Discovery of potent and selective inhibitors of the mammalian target of rapamycin (mTOR) kinase

Article abstract of DOI:10.1021/jm9012642

The mammalian target of rapamycin (mTOR) is a central regulator of cell growth, metabolism, and angiogenesis and an emerging target in cancer research. High throughput screening (HTS) of our compound collection led to the identification of 3-(4-morpholin-4-yl-1-piperidin-4-yl-1H-pyrazolo[3,4-d] pyrimidin-6-yl)phenol (5a), a modestly potent and nonselective inhibitor of mTOR and phosphoinositide 3-kinase (PI3K). Optimization of compound 5a, employing an mTOR homology model based on an X-ray crystal structure of closely related PI3Kγ led to the discovery of 6-(1H-indol-5-yl)-4-morpholin-4-yl-1-[1- (pyridin-3-ylmethyl)piperidin-4-yl]-1H-pyrazolo[3,4-d]pyrimidine (5u), a potent and selective mTOR inhibitor (mTOR IC₅₀ = 9 nM; PI3Kα IC ₅₀ = 1962 nM). Compound 5u selectively inhibited cellular biomarker of mTORC1 (P-S6K, P-4EBP1) and mTORC2 (P-AKT S473) over the biomarker of PI3K/PDK1 (P-AKT T308) and did not inhibit PI3K-related kinases (PIKKs) in cellular assays. These pyrazolopyrimidines represent an exciting new series of mTOR-selective inhibitors with potential for development for cancer therapy. 2009 American Chemical Society.

Full text of DOI:10.1021/jm9012642

J. Med. Chem. 2009, 52, 7081–7089 7081
DOI: 10.1021/jm9012642
Discovery of Potent and Selective Inhibitors of the Mammalian Target of Rapamycin (mTOR) Kinase
Pawel Nowak,*,† Derek C. Cole,† Natasja Brooijmans,§ Matthew G. Bursavich,† Kevin J. Curran,† John W. Ellingboe,†
James J. Gibbons,‡ Irwin Hollander,‡ YongBo Hu,§ Joshua Kaplan,† David J. Malwitz,† Lourdes Toral-Barza,‡
Jeroen C. Verheijen,† Arie Zask,† Wei-Guo Zhang,‡ and Ker Yu‡
†Chemical Sciences and ‡Discovery Oncology and §Structural Biology, Wyeth Research, 401 N. Middletown Road, Pearl River, New York 10965
Received June 23, 2009
The mammalian target of rapamycin (mTOR) is a central regulator of cell growth, metabolism, and
angiogenesis and an emerging target in cancer research. High throughput screening (HTS) of our
compound collection led to the identification of 3-(4-morpholin-4-yl-1-piperidin-4-yl-1H-pyrazolo[3,4-
d]pyrimidin-6-yl)phenol (5a), a modestly potent and nonselective inhibitor of mTOR and phospho-
inositide 3-kinase (PI3K). Optimization of compound 5a, employing an mTOR homology model based
on an X-ray crystal structure of closely related PI3Kγ led to the discovery of 6-(1H-indol-5-yl)-4-
morpholin-4-yl-1-[1-(pyridin-3-ylmethyl)piperidin-4-yl]-1H-pyrazolo[3,4-d]pyrimidine (5u), a potent
and selective mTOR inhibitor (mTOR IC50 = 9 nM; PI3KR IC50 = 1962 nM). Compound 5u selecti-
vely inhibited cellular biomarker of mTORC1 (P-S6K, P-4EBP1) and mTORC2 (P-AKT S473) over the
biomarker of PI3K/PDK1 (P-AKT T308) and did not inhibit PI3K-related kinases (PIKKs) in cellular
assays. These pyrazolopyrimidines represent an exciting new series of mTOR-selective inhibitors with
potential for development for cancer therapy.
Introduction
pathway and cytoskeletal network in human cells. mTORC1
and mTORC2 are critical mediators of the PI3K/AKT and
Ras/MAPK signaling pathways that are frequently deregu-
lated in human tumors.7-9
The mammalian target of rapamycin (mTORa) is an
unconventional high molecular weight serine/threonine pro-
tein kinase that regulates cell growth, proliferation, metabo-
lism, and angiogenesis.1-5 mTOR is the founding member of
the PI3K-related kinase (PIKK) family6 that also includes
DNA-PK, ATM, ATR, and SMG-1. The PIKKs collectively
play important and diverse roles in cell growth and surveil-
lance of the genome and transcriptome. The catalytic sites of
the PIKK family are similar to that of PI3K but differ
significantly from the rest of the human kinome. mTOR
primarily resides in two functional multiprotein comp-
lexes, mTOR complex 1 (mTORC1) and mTOR complex 2
(mTORC2), governing subcellular functions and/or substrate
specificity. mTORC1 is a key regulator of cellular translation,
while mTORC2 is anticipated to regulate the AKT survival
Rapamycins (rapamycin and its analogs) are allosteric
inhibitors that bind (in complex with FKBP12) to mTOR at
the FKBP12-rapamycin binding domain (FRB domain)
adjacent to the catalytic site of mTOR.10,11 These agents are
used clinically for treating a subset of tumors.12-14 However,
only the FRB domain of mTORC1 is inhibited by the
rapamycin-FKBP12 complex while that of mTORC2 is
insensitive to these inhibitors.15,16 Inhibition of mTORC1
by the rapamycins can also induce feedback activation of
PI3K and ERK/MAPK in some settings.7,8,17 Therefore,
although the rapalogs have validated mTOR clinically as a
cancer target, their molecular mechanism of action does not
fully exploit the antitumor potential of mTOR targeting
in cancer. Small molecule ATP-competitive inhibitors of
mTOR kinase should provide global inhibition of both
mTORC1 and mTORC2 and hence minimize the feedback
activation of PI3K signaling (via inhibition of the AKT
pathway) to achieve more robust antitumor efficacy. The
PI3K/mTOR pathway has been a subject of widespread and
intense drug discovery efforts for a number of years and
potent dual/pan-PI3K inhibitors have been reported,18-20
but only recently have reports describing mTOR selective
compounds appeared.21-24
*To whom correspondence should be addressed. Phone: 845 602-
4303. Fax: 845 602-5561. E-mail: nowakp@wyeth.com.
a Abbreviations: mTOR, mammalian target of rapamycin;
mTORC1, mammalian target of rapamycin complex 1; HTS, high-
throughput screening; PI3K, phosphoinositide 3-kinase; PIKK, phos-
phoinositide 3-kinase related kinases; DNA-PK, DNA-dependent pro-
tein kinase; ATM, ataxia-telangiectasia mutated; ATR, ataxia-
telangiectasia and Rad3-related; SMG-1, suppressor of morphogenesis
in genitalia-1; Ras, rat sarcoma; MAPK, mitogen-activated protein
kinase; FRB, FKBP12-rapamycin binding; ERK, extracellular signal-
regulated kinase; DELFIA, dissociation-enhanced lanthanide fluor-
escent immunoassay; PRKCN, protein kinase C, NU; CLK1, CDC-
like kinase 1; STK25, serine/threonine kinase 25; JAK2, Janus kinase 2;
AMPK, AMP-activated protein kinase; PAK2, P21-activated kinase 2;
CSK, c-src tyrosine kinase; FGFR1, fibroblast growth factor receptor 1;
MST4, mammalian sterile twenty-like 4; TEK, TEK tyrosine kinase,
endothelial; NTRK2, neurotrophic tyrosine kinase receptor type 2;
PAK7, P21(CDKN1A)-activated kinase 7; SGKL, serum and gluco-
corticoid-inducible kinase-like kinase; HWT, 17-hydroxywortmannin;
Rapa, rapamycin.
Herein, we report the identification, characterization, and
hit-to-lead optimization of a series of pyrazolopyrimidine
mTOR kinase inhibitors exemplified by 4-(6-(1H-indol-5-yl)-
1-(1-(pyridin-3-ylmethyl)piperidin-4-yl)-1H-pyrazolo[3,4-d]-
pyrimidin-4-yl)morpholine (5u). This small molecule lead
(MW = 495, clogP = 2.1) has low nanomolar mTOR enzyme
inhibitory activity, inhibits signaling functions of both
r
2009 American Chemical Society
Published on Web 10/19/2009
pubs.acs.org/jmc

7082 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Nowak et al.
Scheme 1a
Figure 1. Putative binding mode of 5a with the mTOR homology
model. In this model, the morpholine of 5a forms a key hinge-region
hydrogen bond with the backbone of Val2240. At the rear of the
binding pocket, Asp2195 is shown making a second key hydrogen
bond to the phenol of 5a. Residues within a 3 A radius of 5a that are
conserved between the mTOR active site and PI3KR are shown in
green and include the catalytic Lys2187. The only two residues that
change (highlighted in orange) are Trp2239 and Leu2354. The core
forms hydrophobic contacts with Ile2163 and Leu2185 above the
plane of the inhibitor and with Met2345 and Ile2356 below the plane
of the inhibitor (not shown for clarity).
a Reagents and conditions: (a) POCl3, DMF, reflux, 60%; (b) 1-benz-
yl-4-hydrazinylpiperidine dihydrochloride, Et3N, EtOH, -78 to 0°C,
82%; (c) morpholine, EtOH, 61%; (d) ArB(OH)2 or ArB(OCMe2)2
Pd(PPh3)4, Na2CO3, dioxane, water, 100°C; (e) H2, Pd(OH)2/C (10%),
MeOH; (f) R2COCl, Et3N, THF or R2CHO, NaBH3CN, AcOH (cat.),
MeOH.
mTORC1 and mTORC2 complexes in cellular setting, and is
selective over PI3KR and other kinases.
Table 1. Enzyme Inhibition Data for 5a-f against mTOR and PI3KRa
Chemistry
The synthesis of 5a and related compounds begins with
treatment of barbituric acid (1) with phosphorus oxychloride
(POCl3) in dimethylformamide, resulting in formation of
2,4,6-trichloropyrimidine, which undergoes formylation lead-
ing to aldehyde 2 (Scheme 1). Reaction with substituted
hydrazine produces the dichloropyrazolopyrimidine 3. The
4-chloro substituent on dichloropyrazolopyrimidines 3 is
selectively displaced by morpholine, leading to 4. Finally,
Suzuki coupling with boronic acid derivatives gives the target
compounds 5. In cases where the piperidinyl substitution was
varied, the benzyl protecting group is removed by hydrogena-
tion and the resulting secondary amine acylated or reductively
alkylated (steps e and f). Compounds 5a-v were assayed for
inhibitory activity against mTOR (dissociation-enhanced
lanthanide fluorescent immunoassay assay) and PI3K iso-
forms (fluorescence polarization assay).
Results and Discussion
An mTOR inhibitor high-throughput screen (HTS) led to
the identification of compound 5a, a promising “leadlike”
starting point for further optimization because of its favorable
properties: low MW (297), clogP (1.86), high ligand effici-
ency25 (0.42), low LELP26 (clogP/(ligand efficiency) = 4.4),
and submicromolar potency (IC50 = 215 nM). However,
compound 5a was 6-fold more potent for PI3KR than mTOR
kinase and alerted us to a potential hurdle of achieving
selectivity versus this closely related member of PI3K family
of kinases.
In order to better understand the binding of pyrazolopyr-
imidins to the mTOR binding site, we have constructed an
mTOR binding model based on an X-ray crystal structure of
related PI3Kγ kinase. Thecatalytic sites ofmTOR and PI3KR
differ only by two amino acid residues within a radius of 3 A
around 5a, namely, Trp2239 to Val850 and Leu2354 to
Phe930, respectively (Figure 1). Both these residues are near
a The asterisk (/) indicates that IC50 determinations are the mean of
two to three independent measurements with standard error of <20%.
the morpholine but do not make specific hydrogen bonding
interactions with the compound.
We began modifying our lead 5a by derivatizing the
piperidine NH group. The acetylpiperidine derivative 5b had

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7083
Table 2. Enzyme Inhibition Data for 5g-s against mTOR and PI3KRa
Figure 2. Predicted binding mode of 5u with the mTOR homology
model. The predicted binding mode of 5u with mTOR shows the
hydrogen bonding interaction of the morpholine to Val2240 and the
indole-NH to Asp2195. The pyridin-3-ylmethyl tailpiece of 5u in the
front of the binding site interacts with a region that contains
Leu2249 and Ala2248 in mTOR. The corresponding residues in
PI3KR are Met858 and Gln859, respectively.
activity and, for the first time, more than 10-fold selectivity
versus PI3KR. The corresponding benzyl (5e) and 3-pyridyl-
methyl (5f) analogues were somewhat less potent and selec-
tive.
Simultaneous with the optimization of the piperidine sub-
stitution, the benzylanalogue5ewas chosen asa starting point
to explore the SAR of the pyrazolopyrimidine aryl substitu-
ent. Moving the phenolic hydroxyl group to the 2- or 4-
position of the aryl ring (5g and 5h) led to a loss in activity
(see Table2) which isexplainedbythelossof the key hydrogen
bondto Asp2195(Figure1). Themetabolic liability associated
with the presence of a phenol group, as evidenced by short rat
hepatocyte clearance half-life (t1/2 = 13 min for 5e), prompted
a search for suitable replacements. In the pyridyl and quino-
linyl series (5i-l), compounds having a nitrogen H-bond
acceptor in the 3- position showed modest potency. The
5-benzimidazolonyl derivative 5m was modestly active
and >20-fold selective over PI3KR. Further investigation of
analogous indole substituents (5n-r) led to the identification
of the 5-indolyl derivative 5p, which was equipotent with the
original phenol 5e and had >10-fold selectivity. The N-Me
indole 5s was almost 6-fold less active, suggesting that either
the N-H of 5p is involved in the same hydrogen bonding
interaction as the phenol with Asp2195, as predicted by
modeling (Figure 2), or the N-Me results in a steric clash with
the protein, resulting in the lower activity of 5s.
Having found that the indole substituent leads to potent
and selectivemTORinhibitors, wefurtherexploredpiperidine
substituents (Table3). Compound5t, having an unsubstituent
piperidine group, had only modest potency; however, sub-
stitution of the piperidine with pyridin-3-ylmethyl (5u) and
nicotinoyl (5v) groups gave, as expected (cf. 5d, and 5f,
Table1), compoundsof excellent activityand highlyamplified
selectivity vs PI3KR (>1000-fold). The selectivity increases
associated with modifications of groups in positions 1 and 6 of
the pyrazolopyrimidine appear to be cumulative. This selec-
tivity enhancement may be a result of accessing the more
hydrophobic region in mTOR (Ala2248 and Leu2249 versus
Met858 and Gln859 in PI3KR).
a The asterisk (/) indicates that IC50 determinations are the mean of
two to three independent measurements with standard error of <20%.
5-fold improved activity and almost equal potency in the two
enzymes (see Table 1). Introduction of the benzoyl (5c) and
nicotinyl (5d) groups led to compounds with low nanomolar

7084 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Nowak et al.
Table 3. Enzyme Inhibition Data for 5t-v against mTOR and PI3KRa
Figure 4. Inhibition of cellular mTORC1 and mTORC2 signaling
functions in actively proliferating cancer cells. Renal tumor A498
cells were treated in growth medium for 6 h with DMSO or the
indicated concentrations of 5u or 1 μM rapamycin (Rapa) or 17-
hydroxywortmannin (HWT). Total cell lysates were prepared and
subject to immunoblotting with antibodies against P-S6K1(T389),
P-4EBP1(T70), P-4EBP1(T36/47), P-AKT(S473). The blots were
also stained with Ponceau S, a total protein dye, for sample loading
control.
a The asterisk (/) indicates that IC50 determinations are the mean of
two to three independent measurements with standard error of <20%.
Figure 5. Selective targeting of mTOR versus PI3K in PTEN-
negative cancer cells. U87MG glioma cells were treated in growth
medium for 6 h with DMSO or the indicated concentrations of 5f or
5u or 1 μM rapamycin (Rapa) or 17-hydroxywortmannin (HWT).
Total cell lysates were prepared and subject to immunoblotting with
antibodies against P-S6K1 (T389), P-AKT (S473), and P-AKT
(T308).
Rapamycin inhibited mTORC1 biomarkers P-S6K1(T389)
and P-4EBP1(T70) but not the rapamycin-resistant mTORC1
biomarkerP-4EBP1(T37/46) or the biomarkerP-AKT(S473).
In contrast, the global mTOR inhibitor 5u inhibited both
mTORC1 and mTORC2 biomarkers in a dose-dependent
manner.
Treatment of PTEN-negative, PI3K/AKT-hyperactive
U87MG glioma cells with 5u selectively inhibited P-S6K-
(T389) and P-AKT(S473) but not P-AKT(T308), an
acti-
vity biomarker of PI3K/PDK1. In contrast, a nonselective
inhibitor 5f and the PI3K inhibitors HWT all indiscriminately
inhibited P-AKT(S473) and P-AKT(T308) (Figure 5). Thus,
both the biochemical and cellular results support 5u as an
ATP-competitive and selective inhibitor of mTOR kinase.
Figure 3. ATP competitive inhibition of mTOR kinase activity.
Compound 5u was assayed in an inhibitor versus ATP matrix
competition. The initial enzyme rate (V0) against 0, 0.009, 0.018,
0.027 μM 5u at various concentrations of ATP was measured to
generate the double-reciprocal plot.
Compound 5u was the earliest example in the “indole”
series to display excellent selectivity over PI3KR, and hence, it
was the most fully characterized. In the Invitrogen kinase
panel27 compound 5u had less than 50% inhibition at 2 μM
against 219 of 231 kinases and had modest activity (50-85%
inhibition at 2 μM) against 12 kinases, namely, PRKCN,
CLK1, STK25, JAK2, AMPK, PAK2, CSK, FGFR1,
MST4, TEK, NTRK2, PAK7, and SGKL.
The ATP-competitive nature of inhibition of compound 5u
was confirmed in a Lineweaver-Burk double-reciprocal
dose-response plot (Figure 3) of the mTOR enzyme rates
against the ATP concentration.
Conclusion
Through mTOR inhibitor HTS, we identified a “leadlike”
small molecule inhibitor of mTOR kinase. The hit-to-lead
optimization was guided by a PI3Kγ based mTOR homology
model and led to the identification of an indolopyrazolopyr-
imidine series, exemplified by 4-(6-(1H-indol-5-yl)-1-(1-(pyri-
A498 renal cancer cells treated with 5u or rapamycin
a distinctive biochemical profile (Figure 4).
showed

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7085
din-3-ylmethyl)piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-
4-yl)morpholine (5u), a 9 nM ATP-competitive inhibitor of
mTOR kinase with >200-fold selectivity over PI3KR, which
showed only modest activity against 12 out of the 231 protein
kinases. 5u selectively inhibited mTOR over PI3KR and the
related PIKK21 in cellular settings and inhibited proliferation
of a panel of histologically diverse cancer cell lines.21 The
results of continued optimization and in vivo efficacy studies
of this promising series will be disclosed.
added, and the suspension was stirred for 15 min. The yellow
precipitate was filtered off, dissolved in chloroform (200 mL),
and washed with water. The solution was dried with magnesium
sulfate and filtered through small silica pad with the aid of
dichloromethane. The solvents were evaporated, and the residue
was triturated with hexanes. White crystals were filtered off and
dried in high vacuum (8.00 g, 60%). Mp: 130-132 °C. 1H NMR
(300 MHz, chloroform-d): δ 10.42 (s). 13C NMR (75 MHz,
chloroform-d): δ 184.6, 164.1, 161.6. 122.9. IR: 2964, 1702,
1054. 1036 cm-1. MS (ES-): m/z 211.8 [M]þ (23%), 209.8
[M]þ (26%), 192.8 (69%), 190.8 (100%). Anal. Calcd for
C5HCl3N2O: C 28.40; H 0.48; N 13.25. Found: C 28.51; H
0.77; N 13.28.
Experimental Methods
1-(1-Benzylpiperidin-4-yl)-4,6-dichloro-1H-pyrazolo[3,4-d]pyri-
midine (3). 2,4,6-Trichloro-5-pyrimidinecarboxaldehyde (2)
(5.00 g, 23.65 mmol) was dissolved in ethanol (100 mL). The
solution was cooled to -78 °C and 1-benzyl-4-hydrazinylpiper-
idine dihydrochloride (4.12 g, 23.65 mmol) added followed by
triethylamine (9.9 mL, 70.90 mmol). The solution was stirred
at -78 °C for 0.5 h and then warmed up to 0 °C and stirred at this
temperature for 1.5 h. Ethyl acetate (500 mL) and saturated
sodium bicarbonate (100 mL) were added, and the organic
phase was separated, dried with anhydrous magnesium sulfate,
and concentrated (bath temperature of <15 °C). The residue
was dissolved in ethyl acetate (100 mL), and the solution was
filtered through a plug of silica gel followed by elution with more
ethyl acetate (20 mL). Removal of solvent (rotovap bath tem-
perature of <15 °C) gave a thick yellow oil (7.01 g, 82%) which
was unstable at room temperature and was immediately used in
the next step. Analytical sample was converted to hydrochloride
salt by dissolution in ethanol followed by saturation with
gaseous hydrogen chloride and removal of solvent. The sample
was stable enough to collect analytical data. Mp >230 °C (dec).
1H NMR (300 MHz, DMSO-d6): δ 10.78 (br s, 1H), 8.61 (s, 1H),
7.65-7.59 (m, 2H), 7.51-7.46 (m, 3H), 5.12-5.02 (m, 1H), 4.32
(s, 2H), 3.53-3.43 (m, 2H), 3.61-3.21 (m, 2H), 2.59-2.46 (m,
2H), 2.21-2.12 (m, 2H). 13C NMR (75 MHz, DMSO-d6): δ
155.3, 154.4, 153.9, 133.6, 131.8, 130.0, 129.8, 129.1, 113.2, 59.3,
52.2, 50.4, 28.3. IR: 3432, 1026 cm-1. MS (ESþ): m/z 362.1 [M þ
H]þ (100%), 364.1 [M þ H]þ (48%). Anal. Calcd for
NMR spectra were recorded on a Bruker Avance 300 MHz
spectrometer. All chemical shifts are reported in parts per million
(δ) relative to tetramethylsilane. The following abbreviations are
used to denote signal patterns: s = singlet, d = doublet, t =
triplet, m = multiplet, and br = broad. The mass spectral data
were determined on an Agilent 1100 series LC/MSD, and HRMS
was carried out on a Bruker APEX II 9.4 T mass spectrometer.
Silica gel flash chromatrography was carried out on a Teledyne
Isco, Inc. CombiFlash Companion using RediSep silica gel
cartridges. Reverse phase HPLC purifications were performed
on a Gilson preparative HPLC system controlled by Unipoint
software using either Phenomenex Gemini 100 mm ꢀ 21.2 mm or
Waters Xterra Prep 5 μM, 100 mm ꢀ 19 mm C18 columns and in a
10-90% acetonitrile in water solvent gradient with 0.02% TFA
buffer or with 0.02% ammonium hydroxide buffer. The purity
of all compounds was g95%. The purity was analyzed using
Agilent Technologies 1200 series LCMS with Agilent XDB-C18
30 mm ꢀ 2.1 mm, 1.8 μm particle size column: solvent gradient =
90% A at 0 min, 5% A at 2.6 min, 5% A at 3 min; solvent
A = 0.05% modifier in water; solvent B = 0.05% modifier in
ACN; flow rate 1 mL/min. Modifiers used were NH4OH or
HCO2H.
1-Benzyl-4-hydrazinylpiperidine Dihydrochloride.28 Benzoic
hydrazide (5.47 g, 40.2 mmol) was dissolved in methanol
(30 mL) and 1-benzylpiperidin-4-one (7.60 g, 40.2 mmol) added,
and the solution was stirred for 1 h at 30 °C and for 4 h at 60 °C.
The solution was cooled in an ice bath, and sodium borohydride
(1.52 g, 40.2 mmol) was slowly added. After 2 h the solvent was
evaporated and the residue was partitioned between chloroform
(50 mL) and saturated sodium bicarbonate (50 mL). The chloro-
form fraction was separated, dried with magnesium sulfate, and
concentrated giving yellow oil (20.0 g). Water (16 mL) and
concentrated hydrochloric acid (28 mL) were added. The solu-
tion was stirred for 15 min and the residual chloroform removed.
The aqueous solution was refluxed overnight and then cooled in
an ice bath, and the precipitated benzoic acid was filtered off.
The filtrate was concentrated (∼20 mL volume), and ethanol
(100 mL) was added. The solution was cooled, and white crystals
were filtered off and washed with a small portion of ethanol
followed by diethyl ether. The solid was dried in high vacuum
overnight (5.30 g, 76%). Mp: 200 °C. 1H NMR (300 MHz,
DMSO-d6): δ 11.19-11.04 (br s, 1H), 7.67-7.60 (m, 2H),
7.47-7.42 (m, 3H), 4.31-4.22 (m, 2H), 3.40-3.28 (m, 2H),
3.18-3.03 (m, 2H), 3.02-2.89 (m, 1H), 2.17-1.99 (m, 3H),
1.85-1.72 (m, 1H). 13C NMR (75 MHz, DMSO-d6): δ 131.8,
131.7, 130.2, 130.3, 129.7, 129.0, 58.7, 53.8, 49.6, 46.2, 26.9, 24.0.
IR: 2964, 2464, 1063, 1037 cm-1. MS (ESþ): m/z 206.2 [M þ H]þ
(100%). HRMS: m/z 206.164 46, calcd [M þ H]þ 206.164 82, rel
intens 100%, error -0.36 mAmu.
C17H18Cl3N5 0.1EtOH: C 51.22; H 4.65; N 17.36. Found: C
3
51.58; H 4.28; N 16.87.
4-(1-(1-Benzylpiperidin-4-yl)-6-chloro-1H-pyrazolo[3,4-d]pyrimi-
din-4-yl)morpholine (4). 1-(1-Benzylpiperidin-4-yl)-4,6-dichloro-
1H-pyrazolo[3,4-d]pyrimidine (3) (7.00 g 19.32 mmol) was dis-
solved in ethanol (100 mL). The solution was cooled to 0 °C, and
morpholine (1.68 mL, 19.32 mmol) was added. The solution was
stirred for 0.5 h at 0 °C and 0.5 h at room temperature, the
solvent was evaporated, and the residue was dissolved in ethyl
acetate (200 mL). The solution was washed with saturated
sodium bicarbonate (50 mL), the insoluble particles were re-
moved by filtration and the phases separated, and the organic
solution was dried with magnesium sulfate. The solvents
were evaporated, and the residue was redissolved in ethyl
acetate (50 mL) and filtered through a small (5 g) pad of
silica with ethyl acetate elution. The solvent was evaporated
leaving thick oil. The oil was dissolved in diethyl ether (50 mL)
which led to spontaneous crystallization. The yellow crystals
were filtered off and dried in high vacuum overnight (7.98 g,
61%). Mp 144-146 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.35
(s, 1H), 7.35-7.31 (m, 4H), 7.30-7.27 (m, 1H), 4.64-4.50
(m, 1H), 3.91-3.85 (m, 4H), 3.73-3.69 (m, 4H), 3.53 (s, 2H),
2.97-2.84 (m, 2H), 2.22-2.02 (m, 4H), 1.87-1.78 (m, 2H). 13C
NMR (75 MHz, DMSO-d6): δ 156.5, 156.0, 153.9, 138.4, 133.2,
128.5, 128.0, 126.7, 98.7, 65.6, 61.7, 53.8, 51.9, 45.8 (low
intensity), 30.8. IR: 32954, 1554, 1305, 945 cm-1. MS (ESþ):
m/z 413.2 [M þ H]þ (100%). Anal. Calcd for C21H25ClN6O: C
61.08; H 6.10; N 20.35. Found: C 60.98; H 5.94; N 20.33.
4-(1-(1-Benzylpiperidin-4-yl)-6-(1H-indol-5-yl)-1H-pyrazolo[3,
4-d]pyrimidin-4-yl)morpholine (5p). 4-(1-(1-Benzylpiperidin-4-yl)-
2,4,6-Trichloropyrimidine-5-carbaldehyde (2).29 Phosphorus
oxychloride (46 mL) was added dropwise over 20 min to
dimethylformamide cooled in an ice bath. The solution was
stirred for further 20 min after, and barbituric acid (8.00 g,
62.4 mmol) was added in one portion. The solution was stirred
for 1 h at room temperature, and then it was heated to 100 °C
for 4 h. The solution was evaporated to half-volume, and while
still warm it was poured onto ice (200 g). Water (200 mL) was

7086 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Nowak et al.
6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)morpholine (4) (1.30 g,
3.15 mmol) was dissolved in dioxane (5 mL). 1H-Indol-5-ylboronic
acid (633 mg, 3.94 mmol) followed by tetrakis(triphenylpho-
sphine)palladium(0) (273 mg, 0.24 mmol) and 2 M aquieous
sodium carbonate (5 mL) was added. The solution was degassed
with a stream of nitrogen, and it was heated in a microwave reactor
for 3 min at 120 °C. The reaction mixture was partition between
ethyl acetate (25 mL) and 1 M aqueous sodium hydroxide (25 mL),
and the organic fraction was separated, dried with magnesium
sulfate, and evaporated. The oily residue was purified by flash
chromatography (gradient of ethyl acetate to ethyl acetate/
methanol/triethylamine, 8:1:1). Removal of solvent and drying in
high vacuum gave an off-white, amorphous solid (1.31 g, 90%). 1H
NMR (300 MHz, DMSO-d6): δ 11.25 (s, 1H), 8.71 (s, 1H),
8.31-8.22 (m, 2H), 7.46 (d, J = 9.0 Hz, 1H), 7.41-7.23 (m, 6H),
6.57 (s, 1H), 4.90-4.86 (m, 1H), 4.07-3.98 (m, 4H), 3.83-3.76 (m,
4H), 3.56 (s, 2H), 3.02-2.94 (m, 2H), 2.31-2.13 (m, 4H),
1.96-1.85 (m, 2H). 13C NMR (75 MHz, DMSO-d6): δ 160.7,
156.5, 154.6, 138.4, 137.2, 132.5, 128.8, 128.6, 128.0, 127.4, 126.8,
126.0, 121.6, 120.6, 110.8, 102.1, 98.4, 65.8, 61.8, 53.3, 52.1, 44.9
(low intensity), 31.0. IR 2928, 1580, 1065, 1030, 1005 cm-1. MS
(ESI): m/e 247.6 (7%), 494.3 [M þ H]þ (100%). Anal. Calcd for
mL, 0.60 mmol). The solution was stirred for 1 h. Ethyl acetate
(10 mL) and saturated aqueous sodium bicarbonate (10 mL)
were added, and the organic fraction was separated, dried with
anhydrous magnesium sulfate, and evaporated. The residue was
purified by preparative HPLC.
3-(4-Morpholin-4-yl-1-piperidin-4-yl-1H-pyrazolo[3,4-d]pyrimi-
din-6-yl)phenol (5a). This compound was prepared in an analo-
gous manner to 5p and 5v, using 3-hydroxyphenylboronic acid
instead of 1H-indol-5-ylboronic acid. Purification was by pre-
parative reverse phase HPLC with 0.02% ammonium hydroxide
buffer. 1H NMR (300 MHz, DMSO-d6): δ 9.57 (br s, 1H),
8.31-8.27 (m, 1H), 7.90-7.86 (m, 2H), 7.66-7.54 (m, 1H), 7.28
(dd, J = 8.0, 8.0 Hz, 1H), 6.90-6.84 (m, 1H), 4.93-4.80 (m, 1H),
4.03-3.69 (m, 8H), 3.18-3.03 (m, 2H), 2.79-2.65 (m, 2H),
2.14-1.78 (m, 4H). MS (ESI): m/e 381.2 [M þ H]þ (100%).
HRMS m/z 381.203 63, calcd [M þ H]þ 381.203 35, rel intens
100%, error 0.28 mAmu.
3-[1-(1-Acetylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,
4-d]pyrimidin-6-yl]phenol (5b). Procedure C was used. The pi-
peridine substrate for procedure C was prepared in an analo-
gous manner to 5p and 5t, using 3-hydroxyphenylboronic acid
instead of 1H-indol-5-ylboronic acid. The product was purified
by preparative reverse phase HPLC with 0.02% ammonium
hydroxide buffer, giving an off-white amorphous solid. 1H
NMR (300 MHz, DMSO-d6): δ 8.33-8.29 (m, 2H), 7.83-7.74
(m, 1H), 7.66-7.53 (m, 1H), 7.23-7.16 (m, 1H), 6.84-6.78 (m,
1H), 5.09-4.90 (m, 1H), 4.54-4.44 (m, 1H), 4.02-3.87 (m, 5H),
3.81-3.66 (m, 6H), 2.05 (s, 3H), 2.08-1.80 (m, 4H). MS (ESI):
m/e 423.2 [M þ H]þ (100%), (M þ K)þ 461.2 (6%). HRMS m/z
423.213 69, calcd [M þ H]þ 423.213 92, rel intens 100%, error
0.23 mAmu.
3-[1-(1-Benzoylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo-
[3,4-d]pyrimidin-6-yl]phenol (5c). Procedure C was used. The
chloro substrate for procedure C was prepared in an analogous
manner to 5p and 5t, using 3-hydroxyphenylboronic acid in-
stead of 1H-indol-5-ylboronic acid. The product was purified
by preparative reverse phase HPLC with 0.02% ammonium
hydroxide buffer, giving an off-white amorphous solid. 1H
NMR (300 MHz, DMSO-d6): δ 9.55 (s, 1H), 8.34 (s, 1H),
7.94-7.87 (m, 2H), 7.48 (s, 5H), 7.29 (dd, J = 8.0, 8.0 Hz,
1H), 6.89 (dd, J = 2.0, 8.5 Hz, 1H), 5.19-5.04 (m, 1H),
4.74-4.55 (m, 4H), 4.06-3.97 (m, 5H), 3.21-3.03 (m, 1H),
2.20-1.87 (m, 5H). MS (ESI): m/e 485.2 [M þ H]þ (100%).
HRMS m/z 485.229 07, calcd [M þ H]þ 485.229 57, rel intens
100%, error 0.50 mAmu.
C29H31N7O 2/3H2O: C 68.89; H 6.45; N 19.39. Found: C 68.88; H
3
5.92; N 19.57.
4-(6-(1H-Indol-5-yl)-1-(piperidin-4-yl)-1H-pyrazolo[3,4-d]pyri-
midin-4-yl)morpholine (5t). 4-(1-(1-Benzylpiperidin-4-yl)-6-(1H-
indol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)morpholine (5p)
(1.00 g, 20.3 mmol) was dissolved in tetrahydrofuran-methanol
(50 mL, 1:1). Palladium hydroxide (0.75 g, 20% on carbon) was
added, and the solution was hydrogenated at atmospheric
pressure overnight. The catalyst was filtered off on a Cellite
pad, and the solvent was evaporated. The residue was purified
by flash chromatography (gradient of dichloromethane to
dichloromethane/methanol/triethylamine, 3:1:1). Removal of
solvent and drying in high vacuum gave an off-white, amor-
phous solid (0.45 g, 55%). 1H NMR (300 MHz, DMSO-d6): δ
11.25 (s, 1H), 8.71 (s, 1H), 8.31-8.24 (m, 2H), 7.45 (d, J = 8.5
Hz, 1H), 7.39 (dd, J = 2.5, 2.5 Hz, 1H), 6.57 (s, 1H), 4.94-4.81
(m, 1H), 4.05-3.97 (m, 4H), 3.49-3.77 (m, 4H), 3.15-3.05 (m,
2H), 2.77-2.64 (m, 2H), 2.13-1.96 (m, 3H), 1.88-1.78 (m, 2H).
13C NMR (75 MHz, DMSO-d6): δ 160.6, 156.5, 154.4, 137.3,
132.4, 128.9, 127.4, 126.0, 121.6, 120.6, 110.8, 102.1, 98.4, 65.9,
53.9, 45.3, 45.0 (low intensity), 32.5. IR: 3260, 2930, 1570 cm-1
.
MS (ESI): m/e 404.2 [M þ H]þ (100%). Anal. Calcd for
C22H25N7O 2H2O: C, 60.12; H, 6.65; N, 22.31. Found: C
3
60.53; H 6.25; N 22.5.
3-{4-Morpholin-4-yl-1-[1-(pyridin-3-ylcarbonyl)piperidin-4-yl]-
1H-pyrazolo[3,4-d]pyrimidin-6-yl}phenol (5d). Procedure C was
used. The chloro substrate for procedure C was prepared in an
analogous manner to 5p and 5t, using 3-hydroxyphenylboronic
acid instead of 1H-indol-5-ylboronic acid. The product was
purified by preparative reverse phase HPLC with 0.02% ammo-
nium hydroxide buffer, giving an off-white amorphous solid. 1H
NMR (300 MHz, DMSO-d6): δ 9.53 (s, 1H), 8.69-8.65 (m, 2H),
8.33 (s, 1H), 7.94-7.85 (m, 3H), 7.51 (dd, J = 4.5, 8.0 Hz, 1H),
7.28 (dd, 7.5, 7.5, 1H), 6.87 (dd, J = 3.5, 8.0 Hz, 1H), 5.17-5.07
(m, 1H), 4.68-4.57 (m, 1H), 4.04-3.97 (m, 4H), 3.82-3.75
(m, 4H), 3.25-3.11 (m, 3H), 2.33-1.85 (m, 4H). MS (ESI): m/e
157.0 (15%), 486.2 [M þ H]þ (100%). HRMS m/z 486.22447,
calcd [M þ H]þ 486.224 82, rel intens 100%, error 0.35.
3-[1-(1-Benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,
4-d]pyrimidin-6-yl]phenol (5e). Procedure A was used. Purifica-
tion was by preparative reverse phase HPLC with 0.02%
ammonium hydroxide buffer, giving an off-white amorphous
solid. 1H NMR (300 MHz, DMSO-d6): δ 9.51 (s, 1H), 8.30
(s, 1H), 7.89-7.84 (m, 2H), 7.38-7.32 (m, 4H), 6.89-6.85 (m,
1H), 4.83-4.74 (m, 1H), 4.03-3.96 (m, 4H), 3.81-3.76 (m, 4H),
3.57 (s, 2H), 3.02-2.94 (m, 2H), 2.27-2.15 (m, 4H), 1.96-1.85
(m, 2H). MS (ESI): m/e 413.2 (13%), 471.2 [M þ H]þ (100%).
HRMS m/z 471.25107, calcd [M þ H]þ 471.250 30, rel intens
100%, error 0.77 mAmu.
General Procedures. Procedure A: Suzuki Coupling. The
piperidine compound (0.25 mmol) was dissolved in dioxane
(2 mL), and boronic acid (0.25 mmol) followed by tetrakis-
(triphenylphosphine)palladium(0) (28 mg, 0.025 mmol) and 2 M
aquieous sodium carbonate (2 mL) was added. The solution was
degassed with a stream of nitrogen, and it was heated in a
microwave reactor for 3 min at 120 °C. The reaction mixture was
partitioned between ethyl acetate (10 mL) and saturated aqu-
eous sodium bicarbonate (10 mL). The organic fraction was
separated, dried with anhydrous magnesium sulfate, and eva-
porated. The residue was purified by preparative HPLC.
Procedure B: Reductive Amination. The piperidine compound
(0.25 mmol) was dissolved in methanol (10 mL), and 3-pyridi-
necarboxaldehyde (80 mg, 0.75 mmol) was added followed by
sodium cyanoborohydride (62 mg, 1.00 mmol) and acetic acid
(0.05 mL). The solution was stirred overnight. Solvent was
evaporated, and the residue was partitioned between ethyl
acetate (10 mL) and saturated aqueous sodium bicarbonate
(10 mL). The organic fraction was separated, dried with anhy-
drous magnesium sulfate, and evaporated. The residue was
purified by preparative HPLC.
Procedure C: Conversion to Amide. The piperidine compound
(0.25 mmol) was dissolved in tetrahydrofuran (5 mL), and acid
chloride (0.30 mmol) was added followed by triethylamine (0.09

Article
3-{4-Morpholin-4-yl-1-[1-(pyridin-3-ylmethyl)piperidin-4-yl]-
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7087
6-[1-(1-Benzylpiperidin-4-yl)-4-(morpholin-4-yl)-1H-pyrazolo-
[3,4-d]pyrimidin-6-yl]quinoline Trifluoroacetic Acid Salt (5l). Pro-
cedure A was used. Purification was by preparative reverse phase
HPLC with 0.02% TFA buffer, giving an off-white amorphous
solid. 1H NMR (300 MHz, DMSO-d6): δ 9.71 (s. 1H), 9.08 (d,
J = 2.0 Hz, 1H), 8.99 (dd, J = 1.5, 4.5 Hz, 1H), 8.86 (dd, J = 2.0,
9.0 Hz, 1H), 8.59 (d, J = 8.0 Hz, 1H), 8.42 (s, 1H), 8.15 (d, J =
7.5 Hz, 1H), 7.64 (dd, J = 4.0, 8.5 Hz, 1H), 7.60-7.49 (m, 5H),
2.29-5.15 (m, 1H), 4.45-4.38 (m, 2H), 4.14-4.02 (m, 4H),
3.87-3.76 (m, 4H), 3.63-3.53 (m, 2H), 3.46-3.31 (m, 2H),
2.53-2.35 (m, 2H), 2.30-2.18 (m, 2H). MS (ESI): m/e 253.6
(66%), 506.3 [M þ H]þ (100%). HRMS m/z 506.267 21, calcd
[M þ H]þ 506.266 29, rel intens 100%, error 0.92.
1H-pyrazolo[3,4-d]pyrimidin-6-yl}phenol (5f). Procedure A was
used. The piperidine substrate for procedure B was prepared in
an analogous manner to 5p and 5t, using 3-hydroxyphenyl-
boronic acid instead of 1H-indol-5-ylboronic acid. The product
was purified by preparative reverse phase HPLC with 0.02%
ammonium hydroxide buffer, giving an off-white amorphous
solid. 1H NMR (300 MHz, DMSO-d6): δ 9.52 (s, 1H),
8.57-8.46 (m, 2H), 8.31 (s, 1H), 7.90-7.75 (m, 3H), 7.39 (dd,
J = 4.5, 7.0 Hz, 1H), 7.27 (dd, J = 8.0, 8.0 Hz, 1H), 6.89-6.87
(m, 1H), 4.85-4.72 (m, 1H), 4.04 (m, 4H), 3.83 (m, 4H), 3.61 (s,
2H), 3.03-2.93 (m, 2H), 2.32-2.13 (m, 4H), 1.97-1.89 (m, 2H).
MS (ESI): m/e 236.5 (5%), 472.2 [M þ H]þ (100%). HRMS m/z
472.245 56, calcd [M þ H]þ 472.245 55, rel intens 100%, error
0.03 mAmu.
2-[1-(1-Benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,
4-d]pyrimidin-6-yl]phenol (5g). Procedure A was used. Purifica-
tion was by preparative reverse phase HPLC with 0.02% TFA
buffer, giving an off-white amorphous solid. 1H NMR (300
MHz, DMSO-d6): δ 13.46 (m, 1H), 9.73 (m, 1H), 8.48-8.41
(m, 2H), 7.59-7.35 (m, 5H), 6.98-6.90 (m, 2H), 6.56 (m, 1H),
5.11-4.98 (m, 1H), 4.46-4.34 (m, 2H), 4.02-3.93 (m,
4H), 3.85-3.75 (m, 4H), 3.60-3.23 (m, 4H), 2.47-2.11 (m,
4H). MS (ESI): m/e 471.2 [M þ H]þ (100%). HRMS
m/z 471.250 19, calcd [M þ H]þ 471.250 30, rel intens 100%,
error 0.11 mAmu.
4-[1-(1-Benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,
4-d]pyrimidin-6-yl]phenol Trifluoroacetic Acid Salt (5h). Proce-
dure A was used. Purification was by preparative reverse phase
HPLC with 0.02% TFA buffer, giving an off-white amorphous
solid. 1H NMR (300 MHz, DMSO-d6): δ 9.29 (br s, 1H),
8.34-8.27 (m, 3H), 7.59-7.46 (m, 5H), 6.89-6.82 (m, 2H),
5.17-5.02 (m, 1H), 4.46-4.33 (m, 2H), 4.03-3.91 (m, 4H),
3.82-3.72 (m, 4H), 3.62-3.23 (m, 4H), 2.49-2.10 (m, 4H).
MS (ESI): m/e 471.2 [M þ H]þ (100%). HRMS m/z
471.250 97, calcd [M þ H]þ 471.250 30, rel intens 100%, error
0.67 mAmu.
1-(1-Benzylpiperidin-4-yl)-4-morpholin-4-yl-6-pyridin-3-yl-1H-
pyrazolo[3,4-d]pyrimidine (5i). Procedure A was used. Purifica-
tion was by preparative reverse phase HPLC with 0.02% TFA
buffer, giving an off-white amorphoussolid. 1H NMR (300 MHz,
DMSO-d6): δ 9.95 (br s, 1H), 9.61 (s, 1H), 8.40 (s, 1H), 8.77-8.68
(m, 2H), 7.62-7.46 (m, 6H), 5.21-5.07 (m, 1H), 4.43-4.35 (m,
2H), 4.08-3.98 (m, 4H), 3.83-3.74 (m, 4H), 3.61-3.24 (m, 4H),
2.51-2.14 (m, 4H). MS (ESI): m/e 228.2 (29%), 456.2 [M þ H]þ
(100%). HRMS m/z 456.251 46, calcd [M þ H]þ 456.25064, rel
intens 100%, error 0.82 mAmu.
5-[1-(1-Benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,
4-d]pyrimidin-6-yl]-1,3-dihydro-2H-benzimidazol-2-one Trifluoro-
acetic Acid Salt (5m). Procedure A was used. Purification was by
preparative reverse phase HPLC with 0.02% TFA buffer, giving
an off-white amorphous solid. 1H NMR (300 MHz, DMSO-d6):
δ 10.88 (s, 1H), 10.74 (s, 1H), 8.34 (s, 1H), 8.16 (dd, J = 1.5, 5.5
Hz, 1H), 8.0 (s, 1H), 7.59-7.46 (m, 5H), 7.0 (s, 1H), 5.20-5.03
(m, 1H), 4.45-4.35 (m, 2H), 4.05-3.94 (m, 4H), 3.84-3.74
(m, 4H), 3.60-3.25 (m, 4H), 2.48-2.11 (m, 4H). MS (ESI):
m/e 511.2 [M þ H]þ (100%). HRMS m/z 511.256 42, calcd [M þ
H]þ 511.256 45, rel intens 100%, error 0.03 mAmu.
1-(1-Benzylpiperidin-4-yl)-6-(1H-indol-2-yl)-4-morpholin-4-yl-
1H-pyrazolo[3,4-d]pyrimidine (5n). Procedure A was used. Pur-
ification was by preparative reverse phase HPLC with 0.02%
ammonium hydroxide buffer, giving an off-white amorphous
solid. 1H NMR (300 MHz, DMSO-d6): δ 11.61 (s, 1H),
8.62-8.56 (m, 1H), 8.25-8.20 (m, 2H), 7.49-7.14 (m, 8H),
4.82-4.69 (m, 1H), 4.02-3.95 (m, 4H), 3.82-3.76 (m, 4H),
5.58 (s, 2H), 3.12-2.94 9 m, 4H), 2.34-1.89 (m, 4H). MS
(ESI): m/e 247.6 (7%), 494.3 [M þ H]þ (100%). HRMS m/z
494.267 07, calcd [M þ H]þ 494.266 29, rel intens 100%, error
0.78 mAmu.
1-(1-Benzylpiperidin-4-yl)-6-(1H-indol-4-yl)-4-morpholin-4-yl-
1H-pyrazolo[3,4-d]pyrimidine Trifluoroacetic Acid Salt (5o). Pro-
cedure A was used. Purification was by preparative reverse phase
HPLC with 0.02% TFA buffer, giving an off-white amorphous
1
solid. H NMR (300 MHz, DMSO-d6): δ 11.29 (s, 1H), 9.68
(s, 1H), 8.37 (s, 1H), 8.18 (d, J = 7.0 Hz, 1H), 7.60-7.38 (m, 7H),
7.24-7.16 (1H), 6.56 (s, 1H), 5.22-5.10 (m, 1H), 4.46-4.37
(m, 2H), 4.08-3.98 (m, 4H), 3.85-3.75 (m, 4H), 3.60-3.51 (m,
2H), 2.52-2.33 (m, 2H), 2.30-2.17 (m, 4H). MS (ESI): m/e 247.6
(17%), 494.3 [M þ H]þ (100%). HRMS m/z 494.266 35, calcd
[M þ H]þ 494.266 29, rel intens 100%, error 0.06.
1-(1-Benzylpiperidin-4-yl)-6-(1H-indol-6-yl)-4-morpholin-4-yl-
1H-pyrazolo[3,4-d]pyrimidine Trifluoroacetic Acid Salt (5q). Pro-
cedure A was used. Purification was by preparative reverse phase
HPLC with 0.02% TFA buffer, giving an off-white amorphous
solid. 1H NMR(300MHz, DMSO-d6): δ 11.29 (a, 1H), 9.71(br s,
1H), 8.53 (s, 1H), 8.34 (s, 1H), 8.21 (dd, J = 1.5, 8.5 Hz, 1H),
7.64-7.46 (m, 7H), 6.48 (s, 1H), 5.23-5.09 (m, 1H), 4.49-4.36
(m, 2H), 4.09-3.98 (m, 4H), 3.86-3.77 (m, 4H), 3.75-3.50 (m,
2H), 2.57-2.32 (m, 4H), 2.30-2.15 (m, 2H). MS (ESI): m/e 247.6
(11%), 494.3 [M þ H]þ (100%). HRMS m/z 494.265 80, calcd
[M þ H]þ 494.266 29, rel intens 100%, error 0.49 mAmu.
1-(1-Benzylpiperidin-4-yl)-6-(1H-indol-7-yl)-4-morpholin-4-yl-
1H-pyrazolo[3,4-d]pyrimidine (5r). Procedure B was used. Puri-
fication was by preparative reverse phase HPLC with 0.02%
ammonium hydroxide buffer, giving an off-white amorphous
solid. 1H NMR (300 MHz, DMSO-d6): δ 11.14 (s, 1H), 9.95 (br s,
1H), 8.40 (s, 1H), 8.31 (d, J = 8.0 Hz, 1H), 7.45 (d, J = 8.0 Hz,
1H), 7.65-7.45 (m, 7H), 7.16 (dd, J = 7.0, 7.0 Hz, 1H), 6.60
(s, 1H), 5.47-5.34 (m, 1H), 4.47-4.38 (m, 2H), 4.08-4.00 (m,
4H), 3.85-3.78 (m, 4H), 3.62-3.30 (m, 4H), 2.49-2.16 (m, 4H).
MS (ESI): m/e 494.3 [M þ H]þ (100%). HRMS m/z 494.266 41,
calcd [M þ H]þ 494.266 29, rel intens 100%, error 0.12 mAmu.
1-(1-Benzylpiperidin-4-yl)-6-(1-methyl-1H-indol-5-yl)-4-mor-
pholin-4-yl-1H-pyrazolo[3,4-d]pyrimidine (5s). Procedure A was
1-(1-Benzylpiperidin-4-yl)-4-morpholin-4-yl-6-pyridin-4-yl-1H-
pyrazolo[3,4-d]pyrimidine Trifluoroacetic Acid Salt (5j). Proce-
dure A was used. Purification was by preparative reverse phase
HPLC with 0.02% TFA buffer, giving an off-white amorphous
solid. 1H NMR (300 MHz, DMSO-d6): δ 9.63 (br s, 1H),
8.77-8.73 (m, 2H), 8.43 (s, 1H), 8.34-8.30 (m, 2H), 7.57-7.49
(m, 5H), 5.20-5.09 (m, 1H), 4.17-4.35 (m, 2H), 4.07-3.98
(m, 4H), 3.92-3.47 (m, 6H), 2.39-2.10 (m, 4H). MS (ESI): m/e
228.6 (32%), 379.2 (17%), 456.2 [M þ H]þ (100%). HRMS m/z
456.251 37, calcd [M þ H]þ 456.250 64, rel intens 100%, error
0.73 mAmu.
3-[1-(1-Benzylpiperidin-4-yl)-4-morpholin-4-yl-1H-pyrazolo[3,
4-d]pyrimidin-6-yl]quinoline Trifluoroacetic Acid Salt (5k). Pro-
cedure A was used. Purification was by preparative reverse phase
HPLC with 0.02% TFA buffer, giving an off-white amorphous
solid. 1H NMR (300 MHz, DMSO-d6): δ 9.94 (s, 1H), 9.30
(s, 1H), 8.42 (s, 1H), 8.14 (dd, J = 8.0, 21.5 Hz, 2H), 7.85 (dd,
J = 7.5, 7.5 Hz, 1H), 7.7- (dd, J = 7.5, 7.5 Hz, 1H), 7.59-7.50
(m, 6H), 5.27-5.15 (m, 1H), 4.47-4.37 (m, 2H), 4.13-4.03 (m,
4H), 3.87-3.75 (m, 4H), 3.61-3.30 (m, 4H), 2.48-2.17 (m, 4H).
MS (ESI): m/e 253.6 (62%), 506.3 [M þ H]þ (100%). HRMS m/z
506.266 99, calcd [M þ H]þ 506.266 29, rel intens 100%, error
0.70 mAmu.

7088 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Nowak et al.
used. Purification was by preparative reverse phase HPLC with
0.02% ammonium hydroxide buffer, giving an off-white amor-
phous solid. H NMR (300 MHz, DMSO-d6): δ 8.71 (s, 1H),
Table 4. HPLC Retention Times and Purity Data for 5a-v
method Aa method Bb
1
compd
tR (min)
purity (%)
tR (min)
purity (%)
8.32 (d, J = 9.5 Hz, 1H), 8.27 (s, 1H), 7.49 (d, J = 8.5 Hz, 1H),
7.40-7.31 (m, 5H), 7.31-7.24 (m, 1H), 6.57 (d, J = 3.0 Hz, 1H),
4.91-4.77 (m, 1H), 4.07-3.98 (m, 4H), 3.83 (s, 3H), 3.84-3.75
(m, 4H), 3.57 (s, 2H), 3.04-2.93 (m, 2H), 2.32-2.13 (m, 4H),
1.96-1.85 (m, 2H). MS (ESI): m/e 254.6 (13%), 508.3 [M þ H]þ
(100%). HRMS m/z 508.281 44, calcd [M þ H]þ 508.281 94, rel
intens 100%, error 0.50 mAmu.
6-(1H-Indol-5-yl)-4-morpholin-4-yl-1-[1-(pyridin-3-ylmethyl)-
piperidin-4-yl]-1H-pyrazolo[3,4-d]pyrimidine Trifluoroacetic Acid
Salt (5u). Procedure B was used. Purification was by preparative
reverse phase HPLC with 0.02% TFA buffer, giving an off-
white amorphous solid. 1H NMR (300 MHz, DMSO-d6): δ 1.85
(brs, 1H), 11.35 (s, 1H), 9.19 (s, 1H), 8.97 (d, J = 5.4 Hz, 1H),
8.77 (d, J = 8.5 Hz, 1H), 8.74 (s, 1H), 8.32 (s, 1H), 8.30 (dd, J =
7.5, 0.5 Hz, 1H), 8.06 (dd, J = 5.5, 8.5 Hz, 1H), 7.46 (d, J = 9.0
Hz, 1H), 7.41 (dd, J = 2.5, 2.5 Hz, 1 h), 6.56 (s, 1H), 5.21-5.08
(m, 1H), 4.60 (s, 1H), 4.03 (br s, 4H), 3.08 (br s, 4H), 3.64-3.53
(m, 2H), 3.47-3.53 (m, 2H), 2.71-2.54 (m, 2H), 2.23-2.11 (m,
2H). MS (ESI): m/e 248.1 (24%), 495.3 [M þ H]þ (100%).
HRMS m/z 495.261 73, calcd [M þ H]þ 495.261 54, rel intens
100%, error 0.19 mAmu.
5a
5b
5c
5d
5e
5f
1.86
2.34
2.07
2.33
2.12
1.90
2.26
2.08
1.92
1.87
2.26
2.13
2.00
2.16
2.26
2.15
2.31
2.12
2.37
2.04
2.02
2.45
95
95
1.25
1.28
2.81
1.38
2.34
1.85
2.91
2.25
2.21
2.29
3.51
2.57
1.93
2.51
2.47
2.54
2.65
2.50
2.84
1.66
1.80
1.77
95
96
99
99
99
98
100
97
95
94
99
98
95
99
95
100
94
97
99
100
92
96
95
95
97
100
100
96
5g
5h
5i
98
5j
100
95
5k
5l
99
95
5m
5n
5o
5p
5q
5r
5s
5t
99
90
100
98
95
99
100
100
95
6-(1H-Indol-5-yl)-4-morpholin-4-yl-1-[1-(pyridin-3-ylcarbonyl)-
piperidin-4-yl]-1H-pyrazolo[3,4-d]pyrimidine (5v). Procedure C
was used. Purification was by preparative reverse phase HPLC
with 0.02% ammonium hydroxide buffer, giving an off-white
amorphous solid. 1H NMR (300 MHz, DMSO-d6): δ 11.26
(s, 1H), 8.74-8.65 (m, 2H), 8.31-8.26 (m, 2H), 7.94-7.88 (m,
1H), 7.66 -7.50 (m, 4H), 6.56 (s, 1H), 5.21-5.11 (m, 1H),
4.69-4.59 (m, 1H), 4.06-3.99 (m, 1H), 3.84-3.77 (m, 4H),
3.31-3.12 (m, 3H), 2.41-1.90 (m, 4H). MS (ESI): m/e 279.1
(19%), 509.2 [M þ H]þ (100%). HRMS m/z 509.241 37, calcd
[M þ H]þ 509.240 80, rel intens 100%, error 0.57.
5u
5v
a HPLC method A: solvent gradient = 90% A at 0 min, 5% A at
2.6 min, 5% A at 3 min; solvent A = 0.05% HCO2H in water; solvent
B = 0.05% HCO2H in ACN; flow rate = 1 mL/min; column = Agilent
XDB-C18 30 mm ꢀ 2.1 mm, 1.8 μm particle size; temperature = 70 °C.
b HPLC method B: 90% A at 0 min, 5% A at 2.6 min, 5% A at 3 min;
solvent A = 0.05% NH4OH in water; solvent B = 0.05% NH4OH in
ACN; flow rate = 1 mL/min; column = Agilent SB-C18, 50 mm ꢀ 2.1
mm, 1.8 μm particle size; temperature = 70 °C.
HPLC Data for 5a-v. Table 4 lists the HPLC retention times
and purities for 5a-v.
build the initial model, which was further optimized using
€
EMBRACE (MacroModel, version 9.1, Schrodinger, LLC,
New York, NY, 2005) minimizations in complex with the
cocrystallized ligand. Subsequent docking studies were per-
Assays of mTOR, PI3K, and Other Kinases. mTOR assays
were performed in 96-well plates for 2 h at room temperature in
25 μL containing 6 nM Flag-TOR(3.5), 1 μM His6-S6K1, and
100 μM ATP. The assays were performed and detected by
DELFIA employing an Eu-phospho-p70S6K T389 antibody
as described previously.30 For inhibitor versus ATP matrix
competition, mTOR kinase reactions were carried out with
varying concentrations of ATP in combination with varying
concentrations of inhibitor. The assays contained 12 nM Flag-
TOR(3.5) and were incubated for 30 min. The assay results were
similarly detected by DELFIA and processed for generation of
double-reciprocal plots. PI3K assays were performed in a
fluorescence polarization (FP) assay with 25 μM ATP as
described.31 The assays of 5u (2 μM) against a broad panel of
protein kinases were performed by Invitrogen SelectScreen
profiling.27
Inhibition of Cellular mTOR Signaling. Human A498 renal
cancer and U87MG glioma cells were cultured employing
standard cell culture methods, treated with DMSO or
inhibitors for 6 h. Total cell lysates were prepared using
NuPAGE-LDS sample buffer (Invitrogen), quantified, and
subject to immonoblotting analysis using NuPAGE electro-
phoresis system (Invitrogen). Antibodies against P-S6K1
(T389), P-4EBP1 (T70), P-4EBP1 (T37/46), P-AKT (S473),
and P-AKT (T308) were purchased from Cell Signaling Tech-
nology.
€
formed using Glide 4.0 (Glide, version 4.0, Schrodinger, LLC,
New York, NY, 2005) and 4.5 (Glide, version 4.5, Schrodinger,
€
LLC, New York, NY, 2005) with the Single Precision (SP)
scoring function and a pregenerated docking grid. The box size
used for docking was 10 A in each direction around the centroid
of the ligand. To minimize the conformational search space, a
hydrogen bonding constraint to valine-2240 of the hinge region
was used during the docking.
Acknowledgment. We thank the Wyeth HTS group for
mTOR HTS, Xidong Feng for HRMS data, Michael Kelly
for HPLC purity analyses, Frank Loganzo for kinase panel
assay results, Li Di for rat hepatocytes clearance measure-
ments, and Tarek S. Mansour, Dennis Powell, and Semiramis
Ayral-Kaloustian for helpful suggestions.
Note Added after ASAP Publication. This paper was pub-
lished on October 19, 2009 with an incomplete author list. The
revised version was published on October 22, 2009.
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