The imidazo[1,2-a]pyridine ring system as a scaffold for potent dual phosphoinositide-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.08.016

Based on lead compound 1, which was discovered from a high-throughput screen, a series of PI3Kα/mTOR inhibitors were evaluated that contained an imidazo[1,2-a]pyridine as a core replacement for the benzimidazole contained in 1. By exploring various ring systems that occupy the affinity pocket, two fragments containing a methoxypyridine were identified that gave <100 nM potency toward PI3Kα in enzyme and cellular assays with moderate stability in rat and human liver microsomes. With the two methoxypyridine groups selected to occupy the affinity pocket, analogs were prepared with various fragments intended to occupy the ribose pocket of PI3Kα and mTOR. From these analogs, tertiary alcohol 18 was chosen for in vivo pharmacodynamic evaluation based on its potency in the PI3Kα cellular assay, microsomal stability, and in vivo pharmacokinetic properties. In a mouse liver pharmacodynamic assay, compound 18 showed 56% inhibition of HFG-induced AKT (Ser473) phosphorylation at a 30 mg/kg dose.

Full text of DOI:10.1016/j.bmcl.2015.08.016

Accepted Manuscript
The imidazo[1,2-a]pyridine ring system as a scaffold for potent dual phosphoi-
nositide-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitors
Markian M. Stec, Kristin L. Andrews, Yunxin Bo, Sean Caenepeel, Hongyu
Liao, John McCarter, Erin L. Mullady, Tisha San Miguel, Raju Subramanian,
Nuria Tamayo, Douglas A. Whittington, Ling Wang, Tian Wu, Leeanne P.
Zalameda, Nancy Zhang, Paul E. Hughes, Mark H. Norman
PII:
S0960-894X(15)00840-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.08.016
BMCL 23010
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 June 2015
2 August 2015
6 August 2015
Please cite this article as: Stec, M.M., Andrews, K.L., Bo, Y., Caenepeel, S., Liao, H., McCarter, J., Mullady, E.L.,
Miguel, T.S., Subramanian, R., Tamayo, N., Whittington, D.A., Wang, L., Wu, T., Zalameda, L.P., Zhang, N.,
Hughes, P.E., Norman, M.H., The imidazo[1,2-a]pyridine ring system as a scaffold for potent dual
phosphoinositide-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitors, Bioorganic & Medicinal
Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.08.016
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The imidazo[1,2-a]pyridine ring system as a
scaffold for potent dual phosphoinositide-3-
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rapamycin (mTOR) inhibitors
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Markian M. Stec,* Kristin L. Andrews, Yunxin Bo, Sean Caenepeel, Hongyu Liao, John McCarter, Erin L.
Mullady, Tisha San Miguel, Raju Subramanian, Nuria Tamayo, Douglas A. Whittington, Ling Wang, Tian Wu,
Leeanne P. Zalameda, Nancy Zhang, Paul E. Hughes, and Mark H. Norman

Bioorganic & Medicinal Chemistry Letters
The imidazo[1,2-a]pyridine ring system as a scaffold for potent dual
phosphoinositide-3-kinase (PI3K)/mammalian target of rapamycin (mTOR)
inhibitors
Markian M. Steca,*, Kristin L. Andrewsd, Yunxin Boa, Sean Caenepeelb, Hongyu Liaoa, John McCartere,
Erin L. Mulladyf, Tisha San Miguele, Raju Subramanianc, Nuria Tamayoa, Douglas A. Whittingtond, Ling
Wangb, Tian Wug, Leeanne P. Zalamedae, Nancy Zhangb, Paul E. Hughesb, and Mark H. Normana
aDepartment of Medicinal Chemistry, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320-1799, USA
bDepartment of Oncology, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320-1799, USA
cDepartment of Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320-1799, USA
dDepartment of Molecular Structure, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320-1799, USA and 360 Binney St., Cambridge,
Massachusetts 02142, USA
eDepartment of High-Throughput Screening/Molecular Pharmacology, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320-1799, USA
fDepartment of High-Throughput Screening / Molecular Pharmacology, Amgen Inc., 360 Binney Street, Cambridge, Massachusetts 02142
gDepartment of Basic Pharmaceutics, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320-1799, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Based on lead compound 1, which was discovered from a high-throughput screen, a series of
PI3K/mTOR inhibitors were evaluated that contained an imidazo[1,2-a]pyridine as a core
replacement for the benzimidazole contained in 1. By exploring various ring systems that
occupy the affinity pocket, two fragments containing a methoxypyridine were identified that
gave <100 nM potency toward PI3K in enzyme and cellular assays with moderate stability in
rat and human liver microsomes. With the two methoxypyridine groups selected to occupy the
affinity pocket, analogs were prepared with various fragments intended to occupy the ribose
pocket of PI3K and mTOR. From these analogs, tertiary alcohol 18 was chosen for in vivo
pharmacodynamic evaluation based on its potency in the PI3K cellular assay, microsomal
stability, and in vivo pharmacokinetic properties. In a mouse liver pharmacodynamic assay,
compound 18 showed 56% inhibition of HFG-induced AKT (Ser473) phosphorylation at a 30
mg/kg dose.
Keywords:
Phosphoinositide 3-kinase
Imidazo[1,2-a]pyridine
Ribose pocket
Affinity pocket
Kinase inhibitor
mTOR
2009 Elsevier Ltd. All rights reserved.
The study of phosphoinositide 3-kinases (PI3Ks) dates back to
the 1980s with publications from multiple research groups on the
phosphorylation of phosphatidylinositol at the 3-hydroxyl
position through kinase activity.1-4 Since that time, various
classes of PI3Ks have been elucidated including class I, II, and
III that differ in their structural characteristics and substrate
specificity. The class I PI3Ks are further divided into class Ia,
which include PI3K, , and  and the class Ib subgroup which
includes PI3K. When class I PI3Ks are activated, a signal
transduction cascade leads to the phosphorylation of AKT (also
known as PKB). Phosphorylated AKT mediates multiple cellular
functions including cell cycle progression, survival, transcription,
and glycogen synthesis.5,6
subunit of PI3K, p110 were found to be one of the most
common mutations in human solid tumors.7,8 Numerous PI3K
inhibitors have been developed that have shown preclinical tumor
growth inhibition with varying selectivity profiles from PI3K
isoform-selective to pan-class
I PI3K-selective to dual
PI3K/mTOR compounds.9-13 Mammalian target of rapamycin
(mTOR) is a PI3K-related kinase (PIKK) in the PI3K/AKT
pathway, which integrates signals from growth factors and
nutrients, and is also an important enzyme involved in the
promotion of cancer cell growth and survival.14,15 At least
seven dual PI3K/mTOR inhibitors, six pan-PI3K inhibitors, and
ten isoform-selective PI3K inhibitors are in, or have completed,
phase I or phase II clinical trials for cancer, either alone or in
combination therapy.16 It appears that the various tissue specific
and compensatory mechanisms present in the PI3K/AKT
pathway pose a challenge to patient and tumor type
Approximately 20 years after these initial discoveries,
definitive links between PI3K and cancer were identified when
mutations of the oncogene, PIK3CA, which encodes the catalytic
* Corresponding author. Tel.: +1 805 447 8006 (O); fax: +1 805 480 3015.
E-mail address: mstec@amgen.com (M. M. Stec).

In addition to the successful pyridine core derivatives,
alternative replacements to the benzimidazole core were sought
that would provide metabolically stable yet potent and
efficacious compounds. We postulated that the imidazo[1,2-
a]pyridine core may provide the desired potency, efficacy, and
stability and would also be amenable to the substitution patterns
required for our established SAR. Therefore, this disclosure
explores imidazo[1,2-a]pyridines as an alternative 5,6-heterocylic
core utilizing information that has been acquired in our PI3K
and mTOR23,25 programs to discover potent and efficacious
PI3K/mTOR dual inhibitors. The compounds described herein
are represented by general structure 3. This scaffold maintains
the aminotriazine moiety to interact with the hinge region of the
protein, utilizes aromatic amines (Ar) to occupy the affinity
pocket, and provides a position on the imidazopyridine core (R)
to incorporate groups to occupy the ribose pocket of PI3K.
At the time these studies were performed, structures of
inhibitors bound in PI3K were, and still are, relatively rare and
more difficult to obtain.26-29 However, because of the high
homology between PI3K and PI3K, particularly at the ATP
binding site, the use of PI3K provided good correlation between
observations made from crystallographic studies and
experimentally observed SAR. An overlay of compounds 1 and
2 bound to PI3K is shown in Figure 2. Two hydrogen bond
interactions from the triazine to Val882 are present for both
compounds in the “hinge region” of the kinase. In the affinity
pocket region of the protein, a water molecule bridges the
pyridine nitrogen of 2 with Asp841 and Tyr867 replacing the
phenol group of 1. This water molecule shifts the aromatic ring
that occupies the affinity pocket away from Asp841 and Tyr867.
The ribose pocket is minimally occupied in 1, while compound 2
projects the piperazine methylsulfonamide moiety up towards the
k3-k4 loop (P-loop) in a trajectory that is orthogonal to the
core. The bicyclic nature of the benzimidazole core inherently
projects further into the ribose pocket than the monocyclic
pyridine core. As a result, we postulated that shorter substituents
(e.g., without the benzylic methylene spacer) may be required to
occupy the ribose pocket on an imidazopyridine core relative to a
monocyclic pyridine core.
Figure 1. Lead compound 1 and PI3K series arising from 1. Amino acid
numbering reflects that of the PI3K sequence.
selection; however, preliminary clinical benefits have been
observed.11-13,17-21
We have previously reported the results of a high-throughput
screening effort that identified benzimidazole-triazine 1 (Figure
1) to be a moderately potent inhibitor of PI3K (IC50 = 350 nM)
and mTOR (IC50 = 93 nM).22,23 Compound 1 also showed
moderate potency in the U87 MG pAKT cellular assay; however,
it was metabolically unstable and showed high in vitro clearance
in rat liver microsomes (CLint = 291 L/min/mg). Rat bile duct
cannulation studies determined that oxidation on the
benzimidazole core was one route of metabolism.23 In addition
to poor metabolic stability, benzimidazole-triazine 1 was not
selective over protein kinases such as B-raf (V600EB-Raf IC50 = 3
nM). However, we believed that compound 1 was a reasonable
lead as a PI3K inhibitor and, with X-ray co-crystal and
modeling studies, we sought for ways to improve its potency,
selectivity, and metabolic stability. One approach we have taken
was to replace the benzimidazole core of 1 with 6-membered
rings such as pyridine.22,24 These modifications culminated in the
discovery of clinical candidate 2 (AMG 511), which was a potent
(PI3K = 4 nM) and selective (e.g., 2500-fold over mTOR and
>250-fold over B-Raf) pan-PI3K inhibitor. Compound 2 was
efficacious in the mouse U87 MG xenograft tumor growth
inhibition model with an ED50 of 0.6 mg/kg.24
Figure 2. X-ray co-crystal structures of compounds 1 (blue) and 2 (AMG
511, green) in PI3K (PDB codes 3QAQ and 4FLH, respectively).

The compounds described in this paper were screened in an in
vitro AlphaScreen assay to determine PI3K inhibition of the four
isoforms (, , , and ) and mTOR.30 Inhibition of the ,  and
 isoforms will only be reported for the compound that
progressed into in vivo studies. Cellular activity was determined
using a SureFire detection kit to measure downstream inhibition
of AKT (Ser473) phosphorylation in U87 MG human
glioblastoma cells.31
potency in the cellular assay diminished modestly to 86 nM.
Compound 6 did show some selectivity (14-fold) over mTOR.
Various 5,6- and 6,6-bicyclic groups were also explored in
the affinity pocket region. These bicyclic heterocycles were
selected to probe the hydrogen bonding network within the
affinity pocket by placing nitrogen atoms at several different
positions within these derivatives.32 The isoquinoline, quinoline,
and indazole analogs 8, 9, and 10 were found to be significantly
more potent than quinoline 7 in the enzyme assay. By examining
overlays of compounds 7-10 with 1, it appears that compounds 8
and 9 can place the quinoline nitrogen in a similar position to the
phenol oxygen of 1 in the affinity pocket compared to quinoline
7. With the indazole analog 10, modeling suggested that both
indazole nitrogens projected towards Asp841 and Tyr867 thereby
establishing a good hydrogen bonding network in the affinity
pocket. Although the majority of these bicylic-heterocycle
derivative showed good activity in the enzyme assay, compounds
8, 9, and 10 showed high microsomal clearance.33
In our initial SAR investigations, we explored a select group
of aromatic substituents that occupied the affinity pocket of the
kinase with fragments that afforded potent analogs in the
pyridine core series (Table 1).
The 2-methoxypyridine
substituted compound (4) showed good potency towards PI3K
(29 nM), mTOR (37 nM) and in the pAKT cellular assay (40
nM). Replacing the methoxy group of 4 with an ethoxy group
(compound 5) resulted in a sixfold loss in potency in both the
PI3K enzyme assay and cellular assay. This suggested that
substituents could not be extended further into the affinity
pocket. The 3-fluoro-2-methoxypyridine compound (6) showed
a twofold gain in PI3K potency (14 nM) over 4; however,
Table 1. In vitro potency and microsomal stability of PI3K/mTOR inhibitors with an imidazopyridine core.
Liver Microsomes (Clint, L/min/mg)
PI3K
mTOR
pAKT (U87 MG)
(IC50, nM)b
Compound
Ar
Rat
Human
(Ki, nM)a
(IC50, nM)a
4
29
37
57
40
101
65
5
6
148
229
105
71
49
66
14
198
86
7
8
9
529
54
277
12
>780
N/A
18
62
111
105
133
238
144
43
1.7
10
5.6
16
32
175
70
aData represents an average of at least two determinations unless indicated otherwise. A statistical analysis of this data may be found in the supplementary
material. bData represents a single measurement.
Based on the potency and both rat and human microsomal
stability found in compounds 4 and 6,34,35 a focused series of
analogs was explored that contained the 2-methoxypyridine or
the 3-fluoro-2-methoxypyridine fragment in the affinity pocket
region of PI3K but that also contained an additional substituent
intended to occupy the ribose pocket (Table 2). This substituent
was included with the goal of improving potency and to
potentially block sites of metabolism on the imidazopyridine ring
system. As can be seen in the X-ray structures in Figure 2, the
piperazine sulfonamide of compound 2 bisects the aromatic C-C

bond of the phenyl ring distal to the 5-membered ring.
Therefore, based on crystal structure overlays, both the 6- and 7-
positions of the imidazopyridine appeared to be viable options
for the incorporation of ribose pocket-occupying substituents. A
limited numbers of compounds were made with substitution at
the 7-position; however, these derivatives provided no advantage
in potency or synthetic tractability compared to the 6-position
found to have <50 nM enzymatic potency. These compounds
showed cellular potencies of <70 nM, with compound 13 being
the only exception with near 300 nM cellular potency.
Substitution at the 6-position improved microsomal stability with
all of the compounds assayed having rat and human liver
microsome clearance values below 100 L/min/mg with most
having values of <14 L/min/mg.
analogs.
Based on this observation, only analogs with
substitution at the 6-position will be discussed in this report.
As shown in Table 2, analogs with piperazine substituents at
the 6-position of the imidazopyridine ring system (11-16) were
Table 2. In vitro potency and microsomal stability of PI3K/mTOR inhibitors containing substituents occupying the ribose
pocket.
Liver Microsomes (CLint, L/min/mg)b
PI3K
mTOR
pAKT (U87 MG)
(IC50, nM)b
Compound
X
F
R
Rat
Human
(Ki, nM)a
(IC50, nM)a
11
5.7
43
62
20
41
69
<14
<14
12
13
14
<14
<14
<14
<14
<14
28
H
F
F
6.5
6.3
412
121
294
26
15
16
8.3
6.1
34
34
20
30
73
67
H
F
157b
<14
17
18
26
11
36
45
25
17
H
F
206
4.5
<14
<14
aData represents an average of at least two determinations unless indicated otherwise. A statistical analysis of this data may be found in the supplementary
material.bData represents a single measurement.
Other substituents that were used to occupy the ribose pocket
included tertiary alcohols, such as in compounds 17 and 18.36
These alcohols were comparable to the piperazine analogs in
enzyme potency. While 18 was of similar potency in the enzyme
assay (11 nM) to many of the piperazines, it did have the best
combination of excellent cellular potency (4.5 nM) and rat and
liver microsome stability (<14 L/min/mg). The 3-
fluoropyridine analog 18 demonstrated 19-fold selectivity over
mTOR in contrast to compound 17 which showed less than
twofold selectivity. In addition, with the incorporation of a

fluorine atom on the methoxypyridine ring, an improvement in
cellular potency (17, 45 nM vs 18, 4.5 nM) was obtained.
Compound 18 was potent towards the PI3K,  and ,
isoforms in the enzyme assay with Ki values of 17 nM, 5.3 nM
and 0.6 nM, respectively. This lack of isoform selectivity was
generally observed for the compounds described herein.
Based on its excellent potency and in vitro stability, tertiary
alcohol 1837 was further evaluated in pharmacokinetic and
pharmacodynamic (PD) studies. In rats, intravenous dosing of
compound 18 demonstrated a clearance of 1.7 L/h/kg; (52% of
liver blood flow) and a volume of distribution of 4.2 L/kg with a
terminal elimination half-life of 6.3 hours. Compound 18 dosed
orally in rats at 2 mg/kg also showed good oral bioavailability
(51%) and moderate plasma exposure (AUC = 640 ng·h/mL).
The in vivo activity of compound 18 was evaluated in a mouse
liver pharmacodynamic assay that measured inhibition of
hepatocyte growth factor (HGF)-induced AKT (Ser473)
phosphorylation (Figure 3). In this assay, mice were dosed
orally with compound 18 at 3, 10, and 30 mg/kg and, after 6
hours, PI3K dependent AKT phosphorylation was stimulated by
HGF administration. AKT (Ser473) phosphorylation levels were
measured 5 minutes after HGF administration by a quantitative
electrochemiluminescence immunoassay. Imidazopyridine 18
showed significant inhibition of AKT phosphorylation at all three
doses, with a maximum inhibition of 56% at the 30 mg/kg dose.
The calculated ED50 relative to vehicle for 18 in this study was 11
mg/kg.38
Scheme 1. Reagents and conditions: (a) Ar-NH2, BrettPhos Precatalyst,
NaOtBu, 1,4-dioxane, 90 °C, 1-16 h, 22-77%; (b) 21, Pd(OAc)2, PPh3,
K2CO3, 1,4-dioxane, 140 °C, 3 h, microwave, 14-85%; (c) TfOH, TFA, 25-70
°C, 1-12 h, 12-95%.
The synthesis of compounds with substituents in the 6-
position of the imidazopyridine ring (11-18) began with 6-
bromo-2-chloroimidazo[1,2-a]pyridine (24)43 in which the
bromine atom was utilized to incorporate the ribose pocket
substituents (Schemes 2 and 3). For the preparation of
compounds 11-16, imidazopyridine 24 underwent a Pd-catalyzed
C-N coupling reaction with N-Boc-piperazine selectively at the
bromide position followed by a second Pd-catalyzed C-N
coupling reaction at the chloride position with aminopyridines 25
and 26 to afford intermediates 27 and 28, respectively. Pd-
catalyzed arylation on the 5-membered ring with chlorotriazine
21 afforded 29 and 30.
Figure 3. Inhibition of hepatocyte growth factor (HGF)-induced AKT
(Ser473) phosphorylation in the liver by compound 18. Mice were dosed
orally with either vehicle alone (15% HPBCD, 2% HPMC, 1% Pluronic F68,
adjusted to pH 2.2 w/MSA) or with 18. HGF-induced AKT (Ser473)
phosphorylation was measured 6 hours post-dose. Asterisks denote p < 0.05
compared with the vehicle/HGF group. Statistical significance was evaluated
by Dunnett’s method. Bars represent the average  SD (n = 3). Red
diamonds represent mean plasma concentrations.31
Compounds of general structure 3 in which R = H (4-10),
were synthesized as shown in Scheme 1 beginning with 2-
chloroimidazo[1,2-a]pyridine (19).39
For example, through
Method A, 19 underwent a Pd-catalyzed C-N coupling reaction40
with various aryl amines to afford 20 in modest-to-good yields.
Imidazopyridines 20 then advanced into Pd-catalyzed arylation
reactions41,42 with chlorotriazine 21 to install the protected
amino-triazine moiety of 22. Cleavage of the PMB protecting
groups of 22 under acidic conditions afforded final compounds 4,
7, 9, and 10. Alternatively, analogs 5, 6, and 8 were made by
Method B through initial incorporation of the triazine moiety
which gave 23 followed by introduction of the aryl amine groups
to give 22. Subsequent PMB removal afforded the final
compounds.
Scheme 2. Reagents and conditions: (a) N-Boc-piperazine, Pd2(dba)3, NaOt-
Bu, rac-BINAP, tol., 120 °C, 30 min, microwave, 72%; (b) 25 or 26,
BrettPhos Precatalyst, NaOt-Bu, 1,4-dioxane, 90 °C, 15 h, 19-40%; (c) 21,
Pd(OAc)2, PPh3, Cs2CO3, NEt3, 1,4-dioxane, 140 °C, 2 h, microwave, 63-
81%; (d) TFA, CH2Cl2, rt, 50-77%; (e) TFA, 75 °C, 45-76%; (f) TfOH, TFA,
70 °C, 1 h, 47-97%; (g) MeI, NaHCO3, 56%; (h) Acetone, NaBH(OAc)3,
AcOH, 82%; (i) 2-Bromoethyl methyl ether, DMF, rt, 24 h, 59%; (j)
Me2NCOCl, NEt3, CH2Cl2, rt, 1 h, 50-64%.

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3.
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Freeman, D. J.; Wang, L.; Zhang, N.; Wu, T.; Hughes, P. E.;
Norman, M. H. J. Med. Chem. 2012, 55, 5188.
23. Peterson, E. A.; Andrews, P. S.; Be, X.; Boezio, A. A.; Bush, T.
L.; Cheng, A. C.; Coats, J. R.; Colletti, A. E.; Copeland, K. W.;
DuPont, M.; Graceffa, R.; Grubinska, B.; Harmange, J.-C.; Kim, J.
L.; Mullady, E. L.; Olivieri, P.; Schenkel, L. B.; Stanton, M. K.;
Teffera, Y.; Whittington, D. A.; Cai, T.; La, D. S. Bioorg. Med.
Chem. Lett. 2011, 21, 2064.
Scheme 3. Reagents and conditions: (a) tributyl(1-ethoxyvinyl)tin, Pd(OAc)2,
CsF, XPhos, 1,4-dioxane, 120 °C, 30 min, microwave, 79%; (b) (i) 25 or 26,
BrettPhos Precatalyst, NaOtBu, 1,4-dioxane, 90 °C, 15 h, (ii) 1 N HCl,
CH2Cl2, 15-68%; (c) 21, Pd(OAc)2, PPh3, Cs2CO3, NEt3, 1,4-dioxane, 140 °C,
2 h, microwave, 32-88%; (d) TfOH, TFA, 70 °C, 1 h, 56-100%; (e) MeMgBr,
THF, 18-86%.
24. 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.
25. Peterson, E. A.; Boezio, A. A.; Andrews, P. S.; Boezio, C. M.;
Bush, T. L.; Cheng, A. C.; Choquette, D.; Coats, J. R.; Colletti, A.
E.; Copeland, K. W.; DuPont, M.; Graceffa, R.; Grubinska, B.;
Kim, J. L.; Lewis, R. T.; Liu, J.; Mullady, E. L.; Potashman, M.
H.; Romero, K.; Shaffer, P. L.; Stanton, M. K.; Stellwagen, J. C.;
Teffera, Y.; Yi, S.; Cai, T.; La, D. S. Bioorg. Med. Chem. Lett.
2012, 22, 4967.
In conclusion, based on benzimidazole PI3K/mTOR
inhibitor 1, a series of analogs were screened with an
imidazopyridine core replacing the benzimidazole. Initially,
various substituted aromatic rings were explored to engage the
affinity pocket of PI3K and a 2-methoxypyridine (4) and 3-
fluoro-2-methoxypyridine derivative (6) were found to give the
best combination of potency and in vitro stability. Further
optimization by screening a select group of substituents to
occupy the ribose pocket of PI3K led to the identification of
imidazopyridine 18 that was found to be a potent pan class I
PI3K/mTOR inhibitor and was efficacious in a mouse liver
pharmacodynamic assay.
26. The difficulty is due to limits in the generation of pure and stable
protein and identifying protein constructs that are amenable to
crystallization.
Acknowledgments
27. Huang, C.-H.; Mandelker, D.; Schmidt-Kittler, O.; Samuels, Y.;
Velculescu, V. E.; Kinzler, K. W.; Vogelstein, B.; Gabelli, S. B.;
Amzel, L. M. Science 2007, 318, 1744.
28. Amzel, L. M.; Huang, C.-H.; Mandelker, D.; Lengauer, C.;
Gabelli, S. B.; Vogelstein, B. Nat. Rev. Cancer 2008, 8, 665.
29. 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.
We would like to thank Daniel J. Freeman for his input on in
vivo pharmacology studies. The support of this research program
by Randy Hungate and Rick Kendall was greatly appreciated.
References and notes
1.
Toker, A. Subcell. Biochem. 2012, 58, 95.

30. The experimental procedure for the mTOR assay is described in
the Supplementary Material.
31. D'Angelo, N. D.; Kim, T.-S.; Andrews, K.; Booker, S. K.;
Caenepeel, S.; Chen, K.; Freeman, D.; Jiang, J.; Liu, L.; McCarter,
J. D.; San Miguel, T.; Mullady, E. L.; Schrag, M.; Subramanian,
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32. The indazole group was also selected as an affinity pocket moiety,
since it has been successfully employed in this region of the
enzyme by Genentech in their PI3K inhibitor, Pictilisib (GDC-
094). See: Sarker, D.; Ang, J. E.; Baird, R.; Kristeleit, R.; Shah,
K.; Moreno, V.; Clarke, P. A.; Raynaud, F. I.; Levy, G.; Ware, J.
A.; Mazina, K.; Lin, R.; Wu, J.; Fredrickson, J.; Spoerke, J. M.;
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M. K.; Workman, P.; de Bono, J. S. Clin. Cancer Res. 2015, 21,
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33. CLint (L/min/mg) estimated from parent compound (1 M)
remaining following a 30 min incubation in liver microsome (rat
and human; 0.25 mg/mL) in potassium phosphate buffered (66.7
mM) with NADPH (1 mM) at 37 °C for 30 min. Under these
conditions, a cut-off of approximately 100 L/min/mg-protein or
less was considered desirable.
34. In addition to cell potency and microsomal stability, we examined
the cLogD (pH 7.4) values for the compounds listed in Table 1
(which ranged from 1.7 to 2.5). However, no correlation was
observed between cLogD (pH 7.4) and activity in the U87 cellular
assay (see Supplementary Information).
35. While indazole 10 showed good activity in the pAKT cellular
assay and fairly good stability in human liver microsomes, it was
metabolized rapidly in rat liver microsomes (CLint, = 175
L/min/mg). Since the subsequent evaluation of the compounds
would be in rat, this compound was not selected for further SAR
development.
36. The dimethyl hydroxymethyl group was selected at this position
based on some of our earlier SAR work in the pyridine series. In
our efforts to identify an alternative to the piperazine sulfonamide
portion of AMG 511, we were able to replace this ‘ribose-pocket’
group with a 2-hydroxypropyl group providing a compound that
retained the favorable pharmacokinetic properties of AMG 511
and exhibited similar in vivo efficacy in a mouse liver
pharmacodynamics assay. See: Lanman, B. A.; Reed, A. B.; Cee,
V. J.; Hong, F.-T.; Pettus, L. H.; Wurz, R. P.; Andrews, K. L.;
Jiang, J.; McCarter, J. D.; Mullady, E. L.; San Miguel, T.;
Subramanian, R.; Wang, L.; Whittington, D. A.; Wu, T.;
Zalameda, L.; Zhang, N.; Tasker, A. S.; Hughes, P. E.; Norman,
M. H. Bioorg. Med. Chem. Lett. 2014, 24, 5630.
37. The experimental procedure for the synthesis of compound 18 is
described in the Supplementary Material.
38. With a corresponding EC50 of 542 ng/mL and a fraction unbound
(fu) of 0.066 in mouse plasma, this results in an unbound plasma
concentration of 84 nM or 7.6-fold over the IC50.
39. Maxwell, B. D.; Boye, O. G.; Ohta, K. J. Labelled Compd.
Radiopharm. 2005, 48, 397.
40. Fors, B. P.; Watson, D. A.; Biscoe, M. R.; Buchwald, S. L. J. Am.
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