Design, synthesis and biological evaluation of thiazolidinone derivatives as potential EGFR and HER-2 kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.10.051

Two series of thiazolidinone derivatives designing for potential EGFR and HER-2 kinase inhibitors have been discovered. Some of them exhibited significant EGFR and HER-2 inhibitory activity. Compound 2-(2-(5-bromo-2-hydroxybenzylidene)hydrazinyl)thiazol-4(5H)-one (12) displayed the most potent inhibitory activity (IC₅₀ = 0.09 μM for EGFR and IC₅₀ = 0.42 μM for HER-2), comparable to the positive control erlotinib. Docking simulation was performed to position compound 12 into the EGFR active site to determine the probable binding model. Antiproliferative assay results indicating that some of the thiazolidinone derivatives own high antiproliferative activity against MCF-7. Compound 12 with potent inhibitory activity in tumor growth inhibition would be a potential anticancer agent.

Full text of DOI:10.1016/j.bmc.2009.10.051

Bioorganic & Medicinal Chemistry 18 (2010) 314–319
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of thiazolidinone derivatives
as potential EGFR and HER-2 kinase inhibitors
Peng-Cheng Lv, Chang-Fang Zhou, Jin Chen, Peng-Gang Liu, Kai-Rui Wang, Wen-Jun Mao,
*
Huan-Qiu Li, Ying Yang, Jing Xiong, Hai-Liang Zhu
State Key Laboratory of Pollution Control and Resource Reuse, State Key Laboratory of Pharmaceutical Biotechnology,
Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of thiazolidinone derivatives designing for potential EGFR and HER-2 kinase inhibitors have
been discovered. Some of them exhibited significant EGFR and HER-2 inhibitory activity. Compound 2-
(2-(5-bromo-2-hydroxybenzylidene)hydrazinyl)thiazol-4(5H)-one (12) displayed the most potent inhibitory
Received 27 August 2009
Revised 26 October 2009
Accepted 27 October 2009
Available online 30 October 2009
activity (IC50 = 0.09 lM for EGFR and IC50 = 0.42 lM for HER-2), comparable to the positive control erloti-
nib. Docking simulation was performed to position compound 12 into the EGFR active site to determine
the probable binding model. Antiproliferative assay results indicating that some of the thiazolidinone
derivatives own high antiproliferative activity against MCF-7. Compound 12 with potent inhibitory activ-
ity in tumor growth inhibition would be a potential anticancer agent.
Keywords:
Thiazolidinone derivatives
SAR
EGFR
Ó 2009 Elsevier Ltd. All rights reserved.
HER-2
Inhibitors
Anticancer
1. Introduction
has been most thoroughly studied in breast cancer, where it is
overexpressed in 25–30% of cases and is correlated with a poor
Cancer chemotherapy has entered a new era of molecularly
targeted therapeutics, which is highly selective and not associated
with the serious toxicities of conventional cytotoxic drugs.1 The
first group of these novel anticancer drugs is those targeting mu-
tant or aberrantly expressed oncogenic growth factor receptor and
non-receptor tyrosine kinases involved in mitogenic or prolifera-
tive signal transduction pathways in cancer cells.2 Receptor pro-
tein tyrosine kinases play a key role in signal transduction
pathways that regulate cell division and differentiation. Among
the growth factor receptor kinases that have been identified as
being important in cancer is epidermal growth factor receptor
(EGFR) kinase (also known as erb-B1 or HER-1) and the related
human epidermal growth factor receptor HER-2 (also known as
erbB-2). Deregulation of growth-factor signaling due to hyperacti-
vation of the ErbB receptors (primarily EGFR and HER-2) is seen in
several cancer types.3,4 Activation of EGFR may be because of
overexpression, mutations resulting in constitutive activation, or
autocrine expression of ligand. In contrast, activation of HER-2 oc-
curs mainly by overexpression, which leads to spontaneous
homodimerization and activation of downstream signaling events
in a ligand-independent manner.5,6 The role of EGFR and HER-2
prognosis. EGFR and HER-2 overexpression is also seen in ovarian
cancer,7 lung cancer (especially lung adenocarcinomas)8–10 and in
hormone-refractory prostate cancer.11 Compounds that inhibit the
kinase activity of EGFR and/or HER-2 after binding of its cognate
ligand are of potential interest as new therapeutic antitumor
agents.12,13
Thiazolidinone derivatives have been investigated for a range
of pharmacologic indications such as anti-inflammatory,14 anti-
microbial,15 antiproliferative,16,17 antiviral,18 anticonvulsant,19,20
antifungal,21 and antibacterial22 activities but their anticancer ef-
fects have been less widely documented.23 Since the tyrosine
phosphorylation event catalyzed by EGFR or HER-2 propagates
the signal for cell division and since deregulation of these kinases
has been associated with disease, an inhibitor of this event may
have potential therapeutic value. Moreover, these two receptor
kinases have a high sequence homology in their catalytic do-
mains. In view of the facts above mentioned, in this study, we
describe the synthesis and the SAR of two series of thiazolidi-
none derivatives. Biological evaluation indicated that some of
the synthesized compounds are potent inhibitors of EGFR and
HER-2. Docking simulations were performed using the X-ray
crystallographic structure of the EGFR in complex with an inhib-
itor to explore the binding modes of these compounds at the ac-
tive site.
* Corresponding author. Tel./fax: +86 25 8359 2672?
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.10.051

P.-C. Lv et al. / Bioorg. Med. Chem. 18 (2010) 314–319
315
2. Results and discussion
2.1. Chemistry
n
i
n
H
N
NH2
N
O
The synthesis of compounds 3–21 followed the general path-
way outlined in Scheme 1. Firstly, to a stirred solution of thiosem-
icarbazide in ethanol were added substituted aldehydes. The
mixture was refluxing for 2–6 h. Then, thiosemicarbazones (com-
pound 2) were obtained with yields of 76–85%. In the next step,
treatment of thiosemicarbazones with ethyl bromoacetate in
refluxing ethanol containing a catalytic amount of freshly fused so-
dium acetate afforded the thiazolidinone derivatives 3–21. Simi-
larly, the synthesis of compounds 24–28 followed the general
pathway outlined in Scheme 2. In this series, cyclobutanone and
its analogues were introduced to react with thiosemicarbazide in-
stead of substituted aldehydes. The mixture was refluxing for 4–
8 h. Then, the thiosemicarbazones (compound 23) were obtained
with yields of 75–87%. Subsequently, compounds 24–28 were ob-
tained by treatment of thiosemicarbazones with ethyl bromoace-
tate in refluxing ethanol containing a catalytic amount of freshly
fused sodium acetate. All of the synthetic compounds gave satis-
factory analytical and spectroscopic data, which were in full accor-
dance with their depicted structures.
23
22
S
N
n
H
ii
N
N
O
S
24-28
24 n= 1
25 n= 2
26 n= 3
27 n= 4
28 n= 5
Scheme 2. General synthesis of compounds 24–28. Reagents and conditions: (i)
thiosemicarbazide, 2-propanol, acetic acid; (ii) BrCH2COOEt, AcONa, EtOH, reflux.
2 kinases using a solid-phase ELISA assay. The results were sum-
marized in Table 1. For the given compounds, we observed that
the IC50 value for inhibition of HER-2 kinase is, in general, higher
than that observed for EGFR kinase but have the same trends. This
is possibly due, in part, to the fact that in the enzyme assays we
used higher concentration of the purified HER-2 kinase than EGFR
kinase. It is evident that there is also a reasonable correlation be-
2.2. Biological activity
The synthesized thiazolidinone derivatives were evaluated for
their ability to inhibit the autophosphorylation of EGFR and HER-
R2
R2
R3
R4
R1
R3
R4
R1
i
S
H
H
N
N
O
NH2
2
1
R2
R3
R4
R1
ii
O
N
S
H
N
N
3~21
3 R1= H, R2= H, R3= F, R4= H
4 R1= H, R2= H, R3= Cl, R4= H
5 R1= H, R2= H, R3= Br, R4= H
6 R1= H, R2= H, R3= CH3, R4= H
7 R1= H, R2= H, R3= OCH3, R4= H
8 R1= H, R2= H, R3= NO2, R4= H
9 R1= H, R2= H, R3= OH, R4= H
10 R1= OH, R2= H, R3= H, R4= F
11 R1= OH, R2= H, R3= H, R4= Cl
12 R1= OH, R2= H, R3= H, R4= Br
13 R1= OH, R2= Cl, R3= H, R4= Cl
14 R1= OH, R2= Br, R3= H, R4= Br
15 R1= H, R2= F, R3= H, R4= H
16 R1= H, R2= Cl, R3= H, R4= H
17 R1= H, R2= Br, R3= H, R4= H
18 R1= H, R2= CH3, R3= H, R4= H
19 R1= H, R2= OCH3, R3= H, R4= H
20 R1= H, R2= NO2, R3= H, R4= H
21 R1= H, R2= OH, R3= H, R4= H
Scheme 1. General synthesis of compounds 3–21. Reagents and conditions: (i) thiosemicarbazide, 2-propanol, acetic acid; (ii) BrCH2COOEt, AcONa, EtOH, reflux.

316
P.-C. Lv et al. / Bioorg. Med. Chem. 18 (2010) 314–319
Table 1
Inhibition (IC50) of EGFR and HER-2 kinases and inhibition (IC50) of cell proliferation
Compounds
Enzyme assays (IC50
,
l
M)
MCF-7 (IC50, lM)
EGFR
HER-2
3
4
5
6
7
8
9
2.18
1.94
1.72
3.26
5.78
6.82
4.71
0.97
0.56
0.09
1.46
1.24
13.53
10.78
7.46
23.49
31.25
26.61
19.32
>50
3.89
2.75
2.43
5.83
9.17
8.46
6.93
3.59
1.78
0.42
4.51
3.73
21.75
14.34
10.26
31.78
45.51
37.64
27.83
>50
1.58 0.24
1.82 0.45
0.97 0.08
2.05 0.17
3.16 0.84
2.77 0.56
1.67 0.34
0.27 0.09
0.18 0.05
0.06 0.02
1.56 0.18
0.62 0.12
7.78 0.43
4.31 0.59
2.42 0.12
8.19 0.68
9.73 1.12
12.57 2.41
11.79 1.97
46.3 4.78
38.5 3.65
32.1 3.29
27.6 2.94
18.4 1.78
10
11
12
13
14
15
16
17
18
19
20
21
24
25
26
27
28
Erlotinib
Figure 1. Molecular docking modeling of compound 12 with EGFR kinase: the
hydroxyl group of compound 12 forms hydrogen bond with mercapto group of Met
769, and the nitrogen atom of thiazolidinone ring of compound 12 also forms
hydrogen bond with the side chain mercapto group of Cys 751.
Besides, the in vitro antiproliferative activity of the synthesized
thiazolidinone derivatives was studied on a panel of one human tu-
mor cell line (MCF-7), which overexpresses EGFR and, to a less ex-
tent HER-2. Compounds 10–14, which have potent inhibitory
activity of EGFR and HER-2 showed high antiproliferative activity
>50
41
36
27
>50
>50
47
31
0.03
0.16
0.02 0.005
against MCF-7 with IC50 ranging from 0.06 to 1.56 lM, indicating
that these thiazolidinone derivatives were potent inhibitor of EGFR
and HER-2 as antitumor agents. In particular, compound 12 has
demonstrated significant inhibitory activity in tumor growth inhi-
bition and displayed favorable EGFR and HER-2 inhibitory activity.
tween the EGFR and HER-2 inhibitory activities; thus, this is not
surprising in view of the high sequence homology of the catalytic
domains of these two kinases. As shown in Table 1, a number of
thiazolidinone derivatives exhibited significant EGFR and HER-2
inhibitory activity. Among them, compound 12 displayed the
3. Conclusions
most potent inhibitory activity (IC50 = 0.09
IC50 = 0.42 M for HER-2), comparable to the positive control erl-
otinib (IC50 = 0.03 M for EGFR).
lM for EGFR and
Two series of thiazolidinone derivatives that may function as
inhibitors of EGFR and HER-2 kinases have been prepared, and
some of the synthesized compounds displayed potent EGFR and
HER-2 inhibitory. Compound 12 displayed the most potent inhibi-
l
l
Structure–activity relationships in these thiazolidinone deriva-
tives demonstrated that compounds with aromatic ring (com-
pounds 3–21) showed better inhibitory activity than those with
aliphatic ring (compounds 24–28). Among the former, com-
pounds with substitution on the 3-position of benzene ring
(compounds 15–21) displayed less inhibitory activity than those
with 4-position (compounds 3–9). Besides, compounds 10–14
exhibited potent EGFR inhibitory activity with IC50 ranging from
tory activity (IC50 = 0.09 lM for EGFR and IC50 = 0.42 lM for HER-
2), comparable to the positive control erlotinib. The EGFR molecu-
lar docking model suggested that compound 12 is nicely bound to
the region with the hydroxyl group project toward the mercapto
group of Met 769, with the hydroxyl group forming a more optimal
H-bond interaction, and nitrogen atom of thiazolidinone ring of
compound 12 also forms hydrogen bond with the side chain mer-
capto group of Cys 751. Antiproliferative assay results indicated
that these thiazolidinone derivatives own high antiproliferative
activity against MCF-7; In particular, compound 12 has demon-
strated significant EGFR and HER-2 inhibitory activity and inhibi-
tory activity in tumor growth inhibition as a potential anticancer
agent.
0.09 to 1.46 lM. All of them have one hydroxyl group on the 2-
position of benzene ring. Among them, a comparison of the sub-
stitution on benzene ring demonstrated that 4-position-halogen-
substituted derivatives (compounds 10–12) have more potent
EGFR inhibitory activity than others (compounds 13 and 14),
and the potency order is F < Cl < Br. Compound 12 displayed
the most potent EGFR inhibitory activity with IC50 of 0.09
which was comparable to the positive control.
lM,
4. Experimental section
4.1. Chemistry
In addition, in order to help understand the SARs observed at
the EGFR and guide further SAR studies, molecular docking of the
most potent inhibitor 12 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
12 and EGFR is depicted in Figure 1. In the binding model, com-
pound 12 is nicely bound to the region with the hydroxyl group
project toward the mercapto group of Met 769, with the
hydroxyl group forming a more optimal H-bond interaction,
and nitrogen atom of thiazolidinone ring of compound 12 also
forms hydrogen bond with the side chain mercapto group of
Cys 751. This molecular docking result, along with the enzyme
assay data, suggesting that compound 12 is a potential inhibitor
of EGFR.
Separation of the compounds by column chromatography was
carried out with Silica Gel 60 (200–300 mesh ASTM, E. Merck).
The quantity of silica gel used was 50–100 times the weight
charged on the column. Then, the eluates were monitored using
TLC. Melting points (uncorrected) were determined on a XT4 MP
apparatus (Taike Corp., Beijing, China). ESI mass spectra were ob-
tained on a Mariner System 5304 mass spectrometer, and 1H
NMR spectra were recorded on a Bruker PX500 or DPX300 spec-
trometer at 25 °C with TMS and solvent signals allotted as internal
standards. Chemical shifts were reported in ppm (d). Elemental

P.-C. Lv et al. / Bioorg. Med. Chem. 18 (2010) 314–319
317
analyses were performed on a CHN–O-Rapid instrument and were
within 0.4% of the theoretical values.
4.2.7. 2-(2-(4-Hydroxybenzylidene)hydrazinyl)thiazol-4(5H)-
one (9)
White powder. Yield: 75%; mp: 149–151 °C; 1H NMR (300 MHz,
DMSO-d6): 4.46 (s, 2H); 7.27–7.29 (d, J = 7.86 Hz, 2H); 7.69–7.71(d,
J = 8.23 Hz, 2H); 8.41 (s, 1H); 10.78 (s, 1H). MS (ESI): 236.0
(C10H10N3O2S, [M+H]+). Anal. Calcd for C10H9N3O2S: C, 51.05; H,
3.86, N, 17.86. Found: C, 51.18; H, 3.64; N, 17.64.
4.2. General method of synthesis of compounds 3–21
To a solution of compound 1 (0.01 mol) in ethanol (20 mL) was
added an equimolar amount of thiosemicarbazide (0.91 g,
0.01 mol). The reaction mixture was heated under reflux for 2–
6 h, partially concentrated and cooled. The separated solid product
was filtered off, dried, and recrystallized from ethanol to give com-
pound 2. A mixture of compound 2 (0.005 mol), ethyl bromoace-
tate (0.83 g, 0.005 mol), anhydrous sodium acetate (1.64 g,
0.02 mol), and absolute ethanol (30 mL) was refluxed for 8–12 h.
The product obtained upon cooling was collected by filtration,
washed with water, dried, and recrystallized from a mixture of
EtOH to give compounds 3–21.
4.2.8. 2-(2-(5-Fluoro-2-hydroxybenzylidene)hydrazinyl)thiazol-
4(5H)-one (10)
White powder. Yield: 78%; mp: 157–159 °C; 1H NMR
(300 MHz, DMSO-d6): 4.53 (s, 2H); 6.94–6.96 (d, J = 8.51 Hz,
1H); 7.31–7.34(m, 1H); 7.63–7.65 (m, 1H); 8.69 (s, 1H); 10.79
(s, 1H). MS (ESI): 254.0 (C10H9FN3O2S, [M+H]+). Anal. Calcd for
C10H8FN3O2S: C, 47.43; H, 3.18; N, 16.59. Found: C, 47.57; H,
3.32; N, 16.73.
4.2.1. 2-(2-(4-Fluorobenzylidene)hydrazinyl)thiazol-4(5H)-one
(3)
4.2.9. 2-(2-(5-Chloro-2-hydroxybenzylidene)hydrazinyl)
thiazol-4(5H)-one (11)
White powder. Yield: 80%; mp: 134–136 °C; 1H NMR (300 MHz,
DMSO-d6): 4.49 (s, 2H); 7.26–7.28 (d, J = 7.84 Hz, 2H); 7.65–7.67
(d, J = 8.26 Hz, 2H); 8.38 (s, 1H). MS (ESI): 238.0 (C10H9FN3OS,
[M+H]+). Anal. Calcd for C10H8FN3OS: C, 50.62; H, 3.40; N, 17.71.
Found: C, 50.87; H, 3.27; N, 17.59.
White powder. Yield: 75%; mp: 163–165 °C; 1H NMR
(300 MHz, DMSO-d6): 4.51 (s, 2H); 6.97–6.98 (d, J = 8.55 Hz,
1H); 7.36–7.38(m, 1H); 7.68–7.69 (m, 1H); 8.71 (s, 1H); 10.81
(s, 1H). MS (ESI): 270.0 (C10H9ClN3O2S, [M+H]+). Anal. Calcd for
C10H8ClN3O2S: C, 44.53; H, 2.99; N, 15.58. Found: C, 44.71; H,
3.12; N, 15.67.
4.2.2. 2-(2-(4-Chlorobenzylidene)hydrazinyl)thiazol-4(5H)-one
(4)
4.2.10. 2-(2-(5-Bromo-2-hydroxybenzylidene)hydrazinyl)
thiazol-4(5H)-one (12)
White powder. Yield: 83%; mp: 138–139 °C; 1H NMR (300 MHz,
DMSO-d6): 4.47 (s, 2H); 7.29–7.31 (d, J = 7.86 Hz, 2H); 7.70–7.72
(d, J = 8.29 Hz, 2H); 8.41 (s, 1H). MS (ESI): 254.0 (C10H9ClN3OS,
[M+H]+). Anal. Calcd for C10H8ClN3OS: C, 47.34; H, 3.18; N, 16.56.
Found: C, 47.12; H, 3.31; N, 16.73.
White powder. Yield: 80%; mp: 169–170 °C; 1H NMR (300 MHz,
DMSO-d6): 4.50 (s, 2H); 6.92–6.93 (d, J = 8.53 Hz, 1H); 7.48–7.49
(m, 1H); 7.80–7.81 (m, 1H); 8.62 (s, 1H); 10.84 (s, 1H). MS (ESI):
313.9 (C10H9BrN3O2S, [M+H]+). Anal. Calcd for C10H8BrN3O2S: C,
38.23; H, 2.57; N, 13.38. Found: C, 38.47; H, 2.78; N, 13.46.
4.2.3. 2-(2-(4-Bromobenzylidene)hydrazinyl)thiazol-4(5H)-one
(5)
4.2.11. 2-(2-(3,5-Dichloro-2-hydroxybenzylidene)hydrazinyl)
thiazol-4(5H)-one (13)
White powder. Yield: 78%; mp: 132–134 °C; 1H NMR (300 MHz,
DMSO-d6): 4.45 (s, 2H); 7.23–7.26 (d, J = 7.81 Hz, 2H); 7.64–7.67
(d, J = 8.24 Hz, 2H); 8.38 (s, 1H). MS (ESI): 297.0 (C10H9BrN3OS,
[M+H]+). Anal. Calcd for C10H8BrN3OS: C, 40.28; H, 2.70; N, 14.09.
Found: C, 40.39; H, 2.56; N, 14.16.
White powder. Yield: 77%; mp: 161–163 °C; 1H NMR (300 MHz,
DMSO-d6): 4.52 (s, 2H); 7.69–7.70 (m, 1H); 7.80 (s, 1H); 8.73 (s,
1H); 11.63 (s, 1H). MS (ESI): 303.9 (C10H8Cl2N3O2S, [M+H]+). Anal.
Calcd for C10H7Cl2N3O2S: C, 39.49; H, 2.32; N, 13.82. Found: C,
39.23; H, 2.48; N, 13.74.
4.2.4. 2-(2-(4-Methylbenzylidene)hydrazinyl)thiazol-4(5H)-one
(6)
4.2.12. 2-(2-(3,5-Dibromo-2-hydroxybenzylidene)hydrazinyl)
thiazol-4(5H)-one (14)
White powder. Yield: 82%; mp: 125–127 °C; 1H NMR (300 MHz,
DMSO-d6): 2.35 (s, 3H); 4.49 (s, 2H); 7.27–7.29 (d, J = 7.86 Hz, 2H);
7.66–7.68 (d, J = 8.25 Hz, 2H); 8.39 (s, 1H). MS (ESI): 234.0
(C11H12N3OS, [M+H]+). Anal. Calcd for C11H11N3OS: C, 56.63; H,
4.75; N, 18.01. Found: C, 56.76; H, 4.61; N, 18.19.
White powder. Yield: 79%; mp: 173–175 °C; 1H NMR (300 MHz,
DMSO-d6): 4.51 (s, 2H); 7.64–7.66 (m, 1H); 7.83 (s, 1H); 8.71 (s,
1H); 11.80 (s, 1H). MS (ESI): 391.8 (C10H8Br2N3O2S, [M+H]+). Anal.
Calcd for C10H7Br2N3O2S: C, 30.56; H, 1.80; N, 10.69. Found: C,
30.74; H, 1.98; N, 10.83.
4.2.5. 2-(2-(4-Methoxybenzylidene)hydrazinyl)thiazol-4(5H)-
one (7)
4.2.13. 2-(2-(3-Fluorobenzylidene)hydrazinyl)thiazol-4(5H)-
one (15)
White powder. Yield: 84%; mp: 129–131 °C; 1H NMR (300 MHz,
DMSO-d6): 3.76 (s, 3H); 4.47 (s, 2H); 7.25–7.27 (d, J = 7.81 Hz, 2H);
7.64–7.67 (d, J = 8.26 Hz, 2H); 8.37 (s, 1H). MS (ESI): 250.0
(C11H12N3O2S, [M+H]+). Anal. Calcd for C11H11N3O2S: C, 53.00; H,
4.45; N, 16.86. Found: C, 53.21; H, 4.63; N, 16.97.
White powder. Yield: 74%; mp: 177–178 °C; 1H NMR (300 MHz,
DMSO-d6): 4.48 (s, 2H); 7.37 (s, 1H); 7.53–7.56 (m, 1H); 7.63–7.66
(m, 2H); 8.41 (s, 1H). MS (ESI): 238.0 (C10H9FN3OS, [M+H]+). Anal.
Calcd for C10H8FN3OS: C, 50.62; H, 3.40; N, 17.71. Found: C, 50.78;
H, 3.57; N, 17.63.
4.2.6. 2-(2-(4-Nitrobenzylidene)hydrazinyl)thiazol-4(5H)-one
(8)
4.2.14. 2-(2-(3-Chlorobenzylidene)hydrazinyl)thiazol-4(5H)-
one (16)
White powder. Yield: 77%; mp: 145–146 °C; 1H NMR (300 MHz,
DMSO-d6): 4.48 (s, 2H); 7.35–7.37 (d, J = 7.83 Hz, 2H); 7.73–7.75(d,
J = 8.27 Hz, 2H); 8.46 (s, 1H). MS (ESI): 265.0 (C10H9N4O3S, [M+H]+).
Anal. Calcd for C10H8N4O3S: C, 45.45; H, 3.05; N, 21.20. Found: C,
45.67; H, 3.24; N, 21.34.
White powder. Yield: 72%; mp: 182–184 °C; 1H NMR (300 MHz,
DMSO-d6): 4.49 (s, 2H); 7.39 (s, 1H); 7.56–7.59 (m, 1H); 7.67–7.69
(m, 2H); 8.45 (s, 1H). MS (ESI): 254.0 (C10H9ClN3OS, [M+H]+). Anal.
Calcd for C10H8ClN3OS: C, 47.34; H, 3.18; N, 16.56. Found: C, 47.51;
H, 3.29; N, 16.79.

318
P.-C. Lv et al. / Bioorg. Med. Chem. 18 (2010) 314–319
4.2.15. 2-(2-(3-Bromobenzylidene)hydrazinyl)thiazol-4(5H)-
one (17)
4.3.3. 2-(2-Cyclohexylidenehydrazinyl)thiazol-4(5H)-one (26)
White powder. Yield: 81 %; mp: 86–88 °C; 1H NMR (300 MHz,
DMSO-d6): 1.53–1.56 (m, 4H); 1.63–1.64 (m, 2H); 2.26–2.28 (m,
2H); 2.49–2.52 (m, 2H); 4.41 (s, 2H). MS (ESI): 211.0 (C9H14N3OS,
[M+H]+). Anal. Calcd for C9H13N3OS: C, 51.16; H, 6.20. Found: C,
51.31; H, 6.43.
White powder. Yield: 76%; mp: 179–181 °C; 1H NMR (300 MHz,
DMSO-d6): 4.47 (s, 2H); 7.36 (s, 1H); 7.51–7.55 (m, 1H); 7.62–7.67
(m, 2H); 8.43 (s, 1H). MS (ESI): 297.9 (C10H9BrN3OS, [M+H]+). Anal.
Calcd for C10H8BrN3OS: C, 40.28; H, 2.70; N, 14.09. Found: C, 40.42;
H, 2.97; N, 14.18.
4.3.4. 2-(2-Cycloheptylidenehydrazinyl)thiazol-4(5H)-one (27)
White powder. Yield: 83 %; mp: 89–90 °C; 1H NMR (300 MHz,
DMSO-d6): 1.50–1.54 (m, 4H); 1.68–1.69 (m, 2H); 2.17–2.18 (m,
2H); 2.24–2.26 (m, 2H); 2.45–2.48 (m, 2H); 4.43 (s, 2H). MS
(ESI): 226.0 (C10H16N3OS, [M+H]+). Anal. Calcd for C10H15N3OS: C,
53.31; H, 6.71; N, 19.89. Found: C, 53.46; H, 6.84; N, 19.76.
4.2.16. 2-(2-(3-Methylbenzylidene)hydrazinyl)thiazol-4(5H)-
one (18)
White powder. Yield: 70%; mp: 188–189 °C; 1H NMR (300 MHz,
DMSO-d6): 2.36 (s, 3H); 4.46 (s, 2H); 7.34 (s, 1H); 7.53–7.57 (m,
1H); 7.64–7.69 (m, 2H); 8.47 (s, 1H). MS (ESI): 234.0 (C11H12N3OS,
[M+H]+). Anal. Calcd for C11H11N3OS: C, 56.63; H, 4.75; N, 18.01.
Found: C, 56.82; H, 4.56; N, 18.15.
4.3.5. 2-(2-Cyclooctylidenehydrazinyl)thiazol-4(5H)-one (28)
White powder. Yield: 78 %; mp: 92–94 °C; 1H NMR (300 MHz,
DMSO-d6): 1.56–1.59 (m, 4H); 1.64–1.68 (m, 2H); 1.92–1.95 (m,
2H); 2.14–2.16 (m, 2H); 2.28–2.30 (m, 2H); 2.41–2.44 (m, 2H);
4.41 (s, 2H). MS (ESI): 239.1 (C11H18N3OS, [M+H]+). Anal. Calcd
for C11H17N3OS: C, 55.20; H, 7.16; N, 17.56. Found: C, 55.42; H,
7.34; N, 17.72.
4.2.17. 2-(2-(3-Methoxybenzylidene)hydrazinyl)thiazol-4(5H)-
one (19)
White powder. Yield: 72%; mp: 185–187 °C; 1H NMR (300 MHz,
DMSO-d6): 3.76 (s, 3H); 4.43 (s, 2H); 7.36 (s, 1H); 7.57–7.61 (m,
1H); 7.72–7.75 (m, 2H); 8.51 (s, 1H). MS (ESI): 250.0 (C11H12N3O2S,
[M+H]+). Anal. Calcd for C11H11N3O2S: C, 53.00; H, 4.45; N, 16.86.
Found: C, 53.23; H, 4.64; N, 16.67.
4.4. Preparation, and purification of HER-2 and EGFR and
inhibitory assay
4.2.18. 2-(2-(3-Nitrobenzylidene)hydrazinyl)thiazol-4(5H)-one
(20)
A 1.7 Kb cDNA encoded for human HER-2 cytoplasmic domain
(HER-2-CD, amino acids 676–1245) and 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 se-
quence that encodes (His)6 was located at the 50 upstream to the
HER-2 and 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,
White powder. Yield: 78%; mp: 191–193 °C; 1H NMR (300 MHz,
DMSO-d6): 4.45 (s, 2H); 7.39 (s, 1H); 7.66–7.68 (m, 1H); 7.75–7.78
(m, 2H); 8.53 (s, 1H). MS (ESI): 265.0 (C10H9N4O3S, [M+H]+). Anal.
Calcd for C10H8N4O3S: C, 45.45; H, 3.05; N, 21.20. Found: C,
45.62; H, 3.18; N, 21.37.
4.2.19. 2-(2-(3-Hydroxybenzylidene)hydrazinyl)thiazol-4(5H)-
one (21)
White powder. Yield: 77%; mp: 196–197 °C; 1H NMR (300 MHz,
DMSO-d6): 4.41 (s, 2H); 7.33 (s, 1H); 7.57–7.61 (m, 1H); 7.67–7.69
(m, 2H); 8.57 (s, 1H); 10.78 (s, 1H). MS (ESI): 236.0 (C10H10N3O2S,
[M+H]+). Anal. Calcd for C10H9N3O2S: C, 51.05; H, 3.86; N, 17.86.
Found: C, 51.19; H, 3.78; N, 17.64.
10
l
M ammonium molybdate, 100
g/mL leupeptin, 10
l
M sodium vanadate, 10
l
l
g/mL
g/mL
aprotinin, 10
l
lg/mL pepstatin, and 16
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 imid-
azole and dialyzed against 50 mM NaCl, 20 mM HEPES, 10% glyc-
4.3. General method of synthesis of compounds 24–28
To a solution of compound 22 (0.01 mol) in ethanol (20 mL) was
added an equimolar amount of thiosemicarbazide (0.91 g,
0.01 mol). The reaction mixture was heated under reflux for 2–
6 h, partially concentrated and cooled. The separated solid product
was filtered off, dried, and recrystallized from ethanol to give com-
pound 23. A mixture of compound 23 (0.005 mol), ethyl bromoace-
tate (0.83 g, 0.005 mol), anhydrous sodium acetate (1.64 g,
0.02 mol), and absolute ethanol (30 mL) was refluxed for 8–12 h.
The product obtained upon cooling was collected by filtration,
washed with water, dried, and recrystallized from a mixture of
EtOH to give compounds 24–28.
erol, and 1 lg/mL each of aprotinin, leupeptin, and pepstatin for
2 h. The entire purification procedure was performed at 4 °C or
on ice.24
Both EGFR and HER-2 kinase assays were set up to assess the le-
vel of autophosphorylation based on DELFIA/Time-Resolved Fluo-
rometry. Compounds 3–21 and 24–28 were dissolved in 100%
DMSO and diluted to the appropriate concentrations with 25 mM
HEPES at pH 7.4. In each well, 10
with 10 L (12.5 ng for HER-2 or 5 ng for EGFR) of recombinant en-
zyme (1:80 dilution in 100 mM HEPES) for 10 min at room temper-
ature. Then, 10
L of 5 ꢀ buffer (containing 20 mM HEPES, 2 mM
MnCl2, 100 M Na3VO4, and 1 mM DTT) and 20 L of 0.1 mM
lL of compound was incubated
l
l
l
l
4.3.1. 2-(2-Cyclobutylidenehydrazinyl)thiazol-4(5H)-one (24)
White powder. Yield: 80%; mp: 80–82 °C; 1H NMR (300 MHz,
DMSO-d6): 1.65–1.69 (m, 4H); 2.24–2.27 (m, 2H); 4.41 (s, 2H).
MS (ESI): 184.0 (C7H10N3OS, [M+H]+). Anal. Calcd for C7H9N3OS:
C, 45.88; H, 4.95; N, 22.93. Found: C, 45.64; H, 4.82; N, 22.82.
ATP-50 mM MgCl2 was added for 1 h. Positive and negative con-
trols were included in each plate by incubation of enzyme with
or without ATP–MgCl2. At the end of incubation, liquid was aspi-
rated, and plates were washed three times with wash buffer. A
75 lL (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)]. The IC50 was obtained from curves of percentage
4.3.2. 2-(2-Cyclopentylidenehydrazinyl)thiazol-4(5H)-one (25)
White powder. Yield: 84 %; mp: 82–83 °C; 1H NMR (300 MHz,
DMSO-d6): 1.67–1.72 (m, 4H); 2.31–2.39 (m, 4H); 4.40 (s, 2H).
MS (ESI): 197.0 (C8H12N3OS, [M+H]+). Anal. Calcd for C8H11
N3OS: C, 48.71; H, 5.62; N, 21.30. Found: C, 48.58; H, 5.75; N,
21.45.

P.-C. Lv et al. / Bioorg. Med. Chem. 18 (2010) 314–319
319
inhibition with eight concentrations of compound. As the contam-
inants in the enzyme preparation are fairly low, the majority of the
signal detected by the anti-phosphotyrosine antibody is from EGFR
or HER-2.
Major Projects on Control and Rectification of Water Body Pollu-
tion (Water Special Project) (No. 2008ZX07526-001).
References and notes
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Molecular docking of compound 12 into the three-dimensional
EGFR complex structure (1M17.pdb, downloaded from the PDB)
was carried out using the AutoDock software package (version
4.0) as implemented through the graphical user interface Auto-
DockTools (ADT 1.4.6).
Acknowledgment
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This work was supported by National Basic Research Programs
(973) of China (No. 2008CB418004 & No. 2008CB418201) and the