Design, synthesis and biological evaluation of indole derivatives as novel inhibitors targeting B-Raf kinase

Article abstract of DOI:10.1016/j.cclet.2013.11.006

A series of novel indole derivatives were designed and synthesized and their inhibitory activity against B-Raf and HepG2 cell were also described. Among them, compounds 7a and 7b exhibited excellent potency, which showed the potential for further research

Full text of DOI:10.1016/j.cclet.2013.11.006

Chinese Chemical Letters 25 (2014) 351–354
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Design, synthesis and biological evaluation of indole derivatives as
novel inhibitors targeting B-Raf kinase
Zeng Wu a, Ming Yan b, Shi-He Hu a, Zhi-Cheng Yu a, Yong Zhu a, Ya-Dong Cheng c,
Hai-Chun Liu c, Yan-Min Zhang c, Si-Hui Yao c, Wei-Fang Tang a, , Tao Lu
*
d,e,**
a Department of Organic Chemistry, China Pharmaceutical University, Nanjing 210009, China
b National Nanjing Center for Drug Screening, China Pharmaceutical University, Nanjing 210009, China
c Laboratory of Molecular Design and Drug Discovery, School of Sciences, China Pharmaceutical University, Nanjing 210009, China
d State Key laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China
e The Headmaster’s Office, China Pharmaceutical University, Nanjing 210009, 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 indole derivatives were designed and synthesized and their inhibitory activity against
B-Raf and HepG2 cell were also described. Among them, compounds 7a and 7b exhibited excellent
potency, which showed the potential for further research as lead compounds.
ß 2013 Wei-Fang Tang and Tao Lu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All
rights reserved.
Received 10 July 2013
Received in revised form 27 September 2013
Accepted 18 October 2013
Available online 23 November 2013
Keywords:
B-Raf kinase inhibitors
Receptor-based drug discovery
Indole derivatives
Biological evaluation
1. Introduction
was approved to treat patients with advanced or metastatic
melanoma. So the B-Raf kinase deserves more attention.
The RAS-RAF-MEK-ERK signal transduction pathway plays a
critical role in tumor biology. This pathway is controlled by
extracellular signals through membrane receptors such as receptor
tyrosine kinases and is activated by oncogenic mutations in many
types of cancer [1–4]. The most common mutation, V600E mutant
(B-RafV600E) has been demonstrated in vitro to be constitutively
active in carcinoma cells and to increases cell proliferation and
survival [5–7]. Over the past decade extensive efforts have been
devoted to developing B-Raf kinase inhibitors and there were three
successful examples (Fig. 1). Sorafenib (Bayer/Onyx) was approved
by the US Food and Drug Administration (FDA) for the treatment of
hepatocellular carcinoma and renal cell carcinoma; Regorafenib
was also approved for the treatment of advanced colorectal cancer
several months ago and marketed by Bayer and Onyx, and its
structure is very similar to that of Sorafenib. Different from
Sorafenib and Regorafenib as multi-kinase inhibitors, Vemurafenib
(Plexxikon/Recho) is a selective B-RafV600E kinase inhibitor, which
Employing receptor-based drug discovery approaches, our
group found that there was a small hydrophobic pocket in B-Raf
kinase beside the spot where the center ring of Sorafenib binds in a
binding model, so a methyl or ethyl group was attempted at this
position to increase the activity. The results evaluated by Discovery
Studio were shown in Fig. 2a. They both superimpose well with
Sorafenib. Then, we discovered that using an indole to replace the
phenyl ring could better occupy the hydrophobic pocket, because
the volume of pyrrole was similar to that of an ethyl group.
Therefore, the compound A (Fig. 3) was synthesized. Furthermore,
we found that if a methylene was introduced between the indole
ring and the pyridine ring, the overlay of our hit compound and
Sorafenib was better compared with compound A (Fig. 2b), though
only one rather than two NH in the urea moiety formed hydrogen
bond with side residue in Glu501. Moreover, many articles
reported that only two hydrogen bonds were sufficient to produce
the targeted binding affinity [8]. The design procedure was
exhibited in Fig. 3.
2. Experimental
*
Corresponding author.
**
Corresponding author at: The Headmaster’s Office, China Pharmaceutical
The commercially available ethyl isonicotinate
1 (5.00 g,
University, Nanjing 210009, China.
E-mail addresses: tangwf126@126.com (W.-F. Tang), lutao@cpu.edu.cn (T. Lu).
33.08 mmol) was dissolved in N-methylformamide (20 mL), and
1001-8417/$ – see front matter ß 2013 Wei-Fang Tang and Tao Lu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2013.11.006

352
Z. Wu et al. / Chinese Chemical Letters 25 (2014) 351–354
F
CF
O
3
CF
O
3
Cl
O
Cl
F
O
Cl
O
O
O
N
H
O
N
H
S
N
N
H
N
N
H
N
H
F
N
H
N
H
O
N
N
H
Regorafenib
Sorafenib
Vemurafenib
Fig. 1. Structures of Sorafenib, Regorafenib and Vemurafenib.
cold 98% H2SO4 (3.24 g, 33.08 mmol) was added drop wise and the
temperature was kept below 5 8C. After the addition of H2SO4, 30%
H2O2 and a saturated solution of FeSO4 were added to the reaction
mixture at 10–15 8C [9] until compound 1 disappeared. After
silica chromatography (eluting with 80% acetoacetate in N-hexane)
to afford the compound 4 (477 mg, 2.09 mmol, 52.34%) [11].
A mixture of 5-nitro-1H-indole (200 mg, 1.23 mmol) or its
derivatives and dry DMF (5 mL) was cooled to 0 8C and NaH (60 mg,
1.23 mmol) was added. After the addition, the reaction mixture
extraction, washing, recrystallization, the pure compound
(4.04 g, 19.40 mmol, 58.64%) was obtained.
2
was stirred at r.t. for 0.5–1 h, then compound
4 (282 mg,
A mixture of compound 2 (2.08 g, 10 mmol) and sodium
borohydride (1.16 g, 30 mmol) in ethylalcohol (50 mL) was
allowed to stir overnight at room temperature, then the mixture
was concentrated in vacuo and the purification of the curde
product using silica gel chromatography, eluting with 80%
acetoacetate in N-hexane afforded the compound 3 (1.58 g,
9.49 mmol, 95.03%) [10].
Compound 3 (664 mg, 4 mmol) was dissovled in anhydrous THF
(20 mL), then triphenylphosphine (1.10 g, 4.20 mmol) and carbon
tetrabromide (1.39 g, 4.20 mmol) were added. The reaction
mixture was stirred at room temperature for 3 h. The mixture
was then filtered to remove the precipitates and the filtrate was
concentrated to an oil. The crude product was purified using flash
1.23 mmol) was added, and the reaction mixture was allowed to
stirred for an additional 2 h. After a sequence of extraction, wash,
and concentration, the crude product compound 5 (222 mg,
0.72 mmol, 58.30%) was obtained and could be used in next step
without further purifications [12].
Compound 5 (222 mg, 0.72 mmol) was dissolved in MeOH
(10 mL), and 5%Pd/C (10 mg) was added, then H2 was ventilated.
After 0.5 h the reaction completed. Pd/C was removed by filtration,
the filtrate was condensed in vacuo and chromatographic
purification of the curde product over silica gel, eluting with
50% acetoacetate in N-hexane afforded the compound 6 (194 mg,
0.69 mmol, 96.5%).
A mixture of 4-chloro-3-(trifluoromethyl)aniline or its deriva-
tives and CDI in dry CH2Cl2 (5 mL) was stirred for 1 h at room
temperature, then compound
6 (194 mg, 0.69 mmol) or its
derivatives was added. The mixture was allowed to stir for an
additional 1 h to afford white precipitates. The solid was collected
by filtration under vacuum, washed with water for three times and
dried under vaccum to give compound 7a–7m and 7p–7r [13], but
7m and 7n was synthesis by a condensation reaction between
benzoic acid and compound 6 promoted by EDCI. The yields of all
compounds were between 65.7% and 82.4%. The general synthetic
route is depicted in Scheme 1.
7a: 273 mg, 78.6%, mp 209 8C. 1H NMR (300 MHz, DMSO-d6):
d
2.77 (d, 1H), 5.57 (s, 2H,), 6.50 (d, 1H, J = 2.94 Hz), 7.09 (d, 1H,
J = 8.97 Hz), 7.27 (m, 2H, J = 3.00 Hz, J = 8.97 Hz), 7.51 (m, 1H), 7.58
(m, 2H), 7.73 (d, 2H,), 8.12 (s, 1H), 8.53 (dd, 1H, J = 6.15 Hz,
J = 2.85 Hz), 8.60 (s, 1H), 8.73 (dd, 1H, J = 6.15 Hz, J = 2.85 Hz), 9.06
(s, 1H); ESI (m/z): 501.6 [M+H]. Anal. Calcd. for C24H19ClF3N5O2: C,
57.43; H, 3.82; N, 13.95. Found: C, 57.67; H, 3.94; N: 13.79.
7b: 267 mg, 75.9%, mp 248 8C. 1H NMR (300 MHz, DMSO-d6):
d
2.78 (d, 1H), 5.53 (s, 2H), 7.13 (d, 1H, J = 9.32 Hz), 7.25 (m, 1H), 7.37
(m, 1H, J = 9.32 Hz), 7.51 (m, 1H), 7.59 (m, 1H), 7.63 (m, 2H), 7.76
(m, 2H, J = 2.22 Hz, J = 5.28 Hz), 8.12 (d, 1H, J = 2.22 Hz), 8.56 (m,
1H, J = 5.28 Hz), 8.72 (m, 2H), 9.10 (s, 1H); ESI (m/z): 520.1 [M+H].
Anal. Calcd. for C24H18ClF4N5O2: C, 55.45; H, 3.49; N, 14.62. Found:
C, 55. 54; H, 4.02; N: 14.63.
B-Raf kinase assay: Kinase activity was measured as the
percentage of ATP consumed following the kinase reaction using
luciferase-luciferin-coupled chemiluminescence system. Reac-
tions were conducted in a 384-well plate (PerkinElmer). B-Raf
kinase reactions were initiated by adding test compounds (1
mL/
well) and B-Raf kinase (4 L/well, Signalchem) to the 384-well
m
plate. The assay plate was centrifuged with 1000 rpm for 1 min to
mix them and then pre-incubated at 30 8C for 30 min. ATP-
Unactive MEK1 mixture (5
mL/well, obtained by mixing an equal
Fig. 2. (a) Predicted binding model (PDB: 1UWH) of Sorafenib (gray) and its
derivatives with a methyl (brown) or ethyl (yellow) substitution center phenyl ring.
(b) Overlap binding model of our hit compound (green) with compound A (purple),
volume of ATP and inactive MEK1, Sigma and Signalchem,
respectively) was added to the assay plate. The assay plate was
centrifuged with 1000 rpm for 1 min and incubated at 30 8C for 1 h.
ADP-Glo reagent was added to each well and the assay plate was
incubated at 27 8C for 30 min. Finally, the luminescence signal was
Sorafenib (blue) and its derivatives with
a methyl (red) or ethyl (brown)
substitution center phenyl ring. (c) The binding model of compound 7b. (For
interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)

Z. Wu et al. / Chinese Chemical Letters 25 (2014) 351–354
CF3
353
O
Cl
O
O
N
H
N
N
H
N
H
CF3
O
CF3
O
Cl
O
Cl
O
O
N
H
O
N
H
N
N
N
N
N
H
N
H
H
H
H
H
F3C
Cl
N
N
O
N
A
H
N
N
O
O
H
H
F3C
Cl
N
N
O
N
NH
hit compound
N
Fig. 3. The design procedure of the hit compound.
read with Envision (PerkinElmer). The IC50 value of each compound
was calculated by Prism 5.0, using the inhibition rate.
compound 7b (the binding model was exhibited in Fig. 2c)
showed better inhibitory activity in HepG2 cells than 7a, with an
IC50 value of 2.50
although its IC50 value against B-Raf was on1y 1.36
m
mol/L and 6.63
m
mol/L, respectively,
mol/L.
3. Results and discussion
m
Interestingly, both of them showed lower inhibitory activity
against B-Raf than Sorafenib and better inhibitory activity in
HepG2 than Sorafenib.
Altering the substitution groups at the 3-position of the indole
with F, Cl, Br systematically (7b, 7c, 7d) while maintaining the rest
Thus, a series of novel indole analogs 7a–7r were prepared
(Table 1). All compounds were evaluated against B-Raf kinase
and HepG2 cells. Compound 7a was the most potent inhibitor
with an IC50 value of 0.51
mmol/L against B-Raf. However,
Table 1
B-Raf inhibitory activities of compounds 7a–7r.
R3
H
N
H
N
R2
R1
n
O
N
O
N
NH
Compd.
R1
R2
R3
n
B-Raf
HepG2
MDA-MB-453
IRb,c,d (%)
SW480
IRb,c,d (%)
H522
IRb,c,d (%)
a
a,b,c
IC50
(m
mol/L)
IC50
(
m
mol/L)
7a
Cl
Cl
Cl
Cl
H
CF3
CF3
CF3
CF3
CF3
H
H
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
1
1
1
0.51
1.36
1.90
2.03
6.63
2.50
21.9
31.1
40.1
32.3
40.4
3.2
13.5
10.1
21.1
7.7
À3.7
À0.2
9.6
7b
7c
F
Cl
Br
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
9.01
7d
7e
10.29
9.94
À5.6
9.3
>20
12.1
À3.7
À1.1
0.6
7f
Cl
Cl
Cl
Cl
H
1.38
8.88
1.28
6.10
29.63
11.82
>50
À5.4
20.2
10.4
10.2
13.4
17.8
10.7
24.1
11.8
22.7
11.0
À19.2
10.6
39.4
7g
F
À2.6
0.7
7h
7i
Cl
CH3
CH3
F
>50
21.6
3.5
À1.2
0.9
7j
>20
>50
7k
7l
H
7.68
5.56
43.62
8.48
9.8
1.7
H
Cl
21.2
12.8
28.8
41.4
12.7
17.8
36.7
56.9
3.3
7m
7n
7o
7p
7q
7r
H
Br
>20
41.37
40.66
41.54
45.96
>50
1.7
Cl
H
CF3
CF3
OCH3
CN
H
>20
>20
39.1
28.8
À1.2
À9.1
11.0
28.1
H
8.46
>20
5.52
0.032
H
CF3
>50
14.95c
Sorafenib
a
Values are means of two or more experiments.
b
HepG2, MDA-MB-453, SW480, H522 were evaluated firstly (10
HepG2 cell to acquire IC50
mmol/L). However, only HepG2 cell show preferable inhibitory activity. So we only continued to evaluate
.
c
Cell assay was employed from Ref. [10], and the authors reported that in vitro, the IC50 value of sorafenib inhibiting HepG2 cell proliferation was 4.5 mmol/L.
IR: inhibition ratio.
d

354
Z. Wu et al. / Chinese Chemical Letters 25 (2014) 351–354
O
O
Br
O
O
OH
5-nitro-1H-indole
NaBH4/EtOH
N-methylformamide/H2O2/FeSO4
or its derivatives
CBr4/PPh3/THF
H
N
H
N
H
N
NaH/DMF
N
N
N
N
O
O
3
O
1
2
4
R3
R3
R3
H
N
H
N
H2N
R2
O2N
O
n
aniline derivatives/CDI/CH2Cl2 or
benzoic acid derivatives/EDCI/DMF
5%Pd/C/H2/MeOH
O
N
O
O
N
NH
N
N
NH
NH
R1
N
N
5
7a-r
6
Scheme 1. Synthetic route of compounds 7a–7r.
of the molecule intact, we found the activity against B-Raf
decreased in that order, and the same trend was observed in
HepG2 cell assay except compound 7b. Using other groups (7f, 7g,
7h, 7i) instead of a trifluoromethyl group to determine its
importance, the results showed that there were salient activity
decrease in both enzymatic and cellular assays except compound
7h, which showed better B-Raf inhibitory activity than compound
7c. So the trifluoromethyl group in the meta position was crucial
for the activity. Replacing the chlorine atom with a hydrogen (7e),
the B-raf inhibitory activity decreased significantly, but the HepG2
cell activity was equivalent to that of compound 7c. Changing the
moieties at both the meta and para positions of the phenyl ring (7j,
7k, 7l, 7m, 7p, 7q, 7r) lead to a remarkable destruction of inhibitory
activity in both B-Raf and HepG2 evaluation.
Natural Science Foundation of China (No. 30973609), Fundamental
Research Funds for the Central Universities (No. JKZ2011004) and
Specialized Research Fund for the Doctoral Program of Higher
Education (No. 20100096110007).
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4. Conclusion
In summary, using computational approaches, we designed,
synthesized and evaluated a series of indole derivatives, and some
compounds showed moderate activity against B-Raf. Moreover,
compound 7a and 7b showed better potency than Sorafenib in the
HepG2 assay with about 2- and 6-fold improvement, respectively.
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This work was financially supported by the Natural Science
Foundation of Jiangsu Province (No. BK2009303), the National