Isoform-specific phosphoinositide 3-kinase inhibitors from an arylmorpholine scaffold

Article abstract of DOI:10.1016/j.bmc.2004.06.022

Phosphoinositide 3-kinases (PI3-Ks) are an ubiquitous class of signaling enzymes that regulate diverse cellular processes including growth, differentiation, and motility. Physiological roles of PI3-Ks have traditionally been assigned using two pharmacological inhibitors, LY294002 and wortmannin. Although these compounds are broadly specific for the PI3-K family, they show little selectivity among family members, and the development of isoform-specific inhibitors of these enzymes has been long anticipated. Herein, we prepare compounds from two classes of arylmorpholine PI3-K inhibitors and characterize their specificity against a comprehensive panel of targets within the PI3-K family. We identify multiplex inhibitors that potently inhibit distinct subsets of PI3-K isoforms, including the first selective inhibitor of p110β/p110δ (IC₅₀ p110β = 0.13 μM, p110δ = 0.63 μM). We also identify trends that suggest certain PI3-K isoforms may be more sensitive to potent inhibition by arylmorpholines, thereby guiding future drug design based on this pharmacophore.

Full text of DOI:10.1016/j.bmc.2004.06.022

Bioorganic & Medicinal Chemistry 12 (2004) 4749–4759
Isoform-specific phosphoinositide 3-kinase inhibitors from an
arylmorpholine scaffold
Zachary A. Knight,a Gary G. Chiang,b Peter J. Alaimo,c Denise M. Kenski,a
c,d,
Caroline B. Ho,b Kristin Coan,a Robert T. Abrahamb andKevan M. Shokat
*
aProgram in Chemistry and Chemical Biology, University of California, San Francisco, CA 94143, USA
bProgram in Signal Transduction Research, The Burnham Institute, La Jolla, CA 92037, USA
cDepartment of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143, USA
dDepartment of Chemistry, University of California, Berkeley, CA 94720, USA
Received9 April 2004; accepted10 May 2004
Available online 20 July 2004
Abstract—Phosphoinositide 3-kinases (PI3-Ks) are an ubiquitous class of signaling enzymes that regulate diverse cellular processes
including growth, differentiation, and motility. Physiological roles of PI3-Ks have traditionally been assigned using two pharmaco-
logical inhibitors, LY294002 and wortmannin. Although these compounds are broadly specific for the PI3-K family, they show little
selectivity among family members, and the development of isoform-specific inhibitors of these enzymes has been long anticipated.
Herein, we prepare compounds from two classes of arylmorpholine PI3-K inhibitors and characterize their specificity against a com-
prehensive panel of targets within the PI3-K family. We identify multiplex inhibitors that potently inhibit distinct subsets of PI3-K
isoforms, including the first selective inhibitor of p110b/p110d (IC50 p110b=0.13lM, p110d=0.63lM). We also identify trends that
suggest certain PI3-K isoforms may be more sensitive to potent inhibition by arylmorpholines, thereby guiding future drug design
basedon this pharmacophore.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
stream activation, the physiological roles of individual
PI3-K isoforms remain largely unassigned, and dissect-
ing the unique functions of members of this family is a
major focus of ongoing research.2
Phosphatidylinositol 3-kinases are activated by a wide
range of cell surface receptors to generate the lipidsec-
ondmessengers phosphatidylinositol 3,4-bisphosphate
(PIP2) andphosphatyidlinositol 3,4,5-trisphosphate
(PIP3). In the appropriate cellular context, these two
lipids can regulate a remarkably diverse array of physio-
logical processes, including glucose homeostasis, cell
growth, differentiation, and motility.1 These distinct
functions are carriedout by a family of eight related
In addition to sequence homology within their catalytic
domain, PI3-Ks share sensitivity to two small molecule
inhibitors, wortmannin andLY294002. Wortmannin is
a fungal natural product that irreversibly inhibits PI3-
Ks at low nanomolar concentrations,3,4 whereas
LY294002 is a synthetic chromone that reversibly inhib-
its most PI3-Ks at low micromolar concentrations.5 To-
gether, these two compounds have served as powerful
probes for implicating PI3-Ks in a wide range of physi-
ological processes, and much of our understanding of
PI3-K action in cells derives from the use of these two
reagents.
PI3-Ks in vertebrates that possess unique substrate spe-
1
cificities, localization, andmodes of regulation.
These
include the class IA PI3-Ks (p110a, p110b, p110d),
which are activatedby receptor tyrosine kinases, the
class IB PI3-K (p110c), which is activatedby heterotri-
meric G-proteins, andthe class II PI3-Ks (PI3KC2 a,
PI3KC2b, PI3KC2c) whose regulation remains poorly
understood. Despite these known differences in up-
Although wortmannin andLY294002 are broadly active
against the PI3-K family, they show little specificity
among PI3-K family members. Since these compounds
do not pharmacologically discriminate between PI3-K
isoforms, comparatively little is known about the spe-
cific signaling functions of individual PI3-Ks. Knockout
Keywords: PI3-K; Inhibitor; Isoform; Phosphatidylinositol.
*
Corresponding author. Tel.: +1-415-514-0472; fax: +1-415-514-
0822; e-mail: shokat@cmp.ucsf.edu
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.06.022

4750
Z. A. Knight et al. / Bioorg. Med. Chem. 12 (2004) 4749–4759
mice have defined essential roles for p110c andp110 d in
leukocyte function, including signaling from the B and T
be quite selective for lipidkinases relative to protein kin-
ases15 andto possess broadactivity within the PI3-K
family. Indeed, until very recently, LY294002 was the
only reversible inhibitor that hadbeen reportedto target
the PI3-K family––even though this compoundwas
originally described 10years ago.5 Given the importance
of PI3-Ks in signal transduction, it is remarkable that
there has not been a single subsequent structure–activity
study published investigating compounds of this class.
Our comprehensive selectivity analysis of a panel of
arylmorpholines identifies PI3-K isoforms that tend to
be sensitive to this pharmacophore, as well as others
that are more resistant. This information shouldprove
useful in guiding future drug design based on this core
structure.
cell receptors6,7 (p110d) as well as chemotaxis of neu-
8,9
trophils andmacrophages
(p110c). Furthermore,
microinjection of isoform-specific inhibitory antibodies
has demonstrated that individual class I PI3-Ks can re-
lay unique downstream signals following activation of a
common upstream receptor. For example, colony-stim-
ulating factor-1 treatment of macrophages induces
DNA synthesis through p110a, whereas actin cytoskele-
ton rearrangement andcell migration require p110 b and
p110d.10 These studies suggest that PI3-K isoforms
likely possess nonredundant functions downstream of
the wide range of receptors that are known to activate
PI3-Ks. In general, however, the systematic analysis of
PI3-K isoform action in cells awaits the discovery of
small molecule inhibitors that can selectively target
PI3-K family members. For this reason, the develop-
ment of isoform-specific inhibitors of PI3-Ks has been
long anticipated.2,11–13
2. Results
2.1. LY294002 analogs
Recently, patent disclosures have described new inhibi-
tors basedon the LY294002 arylmorpholine pharmaco-
phore,11,12,14 although the detailed isoform selectivity of
these compounds has not been reported. Since these
compounds are potentially useful probes for PI3-K iso-
form activity in cells, we sought to determine their target
specificity by characterizing their activity against a com-
prehensive panel of PI3-Ks. In this regard, a recent char-
acterization of 28 common protein kinase inhibitors
against a panel of 24 protein kinases has significantly
challengedmany preconceptions about the true selectiv-
ity of protein kinase inhibitors.15 The analysis we report
here identifies several compounds that inhibit distinct
subsets of the PI3-K family, including the first selective
inhibitors of the p110d/p110b isoforms.
Thrombogenix has disclosed a series of LY294002 ana-
logs that differ in the substitution pattern of heteroatoms
within the chromone core,14 as well as through replace-
ment of the phenyl group at the eight position of
LY294002 with more extended aromatic substituents
(Fig. 1). Comparison of the co-crystal structures of
16
17
p110c boundto LY294002
andATP
suggests that
the adenine ring of ATP and the morpholino-chromone
core of LY294002 occupy essentially the same space in
the interior of the ATP binding pocket, with both rings
anchored by a hydrogen bond to the backbone amide of
V882 (Fig. 1A). Likewise, the 8-phenyl moiety of
LY294002 andthe ribose sugar of ATP occupy a similar
space at the entrance to the pocket andproject outward
towardsolvent. To understandhow LY294002 analogs
with extended substitutents at C8 might interact differen-
tially with PI3-K isoforms, we identified all of the residues
in p110c whose side chains possess a rotamer that can ex-
We also sought to explore the potential of the morpho-
lino chromone scaffoldas a starting point for PI3-K
inhibitor optimization. LY294002 has been shown to
˚
tendwithin 4A of either the adenine/LY294002 chromone
Figure 1. ATP binding site conservation among the class I PI3-Ks. (A) Schematic of the hydrogen bonds made by ATP (top) and LY294002
(bottom) in the active site of p110c. Pocket interior (red) and entrance (yellow) are marked for reference. (B) Sequence of alignment residues in the
interior (left) and exterior (right) of the ATP binding pocket. Residue coloring: hydrophobic aliphatic (green), hydrophobic aromatic (light blue),
small (yellow), polar uncharged (dark blue), basic (purple), and acidic (orange). Residue number is for porcine p110c. (C) Model of TGX115 bound
to p110c, basedon the LY294002-p110 c co-crystal structure.16

Z. A. Knight et al. / Bioorg. Med. Chem. 12 (2004) 4749–4759
4751
core (Fig. 1, red) or the ATP ribose/8-phenyl of LY294002
In contrast to LY294002, TGX115 andTGX126 were
(Fig. 1, yellow). This first set of residues defines the inte-
rior of the ATP binding pocket, while the second set mark
the entrance to that pocket (Fig. 1C). We then aligned
these residues with the corresponding residues from each
of the class I PI3-Ks, and shaded them according to their
physiochemical properties (hydrophobicity, size, and
charge) (Fig. 1B). This alignment reveals that the interior
of the ATP binding pocket is highly conserved within the
class I PI3-Ks; the only differences are two conservative
substitutions that distinguish p110c from the class IA
PI3-Ks (Fig. 1B, left). By contrast, the residues that line
the entrance to the ATP binding pocket are divergent,
with major differences between isoforms in their size and
charge (Fig. 1B, right). LY294002 analogs with larger
substituents at C8 wouldbe expectedto extendoutside
the ATP binding pocket and potentially make extensive
contacts with these less conservedresidues ( Fig. 1C). This
suggests that it may be possible for such extended analogs
to target specific PI3-K isoforms, although it is not imme-
diately apparent how to design compounds that would
exploit these differences.
significantly more potent andselective. Surprisingly,
we findthat TGX115 inhibits p110 b andp110 d at nano-
molar concentrations, but p110a andp110 c only at con-
centrations more than 100-foldhigher ( Fig. 2). TGX126
showeda more modest specificity profile, although this
compoundwas still ꢀ50-foldmore potent against
p110b/p110d than against p110a/p110c. Against the
other 10 enzymes in this panel, TGX115 andTGX126
showedsignificant activity only against DNA-PK, with
high nanomolar IC50 values. Thus, within the lipidkin-
ases, these two compounds represent the first selective
inhibitors of the p110b/p110d isoforms. By contrast,
the unsubstitutedanalogs TGX066 andLY292223 were
significantly less potent against all PI3-Ks, although
they retainedstrong activity against DNA-PK ( Fig. 2).
This suggests that presence of an aromatic substituent
at C8 plays an essential role in achieving binding affinity
for PI3-Ks, but may be dispensable for DNA-PK inhibi-
tion.
On the basis of this initial data, seven additional com-
pounds were prepared by palladium catalyzed coupling
of different aromatic substituents to TGX066 (Supple-
mentary Scheme 1) to explore the chemical space
surrounding TGX115 and TGX126. These com-
pounds, termed morpholino chromone analogs 49–55
(MCA049–MCA055), all contain the pyrimidone heter-
ocycle scaffoldof TGX126 anddiffer in their aromatic
substitution at the position analogous to C8 in
LY294002. They include compounds that substitute
the pyrimidone core with anilines (MCA50–MCA52,
MCA54), benzylamines (MCA53 andMCA55), anda
biaryl linkage (MCA49) that mimics LY294002 in this
new scaffold, and were prepared.
We initially prepared( Supplementary Schemes 1 and2 )
andtestedtwo compounds from this series (TGX115
andTGX126) as well as a synthetic intermediate lacking
the aromatic substituent at C8 (TGX066) andcompared
these compounds to LY294002 and the related
LY292223 (which lacks the C8 phenyl group). To obtain
a detailed picture of the target specificity of these com-
pounds, we determined IC50 values in vitro against a
panel of 14 enzymes selectedto span the most relevant
targets for these molecules. These include seven mam-
malian PI3-Ks (p110a, p110b, p110d, p110c, PI3KC2a,
PI3KC2b, andPI3KC2 c), four protein kinases in the
PI3-K family (DNA-PK, ATM, ATR, mTOR), PI4-kin-
ase b (a PI3-K family member reportedto be weakly
inhibitedby LY294002 18), casein kinase II (the only pro-
tein kinase family member known to be inhibitedby
LY29400215), andthe unrelatedserine-threonine kinase
G-protein coupledreceptor kinase 2 (GRK2). These 14
proteins include all of the known targets of LY294002
andmost of the proteins that contain sequence homol-
ogy to the PI3-K catalytic domain. To facilitate compar-
ison of IC50 values across kinases with different
substrate affinities, all assays were carriedout in the
presence of 100lM ATP.
The specificity profile of these compounds was deter-
mined, and several structure–activity trends were identi-
fied( Fig. 2). Compounds containing a benzylamine
substitution to the pyrimidone core exhibit potent
activity against the class IA PI3-Ks p110a/b/d
(IC50 =0.1ꢁ2lM), but display comparatively less
activity against the class IB PI3-K p110c
(IC50 =25ꢁ100lM). Comparedto the closely related
TGX126, these methyl-substitutedcompounds possess
modestly enhanced activity toward p110a anddimin-
ishedactivity against p110 c. MCA55 showedthe largest
selectivity between p110a andp110 c of any compound
in our panel (ꢀ50-fold) and this compound can be
regarded as a multiplex inhibitor of the growth factor
regulatedclass IA PI3-Ks with little activity against
the G-protein regulatedclass IB enzyme, p110 c.
As has been previously reported, LY29002 exhibits a
very broadspecificity profile, inhibiting the class I PI3-
Ks, PI3KC2b, PI3KC2c, mTOR, casein kinase 2, and
DNA-PK all with IC50 values in the low micromolar
range. Within the class I PI3-Ks, LY294002 exhibits a
maximum of approximately 10-foldselectivity between
any two isoforms, with p110b being most sensitive
(2.9lM) andp110 c the least sensitive (38lM). Interest-
ingly, LY294002 did not show activity at 100lM against
the PI3-K relatedprotein kinases ATM andATR.
Although we are not aware of any reports that
LY294002 inhibits these two enzymes at micromolar
concentrations,19 the effects of LY294002 treatment of
cells has been interpretedto imply a requirement for
ATM or ATR kinase activity in several studies.20,21
In contrast, compounds containing an aniline substitu-
tion on the pyrimidone scaffold all showed a 10–100-fold
loss of activity against the PI3-K isoforms p110d and
p110b relative to the parent compounds TGX115 and
TGX126, while retaining potent DNA-PK inhibition.
This trendincludes MCA51, a compoundthat is iso-
steric to TGX115 within the pyrimidone scaffold. Com-
paredto TGX115, this compoundis
potent against p110d/p110b and ꢀ10-foldmore
ꢀ20-foldless
potent against DNA-PK. This effect appears to be a

4752
(A)
Z. A. Knight et al. / Bioorg. Med. Chem. 12 (2004) 4749–4759
(B)
100
75
50
25
0
LY294002
TGX115
LY292223
TGX066
O
O
O
O
N
O
N
N
H
N
O
N
N
N
O
O
O
O
Br
O
TGX126
MCA55
MCA53
MCA51
O
O
O
O
N
N
N
N
-10
-9
-8
-7
-6
-5
-4
-3
log [LY294002]
N
N
N
N
N
N
N
N
NH
O
NH
O
NH
O
NH
O
100
75
50
25
0
MCA52
MCA50
MCA54
MCA49
O
O
O
O
N
N
N
N
N
N
N
N
N
N
OMe
N
N
NH
O
O
NH
O
NH
O
H
N
2
-10
-9
-8
-7
-6
-5
-4
-3
log [TGX115]
p110α p110β p110δ p110γ PIKC2α PIKC2β PIKC2γ PI4Kβ
DNA-PK ATM
ATR
mTOR CK2
GRK2
(C)
TGX115
61
8.1
58
14
~50
~9
~10
1.8
6.2
1.1
45
0.13
0.16
9.9
18
~20
3.3
14
0.99
3.5
0.11
7.5
2.9
0.63
0.15
8.5
6.9
~10
3.8
~5
0.39
3.3
0.87
13
6.0
~100
~10
>100
38
>100
>100
>100
~25
~100
~100
~100
38
>100
ND
ND
~50
ND
ND
>100
>100
>100
ND
~10
ND
~100
ND
ND
>100
>100
>100
ND
>100
>100
>100
>100
>100
>100
>100
~100
1.2
0.28
1.3
>100
ND
>100
ND
>100
>100
>100
>100
>100
>100
ND
>100
ND
ND
ND
ND
ND
ND
ND
ND
>100
ND
>100
ND
>100
>100
>100
>100
>100
>100
ND
>100
>100
>100
ND
>100
>100
>100
>100
>100
>100
>100
12.2
>100
>100
>100
ND
>100
>100
>100
>100
>100
>100
>100
>100
TGX126
TGX066
MCA49
MCA50
MCA51
MCA52
MCA53
MCA54
MCA55
LY292223
LY294002
ND
ND
7.1
>100
>100
>100
>100
>100
>100
ND
>100
~50
~50
>100
~50
~50
~20
40
0.19
0.15
0.55
0.38
0.36
0.27
0.42
0.66
2.8
~100
5.7
>100
ND
>100
9.3
~100
>100
8.9
Figure 2. IC50 values of LY294002 analogs against protein andlipidkinases. (A) Structures of LY294002 analogs. (B) Dose response curves for
LY294002 (top) andTGX115 (bottom) against different PI3-K family members. DNA-PK (purple), p110 a (red), p110b (blue), p110c (green), p110d
(black), andCK2 (yellow). (C) IC values (lM) measuredin the presence of 100 lM ATP.
50
consequence of the aniline substitution, rather than the
pyrimidone core itself, because TGX126 and related
We findthat IC60211 andIC86621 are high nanomolar
inhibitors of DNA-PK, with IC50 values against class I
PI3-Ks in the low to mid-micromolar range, consistent
with an earlier report.22 In this regard, these compounds
exhibit a specificity profile that mirrors LY294002, with
modestly enhanced DNA-PK activity (1.5–5-fold).
Nonetheless, we findthat these compounds are more
selective than LY294002 in that they show no activity
against the secondary targets mTOR, casein kinase II
or any of the class II PI3-Ks. Thus, within the lipidkin-
ases, this structural class is selective for the class I PI3-
Ks. Arylmorpholine analog 37 (AMA37) is the most
DNA-PK selective of these compounds, with ꢀ10-fold
specificity relative to p110b and ꢀ100-foldspecificity
relative to the other class I PI3Ks. This potency and
selectivity is comparable to the most DNA-PK selective
compounds identified from our panel of anilino pyrim-
idones.
compounds containing
a benzylamine substitution
remain highly potent against p110d/p110b. This suggests
that very subtle changes to the aromatic substituent can
significantly shift inhibitor specificity among PI3-K fam-
ily members. Indeed, we find that several anilino-pyrim-
idones inhibit DNA-PK at nanomolar concentrations
anddisplay significant selectivity (10–100-fold) relative
to all other members of the PI3-K family. These com-
pounds may prove useful as selective inhibitors of
DNA-PK, or as leadstructures for the development of
more highly specific compounds.
2.2. Arylmorpholine DNA-PK inhibitors
ICOS has recently disclosed a set of arylmorpholines22,23
that are potent inhibitors of DNA-PK (Fig. 3). These
compounds are trisubstituted benzene derivatives with
hydroxy and carbonyl moieties meta and para to the
morpholine ring, respectively. As these molecules are
structurally reminiscent of LY294002, we prepareda
small set of eight arylmorpholines (Supplementary
Scheme 3) andedterminedtheir specificity profile
against our enzyme panel.
We testeda series of analogs of these compounds to
probe the requirements for DNA-PK inhibition, and
confirm that both the hydrogen bond acceptor at the
1-position andthe donor at the 2-position are required
for potent DNA-PK inhibition (Fig. 3). For example,
AMA57, which differs from AMA37 by the substitution

Z. A. Knight et al. / Bioorg. Med. Chem. 12 (2004) 4749–4759
4753
(A)
(B)
IC60211
AMA48
AMA30
AMA37
100
75
50
25
0
OH
O
OH
O
NH
OH
O
2
Cl
NO
2
H
N
N
N
N
O
O
O
O
IC86621
AMA58
AMA56
AMA57
OH
OH
O
OH
O
NO
2
N
N
N
N
-10
-9
-8
-7
-6
-5
-4
-3
O
O
O
O
log [AMA37]
p110α p110β p110δ p110γ PIKC2α PIKC2β PIKC2γ PI4Kβ
(C)
DNA-PK ATM
ATR
mTOR
CK2
GRK2
~100
AMA30
IC60211
IC86621
AMA37
AMA48
AMA56
AMA57
AMA58
~100
~10
16
23
2.8
24
5.1
3.8
22
ND
>100
ND
ND
>200
~200
~200
ND
ND
~50
>100
~50
ND
>100
>100
>100
>100
>100
ND
2.3
0.43
0.17
0.27
1.1
>100
>100
>100
>100
>100
ND
ND
ND
>100
>100
>100
>100
>100
ND
>100
>100
>100
>100
>100
ND
>100
>100
>100
>100
>100
ND
37
10
0.99
3.7
ND
32
~100
~70
ND
>100
ND
>100
ND
30
ND
ND
ND
ND
4.8
ND
ND
ND
ND
ND
ND
ND
ND
ND
1.2
ND
ND
ND
ND
ND
ND
14.7
~100
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Figure 3. IC50 values of arylmorpholines against protein andlipidkinases. (A) Structures of arylmorpholine analogs. (B) Dose response curves for
AMA37 against different PI3-K family members (color coded as in Fig. 2). (C) IC50 values (lM) measuredin the presence of 100 lM ATP.
of a hydrogen for the 2-hydroxyl, is ꢀ80-foldless
potent, whereas AMA58, which differs from IC60211
potent. However, the specific identity of hydrogen bond
donor and acceptor is not a requirement for potent
DNA-PK inhibition. For example, AMA56, which dif-
fers from IC60211 by the replacement of the aldehyde
with a nitro group, is only ꢀ3-foldless potent. Likewise,
AMA30, which differs from AMA56 by the further sub-
stitution of the 2-hydroxyl group with an amine, is only
ꢀ2-foldless potent.
activity is significantly reduced compared to more substi-
tutedanalogs. Moreover, a thirdclass of DNA-PK
inhibitors basedon a limitedarylmorpholine scaffold
have recently been reported,24 suggesting that diverse
compounds containing this core structure can exhibit po-
tent andselective DNA-PK inhibition.
Selectivity trends were also observed within the class I
PI3-Ks. To visualize these differences, we plotted the
IC50 values of all of the compounds for inhibition of
p110b (x-axis) against the IC50 values for inhibition of
the other class I PI3-Ks andDNA-PK ( y-axis), such that
each data point represents the intersection of IC50 values
for the same compoundagainst two different enzymes
(Fig. 4). In this representation, compounds that lie along
3. Discussion
We have measuredthe activity of a panel of compounds
that share the arylmorpholine pharmacophore first iden-
tifiedin LY294002 against a comprehensive set of targets
within the PI3-K family. Analysis of this data suggests
several trends in selectivity among different PI3-K family
members. First, while we confirm that LY294002 inhibits
three protein kinases at low micromolar concentrations
(DNA-PK, mTOR, CK2), we findthat two of these kin-
ases (mTOR andCK2) are resistant to all other aryl-
morpholines in our panel. DNA-PK, by contrast, is
potently inhibitedby almost every compoundtested,
andshows very broadSAR with respect different analogs
in the same series. In part, we believe these differences re-
flect the fact that DNA-PK binds with high affinity to the
core arylmorpholine pharmacophore, whereas the other
protein kinases (andto a lesser extent, the PI3-Ks) re-
quire more extensive interactions with other regions of
the molecule for high affinity binding. Consistent with
this view, the most structurally simple compounds in
our panel (TGX066, LY292223, andIC60211) all exhibit
potent inhibition of DNA-PK, even though their PI3-K
100
10
1
0.1
0.1
1
10
value (µM)
100
p110β
50
IC
Figure 4. Correlation plot for inhibition of different PI3-K isoforms.
IC50 values (lM) for p110b (x-axis) plottedagainst IC values for
50
DNA-PK (purple), p110a (red), p110c (green), andp110 d (black).

4754
Z. A. Knight et al. / Bioorg. Med. Chem. 12 (2004) 4749–4759
the diagonal show equipotent inhibition of p110b
andthe secondkinase, whereas those that lie above or
below the diagonal are more or less potent, respectively,
against the secondenzyme than p110 b. Virtually all of
the DNA-PK IC50 values (purple) occupy the lower sec-
tion of the plot, reflecting the fact that DNA-PK is more
sensitive to arylmorpholines than the class I PI3Ks.
p110d IC50 values (black) cluster aroundthe diagonal
as few compounds within this series show differential
activity against p110d andp110 b, whereas p110a (red)
tends to be inhibited less potently. p110c (green) was
unexpectedly resistant to inhibition by a large number
of arylmorpholines, although this insensitivity is consis-
tent with p110cÕs underlying poor sensitivity to
LY294002 relative to the other class I isoforms.
the potency andisoform selectivity of compounds such
as TGX115, it does suggest that smaller differences
between the class I isoforms (e.g., as observedfor
LY294002) likely reflect differential competition from
ATP substrate between isoforms. This suggests that
the PI3-K family members for which selective inhibitors
have been identified to date (DNA-PK, and, to a lesser
extent, p110b andp110 d) may be inherently easier to
target with ATP competitive small molecules than other
isoforms.
It has long been anticipatedthat the development of iso-
form-specific PI3-K inhibitors wouldmake it possible to
dissect the unique contributions of individual PI3-K iso-
2,11–13
forms in well-characterizedsignaling pathways.
Recently, Sadhu et al. described the first isoform selec-
tive PI3-K inhibitor,25 IC87114, which targets p110d,
andthis compoundhas foundrapiduse in identifying
essential roles for p110d in chemotaxis andinflamma-
tory responses of neutrophils,25–27 spontaneous tone of
arteries28 andEGF rdiven migration of cancer cell
lines.29 TGX115, when usedeither in conjunction with
IC87114 or in cells that do not express p110d, should
prove equally effective in elucidating physiological roles
for p110b, andwe have begun to use this compoundto
explore p110b mediated signaling in several systems
(Z.A.K. andK.M.S., unpublishedresults). As other mu-
tliplex inhibitors we describe in this report also possess
activity against unique combinations of PI3-K family
members (Fig. 5), it may be possible to use these com-
pounds in combination in order to interrogate addi-
tional PI3-K isoforms. In this way, we envision a
route to begin to pharmacologically dissect the distinct
contributions of members of this important family of
signaling enzymes.
While we have not been able to rationalize these differ-
ences in inhibitor sensitivity in terms of specific residues
that differ between PI3-K isoforms, several observations
are consistent with these IC50 trends. First, the similarity
in the sensitivity of p110d andp110 b to diverse com-
pounds is likely a reflection of the fact that these two
kinases are more closely relatedin primary sequence
than any other members of the PI3-K family. For this
reason, it may be challenging to findanalogs of potent
p110b/p110d inhibitors such as TGX115 containing
the arylmorpholine core that can distinguish between
these two isoforms. Second, we find that the overall
trendin inhibitor sensitivity among these targets
(p110c<p110a<p110b, p110d<DNA-PK) roughly
mirrors the KM for ATP of these proteins. That is, we
have foundthat p110 c has the lowest KM for ATP
among these enzymes (7.4lM), whereas DNA-PK has
the highest (192lM), andthe class IA PI3-Ks exhibit
intermediate values. While this alone cannot explain
Me
(A)
O
O
H
OH
O
Me
O
N
OMe
Me
O
H
N
O
O
Me
O
MeO
O
N
O
Me
O
H
OMe
OH
N
Me
O
O
N
O
OH
Me
N
N
O
Me
OMe Me
Me
NH
2
Rapamycin
IC87114
Wortmannin
p110α p110β p110δ p110γ C2α C2β C2γ DPK ATM ATR TOR
(B)
Wortmannin (mid µM)
Wortmannin (mid nM)
LY294002
IC60211
MCA055
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
TGX115
IC87114, others
AMA037, others
Rapamycin
X
X
Figure 5. Spectrum of characterizedPI3-K inhibitor selectivities. (A) Structures of reportedinhibitors of PI3-K family members. (B) Selectivity
profile of well-characterized PI3-K inhibitors. Data for wortmannin, IC87114, and rapamycin is drawn from published reports. Compounds
characterized in this study are highlighted in red.

Z. A. Knight et al. / Bioorg. Med. Chem. 12 (2004) 4749–4759
4755
4. Experimental
4.1. Lipid kinase expression
fer (10mM HEPES (pH7.4), 50mM NaCl, 50mM
b-glycerophosphate, 10% glycerol). N-terminal His6-
taggedGRK2 was expressedin Sf9 insect cells and
purified using Ni-NTA beads (Qiagen) as described.30
Casein kinase 2 was obtainedfrom Upstate Biotechnol-
ogy, andDNA-PK was obtainedfrom Promega.
Epitope taggedp110 a, p110b, p110d, PI3KC2a,
PI3KC2b, andPI3KC2 c were expressedby transient
transfection of cos-1 cells. Cells were lyzedin lysis buffer
(50mM Tris (pH7.4), 300mM NaCl, 5mM EDTA,
0.02% NaN3, 1% Triton X-100, andprotease inhibitors)
andthe kinase immunoprecipitatedwith the appropriate
antibody-protein G complex. Immunoprecipitates were
washedtwice with buffer A (PBS, 1mM EDTA, 1% Tri-
ton X-100), twice with buffer B (100mM Tris (pH7.4),
500mM LiCl, 1mM EDTA), andtwice with buffer C
(50mM Tris (pH7.4), 100mM NaCl). GST-PI4Kb was
expressedin BL21 E. coli andpurifiedby glutathione
chromatography essentially as described.18 Recombi-
nant p110c was obtainedfrom Sigma. In control exper-
iments, no differences in inhibitor sensitivity were
observedbetween p110 a immunoprecipitatedfrom
cos-1 cells andprotein expressedin Sf9 cells using a
baculovirus system.
4.4. Protein kinase assays
ATM, ATR, andmTOR immunoprecipitates were re-
suspended in kinase assay buffer (10mM HEPES
(pH7.4), 50mM NaCl, 50mM b-glycerophosphate,
10% glycerol, 10mM MnCl2, 1mM DTT) andincubated
with inhibitor for 30min prior to the kinase reaction.
Kinase reactions were initiatedby the aiddtion of
100lM ATP, 10lCi (c-32P)-ATP and1 lg of either a
GST-p70S6K fragment (amino acids 332–414) for
mTOR, or 1lg of a GST-p53 fragment (amino acids
1–70) for ATM or ATR. Reactions were incubatedat
30ꢁC for 20min andterminatedby the addition of 2
·
SDS-PAGE sample buffer. Samples were resolvedby
SDS-PAGE andtransferredto PVDF membranes. The
portion of the membrane containing the 32P-labeledsub-
strate was quantitatedby PhosphorImager, while the
portion of the membrane containing ATM, ATR, or
mTOR was subjectedto Western blotting with anti-
HA, anti-FLAG, or anti-AU1 antibody, respectively.
4.2. Lipid kinase assays
All kinase assays were conducted at a final concentra-
tion of 100lM ATP and2% DMSO. PI3-K andPI4-
K assays were carried out essentially as described.18
Briefly, a reaction mixture was preparedcontaining kin-
ase, inhibitor, buffer (25mM HEPES (pH7.4), 10mM
MgCl2), andfreshly sonicatedphosphatiydlinositol
(200lg/mL). Reactions were initiated by the addition
of ATP containing 10lCi of c-32P-ATP to a final con-
centration 100lM. Reactions were incubated15min at
rt and then terminated by the addition of 105lL 1N
HCl followedby 160 lL CHCl3–MeOH (1:1). The bi-
phasic mixture was then vortexed, briefly centrifuged,
andthe organic phase transferredto a new tube using
a gel loading pipette tip precoated with CHCl3. This ex-
tract was spottedon TLC plates anddevelopedfor 3–4h
in a 65:35 solution of n-propanol–2M AcOH. The TLC
plates were then dried and quantitated using a Phos-
phorImager (Molecular Dynamics). For each com-
pound, kinase activity was measured at 12–15 inhibitor
concentrations representing twofolddilutions from the
highest concentration tested(typically, 200 lM). For
compounds showing significant activity, IC50 determina-
tions were repeatedtwo to six times, andthe reported
value is the average of these independent measurements.
The amount of 32P incorporation in the substrate was
normalizedto the amount of ATM or mTOR present.
GRK2 assays were carriedout using tubulin as substrate
in 20mM HEPES, pH7.4, 2mM EDTA, 10mM MgCl2
containing 100lM ATP essentially as described.30
DNA-PK assays were carriedout using the DNA-PK
assay system (Promega) as directed by the manufac-
turer. Casein kinase 2 assays were carriedout using
the casein kinase 2 assay kit (Upstate Biotechnology)
as directed by the manufacturer.
4.5. Synthesis
4.5.1. General. The following reagents were obtained
commercially: 2-amino-20-methyldiphenyl ether (TCI
America), 3-morpholinophenol (Avacado Research), 4-
morpholinobenzophenone (Sigma), 5-morpholino-2-ni-
trophenol (Alfa Aesar), LY294002 (Calbiochem), and
MnO2 andPdCl
(dppf) (Strem Chemicals). All other
2
reagents were from Aldrich, were of the highest grade
commercially available, andwere usedas suppliedby
the manufacturer without further purification. Reversed
phase high performance liquidchromatography (RP-
HPLC) was performedon a Ranin SD-200 solvent deliv-
ery system equippedwith a Zorbax 300-SB C18 column
using a MeCN/H2O/0.1% TFA gradient (0–100%) as the
mobile phase.
4.3. Protein kinase expression
HA-ATM, FLAG-ATR, andAU-1 mTOR were ex-
pressedby transient transfection of HEK293 cells. The
cells were lyzedin lysis buffer (50mM Tris–Cl (pH7.4),
100mM NaCl, 50mM b-glycerophosphate, 10% glycerol
(w/v), 1% Tween-20, 1mM EDTA, 25mM NaF, and
protease inhibitors), andlysates were subjectedto im-
munoprecipitation with the appropriate epitope-tag
antibody. Immune complexes were collected on rabbit
anti-mouse Sepharose (Sigma) andwashedthree times
in lysis buffer, once in high salt buffer (100mM Tris–
Cl (pH7.4), 500mM LiCl), andonce in kinase wash buf-
4.5.2. 5-(Bis-methylsulfanyl-methylene)-2,2-dimethyl-
(1,3)dioxane-4, 6-dione (1). Triethylamine (9.6mL, 69
mmol) andCS (2.05mL, 34.7mmol) were added con-
2
secutively to a stirredsolution of Melrdum Õs acid
(5.0g, 35mmol) in DMSO (50mL) at rt.31 After 1h,
the reaction was cooledon ice, MeI (4.3mL, 69mmol)
was added, and the reaction was allowed to proceed

4756
Z. A. Knight et al. / Bioorg. Med. Chem. 12 (2004) 4749–4759
overnight at rt. After 18h, the reaction was terminated
by adding ice water, and the precipitate was chromato-
graphedon silica gel (50% EtOAc/hexanes) to yield
1.962g (23%) of a yellow solid. H NMR (400MHz,
CDCl3) d 2.45 (6H, s), 1.53 (6H, s).
(1.5mL, 17mmol) was added to this solution and the
reaction was heatedto reflux for 1h. After 1h, the reac-
tion was allowedto cool to rt, yielding a white precipi-
tate. The precipitate was collectedby filtration and
rinsedwith coldEtOH to yield737mg (38.8%) of a
white solid. 1H NMR (400MHz, DMSO-d6) d 8.56
(1H, s), 8.14 (1H, s), 5.57 (1H, s), 3.63 (4H, s), 3.60
(4H, s), 2.26 (3H, s); 13C NMR (100MHz, CDCl3)
160.6, 159.0, 142.4, 125.2, 122.3, 119.2, 118.9, 81.1,
65.8, 65.7, 44.8, 44.7, 18.0; HR-EI MS (M)+ m/z calcd
for C13H14BrN3O2 323.0269, found323.0259.
1
4.5.3. 2,2-Dimethyl-5-(1-methylsulfanyl-2-(2-o-tolyloxy-
phenyl)-ethylidene)-(1,3)dioxane-4,6-dione (2). 2-Amino-
20-methyldiphenyl ether (802mg, 4.03mmol) was added
to a solution of 2 (1.0g, 4.0mmol) in EtOH (9mL) and
stirred at reflux for 9h essentially as described.14,32
When the reaction was complete, the solvent was re-
movedin vacuo andthe proudct chromatographed
twice on silica (10% EtOAc/hexanes followedby 50%
EtOAc/hexanes) to yield1.54g (95.8%). 1H NMR
(400MHz, CDCl3) d 7.39 (1H, d, J=8Hz), 7.00–7.24
(5H, m), 6.78–6.81 (2H, m), 5.24 (1H, s), 2.27 (3H, s),
2.21 (3H, s), 1.66 (6H, s); 13C NMR (100MHz,
CDCl3) d 178.5, 153.8, 151.3, 131.8, 129.8, 129.5,
128.0, 127.7, 127.3, 124.7, 123.1, 118.9, 117.9, 117.1,
103.2, 86.8, 26.5, 19.0, 16.3.
4.5.7. 9-Benzylamino-7-methyl-2-morpholin-4-yl-pyrido-
(1,2-a)pyrimidin-4-one (TGX126). A stirredsolution of
TGX66 (15mg, 0.046mmol), PdCl2 (dppf) (1.9mg,
0.0023mmol), potassium tert-butoxide (10.4mg,
0.0928mmol), andbenzylamine (4.97mg, 0.0464) in
THF (1mL) was heatedto reflux for 24h. 14 The product
was purifiedby chromatography on silica gel (5%
MeOH/CH2Cl2), followedby RP-HPLC, to yield
10mg (61%) of a white solid. 1H NMR (400MHz,
CDCl3) d 8.10 (1H, s), 7.22–7.31 (m), 6.35 (1H, s),
5.73 (1H, s), 4.47 (2H, s), 3.78 (4H, t, J=4Hz), 3.59
(4H, t, J=4Hz), 2.22 (3H, s); HR-EI MS (M)+ m/z calcd
for C20H22N4O2 350.1743, found350.1749.
4.5.4. 2-Morpholin-4-yl-8-o-tolyloxy-1H-quinolin-4-one
(TGX115). Morpholine (0.44mL, 5.0mmol) was added
to a solution of 2 (900mg, 2.25mmol) in THF
(12mL).14 The reaction was heatedto reflux for 24h.
After cooling to rt, the solvent was removedin vacuo,
the solidwashedwith Et 2O, andthen idssolvedin
Ph2O (10mL). The reaction was heatedto 265 ꢁC for
15min, andthen cooledto rt. The product was purified
by chromatography on silica gel twice (50% EtOAc/hex-
anes followedby 10% MeOH/EtOAc) to yield227mg
4.5.8. 9-(3-Amino-phenyl)-7-methyl-2-morpholin-4-yl-pyr-
ido[1,2-a]pyrimidin-4-one (MCA49). Preparedfollowing
the general procedure for TGX126, using TGX66
(50mg, 0.16mmol), 3-aminophenylboronic acid(96mg,
0.62mmol), andNa CO3 (164mg, 1.55mmol) in place
2
of potassium tert-butoxide. The product was purified
by chromatography on silica gel (5% MeOH/EtOAc)
to yield30mg (58%) of a white soli.d LR-ESI MS
(M+H)+ m/z calcdfor C 20H22N4O3 337.2, found
337.0.
1
(33%) of a white solid. H NMR (400MHz, CDCl3) d
8.51 (1H, s), 7.88 (1H, d, J=8Hz), 7.11–7.27 (3H, m),
7.05 (1H, t, J=16Hz), 6.94 (1H, d, J=8Hz), 6.67 (1H,
d, J=8Hz), 5.73 (1H, s), 3.81 (4H, s), 3.29 (4H, t,
J=4Hz), 2.18 (3H, s); 13C NMR (100MHz, DMSO-
d6) d 161.6, 157.7, 157.0, 149.7, 140.9, 130.6, 126.8,
126.6, 121.7, 120.6, 119.6, 118.9, 117.6, 115.9, 91.1,
65.8, 44.8, 16.1; HR-EI MS (M)+ m/z calcdfor
C20H20N2O3 336.1474, found336.1457.
4.5.9. 9-(2-Methoxy-phenylamino)-7-methyl-2-morpholin-
4-yl-pyrido(1,2-a)pyrimidin-4-one (MCA50). Prepared
following the general procedure for TGX126, using
TGX66 (50mg, 0.16mmol) and o-anisidine (0.017mL,
0.155mmol). The product was purified by chromatogra-
phy on silica gel (2% MeOH/hexanes) followedby RP-
4.5.5. 9-Bromo-2-hydroxy-7-methyl-pyrido(1,2-a)pyrim-
idin-4-one (3). A stirredsolution of 2-amino-3-bromo-
5-methylpyridine (9.85g, 52.7mmol) in diethylmalonate
(20mL, 130mmol) was heatedto 200 ꢁC for 3.5h.14 The
heat was then removedandthe diethylmalonate was
removedunder a stream of argon as the reaction was
allowedto cool to rt. The solidproduct was purified
by chromatography on silica gel twice (50% EtOAc/hex-
anes, followedby 5% MeOH/CH 2Cl2) to yield1.7g
(12.7%) of a yellow solid. 1H NMR (400MHz,
DMSO-d6) d 11.71 (1H, br), 8.69 (1H, s), 8.25 (1H, s),
5.46 (1H, s), 2.30 (3H, s); HR-EI MS (M)+ m/z calcd
for C9H7BrN2O2 253.9691, found253.9674.
HPLC to yield3.8mg (6.7%) of a white soli.d
1H
NMR (400MHz, DMSO-d6) d 8.37 (1H, s), 8.04 (1H,
s), 7.52 (1H, d, J=8Hz), 7.17 (1H, s), 6.98–7.09 (4H,
m), 3.83 (3H, s), 3.70 (4H, t, J=4Hz), 3.62 (4H, t,
J=4Hz), 2.23 (3H, s); HR-EI MS (M)+ m/z calcdfor
C20H22N4O3 366.1692, found366.1701.
4.5.10. 7-Methyl-2-morpholin-4-yl-9-o-tolylamino-pyri-
do(1,2-a)pyrimidin-4-one (MCA51). Preparedfollowing
the general procedure for TGX126, using TGX66
(50mg, 0.16mmol) and o-toluidine (0.017mL,
0.16mmol). The product was purified by chromatogra-
phy on silica gel (2% MeOH/hexanes) followedby RP-
HPLC to yield6.8mg (12.6%) of an off-white solid. 1H
NMR (400MHz, DMSO-d6) d 7.99 (1H, s), 7.82 (1H,
s), 7.23–7.34 (3H, m), 7.12 (1H, t, J=7.2Hz), 6.48
(1H, s), 5.59 (1H, s), 3.64 (8H, br), 2.18 (3H, s), 2.15
(3H, s); HR-EI MS (M)+ m/z calcdfor C 20H22N4O2
350.1743, found350.1754.
4.5.6. 9-Bromo-7-methyl-2-morpholin-4-yl-pyrido(1,2-a)-
pyrimidin-4-one (TGX66). A stirredsolution of 3 (1.7g,
6.7mmol) in POCl3 (20.0mL, 215mmol) was heatedto
reflux overnight.14 After 18h, the reaction was quenched
by pouring onto ice. The aqueous phase was extracted
three times with CH2Cl2, concentratedto dryness in
vacuo, andidssolvedin EtOH (25mL). Morpholine

Z. A. Knight et al. / Bioorg. Med. Chem. 12 (2004) 4749–4759
4757
4.5.11. 9-(3,4-Dimethyl-phenylamino)-7-methyl-2-morph-
olin-4-yl-pyrido(1,2-a)pyrimidin-4-one (MCA52). Pre-
paredfollowing the general proceudre for TGX126,
using TGX66 (50mg, 0.16mmol) and3,4-dimethylani-
line (18.8mg, 0.155mmol). The product was purified
by chromatography on silica gel (2% MeOH/hexanes)
followedby RP-HPLC to yield13.4mg (23.9%) of a yel-
(2H, s), 6.34 (1H, d, J=10Hz), 6.18 (1H, s), 3.67 (4H,
t, J=5Hz), 3.23 (4H, t, J=5Hz); 13C NMR (100MHz,
DMSO-d6) d 155.9, 148.9, 127.8, 123.9, 105.8, 98.3,
66.4, 44.1; HR-EI MS (M)+ m/z calcdfor C 10H13N3O3
223.0957, found223.0954.
4.5.16. 2-Hydroxy-4-morpholin-4-yl-benzaldehyde (IC60-
211). DMF (10mL) was cooledon ice andPOCl
1
low solid. H NMR (400MHz, DMSO-d6) d 8.00 (1H,
3
s), 7.85 (1H, br), 7.05–7.13 (3H, m), 6.94 (1H, s), 5.58
(1H, s), 3.64 (8H, br), 2.18–2.21 (9H, m); HR-EI MS
(M)+ m=z calcdfor C 21H24N4O2 364.1899, found
364.1909.
(1.4mL, 15mmol) was added dropwise. 3-Morpholino-
phenol (2.5g, 14mmol) was slowly added to the stirred
solution at rt, incubated at rt for an additional 40min,
andthen heatedto 100 ꢁC for 9h essentially as de-
scribed.23 The reaction was terminatedby dumping into
1M NaOAc (40mL). The solidwas filtered, rinsedthree
times with H2O, andpurifiedby chromatography on sil-
ica gel (25% EtOAc/hexanes) to yield802mg (28%) of an
4.5.12. 7-Methyl-9-(3-methyl-benzylamino)-2-morpholin-
4-yl-pyrido(1,2-a)pyrimidin-4-one (MCA53). Prepared
following the general procedure for TGX126, using
TGX66 (50mg, 0.16mmol) and3-methylbenzylamine
(0.019mL, 0.16mmol). The product was purified by
chromatography on silica gel (2% MeOH/hexanes) fol-
lowedby RP-HPLC to yield12.3mg (22%) of an off-
white solid. 1H NMR (400MHz, DMSO-d6) d 7.85
(1H, s), 7.09–7.19 (3H, m), 7.01 (1H, d, J=8Hz), 6.92
(1H, br), 6.32 (1H, s), 5.54 (1H, s), 4.43 (2H, s), 3.65
(4H, t, J=5Hz), 3.61 (4H, t, J=5Hz), 2.25 (3H, s),
2.11 (3H, s); HR-EI MS (M)+ m/z calcdfor
C21H24N4O2 364.1899, found364.1904.
1
off-white solid. H NMR (400MHz, DMSO-d6) d 11.07
(1H, s), 9.72 (1H, s), 7.46 (1H, d, J=8Hz), 6.55 (1H, dd,
J=9Hz, 2.4Hz), 6.28 (1H, d, J=2.4Hz), 3.66 (4H, t,
J=5Hz), 3.30 (4H, t, J=5Hz); 13C NMR (100MHz,
DMSO-d6) d 192.0, 163.7, 157.2, 133.9, 113.8, 106.8,
99.5, 66.4, 47.1; HR-EI MS (M)+ m/z calcdfor
C11H13NO3 207.0895, found207.0893.
4.5.17. 1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-ethanone
(IC86621). MeLi (2.4mL of 1.6M solution in Et2O,
3.9mmol) was added dropwise to a solution of
IC60211 (200mg, 0.996mmol) in THF (4mL) at
ꢁ78ꢁC.23 The reaction was allowedto warm to rt and
proceedovernight, at which point the reagent was
quenchedwith satdNH 4Cl (1mL), extractedsix times
with Et2O, andconcentratedin vacuo. The product
was chromatographedon silica gel (25% EtOAc/hexa-
nes) to yielda white solid. The solidwas dissolvedin
10mL MeCN, MnO2 (391mg, 4.5mmol) was added,
andthe reaction was allowedto proceedfor three days
at rt under an inert atmosphere. The product was chro-
matographedon silica gel (50% EtOAc/hexanes) to yield
17.8mg (18%) of a white solid. 1H NMR (400MHz,
CDCl3) d 12.71 (1H, s), 7.55 (1H, d, J=9Hz), 6.35
(1H, d, J=9Hz), 6.26 (1H, s), 3.79 (4H, t, J=5Hz),
3.30 (4H, t, J=5Hz), 2.50 (3H, s); 13C NMR
(100MHz, CDCl3) d 201.7, 165.0, 156.7, 132.5, 105.5,
100.6, 66.6, 47.2; HR-EI MS (M)+ m/z calcdfor
C12H15NO3 221.1052, found221.1049.
4.5.13. 9-(2,3-Dimethyl-phenylamino)-7-methyl-2-morph-
olin-4-yl-pyrido(1,2-a)pyrimidin-4-one (MCA54). Pre-
paredfollowing the general proceudre for TGX126,
using TGX66 (50mg, 0.16mmol) and2,3-dimethylani-
line (19mg, 0.16mmol). The product was purified by
chromatography on silica gel (2% MeOH/hexanes) fol-
lowedby RP-HPLC to yield12.3mg (22%). 1H NMR
(400MHz, DMSO-d6) d 7.96 (1H, s), 7.83 (1H, s),
7.05–7.14 (3H, m), 6.30 (1H, s), 5.58 (1H, s), 3.64 (8H,
br), 2.27 (3H, s), 2.13 (3H, s), 2.06 (3H, s); HR-EI MS
(M)+ m=z calcdfor C 21H24N4O2 364.1899, found
364.1910.
4.5.14. 7-Methyl-9-(2-methyl-benzylamino)-2-morpholin-
4-yl-pyrido(1,2-a)pyrimidin-4-one (MCA55). Prepared
following the general procedure for TGX126, using
TGX66 (50mg, 0.16mmol) and2-methylbenzylamine
(0.019mL, 0.16mmol). The product was purified by
chromatography on silica gel (2% MeOH/hexanes) fol-
lowedby RP-HPLC to yield12.9mg (23%) of an off-
white solid. 1H NMR (400MHz, DMSO-d6) d 7.87
(1H, s), 7.09–7.17 (4H, m), 6.76 (1H, br), 6.31 (1H, s),
5.55 (1H, s), 4.44 (2H, s), 3.64 (4H, t, J=5Hz), 3.60
(4H, t, J=5Hz), 2.32 (3H, s), 2.11 (3H, s); HR-EI MS
(M)+ m/z calcdfor C 21H24N4O2 364.1899, found
364.1905.
4.5.18. 1-(2-Hydroxy-4-morpholin-4-yl-phenyl)-phenyl-
methanone (AMA37). Preparedaccording to the general
procedure of IC86621 using PhMgBr (1.1mL, 3.4mmol)
andIC60211 (150mg, 0.724mmol), except that the
product of PhMgBr addition was taken on for oxidation
by MnO2 (249mg, 3.38mmol) without intervening chro-
matographic purification. Chromatography of the final
product on silica gel (50% EtOAc/hexanes) yielded
4.5.15. 5-Morpholin-4-yl-2-nitro-phenylamine (AMA30).
Morpholine (2.03mL, 23.2mmol) was added to a stirred
solution of 5-chloro-2-nitroaniline (2.0g, 12mmol) in
DMSO (10mL) andheatedto 90 ꢁC for 18h.23 When
the reaction was complete, the product was precipitated
by pouring into H2O (150mL), andthen purifiedby
chromatography on silica gel (50% EtOAc/hexanes) to
(28.9mg, 14.1%) of
a
yellow solid. 1H NMR
(400MHz, CDCl3) d 12.73 (1H, s), 7.41–7.61 (5H, m),
6.35 (1H, d, J=2.4Hz), 6.31 (1H, dd, J=9Hz, 2.8Hz),
3.81 (4H, t, J=8Hz), 3.31(4H, t, J=8Hz); 13C NMR
(100MHz, CDCl3) d 199.1, 166.1, 156.6, 138.8, 135.5,
131.4, 129.0, 128.4, 119.2, 111.3, 105.3, 100.6, 66.7,
47.1; HR-EI MS (M)+ m/z calcdfor C 17H17NO3
283.1208, found283.1195.
yield660mg (25.4%) of a yellow soli.d
(400MHz, DMSO-d6) d 7.79 (1H, d, J=8Hz), 7.25
1H NMR

4758
Z. A. Knight et al. / Bioorg. Med. Chem. 12 (2004) 4749–4759
4.5.19. 2-Chloro-1-(2-hydroxy-4-morpholin-4-yl-phenyl)-
ethanone (AMA48). A solution of 3-morpholinophenol
(5.0g, 28mmol) andchloroacetonitrile (2.1mL,
34mmol) in chloroethane (150mL) in a 3-neck flask
Research. Mass spectra were provided by the Center for
Mass Spectrometry at the University of California, San
Francisco, supportedby the NIH division of Research
Resources.
was cooledin a ice bath, andBCl
(100mL, 1M in
3
CH2Cl2) was slowly added by an addition funnel essen-
tially as described.23 AlCl3 (1.9g, 14mmol) was then
added to this solution, the flask was equipped with a re-
flux condenser, and the reaction was heated to 60ꢁC for
24h. The reaction was then cooledto 0 ꢁC and100mL
2N HCl was added by addition funnel. Following acid-
ification, the organic phase was collectedandrinsed
twice with 2N HCl (100mL) andonce with H 2O
(100mL). The organic phase was concentratedin vacuo
andthe product purifiedby chromatography on silica
gel (30% EtOAc/hexanes) to yield775mg (10.9%). 1H
NMR (400MHz, DMSO-d6) d 11.91 (1H, s), 7.66 (1H,
d, J=9Hz), 6.53 (1H, dd, J=9Hz, 2.4Hz), 4.93 (2H,
s), 3.66 (4H, t, J=5Hz), 3.31 (4H, t, J=5Hz); 13C
NMR (100MHz, DMSO-d6) d 193.0, 163.6, 156.5,
132.1, 109.1, 105.8, 99.1, 65.7, 46.4, 46.3; HR-EI MS
(M)+ m/z calcdfor C 12H14ClNO3 255.0662, found
255.0670.
Supplementary material
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2004.06.022.
References and notes
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4.5.20. LY292223. Preparedaccording to the general
procedure described for the synthesis of 2-aminochro-
mones.33 20-Hydroxyacetophenone (0.50mL, 4.2mmol)
was dissolved in 11mL CH2Cl2, cooledto ꢁ78ꢁC and
TiCl4 (6.5mL, 1M solution in THF, 6.5mmol) was
added. After 1h, DIPEA (2.7mL, 16mmol) was added
at ꢁ78ꢁC andthe reaction was allowedto proceedfor
an additional 1h. 4-Dichloromethylenemorpholin-4-
ium34 (1.2g, 5.0mmol) was then added and the reaction
was allowedto proceedat ꢁ78ꢁC for 20min. The reac-
tion was warmedto 0 ꢁC, MeOH (30mL) was added,
andthe reaction was allowedto warm to rt overnight.
The reaction was then concentratedin vacuo, washed
once with satdNaHCO 3, andthe NaHCO was ex-
3
tractedthree times with CH Cl2. The combinedorganic
2
phase was dried over MgSO4, filtered, and solvent re-
movedin vacuo. The product was purifiedby chroma-
tography on silica gel (10% MeOH/EtOAc) to yield
1
344mg of a white powder (36%). H NMR (400MHz,
CDCl3) d 7.95 (1H, s), 7.35 (1H, s), 7.14 (1H, s), 7.08
(1H, s), 5.27 (1H, s), 3.63 (4H, t, J=5Hz), 3.30 (4H, t,
J=5Hz); 13C NMR (100MHz, CDCl3) d 176.8, 162.5,
153.5, 132.2, 125.2, 124.6, 122.8, 116.3, 87.0, 65.8,
44.5; IR: 1616, 1555, 1418, 1300, 1251, 1117, 985, 766;
HR-EI MS (M)+ m/z calcdfor C 13H13NO3 231.0895,
found231.0887.
Acknowledgements
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cDNA encoding PI3KC2b, Dr. Tamas Balla for cDNA
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