Design, synthesis, and biological evaluation of new B-RafV600E kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.03.038

The association of deregulated signal pathways with various diseases has long been a research hotspot. One of the most important signal pathways, the MAPK (mitogen-activated protein kinase) signal pathway, plays a vital role in transducing extracellular signals into vital intracellular mechanisms. While mutations on its key component Raf kinase lead to sever diseases, targeted inhibition has thereby become an attractive therapeutic strategy. Several drugs have been approved for the treatment of Raf relevant diseases, yet more candidates are ever needed as the known drugs have confronted resistance and side effects. In the present study, we primarily investigated the binding modes of type I/II and type II inhibitors with B-Raf kinase. Based on the current knowledge, these ligands were fragmented and recombined to provide new interesting insights. Afterwards, a series of derivatives has been synthesized after the validation of hit compound. In addition, in vitro assays were carried out to profile the pharmacological properties of all the entities. Of all the compounds, compound 5h showed the best profile and may be used in the future study.

Full text of DOI:10.1016/j.bmc.2018.03.038

Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of new B-RafV600E kinase
inhibitors
a
a
a,b
a
a
a,
⇑
Peng-Fei Wang , Yong-Jiao Zhang , Dong Wang , Hui-Min Hu , Zhong-Chang Wang , Chen Xu ,
a,
⇑
a,
⇑
Han-Yue Qiu , Hai-Liang Zhu
a
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210023, People’s Republic of China
State Key Laboratory of Natural Medicines, Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, People’s Republic of China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
The association of deregulated signal pathways with various diseases has long been a research hotspot.
One of the most important signal pathways, the MAPK (mitogen-activated protein kinase) signal path-
way, plays a vital role in transducing extracellular signals into vital intracellular mechanisms. While
mutations on its key component Raf kinase lead to sever diseases, targeted inhibition has thereby become
an attractive therapeutic strategy. Several drugs have been approved for the treatment of Raf relevant
diseases, yet more candidates are ever needed as the known drugs have confronted resistance and side
effects. In the present study, we primarily investigated the binding modes of type I/II and type II inhibi-
tors with B-Raf kinase. Based on the current knowledge, these ligands were fragmented and recombined
to provide new interesting insights. Afterwards, a series of derivatives has been synthesized after the val-
idation of hit compound. In addition, in vitro assays were carried out to profile the pharmacological prop-
erties of all the entities. Of all the compounds, compound 5h showed the best profile and may be used in
the future study.
Received 13 December 2017
Revised 18 March 2018
Accepted 24 March 2018
Available online xxxx
Keywords:
B-Raf
Molecular dynamics
Docking
Kinase inhibitor
Drug design
Ó 2018 Elsevier Ltd. All rights reserved.
1
. Introduction
strategy.10 Compared with other known drugs (Fig. 1), such as dab-
rafenib and sorafenib, vemurafenib has a significant advantage in
1
1,12
The Raf kinase is a key ingredient in the ERK signaling branch of
the Raf kinase selectivity.
effect towards B-Raf kinase over the other two subtypes. Hence,
It exhibits a much stronger inhibitory
13
MAPK cascade and holds a central position in the signal transduc-
tion machinery. Of its three family members, B-Raf kinase dis-
1
it’s widely employed in the treatment of diseases caused by
V600E
tributes more widely and functions more actively than the other
B-Raf
. However, cases of relapse and resistance gradually
two subtypes, i.e. A-Raf and c-Raf.2 When pathological mutation
increase with the use of vemurafenib.
14,15
The adverse effect caused
3
16
occurs to the Raf kinase, it usually refers to the B-Raf subtype
by vemurafenib, on the other side, is also unneglectable. As a clas-
sic type I/II kinase inhibitor, vemurafenib induces paradoxical effect
in normal cells, i.e. enhancing the activity of wild type B-Raf kinase
Especially, when the VAL residue at site 600 mutated into an
GLU residue, the kinase will mimic its activated state and persis-
tently trigger downstream pathways.4 Various malignant tumors
are found to be related to mutated B-Raf kinase, for instance, most
,5
17
in low doses while suppressing it in high doses. Hitherto, exten-
sive studies have been carried out to reveal the underlying mecha-
V600E
6
18,19
melanoma cases are caused by the B-Raf
mutation. Other
nism and to design new paradoxical breakers.
somatic tumors, such as colorectal carcinoma and lung cancer,
are also validated to be involved with the mutated B-Raf kinase.
It’s of interest to note that among all the three types of inhibi-
tors (targeting type I, type II and type I/II conformational kinases),
the type II inhibitors have the minimal paradoxical inducing
effects. On this basis, modifying the other types of inhibitors into
type II may yield optimized agents without paradoxical inducing
effect. In this study, we primarily investigated the binding modes
of type I/II and type II protein–ligand complexes. It has been noted
that the major differences between them lie in the back pocket
region, rather than in the front pocket region. Based on the analy-
sis, the shared components for both two types’ inhibitors in the
7
,8
So far, great efforts have been dedicated to the development of
potent B-Raf inhibitors, and several drugs have already been
2
0
9
approved for the treatment of Raf-related diseases. The most suc-
cessful case is the gradual optimization for the final drug vemu-
rafenib employing FBDD (Fragment-based Drug Design)
⇑
Corresponding authors.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
https://doi.org/10.1016/j.bmc.2018.03.038
968-0896/Ó 2018 Elsevier Ltd. All rights reserved.
0
Please cite this article in press as: Wang P.-F., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.03.038

2
P.-F. Wang et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
O
S
O
HN
F
F
N
O
F
H
N
O
S
Cl
O
O
Cl
S
N
H
O
O
N
N
F
N
H
N
H
CF3
F
NH2
N
N
N
H
Dabrafenib
Sorafenib
Vemurafenib
Fig. 1. Chemical structures of dabrafenib, sorafenib and vemurafenib.
front pocket region were validated. The recombination was then
achieved to furnish several hybrids. Taking the binding score and
synthesis accessibility into consideration, hit compound was
selected and structurally modified. In addition, the validating
molecular dynamics simulation helped to give further information.
After chemical synthesis, 20 new entities were acquired and tested
by in vitro assays. The results suggest some of the new compounds
have good activity towards B-RafV600E kinase. Meanwhile, the same
workflow may also be employed in other kinase inhibitor studies
to avoid paradoxical effects with high efficiency.
water and extracted with ethyl acetate. The organic layer was
washed with aqueous saturated sodium bicarbonate, dried by
sodium sulfate, filtered and concentrated in vacuo. Purification by
flash chromatography gave the desired products 3a-3b.
2.2.3. Synthesis of 4a-4b
To a solution of 3a-3b (1.8 mmol) in 2 mL ethanol was added
0% hydrazine hydrate (1 mL) and 10% palladium charcoal (0.03
8
g). The reaction was refluxed for 10 min and filtered by Celite.
The filtrate was dried by sodium sulfate, and concentrated in vacuo
to afford compounds 4a-4b, which were used without further
purification.
2
. Material and methods
2.1. Materials
2.2.4. Synthesis of 5a-5t
Under stirring, substituted nitrobenzoic acids (1.5 mmol), EDC
All chemicals (reagent grade) used were purchased from Nan-
(
1.5 mmol, 0.38 g) and HOBT (1.5 mmol, 0.2 g) were added in 4
mL DMF. After reaction at rt for 30 min, the solution was added
with DMAP (1.5 mmol, 0.18 g), 4a-4b (1.5 mmol) and Et N (0.8
jing Chemical Reagent Co. Ltd. (Nanjing, China). Sorafenib was pur-
1
chased from Sigma-Aldrich (St. Louis, MO). All the H NMR spectra
3
were recorded on a Bruker DPX 400 model spectrometer in
mL). The reaction mixture was stirred at rt for 18 h, before diluted
with 20 mL of water and extracted with ethyl acetate. The organic
layer was washed with aqueous saturated sodium bicarbonate,
dried by sodium sulfate, filtered and concentrated in vacuo. Purifi-
cation by flash chromatography gave the desired products 5a-5t.
DMSO d
6
, and chemical shifts (d) are reported as parts per million
(
ppm). ESI-MS spectra were recorded by a Mariner System 5304
Mass spectrometer. Elemental analyses were performed on a
CHN-O-Rapid instrument and were within 0.4% of the theoretical
values. Melting points were determined on a XT4 MP apparatus
(
Taike Corp, Beijing, China). Thin layer chromatography (TLC) was
2
.2.4.1. 3-Benzamido-N-(1H-indazol-5-yl)benzamide (5a). White
performed on silica gel plates (Silica Gel 60 GF254) and visualized
in UV light (254 nm and 365 nm). Column chromatography was
performed using silica gel (200–300 mesh) and eluting with ethy-
lacetate and petroleum ether (bp. 30–60 °C). A375 cells were
kindly provided by Stem Cell Bank, Chinese Academy of Sciences.
The other cell lines (WM266-4, WM1361, HT29 and HCT116) were
preserved in the State Key Laboratory of Pharmaceutical Biotech-
nology of Nanjing University. The AnnexinV-FITC cell apoptosis
assay kit was purchased from Vazyme Biotechco., Ltd (Nanjing,
China). The Raf kinase was purchased from Invitrogen (US). All
antibodies were obtained from WanleiBio (Shenyang, China).
1
solid, yield 88.1%, m.p. 285.2–286.9 °C.
DMSO d ) d 13.03 (s, 1H, NH), 10.48 (s, 1H, NH), 10.32 (s, 1H,
NH), 8.35 (s, 1H, ArH), 8.27 (s, 1H, CH), 8.04 (dd, J = 20.2, 9.5 Hz,
4
2
H NMR (400 MHz,
6
H, ArH), 7.73 (d, J = 7.7 Hz, 1H, ArH), 7.64 (dd, J = 19.6, 8.1 Hz,
+
16 4 2
H, ArH), 7.59–7.51 (m, 4H, ArH). MS EI : 356.39 (C21H N O ,
+
[
4
M] ). Anal. Calcd: C, 70.77; H, 4.53; N, 15.72; Found: C, 70.57; H,
.68; N, 15.83.
2.2.4.2.
N-(3-((1H-Indazol-5-yl)carbamoyl)phenyl)-3-fluorobenza-
1
mide (5b). White solid, yield 73.5%, m.p. 293.6–295.2 °C. H NMR
(
(
(
6
600 MHz, DMSO d ) d 13.03 (s, 1H, NH), 10.54 (s, 1H, NH), 10.32
s, 1H, NH), 8.30 (d, J = 42.5 Hz, 2H, ArH), 8.08 (s, 1H, CH), 8.04
d, J = 8.0 Hz, 1H, ArH), 7.87 (d, J = 7.8 Hz, 1H, ArH), 7.83 (d, J =
2
2
8
.2. Chemistry
9
.7 Hz, 1H, ArH), 7.75 (d, J = 7.7 Hz, 1H, ArH), 7.67–7.60 (m, 2H,
.2.1. Synthesis of 2a-2b
To a solution of 1a-1b (4.5 mmol) in 4 mL ethanol was added
0% hydrazine hydrate (2 mL) and 10% palladium charcoal (0.08
ArH), 7.57–7.51 (m, 2H, ArH), 7.48 (td, J = 8.5, 2.2 Hz, 1H, ArH).
MS EI : 374.38 (C21
4
+
+
H
15FN
4
O
2
, [M] ). Anal. Calcd: C, 67.37; H,
.04; N, 14.97; Found: C, 67.53; H, 4.13; N, 15.21.
g). The reaction was refluxed for 10 min and filtered by Celite.
The filtrate was dried by sodium sulfate, and concentrated in vacuo
to afford compounds 2a-2b, which were used without further
purification.
2
.2.4.3.
N-(3-((1H-Indazol-5-yl)carbamoyl)phenyl)-3-chlorobenza-
1
mide (5c). Grey solid, yield 80.8%, m.p. 282.3–284.1 °C. H NMR
600 MHz, DMSO d ) d 13.03 (s, 1H, NH), 10.71 (s, 1H, NH), 10.33
(
6
2.2.2. Synthesis of 3a-3b
(s, 1H, NH), 8.97 (s, 1H, ArH), 8.32–8.25 (m, 3H, ArH and CH),
Under stirring, 3-nitrobenzoic acid (2 mmol, 0.33 g), EDC (2
8.08 (s, 1H, ArH), 8.03 (d, J = 8.0 Hz, 1H, ArH), 7.88 (d, J = 8.3 Hz,
mmol, 0.38 g) and HOBT (2 mmol, 0.27 g) were added in 5 mL
DMF. After reaction at rt for 30 min, the solution was added with
1H, ArH), 7.77 (d, J = 7.7 Hz, 1H, ArH), 7.65 (d, J = 10.3 Hz, 1H,
+
ArH), 7.57–7.52 (m, 2H, ArH). MS EI : 390.83 (C21
H
15ClN
4
O
2
,
+
DMAP (2 mmol, 0.24 g), 2a-2b (2 mmol) and Et
3
N (1 mL). The reac-
[M] ). Anal. Calcd: C, 64.54; H, 3.87; N, 14.34; Found: C, 64.32; H,
3.83; N, 14.42.
tion mixture was stirred at rt for 18 h, before diluted with 20 mL of
Please cite this article in press as: Wang P.-F., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.03.038

P.-F. Wang et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
3
+
+
2
.2.4.4. 3-(4-Fluorobenzamido)-N-(1H-indazol-5-yl)benzamide (5d).
EI : 357.37 (C20
H
15
N
5
O
2
, [M] ). Anal. Calcd: C, 67.22; H, 4.23; N,
1
White solid, yield 71.8%, m.p. 294.7–296.5 °C. H NMR (600 MHz,
DMSO d ) d 13.04 (s, 1H, NH), 10.41 (d, J = 105.6 Hz, 2H, NH),
.34 (s, 1H, ArH), 8.27 (s, 1H, CH), 8.18–7.97 (m, 4H, ArH), 7.74
d, J = 7.4 Hz, 1H, ArH), 7.66 (d, J = 8.7 Hz, 1H, ArH), 7.53 (t, J =
19.60; Found: C, 67.27; H, 4.29; N, 19.53.
6
8
(
8
H
2.2.4.11. N-(3-((1H-Indazol-5-yl)carbamoyl)phenyl)-6-methylpicoli-
namide (5k). Yellow solid, yield 77.4%, m.p. 291.5–292.7 °C.
NMR (600 MHz, DMSO d
10.34 (s, 1H, NH), 9.06 (s, 1H, ArH), 8.34 (s, 1H, ArH), 8.29–8.25
(m, 2H, ArH and CH), 8.07 (s, 1H, ArH), 8.05–8.02 (m, 1H, ArH),
1
H
+
.0 Hz, 2H, ArH), 7.40 (t, J = 8.8 Hz, 2H, ArH). MS EI : 374.38 (C21
-
6
) d 13.06 (s, 1H, NH), 10.61 (s, 1H, NH),
+
4
15FN O
2
, [M] ). Anal. Calcd: C, 66.37; H, 4.04; N, 14.97; Found:
C, 66.06; H, 4.12; N, 14.96.
7
7
.76 (d, J = 7.7 Hz, 1H, ArH), 7.66 (d, J = 10.0 Hz, 1H, ArH), 7.56–
.51 (m, 2H, ArH), 7.44 (d, J = 8.1 Hz, 1H, ArH), 2.57 (s, 3H, CH).
2
.2.4.5. 3-(4-Chlorobenzamido)-N-(1H-indazol-5-yl)benzamide (5e).
+
+
1
MS EI : 371.40 (C21
N, 18.86; Found: C, 67.88; H, 4.63; N, 18.82.
17 5 2
H N O , [M] ). Anal. Calcd: C, 67.91; H, 4.61;
White solid, yield 76.1%, m.p. 296.5–298.6 °C. H NMR (600 MHz,
DMSO d ) d :13.03 (s, 1H, NH), 10.54 (s, 1H, NH), 10.32 (s, 1H,
6
NH), 8.33 (s, 1H, ArH), 8.26 (s, 1H, CH), 8.08 (s, 1H, ArH), 8.04 (t,
2
.2.4.12. N-(3-((1H-Indazol-5-yl)carbamoyl)phenyl)-6-chloropicoli-
J = 7.4 Hz, 3H, ArH), 7.74 (d, J = 7.8 Hz, 1H, ArH), 7.65 (t, J = 8.7
1
+
namide (5l). Yellow solid, yield 59.8%, m.p. 290.5–292.6 °C.
NMR (600 MHz, DMSO d ) d 13.05 (s, 1H, NH), 10.75 (s, 1H, NH),
1
2
H
Hz, 3H, ArH), 7.53 (t, J = 7.9 Hz, 2H, ArH). MS EI : 406.83(C21
ClN O
4 3
H
15
-
+
, [M] ). Anal. Calcd: C, 64.54; H, 3.87; N, 14.34; Found: C,
6
0.34 (s, 1H, NH), 9.01 (d, J = 2.4 Hz, 1H, ArH), 8.42 (dd, J = 8.3,
.5 Hz, 1H, ArH), 8.32 (s, 1H, ArH), 8.26 (s, 1H, CH), 8.08 (s, 1H,
6
4.81; H, 3.83; N, 13.91.
ArH), 8.03 (d, J = 9.3 Hz, 1H, ArH), 7.78 (d, J = 7.7 Hz, 1H, ArH),
7.74 (d, J = 8.3 Hz, 1H, ArH), 7.65 (d, J = 8.9 Hz, 1H, ArH), 7.57–
2
.2.4.6. 3-(4-Bromobenzamido)-N-(1H-indazol-5-yl)benzamide (5f).
1
Grey solid, yield 82.3%, m.p. 286.7–287.9 °C. H NMR (600 MHz,
DMSO d ) d 13.03 (s, 1H, NH), 10.54 (s, 1H, NH), 10.31 (s, 1H,
NH), 8.32 (s, 1H, ArH), 8.26 (s, 1H, CH), 8.08 (s, 1H, ArH), 8.03 (d,
J = 8.0 Hz, 1H, ArH), 7.97 (d, J = 8.4 Hz, 2H, ArH), 7.78 (d, J = 8.4
Hz, 2H, ArH), 7.76–7.73 (m, 1H, ArH), 7.65 (d, J = 8.8 Hz, 1H, ArH),
+
+
7
5 2
.53 (m, 2H, ArH). MS EI : 391.08 (C20H14ClN O , [M] ). Anal. Calcd:
6
C, 61.31; H, 3.60; N, 17.87; Found: C, 61.36; H, 3.54; N, 17.79.
2
.2.4.13. N-(3-((1H-Indazol-5-yl)carbamoyl)phenyl)-5-bromopicoli-
1
+
+
namide (5m). Grey solid, yield 56.8%, m.p.297.8–299.6 °C.
NMR (600 MHz, DMSO d ) d 13.03 (s, 1H, NH), 10.72 (s, 1H, NH),
0.33 (s, 1H, NH), 9.12 (s, 1H), 8.94 (s, 1H, ArH), 8.61 (s, 1H,
H
7
.56–7.51 (m, 2H, ArH). MS EI : 435.28 (C21
4 2
H15BrN O , [M] ). Anal.
6
Calcd: C, 57.95; H, 3.47; N, 12.87; Found: C, 57.91; H, 3.53; N,
1
1
2.75.
ArH), 8.31 (s, 1H, ArH), 8.26 (s, 1H, CH), 8.08 (s, 1H, ArH), 8.04 (d,
J = 7.9 Hz, 1H, ArH), 7.78 (d, J = 7.7 Hz, 1H, ArH), 7.66 (d, J = 8.8
2
.2.4.7. 3-(4-(Tert-butyl)benzamido)-N-(1H-indazol-5-yl)benzamide
+
Hz, 1H, ArH), 7.58–7.53 (m, 2H, ArH). MS EI : 436.27 (C20
H
14BrN
5
-
1
(5g). White solid, yield 73.3%, m.p. 253.1–255.0 °C. H NMR (600
+
O
2
, [M] ). Anal. Calcd: C, 55.06; H, 3.23; N, 16.05; Found: C, 54.97;
MHz, DMSO d
1
8
6
) d 13.03 (s, 1H, NH), 10.39 (s, 1H, NH), 10.31 (s,
H, NH), 8.34 (s, 1H, ArH), 8.27 (s, 1H, CH), 8.08 (s, 1H, ArH),
.04 (d, J = 7.7 Hz, 1H, ArH), 7.95 (d, J = 8.4 Hz, 2H, ArH), 7.72 (d,
H, 3.41; N, 15.89.
2
.2.4.14 N-(3-((1H-Indazol-5-yl)carbamoyl)phenyl)-4-chloropicoli-
J = 7.7 Hz, 1H, ArH), 7.66 (d, J = 8.8 Hz, 1H, ArH), 7.57 (d, J = 8.4
Hz, 2H, ArH), 7.55–7.51 (m, 2H, ArH), 1.34 (s, 9H, CH). MS EI :
3
1
1
namide (5n). White solid, yield 74.5%, m.p. 296.4–298.2 °C.
NMR (600 MHz, DMSO d ) d 13.05 (s, 1H, NH), 10.56 (s, 1H, NH),
0.33 (s, 1H, NH), 8.33 (s, 1H, ArH), 8.26 (s, 1H, CH), 8.05 (q, J =
.2 Hz, 4H, ArH), 7.64 (d, J = 8.6 Hz, 3H, ArH), 7.53 (t, J = 8.0 Hz,
H
+
+
6
56.39 (C25
H
24
N
4
O
2
, [M] ). Anal. Calcd: C, 72.80; H, 5.86; N,
1
9
2
6
3.58; Found: C, 72.68; H, 5.89; N, 13.62.
+
5 2
H, ArH). MS EI+: 391.08 (C20H14ClN O , [M] ). Anal. Calcd: C,
2.2.4.8. N-(1H-Indazol-5-yl)-3-(4-(trifluoromethyl)benzamido)benza-
1.31; H, 3.60; N, 17.87; Found: C, 61.36; H, 3.58; N, 17.84.
1
mide (5h). Yellow solid, yield 52.9%, m.p. 285.2–287.1 °C. H NMR
(
1
600 MHz, DMSO d
H, NH), 8.36 (s, 1H, ArH), 8.27 (s, 1H, CH), 8.22 (d, J = 8.1 Hz, 2H,
ArH), 8.08–8.04 (m, 2H, ArH), 7.94 (d, J = 8.4 Hz, 2H, ArH), 7.77
6
) d 13.06 (s, 1H, NH), 10.74 (s, 1H, NH), 10.35 (s,
2
.2.4.15. 3-(((3-Chlorobenzoyl)oxy)amino)-N-(1H-indazol-5-yl)ben-
1
zamide (5o). White solid, yield 72.2%, m.p. 294.8–296.3 °C.
NMR (600 MHz, DMSO d
1
H
6
) d 13.04 (s, 1H, NH), 10.55 (s, 1H, NH),
(
d, J = 7.8 Hz, 1H, ArH), 7.66 (d, J = 8.6 Hz, 1H, ArH), 7.55 (t, J =
0.33 (s, 1H, NH), 8.29 (t, J = 2.0 Hz, 1H, ArH), 8.24 (d, J = 1.5 Hz,
+
+
8
15 3 4 2
.0 Hz, 2H, ArH). MS EI : 424.38 (C22H F N O , [M] ). Anal. Calcd:
1
H, CH), 8.11–8.04 (m, 2H, ArH), 7.99 (dt, J = 7.5, 2.1 Hz, 1H,
C, 62.26; H, 3.56; N, 13.20; Found: C, 62.32; H, 3.48; N, 13.21.
+
ArH), 7.72–7.61 (m, 3H, ArH), 7.29–7.20 (m, 4H, ArH). MS EI :
4
1
+
4 3
06.83 (C21H15ClN O , [M] ). Anal. Calcd: C, 62.00; H, 3.72; N,
2
.2.4.9. N-(1H-Indazol-5-yl)-3-(4-phenoxybenzamido)benzamide (5i).
3.77; Found: C, 62.48; H, 3.57; N, 13.71.
1
White solid, yield 85.1%, m.p. 298.2–299.3 °C. H NMR (600 MHz,
DMSO d ) d 13.04 (s, 1H, NH), 10.48 (s, 1H, NH), 10.25 (s, 1H,
NH), 8.16 (d, J = 1.4 Hz, 1H, CH), 8.07 (d, J = 7.7 Hz, 2H, ArH),
7
7
2
6
2
.2.4.16. 3-Benzamido-N-(1H-indol-5-yl)benzamide (5p). White
1
solid, yield 57.9%, m.p. 175.5–177.3 °C.
DMSO d
NH), 8.33 (s, 1H, ArH), 8.07–7.97 (m, 4H, ArH), 7.73 (d, J = 7.7 Hz,
1
7
7
1
H NMR (600 MHz,
.96–7.89 (m, 2H, ArH), 7.86 (dd, J = 7.5, 1.6 Hz, 1H, ArH), 7.76–
.64 (m, 3H, ArH), 7.45 (t, J = 7.5 Hz, 1H, ArH), 7.39 (t, J = 7.5 Hz,
6
) d 11.05 (s, 1H, NH), 10.46 (s, 1H, NH), 10.13 (s, 1H,
H, ArH), 7.23–7.18 (m, 2H, ArH), 7.14 (tt, J = 7.3, 2.0 Hz, 1H,
H, ArH), 7.62 (t, J = 7.3 Hz, 1H, ArH), 7.56 (t, J = 7.5 Hz, 2H, ArH),
.52 (t, J = 7.9 Hz, 1H, ArH), 7.41 (dd, J = 8.7, 1.7 Hz, 1H, ArH),
.37 (d, J = 8.6 Hz, 1H, ArH), 7.34 (t, J = 2.7 Hz, 1H, CH), 6.43 (s,
+
ArH), 7.05 (dd, J = 7.6, 2.0 Hz, 2H, ArH). MS EI : 448.48
+
(
C
27
H
20
N
4
O
3
, [M] ). Anal. Calcd: C, 72.31; H, 4.50; N, 12.49; Found:
+
+
C,71.31; H, 4.63; N, 12.57.
19 3 2
H, CH). MS EI : 357.41 (C22H N O , [M] ). Anal. Calcd: C, 73.93;
H, 5.36; N, 11.76; Found: C, 73.81; H, 4.98; N, 11.51.
2
.2.4.10. N-(3-((1H-Indazol-5-yl)carbamoyl)phenyl)picolinamide (5j).
1
White solid, yield 67.3%, m.p. 294.8–296.1 °C. H NMR (600 MHz,
DMSO d ) d 13.06 (s, 1H, NH), 10.71 (s, 1H, NH), 10.34 (s, 1H,
2.2.4.17. 3-(4-Chlorobenzamido)-N-(1H-indol-5-yl)benzamide (5q).
1
6
White solid, yield 65.5%, m.p. 214.1–216.3 °C. H NMR (600 MHz,
NH), 9.17 (d, J = 1.8 Hz, 1H, ArH), 8.79 (dd, J = 4.8, 1.5 Hz, 1H,
ArH), 8.38–8.34 (m, 2H, ArH), 8.27 (s, 1H, CH), 8.08–8.03 (m, 2H,
ArH), 7.77 (d, J = 7.7 Hz, 1H, ArH), 7.66 (d, J = 7.7 Hz, 1H, ArH),
6
DMSO d ) d 11.05 (s, 1H, NH), 10.52 (s, 1H, NH), 10.12 (s, 1H,
NH), 8.31 (s, 1H), 8.07–7.99 (m, 4H, ArH), 7.74 (d, J = 7.6 Hz, 1H,
ArH), 7.64 (d, J = 8.5 Hz, 2H, ArH), 7.52 (t, J = 7.9 Hz, 1H, ArH),
7.41 (d, J = 8.7 Hz, 1H, ArH), 7.37 (d, J = 8.6 Hz, 1H, ArH), 7.34 (t, J
7
.60 (dd, J = 7.8, 4.8 Hz, 1H, ArH), 7.55 (t, J = 7.7 Hz, 2H, ArH). MS
Please cite this article in press as: Wang P.-F., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.03.038

4
P.-F. Wang et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
+
=
[
4
2.6 Hz, 1H, CH), 6.43 (s, 1H, CH). MS EI : 374.38 (C22
H16ClN
3
O
2
,
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.
+
M] ). Anal. Calcd: C, 67.78; H, 4.14; N, 10.78; Found: C, 67.49; H,
.23; N, 10.51.
2
.2.4.18. N-(1H-Indol-5-yl)-3-(4-phenoxybenzamido)benzamide (5r).
1
White solid, yield 54.9%, m.p. 148.8–150.6 °C. H NMR (600 MHz,
2.4.2. Cell culture
DMSO d
6
) d 11.05 (s, 1H, NH), 10.40 (s, 1H, NH), 10.12 (s, 1H,
Four cancer cell lines were used in this work: human melanoma
V600E
V600E
NH), 8.31 (s, 1H, ArH), 8.06 (d, J = 8.8 Hz, 2H, ArH), 8.04–7.99 (m,
cells A375 (B-Raf
mutated), WM266-4 (B-Raf
mutated)
WT
2
3
H, ArH), 7.72 (d, J = 7.7 Hz, 1H, ArH), 7.49 (dt, J = 21.9, 8.2 Hz,
H, ArH), 7.41 (dd, J = 8.7, 1.7 Hz, 1H, ArH), 7.37 (d, J = 8.7 Hz, 1H,
and WM1361 (B-Raf ), human colon cancer cells HT29 (B-
V600E
WT
Raf
mutated) and HCT116 (B-Raf ). All cell lines were grown
ArH), 7.34 (t, J = 2.7 Hz, 1H, ArH), 7.24 (t, J = 7.4 Hz, 1H, ArH),
in Dulbecco’s modified Eagle’s medium (DMEM Hyclone), except
for A375, which was maintained in DMEM 12,430 (Invitrogen)
with Sodium Pyruvate (Invitrogen, 11360–070) added. All media
were supplemented with 10% foetal bovine serum (FBS, BI), 2
mmol/L of l-glutamine, 100 units/mL of penicillin–streptomycin
(Sigma-Aldrich), 100 mg/mL streptomycin (Hyclone) and incu-
+
7
.13 (t, J = 8.7 Hz, 4H, CH), 6.43 (s, 1H, CH). MS EI : 449.51
+
(
C
28
H
23
N
3
O
3
, [M] ). Anal. Calcd: C, 74.82; H, 5.16; N, 9.35; Found:
C, 74.66; H, 5.03; N, 9.28.
2
.2.4.19. N-(3-((1H-Indol-5-yl)carbamoyl)phenyl)picolinamide (5s).
1
White solid, yield 88.6%, m.p. 204.5–206.4 °C. H NMR (600 MHz,
DMSO d ) d 11.05 (s, 1H, NH), 10.65 (s, 1H, NH), 10.14 (s, 1H,
2
bated at 37 °C in a humidified atmosphere containing 5% CO .
6
NH), 9.16 (d, J = 1.7 Hz, 1H, ArH), 8.79 (dd, J = 4.8, 1.6 Hz, 1H,
ArH), 8.35 (dt, J = 7.9, 1.9 Hz, 1H, ArH), 8.32 (s, 1H, ArH), 8.09–
2.4.3. Anti-proliferation assay
The anti-proliferative activities of the prepared compounds
against the A375, WM266-4, WM1361, HT-29 and HCT116 were
evaluated using a standard (MTT)-based colorimetric assay with
some modification. Cell lines were grown to log phase in DMEM
supplemented with 10% foetal bovine serum. Cell suspensions
7
4
.95 (m, 2H, ArH), 7.75 (d, J = 7.8 Hz, 1H, ArH), 7.60 (dd, J = 7.9,
.8 Hz, 1H, ArH), 7.53 (t, J = 7.9 Hz, 1H, ArH), 7.41 (dd, J = 8.7, 1.8
Hz, 1H, ArH), 7.37 (d, J = 8.6 Hz, 1H, ArH), 7.34 (t, J = 2.7 Hz, 1H,
+
+
CH), 6.43 (s, 1H, CH). MS EI : 356.39 (C21
16 4 2
H N O , [M] ). Anal.
Calcd: C, 70.38; H, 5.06; N, 15.63; Found: C, 70.33; H, 4.97; N,
were prepared and 100
l
L/well dispensed into 96-well plates to
give 10 cells/well. After incubation for 24 h, cells were treated
with the target compounds at 0.01, 0.1, 1, 10 and 100 M in the
4
1
5.52.
l
2
.2.4.20.
N-(3-((1H-Indol-5-yl)carbamoyl)phenyl)-6-chloropicoli-
presence of 10% FBS for 48 h. Afterwards, cell viability was
assessed by the conventional 3-(4, 5-dimethylthiazol-2-yl)-2, 5-
diphenyltetrazolium bromide (MTT) reduction assay carried out
strictly according to the manufacturer’s instructions (Sigma). The
absorbance (OD570) was read on an ELISA reader (Tecan, Austria).
In all experiments, three replicate wells were used for each drug
concentration. Each assay was performed at least three times.
1
namide (5t). White solid, yield 55.8%, m.p. 169.1–171.3 °C.
NMR (600 MHz, DMSO d ) d 11.06 (s, 1H, NH), 10.70 (s, 1H, NH),
0.15 (s, 1H, NH), 9.01 (s, 1H, ArH), 8.41 (dd, J = 8.3, 2.5 Hz, 1H,
H
6
1
ArH), 8.30 (s, 1H, ArH), 8.01 (q, J = 8.4, 7.3 Hz, 2H, ArH), 7.76 (dd,
J = 18.6, 8.1 Hz, 2H, ArH), 7.54 (t, J = 7.9 Hz, 1H, ArH), 7.41 (d, J =
8
.8 Hz, 1H, ArH), 7.37 (d, J = 8.6 Hz, 1H, ArH), 7.35–7.33 (m, 1H,
+
+
4 2
CH), 6.44–6.42 (m, 1H, CH). MS EI : 390.83 (C21H15ClN O , [M] ).
Anal. Calcd: C, 64.54; H, 3.87; N, 14.34; Found: C, 65.51; H, 3.85;
N, 14.39.
2.4.4. Cell apoptosis
5
Approximately 10 cells/well were seeded in a 24-well plate
and allowed to adhere. Subsequently, the medium was replaced
with fresh culture medium containing the target compound at final
2.3. Virtual simulation
concentrations of 0, 1, 2 and 4 lM. Non-treated wells received an
The docking simulation was achieved by Accelrys Discovery
equivalent volume of ethanol (<0.1%). After 24 h, the cells were
Studio version 3.5 and complemented by Maestro 10.1. All the
new compounds were docked into B-Raf (PDB code: 3IDP and
trypsinized, washed twice with PBS and centrifuged at 2000 rpm
for 5 min. The pellet was resuspended in 500
lL staining solution
3
C4C) binding site by CDOCKER. The binding results were further
(containing 5 L AnnexinV-FITC and 5 L PI in Binding Buffer),
l
l
checked by SP, XP Glide and Flexible Dock according to the proto-
cols. To verify whether the binding models were reliable, MD sim-
ulations were carried out using the GROMACS package (version
mixed gently and incubated for 15 min at room temperature in
dark. The cells were then analysed by a FACSCalibur flow cytome-
ter (Becton Dickinson, San Jose, CA, USA).
5
.1.2) with GPU-accelerating support. The parameter setting and
21,22
operating procedure can be seen in previous reports.
2.4.5. Western blot analysis
Cells on 6-well plates were rinsed twice with cold PBS and lysed
in RIPA lysis buffer containing a protease inhibitor (PMSF) mixture
at 1:100 dilution on ice for 30 min. The insoluble components of
cell lysates were removed by centrifugation (4 °C, 12,000Âg, 10
min), and protein concentrations were measured using a Pierce
BCA protein assay kit. Proteins were separated by sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and trans-
ferred onto polyvinylidene difluoride (PVDF) membranes. Mem-
branes were blocked with 5% skim milk in 1ÂTBST buffer for 1 h
at room temperature, and then incubated with corresponding pri-
mary antibodies diluted in 5% milk-TBST solution (1:500–1:1000
dilution) at 4 °C with gentle shaking overnight. After washing five
times (5 min each) with 1ÂTBST, corresponding HRP-conjugated
secondary antibodies (1:5000–1:10000 dilution) were incubated
for 1 h at room temperature. Immunoreactive bands were visual-
ized using an ECL detection kit (Invitrogen, USA) following the
manufacturer’s instruction.
2
2
.4. Biological assays
.4.1. Kinase activity inhibition
The 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 B-Raf V600E was pre-incubated at room
temperature for 1 h with 1 mL drug and 4 mL assay dilution buffer.
The kinase assay was initiated when 5 mL of a solution containing
2
00 ng recombinant human full length, N-terminal His-tagged
MEK1, 200 mM ATP, and 30 mM MgCl in assay dilution buffer
2
was added. The kinase reaction was allowed to continue at room
temperature for 25 min and was then quenched with 5 mL 5 Â pro-
tein denaturing buffer (LDS) solution. Protein was further dena-
tured by heating for 5 min at 70 °C. 10 mL of each reaction was
loaded into a 15-well, 4–12% precast NuPage gel and run at 200
V, and upon completion, the front, which contained excess hot
Please cite this article in press as: Wang P.-F., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.03.038

P.-F. Wang et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
5
3
. Results and discussion
inary docking simulation was carried out using both type II (PDB
code: 3IDP) and type I/II (PDB code: 3C4C) B-Raf kinases as the
receptors. Unsurprisingly, the binding affinities showed significant
variation when the new compound docked into these two recep-
tors (-Glide score for type II is 11.14 and for type I/II is 7.48). Thus,
the result favored our design intention of recombining known inhi-
bitors into new type II inhibitors.
As the preliminary simulation indicates the new compound
may be a promising starting point, it was further substituted to
afford a series of compounds. We also carried out docking simula-
tion towards all the new compounds, as listed in Table 1. Overall,
most compounds exhibited potent binding affinity to the receptor
and especially compound 5h (Fig. 3) has the most potent binding
energy. Typically, compound 5h hydrogen bonds with CYS532,
THR529 and GLU501. Similar to the other type II compounds, resi-
dues VAL504, ILE572, HIS 574 and ASP594 also contribute to the
binding affinity of 5h with the target protein.
It is well acknowledged that docking simulation work should be
employed with caution, for it generally focuses on a static binding
mode prediction under vacuum condition. In light of this consider-
ation, we utilized molecular dynamics simulation to evaluate the
stabilization of the binding model. The results of root-mean-square
deviation (RMSD) values were depicted in Fig. 4A. As shown, both
the system and the ligand settled into equilibrium state soon, with
smooth fluctuations under 0.3 nm and 0.1 nm, respectively.
Also, the gyration radius values of the protein were recorded in
Fig. 4B. Gyration radius (Rg) is an important measurement
criterion to evaluate the compacting degree of protein and lower
value reflects compacter state. The recorded total Rg slightly
declined and remained stable during the simulation, hinting the
protein kept a stable and compact state during the whole
simulation. Together, the MD simulation provides a reliable
verification of the binding model.
3.1. Data retrieval and analysis
As a typical kinase, B-Raf protein generally takes on three differ-
ent conformations, which are type I, type II and type I/II states.
While the front pocket (FP) region basically maintains the same
when the kinase transforms from a conformation into another,
the back pocket (BP) region is rather changeable in volume, shape
and number of sub-pockets.23 Accordingly, known inhibitors tar-
geting B-Raf can be classified as three types depending on the pro-
tein conformations. Based on this theory of knowledge, we
gathered and grouped all the known co-crystals of B-Raf protein
with ligand. Afterwards, all the complexes were superimposed by
a same template protein which has the finest resolution (PDB code:
2
4
3
IDP). It’s not surprising that many inhibitors of the same type
share similar structural features and interaction mode (Fig. 2).
Notably, for the type I/II inhibitors, many bear a substituted sulfon-
amide group in the BP region interacting with residues LEU505,
LEU 514, ASP594 and PHE595; while for the type II inhibitors,
the mainstream chemical group in the BP region is a phenylurea
or a benzamide group interacting with residues GLU501, VAL504,
ILE572, HIS 574 and ASP594. Besides, these two types of ligands
possess an overlapping phenyl group in space. Through this phenyl
group, these ligands could be hybridized to provide new com-
pounds. In this study, we intended to remold the known potent
type I/II inhibitors into new type II inhibitors by replacing the BP
fragments (Fig. 2). The combination gave several choices, and as
listed in Table 1, we selected compound 5a for further study.
3.2. Virtual simulation
Since the new compound is originally extracted and recom-
bined from two types of ligands, docking simulation was primarily
performed to predict the binding affinity difference between the
ligands and the two types of proteins. In our assumption, the alter-
ation of BP fragments shall lead to the increased binding affinity of
ligand towards type II other than type I/II protein. Hence a prelim-
3.3. Chemical synthesis
The synthesis of target compounds followed the general path-
way outlined in Fig. 5. All of the synthetic compounds are being
Fig. 2. Design of type II inhibitor based on type I/II inhibitor. (A) Type I/II inhibitors extracted from superimposed co-crystals. (B) Alignment of typical type I/II and type II
inhibitors extracted from superimposed co-crystals. (C) Indication for the recombination of type I/II and type II inhibitors.
Please cite this article in press as: Wang P.-F., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.03.038

6
P.-F. Wang et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Table 1
The structures of compounds 5a-5t and their binding scores on 3IDP and 3C4C
H
N
O
H
X
R
N
N
H
O
Compd
X
R
Glide scores
Compd
X
R
Glide scores
3IDP
3
IDP
3C4C
7.48
3C4C
8.12
5
5
5
5
a
b
c
N
N
N
N
11.14
10.49
10.55
11.41
5k
5L
N
N
N
N
N
11.51
F
8.59
9.17
9.51
Cl
Br
N
N
11.38
11.17
11.16
8.30
Cl
5m
5n
10.56
8.53
d
N
F
Cl
Cl
5
e
N
11.23
9.77
5o
N
O
10.77
8.89
Cl
Br
5
5
f
N
N
11.62
12.02
8.84
8.68
5p
5q
C
C
11.46
11.10
8.74
8.67
g
Cl
5
h
N
12.08
8.46
5r
C
10.67
8.21
F
F
O
F
5
5
i
j
N
N
10.80
10.76
8.18
5s
5t
C
C
N
11.25
10.20
8.97
7.77
O
N
10.41
Cl
N
Fig. 3. The 3D (A) and 2D (B) binding modes of compound 5 h with type II B-RafV600E kinase 3IDP.
reported for the first time and gave satisfactory analytical and
spectroscopic data. HNMR, ESI-MS and element analysis spectra
were in full accordance with the assigned structures.
The results were summarized in Table 2. According to the results,
1
most of the target compounds displayed good to excellent enzy-
V600E
matic inhibition to B-Raf
kinase. Generally speaking, the
kinase inhibitory effects of the tested compounds corresponded
to their binding affinities with the type II protein 3IDP. While the
meta- substitutions on the aryl R make no contribution to the
activities (5b and 5c), the para- substitutions usually give rise to
an enhanced inhibition (5d-5h), compared to the starting com-
pound 5a. Especially, compound 5h showed the best enzymatic
V600E
3
.4. Compound 5 h potently inhibited B-Raf
kinase activity and
proliferation of cancer cells
The inhibitory effects of all obtained compounds on kinase
B-Raf were evaluated, with sorafenib as a positive control drug.
WT
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P.-F. Wang et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
7
Fig. 4. Molecular dynamics simulation for compound 5 h binding with receptor. (A) The trajectory RMSD values for both system and ligand. (B) The gyration radius values for
the receptor.
H
H
N
H
N
N
O
a
b
X
X
X
O N
2
N
H
O N
H N
2
2
1
a-1b
2a-2b
3a-3b
H
N
H
N
O
O
c
d
X
H
N
X
H N
R
2
N
H
N
H
O
4
a-4b
5a-5t
Fig. 5. The synthetic route for compounds 5a-5 t. (a) 80% Hydrazine hydrate, 10% Palladium charcoal, Ethanol, 80 °C, 10 min; (b) EDC, HOBT, DMAP, Et
0% Hydrazine hydrate, 10% Palladium charcoal, Ethanol, 80 °C, 10 min; (d) EDC, HOBT, DMAP, Et N, DMF, overnight.
3
N, DMF, overnight; (c)
8
3
Table 2
V600E
In vitro antiproliferation activity of 5a-5 t towards cancer cells and inhibition activity towards B-Raf
kinase.
Compounds
IC50 ± SD (lM)
HT29
HCT116
A375
WM-1361
B-Raf V600E
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
16.11 ± 1.81
19.15 ± 1.75
20.54 ± 1.92
12.23 ± 1.86
17.85 ± 1.93
14.40 ± 0.95
10.86 ± 0.88
4.41 ± 0.35
18.52 ± 1.65
17.19 ± 1.98
11.51 ± 1.27
12.24 ± 1.34
9.65 ± 0.82
8.99 ± 0.67
16.70 ± 1.33
10.62 ± 1.88
11.11 ± 1.52
21.09 ± 2.86
10.58 ± 1.64
19.56 ± 1.81
8.44 ± 0.49
30.86 ± 2.15
20.54 ± 1.47
26.16 ± 2.72
36.25 ± 3.51
32.47 ± 3.05
29.19 ± 2.50
26.18 ± 2.33
18.69 ± 1.91
37.38 ± 3.01
35.85 ± 2.78
25.12 ± 2.02
24.87 ± 1.25
22.22 ± 2.49
19.67 ± 1.78
24.54 ± 2.41
21.18 ± 1.70
22.34 ± 2.35
35.25 ± 2.74
23.76 ± 2.53
35.52 ± 2.57
23.31 ± 1.38
9.12 ± 1.12
12.21 ± 1.25
9.87 ± 0.98
7.15 ± 0.69
7.62 ± 0.73
5.74 ± 0.59
2.85 ± 0.31
1.97 ± 0.11
9.45 ± 0.88
8.29 ± 0.94
7.46 ± 0.79
8.83 ± 0.97
7.58 ± 0.65
4.56 ± 0.57
10.18 ± 1.54
6.49 ± 0.82
6.72 ± 0.73
10.45 ± 1.22
5.78 ± 0.62
8.46 ± 0.78
3.36 ± 0.54
35.15 ± 2.70
38.35 ± 2.86
25.47 ± 1.75
29.25 ± 1.94
30.80 ± 2.58
24.57 ± 2.05
18.38 ± 1.41
12.75 ± 1.22
29.57 ± 2.06
28.16 ± 1.98
21.29 ± 2.33
27.15 ± 2.17
19.57 ± 1.72
16.21 ± 1.11
32.24 ± 3.27
24.57 ± 2.54
19.57 ± 1.75
21.64 ± 2.07
17.37 ± 1.62
19.22 ± 1.84
16.44 ± 1.35
5.22 ± 0.65
8.71 ± 0.79
4.94 ± 0.58
1.57 ± 0.27
3.04 ± 0.44
2.78 ± 0.26
0.75 ± 0.57
0.18 ± 0.03
5.27 ± 0.62
4.11 ± 0.54
0.81 ± 0.09
1.05 ± 0.25
0.46 ± 0.06
1.14 ± 0.12
5.32 ± 0.52
2.11 ± 0.41
2.61 ± 0.35
5.72 ± 0.68
3.52 ± 0.34
6.57 ± 0.77
0.13 ± 0.01
j
k
L
m
n
o
p
q
r
s
t
Sorafenib
inhibition with IC50 values of 0.18 ± 0.03
l
M. However, bulky sub-
compounds with carbon atom (5p-5t) were less active than those
with nitrogen atom (5a, 5e, 5i, 5j and 5L), suggesting a preference
for the indazole instead of indole in structure.
station group on the same position seems to attenuate the activity
as 5i only has a moderate activity. The same tendency is also true
for the compounds with pyridine R group or carbon X group. In
addition, it reveals that the pyridine R group shall be a better
choice for compounds 5j-5n showed better potential than their
aryl analogs 5a-5f. Hence, further modification may start with
compounds bearing pyridine R group. As to the X group, most
Subsequently, the entities were evaluated by the MTT assay for
anti-proliferation activity against four cancer cell lines, A375
V600E
WT
V600E
WT
(B-Raf
), HT29 (B-Raf
), WM1361 (B-Raf ), and HCT116
(B-Raf ). The IC50 values were calculated as listed in Table 2. To
compound 5h, it showed better suppression when exerting on
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8
P.-F. Wang et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Fig. 6. Compound 5 h induced A375 cell apoptosis. A375 cells treated with 0, 1, 2 and 4 lM 5 h for 24 h were collected and analyzed. The percentage of early apoptotic cells
was shown in the lower right quadrant (Annexin V-FITC positive/PI negative cells), and late apoptotic cells are located in the upper right quadrant (Annexin V-FITC positive/PI
positive cells).
the A375 cells and HT29 cells for 48 h. To WM1361 and HCT116
WT
cells expressing B-Raf , the cell viability of cells treated with
compound 5h was significantly higher than that of cells carrying
V600E
B-Raf
. The MTT assays suggested that compound 5h is a
V600E
potential B-Raf
liferation of B-Raf
inhibitor and could efficiently inhibit the pro-
V600E
-harbored cell lines.
3.5. Compound 5 h induced cell apoptosis in a dose-dependent manner
To determine whether compound 5h induced cell apoptosis, an
Annexin V/PI double staining assay was carried out. A375 cells
were treated with increasing concentrations (0, 1, 2 and 4 M) of
h for 24 h, followed by Annexin V/PI double staining and flow
l
5
cytometric measurement. As shown in Fig. 6, the percentage of
apoptotic cells dose-dependently elevated with the treatment of
compound 5h, and cell apoptotic rate reached up to 28.3% (sum
of Q2 and Q3) when drug concentration increased to 4 lM. In
short, compound 5h could induce A375 cells apoptosis in a dose-
Fig. 7. Compound 5 h blocked the ERK and MEK pathway in A375 cells. (A) 5 h
inhibited the phosphorylation of ERK and MEK in A375 melanoma cells bearing B-
V600E
Raf
; (B) 5 h slightly inhibited the phosphorylation of ERK and MEK in WM1361
WT
melanoma cells bearing B-Raf
.
dependent manner.
while the expression of total ERK or MEK protein levels was not
V600E
3.6. Compound 5 h could interfere the B-Raf
-mediated pathways
affected. Subsequently, the inhibitory effect of 5h to MEK and
WT
ERK phosphorylation in B-Raf
cell line WM1361 was assessed.
While the abnormally activated ERK pathway caused by
The paradoxical effect of vemurafenib at different concentrations
V600E
WT
B-Raf
increased, effective B-Raf
and down-regulate the amount of pERK and pMEK. The effect of
h on ERK and MEK phosphorylation was investigated in A375 cell
results in the phosphorylated level of MEK and ERK
was reported as in the B-Raf
cells lower doses of vemurafenib
V600E
inhibitors shall block the pathway
give rise to the enhancing phosphorylation of ERK and MEK while
higher doses block the phosphorylation of ERK and MEK. However,
the treatment to WM1361 cells with lower doses of 5h showed no
significant inducing effect on the phosphorylation of ERK and MEK,
as shown in Fig. 7(B). Moreover, treatment with higher dose of 5h
slightly inhibited the phosphorylation of ERK and MEK. Together,
5
V600E
line expressing B-Raf
and WM1361 cell line expressing
WT
B-Raf . As shown in Fig. 7(A), 5h dose-dependently reduced the
expression of the phosphorylation of ERK and MEK in A375 cells,
Please cite this article in press as: Wang P.-F., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.03.038

P.-F. Wang et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
9
the results suggested that compound 5h doesn’t induce paradoxi-
cal effect in the normal cells.
signaling cascade effectively inhibits melanoma lung metastases. Cancer Res.
006;66:8200–8209.
Yang Y-S, Zhang F, Tang D-J, Yang Y-H, Zhu H-L. Modification, Biological
Evaluation and 3D QSAR Studies of Novel 2-(1, 3-Diaryl-4, 5-Dihydro-1H-
Pyrazol-5-yl) Phenol Derivatives as Inhibitors of B-Raf Kinase. PLoS ONE.
2
5.
4
. Conclusion
2014;9:e95702.
6
.
.
Long GV, Stroyakovskiy D, Gogas H, et al. Combined BRAF and MEK inhibition
versus BRAF inhibition alone in melanoma. N Engl J Med. 2014;371:1877–1888.
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ular dynamics to fulfill the design and validation of hit compound,
based on which we further synthesized a series of derivatives and
7
evaluated their biological activities. Preliminary results showed
that a few of the compounds displayed good inhibitory BRAFV600E
8.
Grbovic O, Basso A, Sawai A, et al. V600E B-Raf requires the Hsp90 chaperone
for stability and is degraded in response to Hsp90 inhibitors. PNAS.
activities and antiproliferation activities against A375 cell lines.
2006;103:57–62.
V600E
Among these compounds, 5h showed the most potent BRAF
9. Li J, Feng J, Wang Y, et al. The B-RafV600E inhibitor dabrafenib selectively
inhibits RIP3 and alleviates acetaminophen-induced liver injury. Cell Death Dis.
inhibition activities with IC50 value of 0.18 ± 0.03 mM and antipro-
liferation activities with IC50 value of 1.97 ± 0.11 mM to A375 cells.
2014;5:e1278.
10. Murray CW, Rees DC. Opportunity Knocks: Organic Chemistry for Fragment-
Furthermore, mechanism of action studies proved that 5h could
Based Drug Discovery (FBDD). Angew Chem Int Ed. 2016;55:488–492.
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vemurafenib and other therapies. Clin Pharmacol Ther. 2014;95:24–31.
2. Wilhelm S, Carter C, Lynch M, et al. Discovery and development of sorafenib: a
V600E
induce apoptosis in A375 cells and interfere the B-Raf
-medi-
ated pathways. Our findings indicate that compound 5h could be
1
V600E
a lead molecule for further research into BRAF
. Disclosure of potential conflicts of interest
The authors disclose that there is no potential conflict of
inhibitors.
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The work was financed by the Public Science and Technology
Research Funds Projects of Ocean (No. 201505023), the Interna-
tional Postdoctoral Exchange Fellowship Program 2017 (No.
2
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