Phosphoinositide-3-kinase inhibitors: Evaluation of substituted alcohols as replacements for the piperazine sulfonamide portion of AMG 511

Article abstract of DOI:10.1016/j.bmcl.2014.10.085

Replacement of the piperazine sulfonamide portion of the PI3Kα inhibitor AMG 511 (1) with a range of aliphatic alcohols led to the identification of a truncated gem-dimethylbenzylic alcohol analog, 2-(5-(4-amino-6-methyl-1,3,5-triazin-2-yl)-6-((5-fluoro-6-methoxypyridin-3-yl)amino)pyridin-3-yl)propan-2-ol (7). This compound possessed good in vitro efficacy and pharmacokinetic parameters and demonstrated an EC₅₀ of 239 ng/mL in a mouse liver pharmacodynamic model measuring the inhibition of hepatocyte growth factor (HGF)-induced Akt Ser473 phosphorylation in CD1 nude mice 6 h post-oral dosing.

Full text of DOI:10.1016/j.bmcl.2014.10.085

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5630–5634
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Phosphoinositide-3-kinase inhibitors: Evaluation of substituted
alcohols as replacements for the piperazine sulfonamide portion
of AMG 511
a
a
a
a
a
Brian A. Lanman a, , Anthony B. Reed , Victor J. Cee , Fang-Tsao Hong , Liping H. Pettus , Ryan P. Wurz ,
Kristin L. Andrews b, Jian Jiang c, John D. McCarter d, Erin L. Mullady g, Tisha San Miguel d,
Raju Subramanian c, Ling Wang e, Douglas A. Whittington h, Tian Wu f, Leeanne Zalameda d,
Nancy Zhang e, Andrew S. Tasker a, Paul E. Hughes e, Mark H. Norman a
⇑
a Department of Medicinal Chemistry, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, United States
b Department of Molecular Structure, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, United States
c Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, United States
d Department of High-Throughput Screening/Molecular Pharmacology, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, United States
e Department of Oncology Research, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, United States
f Department of Pharmaceutics, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, United States
g Department of High-Throughput Screening/Molecular Pharmacology, Amgen Inc., 360 Binney Street, Cambridge, MA 02142, United States
h Department of Molecular Structure, Amgen Inc., 360 Binney Street, Cambridge, MA 02142, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Replacement of the piperazine sulfonamide portion of the PI3Ka inhibitor AMG 511 (1) with a range of
Received 26 August 2014
Revised 24 October 2014
Accepted 27 October 2014
Available online 1 November 2014
aliphatic alcohols led to the identification of a truncated gem-dimethylbenzylic alcohol analog, 2-(5-(4-
amino-6-methyl-1,3,5-triazin-2-yl)-6-((5-fluoro-6-methoxypyridin-3-yl)amino)pyridin-3-yl)propan-2-
ol (7). This compound possessed good in vitro efficacy and pharmacokinetic parameters and demonstrated
an EC50 of 239 ng/mL in a mouse liver pharmacodynamic model measuring the inhibition of hepatocyte
growth factor (HGF)-induced Akt Ser473 phosphorylation in CD1 nude mice 6 h post-oral dosing.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Phosphoinositide-3-kinase-alpha
Kinase inhibitor
Protein kinase B
Ribose pocket
Ligand efficiency
Phosphatidylinositol-3-kinases (PI3Ks) are intracellular lipid
kinases that catalyze the phosphorylation of the eponymous
inositol-containing lipids to generate intracellular secondary mes-
sengers (e.g., phosphatidylinositol (3,4,5)-trisphosphate (PIP3)) in
response to external stimulation, such as receptor tyrosine kinase
activation in response to growth factor binding.1 Extensive
research over the past decade has revealed the PI3K signaling
pathway to be a key regulator of cellular survival, growth, and
proliferation.2 Mutations in components of the PI3K signaling
pathway have been estimated to be associated with up to 30% of
all human malignancies.2,3 As a result, significant effort has been
directed toward the discovery and development of PI3K inhibitors
as potential cancer therapeutics.4
Class I PI3Ks—defined both by their dimeric p110/p85 catalytic/
regulatory subunit structure and phosphatidylinositol (4,5)-
bisphosphate substrate specificity—in particular, have been the focus
of these efforts, as these PI3Ks are the most strongly implicated in
cancer;5 aberrant activation of all four p110 isoforms (
a, b, c, and
d) has demonstrated oncogenic potential in in vitro studies.6 As
in vitro studies have also demonstrated the functional redundancy
of these p110 isoforms,7 our efforts in this area have focused on the
identification of pan-isoform class I PI3K inhibitors.
In a prior publication,8 we reported the discovery of AMG 511
(1; Fig. 1), a biaryl aminotriazine inhibitor which demonstrated
excellent pharmacodynamic and pharmacokinetic effects in
murine animal models. We have also previously detailed SAR
investigations leading to the identification of the methoxypyridine
and piperazinyl sulfonamide fragments of 1.8,9 In the present
communication, we expand on these studies, providing the results
⇑
Corresponding author. Tel.: +1 805 313 5777; fax: +1 805 480 1337.
E-mail address: blanman@amgen.com (B.A. Lanman).
http://dx.doi.org/10.1016/j.bmcl.2014.10.085
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

B. A. Lanman et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5630–5634
5631
Table 1
F
PI3Ka, mTOR, and pAkt activity of inhibitors possessing aliphatic alcohol ribose-
pocket binding groupsa
H3CO
N
N
N
F
N
N
N
H
S
5
H3CO
R
O O
N
N
N
N
N
H
N
NH2
N
Figure 1. AMG 511 (1).
N
NH2
Compd
R
PI3K
Ki
a
mTOR
IC50
U87 pAkt HLM/RLM
IC50 (lM) CLint (lL/
(l
M)
(l
M)
min/mg)
1
N
0.004
14.8
0.004
22/20
NMs
H
2
3
0.048
0.056
>22
>15
0.042
0.044
99/36
–/51
OH
OH
OH
4b
5b
0.040
0.048
>25
>25
0.020
0.026
22/45
<14/<14
CF3
( )
6
7
8
0.030
0.023
0.016
2.2
0.034
0.013
0.018
18/34
<14/23
39/79
OH
7.8
3.7
OH
OH
OH
Figure 2. Overlay of X-ray co-crystal structures of AMG 511 (1; blue) and ATP
(pink) bound to PI3Kc 11
Hydrogen-bonding contacts to K802 and A805 are
indicated in black.
.
9b
0.040
0.026
0.040
>15
0.031
0.025
0.037
<14/37
14/36
34/34
of SAR investigations aimed at addressing potential shortcomings
of the piperazine sulfonamide portion of 1.
Figure 2 depicts an overlay of the X-ray co-crystal structures of
AMG 511 and ATP bound to PI3Kc. The piperazine sulfonamide
10b
11
11.1
>17
OH
OH
portion of 1 projects out of the ATP binding site in the ribose-bind-
ing region (‘ribose pocket’) and occupies a lipophilic groove formed
by the bottom face of the P-loop, with the sulfonamide oxygens
a
Data represent an average of at least two determinations. ‘>[Highest concen-
tration tested]’ is reported for compounds that failed to inhibit >10% of control
activity in one or more replicate measurements. A statistical analysis of this data
may be found in the Supplementary material.
engaging in hydrogen-bonds with Lys802 (x-amine) and Ala805
(backbone NH). A comparison of 1 with 2, a compound lacking
the ethylpiperazine sulfonamide fragment (Table 1), shows that
this moiety provides a 10-fold improvement in enzymatic and
cellular potency as well as a significant reduction in oxidative
metabolism (as measured by intrinsic microsomal clearance
(CLint)). Despite these improvements to the core scaffold 2, we con-
tinued to search for alternatives to the piperazine sulfonamide for
several reasons: this group added significantly to both molecular
weight (327 Da for 2 vs 518 Da for 1) and to an already high topo-
logical polar surface area (tPSA; 109 Å2 for 2 and 150 Å2 for 1).10
Additionally, metabolite ID studies using human and rat hepato-
cytes revealed this group to be the major site of metabolism of 1,
with oxidative metabolism of this group accounting for ꢀ70% of
all metabolism in rat hepatocytes and >95% of all metabolism in
human hepatocytes.
b
Absolute stereochemistry not determined.
LanthaScreen™ FRET assay15 was used to monitor the effect of
ribose pocket modifications on inhibition of the structurally
related kinase, mTOR, to determine whether new analogs retained
the PI3K-selectivity of compound 1. Functional activity was
subsequently confirmed by measuring the inhibition of Akt S473
phosphorylation in U87 MG cells.16
As seen in Table 1, introducing a hydroxymethyl group into ana-
log 2 failed to improve PI3K
Ki = 56 nM), as did further -methylation or
of the hydroxymethyl substituent (compounds 4–6). gem-Dimethy-
lation of the hydroxymethyl fragment, however, resulted in 2–3-
a
inhibitory activity (compound 3;
-trifluoromethylation
a
a
fold enhancements in both PI3K
a activity and U87 pAkt IC50
In our efforts to identify an alternative to the piperazine
sulfonamide portion of 1, we chose to focus on small aliphatic sub-
stituents bearing an alcohol group. In so doing, we aimed to mimic
the hydroxylic functionality present in the ribose portion of the
endogenous ligand and, thereby, preserve favorable interactions
with loosely ordered water molecules along the solvent-exposed
face of the ATP binding site. Initial results from these investigations
are found in Table 1.12 New analogs were initially assayed for PI3K
(compound 7). Compound 7 additionally demonstrated signifi-
cantly enhanced microsomal stability relative to the unsubstituted
analog, 2. Although replacement of the piperazine sulfonamide por-
tion of 1 with the 2-hydroxypropyl fragment also led to slightly
enhanced inhibition of mTOR, compound 7 remained highly PI3K-
selective, with greater than 300-fold selectivity for PI3Ka. The
cyclopropyl analog of 7, compound 8, preserved the properties of
the gem-dimethyl analog, retaining potency in both enzymatic
activity against PI3Ka A
using a modified AlphaScreenÒ assay.13,14

5632
B. A. Lanman et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5630–5634
Table 2
PI3Ka
, mTOR, and pAkt activity of inhibitors bearing varied affinity pocket substituentsa
N
OH
R
N
H
N
N
N
NH2
Compd
R
PI3K
a
Ki (l
M)
mTOR IC50
(l
M)
U87 pAkt IC50
0.013
(lM)
HLM/RLM CLint
<14/23
(lL/min/mg)
F3
2
H3CO
7
0.023
0.089
0.087
7.8
N
H3CO
F
12
13
0.79
2.5
0.048
0.097
<14/19
<14/15
N
N
H3CO
14
15
0.118
0.090
7.8
0.049
0.146
<14/ 22
n.a.
N
N
CF3
H3CO
H3CO
H3CO
>25
16
17
0.469b
0.153
15.8b
0.470
0.414
n.a.
n.a.
N
>25
N
SO2CH3
SO2Ph
18
19
0.801
0.224
>16
1.16
<14/<14
n.a.
N
N
6.7
0.766
OCH3
H3CO
20
21
0.030
0.089
10.6
2.1
0.010
0.071
<14/<14
17/19
N
OCH3
N
a
Data represent an average of at least two determinations, unless otherwise noted. ‘>[Highest concentration tested]’ is reported for compounds that failed to inhibit >10%
of control activity in one or more replicate measurements. A statistical analysis of this data may be found in the Supplementary material.
b
Data from a single measurement.
(Ki = 16 nM) and cellular assays (IC50 = 18 nM), while again
demonstrating modestly enhanced mTOR activity (IC50 = 3.7 M).
Compound 8, however, demonstrated significantly reduced
hydroxyethyl analogs 4 and 5 (i.e., compounds 9 and 10) generally
l
mirrored the activity of their non-homologated counterparts.
Homologation of hydroxypropyl analog 7, however, led to reduced
activity in both enzymatic (Ki = 40 nM) and cellular assays
microsomal stability relative to 7. Homologated versions of the

B. A. Lanman et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5630–5634
5633
pocket known as the ‘affinity pocket,’17 with the pyridyl nitrogen
engaging in a hydrogen bond with an ordered water molecule
bridging Asp841 and Tyr867, and the methoxy substituent engag-
ing in a polar interaction with Lys833. Our prior studies revealed
the C2 and C3 positions of the pyridyl ring to be the most tolerant
of modification without loss of enzymatic activity; accordingly, we
targeted these two positions for modification in an effort to
Table 3
Pharmacokinetic parameters for 1, 7, and 20a
Compd
CL (L/h/kg)
Vss (L/kg)
t1/2 (h)
%F
Fu
Efflux ratiob
1
7
20
0.45
1.1
1.3
1.7
1.7
4.2
3.3
3.1
4.8
57
37
n.a.
0.08
0.06
n.a.
9.7
2.4
25
a
Male Sprague-Dawley rat (iv: 1 mg/kg, DMSO, n = 3; p.o.: 2 mg/kg, 1% Pluronic
F68, 2% HPMC, 15% HPbCD, pH = 2.2, n = 3).
Measured in LLC-PK1 cells stably over-expressing rat P-gp. 5
0.1% BSA.
enhance the PI3Ka activity of 7. Results of these investigations
b
l
M compound,
are detailed in Table 2.
Removal of either the 3-fluoro or 2-methoxy substituent from
compound 7 (compounds 12, Ki = 89 nM, and 13, Ki = 87 nM,
respectively), led to nearly 4-fold decreases in PI3Ka activity. Like-
40000
35000
30000
25000
900
800
700
600
500
400
300
200
100
0
wise, replacement of the 3-fluoro substituent with more sterically
demanding substituents (methyl, trifluoromethyl, ethyl, or isopro-
pyl; compounds 14–17) or bulky polar substituents such as meth-
ylsulfone (18) or phenylsulfone (19) produced analogs with
reduced PI3Ka activity or pAkt IC50 values. A 3-methoxy group
*
20000
15000
10000
5000
0
was tolerated as a replacement for the 3-fluoro substituent (com-
pound 20; Ki = 30 nM); however, as in the case of compound 7,
retention of the 2-methoxy group was required to maintain potent
enzymatic activity (cf., compound 21).
**
The pharmacokinetic properties of two of the most promising
analogs to emerge from these studies (compounds 7 and 20) were
subsequently investigated by iv and p.o. dosing in male Sprague-
Dawley rats (Table 3). Compound 7 demonstrated a similar phar-
macokinetic profile to that of AMG 511 (1). Oral bioavailability
was slightly reduced (37% vs 57%) and clearance elevated (1.1 vs
0.45 L/h/kg); however, volume of distribution, terminal half-life,
and plasma protein binding remained nearly unchanged. Com-
pound 20, in contrast, demonstrated a slightly extended terminal
half-life (4.8 h), with increased clearance (1.3 L/h/kg) offset by an
increased volume of distribution (4.2 L/kg). Compound 20 addi-
tionally proved to be a fairly strong P-gp efflux substrate.
On the basis of these results, compound 7 was further evaluated
in a mouse liver pharmacodynamic model that measured the
inhibition of hepatocyte growth factor (HGF)-induced Akt
phosphorylation at Ser473 in female CD1 nude mice. In this study,
compound 7 was dosed orally at 3, 10, and 30 mg/kg. After 6 h,
mice were administered HGF to activate PI3K-dependent Akt phos-
phorylation in the liver, and five minutes later, phosphorylated
(Ser473) Akt levels were determined by an electrochemical
luminescence immunoassay. As depicted in Figure 3, compound
Vehicle Vehicle 3 mg/kg 10 mg/kg 30 mg/kg
+HGF
Figure 3. Mouse liver pharmacodynamic assay. Orally dosed
7 reduced liver
p(S473)Akt levels in female CD1 nude mice 6 h post-dose. (N = 3/group; bars
represent average pAkt/total Akt ratio + SE; circles represent average plasma
concentration SE; ⁄P <0.05 versus vehicle + HGF by ANOVA/Dunnett’s Multiple
Comparison Test; ⁄⁄P <0.0001 versus vehicle + HGF by ANOVA/Dunnett’s Multiple
Comparison Test; vehicle = 1% Pluronic F68, 2% HPMC, 15% HPbCD, pH = 2.2.)
(IC50 = 37 nM), as well as in the mTOR FRET assay (IC50 >17 lM) (cf.,
compound 11).
With an initial screen of small, hydroxylated aliphatic substitu-
ents having revealed the isopropoxy group of analog 7 to be the
most promising candidate for a piperazine sulfonamide replace-
ment, we next turned our attention to re-optimization of the 2-
methoxy-3-fluoropyridyl fragment of 7 to exploit any alteration
in the pyridine binding mode brought about by changes to the
ribose pocket moiety. As we have detailed in prior publications,8,9
the methoxypyridyl fragment projects into a generally lipophilic
Cl
N
N
N
OEt
Cl
Cl
N
a
b
F
F
N
N
+
F
N
N
O
N
NH2
N
B(OH)2
Bu3Sn OEt
N
NH2
N
NH2
22
23
25
24
26
F
F
H3CO
H3CO
N
N
N
OH
c
d
N
N
N
H
N
H
F
N
N
N
H3CO
N
N
NH2
N
NH2
NH2
27
28
7
Scheme 1. Synthesis of 7. Reagents and conditions: (a) PdCl2(AmPhos)2, KOAc, dioxane/water, 100 °C, 38%; (b) Pd(OAc)2, XPhos, CsF, dioxane/DMF, 110 °C, 99%; (c) NaHMDS,
THF, 0 °C, 56%; (d) MeMgBr, THF, 0 °C, 34%.

5634
B. A. Lanman et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5630–5634
7 significantly suppressed PI3K signaling at 10 and 30 mg/kg,
bringing about dose-dependent decrease in p(S473)Akt.
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmcl.2014.10.085.
a
A
nonlinear regression analysis established an EC50 of 239 ng/mL,
comparable to that obtained from compound 1 (EC50 = 240 ng/
mL) in a prior study.8 Separate in vitro studies also confirmed com-
pound 7, like compound 1, to be a pan-isoform PI3K inhibitor,
References and notes
1. Cantley, L. C. Science 2002, 296, 1655.
exhibiting single-digit nanomolar Ki values for the b-,
d-isoforms of PI3K.
c-, and
2. Engelman, J. A.; Luo, J.; Cantley, L. C. Nat. Rev. Genet. 2006, 7, 606.
3. Shaw, R. J.; Cantley, L. C. Nature 2006, 441, 424.
4. Rodon, J.; Dienstmann, R.; Serra, V.; Tabernero, J. Nat. Rev. Clin. Oncol. 2013, 10,
143.
5. Vanhaesebroeck, B.; Guillermet-Guibert, J.; Graupera, M.; Bilanges, B. Nat. Rev.
Mol. Cell Biol. 2010, 11, 329.
6. Kang, S.; Denley, A.; Vanhaesebroeck, B.; Vogt, P. K. Proc. Natl. Acad. Sci. U.S.A.
2006, 103, 1289.
The synthesis of compound 7 is depicted in Scheme 1 and
commenced with the Suzuki coupling of chlorotriazine 229 and
commercial fluoropyridine boronic acid 23 to provide the biaryl
triazine 24 in moderate yield. Stille coupling of 24 with tribu-
tyl(1-ethoxyvinyl)tin (25) subsequently furnished enol ether 26
in excellent yield. The affinity pocket fragment (27) was introduced
by nucleophilic substitution of the 2-fluoro group of 26 using NaH-
MDS as a promoter, providing acetylpyridine 28 in 56% yield
following hydrolysis of the enol ether during chromatographic
purification. Compound 7 was subsequently obtained from 28 by
the addition of methyl Grignard.
As revealed in these studies, replacement of the piperazine
sulfonamide portion of AMG 511 (1) with a 2-hydroxypropyl group
provided a compound (7) that retained the favorable pharmacoki-
netic properties of AMG 511 and exhibited similar in vivo efficacy
(EC50 = 239 ng/mL vs 240 ng/mL) in a mouse liver pharmacody-
namic assay. Although compound 7 was unable to fully replicate
the superb enzymatic and cellular potency of 1, the activity of
the significantly truncated analog (7; 385 Da) relative to AMG
511 (1; 518 Da) highlights both the efficiency of the 2-hydroxypro-
pyl group as a ribose pocket surrogate as well as the unique
effectiveness of the piperazinyl sulfonamide group in the context
of the triazinyl pyridine scaffold.18
7. Chaussade, C.; Rewcastle, G. W.; Kendall, J. D.; Denny, W. A.; Cho, K.; Grønning,
L. M.; Chong, M. L.; Anagnostou, S. H.; Jackson, S. P.; Daniele, N.; Shepherd, P. R.
Biochem. J. 2007, 404, 449.
8. Norman, M. H.; Andrews, K. L.; Bo, Y. Y.; Booker, S. K.; Caenepeel, S.; Cee, V. J.;
D’Angelo, N. D.; Freeman, D. J.; Herberich, B. J.; Hong, F.-T.; Jackson, C. L. M.;
Jiang, J.; Lanman, B. A.; Liu, L.; McCarter, J. D.; Mullady, E. L.; Nishimura, N.;
Pettus, L. H.; Reed, A. B.; San Miguel, T.; Smith, A. L.; Stec, M. M.; Tadesse, S.;
Tasker, A.; Aidasani, D.; Zhu, X.; Subramanian, R.; Tamayo, N. A.; Wang, L.;
Whittington, D. A.; Wu, B.; Wu, T.; Wurz, R. P.; Yang, K.; Zalameda, L.; Zhang,
N.; Hughes, P. E. J. Med. Chem. 2012, 55, 7796.
9. Smith, A. L.; D’Angelo, N. D.; Bo, Y. Y.; Booker, S. K.; Cee, V. J.; Herberich, B.;
Hong, F.-T.; Jackson, C. L. M.; Lanman, B. A.; Liu, L.; Nishimura, N.; Pettus, L. H.;
Reed, A. B.; Tadesse, S.; Tamayo, N. A.; Wurz, R. P.; Yang, K.; Andrews, K. L.;
Whittington, D. A.; McCarter, J. D.; San Miguel, T.; Zalameda, L.; Jiang, J.;
Subramanian, R.; Mullady, E. L.; Caenepeel, S.; Freeman, D. J.; Wang, L.; Zhang,
N.; Wu, T.; Hughes, P. E.; Norman, M. H. J. Med. Chem. 2012, 55, 5188.
10. High compound molecular weight (>500 Da) and PSA (>140 Å2) have been
correlated with reduced oral bioavailability (and membrane permeability, in
the case of PSA): see Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.
Adv. Drug Delivery Rev. 1997, 23, 3 (MW) and Veber, D. F.; Johnson, S. R.; Cheng,
H. -Y.; Smith, B. R.; Ward, K. W.; Kopple, K. D. J. Med. Chem. 2002, 45, 2615
(PSA), and references therein.
11. PDB accession codes: 4FLH (AMG 511); 1E8X (ATP).
12. Detailed synthetic procedures for new analogs may be found in the
Supplementary material.
13. PerkinElmer Inc., 940 Winter Street, Waltham, MA 02451. See Supplementary
material for details.
14. Prior experience with the triazinyl pyridine scaffold (see Refs. 8 and 9) revealed
Acknowledgments
minimal selectivity between p110 isoforms (<10Â), and p110
a activity was
Thanks to Paul Andrews for assistance with in vitro cellular
assays, Sean Caenepeel for assay development, Ronya Primack for
performing rat PK studies, and Jin Tang and Peter Yakowec for
deemed most relevant clinically, given this isoform’s association with human
cancer (see Ref. 5).
15. Life Technologies, 3175 Staley Road, Grand Island, NY 14072. See
Supplementary material for details.
the expression and purification of PI3Kc. We also thank Daniel J.
16. U87 MG is
a PTEN-null human primary glioblastoma cell line, which
Freeman for input on in vivo pharmacology studies, Divesh
Aidasani and Xiaochun Zhu for metabolite ID studies, and Wes
Barnhart, Samuel Thomas, Fang Xia, Zheng Hua, and Kyung Gahm
for support in compound purification.
demonstrates aberrant Akt inactivation in response to PI3K signaling. See
Supplementary material for assay details.
17. Knight, Z. A.; Gonzalez, B.; Feldman, M. E.; Zunder, E. R.; Goldenberg, D. D.;
Williams, O.; Loewith, R.; Stokoe, D.; Balla, A.; Toth, B.; Balla, T.; Weiss, W. A.;
Williams, R. L.; Shokat, K. M. Cell 2006, 125, 733.
18. It is notable that parallel efforts to identify a replacement for the piperazine
sulfonamide group of GDC-0941 likewise concluded in the identification of the
2-hydroxypropyl fragment: Sutherlin, D. P.; Sampath, D.; Berry, M.; Castanedo,
G.; Chang, Z.; Chuckowree, I.; Dotson, J.; Folkes, A.; Friedman, L.; Goldsmith, R.;
Heffron, T.; Lee, L.; Lesnick, J.; Lewis, C.; Mathieu, S.; Nonomiya, J.; Olivero, A.;
Pang, J.; Prior, W. W.; Salphati, L.; Sideris, S.; Tian, Q.; Tsui, V.; Wan, N. C.;
Wang, S.; Wiesmann, C.; Wong, S.; Zhu, B.-Y. J. Med. Chem. 2010, 1086, 53.
Supplementary data
Supplementary data (statistical analysis of data presented in
Tables 1 and 2, synthetic routes for the preparation of compounds
2–21, and details of in vitro and in vivo assays) associated with this