Identification of 4-(4-aminopiperidin-1-yl)-7H-pyrrolo[2,3-d]pyrimidines as selective inhibitors of protein kinase B through fragment elaboration

Article abstract of DOI:10.1021/jm701437d

Fragment-based screening identified 7-azaindole as a protein kinase B inhibitor scaffold. Fragment elaboration using iterative crystallography of inhibitor-PKA-PKB chimera complexes efficiently guided improvements in the potency and selectivity of the compounds, resulting in the identification of nanomolar 6-(piperidin-1-yl)purine, 4-(piperidin-1-yl)-7-azaindole, and 4-(piperidin-1-yl)pyrrolo[2,3-d]pyrimidine inhibitors of PKBβ with antiproliferative activity and showing pathway inhibition in cells. A divergence in the binding mode was seen between 4-aminomethylpiperidine and 4-aminopiperidine containing molecules. Selectivity for PKB vs PKA was observed with 4-aminopiperidine derivatives, and the most PKB-selective inhibitor (30-fold) showed significantly different bound conformations between PKA and PKA-PKB chimera.

Full text of DOI:10.1021/jm701437d

J. Med. Chem. 2008, 51, 2147–2157
2147
Identification of 4-(4-Aminopiperidin-1-yl)-7H-pyrrolo[2,3-d]pyrimidines as Selective Inhibitors
of Protein Kinase B through Fragment Elaboration
John J. Caldwell,† Thomas G. Davies,§ Alastair Donald,† Tatiana McHardy,† Martin G. Rowlands,† G. Wynne Aherne,†
Lisa K. Hunter,† Kevin Taylor,† Ruth Ruddle,† Florence I. Raynaud,† Marcel Verdonk,§ Paul Workman,† Michelle D. Garrett,†
and Ian Collins*,†
Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, U.K.,
and Astex Therapeutics Ltd., 436 Cambridge Science Park, Milton Road, Cambridge CB4 0QA, U.K.
ReceiVed NoVember 14, 2007
Fragment-based screening identified 7-azaindole as a protein kinase B inhibitor scaffold. Fragment elaboration
using iterative crystallography of inhibitor-PKA-PKB chimera complexes efficiently guided improvements
in the potency and selectivity of the compounds, resulting in the identification of nanomolar 6-(piperidin-
1-yl)purine, 4-(piperidin-1-yl)-7-azaindole, and 4-(piperidin-1-yl)pyrrolo[2,3-d]pyrimidine inhibitors of PKBꢀ
with antiproliferative activity and showing pathway inhibition in cells. A divergence in the binding mode
was seen between 4-aminomethylpiperidine and 4-aminopiperidine containing molecules. Selectivity for
PKB vs PKA was observed with 4-aminopiperidine derivatives, and the most PKB-selective inhibitor (30-
fold) showed significantly different bound conformations between PKA and PKA-PKB chimera.
Introduction
flavins,17 isoquinoline-5-sulfonamides,18,19 6-phenylpurines,20
and 4-phenylpyrazoles.21 The application of structure-based
design using crystallographic data from PKA14b,f,g,18 or
PKA-PKB chimeras15,20,21 has been featured in the develop-
ment of some of these chemical series. The high sequence
homology between PKA and PKB in the ATP binding site
(∼80%) supports the use of PKA or PKA-PKB chimeric
proteins as surrogates of PKB. In this present work, a PKA-PKB
chimera with three PKA f PKB mutations in the kinase active
site of PKA was constructed (Val123 f Ala, Val104 f Thr,
Leu173 f Met). An additional mutation, Gln181 f Lys, was
incorporated outside the ATP-binding site to ameliorate the
potential for Gln181 to disturb inhibitor binding in the presence
of the Val123 f Ala substitution.22 We have recently shown
that a PKB-selective inhibitor10 exhibits similar binding modes
in both PKB and this PKA-PKB chimera, with a distinct
binding conformation seen in PKA.23 The major difference in
binding mode was the repositioning of an inhibitor indole
substituent within the ribose-binding pocket of the kinases,
resulting from the Leu173 f Met switch between PKA and
PKB. Although caution is required in translating crystallographic
data observed with surrogates such as PKA or PKA-PKB
chimeras to the interpretation of the structure–activity relation-
ships of selective PKB ligands, the surrogates have nevertheless
proved to be useful tools for the discovery of new inhibitor
scaffolds. For convenience, a PKB-PKA chimera was therefore
used for the iterative crystallographic studies described in this
work. We have described the identification of 7-azaindole as a
very low affinity, ATP-competitive ligand (PKBꢀ IC50 > 100
µM) in a crystallographic fragment screen.20 The fragment was
elaborated through structure-based design to the 6-phenylpurine
1 and subsequently to the potent PKB/PKA inhibitor 2 with
activity in cells (Chart 1). The lead compound 2 and structurally
related 4-phenylpyrazoles21 were unselective for PKB inhibition
over PKA. In this paper we describe the development of the
6-phenylpurine 1 into an alternative series of 4-piperidin-1-yl-
7H-pyrrolo[2,3-d]pyrimidines with nanomolar potency and high
ligand efficiency,24 using iterative ligand-protein crystal-
lography with the PKA-PKB chimera to guide improvements
Signaling through the PI3K-PKB-mTORacascade of intra-
cellular kinases is a major component in the control of cell
proliferation and survival.1–4 It is clear that deregulation of this
pathway is fundamentally associated with the development of
several human cancers. For example, overexpression of the
upstream tyrosine receptor kinase ERBB2 in breast and other
tumors leads to constitutive signaling through protein kinase
B.5 Amplification or mutation of the gene encoding the PI3K
110R catalytic subunit, again leading to overactivation of the
pathway, is frequently observed in human tumors, particularly
ovarian and cervical carcinoma.1,6 Deletion at the genetic level
of the lipid phosphatase PTEN removes a negative regulator
that normally functions to dephosphorylate the 3′-phosphati-
dylinositol substrates of PI3K and prevent PKB activation.
Deletion of PTEN is frequently observed in human tumors,
especially glioblastoma, endometrial, and prostate cancers.1,4,7
PKB, particularly the ꢀ isoform, is itself commonly amplified
at the genetic level, overexpressed, or overactivated.1,8 Evidence
of the therapeutic effect of inhibitors targeting this pathway has
been reported. Analogues of the natural product rapamycin that
inhibit mTOR function have shown antitumor efficacy in early
clinical trials.2 Dual inhibitors of PI3K/mTOR kinase function
have shown antitumor activity in in vivo models.9 An ATP-
competitive inhibitor of PKB has also shown efficacy in animal
models.10 As a result of these observations, the PI3K-PKB-
mTOR pathway is established as a promising locus of action
for new molecularly targeted anticancer agents.3,11–13
There is significant interest in the discovery of new small
molecule inhibitors of PKB, which may be ATP-competitive
or may interact with regulatory domains in the protein.11 Several
classes of ATP-competitive inhibitors have been described,
including pyridines,10,14 azapanes,15 indazole-4,7-diones,16 toxo-
* To whom correspondence should be addressed. Phone: +44 (0)208722-
4317. Fax: +44 (0)2087224126. E-mail: Ian.Collins@icr.ac.uk.
†
The Institute of Cancer Research.
Astex Therapeutics Ltd.
§
a Abbreviations: PKB, protein kinase B (also known as Akt); PI3K,
phosphatidylinositol-3 kinase; mTOR, mammalian target of rapamycin;
PKA, protein kinase A.
10.1021/jm701437d CCC: $40.75
2008 American Chemical Society
Published on Web 03/18/2008

2148 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Chart 1. Development of 6-Phenylpurine PKBꢀ Inhibitors20
Caldwell et al.
As expected from other PKB inhibitor structures, the amine
groups of 5 and 6 occupied a negatively charged region lined
with acceptors. In the benzylamine 1 the secondary amine
interacted directly with the Glu127 carboxylate and the backbone
amide carbonyl of Glu170, while a water-mediated hydrogen
bond was formed to the side chain of Asn171 (interatomic
distances less than 3 Å). The side chain of Asp184 was oriented
away from the inhibitor in this structure, forming a salt bridge
with Lys72. In contrast, the amine of the aminomethylpiperidine
5 occupied the position of the water molecule seen in
1-PKA-PKB. The side chain of Asp184 now adopted an
alternative rotamer that brought the acid group into contact with
the inhibitor. Direct hydrogen bonding to the backbone carbonyl
of Glu170 was maintained, and a contact was made with the
side chain of Asn171, but the hydrogen-bonding interaction with
the acid of Glu127 was lost. When the terminal amine was
attached directly to the piperidine in 6, the amine interactions
resembled that of 1-PKA-PKB, with direct contacts to the
Glu127 acid and the Glu170 carbonyl, and a water-mediated
interaction to Asn171. As in 1-PKA-PKB, the side chain of
Asp184 was rotated away from the inhibitor in this structure.
These divergent binding patterns in the ribose pocket were also
observed for isoquinoline-5-sulfonamide inhibitors and reflect
a broad region of energetically acceptable positions within this
acceptor-rich subsite.18
to inhibitor activity. Within this series, selective and unselective
compounds with respect to PKB vs PKA were identified.
Results and Discussion
The addition of a benzylamine substituent onto the bicyclic
hinge-binding fragment was an important step leading to 2.20
Our studies with 6-phenylpurines and isoquinoline-5-sulfon-
amide inhibitors of PKB18 had shown that several productive
binding positions were possible for the basic amine, reflecting
the presence of two prominent acidic residues, Asp184 and
Glu127 (PKA-PKB chimera numbering), in the ribose binding
site of PKB.25 We therefore sought to investigate alternatives
to the 6-phenyl group as spacers between the purine bicycle
and the amine in 1 and 2. Changes to the heteroaromatic bicycle
were also explored, focusing on replacing the heteroatoms to
maintain the classical bidentate hydrogen-bonding to the hinge
region of the kinase while modulating the lipophilicity and polar
surface area of the compounds. Initial studies in the development
of 2 had suggested that the balance of lipophilicity and
hydrophilicity was a major determinant of cellular activity,
presumably through control of penetration through the cell
membrane.20
Replacement of the 6-phenyl spacer in 1 by piperidine was
first targeted (Table 1). The importance of the basic amine was
confirmed by the >10-fold drop in activity of the unsubstituted
piperidine 3 and the 4-carboxamidopiperidine 4. Gratifyingly,
introduction of either a 4-aminomethyl or 4-amino substituent
to the piperidine to give 5 or 6, respectively, produced
significantly more potent inhibitors of PKBꢀ than 1. Both
compounds showed very high ligand efficiency24 (LE > 0.5
kcal mol-1 per non-H atom) indicating an excellent fit to the
ATP-binding site. Although 5 and 6 were equipotent against
PKBꢀ, a degree of selectivity (3- to 20-fold) versus PKA was
observed, in contrast to the original hit 1 measured in the same
assay protocols.
The third point of the canonical PKB pharmacophore, an
aromatic group capable of filling the lipophilic pocket in the
P-loop near Phe54, was not present in 5 and 6, and this region
of protein exhibited varying amounts of disorder for these
structures. In the structure of 1-PKA-PKB the aromatic ring
of Phe54 pointed toward the inhibitor binding site. Addition of
an appropriate aromatic group to the inhibitor 1, displacing
Phe54, was an important step in the improvement of 1 to 2,20
and overlays of 5 and 6 with 2 suggested that potential vectors
for further substitution with lipophilic groups analogous to the
4-chlorophenyl of 2 could originate either from the 4-position
of the piperidine or from the carbon atom of the aminomethyl
group of 5 (Figure 3A).
Despite submicromolar enzyme inhibitory potency for the
hits, neither 5 nor 6 showed antiproliferative activity in cells.
This was addressed through simple modifications of the purine
heterocycle to decrease the hydrophilicity of the ligands (Table
1). Cellular potency was assayed by antiproliferative activity26
in the PC3M human prostate cancer cell line, which is known
to have an activated PI3K-PKB pathway and PTEN deletion.27
A specific readout of target PKB inhibition in the cancer cells
was also obtained by quantifying inhibition of phosphorylation
of the downstream substrate GSK3ꢀ using a cell ELISA.28 The
bidentate hydrogen bonding potential of the heterocycle was
maintained in all the compounds considered for synthesis. The
8-methylpurine 7 was less active than the parent purine 6.
Rearrangement of the purine to the allopurines 8 and 9, with
similar calculated29,30 log P and topological polar surface area
to 5 and 6, was broadly tolerated in terms of PKBꢀ activity but
did not result in significant cellular activity. However, replace-
ment of one ring nitrogen from the five-membered ring by
carbon to give the pyrrolo[2,3-d]pyrimidines 10 and 11 increased
the calculated log P/TPSA ratio and led to observable antipro-
liferative activity and biomarker inhibition for both compounds.
Substitution of a further nitrogen from the six-membered ring
by carbon to give the azaindole 12 did not increase the cellular
activity. The 4-aminopiperidine 10 was particularly promising,
with reasonable PKBꢀ activity and some selectivity (8-fold) over
PKA. A comparison of the structures of 6 and 10 complexed
The binding modes of 1 (PDB code 2UVY), 5, and 6 to the
PKA-PKB chimera were compared (parts A, B, and C of Figure
1, respectively, and Figure 2). In all cases the bidentate hydrogen
bonding of the purine to the hinge region of the kinase was
maintained, with interactions seen between the N3 acceptor and
N9 donor atoms of the inhibitor, and the backbone amides of
Ala123 and Glu121, respectively. However, the position of the
ligand’s terminal amine was significantly different between 1,
5, and 6 because of the nonplanar structure of the piperidine
spacer (Figure 2). In the structure of 1-PKA-PKB the terminal
methyl substituent of Met173 was packed directly under the
benzene ring of 1, forming favorable hydrophobic contacts. In
contrast, for both 5 and 6 the chairlike conformation of the
piperidine ring caused the ligands to intrude into this space,
packing closely with the sulfur atom of Met173 and displacing
the Cꢀ methyl group by about 180° torsion about the Sδ-Cꢀ
bond.

SelectiVe Inhibitors of Protein Kinase B
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2149
Table 1. Development of 6-(Piperidin-1-yl)purines and Variation of the Bicyclic Heteroaromatic Group
PKBꢀ IC50
PKA IC50
(nM)b
SRB (PC3M) IC50
GSK3ꢀ ELISA (PC3M) IC50
(µM)d
compd
R1
R2
(nM)a
(µM)c
TPSA (A2)e
log Pf
3
4
5
6
7
8
9
10
11
12
-H
-H
-H
-H
-H
-Me
59000g
ndh
ndh
ndh
>50
>50
ndh
38
>50
48
32
31
ndh
ndh
ndh
ndh
ndh
>30
>30
15
52
95
78
78
78
78
78
66
66
54
1.4
-0.13
0.17
-0.33
0.34
0.05
0.55
0.44
0.94
-CONH2
-CH2NH2
-NH2
41000g
ndh
290 ((12)
270 ((25)
110000g
830 ((12)
920 ((80)
180 ((6)
770 ((19)
880 ((107)
890 ((9)
4100 ((800)
ndh
7300 ((627)
2400 ((340)
1550 ((120)
460 ((32)
1933 ((97)
-NH2
-NH2
-CH2NH2
-NH2
-CH2NH2
-NH2
11
ndh
0.32
a Inhibition of PKBꢀ kinase activity in a radiometric filter binding assay. Mean ((SEM) for n ) 3 determinations. Standard isoquinoline-5-sulfonamide
inhibitor H-8918 gave IC50 ) 0.59 µM, SD ( 37% (n ) 20) in this assay. b Inhibition of PKA kinase activity in a radiometric filter binding assay. Mean
((SEM) for n ) 3 determinations. Standard isoquinoline-5-sulfonamide inhibitor H-818 gave IC50 ) 5.3 µM, SD ( 38% (n ) 14) in this assay. c Cell
growth inhibition (sulforhodamine B colorimetric assay) determined in PC3M human prostate cancer cells. Mean of two independent determinations. Standard
isoquinoline-5-sulfonamide inhibitor H-8918 gave IC50 ) 18 µM, SD ( 34% (n ) 20) in this assay. d Single determination. Standard isoquinoline-5-
sulfonamide inhibitor H-8918 gave IC50 ) 15 ((2) µM. e Calculated TPSA.29 f Calculated log P.29,30 g Mean of two independent determinations. h nd ) not
determined.
improved cellular activity in the PC3M cell line and also in
U87MG glioblastoma cells compared to 11 and 13, consistent
with antiproliferative activity occurring through target modula-
tion. The U87MG human tumor cell line has been shown to
have elevated signaling through the PI3K-PKB-mTOR path-
way and is especially relevant for the assessment of molecular
therapeutics targeted to the pathway.9 The improvement in
cellular activity was attributed to both the increased enzyme
inhibition and the increased lipophilicity of 14 (calculated log P
) 3.2; TPSA ) 66 Å2). Switching back to the more polar purine
hinge-binding motif 15 also caused a small (2.5-fold) drop in
inhibitory potency, which together with reduced cell penetration
may be reflected in the reduced antiproliferative effect in PC3M
cells. Conversely, changing the pyrrolo[2,3-d]pyrimidine to the
7-azaindole 16 maintained the enzyme inhibitory activity and
was accompanied by an increase in the cellular inhibition of
PKB as measured by cell ELISA. Compounds 13-16 were
prepared and tested as racemic mixtures. At this stage alterna-
tive, achiral scaffolds were targeted by applying the structure–
activity relationships and crystallographic binding modes from
the foregoing studies to the elaboration of geminal-substituted
4-piperidines. In general, the pyrrolo[2,3-d]pyrimidine hinge-
binding motif was preferred over 7-azaindole because it offered
a useful combination of enzyme potency, cellular activity, and
synthetic accessibility.
Figure 1. X-ray crystallographic structures of the ATP-binding site
for complexes of 1-PKA-PKB (A), 5-PKA-PKB (B), 6-PKA-PKB
(C), 18-PKA-PKB (D), 21-PKA-PKB (E), and 25-PKA-PKB (F).
The final 2mFo - DFc electron density for inhibitors, contoured at 1σ, is
shown in blue. Hydrogen bonds between the inhibitors and the hinge
region of the kinase are denoted by dashed lines.
with the PKA-PKB chimera (data not shown) showed the
ligand and protein conformations in the ATP-binding site to be
identical. Although the selectivity for PKB over PKA in these
compounds was low, the 4-aminopiperidine fragment was
generally associated with more selectivity than the 4-amino-
methylpiperidine moiety (compare 6 vs 5, 8 vs 9, and 10 vs
11).
To improve the PKBꢀ inhibitory activity of the series, the
addition of a lipophilic group to make contact with the P-loop
was targeted. Initially the unselective 4-aminomethylpiperidine
scaffold 11 was investigated by analogy with the development
of 6-phenylpurine and 4-phenylpyrazole inhibitors.20,21 Ap-
pending a phenyl ring to the aminomethyl group in 13 gave a
moderate increase in activity (Table 2). A much more significant
10-fold increase was seen when 4-chloro substitution was
introduced to the aromatic ring in 14, recapitulating structure–
activity relationships observed in the evolution of 220 and
isoquinoline-5-sulfonamide PKB inhibitors.18 Although unse-
lective versus PKA, the more potent compound 14 showed
Moving the aryl group of 14 to the 4-position of the
4-aminomethylpiperidine gave the geminally substituted com-
pound 18 and its purine and 7-azaindole congeners 17 and 19,
respectively. Good PKBꢀ inhibitory activity was seen in vitro
with no selectivity over PKA. The antiproliferative and cellular
target inhibition activities of the pyrrolo[2,3-d]pyrimidine 18
were at or below 1 µM for the first time in this series. However,
determination of general kinase selectivity indicated that some
of this improvement in antiproliferative activity might reflect
off-target effects (see below). Homologation of the lipophilic
substituent to generate the 4-(4-chlorophenylmethyl)-4-ami-
nomethylpiperidines 20 and 21 gave potent PKBꢀ inhibitors,
again with no selectivity over PKA but with decreased cellular

2150 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Caldwell et al.
Figure 2. Detail of the X-ray crystallographic structures for the complexes of 1-PKA-PKB (A), 5-PKA-PKB (B), and 6-PKA-PKB (C)
showing the ribose-binding pocket. The inhibitors and key residues are represented as solid sticks coloured by atom type (carbon ) green). Key
water molecules are represented as red spheres, and polar contacts discussed in the text are indicated by dashed lines.
exact orientation and conformation of the piperidine and terminal
amine were seen between the compounds. The conformationally
flexible piperidine spacer adapted to optimally position the
amine and lipophilic group as the connectivities between these
functionalities were varied (Figure 3B). The 4-amino-
methylpiperidine 18 adopted a similar conformation to the
unsubstituted precursor 5, with near-planar geometry at the
piperidine nitrogen. In contrast, both the 4-aminomethylpiperi-
dine 21 and the 4-aminopiperidine 25 showed a more planar
orientation of the piperidine ring relative to the plane of the
aromatic heterocycle, with a more pronounced pyramidal
Figure 3. (A) Overlay of 2-PKA-PKB (gray), 5-PKA-PKB (red),
and 6-PKA-PKB (green) in the ATP-binding site. The ATP site of
geometry at the piperidine nitrogen. This change in geometry
is presumably required so that the more extended 4-chlorobenzyl
substituent can still occupy the P-loop binding pocket. The
possibility of varying degrees of conjugation between the
piperidine nitrogen lone pair and the heteroaromatic substituent,
and the subsequent variation in geometry of the piperidine
nitrogen in these inhibitors, allows the saturated ring to respond
to varying substitution patterns with a range of energetically
accessible bound conformations. The same divergent pattern in
amine binding was seen for the piperidine scaffolds in these
elaborated compounds as was seen with 5 and 6. Thus, the
4-aminomethylpiperidines 18 and 21 interacted with Asp184,
Asn171, and Glu170, whereas for the 4-aminopiperidine 25 the
major interactions were to Glu127 and Glu170, with Asp184
adopting the rotamer for interaction with Lys72. The lipophilic
4-chlorophenyl and 4-chlorobenzyl substituents were directed
into a hydrophobic pocket defined principally by the side chains
of Thr51, Gly52, Phe54, Val57, Lys72, Leu74 and the backbone
carbonyls of Thr51 and Gly55. However, the 4-chlorobenzyl
substituents of 21 and 25 projected more deeply into this pocket
than the 4-chlorophenyl group of 18.
We have previously shown how the 40-fold selectivity for
PKB over PKA found for an indazole pyridine PKB ligand10
could be rationalized by comparing the interactions formed by
the inhibitor bound with PKA, PKB, and PKA-PKB chimera.23
In that case, the PKA-PKB chimera was found to bind the
ligand in a pose more closely resembling that of PKB than PKA.
For the selective PKB inhibitor 25, we were unable to obtain
sufficiently high-resolution data for the inhibitor-PKB complex.
Nevertheless, the structures of 25 bound to PKA and the
PKA-PKB chimera were compared to help understand the
origin of its selectivity. Although the pyrrolo[2,3-d]pyrimidines
overlaid well, a marked difference in the conformations of the
piperidine rings and consequently in the position of the
4-chlorobenzyl groups was seen such that the aryl groups were
at approximately 90° to one another in the two complexes.
2-PKA-PKB is depicted as a gray surface. (B) Overlay of
18-PKA-PKB (gray), 21-PKA-PKB (gold), and 25-PKA-PKB
(green) in the ATP-binding site. The protein structure for 25-PKA-PKB
is shown as a cartoon representation.
activity (e.g., compare 21 vs 18). The alternative geminal
substituted 4-aminopiperidines showed a different profile. With
the aryl group directly attached to the 4-position, compounds
22 and 23 were potent PKBꢀ inhibitors with no selectivity over
PKA. However, homologation of the 4-chlorophenyl substituent
to the 4-(4-chlorophenylmethyl) group in 24 and 25 introduced
selectivity (11- to 30-fold) for PKBꢀ over PKA. The contrast
between the very poor cellular activities of the purines and the
better activities of the pyrrolo[2,3-d]pyrimidines in compounds
22–25 was more marked than had been seen in the 4-amino-
methylpiperidine compound set 17–21. Comparison of the
enzyme inhibition and cellular effects of the potent, unselective
inhibitor 18 and the most selective inhibitor 25 implied that
the high antiproliferative activity of 18 could be due to off-
target inhibitory effects. The kinase inhibitory profiles of 18
and 25 in a panel of 21 enzymes were investigated.31 Compound
18 showed activity (>60% inhibition at 1 µM) toward 10
kinases, including receptor and cytoplasmic tyrosine kinases
(e.g., FLT3, SRC) and non-AGC serine/threonine kinases (e.g.,
CHK1, CHK2) as well as other AGC kinases (e.g., PKC,
ROCK2, RSK2, p70S6K). In contrast compound 25 was much
less promiscuous with activity seen only against two other
enzymes, both in the AGC family (p70S6K, ROCK2). The
inhibition of p70S6K by 25 as measured in a radiometric filter
binding assay was IC50 ) 120 nM, indicating a 20-fold
selectivity for inhibition of PKBꢀ over p70S6K.
The structures of 18, 21, and 25 bound to the PKA-PKB
chimera were determined (parts D, E, and F of Figure 1,
respectively). The binding modes were found to be consistent
with the earlier derivatives with the heteroaromatic bicycle
serving to anchor the molecules in the ATP-binding site via
hydrogen-bonding interactions with the hinge. Variations in the

SelectiVe Inhibitors of Protein Kinase B
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2151
Table 2. Addition of the Lipophilic Group to the 4-Aminomethyl- and 4-Aminopiperidines
PKBꢀ IC50
PKA IC50
(nM)b
SRB PC3M GSK3ꢀ ELISA PC3M SRB U87MG
GSK3ꢀ ELISA
compd
X
Y
R1
(nM)a
IC50 (µM)c
IC50 (µM)d
IC50 (µM)e
U87MG IC50 (µM)f
13
14
15
16
17
18
19
20
21
22
23
24
25
N
N
N
CH Ph-
246 ((33)
25 ((4)
71 ((17)
12 ((2)
16 ((9)
8 ((3)
25 ((2)
3 ((0.9)
5 ((2)
726 ((67)
54 ((11)
27 ((4)
3 ((0.5)
3 ((0.5)
5 ((0.8)
3 ((0.7)
7 ((1.5)
9 ((2)
38
7.4
23
5.3
5.7
1.0
16
>30
7.5
ndg
2.0
1.3
0.5
1.4
1.7
1.4
ndg
0.6
ndg
3.0
ndg
6.0
ndg
3.5
ndg
0.52
ndg
ndg
4.3
ndg
4.7
ndg
5.0
ndg
CH 4-Cl-Ph-
2.0
N
4-Cl-Ph-
ndg
<1
CH CH 4-Cl-Ph-
N
N
N
4-Cl-Ph-
ndg
0.59
ndg
ndg
0.45
ndg
<1
CH 4-Cl-Ph-
CH CH 4-Cl-Ph-
N
N
N
N
N
N
N
4-Cl-(C6H4)CH2-
CH 4-Cl-(C6H4)CH2-
4-Cl-Ph-
20
15
N
7 ((1.4)
7 ((0.9)
25 ((3)
23 ((4)
15 ((2)
286 ((42)
>50
CH 4-Cl-Ph-
8
N
4-Cl-(C6H4)CH2-
>50
12
ndg
0.66
CH 4-Cl-(C6H4)CH2-
6 ((1.5) 168 ((36)
a Inhibition of PKBꢀ kinase activity in a radiometric filter binding assay. Mean ((SEM) for n ) 3 determinations. Standard isoquinoline-5-sulfonamide
inhibitor H-8918 gave IC50 ) 0.59 µM, SD ( 37% (n ) 20) in this assay. b Inhibition of PKA kinase activity in a radiometric filter binding assay. Mean
((SEM) for n ) 3 determinations. Standard isoquinoline-5-sulfonamide inhibitor H-818 gave IC50 ) 5.3 µM, SD ( 38% (n ) 14) in this assay. c Cell
growth inhibition (sulforhodamine B colorimetric assay) determined in PC3M human prostate cancer cells. Mean of two independent determinations. Standard
isoquinoline-5-sulfonamide inhibitor H-8918 gave IC50 ) 18 µM, SD ( 34% (n ) 20) in this assay. d Single determination. Standard isoquinoline-5-
sulfonamide inhibitor H-8918 gave IC50 ) 15 ((2) µM. e Cell growth inhibition (sulforhodamine B colorimetric assay) determined in U87MG human
glioblastoma cancer cells. Mean of two independent determinations. Standard isoquinoline-5-sulfonamide inhibitor H-8918 gave IC50 ) 15 µM, SD ( 15%
(n ) 19) in this assay. f Single determination. Standard LY294002 gave IC50 ) 8.1 ((3.0) µM. g nd ) not determined.
amine functionality because of its inability to compensate for
the associated dehydration penalty. To accommodate the inhibi-
tor in this case, the piperidine adopts an alternative conformation
in which the amino group is displaced by >1 Å from its position
in PKA-PKB while still maintaining its interaction with
residues lining the basic pocket. However, this conformation
no longer provides a suitable vector by which the 4-chlorobenzyl
substituent can form the favorable P-loop interactions required
for high affinity binding. In contrast to its binding to PKA-PKB,
the aryl group is partially disordered, as judged by relatively
weak electron density and high B-factors, with the dominant
conformation projecting parallel to the P-loop into a solvent
exposed site (Figure 4B). Consistent with the lack of interactions
formed with this region of PKA, the tip of the P-loop also
exhibits disorder, with broken electron density indicating a range
of possible conformations.
Figure 4. (A) Overlay of 25-PKA-PKB (green) and 25-PKA (gold)
in the ATP site. The surface of PKA-PKB is shown in gray, the surface
of 25 when bound to PKB is shown in green, and the putative S· · ·N+
interaction is denoted by a dashed line. (B) Structure of 25-PKA
in the region of the ATP site. The final 2mFo - DFc electron density
for the inhibitor, contoured at 1σ, is shown in blue. Hydrogen bonds
to the hinge region of the kinase are denoted by dashed lines.
The structure–activity and X-ray crystallographic data pre-
sented here suggest that the basic amine and lipophilic binding
elements in this chemical series may influence the selectivity
of the inhibitors for PKB vs PKA. The flexible conformations
afforded by the piperidine linker and the variable hybridization
of the piperidine nitrogen appear to be key to exploiting the
relatively subtle differences between PKA and PKB in this part
of the kinase domain. Selectivity for inhibition of PKB over
PKA may be desirable because PKA activity has been described
as both oncogenic and tumor-suppressing, depending on cellular
context.32,33 Selective ATP-competitive inhibitors of PKB vs
PKA have been recently reported.10,14f,g High specificity for
PKB over PKA has also been achieved with ligands interacting
with the PH domain of PKB.34,35
Importantly in PKA-PKB (and presumably PKB), but not in
PKA, 25 exhibited productive binding of the chlorobenzyl with
the lipophilic pocket at the P-loop (Figure 4A), potentially
providing an explanation for the observed selectivity. The
differences in binding between PKA and PKA-PKB appear to
arise because of the Leu173 f Met change at the base of the
ATP cleft. The ability of the 4-chlorobenzyl substituent to access
the P-loop pocket is dependent on a specific conformation of
the piperidine ring in which the charged amine forms a close
contact with the sulfur of the Met173 side chain (rS· · · N ) 3.6
Å), in addition to interactions with acceptors in the basic pocket.
An analysis of the Cambridge Structural Database (see Sup-
porting Information) suggested that such S · · ·NH3+ interactions
are favorable to binding, and in PKA-PKB the presence of
methionine at this point therefore provides an energetically
suitable environment for the binding of this basic group. In
contrast, the substitution by leucine at this position in PKA does
not provide a favorable environment in which to locate the
The effect of the selective inhibitor 25 on the PI3K-PKB-
mTOR-S6 pathway was assessed in U87MG and PC3M cell
lines (Figure 5). Two divergent arms of the signaling network
downstream of PKB were blocked, with reductions seen in the

2152 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Caldwell et al.
Table 3. Pharmacokinetic Parameters of 25 in Mice
plasma PK parameters
25 iv (25 mg kg-1
)
25 po (25 mg kg-1
)
T1/2 (h)
0.95
0.57
0.5
432
392
Tmax (h)
0.083
6357
4619
0.25
Cmax (nmol L-1
)
AUC (h nmol L-1
)
Vss (L)
Clp (L h-1
)
0.325
measured in human liver microsomes.41 Compound 25 showed
IC50 > 50 µM vs CYP1A2, CYP2A6, CYP2C19, CYP3A4, and
CYP2E1. Very low inhibition (IC50 ) 10–50 µM) was seen for
CYP2C9, while significant inhibition was seen with CYP2D6
(IC50 ) 1–10 µM). The pharmacokinetic (PK) profile of 25 was
determined following iv and oral dosing at 25 mg kg-1 to female
BALB/c mice in 10% DMSO 0.1% Tween-20 in saline (Table
3). Compound 25 was widely distributed (Vss ) 0.25 L) but
cleared very quickly from the general circulation with a plasma
clearance higher than that of mouse liver blood flow. The
compound was short-lived with a plasma half-life of less than
an hour. However, the high volume of distribution was
confirmed by significant tissue distribution with spleen to plasma
concentration ratio of 7:1. Following oral administration of the
same dose of compound 25, plasma concentrations reached
levels of 432 nM. The overall bioavailability was low but
measurable (Foral ) 8.5%). Although clearly requiring optimiza-
tion in terms of increasing the half-life and oral bioavailability,
the PK performance of the 4-(piperidin-1-yl)pyrrolo[2,3-d]py-
rimidine scaffold 25 was encouraging with good distribution
to tissue seen.
Figure 5. Western blot showing the effect of compound 25 on the
phosphorylation state of components of the PI3K-PKB-mTOR
pathway in U87MG glioblastoma and PC3M prostate human cancer
cells. GAPDH was used as the loading control.
levels of pGSK3ꢀ and pS6 ribosomal protein as expected for a
PKB inhibitor. An increase in the levels of pSer473-PKB was
also seen, indicative of increased levels of signaling to PKB as
a compensatory response to blockade of the pathway. This is
consistent with a proposed feedback inhibition pathway from
activated p70S6K downstream of PKB to the upstream regulator
IRS1,36 or an alternative mTORC1-independent route,37 and has
been observed with other inhibitors of PKB.20 Importantly, this
compensatory activation of signaling to PKB is overcome by
the inhibitors such that an overall reduction in signaling
throughput downstream of PKB is still achieved. Targeted
inhibition of the pathway as shown by the Western blots and
cell ELISA assays was seen at concentrations (IC50 ) 0.66–3.1
µM) consistent with the biochemical inhibitory activity of 25.
Given that the Km,ATP for PKBꢀ has been determined as 30
µM 17 and assuming an approximate intracellular ATP concen-
tration of 10 mM and also that Ki ≈ 0.5 IC50 for the ATP-
competitive inhibitor 25 in the biochemical assay,38 then a 1
µM concentration of 25 would be expected to produce ap-
Conclusions
Novel potent 4-(4-substituted-piperidin-1-yl)-7H-pyrrolo[2,3-
d]pyrimidine ATP-competitive inhibitors of PKB were discov-
ered starting from the 6-phenylpurine 1, itself derived from the
identification of 7-azaindole as a weakly binding fragment in a
high-throughput crystallography screen. X-ray crystallography
of inhibitor-PKA-PKB chimera complexes was coupled with
the measurement of compound activity in enzyme assays and
relevant cellular mechanistic assays to efficiently guide im-
provements in the potency and selectivity of the compounds,
resulting in the identification of nanomolar inhibitors of PKBꢀ.
The cellular activity of the purine inhibitors was improved
through modulation of the lipophilicity of the purine heterocycle
by replacement with less polar 7H-pyrrolo[2,3-d]pyrimidine and
7-azaindole. A divergence in the binding mode of the basic
amine was seen between 4-aminomethylpiperidine and 4-ami-
nopiperidine containing molecules. Selectivity for PKB vs PKA
was observed with 4-aminopiperidine derivatives, and the most
PKB-selective (30-fold) inhibitor 25 showed a significantly
different binding mode between PKA and PKA-PKB chimera.
Compound 25 showed inhibition of relevant molecular bio-
markers on the PI3K-PKB-mTOR pathway in cells. With
excellent ligand efficiency (LE ) 0.47 kcal mol-1 per non-H
atom), 25 is a potential lead and a useful tool for investigation
of inhibition of the PKB pathway.
proximately 50% inhibition of intracellular PKB (ν/Vmax
)
[S]/([S] + Km(1 + [I]/Ki)). The inhibition of cell growth by 25
and other compounds in this series generally required higher
concentrations than this. Whereas the cellular ELISA and other
pharmacodynamic assays demonstrate that the compounds do
inhibit the target pathway, the sensitivity of tumor cells to that
blockade is a function of cellular dependence on the pathway,
which may vary considerably between cell types.12,39 It is
possible that profound inhibition of signaling through the
pathway is required to provoke an antiproliferative or apoptotic
effect. Taking into account potential differences in compound
permeability and selectivity, the structure–activity relationships
in this series of inhibitors with respect to biochemical inhibition
of PKBꢀ generally track with the cellular biomarker effects and
antiproliferation.
Measurement of the in vitro metabolism of 25 was made by
tandem liquid chromatography–mass spectrometry (LC-MS)
following incubation in mouse and human liver microsomes.40
Compound 25 was moderately metabolized in mouse liver
microsomes competent for phase I and phase II metabolism,
with 87% of the parent compound remaining after 30 min of
incubation at a concentration of 10 µM. In human liver
microsomes 79% of 25 remained after 30 min of incubation at
a concentration of 10 µM in the presence of cofactors suitable
for phase I metabolism. Identification of metabolites in these
incubations revealed an oxidation product (M + 16). The
potential for inhibition of cytochrome P450 isoforms by 25 was
Experimental Section
Synthetic Chemistry. Simple 6-(piperidin-1-yl)purine analogues
3-6 of the hit compound 2 were prepared by direct nucleophilic
substitution of 6-chloropurine 26 (Scheme 1). Alternative bicyclic
hinge-binding groups were incorporated into compounds 7–12 by
similar reactions (Scheme 2). The use of ethanol rather than
n-butanol as solvent and lower temperatures gave higher yields for
the displacement of 4-chloro-1H-pyrazolo[3,4-d]pyrimidine42 30

SelectiVe Inhibitors of Protein Kinase B
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2153
Scheme 3a
Scheme 1a
a Reagents and conditions: (a) 4-substituted piperidine, Et3N, n-BuOH,
100 °C; (b) 2 M HCl, room temp.
Scheme 2a
a Reagents and conditions: (a) (Boc)2O, Et3N, MeCN, room temp; (b)
NH4OAc, NaCNBH3, MeOH, reflux; (c) 2 M HClaq, MeOH, room temp;
(d) 26 or 31, Et3N, n-BuOH, 100°C; (e) 32, NMP, microwave, 155°C.
Scheme 4a
a Reagents and conditions: (a) NaH, (ClCH2CH2)2NBoc, DMF, 60°C;
(b) Raney Ni, H2 (1 atm), EtOH, room temp; (c) 2 M HClaq, MeOH, room
temp; (d) 26, Et3N, n-BuOH, 100°C; (e) 31, Et3N, n-BuOH, 100°C; (f) 32,
Et3N, NMP, microwave, 155°C; (g) (i) 6 M HClaq, reflux; (ii) NaOH,
(Boc)2O, room temp; (h) (i) i-BuOCOCl, Et3N, THF, room temp; (ii) NaN3,
H2O-THF, room temp; (iii) toluene, 90°C; (j) LDA, (4-ClC6H4)CH2Cl,
THF, -78 °C; (k) (i) 4 M HCl-dioxane, MeOH, room temp; (ii) 1 M
BH3-THF, THF, room temp.
a Reagents and conditions: (a) 4-(Boc-amino)piperidine, Et3N, n-BuOH,
100°C; (b) 2 M HClaq, room temp; (c) 4-(Boc-amino)piperidine or 4-N-
(Boc-aminomethyl)piperidine, Et3N, EtOH, 80°C; (d) 4 M HCl-dioxane,
room temp; (e) 4-(Boc-amino)piperidine, NMP, microwave, 160°C; (f)
CF3CO2H, CH2Cl2, 0°C.
chlorophenyl)acetonitrile 35 with the nitrogen mustard bis-(2-
chloroethyl)carbamic acid tert-butyl ester45 gave the common
intermediate nitrile 36.46 Reduction of the nitrile 36 with Raney
nickel produced the 4-chlorophenyl-4-aminomethylpiperidine frag-
ment 37 from which compounds 17–19 were prepared using the
displacement reactions described before. Alternatively, the inter-
mediate nitrile 36 was hydrolyzed to the carboxylic acid 38. The
acidic hydrolysis removed the carbamate protecting group that was
reinstalled in a separate step. A Curtius rearrangement sequence
via the acyl azide47 was used to transform the acid 38 into the
4-chlorophenyl-4-aminopiperidine39, whichwascoupledto6-chloro-
by amines. The less reactive 4-chloro-1H-pyrrolo[2,3-b]pyridine43
32 required microwave heating for successful reaction.44 Elaboration
of the 4-(aminomethyl)piperidine scaffold to the aryl substituted
compounds 13–16 was achieved by prior construction of the
substituted piperidine through reductive amination of commercially
available 4-ketopiperidines before coupling to the heteroaromatic
bicycle (Scheme 3).
The geminal substituted piperidines 17–25 were prepared by two
related divergent routes (Scheme 4). Double alkylation of (4-

2154 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Caldwell et al.
purine 26 and 4-chloro-7H-pyrrolo[2,3-d]pyrimidine48 31 to give
compounds 22 and 23, respectively.
LC-MS (10 min) m/z 219 [M + H+], tR ) 0.71 min, purity >98%;
found [M + H+] 219.1353, C10H15N6 requires 219.1358; HPLC
1
purity >98% AUC; H NMR (250 MHz, MeOD) δ 8.20 (s, 1H),
To prepare the piperidines substituted at the 4-position with
benzylic groups, 4-cyanopiperidine-1-carboxylic acid tert-butyl ester
40 was alkylated49 with 4-chlorobenzyl chloride to give the
intermediate nitrile 41. N-Deprotection of 41 followed by reduction
of the nitrile with borane-THF gave 4-(4-chlorophenyl)-4-ami-
nomethylpiperidine 42, which was coupled to the heteroaromatic
chlorides as before to give compounds 20 and 21. The acid
hydrolysis and Curtius rearrangement sequence was applied to
intermediate 41 to yield the 4-(4-chlorobenzyl)-4-aminopiperidine
44. The piperidine 44 was coupled to 6-chloropurine 26 and
4-chloro-7H-pyrrolo[2,3-d]pyrimidine 31 to provide inhibitors 24
and 25, respectively. We have recently described a more efficient
and direct one-pot route to 4-benzyl-4-aminopiperidines such as
44, which proceeds through the addition of benzylic Grignard
reagents to the sulfinimine formed in situ from the condensation
of 4-oxopiperidine-1-carboxylic acid tert-butyl ester and N-tert-
butylsulfinamide.47
8.00 (s, 1H), 5.47–5.42 (m, 2H), 3.13 (dt, J 13.0, 2.5 Hz, 2H),
2.60 (d, J 6.5 Hz, 2H), 1.96–1.89 (m, 2H), 1.85–1.71 (m, 1H), 1.27
(dq, J 13.0, 4.5 Hz, 2H); 13C NMR (DMSO) δ 153.1, 151.7, 151.6,
138.0, 118.7, 47.4, 44.8, 39.1, 29.7 ppm.
General Method for the Preparation of 4-(Piperidin-1-yl)-
1H-pyrazolo[3,4-d]pyrimidines. 1-(1H-Pyrazolo[3,4-d]pyrimidin-
4-yl)piperidin-4-amine (8). A solution of 4-chloro-1H-pyrazolo[3,4-
d]pyrimidine (30)42 (0.059 g, 0.38 mmol), tert-butyl piperidin-4-
ylcarbamate (0.134 g, 0.67 mmol), and Et3N (0.100 mL, 0.72 mmol)
in EtOH (2 mL) was stirred at 80 °C for 3 h. The solution was
cooled and evaporated to dryness, and the residue was purified by
recrystallization (iPrOH) to give tert-butyl 1-(1H-pyrazolo[3,4-
d]pyrimidin-4-yl)piperidin-4-ylcarbamate (0.032 g, 26%). 1H NMR
(250 MHz, CDCl3) δ 8.46 (1H, s), 8.06 (1H, s), 4.75 (2H, d, J 12
Hz), 4.55 (1H, d, J 8 Hz), 3.90–3.80 (1H, m), 3.37 (2H, dd, J )
12, 12 Hz), 2.21–2.17 (2H, m), 1.62–1.43 (2H, m), 1.49 (9H, s).
HCl (4 M) in dioxane (1 mL, 4 mmol) was added to a portion of
the material (0.028 g, 0.088 mmol). The suspension was stirred at
room temperature for 1 h and then diluted with Et2O (4 mL). The
precipitate was washed with Et2O, and the solid was collected and
dried in vacuo. Purification by ion exchange chromatography on
SCXII acidic resin, eluting with MeOH and then 2 M NH3-MeOH,
gave 8 (0.011 g, 57%). LC-MS (15 min) m/z 219 [M + H+], tR
) 0.86 min, purity 95%; 1H NMR (250 MHz, DMSO) δ 8.30 (1H,
s), 8.23 (1H, s), 4.75–4.50 (1H, m), 4.00–3.10 (4H, m), 1.95 (2H,
d, J 12 Hz), 1.50–1.20 (2H, m); 13C NMR (DMSO) δ 156.1, 155.6,
154.7, 133.5, 99.2, 47.4, 39.8, 30.0 ppm.
General Synthetic Chemistry Experimental Section. Reactions
were carried out under N2. Organic solutions were dried over
MgSO4 or Na2SO4. Starting materials and solvents were purchased
from commercial suppliers and were used without further purifica-
tion. Flash silica chromatography was performed using Merck silica
gel 60 (0.025–0.04 mm). Ion exchange chromatography was
performed using Isolute Flash SCX-II (acidic) or Flash NH2 (basic)
resin cartridges. 1H NMR spectra were recorded on Bruker AC250
or Bruker AMX500 instruments using internal deuterium locks. 13
C
NMR spectra were recorded at 126 MHz on a Bruker AMX500
instrument using an internal deuterium lock. Chemical shifts (δ)
are reported relative to TMS (δ ) 0) and/or referenced to the solvent
in which they were measured. Combined HPLC-MS analyses were
recorded using a Waters Alliance 2795 separations module and
Waters/Micromass LCT mass detector with electrospray ionization
(+ve or -ve ion mode as indicated) and with HPLC performed
using a Supelco DISCOVERY C18 5 cm × 4.6 mm i.d., 5 µm
column, with gradient elution of 10-90% MeOH/0.1% aqueous
formic acid at a flow rate of 1 mL min-1 and a run time of 6, 10,
or 15 min as indicated. Compounds were detected at 254 nm using
a Waters 2487 dual λ absorbance detector. Additional HPLC purities
were determined on a Surveyor HPLC using a Phenomenex gemini
5 µm C18 25 cm × 4.6 mm i.d. column, with gradient elution of
10-90% MeOH/0.1% aqueous formic acid at a flow rate of 1 mL
min-1 and a run time of 10 min. High-resolution mass spectra were
measured on an Agilent 6210 ToF MS with a Phenomenex Gemini
3 µm C18 (3 cm × 4.6 mm i.d) column. Synthetic intermediates
were characterized by 1H NMR spectra and MS (where electrospray
ionization permitted observation of a relevant ion) for identity and
General Method for the Preparation of 4-(Piperidin-1-yl)-
7H-pyrrolo[2,3-d]pyrimidines. 1-(7H-Pyrrolo[2,3-d]pyrimidin-
4-yl)piperidin-4-amine (10). A solution of 4-chloro-7H-pyrrolo[2,3-
d]pyrimidine (31)48 (0.074 g, 0.48 mmol), tert-butyl piperidin-4-
ylcarbamate (0.106 g, 0.53 mmol), and Et3N (0.20 mL, 1.43 mmol)
in EtOH (1 mL) was heated at 80 °C for 4 h. The mixture was
cooled to room temperature and the resulting precipitate was
collected and washed with EtOH (2 mL), then dried in vacuo to
give tert-butyl 1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-yl-
carbamate (0.057 g, 0.180 mmol, 38%). LC-MS (15 min) m/z 318
1
[M + H+], tR ) 4.57 min; H NMR (250 MHz, CDCl3) δ 9.60
(1H, br s), 8.35 (1H, s), 7.09 (1H, d, J 3 Hz), 6.54 (1H, d, J ) 3
Hz), 4.75–4.69 (2H, m), 4.50–4.45 (1H, m), 3.90–3.70 (1H, m),
3.29 (2H, ddd, J ) 12, 12, 5 Hz), 2.15–2.11 (2H, m), 1.58–1.45
(2H, m), 1.49 (9H, s). A solution of the material in 4 M
HCl-dioxane (1 mL, 4 mmol) was stirred at room temperature for
1 h and then diluted with Et2O (4 mL). The resulting precipitate
was washed with Et2O and dried in vacuo to give 10, hydrochloride
salt (0.027 g, 0.107 mmol, 59%). A sample of the free base was
prepared by ion exchange chromatography on SCXII acidic resin,
eluting with 1 M NH3-MeOH. LC-MS (15 min) m/z 218 [M +
H+], tR ) 0.81 min, purity >98%; 1H NMR (250 MHz, DMSO) δ
12.82 (1H, s), 8.40 (1H, s), 8.31 (2H, br s), 7.51 (1H, br s), 7.00
(1H, br s), 4.65 (2H, d, J 12 Hz), 3.53–3.35 (3H, m), 2.17 (2H, d,
J 10 Hz), 1.80–1.60 (2H, m); 13C NMR (MeOD) δ 158.3, 152.5,
151.7, 122.3, 104.2, 102.5, 46.2, 35.7 ppm.
1
were assessed by H NMR for homogeneity. Key compounds for
biological testing were additionally characterized by 13C NMR and
HRMS for identity and were assessed by HPLC (area-under-curve,
UV detection) for homogeneity.
General Method for the Preparation of 6-(Piperidin-1-yl)-
purines. (1-(9H-Purin-6-yl)piperidin-4-yl)methanamine (5). A
solution of 6-chloropurine (26) (0.100 g, 0.646 mmol), tert-butyl
piperidin-4-ylmethylcarbamate (0.277 mg, 1.29 mmol), and Et3N
(0.450 mL, 3.23 mmol) in n-BuOH (6.5 mL) was stirred at 100 °C
for 15 h. The mixture was cooled and concentrated, and the residues
were triturated with MeOH (10 mL). The white solid was collected
and dried in vacuo to give tert-butyl (1-(9H-purin-6-yl)piperidin-
4-yl)methylcarbamate (27) (0.124 mg, 0.373 mmol, 58%). LC-MS
(15 min) m/z 333 [M + H+], tR ) 5.42 min; 1H NMR (500 MHz,
DMSO) δ 12.95 (br s, 1H), 8.18 (s, 1H), 8.09 (s, 1H), 6.89–6.86
(m, 1H), 5.36 (br s, 2H), 3.04 (br s, 2H), 2.84 (t, J 6.0 Hz, 2H),
1.75–1.70 (m, 3H), 1.38 (s, 9H), 1.15–1.05 (m, 2H); 13C NMR
(DMSO) δ 155.8, 153.1, 151.8, 151.4, 137.8, 118.7, 77.4, 45.3,
44.5, 36.4, 29.6, 28.3 ppm. A solution of 27 (0.112 g, 0.337 mmol)
in 2 M HCl (4 mL) was stirred at room temperature for 17 h. The
solution was concentrated and the crude product was purified by
ion exchange chromatography on acidic resin, eluting with MeOH
and then 1 M NH3-MeOH, to give 5 (0.078 g, 0.336 mmol, 99%).
General Method for the Preparation of 4-(Piperidin-1-yl)-
1H-pyrrolo[2,3-b]pyridines. 1-(1H-Pyrrolo[2,3-b]pyridin-4-yl)pi-
peridin-4-amine (12). A mixture of 4-chloro-1H-pyrrolo[2,3-
b]pyridine (32)43 (0.10 g, 0.64 mmol), tert-butyl piperidin-4-
ylcarbamate (0.453 g, 2.24 mmol), and N-methylpyrrolidinone (0.2
mL) was heated at 160 °C in a microwave reactor for 1 h. The
cooled solution was diluted with MeOH and purified by ion
exchange on SCXII acidic resin, eluting with MeOH and then with
3 M NH3-MeOH. The crude product was further purified by flash
silica column chromatography, eluting with 8% MeOH-CH2Cl2,
to give tert-butyl 1-(1H-pyrrolo[2,3-b]pyridin-4-yl)piperidin-4-
ylcarbamate (0.56 g, 0.18 mmol, 28%). LC-MS (15 min) m/z 316
1
[M]+, tR ) 4.64 min; H NMR (250 MHz, MeOD) δ 7.94–7.92
(d, J ) 5 Hz, 1H), 7.20–7.18 (d, J ) 5 Hz, 1H), 6.52–6.49 (m,
2H), 4.08–4.03 (m, 2H), 3.70–3.55 (m, 1H), 3.15–3.05 (m, 2H),

SelectiVe Inhibitors of Protein Kinase B
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2155
2.05–2.01 (m, 2H), 1.74–1.64 (m, 2H), 1.48 (s, 9H). CF3CO2H (1
mL) was added dropwise to a portion of the material (0.19 g, 0.06
mmol) dissolved in CH2Cl2 (1 mL) and stirred at 0 °C. After 2.5 h
solvents were removed by evaporation and the crude product was
purified by ion exchange on NH2 basic resin, eluting with MeOH,
to give 12 (0.013 mg, 0.058 mmol, 96%). LC-MS (15 min) m/z
65 °C for 1 h and then stirred at room temperature for 89 h. The
reaction mixture was poured into ice–water (60 mL) and extracted
with EtOAc (2 × 100 mL). The combined organic extracts were
washed with water and brine, dried, filtered, and concentrated. Flash
silica column chromatography, eluting with hexane-CH2Cl2
-EtOAc (8:1:1), gave 4-(4-chlorophenyl)-4-cyanopiperidin-1-car-
boxylic acid tert-butyl ester46 (36) (5.60 g, 17.5 mmol, 70%) as a
1
217 [M + H]+, tR ) 0.95 min, purity 90%; H NMR (250 MHz,
1
MeOD) δ 8.15–8.13 (d, J ) 5 Hz, 1H), 7.20–7.18 (d, J ) 5 Hz,
1H), 6.53–6.50 (m, 2H), 4.12–4.07 (m, 2H), 3.09–2.91 (m, 2H),
2.03–2.00 (m, 2H), 1.68–1.54 (m, 3H); 13C NMR (MeOD) δ 153.3,
150.6, 144.4, 131.9, 123.1, 117.1, 112.0, 102.9, 101.2, 49.5, 35.3
ppm.
white solid. LC-MS (15 min) m/z 320 [M +], tR ) 7.71 min; H
NMR (250 MHz, CDCl3) δ 7.43–7.41 (m, 4H), 4.31 (br d, J ) 13
Hz, 2H), 3.22 (br t, J ) 13 Hz, 2H), 2.14–2.05 (m, 2H), 1.93 (dt,
J ) 13, 4.5 Hz, 2H), 1.51 (s, 9H); 13C NMR (CDCl3) δ 154.4,
138.2, 134.4, 129.3, 127.0, 121.0, 80.3, 42.6, 41.2 (br), 36.2, 28.4
ppm. Raney nickel (Raney nickel suspension 2800, 1 mL) was
added at room temperature to a stirred solution of 36 (0.355 g,
1.11 mmol) in EtOH (20 mL), and the suspension was stirred under
H2 (1 atm) for 20 h. The suspension was filtered through Celite,
and the filtrate was concentrated to give 4-aminomethyl-4-(4-
chlorophenyl)piperidine-1-carboxylic acid tert-butyl ester (0.258 g,
0.794 mmol, 72%) as an oil. LC-MS (15 min) m/z 324 [M - tBu
- NH2+], tR ) 5.02 min; 1H NMR (250 MHz, CDCl3) δ 7.40–7.35
(m, 2H), 7.28–7.24 (m, 2H), 3.80–3.70 (m, 2H), 3.06 (ddd, J )
13.5, 10.5, 3.0 Hz, 2H), 2.78 (s, 2H), 2.20–2.12 (m, 2H), 1.72 (ddd,
J ) 14.0, 10.5, 4.0 Hz, 2H), 1.47 (s, 9H). HCl (2 M, 10 mL) was
added at room temperature to a solution of 4-aminomethyl-4-(4-
chlorophenyl)piperidine-1-carboxylic acid tert-butyl ester (0.258 g,
0.794 mmol) in MeOH (10 mL). After 18 h the solution was
concentrated to dryness to give C-(4-(4-chlorophenyl)piperidin-4-
yl)methylamine hydrochloride (37) (0.232 g, 0.778 mmol, 98%).
C-(4-Chlorophenyl)-C-[1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pi-
peridin-4-yl]methylamine (14). Boc2O (1.00 g, 4.59 mmol) was
added at room temperature to a stirred mixture of (4-chlorophe-
nyl)piperidin-4-ylmethanone hydrochloride (33) (0.996 g, 3.83
mmol) and Et3N (2.7 mL, 19.1 mmol) in MeCN (15 mL). After
16 h the mixture was evaporated to dryness and partitioned between
EtOAc (50 mL) and 1 M HCl (20 mL). The organic phase was
separated and washed successively with saturated aqueous NaHCO3
(20 mL) and brine (20 mL), then dried and evaporated to dryness.
Flash silica column chromatography, eluting with 60% Et2O-
hexane, gave tert-butyl 4-(4-chlorobenzoyl)piperidine-1-carboxylate
as an oil (1.12 g, 3.48 mmol, 91%). LC-MS (15 min) m/z 346 [M
+ Na+], tR ) 7.42 min; 1H NMR (250 MHz, CDCl3) δ 7.93–7.87
(m, 2H), 7.50–7.45 (m, 2H), 4.22–4.16 (m, 2H), 3.44–3.32 (m, 1H),
2.98–2.86 (m, 2H), 1.89–1.68 (m, 4H), 1.49 (s, 9H). NaCNBH3
(0.866 g, 13.8 mmol) was added at room temperature to a stirred
mixture of tert-butyl 4-(4-chlorobenzoyl)piperidine-1-carboxylate
(1.12 g, 3.48 mmol) and ammonium acetate (3.19 g, 41.4 mmol)
in MeOH (34 mL). After refluxing for 20 h the mixture was cooled,
concentrated, and stirred with 1 M NaOH aq (100 mL). The aqueous
phase was extracted with Et2O (3 × 75 mL). The combined organic
extracts were dried, filtered and concentrated. Flash silica column
chromatography, eluting with 15% MeOH-CH2Cl2, gave 4-(amino-
(4-chlorophenyl)methyl)piperidine-1-carboxylic acid tert-butyl ester
(0.913 g, 2.85 mmol, 82%). LC-MS (15 min) m/z 208 [M - Boc
- NH2+], tR ) 5.56 min; 1H NMR (250 MHz, CDCl3) δ 7.35–7.22
(m, 4H), 4.22–4.03 (m, 2H), 3.65 (d, J 7.5 Hz, 1H), 2.73–2.53 (m,
2H), 1.94–1.87 (m, 1H), 1.65–1.54 (m, 1H), 1.46 (s, 9H), 1.34–0.98
(m, 3H). HCl (2 M, 6 mL) was added at room temperature to a
solution of 4-[amino-(4-chlorophenyl)methyl]piperidine-1-carboxy-
lic acid tert-butyl ester (0.192 g, 0.591 mmol) in MeOH (6 mL).
After being stirred for 16 h, the solution was evaporated to dryness
to give C-(4-chlorophenyl)-C-piperidin-4-ylmethylamine dihydro-
chloride (34a) (0.174 g, 0.583 mmol, 99%) as a white foam.
t
1
LC-MS (6 min) m/z 324 [M - Bu - NH2+], tR ) 0.59 min; H
NMR (250 MHz, MeOD) δ 7.59–7.51 (4H, m), 3.46–3.37 (2H,
m), 3.24 (2H, s), 3.02–2.92 (2H, m), 2.66–2.60 (2H, m), 2.22–2.10
(2H, m); 13C NMR (MeOD) determined as the free base δ 143.7,
133.2, 130.2, 129.8, 54.6, 43.2, 43.1, 34.5 ppm. A solution of 37
(0.060 g, 0.202 mmol), 31 (0.031 g, 0.202 mmol), and Et3N (0.14
mL, 1.01 mmol) in n-BuOH (2 mL) was heated at 100 °C for 48 h.
The reaction mixture was evaporated to dryness and purified by
ion exchange chromatography on SCX-II acidic resin, eluting with
MeOH and then 1 M NH3-MeOH, to give the crude amine. Flash
silica column chromatography, eluting with 15-20% MeOH-
CH2Cl2, gave 18 (0.018 g, 0.053 mmol, 26%) as an off-white foam.
LC-MS (10 min) m/z 342 [M + H+], tR ) 2.70 min, purity 93%;
found [M + H+] 342.1485, C18H21N5Cl requires 342.1485; HPLC
purity 90%; 1H NMR (500 MHz, MeOD) δ 8.12 (s, 1H), 7.50–7.44
(m, 4H), 7.12 (d, J ) 3.5 Hz, 1H), 6.63 (d, J ) 3.5 Hz, 1H), 4.38
(dt, J ) 13.5, 4.5 Hz, 2H), 3.49 (ddd, J ) 13.5, 10.5, 3.0 Hz, 2H),
2.84 (s, 2H), 2.40–2.35 (m, 2H), 1.95–1.90 (m, 2H); 13C NMR
(MeOD) δ 158.3, 152.4, 151.6, 142.6, 133.7, 130.3, 130.1, 122.3,
104.3, 102.6, 53.7, 43.7, 43.1, 33.9 ppm.
1
LC-MS (6 min) m/z 208 [M - NH2+], tR ) 0.56 min; H NMR
(250 MHz, MeOD) δ 7.61–7.53 (4H, m), 4.22 (1H, d, 9.5 Hz),
3.61–3.35 (2H, m), 3.17–2.90 (2H, m), 2.50–2.22 (2H, m),
1.82–1.40 (2H, m); 13C NMR (MeOD) δ 136.6, 135.4, 130,7, 130.6,
60.0, 44.5, 44.4, 38.8, 27.0, 26.4 ppm. A solution of 34a (0.050 g,
0.168 mmol), 31 (0.026 g, 0.168 mmol), and Et3N (0.117 mL, 0.840
mmol) in n-BuOH (1.7 mL) was heated at 100 °C for 48 h. The
mixture was concentrated and purified by ion exchange chroma-
tography on NH2 basic resin, eluting with MeOH. Flash silica
column chromatography, eluting with 15% MeOH-CH2Cl2, gave
14 (30 mg, 0.088 mmol, 52%) as an off-white solid. LC-MS (6
min) m/z 342 [M + H+], tR ) 1.84 min, purity >98%; found [M
+ H+] 342.1473, C18H21N5Cl requires 342.1485; HPLC purity
>98%; 1H NMR (250 MHz, MeOD) δ 8.12 (s, 1H), 7.38–7.31
(m, 4H), 7.11 (s, J ) 3.5 Hz, 1H), 6.59 (s, J ) 3.5 Hz, 1H),
4.95–4.65 (m, 2H), 3.60 (d, J ) 8.5 Hz, 1H), 3.18–2.95 (m, 2H),
2.16–2.08 (m, 1H), 1.99–1.85 (m, 1H), 1.47–1.16 (m, 3H); 13C
NMR (MeOD) δ 158.2, 152.4, 151.7, 143.8, 134.0, 131.2, 130.15,
130.0, 129.6, 122.2, 61.5, 47.3, 47.2, 44.7, 30.5, 30.2 ppm.
C-(4-(4-Chlorophenyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pi-
peridin-4-yl)methylamine (18). NaH (60% dispersion in mineral
oil, 2.90 g, 72.3 mmol) was added in small portions over 1 h to a
solution of bis-(2-chloroethyl)carbamic acid tert-butyl ester45 (6.74
g, 28.0 mmol) and 4-chlorobenzyl cyanide (35) (3.80 g, 25 mmol)
in anhydrous DMF (25 mL). The reaction mixture was heated at
4-(4-Chlorobenzyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piper-
idin-4-ylamine (25). n-BuLi (36.2 mL, 2.3 M solution in hexanes,
84 mmol) was added at -78 °C to a solution of iPr2NH (11.8 mL,
84 mmol) in THF (230 mL). After 10 min a solution of 4-cyano-
piperidine-1-carboxylic acid tert-butyl ester (40)49 (15.4 g, 73.1
mmol) in THF (92 mL) was added at -78 °C. After 1 h a solution
of 4-chlorobenzyl chloride (11.1 mL, 88 mmol) in THF (40 mL)
was added and the solution was warmed to room temperature over
15 h. Water (500 mL) was added, and the mixture was extracted
with Et2O (500 mL). The organic extract was dried and concentrated
to give a solid. Recrystallization from Et2O-hexane gave 4-(4-
chlorobenzyl)-4-cyanopiperidine-1-carboxylic acid tert-butyl ester
(41) (11.0 g, 32.3 mmol, 44%) as a white solid. LC-MS (10 min)
1
m/z 357 [M + Na+], 235 [M - Boc]+, tR ) 8.02 min; H NMR
(CDCl3) δ 7.35–7.32 (m, 2H), 7.24–7.21 (m, 2H), 4.15 (br s, 2H),
3.00 (br t, J ) 12.0 Hz, 2H), 2.84 (s, 2H), 1.84 (br d, J ) 13.0 Hz,
2H), 1.52–1.46 (m, 2H), 1.47 (s, 9H); 13C NMR (CDCl3) δ 154.4,
133.7, 132.7, 131.5, 128.7, 121.6, 80.1, 45.2, 40.9 (br), 39.1, 34.7,
28.4 ppm. A solution of 41 (6 g, 17.9 mmol) in 9 M HCl (180
mL) was refluxed for 3 days. Additional 12 M HCl (100 mL) was
added, and reflux was continued for 2 days. The solution was
evaporated to dryness, then diluted with 2 M NaOH (360 mL).
Boc2O (4.69 g, 21.5 mmol) was added, and the mixture was stirred

2156 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Caldwell et al.
at room temperature for 24 h. The acidity was adjusted to pH 6
using 2 M HCl, and the mixture was extracted with Et2O (2 × 200
mL). The extracts were concentrated. Flash column chromatogra-
phy, eluting with 5% MeOH-CH2Cl2, gave 1-(tert-butoxycarbo-
nyl)-4-(4-chlorobenzyl)piperidine-4-carboxylic acid (43) (4.29 g,
compounds 3, 4, 6, 7, 9, 11, 13, 15–17, 19–24, 28, 38, 39, and 42.
This material is available free of charge via the Internet at http://
pubs.acs.org.
References
1
68%). LC-MS (15 min) m/z 376 [M + Na+], tR ) 7.62 min; H
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NMR (500 MHz, MeOD) δ 7.28–7.25 (m, 2H), 7.16–7.13 (m, 2H),
3.92 (dt, J 13.5, 3.5 Hz, 2H), 2.92 (br s, 2H), 2.85 (s, 2H), 2.03 (br
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46.5, 42.7 (br), 34.4, 28.7 ppm. Isobutyl chloroformate (0.812 mL,
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mmol) and Et3N (1.15 mL, 8.25 mmol) in THF (41 mL). After 1 h
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added and the solution was warmed to room temperature overnight.
Water (100 mL) was added, and the mixture was extracted with
Et2O (3 × 50 mL). The organic extracts were combined, washed
with saturated aqueous NaHCO3 (50 mL), and dried. Toluene (100
mL) was added, and the overall volume was reduced by evaporation
to approximately 90 mL. The solution was warmed to 90 °C for
2 h, then cooled and poured into 10% HCl (70 mL). The biphasic
mixture was warmed to 90 °C for 24 h. The organic phase was
separated and concentrated to dryness to give the crude amine salt
(1.11 g). The material was dissolved in 2 M NaOH (20 mL), and
Boc2O (1.61 g, 7.39 mmol) was added. After 2 days the mixture
was extracted with Et2O (2 × 50 mL). The organic extracts were
combined, washed with 1 M HCl (20 mL), saturated aqueous
NaHCO3 (20 mL), and brine (20 mL), then dried and concentrated.
Flash silica column chromatography, eluting with 50% Et2O-
hexane, gave the doubly Boc-protected amine (0.685 g), which was
deprotected by stirring with 4 M HCl in dioxane (10 mL) and
MeOH (10 mL) at room temperature for 2 days. Concentration of
the solution gave 4-(4-chlorobenzyl)piperidin-4-yl amine hydro-
chloride (44) (0.492 g, 1.65 mmol, 40% from 43). LC-MS (10
1
min) m/z 208 [M - NH2+], tR ) 0.60 min; H NMR (500 MHz,
MeOD) δ 7.44 (d, J ) 8.5 Hz, 2H), 7.33 (d, J ) 8.5 Hz, 2H),
3.49–3.46 (m, 4H), 3.20 (s, 2H), 2.16–2.13 (m, 4H); 13C NMR
(MeOD) δ 135.3, 133.6, 133.0, 130.3, 54.4, 41.2, 40.6, 31.0 ppm.
A solution of 44 (0.060 g, 0.202 mmol), 31 (0.031 g, 0.202 mmol),
and Et3N (0.140 mL, 1.01 mmol) in n-BuOH (2.0 mL) was heated
at 100 °C for 24 h. The mixture was concentrated and purified by
preparative silica TLC, eluting with 10% MeOH-CH2Cl2, to give
25 (0.034 g, 49%). Recrystallization from MeOH gave a white
powder, mp 214–216 °C. LC-MS (10 min) m/z 342 [M + H+], tR
) 2.75 min, purity >98%; found [M + H+] 342.1494, C18H21N5Cl
requires 342.1485; HPLC purity >98%; 1H NMR (MeOD) δ 8.12
(s, 1H), 7.33–7.30 (m, 2H), 7.24–7.22 (m, 2H), 7.11 (d, J ) 3.5
Hz, 1H), 6.61 (d, J ) 3.5 Hz, 1H), 4.28 (dt, J ) 14.0, 5.0 Hz, 2H),
3.78 (ddd, J ) 14.0, 10.0, 3.0 Hz, 2H), 2.78 (s, 2H), 1.75 (ddd, J
) 14.0, 10.0, 4.0 Hz, 2H), 1.57–1.53 (m, 2H); 13C NMR (MeOD)
δ 158.3, 152.4, 151.7, 136.9, 133.6, 133.4, 129.3, 122.2, 104.2,
102.6, 51.4, 48.7, 43.2, 38.2 ppm.
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Acknowledgment. This work was supported by Cancer
Research UK [CUK] Grant No. C309/A2187. The authors thank
B. Graham and S. Saalau-Bethell for protein purification, W.
Blakemore for assistance with PKA-PKB crystallography, and
A. Mirza and M. Richards for assistance with compound
characterization.
Supporting Information Available: Details of the X-ray
crystallography data collection and refinement (5-PKA-PKB,
6-PKA-PKB, 18-PKA-PKB, 21-PKA-PKB, 25-PKA-PKB,
25-PKA); detailed results of the search for S · · ·NH3+ contacts in
the Cambridge Structural Database; HPLC purities of key com-
pounds; experimental procedures for PKBꢀ and PKA biochemical
assays; experimental procedures for SRB and ELISA cellular assays;
experimental procedures for the measurement of in vivo PK
properties (25); synthetic procedures and characterization of

SelectiVe Inhibitors of Protein Kinase B
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CDK2/cyclin2, CHK1, CHK2, CK2R1, EGFR, FGFR1, FLT3, GSK3ꢀ,
IGFR1, JAK3, KDR, MAPK1, MAPKAPK2, PKCR, PKCδ, PKCγ,
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