Studies leading to the identification of ZD1839 (Iressa): An orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor targeted to the treatment of cancer

Article abstract of DOI:10.1016/S0960-894X(01)00344-4

This paper describes the development of the epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 from a lead series of 4-anilinoquinazoline compounds. ZD1839 has suitable properties for use as a clinically effective drug and shows activity against human tumours. In particular, the use of pharmacokinetic data in the development of ZD1839 is discussed.

Full text of DOI:10.1016/S0960-894X(01)00344-4

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1911–1914
Studies Leading to the Identification of ZD1839 (IressaTM): An
Orally Active, Selective Epidermal Growth Factor Receptor
Tyrosine Kinase Inhibitor Targeted to the Treatment of Cancer
Andrew J. Barker, Keith H. Gibson,* Walter Grundy, Andrew A. Godfrey,
Jeffrey J. Barlow, Mark P. Healy, James R. Woodburn, Susan E. Ashton,
Brenda J. Curry, Lynn Scarlett, Lianne Henthorn and Laura Richards
AstraZeneca, Cancer and Infection Research, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK
Received 14 February 2001; revised 4 May 2001; accepted 7 May 2001
Abstract—This paper describes the development of the epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 from a
lead series of 4-anilinoquinazoline compounds. ZD1839 has suitable properties for use as a clinically effective drug and shows
activity against human tumours. In particular, the use of pharmacokinetic data in the development of ZD1839 is discussed. # 2001
Elsevier Science Ltd. All rights reserved.
Introduction
oxidation products. Oxidation of the methyl group
produced the corresponding benzyl alcohol 2 and
oxidation at the para-position of the aniline moiety
produced the corresponding phenol metabolite 3.
The discovery of 4-anilinoquinazolines as a lead series
for the inhibition of epidermal growth factor receptor
tyrosine kinase (EGFR-TK) activity,1,2 and the ration-
ale behind the inhibition of this enzyme as a potential
method for treating human cancer,3,4 are well docu-
mented. Conventional structure–activity relationship
studies have established the substitution patterns within
anilinoquinazolines which provide the most potent
compounds against EGFR-TK in vitro.5,6 Compound
1, identified early in these studies, is a potent inhibitor
of EGFR-TK (IC50=5 nM) and a very potent inhibitor
of EGF-stimulated human tumour cell growth [IC50=50
nM (KB oral carcinoma cells)]. Compound 1 contains
all of the elements required in an anilinoquinazoline for
highly potent enzyme inhibition: electron-donating sub-
stituents at the 6- and 7-positions of the quinazoline; a
small lipophilic substituent at the meta-position of the
aniline; a free NH at the 4-position and a free CH at the
2-, 5- and 8-positions. On oral dosing, compound 1
showed evidence of antitumour activity against A431
human tumour xenografts in nude mice, but appeared
to be rapidly metabolised (half-life approximately 1 h).
Two major metabolites were identified; both were
Substitution with chlorine in place of the methyl group
and introduction of a fluorine at the para-position of the
aniline gave compound 4, which was resistant to these
routes of metabolism. Although this compound did
*Corresponding author. Tel.: +44-1625-513349; fax: +44-1625-
513910; e-mail: keith.gibson@astrazeneca.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00344-4

1912
A. J. Barker et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1911–1914
show a slight loss of potency in vitro, relative to com-
pound 1 (EGFR-TK enzyme test, IC50=9 nM; EGF-
stimulated KB cell growth, IC50=80 nM), it never-
theless showed a better in vivo profile, having improved
efficacy on oral dosing and also reduced clearance. No
major metabolites were detected and the half-life in mice
improved to approximately 3 h. A related compound 5,
although weaker in the in vitro assays, revealed sig-
nificant antitumour activity for the first time, at well-
tolerated doses in a related model (the KB xenograft).3
This was associated with a much improved pharmaco-
kinetic profile which highlighted the importance of
inhibiting EGFR-TK for a large part of the 24 h in a
day, when using once-daily dosing, in achieving good
activity in this model. A key structural change in
this regard was the substitution at the 6-position of the
quinazoline.
with methanolic aqueous ammonia to remove the acetyl
group and give the required intermediate 11.
As shown in Scheme 2, the 6-hydroxy intermediate (11)
was used as the starting point for three different routes
by which the required side chains were introduced.
First, direct alkylation introduced the preformed side
chain in one step (Route 1). Second, introduction of a
bromoalkyl group (with dibromoethane or dibromo-
propane), followed by displacement of the second bro-
mine by an appropriate amine, produced the required
compound (Route 2). Third, for side chains containing
a 2-hydroxypropyl group, introduction of an epoxy-
Compound 4 became the focus of further attempts to
improve in vivo activity because of its potency and
metabolic stability, and also provided a useful starting
point for the discovery of different structural types of
EGFR-TK inhibitors.7 Efforts to synthesise irreversible
inhibitors have used similar starting points and strate-
gies.8,9 One approach aimed at improving in vivo activ-
ity, which eventually proved successful, involved
modification of the alkyl groups of the methoxy side
chains. This method of varying the physical properties
of the compounds was predicted to favourably influence
their pharmacokinetic properties. In particular, basic
groups were introduced into the alkoxy side chains, as
this was a versatile way to modify physical properties
over a wide range of basicity, lipophilicity and solubi-
lity. This approach had the advantage of retaining both
the potency-enhancing features of the 6- and 7-alkoxy
groups, and the metabolic stability associated with the
3-chloro-4-fluoroaniline moiety. In this paper, the
effects of modifications to the 6-alkoxy group, involving
the addition of nitrogen-containing side chains are
reported.
Scheme 1. Synthesis of intermediate 11. Reagents: (i) methionine/
MeSO3H/100 ꢀC/3 h; (ii) Ac2O/pyridine; (iii) SOCl2; (iv) 3-chloro-4-
fluoroaniline; (v) NH4OH/MeOH.
It was believed that good activity in an in vivo tumour
model (following once-daily, oral dosing) would require
concentrations of drug in the blood sufficient to provide
sustained 24-h inhibition of EGFR-TK. Therefore,
studies were designed to investigate the blood con-
centrations in mice at various times following oral doses
of the candidate drugs.
Chemistry
The efficient synthesis of a wide range of 6-alkoxy ana-
logues required a suitable late-stage intermediate, the 6-
hydroxy derivative 11 (Scheme 1); details of this synth-
esis have been reported previously.7 The key step was
the selective removal of the methyl group from the 6-
methoxy group of 6, by means of the methionine in
methanesulphonic acid, to give 7. In the next step, the
free phenol group of the intermediate 7 was protected as
the acetyl derivative 8, which was then chlorinated with
thionyl chloride to give 9, followed by reaction with 3-
chloro-4-fluoroaniline to give 10 and finally deprotection
Scheme 2. Routes to final products from intermediate 11.

A. J. Barker et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1911–1914
1913
propyl group followed by opening of the epoxide with
the appropriate amine was used (Route 3).
with 200 mg/kg of compound and blood samples were
collected at one or more time points (2, 6 or 24 h) post-
dosing. Samples were assayed for EGFR-TK inhibitory
activity in the TKI assay. The concentration of com-
pound in the blood was estimated in mM by interpola-
Results and Discussion
tion from
a standard curve, relating percentage
The results are shown in Tables 1–3. The ‘TKI’ result is
the IC50 (mM) for the inhibition of EGFR-TK from
A431 vulval squamous carcinoma cells. The ‘KB2’
result is the IC50 (mM) for the inhibition of EGF-stimu-
lated proliferation of KB cells in culture. Details of
these tests have been described previously.1
inhibition to compound concentration, prepared with
‘spiked’ blood.
Tables 1–3 show different sub-classes of structural
types. Table 1 shows compounds with two carbon
atoms between the oxygen atom and the nitrogen atom
of the Rgroup. Table 2 shows compounds with three
carbon atoms between the oxygen atom and the nitro-
gen atom of the Rgroup. Table 3 shows compounds
with three atoms between the oxygen atom and the
nitrogen atom of the Rgroup but with a hydroxyl
group at the 2-position of the carbon chain.
Blood levels of candidate drugs in mice were estimated
by a bioassay procedure. Mice (male Alderley Park
mice, 30–40 g, three per time point) were dosed orally
Table 1. Compounds with two carbon atoms between the 6-oxygen
and the side-chain nitrogen atom
The parent compound 4, the 6,7-dimethoxy analogue,
gave blood concentrations in the TKI assay of 38, 16
and 0.3 mM, at the 2-, 6- and 24-h time points,
respectively (Table 1).
The TKI results show that all of the compounds repor-
ted here are potent enzyme inhibitors (IC50 <400 nM);
compound 17 (IC50=2 nM) was the most potent inhi-
bitor. In the EGF-stimulated cell proliferation test, all
of the compounds had IC50 <1 mM; compound 29
(IC50=80 nM) was the most potent inhibitor of tumour
cell growth.
In vitroa
KB2 2 h
In vivob
24 h
Compd
RTKI
6 h
0.08
4
0.009
0.069
38
—
16
0.1
0.3
0.3
12
0.91
13
14
15
(HOCH2CH2)2N
MeNH
MeOCH2CH2NH
0.035
0.008
0.008
0.46
0.35
0.18
—
—
—
1.0
1.6
—
0.1
—
Table 2. Compounds with three carbon atoms between the 6-oxygen
and the side-chain nitrogen atom
11
164
1.1
16
0.066
0.64
—
7.9
17
18
Me2NCH2CH2NH
Me2N
0.002
0.007
0.15
0.12
—
82
0.4
—
52
19
20
21
0.01
0.061
0.31
0.38
0.17
0.22
—
—
—
156
5.6
1.7
4.7
In vitroa
KB2
0.049
In vivob
24 h
(MeOCH2CH2)2N
7.2
Compd
RTKI
Me2N
2 h
6 h
—
0.4
7.1
0.1
8.2
2.8
27
0.15
0.16
34
—
22
23
24
0.01
0.1
0.09
0.32
0.73
—
—
—
2.4
0.1
3.5
2.3
28
29
0.084
0.023
—
24
38
1.3
0.08
6.8
5.7
0.087
30
31
Et2N
0.057
0.071
0.16
0.12
—
—
5.4
8.8
0.9
3.7
25
26
Et2N
0.093
0.098
0.31
0.19
—
—
16
35
32
0.079
0.12
—
5.2
2.3
— indicates measurement not taken or replicates were considered
unsuitable to give a reliable value.
— indicates measurement not taken or replicates were considered
unsuitable to give a reliable value.
aValues are IC50 in mM.
aValues are IC50 in mM.
bValues are concentrations in blood (mM) and are the average of three
separate determinations.
bValues are concentrations in blood (mM) and are the average of three
separate determinations.

1914
A. J. Barker et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1911–1914
Table 3. Compounds with three carbon atoms between the 6-oxygen
and the side-chain nitrogen atom, and containing a 2-hydroxy sub-
stituent
mately 70 times that of the IC50 in the KB2 cell test
(0.08 mM). As well as ZD1839, several other compounds
reported here showed high and sustained blood con-
centrations; these results were used to prioritise candi-
dates for further antitumour testing in xenograft
models.
ZD1839 was identified as being of particular interest. As
well as having good oral bioavailability, ZD1839 inhi-
bits the growth of a broad range of human solid tumour
xenografts in a dose-dependent manner (range 12.5–200
mg/kg, po once daily) with marked regressions seen in
some tumours. Treatment for up to 4 months in nude
mice was well tolerated.10 ZD1839 has demonstrated a
long half-life in humans compatible with once-daily oral
dosing and extensive Phase I clinical trials have shown
biomarker evidence for inhibition of the EGFRsignal
transduction pathway and antitumour activity.
In vitroa
2 h
In vivob
24 h
Compd
RTKI
KB2
6 h
33
34
35
(HOCH2CH2)2N
H2N
MeNH
0.005
0.006
0.005
0.85
0.39
0.22
—
—
1.1
0.2
0.1
0.5
—
—
—
36
0.008
0.095
0.067
0.38
0.16
0.13
—
—
—
0.04
0.03
3.0
2-S-37
2-R-38
23
15
0.43
References and Notes
— indicates measurement not taken or replicates were considered
unsuitable to give a reliable value.
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Holdgate, G. A.; Green, L. R. Biochem. Pharmacol. 1994, 48,
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2. Wakeling, A. E.; Barker, A. J.; Davies, D. H.; Brown, D. S.;
Green, L. R.; Cartlidge, S. A.; Woodburn, J. R. Breast Cancer
Res. Treat. 1996, 38, 67.
aValues are IC50 in mM.
bValues are concentrations in blood (mM) and are the average of three
separate determinations.
3. Wakeling, A. E.; Barker, A. J.; Davies, D. H.; Brown, D. S.;
Green, L. R.; Cartlidge, S. A.; Woodburn, J. R. Endocr. Relat.
Cancer 1997, 4, 351.
4. Woodburn, J. R. Pharmacol. Ther. 1999, 82, 241.
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Zhou, H. R.; Showalter, H. D. H.; Winters, R. T.; Leopold,
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Drug concentrations in blood varied over a wide range
(0.1–164 mM at 6 h, Tables 1–3). The imidazo-ethyl
derivative 16, and the morpholino-ethyl derivative 19
produced the highest concentrations at the 6-h time
point. Amongst the structural types described here, the
morpholino derivatives consistently produced high
blood concentrations at the 6-h time point. These com-
pounds also sustained a higher concentration at 24 h
than the parent compound 4. Compounds containing a
2-hydroxy substituent (Table 3) generally achieved low
blood concentrations.
Table 2 includes the compound that was eventually
chosen as a drug development candidate (29; ZD1839).
In vitro, ZD1839 is not the most potent compound
reported here, but achieves high and sustained blood
levels, in vivo, over a 24-h period. At the 24-h time
point, the concentration of drug in the blood (5.7 mM)
from this dosing regime (200 mg/kg po) is approxi-
9. Fry, D. W. Anti-Cancer Drug Des. 2000, 15, 3.
10. Woodburn, J. R.; Morris, C. Q.; Kelly, H.; Laight, A.
Cell. Mol. Biol. Lett. 1998, 3, 348.