Design, biological evaluation and 3D QSAR studies of novel dioxin-containing triaryl pyrazoline derivatives as potential B-Raf inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.05.012

A series of novel dioxin-containing triaryl pyrazoline derivatives C1-C20 have been synthesized. Their B-Raf inhibitory and anti-proliferation activities were evaluated. Compound C6 displayed the most potent biological activity against B-Raf^V600E

Full text of DOI:10.1016/j.bmc.2016.05.012

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, biological evaluation and 3D QSAR studies of novel dioxin-
containing triaryl pyrazoline derivatives as potential B-Raf inhibitors
a,
Yu-Shun Yang a,b,y, Bing Yang b,y, Yan Zou a,b, Guigen Li b, , Hai-Liang Zhu
⇑
⇑
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210023, China
b Institute of Chemistry and BioMedical Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel dioxin-containing triaryl pyrazoline derivatives C1–C20 have been synthesized. Their B-
Raf inhibitory and anti-proliferation activities were evaluated. Compound C6 displayed the most potent
biological activity against B-RafV600E and WM266.4 human melanoma cell line with corresponding IC50
Received 17 April 2016
Revised 10 May 2016
Accepted 11 May 2016
Available online xxxx
value of 0.04 lM and GI50 value of 0.87 lM, being comparable with the positive controls and more potent
than our previous best compounds. Moreover, C6 was selective for B-RafV600E from B-RafWT, C-Raf and
EGFR and low toxic. The docking simulation suggested the potent bioactivity might be caused by breaking
the limit of previous binding pattern. A new 3D QSAR model was built with the activity data and binding
conformations to conduct visualized SAR discussion as well as to introduce new directions. Stretching the
backbone to outer space or totally reversing the backbone are both potential orientations for future
researches.
Keywords:
Break limit
Triaryl pyrazoline
Dioxin
Antitumor activity
B-Raf
Ó 2016 Elsevier Ltd. All rights reserved.
Selective
3D QSAR
1. Introduction
secretion.13,14 Therefore B-Raf has been considered as a hotspot
in designing anticancer agents.15–17
Thousands of times researchers claimed breakthroughs on
treating cancer, yet cancer continues to play the role of health ris-
ker all over the world, ranked second in causing mortality.1 Still,
people are looking forward to novel anticancer agents and thera-
peutic methods although huge progress in medicine has been
made.
Now Vemurafenib is the top B-Raf inhibitor with FDA approval,
but attempts in seeking alternative backbones to break the fixed
structural limitation never stop.18 Previous reporters picked out
triarylimidazole derivative SB-590885 as
a
potent B-Raf
inhibitor.19 There exists a key interaction between heterocyclic
rings (both imidazole and pyridine) of SB-590885 and PHE583 of
B-Raf.19 To avoid the 7-azaindole moiety of Vemurafenib and elim-
inate the corresponding side effect, dihydropyrazole derivatives
have been screened and convinced to be potent and selective inhi-
As an important pathway for cell proliferation and survival,2
Ras-Raf-MEK-ERK serine threonine kinase cascade, also called
ERK/MAP kinase pathway or ‘classical’ MAPK pathway, has been
reported to be hyper-activated in up to 30% of human cancers.3–6
Especially, activating mutations in Raf have been observed most,
50–70% of cell lines and tumors in melanoma, then 40–70% in thy-
roid cancer, 50–70% in ovarian cancer.7–9 Researchers regard B-Raf
as a most important isoform of Raf kinases because approximately
90% of its activating mutations in cancers are valine for glutamic
acid substitution (V600E, formally defined as V599E),7,10,11 which
can cause a 500-fold increase in the basal rate of MEK phosphory-
lation over wild-type B-Raf12 and consequently result in stimulat-
ing tumor growth and vascular endothelial growth factor
bitors of B-RafV600E 20,21
.
In our previous studies, a certain pattern of 1,3,5-triaryl-pyrazo-
line derivatives as B-Raf inhibitor has been screened out.22,23
Although the top compound was quite close to the positive control,
the refined substitutes on 1, 3, 5 positions inferred the worry that
this pattern might be reaching its own limitation. Meanwhile, we
cannot ignore the dioxin moiety because it was the best choice
on 1-position (ring C) of pyrazoline. Fortunately, in this pattern
3-position (ring A) and 1-position (ring C) were nearly in one plane
while 5-position (ring B) had an angle with this plane. Meanwhile,
a parallel report suggested the possibility of defaulting carbonyl.24
Thus, here we do the challenging work to interchange ring A and
ring C to break the limitation of former backbone, hoping to
approach an appreciating situation.
⇑
Corresponding authors. Tel./fax: +86 25 89682572.
E-mail addresses: guigenli@nju.edu.cn (G. Li), zhuhl@nju.edu.cn (H.-L. Zhu).
Both authors contributed equally to the work.
y
http://dx.doi.org/10.1016/j.bmc.2016.05.012
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yang, Y.-S.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.05.012

2
Y.-S. Yang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
a same substitute on para-position seemed more potent than on
ortho-position (C10 > C4). As for meta-position, slight
tendency was ACl > AOMe, indicating that an electron-
2. Results and discussion
2.1. Chemistry
a
withdrawing group might be better choice here. The correspond-
ing compounds were C7 (IC50 = 0.15 lM) > C5 (IC50 = 0.21 lM).
3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-aryl-1-phenyl-4,5-
dihydro-1H-pyrazole derivatives C1–C20 were synthesized and
screened for their antitumor activity. All of them were synthesized
for the first time except C1 whose synthesis was reported by Jon-
aitis group and Dauksas group respectively in 1960s.25,26 Their
bioactivities were tested for the first time. The general synthesis
method and the structures of compounds C1–C20 were organized
in Scheme 1. They were all prepared in two steps. Firstly, different
substituted benzaldehydes on treatment with 1-(2,3-dihydrobenzo
[b][1,4]dioxin-6-yl)ethan-1-one in presence of 50% NaOH were
stirred at room temperature till reactions completed, yielding dif-
ferent analogues of chalcones (B). Secondly, phenylhydrazine was
added to participate the cyclization of the obtained powder, lead-
ing to the corresponding target compounds C1–C20 3-(2,3-dihy-
drobenzo[b][1,4]dioxin-6-yl)-5-aryl-1-phenyl-4,5-dihydro-1H-
pyrazoles. Subsequent purification with recrystallisation was con-
ducted and the refined compounds were finally obtained. All of the
synthetic compounds gave satisfactory analytical and spectro-
scopic data, which were in full accordance with their depicted
structures.
As for para-position, the inferred order was AN(Me)2 >
AMe > ABr > AI > AF > ASMe > AH > ACl > ACF3 > AOMe, indicat-
ing that a steric suitable substitute with slighter electronic influ-
ence should be recommended in future designing. The
corresponding compounds were C16 (IC50 = 0.07 lM) > C8
(IC50 = 0.61
(IC50 = 3.88
(IC50 = 10.7
l
l
l
M) > C9 (IC50 = 0.86
M) > C14 (IC50 = 4.27
M) > C17 (IC50 = 36.3
l
l
M) > C13 (IC50 = 1.14
M) > C1 (IC50 = 8.05
l
M) > C10
lM) > C11
l
M) > C12 (IC50 = 118 lM).
4-Dimethylamino showed an accidentally potency might because
it was more like meta-substitutes. Meanwhile, as for multi substi-
tutes, if they were on both para- and meta-positions, the effect was
between a single meta-one and a single para-one. The correspond-
ing compounds with ACl and AOMe were C7 (IC50 = 0.15
> C18 (IC50 = 0.27 M) > C11 (IC50 = 10.7 M) and C5 (IC50 = 0.21 -
M) > C19 (IC50 = 0.54 M) > C12 (IC50 = 118 M), respectively.
lM)
l
l
l
l
l
Secondly, naphthalene was not a fine group and Naphthalen-2-yl
group showed better effect than Naphthalen-1-yl group as C3
(IC50 = 6.85 lM) > C2 (IC50 > 300 lM). This result agreed with the
order of single substitutes (meta- > para- > ortho-). Thirdly, a hete-
rocyclic ring (here it was furan) also seemed worse than benzene
2.2. Biological activity
ring with the corresponding result C1 (IC50 = 8.05
lM) > C20
(IC50 = 83.6 M). Finally, as for benzyloxyphenyl group, both 3-
l
All the synthesized compounds C1–C20 were evaluated for their
anti-proliferation effect and B-RafV600E inhibitory activity with a
general method. The results were expressed as concentrations of
IC50 (the half maximal inhibitory concentration of B-RafV600E medi-
ated MEK phosphorylation) and GI50 (the half maximal inhibitory
concentration of WM266.4 human melanoma cell line27 growth),
presented in Table 1. To make comparison with previous
researches, we chose the same cell line WM266.4 and took two
previous best compounds C0A (named C14 in previous work)23
and C0B (named C6 in previous work)24 into the same evaluation
(both their test results and literature values). As shown in Table 1,
a majority of the compounds still kept potent B-RafV600E inhibitory
activity. It seemed that interchange ring A and ring C was an avail-
able and rational method.
OBn and 4-OBn affected the backbone. However, 3-OBn only
stretched the backbone while 4-OBn made the backbone reversed.
Despite this difference and the slightly inferior ADMET properties
(logP = 6.408, PSA = 41.666), both 3-OBn and 4-OBn showed acci-
dentally admirable activity with the corresponding results C6
(IC50 = 0.04 lM) and C15 (IC50 = 0.11 lM) respectively. That meant
the challenging design might break the limit on at least two orien-
tations which could be interesting in future study. The data were
visualized as maps and a more brief SAR analysis was displayed
in the 3D QSAR part below.
To evaluate the selectivity of this series, the kinase inhibitory
activities against B-RafWT (wild type B-Raf), C-Raf (also called
Raf1), EGFR and the anti-proliferation effect against MCF-7 human
breast cancer cell line of representative compounds (C6, C16, C15,
C7, C5 and C18) were tested. The results were provided in Table 2.
For the most potent C6, the IC50 value against B-RafV600E was
The linear regression between the GI50 values of these com-
pounds shared a similar tendency with their relevant IC50 values
0.04 l lM. This result sug-
M while that against B-RafWT was 1.06
(R square = 0.879,
a normal level) indicated the correlation
gested that C6 was relatively selective between B-RafV600E and B-
RafWT. This scale of selectivity might be further considered in ther-
apeutic method but was acceptable with appropriate dosage. From
C-Raf or EGFR, all representative compounds indicated obvious
selectivity for B-RafV600E. The anti-proliferation against MCF-7 in
which V600E mutant merely happened also convinced the selectiv-
ity of representative compounds. The moderate anti-proliferation
effect against MCF-7 might be from the moderate inhibitory activ-
ities against B-RafWT and C-Raf.
between the anti-proliferative effect and the B-Raf inhibitory
activity.
The most potent compound C6 displayed comparable activity
(IC50 = 0.04
rafenib (IC50 = 0.03
M; GI50 = 8.14 M). The B-Raf inhibitory activity of C6 seemed
slightly better than the previous best compounds C0A (IC50 = 0.11 -
M in test; IC50 = 0.11 M in literature) and C0B (IC50 = 0.14 M in
test; IC50 = 0.15 M in literature). An explanation for being less
l
M; GI50 = 0.87
l
M) with the positive controls Vemu-
l
M; GI50 = 0.21
l
M) and Erlotinib (IC50 = 0.06 -
l
l
l
l
l
l
potent than C0A on anti-proliferation might be the influence of
logP and PSA (polar surface area) by defaulting carbonyl. The
ADMET properties of C1–C20 were shown in Figure 1, in which
the properties of C6 was not top class but good enough. Introduc-
ing appropriate pharmacokinetics groups could cover this
disadvantage.
The cytotoxic activity of the compounds were evaluated on a
mouse embryonic fibroblast cell line (NIH-3T3) using the MTT
assay. The results were summarized in Table 2. It could be con-
cluded that the selected compounds with potent inhibitory activity
and high selectivity were low toxic.
After molecular overlap (Fig. 2) and bioassay results being
examined, preliminary SAR studies were provided to discuss how
structure variation might affect anticancer activity. Firstly, we
fixed R group with substituted phenyl but temporarily skipped
benzyloxyphenyl group for the tortuosity of steric backbone. A sin-
gle substitute on meta-position showed better effect than on para-
position (C7 > C11; C5 > C12, here ‘>’ means ‘better than’); whereas
2.3. Molecular docking
To visualize the possible binding model of interactions between
a protein (enzyme) and small molecules (ligands) were the molec-
ular docking techniques used.28 Molecular modelling in this study
was conducted by using CDOCKER protocol in Discovery Studio 3.5
(Discovery Studio 3.5, Accelrys, Inc. San Diego, CA). All twenty
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Y.-S. Yang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
Scheme 1. General synthesis of compounds (C1–C20). Reagents and conditions: (i) EtOH, 50% NaOH, TLC; (ii) EtOH, phenylhydrazine, reflux, overnight.
Table 1
B-RafV600E inhibitory activity and anti-proliferation activity of the synthesized compounds (C1–C20) as well as previous compounds C0A and C0B
Compounds
IC50
(
l
M)
GI50
WM266.4
(
l
M)
Compounds
IC50
(
l
M)
GI50 (lM)
WM266.4
B-RafV600E
B-RafV600E
C1
C2
C3
C4
C5
C6
C7
C8
8.05 0.73
>300
22.4 1.88
>100
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C0B
C0B(lit)
Vemurafenib
10.7 1.01
118 9.85
1.14 0.10
4.27 0.41
0.11 0.01
0.07 0.005
36.3 3.17
0.27 0.02
0.54 0.04
83.6 7.85
0.14 0.01
0.15 0.01
0.03 0.004
38.6 3.42
>100
6.85 0.57
5.17 0.49
0.21 0.02
0.04 0.003
0.15 0.01
0.61 0.05
0.86 0.13
3.88 0.34
0.11 0.01
0.11 0.02
0.06 0.01
13.9 1.16
9.12 0.83
1.81 0.15
0.87 0.07
1.75 0.16
2.53 0.22
3.01 0.28
6.05 0.56
0.75 0.06
0.58 0.07
8.14 0.78
3.47 0.29
6.87 0.64
1.13 0.11
0.97 0.08
>100
1.97 0.17
2.16 0.19
>100
C9
C10
C0A
C0A(lit)
Erlotinib
1.57 0.11
1.75 0.12
0.21 0.02
compounds were docked into the active site of the receptor B-Raf.
According to previous researches, two crystal structures of B-Raf
(PDB Code: 3PSD.pdb29 and 2FB8.pdb19) were chosen. The receptor
and ligands were prepared and the site sphere was chosen due to
the ligand binding location. The same as previous study,23 the
results of models using 3PSD and 2FB8 were almost the same
due to the generation of random conformations and the similarity
of the active sites. The CDocker Interaction Energy (interaction
energy between the ligand and the receptor) agreed with the B-
Raf inhibitory trend for all the synthesized compounds. Both 2D
and 3D maps of the most potent compound C6 with 3PSD were
depicted in Figure 3 with 2D maps of three comparisons C7, C16
and C15.
to the previous best compounds while C6 broke the limit by
stretching the backbone. Actually, C16 was also this pattern
because PHE583 was between its ring A and ring C. Although the
interaction was not so strong to form p–p interaction, a hydrogen
bonding interaction (Oꢀ ꢀ ꢀHAN: 2.15 Å, 164.566°) between dioxin
moiety and ASN580 remedied this weakness. C15 belonged to a
totally different pattern for PHE583 escaped from the plane of ring
A and ring C. The nice bioactivity might, instead, rely on p–cation
interaction between ring B and LYS483 (distance: 5.19 Å) and
hydrogen bonding interaction (Oꢀ ꢀ ꢀHAN: 2.30 Å, 126.234°)
between dioxin moiety and ASN580.
According to receptor surface model shown in Figure 4, the
molecules were well embedded in the active pocket including
VAL471, PHE583, ALA481, THR529, LEU514 and ASN581. The
stretching and distortion on ring B of C6 resulted in a deeper occu-
pation into the active pocket.
The binding pattern of C6 and C7 was similar to previous
researches with the typical
PHE583. Compound C6 formed this
p
–
p
interaction between ring A/C and
interaction between ring
p–p
A and PHE583 (distance: 6.41 Å) while the stretched ring B pos-
sessed interactions with more residues, improving the binding sit-
2.4. 3D QSAR model
uation. Compound C7 formed this
and PHE583 (distance: 4.47 Å) with
C and TRP531 (distance: 5.29 Å) and
p
–
p
p
p
–
interaction between ring C
interaction between ring
–cation interaction between
p
Since the challenging design affected the backbone, a new 3D
QSAR model was built. With the same method as our previous
researches,30 Create 3D QSAR protocol of Discovery Studio 3.5
ring B and LYS483 (distance: 5.68 Å). Thus, C7 was the nearest one
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Y.-S. Yang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Figure 1. ADMET properties predicted for the twenty compounds C1–C20. Compounds located inside the innermost oval are better for this parameter. The three compounds
outside the innermost oval were C6, C15 and C18.
The 3D QSAR model was acceptable with the correlation coeffi-
cient r2 between observed activity of testing set and training set
found to be 0.884. Molecules aligned with the iso-surfaces of the
model coefficients on electrostatic potential grids (Fig. 5-left) and
Van der Waals grids (Fig. 5-right) were listed. Electrostatic map
indicated regions where electron-withdrawing (red) or electron-
donating (blue) groups would increase activity while steric map
indicated areas where steric bulk would increase (green) or
decrease (yellow) activity. According to the maps, ring A contain-
ing dioxin had reached a relatively ideal situation while ring C
would still have large potential to be modified. A promising orien-
tation was making ring C slightly larger with electron-donating
substitutes. As for ring B, meta-position requested a larger elec-
tron-withdrawing group while para-position asked for a group
Figure 2. Molecular overlap of compounds C1–C20. C15 has
conformation.
a reversed
with appropriate size and slighter electronic influence. However,
after skipping out the residues around ring B, the outer space could
bear further modifications.
was utilized to perform the 3D QSAR of all twenty compounds
based on the definite IC50 values. By convention, the values were
changed into pIC50 scale (ꢁlogIC50). Diverse Molecules method in
Discovery Studio 3.5 was utilized to choose training set and test
set. The alignment conformation of each molecule with lowest
energy in the docked results of CDOCKER protocol was aligned
by the substructure C1. The maps of 3D QSAR model were shown
in Figure 5.
3. Conclusions
To sum up, a series of compounds (C1–C20) 3-(2,3-dihy-
drobenzo[b][1,4]dioxin-6-yl)-5-aryl-1-phenyl-4,5-dihydro-1H-pyra-
zoles have been synthesized. Their B-Raf inhibitory and
anti-proliferation activities were evaluated. Compound C6 dis-
played the most potent biological activity against B-RafV600E and
Table 2
Selectivity (against B-RafWT, C-Raf, EGFR and MCF-7 cell line) and cytotoxicity (against NIH3T3 cell line) of representative compounds (C6, C16, C15, C7, C5 and C18)
Compounds
IC50
(
l
M)
IC50
C-Raf
(
l
M)
IC50
EGFR
(
lM)
GI50
MCF-7
(
l
M)
GI50 (lM)
NIH3T3
B-RafWT
C6
1.06 0.09
0.87 0.06
2.51 0.23
1.83 0.15
2.62 0.22
3.37 0.32
6.80 0.55
5.48 0.51
5.17 0.45
14.6 1.35
22.8 2.10
8.35 0.81
7.92 0.65
>300
9.04 0.87
81.2 7.35
>300
14.8 1.20
12.1 0.95
23.6 2.21
55.9 5.15
>100
221.5 3.35
256.4 3.52
228.5 2.95
172.4 2.03
284.7 3.16
156.3 2.07
C16
C15
C7
C5
C18
192 15.4
38.3 3.51
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Y.-S. Yang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Figure 3. Docking models of representative compounds (A) 2D molecular docking modeling of compound C6 with 3PSD. (B) 3D model of the interaction between compound
C6 and 3PSD bonding site. (C) 2D molecular docking modeling of compound C7 with 3PSD. (D) 2D molecular docking modeling of compound C16 with 3PSD. (E) 2D molecular
docking modeling of compound C15 with 3PSD. The H-bonds are displayed as dotted lines. The
p–cation interactions and p–p interactions are shown as orange lines with
their corresponding amino acids labeled in yellow. Other important amino acids are labeled in blue.
WM266.4 human melanoma cell line with corresponding IC50
value of 0.04 M and GI50 value of 0.87 M, being comparable with
4. Experimental section
4.1. Chemistry
l
l
the positive controls and more potent than our previous best com-
pounds. Moreover, C6 was selective for B-RafV600E from B-RafWT, C-
Raf and EGFR and low toxic. The docking simulation suggested the
potent bioactivity might be caused by breaking the limit of previ-
ous binding pattern. A new 3D QSAR model was built with the
activity data and binding conformations to conduct visualized
SAR discussion as well as to introduce new directions. Stretching
the backbone to outer space or totally reversing the backbone are
both potential orientations for future researches.
4.1.1. General
All commercially available chemicals were used as received
without further purification. Melting points were determined on
a WRS-1C digital melting point apparatus and were uncorrected.
1H NMR, 13C NMR and 19F NMR spectra were recorded on a Bruker
Avance II 400 spectrometer. Shifts are reported in parts per million
based on residual solvent peaks (for 1H or 13C/CDCl3). Shifts in 19F
Please cite this article in press as: Yang, Y.-S.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.05.012

6
Y.-S. Yang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
126.6, 127.6, 129.0, 129.2, 142.9, 143.7, 144.4, 145.2, 146.6; HRMS
(ESI-TOF) m/z: [M+H]+ Calcd for C23H21N2O2 357.1598, Found
357.1594.
4.1.2.4. 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(naphthalen-
1-yl)-1-phenyl-4,5-dihydro-1H-pyrazole (C2).
Yellow solid;
mp 112–114 °C; yield: 81%; 1H NMR (400 MHz, CDCl3) d 3.10
(dd, 1H, J = 16.4, 7.6 Hz), 4.01 (dd, 1H, J = 16.8, 12.8 Hz), 4.27 (s,
4H), 5.89–5.93 (m, 1H), 6.76 (t, 1H, J = 7.2 Hz), 6.85 (d, 1H,
J = 8.4 Hz), 7.03 (d, 2H, J = 8.0 Hz), 7.15 (t, 2H, J = 7.6 Hz), 7.22–
7.26 (m, 2H), 7.35–7.44 (m, 2H), 7.54–7.64 (m, 2H), 7.78 (d, 1H,
J = 8.0 Hz), 7.94 (d, 1H, J = 8.0 Hz), 8.10 (d, 1H, J = 8.4 Hz); 13C
NMR (100 MHz, CDCl3) d 43.0, 64.5, 64.7, 113.4, 114.9, 117.5,
119.0, 119.5, 123.1, 125.9, 126.1, 126.5, 126.6, 128.1, 129.1,
129.4, 130.1, 134.5, 136.9, 143.7, 144.5, 145.3, 147.1; HRMS (ESI-
TOF) m/z: [M+H]+ Calcd for C27H23N2O2 407.1754, Found 407.1751.
Figure 4. The receptor surface model with C6 in 3PSD.
NMR spectra were reported based on an external hexafluoroben-
zene reference. NMR data were resolved with MestreNova soft-
ware. Mass spectra were obtained from an Agilent 6540 UHD
Accurate Mass Q-TOF LC/MS.
4.1.2.5. 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(naphthalen-
2-yl)-1-phenyl-4,5-dihydro-1H-pyrazole (C3).
Yellow solid;
mp 171–172 °C; yield: 88%; 1H NMR (400 MHz, CDCl3) d 3.15
(dd, 1H, J = 17.2, 7.6 Hz), 3.85 (dd, 1H, J = 17.2, 12.4 Hz), 4.28 (s,
4H), 5.38 (dd, 1H, J = 17.2, 12.4 Hz), 6.75 (t, 1H, J = 7.2 Hz), 6.88
(d, 1H, J = 8.0 Hz), 7.08–7.17 (m, 4H), 7.24–7.26 (m, 2H), 7.43–
7.49 (m, 3H), 7.79–7.84 (m, 4H); 13C NMR (100 MHz, CDCl3) d
43.9, 64.5, 64.7, 65.0, 113.5, 114.9, 117.6, 119.1, 119.5, 124.0,
124.8, 126.1, 126.5, 126.6, 127.9, 128.0, 129.0, 129.4, 133.1,
133.7, 140.3, 143.7, 144.5, 145.4, 146.7; HRMS (ESI-TOF) m/z: [M
+H]+ Calcd for C27H23N2O2 407.1754, Found 407.1753.
4.1.2. Compounds
4.1.2.1. General method of synthesis of (E)-chalcones
(B).
To 1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)ethan-1-one
(A) (1 mmol) alcohol solution (5 mL) was added substituted ben-
zaldehyde (1 mmol). After dissolution, 50% NaOH (0.5 mL) was
added. After confirming the completion of the reaction by thin
layer chromatography, the sediment was filtered, washed with
ethanol and dried to obtain chalcone (B).
4.1.2.6.
3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(2-fluo-
Yellow
rophenyl)-1-phenyl-4,5-dihydro-1H-pyrazole (C4).
solid; mp 139–141 °C; yield: 82%; 1H NMR (400 MHz, CDCl3) d
3.06 (dd, 1H, J = 16.8, 6.4 Hz), 3.84 (dd, 1H, J = 17.2, 12.4 Hz), 4.28
(s, 4H), 5.54 (dd, 1H, J = 12.4, 6.8 Hz), 6.77–6.80 (m, 1H), 6.87 (d,
1H, J = 8.4 Hz), 7.01–7.04 (m, 3H), 7.09–7.13 (m, 1H), 7.17–7.26
(m, 5H); 13C NMR (100 MHz, CDCl3) d 42.5, 58.7 (d, J = 3.0 Hz),
64.5, 64.7, 113.2, 114.9, 115.6, 115.8, 117.5, 119.1, 119.5, 124.9
(d, J = 4.0 Hz), 126.4, 127.7 (d, J = 4.0 Hz), 129.1 (d, J = 4.0 Hz),
129.3 (d, J = 6.0 Hz), 143.7, 144.5, 144.9, 147.1, 158.7, 161.1; 19F
NMR (376.38 MHz, CDCl3) d ꢁ118.90; HRMS (ESI-TOF) m/z: [M
+H]+ Calcd for C23H20FN2O2 375.1503, Found 375.1502.
4.1.2.2. General method of synthesis of 3-(2,3-dihydrobenzo[b]
[1,4]dioxin-6-yl)-5-aryl-1-phenyl-4,5-dihydro-1H-pyrazole (C1–
C20).
Chalcone B (0.5 mmol) and phenylhydrazine (0.5 mmol)
in ethanol (5 mL) were refluxed overnight. After the reaction com-
pleted, the resultant solid was filtered, washed with ethanol and
dried to obtain the corresponding target compound C. In some
cases, ultrasonic vibration can contribute to precipitation.
4.1.2.3. 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1,5-diphenyl-
4,5-dihydro-1H-pyrazole (C1).
Yellow solid; mp 135–
137 °C; yield: 87%; 1H NMR (400 MHz, CDCl3) d 3.08 (dd, 1H,
J = 16.8, 7.2 Hz), 3.78 (dd, 1H, J = 16.8, 12.4 Hz), 4.29 (s, 4H), 5.22
(dd, 1H, J = 12.4, 7.2 Hz), 6.76 (t, 1H, J = 7.2 Hz), 6.87 (d, 1H,
J = 8.4 Hz), 7.04 (d, 2H, J = 8.0 Hz), 7.14–7.18 (m, 2H), 7.22–7.27
(m, 3H), 7.30–7.35 (m, 4H); 13C NMR (100 MHz, CDCl3) d 43.9,
64.5, 64.7 (d, J = 2.0 Hz), 113.4, 114.9, 117.5, 119.0, 119.5, 126.0,
4.1.2.7. 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(3-methoxy-
phenyl)-1-phenyl-4,5-dihydro-1H-pyrazole
(C5).
Yellow
solid; mp 125–127 °C; yield: 85%; 1H NMR (400 MHz, CDCl3) d
3.08 (dd, 1H, J = 16.8, 7.2 Hz), 3.73–3.80 (m, 4H), 4.28 (s, 4H),
5.17 (dd, 1H, J = 12.0, 7.2 Hz), 6.75–6.81 (m, 2H), 6.86–6.92 (m,
Figure 5. 3D-QSAR of compounds C1–C20. In the maps red contours mean electron-withdrawing group is expected to increase activity while blue contours mean electron-
donating group is better. Green areas mean steric bulk is better while yellow areas mean small groups are helpful.
Please cite this article in press as: Yang, Y.-S.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.05.012

Y.-S. Yang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
3H), 7.05 (d, 2H, J = 8.4 Hz), 7.17 (t, 2H, J = 8.0 Hz), 7.22–7.26 (m,
3H); 13C NMR (100 MHz, CDCl3) d 43.9, 55.4, 64.5, 64.7, 64.8,
111.5, 113.1, 113.5, 114.9, 117.5, 118.3, 119.1, 119.5, 126.6,
129.0, 130.3, 143.7, 144.4, 144.6, 145.4, 146.7, 160.4; HRMS
(ESI-TOF) m/z: [M+H]+ Calcd for C24H23N2O3 387.1703, Found
387.1707.
4.1.2.13.
dioxin-6-yl)-1-phenyl-4,5-dihydro-1H-pyrazole
(C11).
White solid; mp 116–118 °C; yield: 85%; 1H NMR
(400 MHz, CDCl3) d 3.03 (dd, 1H, J = 16.8, 7.2 Hz), 3.81 (dd, 1H,
J = 17.2, 12.4 Hz), 4.28 (s, 4H), 5.20 (dd, 1H, J = 12.0, 7.2 Hz), 6.78
(t, 1H, J = 7.2 Hz), 6.87 (d, 1H, J = 8.0 Hz), 7.01 (d, 2H, J = 8.0 Hz),
7.15–7.31 (m, 8H); 13C NMR (100 MHz, CDCl3) d 43.8, 64.0, 64.5,
64.7, 113.4, 114.9, 117.6, 119.3, 119.5, 126.4, 127.5, 129.1, 129.4,
133.4, 141.3, 143.7, 144.5, 145.0, 146.6; HRMS (ESI-TOF) m/z: [M
+H]+ Calcd for C23H20ClN2O2 391.1208, Found 391.1211.
5-(4-Chlorophenyl)-3-(2,3-dihydrobenzo[b][1,4]-
4.1.2.8. 5-(3-(Benzyloxy)phenyl)-3-(2,3-dihydrobenzo[b][1,4]-
dioxin-6-yl)-1-phenyl-4,5-dihydro-1H-pyrazole
(C6).
Brown solid; mp 63–65 °C; yield: 79%; 1H NMR
(400 MHz, CDCl3) d 3.07 (dd, 1H, J = 17.2, 7.6 Hz), 3.76 (dd, 1H,
J = 17.2, 12.4 Hz), 4.28 (s, 4H), 5.00 (s, 2H), 5.17 (dd, 1H, J = 12.0,
7.2 Hz), 6.77 (t, 1H, J = 7.2 Hz), 6.87 (d, 2H, J = 8.0 Hz), 6.93 (d,
2H, J = 8.4 Hz), 7.05 (d, 2H, J = 8.0 Hz), 7.15–7.40 (m, 11H); 13C
NMR (100 MHz, CDCl3) d 43.9, 64.5, 64.7 (d, J = 2.0 Hz), 70.1,
112.4, 113.4, 114.0, 114.9, 117.5, 118.6, 119.0, 119.5, 126.6,
127.8, 128.1, 128.7, 129.0, 130.4, 136.9, 143.6, 144.4, 144.7,
145.3, 146.7, 159.6; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for
C30H27N2O3 463.2016, Found 463.2010.
4.1.2.14. 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(4-methox-
yphenyl)-1-phenyl-4,5-dihydro-1H-pyrazole (C12).
Yellow
solid; mp 113–115 °C; yield: 80%; 1H NMR (400 MHz, CDCl3) d
3.05 (dd, 1H, J = 17.2, 7.2 Hz), 3.71–3.78 (m, 4H), 4.28 (s, 4H),
5.18 (dd, 1H, J = 12.0, 7.2 Hz), 6.75 (t, 1H, J = 7.2 Hz), 6.84–6.88
(m, 3H), 7.05 (d, 2H, J = 8.0 Hz), 7.14–7.26 (m, 6H); 13C NMR
(100 MHz, CDCl3) d 44.0, 55.4, 64.2, 64.5, 64.7, 113.5, 114.6,
114.8, 117.5, 119.0, 119.5, 127.2, 129.0, 134.9, 143.7, 144.4,
145.3, 146.4, 159.1; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for
4.1.2.9. 5-(3-Chlorophenyl)-3-(2,3-dihydrobenzo[b][1,4]dioxin-
C24H23N2O3 387.1703, Found 387.1699.
6-yl)-1-phenyl-4,5-dihydro-1H-pyrazole (C7).
Brown solid;
mp 66–68 °C; yield: 72%; 1H NMR (400 MHz, CDCl3) d 3.06 (dd,
1H, J = 16.8, 7.2 Hz), 3.79 (dd, 1H, J = 17.2, 12.4 Hz), 4.28 (s, 4H),
5.18 (dd, 1H, J = 12.4, 7.2 Hz), 6.79 (t, 1H, J = 7.2 Hz), 6.87 (d, 1H,
J = 8.0 Hz), 7.02 (d, 2H, J = 8.4 Hz), 7.16–7.26 (m, 7H), 7.33 (s,
1H); 13C NMR (100 MHz, CDCl3) d 43.8, 64.2, 64.5, 64.7, 113.4,
114.9, 117.6, 119.3, 119.5, 124.2, 126.2, 126.4, 128.0, 129.1,
130.6, 135.1, 143.7, 144.6, 145.1(t, J = 4.0 Hz), 146.6; HRMS (ESI-
TOF) m/z: [M+H]+ Calcd for C23H20ClN2O2 391.1208, Found
391.1197.
4.1.2.15. 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(4-iodophe-
nyl)-1-phenyl-4,5-dihydro-1H-pyrazole (C13). Yellow solid;
mp 150–152 °C; yield: 80%; 1H NMR (400 MHz, DMSO-d6) d 3.03
(dd, 1H, J = 17.2, 7.2 Hz), 3.77 (dd, 1H, J = 17.2, 12.4 Hz), 4.28 (s,
4H), 5.17 (dd, 1H, J = 12.4, 7.2 Hz), 6.78 (t, 1H, J = 7.2 Hz), 6.87 (d,
1H, J = 8.0 Hz), 7.01 (d, 2H, J = 8.4 Hz), 7.07 (d, 2H, J = 7.6 Hz),
7.15–7.26 (m, 4H), 7.65 (d, 2H, J = 8.0 Hz); 13C NMR (100 MHz,
DMSO-d6) d 43.7, 64.1, 64.5, 64.7, 93.0, 113.4, 114.9, 117.6, 119.3,
119.5, 125.4, 126.3, 128.1, 129.1, 130.5, 137.8, 138.3, 142.6,
143.7, 144.5, 145.0, 146.6; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for
4.1.2.10. 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1-phenyl-5-
C23H20IN2O2 483.0564, Found 483.0558.
(p-tolyl)-4,5-dihydro-1H-pyrazole (C8).
Yellow solid; mp
133–135 °C; yield: 83%; 1H NMR (400 MHz, CDCl3) d 2.32 (s, 3H),
3.06 (dd, 1H, J = 17.2, 7.6 Hz), 3.76 (dd, 1H, J = 16.8, 12.0 Hz), 4.28
(s, 4H), 5.19 (dd, 1H, J = 12.0, 7.2 Hz), 6.75 (t, 1H, J = 7.2 Hz), 6.87
(t, 1H, J = 8.4 Hz), 7.05 (d, 2H, J = 8.4 Hz), 7.12–7.26 (m, 8H); 13C
NMR (100 MHz, CDCl3) d 21.2, 44.0, 64.5, 64.7, 113.4, 114.8,
117.5, 118.9, 119.5, 126.0, 126.7, 129.0, 129.9, 137.3, 139.9,
143.7, 144.4, 146.3, 146.6; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for
4.1.2.16.
(methylthio)phenyl)-1-phenyl-4,5-dihydro-1H-pyrazole
(C14).
Yellow solid; mp 182–184 °C; yield: 92%; 1H NMR
3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(4-
(400 MHz, DMSO-d6) d 2.46 (s, 3H), 3.05 (dd, 1H, J = 16.8, 7.2 Hz),
3.76 (dd, 1H, J = 16.8, 12.4 Hz), 4.28 (s, 4H), 5.19 (dd, 1H, J = 12.4,
7.2 Hz), 6.76 (t, 1H, J = 7.2 Hz), 6.87 (d, 1H, J = 8.4 Hz), 7.03 (d,
2H, J = 8.0 Hz), 7.15–7.26 (m, 8H); 13C NMR (100 MHz, DMSO-d6)
d 16.0, 43.8, 64.3, 64.5, 64.7, 113.5, 114.9, 117.6, 119.1, 119.5,
126.6, 127.4, 129.0, 137.8, 139.8, 143.7, 144.5, 145.2, 146.6; HRMS
(ESI-TOF) m/z: [M+H]+ Calcd for C24H23N2O2S 403.1475, Found
403.1480.
C24H23N2O2 358.1676, Found 358.1681.
4.1.2.11.
5-(4-Bromophenyl)-3-(2,3-dihydrobenzo[b][1,4]-
Yel-
dioxin-6-yl)-1-phenyl-4,5-dihydro-1H-pyrazole (C9).
low solid; mp 126–128 °C; yield: 87%; 1H NMR (400 MHz, CDCl3)
d 3.03 (dd, 1H, J = 16.8, 7.2 Hz), 3.78 (dd, 1H, J = 16.8, 12.4 Hz),
4.28 (s, 4H), 5.19 (dd, 1H, J = 12.4, 7.2 Hz), 6.78 (t, 1H, J = 7.2 Hz),
6.87 (d, 1H, J = 8.0 Hz), 7.01 (d, 2H, J = 8.0 Hz), 7.15–7.26 (m, 6H),
7.45 (d, 2H, J = 8.0 Hz); 13C NMR (100 MHz, CDCl3) d 64.5, 64.7,
105.2, 114.9, 117.6, 119.3, 122.7, 125.4, 126.7, 127.7, 129.2,
129.7, 130.4, 131.8, 140.0, 143.2, 143.8 (d, J = 3.0 Hz), 151.9; HRMS
(ESI-TOF) m/z: [M+H]+ Calcd for C23H20BrN2O2 435.0703, Found
435.0702.
4.1.2.17. 5-(4-(Benzyloxy)phenyl)-3-(2,3-dihydrobenzo[b][1,4]-
dioxin-6-yl)-1-phenyl-4,5-dihydro-1H-pyrazole (C15).
Yel-
low solid; mp 136–138 °C; yield: 90%; 1H NMR (400 MHz, CDCl3)
d 3.05 (dd, 1H, J = 16.8, 7.2 Hz), 3.75 (dd, 1H, J = 16.8, 12.0 Hz),
4.28 (s, 4H), 5.02 (s, 2H), 5.18 (dd, 1H, J = 12.0, 7.2 Hz), 6.76 (t,
1H, J = 7.2 Hz), 6.87 (d, 1H, J = 8.8 Hz), 6.93 (d, 2H, J = 8.4 Hz),
7.05 (d, 2H, J = 7.6 Hz), 7.16 (t, 2H, J = 8.4 Hz), 7.22–7.26 (m, 3H),
7.30–7.43 (m, 6H); 13C NMR (100 MHz, CDCl3) d 43.9, 64.2, 64.5
(d, J = 5.0 Hz), 64.6 (d, J = 3.0 Hz), 70.2, 104.6, 113.4, 114.8, 115.0
(d, J = 3.0 Hz), 115.5, 117.5 (d, J = 5.0 Hz), 119.0, 119.4 (d,
J = 8.0 Hz), 125.5, 126.7, 127.7 (d, J = 2.0 Hz), 128.1, 128.3, 128.7
(d, J = 2.0 Hz), 129.0 (d, J = 5.0 Hz), 130.2, 135.2, 137.1, 143.6,
143.8, 144.4, 145.2, 146.6, 158.3, 159.0; HRMS (ESI-TOF) m/z: [M
+H]+ Calcd for C30H27N2O3 463.2016, Found 463.2019.
4.1.2.12.
3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(4-fluo-
Brown
rophenyl)-1-phenyl-4,5-dihydro-1H-pyrazole (C10).
solid; mp 75–77 °C; yield: 86%; 1H NMR (400 MHz, CDCl3) d 3.04
(dd, 1H, J = 16.8, 6.8 Hz), 3.77 (dd, 1H, J = 16.8, 12.4 Hz), 4.28 (s,
4H), 5.21 (dd, 1H, J = 12.0, 6.8 Hz), 6.78 (t, 1H, J = 7.2 Hz), 6.87 (d,
1H, J = 8.8 Hz), 6.99–7.03 (m, 4H), 7.15–7.30 (m, 6H); 13C NMR
(100 MHz, CDCl3) d 43.8, 64.0, 64.5, 64.7, 113.4, 114.9, 116.0,
116.2, 117.6, 119.2, 119.5, 126.4, 127.7 (d, J = 8.0 Hz), 129.0,
138.5 (d, J = 3.0 Hz), 143.7, 144.5, 145.0, 146.6, 161.0, 163.4; 19F
NMR (376.38 MHz, CDCl3) d ꢁ114.93; HRMS (ESI-TOF) m/z: [M
+H]+ Calcd for C23H20FN2O2 375.1503, Found 375.1501.
4.1.2.18. 4-(3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1-phenyl-
4,5-dihydro-1H-pyrazol-5-yl)-N,N-dimethylaniline
(C16).
Yellow solid; mp 168–170 °C; yield: 80%; 1H NMR
(400 MHz, CDCl3) d 2.92 (s, 6H), 3.06 (dd, 1H, J = 16.8, 7.2 Hz),
3.72 (dd, 1H, J = 16.8, 12.0 Hz), 4.28 (s, 4H), 5.14 (dd, 1H, J = 12.4,
Please cite this article in press as: Yang, Y.-S.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.05.012

8
Y.-S. Yang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7.6 Hz), 6.66–6.70 (m, 2H), 6.72–6.76 (m, 1H), 6.85–6.88 (m, 1H),
7.06–7.09 (m, 2H), 7.14–7.26 (m, 6H); 13C NMR (100 MHz, CDCl3)
d 40.7, 43.9, 64.4, 64.5, 64.7, 113.1, 113.5, 114.8, 117.5, 118.8,
119.4, 126.9, 128.9, 130.5, 143.6, 144.2, 145.4, 146.6, 150.1; HRMS
(ESI-TOF) m/z: [M+H]+ Calcd for C25H26N3O2 400.2020, Found
400.2015.
medium giving 200
quots (100 L) of each dilution were added to the wells, giving
doses ranging from 100 M to 0.005 M. After a further incubated
at 37 °C for 24 h in a humidified atmosphere with 5% CO2, the cell
viability was assessed by the conventional 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay and
carried out strictly according to the manufacturer instructions
(Sigma). The absorbance at 590 nm was recorded using LX300
Epson Diagnostic micro-plate reader. Then GI50 was calculated
using SPSS 13.0 software.
l
M, and 10 serial dilutions of 3ꢂ prepared. Ali-
l
l
l
4.1.2.19. 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1-phenyl-5-
(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazole
(C17).
Yellow solid; mp 111–113 °C; yield: 77%; 1H NMR
(400 MHz, CDCl3) d 3.05 (dd, 1H, J = 16.8, 7.2 Hz), 3.82 (dd, 1H,
J = 17.2, 12.4 Hz), 4.28 (s, 4H), 5.29 (dd, 1H, J = 12.0, 6.8 Hz), 6.79
(t, 1H, J = 7.6 Hz), 6.88 (d, 1H, J = 8.4 Hz), 6.99–7.02 (m, 2H),
7.16–7.26 (m, 4H), 7.44 (d, 2H, J = 8.0 Hz), 7.59 (d, 2H, J = 8.4 Hz);
13C NMR (100 MHz, CDCl3) d 43.7, 64.1, 64.5, 64.7, 113.4, 114.9,
117.6, 119.4, 119.5, 126.2, 126.3 (q, J = 3.0 Hz), 126.5, 129.2,
143.7, 144.6, 144.9, 146.6, 146.8 (d, J = 1.0 Hz); 19F NMR
(376.38 MHz, CDCl3) d ꢁ62.53; HRMS (ESI-TOF) m/z: [M+H]+ Calcd
for C24H20F3N2O2 425.1471, Found 425.1468.
MCF-7 human breast cancer cells were evaluated by the same
method as WM266.4 melanoma cells with the results presented
in GI50 values.
NIH3T3 mouse fibroblast cells were cultured in a 96-well plate
at a density of 5 ꢂ 105 cells and different concentrations of com-
pounds were respectively added to each well. The incubation
was permitted at 37 °C, 5% CO2 atmosphere for 24 h before the
cytotoxicity assessments. 20 lL MTT reagent (4 mg/mL) was added
per well 4 h before the end of the incubation. Four hours later, the
plate was centrifuged at 1200 rcf for 5 min and the supernatants
4.1.2.20. 5-(3,4-Dichlorophenyl)-3-(2,3-dihydrobenzo[b][1,4]-
dioxin-6-yl)-1-phenyl-4,5-dihydro-1H-pyrazole
(C18).
were removed, each well was added with 200 lL DMSO. The absor-
bance was measured at a wavelength of 490 nm (OD 490 nm) on
an ELISA microplate reader. Three replicate wells were used for
each concentration and each assay was measured three times, after
which the average of GI50 was calculated.
Brown solid; mp 65–67 °C; yield: 75%; 1H NMR
(400 MHz, CDCl3) d 3.03 (dd, 1H, J = 17.2, 7.2 Hz), 3.79 (dd, 1H,
J = 17.2, 12.4 Hz), 4.28 (s, 4H), 5.16 (dd, 1H, J = 12.4, 7.2 Hz), 6.80
(t, 1H, J = 7.2 Hz), 6.87 (d, 1H, J = 8.4 Hz), 7.00 (d, 2H, J = 8.0 Hz),
7.14–7.26 (m, 5H), 7.32–7.43 (m, 3H); 13C NMR (100 MHz, CDCl3)
d 43.7, 63.7, 64.5, 64.7, 113.4, 114.9, 117.6, 119.5, 125.4, 126.1,
128.1, 129.2, 131.3, 131.7, 133.4, 143.2, 143.7, 144.6, 144.9,
146.7; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C23H19Cl2N2O2
425.0818, Found 425.0820.
4.2.2. Kinase inhibitory assay
This V600E mutant B-Raf kinase assay was performed in tripli-
cate for each tested compound in this study. Briefly, 7.5 ng Mouse
Full-Length GST-tagged BRAFV600E (Invitrogen, PV3849) was pre-
incubated at room temperature for 1 h with 1
assay dilution buffer. The kinase assay was initiated when 5
a solution containing 200 ng recombinant human full length, N-
terminal His-tagged MEK1 (Invitrogen), 200 M ATP, and 30 mM
lL drug and 4
lL
l
L of
4.1.2.21.
dimethoxyphenyl)-1-phenyl-4,5-dihydro-1H-pyrazole
(C19).
Yellow solid; mp 145–147 °C; yield: 81%; 1H NMR
3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(3,4-
l
MgCl2 in assay dilution buffer was added. The kinase reaction
(400 MHz, CDCl3) d 3.07 (dd, 1H, J = 17.2, 8.0 Hz), 3.72–3.86 (m,
7H), 4.28 (s, 4H), 5.14 (dd, 1H, J = 12.0, 7.6 Hz), 6.76–6.83 (m,
3H), 6.86–6.89 (m, 2H), 7.05–7.07 (m, 2H), 7.15–7.19 (m, 2H),
7.22–7.26 (m, 2H); 13C NMR (100 MHz, CDCl3) d 44.0, 56.1 (d,
J = 3.0 Hz), 64.5, 64.7, 64.8, 108.8, 111.6, 113.6, 114.8, 117.5,
118.2, 119.1, 119.5, 126.6, 129.0, 135.5, 143.6, 144.4, 145.5,
146.8, 148.5, 149.7; HRMS (ESI-TOF) m/z: [M+H]+ Calcd for
C25H25N2O4 417.1809, Found 417.1802.
was allowed to continue at room temperature for 25 min and
was then quenched with 5
solution. Protein was further denatured by heating for 5 min at
70 °C. 10 L of each reaction was loaded into a 15-well, 4–12% pre-
lL 5ꢂ protein denaturing buffer (LDS)
l
cast NuPage gel (Invitrogen) and run at 200 V, and upon comple-
tion, the front, which contained excess hot ATP, was cut from the
gel and discarded. The gel was then dried and developed onto a
phosphor screen. A reaction that contained no active enzyme was
used as a negative control, and a reaction without inhibitor was
used as the positive control.
Detection of the effect of compounds on cell based pERK1/2
activity in WM266.4 cells was performed using ELISA kits (Invitro-
gen) and strictly according to the manufacturer instructions.
The wild type B-Raf kinase and C-Raf kinase were evaluated use
the same method as V600E mutant B-Raf kinase.
A 1.6 kb cDNA encoded for the EGFR cytoplasmic domain
(EGFR-CD, amino acids 645–1186) was cloned into baculoviral
expression vectors pBlueBacHis2B and pFASTBacHTc (Huakang
Company China), separately. A sequence that encodes (His)6 was
located at the 50 upstream to the EGFR sequences. Sf-9 cells were
infected for 3 days for protein expression. Sf-9 cell pellets were sol-
ubilized at 0 °C in a buffer at pH 7.4 containing 50 mM HEPES,
4.1.2.22.
3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(furan-2-
Brown solid;
yl)-1-phenyl-4,5-dihydro-1H-pyrazole (C20).
mp 55–57 °C; yield: 80%; 1H NMR (400 MHz, CDCl3) d 3.26 (dd,
1H, J = 16.8, 6.8 Hz), 3.64 (dd, 1H, J = 16.8, 12.4 Hz), 4.28 (s, 4H),
5.30 (dd, 1H, J = 12.0, 6.8 Hz), 6.21 (d, 1H, J = 3.2 Hz), 6.29 (dd,
1H, J = 3.2, 2.0 Hz), 6.81 (t, 1H, J = 7.2 Hz), 6.87–6.90 (m, 1H), 7.14
(d, 2H, J = 7.6 Hz), 7.20–7.26 (m, 4H), 7.35 (d, 1H, J = 1.2 Hz); 13C
NMR (100 MHz, CDCl3) d 40.4, 58.3, 64.5, 64.7, 107.0, 110.6,
113.7, 114.9, 117.5, 119.5 (d, J = 10.0 Hz), 126.4, 129.0, 142.3,
143.6, 144.5, 145.4, 147.3, 153.9; HRMS (ESI-TOF) m/z: [M+Na]+
Calcd for C21H18N2O3Na 369.1210, Found 369.1209.
4.2. Biological assay
10 mM NaCl, 1% Triton, 10
l
M ammonium molybdate, 100
g/mL aprotinin, 10 g/mL leupeptin, 10
g/mL benzamidine, HCl for 20 min followed
lM
lg/
4.2.1. Anti-proliferation assay
sodium vanadate, 10
mL pepstatin, and 16
l
l
l
WM266.4 melanoma cells27 were cultured in DMEM/10% fetal
bovine serum, in 5% CO2 water saturated atmosphere at 37 °C. Cell
suspensions (10,000/mL) were prepared and 100 lL/well dis-
pensed into 96-well plates (Costar) giving 1000 cells/well. The
plates were returned to the incubator for 24 h to allow the cells
to reattach. These compounds were initially prepared at 20 mM
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 non-
specifically bound material. Histidine-tagged proteins were eluted
with 250 and 500 mM imidazole and dialyzed against 50 mM NaCl,
in DMSO. Aliquots (200
lL) were diluted into 20 mL culture
20 mM HEPES, 10% glycerol and 1 lg /mL each of aprotinin,
Please cite this article in press as: Yang, Y.-S.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.05.012

Y.-S. Yang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
leupeptin, and pepstatin for 2 h. The entire purification procedure
was performed at 4 °C or on ice.
low-energy orientations is obtained, or the test times of
bad orientations reached the maximum number.
EGFR kinase assays was set up to assess the level of autophos-
phorylation based on DELFIA/Time-Resolved Fluorometry. Com-
pounds C1–C20 were dissolved in 100% DMSO and diluted to the
appropriate concentrations with 25 mM HEPES at pH 7.4. In each
(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.
well, 10
of recombinant enzyme (1:80 dilution in 100 mM HEPES) for 10 min
at room temperature. Then, 10
L of 5ꢂ buffer (containing 20 mM
HEPES, 2 mM MnCl2, 100 M Na3VO4, and 1 mM DTT) and 20
lL of compound was incubated with 10 lL (5 ng for EGFR)
(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 B-Raf 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.
l
l
lL
of 0.1 mM ATP–50 mM MgCl2 was added for 1 h. Positive and nega-
tive controls were included in each plate by incubation of enzyme
with or without ATP–MgCl2. At the end of incubation, liquid was
aspirated and plates were washed three times with wash buffer. A
75 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 emis-
sion 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 inhibition with
eight concentrations of compound. As the contaminants in the
enzyme preparation are fairly low, the majority of the signal
detected by the anti-phosphotyrosine antibody is from EGFR. The
experiment was performed in triplicate.
4.5. Experimental protocol of QSAR model
4.3. Experimental protocol of ADMET study
Among all the 20 compounds, 80% (that is 16) were utilized
as a training set for QSAR modeling. The remaining 20% (that is
4) were chosen as an external test subset for validating the reli-
ability of the QSAR model by the Diverse Molecules protocol in
Discovery Studio 3.5. The selected test compounds were: C5, C8,
C10 and C15.
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.
The three-dimensional structures of the aforementioned com-
pounds were constructed using Chem. 3D ultra 12.0 software
[Chemical Structure Drawing Standard; Cambridge Soft corpora-
tion, USA (2010)]. Then they were minimized a CHARMm based
force field the same as in docking study. The ADMET study was
conducted using the Calculate Molecular Properties in Small Mole-
cules module of the Discovery Studio (version 3.5). The ADMET
properties map and data were provided by the ADMET Descriptors
tool.
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
constant is used to mimic the solvent effect. As for the Van der
Waals potential, 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 3.5.
4.4. Experimental protocol of docking study
The three-dimensional structures of the aforementioned com-
pounds were constructed using Chem. 3D ultra 12.0 software
[Chemical Structure Drawing Standard; Cambridge Soft corpora-
tion, USA (2010)]. The crystal structures of B-Raf kinase domain
bound to SB-590885 (PDB code: 2FB8) and bound to SM7 (PDB
code: 3PSD) complex were retrieved from the RCSB Protein Data
Bank (http://www.rcsb.org/pdb/home/home.do). All bound waters
and ligands were eliminated from the protein and the polar hydro-
gen was added to the proteins. Molecular docking of all twenty
compounds as well as C0A and C0B was then carried out using
the Discovery Studio (version 3.5) as implemented through the
graphical user interface CDOCKER protocol.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.05.012.
CDOCKER is an implementation of a CHARMm based molecular
docking tool using a half-flexible receptor,30 including the follow-
ing steps:
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