Synthesis and SAR of potent EGFR/erbB2 dual inhibitors

Article abstract of DOI:10.1016/j.bmcl.2003.10.010

A series of 6-alkoxy-4-anilinoquinazoline compounds was prepared and evaluated for in vitro inhibition of the erbB2 and EGFR kinase activity. The IC₅₀ values of the best compounds were below 0.10 uM. Further, several of these compounds inhibit the growth of erbB2 and EGFR over-expressing tumor cell lines at concentrations below 1 uM.

Full text of DOI:10.1016/j.bmcl.2003.10.010

Bioorganic & Medicinal Chemistry Letters 14 (2004) 111–114
Synthesis and SAR of potent EGFR/erbB2 dual inhibitors
Yue-Mei Zhang,y Stuart Cockerill,{ Stephen B. Guntrip, David Rusnak,
Kathryn Smith, Dana Vanderwall, Edgar Wood and Karen Lackey*
GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, NC 27709, USA
Received 28 August 2003; revised 29 September 2003; accepted 4 October 2003
Abstract—A series of 6-alkoxy-4-anilinoquinazoline compounds was prepared and evaluated for in vitro inhibition of the erbB2
and EGFR kinase activity. The IC50 values of the best compounds were below 0.10 uM. Further, several of these compounds
inhibit the growth of erbB2 and EGFR over-expressing tumor cell lines at concentrations below 1 uM.
# 2003 Elsevier Ltd. All rights reserved.
Overexpression of the epidermal growth factor receptor
family members EGFR (also erbB1) and erbB2 kinases
is reported in a variety of human tumors1 and is asso-
ciated with poor prognosis.2 Within the signaling net-
work of the type I tyrosine kinase receptor family,
erbB2 plays a central role because it is the preferred
hetero-dimerization partner for the other members.3
Therefore, inhibition of EGFR and erbB2 kinase activ-
ity has emerged as a promising new approach to cancer
therapy. This was validated by antibody-based therapy4
(C225, HerceptinTM) which is shown to increase mean
survival time in metastatic breast cancer patients over-
expressing EGFR and erbB2, respectively. Small mol-
ecule tyrosine kinase (TK) inhibitors are another class of
promising new anticancer drugs5 and several chemical
series such as 4-anilinoquinazolines,6 4-anilinopyr-
ido[d]pyrimidines,7 4-anilinopyrazolo[3,4-d]pyrimidines8
and dianilinophthalimides9 have been reported as EGFR
TK inhibitors. Figure 1 includes some examples in the
quinazoline series (IressaTM, TarcevaTM, GW572016)
that are currently approved drugs or in clinical trials.10
highlight a potential binding mode with useful interac-
tions in the ATP binding site. Finally, the cellular
activity in relevant tumor lines will be discussed.
Our chemical plan was to prepare a number of quina-
zoline derivatives with a variety of C-4 anilines and
various C-6 ether linkers. Scheme 1 illustrates a six-step
synthesis which allowed us to diversify position 6 via the
key intermediate 4. The synthesis and SAR of the
6-position side-chain modifications will initially be
described where Ar=4-benzyloxyaniline (labeled a).
The remaining anilines studied are listed in Table 2 and
are labeled b–f.
Chlorination of 6-hydroxy-4-quinazolinone 1 was initi-
ally attempted using classic conditions such as POCl3
Here we wish to report the synthesis and biological
activity of a series of dual EGFR and erbB2 TK inhibi-
tors with an ether linker at the 6-position of quinazoline
core coupled with a selection of optimal 4-anilino
groups. We will describe the in vitro enzyme SAR and
* Corresponding author. Tel.: +1-919-483-6240; fax: +1-919-315-
1092; e-mail: karen.e.lackey@gsk.com
y
Current address: J&J Pharm, 1000 Route 202, Raritan, NJ 08869,
USA.
Current address: Arrow Therapeutics, 7 Trinity Street, London SE1
1DA, UK.
{
Figure 1. Leading erbB family TK inhibitors.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.10.010

112
Y.-M. Zhang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 111–114
Figure 2. 8a docked into homology model of erbB2.
1. Biological activity
Exploration of the SAR focused on C6 substitutions and
C4 anilino variations to improve both EGFR and erbB2
activities based on results fromprevious work in the ser-
ies and molecular modeling studies.13 The in vitro enzyme
assay methods were used as reported in the literature.13
Scheme 1.
Means values of greater than three experiments are used
for enzyme assays of EGFR/erbB2.
1.1. Ether linker at the 6-position of the quinazolines
The binding mode for the compounds described was
proposed based on docking ligands into a homology
model for erbB2. In this model, shown in Figure 2, the
anilino portion is oriented deep in a hydrophobic region
in the back of the active site not occupied by ATP. The
6-ether linker extends out of the ATP pocket into the
solvent exposed region at the junction of the C- and
N-terminal lobes of the kinase region. This proposal
suggests that there is some room for modifications at
the C6 position aimed at improving the PK properties
and solubility. Therefore, several quinazolines with dif-
ferent ether linkers at C6 were prepared and evaluated
for their inhibition of phosphorylation of the isolated
EGFR and erbB2 enzymes, respectively (Table 1).
Scheme 2.
and SOCl2, but only mixtures containing an unidentified
by-product along with the desired product resulted.
However, protection of the hydroxy group of quinazo-
linone 111 and subsequent chlorination with thionyl
chloride and DMF gave 4-chloroquinazoline 3 in high
yield. Displacement with appropriate anilines and
hydrolysis of the acetic ester afforded quinazolines 4.
These intermediates were O-alkylated with bromide 5
and then hydrolyzed to give quinazolines 7. Scheme 2
depicts the synthesis of methylsulfonyl ethylamide 5 (a
novel side chain previously developed in our group12)
and amines 6 via N-acylation of methylthioethylamine
with TFA anhydride and subsequent oxidation of sul-
For the compounds not containing a water-solubilizing
sulfonyl amine group (4a, 9a, 10a), the inhibition of
erbB2 enzyme activity IC50 values fall in a range of
0.25–0.35 uM and for EGFR in a range of 0.068–0.098
uM. However, N,N,N-trisubstitution (both electron
withdrawing TFA group and electron donating methyl
group) on the side chain (7a and 13a) decreased both
EGFR and erbB2 enzyme potency 3.5- to 10-fold com-
pared to N,N-disubstituted compounds (8a, 12a, 14a).
In addition, erbB2 activity may be influenced by the
linker length. A modest improvement (ꢀ2.4-fold) can
be observed in the 4 carbon linker over the three-carbon
linker. Several possibilities for specific hydrogen bonds
with the inhibitor are suggested, but are speculative due
to uncertainty in the protein conformation in this
region. The best compound 8a in Table 1 provided an
excellent dual inhibitor enzyme profile (IC50 EGFR:
0.074 uM, erbB2: 0.095 uM).
TM
fur with OXONE , followed by alkylation with 1,4-
dibromobutane gave 2-methyl sulfonylethyl tri-
fluoroacetamide 5.
Alkylation of secondary methylsulfonylethyl-trifluo-
racetamide with commercially available alkylhalides
and basic hydrolysis provided amines 6. The alternative
synthesis for three carbon linker analogues 12 and 13
involved alkylation of key intermediate 4 with alkylhal-
ides or amines 6 with 11.

Y.-M. Zhang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 111–114
113
Table 1. Enzyme inhibitory activity of quinazolines with various C6
substituents
Table 2. Enzyme inhibitory activity of quinazolines with various C4
anilines
Compd
Ar
Enzyme assay IC50, nM
Compd
4a
R
H
Enzyme assay IC50, nMa
EGFR
ErbB2
EGFR
98
ErbB2
263
8a
74
95
7a
263
977
8b
8c
8d
8e
8f
11
20
93
27
24
234
210
147
10
8a
74
68
79
95
309
347
9a
CH3
10a
12a
13a
14a
104
830
74
234
1778
117
19
Mean values of greater than three experiments are used for enzyme
assays of EGFR/erbB2.
a IC50 values are generated by measuring inhibition of phosphorylation
of a peptide substrate added to enzyme reaction.14
erbB2 inhibition data suggests that the anilino portion
may play a strong role in determining the potency and
selectivity of the quinazoline series. This observation
supports our proposed binding mode in which the large
substituted benzyloxyanilino group occupies the back
pocket.
1.2. Anilino ring at the 4-position of quinazolines
Given the proposed binding mode, it is readily apparent
that the target affinity could be modulated to a greater
extent via the 4-anilino substitutions. Therefore, the
most potent side chain with a four-carbon linker was
retained at the C-6 position combined variety of ani-
lines. For comparison, 3-ethynylaniline and 3-chloro-4-
fluoroaniline were introduced, which were reported to
afford potent EGFR inhibitory activity.10 The results
are shown in Table 2.
1.3. Cellular efficacy of EGFR/erbB2 dual inhibitors
Representative examples of cellular activity are shown
in Table 3. The cellular assay methods were used as
reported in the literature.14 Consistent with the erbB
family inhibition profile, compound 8b and 8c inhibit the
proliferation of the HN5 cell line overexpressing EGFR,
but are less active in a BT474 cell line overexpressing
All compounds in Table 2 showed relatively potent
EGFR inhibition, of which 3-ethynylaniline appears to
be the best EGFR inhibitor. Bicyclic anilines containing
a benzyl group greatly improved erbB2 inhibition.
Replacement of 40-fluorine in 8c (210 nM) with benzyl-
oxy group in 8e (10 nM) increased erbB2 potency 21
fold. The utility of small lipophilic groups in the
30-position was reported for EGFR inhibition.13 In our
case, 30-substitution also seems important for erbB2
inhibition (8a, 95 nM vs 8e, 10 nM). The fusion of 30-
and 40-substitutuents in the N1-benzylindazole 8f
retained excellent dual potency.
Table 3. Cellular efficacy of EGFR/erbB2 dual inhibitors
Compd
Tumor cell IC50, mMa
Cellular selectivityb
HFF/avge tumor
HN5
BT474
8b
8c
8f
0.045
0.071
0.63
1.68
1.38
0.39
>35ꢁ
>42ꢁ
>59ꢁ
a Cell line BT474 (breast carcinoma) overexpresses erbB2; cell line
NH5 (head/neck carcinoma) overexpresses EGFR.
b The value for cellular selectivity is calculated by dividing the IC50
value for the normal fibroblasts (not shown) by the average tumor
cell IC50 value.
Although multiple binding modes in the ATP site are
possible as substitutes change on kinase inhibitors, the

114
Y.-M. Zhang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 111–114
4. For Herceptin: 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. For C225: Waksal, H. W. Cancer Meta-
stasis Rev. 2000, 18, 427.
5. Traxler, P. M. Exp. Opin. Ther. Pat. 1997, 7, 571. Traxler,
P. M. Exp. Opin. Ther. Pat. 1998, 8, 1599.
6. Smaill, J. B.; Rewcastle, G. W.; Loo, J. A.; Greis, K. D.;
Chan, O. H.; Reyner, E. L.; Lipka, E.; Showalter,
H. D. H.; Vincent, P. W.; Elliott, W. L.; Denny, W. A.
J. Med. Chem. 2000, 43, 1380, and references cited
therein.
7. Smaill, J. B.; Palmer, B. D.; Rewcastle, G. W.; Denny,
W. A.; McNamara, D. J.; Dobrusin, E. M.; Bridges, A. J.;
Zhou, H.; Showalter, H. D. H.; Winters, R. T.; Leopold,
W. R.; Fry, D. W.; Nelson, J. M.; Slintak, V.; Elliott,
W. L.; Roberts, B. J.; Vincent, P. W.; Patmore, S. J. J.
Med. Chem. 1999, 42, 1803, and references cited therein.
8. Traxeler, P.; Bold, G.; Frei, J.; Lang, M.; Lydon, N.;
Mett, H.; Buchdunger, E.; Meyer, T.; Mueller, M.; Furet,
P. J. Med. Chem. 1997, 40, 3601, and references cited
therein.
9. Trinks, U.; Buchdunger, E.; Furet, P.; Kump, W.; Mett,
H.; Meyer, Th.; Muller, M.; Regenass, U.; Rihs, G.;
Lydon, N. B.; Tralexer, P. J. Med. Chem. 1994, 37, 1015.
10. Iwata, K.; Miller, P. E.; Barbacci, E. G.; Arnold, L.;
Doty, J.; Diorio, C. I.; Pustilnik, L. R.; Reynolds, M.;
Thelemann, A.; Sloan, D.; Moyer, J. D. Proc. Am. Assoc.
Cancer Res. 1997, 38, 633 (abstract 4248). Woodburn,
J. R.; Barker, A. J.; Gibson, K. H.; Ashton, S. E.;
Wakeling, A. E.; Curry, B. J.; Scarlett, L.; Henthorn, L. R.
Proc. Am. Assoc. Cancer Res. 1997, 38, 633. Spector, N.;
Raefsky, E.; Hurwitz, H.; Hensing, T.; Dowlati, A.; Dees,
C.; O’Neil, B.; Koch, K.; Smith, D. A.; Mangum, S.;
Burris, H. A. Safety, Clinical Efficacy, and Biologic
Assessments from EGF10004: A Randomized Phase Ib
Study of GW572016 for Patients with Metastatic Carci-
nomas Overexpressing EGFR or ErbB2. ASCO 2003.
Proc. Am. Soc. Clin. Oncol. 22: page 193, 2003 (abstract
772).
Figure 3. SAR summary.
erbB2. Compound 8f showed potent EGFR/erbB2 inhi-
bitory activity as well as excellent in-vitro efficacy (avge
IC50=0.54 uM) against tumor cell lines. The cellular
selectivity (normal cell vs tumor cell lines) also exceeded
50-fold. Very little difference was observed in the cellular
assay systems between the trifluoracetylated side chains
versus. the secondary amine comparators. Other com-
pounds in the series, for example the three carbon
linked ethers, appeared significantly less attractive in
terms of cellular efficacy.
2. Conclusions
A series of 6-alkoxy-4-anilino as erbB2/EGFR TK
inhibitors quinazolines were prepared via two six-step
synthetic routes with a common intermediate 4 and
assessed for their biological activity. The SAR of the
4-anilino portion and the 6-position was discussed and
is summarized in Figure 3. A binding mode of these
compounds docked into an erbB2 horology model is
helpful for explaining the SAR of 4-aniline but less
predictable at the 6-ether linker region.
11. Maillard, J.; Benard, M.; Vincent, M.; Vo-Van-Tri; Jolly,
R.; Morin, R.; Benharkate, M.; Menillet, C. Chim. Ther.
1967, 2, 231.
12. Carter, M. C.; Cockerill, G. S.; Guntrip, S. B.; Lackey, K.
E.; Smith, K. J. WO 9935146 A1.
References and notes
1. Aaronson, S. A. Science 1991, 254, 1146. Hung, M.; Lau,
Y. Semin. Oncol. 1999, 26, 51.
13. Brignola, P. S.; Lackey, K.; Kadwell, S. H.; Hoffman, C.;
Horne, E.; Carter, H. L.; Stuart, J. D.; Blackburn, K.;
Moyer, M. B.; Alligood, K. J.; Knight, W. B.; Wood,
E. R. J. Biol. Chem. 2002, 277, 1576.
14. Rusnak, D. W.; Lackey, K.; Affleck, K.; Wood, E. R.;
Alligood, K. J.; Rhodes, N. R.; Keith, B.; Murray, D. M.;
Mullin, R. J.; Knight, W. B.; Gilmer, T. M. Mol. Cancer
Ther. 2001, 1, 85.
2. Kersemaekers, A. M. F.; Fleuren, G. J.; Kenter, E. G.;
Van den Broek, L. J. C. M.; Uljee, S. M.; Hermans, J.;
Van de Vijver, M. J. Clin. Cancer Res. 1999, 5, 577.
3. Earp, H. S.; Dawson, T. L.; Li, X.; Yu, H. Breast Cancer
Res. Treat. 1995, 35, 115. GRAU-Porta, D.; Beerli, R. R.;
Daly, J. M.; Hynes, N. E. EMBO J. 1997, 16, 1647.