A chemical tuned strategy to develop novel irreversible EGFR-TK inhibitors with improved safety and pharmacokinetic profiles

Article abstract of DOI:10.1021/jm5014659

Gatekeeper T790 M mutation in EGFR is the most prevalent factor underlying acquired resistance. Acrylamide-bearing quinazoline derivatives are powerful irreversible inhibitors for overcoming resistance. Nevertheless, concerns about the risk of nonspecific covalent modification have motivated the development of novel cysteine-targeting inhibitors. In this paper, we demonstrate that fluoro-substituted olefins can be tuned to alter Michael addition reactivity. Incorporation of these olefins into the quinazoline templates produced potent EGFR inhibitors with improved safety and pharmacokinetic properties. A lead compound 5a was validated against EGFR^WT, EGFR^T790M as well as A431 and H1975 cancer cell lines. Additionally, compound 5a displayed a weaker inhibition against the EGFR-independent cancer cell line SW620 when compared with afatinib. Oral administration of 5a at a dose of 30 mg/kg induced tumor regression in a murine-EGFR^L858R/T790M driven H1975 xenograft model. Also, 5a exhibited improved oral bioavailability and safety as well as favorable tissue distribution properties and enhanced brain uptake. These findings provide the basis of a promising strategy toward the treatment of NSCLC patients with drug resistance.

Full text of DOI:10.1021/jm5014659

Article
pubs.acs.org/jmc
A Chemical Tuned Strategy to Develop Novel Irreversible EGFR-TK
Inhibitors with Improved Safety and Pharmacokinetic Profiles
Guangxin Xia,‡,∥ Wenteng Chen,†,∥ Jing Zhang,‡ Jiaan Shao,† Yong Zhang,‡ Wei Huang,† Leduo Zhang,‡
Weixing Qi,† Xing Sun,‡ Bojun Li,‡ Zhixiong Xiang,‡ Chen Ma,‡ Jia Xu,‡ Hailin Deng,‡ Yufeng Li,‡
Ping Li,‡ Hong Miao,‡ Jiansheng Han,‡ Yanjun Liu,‡ Jingkang Shen,*,‡,§ and Yongping Yu*,†
†Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Science, Zhejiang University, 866
Yuhangtang Road Zijin Campus, Hangzhou 310058, China
‡Central Research Institute, Shanghai Pharmaceutical Holding Co., Ltd., Building 4, No. 898 Ha Lei Road, Zhangjiang Hi-Tech Park,
Shanghai 201203, China
§State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, No. 555 Zuchongzhi
Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China
S
* Supporting Information
ABSTRACT: Gatekeeper T790 M mutation in EGFR is the
most prevalent factor underlying acquired resistance. Acryl-
amide-bearing quinazoline derivatives are powerful irreversible
inhibitors for overcoming resistance. Nevertheless, concerns
about the risk of nonspecific covalent modification have
motivated the development of novel cysteine-targeting
inhibitors. In this paper, we demonstrate that fluoro-
substituted olefins can be tuned to alter Michael addition
reactivity. Incorporation of these olefins into the quinazoline
templates produced potent EGFR inhibitors with improved
safety and pharmacokinetic properties. A lead compound 5a
was validated against EGFRWT, EGFRT790M as well as A431
and H1975 cancer cell lines. Additionally, compound 5a displayed a weaker inhibition against the EGFR-independent cancer cell
line SW620 when compared with afatinib. Oral administration of 5a at a dose of 30 mg/kg induced tumor regression in a murine-
EGFRL858R/T790M driven H1975 xenograft model. Also, 5a exhibited improved oral bioavailability and safety as well as favorable
tissue distribution properties and enhanced brain uptake. These findings provide the basis of a promising strategy toward the
treatment of NSCLC patients with drug resistance.
In this study, we sought to exploit novel irreversible EGFR
inhibitors to overcome the problem of drug resistance and to
reduce the risk of nonspecific covalent binding.13 To this end,
we focused on replacement of the acrylamide function by
alternative and less reactive electrophiles. A novel quinazoline
analogue bearing fluoro-substituted olefins was designed and
synthesized. Their EGFR kinase inhibitory activity, in vitro
antiproliferative effects as well as preliminary pharmacokinetic
studies and in vivo antitumor efficacy were evaluated.
Consequently some compounds with greater potency and
superior safety index were identified. A lead compound 5a was
found to effectively inhibit EGFRWT, EGFRT790M as well as
A431 and H1975 cancer cell lines. Oral administration of 5a led
to tumor regression in a murine-EGFRL858R/T790M driven H1975
xenograft model. Also, 5a exhibited improved oral bioavail-
ability, as well as favorable tissue distribution properties,
increased safety index, and enhanced brain uptake. These
INTRODUCTION
■
The ErbB family kinases play a crucial regulatory role associated
with a variety of malignancies, making this protein family an
attractive drug target.1−4 Despite the demonstrated clinical
efficacy of reversible EGFR-TKIs,5,6 not all patients with cancer
respond to such treatment. The short-lived clinical outcome is
partly due to the acquired drug resistant mutations in EGFR,
which affect roughly 50% of the treated population.7,8 The
elucidated resistance mechanisms may be circumvented by
irreversible kinase inhibitors. These inhibitors all rely on an
acrylamide electrophilic warhead that forms a covalent bond
with a conserved cysteine residue in EGFR (Cys797). The
results enable a greater occupancy of the ATP binding site than
is found with the reversible inhibitors, thus providing the ability
to prolong the time of exposure and circumvent drug
resistance.9,10Afatinib, an irreversible EGFR-TKI, was approved
by the FDA for the treatment of patients with metastatic
NSCLC in 2013.11 However, these irreversible kinase inhibitors
all contain with a reactive “warhead” which can irreversibly bind
to proteins other than the target, creating a toxicity burden.12
Received: July 1, 2014
© XXXX American Chemical Society
A
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Figure 1. GSH conjugate addition to fluoro-substituted olefins. (1-a) model reaction between N-phenylacrylamide (A) or 2-fluoro-N-
phenylacrylamide (B) and reduced GSH in PBS buffer. (1-b) Conjugate addition reactions of GSH (10 mM) with A (1 mM) or B (1 mM) at various
time points (0−1440 min) and monitored by HPLC-HRMS. (1-c) B (1 mM) was treated with increasing concentrations of GSH (10−200 mM) and
monitored by HPLC-HRMS.
findings provide a promising strategy for the development of
novel treatments for NSCLC patients with drug resistance.
changes in cell uptake, tissue distribution, metabolic stability,
and improved pharmacokinetic properties.15−18 Consequently,
we focused our efforts on the development of the fluoro-
substituted olefin as a novel cysteine capture group.
RESULTS AND DISCUSSION
■
Using a structure-based approach, quinazoline analogues
bearing different geometric α-fluoro acrylamides were designed
and synthesized. Scheme 1 depicts the synthetic route for
compounds 1−12. Compounds IV are synthesized by coupling
their precursor amines (III) with the desired carboxylic acid
(II). 6-Amino-4-anilinoquinazoline (III) is prepared in six steps
from commercially available 2-amino-4-fluorobenzoic acid as
previously reported.19,20 The selective hydrolysis of phosphate
(I) affords the corresponding 2-(diethoxyphosphoryl)-2-fluoro-
acetic acid (II) in excellent yield.21 A Horner−Wadsworth−
Emmons olefination of the quinozoline intermediates (IV) and
the substituted aldehydes (VIII) provides a mixture of isomers
(1−12) in good yield.22 The corresponding aldehydes are
prepared in two steps from secondary amines (V) by
substitution with 2-bromo-1,1-dimethoxyethane (VI) and
deprotection of the acetal in conc HCl.23 The isomers (1−
12) were separated by semiprep HPLC or Thar SFC Pre80 for
biological testing.
Chemistry. Attenuated electrophiles have a reduced like-
lihood of reacting irreversibly with off-target proteins.14a,b
Because of the high electronegativity, small size, and special
chemical reactivity with respect to hydrogen, we hypothesized
the replacement of hydrogen at the α-position of acrylamide
with fluorine would chemically tune the Michael-type conjugate
addition. To test this hypothesis, two simple Michael acceptors,
N-phenylacrylamide (A) and 2-fluoro-N-phenylacrylamide (B),
were synthesized and evaluated the Michael addition reaction
rate with reduced glutathione (GSH) (Figure 1). Each
compound was reacted separately with 10.0 equiv of GSH in
PBS buffer at pH = 7.0, and the reactions were monitored by
HPLC-HRMS. The results indicate that the conjugation
reaction of A and GSH is significantly faster than that of B,
with the production of the thioether adduct being 95.4% and
20.7%, respectively (Figure 1). Increasing concentrations of
GSH was found to cause a stepwise decrease in the prominent
UV−visible absorption band of B (λ = 214 nm). Fitting the
titration data provides an apparent kd of 33.3 mM (Figure 1-c).
We anticipated that the slower model reaction between GSH
and B would possibly result in less nonspecific covalent
modification during binding to target proteins.14c Also,
incorporation of fluorine into small molecules has been
previously employed to optimize drug-like properties, such as
Compound 5a was selected to react with GSH under
physiological conditions for 120 min (Figure 2). In comparison
to afatinib, compound 5a produced the conjugate adduct
slowly, only 3% of thioether was detected (afatinib: 66.5%).
This result revealed that compound 5a reacted more slowly
with the thiol of GSH, potentially minimizing its chemical
reactivity toward nonspecific targets.
B
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Scheme 1. Synthesis of Compounds 1−12
a
Conditions and reagents: (a) NaOH, EtOH, 0−5 °C; (b) (COCl2), Et3N, DCM/DMF, 0 °C−rt; (c) K2CO3, DMF, 90 °C; (d) conc HCl, relux;
(e) NaOH, EtOH−H2O, rt.
Figure 2. GSH conjugate addition of afatinib or compound 5a.
Biological Evaluation. To determine the in vitro potency,
compounds 1−12 were screened against wild-type EGFR,
T790 M mutant EGFR, and HER2. Significant potency
differences were observed among the compounds 1−12, with
distinct patterns of inhibition noted for the two types of EGFR
kinases (IC50 values range from 0.18 to 59.8 nM) (Table 1).
The biological assay results show some differences in activity
based on the geometric arrangement of the analogues. For
instance, the IC50 values for compounds 5a and 5b on
EGFRT790M were 5.52 and 25.8 nM, respectively. The same
trend was observed for 4a/4b (4a, 21.3 nM; 4b, 42.9 nM). The
trans-isomers displayed more potent than the cis-isomers in the
EGFRT790M kinase assay. On the other hand, the profile against
HER2 shows that quinazoline analogues bearing α-fluoro
17.3 nM), with the exception of compound 3a (IC50 = 1.38
nM). Activities of compounds 1−12 against tumor cells
expressing EGFR mutations are demonstrated in two cancer
cell lines (Table 1). Gefitinib-sensitive cell line A431 is an
epidermoid cell that is driven by amplified wild-type EGFR.
Gefitinib-resistant cell line NCI-H1975 harbors the
EGFRL858R/T790M double mutation. The most enzymatically
potent compounds, 1a, 3a, and 5a, also exhibit potent growth
inhibitory activities in the A431 cells with IC50 values of 0.55,
0.34, and 0.13 μM, respectively, and are more potent than
gefitinib. More importantly, compounds 1a, 3a, and 5a display
high inhibitory activities against the growth of gefitinib-resistant
H1975 cells, similar to the effects of afatinib. Together, six hit
compounds were identified that retained similar in vitro
potency to afatinib (Figure 3).
acrylamide are 2.5−89-fold less potent than afatinib (IC50
=
C
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Table 1. In Vitro Antiproliferative Activities against Gefitinib-Sensitive and -Resistant Cell Lines and the EGFR-TK Inhibitory
Activities of Compounds 1−12
a
The antiproliferative activities of the compounds were evaluated using the CellTiter-Glo (Promega) Kit assay. The data shown are the means from
b
three independent experiments. The kinase inhibitory activities of the compounds were evaluated using the Homogeneous time-resolved
fluorescence (HTRF) method (Cisbio) assay. The data shown are the means from three independent experiments.
D
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findings confirm that attenuated electrophiles conjugated to
quinazolines would have a reduced likelihood of reacting with
nonspecific proteins.
These promising enzymatic and cellular potencies promoted
us to further investigate the pharmacokinetic profiles. The
influence of geometric isomerism on the compound clearance
(Clint) induced in various microsomes demonstrated remark-
able differences between cis- and trans- isomer pairs
(Supporting Information, Table S1 and Table 3). The cis-
Table 3. Microsomal Stability and CYPs Inhibition Study of
Selected Compounds
Clint (μL/min/mg protein)
IC50 (μM)
CYPs inhibition
mouse
LM
dog
LM
monkey (3A4, 2D6, 2C9,
compd
1a
HLM RLM
LM
1A2, and 2C19)
14
37
27
2
18
11
45
31
47
12
5
194
34
95
39
26
9
128
102
97
>10
>10
>10
>10
2a
3a
33
5a
14
31
canertinib
afatinib
50
8
105
12
221
24
6
isomer analogues suffer a 5−10-fold reduction in human, rat,
and mouse liver microsomal stability when compared to the
corresponding trans-isomers. For instance, the Clint values for
compounds 5a and 5b in human microsome were 2 μL/min/
mg protein and 60 μL/min/mg protein, respectively. The same
trend was observed for 2a/2b (2a, 37 μL/min/mg protein; 2b,
151 μL/min/mg protein), 4a/4b (4a, 22 μL/min/mg protein;
4b, 124 μL/min/mg protein) and 6a/6b (6a, 18 μL/min/mg
protein; 6b, 190 μL/min/mg protein). Replacing the N,N-
dimethyl group of compound 1a with a polar piperidine
obtained compound 2a with an increasing dog LM stability (1a,
194 μL/min/mg protein, vs 2a, 34 μL/min/mg protein).
Modifying the 7-position of compound 1a with (S)-
tetrahydrofuran-3-ol led to compound 5a with a remarkable
increase in microsomal stability (HLM, RLM, mouse LM, dog
LM, and monkey LM). These compounds were also evaluated
for their ability to inhibit the major drug metabolizing enzymes.
The inhibition of cytochrome P450 enzymes (CYPs) was
evaluated by determining the activity of the compounds against
CYPs 3A4, 2D6, 2C9, 1A2, and 2C19 in human liver
microsomes. The results reveal essentially no inhibition, with
IC50 values >10 μM (Supporting Information, Table S2 and
Table 3).
Selected trans-isomers were further evaluated for their great
potency. The pharmacokinetic profiles in BALB/c mice are
summarized in Supporting Information, Table S3, and Table 4.
Encouragingly, our studies demonstrated that the selected
trans-isomers possessed greater oral bioavailability, higher
maximal plasma concentration, and lower clearance rates than
that of afatinib at an oral dose of 10 mg/kg. Also, the cis-isomer
2b exhibited a reduced oral bioavailability (F = 16.5%)
compared with its trans-isomer pair 2a.
The concentration of selected compounds in various tissues
was detected at multiple time points over 6 h following a 30
mg/kg oral dose in BALB/c mice (Table 4 and Supporting
Information, Table S4−S7). The mean drug concentration of
all tested compounds in the lung is 10−25-fold higher than in
plasma. Additionally, the concentration in the brain is higher
than that of afatinib, especially with compounds 2a and 5a. This
Figure 3. Antiproliferative activities and the kinase inhibitory activities
of selected compounds. IC50 values for A431 (4-a), NSCLC H1975
cell (4-b), EGFRWT (4-c), EGFRT790M (4-d), and HER2 (4-e), treated
with indicated drugs. Values were calculated from at least eight data
points. In general three independent determinations were performed.
The specificity of one of the most potent compounds, 1a,
was profiled against a commercial panel of 16 kinases (Table
2). These kinases contain a cysteine residue that is sufficiently
sensitive for electrophilic modification by many drugs, such as
Akt1 (Cy296/Cys310), PDGFRα (Cys814), FGFR2 (Cys486),
and Src (Cys345).24 As shown in Table 2, compound 1a
demonstrates a favorable selectivity profile. EGFRWT and
EGFRT790M are the only two kinases that experience marked
inhibition (IC50 of 0.20 and 6.67 nM, respectively). These
Table 2. Selectivity Profiles for Compound 1a versus a Panel
a
of 16 Kinase Targets
kinase
EGFR
IC50 (nM)
kinase
IC50 (nM)
0.20
6.67
AlK
>10000
>10000
>10000
>10000
>10000
6810
EGFRT790M
AKT1
AKT2
c-Met
FGFR2
FLT3
ABL
HER2
75.0
HER4
23.0
PDGFRα
PDGFRβ
JAK2
3596
2102
>10000
>10000
2262
SYK
SRC
1731
a
The kinase inhibitory activities of the compounds were evaluated
using the homogeneous time-resolved fluorescence (HTRF) method
(Cisbio) assay. The data shown are the means from three independent
experiments.
E
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Table 4. Pharmacokinetic Properties of Selected Compounds in BALB/c Mice
c
compd
AUC(0−∞) (PO, μM·min)
Cmax (PO, μM)
CLz (L/min/kg)
Vz (L/kg)
F (%)
lung/plasma/brain ratio
a
d
1a
460
306
14.7
994
625
29.4
0.79
0.83
0.09
2.66
0.94
0.13
0.05
0.04
0.12
0.02
0.03
0.27
14.0
7.14
12.2
3.94
9.69
36.2
98.5
105
nd
b
2a
20/1.0/2.31
b
d
2b
16.5
89.4
84.7
37.4
nd
a
3a
5a
11/1.0/0.29
25/1.0/0.79
18/1.0/0.14
a
a
afatinib
a
b
Compound 1a, 3a, 5a, and afatinib were administered PO to fed male BALB/c mice at a dose of 10 mg/kg. Compound 2a and 2b were
c
administered PO to fed male BALB/c mice at a dose of 5 mg/kg. Compounds 2a, 3a, 5a, and afatinib were administered PO at a dose of 30 mg/kg.
d
Not detected.
indicates that the quinazoline analogues bearing fluoro-
substituted olefins experience enhanced brain uptake thereby
rendering them viable for the treatment of NSCLC patients
with brain metastasis. Also, the hERG potassium channel patch
clamp assay25 shows that the selected compounds have an IC50
> 10 μM. These results indicate that these analogues have
minimal potential for cardiac toxicity (Table 5).
independent cell line SW620 when compared to afatinib and
doxorubicin.
Given the potent inhibition of EGFRT790M kinase and H1975
cell growth, together with the promising pharmacokinetic
properties, the in vivo antitumor efficacy of compounds 2a, 3a,
and 5a were evaluated using an EGFRL858R/T790M-driven human
H1975 xenograft mouse model. The tumor growth values (T/C
%) of compounds 2a, 3a, 5a, and afatinib were found to be
67.4%, 15.6%, 19.5%, and 30.3% at a dose of 30 mg/kg/day,
respectively (Figure 5 and Supporting Information, Figure S1).
Table 5. Inhibition of hERG Currents by Compounds 1a, 2a,
3a, 4a, 5a, and 5b
no. of cells
tested
% inhibition at
compd
1a
% inhibition at 1 μM
10 μM
4
4
4
4
3
4
2
2.55 3.92
0.71 1.22
18.4 3.71
11.8 5.98
3.06 3.49
11.1 2.34
14.8 4.15
39.5 4.76
2a
3a
0
0
4a
0.94 1.64
5a
0
0
5b
6.37 7.28
cisapride
92.4 3.47 at 1 μM (65.5 0.1
at 0.1 μM)
The cytotoxicity of compound 5a, afatinib, and doxorubicin
against the colon cancer cell line SW620 was also evaluated.
The colon cancer cell line SW620 expresses neither EGFR nor
HER-2 to a significant extent and therefore is a good
counterscreen cell line for EGFR-targeting inhibitors. As
shown in Figure 4, compound 5a exhibited only an 11.5%
growth inhibition of the cancer cell line SW620 at a
concentration of 5 μM. In contrast, at the concentration of 5
μM, both afatinib and doxorubicin exhibited inhibition of
78.0% and 79.8%, respectively. These results indicate that
compound 5a possesses less cytotoxicity against the EGFR-
Figure 5. Effect on tumor volume in xenograft model of H1975 of
compounds 2a, 3a, 5a, and afatinib. (T/C (%) = mean RTV of the
treated group/mean RTV of the control group × 100%. The individual
relative tumor volume (RTV) was calculated as follows: RTV = Vt/V0,
where Vt is the volume on each day of measurement, and V0 is the
volume on the day of initial treatment.
In addition, compound 5a displays dose-dependent inhibition
of tumor growth. The T/C values are 42.5%, 19.5%, and 9.4%
at dosages of 15, 30, and 60 mg/kg/day, respectively (Figure
5). Importantly, the compound 5a is well-tolerated, with no
mortality or significant loss of body weight observed during
treatment at a dose of 30 mg/kg.
CONCLUSION
■
In this study, we report a new chemically tuned strategy to alter
the overall biological profile of covalent EGFR inhibitors. The
lead compound 5a exhibited potent inhibition of EGFR
Figure 4. Cytotoxicity comparison of compound 5a and afatinib in
EGFR-independent cancer cell line SW620.
F
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The reaction mixture was evaporated under reduced pressure at room
temperature to give the crude product, and the crude was dissolved in
acetone to precipitate the salt, filtered, and the filtrate evaporated
under reduced pressure to give the corresponding acid as white waxy
solid; yield 95% (3.78 g). 1H NMR (500 MHz, CDCl3) δ 5.27 (dd, J =
47.0, 13.0 Hz, 1H), 4.36−4.26 (m, 4H), 1.42−1.36 (m, 6H). HRMS
(ESI): m/z calcd for (C6H12FO5P + H)+, 215.0484; found, 215.0494.
(S)-N4−(3-Chloro-4-fluorophenyl)-7-((tetrahydrofuran-3-yl) oxy)-
quinazoline-4,6-diamine (III-a). To a solution of (S)-4-(3-chloro-4-
fluorophenyl)-7-((tetrahydrofuran-3-yl) oxy)-6-nitro-quinazoline
(2.02 g, 5.0 mmol) and NiCl2·6H2O (2.38 g, 10 mmol) in 40 mL
of MeOH/DCM (V/V = 1:4) was added NaBH4 (0.76 g, 20 mmol) at
0 °C. The mixture was stirred at 0 °C for 30 min then at room
temperature for 30 min. The mixture was filtered, and the filtrate was
evaporated to dryness under reduce pressure. The crude product was
purified by flash column chromatography on silica gel eluting with
mutation T790 M as well as gefitinib-resistant cell line H1975.
Additionally, compound 5a displayed weak inhibition against
SW620, an EGFR-independent cell line. Further studies
showed that compound 5a possessed good pharmacokinetic
profiles, favorable tissue distribution as well as an acceptable
safety index. An in vivo antitumor efficacy study demonstrated
that 5a significantly inhibited tumor growth in an
EGFRL858R/T790M-driven human NSCLC xenograft nude
mouse model of H1975 at a dose of 30 mg/kg/day. The
biological results demonstrate that compound 5a, a quinazoline
derivative bearing fluoro-substituted olefin tuned to alter
Michael addition reactivity, is more potent and less toxic than
afatinib. These findings provide the basis of a promising
strategy toward the development of novel inhibitors with
enhanced biological profiles.
1
CH2Cl2/MeOH = 10:1; yellow solid; yield 90% (1.68 g). H NMR
(500 MHz, DMSO) δ 10.26 (s, 1H), 8.56 (s, 1H), 8.11 (s, 1H), 7.76
(s, 1H), 7.57 (s, 1H), 7.46 (t, J = 9.0 Hz, 1H), 7.24 (s, 1H), 5.70 (s,
2H), 5.22 (s, 1H), 4.02−3.99 (m, 2H), 3.95−3.91 (m, 1H), 3.81−3.80
(m, 1H), 2.36−2.32 (m, 1H), 2.15−2.13 (m, 1H). HRMS (ESI): m/z
calcd for (C18H16ClFN4O2 + H)+, 375.1024; found, 375.1012.
Diethyl (2-((4-((3-Chloro-4-fluorophenyl)amino)-7-(((S)-tetrahy-
drofuran-3-yl)oxy)quinazolin-6-yl)amino)-1-fluoro-2-oxoethyl)-
phosphate (IV-a). To a solution of the above compound (III-a) (0.37
g, 1.0 mmol) and Et3N (0.15 g, 1.5 mmol) in DMF (10 mL) was
added dropwise a solution of compound (II) (0.32 g, 1.5 mmol) in
DMF (5 mL) at ice bath. The reaction mixture was stirred at ice bath
for 30 min and then warmed to room temperature for overnight. The
completion of the reaction was detected via LC-MS. After completion,
the reaction was quenched with saturated aqueous NaHCO3 and then
extracted with EtOAc (30 mL × 3) three times. The organic layer was
dried over anhydrous Na2SO4 and evaporated under reduced pressure.
The crude product was purified by silica gel with DCM/MeOH =
40:1; yellow solid; yield 60% (0.34 g). 1H NMR (500 MHz, DMSO) δ
9.94 (s, 1H), 9.64 (s, 1H), 8.89 (s, 1H), 8.55 (s, 1H), 8.09 (dd, J = 6.5,
2.5 Hz, 1H), 7.78−7.75 (m, 1H), 7.43 (t, J = 9.0 Hz, 1H), 7.34 (s,
1H), 6.02 (dd, J = 45.0, 11.0 Hz, 1H), 5.22 (s, 1H), 4.20 (dd, J = 15.0,
7.0 Hz, 4H), 4.02−3.99 (m, 2H), 3.95−3.91 (m, 1H), 3.81−3.80 (m,
1H), 2.36−2.32 (m, 1H), 2.15−2.13 (m, 1H), 1.29 (dt, J = 10.0, 7.0
Hz, 6H). HRMS (ESI): m/z calcd for (C24H26ClF2N4O6P + H)+,
571.1325; found, 571.1335.
EXPERIMENTAL SECTION
■
General Procedure. Unless otherwise noted, all solvents and
chemicals were used as purchased without further purification. 1H
NMR spectra were recorded on a Bruker DRX-500 [Bruker Biospin,
Germany], Bruker AVANCE III-400, and Varian Mercury-300.
Chemical shifts are reported in ppm relative to the residual solvent
peak (CDCl3, TMS: 0.00). Multiplicity was indicated as follows: s
(singlet); d (doublet); t (triplet); q (quartet); m (multiplet); dd
(doublet of doublet); dt (triplet of doublet); td (doublet of triplet);
brs (broad singlet), etc. Intermediates were purified by column
chromatography on silica gel (200−300 mesh), and the separation of
the Z−E isomers were performed on GILSON 215 semiprep HPLC
using PhenomenexGimini 10 μm C18, 30 mm × 250 mm column at a
flow rate of 30 mL/min or Thar SFC Pre80 (supercritical fluid
chromatography, SFC) using an Agela AD-H 5 μm, 20 mm × 250 mm
column at a flow rate of 50 g/min using gradients MeOH (containing
0.1% DEA): CO2 = 40:60. All reported yields are isolated yields after
column chromatography. Purity of all biologically evaluated com-
pounds was determined by HPLC analysis to be >95%. HPLC analysis
was performed on Agilent 1200 using Agilent Eclipse XDB-C18 5 μm
4.6 mm × 150 mm at a flow rate of 1 mL/min using the listed
gradients: (mobile phase A, MeOH; mobile phase B, MeCN; mobile
phase C, 0.01 mol/L KH2PO4, pH = 3.0) (Table 6).
Diethyl (2-((4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyqui-
nazolin-6-yl)amino)-1-fluoro-2-oxoethyl)phosphonate (VI-b). The
preparation was according to the compound (VI-a). The crude
product was purified by silica gel with DCM/MeOH = 40:1; yellow
Table 6
t/min mobile phase A (%) mobile phase B (%) mobile phase C (%)
0
5
5
0
0
95
95
70
70
50
35
35
95
95
1
solid; yield 70% (0.36 g). H NMR (500 MHz, DMSO) δ 9.94 (s,
1.6
8.5
1H), 9.64 (s, 1H), 8.89 (s, 1H), 8.55 (s, 1H), 8.09 (dd, J = 6.5, 2.5 Hz,
1H), 7.77 (ddd, J = 9.0, 4.0, 2.5 Hz, 1H), 7.43 (t, J = 9.0 Hz, 1H), 7.34
(s, 1H), 6.02 (dd, J = 45.0, 11.0 Hz, 1H), 4.20 (dd, J = 15.5, 8.0 Hz,
4H), 4.03 (s, 3H), 1.29 (dt, J = 10.0, 7.5 Hz, 6H).
10
10
15
15
15
5
20
20
35
50
50
0
13
18
30
Diethyl (2-((4-((3-Chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-
7-ethoxyquinazolin-6-yl)amino)-1-fluoro-2-oxoethyl)phosphonate
(VI-c). The preparation was according to the compound (VI-a). The
crude product was purified by silica gel with DCM/MeOH = 40:1;
yellow solid; yield 50% (0.31 g). 1H NMR (500 MHz, DMSO) δ 9.81
(s, 1H), 9.49 (s, 1H), 8.87 (s, 1H), 8.60 (d, J = 4.0 Hz, 1H), 8.49 (s,
1H), 7.94 (d, J = 2.0 Hz, 1H), 7.89 (td, J = 8.0, 2.0 Hz, 1H), 7.65 (dd,
J = 9.0, 2.5 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.40−7.35 (m, 1H), 7.29
(s, 1H), 7.25 (d, J = 9.0 Hz, 1H), 6.03 (dd, J = 45.0, 11.5 Hz, 1H), 5.29
(s, 2H), 4.30 (q, J = 7.0 Hz, 2H), 4.26−4.14 (m, 4H), 1.44 (t, J = 7.0
Hz, 3H), 1.29 (dt, J = 13.5, 7.0 Hz, 6H).
Diethyl (2-((4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-
7-methoxyquinazolin-6-yl)amino)-1-fluoro-2-oxoethyl)-
phosphonate (VI-d). The preparation was according to the compound
(VI-a). The crude product was purified by silica gel with DCM/
MeOH = 40:1; yellow solid; yield 50% (0.31 g). 1H NMR (500 MHz,
DMSO) δ 9.84 (s, 1H), 9.66 (s, 1H), 8.86 (s, 1H), 8.50 (s, 1H), 7.94
(s, 1H), 7.67−7.65(m, 1H), 7.48−7.46 (m, 1H), 7.34−7.31 (m, 3H),
7.25 (d, J = 9.0 Hz, 2H), 7.21−7.18 (m, 1H), 6.03 (dd, J = 45.0, 11.0
35
36.5
40
5
0
The HPLC-HRMS of all compounds was confirmed on a Agilent
1290 HPLC-6224 time-of-flight mass spectrometer using Phenomen-
exLuna 5 μ C18, 100 Å, 150 mm × 4.60 mm 5 μm column at a flow
rate of 0.5 mL/min using liner gradients buffer B in A (B, CH3OH
containing 0.1% formic acid; A, H2O containing 0.1% formic acid).
Mobile phase B was increased linearly from 5% to 95% over 7 min and
95% over the next 2 min, after which the column was equilibrated to
5% for 1 min.
2-(Diethoxyphosphoryl)-2-fluoroacetic Acid (II). To a solution of
2-(diethoxyphosphinyl)-2-fluoroacetic acid ethyl ester (4.5 g, 18.6
mmol) in 180 mL of ethanol was added a solution of sodium
hydroxide (3.72 g, 93 mmol) in water (180 mL) The reaction mixture
was stirred for 2 h at −5 °C and acidified to pH = 2−3 with 4 N HCl.
G
dx.doi.org/10.1021/jm5014659 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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Hz, 1H), 5.26 (s, 2H), 4.20 (dd, J = 14.0, 7.0 Hz, 4H), 4.02 (s, 3H),
1.29 (dd, J = 14.0, 7.0 Hz, 6H).
HRMS (ESI): m/z calcd for (C24H24ClF2N5O2 + H)+, 488.1665;
found, 488.1656.
Diethyl (2-((4-((3-Bromophenyl)amino)-7-methoxyquinazolin-6-
yl)amino)-1-fluoro-2-oxoethyl)phosphonate (VI-e). The preparation
was according to the compound (VI-a). The crude product was
purified by silica gel with DCM/MeOH = 40:1; yellow solid; yield
(E)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazo-
lin-6-yl)-2-fluoro-4-(piperidin-1-yl)but-2-enamide (2b). The reten-
tion time of the E-isomer is 4.65 min; light-yellow solid; yield 28.2%.
1H NMR (400 MHz, CDCl3) δ 9.23 (s, 1H), 8.96 (s, 1H), 8.67 (s,
1
75% (0.41 g). H NMR (500 MHz, DMSO) δ 9.94 (s, 1H), 9.67 (s,
1H), 7.93 (dd, J = 6.8, 2.8 Hz, 1H), 7.73 (s, 1H), 7.57 (ddd, J = 9.0,
4.0, 2.8 Hz, 1H), 7.28 (s, 1H), 7.16 (t, J = 8.8 Hz, 1H), 6.20 (dt, J =
22.8, 5.6 Hz, 1H), 4.07 (s, 3H), 3.79−3.74 (m, 2H), 2.64−2.61 (m,
4H), 1.73−1.68 (m, 4H), 1.54−1.47 (m, 2H). HRMS (ESI): m/z
calcd for (C24H24ClF2N5O2 + H)+, 488.1665; found, 488.1656.
N-(4-((3-Chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-7-ethoxy-
quinazolin-6-yl)-4-(dimethylamino)-2-fluorobut-2-enamide (3a
+3b). The preparation was according to the compound (1a+1b).
TLC Rf = 0.53, 0.56 (DCM/MeOH = 10:1, the two isomers could be
separated). The separation of the Z−E isomers was performed
according to compound (2a+2b).
(Z)-N-(4-((3-Chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-7-
ethoxyquinazolin-6-yl)-4-(dimethylamino)-2-fluorobut-2-enamide
(3a). The retention time of Z-isomer is 5.12 min; light-yellow solid;
yield 34.2%. 1H NMR (400 MHz, CDCl3) δ 9.00 (s, 1H), 8.98 (d, J =
5.6 Hz, 1H), 8.63 (s, 1H), 8.59 (d, J = 4.4 Hz, 1H), 7.88 (d, J = 2.8 Hz,
1H), 7.79−7.78 (m, 1H), 7.75 (d, J = 7.6, 1.6 Hz, 1H), 7.67 (d, J = 8.0
Hz, 1H), 7.52 (dd, J = 8.8, 2.8 Hz, 1H), 7.25 (s, 1H), 7.01 (d, J = 8.8
Hz, 1H), 6.39 (dt, J = 36.4, 7.2 Hz, 1H), 5.30 (s, 2H), 4.30 (q, J = 6.8
Hz, 2H), 3.26 (dd, J = 7.2, 2.4 Hz, 2H), 2.32 (s, 6H), 1.57 (t, J = 7.2
Hz, 4H). HRMS (ESI): m/z calcd for (C28H28ClFN6O3 + H)+,
551.1973; found, 551.1967.
(E)-N-(4-((3-Chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-7-
ethoxyquinazolin-6-yl)-4-(dimethylamino)-2-fluorobut-2-enamide
(3b). The retention time of the E-isomer is 6.06 min; light-yellow
solid; yield 25.8%. 1H NMR (400 MHz, CDCl3) δ 9.19 (d, J = 4.8 Hz,
1H), 8.98 (s, 1H), 8.65 (s, 1H), 8.60 (dd, J = 5.6, 0.8 Hz, 1H), 7.86 (d,
J = 2.8 Hz, 1H), 7.78−7.74 (m, 1H), 7.7−7.66 (m, 1H), 7.55−7.52
(m, 1H), 7.48−7.47 (m, 1H), 7.03 (d, J = 9.2 Hz, 1H), 6.25−6.19 (m,
1H), 5.35 (m, 1H), 5.30 (s, 2H), 4.31 (q, J = 7.2 Hz, 2H), 3.96−3.92
(m, 2H), 2.59−2.55 (s, 6H), 1.57 (t, J = 6.8 Hz, 3H). HRMS (ESI):
m/z calcd for (C28H28ClFN6O3 + H)+, 551.1973; found, 551.1969.
(S)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-
3-yl)oxy)quinazolin-6-yl)-2-fluoro-4-morpholinobut-2-enamide (4a
+4b). The preparation was according to the compound (1a+1b). TLC
Rf = 0.52, 0.56 (DCM/MeOH = 10:1, the two isomers could be
separated) The separation of the Z−E isomers was performed
according to compound (2a+2b).
1H), 8.90 (s, 1H), 8.57 (s, 1H), 8.13 (s, 1H), 7.83 (d, J = 8.5 Hz, 1H),
7.37−7.32 (m, 2H), 7.29 (d, J = 8.0 Hz, 1H), 6.03 (dd, J = 45.0, 11.0
Hz, 1H), 4.21 (dd, J = 15.0, 7.5 Hz, 4H), 4.03 (s, 3H), 1.29 (dt, J =
9.5, 7.0 Hz, 6H).
1-(2,2-Dimethoxyethyl)piperidine (VII-a). A mixture of 2-bromoa-
cetaldehyde diethyl acetal (4.18 g, 25 mmol), piperidine (2.13 g, 25
mmol), and K2CO3 (6.9 g, 50 mmol) was stirred at 120 °C for 16 h.
The reaction was cooled to room temperature, diluted with water (20
mL), and extracted with DCM (3 × 30 mL) three times. The
combined organic layer was washed with saturated aqueous NaHCO3
and brine, dried over anhydrous Na2SO4, filtered, and concentrated to
get colorless oil directly for further reaction. HRMS (ESI): m/z calcd
for (C9H19NO2 + H)+, 174.1494; found, 174.1498.
2-(Piperidin-1-yl)acetaldehyde Hydrochloride (VIII-a). A solution
of intermediate (VII-a) (1.73 g, 10 mmol) dissolved in concentrated
aqueous HCl (20 mL) was refluxed at 110 °C for 4.5 h. The reaction
mixture was concentrated in reduced pressure to obtain dark oil, which
was used without purification. HRMS (ESI): m/z calcd for (C7H13NO
+ H)+, 128.1920; found, 128.1930.
N-(4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
yl)-4-(dimethylamino)-2-fluorobut-2-enamide (1a+1b). NaOH
(0.32 g, 8.0 mmol) was dissolved in EtOH−H2O (10 mL:1 mL).
The phosphonate (0.24 g, 1.0 mmol) was added to the above solution.
After the mixture became clear, the corresponding aldehyde (2.0
mmol) was added at 0−5 °C in an ice bath. The reaction mixture was
warmed to room temperature and stirred at room temperature for the
indicated time. The reaction mixture was adjusted to pH = 1−2 with 2
N HCl and washed with EtOAc (3 × 30 mL) three times. The
aqueous layers were adjusted to pH = 10.0 with 4N NaOH and
extracted with EtOAc (3 × 30 mL) three times. The combined organic
layer was washed with brine, dried over anhydrous Na2SO4, and
concentrated in reduced pressure to obtain the crude product. TLC Rf
= 0.30 (EtOAc/MeOH = 5:1). The two isomers were not separated
on the TLC plate. The separation of the Z−E isomers were performed
on GILSON 215 semiprep HPLC using PhenomenexGimini 10 μ
C18, 30 mm × 250 mm column at a flow rate of 30 mL/min using
liner gradients mobile phase B in A (B, CH3CN containing 0.05%
ammonium hydroxide; A, H2O). Mobile phase B was increased
linearly from 51% to 100% over 15.5 min and 100% over the next 2.5
min, after which the column was equilibrated to 51% for 2 min.
(Z)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazo-
lin-6-yl)-4-(dimethylamino)-2-fluorobut-2-enamide (1a). The reten-
(Z)-(S)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-((tetrahydrofur-
an-3-yl)oxy)quinazolin-6-yl)-2-fluoro-4-morpholinobut-2-enamide
(4a). The retention time of the Z-isomer is 5.52 min; light-yellow
1
solid; yield 24.0%. H NMR (400 MHz, CDCl3) δ 9.03 (s, 1H), 8.91
(d, J = 4.4 Hz, 1H), 8.67 (s, 1H), 7.97 (dd, J = 6.4, 2.4 Hz, 1H), 7.75
(s, 1H), 7.59−7.55 (m, 1H), 7.22 (s, 1H), 7.17 (t, J = 8.8 Hz, 1H),
6.40 (dt, J = 36.4, 6.4 Hz, 1H), 5.21 (s, 2H), 4.14−4.05 (m, 4H),
4.00−3.94 (m, 1H), 3.76−3.74 (m, 4H), 3.31 (dd, J = 7.2, 2.4 Hz,
2H), 2.55−2.53 (m, 4H). HRMS (ESI): m/z calcd for
(C26H26ClF2N5O4 + H)+, 546.1719; found, 546.1706.
1
tion time of Z-isomer is 14.5 min; light-yellow solid; yield 15%. H
NMR (300 MHz, DMSO) δ 11.41(s, 1 H), 10.30 (s, 1 H), 8.99 (s, 1
H), 8.88 (s, 1 H), 8.04 (dd, J = 2.7 Hz, 1 H), 7.77−7.70 (m, 1 H), 7.58
(s, 1 H), 7.56 (dd, J = 2.7 Hz, 1 H), 6.47 (td, J = 33.6, 7.5 Hz, 1 H),
4.03 (s, 3 H), 3.99 (br, 2 H), 2.79 (s, 6 H).
N-(4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-
yl)-2-fluoro-4-(piperidin-1-yl)but-2-enamide (2a+2b). The prepara-
tion was according to the compound (1a+1b). TLC Rf = 0.53, 0.56
(DCM/MeOH = 10:1, the two isomers can be separated). The
separation of the Z−E isomers were performed on Thar SFC Pre80
(supercritical fluid chromatography, SFC) using Agela AD-H 5 μ, 20
mm × 250 mm column at a flow rate of 50 g/min using gradients
MeOH (containing 0.1% DEA): CO2 = 40:60.
(Z)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazo-
lin-6-yl)-2-fluoro-4-(piperidin-1-yl)but-2-enamide (2a). The reten-
tion time of the Z-isomer is 6.85 min; light-yellow solid; yield 35.4%.
1H NMR (400 MHz, CDCl3) δ 9.01 (s, 1H), 8.88 (d, J = 4.4 Hz, 1H),
(E)-(S)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-((tetrahydrofur-
an-3-yl)oxy)quinazolin-6-yl)-2-fluoro-4-morpholinobut-2-enamide
(4b). The retention time of the E-isomer is 3.11 min; light-yellow
1
solid; yield 17.4%. H NMR (400 MHz, CDCl3) δ 9.02 (s, 1H), 8.98
(d, J = 6.0 Hz, 1H), 8.67 (s, 1H), 7.96 (dd, J = 6.4, 2.8 Hz, 1H), 7.59−
7.55 (m, 2H), 7.22 (s, 1H), 7.19 (d, J = 8.8 Hz, 1H), 6.08 (dt, J = 23.6,
6.4 Hz, 1H), 5.20 (s, 2H), 4.10−4.04 (m, 4H), 4.00−3.96 (m, 1H),
3.76−3.74 (m, 4H), 3.71 (dd, J = 6.8, 2.4 Hz, 2H), 2.58−2.55 (m,
4H). HRMS (ESI): m/z calcd for (C26H26ClF2N5O4 + H)+, 546.1719;
found, 546.1706.
(S)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-
3-yl)oxy)quinazolin-6-yl)-4-(dimethylamino)-2-fluorobut-2-enam-
ide (5a+5b). The preparation was according to the compound (1a
+1b). TLC Rf = 0.53, 0.58 (DCM/MeOH = 10:1, the two isomers
could be separated). The separation of the Z−E isomers was
performed according to compound (2a+2b).
8.66 (s, 1H), 7.95 (dd, J = 6.8, 2.8 Hz, 1H), 7.81 (dd, J = 3.2, 0.5 Hz,
1H), 7.55 (ddd, J = 9.0, 4.0, 2.8 Hz, 1H), 7.27 (s, 1H), 7.14 (t, J = 8.8
Hz, 1H), 6.45 (dt, J = 36.0, 7.2 Hz, 1H), 4.07 (s, 3H), 3.34 (d, J = 7.2
Hz, 2H), 2.54−2.50 (m, 4H), 1.68−1.64 (m, 4H), 1.50−1.46 (m, 2H).
H
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Journal of Medicinal Chemistry
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(Z)-(S)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-((tetrahydrofur-
an-3-yl)oxy)quinazolin-6-yl)-4-(dimethylamino)-2-fluorobut-2-en-
amide (5a). The retention time of the Z-isomer is 5.20 min; light-
yellow solid; yield 45.5%. 1H NMR (400 MHz, CDCl3) δ 9.05 (s, 1H),
8.91 (d, J = 6.8 Hz, 1H), 8.67 (s, 1H), 7.98 (dd, J = 6.8, 2.8 Hz, 1H),
7.73 (s, 1H), 7.59−7.55 (m, 1H), 7.22 (s, 1H), 7.18 (t, J = 8.8 Hz,
1H), 6.40 (dt, J = 36.4, 7.2 Hz, 1H), 5.22−5.19 (m, 1H), 4.12−4.07
(m, 4H), 4.00−3.94 (m, 1H), 3.26 (dd, J = 7.6, 2.8 Hz, 2H), 2.33 (s,
6H). HRMS (ESI): m/z calcd for (C24H24ClF2N5O3 + H)+, 504.1614;
found, 504.1605.
HRMS (ESI): m/z calcd for (C27H28ClF2N5O3 + H)+, 544.1927;
found, 544.1914.
N-(4-((3-Chloro-4-((6-methylpyridin-3-yl)oxy)phenyl)amino)-7-
ethoxyquinazolin-6-yl)-2-fluoro-4-(piperidin-1-yl)but-2-enamide
(8a+8b). The preparation was according to the compound (1a+1b).
TLC Rf = 0.52, 0.54 (DCM/MeOH = 10:1, the two isomers could be
separated). The separation of the Z−E isomers was performed
according to compound (2a+2b).
(Z)-N-(4-((3-Chloro-4-((6-methylpyridin-3-yl)oxy)phenyl)amino)-
7-ethoxyquinazolin-6-yl)-2-fluoro-4-(piperidin-1-yl)but-2-enamide
(8a). The retention time of Z-isomer is 15.85 min; light-yellow solid;
(E)-(S)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-((tetrahydrofur-
an-3-yl)oxy)quinazolin-6-yl)-4-(dimethylamino)-2-fluorobut-2-en-
amide (5b). The retention time of the E-isomer is 4.53 min; light-
1
yield 33.2%. H NMR (400 MHz, DMSO) δ 9.84 (s, 1H), 9.64 (s,
1H), 8.77 (s, 1H), 8.58 (s, 1H), 8.22 (s, 2H), 7.84 (dd, J = 8.8, 2.4 Hz,
1H), 7.31 (s, 1H), 7.27 (m, 2H), 7.21 (d, J = 8.8 Hz, 1H), 6.21 (dt, J =
36.8, 7.2 Hz, 1H), 4.28 (q, J = 6.8 Hz, 2H), 3.20 (d, J = 7.2 Hz, 2H),
2.45 (s, 3H), 2.39 (m, 4H), 1.52−1.41 (m, 9H). HRMS (ESI): m/z
calcd for (C31H32ClFN6O3 + H)+, 591.2286; found, 591.2266.
(E)-N-(4-((3-Chloro-4-((6-methylpyridin-3-yl)oxy)phenyl)amino)-
7-ethoxyquinazolin-6-yl)-2-fluoro-4-(piperidin-1-yl)but-2-enamide
(8b). The retention time of the E-isomer is 8.48 min; light-yellow
solid; yield: 17.0%. 1H NMR (400 MHz, DMSO) δ 9.84 (s, 1H), 8.77
(s, 1H), 8.58 (s, 1H), 8.21 (d, J = 2.8 Hz, 2H), 7.83 (d, J = 8.8 Hz,
1H), 7.31 (s, 1H), 7.27 (m, 2H), 7.20 (d, J = 8.4 Hz, 1H), 6.09 (dt, J =
23.6, 7.0 Hz, 1H), 4.32−4.26 (m, 2H), 3.45−3.44 (m, 2H), 2.45 (s,
3H), 2.40 (m, 4H), 1.49−1.41 (m, 9H). HRMS (ESI): m/z calcd for
(C31H32ClFN6O3 + H)+, 591.2286; found, 591.2280.
1
yellow solid; yield 24.5%. H NMR (400 MHz, CDCl3) δ 9.26 (brs,
1H), 9.00 (s, 1H), 8.67 (s, 1H), 7.95 (dd, J = 6.4, 2.8 Hz, 1H), 7.62 (s,
1H), 7.59−7.54 (m, 1H), 7.22 (s, 1H), 7.18 (t, J = 8.8 Hz, 1H), 6.09
(dt, J = 24.8, 6.8 Hz, 1H), 5.20−5.16 (m, 2H), 4.10−4.04 (m, 4H),
3.98−3.95 (m, 1H), 3.60 (dd, J = 7.2, 2.8 Hz, 2H), 2.34 (s, 6H).
HRMS (ESI): m/z calcd for (C24H24ClF2N5O3 + H)+, 504.1614;
found, 504.1603.
N-(4-((3-Chloro-4-fluorophenyl) amino)-7-methoxyquinazolin-6-
yl)-2-fluoro-4-(piperidin-1-yl)but-2-enamide (6a+6b). The prepara-
tion was according to the compound (1a+1b). TLC Rf = 0.52, 0.60
(DCM/MeOH = 10:1, the two isomers could be separated). The
separation of the Z−E isomers was performed according to compound
(2a+2b).
(Z)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-methoxyquinazo-
lin-6-yl)-2-fluoro-4-(piperidin-1-yl)but-2-enamide (6a). The reten-
tion time of the Z-isomer is 3.82 min; light-yellow solid; yield 27.0%.
1H NMR (400 MHz, CDCl3) δ 9.01 (s, 1H), 8.91 (d, J = 4.8 Hz, 1H),
8.67 (s, 1H), 7.95 (dd, J = 6.4, 2.4 Hz, 1H), 7.75 (s, 1H), 7.57−7.54
(m, 1H), 7.29 (s, 1H), 7.15 (t, J = 8.8 Hz, 1H), 6.38 (dt, J = 36.0, 7.2
Hz, 1H), 4.08 (s, 3H), 3.75 (t, J = 4.4 Hz, 4H), 3.31 (dd, J = 7.2, 2.4
Hz, 2H), 2.54 (t, J = 4.4 Hz, 4H). HRMS (ESI): m/z calcd for
(C23H22ClF2N5O3 + H)+, 490.1457; found, 490.1447.
(E)-N-(4-((3-Chloro-4-fluorophenyl) amino)-7-methoxyquinazo-
lin-6-yl)-2-fluoro-4-(piperidin-1-yl)but-2-enamide (6b). The reten-
tion time of the E-isomer is 2.67 min; light-yellow solid; yield 18.6%.
1H NMR (400 MHz, CDCl3) δ 9.05 (d, J = 5.2 Hz, 1H), 9.00 (s, 1H),
8.68 (s, 1H), 7.95 (dd, J = 6.4, 2.8 Hz, 1H), 7.58−7.54 (m, 2H), 7.30
(s, 1H), 7.17 (t, J = 8.4 Hz, 1H), 6.07 (dt, J = 23.6, 6.8 Hz, 1H), 4.08
(s, 3H), 3.75 (t, J = 4.8 Hz, 4H), 3.70 (dd, J = 6.8, 2.4 Hz, 2H), 2.57 (t,
J = 4.8 Hz, 4H). HRMS (ESI): m/z calcd for (C23H22ClF2N5O3 + H)+,
490.1457; found, 490.1451.
N-(4-((3-Chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-7-ethoxy-
quinazolin-6-yl)-2-fluoro-4-(piperidin-1-yl)but-2-enamide (9a+9b).
The preparation was according to the compound (1a+1b). TLC Rf =
0.52, 0.53 (DCM/MeOH = 10:1, the two isomers could be separated).
The separation of the Z−E isomers was performed according to
compound (2a+2b).
N-(4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-7-methox-
yquinazolin-6-yl)-4-(dimethylamino)-2-fluorobut-2-enamide (9a
+9b). The preparation was according to the compound (1a+1b).
TLC Rf = 0.53, 0.56 (DCM/MeOH = 10:1, the two isomers could be
separated) The separation of the Z−E isomers was performed
according to compound (3a+3b).
(Z)-N-(4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-7-me-
thoxyquinazolin-6-yl)-4-(dimethylamino)-2-fluorobut-2-enamide
(9a). The retention time of the Z-isomer is 8.61 min; light-yellow
1
solid; yield 40.3%. H NMR (400 MHz, DMSO) δ 9.75 (s, 1H), 9.69
(s, 1H), 8.68 (s, 1H), 8.53 (s, 1H), 7.99 (d, J = 2.8 Hz, 1H), 7.71 (dd, J
= 9.2, 2.8 Hz, 1H), 7.50−7.44 (m, 1H), 7.34−7.30 (m, 3H), 7.25 (d, J
= 9.2 Hz, 1H), 7.20−7.16 (m, 1H), 6.19 (dt, J = 36.0, 7.2 Hz, 1H),
5.25 (s, 2H), 3.99 (s, 3H), 3.18−3.15 (m, 2H), 2.19 (s, 6H). HRMS
(ESI): m/z calcd for (C26H26ClF2N5O3 + H)+, 554.1770; found,
554.1780.
(E)-N-(4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-7-me-
thoxyquinazolin-6-yl)-4-(dimethylamino)-2-fluorobut-2-enamide
(9b). The retention time of the E-isomer is 5.09 min; light-yellow
solid; yield 21.7%. 1H NMR (400 MHz, DMSO) δ 10.92 (s, 1H), 9.70
(s, 1H), 8.78 (s, 1H), 8.51 (s, 1H), 7.97 (d, J = 2.4 Hz, 1H), 7.69 (dd, J
= 8.8, 2.8 Hz, 1H), 7.50−7.44 (m, 1H), 7.34−7.30 (m, 3H), 7.25 (d, J
= 9.2 Hz, 1H), 7.21−7.16 (m, 1H), 6.11 (dt, J = 23.6, 6.8 Hz, 1H),
5.25 (s, 2H), 4.01 (s, 3H), 3.37 (s, 2H), 2.23 (s, 6H). HRMS (ESI):
m/z calcd for (C26H26ClF2N5O3 + H)+, 554.1770; found, 554.1784.
N-(4-((3-Chloro-4-((6-methylpyridin-3-yl)oxy)phenyl)amino)-7-
ethoxyquinazolin-6-yl)-4-(dimethylamino)-2-fluorobut-2-enamide
(10a+10b). The preparation was according to the compound (1a
+1b). TLC Rf = 0.52, 0.56 (DCM/MeOH = 10:1, the two isomers
could be separated). The separation of the Z−E isomers was
performed according to compound (2a+2b).
(S)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-((tetrahydrofuran-
3-yl)oxy)quinazolin-6-yl)-2-fluoro-4-(piperidin-1-yl)but-2-enamide
(7a+7b). The preparation was according to the compound (1a+1b).
TLC Rf = 0.52, 0.53 (DCM/MeOH = 10:1, the two isomers could be
separated). The separation of the Z−E isomers was performed
according to compound (2a+2b).
(Z)-(S)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-((tetrahydrofur-
an-3-yl)oxy)quinazolin-6-yl)-2-fluoro-4-(piperidin-1-yl)but-2-enam-
ide (7a). The retention time of the Z-isomer is 13.05 min; light-yellow
1
solid; yield 38.1%. H NMR (400 MHz, DMSO) δ 9.84 (s, 1H), 9.64
(s, 1H), 8.75 (s, 1H), 8.57 (s, 1H), 8.15 (dd, J = 6.4, 2.0 Hz, 1H),
7.82−7.79 (m, 1H), 7.44 (t, J = 9.2 Hz, 1H), 7.31 (s, 1H), 6.20 (dt, J =
36.8, 6.8 Hz, 1H), 5.33 (t, J = 5.6 Hz, 1H), 3.99 (dd, J = 10.4, 4.4 Hz,
1H), 3.88 (d, J = 8.8 Hz, 2H), 3.82−3.77 (m, 1H), 3.19 (d, J = 6.0 Hz,
2H), 2.38−2.30 (m, 4H), 2.07−1.96 (m, 2H), 1.53−1.49 (m, 4H),
1.40−1.39 (m, 2H). HRMS (ESI): m/z calcd for (C27H28ClF2N5O3 +
H)+, 544.1927; found, 544.1914.
(E)-(S)-N-(4-((3-Chloro-4-fluorophenyl)amino)-7-((tetrahydrofur-
an-3-yl)oxy)quinazolin-6-yl)-2-fluoro-4-(piperidin-1-yl)but-2-enam-
ide (7b). The retention time of the E-isomer is 11.19 min; light-yellow
solid; yield 17.5%. 1H NMR (400 MHz, DMSO) δ 10.06 (s, 1H), 9.84
(s, 1H), 8.74 (s, 1H), 8.57 (s, 1H), 8.14 (dd, J = 6.8, 2.4 Hz, 1H),
7.82−7.79 (m, 1H), 7.44 (t, J = 9.2 Hz, 1H), 7.32 (s, 1H), 6.09 (dt, J =
23.6, 13.6 Hz, 1H), 5.34 (t, J = 5.6 Hz, 1H), 4.00−3.97 (m, 1H),
3.90−3.85 (m, 2H), 3.82−3.79 (m, 1H), 3.47 (m, 2H), 2.41−2.33 (m,
4H), 2.07−1.96 (m, 2H), 1.51−1.48 (m, 4H), 1.39−1.37 (m, 2H).
(Z)-N-(4-((3-Chloro-4-((6-methylpyridin-3-yl)oxy)phenyl)amino)-
7-ethoxyquinazolin-6-yl)-4-(dimethylamino)-2-fluorobut-2-enam-
ide (10a). The retention time of the Z-isomer is 5.77 min; light-yellow
solid; yield 45%. 1H NMR (400 MHz, DMSO) δ 9.84 (s, 1H), 9.63 (s,
1H), 8.78 (s, 1H), 8.57 (s, 1H), 8.25−8.21 (s, 2H), 7.84 (dd, J = 9.2,
2.4 Hz, 1H), 7.30 (s, 1H), 7.27−7.26 (m, 2H), 7.20 (d, J = 8.8 Hz,
1H), 6.20 (dt, J = 36.4, 7.2 Hz, 1H), 4.27 (q, J = 6.8 Hz, 2H), 2.45 (s,
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3H), 2.19 (s, 6H), 1.42 (t, J = 6.8 Hz, 3H). HRMS (ESI): m/z calcd
for (C28H28ClFN5O3 + H)+, 551.1973; found, 551.1983.
to acquiring absorption spectra (214 nm). Formation of the thioether
was quantified by area normalization method based on the peak area
on HPLC spectrum. Data were fit using Excel to obtain equilibrium
dissociation constants.
2. Determination of GSH Conjugation with Compound 5a or
Afatinib. Test reactions of compound 5a or afatinib with GSH were
also carried out. Reactions were initiated as described above by mixing
equal volumes of compound 5a or afatinib (100 mM in ACN) with
solutions of GSH (1 M in PBS, pH = 7.4). After incubation, conjugate
reaction was observed at various time points (0−120 min) by an
Agilent 1290 HPLC-6224 time-of-flight mass spectrometer.
(E)-N-(4-((3-Chloro-4-((6-methylpyridin-3-yl)oxy)phenyl)amino)-
7-ethoxyquinazolin-6-yl)-4-(dimethylamino)-2-fluorobut-2-enam-
ide (10b). The retention time of the E-isomer is 3.69 min; light-yellow
1
solid; yield 24.3%. H NMR (400 MHz, DMSO) δ 9.84 (s, 1H), 8.81
(s, 1H), 8.56 (s, 1H), 8.22−8.20 (m, 2H), 7.83 (dd, J = 8.8, 2.4 Hz,
1H), 7.30 (s, 1H), 7.27−7.26 (m, 2H), 7.20 (d, J = 8.8 Hz, 1H), 6.11
(dt, J = 23.2, 7.2 Hz, 1H), 4.29 (q, J = 6.8 Hz, 2H), 2.45 (s, 3H), 2.22
(s, 6H), 1.43 (t, J = 6.8 Hz, 3H). HRMS (ESI): m/z calcd for
(C28H28ClFN5O3 + H)+, 551.1973; found, 551.1988.
N-(4-((3-Chloro-4-(cyclopropylmethoxy)phenyl)amino)-7-ethoxy-
quinazolin-6-yl)-4-(dimethylamino)-2-fluorobut-2-enamide (11a
+11b). The preparation was according to the compound (1a+1b).
TLC Rf = 0.52, 0.53 (DCM/MeOH = 10:1, the two isomers could be
separated). The separation of the Z−E isomers were performed
according to compound (2a+2b).
Cell Proliferation Assay. The human epidermal carcinoma cell line
A431 and human nonsmall cell lung cancer cell line NCI-
H1975L858R/T790M were used to evaluate the potency of synthesized
analogues in cell-based level. Both cell lines were purchased from
American Type Culture Collection (ATCC). A431 was cultured with
RPMI 1640 (GIBCO), and NCI-H1975L858R/T790M was cultured with
Dulbecco’s Modified Eagle’s Medium (GIBCO). Both mediums were
supplemented with penicillin, streptomycin, and 10% fetal bovine
serum. The assays were performed using the CellTiter-Glo (Promega)
Kit. A431 and NCI-H1975L858R/T790M cells were seeded in density of
2000 cells/well and 1500 cells/well, respectively, in 384-well plates
(Corning) for 24 h. Duplicate wells were treated with test or reference
compounds for 48 h at various concentrations or DMSO (Sigma) as
control. Plates were incubated at 37 °C in 5% CO2 atmosphere. Cell
proliferation was measured according to the manufacturer’s protocol.
The IC50 was calculated using GraphPad Prism 5.0.
Kinase Inhibition Assay. The assays were performed in vitro using
the homogeneous time-resolved fluorescence (HTRF) method
(Cisbio). EGFR was purchased from Sigma. The kinases and
substrates were incubated first with synthesized analogues for 5 min
in enzymatic buffer (for EGFR). Then ATP (Sigma) was added into
the reaction mixture to start the enzyme reaction. The ATP
concentrations used in each enzyme reaction were 1.65 μM for
EGFR, equivalent to the Km of ATP for the corresponding enzyme in
this assay condition. The assays were conducted at room temperature
for 30 min and stopped by detection reagents which contain EDTA.
The detection step lasted for 1 h. The IC50 was calculated using
GraphPad Prism 5.0.
Microsomal Stability Assay. All assays were conducted in single
sample. The incubation mixtures were prepared in E-tube and were
contained with 1 μM test analogues, 0.5 mg/mL hepatic microsomes
(from rat, mouse, dog, monkey, and human), and 1 mM NADPH in
100 mM potassium phosphate buffer solution (pH = 7.4). Reactions
were initiated by the addition of NADPH and kept in a shaking water
bath at 37 °C. After 0.5, 10, and 30 min incubations at 37 °C, the
reactions were terminated by the addition of cold acetonitrile
equivalent to the volume of the reaction mixture. The samples were
vortexed for 10 min and then centrifuged at 10000 rpm for 10 min.
The supernatant was subjected to LC/MS/MS (Waters UPLC/
API4000 Q Trap). In the determination of the in vitro t1/2 (half-life,
HL), the analyte peak areas were converted to percentage of drug
remaining, using the T = 0 peak area values as 100%. The slope of the
linear regression from log percentage remaining versus incubation time
(Z)-N-(4-((3-Chloro-4-(cyclopropylmethoxy)phenyl)amino)-7-
ethoxyquinazolin-6-yl)-4-(dimethylamino)-2-fluorobut-2-enamide
(11a). The retention time of the Z-isomer is 4.07 min; light-yellow
1
solid; yield 43.4%. H NMR (400 MHz, DMSO) δ 9.69 (s, 1H), 9.65
(s, 1H), 8.74 (s, 1H), 8.51 (s, 1H), 7.96 (d, J = 2.4 Hz, 1H), 7.69 (dd, J
= 8.8, 2.4 Hz, 1H), 7.28 (s, 1H), 7.15 (d, J = 8.8 Hz, 1H), 6.20 (dt, J =
36.4, 7.2 Hz, 1H), 4.27 (q, J = 7.2 Hz, 2H), 3.92 (d, J = 6.8 Hz, 2H),
3.18 (dd, J = 7.2, 2.0 Hz, 2H), 2.20 (s, 6H), 1.42 (t, J = 7.2 Hz, 3H),
1.12 (t, J = 7.2 Hz, 1H), 0.63−0.56 (m, 2H), 0.39−0.33 (m, 2H).
HRMS (ESI): m/z calcd for (C26H29ClFN5O3 + H)+, 514.2021;
found, 514.2017.
(E)-N-(4-((3-Chloro-4-(cyclopropylmethoxy)phenyl)amino)-7-
ethoxyquinazolin-6-yl)-4-(dimethylamino)-2-fluorobut-2-enamide
(11b). The retention time of the E-isomer is 3.16 min; light-yellow
solid; yield 26.6%. 1H NMR (400 MHz, DMSO) δ 10.52 (s, 1H), 9.69
(s, 1H), 8.77 (s, 1H), 8.50 (s, 1H), 7.94 (d, J = 2.4 Hz, 1H), 7.68 (dd, J
= 8.8, 2.4 Hz, 1H), 7.28 (s, 1H), 7.15 (d, J = 9.2 Hz, 1H), 6.12 (dt, J =
23.2, 6.8 Hz, 1H), 4.28 (q, J = 7.0 Hz, 2H), 3.92 (d, J = 6.8 Hz, 2H),
3.37−3.38 (m, 2H), 2.22 (s, 6H), 1.43 (t, J = 7.2 Hz, 3H), 1.27−1.24
(m, 1H), 0.66−0.56 (m, 2H), 0.37−0.36 (m, 2H). HRMS (ESI): m/z
calcd for (C26H29ClFN5O3 + H)+, 514.2021; found, 514.2017.
N-(4-((3-Bromophenyl)amino)-7-methoxyquinazolin-6-yl)-4-(di-
methylamino)-2-fluorobut-2-enamide (12a+12b). The preparation
was according to the compound (1a+1b). TLC Rf = 0.52, 0.53
(DCM/MeOH = 10:1, the two isomers could be separated). The
separation of the Z−E isomers were performed according to
compound (2a+2b).
(Z)-N-(4-((3-Bromophenyl)amino)-7-methoxyquinazolin-6-yl)-4-
(dimethylamino)-2-fluorobut-2-enamide (12a). The retention time
1
of the Z-isomer is 7.40 min; light-yellow solid; yield 38.5%. H NMR
(400 MHz, DMSO) δ 9.79 (s, 2H), 8.73 (s, 1H), 8.60 (s, 1H), 8.19 (t,
J = 1.6 Hz, 1H), 7.88 (d, J = 8.8 Hz, 1H), 7.36−7.27 (m, 3H), 6.19 (dt,
J = 36.4, 7.2 Hz, 1H), 4.00 (s, 2H), 3.17 (dd, J = 7.2, 2.0 Hz, 1H), 2.20
(s, 3H). HRMS (ESI): m/z calcd for (C21H21BrFN5O2 + H)+,
474.0941; found, 476.0925.
(E)-N-(4-((3-Bromophenyl)amino)-7-methoxyquinazolin-6-yl)-4-
(dimethylamino)-2-fluorobut-2-enamide (12b). The retention time
1
of the E-isomer is 5.19 min; light-yellow solid; yield 31.5%. H NMR
relationships (−k) was used in the conversion to the in vitro t1/2
,
(400 MHz, DMSO) δ 10.99 (s, 1H), 9.81 (s, 2H), 8.83 (s, 1H), 8.60
(s, 1H), 8.16 (s, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.38−7.28 (m, 4H),
6.12(td, J = 23.2, 6.8 Hz, 1H), 4.02 (s, 3H), 3.27−3.25 (m, 2H), 2.22
(s, 6H). HRMS (ESI): m/z calcd for (C21H21BrFN5O2 + H)+,
474.0941; found, 476.0925.
Biological Section. Determination of the GSH Conjugates
Addition to Substituted Olefins. 1. Determination of GSH
Conjugation with N-Phenylacrylamide (A) or 2-Fluoro-N-phenyl-
acrylamide (B). Reactions of N-phenylacrylamide (A) or 2-fluoro-N-
phenylacrylamide (B) with GSH were monitored with an Agilent 1290
HPLC-6224 time-of-flight mass spectrometer. Reactions were initiated
by mixing various volumes of the Michael acceptor (100 mM in ACN)
with the GSH (1.0 M GSH in PBS, pH = 7.4). Final solutions in 200
μL of PBS (Costar flat-bottom clear 96-well plate, 300 μL per well),
containing 1 mM Michael acceptor and increasing concentrations of
GSH (10−200 mM), were incubated for 1440 min at room temp prior
values by the in vitro t1/2= −0.693/k. The percent remaining of test
compound was calculated compared to the initial quantity at 0 time.
Measurement of CYP Inhibition Assay. Inhibition activity of CYP
was evaluated by incubating 100 μM human hepatic microsomes in the
presence of 10 μM test compound. The incubation mixture was
allowed to stand for 20 min at 37 °C. The incubation was terminated
by the addition of acetonitrile equivalent to the volume of the
incubation mixture. After the centrifugation, the supernatant was
subjected to LC/MS/MS (Waters UPLC/API4000 Q Trap). The
relative CYP activity was calculated via the percentage of metabolite
product.
Pharmacokinetic Assay. Male rats (Sprague−Dawley rats, body-
weight range of 180−220 g, iv, n = 2, po, n = 3) were administered
analogue intravenously via the tail vein at 3 mg/kg, respectively, or
orally at 10 mg/kg, respectively. At predetermined times 24 h or more
J
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after dosing, 0.4 mL blood was collected and the plasma was separated
by centrifugation (8000 rpm, 5 min, Sigma 3K15). The concentrations
of the compound were measured in the plasma using LC/MS/MS
after protein precipitation with acetonitrile. The relevant estimated
pharmacokinetic parameters for plasma were derived using DAS 2.0.
Mouse Tumor Xenograft Efficacy Study. The efficacy study was
conducted in strict accordance with protocols approved by the
Institutional Animal Care and Use Committee of the Central Research
Institute, Shanghai Pharmaceuticals Holding Co., Ltd. H1975 NSCLC
xenografts were established by 3.0 × 107 cells subcutaneously
inoculated in nude mice. Treatments were initiated when tumors
reached a mean group size of 50−150 mm3. The mice were
randomized to Control (10 mL/kg 0.5% CMC-Na + 0.4%
Tween80, ig administration), afatinib (30 mg/kg, ig administration),
2a (30 mg/kg, ig administration), 3a (30 mg/kg, ig administration), 5a
(30 mg/kg, ig administration), 5a (60 mg/kg, ig administration), and
5a (15 mg/kg, ig administration) every 2 days for 9 days. The size of
tumors were measured individually twice per week with microcalipers.
Tumor volume (V) was calculated as V = (length × width)2/2. The
individual relative tumor volume (RTV) was calculated as follows:
RTV = Vt/V0, where Vt is the volume on each day of measurement and
V0 is the volume on the day of initial treatment. Therapeutic effect of
compound was expressed in terms of T/C% and the calculation
formula is T/C (%) = mean RTV of the treated group/mean RTV of
the control group × 100%.
hERG, human ether-a-go-go-related gene; JAK2, Janus kinase
2; NSCLC, nonsmall-cell lung cancer; PDGFR, platelet-derived
growth factor receptor; SFC, supercritical fluid chromatog-
raphy; Src, Rous sarcoma oncogene cellular homologue; SYK,
spleen tyrosine kinase
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ASSOCIATED CONTENT
■
S
* Supporting Information
Additional figures and tables illustrating inhibition data, purity
1
data, and H NMR spectra. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
■
Moreno, M. A.; Terrasa, J.; Munoz-Langa, J.; Valdivia, J.; Isla, D.;
̃
*For Y. Yu: phone, 86-571-8820 8452; fax, 86-571-8820 8452;
E-mail, yyu@zju.edu.cn.
Domine, M.; Molinier, O.; Mazieres, J.; Baize, N.; Garcia-Campelo, R.;
Robinet, G.; Rodriguez-Abreu, D.; Lopez-Vivanco, G.; Gebbia, V.;
Ferrera-Delgado, L.; Bombaron, P.; Bernabe, R.; Bearz, A.; Artal, A.;
Cortesi, E.; Rolfo, C.; Sanchez-Ronco, M.; Drozdowskyj, A.; Queralt,
C.; de Aguirre, I.; Ramirez, J. L.; Sanchez, J. J.; Molina, M. A.; Taron,
M.; Paz-Ares, L. Spanish Lung Cancer Group in collaboration with
*For J. Shen: E-mail, jkshen@mail.shcnc.ac.cn.
Author Contributions
∥Guangxin Xia and Wenteng Chen contributed equally to this
work.
Notes
́
Groupe Franca̧ is de Pneumo-Cancerologie and Associazione Italiana
Oncologia Toracica. Erlotinib versus standard chemotherapy as first-
line treatment for European patients with advanced EGFR mutation-
positive non-small-cell lung cancer (EURTAC): a multicentre, open-
label, randomized phase 3 trial. Lancet Oncol. 2012, 13, 239−246.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Marc A. Giulianotti in Torrey Pines Institute for
Molecular Studies, USA, for critical reading this manuscript and
Xiaoming Xie in Shanghai Pharmaceutical Holding Co., Ltd.,
for performing NMR spectrometry for structure elucidation.
This project was supported from the National Natural Science
Foundation of China (no. 81273356), Program for Zhejiang
Leading Team of S&T Innovation, and the Osteoporosis and
Breast Cancer Research Center, USA, to Y. Yu, and the China
Postdoctoral Science Foundation Funded Project
(2014M550331) to W. Chen. We are also grateful for
additional financial support from New Drug Creation Project
of the National Science and Technology Major Foundation of
China (no. 2010ZX09401-404) to Shanghai Pharmaceutical
Holding Co., Ltd.
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ABBREVIATIONS USED
■
Alk, anaplasticlymphoma kinase; EGFR, epidermal growth
factor receptor; EGFR-TK, epidermal growth factor receptor
tyrosine kinase; FLT, FMS like tyrosine kinase; GSH,
glutathione; HER, human epidermal growth factor receptor;
K
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Journal of Medicinal Chemistry
Article
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dx.doi.org/10.1021/jm5014659 | J. Med. Chem. XXXX, XXX, XXX−XXX