2-Anilino-4-aryl-8H-purine derivatives as inhibitors of PDK1

Article abstract of DOI:10.1016/j.bmcl.2012.02.058

A series of 2-anilino substituted 4-aryl-8H-purines were prepared as potent inhibitors of PDK1, a serine-threonine kinase thought to play a role in the PI3K/Akt signaling pathway, a key mediator of cancer cell growth, survival and tumorigenesis. The synthesis, SAR and ADME properties of this series of compounds are discussed culminating in the discovery of compound 6 which possessed sub-micromolar cell proliferation activity and 65% oral bioavailability in mice.

Full text of DOI:10.1016/j.bmcl.2012.02.058

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2880–2884
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Anilino-4-aryl-8H-purine derivatives as inhibitors of PDK1
⇑
Stéphanie Blanchard , Chang Kai Soh, Chai Ping Lee, Anders Poulsen, Zahid Bonday, Kay Lin Goh,
Kee Chuan Goh, Miah Kiat Goh, Mohammed Khalid Pasha, Haishan Wang, Meredith Williams,
Jeanette M. Wood, Kantharaj Ethirajulu, Brian W. Dymock
S⁄BIO Pte. Ltd, 1 Science Park Road, #05-09 The Capricorn, Singapore Science Park II, Singapore 117528, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-anilino substituted 4-aryl-8H-purines were prepared as potent inhibitors of PDK1, a serine-
threonine kinase thought to play a role in the PI3K/Akt signaling pathway, a key mediator of cancer cell
growth, survival and tumorigenesis. The synthesis, SAR and ADME properties of this series of compounds
are discussed culminating in the discovery of compound 6 which possessed sub-micromolar cell prolif-
eration activity and 65% oral bioavailability in mice.
Received 6 November 2011
Revised 16 February 2012
Accepted 20 February 2012
Available online 27 February 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
3-Phosphoinositide-dependent kinase 1
(PDK1)
PI3K
Akt
CDK2
4-Aryl-purine
Mutations or altered expression of most of the components of
the PI3K/Akt pathway have been strongly implicated in oncogene-
sis. Gene amplification of p110 occurs in some cases of human
ovarian cancer,1 and amplification of Akt is found in ovarian,
breast, and colon cancer.1–3 In addition, activating mutations in
p85 have been identified in ovarian and colon cancer.1,2,4 PDK1
(3-phosphoinositide-dependent kinase 1) is a 63 kDa Ser/Thr ki-
nase over-expressed in the majority of human cancer cell lines
and in the tissue specimens of breast, prostate, stomach and pan-
creatic cancer patients.5–7 Expression of PDK1 alone was sufficient
to transform mouse mammary epithelial cells, a process believed
to depend on PDK1-mediated activation of protein kinase C rather
than activation of Akt1.8 PDK1 has been shown to serve as a master
regulator of a group of protein kinases known as the AGC kinase
super-family which play important roles in the progression of can-
cer, such as activation of Akt, S6 kinase (S6K, SGK, and PKA), and
protein kinase C (PKC). Most importantly PTEN, a phosphatase up-
stream of PDK1, has been identified as a major tumor suppressor in
humans and loss-of-function mutations in the PTEN gene are ex-
tremely common among sporadic glioblastomas, melanomas, pros-
high percentage of human cancers possess activated PI3K
signaling.
PDK1 presents an attractive anti-cancer target because it is lo-
cated downstream of PI3K in the signaling pathway.10 Thus,
PDK1 inhibition, might avoid possible adverse effects resulting
from inhibiting PI3K which is upstream of a wide range of signaling
cascades.
Considerable effort has been expended in the search for inhibi-
tors of PDK1, most of which target the ATP binding site.10 Ad-
vanced programs have led to new PDK1 inhibitors recently
entering the clinic.11 However these compounds often inhibit
many kinases and in many cases are not optimal for oral dosing.
We sought to develop a small molecule selective PDK1 inhibitor
suitable for oral administration.
Our strategy was to carry out a virtual screen of our in-house
chemical libraries and test a subset of hit compounds in an isolated
ATPase enzyme assay. In parallel, we set out to screen our fragment
library by NMR with follow-up of competitive binders in the ATP-
OMe
tate cancers, and endometrial carcinomas, and
a significant
O
R3
percentage of breast tumors, lung cancers, and lymphomas also
bear PTEN mutations.9 Thus, through a variety of mechanisms, a
N
R2
4
N
N
N
N
8
N
H
N
H
N
N
H
N
N
H
1
2
⇑
Corresponding author.
E-mail address: stephanie_blanchard@sbio.com (S. Blanchard).
Figure 1. PDK1 hits discovered by focused screening.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.058

S. Blanchard et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2880–2884
2881
Table 1
2-Anilinophenyl and 4-aryl groups of 2 produced via Scheme 1
R1
R2
N
N
N
H
N
N
H
Compound
R1
R2
Cl
PDK1 IC50
(l
M)15
CDK2 IC50
>10
(l
M)16
Solubility (
l
g/ml)*
OMe
OMe
3
0.97
N.T.
OH
4
5
6
Cl
Cl
1.6
>10
>10
>10
N.T.
N.T.
25
N
0.26
0.15
N
O
OMe
OMe
N
N
H
O
O
OMe
N
H
N
N
7
0.17
>10
N.T.
O
O
N
H
8
9
0.28
1.2
>10
N.T.
>10
23
13
29
N
F
O
O
N
H
N
N
N
N
H
10
0.4
NH
O
2
OMe
11
0.41
>10
65
N
H
N
*
MTS dilution based turbidimetric assay using DMSO stock solution; N.T. = not tested.
ase assay.12 From our initial hits we identified several different
classes of compounds that were optimized using structural models
of the hit structures docked into the PDK1 active site. This focused
screening approach resulted in the discovery of compound 1
attention focused on the 2-position where meta-substituted ani-
lines were preferred. Hence the strategy focused on development
of 2-anilinophenyl side-chains and parallel optimization of the 4-
aryl group (R1). Introduction of various R1 groups combined with
meta-substituted aniline groups was the first major area investi-
gated in this series (Table 1).
(Fig. 1) as a potent inhibitor of PDK1 with an IC50 of 0.39
our ATPase assay.
lM in
Macrocycle 1 was originally synthesized as part of a cyclin-
dependent protein kinase (CDK) inhibitor program where it had
been shown to inhibit CDK2 at low micromolar concentrations.
In order to identify potentially selective PDK1 inhibitors it was nec-
essary to deconstruct 1 to eliminate the linker and basic centre
which were known to be responsible for the unwanted CDK activ-
ity. The subsequent hit to lead process was heavily influenced by
prior art in this congested area of kinase inhibitors and led to the
design of novel 4-aryl purines (2), where A, B or D are either carbon
or nitrogen (Fig. 1).
Herein, we disclose our optimization strategy applied to the 4-
aryl-purine scaffold leading to potent PDK1 inhibitors with good
physicochemical and pharmacological properties and devoid of
CDK activity. A counter-screen against CDK2 was introduced at
an early stage of our screening cascade to ensure this unwanted
activity was not re-introduced.
Aromatic or heteroaromatic moieties at R1 conferred novelty
and encouraging initial potency and selectivity: dimethoxy 3 with
IC50 of 0.97 lM was also selective over CDK2, a key objective in our
target profile; the bulky basic methylpiperazine (5) improved tar-
get potency while maintaining selectivity. Structure-based design
(see Ref. 15) suggested amides or ureas as R2 substituents which
turned out to be beneficial (compare fivefold potency difference
between 7 and 3): methoxy, and a range of other ether substituents
(6, 7, 8 and 11) gave good potency with IC50s at or below 400 nM.
Introduction of polar groups in the para position, such as phenol
moiety 4 or pyridyl 9, resulted in a significant reduction in potency.
meta Substituents on phenyl ring R1, such as benzyl methyl piper-
azine 5, generally gave good potency. Similarly, the methoxy group
of compound 11 appears to be responsible for its potency, expected
to be lower due to the polar amine group in the para position, as
discussed above. These promising results indicate that meta sub-
stituents, including larger basic groups, likely to be more soluble,
were tolerated in this region of the active site. In general, this first
It was clear from initial SAR, and supported by modeling stud-
ies, that substitutions on either 9-N or C-8 were not tolerated so

2882
S. Blanchard et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2880–2884
Table 2
2-[3-Anilinophenyl]-4-[4-methoxyaryl] analogues produced via Scheme 1
OMe
R3
R2
N
N
N
H
N
N
H
Compound
R3
R2
PDK1 IC50
(
l
M)15
CDK2 IC50
>10
(l
M)16
Solubility (
lg/ml)
NH
NH
O
O
O
O
O
12
OMe
0.11
175
N
N
H
H
13
14
15
16
17
18
H
0.046
0.084
0.075
0.22
>10
>10
>10
N.T.
N.T.
>10
168
186
148
N.T.
N.T.
175
N
H
N
H
O
O
N
N
OMe
H
N
H
N
H
N
H
N
H
H
N
H
N
H
N
OMe
OMe
4.5
O
NH
O
0.079
N
H
NH
NH
H
N
19
OMe
3.3
N.T.
171
O
OMe
OMe
N
OMe
N
O
O
Cl
Cl
HN
N
HN
N
N
d
N
b
a
N
N
N
N
c
N
N
N
N
N
N
H
N
Cl
N
H
N
Cl
N
Cl
N
N
N
N
H
H
20
21
OMe
22
OMe
23
6
OMe
Scheme 1. Synthesis of PDK1 inhibitor 6. Reagents and conditions: (a) 1-Chloromethyl-4-methoxy-benzene, K2CO3, DMSO, 80 °C (52% yield); (b) 4-methoxyphenylboronic
acid,[1,10-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane, K2CO3, dioxane/water (75% yield); (c) pyrrolidine-1-carboxylic acid
(3-amino-phenyl)-amide, Pd2(dba)3, NaOtBu, dioxane (35% yield); (d) TFA (53% yield).
series of compounds showed good initial potency towards PDK1
while achieving excellent selectivity over CDK2. To simplify the
SAR in moving forward we chose to fix R1 as a 3,4-dimethoxy-
phenyl or 4-methoxyphenyl group. To guide the optimization,
modeling studies were carried out using published X-ray struc-
tures of PDK1.13,14
Purines from Tables 1 and 2 were accessible using a straight for-
ward and high yielding procedure: the synthesis of representative
PDK1 inhibitor 6 is outlined in Scheme 1 and its docking to the ATP
binding site is illustrated in Fig. 2. Commercially available 2,4-
dichloropurine 20 was first protected using 1-chloromethyl-4-
methoxy-benzene under basic conditions giving 21 followed by a
Suzuki coupling to give 4-aryl-purine intermediate 22. Reaction
with pyrrolidine-1-carboxylic acid (3-amino-phenyl)-amide was
performed under Buchwald–Hartwig cross coupling conditions. Fi-
nal deprotection with trifluoroacetic acid yielded compound 6.
Modification of R2 was investigated with a view to increasing
potency as well as improving the solubility of the compounds. meta
Substitution was preferred (SAR not shown). Based on the urea
analogues 6 and 7, which gave good potency against PDK1 as well
as in cellular assays (described below) whilst also achieving selec-
tivity over CDK2, but with low solubility, we decided to investigate
the introduction of various ureas and amides with appended solu-
bility groups (Table 2).
Docking of compound 6 suggests key hydrogen bonds form
from the aminopurine with the hinge region (Tyr161, Ala162)
and the urea with Leu88. Addition of hydrogen bond donors (12
and 13) or a morpholine group on the urea linker (14 and 15) ren-
dered the compounds more soluble and enhanced PDK1 potency
with IC50s now below 100 nM. Further studies indicated that the
basic nitrogen of the pyrrolidine moiety in 13 may form an addi-
tional salt bridge to Glu166 explaining its increased potency.

S. Blanchard et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2880–2884
2883
100000
IV (5 mg/kg, n=3)
PO ( 50 mg/kg, n=3)
10000
1000
100
10
1
Figure 2. Compound 6 (bold tube with green carbon) docked into the PDK1 ATP-
binding site (thin tube with grey carbon). Hydrogen bonds are shown as yellow
dashed lines.
0
4
8
12
16
20
24
Time (h)
Figure 3. Oral and intravenous pharmacokinetics of compound 6.
Replacement of the urea linker by an amide (18) had little effect
on potency whereas reversal of the amide group, as seen in 17 and
19, results in a dramatic loss of activity for PDK1, probably due to
the destruction of the NH—Leu88 H-bond. Comparison of 7 and the
ring expanded analogue 16 implies that small steric changes in the
urea do not affect the potency. For this second iteration of com-
pounds, although improved potency and solubility were achieved
permeability was seriously compromised (data not shown) render-
ing these compounds useful only as in vitro ligands with negligible
utility in cells. In our experience loss of permeability when the
number of H-bond donors exceeds three appears to be a particular
danger in PDK1 inhibitor design.11
Table 4
Pharmacokinetic parameters of compound 6
PK parameters
Cmax g/ml)
po (50 mg/kg)
iv (5 mg/kg)
(l
22
1
—
—
1.48
0.41
0.9
12
Tmax (h)
T1/2 (h)
1.84
CL (L/h/kg)
Vdz (L/kg)
0.63
1.7
AUC0–last
F %
(l
g h/ml)
79
65
—
Selected compounds were evaluated in cellular proliferation as-
says, metabolic stability in mouse and human liver microsomes
and Caco-2 permeability. All compounds had very good in vitro
metabolic stability (t1/2) as assessed in human and mouse liver
microsomes. Compounds 6 and 18 showed encouraging activity
against human tumor cell lines sensitive to the PI3K/Akt pathway,
such as the prostate cancer cell line PC3 (Table 3). However an
additional hydrogen bond and high polar surface area (128 Å2) ren-
dered 13 much less active than 6 or 18 in PC3 cells. This was not
unexpected from its negligible permeability which serves to ex-
plain the poor cellular activity. Although not apparently permeable
18 was still active in PC3 cells perhaps due to active transport, fur-
ther studies would be required to fully understand this activity.
From this data it is clearly preferred to maintain a maximum H-
bond donor count of three in these polar molecules and ensure glo-
bal polar surface area is below 120 Å2.
Compound 6 was also potent in another prostate cell line,
DU145, and in a breast cancer cell line, MDA-MB468, leading us
to select it for pharmacokinetic studies in the mouse. The results
are represented in Figure 3 and Table 4 and show that 6 has an
excellent pharmacokinetic profile in mice. A single oral dose re-
sults in rapid exposure with a high Cmax and good oral bioavailabil-
ity of 65%.
inhibition and a good understanding of the structural elements re-
quired for desirable drug-like properties.
Compound 6 represents a promising lead with good ADME
properties, cell proliferation inhibition and good oral bioavailabil-
ity in the mouse. Further studies of PDK1 inhibitors are under
investigation to fully elucidate their intracellular mechanism of
action.17
References and notes
1. Campbell, I. G.; Russell, S. E.; Choong, D. Y. H.; Montgomery, K. G.; Ciavarella, M.
L.; Hooi, C. S. F.; Cristiano, B. E.; Pearson, R. B.; Phillips, W. A. Cancer Res. 2004,
64, 7678.
2. Levine, D. A.; Bogomolniy, F.; Yee, C. Y.; Lash, A.; Barakat, R. R.; Borgen, P. I.;
Boyd, J. Clin. Cancer Res. 2005, 11, 2875.
3. Vivanco, I.; Sawyers, C. L. Nat. Rev. Cancer 2002, 2, 489.
4. Courtney, K. D.; Corcoran, R. B.; Engelman, J. A. J. Clin. Oncol. 2010, 28, 1075.
5. Liang, K.; Lu, Y.; Li, X.; Zeng, X.; Glazer, R. I.; Mills, G. B.; Fan, Z. Mol. Pharmacol.
2006, 70(3), 1045.
6. Xie, Z.; Yuan, H.; Yin, Y.; Zeng, X.; Bai, R.; Glazer, R. I. BMC Cancer 2006, 6, 77.
7. Maurer, M.; Su, T.; Saal, L. H.; Koujak, S.; Hopkins, B. D.; Barkley, C. R.; Wu, J.;
Nandula, S.; Dutta, B.; Xie, Y.; Chin, Y. R.; Kim, D.-I.; Ferris, J. S.; Gruvberger-
Saal, S. K.; Laakso, M.; Wang, X.; Memeo, L.; Rojtman, A.; Matos, T.; Yu, J. S.;
Cordon-Cardo, C.; Isola, J.; Terry, M. B.; Toker, A.; Mills, G. B.; Zhao, J. J.; Murty,
V. V. V. S.; Hibshoosh, H.; Parsons, R. Cancer Res. 2009, 69, 6299.
8. Lawlor, M. A.; Mora, A.; Ashby, P. R.; Williams, M. R.; Murray-Tait, V.; Malone,
L.; Prescott, A. R.; Lucocq, J. M.; Alessi, D. R. EMBO J. 2002, 21, 372.
9. Lim, M. A.; Yang, L.; Zheng, Y.; Wu, H.; Dong, L. Q.; Liu, F. Oncogene 2004, 23,
9348.
In conclusion we have designed and synthesized a series of 2-
anilino-4-aryl-8H-purine derivatives with potent PDK1 in vitro
Table 3
Cellular, microsomal and physicochemical profile of preferred compounds
Compound PC3 IC50
(
lM) DU145 IC50
(
lM) MDA-MB468 IC50
(l
M) MLM t1/2 (min) HLM t1/2 (min) PAPP (ꢀ10ꢁ6 cm/s) TPSA (Å2) H-bond donor count
6
13
18
0.58
44
1.07
0.83
N.T.
N.T.
1.3
N.T.
N.T.
>60
>60
>60
>60
>60
N.T.
2.07
0
0
104
128
121
3
5
4

2884
S. Blanchard et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2880–2884
10. Engelman, J. A. Nat. Rev. Cancer 2009, 9, 550.
11. Peifer, C.; Alessi, D. R. ChemMedChem 1810, 2008, 3.
12. Lee, A. C. H.; Murugappan, S. R.; Poulsen, A.; Williams, M.; Blanchard, S.; Ma, D.
M.; Bonday, Z.; Goh, K. L.; Goh, K. C.; Goh, M. K.; Wood, J. M.; Dymock, B. W.
Bioorg. Med. Chem. Lett., in preparation.
13. All computational chemistry software used is from Schrödinger (http://
www.schrodinger.com). The PDK1 and CDK2 X-ray structures (1Z5M and
1AQ1, respectively) were downloaded from the Protein Data Bank (http://
www.pdb.org) and prepared with the Protein Preparation Wizard as
recommended by Schrodinger. Ligands were prepared with Ligprep and
docked into the protein structures using Glide and the XP scoring function
with standard settings. PDB entry 1Z5M. Feldman, R. I.; Wu, J. M.; Polokoff, M.
A.; Kochanny, M. J.; Dinter, H.; Zhu, D.; Biroc, S. L.; Alicke, B.; Bryant, J.; Yuan, S.;
Buckman, B.; Lentz, D.; Ferrer, M.; Whitlow, M.; Adler, M.; Finster, S.; Chang, Z.;
Arnaiz, D. O. J. Biol. Chem. 2005, 280, 19867.
1 h incubation at room temperature, the reaction was terminated with 25
stop buffer (45 mM EDTA, 50 mM ATP, 0.1% Triton-X, 30 mM Hepes pH 7.5)
and 60 L of the stopped reaction was transferred to a 384-well Streptavidin
FlashPlate HTS PLUS (Perkin Elmer Cat # SMP410). After minimum 30 min
L/
lL of
l
incubation at room temperature, the FlashPlate was washed 3ꢀ with 90
l
well of H2O/0.02% Tween 20, air-dried and counted on a Wallac MicroBeta
(Perkin–Elmer) scintillation counter.
16. CDK2 kinase assay was carried out in 384-well white microtiter plates using
the PKLight assay system from Cambrex. For CDK2/Cyclin A assay, the reaction
mixture consisted of the following components in 25
Hepes pH 7.5, 10 mM MgCl2, 5 mM MnCl2, 5 mM BGP, 1 mM DTT, 0.1 mM
sodium orthovanadate), 1.4 g/ml of CDK2/Cyclin A complex, 0.5 M of RbING
substrate (Invitrogen, Cat # PV2939) and 0.5 M of ATP. The reaction was
lL assay buffer (50 mM
l
l
l
incubated at room temperature for 2 h in each case. Thirteen microliter of
PKLight ATP detection reagent was added and the reaction was incubated for
10 min. Luminescence signals were detected on a multi-label plate reader
(Victor2 V 1420, Perkin–Elmer). The analytical software, Prism 4.0 (GraphPad
Software Pte Ltd) was used to generate IC50 values from the data.
17. Tan, J.; Lee, P. L.; Li, Z.; Jiang, X.; Lim, Y. C.; Hooi, S. C.; Yu, Q. Cancer cell 2010, 18,
459.
14. Poulsen, A.; Blanchard, S.; Soh, C. K.; Lee, C.; Williams, M.; Wang, H.; Dymock,
B. Bioorg. Med. Chem. Lett. 2012, 22, 305.
15. PDK1 assay: The kinase reactions were initiated by adding 25
solution (2 M biotin–casein, 4 M ATP, 10 Ci/ml [
33P] ATP in 1x assay
buffer: 30 mM Hepes pH 7.5, 5 mM MgCl2, 30 mM KCl, 1 mM DTT and 0.2 mg/
ml BSA) to 25 L of 2x enzyme solution (100 nM PDK1 in 1x assay buffer). After
lL of 2x substrate
l
l
l
c-
l