Synthesis and biological evaluation of crown ether fused quinazoline analogues as potent EGFR inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.06.067

Crown ether fused anilinoquinazoline analogues were synthesized as novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors. Representative compounds showed potent and selective EGFR inhibitory activities in an in vitro EGFR kinase assay a

Full text of DOI:10.1016/j.bmcl.2012.06.067

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6301–6305
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of crown ether fused quinazoline
analogues as potent EGFR inhibitors
Shaojing Hu b, , Guojian Xie a, Don X. Zhang a, Charles Davis a, Wei Long b, Yunyan Hu b, Fei Wang b,
⇑
Xinshan Kang b, Fenlai Tan b, Lieming Ding b, Yinxiang Wang b,
⇑
a Betapharma USA, 31 Business Park Dr. Branford, CT 06405, USA
b Zhejiang Betapharma, 589 Hongfong Rd., YuhangDistrict, Zhejiang 311100, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Crown ether fused anilinoquinazoline analogues were synthesized as novel epidermal growth factor
receptor (EGFR) tyrosine kinase inhibitors. Representative compounds showed potent and selective EGFR
inhibitory activities in an in vitro EGFR kinase assay and an EGFR-mediated intracellular tyrosine phos-
phorylation assay. The synthesis and preliminary biological, physical, and pharmacokinetic evaluation of
these fused quinazoline compounds is reported.
Received 4 January 2012
Revised 16 June 2012
Accepted 21 June 2012
Available online 28 June 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
EGFR inhibitor
Crown ether fused anilinoquinazoline
Synthesis
The epidermal growth factor receptor (EGFR), as a transmem-
brane glycoprotein, belongs to the erbB family of closely related
cell membrane receptors that includes EGFR (erbB-1 or HER1),
erbB-2 (HER2), erbB-3 (HER3), and erbB-4 (HER4).1 Expression,
overexpression, or dysregulated function of EGFR is observed in
many human solid tumors, including breast, ovarian, non–small-
cell lung (NSCLC), colorectal, and head and neck cancers.2–5 In sup-
port of its important role in tumor biology, EGFR activation may as-
sist tumor growth by increasing cell proliferation, motility,6
adhesion, and invasive capacity7 and by blocking apoptosis.8
EGFR-dependent aberrant signaling, such as overexpression and
dysregulation, is associated with indices of poorer prognosis in pa-
tients and is associated with metastasis, late-stage disease, and
resistance to chemotherapy, hormonal therapy, and radiother-
apy.2,4,9–12 Small molecule EGFR inhibitors have been shown to
be effective antitumor agents.1 For example, two closely related
anilinoquinazoline-containing EGFR inhibitors, gefitinib (Iressa™,
1)2 and erlotinib (Tarceva™, 2)3 have efficacy against several types
of cancers in human clinical trials and were approved for the treat-
ment of NSCLC and colon cancers. The dual EGFR/HER2 inhibitors
lapatinib (Tykerb™, also known as GW-572016, 3) was recently
approved for the treatment of HER2-positive metastatic breast
cancer.4 Many more EGFR inhibitors including antibodies are either
still under evaluation in clinical trials or been approved for the
treatment of cancer.1
F
HN
N
Cl
O
HN
O
O
O
O
N
N
O
O
N
N
Tarceva, 2
Iressa, 1
O
F
S
O
NH
O
HN
N
Cl
O
N
Lapatinib, 3
There are numerous crystal structures of EGFR available, includ-
ing the co-crystal structures of EGFR with erlotinib or gefitinib,
which have provided a rich set of structural information for our
drug discovery effort. The structure–activity relationship (SAR) of
these EGFR inhibitors are mostly well understood.13 As shown in
Figure 1, binding of erlotinib depends on several key interactions
with the ATP binding pocket of EGFR, including the critical H-bond
with the hinge region using its quinazoline core, the filling up of
the so-called ‘back pocket’ by the ethynylphenyl substitution,
and the interactions in the tail region. There is also a hydrophobic
⇑
Corresponding authors.
E-mail address: shaojing.hu@betapharma.com.cn (S. Hu).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.067

6302
S. Hu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6301–6305
Figure 1. Crystal structure of erlotinib (gold) in EGFR (1M17 pdb). Compound 12k (cyan) was modeled in for comparison. The protein surface of the ATP binding pocket of
EGFR was shown, with red representing the hydrophobic areas and blue the hydrophilic areas.
O
O
O
Lg
Lg
O
O
HO
HO
O
O
OCH3
5a-g
OCH3
OCH3
b
B
B
NO2
4
6a-g
a
7a-g
O
OH
O
O
O
O
OCH3
e
c
N
d
B
B
9a-g
NH2
8a-g
N
R
Cl
N
R
HN
N
O
O
H2N
N
O
O
B
N
11a-d
B
12a-k
10a-g
f
Scheme 1. General synthetic approach to fused quinazolines. Reagents and conditions: (a) K2CO3/DMF, 90 °C, 3 h, 24–45%; (b) HNO3/H2SO4, HOAc, 72%; (c) H2, Pd/C, 96%; (d)
HCONH2/HCO2NH4, 165 °C, 80%; (e) POCl3, 77%; (f) i-PrOH/DMF,72%.
patch a little further away from the hinge, which may also provide
additional opportunities for ligand optimization. In particular, the
tail region is able to accommodate a variety of functional groups
without affecting the activities of the compounds too much. The
various substituents at the 6 and 7 positions of the quinazolines
ring which occupies the tail region are able to significantly modu-
late the properties of the compounds, and such differences be-
tween gefitinib, erlotinib and lapatinib contribute in part to the
distinct profiles of these three EGFR inhibitors. Therefore this is a
major area to further optimize good lead molecules.
tural modified quinazolines compounds by fused with numerous
ring systems, aimed at identifying potent, selective, and bioavail-
able EGFR inhibitors as anti-cancer agents. Herein we report their
synthesis and preliminary biological evaluation.
A general approach to synthesize the designed crown ether
fused quinazolines compounds 12a–k is shown in Scheme 1, start-
ing from commercially available methyl 2,3-dihydroxybenzoate 4.
The key intermediates, methyl benzoates 6a–g with different ring
system were synthesized from the reaction of methyl 2,3-
dihydroxybenzoate 4 with various a,x-dihalogens or a,x-tosylates
Our computer model study provides us much needed informa-
tion to design new EGFR inhibitors. We have focused our develop-
ment on the modification of tail region by attaching cyclic systems
to the quinazoline ring. Therefore we synthesized a series of struc-
5a–g in the presence of abase such as potassium carbonate at ele-
vated temperature. The standard nitration procedure is employed
for the nitration of methyl benzoates 6a–g, which was reduced
by hydrogenation in methanol to give anilines 8a–g. Compounds
R
R
HN
R
Lg
Lg
HN
HN
O
5a-g
N
HO
HO
a
O
O
N
N
B
O
N
N
N
b
13
14
12a-k
Scheme 2. Alternative synthetic approach to fused quinazolines. Reagents: (a) BBr3, THF, 63%; (b) K2CO3/DMF, 20–36%.

S. Hu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6301–6305
6303
8a–g were treated with formamide and ammonium formate to
provide substituted quinazolin-4-ones 9a–g. Using 5 equiv of
POCl3 yielded the key intermediates 10a–g. Coupling 10a–g with
substituted anilines 11a–d in a solvent such as isopropanol,
diglyme, or butoxyethanol gave the target compounds, the crown
ether fused quinazolines 12a–k in six steps of 11–20% yield.
An alternative approach to synthesize the desired compounds
12a–k is shown in Scheme 2. The known 4-anilinoquinazoline class
of compounds, (6,7-dimethoxy-quinazolin-4-yl)phenyl-amines
Table 1
Structure and EGFR protein kinase inhibitory activity of the fused quinazolines 12a–k
a
b
Compound
Structure
EGFR IC50 (nM)
Tyrphospor-IC50 (nM)
Br
HN
N
12a
85
120
55
N/Dc
N/Dc
N/Dc
O
O
N
Br
HN
N
12b
12c
O
N
O
HN
Br
O
N
O
N
O
Br
HN
N
O
O
S
O
N
12d
12e
150
N/Dc
N
O
HN
Br
5
50
O
O
O
O
N
N
Br
HN
N
12f
12g
12h
12i
5
7
2
8
50
O
O
O
N
O
S
O
Br
Cl
HN
>1000
O
O
N
N
N
HN
55
O
O
O
O
N
Br
HN
N
>1000
O
S
N
O
(continued on next page)

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S. Hu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6301–6305
Table 1 (continued)
a
b
Compound
Structure
EGFR IC50 (nM)
Tyrphospor-IC50 (nM)
F
Cl
HN
N
12j
2
45
45
O
O
O
O
N
N
HN
N
12k
2
O
O
O
O
a
In vitro kinase assay according to Ref. 15.
EGFR-mediated intracellular tyrosine phosphorylation assay according to Ref. 14.
Not determined (N/D).
b
c
13,13c were treated with boron tribromide to give the (6,7-dihy-
droxy-quinazolin-4-yl)-phenylamines 14. The final ring formation
In summary, a series of fused anilino-quinazoline analogues
showed modest to potent EGFR inhibition, with IC50 values ranging
from 2 nM to 150 nM. SAR studies of the series revealed that oxy-
gen containing heterocycles with ring size higher than 12 member
are the favorable fused anilinoquinazolines, and the preferred sub-
stituent on the 7-anilino is a halogen such as chlorine, bromine or
ethynyl group at the meta-position. A subset of the selected com-
pounds proved to be active in an EGFR-mediated intracellular tyro-
sine phosphorylation assay in human tumor cell line A431.
Compound 12k is shown as a potent inhibitor of EGFR with excel-
lent selectivity against Abl, Arg and other kinases, exhibits a favor-
able pharmacokinetic profile in the preclinical studies, and inhibits
the growth of a broad range of human solid tumor xenografts in a
dose-dependent manner (range 50–100 mg/kg, po once daily). 12k
was completed by the reaction of intermediate 14 with
a,x-dihalo-
gen or - tosylates 5a–g in the presence of base such as potas-
a,x
sium carbonate to provide crown ether fused quinazolines 12a–k
in total 20–36% yield.
Table 1 summarizes the structures and EGFR inhibitory activi-
ties for the series of fused quinazolines 12a–k. SAR studies of the
series were first focused on the variation of different ring size. A
ring size of 12 or higher is clearly more preferred than 7 or 9 mem-
ber ring (IC50 = 2–7 nM vs >50 nM). Heteroatoms other than oxy-
gen in the ring system slightly reduced EGFR potency (oxygen
with sulfur, IC50 = 2–5 nM and 7–9 nM, respectively). However
introduction of a sulfur atom in the ring system almost completely
lost inhibition activity in the cell assay (compound 12g and com-
pound 12i). A nitrogen atom capped with a sulfonyl group embed-
ded in the ring (or something similar) drastically reduced EGFR
potency (compound 12d vs compound 12c, IC50 = 150 vs 55 nM).
As for aniline, which occupied the ‘back pocket’ or hydrophobic
pocket of EGFR, like gefitinib and erlotinib, small non-polar substit-
uents at the anilines, such as chloro, bromo, or ethynyl, are well
tolerated and more preferred at the meta-position, as in com-
pounds 12h–k (IC50 = 2–8 nM).
Table 2
Pharmacokinetic properties of the selected compound 12k
F (%) (rat)
Cmax (po)
AUC (po)
T1/2 (po)
Cl
52
6.12
49.4
2.8 h
21 mL/min/kg
3.9 L/kg
l
l
g/mL
g/mL
Vss
Compounds 12e–12k with IC50 6 5 nM in the in vitro kinase as-
say were selected for further evaluation in an EGFR-mediated
intracellular tyrosine phosphorylation assay in human epidermoid
A431 carcinoma cell line .14
MRT
6.9 h
Fused quinazolines with oxygen substituted heterocycles and
ring size higher than 12 members are shown to be the most potent
EGFR inhibitors in this series. Representative compounds 12e,12f,
12h, 12j and 12k showed potent inhibition of EGFR kinase in an
A431 cell with IC50 values in 45–55 nM. Perhaps because of lack
cell permeability, 12g and 12i are not potent at this assay.
Compound 12k and other selected compounds were profiled in
a panel of kinase assays. No inhibition of Abl, Arg and c-Src tyrosine
kinases was observed even at concentrations higher than 1000 nM
by 12k (unpublished data).
Preliminary pharmacokinetic studies were conducted in rats with
selected compound 12k (Table 2). Pharmacokinetic parameters were
determined after single oral (35 mg/kg) and intravenous (35 mg/kg)
dosing. Compound 12k demonstrated good oral bioavailability (52%)
and exposure level (Cmax and AUC) in rats. Therefore 12k was se-
lected for the advanced preliminary pharmacological study. After
oral administration, 12k inhibits the growth of a broad range of
human solid tumor xenografts in a dose-dependent manner (range
50–120 mg/kg, po once daily). Figure 2 shows the effects of
compound 12k on the tumor size of A431 xenograft.
Figure 2. Effects of Compound 12k on the tumor size of A431 xenografts. On
tumors achieving volumes of approximately 20 mm3, tumor-bearing mice were
randomized into treatment groups (n = 10/group) and po qd Icotinib at doses of 30,
60, and 120 mg/kg in a vehicle of 0.5% CMC-Na solution for 30, 18, 18, 27 days, Taxol
ip qw at dose of 30 mg/kg as positive control.

S. Hu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6301–6305
6305
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15. In vitro kinase assay: EGFR protein (2.4 ng/
with GST-Crk (32 ng/ l) in 25 l kinase reaction buffer containing 1
ATP and 1 Ci 32P- -ATP. The mix was incubated with BPI-2009 at 0, 0.5, 2.5,
l
l, 14.5 units/
l
g, Sigma) was mixed
l
l
lM cold
l
c
12.5 and 62.5 nM on ice for 10 min, then switched to 30 °C for another 20 min.
After quenching by SDS sample buffer at 100 °C for 4 min, the protein mix was
separated by 10% SDS–PAGE gel. The dried gel was then exposed to
Phosphorimager for quantification. The phosphorylated Crk was plotted
against the concentration of the compound.
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