Use of core modification in the discovery of CC214-2, an orally available, selective inhibitor of mTOR kinase

Article abstract of DOI:10.1016/j.bmcl.2013.01.110

We report here the discovery of a novel series of selective mTOR kinase inhibitors and the identification of CC214-2, a compound with demonstrated anti-tumor activity upon oral dosing in a PC3 prostate cancer xenograft model. A series of 4,6-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones were discovered through a core modification of our original compound series. Analogs from this series have excellent mTOR potency and maintain selectivity over the related PI3Kα lipid kinase. Compounds such as CC214-2 were found to block both mTORC1(pS6) and mTORC2(pAktS473) signaling in PC3 cancer cells, in vitro and in vivo.

Full text of DOI:10.1016/j.bmcl.2013.01.110

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1588–1591
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Use of core modification in the discovery of CC214-2, an orally
available, selective inhibitor of mTOR kinase
a
a
a
a
Deborah S. Mortensen a, , John Sapienza , Branden G. S. Lee , Sophie M. Perrin-Ninkovic , Roy Harris ,
Graziella Shevlin a, Jason S. Parnes a, Brandon Whitefield a, Matt Hickman b, Gody Khambatta b,
Rene R. Bisonette c, Sophie Peng d, Jim C. Gamez d, Jim Leisten d, Rama Krishna Narla d, Kimberly E. Fultz c,
Sabita Sankar c
⇑
a Department of Medicinal Chemistry, Celgene Corporation, 4550 Towne Centre Court, San Diego, CA 92121, United States
b Department of Biochemistry, Celgene Corporation, 4550 Towne Centre Court, San Diego, CA 92121, United States
c Department of Oncology Research, Celgene Corporation, 4550 Towne Centre Court, San Diego, CA 92121, United States
d Department of Pharmacology, Celgene Corporation, 4550 Towne Centre Court, San Diego, CA 92121, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
We report here the discovery of a novel series of selective mTOR kinase inhibitors and the identification
of CC214-2, a compound with demonstrated anti-tumor activity upon oral dosing in a PC3 prostate can-
cer xenograft model. A series of 4,6-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones were
discovered through a core modification of our original compound series. Analogs from this series have
Received 30 November 2012
Revised 15 January 2013
Accepted 22 January 2013
Available online 30 January 2013
excellent mTOR potency and maintain selectivity over the related PI3K
a lipid kinase. Compounds such
as CC214-2 were found to block both mTORC1(pS6) and mTORC2(pAktS473) signaling in PC3 cancer cells,
in vitro and in vivo.
Keywords:
Mammalian target of rapamycin
mTOR kinase
Ó 2013 Elsevier Ltd. All rights reserved.
Kinase inhibitors
PI3K/Akt/mTOR pathway
Oncology
The mammalian target of rapamycin (mTOR) is a serine/threo-
nine kinase member of the phosphatidylinositol 3-kinase-like ki-
nase superfamily. mTOR regulates cell growth, metabolism,
proliferation and survival through integration of growth factor sig-
naling with cellular nutritional status and energy use.1 The mTOR ki-
nase exists within two distinct multiprotein complexes, mTOR
complex-1 (mTORC1) and mTOR complex-2 (mTORC2).2 Both
mTORC1 and mTORC2 are critical mediators of the PI3K/AKT path-
way, which is frequently mutated in many cancers, leading to hyper-
activation of mTOR signaling.3,4 While rapamycin analogs such as
temsirolimus and everolimus, compounds that target only the
mTORC1 complex, have shown some clinical activity; it is hypothe-
sized that mTOR kinase inhibitors, blocking both mTORC1 and
mTORC2 signaling, will have expanded therapeutic potential.5 We
have previously described the identification and initial SAR explora-
tion of a potent series of 1,6-substituted imidazo[4,5-b]pyrazin-2-
ones as mTOR kinase inhibitors.6 Here we describe the use of core
modification in the identification of a new series of 4,6-disubsti-
tuted-3,4-dihydropyrazino[2,3-b]pyrazin-2(1H)-one mTOR kinase
inhibitors, leading to the identification of CC214-2, an orally
O
A
B
HN
N
N
HO
N
N
N
N
N
N
O
O
N
H
N
H
1
2
mTORIC50 = 0.002 μM
PI3Kα IC50 = 2.6 μM
mTORIC50 = 0.03 μM
PI3Kα IC50 = 21.9 μM
R1
X
R1
R2
R2
N
core
modification
N
X
N
N
N
O
N
H
N
N
H
O
Figure 1. (A) Representative analogs from initial compound series. (B) Core
modification of the imidazo[4,5-b]pyrazin-2-ones to give 3,4-dihydropyrazi-
no[2,3-b]pyrazin-2(1H)-ones.
available mTOR kinase inhibitor with demonstrated anti-tumor
activity in mice with PC3 human prostate cancer tumors.
Our original SAR efforts in the imidazo[4,5-b]pyrazin-2-one ser-
ies lead to the identification of analogs such as 1 and 2 (Fig. 1A).
⇑
Corresponding author. Tel.: +1 858 795 4951; fax: +1 858 795 4719.
E-mail address: dmortens@celgene.com (D.S. Mortensen).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.110

D. S. Mortensen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1588–1591
1589
b]pyrazin-2-one core. Ring-expansion of the imidazo-ring, through
insertion of a methylene unit, led to the identification of a new ser-
ies of potent and selective mTOR kinase inhibitors (Fig. 1B).
Compounds in the ring-expansion series were synthesized
through the route outlined in Scheme 1. Treatment of 3,5-dibromo-
pyrazin-2-amine 3 with either bromo- or chloro-acetic anhydride
gave 4 or 5, respectively. Chloro intermediate 5 was converted to
the corresponding iodide 6 using sodium iodide. This conversion
was necessary as the chloro-intermediate proved unreactive in
the subsequent amine addition reactions. Amine additions to 4 or
6 afforded halogen displacement and intramolecular ring closure
to give intermediates 7a–b. Compound 10a (see Table 1) was syn-
thesized through a Suzuki coupling of 4-(1-(tetrahydro-2H-pyran-
2-yl)-1H-1,2,4-triazol-3-yl)phenyl-boronic acid pinacol ester to 7a,
followed by acidic deprotection. Stille coupling of 2-(5-(trimethyl-
stannyl)pyridin-2-yl)propan-2-ol with 7b afforded 10b. Com-
pounds 9a and 9b from the imidazo[4,5-b]pyrazin-2-one series
were synthesized following methods described previously.6
Compounds from the newly identified series provided similar
potency relative to the parent imidazo[4,5-b]pyrazin-2-ones (Ta-
ble 1). These analogs retained selectivity for mTOR over the related
Br
N
N
Br
Br
N
Br
a
c
O
X
N
H
N
NH2
3
4 X=Br
b
5 X=Cl
6 X=I
R2
N
R2
N
Br
N
R1
N
N
d, e
or f
N
N
H
O
N
H
O
7 a-b
10 a-b
Scheme 1. Reagents and conditions: (a) 2-bromoacetic anhydride or 2-chloroacetic
anhydride, acetonitrile, 70 °C, 79–85%; (b) sodium iodide, acetone, 77%; (c) desired
amine, acetonitrile, diisopropylethyl amine, rt or 40 °C, 50–79%; (d) 4-(1-(tetrahy-
dro-2H-pyran-2-yl)-1H-1,2,4-triazol-3-yl)phenyl-boronate ester, sodium carbon-
ate, PdCl2(dppf)2–CH2Cl2, dioxane and water, 120 °C, 48%; (e) 4 N HCl in dioxane,
ethanol,
rt,
22%;
(f)
2-(5-(trimethyl-stannyl)pyridin-2-yl)propan-2-ol,
PdCl2(dppf)2–CH2Cl2, DMF, 110 °C, 29%.
lipid kinase PI3Ka, maintaining 65- to >500-fold selectivity for
These compounds were potent and selective inhibitors of mTOR ki-
nase and showed inhibition of pathway biomarkers and prolifera-
tion in a PC3 prostate cancer cell line.6
As we continued our medicinal chemistry exploration of this
series, we also sought to examine changes to the imidazo[4,5-
mTOR. In PC3 prostate cancer cell lines, the compounds proved to
be inhibitors of both mTOR complexes as measured by inhibition
of pS6 (mTORC1) and pAktS473 (mTORC2), as would be expected
for mTOR kinase domain inhibitors. We also observed inhibition
Table 1
mTOR and PI3K
a
potency and mTOR pathway biomarker and antiproliferation data in PC3 cancer cellsa
b
b
Analog
Structure
mTOR IC50
(
lM)
PI3K
a
IC50
(
lM)
PC3 cellular activity
b
b
b
pS6 IC50
(
lM)
pAkt IC50
(
lM)
Prolif IC50 (lM)
O
HN
N
N
9a CC214-1
0.002
1.38
0.040
0.018
0.224
N
N
N
O
N
H
O
HN
N
N
10a
0.008
0.526
0.046
0.034
0.148
N
N
N
O
N
H
O
O
HO
HO
9b
0.176
0.106
>30c
2.41
2.55
4.18c
N
N
N
N
N
N
H
O
10b CC214-2
N
N
N
>30c
0.386
0.315
1.55
N
H
O
a
b
c
See Supplementary data for assay details.
Average of two or more experiments.
Single experiment.

1590
D. S. Mortensen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1588–1591
Figure 3. PK/PD relationship of CC214-2 in mice with PC3 tumors with a single oral
dose of 30 mg/kg. Inhibition of pS6 (A) and pAktS473 (B) in the tumors was
correlated with the compound levels in both plasma and tumors.
10
l
M. Generation of concentration-response curves for the one ki-
M.
nase, FMS, yielded an IC50 value of 2.70
l
The antitumor activity of CC214-2 in PC3 xenograft model was
initially determined using a number of oral dosing paradigms;
once or twice daily or every second day.9 CC214-2 significantly
inhibited PC3 tumor growth under these dosing regimens
(Fig. 2A). The activity was further explored using a number of dose
levels with a once daily dosing regimen.10 In this study, CC214-2
significantly inhibited tumor growth in a dose dependent manner,
demonstrating 18%, 63% and 84% tumor volume inhibition com-
pared to vehicle control at 10, 25 and 50 mg/kg, respectively
(Fig. 2B). The compound was well tolerated at all dose levels and
schedules following 3 weeks of treatment.
In order to determine the extent of mTOR pathway inhibition
and PK/PD relationship, PC3 tumor-bearing mice were adminis-
tered with a single dose of CC214-2, and plasma and tumor samples
were collected at various time points for analysis. Significant inhi-
bition of mTOR pathway markers pS6 and pAktS473 was observed,
indicating that the antitumor activity was mediated through the
inhibition of both mTORC1 (pS6) and mTORC2 (pAktS473). When
dosed at 100 mg/kg, CC214-2 achieved full biomarker inhibition
in the PC3 tumor model through 24 h and compound levels in both
tumor and plasma were more than 10-fold above the cellular bio-
marker IC50 values at 24 h.11 Given the CC214-2 mouse plasma pro-
tein binding of 65%, the free plasma concentration at 24 h was 4.6-
to 5.7-fold above the pS6 and pAktS473 cellular IC50 values. When
CC214-2 was dosed at 30 mg/kg, pS6 inhibition was maintained
at 87–93% from 2 to 8 h, with 43% inhibition at 24 h (Fig. 3A). Sim-
ilarly, inhibition of pAktS473 from 2 to 8 h was 53–77% and fell to
28% at 24 h (Fig. 3B). The level of biomarker inhibition correlated
well with the plasma and tumor level of compound, which was al-
most completely cleared by 24 h (Fig. 3). In the 30 mg/kg study, sig-
nificant biomarker inhibition was observed in tumors at time points
Figure 2. Antitumor activity of CC214-2 in PC3 prostate cancer xenograft model.
Activity was determined at various dosing schedules (A) and dose levels with once
daily dosing (B).
of cellular proliferation as a functional effect of mTOR pathway
inhibition in these cells.
While analogs such as 9a and 10a demonstrated low nanomolar
on-target potency and corresponding cellular efficacy, these gener-
ally suffered from poor oral bioavailability.7 Compound 9a, CC214-
1, proved to be useful as an in vitro tool compound for the explo-
ration of mTOR kinase biology.
Comparison of matched-pair 9b and 10b shows the ring-ex-
panded core 10b provides improved cellular efficacy, as assessed
by both biomarker and proliferation assays, relative to the parent
imidazo[4,5-b]pyrazin-2-one 9b. Compound 10b, also showed
excellent oral bioavailability in rodent, allowing for the exploration
of mTOR kinase in in vivo sytems.7
Compound 10b, CC214-2, was further profiled for overall kinase
selectivity. When tested in a single point assay against 249 ki-
nases,8 only one kinase other than mTOR was inhibited >80% at

D. S. Mortensen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1588–1591
1591
where free plasma concentrations were maintained 7.5- to 2-fold
above the cellular biomarker IC50 values (2–8 h).
110. These data include MOL files and InChiKeys of the most
important compounds described in this article.
In summary, through use of a ring-expansion core modification
to our initial compound series, we have discovered a novel series of
4,6-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazin-2(1H)-one
mTOR kinase inhibitors, with exquisite kinase selectivity. This
work led to the identification of CC214-2, a selective and potent
inhibitor of mTOR kinase. CC214-2 significantly inhibited PC3 tu-
mor growth in a dose and schedule-dependent manner and has
further shown, in vitro and in vivo, potent inhibition of both
mTORC1 (pS6) and mTORC2 (pAktS473).
References and notes
1. Laplante, M.; Sabatini, D. M. J. Cell Sci. 2009, 122, 3589.
2. Shor, B.; Gibbons, J. J.; Abraham, R. T.; Yu, K. Cell Cycle 2009, 8, 3831.
3. Engelman, J. A. Nat. Rev. Cancer 2009, 9, 550.
4. Vivanco, I.; Sawyers, C. L. Nat. Rev. Cancer 2002, 2, 489.
5. Guertin, D. A.; Sabatini, D. M. Trends Mol. Med. 2005, 11, 353.
6. Mortensen, D. S.; Perrin-Ninkovic, S. M.; Harris, R.; Lee, B. G. S.; Shevlin, G.;
Hickman, M.; Khambatta, G.; Bisonette, R. R.; Fultz, K. E.; Sankar, S. Bioorg. Med.
Chem. Lett. 2011, 21, 6793.
7. Compound 9a: rat oral F% 0.85. Compound 10a: rat oral F% 0 (BLQ). Compound
10b: rat oral F% 100, rat iv CL 8.1 mL/min/kg.
Acknowledgments
8. SelectScreenÒ Kinase Profiling Services: Life Technologies, Inc., Carlsbad CA.
9. CB17 SCID mice were inoculated subcutaneously with 2 Â 106 PC3 prostate
cancer cells. On day 11 when the tumors were established and reached
approximately 175 mm3, the mice were randomized and treated either once or
twice daily or every second day orally with vehicle or CC214-2 at a dose
volume of 5 mL/kg. CC214-2 was formulated as a suspension in 0.5% CMC/
0.25% Tween 80 in water.
The authors thank the Celgene San Diego DMPK department for
plasma and tumor compound level analysis and Drs Peter Worland,
Heather Raymon, Brian Cathers, Mehran Moghaddam and Stacie
Canan for project advice and support.
10. CB17 SCID mice were inoculated subcutaneously with 2 Â 106 PC3 prostate
cancer cells. On day 11 when the tumors were established and reached
approximately 125 mm3, the mice were randomized and treated once daily
orally with vehicle or CC214-2 at a dose volume of 5 mL/kg. CC214-2 was
formulated as a suspension in 0.5% CMC/0.25% Tween 80 in water.
11. CB17 SCID mice with PC3 tumors were administered with a single dose of
CC214-2 at 30 or 100 mg/kg orally. CC214-2 was formulated as a suspension in
0.5% CMC/0.25% Tween 80 in water. Plasma and tumor samples were collected
at 2, 4, 8 and 24 h and processed for compound exposure analysis. Tumors
were also processed for biomarker inhibition using MSD pS6RP(S234/236) and
pAktS473.
Supplementary data
Supplementary data (biological data with standard deviation
and rat PK for CC214-2 (10b) and kinase selectivity panel for
CC214-1 (9a) and CC214-2 (10b), synthetic experimental proce-
dures and assay protocols associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.01.