5-Substituted 4-anilinoquinazolines as potent, selective and orally active inhibitors of erbB2 receptor tyrosine kinase

Article abstract of DOI:10.1016/j.bmcl.2005.06.068

Starting from a 6,7-substituted quinazoline lead 4, optimisation of 5-substituted quinazolines containing an extended aniline motif led to potent and selective inhibitors of erbB2 receptor tyrosine kinase, and a representative compound 12a inhibited tumour growth in a mouse xenograft model.

Full text of DOI:10.1016/j.bmcl.2005.06.068

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4226–4229
5-Substituted 4-anilinoquinazolines as potent, selective and
orally active inhibitors of erbB2 receptor tyrosine kinase
Peter Ballard,a Robert H. Bradbury,a,* Laurent F. A. Hennequin,b D. Mark Hickinson,a
Paul D. Johnson,a Jason G. Kettle,a Teresa Klinowska,a Remy Morgentin,b
Donald J. Ogilviea and Annie Olivierb
aCancer and Infection Research, AstraZeneca, Mereside, Alderley Park, Macclesfield SK10 4TG, UK
bAstraZeneca, Centre de Recherches, Z.I. la Pompelle, BP1050, 51689 Reims Cedex 2, France
Received 6 May 2005; revised 22 June 2005; accepted 23 June 2005
Available online 1 August 2005
Abstract—Starting from a 6,7-substituted quinazoline lead 4, optimisation of 5-substituted quinazolines containing an extended ani-
line motif led to potent and selective inhibitors of erbB2 receptor tyrosine kinase, and a representative compound 12a inhibited
tumour growth in a mouse xenograft model.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Following the discovery that over-expression of the
EGFR (erbB1) and erbB2 receptor tyrosine kinases is
found in a number of cancers and is associated with
poor prognosis in patients, blockade of this signalling
pathway has emerged as a promising approach to selec-
tive targeting of tumour cells.1 The clinical utility of the
EGFR inhibitor gefitinib 1 (Fig. 1) in non-small cell
lung cancer has been demonstrated,2 while the monoclo-
nal antibody trastuzumab has been shown to increase
survival time in patients with metastatic breast tumours
that over-express erbB2.3 Among compounds currently
undergoing clinical trials are the mixed EGFR-erbB2
receptor tyrosine kinase inhibitor lapatinib (GW2016)
2,4 the erbB2-selective inhibitor CP-724714 3,5 and the
irreversible inhibitors canertinib (CI-1033),6 EKB-5697
and HKI-272.8
Figure 1. Screening lead 4 and examples of EGFR and erbB2 receptor
tyrosine kinase inhibitors in clinical use or undergoing trials: gefitinib
1; lapatinib 2; CP-724714 3.
X-ray crystal structures of EGFR enzyme-inhibitor
complexes provide clear evidence that the mechanism
of action of receptor tyrosine kinase inhibitors such as
1–3 involves reversible displacement of ATP at the
moderately potent inhibitor of erbB2 receptor tyrosine
kinase active site, through binding to either the active9
kinase. Like other documented quinazoline-based erbB2
or the inactive10 form of the kinase. As part of the pro-
gramme of work that led to the discovery of gefitinib, we
found 6,7-substituted quinazoline 411 (Fig. 1) to be a
inhibitors, compound 4 contains an extended aniline
motif, which we assumed to interact with the selectivity
pocket at the erbB2 active site.10 From consideration of
an in-house homology model12 and related work on
inhibitors of c-Src,13 we reasoned that switching of the
quinazoline substituent from the 6- to the 5-position
could improve erbB2 affinity through occupation of
the kinase sugar pocket. In this publication, we describe
Keywords: Anilinoquinazoline; erbB2; Receptor tyrosine kinase.
*
Corresponding author. Tel.: +44 1625 515708; fax: +44 1625
513910; e-mail: rob.bradbury@astrazeneca.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.068

P. Ballard et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4226–4229
4227
how this work led to potent, selective and orally active
inhibitors of erbB2.
Representative compounds prepared during the course
of this work are listed in Table 1, and synthetic routes
are outlined in Scheme 1–4.14 Initial variant 7 was
prepared (Scheme 1) from the appropriate extended
aniline11 and chloroquinazoline 6, in turn obtained from
5,7-dimethoxy-4(3H)-quinazolone 514b via a sequence
involving as the key steps selective demethylation,13
protection of quinazolone nitrogen and Mitsunobu
alkylation.
Compounds 10a–c were likewise prepared (Scheme 2)
from the appropriate anilines11,15 and the corresponding
chloroquinazolines 9a–b, readily accessible in two steps
from commercially available 5-fluoro-4(3H)-quinazo-
lone 8 via alkoxide displacement and chlorination.
Scheme 2. Synthesis of compounds 10a–c. Reagents and conditions:
(a) NaH, 4-hydroxy-N-methylpiperidine or 4-hydroxytetrahydropy-
ran, MeCONMe2, 80 ꢁC (work up: DOWEX H+/MeOH/NH3, 20 ꢁC);
(b) POCl3, i-Pr2NEt, CH2Cl2, reflux (work up: aq NaHCO3, 0 ꢁC); (c)
H2NAr,11,15 i-PrOH, reflux.
More versatile routes enabled introduction of the aniline
para substituent or the quinazoline 5-substituent at the
last stage of the synthesis. Reaction of chloroquinazo-
line 9a with the appropriate amino phenol gave interme-
diates 11a–b, which were then alkylated to provide
Table 1. erbB2 and EGFR kinase inhibition data18 for compounds 4,
7, 10a–c, 12a–d, 14
Entry
IC50 (lM)a
erbB2
0.056 ( 0.003)
EGFR
Scheme 3. Synthesis of compounds 12a–d. Reagents and conditions:
(a) 4-amino-2-chlorophenol or 4-aminophenol, i-PrOH, reflux; (b)
ClCH2Y,16 K2CO3, 18-crown-6, MeCN, reflux.
4
0.059 ( 0.003)
0.005 ( 0.003)
0.005 ( 0.002)
1.8 ( 0.25)
7
0.005 ( 0)
<0.002
10a
10b
10c
12a
12b
12c
12d
14
1.1 ( 0.085)
0.024 ( 0.001)
<0.002b
2.4 ( 0.41)
0.14 ( 0.048)
0.69 ( 0.18)
0.28( 0.08)
<0.002
<0.002
0.070 ( 0.014)
0.024 ( 0.010)
2.1 ( 0.11)
0.44 ( 0.026)
a n P 3, standard error is given in parentheses.
b IC50 7.6 lM (Kdr), 23 lM (Src).
Scheme 4. Synthesis of compound 14. Reagents and conditions: (a)
SOCl2, cat HCONMe2, reflux (work up: aq NaHCO3, 0 ꢁC);17 (b)
H2NAr,11 i-PrOH, reflux; (c) NaOMe, 15-crown-5, MeOH, dioxan,
reflux.
extended aniline variants 12a–d (Scheme 3). Alternative-
ly (Scheme 4) the fluoroquinazoline 13, readily obtained
from fluoroquinazolone 8,17 underwent microwave
assisted displacement by alkoxide to give 5-methoxy
quinazoline derivative 14.
The compounds listed in Table 1 were evaluated in
erbB2 and EGFR kinase assays measuring inhibition
of phosphorylation of a synthetic peptide substrate at
Km ATP concentration for each enzyme.18 Lead com-
pound 4 showed moderate inhibition in the erbB2 kinase
assay (IC50 0.056 lM). Transposition of the quinazoline
substituent from the 6- to the 5-position, introduction
of a cyclic amine and changing the meta substituent in
the extended aniline from methyl to chlorine led to
Scheme 1. Synthesis of compound 7. Reagents and conditions: (a)
MgBr2, pyridine, reflux; (b) NaH, chloromethyl pivalate, DMF, 0–
20 ꢁC; (c) 4-hydroxy-N-methylpiperidine, Ph3P, (t-BuO2C)2N2,
CH2Cl2, 20 ꢁC; (d) NH3, MeOH, 20 ꢁC; (e) POCl3, i-Pr2NEt,
ClCH2CH2Cl, reflux; (f) H2NAr,11 i-Pr2NEt, i-PrOH, reflux.

4228
P. Ballard et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4226–4229
compound 7 with significantly improved affinity for
erbB2 and EGFR. Removal of the 7-methoxy substitu-
ent then provided compound 12a with favourable erbB2
kinase selectivity versus EGFR (IC50 0.002 lM vs
0.11 lM). Compound 12a also showed a clean selectivity
profile versus in-house and external kinase panels
(data not shown).19 Thus, for example, 12a gave
selectivity ratios of >3800 and >11,500, respectively,
versus the receptor tyrosine kinases Kdr and Src (Table
1, footnote b).
dosing at 100 mg/kg po, and calculation of plasma free
concentrations showed the free levels of 12a to compare
favourably with the IC50 value in the Clone 24 phos-
phorylation assay (Table 4).25
Encouraged by these pharmacokinetic data, we evaluat-
ed compound 12a at 100 mg/kg po q.d. and b.i.d. and at
200 mg/kg po q.d. for inhibition of growth of a BT474C
tumour xenograft in a 28-day study in athymic mice.22
No significant reduction in body weight was observed
for any animal group in this study. As shown in Figure
2, compound 12a gave statistically significant inhibition
of tumour growth with all dosing schedules. Analysis of
excised tumour samples confirmed significant inhibition
of phospho-erbB2 (e.g., >90% inhibition 4 and 24 h
after the last dose at 100 mg/kg b.i.d.).
The remaining compounds included in Table 1 illustrate
SAR for the series of 5-substituted quinazolines. Alter-
native extended anilines (10a, 12b–c) gave comparable
ErbB2 activity but differing selectivity versus EGFR.
More drastic aniline modifications (10b, 12d) gave re-
duced affinity, consistent with interaction of the extend-
ed aniline with the selectivity pocket at the erbB2 active
site10 and with published work highlighting the impor-
tance of the meta substituent.20 Alternative 5-substitut-
ed quinazolines (10c, 14) also showed reduced affinity
relative to 12a.
Although the BT474C mouse xenograft model responds
to both erbB222 and EGFR inhibitions,26 the weak
activity of compound 12a in the KB cell assay (Table
2) is consistent with xenograft activity reflecting inhibi-
tion of erbB2. Further support for this hypothesis is pro-
vided by the lack of activity of 12a at 100 mg/kg po in
the EGFR-driven mouse LoVo xenograft model18 (data
not shown).
A number of assays18,21–24 were used to determine the
cellular profile of selected compounds (Table 2).
ErbB2-selective inhibitor 12a and mixed erbB2-EGFR
inhibitor 10a were shown to inhibit erbB2 autophospho-
rylation in a MCF7 breast carcinoma cell line engi-
neered to overexpress erbB2 (ÔClone 24Õ assay).21 The
cellular activity of compounds 10a and 12a was con-
firmed in a proliferation assay using the BT474C cell
line.22,23 Compounds 10a and 12a were shown to be only
weak to moderate inhibitors of growth of the KB cell
line when stimulated with EGF,18 and good separation
was also seen between BT474C antiproliferative activity
and effect on basal growth in KB cells,18 consistent with
a cellular mode of action involving inhibition of erbB2.
The moderate EGFR cellular potency of compound 10a
was confirmed using an EGFR autophosphorylation as-
say,24 reflecting contrasting enzyme and cellular profiles
for this compound.
In summary, optimisation of 5-substituted quinazolines
containing an extended aniline motif led to potent and
selective inhibitors of erbB2 receptor tyrosine kinase,
and a representative compound 12a inhibited tumour
growth in the mouse BT474C xenograft model. Further
work describing a wider range of 5-substituted quinazo-
lines as selective inhibitors of erbB2 will be reported in
due course.
Table 4. Plasma concentrations for compound 12a after dosing at
100 mg/kg po,a and ratios of free plasma levelsb to IC50 value in Clone
24 cellular assay
Time (h)
2
4
6
16
24
Table 3 summarises pharmacokinetic parameters for
compound 12a determined following dosing to immuno-
competent Alderley Park mice at 2 mg/kg iv and 5
mg/kg po. Exposure of the compound scaled well on
Plasma concentration 12a (lM) 9.2 7.2 6.9 2.2
Ratio free 12a/Clone 24 IC50
1.1
4.1 3.2 3.1 0.97 0.48
a AUC 98.5 lM.h.
b Mouse free drug 2.72%.
Table 2. Cell assay data18,21–23 for compounds 10a and 12a
Entry
IC50 (lM)a
BT474C KB
Clone 24
KB Basal
10a
12a
0.056 ( 0.007) 0.027 ( 0.007) 0.46b( 0.10) 1.7 ( 0.46)
0.061 ( 0.009) 0.041 ( 0.004) 2.6 ( 0.81) 11.9 ( 4.1)
a n P 3, standard error is given in parentheses.
b IC50 2.5 ( 0.75) lM for inhibition of phospho-EGFR in KB cells.24
Table 3. Pharmacokinetic parameters for compound 12a, determined
by dosing to immunocompetent mice at 2 mg/kg iv and 5 mg/kg po
Entry Vdss (l/kg) Cl
po AUC Bioavailability %
(ml/min/kg) (lM.h)
Figure 2. Inhibition of growth of BT474C xenograft22 in athymic mice
dosed po with compound 12a.
12a
2.6
15 5.5
50

P. Ballard et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4226–4229
4229
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Acknowledgments
We thank Martin Pass and Andrew Thomas for helpful
discussions at the outset of this work, and Sarah Beck,
Rowena Callis, Sara Davenport, Frederic Ducrozet,
Paul Farrington, Janet Jackson, Santosh Nandan, Helen
Ross, James Scott, Cath Trigwell, John Vincent, Ingrid
Wilson and Lisa Woods for technical assistance.
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