4-Amino-6-arylamino-pyrimidine-5-carbaldehyde hydrazones as potent ErbB-2/EGFR dual kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.07.020

Members of a novel class of 4-amino-6-arylamino-pyrimidine-5-carbaldehyde hydrazones were identified as potent dual ErbB-2/EGFR kinase inhibitors using concept-guided design approach. These compounds inhibited the growth of ErbB-2 over-expressing human tumor cell lines (BT474, N87, and SK-BR-3) in vitro. Compound 15 emerged as a key lead and showed significant ability to inhibit growth factor-induced receptor phosphorylation in SK-BR-3 cells (IC₅₀ = 54 nM) and cellular proliferation in vitro (IC₅₀ = 14, 58, and 58 nM for BT474, N87, and SK-BR-3 respectively). The X-ray co-crystal structure of EGFR with a close analog (17) was determined and validated our design rationale.

Full text of DOI:10.1016/j.bmcl.2008.07.020

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4615–4619
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
4-Amino-6-arylamino-pyrimidine-5-carbaldehyde hydrazones as potent
ErbB-2/EGFR dual kinase inhibitors
*
Guozhang Xu , Marta C. Abad, Peter J. Connolly, Michael P. Neeper, Geoffrey T. Struble, Barry A. Springer,
Stuart L. Emanuel, Niranjan Pandey, Robert H. Gruninger, Mary Adams, Sandra Moreno-Mazza,
Angel R. Fuentes-Pesquera, Steven A. Middleton
Johnson & Johnson Pharmaceutical Research and Development, Medicinal Chemistry, 8 Clarke Drive, Cranbury, NJ 08512, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Members of a novel class of 4-amino-6-arylamino-pyrimidine-5-carbaldehyde hydrazones were identi-
fied as potent dual ErbB-2/EGFR kinase inhibitors using concept-guided design approach. These com-
pounds inhibited the growth of ErbB-2 over-expressing human tumor cell lines (BT474, N87, and SK-
BR-3) in vitro. Compound 15 emerged as a key lead and showed significant ability to inhibit growth fac-
tor-induced receptor phosphorylation in SK-BR-3 cells (IC50 = 54 nM) and cellular proliferation in vitro
(IC50 = 14, 58, and 58 nM for BT474, N87, and SK-BR-3 respectively). The X-ray co-crystal structure of
EGFR with a close analog (17) was determined and validated our design rationale.
Received 24 April 2008
Revised 7 July 2008
Accepted 8 July 2008
Available online 10 July 2008
Keywords:
Receptor tyrosine kinase
ErbB-2
Ó 2008 Elsevier Ltd. All rights reserved.
EGFR
Hydrazones
Quinazoline
Bioisostere
The type I receptor tyrosine kinases (RTK) are involved in vari-
ous aspects of cell growth, survival, and differentiation.1 Among
the known RTKs, the epidermal growth factor receptor (EGFR)
and ErbB-2 (HER-2) are two widely studied proteins that are pro-
totypic members of the ErbB family that also includes ErbB-3
(Her-3) and ErbB-4 (HER-4).2 Over-expression of ErbB-2 and EGFR
has been associated with aggressive disease and poor patient prog-
nosis in a range of human tumor types (e.g. breast, lung, ovarian,
prostate, and squamous carcinoma of head and neck).3 Disruption
of signal transduction of these kinases has been shown to have an
antiproliferative and therapeutic effect.4 Various approaches have
been developed to target the ErbB signaling pathways including
monoclonal antibodies (trastuzumab/HerceptinÒ and cetuximab/
ErbituxÒ) directed against the receptor and synthetic tyrosine ki-
nase inhibitors (gefitinib/IressaÒ, 2, and erlotinib/TarcevaÒ, 4).5
Since many tumors over-express ErbB receptors and/or ligands,
simultaneous targeting of multiple erbB receptors therefore be-
comes a promising approach to cancer treatment. Lapatinib (Tyk-
ErbB family. The quinazoline moiety fits into the ATP-binding
pocket in the kinase domain, while the aniline ring fills an adjacent
lipophilic pocket.
In an effort to develop non-anilinoquinazoline small molecular
ATP-competitive ErbB-2/EGFR inhibitors as cancer therapeutics,
we recently reported a novel series of 4-aminopyrimidine-5-carb-
aldehyde oximes (6)8 that are potent dual inhibitors of EGFR and
ErbB-2 tyrosine kinases (Fig. 2). This scaffold effectively mimics
the well-known quinazoline kinase template by forming a pseu-
do-bicyclic structure with the help of an intramolecular hydrogen
bond between the 4-amino group and the oxime nitrogen atom.
Further bioisosteric replacement of the oxime moiety with hydra-
zone leads to aminopyrimidine hydrazone (7), in which the intra-
molecular NH. . .N@C hydrogen bond is maintained and would
function as mimics of the phenyl ring of the quinazoline (Fig. 2).
Although the hydrazone side chain is expected to orient toward
the solvent front (vide infra), a cyclic N,N-disubstituted hydrazone
might gain additional kinase potency due to the restricted bond
rotation of the side chain. We report here the synthesis and struc-
ture–activity relationships (SAR) of this series as dual ErbB-2/EGFR
kinase inhibitors and the X-ray crystal structure of representative
compound 17 in complex with EGFR.
erbÒ, 3),
a potent dual EGFR/ErbB-2 inhibitor, was recently
approved for the treatment of ErbB-2 positive breast cancer.6
Among various scaffolds employed as ErbB-2/EGFR kinase
inhibitors (Fig. 1),5c,6,7 the 4-anilinoquinazoline scaffold (2, 3, and
4) is the most commonly utilized template for inhibition of the
4-Amino-6-arylaminopyrimidine-5-carbaldehyde hydrazones
were synthesized according to the two-step sequence outlined in
Scheme 1. Treatment of 4-amino-6-chloro-pyrimidine-5-carbalde-
hyde (8)9 with the appropriate aniline in DMSO at 100 °C for 3 h
* Corresponding author. Tel.: +1 609 655 6915; fax: +1 609 655 6930.
E-mail address: gxu4@prdus.jnj.com (G. Xu).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.020

4616
G. Xu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4615–4619
F
N
N
F
HN
O
HN
N
Cl
O
H
N
N
N
O
O
O
N
N
N
H
N
O
BMS-599626 (1)
ErbB-2/EGFR TK inhibitor
gefitinib (2)
EGFR TK inhibitor
O
F
H
N
HN
N
Cl
HN
O
N
O
O
N
O
S
O
O
O
N
lapatinib (3)
ErbB-2/EGFR TK inhibitor
erlotinib (4)
EGFR TK inhibitor
Figure 1. Examples of ErbB family TK inhibitors currently approved or in clinical trials for anti-cancer therapy.
Ar
N
Ar
N
R1
N
HN
6
Ar
N
HN
6
HN
4
O
R2
N
H
O
O
1
1
R
N
H
R
R
5
5
2
2
2
N
H
N
3
N
H
N
3
4
4
N
1
6
7
5
Figure 2. Quinazoline (5), 4-amino-6-arylaminopyrimidine-5-carbaldehyde oxime (6),8 and hydrazone (7) templates.
Ar
N
Ar
N
Cl
N
HN
N
R1
N
HN
N
N
R2
N
O
O
(a)
(b)
H2N
H2N
H2N
8
9
10
Scheme 1. Reagents and conditions: (a) ArNH2, DIPEA, DMSO; (b) R2R1NHNH2, MeOH.
provided the 4-amino-6-arylamino-pyrimidine-5-carbaldehyde
(9). Condensation of (9) with various hydrazines in MeOH afforded
the aminopyrimidine hydrazones (10). In all cases the E-isomer
was either the major or exclusive product obtained.
Table 1
Inhibitory activity of oxime and primary hydrazones–SAR at the C-5 position
In the quinazoline and pyrrolo[2,1-f][1,2,4]triazine series,10,11 it
has been demonstrated that larger aniline substitutions confer
greater dual ErbB-2 and EGFR tyrosine kinase inhibition. A similar
SAR trend was also seen in our 4-aminopyrimidine 5-carbaldehyde
oxime scaffold, where increasing size at the C-6 aniline position
substantially improved ErbB-2 potency.8 In pursuit of our goal to
improve dual ErbB-2/EGFR activities in the 4-aminopyrimidine
hydrazone series, we selected 1-(3-fluorobenzyl)-indazol-5-amino
at the C-6 position for our SAR study. Our initial effort focused on
the primary hydrazones and Table 1 summarizes the inhibitory
activities of these compounds against ErbB-2/EGFR kinases and
ErbB-2 receptor phosphorylation in SK-BR-3 cells.12,13
R
N
H
N
F
5
6
NH2
4
1
3
N
N
N
N
2
Compound
R
ErbB-2
IC50
EGFR
IC50
ErbB-2 phosphorylation
a
a
a
(
lM)
(
l
M)
IC50 (lM)
118
12
13
14
CH3O
CH3NH
CF3CH2NH
4-CH3O–C6H4
0.012
0.084
0.020
0.118
0.008
0.012
0.052
0.049
0.301
0.361
0.258
1.291
In terms of ErbB-2 kinase activity, the primary hydrazone 12 is
7-fold less potent than the corresponding methyl oxime 11. How-
ever, both compounds significantly inhibit ErbB-2 receptor phos-
phorylation in SK-BR-3 cells. The aryl-substituted primary
a
Mean values of three experiments are used for ErbB-2 and EGFR enzyme assays
and ErbB-2 cellular phosphorylation assay. IC50 values are reported as
concentrations.
lM

G. Xu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4615–4619
4617
hydrazone 14 is much less potent in both ErbB-2 and EGFR enzyme
assays. Trifluoroethylhydrazone 13 maintains decent ErbB-2 ki-
nase potency as well as significant inhibition of ErbB-2 receptor
phosphorylation in SK-BR-3 cells but is less potent against EGFR
kinase.
hydrazones 16–21 all remain very potent (IC50 within the range
of 2 to72 nM) against both ErbB-2 and EGFR kinases. Compounds
16, 17, and 19 exhibit very potent antiproliferative activities in
BT474, SK-BR-3, and N87 cell lines. All compounds tested do not
inhibit the growth of the non-ErbB-2-dependent HeLa cell line
We next turned our attention to di-substituted hydrazones,
especially the cyclic N,N-disubstituted hydrazones. Table 2 sum-
marizes the inhibitory activities of these compounds against
ErbB-2/EGFR kinases and ErbB-2 receptor phosphorylation in SK-
BR-3 cells. Antiproliferative activity of selected compounds against
four cancer cell lines, BT474 (breast tumor), N87 (gastric tumor),
SK-BR-3 (breast carcinoma), and HeLa (cervical adenocarcinoma)
are also summarized in Table 2.14
The N,N-dimethylhydrazone 15 is 42-fold more potent than N-
methylhydrazone 12 in the ErbB-2 kinase assay and 3-fold more
potent in inhibiting the ErbB-2 receptor phosphorylation. It is also
6-fold more potent than the methyl oxime 11 in the ErbB-2 kinase
assay. In the cell antiproliferative assays, N,N-dimethylhydrazone
15 significantly inhibits the growth of ErbB-2 over-expressing
BT474 (63 nM) and SK-BR-3 (148 nM) cell lines as well as N87
(131 nM) cell line, which over-expresses both ErbB-2 and EGFR.
We also tested the oxime 11 in the BT474 and SK-BR-3 antiprolif-
erative assays. It has IC50 of 254 nM against BT474 cell line and IC50
of 260 nM against SK-BR-3 cell line. The cyclic N,N-disubstituted
(IC50 > 10 lM), consistent with a cellular mode of action involving
inhibition of ErbB-2/EGFR. For reference, lapatinib (3) has an IC50 of
25 nM in the BT474 cell line. The high potency seen for the cyclic
N,N-disubstituted hydrazones in both enzyme and cell assays is be-
lieved to result in part from the restricted bond rotation of the
hydrazone side chain, which contributes to good Caco-2 perme-
ability properties of these molecules (data not shown).
To determine kinase selectivity for this series of dual ErbB-2/
EGFR inhibitors, analogs 16 and 17 were screened against a panel
of 100 kinases at the concentration of 3
lM in the presence of
100
l
M ATP.15 Both compounds were highly selective versus the
kinase panel, inhibiting only Lck (71% and 60%, respectively) and
c-Raf (47% and 50%, respectively) in addition to EGFR and ErbB-2.
To further validate our design rationale, compound 17 was suc-
cessfully crystallized with EGFR protein and the X-ray crystal
structure of the complex was determined to a resolution of
2.0 Å.16 The structure reveals that compound 17 binds to the
ATP-binding cleft with the amino-pyrimidine ring hydrogen-
bonded to the kinase hinge region. Details of the interactions be-
Table 2
Inhibitory activity of N,N-disubstituted hydrazones at the C-5 position
R1
R2
N
N
H
N
F
5
6
NH2
4
1
3
N
N
N
N
2
a
a
a
Compound
R2R1NErbB-2 IC50
(
lM)
EGFR IC50
0.008
(
lM)
ErbB-2 phosphorylation IC50
(lM)
BT474b
0.063
(l
M)
N87c
(lM)
SK-BR-3d
0.148
(
lM)
HeLae
>100
(lM)
H3C
N
H3C
15
0.002
0.007
0.018
0.008
0.008
0.039
0.105
0.131
16
17
18
19
20
0.016
0.030
0.016
0.009
0.026
0.083
0.148
0.509
0.054
0.426
0.031
0.049
NDf
0.091
0.365
NDf
0.122
0.691
NDf
>100
>10
O
N
N
NDf
H3C
HO
N
N
N
N
0.014
NDf
0.058
NDf
0.058
NDf
>100
NDf
OCH3
OCH3
N
N
21
0.072
0.031
NDf
NDf
NDf
NDf
NDf
a
Mean values of three experiments are used for ErbB-2 and EGFR enzyme assays and ErbB-2 cellular phosphorylation assay. IC50 values are reported as micromolar
concentrations. IC50 values listed as >10 or >100 indicate that 50% inhibition was not reached at the highest dose tested (10 or 100
maximum observed.
lM, respectively), nor was an inhibition
b
Breast tumor line over-expressing ErbB-2.
Gastric tumor line over-expressing ErbB-2.
Breast Carcinoma cell line over-expressing ErbB-2.
Cervical adenocarcinoma.
c
d
e
f
Not determined.

4618
G. Xu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4615–4619
binds in a similar fashion to lapatinib (3). The overlay between pro-
teins in both crystal structures showed an alignment throughout
the protein with a rms deviation value of 0.01096. This comparison
also shows a near-perfect alignment between both compounds
with the exception of the orientation of the hydrazone moiety in
17 compared to the furan of lapatinib.
Compounds 15, 16, and 17 were selected for fast pharmacoki-
netic evaluation (at four time points: 0.5, 1, 2, and 4 h) in Spra-
gue–Dawley rats, all compounds exhibited very low plasma
levels (Table 3). Although most compounds in the series displayed
good Caco-2 properties and modest in vitro rat microsomal stabil-
ity (data not shown), aqueous solubility for most of the 4-amino-
pyrimidine hydrazones synthesized was very low, which could
account for the poor rat PK profile for 15, 16, and 17. Solubility
could be improved by appropriate modification of the R1 and R2
substituents on the hydrazone moiety since this region is posi-
tioned toward the solvent interface, as shown in the crystal struc-
ture of 17 and EGFR.
In summary, 4-amino-6-arylaminopyrimidine-5-carbaldehyde
hydrazones have been explored as dual ErbB-2/EGFR kinase inhib-
itors. The N,N-disubstituted hydrazone compounds possess excel-
lent in vitro tyrosine kinase inhibition as well as cellular
antiproliferative activity in the ErbB-2 over-expressing BT474,
N87 and SK-BR-3 tumor cell lines. One of the optimized com-
pounds, 15, exhibited IC50 values of 8 and 9 nM against ErbB-2
and EGFR, respectively, in the kinase assay, and showed potent
antiproliferative effect in the ErbB-2 over-expressing tumor cell
lines (IC50 = 14, 58, and 58 nM for BT474, N87, and SK-BR-3 respec-
tively). Consequently, its close analog 17 was co-crystallized in
complex with EGFR and the X-ray crystal structure was deter-
mined. The X-ray structure showed a nearly identical binding
mode as lapatinib (3), except that lapatinib makes hinge contact
at Met 793,17 whereas 17 forms a bidentate interaction with NH
and the carbonyl oxygen of Met793.
Figure 3. Interactions between EGFR and compound 17. Compound 17 is shown in
green, protein residues are colored gray and atoms are colored by element for both
molecules with nitrogen blue, oxygen red and fluorine light green.
tween the protein and 17 are shown in Figure 3. The pyrimidine
ring nitrogen N3 interacts with the backbone NH of Met793 (not
shown), and the amino group at the C-4 position is engaged in a
second hydrogen bond with the backbone C@O of Met793. The
1-(3-fluorobenzyl)indazolylamino group is oriented deep in the
back of the ATP binding site and makes predominantly hydropho-
bic interactions with the protein. The 3-fluorobenzyl group occu-
pies a pocket formed by the side chains of Met766, Leu777,
Thr790, Thr854 (not shown), and Phe856 (not shown). The 3-fluoro
on the benzyl group interacts with both backbone NH groups of
Arg776 and Thr790 (3.2 Å and 3.19 Å, respectively). The N-2 atom
of the indazole ring forms a weak hydrogen bond to the C@O of Leu
788 (3.9 Å).
Table 3
Fast pharmacokinetic evaluation for compounds 15, 16, and 17 in Sprague–Dawley
rats at 10 mg/kg po
The aniline nitrogen and the N-1 atom of the indazole ring are
not involved in any direct hydrogen-bonding interactions with
the protein. The piperidine group of the hydrazone moiety is ex-
tended to the solvent-exposed region. The C-4 amino group on
the pyrimidine ring clearly forms an intramolecular hydrogen bond
with the hydrazone nitrogen atom as we anticipated, therefore
mimicking the quinazoline phenyl ring.
Compound Concn (ng/mL)
at 0.5 h
Concn (ng/mL) Concn (ng/mL) Concn (ng/mL)
at 1 h
at 2 h
at 4 h
15a
16b
17b
10.1
26.1
23.4
6.3
21.6
12.6
13.2
1.5
32.4
4.6
0
0
An overlay of the EGFR-bound conformations of lapatinib (3)17
and compound 17 is shown in Figure 4, which confirms that 17
a
Vehicle = 10% solutol in D5W.
Vehicle = 0.5% hydroxypropyl methylcellulose.
b
Figure 4. Overlay of lapatinib (3) onto compound 17. Compound 17 is shown in green, Lapatinib is colored pink, and atoms are colored by element for both molecules with
nitrogen blue, oxygen red, fluorine magenta, chlorine light green, and sulfur yellow.

G. Xu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4615–4619
4619
chromogenic substrate, tetra-methylbenzidine (TMB) was used to measure
the absorbance on a spectrophotometer at 450 nm.
Acknowledgments
14. Antiproliferative activity of dual EGFR/ErbB-2 kinase inhibitors was assessed in
monolayer cultures by 14C-thymidine incorporation into cellular DNA as
described (Emanuel S. et al. Mol. Pharm. 2004, 66, 635) except that total time
cells were exposed to drug was 96 h.
Use of the IMCA-CAT beamline 17-ID (or 17-BM) at the Ad-
vanced Photon Source was supported by the companies of the
Industrial Macromolecular Crystallography Association through a
contract with the Center for Advanced Radiation Sources at the
University of Chicago.
15. Upstate Cell Signaling Solutions, Charlottesville, VA, USA.
16. (a) PDB code is 2RGP. (b) EGFR cloning. The DNA sequence encoding amino
acids S671–G998 of human EGFR mature protein (Genbank Accession No.
NM_005228) was amplified by polymerase chain reaction (PCR) and inserted
into the vector pDEST8 (Invitrogen) modified to include sequences encoding an
in-frame hexameric histidine sequence and a thrombin cleavage site. The
References and notes
resulting construct thus encoded MHHHHHHVDLVPRGSHMA-(EGFRS671–G998
)
1. Olayioye, M. A.; Neve, R. M.; Lane, H. A.; Hynes, N. E. EMBO J. 2000, 19, 3159.
2. (a) Salomon, D. S.; Bradt, R.; Ciardiello, F.; Normanno, N. Crit. Rev. Oncol.
Hematol. 1995, 19, 183; (b) Black, J. D.; Brattain, M. G.; Krishnamurthi, S. A.;
Dawson, D. M.; Willson, J. K. Curr. Opin. Invest. Drugs 2003, 4, 1451.
3. (a) Gullick, W. J. Br. Med. Bull. 1991, 47, 87; (b) Woodburn, J. R. Pharmacol. Ther.
1999, 82, 241.
which was confirmed by DNA sequencing. This expression construct was then
integrated into bacmid DNA and a recombinant isolate used to transfect SF9
insect cells as recommended by the vendor. The resulting baculovirus was then
used to infect additional SF9 cells to increase viral titer and volume. (c) EGFR
Purification. All purification processes were carried out on an ÄKTA FPLC system
(GE Healthcare) at 4 °C. The purification protocol was performed as described
previously [J. Biol. Chem. 2002, 277, 46265–46272]. Briefly, frozen cells were
thawed and resuspended in 50 mM Tris, pH 7.5, 200 mM NaCl, 1% glycerol,
5 mM BME, 1Â complete EDTA-free protease inhibitor cocktail (Roche).
Resuspended cells were dounce homogenized and mechanically lysed with an
Emulsiflex-C5 (Avestin) at 10,000–15,000 psi. The lysate was clarified by
centrifugation at 40,000g for 1 h. The supernatant was filtered through a 0.8-
4. Seymour, L. Curr. Opin. Invest. Drugs 2003, 4, 658.
5. (a) Baselga, J.; Tripathy, D.; Mendelsohn, J.; Baughman, S.; Benz, C. C.; Dantis, L.;
Sklarin, N. T.; Seidman, A. D.; Hudis, C. A.; Moore, J.; Rosen, P. P.; Twaddell, T.;
Henderson, I. C.; Norton, L. J. Clin. Oncol. 1996, 14, 737; (b) Waksal, H. W. Cancer
Metastasis Rev. 2000, 18, 427; (c) Gschwind, A.; Fisher, O. M.; Ullrich, A. Nature
2004, 361.
6. Rusnak, D. W.; Lackey, K.; Affleck, K.; Wood, E. R.; Alligood, K. J.; Rhodes, N.;
Keith, B. R.; Murray, D. M.; Knight, W. B.; Mullin, R. J.; Gilmer, T. M. Mol. Cancer
Ther. 2001, 1, 85.
lm vacuum filter and mixed to a TALON metal affinity resin (BD Biosciences)
overnight at 4 °C. The lysate-TALON mixture was loaded into a column and
washed with 10 column volumes of Buffer A (50 mM Tris, pH 8.0, 500 mM NaCl,
5 mM imidazole). EGFR was eluted using a 10-column volume linear gradient
going from Buffer A to Buffer B (50 mM Tris, pH 8.0, 300 mM NaCl, 250 mM
imidazole). Fractions containing EGFR, as assayed by SDS–PAGE, were pooled.
Thrombin was added to the pooled protein to remove the histidine tag (0.005 U
7. (a) Vite, G. D.; Gavai, A. V.; Fink, B. E.; Mastalerz, H.; Kadow, J. F. PCT Int. Appl.
WO2004054514 A2, 2004.; (b) Borman, S. Chem. Eng. News 2005, 83, 40.
8. Xu, G.; Searle, L. L.; Hughes, T. V.; Beck, A. K.; Connolly, P. J.; Abad, M. C.;
Neeper, M. P.; Struble, G. T.; Springer, B. A.; Emanuel, S. L.; Gruninger, R. H.;
Pandey, N.; Adams, M.; Moreno-Mazza, S.; Fuentes-Pesquera, A. R.; Middleton,
S. A.; Greeberger, L. M. Bioorg. Med. Chem. Lett. 2008, 18, 3495.
9. Gomtsyan, A.; Didomenico, S.; Lee, C.; Matulenko, M. A.; Kim, K.; Kowaluk, E.
A.; Wismer, C. T.; Mikusa, J.; Yu, H.; Kohlhaas, K.; Jarvis, M. F.; Bhagwat, S. S. J.
Med. Chem. 2002, 45, 3639.
10. Gaul, M. D.; Guo, Y.; Affleck, K.; Cockerill, G. S.; Gilmer, T. M.; Griffin, R. J.;
Guntrip, S.; Keith, B. R.; Knight, W. B.; Mullin, R. J.; Murray, D. M.; Rusnak, D.
W.; Smith, K.; Tadepalli, S.; Wood, E. R.; Lackey, K. Bioorg. Med. Chem. Lett. 2003,
13, 637.
thrombin/
buffer, pH 8.0, 250 mM NaCl, 1 mM DTT. Cleaved EGFR was filtered through a
0.2- m SFCA cartridge filter, concentrated, and loaded onto a Superdex 200 HR
lg EGFR). The reaction was dialyzed overnight against 50 mM Tris
l
10/30 column (GE Healthcare,) preequilibrated with 50 mM Tris, pH 8.0,
500 mM NaCl, 1 mM DTT. Fractions containing EGFR, as assayed by SDS–PAGE,
were pooled and dialyzed against 10 mM Tris, pH 8.0, 1 mM DTT, 1 mM sodium
azide, 0.1 mM benzamidine. (d) Crystallization, data collection, molecular
replacement, and structure refinement of the EGFR-compound 17 complex.
Purified human EGFR was concentrated to 4 mg/ml, complexed with compound
17 in a 1:2 ratio, and crystallized from a solution containing 2 M Na/K3PO4,
0.1 M Caps, pH 9.0, and 0.2 M Li2SO4.16 X-ray diffraction data to a resolution of
2.0 Å were collected at the IMCA-CAT ID-17 beamline at the Argonne National
Laboratory. Diffraction data were indexed, integrated, and scaled using the
HKL2000 suite. The EGFR crystals belong to the P21212 space group, with unit
cell parameters a = 45.85 Å, b = 68.03 Å, and c = 103.86 Å. The structure was
determined by molecular replacement with CNX [Brunger, A. T.; Adams, P. D.;
Clore, G. M.; DeLano, W. L.; Gros, P.; Grosse-Kunstleve, R. W.; Jiang, J. S.;
Kuszewski, J.; Nilges, M.; Pannu, N. S.; Read, R. J.; Rice, L. M.; Simonson, T.;
Warren, G. L. Acta Crystallogr. D 1998, 54, 905–921] using the structure of EGFR
complexed with lapatinib as a search model (PDB ID 1XKK). All model building
was done using O [Jones, T. A.; Zou, J. Y.; Cowan, S. W.; Kjeldgaard, M. Acta
Crystallogr. A 1991, 47, 110–119], and refinement and map calculations were
carried out using CNX [Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.;
Gros, P.; Grosse-Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges, M.; Pannu, N.
S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren, G. L. Acta Crystallogr. D 1998,54,
905–921]. The final structure was refined to an Rfactor of 22.7 and Rfree of 26.7.
17. Wood, E. R.; Truesdale, A. T.; McDonald, O. B.; Yuan, D.; Hassell, A.; Dickerson,
S. H.; Ellis, B.; Pennisi, C.; Horne, E.; Lackey, K.; Alligood, K. J.; Rusnak, D. W.;
Gilmer, T. M.; Shewchuk, L. A. Cancer Res. 2004, 64, 6652. PDB ID 1XKK.
11. Fink, B. E.; Vite, G. D.; Mastalerz, H.; Kadow, J. F.; Kim, S.; Leavitt, K. J.; Du, K.;
Crews, D.; Mitt, T.; Wong, T. W.; Hunt, J. T.; Vyas, D. M.; Tokarski, J. S. Bioorg.
Med. Chem. Lett. 2005, 15, 4774.
12. The reaction was incubated for 60 min at 30 °C for ErbB-2 (in 60 nM Hepes pH
7.5, 3 nM magnesium chloride, 3 mM manganese chloride, 0.003 mM sodium
vanadate, 1.2 mM DTT, 50 g/mL PEG 20,000, 0.001 mM ATP, 1.5 ng/mL
biotinylated polyGluTyr and 0.2
pH 8.0, 10 mM manganese chloride, 0.1 mM sodium vanadate, 1 mM DTT,
0.005 mM ATP, 1.5 ng/mL biotinylated polyGluTyr and 0.2 -ATP) in
33P-
lC c-ATP) and for EGFR (in 50 mM Tris,
33P-
l
C
c
streptavidin coated FlashPlates (NEN, Boston, MA). Plates were sealed and read
on the TopCount scintillation counter. Each measurement was performed at
least in duplicate and the IC50 values were calculated with standard deviation
from two to eight separate experiments.
13. A sandwich ELISA was developed utilizing two mouse monoclonal antibodies
in a 96-well format. SK-BR-3 cells were treated for 24 h with serial dilutions of
test compound or vehicle, and cell lysates were prepared. Lysates were
transferred to a capture plate coated with a primary antibody-specific for the
human extracellular domain of the ErbB-2 receptor. A horseradish peroxidase-
conjugated anti-phosphotyrosine secondary antibody was used to detect the
extent of phosphorylation of the immobilized ErbB-2 receptor. The