Indolinone based phosphoinositide-dependent kinase-1 (PDK1) inhibitors. Part 2: Optimization of BX-517

Article abstract of DOI:10.1016/j.bmcl.2007.05.060

Based on the lead compound BX-517, a series of C-4′ substituted indolinones have been synthesized and evaluated for PDK1 inhibition. Modification at C-4′ of the pyrrole afforded potent compounds (7b and 7d) with improved solubility and ADME properties. In this letter, we describe the synthesis, selectivity profile, and pharmacokinetic data of selected compounds.

Full text of DOI:10.1016/j.bmcl.2007.05.060

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3819–3825
Indolinone based phosphoinositide-dependent kinase-1
(PDK1) inhibitors. Part 2: Optimization of BX-517
Imadul Islam,* Greg Brown, Judi Bryant, Paul Hrvatin, Monica J. Kochanny,
Gary B. Phillips, Shendong Yuan, Marc Adler, Marc Whitlow, Dao Lentz,
Mark A. Polokoff, James Wu, Jun Shen, Janette Walters, Elena Ho,
Babu Subramanyam, Daguang Zhu, Richard I. Feldman and Damian O. Arnaiz
Berlex Biosciences, 2600 Hilltop Dr. Richmond, CA 94804, USA
Received 8 March 2007; revised 17 May 2007; accepted 18 May 2007
Available online 23 May 2007
Abstract—Based on the lead compound BX-517, a series of C-40 substituted indolinones have been synthesized and evaluated for
PDK1 inhibition. Modification at C-40 of the pyrrole afforded potent compounds (7b and 7d) with improved solubility and ADME
properties. In this letter, we describe the synthesis, selectivity profile, and pharmacokinetic data of selected compounds.
Ó 2007 Elsevier Ltd. All rights reserved.
The phosphoinositide-dependent kinase-1 (PDK1) is a
Ser/Thr kinase that is a key activator of a number of
protein kinases in the AGC kinase super-family that
play important roles in the progression of cancer, such
as Akt, S6 kinase, and protein kinase C.1a,b The best
characterized cellular substrate for PDK1 is Akt.2
PDK1 activates each of the three isoforms of Akt
(AKT1, AKT2, and AKT3) through phosphorylation
of a regulatory Threonine residue within the activation
loop (e.g., on Thr308 of AKT1). This Thr residue func-
tions in Akt and other AGC super-family kinases to
control access of substrates to the active site.1a,b Binding
of the phosphoinositide 3-kinase (PI3K) products
PtdIns-3,4-P2 or PtdIns-3,4,5-P3 to the pleckstrin
homology domains of Akt and PDK1, recruits these
proteins to the plasma membrane and is necessary for
Akt activation. A large number of studies implicate
Akt in cancer progression through its role in the
promotion of tumor cell growth, metabolism, survival,
epithelial to mesenchymal transition (EMT), and angio-
genesis.3 S6 kinase activity also depends on several mod-
ifications downstream of PDK1.1a As in the case of Akt,
PDK1 phosphorylates a critical regulatory site on the
activation loop of S6 kinase (Thr229) that is required
for catalytic activity. In addition, PDK1-mediated acti-
vation of Akt results in phosphorylation of Thr389 on
S6 kinase, which is also required for activation. S6 ki-
nase promotes growth and survival of cancer cells and
is a key target of rapamycin analogs (e.g., CCI-779/
temsirolimus) which are currently being tested clinically
as anticancer agents.4
The PI3K/PDK1/Akt/S6 kinase signaling pathway is
commonly elevated in tumors through the constitutive
activation or over-expression of a variety of upstream
signaling molecules such as growth factor receptors
(e.g., ErbB2, EGFR, c-Met), Src, Ras, or through the
elevated expression of PI3K and AKT proteins.3 The
pathway is also activated through the frequent loss of
the tumor suppressor PTEN/MMAC1, whose D-3 inosi-
tol phosphatase activity removes the phosphorylation
products of PI3K.5 PDK1 has also been implicated in
the activation of other potential cancer targets, such as
protein kinase C.1a,b The role of PDK1 in several key
signaling pathways important in the progression of can-
cer led us to investigate the use of PDK1 inhibitors as
anticancer agents.
In the preceding paper, we described our discovery of
indoline based inhibitors of PDK1 and their optimiza-
tion to yield BX-517. BX-517 is a potent and selective
inhibitor of PDK1 that binds to the ATP binding pocket
of the protein. Furthermore, BX-517 blocks activation
of Akt in tumor cells. Herein, we describe additional
Keywords: Kinases; PDK-1; AKT; PI 3-kinase; Cancer; Antitumor
agent.
*
Corresponding author. Tel.: +1 510 799 9512; e-mail: imadulislam@
yahoo.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.060

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I. Islam et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3819–3825
G2
Synthesis of aryl, heteroaryl, and substituted aryl ana-
3'
logs (7a–7l) is shown in Scheme 2. 4-Bromo-2-formyl-
pyrrole (4) was protected7 with toluenesulfonyl
chloride and subsequent Suzuki coupling8 with boronic
acids gave intermediate 6. Condensation with 5-ureido-
indolin-2-one under basic conditions afforded intermedi-
ate 7. Reaction of 7c with amines under standard condi-
tions for amide formation gave compounds 7d and 7e.
Demethylation of analog 7g using BBr3 furnished ana-
log 7f.
G1
G1
5'
H
N
H
H
N
H2N
N
H2N
N
H
O
O
O
O
N
N
H
H
Figure 1. Optimization plan.
optimization of BX-517, focused on addressing ADME
and solubility liabilities of the compound. This effort
led to a series of novel and potent PDK1 inhibitors
showing superior physicochemical properties compared
to BX-517.
The analogs (13–22) derived from 4-aminomethyl pyr-
roles and bearing a methyl substituent on the olefin were
prepared as described in Scheme 3. 2-Acetylpyrrole-4-
carbonitrile (8)9 was reduced and protected using Boc
anhydride in one pot. Condensation of 9 with 5-nitroin-
dolin-2-one under basic conditions followed by reduc-
tion of the nitro group with Tin chloride provided 11.
Urea formation from 11 with trimethylsilyl isocyanate
and subsequent cleavage of the Boc group with HCl
afforded 13. Acylation of intermediate 13 with acetyl
chloride gave analog 14. Coupling of 13 with pyridine
carboxylic acids in presence of EDC afforded analogs
15 and 16. Coupling with substituted acids and subse-
quent deprotection under standard conditions furnished
analogs 17–22.
Despite the high enzymatic potency of BX-517, it pos-
sessed poor solubility and pharmacokinetic properties
that prohibited further development (Fig. 1). Exami-
nation of the structure of BX-517 co-crystallized with
PDK1 suggests that substitution at the 4-position of
the pyrrole ring would be tolerated as it points to-
ward the solvent. To improve the solubility we intro-
duced hydrophilic groups onto the 4-position of
pyrrole ring. We first incorporated a carboxylic acid
which was further elaborated into amides. Another
approach to improve physicochemical properties was
to synthesize heteroaryl and aryl linked solubilizing
groups. Finally, inhibitors possessing a 4-aminomethyl
pyrrole were synthesized and derivatized via amide
bond formation.
The in vitro data for acid substituted analogs and their
corresponding amides are shown in Table 1. While the
analogs showed potency in the enzyme assay within
about 2-fold of the parent compound, BX-201
(IC50 = 20 nM), they all displayed lower cell based activ-
ity. At the highest dose tested (10 lM) none of the ana-
logs were active, and therefore, were at least 10-fold less
potent than BX-517 in cells (IC50 = 0.1–1.0 lM). Indeed,
some of the analogs slightly stimulated the cell based
assay, which can occur through apparent feedback acti-
vation of the AKT signaling pathway.10 We observed
such stimulatory effects in PC-3 cells to be a property
Syntheses of the compounds having a carboxylate at the
4-position of the pyrrole are shown in Scheme 1.
Carboxylic acids 1 were either prepared according to
published procedures6 or purchased. Compounds
2a–2d in Table 1 were prepared by condensation of
the appropriate carboxylic acids with 5-ureidoindolin-
2-one. The subsequent amide formation afforded com-
pounds 3a–3f (Table 1).
OH
n
R1
O
R2
H
H2N
N
OH
O
n
N
H
O
H
N
R1
O
H2N
N
H
O
H
i
R2
O
N
N
H
H
O
2
1
n = 0, 2
R1, R2 = H or Me
R3
n
R4
N
R1
O
R2
N
H
H
H2N
N
ii
O
O
N
H
3
Scheme 1. Reagents and conditions: (i) cat. piperidine, ethanol, reflux (40–80%); (ii) amine, HATU (60–75%).

I. Islam et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3819–3825
3821
Table 1. Activity of PDK-1 inhibitors
R
H
H2N
N
O
O
N
H
Compound
R
cAKT211 IC50 (nM)
P-AKT in PC-3 cells12 IC50 (lM)
BX-201
20
3–10
>10a
N
H
O
O
OH
OH
2a
2b
2c
2d
9
9
N
H
No inhibition
N
H
O
OH
OH
39
57
ND
N
H
O
>10 (26% inhibition at 10 lM)
N
H
O
O
N
N
H
3a
3b
3c
3d
3e
3f
27
11
29
31
14
17
(62% inhibition at 10 lM)
N
H
N
N
1.0
N
H
N
H
O
O
O
No inhibition
No inhibition
No inhibitiona
No inhibitiona
N
H
N
H
N
N
N
H
N
O
O
N
H
N
H
N
H
N
N
H
N
H
For assay descriptions see Refs. 11 and 12.
a Showed some stimulation at highest dose (10 lM).
of some weak PDK1 inhibitors. While introduction of a
carboxylic acid group at C-40 (2a) or a methyl group at
C-30 did not affect enzyme potency, compounds with a
propionic acid side chain at C-40 and mono- or
cdimethyl substitution at C-30 (2c) and C-50 (2d) were
4- to 6-fold less potent than 2a which had weak activity
in cells.
Amidation of analog 2a either maintained or reduced
potency (3a–3f) but reduced cellular activity in all cases
tried.
In vitro potency of aryl and heteroaryl analogs is shown
in Table 2. Phenyl (7a) substitution at C-40 was tolerated
as were substituted phenyl compounds. Substituents at

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I. Islam et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3819–3825
compound (13) and its analogs (14–22) were all highly
Ar
Br
Br
potent in the in vitro cAKT assay, the cellular activity
of these compounds was lower than that BX-517 or
7b and 7d. Compound 17 displays a solubility similar
to 7d.
ii
H
i
H
H
N
N
N
H
O
O
Tos
Tos
O
4
5
6
An X-ray structure of 7e bound to PDK1 was obtained
during optimization (Fig. 2). The inhibitor forms hydro-
Ar
H
H2N
N
˚
gen bonds to the backbone C@O of Ser160 (2.8 A), the
O
˚
˚
H
N
N
NH of Ala162 (2.8 A) , the C@O of Ala162 (3.0 A), and
O
N
H
H2N
H
˚
˚
the side chains of Lys111 (2.8 A) and Thr222 (2.8 A).
The hydrogen bonds to Ser160 and Ala162 mimic the
interaction of adenosine with the hinge region of
PDK1. The hydrogen bond to Thr222 (3.0 A) and the
˚
hydrophobic interaction with Tyr170 (4.0 A) are se-
O
O
N
H
iii
˚
7
Scheme 2. Reagents and conditions: (i) LDA, toluenesulfonyl chlo-
ride, THF, ꢀ5 °C (40%); (ii) ArB(OH)2, Pd(PPh3)4 NaOtBu, toluene
(40–80%); (iii) catalytic piperidine, ethanol, 110 °C (55–85%).
quence specific interactions that augment the selectivity
of the compound.
Selectivity of key analogs was assessed against a
highly structurally related kinase, protein kinase A
(PKA).
the 3-position of the phenyl ring (7a) such as acid (7c),
methoxy (7g), fluoro (7h), and methyl (7i) afforded
similar potency to the unsubstituted phenyl compound.
The 3-pyridyl analog (7b) was highly potent with good
cellular activity (8 nM, and 0.10–1.0 lM, respectively).
Amide analogs 7d and 7e were also potent PDK1 inhib-
itors with good cellular activity. The hydroxy and meth-
oxy analogs (7f and 7g) were very potent in cells, but
showed high clearance when dosed in rats (data not
shown).
BX-517 displayed good selectivity (320-fold), while
compounds 7b, 7d, 7e, and 16 had variable degrees
of selectivity against PKA (2-, 78-, 35-, and 21-fold,
respectively). These and other data indicated that,
among the analogs we tested, BX-517 was the most
selective.
Some of the major issues with further developing BX-
517 as an antitumor agent were its poor pharmaceutical
properties including short half-life, low metabolic stabil-
ity, and poor solubility. As shown in Table 4, selected
analogs show longer half-life, better solubility, and
A further strategy to improve solubility was installing
an aminomethyl group at the C-40 of the pyrrole
which could serve as a linker to attach water solubiliz-
ing groups (Table 3). Although the aminomethyl
NHBoc
NHBoc
CN
N
O2N
H
i
ii
O
N
N
N
H
H
O
O
H
8
9
10
NHBoc
NHBoc
N
H
N
H
O
H2N
H
H2N
N
iv
iii
O
O
N
N
H
H
12
11
NH2
H
N
H
O
H2N
N
v
O
N
H
13
Scheme 3. Reagents and conditions: (i) Pd/C, methanol, (Boc)2O, H2, (40PSI, 73%); (ii) 5-nitro indolin-2-one, catalytic piperidine, ethanol, 110 °C
(20–35%); (iii) SnCl4, pyridine, ethyl acetate, reflux (65%); (iv) (CH3)3SiNCO, THF, DMF (58%); (v) 4 N HCl in dioxane (80%).

I. Islam et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3819–3825
3823
Table 2. Activity of PDK-1 inhibitors
R
H
N
H2N
N
H
O
O
N
H
Compound
R
H
cAKT2 IC50 (nM)
20
P-AKT in PC-3 cells IC50 (lM)
BX-201
3–10
7a
7b
21
8
ND
0.10–1.0
N
OH
7c
7d
7e
45
4
>10 (34% inhibition at 10 lM)
O
O
O
H
N
0.10–1.0
1.0
N
N
H
N
4
7f
7g
7h
7i
6
19
51
37
0.02–2.0
0.01–0.1
ND
OH
O
F
ND
>1.0a
NH2
7j
5
O
O
7k
8
3
1.0–1.0
N
H
7l
(65% inhibition at 10 lM)
NH2
a Phosphorylation was stimulated at high doses.
better metabolic stability (except for 7d) compared to
BX-517. Plasma clearance and volume of distribution
were comparable, however.
soft agar with an estimated IC50 of 1 lM, while not
blocking growth on tissue culture plastic at concentra-
tions below 10 lM. Compound 7d showed a similar
pattern, blocking growth of MDA-468 cells in soft
agar with an estimated IC50 of 0.3 lM, and not block-
ing growth on plastic at concentrations below 10 lM.
These data are consistent with a significant role of
PDK1/Akt signaling in growth and/or survival of tu-
mor cells under certain conditions, such as anchor-
age-independent growth on soft agar. Whether this
activity is sufficient to block tumor progression or
Based on their superior overall profile in enzyme and
cellular assays and their improved PK properties, we
further assessed the in vitro efficacy of compounds
7b and 7d by evaluating their effect on MDA-468
breast cancer cell growth in soft agar (transforma-
tion-dependent growth) and on tissue culture plastic.
Compound 7b blocked the growth of tumor cells in

3824
I. Islam et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3819–3825
Table 3. Activity of PDK-1 inhibitors
R
H3C
H
N
H2N
N
H
O
O
N
H
Compound
R
H
cAKT2 IC50 (nM)
5
P-AKT in PC-3 cells IC50 (lM)
BX-517
0.1–1.0
13
14
10
8
ND
1.0
NH2
O
N
H
O
15
5
(29% inhibition at 10 lM)
N
H
N
O
16
17
10
6
1.0–10.0
N
H
N
O
(33% inhibition at 10 lM)
N
H
NH
N
O
18
19
4
5
(28% inhibition at 10 lM)
N
H
O
H
N
N
H
No inhibition
N
H
O
20
21
22
NH2
5
10
3
No inhibitiona
1.0–0.0
N
H
O
OH
N
H
O
1.0–10.0
N
H
N
a Phosphorylation was stimulated at high doses.
Figure 2. A divergent stereo view of the crystal structure of compound 7e bound to PDK1, PDB entry 2PE2.

I. Islam et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3819–3825
Table 4. PK profile of select PDK-1 inhibitors in rat
3825
Cla (mL/min/kg)
Vssa L/kg
hLM stability (% remaining 1 h)
Solubility (mg/L in PBS)
a
t1/2 (h)
Compound
BX-517
7b
7d
0.4
1.6
3.7
2.0
2.4
30.8
37.1
41.8
22.0
8.8
0.9
3.8
3.6
0.8
0.8
27
82
30
80
nd
2
29
13
nd
17
16
17
a 5 mg/kg iv.
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11. cAKT2 (33P-SPA). The coupled assay can detect inhib-
itors of AKT2 activation, as well as direct inhibition of
PDK1 or AKT2. In active AKT2 is activated in situ by
incubating with PDK1 and PtdIns-3,4-P2 in presence of
compound, 33P-ATP, and a biotinylated peptide substrate
for AKT2. The peptide is captured on streptavidin-coated
SPA beads for detection.
In summary, we optimized the pharmaceutical proper-
ties of BX-517 by exploring substitution at the C-40
position of the pyrrole. Carboxylic acids and amides
had improved solubility but decreased cellular po-
tency. 3-Pyridyl substitution afforded a compound
(7b) with improved pharmacokinetic properties. Com-
pound 7d showed excellent half-life and good solubil-
ity but poor in vitro liver microsome stability.
Compounds 16 and 17 also had better pharmacoki-
netic properties compared to BX-517. Discovery of
these compounds will provide tools for further evalu-
ation of the role of the PDK1/Akt signaling in the
treatment of various cancers and the development of
new therapeutic agents.
References and notes
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12. P-AKT in PC 3-cells. Inhibition of Akt phosphorylation in
cells is measured by incubating compounds with cells,
followed by lysis and standard immunoblotting techniques
using phosphospecific antibodies to Akt P-Thr 308.