Discovery of 3,3′-(2,4-diaminopteridine-6,7-diyl)diphenol as an isozyme-selective inhibitor of PI3K for the treatment of ischemia reperfusion injury associated with myocardial infarction

Article abstract of DOI:10.1021/jm051056c

In studies aimed toward identifying effective and safe inhibitors of kinase signaling cascades that underlie ischemia/reperfusion (I/R) injury, we synthesized a series of pteridines and pyridopyrazines. The design strategy was inspired by the examination

Full text of DOI:10.1021/jm051056c

J. Med. Chem. 2007, 50, 4279-4294
4279
Discovery of 3,3′-(2,4-Diaminopteridine-6,7-diyl)diphenol as an Isozyme-Selective Inhibitor of
PI3K for the Treatment of Ischemia Reperfusion Injury Associated with Myocardial Infarction
Moorthy S. S. Palanki,*,† Elena Dneprovskaia,† John Doukas,† Richard M. Fine,‡ John Hood,† Xinshan Kang,‡, Dan Lohse,†
Michael Martin,† Glenn Noronha,† Richard M. Soll,† Wolfgang Wrasidlo,† Shiyin Yee,† and Hong Zhu†
TargeGen Inc., 9380 Judicial DriVe, San Diego California 92121, and BioPredict, Inc., Suite 201, 660 Kinderkamack Road,
Oradell, New Jersey 07649
ReceiVed October 19, 2005
In studies aimed toward identifying effective and safe inhibitors of kinase signaling cascades that underlie
ischemia/reperfusion (I/R) injury, we synthesized a series of pteridines and pyridopyrazines. The design
strategy was inspired by the examination of naturally occurring PI3K inhibitors such as wortmannin and
quercetin, and building a pharmacophore-based model used for optimization. Structural modifications led
to hybrid molecules which incorporated aminopyrimidine and aminopyridine moieties with ATP mimetic
characteristics into the pharmacophore motifs to modulate kinase affinity and selectivity. Elaborations
involving substitutions of the 2 and 4 positions of the pyrimidine or pyridine ring and the 6 and 7 positions
of the central pyrazine ring resulted in in ViVo activity profiles which identified potent inhibitors of vascular
endothelial growth factor (VEGF) induced vascular leakage. Pathway analysis identified a diaminopteridine-
diphenol as a potent and selective phosphatidylinositol-3-kinase (PI3K) inhibitor. The structure-activity
relationship studies of various analogues of diaminopteridine-diphenol-based on biochemical assays resulted
in potent inhibitors of PI3K.
Introduction
The vascular endothelium plays a key role in physiological
homeostasis by regulating functions such as blood plasma-
interstitial fluid balance via vascular permeability control.
Ischemic tissue damage results in part from the disruption of
this homeostatic balance. As first shown by Dvorak et al.,1 an
ischemic environment triggers upregulation of vascular endot-
helial growth factor (VEGF) expression, followed by VEGF-
mediated vascular leakage. This process is regulated through
complex interactions between VEGF and structural proteins in
the endothelium that form the intercellular junctions between
neighboring endothelial cells, VE-cadherin and â-catenin.
Phosphorylation of these junctional proteins induces confor-
mational changes in these molecules, with subsequent losses in
cell-cell adhesion.2 This in turn leads to a reduction in the
vascular barrier that controls fluid movement from the blood
compartment to the interstitium. The importance of kinases in
regulating major cellular signaling events such as cell survival,3-6
cell migration,7-10 and transformation and apoptosis11-14 has
long been recognized, and perturbation in these pathways has
been implicated in a number of diseases including cancer and
inflammation.15,16 The critical roles that kinases play in regulat-
ing the vascular barrier have been extensively researched.17-19
These studies suggest that the inhibition of the implicated
signaling pathways might prove therapeutically effective for the
treatment of diseases and injuries associated with microvascular
barrier function such as ischemic conditions where edema
accumulation is believed to contribute to tissue damage.
In the regulation of microvascular processes, two major
signaling pathways, the Src family and the PI3K signaling
cascade in endothelial cells (Figure 1), are of pivotal importance.
A frequent target for intervention downstream of receptor
Figure 1. Kinase pathways regulating endothelial cell junction
integrity. The two major signal transduction pathways in the regulation
of intercellular integrity shown in this figure are Src and PI3K. PI3K
can be activated via multiple growth factors or through G protein
coupled PAFs. Signaling through Src can also affect gene transcription
via ERK activation.
tyrosine kinases is Src,20-23 but investigations on PI3K inhibitors
for a while was confined to natural products such as wortmannin,
analogues of wortmannin,24 semisynthetic viridine analogues,
quercetin, LY294002 (a quercetin analogue), and staurosporin.25-27
Even though wortmannin is very potent inhibitor of PI3K, its
development was hampered by its toxicity, insolubility, and
aqueous instability. Several analogues of wortmannin were
developed to circumvent these challenges. One such analogue
is a pegylated wortmannin derivative, PWT-458, currently being
developed by Wyeth Research.28 This pegylation of wortmannin
resulted in a compound with improved plasma stability and
reduced toxicity. Studies with synthetic, small molecules have
focused primarily on the catalytic domain and specifically the
ATP binding site of these proteins. Several inhibitors of PI3K
were reported recently including 1-benzopyran-4-ones,29 ben-
zoxazines,30 benzothiophenes,31 tetrazole benzofurancarboxa-
mides,32 pyrimidine carboxamides,33 and fused azolepyrim-
idines.34
* Author to whom correspondence should be addressed. Telephone:
(858)-678-0760. Fax: (858)-678-0762. E-mail: Palanki@targegen.com.
† TargeGen Inc.
‡ BioPredict.
10.1021/jm051056c CCC: $37.00 © 2007 American Chemical Society
Published on Web 08/09/2007

4280 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Palanki et al.
Figure 2. Six representative polyphenolic structures selected from
different families and origins, all of which have a common motif shown
in blue. The pharmacophore identified from these structures served as
template for structural elaboration.
Thus, many drug design schemes have concentrated on
heterocyclic ring structures mimicking the pyrimidine portion
of ATP and being capable of affinity competition with ATP. In
this paper we report for the first time the discovery of a highly
selective and potent PI3K inhibitor, 6,7-bis(3-hydroxyphenyl)-
pteridine-2,4-diamine, and the evolution of this lead via
structure-in ViVo activity correlations. We discovered common
structural motifs from natural products such as polyphenols,
including flavonoids which are known to have vascular protec-
tive properties35-40 (Figure 2) and combined with ATP mimics
to design new structural class of compounds for targeting PI3K.
Another important aspect of the design involved the incorpora-
tion of a phenolic group in the inhibitor. In general, the phenolic
group in a drug molecule is considered less desirable due to its
ability to undergo Phase II metabolism and elimination.
However, the rapid elimination of the drug from a body is a
desirable property in developing compounds with acute inter-
ventions, where a short time window for action followed by
rapid clearance may be desirable, as in the case of targeting
myocardial infarction.
Our design concept was to incorporate ATP-like structures
(aminopyrimidine or aminopyridine) into the natural product
pharmacophore to generate novel hybrid molecules with the
expectation of combining high affinity for the ATP binding site
of kinases and high selectivity from a balance between the ATP-
like and hydrophobic interactions of the new structures (Figure
3).
Figure 3. Evolution of an ATP-phenolic hybrid as an example for the
design of pteridines incorporating the basic pharmacophore with the
correct regioisomeric configuration.
Functionalization through either the 2 and 4 position (X, Y)
of the pyrimidine or pyridine and the 6 and 7 positions (R1,
R2) (pteridine class) or 2 and 3 position (pyridopyrazine class)
of the pyrazine ring (Figure 4) allowed for structural elaboration
and enabled us to systematically generate structure-activity
profiles.
In this paper we wish to communicate the results of this drug
discovery effort to the point of lead selection and target
verification. Preclinical myocardial infarct model studies and
details of pharmacology were reported recently.54
Design and Synthesis. The new pteridines were synthesized
from substituted 5,6-diaminopyrimidines by condensation reac-
tions with substituted phenylglyoxals or benzils. Reactions using
phenylglyoxals were carried out under conditions which yielded
regioselective isomers as shown in Scheme 1.
Figure 4. Pteridine and pyridopyrazine templates for structural
elaboration.
All benzils and glyoxals, with the exception of 3,3-dihydroxy-
and 3,3′,4,4′-tetrahydroxybenzils, 3′,4′-dihydroxyphenylglyoxal,
and 3-hydroxyphenylglyoxal were commercially available.
Certain noncommercial glyoxals, 3,4,5-triaminopyridine,41 and
2,3,6-triaminopyridine42 were synthesized based on published
procedures.
Condensation of 2,3-diamino- or 3,4-diaminopyridines with
substituted benzils or phenylglyoxals affords 2,3-subsituted
pyrido[2,3-b]pyrazin-6-ylamines (Scheme 2) or 2,3-substituted
pyrido[3,4-b]pyrazin-8-ylamines (Scheme 3).
Results and Discussion
1. Synthesis. The synthetic strategy was based on a modular
approach, starting with prefunctionalized intermediates with up

Isozyme-SelectiVe Inhibitor of PI3K
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4281
Scheme 1a
a (a) NaHCO3, MeOH-water; (b) Me2CdNOH, HCl, water, pH ) 3-4; (c) NaOH, MeOH-water, pH ) 9-10
Scheme 2a
a (a) NaHCO3, MeOH-water; (b) NaOH, MeOH-water, pH ) 9-10; (c) Me2CdNOH, HCl, water, pH ) 3-4.
Scheme 3
a (a) NaHCO3, MeOH-water; (b) NaOH, MeOH-water, pH ) 9-10; (c) Me2CdNOH, HCl, water, pH ) 3-4.
to 14 positions available for structural elaborations. The
cyclocondensation reaction of the aminosubstituted heterocycles
with either substituted benzils or glyoxals forming the central
pyrazine ring in a single step resulted in final products in nearly
quantitative yields and in high purity. A large variety of
substituted diaminopyrimidines, pyridines, benzils, and glyoxals
were commercially available for initial synthesis. However,
several benzils and glyoxals are not commercially available and
synthesized based on published procedures. Reaction conditions
involving the condensation of o-diamines with glyoxals were
developed to obtain either the 6- or 7-substituted regioisomers
selectively. The ability to obtain regioisomers proved to be

4282 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Palanki et al.
Scheme 4
a (a) (CN)2CHNH2·TosOH, 2-propanol, Rt; (b) PCl3, (MeO)2CHNMe2; (c) NH4OAc, AcOH, reflux; (d) NaHCO3, 1:1 dioxane/water, pH ∼ 9-10; (e)
1
MeOH/H2O, reflux. Analysis of intermediates with H NMR in DMSO-d6, 500 MHz Bruker NMR.
Figure 5. Miles assay for determining the inhibition of VEGF induced vascular permeability in Sprague-Dawley rats. (For details, see Experimental
Section).
valuable during the biological screening phase of the project.
Unequivocal preparation of 6-substituted pteridines unac-
companied by the 7-substituted isomer was first reported by
Taylor et al.43 Since then, the regioselective syntheses of 6- and
7-substituted pterines have been reported by several groups.44,57
Generally, the regioselective synthesis of 6-substituted pteridines
is achieved by condensation of 4,5,6-triamino- and 2,4,5,6-
tetraaminopyrimidines with substituted phenylglyoxal mo-
nooximes in MeOH.45,57 7-Substituted pteridines were previously
reported via pH-controlled condensation of 2,4,5,6-tetraami-
nopyrimidine with substituted benzylglyoxals.46 Above a pH
of 8 only the 5-amino group of 2,4,5,6-tetraaminopyrimidine is
nucleophilic enough to react with aldehyde groups of benzylg-
lyoxals, thus resulting in 7-substituted pteridines. In the prepara-
tion of 6- and 7-substituted pteridines, we were interested in a
coupled receptors such as platelet-activating factor (PAF),48 (2)
hypoxia-inducible factor (HIF) induced upregulation of VEGF,49
and (3) the influence of nitric oxide on vascular tone. A complex
network of signal transduction pathways are triggered (Figure
1) by the activation of G protein coupled receptor using either
VEGF, PAF, or histamine. This leads to the dissociation of a
complex of intercellular junctional proteins and ultimately to a
leaky endothelium. For the inhibition of VP, proteins other than
growth factors and downstream kinases, for example the
destabilization of HIF-1R50 or nitric oxide synthase,51,57 have
also been suggested as potential targets for developing new
drugs. Given the complex nature and multiple targets likely to
be involved in VP, we selected an in ViVo screen, the Miles
assay, instead of a biochemical kinase inhibition screen for initial
structure-activity profiling.58 This simple assay consists of
injecting rats intravenously with test agent followed by intra-
venous injection of Evans blue. Evans blue binds to plasma
proteins and thus allows for visual assessment of vascular
leakage. The intradermal injection of VEGF on each flank
produces a blue stain (Figure 5). On the basis of the area and
intensity of this stain, we graded the edema at the intradermal
injection site according to a 12 point scale and thus were able
to test the ability of agents to inhibit vascular leakage directly.
In each rat, the scores for both the right and left flank injection
sites were summed to yield a composite score. A 4-point scale
was utilized, where 3 ) background response (bluing equivalent
to intradermal saline controls), 2 ) minimal bluing (surrounding
area <25% intensity of injection site), 1 ) midlevel bluing
(surrounding area about 50% of intensity of injection site), and
0 ) maximal bluing (surrounding area >75% of intensity of
injection site). Scores for four reaction sites were summed to
1
rapid elucidation of the regioisomeric structure by H NMR
spectroscopy. Thus, the 4-amino-6-phenylpteridine, prepared by
the unequivocal route reported by Taylor et al.,43 was in all
respects identical to the compound prepared by the condensation
of 4,5,6-triaminopyrimidine with oximinoacetophenone in MeOH.
A comparison of the 1H NMR spectrum of 4-amino-7-phenylp-
teridine, prepared by the condensation of 4,5,6-triaminopyri-
midine with phenylglyoxal, with the 1H NMR spectrum of
4-amino-6-phenylpteridine showed that the C-7-H is shifted
downfield to 9.73 ppm compared to C-6-H at 9.43 ppm. The
C-2-H of 4-amino-6-phenylpteridine, however, is slightly shifted
upfield to 8.54 ppm compared to C-2-H of 4-amino-7-phenylp-
teridine at 8.57 ppm, as shown in Scheme 4.
2. Lead Identification from In ViWo Screen. The occurrence
of vascular permeability (VP) results from (1) edema from
receptor tyrosine kinases such as VEGFR247 and G-protein

Isozyme-SelectiVe Inhibitor of PI3K
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4283
Table 1. Inhibition of Vascular Permeability of a Selected Number of
Pyrazines and Quercetin
Figure 6. Western blot of lysates from VEGF-induced increase in VE-
cadherin in HUVEC cells. The figure shows the prevention of VEGF-
induced phosphorylation of VE-cadherin by compound 6. Serum-starved
HUVEC cells (Cambrex BioScience, Walkersville, MD) were treated
for 15 min with compound 6 (0.125 to 10 µM) or vehicle, followed by
a 5-min treatment with 20 ng/mL recombinant VEGF (PeproTech,
Rocky Hill, NJ) or vehicle. Cell pellets were suspended in RIPA buffer
(10 to 1 v/v, Pierce) on ice for 10 min. The cell suspension was passed
through a 23 gauge needle (3×) to complete the lysis process. Lysed
cells were centrifuged at 16 000 rcf at 4 °C for 10 min. The supernant
was removed, and the protein was assayed using the BCA method
(Pierce). After calculating protein concentration, RIPA buffer was added
to adjust the concentration. Laemmli buffer (Pierce) was added to the
samples and heated at 100 °C for 10 min. Samples were loaded onto
an SDS polyacralymide gel. Gels were probed with antibody specific
for phosphorylated VE-cadherin. The antibody was removed and probed
with the antibody for total VE-cadherin. The antibodies for both
phosphorylated VE- cadherin and unphosphorylated VE-cadherin were
purchased from Cell Signaling Technology, Inc. (Danvers, MA).
a control, also showed inhibition of vascular leak in this screen,
although, as the score indicated, at a considerable lower level
than the corresponding hybrid structures (5, 6, and 7). In
summary, the in ViVo-SAR studies identified basic structural
elements in these molecules which are required for high
inhibition (Miles score above 7) of vascular permeability. It was
clearly shown that these novel hybrid molecules incorporating
the aminopyrimidino or pyridinopyrazine scaffold in place of
the chromenone-fused ring structure of flavonoids dramatically
improved the potency. The selection of 6 as a lead compound
was the result of this in ViVo screen. In the following discussion,
we present the data in support of identifying a molecular target
and the cellular signaling path for the inhibitory activity of this
lead compound.
3. Target-Path Verification of the Lead Compound.
Endothelial junctions are formed by transmembrane adhesion
proteins. These include members of the claudin family, occludin,
junctional adhesion molecules (JAM) A, B, and C, and
endothelial cell selection adhesion molecules (ESAM). At
adherens junctions, the main adhesion protein is VE-cadherin.
Phosphorylation of VE-cadherin has been reported to regulate
VEGF-induced changes in microvascular permeability52 in a
manner that can be blocked by inhibitors of kinases such as
Src family kinases.18 In order to evaluate the effect of compound
6 on this process, human umbilical vein endothelial cells
(HUVEC) were exposed to the compound, and VE-cadherin
was monitored by immunoprecipitation of cell lysates. The
results of western blots are shown in Figure 6, where a VEGF-
induced increase in total level of VE-cadherin was observed,
and this process was inhibited by prior exposure of cells to 10
µM of 6. Before Western Blot analysis, protein concentrations
in each well was measured using the bicinchoninic acid (BCA)
method (using reagents from Pierce). Using the information
obtained on amount of protein from each well, the gel loading
was adjusted to ensure equal amounts of protein. â-Actin was
used as second control to ensure that equal amount of protein
was loaded on to the gel. The gel was probed with an antibody
targeting phosphorylated VE-cadherin. Further, the antibody was
removed, and the gel was probed with an antibody targeting
VE-cadherin for analysis. On the basis of our experiments, we
are confident that the compound is not degrading VE-cadherin
protein but merely inhibiting phosphorylation of VE-cadherin.53
produce a final score rating from 0 (maximal vascular perme-
ability) to 12 (background edema response). Overall responses
are ranked according to the following scale: score 0-4 ) low
to no activity, 5-8 ) moderate activity, 9-12 ) high activity.
Results of a representative number of compounds tested this
way are shown in Table 1.
We started with the simplest, nonfunctionalized phenylqui-
noxaline 1 which constituted the basic scaffold identified from
our natural product search (Figure 2) and then systematically
added structural complexity according to our design scheme in
Figure 3. From the results of the Miles score it became
immediately apparent that inhibition of vascular permeability
in these heterocyclic compounds depended predominantly on
the nature of substituents on both sides of the hybrid structures.
For example, the change of structure from a quinoxaline 2 to a
pteridine 3 was crucial in the development of inhibitory activity.
Nearly maximum activity (score 10-11) as obtained for
compounds 6 and 7, which required extensive functionalization
of both the heterocyclic ring structure and aromatic groups to
the right of the pyrazine ring. Particularly, the presence of
phenolic hydroxyls added substantially to increases in the
potency of these pteridines. The flavonoid quercetin, tested as

4284 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Palanki et al.
Figure 8. Western blots of lysates from FGF-stimulated HUVEC cells,
where the inhibition of phosphorylation of Akt was observed in a dose-
dependent manner. Serum-starved HUVECs (Cambrex BioScience,
Walkersville, MD) were treated for 15 min with compound 6 (0.125
to 10 µM) or vehicle, followed by a 5-min treatment with 20 ng/mL
recombinant bFGF (PeproTech, Rocky Hill, NJ) or vehicle. Cell pellets
were suspended in RIPA buffer (10 to 1 v/v, Pierce) on ice for 10 min.
The cell suspension was passed through a 23 gauge needle (3×) to
complete the lysis process. Lysed cells were centrifuged at 16 000 rcf
at 4 °C for 10 min. The supernant was removed, and the protein was
assayed using the BCA method (Pierce). After calculating protein
concentration, RIPA buffer was added to adjust the concentration.
Laemmli buffer (Pierce) was added to the samples and heated at
100 °C for 10 min. Samples were loaded to SDS polyacralymide gel.
Gels were probed with antibody specific for phosphorylated Akt. The
antibody was removed and probed with the antibody for total Akt.
Control lanes show unphosphorylated Akt. The antibodies for both
phosphorylated Akt and unphosphorylated Akt were purchased from
Cell Signaling Technology, Inc. (Danvers, MA).
Figure 7. Selected results from the Kinexus screen of 73 kinase targets.
The figure shows the inhibition of VEGF induced mTOR and p70s6S
kinases in HUVEC cell lysates. While LY294002 and wortmannin
inhibits c-Fos (data not shown), compound 6 does not. Basal activation
of cell signaling proteins in HUVECs was first down-regulated by
starving the cells for 4 h, followed by exposure to 20 ng/mL VEGF in
the absence or presence of 10 mM 6 for 10 min. HUVECs were rinsed
with ice-cold phosphate-buffered saline and then lysed by addition of
RIPA buffer (100 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1%
deoxycholic acid, 1% Triton X-100, 0.1% SDS, 2 mM PMSF, 500
mM NaF, 1 mM vanadate). Protein concentration was determined using
the BCA method according to the manufacturer’s instructions (Pierce,
Rockford, IL). Cell lysates were adjusted to equivalent protein
concentrations and then sent to Kinexus Bioinformatics for screening
against four antibody panels representing 73 distinct phosphorylated
kinases.
Table 2. Inhibition of PI3K Isoforms of Compound 6 in a Commercial
Screen of 140 Kinases
kinase
IC50 (nM)
PI3KR
PI3Kâ
PI3Kδ
PI3Kγ
1300
1200
235
83
Only gel that is exposed to antibody targeting phosphorylated
VE-cadherin is shown.
Cell lysates from this experiment were also used to determine
phosphorylated ERK (data not shown). Treatment of the cells
with VEGF stimulates VE-cadherin through the PI3K pathway
and ERK through the Src pathway. The inhibitors targeting PI3K
do not affect VEGF stimulated ERK pathway. Importantly we
found that compound 6 does not affect this path, thus potentially
avoiding inhibition of endothelial cell proliferation. The latter
result was confirmed in cell culture, where no inhibition of
endothelial cell proliferation was observed (data not shown).54
Cell lysates prepared similarly to those of the previous assay
were also confirmed via commercial screen (Kinexus Bioin-
formatics Co., Vancouver, BC, Canada) of 73 kinase targets.55
Results from this screen show that compound 6 inhibited VEGF
mediated phosphorylation of mTOR, p70S6 kinase as shown
in Figure 7. Both p70S6K and mTOR are downstream of PI3K
and were inhibited as expected. The biochemical profile of
compound 6 contrasts with two commonly used PI3K targeting
reagents, wortmannin and LY294002. This was confirmed by
6’s inhibition of PI3K pathway signaling but (unlike wortmannin
and LY294002) its sparing of ERK and cFos phosphorylation.
One of the disadvantages of LY294002 and wortmannin is their
effect on c-Fos which was inhibited by these two compounds
(data not shown). C-Fos, a family of intermediate early genes,
is present in many tissues in very low levels under basal
conditions. C-Fos plays a dual role; in some cases their ex-
pression is protective to cells and involved in cellular prolifera-
tion, but they are also implicated in apoptosis.56 It is beneficial
to have a PI3K inhibitor that does not inhibit this key proto-
oncogene.
of Akt by 6, and an Akt loading control for comparison. We
observed inhibition of Akt at the background level of the vehicle
control at the highest concentration, following blot intensities
in a dose dependent manner.
Direct measurements of PI3K inhibition by 6 were done in a
biochemical assay, which showed in an ATP competitive mode
an IC50 value of 83 nM for the gamma isoform of this enzyme.
This isoform has previously been shown to play a central role
in relaying inflammatory signals in general and specifically been
implicated in edemas.59 Taken together the results of cell-based
and biochemical analysis strongly support the inhibition of PI3K
signaling as a primary mechanism of drug action. We also tested
compound 6 in a commercial biochemical kinase screen. The
results showed inhibitory activity for only 8 out of a panel of
140 kinases, demonstrating the high target selectivity of this
compound.60 Inhibition data for the 4 isoforms is shown in
Table 2.
4. Structure-Activity Correlations. Further insights into
the structural features necessary for PI3K inhibitor activity were
obtained from SAR of a selected number of structurally related
analogues. The results are shown in Tables 3, 4, and 5. Table
3 gives IC50 values for derivatives 8-16 in which the hydro-
phobic phenolic region including the pyrazine core structure of
6 was unchanged, and only the pyrimidine/pyridine region of
the molecule was modified. The nature of substituents in this
part of the molecule proved to be very important for binding to
PI3K. In general, the data shows that any structural deviation
in this region from compound 6 results in a loss of binding
activity up to 4 orders of magnitude. Substitution of hydroxyl
for amino groups as in compounds 12 and 13 results in nearly
complete loss of PI3K binding. One reason for this loss may
be that these molecules tautomerize, and that the amide form
which is expected to dominate in these molecules would exhibit
incompatible hydrogen bond donor/acceptor geometry for
The pathway data presented here established that 6 altered
VEGF-mediated changes in phosphorylation but did not directly
confirm what molecular targets it acts on to bring about these
alterations. However, strong inhibition (80%) of mTOR phos-
phorylation suggested the PI3K/Akt path, since mTOR is
directly phosphorylated by Akt. Figure 8 shows the inhibition

Isozyme-SelectiVe Inhibitor of PI3K
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4285
Table 4. Structure-Activity Correlations of Compounds 17-28
Table 3. Structure-Activity Correlations of Compounds 8-16
binding at the ATP site. Either mono- or disubstitution of the
primary amino groups with solubilizing moieties such as shown
in the examples of structure 15 and 16 results in essentially
inactive compounds. These relatively bulky side groups appar-
ently cannot be accommodated in the space available at the ATP
site, an observation which was confirmed from the modeling
studies (see section 5). The elimination of one amino group as
in structure 14 or both amino groups as in structure 9 results in
a loss in binding of nearly 2 orders of magnitude. Thus, the
data in Table 3 clearly shows that the triplet HB-donor-
acceptor-donor motif in these compounds is utilized by these
compounds in binding to PI3K.
modified. Several important structural correlations were ob-
served. For example, comparison of the two regioisomers (17
and 21) showed the 6′ and 7′-substituted isomer to give IC50
value of 40 nM and 3000 nM, respectively, a nearly 2 order of
magnitude difference. It is interesting to note that the model
Table 4 summarizes the results for the compounds 17-28,
in which the 2,4-diaminopyrimidine portion of 6 was kept
constant, and only the right side of the core structure was

4286 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Table 5. Structure-Activity Correlations of Compounds 29-35
Palanki et al.
structures of natural products, which served as templates for
our initial design (Figure 2), suggested a preferential 6′ over a
7′ substitution. Another important requirement for high affinity
binding to PI3K was the presence of meta-substituted hydroxyl
groups (see comparison of structure 17 with 19, and 6 with 20).
In fact the compounds listed in this table can be categorized
into two subclasses, 6′-substituted phenols with meta-substituted
hydroxyl groups (compounds 17, 6, 7, and 18), which gave
nanomolar IC50 values, and compounds 19-28 with other
phenol substitution patterns, which fall into the micromolar
binding range. The introduction of the solubilizing groups on
the m-hydroxyphenol moiety of compound 6 resulted in
essentially inactive derivatives (26-28).
Table 5 shows SAR results for compounds 29-35. A com-
parison of the IC50 values of these compounds clearly confirms
the importance of the second nitrogen in the pyrimidine ring in
contributing to PI3K binding. Compounds 30, 31, and 32 and
compounds 10 and 11 lacking a nitrogen in the second ring
failed to exhibit any binding activity to PI3K, even if all other
structural requirements for high affinity binding are fulfilled.
In summary, the structural requirements for high binding of
pteridines to PI3K are (1) the presence of the 2,4-diaminopy-
rimidine moiety, (2) 6′-regioselectivity, and (3) m-hydroxyphe-
nyl substitution in addition to requirement 2. The minimum
structure which satisfies all three requirements is shown in
compound 17, which so far has also exhibited the best PI3K
binding activity. It is interesting that the SAR profiles generated
in Table 3, 4 and 5 correlate quite well with the results of the
Figure 9. Model structures of the human PI3Kγ kinase showing the
location of compound 6 in the catalytic domain. (A) An expanded view
of compound 6 in the ATP pocket of the enzyme, with specific
interactions shown. (B) Compound 6 in the ATP binding site overlapped
with ATP from 1E8X. Complexes were superimposed using a method
due to Lesk68 that superimposes corresponding carbon alpha atoms in
residues near the ATP adenine ring. (C) Compound 6 docked in an
alternative binding mode. Renderings in (A) through (C) were generated
using the software package Insight II.
initial in ViVo screen, further supporting the involvement of PI3K
as a target for the inhibition of vascular permeability in these
compounds.

Isozyme-SelectiVe Inhibitor of PI3K
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4287
Table 6. Mean Pharmacokinetic Parameters Following Intravenous Doses of 6 in Rats
dose (mg/kg)
C0 (µg/mL)
Cmax (µg/mL)
AUC(0-inf) (h µg/mL)
t1/2 (hr)
CL (mL/min/ kg)
Vdss (L/ kg)
0.5
5.0
0.713 ( 0.266
7.73 ( 2.53
0.363 ( 0.105
3.33 ( 0.44
0.124 ( 0.033
1.38 ( 0.56
0.4 ( 0.1
0.8 ( 0.3
71 ( 20
66 ( 23
2.3 ( 0.7
4.4 ( 0.5
binding mode (Figure 9c) shows only one phenolic group is
involved in hydrogen bonding and not the other. Both binding
modes explain the SAR results. Based on the modeling work
on this series, it appears that the binding mode depicted in Figure
9a provides better explanation of the data.
6. Pharmacokinetic Studies in Rats. The in ViVo pharma-
cological work using compound 6 was completed in rats and
shown below. We also completed the pharmacodymanic work
using compound 6 using Sprague-Dawley rats.54 In order to
fully elucidate the properties of 6, the pharmacokinetic (PK)
parameters of 6 were studied in Sprague-Dawley rats following
intravenous administration of either 0.5 or 5.0 mg/kg. The mean
intravenous PK parameters for 6 in the rat are presented in Table
6. After single intravenous administration, 6 displayed an
approximate first-order decline in plasma concentrations with
respect to time (Figure 10). Using a noncompartmental model,
the intravenous plasma pharmacokinetics of 6 at doses of either
0.5 or 5.0 mg/kg were characterized by high clearance (CL
approximately equal to hepatic blood flow), a moderate volume
of distribution (3-fold to 6-fold total body water), and a short
plasma half-life (t1/2) ranging from 0.4 h to 0.8 h. Dose-
proportional pharmacokinetic parameters were observed fol-
lowing doses of 0.5 to 5.0 mg/kg. A 10-fold increase in dose
resulted in a 10-fold increase in plasma exposure parameters
(C0, Cmax, and AUCinf).
7. Summary. Starting from a natural product search of
vasoprotective agents with kinase inhibitory activities, we
identified a pharmacophore useful for the design of novel hybrid
molecules which incorporated structures mimicking the ATP
binding motif for competitive inhibition. The synthesis of an
initial set of compounds followed by SAR profiling in an in
ViVo screen identified a lead, 6, for clinical development.
Pathway analysis identified this lead as a potent and selective
inhibitor of PI3K. Structural reiteration, combined with molec-
ular modeling studies, elucidated the molecular details necessary
for high affinity binding and validated both our initial hybrid
design concept and in ViVo targeting approach.
The biochemical profile of compound 6 contrasts with two
commonly used PI3K inhibitors, wortmannin (an equipotent but
pan-isoform inhibitor) and LY294002 (a weak inhibitor). These
distinctions suggest the three compounds should differently
influence cellular processes. This was confirmed by 6’s inhibi-
tion of PI3K pathway signaling but (unlike wortmannin and
LY294002) its sparing of ERK and c-Fos phosphorylation.
Additionally 6 had no influence on mitogenesis whereas the
other compounds inhibited cell proliferation and even survival.
Wortmannin’s effects might arise from its inhibition of PI3KR
and/or â. Alternatively this may represent off-target hits, as
wortmannin inhibits MAP kinase, myosin light chain kinase,
and mTOR in the sub-µM range.65 LY294002’s cytotoxicity
likely reflects off-target hits such as Raf or GSK3â inhibition,66
or non-PI3K-dependent pro-apoptotic effects mediated via
intracellular H2O2.67 Of most relevance to cardioprotection,
however, is that 6 inhibited VEGF-induced pro-inflammatory
events (VE-cadherin phosphorylation) while sparing those that
control mitogenesis. VEGF plays both positive (pro-angiogenic)
and negative (pro-edema) roles in I/R injury, and differentiating
between these processes would be beneficial in a proposed MI
therapy.
Figure 10. Mean plasma concentration-time profiles following
intravenous doses of 6 in rats.
5. Molecular Modeling. The SAR data was used together
with molecular models of the interaction between compound 6
and PI3K to gain insights into how it might inhibit this kinase.
Compound 6 was docked into the active site of the enzyme
[1E8Z] using a combination of interactive modeling using the
Insight II program61 and automated docking software.62 The
resulting complex was then minimized in the molecular
mechanics program DISCOVER using the CFF force field with
a distance-dependent dielectric.61 In optimizing the complex,
all atoms were allowed to move in residues with at least one
atom within 8 Å of any atom of the ligand. The modeling
experiments suggest a mode of binding that is summarized in
Figures 9a through 9c. Two strong hydrogen bonds are formed
from the ligand to the backbone of the protein in the hinge
region of PI3Kγ: the first is from the N-3 nitrogen of the
pteridine ring serving as an acceptor to the backbone NH of
Val 882 and the second is from 4-primary amine group serving
as a donor to the backbone carbonyl of the same residue (Figure
9a). While the second NH2 group on compound 6 is positioned
adjacent to the backbone carbonyl of Glu 880, the geometry
for formation of a hydrogen bond is poor. For reference, ATP
from the ATP/PI3Kγ (Sus scrofa) complex in 1E8X is shown
overlaid on the model in Figure 9b. The pattern of hydrogen
bonds observed in 1E8X to the backbone is similar, with a strong
hydrogen bond formed to the NH of Val 882 and a second
potential hydrogen bond to the backbone carbonyl of Glu 880
either geometrically weak or absent. A pocket is evident at the
back of the ATP binding site that accommodates the 6′-
substituted m-phenol of compound 6 (Figure 9c). Asp 841, on
the kR3 helix, lies at the back of this pocket and can form a
hydrogen bond with the OH group of the 6′-substituted
m-phenol. Additional stabilization of the phenolic group can
come from the side chain of Tyr 867, which can swing to assist
in the coordination. The second phenolic group can form an
interaction with the side chain of Asp 964 that is part of the
DFG loop.
Overall the modeling data is consistent with nearly all of the
SAR profiling results in Table 4. Using the first binding mode
(Figure 9a), one can see that both phenolic groups are involved
in the hydrogen bond. The first binding mode also explains the
activity of both compounds 6 and 17. However, the second

4288 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Palanki et al.
Preparation of Cell Lysates. Cell lysates were produced from
human umbilical vein endothelial cells (HUVEC, Cambrex, Bio-
science, Walkersville, MD) between passage 2 and 4 by incubation
for 4 h with serum free medium (EBM-2 medium Cambrex
BioScience) in order to downregulate basal activation of cell
signaling proteins. This was followed by exposure for 10 min of
20 ng/mL recombinant human VEGF (catalog no. 100-20 Pepro-
Tech, Rocky Hill, NJ) in the absence or presence of compound 6
(10 µM in dimethyl sulfoxide) into EBM-2 medium. HUVEC cells
were then rinsed with ice cold PBS by the addition of 300 µL RIPA
buffer (100 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1%
deoxycholic acid, 1% Triton X-100, 0.1% SDS, 2 mM PMSF, 500
mM NaF, 1 mM vanadate). Protein concentrations were determined
using the bicinchronic acid method according to the manufactures
instructions (Pierce, Chemical, Rockford, IL). These lysates were
used in various cell-based assays. Cell lysates were adjusted to
equivalent protein concentrations and then sent to Kinexus Bioin-
formatics (Kinexus Bioinformatics Corp., Vancouver, British
Columbia, Canada) for screening against four antibody panels
representing 73 distinct phosphorylated kinases, which analyses cell
lysates using a Western-blot-based protocol in order to identify
phosphorylated signaling proteins in a quantitative manner.
Ki Value Determination. The Ki value for 6 was determined
against PI3-kinase (p120γ) using a fluorescence polarization-based
assay. Briefly, assays were run at room temperature and a final
volume of 50 µL containing buffer, an appropriate amount of the
PI3K such that the assay was linear over 60 min, and varying
amounts of ATP in the presence of a fixed varied amount of 6.
The reaction was initiated by the addition of PIP2 at a final
concentration of 50 µM. After the reaction was allowed to proceed
for 60 min, fluorescence polarization reagent was added to terminate
the kinase reaction and then allowed to proceed for an additional
10 min to maximize the signal. Control reactions containing no
ATP were used for each varied ATP concentration. Enzyme reaction
rates were derived by calculating the difference between kinase
catalyzed and noncatalyzed reactions at a specific 6 concentration.
The Ki value was derived from rate data using competitive enzyme
kinetics curve fitting. On the basis of the data analysis of curve
fitting, the compound was judged to be an ATP-competitive
inhibitor.
Molecular Modeling. Modeling studies were conducted in
collaboration with BioPredict Inc., Oradell NJ. Protein coordinates
for human PI3Kγ were taken from the PDB entry 1E8Z, a cocrystal
of human PI3Kγ @ 2.4 Å with staurosporin. Additional structures
of human and Sus scrofa PI3Kγ available from the PDB were
superimposed to examine potential protein flexibility and modes
of binding to other ligands (1E7U, 1E7V, 1E8W, and 1E8Y). Sus
scrofa PI3Kγ is 95% identical in sequence to human PI3Kγ; all
residues within 8 Å of staurosporin in 1E8Z are identical.
Pharmacokinetic Studies of 6. All animals were housed
separately and had continual access to food and water. All doses
were administered as aqueous solutions containing 9.5% Captisol
(Cydrex). Heparinized whole blood samples were obtained and
centrifuged to separate plasma, and the plasma was stored at
-70 °C until bioanalysis. Male Sprague-Dawley rats, body weight
200 to 250 g, from Charles River, received an intravenous 6 bolus
dose of either 0.5 or 5 mg/kg into the tail. Serial plasma samples
were isolated from rats (N ) 5 animals per dose route) over the
following time course: 5, 15, and 30 min and 1, 2, 4, 6, 8, and 24
h. Blood samples (0.25 mL) were isolated through an indwelling
jugular vain catheter. Plasma samples were extracted by addition
of a 2-fold excess of acetonitrile containing internal standard
followed by centrifugation. The supernatants were isolated for
analysis. Processed plasma samples were quantitated by LC/MS/
MS against external calibration standards prepared in blank rat
plasma. The performance of each analytical run was assessed by
quality control samples prepare in plasma matrix. The standard
calibration curves ranged from 1 to 10 000 ng/mL. The LC/MS/
MS system consisted of an Sciex API4000 triple quadrupolar mass
spectrometer (MDS Sciex), an Agilent 1100 HPLC system (Agilent
Technologies, Inc), and a CTC autosampler (Leap Technologies).
Several PI3K inhibitors are currently in development as
antitumor drugs. However, the role of PI3K in myocardial
infarction was not fully exploited for drug discovery efforts. A
biphasic I/R injury to the heart is caused by myocardial
infarction. Despite a great understanding of the cause, very little
progress has been made in this area due to the fact that an
antiischemia theraphy is required early in MI pathogenesis, a
time when the great majority of patients are inaccessible. There
is a large body of evidence suggesting that PI3K is an attractive
target for developing compounds for ischemia. Several com-
monly known PI3K inhibitors reduce inflammatory events in
animal models but fail to reduce infarct size when delivered
after reperfusion. We have developed a PI3Kγ/δ inhibitor to
interrupt the reperfusion phase of I/R injury, that shows reduced
infarct development and improved myocardial function.54
Compound 6 also offered cardioprotection upon delivery up to
several hours after reperfusion.54 On the basis of this promising
profile in models of leakage and ischemia reperfusion injury, 6
(TG100-115) has been selected for clinical development.63
Experimental Section
Miles Assay. Experiments were conducted according to NIH
Guidelines for the use of laboratory animals. Non-fasted male
Sprague-Dawley rats (Harlan, Indianapolis, IN), weighing between
175 and 200 g, were injected via tail vein with either vehicle or
200 µL of test compound (from 10 mM stock formulations).
Treatment groups sizes were N ) 2-4. Two hours posttreatment,
rats were anesthetized with ip injection and placed on a heating
pad set to 35-37 °C. Evans blue dye (500 µL of a 2% saline, 0.2
µm sterile filtered) was administered iv. Immediately following dye
injections, animals received two intradermal injections of VEGF
(200 ng total) in a volume of 100 µL. Thirty minutes after this
challenge, vascular permeability was visually assessed by the extent
of Evans blue tattooing at the injection sites. Agonist injection sites
were scored based on the degree of extravasation into the sur-
rounding dermis and compared to the neighboring PBS injection
site. In each rat, the scores for both the right and left flank injection
sites were summed to yield a composite score. A 4-point scale was
utilized, where 3 ) background response (bluing equivalent to
intradermal saline controls), 2 ) minimal bluing (surrounding area
<25% intensity of injection site), 1 ) midlevel bluing (surrounding
area about 50% of intensity of injection site) and 0 ) maximal
bluing (surrounding area >75% of intensity of injection site). Scores
for four reaction sites were summed to produce a final score rating
from 0 (maximal vascular permeability) to 12 (background edema
response). Overall responses are ranked according to the following
scale: score 0-4 ) low to no activity, 5-8 ) moderate activity,
9-12 ) high activity.
Determination of PI3K Activity. For the determination of IC50
values of the compounds shown in Table 3, 4, and 5, we used a
luminescence-based kinase assay with recombinant phosphatidyli-
nositol 3-kinase- p-120 gamma, alpha, beta, and delta (Upstate Cell
Signaling Solutions). In white, flat-bottom, 96-well plates (Nunc)
parallel assays were run at ambient temperature at final volumes
of 50 µL. Each well contained 40 µL of buffer consisting of 20
mM Tris buffer, pH 7.4, containing 4 mM MgCl2, 10 mM NaCl,
50 µM d-myo-phosphatidylinositol-4,5-bisphosphate substrate (Ech-
elon Bioscience, Inc.) and an appropriate amount of PI3K (250-
500 ng/well). The final concentration of compound in these
experiments ranged from 100 to 0.001 µM and was done by direct
addition of compound in 2.5 µL of DMSO. Then 10 µL of ATP
was added to the wells from a stock of 3 µM. After 90 min, 50 µL
of Kinase-Glo reagent (Promega) was added to terminate the
reaction, and the well content was allowed to equilibrate for an
additional 10 min. Luminosity was measured using an Ultra 384
instrument (Teca). IC50 values were calculated from the lumines-
cence-concentration curves using the nonlinear curve fitting
capabilities of Prism (Version4, GraphPad software).

Isozyme-SelectiVe Inhibitor of PI3K
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4289
The LC separations were performed on a Waters Xterra, C18, 15 ×
2.1 mm, 2.5 µm reverse phase HPLC column (Waters Corporation).
The column temperature was keep at 60 °C. Mobile phase A
consisted of 1.0% formic acid (FA) in water and mobile phase B
consisted of 1.0% FA in acetonitrile. The flow rate was kept
constant at 0.5 mL/min. Following a 20 µL sample injection, mobile
B was held at 5% for 0.5 min followed by a linear increase to 95%
mobile phase B over 1.6 min. The mass spectrometric detection of
6 and internal standard was achieved using electrospray ionization
operating in positive ionization mode. The molecular ion transitions
were monitored in MRM mode. The maximum plasma concentra-
tions following intravenous dosing (C0) were extrapolated from the
apparent distribution phase for individual animals. Plasma clearance
(CL) was calculated as dose/AUC(0-inf) while the volume of
distribution at steady state (Vdss) was calculated as Cl × MRT,
3,3′-(2,4-Diaminopteridine-6,7-diyl)diphenol (6). 2,4,5,6-Tet-
raaminopyrimidine sulfate (4.76 g, 20.0 mmol) was added in small
portions to a solution of sodium bicarbonate (3.36 g, 40.0 mmol)
in 100 mL of water with vigorous stirring. A brisk evolution of
CO2 gas was observed. The resulting suspension was heated to
80 °C and 3,3′-dihydroxybenzil (4.84 g, 20.0 mmol) was added to
the mixture. The reaction mixture was refluxed for 3 h, at which
point a bright-yellow precipitate was formed in abundance. The
precipitate was filtered, washed sequentially with water, methanol,
and diethyl ether and dried in Vacuo to give 6.46 g (93.3% yield)
of the title product as bright-yellow solid, 98.10% purity by LC/
1
MS (230 DAD). MS m/z [M + H+] ) 347.7. H NMR (DMSO-
d6) δ 6.75-6.77 (dd, J1 ) 8.2 Hz, J2 ) 2.0, 1H), 6.79-6.80 (dd,
J1 ) 8.2 Hz, J2 ) 2.0, 1H), 6.81-6.83 (d, J ) 7.8 Hz, 2H), 6.86-
6.87 (d, J ) 7.8 Hz, 2H), 6.93 (s, 1H), 6.97 (s, 1H), 7.08-7.10 (t,
J ) 7.8 Hz, 1H), 7.09-7.11 (t, J ) 7.8 Hz, 1H), 7.75 (br s, 1H),
8.02 (br s, 1H), 9.56 (br s, 2H). 13C NMR (DMSO-d6) δ 115.2,
116.1, 116.5, 116.6, 120.5, 120.5, 120.6, 129.0, 129.0, 140.1, 140.3,
145.4, 154.3, 157.0, 157.1, 157.6, 163.0, 163.3. Anal. (C18H14N6O2)
C, H, N.
4,4′-(2,4-Diaminopteridine-6,7-diyl)dibenzene-1,2-diol (7): 96.5%
yield of the product as yellow, fluffy powder. 97% purity by LC/
MS (230 DAD). MS m/z [MH+] ) 379.3. 1H NMR (MeOH-d4) δ
6.68-6.70 (d, J ) 8.1 Hz, 1H), 6.71-6.73 (d, J ) 8.1 Hz, 1H),
6.79-6.81 (dd, J1 ) 8.1 Hz, J2 ) 2.1 Hz, 1H), 6.84-6.86 (dd, J1
) 8.1 Hz, J2 ) 2.0 Hz, 1H), 6.93 (d, J ) 2.0 Hz, 1H), 7.03 (d, J
) 2.1 Hz, 1H). 1H NMR (DMSO-d6) δ 6.47 (br s, 2H), 6.57-6.59
(d, J ) 8.1 Hz, 1H), 6.59-6.61 (d, J ) 8.6 Hz, 1H), 6.64-6.66
(dd, J1 ) 8.1 Hz, J2 ) 1.8 Hz, 1H), 6.65-6.67 (dd, J1 ) 8.6 Hz,
J2 ) 1.8 Hz, 1H), 6.90-6.91 (d, J ) 1.8 Hz, 1H), 6.95 (d, J ) 1.8
Hz, 1H), 7.41 (br s, 2H).
3,3′-(6-Aminoquinaxoline-2,3-diyl)diphenol Hydrochloride Salt
(8). 1,2,4-benzenetriamine dihydrochloride (40.4 mg, 0.206 mmol)
and 3,3′-dihydroxybenzil (50.0 mg, 0.20 mmol) were dissolved in
2 mL of 1:1 mixture of dioxane-water. The reaction was refluxed
for 3 h. Then solvent was removed in Vacuo. The residue was
dissolved in 2 mL of methanol, and this solution was added
dropwise to 40 mL of diethyl ether with stirring. The formed dark-
red precipitate was collected, washed with diethyl ether and dried
in Vacuo to give the title compound 8 (69.8 mg, 85.4% yield) as
red solid, 97.6% purity by LC/MS (230 DAD). MS m/z [MH+]
)330.8. 1H NMR (MeOH-d4) δ 6.81-6.83 (dd, J1 ) 8.0 Hz, J2 )
1.8 Hz, 1H), 6.85-6.87 (dd, J1 ) 8.0 Hz, J2 ) 1.2 Hz, 1H), 6.96-
6.98 (m, 3H), 6.98 (d, J ) 1.8 Hz, 1H), 7.10 (d, J ) 2.3 Hz, 1H),
7.13-7.16 (t, J ) 8.0 Hz, 1H), 7.28-7.31 (t, J ) 8.0 Hz, 1H),
7.56-7.58 (dd, J1 ) 9.5 Hz, J2 ) 2.3 Hz, 1H), 8.04-8.06 (d, J )
9.5 Hz, 1H). 1H NMR (DMSO-d6) δ 5.9 (br s, 3H), 6.74-6.77 (m,
2H), 6.83-6.86 (m, 2H), 6.92-6.93 (t, J ) 2.0 Hz, 1H), 6.97-
6.98 (t, J ) 2.0 Hz, 1H), 7.07-7.10 (t, J ) 7.9 Hz, 1H), 7.15-
7.18 (t, J ) 7.9 Hz, 1H), 7.31 (d, J ) 1.7 Hz, 1H), 7.51-7.54 (dd,
J1 ) 9.1 Hz, J1 ) 2.4 Hz, 1H), 7.97-7.99 (d, J ) 9.1 Hz, 1H).
Anal. (C20H15N3O2‚HCl‚1.7H2O) C, H, N.
3,3′-Pteridine-6,7-diyldiphenol (9). A 5 mL reaction vial
equipped with a stirring vane was charged with 4,5-diaminopyri-
midine (110 mg, 1.00 mmol, 1.00 equiv), 3,3′-dihydroxybenzil (242
mg, 0.500 mmol, 1.00 equiv), and m-cresol (0.750 mL). The slurry
was heated to ca. 200 °C and within 10 min gave rise to a dark
colored solution. A dull orange precipitate started to form in ca.
20 min and remained through the rest of the heating. The reaction
was cooled to room temperature and was triturated by pouring into
diethyl ether (30 mL) to yield a dark green precipitate. The solid
was isolated and washed with 3 × 30 mL of diethyl ether and dried
in Vacuo to yield the title compound as a 1/1 complex with m-cresol
(341 mg). MS m/z [MH+] ) 365.1. 1H NMR (DMSO-d6) δ 6.88-
6.90 (m, 4H), 6.95 (app t, J ) 8.0 Hz, 2 H), 7.05 (dd, J1 ) 2.0 Hz,
J2 ) 2.0 Hz, 1H), 7.14 (dd, J1 ) 2.0 Hz, J2 ) 2.0 Hz, 1H), 7.17-
7.22 (q, J ) 7.9 Hz, 2 H), 8.73 (d, J ) 7.3 Hz, 1 H), 9.46 (s, 1H),
9.67 (br s, 1 H), 9.71 (br s, 1H).
where MRT (mean residence time) was defined as AUMC(0-inf)
/
AUC(0-inf). The area under the curve, AUC(0-inf), from individual
concentration data were calculated using the linear trapezoidal rule.
Chemistry. All solvents and reagents were obtained from
commercial sources and used without further purification. 3,3′-
Dihydroxybenzil, 4,4′-dihydroxybenzil, and 3,3′,4,4′-tetrahydroxy-
benzyl, 3-hydroxyphenylglyoxal hydrate, and 4-hydroxyphenylg-
1
lyoxal hydrate were purchased from Midori Kagaku Co Ltd. H
nuclear magnetic resonance (NMR) spectra were recorded on a 500
MHz Bruker NMR. Chemical shifts are reported in parts per million
(δ) downfield from the tetramethylsilane resonance in the indicated
solvent. Coupling constants are reported in hertz (Hz). Mass spectra
were obtained from a Waters LC/MS spectrometer. This system
includes a 2795 separation module, a 996 photodiode array detector
and a ZQ2000 mass spectrometer. A Zorbax SB column (150 ×
4.6 mm 3.5 µm, Agilent Technologies) is used for the LC. Column
temperature is 40 °C. Compounds are separated using gradient
elution with mobile phases of water (0.05% TFA (A)) and
acetonitrile (0.05% TFA (B)). The mass spectrometer is equipped
with an electrospray probe. Source temperature is 120 °C. All of
the compounds were identified using the positive mode with mass
scan range from 100 to 800. Elemental analyses were performed
at NuMega Laboratories (San Diego) on a Perkin-Elmer Series II
- 2400 CHNS analyzer, and the results are within 0.4% of the
calculated values unless otherwise stated.
6-Phenylpteridin-4-amine (3). Ammonium acetate (192.7 mg,
2.5 mmol) was suspended in 3 mL of glacial acetic acid, and the
mixture was heated to ca. 100 °C. N′-(3-cyano-5-phenylpyrazin-
2-yl)-N,N′-dimethylimidoformamide (125.6 mg, 0.5 mmol) was
added, and the reaction mixture was refluxed for 1 h. Then it was
cooled down to ambient temperature, and solvent was removed in
Vacuo. The resulting residue was washed once with 10 mL of
methanol and twice with 40 mL of diethyl ether and dried in Vacuo
1
to give the product as a light-yellow solid. H NMR (DMSO-d6):
δ 7.55-7.60 (m, 3 H), 8.31 (br s, 1 H), 8.46 (br s, 1 H), 8.49 (dd,
J1 ) 8.2 Hz, J2 ) 1.9 Hz, 2 H), 8.54 (s, 1 H), 9.73 (s, 1 H).
6,7-Diphenylpteridin-4-amine (4) was prepared according to
the literature.42
4,4′-(4-Aminopteridine-6,7-diyl)diphenol (5). 4,5,6-Triami-
nopyrimidine sulfate (2.23 g, 10.0 mmol) was added in small
portions to a solution of sodium bicarbonate (1.68 g, 20.0 mmol)
in 50 mL of DI water with vigorous stirring. A brisk evolution of
CO2 gas was observed. The resulting suspension was heated to
80 °C, and 4,4′-dihydroxybenzil (2.42 g, 10.0 mmol) was added to
the mixture. The reaction mixture was refluxed for 3 h, at which
point a light-yellow precipitate was formed in abundance. The
precipitate was filtered, washed sequentially with water (2 × 40
mL), methanol (1 × 20 mL), and diethyl ether (2 × 40 mL) and
dried in Vacuo to give 3.14 g of the title product (93.3% yield) as
light-yellow solid. 95.5% purity by LC/MS (230 DAD). MS m/z
1
[MH+] ) 332.8. H NMR (DMSO-d6) δ 6.72-6.73 (d, J ) 8.7
Hz, 2H), 6.74-6.75 (d, J ) 8.7 Hz, 2H), 7.35-7.37 (d, J ) 8.6
Hz, 2H), 8.40-7.42 (d, J ) 8.6 Hz, 2H), 8.06 (br s, 1H), 8.15 (br
s, 1H), 8.50 (1H, s), 9.77 (br s, 1H), 9.87 (br s, 1H). Anal.
(C18H13N5O2‚0.1H2O) C, H, N.
3,3′-(8-Aminopyrido[3,4-b]pyrazine-2,3-diyl)diphenol Hydro-
chloride Salt (10). 3,4,5-Triaminopyridine dihydrochloride (60.0
mg, 0.304 mmol) and 3,3′-hydroxybenzil (99.6 mg, 0.41 mmol)

4290 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Palanki et al.
were heated at 190 °C in 1.0 mL of m-cresol for 1 h. Then the
mixture was cooled down to ambient temperature and mixed
thoroughly with 35 mL of diethyl ether. The formed brown
precipitate was collected, washed repeatedly with diethyl ether, and
dried in Vacuo to give the title compound 10 (93.9 mg, 78.4% yield)
7.09 (t, J ) 7.9 Hz, 1H), 7.09-7.12 (t, J ) 7.9 Hz, 1H), 6.95 (br
s, 2H), 9.47 (br s, 1H), 9.52 (br s, 1H), 11.50 (br s, 1H). Anal.
(C18H12N4O4‚0.9H2O) C, H, N.
3,3′-(4-Aminopteridine-6,7-diyl)diphenol (14). 4,5,6-Triami-
nopyrimidine sulfate (2.23 g, 10.0 mmol) was added in small
portions to a solution of sodium bicarbonate (1.68 g, 20.0 mmol)
in 50 mL of water with vigorous stirring. A brisk evolution of CO2
gas was observed. The resulting suspension was heated to 80 °C,
and 3,3′-dihydroxybenzil (2.42 g, 10.0 mmol) was added to the
mixture. The reaction mixture was refluxed for 1 h, during which
time the starting materials completely dissolved and the product
precipitated out as a light-yellow solid. The precipitate was
collected, washed with water and then with methanol followed by
diethyl ether, and dried in Vacuo to give 3.14 g of the title product
14 (94.8% yield) as light-yellow solid, 100% purity by LC/MS (230
DAD). MS m/z [MH+] ) 332. 1H NMR (DMSO-d6) δ 6.80-6.83
(m, 2H), 6.84-6.86 (d, J ) 7.8 Hz, 1H), 6.93-6.94 (d, J ) 7.8
Hz, 1H), 7.01-7.03 (m, 2H), 7.11-7.14 (dt, J1) 7.8 Hz, J2 ) 1.9
Hz, 2H), 8.23 (br s, 1H), 8.30 (br s, 1H), 8.59 (s, 1H), 9.63 (br s,
2H). Anal. (C18H13N5O2‚0.55H2O) C, H, N.
1
as greenish-brown powder. MS m/z [MH+] ) 331.4. H NMR
(MeOH-d4) δ 6.88-6.91 (m, 2H), 6.99-7.01 (m, 1H), 7.07-7.10
(m, 2H), 7.13-7.14 (t, J ) 2.1 Hz, 1H), 7.18-7.22 (dt, J1 ) 8.0
Hz, J2 ) 3.6 Hz, 2H), 8.03 (s, 1H), 8.82 (s, 1H). 1H NMR (DMSO-
d6) δ 6.85-6.87 (m, 3H), 6.98-7.01 (m, 1H), 7.02-7.03 (t, J )
2.0 Hz, 1H), 7.09-7.10 (t, J ) 2.0 Hz, 1H), 7.14-7.16 (t, J ) 7.9
Hz, 1H), 7.15-7.17 (t, J ) 7.9 Hz, 1H), 8.08 (d, J ) 0.74 Hz,
1H), 8.94 (d, J ) 0.74 Hz, 1H), 9.78 (br s, 2H). Anal. (C19H14N4O2‚
HCl‚1.5H2O) C, H, N.
3,3′-(6-Aminopyrido[2,3-b]pyrazine-2,3-diyl)diphenol Hydro-
chloride Salt (11). 2,3,6-Triaminopyrimidine dihydrochloride
(197.0 mg, 1.0 mmol) and 3,3′-dihydroxybenzil (242.4 mg, 1.0
mmol) were dissolved in 3.0 mL of 1:1 mixture of dioxane-water.
The reaction mixture was refluxed for 3 h, and then solvent was
removed in Vacuo. The resulting greenish solid was dissolved in 3
mL of MeOH, and this solution was added to 40 mL of diethyl
ether with vigorous stirring. The formed precipitate was collected,
washed with diethyl ether, and dried in Vacuo to give 342.9 mg
(90.4% yield) of the product as light-green powder, 99.0% purity
by LC/MS (230 DAD). MS m/z [MH+] ) 331.8. 1H NMR (MeOH-
d4) δ 6.83-6.85 (m, 2H), 6.88-6.90 (m, 1H), 6.95-6.97 (m, 2H),
7.02-7.03 (t, J ) 1.9 Hz, 1H), 7.14-7.17 (t, J ) 8.0 Hz, 1H),
7.15-7.18 (t, J ) 7.8 Hz, 1H), 7.36-7.38 (d, J ) 9.5 Hz, 1H),
3,3′-{2-Amino-4-[(3-morpholin-4-ylpropyl)amino]pteridine-
6,7-diyl}diphenol Trifluoroacetic Acid Salt (15). 3,3′-(2,4-Di-
aminopteridine-6,7-diyl)diphenol (1.0 g, 2.88 mmol) was dissolved
in 5 mL of 4-(3-aminopropyl)morpholine, and sulfamic acid (0.530
g, 5.45 mmol) was added to this solution. The reaction mixture
was refluxed for 18 h (ca. 160 °C). Then it was cooled down to
ambient temperature, filtered through 0.22 µm syringe filter, and
purified by reverse-phase preparative HPLC using a gradient of
acetonitrile/water mixture with 0.1% of TFA. Fractions, containing
the product, were collected. Solvent was removed in Vacuo to afford
33.0 mg of the title product 15 as a brown viscous oil. MS m/z
1
8.43-8.46 (d, J ) 9.5 Hz, 1H). H NMR (DMSO-d6) δ 6.76-
6.80 (m, 4H), 6.91-6.92 (t, J ) 2.0 Hz, 1H), 6.98-6.99 (t, J )
2.0 Hz, 1H), 7.11-7.14 (t, J ) 7.8 Hz, 1H), 7.12-7.15 (t, J ) 7.8
Hz, 1H), 7.46-7.48 (d, J ) 9.5 Hz, 1H), 8.43-8.45 (d, J ) 9.5
Hz, 1H), 8.83 (br s, 1H), 9.71 (br s, 2H), 9.90 (br s, 1H). Anal.
(C19H14N4O2‚0.6HCl‚1.5H2O) C, H, N.
1
[MH+] ) 474.5. H NMR (DMSO-d6) δ 3.07 (m, 2H), 3.20 (m,
2H), 3.44 (m, 2H), 3.59-3.80 (m, 6H), 3.97 (m, 2H), 6.78-6.85
(m, 4H), 6.91-6.92 (m, 2H), 7.13-7.17 (t, J ) 7.9 Hz, 1H), 7.14-
7.18 (t, J ) 7.9 Hz, 1H).
6,7-Bis(3-hydroxyphenyl)pteridine-2,4-diol (12). 5,6-Diamino-
2,4-dihydroxypyrimidine sulfate (240.1 mg, 1.0 mmol) was sus-
pended in 25 mL of DI water, and solid sodium bicarbonate (168.0
mg, 2.0 mmol) was added to this suspension. The reaction mixture
was stirred at ambient temperature for 15 min. Then a solution of
3,3′-dihydroxybenzil (242.4 mg, 1.0 mmol) in 10 mL of methanol
was added. The reaction mixture was brought to reflux and refluxed
for 3 h. At this point it formed an orange solution with a yellow
precipitate. The reaction mixture was cooled down to ambient
temperature, and the resulting precipitate was centrifuged down and
washed sequentially with 2 × 40 mL of DI water, 1 × 10 mL of
MeOH, and 2 × 40 mL of diethyl ether and dried in Vacuo to give
220.2 mg of the title compound as light-yellow fluffy solid, 63.2%
yield, 99.3% purity by LC/MS (230 DAD). MS m/z [MH+] ) 349.0.
1H NMR (DMSO-d6) δ 6.68-6.70 (d, J ) 7.9 Hz, 1H), 6.72-
6.74 (dd, J1 ) 7.9 Hz, J2 ) 1.6 Hz, 1H), 6.77-6.80 (m, 2H), 6.88
(t, J ) 1.1 Hz, 2H), 7.08-7.11 (t, J ) 7.9 Hz, 1H), 7.11-7.13 (t,
J ) 7.9 Hz, 1H), 9.52 (br s, 1H), 9.60 (br s, 1H), 11.66 (br s, 1H),
11.97 (br s, 1H). Anal. (C18H12N4O4‚H2O) C, H, N.
3,3′-{2,4-Bis[(3-morpholin-4-ylpropyl)amino]pteridine-6,7-
diyl}diphenol Trifluoroacetic Acid Salt (16). 3,3′-(2,4-Diami-
nopteridine-6,7-diyl)diphenol (1.0 g, 2.88 mmol) was dissolved in
5 mL of 4-(3-aminopropyl)morpholine, and sulfamic acid (0.530
g, 5.45 mmol) was added to this solution. The reaction mixture
was refluxed for 18 h (ca. 160 °C). Then it was cooled down to
ambient temperature, filtered through 0.22 µm syringe filter, and
purified by reverse-phase preparative HPLC using gradient of
acetonitrile/water mixture with 0.1% of TFA. Fractions, containing
the product, were collected. Solvent was removed in Vacuo to afford
800 mg of the title product 16 as a brown viscous oil. MS m/z
1
[MH+] ) 601.6. H NMR (DMSO-d6): δ 3.07 (m, 4H), 3.19 (m,
4H), 3.42 (m, 4H), 3.59-3.80 (m, 12H), 3.97 (m, 4H), 6.75-6.84
(m, 4H), 6.90 (m, 2H), 7.13-7.18 (m, 2H).
3-(2,4-Diaminopteridin-6-yl)phenol (17). 3-Hydroxyphenylg-
lyoxal (2.1 g, 12.48 mmol) was dissolved in 100 mL of DI water.
Acetone oxime (0.912 g, 12.48 mmol) was added, followed by 3
drops of 1 N aqueous hydrochloric acid to bring the pH to ca. 2-3.
This solution was stirred at 50 °C for 1 h, 2,4,5,6-tetraaminopyri-
midine sulfate (2.67 g, 11.23 mmol) was added, and the reaction
mixture was stirred at ambient temperature for 3 h and refluxed
for 6 h. At this point it formed a thick yellow suspension. The
reaction was allowed to cool down to ambient temperature.
Saturated aqueous sodium bicarbonate was added dropwise to bring
the pH to ca. 6-7. The precipitate was collected, washed
extensively with water, with 60 mL of methanol, and repeatedly
with diethyl ether, and dried in Vacuo to give 2.75 g of the title
compound 17 (96.5% yield) as light-yellow solid. 99.5% purity by
LC/MS (230 DAD). MS m/ z [MH+] ) 254.9. 1H NMR (DMSO-
d6) δ 6.88-6.90 (dd, J1 ) 7.9 Hz, J2 ) 2.3 Hz, 1H), 7.16 (br s,
2H), 7.29-7.32 (t, J ) 7.9 Hz, 1H), 7.66-7.67 (m, 1H), 7.72-
7.73 (d, J ) 7.9 Hz, 1H), 8.21 (br s, 1H), 8.27 (br s, 1H), 9.28 (s,
1H), 9.63 (s, 1H). Anal. (C12H10N6O‚1.7H2O) C, H, N.
2-Amino-6,7-bis(3-hydroxyphenyl)pteridin-4-ol (13). 6-Hy-
droxy-2,4,5-triaminopyrimidine sulfate (239.2 mg, 1.0 mmol) was
suspended in 25 mL of DI water, and solid sodium bicarbonate
(168.0 mg, 2.0 mmol) was added to this suspension. The reaction
mixture was stirred at ambient temperature for 15 min. Then a
solution of 3,3′-dihydroxybenzil (242.4 mg, 1.0 mmol) in 10 mL
of methanol was added. The reaction mixture was brought to reflux
and refluxed for 3 h. At this point it formed a yellow solution with
a yellow precipitate. The reaction mixture was cooled down to
ambient temperature, and the resulting precipitate was centrifuged
down and washed sequentially with 2 × 40 mL of DI water, 1 ×
10 mL of MeOH, and 2 × 40 mL of diethyl ether and dried in
Vacuo to give 231.2 mg of the title compound 13 as reddish-yellow
solid, 66.5% yield, 99.1% purity by LC/MS (230 DAD). MS m/z
1
[MH+] ) 348.0. H NMR (DMSO-d6) δ 6.68-6.70 (d, J ) 7.9
Hz, 1H), 6.69-6.72 (dd, J1 ) 7.9 Hz, J2 ) 2.0 Hz, 1H), 6.75-
6.77 (dd, J1 ) 7.9 Hz, J2 ) 2.4 Hz, 1H), 6.77-6.79 (d, J ) 7.9
Hz, 1H), 6.88 (t, J ) 2.0 Hz, 1H), 6.90 (t, J ) 2.0 Hz, 1H), 7.05-
4-(2,4-Diaminopteridin-6-yl)benzene-1,2-diol (18). The same
synthetic procedure as for compound 19, 97.1% purity (97.5:2.5
ratio of regioisomers) by LC/MS (230 DAD). ELSD purity is 100%.

Isozyme-SelectiVe Inhibitor of PI3K
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4291
1
MS m/z [MH+] ) 271.6. H NMR (DMSO-d6) for major regio-
dimethoxybenzil (135 mg, 0.50 mmol, 1.0 equiv), and m-cresol
(1.0 mL). The slurry was heated to ca. 200 °C and within 10 min
gave rise to a dark-green solution. A dull green precipitate started
to form in ca. 20 min and remained through the rest of the heating.
The reaction was cooled to room temperature and triturated by
pouring into diethyl ether (30 mL) to yield a dull green precipitate.
The solid was isolated and washed with 3 × 30 mL of diethyl ether
and dried in Vacuo to yield the title compound as green solid as a
1/1 complex with m-cresol (210 mg). The solid obtained as the
sulfate salt as a complex with m-cresol (133 mg; 0.281 mmol; 1.0
equiv) was suspended in methanol (15 mL) in a reaction vessel
equipped with a stirring bar. Amberlite chloride resin (GFS
Chemical; 3.2 g) was added to the suspension. The resulting green-
brown suspension was stirred at ca. 40 °C for 4 h, cooled to room
temperature, filtered to remove the resin, and concentrated to yield
an olive green solid. The solid was taken in ca. 4 mL of methanol
and precipitated by adding into 40 mL of ether. The precipitate
was washed with ether (2 × 40 mL) and dried in the air and then
in Vacuo to yield the title compound 25 as a grayish-green solid
(58 mg, 55% yield), 100% purity by LC/MS (230 DAD), 98.4%
purity by ELSD. MS m/z [MH+] ) 375.5. 1H NMR (DMSO-d6) δ
3.64 (s, 3H), 3.67 (s, 3H), 6.93 - 6.98 (m, 2H), 6.98 - 6.99 (m,
3H), 7.10 - 7.12 (m, 1H), 7.23 (t, J ) 8.5 Hz, 1H), 7.29 (t, J )
8.0 Hz, 1 H), 7.70 (br s, 1H), 8.68 (br s, 1H), 9.20 (br s, 1H), 9.28
(br s, 1 H).
isomer δ 6.85-6.88 (d, J ) 8.34 Hz, 1H), 7.03 (br s, 2H), 7.61-
7.63 (dd, J1 ) 7.9 Hz, J2 ) 2.1 Hz, 1H), 7.68 (d, J ) 2.1 Hz, 1H),
8.18 (br s, 2H), 8.98 (s, 1H), 9.20 (s, 1H), 9.51 (s, 1H).
4-(2,4-Diaminopteridin-6-yl)phenol (19). A 10-mL reaction
vessel equipped with a stirring bar and a reflux condenser was
charged with 3-hydroxyphenylglyoxal (37.5 mg, 0.27 mmol; 1.00
equiv) was dissolved in 5 mL of methanol. Hydroxylamine
hydrochloride (20 mg, 0.29 mmol) was added. This solution was
stirred at 50 °C for 15 min, 2,4,5,6-tetraaminopyrimidine sulfate
(60 mg; 0.25 mmol) was added, and the reaction mixture was heated
to reflux for 3 h. The reaction was cooled, and a yellow precipitate
started to form. The solution was neutralized using NaHCO3 (satd,
aqueous) to bring the pH to ca. 7. The solution was filtered, washed
with water (1 × 30 mL), then with methanol (1 × 30 mL) and
finally with ether (1 × 30 mL) and dried in the air, followed by
vacuum to give the title compound 19 (32.0 mg, 50% yield) as a
yellow solid, 100% purity by LC/MS (230 DAD), 100% purity by
ELSD. [MH+] ) 255.1. 1H NMR (DMSO-d6) δ 6.90 (d, J ) 1H),
6.88 (d, J ) 8.5 Hz, 2 H), 7.62 (br s, 1H), 7.79 (br s, 1H), 8.15 (d,
J ) 8.5 Hz, 2H), 9.24 (s, 1H), 9.79 (s, 1H).
4,4′-(2,4-Diaminopteridine-6,7-diyl)diphenol (20). Same pro-
cedure as for compound 6, 96.5% purity by LC/MS (230 DAD).
MS m/z [MH+] ) 347.7. 1H NMR (DMSO-d6): δ 6.58 (br s, 2H),
6.67-6.69 (d, J ) 8.8 Hz, 2H), 6.69-6.72 (d, J ) 8.8 Hz, 2H),
7.27-7.31 (m, 4H), 7.55 (br s, 1H), 7.64 (br s, 1H), 9.61 (s, 1H),
9.77 (s, 1H). Anal. (C18H14N6O2‚2.2H2O) C, H, N.
3,3′-[(2,4-Diaminopteridine-6,7-diyl)bis(3,1-phenyleneoxy)]-
dipropane-1,2-diol Trifluoroacetic Acid Salt (26). To a solution
of solketal (524 uL, 4.2 mmol), triphenylphosphine (1.10 g, 4.2
mmol), and diisopropyl azodicarboxylate (DIAD, 827 µL, 4.2
mmol) in 5 mL of anhydrous THF was added a solution of 3,3′-
dihydroxybenzil (485 mg, 2.0 mmol) in 5 mL of anhydrous THF.
The mixture was stirred at room temperature for 12 h. The solvent
was removed, and the crude product was purified by silica gel
column chromatography (Rf ) 0.8, CH2Cl2/ MeOH, 95:5) to give
1,2-bis[3-(2,2-dimethyl-1,3dioxolan-4-ylmethoxy)phenyl]ethane-1,2-
dione as a yellow solid (378 mg, 40%). To a solution of 2,4,5,6-
tetraaminopyrimidine sulfate (238 mg, 1.0 mmol) and sodium
bicarbonate (168 mg, 2.0 mmol) in 5 mL water was added a solution
of 1,2-bis[3-(2,2-dimethyl-1,3dioxolan-4-ylmethoxy)phenyl]ethane-
1,2-dione (350 mg, 0.74 mmol) in 3 mL of methanol. The mixture
was heated to 60 °C for 18 h, cooled to room temperature, and
acidified with 1 N HCl to pH ) 3. It was stirred at room temperature
for 3 h, the solvent was removed, and the crude product was purified
by preparative, reverse phase HPLC to give trifluoroacetate salt of
the title compound 26 as a yellow solid (92.4 mg, 15%), 97.4%
3-(2,4-Diaminopteridin-7-yl)phenol (21). 2,4,5,6-Tetraaminopy-
rimidine sulfate (476.4 mg, 2.0 mmol) was suspended in 10 mL of
DI water, and 7.5 M aqueous sodium hydroxide was added until
the measured pH was ca. 9-10. Then 3-hydroxyphenylglyoxal
(336.3 mg, 2.0 mmol) was added. The reaction mixture was stirred
at ambient temperature for 3 h and then brought to reflux and
refluxed for 3 h. At this point it formed a thick yellow suspension.
The reaction was allowed to cool down to ambient temperature
and 1 N aqueous hydrochloric acid was added dropwise to bring
the pH to ca. 6-7. The precipitate was collected, washed
extensively with water, with 60 mL of methanol, and repeatedly
with diethyl ether and dried in Vacuo to give the title compound
21 (469.3 mg, 92.3% yield) as a bright-yellow solid, 98.5% purity
by LC/MS (230 DAD). MS m/z [MH+] ) 254.9. 1H NMR (DMSO-
d6) δ 6.83 (br s, 2H), 6.93-6.96 (dd, J1 ) 8.0 Hz, J2 ) 2.1 Hz,
1H), 7.33-7.36 (t, J ) 7.9 Hz, 1H), 7.62-7.63 (m, 1H), 7.65 (d,
J ) 0.83 Hz, 1H), 7.83 (br s, 1H), 7.87 (br s, 1H), 8.85 (s, 1H),
9.77 (br s, 1H). Anal. (C12H10N6O‚1.4H2O) C, H, N.
4-(2,4-Diaminopteridin-7-yl)phenol (22). This compound is
made by stirring a 1:1 ratio of 4-hydroxyphenylglyoxal with the
free base of the 2,4,5,6-tetraaminopyrimidine in water at a pH of
7 for ca. 3 h. The product is isolated by filtering the precipitated
free base, washing sequentially with water (2 × 40 mL), methanol
(1 × 40 mL), and ether (2 × 40 mL), and drying in vacuum
desiccator. 1H NMR (DMSO-d6): δ 6.57 (br s, 2 H), 6.91 (d, J )
8.5 Hz, 2 H), 7.55 (br s, 1 H), 7.62 (br s, 1 H), 8.09 (d, J ) 8.5 Hz,
2 H), 8.81 (s, 1 H), 10.0 (br s, 1 H).
1
purity by LC/MS (230 DAD). MS m/z [MH+] ) 495.5. H NMR
(MeOH-d4): δ 3.61-3.65 (m, 4H), 3.83-3.98 (m, 6H), 7.01-7.06
(m, 3H), 7.09-7.12 (m, 3H), 7.24-7.27 (m, 2H).
6,7-Bis{3-[2-(diethylamino)ethoxy]phenyl}pteridine-2,4-di-
amine (27). A solution of 2-diethylaminoethyl chloride hydrochlo-
ride (1.03 g, 6.0 mmol), Cs2CO3 (3.9 g, 12 mmol), and 3,3′-
dihydroxybenzil (727 mg, 3.0 mmol) in 30 mL of acetone was
heated to reflux for 1.5 h and then cooled to ambient temperature.
The solvent was removed in Vacuo, and the crude product was
purified by silica gel column chromatography (Rf ) 0.2, MeOH/
Et3N, 95.5:0.5) to give 1,2-bis[3-(2-diethylaminoethoxy)phenyl]-
ethane-1,2-dione as a yellow solid (823 mg, 62%). To a suspension
of 2,4,5,6-tetraaminopyrimidine sulfate (434 mg, 1.82 mmol) and
sodium bicarbonate (306 mg, 3.64 mmol) in 4 mL of water and 1
mL of 1,4-dioxane was added a solution of 1,2-bis[3-(2-diethy-
laminoethoxy)phenyl]ethane-1,2-dione (802 mg, 1.82 mmol) in 4
mL of methanol. The mixture was heated to reflux for 10 h, and
then solvent was removed in Vacuo. The crude product was purified
by reverse phase preparative HPLC to give trifluoroacetate salt of
the title product 27 as yellow solid (848 mg, 71%), 100% purity
by LC/MS (230 DAD). MS m/z [MH+] ) 545.6. 1H NMR (MeOH-
d4): δ 1.34-1.38 (t, J ) 7.4 Hz, 12H), 3.31-3.34 (q, J ) 7.4 Hz,
8H), 3.58-3.60 (t, J ) 4.8 Hz, 4H), 4.28-4.33 (dt, J1 ) 11.6 Hz,
J2 ) 4.8 Hz, 4H), 7.09-7.13 (m, 4H), 7.23-7.24 (m, 2H), 7.27-
7.30 (m, 2H).
4-(2,4-Diaminopteridin-7-yl)benzene-1,2-diol (23). This com-
pound is made by stirring a 1:1 ratio of 3,4-dihydroxyphenylglyoxal
with the free base of 2,4,5,6-tetraaminopyrimidine in water at a
pH of 7 for ca. 3 h. The product is isolated by filtering the
precipitated free base, washing sequentially with water (2 × 40
mL), methanol (1 × 40 mL), and ether (2 × 40 mL), and drying
1
in a vacuum desiccator. H NMR (500 MHz; DMSO-d6): δ 6.52
(br s, 2 H), 6.84 (d, J ) 8.3 Hz, 1 H), 7.53 (dd, J1) 8.3 Hz, J2 )
2.1 Hz, 1 H), 7.53-7.56 (br s, 2 H), 7.64 (d, J ) 2.3 Hz, 1 H),
8.71 (s, 1 H).
6,7-Diphenylpteridine-2,4-diamine (24) was prepared according
to literature.42 97.5% purity by LC/MS (230 DAD). MS m/z [MH+]
) 315.6. 1H NMR (DMSO-d6): δ 6.72 (br s, 2H), 7.28-7.44 (m,
10H), 7.69 (br s, 1H), 7.75 (br s, 1H).
6,7-Bis(3-methoxyphenyl)pteridine-2,4-diamine (25). A 5 mL
reaction vial equipped with a stirring vane was charged with 2,4,5,6-
tetraaminopyrimidine sulfate (119 mg, 0.50 mmol, 1.0 equiv), 3,3′-

4292 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Palanki et al.
(2,4-Diaminopteridine-6,7-diyl)di-3,1-phenylene Tetraethyl
Bis(phosphate) (28). A 50-mL one-necked round-bottomed flask
with a stirring bar and a septum was charged with 3,3′-dihydroxy-
benzil (512 mg; 2.11 mmol; 1.00 equiv) and acetonitrile (10 mL).
To this partially dissolved mixture were added triethylamine (1.06
g; 14.9 mmol; 7.06 equiv), dimethylaminopyridine (DMAP) (506
mg; 4.13 mmol; 1.96 equiv), and dichloromethane (DCM, 10 mL)
as cosolvents. Diethyl chlorophosphate (1.05 g; 6.09 mmol; 2.88
equiv) was added via a pipet over ca. 30 s A white precipitate
immediately formed and remained through the course of the reaction
in the yellow solution. The reaction mixture was stirred for 3 d at
room temperature after which it was concentrated by rotary
evaporation to yield an off- white slurry. This oily slurry was
partitioned between sodium bicarbonate (satd aqueous) and DCM.
The aqueous layer was rewashed with 2 × 5 mL DCM, followed
by extraction of the combined organics with 10 mL of 1 M HCl
(aqueous). The combined organic layer was dried over anhydrous
MgSO4, filtered, and concentrated by rotary evaporation to yield
the desired bis-phosphate ester material as light yellow slightly
viscous oil. The compound does not require any purification but is
easily purified by column chromatography using DCM-EtOAc (1:
1) if needed. The chromatographically purified material is a yellow
oil (911 mg; 89%). 1H NMR (500 MHz; DMSO-d6): δ 8.01 (d, J
) 8.6 Hz, 4 H), 7.45 (d, J ) 8.5 Hz, 4 H), 4.18-4.21 (m, 8 H),
1.28 (app t, J ) 5.0 Hz, 12 H). A 60-mL reaction vessel with a
stirring bar was charged with 2,4,5,6-tetraaminopyrimidine sulfate
(188 mg; 0.791 mmol; 1.0 equiv) and sodium bicarbonate (10 mL
solution containing 134 mg; 0.80 mmol; 1.01 equiv). The solution
was heterogeneous at this point. After 10 min, the bis(phosphate)
ester (499 mg; 0.971 mmol; 1.23 equiv) was added as a solution in
methanol (9 mL). The solution was heated to ca. 60 °C and appeared
turbid and yellow. The reaction was monitored to completion by
HPLC in ca. 3 h time. The reaction mixture was cooled to room
temperature and concentrated by rotary evaporation. The resulting
yellow solid was dissolved in methanol, precipitated into ether,
washed with 3 × 30 mL ether, and dried in the air, followed by
drying in Vacuo, resulting in the title compound 28 as a light yellow
solid (0.20 g; 41%), 96.5% purity by LC/MS (230 DAD), 98.4%
purity by ELSD. MS m/z [MH+] ) 619.4. 1H NMR (DMSO-d6) δ
1.21-1.25 (m, 12H), 4.06-4.11 (m, 8H), 7.23-7.26 (m, 2H),
7.30-7.34 (m, 4H), 7.34-7.43 (m, 2H).
(d, J ) 2.1 Hz, 1H), 7.34-7.35 (d, J ) 9.5 Hz, 1H), 8.37-8.39
(d, J ) 9.5 Hz, 1H), 8.56 (br s, 1H), 9.65 (br s, 1H). Anal.
(C19H14N4O4‚2HCl‚1.5H2O) C, H, N.
4,4′-(8-Aminopyrido[3,4-b]pyrazine-2,3-diyl)diphenol Hydro-
chloride Salt (31). 3,4,5-Triaminopyridine trihydrochloride (2.6 g,
11.1 mmol) and 4,4′-dihydroxybenzil (2.83 g, 11.7 mmol) were
dissolved in 60 mL of 1:1 mixture of dioxane/water. The reaction
mixture was refluxed for 18 h. Then solvent was removed in Vacuo.
The resulting dark-brown residue was dissolved in ca. 50 mL of
MeOH, and this solution was added dropwise to 1 L of diethyl
ether with vigorous stirring. The formed solid was centrifuged down,
washed repeatedly with diethyl ether, and dried in Vacuo to give
the title compound 31 (4.11 g, 91.9% yield) as dark-yellow solid,
97.7% purity by LC/MS (ELSD). MS m/z [MH+] ) 331.5. 1H NMR
(DMSO-d6) δ 6.78-6.83 (m, 4H), 7.40-7.43 (m, 2H), 7.57-7.60
(m, 2H), 7.98 (d, J ) 0.76 Hz, 1H), 8.85 (d, J ) 0.76 Hz, 1H),
10.2 (br s, 2H). Anal. (C19H14N4O2‚1HCl‚2H2O) C, H, N.
4,4-(6-Aminopyrido[2,3-b]pyrazine-2,3-diyl)diphenol (32). 2,3,6-
Triaminopyridine dihydrochloride (1.97 g, 10.0 mmol) and 4,4′-
dihydroxybenzil (2.42 g, 10.0 mmol) were dissolved in 60 mL of
1:1 mixture of dioxane/water. The reaction mixture was refluxed
for 18 h. Then solvent was removed in Vacuo. The resulting dark-
brown residue was dissolved in ca. 20 mL of MeOH, and this
solution was added dropwise to 1 L of diethyl ether with vigorous
stirring. The formed solid was centrifuged down, washed repeatedly
with diethyl ether, and dried in Vacuo to give the title compound
32 (3.29 g, 85.4% yield) as dark-yellow solid. 94.7% purity by
LC/MS (230 nm). MS m/z [MH+] ) 331.5, 332.5. 1H NMR
(MeOH-d4) δ 6.74-6.78 (m, 4H), 7.29-7.31 (d, J ) 9.5 Hz, 1H),
7.34-7.36 (m, 2H), 7.43-7.45 (m, 2H), 8.39-8.40 (d, J ) 9.5
1
Hz, 1H). H NMR (DMSO-d6) δ 6.75-6.77 (d, J ) 5.9 Hz, 2H),
6.77-6.78 (d, J ) 5.9 Hz, 2H), 7.23-7.24 (d, J ) 8.7 Hz, 2H),
7.29-7.31 (d, J ) 8.7 Hz, 2H), 7.38-7.40 (d, J ) 9.5 Hz, 1H),
8.38-8.40 (d, J ) 9.5 Hz, 1H), 8.68 (br s, 1H), 9.71 (br s, 1H),
9.87 (br s, 1H), 10.07 (br s, 1H). Anal. (C19H14N4O2‚1.5HCl) C,
H, N.
4-(4-Aminopteridin-7-yl)benzene-1,2-diol (33). This compound
is made by stirring a 1:1 ratio of 3,4-dihydroxyphenylglyoxal with
the free base of 2,4,5,6-tetraaminopyrimidine in water at a pH of
7 for ca. 3 h. The product was isolated by filtering the precipitated
free base, washing sequentially with water (2 × 40 mL), methanol
(1 × 40 mL), and ether (2 × 40 mL), and drying in a vacuum
4,4′-[4-Aminopteridine-6,7-diyl]dibenzene-1,2-diol (29). Same
1
procedure as for compound 6. MS m/z [MH+] ) 364.8. H NMR
1
desiccator. H NMR (500 MHz; DMSO-d6): δ 6.92 (d, J ) 8.3
(MeOH-d4) δ 6.70-6.72 (d, J ) 8.1 Hz, 1H), 6.73-6.75 (d, J )
8.1 Hz, 1H), 6.91-6.93 (dd, J1 ) 8.1 Hz, J2 ) 2.18 Hz, 1H), 6.93-
6.95 (dd, J1 ) 8.1 Hz, J2 ) 2.18 Hz, 1H), 7.03 (d, J ) 2.18 Hz,
1H), 7.12 (d, J ) 2.18 Hz, 1H), 8.49 (s, 1H). 1H NMR (DMSO-d6)
6.63-6.65 (d, J ) 8.4 Hz, 1H), 6.66-6.68 (d, J ) 7.9 Hz, 1H),
6.74-6.76 (dd, J1 ) 8.4 Hz, J2 ) 1.85 Hz, 1H), 6.85-6.87 (dd, J1
) 7.9 Hz, J2 ) 1.85 Hz, 1H), 7.00 (d, J ) 1.85 Hz, 1H), 7.06 (d,
J ) 1.9 Hz, 1H), 7.93 (br s, 2H), 8.47 (s, 1H).
Hz, 1 H), 7.71 Hz, (dd, J ) 8.4 Hz, J ) 2.3 Hz, 1 H), 7.80 (d, J
) 2.3 Hz, 1 H), 8.17 (br s, 1 H), 8.12 (br s, 1 H), 8.51 (s, 1 H),
9.28 (s, 1 H), 9.40 (br s, 1 H), 9.72 (s, 1 H).
4-(4-Aminopteridin-6-yl)benzene-1,2-diol (34). The same syn-
thetic procedure as for compound 19, 98.7% purity (92:8 ratio of
regioisomers) by LC/MS (230 DAD), ELSD purity 100%. MS m/z
1
[MH+] ) 256.1. H NMR (DMSO-d6) for major regioisomer δ
6.89 (dd, J1 ) 7.9 Hz, J2 ) 2.1 Hz, 1H), 7.82 (d, J ) 2.1 Hz, 1H),
8.11 (br s, 1H), 8.22 (br s, 1H), 8.49 (br s, 1H), 9.08 (br s, 1H),
9.52 (s, 1H), 9.70 (br s, 1H).
4,4-(6-Aminopyrido[2,3-b]pyrazine-2,3-diyl)dibenzene-1,2-
diol Dihydrochloride Salt (30). 2,3,6-Triaminopyridine dichloride
(1.97 g, 10.0 mmol) and 3,3′4,4′-tetrahydroxybenzil (2.74 g, 10.0
mmol) were dissolved in 60 mL of 1:1 mixture of dioxane/water.
The reaction mixture was refluxed for 18 h. Then solvent was
removed in Vacuo to give a dark-brown residue. The residue was
dissolved in 20 mL of methanol and added dropwise with vigorous
stirring to 500 mL of diethyl ether. The resulting brown precipitate
was collected, redissolved in 20 mL of MeOH, and added again
dropwise to 500 mL of anhydrous diethyl ether. The formed dark-
yellow precipitate was centrifuged down, washed repeatedly with
diethyl ether, and dried in Vacuo to give the title compound 30
(3.96 g, 85.6%) as yellow solid, 98.2% purity by LC/MS (230
Acknowledgment. We thank Chris Jacob, Antonio Boccia,
and Jann Key for assistance in the animal experiments and Kathy
Barrett and Tessa Chung for assistance in the execution of
various cell culture and biochemical assays.
Supporting Information Available: Elemental analyses data.
This material is available free of charge via the Internet at http://
pubs.acs.org.
References
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(60) We have tested compound 6 against 140 kinases at a single
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Kinases inhibited at greater than 90% were selected for IC50
determinations. Compound 6 potently inhibited the â, δ, and γ
isoforms of PI3K. Apart from the PI3K isoforms, compound 6 also
inhibited AAk1, BIKE, CLK1, CLK2, CLK3, PKCµ, RIPK2, and
Tie2. This information is provided as supplementary material
(Appendix A).
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(61) DISCOVER, Insight II and the CFF force field are trademarked
software offerings from Accelrys Inc., San Diego, CA.
(62) Manuscript in preparation.
(63) http://clinicaltrials.gov/ct/show/NCT00103350?order)1.
(64) YASARA is a trademarked software offering of Yasara Biosciences,
Austria.
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(53) The antibodies used in this study for Western blotting were from
Santa Cruz Biotechnology; catalog no. sc-6548, Santa Cruz, CA
(antibody to VE-Cadherin), and Chemicon; catalog no. APT-184,
Temecula, CA (Omni-Phos, and pan-phosphorylated proteins).
(54) Doukas, J.; Wrasidlo, W.; Noronha, G.; Dneprovskaia, E.; Fine, R.;
Weis, S.; Hood, J.; Demaria, A.; Soll, R.; Cheresh, D. Phosphoi-
nositide 3-kinase gamma/delta inhibition limits infarct size after
myocardial ischemia/reperfusion injury. Proc. Natl. Acad. Sci.
U.S.A. 2006, 103, 19866-71. Briefly, cell proliferation assays was
carried out using human umbilical vein EC plated in 96-well
cluster plates (5000 cells/well) were cultured in assay medium
(containing 0.5% serum and 50 ng/ml VEGF) in the presence
or absence of test compounds (10 µM), and cell numbers were
quantified by XTT assay (Promega, Madison, WI) after 24, 48, or
72 h later.
(55) Kinexus provides antibody microarray services that would examine
cell lysates using 377 pan-specific and 273 phospho-site-specific
antibodies. The proteomics service provides information on protein
expression, phosphorylation, and protein-protein expression from
cell lysates. This technique would allow one to examine the kinases
that were affected by a compound. The complete details are provided
at their web site (www.kinexus.ca). Kinexus technology would allow
one to examine numerous pathways. Visit their web site for more
information. The concentration of compound 6 was 10 µM in cell
assays.
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