Discovery of a novel and potent series of dianilinopyrimidineurea and urea isostere inhibitors of VEGFR2 tyrosine kinase

Article abstract of DOI:10.1016/j.bmcl.2005.05.096

A series of dianilinopyrimidineureas demonstrate potency as VEGFR2 kinase inhibitors.

Full text of DOI:10.1016/j.bmcl.2005.05.096

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3519–3523
Discovery of a novel and potent series of dianilinopyrimidineurea
and urea isostere inhibitors of VEGFR2 tyrosine kinase
Douglas M. Sammond,* Kristen E. Nailor, James M. Veal, Robert T. Nolte, Liping Wang,
Victoria B. Knick, Sharon K. Rudolph, Anne T. Truesdale, Eldridge N. Nartey,
Jeffrey A. Stafford, Rakesh Kumar and Mui Cheung
GlaxoSmithKline, Five Moore Drive, Research Triangle Park, NC 27709, USA
Received 28 March 2005; revised 23 May 2005; accepted 25 May 2005
Abstract—A series of dianilinopyrimidineureas demonstrate potency as VEGFR2 kinase inhibitors.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Angiogenesis is the process of forming new blood vessels
from existing ones and is tightly controlled by the bal-
ance of a number of angiogenic growth factors and
endogenous inhibitors.1 Disruption of this balance is
thought to have implications for a number of diseases
including diabetic retinopathy and macular degenera-
tion,2 rheumatoid arthritis,3 coronary artery disease4,
and stroke.5 Furthermore, angiogenesis is required for
the establishment and maintenance of solid tumors.6 It
Figure 1.
has been postulated that inhibition of angiogenesis
activity on human umbilical vein endothelial cells11
through the blockade of the tyrosine kinase VEGFR2
(HUVECs). In addition, we have used homology model-
(FLK-1/KDR) signaling pathway could starve develop-
ing to identify key residues in the ATP-binding site that
ing cancers of required blood flow by limiting vascula-
we propose are responsible for optimal interaction with
ture to the growth site.7 This approach is supported by
our inhibitors. This urea series was discovered during
recent phase III clinical data from combination studies
our comprehensive exploration of the dianilinopyrimi-
with AvastinTM, the monoclonal antibody to VEGF,
dine scaffold as a kinase inhibitor.12 In this paper, we
and standard chemotherapy.8
report SAR for our urea and urea isostere series.
Clinical validation for VEGF as an antitumor target has
All of the ureas and isosteres presented here were
generated considerable interest in developing inhibitors
synthesized by acylation of aniline 6 (see Scheme 1).
of the VEGFR2 kinase.9 We have investigated a series
The synthesis of 6 started with the displacement of
of dianilinopyrimidine ureas and urea isosteres10 (Fig.
the 4-chloro group of 2,4-dichloropyrimidine with
1). These compounds are low nanomolar inhibitors
the acetamide-protected dianiline 2. The resulting
of the VEGFR2 enzyme and have anti-proliferative
aminopyrimidine 3 was methylated with methyl iodide
and cesium carbonate. The methylated adduct 4 was
heated to reflux in isopropanol with substituted aniline
10a and a catalytic amount of hydrochloric acid
to produce the protected dianilinopyrimidine interme-
diate 5. Aniline 6 was revealed by heating 5 in 6 N
hydrochloric acid.
Keywords: VEGFR2; FLK-1; KDR; Kinase inhibitor.
*
Corresponding author. Tel.: +1 919 483 4279; fax: +1 919 483
6053; e-mail: douglas.m.sammond@gsk.com
Present address: Syrrx, Inc., 10410 Science Center Drive, La Jolla,
CA 92121, USA.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.05.096

3520
D. M. Sammond et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3519–3523
Table 1. SAR of urea isosteres
Compound
R
VEGFR2
a
IC50 (nM)
HUVEC-v cell
b
IC50 (nM)
1
18
176
95
0.2
49
11
12
13
14
15
224
6
117
269
324
Scheme 1. Reagents and conditions: (a) 2, 4-dichloropyrimidine,
NaHCO3, 1:3 THF/EtOH, 75 °C, 95%; (b) MeI, Cs2CO3, DMF,
84%; (c) 10a, HCl(cat.), i-PrOH, 80 °C; (d) 6 N HCl, 90 °C, 4 h, 67%
over steps (c and d); (e) PhNCO, DMF, 26%; phenylacetylchloride,
DMF, 21%; phenylchloroformate, DMF, 69%; phenylthioisocyanate,
33%; a-toluenesolfonylchloride, DMF, 21%; 13, ammonium hydroxide
(30% in water), sodium periodate, 80 °C, 39%.
350c
596c
Compounds 1 and 11–14 were prepared by acylation of
6 with phenylisocyanate, phenylacetylchloride, phenyl-
chloroformate, phenylthioisocyanate, and a-toluene-
sulfonyl chloride, respectively. Guanidine derivative 15
was prepared by heating 13 with aqueous ammonium
hydroxide and sodium periodate.13
a All values represent the average of two or more experiments except
where noted.
b Inhibition of VEGF induced proliferation of HUVECs.
c Value reported from one experiment.
The SAR for a representative sample of our urea com-
pounds is presented in Table 2.14 The data suggest that
a variety of groups are accommodated at the urea.
Examples of straight chain alkyl (27–29), cycloalkyl
(30) and phenyl (1 and 16–25) substituted ureas all dem-
onstrated low nanomolar potency against the VEGFR2
enzyme. The most potent compounds in this series ap-
pear to be the diphenylureas and the data indicate toler-
ance for a wide range of substituents and positions
around the distal ring (1 and 16–25). Furthermore, of
the examples modified at the end opposite the urea, those
with a sulfonamide substituent at the 3- or 4-position (16,
19, 20, 23, and 24) exhibited superior enzyme inhibition
to those presenting the corresponding sulfone substitu-
ents (1, 17, 18, and 22). However, in most cases, the sul-
fonamides were less potent in the HUVEC assay, which
may be due to reduced cell permeability of the hydrogen
bond donating the sulfonamide group. This SAR is con-
sistent with our understanding of the VEGFR2 enzyme
binding site. X-ray crystallographic work in our labora-
tories15 and work by others16 suggest that binding orien-
tation for our compounds projects the urea substituent
into the back pocket of the enzyme and the opposite
end is directed toward the solvent interface.
Synthesis of the sulfone aniline tails was achieved start-
ing from the commercially available 3- or 4-nitrobenzyl
chloride (Scheme 2). Chloromethylnitrobenzene 7 was
treated with sodium methylsulfide to afford the methyl
thioether 8. The thioether was oxidized to sulfone 9 with
3-chloroperbenzoic acid. Reduction of the nitro group
by hydrogenation with 10% palladium on carbon pro-
duced the sulfone aniline tail 10.
The SAR for a series of urea isosteres is presented in
Table 1. No improvement in enzyme or cell potency
was observed with the urea isosteres examined. In this
series, urea 1 gave the best enzyme and cell potency.
Based on these data, we chose to continue our investiga-
tion on the urea class of compounds.
Although lipophilic residues dominate the back pocket
of VEGFR2 ATP-binding site,17 Schindler and co-
workers have reported that the Abelson (Abl) tyrosine
kinase inhibitor STI-571 (GleevecTM) benefits from pro-
tein backbone hydrogen bonding interactions with the
Scheme 2. Reagents and conditions: (a) NaSMe, EtOH, 85%; (b) 3-
chloroperbenzoic acid, CH2Cl2, 0 °C, 63%; (c) 10% Pd/C, H2 (55 psi),
EtOAc, 79%.

Table 2. SAR of urea derivatives
Compound
R1
R2
VEGFR2
a
IC50 (nM)
HUVEC-v
b
IC50 (nM)
Compound
R1
R2
VEGFR2
a
IC50 (nM)
HUVEC-v
b
IC50 (nM)
1
16
3-MeSO2CH2–
3-H2NSO2–
18
5c
0.2
2
29
30
3-MeSO2CH2–
3-MeSO2CH2–
–n-Bu
29
31
2
1
–Ph
17
18
19
20
3-MeSO2CH2–
4-MeSO2CH2–
3-H2NSO2–
35
2
1c
1c
7c
2
31
32
3-MeSO2CH2–
4-MeSO2CH2–
681
3114
530c
2982
10
8
4-H2NSO2–
21
3-MeSO2CH2–
3
2
33
34
4-MeSO2CH2–
4-MeSO2CH2–
990
471c
850c
22
23
24
3-MeSO2CH2–
3-H2NSO2–
4-H2NSO2–
6
7
5
2
2c
4490
37
25
26
3-MeSO2CH2–
3-MeSO2CH2–
65
29
35
36
4-MeSO2CH2–
4-MeSO2CH2–
4c,d
150
2
11c
10c
516c
27
28
3-MeSO2CH2–
4-MeSO2CH2–
17
26c
3
27c
–Et
a All values represent the average of two or more experiments except where noted.
b Inhibition of VEGF induced proliferation of HUVECs.
c Value reported from one experiment.
d Ki = 11 nM.

3522
D. M. Sammond et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3519–3523
Supplementary data
Supplementary data associated with this article can be
found in the online version at doi:10.1016/j.bmcl.2005.
05.096.
References and notes
1. (a) Tonini, T.; Rossi, F.; Claudio, P. P. Oncogene 2003, 22,
6549; (b) Carmeliet, P.; Jain, R. K. Nature 2000, 407, 249.
2. Kaplan, H. J.; Leibole, M. A.; Tezel, T.; Ferguson, T. A.
Nat. Med. 1999, 5, 292.
3. Koch, A. E. Ann. Rheum. Dis. 2000, 59, i65.
4. Freedman, S. B.; Isner, J. M. Ann. Intern. Med. 2002, 136,
54.
5. Zhang, Z. G.; Zhang, L.; Jiang, Q.; Zhang, R.; Davies, K.;
Powers, C.; van Bruggen, N.; Chopp, M. J. Clin. Invest.
2000, 106, 829.
Figure 2. The VEGFR2 kinase inhibitor 35 bound in the enzyme
active site as proposed based on homology modeling.
back of the related Abl kinase pocket.18 In light of this
finding, we were encouraged to pursue finding an analo-
gous interaction in the VEGFR2 back pocket. A num-
ber of compounds were synthesized that tethered polar
functionalities at the end of extended aryl and alkyl link-
ers (31–36). Generally, the SAR did not reward this
exercise. However, it appears that in the case of methyl-
piperazine 35 a stabilizing interaction may have been
discovered, as the enzyme potency is maintained in the
range of the best compounds in this series. The same
benzylic methylpiperazine is found on the Bcr-Abl, ki-
nase inhibitor STI-571, and based on our modeling ap-
pears to be picking up a similar hydrogen bond
interaction with a backbone carbonyl at the back of
the pocket.
6. Folkman, J. Curr. Mol. Med. 2003, 3, 643.
7. (a) Hood, J. D.; Cheresh, D. A. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 8624; (b) Veikkola, T.; Karkkainen,
M.; Claesson-Welsh, L.; Alitalo, K. Cancer Res. 2000,
60, 203; (c) Folkman, J. N. Engl. J. Med. 1971, 285,
1182.
8. Hurwitz, H.; Fehrenbacher, L.; Cartwright, T.; Hains-
worth, J.; Heim, W.; Berlin, J.; Baron, J.; Griffing, S.;
Holmgren, E.; Ferrara, N.; Fyfe, G.; Rogers, B.; Ross, R.;
Kabbinavar, F. N. Engl. J. Med. 2004, 23, 2335.
9. (a) Miyazaki, Y.; Matsunaga, S.; Tang, J.; Maeda, Y.;
Nakano, M.; Philippe, R. J.; Shibahara, M.; Liu, W.;
Sato, H.; Wang, L.; Nolte, R. T. Bioorg. Med. Chem. Lett.
2005, 15, 2203; (b) Curtin, M. L.; Frey, R. R.; Heyman, H.
R.; Sarris, K. A.; Steinman, D. H.; Holmes, J. H.;
Bousquet, P. F.; Cunha, G. A.; Moskey, M. D.; Ahmed,
A. A.; Pease, L. J.; Glaser, K. B.; Stewart, K. D.;
Davidson, S. K.; Michaelides, M. R. Bioorg. Med. Lett.
2004, 14, 4505; (c) Manley, P. J.; Balitza, A. E.; Bilodeau,
M. T.; Coll, K. E.; Hartman, G. D.; McFall, R. C.;
Rickert, K. W.; Rodman, L. D.; Thomas, K. A. Bioorg.
Med. Chem. Lett. 2003, 13, 1673.
10. Cheung, M.; Nailor, K. E.; Sammond, D. M.; Veal, J. M.
U.S. PCT Application WO 03066601, 2003; Chem. Abstr.
2003, 139, 180077.
11. Enzyme and cellular assays were performed as described
previously: Kumar, R.; Miller, C. G.; Johnson, J. H.;
Crosby, R. M.; Hopper, T. M.; Liu, W.; Epperly, A. H.;
Davis-Ward, R. G.; Harris, P. A.; Mook, R. A., Jr.; Veal,
J. M.; Stafford, J. A.; Luttrell, D. K. Proc. Am. Assoc.
Cancer Res. 2001, 42, 587, # 3154.
Our modeling of the urea series inhibitor is exemplified
by the methylpiperazine example 35 (Fig. 2). The mod-
el illustrates that the pyrimidine core nitrogen is sup-
ported by hydrogen bonding to the enzyme backbone
residue CYS917. This interaction with the b strand
ÔhingeÕ region is flanked by a hydrogen bond between
the inhibitor tail sulfone and the backbone nitrogen
of ASN921, occurring close to the solvent interface
of the protein. Extending toward the back pocket of
the enzyme, the inhibitor pushes through a narrow pas-
sage lined with the residues GLU883, which accepts a
hydrogen bond from a urea nitrogen, and ASP1044,
which makes a backbone interaction with the urea car-
bonyl. In the case of the methylpiperazine, one addi-
tional point interaction is proposed between the distal
piperazine nitrogen and the backbone carbonyl of res-
idue ILE1023.
12. (a) Chamberlain, S. D.; Cheung, M.; Emerson, H. K.;
Johnson, N. W.; Nailor, K, E.; Sammond, D. M.;
Semones, M. PCT Int. Appl. WO 03074515, 2003; Chem.
Abstr. 2003, 139, 246042; (b) Boloor, A.; Cheung, M.;
Davis, R.; Harris, P. A.; Hinkle, K.; Mook, R. A.;
Stafford, J. A.; Veal, J. M.; PCT Int. Appl. WO 0259110,
2002; Chem. Abstr. 2002, 137, 140534.
13. Ramadas, K.; Janarthanan, N.; Pritha, R. Synlett 1997, 9,
1053.
2. Conclusion
14. For enzyme and cell selectivity profiles see Supplementary
data.
15. Miyazaki, Y.; Matsunaga, S.; Tang, J.; Maeda, Y.;
Nakano, M.; Philippe, R. J.; Shibahara, M.; Liu, W.;
Sato, H.; Wang, L.; Nolte, R. T. Bioorg. Med. Chem. Lett.
2005, 15, 2203 (PDB entry 1YWN).
We have reported a SAR for a potent series of VEGFR2
kinase inhibitors that demonstrate potency against both
the enzyme and the HUVEC line. Based on published
X-ray crystal analysis and homology modeling of inhib-
itors bound to the VEGFR2 enzyme, we have proposed
a likely binding mode for a representative compound
from our urea series.
16. Curtin, M. L.; Frey, R. R.; Heyman, H. R.; Sarris, K. A.;
Steinman, D. H.; Holmes, J. H.; Bousquet, P. F.; Cunha,

D. M. Sammond et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3519–3523
3523
G. A.; Moskey, M. D.; Ahmed, A. A.; Pease, L. J.; Glaser,
K. B.; Stewart, K. D.; Davidson, S. K.; Michaelides, M.
R. Bioorg. Med. Chem. Lett. 2004, 14, 4505.
R.; Mroczkowski, B.; Kan, C.; Villafranca, J. E.; Appelt,
K. Structure 1999, 7, 319 (PDB entry 1vr2).
18. Nagar, B.; Bornmann, W. G.; Pellicena, P.; Schindler, T.;
Weach, D. R.; Miller, W. T.; Clarkson, B.; Kuriyan, J.
Cancer Res. 2002, 62, 4236.
17. McTigue, M. A.; Wickersham, J. A.; Pinko, C.; Showalter,
R. E.; Parast, C. V.; Tempczyk-Russell, A.; Gehring, M.