Discovery of new aminopyrimidine-based phosphoinositide 3-kinase beta (PI3Kβ) inhibitors with selectivity over PI3Kα

Article abstract of DOI:10.1016/j.bmcl.2011.09.118

Phosphatidylinositol-3-kinase beta (PI3Kβ) is an important therapeutic target in arterial thrombosis and special types of cancer. In this study, a new series of aminopyridine-based PI3Kβ selective inhibitors have been developed by the structure-based design strategy. When incorporated with the phenyl ring on sulfonamide moiety, aminopyrimidine analogs showed good potency on PI3Kβ and selectivity over PI3Kα. Intriguingly, replacement of phenyl group on sulfonamide with naphthyl group enhanced selectivity over PI3Kα while retaining submicromolar PI3Kβ potency. Molecular modeling suggests that increased PI3Kβ specificity is caused by the interaction with salt bridge (Lys782-Asp923) and Asp862 that creat a unique pocket in PI3Kβ. These results clearly provide useful insight in the design of new PI3Kβ inhibitors with high potency and selectivity.

Full text of DOI:10.1016/j.bmcl.2011.09.118

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6977–6981
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of new aminopyrimidine-based phosphoinositide 3-kinase beta
(PI3Kb) inhibitors with selectivity over PI3K
a
⇑
Jinhee Kim, Seunghee Hong, Sungwoo Hong
Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Phosphatidylinositol-3-kinase beta (PI3Kb) is an important therapeutic target in arterial thrombosis and
special types of cancer. In this study, a new series of aminopyridine-based PI3Kb selective inhibitors have
been developed by the structure-based design strategy. When incorporated with the phenyl ring on sul-
Received 6 September 2011
Revised 27 September 2011
Accepted 28 September 2011
Available online 8 October 2011
fonamide moiety, aminopyrimidine analogs showed good potency on PI3Kb and selectivity over PI3K
Intriguingly, replacement of phenyl group on sulfonamide with naphthyl group enhanced selectivity over
PI3K while retaining submicromolar PI3Kb potency. Molecular modeling suggests that increased PI3Kb
a.
a
Keywords:
Phosphatidylinositol-3-kinase beta
PI3Kb inhibitor
Structure-based design
Aminopyrimidine
specificity is caused by the interaction with salt bridge (Lys782–Asp923) and Asp862 that creat a unique
pocket in PI3Kb. These results clearly provide useful insight in the design of new PI3Kb inhibitors with
high potency and selectivity.
Ó 2011 Elsevier Ltd. All rights reserved.
Phosphoinositide 3-kinases (PI3Ks) are a family of evolution-
arily conserved lipid kinases that catalyze phosphorylation of the
3-hydroxyl position of the inositol ring of phosphatidylinositol
4,5-diphosphate (PIP2) to produce phosphatidylinositol 3,4,5-tri-
phosphate (PIP3). The resulting second messenger, PIP3 mediates
a variety of physiological processes, including cell growth, differen-
tiation, survival, and motility.1 The PI3K signaling pathway is
negatively regulated by the lipid-phosphatase PTEN (phosphatase
and tension homolog deleted on chromosome 10). Aberrant upreg-
ulation of the PI3K pathway leads to an extraordinarily elevated
PIP3 level and downstream activation of Akt, which has been
known to be responsible for cancer, inflammation, immune
disorders, and cardiovascular diseases.2
subtype of PI3Ks, the p110b isoform potentiates integrin-mediated
platelet aggregation and arterial thrombosis.4 In addition, recent
studies on genetically engineered mouse models and chemical
inhibitors indicate that tumors driven by loss of PTEN may be more
sensitive to inhibition of p110b rather than p110a 5
. Therefore, it
would be beneficial to consider the generation of p110b selective
compounds for special type of cancer or cardiovascular treatment
in order to minimize the risk of potential toxicity and insulin resis-
tance associated with the inhibition of PI3Ka. Although many PI3K
inhibitors have been identified, only a few isoform-specific PI3Kb
inhibitors have been reported. Recently, patent disclosure has de-
scribed TGX series as selective PI3Kb inhibitors (patent WO
2004016607).6 The structure of TGX-221 was modified based on
structure and function analysis of LY294002 (Fig. 1). Here, we re-
port the identification of a new series of aminopyrimidine-based
PI3Kb specific inhibitors.
The recent availability of the three-dimensional structure of
therapeutic targets has enhanced opportunities for the rapid devel-
opment of active compounds utilizing structure-based design. For
Class I PI3Ks are subdivided into class IA and class IB based on
different regulatory subunits and activation mechanisms. Class IA
includes three isoforms, PI3K
one isoform, PI3K . PI3K and d isoforms are activated by receptor
tyrosine kinases/cytokine receptor activation, while PI3K is acti-
vated by G protein b subunits (Gb ), which are usually derived
a, b, and d. Class IB PI3K contains only
c
a
c
c
c
from G-protein-coupled receptors (GPCRs). In contrast, the PI3Kb
isoform is regulated by both receptor tyrosine kinases and by
Gb
with cancer progress because the p110
is overexpressed or frequently mutated in many human cancers.3
The PI3Kd and PI3K isoforms are mainly restricted to
c
-subunits. The p110
a
is reported to correlate most strongly
O
N
O
O
a
-encoding PIK3CA gene
N
c
N
N
hematopoietic systems and their impairment has been linked to
immunodeficiency and inflammatory disorder. Among different
O
O
N
H
TGX-221
LY294002
⇑
Corresponding author. Tel.: +82 42 350 2811; fax: +82 42 350 2810.
E-mail address: hongorg@kaist.ac.kr (S. Hong).
Figure 1. Structure of selective PI3Kb inhibitor, TGX-221.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.118

6978
J. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6977–6981
inhibiting kinases, the most exemplified approach targets the
kinase domain by competitive inhibition of the ATP binding site.
Some heterocyclic fragments mimicking the purine portion of
ATP can bind to the hinge region of the kinase via hydrogen bonds
with the backbone. During the past decade, substantial structural
and biochemical information on PI3Ks has greatly improved the
potency and selectivity of PI3K inhibitors. Based on these premises,
our aim was to identify new structural class of PI3Kb selective
inhibitors by a pharmacophore-directed design. First, we planned
to identify the template of efficient PI3Kb inhibitors, which would
offer opportunities for future optimization of selectivity by exploit-
ing differences in the conformational plasticity of PI3K isoforms. To
assess the back pocket (DFG-motif, gate keeper and catalytic
lysine), we took advantage of the information in the literature
and combine a suitable pharmacophore to yield potent PI3Kb
inhibitor. Recently, it is reported that a number of PI3K inhibitors
have pyridyl sulfonamides to enhance potency by filling this pock-
et.7 While fixing a N-(pyridin-3-yl)benzenesulfonamide moiety,
our initial round of analogues was focused around incorporation
of a variety of heterocycles to generate potent PI3Kb inhibitors (
Fig. 2). With the identified PI3Kb inhibitors, we planned to enhance
selectivty by exploiting extended interactions with PI3Kb unique
residues (Lys782, Asp923, and Asp862) at the selectivity pocket.
The preparation of the target compounds is described in
Scheme 1. The synthesis begins with treatment of the commer-
cially available bromopyridine amine (1) with benzene sulfonyl
chloride in CH2Cl2, followed by borylation with bis(pinacolato)
diboron to afford the corresponding boronic ester 2. Finally, vari-
ous heteroaryl groups were then attached to the pyridyl sulfonyl
moiety using a palladium-catalyzed cross-coupling to furnish the
target compounds 3 as shown in Scheme 1.
O
O
O
O
S
N
S
N
NH
NH
NH2
c
a,b
O
N
B
Br
Het
O
1
2
3
Scheme 1. Reagents and conditions: (a) PhSO2Cl, Pyridine, CH2Cl2, rt;
(b) bis(pinacolato)diboron, Pd(dppf)Cl2, KOAc, 1,4-dioxane, 100 °C; (c) Aryl bro-
mide, Pd(dppf)Cl2, K2CO3, 1,4-dioxane/water = 3:1, 100 °C.
Table 1
Array of various heterocycles and inhibition data against PI3K
a and PI3Kb
a
a
Compd
Het
PI3Kb (POC)
PI3K
0
a (POC)
N
4
0
N
NH2
5
4.9
56
N
H
N
N
N
6
7
8
9
0.6
39
4
95
52
60
The resulting compounds in which various heterocycles were
grown from the pyridyl sulfonamide moiety, were tested over
N
N
PI3Kb and PI3K
a at 10 lM concentration in a high-throughput
binding assay (KINOMEscan, Ambit Biosciences).8 To our delight,
several compounds were discovered as hits for PI3Kb [POC (percent
of control) values <10] as shown in Table 1. Of particular signifi-
cance is the observation that a profound potency on PI3Kb and
N
N
N
7.4
1.4
selectivity over PI3K
nopyrimidine with the pyridyl sulfonamide moiety (9, PI3Kb,
PI3K : POC = 1.4, 60, respectively). Considering tolerance within
the PI3Kb active site, selectivity over PI3K and low molecular
a was achieved by the combination of 2-ami-
N
N
a
NH2
NH2
a
weight of 327, compound 9 was expected to serve as a good
scaffold from which much more potent and selective inhibitors
can be derivatized. For these reasons, compound 9 was selected
for further optimization to embark upon in-depth structural
modification.
10
11
5.2
18
66
89
N
N
N
NH2
N
N
12
13
5.2
66
39
N
N
NHMe
100
NMe2
O
N
14
34
100
N
N
H
a
Lower numbers of POC (percent of control) indicate stronger hits. Values show
an average of duplicate measurements.
Building upon the success observed with aminopyrimidine scaf-
fold, a further investigation of the effect of sulfonamide subunit
was then conducted (Table 2). The sulfonamide moiety was found
to be essential for enhanced potency, and replacement of
Figure 2. Design of aminopyrimidine scaffolds as PI3Kb inhibitors, and
opportunities for modification.

J. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6977–6981
6979
sulfonamide group with hydrogen or amide group resulted in a sig-
nificant loss of activity over all PI3Ks. Introduction of small size
sulfonamide, such as methylsulfonamide also decreased all PI3Ks
inhibitory activity, implying the importance of the role of aryl ring
for PI3K inhibition. The increase in activity afforded by toluene
As the structure of PI3Kb has not been established, a homology
model was built based on p110 complex with staurosporine
c
(PDB code: 1E8Z) as the template using the program MODELER
to mimic the conformational rearrangement. Subsequent docking
studies were performed using AutoDock 4.0.9 Docking studies were
either performed without constraints or with a hydrogen bonding
constraint to the backbone-NH of Val854. In the calculated
PI3Kb–9 complex shown in Figure 3, compound 9 appeared to be
in close contact with residues Val854–Val853, Tyr839–Asp813,
Lys805, Asp862, Ser781, and Lys782–Asp923, respectively. From
the overall structural features derived from docking simulations,
the inhibitory activity of 9 is likely to stem from the multiple
hydrogen bonds and hydrophobic interactions. Thus, examination
of the hinge-region/aminopyrimidine interaction in the PI3Kb
homology model showed that the 2-aminopyrimidine of 9 main-
tains the hydrogen-bonding pattern with Val854. The pyridyl
group is anchored by a hydrogen bond to the Tyr839 and the
sulfonamide participates in hydrogen bonding with the catalytic
lysine (Lys805), as shown in Figure 3. These three hydrogen bonds
seem to play a role of anchor for binding of 9 with PI3Kb.
sulfonamide group (Kd of 22 = 0.1 lM) prompted further investiga-
tion through synthesis of a series of substituted aryl sulfonamide
analogs. The presence of meta-, or para-substituents on aryl ring
resulted in compounds which were generally of equivalent or bet-
ter enzyme potency than the corresponding unsubstituted deriva-
tives. Compound 23, having
selectivity verses PI3K . Intriguingly, profound effect on selectivity
for PI3Kb over PI3K was achieved with compound incorporating
the naphthyl group, leading to 24 with excellent specificity profiles
(PI3Kb, PI3K : POC = 1.0, 92; Kd = 0.23, >40 M, respectively). In
a p-OMe group had amplified
a
a
a
l
addition, this series of PI3Kb inhibitors possess excellent selectivity
over mTOR.
To obtain structural insight into the inhibitory and selectivity
mechanisms of the identified PI3Kb inhibitors, their binding modes
in the ATP-binding site were investigated in a comparative fashion.
Table 2
Structure–activity relationship with R groups and selectivity data against PI3K isoforms
a
Compd
R
PI3Kb Kd
(l
M)
POC
PI3Kb/
a
/
c
/d
mTOR
15
16
17
9
–H
>40
>20
5.2
86/98/100/82
69/97/98/65
28/62/43/38
1.4/60/24/4.2
53
79
67
74
–COMe
–SO2Me
–SO2Ph
0.36
O
S
18
19
0.43
0.22
1.2/30/9.6/0.9
0.7/22/7.2/4.7
41
56
CF3
F
O
O
S
O
F
O
S
20
21
0.25
0.10
0.7/42/5.3/0.1
0.5/17/8.2/3.4
78
76
NO2
O
O
S
O
NO2
O
S
22
0.55
1.6/18/6.7/9.5
68
O
O
O
S
23
24
0.50
0.23
1.7/87/20/4.3
1.0/92/25/4.2
39
96
O
O
S
O
a
Lower numbers of POC (percent of control) indicate stronger hits. Values show an average of duplicate measurements.

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J. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6977–6981
Table 3
Inhibition of breast cancer cell (T47D) proliferation by representative
aminopyrimidine derivatives
9
19
21
T47D IC50
(lM)
9.6
8.5
4.7
deeper pocket in PI3Kb. These results clearly provide useful insight
in the design of new PI3Kb inhibitors with high potency and
selectivity.
Acknowledgments
This research was supported by National Research Foundation
of Korea (NRF) funded by the Ministry of Education, Science and
Technology (NRF-2011-0003609, 2011-0026680, 2011-0016436
and 2011-0020322).
References and notes
Figure 3. Calculated binding mode of 9 in the ATP-binding site of the PI3Kb
homology model. In this model, the aminopyrimidine unit forms a key hinge region
hydrogen bond with the backbone of Val854. Selected residues (Try839 and Lys805)
are shown for possible hydrogen bonding interaction with pyridyl sulfonamide
subunit. Each dotted line indicates a hydrogen bond.
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Compound 9 may be further stabilized in the ATP-binding site via
the hydrophobic interactions among its nonpolar groups with the
side chains in the back pocket (DFG-motif, gate keeper and cata-
lytic lysine). The selectivity enhancement of 9 seems to be the re-
sult of accessing the more hydrophobic region within PI3Kb.
Docking modeling suggests that the difference of several residues
between PI3Kb and PI3Ka causes a difference in the depth of the
phenyl group binding pockets that is responsible for the increased
selectivity observed for the aminopyrimidine analogs with aryl-
sulfonamide subunit. It was expected that the presence of aryl ring
on sulfonamide group (in red circle, Fig. 3) would allow for a favor-
able hydrophobic interaction with salt bridge (Lys782–Asp923)
which is missing in alpha isoform (PI3K
This hydrophobic interaction seems to introduce affinity for PI3Kb
and explains weaker binding affinity for PI3K . Moreover, phenyl
group is also directed toward a hydrophobic pocket located behind
Asp862 whch is unique to PI3Kb ( , Gln859; d, Asn836;
a
: Ala775 and Ser919).10
a
a
c,
Lys890).10 These amino acid residues may create a deeper binding
pocket in PI3Kb, which accommodates the aryl ring of sulfonamide
derivatives without causing unfavorable steric contacts. The mod-
eling analysis similarly applies to analogs with larger aryl groups
(e.g., naphthyl group) which seem to be accommodated by this
binding pocket in PI3Kb.
6. Jackson, S. P.; Schoenwaelder, S. M.; Goncalves, I.; Nesbitt, W. S.; Yap, C. L.;
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Sarpong, M. A.; Schmidt, S. J.; Van Aller, G. S.; Carson, J. D.; Diamond, M. A.;
Elkins, P. A.; Gardiner, C. M.; Garver, E.; Gilbert, S. A.; Gontarek, R. R.; Jackson, J.
R.; Kershner, K. L.; Luo, L.; Raha, K.; Sherk, C. S.; Sung, C. M.; Sutton, D.;
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Given the impressive enzyme activity profiles, several com-
pounds from this series were further tested for cellular prolifera-
tion activity. To measure the inhibitory effect of compounds on
cell growth, cell viability was tested by 3-(4,5-dimethylthiazol-2-
yl-2,5-diphenyltetrazolium bromide (MTT) assay in T47D human
breast cancer cell cultures. Notably, compounds 9, 19, and 21
showed promising inhibitory activity against T47D at micromolar
concentration (Table 3).
In conclusion, a new series of aminopyridine-based PI3Kb inhib-
itors Ref.11 have been developed by the structure-based design.
When incorporated with the phenyl sulfonamide moiety, amino-
pyrimidine analogs showed good potency on PI3Kb and selectivity
over PI3K
amide with larger groups, such as naphtyl group enhanced selec-
tivity over PI3K while retaining submicromolar PI3Kb potency.
a. Intriguingly, replacement of phenyl group on sulfon-
a
Molecular modeling suggests that increased PI3Kb selectivity is
caused by salt bridge (Lys782–Asp923) and Asp862 that creat a
10. (a) Frazzetto, M.; Suphioglu, C.; Zhu, J.; Schmidt-Kittler, O.; Jennings, I. G.;
Cranmer, S. L.; Jackson, S. P.; Kinzler, K. W.; Vogelstein, B.; Thompson, P. E.

J. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6977–6981
6981
Biochem. J. 2008, 414, 383; (b) Zvelebil, M. J.; Waterfield, M. D.; Shuttleworth, S.
J. Arch Biochem. Biophys. 2008, 477, 404.
(300 MHz, DMSO-d6) d 2.33 (s, 3H), 6.92 (s, 2H), 7.34 (s, 1H), 7.36 (s, 1H), 7.59
(t, J = 2.2, 1H), 7.67 (s, 1H), 7.70 (s, 1H), 8.17 (d, J = 2.4, 1H), 8.46 (s, 2H), 8.48 (d,
J = 2.0, 1H), 10.60 (s, 1H). 22: 1H NMR (300 MHz, MeOD-d6) d 7.71 (t, J = 2.2,
1H), 8.01 (m, 2H), 8.19 (d, J = 2.3, 1H), 8.31 (m, 2H), 8.44 (d, J = 1.9, 1H), 8.45 (s,
2H). 23: 1H NMR (300 MHz, MeOD-d6) d 3.76 (s, 3H), 6.96 (dd, J = 2.0, 6.9, 2H),
7.67 (m, 3H), 8.15 (d, J = 2.3, 1H), 8.37 (d, J = 1.9, 1H), 8.42 (s, 2H). 24: 1H NMR
(300 MHz, DMSO-d6) d 6.91 (s, 2H), 7.66 (m, 3H), 7.80 (dd, J = 1.7, 8.7, 1H), 7.99
(d, J = 7.6, 1H), 8.12 (m, 2H), 8.20(d, J = 2.3, 1H), 8.42 (s, 3H), 8.51 (s, 1H), 10.74
(s, 1H).
11. Spectra data of representative compounds. 9: 1H NMR (300 MHz, MeOD-d6)
d 7.50 (m, 4H), 7.65 (s, 1H), 7.75 (s, 1H), 7.78 (s, 1H), 8.16 (s, 1H), 8.41 (m, 2H).
18: 1H NMR (300 MHz, MeOD-d6) d 7.68 (m, 2H), 7.87 (d, J = 7.4, 1H), 8.01 (d,
J = 7.6, 2H), 8.15 (d, J = 2.3, 1H), 8.43 (d, J = 2.6, 3H). 19: 1H NMR (300 MHz,
DMSO-d6) d 6.93 (s, 2H), 7.25 (m, 1H), 7.53 (m, 1H), 7.61 (s, 1H), 7.92 (m, 1H),
8.21 (d, J = 1.9, 1H), 8.47 (s, 2H), 8.51 (s, 1H), 11.02 (s, 1H). 20: 1H NMR
(300 MHz, MeOD-d6) d 2.53 (s, 3H), 7.55 (d, J = 8.2, 1H), 7.68 (m, 1H), 7.88 (dd,
J = 2.0, 8.1, 1H), 8.17 (s, 1H), 8.30 (d, J = 1.8, 1H), 8.42(m, 3H). 21: 1H NMR