Design of selective, ATP-competitive inhibitors of akt

Article abstract of DOI:10.1021/jm1003842

This paper describes the design and synthesis of novel, ATP-competitive Akt inhibitors from an elaborated 3-aminopyrrolidine scaffold. Key findings include the discovery of an initial lead that was modestly selective and medicinal chemistry optimization o

Full text of DOI:10.1021/jm1003842

J. Med. Chem. 2010, 53, 4615–4622 4615
DOI: 10.1021/jm1003842
Design of Selective, ATP-Competitive Inhibitors of Akt†
Kevin D. Freeman-Cook,* Christopher Autry, Gary Borzillo, Deborah Gordon, Elsa Barbacci-Tobin, Vincent Bernardo,
David Briere, Tracey Clark, Matthew Corbett, John Jakubczak, Shefali Kakar, Elizabeth Knauth, Blaise Lippa,
Michael J. Luzzio, Mahmoud Mansour, Gary Martinelli, Matthew Marx, Kendra Nelson, Jayvardhan Pandit,
Francis Rajamohan, Shaughnessy Robinson, Chakrapani Subramanyam, Liuqing Wei, Martin Wythes, and Joel Morris
Pfizer Global Research and Development, Eastern Point Road, Groton, Connecticut 06340
Received March 26, 2010
This paper describes the design and synthesis of novel, ATP-competitive Akt inhibitors from an
elaborated 3-aminopyrrolidine scaffold. Key findings include the discovery of an initial lead that was
modestly selective and medicinal chemistry optimization of that lead to provide more selective
analogues. Analysis of the data suggested that highly lipophilic analogues would likely suffer from
poor overall properties. Central to the discussion is the concept of optimization of lipophilic efficiency
and the ability to balance overall druglike propeties with the careful control of lipophilicity in the lead
series. Discovery of the nonracemic amide series and subsequent modification produced an advanced
analogue that performed well in advanced preclinical assays, including xenograft tumor growth
inhibition studies, and this analogue was nominated for clinical development.
Introduction
therapeutic approaches have been reported,12-14 including the
use of rapamycin and small molecule analogues as mTOR inhi-
bitors,15-18 PI3K inhibitors,19,20 PI3K/mTOR dual inhibi-
tors,21-23 and inhibitors that modulate Akt activity in both a
direct ATP-competitive24-30 and an indirect (allosteric)31-38
fashion. Continued reports of tumor growth inhibition with
Akt inhibitors in animal models reflect ongoing attempts to
interfere with this pathway and in some cases to include Akt
inhibitors in combination with other treatment strategies.26,39,40
Akt (PKB)a is a serine/threonine kinase that plays a central
role in promoting cell survival. Akt denotes a family of kinases
(Akt1, Akt2, Akt3) with tissue-specific patterns of expression that
can include the coexpression of multiple family members in nor-
mal tissues and tumors.1-3 Akt is part of a pathway that encom-
passes multiple receptor tyrosine kinases (PI3K and mTOR) and
integrates extracellular signals with cell growth, proliferation, and
survival. Akt activity is often deregulated in malignant cells, and
evidence continues to accumulate that Akt is a promising point of
therapeutic intervention for many cancers.4,5 Akt is located at a
critical junction of multiple oncogenic and tumor suppression
signaling pathways. Deregulation of Akt through inactivation of
PTEN,6 point mutation, or overexpression can result in aberrant
signaling.7 High levels of Akt activity are observed in a large
number of human cancers and are correlated with poor prognosis
and resistance to chemotherapy.8,9
Results and Discussion
Our work started with high-throughput screening against
the kinase domain of Akt1 in an effort to identify ATP-
competitive inhibitors for further medicinal chemistry opti-
mization. A description of this approach, which led to the
discovery of 1 and its subsequent optimization to 2 (Figure 1),
was recently published.39
While spiroindoline 2 represented a substantial improvement
in potency over the initial leads and an important investigational
compound demonstrating tumor growth inhibition (TGI), it
had several undesirable properties. These included a distinct
lack of selectivity against many other kinases (particularly vs the
Because it encompasses such promising targets for cancer
treatment, the PI3K-mTOR pathway in general, and Akt
specifically, has generated substantial interest in the scientific
community.10,11 Withinthe pharmaceutical industry, multiple
†The X-ray coordinates of compound 5 bound to Akt1 and PKA have
been deposited in the Protein Data Bank with accession numbers 3MV5
and 3MVJ. The X-ray coordinates of compound 42 bound to Akt1 have
been deposited in the Protein Data Bank with accession number 3MVH.
*To whom correspondence should be addressed. Phone: 858-526-
4844. Fax: 860-686-5036. E-mail: kevin.freeman-cook@pfizer.com.
aAbbreviations: ACN, acetonitrile; Akt, protein kinase B (encom-
passing Akt1, Akt2, and Akt3); BOC, tert-butyl carbamate; DCM,
dichloromethane; DIPEA, diisopropylethylamine; DMF, dimethylforma-
mide; DMSO, dimethyl sulfoxide; GSK-3, glycogen synthase kinase 3;
hERG, human ether-a-go-go-related gene; HLM Er, human liver micro-
some extraction ratio; HOBt, 1-hydroxybenzotriazole; LAH, lithium alu-
minum hydride; LipE, lipophilic efficiency; MeOH, methanol; mTor,
mammalian target of rapamycin; PI3K, phosphoinositide 3-kinases;
PKA, protein kinase A; PKB, protein kinase B (Akt); PTEN, phosphatase
and tensin homologue; TEA, triethylamine; TFA, trifluoroacetic acid.
Figure 1. Aniline triazole 1 and spiroindoline 2.
r
2010 American Chemical Society
Published on Web 05/19/2010
pubs.acs.org/jmc

4616 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12
Table 1. New Scaffold Identificationa
Freeman-Cook et al.
Table 3. Amine substituent SAR expansiona
a The values are reported as the geometric mean of at least two separate
determinations, with a typical standard variation of less than (30%. The
PKA selectivity ratio is calculated using the following formula: PKA
selectivity = (PKA IC50)/(Akt IC50).
a The values are reported as the geometric mean of at least two separate
determinations, with a typical standard variation of less than (30%.
be utilized for further elaboration. In general, even when
potent starting points (e.g., 6 and 7) were identified, they
exhibited a disappointing level of selectivity for Akt over
PKA. In contrast, the unique selectivity displayed by the
3-aminopyrrolidine series of analogues provided a compelling
entry for further investigation.
Table 2. Initial Kinase Selectivity Dataa
The (3R)-aminopyrrolidine enantiomer 4 was both potent
and 19-fold selective for Akt over PKA. The (3S)-enantiomer 3,
on the other hand, was somewhat less active (240 nM) and
displayed equivalent activity against Akt and PKA. The fact
that 5 displayed a similar level of selectivity to 4 suggested that
the (3R)-aminopyrrolidine was responsible for conferring sel-
ectivity, regardless of small modifications at C-5 of the core
pyrrolopyrimidine. Compounds 2 and 5 were subjected to
further broad kinase profiling, which demonstrated an overall
high level of selectivity for compound 5, as shown in Table 2.
Our initial approach to elaboration of the (3R)-aminopyr-
rolidine scaffold involved derivatization of the basic amine.
By use of standard parallel chemistry transformations, amides,
sulfonamides, and ureas were prepared; the amine was also
alkylated via reductive amination reactions.
Disappointingly, all of these compounds, a sample of which is
shown in Table 3, lost potency against Akt. More significantly,
they exhibited reduced or reversed selectivity versus PKA. The
loss of potency alone may not have been impossible to overcome;
however, the difficulty in achieving selectivity against this target
in general argued that these modifications were disrupting an
important interaction, and thus, it would be extremely challen-
ging to reconstruct a reasonable level of selectivity. Ultimately,
crystal structures of 5 in the kinase domains of both Akt and
PKA (Figure 2a and 2b, respectively) provided a structural
explanation for the observed loss of selectivity.
a Values shown are % inhibition data at 1 M, and reflect the mean of
at least two experiments.
highly homologous family member PKA) and a generally poor
safety profile in animal studies. This compound exhibited
dramatic effects on the GI tract and consequently had a very
low projected therapeutic index (TI). An early hypothesis linked
its low overall kinase selectivity with its low TI, supported by the
finding that a structurally similar but kinase-inactive homologue
of 2had significantly diminished GI toxicity.41 Thus, an effort to
discover new, more selective kinase inhibitors was initiated
utilizing high speed parallel chemistry.39
In this effort, potential new scaffolds were investigated by
replacing the spiroindoline substituent of 2 by thousands of
structurally diverse amines. The data for selected analogues
are shown in Table 1. Most scaffolds were deliberately chosen
to incorporate basic primary or secondary amines that could
The X-ray structure of 5 in the active site of Akt (Figure 2a)
revealed that the C-3 primary amine is displayed in a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12 4617
Figure 2. X-ray structures of 5: (a, left) bound to Akt1; (b,right) bound to PKA.
Scheme 1. Synthesis of 3,3-Disubstituted Analogues
Table 4. Initial SAR in the 3-Amino-3-Anilinomethylpyrrolidine Series
Akt kinase
IC50
chirality (nM)a
PKA
selectivity
ratio
HLM
Erb
compd
R
clogP LipE
19
-H
rac
ent1
ent2
rac
rac
rac
rac
160
3500
56
15.7
2.7
2.3 4.5
2.3 3.2
2.3 5.0
2.8 4.1
3.3 3.9
3.7 3.5
3.7 3.6
4.4 3.4
0.64
0.75
ND
0.60
0.61
0.75
0.73
0.73
19a -H
19b -H
20
21
22
23
24
22.7
31.4
37.5
20
-Me
-Cl
120
58
-CF3
61
50
-iPr
50.9
51.2
pseudoaxial orientationand forms a salt bridge to Glu-234, an
interaction that would not be possible for the bulkier deriva-
tized analogues. On the basis of this structural information,
we proposed that introduction of substitution at the C-3
methine would lock the amine into the required pseudoaxial
conformation while simultaneously providing an additional
vector for optimization of binding potency.
-OPh rac
16
a The values are reported as the geometric mean of at least two separate
determinations, with a typical standard variation of less than (30%. b Er is
the in vitro hepatic extraction ratio, which is obtained from dividing
estimated hepatic blood clearance of test compounds by the human hepatic
blood flow of 20 (mL/min)/kg. Protocols for measuring half-lives in HLM
and further scaling to blood clearance have been published (see ref 44).
Analysis of the crystal structure of PKA with 5 (Figure 2b)
supported the hypothesis that this would be an effective strategy
to maintain selectivity. Examination of the C-3 pyrrolidine
methine proton in the PKA structure indicated that any exten-
sion at that position would likely result in a steric clash with
nearby residues. Modeling the effect of adding even a single
methyl group in place of the C-3 methine revealed a dramatic
decrease in predicted binding affinity for PKA, while the
corresponding binding in Akt was undisturbed. Together, these
observations provided a compelling rationale to prepare the C-3
substituted pyrrolidine analogues.
The approach to derivatization at the 3-position utilized
BOC-protected aminopyrroline 16 bearing a 3-hydroxy-
methyl group (Scheme 1).42 Addition to chloropyrrolopyri-
midine 15 proceeded smoothly. After oxidation and reductive
amination, the Boc was removed to reveal the elaborated
aminopyrrolidine analogues 18.
Figure 3. Pie charts showing the probability of success in higher log P
space: (A) HLM stability. Stable compounds (HLM Er < 0.5) are
represented in green, and the overall percentages of stable compounds
are shown below the respective pie charts. (B) Dofetilide activity. Low
dofetilide activity compounds (Dof , <20% at 3 μM) are represented
in green, and the overall percentages of compounds with low dofetilide
activity are shown below their respective pie charts.
In the first set of compounds, anilines were installed during
the reductive amination step (Table 4). Unsubstituted ani-
line 19 was found to be similarly potent and selective com-
pared to the original aminopyrrolidine 5, even when 19 was
tested as a racemate. When 19 was separated into its two
enantiomers (19a and 19b), one was both more potent and
more selective while the other was substantially poorer in both
respects.43
Since the enantiomerically pure substituted 3-aminopyrroli-
dine confirmed our structural design hypothesis, we undertook

4618 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12
Freeman-Cook et al.
Scheme 2. Modified Synthesis for Preparation of Racemic Amides
Table 5. SAR of Racemic Amide Analoguesa
Akt kinase
IC50 (nM)
compd
R
substituent
substituent position
clogP
LipE
HLM Er
26
27
28
29
30
Me
Ph
Ph
Ph
Ph
none
none
-F
-F
-F
765
62
0
6.1
5.5
5.7
5.4
6.1
ND
ND
0.39
0.39
0.46
1.7
1.6
2
ortho
meta
para
44
38
7.4
2
a The values are reported as the geometric mean of at least two separate determinations, with a typical standard variation of less than (30%.
the synthesis of additional analogues (Table 4). To illustrate a
trend, only a subset of ortho-substituents is shown. These modi-
fications consistently showed improved potency as a result of
increased lipophilicity. Unfortunately, the Akt potency gain was
offset by a corresponding loss in human microsomal stability
relative to 3, a low clearance compound with HLM Er < 0.30.44
Further examination of the data revealed that this was generally
true in this series and not specifically related to the SAR of the
ortho-position of the aniline. Lipophilic substituents generally
provided potency enhancements. However, the net effects of
increased clearance and increased hERG binding45-47 made
these lipophilic analogues unattractive for further study. An
analysis of the trends for in vitro clearance (Figure 3A) and
dofetilide binding (Figure 3B), made evident by binning the
data, revealed that subsequent designs would likely need to be
targeted at clogP < 3. At clogP >3 there was little chance of
achieving both low clearance and low hERG binding.
At this point, the focus changed from identifying the most
potent compounds to identifying those compounds with the
most activity per unit of lipophilicity (the highest LipE).48,49
Theoretically, these would provide the best starting points for
optimization with respect to the logP parameters outlined
above and directly address multiple liabilities of this series.
Additionally, consideration of LipE allows the identification
of truly beneficial SAR as opposed to potency gains that are
simply the consequence of large lipophilicity increases.
hydride produced the valuable amine intermediate 25, which
could be manipulated in a variety of standard transformations.
To reduce the lipophilicity of the series, amide analogues were
prepared by coupling of carboxylic acids followed by TFA-
mediated cleavage of the dimethoxybenzyl protecting group.
Acetamide 26, prepared in racemic form (Table 5), emerged as
a notable analogue from this effort. This compound, while 300-
fold weaker than derivatives that were being prepared in parallel
in the aniline series, was a significant step forward in terms of
LipE (6.1 vs 4.5 for aniline 19). In contrast to compound 19, the
high LipE of 26 coupled with its clogP of 0 suggested that the
judicious addition of lipophilicity within the amide framework
could drive an increase in potency while still remaining within the
defined high probability space (clogP < 3).
The synthesis of 27 suggested that this concept was correct.
The phenyl ring of 27 conferred a 10-fold potency improve-
ment relative to the methyl substituent. This provided the
impetus to begin a full scale effort to optimize the amide in
conjunction with the pyrrolopyrimidine C-5 substituent37
while maintaining an emphasis on lipophilic efficiency.
We engaged in a focused incorporation of simple substitu-
ents expected to maintain clogP < 3. In particular, the ortho
and para positions of the benzamide provided efficient loca-
tions to introduce substituents; this was most apparent with the
fluoro analogues 28, 29, and 30. In all cases, the fluorine
substituent provided an increase in potency relative to the
unsubstituted phenyl group of 27. In the ortho-position, this
improvement incurred no additional lipophilicity, while in the
para-position, the increase in potency was large enough to
compensate for the heightened lipophilicity. The higher LipE
values for both 28 and 30 reflect the role of the ortho- and para-
positions in potency improvement for this series. The small
decrease in LipE for 29 versus 27 indicated that the meta-
position was potentially less promising for manipulation.
Modification of the synthetic route (Scheme 2) gave access to
the valuable amine intermediate 25 in racemic form. Addition
of 3-pyrrolidinol to 4-chloro-5-methyl-7H-pyrrolopyrimidine
proceeded smoothly in the presence of Hunig’s base. Oxidation
of the resulting alcohol to the corresponding ketone with SO3-
pyridine complex was followed by a modified Strecker reaction
with dimethoxybenzylamine using TMS-CN and ZnCl2 as a
Lewis acid.50 Reduction of the nitrile with lithium aluminum

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12 4619
Table 6. SAR of Nonracemic (3S)-Amino-aminomethylbenzamide Analoguesa
Akt kinase
IC50 (nM)
Akt cell
IC50(μM)
compd
R
Ph substituent
PKA selectivity ratio
LipE
HLM Er
clogP
31
32
33
34
35
36
37
38
39
40
41
42
CN
Me
Cl
4-F
4-F
7.8
3.0
2.6
1.4
17.5
11.4
3.3
1.8
7.7
1.0
1.1
0.5
5.74
1.46
0.36
0.65
6.34
1.95
0.35
0.92
1.86
0.31
0.28
0.31
120.3
522
7
0.26
0.36
0.55
0.63
0.38
0.62
0.61
0.67
0.26
0.39
0.47
0.56
1.1
2
6.5
6.3
6.3
6.1
5.3
5.6
5.6
7.3
7.2
6.9
6.9
4-F
4-F
115.4
645
2.3
2.6
1.6
2.6
2.9
3.1
0.9
1.8
2.1
2.4
Et
CN
Me
Cl
4-Cl
4-Cl
ND
51.8
39.8
ND
88.4
ND
50.9
900
4-Cl
4-Cl
Et
CN
Me
Cl
2,4-diF
2,4-diF
2,4-diF
2,4-diF
Et
a The kinase and cell values are reported as the geometric mean of at least two separate determinations, with a typical standard variation of less than (30%.
Once this scan of substituents was complete, sets of com-
pounds were prepared with enantiomerically pure amino-
pyrrolidines51 using multiple pyrrolopyrimidine C-5 core
variations (-CN, -Cl, -Me, -Et). Again, a key aspect of
the design was to keep the clogP below 3. For a given amide,
the trends for potency, selectivity, and in vitro clearance are
very consistent (Table 6). Use of a polar core substituent
(-CN) at C-5 reduced the log P and provided good micro-
somal stability but attenuated the potency (entries 31, 35,
and 39). Compromised potency combined with an apparent
permeability issue (in part related to the higher total polar
surface area) gave weaker cellular activity for these com-
pounds in an assay measuring the phosphorylation of a
known Akt substrate, GSK-3. As the core substituents be-
came more lipophilic (-Me, -Cl, -Et), potency improved
Figure 4. Cocrystal structure of 42 bound to the Akt1 kinase domain.
and stability was diminished. For benzamide substitution, the
4-Cl analogues (35-38) all had lower lipophilic efficiency
xenograft tumor progression. On the basis of these data, 42 was
chosen as the most promising molecule to advance to clinical
development. In vitro, 42 proved highly permeable, and in dog
pharmacokinetic studies (oral at 2 mg/kg, iv at 1.2 mg/kg) the
compound was well absorbed (F = 54%), with moderate
clearance (11.6 (mL/min)/kg) and volume of distribution
(4.8 L/kg), and a half-life of 4.4 h. Compound 42 was subse-
quently evaluated for modulation of Akt in tumors53 and in
multiple in vivo mouse models of antitumor efficacy. It was
active in a PC3 prostate carcinoma xenograft experiment, with
75% TGI observed at 100 mg/kg b.i.d. dosing for 10 days. In a
colorectal carcinoma (Colo205) xenograft study, 42 produced
60% TGI at 150 mg/kg b.i.d. after 10 days. Most intriguingly,
in combination with rapamycin (10 mg/kg, ip), 75 mg/kg b.i.d.
(10 days) of 42 resulted in 98% TGI in an additional PC3
prostate carcinoma xenograft study compared to 56% TGI and
66% TGI with 42 and rapamycin as single agents. There were
no significant toxicological side effects observed in these studies
beyond modest weight loss in the highest dosing groups.
compared with the corresponding 4-F compounds (31-34),
similar to the results observed in the racemic series. Of the
substituents examined, the 2,4-difluoro analogues (39-42)
exhibited the best overall potency and metabolic profile.
All of the compounds in Table 6 displayed good selectivity
for PKA, and some had extraordinary selectivity. Particularly
notable was compound42, which achieved 900-fold selectivity
for Akt over PKA, presumably because of the increased steric
demand of the ethyl core substituent being better tolerated in
Akt than in PKA. This, in conjunction with the beneficial effect
ofthe2,4-difluorosubstitutionforAkt, providedanextremely
selective analogue. After submission of 42 to a panel of 226
kinases, only seven (CDK7, MSK1, MSK2, PKG1R, RSK2,
RSK3, and PKA) showed >50% inhibition at 1 μM. After
determinination of IC50 values, 42 demonstrated >100-fold
selectivity against Akt for all seven kinases.52 Cocrystallization
of 42 withAkt (Figure4) showed thatthe C-3 amine was enga-
ged in the Glu-234 salt bridge, similar to the early analogues.
As expected, the 2,4-difluorobenzamide was extended into the
accessible pocket in Akt.
Conclusion
On the basis of these in vitro data, a small set of these ad-
vanced analogues were put into further studies, including rat
and dog pharmacokinetic studies, as well as in vivo models of
In summary, we have outlined the optimization of 2-4 to
advanced molecules such as 42 using a combination of both

4620 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12
Freeman-Cook et al.
structure- and physical-property-based design parameters.
Substituents in place of the C-3 methine proton of the amino-
pyrrolidine lead 4 served to lock the pseudoaxial disposition of
the amine and to provide a platform for optimizing the physical
properties of the resulting molecules. Critical in this effort was
the understanding of overall lipophilicity and its effect on both
potency and in vitro ADME properties. During lead optimiza-
tion, changes in LipE highlighted valuable structural modifica-
tions and discouraged the overuse of lipophilicity, which ulit-
mately provided more balanced molecules. Compound 42 pro-
vided significantly enhanced selectivity for Akt relative to
earlier leads such as spiroindoline 2. As a result, 42 was nomi-
nated for clinical development and represents a broadly selec-
tive, potent, ATP-competitive Akt inhibitor.
peptide), and 5 μL of recombinant PKA catalytic subunit (Up-
state Biotechnology, catalog number 14-440) in reaction buffer
are combined. After a 90 min incubation, IMAP beads are
added and plates are read (lamp filter, 544 nm; emission filter,
615 nm). All IC50 values are the geometric mean of at least n=2
determinations.
Akt Cell Assay. The phosphorylation status of an Akt sub-
strate, GSK-3R, in the U87 glioblastoma line was measured as
an indicator of cellular activity. U87 cells (10 000) were plated
overnight in DMEM medium containing 15% fetal calf serum
(FCS) and incubated with compound (seven concentrations plus
vehicle) for 2 h in DMEM medium lacking FCS. After cell lysis,
45% of the lysate was transferred to a 96-well, flat bottom, black
polystyrene plate (Costar, catalog number 3295) previously
coated in a stepwise fashion with 100 μL/well of goat antirabbit
IgG (Pierce Corp., catalog number 31210) diluted to 5 μg/mL in
carbonate-bicarbonate buffer (Sigma, catalog number C-3041),
followed by TBST washing (Sigma, catalog number T-9039),
blocking in 5% BSA/TBST, and addition of 100 μL/well of a
phosphospecific antibody to GSK-3R/β (Cell Signaling Tech-
nology, catalog number 9331) used at 0.06 μg/mL. After sub-
sequent washing and blocking steps, cell lysate was added and
incubated for 1 h at ambient temperature with shaking. The
plates were then washed four times with TBST. To detect bound
phospho-GSK-3R, 70 μL of a 1:1000 dilution of anti-GSK-3R
was added (Santa Cruz, catalog number SC-5264). Plates were
incubated for 1 h at ambient temperature with shaking, and
washed four times with TBST. After washing, 70 μL/well of a
1:5000 dilution of goat antimouse-HRP (Jackson Labs, catalog
number 115-035-146) in 5% BSA/TBST was added. Plates were
incubated for 1 h at ambient temperature with shaking, washed,
and 70 μL/well of SuperSignal ELISA PICO Maximum Sensi-
tivity Substrate (Pierce Corp., catalog number 37075) was
added. Signal was allowed to develop for 1 min, and plates were
read on a Victor luminometer. The signal obtained from wells
containing lysis buffer only (no cell control) was subtracted
before IC50 values were calculated. All IC50 values are the
geometric mean of at least n = 2 determinations.
Experimental Section
General Experimental Details. All solvents and reagents were
obtained from commercially available sources and used without
further purification. Reactions were carried out under nitrogen
atmosphere unless otherwise described. Flash chromatography
purifications were performed on prepacked silica gel cartridges
on ISCO CombiFlash Companion systems using the indi-
cated mobile phases. HPLC analysis was performed on an
Agilent HP1100 system with an Agilent Zorbax SB-CN column
(4.6 mm ꢀ 75 mm, 3.5 μm, 1.5 mL/min flow rate), mobile phase
(H2O/0.1% HClO4)/(ACN/0.1% HClO4) (90:10 at 0 min to
10:90 at 5 min). All tested compounds prepared as solids were
determined to be >95% pure at 210 nm. LC/MS STD refers to a
Polaris C18A column (2.0 mm ꢀ 20 mm, 5 μm, 1.0 mL/min flow
rate); mobile phase A of 94.95% H2O, 5% CAN, 0.05% formic
acid; mobile phase B of 99.95% CAN, 0.05% formic acid; A/B
ratios of 95:5 at 0 min, 80:20 at 1 min, 50:50 at 2.3 min, and 0:100
at 3.6 min, with compound detection by UV and MS. LC/MS
Polar refers to a Polaris C18A column (2.0 mm ꢀ 20 mm, 5 μm,
1.0 mL/min flow rate), mobile phase A of 94.95% H2O, 5%
ACN, 0.05% formic acid; mobile phase B of 99.95% ACN,
0.05% Fformic acid; A/B ratios of 95:5 at 0 min, 80:20 at 2 min,
50:50 at 2.3 min, and 0:100 at 3.6 min, with compound detec-
tion by UV and MS. Mass spectral data were collected on a
Micromass ADM atmospheric pressure chemical ionization
instrument (LRMS APCI). NMR spectra were generated on
a Varian 400 MHz or a Varian 500 MHz instrument as indi-
cated. Chemical shifts were recorded in ppm relative to tetra-
methylsilane (TMS) with multiplicities given as s (singlet), bs
(broad singlet), d (doublet), t (triplet), dt (double of triplets), m
(multiplet).
Assay Description. Akt1 Kinase Assay. A fluorescence polar-
ization IMAP type assay is used. An amount of 15 μL of diluted
test compound in DMSO is mixed with 60 μL of reaction buffer
(10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.1 mM EGTA,
0.01% Triton-X100, 1 mM DTT). Then 5 μL of the compound/
buffer mixture, 10 μL of a solution containing 4 μM ATP and
40 nM fluorescent-labeled Crosstide (Tamara-labeled GRPR-
TSSFAEG peptide), and 5 μL of Akt1 protein (lacking the
pleckstrin homology (PH) domain, containing an Asp at posi-
tion 473, and prephosphorylated at Thr 308) in reaction buffer
are combined. After a 90 min incubation, IMAP beads are
added and plates are read (lamp filter, 544 nm; emission filter,
615 nm). The same procedure can be applied to full length Akt1
to provide similar results. All IC50 values are the geometric mean
of at least n = 2 determinations.
1-(5-Methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-3((phenylamino)-
methyl)pyrrolidin-3-amine (19). A mixture of aniline (0.16 mL,
˚
1.77 mmol) and 3 A molecular sieves (0.5 g) was added to a
solution of tert-butyl 3-formyl-1-(5-methyl-7H-pyrrolo[2,3-d]pyri-
midin-4-yl)pyrrolidin-3-ylcarbamate 17 (0.5 g, 1.45 mmol),
glacial acetic acid (1 mL), and MeOH (9 mL). The resulting
reaction mixture was stirred at 25 °C overnight, treated with
MP-cyanoborohydride (1.45 g, 2.5 mmol/g, 3.65 mmol), and
stirred for an additional 5 h. The mixture was filtered, and the
solids were rinsed with MeOH. The combined organic phases
were concentrated to provide tert-butyl 1-(5-methyl-7H-pyrrolo-
[2,3-d]pyrimidin-4-yl)-3-((phenylamino)methyl)pyrrolidin-3-yl-
carbamate, which was treated directly with DCM (10 mL) and
TFA (10 mL), and the resultant reaction mixture was stirred at
25 °C for 3 h. The mixture was concentrated, and the residue was
purified by chromatography on silica gel (eluting with dichlor-
omethane, with a gradient ramp to 1% aqueous NH4OH, 29%
MeOH, 70% DCM) to provide 1-(5-methyl-7H-pyrrolo[2,3-
d]pyrimidin-4-yl)-3-((phenylamino)methyl)pyrrolidin-3-amine
(19). Yield: 325 mg, 1.01 mmol, 70%. APCI MSþ (M þ 1)=
323.2. 1H NMR (500 MHz, methanol-d4) δ ppm 2.03 (1H, m),
2.21 (1H, m), 2.38 (3H, s), 3.37 (2H, m), 3.68 (1H, d), 3.89 (1H,
d), 3.91(1H, m), 4.05 (1H, q), 6.60(1H, t), 6.69 (2H, d), 6.92 (1H,
s), 7.08 (2H, m), 8.08 (1H, s).
N-((3-Amino-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-
pyrrolidin-3-yl)methyl)benzamide (27). A solution of 3-(amino-
methyl)-N-(2,4-dimethoxybenzyl)-1-(5-methyl-7H-pyrrolo[2,3-
d]pyrimidin-4-yl)pyrrolidin-3-amine (50 mg, 0.13 mmol) in
DMF (1.2 mL) was treated with 1-hydroxybenzotriazole (HOBt)
(26 mg, 0.19 mmol), benzoic acid (16 mg, 0.13 mmol), and PS-
carbodiimide (160 mg, 0.25 mmol). The resultant reaction mixture
was stirred at 25 °C for 6 h, treated with MP-carbonate (160 mg,
PKA Kinase Assay. A fluorescence polarization IMAP (Mole-
cular Devices Corp.) type assay is used. An amount of 15 μL of
diluted test compound in DMSO is mixed with 60 μL of reaction
buffer (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.1 mM EGTA,
0.01% Triton-X100, 1 mM DTT). Then 5 μL of the compound/
buffer mixture, 10 μL of a solution containing 4 μM ATP and
40 nM fluorescent-labeled Kemptide (Tamara-labeled LRRASLG

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12 4621
0.5 mmol), and stirred overnight. The mixture was filtered, and the
solids were rinsed with MeOH. The combined filtrates were evapo-
rated, and the residue was treated with TFA (0.5 mL) and heated at
80 °C for 3 h. The reaction mixture was concentrated, and the
residue was purified by preparative RP-HPLC (TFA/ACN/H2O)
to provide N-((3-amino-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-
yl)pyrrolidin-3-yl)methyl)benzamide (27). Yield: 12 mg, 26% for
two steps. MSþ (M þ 1)þ: 351.3. tR (LCMS standard): 0.7 min. 1H
NMR (400 MHz, methanol-d4) δ ppm 8.03 (s, 1 H), 7.82 (d, J =
7.48 Hz, 2 H), 7.53 (t, J=7.06Hz, 1H),7.40-7.49 (m, 2 H), 6.90 (s,
1 H), 3.85-4.05 (m, 3 H), 3.54-3.68 (m, 3 H), 3.31 (s, 2 H), 2.39 (s,
3 H), 2.08-2.19 (m, 1 H), 1.92-2.01 (m, 1 H).
N-((3-Amino-1-(5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyr-
rolidin-3-yl)methyl)-4-fluorobenzamide (30). Compound 30 was
prepared by an identical sequence of reactions as was used to
prepare 27 but using 4-fluorobenzoic acid in place of benzoic
acid. 1H NMR (DMSO-d6) δ ppm 11.3 (bs, 1H), 8.56 (t, J = 5.7
Hz, 1H), 8.04 (s, 1H), 7.94 (dd, J = 8.8, 5.7 Hz, 2H), 7.30 (dd,
J = 8.8, 8.8 Hz, 2H), 6.93 (s, 1H), 3.88-3.82 (m, 1H), 3.75-3.71
(m, 1H), 3.68 (d, J = 10.9 Hz, 1H), 3.47 (m, 3H), 2.31 (s, 3H),
1.95-1.73 (m, 4H).
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C.; Coppola, D.; Nicosia, S. V.; Cheng, J. Q. Frequent activation of
AKT2 and induction of apoptosis by inhibition of phosphoinosi-
tide-3-OH kinase/Akt pathway in human ovarian cancer. Onco-
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(S)-N-((3-Amino-1-(5-ethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-
pyrrolidin-3-yl)methyl)-2,4-difluorobenzamide (42). A mixture of
(R)-N-((3-aminopyrrolidin-3-yl)methyl)-2,4-difluorobenzamide
(16 g, 49 mmol), 4-chloro-5-ethyl-7H-pyrrolo[2,3-d]pyrimidine
(8.9 g, 49 mmol), and sodium bicarbonate (20.5 g, 244 mmol) in
ethanol (150 mL) was refluxed for 10 h. The mixture was then
filtered hot through Celite, and the filtrate was concentrated
under reduced pressure. The resultant residue was partitioned
between EtOAc (100 mL) and water (200 mL), and the organic
phase was collected. The aqueous phase was extracted with ethyl
acetate (2 ꢀ 100 mL), and the combined organic extracts were
washed with water (2 ꢀ 100 mL), brine (80 mL), dried over Na2-
SO4, filtered, and concentrated under reduced pressure to
provide (S)-N-((3-amino-1-(5-ethyl-7H-pyrrolo[2,3-d]pyrimi-
din-4-yl)pyrrolidin-3-yl)methyl)-2,4-difluorobenzamide (42) as
a pale-yellow solid. Yield: 17.4 g, 89.3% . TLC Rf = 0.42 (10%
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Zask, A.; Chaudhary, I.; Wang, J.; Di, L.; Young, M.; Ruppen, M.;
Mansour, T. S.; Gibbons, J. J.; Yu, K. Hybrid inhibitors of
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of rapamycin (mTOR): design, synthesis, and superior antitumor
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1
methanol/CH2Cl2). APCI MSþ (M þ 1) = 402.1. H NMR
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(500 MHz, methanol-d4) δ ppm 8.07 (s, 1 H), 7.80 (q, J = 8.64
Hz, 1 H), 7.10 (q, J = 8.12 Hz, 2 H), 6.94 (s, 1 H), 3.98 (m, 3 H),
3.65 (m, 3 H), 2.87 (q, J = 7.08 Hz, 2 H), 2.17 (m, 1 H), 1.97 (m, 1
H), 1.30 (t, J = 7.26 Hz, 3 H).
(18) Chresta, C. M.; Davies, B. R.; Hickson, I.; Harding, T.; Cosulich,
S.; Critchlow, S. E.; Vincent, J. P.; Ellston, R.; Jones, D.; Sini, P.;
James, D.; Howard, Z.; Dudley, P.; Hughes, G.; Smith, L.;
Maguire, S.; Hummersone, M.; Malagu, K.; Menear, K.; Jenkins,
R.; Jacobsen, M.; Smith, G. C. M.; Guichard, S.; Pass, M.
AZD8055 is a potent, selective, and orally bioavailable ATP-
competitive mammalian target of rapamycin kinase inhibitor
with in vitro and in vivo antitumor activity. Cancer Res. 2010, 70,
288–298.
Acknowledgment. The authors want to thank Katherine
Brighty for extremely helpful advice in manuscript prepara-
tion and editing.
(19) Marone, R.; Cmiljanovic, V.; Giese, B.; Wymann, M. P. Targeting
phosphoinositide 3-kinase;moving towards therapy. Biochim.
Biophys. Acta, Proteins Proteomics 2008, 1784, 159–185.
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S. A.; Friedman, L. S.; Hayes, A.; Hancox, T. C.; Kugendradas,
A.; Lensun, L.; Moore, P.; Olivero, A. G.; Pang, J.; Patel, S.;
Pergl-Wilson, G. H.; Raynaud, F. I.; Robson, A.; Saghir, N.;
Salphati, L.; Sohal, S.; Ultsch, M. H.; Valenti, M.; Wallweber,
H. J. A.; 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,
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Supporting Information Available: Experimental details for
key intermediates and for compounds 31-41; details of the
X-ray crystallography and pharmacokinetic and pharmacody-
namic studies. This material is available free of charge via the
Internet at http://pubs.acs.org.
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