Design, synthesis, and evaluation of 5-methyl-4-phenoxy-5H-pyrrolo[3,2-d] pyrimidine derivatives: Novel VEGFR2 kinase inhibitors binding to inactive kinase conformation

Article abstract of DOI:10.1016/j.bmc.2010.08.017

We synthesized a series of pyrrolo[3,2-d]pyrimidine derivatives and evaluated their application as type-II inhibitors of vascular endothelial growth factor receptor 2 (VEGFR2) kinase. Incorporation of a diphenylurea moiety at the C4-position of the pyrrolo[3,2-d]pyrimidine core via an oxygen linker resulted in compounds that were potent inhibitors of VEGFR2 kinase. Of these derivatives, compound 20d showed the strongest inhibition of VEGF-stimulated proliferation of human umbilical vein endothelial cells (HUVEC). The co-crystal structure of 20d and VEGFR2 revealed that 20d binds to the inactive form of VEGFR2. Further studies indicated that 20d inhibited VEGFR2 kinase with slow dissociation kinetics and also inhibited PDGFR and Tie-2 kinases. Oral administration of the hydrochloride salt of 20d at 3 mg/kg/day showed potent inhibition of tumor growth in a DU145 human prostate cancer cell xenograft nude mouse model.

Full text of DOI:10.1016/j.bmc.2010.08.017

Bioorganic & Medicinal Chemistry 18 (2010) 7260–7273
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and evaluation of 5-methyl-4-phenoxy-5H-pyrrolo-
[3,2-d]pyrimidine derivatives: Novel VEGFR2 kinase inhibitors binding
to inactive kinase conformation
a
b
b
b
Yuya Oguro a, , Naoki Miyamoto , Kengo Okada , Terufumi Takagi , Hidehisa Iwata ,
*
Yoshiko Awazu a, Hiroshi Miki b, Akira Hori a, Keiji Kamiyama a, Shinichi Imamura a
a Pharmaceutical Research Division, Takeda Pharmaceutical Co., Ltd, 10, Wadai, Tsukuba, Ibaraki 300-4293, Japan
b Pharmaceutical Research Division, Takeda Pharmaceutical Co., Ltd, 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka 532-8686, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized a series of pyrrolo[3,2-d]pyrimidine derivatives and evaluated their application as type-II
inhibitors of vascular endothelial growth factor receptor 2 (VEGFR2) kinase. Incorporation of a diphenyl-
urea moiety at the C4-position of the pyrrolo[3,2-d]pyrimidine core via an oxygen linker resulted in com-
pounds that were potent inhibitors of VEGFR2 kinase. Of these derivatives, compound 20d showed the
strongest inhibition of VEGF-stimulated proliferation of human umbilical vein endothelial cells (HUVEC).
The co-crystal structure of 20d and VEGFR2 revealed that 20d binds to the inactive form of VEGFR2. Fur-
ther studies indicated that 20d inhibited VEGFR2 kinase with slow dissociation kinetics and also inhib-
ited PDGFR and Tie-2 kinases. Oral administration of the hydrochloride salt of 20d at 3 mg/kg/day
showed potent inhibition of tumor growth in a DU145 human prostate cancer cell xenograft nude mouse
model.
Received 28 June 2010
Revised 6 August 2010
Accepted 7 August 2010
Available online 13 August 2010
Keywords:
VEGFR
Type-II inhibitor
Pyrrolo[3,2-d]pyrimidine
diphenylurea
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
chemotactic effects on endothelial cells.12 VEGF overexpression
correlates with poor prognosis and clinical staging in the majority
Angiogenesis, the formation of new capillary blood vessels from
preexisting vasculature, is the process by which solid tumors are
supplied with oxygen and nutrients.1 Angiogenesis is increased
in various types of cancers, and high microvessel density in the tu-
mor correlates with poor prognosis in patients.2,3 Therefore, the
inhibition of tumor angiogenesis is considered to be a promising
approach for the treatment of many human malignancies.4
The vascular endothelial growth factor (VEGF) family is a large
family of angiogenic and lymphangiogenic growth factors, and
VEGF plays an important role in tumor angiogenesis.5 In solid tu-
mors, VEGF expression is upregulated by cancer-related changes,
such as proto-oncogene activation,6 loss of tumor suppressor func-
tion,7,8 growth factor stimuli,9 and hypoxic status.10,11 Angiogene-
sis is triggered by the binding of VEGF to vascular endothelial
growth factor receptor (VEGFR); the different subtypes of VEGFR
include VEGFR1 (also known as Flt1), VEGFR2 (also known as
KDR), and VEGFR3 (also known as Flt4).5 Binding of VEGF to VEGFR
induces conformational changes in VEGFR followed by receptor
dimerization, autophosphorylation of tyrosine residues in the
intracellular kinase domain and exerts potent mitogenic and
of solid tumor patients.13–15 VEGF/VEGFR signaling is, therefore, re-
garded as an attractive therapeutic target for inhibition of tumor
angiogenesis. Bevacizumab, a recombinant humanized monoclonal
antibody against VEGF, has been approved as first-line therapy for
various conditions such as metastatic colorectal cancer.16–18
The majority of known kinase inhibitors such as gefitinib are
classified as type-I kinase inhibitors, which bind in and around
the region occupied by the adenine ring of ATP.19 On the other
hand, type-II kinase inhibitors (e.g., imatinib) induce the DFG-out
conformation of the activation loop, enabling them to occupy the
adenine binding site and an adjacent hydrophobic pocket created
by this rearrangement.20 The X-ray co-crystal structures of ki-
nase-bound type-II kinase inhibitors also generally reveal two con-
served hydrogen-bond interactions between the ligand and
protein: one with the side chain of a conserved glutamic acid in
the aC-helix (Glu885 in VEGFR2), and the other with the backbone
amide of aspartic acid in the DFG motif (Asp1046 in VEGFR2).
Type-II kinase inhibitors have several advantages over type-I ki-
nase inhibitors, including improved kinase selectivity and slower
off-rates.21,22 Therefore, we focused on the identification of novel
type-II VEGFR kinase inhibitors. Studies to identify novel kinase
inhibitors showed that the pyrrolo[3,2-d]pyrimidine scaffold could
serve as a novel kinase hinge binding template.23 This finding
* Corresponding author. Tel.: +81 29 864 6384; fax: +81 29 864 6308.
E-mail address: Ooguro_Yuuya@takeda.co.jp (Y. Oguro).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.017

Y. Oguro et al. / Bioorg. Med. Chem. 18 (2010) 7260–7273
7261
encouraged us to generate novel type-II kinase inhibitors by incor-
porating a substituted benzene ring at the C4-position of the
pyrrolo[3,2-d]pyrimidine core via an appropriate linker (X)
(Fig. 1). To rationalize the designed compound as a good starting
point for type-II inhibitors, the binding model of VEGFR2 with
the pyrrolo[3,2-d]pyrimidine derivative (A) (X = O, R0 = H, Y-R = H
in Fig. 1) was generated. The binding model suggested the N1-
nitrogen of the pyrrolo[3,2-d]pyrimidine interacts with the main
chain NH group of Cys919 in the hinge region and the phenoxy
group extended toward the back hydrophobic pocket. Thus, we de-
signed the compounds possessing a hydrogen bond donor–accep-
tor pair (Y) and a lipophilic group (R) on the benzene ring,
aiming interaction with Glu885, Asp1046, and the hydrophobic
pocket. In this paper, we report the synthesis, structure–activity
relationships (SAR), and characterization of these new inhibitors.
coupling of 2 and 4-aminothiophenol in a manner similar to the
synthesis of ether 6. In contrast, 17b,c, which have nitrogen linkers,
were not obtained under the same conditions as the synthesis of
17a. Consequently, coupling of 2 with 4-nitroaniline under acidic
conditions followed by hydrogenation successfully provided 17b.
In the same manner, coupling of 2 with N-methyl-4-nitroaniline
using sodium hydride and subsequent hydrogenation gave 17c.
Treatment of anilines 17a–c with the appropriate phenyl isocya-
nates provided the corresponding ureas 18a–c.
Urea derivatives possessing various substituents on the central
phenyl ring (20a–d) were prepared as shown in Scheme 4. While
direct coupling of 2 with the corresponding aminophenols in the
presence of K2CO3 provided the 2-chloro-, 3-chloro-, and 2-meth-
oxyanilines 19a–c in good yield, synthesis of the 2-fluoroaniline
derivative 19d was unsuccessful under these basic conditions
due to the self-coupling of 3-fluoro-4-aminophenol. Thus, 19d
was prepared by coupling of 2 with 3-fluoro-4-nitrophenol with-
out base followed by reduction with zinc. The anilines 19a–d were
converted to the corresponding ureas 20a–d by treatment with 3-
(trifluoromethyl)phenyl isocyanate.
2. Chemistry
A
general synthesis of 4-phenoxy-pyrrolo[3,2-d]pyrimidine
derivatives possessing urea, amide, thiourea, and benzimidazole
functionalities is shown in Schemes 1 and 2. Synthesis was initiated
by treating pyrrolo[3,2-d]pyrimidine 124,25 with methyl methane-
sulfonate to afford 2, which was coupled with 3- or 4-aminophenol
in the presence of potassium carbonate to provide aniline 3 or 6,
respectively. Aniline 3 was converted to the corresponding urea 4
or amide 5 using phenyl isocyanate or benzoyl chloride, respec-
tively. Aniline 6 was also converted to ureas 7a–j, amides 8 and 9,
thiourea 10, and benzimidazole 11. The ureas 7a–j were formed by
reaction of 6 and the corresponding amines with N,N-carbonyldiim-
idazole (CDI), or by reaction with the corresponding isocyanates. The
amides 8 and 9, and thiourea 10 were obtained by treatment of 6
with benzoyl chloride, phenylacetyl chloride, or phenyl isothiocya-
nate, respectively. In addition, the benzimidazole 11 was synthe-
sized via reaction of 6 with 2-chlorobenzimidazole.
Benzoic acids 12a,b and ester 14, which were prepared by cou-
pling 2 with the corresponding phenols, were used for the synthe-
sis of amides 13a,b and 16 (Scheme 2). Condensation of 12a,b with
aniline using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDCIꢀHCl) and 1-hydroxybenzotriazole (HOBt) pro-
vided 13a,b, respectively. Similarly, the phenylacetic acid deriva-
tive 15, which was obtained by hydrolysis of 14, was converted
to amide 16.
The synthesis of the pyrrolo[3,2,1-de]pteridine derivative 23 is
presented in Scheme 5. Pyrrolo[3,2-d]pyrimidine 1 was alkylated
with 2-bromo-1-chloroethane to give 21, which was converted to
22 by coupling with aniline 25. Compound 22 was then cyclized
in the presence of K2CO3 to provide pyrrolo[3,2,1-de]pteridine 23.
Aniline 25 was synthesized by reaction of the 4-nitroaniline 24
with 3-(trifluoromethyl)phenyl isocyanate and subsequent hydro-
genation using Pd/C.
3. Results and discussion
The pyrrolo[3,2-d]pyrimidine derivatives depicted in Tables 1–4
were evaluated for their inhibitory activity against human VEGFR2
kinase by a non-RI assay using the amplified luminescent proxim-
ity homogeneous assay (AlphaScreenÒ) system. AlphaScreenÒ is
based on the transfer of energy from donor to acceptor microbeads
brought together by a biomolecular interaction.26 In this system,
an anti-phosphotyrosine antibody is immobilized with acceptor
beads, and the biotinylated poly-GluTyr (4:1) is conjugated with
streptavidin donor beads. The amount of phosphorylated substrate
is measured as a signal of AlphaScreenÒ. Subsequently, a cell pro-
liferation assay was performed to investigate the potent VEGFR2
kinase inhibitors among these compounds for their ability to inhi-
bit VEGF-stimulated proliferation of human umbilical vein endo-
thelial cells (HUVEC).
Modification of the linker atoms between the pyrrolo[3,2-
d]pyrimidine core and the central phenyl ring was accomplished
as shown in Scheme 3. Thioether 17a was readily prepared by
Figure 1. Design of pyrrolo[3,2-d]pyrimidine derivatives. Molecular modeling study of the pyrrolo[3,2-d]pyrimidine derivative A was carried out using the program GOLD
and the X-ray structure of VEGFR2 (PDB code: IVR2).

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Y. Oguro et al. / Bioorg. Med. Chem. 18 (2010) 7260–7273
Scheme 1. Reagents and conditions: (a) MsOMe, Cs2CO3, DMF, rt; (b) 3-aminophenol, K2CO3, NMP, 110 °C; (c) 4-aminophenol, K2CO3, NMP, 110 °C; (d) phenyl isocyanates,
Et3N, THF, rt; (e) PhCOCl, Et3N, THF, rt; (f) amines, CDI, DMF, rt; (g) PhCH2COCl, Et3N, THF, rt; (h) PhNCS, Et3N, THF, rt; (i) 2-chlorobenzimidazole, NMP, 120 °C.
Scheme 2. Reagents and conditions: (a) 3- or 4-Hydroxybenzoic acid, Cs2CO3, DMSO, 100 °C; (b) aniline, EDCIꢀHCl, HOBt, DMF, rt; (c) ethyl 4-hydroxyphenylacetate, K2CO3,
NMP, 110 °C; (d) NaOH, H2O, MeOH, rt.
We introduced appropriate substituents into the phenoxy
group at C4-position of pyrrolo[3,2-d]pyrimidine considered the
interactions between Glu885/Asp1046 and the hydrophobic pocket
of VEGFR2, which is seen in many type-II kinase inhibitors (Table
1). The initial compounds evaluated from this series were the
30-phenylurea and 30-phenylamide derivatives 4, 5, and 13a, which
showed inhibitory activity toward VEGFR2 kinase with IC50 values
of around 1000 nM. The moderate potencies shown by 4, 5, and
13a suggested that they might serve as good starting points for
the development of potent type-II VEGFR2 kinase inhibitors.

Y. Oguro et al. / Bioorg. Med. Chem. 18 (2010) 7260–7273
7263
Scheme 3. Reagents and conditions: (a) 4-Aminothiophenol, K2CO3, NMP, 110 °C; (b) (1) 4-nitroaniline, 2-propanol, 4 M HCl/AcOEt, 80 °C; (2) H2, Pd/C, MeOH, rt;
(c) N-methyl-4-nitroaniline, NaH, DMF, 0 °C to rt; (2) H2, Pd/C, MeOH, rt; (d) phenyl isocyanates, Et3N, THF, rt.
Scheme 4. Reagents and conditions: (a) Aminophenols, K2CO3, NMP, 110 °C; (b) (1) 3-fluoro-4-nitrophenol, o-xylene, 100 °C; (2) Zn, NH4Cl, MeOH, reflux; (c) 3-
(trifluoromethyl)phenyl isocyanate, Et3N, THF, rt.
Scheme 5. Reagents and conditions: (a) 2-Bromo-1-chloroethane, Cs2CO3, DMF, rt; (b) 25, 2-propanol, 80 °C; (c) K2CO3, DMF, 80 °C; (d) (1) 3-(trifluoromethyl)phenyl
isocyanate, Et3N, THF, rt; (2) H2, Pd/C, MeOH, rt; (3) 4 M HCl/AcOEt, AcOEt, rt.
Further investigation revealed that moving the phenylureido group
of 4 from the 30-position to the 40-position (7a) significantly in-
creased potency (7a: IC50 = 33 nM), while the potency did not in-
creased by similar modification of 30-phenylamides 5 and 13a to
the corresponding 40-phenylamides 8 and 13b (8: IC50 = 9400 nM,
13b: IC50 >10,000 nM). The importance of the urea linker was also
shown by the significant loss of potency observed for the amides 9
and 16 and for the thiourea 10. The amide derivatives 9 and 16,
which have a carbon in place of the ureido nitrogen of the urea
7a, were >300- and 100-fold less active than 7a, respectively. Thio-
urea 10 also exhibited an approximate 200-fold loss of potency.
These results indicated that all of the NH groups and the oxygen
in the urea of 7a might be involved in hydrogen bonding with
VEGFR2. The benzimidazole derivative 11 was designed to exam-
ine the potential of using the benzimidazole moiety as an isosteric
replacement for the biphenylurea side chain. However, 11 was
found to be less potent than 7a (11: IC50 = 4200 nM), revealing that
it was less suited for hydrogen bonding with VEGFR2 than 7a.

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Y. Oguro et al. / Bioorg. Med. Chem. 18 (2010) 7260–7273
Table 1
was achieved by the introduction of a trifluoromethyl group (7g:
IC50 = 5.3 nM), bromine atom (7h: IC50 = 4.4 nM), or methyl group
(7j: IC50 = 2.7 nM) although the smaller fluorine atom (7i:
IC50 = 19 nM) showed slightly decreased activity. It is noteworthy
that the meta-CF3 derivative (7g) yielded cellular potency (HUVEC
IC50 = 22 nM) quite close to the inhibitory activity against VEGFR2
kinase.
Inhibitory activity of pyrrolo[3,2-d]pyrimidine derivatives against vascular endothe-
lial growth factor receptor 2 kinasea
Optimization of the linker atom between the pyrrolo[3,2-d]-
pyrimidine core and the central phenyl ring revealed that an oxygen
linker is preferred to sulfur or nitrogen (Table 3). In fact, the sulfur
linker derivative (18a: IC50 = 110 nM) was threefold less active than
the oxygen linker derivative (7a), while the amino linker derivative
(18b: IC50 = 1400 nM) exhibited 40-fold loss of potency. Based on
their decreased enzymatic activity, 18a,b showed the declined HU-
VEC inhibitory activity. On the other hand, a significant reduction
in activity was observed with an N-methylamino linker (18c: IC50
>10000 nM), suggesting that steric hindrance between the methyl
group on the linker nitrogen and the methyl group at the N5-posi-
tion of the pyrrolo[3,2-d]pyrimidine core causes destabilization of
the active conformation. The recovery of activity by cyclization be-
tween the nitrogen linker and the N-5 nitrogen (23: IC50 = 7.1 nM)
supports this hypothesis.
Compd
R3
R4
VEGFR2 IC50 (nM)
4
5
NHCONHPh
NHCOPh
CONHPh
H
H
H
H
H
H
H
H
H
1800 (1600–2200)
940 (770–1100)
870 (740–1000)
33 (27–40)
9400 (7000–13,000)
>10,000
>10,000
3300 (2400–4500
6700 (5200–8800))
13a
7a
8
13b
9
NHCONHPh
NHCOPh
CONHPh
NHCOCH2Ph
CH2CONHPh
NHCSNHPh
16
10
11
H
4200 (3200–5600)
The effects of substituents on the central phenyl group were
also investigated (Table 4). Introduction of a chlorine atom into
the central phenyl ring of 7g revealed that substitution at the 2-po-
sition was more promising than the 3-position. The 2-chloro com-
pound 20a was twofold more potent in cellular assay compared to
7g, whereas the 3-chloro compound 20b exhibited a decreased po-
tency in both kinase and cellular assays. As a result of further
investigations, the 2-fluorine substituted compound 20d was
found to show the strongest HUVEC inhibitory activity with an
IC50 value of 4.4 nM. On the other hand, replacement of the fluorine
(20d) with a methoxy group (20c) resulted in decreased enzyme
and cell activity. There are three possible explanation for decreased
potency observed for 20c: the methoxy group may relatively too
big to occupy the binding pocket around the central phenyl ring,
the steric hindrance between the methoxy group and the ureido
moiety of 20c may destabilize the active conformation, the low-
ered acidity of ureido moiety may cause the decreased interaction
between the ureido moiety and amino acid residues in the protein.
Pharmacokinetic parameters for the compounds were obtained by
cassette dosing in mice. After oral administration (10 mg/kg), com-
pound 20d showed an area under the plasma concentration-time
a
Numbers in parentheses represent 95% confidence interval.
Table 2
Effect of urea substituents on vascular endothelial growth factor receptor 2 kinase
and cellular growth inhibitiona
Compd
R
VEGFR2 IC50 (nM)
HUVECb IC50 (nM)
7a
7b
7c
7d
7e
7f
7g
7h
7i
Ph
Me
Pr
2-ClPh
3-ClPh
4-ClPh
3-CF3Ph
3-BrPh
3-FPh
3-MePh
33 (27–40)
810 (630–1000)
24,000 (21,000–27,000)
24,000 (21,000–27,000)
2700 (2300–3200)
110 (84–130)
1100 (940–1300)
22 (16–30)
44 (31–63)
6300 (4800–8400)
3900 (3300–4600)
340 (280–400)
4.1 (3.4–5.0)
32 (26–40)
5.3 (4.4–6.3)
4.4 (3.9–5.1)
19 (16–22)
curve (AUC0–8 h) equal to 10.9
concentration of 20d at 8 h after administration (C8 h) was 410-fold
higher than its IC50 value in HUVEC assay (C8 h = 0.803 g/
mL = 1.8 M). On the basis of these results, we selected 20d for fur-
ther evaluation.
lg h/mL. Furthermore, the plasma
290 (230–370)
62 (38–110)
7j
2.7 (2.3–3.1)
l
a
Numbers in parentheses represent 95% confidence interval.
Growth inhibition of human umbilical vein endothelial cells (HUVEC).
l
b
In order to confirm the binding mode of 20d, the co-crystal struc-
ture of the complex between 20d and VEGFR2 was obtained (Fig. 2).
The kinase adopts an inactive conformation (DFG-out) so that the
urea portion occupies the back hydrophobic pocket with additional
hydrogen-bonding interactions. The N1-nitrogen of the pyrrolo[3,2-
d]pyrimidine core interacts with the main chain NH group of Cys919
in the hinge region. The urea moiety binds to the protein through
two hydrogen-bonding interactions: both urea NH groups interact
with Glu885, and the carbonyl interacts with Asp1046. Compounds
missing one of the NH groups (9 or 16) or the carbonyl oxygen (10)
bind more weakly to the protein, suggesting that all of these hydro-
gen bonds contribute significantly to the binding affinity. The exter-
nal 3-(trifluoromethyl)phenyl moiety occupies the hydrophobic
pocket created by the conformational rearrangement of Phe1047
(DFG-out). We can rationalize that the significant loss of activity
caused by replacement of phenyl (7a: VEGFR2 IC50 = 33 nM) with
methyl (7b: VEGFR2 IC50 = 6300 nM) is probably due to insufficient
occupation of this extended hydrophobic pocket.
Thus, we concluded that a 40-urea substituent on the 4-phenoxy-
5H-pyrrolo[3,2-d]pyrimidine scaffold was optimal for the inhibition
of VEGFR2 kinase, and then, we examined the urea substituents. The
results in Table 2 show that phenyl groups seemed to be more pre-
ferred as urea substituents than smaller aliphatic groups. Replace-
ment of phenyl (7a) with methyl (7b) or propyl (7c) brought >100-
fold loss of inhibitory activity against VEGFR2 kinase, indicating that
the small lipophilic substituents were insufficient to occupy the
hydrophobicpocket. Systematic investigation of substituents placed
at the ortho-, meta-, and para-positions of the urea phenyl ring re-
vealed that a meta-substituted phenyl was optimal for inhibitory
activity against VEGFR2 kinase (7e vs 7d,f). In addition to the in-
creased enzymatic activity, 7e also displayed moderate cellular
potency (HUVEC IC50 = 110 nM) in the VEGF-driven HUVEC prolifer-
ation assay. Optimization of substituents at the meta-position re-
vealed that very potent inhibitory activity against VEGFR2 kinase

Y. Oguro et al. / Bioorg. Med. Chem. 18 (2010) 7260–7273
7265
Table 3
Effect of linker atoms on vascular endothelial growth factor receptor 2 kinase and cellular growth inhibitiona
Compd
X
R
VEGFR2 IC50 (nM)
HUVECb IC50 (nM)
7a
7g
18a
18b
18c
O
O
S
NH
H
CF3
H
H
CF3
33 (27–40)
810 (630–1000)
22 (16–30)
35% inhibition at 1 lM
<10% inhibition at 1
<10% inhibition at 1
5.3 (4.4–6.3)
110 (65–170)
1400 (1200–1600)
>10,000
l
M
N(Me)
lM
23
7.1 (6.3–8.1)
230 (170–310)
a
Numbers in parentheses represent 95% confidence interval.
Growth inhibition of human umbilical vein endothelial cells (HUVEC).
b
Table 4
Effects of central phenyl ring substituents on in vitro and pharmacokinetic propertiesa
Compd
R
VEGFR2 IC50 (nM)
HUVECb IC50 (nM)
Mice PKc
d
e
AUC0–8 h
(lg h/mL)
C8h (lg/mL)
7g
H
5.3 (4.4–6.3)
3.7 (3.4–4.1)
30 (25–36)
14 (11–17)
6.2 (4.7–8.3)
22 (16–30)
13 (8.6–20)
39 (25–60)
87 (74–103)
4.4 (3.0–6.4)
11.7
10.5
9.10
14.7
10.9
0.570
0.843
0.584
0.572
0.803
20a
20b
20c
20d
2-Cl
3-Cl
2-OMe
2-F
a
b
c
Numbers in parentheses represent 95% confidence interval.
Growth inhibition of human umbilical vein endothelial cells (HUVEC).
Compounds were orally administered at a dose of 10 mg/kg by cassette dosing.
d
e
Area under the plasma concentration-time curve (
lg h/mL).
Plasma concentration at 8 h after administration.
To characterize the mode of inhibitory activity of 20d, we
Compound 20d was tested for selectivity with respect to other ki-
nases (Table 5). Compound 20d inhibited VEGFR2, tyrosine kinase
with Ig and EGF homology domains-2 (Tie-2), and platelet-derived
growth factor receptors b (PDGFRb) kinases with IC50 values of 6.2,
20, and 96 nM, respectively. These kinases are close to each other
in the kinome tree.31 In addition to VEGF/VEGFR, angiopoietin/Tie-
2 and platelet-derived growth factors (PDGF)/PDGFR signals play
important roles in angiogenesis. Tyrosine phosphorylation of Tie-2
by its ligand angiopoietin-1 (Ang-1) is involved in more mature
angiogenic processes, such as vessel branching, sprouting, remodel-
ing, maturation, and stability.32 Activation of PDGF/PDGFR signal
also induce proliferation and migration of pericytes, which leads
to the formation of thicker and more stable blood vessels.33,34 There-
fore, these additional kinase inhibitory activities of 20d may be ben-
eficial for its antiangiogenic activity. On the other hand, IC50 values
against other protein kinases such as fibroblast growth factor recep-
tor 1 (FGFR1), human epidermal growth factor receptor 2 (HER2),
measured the recovery of enzymatic activity after a large dilution
of the VEGFR2-20d complex (Fig. 3).27,28 After the preformed
complex of 20d and VEGFR2 was diluted to one tenth of IC50 with
buffer including 1 mM of ATP, the recovery of kinase activity was
much slower than control (no inhibitor). The progress curve for
the control reaction (no inhibitor) was essentially linear for
2000 second after dilution. In contrast, the activity of VEGFR2 ki-
nase pre-incubated with 20d was very low at that time, indicat-
ing a unique mode of 20d to inhibit VEGFR2 kinase with very
slow dissociation kinetics. This result suggests DFG-out conforma-
tion of VEGFR2 induced by 20d seems very stable. Slow dissocia-
tion kinetics and lack of biochemical evidence for the formation
of an irreversible covalent bond is called pseudo-irreversible inhi-
bition.29,30 Thus, 20d is pseudo-irreversible inhibitor and may
show prolonged action against VEGFR2 kinase in both in vitro
and in vivo settings.

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Y. Oguro et al. / Bioorg. Med. Chem. 18 (2010) 7260–7273
Figure 2. X-ray co-crystal structure of 20d in complex with VEGFR2.
Figure 4. Efficacy of the hydrochloride of 20d in DU145 tumor xenograft model.
DU145 tumor-bearing mice were orally administered with the hydrochloride of 20d
(1.5, 3, 6, and 12 mg/kg) twice daily for 21 days. Data are shown as mean (standard
deviation, SD) (n = 5). *: p 6 0.025 versus vehicle control as determined by a one-
tailed Williams’ test. Antitumor effects expressed as T/C (growth of treated tumor/
growth of control tumor) ꢂ 100.
genesis-related multikinase inhibitory activity of 20d may contrib-
ute to its potent antitumor activity.
4. Conclusion
In order to generate novel type-II inhibitors of VEGFR kinase, we
designed and synthesized a series of pyrrolo[3,2-d]pyrimidine
derivatives. Incorporation of a diphenylurea moiety at the C4-posi-
tion of the pyrrolo[3,2-d]pyrimidine core via an oxygen linker
afforded potent VEGFR2 kinase inhibitors. Moreover, meta-substi-
tution of the urea terminal benzene ring and substitution with a
small lipophilic group at the 2-position of the central benzene ring
enhanced HUVEC inhibitory activity. In particular, compound 20d
was extremely potent, with an IC50 value of 4.4 nM. A co-crystal
structure between 20d and VEGFR2 revealed that 20d binds to
the inactive conformation of VEGFR2, in which its diphenylurea
moiety occupies the back hydrophobic pocket created by the con-
formational change of DFG motif (DFG-out). Results of further
studies indicated that 20d inhibited PDGFR and Tie-2 kinases in
addition to VEGFR kinase and displayed slow dissociation kinetics.
Oral treatment with the hydrochloride salt of 20d showed potent
antitumor effects in a DU145 mouse xenograft model. Thus, the
slow dissociation kinetics and potent angiogenesis-related multi-
kinase inhibitory activities of this compound class hold great
promise as anticancer agents. Further modification using this urea
series will be reported in due course.
Figure 3. Dilution assay of VEGFR2-20d complex. Phosphorylation of peptide
substrate as a function of time is shown. The reaction was initiated by diluting a
preformed enzyme–inhibitor complex to one tenth of IC50 with reaction buffer
containing 1 mM ATP and 0.1 lg/mL biotinylated poly-GluTyr (4:1). The recovery of
activity was measured using the AlphaScreenÒ system.
Table 5
Kinase inhibition profile of 20da,b
Kinase
IC50 (nM)
Kinase
IC50 (nM)
VEGFR2
VEGFR1
6.2 (4.7–8.3)
15 (14–17)
35 (27–43)
96 (76–120)
>10,000
20 (17–23)
>10,000
>10,000
IGF1-R
c-kit
Src
>10,000
170 (120–240)
>10,000
2600 (2000–3400)
900 (660–1200)
>10,000
>10,000
>10,000
1050 (660–1700)
PDGFR
a
PDGFRb
FGFR1
Tie-2
HER2
EGFR
IR
FAK
B-raf
ERK1
PKCh
GSK3b
AurolaA
>10,000
a
Abbreviations of kinases are described in Section 5.
Numbers in parentheses represent 95% confidence interval.
b
5. Experimental
epidermal growth factor receptor (EGFR), insulin receptor (IR), and
protein kinase C h (PKCh) were over 10,000 nM.
Melting points were determined on a BÜCHI Melting Point B-545,
and were not corrected. Proton nuclear magnetic resonance (1H
NMR) spectra were recorded on Varian Mercury 300 (300 MHz) or
Bruker DPX300 (300 MHz) instruments. Chemical shifts are re-
ported as d values (ppm) downfield from internal tetramethylsilane
of the indicated organic solution. Peak multiplicities are expressed
as follows: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet
of doublet; ddd, doublet of doublet of doublets; dt, doublet of triplet;
br s, broad singlet; m, multiplet. Coupling constants (J values) are gi-
ven in hertz (Hz). Element analyses were carried out by Takeda Ana-
lytical Laboratories. Reaction progress was determined by thin layer
chromatography (TLC) analysis on Silica Gel 60 F254 plate (Merck)
Finally, we evaluated the antitumor effects of the hydrochloride
salt of 20d in a xenograft nude mouse model with the human
DU145 prostate cancer cell line which is known to produce VEGF
(Fig. 4).35 Consistent with its potent in vitro activities and good oral
exposure levels, oral administration of the hydrochloride salt of
20d twice daily at 1.5, 3, 6, and 12 mg/kg for 21 days led to signif-
icant antitumor effects with T/C (treatment over control) values of
38%, 15%, 1%, and ꢁ8%, respectively. Under these conditions, nei-
ther body weight loss nor obvious signs of toxicity were observed
(data not shown). The slow dissociation kinetics and potent angio-

Y. Oguro et al. / Bioorg. Med. Chem. 18 (2010) 7260–7273
7267
or NH TLC plates (Fuji Silysia Chemical Ltd). Chromatographic puri-
fication was carried on Silica Gel columns 60 (0.063–0.200 mm or
0.040–0.063 mm, Merck), basic silica gel (ChromatorexNH, 100–
200 mesh, Fuji Silysia Chemical Ltd) or Purif-Pack (SI 60 lM or NH
60 lM, Fuji Silysia Chemical Ltd). Commercial reagents and solvents
were used without additional purification. Abbreviations are used as
follows: CDCl3, deuterated chloroform; DMSO-d6, dimethyl sulfox-
ide-d6; AcOEt, ethyl acetate; DMF, N,N-dimethylformamide; MeOH,
methanol; THF, tetrahydrofuran; EtOH, ethanol; DMSO, dimethyl
sulfoxide; NMP, N-methylpyrrolidone.
6.61 (1H, d, J = 3.0 Hz), 7.04–7.07 (1H, m), 7.44 (1H, t, J = 8.1 Hz),
7.50–7.68 (4H, m), 7.80 (1H, d, J = 3.0 Hz), 7.84 (1H, t, J = 2.3 Hz),
7.93–7.97 (2H, m), 8.30 (1H, s), 10.41 (1H, s); Anal. Calcd for
C20H16N4O2: C, 69.76; H, 4.68; N, 16.27. Found: C, 69.58; H, 4.53;
N, 16.07.
5.5. 4-[(5-Methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)oxy]aniline
(6)
The compound 6 was prepared from 2 in a manner similar to
that described for 3 to yield a pale yellow solid (60%): 1H NMR
(DMSO-d6) d 4.06 (3H, s), 5.04 (2H, br s), 6.53 (1H, d, J = 3.0 Hz),
6.58 (2H, d, J = 8.7 Hz), 6.91 (2H, d, J = 8.7 Hz), 7.70 (1H, d,
J = 3.0 Hz), 8.21 (1H, s).
5.1. 4-Chloro-5-methyl-5H-pyrrolo[3,2-d]pyrimidine (2)
A mixture of 1 (5.01 g, 32.5 mmol), methyl methanesulfonate
(3.07 g, 34.2 mmol), cesium carbonate (21.2 g, 65.2 mmol), and
DMF (50 mL) was stirred at room temperature for 15 h. The mix-
ture was diluted with water and extracted with AcOEt. The extract
was washed with brine, dried over anhydrous magnesium sulfate,
and concentrated under reduced pressure to give 2 (4.36 g, 80%) as
a pale yellow solid: 1H NMR (DMSO-d6) d 4.09 (3H, s), 6.67–6.68
(1H, m), 7.95 (1H, d, J = 3.0 Hz), 8.57 (1H, s).
5.6. 1-{4-[(5-Methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)oxy]-
phenyl}-3-phenylurea (7a)
To a solution of 6 (150 mg, 0.624 mmol) in DMF (3 mL) was
added N,N0-carbonyldiimidazole (101 mg, 0.624 mmol). After being
stirred at room temperature for 1 h, aniline (61.0 mg, 0.655 mmol)
was added and the resulting mixture was stirred at room temper-
ature for 15 h. The mixture was diluted with water and extracted
with AcOEt. The extract was washed with brine, dried over anhy-
drous magnesium sulfate, and concentrated under reduced pres-
sure. The residue was purified by basic silica gel column
chromatography (AcOEt/hexane) followed by recrystallization
from AcOEt–hexane to give 7a as a white solid: mp 183–186 °C;
1H NMR (DMSO-d6) d 4.04 (3H, s), 6.53 (1H, d, J = 2.9 Hz), 6.90
(1H, t, J = 7.4 Hz), 7.14–7.26 (4H, m), 7.39–7.49 (4H, m), 7.71 (1H,
d, J = 2.9 Hz), 8.21 (1H, s), 8.68 (1H, br s), 8.75 (1H, br s); Anal. Calcd
for C20H17N5O2ꢀ0.2H2O: C, 66.18; H, 4.83; N, 19.29. Found: C, 66.16;
H, 4.73; N, 19.23.
5.2. 3-[(5-Methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)oxy]aniline
(3)
A mixture of 2 (2.08 g, 9.83 mmol), 3-aminophenol (1.29 g,
11.8 mmol), potassium carbonate (3.26 g, 23.6 mmol), and NMP
(15 mL) was stirred at 110 °C for 2 h. The mixture was diluted with
water and extracted with AcOEt. The extract was washed with
brine, dried over anhydrous magnesium sulfate, and concentrated
under reduced pressure to give 3 (1.46 g, 62%) as a white solid: 1H
NMR (DMSO-d6) d 4.07 (3H, s), 5.28 (2H, br s), 6.34–6.50 (3H, m),
6.58 (1H, d, J = 3.2 Hz), 7.06 (1H, t, J = 7.8 Hz), 7.76 (1H, d,
J = 3.2 Hz), 8.28 (1H, s).
The following compounds 7b–h were prepared using a proce-
dure similar to that described for 7a from 6.
5.3. 1-{3-[(5-Methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)oxy]-
phenyl}-3-phenylurea (4)
5.7. 1-Methyl-3-{4-[(5-methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)-
oxy]phenyl}urea (7b)
To a solution of 3 (579 mg, 2.41 mmol), triethylamine (1.00 mL,
7.22 mmol) in THF (10 mL) was added phenyl isocyanate (314
lL,
Yield 26%: mp 190–196 °C (AcOEt–hexane); 1H NMR (DMSO-d6)
d 2.59 (3H, d, J = 4.8 Hz), 4.03 (3H, s), 5.99 (1H, d, J = 4.8 Hz), 6.51
(1H, d, J = 2.4 Hz), 7.08 (2H, d, J = 8.8 Hz), 7.39 (2H, d, J = 8.8 Hz),
7.70 (1H, d, J = 2.4 Hz), 8.19 (1H, s), 8.54 (1H, br s); Anal. Calcd
for C15H15N5O2ꢀ0.15H2O: C, 60.05; H, 5.14; N, 23.34. Found: C,
60.16; H, 5.09; N, 23.09.
2.89 mmol), and the mixture was stirred at room temperature for
15 h. The mixture was diluted with AcOEt and washed with water,
brine, dried over anhydrous magnesium sulfate, and concentrated
under reduced pressure. The residue was purified by basic silica gel
column chromatography (AcOEt/hexane) followed by recrystalliza-
tion from AcOEt–hexane to give 4 (690 mg, 80%) as a white solid:
mp 197–199 °C; 1H NMR (DMSO-d6) d 4.10 (3H, s), 6.60–6.61
(1H, m), 6.88–6.98 (2H, m), 7.20–7.30 (3H, m), 7.35 (1H, t,
J = 8.1 Hz), 7.43 (2H, d, J = 8.1 Hz), 7.57 (1H, t, J = 2.1 Hz), 7.79
(1H, d, J = 3.0 Hz), 8.29 (1H, s), 8.72 (1H, s), 8.86 (1H, s); Anal. Calcd
for C20H17N5O2ꢀ0.1H2O: C, 66.51; H, 4.80; N, 19.39. Found: C, 66.35;
H, 4.77; N, 19.25.
5.8. 1-{4-[(5-Methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)oxy]-
phenyl}-3-propylurea (7c)
Yield 17%: mp 184–187 °C (AcOEt–hexane); 1H NMR (DMSO-d6)
d 0.86 (3H, t, J = 7.5 Hz), 1.37–1.46 (2H, m), 2.99–3.06 (2H, m), 4.07
(3H, s), 6.15 (1H, t, J = 6.0 Hz), 6.55 (1H, d, J = 3.0 Hz), 7.13 (2H, d,
J = 8.7 Hz), 7.43 (2H, d, J = 8.7 Hz), 7.74 (1H, d, J = 3.0 Hz), 8.23
(1H, s), 8.48 (1H, br s); Anal. Calcd for C17H19N5O2: C, 62.75; H,
5.89; N, 21.52. Found: C, 62.56; H, 5.75; N, 21.36.
5.4. N-{3-[(5-Methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)oxy]phenyl}-
benzamide (5)
To a solution of 3 (415 mg, 1.73 mmol), triethylamine (719
5.19 mmol) in THF (10 mL) was added benzoyl chloride (221
l
l
L,
L,
5.9. 1-(2-Chlorophenyl)-3-{4-[(5-methyl-5H-pyrrolo[3,2-d]pyri-
midin-4-yl)oxy]phenyl}urea (7d)
1.90 mmol) at 0 °C, and the mixture was stirred at room tempera-
ture for 15 h. The mixture was diluted with AcOEt and washed
with water, brine, dried over anhydrous magnesium sulfate, and
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (AcOEt/hexane) followed by
recrystallization from AcOEt–hexane to give 5 (262 mg, 44%) as a
white solid: mp 209–212 °C; 1H NMR (DMSO-d6) d 4.12 (3H, s),
Yield 7.0%: mp 196–200 °C (AcOEt–hexane); 1H NMR (DMSO-
d6) d 4.05 (3H, s), 6.53 (1H, d, J = 2.9 Hz), 6.94–7.01 (1H, m),
7.16–7.28 (3H, m), 7.38–7.50 (3H, m), 7.72 (1H, d, J = 2.9 Hz),
8.09–8.13 (1H, m), 8.21 (1H, s), 8.32 (1H, br s), 9.56 (1H, br s); Anal.
Calcd for C20H16ClN5O2ꢀ0.2H2O: C, 60.44; H, 4.16; N, 17.62. Found:
C, 60.40; H, 4.15; N, 17.66.

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Y. Oguro et al. / Bioorg. Med. Chem. 18 (2010) 7260–7273
5.10. 1-(3-Chlorophenyl)-3-{4-[(5-methyl-5H-pyrrolo[3,2-d]pyr-
imidin-4-yl)oxy]phenyl}urea (7e)
(1H, m), 7.30 (2H, d, J = 8.7 Hz), 7.52–7.63 (3H, m), 7.79 (1H, d,
J = 3.0 Hz), 7.85 (2H, d, J = 8.7 Hz), 7.98 (2H, d, J = 7.5 Hz), 8.28
(1H, s), 10.36 (1H, s); Anal. Calcd for C20H16N4O2ꢀ0.1H2O: C,
69.39; H, 4.72; N, 16.18. Found: C, 69.15; H, 4.59; N, 16.40.
Yield 14%: mp 184–186 °C (AcOEt–hexane); 1H NMR (DMSO-d6)
d 4.04 (3H, s), 6.53 (1H, d, J = 2.8 Hz), 6.94–6.98 (1H, m), 7.15–7.25
(4H, m), 7.46 (2H, d, J = 8.8 Hz), 7.66 (1H, s), 7.71 (1H, d, J = 2.8 Hz),
8.21 (1H, s), 8.78 (1H, br s), 8.85 (1H, br s); Anal. Calcd for
5.17. N-{4-[(5-Methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)oxy]-
phenyl}-2-phenylacetamide (9)
C20H16ClN5O2: C, 60.99; H, 4.09; N, 17.78. Found: C, 60.71; H,
4.21; N, 17.84.
The compound 9 was prepared from 6 in a manner similar to
that described for 5 to yield a white solid (84%): mp 164–165 °C
(AcOEt–hexane); 1H NMR (DMSO-d6) d 3.66 (2H, s), 4.09 (3H, s),
6.58 (1H, d, J = 3.0 Hz), 7.22–7.38 (7H, m), 7.67 (2H, d, J = 9.0 Hz),
7.77 (1H, d, J = 3.0 Hz), 8.26 (1H, s), 10.26 (1H, br s); Anal. Calcd
for C21H18N4O2ꢀ0.2H2O: C, 69.68; H, 5.12; N, 15.48. Found: C,
69.80; H, 5.15; N, 15.20.
5.11. 1-(4-Chlorophenyl)-3-{4-[(5-methyl-5H-pyrrolo[3,2-d]pyr-
imidin-4-yl)oxy]phenyl}urea (7f)
Yield 18%: mp 214–218 °C (AcOEt–hexane); 1H NMR (DMSO-d6)
d 4.04 (3H, s), 6.53 (1H, d, J = 3.1 Hz), 7.16 (2H, d, J = 8.8 Hz), 7.26
(2H, d, J = 8.6 Hz), 7.24–7.48 (4H, m), 7.71 (1H, d, J = 3.1 Hz), 8.21
(1H, s), 8.73 (1H, br s), 8.78 (1H, br s); Anal. Calcd for C20H16ClN5O2:
C, 60.99; H, 4.09; N, 17.78. Found: C, 60.80; H, 4.13; N, 17.83.
5.18. 1-{4-[(5-Methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)oxy]-
phenyl}-3-phenylthiourea (10)
5.12. 1-{4-[(5-Methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)oxy]-
To a solution of 6 (1.10 g, 4.58 mmol), triethylamine (1.90 mL,
phenyl}-3-[3-(trifluoromethyl)phenyl]urea (7g)
13.7 mmol) in THF (10 mL) was added phenyl isothiocyanate
(657 lL, 5.44 mmol), and the mixture was stirred at room temper-
Yield 14%: mp 188–190 °C (AcOEt–hexane); 1H NMR (DMSO-d6)
d 4.04 (3H, s), 6.53 (1H, d, J = 2.8 Hz), 7.15–7.27 (3H, m), 7.41–7.51
(4H, m), 7.71 (1H, d, J = 2.8 Hz), 7.98 (1H, s), 8.21 (1H, s), 8.94 (1H,
br s), 9.14 (1H, br s); Anal. Calcd for C21H16F3N5O2: C, 59.02; H,
3.77; N, 16.39. Found: C, 58.77; H, 3.74; N, 16.22.
ature for 15 h. The mixture was diluted with AcOEt and washed
with water, brine, dried over anhydrous magnesium sulfate, and
concentrated under reduced pressure. The residue was purified
by basic silica gel column chromatography (AcOEt/hexane) fol-
lowed by recrystallization from AcOEt–hexane to give 10 (1.29 g,
75%) as a white solid: mp 155–157 °C; 1H NMR (DMSO-d6) d 4.11
(3H, s), 6.60 (1H, d, J = 3.2 Hz), 7.10–7.16 (1H, m), 7.24–7.38 (4H,
m), 7.47–7.58 (4H, m), 7.78 (1H, d, J = 3.2 Hz), 8.29 (1H, s), 9.84
(2H, s); Anal. Calcd for C20H17N5OS: C, 63.98; H, 4.56; N, 18.65.
Found: C, 63.67; H, 4.60; N, 18.52.
5.13. 1-(3-Bromophenyl)-3-{4-[(5-methyl-5H-pyrrolo[3,2-d]pyr-
imidin-4-yl)oxy]phenyl}urea (7h)
Yield 16%: mp 207–211 °C (AcOEt–hexane); 1H NMR (DMSO-d6)
d 4.08 (3H, s), 6.57 (1H, d, J = 3.0 Hz), 7.13 (1H, d, J = 8.0 Hz), 7.20–
7.24 (3H, m), 7.31 (1H, d, J = 8.0 Hz), 7.51 (2H, d, J = 9.0 Hz), 7.75
(1H, d, J = 3.0 Hz), 7.85 (1H, t, J = 2.1 Hz), 8.25 (1H, s), 8.88 (1H,
br s), 8.95 (1H, br s); Anal. Calcd for C20H16BrN5O2: C, 54.81; H,
3.68; N, 15.98. Found: C, 54.76; H, 3.71; N, 15.84.
5.19. N-{4-[(5-Methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)oxy]-
phenyl}-1H-benzimidazol-2-amine (11)
A mixture of 6 (475 mg, 1.98 mmol), 2-chloro-1H-benzimid-
azole (322 mg, 2.18 mmol), and NMP (5 mL) was stirred at 120 °C
for 15 h. The mixture was diluted with water and extracted with
AcOEt. The extract was washed with brine, dried over anhydrous
magnesium sulfate, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography
(AcOEt/hexane) followed by recrystallization from AcOEt–hexane
to give 11 (66.0 mg, 9.3%) as a white solid: mp 219–222 °C; 1H
NMR (DMSO-d6) d 4.12 (3H, s), 6.59 (1H, d, J = 3.0 Hz), 6.94–7.04
(2H, m), 7.22–7.38 (4H, m), 7.77 (1H, d, J = 3.0 Hz), 7.83 (2H, d,
J = 9.0 Hz), 8.27 (1H, s), 9.52 (1H, s), 10.97 (1H, s); Anal. Calcd for
5.14. 1-(3-Fluorophenyl)-3-{4-[(5-methyl-5H-pyrrolo[3,2-d]pyr-
imidin-4-yl)oxy]phenyl}urea (7i)
The compound 7i was prepared from 6 in a manner similar to
that described for 4 to yield a white solid (59%): mp 189–191 °C
(AcOEt–hexane); 1H NMR (DMSO-d6) d 4.08 (3H, s), 6.56–6.57
(1H, m), 6.73–6.57 (1H, m), 7.10–7.13 (1H, m), 7.20–7.32 (3H,
m), 7.46–7.52 (3H, m), 7.75 (1H, d, J = 3.3 Hz), 8.25 (1H, s), 8.81
(1H, s), 8.91 (1H, br s); Anal. Calcd for C20H16FN5O2: C, 63.65; H,
4.27; N, 18.56. Found: C, 63.54; H, 4.11; N, 18.49.
C20H16N6O: C, 67.40; H, 4.53; N, 23.58. Found: C, 67.11; H, 4.43;
N, 23.52.
5.15. 1-(3-Methylphenyl)-3-{4-[(5-methyl-5H-pyrrolo[3,2-d]pyr-
imidin-4-yl)oxy]phenyl}urea (7j)
5.20. 3-[(5-Methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)oxy]benzoic
acid (12a)
The compound 7j was prepared from 6 in a manner similar to
that described for 4 to yield a white solid (76%): mp 174–176 °C
(AcOEt–hexane); 1H NMR (DMSO-d6) d 2.26 (3H, s), 4.09 (3H, s),
6.57 (1H, d, J = 3.2 Hz), 6.77 (1H, d, J = 7.2 Hz), 7.11–7.29 (5H, m),
7.48–7.51 (2H, m), 7.75 (1H, d, J = 3.2 Hz), 8.25 (1H, s), 8.59 (1H,
s), 8.71 (1H, br s); Anal. Calcd for C21H19N5O2: C, 67.55; H, 5.13;
N, 18.76. Found: C, 67.32; H, 5.17; N, 18.50.
A mixture of 2 (2.23 g, 13.3 mmol), 3-hydroxybenzoic acid
(2.02 g, 14.6 mmol), cesium carbonate (13.0 g, 39.9 mmol), and
DMSO (20 mL) was stirred at 100 °C for 4 h. The mixture was acid-
ified with 1 M HCl and the resulting solid was collected to give 12a
(3.48 g, 76%) as a white solid: 1H NMR (DMSO-d6) d 4.12 (3H, s),
6.62 (1H, d, J = 3.0 Hz), 7.58–7.64 (2H, m), 7.80–7.91 (3H, m),
8.30 (1H, s), 13.21 (1H, br s).
5.16. N-{4-[(5-Methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)oxy]-
phenyl}benzamide (8)
5.21. 4-[(5-Methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)oxy]benzoic
acid (12b)
The compound 8 was prepared from 6 in a manner similar to
that described for 5 to yield a white solid (7.6%): mp 209–212 °C
(AcOEt–hexane); 1H NMR (DMSO-d6) d 4.12 (3H, s), 6.59–6.60
The compound 12b was prepared from 2 in a manner similar to
that described for 12a to yield a white solid (72%): 1H NMR (DMSO-

Y. Oguro et al. / Bioorg. Med. Chem. 18 (2010) 7260–7273
7269
d6) d 4.10 (3H, s), 6.62 (1H, d, J = 3.3 Hz), 7.44 (2H, d, J = 8.9 Hz),
7.81 (1H, d, J = 3.3 Hz), 8.04 (2H, d, J = 8.9 Hz), 8.31 (1H, s), 13.02
5.27. 4-[(5-Methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)sulfanyl]-
aniline (17a)
(1H, br s).
The compound 17a was prepared from 2 in a manner similar to
that described for 3 to yield a white solid (39%): 1H NMR (DMSO-
d6) d 4.16 (3H, s), 5.56 (2H, s), 6.52 (1H,d, J = 3.0 Hz), 6.62 (2H, d,
J = 8.7 Hz), 7.19 (2H, d, J = 8.7 Hz), 7.74 (1H, d, J = 3.0 Hz), 8.37
(1H, s).
5.22. 3-[(5-Methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)oxy]-N-
phenylbenzamide (13a)
To a solution of 12a (552 mg, 2.05 mmol), 1-hydroxybenzotria-
zole (415 mg, 2.05 mg), and aniline (187 lL, 2.05 mmol) in DMF
(5 mL) was added 1-ethyl-3-(3-dimethylaminopropyl) carbodiim-
ide hydrochloride (589 mg, 3.07 mmol) at 0 °C, and the mixture
was stirred at room temperature for 15 h. The mixture was diluted
with water and extracted with AcOEt. The extract was washed with
brine, dried over anhydrous magnesium sulfate, and concentrated
under reduced pressure. The residue was purified by basic silica gel
column chromatography (AcOEt/hexane) followed by recrystalliza-
tion from AcOEt–hexane to give 13a (496 mg, 70%) as a white so-
lid: mp 223–228 °C; 1H NMR (DMSO-d6) d 4.14 (3H, s), 6.62 (1H,
d, J = 3.0 Hz), 7.07–7.13 (1H, m), 7.32–7.38 (2H, m), 7.54–7.67
(2H, m), 7.75–7.82 (3H, m), 7.89–7.92 (2H, m), 8.30 (1H, s), 10.31
(1H, s); Anal. Calcd for C20H16N4O2: C, 69.76; H, 4.68; N, 16.27.
Found: C, 69.48; H, 4.59; N, 16.03.
5.28. N-(5-Methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)benzene-1,4-
diamine (17b)
A mixture of 2 (2.06 g, 12.3 mmol), 4-nitroaniline (2.04 g,
14.7 mmol), 4 M HCl in AcOEt (5.5 mL), and 2-propanol (10 mL)
was stirred at 80 °C for 1 h. The mixture was diluted with AcOEt
and washed with water, brine, dried over anhydrous magnesium
sulfate, and concentrated under reduced pressure. The residue was
dissolved in MeOH (10 mL) and 10% palladium on carbon (water
ꢃ50%, 100 mg) was added. The resulting mixture was stirred under
a hydrogen atmosphere at room temperature for 3 h. The catalyst
wasfilteredoff, and thefiltrate wasconcentratedinvacuo. Theresid-
ual solid was collected and washed with AcOEt–hexane to give 17b
(867 mg, 88%) as a white solid: 1H NMR (DMSO-d6) d 4.10 (3H, s),
4.90 (2H, s), 6.33 (1H, d, J = 3.0 Hz), 6.55 (2H, d, J = 8.4 Hz), 7.17
(2H, d, J = 8.4 Hz), 7.44 (1H, d, J = 3.0 Hz), 7.99 (1H, s), 8.08 (1H, s).
5.23. 4-[(5-Methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)oxy]-N-
phenylbenzamide (13b)
The compound 13b was prepared from 12b in a manner similar
to that described for 3 to yield a white solid (28%): mp 157–160 °C
(AcOEt–hexane); 1H NMR (DMSO-d6) d 4.12 (3H, s), 6.62 (1H, d,
J = 3.0 Hz), 7.10 (1H, t, J = 7.7 Hz), 7.36 (2H, t, J = 7.7 Hz), 7.48 (2H,
d, J = 8.6 Hz), 7.78–7.83 (3H, m), 8.06 (2H, d, J = 8.6 Hz), 8.31 (1H,
s), 10.30 (1H, s); Anal. Calcd for C20H16N4O2: C, 69.76; H, 4.68; N,
16.27. Found: C, 69.74; H, 4.63; N, 16.34.
5.29. N-Methyl-N-(5-methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)-
benzene-1,4-diamine (17c)
To
a suspension of sodium hydride (60% in oil, 300 mg,
7.5 mmol) in DMF (10 mL) was added N-methyl-4-nitroaniline
(761 mg, 5.0 mmol) at 0 °C, and the mixture was stirred. After
30 min, 2 was added to the mixture, and the resulting mixture
was stirred at room temperature for 18 h. The mixture was diluted
with AcOEt and washed with water, brine, dried over anhydrous
magnesium sulfate, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography
(AcOEt/hexane) to give a yellow solid. The solid thus obtained
was dissolved in MeOH (30 mL) and 10% palladium on carbon
(water ꢃ50%, 162 mg) was added. The resulting mixture was stir-
red under a hydrogen atmosphere at room temperature for 6 h.
The catalyst was filtered off, and the filtrate was concentrated in
vacuo. The residue was purified by basic silica gel column chroma-
tography (AcOEt/hexane). The residual solid was collected and
washed with AcOEt–hexane to give 17c (328 mg, 1.29 mmol,
91%) as a white solid: 1H NMR (DMSO-d6) d 3.08 (3H, s), 3.36
(3H, s), 5.06 (2H, s), 6.39 (1H, d, J = 3.0 Hz), 6.51 (2H, d,
J = 8.7 Hz), 6.68 (2H, d, J = 8.7 Hz), 7.36 (1H, d, J = 3.0 Hz), 8.39
(1H, s).
5.24. Ethyl {4-[(5-methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)oxy]-
phenyl}acetate (14)
The compound 14 was prepared from 2 in a manner similar to
that described for 12a to yield a white solid (88%): 1H NMR
(DMSO-d6) d 1.28 (3H, t, J = 7.2 Hz), 3.65 (2H, s), 4.14 (3H, s), 4.17
(2H, q, J = 7.2 Hz), 6.65 (1H, d, J = 3.0 Hz), 7.22 (2H, d, J = 8.4 Hz),
7.32 (1H, d, J = 3.0 Hz), 7.39 (2H, d, J = 8.4 Hz), 8.44 (1H, s).
5.25. {4-[(5-Methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)oxy]-
phenyl}acetic acid (15)
A mixture of 14 (990 mg, 3.2 mmol), 8 M NaOH (1 mL), and
MeOH (10 mL) was stirred at room temperature for 18 h. The mix-
ture was acidified with 1 M HCl and the resulting solid was col-
lected followed by recrystallization from MeOH to give 15
(669 mg, 74%) as a white solid: 1H NMR (DMSO-d6) d 3.62 (2H,
s), 4.10 (3H, s), 6.59 (1H, d, J = 3.2 Hz), 7.25 (2H, d, J = 8.6 Hz),
7.35 (2H, d, J = 8.6 Hz), 7.78 (1H, d, J = 3.2 Hz), 8.28 (1H, s), 12.37
(1H, br s).
5.30. 1-{4-[(5-Methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)sulfanyl]-
phenyl}-3-phenylurea (18a)
The compound 18a was prepared from 17a in a manner similar
to that described for 4 to yield a white solid (75%): mp 204–207 °C
(AcOEt–hexane); 1H NMR (DMSO-d6) d 4.19 (3H, s), 6.55 (1H, d,
J = 3.0 Hz), 6.95–7.01 (1H, m), 7.26–7.32 (2H, m), 7.45–7.60 (6H,
m), 7.79 (1H, d, J = 3.0 Hz), 8.40 (1H, s), 8.78 (1H, s), 8.94 (1H, s);
Anal. Calcd for C20H79N5OS: C, 63.98; H, 4.56; N, 18.65. Found: C,
64.11; H, 4.53; N, 18.63.
5.26. 2-{4-[(5-Methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)oxy]-
phenyl}-N-phenylacetamide (16)
The compound 16 was prepared from 15 in a manner similar to
that described for 13a to yield a white solid (81%): mp 236–240 °C
(AcOEt–hexane); 1H NMR (DMSO-d6) d 3.69 (2H, s), 4.10 (3H, s),
6.59 (1H, d, J = 3.0 Hz), 7.05 (1H, t, J = 7.5 Hz), 7.26 (2H, d,
J = 8.7 Hz), 7.32 (2H, d, J = 7.5 Hz), 7.42 (2H, d, J = 8.7 Hz), 7.61
(2H, d, J = 7.5 Hz), 7.78 (1H, d, J = 3.0 Hz), 8.27 (1H, s), 10.18 (1H,
br s); Anal. Calcd for C21H18N4O2: C, 70.38; H, 5.06; N, 15.63.
Found: C, 70.21; H, 5.02; N, 15.70.
5.31. 1-{4-[(5-Methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)amino]-
phenyl}-3-phenylurea (18b)
The compound 18b was prepared from 17b in a manner similar
to that described for 4 to yield a white solid (13%): mp 207–210 °C

7270
Y. Oguro et al. / Bioorg. Med. Chem. 18 (2010) 7260–7273
(AcOEt–hexane); 1H NMR (DMSO-d6) d 4.14 (3H, s), 6.40 (1H, d,
J = 3.3 Hz), 6.92–6.99 (1H, m), 7.24–7.56 (8H, m), 8.20 (1H, s),
8.29 (1H, s), 8.55–8.66 (3H, m); Anal. Calcd for C20H18N6Oꢀ0.5H2O:
C, 65.38; H, 5.21; N, 22.87. Found: C, 65.59; H, 5.01; N, 22.87.
J = 3.2 Hz), 6.77–6.89 (2H, m), 7.05 (1H, dd, J = 12.0, 2.1 Hz), 7.75
(1H, d, J = 3.2 Hz), 8.26 (1H, s).
5.37. 1-{2-Chloro-4-[(5-methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)-
oxy]phenyl}-3-[3-(trifluoromethyl)phenyl]urea (20a)
5.32. 1-{4-[Methyl(5-methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)-
amino]phenyl}-3-[3-(trifluoromethyl)phenyl]urea (18c)
The compound 20a was prepared from 19a in a manner similar
to that described for 4 to yield a white solid (61%): mp 192–194 °C
(AcOEt–hexane); 1H NMR (DMSO-d6) d 4.11 (3H, s), 6.61 (1H, d,
J = 3.0 Hz), 7.30–7.36 (2H, m), 7.52–7.58 (3H, m), 7.80 (1H, d,
J = 3.0 Hz), 8.06 (1H, br s), 8.18 (1H, d, J = 8.7 Hz), 8.31 (1H, s),
8.45 (1H, br s), 9.73 (1H, br s); Anal. Calcd for C21H15ClF3N5O2: C,
54.61; H, 3.27; N, 15.16. Found: C, 54.53; H, 3.25; N, 15.17.
The compound 18c was prepared from 17c in a manner similar
to that described for 4 to yield a white solid (80%): mp 170–174 °C
(AcOEt–hexane); 1H NMR (DMSO-d6) d 3.18 (3H, s), 3.47 (3H, s),
6.48 (1H, d, J = 3.0 Hz), 6.88 (2H, d, J = 8.9 Hz), 7.30 (1H, d,
J = 7.5 Hz), 7.40 (2H, d, J = 8.9 Hz), 7.45–7.65 (3H, m), 8.01 (1H, s),
8.50 (1H, s), 8.80 (1H, s), 9.03 (1H, s); Anal. Calcd for C22H19F3N6O:
C, 60.00; H, 4.35; N, 19.08. Found: C, 59.99; H, 4.30; N, 18.92.
5.38. 1-{3-Chloro-4-[(5-methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)-
oxy]phenyl}-3-[3-(trifluoromethyl)phenyl]urea (20b)
5.33. 2-Chloro-4-[(5-methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)-
oxy]aniline (19a)
The compound 20b was prepared from 19b in a manner similar
to that described for 4 to yield a white solid (85%): mp 241–242 °C
(AcOEt–hexane); 1H NMR (DMSO-d6) d 4.10 (3H, s), 6.59–6.60 (1H,
m), 7.31 (1H, d, J = 8.0 Hz), 7.37–7.43 (2H, m), 7.51 (1H, t,
J = 8.0 Hz), 7.59 (1H, d, J = 8.7 Hz), 7.79 (1H, d, J = 3.3 Hz), 7.84–
7.85 (1H, m), 8.01 (1H, s), 8.25 (1H, s), 9.06 (1H, s), 9.16 (1H, br
s); Anal. Calcd for C21H15ClF3N5O2: C, 54.61; H, 3.27; N, 15.16.
Found: C, 54.59; H, 3.29; N, 15.03.
A mixture of 2 (168 mg, 1.0 mmol), 4-amino-3-chlorophenol
(215 mg, 1.5 mmol), potassium carbonate (415 mg, 3.0 mmol),
and NMP (3 mL) was stirred at 120 °C for 18 h. The mixture was
diluted with water and extracted with AcOEt. The extract was
washed with brine, dried over anhydrous magnesium sulfate, and
concentrated under reduced pressure. The residue was purified
by basic silica gel column chromatography (AcOEt/hexane) to give
19a (100 mg, 36%) as a white solid: 1H NMR (DMSO-d6) d 4.05 (2H,
br s), 4.13 (3H, s), 6.64 (1H, d, J = 3.0 Hz), 6.84 (1H, d, J = 8.7 Hz),
7.00 (1H, dd, J = 8.7, 2.4 Hz), 7.20 (1H, d, J = 2.4 Hz), 7.31 (1H, d,
J = 3.0 Hz), 8.45 (1H, s).
5.39. 1-{2-Methoxy-4-[(5-methyl-5H-pyrrolo[3,2-d]pyrimidin-
4-yl)oxy]phenyl}-3-[3-(trifluoromethyl)phenyl]urea (20c)
The compound 20c was prepared from 19c in a manner similar
to that described for 4 to yield a white solid (54%): mp 173–177 °C
(AcOEt–hexane); 1H NMR (DMSO-d6) d 3.38 (3H, s), 4.12 (3H, s),
6.60 (1H, d, J = 3.3 Hz), 6.86 (1H, dd, J = 8.9, 2.3 Hz), 7.07 (1H, d,
J = 2.3 Hz), 7.30–7.34 (1H, m), 7.52–7.54 (2H, m), 7.78 (1H, d,
J = 3.3 Hz), 8.06 (1H, s), 8.15 (1H, d, J = 8.9 Hz), 8.29 (1H, s), 8.33
(1H, s), 9.67 (1H, s); Anal. Calcd for C22H18F3N5O3ꢀ0.2H2O: C,
57.32; H, 4.02; N, 15.19. Found: C, 57.22; H, 3.85; N, 15.12.
5.34. 3-Chloro-4-[(5-methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)-
oxy]aniline (19b)
The compound 19b was prepared from 2 in a manner similar to
that described for 19a to yield a white solid (32%): 1H NMR (DMSO-
d6) d 4.07 (3H, s), 5.35 (2H, br s), 6.56–6.58 (2H, m), 6.69–6.70 (1H,
m), 7.07 (1H, d, J = 8.7 Hz), 7.74 (1H, d, J = 2.7 Hz), 8.22 (1H, s).
5.40. 1-{2-Fluoro-4-[(5-methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)-
oxy]phenyl}-3-[3-(trifluoromethyl)phenyl]urea (20d)
5.35. 2-Methoxy-4-[(5-methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)-
oxy]aniline (19c)
The compound 20d was prepared from 19d in a manner similar
to that described for 4 to yield a white solid (87%): mp 196–198 °C
(AcOEt–hexane); 1H NMR (DMSO-d6) d 4.10 (3H, s), 6.61 (1H, d,
J = 3.0 Hz), 7.12–7.19 (1H, m), 7.30–7.44 (2H, m), 7.50–7.60 (2H,
m), 7.79 (1H, d, J = 3.0 Hz), 8.05 (1H, s), 8.13 (1H, t, J = 9.3 Hz),
8.30 (1H, s), 8.67 (1H, s), 9.41 (1H, s); Anal. Calcd for C21H15F4N5O2:
C, 56.63; H, 3.39; N, 15.72. Found: C, 56.62; H, 3.24; N, 15.78.
The compound 19c was prepared from 2 in a manner similar to
that described for 19a to yield a white solid (61%): 1H NMR (DMSO-
d6) d 3.74 (3H, s), 4.08 (3H, s), 4.67 (2H, s), 6.55–6.67 (3H, m), 6.78
(1H, d, J = 2.1 Hz), 7.73 (1H, d, J = 3.0 Hz), 8.24 (1H, s).
5.36. 2-Fluoro-4-[(5-methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)-
oxy]aniline (19d)
5.41. 4-Chloro-5-(2-chloroethyl)-5H-pyrrolo[3,2-d]pyrimidine (21)
A mixture of 2 (550 mg, 3.28 mmol), 3-fluoro-4-nitrophenol
(619 mg, 3.94 mmol), and o-xylene (20 mL) was stirred at 100 °C
for 48 h. The mixture was diluted with AcOEt and washed with
water, brine, dried over anhydrous magnesium sulfate, and con-
centrated under reduced pressure. The residual solid was collected
and washed with AcOEt–hexane to give 4-(3-fluoro-4-nitrophen-
oxy)-5-methyl-5H-pyrrolo[3,2-d] pyrimidine (725 mg, 77%) as a
white solid. A mixture of 4-(3-fluoro-4-nitrophenoxy)-5-methyl-
5H-pyrrolo[3,2-d] pyrimidine (715 mg, 0.867 mmol), zinc (1.62 g,
24.8 mmol), ammonium chloride (531 mg, 9.92 mmol) and MeOH
(10 mL) was stirred at reflux for 1 h. The mixture was filtered
through Celite, and the filtrate was concentrated under reduced
pressure. The residual solid was collected and washed with
AcOEt–hexane to give 19d (256 mg, 40%) as a white solid: 1H
The compound 21 was prepared from 1 in a manner similar to
that described for 2 to yield a white solid (54%): 1H NMR (DMSO-
d6) d 4.10 (2H, t, J = 5.9 Hz), 4.72 (2H, t, J = 5.9 Hz), 6.51 (1H, d,
J = 3.0 Hz), 7.22 (1H, d, J = 3.0 Hz), 8.75 (1H, s).
5.42. 1-(4-{[5-(2-Chloroethyl)-5H-pyrrolo[3,2-d]pyrimidin-4-yl]-
amino}phenyl)-3-[3-(trifluoromethyl)phenyl]urea (22)
A mixture of 21 (318 mg, 1.47 mmol), 25 (493 mg, 1.49 mmol),
and 2-propanol (10 mL) was stirred at 80 °C for 1 h. The mixture
was diluted with AcOEt and washed with water, brine, dried over
anhydrous magnesium sulfate, and concentrated under reduced
pressure. The residue was purified by basic silica gel column chro-
matography (AcOEt/MeOH) to give 22 (466 mg, 67%) as a white so-
lid: 1H NMR (DMSO-d6) d 4.28–4.32 (2H, m), 4.48–4.53 (2H, m),
NMR (DMSO-d6)
d 4.08 (3H, s), 5.11 (2H, s), 6.57 (1H, d,

Y. Oguro et al. / Bioorg. Med. Chem. 18 (2010) 7260–7273
7271
6.58 (1H, d, J = 2.9 Hz), 7.32–7.59 (8H, m), 7.82 (1H, d, J = 2.9 Hz),
8.02 (1H, s), 8.49 (1H, s), 9.26 (1H, s), 9.44 (1H, s).
pooled and then the tag was removed by cleavage with rTEV prote-
ase, followed by a Mono-Q anion exchange column pre-equilibrated
in 20 mM Tris–HCl pH 8.0, 50 mM NaCl and 5 mM DTT and eluted
using a linear gradient to 20 mM Tris–HCl pH 8.0, 300 mM NaCl
and 5 mM DTT. Fractions containing the non-phosphorylated pro-
tein species were pooled and exchanged its buffer to 50 mM HEPES
pH 7.5, 25 mM NaCl, 10 mM DTT and 10% glycerol by a HiPrep
desalting column. Inhibitor in DMSO solution was added to a solu-
tion of approximately 8 mg/mL protein with gentle stirring to give
a final concentration of 0.5 mM, and the enzyme–inhibitor complex
was then incubated for 3 h on ice.
Crystallization experiments were performed at 4 °C by mixing
100 nL of enzyme/inhibitor (20d) solution with 100 nL of precipi-
tant solution containing 1.3 M tri-sodium citrate. Crystals were
harvested by mixing precipitant solution with the cryoprotectant
glycerol to a final concentration of 24% and flash-frozen by direct
immersion in liquid nitrogen. X-ray diffraction data of the crystals
5.43. 1-[4-(5,6-Dihydro-4H-pyrrolo[3,2,1-de]pteridin-4-yl)-
phenyl]-3-[3-(trifluoromethyl)phenyl]urea (23)
A mixture of 22 (450 mg, 0.948 mmol), potassium carbonate
(392 mg, 2.84 mmol), and DMF (10 mL) was stirred at 80 °C for
15 h. The mixture was diluted with AcOEt and washed with water,
brine, dried over anhydrous magnesium sulfate, and concentrated
under reduced pressure. The residue was purified by basic silica gel
column chromatography (AcOEt) followed by recrystallization
from AcOEt–hexane to give 23 (260 mg, 63%) as a white solid:
mp 197–200 °C; 1H NMR (DMSO-d6) d 4.16–4.20 (2H, m), 4.39–
4.43 (2H, m), 6.45 (1H, d, J = 2.7 Hz), 7.31 (1H, d, J = 7.5 Hz),
7.43–7.60 (7H, m), 8.03 (1H, s), 8.20 (1H, m), 8.89 (1H, s), 9.09
(1H, s); Anal. Calcd for C22H17F3N6O: C, 60.27; H, 3.91; N, 19.17.
Found: C, 60.17; H, 3.80; N, 19.21.
were collected at 100 K with RIGAKU in-house CuKa X-ray gener-
ator and RAXIS image-plate system, and at SPring-8 in Harima, Ja-
pan on beam-line BL32B2 at a wavelength of 1 Å, respectively. The
crystals belong to the monoclinic space group C2 with approximate
cell dimensions 135 ꢂ 56 ꢂ 52 Å, b = 95°, diffracting to 1.55 Å res-
olution. The structure was solved by molecular replacement with
MOLREP in the CCP4 program suite using the apo structure of VEG-
FR2 and refined with CNX2002. The final refined crystallographic
statistics for the co-crystal structure with 20d are R = 18.6%
(Rfree = 20.9%) with root-mean-square deviations (RMSD) in bond
lengths and angles of 0.007 Å and 1.2°, respectively.
5.44. 1-(4-Aminophenyl)-3-[3-(trifluoromethyl)phenyl]urea
hydrochloride (25)
To a solution of 4-nitroaniline (2.02 g, 14.6 mmol), triethyl-
amine (6.07 mL, 43.8 mmol) in THF (10 mL) was added 3-(trifluo-
romethyl)phenyl isocyanate (2.45 mL, 17.5 mmol), and the
mixture was stirred at room temperature for 15 h. The mixture
was diluted with AcOEt and washed with water, brine, dried over
anhydrous magnesium sulfate, and concentrated under reduced
pressure. The residue was dissolved in MeOH (10 mL) and 10% pal-
ladium on carbon (water ꢃ50%, 100 mg) was added. The resulting
mixture was stirred under a hydrogen atmosphere at room tem-
perature for 3 h. The catalyst was filtered off, and 4 M HCl/AcOEt
(4 mL) was added to the filtrate. The resulting solid was collected
and washed with AcOEt–hexane to give 25 (1.42 g, 29%) as a white
solid: 1H NMR (DMSO-d6) d 7.26–7.32 (3H, s), 7.47–7.59 (4H, m),
8.00 (1H, s), 9.49 (1H, s), 9.65 (1H, s), 9.93 (3H, br s).
5.47. Measurement of inhibitory activities against VEGFR2,
PDGFRb and FGFR1 kinases
VEGFR2, PDGFRb and FGFR1 kinase activities were determined
by use of an anti-phosphotyrosine antibody with quantitation per-
formed through the AlphaScreenÒ system (PerkinElmer, USA). For
VEGFR2, enzyme reactions were performed in 50 mM Tris–HCl
pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.01% Tween-20 and 2 mM
5.45. Docking model of VEGFR2
DTT, containing 10 lM ATP, 0.1 lg/mL biotinylated poly-GluTyr
(4:1) and 0.1 nM of VEGFR2 (Millipore, UK). For PDGFRb, the kinase
Pyrrolo[3,2-d]pyrimidine derivative (A) was docked into the
crystal structure of VEGFR2 (PDB ID: 1VR2) by using GOLD (version
3.2, 2007, The Cambridge Crystallographic Data Centre, UK). After
docking calculation, obtained complex model structure was ener-
getically optimized on MOE platform (version 2006.0804, 2006,
Chemical Computing Group, Canada). Optimization was performed
on the following conditions: MMFF94s for force field setting, 4 ꢂ r
(r = interatomic distance) for the dielectric constant setting, AM-
BER99 for the partial charge setting for all atoms of protein, and
AM1-BCC for the partial charge setting for ligand.
assay was performed as described above with 0.8 nM of PDGFRb
(Millipore) and 20
formed as described above with 0.1 nM of FGFR1 (ProQinase
GmbH, Germany) and 0.2 M of ATP.
lM of ATP. For FGFR1, kinase assay was per-
l
Prior to catalytic initiation with ATP, compound and enzyme
were incubated for 5 min at room temperature. The reactions were
quenched by the addition of 25
lL of 100 mM EDTA, 10
lg/mL
AlphaScreen streptavidine donor beads and 10
l
g/mL acceptor
beads in 62.5 mM HEPES pH 7.4, 250 mM NaCl, and 0.1% BSA.
Plates were incubated in the dark overnight and then read by EnVi-
sion 2102 Multilabel Reader (PerkinElmer). Wells containing the
substrate and the enzyme without compound were used as total
reaction control. Wells containing biotinylated poly-GluTyr (4:1)
and enzyme without ATP were used as basal control. The concen-
tration of inhibitor producing 50% inhibition of the kinase activities
(IC50 values) and 95% confidence intervals (95% CI) for VEGFR2,
PDGFRb and FGFR1 were analyzed using GraphPad Prism version
5.01, GraphPad Software (USA). Sigmoidal dose–response (variable
slope) curves were fitted using non-linear regression analysis, with
the top and bottom of the curve constrained at 100 and 0,
respectively.
5.46. Expression, purification, crystallization and structure
determination
The gene for human VEGFR2 (residue 806–1171 except a kinase
insertion domain (KID) region (940–989)) was cloned into a pFast-
BacHT (Invitrogen, USA) baculovirus expression vector with an
N-terminal 6-Histidine tag containing an recombinant Tobacco Etch
Virus (rTEV) protease cleavage site. The protein was expressed in Sf9
insect cells (Invitrogen) and purified by immobilized metal-chelate
affinity chromatography (IMAC). Protein bound to the IMAC column
was eluted with a buffer containing 20 mM Tris–HCl pH 8.0, 0.5 M
NaCl, 10% glycerol, 5 mM DTT and 250 mM imidazole. The eluent
from the IMAC column was loaded onto a Sephacryl S-200 size
exclusion column pre-equilibrated in 20 mM Tris–HCl pH 8.0,
50 mM NaCl, 5 mM DTT and 10% glycerol and eluted with the same
buffer. Fractions containing the monomeric protein species were
5.48. Dilution assay of VEGFR2-20d complex
To confirm slow dissociative property of the compound, en-
zyme–inhibitor dilution assay was performed.26,27 The recovery
of enzyme activity from a preformed enzyme–inhibitor complex

7272
Y. Oguro et al. / Bioorg. Med. Chem. 18 (2010) 7260–7273
was measured using the AlphaScreenÒ system as described above.
VEGFR2 at 100-fold higher concentration than the standard reac-
tion condition (Section 5.46) and 20d at 10-fold higher concentra-
tion than IC50 value were incubated together in reaction buffer for
60 min at ambient temperature to form enzyme–inhibitor com-
plex. This complex was diluted 1:100 into reaction buffer contain-
samples were collected. The blood samples were centrifuged to ob-
tain the plasma fraction. The plasma samples were deproteinized
with acetonitrile containing an internal standard. After centrifuga-
tion, the supernatant was diluted with a mixture of 0.01 mol/L
ammonium formate solution and acetonitrile (9:1, v/v) and centri-
fuged again. The compound concentrations in the supernatant
were measured by LC/MS/MS.
ing 1 mM ATP and 0.1 lg/mL biotinylated poly-GluTyr (4:1), to
initiate the kinase reaction.
5.52. Xenograft nude mouse model
5.49. Kinase profiling by IC50 measurement
Kinase profiling was performed as described previously.36
Human prostate carcinoma cell line DU145 (ATCC No. HTB-81)
was obtained from American Type Culture Collection (Manassas,
USA). The cells were proliferated in RPMI1640 (Invitrogen Corp)
supplemented with 10% heat-inactivated fetal bovine serum (Hy-
Briefly, kinase activities of VEGFR1, PDGFRa, Tie-2, epidermal
growth factor receptor (EGFR), insulin receptor (IR), insulin-like
growth factor 1 receptor (IGF1-R), c-kit, Src and focal adhesion ki-
nase (FAK) were determined by use of the AlphaScreenÒ system.
For VEGFR1, kinase assay was performed with 20 ng/mL VEGFR1
Clone) and antibiotics (100 units/mL penicillin G and 100 lg/mL
streptomycin, Wako Pure Chemical, Japan). The cells were cultured
in tissue culture dishes in a humidified incubator at 37 °C in an
atmosphere of 5% CO2 and 95% air. The hydrochloride salt of 20d
was suspended in vehicle solution including 1% citric acid (Wako
Pure Chemical) and 1% gum arabic (Suzu Pharmaceutical, Japan)
solution.
(Millipore) and 0.5
with 50 ng/mL EGFR and 2
performed with 10 ng/mL IGF1-R (BIOMOL, USA) and 10
l
M ATP. For EGFR, kinase assay was performed
M ATP. For IGF1-R, kinase assay was
M ATP.
l
l
For FAK, kinase assay was performed with 62 ng/mL FAK (N-termi-
nal FLAG-tagged recombinant proteins using the baculovirus
expression system) and 2 lM of ATP. In these assays, wells con-
taining the substrate and the enzyme without the compound were
used as total reaction control. Wells containing the biotinylated
poly-GluTyr (4:1) and the enzyme without ATP were used as basal
control. B-raf, extracellular signal-regulated kinases (ERK1), pro-
tein kinase Ch (PKCh), glycogen synthase kinase-3b (GSK-3b) and
Aurora A kinase activity was determined by use of radio labeled
Six-week old male athymic nude mice (BALB/cA Jcl-nu/nu, Ja-
pan Clea, Japan) received subcutaneous injections into the hind
flank with 3 ꢂ 106 DU145 cells in 100
lL of 1:1 volume mixture
of Hanks’ balanced salt solution (GIBCO, Invitrogen Corp, USA)
and Matrigel (BD Biosciences, CA, USA). When tumors reached a
volume of 150–210 mm3, mice were randomized into groups of 5
(n = 5). Then, mice were orally given vehicle or the hydrochloride
salt of 20d (1.5, 3, 6 and 12 mg/kg) twice-daily for 3 weeks.
Tumor volumes were assessed by bilateral vernier caliper mea-
surement twice-weekly after inoculation and calculated as vol-
ume = length ꢂ width2 ꢂ 1/2, where length was taken to be the
longest diameter across the tumor and width the corresponding
perpendicular. Treatment over control (T/C, %), an index of antitu-
mor efficacy, was calculated by comparison of the mean change in
tumor volume over the treatment period for the control and trea-
ted groups. Body weight was also measured on the day of tumor
volume assessment. Effects of the hydrochloride salt of 20d on tu-
mor growth and body weight were statistically analyzed by a one-
tailed Williams’ test. Differences were considered significant at
p 6 0.025.
[
c
-
33P] ATP (GE Healthcare, USA). For GSK-3b, kinase assay was
performed with 2 g/mL GSK-3b, 4 g/mL of pGS peptide and
0.5 M ATP (0.1 Ci/50 L/well of [
33P] ATP). For Aurora A,
kinase assay was performed with 10 g/mL Aurora A, 30 mol/L
Aurora substrate peptide and 0.5 M ATP (0.2 Ci/50 L/well of
33P] ATP). HER2 kinase activity was determined by use of radio
labeled [
32P] ATP (GE Healthcare). In these assays, wells contain-
l
l
c-
l
l
l
l
l
l
l
l
[c-
c
-
ing the substrate and the enzyme without the compound were
used as total reaction control. Wells containing the substrate and
the radio labeled ATP without the enzyme was used as basal con-
trol. The concentration of inhibitor producing 50% inhibition of the
kinase activities (IC50 values) and 95% confidence intervals (95% CI)
were analyzed as described above.
Acknowledgments
5.50. Cell proliferation assay
The authors thank Mr. T. Hirayama, Dr. T. Ichikawa, and Dr. Y.
Nishikimi for synthetic support; Ms. T. Yoshida, Dr. A. Mizutani,
and Ms. Y. Nagase for in vitro and in vivo assays. The authors thank
Mr. K. Iwamoto and Ms. A. Hirokawa for molecular biology and
protein expression. The authors thank Mr. S. Yamasaki and Ms. Y.
Watanabe for PK evaluation.
HUVECs (Cambrex, USA) were seeded into a 96-well plate at
3000 cells/well in Human Endothelial-SFM Growth Medium (Invit-
rogen) containing 3% fetal bovine serum (FBS) (Hyclone, USA) and
were incubated overnight at 37 °C in a 5% CO2 incubator. Various
concentrations of the test compounds were added in the presence
of 60 ng/mL VEGF (R&D systems, USA), and the cells were cultured
for a further 5 days. Cellular proliferation was determined by the
WST-8 formazan assay using Cell Counting Kit-8 (DOJINDO Labora-
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