Structure-based optimization of potent and selective inhibitors of the tyrosine kinase erythropoietin producing human hepatocellular carcinoma receptor B4 (EphB4)

Article abstract of DOI:10.1021/jm9009444

The tyrosine kinase EphB4 is an attractive target for drug design because of its recognized role in cancer-related angiogenesis. Recently, a series of commercially available xanthine derivatives were identified as micromolar inhibitors of EphB4 by high-throughput fragment-based docking into the ATP-binding site of the kinase domain. Here, we have exploited the binding mode obtained by automatic docking for the optimization of these EphB4 inhibitors by chemical synthesis. Addition of only two heavy atoms, methyl and hydroxyl groups, to compound 4 has yielded the single-digit nanomolar inhibitor 66, with a remarkable improvement of the ligand efficiency from 0.26 to 0.37 kcal/(mol per non-hydrogen atom). Compound 66 shows very high affinity for a few other tyrosine kinases with threonine as gatekeeper residue (Abl, Lck, and Src). On the other hand, it is selective against kinases with a larger gatekeeper. A 45 ns molecular dynamics (MD) simulation of the complex of EphB4 and compound 66 provides further validation of the binding mode obtained by fragment-based docking. 2009 American Chemical Society.

Full text of DOI:10.1021/jm9009444

J. Med. Chem. 2009, 52, 6433–6446 6433
DOI: 10.1021/jm9009444
Structure-Based Optimization of Potent and Selective Inhibitors of the Tyrosine Kinase Erythropoietin
Producing Human Hepatocellular Carcinoma Receptor B4 (EphB4)
Karine Lafleur,†,‡ Danzhi Huang,*,† Ting Zhou,† Amedeo Caflisch,*,† and Cristina Nevado*,‡
†Department of Biochemistry and ‡Department of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057,
Zurich, Switzerland
Received June 26, 2009
The tyrosine kinase EphB4 is an attractive target for drug design because of its recognized role in cancer-
related angiogenesis. Recently, a series of commercially available xanthine derivatives were identified as
micromolar inhibitors of EphB4 by high-throughput fragment-based docking into the ATP-binding site
of the kinase domain. Here, we have exploited the binding mode obtained by automatic docking for the
optimization of these EphB4 inhibitors by chemical synthesis. Addition of only two heavy atoms,
methyl and hydroxyl groups, to compound 4 has yielded the single-digit nanomolar inhibitor 66, with a
remarkable improvement of the ligand efficiency from 0.26 to 0.37 kcal/(mol per non-hydrogen atom).
Compound 66 shows very high affinity for a few other tyrosine kinases with threonine as gatekeeper
residue (Abl, Lck, and Src). On the other hand, it is selective against kinases with a larger gatekeeper.
A 45 ns molecular dynamics (MD) simulation of the complex of EphB4 and compound 66 provides
further validation of the binding mode obtained by fragment-based docking.
1. Introduction
N-terminal domain necessary for ligand binding, whereas its
intracellular domain includes a C-terminal domain and a tyrosine
kinase domain. Despite the potential therapeutic importance of
EphB4, only four series of small molecule inhibitors are currently
known (Figure 1).6-9 In 2007, Miyazaki and co-workers reported
the synthesis of 3-[4-amino-3-(3-chloro-4-fluorophenyl)thieno-
[3,2-c]pyridin-7-yl]-benzenesulfonamide (1), which is a potent
EphB4 inhibitor.7 One year later, 2,4-bis-anilinopyrimidine deri-
vatives such as 2 showed also high potency as EphB4 inhibitors,
and their cocrystallization with human EphB4 highlighted their
dual binding mode (Figure 1).10 The marketed drug dasatinib,
with Abl1 and Src as primary targets, also showed a very high
affinity to Eph kinases.11
High throughput docking is a computational tool frequently
used to discover small-molecule inhibitors of enzymes or recep-
tors of known three-dimensional structures.12,13 Recently, we
have developed an efficient computational method (termed
ALTA for anchor-based library tailoring) to focus a chemical
library by docking and prioritizing molecular fragments accord-
ing to their binding energy.6 From a collection of about 700 000
compounds, ALTA generated a focused library of 21 418 mole-
cules, each containing at least one fragment predicted to bind to
the ATP-binding site of EphB4. Automatic docking of these
21 418 molecules yielded two series of micromolar inhibitors, one
of them based on a xanthine scaffold predicted to be involved in
two hydrogen bonds with the hinge region that connects the
N-terminal and C-terminal lobes of the kinase domain. Further
characterization of the commercially available 3 (Figure 1)
indicated that this molecule binds to the ATP-binding site, as
predicted by the docking calculations.6 In addition, an analogue
with a pendent anisidine chain (4, Figure 1) showed similar
inhibition properties to 3 and was active in a cell-based assay.
Here, we present a medicinal chemistry campaign aimed at
improvingthe affinityofthe micromolar hits3and4 identified
by in silico screening. The optimization was carried out using
Angiogenesis, the formation of new blood vessels from pre-
existing ones, has been identified as one of the key steps in
human carcinogenesis. In fact, nutrient supply and waste
elimination are required for cell proliferation. Because of low
toxicity and resistance potential,1 as well as the possibility of
treating a large spectrum of solid tumor types,2 angiogenesis
inhibition is considered a promising target in anticancer thera-
pies. Several studies have implicated erythropoietin-producing
human hepatocellular carcinoma receptor (Epha) signaling in
sprouting angiogenesis and blood vessel remodeling during
vascular development.3 Furthermore, overexpression of several
of the 14 Eph receptors has been linked to tumors and the
associated vasculature, suggesting a critical role in tumor-
related angiogenesis. In fact, inhibition of binding of EphB4
to its natural ligand EphrinB2 using soluble extracellular
domains of EphB4 has been shown to reduce tumor growth
in murine tumor xenograft models.4,5 Thus, inhibition of Eph
angiogenic activity has been recognized as an effective strategy
for blocking tumor progression and metastasis.
Like all receptor tyrosine kinases, EphB4 is a type-I trans-
membrane protein. Its extracellular domain is composed of an
*To whom correspondence should be addressed. For D.H.: phone,
(41) 446355568; fax, (41) 446356862; e-mail, dhuang@bioc.uzh.ch. For
A.C.: phone, (41) 446355521; fax, (41) 446356862; e-mail, caflisch@
bioc.uzh.ch. For C.N.: phone, (41) 446353945; fax, (41) 446353948;
e-mail, nevado@oci.uzh.ch.
a Abbreviations: Abl, Abelson murine leukemia viral oncogene
homologue; ALTA, anchor-based library tailoring; ATP, adenosine tri-
phosphate; DIPEA, diisopropylethylamine; DMF, dimethylformamide;
DMSO, dimethyl sulfoxide; Eph, erythropoietin-producing human
hepatocellular carcinoma receptor; FRET, fluorescence-resonance
energy transfer; GCK, germinal center kinase; Lck, lymphocyte-specific
kinase; MD, molecular dynamics; MLK, mixed-lineage kinase; rmsd,
root-mean-square deviation; SAR, structure-activity relationship;
THF, tetrahydrofuran.
r
2009 American Chemical Society
Published on Web 09/29/2009
pubs.acs.org/jmc

6434 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Lafleur et al.
the binding mode obtained by automatic docking into EphB4.
Chemical synthesis of about 30 derivatives of 3 and 4 yielded
six nanomolar inhibitors, one of which (compound 66) has
an IC50 of 2-5 nM and shows good selectivity against other
protein kinases. The addition of only two heavy atoms (-CH3
and -OH substituents at the phenyl ring, 4 vs 66) has resulted
in a ∼1000 times improvement of affinity, which is a remark-
able example of the usefulness of structure-based hit modifi-
cations.
2. Synthesis
Our foreseen modifications to the scaffold of compound 4
(R1-7) for the structure-activity relationship study (SAR)
have been summarized at the top of Table 1. Such studies
demand a rapid, reliable, and flexible access to a wide variety of
potentially active structures with a minimum synthetic varia-
tion cost. To succeed in such a goal, a modular synthetic
approach was developed. Thus, methyl and benzyl substituted
derivatives at R1 were prepared in parallel as summarized in
Scheme 1. The synthesis started by condensation of cyano-
acetic acid with commercially available methylurea or benzyl-
urea to give the corresponding cyanoacetylurea intermediates,
which upon treatment with base afforded the desired 1-alkyl-
6-aminouracils (5,6).14 Nitrosation at C5 of the pyrimidine ring
with sodium nitrite in acetic acid furnished compounds 7 and 8,
which were subsequently reduced with sodium dithionite to
give 1-alkyl-5,6-diaminouracils 9 and 10.15 The diamino com-
pounds were immediately refluxed with formic acid to give an
amide intermediate, followed by cyclization in basic media, to
afford the desired xanthines 11and 12. Bromination at C8 of the
xanthine core with Br2 and sodium acetate in acetic acid led to
the formation of the key 8-bromoxanthines 13 and 14 in
excellent overall yield.16
The synthesis of the noncommercially available R-halo
ketones is summarized in Scheme 2. First, commercially
available 1,3-benzodioxole-5-carboxaldehyde (piperonal, 15)
Figure 1. Previously known EphB4 inhibitors.6-9
Table 1. EphB4 Inhibition Data for Xanthine Derivatives
compd
R1
R2
R3
R4
R5
R6
R7
IC50 (nM)a
3
Me
Me
Bn
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4-hydroxybutyl
o-methoxyphenyl
o-methoxyphenyl
o-methoxyphenyl
o-methoxyphenyl
o-methoxyphenyl
4-hydroxybutyl
o-methoxyphenyl
butyl
7000 (5680)
3300 (4350)
>10000
4
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
45
46
69
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
H
H
Bn
H
F
H
>10000
5400
>20000
F
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
1,3-dioxol
1,3-dioxol
H
H
H
H
42% at 10 μM
30% at 10 μM
>10000
1,3-dioxol
OMe
H
H
H
H
H
H
H
OMe
H
H
H
butyl
butyl
36% at 10 μM
>10000
180 (47)
H
OMe
H
H
Me
H
H
H
o-methoxyphenyl
o-methoxyphenyl
butyl
Me
Me
H
H
H
>10000
H
H
H
37% at 10 μM
38% at 10 μM
64% at 10 μM
1600
H
Me
H
H
butyl
o-methoxyphenyl
butyl
OH
OH
H
H
H
H
H
H
H
H
OH
OH
H
o-methoxyphenyl
butyl
368 (213)
691
H
H
H
H
H
OH
OH
H
o-methoxyphenyl
butyl
59% at 10 μM
558
1200
H
H
H
Me
Me
Me
H
OH
H
H
o-methoxyphenyl
o-methoxyphenyl
o-methoxyphenyl
o-methoxyphenyl
OH
H
H
236
5 (1.6)
5000
H
OH
OH
H
H
a IC50 values were measured by a FRET based enzymatic assay while values in parentheses are IC50 values determined by an enzymatic assay with
radioactive ATP (see Experimental Section).

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6435
was reacted with methylmagnesium bromide to give secondary
alcohol 16, which was subsequently oxidized in the presence of
manganese oxide to give methyl ketone 17. R-Bromination at
the methyl position took place in the presence of phenyltri-
methylammonium tribromide to give alkylating agent 18 in
only three steps. 2-Bromo-20-methyl-30-hydroxyacetophenone
(24) and 2-bromo-20-methyl-40-hydroxyacetophenone (25)
were prepared by treatment of the corresponding carboxylic
acids with methyllithium to afford methyl ketones 21 and 23,
respectively (Scheme 2). Demethylation of 21 using AlCl3
yielded phenolic derivative 22. Finally, R-bromination at the
methyl group was achieved in the presence of copper(II)
bromide in chloroform to give monohalogenated ketones 24
and 25.
Alkylation at the N7 position of the xanthine core with
the appropriate R-haloketone was accomplished using N,N-
diisopropylethylamine in dimethylformamide, providing the
8-bromo-3-alkyl-7-(2-oxo-2-phenylethyl)xanthine derivatives
26-44 in good yields. Treatment of these intermediates with
primary alkylamines or aromatic amines in a sealed tube
at 180 °C in ethanol as solvent afforded the desired imidazo-
[1,2-f ]xanthine derivatives 45-68 (Scheme 3).
Scheme 1a
Two more derivatives were prepared as chemical probes
to address the binding mode of these molecules to EphB4
(Scheme 4). First, the benzyl group in 46 was removed in
the presence of Pd/C with ammonium formate to give 69.
Alternatively, the nitrile group in 68 was transformed into
the corresponding carboxylic acid (70) by hydrolysis with
sulfuric acid.
3. Results and Discussion
The inhibitory activity of the compounds prepared in
Schemes 3 and 4 was measured by a fluorescence resonance
energy transfer (FRET) based enzymatic assay that quantifies
inhibition of phosphorylation of a synthetic substrate of EphB4
at Km concentration of ATP (see Experimental Section).
Experimental Validation of the Binding Mode. The binding
mode of compound 3 obtained by automatic docking6
a Reagents and conditions: (a) (i) NCCH2CO2H, Ac2O, 60 °C, 1.5 h;
(ii) NaOH, 90 °C, 30 min; (b) NaNO2, AcOH-H2O, 25 °C, 12 h;
(c) Na2S2O4, NH4OH, 50 °C, 1 h, then 25 °C, 8 h; (d) (i) formic acid,
reflux, 3 h; (ii) NaOH, reflux, 1 h; (e) Br2, NaOAc, AcOH, 65 °C, 2 h.
Scheme 2a
Scheme 4a
a Reagents and conditions: (a) MeMgBr, THF, -10 °C, 1 h; (b)
MnO2, Et2O, 25 °C, 48 h; (c) phenyltrimethylammonium tribromide,
THF, 25 °C, 16 h; (d) MeLi, Et2O, 25 °C, 3.5 h; (e) AlCl3, PhCl, reflux,
6 h; (f) CuBr2, CHCl3, EtOAc, reflux, 15 h.
aReagents and conditions: (a) Pd/C, ammonium formate, MeOH, 140
°C, 3 h; (b) H2SO4, H2O, 120 °C, 2 h.
Scheme 3a
a Reagents and conditions: (a) R-halo ketone, DIPEA, DMF, 25 °C, 17 h; (b) primary amine, EtOH, sealed tube, reflux, 15 h.

6436 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Lafleur et al.
Figure 2. Binding modes of compound 3. Poses A (left) and B (right) were generated by automatic6 and manual docking, respectively. In the
schematic view (bottom), the side chain of Phe695 is not shown for clarity.
Table 2. EphB4 Inhibition Data for Compounds Synthesized To
substituents at the para position of the phenyl ring. Deriva-
Discriminate between Two Putative Binding Modes
tives with a nitro (47), a cyano (68), or a highly hydrophilic
carboxylic group (70) were prepared according to the meth-
od described in Schemes 3 and 4. These three derivatives were
inactive (Table 2). Furthermore, a hydroxyl substituent at
R4 reduced the affinity by a factor of about 2-3 (compare 4
and 62, Table 1). These results indicate that the phenyl ring of
compound 3 more likely fits into the hydrophobic pocket of
the ATP-binding site, as suggested by automatic docking
(binding mode A in Figure 2).
compd
R4
IC50 (nM)
Lead Optimization Strategy. Once sufficient evidence
about the binding mode had been gathered, we started our
optimization campaign from commercially available 4 (com-
pound 5 in ref 6). Compounds 3 and 4 differ only at R7 (alkyl
vs aromatic) and have similar IC50 values in the enzymatic
assay (Table 1), but only the latter showed activity in a cell-
based assay (Table 1 in ref 6). According to the binding
mode, the substituent at N3 of the pyrimidine ring could be
involved in additional interactions with Ala700 (Figure 2) so
that we decided to start the chemical edition of the molecule
at R1. The methyl group was replaced by a benzyl substitu-
ent, causing a major loss in the inhibitory activity of these
molecules (45 and 46 vs 4). Compound 69, with a N3-H
bond, was not more potent than 4. Since no improvement
was achieved by modification of the pyrimidine ring, we
decided to focus our efforts on a different region of the
molecule.
47
68
70
NO2
CN
>20000
>20000
>20000
CO2H
indicated that the carbonyl C6dO and amide group N1-H
are involved in hydrogen bonds with the backbone polar
groups of Met696 (Figure 2, left). In this model, the phenyl
ring of 3 is buried in the hydrophobic pocket, while the
4-hydroxybutyl lateral chain at R7 points toward the sol-
vent. Visual inspection of such binding mode suggested that
another pose could be obtained by a 180° rotation of the first
one (binding mode B, Figure 2, right) so that the phenyl ring
points toward the solvent. In pose B, the C6dO and N1-H of
compound 3 interact with the NH of Met696 and the back-
bone CO of Glu694, respectively. To discriminate between
these two binding modes, we decided to introduce polar

Article
The ATP-binding site of protein kinases can be divided
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6437
Table 3. Local Selectivity of Compound 66a
into five different subpockets.17 The size of the hydrophobic
pocket is controlled by a so-called gatekeeper residue, and it
is well-known that not only affinity but also selectivity can be
improved by fully exploring this site.17 In fact, only around
20% of the 518 human kinases have a small gatekeeper
residue. Since EphB4 belongs to this class having a threonine
gatekeeper (Thr693), we envisioned that a straightforward
strategy to improve affinity would stem from modifications
of the substitution pattern at the phenyl ring. In sharp
contrast to compound 2 and other previously developed
inhibitors of EphB4,8,10 a dioxole ring in relative positions
R3, R4 (48 and 49) or R2, R3 (50) of the aromatic ring signi-
ficantly reduced the activity. A methoxy group with different
substitution patterns (51, 52, 53) also suppressed the inhibi-
tory activity of the corresponding molecules. According to
binding mode A (Figure 2), the space around the phenyl ring
is rather limited, thus restricting the size of the substituents
that might improve steric complementarity. Furthermore,
the phenyl ring is close to the carbonyl group of Glu664, the
hydroxyl and carbonyl groups of Ser757, and the NH group
of Asp758, which can act as hydrogen bonding partners. On
the basis of these observations, we decided to examine less
sterically demanding substituents such as methyl or hydroxyl
groups, which are expected to fulfill the nearby hydrogen
bonding capacity.
Notably, a methyl substituent at position R2 of the
benzene ring (54) showed an IC50 value close to 100 nM. In
contrast, R3 and R4 methyl substituted derivatives (55-57)
were almost inactive. Furthermore, introduction of a hydro-
xyl group at R5 (60) was also beneficial with an IC50 of about
200-400 nM. Some inhibition activity was also detected for
compounds bearing the OH group at R2 (58) and R4 (62) of
the phenyl ring. In these cases, replacing the anisidine lateral
chain at R7 (58, 60, and 62) for an alkyl one such as butyl
(59, 61, and 63) seemed to have only a limited influence in the
inhibitory activity of these molecules as previously observed
for the commercially available compounds with a propyl or a
butyl chain at R7.6 With these results in hand, we decided to
explore how the combination of the most favored substitu-
tion patterns could influence the inhibitory activity. Thus,
the methyl substituent was kept at R2 and a hydroxyl group
was added at the relative positions R3 (64), R4 (65), and R5
(66) of the benzene ring. Strikingly, a combination of a
methyl and a hydroxyl group at R2 and R5, respectively,
yielded compound 66, which has a ∼1000-fold higher affinity
compared to the original hits obtained by docking, i.e.,
compounds 3 and 4.
kinases
IC50 (nM)
EphA1
EphA2
EphA3
EphA4
EphA5
EphA7
EphA8
EphB1
EphB2
EphB3
EphB4
2.9
2.3
40
3.3
3.0
1118
4.5
1.1
1.2
15
1.6
a These IC50 values were measured at Reaction Biology Corporation.
for 60, and 0.143 kcal/mol for 66), which suggests a quasi-
equal distribution of the population in each of the four
conformers. Notably, the presence of a methyl group at R2
seems to restrict the accessible conformations more than a
hydroxyl group at R5 (compare the plots obtained for 54 and
66 and for 4 and 60 in Figure S1). In addition, the conforma-
tional strain, which is the energy difference between the
minimized bound conformation and the lowest energy con-
formation of the isolated ligand, was evaluated for each
molecule. The similar values obtained for the strain energy of
54 (0.5 kcal/mol) and 4 (0 kcal/mol), as well as 66 (0.7 kcal/
mol) and 60 (0.3 kcal/mol), indicate that the methyl group
contributes to a gain in intermolecular van der Waals energy
rather than in strain energy (see also the subsection Binding
Mode of Compound 66 Investigated by MD Simulations).
During the preparation of this manuscript we discovered
in the literature a series of inhibitors of the tyrosine kinase
Lck with a 2,4-dianilinopyrimidine scaffold which have a
very similar SAR for the phenyl substituents to the one
observed for compounds 4, 54, 60, and 66.18 Note that the
phenyl substituent in both series of compounds is located in
the hydrophobic pocket, but it is connected to the 2-anilino-
pyrimidine core by a -NH- linker in the Lck inhibitors
whereas the phenyl ring is attached directly to the xanthine
scaffold in the EphB4 inhibitors described here.
Selectivity Profile. To assess the specificity of kinase
inhibitors, it is useful to distinguish between local and global
selectivity profiles, which reflect the inhibitory activity of the
tested compound on a single branch of the kinome dendro-
gram and on the whole kinome, respectively. The local
selectivity of compound 66 was tested against a panel of 11
Eph receptor kinases by an enzymatic assay with [γ-33P]ATP
(Reaction Biology Corporation). The IC50 values measured
for this compound against 10 of the 11 Ephs are in the low
nanomolar range (1-40 nM), while an IC50 value of 1.1 μM
is observed for EphA7 (Table 3). These IC50 values are
consistent with the very high sequence identity (60-90%)
of Ephs and the bulkier gatekeeper residue in EphA7
(isoleucine) with respect to the other Eph kinases
(threonine). To evaluate the global selectivity, enzymatic
assays (at single concentration of inhibitor) were performed
for compounds 66 and 54 using a panel of 85 kinases
(National Centre for Protein Kinase Profiling at the Uni-
versity of Dundee, Figure 3). Out of these 85 kinases, only
five (EphA2, EphB3, Src, Lck, and Yes1) and three (EphA2,
Lck, Yes1) are very strongly inhibited by compounds 66 and
54, respectively (less than 10% activity remaining compared
to a DMSO control at 1 μM of 66 and 3 μM of 54; see
Supporting Information). It is important to note that these
As mentioned above, the addition of only one heavy atom,
CH3 at R2, resulted in a factor of 20-100 improvement (54 vs
4, or 66 vs 60). This observation led us to further investigate
the role of the methyl group, in particular if it stabilizes the
orientation of the phenyl ring required for binding. Con-
formational analysis was performed on 4, 54, 60, and 66 by
exhaustive sampling of the dihedral angles involved in the
rotation of the phenyl and o-methoxyphenyl rings (γ1 and γ2
in Figure S1 of the Supporting Information), followed by
geometry optimization of the resulting structures using
quantum mechanics. For each of the four inhibitors, the
local minima are distributed into four sets of conformers,
which are separated by rotation barriers. The docked con-
formation lies in one of these four basins whose local minima
have similar energy values (maximal difference in energy of
0.082 kcal/mol for 4, 0.219 kcal/mol for 54, 0.257 kcal/mol

6438 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Lafleur et al.
Figure 4. Time series of the CR root-mean-square deviation (rmsd)
of residues 613-883 of the kinase domain of EphB4 with respect to
the X-ray structure (PDB code 2VWX). The loop 772-778 was not
taken into account because it is not present in the crystal structure.
dendrogram, in agreement with the 100-1000 lower affinity
with respect to the other 10 Eph kinases.
The profiles provide evidence for a good specificity of
compound 66, as it shows significant inhibition for a relati-
vely small fraction of the human kinome, which is compar-
able to, or even more specific than some type I kinase
inhibitors currently used as drugs, e.g., dasatinib21 and
sunitinib.9 The high selectivity may originate from fully
exploring the inner hydrophobic pocket whose size is con-
trolled by the gatekeeper. In addition, the rigidity of this
compound may contribute to its selectivity.
Figure 3. Selectivity profile of compound 66. The circles corres-
pond to all kinases tested. Inhibition of activity was measured in
enzymatic assays with radiolabeled ATP at Reaction Biology
Corporation and University of Dundee for 11 and 85 kinases,
respectively, while binding affinity of 50 kinases was measured at
Ambit Biosciences Corporation. Enzymatic assays: high, medium,
low, and no compound affinity for kinase activity (with respect to
DMSO control) of <10%, 10-30%, 30-60%, and >60%, respec-
tively. Binding assay: high, medium, low, and no compound affi-
nity for kinase activity (with respect to DMSO control) of <1%,
1-10%, 10-30%, and >30%, respectively. Kinome diagram is
reproduced courtesy of Cell Signaling Technology, Inc. (www.
cellsignal.com).44
Binding Mode of Compound 66 Investigated by MD Simu-
lations. Although the SAR of the 28 xanthine derivatives
(Tables 1 and 2) is consistent with the pose of compounds 3
and 4 predicted by automatic docking,6 explicit water mole-
cular dynamics (MD) simulations were carried out to further
validate the binding mode of compound 66, which is the most
potent of all derivatives. The MD run started from the pose
of 66 obtained by automatic flexible ligand docking (see
computational methods section). The structure of EphB4 is
stable in the 45 ns simulation, as indicated by the time
series of the CR rmsd (Figure 4). Importantly, the hydrogen
bonds with the hinge region (i.e., between the backbone
polar groups of Met696 and the pyrimidine ring of com-
pound 66) are conserved during the entire run except for
some transient ruptures of the one involving the carbonyl
group of Met696 (Figure 5A,B). Furthermore, the side chain
of Ser757 is involved in a rather stable hydrogen bond with
the methoxy oxygen of compound 66 (Figure 5C). On the
other hand, a change in the orientation of the hydroxyl group
of compound 66 was observed after about 12 ns. The two
hydrogen bonds with the side chain of Glu664 and the
backbone NH of Asp758 broke apart with concomitant
formation of a rather stable hydrogen bond with the back-
bone CO of Ser757 (Figure 5D). This change in the hydrogen
bonding pattern of the hydroxyl group occurred immediately
after the rotation of the peptide bond between Ser757 and
Asp758 (Figure 5) and persisted until the end of the MD run.
In other words, the reorientation of the Ser757 CO and
Asp758 NH groups promoted the new hydrogen bond
pattern.
five kinases have a threonine as gatekeeper, as well as CSK,
BTK, and HER-4, which show relatively strong inhibition by
66 (between 10% and 50% activity remaining compared to a
DMSO control).
The local and global selectivity profiles prompted us to test
the binding of compound 66 against a set of 49 kinases
(Ambit Biosciences Corporation) selected among the nearly
100 that are predicted to have a small gatekeeper based on
sequence and structure analysis.19 Interestingly, in competi-
tion binding assays, compound 66 shows significant affinity
(kinase activity <10% with respect to DMSO control) to
only 15 of these 49 kinases (see Supporting Information).
Considering all these selectivity tests (Figure 3), com-
pound 66 has been profiled against a total of 143 kinases
(three kinases, EphA2, EphB3, and EphB4, were tested
twice) about half of which were chosen because of their small
gatekeeper residue. Only 21 kinases with a threonine gate-
keeper showed high inhibition by 66, and 10 of them are Eph
kinases (Figure 3). Among the kinases with a gatekeeper
different from threonine, only GCK and MLK1 (both with
methionine as a gatekeeper) are inhibited by compound 66.
Interestingly, the selectivity of 66 is consistent with the
recently published kinase tree constructed by using SAR
data,20 as the kinases that bind compound 66 colocalize
in a small branch of the SAR-based dendrogram (Abl, Src,
and Eph families; see Supporting Information). Moreover,
EphA7 is located in another branch of the SAR-based
Overall, the 45 ns MD simulation of the complex of EphB4
with compound 66 shows that the major features of the
binding mode predicted by docking are stable. These include
the orientation of the phenyl in the hydrophobic pocket and
the two hydrogen bonds with the hinge region. It is interest-
ing to note that 66 forms by its hydroxyl substituent one or
two additional hydrogen bonds compared to compound 54,
which is consistent with its ∼30-35 higher affinity than
that of 54 (Table 1). Furthermore, the methyl group at

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6439
group and a hydroxyl group on the same benzene ring), and
these small but very effective substituents were suggested by
analysis of the binding mode obtained by automatic docking.
Furthermore, the predicted binding mode of compound 66
into the ATP-binding site of EphB4 is stable in a 45 ns MD
simulation with explicit solvent. Analysis of the MD trajec-
tory suggests that the higher potency of 66 with respect to 4 is
due to favorable van der Waals interactions of the methyl
group with the side chains of Val629, Ala645, and the gate-
keeper residue (Thr693), as well as a hydrogen bond between
the hydroxyl group and the backbone CO of Ser757. In
addition, quantum mechanics calculations indicate that the
beneficial effect of the methyl group is not due to a lower
conformational strain upon binding but rather to the afore-
mentioned van der Waals interactions. Although the SAR of
all the compounds synthesized in this work is consistent with
the binding mode of compound 66 obtained by docking and
slightly refined by explicitsolventMD, itsdefinitivevalidation
awaits for X-ray crystallography, which is being pursued
vigorously and will be reported in due course. Finally, com-
pound 66 shows a relatively good selectivity profile, which is
an important requirement for further consideration as a lead
compound.
5. Experimental Section
Docking. The flexible ligand docking employed here for
compounds 3 and 66 is a fragment-based approach, as it exploits
the optimal binding modes of mainly rigid fragments. The
approach consists of four consecutive steps which have been
detailed in a previous work.23 Briefly, the four steps are: (1)
decomposition of the ligand into mainly rigid fragments by the
program DAIM,24 (2) fragment docking with evaluation of
electrostatic solvation25,26 by the program SEED,27,28 (3) flex-
ible docking using the position and orientation of its fragments
as anchors by the program FFLD,29,30 and (4) final minimiza-
tion by CHARMM.31,32 The protein structure (PDB file
2VWX) was kept rigid in all steps.
Parametrization of Compound 66 and MD Simulations. Initial
Mulliken partial charges of 1,7,8-trimethyl-1H-imidazo[2,1-f ]-
purine-2,4(3H,8H)-dione were obtained by optimization with
Gaussian 03 at the HF/6-31G(d) level.33 Partial charges of
hydrogen atoms in the methyl groups were set to 0.09e, and
the excessive positive charge was added to the adjacent carbon
atom. To determine the partial charges on the remaining atoms,
interaction energies between each polar group and a TIP3
water molecule34 were evaluated as the difference between the
supermolecule energy and the sum of the individual monomer
energies. The partial charges on the polar groups were then
manually adjusted to fit the interaction energies calculated by
CHARMM to the ab initio values. Bond lengths and angle
values were also modified to reproduce the quantum mechani-
cally obtained geometries. Normal mode contributions gener-
ated with the MOLVIB program were adjusted to scaled HF/
6-31G(d) values. Final steps included introduction of the pre-
viously parametrized phenol and methyl moieties,33 followed by
optimization of the remaining missing parameters.
Figure 5. Hydrogen bonds between compound 66 and the ATP-
binding site of EphB4: (A) schematic illustration; (B-D) time series
of hydrogen-bond distances, i.e., the distance between the donor and
acceptor atoms. The colors are consistent with those used in part A.
R2 is involved in favorable van der Waals interactions with
the side chains of Val629, Ala645, and Thr693 throughout
the simulation (see Supporting Information). These van der
Waals interactions contribute about -1.5 kcal/mol to the
binding energy. Compounds 66 and 60 differ only by this
methyl group so that the former has a more favorable
desolvation energy upon binding, which, taken together with
the additional van der Waals interactions with EphB4,
explains the 100 times higher potency of compound 66 vs
60 (Table 1).
4. Conclusions
The coordinates of EphB4 were downloaded from the PDB
database (2VWX). As the X-ray structure contains only residues
608-888, the -COCH3 group and -NHCH3 group were added
to the N-terminal Lys608 and C-terminal Ala888, respectively.
To reproduce neutral pH conditions, the side chains of aspar-
tates and glutamates were negatively charged, those of lysines
and arginines were positively charged, and histidines were
considered neutral. The protein was immersed in an orthorhom-
bic box of pre-equilibrated water molecules. The size of the box
We had previously discovered a series of micromolar in-
hibitors of the tyrosine kinase EphB4 by fragment-based
high-throughput docking of a library of about 700 000 com-
pounds.6 Here, we describe the structure-guided improvement
of these relatively weak inhibitors by chemical synthesis which
has culminated into the discovery of a single-digit nanomolar
inhibitor 66, with a remarkable increase in ligand efficiency22
from 0.26 to 0.37 kcal/mol per non-hydrogen atom (for
inhibitors 4 and 66, respectively). Notably, compound 66
has only two non-hydrogen atoms more than 4 (a methyl
˚
was chosen to have a minimal distance of 13 A between the
boundary and any atom of the protein. The program VMD35

6440 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Lafleur et al.
was used for setting up the simulation system, while minimization,
heating, and production runs were performed with NAMD36 using
the CHARMM22 force field33 and the TIP3P model of water.
Periodic boundary conditions were applied, and the particle-mesh
Ewald approach37 was used for the long-range electrostatics. The
grade, Scharlau, E-Barcelona, Spain) as internal standard.
The purity of all tested compounds was determined by HPLC
on a Finnigan Voyager GC8000 Top spectrometer using an
Interchim Strategy (2.2 μm, 2.1 mm ꢀ 100 mm) column by
running a 5-80% gradient for water (þ0.1% HCOOH þ 0.01%
TFA)/CH3CN. Flow rate was 200 μL/min, and UV detection
was set to 254 nm. Unless otherwise stated, all the compounds
showed g95% purity.
˚
van der Waals interactions were truncated at a cutoff of 12 A, and a
˚
switch function is applied starting at 10 A. The MD simulation
was performed at constant temperature of 310 K by applying
the Langevin thermostat and at constant pressure of 1 atm using
the Nose-Hoover Langevin piston pressure control.38,39 The time
step was 2 fs, and the SHAKE algorithm40 was used to fix the
length of covalent bonds involving hydrogen atoms. Coordinate
sets were saved every 2 ps.
1-(Benzo[d ][1,3]dioxol-4-yl)ethanol (16).43
A solution of
MeMgBr in THF (1 M, 9.9 mL, 9.9 mmol, 1.5 equiv) was
diluted with THF (10 mL) and cooled to -10 °C. A solution
of benzo[d ][1,3]dioxole-4-carbaldehyde 15 (1.0 g, 6.6 mmol) in
9.9 mL of THF was slowly added, and the mixture was stirred
for 1 h. The reaction mixture was then quenched by pouring
it into 100 mL of ice cold saturated ammonium chloride, and
the aqueous layer was then extracted with Et2O. The organic
extracts were dried over MgSO4 and evaporated under reduced
pressure to provide the desired compound as a colorless oil (971
mg, 88% yield). 1H NMR (400 MHz, CDCl3): δ = 6.87 (ddd,
J = 7.8, 1.4, 0.4 Hz, 1H), 6.82 (dd, J = 7.8, 7.5 Hz, 1H), 6.75
(dd, J = 7.5, 1.4 Hz, 1H), 5.96 (d, J = 1.4 Hz, 1H), 5.95 (d, J =
1.4 Hz, 1H), 4.99 (q, J = 6.5 Hz, 1H), 1.52 (d, J = 6.5 Hz, 3H),
OH not observed. 13C NMR (100 MHz, CDCl3): δ = 147.3,
143.9, 127.3, 121.7, 118.7, 107.7, 100.8, 66.2, 23.3. IR (film):
ν~=3359, 2972, 2887, 1455, 1245, 1039, 727 cm-1. MS (ESI),
m/z: calcd for C9H10NaO3, 189.1; found, 189.0 [M þ Na]þ.
1-(Benzo[d ][1,3]dioxol-4-yl)ethanone (17).43 A mixture of
1-(benzo[d][1,3]dioxol-4-yl)ethanol 16 (971 mg, 5.84 mmol)
and MnO2 (5.08 g, 58.4 mmol) in Et2O (36 mL) was stirred
vigorously for 48 h. The reaction mixture was then filtered
through a path of Celite and evaporated under reduced pressure
to give the desired compound as a white solid (679 mg, 70%
Evaluation of Strain Energy by Quantum Mechanics. System-
atic rotation of rotatable bonds in increments of 10° and 20° for
the two phenyls and the methoxy, respectively, was performed
using CHARMM, and the resulting conformations were mini-
mized with Gaussian 03 using the AM1 Hamiltonian. Removal
of duplicate conformations was done using the Ultrafast Shape
Recognition protocol41 supplemented by a chirality descriptor
that distinguishes molecules that are mirror images of each
other. Geometry optimization of the resulting structures was
done at the B3LYP level using the PC GAMESS version of the
GAMESS quantum chemical package.42 Redundant conforma-
tions were discarded by a second Shape Recognition analysis.
The strain energy was calculated as the energy difference
between the minimized bound conformation and the global
minimum obtained by the systematic search.
Chemistry. All reactions, unless otherwise stated, were carried
out under a nitrogen atmosphere using standard Schlenk tech-
niques. All reagents were used as received unless otherwise
noted. Solvents were purchased in the best quality available,
degassed by purging thoroughly with nitrogen, and dried over
activated molecular sieves of appropriate size. Alternatively,
they were purged with argon and passed through alumina
columns in a solvent purification system (Innovative Techno-
logy). Reactions were monitored by thin layer chromatography
(TLC) using Merck TLC silica gel 60 F254. Flash column
chromatography was performed over silica gel (230-400 mesh).
NMR spectra were recorded on a AV2 400 or AV2 500 MHz
Bruker spectrometer. Chemical shifts are given in ppm. The spectra
1
yield). Mp 95-97 °C. H NMR (400 MHz, CDCl3): δ=7.37
(dd, J=8.2, 1.3 Hz, 1H), 6.97 (dd, J=7.6, 1.3 Hz, 1H), 6.87 (dd,
J=8.2, 7.6 Hz, 1H), 6.09 (s, 2H), 2.60 (s, 3H). 13C NMR (100
MHz, CDCl3): δ=195.4, 148.6, 147.9, 121.4, 121.2, 120.3, 112.5,
101.5, 30.2. IR (film): ν~=3068, 2908, 1671, 1625, 1450, 1361,
1283, 1240, 1179, 1060, 1022, 950, 727 cm-1. MS (EI), m/z: calcd
for C9H8O3, 164.1; found, 163.9 [M]þ.
1-(Benzo[d ][1,3]dioxol-4-yl)-2-bromoethanone (18).43 To a solu-
tion of 1-(benzo[d][1,3]dioxol-4-yl)ethanone 17 (679 mg, 4.14
mmol) in anhydrous THF (19 mL) at 0 °C was added phenyl-
trimethylammonium tribromide (1.55 g, 4.14 mmol). The reaction
mixture was stirred at room temperature for 16 h, concentrated,
and redissolved in EtOAc. The organic layer was washed with
water (2 ꢀ 50 mL) and brine (1 ꢀ 50 mL), dried over MgSO4,
filtered, and evaporated under reduced pressure. Purification by
column chromatography on silica gel (toluene/hexane, 1:4), fol-
lowed by recrystallization in hexane/EtOAc afforded the desired
compound as a white solid (309 mg, 30% yield). Mp 75-77 °C.
1H NMR (500 MHz, CDCl3): δ=7.42 (dd, J=8.2, 1.2 Hz, 1H),
7.01 (dd, J=7.6, 1.2 Hz, 1H), 6.91 (dd, J=8.2, 7.6 Hz, 1H), 6.11
(s, 2H), 4.48 (s, 2H). 13C NMR (125 MHz, CDCl3): δ=188.2,
148.5, 147.8, 121.9, 121.7, 117.1, 113.3, 101.8, 34.9. IR (film): ν~=
3002, 2942, 2917, 1693, 1624, 1451, 1387, 1248, 1204, 1177, 1048,
942, 768, 723 cm-1. MS (ESI), m/z: calcd for C9H7BrNaO3, 264.9;
found, 265.0 [M þ Na]þ.
1
are calibrated to the residual H and 13C signals of the solvents.
Multiplicities are abbreviated as follows: singlet (s), doublet (d),
triplet (t), quartet (q), doublet-doublet (dd), quintet (quint), septet
(sept), multiplet (m), and broad (br). Melting points were deter-
mined on a Buchi melting point B-540 instrument. Methylurea,
benzylurea, benzo[d][1,3]dioxole-4-carbaldehyde (piperonal 15),
2-methyl-3-methoxybenzoic acid (19), and 2-methyl-5-hydro-
xybenzoic acid (20) were purchased from Fluka. The following
compounds 2-bromo-1-phenylethanone, 2-bromo-1-(4-fluoro-
phenyl)ethanone, 2-bromo-1-(4-methoxyphenyl)ethanone,
2-bromo-1-(3-methoxyphenyl)ethanone, 2-bromo-1-(2-methoxy-
phenyl)ethanone, 2-chloro-1-(4-hydroxyphenyl)ethanone, 2-bromo-
1-(3-hydroxy.phenyl)ethanone, 2-bromo-1-(2-hydroxyphenyl)-
ethanone, 2-chloro-1-(3,4-dihydroxyphenyl)ethanone, 2-bromo-
1-(4-nitrophenyl)ethanone, 2-bromo-40-cyanoacetophenone,
2-chloro-1-(4-hydroxy-2-methylphenyl)ethanone, and 2-bromo-
1-(4-methylphenyl)-1-ethanone were purchased from Fluka.
2-Bromo-1-(3-methylphenyl)-1-ethanone and 2-bromo-1-(2-methyl-
phenyl)-1-ethanone were purchased from Synchem, and 1-(1,3-
benzodioxol-5-yl)-2-bromo-1-ethanone was purchased from Acros
Organics.
1-(3-Methoxy-2-methylphenyl)ethanone (21). To a solution of
2-methyl-3-methoxybenzoic acid 19 (1.5 g, 9.02 mmol) in dry
diethyl ether (68 mL) at 0 °C, methyllithium (1.6 M in Et2O,
22 mL, 36.1 mmol) was added dropwise with vigorous stirring.
The mixture was allowed to warm to room temperature and
stirred for an additional 3.5 h. The reaction was quenched by
pouring the mixture into ice cold 10% HCl (50 mL), and the
aqueous layer was then extracted with Et2O (3 ꢀ 50 mL). The
organic solution was washed with brine, dried over MgSO4,
filtered, and concentrated under reduced pressure. Purification
by column chromatography on silica gel (hexane/EtOAc, 16:1)
afforded the desired compound as a colorless oil (613 mg, 41%).
1H NMR (500 MHz, CDCl3): δ=7.22 (dd, J=8.1, 7.6 Hz, 1H),
High-resolution electrospray ionization mass spectrometry
was performed on a Finnigan MAT 900 (Thermo Finnigan, San
Jose, CA) double-focusing magnetic sector mass spectrometer.
Ten spectra were acquired. A mass accuracy of e2 ppm was
obtained in the peak matching acquisition mode by using
a solution containing 2 μL of PEG200, 2 μL of PPG450, and
1.5 mg of NaOAc (all obtained from Sigma-Aldrich, Buchs,
Switzerland) dissolved in 100 mL of MeOH (HPLC Supra

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6441
7.15 (dd, J=7.6, 1.1 Hz, 1H), 6.95 (d, J=8.1 Hz, 1H), 3.85
(s, 3H), 2.55 (s, 3H), 2.32 (s, 3H). 13C NMR (125 MHz, CDCl3):
δ=203.1, 158.2, 140.6, 126.1, 125.9, 120.0, 112.7, 55.8, 30.4,
12.6. IR (film): ν~=3001, 2960, 2938, 2836, 1684, 1577, 1456,
1258, 1051, 781 cm-1. MS (ESI), m/z: calcd for C10H12NaO2,
187.1; found, 187.0 [M þ Na]þ.
stirred for 5 min at room temperature, R-bromoacetophenone
(1 equiv) was slowly added. The mixture was stirred at room
temperature for 1-3 days. The mixture was then poured into
EtOAc/HCl (10%), and the formed precipitate was filtered and
washed with water to afford 26-42 in pure form.
3-Benzyl-8-bromo-3,7-dihydro-7-(2-oxo-2-phenylethyl)-1H-purine-
2,6-dione (26). White solid. Yield: 76%. Mp 249-252 °C. 1H NMR
(500 MHz, DMSO-d6): δ=11.43 (s, 1H), 8.10 (d, J=7.4 Hz, 2H),
7.77 (t, J=7.4 Hz, 1H), 7.63 (t, J=7.4 Hz, 2H), 7.27-7.38 (m, 5H),
5.94 (s, 2H), 5.11 (s, 2H). 13C NMR (125 MHz, DMSO-d6): δ=
191.2, 153.9, 150.2, 148.5, 136.5, 134.5, 133.5, 129.2, 129.0, 128.4,
128.2, 127.3, 127.3, 109.0, 52.9, 44.9. IR (film): ν~=3190, 3059, 2978,
1-(3-Hydroxy-2-methylphenyl)ethanone (22). A solution of
1-(3-methoxy-2-methylphenyl)ethanone 21 (450 mg, 2.74 mmol)
in chlorobenzene (11 mL) was treated with AlCl3 (475 mg, 3.56
mmol) at room temperature and then refluxed for 5 h. A second
portion of AlCl3 (475 mg, 3.56 mmol) was added, and the resulting
mixture was refluxed for an additional hour until no starting
material was detected by TLC. The reaction mixture was carefully
treated with 1 M HCl at 0 °C. The aqueous phase was extracted
with EtOAc, and the combined organic layers were dried with
MgSO4. Evaporation of the solvent and column chromatography
on silica gel (hexane/EtOAc, 6:1) afforded the desired compound
as a yellow solid (328 mg, 79% yield). Mp 122-124 °C. 1H NMR
(400 MHz, CDCl3): δ=7.18 (dd, J=7.8, 1.2 Hz, 1H), 7.12 (t, J=
7.8 Hz, 1H), 6.93 (dd, J=7.8, 1.2 Hz, 1H), 5.34 (s, 1H), 2.57 (s, 3H),
2.36 (s, 3H). 13C NMR (100 MHz, CDCl3): δ=203.4, 154.7, 140.4,
126.2, 123.3, 120.9, 118.0, 30.1, 12.4. IR (film): ν~=3221, 1652,
1577, 1466, 1360, 1277, 782 cm-1. MS (ESI), m/z: calcd for
C9H10NaO2, 173.1; found, 173.0 [M þ Na]þ.
2936, 1707, 1697, 1669, 1592, 1536, 1446, 1358, 1260, 742 cm-1
.
HRMS (ESI), m/z: calcd for C20H16BrN4O3, 439.0; found, 439.1
[M þ H]þ.
3-Benzyl-8-bromo-7-[2-(4-fluorophenyl)-2-oxoethyl]-3,7-dihydro-
1H-purine-2,6-dione (27). White solid. Yield: 62%. Mp 258-
261 °C. 1H NMR (500 MHz, DMSO-d6): δ = 11.43 (s, 1H),
8.18-8.22 (m, 2H), 7.45-7.49 (m, 2H), 7.27-7.38 (m, 5H), 5.93
(s, 2H), 5.11 (s, 2H). 13C NMR (125 MHz, DMSO-d6): δ=189.9,
153.9, 150.2, 148.6, 136.5, 131.4 (d, J=9.8 Hz), 130.3 (d, J=2.7 Hz),
129.2, 128.4, 127.5, 127.3, 127.3, 116.1 (d, J=22.1 Hz), 109.0, 52.8,
44.9. IR (film): ν~=3184, 3068, 2972, 2934, 1697, 1665, 1592, 1536,
1359, 1234, 834, 742 cm-1. HRMS (ESI), m/z: calcd for
C20H14BrFN4NaO3, 479.0131; found, 479.0137 [M þ Na]þ.
8-Bromo-3,7-dihydro-3-methyl-7-[2-(4-nitrophenyl)-2-oxoethyl]-
1H-purine-2,6-dione (28). Yellow solid. Yield: 63%. Mp 290-
292 °C. 1H NMR (500 MHz, DMSO-d6): δ=11.32 (s, 1H), 8.42
(d, J=8.9 Hz, 2H), 8.34 (d, J=8.9 Hz, 2H), 6.01 (s, 2H), 3.37
(s, 3H). 13C NMR (125 MHz, DMSO-d6): δ=190.9, 153.9, 150.6,
150.4, 149.0, 138.2, 129.8, 129.0, 124.0, 108.8, 53.2, 28.5. IR (film):
ν~=3158, 3037, 2939, 2831, 1674, 1523, 1369, 1346, 1220, 1203,
852 cm-1. HRMS (ESI), m/z: calcd for C14H11BrN5O5, 407.9;
found, 408.1 [M þ H]þ.
1-(5-Hydroxy-2-methylphenyl)ethanone (23). To a solution
of 2-methyl-5-hydroxybenzoic acid 20 (900 mg, 5.9 mmol) in
THF (6.5 mL) at -78 °C was added MeLi (1.6 M in ether,
11 mL, 17.7 mmol). The mixture was stirred for 17 h while slowly
warming up to room temperature. EtOAc (10 mL) was slowly
added to the reaction mixture, which was subsequently washed
(10 mL of 1 M HCl, 10 mL of water, and 10 mL of brine). After
drying of the organic layer over MgSO4 and evaporation of the
solvent under reduced pressure, the residue was purified by flash
chromatography on silica gel (EtOAc/hexane, 1:10) to afford
desired compound as a white solid (608 mg, 68% yield). Mp
7-[2-(Benzo[d][1,3]dioxol-5-yl)-2-oxoethyl]-8-bromo-3,7-dihydro-
3-methyl-1H-purine-2,6(3H,7H)-dione (29). White solid. Yield:
1
129-131 °C. H NMR (400 MHz, CDCl3): δ = 7.17 (d, J =
1
2.6 Hz, 1H), 7.10 (d, J=8.2 Hz, 1H), 6.88 (dd, J=8.2, 2.6 Hz,
1H), 5.32-5.37 (m, 1H), 2.55 (s, 3H), 2.43 (s, 3H). 13C NMR
(100 MHz, CDCl3): δ=201.9, 153.4, 138.5, 133.1, 130.2, 118.7,
116.1, 29.5, 20.6. IR (film): ν~=3188, 2929, 1657, 1603, 1456,
1434, 1302, 1221, 734 cm-1. MS (ESI), m/z: calcd for
C9H10NaO2, 173.1; found, 173.0 [M þ Na]þ.
61%. Mp 297-299 °C. H NMR (500 MHz, DMSO-d6): δ =
11.23 (s, 1H), 7.76 (dd, J=8.2, 1.5 Hz, 1H), 7.56 (d, J=1.5 Hz, 1H),
7.13 (d, J=8.2 Hz, 1H), 6.19 (s, 2H), 5.83 (s, 2H), 3.37 (s, 3H). 13C
NMR (100 MHz, DMSO-d6): δ=189.0, 153.9, 152.5, 150.4, 148.9,
148.1, 129.1, 127.9, 125.0, 108.9, 108.4, 107.3, 102.3, 52.6, 28.5. IR
(film): ν~=3155, 3039, 2942, 2811, 1674, 1447, 1357, 1258, 1035,
930, 869, 817 cm-1. HRMS (ESI), m/z: calcd for C15H11BrN4-
NaO5, 428.9811; found, 428.9808 [M þ Na]þ.
General Procedure for the r-Bromination of Acetophenones. A
solution of acetophenone (1 equiv) in CHCl3 (0.18 M) was
added to a refluxing solution of copper(II) bromide (1.99 equiv)
in EtOAc (0.45 M). The mixture was then refluxed for 10 h. The
solution was filtered through Celite and concentrated under
reduced pressure to afford a green solid. The solid was purified
by flash chromatography on silica gel (toluene) to afford the
corresponding products in pure form. This method was used to
obtain 24 and 25.
7-[2-(Benzo[d][1,3]dioxol-4-yl)-2-oxoethyl]-8-bromo-3,7-dihydro-
3-methyl-1H-purine-2,6-dione (30). White solid. Yield: 67%. Mp
306-308 °C. 1H NMR (500 MHz, DMSO-d6): δ=11.31 (s, 1H),
7.35 (d, J=7.9 Hz, 1H), 7.27 (d, J=7.9 Hz, 1H), 7.02 (t, J=7.9 Hz,
1H), 6.28 (s, 2H), 5.69 (s, 2H), 3.36 (s, 3H). 13C NMR (125 MHz,
DMSO-d6):δ=188.1, 153.9, 150.4, 148.9, 148.7, 148.4, 129.0, 122.0,
119.9, 115.9, 113.6, 108.9, 102.4, 55.0, 28.5. IR (film): ν~=3170, 3057,
2831, 1680, 1454, 1352, 1235, 1064, 946,745 cm-1. HRMS (ESI), m/
z: calcd for C15H11BrN4NaO5, 428.9811; found, 428.9808 [M þ
Na]þ.
2-Bromo-1-(3-hydroxy-2-methylphenyl)ethanone (24). White
solid. Yield: 61%. 1H NMR (400 MHz, CDCl3): δ = 7.15-
7.17 (m, 2H), 6.95-6.98 (m, 1H), 5.14 (s, 1H), 4.39 (s, 2H), 2.34
(s, 3H). 13C NMR (100 MHz, CDCl3): δ=195.1, 154.7, 137.2,
126.5, 124.4, 120.6, 118.8, 34.2, 12.4. IR (film): ν~=3420, 2933,
1683, 1581, 1466, 1327, 796 cm-1. MS (ESI), m/z: calcd for
C9H9BrNaO2, 250.9; found, 251.0 [M þ Na]þ.
8-Bromo-3,7-dihydro-7-[2-(2-methoxyphenyl)-2-oxoethyl]-
3-methyl-1H-purine-2,6-dione (31). White solid. Yield: 75%. Mp
294-296 °C. 1H NMR (500 MHz, DMSO-d6): δ=11.29 (s, 1H),
7.78 (d, J=7.4 Hz, 1H), 7.69 (t, J=7.4 Hz, 1H), 7.30 (d, J=
7.4 Hz, 1H), 7.12 (t, J=7.4 Hz, 1H), 5.68 (s, 2H), 4.02 (s, 3H),
3.36 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ=191.1, 159.6,
153.9, 150.4, 148.9, 135.9, 130.2, 129.0, 123.2, 120.8, 112.8,
108.8, 56.4, 56.1, 28.5. IR (film): ν~=3170, 2987, 2900, 1673,
1462, 1357, 1204, 758 cm-1. HRMS (ESI), m/z: calcd for
C15H13BrN4NaO4: 415.0018; found, 415.0019 [M þ Na]þ.
8-Bromo-3,7-dihydro-7-[2-(3-methoxyphenyl)-2-oxoethyl]-
3-methyl-1H-purine-2,6-dione (32). White solid. Yield: 75%.
2-Bromo-1-(5-hydroxy-2-methylphenyl)ethanone (25). White
solid. Yield: 50%. Mp 63-65 °C. 1H NMR (400 MHz, CDCl3):
δ = 7.14-7.16 (m, 2H), 6.91 (dd, J = 8.2, 2.7 Hz, 1H), 4.92
(s, 1H), 4.37 (s, 2H), 2.43 (s, 3H). 13C NMR (100 MHz, CDCl3):
δ=194.1, 153.3, 135.4, 133.4, 131.5 119.4, 115.7, 33.5, 20.3. IR
(film): ν~=3232, 2962, 2924, 1662, 1562, 1492, 1294, 1198, 824,
632 cm-1. MS (GC/MS), m/z: calcd for C9H10BrO2, 230.1;
found, 230.2 [M þ H]þ.
1
General Method for the Preparation of Alkylated Xanthines
26-40, 42, 44. 3-Alkyl-8-bromoxanthine (0.55 mmol) was dis-
solved in anhydrous DMF (4 mL), and distilled N,N-diisopro-
pylethylamine (1.5 equiv) was added. After the mixture was
Mp 263-265 °C. H NMR (400 MHz, DMSO-d6): δ=11.30
(s, 1H), 7.71 (ddd, J=7.7, 1.5, 0.9 Hz, 1H), 7.57 (dd, J=2.6,
1.5 Hz, 1H), 7.54 (dd, J=8.1, 7.7 Hz, 1H), 7.33 (ddd, J=8.1, 2.6,
0.9 Hz, 1H), 5.92 (s, 2H), 3.86 (s, 3H), 3.37 (s, 3H). 13C NMR

6442 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Lafleur et al.
(100 MHz, DMSO-d6): δ = 191.0, 159.5, 153.9, 150.4, 148.9,
134.8, 130.2, 129.1, 120.7, 120.6, 112.5, 108.9, 55.4, 53.0, 28.5.
IR (film): ν~ = 3145, 3027, 2807, 1679, 1535, 1365, 1261, 748
cm-1. HRMS (ESI), m/z: calcd for C15H13BrN4NaO4, 415.0018;
found, 415.0018 [M þ Na]þ.
108.9, 52.4, 28.5. IR (film): ν~=3154, 2818, 1680, 1577, 1360,
1208, 1166, 759 cm-1
. HRMS (ESI), m/z: calcd for
C14H11BrN4NaO4, 400.9861; found, 400.9858 [M þ Na]þ.
8-Bromo-3,7-dihydro-3-methyl-7-[2-(2-methyl-3-hydroxyphenyl)-
2-oxoethyl]-1H-purine-2,6-dione (40). White solid. Yield: 72%.
1
Mp 277-279 °C. H NMR (400 MHz, DMSO-d6): δ=11.32
8-Bromo-3,7-dihydro-7-[2-(4-methoxyphenyl)-2-oxoethyl]-
3-methyl-1H-purine-2,6-dione (33). White solid. Yield: 73%.
(s, 1H), 9.80 (s, 1H), 7.37 (dd, J=7.8, 0.8 Hz, 1H), 7.22 (t, J=
7.8 Hz, 1H), 7.07 (dd, J=7.8, 0.8 Hz, 1H), 5.70 (s, 2H), 3.36 (s, 3H),
2.18 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ=195.1, 156.1,
153.9, 150.4, 148.9, 136.0, 129.0, 126.4, 123.6, 118.9, 118.6, 108.8,
54.7, 28.5, 12.2. IR (film): ν~=3411, 3145, 3023, 2823, 1663, 1535,
1366, 1274, 1205, 785 cm-1. HRMS(ESI), m/z: calcd C15H13BrN4-
NaO4, 415.0018; found, 415.0011 [M þ Na]þ.
1
Mp 280-282 °C. H NMR (500 MHz, DMSO-d6): δ=11.29
(s, 1H), 8.08 (d, J=8.9 Hz, 2H), 7.13 (d, J=8.9 Hz, 2H), 5.86
(s, 2H), 3.88 (s, 3H), 3.37 (s, 3H). 13C NMR (125 MHz, DMSO-
d6): δ= 189.2, 164.1, 153.9, 150.4, 148.9, 130.6, 129.1, 126.4,
114.3, 108.9, 55.6, 52.5, 28.5. IR (film): ν~=3153, 3034, 2941,
2838, 1680, 1600, 1364, 1173, 834 cm-1. MS (ESI), m/z: calcd for
C15H14BrN4O4, 393.2; found, 393.1 [M þ H]þ.
8-Bromo-3,7-dihydro-3-methyl-7-[2-(2-methyl-5-hydroxyphenyl)-
2-oxoethyl]-1H-purine-2,6-dione (42). White solid. Yield: 35%.
8-Bromo-3,7-dihydro-3-methyl-7-[2-(2-methylphenyl)-2-oxo-
ethyl]-1H-purine-2,6-dione (34). White solid. Yield: 58%. Mp
242-244 °C. 1H NMR (500 MHz, DMSO-d6): δ=11.32 (s, 1H),
8.04 (dd, J=7.6, 1.1 Hz, 1H), 7.56 (dt, J=7.6, 1.1 Hz, 1H), 7.44
(t, J=7.6 Hz, 1H), 7.40 (d, J=7.6 Hz, 1H), 5.79 (s, 2H), 3.37
(s, 3H), 2.42 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ=194.1,
153.9, 150.4, 148.9, 138.3, 133.7, 132.7, 131.9, 129.1, 129.0,
126.1, 108.8, 54.3, 28.5, 20.6. IR (film): ν~=3154, 3025, 2814,
1676, 1535, 1364, 1208, 768 cm-1. HRMS (ESI), m/z: calcd for
C15H13BrN4NaO3, 399.0069; found, 399.0068 [M þ Na]þ.
8-Bromo-3,7-dihydro-3-methyl-7-[2-(3-methylphenyl)-2-oxo-
ethyl]-1H-purine-2,6-dione (35). White solid. Yield: 75%. Mp
280-282 °C. 1H NMR (500 MHz, DMSO-d6): δ=11.30 (s, 1H),
7.93 (s, 1H), 7.90 (d, J=7.6 Hz, 1H), 7.58 (d, J=7.6 Hz, 1H), 7.51
(t, J=7.6 Hz, 1H), 5.91 (s, 2H), 3.37 (s, 3H), 2.42 (s, 3H). 13C
NMR (125 MHz, DMSO-d6): δ = 191.2, 153.9, 150.4, 148.9,
138.6, 135.1, 133.5, 129.1, 128.9, 128.5, 125.3, 108.9, 52.9, 28.5,
20.7. IR (film): ν~=3149, 3024, 2813, 1678, 1536, 1360, 1164,
715 cm-1. HRMS (ESI), m/z: calcd for C15H13BrN4NaO3,
399.0069; found, 399.0070 [M þ Na]þ.
1
Mp 271-274 °C. H NMR (400 MHz, DMSO-d6): δ=11.32
(s, 1H), 9.73 (s, 1H), 7.34 (d, J=2.6 Hz, 1H), 7.17 (d, J=8.3 Hz,
1H), 6.96 (dd, J=2.6, 8.3 Hz, 1H), 5.71 (s, 2H), 3.37 (s, 3H), 2.29
(s, 3H). 13C NMR (100 MHz, DMSO-d6): δ = 194.1, 155.3,
153.9, 150.4, 148.9, 134.5, 132.9, 129.1, 127.9, 119.6, 115.4,
108.8, 54.3, 28.5, 19.6. IR (film): ν~=3370, 3145, 3025, 2829,
1677, 1535, 1365, 1293, 1183, 749 cm-1. HRMS (ESI), m/z: calcd
for C15H13BrN4NaO4, 415.0018; found, 415.0007 [M þ Na]þ.
8-Bromo-7-[2-(4-cyanophenyl)-2-oxoethyl]-3,7-dihydro-3-methyl-
1H-purine-2,6-dione (44). White solid. Yield: 79%. Mp 281-
284 °C. 1H NMR (500 MHz, DMSO-d6): δ=11.31 (s, 1H), 8.25
(d, J=8.5 Hz, 2H), 8.11 (d, J=8.5 Hz, 2H), 5.98 (s, 2H), 3.36
(s, 3H). 13C NMR (125 MHz, DMSO-d6): δ = 191.1, 153.9,
150.4, 149.0, 136.7, 132.9, 129.0, 128.9, 117.9, 116.3, 108.8, 53.1,
28.5. IR (film): ν~=3162, 3043, 2963, 2932, 1705, 1671, 1540,
1369, 1204, 834 cm-1. HRMS (ESI), m/z: calcd for C15H11-
BrN5O3, 388.0; found, 388.0 [M þ H]þ.
8-Bromo-3,7-dihydro-3-methyl-7-[2-(2-methyl-4-hydroxyphenyl)-
2-oxoethyl]-1H-purine-2,6-dione (41). 8-Bromo-3,9-dihydro-
3-methyl-1H-purine-2,6-dione (13, 400 mg, 1.41 mmol) was
added to a solution of KOH (79 mg, 1.41 mmol) in EtOH
(3 mL). The resulting mixture was then heated to reflux for 2 h.
EtOH was then removed under reduced pressure, and the
resulting solid was washed with cold EtOH and filtered off
to afford a light-yellow solid. A mixture of this solid (250 mg,
0.88 mmol) and R-bromo-4-hydroxy-2-methylacetophenone
(202 mg, 0.88 mmol) in anhydrous DMF (2.5 mL) was stirred
at room temperature for 16 h. The mixture was then poured into
EtOAc/HCl (10%), and the formed precipitate was filtrated off
and washed with water to afford the desired product as a white
solid (177 mg, 51% yield). Mp 313-315 °C. 1H NMR (400
MHz, DMSO-d6): δ=11.28 (s, 1H), 10.45 (s, 1H), 8.00 (d, J=
8.6 Hz, 1H), 6.78 (dd, J=8.6, 2.4 Hz, 1H), 6.73 (d, J=2.4 Hz,
1H), 5.73 (s, 2H), 3.36 (s, 3H), 2.39 (s, 3H). 13C NMR (100 MHz,
DMSO-d6): δ=190.6, 161.6, 153.9, 150.4, 148.9, 142.6, 132.6,
129.2, 124.2, 118.9, 112.8, 108.9, 53.6, 28.5, 21.8. IR (film): ν~=
8-Bromo-3,7-dihydro-3-methyl-7-[2-(4-methylphenyl)-2-oxo-
ethyl]-1H-purine-2,6-dione (36). White solid. Yield: 68%. Mp
293-295 °C. 1H NMR (500 MHz, DMSO-d6): δ=11.29 (s, 1H),
8.00 (d, J=7.8 Hz, 2H), 7.43 (d, J=7.8 Hz, 2H), 5.89 (s, 2H), 3.37
(s, 3H), 2.42 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ=190.6,
153.9, 150.4, 148.9, 145.2, 131.0, 129.5, 129.1, 128.2, 108.9, 52.7,
28.5, 21.2. IR (film): ν~=3155, 3002, 2819, 1677, 1538, 1369,
1203, 751 cm-1. MS (ESI), m/z: calcd for C15H14BrN4O3, 377.0;
found, 377.1 [M þ H]þ.
8-Bromo-3,7-dihydro-7-[2-(2-hydroxyphenyl)-2-oxoethyl]-
3-methyl-1H-purine-2,6-dione (37). White solid. Yield: 48%. Mp
289-291 °C. 1H NMR (400 MHz, DMSO-d6): δ=11.28 (s, 1H),
11.19 (s, 1H), 7.80 (dd, J=7.9, 1.8 Hz, 1H), 7.55 (ddd, J=8.3,
7.2, 1.8 Hz, 1H), 7.08 (dd, J=8.3, 0.9 Hz, 1H), 6.98 (ddd, J=7.9,
7.2, 0.9 Hz, 1H), 5.76 (s, 2H), 3.37 (s, 3H). 13C NMR (100 MHz,
DMSO-d6): δ=191.8, 159.2, 153.9, 150.4, 148.9, 136.1, 130.0,
129.1, 120.5, 119.5, 117.5, 108.9, 55.8, 28.5. IR (film): ν~=3150,
3034, 2807, 1680, 1535, 1366, 1208, 748 cm-1. HRMS (ESI),
m/z: calcd for C14H11BrN4NaO4, 400.9861; found, 400.9856
[M þ Na]þ.
3225, 3145, 3025, 2987, 1673, 1540, 1371, 1204, 1065, 743 cm-1
.
HRMS (ESI), m/z: calcd for C15H13BrN4NaO4, 415.0018;
found, 415.0015 [M þ Na]þ.
8-Bromo-7-[2-(3,4-dihydroxyphenyl)-2-oxoethyl]-3,7-dihydro-
3-methyl-1H-purine-2,6-dione (43). 8-Bromo-3,9-dihydro-3-
methyl-1H-purine-2,6-dione (13, 500 mg, 2.04 mmol) was dis-
solved in anhydrous DMF (5 mL), and distilled diisopropylethyl-
amine (0.534 mL, 3.06 mmol) was added. After the mixture was
stirred at 5 min at room temperature, R-chloro-3,4-dihydroxy-
acetophenone (380 mg, 2.04 mmol) was slowly added. The reaction
mixture was heated to 70 °C for 5 h. The mixture was then poured
into EtOAc/HCl (10%), and the organic layer was concentrated
under reduced pressure. The resulting suspension was filtered and
washed with MeOH to afford the desired product as a white solid
(294 mg, 36% yield). Mp 311-314 °C. 1H NMR (400 MHz,
DMSO-d6): δ=11.29 (s, 1H), 10.16 (s, 1H), 9.52 (s, 1H), 7.53 (dd,
J=8.2, 1.9 Hz, 1H), 7.40 (d, J=1.9 Hz, 1H), 6.90 (d, J=8.2 Hz,
1H), 5.77 (s, 2H), 3.36 (s, 3H). 13C NMR (100 MHz, DMSO-d6):
δ= 188.8, 153.9, 151.9, 150.4, 148.9, 145.6, 129.2, 125.3, 121.7,
8-Bromo-3,7-dihydro-7-[2-(3-hydroxyphenyl)-2-oxoethyl]-
3-methyl-1H-purine-2,6-dione (38). White solid. Yield: 75%. Mp
299-305 °C. 1H NMR (400 MHz, DMSO-d6): δ=11.30 (s, 1H),
9.97 (s, 1H), 7.57 (d, J=8.1 Hz, 1H), 7.39-7.44 (m, 2H), 7.15
(ddd, J=8.1, 2.5, 0.7 Hz, 1H), 5.86 (s, 2H), 3.37 (s, 3H). 13C
NMR (100 MHz, DMSO-d6): δ = 191.0, 157.8, 153.9, 150.4,
148.9, 134.8, 130.2, 129.1, 121.6, 119.1, 114.1, 108.9, 52.9,
28.5. IR (film): ν~=3370, 3008, 2822, 1676, 1534, 1364, 1209,
750 cm-1. HRMS (ESI), m/z: calcd for C14H11BrN4NaO4,
400.9861; found, 400.9860 [M þ Na]þ.
8-Bromo-3,7-dihydro-7-[2-(4-hydroxyphenyl)-2-oxoethyl]-
3-methyl-1H-purine-2,6-dione (39). Yellow solid. Yield: 74%.
1
Mp 320-322 °C. H NMR (500 MHz, DMSO-d6): δ=11.28
(s, 1H), 10.64 (s, 1H), 7.97 (d, J=8.6 Hz, 2H), 6.93 (d, J=8.6 Hz,
2H), 5.81 (s, 2H), 3.36 (s, 3H). 13C NMR (125 MHz, DMSO-d6):
δ=188.8, 163.2, 153.9, 150.4, 148.9, 130.8, 129.2, 125.0, 115.6,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6443
1
115.3, 114.7, 108.9, 52.3, 28.5. IR (film): ν~=3460, 3233, 3044, 2945,
1698, 1666, 1357, 1289, 1186, 1065, 763 cm-1. HRMS (ESI), m/z:
calcd for C14H11BrN4NaO5, 416.9808; found, 416.9805 [M þ Na]þ.
General Method for the Cyclization of Alkylated Xanthines
45-50, 54, 55, 58, 60, 62, 64-68. A mixture of 3-alkyl-8-bromo-
3,7-dihydro-7-(2-oxo-2-phenyl-ethyl)-1H-purine-2,6-dione (1.0
equiv) and the corresponding primary amine (4.0 equiv) in
EtOH (0.1 M) was heated in a sealed tube at 175 °C for 12 h.
The mixture was cooled to room temperature and the solid was
filtered off and washed with water, affording the corresponding
products in pure form. This method was used to obtain com-
pounds 45-50, 54, 55, 58, 60, 62, 64-68.
General Method for the Cyclization of Alkylated Xanthines
51-53, 56, 57, 59, 61, 63. A mixture of 3-alkyl-8-bromo-3,7-
dihydro-7-(2-oxo-2-phenylethyl)-1H-purine-2,6-dione (1.0 equiv)
and butylamine (4.0 equiv) in n-PrOH (0.1 M) was heated in a
sealed tube at 175 °C for 12 h. The mixture was cooled to room
temperature, and the solid was filtered off and washed with water.
Subsequent recrystallization in EtOH afforded the corresponding
products in pure form. This method was used to obtain compounds
51-53, 56, 57, 59, 61, 63.
12%. Mp 267-270 °C. H NMR (400 MHz, DMSO-d6): δ=
10.85 (s, 1H), 7.61 (s, 1H), 7.16-7.17 (m, 1H), 7.05-7.06 (m,
2H), 6.12 (s, 2H), 4.35 (s, 1H), 4.09 (t, J=7.4 Hz, 2H), 3.40 (s,
3H), 3.26 (t, J=7.4 Hz, 2H), 1.67 (quint, J=7.4 Hz, 2H), 1.24
(quint, J=7.4 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ=
153.2, 152.7, 150.9, 147.9, 147.7, 147.6, 131.9, 123.1, 121.4,
109.3, 108.6, 104.8, 101.5, 98.8, 59.8, 43.6, 29.1, 28.8, 25.0. IR
(film): ν~=3575, 3369, 3161, 3012, 2936, 2822, 1676, 1498, 1451,
1243, 1028, 742 cm-1. HRMS (ESI), m/z: calcd for C19H19-
N5NaO5, 420.1284; found, 420.1289 [M þ Na]þ.
7-(Benzo[d ][1,3]dioxol-4-yl)-8-(2-methoxyphenyl)-1-methyl-
1H-imidazo[1,2-f ]purine-2,4(3H,8H)-dione (50). White solid.
1
Yield: 38%. Mp 347-349 °C. H NMR (500 MHz, DMSO-
d6): δ=11.01 (s, 1H), 7.87 (s, 1H), 7.51-7.53 (m, 2H), 7.20 (d, J=
8.5 Hz, 1H), 7.11 (t, J=7.5 Hz, 1H), 6.85 (d, J=7.9 Hz, 1H), 6.68
(t, J=7.9 Hz, 1H), 6.44 (d, J=7.9 Hz, 1H), 5.99 (s, 1H), 5.95 (s,
1H), 3.59 (s, 3H), 3.29 (s, 3H). 13C NMR (125 MHz, DMSO-d6):
δ=154.8, 153.3, 152.9, 150.9, 147.7, 147.2, 144.6, 131.2, 129.5,
126.9, 122.6, 121.4, 120.8, 119.9, 112.9, 110.3, 108.5, 106.7,
101.1, 99.0, 55.7, 28.8. IR (film): ν~=3520, 3151, 3000, 2822,
1667, 1600, 1507, 1438, 1025, 739 cm-1. HRMS (ESI), m/z: calcd
for C22H17N5NaO5, 454.1127; found, 454.1113 [M þ Na]þ.
8-(Butyl)-1-methyl-7-o-methoxyphenyl-1H-imidazo[2,1-f ]purine-
2,4(3H,8H)-dione (51). White solid. Yield: 28%. Mp 242-
1-Benzyl-8-(2-methoxyphenyl)-7-phenyl-1H-imidazo[2,1-f ]-
purine-2,4(3H,8H)-dione (45). White solid. Yield: 40%. Mp
301-303 °C. 1H NMR (400 MHz, DMSO-d6): δ = 11.08
(s, 1H), 8.00 (s, 1H), 7.46-7.50 (m, 2H), 7.23-7.29 (m, 10H),
7.18 (d, J=7.8 Hz, 1H), 7.06 (t, J=7.8 Hz, 1H), 5.06 (s, 2H), 3.54
(s, 3H). 13C NMR (100 MHz, DMSO-d6, CDCl3): δ=154.7,
153.3, 152.4, 150.6, 147.8, 136.9, 132.9, 131.1, 129.5, 128.3,
128.2, 128.1, 127.5, 127.1, 127.1, 122.4, 120.9, 113.0, 105.4,
99.1, 55.6, 44.9, 1C missing due to overlapping. IR (film): ν~=
1
244 °C. H NMR (500 MHz, DMSO-d6): δ = 10.89 (s, 1H),
7.53 (t, J=7.8 Hz, 1H), 7.50 (s, 1H), 7.38 (d, J=7.8 Hz, 1H), 7.19
(d, J=7.8 Hz, 1H), 7.08 (t, J=7.8 Hz, 1H), 3.84 (t, J=7.3 Hz,
2H), 3.81 (s, 3H), 3.39 (s, 3H), 1.60 (quint, J=7.3 Hz, 2H), 1.08
(sext, J=7.3 Hz, 2H), 0.69 (t, J=7.3 Hz, 3H). 13C NMR (125
MHz, DMSO-d6): δ=157.3, 153.2, 152.7, 150.9, 147.3, 132.3,
131.5, 129.0, 120.6, 116.3, 111.4, 105.3, 98.8, 55.4, 43.4, 29.9,
28.8, 18.8, 12.9. IR (film): ν~ = 3145, 2999, 2934, 2837, 1674,
1514, 1461, 1246, 745 cm-1. HRMS (ESI), m/z: calcd for
C19H21N5NaO3, 390.1542; found, 390.1545 [M þ Na]þ.
3151, 3030, 2817, 1669, 1488, 1263, 1155, 1024, 754, 529 cm-1
.
HRMS (ESI), m/z: calcd for C27H21N5NaO3, 486.1542; found,
486.1540 [M þ Na]þ.
1-Benzyl-7-p-fluorophenyl-8-(2-methoxyphenyl)-1H-imidazo-
[2,1-f ]purine-2,4(3H,8H)-dione (46). White solid. Yield: 19%.
8-(Butyl)-1-methyl-7-m-methoxyphenyl-1H-imidazo[2,1-f ]purine-
2,4(3H,8H)-dione (52). White solid. Yield: 52%. Mp 206-
1
Mp 251-253 °C. H NMR (500 MHz, DMSO-d6): δ=11.09
1
(s, 1H), 7.99 (s, 1H), 7.46-7.49 (m, 2H), 7.25-7.29 (m, 6H),
7.20-7.23 (m, 1H), 7.12-7.17 (m, 3H), 7.04-7.08 (m, 1H), 5.05
(s, 2H), 3.54 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ =
161.9 (d, J=246.7 Hz), 154.7, 153.4, 152.4, 150.7, 147.8, 136.9,
132.0, 131.3, 129.9 (d, J=8.4 Hz), 129.6, 128.3, 127.1, 127.1,
124.6 (d, J=3.1 Hz), 122.2, 121.0, 115.4 (d, J=21.8 Hz), 113.1,
105.6, 99.1, 55.7, 44.9. IR (film): ν~=3160, 3025, 2822, 1671,
1487, 1225, 1157, 841, 744, 529, 409 cm-1. MS (ESI), m/z: calcd
for C27H20FN5NaO3, 504.4; found, 505.0 [M þ Na]þ.
208 °C. H NMR (500 MHz, DMSO-d6): δ = 10.91 (s, 1H),
7.70 (s, 1H), 7.44 (t, J=8.2 Hz, 1H), 7.13-7.14 (m, 2H), 7.05 (dd,
J=8.2, 2.4 Hz, 1H), 4.11 (t, J=7.3 Hz, 2H), 3.82 (s, 3H), 3.39
(s, 3H), 1.63 (quint, J=7.3 Hz, 2H), 1.13 (sext, J=7.3 Hz, 2H),
0.74 (t, J=7.3 Hz, 3H). 13C NMR (125 MHz, DMSO-d6): δ=
159.4, 153.2, 152.7, 150.9, 147.9, 132.0, 130.0, 129.2, 120.8,
114.8, 114.1, 105.2, 98.8, 55.2, 43.5, 30.2, 28.8, 18.9, 13.1.
IR (film): ν~=3141, 3044, 2957, 2817, 1679, 1509, 1467, 1156,
716 cm-1. HRMS (ESI), m/z: calcd for C19H21N5NaO3,
390.1542; found, 390.1544 [M þ Na]þ.
8-(2-Methoxyphenyl)-1-methyl-7-p-nitrophenyl-1H-imidazo-
[2,1-f ]purine-2,4(3H,8H)-dione (47). Yellow solid. Yield: 63%.
8-(Butyl)-1-methyl-7-p-methoxyphenyl-1H-imidazo[2,1-f ]purine-
2,4(3H,8H)-dione (53). White solid. Yield: 89%. Mp 272-
1
Mp 323-325 °C. H NMR (400 MHz, DMSO-d6): δ = 11.06
1
(s, 1H), 8.35 (s, 1H), 8.14 (d, J=8.7 Hz, 2H), 7.64 (dd, J=7.6,
1.1 Hz, 1H), 7.55 (dt, J=7.6, 1.1 Hz, 1H), 7.51 (d, J=8.7 Hz, 2H),
7.21 (d, J=7.6 Hz, 1H), 7.16 (t, J=7.6 Hz, 1H), 3.54 (s, 3H), 3.30
(s, 3H). 13C NMR (100 MHz, DMSO-d6): δ=154.4, 153.3, 153.2,
150.9, 148.4, 146.5, 134.8, 131.5, 130.8, 129.5, 127.7, 123.6, 122.1,
121.2, 113.1, 107.6, 99.2, 55.7, 28.8. IR (film): ν~=3012, 2826, 1680,
1501, 1016, 844, 720, 600, 432 cm-1. HRMS (ESI), m/z: calcd for
C21H16N6NaO5, 455.1080; found, 455.1085 [M þ Na]þ.
274 °C. H NMR (500 MHz, DMSO-d6): δ = 10.90 (s, 1H),
7.60 (s, 1H), 7.51 (d, J=8.7 Hz, 2H), 7.09 (d, J=8.7 Hz, 2H), 4.07
(t, J=7.3 Hz, 2H), 3.83 (s, 3H), 3.40 (s, 3H), 1.62 (quint, J=
7.3 Hz, 2H), 1.12 (sext, J=7.3 Hz, 2H), 0.75 (t, J=7.3 Hz, 3H).
IR (film): ν~=3144, 3051, 2963, 2864, 2802, 1671, 1507, 1247,
831, 742 cm-1. HRMS (ESI), m/z: calcd for C19H21N5NaO3,
390.1542; found, 390.1541 [M þ Na]þ.
8-(2-Methoxyphenyl)-1-methyl-7-o-methylphenyl-1H-imidazo-
[2,1-f ]purine-2,4(3H,8H)-dione (54). White solid. Yield: 64%.
Mp 301-303 °C. 1H NMR (400 MHz, DMSO-d6): δ=10.98 (s,
1H), 7.81 (s, 1H), 7.49 (dd, J=7.7, 1.6 Hz, 1H), 7.39 (ddd, J=8.6,
8.3, 1.6 Hz, 1H), 7.21-7.22 (m, 2H), 7.13-7.15 (m, 1H),
7.04-7.08 (m, 2H), 7.00 (ddd, J=8.6, 7.7, 1.2 Hz, 1H), 3.57
(s, 3H), 3.31 (s, 3H), 2.27 (s, 3H). 13C NMR (100 MHz, DMSO-
d6): δ= 154.8, 153.3, 152.9, 150.9, 147.7, 147.2, 144.6, 131.2,
129.5, 126.9, 122.6, 121.4, 120.8, 119.9, 112.9, 110.3, 108.5,
106.7, 101.1, 99.0, 55.7, 28.8. IR (film): ν~=3166, 3007, 2811,
1666, 1490, 1157, 744 cm-1. HRMS (ESI), m/z: calcd for
C22H19N5NaO3, 424.1386; found, 424.1389 [M þ Na]þ.
8-(2-Methoxyphenyl)-1-methyl-7-m-methylphenyl-1H-imidazo-
[2,1-f ]purine-2,4(3H,8H)-dione (55). White solid. Yield: 72%.
7-(Benzo[d ][1,3]dioxol-5-yl)-8-(2-methoxyphenyl)-1-methyl-1H-
imidazo[1,2-f ]purine-2,4(3H,8H)-dione (48). White solid. Yield:
1
33%. Mp 323-328 °C. H NMR (400 MHz, DMSO-d6): δ=
10.97 (s, 1H), 7.90 (s, 1H), 7.50-7.54 (m, 2H), 7.20 (dd, J=8.9,
1.2 Hz, 1H), 7.10 (ddd, J = 8.8, 7.7, 1.2 Hz, 1H), 6.82-6.84
(m, 2H), 6.71 (dd, J = 8.1, 1.8 Hz, 1H), 6.00 (s, 2H), 3.62
(s, 3H), 3.29 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ =
154.9, 153.3, 152.7, 150.9, 147.8, 147.4, 147.1, 132.8, 131.3,
129.9, 122.5, 121.7, 121.6, 120.9, 112.9, 108.2, 107.9, 104.9,
101.2, 99.0, 55.7, 28.8. IR (film): ν~=3142, 3007, 2820, 1674,
1484, 1448, 1235, 1024, 737 cm-1. HRMS (ESI), m/z: calcd for
C22H17N5NaO5, 454.1127; found, 454.1125 [M þ Na]þ.
7-(Benzo[d ][1,3]dioxol-5-yl)-8-(4-hydroxybutyl)-1-methyl-1H-
imidazo[1,2-f ]purine-2,4(3H,8H)-dione (49). White solid. Yield:
1
Mp 313-315 °C. H NMR (500 MHz, DMSO-d6): δ=10.98

6444 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Lafleur et al.
(s, 1H), 7.95 (s, 1H), 7.51 (d, J = 7.2 Hz, 2H), 6.98-7.21
(m, 6H), 3.58 (s, 3H), 3.30 (s, 3H), 2.20 (s, 3H). 13C NMR (125
MHz, DMSO-d6): δ = 154.9, 153.3, 152.7, 150.9, 147.9, 137.5,
133.0, 131.2, 129.8, 128.9, 128.2, 128.1, 128.0, 124.3, 122.6, 120.9,
112.9, 105.2, 99.1, 55.7, 28.8, 20.8. IR (film): ν~=3160, 3028, 2822,
1704, 1671, 1505, 1435, 1160, 738 cm-1. HRMS (ESI), m/z: calcd
for C22H19N5NaO3, 424.1386; found, 424.1383 [M þ Na]þ.
8-(Butyl)-1-methyl-7-m-methylphenyl-1H-imidazo[2,1-f ]purine-
2,4(3H,8H)-dione (56). White solid. Yield: 42%. Mp 238-
7.3 Hz, 2H), 3.38 (s, 3H), 1.63 (quint, J=7.3 Hz, 2H), 1.12 (sext,
J=7.3 Hz, 2H), 0.74 (t, J=7.3 Hz, 3H). 13C NMR (125 MHz,
DMSO-d6): δ=157.6, 153.2, 152.7, 150.9, 147.8, 132.3, 129.9,
129.0, 119.5, 116.0, 115.6, 104.7, 98.8, 43.3, 30.2, 28.8, 18.8, 13.1.
IR (film): ν~=3297, 3166, 3046, 2963, 2864, 1675, 1512, 1459,
1312, 1148, 722 cm-1. HRMS (ESI), m/z: calcd for C18H19N5-
NaO3, 376.1386; found, 376.1388 [M þ Na]þ.
8-(2-Methoxyphenyl)-1-methyl-7-p-hydroxyphenyl-1H-imidazo-
[2,1-f ]purine-2,4(3H,8H)-dione (62). White solid. Yield: 3%.
1
1
Mp 364-366 °C. H NMR (500 MHz, DMSO-d6): δ=10.94
240 °C. H NMR (500 MHz, DMSO-d6): δ = 10.91 (s, 1H),
(s, 1H), 9.66 (s, 1H), 7.78 (s, 1H), 7.50 (ddd, J=8.4, 7.6, 1.7 Hz,
1H), 7.47 (dd, J=7.7, 1.7 Hz, 1H), 7.19 (dd, J=8.4, 1.1 Hz, 1H),
7.05-7.10 (m, 3H), 6.65 (d, J=8.7 Hz, 2H), 3.60 (s, 3H), 3.29 (s,
3H). 13C NMR (125 MHz, DMSO-d6): δ=157.6, 155.0, 153.3,
152.5, 150.9, 147.7, 133.4, 131.1, 129.9, 129.2, 122.6, 120.9,
118.7, 115.1, 112.9, 104.1, 99.0, 55.7, 28.7. IR (film): ν~=3326,
7.65 (s, 1H), 7.40-7.43 (m, 2H), 7.37 (d, J=7.6 Hz, 1H), 7.31 (d,
J=7.6 Hz, 1H), 4.10 (t, J=7.3 Hz, 2H), 3.40 (s, 3H), 2.38 (s, 3H),
1.62 (quint, J=7.3 Hz, 2H), 1.12 (sext, J=7.3 Hz, 2H), 0.74 (t,
J=7.3 Hz, 3H). 13C NMR (125 MHz, DMSO-d6): δ=153.2,
152.7, 150.9, 147.9, 138.3, 132.3, 129.6, 129.3, 128.8, 127.9,
125.8, 104.9, 98.8, 43.3, 30.1, 28.8, 20.8, 18.8, 13.0. IR (film):
ν~=3160, 3044, 2949, 2802, 1672, 1509, 1147, 721 cm-1. HRMS
(ESI), m/z: calcd for C19H21N5NaO2, 374.1593; found, 374.1584
[M þ Na]þ.
3140, 3005, 2820, 1678, 1508, 1438, 1226, 1159, 838, 742 cm-1
.
HRMS (ESI), m/z: calcd for C21H17N5NaO4, 426.1178; found,
426.1177 [M þ Na]þ.
8-(Butyl)-1-methyl-7-p-hydroxyphenyl-1H-imidazo[2,1-f ]purine-
2,4(3H,8H)-dione (63). White solid. Yield: 10%. Mp 277-
8-(Butyl)-1-methyl-7-p-methylphenyl-1H-imidazo[2,1-f ]purine-
2,4(3H,8H)-dione (57). White solid. Yield: 24%. Mp 227-
1
1
281 °C. H NMR (400 MHz, DMSO-d6): δ = 10.89 (s, 1H),
229 °C. H NMR (500 MHz, DMSO-d6): δ = 10.91 (s, 1H),
9.85 (s, 1H), 7.52 (s, 1H), 7.36 (d, J=8.7 Hz, 2H), 6.89 (d, J=
8.7 Hz, 2H), 4.04 (t, J=7.3 Hz, 2H), 3.39 (s, 3H), 1.62 (quint, J=
7.3 Hz, 2H), 1.12 (sext, J=7.3 Hz, 2H), 0.74 (t, J=7.3 Hz, 3H).
13C NMR (100 MHz, DMSO-d6): δ=158.2, 153.2, 152.6, 150.9,
147.6, 132.5, 130.5, 118.3, 115.6, 104.1, 98.8, 43.1, 30.2, 28.8,
18.8, 13.1. IR (film): ν~=3283, 3160, 3043, 2973, 1676, 1510,
1226, 1148, 843, 716 cm-1. HRMS (ESI), m/z: calcd for C18H19-
N5NaO3, 376.1386; found, 376.1381 [M þ Na]þ.
7.63 (s, 1H), 7.46 (d, J=7.9 Hz, 2H), 7.34 (d, J=7.9 Hz, 2H), 4.09
(t, J=7.3 Hz, 2H), 3.40 (s, 3H), 2.38 (s, 3H), 1.62 (quint, J=
7.3 Hz, 2H), 1.11 (sext, J=7.3 Hz, 2H), 0.74 (t, J=7.3 Hz, 3H).
13C NMR (125 MHz, DMSO-d6): δ=153.2, 152.7, 150.9, 147.8,
138.6, 132.2, 129.5, 128.8, 125.0, 104.7, 98.8, 43.3, 30.2, 28.8,
20.7, 18.8, 13.1. IR (film): ν~ = 3169, 3051, 2958, 2858, 2785,
1671, 1506, 1294, 1147, 825 cm-1. HRMS (ESI), m/z: calcd for
C19H21N5NaO2, 374.1593; found, 374.1589 [M þ Na]þ.
8-(2-Methoxyphenyl)-1-methyl-7-o-hydroxyphenyl-1H-imidazo-
[2,1-f ]purine-2,4(3H,8H)-dione (58). Yellow solid. Yield: 50%.
8-(2-Methoxyphenyl)-1-methyl-7-(20-methyl-30-hydroxyphenyl-
1H-imidazo[2,1-f ]purine-2,4(3H,8H)-dione (64). Light-yellow
solid. Yield: 56%. Mp 372-374 °C. 1H NMR (400 MHz,
DMSO-d6): δ=10.96 (s, 1H), 9.43 (s, 1H), 7.71 (s, 1H), 7.38-
7.42 (m, 2H), 7.09 (d, J=8.1 Hz, 1H), 6.99 (t, J=7.6 Hz, 1H),
6.89 (t, J=7.6 Hz, 1H), 6.75 (d, J=8.1 Hz, 1H), 6.64 (d, J=
7.6 Hz, 1H), 3.60 (s, 3H), 3.30 (s, 3H), 2.01 (s, 3H). 13C NMR
(100 MHz, DMSO-d6): δ = 155.1, 154.7, 153.3, 152.7, 150.9,
147.4, 132.1, 130.8, 129.6, 128.3, 125.4, 124.5, 122.2, 121.9,
120.5, 115.3, 112.5, 106.1, 99.0, 55.5, 28.7, 12.9. IR (film): ν~=
1
Mp 350-352 °C. H NMR (500 MHz, DMSO-d6): δ=10.95
(s, 1H), 9.75 (s, 1H), 7.68 (s, 1H), 7.40-7.44 (m, 2H), 7.10-7.14
(m, 2H), 7.00-7.04 (m, 2H), 6.81 (dd, J=8.2, 0.9 Hz, 1H), 6.67
(ddd, J=8.4, 7.5, 0.9 Hz, 1H), 3.58 (s, 3H), 3.30 (s, 3H). 13C
NMR (125 MHz, DMSO-d6): δ = 155.5, 154.8, 153.3, 152.7,
150.9, 147.5, 130.7, 130.6, 130.1, 129.7, 129.4, 122.8, 120.5,
118.3, 115.5, 114.7, 112.7, 106.4, 98.9, 55.5, 28.7. IR (film):
ν~=3251, 3166, 2821, 1677, 1486, 1445, 1159, 744 cm-1. HRMS
(ESI), m/z: calcd for C21H17N5NaO4, 426.1178; found, 426.1174
[M þ Na]þ.
3326, 3145, 3062, 2963, 2817, 1669, 1508, 1280, 1157, 747 cm-1
.
HRMS (ESI), m/z: calcd for C22H19N5NaO4, 440.1335; found,
440.1332 [M þ Na]þ.
8-(Butyl)-1-methyl-7-o-hydroxyphenyl-1H-imidazo[2,1-f ]purine-
2,4(3H,8H)-dione (59). White solid. Yield: 22%. Mp 209-
8-(2-Methoxyphenyl)-1-methyl-7-(20-methyl-40-hydroxyphenyl-
1H-imidazo[2,1-f ]purine-2,4(3H,8H)-dione (65). White solid.
1
211 °C. H NMR (400 MHz, DMSO-d6): δ = 10.88 (s, 1H),
1
Yield: 52%. Mp 330-333 °C. H NMR (400 MHz, DMSO-
9.99 (s, 1H), 7.48 (s, 1H), 7.35 (t, J=7.8 Hz, 1H), 7.30 (d, J=
7.8 Hz, 1H), 7.00 (d, J=7.8 Hz, 1H), 6.93 (t, J=7.8 Hz, 1H), 3.92
(t, J=7.3 Hz, 2H), 3.40 (s, 3H), 1.60 (quint, J=7.3 Hz, 2H),
1.08 (sext, J=7.3 Hz, 2H), 0.70 (t, J=7.3 Hz, 3H). 13C NMR
(100 MHz, DMSO-d6): δ = 155.8, 153.2, 152.6, 150.9, 147.4,
132.2, 131.2, 129.6, 119.2, 115.7, 114.8, 105.1, 98.8, 43.4, 30.1,
28.8, 18.8, 13.0. IR (film): ν~ = 3138, 3044, 2953, 2870, 2811,
1683, 1509, 1200, 739 cm-1. HRMS (ESI), m/z: calcd for C18-
H19N5NaO3, 376.1386; found, 376.1383 [M þ Na]þ.
d6): δ=10.94 (s, 1H), 9.55 (s, 1H), 7.66 (s, 1H), 7.38-7.43 (m,
2H), 7.09 (d, J=7.5 Hz, 1H), 7.00 (t, J=7.5 Hz, 1H), 6.94 (d, J=
8.3 Hz, 1H), 6.57 (s, 1H), 6.44 (d, J=8.3 Hz, 1H), 3.60 (s, 3H),
3.30 (s, 3H), 2.14 (s, 3H). IR (film): ν~=3208, 3048, 2942, 2838,
1675, 1596, 1455, 1205, 1153, 747 cm-1. HRMS (ESI), m/z: calcd
for C22H19N5NaO4, 440.1335; found, 440.1330 [M þ Na]þ.
8-(2-Methoxyphenyl)-1-methyl-7-(20-methyl-50-hydroxyphenyl-
1H-imidazo[2,1-f ]purine-2,4(3H,8H)-dione (66). White solid.
1
Yield: 45%. Mp 350-352 °C. H NMR (400 MHz, DMSO-
8-(2-Methoxyphenyl)-1-methyl-7-m-hydroxyphenyl-1H-imidazo-
[2,1-f ]purine-2,4(3H,8H)-dione (60). White solid. Yield: 59%.
d6): δ=10.96 (s, 1H), 9.22 (s, 1H), 7.74 (s, 1H), 7.39-7.44 (m,
2H), 7.10 (dd, J=8.4, 0.9 Hz, 1H), 6.97-7.04 (m, 2H), 6.62 (dd,
J=8.3, 2.6 Hz, 1H), 6.56 (d, J=2.6 Hz, 1H), 3.61 (s, 3H), 3.30 (s,
3H), 2.12 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ=154.6,
154.4, 153.3, 152.7, 150.9, 147.4, 131.8, 130.8, 130.7, 129.5,
127.8, 127.6, 122.2, 120.5, 117.7, 116.2, 112.6, 106.1, 99.0,
55.5, 28.7, 18.6. IR (film): ν~ = 3296, 3145, 1698, 1670, 1593,
1505, 1282, 1153, 1017, 751 cm-1. HRMS (ESI), m/z: calcd for
C22H19N5NaO4, 440.1335; found, 440.1333 [M þ Na]þ.
1
Mp 353-355 °C. H NMR (500 MHz, DMSO-d6): δ=10.97
(s, 1H), 9.51 (s, 1H), 7.88 (s, 1H), 7.47-7.52 (m, 2H), 7.20 (d, J=
8.2 Hz, 1H), 7.06-7.11 (m, 2H), 6.68-6.70 (m, 2H), 6.64 (s, 1H),
3.60 (s, 3H), 3.29 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ=
157.1, 154.9, 153.3, 152.7, 150.9, 147.9, 133.1, 131.1, 129.7,
129.4, 129.2, 122.6, 120.9, 118.3, 115.4, 114.4, 112.9, 105.1,
99.1, 55.7, 28.8. IR (film): ν~ = 3147, 3030, 2817, 1676, 1482,
1299, 1159, 758 cm-1. HRMS (ESI), m/z: calcd for C21H17N5-
NaO4, 426.1178; found, 426.1181 [M þ Na]þ.
8-(2-Methoxyphenyl)-1-methyl-7-(20,50-dihydroxyphenyl-1H-
imidazo[2,1-f ]purine-2,4(3H,8H)-dione (67). White solid. Yield:
8-(Butyl)-1-methyl-7-m-hydroxyphenyl-1H-imidazo[2,1-f ]purine-
2,4(3H,8H)-dione (61). White solid. Yield: 41%. Mp 256-
1
34%. Mp 333-336 °C. H NMR (400 MHz, DMSO-d6): δ=
1
258 °C. H NMR (500 MHz, DMSO-d6): δ = 10.89 (s, 1H),
10.94 (s, 1H), 9.18 (s, 1H), 8.96 (s, 1H), 7.69 (s, 1H), 7.50 (t, J=
7.6 Hz, 1H), 7.42 (d, J=7.6 Hz, 1H), 7.20 (d, J=7.6 Hz, 1H), 7.07
(t, J=7.6 Hz, 1H), 6.61-6.63 (m, 2H), 6.53 (dd, J=7.8, 1.8 Hz,
9.75 (s, 1H), 7.59 (s, 1H), 7.31 (t, J=7.8 Hz, 1H), 6.96 (d, J=
7.8 Hz, 1H), 6.93 (s, 1H), 6.88 (d, J=7.8 Hz, 1H), 4.07 (t, J=

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6445
1H), 3.64 (s, 3H), 3.28 (s, 3H). 13C NMR (100 MHz, DMSO-d6):
δ=155.1, 153.3, 152.5, 150.9, 147.7, 145.9, 144.9, 133.7, 131.1,
129.9, 122.7, 120.8, 119.4, 118.9, 115.4, 115.3, 112.9, 103.9, 99.0,
55.7, 28.8. IR (film): ν~=3446, 3243, 3062, 1714, 1656, 1506,
1464, 1276, 1148, 749 cm-1. HRMS (ESI), m/z: calcd for
C21H17N5NaO5, 442.1121; found, 442.1122 [M þ Na]þ.
for 30 min using Multidrop Micro reagent dispensers (Thermo
Electron Corporation, Waltham, MA) in a 96-well format. The
concentration of magnesium acetate in the assays was 10 mM, and
[γ-33P]ATP (800 cpm/pmol) was used at 5, 20, or 50 μM as
indicated in Supporting Information, in order to be at or below
the Km for ATP for each kinase. The assays were initiated with
MgATP, stopped by the addition of 5 μL of 0.5 M orthophos-
phoric acid, and spotted on to P81 filter plates using a unifilter
harvester (PerkinElmer, Boston, MA). IC50 values were deter-
mined after carrying out assays at 10 different concentrations
between 10 μM and 110 pM. The data are presented as mean
percentage activity of duplicate assays at single concentration
compared to DMSO controls. A similar protocol was used at
Reaction Biology Corporation to measure the inhibitory activity of
compound 66 against 11 Eph kinases.
Phage Display Based Binding Assay. The experiments were
performed at Ambit Biosciences Corporation using binding
assays as previously described.11 Briefly, kinases were expressed
as fusion proteins to T7 phage. In general, full-length constructs
were used for small kinases and catalytic domains for large
kinases. T7-kinase-tagged phage strains were mixed with known
kinase inhibitors immobilized on streptavidin-coated magnetic
beads and with test compounds at a single concentration of
1 μM. Test compounds that bind to the kinase ATP site displace
the immobilized ligand from the kinase/phage, which is detected
using quantitative PCR. The results are reported as the percen-
tage of kinase/phage remaining bound to the ligand/beads,
relative to a control (DMSO lacking a test compound). High
affinity compounds have % control=0, while weaker binders
have higher % control values. Results are reported for screening
against 50 human kinases.
8-(2-Methoxyphenyl)-1-methyl-7-p-cyanophenyl-1H-imidazo-
[2,1-f ]purine-2,4(3H,8H)-dione (68). White solid. Yield: 68%.
1
Mp 303-305 °C. H NMR (400 MHz, DMSO-d6): δ=11.04
(s, 1H), 8.28 (s, 1H), 7.77 (d, J=8.4 Hz, 2H), 7.62 (dd, J=7.6,
1.2 Hz, 1H), 7.55 (dt, J=7.6, 1.2 Hz, 1H), 7.43 (d, J=8.4 Hz,
2H), 7.20 (dd, J=7.6, 1.2 Hz, 1H), 7.15 (dt, J=7.6, 1.2 Hz, 1H),
3.53 (s, 3H), 3.30 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ=
154.4, 153.3, 153.1, 150.9, 148.3, 132.9, 132.3, 131.5, 131.1,
129.5, 127.4, 122.2, 121.2, 118.3, 113.1, 110.5, 107.2, 99.2,
55.6, 28.8. IR (film): ν~ = 3010, 2835, 1680, 1497, 745, 605,
445 cm-1. HRMS (ESI), m/z: calcd for C22H16N6NaO3,
435.1182; found, 435.1187 [M þ Na]þ.
7-p-Fluorophenyl-8-(2-methoxyphenyl)-1H-imidazo[2,1-f ]purine-
2,4(3H,8H)-dione (69). A mixture of 46 (528 mg, 1.09 mmol),
10% Pd-C (339 mg), and dry ammonium formate (692 mg,
10.9 mmol) in absolute methanol (16 mL) was heated in a
sealed tube at 140 °C for 3 h. The mixture was cooled to room
temperature, and the mixture was filtered over a short path of
Celite. The residue was evaporated under reduced pressure,
affording the desired compound as a white solid (121 mg, 28%
1
yield). Mp 330-333 °C. H NMR (400 MHz, DMSO-d6): δ=
11.53 (s, 1H), 10.71 (s, 1H), 7.96 (s, 1H), 7.47-7.51 (m, 2H),
7.28-7.31 (m, 2H), 7.13-7.18 (m, 3H), 7.06-7.10 (m, 1H), 3.56
(s, 3H). 13C NMR (100 MHz, DMSO-d6): δ = 162.4 (d, J =
246.1 Hz), 155.2, 154.7, 152.7, 151.7, 148.9, 132.3, 131.7, 130.2
(d, J=8.4 Hz), 130.1, 125.3 (d, J=3.2 Hz), 122.9, 121.4, 115.9
(d, J=21.8 Hz), 113.4, 105.9, 99.5, 56.2. IR (film): ν~=3159,
2900, 1668, 1500, 1393, 1242, 1065, 750, 536 cm-1. MS (ESI),
m/z: calcd for C20H14FN5NaO3: 414.3; found, 414.2 [M þ Na]þ.
8-(2-Methoxyphenyl)-1-methyl-7-p-carboxyphenyl-1H-imidazo-
[2,1-f ]purine-2,4(3H,8H)-dione (70). To a solution of 8-(2-methoxy-
phenyl)-1-methyl-7-p-cyanophenyl-1H-imidazo[2,1-f ]purine-
2,4(3H,8H)-dione (68, 85 mg, 0.206 mmol) in water (0.910 mL)
was added concentrated H2SO4 (0.803 mL), and the mixture was
refluxed for 2 h. The mixture was cooled to room temperature,
water (1 mL) was added, and the precipitate formed was filtered
off, affording the desired product as a white solid (60 mg, 67%
yield). 1H NMR (400 MHz, DMSO-d6): δ=11.02 (s, 1H), 8.16 (s,
1H), 7.82(d, J=8.3 Hz, 2H), 7.58 (dd, J=7.7, 1.4 Hz, 1H), 7.53 (dt,
J = 7.7, 1.4 Hz, 1H), 7.37 (d, J = 8.3 Hz, 2H), 7.19 (d, J =
7.6 Hz, 1H), 7.14 (t, J=7.6 Hz, 1H), 3.54 (s, 3H), 3.30 (s, 3H),
CO2H not observed. 13C NMR (100 MHz, DMSO-d6): δ=166.6,
154.6, 153.3, 152.9, 150.9, 148.2, 132.4, 131.9, 131.3, 130.3, 129.6,
129.2, 127.0, 122.4, 121.1, 113.0, 106.4, 99.1, 55.6, 28.8. IR (film):
Acknowledgment. We thank Marino Convertino and Dr.
Peter Kolb for interesting discussions. The calculations were
performed on the Matterhorn Beowulf cluster at the Infor-
matikdienste of the University of Zurich. This work was
supported in part by a grant of the Swiss National Science
Foundation to A.C. and the Organic Chemistry Institute of
the University of Zurich to C.N.
Supporting Information Available: Biological activity of
compounds 54 and 66 on the selected kinases, conformation
analysis data, times series of van der Waals and electrostatic
interaction energies, SAR-based dendrogram, experimental
protocols for 5-14, and analytical data for final compounds
45-70 and intermediates 5-14, 16-18, and 21-44. This ma-
terial is available free of charge via the Internet at http://pubs.
acs.org.
ν~ = 3013, 2839, 1681, 1547, 1264, 1158, 1013, 745, 570 cm-1
.
HRMS (ESI), m/z: calcd for C22H17N5NaO5, 454.1127; found,
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