Lead optimization of n-3-substituted 7-morpholinotriazolopyrimidines as dual phosphoinositide 3-kinase/mammalian target of rapamycin inhibitors: Discovery of PKI-402

Article abstract of DOI:10.1021/jm9014982

Herein we describe the identification and lead optimization of triazolopyrimidines as a novel class of potent dual PI3K/mTOR inhibitors, resulting in the discovery of 3 (PKI-402). Compound 3 exhibits good physical properties and PK parameters, low nanomolar potency against PI3KR and mTOR, and excellent inhibition of cell proliferation in several human cancer cell lines. Furthermore, in vitro and in vivo biomarker studies demonstrated the ability of 3 to shut down the PI3K/Akt pathway and induce apoptosis in cancer cells. In addition, 3 showed excellent in vivo efficacy in various human cancer xenografts, validating suppression of PI3K/mTOR signaling as a potential anticancer therapy. 2009 American Chemical Society.

Full text of DOI:10.1021/jm9014982

798 J. Med. Chem. 2010, 53, 798–810
DOI: 10.1021/jm9014982
Lead Optimization of N-3-Substituted 7-Morpholinotriazolopyrimidines as Dual
Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Inhibitors: Discovery of PKI-402
Christoph M. Dehnhardt,*,† Aranapakam M. Venkatesan,† Efren Delos Santos,† Zecheng Chen,† Osvaldo Santos,†
Semiramis Ayral-Kaloustian,† Natasja Brooijmans,‡ Robert Mallon,§ Irwin Hollander,§ Larry Feldberg,§ Judy Lucas,§
Inder Chaudhary, Ker Yu,§ Jay Gibbons,§ Robert Abraham,§ and Tarek S. Mansour†
†Discovery Medicinal Chemistry, ‡Computational Chemistry, §Oncology, and Drug Safety and Metabolism, Wyeth Research, Pearl River,
New York 10965
Received October 8, 2009
Herein we describe the identification and lead optimization of triazolopyrimidines as a novel class of
potent dual PI3K/mTOR inhibitors, resulting in the discovery of 3 (PKI-402). Compound 3 exhibits
good physical properties and PK parameters, low nanomolar potency against PI3KR and mTOR, and
excellent inhibition of cell proliferation in several human cancer cell lines. Furthermore, in vitro and in
vivo biomarker studies demonstrated the ability of 3 to shut down the PI3K/Akt pathway and induce
apoptosis in cancer cells. In addition, 3 showed excellent in vivo efficacy in various human cancer
xenografts, validating suppression of PI3K/mTOR signaling as a potential anticancer therapy.
Introduction
which exists as either the mTOR complex-1 (TORC1) or
mTOR complex-2 (TORC2) enzyme complexes. TORC1
regulates such processes as protein biosynthesis, while
TORC2 fully activates Akt kinase by phosphorylating it at
serine 473 (S473).1,2 A dual PI3K/mTOR inhibitor could both
prevent cancer cell proliferation and induce programmed cell
death (apoptosis) by fully suppressing Akt activation.1,4,7
Therefore, our goal was to identify potent small molecule
dual PI3K/mTOR inhibitors, which we hypothesized would
exert antitumor activity in a broad arrayof preclinical models.
This hypothesis was validated by the Novartis dual PI3K/
mTOR inhibitor 1 (NVP-BEZ-235, Figure 1).3 Compound 1
is the most advanced PI3K/mTOR inhibitor reported and is
currently undergoing phase II clinical trials.4 In this publica-
tion, we describe the discovery of triazolopyrimidines and
their lead optimization, culminating in the novel dual PI3K/
mTOR inhibitor 3 (PKI-402).5 Compound 3 displayed potent
inhibition in all class I PI3Ks (e.g., IC50 vs PI3KR = 1 nM)
and mTOR. Compound 3 inhibited growth of a diverse set of
human tumor lines in vitro, and it proved to be efficacious in
three human tumor xenograft models.
The versatility of fused 4-morpholino-2-arylpyrimidines as
PI3K/mTOR inhibitors has been demonstrated impressively
by the discovery of 2 (GDC-0941), a PI3KR isoform selective
inhibitor built on a thieno[3,2,d ]pyrimidine core,6 and by the
introduction of pyridofuranopyrimidines,7 imidazolopyrimi-
dines,8 pyrrolopyrimidines9 as dual PI3K/mTOR inhibitors
or pyrazolopyrimidines10 that function as highly selective
mTOR inhibitors. However, despite the fact that highly
selective PI3KR6 and mTOR10 inhibitors have been intro-
duced recently, only one preclinically validated dual PI3K/
mTOR inhibitor, utilizing a fused pyrimidine scaffold bearing
a morpholine, has been disclosed to date.11 Herein, we report
the discovery of the novel triazolopyrimidine scaffold and our
lead optimization in this series, resulting in the discovery of 3,
a highly in vivo efficacious dual PI3K/mTOR inhibitor.
The phosphoinositide 3-kinases (PI3Ka) are a group of
enzymes that phosphorylate the 30-OH position of the inositol
ring of phosphatidylinositol bisphosphate (4,5) (PIP2) to
phosphatidylinositol triphosphate (3,4,5) (PIP3). Class I
PI3Ks are divided into two groups, class IA (p110R, P110β,
and p110δ isoforms) and class IB (p110γ). These isoforms are
structurally similar but differ in subunit association and mode
of activation (e.g., receptor tyrosine kinase vs G-protein-
coupled receptor). Aberrant PI3K signaling caused by phos-
phoinositide 3-kinase R encoded gene (PIKCA) amplification
or mutation, PIK3R1 mutation, phosphatase and tensin
homologue (PTEN) loss, or receptor tyrosine kinase (RTK)
dependent activation occurs in about 50% of all known solid
tumors. Deregulated PI3K activity leads to elevated levels of
PIP3, which acts as a second messenger responsible for the
activation of the downstream kinase protein kinase B (Akt).1
PIP3 is bound by the PH (pleckstrin homology) domains of
both PDK1 and Akt serine/threonine (S/T) kinases resulting
in their proximity at the inner cell membrane where 3-phos-
phoinositide-dependent kinase 1 (PDK1) phosphorylates and
activates Akt (at T308).2 Aberrantly activated Akt promotes
growth, survival, proliferation, enhanced migration, and ad-
hesion in cancer cells. Many Akt effects are mediated through
the S/T kinase mammalian target of rapamycin (mTOR),
*To whom correspondence should be addressed. Phone: 1-845-602-
2146. Fax: 1-845-602-5561. E-mail: dehnhac@wyeth.com.
a Abbreviations: PI3K, phosphoinositide 3-kinase; mTOR, mamma-
lian target of rapamycin; Akt, protein kinase B; PIP2, phosphatidylino-
sitol bisphosphate (4,5); PIP3, phosphatidylinositol triphosphate
(3,4,5); PTEN, phosphatase and tensin homologue; RTK, receptor
tyrosine kinase; PH, pleckstrin homology; mTORC1, mammalian target
of rapamycin complex 1; mTORC2, mammalian target of rapamycin
complex 2; PDK1, 3-phosphoinositide-dependent kinase 1; S, serine; T,
threonine; ATP, adenosine 50-triphosphate; Her2þ, human epidermal
growth factor receptor 2þ; ELISA, enzyme-linked immunosorbent
assay; DELFIA, dissociation-enhanced lanthanide fluorescent.
r
pubs.acs.org/jmc
Published on Web 12/07/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 799
Figure 2. Structure and biological profile of initial discovery lead 4
and the triazolopyrimidine lead 5.
primary optimization was directed at improving potency
against PI3KR. However, we are showing potency against
PI3Kγ to illustrate the selectivity of our compounds over class
IB isoforms, while the possible role of PI3Kγ in cancer
remains unclear.1 Our molecular modeling is based on cocrys-
tals of inhibitors with PI3Kγ. Hence, predictions using mo-
lecular modeling only make sense when the series has good
potency against PI3KR and PI3Kγ. For mTOR enzyme
potency, we used a dissociation-enhanced lanthanide fluore-
scent immunoassay (DELFIA) platform enzyme-linked im-
munosorbent assay (ELISA).10 Cell growth inhibition was
evaluated in MDA-361 [breast, human epidermal growth
factor receptor (Her)2þ/PI3KCA mutant] and PC3
(prostate, PTEN mutant) human tumor cell lines.
Because of the high degree of homology in the adenosine 50-
triphosphate (ATP) binding pocket and the similarity of their
cores, all 4-morpholino-2 arylpyrimidines are expected to
bind in the ATP binding pocket of mTOR, p110R, and
p110γ in a similar binding mode, with the morpholine oxygen
binding to the hinge region Val851 and the substituents at the
aryl group in the 2-position interacting with Asp810 and
Lys883. X-ray crystallography studies with PI3Kγ, molecular
modeling and unpublished SAR results indicated that, in fact,
those three interactions are crucial for potency against PI3KR
and mTOR. Therefore, we felt that modifying fused pyrimi-
dines would give us the best chance to successfully develop a
dual PI3K/mTOR inhibitor.
Imidazolopyrimidine 4 was identified as a potent and
selective PI3KR inhibitor. Replacing the “CH” of the
imdazole ring 4 with a “N” led to triazolopyrimidine 5,
which exhibited a lower cLogP value and slightly higher
topological polar surface area (TPSA) than 4. In addition, 5
showed similar potency against PI3KR but better potency
against PC3 and MDA-361 cells relative to compound 4.
However, compound 5 showed limited solubility and poor
microsomal stability caused by the OH group as a metabolic
hot spot. A plethora of OH bioisosteres have already been
published for fused pyrimidines.6,10,16 Recently, it has been
shown by Zask et al.10 that incorporation of a urea appen-
dage in the pyrazolopyrimidine core not only improved the
potency against PI3KR and mTOR but also increased
metabolic stability. Hence, we explored this ureido appen-
dage with triazolopyrimidines carrying small alkyl substi-
tutents at the N-3 position to obtain potent PI3K/mTOR
inhibitors with low molecular weight and good microsomal
stability.
Figure 1. Structures of known PI3K inhibitors (1 and 2) and of
novel compound 3.
Chemistry
The synthesis of triazolopyrimidine 5 (Figure 2) is shown in
Scheme 1. Nitrobarbituric acid 612 was chlorinated using
POCl3 and DMA to give 5-nitrotrichloropyrimidine 7 in
13% yield.13 Pyrimidine 7 was reacted with morpholine at
0 °C to give the 4-morpholino-5-nitro-2,6-dichloropyrimidine
8, which was aminated with 4-amino-1-benzylpiperidine in
THF in the presence of NEt3 to give 9. Reduction14of 9 with
Raney Ni and N2H4 gave the bis-amino analogue 10, which
was cyclized14 using NaNO2 in AcOH at 0 °C to yield
triazolopyrimidine 11.
For the introduction of C-2 aryl groups, Suzuki reaction of
11 with 3-hydroxymethylphenyl boronate was used to give 5
in good yield. This synthetic sequence can be applied to the
synthesis of any 3-substituted 5-aryltriazolopyrimidine by
varying the primary amine or arylboronic acid or ester.
A similar route was used to synthesize N-3-alkyltriazolopyri-
midines 14-32 (Scheme 2). Compound 8 was reacted with the
appropriate primary amines (in this case MeNH2, EtNH2,
i-PrNH2, and c-PrNH2) and subsequently converted to 12a-d
as outlined in Scheme 1 for compound 11. Then 12a-d were
converted to anilines 13a-d by reaction with 4-aminophenyl-
boronic acid pinacol ester under Suzuki conditions. Intermedi-
ates 13a-d were condensed with the appropriate isocyanates to
yield urea derivatives 14-32. Alternatively, compounds 13a-d
were reacted with triphosgene and the appropriate aromatic and
heteroaromatic amines to form ureido analogues 14-30.
The synthesis of substituted amides is shown in Scheme 3.
Compound 29 was saponified using NaOH in THF to obtain
acid 31, which was coupled with the appropriately substituted
amines via HOBT and EDCI (or alternatively HBTU) activa-
tion to yield amides 3 and 32-38.
Results and Discussion
All final compounds 3 and 14-38 were tested in vitro
against PI3KR, PI3Kγ, and mTOR.
Our primary assay for inhibition of PI3KR and PI3Kγ
enzymes was a fluorescence polarization format assay.15 Our

800 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Scheme 1. Synthesis of Triazolopyrimidine 5a
Dehnhardt et al.
a Reagents and conditions: (a) POCl3, Me2NC6H5, T < 30 °C, 13%; (b) NEt3, morpholine, 0 °C, 92%; (c) 4-amino-1-benzylpiperidine, NEt3, CH2Cl2,
78%; (d) Raney Ni, MeOH, N2H4, 63%; (e) NaNO2, AcOH, 0 °C, 54%; (f ) (Ph3P)4Pd, 3-hydroxymethylphenylboronic acid, Na2CO3, DME, 140 °C, 1 h
microwave, 45%.
Scheme 2. Synthesis of N-3-Alkyl-2-arylureidotriazolopyrimidines 14-30a
a Reagents and conditions: (a) 4-aminophenylboronic acid pinacol ester, (Ph3P)4Pd, Na2CO3, DME, 140 °C, 1 h, microwave, 57%; (b) triphosgene,
R2NH2, Et3N, THF or R2NCO, THF.
The biological data for analogues 14-24 are compiled in
Table 1. All heteroaryl derivatives (14-24) with small groups
at N-3 showed potent inhibition of PI3KR enzyme activity
and potent inhibition of MDA-361/PC3-cell growth. The 4-
pyridyl substituted urea analogues 14-16 exhibited good
activityagainst PI3KR, mTOR, PC3, and MDA-361. In terms
of potent inhibition of PI3KR enzyme activity and potent
inhibition of MDA-361/PC3 cell growth, compounds are
ranked in the order Et = c-Pr > i-Pr. However, the ethyl
analogue 14 showed the best solubility compared to 15 and
16. Compounds 14-16 also showed acceptable microsomal
stability. Almost equally good potency was observed for the
3-pyridyl analogues 17-20. The i-Pr analogue 18 exhibited
the best cell potency in this subset, while c-Pr (20) and Et (17)
were equipotent in enzyme and cells, and the Me substituted
analogue 19 exhibited decreased potency against PI3KR and
cells. Interestingly, when an additional methyl group was
introduced to the 3-pyridine core in 24, we observed decreased
cell potency compared to 17. The 2-thiophene analogues
21-23 were highly potent against PI3KR, mTOR, and cells.
Here, ethyl analogue 22 showed the best potency against
PI3KR and against cells, establishing the order Et > c-Pr >
Me. However, the thiophene analogues 21-23 showed
only modest microsomal stability and poor solubility at

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 801
Scheme 3. Synthesis of 4-Benzamidoureido Analogues 3 and 32-38a
a Reagents and conditions: (a) NaOH, THF, H2O, 85%; (b) HOBT, EDCI (or HBTU), Et3N, RR0NH, THF.
Table 1. Biological Data and Physical Properties for Heteroarylureidotriazolopyrimidines 14-24
IC50 [nM]a
compd
PI3KR
PI3Kγ
mTOR
MDA-361
PC3
sol.b at pH 7.4
t1/2(rat) [min]c
14
15
16
17
18
19
20
21
22
23
24
5.0
5.0
18.5
21.5
13.5
25.0
48.5
49.0
48.5
22.0
11.5
19.5
37.0
0.63
0.51
0.84
0.89
0.57
2.95
0.57
3.70
3.00
3.05
21.00
35
54
49
67
18
0
0
0
1
1
1
2
1
0
3
27
>30
21
6.0
9.5
31
74
70
96
19
24
10.5
10.5
10.5
6.5
39
91
198
67
281
90
12
24
184
81
366
133
149
252
9
10
2.5
3.0
73
196
15
11
8.0
a The values are an average of at least two separate determinations with a typical variation of less than (30%. b In μg/mL. c Half-life of drug when
incubating with rat liver microsomes.
Table 2. Biological Data and Physical Properties for 4-Arylureidotriazolopyrimidines 25-30
IC50 [nM]a
compd
PI3KR
PI3Kγ
mTOR
MDA-361
PC3
sol.b at pH 7.4
t1/2(rat) [min]c
25
26
27
28
29
30
23.5
3.5
69.0
24.8
12.2
17.5
84.5
78.0
1.60
0.32
0.39
0.31
2.55
0.35
84
<30
28
171
55
0
0
6
0
0
1
7
21
21
9
2.4
3.6
65
57
38
23.3
25.0
199
47
285
110
15
25
a The values are an average of at least two separate determinations with a typical variation of less than (30%. b In μg/mL. c Half-life of drug when
incubating with rat liver microsomes.
physiological pH. Other heteroaryl analogues such as 3-
pyrrole, isoxazole, or 2-thiazole were screened as well, but
they showed only moderate potency against PI3KR and in
cells and are not shown here. A number of compounds where
R2 = Ph demonstrated excellent potency against PI3KR,
mTOR, and PC3/MDA-361 cells. Especially, compound 26
appeared to be a potent inhibitor (Table 2). However, 26
showed poor solubility and only moderate microsomal stabi-
lity. Amides 27 and 28 showed similar good cell and enzyme
potency relative to 26. In vitro biomarker studies revealed
excellent suppression of Akt T308 phosphorylation (IC50 = 8
nM) in MDA-361 cells after 4 h of incubation with drug,
which confirms correlation of the antiproliferative effect of 27
via PI3K/Akt pathway inhibition. For this study, MDA-361
cells were incubated with 27 for 4 h. In lieu of directly
monitoring cellular PIP3 levels, compound inhibition of
PI3K in MDA-361cells was demonstrated by potent suppres-
sion of Akt phosphorylation at T308. Suppression of pAkt-
T308 linked the antiproliferative effect of 27 with PI3K/Akt
pathway inhibition. Suppression of pAkt (T308) by 27 was

802 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Dehnhardt et al.
Figure 3. Modeling studies of analogue 27 docked in a homology model of PI3KR, based on PI3Kγ crystal structures.10
Table 3. Biological Data and Physical Properties for 4-Benzamido Analogues 3 and 32-38
IC50 [nM]a
sol.b at
t1/2 (min)c
compd
PI3KR
PI3Kγ
mTOR
MDA-361
PC3
Akt T308d
pH 3.0
pH 7.4
human
nude mouse
rat
3
1.4
3.0
1.4
6.0
1.0
6.8
3.2
2.4
9.2
1.70
0.56
0.75
2.70
0.50
1.21
1.10
2.20
8
21
33
17
96
52
88
67
116
5
100
0
>30
>30
>30
>30
>30
16
>30
29
15
16
14
21
29
23
17
26
32
33
34
35
36
37
38
17.2
12.3
33.0
12.0
22.0
25.0
9.0
29
16
68
13
36
24
41
27
8
ND
ND
ND
>100
4.0
1
1
ND
27
47
16
65
74
44
0
>100
28
19
1
100
100
1
100
ND
30
28
26
a The values are an average of at least two separate determinations with a typical variation of less than (30%. b In μg/mL. c Half-life of drug when
incubating with liver microsomes of the species shown. d Phosphoblot IC50 values were determined by densitometric scans of Western blots.
measured by Western blot, using a phosphorylated Akt
specific antibody (Cell Signaling Technology). Phosphoblot
IC50 values were determined by densitometric scans of
Western blots.
PI3KR, mTOR, and cells was observed for N,N-dimethyl-
aminoethyleneamide 35. This compound showed good aqu-
eous solubility at pH 3 and pH 7 and very good stability in rat,
nude mouse, and especially human microsomes. The piper-
azineamide 3 exhibited potency comparable to that of 35
against PI3KR and mTOR. However, cell potency was further
enhanced by 2-fold for 3, with an IC50 value well below 10 nM
against MDA-361 cells and an IC50 value of 21 nM against
PC3 cells. Compound 3 also exhibited good pharmaceutical
properties, such as good stability in nude mouse and human
microsomes and aqueous solubility at pH 3. Replacing the N,
N-dimethylaminoethyleneamide (35) with N,N-dimethylami-
nopropyleneamide (38) led to decreased (2- to 3-fold) potency
in cell proliferation assays. Also, substituting the N,N-di-
methylamino group with a 2-pyridyl group (36) or a 4-
methylpiperazinyl group (37) led to lower potency against
cells (MDA-361, PC3). In addition, 36 exhibited weaker
potency against PI3KR and mTOR than 35, as well as low
aqueous solubility and only modest microsomal stability in all
species.
Attaching other solubilizing groups such as Me2N (25) or
morpholine (30) in the 4-position led to only moderately
potent PI3KR inhibitors with high potency against mTOR.
Among the various 4-substituted phenyl analogues, 27 and
28 looked the most interesting because of their high potency in
enzyme and cell assays. Molecular modeling of compound 27
(Figure 3) shows the crucial hydrogen bonding interactions of
the morpholine oxygen to the hinge region Val851 and of the
urea to Asp810 and Lys802. Since the amide moiety is
pointing away from the binding pocket toward the solvent,
this is a good position to improve solubility and/or metabolic
stability, with minimal impact on enzyme and cell potency.
Table 3 shows the biological data for amido analogues 3 and
32-38 synthesized to probe this hypothesis.
As can be seen from the onset, introduction of an amido
moiety at the 4-position of the phenyl in the urea appendage
improved the enzyme and cell potencies and the microsomal
stability. A comparison of the N-alkyamides 32 and 34 shows
that introduction of alkyl substituents can increase cell po-
tency of the amido analogues. However, this effect depends on
the size of the alkyl group, so the n-Bu derivate (34) showed
about 2-fold lower cell and enzyme potency relative to 32. The
tertiary amide 33 demonstrated increased cell potency over 32
but exhibited a similar stability profile. Excellent potency in
Moreimportantlyforamidoanalogues3 and 32-38the cell
potency translated to the suppression of pAkt-T308, the most
relevant PI3KR specific biomarker. Among compounds 3 and
32-38, the best in vitro biomarker suppression was found for
35 and 3. Therefore, 35 and 3 were taken forward for in vivo
biomarker studies in MDA-361 xenograft bearing mice.
Figures 4 and 5 show both compounds 35 and 3 given at 50
mpk, iv, suppressed pAkt-T308, pAkt-S473, P70S6K, and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 803
pS6 (pAkt-S473, P70S6K, and pS6 are mTOR specific
biomarkers). Compound 3 administered at 50 and 100 mpk,
iv, not only suppressed all the PI3K/mTOR biomarkers for at
least 8 h (Figure 5) but also induced cleaved poly ADP-ribose
polymerase (PARP), a marker for cells undergoing apopto-
sis.17
Pharmacokinetic analysis showed that the plasma concen-
tration of 35 was 1229 ng/mL and tumor concentration was
2130 ng/mL at 4 h after a single 50 mg/kg iv dose. For
compound 3, plasma level was 525 ng/mL and tumor level
was 2219 ng/mL at 8 h after a single 50 mg/kg iv dose. For
both compounds 3 and 35 an excellent correlation between
drug concentration in the plasma and tumor and biochemical
response (biomarker pattern) was observed. However, 3
showed a better PK profile and longer lasting biomarker
suppression in vivo than 35, so 3 was taken forward for in
vivo efficacy studies.
Evaluation of efficacy was performed in nude mice bearing
MDA-361 human breast cancer tumors (Figure 6). When 100
mpk of 3 was given once daily for 5 days (one round), tumor
regression was observed with no tumor regrowth until day 70
(not shown). At lower doses (25, 50 mg/kg), initial tumor
regression was also observed, but tumor regrowth occurred
between day 16 and day 20. Compound 3 also showed in vivo
efficacy against other human tumor xenograft models, includ-
ing U87 (glioma) and A597 (non-small-cell lung). In these
studies, compound 3 was well tolerated, with no adverse side
effects such as weight loss.
Additional analysis of compound 3 showed that it was
Ames negative and selective for class I PI3Ks and mTOR.
Selectivity of 3 was assessed against a panel of 236 human
kinases (Invitrogene), where no off-target activity (>50%
inhibition) was detected at 10 μM. Compound 3 is one of
several compounds undergoing preclinical evaluation, and we
will report further efficacy data in due course.
Figure 4. In vivo biomarker analysis of 35 given at 50 mpk (iv) to
MDA-361 tumor bearing nude mice.
Conclusions
We have introduced a novel class of PI3K/mTOR inhibi-
tors, namely, the triazolopyrimidines, which showed better
potency than our related imidazolopyrimidines. Studies of
various N-3-substitutions established the ethyl group as the
best choice. Various aryl- and heteroarylureido appendages
were screened resulting in the discovery of 4-benzamido
analogues. This set of compounds showed especially high
potency against PI3KR and mTOR enzyme activity and
excellent potency in tumor cell proliferation assays. By use
Figure 5. In vivo biomarker analysis at 8 h after 3 was administered
at 25, 50, and 100 mpk (iv) to MDA-361 tumor bearing nude mice.
Figure 6. Antitumor efficacy of 3 in the MDA-361 (breast, Her2þ/PIK3CA mutant) xenograft model.

804 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Dehnhardt et al.
of molecular modeling, water solubilizing amino substituents
were designed and introduced, leading to the discovery of
compounds 3 and 35. These compounds had single digit
nanomolar IC50 values in the PI3KR and mTOR enzyme
assays and in the tumor cell growth inhibition assays. For
both compounds 3 and 35 cell growth inhibition translated
well into both in vitro and in vivo biomarker suppression of
pAkt-T308, pAkt-S473, P70S6K, and pS6. Additionally, both
compounds 3 and 35 induced cleaved PARP (a marker for
cells undergoing apoptosis) in vitro. Compound 3 demon-
strated superior in vivo biomarker suppression profile
(Figure 5) in terms of both duration (8 h) and induction of
cleaved PARP. These data suggest that complete and sus-
tained pAkt suppression induced apoptosis in MDA-361
tumor cells in vivo. Subsequently, an in vivo efficacy study
with MDA-361 xenograft tumors showed that compound 3
administered at 100 mg/kg, iv, dx5 (one round) caused both
initial tumor regression and no tumor regrowth for 70 days.
We are currently evaluating the antitumor efficacy of com-
pound 3 in other models.
mixture was heated for another 30 min to 45 °C. The reaction
mixture was cooled to room temperature and poured onto 600 g
of ice-water. The resulting mixture was stirred for 5 min and
extracted with Et2O (3 ꢀ 800 mL). The combined ethereal layers
were dried over MgSO4. Filtration for removal of MgSO4 and
evaporation of Et2O gave a red oil, which was purified by
column chromatography on silica gel using 100% CH2Cl2.
The product fractions were unified and the solvent was evapo-
rated to give 7 as a white solid (3.45 g, 13% yield). 13C NMR
(CDCl3) δ 154.4, 159.3 ppm. MS (EI) m/z 228.
Preparation 2,6-Dichloro-5-nitro-4-morpholinopyrimidine (8).
To a solution of 2,4,6-trichloronitropyrimidine 7 (6.20 g, 27.2
mmol) in CH2Cl2 (170 mL) at 0 °C was added a solution of
morpholine (2.34 g, 27.2 mmol) and NEt3 (2.74 g, 27.2 mmol) in
CH2Cl2 (70 mL) over a period of 1 h. The reaction mixture was
stirred for another 1 h at 0 °C and allowed to warm to 20 °C and
stirred for 12 h to drive the reaction to completion. For
purification, silica gel (20 g) was added to the reaction mixture
and the solvent was removed so that product was adsorbed on
the silica gel. The resultant residue was placed on a silica gel
column, and the crude material was purified by flash chroma-
tography using CH2Cl2 eluent. After concentration, the product
1
8 was obtained as yellow solid (6.90 g, 91% yield). H NMR
(CDCl3) δ 3.62 (m, 4H), 4.17 (m, 4H) ppm. MS (ESI) m/z 279.
Prepration of 5-Chloro-3-ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo-
[4,5-d]pyrimidine (12b). Step 1. To a cooled solution of ethylamine
(2 M solution in THF) (3.94 mL, 7.89 mmol) and NEt3 (796 mg, 7.89
mmol) in CH2Cl2 (10 mL) at 0 °C was added a solution of 2,6-
dichloro-5-nitro-4-morpholinopyrimidine 8 (2.0 g, 7.2 mmol) dis-
solved in CH2Cl2 (10 mL) over a period of 0.5 h. The reaction mixture
was stirred for another 1 h at 0 °C and allowed to warm to 20 °C and
stirred for 1-4 h to drive the reaction to completion. The product was
purified by flash chromatography on silica gel by eluting it with
CH2Cl2 followed by CH2Cl2/EA (10:1) to give the product as yellow
solid (2.1 g, 100% yield). MS (ESI) m/z 288.
Experimental Section
General Methods. Melting points were determined in open
capillary tubes on a Mel-temp melting point apparatus and are
1
uncorrected. H NMR spectra were determined with a Bruker
DPX-400 spectrometer at 400 MHz. Chemical shifts are re-
ported in parts per million (δ) relative to residual chloroform
(7.26 ppm), TMS (0 ppm), or dimethyl sulfoxide (2.49 ppm) as
an internal reference with coupling constants (J ) reported in
hertz (Hz). The peak shapes are denoted as follows: s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; br, broad. Electro-
spray (ES) mass spectra were recorded in positive or negative
mode on a Micromass Platform spectrometer. Electron impact
(EI) and high-resolution mass spectra were obtained on a
Finnigen MAT-90 spectrometer. Combustion analyses were
obtained using Perkin-Elmer series II 2400 CHNS/O analyzer.
The purity of compound 3 was determined by combustion
analysis. The purity of compounds 18, 25, 28, 30, 34, 37 was
determined by analytical LC/MS using an Agilent 1100 LC,
equipped with an Agilent MSD mass spectrometer, and by
analytical LC using X-Bridge BEH column, MeOH/H2O eluent
at 1 mL/min flow (containing 0.05% NH4OAc), 20 min gradient
of 10% MeOH to 90% ACN, monitored by UV absorption at
215 nm. The purity of compounds 3, 5, 13a-d, 14-17, 19-24,
26, 27, 29, 31-33, 35, 36, and 38 was determined by analytical
LC on a Aquasil C18 column, using MeCN/H2O eluent at 0.8
mL/min flow (containing 0.1% formic acid), 5.5 min gradient of
0% MeCN to 100% MeCN, monitoring UV absorption at 254
nm. The purity of all the above-mentioned compounds 3, 5,
13a-d, 14-38 was found to be >95%. Reverse phase HPLC
purifications were performed on a Gilson preparative HPLC
system controlled by Unipoint software using a Phenomenex
Gemini 100 mm ꢀ 30.0 mm. Thin-layer chromatography (TLC)
was performed on Merck PLC prescored plates 60F254.The
terms “concentrated” and “evaporated” refer to removal of
solvents using a rotary evaporator at water aspirator pressure
with a bath temperature equal to or less than 40 °C. Unless
otherwise noted, reagents were obtained from commercial
sources and were used without further purification.
Step 2. In a three-neck flask was suspended under nitrogen
atmosphere 2-chloro-N 4-ethyl-6-morpholin-4-ylpyrimidine-4,
5-diamine (6 g, 21.87 mmol) and Raney Ni (15 g) in methanol
(600 mL). To the stirring reaction mixture was added slowly
N2H4 H2O (3 mL, 9 mmol, 9 equiv), and the stirring was
3
continued for 1 h to drive the reduction to completion. The
reaction mixture was filtered over Celite and the filtrate was
evaporated to obtain the product (N4-ethyl-2-chloro-6-morpho-
lin-4-ylpyrimidine-4,5-diamine) as light-brown solid (4.1 g, 73%
yield). MS (ESI) m/z 258.
Step 3. To a stirred solution of N 4-ethyl-2-chloro-6-morpho-
lin-4-ylpyrimidine-4,5-diamine (4.1 g, 15.89 mmol) in acetic
acid/water (1:1) (82 mL) at 0 °C was added aqueous (0.5 N)
NaNO2 solution (41 mL, 20.5 mmol), and the reaction mixture
was allowed to stir for 2 h. The off-white solid was collected by
filtration and dried in vacum to give the 5-chloro-3-methyl-7-
morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidine 12b (3.26 g,
76% yield). 1H NMR (DMSO) δ 1.47 (t, J = 7.3 Hz, 3H), 3.74
(m, 2H), 3.81 (m, 2H), 3.94 (m, 2H), 4.53 (q, 7.3 Hz, 2H), 4.56
(m, 2H) ppm. MS (ESI) m/z 269. Analytical LC, using Prodigy
ODS3 column, ACN/H2O eluent at 1 mL/min flow (containing
0.05% TFA), 20 min gradient 5% ACN to 95% ACN, moni-
tored by UV absorption at 215 nm, showed 98.9% purity.
Preparation of 5-Chloro-3-methyl-7-morpholin-4-yl-3H-[1,2,3]tri-
azolo[4,5-d]pyrimidine (12a). 5-Chloro-3-methyl-7-morpholin-4-yl-
3H-[1,2,3]triazolo[4,5-d]pyrimidine (12a) was synthesized starting
from 2,6-dichloro-5-nitro-4-morpholinopyrimidine 8 (2.75 g, 10
mmol) and methylamine in THF solution (2.5 mL, 10 mmol) as
outlined for compound 12b. 5-Chloro-3-methyl-7-morpholin-4-yl-
3H-[1,2,3]triazolo[4,5-d]pyrimidine 12a was obtained as yellow solid
(1.3 g, 51% yield). Mp 168 °C. 1H NMR (CDCl3) δ 3.87 (m, 4H),
4.07 (m, 2H), 4.17 (s, 3H), 4.68 (m, 2H) ppm. MS (APCI) m/z 255.2.
Preparation of 5-Chloro-3-methyl-7-morpholin-4-yl-3H-[1,2,3]tri-
azolo[4,5-d]pyrimidine (12c). 5-Chloro-3-methyl-7-morpholin-4-yl-
3H-[1,2,3]triazolo[4,5-d]pyrimidine (12c) was synthesized starting
Preparation of 5-Nitro-2,4,6-trichloropyrimidine (7). An
amount of 19.2 g (111 mmol) of anhydrous 5-nitrobarbituirc
acid (6) was suspended in 77 mL (833 mmol, 7.5 equiv) of POCl3.
To the suspension were added slowly 53 mL (333 mmol, 3 equiv)
of N,N-diethylaniline so that the reaction temperature never
exceeded 45 °C. A water bath at 28 °C was used to cool the
reaction mixture during the addition. (Caution, reaction is
strongly exothermic!!!). When addition was completed, the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 805
from 2,6-dichloro-5-nitro-4-morpholinopyrimidine (2 g, 7.19 mmol)
and isopropylamine (424 mg, 7.19 mmol) as outlined for compound
12b to obtain 5-chloro-3-isopropyl-7-morpholin-4-yl-3H-[1,2,3]tri-
azolo[4,5-d]pyrimidine 12c as an off-white solid. The product was
found to be pure enough for further transformations (1.5 g, 74%
yield). 1H NMR (DMSO) δ 1.58 (d, J = 6.5 Hz, 6H), 3.75 (m, 2H),
3.81 (m, 2H), 3.93 (m, 2H), 4.56 (m, 2H), 5.03 (hep, J = 6.5 Hz, 1H)
ppm. MS (ESI) m/z 283.2. Analytical LC, using Prodigy ODS3
column, ACN/H2O eluent at 1 mL/min flow (containing 0.05%
TFA), 20 min gradient 5% ACN to 95% ACN, monitored by UV
absorption at 215 nm, showed 99.5% purity.
Preparation of 5-Chloro-3-methyl-7-morpholin-4-yl-3H-[1,2,3]-
triazolo[4,5-d ]pyrimidine (12d). Starting from 2,6-dichloro-5-ni-
tro-4-morpholinopyrimidine 8 (1.38 g, 5 mmol) and cyclopropyl-
amine (54 mg, 6 mmol) and following the procedures as outlined
for compound 12b, 5-chloro-3-cyclopropyl-7-morpholin-4-yl-
3H-[1,2,3]triazolo[4,5-d]pyrimidine 12d was obtained as an off-
white solid (940 mg, 61%). MS (ESI) m/z 283.2.
m/z 340. Analytical LC, using Prodigy ODS3 column, ACN/
H2O eluent at 1 mL/min flow (containing 0.05% TFA), 20 min
gradient 5% ACN to 95% ACN, monitored by UV absorption
at 215 nm, showed 95.3% purity.
Preparation of (3-Cyclopropyl-7-morpholino-3H-[1,2,3]triazolo-
[4,5-d]pyrimidin-5-yl)aniline (13d). 4-(3-Cyclopropyl-7-morpholi-
no-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)aniline 13d was prepared
from 4-(5-chloro-3-cyclopropyl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-
7-yl)morpholine 12d (600 mg., 2.14 mmol) and 4-(4,4,5,5-tetra-
methyl-1,3,2-dioxaborolan-2-yl)aniline following the general Suzuki
procedure to give the product as an off-white solid (700 mg, 97%
yield). 1HNMR(DMSO) δ1.24 (m, 2H), 1.35 (m, 2H),3.52 (m, 2H),
3.79 (m, 4H), 3.99 (hep, J = 3.8 Hz, 1H) 4.12 (m, 2H), 4.52 (m, 2H),
6.66 (d, J = 9.0 Hz, 2H), 8.17 (d, J = 9.0 Hz, 2H) ppm. MS (ESI)
m/z = 338.3. Analytical LC, using Prodigy ODS3 column, ACN/
H2O eluent at 1 mL/min flow (containing 0.05% TFA), 20 min
gradient 5% ACN to 95% ACN, monitored by UV absorption at
215 nm, showed 96.6% purity.
General Procedure for the Preparation of 2-Aryl-7-morpholi-
notriazolopyrimidines by Suzuki Reaction. To a microwave
processing tube was added dimethoxyethane (1.6 mL), aqueous
Na2CO3 (2 M solution) (0.4 mL, 0.8 mmol, 2 equiv), (Ph3P)4Pd
(46 mg, 0.08 mmol), and the appropriately substituted boronic
acid or ester (0.75 mmol, 2 equiv) and the 2 chloro-3-alkyl-7-
morpholinotriazolopyrimidines (0.38 mmol), and the vessel was
sealed. The mixture was heated to 140 °C for 45 min. The
solvents were distilled on a rota-evaporator, and the crude
compound was purified by silica gel chromatography CH2Cl2/
EA (10:1) to give the product as an off-white solid (76-97%
yield).
Preparation of 4-(3-Ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-
d ]pyrimidin-5-yl)phenylamine (13b). 4-(3-Ethyl-7-morpholin-4-yl-
3H-[1,2,3]triazolo[4,5-d ]pyrimidin-5-yl)phenylamine 13b was
prepared from 5-chloro-3-ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo-
[4,5-d]pyrimidine 12b (1.45 g, 5.40 mmol) and 4-(4,4,5,5-tetramethyl-
[1,3,2]dioxaborolan-2-yl)phenylamine (1.53 g, 7.03 mmol) following
the above general Suzuki procedure to give the titled product 13b (1.63
g, 92% yield). 1H NMR (DMSO) δ1.52 (t, J = 7.6 Hz, 3H), 3.79 (m,
4H), 4.12 (m, 2H), 4.58 (q, J= 7.6 Hz, 2H), 4.59 (m, 2H), 5.65 (s, 2H),
6.62(d,J= 8.3 Hz, 2H), 8.15 (d, J= 8.3 Hz, 2H) ppm. MS (ESI) m/z
326. Analytical LC, using Prodigy ODS3 column, ACN/H2Oeluentat
1 mL/min flow (containing 0.05% TFA), 20 min gradient 5% ACN to
95% ACN, monitored by UV absorption at 215 nm, showed 99.5%
purity.
Preparation of 4-(3-Methyl-7-morpholin-4-yl-3H-[1,2,3]triazolo-
[4,5-d]pyrimidin-5-yl)phenylamine (13a). 4-(3-Methyl-7-morpholin-
4-yl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)phenylamine 13a was
prepared from 5-chloro-3-methyl-7-morpholin-4-yl-3H-[1,2,3]tri-
azolo[4,5-d]pyrimidine 12a (1.3 g, 5.1 mmol) and 4-(4,4,5,5-tetra-
methyl[1,3,2]dioxaborolan-2-yl)phenylamine (2.2 g, 10 mmol) fol-
lowing the general Suzuki procedure to give 4-(3-methyl-7-morpho-
lin-4-yl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)aniline 13a as a
brown solid (900 mg, 56%yield). Mp 153 °C. 1H NMR (CDCl3)
δ 3.91 (m, 6H), 4.23 (m, 1H), 4.23 (s, 3H), 4.69 (m, 2H), 6.74 (d, J =
8.8 Hz, 2H), 8.31 (d, J = 8.8 Hz, 2H) ppm. MS (ESI) m/z 312.3.
Analytical LC, using Prodigy ODS3 column, ACN/H2O eluent at 1
mL/min flow (containing 0.05% TFA), 20 min gradient 5% ACN
to 95% ACN, monitored by UV absorption at 215 nm, showed
97.3% purity.
Preparation of 4-(3-Isopropyl-7-morpholin-4-yl-3H-[1,2,3]-
triazolo[4,5-d ]pyrimidin-5-yl)phenylamine (13c). 4-(3-Isopro-
pyl-7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidin-5-yl)-
phenylamine 13c was prepared from 5-chloro-3-isopropyl-7-
morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidine 12c (1.50
g, 5.3 mmol) and 4-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-
yl)phenylamine (1.74 g, 7.97 mmol) following the general
Suzuki procedure to give the titled product (1.22 g, 74% yield).
1H NMR (DMSO) δ 1.64 (d, J = 6.8 Hz, 6H), 3.80 (m, 4H), 4.11
(m, 2H), 4.55 (m, 4H), 5.14 (hep, J = 6.8 Hz, 1H), 6.72 (d,
J = 9.5 Hz, 2H), 8.19 (d, J = 9.5 Hz, 2H) ppm. MS (ESI)
Preparation of Methyl 4-({[4-(3-Ethyl-7-morpholin-4-yl-3H-[1,2,3]-
triazolo[4,5-d]pyrimidin-5-yl)phenyl]carbamoyl}amino)benzoate (29).
To a stirred solution of 4-(3-ethyl-7-morpholin-4-yl-3H-[1,2,3]-
triazolo[4,5-d]pyrimidin-5-yl)phenylamine 13b (2.6 g, 8.0 mmol)
in anhydrous CH2Cl2 (65 mL) was added methyl 4-isocyanato-
benzoate (1.55 g, 8.8 mmol) in one portion. The mixture was stirred
for 8 h, hexane (50 mL) was added, and the solid was collected by
filtration. The filter cake was washed with hexane (10 mL) and
dried in a vacuum oven to give the product 29 as an off-white solid
(3.06 g, 76% yield). 1H NMR (DMSO) δ 1.55 (t, J = 7.3 Hz, 3H),
3.83 (s, 7H), 4.16 (m, 2H), 4.61 (m, 2H), 4.63 (q, J = 7.3 Hz, 2H),
7.61 (m, 4H), 7.91 (d, J = 9.3 Hz, 2H), 8.39 (d, J = 8.6 Hz, 2H),
9.13 (s, 1H), 9.18 (s, 1H) ppm. MS (ESI) m/z 503.3. Analytical LC,
using Prodigy ODS3 column, ACN/H2O eluent at 1 mL/min flow
(containing 0.05% TFA), 20 min gradient 5% ACN to 95% ACN,
monitored by UV absorption at 215 nm, showed 95.7% purity.
Preparation of 4-({[4-(3-Ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo-
[4,5-d]pyrimidin-5-yl)phenyl]carbamoyl}amino)benzoic Acid (31). In a
one-neck flask equipped with reflux condenser were suspended
methyl 4-({[4-(3-ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d]-
pyrimidin-5-yl)phenyl]carbamoyl}amino)benzoate 29 (3.54 g, 7.1
mmol) in THF (20 mL), methanol (5 mL), and NaOH (5N)(5 mL,
25 mmol). The mixture was refluxed for 2 h and cooled to 0 °C and
acidified (pH < 1) with HCl (6 N). During the acidification a white
solid was formed, which was collected by filtration. The filter cake
was washed with water (10 mL) and dried in a vacuum oven to give
the product 31 as an off-white solid (3.34 g, 98% yield). 1H NMR
(DMSO) δ 1.55 (t, J = 7.3 Hz, 3H), 3.83 (m, 4H), 4.16 (m, 2H),
4.62 (m, 2H), 4.63 (q, J = 7.3 Hz, 2H), 7.60 (m, 4H), 7.88 (d, J =
8.3 Hz, 2H), 8.39 (d, J = 8.8 Hz, 2H), 9.24 (s, 1H), 9.27 (s, 1H)
ppm. MS (ESI) m/z 489.3. Analytical LC, using Prodigy ODS3
column, ACN/H2O eluent at 1 mL/min flow (containing 0.05%
TFA), 20 min gradient 5% ACN to 95% ACN, monitored by UV
absorption at 215 nm, showed 97.3% purity.
Preparation of 1-[4-(3-Ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo-
[4,5-d]pyrimidin-5-yl)phenyl]-3-{4-[(4-methylpiperazin-1-yl)carbonyl]-
phenyl}urea (3). 4-({[4-(3-Ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo-
[4,5-d ]pyrimidin-5-yl)phenyl]carbamoyl}amino)benzoic acid
31 (1.26 g, 2.57 mmol), 1-methylpiperazine (335 mg, 3.35
mmol), NEt3 (464 μL, 3.35 mmol), and HOBT (452 mg, 3.35
mmol) were suspended in anhydrous THF (8 mL), and EDCI
(640 mg, 3.35 mmol) was added. The mixture was stirred for a
minimum of 6 h. The solvent was removed on a rota-evapora-
tor, and the crude mixture was dissolved in DMSO (15 mL)
and purified by semipreparative HPLC using (ACN/water/
NH3) to give after combining product fractions and solvent
removal (1.15 g, 79% yield) 1-[4-(3-ethyl-7-morpholin-4-yl-
3H-[1,2,3]triazolo[4,5-d ]pyrimidin-5-yl)phenyl]-3-{4-[(4-methyl-
piperazin-1-yl)carbonyl]phenyl}urea 3 as a white solid. 1H NMR
(DMSO) δ 1.55 (t, J = 7.3 Hz, 3H), 2.20 (s, 3H), 2.33 (m, 4H),
3.50 (m, 4H), 3.83 (m, 4H), 4.15 (m, 2H), 4.62 (m, 2H), 4.63 (q,
J= 7.3 Hz, 2H), 7.35(d, J=8.6Hz, 2H), 7.54(d, J =8.6Hz, 2H),

806 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Dehnhardt et al.
7.61 (d, J = 8.8 Hz, 2H), 8.39 (d, J = 8.8 Hz, 2H), 8.97 (s, 1H),
9.07 (s, 1H) ppm. Anal. Calcd for C29H34FN10O3 (%): C, 61.04; H,
6.01; N, 24.54. Found: C, 60.85; H, 6.40; N, 24.25. MS (ESI) m/z
571.3. HRMS: calcd 571.288 82, found 571.287 38. Analytical LC,
using Prodigy ODS3 column, ACN/H2O eluent at 1 mL/min flow
(containing 0.05% TFA), 20 min gradient 5% ACN to 95% ACN,
UV absorption at 215 nm, showed 99.3% purity.
The reaction mixture was stirred for 15 min, and 4-aminopyridine
(40 mg, 0.46 mmol) and NEt3 (64 μL, 0.46 mmol) were added. The
reaction mixture was stirred for additional 1 h. The solvents were
distilled on a rota-evaporator and the crude mixture was purified by
semipreparative HPLC (NH3-method) to give 1-[4-(3-ethyl-7-mor-
pholin-4-yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidin-5-yl)phenyl]-
1
3-pyridin-4-ylurea 14 (22 mg, 11% yield). H NMR (DMSO) δ
Preparation of (1-Benzylpiperidin-4-yl)-(2-chloro-6-morpho-
lin-4-yl-5-nitropyrimidin-4-yl)amine (9). To a cooled solution
of 4-amino-1-benzylpiperidine (1.25 g, 6.58 mmol) and NEt3
(796 mg, 7.89 mmol) in CH2Cl2 (10 mL) at 0 °C was added, over
a period of 0.5 h, a solution of 2,6-dichloro-5-nitro-4-morpho-
linopyrimidine 8 (1.5 g, 6.58 mmol) dissolved in CH2Cl2 (10
mL). The reaction mixture was stirred for another 1 h at 0 °C and
allowed to warm to 20 °C and stirred for 4 h to drive the reaction
to completion. The product was purified by silica gel column
chromatography by eluting it with CH2Cl2 followed by CH2Cl2/
EA (10:1) to give the product 9 as a yellow solid (2.0 g, 70%
yield). 1H NMR (CDCl3) δ 1.62 (m, 2H), 2.01 (m, 2H), 2.24 (t,
J = 10.8 Hz, 2H), 2.81 (m, 2H), 3.54 (m, 6H), 3.77 (m, 4H), 4.18
(m, 1H), 7.33 (m, 5H), 8.44 (d, J = 7.8 Hz, 1H) ppm. MS (ESI)
m/z 433.1.
1.55 (t, J = 7.3 Hz, 3H), 3.83 (m, 4H), 4.16 (m, 2H), 4.62 (m, 2H),
4.64 (q, 7.3 Hz, 2H), 7.48 (d, J = 5.8 Hz, 2H), 7.61 (d, J = 7.8 Hz,
2H), 7.37-7.42 (m, 4H), 9.23 (s, 1H), 9.27 (s, 1H) ppm. MS (ESI)
m/z 446. Analytical LC, using Prodigy ODS3 column, ACN/H2O
eluent at 1 mL/min flow (containing 0.05% TFA), 20 min gradient
5% ACN to 95% ACN, monitored by UV absorption at 215 nm,
showed 99.2% purity.
Preparation of 1-[4-(3-Isopropyl-7-morpholin-4-yl-3H-[1,2,3]-
triazolo[4,5-d ]pyrimidin-5-yl)phenyl]-3-pyridin-4-ylurea (15). To
a stirred solution of triphosgene (39 mg, 0.13 mmol) in CH2Cl2
(1 mL) was added 4-(3-isopropyl-7-morpholin-4-yl-3H-[1,2,3]-
triazolo[4,5-d ]pyrimidin-5-yl)phenylamine 13c (50 mg, 0.14
mmol) at 25 °C. The reaction mixture was stirred for 15 min,
4-aminopyridine (42 mg, 0.44 mmol) and NEt3 (62 μL, 0.44
mmol) were added, and the reaction mixture was stirred for
an additional 1 h. The solvents were removed in a nitrogen
stream and the crude mixture was purified by semipreparative
HPLC (TFA method) to give 1-[4-(3-isopropyl-7-morpholin-4-
yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidin-5-yl)phenyl]-3-pyridin-
4-ylurea 15 (22 mg, 57% yield). MS (ESI) m/z 460. HRMS: calcd
460.220 40, found 460.219 84. Analytical LC/MS, using Aquasil
C18 column, ACN/H2O eluent at 0.8 mL/min flow (containing
0.1% formic acid), 5.5 min gradient 0% ACN to 100% ACN,
monitored by UV absorption at 254 nm, showed 99.5% purity.
Analytical LC, using X-Bridge BEH column, MeOH/H2O
eluent at 1 mL/min flow (containing 0.05% NH4OAc), 20 min
gradient 10% MeOH to 90% ACN, monitored by UV absorp-
tion at 215 nm, showed 99.4% purity.
Preparation of N 4-(1-Benzylpiperidin-4-yl)-2-chloro-6-mor-
pholin-4-ylpyrimidine-4,5-diamine (10). In a three-neck flask
was suspended under nitrogen atmosphere (1-benzylpiperidin-
4-yl)-(2-chloro-6-morpholin-4-yl-5-nitropyrimidin-4-yl)amine
(1.0 g, 2.3 mmol) and Raney Ni (1 g) in methanol (100 mL). To
the stirring reaction mixture was added slowly N2H4 H2O (1.2
3
mL, 21 mmol, 9 equiv), and the stirring was continued for 1 h to
drive the reduction to completion. The reaction mixture was
filtered over Celite and the filtrate was evaporated to obtain the
product 10 as light-brown solid (900 mg, 97% yield). 1H NMR
(DMSO) δ 1.50 (m, 2H), 1.87 (m, 2H), 2.05 (m, 2H), 2.82 (m,
2H), 2.96 (m, 4H), 3.48 (m, 2H), 3.71 (m, 4H), 4.31 (m, 2H), 6.45
(m, 1H), 7.32 (m, 2H) ppm. MS (ESI) m/z 403.1.
Preparation of 1-(4-(3-Cyclopropyl-7-morpholino-3H-[1,2,3]-
triazolo[4,5-d ]pyrimidin-5-yl)phenyl)-3-(pyridin-4-yl)urea (16).
To a solution of triphosgene (66 mg, 0.223 mmol) in CH2Cl2
(1 mL) was added 4-(3-cyclopropyl-7-morpholino-3H-[1,2,3]-
triazolo[4,5-d ]pyrimidin-5-yl)aniline 13d (150 mg, 0.445 mmol),
and the mixture was stirred for 30 min. Then pyridin-4-amine
(126 mg, 1.34 mmol) and Et3N (187 uL, 1.34 mmol) in CH2Cl2-
(1.5 mL) were added and the mixture was stirred overnight. The
solvents were removed in a nitrogen stream, and the residue was
purified by semipreparative HPLC (TFA method) to give the
product 16 as a white solid (TFA salt) (120 mg, 47% yield). 1H
NMR (DMSO) δ 1.28(m, 2H), 1.39 (m, 2H), 3.82 (m, 4H), 4.04
(hep, J =3.8Hz, 1H), 4.15(m, 2H), 4.58(m, 2H), 7.67 (d, J = 9.0
Hz, 2H), 7.92 (d, J = 7.0 Hz, 2H), 8.43 (d, J = 9.0 Hz, 2H), 8.61
(d, J = 7.0 Hz, 2H), 9.64 (s, 1H), 10.66 (s, 1H) ppm. MS (ESI)
m/z = 458.3. Analytical LC, using Prodigy ODS3 column,
ACN/H2O eluent at 1 mL/min flow (containing 0.05% TFA),
20 min gradient 5% ACN to 95% ACN, monitored by UV
absorption at 215 nm, showed 96.3% purity.
Preparation of 1-[4-(3-Ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo-
[4,5-d]pyrimidin-5-yl)phenyl]-3-pyridin-3-ylurea (17). To a stirred
solution of 4-(3-ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d]-
pyrimidin-5-yl)phenylamine 13b (150 mg, 0.46 mmol) in anhydrous
CHCl3 (2 mL) were added pyridine 3-isocyanate (83 mg, 0.69 mmol)
and NEt3 (97 μL, 0.69 mmol). The mixture was stirred for 18 h and
the solvents were removed in vacuo to obtain the crude product,
which was purified by semipreparative HPLC (NH3 method), to
give 1-[4-(3-ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d ]pyri-
midin-5-yl)phenyl]-3-pyridin-3-ylurea 17 as an off-white solid (55
mg, 26% yield). 1H NMR (DMSO) δ 1.55 (t, J = 7.6 Hz, 3H), 3.83
(m, 4H), 4.18 (m, 2H), 4.62 (m, 2H), 4.64 (q, J = 7.6 Hz, 2H), 7.33
(dd, J = 8.3, 4.0 Hz, 1H), 7.60 (d, J = 8.8 Hz, 2H), 7.97 (ddd, J =
8.3, 2.5, 1.5 Hz, 1H), 8.21 (dd, J = 4.8, 1.5 Hz, 1H), 8.39 (d, J = 8.8
Hz, 2H), 8.62 (d, J = 2.5 Hz, 1H), 8.93 (s, 1H), 9.13 (s, 1H) ppm.
MS (ESI) m/z 446.4. Analytical LC, using Prodigy ODS3 column,
Preparation of 3-(1-Benzylpiperidin-4-yl)-5-chloro-7-morpho-
lin-4-yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidine (11). To a stirred
solution of N4-(1-benzylpiperidin-4-yl)-2-chloro-6-morpholin-
4-ylpyrimidine-4,5-diamine 10 (500 mg, 1.24 mmol) in acetic
acid/water (1:1) (5 mL) at 0 °C was added aqueous (0.5 N)
NaNO2 solution (5 mL, 2.5 mmol), and the reaction mixture was
allowed to stir for 2 h. The off-white solid was collected by
filtration and dried in vacuum to give 11 (510 mg, 100% yield).
MS (ESI) m/z 414.2
Preparation of {3-[3-(1-Benzylpiperidin-4-yl)-7-morpholin-4-
yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidin-5-yl]phenyl}methanol (5).
{3-[3-(1-Benzylpiperidin-4-yl)-7-morpholin-4-yl-3H-[1,2,3]triazolo-
[4,5-d]pyrimidin-5-yl]phenyl}methanol 5 was prepared from 3-(1-
benzylpiperidin-4-yl)-5-chloro-7-morpholin-4-yl-3H-[1,2,3]triazolo-
[4,5-d]pyrimidine (145 mg, 0.35 mmol) and 3-hydroxymethylphe-
nylboronic acid (106 mg, 0.7 mmol) following the above general
procedure for Suzuki reactions. The crude product was dissolved in
DMSO (2 mL), filtered, and purified by semipreparative HPLC
using ACN/water/NH3 as mobile phase. After unification of the
product fraction and solvent removal, the product was obtained as a
white solid (80 mg, 47% yield). 1H NMR (DMSO) δ 2.10 (m, 2H),
2.31 (m, 4H), 2.99 (m, 2H), 3.58 (s, 2H), 3.82 (m, 4H), 4.16 (m, 2H),
4.60 (m, 2H), 4.60 (d, J = 5.8 Hz, 2H), 4.86 (m, 1H), 5.32 (t, J = 5.8
Hz, 1H), 7.27 (m, 1H), 7.34-7.40 (m, 4H), 7.47 (d, J = 5.1 Hz, 2H),
8.32 (td, J= 5.8, 2.1 Hz, 1H), 8.39 (s, 1H) ppm. MS (ESI) m/z486.4.
HRMS: calcd 486.261 75, found 486.2607. Analytical LC, using
Prodigy ODS3 column, ACN/H2O eluent at 1 mL/min flow
(containing 0.05% TFA), 20 min gradient 5% ACN to 95%
ACN, monitored by UV absorption at 215 nm, showed 97.7%
purity.
Preparation of 1-[4-(3-Ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo-
[4,5-d]pyrimidin-5-yl)phenyl]-3-pyridin-4-ylurea (14). To a stirred
solution of triphosgene (68 mg, 0.23 mmol) in CH2Cl2 (5 mL)
was added 4-(3-ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d]-
pyrimidin-5-yl)phenylamine 13b (100 mg, 0.46 mmol) at 0 °C.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 807
ACN/H2O eluent at 1 mL/min flow (containing 0.05% TFA), 20
min gradient 5% ACN to 95% ACN, monitored by UV absorption
at 215 nm, showed 99.1% purity.
solution of 4-(3-ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d]-
pyrimidin-5-yl)phenylamine 13b (150 mg, 0.46 mmol) in anhydrous
CHCl3 (2 mL) was added thienyl 2-isocyanate (87 mg, 0.69 mmol)
and NEt3 (97 μL, 0.69 mmol). The mixture was stirred for 18 h and
the solvent was evaporated in a N2 stream to obtain the crude
product, which was purified by semipreparative HPLC (NH3
method), to give 1-[4-(3-ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo-
[4,5-d]pyrimidin-5-yl)phenyl]-3-(2-thienyl)urea 22 as an off-white
solid (90 mg, 43% yield). 1H NMR (DMSO) δ 1.55 (t, J = 7.3 Hz,
3H), 3.83 (m, 4H), 4.16 (m, 2H), 4.62 (m, 2H), 4.63 (q, J = 7.3 Hz,
2H), 6.56 (dd, J = 3.5, 1.5 Hz, 1H), 6.83 (dd, J = 5.5, 3.5 Hz, 1H),
6.89 (dd, J = 5.5, 1.5 Hz, 1H), 7.59 (d, J= 8.8 Hz, 2H), 8.38 (d, J=
8.8 Hz, 2H), 9.03 (s, 1H), 9.67 (s, 1H) ppm. MS (ESI) m/z 451.4.
HRMS: calcd 451.165 92, found 451.165 62. Analytical LC, using
Prodigy ODS3 column, ACN/H2O eluent at 1 mL/min flow
(containing 0.05% TFA), 20 min gradient 5% ACN to 95%
ACN, monitored by UV absorption at 215 nm, showed 97.8%
purity.
Preparation of 1-(4-(3-Cyclopropyl-7-morpholino-3H-[1,2,3]-
triazolo[4,5-d ]pyrimidin-5-yl)phenyl)-3-(thiophen-2-yl)urea (23).
To a solution of 4-(3-cyclopropyl-7-morpholino-3H-[1,2,3]-
triazolo[4,5-d]pyrimidin-5-yl)aniline 13d (75 mg, 0.222 mmol) in
CHCl3 (1 mL) was added Et3N (46 uL, 0.0.333 mmol) and
2-isocyanatothiophene (42 mg, 0.333 mmol). The mixture was
stirred overnight, and the solvent was evaporated in a N2 stream.
The crude material was purified by HPLC (NH3 method) to give
the product as a white solid (51 mg, 50% yield). 1H NMR (DMSO)
δ 1.27 (m, 2H), 1.38 (m, 2H), 3.82 (m, 4H), 4.04 (hep, J = 3.8 Hz,
1H), 4.15 (m, 2H), 4.57 (m, 2H), 6.56 (dd, J = 3.5, 1.5 Hz, 1H),
6.83 (dd, J = 5.5, 3.5 Hz, 1H), 6.90 (dd, J = 5.5, 1.5 Hz, 1H), 7.60
(d, J = 8.8 Hz, 2H), 8.38 (d, J = 8.8 Hz, 2H), 9.05 (s, 1H), 9.69 (s,
1H) ppm. MS (ESI) m/z = 463.3. Analytical LC, using Prodigy
ODS3 column, ACN/H2O eluent at 1 mL/min flow (containing
0.05% TFA), 20 min gradient 5% ACN to 95% ACN, monitored
by UV absorption at 215 nm, showed 95.7% purity.
Preparation of 1-[4-(3-Isopropyl-7-morpholin-4-yl-3H-[1,2,3]-
triazolo[4,5-d ]pyrimidin-5-yl)phenyl]-3-pyridin-3-ylurea (18). The
compound was prepared as described for compound 16 using
triphosgene (39 mg, 0.13 mmol), 4-(3-isopropyl-7-morpholin-4-
yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidin-5-yl)phenylamine (50 mg,
0.14 mmol), 3-aminopyridine (42 mg, 0.44 mmol), and NEt3 (62
μL, 0.44 mmol) in CH2Cl2 (1 mL) to give 1-[4-(3-isopropyl-7-
morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidin-5-yl)phenyl]-
3-pyridin-3-ylurea 18 (18 mg, 47% yield). MS (ESI) m/z 460.
HRMS: calcd 460.220 40, found 460.219 78. Analytical LC/MS,
using Aquasil C18 column, ACN/H2O eluent at 0.8 mL/min flow
(containing 0.1% formic acid), 5.5 min gradient 0% ACN to
100% ACN, monitored by UV absorption at 254 nm, showed
>99.5% purity. Analytical LC, using X-Bridge BEH column,
MeOH/H2O eluent at 1 mL/min flow (containing 0.05%
NH4OAc), 20 min gradient 10% MeOH to 90% ACN, moni-
tored by UV absorption at 215 nm, showed 96.4% purity.
Preparation of 1-[4-(3-Methyl-7-morpholin-4-yl-3H-[1,2,3]-
triazolo[4,5-d ]pyrimidin-5-yl)phenyl]-3-pyridin-3-ylurea (19). 4-
(3-Methyl-7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-
yl)aniline 13a (60 mg, 0.19 mmol) and 3-pyridylisocyanate (25 mg,
0.20 mmol) were suspended in THF (2 mL) to obtain 1-[4-(3-
methyl-7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)-
phenyl]-3-pyridin-3-ylurea as a solid. The solid was suspended in
diethylether and filtered. It was found to be pure enough (60 mg,
72% yield). Mp 272 °C. 1H NMR (DMSO) δ 3.82 (m, 4H), 4.18
(m, 2H), 4.20 (s, 3H), 4.58 (m, 2H), 7.33 (dd, J = 8.8, 4.8 Hz, 1H),
7.61 (d, J = 8.8 Hz, 2H), 7.97 (m, 1H), 8.21 (m, 1H), 8.40 (d, J =
8.8 Hz, 2H), 8.62 (s, 1H), 8.92 (s, 1H), 9.13 (s, 1H) ppm. MS (ESI)
m/z 432.46. Analytical LC, using Prodigy ODS3 column, ACN/
H2O eluent at 1 mL/min flow (containing 0.05% TFA), 20 min
gradient 5% ACN to 95% ACN, monitored by UV absorption at
215 nm, showed 97.8% purity.
Preparation of 1-[4-(3-Ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo-
[4,5-d]pyrimidin-5-yl)phenyl]-3-(2-methylpyridin-4-yl)urea (24). The
compound was prepared as described for 16 using triphosgene (74
mg, 0.25 mmol), 4-(3-ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-
d]pyrimidin-5-yl)phenylamine 13b (100 mg, 0.31 mmol), 4-amino-
2-methylpyridine (100 mg, 0.93 mmol), and NEt3 (430 μL, 0.44
mmol) in CH2Cl2 (3 mL) to give 1-[4-(3-ethyl-7-morpholin-4-
yl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)phenyl]-3-(2-methylpyri-
din-4-yl)urea 24 (13 mg, 9% yield). 1H NMR (DMSO) δ 1.55 (t,
J = 7.6 Hz, 3H), 2.63 (s, 3H) 3.83 (m, 4H), 4.16 (m, 2H), 4.62 (m,
2H), 4.64 (q, J = 7.6 Hz, 2H), 7.66 (d, J = 8.8 Hz, 2H), 7.77 (m,
1H), 7.81 (m, 1H), 8.44 (d, J = 8.8 Hz, 2H), 8.50 (d, J = 6.5 Hz,
1H), 9.88 (s, 1H), 10.53 (s, 1H) ppm. MS (ESI) m/z 460. Analytical
LC, using Prodigy ODS3 column, ACN/H2O eluent at 1 mL/min
flow (containing 0.05% TFA), 20 min gradient 5% ACN to 95%
ACN, monitored by UV absorption at 215 nm, showed 95.0%
purity.
Preparation of 1-(4-Dimethylaminophenyl)-3-[4-(3-isopropyl-
7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidin-5-yl)phenyl]-
urea (25). The compound was prepared as described for com-
pound 16 using triphosgene (39 mg, 0.13 mmol), 4-(3-isopropyl-
7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidin-5-yl)phe-
nylamine 13c (50 mg, 0.14 mmol), 4-N,N-dimethylaniline (60 mg,
0.44 mmol), and NEt3 (62 μL, 0.44 mmol) in CH2Cl2 (1 mL) to
give 1-(4-dimethylaminophenyl)-3-[4-(3-isopropyl-7-morpholin-
4-yl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)phenyl]urea 25 (26
mg, 63% yield). Analytical LC/MS, using Aquasil C18 column,
ACN/ H2O eluent at 0.8 mL/min flow (containing 0.1% formic
acid), 5.5 min gradient 0% ACN to 100% ACN, monitored by UV
absorption at 254 nm, showed 99.5% purity. Analytical LC, using
X-Bridge BEH column, MeOH/H2O eluent at 1 mL/min flow
(containing 0.05% NH4OAc), 20 min gradient 10% MeOH to
90% ACN, monitored by UV absorption at 215 nm, showed
96.7% purity. MS (ESI) m/z 502. HRMS: calcd 502.267 35, found
502.265 93.
Preparation of 1-(4-(3-Cyclopropyl-7-morpholino-3H-[1,2,3]-
triazolo[4,5-d ]pyrimidin-5-yl)phenyl)-3-(pyridin-3-yl)urea (20).
To a solution of 4-(3-cyclopropyl-7-morpholino-3H-[1,2,3]-
triazolo[4,5-d ]pyrimidin-5-yl)aniline 13d (120 mg, 0.35 mmol)
in CHCl3 (2 mL) were added Et3N (93 μL, 0.668 mmol) and
3-isocyanatopyridine (80 mg, 0.668 mmol). The mixture was
stirred overnight and the solvent was evaporated and purified
by HPLC (TFA method) to give the product 20 as a white solid
(TFA salt) (112 mg, 55% yield). 1H NMR (DMSO) δ 1.27 (m,
2H), 1.38 (m, 2H), 3.82 (m, 4H), 4.04 (hep, J = 3.8 Hz, 1H),
7.63 (d, J = 8.8 Hz, 2H), 7.66 (m, 1H), 8.19 (m, 1H), 8.38 (m,
1H), 8.40 (d, J = 8.8 Hz, 2H), 8.89 (m, 1H), 9.42 (s, 1H), 9.43 (s,
1H) ppm. MS (ESI) m/z = 458.3. Analytical LC, using Prodigy
ODS3 column, ACN/H2O eluent at 1 mL/min flow (containing
0.05% TFA), 20 min gradient 5% ACN to 95% ACN, moni-
tored by UV absorption at 215 nm, showed 95.7% purity.
Preparation of [4-(3-Methyl-7-morpholin-4-yl-3H-[1,2,3]triazolo-
[4,5-d]pyrimidin-5-yl)phenyl]-3-(2-thienyl)urea (21). To a solution of
4-(3-methyl-7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-
5-yl)aniline 13a (60 mg, 0.19 mmol) in CHCl3 (2 mL) was added 2-
thienyl isocyanate (20 mg, 0.20 mmol). The mixture was stirred
overnight. The solvent was evaporated in a N2 stream and the crude
mixture was purified by semipreparative HPLC (NH3 method) to
give the product 21 as a white solid (62 mg, 72% yield). Mp 182 °C.
1H NMR (DMSO) δ 3.83 (m, 4H), 4.16 (m, 2H), 4.19 (s, 3H), 4.59
(m, 2H), 6.56 (dd, J = 3.5, 1.5 Hz, 1H), 6.82 (dd, J = 5.5, 3.5 Hz,
1H), 6.89 (dd, J= 5.5, 1.5 Hz, 1H), 7.59 (d, J= 8.8 Hz, 2H), 8.39 (d,
J = 8.8 Hz, 2H), 9.35 (s, 1H), 9.99 (s, 1H) ppm. MS (ESI) m/z =
437.5. Analytical LC, using Prodigy ODS3 column, ACN/H2O
eluent at 1 mL/min flow (containing 0.05% TFA), 20 min gradient
5% ACN to 95% ACN, monitored by UV absorption at 215 nm,
showed 96.1% purity.
Preparation of 1-[4-(3-Ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo-
[4,5-d]pyrimidin-5-yl)phenyl]-3-(2-thienyl)urea (22). To a stirred

808 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Dehnhardt et al.
Preparation of 1-(4-Hydroxymethylphenyl)-3-[4-(3-isopropyl-
7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)phenyl]urea
(26). The compound was prepared as described for compound 16
using triphosgene (39 mg, 0.13 mmol), 4-(3-isopropyl-7-morpholin-
4-yl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)phenylamine 13c (50
mg, 0.14 mmol), 4-aminobenzyl alcohol (54 mg, 0.44 mmol), and
NEt3 (62 μL, 0.44 mmol) in CH2Cl2 (1 mL) to give 1-[4-(3-
isopropyl-7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidin-
5-yl)phenyl]-3-pyridin-3-ylurea 26 (18 mg, 47% yield). 1H NMR
(DMSO) δ 1.66 (d, J = 6.3 Hz, 6H), 3.83 (m, 4H), 4.16 (m, 2H),
4.43 (s, 3H), 4.60 (m, 2H), 5.19 (hep, J = 6.3 Hz, 1H), 7.24 (d,
J = 8.8 Hz, 2H), 7.42 (d, J = 8.8 Hz, 2H), 7.59 (d, J = 8.8 Hz,
2H), 8.37 (d, J = 8.8 Hz, 2H), 8.70 (s, 1H), 8.96 (s, 1H) ppm. MS
(ESI) m/z 489. HRMS: calcd 489.235 72, found 489.236 10.
Analytical LC/MS, using Aquasil C18 column, ACN/H2O
eluent at 0.8 mL/min flow (containing 0.1% formic acid), 5.5
min gradient 0% ACN to 100% ACN, monitored by UV
absorption at 254 nm, showed 99.5% purity.
215 nm, showed 97.1% purity. MS (ESI) m/z 544. HRMS: calcd
544.277 92, found 544.277 21.
Preparation N-Ethyl-4-({[4-(3-ethyl-7-morpholin-4-yl-3H-[1,2,3]-
triazolo[4,5-d]pyrimidin-5-yl)phenyl]carbamoyl}amino)benzamide (32).
A solution of 4-({[4-(3-ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-
d]pyrimidin-5-yl)phenyl]carbamoyl}amino)benzoic acid 31 (50 mg,
0.102 mmol), Hunig’s base (79 mg, 0.612 mmol), HBTU (116 mg,
0.306 mmol), and NMP (1 mL) was stirred for 1 h at room
temperature, and ethylamine (2 M in THF) (77 μL, 0.15 mmol) was
added. The stirring was continued overnight. The solvents were
removed in a nitrogen stream and the crude mixture was purified by
semipreparative HPLC (TFA method) to give the product 32 as a
white solid (16 mg, 30% yield). 1H NMR (DMSO) δ 1.12 (t, J = 7.3
Hz, 3H), 1.55 (t, J= 7.6 Hz, 3H), 3.28 (m, 1H), 3.83 (m, 4H), 4.16 (m,
2H), 4.62 (m, 2H), 4.64 (q, J = 7.6 Hz, 2H), 7.54 (d, J = 8.8 Hz, 2H),
7.60 (d, J = 8.8 Hz, 2H), 7.80 (d, J = 8.8 Hz, 2H), 8.30 (t, J = 53 Hz,
3H),8.39(d,J= 8.8 Hz, 2H), 9.00 (s, 1H), 9.08 (s, 1H) ppm. MS (ESI)
m/z 516.2. Analytical LC, using Prodigy ODS3 column, ACN/H2O
eluent at 1 mL/min flow (containing 0.05% TFA), 20 min gradient 5%
ACN to 95% ACN, monitored by UV absorption at 215 nm, showed
96.3% purity.
Preparation of 4-({[4-(3-Ethyl-7-morpholin-4-yl-3H-[1,2,3]-
triazolo[4,5-d]pyrimidin-5-yl)phenyl]carbamoyl}amino)-N,N-dimethyl-
benzamide (33). A solution of 4-({[4-(3-ethyl-7-morpholin-4-yl-
3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)phenyl]carbamoyl}amino)-
benzoic acid 31(50 mg, 0.102 mmol), Hunig’s base (79 mg, 0.612
mmol), HBTU (116 mg, 0.306 mmol), and NMP (1 mL) was stirred
for 1 h at room temperature, and HNMe2 (2MinTHF) (77μL, 0.15
mmol) was added. The stirring was continued overnight. The
solvents were removed in a nitrogen stream and the crude mixture
was purified by semipreparative HPLC (TFA method) to give the
product 33 as a white solid (9 mg, 17% yield). 1H NMR (DMSO) δ
1.55 (t, J = 7.6 Hz, 3H), 2.96 (s, 6H), 3.83 (m, 4H), 4.16 (m, 2H),
4.62 (m, 2H), 4.63 (q, J= 7.6 Hz, 2H), 7.37 (d, J= 8.8 Hz, 2H), 7.52
(d, J = 8.8 Hz, 2H), 7.60 (d, J = 8.8 Hz, 2H), 8.39 (d, J = 8.8 Hz,
2H), 8.94 (s, 1H), 9.05 (s, 1H) ppm. MS (ESI) m/z 516.3. Analytical
LC, using Prodigy ODS3 column, ACN/H2O eluent at
1 mL/min flow (containing 0.05% TFA), 20 min gradient 5%
ACN to 95% ACN, monitored by UV absorption at 215 nm,
showed 99.0% purity.
Preparation of N-Butyl-4-({[4-(3-ethyl-7-morpholin-4-yl-3H-[1,2,3]-
triazolo[4,5-d]pyrimidin-5-yl)phenyl]carbamoyl}amino)benzamide (34).
A solution of 4-({[4-(3-ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-
d]pyrimidin-5-yl)phenyl]carbamoyl}amino)benzoic acid 31 (50 mg,
0.102 mmol), Hunig’s base (79 mg, 0.612 mmol), HBTU (116 mg,
0.306 mmol), and NMP (1 mL) was stirred for 1 h at room
temperature, and n-butylamine (14 mg, 0.15 mmol) was added. The
stirring was continued overnight. The solvents were removed in a
nitrogen stream and the crude mixture was purified by semipreparative
HPLC (TFA method) to give the product 34 as a white solid (30 mg,
54% yield). Analytical LC/MS, using Aquasil C18 column, ACN/H2O
eluent at 0.8 mL/min flow (containing 0.1% formic acid), 5.5 min
gradient 0% ACN to 100% ACN, monitored by UV absorption at 254
nm, showed 95.5% purity. Analytical LC, using X-Bridge BEH
column, MeOH/H2O eluent at 1 mL/min flow (containing 0.05%
NH4OAc), 20 min gradient 10% MeOH to 90% ACN, monitored by
UV absorption at 215 nm, showed 95.5% purity. MS (ESI) m/z 544.3.
HRMS: calcd 544.277 92, found 544.278 98.
Preparation of N-[2-(Dimethylamino)ethyl]-4-({[4-(3-ethyl-7-
morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidin-5-yl)phenyl]-
carbamoyl}amino)benzamide (35). A suspension of 4-({[4-(3-
ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidin-5-
yl)phenyl]carbamoyl}amino)benzoic acid 31 (200 mg, 0.40
mmol), N,N-dimethylethylendiamine (87 μL, 0.8 mmol), NEt3
(112 μL, 0.8 mmol), HOBT (110 mg, 0.8 mmol), and EDCI (154
mg, 0.8 mmol) in anhydrous THF (3 mL) gave N-[2-(di-
methylamino)ethyl]-4-({[4-(3-ethyl-7-morpholin-4-yl-3H-[1,2,3]-
triazolo[4,5-d]pyrimidin-5-yl)phenyl]carbamoyl}amino)benzamide
35 as free base. The free base was treated with MeOH/HCl to form
the HCl salt of 35 (89 mg, 37% yield).1H NMR (DMSO) δ 1.55
Preparation of 1-[4-(3-Ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo-
[4,5-d]pyrimidin-5-yl)phenyl]-3-pyridin-4-ylurea (27). To a stirred
solution of triphosgene (39 mg, 0.13 mmol) in CH2Cl2 (1 mL)
was added 4-(3-ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d]-
pyrimidin-5-yl)phenylamine 13b (50 mg, 0.14 mmol) at 25 °C.
The reaction mixture was stirred for 15 min, 4-aminobenzamide
(59 mg, 0.44 mmol) and NEt3 (62 μL, 0.44 mmol) were added, and
the reaction mixture was stirred for an additional 1 h. The solvents
were removed in a nitrogen stream and the crude mixture was
purified by semipreparative HPLC (NH3 method) to give 1-[4-(3-
ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidin-
1
5-yl)phenyl]-3-pyridin-4-ylurea 27 (22 mg, 57% yield). H NMR
(DMSO) δ1.55(t, J= 7.6 Hz, 3H), 3.83 (m, 4H), 4.16 (m, 2H), 4.62
(m, 2H), 4.64 (q, J = 7.6 Hz, 2H), 7.20 (s, 1H), 7.53 (d, J = 8.8 Hz,
2H), 7.60 (d, J = 8.8 Hz, 2H), 7.83 (d, J = 8.8 Hz, 3H), 8.39 (d, J =
8.8 Hz, 2H), 8.99 (s, 1H), 9.07 (s, 1H) ppm. MS (ESI) m/z 488.
Analytical LC, using Prodigy ODS3 column, ACN/H2O eluent at
1 mL/min flow (containing 0.05% TFA), 20 min gradient 5% ACN
to 95% ACN, monitored by UV absorption at 215 nm, showed
96.4% purity.
Preparation of 4-{3-[4-(3-Isopropyl-7-morpholin-4-yl-3H-[1,2,3]-
triazolo[4,5-d]pyrimidin-5-yl)phenyl]ureido}benzamide (28). The
compound was prepared as described for compound 16 using
triphosgene (100 mg, 0.33 mmol), 4-(3-isopropyl-7-morpholin-4-
yl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)phenylamine (140 mg,
0.41 mmol), 4-aminobenzamide (163 mg, 1.2 mmol), and NEt3
(567μL, 4.1 mmol) in CH2Cl2 (5 mL) togive 4-{3-[4-(3-isopropyl-7-
morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidin-5-yl)phenyl]-
ureido}benzamide 28 (68 mg, 33% yield). 1H NMR (DMSO) δ
1.66(d, J = 6.3 Hz, 6H), 3.83 (m, 4H), 4.16 (m, 2H), 4.60 (m, 2H),
5.19 (hep, J = 6.3 Hz, 1H), 7.20 (s, 1H), 7.53 (d, J = 8.8 Hz, 2H),
7.60 (d, J = 8.8 Hz, 2H), 7.83 (d, J = 8.8Hz, 3H), 8.39 (d, J = 8.8
Hz, 2H), 9.00 (s, 1H), 9.08 (s, 1H) ppm. MS (ESI) m/z 502.
Analytical LC, using Prodigy ODS3 column, ACN/H2O eluent
at 1 mL/min flow (containing 0.05% TFA), 20 min gradient 5%
ACN to 95% ACN, monitored by UV absorption at 215 nm,
showed 95.4% purity.
Preparation of 1-[4-(3-Isopropyl-7-morpholin-4-yl-3H-[1,2,3]-
triazolo[4,5-d]pyrimidin-5-yl)phenyl]-3-(4-morpholin-4-ylphenyl)urea
(30). The compound was prepared as described for compound 16
using triphosgene (39 mg, 0.13 mmol), 4-(3-isopropyl-7-morpholin-
4-yl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)phenylamine 13c (50
mg, 0.14 mmol), 4-morpholinylaniline (79 mg, 0.44 mmol), NEt3
(62 μL, 0.44 mmol) in CH2Cl2 (1 mL) to give 1-[4-(3-isopropyl-7-
morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidin-5-yl)phenyl]-
3-(4-morpholin-4-ylphenyl)urea 30 (14 mg, 31% yield). Analytical
LC/MS, using Aquasil C18 column, ACN/H2O eluent at 0.8 mL/
min flow (containing 0.1% formic acid), 5.5 min gradient 0% ACN
to 100% ACN, monitored by UV absorption at 254 nm, showed
98.5% purity. Analytical LC, using X-Bridge BEH column, MeOH/
H2O eluent at 1 mL/min flow (containing 0.05% NH4OAc), 20 min
gradient 10% MeOH to 90% ACN, monitored by UV absorption at

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 809
(t, J = 7.6 Hz, 3H), 2.84 (d, J = 5.0 Hz, 6H), 3.26 (dd, J = 11.3 5.0
Hz, 2H), 3.61 (dd, J = 11.3 5.0 Hz, 1H), 3.83 (m, 4H), 4.16 (m, 2H),
4.62 (m, 2H), 4.63 (q, J=7.6Hz,2H),7.58(d,J= 8.8 Hz, 2H), 7.61
(d, J = 8.8 Hz, 2H), 7.86 (d, J = 8.8 Hz, 2H), 8.39 (d, J = 8.8 Hz,
2H), 8.63 (t, J = 5 Hz, 1H), 9.48 (m, 2H), 9.68 (s, 1H) ppm. MS
(ESI) m/z 559.3. Analytical LC, using Prodigy ODS3 column,
ACN/H2O eluent at 1 mL/min flow (containing 0.05% TFA),
20 min gradient 5% ACN to 95% ACN, monitored by UV
absorption at 215 nm, showed 99.0% purity.
1 mL/min flow (containing 0.05% TFA), 20 min gradient 5% ACN
to 95% ACN, monitored by UV absorption at 215 nm, showed
98.4% purity.
Biology Methods. Enzyme Assays. Enzyme assays were done
in fluorescent polarization (FP) format, adapted from the
Echelon K-1100 PI3K FP assay kit protocol (1). PIP2 and
TAMRA-labeled detector (PIP3-analogue) were purchased
from Echelon. Human PI3KR and PI3Kγ were expressed in
SF9 insect cells and purified by GST and/or FLAG affinity
columns. Human PI3Kβ, PI3Kδ and the two most common
mutant forms of PI3KR (E545K and H1047R) were purchased
from Upstate Biotech. Mouse GRP1 was produced as a GST
fusion protein in E. coli and isolated by GST-Sepharose. Assay
reaction buffer was 20 mM Hepes, pH 7.5, 2 mM MgCl2, 0.05%
CHAPS, and 0.01% βME. Assay STOP/detection buffer was
100 mM Hepes, pH 7.5, 4 mM EDTA, 0.05% CHAPS. FP
assays were run in Nunc 384-well black polypropylene fluor
plates. The FP reaction was run in 20 μL of reaction buffer
containing 20 μM PIP2, 25 μM ATP, and >4% DMSO
(compound solvent). The reaction was run for 30 min at room
temp. The reaction was stopped with 20 μL of STOP/detection
buffer containing 10 nM probe and 40 nM GST-GRP. Assay
plates were incubated for 2 h, and fluorescence polarization was
measured in a Perkin-Elmer Envision plate reader with TAM-
RA-FP filters.
Preparation of 4-({[4-(3-Ethyl-7-morpholin-4-yl-3H-[1,2,3]-
triazolo[4,5-d ]pyrimidin-5-yl)phenyl]carbamoyl}amino)-N-(2-pyr-
idin-2-ylethyl)benzamide (36). A solution of 4-({[4-(3-ethyl-7-
morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidin-5-yl)phenyl]-
carbamoyl}amino)benzoic acid 31 (50 mg, 0.102 mmol), Hunig’s
base (79 mg, 0.612 mmol), and HBTU (116 mg, 0.306 mmol) in
NMP (1 mL) was stirred for 1 h at room temperature, and 2-(2-
aminoethyl)pyridine (19 mg, 0.15 mmol) was added. The stirring
was continued overnight. The solvents were removed in a nitro-
gen stream andthe crude mixturewas purified bysemipreparative
HPLC (NH3 method) to give the product 36 as a white solid (44
mg, 61% yield). 1H NMR (DMSO) δ 1.55 (t, J = 7.5 Hz, 3H),
3.01(t, J = 6.8 Hz, 2H), 3.61 (q, J = 6.8 Hz, 2H), 3.83 (m, 4H),
4.16 (m, 2H), 4.62 (m, 2H), 4.63 (q, J = 7.5 Hz, 2H), 7.28 (m, 1H),
7.31 (d, J = 8.3 Hz, 1H), 7.54 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 8.8
Hz, 2H), 7.77 (m, 3H), 8.39 (d, J = 8.8 Hz, 2H), 8.46 (t, J = 5.7
Hz, 1H), 8.56 (d, J = 5.0 Hz, 1H), 9.02 (m, 2H), 9.10 (s, 1H) ppm.
MS (ESI) m/z 593.3. HRMS: calcd 593.2732, found 593.2727.
Analytical LC, usingProdigy ODS3column, ACN/H2O eluent at
1 mL/min flow (containing 0.05% TFA), 20 min gradient 5%
ACN to 95% ACN, monitored by UV absorption at 215 nm,
showed 98.2% purity.
Preparation of 4-({[4-(3-Ethyl-7-morpholin-4-yl-3H-[1,2,3]-
triazolo[4,5-d ]pyrimidin-5-yl)phenyl]carbamoyl}amino)-N-[2-(4-
methylpiperazin-1-yl)ethyl]benzamide (37). The compound was
prepared as described in the example above using 4-({[4-(3-
ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidin-5-yl)-
phenyl]carbamoyl}amino)benzoic acid 31 (50 mg, 0.1 mmol),
4-methylpiperazinylethanamine (20 mg, 0.2 mmol), NEt3 (30 μL,
0.2 mmol), HOBT (30 mg, 0.2 mmol), and EDCI (40 mg, 0.2
mmol) in anhydrous THF (1 mL) to give 4-({[4-(3-ethyl-7-
morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidin-5-yl)phenyl]-
carbamoyl}amino)-N-[2-(4-methylpiperazin-1-yl)ethyl]benzamide
38 (34 mg, 55% yield). Analytical LC/MS, using Aquasil C18
column, ACN/H2O eluent at 0.8 mL/min flow (containing 0.1%
formic acid), 5.5 min gradient 0% ACN to 100% ACN, monitored
by UV absorption at 254 nm, showed >99.5% purity. Analytical
LC, using X-Bridge BEH column, MeOH/H2Oeluentat1mL/min
flow (containing 0.05% NH4OAc), 20 min gradient 10% MeOH
to 90% ACN, monitored by UV absorption at 215 nm, showed
98.4% purity. MS (ESI) m/z 614.3. HRMS: calcd 614.3310, found
614.3303.
Preparation of N-[3-(Dimethylamino)propyl]-4-({[4-(3-ethyl-
7-morpholin-4-yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidin-5-yl)phenyl]-
carbamoyl}amino)benzamide (38). The compound was prepared
as described in the example above using 4-({[4-(3-ethyl-7-mor-
pholin-4-yl-3H-[1,2,3]triazolo[4,5-d ]pyrimidin-5-yl)phenyl]-
carbamoyl}amino)benzoic acid 31 (100 mg, 0.2 mmol), N,N-
dimethylpropyldiamine (40 mg, 0.4 mmol), NEt3 (56 μL, 0.4
mmol), HOBT (55 mg, 0.4 mmol), and EDCI (77 mg, 0.4
mmol) in anhydrous THF (2 mL) to give N-[3-(dimethyl-
amino)propyl]-4-({[4-(3-ethyl-7-morpholin-4-yl-3H-[1,2,3]triazolo-
[4,5-d]pyrimidin-5-yl)phenyl]carbamoyl}amino)benzamide 38 as
free base. The free base was treated with MeOH/HCl to form the
HCl salt of 38 (39 mg, 32% yield). 1H NMR (DMSO) δ 1.55 (t, J =
7.3 Hz, 3H), 1.90 (m, 2H), 2.76 (d, J = 4.8 Hz, 6H), 3.08 (m, 2H),
3.32 (6, J = 6.3 Hz, 2H), 3.83 (m, 4H), 4.16 (m, 2H), 4.62 (m, 2H),
4.63 (q, J = 7.3 Hz, 2H), 7.56 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 8.8
Hz, 2H), 7.83 (d, J = 8.8 Hz, 2H), 8.38 (d, J = 8.8 Hz, 2H), 8.54 (t,
J= 5.8 Hz, 1H), 9.58 (s, 2H), 9.89 (s, 1H) ppm. MS (ESI) m/z573.4.
Analytical LC, using Prodigy ODS3 column, ACN/H2O eluent at
Selectivity of 3 was evaluated in a 236 human kinase panel
(Invitrogen) at ATP Km for each enzyme.
Cell Culture and Growth Inhibition Assay. The following cell
lines were obtained from ATCC: MDA361, U87MG, A549, and
PC3. All cell lines were propagated at 37 °C in 5% CO2
incubators in growth media recommended by ATCC supple-
mented with penicillin/streptomycin and 10% fetal calf serum.
Cell growth inhibition was determined using the CellTiter 96
Aqueous nonradioactive cell proliferation assay from Promega.
This homogeneous colorimetric method determined the number
of viable cells in proliferation assays. The assay was carried out
in 96-well format following manufacturer’s instructions, with
cell number numbers per well being adjusted on the basis of
growth characteristics of the various cell lines used. Assay end
point data were quantitated after 72 h of compound exposure
using a Victor2 V (Wallac) model 1420 multilabel HTS counter.
Cell Lysis and Western Blotting. Cell lysis was done to enable
biochemical analysis of PI3K/mTOR signaling pathway pro-
teins after exposure of various cell lines to 3. Briefly, 3 ꢀ 106 cells
were seeded onto 6-well microtiter plates (Nunc) 24 h prior to
being exposed to these compounds in complete growth medium.
Cells were exposed to these compounds for 4 h (unless indicated
otherwise in the figures). After exposure to compounds, cell
growth medium was removed and cells were washed twice with
cold (4 °C) PBS. Cell lysis buffer (0.2 mL) was then added to
each microtiter plate well with sufficient mixing to ensure
complete cell lysis. Cell lysis buffer consisted of 20 mM Tris-
HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA,
1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycero-
phosphate, 1 mM Na3VO4, and 1 μg/mL leupeptin. Cell lysates
were then spun in a microfuge for 30 s at 14 000 rpm. Super-
natant (75 μL) was then combined with 30 μL of 3ꢀ protein gel
loading buffer [187.5 mM Tris-HCl (pH 6.8), 6% (w/v) SDS,
30% glycerol, 0.03% (w/v) bromophenol blue, and 125 mM
DTT]. Samples were boiled for 5 min and then subjected to
SDS-PAGE, transferred to nitrocellulose, and probed with
various antibodies specific to the protein and phosphoprotein
components of the PI3K/Akt/mTOR signaling pathway. Anti-
bodies were obtained from Cell Signaling Technology, and they
were anti(R)-Akt, R-phospho(p)-Akt at T308, R-p-Akt at S473,
R-4EBP1, R-p-4EBP1 at T37/46, R-p70 S6 kinase, R-p-p70 S6
kinase at T389, R-PARP, R-cleaved PARP at D214, R-p-ENOS
at S1177, R-p-GSK3R/β at S21/9, and R-p-PRAS40 at T246. All
antibodies were used as recommended by the manufacturer, and
specific antigen/antibody interactions were identified by a

810 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Dehnhardt et al.
horseradish peroxidase (HRP) conjugate secondary antibody
(Ab) that enabled chemiluminescent detection. 3 inhibition of
protein phosphorylation was quantified from Western blots
analyzed on the BioRad Fluor-S MultiImager (Hercules, CA),
using Quantity One Analysis software.
(5) Mallon, R.; Hollander, I.; Feldberg, L. Lucas, J.; Gibbons,
J.; Abraham, R.; Dehnhardt, C. M.; Venkatesan, A. M.;
Delos Santos, E.; Chen, Z.; Santos, O.; Ayral-Kaloustain, S.;
Brooijmans, N.; Mansour T. S. PKI-402, a Dual PI3K/mTOR
Inhibitor. Molecular Targets and Cancer Therapeutics, Boston, MA,
Nov 15-19, 2009; AACR: Philadelphia, PA, 2009; Abstract B142.
(6) Folkes, A. J.; Ahmadi, K.; Alderton, W. K.; Alix, S.; Baker, S. J.;
Box, G.; Chuckowree, I. S.; Clarke, P. A.; Depledge, P.; Eccles,
S. A.; Friedman, L. S.; Hayes, A.; Hancox, T. C.; Kugendradas, A.;
Lensun, L.; Moore, P.; Olivero, A. G.; Pang, J. S.; Patel, G.; Pergl-
Wilson, H.; Raynaud, F. I.; Robson, A.; Saghir, N.; Salphati, L.;
Sohal, S.; Ultsch, M. H; Valenti, M.; Wallweber; Wan, N. C.;
Wiesmann, C.; Workman, P.; Zhyvoloup, A.; Zvelebil, M. J.;
Shuttleworth, S. J. The identification of 2-(1H-indazol-4-yl)-6-(4-
methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-
d ]pyrimidine(GDC 0941) as a potent, selective, orally bioavailable
inhibitor of class I PI3 kinase for the treatment of cancer. J. Med.
Chem. 2008, 51, 5522–5532.
(7) Hayakawa, M.; Kaizawa, H.; Moritomo, H.; Koizumi, T.; Ohishi,
T.; Yamano, M.; Okada, M.; Ohta, M.; Tsukamoto, S.; Raynaud,
F. I; Workman, P.; Waterfield, M. D.; Parker, P. Synthesis and
biological evaluation of pyrido[30,20:4,5]furo[3,2-d ]pyrimidine de-
rivatives as novel PI3 kinase p110R inhibitors. Bioorg. Med. Chem.
Lett. 2007, 17, 2438–2442.
(8) Dehnhardt, C. M.; Venkatesan, A. M.; Chen, Z.; Delos Santos, E.;
Santos, O.; Bursavich, M.; Brooijmans, B.; Mallon, R.; Hollander,
I.; Feldberg, L.; Lucas, J.; Yu, K.; Ayral-Kaloustian, S.; Gibbons,
J.; Abraham, R.; Mansour, T. S. Novel Imidazolopyrimidines as
Dual PI3-Kinase/mTor Inhibitors. Proceedings of the 100th Annual
Meeting of the American Association for Cancer Research, Denver,
CO, Apr 18-22, 2009; AACR: Philadelphia, PA, 2009; Abstract 2017.
(9) Chen, Z.; Venkatesan, A. M.; Dehndardt, C. M.; Ayral-Kalous-
tian, S.; Mansour, T. S.; Brooijmans, N.; Mallon, R.; Hollander, I.;
Yu, K. Discovery of Novel Pyrrolo[3,2-d ]pyrimidine Derivatives
as PI3-Kinase Inhibitors. Abstracts of Papers, 238th National Meet-
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2009; American Chemical Society: Washington, DC, 2009.
Establishment of Xenograft Tumors, Efficacy Studies, and
Biomarker Analysis. Establishment of tumors, group randomi-
zation, tumor, and body weight recording during efficacy
studies was described elsewhere.17 Briefly, all in vivo studies
using nude mice were conducted under an approved Institu-
tional Animal Care and Use Committee protocol. Generally,
tumor cells were suspended at 5 ꢀ 107 cells/mL in cell growth
medium and the cell suspension (0.2 mL) was injected subcuta-
neously (sc) into the flank of a ∼8 week old female nude mice
(Charles River, Wilmington, MA). Mice with tumors greater
than 100 mm3 after 1 week were administered vehicle or
compound by intravenous (iv) tail vein injection at various dose
levels. Injection volumes for 3 were 0.2 mL in vehicle containing
0.5% methylcellulose and 0.4% polysorbate 80 (Tween-80),
with vehicle pH adjusted to 3.5 by lactic acid to fully solubilize
3. In tumor bearing mice tumor weight and body weight were
monitored two or three times weekly. Tumor weight was
calculated by the following formula: tumor weight (mg) = (d 2
ꢀ d /2), where d and d are the shortest and longest diameters of
the tumor, respectively, measured in millimeters. Statistical
analysis (Student’s t test) of the log of the relative tumor growth
compared treated groups with the control group. Statistically
significant reduction in the tumor growth of treated groups
compared to controls (vehicle) was defined as p < 0.05.
Pharmacodynamic (biomarker) measurements were done on
tumor bearing female nude mice administered 3 or vehicle by iv
injections as above. Tumor tissue and normal tissue samples
were collected from euthanized animals, and tissue was homo-
genized, washed twice with cold (4 °C) PBS, and then treated
with cell lysis buffer. Cell lysis buffer was the same as that
described above. As with in vitro samples, cell lysates combined
with gel loading buffer were boiled for 5 min and then subjected
to SDS-PAGE, transferred to nitrocellulose, and probed with
various antibodies specific to the protein and phosphoprotein
components of the PI3K/Akt/mTOR signaling pathway.
(10) Zask, A.; Verheijen, J. C.; Curran, K.; Kaplan, J.; Richard, D. J.;
Nowak, P.; Malwitz, D. J.; Brooijmans, N.; Bard, J.; Svenson, K.;
Lucas, J.; Toral-Barza, L.; Zhang, W.; Hollander, I.; gibbons, J. J.;
Abraham, R. T.; Ayral-Kaloustian, S.; Mansour, T. S.; Yu, K.
ATP-competitive inhibitors of the mammalian target of rapamy-
cin: design and synthesis of highly potent and selective pyrazolo-
pyrimidines. J. Med. Chem. 2009, 52, 5013–5016.
(11) Fan, Q.; Knight, Z. A.; Goldenberg, D. D.; Yu, W.; Mostov, K. E.;
Stokoe, D.; Shokat, K. M.; Weiss, W. A. A dual PI3 kinase/mTOR
inhibitor reveals emergent efficacy in glioma. Cancer Cell 2006, 9,
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(12) Gillman, H.; Blatt, H. Organic Syntheses; Wiley & Sons: New York,
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(13) Robins, R. K.; Dille, K. L.; Christensen, B. E. J. Org. Chem. 1954,
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(14) Fukskovka, K.; Halishek, L.; Krystof, V.; Lenobel, R.; Strnad, M.
Acknowledgment. The authors thank GVK-Bioscience
and Dr. Rocco Galante and Thiru Singanallore for the
scale-up of 5-nitro-2,4,6-trichloropyrimidine 7 and Dr. Anja
Dilley for the scale-up compound 3. Furthermore, we thank
the Pearl River Chemical Technologies group for its support.
PCT Int. Patent. Appl. WO2004/018473, 2004.
(15) Yang, X.; Li, P.; Feldberg, L.; Kim, S. C.; Bowman, M.; Hollander,
I.; Mallon, R.; Wolf, S. F. A directly labeled TR-FRET assay for
monitoring phosphoinositide-3-kinase activity. Comb. Chem. High
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