Rapid evolution of 6-phenylpurine inhibitors of protein kinase B through structure-based design

Article abstract of DOI:10.1021/jm0700924

6-Phenylpurines were identified as novel, ATP-competitive inhibitors of protein kinase B (PKB/Akt) from a fragment-based screen and were rapidly progressed to potent compounds using iterative protein-ligand crystallography with a PKA-PKB chimeric protein.

Full text of DOI:10.1021/jm0700924

J. Med. Chem. 2007, 50, 2289-2292
2289
Chart 1
Rapid Evolution of 6-Phenylpurine Inhibitors of
Protein Kinase B through Structure-Based Design
Alastair Donald,† Tatiana McHardy,† Martin G. Rowlands,†
Lisa-Jane K. Hunter,† Thomas G. Davies,‡ Valerio Berdini,‡
Robert G. Boyle,‡ G. Wynne Aherne,†
Michelle D. Garrett,† and Ian Collins*,†
Cancer Research UK Centre for Cancer Therapeutics, The Institute
of Cancer Research, 15 Cotswold Road, Sutton, Surrey, United
Kingdom SM2 5NG, and Astex Therapeutics Ltd., 436 Cambridge
Science Park, Milton Road, Cambridge, United Kingdom CB4 0QA
f Lys, outside the ATP binding site, was made due to the
potential for Gln181 to perturb ligand binding in the presence
of the Val123 f Ala substitution.15 We have recently shown
that the binding modes of the unselective inhibitor 1 are identical
in PKA, PKB, and the PKA-PKB chimera.16 Additionally, for
one PKB-selective inhibitor,6 the chimera and PKB binding
modes were similar, and distinct from the binding to PKA.16
Although the use of PKB crystallography may be preferable
for developing PKB selective ligands, the PKA-PKB chimera
is suitable for guiding the de novo construction of inhibitor
scaffolds.
ReceiVed January 23, 2007
Abstract: 6-Phenylpurines were identified as novel, ATP-competitive
inhibitors of protein kinase B (PKB/Akt) from a fragment-based screen
and were rapidly progressed to potent compounds using iterative
protein-ligand crystallography with a PKA-PKB chimeric protein.
An elaborated lead compound showed cell growth inhibition and effects
on cellular signaling pathways characteristic of PKB inhibition.
7-Azaindole (2 (pdb code 2UVX), Chart 1) was one fragment
found to occupy the ATP-binding site, interacting through
bidentate hydrogen bonding of the heterocylic N and N-H to
the backbone amides of Glu121 and Val123 (PKA-PKB
chimera numbering) in the hinge region of the kinase (Figure
1a). Comparison with the crystallographic data11 for 1 showed
that this binding mode of 2 corresponded to the interactions of
the isoquinoline nitrogen and adjacent C1-H. The other main
determinants of binding for 1 to PKA and PKB are the basic
amine, which interacts with acid side chains in the ribose binding
pocket and the terminal lipophilic group that contacts the
P-loop.11
The PI3K-PKB-mTOR cascade is an important cellular
signaling pathway that regulates cell growth and proliferation,
cell survival, protein synthesis, glucose uptake, and glycogen
metabolism.1,2 Protein kinase B (PKB or Akt) has a pivotal
position in the pathway and is a well-validated anticancer target.3
PKB is downstream of the tumor suppressor PTEN, which is
deleted in many tumor types.4 Upstream receptor tyrosine
kinases, the lipid kinase PI3K and PKB itself, particularly the
â-isoform, are commonly amplified or mutated at the genetic
level, overexpressed, or overactivated in human tumors.1,4
Analogues of the natural product rapamycin, which inhibit the
signaling pathway at the downstream level of mTOR, have
shown efficacy in early clinical trials.2 In preclinical studies,
tumor growth reduction in animal models was observed with a
dual PI3K/mTOR inhibitor,5 and a recently reported ATP-
competitive inhibitor of PKB.6 There is, therefore, considerable
interest in the discovery of new small molecule inhibitors of
PKB kinase activity, which may act through competition with
ATP or through interaction with regulatory domains in the
protein.7 Some classes of ATP-competitive inhibitors have been
reported, including pyridines,6,8 azapanes,9 toxoflavins,10 and
isoquinoline-5-sulfonamides11,12 such as 111 (Chart 1). We show
here the use of iterative protein-ligand crystallography and
structure-based design to rapidly identify novel, potent 6-phen-
ylpurine PKBâ inhibitors.
A virtual screen of approximately 300 000 available low
molecular weight compounds13 (<250 kDa) was carried out
using the published PKBâ coordinates.14 Selected virtual hits
with promising predicted binding modes, particularly occupation
of the adenine binding pocket, were validated through bioassay
and crystallography with a PKA-PKB chimera.15 The use of
PKA9a,11 or PKA-PKB chimeras9b as surrogates for PKB is
well established and is successful due to the high sequence
homology of PKA and PKB in the ATP binding site (ca. 80%).
In this work, a chimera with three PKA f PKB mutations in
the kinase active site of PKA was used, namely, Val123 f Ala,
Val104 f Thr, and Leu173 f Met. A fourth mutation, Gln181
We initially replaced the 7-azaindole with purine. This offered
more flexible synthetic access to the required analogues, while
potentially retaining the hydrogen bonding to the hinge region.
A limited range of simple benzylamine and phenethylamine
substituents were added to the purine core, aiming to access
acidic residues in the ribose binding pocket. Suzuki couplings
to the protected 6-chloropurine 317 attached either benzaldehyde
(4, 5) or (cyanomethyl)phenyl (8, 9) groups, which were
elaborated by reductive amination and reduction, respectively
(Scheme 1). The increase in PKBâ enzyme inhibition seen for
compounds such as 6 (pdb code 2UVY), 7, and 11 (Table 1)
indicated that additional binding interactions were present, which
was confirmed by crystallography with the chimera (Figure 1b).
The ligand 6 occupied the ATP cleft, with bidentate hydrogen
bonding of the purine mimicking the binding mode of 2, as
anticipated, and the basic nitrogen forming favorable electro-
static interactions with the acidic residue Glu127 and the
backbone carbonyl of Glu170.11 Several water molecules were
also displaced by the 6-phenyl group relative to the structure
of 2. Compound 6 had good inhibitory activity for a small
molecule, with a high ligand efficiency18 (∆g ) 0.40 kcal mol-1
per non-H atom), indicating an excellent start point for hit-to-
lead evaluation. Unsurprisingly, 6 was equipotent against PKA
(Table 1).
Comparison of the crystallographic data for 6 with that for 1
indicated that the benzylic position of the 4-benzylamine 6
provided a suitable attachment point for lipophilic substitution.
This was achieved quickly through addition of Grignard reagents
to the N-sulfinylimine19 derived from the aldehyde 4 (Scheme
1). The resulting phenyl and benzyl substituted compounds 13
* To whom correspondence should be addressed. Tel.: +44 2087224317.
Fax: +44 02087224126. E-mail: ian.collins@icr.ac.uk.
† The Institute of Cancer Research.
‡ Astex Therapeutics Ltd.
10.1021/jm0700924 CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/24/2007

2290 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Letters
Figure 1. X-ray crystal structures of (a) 2, (b) 6, (c) 13, and (d) 20 with PKA-PKB chimeric kinase domain. A detail of the ATP binding cleft
is shown, oriented with the N-terminal domain and P-loop at the top of the panel, with the protein surface colored by electrostatic charge (red
) negative, blue ) positive, white ) neutral). The prominent areas of negative charge are the side chains of Glu127 (left) and Asp184 (right).
Scheme 1a
Table 1. Enzyme Inhibition, Cellular Growth Inhibition, and Calculated
Physicochemical Properties of PKB Inhibitors
PKBâa IC50
(µM)
PKAb IC50 PC3 SRBc
TPSA
(A2)
No.
(µM)
GI50 (µM)
ClogP
H-89 0.59 (( 0.07)
0.073
0.17
n.d.d
5.2
5.5
n.d.
n.d.
n.d.
0.61
n.d.
0.015
0.033
18 (( 1.4)
25 (( 1)
n.d.
>50
>50
n.d.
>50
32
32
28
4.44
3.95
1.18
1.25
1.25
1.17
1.17
2.18
2.71
2.89
3.47
4.81
71
80
24
61
61
75
75
75
75
75
61
61
1
0.26 (( 0.02)
>100
2
6
7
6.9 (( 1.3)
1.6 (( 0.4)
10
11
13
14
16
18
20
9.5 (( 2.3)
3.2 (( 0.4)
0.40 (( 0.06)
0.43 (( 0.12)
0.009 (( 0.003)
0.010 (( 0.003)
0.020 (( 0.003)
16
4.5
a Inhibition of PKBâ kinase activity in a radiometric filter binding assay.
Mean ((SEM) for n ) 3 determinations. b Inhibition of PKA kinase activity
in a radiometric filter binding assay. Single determinations from duplicate
points. Standard isoquinoline-5-sulfonamide inhibitor H-811 gave IC50
)
7.6 µM, SD ( 38% (n ) 14) in this assay. c Cell growth inhibition
(sulforhodamine B colorimetric assay) determined in PC3M prostate cancer
cells. Mean of two independent determinations or mean ((SEM) for n >
2 determinations. Standard isoquinoline-5-sulfonamide inhibitor H-8911 gave
SD ( 34% (n ) 20) in this assay. d n.d. ) not determined.
philic substituent was able to generate potent PKB inhibitors.
However, compound 16 did not show an improvement in cell
growth inhibition. Comparison of calculated values20 for lipo-
philicity (ClogP) and polar surface area (TPSA) for the
isoquinoline and purine inhibitors (Table 1) suggested that the
purines had a lower ratio of lipophilicity to polar surface area,
which we considered might impair cellular penetration. To probe
this, we examined minor structural variations to 16 that increased
the ratio of ClogP to TPSA.
a Reagents: (i) ArB(OH)2, Pd(PPh3)4, K2CO3, DME; (ii) MeNH2, EtOH,
then NaBH4, MeOH; (iii) HCl, EtOH; (iv) HCO2NH4, Pd/C, MeOH; (v)
H2NSOtBu, PPTS, MgSO4, CH2Cl2; (vi) RMgBr, Et2O-CH2Cl2.
(pdb code 2UVZ) and 14 showed a greater than 15-fold increase
in PKB enzyme inhibition. The compounds were prepared and
tested as racemates, although the crystal structure of 13 with
the PKA-PKB chimera (Figure 1c) indicated that the S-
enantiomer was binding preferentially. The position of the amine
was observed to shift slightly relative to 6 and to make contacts
with Asp184 and Asn171. The crystallographic data for 13
showed a change in the structure of the P-loop relative to that
seen with 6, such that the additional phenyl ring of 13 displaced
the side chain of Phe54 and occupied the corresponding space.
Potential for further substitution in this region was also evident.
Importantly, cell growth inhibition, albeit weak, was now
observed for 13 and 14, which had similar profiles to the
isoquinoline 1 (Table 1).
By analogy with the optimal 4-chlorophenyl group identified
for 1, 4-chloro substitution of 13 was investigated. A substituted
arylboronate was constructed from the benzyl alcohol 15 before
coupling to the 6-chloropurine 3 (Scheme 2). A further 40-fold
increase in enzyme activity was observed, producing a nano-
molar inhibitor of PKB, 16 (IC50 ) 9 nM). This confirmed that
recapitulation of the 3-D pharmacophore defined by the biden-
tate hydrogen-bonding group, basic amine, and terminal lipo-
Elaboration of the phenethylamine scaffold 1721 to prepare
the N-methylated homologue 18 (Scheme 2) increased the
lipophilicity somewhat and led to a 2-fold improvement in
cellular activity. Compound 18 was prepared and tested as the
racemate, and the individual enantiomers were not separated.
We had previously observed11 that there was some tolerance in
the exact positioning of the basic amine in the ribose pocket
with isoquinoline ligands, reflecting the possibility for produc-
tive binding interactions with either or both of the acid side
chains Glu127 and Asp184, as seen for 6 versus 13 (Figure
1b,c and Figure 2a). Among other alterations to the inhibitor
structures in the region of the basic amine, we prepared the
4-substituted piperidine 20 (pdb code 2UW0) from the bromide
1922 (Scheme 2). Compound 20, which was substantially more
lipophilic than 16 or 18, maintained good enzyme inhibitory
activity and ligand efficiency (∆g ) 0.38 kcal mol-1 per non-H
atom) and now showed much improved cell growth inhibition.
Comparison of the crystallographic data for 1 and 20 bound
to the PKA-PKB chimera shows the close correspondence of
the key pharmacophoric groups (Figure 2b). Several factors may
explain the better PKB affinity of 20 relative to 1. First, the
hydrogen bonding to the hinge region of the kinase is supplied

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2291
Table 2. Growth Inhibition and Target Modulation in Cells by 20
Scheme 2a
growth inhibition
pGSK3â ELISAb
IC50 (µM)
cell line
SRBa GI50 (µM)
PC3M (prostate)
HCT116 (colon)
U87MG (brain)
4.5
5.7
8.7 (( 0.5)
6.0
n.d.c
1.9 (( 0.7)
a See footnote to Table 1 for details of assay variability. b Single
determination or mean ((SEM) for n ) 3. Standard isoquinoline-5-
sulfonamide inhibitor H-8911 gave IC50 15 (( 2) µM in PC3M cells. c n.d.
) not determined.
Figure 3. Western blot showing titration of the effect of compound
20 on the phosphorylation state of components of the PI3K-PKB-
mTOR pathway in U87MG glioblastoma cells after 1 h treatment. D
) DMSO control.
a Reagents: (i) phthalimide, DIAD, PPh3, THF; (ii) bis(pinacolato)di-
boron, Pd2(dba)3, (c-hexyl)3P, KOAc, dioxane; (iii) 3, PdCl2(PPh3)2, K2CO3,
DME; (iv) NH2NH2, EtOH; (v) HCl-dioxane; (vi) (Boc)2O, Et3N, CH2Cl2;
(vii) n-BuLi, B(Oi-Pr)3, THF.
penetration is unoptimized for these compounds or may reflect
the degree to which the pathway is sensitive to inhibition of
PKB in these cell lines.
The effects of 20 on the PI3K-PKB-mTOR and associated
pathways in U87MG glioblastoma cells was examined by
Western blot using phospho-specific antibodies (Figure 3). This
cell line has recently been shown to be particularly relevant for
the characterization of inhibitors of the PI3K-PKB-mTOR
pathway.5
At similar concentrations to the IC50 for cell growth inhibition,
compound 20 showed inhibition of signaling through PKB.
Effects were seen in multiple branches of the downstream
pathway, with decreases in the levels of pGSK3â, pS6, and
pFKHR proteins. An increase in the levels of pSer473-PKB
was observed at low concentrations of 20, suggesting increased
activation of PKB in response to pathway inhibition. This is
consistent with the recently identified feedback inhibition
pathway from activated p70S6K, downstream of PKB, to the
upstream regulator IRS1.5,25 Although the release of this
feedback through inhibition of PKB may elicit a compensatory
upregulation of signaling in the pathway, it is important to note
that overall inhibition of downstream signaling, and cell growth,
is seen with compounds such as 20.
To further assess the potential of the 6-phenylpurine scaffold
as a lead PKB inhibitor, the inhibitory effect of 20 on important
cytochrome P450 isoforms in vitro was measured. Compound
20 showed IC50 >10 µM for CYP1A2, CYP2C9, and CYP2D6,
while some inhibition of CYP3A4 (IC50 ∼ 10 µM) and
CYP2C19 (IC50 ) 1.6 µM) was observed.
Figure 2. Overlay of bound conformations of (a) 6 (cyan), 13 (gold),
and 20 (gray) and (b) 1 (gold) and 20 (gray), as determined by
crystallography with PKA-PKB chimera.
by two overt hydrogen bonds for 20, whereas one of these is a
weaker C-H‚‚‚O type interaction in 1. Second, it is clear from
the overlay that the more rigid structure 20 delivers the key
functional groups to the protein’s surface in a more efficient
manner than 1. In particular, more rotatable bonds must be
frozen for the binding of 1, leading to a higher entropic penalty,
which would compromise the overall binding affinity. The
enzyme inhibitory activity and binding mode of 20 was
comparable to that of a related 4-phenylpyrazole discovered
through parallel optimization of a pyrazole fragment hit.23
Compound 20 was examined for growth inhibition in other
cell lines and showed a similar level of activity (Table 2). For
some of these, the cellular mechanism of action of 20 was
probed using a cellular ELISA protocol to examine phosphor-
ylation of GSK3â, an immediate downstream substrate for
PKB.24 This quantification of the inhibitory potency of the
compound in cells indicated that modulation of the target activity
was occurring at similar concentrations to the cell growth
inhibition. The difference between the enzymic inhibitory
activity of 20 and the cellular activity may indicate that cell
In summary, elaboration of the very low affinity fragment
hit 2, using iterative protein-ligand crystallography, allowed
us to rapidly identify novel PKB inhibitors based on the

2292 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
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physicochemical properties consistent with further optimization.
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PKA pharmacophore. Selectivity for inhibition of PKB over
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Acknowledgment. This work was supported by Cancer
Research UK [CUK] Grant Number 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: Experimental details for
the preparation of 13, 14, 16, 18, and 20, experimental details for
pGSK3â ELISA and SRB cellular assays, and X-ray crystallography
statistics (2, 6, 13, 20). This material is available free of charge
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