Novel anti-cancer agents: design, synthesis, biological activity, molecular docking, and MD simulations of 2, 3, 4, 5-tetrahydro-1H-pyrido-[4,3-b]indole derivatives

Article abstract of DOI:10.1007/s00044-018-2271-0

In the previous research, our group designed and synthesized 2,3,4,5-tetrahydro-1H-pyrido-[4,3-b] indoles, which showed high anti-tumor activity. In this study, a series of novel 2,3,4,5-tetrahydro-1H-pyrido-[4,3-b] indole derivatives were designed by int

Full text of DOI:10.1007/s00044-018-2271-0

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2271-0
ORIGINAL RESEARCH
Novel anti-cancer agents: design, synthesis, biological activity,
molecular docking, and MD simulations of 2, 3, 4, 5-tetrahydro-1H-
pyrido-[4,3-b]indole derivatives
Yu Feng1 Xingxing Teng Jinhua Gu Bangwei Yu Yan Luo Lianbao Ye1,3
1 ●
1 ●
2 ●
1 ●
●
Received: 27 March 2018 / Accepted: 4 December 2018
©
Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
In the previous research, our group designed and synthesized 2,3,4,5-tetrahydro-1H-pyrido-[4,3-b] indoles, which showed
high anti-tumor activity. In this study, a series of novel 2,3,4,5-tetrahydro-1H-pyrido-[4,3-b] indole derivatives were
designed by introducing an alkyl or aralkyl and a sulfonyl group, which are considered as the pharmacophores of some
antitumor drugs based on the combination principles, and synthesized. The antiproliferative activity of all the target
compounds were evaluated against Hela, A549, HepG2, and MCF-7 cell lines using the MTT assay in vitro. The results were
represented by IC50 values. All compounds showed moderate to excellent antiproliferative activity with IC50 values between
0
μM and 100 μM against cancer cells. The proliferations of Hela, A549, HepG2, and MCF-7 cell lines were inhibited in a
dose-dependent manner, and the cytolytic activity was markedly inhibited at the same time. The IC50 values of intermediate
inhibited against Hela, A549, HepG2, and MCF-7 cell lines were 52.75, 50.30, 60.31, and 54.39 μM, respectively, which
3
were higher than the new compounds that we expected. The compounds 4a–4d bearing sulfonyl, substituted by electron
donating group, showed moderate to significant antiproliferative activity, in which compound 4c was the best with the IC50
values of 13.71, 9.42, 15.06, and 14.77 μM, and these results suggested that the introduction of sulfonyl could increase the
antiproliferative activity of 2,3,4,5-tetrahydro-1H-pyrido-[4,3-b]indole. Compounds 4e–4g bearing alkyl, phenyl, and
arylated alkyl produced good antiproliferative activity and the IC50 value of 4g was lower than 30 μM. The target compounds
were more potent against A549 compared to the other three cell lines. Molecular docking studies revealed the binding
orientations of all the synthesized compounds in the active site of c-Met. Moreover, molecular dynamics simulations have
been performed to evaluate the binding stabilities between the synthesized compounds and their receptors.
●
●
●
Keywords 2,3,4,5-Tetrahydro-1H-pyrido-[4,3-b]indole derivatives Design and synthesis Biological activities Molecular
●
docking Molecular dynamics simulations
Introduction
Finding the effective and specific anti-tumor drug has
epoch-making significance since malignant tumor is a
serious threat to human health. There are plenty of studies
on the treatment of cancer by chemotherapeutic agents
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-018-2271-0) contains supplementary
material, which is available to authorized users.
that can act directly on DNA or interfere with the
*
Lianbao Ye
yelb7909@163.com
Ordinary Universities of Guangdong Province, Guangzhou Key
Laboratory of Construction and Application of New Drug
Screening Model Systems, Guangzhou Key Laboratory of
Construction and Application of New Drug Screening Model
Systems and Guangdong Province Engineering Technology
Center for Molecular Probes & Biomedical Imaging, Guangdong
Pharmaceutical University, Guangzhou, 510006
1
2
3
School of Pharmacy, Guangdong Pharmaceutical University,
5
10006 Guangzhou, China
Institute of Traditional Chinese Medicine, Guangdong
Pharmaceutical University, 510006 Guangzhou, China
Guangdong, China
Key Laboratory of New Drug Discovery and Evaluation of

Medicinal Chemistry Research
synthesis of DNAs to inhibit the proliferation and
metastasis of tumor cells (Johnson 2000; Lee et al. 2014;
Bellacosa et al. 2013). Tumor-targeting therapies have
significantly changed cancer treatments, which depend on
specific vectors to kill the tumor cells so as to improve the
efficacy and reduce the toxic side effects on normal cells
products such as coumarins, in which a sulfonamide
group was introduced, have antiproliferative activity
(Bhat et al. 2006; Luo et al. 2001; Farahi et al. 2015;
Kovác et al. 2001; Thaisrivongs et al. 1994; Dang et al.
2010; Chandak et al., 2016; Desai et al. 2014). The sul-
fonamide group has substituted for structurally similar
amino sulfonic esters, and has shown well anti-
proliferative activity in vivo and in vitro according to the
biological isostere principles. Additionally, we expected
that bearing an alkyl or aralkyl in position N-5 would both
be absorbed by various tissues quickly and increase the
antiproliferative activity. Our group reported that a series
of small molecule inhibitors of c-Met, which are prepared
by sulfonic acids and natural products, inhibited the pro-
liferation of many tumor cell lines excellently (Ye et al.
2017). It is hoped that the introduction of sulfonyl, alkyl,
or aralkyl (Fig. 2) can increase the antiproliferative
activity of 2,3,4,5-tetrahydro-1H-pyrido-[4,3-b]indole.
Hopefully, it was quickly and effectively absorbed by the
creased polarity and increased the lipid solubility.
(
Kircheis et al. 2002; Hofmeister et al. 2008). In the
previous research, our group designed and synthesized a
new compound 1 (Fig. 1) bearing active skeleton 2,3,4,5-
tetrahydro-1H-pyrido-[4,3-b]indole, which showed high
antiproliferative activity and c-Met inhibitory potency (Ye
et al. 2012). Additionally, molecular docking studies
showed that the main binding mode of 2,3,4,5-tetrahydro-
1
H-pyrido-[4,3-b]indole with c-Met kinase site was the
hydrophobic region (Ye et al. 2016). In an ongoing effort
to discover novel anti-tumor agents, we were going to
design and synthesize novel 2,3,4,5-tetrahydro-1H-pyr-
ido-[4,3-b]indoles using compound 1 as the leading
compound based on combination principles.
Related references reported that the sulfonamide group
was the pharmacophore of some anti-tumor drugs. Natural
In this study, all compounds synthesized were evaluated
for their antiproliferative activity in vitro against Hela,
A549, HepG2, and MCF-7 cells. In addition, molecular
docking study simulated the interaction between small
molecule ligands and receptor macromolecules at the
molecular level. Molecular dynamics (MD) experiments
were performed to evaluate the binding stabilities between
the compounds and their receptors.
H
N
N
N
N
N
N
N
O
H
Fig. 1 Structure of 1
Fig. 2 Target compounds
General Structure
Compound
R
O
S
4
a
O
O
O
S
4b
O
S
R
N
4c
O
Cl
Br
O
N
O
4d
O
S
NO2
O
4e
CH3
4f
4g

Medicinal Chemistry Research
Material and methods
stirred for 2.5 h at r.t. under nitrogen. The solution was
poured into H O and ethyl acetate was added to the sus-
2
Unless otherwise noted, all the chemicals were obtained
from Aladdin or J&K Scientific Ltd., China. The solvents
were purified and dried based on standard procedures and
stored over 3A molecular sieves. The reactions progress
was determined by thin layer chromatography (TLC) ana-
lysis on silica gel F254 plate (Merck). Chromatography
purification was run on silica gel (200–300 mesh) from
pension, dried over anhydrous sodium sulfate, filtered, and
evaporated. The crude product was purified by column
chromatography (petroleum ether/ethyl acetate, 10/4), to
afford the compound (393 mg, 78%) as a yellow solid.
Synthesis of 4b–4d
1
Qingdao Ocean Chemical (Qingdao, Shandong, China). H
Compounds were prepared in analogy to 4a.
Synthesis of 4e
1
3
NMR and C NMR spectra were recorded on a Bruker
Digital NMR Spectrometer, rep, δ (ppm), J in Hz, using
tetramethylsilane (TMS) as an internal standard and CDCl3
or DMSO-d as solvent. The chemical structures of the
To a solution of 3 (351 mg, 1 mmol) in N,N-Dimethylfor-
6
target compounds were determined by Electron Impact
Mass Spectra (EI-MS) using a Waters ZQ400 instrument.
RPMI-1640 culture medium and new-born calf serum were
purchased from Gibco (Grand Island, NY), and methyl
thiazolyl tetrazolium (MTT) was purchased from Amresco
mamide (DMF) (8 ml) at 0 °C under N , sodium hydride
2
(60% weight dispersion in mineral oil) was added, the
mixture was stirred at 0 °C for 30 min. Methyl iodide (284
mg, 2 mmol) was added, and the reaction mixture was stirred
overnight at ambient temperature, quenched by adding a
saturated solution of ammonium chloride. The mixture was
extracted three times with equivalent dichloromethane and
the combined extracts dried over sodium sulfate. After
concentration in vacuum, the residue was purified by column
chromatography (petroleum ether/ethyl acetate, 10:4) to
obtain the product (223 mg, 61%) as a yellow solid.
(
Solon, OH).
Synthesis of compounds
Synthesis of 2
4
-Bromophenylhydrazine hydrochloride (224 mg, 1 mmol)
and N-tert-butoxycarbonyl-4-piperidone (298.5 mg, 1.5
mmol) were dissolved in 10 ml ethanol solution and satu-
rated hydrochloric acid, and the mixture was stirred at 95 °C
for 8 h. The solution was filtered; the filtered cake was
rinsed with anhydrous ethanol and dried in vacuo to give 2
Synthesis of 4f–4g
Compounds were prepared in analogy to 4e.
Cell assay
(
151 mg, 60%) as a white solid.
The anti-proliferative activities of compounds 4a–4g were
evaluated against Hela, A549, HepG2, and MCF-7 cell lines
using the standard MTT assay in vitro. The cancer cell lines
were cultured in minimum essential medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) and 100
Synthesis of 3
The compound 2 (251 mg, 1 mmol) and di-tert butyl
dicarbonate (262 mg, 1.2 mmol) were dissolved in dichlor-
omethane (8 ml). Then N, N-disopropylamine (268 mg, 2
mmol) was added slowly under stirring. The solution was
−
1
4
−1
μg ml penicillin. Approximately 1.0 × 10 cells ml with
200 μl of DMEM medium were plated onto each well of a
poured into H O, and extracted with dichloromethane, then
96-well plate and incubated in 5% CO at 37 °C for 24 h.
2
2
dried over anhydrous sodium sulfate, filtered, and evapo-
rated. The crude product was purified by column chroma-
tography (dichloromethane/methanol, 23/1) to give 3 (299
mg, 88.9%) as a faint yellow solid.
The target products at indicated final concentrations (0–800
μM) or 0.1% Dimethyl sulfoxide (DMSO) were added to
the medium for 24 and 48 h. Fresh MTT (10 μl) was added
to each well and at a terminal concentration of 5 μg ml−
and incubated with the cells at 37 °C in dark for 4 h. The
formazan crystals were dissolved in 100 μl of DMSO in
each well and shaken for 15 min to dissolve it completely.
The absorbance at 492 nm was measured with a Spec-
traMAX190 micro plate reader (Molecular Devices, USA).
All target products were tested three times in each of the cell
lines. The results expressed as IC50 (inhibitory concentra-
tion 50%) were the averages of three determinations and
were calculated by using the GraphPad Prism 5 software.
1
Synthesis of 4a
The compound 3 (351 mg, 1 mmol), (Bu N) SO (50 wt%,
4
2
4
solution in H O, 0.4 ml), and 6 N NaOH solution (0.32 ml)
2
were dissolved in toluene (8 ml) under an ice bath and
stirred for 15 min. Benzenesulfonyl chloride (176 mg, 1
mmol) was added slowly. The mixture was vigorously

Medicinal Chemistry Research
H
N
Fig. 3 Synthesis of compounds,
reagents, and conditions: a
H
N
O
O
a
NH2
EtOH/HCl, 90 °C, 8 h; b DIEA,
DCM, rt, 5 h; c (Bu4N) SO , 6
O
N
Br
2
4
NH
2
Br
N NaOH, toluene, rt, 4 h; d
NaH, DMF, rt, overnight
R
N
H
N
b
c
Br
Br
N
d
N
O
O
O
O
4
3
Molecular docking
electrostatic potential (RESP).The simulations were per-
formed at a neutral pH and the hydrogen bonds were con-
strained using SHAKE algorithm. The residue-based cut-off
10 Å was used for nonbonded interaction and the time step
was set to 2 fs. The G-quadruplex complex and inner ions
Molecular docking was performed according to the related
references (Pirali et al. 2010; Huang et al. 2013; Ye et al.
2
015). To further elucidate the binding mode of compounds
−
1
in the tyrosine kinase ligand binding region, the CDOKER
program which connected with Accelrys Discovery Studio
were initially fixed with force constants of 100 kcal mol .
The system was heated from 0 to 300 K in 100 ps with
solutes constrained at a weak harmonic constraint of 10
kcal mol− and then equilibrated for 100 ps after all the
minimization steps contained 2000 cycles of steepest des-
cent minimization, followed by 2000 cycles of conjugated
gradient minimization. Finally, periodic boundary dynamics
simulations of 8 ns were carried out in an NPT ensemble at
1 atm and 300 K in the production step. The output trajec-
tory files were saved every 2 ps for subsequent analysis.
2
.5.5 was used to simulate. CDOKER can offer all the
1
advantages of full ligand flexibility (including bonds,
angels, and dihedrals), the CHARMm family of force field,
the flexibility of CHARMm engine, and reasonable com-
putation times, which has an important advantage of
introducing the soft-core potentials. In this work, we
maintained the root-mean-square deviation (RMSD) value
of the optimum pose less than 1Å of the related crystal pose
and optimized the crystal structure of 3EFJ with polar
hydrogen atoms and CHARMm force field, but not water.
We set the radius of the input site sphere as 10 Å from the
center of the binding site, and each ligand generated 20
random conformations. The ligands’ other parameters were
default values. The optimized ligands were docked into the
corresponding proteins active binding site according to the
protocol. The structure of the receptor was minimized to
Results and discussion
Chemistry
The synthesis of the key intermediate of tert-butyl-8-bromo-
2, 3,4,5-tetrahydro-1H-pyrido-[4,3-b]indole carboxylate was
achieved according to previous reported general procedure
using commercially available 4-bromophenylhydrazine
hydrochloride and N-tert-butoxycarbonyl-4-piperidone as
starting materials based on Fischer Indolizations (Fig. 3).
Different benzene sulfonyl groups were added to the N of 9-
position of tert-butyl-8-bromo-2,3,4,5-tetra-hydro-1H-pyr-
ido-[4,3-b]indole carboxylate to obtain tert-butyl-8-bromo-
5-(phenyl-sulfonyl)-3,4-dihydro-1H-pyrido-[4,3-b]indole-2-
carboxylate (4a), tert-butyl-8-bromo-5-tosyl-3,4-dihydro-
1H-pyrido-[4,3-b]indole-2-carboxylate (4b), tert-butyl-8-
bromo-5-[(4-chlorophenyl)sulfonyl]-3,4-dihydro-1H-pyrido-
[4,3-b]indole-2-carboxylate (4c), tert-butyl-8-bromo-5-[(4-
nitro-phenyl)sulfonyl]-3,4-dihydro-1H-pyrido-[4,3-b]indole-
2-carboxylate (4d) via nucleophilic substitution with yields
of 63%, 61%, 58%, and 52%, respectively. This reaction
provided good yields using sodium hydroxide as a catalyst
and (Bu N) SO as a phase transfer catalyst and the reaction
1
0,000 cycles using the Powell method in DS2.5.5. The
−
1
convergence criterion was identified as 0.001 kcal mol .
MD simulations
MD is a general simulation technique that is included in
many molecular modeling packages such as CHARMm and
AMBER. Applying MD to evaluate the binding stabilities
between all the compounds and the 3EFJ due to the possible
binding mode, which was predicted by molecular docking
studies between a ligand and receptor, may be not reason-
able or stable (Tian et al. 2014; Yuan et al. 2014; Hou et al.
2
012; Yan et al., 2016; He et al. 2010). In this work, we
used AMBER 10.0 for ligands and AMBER ff03 for pro-
teins on the basis of molecular docking results. The Gaus-
sian 0.3 program was used to calculate partial atomic
charges of the ligand by using the HF/6-31G* basis set and
the Antechamber module was used to fit the restricted
4
2
4
was completed within 10 h due to the hydrolysis of sulfonyl

Medicinal Chemistry Research
chloride when it was treated with water. Benzene sul-
fochloride substituted by electron donating group would
obtain high yield. Furthermore, the compounds tert-butyl-8-
bromo-5-methyl-3,4-dihydro-1H-pyrido-[4,3-b]indole-2-car-
boxylate(4e), tert-butyl-8-bromo-5-benzyl-3,4-dihydro-1H-
pyrido-[4,3-b]indole-2-carboxylate (4f), and tert-butyl-8-
bromo-5-phenethyl-3,4-dihydro-1H-pyrido-[4,3-b]indole-2-
carbo-xylate (4g) were synthesized with yields of 69%, 63%,
H, 4.72; Br, 16.26; N, 5.70; O, 13.02; S, 6.52. Found: C,
53.73; H, 4.70; Br, 16.23; N, 5.70; O, 13.00; S, 6.49.
Tert-butyl-8-bromo-5-tosyl-3,4-dihydro-1H-pyrido-[4,3-b]
indole-2-carboxylate (4b)
1
Yield: 61%, H NMR (400 MHz, CDCl ) δ 8.44 (dd, J = 4.5,
3
1.8 Hz, 1H), 7.58 (dd, J = 7.8 Hz, 2H), 7.36 (dd, J = 8.4, 1.6
Hz, 1H), 7.30 (d, J = 7.6 Hz, 2H), 7.21 (dd, J = 8.5, 4.7 Hz,
1H), 4.77 (s, 2H), 3.76 (t, J = 5.1 Hz, 2H), 3.27–3.15 (m,
6
1%, respectively. Sodium hydride was used as a strong
base to make a compound alkylated by deprotonation and N,
N-dimethylformamide was used as a solvent.
1
3
2H), 2.47 (s, 3H), 1.37 (s, 9H). C NMR (100 MHz, (D )
6
DMSO) δ 154.69 (C-15), 144.28 (C-31), 139.96 (C-8),
8
-Bromo-2,3,4,5-tetrahydro-1H-pyrido-[4,3-b]indole (2)
135.96 (C-5), 135.00 (C-25), 129.42 (C-27, C-29), 127.11 (C-
2
6, C-30), 121.75 (C-3), 114.98 (C-6), 113.79 (C-2), 107.82
1
Yield: 60%, H NMR (400 MHz, CDCl ) δ 7.38 (s, 1H),
(C-7), 81.41 (C-17), 51.11 (C-10), 49.57 (C-12), 28.22 (C-18,
C-19, C-20), 25.68 (C-13), 21.27 (C-31). EI-MS: 505.43 [M
+H ]. Anal. calcd. for C H BrN O S (504.07): C, 54.66;
3
7
1
.20 (dd, J = 8.2, 1.3 Hz, 1H), 6.98 (dd, J = 8.2, 1.3 Hz,
H), 3.65 (s, 2H), 3.00 (m, 4H). C NMR (100 MHz, (D6)
13
+
23
25
2 4
DMSO) δ 136.35 (C-5), 131.91 (C-8), 126.74 (C-4), 123.58
C-1), 121.75 (C-3), 113.79 (C-2), 113.10 (C-6), 107.82 (C-
H, 4.99; Br, 15.81; N, 5.54; O, 12.66; S, 6.34. Found: C,
54.64; H, 4.99; Br, 15.80; N, 5.52; O, 12.65; S, 6.31.
(
7
2
5
4
), 44.43 (C-10), 43.09 (C-12), 25.68 (C-13). EI-MS:
+
51.33 [M+H ]. Anal. calcd. for C H BrN (250.01): C,
Tert-butyl-8-bromo-5-[(4-chlorophenyl)sulfonyl]-3,4-
dihydro-1H-pyrido-[4,3-b]indole-2-carboxylate (4c)
11
11
2
2.61; H, 4.42; Br, 31.82; N, 11.16. Found: C, 52.59; H,
.42; Br, 31.81; N, 11.13.
1
Yield: 58%, H NMR (400 MHz, CDCl ) δ 8.43 (dd, J = 4.4,
3
Tert-butyl-8-bromo-2,3,4,5-tetrahydro-1H-pyrido-[4,3-b]
indole carboxylate (3)
1.6 Hz, 1H), 7.71 (dd, J = 7.9 Hz, 2H), 7.58 (dd, J = 7.9 Hz,
2H), 7.35 (dd, J = 8.3, 1.5 Hz, 1H), 7.20 (dd, J = 8.5, 4.4 Hz,
1
H), 4.75 (s, 2H), 3.76 (t, J = 5.1 Hz, 2H), 3.29–3.13 (m,
1
13
Yield: 78%, H NMR (400 MHz, CDCl ) δ 7.39 (t, J = 1.6
2H), 1.37 (s, 9H). C NMR (100 MHz, (D ) DMSO) δ
3
6
Hz, 1H), 7.20 (dd, J = 8.2, 1.6 Hz, 1H), 6.98 (dd, J = 8.1,
154.69 (C-15), 154.69 (C-8), 139.96 (C-5), 135.18 (C-28),
130.01 (C-27, C-29), 129.12 (C-1), 128.37 (C-26, C-30),
126.74 (C-4), 121.75 (C-3), 114.98 (C-6), 113.79 (C-2),
107.82 (C-7), 81.41 (C-17), 51.11 (C-10), 49.57 (C-12),
28.22 (C-18, C-19, C-20), 25.68 (C-13). EI-MS: 525.84 [M
1
3
.5 Hz, 1H), 4.49 (s, 2H), 3.70 (dd, J = 9.6, 4.7 Hz, 2H),
.07 (dd, 2H), 1.41 (s, 9H). 1 C NMR (100 MHz, (D6)
3
DMSO) δ 154.69 (C-15), 136.35 (C-5), 131.91 (C-8),
1
1
4
26.74 (C-4), 123.58 (C-1), 121.75 (C-3), 113.79 (C-2),
13.10 (C-6), 107.82 (C-7), 81.41 (C-17), 51.11 (C-10),
+
+H ]. Anal. calcd. for C H BrClN O S (524.02): C, 53.88;
2
2
22
2 4
9.57 (C-12), 28.22 (C-18, C-19, C-20), 23.21 (C-13). EI-
H, 4.52; Br, 16.29; N, 5.71; O, 13.05; S, 6.54. Found: C,
53.87; H, 4.50; Br, 16.30; Cl, 6.02; N, 5.70; O, 13.03; S, 6.54.
+
MS: 351.24 [M+H ]. Anal. calcd. for C H BrN O
16
19
2
2
(
350.06): C, 54.71; H, 5.45; Br, 22.75; N, 7.98; O, 9.11.
Found: C, 54.70; H, 5.45; Br, 22.73; N, 7.99; O, 9.10.
Tert-butyl-8-bromo-5-[(4-nitrophenyl)sulfonyl]-3,4-dihydro-
1
H-pyrido-[4,3-b]indole-2-carboxylate (4d)
Tert-butyl-8-bromo-5-(phenylsulfonyl)-3,4-dihydro-1H-
pyrido-[4,3-b]indole-2-carboxylate (4a)
1
Yield: 52%, H NMR (400 MHz, CDCl ) δ 8.45 (dd, J =
3
4
.7, 1.6 Hz, 1H), 8.22 (dd, 1H), 8.07 (dd, J = 7.9, 3.7 Hz,
1
Yield: 63%, H NMR (400 MHz, CDCl ) δ 8.44 (dd, J =
4
2H), 7.37 (dd, J = 8.3, 1.8 Hz, 1H), 7.21 (dd, J = 8.3, 4.6
Hz, 1H), 4.80 (s, 2H), 3.76 (t, J = 5.0 Hz, 2H), 3.21 (t, 2H),
1.37 (s, 9H). C NMR (100 MHz, (D ) DMSO) δ 154.69
3
.5, 1.5 Hz, 1H), 7.82 (dd, J = 8.1 Hz, 2H), 7.74 (t, J = 7.7
Hz, 1H), 7.50 (t, J = 7.7 Hz, 2H), 7.36 (dd, J = 8.3, 1.8 Hz,
1
3
6
1
3
H), 7.21 (dd, J = 8.4, 4.7 Hz, 1H), 3.76 (t, J = 5.1 Hz, 2H),
.31–3.13 (m, 2H), 1.37 (s, 9H). 1 C NMR (100 MHz, (D6)
(C-5), 140.48 (C-28), 139.96 (C-8), 135.96 (C-5), 135.11
(C-25), 129.12 (C-1), 128.57 (C-26, C-30), 126.74 (C-4),
121.75 (C-3), 117.31 (C-27, C-29), 114.98 (C-6), 113.79 (C-
2), 107.82 (C-7), 81.41 (C-17), 51.11 (C-10), 49.57 (C-12),
28.22 (C-18, C-19, C-20), 25.68 (C-13). EI-MS: 536.40 [M
3
DMSO) δ 154.69 (C-15), 139.96 (C-8), 137.86 (C-25),
35.96 (C-5), 131.46 (C-28), 129.17 (C-27, C-29), 126.80
C-26, C-30), 121.75 (C-3), 114.98 (C-6), 113.79 (C-2),
1
(
+
1
2
07.82 (C-7), 81.41 (C-17), 51.11 (C-10), 49.57 (C-12),
8.22 (C-18, C-19, C-20), 25.68 (C-13). EI-MS: 491.40 [M
+H ]. Anal. calcd. for C H BrN O S (535.04): C, 49.26;
2
2
22
3
6
H, 4.13; Br, 14.90; N, 7.83; O, 17.90; S, 5.98. Found: C,
49.24; H, 4.11; Br, 14.89; N, 7.83; O, 17.88; S, 5.97.
+
+
H ]. Anal. calcd. for C H BrN O S (490.06): C, 53.77;
22
23
2
4

Medicinal Chemistry Research
Table 1 IC50 values of all compounds against Hela, A549, HepG2, and MCF-7 cell lines in vitro
Compound
Hela IC50 (μM) ± SD
A549 IC50 (μM) ± SD
HepG2 IC50 (μM) ± SD
MCF-7 IC50 (μM) ± SD
2
4 h
48 h
24 h
48 h
24 h
48 h
24 h
48 h
2
3
57.21 ± 0.40
52.75 ± 0.35
47.21 ± 0.56
19.6 ± 0.27
13.71 ± 0.15
29.1 ± 0.21
38.99 ± 0.31
28.01 ± 0.28
20.97 ± 0.18
60.00 ± 0.52
56.21 ± 0.41
49.89 ± 0.27
20.64 ± 0.20
13.5 ± 0.09
54.28 ± 0.49
50.3 ± 0.28
38.72 ± 0.16
17.03 ± 0.11
9.42 ± 0.08
25.06 ± 0.15
31.73 ± 0.19
26.14 ± 0.33
18.4 ± 0.10
59.27 ± 0.36
56.18 ± 0.31
40.61 ± 0.19
17.92 ± 0.15
8.3 ± 0.07
62.37 ± 0.47
60.31 ± 0.47
46.88 ± 0.43
20.01 ± 0.16
15.06 ± 0.14
30.61 ± 0.27
31.97 ± 0.40
30.58 ± 0.24
21.6 ± 0.14
69.13 ± 0.40
63.77 ± 0.49
49.49 ± 0.27
22.8 ± 0.10
57.72 ± 0.38
54.39 ± 0.29
42.5 ± 0.37
19.63 ± 0.19
14.77 ± 0.08
20.99 ± 0.16
30.65 ± 0.24
29.5 ± 0.15
18.27 ± 0.19
68.36 ± 0.60
62.9 ± 0.40
49.72 ± 0.29
19.01 ± 0.13
17.91 ± 0.17
21.7 ± 0.11
33.08 ± 0.52
31.27 ± 0.32
20.51 ± 0.16
4
4
4
4
4
4
4
a
b
c
d
e
f
15.81 ± 0.20
32.04 ± 0.31
35.69 ± 0.19
33.18 ± 0.49
25.01 ± 0.21
30.51 ± 0.27
38.72 ± 0.24
29.99 ± 0.17
23.62 ± 0.22
25.74 ± 0.22
33.05 ± 0.22
26.03 ± 0.28
19.33 ± 0.21
g
Tert-butyl-8-bromo-5-methyl-3,4-dihydro-1H-pyrido-[4,3-b]
indole-2-carboxylate (4e)
Hz, 2H), 3.62 (t, 2H), 3.33 (t, J = 7.4 Hz, 2H), 3.13 (t, J =
1
3
4.43 Hz, 2H), 1.39 (s, 9H). C NMR (100 MHz, (D6)
DMSO) δ 154.69 (C-15), 138.68 (C-24), 136.31 (C-5),
136.16 (C-8), 131.34 (C-1), 128.94 (C-27), 128.72 (C-26,
C-28), 128.49 (C-25, C-29), 126.74 (C-4), 121.75 (C-3),
116.21 (C-6), 113.79 (C-2), 107.82 (C-7), 81.41 (C-17),
51.11 (C-10), 49.57 (C-12), 35.70 (C-23), 28.22 (C-18, C-
1
Yield: 69%, H NMR (400 MHz, CDCl ) δ 7.40 (dd, J =
3
7
1
2
.9, 2.2 Hz, 1H), 7.27 (t, J = 1.9 Hz, 1H), 6.81 (dd, J = 8.1,
.8 Hz, 1H), 4.50 (s, 2H), 3.74 (s, 3H), 3.66 (t, J = 5.1 Hz,
13
H), 3.20–3.06 (m, 2H), 1.41 (s, 9H). C NMR (100 MHz,
+
(
D ) DMSO) δ 154.69 (C-15), 136.37 (C-5),131.34 (C-1),
6
19, C-20), 25.68 (C-13). EI-MS: 455.40 [M+H ]. Anal.
1
1
3
26.74 (C-4), 121.75 (C-3), 116.21 (C-6), 113.79 (C-2),
07.82 (C-7), 81.41 (C-17) 51.11 (C-10), 49.57 (C-12),
calcd. for C H BrN O (454.13): C, 63.30; H, 5.98; Br,
17.55; N, 6.15; O, 7.03. Found: C, 63.28; H, 5.99; Br,
17.56; N, 6.17; O, 7.01.
2
4
27
2
2
1.40 (C-22), 28.22 (C-18, C-19, C-20), 25.68 (C-13). EI-
+
MS: 365.27 [M+H ]. Anal. calcd. for C H21 N O
17
Br
2
2
(
364.06): C, 55.90; H, 5.80; Br, 21.88; N, 7.67; O, 8.76.
Evaluation of the biological activity
Found: C, 55.93; H, 5.79; Br, 21.89; N, 7.67; O, 8.74.
The antiproliferative activity of all the target compounds
was evaluated against Hela, A549, HepG2, and MCF-7 cell
lines using the MTT assay in vitro.The results were repre-
sented by IC50 values as shown in Table 1. The IC50 values
were obtained by at least three independent trials. All
compounds showed moderate to excellent antiproliferative
activity with IC50 values between 0 μM and 100 μM against
cancer cells as illustrated in Fig. 4. The proliferations of
Hela, A549, HepG2, and MCF-7 cell lines were inhibited in
a dose-dependent manner, and the cytolytic activity was
markedly inhibited at the same time. Moreover, there was
no significant difference between the inhibitory effects of
the all compounds on the growth of the tumor cells for 24 h
and 48 h; thus, there was no significant influence on the
antiproliferation effect of compounds on Hela cells, A549
cells, HepG2 cells, and MCF-7 cells with an extended
treatment time after 24 h. The IC50 values of intermediate 3
inhibited against Hela, A549, HepG2, and MCF-7 cell lines
were 52.75, 50.30, 60.31, 54.39 μM, respectively, which
were higher than the new compounds that we expected. The
compounds 4a–4d bearing sulfonyl, which was substituted
by the electron donating group, showed moderate to sig-
nificant antiproliferative activity, in which compound 4c
was the best with the IC50 values of 13.71, 9.42, 15.06,
Tert-butyl-8-bromo-5-benzyl-3,4-dihydro-1H-pyrido-[4,3-b]
indole-2-carbox-ylate (4f)
1
Yield: 63%, H NMR (400 MHz, CDCl ) δ 7.25 (ddd, J =
3
11.1, 10.5, 5.8 Hz, 4H), 6.94 (m, 2H), 6.83 (d, J = 8.1 Hz,
1H), 5.23 (s, 2H), 4.53 (s, 2H), 3.62 (t, J = 5.5 Hz, 2H),
3
.13 (t, 2H), 1.39 (s, 9H). 13C NMR (100 MHz, (D6)
DMSO) δ 154.69 (C-15), 136.85 (C-23), 136.31 (C-5),
36.16 (C-8), 131.34 (C-1), 128.94 (C-26), 128.60 (C-25,
C-27), 127.99 (C-24, C-28), 126.74 (C-4), 121.75 (C-3),
1
1
5
1
16.21 (C-6), 113.79 (C-2), 107.82 (C-7), 81.41 (C-17),
1.11 (C-10), 49.57 (C-12), 47.01 (C-22), 28.22 (C-18, C-
9, C-20), 25.68 (C-13). EI-MS: 441.37 [M+H ]. Anal.
+
calcd. for C H BrN O (440.11): C, 62.59; H, 5.71; Br,
2
3
25
2
2
1
1
8.10; N, 6.35; O, 7.25. Found, C, 62.58; H, 5.72; Br,
8.08; N, 6.33; O, 7.26.
Tert-butyl-8-bromo-5-phenethyl-3,4-dihydro-1H-pyrido-
[
4,3-b]indole-2-car-boxylate (4g)
1
Yield: 61%, H NMR (400 MHz, CDCl ) δ 7.25 (ddd, J =
3
8
.1, 6.0, 2.6 Hz, 3H), 7.21–7.16 (m, 1H), 7.15–7.06 (m,
1
H), 6.82 (d, J = 8.1 Hz, 1H), 4.56 (s, 2H), 4.26 (t, J = 7.4

Medicinal Chemistry Research
Fig. 4 The inhibition of all
compounds on Hela a, A549 b,
HepG2 c, and MCF-7 d cells
could initially confirm that the target compounds might well
repay investigation. In conclusion, the compounds synthe-
sized by our experimental scheme had a good inhibitory
effect on cancer cells, and they could be used as the leading
compounds for the development of new anticancer inhibi-
tors, and this study was of great significance to the estab-
lishment of the chemical library and further research
studies.
Molecular docking
In pre-experiment, we carried out the docking experiment
by using several pdb proteins including 3DKF, 3EFJ, 3F82,
3WGJ, and 3RTO. Only 3EFJ interacted with compound 2;
Fig. 5 Compact binding modes of all compounds (PDB:3EFJ)
compound 3 and derivatives achieved good binding effect
owing to the stable indole fused ring structure, so we chose
it for the docking experiment. The 3EFJ is the crystal
structure of c-Met kinase in complex with ATP and its
information is seen from http://www.rcsb.org/pdb/explore/
explore.do?structureId=3EFJ.
Molecular docking suggested that these compounds were
bound to the active site of the protein 3EFJ (shown in Fig.
5). Docking experiments were performed to elucidate the
binding model of the strongest compounds 4c with c-Met
kinase, as shown in Fig. 6. Compound 4c was docked into
the binding site of c-Met kinase using CDOCKER (dock
ligands into an active site using CHARMm) program con-
ducted through Discovery Studio 2.5.5. The docking
experiments suggested that compound 4c would dock
1
4.77 μM, and these results suggested that the introduction
of sulfonyl group could increase the antiproliferative
activity of 2,3,4,5-tetrahydro-1H-pyrido-[4,3-b]indole.
Compounds 4e–4g bearing alkyl, phenyl, and arylated alkyl
groups produced good antiproliferative activity and the IC50
value of 4g was lower than 30 μM. In general, the target
compounds were more potent against A549 compared to the
other three cell lines. In addition, DMSO alone did not show
obvious inhibitory effect compared to untreated cells. These
results revealed that this series of compounds possessed
selectivity for A549 cancer cell lines, and had the makings
of good drugs for lung cancer. Based on the molecular
docking, MD simulations, and preliminary activity tests, we

Medicinal Chemistry Research
strongly into the ATP-binding site of c-Met. The binding
energies of complexes between all compounds and 3EFJ are
shown in Table 2. The strong interaction of 4c with c-Met
was attributed to two H-bonds between two oxygen atoms
on the sulfonyl group and THR1257 and GLN1256; in
addition, there was also a H-bond between the chlorine
atom and hydroxyl of THR1293. The π–π stacking inter-
action between the indole ring and the benzene sulfonyl
would make the binding more firm between small 4c and
of indole and PHE1223, H-bond between carbonyl oxygen
and GLN1258. Thus, it was illustrated that the compounds
4a–4d, bearing a benzene ring substituted by the electron
donating group, showed higher binding energies and com-
pounds 4e–4g, bearing arylated alkyl, produced the highest
activity.
MD simulations
3
EFJ. Moreover, 4c showed highest activity owing to the π–
Molecular docking studies were first carried out to predict
the plausible interactions between all compounds and 3EFJ.
On the basis of the docking results, MD simulation may
provide information on rearrangement and transition states.
The crystal structures of 3EFJ complex with compounds 2,
π stacking interaction between the pyrrole ring and phenyl
3
, and 4a–4g were used to evaluate the reliability of MD
simulations. As a result, the MD models appeared to reach a
stable state after 1 ns equilibration and the RMSD values
converged below 2.5 Å, especially the binding state
between 4c and 3EFJ was relatively stable and the trajec-
tories were smooth (Fig. 7). The MD parameters we chose
were appropriate for the MD simulations.
Most compounds gave stable RMSD curves during their
simulations (shown in Fig. 8). The binding free energies
ΔGpred were computed by means of MM-PBSA inside the
Amber 10 program, where values more negative than −20
kcal mol− were selected to assess the inhibitory activity for
1
Fig. 6 Docking model of compounds 4c (red) with c-Met (3EFJ)
Table 2 Binding energies of complexes between all compounds and 3EFJ
-
CDOCKER energy
Compounds
R
-1
(kcal mol )
2
3
_
21.8334
25.7259
-Boc
4
a
32.2177
4
b
53.7629
4
c
59.4801
40.6372
4
d
4
e
44.8160
47.0279
4
f
4
g
48.9527

Medicinal Chemistry Research
Fig. 7 Plot of RMSD for all the backbond atoms (Å) vs simulation
time (ns) for 3EFJ in complex with 4c
Fig. 8 Plot of RMSD for all the backbond atoms (Å) vs simulation
time (ns) for 3EFJ in complex with compounds 4a–4g
Table 3 Predicted binding free energies (ΔGpred, kcal mol−1) of 2–4g inhibitors selected for the bioassay
Compound
R
_
ΔGpred
-11.48
-15.62
std
2
3
±2.86
±2.51
-Boc
4
a
-16.94
-26.81
±3.04
±3.10
4
b
4
c
-30.75
-21.06
±2.73
±2.09
4
d
4
e
-20.96
23.83
±2.81
±3.17
4
f
4
g
-23.75
±2.95
3
EFJ. The predicted ΔGpred values of all compounds are
(2015A03031356), Science and Technology Planning Project of
Guangdong Province (2017ZC0199), Guangdong Provincial Applied
Scientific and Technological Project (2015B020234009), National
Scientific and Technological Project of Traditional Chinese Medicine
Industry (201507004), Guangdong Pharmaceutical University’ School
Powered by Innovation Foundation of China (2016KZDXM039), and
Guangdong special training fund for university students’ scientific and
technological innovation (51328004). The authors are grateful to Mr.
Tan and Sun Yet-Sen University for molecular docking experiment.
listed in Table 3.
MM-PBSA estimation of binding free energy ΔGpred
For each system, the values were calculated using
1
00 snapshots recorded from the last with 1 ns trajectory at
an interval of 10 ps by Molecular Mechanics Poisson–
Boltzmann Surface Area method.
Compliance with ethical standards
Acknowledgements The work was supported by the Special Innova-
tion Project of Guangdong Education Department (Natural Science)
Conflict of interest The authors declare that they have no conflict of
interest.
(
2017KTSCX107), Guangdong Natural Science Foundation

Medicinal Chemistry Research
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