Synthesis, biological evaluation and 3D-QSAR study of novel 4,5-dihydro-1H-pyrazole thiazole derivatives as BRAFV600E inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.11.029

Many reports implied that the BRAF serine/threonine kinase was mutated in various types of human tumors, which were related with cell growth, survival and differentiation. To provide new therapeutic opportunities, a series of novel 4,5-dihydro-1H-pyrazole

Full text of DOI:10.1016/j.bmc.2014.11.029

Accepted Manuscript
Synthesis, biological evaluation and 3D-QSAR study of novel 4,5-dihy-
dro-1H-pyrazole thiazole derivatives as BRAFV600E inhibitors
Meng-Yue Zhao, Yong Yin, Xiao-Wei Yu, Chetan B. Sangani, Shu-Fu Wang,
Ai-Min Lu, Li-Fang Yang, Peng-Cheng Lv, Ming-Guo Jiang, Hai-Liang Zhu
PII:
S0968-0896(14)00816-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.11.029
BMC 11918
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 September 2014
19 November 2014
20 November 2014
Please cite this article as: Zhao, M-Y., Yin, Y., Yu, X-W., Sangani, C.B., Wang, S-F., Lu, A-M., Yang, L-F., Lv,
P-C., Jiang, M-G., Zhu, H-L., Synthesis, biological evaluation and 3D-QSAR study of novel 4,5-dihydro-1H-
pyrazole thiazole derivatives as BRAFV600E inhibitors, Bioorganic & Medicinal Chemistry (2014), doi: http://
dx.doi.org/10.1016/j.bmc.2014.11.029
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Synthesis, biological evaluation and 3D-QSAR study of novel 4, 5-dihydro-1H-pyrazole
thiazole derivatives as BRAFV600E inhibitors
Meng-Yue Zhao+a, Yong Yin+a, Xiao-Wei Yua, Chetan B. Sangania, Shu-Fu Wanga, Ai-Min Lub,
Li-Fang Yangac, Peng-Cheng Lv*a, Ming-Guo Jiang*ac, Hai-Liang Zhu*a
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093,
People’s Republic of China
b College of Science, Nanjing Agriculture University, Nanjing 210095, People’s Republic of
China
cGuangxi Colleges and Universities Key Laboratory of Utilization of Microbial and Botanical Re
sources, Guangxi Universitiy for nationalities，530006
+ These authors contributed equally to this work.
* Corresponding authors. Tel.: +86 25 83592572; Fax: +86 25 83592672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
1

Abstract
Many reports implied that the BRAF serine /threonine kinase was mutated in various types
of human tumors, which were related with cell growth, survival and differentiation. To provide
new therapeutic opportunities, a series of novel 4, 5-dihydro-1H-pyrazole derivatives (6a-10d)
containing thiazole moiety as potential V600E mutant BRAF kinase (BRAFV600E) inhibitors were
designed and synthesized. All compounds were evaluated in vitro for anticancer activities against
WM266.4 human melanoma cell line and breast cancer MCF-7 cell line. Compound 10d
displayed the most potential antiproliferative activity with an IC50 value of 0.12 µM against cell
line WM266.4 and 0.16 µM against MCF-7 with positive control Sorafenib. Results of the
inhibitory activity against BRAFV600E revealed that compound 10d was bearing the best
bioactivity with IC50 of 0.05 µM as well. On the basis of the result of flow cytometry,with the
dose of compound 10d increasing, more and more cancer cell gradually encountered apoptosis or
died, which indicated the compound 10d could induce remarkable apoptosis of MCF-7 and
WM266.4 cells in a dose dependent manner. Furthermore, docking simulation of inhibitor
analogues and 3D-QSAR modeling provided protential binding model and further knowledge of
pharmacophore.
Keywords:
4, 5-dihydro-1H-pyrazole
Anticancer activity
BRAFV600E inhibition
Molecular docking
2

1. Introduction
The Ras/RAF/MEK/ERK mitogen-activated protein kinase (MAPK) signaling pathway
plays an important role in the transduction of signals from cell surface receptors to the nucleus,
like regulating cell growth, survival, differentiation and proliferation in response to external
stimuli (growth factors, cytokines or hormones).[1] Phosphorylation of RAF stimulates its
serine/threonine activity, triggering and activating the protein kinases MEK and ERK
sequentially.[2] ERK phosphorylates several transcription factors such as ELK-1 and controls
gene expression, cytoskeletal rearrangements and metabolism, causing cell transformation and
contributing to tumor initiation and maintenance. Therefore, targeting this pathway, for example
through inhibition of BRAF, becomes important in many current human cancer therapeutics
approaches.
A number of activating mutations of the BRAF gene have been identified, but the most
common chance in human cancer is the substitution of a valine for glutamic acid at position 600
(V600E; formally identified as V599E).[3] This single mutation accounts for a 500-fold activation
compared to the wild type in melanoma, providing cancer cells with proliferation, survival
signals, growth and maintenance functions. The large-scale genomic screens have detected that
BRAF was mutated in approximately 7% of human tumors,[4, 5] colorectal cancer (5–22%),[4]
papillary thyroid cancer (29–83%),[4-7] cholangiocarcinoma (22%),[8] and ovarian cancer (30%),
especially in melanoma (50–70%).[9,10]
The RAF family is serine/threonine kinases, which phosphorylate and activate downstream
MEK1/2, including A-RAF, B-RAF and C-RAF (also known as RAF-1) with different
biochemical potencies (B-Raf > C-Raf > A-Raf). Several drug development programs in the field
of small molecule kinase inhibitors have been initiated, with some representatively marketed
compounds such as SB-590885,[11] AZ628,[12] PLX4032,[13] and RAF265.[14] A detailed account
of the discovery and clinical development of PLX4032, as selective BRAFV600E inhibitor that
binds to the active conformation of the kinase, has recently been published.[15,16]It uses a
common scaffold that comprises a terminal aromatic group (phenyl or pyrazole) that fills the
allosteric pocket. The relevant compounds were created by the displacement of the DFG loop
and an amide linker which couples an aryl group in the hydrophobic pocket to a hinge-binding
heterocycle. Linkage via an amide bond would allow for rapid and efficient screening of several
3

potential hinge-binding groups.[17,18] Pyrazole and its derivatives have attracted constant interest
over the past decades because of their wide range of pharmacological activities, such as
antitumor, anti-inflammatory, antibacterial, analgesic, fungistatic, and anti-hyperglycemic.[19-21]
SB-590885 has the hottest spots, containing the pyrazolyl ring, which inhibits BRAFV600E with a
Ki of 0.16 nmol/L. In consequence, the compounds encompassing pyrazole ring as antineoplastic
leads is a reasonable attempt for potential inhibitors of BRAFV600E Based on the studies
.
generalized, thiophene and thiazole group manifest a wide range of pharmaceutical of
antibacterial, anti-inflammatory and antiviral property when exposed to the native
compounds.[22,23] With the development of scientific research, more reports reveal anticancer
activities of the compounds containing thiophene and thiazole group,[24-26]which inhibits BRAF
kinase activity. As a consequence, the combined substructures (thiophene and thiazole group)
without wrecking their original effective characteristics, might exhibit synergistic effects to
improve anticancer activities[27-29]
.
In this study, a series of novel pyrazole-based derivatives were recently synthesized as
potential anticancer agents targeting BRAF in our group, and some of them demonstrated
potential antitumor activity especially about compound 10d with an IC50 value of 0.12 µM
against cell line WM266.4 and 0.16 µM against MCF-7, which equally shows the most potential
antiproliferative activity of apoptosis by flow cytometry experiments. Based on the active data,
QSAR model was built to examine the interaction of all the compounds with SB-590885
(2FB8.pdb), and to guide the further study. Docking simulations were performed using the X-ray
crystallographic structure of the BRAF in complex with an inhibitor to explore the binding
modes of these compounds at the active site.
2. Results and Discussion
2.1. Chemistry
The synthetic approach of 4,5-dihydro-1H-pyrazole derivatives adopted is depicted in
Scheme1. First of all, to a solution of thiophene-2-carbaldehyde and different substituted
acetophenones, saturated solution of KOH was added slowly. The reaction mixture was stirred
until the solids fully formed. Secondly, conduct chalcone derivatives in refluxing KOH ethanol
4

solution with thiosemicarbazide which leads to the cyclization of the obtained (E)-1-phenyl-3-
(thiophen-2-yl)prop-2-en-1-one,eventually to get 3-phenyl-5-(thiophen-2-yl)-4,5-dihydro-1H-
pyrazole-1-carbothioamides. Finally, to DMF solution of 3-phenyl-5-(thiophen-2-yl)-4, 5-
dihydro-1H-pyrazole-1-carbothioamides and 2-Bromoacetophenone, the mixture was allowed to
stir. Afterwards, a saturated sodium chloride solution was then slowly added to the reaction flask
via a self-equalizing addition funnel. Most commonly, a precipitate formed and was then
collected by suction filtration, which was purified by silica gel chromatography after drying out.
All of the synthetic compounds gave satisfactory analytical and spectroscopic data, which were
in full accordance with their depicted structures.
2.2. Bioassay
As described above, the BRAFV600E was considered as an important target for the
development of small molecule inhibitors in the treatment of human cancers. All compounds
were tested against BRAFV600E kinases as well as against WM266.4 and MCF-7 cell lines. The
results were expressed as concentrations of IC50 (the half maximal inhibitory concentration of B-
RafV600E mediated MEK phosphorylation).
Upon investigation of antiproliferative activity of compounds 6a-10d, it has been observed
against WM266.4. Compounds 10d (IC50 = 0.12 µM), 7a (IC50 = 0.22 µM) and 7b (IC50 = 0.17
µM) showed most potent activity as compared to other compounds of the series and less
comparable to the positive control Sorafenib (IC50 = 0.06 µM).while performed against MCF-7
compounds 10d (IC50 = 0.16 µM) showed most potent activity; in addition, compounds 7d (IC50
= 1.33 µM) 9c (IC50 = 0.68 µM) and 10b (IC50 = 0.19 µM) showed good activity as compared to
other compounds of the series and comparable to the positive control Sorafenib (IC50 = 0.19
µM).
As shown in Table 1, inhibitory activity conducted against BRAFV600E kinases, compound
10d (IC50 = 0.05 µM) displayed the most potent inhibitory activity. As well, compounds 7b (IC50
= 0.67 µM), 8a (IC50 = 0.56 µM), 8c (IC50 = 0.11 µM) and 9c (IC50 =0.08 µM) showed excellent
activity as compared to other compounds of the series and less to the positive control Sorafenib
(IC50 = 0.03 µM).
5

Structure activity relationship (SAR) was carried out of BRAFV600E kinase. According to the
activity data, it has been observed that the change in R1 and R2 substitution may lead to change
in the activity against employed cancer cells as well as BRAFV600E kinase. Compounds
containing R1=Cl and R2=CF3 (Compound 10d) have proved as most effective members against
BRAFV600E kinase and two cancer cell lines WM266.4 and MCF-7. Against BRAFV600E kinase,
compounds 6d (IC50 =10.90µM) and 8d (IC50 =6.18µM) owning a substitution at R1 position is
electron releasing group (R1 = OCH3, CH3) but when it is replaced by electron withdrawing group
(R1 = F, Cl, Br) in compounds 7d (IC50 =1.01 µM), 9d (IC50 =2.09 µM) and 10d (IC50 =0.05 µM)
the activity increases. The activity in deceasing order for R1 substitution is Cl>Br>F>CH3>
OCH3 and for R2 substitution is CF3>H>OCH3>Br, while against for cancer cell lines WM266.4
and MCF-7 the activity in increasing order for R1 substitution is OCH3<CH3<Br<F<Cl and for
R2 substitution is OCH3<H<Br<CF3. The above results indicated that the pyrazolyl core, as well
as the phenyl thiazole motif, plays an important role in the BRAFV600E inhibition. The activity of
the compounds was slightly influenced by the changes of substituents on the benzene thiazole
rings.Moreover, reviewing and comparing the activity data, it is worthy to mention that the
anticancer activity and inhibition against BRAFV600E kinases of the target compounds depends
not only on the bicyclic heteroaromatic pharmacophore, but also on the nature of the
substituents.
According to the apoptosis expression by flow cytometry, the 4, 5-dihydro-1H-pyrazole
derivatives show cell proliferation activity in another way. To examine whether compound 10d
can induce apoptosis of MCF-7 and WM266.4, flow cytometry method of operation was
applied .We observed an obvious reduction in 1 and 3 µM concentration respectively. In contrast,
no significant change was seen in control group. When the concentration increased to 3 µM,
apoptosis emerges more evidently in Fig. 1
2.3. Molecular docking
To gain a better understanding of the potency of the synthesized compounds and guide
further SAR studies, we proceeded to examine the interaction of the 4, 5-dihydro-1H-pyrazole
derivatives with the BRAFV600E crystal structure (PDB code: 2FB8). The molecular docking was
performed by inserting the compound into the SB-590885 binding site of BRAFV600E. All
6

docking runs were performed by Discovery Studio 3.5(Discovery Studio 3.5, Accelrys, Inc., San
Diego, CA).
Residues within a distance of 10 Å around the BRAFV600E inhibitor SB-590885 were
isolated for the construction of the grid for docking screening. This grid was large enough to
include every residue of the BRAFV600E kinase ATP binding pocket. The binding models of
compound 10d and BRAFV600E are depicted in Fig. 2 and Fig. 3 and binding energy of
compounds 6a-10d with BRAFV600E demonstrated the possibility of inhibition in Table 2. The
amino acid residues which interact with BRAFV600E are labeled. In the binding mode, compound
10d binds to BRAFV600E via two π–π interaction. Among them is one π–π interaction are formed
between the thiazole rings of compound 10d and PHE583 having distance = 5.2 Å and second π–
π interaction are formed between the benzene rings of compound 10d and HIS539 having
distance = 5.2 Å. Extensive hydrophobic interactions are formed between compound 10d and
residues VAL471, ALA481, LYS483, LEU514, ILE527, PHE583, suggesting that the
introduction of the hydrophobic group from the phenyl isocyanate has enhanced the affinity of
the receptor BRAFV600E for the ligand compound 10d. This molecular docking result, along with
the enzyme assay data, suggests that compound 10d is a potential inhibitor of BRAFV600E
.
2.4. 3D QSAR model
To evaluate the synthesized compounds 4, 5-dihydro-1H-pyrazole derivates as BRAF
inhibitors and to explore more potent inhibitors, 3D QSAR models were built using the Create
3D QSAR protocol of Discovery Studio 3.5. In this study, 20 compounds bearing 4, 5-dihydro-
1H-pyrazole core with definite IC50 values were selected as the model dataset. By convention,
the pIC50 scale (-log IC50), which higher values indicate exponentially greater potency, which is
used as a method to measure inhibitory activity. The training and test set was chosen by the
Diverse Molecules method in Discovery Studio. Because a good alignment is very important for
the analysis of molecular fields, we applied the CDOCKER protocol to explore each molecule
with the lowest energy before alignment conformation. 4, 5-dihydro-1H-pyrazole was selected as
substructure to build the alignment conformation before building the QSAR model.
The correlation coefficient r2, between the observed and predicted activity of the training set
and test set, proved that the built QSAR model was acceptable. Predicted pIC50 values and
7

residual errors for the 20 compounds by this QSAR model are given in Table 3. The good
agreement between predicted and experimental pIC50 values for both test sets and training sets is
shown in Fig. 4.
The molecules aligned with the iso-surfaces of the 3D-QSAR model coefficients on van der
Waals grids (Fig. 5a) and electro-static potential grids (Fig. 5b) were also listed. The electrostatic
map has red contours around the regions where high electron density (negative charge) is
expected to increase activity, and blue contours around areas where low electron density (partial
positive charge) is expected to increase activity. Similarly, the steric map has areas where steric
bulk is predicted to increase (green) or decrease (yellow) activity.
According to the maps, it is suggested that the compound with high negative charged and
small R1 group, would show higher activity, attesting that chlorine substituent from being a
better choice. Whereas, a low negative charged and bulky R2 group would help obtain sound
activity, validating the trifluoromethyl substituent could be more effective. As a result, data
summarized above demonstrate that compound 10d, the most potent BRAF inhibitor (IC50
against WM266.4=0.12 µM, IC50 against MCF-7=0.16 µM), containing favored substituents has
an outstanding activity. The 3D QSAR models fit the inhibitory activity well, which thus
provides us with a direction for further modification.
3. Conclusion
In this study, a series of 4, 5-dihydropyrazole derivatives, containing thiazole and thiophene
moiety, as potential V600E mutant BRAF kinase (BRAFV600E) inhibitors were designed and
synthesized. According to the biological active data, it could be concluded that majority of the
compounds showed effective BRAFV600E inhibitory activity and antiproliferative activity against
WM266.4 and MCF-7 cell lines. Among these compounds, 10d showed the most potent
BRAFV600E inhibitory activity (IC50 = 0.05 µM) and antiproliferative activity (IC50 = 0.12 µM for
WM266.4 and IC50 = 0.16 µM for MCF-7). From the result of flow cytometry, more and more cancer
cell gradually encountered apoptosis or died with the dose of compound 10d increasing, which
indicated the compound 10d could induce remarkable apoptosis of MCF-7 and WM266.4 cells.
Molecular docking studies provided further insight into interactions between the BRAFV600E
protein and its ligand. After analyzing the binding model of compound 10d with BRAF, we
8

found that one π–π interaction and the hydrophobic interactions with the protein residues in the
ATP binding site might play essential roles in its BRAFV600E inhibition and antiproliferative
activity. Therefore, it can be concluded that compound 10d as well as the other pyrazole
derivatives, containing effective thiazole and thiophene pharmacophore, are promising subjects
for further study as potential anticancer agents. The results of this study might help with the
search for a promising BRAFV600E inhibitor with potent activity.
4. Experimental
4.1. Materials and measurements
All chemicals and reagents used in the current study were of analytical grade. Melting
points (uncorrected) were determined on an XT4 MP apparatus (Taike Corp., Beijing, China).
13
All the 1H NMR and C NMR spectra were recorded on a Bruker DPX400 model Spectrometer
in DMSO-d6 and chemical shifts were reported in ppm (d). FTIR spectra (KBr) were run on a
Nexus 870 FT-IR spectrophotometer. ESI-MS spectra were recorded on a Mariner System 5304
mass spectrometer. Elemental analyses were performed on a CHN-O-Rapid instrument. TLC
was performed on the glass backed silica gel sheets (Silica Gel 60 GF254) and visualized in UV
light (254 nm).
4.2. General procedure for synthesis of chalcones
To a stirred solution of acetophenone derivatives (10 mmol) and a thiophene-2-
carbaldehyde (10 mmol) in ethanol (30 mL), a saturated solution of KOH (6 mol/4 L) was added
slowly. The reaction mixture was stirred until the solids fully formed. The products were filtrated
and washed carefully with ice water and cool ethanol, then purified by crystallization from
ethanol in refrigerator to give chalcones.
4.3. General procedure for synthesis of 3-phenyl-5-(thiophen-2-yl)-4,5-dihydro-1H-
pyrazole-1-carbothioamide
9

To ethanol (45 mL) solution of equimolar quantities thiosemicarbazide (15 mmol) and
chalcone ketones (15 mmol) obtained from step one, KOH (15-18mmol) was slowly added with
stirring. The mixture was refluxed by 2 hours at 80ºC. After completion of the reaction, the
contents were cooled. The product was filtered and purified by recrystallization from ethanol.
4.4. General procedure for synthesis of 4-phenyl-2-(3-phenyl-5-(thiophen-2-yl)-4,5-dihydro-
1H-pyrazol-1-yl)thiazole
To DMF (45mL) solution of 3-phenyl-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-
carbothioamide (10 mmol) and 2-Bromoacetophenone (10 mmol), the mixture was allowed to
stir for 3 hours at 25 ºC. After completion of the reaction, a 40 mL aliquot of a saturated sodium
chloride solution was then slowly added to the reaction flask via a self-equalizing addition funnel.
Most commonly, a precipitate formed and was then collected by suction filtration. After drying at
room temperature, the residue was purified by silica gel chromatography (PE/EtOAc=10:1) to
afford pure compounds.
4.4.1.
4-(4-Bromophenyl)-2-(3-(4-methoxyphenyl)-5-(thiophen-2-yl)-4,5-dihydro-1H-
pyrazol-1-yl)thiazole (6a).
1
Yield 76%; mp. 211-213 ℃. H NMR (400 MHz, DMSO): δ 3.50-3.56 (m, 1H, CH2); 3.82
(s, 3H, OCH3); 3.98-4.06 (m, 1H, CH2); 5.95-5.99 (m, 1H, CH); 6.99 (t, J = 3.60 Hz, 1H, ArH);
7.04 (d, J = 3.60 Hz, 1H, ArH);7.06 (d, J = 3.60 Hz, 1H, ArH); 7.24 (s, 1H, ArH); 7.44 (d, J =
3.20 Hz, 2H, ArH); 7.59 (d, J = 8.40 Hz, 2H, ArH); 7.77 (t, J = 17.70 Hz, 4H, ArH). ESI-MS:
497.39 (C23H19BrN3OS2, [M+H]+). Anal. Calcd for C23H18BrN3OS2: C, 55.65; H, 3.65; N, 8.46;
Found: C, 55.45; H, 3.45; N, 8.56.
4.4.2.
2-(3-(4-Methoxyphenyl)-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-4-
phenylthiazole (6b).
Yield 69%; mp. 174-176 ℃. 1H NMR (400 MHz, DMSO): δ 3.49-3.55 (m, 1H, CH2); 3.82
(s, 3H, OCH3); 3.98-4.06 (m, 1H, CH2); 5.95-5.99 (m, 1H, CH); 6.99 (t, J = 3.60 Hz, 1H, ArH);
7.04 (d, J = 3.60 Hz, 1H, ArH); 7.06 (d, J = 3.60 Hz, 1H, ArH); 7.25 (d, J=3.50 Hz, 1H, ArH);
7.29 (d, J = 7.30 Hz, 1H, ArH); 7.37 (t, J = 13.20 Hz, 3H, ArH); 7.41-7.45 (m, 1H, ArH); 7.76
10

(d, J = 8.80 Hz, 2H, ArH); 7.82-7.84 (d, J = 7.20 Hz, 2H, ArH). ESI-MS: 418.58 (C23H20N3OS2,
[M+H]+). Anal. Calcd for C23H19N3OS2: C, 66.16; H, 4.59; N, 10.06; Found: C, 66.11; H, 4.49;
N, 10.16.
4.4.3.
4-(4-Methoxyphenyl)-2-(3-(4-methoxyphenyl)-5-(thiophen-2-yl)-4,5-dihydro-1H-
pyrazol-1-yl)thiazole (6c).
Yield 59%; mp. 198-201 ℃. 1H NMR (400 MHz, DMSO): δ 3.35-3.54 (m, 1H, CH2); 3.77
(s, 3H, OCH3); 3.82 (s, 3H, OCH3); 3.98-4.05 (m, 1H, CH2); 5.93-5.98 (m, 1H, CH); 6.94 (d, J =
8.80 Hz, 2H, ArH); 6.99 (t, J = 3.60 Hz, 1H, ArH); 7.04 (d, J = 3.60 Hz, 1H, ArH); 7.06 (d, J =
3.60 Hz, 1H, ArH); 7.18 (s, 1H, ArH); 7.24 (d, J = 1.20 Hz, 1H, ArH); 7.43-7.45 (m, 1H, ArH);
7.76 (d, J = 8.00 Hz, 4H, ArH). ESI-MS: 448.48 (C24H22N3O2S2, [M+H]+). Anal. Calcd for
C24H21N3O2S2: C, 64.40; H, 4.73; N, 9.39; Found: C, 64.20; H, 4.63; N, 9.45.
4.4.4.
2-(3-(4-Methoxyphenyl)-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-4-(4-
(trifluoromethyl)phenyl)thiazole (6d).
Yield 49%; mp. 208-209 ℃. 1H NMR (400 MHz, DMSO): δ 3.51-3.58 (m, 1H, CH2); 3.82
(d, J = 8.40 Hz, 3H, OCH3); 4.00-4.07 (m, 1H, CH2); 5.98-6.02 (m, 1H, CH); 6.98-7.00 (m, 1H,
ArH); 7.05 (d, J = 3.60 Hz, 1H, ArH); 7.07 (d, J = 3.60 Hz, 1H, ArH); 7,26 (d, J = 10.80 Hz, 1H,
ArH); 7.44 (d, J = 4.00 Hz, 1H, ArH); 7.62 (d, J = 11.60 Hz, 1H, ArH); 7.75-7.78 (m, 4H, ArH);
8.03 (d, J = 2.00 Hz, 2H, ArH). ESI-MS: 486.61 (C24H19F3N3OS2, [M+H]+). Anal. Calcd for
C24H18F3N3OS2: C, 59.37; H, 3.74; N, 8.65; Found: C, 59.17; H, 3.72; N, 8.45.
4.4.5. 4-(4-Bromophenyl)-2-(3-(4-fluorophenyl)-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-
1-yl)thiazole (7a).
Yield 67%; mp. 199-201 ℃. 1H NMR (400 MHz, DMSO): δ 3.55-3.61 (m, 1H, CH2); 4.02-
4.10 (m, 1H, CH2); 6.00-6.04 (m, 1H, CH); 7.00 (t, J = 8.40 Hz, 1H, ArH); 7.26 (d, J = 0.80 Hz,
1H, ArH); 7.35 (t, J = 17.20 Hz, 2H, ArH); 7.45 (d, J = 7.60 Hz, 2H, ArH); 7.60 (d, J = 8.40 Hz,
2H, ArH); 7.78-7.80 (d, J = 8.40 Hz, 2H, ArH); 7.86-7.90 (m, 2H, ArH). ESI-MS: 485.44
(C22H16BrFN3S2, [M+H]+). Anal. Calcd for C22H15BrFN3S2: C, 54.55; H, 3.12; N, 8.67; Found:
C, 54.45; H, 3.21; N, 8.68.
11

4.4.6.
2-(3-(4-Fluorophenyl)-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-4-
phenylthiazole (7b).
Yield 54%; mp. 156-157 ℃. 1H NMR (400 MHz, DMSO): δ 3.53-3.59 (m, 1H, CH2); 4.01-
4.08 (m, 1H, CH2); 5.99-6.03 (m, 1H, CH); 7.00 (t, J = 8.40 Hz, 1H, ArH); 7.26-7.32 (m, 2H,
ArH); 7.34 (s, 5H, ArH); 7.37-7.45 (m, 1H, ArH); 7.83-7.89 (m, 4H, ArH). ESI-MS: 406.53
(C22H17FN3S2, [M+H]+). Anal. Calcd for C22H16FN3S2 : C, 65.16; H, 3.98; N, 10.36; Found: C,
65.25; H, 3.79; N, 10.46.
4.4.7.
2-(3-(4-Fluorophenyl)-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-4-(4-
methoxyphenyl)thiazole (7c).
Yield 65%; mp. 181-183 ℃. 1H NMR (400 MHz, DMSO): δ 3.5-3.59 (m, 1H, CH2); 3.79
(s, 3H, OCH3); 4.02-4.09 (m, 1H, CH2); 5.99-6.03 (m, 1H, CH); 6.97 (t, J = 22.80 Hz, 3H, ArH);
7.20 (s, 1H, ArH); 7.26(s, 1H, ArH); 7.35 (t, J = 8.40 Hz, 2H, ArH); 7.45(d, J = 4.80 Hz, 1H,
ArH); 7.77 (d, J = 8.40 Hz, 2H, ArH); 7.88 (t, J = 13.20 Hz, 2H, ArH). ESI-MS: 436.44
(C23H19FN3OS2, [M+H]+). Anal. Calcd for C23H18FN3OS2: C, 63.43; H, 4.17; N, 9.65; Found: C,
63.53; H, 4.23; N, 9.85.
4.4.8.
2-(3-(4-Fluorophenyl)-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-4-(4-
(trifluoromethyl)phenyl)thiazole (7d).
Yield 52%; mp. 198-200 ℃. 1H NMR (400 MHz, DMSO): δ 3.55-3.61 (m, 1H, CH2); 4.02-
4.10 (m, 1H, CH2); 6.01-6.05 (m, 1H, CH); 7.00 (t, J = 8.40 Hz, 1H, Ar-H); 7.27 (d, J = 3.20 Hz,
1H, ArH); 7.43 (t, J = 16.80 Hz, 2H, ArH); 7.44 (d, J = 4.80 Hz, 1H, ArH); 7.63 (d, J = 14.80 Hz,
1H, ArH); 7.75 (d, J = 8.00 Hz, 2H, ArH); 7.78 (d, J = 24.00 Hz, 2H, ArH); 8.04(d, J = 8.10 Hz,
2H, ArH). ESI-MS: 474.49 (C23H16F4N3S2, [M+H]+). Anal. Calcd for C23H15F4N3S2: C, 58.34; H,
3.19; N, 8.87; Found: C, 58.45; H, 3.32; N, 8.81.
4.4.9.
4-(4-Bromophenyl)-2-(5-(thiophen-2-yl)-3-p-tolyl-4,5-dihydro-1H-pyrazol-1-
yl)thiazole (8a).
12

Yield 72%; mp. 209-210 ℃. 1H NMR (400 MHz, DMSO): δ 2.37 (s, 3H, CH3); 3.51-3.56
(m, 23.6 Hz, 1H, CH2); 3.99-4.07 (m, 1H, CH2); 5.97-6.02 (m, 1H, CH); 6.98-7.00 (m, 1H,
ArH); 7.25 (d, J = 3.60 Hz, 1H, ArH); 7.31 (d, J = 8.00 Hz, 2H, ArH); 7.45 (t, J = 8.00 Hz, 2H,
ArH); 7.59 (d, J = 15.80 Hz, 2H, ArH); 7.71 (d, J = 8.10 Hz, 2H, ArH); 7.78 (d, J = 8.60 Hz, 2H,
ArH). ESI-MS: 481.49 (C23H19BrN3S2, [M+H]+). Anal. Calcd for C23H18BrN3S2: C, 57.50; H,
3.78; N, 8.75; Found: C, 57.60; H, 3.88; N, 8.78.
4.4.10. 4-Phenyl-2-(5-(thiophen-2-yl)-3-p-tolyl-4,5-dihydro-1H-pyrazol-1-yl)thiazole(8b).
Yield 61%; mp. 190-192 ℃. 1H NMR (400 MHz, DMSO): δ 2.37 (s, 3H, CH3); 3.50-3.56
(m, 1H, CH2); 3.99-4.06 (m, 1H, CH2); 5.97-6.01 (m, 1H, CH); 6.99 (t, J = 8.40 Hz, 1H, ArH);
7.25-7.30 (m, 4H, ArH); 7.32 (s, 3H, ArH); 7.37-7.45 (m, 1H, ArH). 7.71 (d, J = 8.00 Hz, 2H,
ArH); 7.83-7.84 (d, J = 7.60 Hz, 2H, ArH). ESI-MS: 402.59 (C23H20N3S2, [M+H]+). Anal. Calcd
for C23H19N3S2: C, 68.80; H, 4.77; N, 10.46; Found: C, 68.70; H, 4.79; N, 10.66.
4.4.11.
4-(4-Methoxyphenyl)-2-(5-(thiophen-2-yl)-3-p-tolyl-4,5-dihydro-1H-pyrazol-1-
yl)thiazole (8c).
Yield 48%; mp. 198-200 ℃. 1H NMR (400 MHz, DMSO): δ 2.37 (s, 3H, CH3); 3.49-3.55
(m, 1H, CH2); 3.77 (s, 3H, OCH3); 3.99-4.06 (m, 1H, CH2); 5.96-6.00 (m, 1H, CH); 6.94-7.00
(m, 3H, ArH); 7.19 (s, 1H, ArH); 7.25 (d, J = 3.20 Hz, 1H, ArH); 7.31 (d, J = 4.00 Hz, 2H,
ArH); 7.44 (d, J = 17.20 Hz, 1H, ArH); 7.70-7.77 (m, 4H, ArH). ESI-MS: 432.61(C24H22N3OS2,
[M+H]+). Anal. Calcd for C24H21N3OS2: C, 66.79; H, 4.90; N, 9.74; Found: C, 66.89; H, 4.95; N,
9.84.
4.4.12.
2-(5-(Thiophen-2-yl)-3-p-tolyl-4,5-dihydro-1H-pyrazol-1-yl)-4-(4-
(trifluoromethyl)phenyl)thiazole (8d).
Yield 67%; mp. 208-209 ℃. 1H NMR (400 MHz, DMSO): δ 2.35 (s, 3H, CH3); 3.56-3.58
(d, J = 6.00 Hz, 1H, CH2); 4.00-4.07 (m, 1H, CH2); 5.99-6.03 (m, 1H, CH); 6.98-7.00 (m, 1H,
ArH); 7.26 (d, J = 3.20 Hz, 1H, ArH), 7.31 (d, J = 8.00 Hz, 2H, ArH); 7.43-7.45 (m, 1H, ArH);
7.62 (s, 1H, ArH); 7.71 (d, J = 8.00 Hz, 2H, ArH); 7.75 (d, J = 8.40 Hz, 2H, ArH); 8.04 (d, J =
13

8.00 Hz, 2H, ArH). ESI-MS: 470.60 (C24H19F3N3S2, [M+H]+). Anal. Calcd for C24H18F3N3S2: C,
61.39; H, 3.86; N, 8.95; Found: C, 61.59; H, 3.66; N, 8.96.
4.4.13.
4-(4-Bromophenyl)-2-(3-(4-bromophenyl)-5-(thiophen-2-yl)-4,5-dihydro-1H-
pyrazol-1-yl)thiazole (9a).
Yield 59%; mp. 219-201 ℃. 1H NMR (400 MHz, DMSO): δ 3.55-3.61 (m, 1H, CH2); 4.02-
4.09 (m, 1H, CH2); 6.03-6.07 (m, 1H, CH); 7.00 (t, J = 8.40 Hz, 1H, ArH); 7.27-7.28 (d, J = 3.20
Hz, 1H, ArH); 7.44-7.46 (d, J = 5.20 Hz, 1H, ArH); 7.64 (s, 1H, ArH); 7.70-7.72 (d, J = 8.40 Hz,
2H, ArH); 7.75-7.77 (d, J = 8.80 Hz, 4H, ArH); 8.03-8.05 (d, J = 8.10 Hz, 2H, ArH). ESI-MS:
546.38 (C22H16Br2N3S2, [M+H]+). Anal. Calcd for C22H15Br2N3S2: C, 48.46; H, 2.77; N, 7.71;
Found: C, 48.56; H, 2.84; N, 7.81.
4.4.14.
2-(3-(4-Bromophenyl)-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-4-
phenylthiazole (9b).
Yield 66%; mp. 180-182 ℃. 1H NMR (400 MHz, DMSO): δ 3.53-3.59 (m, 1H, CH2); 4.00-
4.07 (m, 1H, CH2); 6.00-6.05 (m, 1H, CH); 6.99-7.01 (m, 1H, ArH); 7.26-7.30 (m, 2H, ArH);
7.39 (t, J = 15.20 Hz, 3H, ArH); 7.44-7.46 (m, 1H, ArH); 7.69-7.76 (m, 4H, ArH); 7.84 (t, J =
8.40 Hz, 2H, ArH). ESI-MS: 467.48 (C22H17BrN3S2, [M+H]+). Anal. Calcd for C22H16BrN3S2: C,
56.65; H, 3.46; N, 9.01; Found: C, 56.75; H, 3.49; N, 9.00.
4.4.15.
2-(3-(4-Bromophenyl)-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-4-(4-
methoxyphenyl)thiazole (9c).
Yield 45%; mp. 196-198 ℃. 1H NMR (400 MHz, DMSO): δ 3.51-3.57 (m, 1H, CH2); 3.77
(s, 3H, OCH3); 3.95-4.07 (m, 1H, CH2); 5.99-6.03 (m, 1H, CH); 6.95 (d, J = 8.40 Hz, 2H, ArH);
6.99 (t, J = 8.40 Hz, 1H, ArH); 7.20 (s, 1H, ArH); 7.25-7.26 (d, J = 3.20 Hz, 1H, ArH); 7.44 (d, J
= 5.20 Hz, 1H, ArH); 7.68-7.71 (d, J = 7.60 Hz, 2H, ArH); 4.09 (s, 4H, ArH). ESI-MS: 497.49
(C23H19BrN3OS2, [M+H]+). Anal. Calcd for C23H18BrN3OS2 C, 55.65; H, 3.65; N, 8.46; Found:
C, 55.75; H, 3.68; N, 8.52.
4.4.16.
2-(3-(4-Bromophenyl)-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-4-(4-
(trifluoromethyl)phenyl)thiazole (9d).
14

Yield 49%; mp. 196-197 ℃. 1H NMR (400 MHz, DMSO): δ 3.52-3.58 (m, 1H, CH2); 3.99-
4.07 (m, 1H, CH2); 5.99-6.04 (m, 1H, CH); 6.94-7.01 (m, 3H, ArH); 7.25 (t, J = 19.20 Hz, 2H,
ArH); 7.45 (d, J = 4.80 Hz, 1H, ArH); 7.69-7.77 (m, 6H, ArH). ESI-MS: 535.51
(C23H16BrF3N3S2, [M+H]+). Anal. Calcd for C23H15BrF3N3S2: C, 51.69; H, 2.83; N, 7.86; Found:
C, 51.79; H, 2.93; N, 7.90.
4.4.17. 4-(4-Bromophenyl)-2-(3-(4-chlorophenyl)-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-
1-yl)thiazole (10a).
Yield 54%; mp. 218-220 ℃. 1H NMR (400 MHz, DMSO): δ 3.54-3.60 (m, 1H, CH2); 4.05-
4.078 (m, 1H, CH2); 6.01-6.05 (m, 1H, CH); 6.93-7.00 (m, 1H, ArH); 7.26 (d, J = 3.20 Hz, 1H,
ArH); 7.45 (t, J = 14.00 Hz, 2H, ArH); 7.58 (t, J = 17.30 Hz, 4H, ArH); 7.77-7.84 (m, 4H, ArH).
ESI-MS: 501.91 (C22H16ClN3S2, [M+H]+). Anal. Calcd for C22H15ClN3S2: C, 52.76; H, 3.02; N,
8.39; Found: C, 52.79 H, 3.02;N, 8.30.
4.4.18.
2-(3-(4-Chlorophenyl)-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-4-
phenylthiazole (10b).
Yield 43 %; mp. 160-163 ℃. 1H NMR (400 MHz, DMSO): δ 3.54-3.60 (m, 1H, CH2); 4.01-
4.08 (m, 1H, CH2); 6.01-6.05 (m, 1H, CH); 6.99-7.01 (m, 1H, ArH); 7.26-7.30 (m, 2H, ArH);
7.37-7.42 (m, 3H, ArH); 7.46 (t, J = 12.00 Hz, 1H, ArH); 7.58 (t, J = 17.20 Hz, 2H, ArH); 7.82-
7.84 (m, 4H, ArH). ESI-MS: 422.87 (C22H17ClN3S2, [M+H]+). Anal. Calcd for C22H16ClN3S2: C,
62.62; H, 3.82; N, 9.96; Found: C, 62.72;H, 3.89; N, 9.99.
4.4.19.
2-(3-(4-Chlorophenyl)-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-4-(4-
methoxyphenyl)thiazole (10c).
Yield 51%; mp. 185-186 ℃. 1H NMR (400 MHz, DMSO): δ 3.52-3.58 (m, 1H, CH2); 3.77
(s, 3H, OCH3); 4.00-4.07 (m, 1H, CH2); 5.99-6.04 (m, 1H, CH); 6.94-7.01 (m, 3H, ArH); 7.25 (t,
J = 18.00 Hz, 2H, ArH); 7.45(d, J = 4.40 Hz, 1H, ArH); 7.56-7.58 (d, J = 8.40 Hz, 2H, ArH);
7.75-7.77 (d, J = 8.60 Hz, 2H, ArH); 7.81-7.83 (d, J = 8.40 Hz, 2H, ArH). 13C NMR (DMSO-d6,
100 MHz) d: 21.53, 43.36, 60.04, 107.91, 126.02, 126.11(2), 126.44(2), 126.57, 126.97, 127.21,
127.90, 128.49(2), 129.94(2), 138.67, 140.55, 144.25, 149.39, 153.96, 165.17. ESI-MS:
15

452.91(C23H19ClN3OS2, [M+H]+). Anal. Calcd for C23H18ClN3OS2: C, 61.12; H, 4.01; N, 9.30;
Found: C, 61.17; H, 4.21; N, 9.37.
4.4.20.
2-(3-(4-Chlorophenyl)-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-4-(4-
(trifluoromethyl)phenyl)thiazole (10d).
Yield 49%; mp. 209-211 ℃. 1H NMR (400 MHz, DMSO): δ 3.55-3.61 (m, 1H, CH2); 4.01-
4.09 (m, 1H, CH2); 6.02-6.07 (m, 1H, CH); 6.99-7.01 (m, 1H, ArH); 7.28 (d, J = 2.80 Hz, 1H,
ArH); 7.44-7.48 (m, 1H, ArH); 7.57 (d, J = 8.60 Hz, 2H, ArH); 7.64 (s, 1H, ArH); 7.76 (d, J =
13
8.30 Hz, 2H, ArH);7.83 (d, J = 5.00 Hz, 2H, ArH); 8.04 (d, J = 8.00 Hz, 2H, ArH). C NMR
(DMSO-d6, 100 MHz) d: 40.82, 59.78, 105.60, 124.22, 125.74(2), 125.96(2), 127.10, 127.54,
127.88, 128.31(2), 129.02(2), 129.55, 130.94, 134.94, 136.44, 136.74, 151.08, 152.08, 169.02.
ESI-MS: 490.87 (C23H16ClF3N3S2, [M+H]+). Anal. Calcd for C23H15ClF3N3S2: C, 56.38; H, 3.09;
N, 8.58; Found: C, 56.39; H, 3.19; N, 8.67.
4.5. Antiproliferation activity
The antiproliferative activities of the prepared compounds against MCF-7and WM266.4
cell lines were evaluated as described elsewhere with some modifications.[30] Target tumor cell
lines were grown to log phase in RPMI 1640 medium supplemented with 10% fetal bovine
serum. After diluting to 2 × 104 cells per mL with the complete medium, 100 µL of the obtained
cell suspension were added to each well of 96-well culture plates. The subsequent incubation
was left at 37 °C, 5% CO2 atmosphere for 24 h before the cytotoxicity assessments. Tested
samples at pre-set concentrations were added to six wells with sorafenib as positive control.
After 48 h exposure period, 40 µL of PBS containing 2.5 mg mL-1 of MTT (3-(4, 5
dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) were added to each well. Four hours
later, 100 µL extraction solution (10% SDS-5% isobutyl alcohol-0.01 M HCl) were added. After
overnight incubation at 37 °C, the optical density was measured at a wavelength of 570 nm on an
ELISA microplate reader. In all experiments three replicate wells were used for each drug
concentration. Each assay was carried out for at least three times.
4.6. Kinase assay
16

The BRAF enzymatic assay was described by J. Dietrich et al.[31] The assay was performed
in triplicate for each tested compound in this study. Briefly, 7.5 ng Mouse Full-Length
GSTtagged BRAFV600E (Invitrogen, PV3849) were pre-incubated at room temperature for 1 h
with 1 µL drug and 4 µL assay dilution buffer. The kinase assay was initiated when 5 µL of a
solution containing 200 ng recombinant human full length, N-terminal His-tagged MEK1
(Invitrogen), 200 µM ATP (0.8 µCi hot ATP), and 30 mM MgCl2 in dilution buffer were added.
The kinase reaction was allowed to continue at room temperature for 25 min and then quenched
with 5 µL 5 × protein denaturing buffer (LDS) solution. The protein was further denatured by
heating for 5 min at 70 °C. 10 µL of each reaction were loaded into a 15-well, 4-12% precast
NuPage gel (Invitrogen) and run at 200 V, and upon completion, 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. The reaction that contained no active enzyme was used as the negative
control, and the reaction without inhibitor was used as the positive control.
4.7. Cell apoptosis assay by flow cytometry
MCF-7 cells were seeded in 16-well plates at a density of 1 × 106cells/well in RPMI 1640
medium and treated with 1, 3 µM 10d for 48 h. Cultured cells were stained with Annexin V-
FITC and propidium iodide (PI) in the dark at 4 °C for 30 min and analyzed by FACS Calibur
flow cytometer (Becton Dickinson, San Jose, CA) using Cell Quest software.
4.8. Docking simulations
The three-dimensional X-ray structure of BRAFV600E (PDB code: 2FB8) was chosen as the
template for the modeling study of compound 10d. The pdb file about the crystal structure of the
BRAFV600E kinase domain bound to SB-590885 (2FB8.pdb) was obtained from the RCSB
protein data bank (http://www.pdb.org). The molecular docking procedure was performed using
the CDOCKER protocol for the receptor–ligand interactions section of Discovery Studio 3.5
(Accelrys Software Inc, San Diego, CA).[32,33] All bound water and ligands were eliminated from
the protein and the polar hydrogen was added. The whole BRAFV600E complex was defined as a
receptor and the site sphere was selected based on the ligand binding location of SB-590885,
17

then the SB-590885 molecule was removed and replaced with compound 10d during the
molecular docking procedure. The types of interactions of the docked protein with ligand were
analyzed after the end of molecular docking.
4.9. QSAR model
A series of 20 compounds was conducted as a training set for QSAR modeling. Since it is
essential to assess the predictive power of the resulting QSAR models on an external set of
inhibitors, 80% (that is, 16) were utilized as a training set for QSAR modeling and the remaining
20%(that is, 4) were chosen as an external test subset for validating the reliability of the QSAR
model by the Diverse Molecules protocol in Discovery Studio 3.5. The selected test compounds
were: 10b, 6d, 9a, 9d.
The inhibitory abilities of the compounds in these literatures [IC50 (mol/L)] was changed to
the minus logarithmic scale [pIC50 (mol/L)] and then used for subsequent QSAR analyses as the
response variable.
In Discovery Studio, the CHARMm force field is used and the electrostatic potential and
the van der Waals potential are treated as separate terms. A +1e point charge is used as the
electrostatic potential probe and distance-dependent dielectric constant is used to mimic the
solvation effect. For the van der Waals potential a carbon atom with a 1.73 Å radius is used as a
probe. The truncation for both the steric and the electrostatic energies was set to 30 kcal/mol.
The standard parameters implemented in Discovery Studio 3.5 were used.
A Partial Least-Squares (PLS) model is built using energy grids as descriptors. QSAR
models were built using the created 3D QSAR protocol of Discovery Studio 3.5.
Acknowledgments
This work was supported by Natural Science Foundation of Jiangsu Province (No.
BK20130554) and supported by Major Projects on Control and Rectification of Water Body
Pollution (No. 2011ZX07204-001-2214 004).
18

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21

Figure captions
Scheme 1. Synthesis of compound 6a-10d. Reagents and conditions: (i) ethanol, 25 °C, 10
minutes; (ii) thiosemicarbazide EtOH, reflux,2h; (iii) DMF, 25 °C.
Table 1. Inhibition (IC50) of MCF-7 and WM266.4 cells proliferation and inhibition of
BRAFV600E by compounds 6a-10d (µM).
Table 2. Binding energy of compounds 6a-10d with BRAFV600E
.
Table 3. Experimental, predicted inhibitory activity of compounds 6a-10d by 3D-QSAR models
based upon active conformation achieved by molecular docking.
Figure 1. Apoptosis result by flow cytometry method of operation.
Figure 2. 2D binding model of compound 10d into the active pocket of BRAFV600E
.
Figure 3. 3D binding model of compound 10d into the active pocket of BRAFV600E
.
Figure 4. Plot of experimental versus predicted BRAF inhibitory activities of training set and
test set.
Figure 5. (a) 3D-QSAR model coefficients on electrostatic potential grids. Blue represents
positive coefficients; red represents negative coefficients. (b) 3D-QSAR model coefficients on
van der Waals grids. Green represents positive coefficients; yellow represents negative
coefficients.
22

O
O
Ethanol
25 °C 10 min
KOH
S
CHO
S
H3C
R1
R1
2a,2b,2c,2d,2e
3a,3b,3c,3d,3e
1
H
N
NH2
KOH
80 ºC 2 hr
H2N
R1
S
S
R1
5a,5b,5c,5d
R2
N
N
S
S
Br
N
N
O
N
DMF
R1 = OCH3, F, CH3, Br, Cl
R2 = Br, H, OCH3, CF3
S
6a -10d
H2N
25 ºC
3 hr
4a,4b,4c,4d,4e
R2
R1
R2
R1
R2
Comp.
6a
Comp.
8c
OCH3
OCH3
OCH3
OCH3
CH3
CH3
OCH3
Br
H
CF3
6b
8d
OCH3
CF3
6c
9a
Br
Br
Br
Br
Cl
Cl
Cl
Cl
Br
H
6d
9b
OCH3
CF3
7a
9c
F
F
Br
H
7b
9d
OCH3
CF3
7c
10a
10b
10c
10d
F
Br
H
7d
F
CH3
CH3
OCH3
CF3
8a
Br
H
8b
Scheme 1. Synthesis of compound 6a-10d. Reagents and conditions: (i) ethanol, 25 °C, 10
minutes; (ii) thiosemicarbazide EtOH, reflux, 2h; (iii) DMF, 25 °C.
23

Table 1. Inhibition (IC50) of MCF-7 and WM266.4 cells proliferation and inhibition of
BRAFV600E by compounds 6a-10d (µM).
IC50 SD (µM)
Compound
R1
R2
BRAFV600E
4.01 0.13
1.11 0.21
5.23 0.31
10.90 0.56
1.86 0.11
0.67 0.03
5.12 0.32
1.01 0.09
0.56 0.06
3.88 0.27
0.11 0.01
6.18 0.07
8.87 0.45
2.19 0.23
0.08 0.01
2.09 0.02
4.78 0.04
0.09 0.01
0.19 0.02
0.05 0.01
0.03 0.005
WM266.4a
5.87 0.49
2.12 0.32
31.60 1.12
8.11 0.78
0.22 0.03
0.17 0.02
4.42 0.45
5.12 0.39
18.36 1.15
7.52 0.58
3.77 0.44
2.11 0.26
12.98 0.89
1.87 0.21
2.01 0.19
27.38 0.81
1.45 0.22
2.19 0.34
1.31 0.17
0.12 0.01
0.06 0.01
MCF-7a
OCH3 Br
21.17.17 0.67
9.01 0.83
16.19 0.76
45.67 1.01
8.28 0.57
2.34 0.31
14.16 0.45
1.33 0.11
2.01 0.09
18.56 0.32
4.77 0.39
31.48 0.79
28.29 0.61
6.79 0.41
0.68 0.02
7.81 0.51
14.67 0.61
0.19 0.01
2.48 0.02
0.16 0.01
0.19 0.02
6a
OCH3
H
6b
OCH3 OCH3
OCH3 CF3
6c
6d
F
Br
7a
F
H
7b
F
OCH3
CF3
Br
7c
F
7d
CH3
CH3
CH3
CH3
Br
Br
Br
Br
Cl
8a
H
8b
OCH3
CF3
Br
8c
8d
9a
H
9b
OCH3
CF3
Br
9c
9d
10a
10b
10c
10d
Sorafenibb
Cl
H
Cl
OCH3
CF3
Cl
a BRAFV600E cell lines.
b Used as a positive control.
24

Table 2. Binding energy of compounds 6a-10d with BRAFV600E
.
Comp.
6a
Binding energy ∆Gb
-42.7731
Comp.
8c
Binding energy ∆Gb
-41.7160
-44.5859
-42.8494
6b
8d
-45.2577
-44.7631
6c
9a
-44.3719
-41.6707
6d
9b
-41.0285
-42.8896
7a
9c
-41.0247
-43.0418
7b
9d
-41.6267
-44.2035
7c
10a
10b
10c
10d
-42.6731
-43.5537
7d
-42.7915
-41.8499
8a
-42.7272
-45.6649
8b
25

Table 3 Experimental, predicted inhibitory activity of compounds 6a –10d by 3D-QSAR models
based upon active conformation achieved by molecular docking
BRAFV600E
Residual
Compound
error
Experimental pIC50 Predicted pIC50
4.674
5.045
4.791
4.340
5.082
5.361
4.849
5.876
5.696
4.736
5.321
4.502
4.548
5.168
6.167
5.107
4.834
6.721
5.606
6.796
4.583
5.068
4.713
4.754
5.069
5.371
4.875
5.907
5.602
4.787
5.353
4.473
5.476
5.277
6.164
5.141
4.888
5.393
5.622
6.751
0.091
-0.023
0.078
-0.414
0.012
-0.010
-0.026
-0.031
0.094
-0.051
-0.032
0.029
-0.928
-0.109
0.003
-0.034
-0.054
1.327
-0.016
0.045
6a
6b
6c
6d
7a
7b
7c
7d
8a
8b
8c
8d
9a
9b
9c
9d
10a
10b
10c
10d
26

Figure 1. Apoptosis result by flow cytometry method of operation.The apoptosis qualitative
effect was demonstrated in this two group of figures. A and B respectively represents the
apoptosis qualitative effect of MCF-7 and WM 266.4 conducted by compound 10d, which
contains control group. A1, B1 equally were conducted with 1 µM and A2, B2 were equally
conducted with 3 µM. With the dose of compound 10d increasing, the WM 266.4 and MCF-7
were dyed more and more Propidum Iodide and Annexin V-FITC, which indicated more and
more cancer cell gradually encountered apoptosis or died. When conducted with 3 µM, most of
the WM 266.4 and MCF-7 were caught in apoptosis and even died. This result explains
compound 10d could induce the apoptosis of MCF-7 and WM266.4 by qualitative analysis.
27

Figure 2. 2D binding model of compound 10d into the active pocket of BRAFV600E
28

Figure 3. 3D binding model of compound 10d into the active pocket of BRAFV600E
.
29