Discovery and SAR exploration of a novel series of imidazo[4,5-b]pyrazin-2- ones as potent and selective mTOR kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.09.035

We report here the discovery of a novel series of selective mTOR kinase inhibitors. A series of imidazo[4,5-b]pyrazin-2-ones, represented by screening hit 1, was developed into lead compounds with excellent mTOR potency and exquisite kinase selectivity. Potent compounds from this series show >1000-fold selectivity over the related PI3Kα lipid kinase. Further, compounds such as 2 achieve mTOR pathway inhibition, blocking both mTORC1 and mTORC2 signaling, in PC3 cancer cells as measured by inhibition of pS6 and pAkt (S473).

Full text of DOI:10.1016/j.bmcl.2011.09.035

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6793–6799
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and SAR exploration of a novel series of imidazo[4,5-b]pyrazin-2-ones
as potent and selective mTOR kinase inhibitors
Deborah S. Mortensen a, , Sophie M. Perrin-Ninkovic a, Roy Harris a, Branden G.S. Lee a, Graziella Shevlin a,
⇑
Matt Hickman b, Gody Khambatta b, Rene R. Bisonette c, Kimberly E. Fultz c, Sabita Sankar c
a Medicinal Chemistry, Celgene Corporation, 4550 Towne Centre Court, San Diego, CA 92121, USA
b Biochemistry, Celgene Corporation, 4550 Towne Centre Court, San Diego, CA 92121, USA
c Oncology Research, Celgene Corporation, 4550 Towne Centre Court, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 July 2011
Revised 9 September 2011
Accepted 12 September 2011
Available online 16 September 2011
We report here the discovery of a novel series of selective mTOR kinase inhibitors. A series of imi-
dazo[4,5-b]pyrazin-2-ones, represented by screening hit 1, was developed into lead compounds with
excellent mTOR potency and exquisite kinase selectivity. Potent compounds from this series show
>1000-fold selectivity over the related PI3Ka lipid kinase. Further, compounds such as 2 achieve mTOR
pathway inhibition, blocking both mTORC1 and mTORC2 signaling, in PC3 cancer cells as measured by
inhibition of pS6 and pAkt (S473).
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Mammalian target of rapamycin
mTOR kinase
Kinase inhibitors
PI3K/Akt/mTOR pathway
Oncology
The mammalian target of rapamycin (mTOR) is a serine/threo-
nine kinase that regulates cell growth, metabolism, proliferation
and survival by integrating growth factor signaling with cellular
nutritional status and energy use.1 The mTOR kinase exists within
two distinct multiprotein complexes, mTOR complex-1 (mTORC1)
and mTOR complex-2 (mTORC2).2 mTORC1 is responsible for reg-
ulating protein synthesis and growth,3 whereas mTORC2 has been
shown to phosphorylate and activate AKT (also known as protein
kinase B),4 a key kinase in the control of cell growth, metabolism
and survival. mTOR is activated by the phosphatidylinositol-3
kinase (PI3K)/AKT pathway and as a result resides both down-
stream and upstream of AKT activation. The PI3K/AKT pathway is
frequently mutated in many cancers, leading to hyperactivation
of mTOR signaling,5,6 making mTOR an attractive drug target. Here
we describe our initial efforts in the identification and SAR explo-
ration of a potent series of mTOR kinase inhibitors. We conducted a
high-throughput screen and identified a series of 1,6-substituted
imidazo[4,5-b]pyrazin-2-ones, represented by screening hit 1, with
O
N
O
HO
O
N
N N1
HO
N
N
N
C6
N
O
N
H
1
PI-103
With little known about the structural requirements for mTOR
potency, we looked to explore the SAR of our series with respect
to mTOR kinase inhibition. Intermediate 9 allowed us to rapidly ex-
plore the SAR at the N1 position, while holding the C6 phenol
constant. Synthesis of intermediate 9 began with TIPS protection
of 4-bromophenol (Scheme 1). The protected phenol 4 was con-
verted to the corresponding boronate ester 5, followed by Suzuki
coupling with 4-bromopyrizine amine to afford the desired prod-
uct 6 in good yield. Compound 6 was brominated with NBS to af-
ford 7, with minimal competing bromination on the phenol ring,
presumably due to the steric bulk of the TIPS group. Addition of
the Boc-groups to the amine followed by deprotection of the phe-
nol gave desired intermediate 9.
an mTOR potency of 1.27 lM.
Though a number of ATP competitive mTOR kinase inhibitors
have been recently disclosed,7–20 at the time we initiated this
exploration the only reported inhibitor of the mTOR kinase domain
other than LY-294002 was PI-103, a dual PI3K/mTOR inhibitor.21
The first approach to explore N1 substituents is shown in
Scheme 2. With intermediate 9 in hand, addition of amines and
⇑
Corresponding author. Tel.: +1 858 558 7500; fax: +1 858 795 4719.
E-mail address: dmortens@celgene.com (D.S. Mortensen).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.035

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D. S. Mortensen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6793–6799
TIPSO
HO
TIPSO
TIPSO
a
b
c
N
N
O
Br
Br
B
O
NH2
3
4
5
6
TIPSO
TIPSO
HO
d
e
f
N
N
Br
NH2
N
Br
N
N
Br
N
Boc
Boc
N
N
Boc
Boc
7
8
9
Scheme 1. Reagents and conditions: (a) imidazole, triisopropylsilyl chloride, CH2Cl2, quant; (b) bis(pinacolato)diboron, potassium acetate, Cl2(dppf)2Pd–CH2Cl2 complex,
dioxane, 100 °C, 84%; (c) 5-bromopyrazin-2-amine, Cl2(dppf)2Pd–CH2Cl2 complex, 1 M Na2CO3, dioxane, 120 °C, microwave irradiation, 80%; (d) N-bromosuccininimide, DMF,
56%; (e) di-t-butyl dicarbonate, 4-dimethylaminopyridine, acetonitrile, 50 °C, 99%; (f) tetrabutylammonium fluoride, THF, 92%.
superior potency and potentially improved properties in the series.
HO
HO
We found that most alkyl substituents were tolerated, with even
small groups such as isopropyl (15) maintaining similar potency
to the original screening hit 1.
R
N
N
N
Br
N
a
N
N
O
Boc
R
NH2
N
H
Comparisons of the methyl-cyclohexyl (14) or cyclohexyl (20)
compounds to the corresponding tetrahydropyran derivatives (18
and 21) show a small improvement in potency, arguing for a
possible favorable H-bond accepting or polar interaction in the
binding pocket. Of the analogs explored, only the corresponding
N-methyl-piperidine (19) resulted in a dramatic loss in potency.
The cis- and trans-cyclohexyl carboxamides (22 and 23), which
provided the highest potency seen in this exploration of the C6-
phenol series, further supported the importance of H-bonding
interactions in this pocket. Unfortunately, these analogs were inac-
tive in cellular assays, presumably due to cellular permeability
issues.23
Boc
9
14-17, 19-21
Scheme 2. Reagents and conditions: (a) desired amine, ethanol or DMF or N-
methylpyrrolidinone, 150 °C microwave irradiation, 16–54%.
subsequent ring closure gave the desired final compounds (Scheme
2). Interestingly, ring closure was initiated by amine addition to
one of the Boc-groups, followed by bromide displacement to close
the ring.22 These reactions required an excess of the amine and
gave poor to moderate yields of the desired products.
An alternate synthesis, while less amenable to rapid SAR at the
N1 position, was also used to make analogs in this series (Scheme
3). In this case, addition of amines to 3,5-dibromopyrazin-2-amine
24 afforded selective amine addition at the desired 3-position (25).
Ring closure was achieved through use of either CDI or urea to pro-
vide intermediate bromides (26). Suzuki couplings with 4-
hydroxyphenyl boronic acid afforded the final compounds in rea-
sonable yields.
While most N1 benzyl analogs gave moderate potency, we
found that the saturated version, or the methyl-cyclohexyl analog
14 to have similar potency to the more potent benzyl substituents
(Table 1). We focused our efforts on N1 alkyl compounds, for both
We chose to explore the mTOR potency requirements of the C6-
position holding either methyl-cyclohexyl or methyl-THP constant
at N1. For rapid exploration of the C6-position, we synthesized
common intermediates 29 and 30, by an analogous route to that
in Scheme 3. Here the amine additions to 3,5-dibromopyrazin-2-
amine 24 afforded selective addition to give the 2,3-diamino-
bromopyrazines 27 and 28 in good yield. Ring closure using CDI
in THF gave the desired coupling partners to allow for C6 SAR
exploration (Scheme 4).
For the compounds in the initial C6 SAR assessment, intermedi-
ate 29 was coupled with the desired boronic acid or bornate ester
under standard Suzuki coupling conditions to afford compounds
31–37 (Scheme 5, Table 2). Methyl ester 37, was subjected to Grig-
nard reaction conditions to give the isopropyl alcohol product 39,
or alternately converted to the corresponding isopropyl amide 38
by hydrolysis and subsequent amide coupling.
R
Br
N
Br
Br
N
a
NH
Comparison of phenol 14 with phenyl 31 clearly indicates the
OH group makes a key interaction with the mTOR binding pocket
(Table 2). Extension of the OH by a single carbon, to either benzylic
alcohol 32 or carboxylic acid 33 showed an improvement in po-
tency. The corresponding benzylic amine 34 resulted in a dramatic
loss of potency, though the primary amide 35 improved in potency
compared to carboxylic acid 33. Substitution of the amide with iso-
propyl 38 resulted in a loss of potency, suggesting there is a size
limitation in this area of the binding pocket. There was room how-
ever, for small substitution on the benzylic carbon, as evidenced by
the potency for the isopropanol analog 39. This compound was also
of interest as it provides improved metabolic stability relative to
compound 32.24
N
24
NH2
N
NH2
25
b
HO
R
N
R
N
c
N
N
Br
N
O
O
N
H
N
H
N
10, 12-13, 22-23
26
Scheme 3. Reagents and conditions: (a) desired amine, neat or DMSO and/or
diisopropyethyl amine, 100–150 °C, 30–50%; (b) CDI, dioxane 100 °C or urea 200 °C,
30–65%; (c) 4-hydroxyphenylboronic acid, Cl2(dppf)2Pd–CH2Cl2 complex, 1 M
sodium carbonate, dioxane, 120–150 °C, microwave irradiation, 20–60%.
We next looked to explore carboxylic acid isosteres, hoping to
maintain or improve upon the potency of 33, and overcome the

D. S. Mortensen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6793–6799
6795
Table 1
Table 2
mTOR potency and SAR of N1 substituentsa
mTOR potency and initial SAR of C6 substituentsa
HO
R
N
N
N
O
R
N
N
N
N
H
O
N
H
b
Compound
R
mTOR IC50
2.44c
(lM)
b
O
Compound
R
mTOR IC50
(lM)
10
31
>10c
O
1
1.27 0.30
0.63c
HO
14
32
0.78 0.50
0.06 0.01
11
12
O
HO
OH
1.05 0.13
O
33
34
35
0.13c
8.3c
HO
13
14
15
1.93c
0.78 0.50
1.04c
H2N
O
O
16
2.38c
0.07c
H2N
17
18
19
1.02c
O
O
O
O
N
0.34 0.19
14.1c
36
37
0.12 0.05
N
H
2.88c
1.67c
>10c
20
21
O
O
38
39
8.9c
O
N
H
0.179c
0.178c
NH2
NH2
22
23
O
HO
0.03 0.007
a
b
c
See Supplementary data for assay details.
Average of two or more experiments.
Single experiment.
a
b
c
See Supplementary data for assay details.
Average of two or more experiments.
Single experiment.
standard Stille coupling conditions. Two approaches were taken,
in the first; coupling intermediate 29 or 30 with the desired aryl-
stannanes gave the desired analogs. Alternately, the conversion
of 29 or 30 to stannane derivatives 40 or 41 and coupling with
issues that often plague carboxylic acid containing compounds in
drug discovery.25,26 These compounds were synthesized from the
common intermediates 29 or 30, and most were realized using
X
X
Br
N
N
R
N
N
a
N
O
O
N
H
N
H
N
31-37
b
Br
N
Br
Br
Br
N
N
NH
N
a
b
N
29
O
N
H
N
NH2
NH2
N
37
37
38
39
c
24
27
28 X = O
29
30 X = O
X = CH2
X = CH2
Scheme 5. Reagents and conditions: (a) desired boronic acid or boronate ester,
Cl2(dppf)2Pd–CH2Cl2, potassium phosphate, DMF and water, 85–95 °C, 15–50%; (b)
LiOH, THF/water followed by amide coupling with isopropyl amine, 23% two steps;
(c) methylmagnesium bromide, THF, 73%.
Scheme 4. Reagents and conditions: (a) cyclohexylmethanamine or (tetrahydro-
2H-pyran-4-yl)methanamine, diisopropyethyl amine, n-butanol 120–130 °C, 64–
84%; (b) CDI, THF, 95–100 °C, 67–89%.

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D. S. Mortensen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6793–6799
Table 3
X
X
mTOR potency and SAR for carboxylic acid isosteres at C6a
X
Br
N
N
R
N
N
a
N
O
O
N
H
R
N
H
N
N
N
O
29
X = CH2
44-52
N
H
30 X = O
N
X
b
c
b
Compound
R
X
mTOR IC50
(lM)
O
N
N
Sn
N
N
N
33
CH2
0.13c
HO
O
N
H
HN
N
40
X = CH2
41 X = O
42
43
2
CH2
CH2
O
1.59c
N
Scheme 6. Reagents and conditions: (a) desired stannane, Cl2(PPh3)2Pd (II), DMF,
90–115 °C, 1–40%; (b) hexamethylditin, (PPh3)4Pd (0), toluene, 100 °C, 79%; (c)
desired aryl bromide, Cl2(PPh3)2Pd (II), DMF, 90 °C, 3–10%.
HN
0.009 0.001
0.002 0.001
N
the desired aryl-halides also afforded the target compounds
(Scheme 6).
The resulting set of azole compounds provided a good platform
to explore possible hydrogen-bonding interactions in the binding
pocket. In agreement with the initial aryl SAR, the results suggest
a preference for a hydrogen bond donating substituent, as evi-
denced by the lesser potency of analogs such as 47–49 (Table 3).
The positioning of this H-bond donor also influences potency, as
the most potent compounds identified were imidazole 44 and
the 1,2,4-triazoles 2 and 43.
We next explored 6,5-fused heterocyclic systems as phenol
replacements and/or carboxylic acid isosteres (Table 4). These
compounds were synthetically accessible by the routes outlined
in Schemes 5 and 6. In general most of these 5,6-fused systems
were tolerated and provided analogs of moderate to excellent po-
tency. Amino-imidazole compounds 57–59 gave excellent mTOR
potencies, at 10 nM or below. A preferred orientation of H-bond
donor within the binding site is evidenced here by the two iso-
meric indole analogs 61 and 62.
HN
N
N
N
44
45
N
H
CH2
0.009 0.002
0.005 0.002
N
N
H
O
N
46
47
48
O
2.02c
0.58c
>10
N
H
N
N
While no protein crystal structures of the mTOR kinase binding
domain have been reported to date, based on the results of the ini-
tial SAR exploration on this series, the compounds were docked
into a homology model of the ATP binding site of mTOR, based
CH2
CH2
N
N
N
N
upon the PI3K-c
crystal structure27 (Fig. 1). In the model, the 3-
NH and 4-N of the core are thought to be the donor–acceptor
hinge-binding motif and the C6 aryl substituents extend into the
‘back-pocket’ of the active site, interacting with the catalytic lysine
or other residues. The N1 substituents extend out toward the sol-
vent exposed region or may reach into the ribose binding pocket.
Compounds from this series were evaluated for potency against
49
50
51
52
CH2
CH2
CH2
O
1.36c
1.69c
0.12c
0.17c
O
the related lipid kinase PI3Ka. We selected a cross section of C6-aryl
N
groups to evaluate the possible influence of this substituent on PI3K
selectivity. No analogs from this series were found to be potent
inhibitors of PI3K (Table 5). Even the analog found to be most active
N
H
HN
N
against PI3Ka, 2, maintained >1000-fold selectivity for mTOR. A
sub-set of these compounds (2, 39, and 56) were further profiled
against a panel of more than 50 protein kinases at Invitrogen.28
Upon assessment of single point inhibitory activity at 10
GSK3 was identified with significant (P80%) inhibition (39 and
56). The results of the follow-up IC50 determinations on GSK3
show the compounds maintained P20-fold selectivity for mTOR,
with GSK3 IC50s of 0.60 M (39) and 0.99 M (56). Our investiga-
lM, only
N
N
a
a
l
l
a
b
c
See Supplementary data for assay details.
Average of two or more experiments.
Single experiment.
tions have shown this series of compounds to be potent mTOR
kinase inhibitors with exquisite selectivity over other kinases.

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6797
Table 4
Table 5
mTOR potency and SAR for 5,6-fused heterocycles at C6a
Kinase selectivity data over PI3Kaa
X
X
R
N
N
N
R
N
N
O
O
N
H
N
H
N
b
b
b
Compound
R
X
mTOR IC50
0.19c
(lM)
Compound
R
X
mTOR IC50
PI3Ka
IC50
(
lM)
(lM)
H
N
O
53
CH2
N
33
CH2 0.13c
23.5 0.54
HO
H
N
N
54
55
56
57
58
O
0.39c
O
N
35
32
39
CH2 0.069c
>30
H2N
HO
H
N
O
0.030 0.005
0.021 0.009
0.010 0.003
0.002 0.002
N
CH2 0.06 0.005
CH2 0.03 0.007
>30
H
N
CH2
O
HO
21.9 0.88
N
H
N
HN
N
N
H2N
H2N
HN
N
H
N
2
O
0.002 0.001
2.6 0.75
CH2
HN
N
N
CH2 0.12c
>30
H
51
N
59
60
61
62
O
0.006c
N
H
N
H
53
56
CH2 0.19c
12.6 1.45
20.1 2.95
N
N
O
0.021 0.012
0.063 0.004
3.5c
H
N
H
N
CH2 0.021 0.009
CH2
CH2
N
H
N
59
60
61
O
O
0.006c
>30
>30
>30
HN
N
H
N
H
a
b
c
N
See Supplementary data for assay details.
Average of two or more experiments.
Single experiment.
0.021 0.012
H
N
CH2 0.063 0.004
a
b
c
See Supplementary data for assay details.
Average of two or more experiments.
Single experiment.
With potent and selective inhibitors of mTOR kinase in hand,
we next evaluated the inhibitors effect on the mTOR pathway in
PC3 prostate cancer cells. We assessed inhibition of both mTOR
complexes, measuring inhibition of pS6 (TORC1) and pAkt(S473)
(TORC2). We also assessed proliferation as a functional effect of
mTOR pathway inhibition in these cells. As expected for mTOR ki-
nase domain inhibitors, our compounds showed inhibition of both
pS6 and pAkt, as well as inhibition of cell proliferation (Table 6).
Rapamycin, which directly inhibits only the TORC1 complex,
inhibits pS6 and not pAkt in these assays (also in Table 6). We
found reasonable correlations between mTOR potency and cellular
biomarker inhibition; though some sub-sets of compounds with
Figure 1. Docked binding mode of 2 in the mTOR binding site.

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D. S. Mortensen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6793–6799
Table 6
mTOR pathway biomarker and antiproliferation data in PC3 cancer cellsa
X
R
N
N
O
N
H
N
b
Compound
R
X
mTOR IC50
(lM)
PC3 cellular activity
b
b
b
pS6 IC50
(
lM)
pAkt IC50
(
lM)
Prolif IC50 (lM)
HO
39
45
CH2
0.029 0.007
0.021 0.005
1.35 0.37
0.322 0.12
1.38 0.57
0.916c
N
N
H
O
0.273 0.022
0.273 0.027
0.055 0.024
0.150 0.025
NH
N
2
O
0.002 0.001
1.37 0.38
N
H
N
53
56
58
59
CH2
CH2
CH2
O
0.194c
0.633c
NT
2.12c
N
H
N
0.021 0.009
0.002 0.002
0.006c
0.427 0.235
0.201c
0.137 0.078
0.092c
1.16 0.80
N
H
N
0.566 0.215
H2N
HN
N
H
N
N
>5
NT
>5
H
N
60
O
0.021 0.012
0.231 0.100
<0.01
0.050 0.009
>10
0.602 0.077
<0.01
Rapamycin
a
b
c
See Supplementary data for assay details.
Average of two or more experiments.
Single experiment.
apparent permeability issues within the benzimidazole series were
identified, such as 59.
In summary, we have discovered a novel series of 1,6-substi-
tuted imidazo[4,5-b]pyrazin-2-one mTOR kinase inhibitors, with
exquisite kinase selectivity, including >1000-fold selectivity over
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the related PI3K
a lipid kinase. These compounds have promising
cellular activities and represent good lead compounds for further
elaboration and optimization.
Acknowledgments
The authors thank Dr. Robert Abraham and Dr. Gary Chiang at
the Burnham Institute for advice on our initial mTOR kinase assay
development, Drs Margaret McCarrick and Sogole (Sami) Bahman-
yar for SBDD support and Drs Peter Worland, Heather Raymon,
Brian Cathers and Garrett Hampton for project advice and support.
Supplementary data
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the online version, at doi:10.1016/j.bmcl.2011.09.035.

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10 min showed two peaks: the first eluting peak corresponded to the desired
product, with MS (ESI) m/z 313.4 [M+1]+ and the second eluting peak gave MS
(ESI) m/z 393.0 [M]+ and 395.0 [M+2]+(1:1 ratio), corresponding to loss of one
Boc-group with amine addition to the second Boc-group to afford the
intermediate urea (with the bromide still present). LC–MS at 1 h shows only
the desired product.
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27. A homology model of the active site of mTOR was built using the coordinates of
PI3K-gamma, PDB code 1e7u: Walker, E. H.; Pacold, M.E.; Perisic, O.; Stephens,
L.; Hawkins, P. T.; Wymann, M. P.; Williams, R. L. Mol. Cell. 2000, 6, 909. The
model was constructed by substituting the active site residues of mTOR into
the active site coordinates of PI3K-gamma. More distant residues were left as
in PI3K-gamma due to low sequence identity outside of the ATP binding site.
The binding site was defined as residues with atoms within about 5 A of the
bound ligand (Wortmannin in structure 1e7u).
28. SelectScreenÒ Kinase Profiling Services: Invitrogen Corporation, Carlsbad CA.
For enzymes assessed, see Supplementary data.
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