Novel 4-amino-furo[2,3-d]pyrimidines as Tie-2 and VEGFR2 dual inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.03.034

A novel class of furo[2,3-d]pyrimidines has been discovered as potent dual inhibitors of Tie-2 and VEGFR2 receptor tyrosine kinases (TK) and a diarylurea moiety at 5-position shows remarkably enhanced activity against both enzymes. One of the most active compounds, 4-amino-3-(4-((2-fluoro-5-(trifluoromethyl) phenyl)amino-carbonylamino)phenyl)-2-(4-methoxyphenyl)furo[2,3-d]pyrimidine (7k) is <3 nM on both TK receptors and the activity is rationalized based on the X-ray crystal structure.

Full text of DOI:10.1016/j.bmcl.2005.03.034

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2203–2207
Novel 4-amino-furo[2,3-d]pyrimidines as Tie-2 and VEGFR2
dual inhibitors
Yasushi Miyazaki,a,* Shinichiro Matsunaga,a Jun Tang,a Yutaka Maeda,a
Masato Nakano,a Rocher J. Philippe,a, Megumi Shibahara,a Wei Liu,b,à Hideyuki Sato,a
Liping Wangb and Robert T. Nolteb
aGlaxoSmithKline K.K., Tsukuba Research Laboratories, 43, Wadai, Tsukuba 300-4247, Ibaraki, Japan
bGlaxoSmithKline Inc., Five Moore Drive, Research Triangle Park, NC 27709, USA
Received 19 November 2004; revised 4 March 2005; accepted 10 March 2005
Abstract—A novel class of furo[2,3-d]pyrimidines has been discovered as potent dual inhibitors of Tie-2 and VEGFR2 receptor
tyrosine kinases (TK) and a diarylurea moiety at 5-position shows remarkably enhanced activity against both enzymes. One of
the most active compounds, 4-amino-3-(4-((2-fluoro-5-(trifluoromethyl)phenyl)amino-carbonylamino)phenyl)-2-(4-methoxyphen-
yl)furo[2,3-d]pyrimidine (7k) is <3 nM on both TK receptors and the activity is rationalized based on the X-ray crystal structure.
Ó 2005 Elsevier Ltd. All rights reserved.
Angiogenesis, the formation of new blood vessels by
capillary sprouting from pre-existing vasculature, has
been shown to be involved in many diseases such as dia-
betic retinopathy, psoriasis, rheumatoid arthritis, and
cancer. In particular, it is widely accepted that growth
and metastasis of solid tumors is dependent on angio-
genesis.1 Out of the many factors shown to be involved
in angiogenesis, vascular endothelial growth factor
(VEGF) and angiopoietins are of particular interest as
these are specific factors for endothelial cells and expres-
sion of their receptors is restricted to these cells.2 VEGF
and angiopoietins have been thought to play comple-
mentary and coordinated roles in vascular develop-
ment.3 During development, VEGF and its receptor
VEGFR2 play crucial roles in vessel sprouting and
new vessel initiation in early stages of angiogenesis
through induction of proliferation, migration, and sur-
vival of endothelial cells.4 Ang1 and its receptor Tie-2
play an important role in stabilizing the immature endo-
thelial cell network, attracting pericytes, and maintain-
ing biochemical interactions and vessel integrity, which
are thought to be implemented in secondary stages of
blood vessel formation.5
The development of a dual inhibitor of VEGFR2 and
Tie-2 would be expected to demonstrate synergistic ef-
fects through inhibition of both critical stages of blood
vessel formation and offers the potential for new ap-
proaches for the discovery of anti-angiogenesis agents.6
Herein, we wish to report the synthesis and biological
activity of 4-amino[2,3-d]furopyrimidines and the dis-
covery that diarylurea derivatives show VEGFR2/Tie-
2 dual inhibitory activity both at the enzyme and cell
levels.7 We will also describe an X-ray crystal structure
of an inhibitor complexed with VEGFR2 to rationalize
the enzyme potency of the 4-amino[2,3-d]furo-
pyrimidines.
From the results of a focused screening effort, 5,6-diaryl-
4-amino[2,3-d] furopyrimidines, such as 1 (IC50
=
1.25 lM and 1 lM vs VEGFR2 and Tie-2, respectively),
were identified as compounds with moderate inhibitory
activity against VEGFR2 and Tie-2.8 According to a
pharmacophore model for ATP competitive kinase
inhibitors (see Fig. 1), we can speculate that (1) the
aminopyrimidine moiety of the furopyrimidine core
binds to the hinge region through hydrogen-bond do-
nor–acceptor interactions in a similar manner to the
N1 and N6 in the adenine base of ATP; (2) the 5-aryl
moiety would fill a hydrophobic region not generally
Keywords: Angiogenesis; Anticancer; Protein kinase; VEGFR2; Tie-2.
*
Corresponding author. Tel.: +81 298645544; fax: +81 29645559;
e-mail: yasushi.miyazaki@gsk.com
Present address: Addex Pharmaceuticals SA, 12, Chemin Des Aulx
CH-1228, Plan-les-Ouates, Geneva, Switzerland.
à Present address: Medtronic Vascular, 3576 Unocal Place, Suite
OW19, Santa Rosa, CA 95403, USA.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.034

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Y. Miyazaki et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2203–2207
O
hydrophobic region
O
NH2
hinge region
4
O
N
H
5
H
3 N
2
H
O
H
N
O
N
6
N
O
H
N
N
phosphate binding site
O
N
O
N
N
1
O
O P
N
N
N
1
O
VEGFR2 IC50=1.25uM
Tie-2 IC50=1.0uM
O
O
sugar pocket
Figure 1. Original hit compound 1 and proposed binding mode in protein kinase pharmacophore with ATP (1, blue; ATP, magenta).
occupied by ATP in most protein kinases; (3) the 6-aryl
group would be exposed to the phosphate binding re-
gion. Based on these hypotheses, we focused on the
derivatization of both the 5- and 6-position, in order
to improve the potency.
pyrimidine formation using formamide afforded 10.
The 5-bromo intermediate 11 was prepared by bromin-
ation of 6-aryl-furo[2,3-d]pyrimidine 10 with NBS.
Intermediates 11 were coupled under palladium cata-
lyzed conditions with aryl boronic acids leading to 7.
Synthesis of 5,6-diaryl-furo-4-amino[2,3-d]pyrimidines 7
for exploring the structure–activity relationship (SAR)
of the 5- and 6-positions was accomplished using proce-
dures illustrated in Scheme 1.9 In the procedure to deri-
vatize the 6-position, 1-aryl-2-bromoethanone (2) was
converted to 1-aryl-2-hydroxyethanone (3) using potas-
sium formate and aqueous sodium bicarbonate, which
was treated with malononitrile in the presence of dieth-
ylamine to give furan 4. Cyclization with formamide led
to furo[2,3-d]pyrimidine 5. Bromination with NBS gave
6 and a palladium mediated coupling with a variety of
boronic acids resulted in derivatives of 7. In another
procedure focused on derivatizing the 5-position, 1-
aryl-3,3-dicyanopropan-1-one (8) was prepared from 2
and malononitrile using NaOEt as the base. A double
cyclization, first under acidic conditions followed by
The enzyme inhibitory activities are summarized in
Table 1. As indicated in the data, mono aryl derivatives
5a and 10a show reduced activity compared to that of 1.
Apparently, 5, 6-diaryl moieties are important for po-
tent enzyme activity. With R1 retained as the 4-meth-
oxyphenyl substituent, changes to determine the SAR
at the 6-position were investigated. Compounds with
hydrogen-bond donor–acceptors such as 3-carboxamide
7b modestly improved the enzyme inhibitory activity
compared with that of the initial hit compound 1. How-
ever, a meta-methanesulfonamide 7d introduced a mod-
est 3-fold improvement in Tie-2 activity and a more
dramatic 20-fold improvement in VEGFR2 activity.
When R2 is retained as the 4-methoxyphenyl group
and the 3-methanesulfonamide substituent, the 5-posi-
tion N,N-dimethylaminophenyl and biphenyl derivatives
R1
R1
NH2
O
O
NC
H2N
OH
Br
a
b
c, d
N
R1
R1/R2
R
O
N
O
4
3
5 R = H
6 R = Br
2
e
f
R1
NC
H2N
NH2
O
CN
CN
NH2
R
e
g
c, d
N
N
O
O
R2
O
N
N
R2
R2
R2
8
9
7
10 R = H
11 R = Br
Scheme 1. Reagents and conditions: (a) potassium formate, NaHCO3, H2O–EtOH, 40 °C, 90%; (b) malononitrile, Et2NH, DMF, rt, 87%; (c)
formamide, reflux, 69–74%; (d) NBS, CCl4, 50–88%; (e) aryl boronic acid/ester, Pd(PPh3)4, 2 M Na2CO3, DME, 80 °C; (f) malononitrile, NaOEt,
EtOH, rt, 89%; (g) acetic acid, concd HCl, rt, 56%. Yields are shown for 7 where R1 = R2 = 4-OMe.

Y. Miyazaki et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2203–2207
Table 1. Tie-2 and VEGFR2 kinase enzyme inhibition of 4-NH2 furo[2,3-d]pyrimidines5
2205
NH2
R1
N
R2
O
N
Compound
R1
R2
Tie-2 (lM)
VEGFR2 (lM)
1
4-OMe-phenyl
4-OMe-phenyl
H
4-OMe-phenyl
H
1.0
1.12
5.5
5a
10a
7a
7b
7c
7d
7e
7f
>20
>20
1.78
0.54
1.48
0.30
0.60
0.19
>20
4-OMe-phenyl
3,4-Cl2-phenyl
>17
>20
0.18
ND
0.065
0.35
0.036
ND
4-OMe-phenyl
4-OMe-phenyl
4-OMe-phenyl
4-OMe-phenyl
4-NMe2-phenyl
4-Biphenyl
3-CONH2-phenyl
3-CONMe2-phenyl
3-NHSO2Me-phenyl
4-OMe-phenyl
3-NHSO2Me-phenyl
4-OMe-phenyl
7g
3-NHAc-phenyl
IC50 value are generated by measuring inhibition of peptide substrate added to enzyme reaction in homologous time-resolved fluorescence format
(HTRF). ND = not determined.
(7e and f, respectively), increased enzyme potency. We
envision that this is due to hydrophobic interactions of
the biphenyl group at the 5-position, which is expected
to project into the hydrophobic region that is not fully
occupied by the 4-methoxyphenyl group. Additionally,
interactions of the dimethylamino moiety with Lys866
(see Fig. 3) may have also improved the activity. Based
on the assumption that a large hydrophobic pocket
exists into which the 5-position of the furopyrimidine
core projects and that interactions with Lys866 would
be expected, additional 5-position functionalized ana-
logues were evaluated and are listed in Table 2. Based
on this hypothesis, compound 7j wherein arylurea is
substituted at the para position was prepared and it
had remarkably enhanced Tie-2 and VEGFR2 enzyme
activities. The 2-fluoro-5-trifluoromethyl phenyl-urea
7k is the most active analogue and enhanced potency
by approximately 15-fold against Tie-2 and 20-fold
against VEGFR2, compared with the unsubstituted
phenyl-urea 7j. Amide and sulfonamide derivatives (7h
Figure 2. X-ray structure of compound 7k (orange) complexed with
VEGFR2.
and i) decreased potency.
The crystal structure of VEGFR2 with 7k was deter-
mined at high resolution, as shown in Figure 2.10 The
NH and CO motifs of the urea form interactions with
the backbone of Asp1044 and the carboxylic acid resi-
due of Glu883, respectively. The NH2 and nitrogen of
Table 2. Tie-2 and VEGFR2 kinase enzyme inhibition of 4-NH2 furo[2,3-d]pyrimidines
R
NH2
N
O
O
N
Compound
R
Tie-2 (lM)
VEGFR2 (lM)
7h
7i
–NHCO-(3-fluorophenyl)
–NHSO2-(3-chlorophenyl)
–NHCONH-phenyl
0.501
0.851
0.028
0.002
0.022
0.059
0.275
ND
ND
7j
0.062
0.003
ND
7k
7l
–NHCONH-(2-fluoro-5-trifluorophenyl)
–NHCONH-(4-chlorophenyl)
–NHCONH-cyclohexyl
7m
7n
0.186
0.058
–NHCONH2

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Y. Miyazaki et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2203–2207
Table 3. Cellular inhibitory activity of compound 7k expressed as IC50
value in micromolars
the aminopyrimidine form interactions with Glu915 and
Cys917. Taking into consideration the fact that the en-
zyme potency of 7m and n decreased because of the lack
of a terminal aryl group, the binding potency therefore
relies on strong interactions of the 2-fluoro-5-trifluoro-
methyl phenyl moiety with the hydrophobic area which
exists beyond the hydrophilic region flanked by Lys866,
Glu883, and the NH of Asp1044. As shown in Figure 3,
the terminal aryl ring and its substitutions are accom-
modated into this hydrophobic area composed of resi-
dues Ile886, Leu887, Ile890, Val896, and Leu1017.
Additionally the urea NH and carbonyl groups assisted
in stabilizing the molecule by interaction with the
Lys866-Glu883 salt bridge and the NH of Asp1044. In
a manner similar to the binding in VEGFR2, the termi-
nal aryl group of the urea moiety in 7k would be ex-
pected to reside in the hydrophobic back pocket, and
the CO and NH of the urea moiety would be expected
to interact with the conserved Glu883 and Asp982 of
Tie-2.
Compound
Tie-2 autophosphorylation
(lM)
HUVECv
(lM)
HFF
(lM)
7k
0.0063
0.045
0.79
assays, Compound 7k exhibits potent cellular inhibitory
activity versus proliferation of HUVEC and autophos-
phorylation of the c-fms-Tie-2 receptor. The arylurea
function plays an important role by means of hydropho-
bic and hydrophilic interactions which have been veri-
fied through X-ray analysis.
Acknowledgements
We acknowledge Noriko Tadotsu and Hiroko Toyoda
for providing cellular data. We thank Anne Truesdale,
Hiroshi Sootome, Joseph H. Chan, Karen E. Lackey,
and Stephen V. Frye for their guidance and support.
Compound 7k was evaluated for its ability to inhibit the
growth of human umbilical vein endothelial cells (HU-
VECs) stimulated by VEGF and the autophosphoryl-
ation of c-fms-Tie-2 kinase chimeric receptor
transfected in 3T3 cells. Additionally, a cytotoxic cell as-
say was conducted using the HFF cell line.11 Data are
summarized in Table 3, which show a good correlation
between enzyme and cellular potency. Importantly,
there is >10-fold selectivity between HUVECs and
HFF cell lines suggesting that general cytotoxicity is
not the mechanism for inhibition of HUVEC growth.
References and notes
1. (a) Carmeliet, P.; Jain, R. K. Nature 2000, 407, 249; (b)
Hanahan, D.; Folkman, J. Cell 1996, 86, 353; (c) Folk-
man, J. Nat. Med. 1995, 1, 27.
2. Yancopoulos, G. D.; Davis, S.; Gale, N. W.; Rudge, J. S.;
Wiegand, S. J.; Holash, J. Nature 2000, 407, 242.
3. Maisonpierre, P. C.; Suri, C.; Jones, P. F.; Bartunkova, S.;
Wiegand, S. J.; Radziejewski, C.; Compton, D.; McClain,
J.; Aldrich, T. H.; Papadopoulos, N.; Daly, T. J.; Davis,
S.; Sato, T. N.; Yancopoulos, G. D. Science 1997, 277, 55.
4. (a) Shalaby, F.; Rossant, J.; Yamaguchi, T. P.; Gertsen-
stein, M.; Wu, X. F.; Breitman, M. L.; Schuh, A. C.
Nature 1995, 376, 62; (b) Carmeliet, P.; Ferreira, V.;
Breier, G.; Pollefeyt, S.; Kieckens, L.; Gertsenstein, M.;
Fahrig, M.; Vandenhoeck, A.; Harpal, K.; Eberhardt, C.;
Declercq, C.; Pawling, J.; Moons, L.; Collen, D.; Risau,
W.; Nagy, A. Nature 1996, 380, 435; (c) Ferrara, N.;
Davis-Smyth, T. Endocr. Rev. 1997, 18, 4; (d) For recent
review of small molecule of VEGFR2 inhibitor, see:
Bilodeau, M. T.; Fraley, M. E.; Hartman, G. D. Expert
Opin. Investig. Drugs 2002, 99, 11393; (e) Boyer, S. J. Curr.
Top. Med. Chem. 2002, 2, 973.
In conclusion we have discovered 4-NH2-furo[2,3-d]pyr-
imidines bearing a diarylurea substituent at the 5-posi-
tion as a novel class of highly potent inhibitors of the
angiogenesis receptor type tyrosine kinases, VEGFR2
and Tie-2. In addition to activity in isolated enzyme
5. (a) Davis, S.; Aldrich, T. H.; Jones, P. F.; Acheson, A.;
Compton, D. L.; Jain, V.; Ryan, T. E.; Bruno, J.;
Radziejewski, C.; Maisonpierre, P. C.; Yancopoulos, G.
D. Cell 1996, 87, 1161; (b) Suri, C.; Jones, P. F.; Patan, S.;
Bartunkova, S.; Maisonpierre, P. C.; Davis, S.; Sato, T.
N.; Yancopoulos, G. D. Cell 1996, 87, 1171; For review,
see: Thurston, G. Cell Tissue Res. 2003, 314, 61.
6. Holash, J.; Maisonpierre, P. C.; Compton, D.; Boland, P.;
Alexander, C. R.; Zagzag, D.; Yancopoulos, G. D.;
Wiegand, S. J. Science 1999, 284, 1994.
7. Adams, J. J.; Bryan, D. D.; Feng, Y.; Matsunaga, S.;
Miyazaki, Y.; Nakano, M.; Rocher, J.-P.; Sato, H.;
Semones, M.; Silva, D. J.; Tang, J. PCT Application,
1
WO03022852, 2003. Analytical data of compound 7k: H
NMR (DMSO-d6) ppm 3.75 (s, 3H), 6.95 (d, J = 9.1 Hz,
2H), 7.41 (d, J = 9.1 Hz, 2H), 7.43 (d, J = 8.8 Hz, 2H),
7.53 (dd, J = 8.8 Hz, 10.9 Hz, 1H), 7.66 (d, J = 8.6 Hz,
2H), 8.64 (d, J = 7.1 Hz, 1H), 9.04 (s, 1H), 9.46 (s, 1H).
MS (ESI) m/z 538 (M+H)+. The yields for the preparation
of 7k (R2 = 4-OMe in Scheme 1) are as follows: step (f)
Figure 3. Angle from phosphate binding region. Protein surface was
added and colored by atom (C, gray; N, purple; O, red; S, yellow; F,
cyan).

Y. Miyazaki et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2203–2207
2207
89%, (g) 56%, (c) 69%, (d) 88%, (e) 31%, and the reaction
with corresponding isocyanate is 60%.
0.1 mg/ml BSA and test compound in 1 mM HEPES.
VEGFR2: GST-VEGFR2 was incubated for 40–60 min
with 360 nM peptide, 75 lM ATP, 5 mM MgCl2, 0.1 mM
DTT, 0.1 mg/ml BSA and test compound in 100 mM
HEPES.
8. The enzyme assay was performed by HTRF (homoge-
neous time-resolved fluorescence) method using baculo-
virus-expressed recombinant protein. HTRF is based on
the proximity of a donor label (europium chelate) and
acceptor label (allophycocyanin, APC) which have been
brought together by a specific binding reaction. When the
two entities come into close proximity and upon excita-
tion, energy transfer occurs and APC re-emits a specific
long-lived fluorescence at 665 nm. The kinases were
purified as the intracellular domain of human Tie-2 or
VEGFR2 fused by GST tag. The catalytic activity of each
kinases was detected by a biotinylated synthetic peptide
as a substrate, biontin-C6-LEARLVAYEGWVAGKKK-
amide, and biotin-aminohexyl-EEEEYFELVAKKKK-
NH2 for TIE-2 and VEGFR2, respectively. Phosphoph-
orylated substrate is measured by streptavidin linked-APC
(Molecular Probes) and europium-labeled anti-phosphor-
ylated tyrosine antibody (Perkin Elmer). Assay conditions
are as follows. Tie-2: pre-activated GST-TIE-2 with 2 mM
ATP, 5 mM MgCl2 and 12.5 mM DDT was incubated for
30 min with 1 lM peptide, 80 lM ATP, 10 mM MgCl2,
9. (a) Temnikova, T. I.; Sharanin, Y. U. A.; Karavan, V. S.
J. Org. Chem. USSR (Engl. Trans.) 1967, 651; (b)
Matsuda, T.; Yamagata, K.; Tomioka, Y.; Yamazaki,
M. Chem. Pharm. Bull. 1985, 33, 937; (c) Maeda, Y.;
Nakano, M.; Sato, H.; Miyazaki, Y.; Schweiker, S. L.;
Smith, J. L.; Truesdale, A. T. Bioorg. Med. Chem. Lett.
2004, 14, 3907.
10. The PDB deposition code is 1YWN for this structure.
11. Tie-2-autophosphorylation at the cellular level was mea-
sured by ELISA using recombinant mouse 3T3 cells (TIE-
2/c-fms) that stably overexpress the chimeric protein of
c-fms extracellular domain and Tie-2 intracellular domain.
The test compound was incubated with TIE-2/c-fms cells
for 1 h followed by the activation of TIE-2-c-fms receptor
using c-fms ligand, MCSF (macrophage colony stimulat-
ing factor). For capture ELISA with anti-c-fms antibody,
phosphorylation was detected using phosphotyrosine
antibody and colorimetric substrate.