Design, modification and 3D QSAR studies of novel naphthalin-containing pyrazoline derivatives with/without thiourea skeleton as anticancer agents

Article abstract of DOI:10.1016/j.bmc.2013.01.013

Two series of novel naphthalin-containing pyrazoline derivatives C1-C14 and D1-D14 have been synthesized and evaluated for their EGFR/HER-2 inhibitory and anti-proliferation activities. Compound D14 displayed the most potent activity against EGFR and A549

Full text of DOI:10.1016/j.bmc.2013.01.013

Bioorganic & Medicinal Chemistry 21 (2013) 1050–1063
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, modification and 3D QSAR studies of novel
naphthalin-containing pyrazoline derivatives with/without
thiourea skeleton as anticancer agents
Wen Yang a, , Yang Hu a, , Yu-Shun Yang a, Fei Zhang a, Yan-Bin Zhang a, Xiao-Liang Wang a,
Jian-Feng Tang a, Wei-Qing Zhong b, , Hai-Liang Zhu a,
⇑
⇑
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
b School of Pharmacy, Second Military Medical University, Shanghai 200433, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of novel naphthalin-containing pyrazoline derivatives C1–C14 and D1–D14 have been synthe-
sized and evaluated for their EGFR/HER-2 inhibitory and anti-proliferation activities. Compound D14 dis-
Received 31 October 2012
Revised 4 January 2013
Accepted 6 January 2013
Available online 16 January 2013
played the most potent activity against EGFR and A549 cell line (IC50 = 0.05
being comparable with the positive control Erlotinib (IC50 = 0.03 M and GI50 = 0.03
than our previous compounds C0–A (IC50 = 5.31 M and GI50 = 33.47 M) and C0–B (IC50 = 0.09
GI50 = 0.34 M). Meanwhile, compound C14 displayed the most potent activity against HER-2 and MCF-7
cell line (IC50 = 0.88 M and GI50 = 0.35 M), being a little less potent than Erlotinib (IC50 = 0.16 M and
GI50 = 0.08 M) but far more potent than C0–A (IC50 = 6.58 and GI50 = 27.62 M) and C0–B
(IC50 = 2.77 M and GI50 = 3.79 M). The docking simulation was performed to analyze the probable
l
M and GI50 = 0.11
M) and more potent
M and
lM),
l
l
l
l
l
l
Keywords:
Naphthalin
Thiourea
Modification
Pyrazoline
Antitumor activity
EGFR
l
l
l
l
l
M
l
l
l
binding models and the QSAR models were built for reasonable design of EGFR/HER-2 inhibitors at pres-
ent and in future. The structural modification of introducing naphthalin moiety reinforced the combina-
tion of our compounds and the receptor, resulting in progress of bioactivity. Moreover, the replacement of
thiourea skeleton by using benzene ring resulted in the slight diversity of the two series towards specific
targets.
HER-2
3D QSAR
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
lation of growth-factor signaling due to hyper-activation of the
ErbB receptors (primarily EGFR and HER-2) has been seen in
Cancer, being the second cause of mortality in the world, is
continuing to act as a major problem of health in both developing
and developed countries.1 In spite of the progress in medicine, the
commitment to the laborious task of discovering new anticancer
agents remains critically significant.
A variety of novel anticancer drugs are targeting mutant or
aberrantly expressed oncogenic growth factor receptor and non-
receptor tyrosine kinases involved in mitogenic or proliferative sig-
nal transduction pathways in cancer cells.2 Among the receptor
protein tyrosine kinases (RPTKs), epidermal growth factor receptor
(EGFR) kinase has been identified as a critical role in cancer. EGFR
family comprises four members, including: EGFR (HER-1/ErbB-1),
HER-2 (ErbB-2/neu), HER-3 (ErbB-3) and HER-4 (ErbB-4).3 Deregu-
several cancer types.4–7 Activation of EGFR may be because of
over-expression, mutations resulting in constitutive activation, or
autocrine expression of ligand while in contrast, activation of
HER-2 occurs mainly by over-expression, which leads to spontane-
ous homodimerization and activation of downstream signaling
events in a ligand-independent manner.8,9 Compounds that inhibit
the kinase activity of EGFR and/or HER-2 after binding in its ATP
binding site are of high interest as new therapeutic antitumor
agents.10,11
The thiourea and urea derivatives play important roles in anti-
cancer agents because of their brilliant inhibitory activity against
receptor tyrosine kinases (RTKs), protein tyrosine kinases (PTKs)
and NADH oxidase, which all play critical roles in many aspects
of tumorigenesis.12–14 Meanwhile, with a wide range of pharma-
ceutical and agrochemical activities, 4,5-dihydropyrazoles act as
a significant class of heterocyclic biological agents.15–19 Despite
their anticancer activity through other pathways, some dihydropy-
razole derivatives are also considered to be potent and selective
inhibitors of EGFR and/or HER-2 with sound IC50 values.20
Abbreviations: IC50, half maximal inhibitory concentration; GI50, the concentra-
tion that causes 50% growth inhibition; SI, the selectivity index of HER-2/EGFR;
QSAR, quantitative structure–activity relationship.
⇑
Corresponding authors. Tel./fax: +86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
These authors contributed equally to the work.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.013

W. Yang et al. / Bioorg. Med. Chem. 21 (2013) 1050–1063
1051
a
A
R1
B
R2
N
N
NH2
S
Figure 1. The chemical structure (a) and 3D QSAR of 4,5-dihydropyrazole derivatives containing thiourea skeleton in our previous work. (b) Red contours mean high electron
density is expected to increase activity while blue contours mean low electron density is better. (c) Green areas mean steric bulk is better while yellow areas mean small
groups are helpful.
In our previous research, a series of 4,5-dihydropyrazole deriv-
atives containing thiourea skeleton (Shown in Fig. 1) were synthe-
sized with their EGFR inhibitory activity evaluated.21 As the most
potent compound showed comparable EGFR inhibitory activity
containing thiourea skeleton and naphthalin structure was de-
signed as potential EGFR and/or HER-2 inhibitors and predicted
to have a positive progress with a sound cancer therapeutic bene-
fit. Moreover, we also bring in some additional comparisons with
4,5-dihydro-1H-pyrazole derivatives in which thiourea skeleton
is replaced to broaden the backbone of these inhibitors, to explore
the importance of certain moiety and to make our work
comprehensive.
(IC50 = 0.07 lM) with positive control Erlotinib (IC50 = 0.03 lM),
modification of this potential backbone might be a cogent way to
identify novel inhibitors of EGFR and/or HER-2. We created a 3D
QSAR (Quantitative Structure–Activity Relationship) model of this
former series and the concrete process is detailed in the 3D QSAR
part below. As shown in the result of 3D QSAR in Figure 1, an
increasing activity might be obtained by replacing the substitutes
on ring B with a slightly larger template which can cause a higher
electron density. We supposed four pairs of similar backbones
(Shown in Fig. 2) and at last chose pair IV because compounds with
acetyl were convinced less potent than those with thiourea
skeleton21 while benzodioxan structure actually causes a lower
electron density. Meanwhile, some previous results imply that
the naphthalin ring, which has higher lipophilicility can fix in the
hydrophobic pocket of the tubulin better.22 According to the stud-
ies above, a novel series of 4,5-dihydro-1H-pyrazole derivatives
2. Results and discussion
2.1. Chemistry
Twenty-eight naphthalin-containing 4,5-dihydro-1H-pyrazo-
line derivatives were synthesized to be screened for the antitumor
activity and half of them contain thiourea skeleton. All of the com-
pounds with thiourea skeleton were synthesized for the first time
and six of the rest were also novel. As for the eight compounds re-
ported before, they were only used for structure introduction or
antibacterial experiments23–25 while this time we attempted to
O
R
O
R
R
R
R
R
O
O
O
N
N
N
N
N
N
N
N
N
N
O
N
NH2
NH2
NH2
CH3
CH3
CH3
CH3
S
S
O
O
O
R
R
O
N
N
N
N
O
N
NH2
S
S
O
IV
III
I
II
Figure 2. Four pairs of similar backbones which are potential for exploring EGFR and/or HER-2 inhibitors.

1052
W. Yang et al. / Bioorg. Med. Chem. 21 (2013) 1050–1063
S
NH2
N
N
CHO
ii
R1
O
O
C1-C7
R2
R1
R1
i
N
N
R2
R2
iii
A
B
R1
D1-D7
R2
S
NH2
N
N
ii
CHO
R1
O
O
R1
C8-C14
R2
R1
i
N
R2
R2
N
B
A
iii
R1
D8-D14
R2
Series C
C1
C2
C3
C4
C5
C6
C7
C8
R1
H
F
Cl
Br
OCH3
CH3
Cl
H
F
Cl
Br
R2
H
H
H
H
H
H
Cl
H
H
H
H
H
H
Cl
Series D
D1
D2
D3
D4
D5
D6
D7
D8
R1
H
F
Cl
R2
H
H
H
H
H
H
Cl
H
H
H
H
H
H
Cl
Br
OCH3
CH3
Cl
H
F
Cl
Br
OCH3
CH3
Cl
C9
D9
C10
C11
C12
C13
C14
D10
D11
D12
D13
D14
OCH3
CH3
Cl
Scheme 1. General synthesis of compounds (C1–C14; D1–D14). Reagents and conditions: (i) EtOH, 40% NaOH, reflux, 30 min; (ii) EtOH, Thiosemicarbazide, 80 °C, 5 h;
(iii) EtOH, Phenylhydrazine, 80 °C, 5 h.
involve them in our antitumor research. The synthesis of com-
pounds C1–C14; D1–D14 followed the general pathway outlined
in Scheme 1. They were both prepared in two steps. Firstly, different
substituted acetophenone (A) on treatment with naphthaldehyde in
presence of 40% NaOH yielded different analogues of chalcones (B).
Secondly, the cyclizationofthe obtained shiny powderby subjoining
thiosemicarbazide led to corresponding target compounds C1–C14
5-(naphthalen-1-yl)-3-aryl-4,5-dihydro-1H-pyrazole-1-carbo-
thioamide/5-(naphthalen-2-yl)-3-aryl-4,5-dihydro-1H-pyra-
zole-1-carbothioamide while the cyclization of the analogues of
chalcones by subjoining phenylhydrazine led to D1–D14 5-(naph-
thalen-1-yl)-1,3-diaryl-4,5-dihydro-1H-pyrazole/5-(naphthalen-2-
yl)-1,3-diaryl-4,5-dihydro-1H-pyrazole. The refined compounds
were finally obtained by subsequent purification with recrystallization.

W. Yang et al. / Bioorg. Med. Chem. 21 (2013) 1050–1063
1053
All of the synthetic compounds gave satisfactory analytical and
spectroscopic data, which were full accordance with their depicted
structures.
ence that the anti-proliferative effect was produced by inhibitory
action against EGFR and/or HER-2. Further, as we can see in Fig-
ure 3A, linear regression results indicated that A549 cell line was
basically correlated to EGFR (R square = 0.593) while MCF-7 cell
line showed an obvious positive correlation with HER-2 (R
square = 0.748). This inference agreed with the relative expression
levels of EGFR and HER-2 in the two cell lines.
2.2. Bioassay
All the synthesized compounds C1–C14, D1–D14 were evaluated
for their EGFR and HER-2 inhibitory activity. The results were ex-
pressed as concentrations of IC50 (the half maximal inhibitory con-
centration of EGFR and HER-2 mediated autophosphorylation) and
GI50 (the half maximal inhibitory concentration of MCF-7 human
breast cancer cell line and A549 human lung cancer cell line growth),
presented in Table 1. Meanwhile, we took compounds C0–A and C0–
B as other comparisons to participate in the bioassay to evaluate our
modification. C0–A was a representative of our previous com-
pounds,21 sharing the same ring A with our synthesized compounds
(C7, C14, D7, D14). C0–B was the most potent one in the same pa-
per.21 A general glance of the data as well as the selectivity index
HER-2/EGFR (SI) suggested that most of the synthetic compounds
exhibited significant EGFR or HER-2 inhibitory activity while some
of them were potential inhibitors against both enzymes.
Out of the twenty-eight 4,5-dihydro-1H-pyrazole derivatives,
D14 displayed the most potent activity against EGFR and A549 cell
line (IC50 = 0.05
the positive control Erlotinib (IC50 = 0.03
obviously more potent than C0–A (IC50 = 5.31
33.47 M) and slightly more potent than our previous best com-
pound C0–B (IC50 = 0.09 M and GI50 = 0.34 M). Meanwhile, C14
displayed the most potent activity against HER-2 and MCF-7 cell line
(IC50 = 0.88 M and GI50 = 0.35 M), being a little less potent than
l
M and GI50 = 0.11
l
M), being comparable with
M and GI50 = 0.03 M),
M and GI50
l
l
l
=
l
l
l
l
l
the positive control Erlotinib (IC50 = 0.16
but far more potent than C0–A (IC50 = 6.58
l
M and GI50 = 0.08
l
l
M)
M)
lM and GI50 = 27.62
and C0–B (IC50 = 2.77
l
M and GI50 = 3.79
lM). This fact indicated
that our modification led to favorable effect.
Subsequently preliminary SAR (Structure–Activity Relationship)
studies were conducted to deduce how the structure variation and
modification could affect the anticancer activity. Initially, despite
the particular differences, the general trends of SAR are similar
The same as the previous study,21 the activity data suggested
that GI50 values of these compounds shared a similar tendency
with their relevant IC50 values, which convinced the former infer-
Table 1
EGFR and HER-2 inhibitory activity and selectivity index with anti-proliferation activity of the synthesized compounds (C1–C14, D1–D14) with the structure of C0–A and C0–B
S
S
NH2
NH2
N
N
N
N
H3C
H3C
OCH3
Cl
Cl
OH
C0-B
C0-A
Compounds
IC50
(
lM)
IC50
(l
M)
GI50
(l
M)
GI50
(
lM)
SI
EGFR
HER-2
A549
1.44 0.11
>50
MCF-7
44.48 3.71
>50
11.23 1.05
>50
HER-2/EGFR
3.25
1.29
5.81
1.61
26.12
3.24
32.57
1.89
0.53
7.30
3.46
3.15
2.00
1.33
1.52
3.36
4.51
4.73
30.10
9.55
14.14
7.88
6.70
13.27
1.80
12.08
18.83
20.40
1.24
30.78
5.33
C1
C2
C3
C4
C5
C6
C7
C8
2.23 0.21
8.25 0.65
0.94 0.07
5.68 0.39
0.16 0.01
2.06 0.21
0.21 0.02
3.01 0.26
3.28 0.19
0.53 0.04
0.35 0.03
1.04 0.11
0.52 0.04
0.66 0.05
2.52 0.18
1.07 0.11
0.68 0.07
0.49 0.03
0.10 0.007
0.20 0.013
0.14 0.02
0.24 0.03
0.30 0.02
0.11 0.01
0.71 0.10
0.12 0.014
0.06 0.005
0.05 0.003
5.31 0.32
0.09 0.008
0.03
7.24 0.51
10.63 0.75
5.46 0.33
9.15 0.61
4.18 0.29
6.68 0.36
6.84 0.42
5.69 0.31
1.75 0.13
3.87 0.24
1.21 0.08
3.28 0.23
1.04 0.10
0.88 0.07
3.83 0.31
3.59 0.28
3.07 0.22
2.32 0.21
3.01 0.25
1.91 0.12
1.98 0.13
1.89 0.11
2.01 0.18
1.46 0.09
1.28 0.08
1.45 0.10
1.13 0.11
1.02 0.09
6.58 0.41
2.77 0.19
0.16
0.38 0.04
49.06 3.87
0.17 0.02
1.21 0.09
0.18 0.01
3.21 0.21
4.18 0.28
0.26 0.01
0.23 0.02
0.46 0.03
0.27 0.02
0.31 0.02
1.98 0.17
0.49 0.06
0.33 0.02
0.25 0.01
0.16 0.01
0.19 0.01
0.20 0.02
0.22 0.01
0.23 0.03
0.16 0.007
0.36 0.02
0.16 0.008
0.13 0.005
0.11 0.003
33.47 1.99
0.34 0.02
0.03 0.003
4.18 0.36
29.10 1.98
33.06 2.17
13.79 1.22
0.88 0.11
3.31 0.28
0.44 0.03
2.12 0.15
0.39 0.03
0.35 0.02
3.29 0.27
2.68 0.19
1.82 0.13
1.03 0.12
1.71 0.11
0.74 0.08
0.81 0.06
0.73 0.05
0.85 0.06
0.53 0.03
0.48 0.03
0.55 0.06
0.39 0.02
0.39 0.04
27.62 1.94
3.79 0.25
0.08 0.005
C9
C10
C11
C12
C13
C14
D1
D2
D3
D4
D5
D6
D7
D8
D9
D10
D11
D12
D13
D14
C0–A
C0–B
Erlotinib

1054
W. Yang et al. / Bioorg. Med. Chem. 21 (2013) 1050–1063
Figure 3A. The linear regression results of the acquired data from bioassay.
against both two enzymes, for the top compounds (D14, D13 for
EGFR and C14, D14 for HER-2) and the bottom ones (C4, C2 for
EGFR and C4, C2 for HER-2) are similar. This reminds us of the sim-
ilarity between EGFR and HER-2, indicating our compounds to be
potential bi-targeted inhibitors in spite of the fact that the inhibi-
tory activity against HER-2 is weaker than that against EGFR.
Secondly, D series are basically more potent than C series as for
the topic of EGFR, for example, D14 (IC50 = 0.05 M) > C14 (IC50
0.66 M), D13 (IC50 = 0.06 M) > C13 (IC50 = 0.52 M); whereas
the order is contrary as for the topic of HER-2, for example, C14
l
=
l
l
l
(IC50 = 0.88
(IC50 = 1.13
l
l
M) > D14 (IC50 = 1.02
M). This point infers that the active site of EGFR and
lM), C13 (IC50 = 1.04 lM) > D13
that of HER-2 are actually different. A thiourea skeleton is more
suitable for HER-2 while replacing it with a benzene ring would
make it more favorable for EGFR. For this point, SI has given a more
brief indication of selectivity. Thirdly, after claiming the success of
introducing naphthalin moiety, we found naphthalin-2-yl more
potent than naphthalin-1-yl against both enzymes, generally but
not absolutely. For example, as for EGFR, D10 (IC50 = 0.11
(IC50 = 0.68 M), C11 (IC50 = 0.35 M) > C4 (IC50 = 5.68 M); as for
HER-2, C11 (IC50 = 1.21 M) > C4 (IC50 = 9.15 M), D9 (IC50
2.01 M) > D2 (IC50 = 3.59 M). Finally, we attempted to reveal
lM) > D3
l
l
l
l
l
=
l
l
the influence of the substituent groups on ring A (acquired from
substituted acetophenone). After considering both steric and elec-
tronic complexities, we simplified the situations by locking other
factors as their optimal choice respectively. For the aspect of EGFR,
we only discuss the D series with their ring B (acquired from naph-
thaldehyde) fixed with naphthalin-2-yl. Thus, we could perceive
the tendency that –Me > –Cl > –OMe > –H > –F > –Br, inferring that
an electron-donating substitute with appropriate size should be in-
volved in designing this kind of inhibitors. The corresponding com-
pounds were D13 (IC50 = 0.06
l
M) > D10 (IC50 = 0.11
l
l
M) > D12
M) > D11
(IC50 = 0.12
(IC50 = 0.71
l
l
M) > D8 (IC50 = 0.24 lM) > D9 (IC50 = 0.30
M). For the aspect of HER-2, we turn to C series with
Figure 3B. The linear regression results of cell lines vs kinases to explore the
Figure 3C. Western blotting was performed to detect the effect of compound D14
correlations as well as kinase selectivity of representative compounds.
on p-EGFR, EGFR, p-Akt and Akt.

W. Yang et al. / Bioorg. Med. Chem. 21 (2013) 1050–1063
1055
Figure 3D. Crystal structure of compounds D5, C10.
Table 2
Table 3
Kinases inhibitory activities (VEGFR2, FAK and HER-3) of representative compounds
Crystal data of compounds D5, C10
(C5, C13, C14, D5, D13 and D14)
Crystal parameters Compound D5
Compound C10
Compounds
IC50
VEGFR-2
(
l
M)
IC50
FAK
(
l
M)
IC50(lM)
HER-3
Formula
Formula weight
Crystal system
C
26H22N2O
C20H16ClN3S
365.88
Monoclinic
90
127.913(1)
90
26.3433(10)
8.2568(3)
20.4628(13)
3511(3)
8
378.46
C5
4.1 0.23
2.1 0.12
3.2 0.18
3.4 0.17
1.56
1.83 0.11
13.6 1.1
28.7 1.7
9.9 0.83
10.3 0.85
11.5 1.0
6.2 0.43
55.6 3.5
63.2 4.2
51.7 3.3
40.2 2.9
45.3 2.7
35.9 2.4
Triclinic
100.087(2)
96.024(2)
90.568(2)
8.5525(6)
9.1205(5)
13.1272(9)
1002.15(11)
2
C13
C14
D5
D13
D14
a
(°)
b (°)
(°)
c
0.09
a (Å)
b (Å)
c (Å)
V (Å)
their ring B fixed with naphthalin-2-yl as well. This time the results of
inhibitory activity inferred the order that –Me > –Br > –F > –OMe > –
Cl > –H, indicating that the steric factor might be more important than
the electronic factor. The corresponding compounds were C13
Z
Calculated density
(Mg/cm3)
Mu (MoKa)
1.2542
1.3842
0.077
0.128
0.344
1.414
(mmÀ1
)
(IC50 = 1.04
l
M) > C11 (IC50 = 1.21
l
M) > C9 (IC50 = 1.75
lM) >
Absorption
coefficient
C12 (IC50 = 3.28
l
M) > C10 (IC50 = 3.87
l
M) > C8 (IC50 = 5.69
lM).
(mmÀ1
)
Further, our work in this paper revealed a new inference that substi-
tuent groups on meta position might enhance the inhibitory activity,
for the most potent compounds D14 and C14 share the same ring A
with 3,4-dichloro substitutes. That might be a potential direction of
our future research. A clearer and concise SAR analysis was ex-
pounded in the 3D QSAR part below.
F (000)
h range (°)
hkl limits
400.0
2.4–25.7
À10 6 h 6 10,
À11 6 k 6 10,
À15 6 l 6 14
9649/3199[Rint = 0.046]
1520
2.03–25.70
À31 6 h 6 32,
À9 6 k 6 10,
À24 6 l 6 22
Reflections
collected/
15,101/3316 [Rint = 0.029]
Considering the kinase selectivity, inhibitory activities of repre-
sentative compounds (C5, C13, C14, D5, D13 and D14) against three
kinases were tested. We took six points to fulfill the basic standard of
performing linear regressions. The three kinases we chose were:
VEGFR2 (activating similar pathways and often studied together),
HER-3 (also called erbB-3, belonging to EGFR family but activating
different pathway), FAK (a downstream kinase of EGFR but in a un-
ique pathway). Using the same method as that of studying EGFR/
HER-2, the results were expressed as IC50 values, presented in Ta-
ble 2. To gain a brief understanding of relationships between anti-
proliferation and kinase inhibitory activity, we conducted several
linear regressions. The results were displayed in Figure 3B (While
reducing the scale from 28 points to 6 points, the request of R square
should be raised to larger than about 0.8). The diagrams indicated
two correlations (A549 vs EGFR; MCF-7 vs HER-2), agreeing with
the inference we got from Figure 3A. Meanwhile, other pairs seemed
slightly correlated or even nearly unrelated. These differences fur-
ther validated the selectivity of the compounds to some extent.
Further, the corresponding p-EGFR and EGFR, p-Akt and Akt lev-
els of A549 dealt by D14 were measured by Western blotting
(Fig. 3C). With the increasing of D14 levels, p-EGFR and p-Akt lev-
els showed gradually trend of decrease. It is remarkable that these
inhibitors against phosphorylation of EGFR could decline the level
of p-Akt (the downstream target protein of p-EGFR). Consequently,
compound D14 could directly bind to EGFR, and lower its
unique
Data/restraints/
parameters
R1/wR2[I > 2
R1/wR2 (all data)
2033/0/263
2628/0/226
r(I)]
0.0436/0.0961
0.0922/0.1118
1.022
00,372/0.0859
0.0531/0.0940
1.032
GOF
phosphorylation level. The conclusion supported the molecular
docking results (Besides, the result about C14 and HER-2 was sim-
ilar, so related data is not shown).
2.3. Crystal structure of compound
Among these compounds, crystal structures of compound D5
and C10 were determined by X-ray diffraction analysis. The crystal
data were presented in Table 3 and Figure 3D, giving perspective
views of D5 and C10 with the atomic labeling system.
2.4. Docking study
In molecular docking, molecular modeling techniques are used to
predict how a protein (enzyme) interacts with small molecules (li-
gands).26 In our present study, to explore the interaction between
compounds and EGFR/HER-2 as well as to visualize the probable
binding mode, a docking study was performed using the CDOCKER

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W. Yang et al. / Bioorg. Med. Chem. 21 (2013) 1050–1063
Figure 4. (A) 2D molecular docking model of compound D14 with 1M17. (B) 3D model of the interaction between compound D14 and 1M17 binding site. (C) 2D molecular
docking model of compound C14 with 1M17. (D) 3D model of the interaction between compound C14 and 1M17 binding site. The H-bonds are displayed as dotted lines and
the amino acid they act on are labeled in green. The
p–cation interactions and p–sigma interactions are shown as orange lines with their corresponding amino acids labeled in
yellow. Other important amino acids are labeled in blue.
protocol in Discovery Studio 3.1 (Discovery Studio 3.1, Accelrys, Inc.
San Diego, CA). All the synthetic compounds have been conducted a
docking study into the active site of the receptor EGFR and HER-2.
We chose the crystal structure of EGFR (PDB Code: 1M17.pdb)27 in
which the original ligand was Erlotinib and the crystal structure of
HER-2 (PDB Code: 3PP0.pdb)28 in which the original ligand was
2-{2-[4-({5-chloro-6-[3-(trifluoromethyl)phenoxy]pyridin-3-yl}
amino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethoxy}ethanol (Ligand
Code: 03Q), respectively. They were obtained from the RCSB protein
data bank (http://www.pdb.org). After preparing the receptor and
ligands, the site sphere was selected based on the ligand binding
location. The binding models of the most potent compounds with
corresponding target as well as the other target were depicted in
Figure 4 (D14, C14 with 1M17) and Figure 5 (C14, D14 with 3PP0),
respectively.
the previous work.20,21 To introduce comparison, we put C14 into
the same domain and found that the interactions between receptor
and ring A were almost the same, while the binding of naphthalin
ring was looser. Thiourea structure seemed more likely to form
hydrogen bonds although the situation of overall energy was
worse than D series. On the other hand, compound C14 performs
a nice bonding situation towards 3PP0 mainly via two hydrogen
bonds and one
p–sigma interaction. One of the hydrogen bond is
formed between the amino hydrogen of carbothioamide and the
main chain of GLN799 (N–H. . .O: 2.40 Å, 127.241°) while the other
was formed between N2 of pyrazoline and the side chain of
THR862 (H. . .N: 2.00 Å, 149.003°). The only one
p–sigma interac-
tion is formed between the naphthalin ring and LEU852 (2.91 Å).
This result agrees with the fact that most of our compounds are dif-
ferent against EGFR than against HER-2. A possible factor to influ-
ence the bioactivity might be the shown different binding patterns.
The binding model of D14 and HER-2 was also shown, indicating
the same binding pattern but looser combination.
Thus, we conducted the receptor surface model (Shown in
Fig. 6) to reveal the possible situation of the compounds embedded
in the active pocket. As for the EGFR model, the active pocket
including ASP831, MET769, CYS773, LEU768, ALA719 and
PRO770 is occupied by the naphthalin structure, confirming the
success of structural modification. As for the HER-2 model, almost
the whole molecules are embedded into the active pocket includ-
ing GLN799, THR862, THR798, LEU800, MET801, CYS805, SER783
and LEU796 except the naphthalin ring. This result agrees with
In the binding model, compound D14 is nicely bound to 1M17
via one hydrogen bond, one p–cation interaction and two p–sigma
interaction. The meta-chlorine atom on ring A contributes to the
hydrogen bonding interaction (Cl. . .H–O: 2.29 Å, 131.868°) with
the hydrogen atom on the side chain of THR766, being a probable
explanation for its nice activity. Meanwhile, the two
p–sigma
interaction is formed between the naphthalin ring and LEU694
(2.60 Å and 2.63 Å), suggesting that the introduction of the naph-
thalin structure has enhanced the combination of the receptor
and ligand. The
p–cation interaction between ring A and LYS721,
together with the interactional residues such as ASP831 and
MET769, infer that this docking model is basically accordant with

W. Yang et al. / Bioorg. Med. Chem. 21 (2013) 1050–1063
1057
Figure 5. (A) 2D molecular docking model of compound C14 with 3PP0. (B) 3D model of the interaction between compound C14 and 3PP0 binding site. (C) 2D molecular
docking model of compound D14 with 3PP0. (D) 3D model of the interaction between compound D14 and 3PP0 binding site. The H-bonds are displayed as dotted lines and
the amino acid they act on are labeled in green. The one
amino acids are labeled in blue.
p–sigma interaction is shown as orange line with their corresponding amino acid labeled in yellow. Other important
Figure 6. The receptor surface models with D14 for EGFR (A) and C14 for HER-2 (B) respectively.
the fact that most of our compounds are more potent against EGFR
nite IC50 values against EGFR were selected as the model dataset.
By convention, we used the pIC50 scale (Àlog IC50), in which higher
value indicates exponentially greater potency, to measure the
inhibitory activity. The training set and testing set were chosen
by the Diverse Molecules method in Discovery Studio 3.1. To en-
sure a good alignment, we chose the alignment conformation of
each molecule with lowest energy in the docked results of CDOC-
KER protocol. Besides, we applied the alignment by the substruc-
ture 4,5-dihydro-1H-pyrazole before building the QSAR model.
With the correlation coefficient r2 between observed activity of
testing set and training set found to be 0.735, the primary QSAR
model built by us is acceptable. As shown in Figure 1, the mole-
cules aligned with the iso-surfaces of the 3D QSAR model coeffi-
cients on electrostatic potential grids and Van der Waals grids are
listed. Electrostatic map indicates red contours around regions
where high electron density (negative charge) is expected to
increase activity, and blue contours represent areas where low
than against HER-2, for our structural modification of naphthalin
ring contributes more in EGFR binding model. Moreover, the dock-
ing calculation of all the compounds was also taken into account
(detailed data not provided for a concise consideration). The CDoc-
ker Interaction Energy (interaction energy between the ligand and
the receptor) and the CDocker Energy (energy of the ligand–recep-
tor complexes) agreed with the inhibitory trend for all the synthe-
sized compounds.
2.5. QSAR model
Our original intention is conducting modification of our previ-
ous work and exploring novel anticancer agents. The design of
our compounds in this paper was initially inferred by the 3D QSAR
result of our previous work.21 By using the Create 3D QSAR proto-
col of Discovery Studio 3.1, thirty previous compounds with defi-

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W. Yang et al. / Bioorg. Med. Chem. 21 (2013) 1050–1063
Figure 7. 3D QSAR of 4,5-dihydro-1H-pyrazole derivatives containing naphthalin moiety for EGFR (A, B) and for HER-2 (C, D). Red contours mean high electron density is
expected to increase activity while blue contours mean low electron density is better. Green areas mean steric bulk is better while yellow areas mean small groups are
helpful.
electron density (partial positive charge) is expected to increase
activity. Similarly, steric map indicates areas where steric bulk is
predicted to increase (green) or decrease (yellow) activity. Thus,
we reached to the design concept that an increasing activity might
be obtained by replacing the substitutes on ring B with a slightly
larger template which can cause a higher electron density. We
have chosen the naphthalin structure.
that of EGFR model. The 3D QSAR models agree with the inhibitory
activity well and provide us the direction of further modification
with the similarities and differences between EGFR and HER-2 ta-
ken into consideration.
3. Conclusion
To ensure a more cogent SAR discussion of compounds in this
paper, we abandoned the convenience of using the primary 3D
QSAR model and created a secondary one for the reason that our
modification might have slightly changed the backbone of our pre-
vious inhibitors. Meanwhile, we attempted to take HER-2 into an-
other model this time. With the same protocol and steps, we have
got secondary 3D QSAR models of compounds in this paper. The
correlation coefficient r2 between observed activity of testing set
and training set was found to be 0.666 for the EGFR model and
0.913 for the HER-2 model, which proved that the QSAR models
built by us are both acceptable. The electrostatic and steric maps
are shown in Figure 7. As for EGFR, being agreed with the fact that
D series performed more potent activity than C series, a moiety
with higher electron density would help obtain sound activity
(benzene > thiourea) while the size could be fixed as the present
stage. Meanwhile, on ring B, the present electronic status and gen-
eral size is nice but naphthalin-2-yl showed superiority upon
naphthalin-1-yl in steric situation. On ring A, a slightly bulk and
high negative charged group on the para-position would enhance
the bioactivity while a same one on the meta-position might make
the efficiency better. When we come to the issue of HER-2, it seems
that the efficiency of thiourea skeleton equals that of benzene. The
situation of ring B is similar to that of EGFR model, while a small
difference is that naphthalin-2-yl is recommended to be replaced
by a longer one. As for ring A, still, the suggestions are similar to
To conclude, two series of compounds (twenty-eight in total),
C1–C14 5-(naphthalen-1-yl)-3-aryl-4,5-dihydro-1H-pyrazole-1-
carbothioamide/5-(naphthalen-2-yl)-3-aryl-4,5-dihydro-1H-pyra-
zole-1-carbothioamide and D1–D14 5-(naphthalen-1-yl)-1,3-dia-
ryl-4,5-dihydro-1H-pyrazole/5-(naphthalen-2-yl)-1,3-diaryl-4,5-
dihydro-1H-pyrazole, have been synthesized and evaluated for
their EGFR/HER-2 inhibitory and anti-proliferation activities.
Compound D14 displayed the most potent activity against EGFR
and A549 human lung cancer cell line (IC50 = 0.05
GI50 = 0.11 M), being comparable with the positive control Erloti-
nib (IC50 = 0.03 M and GI50 = 0.03 M), obviously more potent
than our previous representative C0–A (IC50 = 5.31 and
GI50 = 33.47 M) and slightly more potent than our previous best
compound C0–B (IC50 = 0.09 M and GI50 = 0.34 M). Meanwhile,
compound C14 displayed the most potent activity against HER-2
and MCF-7 human breast cancer cell line (IC50 = 0.88 M and
GI50 = 0.35 M), being a little less potent than the positive control
Erlotinib (IC50 = 0.16 M and GI50 = 0.08 M) but far more potent
than C0–A (IC50 = 6.58 and GI50 = 27.62 M) and C0–B
(IC50 = 2.77 M and GI50 = 3.79 M). The results of docking simula-
lM and
l
l
l
lM
l
l
l
l
l
l
l
lM
l
l
l
tion indicate that D14 can bind well into the active site of EGFR
while C14 can be embedded into the active pocket of HER-2. Wes-
tern blotting results showed that the inhibition of D14 against
phosphorylation of EGFR could decline p-Akt, the downstream
target protein of p-EGFR. Most of the synthetic compounds can

W. Yang et al. / Bioorg. Med. Chem. 21 (2013) 1050–1063
1059
be potential inhibitors of EGFR and/or HER-2, showing selectivity
to some extent. QSAR models were built with activity data and
binding conformations to begin our work in this paper as well as
to provide a reliable tool for reasonable design of EGFR/HER-2
inhibitors in future. The structural modification of introducing
naphthalin moiety reinforced the combination of our compounds
and the receptor, resulting in progress of bioactivity. Moreover,
the replacement of thiourea skeleton by using benzene ring results
in the slight diversity of the two series towards specific targets.
J1 = 11.4 Hz, J2 = 3.3 Hz, 1H), 7.01–7.03 (d, J = 6.9 Hz, 1H), 7.39–
7.44 (m, 4H), 7.54–7.64 (m, 2H), 7.79–7.87 (m, 3H), 7.95–7.98 (d,
J = 8.1 Hz, 1H), 8.16–8.19 (d, J = 8.1 Hz, 1H). MS (ESI): 332.11
(C20H18N3S, [M+H]+). Anal. Calcd for C20H17N3S: C, 72.48; H, 5.17;
N, 12.68; S, 9.67. Found: C, 72.31; H, 5.17; N, 12.70. As for C1–
C7, the two active hydrogen atoms of amidogen cannot be shown
in the 1H NMR, which is the same as our previous study.21
4.4.2. 3-(4-Fluorophenyl)-5-(naphthalen-1-yl)-4,5-dihydro-1H-
pyrazole-1-carbothioamide (C2)
White crystal, Yield 78%, mp: 218–220 °C. 1H NMR (CDCl3,
300 MHz) d: 3.12–3.19 (dd, J1 = 17.4 Hz, J2 = 3.7 Hz, 1H), 3.95–
4.05 (dd, J1 = 17.4 Hz, J2 = 11.4 Hz, 1H), 6.36 (s, 1H), 6.79–6.84
(dd, J1 = 11.1 Hz, J2 = 3.3 Hz, 1H), 7.05–7.11 (t, J = 8.7 Hz, 1H),
7.21–7.28 (m, 1H), 7.39–7.45 (t, J = 7.5 Hz, 1H), 7.51–7.62 (m,
2H), 7.67–7.71 (dd, J1 = 9.0 Hz, J2 = 5.4 Hz, 2H), 7.78–7.80 (d,
J = 8.4 Hz, 1H), 7.90–7.92 (d, J = 8.4 Hz, 1H), 8.00–8.03 (d,
J = 8.1 Hz, 1H). MS (ESI): 350.10 (C20H17FN3S, [M+H]+). Anal. Calcd
for C20H16FN3S: C, 68.75; H, 4.62; F, 5.44; N, 12.03; S, 9.18. Found:
C, 68.59; H, 4.63; N, 12.04.
4. Experimental section
4.1. Materials and measurements
All chemicals used were purchased from Aldrich (USA). The elu-
ates were monitored using TLC (Thin layer chromatography). TLC
was run on the silica gel coated aluminum sheets (Silica Gel 60 Å
GF254, E. Merk, Germany) and visualized in UV light (254 nm). Sep-
aration of the compounds by column chromatography was carried
out with silica gel 60 (200–300 mesh ASTM, E. Merck, Germany).
Developed plates were visualized by a Spectroline ENF 260C/F
UV apparatus. The quantity of silica gel used was 50–100 times
the weight charged on the column. Melting points (uncorrected)
were determined on a XT4MP apparatus (Taike Corp., Beijing, Chi-
na). ESI mass spectra were obtained on a Mariner System 5304
mass spectrometer, and 1H NMR spectra were recorded on a
DPX300 spectrometer at 25 °C with TMS and solvent signals allot-
ted as internal standards, Chemical shifts are reported in ppm (d).
Elemental analyses were performed on a CHN–O-Rapid instrument
and were within 0.4% of the theoretical values.
4.4.3. 3-(4-Chlorophenyl)-5-(naphthalen-1-yl)-4,5-dihydro-1H-
pyrazole-1-carbothioamide (C3)
Yellow crystal, Yield 79%, mp: 230–233 °C. 1H NMR (CDCl3,
300 MHz) d: 3.02–3.10 (dd, J1 = 18.0 Hz, J2 = 3.6 Hz, 1H), 4.07–
4.16 (dd, J1 = 18.0 Hz, J2 = 11.4 Hz, 1H), 6.65–6.70 (dd,
J1 = 11.4 Hz, J2 = 3.3 Hz, 1H), 7.00–7.02 (d, J = 7.2 Hz, 1H), 7.39–
7.48 (m, 3H), 7.53–7.64 (m, 2H), 7.79–7.82 (d, J = 8.1 Hz, 1H),
7.86–7.89 (d, J = 8.4 Hz, 2H), 7.95–7.98 (d, J = 8.1 Hz, 1H), 8.15–
8.18 (d, J = 8.1 Hz, 1H). MS (ESI): 366.08 (C20H17ClN3S, [M+H]+).
Anal. Calcd for C20H16ClN3S: C, 65.65; H, 4.41; Cl, 9.69; N, 11.48;
S, 8.76. Found: C, 65.42; H, 4.41; N, 11.50.
4.2. General method of synthesis of (E)-Chalcones (B)
4.4.4. 3-(4-Bromophenyl)-5-(naphthalen-1-yl)-4,5-dihydro-1H-
pyrazole-1-carbothioamide (C4)
Substituted acetophenone (10 mmol) and naphthaldehyde
(10 mmol) in ethanol (25 mL) were refluxed gently for 30 min.
Then 40% NaOH (5 mL) was added and the solid was filtered,
washed with water and dried to obtain shiny solid B.
Yellow crystal, Yield 77%, mp: 138–140 °C. 1H NMR (CDCl3,
300 MHz) d: 3.16–3.20 (dd, J1 = 10.5 Hz, J2 = 2.1 Hz, 1H), 4.00–
4.06 (dd, J1 = 10.5 Hz, J2 = 6.9 Hz, 1H), 6.19 (s, 1H), 6.81–6.83 (d,
J = 6.6 Hz, 1H), 7.08–7.11 (t, J = 5.1 Hz, 2H), 7.41–7.44 (t,
J = 4.6 Hz, 1H), 7.53–7.56 (t, J = 4.2 Hz, 1H), 7.59–7.62 (t,
J = 4.5 Hz, 1H), 7.70–7.73 (m, 2H), 7.79–7.80 (d, J = 4.8 Hz, 1H),
7.91–7.92 (d, J = 4.8 Hz, 1H), 8.01–8.02 (d, J = 5.1 Hz, 1H). MS
(ESI): 410.02 (C20H17BrN3S, [M+H]+). Anal. Calcd for C20H16BrN3S:
C, 58.54; H, 3.93; Br, 19.47; N, 10.24; S, 7.81. Found: C, 58.43; H,
3.94; N, 10.25.
4.3. General method of synthesis of 5-(naphthalen-1-yl)-3-aryl-
4,5-dihydro-1H-pyrazole-1-carbothioamide and 5-(naphthalen-
2-yl)-3-aryl-4,5-dihydro-1H-pyrazole-1-carbothioamide (C1–
C14)
Compound B (5 mmol) and thiosemicarbazide (5 mmol) in eth-
anol (20 mL) were refluxed at 80 °C for 5 h. While the reaction
completed, the resultant solid was filtered, washed with water
and dried to obtain slight yellow solid. The separated solid was
crystallized from mixture of DMF and ethanol (1:9) to obtain the
corresponding compounds C1–C14 as translucent solid.
4.4.5. 3-(4-Methoxyphenyl)-5-(naphthalen-1-yl)-4,5-dihydro-
1H-pyrazole-1-carbothioamide (C5)
White crystal, Yield 79%, mp: 211–214 °C. 1H NMR (CDCl3,
300 MHz) d: 3.15–3.19 (dd, J1 = 10.4 Hz, J2 = 2.1 Hz, 1H), 3.83 (s,
3H), 3.98–4.03 (dd, J1 = 10.5 Hz, J2 = 6.9 Hz, 1H), 6.78–6.80 (d,
J = 4.9 Hz, 1H), 6.89–6.91 (d, J = 5.4 Hz, 2H), 7.21–7.23 (d,
J = 4.3 Hz, 1H), 7.40–7.43 (t, J = 4.5 Hz, 1H), 7.52–7.54 (t,
J = 4.2 Hz, 1H), 7.58–7.61 (t, J = 4.2 Hz, 1H), 7.64–7.66 (d,
J = 5.4 Hz, 2H), 7.77–7.79 (d, J = 4.9 Hz, 1H), 7.90–7.91 (d,
J = 4.8 Hz, 1H), 8.02–8.03 (d, J = 5.1 Hz, 1H). MS (ESI): 362.12
(C21H20N3OS, [M+H]+). Anal. Calcd for C21H19N3OS: C, 69.78; H,
5.30; N, 11.63; O, 4.43; S, 8.87. Found: C, 69.57; H, 5.30; N, 11.65.
4.4. General method of synthesis of 5-(naphthalen-1-yl)-1,3-
diaryl-4,5-dihydro-1H-pyrazole and 5-(naphthalen-2-yl)-1,3-
diaryl-4,5-dihydro-1H-pyrazole (D1–D14)
Compound B (5 mmol) and phenylhydrazine (5 mmol) in etha-
nol (20 mL) were refluxed at 80 °C for 5 h. While the reaction com-
pleted, the resultant solid was filtered, washed with water and
dried to obtain slight yellow solid. The separated solid was crystal-
lized from mixture of DMF and ethanol (1:9) to obtain the corre-
sponding compounds D1–D14 as translucent solid.
4.4.6. 5-(Naphthalen-1-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazole-
1-carbothioamide (C6)
White crystal, Yield 76%, mp: 223–227 °C. 1H NMR (CDCl3,
300 MHz) d: 2.33 (s, 3H), 3.01–3.08 (dd, J1 = 18.0 Hz, J2 = 3.6 Hz,
1H), 4.08–4.18 (dd, J1 = 17.7 Hz, J2 = 11.4 Hz, 1H), 6.65–6.70 (dd,
J1 = 11.4 Hz, J2 = 3.3 Hz, 1H), 7.02–7.05 (d, J = 7.2 Hz, 1H), 7.22–
7.25 (d, J = 8.1 Hz, 1H), 7.40–7.46 (t, J = 8.1 Hz, 1H), 7.56–7.66 (m,
4.4.1. 5-(Naphthalen-1-yl)-3-phenyl-4,5-dihydro-1H-pyrazole-
1-carbothioamide (C1)
White crystal, Yield 81%, mp: 227–230 °C. 1H NMR (CDCl3,
300 MHz) d: 3.02–3.10 (dd, J1 = 18.0 Hz, J2 = 3.6 Hz, 1H), 4.09–
4.19 (dd, J1 = 18.0 Hz, J2 = 11.4 Hz, 1H), 6.65–6.70 (dd,

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W. Yang et al. / Bioorg. Med. Chem. 21 (2013) 1050–1063
2H), 7.75–7.78 (d, J = 8.1 Hz, 2H), 7.81–7.84 (d, J = 8.1 Hz, 1H),
7.98–8.00 (d, J = 7.8 Hz, 2H), 8.17–8.20 (d, J = 8.4 Hz, 1H). MS
(ESI): 346.13 (C21H20N3S, [M+H]+). Anal. Calcd for C21H19N3S: C,
73.01; H, 5.54; N, 12.16; S, 9.28. Found: C, 72.82; H, 5.55; N, 12.17.
3H), 3.92–3.98 (dd, J1 = 10.8 Hz, J2 = 6.9 Hz, 1H), 6.04–6.07 (dd,
J1 = 6.9 Hz, J2 = 2.1 Hz, 1H), 7.00–7.01 (d, J = 5.1 Hz, 2H), 7.28–7.30
(d, J = 5.1 Hz, 1H), 7.45–7.50 (m, 2H), 7.62 (s, 1H), 7.83–7.88 (m,
6H), 7.97 (s, 1H). MS (ESI): 362.12 (C21H20N3OS, [M+H]+). Anal.
Calcd for C21H19N3OS: C, 69.78; H, 5.30; N, 11.63; O, 4.43; S,
8.87. Found: C, 69.56; H, 5.31; N, 11.63.
4.4.7. 3-(3,4-Dichlorophenyl)-5-(naphthalen-1-yl)-4,5-dihydro-
1H-pyrazole-1-carbothioamide (C7)
Yellow crystal, Yield 74%, mp: 210–214 °C. 1H NMR (CDCl3,
300 MHz) d: 3.16–3.25 (dd, J1 = 17.7 Hz, J2 = 3.6 Hz, 1H), 4.05–
4.14 (dd, J1 = 17.7 Hz, J2 = 11.4 Hz, 1H), 6.67–6.73 (dd,
J1 = 11.4 Hz, J2 = 3.3 Hz, 1H), 7.02–7.04 (d, J = 7.2 Hz, 1H), 7.55–
7.60 (m, 3H), 7.64–7.68 (m, 2H), 8.00–8.01 (d, J = 4.8 Hz, 1H),
8.21–8.22 (d, J = 4.2 Hz, 1H), 8.26 (s, 1H), 8.35–8.37 (d, J = 5.1 Hz,
1H). MS (ESI): 400.04 (C20H16Cl2N3S, [M+H]+). Anal. Calcd for
4.4.13. 5-(Naphthalen-2-yl)-3-(p-tolyl)-4,5-dihydro-1H-
pyrazole-1-carbothioamide (C13)
White powder, Yield 75%, mp: 179–182 °C. 1H NMR (CDCl3,
300 MHz) d: 2.34 (s, 3H), 3.20–3.23 (dd, J1 = 10.8 Hz, J2 = 2.4 Hz,
1H), 3.94–4.00 (dd, J1 = 11.1 Hz, J2 = 6.9 Hz, 1H), 6.05–6.08 (dd,
J1 = 6.9 Hz, J2 = 2.4 Hz, 1H), 7.26–7.28 (d, J = 4.8 Hz, 2H), 7.29–7.30
(d, J = 0.9 Hz, 1H), 7.46–7.50 (m, 2H), 7.62 (s, 1H), 7.78–7.80 (d,
J = 5.1 Hz, 2H), 7.85–7.88 (m, 4H), 8.01 (s, 1H). MS (ESI): 346.13
(C21H20N3S, [M+H]+). Anal. Calcd for C21H19N3S: C, 73.01; H, 5.54;
N, 12.16; S, 9.28. Found: C, 72.85; H, 5.54; N, 12.18.
C20H15Cl2N3S: C, 60.00; H, 3.78; Cl, 17.71; N, 10.50; S, 8.01. Found:
C, 59.91; H, 3.78; N, 10.50.
4.4.8. 5-(Naphthalen-2-yl)-3-phenyl-4,5-dihydro-1H-pyrazole-
1-carbothioamide (C8)
4.4.14. 3-(3,4-Dichlorophenyl)-5-(naphthalen-2-yl)-4,5-
dihydro-1H-pyrazole-1-carbothioamide (C14)
White powder, Yield 82%, mp: 202–204 °C. 1H NMR (CDCl3,
300 MHz) d: 3.22–3.26 (dd, J1 = 10.8 Hz, J2 = 2.4 Hz, 1H), 3.97–
4.02 (dd, J1 = 10.8 Hz, J2 = 6.9 Hz, 1H), 6.07–6.10 (dd, J1 = 6.9 Hz,
J2 = 2.4 Hz, 1H), 7.29–7.31 (dd, J1 = 5.1 Hz, J2 = 0.9 Hz, 1H), 7.44–
7.50 (m, 5H), 7.64 (s, 1H), 7.86–7.91 (m, 5H), 7.95–8.06 (d,
J = 33.6 Hz, 2H). MS (ESI): 332.11 (C20H18N3S, [M+H]+). Anal. Calcd
for C20H17N3S: C, 72.48; H, 5.17; N, 12.68; S, 9.67. Found: C, 72.28;
H, 5.18; N, 12.68.
Yellow powder, Yield 75%, mp: 201–204 °C. 1H NMR (CDCl3,
300 MHz) d: 3.25–3.30 (dd, J1 = 10.8 Hz, J2 = 2.4 Hz, 1H), 3.93–
3.99 (dd, J1 = 11.1 Hz, J2 = 7.2 Hz, 1H), 6.07–6.10 (dd, J1 = 6.9 Hz,
J2 = 2.1 Hz, 1H), 7.28–7.29 (d, J = 4.2 Hz, 1H), 7.47–7.50 (m, 2H),
7.63 (s, 1H), 7.71–7.73 (d, J = 5.1 Hz, 1H), 7.80–7.82 (dd,
J1 = 5.1 Hz, J2 = 1.2 Hz, 1H), 7.86–7.88 (m, 3H), 8.14–8.16 (d,
J = 6.6 Hz, 2H), 8.26 (s, 1H). MS (ESI): 400.04 (C20H16Cl2N3S,
[M+H]+). Anal. Calcd for C20H15Cl2N3S: C, 60.00; H, 3.78; Cl,
17.71; N, 10.50; S, 8.01. Found: C, 59.87; H, 3.79; N, 10.51.
4.4.9. 3-(4-Fluorophenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-
pyrazole-1-carbothioamide (C9)
White powder, Yield 79%, mp: 230–234 °C. 1H NMR (CDCl3,
300 MHz) d: 3.15–3.19 (dd, J1 = 10.8 Hz, J2 = 2.1 Hz, 1H), 3.89–
3.95 (dd, J1 = 10.8 Hz, J2 = 6.9 Hz, 1H), 6.03 (m, 1H), 7.45–7.47 (d,
J = 5.1 Hz, 2H), 7.54–7.55 (m, 2H), 7.90–7.96 (m, 4H), 8.06 (m,
1H), 8.11 (s, 1H), 8.16–8.18 (d, J = 5.1 Hz, 1H), 8.22 (s, 2H). MS
(ESI): 350.10 (C20H17FN3S, [M+H]+). Anal. Calcd for C20H16FN3S: C,
68.75; H, 4.62; F, 5.44; N, 12.03; S, 9.18. Found: C, 68.57; H, 4.63;
N, 12.03.
4.4.15. 5-(Naphthalen-1-yl)-1,3-diphenyl-4,5-dihydro-1H-
pyrazole (D1)
White crystal, Yield 78%, mp: 169–172 °C. 1H NMR (CDCl3,
300 MHz) d: 3.03–3.11 (dd, J1 = 17.4 Hz, J2 = 6.0 Hz, 1H), 4.12–
4.22 (dd, J1 = 17.4 Hz, J2 = 12.6 Hz, 1H), 6.16 (m, 1H), 6.68–6.73 (t,
J = 7.2 Hz, 1H), 6.93–6.95 (d, J = 8.1 Hz, 2H), 7.10–7.15 (t,
J = 7.5 Hz, 2H), 7.24 (m, 1H), 7.32–7.42 (m, 4H), 7.57–7.68 (m,
2H), 7.73–7.75 (d, J = 6,6 Hz, 2H), 7.83–7.86 (d, J = 8.1 Hz, 1H),
7.98–8.01 (d, J = 8.4 Hz, 1H), 8.29–8.32 (d, J = 7.5 Hz, 1H). MS
(ESI): 349.16 (C25H21N2, [M+H]+). Anal. Calcd for C25H20N2: C,
86.17; H, 5.79; N, 8.04. Found: C, 85.93; H, 5.78; N, 8.05.
4.4.10. 3-(4-Chlorophenyl)-5-(naphthalen-2-yl)-4,5-dihydro-
1H-pyrazole-1-carbothioamide (C10)
Yellow powder, Yield 78%, mp: 170–173 °C. 1H NMR (CDCl3,
300 MHz) d: 3.21–3.26 (dd, J1 = 11.1 Hz, J2 = 2.4 Hz, 1H), 3.95–
4.01 (dd, J1 = 11.1 Hz, J2 = 6.9 Hz, 1H), 6.07–6.10 (dd, J1 = 6.9 Hz,
J2 = 2.1 Hz, 1H), 7.28–7.30 (d, J = 4.5 Hz, 1H), 7.46–7.50 (m, 3H),
7.52–7.54 (d, J = 5.7 Hz, 1H), 7.63 (s, 1H), 7.85–7.93 (m, 5H),
8.02–8.09 (d, J = 22.2 Hz, 2H). MS (ESI): 366.08 (C20H17ClN3S,
[M+H]+). Anal. Calcd for C20H16ClN3S: C, 65.65; H, 4.41; Cl, 9.69;
N, 11.48; S, 8.76. Found: C, 65.47; H, 4.42; N, 11.49.
4.4.16. 3-(4-Fluorophenyl)-5-(naphthalen-1-yl)-1-phenyl-4,5-
dihydro-1H-pyrazole (D2)
White crystal, Yield 88%, mp: 78–81 °C. 1H NMR (CDCl3,
300 MHz) d: 3.06–3.14 (dd, J1 = 16.5 Hz, J2 = 6.0 Hz, 1H), 4.14–
4.23 (dd, J1 = 17.1 Hz, J2 = 12.6 Hz, 1H), 6.19 (m, 1H), 6.70–6.75 (t,
J = 7.2 Hz, 1H), 6.94–6.97 (d, J = 7.8 Hz, 2H), 7.12–7.17 (t,
J = 7.8 Hz, 2H), 7.22–7.28 (m, 3H), 7.39–7.44 (t, J = 7.5 Hz, 1H),
7.59–7.67 (m, 2H), 7.78–7.88 (m, 3H), 8.00–8.03 (d, J = 7.8 Hz,
1H), 8.30–8.33 (d, J = 8.4 Hz, 1H). MS (ESI): 367.15 (C25H20FN2,
[M+H]+). Anal. Calcd for C25H19FN2: C, 81.94; H, 5.23; F, 5.18; N,
7.64. Found: C, 81.78; H, 5.23; N, 7.65.
4.4.11. 3-(4-Bromophenyl)-5-(naphthalen-2-yl)-4,5-dihydro-
1H-pyrazole-1-carbothioamide (C11)
Yellow powder, Yield 74%, mp: 293–295 °C. 1H NMR (CDCl3,
300 MHz) d: 3.00–3.05 (dd, J1 = 11.9 Hz, J2 = 3.3 Hz, 1H), 4.15–
4.20 (dd, J1 = 10.8 Hz, J2 = 6.6 Hz, 1H), 5.41 (m, 1H), 7.17–7.18 (d,
J = 5.1 Hz, 1H), 7.25–7.36 (m, 4H), 7.38–7.42 (m, 1H), 7.48 (m,
1H), 7.53–7.55 (d, J = 5.1 Hz, 1H), 7.57–7.59 (d, J = 5.1 Hz, 1H),
7.62–7.64 (d, J = 5.1 Hz, 1H), 7.71–7.72 (d, J = 5.1 Hz, 1H), 7.74–
7.76 (m, 1H), 7.89 (s, 1H). MS (ESI): 410.02 (C20H17BrN3S,
[M+H]+). Anal. Calcd for C20H16BrN3S: C, 58.54; H, 3.93; Br, 19.47;
N, 10.24; S, 7.81. Found: C, 58.39; H, 3.94; N, 10.24.
4.4.17. 3-(4-Chlorophenyl)-5-(naphthalen-1-yl)-1-phenyl-4,5-
dihydro-1H-pyrazole (D3)
Yellow crystal, Yield 81%, mp: 149–153 °C. 1H NMR (CDCl3,
300 MHz) d: 3.09 (m, 1H), 4.11–4.21 (dd, J1 = 17.4 Hz, J2 = 12.3 Hz,
1H), 6.20 (m, 1H), 6.69–6.74 (t, J = 7.2 Hz, 1H), 6.93–6.95 (d,
J = 7.8 Hz, 2H), 7.10–7.16 (t, J = 7.8 Hz, 2H), 7.22 (m, 1H), 7.37–
7.46 (m, 3H), 7.59–7.65 (m, 2H), 7.74–7.76 (d, J = 8.4 Hz, 2H),
7.83–7.86 (d, J = 8.1 Hz, 1H), 7.98–8.01 (d, J = 8.4 Hz, 1H), 8.30
(m, 1H). MS (ESI): 383.12 (C25H20ClN2, [M+H]+). Anal. Calcd for
4.4.12. 3-(4-Methoxyphenyl)-5-(naphthalen-2-yl)-4,5-dihydro-
1H-pyrazole-1-carbothioamide (C12)
White powder, Yield 79%, mp: 170–172 °C. 1H NMR (CDCl3,
300 MHz) d: 3.19–3.23 (dd, J1 = 10.8 Hz, J2 = 2.1 Hz, 1H), 3.80 (s,
C25H19ClN2: C, 78.42; H, 5.00; Cl, 9.26; N, 7.32. Found: C, 78.21;
H, 5.01; N, 7.33.

W. Yang et al. / Bioorg. Med. Chem. 21 (2013) 1050–1063
1061
4.4.18. 3-(4-Bromophenyl)-5-(naphthalen-1-yl)-1-phenyl-4,5-
dihydro-1H-pyrazole (D4)
J = 4.5 Hz, 2H), 7.11–7.15 (t, J = 4.8 Hz, 2H), 7.25–7.29 (t,
J = 5.4 Hz, 2H), 7.41–7.43 (dd, J1 = 5.1 Hz, J2 = 0.9 Hz, 1H), 7.47–
7.51 (m, 2H), 7.80–7.82 (m, 2H), 7.86–7.91 (m, 4H). MS (ESI):
367.15 (C25H20FN2, [M+H]+). Anal. Calcd for C25H19FN2: C, 81.94;
H, 5.23; F, 5.18; N, 7.64. Found: C, 81.76; H, 5.24; N, 7.64.
Yellow crystal, Yield 84%, mp: 148–151 °C. 1H NMR (CDCl3,
300 MHz) d: 3.08 (m, 1H), 4.11–4.21 (dd, J1 = 17.7 Hz, J2 = 12.9 Hz,
1H), 6.19 (m, 1H), 6.69–6.74 (t, J = 7.2 Hz, 1H), 6.93–6.95 (d,
J = 7.8 Hz, 2H), 7.10–7.16 (t, J = 7.8 Hz, 2H), 7.22 (m, 1H), 7.37–
7.42 (t, J = 7.8 Hz, 1H), 7.57–7.62 (m, 3H), 7.67–7.70 (m, 3H),
7.83–7.86 (d, J = 8.4 Hz, 1H), 7.98–8.01 (d, J = 8.4 Hz, 1H), 8.30
(m, 1H). MS (ESI): 427.07 (C25H20BrN2, [M+H]+). Anal. Calcd for
4.4.24. 3-(4-Chlorophenyl)-5-(naphthalen-2-yl)-1-phenyl-4,5-
dihydro-1H-pyrazole (D10)
Yellow crystal, Yield 78%, mp: 182–184 °C. 1H NMR (CDCl3,
300 MHz) d: 3.18–3.23 (dd, J1 = 10.8 Hz, J2 = 3.9 Hz, 1H), 3.96–
4.02 (dd, J1 = 10.5 Hz, J2 = 7.5 Hz, 1H), 5.64–5.68 (dd, J1 = 7.2 Hz,
J2 = 3.9 Hz, 1H), 6.70–6.72 (t, J = 4.2 Hz, 1H), 7.05–7.07 (d,
J = 4.5 Hz, 2H), 7.12–7.15 (t, J = 4.8 Hz, 2H), 7.40–7.42 (d,
J = 5.1 Hz, 1H), 7.48–7.50 (m, 4H), 7.77–7.79 (d, J = 5.1 Hz, 2H),
7.86–7.91 (m, 4H). MS (ESI): 383.12 (C25H20ClN2, [M+H]+). Anal.
Calcd for C25H19ClN2: C, 78.42; H, 5.00; Cl, 9.26; N, 7.32. Found:
C, 78.23; H, 5.00; N, 7.33.
C25H19BrN2: C, 70.27; H, 4.48; Br, 18.70; N, 6.56. Found: C, 70.03;
H, 4.48; N, 6.56.
4.4.19. 3-(4-Methoxyphenyl)-5-(naphthalen-1-yl)-1-phenyl-4,5-
dihydro-1H-pyrazole (D5)
White crystal, Yield 79%, mp: 164–167 °C. 1H NMR (CDCl3,
300 MHz) d: 3.01–3.07 (dd, J1 = 10.8 Hz, J2 = 3.6 Hz, 1H), 3.77 (s,
3H), 4.10–4.19 (dd, J1 = 17.1 Hz, J2 = 12.3 Hz, 1H), 6.08 (m, 1H),
6.65–6.70 (t, J = 7.2 Hz, 1H), 6.89–6.97 (m, 4H), 7.08–7.13 (t,
J = 7.8 Hz, 2H), 7.25 (m, 1H), 7.37–7.42 (t, J = 7.8 Hz, 1H), 7.56–
7.70 (m, 4H), 7.82–7.85 (d, J = 8.1 Hz, 1H), 7.98–8.01 (d,
J = 8.1 Hz, 1H), 8.28–8.31 (d, J = 8.1 Hz, 1H). MS (ESI): 379.17
(C26H23N2O, [M+H]+). Anal. Calcd for C26H22N2O: C, 82.51; H,
5.86; N, 7.40; O, 4.23. Found: C, 82.34; H, 5.87; N, 7.41.
4.4.25. 3-(4-Bromophenyl)-5-(naphthalen-2-yl)-1-phenyl-4,5-
dihydro-1H-pyrazole (D11)
Yellow crystal, Yield 81%, mp: 200–201 °C. 1H NMR (CDCl3,
300 MHz) d: 3.18–3.22 (dd, J1 = 10.5 Hz, J2 = 3.9 Hz, 1H), 3.96–
4.02 (dd, J1 = 10.5 Hz, J2 = 7.5 Hz, 1H), 5.64–5.68 (dd, J1 = 7.5 Hz,
J2 = 3.9 Hz, 1H), 6.70–6.72 (t, J = 4.2 Hz, 1H), 7.05–7.07 (d,
J = 4.8 Hz, 2H), 7.12–7.15 (t, J = 4.8 Hz, 2H), 7.40–7.42 (d,
J = 4.8 Hz, 1H), 7.48–7.50 (m, 2H), 7.62–7.63 (d, J = 5.1 Hz, 2H),
7.70–7.72 (d, J = 5.1 Hz, 2H), 7.85–7.91 (m, 4H). MS (ESI): 427.07
(C25H20BrN2, [M+H]+). Anal. Calcd for C25H19BrN2: C, 70.27; H,
4.48; Br, 18.70; N, 6.56. Found: C, 70.09; H, 4.48; N, 6.57.
4.4.20. 5-(Naphthalen-1-yl)-1-phenyl-3-(p-tolyl)-4,5-dihydro-
1H-pyrazole (D6)
White crystal, Yield 77%, mp: 154–157 °C. 1H NMR (CDCl3,
300 MHz) d: 2.30 (s, 3H), 3.00–3.08 (dd, J1 = 17.4 Hz, J2 = 6.3 Hz,
1H), 4.10–4.20 (dd, J1 = 17.4 Hz, J2 = 12.6 Hz, 1H), 6.13 (m, 1H),
6.67–6.71 (t, J = 6.6 Hz, 1H), 6.91–6.94 (d, J = 8.4 Hz, 2H), 7.09–
7.14 (t, J = 7.8 Hz, 2H), 7.19–7.22 (m, 3H), 7.36–7.42 (t, J = 7.8 Hz,
1H), 7.56–7.64 (m, 4H), 7.82–7.85 (d, J = 8.4 Hz, 1H), 7.98–8.01
(d, J = 7.5 Hz, 1H), 8.28–8.30 (d, J = 7.5 Hz, 1H). MS (ESI): 363.18
(C26H23N2, [M+H]+). Anal. Calcd for C26H22N2: C, 86.15; H, 6.12;
N, 7.73. Found: C, 85.98; H, 6.11; N, 7.73.
4.4.26. 3-(4-Methoxyphenyl)-5-(naphthalen-2-yl)-1-phenyl-4,5-
dihydro-1H-pyrazole (D12)
White crystal, Yield 84%, mp: 162–164 °C. 1H NMR (CDCl3,
300 MHz) d: 3.14–3.19 (dd, J1 = 10.5 Hz, J2 = 4.2 Hz, 1H), 3.80 (s,
3H), 3.95–4.00 (dd, J1 = 10.5 Hz, J2 = 7.2 Hz, 1H), 5.55–5.59 (dd,
J1 = 7.2 Hz, J2 = 3.9 Hz, 1H), 6.66–6.69 (t, J = 4.2 Hz, 1H), 6.99–7.03
(m, 4H), 7.10–7.13 (t, J = 4.8 Hz, 2H), 7.41–7.43 (d, J = 4.8 Hz, 1H),
7.47–7.51 (m, 2H), 7.70–7.72 (d, J = 5.4 Hz 2H), 7.86–7.91 (m,
4H). MS (ESI): 379.17 (C26H23N2O, [M+H]+). Anal. Calcd for
4.4.21. 3-(3,4-Dichlorophenyl)-5-(naphthalen-1-yl)-1-phenyl-
4,5-dihydro-1H-pyrazole (D7)
Yellow crystal, Yield 82%, mp: 164–166 °C. 1H NMR (CDCl3,
300 MHz) d: 3.07–3.15 (dd, J1 = 17.7 Hz, J2 = 6.3 Hz, 1H), 4.09–
4.19 (dd, J1 = 17.7 Hz, J2 = 12.9 Hz, 1H), 6.22 (m, 1H), 6.71–6.76 (t,
J = 7.2 Hz, 1H), 6.95–6.98 (d, J = 8.1 Hz, 2H), 7.11–7.16 (t,
J = 7.8 Hz, 2H), 7.22 (m, 1H), 7.36–7.41 (t, J = 7.8 Hz, 1H), 7.57–
7.64 (m, 3H), 7.71–7.74 (dd, J1 = 8.4 Hz, J2 = 1.8 Hz, 1H), 7.83–7.86
(d, J = 8.1 Hz, 1H), 7.92 (s, 1H), 7.98–8.01 (d, J = 8.4 Hz, 1H), 8.26
(m, 1H). MS (ESI): 417.08 (C25H19Cl2N2, [M+H]+). Anal. Calcd for
C26H22N2O: C, 82.51; H, 5.86; N, 7.40; O, 4.23. Found: C, 82.33; H,
5.86; N, 7.41.
4.4.27. 5-(Naphthalen-2-yl)-1-phenyl-3-(p-tolyl)-4,5-dihydro-
1H-pyrazole (D13)
White crystal, Yield 81%, mp: 142–144 °C. 1H NMR (CDCl3,
300 MHz) d: 2.34 (s, 3H), 3.15–3.20 (dd, J1 = 10.5 Hz, J2 = 4.2 Hz,
1H), 3.95–4.01 (dd, J1 = 10.8 Hz, J2 = 7.2 Hz, 1H), 5.58–5.62 (dd,
J1 = 7.2 Hz, J2 = 3.9 Hz, 1H), 6.67–6.70 (t, J = 4.2 Hz, 1H), 7.03–7.05
(d, J = 4.5 Hz, 2H), 7.11–7.14 (t, J = 4.8 Hz, 2H), 7.24–7.26 (d,
J = 4.8 Hz, 2H), 7.40–7.42 (dd, J1 = 5.1 Hz, J2 = 0.9 Hz, 1H), 7.47–
7.51 (m, 2H), 7.66–7.67 (d, J = 4.8 Hz, 2H), 7.86–7.91 (m, 4H). MS
(ESI): 363.18 (C26H23N2, [M+H]+). Anal. Calcd for C26H22N2: C,
86.15; H, 6.12; N, 7.73. Found: C, 85.92; H, 6.12; N, 7.73.
C25H18Cl2N2: C, 71.95; H, 4.35; Cl, 16.99; N, 6.71. Found: C,
71.72; H, 4.36; N, 6.72.
4.4.22. 5-(Naphthalen-2-yl)-1,3-diphenyl-4,5-dihydro-1H-
pyrazole (D8)
White crystal, Yield 83%, mp: 134–136 °C. 1H NMR (CDCl3,
300 MHz) d: 3.18–3.23 (dd, J1 = 10.5 Hz, J2 = 3.9 Hz, 1H), 3.98–
4.04 (dd, J1 = 10.5 Hz, J2 = 7.5 Hz, 1H), 5.62–5.66 (dd, J1 = 7.2 Hz,
J2 = 3.9 Hz, 1H), 6.68–6.71 (t, J = 4.5 Hz, 1H), 7.05–7.07 (d,
J = 4.8 Hz, 2H), 7.12–7.15 (t, J = 4.8 Hz, 2H), 7.36–7.51 (m, 6H),
7.77–7.78 (d, J = 5.1 Hz, 2H), 7.87–7.91 (m, 4H). MS (ESI): 349.16
(C25H21N2, [M+H]+). Anal. Calcd for C25H20N2: C, 86.17; H, 5.79;
N, 8.04. Found: C, 85.99; H, 5.79; N, 8.05.
4.4.28. 3-(3,4-Dichlorophenyl)-5-(naphthalen-2-yl)-1-phenyl-
4,5-dihydro-1H-pyrazole (D14)
Yellow crystal, Yield 83%, mp: 169–172 °C. 1H NMR (CDCl3,
300 MHz) d: 3.21–3.26 (dd, J1 = 10.5 Hz, J2 = 3.9 Hz, 1H), 3.96–
4.02 (dd, J1 = 10.5 Hz, J2 = 7.5 Hz, 1H), 5.69–5.73 (dd, J1 = 7.5 Hz,
J2 = 3.9 Hz, 1H), 6.71–6.74 (t, J = 4.2 Hz, 1H), 7.07–7.09 (d,
J = 4.8 Hz, 2H), 7.13–7.16 (t, J = 4.5 Hz, 2H), 7.39–7.41 (dd,
J1 = 5.1 Hz, J2 = 0.9 Hz, 1H), 7.47–7.51 (m, 2H), 7.67–7.69 (d,
J = 4.8 Hz, 1H), 7.73–7.76 (dd, J1 = 5.1 Hz, J2 = 1.2 Hz, 1H), 7.85–
7.91 (m, 4H), 7.94 (s, 1H). MS (ESI): 417.08 (C25H19Cl2N2,
[M+H]+). Anal. Calcd for C25H18Cl2N2: C, 71.95; H, 4.35; Cl, 16.99;
N, 6.71. Found: C, 71.77; H, 4.35; N, 6.71.
4.4.23. 3-(4-Fluorophenyl)-5-(naphthalen-2-yl)-1-phenyl-4,5-
dihydro-1H-pyrazole (D9)
White crystal, Yield 86%, mp: 125–127 °C. 1H NMR (CDCl3,
300 MHz) d: 3.18–3.23 (dd, J1 = 10.5 Hz, J2 = 4.2 Hz, 1H), 3.97–
4.03 (dd, J1 = 10.5 Hz, J2 = 7.5 Hz, 1H), 5.61–5.65 (dd, J1 = 7.5 Hz,
J2 = 4.2 Hz, 1H), 6.68–6.71 (t, J = 4.2 Hz, 1H), 7.04–7.06 (d,

1062
W. Yang et al. / Bioorg. Med. Chem. 21 (2013) 1050–1063
4.5. Anti-proliferation assay
in the enzyme preparation are fairly low, the majority of the signal
detected by the anti-phosphotyrosine antibody is from EGFR or
HER-2. The experiment was performed in triplicate.
MCF-7 human breast cancer cells and A549 human lung cancer
cells were cultured in DMEM/10% fetal bovine serum, in 5% CO2
water saturated atmosphere at 37 °C. Cell suspensions (10,000/
4.6.2. HER-3 inhibitory assay
mL) were prepared and 100
(Costar) giving 1000 cells/well. The plates were returned to the
incubator for 24 h to allow the cells to reattach. These compounds
l
L/well dispensed into 96-well plates
A 1.8 Kb cDNA encoded for human HER-3 cytoplasmic domain
(HER-3-CD, amino acids 665–1339) was applied to perform the
experiment using the same method as EGFR/HER-2.
were initially prepared at 20 mM in DMSO. Aliquots (200
diluted into 20 mL culture medium giving 200 M, and 10 serial
dilutions of 3Â prepared. Aliquots (100 L) of each dilution were
added to the wells, giving doses ranging from 100 to
0.005 M. After a further incubated at 37 °C for 24 h in a humidi-
lL) were
l
4.6.3. VEGFR-2 inhibitory assay
l
In a 96-well plate, 5 nM/L of either the phosphorylated or
nonphosphorylated form of the VEGFR-2 kinase domain was
incubated with compounds C5, C13, C14, D5, D13, D14 (10-point
l
M
l
fied atmosphere with 5% CO2, the cell viability was assessed by
the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide (MTT) reduction assay and carried out strictly
according to the manufacturer instructions (Sigma). The absor-
bance at 590 nm was recorded using LX300 Epson Diagnostic mi-
cro-plate reader. Then GI50 was calculated using SPSS 13.0
software.
titration ranging from 4 lM to 0.1 lM), 1 lM/L gastrin substrate,
20 mM/L Tris–HCl, pH 7.5, 10 mM/L MgCl2, 100 mM/L NaCl,
1.5 mM/L EGTA, 1 mM/L DTT, 0.2 mM/L sodium orthovanadate,
and 20
concentration of 11.8
l
g/mL BSA for 30 min at 25 °C. ATP was added at a final
M and incubated for 60 min at room
l
temperature. Eu-labeled antiphosphotyrosine pT66 antibody
(Perkin–Elmer) was then used for detection. The fluorescence
at 665 nM was measured with a Wallac Victor plate reader using
4.6. Inhibitory assay
a
time delay of 50
ls. The experiment was performed in
triplicate.
4.6.1. EGFR/HER-2 inhibitory assay
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 5’ 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,
4.6.4. FAK inhibitory assay
Compounds C5, C13, C14, D5, D13 and D14 were tested against
FAK. In a typical study, human-recombinant full-length FAK was
incubated in kinase buffer containing ATP and the substrate for
4 h at room temperature with or without the presence of the cor-
responding coumpounds. The final concentration of drug as 100,
30, 10, 3 lg/mL. The remaining ATP in solution was then quantified
utilizing the Kinase-Glo-luminescence kit (Promega).
10
l
M ammonium molybdate, 100
l
M sodium vanadate, 10
l
l
g/mL
g/mL
4.7. Western blotting
aprotinin, 10
l
g/mL leupeptin, 10
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.
Histidine-tagged proteins were eluted with 250 and 500 mM imid-
azole and dialyzed against 50 mM NaCl, 20 mM HEPES, 10% glyc-
After incubation with compound, cells were harvested and
washed with PBS, then lysed in lysis buffer (30 mm Tris, pH 7.5,
150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4,
1% Nonidet P-40, 10% glycerol, and phosphatase and protease
inhibitors). After centrifugation at 12,000 g for 5 min, the superna-
tant was collected as total protein. The concentration of the protein
was determined by a BCATM protein assay kit (Pierce, Rockford, IL,
USA). The protein samples were separated by 10% SDS–PAGE and
subsequently electrotransferred onto a polyvinylidene difluoride
membrane (Millipore, Bedford, MA, USA). The membrane was
blocked with 5% non-fat milk for 2 h at room temperature. The
blocked membrane was probed with the indicated primary anti-
bodies overnight at 4 °C, and then incubated with a horse radish
peroxidase (HRP)-coupled secondary antibody. P-EGFR, EGFR,
p-Akt (pS473) and Akt were measured as shown in Figure 3C.
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.29
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 C1–C14 and D1–D14 were dissolved in
100% DMSO and diluted to the appropriate concentrations with
25 mM HEPES at pH 7.4. In each well, 10
bated with 10 L (12.5 ng for HER-2 or 5 ng for EGFR) of recombi-
nant enzyme (1:80 dilution in 100 mM HEPES) for 10 min at room
temperature. Then, 10
L of 5 Â buffer (containing 20 mM HEPES,
2 mM MnCl2, 100 M Na3VO4, and 1 mM DTT) and 20 L of
lL of compound was incu-
l
l
4.8. Crystal structure determination
l
l
0.1 mM ATP-50 mM MgCl2 was 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 aspi-
rated and plates were washed three times with wash buffer. A
Crystal structure determination of compounds D5, C10 was car-
ried out on a Nonius CAD4 diffractometer (Fig. 3D). And the data
were listed in Table 3. The structure was solved by direct methods
and refined on F2 by full-matrix least-squares methods using
SHELX-97.31 All non-hydrogen atoms of compounds D5, C10 were
refined with anisotropic thermal parameters. All hydrogen atoms
were placed in geometrically idealized positions and constrained
to ride on their parent atoms.
75 lL (400 ng) sample of europium-labeled 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% À [(negative control)/(positive control À negative
control)]. The IC50 was obtained from curves of percentage inhibi-
tion with eight concentrations of compound. As the contaminants
4.9. Docking study
The three-dimensional structures of the aforementioned com-
pounds were constructed using Chem. 3D ultra 12.0 software

W. Yang et al. / Bioorg. Med. Chem. 21 (2013) 1050–1063
1063
[Chemical Structure Drawing Standard; Cambridge Soft corpora-
tion, 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 domain bound to Erlotinib (PDB
Code: 1M17.pdb)27 and HER-2 domain bound to 03Q (PDB Code:
3PP0.pdb)28 complexes 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 hydro-
gen was added to the proteins. Molecular docking of all twenty-
eight compounds was then carried out using the Discovery Stutio
(version 3.1) as implemented through the graphical user interface
CDocker protocol.
The selected test compounds were: C5, C8, C10, C13, D1, D13 and
D14.
The inhibitory activity of the compounds in literatures [IC50
(mol/L)] was initially changed into the minus logarithmic scale
[pIC50 (mol/L)] and then used for subsequent QSAR analysis as
the response variable.
In Discovery Studio, the CHARMm force field is applied and the
electrostatic potential together with the Van der Waals potential
are treated as separate terms. As the electrostatic potential probe,
A + le point change is used while distance-dependent dielectric con-
stant is used to mimic the solvent effect. As for the Van der Waals po-
tential, a carbon atom with a radius of 1.73 Å is used as a probe.
A Partial Least-Squares (PLS) model is built using energy grids
as descriptors. QSAR models were built by using the Create 3D
QSAR Model protocol in Discovery Studio 3.1.
CDOCKER is an implementation of a CHARMm based molecular
docking tool using a rigid receptor,30 including the following steps:
(1) A series of ligands conformations are generated using high
temperature molecular dynamics with different random
seeds.
Acknowledgement
(2) Random orientations of the conformations are generated by
translating the center of the ligand to a specified position
within the receptor active site, and making a series of ran-
dom rotations. A softened energy is calculated and the orien-
tation is kept when it is less than a specified limit. This
process repeats until either the desired number of low-
energy orientations is obtained, or the test times of bad ori-
entations reached the maximum number.
(3) Each orientation is subjected to simulated annealing molec-
ular dynamics. The temperature is heated up to a high tem-
perature then cooled to the target temperature. A final
energy minimization of the ligand in the rigid receptor using
non-softened potential is performed.
(4) For each of the final pose, the CHARMm energy (interaction
energy plus ligand strain) and the interaction energy alone
are figured out. The poses are sorted according to CHARMm
energy and the top scoring (most negative, thus favorable to
binding) poses are retained. The whole kinase domain
defined as a receptor and the site sphere was selected based
on the original ligand binding location, then the original
ligand was removed and the ligands prepared by us were
placed during the molecular docking procedure. CHARMm
was selected as the force field. The molecular docking was
performed with a simulated annealing method. The heating
steps were 2000 with 700 of heating target temperature. The
cooling steps were 5000 with 300 cooling target tempera-
ture. Ten molecular docking poses saved for each ligand
were ranked according to their dock score function. The pose
with the highest-CDocker energy was chosen as the most
suitable pose.
This work was financed by National Natural Science Foundation
of China (No. J1103512).
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In the primary model, among all the 30 compounds, 80% (that is
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protocol in Discovery Studio 3.1. The selected test compounds
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article).
In the secondary model, 75% (that is 21) of the 28 compounds
were utilized as a training set for QSAR modeling. The remaining
25% (that is 7) were chosen as test subset using the same protocol.
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