Imidazo[1,2-a]pyrazines as novel PI3K inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.01.074

Phosphoinositide-3-kinase (PI3K) is an important target for cancer therapeutics due to the deregulation of its signaling pathway in a wide variety of human cancers. We describe herein a novel series of imidazo[1,2-a]pyrazines as PI3K inhibitors.

Full text of DOI:10.1016/j.bmcl.2012.01.074

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1874–1878
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Imidazo[1,2-a]pyrazines as novel PI3K inhibitors
Sonia Martínez González, Ana Isabel Hernández, Carmen Varela, Sonsoles Rodríguez-Arístegui,
Rosa María Alvarez, Ana Belén García, Milagros Lorenzo, Virginia Rivero, Julen Oyarzabal , Obdulia Rabal ,
à
James R. Bischoff , Maribel Albarrán, Antonio Cebriá, Patricia Alfonso, Wolfgang Link, Jesús Fominaya,
⇑
Joaquín Pastor
Experimental Therapeutics Program, Spanish National Cancer Research Centre (CNIO). C/Melchor Fernández Almagro 3, E-28029 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Phosphoinositide-3-kinase (PI3K) is an important target for cancer therapeutics due to the deregulation
of its signaling pathway in a wide variety of human cancers. We describe herein a novel series of imi-
dazo[1,2-a]pyrazines as PI3K inhibitors.
Received 9 December 2011
Revised 18 January 2012
Accepted 20 January 2012
Available online 28 January 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
P13K inhibitors
p110
-Morpholinyl Imidazo[1,2-a]pyrazines
Cancer treatment
a,b,d,c
8
The lipid phosphoinositide 3-kinase (PI3K) is a central compo-
nent in the PI3K/AKT/mTOR signaling pathway. This enzyme cata-
lyzes phosphorylation of the 3-hydroxyl position of
phosphatidylinositides (PIs) and plays a crucial role in mitogenic
It is important to mention that the expression of PI3Kd is confined
to leukocytes, playing a key role in cell survival in leukaemia and
2
2,23
other haematological malignancies.
Based on these precedents, PI3K has emerged as an attractive
target for the discovery of cancer therapeutics and several groups
are working to identify potent small molecule inhibitors of this sig-
naling pathway.
1
–4
signal transduction.
Various subtypes of PI3K have been inden-
5
–8
tified to date,
Class Ia PI3Ks (p110 , p110b, and p110d), acti-
a
vated by tyrosine kinase receptors, are known to play critical
9
roles in cell growth and survival. Class Ib has one family member,
A considerable number of PI3K inhibitors described in the liter-
ature contains a ‘morpholinyl–pyrimidine’ substructure (Fig. 1);
p110c. It is activated directly by G protein-coupled receptors and
2
4
25
linked mainly with inflammatory, allergic responses, transcription
some of them (e.g., BKM-120 and GDC-0941 ) are currently un-
der evaluation in human clinical trials.
1
0,11
and translation.
All four isoforms of PI3K have different distributions and share
similar cellular functions, which are context dependent. They seem
to be implicated in various degrees in the development of cancer
and are often up-regulated and mutated in cancer cells. In particu-
In order to find new hits for our PI3K program, we carried out, in
parallel, a HTS campaign together with a rational design exercise
based on known PI3K inhibitors structures. The results of the
HTS and the exploration of the corresponding hits have been pre-
viously reported. We would now like to report our findings using
the latter strategy.
2
6
lar, in ovarian, breast, colon, and brain cancers, p110
a
is often up-
regulated, by overexpression, gene amplification, mutation, and/or
PTEN phosphatase deletion.1
2–15
It also contributes to acquired
The replacement of the central core in bioactive molecules is a
common practice in medicinal chemistry to find proprietary and
novel hits. As we mentioned before, the ‘morpholinyl–pyrimidine’
resistance to both targeted anticancer therapies and conventional
1
6–20
cytotoxic agents.
On the other hand, the kinase activity of
2
1
24
p110b is essential in cellular transformation caused by PTEN loss.
substructure as part of monocyclic (e.g., BKM-120) , bicyclic (e.g.,
2
7
28
PI-249) or tricyclic (e.g., PI-103) PI3K inhibitors, has been
2
9
widely used. In particular, for bicyclic and tricyclic inhibitors,
the junctions between the pyrimidine ring and the fused cycle
were always defined by a C–C unit (i.e, C4a–C7a in PI-249 and
GDC-0941). In this context, the design of bicyclic compounds bear-
ing an N atom in bridgehead position was less obvious, and could
⇑
Corresponding author. Tel.: +34 917328000; fax: +34 917328051.
E-mail address: jpastor@cnio.es (J. Pastor).
Present address: Center for Applied Medical Research (CIMA), Small Molecule
Discovery, Avda. Pio XII, 31008 Pamplona, Spain.
à
Johnson & Johnson Pharmaceutical R&D, 2340 Beerse, Belgium.
0
960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.074

S. Martínez González et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1874–1878
1875
O
O
N
N
O
4a
S
N
N
N
HO
HO
N
N
7a
PI-249
PI-103
O
O
N
N
F
F
F
4a
S
N
N
N
HN
N
N
N
N
7a
O
H N
N
N
2
Figure 2. Proposed binding mode of compound 1 in PI3Kc.
SO Me
2
BKM-120
GDC-0941
Figure 1. Chemical structures of PI3K inhibitors.
In order to evaluate the potential of this novel PI3K scaffold, we
synthesized a couple of imidazo[1,2-a]pyrazine probes, com-
pounds 1 and 2 (Table 1), which showed an activity against
Table 1
Inhibition of PI3K
p110
a of 197 and 96 nM, respectively. Both compounds incorpo-
*
a
by imidazo[1,2-a]pyrazine derivatives
rate two fragments, morpholinyl and 3-hydroxyphenyl, that play
2
5,27,30,31
O
an important role in their binding with PI3K.
The proposed binding mode of these compounds in p110
shown in Figure 2. The model was built in p110 based on the high
homology between the and isoforms in the catalytic site, so a
similar binding mode was then expected. The morpholine oxygen
would form a hydrogen bond to the hinge Val882 NH. The phenol
hydroxyl group would make hydrogen bonds with Asp841 and
c
is
N
c
8
N 2
a
c
N
2
R
6
HO
N
3
5
3
R
Tyr867 residues of PI3K
The C –C positions of these structures extend out toward the
so called solvent accessible area in PI3K. The exploration of this re-
c.
Compound
R2
R3
PI3K (p110
IC50
a)
2
3
, lM
1
2
3
4
5
H
Me
Cyclopropyl
CF
CH
H
H
H
H
H
0.197
0.096
0.340
0.108
0.639
2
3
gion was achieved by substitution with different R , R fragments,
including 4-methanesulfonyl-piperazin-1-ylmethyl group, present
in GDC-0941, with reported hydrogen bond interactions to Lys802
3
2
5
2
OH
and Ala805. Selected results of this exploration are shown in
Table 1.
6
7
O
N
H
H
0.444
2.75
Compounds 4 and 9 displayed activity values in PI3K
in the same range than our initial hits (1,2). However, other mod-
ifications at C gave less active inhibitors. Substitution of C with
secondary amines yielded compounds 11–13, with lower inhibi-
tory activity (0.5–1 M).
Among these derivatives, compound 9 showed to be the most
potent inhibitor (IC50 PI3K : 37 nM). However, its in vivo potential
was limited by the presence of the metabolically unstable
-hydroxyphenyl group. In order to overcome this issue, we re-
a better or
N
N
2
3
O
O
8
9
H
H
0.451
0.037
N
O
N
l
S
N
N
a
O
3
1
0
H
0.540
HN
N
placed this fragment by the known isosteric indazole, following a
similar strategy to that applied by the Genentech team for the
2
5
optimization of GDC-0941.
N
1
1
2
H
0.512
0.897
The synthesis of compound 19, within the imidazo[1,2-a]pyra-
zine series is depicted in Scheme 1. Bromination of 2-aminopyr-
azine 14, followed by morpholine introduction gave pyrazine 16.
The condensation with 1,3-dichloroacetone yielded chloride 17,
which reacted with N-mesylpiperazine to give precursor 18.
Finally, Suzuki–Miyaura cross coupling with indazol-4-boronic
acid afforded the target compound.
N
N
1
H
H
HN
O
N
O
S
1
3
0.526
N
Indazole derivative 19 was potent, IC50 PI3K
2.5-fold less active against PI3K than the 3-hydroxyphenyl
counterpart 9. Additionally, it was 30 times less active than the
a: 95 nM, but
*
a
The values are averages of two independent experiments performed in duplicate
with typical variation of less than ± 20%. Assay conditions are described in Ref. 37.
2
5
benchmark GDC-0941 (3.0 nM). When we introduced a bromine
atom in C , the inhibitory activity towards PI3K was improved
-fold (20), meanwhile exploration of C with small groups such
as chlorine and cyano (21,22) produced a slight decrease in
a
potentially afford novel inhibitors. We describe herein the design
and preparation of a novel series of imidazo[1,2-a]pyrazine PI3K
inhibitors.
3
2
5

1
876
S. Martínez González et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1874–1878
O
N
O
N
Br
NH2
N
NH2
a
N
b
c
N
NH2
N
N
N
N
Br
N
N
N
Br
Br
Cl
14
15
16
17
O
N
O
N
N
d
N
N
e
N
HN
N
N
N
Br
N
N
19
18
N
SO Me
2
SO Me
2
i
Scheme 1. Reagents and conditions: (a) Br
2
, Py, CHCl
3 2 3 3
, rt 36%; (b) morpholine, 120 °C, 96%; (c) 1,3-dichloroacetone, PrOH, 60 °C, 38%; (d) N-Ms-piperazine, K CO , CH CN,
.
1
00 °C, 90%; (e) PdCl
2
(dppf) DCM, aq. K
2
CO
3
, indazole-4-boronic acid hydrochloride, DME, 130 °C, 75%.
Table 2
The lower activity of 19 versus GDC-0941 could be explained by
the influence of geometric (thieno-ring versus imidazo-ring) and/
or electronic factors of the imidazo[1,2-a]pyrazine scaffold. The
overlapping of both compounds (Fig. 3), using docking studies
and the active conformation of GDC-0941 from X-ray structural
Inhibition of PI3K
a
and phosphorylation of AKT by imidazo[1,2-a]pyrazine deriva-
*
tives
O
N
data reflects some differences in the orientation of the 4-methane-
N
25,32
N
N
sulfonyl-piperazin-1-yl-methyl side chain.
This fragment in
HN
N
Genentech’s compound is packed against the side chain of
Met804 and the oxygen atoms of the sulfonyl group positioned
in H-bonding distance with the side chain of Lys802 and the amide
nitrogen of Ala805. However, 19 only display potential interactions
with Lys802, which could explain the weaker activity observed for
the imidazopyrazine inhibitor.
As a continuation of the hit to lead exploration, we prepared a
2
series of secondary amide derivatives at the C position. The
synthesis of these imidazo[1,2-a]pyrazines is represented in
Scheme 2 and starts with pyrazine 16, which was refluxed with
ethyl bromopyruvate in a minimum amount of DME to give
compound 23. Suzuki–Miyaura cross coupling of 23 with indazole-
N
3
5
R
R
N
S
O
O
Compond
R3
R5
PI3K(p110
a) IC50
,
lM
pAKT EC50, lM
1
2
2
2
9
0
1
2
H
Br
H
H
H
H
CN
Cl
0.095 ± 0.016
0.047 ± 0.009
0.194 ± 0.018
0.145 ± 0.021
0.093 ± 0.031
0.208 ± 0.072
0.203 ± 0.069
0.495 ± 0.099
*
The values are averages of two independent experiments performed in dupli-
cate ± Standard Deviation. Assay conditions are described in Ref. 37,38.
4
-boronic acid afforded the ester precursor 24 for the final amida-
3
tion reaction, which was carried out under Me Al conditions to
give amides 25 in moderated yields.
Selected results from this exploration are summarized in
Table 3. Compounds with polar groups in the amide side chain
2
5d–h were more potent than compound 19, with IC50 values be-
low 50 nM. More simple amides 25a–c were 2-fold less actives
IC50 ꢀ100 nM). Despite of the good biochemical activity, the cellu-
lar potency was rather disappointing for some of these amides
(
(
25d–f). This could be due to the presence of a basic side chain,
33
which seems to be detrimental for cell membrane permeability.
Nevertheless compounds 25a, 25g, 25h demonstrated a good cellu-
lar activity with values below 200 nM in U2OS cell line for the
inhibition of phosphorylation of S473 residue of AKT.
Selected imidazo[1,2-a]pyrazine inhibitors were evaluated
against the PI3K isoform family and Kiapp data are presented in
Table 4 for the four different isoforms. It is interesting to note that
imidazo [1,2-a] pyrazine scaffold seems to give a more selectivity
behaviour in terms of isoforms profile when compared to the
GDC-0941, reported as modest selective PI3K
compounds showed a slight increase of potency against p110d than
towards p110 , about 2–10 fold, while displaying good levels of
selectivity against p110b and p110 (Kiapp >500 nM). This activity
a
d inhibitor.25 Our
Figure 3. Overlay of GDC-0941(grey) and compound 19 (green) in PI3Kc.
a
c
potency. Modulation of the PI3K pathway with these inhibitors
was confirmed by inhibition of AKT phosphorylation on S473 in
U2OS cell line, displaying compound 19 an EC50 of 93 nM
against PI3Kd isoform can have benefits in some specific type of
cancers as we mentioned previously. In this regard there are two
selective PI3Kd inhibitors in clinical phases, under evaluation for
3
4
(Table 2).
haematological malignancies.

S. Martínez González et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1874–1878
1877
O
N
O
N
O
O
N
O
N
Br
NH2
OEt
b
O
c
O
O
N
O
N
N
N
N
N
N
N
N
a
HN
HN
N
N
N
N
OEt
OEt
N
H
R
Br
Br
16
23
24
25
.
Scheme 2. Reagents and conditions: (a) 1,2-DME, reflux 20 h, 35%; (b) PdCl
Me Al, EtOH, reflux, 6–41%.
2
2 3
(dppf) DCM, aq. K CO , indazole-4-boronic acid hydrochloride, DME, 130 °C, 75%; (c) Amine,
3
Representative PI3K inhibitors were tested against mTOR
mammalian Target of Rapamycin), a member of PI3K class IV,
Table 3
40
(
Inhibition of PI3K
a
and phosphorylation of AKT by imidazo[1,2-a]pyrazine deriva-
displaying high selectivity (IC50 >10.0
l
M, Table 4). GDC-0941
was included in our studies showing an IC50 = 0.42 M for mTOR
M). The use of imi-
*
tives
3
6
l
O
N
25
inhibition (reported in literature Kiapp = 0.58
l
dazo [1,2-a] pyrazine scaffold confers higher selectivity to these
compounds versus mTOR kinase. Compounds were also tested in
N
O
35
N
N
a 24 kinase panel showing a good selectivity profile, with values
HN
under 50% of inhibition at 5–10 lM.
N
N
H
R
In Table 5 is presented preliminary in vitro ADME data gener-
ated for compounds 19, 22, 25g. Our compounds showed minimal
inhibition, less than 25%, of five of the principal cytochrome P450
isoforms, sharing similar results with GDC-0941. Metabolic stabil-
ity was measured in human and mouse liver microsomes with
diverse results depending on the decoration of the compounds.
Compound 19 has a similar behaviour than GDC-0941 (45–54%,
mouse–human, of compound remaining in our assay, and 92% re-
Compound
R
PI3K(p110
a
) IC50
,
lM
pAKT EC50, lM
2
2
2
5a
5b
5c
H
Me
Et
0.107 ± 0.011
0.110 ± 0.022
0.092 ± 0.016
0.138 ± 0.027
0.600 ± 0.187
0.713 ± 0.198
O
2
5d
0.038 ± 0.006
0.452 ± 0.068
N
2
5
ported in literature in different assay conditions). The introduc-
tion of a chlorine atom in C decrease the stability of compound
2, meanwhile the opposite effect was observed with the amide
5g. In addition, there was no significant blockade of the hERG
M) in the Herg binding
N
2
2
5e
5f
0.035 ± 0.008
0.035 ± 0.014
0.429 ± 0.131
0.976 ± 0.201
5
2
2
N
O
2
5g
5h
0.050 ± 0.006
0.031 ± 0.009
0.200 ± 0.037
0.114 ± 0.039
channel for compound 19 (IC50 = 100
l
4
1
2
O
assay.
*
In summary, we have discovered novel PI3K inhibitors by ligand
based rational design. The series of 8-morpholinyl-imidazo[1,2-
The values are averages of two independent experiments performed in dupli-
cate ± Standard Deviation. Assay conditions are described in Ref. 37,38.
a]pyrazines afforded potent compounds against PI3Kd and
a
Table 4
*
mTOR activity and Biochemical selectivity profile
Compound
mTOR IC50
(lM)
p110a
K
iapp (nM)
p110b Kiapp (nM)
p110d Kiapp (nM)
p110c Kiapp (nM)
1
2
2
2
2
2
2
2
9
0
1
2
5b
5d
5e
5h
>10
>10
>10
>10
>10
>10
>10
>10
181 ± 13
86 ± 14
410 ± 50
210 ± 38
102 ± 8
65 ± 5
>500
>500
>500
>500
>500
>500
>500
>500
29 ± 2
8 ± 0.8
43 ± 3
>500
>500
>500
>500
>500
>500
>500
>500
65 ± 4
17.1 ± 1
6.5 ± 0.5
3.6 ± 0.4
2.8 ± 0.3
44 ± 5
60 ± 5
*
Isoforms values in the table correspond to Kiapp (nM) ± Standard Error. Assay conditions are described in Ref. 39,40.
Table 5
ADME profile
Compound
human Metabolic stabilitya (%)
mouse Metabolic stabilitya (%)
CYPs inhibitionb (%)
hERG binding IC50 (lM)
GDC-941
54
48
35
75
45
32
28
62
<25
<25
<25
<25
64
100
1
2
2
9
2
5g
a
Percent remaining after 30 min incubation of 1
l
M compound with 0.7 mg/ml human/mouse liver microsomes. Assays performed at Wuxi in duplicates.
b
Percent of CYPs inhibition at 10
lM test compound. Assays performed at Wuxi in duplicates (CYPs panel: 1A2, 2C9, 2C19, 2D6, 3A4).

1
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S. Martínez González et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1874–1878
isoforms, showing downregulation of inhibition of AKTS473 phos-
phorylation in a U2OS cell line. These compounds show also an im-
proved selectivity vs mTOR kinase. Available data justifies
additional optimization of this chemical series and progress in this
direction shall be reported in due course.
25. Folkes, A. J.; Ahmadi, K.; Alderton, W. K.; Alix, S.; Baker, S. J.; Box, G.;
Chuckowree, I. S.; Clarke, P. A.; Depledge, P.; Eccles, S. A.; Friedman, L. S.; Hayes,
A.; Hancox, T. C.; Kugendradas, A.; Lensun, L.; Moore, P.; Olivero, A. G.; Pang, J.;
Patel, S.; Pergl-wilson, G. H.; Raynaud, F. I.; Robson, A.; Saghir, N.; Salphati, L.;
Sohal, S.; Ultsch, M. H.; Valenti, M.; Wallweber, H. J. A.; Wan, N. C.; Wiesmann,
C.; Workman, P.; Zhyvoloup, A.; Zvelebil, M. J.; Shuttleworth, S. J. J. Med. Chem.
2008, 51, 5522.
2
6. Link, W.; Oyarzabal, J.; Serelde, B. G.; Albarrán, M. I.; Rabal, O.; Cebria, A.;
Alfonso, P.; Fominaya, J.; Renner, O.; Peregrino, S.; Soilán, D.; Ceballos, P. A.;
Hernández, A. I.; Lorenzo, M.; Pevarello, P.; Granda, T. G.; Kurz, G.; Carnero, A.;
Bischoff, J. R. J. Biol. Chem. 2009, 284, 28392.
Acknowledgments
This research was supported by the Ministry of Science and
Innovation (MICINN) of Spain through the project CIT-090100-
2
2
2
7. Hayakawa, M.; Kaizawa, H.; Moritomo, H.; Koizumi, T.; Ohishi, T.; Okada, M.;
Ohta, M.; Tsukamoto, S.; Parker, P.; Workman, P.; Waterfield, M. Bioorg. Med.
Chem. 2006, 14, 6847.
8. Hayakawa, M.; Kaizawa, H.; Moritomo, H.; Koizumi, T.; Ohishi, T.; Yamano, M.;
Okada, M.; Ohta, M.; Tsukamoto, S.; Raynaud, F. I.; Workman, P.; Waterfield, M.
D.; Parker, P. Bioorg. Med. Chem. Lett. 2007, 17, 2438.
2
007-48.
We thank Elena Cendón and Antonio Salgado for compound
purification and characterization. We also thank Isabel Reymundo
and Genoveva Mateos for compound handling and sample prepara-
tion and Manuel Urbano for data management.
9. As far as we know, 93 patents reported PI3K inhibitors with ‘mopholinyl
pyrimidine’ core.
3
3
0. Walker, E. H.; Pacold, M. E.; Perisic, O. Mol. Cell 2000, 6, 909.
1. Knight, Z. A.; Chiang, G. G.; Alaimo, P. G.; Kenski, D. M.; Ho, C. B.; Coan, K.;
Abraham, R. T.; Shokat, K. M. Bioorg. Med. Chem. 2004, 12, 4749.
2. For docking purposes, the protein used was obtained from the crystal of the
PI3K gamma in complex with GDC-0941 (3DBS). Program used was GOLD3.1
from Open Eye Scientific Software.
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3
3
1
.
Leevers, S. J.; Vanhaesebroeck, B.; Waterfield, M. D. Curr. Opin. Cell Biol. 1999,
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1
3. Permeability of some compounds was measured in MDCK cells. Results
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2
3
.
.
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(
Paap <1) than compounds 25a, 25b, 25g with Papp >3.5, where Papp denotes
the apparent permeability coefficient.
5
35.
3
3
4. Norman, P. Expert Opin. Ther. Patents. 2011, 21, 1773.
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4.
5.
6.
7.
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6. GDC-0941 was purchased to Chemexpress (MFCD11616196).
3
3
7. The PI3Ka activity was measured by using the commercial ADP HunterTM Plus
8
9
.
.
Fruman, D. A.; Meyers, R. E.; Cantley, L. C. Annu. Rev. Biochem. 1998, 67, 481.
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assay available from DiscoveRx, homogeneous assay to measure the
accumulation of ADP, a universal product of kinase activity. The enzyme,
1
0. Katso, R.; Okkenhaug, K.; Ahmadi, K., et al Annu. Rev. Cell Dev. Biol. 2001, 17,
15.
1. Wymann, M. P.; Zvelebil, M.; Laffargue, M. Trends Pharmacol. Sci. 2003, 24,
66.
PI3K (p110
done following the manufacturer recommendation with slight modifications in
the kinase buffer (50 mM HEPES, pH 7.5, 3 mM MgCl , 100 mM NaCl, 1 mM
EGTA, 0.04% CHAPS, 2 mM TCEP and 0.01 mg/ml BGG), and working at 10 nM
PI3K (p110 /p85 ) and at 50 M of ATP concentration. Values were plotted
a/p85a) was purchased from Carna Biosciences and the assay was
6
1
2
3
12. Liu, P.; Cheng, H.; Roberts, T. M.; Zhao, J. J. Nat. Rev. Drug Disc. 2009, 8, 627.
13. Ali, I. U.; Schriml, L. M.; Dean, M. J. Natl. Cancer Inst. 1922, 1999, 91.
14. Cross, D. A.; Alessi, D. R.; Cohen, P., et al Nature 1995, 378, 785.
15. Samuels, Y.; Wang, Z.; Bardelli, A.; Silliman, N.; Ptak, J.; Szabo, S.; Yan, H.;
Gazdar, A.; Powell, S. M.; Riggins, G. J.; Willson, J. K.; Markowitz, S.; Kinzler, K.
W.; Vogelstein, B.; Velculescu, V. E. Science 2004, 304, 554; Leslie, N. R.;
Downes, C. B. Biochem. J. 2004, 382, 1.
a
a
a
l
against the inhibitor concentration and fitted to a sigmoid dose-response curve
by using GraphPad Prism version 5.03 (GraphPad Software CA, USA). Values
given are averages of two independent experiments performed in duplicate.
8. Cellular activity was measured as endogenous levels of phospho-Akt1 (Ser473)
protein after serum stimulation in U2OS (osteosarcoma) cells growing in 0.1 %
of FBS. Assay was run under C-Elisa format (Reagent: Supersignal Elisa Femto,
purchased from Pierce). Values were plotted against the inhibitor
concentration and fitted to a sigmoid dose-response curve using GraphPad
Software.
3
3
1
1
1
6. Shayesteh, L.; Lu, Y.; Kuo, W.-L.; Baldocchi, R.; Godfrey, T.; Collins, C.; Pinkel, D.;
Powell, B.; Mills, G. B.; Gray, J. W. Nat. Genet. 1999, 21, 99.
7. Ma, Y.-Y.; Wei, S.-J.; Lin, Y.-C.; Lung, J.-C.; Chang, T.-C.; Whang-Peng, J.; Liu, J.
M.; Yang, D.-M.; Yang, W. K.; Schen, C.-Y. Oncogene 2000, 19, 2739.
8. Parsons, D. W.; Wang, T.-L.; Samuels, Y.; Bardelli, A.; Cummins, J. M.; DeLong,
L.; Silliman, N.; Ptak, J.; Szabo, S.; Willson, J. K.; Markowitz, S.; Kinzler, K. W.;
Wogelstein, B.; Lengauer, C.; Velculescu, V. E. Nature 2005, 436, 792.
9. Campbell, I. G.; Russell, S. E.; Choong, D. Y.; Montgomery, K. G.; Ciavarella, M.
L.; Hooi, C. S.; Cristiano, B. E.; Pearson, R. B.; Phillips, W. A. Cancer Res. 2004, 64,
9. The kinase activity of PI3K isoforms was measured by using the commercial
PI3-kinase (h) HTRF™ assay available from Millipore, following the
manufacturer recommendations. PI3K
p85 were used at 100 pM; PI3Kb (p110b/p85
p110 ) at 500 pM and 4 nM respectively. ATP concentration was 50 times
KMATP: 200 M for PI3K and PI3Kd, 250 M for PI3Kb and 100 M for PI3K
PIP2 was held at 10 M. Values were normalized against the control activity
a
(p110
a
/p85
a
)
and PI3Kd (p110d/
a)
a)
and PI3K isoforms
c
1
(
c
l
a
l
l
c.
7
678.
l
2
2
0. Kang, S.; Bader, A. G.; Vogt, P. K. Proc. Natl. Sci. U.S.A. 2005, 102, 802.
1. Jia, S.; Liu, Z.; Zhang, S.; Liu, P.; Zhang, L.; Lee, S. H.; Zhang, J.; Signoretti, S.;
Loda, M.; Roberts, T. M.; Zhao, J. J. Nature 2008, 454, 776.
included for each enzyme (i.e, 100 % PI3K activity, without compound). These
values were plotted against the inhibitor concentration and were fitted to a
sigmoidal dose-response (variable slope) curve by using GraphPad Software.
The obtained IC50 were converted to Kiapp according to Cheng-Prusoff equation
for competitive inhibitors (Cheng, Y.; Prussoff, W.H. Biochem. Pharmacol. 1973,
2
2. Kok, K.; Nock, G. E.; Verrall, E. A.; Mitchell, M. P.; Hommes, D. W.;
Peppenlenbosch, M. P.; Vanhaesebroeck, B. PLoS One 2009, 4, e5145.
3. Sujobert, P.; Bardet, V.; Cornillet-Lefebvre, P.; Hayflick, J. S.; Prie, N.; Verdier, F.;
Vanhaesebroeck, B.; Muller, O.; Pesce, F.; Ifrah, N.; Hunault-Berger, M. Blood
2
22, 3099).
4
4
0. MTOR (FRAP1), LanthaScreenTM Tb-anti-p4EBP1 (phosphor-threonine 46) and
GFP-4E BP1 were purchased from Invitrogen. Reaction conditions used were
those recommended by the manufacturer. Values given are averages of two
independent experiments performed in duplicate.
1. hERG membrane radioligand binding assay. CHO-K1 cell line stably expressing
hERG channel, radioligand: [3H]-astemizole. Assays performed at Wuxi in
duplicates.
2
005, 106, 1063.
2
4. Burger, M. T.; Pecchi, S.; Wagman, A.; Ni, Z.; Knapp, M.; Hendrickson, T.;
Atallah, G.; Pfister, K.; Zhang, Y.; Bartulis, S.; Frazier, K.; Ng, S.; Smith, A.;
Verhagen, J.; Haznedar, J.; Huh, K.; Iwanowicz, E.; Xin, X.; Menezes, D.; Merritt,
H.; Lee, I.; Wiesmann, M.; Kaufman, S.; Crawford, K.; Chin, M.; Bussiere, D.;
Shoemaker, K.; Zaror, I.; Maira, S.; Voliva, C. F. ACS Med. Chem. Lett. 2011, 2(10),
7
74.