggest that
multikinase inhibitors (MKIs) produce greater benefit over
single kinase inhibition, especially in solid tumors where dif-
ferent kinases and different pathways synergistically contribute
to tumor proliferation.17-19 Indeed, emerging phase II clinical
trial data reveal that dual PI3K/mTOR inhibitors may be more
effective, having the advantage of being less susceptible to
PI3K drug resistance owing to their preserved activity against
mTOR.16
The PI3K/AKT/mTORa cascade is an important cellular
signaling pathway that regulates intersecting biological pro-
cesses such as cell growth and proliferation, cell survival,
protein synthesis, and glycolysis metabolism.1-3 Physiological
stimulation of this cascade occurs through the binding of
growth factors (e.g., insulin or insulin-like growth factor;
IGF-1) to receptor tyrosine kinases (RTK) on the cell surface.
Subsequent activation of the lipid phosphatidylinositol 3-ki-
nase (PI3K) leads to the phosphorylation of phosphatidylino-
sitol 4,5-bisphosphate (PIP2) to produce phosphatidylinositol
3,4,5-triphosphate (PIP3), which in turn interacts with the
pleckstrin homology (PH) domain of AKT and recruits the
kinase to the plasma membrane. Full activation of AKT
requires phosphorylation of two distinct residues: Thr308 by
the upstream kinase PDK1 and Ser473 by the mammalian
target of rapamycin complex 2 (mTORC2).1-3Activated AKT,
among its wide array of downstream effects, increases protein
synthesis rate by phosphorylation at Thr246 of the proline-rich
substrate of 40 kDa (PRAS40). This substrate is further
regulated through phosphorylation at Ser183 by the mTOR
complex 1 (mTORC1). Downstream of mTORC1, signaling
events can go through p70 S6 kinase (S6K1), which in turn
phosphorylates the programmed cell death protein (PDCD4)
at residue Ser457 (Figure 1).
In our drug discovery program we interrogated the PI3K
signaling pathway, and we report on the identification of a
novel scaffold of the 3-ethynylindazole family as multiple
PI3K/PDK1/mTOR inhibitors. Such scaffold represents a
novel lead structure for the PI3K pathway, suitable for further
functionalization and drug development.
*Corresponding author. E-mail: mpellecchia@sanfordburnham.org.
Phone: 858-646-3159. Fax: 858-713-9925.
Results and Discussion
a Abbreviations: PI3K, phosphatidylinositol 3-kinase; PDK1, 3-phos-
phoinositide-dependent protein kinase-1; RTK, receptortyrosine kinases;
AKT, protein kinase B; mTOR, mammalian target of rapamycin; PTEN,
phosphatase and tensin homologue protein; PIP2, phosphatidylinositol
4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-triphosphate; PDCD4,
programmed cell death protein; S6K1, S6 kinase β-1; GFP, green fluorescent
protein.
Compounds 6-19 (Table 1) were prepared as shown in
Scheme 1. Compound 1 was iodinated, and Boc protection
was performed according to the previously reported pro-
cedures.20 Compound 3 (Scheme 1) was coupled with the
appropriate alkyne using the Sonogashira reaction conditions
r
pubs.acs.org/jmc
Published on Web 11/09/2010
2010 American Chemical Society

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8369
Figure 1. Schematic representation of the PI3K/mTOR signaling pathway highlighting the phosphorylation sites tested in our cellular and
biochemical assays.
to give 4 and its analogues in moderate yield (45-65%). Com-
pound 4 was treated with TFA to afford the final compound 5
and its analogues (6-19, Table 1) in good yields (89-95%).
Similarly, compounds 20 and 21 (Table 1) were synthesized
starting from 7-azaindazole instead of indazole and following
the same synthetic procedure reported in Scheme 1 for indazole
derivatives.
We chose to initially screen the synthesized compounds
(shown in Table 1) in a cell-based format using a LanthaScreen
cellular assay technology, recently reported and success-
fully applied to the analysis of the PI3K/AKT/mTOR
pathway.21-23 This assay platform uses individual cell
lines that stably express key kinase substrate within the
cascade as GFP-fusion proteins. Cells are stimulated, in
the presence or absence of an inhibitor, to trigger endo-
genous kinase activity and phosphorylation of the sub-
strate. The phosphorylation status of the GFP substrate is
detected after cell lysis by addition of a terbium- (Tb-)
multiple PI3KR/PDK1/mTOR inhibitors, while compounds
9 and 13 are dual PI3KR /PDK1 inhibitors (Table 1). All
compounds tested were inactive against AKT as measured by
a biochemical kinase assay for AKT1 (data not shown).
Further in vitro displacement assays against the PI3K iso-
forms revealed that compounds 6, 10, and 13 are R-isoform
specific and ATP competitive with about 100-fold selectivity
over the β- and γ-isoforms (Table 2).
In order to rationalize the affinity observed for the lead
structure 10, we studied its binding mode within the ATP-
binding site of PI3KR by using computational docking stud-
ies. A target binding pocket was derived from the X-ray
crystal structure of the ternary complex involving the most
common mutant of PI3K catalytic domain p110R (H1047R),
its regulatory subunit (p85R), and the drug wortmannin (PDB
id: 3hhm).24 From the docked binding pose, compound 10
appears to be deeply inserted in the ATP-binding site (Figure 2A):
the N-2 atom of compound 10 seems to be involved in
hydrogen-bonding interactions with the OH of Tyr836 and
the indazole moiety further stabilized via hydrogen bond
contacts between the NH-1 and the N-2 of the heterocycle
with the backbone carbonyl of Asp810 and the backbone NH
of Asp933, respectively (Figure 2B). Furthermore, the com-
pound binds to the hinge region of the kinase via a hydrogen
bond between the protonated NH of the pyridine ring and the
backbone carbonyl of Val851 (Figure 2). From these studies it
became apparent that compounds of this series capable of
interacting with the hinge region are most effective against
PI3KR. Indeed, the isomer 9, whose protonated nitrogen
could still constitute a suitable hydrogen-bonding donor
group interacting with the hinge residue Val851, showed good
potency (IC50 = 1.85 μM); in compounds 6 and 13, where the
ethynylindazole moiety is linked to an aniline, is the amino
group at the 3- or 4-position on the phenyl ring to be involved
in the hydrogen bond with the backbone carbonyl of Val851.
Indeed, they both showed a low micromolar inhibition (IC50
values of 1.05 and 5.12 μM, respectively).
€
labeled phosphospecific antibody via time-resolved Forster
resonance energy transfer (TR-FRET) signal between Tb and
GFP.21 We used three phosphorylation readouts to interro-
gate pathway signaling: AKT (pThr308) for PI3K/PDK1,
PRAS40 (pThr246) for AKT, and PRAS40 (pSer183) for
mTORC1 activity (Figure 1).
Compounds were incubated with the different cell lines in
10-point dose-response format for 60 min to generate IC50
curves. Among all newly synthesized series of 3-ethynyl-1H-
indazoles (6-19), we found that compounds 6,10,and13 inhib-
ited both AKT and PRAS40 phosphorylation, while com-
pound 9 inhibited only AKT phosphorylation, with potencies
in the low micromolar range (Table 1).
To understand if the inhibition of AKT phosphorylation at
Thr308 was a result of a direct inhibition of PDK1 or PI3K,
we performed a cell-free kinase assay against both kinases. All
of the compounds which displayed inhibition in cell-based
assays (6, 9, 10, 13) resulted in being active against both
kinases, with the highest potency for 10 (IC50=361 nM)
against PI3KR. Similarly, we tested the compounds against
mTORthrougha biochemicalassay. Compounds6 and 10 are
The shift of the amino group at the 2-position on the phenyl
ring (16) does not allow the compound to form the critical

8370 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Table 1. Kinase and Cell-Based Assay Results for Compounds 6-21a
Barile et al.
a Values are means of at least three or more experiments with a typical standard deviation of less than (20%. b LanthaScreen Eu kinase binding assay.
c Z0-LYTE kinase activity assay. d LanthaScreen cellular assays in HEK293E cell line; AKT (pThr308) for PI3K/PDK1 readout and PRAS40 (pSer183)
for mTORC1 readout. ND: not determined (compound was not tested).
interaction with the hinge region, thus explaining its loss of
activity (IC50 > 100 μM). As expected, the simple phenyl (7),
fluorophenyl (8), trifluoromethylphenyl (17), thiophenyl (18),
and 4-cyanophenyl (19) substitutions on 3-ethynylindazole
showed poor activity. Accordingly, aliphatic alkynes such as
cyclopentyl (14), cyclopropyl (15), and N,N-dimethyl (11)
were inactive both in the biochemical and cell-based assays
(Table 1).
Keeping the pyridine group as the most efficient substitu-
ent, we explored possible modifications on the indazole ring
(20 and 21). Isosteric replacement of the indazole with the
pyrazolopyridine ring revealed to be detrimental for PI3KR
inhibition. Indeed, by comparing the most active compound of
the 3-ethynylindazole series (10, IC50 = 361 nM) with the corres-
ponding pyrazolopyridine analogue (20, IC50 = 3.05 μM),
a 10-fold loss of activity was observed, and the same trend was

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8371
followed by compound 9 (IC50 = 1.85 μM) versus compound
21 (IC50 = 9.0 μM). Such decrease in activity is expected given
that the additional nitrogen atom is not engaged in hydrogen-
bonding interactions in the docked structures of compounds
20 and 21 in the binding pocket of PI3KR (Supporting
Information).
Since the ATP-binding pocket of mTOR is highly homo-
logous to that of PI3K, it is not surprising that some of
the compounds inhibit both kinases (Table 1). Indeed, even
previously reported selective PI3K inhibitors show some
degree of activity against mTOR.16 However, noteworthy
compound 10 in our cellular and biochemical assays inhibits
also the less structurally related PDK-1. Inhibition of multiple
kinases on the PI3K pathway may be highly desirable to
suppress oncogenic transformations that result from the
activation of the pathway in cancer cells.
To evaluate the drug likeness of the proposed series on
empirical ground,25,26 we gathered information about physi-
cochemical properties of the compounds, such as solubility,
cell permeability, and ligand efficiency indices,27-29 as well as
plasma and microsomal stability (Table 3 and Supporting
Information). Hence, investigation of these parameters
suggests that the compounds of this novel scaffold series
may be deemed suitable as lead candidates for further drug
optimizations.30
Scheme 1. Synthetic Scheme for Compounds 6-21a
On the basis of their cellular efficacy and favorable bio-
pharmaceutical properties, we further investigated the anti-
proliferative activity of the most promising compounds (6, 9,
10, and 13) against human cancer cell lines including prostatic
(PC3), breast (MD-MA-231 and MCF7), and cervical (HeLa)
cancers. The results are summarized in Table 1. The anti-
tumor activity of compound 10, which displayed the high-
est cellular potency inhibiting PI3KR, mTOR, and PDK1,
has been further evaluated against growth/proliferation
of the glioblastoma U87 cell line when tested in three-
dimensional cultures. The three-dimensional cell culture
system has been chosen because it better recapitulates real
human tissue with respect to oxygen and nutrient levels,
cell-cell contacts, and cellular architecture compared
with conventional two-dimensional cell culture systems.31
It has also been observed that spheroid cultures exhibit
a Reagents and conditions: (a) iodine, KOH, DMF, room tempera-
ture; (b) di-tert-butyl dicarbonate, Et3N, DMAP, CH3CN, room tem-
perature; (c) aryl or aliphatic alkyne, Pd(Ph3P)2Cl2, CuI, Et3N, CH3CN,
room temperature; (d) TFA, CH2Cl2, room temperature.
Table 3. Ligand Efficiency Indices and in Vitro ADME Properties of 10
and 13a
Table 2. Biochemical Selectivity Profile against PI3K Isoforms, IC50
(μM)
parameter
ligand efficiency
binding efficiency index
10
13
0.4
compd ID
PI3KRa
PI3Kγa
PI3Kδa
LE (kcal mol-1
BEI
)
0.5
29.4 22.7
17.5 10.5
6
1.05
0.323
5.12
>100
40
>100
>100
39
>100
surface binding efficiency index SEI
10
13
lipophilic ligand efficiency
microsomal stability
plasma stability
LLE
1/2 (min)
% remaining after 60 min 97
4.2
35 >60
100
2.5
t
a These assays were conducted by Invitrogen’s SelectScreen Biochem-
ical Kinase (SSBK) Profiling Service. Values are means of at least three
or more experiments with a typical standard deviation of less than
(20%.
a Key: LE, ligand efficiency;28 BEI, binding efficiency index;27 SEI,
surface efficiency index;27 LLE, lipophilic ligand efficiency indices.27-29
.
Figure 2. Docking studies of compound 10 within the ATP-binding site of PI3KR (H1047R) mutant (PDB id: 3hhm).24 (A) Surface
representation of the active site of p110R with the ATP mimic compound 10. Surface generated with MOLCAD37 and color coded according to
cavity depth (blue, shallow; yellow, deep). Protein residues involved in hydrogen bonds are highlighted. (B) Ribbon representation of the
PI3KR (H1047R) mutant in complex with compound 10 in the same binding pose of (A). Protein residues involved in hydrogen bonds shown as
capped sticks.

8372 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Barile et al.
Figure 3. U87 cells grown as spheroids generated by the hanging drop method34 and treated with the indicated concentration of 22 or
compound 10 every day for a total of 6 days. Left panel: Phase-contrast photos representative of four independent experiments. Right panel:
Spheroid length and width (measured with an optical micrometer) were used to calculate spheroid volumes (μm3).
Table 4. Kinases Which Showed an Inhibition Higher than 50% at 10
as reported for the wortmannin derivative PX-866 (22)32
μM Concentration of Compound 10
differential sensitivity to known chemotherapeutic agents,
kinases tested
kinases tested
MARK4
MELK
which showed higher potency in multicellular 3D system
than in monolayer.33 We chose U87 glioblastoma cells
because they exhibit deregulated PI3K signaling due to the
functional loss of the tumor suppressor PTEN (Figure 1).
U87 spheroids, generated through the previously reported
hanging drop method,34 were treated daily, for a total of
6 days, with compounds 10 and 22 as control at different
concentrations (Figure 3). Treatment with 10 μM com-
pound 10 every day strongly suppressed cell growth/
proliferation, as proved by detected spheroid volumes,
comparable to those produced by the potent PI3K inhib-
itor 22 (Figure 3).
In summary, we successfully developed a useful synthetic
routetoobtain3-ethynylindazolederivativeswhichleadtothe
identification of a multiple PI3K/PDK1/mTOR inhibitor,
with a nanomolar inhibition against PI3KR. Other indazoles
were recently reported as possible inhibitors of other protein
kinases;35 however, the scaffolds reported here, in particular
compound 10, represent a novel and valuable starting point
for the development of inhibitors of the PI3K pathway and
potentially other kinases (see below). Moreover, due to the
observed drug likeness and in vitro ADME properties of this
novel scaffold (Table 3), we anticipate that further selective
and/or multikinase inhibitors could arise from further deri-
vatization of this novel ATP mimetic. In fact, preliminary
selectivity panels with compound 10 against 314 protein kinases
(Supporting Information) reveal that less than 10% of these
proteins showed g50% inhibition at 10 μM (Table 4). Hence,
ABL1 T3151I
CDK5/p25
CHUK (IKK alpha)
CLK2
DNA-PK
MINK1
MUSK
MYLK2 (skMLCK)
NTRK1 (TRKA)
NTRK2 (TRKB)
NTRK3 (TRKC)
NUAK1 (ARK5)
PDGFRA T674I
PDGFRA V561D
RET V804L
FLT3
FLT3 D835Y
FLT4 (VEGFR3)
GSG2 (Haspin)
IRAK1
JNK1 (MAPK8)
JNK2 (MAPK9)
KDR (VEGFR2)
LRKK2
RET Y791F
SGK (SGK1)
SYK
LRRK2 G2019S
MAP4K4 (HGK)
based on these data, we envision that further elaborations of
compound 10 could lead to further selective or multikinase
inhibitors against a variety of drug targets.
Experimental Section
Chemistry. Unless otherwise indicated, all anhydrous sol-
vents were commercially obtained and stored in Sure-seal
bottles under nitrogen. All other reagents and solvents were
purchased as the highest grade available and used without
further purification. Thin-layer chromatography (TLC) anal-
ysis of reaction mixtures was performed using Merck silica gel 60
F254 TLC plates and visualized using ultraviolet light. NMR

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8373
spectra were recorded on Varian 300 or 500 MHz instruments.
Chemical shifts (δ) are reported in parts per million (ppm)
referenced to H (Me4Si at 0.00). Coupling constants (J) are
7.56 (d, J = 8.4 Hz, 1H), 7.65 (d, J = 7.8 Hz, 1H), 11.65 (br s,
1H, NH); MS m/z 200 (M þ H)þ, 155, 127, 83; HRMS calcd for
C12H14N3 200.1182 (M þ H), found 200.1190.
1
Synthesis of 3-(Pyridin-2-ylethynyl)-1H-indazole (12). Yield
91%; 1H NMR (300 MHz, CD3OD) δ 7.29 (t, J = 6.9 Hz, 1H),
7.40-7.55 (m, 2H), 7.60 (d, J = 8.1 Hz, 1H), 7.77 (d, J = 6.6 Hz,
2H), 7.93 (d, J = 6.9 Hz, 2H), 11.31 (br s, 1H, NH); HRMS
calcd for C14H10N3 220.0875 (M þ H), found 220.0878.
Synthesis of 3-((1H-Indazol-3-yl)ethynyl)aniline (13). Yield
reported in hertz throughout. Mass spectral data were acquired
on a Shimadzu LCMS-2010EV for low resolution and on an
Agilent ESI-TOF for either high or low resolution. Purity of all
compounds was obtained in a HPLC Breeze from Waters Co.
using an Atlantis T3 3 μm 4.6 ꢀ 150 mm reverse-phase column.
The eluant was a linear gradient with a flow rate of 1 mL/min
from 95% A and 5% B to 5% A and 95% B in 15 min followed
by 5 min at 100% B (solvent A, H2O with 0.1% TFA; solvent B,
acetonitrile with 0.1% TFA). The compounds were detected at
λ=254 nm. Purity of key compounds was established by elemen-
tal analysis as performed on a Perkin-Elmer series II-2400 and
HPLC analysis and determined to be >95%. Combustion analysis
was performed by NuMega Resonance Laboratories, San Diego,
CA, USA.
Synthesis of tert-Butyl 3-(Phenylethynyl)-1H-indazole-1-car-
boxylate (4, R=Ph). To a solution of compound 3 (343 mg,
1 mmol) in CH3CN (5 mL) were added phenyl acetylene (0.13 mL,
1.2 mmol), CuI (20 mg, 0.10 mmol), Pd(Ph3P)2Cl2 (70 mg, 0.10
mmol), and Et3N (0.42 mL, 3 mmol), respectively, at room
temperature under nitrogen atmosphere. The reaction mixture
was stirred at room temperature for 16 h; then solvent was
removed in vacuo. The residue was chromatographed over silica
gel (2% ethyl acetate in hexane) to give a Boc-protected product
4 (270 mg, 85%).
Synthesis of 3-(Phenylethynyl)-1H-indazole (7). To a solution
of 4 (150 mg, 0.47 mmol) in CH2Cl2 (3 mL) was added TFA
(1 mL) at room temperature. The resulting mixture was stirred
at room temperature for 3 h; then solvent and TFA were
removed in vacuo. The residue was extracted with CH2Cl2
(50 mL), washed with saturated NaHCO3 aqueous solution
(3 ꢀ 30 mL) and brine (30 mL), dried (MgSO4), and concen-
trated in vacuo. The residue was chromatographed over silica gel
(40% ethyl acetate in hexane) to afford a white solid compound
7 (94 mg, 92%). 1H NMR (300 MHz, CDCl3) δ 7.26-7.34
(m, 1H), 7.40-7.50 (m, 5H), 7.66-7.76 (m, 2H), 7.95 (d, J = 7.8
Hz, 1H), 11.68 (br s, 1H, NH); MS m/z 219 (M þ H)þ, 166, 155,
121, 88; HRMS calcd for C15H11N2 219.0917 (M H), found
219.0918.
1
91%; H NMR (300 MHz, CD3OD) δ 5.26 (br s, 2H, NH2),
6.76 (d, J = 7.8 Hz, 1H), 6.94 (d, J = 7.5 Hz, 1H), 6.97 (s, 1H),
7.13 (t, J = 8.1 Hz, 1H), 7.24 (t, J = 8.1 Hz, 1H), 7.44 (t, J = 8.1
Hz, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.84 (d, J = 8.7 Hz, 1H), 11.32
(br s, 1H, NH); HRMS calcd for C15H12N3 234.1031 (M þ H),
found 234.1025. Anal. Calcd for C15H11N3: C, 77.23; H, 4.75; N,
18.01. Found: C, 77.01; H, 4.89; N, 17.89.
Synthesis of 3-(Cyclopentylethynyl)-1H-indazole (14). Yield
92%; 1H NMR (300 MHz, CDCl3) δ 1.66-1.69 (m, 2H),
1.80-189 (m, 4H), 2.02-2.19 (m, 2H), 2.94-3.08 (m, 1H),
7.22 (t, J = 7.4 Hz, 1H), 7.41 (t, J = 7.4 Hz, 1H), 7.70 (d,
J = 7.8 Hz, 1H), 7.82 (d, J = 7.8 Hz, 1H), 11.84 (br s, 1H, NH);
HRMS calcd for C14H15N2 211.1235 (M þ H), found 211.1233.
Synthesis of 3-(Cyclopropylethynyl)-1H-indazole (15). Yield
90%; 1H NMR (300 MHz, CDCl3) δ 0.92-1.00 (m, 4H),
1.56-1.67 (m, 1H), 7.17-7.27 (m, 1H), 7.36-7.46 (m, 1H), 7.66
(d, J = 8.4 Hz, 1H), 7.82 (d, J = 8.1 Hz, 1H), 9.86 (br s, 1H, NH);
HRMS calcd for C12H11N2 183.0922 (M þ H), found 183.0925.
Synthesis of 2-((1H-Indazol-3-yl)ethynyl)aniline (16). Yield
1
81%; H NMR (300 MHz, CD3OD) δ 5.26 (br s, 2H, NH2),
6.58-6.60 (m, 2H), 7.12-7.15 (m, 1H), 7.24 (d, J = 7.5 Hz, 2H),
7.28-7.46 (m, 2H), 7.70 (d, J = 6.9 Hz, 1H), 11.80 (br s, 1H,
NH); MS m/z 234 (M þ H)þ, 158, 149, 121, 102, 65; HRMS
calcd for C15H12N3 234.1031 (M þ H), found 234.1021.
Synthesis of 3-((3,5-Bis(trifluoromethyl)phenyl)ethynyl)-1H-
1
indazole (17). Yield 90%; H NMR (300 MHz, CDCl3) δ 7.32
(t, J = 8.5 Hz, 1H), 7.49 (t, J = 6.6 Hz, 1H), 7.85-7.94 (m, 2H),
8.07 (s, 2H), 9.56 (br s, 1H, NH); MS m/z 355 (M þ H)þ, 275,
244, 149, 130, 127, 121, 118, 88; HRMS calcd for C17H9F6N2
355.0664 (M þ H), found 355.0664.
Synthesis of 3-(Thiophen-3-ylethynyl)-1H-indazole (18). Yield
92%; 1H NMR (300 MHz, CDCl3) δ 7.22-7.46 (m, 4H),
7.63-7.70 (m, 2H), 7.90 (d, J = 8.1 Hz, 1H), 9.52 (br s, 1H,
NH); MS m/z 225 (M þ H)þ, 130, 121, 102; HRMS calcd for
C13H9N2S 225.0481 (M þ H), found 225.0484.
Following the above-mentioned procedure and the use of the
appropriate starting materials and reagents, compounds 6-16
were prepared. Yields refer to the final deprotection step.
Synthesis of 4-((1H-Indazol-3-yl)ethynyl)aniline (6). Yield
Synthesis of 4-((1H-Indazol-3-yl)ethynyl)benzonitrile (19). Yield
91%; 1H NMR (300 MHz, CD3OD) δ 7.28 (t, J=7.5 Hz, 1H), 7.47
(t, J = 7.8 Hz, 1H), 7.59 (d, J=8.1 Hz, 1H), 7.74-7.76 (m, 4H),
7.85 (d, J = 8.1 Hz, 1H), 10.89 (br s, 1H, NH); MS m/z 244 (M þ
H)þ, 217, 149, 141, 130, 127, 121, 102; HRMS calcd for C16H10N3
244.0869 (M þ H), found 244.0875.
1
65%; H NMR (300 MHz, CD3OD) δ 5.24 (br s, 2H, NH2),
6.57-6.60 (m, 2H), 7.12-7.15 (m, 1H), 7.24 (d, J=7.5 Hz, 2H),
7.28-7.46 (m, 2H), 7.70 (d, J=6.9 Hz, 1H), 11.82 (br s, 1H,
NH); MS m/z 234 (M þ H)þ, 158, 149, 121, 102, 65; HRMS
calcd for C15H12N3 234.1031 (M þ H), found 234.1021.
Synthesis of 3-((3,5-Difluorophenyl)ethynyl)-1H-indazole (8).
Yield 89%; 1H NMR (300 MHz, DMSO) δ7.27 (t, J=7.5 Hz, 1H),
7.36-7.51 (m, 4H), 7.63 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 8.1 Hz,
1H), 13.32 (br s, 1H); MS m/z 255 (M þ H)þ, 149, 121; HRMS
calcd for C15H9F2N2 255.0728 (M þ H), found 255.0726.
Synthesis of 3-(Pyridin-4-ylethynyl)-1H-pyrazolo[3,4-b]pyri-
1
dine (20). Yield 86%; H NMR (300 MHz, DMSO-d6) δ 7.37
(dd, J = 8.1 and 4.5 Hz, 1H), 7.65-7.69 (m, 2H), 8.42 (dd, J =
8.1 and 1.2 Hz, 1H), 8.65 (dd, J = 4.5 and 1.2 Hz, 1H), 8.67-
8.71 (m, 2H), 13.89 (br s, 1H, NH); MS m/z 221 (M þ H)þ, 159,
130, 121, 102, 88, 85, 56; HRMS calcd for C13H9N4 (M þ H)
221.0827, found 221.0822.
Synthesis of 3-(Pyridin-3-ylethynyl)-1H-indazole (9). Yield
1
92%; H NMR (300 MHz, CDCl3) δ 7.24-7.41 (m, 3H), 7.46
(m, 1H), 7.59 (d, J=7.8 Hz, 1H), 7.87-7.99 (m, 2H), 8.63 (s, 1H),
11.32 (br s, 1H, NH); MS m/z 220 (M þ H)þ, 192, 121, 102;
HRMS calcd for C14H10N3 220.0875 (M þ H), found 220.0877.
Synthesis of 3-(Pyridin-4-ylethynyl)-1H-indazole (10). Yield
91%; 1H NMR (400 MHz, DMSO) δ 7.29 (t, J = 7.3 Hz, 1H),
7.45 (t, J = 7.3 Hz, 1H), 7.62-7.67 (m, 3H), 7.89 (d, J = 7.9 Hz,
1H), 8.65 (d, J = 6.1 Hz, 2H), 13.68 (br s, 1H, NH); HRMS
calcd for C14H10N3 220.0875 (M þ H), found 220.0878. Anal.
Calcd for C14H9N3: C, 76.70; H, 4.14; N, 19.17. Found: C, 76.58;
H, 4.29; N, 19.01.
Synthesis of 3-(Pyridin-2-ylethynyl)-1H-pyrazolo[3,4-b]pyri-
1
dine (21). Yield 83%; H NMR (300 MHz, DMSO-d6) δ 7.35
(dd, J = 8.1 and 4.2 Hz, 1H), 7.42 (t, J=5.7 Hz, 1H), 7.79 (d,
J = 7.5 Hz, 1H), 7.90 (t, J=7.7 Hz, 1H), 8.35 (d, J = 8.1 Hz,
1H), 8.60-8.67 (m, 2H), 13.87 (br s, 1H, NH); HRMS calcd for
C13H9N4 (M þ H) 221.0827, found 221.0824.
Assays. LanthaScreen Cellular Assay. Cell-based assays for
both AKT and PRAS40 phosphorylation were carried out using
LanthaScreen cellular assay technology from Invitrogen (Carlsbad,
CA).21 Briefly, the assay protocol for compound screening is as
follows. Cells were plated in white tissue culture-treated 384-well
assay plates at a density of 20000 cells per well in 32 μL of assay
medium (low glucose DMEMþ0.1% bovine serum albumin, BSA).
Synthesis of 3-(1H-Indazol-3-yl)-N,N-dimethylprop-2-yn-
1
1-amine (11). Yield 90%; H NMR (300 MHz, CDCl3) δ 2.47
(s, 6H), 3.67 (s, 2H), 7.18-7.27 (m, 1H), 7.40 (t, J = 7.6 Hz, 1H),

8374 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Barile et al.
After overnight serum starvation, cells were pretreated with
4 μL of compound at the indicated concentrations (10-point dose-
response in duplicate) for 60 min. The cells were then stimulated with
insulin (EC80 concentration of ∼5 ng/mL) for 30 min to activate
PI3K/AKT/mTOR signaling. The assay medium was subsequently
removed via aspiration, and cells were lysed by the addition of 20 μL
of LanthaScreen cellular assay lysis buffer supplemented with
protease and phosphatase inhibitor cocktails (Sigma P8340 and
P2850, respectively) as well as 2 nM Tb-labeled detection antibody,
also from Invitrogen. Following 2 h assay equilibration at room
temperature, the TR-FRET emission ratios were acquired on a
PerkinElmer EnVision fluorescence plate reader (Waltham, MA)
with TRF laser excitation and emission wavelengths of 520 and
495 nm. Data analysis and curve fitting were performed using
XLfit4 and GraphPad Prism4 software.
PI3Kr in Vitro Assay. The lipid kinase PI3K was assayed by
using a LanthaScreen Eu kinase binding assay technology from
Invitrogen. To a solution of the compounds diluted in assay
buffer (25 mM Tris-HCl, 5 mM MgCl2, 0.02% CHAPS) were
added the following components: the kinase tracer (PV6088,
20 nM final concentration), the kinase PIK3CA/PIK3R1 (PV4788,
5 nM final concentration), and Eu-labeled anti-His tag antibody
(PV6089, 2 nM final concentration).
The assay was performed in Corning 3673 white 384-well
assay plates. Following 1 h assay equilibration at room tem-
perature, the TR-FRET emission ratios were acquired on a
PerkinElmer EnVision fluorescence plate reader (Waltham,
MA) with TRF laser excitation and emission wavelengths of
665 and 615 nm. Data analysis and curve fitting were performed
using XLfit4 and GraphPad Prism4 software.
mTOR in Vitro Assay. Mammalian target of rapamycin
(mTOR) was assayed by using a LanthaScreen Eu kinase bind-
ing assay technology from Invitrogen. To a solution of the com-
pound diluted in assay buffer (25 mM Tris-HCl, 5 mM MgCl2,
0.02% CHAPS) were added the following components: the kinase
tracer (PV6087, 20 nM final concentration), the kinase FRAP1
(PV4753, mTOR, 5 nM final concentration), and Eu-labeled anti-
GST tag antibody (PV5594, 2 nM final concentration).
The assay was performed in Corning 3673 white 384-well assay
plates. Following 1 h assay equilibration at room temperature, the
TR-FRET emission ratios were acquired on a PerkinElmer EnVi-
sion fluorescence plate reader (Waltham, MA) with TRF laser
excitation and emission wavelengths of 665 and 615 nm. Data
analysis and curve fitting were performed using XLfit4 and
GraphPad Prism4 software.
PDK1 in Vitro Assay. The PDK1 kinase was tested by using
the Z0-LYTE biochemical assay technology from Invitrogen
(PV 3180). The assay was performed in Corning 3676 black
384-well assay plates. The final 10 μL of the kinase reaction
consisted of 9.75-49.4 ng of PDK1 and 2 μM substrate Ser/
Thr 07 in 50 mM of HEPES buffer at pH 8.0, containing
0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, and 0.01%
NaN3. ATP concentration at the Km value was used. After the
1 h kinase reaction incubation, 5 μL of a 1:32768 dilution of
development reagent A was added. After the development
reaction, where a site-specific protease recognizes and cleaves
the nonphosphorylated peptide, a ratiometric read-out of
the donor emission (coumarin, 445 nm) over the acceptor
(fluorescein, 520 nm) was detected by a PerkinElmer EnVi-
sion fluorescence plate reader (Waltham, MA). Data analysis
and curve fitting were performed using XLfit4 and GraphPad
Prism4 software.
cultured in RPMI plus GlutaMAX medium (Gibco) plus 10% FBS
and 1% penicillin/streptomycin.
Approximately 3000 cells were seeded into individual wells of
a 96-well tissue culture plate and incubated for 24 h. Cells were
replenished with fresh medium (0.1 mL/well) and exposed
to triplicates of different concentration solutions (from 0.5 to
100 μM) of test compounds. The analyzed inhibitors were
dissolved in DMSO reaching a final DMSO concentration of
0.5%. After incubation for 72 h at 37 °C and 5% CO2, cell
viability was assessed using ATPlite assay from Perkin-Elmer
(Waltham, MA). Viability was normalized to control cells which
were treated with the vehicle, DMSO. The reported IG50 values
were calculated by Prism5 (GraphPad).
3D Culture Assay. U87 cells were induced to form spheroids
via a hanging drop method.34 Cells were plated at 200 cells per
well (20 μL) of a Nunc-60 well microwell MiniTray (polystyrene).
The trays were covered, inverted, and placed in a humidity
chamber for 5 days until one spheroid formed in each well. The
spheroids were then transferred into a 48-well plate coated in
1% low melting point agarose to prevent them from adhering.
The spheroids were measured, and DMSO or drug was added
every 24 h for 6 days. Spheroid length and width (measured
with an optical micrometer) were used to calculate spheroid
volumes (μm3).
Microsomal Stability Assay (Rat Liver Microsome Assay).
Test compound solutions were incubated with rat liver micro-
somes (RLM) for 60 min at 37.5 °C. The final incubation
solutions contained 4 μM test compound, 2 mM NADPH,
1 mg/mL (total protein) microsomes, and 50 mM phosphate
(pH 7.2). Compound solutions, protein, and phosphate were
preincubated at 37.5 °C for 5 min, and the reactions were
initiated by the addition of NADPH and incubated for 1 h at
37.5 °C. Aliquots were taken at 15 min time points and quenched
with the addition of methanol containing internal standard.
Following protein precipitation and centrifugation, the samples
were analyzed by LC-MS. Test compounds were run in dupli-
cate with two control compounds of known half-life.
Plasma Stability Assay. Test compound solution was incu-
bated (1 μM, 2.5% final DMSO concentration) with fresh rat
plasma at 37 °C. The reactions were terminated at 0, 30, and
60 min by the addition of 2 volumes of methanol containing
internal standard. Following protein precipitation and centri-
fugation, the samples were analyzed by LC-MS. The percentage
of parent compound remaining at each time point relative to the
0 min sample is calculated from peak area ratios in relation to
the internal standard. Compounds were run in duplicate with a
positive control known to be degraded in plasma.
Molecular Modeling. Molecular modeling studies were con-
ducted on a Linux workstation and a 64 3.2-GHz CPUs Linux
cluster. Docking studies were performed using the X-ray co-
ordinates of the p110alpha (H1037R) mutant in complex with
niSH2 of p85alpha and the drug wortmannin (PDB id: 3hhm).24
The PI3KR crystal structure was extracted from the Protein
Data Bank, and the complexed ligand was used to define the
binding site for docking of small molecules. The genetic algorithm
(GA) procedure in the GOLD docking software performed flex-
ible docking of small molecules whereas the protein structure was
static.36-38 For each compound, 20 solutions were generated and
subsequently ranked according to GoldScore. Molecular surfaces
were generated with MOLCAD38 and docked structures analyzed
with Sybyl (Tripos Inc., St. Louis, MO).
Acknowledgment. We gratefully acknowledge financial
support from the NIH (Grant P01CA128814 to M.P.).
Antiproliferative Assay. All human cancer cell lines were
obtained from the American Type Culture Collection (Manassas,
VA, USA) and were maintained in 5% CO2 at 37 °C. Human
cervical carcinoma (HeLa) and U87 glioblastoma cells were grown
in Dulbecco’s modified Eagle’s medium (DMEM; Cellgro) supple-
mented with 10% fetal bovine serum (FBS; Omega Scientific) and
1% penicillin/streptomycin (Omega Scientific). MDA-MB-231 and
MCF7 breast cancer cells and PC3 prostate cancer cells were
Supporting Information Available: A list of 314 kinases used
for the selectivity profile, graphs reporting plasma and micro-
somal stability of compounds 10 and 13, and docking studies
with compound 20. This material is available free of charge via
the Internet at http://pubs.acs.org.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8375
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