Synthesis and biological evaluation of novel tricyclic oxazine and oxazepine fused quinazolines. Part 1: Erlotinib analogs

Article abstract of DOI:10.1016/j.bmcl.2013.12.079

Two series of novel tricyclic oxazine and oxazepine fused quinazolines have been designed and synthesized. The in vitro antitumor effect of the title compounds was screened on N87, A431, H1975, BT474 and Calu-3 cell lines. Compared to erlotinib and gefiti

Full text of DOI:10.1016/j.bmcl.2013.12.079

Bioorganic & Medicinal Chemistry Letters 24 (2014) 884–887
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel tricyclic oxazine
and oxazepine fused quinazolines. Part 1: Erlotinib analogs
⇑
Xiangfeng Chen, Youguo Du, Huanliang Sun, Feidong Wang, Lingsheng Kong, Min Sun
Nanjing Haiguang Applied Chemistry Institute, Jiangsu Aosaikang Pharmaceutical Co. Ltd, Nanjing 211112, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of novel tricyclic oxazine and oxazepine fused quinazolines have been designed and synthe-
sized. The in vitro antitumor effect of the title compounds was screened on N87, A431, H1975, BT474 and
Calu-3 cell lines. Compared to erlotinib and gefitinib, compounds 1a–1h were found to demonstrate more
potent antitumor activities. Several derivatives could counteract EGF-induced phosphorylation of EGFR in
cells, and their potency was comparable to the reference compounds. Compounds 1a–1h were chosen for
further evaluation of EGFR and HER2 in vitro kinase inhibitory activity. Compounds 1b–1f, 1h effectively
inhibited the in vitro kinase activity of EGFR and HER2 with similar efficacy as erlotinib and gefitinib.
Ó 2014 Published by Elsevier Ltd.
Received 24 September 2013
Revised 16 December 2013
Accepted 19 December 2013
Available online 25 December 2013
Keywords:
Tricyclic fused quinazolines
Antitumor activity
EGFR
HER2
Lung cancer is the number one cause of cancer mortality in men
globally, with an estimated 13% (1.6 million) of total cases and
accounting for 18% (1.4 million) of total deaths worldwide in
2008.1 Although chemotherapy is the mainstay of cancer therapy,
the use of available chemotherapeutics is often limited mainly
due to undesirable side effects and a limited choice of available
anticancer drugs. This clearly underlies the urgent need of develop-
ing novel chemotherapeutic agents with more potent antitumor
activities.2
EGFR is a member of a family of closely related receptors,
including EGFR (ErbB1), human epidermal growth factor recep-
tor-2 (HER2)/neu (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). EGFR
is overexpressed in the majority of NSCLCs and its expression is in-
versely related to survival outcome.3 The two main signaling path-
ways activated by EGFR are the RAS/RAF/MEK/ERK pathway and
the PI3K/AKT pathway, which lead to evasion of apoptosis (cell sur-
vival) and cell proliferation.4,5
Despite the benefits of reversible EGFR TKIs, the efficacy of these
agents has been limited by the development of resistance in most,
if not all, initially responsive patients, which leads to tumor pro-
gression and relapse after a median time of 12 months.9
Recently, second generation inhibitors, designed to address
resistance, are currently under investigation in clinical trials. The
two most advanced compounds are dacomitinib (PF-00299804,
Pfizer) and afatinib (BIBW-2992, Boehringer–Ingelheim) (Fig. 1)
which are currently approved and in phase III clinical trials.10 Both
of these compounds are structurally very similar to gefitinib and
erlotinib with the exception that they harbor Michael acceptors
in the 6-position side chain of the quinazoline core. This leads to
dacomitinib and afatinib to be irreversible inhibitors of EGFR.11
The results of several phase III clinical trials for dacomitinib and
afatinib are expected to be completed in 2013. Moreover, many
studies have been targeted at finding new structures based on
quinazolines that are potent EGFR inhibitors.12–14
Based on the critical role of the ErbB family of receptors in the
growth and metastases of NSCLC and other human malignancies,
epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors
(TKIs) have been developed as targeted antitumor agents.6 Cur-
rently gefitinib (Iressa™, AstraZeneca) and erlotinib (Tarceva™,
Genentech) (Fig. 1), were approved by the U.S. Food and Drug
Administration (FDA) for the treatment of patients with non–small
cell lung cancer (NSCLC) in May 2003 and November 2004, respec-
tively.7,8 Both are reversible competitive inhibitors at the adeno-
sine triphosphate (ATP) binding site of the EGFR TK domain.
Based on erlotinib as the leading compound, we have devised
and synthesized two series of novel tricyclic oxazine and oxazepine
fused quinazolines through intramolecular cyclization which pos-
sessed a functional Michael acceptor group, with the aim of obtain-
ing agents displaying more potent antitumor activities. The
antitumor effect of all the newly synthesized compounds on the
in vitro growth of five cell lines, namely human gastric carcinoma
cell line NCI-N87 (HER2 overexpression), human epidermoid carci-
noma cell line A431 (EGFR overexpression), human adenocarci-
noma cell line NCI-H1975 (EGFR L858R/T790M mutation), human
breast cancer cell line BT-474 (HER2 overexpression) and human
adenocarcinoma cell line Calu-3(HER2 overexpression), was evalu-
ated. Apparent growth inhibition was observed for most of the
⇑
Corresponding author. Tel.: +86 25 51198557; fax: +86 25 52162777.
E-mail address: sunmin@ask-pharm.com (M. Sun).
0960-894X/$ - see front matter Ó 2014 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2013.12.079

X. Chen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 884–887
885
Figure 1. Selected 1st, and 2nd generation EGFR inhibitors for NSCLC.
compounds, with 1a–1h demonstrating more potent activities
against all five cell lines as compared to gefitinib and erlotinib,
respectively. Furthermore, all the synthesized compounds were as-
sessed the in vitro inhibition of EGF-induced receptor autophos-
phorylation in the KB nasopharyngeal carcinoma cell line. Finally,
compounds 1a–1h were chosen for further evaluation of EGFR
and HER2 in vitro kinase inhibitory activity.
(1a–1h) demonstrated more remarkable inhibition activity against
the four cell lines (IC50: 0.046–2.06 M) compared with erlotinib
(IC50: 0.75–>10 M) and gefitinib (IC50: 0.36–1.00 M). The activ-
ity of compound 1h16 (IC50: 0.046–0.24
M) was found to be 3 to
>210 fold and 1.5 to 22 fold more potent than erlotinib (IC50
0.75–>10 M) and gefitinib (IC50: 0.36–1.00 M) anaginst the four
cell lines, respectively.
l
l
l
l
:
l
l
The synthetic route to tricyclic oxazine and oxazepine fused
quinazolines (1a–1p) was illustrated in Scheme 1. Conversion of
The in vitro sensitivity of NSCLC cell lines to gefitinib was most
closely associated with the presence of activating mutations in
EGFR. However, some EGFR mutations, such as T790M or exon
20 insertion mutations, were associated with gefitinib resistance
in vitro and in vivo.17 Hence the efficacy of target compounds
(1a–1p) to gefitinib and erlotinib in H1975 cell line (EGFR L858R/
T790M mutation) was compared (Table 1). The tricyclic oxazine
compounds (1a–1h) demonstrated more remarkable inhibition
the fluoro group of
2 to the corresponding hydroxylethoxy
(hydroxylpropoxy) compound 3. Treatment of 3 with phosphoryl
chloride and thionyl chloride gave the dichloride 4. Coupling of 4
with 3-aminophenylacetylene 9 gave nitro compound 5. Reduction
of 5 with iron/ammonium chloride gave the corresponding amine
6. Intramolecular cyclization of 6 with potassium carbonate gave
7. Acylation of 7 with the bromocrotonic acid chloride 10 gener-
ated from bromocrotonic acid and oxalyl chloride gave the bromo-
crotonamide 8, which was finally submitted to bromide
displacement with different aliphatic amines to yield the corre-
sponding target compounds 1a–1p.
activity against the H1975 cell line (IC50: 0.30–1.46
lM) compared
with gefitinib (IC50: >10 M) and erlotinib (IC50: 5.51
l
l
M). The
activity of compound 1h was found to be 33 fold and 18 fold more
potent than gefitinib and erlotinib, respectively.
To determine the potency of target compounds against EGFR
autophosphorylation in intact cells, we performed ELISA tests18
with EGFR-specific antibodies and measured levels of receptor
phosphorylation with increasing drug concentrations (Table 1).
Compounds 1a–1h demonstrated potent cellular effects on EGFR
All the 16 newly synthesized erlotinib derivatives (1a–1p) were
tested for cytoxicity15 toward cancer cell lines either with EGFR
(A431) or HER2 (N87, BT474, Calu-3) overexpression. The data
are summarized in Table 1. The tricyclic oxazine compounds
Scheme 1. Synthetic route for the preparation of the target compounds 1aꢀ1p. Reagents and conditions: (a) ethylene glycol, NaH, THF, 75 °C, 20 h, 99% of 3a; (a’) propylene
glycol, NaH, THF, 75 °C, 20 h, 99% of 3b; (b) POCl3, SOCl2, 90 °C, 3 h, 83% of 4a and 4b; (c) 3-aminophenylacetylene (9), isopropanol, 50 °C, 2 h, 99% of 5a and 5b; (d) Fe/NH4Cl,
DMF/H2O, 80 °C, 1 h, 80% of 6a and 76% of 6b; (e) K2CO3, KI, DMF, 110 °C, 24 h, 27% of 7a and 23% of 7b; (f) bromocrotonic acid chloride (10), Et3N, DCM, 35 °C, 24 h, 76% of 8a
and 70% of 8b; (g) aliphatic amines, DMF, r.t, 1 h.

886
X. Chen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 884–887
Table 1
Structure–activity relationships for compounds 1aꢀ1p
O
O
R
R
HN
N
HN
N
N
O
N
O
N
N
1a~1h
1i~1p
Compd
R
Cytotoxicity in different cell lines (IC50
l
M)
EGFR (cellular) IC50 (lM)
N87
0.60
0.12
0.25
H1975
0.77
A431
0.53
0.11
0.40
BT474
1.39
Calu-3
1.01
N
1a
1b
1c
O
0.64
N
0.32
0.33
0.71
0.060
0.082
N
0.89
1.38
1.67
N
1d
0.27
1.23
0.69
1.15
0.95
0.067
N
1e
1f
0.15
0.16
0.22
0.046
>10
0.68
0.87
1.46
0.30
>10
>10
>10
0.19
0.34
0.48
0.23
>10
>10
>10
0.55
0.87
1.33
0.24
>10
>10
>10
0.35
0.55
2.06
0.14
>10
>10
>10
0.042
0.042
0.78
0.046
>10
N
N
N
N
1g
1h
1i
N
N
O
N
N
1j
>10
>10
1k
>10
>10
N
1l
>10
>10
>10
>10
>10
>10
N
1m
1n
1o
1p
>10
>10
>10
>10
8.59
1.74
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
N
N
1.82
2.16
1.75
N
1.86
erlotinib
gefitinib
>10
1.00
5.51
>10
0.75
0.55
>10
0.36
0.93
0.99
0.019
0.025
phosphorylation in line with the in vitro kinase results, and their
potency was comparable to the reference compound erlotinib
and gefitinib.
Table 2
In vitro enzyme inhibition (IC50
)
Perhaps because of lacking cell permeability, all the tricyclic
oxazepine compounds (1i–1p) did not show noticeable inhibition
to all five cell lines and EGF-induced receptor autophosphorylation
Compd
EGFR (
l
M)
HER2 (lM)
1a
1b
1c
1d
1e
1f
1g
1h
0.012
0.003
0.005
0.005
0.005
0.004
0.016
0.003
0.001
0.001
0.93
0.23
0.33
0.29
0.25
0.29
1.11
0.25
0.37
0.28
in the KB nasopharyngeal carcinoma cell line even at 10 lM.
Compounds 1a–1h were chosen for further evaluation of EGFR
and HER2 in vitro kinase inhibitory activity19 (Table 2). Com-
pounds 1b–1f, 1h effectively inhibited the in vitro kinase activity
of EGFR and HER2 with similar efficacy as erlotinib and gefitinib.
We have synthesized two series of novel tricyclic oxazine and
oxazepine fused quinazolines and tested for their antitumor activ-
ities on N87, A431, H1975, BT474 and Calu-3 cell lines. Compounds
1a–1h demonstrated more potent antitumor activities when com-
pared with erlotinib and gefitinib. Moreover, compounds 1b–1f, 1h
effectively inhibited EGFR autophosphorylation in KB cell and the
in vitro kinase activity of EGFR and HER2 with similar efficacy as
erlotinib and gefitinib. From the structure-activity relationships
we may conclude that compounds containing oxazine ring demon-
erlotinib
gefitinib
strated significantly better cytotoxic activities than those with oxa-
zepine ring. The results further supported investigation of these
target compounds in vivo for cancer therapy with amplifications
of ErbB family members.

X. Chen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 884–887
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stimulated with EGF were used to quantify inhibition of EGFR
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phosphorylation. Cell were seeded at 1 Â 105 cells/ml in 100
ll grow
medium per well in 96-well plates. After 4 h, the growth medium was
replaced with 100 ll serum-depleted medium overnight. Test compound was
diluted in 100% DMSO, added to cells at a final DMSO of 0.1% v/v, and incubated
at 37 °C for 1 h. KB cells were stimulated with EGF and incubated for another
6 min. Un-stimulated cells were used as blank and untreated but stimulated
cells as control. The medium was removed, the cells were washed once with
PBS, and then lysed with 100
thoroughly mixed, 100 l supernatant was transferred to ELISA capture plate
and incubated at 37 °C for 2 h. ELISA capture plates were prepared by coating
96-well plates with 100 l/well of 0.2–0.4 g/ml EGFR capture antibody
ll lysis buffer. The diluted cell lysates were
l
l
l
overnight at 4 °C. After 2 h incubation, the plates were washed with PBST for 3
times, then incubated with anti-phosphotyrosine antibody (1:2000) at 37 °C
for 1 h. The plates were washed again and then incubated with HRP goat anti-
mouse IgG (1:4000) at 37 °C for 1 h. The plates were washed a final time and
then incubated with TMB and evaluated. IC50 values were calculated as percent
inhibition of control.
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19. In vitro kinase assay: A Caliper motility shift assay was used to measure the
potency of title compounds against HER2 and EGFR. Compounds were
prepared in DMSO at 10 mM and serially diluted in 96-well plates to obtain
50Â compound solution in 100% DMSO of 10 concentrations ranging from
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15. Cytotoxicity assay: A standard MTT assay was used to measure cell growth.
Tumor cell lines in corresponding growth media recommended by American
Type Culture Collection (ATCC) with 10% fetal bovine serum were plated in 96-
well cell culture plates (5.0 Â 103 cells/well), and allowed to adhere at 37 °C,
5% CO2 for overnight. Test compound was added, cells were incubated at 37 °C,
5% CO2 for 72 h. Cell growth medium was removed afterwards, MTT was added
to each well, and incubation at 37 °C for 4 h. Absorbance was measured at
550 nm with correction at 690 nm. IC50 values were determined by nonlinear
regression analysis of the concentration response data.
10 lM to 1 nM. 100 lL of 100% DMSO was transferred for DMSO control.
Staurosporine was used as the reference compound. Kinase reaction: Add
kinase (HER2 or EGFR) into 1Â kinase base buffer (EGFR: 50 mM HEPES, pH
7.5; 0.0015% Brij-35; 10 mM MgCl2; 2.5 mM DTT. HER2: 20 mM HEPES, pH 7.5;
0.01% Triton X-100, 5 mM MnCl2; 2 mM DTT) to prepare 2.5Â enzyme solution.
Transfer 5
assay plate in duplicate. Transfer 10
the 384-well assay plate. Incubate at room temperature for 10 min. Transfer
10
L of 2.5Â peptide solution (prepared by adding FAM-labeled peptide and
ATP into the 1Â kinase base buffer) to each well of the 384-well assay plate.
Incubate at 28 °C for 40 min. Add 25 L of stop buffer (100 mM HEPES, pH 7.5;
lL of each 5Â compound solution in 10% DMSO to the 384-well
lL of 2.5Â enzyme solution to each well of
16. Analytic data of potent inhibitor 1h: 1H NMR (DMSO-d6, 500MHz) d(ppm):
9.62(s, 1H, Ar-H), 8.52(s, 1H, Ar-H), 8.50(br s, 1H, NH), 7.99(s, 1H, Ar-H), 7.87(d,
J = 8.2Hz, 1H, Ar-H), 7.38(t, J = 7.9Hz, 1H, Ar-H), 7.19–7.21(m, 2H, Ar-H),
6.86(td, J = 5.8, 15.2Hz, 1H, CH=CH), 6.61(d, J = 15.2Hz, 1H, CH=CH), 4.43(t,
J = 4.6Hz, 2H, CH2O), 4.17(s, 1H, ethynyl-H), 4.06–4.08(m, 2H, CH2N), 3.06(d,
l
l
0.015% Brij-35; 0.2% Coating Reagent #3; 50 mM EDTA) to stop the reaction.
Collect conversion data on Caliper EZ Reader II. Convert conversion values to
inhibition values according to percent inhibition = (max-conversion)/(max-
min) Â 100%, in which ‘max’ stands for DMSO control, ‘min’ stands for low
control. Fit the data in XLfit to obtain IC50 values. The equation used is:
Y = Bottom + (Top-Bottom)/(1 + 10^((log IC50 À X) Â HillSlope)).
J = 4.8Hz, 2H, CH2N)
,
2.12(s, 6H, 2ÂCH3); 13C NMR (DMSO-d6, 125MHz)
d(ppm): 163.45, 157.51, 154.72, 151.70, 149.20, 140.15, 134.98, 129.92, 129.30,
126.98, 126.88, 125.16, 122.94, 122.16, 118.58, 112.22, 109.62, 84.03, 80.99,
67.41, 57.22, 45.82, 42.33; MS [ESI]+: m/z: 414[M+H]+; HRMS [ESI]+: m/z
414.1918 [M+H]+ calcd for C24H23N5O2: found 414.1852.
17. Engelman, J. A.; Zejnullahu, K.; Gale, C. M.; Lifshits, E.; Gonzales, A. J.;
Shimamura, T.; Zhao, F.; Vincent, P. W.; Naumov, G. N.; Bradner, J. E.; Althaus, I.