Four-membered heterocycles-containing 4-anilino-quinazoline derivatives as epidermal growth factor receptor (EGFR) kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.07.049

We report herein the design and synthesis of novel azaspirocycle or azetidine substituted 4-anilinoquinazoline derivatives. The EGFR inhibitory activities and in vitro antitumor potency of these newly synthesized compounds against two lung cancer cell lines HCC827 and A549 were evaluated. Most of the target compounds possess good inhibitory potency. In particular, compounds 21g with 2-oxa-6-azaspiro[3.4]octane substituent was found to possess higher EGFR inhibitory activities and similar antitumor potency comparing to the lead compound gefitinib with improved water solubility.

Full text of DOI:10.1016/j.bmcl.2013.07.049

Accepted Manuscript
Four-membered heterocycles-containing 4-anilino-quinazoline derivatives as
epidermal growth factor receptor (EGFR) kinase inhibitors
Feng Zhao, Zhaohu Lin, Feng Wang, Xiufang Zheng, Hongying Yun, Weili
Zhao, Xiaochun Dong
PII:
S0960-894X(13)00894-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.07.049
BMCL 20714
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
21 June 2013
18 July 2013
23 July 2013
Please cite this article as: Zhao, F., Lin, Z., Wang, F., Zheng, X., Yun, H., Zhao, W., Dong, X., Four-membered
heterocycles-containing 4-anilino-quinazoline derivatives as epidermal growth factor receptor (EGFR) kinase
inhibitors, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.2013.07.049
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Four-membered heterocycles-containing 4-
anilino-quinazoline derivatives as epidermal
growth factor receptor (EGFR) kinase
inhibitors
Leave this area blank for abstract info.
Feng Zhao, Zhaohu Lin, Feng Wang, Xiufang Zheng, Hongying Yun, Weili Zhao, Xiaochun Dong*
A series of novel azaspirocycle or azetidine substituted 4-anilinoquinazoline
derivatives were prepared and their EGFR inhibitory activities as well as
in vitro antitumor potency were reported.

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Four-membered heterocycles-containing 4-anilino-quinazoline derivatives as
epidermal growth factor receptor (EGFR) kinase inhibitors
Feng Zhaoa, Zhaohu Lina,b, Feng Wanga, Xiufang Zhengb, Hongying Yunb, Weili Zhaoa, Xiaochun Donga,
a School of Pharmacy, Fudan University, Shanghai, 201203, P. R. China
b Roche Pharma Research and Early Development China, Shanghai, 201203, P. R. China
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
We report herein the design and synthesis of novel azaspirocycle or azetidine substituted 4-
anilinoquinazoline derivatives. The EGFR inhibitory activities and in vitro antitumor potency of
these newly synthesized compounds against two lung cancer cell lines HCC827 and A549 were
evaluated. Most of the target compounds possess good inhibitory potency. In particular,
compounds 21g with 2-oxa-6-azaspiro[3.4]octane substituent was found to possess higher EGFR
inhibitory activities and similar antitumor potency comparing to the lead compound gefitinib
with improved water solubility.
Keywords:
EGFR inhibitors
Anti-tumor agents
Anilinoquinazolines
Four-membered heterocycles
2009 Elsevier Ltd. All rights reserved.
The epidermal growth factor receptor (EGFR, erbB1) is a
member of the erbB family of receptor tyrosine kinases (RTKs).
The epidermal growth factor receptor contains a subfamily of
four closely related receptor tyrosine kinases: EGFR (ErbB-1),
erbB2/HER2, erbB3/HER3, and erbB4/HER4.1,2 Upon ligand
binding, EGFR forms homo- or heterodimers with other
members of the erbB family and undergoes autophosphorylation
of specific tyrosine residues in its intracellular domain. This
event activates a series of downstream signals and regulates
biological processes such as cell proliferation, apoptosis,
angiogenesis, and differentiation.3,4 Deregulation of EGFR
signaling has been observed in many human cancers, including
lung, head and neck, colorectal, ovarian, breast, and bladder
cancers.5,6 The identification of EGFR as an oncogene has led to
the development of many anticancer therapeutics directed against
EGFR.
market place.7-12 These quinazoline derivatives constitute an
important scaffold for EGFR inhibition.
There are two classes of inhibitors of EGFR: (i) monoclonal
antibodies (e.g. Cetuximab and Panitumumab) which block
binding of native EGF ligand to the receptor for treatment of
metastatic colorectal cancer, as well as head and neck cancer; (ii)
small molecules that compete with ATP in the intracellular
tyrosine kinase domain (TKD) and block kinase activity which
don’t need to consider about endogenous ligand binding.6 Small
molecule ATP competitive inhibitors including gefitinib (Iressa,
AstraZeneca, 1), erlotinib (Tarceva, OSI Pharmaceuticals, 2) and
lapatinib (Tykerb, Glaxo-SmithKline, 3) were launched into the
Figure 1. Chemical structures of EGFR-TKIs
The co-crystal structures of EGFR with gefitinib and erlotinib
have provided a rich set of structural information for drug
discovery efforts on EGFR inhibitors. The binding modes of
these EGFR inhibitors are mostly well understood.13 Binding
mode analysis of gefitinib suggests several key interactions with
the ATP binding pocket of EGFR, including the critical H-bond
with the hinge region using its quinazoline moiety, the filling up
of the hydrophobic pocket by the 3-chloro-4-fluoro aniline
substituent, and the occupation of 6-morpholinopropoxy group
———
 Corresponding author. Tel.: +86-21-5198-0123; e-mail: xcdong@fudan.edu.cn

into solvent region.14 The solvent region generally tolerates a
variety of polar functional groups without affecting the efficacy
too much. On the other hand, the modulation of the side chain in
the solvent region may alter the physico-chemical properties of
the inhibitors . The success of icotinib (Conmana, Beta Pharma,
4) which mimics erlotinib with a crown ether fused quinazoline
moiety is a good example of this strategy.15
In recent years, four-membered heterocycles as “compact
modules” have attracted much interest in organic chemistry and
drug discovery.16 Grafting these small four-membered
heterocycles onto a molecular scaffold may lead to significant
impacts on physico-chemical properties. As typical examples,
azaspiro[3.3]heptanes were considered as surrogates for
piperazines, morpholine, thiomorpholine, and piperidine (Figure
2).17,18 These novel frameworks have been incorporated onto
scaffolds of druglike structures, such as fluoroquinolones, to
provide compounds that retain notable activity and populate
chemical space otherwise not previously accessed.17
In this study, we choose gefitinib as our lead compound for
the development of novel EGFR kinase inhibitors by
modification of the morpholine moiety in the solvent region with
four-membered heterocyclic building blocks. Therefore a series
of structural modified quinazolines compounds connected with
different azaspirocycles or azetidines were prepared. Herein, the
syntheses and preliminary biological evaluation results are
communicated.
The designed compounds were synthesized as shown in
Scheme 1.19 The 3-hydroxy-4-methoxy benzaldehyde (5) was
alkylated with 1-bromo-3-chloro propane or 1-bromo-2-
chloroethane to afford the corresponding chlorides 6 or 7,
respectively, which was then converted to methyl benzoate 8 or 9
through oxidation followed by esterification. Compound 10 or 11
obtained by nitration of 8 or 9 was reduced with iron powder in
acetic acid to give 12 or 13 in satisfactory yield. Cyclization of
12 or 13 with formamidine acetate generated hydroxyl
quinazoline 14 or 15 efficiently. Chlorination with phosphorus
oxychloride followed by nucleophilic substitution with 3-chloro-
4-fluoroaniline affords the key precursor 18 or 19. The target
products 20a-f and 21a-g were then obtained by halogen
exchange and then simple alkylation with various four-membered
heterocylces.
Figure 2. Azaspiro[3.3]heptanes as novel building blocks in drug discovery.
Scheme 1. Reagents and conditions: (a) 1-bromo-3-chloropropane or 1-bromo-2- chloroethane, K2CO3, 60°C, 95% for 6 and 99% for 7; (b) KMnO4, acetone,
reflux; (c) SOCl2, MeOH, reflux, 47% for 8 and 45% for 9 (two steps); (d) Ac2O, AcOH, HNO3, 0-5°C, 78% for 10 and 81% for 11; (e) Fe, AcOH, MeOH, 88%
for 12 and 90% for 13; (f) formamidine acetate, ethanol, reflux, 87% for 14 and 88% for 15; (g) POCl3, Et3N, toluene, reflux, 63% for 16 and 61% for 17; (h) 3-
chloro-4-fluoroaniline, i-PrOH, reflux, 75% for 18 and 77% for 19; (i) 1) KI, acetone, reflux, 2) K2CO3, amine, 14-82% (two steps).
The 13 newly synthesized azaspirocycle or azetidine
substituted 4-anilinoquinazoline derivatives 20a-f and 21a-g
were subjected for tyrosine kinase activity inhibitory assay of the
EGFR with Z’-Lyte Kinase Assay Kit, 20 as well as in vitro cell
cytotoxicity against two human lung cancer cell lines, HCC827
and A549, with gefitinib (1) as a positive control. 21 The HCC827
cell line harbors an EGFR-activating mutation (deletion
mutation) and is a representative EGFR-driven cell line. The
A549 cell line was used to evaluate non-EGFR-mediated effects
(“off target” or cellular toxicity effects). The results are collected
in Table 1. Most compounds exhibit good correlation between
EGFR inhibition and in vitro antitumor activities. All the tested
compounds possess potent EGFR inhibition with IC50 values
ranging from 190 nM to 15 nM, as well as good anti-proliferative
activities with low μM IC50 values against HCC827. As expect,
none of compounds, including gefitinib, showed good efficacy
against the EGFR insensitive cell line A549. Compared to
gefitinib which contains a morpholinopropoxy side chain,
compounds 20a and 21a which contain 2-oxa-6-
azaspiro[3.3]heptanes as the most close surrogate of morpholine
possess potent EGFR inhibition and good efficacy against the
HCC827 cell line. Compounds 20b and 21b with 1-oxa-6-
azaspiro[3.3]heptanes also have potent efficacy against HCC827.
Compound 21b matches the potency of gefinitib against EGFR,
however 20b exhibits diminished inhibition compared to
gefitinib. Both quinazoline analogues containing 2-thio-6-
azaspiro[3.3]heptanes (20c and 21c) are effective in enzyme
inhibition of EGFR and cell based efficacy against HCC827.
However, the sulfone derivatives 20d and 21d have diminished
efficacy compared to the thio analogues. It is note worthy that 3-
hydroxy azetidine derivatived quinazoline analogues 20e and 21e
exhibit relatively low anti-proliferative effect against HCC827

although potent inhibition for EGFR was noticed. In a stark
contrast, the methylation of the hydroxyl leads to the recovery of
efficacy against HCC827 (compare 20f and 21f with 20e and
21e). Compound 21g containing 2-oxa-6-azaspiro[3.4]octane
moiety possesses both high activities against EGFR and HCC827
(IC50 = 15 nM for EGFR and 28 nM for HCC827).
region as does the 6-morpholinopropoxy group of gefitinib. The
in silico experiments corroborate well with in vitro results.
Table 1. Inhibition of EGFR Kinase and HCC827 and A549 Cell
Lines by 20a-f, 21a-g in vitro a
Compounds
EGFR-WTb Kinase
inhibition (IC50, μM)
0.016
0.190
0.052
0.158
0.026
0.094
0.056
0.036
0.048
0.024
0.025
0.034
0.015
0.023
HCC827
(IC50, μM)c
0.016
0.023
0.051
0.154
0.116
0.064
0.069
0.039
0.050
0.389
0.352
0.042
0.028
0.012
A549
(IC50, μM)c
>50
>50
4.8
>50
>50
>50
31.8
25.7
9.5
25.2
>50
25.1
>50
20.3
20a
20b
20c
20d
20e
20f
21a
21b
21c
21d
21e
21f
Figure 3. Docking of compound 20a (orange carbons) and 21g (cyan
carbons) within the EGFR kinase domain (2ITY.pdb) in comparison with
gefitinib (magenta carbons).
21g
Gefitinib(1)
a Values are expressed as the mean of three independent experiments and are
mostly within 10% error margins. b WT: wild type kinase. c Determined by
CCK-8 assay.
In summary, a series of azaspirocycle or azetidine substituted
4-anilinoquinazoline derivatives were designed and synthesized.
Cytotoxicity against two cancer cell lines HCC827 and A549 and
EGFR inhibitory activity of these novel compounds were
examined. The preliminary evaluation results demonstrate that
the replacement of the morpholine moiety of gefitinib with four-
membered building blocks can retain the EGFR inhibition and in
vitro antitumor activities by appropriate modifications. The
efficacy of three azaspirocycle substituted compounds 20a, 20b
and 21g are very similar to the parental drug. And the
incorporation of these small four-membered heterocycles may
lead to significant impacts on water solubility, part of the
compounds was found to have greatly improved water solubility
(up to 34-fold) than gefitinib. Compound 21g carrying 2-oxa-6-
azaspiro[3.4]octane module possesses good potency and water
solubility.
Gefitinib was reported to have limited water solubility (3.77
μmol/L).22 We are interested in the effects of the modules on the
water solubility. The aqueous solubility of some selected
compounds in comparison with gefitinib were measured and
summarized in Table 2.23 As a trend, compounds with two carbon
linker are sparingly soluble in water. In a stark contrast,
compounds with three carbon linker possess much higher
solubility (compare 20a/21a, 20b/21b, 20c/21c, and 20d/21d). 2-
Oxa-6-azaspiro[3.3]heptane is advantageous over 1-Oxa-6-
azaspiro[3.3]heptane for the water solubility (compare 20a/20b,
21a/21b). 2-Thio-6-azaspiro[3.3]heptane is less effective than 2-
oxa-6-azaspiro[3.3]heptane (compare 20a/20c, 21a/21c). The
sulfone analogue is advantageous over the thio precursor
(compare 21c/21d). Compound 21g containing 2-oxa-6-
azaspiro[3.4]octane moiety possesses much improved water
solubility in addition to the potent inhibition and efficacy
compared with gefitinib.
Our results indicate the potential applicable value of these
four-membered building blocks in new kinase inhibitor
development.
Acknowledgments
Table 2. Water solubility of selected compounds at 20°C.
Compounds
Water solubility (μg/mL)
20a
20b
20c
20d
21a
21b
1.4
< 1
< 1
< 1
28.3
1.2
This work was supported by Shanghai Municipal Natural
Science Foundation (12ZR1403300). The authors are also
grateful to the financial supports from Roche Pharma Research
and Early Development China.
21c
21d
21g
Gefitinib(1)
25.6
72.3
33.8
2.1
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was carried out using Autodock 4.2 package.24 As shown in
Figure 3, 20a (orange carbons) and 21g (cyan carbons) bind in
almost the same location as that of gefitinib (magenta carbons).
The quinazoline ring is oriented with the 1-N in the back of the
ATP-binding pocket, where it hydrogen bonds with the main
chain amide of Met793 which lies in the hinge region. The 3-
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pocket in the back of the ATP-binding cleft. And the
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20. In vitro kinase assay: Kinase inhibitory ability was determined
using Z’-Lyte Kinase Assay Kit (Invitrogen Corporation, Carlsbad,
CA, USA), following the manufacturer's instructions in a 20 μl,
two-hour, room-temperature reaction. EGFR and ATP
concentrations were 5 nM and 10 μM, respectively. Fluorescence
signals were detected through SpectraMax M5 microplate reader
(Molecular Devices).
21. Cell proliferation assay: Cell lines were plated in triplicate and
incubated with escalating concentrations of compounds for 72 h.
After 72 h, proliferation was measured using a WST-8(2-(2-
methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfo-phenyl)-
2H-tetrazolium)-based viability assay (CCK8). Medium was
replaced with fresh WST8- medium (1 : 10) to cell plates with 1-2
h
in an incubator, and read at 450 nm wavelengths with
SpectraMAX190 (MDS). Determine inhibition rate (IR) of the
tested compounds with following formula: IR (%) = (ODcontrol
-
ODcompound)/ODcontrol ×100%. Plot curve of IR against tested
compound concentrations with SoftMax Pro and this software will
find the concentration corresponding to 50% IR (IC50) on the
curve.
22. McKillop, D.; Partridge, E. A.; Kemp, J. V.; Spence, M. P.;
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23. The compounds were suspended in water and the suspensions
were sonicated for 1.5 h and filtered through 0.22 μM micronic
filter plate. The above fluid was analyzed by HPLC.
24. Autodock 4.2 package was used for performing automated
docking of inhibitors to EGFR. Larmackian genetic algorithm was
used to search docking conformers. During the docking process, a
series of the docking parameters were set on. The number of
generations, energy evaluations, and docking runs about genetic
algorithm were set to 27000, 2500000, and 50. The rate of gene
mutation and crossover is 0.02 and 0.8, respectively. The binding
free energy between inhibitor and EGFR is -7.973 kcal/mol
(Gefitinib, 1), -7.673 kcal/mol (20a), and -7.431 kcal/mol (21g),
respectively.