Discovery of a novel series of potent and orally bioavailable phosphoinositide 3-kinase γ inhibitors

Article abstract of DOI:10.1021/jm300403a

The phosphoinositide 3-kinases (PI3Ks) have been linked to an extraordinarily diversified group of cellular functions making these enzymes compelling targets for the treatment of disease. A large body of evidence has linked PI3Kγ to the modulation of autoimmune and inflammatory processes making it an intriguing target for drug discovery. Our high-throughput screening (HTS) campaign revealed two hits that were nominated for further optimization studies. The in vitro activity of the first HTS hit, designated as the sulfonylpiperazine scaffold, was optimized utilizing structure-based design. However, nonoptimal pharmacokinetic properties precluded this series from further studies. An overlay of the X-ray structures of the sulfonylpiperazine scaffold and the second HTS hit within their complexes with PI3Kγ revealed a high degree of overlap. This feature was utilized to design a series of hybrid analogues including advanced leads such as 31 with desirable potency, selectivity, and oral bioavailability.

Full text of DOI:10.1021/jm300403a

Article
pubs.acs.org/jmc
Discovery of a Novel Series of Potent and Orally Bioavailable
Phosphoinositide 3-Kinase γ Inhibitors
James W. Leahy, Chris A. Buhr, Henry W. B. Johnson,* Byung Gyu Kim, TaeGon Baik, Jonah Cannoy,
Timothy P. Forsyth, Joon Won Jeong, Matthew S. Lee, Sunghoon Ma, Kevin Noson, Longcheng Wang,
Matthew Williams, John M. Nuss, Eric Brooks, Paul Foster, Leanne Goon, Nathan Heald, Charles Holst,
Christopher Jaeger, Scott Lam, Julie Lougheed, Lam Nguyen, Arthur Plonowski, Joanne Song,
Thomas Stout, Xiang Wu, Michael F. Yakes, Peiwen Yu, Wentao Zhang, Peter Lamb, and Olivia Raeber
Department of Drug Discovery, Exelixis, 169 Harbor Way, South San Francisco, California 94083, United States
S
* Supporting Information
ABSTRACT: The phosphoinositide 3-kinases (PI3Ks) have been
linked to an extraordinarily diversified group of cellular functions
making these enzymes compelling targets for the treatment of
disease. A large body of evidence has linked PI3Kγ to the
modulation of autoimmune and inflammatory processes making it
an intriguing target for drug discovery. Our high-throughput
screening (HTS) campaign revealed two hits that were nominated
for further optimization studies. The in vitro activity of the first
HTS hit, designated as the sulfonylpiperazine scaffold, was
optimized utilizing structure-based design. However, nonoptimal
pharmacokinetic properties precluded this series from further
studies. An overlay of the X-ray structures of the sulfonylpiperazine
scaffold and the second HTS hit within their complexes with PI3Kγ revealed a high degree of overlap. This feature was utilized to
design a series of hybrid analogues including advanced leads such as 31 with desirable potency, selectivity, and oral bioavailability.
degranulation, thereby generating interest in this target for the
treatment of autoimmune and inflammatory disorders.14−16
Recently, several reports linking PI3Kγ to cancer,17−21
diabetes,22 cardiovascular disease,23−28 and Alzheimer’s29 have
also been disclosed.
Given the overwhelming evidence linking the PI3K pathway
to various disease states, there have been reports from a
number of research groups targeting both pan-active and
isoform selective inhibitors. These efforts have resulted in a
considerable number of compounds now in the clinic, two of
which were generated by our in-house program.30 Both agents
are pan-active inhibitors of the class I PI3Ks and are
differentiated primarily by their activity against the closely
related PIKK family member mTOR.31,32 Our more recent
exploration has involved targeting isoform selective inhibitors in
attempts to deconvolute the pharmacological activity observed
with pan-active PI3K inhibition.
INTRODUCTION
■
The phosphoinositide 3-kinases (PI3Ks) are attractive targets
for the design of small molecule inhibitors since abnormal PI3K
activity contributes to the transition from typical cell function
to the progression of disease.1−5 The most extensively studied
of the three PI3K classes, class I, includes heterodimeric
proteins consisting of a catalytic subunit (p110α, p110β, p110δ,
and p110γ) and regulatory subunit (p85 in the case of p110α,
p110β, and p110δ; either p84 or p101 in the case of PI3Kγ).5
This target is further grouped into the class IA subset,
comprising PI3Kα, β, and δ, which is predominately activated
by receptor tyrosine kinase signaling and the lone class IB
member, PI3Kγ, which is activated by G-protein coupled
receptors.1,6−9
The primary role of the class I PI3Ks involves catalyzing the
phosphorylation of phosphatidylinositol (3,4)-bisphosphate
(PIP2) to phosphatidylinositol (3,4,5)-triphosphate (PIP3).4
However, despite this unifying characteristic, differences in
tissue distribution among the isoforms allows for differentiation
in a disease setting. Where PI3Kα and PI3Kβ are ubiquitously
expressed, PI3Kδ and PI3Kγ expression is restricted primarily
to the hematopoietic system.6−11 The class IA PI3Ks regulate
many diverse processes and have been studied extensively as
targets for the treatment of cancer and metabolic disorders.12,13
Experimental evidence has uncovered the link between PI3Kγ
and processes such as lymphocyte chemotaxis and mast cell
Fortunately, at the start of our work several reports exploring
the potential of selective PI3Kγ and dual PI3Kγ/δ inhibition as
a target profile for the treatment of inflammatory disease
existed.33−35 Collectively, this large body of evidence provided
a therapeutic rationale for pursuing PI3Kγ as a drug discovery
target and alleviated concerns around the risk of inhibiting this
Received: March 22, 2012
Published: May 1, 2012
© 2012 American Chemical Society
5467
dx.doi.org/10.1021/jm300403a | J. Med. Chem. 2012, 55, 5467−5482

Journal of Medicinal Chemistry
Article
central signaling enzyme. The premise that PI3Kγ inhibition
would cause undesired side effects as a consequence of down-
regulating central signaling pathways, such as mitogen-activated
protein kinase (MAPK),36−39 was largely addressed by
knockout and knock-in studies which indicated that mice
lacking PI3Kγ activity are viable and fertile.40−42 Subsequently,
a groundbreaking report from Camps and co-workers disclosed
the first PI3Kγ selective inhibitor which demonstrated the role
of pharmacological inhibition on this target in a disease
setting.43 In addition to this disclosure, few other examples of
subtype selective PI3Kγ inhibitors have been disclosed.33,44 In
an effort to further investigate the role of PI3Kγ in autoimmune
and inflammatory disorders, we embarked on a campaign to
design a tool compound with selectivity for PI3Kγ vs class IA
isoforms (PI3Kα, β, and δ) and pharmacokinetic properties
amenable to oral dosing. Herein is described our efforts to this
end.
observed in the plasma when dosed orally in mice at 100 mpk
after one and four hours. The second HTS hit 2 was previously
prepared as part of a program aimed at the inhibition of
checkpoint kinases 1 and 2.45 While 2 was not particularly
active in these investigations, its biochemical and selectivity
profile against PI3Kγ was noteworthy. Given that the IC50 of 2
was >10 μM when tested in our cellular assay, we chose to
prioritize our optimization efforts on 1 with the knowledge that
2 could serve as a viable scaffold as well.
The first generation of analogues aimed at improving the
potency and selectivity of 1 utilized key observations gathered
from a cocrystal structure of the ligand bound to the ATP-
binding site of PI3Kγ (Figure 2). Interaction of 1 with the
RESULTS AND DISCUSSION
■
Screening of our extensive ∼4.6 million compound in-house
library revealed hits 1 and 2 that we felt could be useful starting
points from which to initialize lead optimization studies (Figure 1).
Figure 2. Cocrystal structure of 1 in the PI3Kγ active site (PDB code
4anv). Residues nearest 1 in the PI3Kγ active site that are different
among class I PI3K isoforms are displayed in yellow. Hydrogen bonds
of 1 with Val882 and Lys833 are illustrated.
PI3Kγ hinge occurs through hydrogen bonding of the methoxy
phenyl oxygen to the backbone NH of Val882; a fact that was
not surprising based on the significant number of ligands that
engage oxygen with this residue.46 From our structural data, it
appeared likely that converting the methoxyphenyl group
interacting with Val882 to a functional group capable of
forming a bidentate interaction would improve the PI3K
activity. Indeed, introduction of the aminopyrimidine, with
analogue 3, produced a >40-fold improvement in biochemical
activity for PI3Kγ (Table 1). A high degree of homology is
exhibited within the class I PI3Ks; therefore, to improve γ
selectivity priority was given to modifying a part of the scaffold
in close proximity to a less conserved region along the
periphery of the active site.47 Selectivity residues near the ligand
are highlighted in Figure 2. The effectiveness of this strategy
was exemplified with modification of the R group of 3, where
replacing the methoxy group with an isopropyl group, to
provide 4, improved the PI3K selectivity in favor of γ over α
and β (Table 1). Unfortunately, 4 displayed poor plasma
exposure in mice, presumably due to high mouse liver
Figure 1. Data for HTS hits 1 and 2.
Assessment of biochemical selectivity was carried out with a
luciferase-coupled chemiluminescence assay. Compounds were
evaluated in a cellular context by measuring inhibition of AKT
(PKB) phosphorylation with C5a-stimulated Raw 264.7 murine
macrophages in a fixed-cell ELISA format.43 HTS hit 1
possessed moderate in vitro potency and poor selectivity for
PI3Kγ vs α, β, and δ. Further evaluation of the pharmacokinetic
profile of 1 demonstrated that a <1 μM concentration was
microsome (MLM) oxidation (79
2.6% conversion at
30 min). Pyridine 5 displayed lower mouse liver microsome
oxidation (37 2.7% conversion at 30 min) and in-turn higher
mouse plasma exposure than pyrazine 4. However, despite
5468
dx.doi.org/10.1021/jm300403a | J. Med. Chem. 2012, 55, 5467−5482

Journal of Medicinal Chemistry
Article
Table 1. Optimization of Sulfonylpiperazine Derivatives of HTS Hit 1
PI3K selectivity IC50
(nM)
PI3Kγ IC50
PI3Kγ cell IC50
mouse plasma
a,b
b,c
compd
X
Y
R
(nM)
(nM)
α
β
δ
exposure
n.d.
rat PK
3
4
N
N
N
N
OMe
iPr
10
18
264
182
38
1970
387
816
n.d.
n.d.
d
505
>10,000
0.7 0.3 μM (1 h)
d
0.1 0.08 μM (4 h)
e
5
C
N
iPr
18
213
220
3512
456
12.9 1.4 μM (1 h)
AUC(0‑∞) = 0.95 μM•h, t1/2 = 1.82 h, %F
e
= 12.7
3.2 0.9 μM (4 h)
a
b
c
Data are reported as the mean SD. n.d. = not determined. Female Sprague−Dawley rats (n = 3) dosed at 5 mpk (PO) as a solution in 5%
d
EtOH 45% PEG400/water + 1:2 HCl. Male C57BL6 mice (n = 3) dosed at 100 mpk (PO) as a suspension in 0.5% CMC 0.2% Tween80/water.
e
Male C57BL6 mice (n = 3) dosed at 100 mpk (PO) as a turbid solution in 10% EtOH 20% PEG400/water + 1:1 HCl.
Table 2. In Vitro Potency and Selectivity Data for Derivatives of HTS Hit 2
other efforts to improve the pharmacokinetics of the sulfonyl
piperazine series, 5 was our most promising analogue thus far.
The combination of moderate mouse plasma exposure along
with undesirable rat pharmacokinetic properties precluded
further evaluation of this analogue. When considering the poor
oral bioavailability of 5, the low solubility (Ksol <5 μM) was
considered a primary factor. As the analogues in this series had
high molecular weight (>500) and lipophilicity (LogD7.4
>4.0),48 lowering these values was viewed as an attractive
approach to improving the solubility.
Fortunately, HTS hit 2 had considerably higher solubility
(Ksol = 424 μM), lower lipophilicity (LogD7.4 = 0.47), and
molecular weight (MW = 296). Therefore, 2 seemed like a
more reasonable starting point for pharmacokinetic optimiza-
tion since solubility would not likely be a limiting factor in
achieving acceptable oral bioavailability in this series. In the
optimization of 2, our first priority was improving the poor
cellular activity (IC50 >10 μM), and there was evidence to infer
this could be addressed by replacement of the tetrazole
functional group. For example, lower biochemical activity, yet
increased cellular activity, was observed when the tetrazole was
replaced with a trifluoromethyl group (6, Table 2). Fortunately,
our optimization effort was aided by structural data of HTS hit
2 and sulfonyl piperazine 4.
An overlay of the cocrystal structures of 2 and 4 indicated
that these analogues adopted a similar binding mode (Figure 3).
With 4, hydrogen bonding with kinase hinge residue Val882
involved the aminopyrimidine, and with 2, a similar interaction
is formed with the aminopyrazine. Additionally, interaction
with the salt bridge residue Lys833 occurs via the tetrazole of 2
and the sulfonamide oxygen of 4. Therefore, when considering
a surrogate functional group for the tetrazole, a sulfonamide
was deemed appropriate since it would be able to interact with
Lys833 while also allowing for additional structural manipu-
lation. It was also deemed beneficial that the tetrazole of 2
did not reside in close proximity to selectivity residues within
the active site, and therefore, modifying this region would be less
likely to disrupt the desirable PI3Kγ selectivity of this analogue.
Described in Table 2 is a representative sample of our initial
attempts to find a suitable sulfonamide replacement for the
tetrazole of 2. Phenylsulfonamide 7 displayed a similar
biochemical potency and selectivity profile to 2, but with
greatly enhanced cellular activity, thereby providing evidence
that the potency would be retained with an appropriate tetrazole
5469
dx.doi.org/10.1021/jm300403a | J. Med. Chem. 2012, 55, 5467−5482

Journal of Medicinal Chemistry
Article
Figure 3. (a) Overlay of the cocrystal structures of 2/P13Kγ (green/gray) and 4/PI3Kγ (magenta/orange). P-loop removed for clarity. Selectivity
residues in close proximity to the ligands are displayed in yellow (PDB codes 4anu and 4anx). (b) Schematic presentation of the binding interactions
between 2 and 4 in the active site.
replacement. Installation of a chlorine on the central ring generally
led to an increase in cell activity as indicated with 8. This finding
was further explored with reduction of the size of the sulfonamide
group as demonstrated with 9−11 (Table 3). These analogues
Further derivatization involved modification of the scaffold
core (Table 4). Comparison of methoxy analogue 12 with 8
and 13 suggested that electron withdrawing substituents
improved the potency. Aminopyridine analogue 14 displayed
a biochemical profile similar to that of parent 8, and reverse
sulfonamide derivatives 15−16 displayed slightly decreased
PI3K activity compared to that of parent 7. However,
reintroduction of chlorine on the central ring increased the
biochemical potency but decreased the selectivity profile as
shown with a comparison of 16 and 17. Overall, alteration to
the central ring of the aryl sulfonamide series did not lead to an
improvement in the biochemical activity or selectivity profile.
Building upon lead 8, substituted aryl sulfonamide derivatives
were next considered. Changing substituents at the meta
position, with groups exhibiting different electronic and steric
properties, generally did not greatly alter the selectivity profile,
cellular activity, or mouse plasma exposure as indicated by 18−
22 (Table 5). However, walking a chlorine atom around the
aryl group revealed that ortho substitution provided two
significant advantages (22−24, Table 6). First, a >40-fold
increase in plasma exposure in mice was observed for the ortho-
substituted analogue 23 when compared to the meta-
substituted analogue 22. Examination of the in vitro data for
these analogues indicated that the explanation for the drastic
difference in plasma exposure was likely related, at least in part,
to differences in the solubility of 22 (Ksol = <1 μM) and 23
(Ksol = 24 μM). Second, ortho substitution incrementally
improved the PI3Kγ selectivity over other class I PI3Ks. As an
aside, the slight potency improvement realized by placing a
Table 3. In Vitro Potency and Selectivity Data for
Sulfonamide Derivatives of HTS Hit 2
PI3K selectivity IC50 (nM)
compd
R
PI3Kγ IC50 (nM)
α
β
δ
9
Me
H
17
26
65
142
97
1563
701
1073
638
10
11
CH2CF3
752
2511
1418
were biochemically potent and provided evidence that the PI3K
selectivity could be modulated by changing the alkyl group on the
sulfonamide. Although the potency and selectivity was less than
optimal with 11, it displayed acceptable oral plasma exposure in
mice which provided affirmation of our thesis that a sulfonamide
derivative with desirable solubility (Ksol = 211 μM) would improve
the pharmacokinetic properties of this series.49 We were particularly
encouraged by the excellent cellular activity observed with aryl
sulfonamide 8 and adopted it as a standard going forward.
5470
dx.doi.org/10.1021/jm300403a | J. Med. Chem. 2012, 55, 5467−5482

Journal of Medicinal Chemistry
Article
Table 4. Biochemical Activity and PI3K Selectivity Data for Core Modifications of the Aryl Sulfonamide Series
PI3K selectivity IC50 (nM)
compd
R1
X
Y
Z
R2
PI3Kγ IC50 (nM)
α
β
δ
12
13
14
15
16
17
OMe
F
C
C
N
C
C
C
SO2
SO2
SO2
NH
NH
NH
NH
NH
NH
SO2
SO2
SO2
H
H
H
H
Cl
Cl
507
152
53
1807
953
>10,000
3370
362
1202
1582
533
NH2
H
200
167
161
11
3643
3381
90
>10,000
6343
64
3389
2964
100
H
Cl
Table 5. PI3K Selectivity, Cellular Inhibition, and Mouse Plasma Exposure Data for meta-Substituted Aryl Sulfonamide
Derivatives
PI3K selectivity IC50 (nM)
a,b
compd
R
PI3Kγ IC50 (nM)
PI3Kγ cell IC50 (nM)
α
β
δ
mouse plasma exposure
n.d.
18
19
Me
F
48
43
572
171
323
316
2075
1611
568
415
c
1.6 0.3 μM (1 h)
c
1.0 0.1 μM (4 h)
c
20
21
22
OMe
CF3
Cl
31
49
431
552
290
727
174
1656
5070
1130
546
1355
437
0.7 μM (1 h)
c
0.09 μM (4 h)
d
d
e
1.7 0.7 μM (1 h)
0.4 0.2 μM (4 h)
18
184
1.3 0.6 μM (1 h)
0.1 0.04 μM (4 h)
e
a
b
c
Data are reported as the mean SD. n.d. = not determined. Male C57BL6 mice (n = 3) dosed at 100 mpk (PO) as a suspension in 10% EtOH
d
e
40% PEG400/water + 1:1 HCl. Female C57BL6 mice (n = 3) dosed at 100 mpk (PO) as a suspension in 0.75% CMC 0.1% Tween80/water. Male
C57BL6 mice (n = 3) dosed at 100 mpk (PO) as a suspension in 10% EtOH 60% PEG400/water.
chlorine on the central ring with earlier analogues was also
observed with a comparison of 22 and 25.
optimization of the hinge-binding heterocycle utilizing the
difluoroaryl sulfonamide contained in 31.
An intriguing observation was made when considering the
binding modes of 31 and 4 (Figure 4). As mentioned
previously, the isopropyl group of 4 resides in a region
containing multiple selectivity residues and was thought to
contribute to the PI3Kγ selectivity observed with this analogue.
Therefore, a series of derivatives was synthesized to determine
the extent to which the N-methylcarboxamide of 31 contributes
to the PI3K selectivity profile since this group is in close
proximity to the isopropyl group of 4 (Table 8). Analogue 33,
lacking the N-methylcarboxamide, displayed a significantly less
attractive biochemical profile than 31. However, the amino-
pyrimidine analogue 34 exhibited a biochemical profile
similar to that of 31, thereby providing evidence that the
N-methylcarboxamide was not necessary to retain γ
selectivity. Substitution at the 4-position of the pyrimidine
to provide 35 and 36 incrementally increased the activity
and deteriorated the PI3K selectivity. While it was intrigu-
ing that removal of the N-methylcarboxamide did not
In an effort to explore the scope of the encouraging data
surrounding 23, disubstituted aryl derivatives were pursued.
Both 26 and 27 exhibited a profile similar to that of the parent
23 (Table 7). All other bis-substituted derivatives (28−29 and
31−32) also displayed excellent mouse plasma exposure. When
comparing the high oral plasma exposure levels observed in
mice for these analogues, in addition to solubility, permeability
was a differentiating factor. Throughout the evolution of the
program, passive membrane permeability was modeled using
an MDCK cell-based assay, and the aryl sulfonamide series
generally displayed apparent permeability (Papp) values of
100−200 nm/s. Therefore, it was not surprising to observe that
31 with excellent solubility (Ksol = 176 μM) and permeability
(550 nm/s) displayed the highest oral plasma exposure in the
bis-substituted aryl series. Additionally, a comparison of 30 and
31 again verified the potency increase observed when chlorine
was placed on the central ring. Further SAR plans involved
5471
dx.doi.org/10.1021/jm300403a | J. Med. Chem. 2012, 55, 5467−5482

Journal of Medicinal Chemistry
Article
Table 6. PI3K Selectivity, Cellular Inhibition, and Mouse Plasma Exposure Data for Chloroaryl Sulfonamide Derivatives
PI3K selectivity IC50 (nM)
a,b
compd
R1
Cl
R2
Cl
R3
H
R4
H
PI3Kγ IC50 (nM)
PI3Kγ cell IC50 (nM)
α
β
δ
mouse plasma exposure
c
23
20
507
476
2404
1020
59.1 4.0 μM (1 h)
c
9.6 μM 1.2(4 h)
d
22
Cl
H
Cl
H
18
184
174
1130
437
1.3 0.6 μM (1 h)
0.1 0.04 μM (4 h)
n.d.
d
24
25
Cl
H
H
H
H
Cl
H
26
42
394
409
142
705
1263
4774
539
e
Cl
1253
0.6 0.07 μM (1 h)
e
0.4 0.1 μM (4 h)
a
b
c
Data are reported as the mean SD. n.d. = not determined. Male C57BL6 mice (n = 3) dosed at 100 mpk (PO) as a suspension in 5% EtOH
d
e
40% PEG400/water. Male BALB/c mice (n = 3) dosed at 100 mpk (PO) as a suspension in 10% EtOH 60% PEG400/water. Male C57BL6 mice
(n = 3) dosed at 100 mpk (PO) as a suspension in 0.75% CMC 0.1% Tween80/water.
Table 7. PI3K Selectivity, Cellular Inhibition, and Mouse Plasma Exposure Data for Bis-Substituted Aryl Sulfonamide
Derivatives
PI3K selectivity IC50 (nM)
a,b
compd
R1
Cl
R2
Cl
R3
H
R4
F
PI3Kγ IC50 (nM)
PI3Kγ cell IC50 (nM)
α
β
δ
mouse plasma exposure
c
26
5
528
209
1069
340
64.8 7.1 μM (1 h)
19.2 2.1 μM (4 h)
236.7 6.5 μM (1 h)
c
c
27
28
29
Cl
Cl
Cl
Cl
Cl
Cl
F
H
Cl
H
Cl
H
21
8
1575
464
972
323
555
4442
1964
1968
1186
1032
743
c
39.8 6.5 μM (4 h)
d
21.3 1.7 μM (1 h)
d
8.6 μM 2.5 (4 h)
d
18
587
172.9 32.3 μM (1 h)
d
22.1 11.1 μM (4 h)
n.d.
30
31
H
F
F
F
F
H
H
102
18
1877
773
2369
435
5016
2059
2452
690
e
Cl
333.4 8.3 μM (1 h)
119.3 μM 40.6 (4 h)
e
e
32
Cl
Me
F
H
34
805
342
1148
415
13.1 4.1 μM (1 h)
e
6.2 2.1 μM (4 h)
a
b
c
Data are reported as the mean SD. n.d. = not determined. Male C57BL6 mice (n = 3) dosed at 100 mpk (PO) as a suspension in 0.75% CMC
d
e
0.1% Tween80/water. Male C57BL6 mice (n = 3) dosed at 100 mpk (PO) as a suspension in 10% EtOH 20% PEG400/water. Male C57BL6 mice
(n = 3) dosed at 100 mpk (PO) as a suspension in 10% EtOH 40% PEG400/water + 1:1 HCl.
significantly influence γ selectivity, the inability to improve
upon the biochemical profile of 31 prompted the deprioritiza-
tion of effort in this area.
degranulation (AIMCD) mouse in vivo model. Response was
measured by quantitating Evans’ blue dye extravasation at 4 h in
the ears of BALB/c mice. Of note, 26, 28, 31, and 32 exhibit a
response analogous to that observed with PI3Kγ-deficient mice
in this assay.50 Additionally, data for 37 (GDC-0941),51 a well
characterized pan-active PI3K inhibitor currently in clinical
trials, provided evidence that knock-down in this assay was not
enhanced by additional inhibition of other PI3K isoforms. A
comparison of 32 to the potent anti-inflammatory steroid
dexamethasone, indicated that while 32 was less potent, it
induced a significant degranulation response in this assay
(Figure 5).
Select advanced analogues (26, 28, and 31−32) demonstrat-
ing >10-fold selectivity vs other PI3K class I isoforms, potent
cellular activity, and excellent mouse plasma exposure were next
examined in a pharmacodynamic assay (Table 9). Both genetic
and pharmacological inhibitions of PI3Kγ are known to reduce
adenosine-evoked mast cell degranulation in vivo.16,50 To
determine the ability of our advanced PI3Kγ inhibitors to affect
mast cell activation in an inflammatory response, they were
tested in an established adenosine-induced mast cell
5472
dx.doi.org/10.1021/jm300403a | J. Med. Chem. 2012, 55, 5467−5482

Journal of Medicinal Chemistry
Article
Table 9. In Vivo Data for Aryl Sulfonamide Advanced Leads
and Pan-Active PI3K Inhibitor 37
PI3Kγ
IC50
PI3Kγ cell adenosine induced mast mouse plasma
a b
compd
(nM)
IC50 (nM) cell degranulation (%)
exposure (4 h)
c
26
28
31
32
37
5
528
464
773
805
298
61 25
65 15
63 21
72 12
29 19
20.3 5.8 μM
Figure 4. Overlay of cocrystal structures of 31/PI3Kγ (green/gray)
and 4/PI3Kγ (magenta/orange) with selectivity residues displayed in
yellow (PDB codes 4anu and 4anw). The difluorophenyl ring of 31
was found to be present and modeled in two equivalently populated
poses, related by a 180° flip of the ring plane.
d
8
6.5 1.1 μM
c
18
34
42
43.1 7.9 μM
c
9.2 3.1 μM
e
5.3 2.1 μM
a
Response was measured by quantitating Evans′ blue (% absorbance at
620 nM relative to vehicle, mean SD, n ≥ 8) extravasation at 4 h in
Table 8. Hinge-Binding Heterocycle Derivatives of 31
the ears of male BALB/c mice. Analogues were dosed at 100 mpk
b
c
orally. Data are reported as the mean
SD. Male BALB/c mice
(n ≥ 8) dosed at 100 mpk (PO) as a suspension in 10% EtOH 40%
d
PEG400/water + 1:1 HCl. Male BALB/c mice (n = 8) dosed at
100 mpk (PO) as a suspension in 10% EtOH 40% PEG400/water.
e
Male BALB/c mice (n = 10) dosed at 100 mpk (PO) as a suspension
in 0.5% CMC 0.2% Tween80/water.
Given the established link between PI3Kγ in preventing joint
damage and neutrophil recruitment in mouse models of
rheumatoid arthritis,43 an experiment was undertaken to
measure the impact that 32 would have on neutrophil
migration (Figure 6). In mice, intraperitoneal injection of the
chemokine RANTES (CCL5) is known to induce neutrophil
recruitment to the peritoneal cavity.43 Oral administration of 32
resulted in a 50% reduction in neutrophil recruitment, thereby
providing evidence of the effect PI3Kγ inhibition can exhibit on
neutrophil migration in vivo.
On the basis of the desirable profile of 31 and 32 in the
mouse, full pharmacokinetic data was gathered in rat (Table 10).
Generally, the exposure data observed between the mouse and
rat correlated appropriately where 31 demonstrated signifi-
cantly higher plasma levels than 32 in both species. High
plasma exposure levels were observed for both analogues
administered as a solution. Bioavailabilities were >60% at 5 mpk
along with a half-life of >4 h for 31. Figure 7 shows the
pharmacokinetic profile of 31 when dosed as a solution in the
rat at 5 mpk.
Figure 5. Inhibition of adenosine-induced mast cell degranulation
(AIMCD) by 32 dosed orally at 100 mpk. Response was measured by
quantitating Evans′ blue extravasation in the ears of male BALB/c mice
at 4 h. Data are reported as the mean SD (n = 8, *P = <0.05).
Vehicle (Ad-, nonsensitized) and Vehicle (Ad+, adenosine sensitized)
formulation: 10% EtOH 40% PEG400/water + 1:1 HCl. Dexametha-
sone (Dex) dosed PO at 3 mpk in 0.5% CMC/0.2% Tween80.
Analogue 32 was dosed as a suspension in 10% EtOH 40% PEG400/
water + 1:1 HCl.
31 and 32 against the vast majority of kinases tested was
>3600 nM. Not surprisingly, cross-reactivity with the closely
related kinase mTOR was observed in both analogues.54 The
CYP, hERG, and P-glycoprotein inhibition profile of 31 was
also characterized (Table 12). The IC50 inhibition measured
was >20,000 nM with the majority of ADME targets profiled.
Collectively, the attractive off-target selectivity and ADME
profile of 31 will allow this analogue to serve as a useful
pharmacological tool.
Chemistry. Outlined in Scheme 1 is the synthesis of HTS
hit 1. Deprotection of 38 under acidic conditions followed by
coupling with 3-bromobenzene-1-sulfonyl chloride provided
bromide 39. A Suzuki−Miyaura cross-coupling utilizing two
Additionally, advanced leads 31 and 32 were also profiled
against a set of kinase targets using previously described
methods (Table 11).52,53 The IC50 inhibition measured for
5473
dx.doi.org/10.1021/jm300403a | J. Med. Chem. 2012, 55, 5467−5482

Journal of Medicinal Chemistry
Article
Table 11. Kinase Selectivity Profile of 31 and 32
kinase
31 inhibition IC50 (nM) 32 inhibition IC50 (nM)
AKT1
>3600
>3600
>3600
>3600
>3600
>3600
206
>3600
>3600
>3600
>3600
>3600
>3600
1814
Aurora B
c-Met
cdc7 MP
cdk1-cyclinB
Chk1 MP
cRaf-1
cRAF/MEK1/ERK2
EGFR
2313
2043
739
410
FGFR1
>3600
>3600
>3600
>3600
>3600
>3600
>3600
>3600
>3600
>3600
>3600
623
>3600
>3600
>3600
>3600
>3600
>3600
>3600
>3600
>3600
>3600
>3600
1781
Flt-3
hPKA Kem
IRK
JAK2
KDR
p70S6k
Figure 6. Analogue 32 dosed orally in male BALB/c mice at 100 mpk
reduced RANTES-induced peritoneal neutrophil recruitment in vivo
by 50%. Total neutrophil count was quantitated using FACS analysis.
Data are reported as the mean SD (n = 5, *P = <0.05). Vehicle
formulation: 10% EtOH 40% PEG400/water + 1:1 HCl. Dexametha-
sone (Dex) dosed PO at 3 mpk in 0.5% CMC/0.2% Tween80.
Analogue 32 was dosed as a suspension in 10% EtOH 40% PEG400/
water + 1:1 HCl.
PDGFRB
PDK1
PKCbetaII
SRC MP
mTOR/GbL/Raptor
Table 12. CYP450, hERG, and P-gp Inhibition Profile of
Compound 31
Table 10. Oral Pharmacokinetic Parameters for 31 and 32 in
Rat (5 mpk, PO)
a
target
inhibition IC50 (nM)
CYP1A2
>20,000
>20,000
2331
parameter
CYP2C19
CYP2C8
CYP2C9
CYP2D6*1
CYP3A4 (substrate: midazolam)
CYP3A4 (substrate: testosterone)
hERG potassium channel
P-glycoprotein
compd
Cmax (μM)
t1/2 (h)
AUC(0‑∞) (μM·h)
% F
31
32
42.5
9.1
4.44
2.08
449.4
26.77
76.7
66.2
591
>20,000
>20,000
>20,000
17,318
>20,000
a
Analogues were dosed in female Sprague−Dawley rats (n = 3) as a
solution in 10% NMP 60% PEG400/water.
a
Scheme 1
Figure 7. Pharmacokinetic profile of 31 in rat. Dosed as a solution in
female Sprague−Dawley rats at 5 mpk using IV formulation, 10%
NMP 60% PEG400/water + 1:1 HCl, and PO formulation, 10% NMP
60% PEG400/water. Data are reported as the mean SD (n = 3).
equivalents of 4-methoxyphenylboronic acid provided the final
product 1.
a
Reagents: (a) 4 N HCl, 1,4-dioxane, rt; (b) N,N-diisopropylethyl-
The synthesis of 3 was carried out in 3 steps starting from
intermediate 39 (Scheme 2). First, a Suzuki−Miyaura cross-
coupling with one equivalent 4-methoxyphenylboronic acid
provided 40, which was followed by boronic ester formation
and another Suzuki−Miyaura coupling with 5-bromopyrimidin-
2-amine.
amine, CH2Cl2, 3-bromobenzene-1-sulfonyl chloride, 0 °C; (c) 4-
methoxyphenylboronic acid, Pd(dppf)Cl2·CH2Cl2, potassium carbo-
nate, 1,4-dioxane, H2O, 90 °C.
tert-butyl piperazine-1-carboxylate to provide 41, followed by
a Miyaura borylation and Suzuki−Miyaura cross-coupling with
5-bromopyrimidin-2-amine to provide 42. Deprotection under
Analogue 4 was constructed as outlined in Scheme 3. The
3-bromobenzene-1-sulfonyl chloride was first coupled to
5474
dx.doi.org/10.1021/jm300403a | J. Med. Chem. 2012, 55, 5467−5482

Journal of Medicinal Chemistry
Article
a
a
Scheme 2
Scheme 3
a
Reagents: (a) 4-methoxyphenylboronic acid, tetrakis-
(triphenylphosphine)palladium(0), potassium carbonate, 1,4-dioxane,
H2O 70 °C; (b) bis(pinacolato)diboron, Pd(dppf)Cl2·CH2Cl2,
potassium acetate, 1,2-dimethoxyethane, 90 °C; (c) 5-bromopyrimi-
din-2-amine, Pd(dppf)Cl2·CH2Cl2, potassium carbonate, 1,4-dioxane,
H2O, 90 °C.
a
Reagents: (a) N,N-diisopropylethylamine, CH2Cl2, 3-bromobenzene-
1-sulfonyl chloride, 0 °C; (b) bis(pinacolato)diboron, Pd(dppf)-
Cl2·CH2Cl2, potassium acetate, 1,2-dimethoxyethane, 90 °C; (c) 5-
bromopyrimidin-2-amine, tetrakis(triphenylphosphine)palladium(0),
potassium carbonate, 1,4-dioxane, H2O, 90 °C; (d) 4 N HCl, 1,4-
dioxane, rt; (e) 2,3-dichloropyrazine, potassium carbonate, dimethy-
lacetamide, 120 °C; (f) 4-isopropylphenylboronic acid, Pd(dppf)-
Cl2·CH2Cl2, potassium carbonate, 1,4-dioxane, H2O, 90 °C.
acidic conditions followed by coupling with 2,3-dichloropyr-
azine afforded 43, and a Suzuki−Miyaura cross-coupling with 4-
isopropylphenylboronic acid provided the final product 4.
The synthesis of optimized lead 5 is described in Scheme 4.
A Buchwald−Hartwig cross-coupling was used to fuse 3-
bromo-2-chloropyridine to tert-butyl piperazine-1-carboxylate
followed by a Suzuki−Miyaura cross-coupling to provide 44.
Deprotection under acidic conditions followed by coupling
with 3-bromobenzene-1-sulfonyl chloride provided 45. Lastly,
the Miyaura borylation/Suzuki−Miyaura cross-coupling se-
quence used to synthesize 4 was also proficient in providing 5.
The synthesis of 3-aminopyrazine-2-carboxamides 7−13 and
18−32 is outlined in Scheme 5. Analogue 14 was synthesized
using a procedure similar to that outlined in Scheme 5 starting
from the known 2-amino-5-bromopyridine-3-sulfonyl chlo-
ride.55 The noncommercially available sulfonyl chlorides were
obtained via a modified Sandmeyer reaction and subsequently
coupled to the appropriate amine.56 The sulfonamide substrate
was then subjected to a single-flask two-step sequence involving
a Miyaura borylation reaction followed by a Suzuki−Miyaura
cross-coupling reaction with 3-amino-6-bromo-N-methylpyra-
zine-2-carboxamide to yield the corresponding analogue.
The synthesis of reverse sulfonamide derivatives 15−17
outlined in Scheme 6 was carried out by coupling the appro-
priate aniline to the corresponding aryl sulfonyl chloride. In a
similar fashion to the analogues synthesized in Scheme 2, a
Suzuki−Miyaura cross-coupling was utilized to provide the final
compounds. Likewise, the aminopyrimidine derivatives 33−36
shown in Scheme 7 were also synthesized in a manner similar
to that used for the derivatives in Scheme 2 but substituting the
appropriate aminopyrimidine in place of 3-amino-6-bromo-N-
methylpyrazine-2-carboxamide.
CONCLUSIONS
■
A series of novel, highly potent, and selective PI3Kγ inhibitors
are disclosed. The sulfonylpiperazine series was optimized
utilizing structure-based design; however, despite improving the
potency and selectivity of this series, desirable pharmacokinetic
properties could not be achieved. Taking advantage of insight
from cocrystal structures of 4 and HTS hit 2 in PI3Kγ provided
a series of novel hybrid sulfonamide analogues with improved
physical properties and pharmacokinetics. Pharmacodynamic
data for lead analogues provided a response similar to that
observed for PI3Kγ deficient mice. In summary, advanced leads
such as 31 contain a desirable potency, selectivity, and
pharmacokinetic profile that will contribute both biological
and chemical insights into the developing area of PI3K research
for the study of inflammatory disease.
EXPERIMENTAL SECTION
■
All reagents and solvents employed were purchased commercially and
used without further purification unless otherwise indicated. NMR
spectra were recorded on a Varian Mercury Plus 400 MHz instrument.
Reported spectra may appear to contain superfluous signals due to the
existence of rotamers and/or carbon−fluorine coupling. Chemical
shifts are reported in parts per million (ppm) relative to an internal
standard of tetramethylsilane in deuterated dimethyl sulfoxide
(DMSO-d6), deuterated methanol (CD3OD), or deuterated chloro-
form (CDCl3). All final compounds were purified to ≥95% purity as
assayed by analytical HPLC (YMC-Pack Pro 150 × 4.6 mm, 5 μm C18
column, Shimadzu LC-10AT VP system equipped with a Shimadzu
SPD-M10A VP diode array detector) at a 1.5 mL/min flow rate with a
5475
dx.doi.org/10.1021/jm300403a | J. Med. Chem. 2012, 55, 5467−5482

Journal of Medicinal Chemistry
Article
a
a
Scheme 4
Scheme 6
a
Reagents: (a) 3-bromoaniline or 5-bromo-2-chloroaniline, pyridine,
CH2Cl2, rt; (b) bis(pinacolato)diboron, Pd(dppf)Cl2·CH2Cl2, potas-
sium acetate, dioxane, 90 °C; 3-amino-6-bromo-N-methylpyrazine-2-
carboxamide, triethylamine, Pd(dppf)Cl2·CH2Cl2, triethylamine, N,N-
dimethylformamide, H2O, 85 °C.
a
Scheme 7
a
Reagents: (a) tert-butyl piperazine-1-carboxylate, xantphos, tris-
(dibenzylideneacetone)dipalladium(0), sodium tert-butoxide, toluene,
100 °C; (b) 4-isopropylphenylboronic acid, Pd(dppf)Cl2·CH2Cl2,
potassium carbonate, 1,4-dioxane, H2O, 90 °C; (c) 4 N HCl, 1,4-
dioxane, rt (d) N,N-diisopropylethylamine, CH2Cl2, 3-bromobenzene-
1-sulfonyl chloride, rt; (e) bis(pinacolato)diboron, Pd(dppf)-
Cl2·CH2Cl2, potassium acetate, 1,2-dimethoxyethane, 90 °C; (f) 5-
bromopyrimidin-2-amine, Pd(dppf)Cl2·CH2Cl2, potassium carbonate,
1,4-dioxane, H2O, 90 °C.
a
Scheme 5
a
Reagents: (a) NaNO2, H2O, HCl, 0 °C; SO2, CuCl, AcOH, 0 °C to
rt; (b) 2,3-difluoroaniline, pyridine, CH2Cl2, rt; (c) bis(pinacolato)-
diboron, Pd(dppf)Cl2·CH2Cl2, potassium acetate, dioxane, 90 °C; RBr,
Pd(dppf)Cl2·CH2Cl2, triethylamine, N,N-dimethylformamide, H2O,
85 °C.
for 2 h. The reaction mixture was concentrated and used directly for
the next step.
a
Reagents: (a) NaNO2, H2O, HCl, 0 °C; SO2, CuCl, AcOH, 0 °C to
To a stirred mixture of crude piperazine (962 mg, 3.00 mmol), N,N-
diisopropylethylamine (2.61 mL, 15.0 mmol), and CH2Cl2 (20 mL)
was added 3-bromobenzenesulfonyl chloride (770 mg, 3.03 mmol)
dropwise at 0 °C. After stirring for 30 min, saturated sodium
bicarbonate was added, and the resulting solution was extracted with
CH2Cl2 (2×). The combined organic layers were dried over anhydrous
sodium sulfate and concentrated, and the residue was triturated with
ethyl acetate and hexanes to afford 39 (1.04 g, 83%).
rt; (b) R2NH2, pyridine, CH2Cl2, rt; (c) bis(pinacolato)diboron,
Pd(dppf)Cl2·CH2Cl2, potassium acetate, dioxane, 90 °C; 3-amino-6-
bromo-N-methylpyrazine-2-carboxamide, triethylamine, Pd(dppf)-
Cl2·CH2Cl2, triethylamine, N,N-dimethylformamide, H2O, 85 °C.
gradient of 5−95% acetonitrile (containing 0.1% TFA) in 0.1%
aqueous TFA for 25 min and total run time of 27 min. Analogues 2
and 6 were synthesized as previously described.45 Mouse liver
microsome assay data, reported as the mean SD, was carried out
as previously described.57
2-(4-(4′-Methoxybiphenyl-3-ylsulfonyl)piperazin-1-yl)-3-(4-
methoxyphenyl)pyrazine (1). To a stirred solution of 38 (896 mg,
3.00 mmol) in 1,4-dioxane (20 mL) was added HCl (5 mL, 4 N in 1,4-
dioxane), and the reaction mixture was stirred at ambient temperature
A mixture of 39 (125 mg, 0.300 mmol), 4-methoxyphenylboronic
acid (137 mg, 0.901 mmol), potassium carbonate (415 mg, 3.00
mmol), Pd(dppf)Cl2·CH2Cl2 (49.0 mg, 60.0 μmol), 1,4-dioxane
(4 mL), and H2O (1 mL) was stirred at 90 °C for 2 h. The reaction
mixture was concentrated and purified by flash chromatography
(hexanes/ethyl acetate, 2:1) to provide 1 (117 mg, 75%) as a white
1
powder after lyophilization. H NMR (400 MHz, CDCl3) δ 8.14 (d,
J = 2.5 Hz, 1H), 8.01 (d, J = 2.5 Hz, 1H), 7.91 (t, J = 1.7 Hz, 1H),
5476
dx.doi.org/10.1021/jm300403a | J. Med. Chem. 2012, 55, 5467−5482

Journal of Medicinal Chemistry
Article
7.84−7.79 (m, 1H), 7.77−7.72 (m, 2H), 7.70−7.66 (m, 1H), 7.61 (d,
J = 7.7 Hz, 1H), 7.59−7.54 (m, 2H), 7.05−7.01 (m, 2H), 6.89−6.84
(m, 2H), 3.88 (s, 3H), 3.80 (s, 3H), 3.32−3.22 (m, 4H), 3.12−3.00
(m, 4H). MS (EI) for C28H28N4O4S, found 517.2 (MH+).
5-(3-(4-(3-(4-Methoxyphenyl)pyrazin-2-yl)piperazin-1-
ylsulfonyl)phenyl)pyrimidin-2-amine (3). A mixture of 39 (251
mg, 0.603 mmol), 4-methoxyphenylboronic acid (91.0 mg, 0.599
mmol), potassium carbonate (415 mg, 3.00 mmol), tetrakis-
(triphenylphosphine)palladium(0) (69.4 mg, 60.0 μmol), 1,4-dioxane
(4 mL), and H2O (1 mL) was stirred at 70 °C for 2 h. The reaction
mixture was concentrated and purified by flash chromatography
(hexanes/ethyl acetate, 5:1) to provide 40 (251 mg, 85%).
A mixture of 40 (251 mg, 0.510 mmol), bis(pinacolato)diboron
(260 mg, 1.02 mmol), Pd(dppf)Cl2·CH2Cl2 (41.6 mg, 51.0 μmol),
potassium acetate (200 mg, 2.04 mmol), and 1,2-dimethoxyethane
(6 mL) was stirred at 90 °C for 2 h. The reaction mixture was cooled
to ambient temperature, concentrated, and purified by flash
chromatography (hexanes/ethyl acetate, 3:1) followed by trituration
with hexanes to afford 2-(4-methoxyphenyl)-3-(4-(3-(4,4,5,5-tetra-
methyl-1,3,2-dioxaborolan-2-yl)phenylsulfonyl)piperazin-1-yl)pyrazine
(236 mg, 86%) as an off-white solid.
A mixture of 2-(4-methoxyphenyl)-3-(4-(3-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenylsulfonyl)piperazin-1-yl)pyrazine (96.5
mg, 0.180 mmol), 5-bromopyrimidin-2-amine (41.5 mg, 0.240
mmol), potassium carbonate (150 mg, 1.09 mmol), Pd(dppf)-
Cl2·CH2Cl2 (16.3 mg, 20 μmol), 1,4-dioxane (4 mL), and H2O
(1 mL) was stirred at 90 °C for 1 h. The reaction mixture was cooled
to ambient temperature, concentrated, and purified by flash chromatog-
raphy (ethyl acetate/hexanes, 3:1) to afford 3 (54.0 mg, 60%) as a
white powder after lyophilization. 1H NMR (400 MHz, CDCl3) δ 8.57
(s, 2H), 8.15 (d, J = 2.0 Hz, 1H), 8.02 (d, J = 2.0 Hz, 1H), 7.86 (br s
1H), 7.76−7.73 (m, 4H), 7.65 (t, J = 8.4 Hz, 1H), 6.88 (d, J = 8.4 Hz,
2H), 5.29 (s, 2H), 3.83 (s, 3H), 3.30−3.28 (m, 4H), 3.10−3.08 (m,
4H). MS (EI) for C25H25N7O3S, found 504.1 (MH+).
5-(3-(4-(3-(4-Isopropylphenyl)pyrazin-2-yl)piperazin-1-
ylsulfonyl)phenyl)pyrimidin-2-amine (4). To a stirred solution of
tert-butyl piperazine-1-carboxylate (5.59 g, 30.0 mmol) and N,N-
diisopropylethylamine (6.30 mL, 36.0 mmol) in CH2Cl2 (60 mL) was
added 3-bromobenzene-1-sulfonyl chloride dropwise at 0 °C. After
stirring for 30 min, H2O was added, and the resulting solution was
extracted with CH2Cl2 (2×). The combined organic layers were dried
over anhydrous sodium sulfate, concentrated, and purified by flash
chromatography (hexanes/ethyl acetate, 5:1) followed by trituration
with hexanes to provide 41 (11.9 g, 98%) as an off-white solid.
A mixture of 41 (8.10 g, 20.0 mmol), bis(pinacolato)diboron (10.2
g, 40.0 mmol), Pd(dppf)Cl2·CH2Cl2 (820 mg, 1.00 mmol), potassium
acetate (7.85 g, 80.0 mmol), and 1,2-dimethoxyethane (40 mL) was
stirred at 90 °C for 2 h. The reaction mixture was cooled to ambient
temperature, concentrated, and purified by flash chromatography
(hexanes/ethyl acetate, 5:1) followed by trituration with hexanes to
afford tert-butyl 4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
phenylsulfonyl)piperazine-1-carboxylate (9.1 g, quant.) as a pale pink
solid.
A mixture of tert-butyl 4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)phenylsulfonyl)piperazine-1-carboxylate (9.10 g, 20.0 mmol), 5-
bromopyrimidin-2-amine (3.48 g, 20.0 mmol), potassium carbonate
(8.30 g, 60.0 mmol), and tetrakis(triphenylphosphine)palladium(0)
(817 mg, 1.00 mmol), 1,4-dioxane (40 mL), and H2O (10 mL) was
stirred at 90 °C for 1 h. The reaction mixture was cooled to ambient
temperature, concentrated, and purified by flash chromatography
(ethyl acetate/hexanes 3:1) followed by trituration with ethyl acetate
to afford 42 (6.49 g, 77%) as an off-white solid.
at 120 °C overnight. The reaction mixture was cooled to ambient
temperature, diluted with H2O, the precipitate was collected, and the
separated aqueous layer was extracted with ethyl acetate (2×). The
combined organic layers were dried over anhydrous sodium sulfate and
concentrated to give a light-brown solid. The solid and precipitate
were combined, triturated with ethyl acetate, filtered, and dried to
afford 43 (4.5 g). Another crop (0.3 g) was precipitated from the
filtrate to provide a total yield of 72% in this manner.
A mixture of 43 (43 mg, 99.5 μmol), 4-isopropylphenylboronic acid
(24.6 mg, 0.150 mmol), potassium carbonate (69.0 mg, 0.500 mmol),
and Pd(dppf)Cl2·CH2Cl2 (8.2 mg, 10 μmol), 1,4-dioxane (4 mL), and
H2O (1 mL) was stirred at 90 °C for 1 h. The reaction mixture was
cooled to ambient temperature, concentrated, and purified by flash
chromatography (ethyl acetate/hexanes, 4:1) to afford 4 (32.0 mg,
1
62%) as a white powder after lyophilization. H NMR (400 MHz,
DMSO-d6) δ 8.67 (s, 2H), 8.18 (d, J = 2.5 Hz, 1H), 8.11 (d, J = 2.5
Hz, 1H), 8.03 (d, J = 7.9 Hz, 1H), 7.87 (s, 1H), 7.77−7.62 (m, 4H),
7.19 (d, J = 8.2 Hz, 2H), 6.97 (s, 2H), 3.17−3.14 (m, 4H), 2.97−2.95
(m, 4H), 2.85 (dt, J = 13.9, 7.1 Hz, 1H), 1.17 (d, J = 6.9 Hz, 6H). MS
(EI) for C27H29N7O2S, found 516.3 (MH+).
5-(3-(4-(2-(4-Isopropylphenyl)pyridin-3-yl)piperazin-1-
ylsulfonyl)phenyl)pyrimidin-2-amine (5). A solution of 3-bromo-
2-chloropyridine (750 mg, 3.90 mmol), tert-butyl piperazine-1-
carboxylate (559 mg, 3.00 mmol), sodium tert-butoxide (432 mg,
4.50 mmol), tris(dibenzylideneacetone)dipalladium(0) (55.0 mg,
60.0 μmol), xantphos (104 mg, 0.180 mmol), and toluene (15 mL)
was stirred at 100 °C for 2 h. The reaction mixture was cooled to
ambient temperature, diluted with H2O, extracted with ethyl acetate
(2×), and washed with brine. The separated organic layer was dried
over sodium sulfate, concentrated, and purified by flash chromato-
graphy (hexanes/ethyl acetate, 10:1) to give tert-butyl 4-(2-chloro-
pyridin-3-yl)piperazine-1-carboxylate (747 mg, 84%).
A mixture of tert-butyl 4-(2-chloropyridin-3-yl)piperazine-1-carbox-
ylate (505 mg, 1.70 mmol), 4-isopropylphenylboronic acid (335 mg,
2.00 mmol), potassium carbonate (705 mg, 5.10 mmol), Pd(dppf)-
Cl2·CH2Cl2 (139 mg, 0.170 mmol), 1,4-dioxane (15 mL), and H2O
(5 mL) was stirred at 90 °C for 1 h. The reaction mixture was cooled to
ambient temperature, concentrated, and purified by flash chromatography
(hexanes/ethyl acetate, 10:1) to afford 44 (541 mg, 84%).
To a stirred solution of 44 (541 mg, 1.42 mmol) in 1,4-dioxane
(8 mL) was added HCl (5 mL, 4 N in 1,4-dioxane), and the reaction
mixture was stirred at ambient temperature overnight. The reaction
mixture was concentrated and used directly for the next step.
To a stirred mixture of crude 1-(2-(4-isopropylphenyl)pyridin-3-
yl)piperazine and N,N-diisopropylethylamine (1.23 mL, 3.75 mmol) in
CH2Cl2 (6 mL) was added 3-bromophenylsulfonyl chloride (397 mg,
1.55 mmol). After stirring for 30 min at ambient temperature, H2O
was added, and the resulting solution was extracted with CH2Cl2 (2×).
The combined organic layers were dried over anhydrous sodium
sulfate, concentrated, and purified by flash chromatography (hexanes/
ethyl acetate, 3:1) to provide 45 (647 mg, 92% in 2 steps)
A mixture of 45 (375 mg, 0.750 mmol), bis(pinacolato)diboron
(571 mg, 2.25 mmol), Pd(dppf)Cl2·CH2Cl2 (61.2 mg, 75.0 μmol),
potassium acetate (442 mg, 4.50 mmol), and 1,2-dimethoxyethane (8
mL) was stirred at 90 °C for 1 h. The reaction mixture was cooled to
ambient temperature, concentrated, and purified by flash chromatog-
raphy (hexanes/ethyl acetate, 2:1) followed by trituration with hexanes
to afford 1-(2-(4-isopropylphenyl)pyridin-3-yl)-4-(3-(4,4,5,5-tetra-
methyl-1,3,2-dioxaborolan-2-yl)phenylsulfonyl)piperazine (337 mg,
82%) as a white powder.
A mixture of 1-(2-(4-isopropylphenyl)pyridin-3-yl)-4-(3-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)phenylsulfonyl)piperazine (137
mg, 0.250 mmol), 5-bromopyrimidin-2-amine (51.9 mg, 0.300
mmol), potassium carbonate (138 mg, 1.00 mmol), Pd(dppf)-
Cl2·CH2Cl2 (20.4 mg, 25.0 μmol), 1,4-dioxane (4 mL), and H2O
(1 mL) was stirred at 90 °C for 1 h. The reaction mixture was cooled
to ambient temperature, concentrated, and purified by flash chromatog-
raphy (ethyl acetate/hexanes, 3:1) followed by reverse phase HPLC
(acetonitrile/aqueous ammonium acetate buffer solution; 30−100%
gradient) to provide 5 (64.0 mg, 50%) as a white powder after
To a stirred solution of 42 (6.49 g, 15.4 mmol) in 1,4-dioxane
(60 mL) was added HCl (30 mL, 4 N in 1,4-dioxane), and the
resulting mixture was stirred at ambient temperature overnight. The
reaction mixture was concentrated and used for the next step without
further purification.
A mixture of crude 5-(3-(piperazin-1-ylsulfonyl)phenyl)pyrimidin-
2-amine, potassium carbonate (21.3 g, 154 mmol), 2,3-dichloropyr-
azine (3.44 g, 23.1 mmol) in dimethylacetamide (120 mL) was stirred
5477
dx.doi.org/10.1021/jm300403a | J. Med. Chem. 2012, 55, 5467−5482

Journal of Medicinal Chemistry
Article
1
lyophilization. H NMR (400 MHz, DMSO-d6) δ 8.69 (s, 2H), 8.27
7.16 (d, J = 7.6 Hz, 2H), 7.01 (t, J = 7.2 Hz, 1H), 2.85 (d, J = 4.8 Hz,
3H). MS (EI) for C18H16FN5O3S, found 402.1 (MH+).
(dd, J = 4.0, 1.2 Hz, 1H), 8.07 (t, J = 1.2 Hz, 1H), 7.87 (t, J = 1.6 Hz,
1H), 7.75 (t, J = 8.0 Hz, 1H), 7.67−7.65 (m, 3H), 7.47 (dd, J = 8.4, 1.6
Hz, 1H), 7.26 (dd, J = 8.4, 4.8 Hz, 1H), 7.06 (d, J = 8.4 Hz, 2H), 6.98
(s, 2H), 2.91−2.74 (m, 9H), 1.11 (d, J = 7.2 Hz, 6H). MS (EI) for
C28H30N6O2S found 515.1 (MH+).
3-Amino-6-(6-amino-5-(N-phenylsulfamoyl)pyridin-3-yl)-N-
methylpyrazine-2-carboxamide (14). 1H NMR (400 MHz,
DMSO-d6) δ 10.47 (s, 1H), 8.98 (d, J = 2.4 Hz, 1H), 8.76 (q, J =
4.4 Hz, 1H), 8.66 (s, 1H), 8.40 (d, J = 2.4 Hz, 1H), 7.61 (br s, 2H),
7.25−7.21 (m, 2H), 7.12 (d, J = 7.2 Hz, 2H), 7.00 (t, J = 7.4 Hz, 1H),
6.87 (s, 2H), 2.83 (d, J = 4.4 Hz, 3H). MS (EI) for C18H18N6O3S,
found 400.2 (MH+).
3-Amino-6-(4-chloro-3-(N-m-tolylsulfamoyl)phenyl)-N-
methylpyrazine-2-carboxamide (18). A solution of SO2/CuCl/
AcOH was formed by bubbling sulfur dioxide gas into acetic acid (424
mL) for 10 min at 0 °C to saturate and adding CuCl (7.15 g, 73.0
mmol). In a separate flask, an aqueous solution of NaNO2 (25.0 g,
0.362 mol) in H2O (380 mL) was added to a precooled (0 °C)
suspension of 5-bromo-2-chloroaniline (50.0 g, 242 mol) in conc. HCl
(aq., 380 mL). After stirring for 1 h, the reaction mixture was added to
the SO2/CuCl/AcOH solution at 0 °C via an addition funnel over 15
min. The ice bath was removed, and the reaction mixture was allowed
to stir at ambient temperature for 2 h, then diluted with H2O (1200
mL) and filtered. The off-white solid product was concentrated in
vacuo overnight to provide 63.6 g (91%) of 5-bromo-2-chlorobenzene-
1-sulfonyl chloride. 1H NMR (400 MHz, CDCl3) δ 8.28 (d, J = 2.3 Hz,
1H), 7.78 (dd, J = 8.5, 2.3 Hz, 1H), 7.52 (d, J = 8.5 Hz, 1H).
A mixture of 5-bromo-2-chlorobenzene-1-sulfonyl chloride (500
mg, 1.72 mmol), m-toluidine (202 mg, 1.89 mmol), pyridine (1.89
mmol, 153 μL), and CH2Cl2 (5 mL) was stirred for 30 min then
purified directly by flash chromatography (hexanes/ethyl acetate 4:1)
to provide 5-bromo-2-chloro-N-m-tolylbenzenesulfonamide (521 mg,
84%) as an off-white solid.
3-Amino-6-(4-chloro-3-(N-(3-fluorophenyl)sulfamoyl)-
1
phenyl)-N-methylpyrazine-2-carboxamide (19). H NMR (400
MHz, DMSO-d6) δ 11.00 (s, 1H), 8.88 (s, 1H), 8.80 (d, J = 4.8 Hz,
1H), 8.62 (d, J = 2.0 Hz, 1H), 8.46 (dd, J = 8.4, 2.1 Hz, 1H), 8.34−
7.37 (m, 3H), 7.26 (dd, J = 15.1, 8.1 Hz, 1H), 7.10−6.91 (m, 2H),
6.82 (td, J = 8.5, 1.9 Hz, 1H), 2.86 (d, J = 4.8 Hz, 3H). MS (EI) for
C18H15ClFN5O3S, found 436.0 (MH+).
3-Amino-6-(4-chloro-3-(N-(3-methoxyphenyl)sulfamoyl)-
1
phenyl)-N-methylpyrazine-2-carboxamide (20). H NMR (400
MHz, DMSO-d6) δ 10.69 (s, 1H), 8.83 (s, 1H), 8.77 (dd, J = 8.1, 4.9
Hz, 1H), 8.58 (d, J = 2.0 Hz, 1H), 8.42 (d, J = 7.8 Hz, 1H), 8.27−7.33
(m, 3H), 7.10 (t, J = 8.3 Hz, 1H), 6.78−6.62 (m, 2H), 6.61−6.48 (m,
1H), 3.61 (s, 3H), 2.85 (d, J = 4.8 Hz, 3H). MS (EI) for
C19H18ClN5O4S, found 448.0 (MH+).
3-Amino-6-(4-chloro-3-(N-(3-(trifluoromethyl)phenyl)-
sulfamoyl)phenyl)-N-methylpyrazine-2-carboxamide (21). 1H
NMR (400 MHz, DMSO-d6) δ 11.18 (s, 1H), 8.86 (s, 1H), 8.80 (q,
J = 4.5 Hz, 1H), 8.63 (d, J = 2.2 Hz, 1H), 8.46 (dd, J = 8.4, 2.2 Hz, 1H),
8.27−7.52 (m, 3H), 7.54−7.40 (m, 3H), 7.42−7.26 (m, 1H), 2.86 (d, J =
4.8 Hz, 3H). MS (EI) for C19H15ClF3N5O3S, found 486.0 (MH+).
3-Amino-6-(4-chloro-3-(N-(3-chlorophenyl)sulfamoyl)-
A mixture of 5-bromo-2-chloro-N-m-tolylbenzenesulfonamide (521
mg, 1.44 mmol), bis(pinacolato)diboron (403 mg, 1.59 mmol),
Pd(dppf)Cl2·CH2Cl2 (105 mg, 0.129 mmol), potassium acetate (423
mg, 4.32 mmol), and dioxane (10 mL) was heated to 90 °C in a sealed
tube. After 3 h, the reaction mixture was cooled to ambient
temperature, and 3-amino-6-bromo-N-methylpyrazine-2-carboxamide
(100 mg, 0.433 mmol), Pd(dppf)Cl2·CH2Cl2 (42 mg, 0.051 mmol),
triethylamine (238 μL, 1.84 mmol), N,N-dimethylformamide (2 mL),
and H2O (2 mL) were added. The reaction mixture was heated to
85 °C. After 2 h, the mixture was filtered through celite and purified
directly by reverse phase HPLC (acetonitrile/aqueous ammonium
acetate buffer solution; 10−90% gradient) to provide 18 (121 mg, 65%
1
phenyl)-N-methylpyrazine-2-carboxamide (22). H NMR (400
MHz, DMSO-d6) δ 11.00 (s, 1H), 8.86 (s, 1H), 8.81 (q, J = 4.6 Hz,
1H), 8.62 (s, 1H), 8.46 (dd, J = 8.4, 2.2 Hz, 1H), 8.35−7.34 (m, 3H),
7.25−7.19 (m, 2H), 7.14 (ddd, J = 8.2, 2.1, 0.9 Hz, 1H), 7.05 (ddd, J =
8.0, 2.0, 0.9 Hz, 1H), 2.86 (d, J = 4.8 Hz, 3H). MS (EI) for
C18H15Cl2N5O3S, found 452.0 (MH+).
3-Amino-6-(4-chloro-3-(N-(2-chlorophenyl)sulfamoyl)-
1
phenyl)-N-methylpyrazine-2-carboxamide (23). H NMR (400
MHz, DMSO-d6) δ 10.29 (s, 1H), 8.88−8.63 (m, 2H), 8.46 (d, J =
10.2 Hz, 1H), 8.41 (s, 1H), 8.12−7.55 (m, 3H), 7.43 (d, J = 7.8 Hz,
1H), 7.34−7.14 (m, 3H), 2.83 (d, J = 4.8 Hz, 3H). MS (EI) for
C18H15Cl2N5O3S, found 451.7 (MH+).
1
in 2 steps) as an off-white solid. H NMR (400 MHz, DMSO-d6) δ
10.60 (s, 1H), 8.83 (s, 1H), 8.79 (dd, J = 12.0, 4.0 Hz, 1H), 8.57 (d,
J = 2.2 Hz, 1H), 8.41 (dd, J = 8.4, 2.1 Hz, 1H), 7.88 (s, 1H), 7.64−
7.37 (m, 2H), 7.08 (t, J = 7.8 Hz, 1H), 7.02−6.88 (m, 2H), 6.79 (d, J =
7.9 Hz, 1H), 2.85 (d, J = 4.8 Hz, 3H), 2.16 (s, 3H). MS (EI) for
C19H18ClN5O3S, found 431.8 (MH+).
Compounds 7−8, 12−14, and 19−36 were prepared by a
procedure similar to that described for the synthesis of compound 18.
3-Amino-N-methyl-6-(3-(N-phenylsulfamoyl)phenyl)-
pyrazine-2-carboxamide (7). 1H NMR (400 MHz, CD3OD) δ 8.61
(s, 1H), 8.30−8.22 (m, 2H), 7.72−7.70 (m, 1H), 7.55 (t, J = 7.6 Hz,
1H), 7.24−7.20 (m, 2H), 7.12−7.04 (m, 3H), 2.97 (s, 3H). MS (EI)
for C18H17N5O3S, found 382.1 (MH+).
3-Amino-6-(4-chloro-3-(N-(4-chlorophenyl)sulfamoyl)-
1
phenyl)-N-methylpyrazine-2-carboxamide (24). H NMR (400
MHz, DMSO-d6) δ 10.86 (s, 1H), 8.85 (s, 1H), 8.79 (q, J = 4.8 Hz,
1H), 8.57 (d, J = 2.2 Hz, 1H), 8.45 (dd, J = 8.4, 2.2 Hz, 1H), 8.30−
7.43 (m, 3H), 7.28 (d, J = 8.9 Hz, 2H), 7.16 (d, J = 8.9 Hz, 2H), 2.85
(d, J = 4.8 Hz, 3H). MS (EI) for C18H15Cl2N5O3S, found 452.0
(MH+).
3-Amino-6-(3-(N-(3-chlorophenyl)sulfamoyl)phenyl)-N-
methylpyrazine-2-carboxamide (25). 1H NMR (400 MHz,
CD3OD) δ 8.65 (s, 1H), 8.35 (t, J = 1.7 Hz, 1H), 8.32−8.24 (m,
1H), 8.06−7.40 (m, 6H), 7.24−7.13 (m, 2H), 7.09−7.00 (m, 2H),
2.98 (s, 3H). MS (EI) for C18H16ClN5O3S, found 416.1 (MH+).
3-Amino-6-(4-chloro-3-(N-(2-chloro-4-fluorophenyl)-
sulfamoyl)phenyl)-N-methylpyrazine-2-carboxamide (26). 1H
NMR (400 MHz, DMSO-d6) δ 10.34 (s, 1H), 8.76 (s, 2H), 8.48 (d,
J = 6.2 Hz, 1H), 8.38 (s, 1H), 7.97−7.53 (m, 3H), 7.46 (dd, J = 8.5, 2.9
Hz, 1H), 7.29 (dd, J = 9.0, 5.7 Hz, 1H), 7.22−7.14 (m, 1H), 2.83 (d, J =
4.8 Hz, 3H). MS (EI) for C18H14Cl2FN5O3S, found 469.7 (MH+).
3-Amino-6-(4-chloro-3-(N-(2-chloro-3-fluorophenyl)-
sulfamoyl)phenyl)-N-methylpyrazine-2-carboxamide (27). 1H
NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 8.89−8.69 (m, 2H),
8.48 (dd, J = 8.4, 2.1 Hz, 1H), 8.45 (d, J = 2.2 Hz, 1H), 8.14−7.49 (m,
3H), 7.40−7.19 (m, 2H), 7.14 (d, J = 8.0 Hz, 1H), 2.83 (d, J = 4.8 Hz,
3H). MS (EI) for C18H14Cl2FN5O3S, found 469.7 (MH+).
3-Amino-6-(4-chloro-3-(N-phenylsulfamoyl)phenyl)-N-meth-
ylpyrazine-2-carboxamide (8). H NMR (400 MHz, DMSO-d6) δ
1
10.69 (s, 1H), 8.84 (s, 1H), 8.79 (q, J = 4.6 Hz, 1H), 8.57 (d, J = 2.4
Hz, 1H), 8.43 (dd, J = 8.4, 2.4 Hz, 1H), 7.82 (br s, 2H), 7.70 (d, J =
8.0 Hz, 1H), 7.24−7.15 (m, 4H), 7.00−6.96 (m, 1H), 2.85 (d, J = 5.2
Hz, 3H). MS (EI) for C18H16ClN5O3S, found 418.0 (MH+).
3-Amino-6-(4-methoxy-3-(N-phenylsulfamoyl)phenyl)-N-
methylpyrazine-2-carboxamide (12). 1H NMR (400 MHz,
DMSO-d6) δ 10.07 (s, 1H), 8.75 (q, J = 4.6 Hz, 1H), 8.73 (s, 1H),
8.35 (dd, J = 8.4, 2.4 Hz, 1H), 8.31 (d, J = 2.4 Hz, 1H), 7.63 (br s,
2H), 7.23 (d, J = 8.8 Hz, 1H), 7.20−7.11 (m, 4H), 6.96−6.92 (m,
1H), 3.94 (s, 3H), 2.84 (d, J = 4.8 Hz, 3H). MS (EI) for
C19H19N5O4S, found 414.0 (MH+).
3-Amino-6-(4-chloro-3-(N-(2,4-dichlorophenyl)sulfamoyl)-
1
phenyl)-N-methylpyrazine-2-carboxamide (28). H NMR (400
3-Amino-6-(4-fluoro-3-(N-phenylsulfamoyl)phenyl)-N-meth-
ylpyrazine-2-carboxamide (13). 1H NMR (400 MHz, DMSO-d6) δ
10.68 (s, 1H), 8.83−8.79 (m, 2H), 8.50−8.47 (m, 1H), 8.41−8.37 (m,
1H), 7.78 (br s, 2H), 7.51 (t, J = 9.2 Hz, 1H), 7.23 (t, J = 7.6 Hz, 2H),
MHz, DMSO-d6) δ 10.44 (s, 1H), 8.87−8.56 (m, 2H), 8.58−8.28 (m,
2H), 7.91−7.19 (m, 6H), 2.84 (d, J = 4.8 Hz, 3H). MS (EI) for
C18H14Cl3N5O3S, found 483.7 (MH−).
5478
dx.doi.org/10.1021/jm300403a | J. Med. Chem. 2012, 55, 5467−5482

Journal of Medicinal Chemistry
Article
3-Amino-6-(4-chloro-3-(N-(2,3-dichlorophenyl)sulfamoyl)-
(10 mL). The organic solvent was removed in vacuo, and the aqueous
layer was diluted with CH2Cl2 and H2O. The separated aqueous layer
was extracted with CH2Cl2 (2×), and the combined organic layers
were dried over sodium sulfate, filtered, and concentrated in vacuo.
The residue was purified by flash chromatography (hexanes/ethyl
acetate, 3:1) to provide 5-bromo-4-(2,2,2-trifluoroethoxy)pyrimidin-2-
amine (2.43 g, 89%) as an off-white powder. MS (EI) for
C6H5BrF3N3O, found 271.9 (MH+).
Analogue 36 was synthesized using a procedure similar to that
described for the synthesis of compound 18 using 5-bromo-4-(2,2,2-
trifluoroethoxy)pyrimidin-2-amine in place of 3-amino-6-bromo-N-
methylpyrazine-2-carboxamide. 1H NMR (400 MHz, DMSO-d6) δ
10.75 (s, 1H), 8.23 (s, 1H), 8.10 (d, J = 2.0 Hz, 1H), 7.76−7.70 (m,
2H), 7.22−7.20 (m, 1H), 7.09−7.05 (m, 4H), 4.98 (q, J = 9.1 Hz,
2H). MS (EI) for C18H12ClF5N4O3S, found 494.9 (MH+).
1
phenyl)-N-methylpyrazine-2-carboxamide (29). H NMR (400
MHz, DMSO-d6) δ 10.54 (s, 1H), 8.95−8.63 (m, 2H), 8.59−8.33 (m,
2H), 8.02−7.15 (m, 6H), 2.83 (d, J = 4.8 Hz, 3H). MS (EI) for
C18H14Cl3N5O3S, found 485.7 (MH+).
3-Amino-6-(3-(N-(2,3-difluorophenyl)sulfamoyl)phenyl)-N-
methylpyrazine-2-carboxamide (30). 1H NMR (400 MHz,
DMSO-d6) δ 10.78 (s, 1H), 8.96 (s, 1H), 8.93 (q, J = 4.6 Hz, 1H),
8.45 (d, J = 2.3 Hz, 1H), 8.33 (dd, J = 8.5, 2.3 Hz, 1H), 7.97 (t, J = 2.0
Hz, 1H), 7.91−7.45 (m, 4H), 7.49−7.34 (m, 1H), 7.20 (dd, J = 7.6,
1.7 Hz, 1H), 2.84 (d, J = 4.8 Hz, 3H). MS (EI) for C18H15F2N5O3S,
found 419.9 (MH+).
3-Amino-6-(4-chloro-3-(N-(2,3-difluorophenyl)sulfamoyl)-
1
phenyl)-N-methylpyrazine-2-carboxamide (31). H NMR (400
MHz, DMSO-d6) δ 10.80 (s, 1H), 8.83−8.61 (m, 2H), 8.47 (d, J = 2.2
Hz, 1H), 8.40 (d, J = 7.7 Hz, 1H), 8.24−7.36 (m, 3H), 7.15−6.85 (m,
3H), 2.84 (d, J = 4.8 Hz, 3H). MS (EI) for C18H14ClF2N5O3S, found
454.0 (MH+).
3-Amino-6-(4-chloro-3-(N-(3-fluoro-2-methylphenyl)-
sulfamoyl)phenyl)-N-methylpyrazine-2-carboxamide (32). 1H
NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H), 8.86−8.63 (m, 2H),
8.48 (dd, J = 8.4, 2.3 Hz, 1H), 8.39 (d, J = 2.2 Hz, 1H), 8.11−7.30 (m,
3H), 7.11 (dd, J = 15.0, 7.9 Hz, 1H), 7.02 (t, J = 8.8 Hz, 1H), 6.85 (d,
J = 7.9 Hz, 1H), 2.83 (d, J = 4.8 Hz, 3H), 2.12 (d, J = 2.1 Hz, 3H). MS
(EI) for C19H17ClFN5O3S, found 450.0 (MH+).
5-(5-Aminopyrazin-2-yl)-2-chloro-N-(2,3-difluorophenyl)-
benzenesulfonamide (33). 1H NMR (400 MHz, DMSO-d6) δ
10.76 (s, 1H), 8.57 (s, 1H), 8.49 (d, J = 2.2 Hz, 1H), 8.16 (d, J = 7.5
Hz, 1H), 7.96 (d, J = 1.4 Hz, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.20 (s,
1H), 7.15−7.01 (m, 2H), 6.81 (s, 2H). MS (EI) for
C16H11ClF2N4O2S, found 397.0 (MH+).
5-(2-Aminopyrimidin-5-yl)-2-chloro-N-(2,3-difluorophenyl)-
benzenesulfonamide (34). 1H NMR (400 MHz, DMSO-d6) δ 8.45
(s, 2H), 8.06 (d, J = 2.3 Hz, 1H), 7.81−7.10 (m, 3H), 6.98−6.82 (m,
3H), 6.66 (dd, J = 14.0, 6.9 Hz, 1H), 6.44 (s, 1H). MS (EI) for
C16H11ClF2N4O2S, found 397.0 (MH+).
5-(2-Amino-4-methoxypyrimidin-5-yl)-2-chloro-N-(2,3-
difluorophenyl)benzenesulfonamide (35). To a stirred solution
of 2-amino-4-chloropyrimidine (2.58 g, 20.0 mmol) in acetic acid
(40 mL) was added dropwise bromine (1.03 mL, 20.0 mmol) at
ambient temperature. After stirring for 2 h, the precipitate was
collected by filtration. The yellow solid was suspended with H2O, and
the pH of the mixture was adjusted to pH 8−9 using 50% aq. NaOH.
After sonicatation for 10 min, the precipitates were collected by
filtration, washed with H2O, and dried in vacuo to afford 2-amino-5-
bromo-4-chloropyrimidine (3.59 g, 86%).
To a stirred solution of 2-amino-5-bromo-4-chloropyrimidine (627
mg, 3.00 mmol) in 1,4-dioxane (15 mL) and methanol (15 mL) was
added NaOMe (4 mL of a 25 wt % solution in methanol) at ambient
temperature. After stirring for 1.5 h, H2O (20 mL) was added, and the
majority of solvent was removed in vacuo. The residual aqueous layer
was extracted with CH2Cl2 (3×). The combined organic layers were
dried over anhydrous sodium sulfate and concentrated to provide 5-
bromo-4-methoxypyrimidin-2-amine (578 mg, 94%) as a white
powder. MS (EI) for C5H6BrN3O, found 204.0 (MH+).
Compound 9 was prepared by a procedure similar to that described
for the synthesis of compound 18 using 3 equivalents of methyl amine
(2 M in THF) to form the sulfonamide. 3-Amino-6-(4-chloro-3-(N-
methylsulfamoyl)phenyl)-N-methylpyrazine-2-carboxamide (9): 1H
NMR (400 MHz, DMSO-d6) δ 8.87 (s, 1H), 8.85−8.76 (m, 1H),
8.52−8.41 (m, 2H), 7.82−7.71 (m, 4H), 2.86 (d, J = 4.8 Hz, 3H), 2.54
(d, J = 4.8 Hz, 3H). MS (EI) for C13H14ClN5O3S, found 355.8 (MH+).
Compound 10 was prepared by a procedure similar to that
described for the synthesis of compound 18 using neat ammonium
hydroxide to form the sulfonamide. 3-Amino-6-(4-chloro-3-sulfamoyl-
1
phenyl)-N-methylpyrazine-2-carboxamide (10): H NMR (400 MHz,
DMSO-d6) δ 8.87 (s, 1H), 8.83−8.73 (m, 1H), 8.58 (d, J = 2.2 Hz,
1H), 8.41 (dd, J = 8.4, 2.2 Hz, 1H), 8.28−7.23 (m, 5H), 2.86 (d, J =
4.8 Hz, 3H). MS (EI) for C12H12ClN5O3S, found 341.9 (MH+).
Compound 11 was prepared by a procedure similar to that
described for the synthesis of compound 18 using 3 equivalents of
2,2,2-trifluoroethylamine to form the sulfonamide. 3-Amino-6-(4-
chloro-3-(N-(2,2,2-trifluoroethyl)sulfamoyl)phenyl)-N-methylpyra-
1
zine-2-carboxamide (11): H NMR (400 MHz, DMSO-d6) δ 9.01 (s,
1H), 8.88 (s, 1H), 8.85−8.76 (m, 1H), 8.54−8.39 (m, 2H), 8.09−7.42
(m, 3H), 3.90 (q, J = 9.5 Hz, 2H), 2.85 (d, J = 4.8 Hz, 3H). MS (EI)
for C14H13ClF3N5O3S, found 424.0 (MH+).
Compounds 15−17 were synthesized following the reaction
sequence outlined in Scheme 6 and following the procedure used to
synthesize 18.
3-Amino-N-methyl-6-(3-(phenylsulfonamido)phenyl)-
1
pyrazine-2-carboxamide (15). H NMR (400 MHz, DMSO-d6) δ
10.40 (br s, 1H), 8.62 (q, J = 4.8 Hz, 1H), 8.59 (s, 1H), 7.83−7.79 (m,
3H), 7.68 (br s, 2H), 7.66−7.53 (m, 4H), 7.30 (t, J = 8.0 Hz, 1H),
7.08−7.05 (m, 1H), 2.85 (d, J = 5.2 Hz, 3H). MS (EI) for
C18H17N5O3S, found 384.2 (MH+).
3-Amino-6-(3-(3-chlorophenylsulfonamido)phenyl)-N-meth-
ylpyrazine-2-carboxamide (16). 1H NMR (400 MHz, DMSO-d6) δ
10.45 (br s, 1H), 8.65 (q, J = 4.8 Hz, 1H), 8.62 (s, 1H), 7.89−7.86 (m,
1H), 7.81 (t, J = 2.0 Hz, 1H), 7.75−7.68 (m, 3H), 7.70 (br s, 2H),
7.59 (t, J = 4.0 Hz, 1H), 7.34 (t, J = 8.0 Hz, 1H), 7.08−7.05 (m, 1H),
2.85 (d, J = 4.8 Hz, 3H). MS (EI) for C18H16ClN5O3S, found 416.1
(MH+).
3-Amino-6-(4-chloro-3-(3-chlorophenylsulfonamido)-
1
phenyl)-N-methylpyrazine-2-carboxamide (17). H NMR (400
Analogue 35 was synthesized using a procedure similar to that
described for the synthesis of compound 18 using 5-bromo-4-
methoxypyrimidin-2-amine in place of 3-amino-6-bromo-N-methyl-
MHz, CD3OD) δ 8.69−8.62 (m, 1H), 8.64 (s, 1H), 8.10 (d, J = 2.4
Hz, 1H), 7.90 (dd, J = 8.8, 2.4 Hz, 1H), 7.74 (t, J = 2.0 Hz, 1H), 7.64−
7.61 (m, 2H), 7.47 (t, J = 8.0 Hz, 1H), 7.40 (d, J = 8.8 Hz, 1H), 2.98
(d, J = 4.8 Hz, 3H). MS (EI) for C18H15Cl2N5O3S, found 453.1
(MH+).
PI3K Biochemical Assay. All PI3K proteins were expressed and
purified in-house. Human PI3Kγ with boundaries of S144-A1102 and a
C-terminal His-tag was expressed in Sf 9 cells using a baculovirus
expression vector system. Heterodimers of full-length N-His PI3Kα,
PI3Kβ, and PI3Kδ were coexpressed with truncated p85α [M322-
N600] in the same Sf 9 expression system. Purification was done using
Ni-NTA affinity columns. Protein concentration was determined by
the Bradford assay, and identification was confirmed by trypsin
digestion and mass spectrometry. Purity was determined by SDS−
PAGE.
1
pyrazine-2-carboxamide. H NMR (400 MHz, DMSO-d6) δ 10.79 (s,
1H), 8.10 (s, 1H), 8.03 (d, J = 2.2 Hz, 1H), 7.74 (dd, J = 8.4, 2.2 Hz,
1H), 7.68 (d, J = 8.3 Hz, 1H), 7.31−7.18 (m, 1H), 7.18−7.05 (m,
2H), 6.91 (s, 2H), 3.80 (s, 3H). MS (EI) for C17H13ClF2N4O3S, found
427.0 (MH+).
5-(2-Amino-4-(2,2,2-trifluoroethoxy)pyrimidin-5-yl)-2-
chloro-N-(2,3 difluorophenyl)benzenesulfonamide (36). To a
stirred solution of 2-amino-5-bromo-4-chloropyrimidine (2.08 g, 9.95
mmol), 2,2,2,-trifluoroethanol (2.89 mL, 40.0 mmol), and 1,4-dioxane
(80 mL) was added NaH (1.6 g, 40 mmol, 60% in dispersion oil)
portionwise at 0 °C. Upon completion of the addition of NaH, the
reaction mixture was allowed to stir at 80 °C for 2 h. The reaction
mixture was cooled to ambient temperature and quenched with water
5479
dx.doi.org/10.1021/jm300403a | J. Med. Chem. 2012, 55, 5467−5482

Journal of Medicinal Chemistry
Article
Enzyme activity and compound inhibition were determined using
luciferase-luciferin-coupled chemiluminescence and measured as the
percent of ATP consumed following the kinase reaction as described
previously.58 The standard PI3K assay buffer is composed of 50 mM
Tris, pH 7.5, 1 mM EGTA, 1 mM DTT, 10 mM MgCl2 (5 mM for
PI3Kγ), and 0.03% CHAPS. MgCl2 was added only into the enzyme
solution as a 2× concentration. The standard assay concentrations for
ATP and PIP2 were 1 μM and 10 μM, respectively. The standard
enzyme concentrations for the PI3K isoforms were PI3Kα, 3 nM;
PI3Kβ, 10 nM; PI3Kδ, 4 nM; and PI3Kγ, 30 nM. All reactions were
conducted in 384-well white, medium binding microtiter plates.
Generally, 0.5 μL of DMSO containing varying concentrations of
the test compound was mixed with 10 μL of enzyme solution
(2× concentration). Kinase reactions were initiated with the addition
of 10 μL of PIP2 and ATP solution (2× concentration). Following
incubation times of 30−90 min, a 10 μL aliquot of Promega Kinase-
Glo was added and the chemiluminescence signal measured using an
Envision plate reader (Perkin-Elmer). Total ATP consumption was
limited to 40−60%, and IC50 values of control compounds correlated
well with literature references. Data are reported as the mean (n ≥ 2).
Fixed-Cell ELISA Cellular Assay. RAW 264.7 cells (ATCC, TIB-
71) were seeded at 8 × 104 cells/well onto clear bottom black 96-well
tissue culture-treated plates (Corning, Costar 3904) in serum-free
DMEM medium (Cellgro, 10−013-CV) one day before the assay.
Dilution series of test compounds were generated using DMSO as a
diluent. After resuspending diluted compounds in serum-free medium,
compounds were added to the cells and incubated for 90 min. Minimal
signal wells were treated with 10 μM PI-103, a pan-PI3K inhibitor.
Maximal signal wells were treated with 0.3% DMSO only. After
compound treatment, cells were stimulated with 250 ng/mL
Complement Factor 5a (C5a, Sigma, C5788) for 3 min. Cells were
then fixed with 4% formaldehyde (Sigma, F8775) for 30 min at
ambient temperature. Plates were washed four times with Tris-buffered
saline (TBS; 20 mM Tris, 50 mM NaCl) containing 0.1% Triton X-
100 (Sigma, T9284) using the ELx405 plate washer (BioTek),
incubating with the final wash volume for 20 min at ambient
temperature. The wash buffer wash was removed, to be replaced by
Odyssey Blocking Buffer (Li-Cor, 927−40000). Cells were blocked for
1 h at ambient temperature. Cells were stained overnight at 4 °C on a
rocker with Odyssey Blocking Buffer containing rabbit antiphospho-
AKTS473 (1:300 dilution; Cell Signaling Technology, 4060) and mouse
anti-AKT (2 μg/mL; R&D Systems, MAB 2055) antibodies. Plates
were washed four times with TBS containing 0.1% Tween-20 (Bio-
Rad, 161−0781), then incubated for 1 h at ambient temperature with
secondary antibodies (goat antirabbit-IRDye800CW, Li-Cor, 926−
32211; goat antimouse-IRDye680, Li-Cor, 926−32220) diluted 1:400
in Odyssey Blocking Buffer containing 0.1% Tween-20. Plates were
washed three times with TBS with 0.1% Tween-20, followed by two
times with TBS without Tween-20, then scanned using the Odyssey
detector (Li-Cor). Intrawell normalization was accomplished by
dividing the phospho-AKTS473 intensity values from the 800 nm
channel by the total AKT values from the 700 nm channel. IC50 values
were then estimated by comparing the values of compound-treated
samples with averages of the aforementioned minimal and maximal
signal condition wells. Data are reported as the mean (n ≥ 2).
Crystallization and Structure Determination. A construct
representing amino acids 114−1102 of PIK3CG was expressed in Sf-9
cells. The purification of PI3Kγ protein was carried out as previously
reported59 except for the removal of betaine, ethylene glycol, and
CHAPS solutes from all purification buffers. Crystals of PI3Kγ were
grown at 19 °C in hanging drops using vapor diffusion. The complexes
were formed by incubating 49.5 μL of PI3Kγ-His6 (7.2 mg/mL in 20
mM Tris-HCl (pH 7.2), 50 mM (NH4)2SO4, and 1 mM TCEP) with
0.5 μL of ligand (100 mM stock in 100% DMSO) for 30 min at 4 °C.
Following incubation, the complexes were centrifuged at 14,000g to
clarify the protein−inhibitor solutions. Crystals of the binary
complexes were obtained by mixing 1.0 μL of the PI3Kγ:inhibitor
complex with 1.0 μL of a reservoir solution consisting of 16−21% PEG
4K, 0.25 M (NH4)2SO4, and 0.1 M Tris-HCl (pH 7.5). In addition,
0.2 μL of an apo-PI3Kγ crystalline seed stock (1 × 10−3 dilution) was
added to the drop to aid crystal growth. Crystals generally appeared
within one day and grew to full size in approximately 1−2 weeks.
Crystals were transferred for 30 s into a cryoprotectant solution
composed of 21% PEG 4K, 0.25 M (NH4)2SO4, 0.1 M Tris-HCl (pH
7.5), 10% ethylene glycol (v/v), 10% glycerol (v/v), and 500 μM
inhibitor. The structure of the PI3Kγ:inhibitor complexes were solved
by molecular replacement using all protein atoms of a previous
structure of PI3Kγ as a search model. A single unambiguous solution
was found using PHASER, with one molecule in the asymmetric unit.
The resulting model was subsequently refined in REFMAC5 using
rigid body and maximum likelihood procedures. At this stage, Fourier
syntheses with coefficients of Fo − Fc and 2Fo − Fc yielded clearly
interpretable electron density for all atoms of the ligands. The ligands
were built and minimized in Concorde/PRODRG, imported into
Coot, and fit into the difference electron density. The complex was
subsequently refined using maximum likelihood procedures in
REFMAC5. Portions of the protein were adjusted by iterative cycles
of manual rebuilding in Coot and refinement in REFMAC5, and
waters were built and manually inspected in Coot.
ASSOCIATED CONTENT
■
S
* Supporting Information
Materials and methods used in determination of pharmacoki-
netic, AIMCD, hERG, PgP, CYP, MDCK, and Ksol data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Accession Codes
PDB codes are the following: 4anv for the 1/PI3Kγ complex;
4anu for the 2/PI3Kγ complex; 4anx for the 4/PI3Kγ complex;
and 4anw for the 31/PI3Kγ complex.
AUTHOR INFORMATION
■
Corresponding Author
*Exelixis, 169 Harbor Way, South San Francisco CA 94080-
6109, USA. Tel: (650) 837-7006. Fax: (650) 837-8177. E-mail:
hjohnson@exelixis.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Siamak Dailami for analytical support; Eun Ok Kim,
Iris Ngan, Yongchang Shi, Keith Calkins, Vera Shihadeh for
additional support of cell based assays; Erik Gjerstad, Arturo
Picones, Lory Tan, and John Bui for collecting reported ADME
data; Atulkumar Ramaiya, Ron Aoyama, and Scott Womble for
obtaining PK data; Steven Richards and Amy Tsuhako for
editorial comments.
ABBREVIATIONS USED
■
CCL5, chemokine CC motif ligand 5; CHAPS, 3-[(3-
cholamidopropyl)dimethylammonio]-1-propanesulfonate;
C
max, maximum plasma concentration; C5a, complement factor
5a; DMEM, Dulbecco’s modified Eagle’s medium; FACS,
fluorescence-activated cell sortin; Ksol, kinetic solubility;
MDCK, Madin−Darby canine kidney; MLM, mouse liver
microsome; mTOR, mammalian target of rapamycin Pd(dppf)-
Cl2·CH2Cl2, [1,1′ bis(diphenylphosphino)ferrocene]-
dichloropalladium(II) complex with dichloromethane; PIKK,
phosphoinositide kinase-related kinase; RANTES, regulated
upon activation, normal T-cell expressed, and secreted; TCEP,
tris(2-carboxyethyl)phosphine
5480
dx.doi.org/10.1021/jm300403a | J. Med. Chem. 2012, 55, 5467−5482

Journal of Medicinal Chemistry
Article
and tumor growth in a model of colitis-associated cancer. Gastro-
enterology 2010, 138, 1374−1383.
REFERENCES
■
(1) Vivanco, I.; Sawyers, C. L. The phosphatidylinositol 3-kinase
AKT pathway in human cancer. Nature Rev. Cancer 2002, 2, 489−501.
(2) Cantley, L. C. The phosphoinositide 3-kinase pathway. Science
2002, 296, 1655−1657.
(20) Monterrubio, M.; Mellado, M.; Carrera, A. C.; Rodriguez-Frade,
J. M. R.-F. PI3Kγ activation by CXCL12 regulates tumor cell adhesion
and invasion. Biochem. Biophys. Res. Commun. 2009, 388, 199−204.
(21) Attoub, S.; De Wever, O.; Bruyneel, E.; Mareel, M.; Gespach, C.
The transforming functions of PI3-kinase-γ are linked to disruption of
intercellular adhesion and promotion of cancer cell invasion. Ann. N.Y.
Acad. Sci. 2008, 1138, 204−213.
(3) Vanhaesebroeck, B.; Guillermet-Guibert, J.; Graupera, M.;
Bilanges, B. The emerging mechanisms of isoform-specific PI3K
signaling. Nature Rev. Mol. Cell Biol. 2010, 11, 329−341.
(4) Vanhaesebroeck, B.; Leevers, S. J.; Ahmadi, K.; Timms, J.; Katso,
R.; Driscoll, P. C.; Woscholski, R.; Parker, P. J.; Waterfield, M. D.
Synthesis and function of 3-phosphorylated inositol lipids. Annu. Rev.
Biochem. 2001, 70, 535−602.
(22) Kobayashi, N.; Ueki, K.; Okazaki, Y.; Iwane, A.; Kubota, N.;
Ohsugi, M.; Awazaw, M.; Kobayashi, M.; Sasako, T.; Kaneko, K.;
Suzuki, M.; Nishikawa, Y.; Hara, K.; Yoshimura, K.; Koshima, I.;
Goyama, S.; Murakami, K.; Sasaki, J.; Nagai, R.; Kurokawa, M.; Sasaki,
T.; Kadowaki, A. Blockade of class IB phosphoinositide-3 kinase
ameliorates obesity-induced inflammation and insulin resistance. Proc.
Natl. Acad. Sci. U.S.A. 2011, 108, 5753−5758.
(23) Damilano, F.; Franco, I.; Perrino, C.; Schaefer, K.; Azzolino, O.;
Carnevale, D.; Cifelli, G.; Carullo, P.; Ragona, R.; Ghigo, A.; Perino,
A.; Lembo, G.; Hirsch, E. Distinct effects of leukocyte and cardiac
phosphoinositide 3-kinase γ activity in pressure overload-induced
cardiac failure. Circulation 2001, 123, 391−399.
(24) Jin, R.; Song, Z.; Yu, S.; Piazza, A.; Nanda, A.; Penninger, J. M.;
Granger, D. N.; Li, G. Phosphatidylinositol-3-kinase gamma plays a
central role in blood-brain barrier dysfunction in acute experimental
stroke. Stroke 2011, 42, 2033−2044.
(25) Guo, D.; Thiyam, G.; Bodiga, S.; Kassiri, Z.; Oudit, G. Y.
Uncoupling between enhanced excitation-contraction coupling and the
response to heart disease: Lessons from the PI3Kγ knockout murine
model. J. Mol. Cell Cardiol. 2011, 50, 606−612.
(26) Siragusa, M.; Katare, R.; Meloni, M.; Damilano, F.; Hirsch, E.;
Emanueli, C.; Madeddu, P. Involvement of phosphoinositide kinase γ
in angiogenesis and healing of experimental myocardial infarction in
mice. Circ. Res. 2010, 106, 757−768.
(27) Seropian, I. M.; Abbate, A.; Toldo, S.; Harrington, J.; Smithson,
L.; Ockaili, R.; Mezzaroma, E.; Damilano., F.; Hirsch, E.; Van Tassell,
B. W. Pharmacologic inhibition of phosphoinositide 3-kinase gamma
(PI3Kγ) promotes infarct resorption and prevents adverse cardiac
remodeling after myocardial infarction in mice. J. Cardiovasc. Pharm.
2010, 56, 651−658.
(5) Fruman, D. A.; Meyers, R. E.; Cantley, L. C. Phosphoinositide
kinases. Annu. Rev. Biochem. 1998, 67, 481−507.
(6) Krugmann, S.; Hawkins, P. T.; Pryer, N.; Braselmann, S.
Characterizing the interactions between the two subunits of the p101/
p110γ phosphoinositide 3-kinase and their role in the activation of this
enzyme by Gβγ subunits. J. Biol. Chem. 1999, 274, 17152−17158.
(7) Stephens, L.; Smrcka, A.; Cooke, F. T.; Jackson, T. R.; Sternweis,
P. C.; Hawkins, P. T. A novel phosphoinositide 3 kinase activity in
myeloid-derived cells is activated by G-protein βγ subunits. Cell 1994,
77, 83−93.
(8) Stoyanov, B.; Volinia, S.; Hanck, T.; Rubio, I.; Loubtchenkov, M.;
Malek, D.; Stoyanova, S.; Vanhaesebroeck, B.; Dhand, R.; Nurnberg,
B.; Gierschik, P.; Seedorf, K.; Hsuan, J. J.; Waterfield, M. D.; Wetzker,
R. Cloning and characterization of a G-protein activated human
phosphoinositide 3-kinase. Science 1995, 269, 690−693.
(9) Stephens, L. R.; Eguinoa, A.; Erdjument-Bromage, H.; Lui, M.;
Cooke, F.; Coadwell, J.; Smrcka, A. S.; Thelen, M.; Cadwallader, K.;
Tempst, P.; Hawkins, P. T. The Gβγ sensitivity of a PI3K is dependent
upon a tightly associated adaptor, p101. Cell 1997, 89, 105−114.
(10) Okkenhaug, K.; Vanhaesebroeck, B. PI3K in lymphocyte
development, differentiation, and activation. Nature Rev. Immumol.
2003, 3, 317−330.
(11) Wetzker, R.; Rommel, C. Phosphoinositide 3-kinases as targets
for therapeutic intervention. Curr. Pharm. Des. 2004, 10, 1915−1922.
(12) Foukas, L. C.; Claret, M.; Pearce, W.; Okkenhaug, K.; Meek, S.;
Peskett, E.; Sancho, S.; Smith, A. J. H.; Withers, D. J.; Vanhaesebroeck,
B. Critical role for the p110α phosphoinositide-3-OH kinase in growth
and metabolic regulation. Nature 2006, 441, 366−370.
(28) Jin, R.; Yu, S.; Song, Z.; Quillin, J. W.; Deasis, D. P.; Penninger,
J. M.; Nanda, A.; Granger, D. N.; Li, G. Phosphoinositide 3-kinase-
gamma expression is upregulated in brain microglia and contributes to
ischemia-induced microglial activation in acute experimental stroke.
Biochem. Biophys. Res. Commun. 2010, 399, 458−464.
(29) Passos, G. F.; Figueiredo, C. P.; Prediger, R. D. S.; Silva, K. A. B.
S.; Siqueira, J. M.; Duarte, F. S.; Leal, P. C.; Medeiros, R.; Calixto, J. B.
Involvement of phosphoinositide 3-kinase γ in the neuro-inflammatory
response and cognitive impairments induced by β-amyloid 1−40
peptide in mice. Brain Behav. Immun. 2010, 24, 493−501.
(30) Maira, S. M.; Finan, P.; Garcia-Echeverria, C. From the bench to
the bed side: PI3K pathway inhibitors in clinical development. Curr.
Top. Microbiol. Immunol. 2011, 347, 209−239.
(13) 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. High
frequency of mutations of the PIK3Ca gene in human cancers. Science
2004, 304, 554.
(14) Ghigo, A.; Damilano, F.; Braccini, L.; Hirsch, E. PI3K inhibition
in inflammation: Toward tailored therapies for specific diseases.
BioEssays 2010, 32, 185−196.
(15) Reif, K.; Okkenhaug, K.; Sasaki, T.; Penninger, J. M.;
Vanhaesebroeck, B.; Cyster, J. G. Differential roles for phosphoinosi-
tide 3-kinases, p110γ and p110δ, in lymphocyte chemotaxis and
homing. J. Immunol. 2004, 173, 2236−2240.
(16) Laffargue, M.; Calvez, R.; Finan, P.; Trifilieff, A.; Barbier, M.;
Altruda, F.; Hirsch, E.; Wymann, M. P. Phosphoinositide 3-kinase γ is
an essential amplifier of mast cell function. Immunity 2002, 16, 441−
451.
(31) Shapiro, G.; Kwak, E.; Baselga, J.; Rodon, J.; Scheffold, C.; Laird,
A. D.; Bedell, C.; Edelman, G. Phase I dose-escalation study of XL147,
a PI3K inhibitor administered orally to patients with solid tumors.
Presented at ASCO 2009 Annual Meeting, Orlando, FL, May 29−June
2, 2009; Abstract 3500
(17) Martin, D.; Galisteo, R.; Molinolo, A. A.; Wetzker, R.; Hirsch,
E.; Gutkind, J. S. PI3Kγ mediates kaposi’s sarcoma-associated
herpesvirus vGPCR-induced sarcomagenesis. Cancer Cell 2011, 19,
805−813.
(32) Papadopoulos, K. P.; Markman, B.; Tabernero, J.; Patnaik, A.;
Heath, E. I.; DeCillis, A.; Laird, D.; Aggarwal, S. K.; Nguyen, L.;
LoRusso, P. M. A phase I dose-escalation study of the safety,
pharmacokinetics (PK), and pharmacodynamics (PD) of a novel PI3K
inhibitor, XL765, administered orally to patients (pts) with advanced
solid tumors. Presented at ASCO 2009 Annual Meeting, Chicago, IL,
May 30−June 3, 2008; Abstract 3510
(18) Schmid, M. C.; Avraamides, C. J.; Dippold, H. C.; Franco, I.;
Foubert, P.; Ellies, L. G.; Acevedo, L. M.; Manglicmot, J. R. E.; Song,
X.; Wrasidlo, W.; Blair, S. L.; Ginsberg, M. H.; Cheresh, D. A.; Hirsch,
E.; Field, S. J.; Varner, J. A. receptor tyrosine kinases and TLR/IL1Rs
unexpectedly activate myeloid cell PI3Kγ, a single convergent point
promoting tumor inflammation and progression. Cancer Cell 2011, 19,
715−727.
(33) Ruckle, T.; Schwarz, M. K.; Rommel, C. PI3Kγ inhibition:
̈
towards an ’aspirin of the 21st century’? Nat. Rev. Drug Discovery 2006,
5, 903−918.
(19) Gonzalez-Garcia, A.; Sanchez-Ruiz, J.; Flores, J. M.; Carrera, A.
C. Phosphatidylinositol 3-kinase γ inhibition ameliorates inflammation
5481
dx.doi.org/10.1021/jm300403a | J. Med. Chem. 2012, 55, 5467−5482

Journal of Medicinal Chemistry
Article
(34) Rommel, C.; Camps, M.; Ji, H. PI3Kδ and PI3Kγ: partners in
crime in inflammation in rheumatoid arthritis and beyond? Nat. Rev.
Drug Discovery 2007, 7, 191−201.
(35) Ameriks, M. K.; Venable, J. D. Small Molecule inhibitors of
phosphoinositide 3-kinase (PI3K) δ and γ. Curr. Top. Med. Chem.
2009, 9, 738−753.
(36) Pacold, M. E.; Suire, S.; Perisic, O.; Lara-Gonzalez, S.; Davis, C.
T.; Walker, E. H.; Hawkins, P. T.; Stephens, L.; Eccleston, J. F.;
Williams, R. L. Crystal structure and functional analysis of Ras binding
to its effector phosphoinositide 3-kinase γ. Cell 2000, 103, 931−943.
(37) Suire, S.; Hawkins, P.; Stephens, L. Activation of phosphoinosi-
tide 3-kinase γ by Ras. Curr. Biol. 2002, 12, 1068−1075.
(38) Bondeva, T.; Pirola, L.; Bulgarelli-Leva, G.; Rubio, I.; Wetzker,
R.; Wymann, M. P. Bifurcation of lipid and protein kinase signals of
PI3Kγ to the protein kinases PKB and MAPK. Science 1998, 282, 293−
296.
(39) Lopez-Ilasaca, M.; Gutkind, J. S.; Wetzker, R. Phosphoinositide
3-kinase γ is a mediator of Gβγ-dependent Jun kinase activation. J. Biol.
Chem. 1998, 273, 2505−2508.
(40) Li, Z.; Jiang, H.; Xie, W.; Zhang, Z.; Smrcka, A. V.; Wu, D. Roles
of PLC-β2 and -β3 and PI3Kγ in chemoattractant-mediated signal
transduction. Science 2000, 287, 1046−1049.
(41) Hirsch, E.; Katanaev, V. L.; Garlanda, C.; Azzolino, O.; Pirola,
L.; Silengo, L.; Sozzani, S.; Mantovani, A.; Altruda, F.; Wymann, M. P.
Central role for G protein-coupled phosphoinositide 3-kinase γ in
inflammation. Science 2000, 287, 1049−1053.
(51) 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.; Wan, N. C.; Wiesmann, C.;
Workman, P.; Zhyvoloup, A.; Zvelebil, M. J.; Shuttleworth, S. J. The
Identification of 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-
1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941)
as a potent, selective, orally bioavailable inhibitor of class I PI3 kinase
for the treatment of cancer. J. Med. Chem. 2008, 51, 5522−5532.
(52) Gendreau, S. B.; Ventura, R.; Keast, P.; Laird, A. D.; Yakes, F.
M; Zhang, W.; Bentzien, F.; Cancilla, B.; Lutman, J.; Chu, F.; Jackman,
L.; Shi, Y.; Yu, P.; Wang, J.; Aftab, A. T.; Jaeger, C. T.; Meyer, S. M.;
De Costa, A.; Engell, K.; Chen, J.; Martini, J. F.; Joly, A. H. Inhibition
of the T790M gatekeeper mutant of the epidermal growth factor
receptor by EXEL-7647. Clin. Cancer Res. 2007, 13, 3713−3723.
(53) Qian, F.; Engst, S.; Yamaguchi, K.; Yu, P.; Won, K. A.; Mock, L.;
Lou, T.; Tan, J.; Li, C.; Tam, D.; Lougheed, J.; Yakes, M. F.; Bentzien,
F.; Xu, W.; Zaks, T.; Wooster, R.; Greshock, J.; Joly, A. H. Inhibition
of tumor cell growth, invasion, and metastasis by EXEL-2880 (XL880,
GSK1363089), a novel inhibitor of HGF and VEGF receptor tyrosine
kinases. Cancer Res. 2009, 69, 8009−8016.
(54) Zoncu, R.; Efayan, A.; Sabatini, D. M. mTOR: from growth
signal integration to cancer, diabetes and ageing. Nature Rev. Mol. Cell
Biol. 2011, 12, 21−35.
(55) Adams, N. D.; Burgess, J. L.; Darcy, M. G.; Donatelli, C. A.;
Knight, S. D.; Newlander, K. A.; Ridgers, L.; Sarpong, M.; Schmidt, S.
J. Preparation of Quinoline Derivatives as PI3 Kinase Inhibitors. PCT
Int. Appl. WO144463A1, 2008.
(56) Harris, K. J.; Lang, H.-J. ; Mathew, R. M.; Shimshock, S. J.;
Nieduzak, T. R.; Jackson, S.; Yang, Z.; Bordeau, K. J. 2-Phenyl-indoles
as Prostoglandin D2 Receptor Antagonists. PCT Int. Appl.
WO2006081343, 2006.
(57) Ibrahim, M. A.; Johnson, H. W. B.; Jeong, J. W.; Lewis, G. L.;
Shi, X.; Noguchi, R. T.; Williams, M.; Leahy, J. W.; Nuss, J. M.;
Woolfrey, J.; Banica, M.; Bentzien, F.; Chou, Y.-C.; Gibson, A.; Heald,
N.; Lamb, P.; Mattheakis, L.; Matthews, D.; Shipway, A.; Wu, X.;
Zhang, W.; Zhou, S.; Shankar, G. Discovery of a novel class of potent
and orally bioavailable sphingosine 1-phosphate receptor 1 antagonists.
J. Med. Chem. 2012, 55, 1368−1381.
(58) Munagala, N.; Nguyen, S.; Lam, W.; Lee, J.; Joly, A.; McMillan,
K.; Zhang, W. Identification of small molecule ceramide kinase
inhibitors using a homogeneous chemiluminescence high throughput
assay. Assay Drug Dev. Technol. 2007, 5, 65−73.
(42) Sasaki, T.; Irie-Sasaki, J.; Jones, R. G.; Oliveira-dos-Santos, A. J.;
Stanford, W. L.; Bolon, B.; Wakeham, A.; Itie, A.; Bouchard, D.;
Kozieradzki, I.; Joza, J.; Mak, T. W.; Ohashi, P. S.; Suzuki, A.;
Penninger, J M. Function of PI3Kγ in thymocyte development, T cell
activation, and neutrophil migration. Science 2000, 287, 1040−1046.
(43) Camps, M.; Ruckle, T.; Ji, H.; Ardissone, V.; Rintelen, F.; Shaw,
̈
J.; Ferrandi, C.; Chabert, C.; Gillieron, C.; Franco̧ n, B.; Martin, T.;
Gretener, D.; Perrin, D.; Leroy, D.; Vitte, P. A.; Hirsch, E.; Wymann,
M. P.; Cirillo, R.; Schwarz, M. K.; Rommel, C. Blockade of PI3Kγ
suppresses joint inflammation and damage in a murine model of
rheumatoid arthritis. Nature Med. 2005, 11, 936−943.
(44) Pomel, V.; Klicic, J.; Covini, D.; Church, D. D.; Shaw, J. P.;
Roulin, K.; Burgat-Charvillon, F.; Valognes, D.; Camps, M.; Chabert,
C.; Gillieron, C.; Franco
Nichols, A.; Vitte, P. A.; Carboni, S.; Rommel, C.; Schwarz, M. K.;
̧ n, B.; Perrin, D.; Leroy, D.; Gretener, D.;
Ruckle, T. Furan-2-ylmethylene thiazolidinediones as novel, potent,
̈
and selective inhibitors of phosphoinositide 3-kinase γ. J. Med. Chem.
2006, 49, 3857−3871.
(59) Walker, E. H.; Pacold, M. E.; Perisic, O.; Stephens, L.; Hawkins,
P. T.; Wymann, M. P.; Williams, R. L. Structural determinants of
phosphoinositide 3-kinase inhibition by wortmannin, LY294002,
quercetin, myricetin, and staurosporine. Mol. Cell 2000, 6, 909−919.
(45) Buhr, C. A.; Baik, T.-G.; Sunghoon, M.; Tesfai, Z.; Wang, L.;
Co, E. W.; Epshteyn, S.; Kennedy, A. R.; Chen, B.; Dubenko, L.;
Anand, N. K.; Tsang, T. H.; Nuss, J. M.; Peto, C. J.; Rice, K. D.;
Ibrahim, M. A.; Schnepp, K. L.; Shi, X.; Leahy; J. W.; Chen, J.;
Dalrymple, L. E.; Forsyth, T. P.; Huynh, T. P.; Mann, G.; Mann, L. W.;
Takeuchi, C. S.; Lamb, P.; Matthews, D. J.; Miller, N. Protein Kinase
Modulators and Methods of Use. PCT Int. Appl. WO0309329A2,
2003.
(46) Sundstrom, T. J.; Anderson, A. C.; Wright, D. L. Inhibitors of
phosphoinositide-3-kinase: a structure-based approach to under-
standing potency and selectivity. Org. Biomol. Chem. 2009, 7, 840−
850.
(47) Walker, E. H.; Perisic, O.; Ried, C.; Stephens, L.; Williams, R. L.
Structural insights into phosphoinositide 3-kinase catalysis and
signalling. Nature 1999, 402, 313−320.
(48) Calculated using ACD Laboratories software 9.0.
(49) Mouse plasma exposure of 11: 44.4 14.3 and 17.9 3.5 μM
in plasma at 1 and 4 h. respectively. dosed in male BALB/c mice (n =
3) at 100 mpk as a solution in 10% EtOH 60% PEG400/water.
(50) Ali, K.; Camps, M.; Pearce, W. P.; Ji, H.; Kuehn, N.; Pasquali,
C.; Chabert, C.; Rommel, C.; Vanhaesebroeck, B. Isoform-specific
functions of phosphoinositide 3-kinases: p110δ but not p110γ
promotes optimal allergic responses in vivo. J. Immunol. 2008, 180,
2538−2544.
5482
dx.doi.org/10.1021/jm300403a | J. Med. Chem. 2012, 55, 5467−5482