Discovery of 4-amino-l-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4- carboxamides as selective, orally active inhibitors of protein kinase B (Akt)

Article abstract of DOI:10.1021/jm901788j

Protein kinase B (PKB or Akt) is an important component of intracellular signaling pathways regulating growth and survival. Signaling through PKB is frequently deregulated in cancer, and inhibitors of PKB therefore have potential as antitumor agents. The optimization of lipophilic substitution within a series of 4-benzyl-l-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amines provided ATP-competitive, nanomolar inhibitors with up to 150-fold selectivity for inhibition of PKB over the closely related kinase PKA. Although active in cellular assays, compounds containing 4-amino-4-benzylpiperidines underwent metabolism in vivo, leading to rapid clearance and low oral bioavailability. Variation of the linker group between the piperidine and the lipophilic substituent identified 4-amino-l-(7H-pyrrolo[2,3-d]-pyrimidin-4-yl)piperidine-4- carboxamides as potent and orally bioavailable inhibitors of PKB. Representative compounds modulated biomarkers of signaling through PKB in vivo and strongly inhibited the growth of human tumor xenografts in nude mice at well-tolerated doses.

Full text of DOI:10.1021/jm901788j

J. Med. Chem. 2010, 53, 2239–2249 2239
DOI: 10.1021/jm901788j
Discovery of 4-Amino-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamides As Selective,
Orally Active Inhibitors of Protein Kinase B (Akt)†
Tatiana McHardy,‡ John J. Caldwell,‡ Kwai-Ming Cheung,‡ Lisa J. Hunter,‡ Kevin Taylor,‡ Martin Rowlands,‡ Ruth Ruddle,‡
Alan Henley,‡ Alexis de Haven Brandon,‡ Melanie Valenti,‡ Thomas G. Davies,§ Lynsey Fazal,§ Lisa Seavers,§
Florence I. Raynaud,‡ Suzanne A. Eccles,‡ G. Wynne Aherne,‡ Michelle D. Garrett,‡ and Ian Collins*,‡
‡Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey SM2 5NG, U.K., and
§Astex Therapeutics Ltd, 436 Cambridge Science Park, Milton Road, Cambridge CB4 0QA, U.K.
Received December 2, 2009
Protein kinase B (PKB or Akt) is an important component of intracellular signaling pathways regulating
growth and survival. Signaling through PKB is frequently deregulated in cancer, and inhibitors of PKB
therefore have potential as antitumor agents. The optimization of lipophilic substitution within a series
of 4-benzyl-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amines provided ATP-competitive, nano-
molar inhibitors with up to 150-fold selectivity for inhibition of PKB over the closely related kinase
PKA. Although active in cellular assays, compounds containing 4-amino-4-benzylpiperidines under-
went metabolism in vivo, leading to rapid clearance and low oral bioavailability. Variation of the linker
group between the piperidine and the lipophilic substituent identified 4-amino-1-(7H-pyrrolo[2,3-d]-
pyrimidin-4-yl)piperidine-4-carboxamides as potent and orally bioavailable inhibitors of PKB. Repre-
sentative compounds modulated biomarkers of signaling through PKB in vivo and strongly inhibited
the growth of human tumor xenografts in nude mice at well-tolerated doses.
Introduction
number of inhibitor chemotypes with well-defined interaction
to the protein have been described in the literature.7-10 These
cover a range of mechanisms from ATP- or substrate-compe-
titive inhibition through to allosteric modulation of kinase
activity. Several classes of ATP-competitive small molecule
inhibitors of PKB have been described, including pyridines,11
azepanes,12 indazole-4,7-diones,13 isoquinoline-5-sulfona-
mides,14 6-phenylpurines,15 4-phenylpyrazoles,16 pyrrolo[2,3-d]-
pyrimidines,17,18 thiophenecarboxamides,19 and aminofura-
zans.20 However, only a limited number of chemotypes have
been reported to have entered early phase clinical trials,
including the aminofurazan 1 (GSK690693)21 (Figure 1).
A challenge in the development of selective ATP-competitive
inhibitors of PKB has been the extensive conservation of the
ATP binding sites of the AGC kinase family.22 An alternative
approach to achieve highly selective inhibition of PKB has
been developed using ATP noncompetitive inhibitors that
target an allosteric site between the kinase and PH-domains of
the enzyme.7,8,23,24 An allosteric PKB inhibitor is in clinical
development.25
Our laboratory has previously reported the development of
a hit from fragment screening15,16 into 4-(4-chlorobenzyl)-
1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amine 2 (CC-
T128930) (Figure 1), a potent ATP-competitive inhibitor of
PKBβ. Crucially, 2 also showed inhibition of relevant mole-
cular biomarkers in the PI3K-PKB-mTOR pathway in
cells.17 This compound was 28-fold selective for PKB com-
pared to the structurally homologous kinase PKA and
showed good overall selectivity for PKB and other AGC
kinases in a wider kinome profile. Although the selectivity
and cellular potency of 2 were sufficient to merit investigation
of its in vivo profile, the compound had high clearance in vivo
The serine/threonine protein kinase B (PKB,a also known
as Akt) plays an important role in signaling within cells,
promoting both cell proliferation and survival.1 PKB is a key
downstream component in the phosphatidylinositol-3 kinase
(PI3K) signaling pathway.2 The binding of extracellular
growth factors to tyrosine receptor kinases at the cell surface
leads to activation of PI3K, which in turn produces phos-
phatidylinositol-3,4,5 triphosphate (PI(3,4,5)P3) anchored to
theinner side of theplasma membrane. Binding of PKB to PI-
3,4,5-P3 through the pleckstrin homology (PH) domain of the
enzyme promotes activation of the kinase by phosphoryla-
tion on Ser473 and Thr308.3,4 Activated PKB signals through
phosphorylation of several enzyme or transcription factor
substrates, including GSK3β, FKHRL1, BAD, and mTOR,
to promote proliferation, protein translation, progression
through the cell cycle, and antiapoptotic survival.1,2
Unregulated signaling in the PI3K-PKB-mTOR path-
way is a common molecular pathology in many human
cancers.5 PKB itself is overexpressed or activated in several
cancers, such as prostate, breast, and ovarian carcinomas, and
PKB is therefore an attractive target for cancer therapy.6-10
Efforts in targeting PKB have increased in recent years, and a
†PDB ID Codes: 2x37 (10-PKB), 2x39 (21-PKB).
*To whom correspondence should be addressed. Phone þ44 208 722
4317; Fax þ44 208 722 4126. E-mail: ian.collins@icr.ac.uk.
a Abbreviations: AGC, cAMP-dependent, cGMP-dependent and pro-
tein kinase C; ELISA, enzyme-linked immunosorbent assay; mTOR,
mammalian target of rapamycin; PH, pleckstrin homology; PKA, protein
kinase A; PKB, protein kinase B; PI3K, phosphatidylinositol-3 kinase;
PI(3,4,5)P3, phosphatidylinositol-3,4,5triphosphate;PTEN, phosphatase
and tensin homologue.
r
2010 American Chemical Society
Published on Web 02/12/2010
pubs.acs.org/jmc

2240 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
McHardy et al.
Table 1. Inhibition of PKBβ and PKA by Substituted 4-Benzyl-1-(7H-
pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amines
selectivity for
PKB against
PKA (fold)c
PKBβ IC50
PKA IC50
(nM)b
no.
Rn
4-Cl
(nM)a
2
6.0 ((1.5)d
46
168 ((36)d
280
2150 (1700, 2600)f
660
28
3
3-Cl
2-Cl
6
49
4
44 ((35)e
5
4-OCF3
3-OCF3
2-OCF3
3-OMe
2-OMe
4-tBu
35 (19, 50)f
19
19
32
6
600
2800
7
84
15
33
8
720
ndg
48
9
>1000
27 ((18)h
35
8.5 (7, 10)f
16
ndg
126
15
Figure 1. Structures of the ATP-competitive inhibitors 1 and 2.
10
11
12
13
14
15
16
17
18
3400 (3200, 3600)f
520
1300 (1300, 1300)f
770
2,3-Cl2
2,4-Cl2
2,5-Cl2
2,6-Cl2
3,4-Cl2
2-Cl, 4-F
and low oral bioavailability. In this article, we describe
modifications to 2 leading initially to compounds with higher
selectivity for PKB and ultimately to the identification of 4-
amino-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-car-
boxamides as selective and orally bioavailable inhibitors of
PKB with in vivo antitumor activity.
153
48
20
18 (9, 26)f
3300
>300
1200
ndg
165
>16
13
ndg
70
90
25
7.0
490
a Inhibition of PKBβ kinase activity in a radiometric filter binding
assay,17 single determination. Standard inhibitor N-[2-(p-bromocin-
namylamino)ethyl]-5-isoquinoline sulfonamide (H-89) gave mean IC50
((SD) = 590 ((220) nM (n = 20). b Inhibition of PKA kinase activity in
a radiometric filter binding assay,16 single determination. Standard
inhibitor N-(2-methylaminoethyl)-5-isoquinoline sulfonamide (H-8)
gave mean IC50 ((SD) = 5300 ((2010) nM (n = 14). c Ratio of IC50
values (PKA/PKB). d Mean ((SD), n = 3 determinations. e Mean
((SD), n = 4 determinations. f Mean of two determinations, individual
values in parentheses. g nd = not determined. h Mean ((SD), n = 5
determinations.
Results and Discussion
The design of ATP-competitive inhibitors selective for PKB
against PKA is challenging because these enzymes are very
closely related with high sequence homology in the ATP-
binding site (∼80%).22 X-ray crystallographic analysis of the
modes of binding of 2 in PKA and a PKA-PKB chimeric
protein representative of PKB26 suggested that 2 exhibited
productive binding of the chlorobenzyl group within a lipo-
philic pocket formed by P-loop residues in PKB.17 However,
in PKA, the presence of a single amino acid difference
(Met282 PKB, Leu173 PKA) in the ribose binding site
resulted in a change of conformation of the bound ligand,
directing the lipophilic 4-chlorobenzyl group into a less
favorable, solvent exposed region. On the basis of this ex-
planation for the observed selectivity of 2, we attempted the
synthesis of a wider range of substituted analogues to inves-
tigate if higher selectivity could be obtained (Table 1).
Variation of the substituents on the benzyl group of 2 in
general lead to somewhat reduced affinity for PKB. Excep-
tions were the 2,4-dichlorobenzyl and 2-napthyl analogues 12
and 18, respectively, which inhibited PKB with similar poten-
cies to 2. An interesting influence of the substituents on the
selectivity of the compounds for PKB versus PKA was seen.
While translocation of the 4-chloro group of 2 to the 3-
position (3) reduced both affinity and selectivity, approxi-
mately 40-fold selectivity was recovered in the 2-chlorobenzyl
analogue 4. Replacement with more electron-rich 2-, 3-, or 4-
substituents (5-8) gave compounds with selectivities in a
similar range (ca. 20-48-fold), although the 2-methoxy ana-
logue 9 was surprisingly less potent at PKB. Gratifyingly,
combination of the 2- and 4-chloro substituents in the ana-
logue 12 increased the selectivity to ca. 150-fold while retain-
ing nanomolar potency at PKB. The 2,6-dichloro substitution
pattern 14 gave similarly high selectivity for PKB, although
this was not seen with other dihalobenzyl analogues 13, 15,
and 16. Introduction of a larger, lipophilic 4-tert-butyl sub-
stituent 10 also gave a high selectivity for PKB (ca. 126-fold).
An intermediate level of selectivity (ca. 70-fold) was seen for
the 2-napthyl derivative 18.
Where the selectivity of PKB over PKA was increased for
the compounds in Table 1, this was due to reduced inhibitory
activity against PKA rather than an increase in affinity for
PKB and was associated with increased lipophilicity of the
benzyl group. This structure-activity relationship was
broadly consistent with the rationale proposed from the
comparison of 2 bound to PKA and PKA-PKB chimera, in
which the benzyl substituent interacts poorly with PKA
relative to PKB, and is directed toward solvent. The ability
tobind toPKB was minimallycompromisedfortheanalogues
with larger substituents. The X-ray crystal structure of
the PKB-selective analogue 10 bound to PKBβ was deter-
mined and showed a very similar binding mode to that of 217
(Figure 2). The tert-butyl substituent occupied the lipophilic
pocket formed by the P-loop of PKB, with the 4-amino
substituent interacting with Glu236 and the backbone carbo-
nyl of Glu279 in the ribose pocket.
As an alternative to substituent variation in the 4-amino-4-
benzylpiperidine series, we also investigated compounds with

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2241
Figure 2. Crystal structures of (A) 10 bound to PKBβ and (B) 21 bound to PKBβ. (C) Overlay of the bound conformations of 2 (gold),
10 (green), and 21 (blue).
varied chain length between the 4-aminopiperidine and 4-
chlorophenyl groups (Table 2). The ether 19 was as potent as2
against PKB but had no selectivity against PKA, which we
speculated was due to the more flexible linker group. While
the amide 20 had reduced affinity for PKB, the isomeric amide
21 retained activity for PKB and showed some selectivity (ca.
14-fold) over PKA.
fashion to 2 and 10, with the 4-amino group forming inter-
actions with Glu236 and the backbone carbonyl of Glu279,
while the 4-chlorophenyl ring was located in the P-loop
lipophilic pocket (Figure 2B). As observed for 2 and 10, the
inhibitor’s basic amino group formed a favorable close con-
˚
tact with the sulfur of Met282 (ca. 3.5 A), an interaction which
is lost in PKA. It is possible that the proximity of the electron-
rich sulfur residue compensates for loss of hydration of the
protonated amine on binding.17 A possible additional inter-
action was also observed to the amide spacer of 21 with close
A set of analogues of the amide 21 were investigated using
substituent patterns corresponding to those studied for the
4-amino-4-benzylpiperidines (Table 2). Most compounds
were potent against PKB, but selectivity was generally de-
creased against PKA when compared with the 4-benzylpiper-
idines shown in Table 1. Variation of the position of the
chlorine atom in the aromatic ring showed that 4-substitution
as in 21 was optimal. Other 4-substituents (24-27) showed a
decrease in PKB inhibitory activity with increasing size, and
the 4-tert-butyl analogue 27 in particular was less active than
the rest of the analogues in this set. This contrasted with the
structure-activity relationship seen for the 4-benzylpiper-
idines, and we ascribed these differences to the presence of
the longer and relatively inflexible amide spacer which could
result in larger 4-substituents being unable to interact as
favorably with PKB. As with the 4-benzylpiperidines, the
2,4-dichlorobenzyl amide 28 gave improved selectivity (ca.
24-fold) for PKB over PKA. Other less lipophilic 2,4-dihalo-
benzyl amides (30-32) retained activity at PKB but with
reduced selectivity. In some cases, increases in PKA activity
for the benzyl amides were seen relative to nonamide com-
parators. Although constrained by the amide, the longer
linker will allow the lipophilic substituent to attain a different
range of conformations compared to the simple 4-benzyl-
piperidines (Figure 2), resulting in the recovery of productive
contacts to the P-loop of PKA.
˚
approach (ca. 3.3 A) of the amide NH in the inhibitor and
the side chain of Asp293. The 10-fold drop in activity for the
N-methyl amide 33 relative to 21 may reflect the disruption of
this conformation in that complex.
The effect of substituting the pyrrolo[2,3-d]pyrimidine bi-
cycle by 7-azaindole, oxopurine, and pyrazolo[3,4-b]pyridine
was investigated for the most potent and selective piperidine
moieties (Table 3). The bicyclic heteroaromatic groups form
hydrogen bonds to a part of the kinase domain, known as
the hinge region, that links the distinct N- and C-terminal
lobes. 7-Azaindole was the original hinge-binding fragment
from which this compound series was derived.15,17 The car-
bonyl functionality of 8-oxopurine was expected to make
additional interactions with PKB, particularly the residue
Thr213 at the entrance to the hydrophobic pocket of the
kinase which differs between PKB and PKA. For a similar
reason, the pyrazolo[3,4-b]pyridine bicycle was selected to
provide an additional polar atom in the ligand in this region.
The azaindole 36, the direct analogue of 2, showed similar
potency but no selectivity for PKB over PKA. The 4-amido-
piperidine containing azaindole 38 was also unselective. In-
troduction of the 4-tert-butyl substituent to give 37 increased
the selectivity, mirroring the structure-selectivity relation-
ship seen with the pyrrolo[2,3-d]pyrimidines 2 and 27, but
only to ca. 20-fold. The 7-azaindoles were therefore associa-
ted with generally lower selectivity for PKB over PKA than
the pyrrolo[2,3-d]pyrimidines. We believe this reduction in
Methylation of the amide NH of 21 to give compound 33,
and the conformationally constrained tertiary amides 34 and
35, led to loss of potency against PKB. The crystal structure of
21 bound to PKBβ showed the inhibitor bound in very similar

2242 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
McHardy et al.
Table 2. Development of 4-Amino-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-
piperidine-4-carboxamide PKB Inhibitors
Table 3. 4-Azaindole, 8-Oxopurine, and Pyrazolo[3,4-b]pyridine Hinge
Binders Substituted with Selected 4-Aminopiperidines
selectivity for
PKB against
PKA (fold)a
PKBβ IC50
PKA IC50
(nM)a
no.
R
(nM)a
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
8.0
69
2.2 ((1.2)c
7.0
36
13
1.6
ndb
14
ndb
30
4-Cl
3-Cl
82
64
12
2-Cl
4-F
1.8
6.7
10
ndb
ndb
24
14
11
94
110
4-CF3
4-CF3O
4-tBu
29
ndb
210
4.9
5.7
11
ndb
120
2,4-Cl2
3,4-Cl2
2,4-F2
2-Cl, 4-F
2-F, 4-Cl
45
79
7.9
7.2
ndb
ndb
3.4
ndb
ndb
8.0
4.0
25
44% @ 30 nM
60% @ 10 nM
84
250
680
ndb
ndb
a Inhibition of PKB β kinase activity in a radiometric filter binding
assay,17 single determination. Standard inhibitor N-[2-(p-bromocin-
namylamino)ethyl]-5-isoquinolinesulfonamide (H-89) gave mean IC50
((SD)=590 ((220)nM (n=20). b nd=not determined. c Mean ((SD),
n = 3 determinations.
selectivity arises from the replacement of a nitrogen (N-3) in
the pyrrolo[2,3-d]pyrimidines by a carbon in the azaindoles.
This changes the preferred conformation and orientation of
the piperidine ring relative to the bicycle and thus the vectors
of the basic amine and lipophilic substituents. Because selec-
tivity in this series arises from efficiently exploiting a single
amino acid difference (Leu/Met) between PKA and PKB,
selectivity is particularly sensitive to the positioning of the
amine group relative to this residue. The 8-oxopurines 39-41
offered similar or improved selectivity compared to their
pyrrolo[2,3-d]pyrimidine congeners but with somewhat lower
potency at PKB. On the basis of the binding modes of purine
inhibitors in this series,17 the 8-oxopurine carbonyl group is
positioned to accept a hydrogen bond from the side-chain of
Thr213. Because the equivalent residue is valine in PKA, this
would be expected to contribute to selectivity for PKB.
Targeting this difference in the ATP binding sites of PKB
and PKA has been noted to increase the selectivity of other
inhibitor chemotypes.11g The pyrazolo[3,4-b]pyridine hinge-
binding group provided ligands 42 and 43 with intermediate
a Inhibition of PKBβ kinase activity in a radiometric filter binding
assay,17 single determination. Standard inhibitor N-[2-(p-bromocin-
namylamino)ethyl]-5-isoquinoline sulfonamide (H-89) gave mean IC50
((SD) = 590 ((220) nM (n = 20). b Mean of two determinations, indi-
vidual values in parentheses. c nd = not determined.
potency and selectivity between the azaindole and pyrrolo-
[2,3-d]pyrimidine analogues.
The inhibitory profiles of the PKB-selective compounds 2,
10, and 21 were investigated in a wider panel of 22 kinases27
(Figure 3). As previously reported,17 2 mainly showed activity
(>80% at 1uM) against PKB, PKA, and two other enzymes
(ROCK2 and p70S6K), all from the AGC kinase subfamily.
Some activity (40-80% @ 1 μM) was seen against four other
kinases in the panel. The tert-butyl analogue 10 was remark-
ably selective, showing significant activity against only two

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2243
Table 4. Cellular Activities of Selected Compounds in PC3M (Prostate)
and U87MG (Glioblastoma) Human Tumour Cell Linesa
PC3M
ELISA IC50
(μM)c
U87MG
SRB GI50
(μM)b
SRB GI50
(μM)d
ELISA IC50
(μM)e
no.
2
12
8.9 (7.7, 10)f
3.0
4.0
1.9
6.7
ndg
5.0
5.7
5.9
14
0.66
ndg
ndg
ndg
0.59
0.93
ndg
2.8
10
12
14
19
21
28
32
33
36
8.5 (8.0, 8.9)f
20
ndg
>5 0 h
26
5.1
17
2.3 (2.3, 2.3)f
3.8
17
20
ndg
29
ndg
4.2
34
ndg
ndg
9.3
0.37
(0.36, 0.37)f
ndg
4.6
37
38
40
41
42
43
ndg
ndg
ndg
ndg
ndg
ndg
3.7
19
3.2
7.2
8.8
>50
2.7
6.5
ndg
ndg
13
ndg
>50
16
ndg
ndg
7.0
a Inhibition of PKBβ kinase activity in a radiometric filter binding
assay,17 single determination. Standard inhibitor N-[2-(p-bromocinna-
mylamino)ethyl]-5-isoquinoline sulfonamide (H-89) gave mean IC50
((SD) = 590 ((220) nM (n = 20). b Cell growth inhibition by sulforho-
damine B colorimetric assay,33 single determination in PC3 M human
prostate cancer cells. Standard inhibitor H-89 gave mean ((SD) IC50
=
18 ((6.0) μM in this assay. c Cellular ELISA for inhibition of GSK3β
phosphorylation in PC3 M cells,32 Standard inhibitor H-89 gave mean
((SD) IC50 = 15 ((2.0) μM. d Cell growth inhibition by sulforhodamine
B colorimetric assay,32 single determination in U87MG human glio-
blastoma cancer cells. Standard inhibitor H-89 gave mean ((SD)
IC50 = 15 ((2.3) μM in this assay. e Cellular ELISA for inhibition of
GSK3β phosphorylation in U87MG cells.32 Standard inhibitor 2-(4-
Figure 3. Selectivity profiles and Gini coefficients28 (G) of 2 (A), 10
(B), and 21 (C) against a panel of 22 human kinases, measured at
1 μM concentration of the test compound. Kinase dendrogram29
reproduced courtesy of Cell SignalingTechnology, Inc. (www.
cellsignal.com).
morpholino)-8-phenyl-4H-1-benzopyran-4-one (LY294002) gave IC50 =
8.1 ((3.0) μM. f Mean of n = 2 determinations, individual values in
parentheses. g nd not determined. h n = 2 determinations.
kinases, namely PKB and p70S6K, in this panel. The amide 21
had a similar profile to 2, showing some activity (>80% at
1uM) against the same enzymes (PKB, PKA, ROCK2, and
p70S6K). Lower levels of activity were seen for some other
AGC kinases but not for kinases outside this subfamily. The
pattern of selectivity was reflected in the Gini coefficients for
the compounds,28 which takes into account the total levels of
inhibitory activity across the panel, with compound 10 show-
ing a higher overall selectivity (Figure 3).
analogous pyrrolo[2,3-d]pyrimidine 10. The cellular activity
of these compounds, which are highly selective for PKB over
PKA, and over other kinases in the case of 10, argues for the
antiproliferative effects of the inhibitors being primarily
driven by inhibition of PKB. The8-oxopurine amide analogue
41 had no activity in the cellular assays despite good PKB
affinity. It is likely that low cell penetration is encountered for
this scaffold, the most polar of those prepared, an effect
encountered earlier in the evolution of this series.17 Better
cellular activity was seen for the pyrazolo[3,4-b]pyridines 42
and 43, but these compounds offered no advantage over the
activity of 2, 10, and 21.
The inhibitory effect of compounds 2, 10, and 21 toward
five human cytochrome P450 isoforms (1A2, 2D6, 3A4, 2C9,
and 2C19) was assessed in microsomal preparations.34 In
general, no significant inhibition was observed (IC50>10 μM)
for most of the isoforms tested. Compound 2 showed inhibition
of the 2D6 isoform (IC50 = 0.66 μM), but this was not observed
for compounds 10 and 21. Rather, these examples showed
moderate inhibition (IC50 ca. 1 μM) for the 2C9 isoform only.
The pharmacokinetic properties of compounds from this
series were investigated in mice, including the selective
pyrrolo[2,3-d]pyrimidine inhibitors 2, 10, and 21 (Table 5).
The 4-(4-tert-butylbenzyl)-4-aminopiperidine 10 showed a
similar profile to that for 2.17 Following iv dosing at 10 mg/
kg, compound 10 was widely distributed but was very quickly
cleared from the general circulation with a plasma clearance
higher than that of mouse liver blood flow. Although com-
pound 2 had shown low oral bioavailability (Foral = 8.5%),
The antiproliferative activity of selected inhibitors was
assessed in the PC3 M human prostate cancer and U87MG
glioblastoma human cancer cell lines, which are known to
have PTEN deletion and an activated PI3K-PKB pathway30,31
(Table 4). A specific readout of targeted PKB inhibition
in cells was also obtained by quantifying inhibition of phos-
phorylation of the downstream substrate GSK3β by cell
ELISA.32 The majority of the compounds were active in the
antiproliferative assays, although this assay may sometimes
include contributions from off-target activities. The more
selective pyrrolo[2,3-d]pyrimidines 10 and 12 showed similar
potencies in the cellular assays to the lead compound 2. The
potent but unselective ether-linked analogue 19 was also
active, but a fall in antiproliferative activity was observed
for the amides21and 28compared to theirnonamide counter-
parts. N-Methylationof the amide in 33 toreduce polarityand
increase cell permeability did not substantially improve the
cellular activity relative to 21, but the activity of 33 may be
compromised by the 3-fold drop in affinity for PKB (Table 2).
The 7-azaindole analogues 36 and 37 were similar to their
pyrrolo[2,3-d]pyrimidine counterparts 2 and 10, while the
7-azaindole 38 was less active in cells than its comparator
21. The 8-oxopurine 40 retained similar cellular activity to the

2244 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
McHardy et al.
no measurable drug levels were detectable for 10 following
oral dosing. Oxidative metabolites of 2 and 10, corresponding
to masses of M þ 16 and M þ 32, were detected by mass
spectrometry in plasma samples from these experiments. The
fragmentation patterns in the mass spectra of the M þ 16
metabolite of 2, when compared to those for the parent drug,
suggested that oxidation was occurring at C-2 in the piper-
idine ring.
A striking difference was observed in the in vivo pharmaco-
kinetic properties of the inhibitors containing the 4-amino-
4-amidopiperidine moiety, such as 21, compared to the
4-benzyl-4-aminopiperidines 2 and 10. The plasma clearance
of 21 was approximately 3-fold lower than that of 2 and 10,
while the volume of distribution was also reduced for the more
polar amide scaffold. Importantly, compound 21 showed very
good oral bioavailability in mice (Foral = 58%). While lower
first pass metabolism and subsequent reduced clearance may
contribute to the improved oral bioavailabilty of 21, the
difference in basicity between 2 and 21 may also play a part.
Calculated pKa values35 for the protonation of the 4-amino
group varied between 8.8 and 9.3 for 2, depending on the
methodology, compared toa rangeof6.5-7.4 for 21. Thusthe
4-amino-4-amidopiperidines would be expected to be signifi-
cantly less protonated than 2 or 10 in the gut, leading to
enhanced passive absorption. The solubilities of 2 and 21 were
determined in aqueous buffer at pH 7 and 6.5. Interestingly,
the solubility of 2 showed a strong pH dependence, with
S=0.26 mg/mL at pH 6.5 but negligible solubility at pH 7,
suggesting a much greater aqueous solubility for the proto-
nated than the unprotonated form. In contrast, the solu-
bilty of 21 was less affected by pH (S = 0.1 mg/mL at pH 7,
S = 0.04 mg/mL at pH 6.5). Thus better solubility for the
unprotonated form may also contribute to the improved
bioavailability of 21.
Earlier reported studies on the efficacy of some indazole-
derived PKB inhibitors in human tumor xenograft models
had suggested that mechanism-related effects of PKB inhibi-
tion could underlie the toxicity observed with these com-
pounds.12a We were therefore keen to test selective inhibi-
tors from the novel pyrrolo[2,3-d]pyrimidine series in vivo.
The efficacy and pharmacodynamic effects of the orally
bioavailable inhibitor 21 and the close analogue 32 were
studied in mice bearing established subcutaneous U87MG
human glioblastoma xenografts (Figure 4). Doses of 21 up to
200 mg kg-1 (5 days dosing in 7) were well tolerated with no
effects on mouse body weight (not shown). Efficacy was
measured by comparison of the estimated volume of tumors
in treated and control groups during the study (Figure 4) and
by comparison of the final tumor weights in the treated and
control groups (T/C). Very strong inhibition of tumor growth
was seen with T/C = 23%. Additionally, 44% of treated
tumors had regressed in volume at the completion of the
experiment. In a parallel pharmacokinetic and pharmaco-
dynamic study, high levels of 21 were found in plasma and tumor
samples (20 and 43 μM, respectively) at 4 h after a single dose.
Clear inhibition of PKB signaling in the tumors was observed
Table 5. Pharmacokinetic Parameters of Compounds 2, 10, and 21
in Mice
plasma PK
parametersa
2
10
10
21
IV dose (mg kg-1
T1/2 (h)
Vss (L)
)
25
5
0.95
0.25
0.33
8.5
0.79
0.27
0.31
<1c
0.90
0.06
0.08
58
Clp (L h-1
oral (%)b
)
F
a Pharmacokinetic parameters were calculated using the program
WinNonLin, fitted to model 201 with default settings. b Following oral
administration of same dose. c Plasma levels below limit of detection
following oral dosing.
Figure 4. Effect of 21 (A) and 32 (C) (both dosed at 200 mg/kg po 5 days per week) on the growth of U87MG human glioblastoma xenografts in
CrTac:Ncr-Fox1(nu) athymic mice. Effect of 21 (B) and 32 (D) on PKB phosphorylation of GSK3β in U87MG human tumor xenografts at 4 h
(B) or 6 h (D) following a single dose of 200 mg/kg po.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2245
Scheme 1a
aReagents and conditions (yields for R = 4-tBu): (i) LDA, 4-tert-butylbenzyl bromide, THF, -78 °C, 79%; (ii) AcOH, H2SO4, reflux, then Boc2O,
NaOH, dioxane-H2O, 71%; (iii) PhI(TFA)2, MeCN-H2O, 46%; (iv) tert-butylsulfinamide, Ti(OEt)4 (2 equiv), THF, reflux, then 4-tert-butylbenzyl-
magnesium bromide (5 equiv), THF, rt, 26%; (v) 4M HCl, dioxane, MeOH, rt, 100%; (vi) 4-chloro-7H-pyrrolo[2,3-d]pyrimidine, Et3N, n-BuOH,
100 °C, 86%; (vii) 4-fluoro-1-(triisopropylsilyl)-1H-pyrrolo[2,3-b]pyridine, Et3N, NMP, 160 °C, 37%; (viii) 6-chloro-7H-purin-8(9H)-one, Et3N,
n-BuOH 100 °C, 50%; (ix) ethyl 4-chloro-1H-pyrazolo[3,4-b]pyridine-5-carboxylate, Et3N, n-BuOH, 100 °C, 66%; (x) KOH, H2O, 120 °C, 47%.
using an electrochemiluminescence immunoassay to measure
levels of phospho-GSK3β in tumor lysates32 (Figure 4). Thus
despite the somewhat reduced cellular antiproliferative acti-
vity for the more polar scaffold of 21 compared to 2, the good
tolerability and reduced clearance of 21 enabled oral dosing to
achieve drug levels above the concentrations at which me-
chanism-based and antiproliferative effects were seen in vitro
in cells, resulting in inhibition of the target in vivo and
reduction of tumor growth. Measurement of tumor pharmaco-
dynamic changes in other kinase-mediated pathways would
be required to establish if inhibition of other targets can
contribute to the efficacy of the compounds, however the
selectivity profile of the compounds argues for a major
contribution from PKB inhibition. Similar effects on in vivo
biomarkers and reduction in growth (T/C=32%) of U87MG
tumor xenografts were seen following treatment with the
closely related compound 32, also dosed orally at 200 mg/kg
(Figure 4). Details of the efficacy, pharmacodynamic effects,
and tumor pharmacokinetics of 21 (CCT129524) in a broader
range of tumor xenograft models will be reported separately.
good selectivity for inhibition of PKB over a range of other
human kinases, with some activity observed for related AGC
kinases. The observation of strong tumor growth inhibition
and biomarker modulation in vivo with well tolerated doses of
21 supports the further evaluation of compounds from this
series as potential anticancer therapeutics.
Experimental Section
Synthetic Chemistry. Substituted 4-amino-4-benzylpiperidine
intermediates were prepared from 4-cyano-4-benzylpiperidines
as previously described for 2 using a Curtius rearrangement
sequence to install the 4-amino substituent.17 A more conveni-
ent reagent combination for this transformation was found by
treating 4-benzyl-4-carbamoylpiperidines with bis(trifluoro-
acetoxy)iodobenzene,36 as exemplified for the preparation of
10 (Scheme 1). Alternatively, the reactive tert-butyl sulfinimine
formed from N-Boc-piperidin-4-one and tert-butylsulfonamide
was reacted in situ with benzylic Grignard reagents to give the
4-amino-4-benzylpiperidine scaffolds directly.37 Hinge-binding
groups were introduced to the piperidines through SNAr reac-
tion of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine, 6-chloro-7H-
purin-8(9H)-one, or 4-fluoro-1-(triisopropylsilyl)-1H-pyrrolo-
[2,3-b]pyridine,38 which occurred selectively at the more reactive
and less hindered secondary nitrogen atom. Additionally, the
piperidines were reacted with ethyl 4-chloro-1H-pyrazolo[3,4-
b]pyridine-5-carboxylate39 followed by decarboxylation to give
the pyrazolo[3,4-b]pyridine hinge-binder. Through these means
the 4-benzyl-4-aminopiperidine analogues 2-18, 36, 37, 39, 40,
42, and 43 were prepared.
To prepare the ether-linked analogue 19, 1-(tert-butoxy-
carbonyl)-4-(tert-butoxycarbonylamino)piperidine-4-carboxylic
acid 47 was reduced to the alcohol 48 with lithium aluminum
hydride and O-benzylated to give 49 after double N-deprotection
(Scheme 2). The piperidine 49 was reacted with 4-chloro-7H-
pyrrolo[2,3-d]pyrimidine to give the test compound 19. Alterna-
tively, formation of the primary amide from 47 and reduction
with borane in THF gave the 4-aminomethylpiperidine 50.
Acylation with 4-chlorobenzoyl chloride and deprotection pro-
duced the amide 51, which was coupled to the pyrrolopyrimidine
Conclusions
A series of 4-benzyl-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-
piperidin-4-amines provided potent inhibitors of PKBβ. The
selectivity for inhibition of PKBβ over the closely related
kinase PKA was increased by introducing larger lipophilic
substituents to the benzyl group. This strategy exploited the
subtly different binding modes for the ligands between the two
targets, arising from a single amino acid residue difference
within the ATP-binding site of the enzymes. The 4-amino-4-
benzylpiperidine scaffold underwent metabolism in vivo,
leading to rapid clearance and poor oral bioavailability. This
was overcome by modification of the piperidine scaffold to
give orally bioavailable 4-amino-1-(7H-pyrrolo[2,3-d]pyri-
midin-4-yl)piperidine-4-carboxamides, exemplified by the po-
tent and selective PKB inhibitor 21. Compound 21 showed

2246 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
McHardy et al.
Scheme 2a
a Reagents and conditions: (i) LiAlH4, THF, 0 °C then rt, 47%; (ii) NaH, 4-chlorobenzyl bromide, DMF, 0 °C then rt, 22%; (iii) 4M HCl, dioxane,
MeOH, rt; 100%; (iv) 4-chloro-7H-pyrrolo[2,3-d]pyrimidine, Et3N, n-BuOH, 100 °C, 69-82%; (v) 1-hydroxybenzotriazole, 1-ethyl-3-(3-dimethyl-
amino-propyl)carbodiimide, DMF, NH3 aq, rt, 97%; (vi) BH3, THF, 0 °C then 60 °C, 6%; (vii) 4-chlorobenzoyl chloride, Et3N, CH2Cl2, rt, 35%;
(viii) R1R2NH, HATU, iPr2NEt, DMF, rt, (R1R2NH = 4-chlorobenzylamine, 86%).
hinge-binder to give 20. The isomeric amide 21 was prepared
from an initial coupling of 4-chlorobenzylamine and 47 to give
the amide 52. Deprotection to 53 and introduction of the pyr-
rolopyrimidine gave 21. Analogues of 21 with different substitu-
tion of the amide were prepared by varying the amine in the first
step of this sequence. The 4-carbamido-4-aminopiperidine 53
was reacted with 4-fluoro-1-(triisopropylsilyl)-1H-pyrrolo[2,3-
b]pyridine38 and 6-chloro-7H-purin-8(9H)-one to give the ana-
logues 38 and 41, respectively.
General Synthetic Chemistry. Reactions were carried out
under N2. Organic solutions were dried over MgSO4 or Na2SO4.
Starting materials and solvents were purchased from commer-
cial suppliers and were used without further purification. Micro-
wave reactions were carried out using Biotage Initiator 60 or
CEM microwave reactors. 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 a Bruker AMX500 instrument at
500 MHz using internal deuterium locks. 13C NMR spectra
were recorded on a Bruker AMX500 instrument at 125 MHz.
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 (þve or -ve ion
mode as indicated) and with HPLC performed using Supelco
DISCOVERY C18, 50 mm ꢀ 4.6 mm or 30 mm ꢀ 4.6 mm i.d.
columns, at a temperature of 22 °C with gradient elution of
10-90% MeOH/0.1% aqueous formic acid at a flow rate of
1 mL/min and a run time of 3.5 or 10 min as indicated.
Compounds were detected at 254 nm using a Waters 2487 dual
λ absorbance detector. All tested compounds gave >95% purity
as determined by this method. All purified synthetic intermedi-
ates gave >95% purity as determined by this method except
where indicated in the text. High-resolution mass spectra were
measured on an Agilent 6210 ToF HPLC-MS with a Pheno-
menex Gemini 3 μm C18 (3 cm ꢀ 4.6 mm i.d.) column.
General Methods for Preparation of 4-Amino-4-benzylpiper-
idines. 4-(4-tert-Butylbenzyl)piperidin-4-amine (45). Method A.
n-BuLi (2.32 M in hexanes, 11.8 mL, 27.4 mmol) was added to
i
a solution of Pr2NH (3.8 mL, 27.4 mmol) in THF (75 mL) at
-78 °C under N2. After 10 min, a solution of tert-butyl 4-
cyanopiperidine-1-carboxylate (5.00 g, 23.8 mmol) in THF
(30 mL) was added. The cloudy solution was stirred for 1 h
at -78 °C. 1-(Bromomethyl)-4-tert-butylbenzene (5.2 mL,
28.5 mmol) was added and the clear yellow-brown solution
was warmed to rt and stirred for 15 h. Water (500 mL) was
added, and the mixture was extracted with Et2O (2 ꢀ 250 mL).
The organic layers were combined, washed with brine (200 mL),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2247
dried, and concentrated. Recrystallization from Et2O-hexane
gave tert-butyl 4-(4-tert-butylbenzyl)-4-cyanopiperidine-1-car-
boxylate (6.69 g, 79%). LC-MS (3.5 min) m/z 379 [M þ Naþ],
Rt = 2.96 min. 1H NMR (CDCl3) δ 1.33 (s, 9H), 1.47 (s, 9H),
1.48-1.52 (m, 2H), 2.85 (s, 2H), 2.95-3.04 (m, 2H), 4.08-4.16
(m, 2H), 7.20-7.22 (m, 2H), 7.36-7.38 (m, 2H). 13C NMR
(CDCl3) δ 28.4, 31.3, 34.5, 34.7, 39.2, 41.0, 45.4, 80.0, 122.0,
125.4, 130.0, 131.2, 150.5, 154.5 ppm. A solution of tert-butyl
4-(4-tert-butylbenzyl)-4-cyanopiperidine-1-carboxylate (5.40 g,
15.2 mmol) in AcOH (14 mL) and conc H2SO4 (7 mL) was
heated at 50 °C for 3 h and then at 90 °C for 2 h. The mixture was
cooled and cautiously basified to pH 14 by the addition of 2 M
NaOH aq (350 mL). Boc2O (4.96 g, 22.7 mmol) in dioxane
(100 mL) was added, and the mixture was stirred for 24 h. The
mixture was extracted with EtOAc (3 ꢀ 100 mL). The extracts
were dried and concentrated. Flash column chromatography,
eluting with EtOAc, gave tert-butyl 4-(4-tert-butylbenzyl)-4-car-
bamoylpiperidine-1-carboxylate 44 (4.02 g, 71%). LC-MS
(3.5 min) m/z 397 [MþNaþ], Rt=2.86 min. 1H NMR (CDCl3)
δ 1.31 (s, 9H), 1.46 (s, 9H), 1.49-1.56 (m, 2H), 2.81 (s, 2H),
3.00-3.07 (m, 2H), 3.82-3.90 (m, 2H), 7.07-7.08 (m, 2H),
7.29-7.30 (m, 2H). 13C NMR (CDCl3) δ 28.4, 31.3, 33.5,
34.4, 41.0 (br), 45.6, 46.7, 79.6, 125.1, 129.9, 133.1, 149.7, 154.9,
176.9 ppm.
Bis(trifluoroacetoxy)iodobenzene (5.05 g, 11.8 mmol) was
added to a suspension of 44 (4.00 g, 10.7 mmol) in MeCN (17
mL) and water (17 mL). After 17 h, water (70 mL) and conc HCl
(15 mL) were added. After a further 1 h, the mixture was washed
with Et2O (100 mL) and the aqueous layer was concentrated to
give 4-(4-tert-butylbenzyl)piperidin-4-amine bis hydrochloride
45 (1.57 g, 46%). LC-MS (10 min) m/z 247 [M þ Hþ], Rt = 1.25
min. 1H NMR (CD3OD) δ 1.33 (s, 9H), 2.14-2.17 (m, 4H), 3.16
(s, 2H), 3.47-3.49 (m, 4H), 7.24-7.26 (m, 2H), 7.47-7.49 (m,
2H). 13C NMR (CD3OD) δ 30.9, 31.7, 35.4, 40.7, 41.7, 54.5,
127.2, 131.2, 131.7, 152.2 ppm.
Method B. Ti(OEt)4 (0.42 mL, 2.00 mmol) was added to a
solution of tert-butyl 4-oxopiperidine-1-carboxylate (0.205 g,
1.03 mmol) and 2-methylpropane-2-sulfinamide (0.130 g, 1.07
mmol) in THF (5 mL) at rt. The resulting pale-yellow solution
was refluxed for 5 h and then cooled to 0 °C. A solution of (4-
tert-butylbenzyl)magnesium bromide in Et2O, prepared from
Mg (0.121 g, 5.00 mmol), 4-tert-butylbenzyl bromide (0.62 mL,
5.00 mmol), and Et2O (5 mL), was added and the mixture was
stirred at rt for 15 h. The reaction was quenched with methanol
(5 mL) and absorbed onto silica gel. Flash column chromato-
graphy, eluting with EtOAc-hexanes 1:1, gave tert-butyl 4-(4-
tert-butylbenzyl)-4-(1,1-dimethylethylsulfinamido)piperidine-
1-carboxylate 46 (0.121 g, 26%). LC-MS (10 min) m/z 473 [M þ
Naþ], Rt = 8.84 min. 1H NMR (CDCl3) δ 7.32-7.28 (m, 4H),
3.90-3.71 (br m, 2H), 3.35 (br s, 1H), 3.17 (d, J = 13.5 Hz, 1H),
3.15-2.98 (br m, 2H), 2.68 17 (d, J = 13.5 Hz, 1H), 2.36-2.33
(m, 1H), 1.79-1.72 (m, 1H), 1.59-1.54 (m, 1H), 1.46 (s, 9H),
1.42-1.35 (m, 1H), 1.32 (s, 9H), 1.25 (s, 9H). 4 M HCl in dioxane
(4 mL) was added to 46 (0.111 g, 0.246 mmol) in MeOH (4 mL)
at rt. After 16 h, the mixture was concentrated and purified by
ion exchange on acidic resin, eluting with MeOH and then 2 M
NH3 in MeOH to give 45 (free amine) (0.061 g, 100%). LC-MS
(10 min) m/z 247 [M þ Hþ], Rt = 1.25 min. 1H NMR (CD3OD)
δ 1.33 (s, 9H), 1.39-1.41 (m, 2H), 1.60-1.65 (m, 2H), 2.71 (s,
2H), 2.85-2.96 (m, 4H), 7.14-7.15 (m, 2H), 7.34-7.36 (m, 2H).
13C NMR (CD3OD) δ 31.8, 35.2, 39.1, 43.0, 47.8, 50.9, 126.0,
131.5, 131.7, 135.4, 150.8 ppm.
acidic resin, eluting with MeOHand then 2 M NH3 in MeOH,
gave 10 (0.465 g, 86%). LC-MS (10 min) m/z 364 [M þ Hþ], Rt =
4.25 min. Hi-Res LC-MS calcd for C22H30N5 364.2501, found
364.2503. 1H NMR (CD3OD) δ 1.32 (s, 9H), 1.55-1.58 (m, 2H),
1.75-1.81 (m, 2H), 2.78 (s, 2H), 3.79-3.85 (m, 2H), 4.24-4.29
(m, 2H), 6.63 (d, J = 4.0 Hz, 1H), 7.13 (d, J = 4.0 Hz, 1H), 7.37
(d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 8.12 (s, 1H). 13
C
NMR (CD3OD) 31.9, 35.3, 38.2, 43.3, 51.4, 102.7, 104.2, 122.2,
126.2, 131.6, 135.0, 150.6, 151.7, 152.4, 158.3 ppm. One 13C peak
was obscured by solvent.
4-Amino-N-(4-chlorobenzyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-
yl)piperidine-4-carboxamide (21). Dry DMF (1 mL) was added
to a mixture of 47 (151 mg, 0.44 mmol) and HATU (220 mg,
i
0.58 mmol). Pr2NEt (0.38 mL, 2.1 mmol) was added, and the
mixture was stirred for 15 min at rt. 4-Chloro-benzylamine
(70 μL, 0.57 mmol) was added, and the solution was stirred for
23 h. The reaction mixture was partitioned between water
(10 mL) and CH2Cl2 (3 ꢀ 10 mL). The organic extracts were
dried, filtered, and concentrated. Flash column chromato-
graphy, eluting with CH2Cl2-MeOH 96:4, gave 4-tert-butoxycar-
bonylamino-4-(4-chloro-benzylcarbamoyl)-piperidine-1-carbo-
xylic acid tert-butyl ester 52 (177 mg, 0.38 mmol, 86%). LC-MS
(10 min) m/z 490 [M þ Naþ], Rt = 8.07 min. 1H NMR (CDCl3) δ
1.41 (s, 9H), 1.47 (s, 9H), 1.98 (br d, J = 12 Hz, 2H), 2.10-2.16
(m, 2H), 3.10-3.15 (m, 2H), 3.84 (b rd, J = 11 Hz, 2H), 4.42 (d,
J = 5 Hz, 2H), 4.69 (s, 1H), 7.22 (d, J = 9 Hz, 2H), 7.28 (d, J = 9
Hz, 2H). 13C NMR (CDCl3) δ 28.2, 28.4, 32.2, 39.9, 42.9, 57.7,
79.8, 80.9, 128.7, 128.9, 133.2, 136.9, 154.6, 154.9, 173.4 ppm.
HCl (4 M) in dioxane (4.8 mL, 19.2 mmol) was added
dropwise to a solution of 52 (96 mg, 0.20 mmol) in MeOH
(4.8 mL). The solution was stirred at rt for 17 h. The mixture
was concentrated to give 4-amino-piperidine-4-carboxylic acid
4-chloro-benzylamide bis hydrochloride 53 (70 mg, 0.20 mmol,
100%) that was used in the next step without further purifica-
tion. 1H NMR (CD3OD) δ 2.15-2.22 (m, 2H), 2.60-2.69
(m, 2H), 3.34-3.36 (m, 2H), 3.44-3.46 (m, 2H), 4.47 (s, 2H),
7.36 (s, 4H). 13C NMR (CD3OD) δ 29.6, 41.0, 44.2, 57.5, 129.7,
130.6, 134.4, 138.3, 169.4 ppm. A degassed mixture of 53 bis
hydrochloride (48 mg, 0.14 mmol), 4-chloro-7H-pyrrolo[2,3-
d]pyrimidine (21 mg, 0.12 mmol), Et3N (126 μL, 0.9 mmol), and
n-BuOH (1.2 mL) was stirred at 100 °C for 18 h. The mixture was
concentrated and purified by ion exchange chromatography on
acidic resin, eluting with MeOH then 2 M NH3 in MeOH.
Preparative TLC, eluting with CH2Cl2-MeOH 9:1, gave 21
(37 mg, 0.096 mmol, 69%). LC-MS (10 min) m/z 385 [M þ Hþ],
Rt = 2.88 min. Hi Res LC-MS calcd for C19H22N6OCl
385.1544, found 385.1557. 1H NMR (CD3OD) δ 1.61 (br d,
J = 14 Hz, 2H), 2.19-2.25 (m, 2H), 3.65-3.71 (m, 2H), 4.38 (s,
2H), 4.47-4.50 (m, 2H), 6.64 (d, J = 4 Hz, 1H), 7.14 (d, J = 4
Hz, 1H), 7.28 (d, J = 8 Hz, 2H), 7.32 (d, J = 8 Hz, 2H), 8.14 (s,
1H). 13C NMR (CD3OD) δ 35.6, 43.0, 43.4, 57.2, 102.5, 104.2,
122.4, 129.6, 130.0, 133.9, 139.1, 151.6, 152.4, 158.2, 179.4 ppm.
Acknowledgment. We thank Dr A. Mirza, Dr M. Liu, and
M. Richards for assistance in the spectroscopic characterisa-
tion of test compounds and V. Martins for assistance in
analysing PK parameters. This work was supported by fund-
ing from Cancer Research UK [CUK] grant numbers C309/
A2187, C309/A8274, and C309/A8365, and The Institute of
Cancer Research. We acknowledge NHS funding to the
NIHR Biomedical Research Centre. This work was carried
out as part of a funded research collaboration with Astex
Therapeutics and intellectual property arising from the pro-
gramme has been licensed to AstraZeneca plc.
General Method for Preparation of Pyrrolo[2,3-d]pyrimidines. 4-
(4-tert-Butylbenzyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-
4-amine (10). A mixture of 45 bis hydrochloride salt (0.500 g, 1.57
mmol), 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (0.229 g, 1.49
mmol) and Et3N (1.04 mL, 7.46 mmol) in n-BuOH (15 mL)
was heated at 100 °C for 20 h. The mixture was cooled, concen-
trated, and the resulting solid washed with CH2Cl2 (15 mL). The
residual solid was collected. Ion exchange chromatography on
Supporting Information Available: Representative experimen-
tal procedures and characterization data for compounds 3-9,
11-20, 22-43; kinase panel selectivity data for compounds
10 and 21; experimental procedures for PKBβ and PKA

2248 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
McHardy et al.
biochemical assays; experimental procedures for SRB and
ELISA cellular assays; details of the X-ray crystallography data
collection and refinement for 10-PKB, 21-PKB. This material is
available free of charge via the Internet at http://pubs.acs.org.
Design and synthesis of pyridine-pyrazolopyridine-based inhibitors
of protein kinase B/Akt. Bioorg. Med. Chem. 2007, 15, 2441–2452.
(h) Zhu, G. D.; Gandhi, V. B.; Gong, J.; Thomas, S.; Woods, K. W.;
Song, X.; Li, T.; Diebold, R. B.; Luo, Y.; Liu, X.; Guan, R.; Klinghofer,
V.; Johnson, E. F.; Bouska, J.; Olson, A.; Marsh, K. C.; Stoll, V. S.;
Mamo, M.; Polakowski, J.; Campbell, T. J.; Martin, R. L.; Gintant,
G. A.; Penning, T. D.; Li, Q.; Rosenberg, S. H.; Giranda, V. L. Syntheses
of potent, selective, and orally bioavailable indazole-pyridine series of
protein kinase B/Akt inhibitors with reduced hypotension. J. Med.
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