Combination of 4-anilinoquinazoline and rhodanine as novel epidermal growth factor receptor tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.04.065

Abstract A type of novel rhodanine-based 4-anilinoquinazoline, which designed the combination between quinazoline as the backbone and various substituted biological rhodanine groups as the side chain, have been synthesized, and their antiproliferative act

Full text of DOI:10.1016/j.bmc.2015.04.065

Accepted Manuscript
Combination of 4-anilinoquinazoline and rhodanine as novel epidermal growth
factor receptor tyrosine kinase inhibitors
Si-Ning Li, Yun-Yun Xu, Jia-Yu Gao, Hong-ran Yin, Shi-Lei Zhang, Huan-Qiu
Li
PII:
S0968-0896(15)00373-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.04.065
BMC 12277
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 March 2015
22 April 2015
23 April 2015
Please cite this article as: Li, S-N., Xu, Y-Y., Gao, J-Y., Yin, H-r., Zhang, S-L., Li, H-Q., Combination of 4-
anilinoquinazoline and rhodanine as novel epidermal growth factor receptor tyrosine kinase inhibitors, Bioorganic
& Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.04.065
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Combination of 4-anilinoquinazoline and rhodanine as novel
Epidermal Growth Factor Receptor tyrosine kinase
inhibitors
Si-Ning Li, Yun-Yun Xu, Jia-Yu Gao, Hong-ran Yin, Shi-Lei Zhang*, Huan-Qiu
Li*.
College of Pharmaceutical Science, Soochow University, Suzhou 215123, PR
China.; *Corresponding author. Tel.: +86 512 65882090; fax.: +86 512
65882090; e-mail: huanqiuli@suda.edu.cn; zhangshilei@suda.edu.cn
Abstract：A type of novel rhodanine-based 4-anilinoquinazoline，which designed the
combination between quinazoline as the backbone and various substituted biological
rhodanine groups as the side chain, have been synthesized, and their antiproliferative
activities were also evaluated firstly. These compounds displayed good
antiproliferative activity and EGFR-TK inhibitory activity. Among them, compound
8d showed good inhibitory activity (IC50 = 2.7 µM for Hep G2, IC50 = 3.1 µM for
A549) and molecular docking of 8d into EGFR TK active site was also performed,
this inhibitor well fitting the active site might well explain its excellent inhibitory
activity.
Keywords: EGFR-TK inhibitors; 4-anilinoquinazoline; rhodanine ; structure-based
design; molecular docking

1. Introduction
EGFR belongs to the ErbB family of receptor TKs that plays indispensable roles
in cell proliferation, survival, adhesion, migration and differentiation. 1 The role of
EGFR has been most thoroughly studied in breast cancer,2 lung cancer (especially
lung adenocarcinomas)3–5 and in hormone-refractory prostate cancer.6 Compounds
that inhibit the kinase activity of EGFR after binding of its cognate ligand are of
potential interest as new therapeutic antitumor agents. 7-8
Among the EGFR TKIs, the 4-anilinoquinazolines play a key role and has been
extensively studied in the past few years; the most representative compounds are
gefitinib, erlotinib and lapatinib. 9-11 These TKIs generally designed to selectively
inhibit EGF-stimulated signal transduction showed high potency in vitro, inhibiting
EGFR at nanomolar concentration. It was concluded that the quinazoline core was the
best scaffold for the development of EGFR inhibitors because any alteration of the
nitrogen substitution pattern in the bicyclic ring resulted in less active compounds.
From 2010 to present, many gefitinib, erlotinib and lapatinib analogs were reported in
published patents and articles, and the focal points of the chemical space of these new
irreversible and reversible inhibitors were centered on the 4-anilino headgroup moiety,
6-substituents and the bioisosteres of quinazoline ring, which have led to the
development and the marketing of new series of antitumor agents. 12
In our studies of the inhibition of EGFR by reversible and irreversible
4-anilinoquinazolines, many new 4,6-substituted-(diaphenylamino)quinazoline
derivatives have been synthesized and displayed potent EGFR inhibitory
activity,13-15modeling studies of the binding of reversible anilinoquinazoline with
EGFR-TK has been developed. The results suggest that a binding mode whereby the
C-6 position of the bicyclic chromophore point toward the solvent out of the ATP
binding pocket. Also, in recent years, the structure-based approach has become very
powerful for the discovery of novel lead compounds and for lead optimization, and
new rhodanine-based derivatives as potent anticancer angents are reflected in our
recent publication, the reported anti-apoptotic rhodanine-based acylsulfonamide

derivatives exhibited remarkable antitumor activity. 16 Based on our previous results,
in the paper, by combining two distinct pharmacological properties
4-anilinoquinazoline and rhodanine in one molecule with a linker heterocyclic amino
acid Pro, series of novel rhodanine-based 4-anilinoquinazoline derivatives were
designed and synthesized as potent EGFR inhibitors, we postulated that these
rhodanine-based 4-anilinoquinazoline derivatives may serve such a purpose while
being topographically appropriate to maintain high affinity binding to EGFR proteins
with potent antitumor activity and represent a novel approach to cancer therapy.
2. Results and Discussion
2.1 Chemsitry
Eighteen rhodanine-based 4-anilinoquinazoline derivatives 8a-8q, were designed to
perform comparative SAR studies. We designed a more efficient synthesis that
allowed ring cyclization and incorporation of the 4-anilino group in a single step, as
shown in Scheme 1. Commercially available 5-nitroanthranilonitrile 1 was converted
into the corresponding formamidine 2 using DMF acetal. Heating a solution of
formamidine
2
and
3-Cl-aniline
in
HOAc
gave
6-nitro-4-(3-X-phenylamino)quinazolines 3 and. reduction of the nitro group of 3 with
iron in HOAc yielded the intermediate 6-aminoquinazolines 4. One the other hand,
different substituted intermediates 5a–5q and 6a–6q were prepared by the
Knoevenagel reaction of various substitute aldehydes with rhodanine, and methylated
by CH3I. Then, the rhodanine derivatives 7a–7q was synthesized from Pro with
6a–6q and diisopropylethyl amine (DIEA). Finally, rhodanine derivatives 7a–7q,
4-aminoquinazolines
4
and 1-(3-dimethylaminopropyl)-3- thylcarbodiimide
hydrochloride (EDC) were dissolved in CH2Cl2 at room temperature to give the
desired rhodanine-based 4-anilinoquinazoline derivatives 8a-8q.
2.2. Antiproliferative assay
In our former reports, the N1, N3 of the 4-(phenylamino)quinazoline moiety
would interact with active centre probably by hydrogen bond, water bridge or else. 13
So we saved the quinazoline skeleton in the new designed compunds. Recently, we

found that the thioxo-thiazolidinedione (rhodanine) derivatives have potent
antiproliferative activities against HepG2. Thus, we postulated that the
4-(phenylamino)quinazoline core structure with active thioxo-thiazolidinedione
groups may serve such a purpose while being topographically appropriate to maintain
high affinity binding to EGFR protein with potent antiproliferative activity. To
confirm our postulation, the synthesized rhodanine-based 4-anilinoquinazoline
derivatives were evaluated for their antiproliferative activities against Hep G2 and
A549 cells by applying the MTT colorimetric assay. The results were summarized in
Table 1. Compounds were tested over a range of concentrations from 0.01 to 100 µg
/ml, and the calculated IC50 values, that is, the concentration (µg /mL) of a compound
that was able to cause 50% cell death with respect to the control culture, were
reported differently according to different cancer cells. As expected, these compounds
exhibited remarked effects on antiproliferative activities, and generally the results
showed which those applied to Hep G2 cells would performed better than that in
A549
(S,E)-1-(5-((6-bromopyridin-2-yl)methylene)-4-oxo-4,5-dihydrothiazol-2-yl)-N-(4-((3
-chlorophenyl)amino)quinazolin-6-yl)pyrrolidine-2-carboxamide (8d), with
cells.
Especially,
a
bromopyridine group on the rhodanine moiety showed good inhibitory activity (IC50
= 2.7 µM for Hep G2 and IC50 = 3.1 µM for A549), comparable to the positive control
gefitinib (IC50=7.52 µM for Hep G2 and IC50=5.51 µM for A549).
Structure-activity relationships in these new compounds demonstrated that
compounds containing pyridine group on the rhodanine moiety (8c-8f) showed better
activity than others, which is accord with the reported literature that pyridylrhodanine
derivatives have potent anti-apoptotic activity. 17 Compound with substitution at the
meta (8i) or ortho (8h-8k, 8n, 8o) on the phenyl ring at R position showed less potent
activities against A549 cell line than those with substitution at the para position (8m,
8p), and compound 8h-8p displayed similar moderate activity against Hep G2 cell
line, indicating relatively substituents on the phenyl ring at R position didn’t input
substantial effects on the antiproliferative capability against Hep G2 cell line of the
compounds.

2.3. EGFR inhibitory activity and molecular docking
To evaluate the EGFR inhibitory potency of new compounds, their ability to
block EGFR TK was tested in an EGFR TK assay. We chose theeight compounds
behaved well for antiproliferation and their activities positively correlated with
anti-proliferative activity, which they had the same trends (Table 2). This could
indicate that rhodanine-based 4-anilinoquinazoline derivatives basically follow the
EGFR inhibition path against cancer cell. As shown in Table 2, compound 8c and 8d
displayed the most potent inhibitory activity (IC50 = 6.5 µM and 3.1 µM for EGFR).
To help understand the SARs observed at the EGFR and guide further EGFR
inhibitors design, molecular docking of the most potent inhibitor 8d into ATP binding
site of EGFR kinase was performed on the binding model based on the EGFR
complex structure (1M17.pdb). The binding model of compound 8d and EGFR was
depicted in Figure 1. In the binding model, compound 8d is nicely bound to the region
of EGFR, the quinazoline ring be inserted well inside the pocket. Outside the cleft, the
N atom of 4-anilinoquinazoline core structure of 8d forms hydrogen bond with the
MET769 (OD···N: 2.4 Å), which made the combination ability of 8d acting on EGFR.
Meanwhile, the carbonyl group of rhodanine moiety forms hydrogen bond with the
PRO770, the amide group which linked the 4-anilinoquinazoline core structure with
rhodanine moiety forms hydrogen bond with the CYS773 of EGFR protein and play
an important role in stabilizing the three dimensional structure of a protein, these
results showed that the designed rhodanine-based 4-anilinoquinazoline derivatives
were potential inhibitors of EGFR.
3. Conclusion
In summary, by combining two distinct pharmacological properties
4-anilinoquinazoline and rhodanine in one molecule with a linker Pro, a series of
novel rhodanine-based 4-anilinoquinazoline derivatives were designed and
synthesized potent EGFR Inhibitors. Four compounds containing pyridine group on
the rhodanine moiety (8c-8f) showed better activity than others, which is accord with
the reported literature that pyridyl-rhodanine derivatives have potent anti-apoptotic

activity The EGFR molecular docking experiment of compound 8d indicated that 8d
was is well bound to the region of EGFR, the quinazoline ring be inserted nicely
inside the pocket, the carbonyl group of rhodanine moiety forms hydrogen bond with
the PRO770. In conclusion, these findings expand the chemical diverse of EGFR
inhibitors, and similar strategies might be applied to the design of compounds able to
form a covalent bond with a biological target.
4. Experimental
4.1 Chemistry General. All chemicals (reagent grade) used were purchased from
Sigma–Aldrich (USA) and Sinopharm Chemical Reagent Co. Ltd.(China). NMR
spectra were measured on a Varian Unity Inova 300MHz and 400MHz spectrometer
at 25°C and referenced to Me4Si. Chemical shifts are reported in ppm (δ) using the
residual solvent line as internal standard. Splitting patterns are designed as s, singlet;
d, doublet; t, triplet; m, multiplet. HRMS spectra were acquiredon Bruker Esquire
Liquid Chromatography-Ion Trap Mass Spectrometer. Melting points were
determined on a XT4 MP apparatus (Taike Corp. , Beijing, China) and are as read.
Analytical thin-layer chromatography (TLC) was performed on the glass-backed
silica gel sheets (silica gel 60Å GF254). All compounds were detected using UV light
(254 nm or 365 nm).
4.2. General procedure for the preparation of compounds 5a–5q
Rhodanine 1 (1.3 g, 10 mmol), substitute aldehyde (10 mmol) and sodium acetate (2.9
g, 35 mmol) were suspended in acetic acid (100 mL) and the mixture was refluxed for
24 h with N2. The resulting mixture was cooled to room temperature, and then 360
mL water was added. The precipitate that formed was filtered, washed with ethyl
ether, and dried to give 5a–5q (Yield 43%-57%). 5k: 1H NMR (300 MHz, DMSO-d6)
d 7.38–7.58 (m, 3H), 7.64–7.80 (m, 5H), 7.82–7.92 (m, 2H).
4.3. General procedure for the preparation of compounds 6a–6q
Compound 5a-5q (1 mmol), DIEA (0.213 g, 1.5 mmol) were suspended in anhydrous
ethanol (50 mL), then CH3I (0.215 g, 1.5 mmol) was slowly added within ten minutes,
the mixture was stirred at room temperature for 6 h. Then 150 mL water was added,

the precipitate was filtered, and dried to give 6a-6q (Yield: 63%-79%). 6k: 1H NMR
(300 MHz, DMSO-d6) δ 2.85 (s, 3H), 7.38–7.56 (m, 3H), 7.72–7.81 (m, 4H),
7.84–7.92 (m, 3H).
4.4. General procedure for the preparation of compounds 7a–7q
Compound 6a-6q (0.5 mmol), DIEA (0.071 g, 0.5 mmol) and L- proline (0.058 g, 0.5
mmol) were suspended in ethanol (5 mL) and heated at reflux at 70–80 oC for 20 h.
The reaction mixture was cooled to room temperature and concentrated under reduced
pressure. The resulting solid was extracted with ethyl acetate for column
chromatography. Column chromatography was performed using silica gel (200–300
mesh), eluted with ethyl acetate and petroleum ether (1:1, v/v) to give 7a–7q (Yield:
57%-68%). 7k:1H NMR (400 MHz, DMSO-d6) 7.84–7.92 (m, 3H), 7.72–7.81 (m,
4H), 7.38–7.56 (m, 3H), 5.44 (d, J = 4.6 Hz, 1H), 4.04 (s, 1H), 3.86 (d, J = 10.1 Hz,
1H), 2.78 – 2.58 (m, 2H), 2.48 (s, 1H), 2.31 (s, 1H).
4.5. General procedure for the preparation of target compounds 8a–8q
4-anilinoquinazoline 4 was synthesized as our previous report 12. Compound 7a-7q
(0.1 mmol), EDCI (0.040 g, 0.2 mmol), compound 4 (0.014 g, 0.053 mmol) were
suspended in pyridine (15 mL) and stirred at room temperature for 12 h. The mixture
was diluted by EtOAc and washed with H2O and brine, dried over Na2SO4, and
concentrated in vacuo. The residue was subjected to silica gel chromatography or
crystallization if necessary to afford the compounds 8a–8q.
(S,E)-N-(4-((3-chlorophenyl)amino)quinazolin-6-yl)-1-(4-oxo-5-(thiophen-2-ylm
ethylene)-4,5-dihydrothiazol-2-yl)pyrrolidine-2-carboxamide (8a) yellow solid，yield：
35%，Mp 214-215 oC. 1H NMR (400 MHz, CDCl3) δ 10.75 (s, 1H), 8.58 (s, 1H),
8.52 (s, 1H), 8.01 (d, J = 7.6 Hz, 1H), 7.94 (d, J = 4.6 Hz, 2H), 7.69 (d, J = 8.7 Hz,
1H), 7.60 (d, J = 4.9 Hz, 1H), 7.35 (s, 1H), 7.30 (d, J = 8.3 Hz, 1H), 7.22 (d, J = 9.0
Hz, 2H), 7.15 (s, 1H), 7.06 (d, J = 7.6 Hz, 1H), 5.44 (s, 1H), 4.07 (s, 1H), 3.90 (s, 1H),
2.64 (d, J = 23.3 Hz, 2H), 2.50 (s, 1H), 2.30 (s, 1H). 13C NMR (75 MHz, DMSO) δ
178.4, 171.34, 168.95, 157.36, 153.16, 146.80, 141.08, 138.50, 136.58, 133.45,
132.67, 131.46, 129.97, 128.98, 128.63, 127.05, 126.78, 123.77, 122.93, 121.47,

120.44, 115.53, 111.74, 64.19, 49.81, 30.57, 23.69. HRMS: [M+Na+]: Found m/z
583.0751, Calcd m/z 583.0856.
(S,E)-1-(5-((5-bromothiophen-2-yl)methylene)-4-oxo-4,5-dihydrothiazol-2-yl)-N
-(4-((3-chlorophenyl)amino)quinazolin-6-yl)pyrrolidine-2-carboxamide (8b) yellow
solid，yield：52%，Mp 194-195 oC. 1H NMR (400 MHz, CDCl3) δ 10.68 (s, 1H), 8.54
(d, J = 23.0 Hz, 2H), 8.03 – 7.88 (m, 2H), 7.79 (s, 1H), 7.67 (d, J = 9.0 Hz, 1H), 7.30
(d, J = 8.0 Hz, 1H), 7.22 (d, J = 8.7 Hz, 2H), 7.09 (dt, J = 12.5, 5.8 Hz, 3H), 5.39 (dd,
J = 8.1, 4.1 Hz, 1H), 4.09 (dd, J = 19.6, 12.2 Hz, 1H), 3.87 (dd, J = 17.4, 7.2 Hz, 1H),
2.72 – 2.43 (m, 3H), 2.33 – 2.23 (m, 1H). 13C NMR (101 MHz, DMSO) δ 178.25,
171.01, 168.87, 157.35, 153.15, 146.79, 140.73, 136.56, 133.80, 132.69, 132.30,
129.94 , 128.61, 127.64, 127.04, 124.47, 121.43, 120.50, 116.99, 115.53, 111.76,
64.27, 62.86, 49.90, 40.15, 30.60, 23.69. HRMS: [M+Na+]: Found m/z 662.9825,
Calcd m/z 662.9737.
(S,E)-N-(4-((3-chlorophenyl)amino)quinazolin-6-yl)-1-(5-((6-methylpyridin-2-yl
)methylene)-4-oxo-4,5-dihydrothiazol-2-yl)pyrrolidine-2-carboxamide (8c) yellow
solid，yield：23%，Mp 197-198 oC. 1H NMR (400 MHz, CDCl3) δ 10.84 (s, 1H), 8.64
(s, 1H), 8.52 (s, 1H), 8.02 (d, J = 7.5 Hz, 1H), 7.97 (s, 1H), 7.73 (d, J = 9.5 Hz, 1H),
7.67 (s, 1H), 7.61 (t, J = 7.7 Hz, 1H), 7.30 (d, J = 8.1 Hz, 1H), 7.23 (dd, J = 13.7, 8.2
Hz, 4H), 7.11 (d, J = 7.7 Hz, 1H), 7.05 (d, J = 7.6 Hz, 1H), 5.51 (s, 1H), 4.14 (s, 1H),
3.96 (d, J = 9.8 Hz, 1H), 2.66 (s, 3H), 2.51 (s, 3H), 2.29 (s, 1H). 13C NMR (75 MHz,
DMSO) δ 178.33, 172.01, 168.84, 157.35, 153.16, 150.93, 150.17, 146.81, 141.07,
136.56, 135.63, 132.65, 130.87, 129.97, 129.84, 128.65, 127.24, 124.17, 122.92,
121.45, 120.43, 115.53, 111.72, 64.27, 54.95, 49.85, 30.53 , 23.68. HRMS: [M+H+]:
Found m/z 570.1448, Calcd m/z 570.1401.
(S,E)-1-(5-((6-bromopyridin-2-yl)methylene)-4-oxo-4,5-dihydrothiazol-2-yl)-N
-(4-((3-chlorophenyl)amino)quinazolin-6-yl)pyrrolidine-2-carboxamide (8d) yellow
solid，yield：47%，Mp 260-261 oC. 1H NMR (400 MHz, CDCl3) δ 10.75 (s, 1H), 8.59
(s, 1H), 8.51 (s, 1H), 7.96 (d, J = 11.6 Hz, 2H), 7.71 (d, J = 9.1 Hz, 1H), 7.61 – 7.56

(m, 2H), 7.43 (d, J = 7.9 Hz, 1H), 7.37 (d, J = 7.5 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H),
7.22 (d, J = 8.5 Hz, 2H), 7.06 (d, J = 8.0 Hz, 1H), 5.48 (dd, J = 8.3, 4.1 Hz, 1H), 4.14
(d, J = 7.0 Hz, 1H), 3.98 (dd, J = 17.8, 7.6 Hz, 1H), 2.73 – 2.44 (m, 3H), 2.35 – 2.23
(m, 1H). 13C NMR (101 MHz, DMSO) δ 183.93, 181.04, 174.33, 162.69, 158.48,
158.18, 152.13, 146.41, 145.90, 141.93, 139.28, 138.00, 135.29, 133.96, 133.13,
132.35, 131.56, 131.22, 128.25, 126.81, 125.78, 120.87, 117.31, 117.15, 69.56, 54.89,
35.74, 29.12. HRMS: [M+H+]: Found m/z 636.0400, Calcd m/z 635.9340.
(S,E)-N-(4-((3-chlorophenyl)amino)quinazolin-6-yl)-1-(5-((2-chloropyridin-3-
yl)methylene)-4-oxo-4,5-dihydrothiazol-2-yl)pyrrolidine-2-carboxamide (8e) yellow
solid，yield：46%，Mp 188-189 oC. 1H NMR (400 MHz, CDCl3) δ 10.60 (s, 1H), 8.62
(s, 1H), 8.53 (s, 1H), 8.41 (d, J = 4.8 Hz, 1H), 8.08 – 8.01 (m, 2H), 7.93 (d, J = 8.9 Hz,
2H), 7.68 (d, J = 9.1 Hz, 1H), 7.40 – 7.34 (m, 1H), 7.30 (t, J = 7.8 Hz, 1H), 7.25 –
7.18 (m, 2H), 7.07 (d, J = 8.0 Hz, 1H), 5.43 (s, 1H), 4.05 (s, 1H), 3.88 (s, 1H), 2.82 –
2.38 (m, 3H), 2.31 (s, 1H). 13C NMR (101 MHz, DMSO) δ 177.76, 171.97, 168.74,
157.34, 153.18, 150.50, 150.07, 146.82, 141.07, 137.42 , 136.53, 134.34 , 132.67 ,
129.97 , 128.83, 127.03, 124.27, 123.88 , 122.92, 121.43, 120.39, 115.53, 111.76,
64.40 , 62.84, 39.62, 30.52, 23.68. HRMS: [M+H+]: Found m/z 590.0904, Calcd
m/z .590.0854
(S,E)-N-(4-((3-chlorophenyl)amino)quinazolin-6-yl)-1-(4-oxo-5-(pyridin-3-ylme
thylene)-4,5-dihydrothiazol-2-yl)pyrrolidine-2-carboxamide (8f) yellow solid，yield：
38%，Mp 230-231 oC. 1H NMR (400 MHz, DMSO) δ 10.64 (s, 1H), 8.79 (s, 1H), 8.58
(dd, J = 29.4, 10.6 Hz, 3H), 8.01 (d, J = 7.6 Hz, 1H), 7.92 (s, 1H), 7.83 (d, J = 8.2 Hz,
1H), 7.76 (s, 1H), 7.68 (d, J = 8.9 Hz, 1H), 7.41 (dd, J = 8.0, 5.0 Hz, 1H), 7.30 (d, J =
8.2 Hz, 1H), 7.23 (d, J = 8.9 Hz, 2H), 7.06 (d, J = 8.1 Hz, 1H), 5.44 (dd, J = 8.5, 3.9
Hz, 1H), 4.09 (t, J = 14.1 Hz, 1H), 3.89 (dd, J = 17.6, 7.5 Hz, 1H), 2.69 (d, J = 7.5 Hz,
1H), 2.64 (d, J = 11.6 Hz, 1H), 2.51 (dd, J = 17.7, 10.1 Hz, 1H), 2.38 – 2.23 (m, 1H).
13C NMR (75 MHz, DMSO) δ 178.93, 176.71, 169.16, 157.74, 157.36, 153.13,
150.64, 146.78, 141.10, 137.61, 136.66, 132.66, 132.25, 129.96, 128.61, 127.44,
127.02, 124.29, 123.22, 122.91, 121.47, 120.44, 115.54, 111.66, 64.00, 49.39, 30.40,
23.89. HRMS: [M+Na+]: Found m/z 578.1135, Calcd m/z 578.1244.

(S,E)-N-(4-((3-chlorophenyl)amino)quinazolin-6-yl)-1-(4-oxo-5-(quinolin-2-yl
methylene)-4,5-dihydrothiazol-2-yl)pyrrolidine-2-carboxamide(8g)
yellow solid，yield：23%，Mp 217-218 oC. 1H NMR (400 MHz, CDCl3) δ 10.84
(s, 1H), 8.63 (s, 1H), 8.50 (s, 1H), 8.23 (d, J = 8.4 Hz, 1H), 8.17 (d, J = 8.4 Hz, 1H),
7.98 (d, J = 6.2 Hz, 2H), 7.85 – 7.72 (m, 4H), 7.58 (t, J = 7.5 Hz, 1H), 7.51 (d, J = 8.4
Hz, 1H), 7.28 (s, 1H), 7.26 – 7.20 (m, 2H), 7.03 (d, J = 8.1 Hz, 1H), 5.54 (dd, J = 8.3,
4.0 Hz, 1H), 4.21 (dd, J = 14.1, 9.3 Hz, 1H), 4.07 – 3.99 (m, 1H), 2.76 – 2.49 (m, 3H),
2.38 – 2.27 (m, 1H). 13C NMR (101 MHz, DMSO) δ 182.35, 174.69, 162.94, 158.73,
157.60, 152.54, 152.38, 146.66, 142.82, 142.22, 140.09, 138.24, 136.04, 135.53,
134.22, 134.04, 133.61, 132.52, 132.52, 129.80, 128.50, 127.19,127.05, 126.17,
126.03, 121.12, 117.41, 117.26, 69.69, 55.12, 36.01, 29.39. HRMS: [M+H+]: Found
m/z 606.1348, Calcd m/z 606.1401.
(S,E)-1-(5-(2-chlorobenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl)-N-(4-((3-chlor
ophenyl)amino)quinazolin-6-yl)pyrrolidine-2-carboxamide (8h) yellow solid，yield：
54%，Mp 181-182 oC. 1H NMR (400 MHz, CDCl3) δ 10.70 (s, 1H), 8.58 (d, J = 42.3
Hz, 2H), 8.13 (s, 1H), 8.05 (d, J = 8.2 Hz, 1H), 7.95 (s, 1H), 7.66 (dd, J = 22.7, 8.3 Hz,
2H), 7.45 (d, J = 7.6 Hz, 1H), 7.34 (ddd, J = 22.3, 15.7, 7.8 Hz, 3H), 7.21 (d, J = 8.6
Hz, 2H), 7.06 (d, J = 7.9 Hz, 1H), 5.45 (dd, J = 8.5, 4.0 Hz, 1H), 4.06 (t, J = 12.2 Hz,
1H), 3.87 (dd, J = 17.4, 7.5 Hz, 1H), 2.81 – 2.43 (m, 3H), 2.30 (dd, J = 12.4, 5.8 Hz,
1H). 13C NMR (75 MHz, DMSO) δ 178.13, 172.31, 168.84, 157.36, 153.18, 146.82,
141.09, 136.57, 134.29, 132.70, 132.34, 131.97, 131.67, 130.27, 130.12, 128.64,
128.06, 127.06, 125.81, 122.93, 121.46 , 120.42, 115.55, 111.78, 64.30, 49.88, 30.54,
23.69. HRMS: [M+H+]: Found m/z 589.0955, Calcd m/z 589.0902.
(S,E)-1-(5-(3-chlorobenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl)-N-(4-((3-chlor
ophenyl)amino)quinazolin-6-yl)pyrrolidine-2-carboxamide (8i) yellow solid，yield：
23%，Mp 182-183 oC. 1H NMR (400 MHz, CDCl3) δ 10.68 (s, 1H), 8.60 (s, 1H), 8.52
(s, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.94 (s, 1H), 7.68 (d, J = 11.9 Hz, 1H), 7.50 (s, 1H),
7.39 (s, 2H), 7.30 (d, J = 7.9 Hz, 1H), 7.21 (d, J = 10.2 Hz, 2H), 7.06 (d, J = 7.5 Hz,
1H), 5.37 (d, J = 57.4 Hz, 1H), 4.17 – 3.67 (m, 2H), 2.78 – 2.39 (m, 2H), 2.31 (s, 1H),
1.93 (s, 1H). 13C NMR (75 MHz, DMSO) δ 178.42, 172.06, 168.86, 157.36, 153.17,

146.79, 141.07, 136.58, 136.29, 132.67, 132.68, 132.20, 131.32, 130.30, 129.99,
129.04, 128.66, 127.98, 127.03, 122.95, 122.52, 121.47, 120.45, 115.54, 111.70,
64.28, 49.89, 30.55, 23.70. HRMS: [M+Na+]: Found m/z 610.9763, Calcd m/z
611.0902.
(S,E)-1-(5-(2-bromobenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl)-N-(4-((3-chl
orophenyl)amino)quinazolin-6-yl)pyrrolidine-2-carboxamide (8j) yellow solid，yield：
37%，Mp 200-201 oC. 1H NMR (400 MHz, CDCl3) δ 10.68 (s, 1H), 8.61 (s, 1H), 8.53
(s, 1H), 8.10 – 8.03 (m, 2H), 7.96 (s, 1H), 7.72 – 7.58 (m, 3H), 7.42 (t, J = 7.3 Hz,
1H), 7.30 (t, J = 8.2 Hz, 1H), 7.23 (d, J = 8.1 Hz, 3H), 7.07 (d, J = 8.1 Hz, 1H), 5.44
(d, J = 4.6 Hz, 1H), 4.04 (s, 1H), 3.86 (d, J = 10.1 Hz, 1H), 2.78 – 2.58 (m, 2H), 2.48
(s, 1H), 2.31 (s, 1H). 13C NMR (75 MHz, DMSO) δ 178.20, 172.52, 168.83, 157.53,
153.36, 146.99, 141.27, 136.76, 133.95, 133.70, 132.85, 132.68, 131.75, 130.08,
128.97, 128.81, 127.25, 125.31, 123.12, 121.64, 120.61, 115.72, 111.93, 64.47, 50.17,
30.72, 23.88. HRMS: [M+H+]: Found m/z 634.9476, Calcd m/z 634.9460.
(S,E)-N-(4-((3-chlorophenyl)amino)quinazolin-6-yl)-1-(5-(2-fluorobenzylidene
)-4-oxo-4,5-dihydrothiazol-2-yl)pyrrolidine-2-carboxamide (8k) yellow solid，yield：
32%，Mp 182-183 oC. 1H NMR (400 MHz, CDCl3) δ 10.73 (s, 1H), 8.62 (s, 1H),
8.53 (s, 1H), 8.01 (dd, J = 23.3, 17.2 Hz, 3H), 7.73 – 7.55 (m, 2H), 7.41 (dd, J = 12.7,
6.6 Hz, 1H), 7.32 – 7.27 (m, 1H), 7.25 – 7.19 (m, 2H), 7.16 – 7.10 (m, 1H), 7.06 (d, J
= 7.3 Hz, 1H), 5.46 (dd, J = 8.4, 4.1 Hz, 1H), 4.08 (dd, J = 11.8, 6.5 Hz, 1H), 3.90 (t,
J = 8.8 Hz, 1H), 2.78 – 2.44 (m, 3H), 2.36 – 2.25 (m, 1H). 13C NMR (75 MHz,
DMSO) δ 178.70, 172.59, 169.26, 159.25, 157.76, 153.58, 147.23, 141.49, 136.97,
133.08, 131.71, 130.37, 129.06, 128.88, 127.45, 125.72, 123.33 ,122.21, 122.05,
121.86, 120.83, 116.75, 116.46, 115.95, 112.16, 64.69, 60.20, 30.94, 21.20. HRMS:
[M+H+]: Found m/z 574.0360, Calcd m/z 574.0404.
(S,E)-1-(5-(3-bromobenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl)-N-(4-((3-chlorophen
yl)amino)quinazolin-6-yl)pyrrolidine-2-carboxamide (8l)
yellow solid，yield：43%，Mp 184-185 oC. 1H NMR (400 MHz, CDCl3) δ 10.68 (s,
1H), 8.56 (d, J = 22.6 Hz, 2H), 8.07 – 7.90 (m, 2H), 7.68 (d, J = 13.6 Hz, 3H), 7.53 (d,
J = 7.5 Hz, 1H), 7.45 (d, J = 7.4 Hz, 1H), 7.36 – 7.28 (m, 2H), 7.22 (d, J = 9.6 Hz,

2H), 7.07 (d, J = 7.8 Hz, 1H), 5.44 (s, 1H), 4.10 (s, 1H), 3.92 (s, 1H), 2.76 – 2.45 (m,
3H), 2.31 (s, 1H). 13C NMR (75 MHz, DMSO) δ 178.00, 172.32, 168.83, 157.35,
153.17, 146.82, 141.08, 136.57, 133.80, 133.51, 132.66, 132.49, 131.55, 129.98,
128.80, 128.60, 127.06, 125.12, 122.93, 121.45, 120.42, 115.53, 111.74, 64.28, 49.90,
30.53, 23.69. HRMS: [M+H+]: Found m/z 635.0439, Calcd m/z 634.9460.
(S,E)-1-(5-(4-chlorobenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl)-N-(4-((3-chlor
ophenyl)amino)quinazolin-6-yl)pyrrolidine-2-carboxamide (8m) yellow solid，yield：
49%，Mp 172-173 oC. 1H NMR (400 MHz, CDCl3) δ 10.70 (s, 1H), 8.56 (d, J = 28.7
Hz, 2H), 8.03 (d, J = 8.2 Hz, 1H), 7.93 (s, 1H), 7.74 – 7.63 (m, 2H), 7.44 (q, J = 8.7
Hz, 4H), 7.29 (t, J = 8.1 Hz, 1H), 7.20 (d, J = 9.4 Hz, 2H), 7.06 (d, J = 7.8 Hz, 1H),
5.45 (dd, J = 8.5, 3.9 Hz, 1H), 4.13 – 4.02 (m, 1H), 3.89 (dd, J = 17.6, 7.3 Hz, 1H),
2.77 – 2.44 (m, 3H), 2.37 – 2.23 (m, 1H). 13C NMR (75 MHz, DMSO) δ 178.62,
172.10, 168.90, 157.36, 153.17, 146.82, 141.09, 136.59, 134.49, 132.67, 131.21,
131.05, 129.97, 129.34, 128.66, 127.04, 122.93, 121.45, 120.42, 115.55, 111.72,
64.25, 49.80, 30.56, 23.70. HRMS: [M+H+]: Found m/z 589.0959, Calcd m/z
589.0902.
(S,E)-N-(4-((3-chlorophenyl)amino)quinazolin-6-yl)-1-(5-(2-methoxybenzylide
ne)-4-oxo-4,5-dihydrothiazol-2-yl)pyrrolidine-2-carboxamide (8n) yellow solid ，
yield：43%，Mp 252-253 oC. 1H NMR (400 MHz, CDCl3) δ 10.83 (s, 1H), 8.62 (s, 1H),
8.53 (s, 1H), 8.18 (s, 1H), 8.02 (d, J = 7.4 Hz, 1H), 7.96 (s, 1H), 7.70 (s, 1H), 7.53 (d,
J = 8.1 Hz, 1H), 7.38 (d, J = 7.2 Hz, 1H), 7.30 (d, J = 8.1 Hz, 3H), 7.24 – 7.21 (m,
2H), 7.04 (s, 2H), 6.92 (d, J = 8.3 Hz, 1H), 5.43 (s, 1H), 4.03 (s, 1H), 3.84 (s, 3H),
2.54 (d, J = 61.3 Hz, 4H), 2.26 (s, 1H). 13C NMR (75 MHz, DMSO) δ 178.44, 172.07,
168.85, 157.35, 153.15, 146.79, 141.08, 136.58, 136.01, 133.95, 132.67, 131.04,
130.33, 129.95, 129.55, 129.28, 127.62, 127.02, 122.91, 121.46, 120.42, 115.54,
111.72, 64.27, 54.95, 49.85, 30.54, 23.68. HRMS: [M+H+]: Found m/z 585.1452,
Calcd m/z 585.1397.
(S,E)-N-(4-((3-chlorophenyl)amino)quinazolin-6-yl)-1-(5-(2-nitrobenzylidene)-4-oxo-
4,5-dihydrothiazol-2-yl)pyrrolidine-2-carboxamide (8o)

pink solid，yield：53%，Mp 187-188 oC. 1H NMR (400 MHz, Acetone) δ 10.63
(s, 1H), 8.65 (s, 1H), 8.53 (s, 1H), 8.16 – 8.02 (m, 3H), 7.94 (s, 1H), 7.76 – 7.63 (m,
3H), 7.61 – 7.54 (m, 1H), 7.31 (t, J = 8.1 Hz, 1H), 7.22 (d, J = 8.7 Hz, 2H), 7.08 (d, J
= 7.9 Hz, 1H), 5.41 (dd, J = 8.6, 4.1 Hz, 1H), 3.99 (d, J = 7.7 Hz, 1H), 3.79 (dt, J =
9.1, 6.0 Hz, 1H), 2.78 – 2.42 (m, 3H), 2.30 (dd, J = 12.5, 5.6 Hz, 1H). 13C NMR (101
MHz, DMSO) δ 177.55, 172.34, 168.85, 157.37, 153.20, 147.91, 146.84, 141.10,
136.59, 134.48, 133.99, 132.71, 130.65, 130.13, 130.00, 129.32, 128.68, 127.05,
126.87, 125.30, 122.96, 121.47, 120.44, 115.57, 111.75, 64.30, 49.90, 30.55 , 23.71.
HRMS: [M+H+]: Found m/z 600.1198, Calcd m/z 600.1143.
(S,E)-N-(4-((3-chlorophenyl)amino)quinazolin-6-yl)-1-(5-(4-methoxybenzylidene)-4-
oxo-4,5-dihydrothiazol-2-yl)pyrrolidine-2-carboxamide (8p)
yellow solid，yield：33%，Mp 176-177 oC. 1H NMR (400 MHz, CDCl3) δ
10.82 (s, 1H), 8.58 (d, J = 46.2 Hz, 2H), 8.08 – 7.91 (m, 2H), 7.76 – 7.65 (m, 2H),
7.49 (d, J = 8.7 Hz, 2H), 7.30 (d, J = 8.0 Hz, 1H), 7.20 (d, J = 9.3 Hz, 2H), 7.05 (d, J
= 7.7 Hz, 1H), 6.97 (d, J = 8.7 Hz, 2H), 5.45 (d, J = 4.4 Hz, 1H), 4.06 (s, 1H), 3.85 (s,
3H), 2.62 (dd, J = 51.5, 26.0 Hz, 4H), 2.29 (d, J = 5.5 Hz, 1H). 13C NMR (75 MHz,
DMSO) δ 179.06, 172.22, 169.05, 160.64, 157.36, 153.15, 146.79, 141.10, 136.64,
132.68, 131.51, 130.72, 129.97, 128.63, 127.04, 126.16, 125.71, 122.92, 121.47,
120.44, 115.55, 114.83, 111.69, 64.09, 55.47, 49.69 30.58, 23.71. HRMS: [M+H+]:
Found m/z 585.1450, Calcd m/z 585.1397.
(S,E)-1-(5-([1,1'-biphenyl]-4-ylmethylene)-4-oxo-4,5-dihydrothiazol-2-yl)-N-(4-((3-c
hlorophenyl)amino)quinazolin-6-yl)pyrrolidine-2-carboxamide (8q)
yellow solid，yield：42%，Mp 203-204 oC. 1H NMR (400 MHz, CDCl3) δ 10.78 (s,
1H), 8.62 (s, 1H), 8.53 (s, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.95 (s, 1H), 7.83 (s, 1H),
7.74 – 7.66 (m, 3H), 7.62 (d, J = 8.6 Hz, 4H), 7.47 (t, J = 7.4 Hz, 2H), 7.40 (d, J = 7.5
Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.22 (s, 1H), 7.06 (d, J = 7.9 Hz, 1H), 5.48 (s, 1H),
4.12 (d, J = 6.9 Hz, 1H), 3.92 (s, 1H), 2.79 – 2.46 (m, 3H), 2.33 (s, 1H). 13C NMR
(101 MHz, DMSO) δ 178.80, 172.22, 168.96, 157.36, 153.16, 146.82, 141.38, 141.09,
138.99, 136.60, 132.78, 132.68, 130.26, 129.95, 129.08, 128.65, 128.47, 128.12,

127.39, 127.03, 126.78, 126.16, 122.92, 121.46, 120.42, 115.55, 111.72, 64.20, 49.76,
30.55, 23.70. HRMS: [M+Na+]: Found m/z 653.1495, Calcd m/z 653.1605.
4.6. General procedure for preparation, purification of EGFR inhibitory assay
A 1.6 kb cDNA encoded for the EGFR cytoplasmic domain (EGFR-CD, amino
acids 645-1186) were cloned into baculoviral expression vectors pBlueBacHis2B and
pFASTBacHTc (Huakang Company China), separately. A sequence that encodes
(His)6 was located at the 5’ upstream to the EGFR sequences. Sf-9 cells were infected
for 3 days for protein expression. Sf-9 cell pellets were solubilized at 0 °C in a buffer
at pH 7.4 containing 50 mM HEPES, 10 mM NaCl, 1% Triton, 10 µM ammonium
molybdate, 100 µM sodium vanadate, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 10
µg/mL pepstatin, and 16 µg/mL benzamidine HCl for 20 min followed by 20 min
centrifugation. Crude extract supernatant was passed through an equilibrated Ni-NTA
superflow packed column and washed with 10 mM and then 100 mM imidazole to
remove nonspecifically bound material. Histidinetagged proteins were eluted with 250
and 500 mM imidazole and dialyzed against 50 mM NaCl, 20 mM HEPES, 10%
glycerol, and 1 µg/mL each of aprotinin, leupeptin, and pepstatin for 2 h. The entire
purification procedure was performed at 4°C or on ice.18
The EGFR kinase assay was set up to assess the level of autophosphorylation
based on DELFIA/Time-Resolved Fluorometry. Compounds 8a-8d were dissolved in
100% DMSO and diluted to the appropriate concentrations with 25 mM HEPES at pH
7.4. In each well, 10 µL compound was incubated with 10µL (5 ng for EGFR)
recombinant enzyme (1:80 dilution in 100 mM HEPES) for 10 min at room
temperature. Then, 10 µL of 5 × buffer (containing 20 mM HEPES, 2 mM MnCl2,
100 µM Na3VO4, and 1 mM DTT) and 20 µL of 0.1 mM ATP-50 mM MgCl2 were
added for 1 h. Positive and negative controls were included in each plate by
incubation of enzyme with or without ATP-MgCl2. At the end of incubation, liquid
was aspirated, and plates were washed three times with wash buffer. A 75 µL (400 ng)
sample of europiumlabeled anti-phosphotyrosine antibody was added to each well for
another 1 h of incubation. After washing, enhancement solution was added and the
signal was detected by Victor (Wallac Inc.) with excitation at 340 nm and emission at

615 nm. The percentage of autophosphorylation inhibition by the compounds was
calculated using the following equation: 100% - [(negative control)/(positive control –
negative control)]. 15 The IC50 was obtained from curves of percentage inhibition with
eight concentrations of compound. As the contaminants in the enzyme preparation are
fairly low, the majority of the signal detected by the anti-phosphotyrosine antibody is
from EGFR.
4.7 Molecular Docking Modeling.
The three-dimensional structures of the aforementioned compounds were constructed
using Chem. 3D ultra 12.0 software [Chemical Structure Drawing Standard;
Cambridge Soft corporation, USA (2010)], then they were energetically minimized by
using MMFF94 with 5000 iterations and minimum RMS gradient of 0.10. The crystal
structures of EGFR kinase (PDB code: (1M17.pdb)) complex were retrieved from the
RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). All bound waters
and ligands were eliminated from the protein and the polar hydrogen was added to the
proteins. Molecular docking of all twenty compounds was then carried out using the
Discovery Studio (version 3.1) as implemented through the graphical user interface
CDocker protocol.
5. Acknowledgments
The work was supported by National Natural Science Foundation of China (Grant No.
81102316) and the Natural Science Foundation of the Jiangsu Higher Education
Institutions of China (Grant No. 11KJD350002).
References
1. Slamon, D. J.; Clark, G. M.; Wong, S. G.; Levin, W. J.; Ullrich, A.; McGuire, W.
L. Science 1987, 235, 177-182.
2. Traxler, P. M. Exp. Opin. Ther. Pat. 1997, 7, 571-588.
3. Fry, D. W. Annu. Rep. Med. Chem. 1996, 31, 151-160.
4. Fry, D.W.; Kraker, A. J.; McMichael, A.; Ambroso, L. A.; Nelson, J. M.;

Leopold, W. R.; Connors, R. W.; Bridges, A. J. Science. 1994, 265, 1093-1095.
5. Ward, W. H. J.; Cook, P. N.; Slater, A. M.; Davies, D. H.; Holdgate, G. A.; Green,
L. R. Biochem. Pharmacol. 1994, 48, 659-666.
6. Bridges, A. J.; Zhou, H.; Cody, D. R.; Rewcastle, G. W.; McMichael, A.;
Showalter, H. D. H.; Fry, D. W.; Kraker, A. J.; Denny, W. A. J. Med. Chem.
1996, 39, 267-276.
7. Bridges, A. J. Curr. Med.Chem. 1999, 6, 825-843.
8. Boschelli, D. H. Drugs of the Future 1999, 24, 515.-537
9. Iwata, K.; Miller, P. E.; Barbacci, E. G.; Arnold, L.; Doty, J.; DiOrio, C. I.;
Pustilnik, L. R.; Reynolds, M.; Thelemann, A.; Sloan, D.; Moyer, J. D. Proc.
Am. Assoc. Cancer Res. 1997, 38, 633 (Abstract 4248).
10. Woodburn, J. R.; Barker, A. J.; Gibson, K. H.; Ashton, S. E.; Wakeling, A. E.;
Curry, B. J.; Scarlett, L.; Henthorn, L. R. Proc. Am. Assoc. Cancer Res. 1997, 38,
633 (Abstract 4251).
11. Company Press Release, Feb 1, 2010: GSK’s TYKERB receives accelerated
approval for first-line combination treatment of hormone receptor positive,
HER2+/ErbB2+ metastatic breast cancer.
12. Li, S. N.; Li, H. Q. Exp. Opin. Ther. Pat. 2014, 24, 309-321.
13. Xu, Y. Y.; Li, S. N.; Yu, G. J.; Hu, Q. H.; Li, H. Q. Bioorg. Med. Chem. 2013, 21,
6084-6091.
14. Xu, Y. Y.; Gao, Y.; Ma, H. K.; Ao, G. Z.; Li, H. Q. Bioorg. Med. Chem. 2013, 21,
388-394.
15. Li, H. Q.; Li, D. D.; Lu, X.; Zhu, H. L. Bioorg. Med. Chem. 2012, 20, 317-323.
16. Li, H. Q.; Yang, J.; Ma, S. H.; Qiao, C. H. Bioorg. Med. Chem. 2012, 20,
4194-4200.
17. Chai, C. C. L.; Bernardo, P. H., Xu, J. WO 2010/024783 A1. US Patent.
18. Tsou, H.-R.; Mamuya, N.; Johnson, B. D.; Reich, M. F.; Gruber, B. C.; Ye, F.;
Nilakantan, R.; Shen, R.; Discafani, C.; DeBlanc, R.; Davis, R.; Koehn, F. E.;
Greenberger, L. M.; Wang, Y.-F.; Wissner, A. J. Med. Chem. 2001, 44, 2719.

Scheme 1 Synthesis of 4-(phenylamino)quinazoline 4
Scheme 2 Synthetic routes of target rhodanine-based 4-anilinoquinazoline derivatives
8a-8q

Table 1. In vitro antiproliferative effects of synthesized compoundsa 8a-8q
Compound
IC50(µM)
Compound
IC50(µM)
Hep G2 cells
A549 cellsa
6.1 1.5
＞50
Hep G2 cells
4.12 1.2
＞50
A549 cellsa
＞50
8a
8b
8c
8d
8e
8f
8j
8k
24.3 0.2
18.2 1.6
21.3 3.3
10.5 1.4
22.1 0.8
32.5 2.6
19.0 1.5
15.4 3.3
0.12 0.01
34.5 4.2
＞50
8.5 0.9
3.1 0.5
10.8 0.9
10.4 1.3
＞50
4.5 0.2
2.7 0.4
6.4 2.1
8.5 1.7
22.9 4.4
28.1 2.4
27.6 2.7
8l
8m
18.7 0.6
＞50
8n
8o
＞50
8g
8h
8i
8p
31.7 2.7
24.2 1.8
0.13 0.02
8q
＞50
＞50
gefitinib
a Cell growth was measured using the MTT assay. Values are the average of
two independent experiments run in triplicate. Variation was generally 1%.
Table 2 Inhibition (IC50 ) of EGFR kinase
Compound
EGFR inhibition IC50(µM)
8.1 2.1
8a
8b
8c
8d
8e
8f
31.4 5.2
6.5 1.2
3.1 0.8
7.4 1.6
9.8 2.2
8m
8q
7.6 1.7
13.2 3.8

Figure 1. Molecular docking modeling of compounds 8d with EGFR
kinase(1M17.pdb): N atom of 4-anilinoquinazoline moiety of 8d forms hydrogen
bond with the MET769, the carbonyl group of rhodanine moiety forms hydrogen
bond with the PRO770

Combination of 4-anilinoquinazoline and rhodanine as novel
Epidermal Growth Factor Receptor tyrosine kinase
inhibitors
Si-Ning Li, Yun-Yun Xu, Hong-ran Yin, Gui-zhen Ao, Huan-Qiu Li*.
College of Pharmaceutical Science, Soochow University, Suzhou 215123, PR China.;
*Corresponding author. Tel.: +86 512 65882090; fax.: +86 512 65882090; e-mail:
huanqiuli@suda.edu.cn