Pyrazolopyrimidines as dual Akt/p70S6K inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.03.011

Activation of the PI3K/Akt/mTOR kinase pathway is frequently associated with human cancer. Selective inhibition of p70S6Kinase, which is the last kinase in the PI3K pathway, is not sufficient for strong tumor growth inhibition and can lead to activation of upstream proteins including Akt through relief of a negative feedback loop. Targeting multiple sites in the PI3K pathway might be beneficial for optimal activity. In this manuscript we report the design of dual Akt/p70S6K inhibitors and the evaluation of the lead compound 11b in vivo, which was eventually advanced into clinical development.

Full text of DOI:10.1016/j.bmcl.2012.03.011

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2693–2697
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrazolopyrimidines as dual Akt/p70S6K inhibitors
⇑
,
⇑
Kenneth D. Rice, Moon H. Kim , Joerg Bussenius , Neel K. Anand, Charles M. Blazey, Owen J. Bowles,
Lynne Canne-Bannen, Diva S.-M. Chan, Baili Chen, Erick W. Co, Simona Costanzo, Steven C. DeFina,
Larisa Dubenko, Stefan Engst, Maurizio Franzini, Ping Huang, Vasu Jammalamadaka, Richard G. Khoury,
Rhett R. Klein, A. Douglas Laird, Donna T. Le, Morrison B. Mac, David J. Matthews, David Markby,
Nicole Miller, John M. Nuss, Jason J. Parks, Tsze H. Tsang, Amy L. Tsuhako, Yong Wang, Wei Xu
Exelixis, 210 E. Grand Avenue, South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Activation of the PI3K/Akt/mTOR kinase pathway is frequently associated with human cancer. Selective
inhibition of p70S6Kinase, which is the last kinase in the PI3K pathway, is not sufficient for strong tumor
growth inhibition and can lead to activation of upstream proteins including Akt through relief of a neg-
ative feedback loop. Targeting multiple sites in the PI3K pathway might be beneficial for optimal activity.
In this manuscript we report the design of dual Akt/p70S6K inhibitors and the evaluation of the lead com-
pound 11b in vivo, which was eventually advanced into clinical development.
Received 9 February 2012
Revised 29 February 2012
Accepted 2 March 2012
Available online 10 March 2012
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
p70S6K inhibitor
Akt inhibitor
Pyrazolopyrimidine
PI3K pathway
Cancer
Hyperactivated signaling due to dysregulation of the phospho-
inositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin
(mTOR) pathway is observed in many cancers.1 The potential for
therapeutic intervention by inhibiting one or more targets in this
pathway has been extensively discussed in the literature.2–6 Some
of the first compounds to enter the clinic were analogs of rapamy-
cin, which are all highly specific inhibitors of mTORC1.7 Although
these compounds have shown great promise in preclinical models,
and have demonstrated clinical activity in select indications, they
have not shown broad spectrum clinical activity in single agent tri-
als.8 This may be in part due to the presence of a negative feedback
mechanism, whereby inhibition of mTORC1 induces insulin recep-
tor substrate IRS-1 expression leading to upregulation of IGF1R-
dependent signaling, which then leads to activation of PI3K and
its downstream effectors, in particular Akt.9 The upregulation of
S473 phosphorylated Akt was observed in vitro in different cancer
cell lines as well as in vivo in human tumors.9
resulting in lead compound 1a (Table 1).10 While 1a represented
a substantial improvement over the initial HTS hit and demon-
strated tumor growth inhibition in vivo, it also showed a similar
upregulation of pAkt(S473) in pharmacodynamic studies. This ef-
fect can likely be explained by induction of the same negative feed-
back loop, which would then account for the relative weak activity
in the efficacy experiment considering the high potency of com-
pound 1a. These observations encouraged us to determine if we
could compensate for the upregulated Akt activity by reducing
the selectivity of compound 1a and adding Akt inhibitory proper-
ties into the molecule.
The SAR leading to 1a has shown that replacing the 3-position
ethyl group on the pyrazolopyrimidine with a bromine resulted
in less selectivity and higher enzymatic Akt activity (see com-
pounds 2a,b Table 1).11 We then determined the cellular potency
of these compounds in two different cell lines, A549 and PC-3,
which also served as in vivo models. The assays measured the inhi-
bition of phosphorylation of ribosomal protein S6 to quantify
p70S6K activity, and of GSK3b, a known Akt substrate, for quanti-
fying Akt activity.12,13 The higher biochemical Akt activity of the
bromo analogs compared to the ethyl compounds had no effect
on S6 phosphorylation in A549 cells, but did result in higher po-
tency in the PC-3 cell line. The higher enzymatic Akt activity of
the bromo analogs also led to increased cellular Akt potency over
the ethyl compounds in PC-3 cells, as measured by inhibition of
phosphorylation of GSK3b, especially when R1 was a pyrrolidine.
In a recent disclosure we have described the SAR and synthesis
of pyrazolopyrimidines as potent and selective p70S6K inhibitors
⇑
Corresponding authors. Tel.: +82 31 920 2773; fax: +82 31 920 1571 (M.H.K.);
tel.: +1 650 837 7000; fax: +1 650 837 8177 (J.B.).
E-mail addresses: mkim58@ncc.re.kr (M.H. Kim), jbusseni@exelixis.com (J.
Bussenius).
Present address: National Cancer Center, 323 Ilsan-ro, Ilsandong-gu, Goyang-si,
Gyeonggi-do 410-769, Republic of Korea.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.011

2694
K. D. Rice et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2693–2697
Table 1
Activities for compounds 1a,b and 2a,b
H
N
Cl
R1
N
N
R
3
N
N
N
H
N
ID
R
R1
p70S6K IC50
(nM)
Akt1 IC50
(nM)
Cell S6-p IC50
(nM)A549/PC-3
Cell GSK3b-p IC50
(nM)PC-3
Mouse liver microsome stability
(% conversion)a
1a
Et
NMe2
2
371
45/211
9026
29
1b
2a
Et
Br
3
2
434
53
80/832
43/141
3589
4285
18
50
N
NMe2
2b
Br
2
42
94/446
1271
19
N
a
After 30 min incubation at 15 lM compound concentration and 0.5 mg/mL microsome concentration.
As expected, the pyrrolidines 1b and 2b also have the additional
advantage of higher metabolic stability compared to the dimethyl-
amine analogs 1a and 2a expressed by their mouse liver micro-
somal degradation. On the basis of these in vitro data we decided
to use compound 2b as starting point for further SAR optimization
with the help of computer modeling.
We began our study by docking the literature Akt inhibitor 3
(Fig. 1)14 into the crystal structure of active Akt (PDB code 1O6L)
and compared the results with the docking of our early p70S6K
inhibitor 4 in the same structure. The superimposition of the two
compounds showed some overlap, particularly in the proposed
binding site on the kinase hinge region, but also revealed that
the hydrophobic pocket containing the benzophenone moiety of
compound 3 was unoccupied by our scaffold (Fig. 2). Extension
with a hydrophobic group off the 50-position, where the chlorine
substituent is located, should allow access to this pocket. This
motivated us to focus our lead optimization efforts on increasing
the interactions with this pocket and therefore the Akt kinase
potency.
Figure 2. Docking of Akt inhibitor 3 and p70S6K inhibitor 4 into the crystal
structure of Akt.
In order to test our model a number of analogs with different
structural motifs at the 50-position were synthesized and are
shown in Table 2 (compounds 5–11).
The synthesis of compounds 1a,b, 2a, and 4 was described in
our recent disclosure and compound 2b was prepared in a analo-
gous manner.10 Compounds 5a,b and 8 in Table 2 were made as
shown in Scheme 1. Alkylation of phenol 12 with either ethyl or
isobutyl bromide gave the ethers 13a,b. Successive Buchwald–
Hartwig amination with Boc-piperazine followed by 2-pyrroli-
dine-1-yl-ethylamine afforded compounds 15a,b, which were
deprotected and reacted with 3-bromo-4-chloro-1H-pyrazolo[3,4-
d]pyrimidine to give the desired products 5a,b. Successive amina-
tion of phenol 12 as above followed by activation as triflate and
Sonogashira reaction with tert-butylacetylene afforded intermedi-
ate 17, which was converted to the alkylphenyl derivative 18 by
hydrogenolysis. Finally, conversion to compound 8 was achieved
as described above.
The preparation of the remaining compounds 6, 7, 9, 10 and
11a,b in Table 2 is illustrated in Scheme 2. Central starting material
for all compounds was methyl ester 19. Successive Buchwald–Har-
twig amination of 19 with Boc-piperazine and 2-pyrrolidine-1-yl-
ethylamine, followed by deprotection and reaction with 3-bro-
mo-4-chloro-1H-pyrazolo[3,4-d]pyrimidine gave ester 10. Saponi-
fication of 19 followed by conversion to the methyl oxadiazole
23 and then repetition of the above described steps yielded com-
pound 9. Buchwald–Hartwig amination of 19 to intermediate 20,
followed by ester hydrolysis and amide coupling with aniline affor-
ded amide 24. A second amination, then deprotection and final
coupling to the pyrimidine gave compound 6. In a similar manner,
successive aminations of 19, saponification and amide coupling
O
F
Cl
OMe
O
HO
N
N
NH
NH
HN
Br
O
N
N
N
H
N
N
3
4
Figure 1. Structures of Roche Akt inhibitor 3 and compound 4.

K. D. Rice et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2693–2697
2695
Table 2
Biochemical and cellular activities for analogs 2b and 5–11
H
2
5'
N
R
N
N
N
Br
N
N
N
H
N
ID
R2
p70S6K IC50 (nM)
Akt1 IC50 (nM)
Cell S6-p IC50 (nM) A549/PC-3
Cell GSK3b-p IC50 (nM) PC-3
2b
5a
5b
6
7
8
Cl
–OEt
–OiBu
–CONHPh
–CH2OCH2CF3
–CH2CH2tBu
2
2
1
1
2
2
42
56
9
13
8
94/446
197/—
37/214
14/190
17/66
1507
9067
700
7811
730
7
116/203
1982
N
9
1
6
13/47
665
N
O
10
11a
11b
–CO2Me
–CO-CH2CH3
–CO-CH2CH3CF3
2
1
2
5
5
1
44/52
18/25
30/55
1843
976
194
H
H
N
Br
OR3
b
OR3
c
N
OR3
OR3
Br
OH
a
Br
d, e
N
N
Br
N
Br
N
N
N
N
Boc
N
Boc
3
Br
N
12
13a
13b
: R = Et
3
N
: R = iBu
N
N
14a,b
15a,b
H
b, c, f
5a,b
t
Bu
H
N
H
N
H
N
H
N
t
Bu
OTf
t
Bu
N
N
N
N
N
N
N
N
N
N
g
h
d, e
N
N
Br
N
Boc
Boc
Boc
N
N
N
16
17
18
H
8
Scheme 1. Preparation of compounds 5a,b and 8 in Table 2. Reagents and conditions: (a) R3Br, Cs2CO3, DMF, rt, 16 h; (b) Boc-piperazine, toluene, Pd2(dba)3, BINAP, NaOtBu,
110 °C, 16 h; (c) 2-pyrrolidine-1-yl-ethylamine, toluene, Pd2(dba)3, BINAP, NaOtBu, 110 °C, 16 h; (d) HCl, dioxane, MeOH, reflux, 2 min; (e) 3-bromo-4-chloro-1H-
pyrazolo[3,4-d]pyrimidine, iPrOH, DIEA, 60 °C, 2 h; (f) triflic anhydride, CH2Cl2, triethylamine, ꢀ78 °C, 30 min, then rt, 16 h; (g) tert-butylacetylene, DMF, PdCl2(PPh3)2, CuI,
triethylamine, 100 °C, 16 h; (h) hydrogen, Pd/C, ethyl acetate, 2 h.
afforded Weinreb amide 25, which was converted by Grignard
reaction into the ketones 26a,b. Completion of the sequence with
the already described final two steps gave the ketones 11a,b. Final-
ly, reduction of ester 19 to the hydroxymethyl derivative 27 fol-
lowed by Misunobu reaction with trifluoroethanol afforded ether
28, which was converted by the remaining sequence to compound
7.
Interestingly, none of the different substituents affected the bio-
chemical p70S6K activity, but as the model predicted, larger hydro-
phobic groups in the 50-position indeed increased the biochemical
Akt activity. Particularly illustrative were two pairs of compounds,
ethers 5a and 5b, and ketones 11a and 11b, where the increased size
of the substituent resulted in an about five-fold improvement in bio-
chemical Akt activity. For these examples the higher biochemical

2696
K. D. Rice et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2693–2697
H
N
CH2OCH2CF3
Br
CH2OCH2CF3
Br
Br
Br
CH2OH
CO2Me
CO2H
a, b, c, d
N
l
N
N
Br
Br
Br
N
27
28
N
N
N
H
k
7
H
N
H
N
CO2Me
Br
CO2Me
CO2Me
a
b
c, d
N
N
N
N
Br
N
N
N
Boc
N
Boc
Br
N
19
N
N
e
N
20
21
H
10
e, i
e, h
O
O
O
H
N
Br
H
Br
Ph
N
OMe
N
R4
N
N
N
H
Me
22
j
N
N
N
N
N
f, g
N
Boc
Boc
Boc
N
N
26a: R4 = Et
Br
25
24
O
26b: R4 = CH2CH2CF3
Br
b, c, d
c, d
23
a, b, c, d
N
O
O
H
H
N
H
N
N
Ph
N
R4
O
N
N
N
H
N
N
N
N
N
N
N
Br
Br
Br
N
N
N
N
N
N
N
H
N
H
N
H
N
N
N
9
6
11a,b
Scheme 2. Preparation of compounds 6, 7, 9, and 11a,b in Table 2. Reagents and conditions: (a) Boc-piperazine, toluene, Pd2(dba)3, BINAP, NaOtBu, 110 °C, 16 h; (b) 2-
pyrrolidine-1-yl-ethylamine, toluene, Pd2(dba)3, BINAP, NaOtBu, 110 °C, 16 h; (c) HCl, dioxane, MeOH, reflux, 2 min; (d) 3-bromo-4-chloro-1H-pyrazolo[3,4-d]pyrimidine,
iPrOH, DIEA, 60 °C, 2 h; (e) KOH, methanol, water, 60 °C, 2 h; (f) N-hydroxyethanimidamide, DMF, HATU, DIEA, rt, 16 h; (g) TBAF, THF, rt, 15 h; (h) aniline, DMF, HATU, HOAt,
N-methylmorpholine, rt, 17 h; (i) N,O-dimethylhydroxylamine, HATU, HOAt, N-methylmorpholine, rt, 17 h; (j) R4MgBr, THF, 0 °C, then rt, 16 h; (k) NaBH4, EtOH, reflux, 16 h;
(l) CF3CH2OH, benzene, 1,10-(azodicarbonyldipiperidine), tri-n-butylphosphine, rt, 6 h.
activity translated into a similar improvement in cell based potency,
as measured by inhibition of phosphorylation of GSK3b. However,
this correlation of biochemical and cellular activity was not always
observed, although there was clearly a general trend.
For compounds with cellular IC50s less than 1 lM the pharma-
cokinetic profiles in the rat were evaluated (Table 3). Also the
mouse plasma exposures at 100 mg/kg were determined, since this
was the model for further pharmacodynamic and efficacy studies.

K. D. Rice et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2693–2697
2697
Table 3
Rat PK and mouse plasma exposure for compounds 5b, 7, 9, and 11a,b
ID
Species
Dose (mg/kg)
CL (mL/h/kg)
Vd (L/kg)
T1/2 (h)
F (%)
Cmax
(lM)
AUC/dose (lM h kg/mg)
Mouse plasma exposure at 100 mg/kg,
IV/PO
IV/PO
IV/PO
1 and 4 h (lM)
5b
7
9
11a
11b
11b
11b
Rat
Rat
Rat
Rat
Rat
Dog
Monkey
5
4769
3809
4382
2940
2722
1562
1230
14.7
9.7
2.5/1.7
2.1/1.4
2.4/2.9
3.1/2.9
3.5/7.9
7.5/10.4
6.1/6.4
13
11
10
28
35
58
77
1.15/0.07
0.39/0.02
0.37/0.01
1.44/0.14
0.33/0.04
0.21/0.10
0.58/0.40
0.35/0.05
0.43/0.05
0.45/0.05
0.56/0.15
0.64/0.17
0.95/0.54
1.22/0.94
1.2/1.3
1.2/1.2
0.3/0.1a
2.0/2.0
1.6/3.1
2.5
2.5
5
2.5
3
14.2
10.5
12.9
17.0
6.9
3
a
At 30 mg/kg.
9. O’Reilly, K.E.;Rojo, F.; She, Q.-B.;Solit, D.;Mills, G. B.;Smith, D.; Lane,H.; Hofmann,
F.; Hicklin, D. J.; Ludwig, D. L.; Baselga, J.; Rosen, N. Cancer Res. 2006, 66, 1500.
10. Bussenius, J.; Anand, N. K.; Blazey, C. M.; Bowles, O. J.; Canne Bannen, L.; Chan,
D. S. M.; Chen, B.; Co, E. W.; Costanzo, S.; DeFina, S. C.; Dubenko, L.; Engst, S.;
Franzini, M.; Huang, P.; Jammalamadaka, V.; Khoury, R. G.; Kim, M. H.; Klein, R.
R.; Laird, D.; Le, D. L.; Mac, M. B.; Matthews, D. J.; Markby, D.; Miller, N.; Nuss, J.
M.; Parks, J. J.; Tsang, T. H.; Tsuhako, A. L.; Wang, Y.; Xu, W.; Rice, K. D. Bioorg.
Med. Chem. Lett. 2012, 12, 2283.
11. p70S6K and Akt1 assay: Kinase activity was measured as the percent of ATP
consumed following the kinase reaction using luciferase–luciferin-coupled
chemiluminescence. Reactions were conducted in 384-well white, medium
binding microtiter plates (Greiner). Kinase reactions were initiated by
Unfortunately, most compounds like the ether 5b, the fluorinated
ethyloxymethyl analog 7, and the oxadiazole 9 showed low
absorption and oral bioavailability. Only the ketones 11a and 11b
had significantly improved values. The pharmacokinetic profile of
the most active compound, ketone 11b was then also obtained in
the dog and monkey, where it showed even higher oral bioavaila-
bilities of 58% and 77%, respectively.
On the basis of the high biochemical activity and cellular po-
tency combined with good oral bioavailability in different species,
we decided to advance compound 11b into a pharmacodynamic
study, in which the inhibition of S6-, GSK3b-, and PRAS40- (an-
other downstream target of Akt) phosphorylation was measured.
The compound was dosed at 100 mg/kg bid with a total of five
doses before measurement in the PC-3 prostate carcinoma model
and demonstrated 90% inhibition of S6-p, 46% inhibition of
GSK3b-p, and 76% inhibition of PRAS40-p. Compound 11b was sub-
sequently evaluated in a PC-3 xenograft efficacy experiment and
dosed orally at 50 mg/kg qd for 15 days. In this study 11b resulted
in 52% tumor growth inhibition (TGI), compared to 49% TGI for the
selective p70S6K inhibitor 1a at 100 mg/kg.10 The efficacy of com-
pound 11b was also determined in the A549 breast cancer model
and dosed orally at 50 mg/kg qd and 100 mg/kg qd for two days
every 4 days. The compound was highly active in this study with
71% tumor growth inhibition for the 50 mg/kg dose and 96% TGI
for the 100 mg/kg alternate dosing schedule as a single agent.
In summary, we have described the successful optimization of a
selective p70S6K inhibitor 1a into a dual Akt/p70S6K inhibitor 11b.
The additional Akt activity is reflected in better efficacy in xeno-
graft studies compared to 1a. Due to its excellent activity in vitro
and in vivo combined with good oral bioavailability in higher spe-
cies, compound 11b was nominated as clinical candidate and went
into development as an oral agent for use in patients with solid tu-
mors and hematological malignancies under the name XL418.
combining test compounds, 500 nM ATP, and kinase in 20 lL of reaction
buffer (20 mM Hepes pH 7.5, 10 mM MgCl2, 0.03% NP40, 0.03% BSA, 1 mM
DTT). The reaction mixture was incubated at ambient temperature for 3 h after
which 20
lL aliquot of luciferase–luciferin mix (50 mM HEPES, pH 7.8, 67 mM
oxalic acid (pH 7.8),
5
(or 50) mM DTT, 0.4% Triton X-100, 0.25 mg/mL
coenzyme A, 63 mM AMP, 28 mg/mL luciferin and 40,000 units/mL luciferase)
was added and the chemiluminescence signal measured using a Victor2 plate
reader (Perkin–Elmer).
12. pS6 assay in cells: A549 (ATCC, CCL-185) or PC-3 (ATCC, CRL-1435) cells were
seeded in 96-well plates (NUNC) in DMEM (Cellgro) containing heat inactivated
10% FBS (Hyclone), 1% penicillin–streptomycin (Cellgro), and 1% non-essential
aminoacids (Cellgro) at7.5 ꢁ 103cells/well. Cellswere incubatedat 37 °C, 5%CO2
for 48 h. Test compound in DMSO was diluted in DMEM. The DMSO
concentration in cell culture medium was maintained at 0.2% in all wells. Cells
were treated with diluted compound and each concentration assayed in
triplicate at concentrations of 1.2–300 nM. 0.004–1 nM rapamycin (Cell
Signaling Technology, 9904) and 0.12–30 nM staurosporine (Calbiochem,
569396) served as positive controls. Negative control wells were treated with
DMSO. Cells were incubated for 3 h at 37 °C, 5% CO2 for 3 h in the presence of
compound. Post compound treatment cells were fixed as follows: medium was
removed and 100
(Pierce, 28376) was added to each well at room temperature (RT) for 20 min.
Cells were quenched with 100 l 0.6% H2O2 (VWR International, 43038308) in
TBS (Pierce, 28376) + 0.1% Tween20 (Sigma, P-7949) (TBST) for 20 min at rt.
l TBS and blocked with 100 l 5% BSA (Jackson
ll/well of 4% formaldehyde (Sigma–Aldrich, 08920AD) in TBS
l
Plates were washed 3ꢁ with 200
l
l
ImmunoResearch Laboratory, 001-000-162 unless otherwise noted) in TBST for
1 h at rt. Anti-phospho-S6 ribosomal protein antibody (Cell Signaling
Technology, Ser 240/244 2212L) and anti-total-S6 ribosomal protein antibody
(Cell Signaling Technology, 2215L) were diluted 1:200 in 5% BSA (Jackson
ImmunoResearch Laboratory, 64004) in TBST. Fifty microliter primary antibody
solution was added to each well and incubated overnight at 4 °C. Plates were
washed 3ꢁ with 200
ll TBST. Goat anti-rabbit secondary antibody (Chemicon
International, 24070101) was diluted at 1:20000 in 5% non-fat dry milk in TBST.
Fifty microliter of antibody solution was added to each well and incubated for 1 h
Acknowledgment
at rt. Plates were washed 3ꢁ with 200
ll TBST and 2ꢁ 200 ll TBS.
Chemiluminescent substrate (Super Signal Elisa Femto Chemiluminescent
Substrate; Pierce, 37075) was prepared at rt. Hundred microliter of
chemiluminescent substrate per well was added and then the plate was
shaken for 1 min. Luminescence was read immediately on a Wallac plate reader.
13. GSK3b cell assay in PC-3 cells: PC-3 cells were seeded in 96-well plates in
DMEM containing heat-inactivated 10% FBS, 1% penicillin–streptomycin, and
1% non-essential amino acids at 7.5 ꢁ 103cells/well. Cells were incubated at
37 °C, 5% CO2 for 24 h and then starved in serum-free medium for 24 h. Test
compound in DMSO was diluted in DMEM. The DMSO concentration in cell
culture medium was maintained at 0.2% in all wells. Cells were treated with
diluted compound and each concentration assayed in triplicate at
The authors thank Paul Foster for his graphical support.
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Bergsneider, M.; Lai1, A.; Green, R.; Oglesby, T.; Koleto, M.; Trent, J.; Horvath, S.;
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positive control. Negative control wells were treated with DMSO. Cells were
incubated for 3 h at 37 °C, 5% CO2 in the presence of compound followed by
stimulation with 100 ng/ml IGF1 (Upstate Biotechnology, 01-189) for 30 min.
Post compound treatment cell plates were processed as described for the pS6
cell assay except that the primary antibodies were Anti-phospho-GSK3b(S9)
(Cell Signaling Technology, 9336) and anti-total-GSK3b (Cell Signaling
Technology, 9332) diluted 1:200 in 5% BSA in TBST.
14. Breitenlechner, C. B.; Wegge, T.; Berillon, L.; Graul, K.; Marzenell, K.; Friebe, W.-
G.; Thomas, U.; Schumacher, R.; Huber, R.; Engh, R. A.; Masjost, B. J. Med. Chem.
2004, 47, 1375.