4-(Benzimidazol-2-yl)-1,2,5-oxadiazol-3-ylamine derivatives: Potent and selective p70S6 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.07.022

We report herein the design and synthesis of 4-(benzimidazol-2-yl)-1,2,5-oxadiazol-3-amine derivatives as inhibitors of p70S6 kinase. Screening hits containing the 4-(benzimidazol-2-yl)-1,2,5-oxadiazol-3-ylamine scaffold were optimized for p70S6K potency and selectivity against related kinases. Structure-based design employing an active site homology model derived from PKA led to the preparation of benzimidazole 5-substituted compounds 26 and 27 as highly potent inhibitors (K_i <1 nM) of p70S6K, with >100-fold selectivity against PKA, ROCK and GSK3.

Full text of DOI:10.1016/j.bmcl.2009.07.022

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5191–5194
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
4-(Benzimidazol-2-yl)-1,2,5-oxadiazol-3-ylamine derivatives: Potent
and selective p70S6 kinase inhibitors
Upul Bandarage a, Brian Hare a, Jonathan Parsons a, Ly Pham a, Craig Marhefka a, Guy Bemis a, Qing Tang a,
Cameron Stuver Moody a, Steve Rodems b, Sundeep Shah b, Chris Adams c, Jose Bravo c,
Emmanuelle Charonnet c, Vladimir Savic c, Jon H. Come a, Jeremy Green a,
*
a Vertex Pharmaceuticals, Inc., 130 Waverly Street, Cambridge, MA 02139, USA
b Vertex Pharmaceuticals, Inc., 11010 Torreyana Road, San Diego, CA 92121, USA
c Biofocus, Chesterford Research Park, Saffron Walden, Essex CB10 1XL, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
We report herein the design and synthesis of 4-(benzimidazol-2-yl)-1,2,5-oxadiazol-3-amine derivatives
as inhibitors of p70S6 kinase. Screening hits containing the 4-(benzimidazol-2-yl)-1,2,5-oxadiazol-3-yla-
mine scaffold were optimized for p70S6K potency and selectivity against related kinases. Structure-based
design employing an active site homology model derived from PKA led to the preparation of benzimid-
azole 5-substituted compounds 26 and 27 as highly potent inhibitors (Ki <1 nM) of p70S6K, with >100-
fold selectivity against PKA, ROCK and GSK3.
Received 7 May 2009
Revised 29 June 2009
Accepted 2 July 2009
Available online 9 July 2009
Keywords:
p70S6K
Ó 2009 Elsevier Ltd. All rights reserved.
p70S6 Kinase
1,2,5-Oxadiazole
3-Aminofurazan
The 70-kDa ribosomal S6 kinase (p70S6K; S6K1) is a Ser/Thr
kinase that has been implicated in the control of cell growth and cell
size.1 As an effector kinase in the phosphoinositide-3-kinase (PI3K)
pathway, p70S6K activity is regulated by the tuberous sclerosis
complex proteins (TSC1/2) and the mammalian target of rapamycin
(mTOR), in response to multiple types of signals.2 Tuberous sclerosis
complex (TSC) is an autosomal dominant disorder characterized by
benign tumors (hamartomas) in which mutational inactivation of
TSC1/2 leads to constitutive activation of mTOR and p70S6K. Consti-
tutively active p70S6K is also found in many types of cancer cells due
to dysregulation of the PI3K pathway.3 In drosophila, knockout of
TSC1/2 is lethal, but the lethality can be rescued by reduction of
S6K signaling activity.4 Taken together, these data suggest that small
molecule inhibitors of p70S6K may reduce the oncogenic signal
caused by activation of the PI3K pathway and may be an effective
therapy for TSC or other types of cancer.
and stress-activated protein kinase (MSK1)7 (e.g., 5), which, like
p70S6K, are members of the AGC subfamily of Ser/Thr kinases.8
A homology model of the p70S6K active site was constructed
starting from the X-ray crystal structure of PKA
a
(PDB ID: 1jbp).9
Placement of the 3-amino-1,2,5-oxadiazole of 1 in its likely binding
orientation, with hydrogen-bonding contacts between the amino
group and the carbonyl of Glu173, and the oxadiazole N-2 and
the backbone NH of residue Leu175 in the kinase hinge region
showed an opportunity to improve the inhibitors by creating fur-
ther interactions with two different binding pockets (Fig. 2). In this
model, pocket A (lower left of Fig. 2) consists of the sidechains of
residues Met225, Phe382, Glu179 along with the backbone car-
bonyl of Glu222 (lower middle of Fig. 2) and might be accessible
from the benzimidazole nitrogen. Pocket B (upper right of Fig. 2)
is a much smaller pocket, deeper in the active site cleft and is
bounded primarily by the polar side chains of residues Lys123,
Glu143, and Asp236 (upper right of Fig. 2). Access to this region
would be made from the benzimidazole 5-position. The synthesis
of compounds designed to interact with these two binding pockets
is illustrated in Scheme 1.
To identify inhibitor leads for p70S6K, we screened our com-
pound collection and identified several 4-(benzimidazol-2-yl)-
1,2,5-oxadiazol-3-amine derivatives (1) as inhibitors of p70S6K
(Fig. 1).
Molecules bearing the 3-amino-1,2,5-oxadiazole moiety have
been reported as inhibitors of glycogen synthase kinase (GSK3)5
(e.g., 2), as well as Rho kinase (ROCK1)6 (e.g., 3, 4) and mitogen
In this scheme, readily available 2-nitro-4-substituted fluoro-
benzenes (6) were first treated with various amines to afford the
substituted-amino nitrobenzenes in good yield. The products were
reduced to diamines (7) by palladium-catalyzed hydrogenation.
Treatment with the methyl 4-amino-1,2,5-oxadiazolyl-3-carbimi-
date (8)10 gave the desired products (9–27) in good overall yield.
* Corresponding author. Tel.: +1 6174446315.
E-mail address: jeremy_green@vrtx.com (J. Green).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.022

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U. Bandarage et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5191–5194
H2N
H2N
N
N
O
R2
N
N
N
O
N
O
H
N
N
N
O
N
N
N
N
R1
N
N
1
2
HN
O
O
H2N
H2N
N
N
H2N
N
N
O
N
N
N
N
N
O
N
N
N
N
O
N
N
Et
N
HN
3
Et
Et
O
H2N
N
H
4
5
Figure 1. 3-Amino-1,2,5-oxadiazole kinase inhibitors.
Table 1
Activity of N-substituted 4-(benzimidazol-2-yl)-1,2,5-oxadiazol-3-amine derivatives
against p70S6K, PKA
a
, GSK-3b and ROCK-111
H2N
N
N
N
O
N
R1
ID
R1
p70S6K Ki
M)
PKA
a
Ki
GSK-3b Ki
M)
ROCK-1 Ki
M)
(
l
(lM)
(
l
(l
9
Me
Et
iPr
cPr
cBu
cPent
cHex
cPrCH2
cBuCH2
iBu
0.54
>4.0
0.96
>3.3
>3.3
0.54
0.72
0.60
0.38
1.4
7.3
0.70
1.5
>3.3
0.33
1.9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
0.050
0.040
0.095
0.004
0.013
0.012
0.012
0.069
0.022
0.11
0.026
0.80
0.80
0.031
1.9
0.55
>3.3
0.38
0.21
0.24
0.29
0.69
0.39
0.62
>3.3
>2.5
1.0
0.45
0.18
0.34
0.72
0.36
0.66
0.93
0.59
0.81
>2.5
>2.5
1.3
Figure 2. Homology model of the proposed binding mode of 4-(benzimidazol-2-
yl)-1,2,5-oxadiazol-3-amine derivatives to p70S6K.
1.1
1.3
Ph
2-CN–Ph
3-CN–Ph
4-CN–Ph
2-MeO–Ph
3-MeO–Ph
4-MeO–Ph
>5.0
>5.0
>5.0
>5.0
NDa
>4.0
With respect to proposed pocket A, a survey of aliphatic benz-
imidazole N-1 substituents showed that the modest inhibition
exhibited by the methyl group (9) could be improved significantly
as larger substituents gave notably improved inhibition (Table 1).
Directly attached cycloalkyl groups demonstrated the largest
improvements with N-cyclobutyl (13), -cyclopentyl (14) and -
cyclohexyl (15) compounds exhibiting <20 nM inhibition. More
polar substituents generally showed significantly weaker binding
affinity (data not shown). Aromatic benzimidazole N-1 substitu-
ents showed a strong dependency on substituent position. For
example, ortho-aromatic substituents such as compounds 20 and
23, were approximately 20-fold more potent than analogous meta-
or para-substituted systems. In addition compounds 20–25 were
>5.0
>5.0
>5.0
>5.0
2.1
a
Result not determined.
much less active towards GSK3b or ROCK-1. Our model suggests
that the conformational preferences for these N-aryl compounds
(20–25 in Table 1) do not affect the orientation of the 4-(ben-
zimidazol-2-yl)-1,2,5-oxadiazol-3-ylamine relative to the unsub-
stituted parent compound (19 in Table 1) but the Met225
sidechain can move to accommodate and make hydrophobic inter-
H2N
MeO
N
O
H2N
HN
N
X
NO2
F
X
X
NH2
N
N
(a)
8
N
O
N
(b)
NH
R1
R1
6
7
9-27
Scheme 1. Synthesis of 4-(benzimidazol-2-yl)-1,2,5-oxadiazol-3-amine derivatives. Reagents and conditions: (a) (i) R1NH2, DMF, rt, 88–93%; (ii) H2, Pd/C, EtOH, rt, 2 h, 95%;
(b) AcOH, 110 °C, 15 min, 68–82%.

U. Bandarage et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5191–5194
5193
Table 2
or Asp236 whereas PKA
a
and ROCK both require both correspond-
Activity of 5-substituted 4-(benzimidazol-2-yl)-1,2,5-oxadiazol-3-amine derivatives
against p70S6K, PKA
ing acidic side chains to be coordinated with intermolecular H-
bonds. This could be due to a reconfiguration of the Lys123 side
chain. Experimentally determined ligand/kinase complex struc-
tures can help elucidate this potentially useful selectivity lesson.
To better understand the value of the hydrophobic interactions
provided by the benzene portion of the benzimidazole ring, we
removed the ring and substituted the remaining imidazole at the
4-position. The imidazole derivatives were prepared as outlined
in Schemes 2 and 3.
The 4-phenyl imidazole derivative (35) was prepared by initial
condensation of phenylacetamide (33) with imidate (8) under
microwave conditions to afford 34, which was alkylated with
isobutyl iodide to give the desired compound 35 (Scheme 2). The
4-methyl and 4-cyano imidazole derivatives 38 and 40 were pre-
pared as shown in Scheme 3. 4-Amino-1,2,5-oxadiazole-3-carboni-
trile12 was treated with isobutylamine and AlCl3 to afford the
intermediate amidine which was used without characterization.
Condensation with 1-chloropropanone afforded the 4-methyl
a
, GSK-3b and ROCK-111
H2N
R2
N
N
O
N
N
ID
R2
p70S6K Ki
M)
PKA
a
Ki
GSK-3b Ki
M)
ROCK-1 Ki
(lM)
(
l
(l
M)
(l
13
26
27
28
29
30
31
32
H
OH
NH2
OMe
CO2Me
NHCOMe
Me
0.004
<0.001
<0.001
0.039
0.41
2.2
0.019
0.016
0.54
2.8
0.034
2.5
>3.3
>3.3
2.6
0.38
1.1
2.1
0.46
>3.3
>3.3
3.2
0.18
2.2
0.085
0.94
>3.3
>3.3
>3.3
>3.3
CH2OH
1.5
>3.3
H2N
H2N
N
N
H2N
(a)
N
O
(b)
NH2
MeO
HN
N
N
O
+
N
O
N
O
N
H
N
N
8
33
34
35
Scheme 2. Synthesis of 4-(1-isobutyl-4-phenyl-1H-imidazol-2-yl)-1,2,5-oxadiazol-3-amine. Reagents and conditions: (a) EtOH, cat. AcOH, 100 °C,
lW, 5 min, 31%; (b)
isobutyl iodide, NaH, DMF, 19%.
H2N
N
N
O
N
N
H2N
(b)
(c)
H2N
HN
NH
(a)
N
O
38
N
O
NC
N
N
H2N
H2N
NC
(d)
NC
N
N
N
N
36
37
N
N
O
O
N
N
40
39
Scheme 3. Synthesis of 4-methyl and 4-cyano imidazoles. Reagents and conditions: (a) iBuNH2, AlCl3, 1,2-DCE, rt, 20 min, 68%; (b) 1-chloropropanone, K2CO3, DMF, 180 °C,
i
l
W, 20 min, 25%; (c) 2-chloroacrylonitrile, PrNEt2, THF, 160 °C,
l
W, 30 min, 50%; (d) MnO2, toluene, 85 °C, 19%.
actions with small ortho-substituents. However, meta- or para-sub-
stituents are too large to be accommodated as effectively. The
corresponding residues in each of GSK3b, ROCK-1 and PKA
a are
Table 3
all leucines, which are more bulky and less able to accommodate
Activity of 4-substituted imidazoles against p70S6K, PKAa
, GSK-3b and ROCK-111
the phenyl ring substituents.
Compounds designed to interact with pocket B are shown in
Table 2: the benzimidazole substituent is fixed with cyclobutyl.
Unsurprisingly, small polar substituents at the benzimidazole
5-position fared the best, with hydroxyl (26) and amino (27)
groups going below the limit of detection of our assay (Table 2).
However, larger polar groups such as acetamido (30) appear to
be either too large for the pocket or possess unsuitable vectors
for optimal interaction with the protein and displayed significantly
poorer affinity. The off target activities of 26 and 27 raise interest-
ing questions about how similar small polar substituents give rise
to very different SAR. One possibility is that for p70S6K and GSK3b,
the 5-substituent needs to donate a single H-bond to either Glu143
H2N
R
N
N
O
N
N
ID
R
P70S6K Ki
M)
PKAa
Ki
GSK-3b Ki
M)
ROCK-1 Ki
M)
(
l
(
l
M)
(l
(l
35
38
40
Ph
Me
CN
0.089
0.34
1.6
1.2
>3.3
>3.3
>3.3
0.69
>3.3
>3.3
>3.3
>3.3

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U. Bandarage et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5191–5194
derivative (38), while condensation with 2-chloroacrylonitrile and
subsequent oxidation gave the 4-cyano derivative (40).
As shown in Table 3, replacement of the benzimidazole system
with a 4-phenylimidazole (compound 35) maintained the activity
and selectivity profile of the parent compound (cf. 18, Table 1).
Imidazoles bearing smaller 4-substituents (38, 40) were signifi-
cantly less potent.
In summary, a series of low molecular weight, potent and selec-
tive inhibitors of p70S6K have been identified and developed using
a homology model derived from PKA. Selectivity relative to other
AGC-family kinases is good. Compound 13 and the closely analogous
benzimidazole 5-substituted analogs 26 and 27, as well as the
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9. Sequence alignment of p70S6 K to PKAa used blastp (http://
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N-substituted
4-phenylimidazol-2-yl-1,2,5-oxadiazol-3-amines
(e.g., 35) are promising leads for further exploration of p70S6K and
building an understanding of its in vivo role.
Acknowledgements
The authors thank Dr. Jackie Macritchie of Biofocus and Dr.
Scott Harbeson of Vertex for project management in the collabora-
tion and Christine Memmott, Paul Taslimi and Dr. Kirk Tanner for
enzymology support.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.07.022.