Novel anti-cancer agents based on germacrone: design, synthesis, biological activity, docking studies and MD simulations

Article abstract of DOI:10.1039/c6ra26944c

Germacrone is a major activity component found in Curcuma zedoaria oil product, which is extracted from Curcuma zedoaria. In the present study, novel germacrone derivatives were first designed based on the theories of bioalkylating agents, synthesized, and then investigated for their inhibition effects on Bel-7402, HepG2, A549, and HeLa cells. Moreover, the study also evaluated the inhibition of these derivatives on c-Met kinase, which is expressed in a number of cancers. The results indicated that most of the compounds showed a stronger inhibitory effect on cancer cells and c-Met kinase than germacrone. Besides these findings, molecular docking studies were also carried out to analyze the results and explain the molecular mechanism of activities to c-Met kinase. Molecular dynamics simulations have been carried out for the further evaluation of binding stabilities between the compounds and their receptors.

Full text of DOI:10.1039/c6ra26944c

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PAPER
Novel anti-cancer agents based on germacrone:
design, synthesis, biological activity, docking
studies and MD simulations†
a
Cite this: RSC Adv., 2017, 7, 3760
Lianbao Ye, Jie Wu,a Weiqiang Chen,b Yu Fenga and Zhibing Shenc
*
Germacrone is a major activity component found in Curcuma zedoaria oil product, which is extracted from
Curcuma zedoaria. In the present study, novel germacrone derivatives were first designed based on the
theories of bioalkylating agents, synthesized, and then investigated for their inhibition effects on Bel-
7402, HepG2, A549, and HeLa cells. Moreover, the study also evaluated the inhibition of these derivatives
on c-Met kinase, which is expressed in a number of cancers. The results indicated that most of the
compounds showed a stronger inhibitory effect on cancer cells and c-Met kinase than germacrone.
Besides these findings, molecular docking studies were also carried out to analyze the results and explain
the molecular mechanism of activities to c-Met kinase. Molecular dynamics simulations have been
carried out for the further evaluation of binding stabilities between the compounds and their receptors.
Received 17th November 2016
Accepted 19th December 2016
DOI: 10.1039/c6ra26944c
www.rsc.org/advances
cellular responses including proliferation, angiogenesis,
survival, tissue regeneration, wound healing, scattering,
motility, branching morphogenesis and invasion.14–17 There-
fore, c-Met has become an attractive target for anti-tumor
therapy.
Introduction
Curcuma zedoaria (Christm.) Roscoe, also called zedoary, is
a common plant grown in tropical countries and it is used as
a natural avor, spice, and herb in Indonesia, India, and China.
In China, it is traditionally used for the treatment of dyspepsia,
menstrual disorders, atulence, fever, and cough.1,2 Curcuma
zedoaria extract exhibit antitumor, antimicrobial, analgesic,
and anti-allergic activities.3–6 Germacrone is a major activity
component found in zedoary oil product, which is extracted
from Curcuma zedoaria. Germacrone (Fig. 1) has various kinds
of biological activities, including antiulcer, anti-bacterial, anti-
fungal, antitumor, antitussive, antifeedant, depressant, anti-
inammatory, hepatoprotector, vasodilator, and choleretic
effects.7–10
The overexpression and mutation of c-Met and its natural
ligand, hepatocyte growth factor (HGF), have been found in
different types of tumors including colorectal, lung, pancreatic,
breast, gastric, prostate, ovarian, head and neck, glioma,
hepatocellular, renal, melanoma, and a number of sarcomas;
this overexpression and mutation is correlated with advanced
stays and poor prognosis.11–13 Activation of the HGF/c-Met
signaling pathway has been shown to lead to a wide array of
Sulfonates have been widely used as anticancer drugs. In the
alkylation reaction of organic synthesis, the existence of the
sulfonate group can make the C–O bond active and become
a useful alkylation reaction reagent.18 Based on the design
theories of bioalkylating agents and combination principles,
different sulfonate esters at C-8 were introduced on germacrone
to obtain novel derivatives 3a–3f (Fig. 2). These compounds
might form intermediates with electrophilic groups and react
with the rich electron groups of biomacromolecules (such as
DNA, RNA or some important enzymes) to form the covalent
binding in vivo resulting in the loss of activity of the DNA
molecule, thus playing the role of an antitumor agent.18
In this study, we prepared these compounds and evaluated
their inhibitions on Bel-7402, HepG2, A549, and HeLa cells.
Simultaneously, the study evaluated the inhibition of these
derivatives on c-Met kinase, which has been overexpressed in
a number of cancers. Further, molecular docking studies
aMedicinal Chemistry of Department, Guangdong Pharmaceutical University,
Guangzhou 510006, China. E-mail: yelb7909@163.com; Fax: +86-20-39352129; Tel:
+86-20-39352139
bSchool of Basic Courses, Guangdong Pharmaceutical University, Guangzhou 510006,
China
cSchool of Traditional Chinese Medicine, Guangdong Pharmaceutical University,
Guangzhou, Guangdong, 510006, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c6ra26944c
Fig. 1 Structure of germacrone.
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added portion wise to a solution of 2 (21.9 mg, 0.1 mmol),
pyridine (15.8 mg, 0.2 mmol) in THF (15 mL) at 0 ꢁC under
nitrogen. Then, the mixture was stirred for 4 h at r.t. (TLC
control) and the solution was ltered. The solvent was removed
and the crude product was puried by column chromatography
(ethyl/acetate, 80/20) to obtain 3a (19.9 mg, 67%).
Synthesis of 3b, 3c, and 3d. Compounds 3b, 3c, and 3d were
prepared in analogy to 3a.
Synthesis of 3e. A mixture of 2 (21.9 mg, 0.1 mmol), DMAP
(6.1 mg, 0.05 mmol), toluene-4-sulfonic acid (17.2 mg, 0.1
mmol), and DCM (10 mL) was stirred for 30 min under nitrogen
at ꢀ10 ꢁC. A solution of DCC (41.2 mg, 0.2 mmol) in DCM (5 mL)
was slowly added and the mixture was vigorously stirred for 24 h
at r.t. and ltered. The solvent was removed to give a crude
product, which was puried by column chromatography to
obtain the product 3e (23.1 mg, 62%).
Synthesis of 3f. Compound 3f was prepared in analogy to 3e.
(3E,7E)-3,7-Dimethyl-10-(propan-2-ylidene)cyclodeca-3,7-
dien-1-ylmethanesulfonate (3a). Yield: 67%, 1H NMR (400 MHz,
CDCl3) d 5.88 (dd, J ¼ 5.0, 2.0 Hz, 1H), 5.30 (dd, J ¼ 11.0, 9.7 Hz,
1H), 5.15–5.07 (m, 1H), 3.01 (s, 3H), 2.58 (t, J ¼ 3.6 Hz, 1H), 2.54
(t, J ¼ 3.6 Hz, 1H), 2.38 (d, J ¼ 3.4 Hz, 1H), 2.37–2.32 (m, 2H),
2.31 (t, J ¼ 2.5 Hz, 2H), 2.30–2.25 (m, 1H), 1.72 (s, 6H), 1.58 (s,
3H), 1.56 (s, 3H). 13C NMR (100 MHz, (D6) DMSO) d 138.25 (C-
11), 137.17 (C-4), 131.69 (C-10), 129.40 (C-1), 129.24 (C-7),
120.14 (C-5), 74.64 (C-8), 39.24 (C-3), 38.65 (C-9), 38.60 (C-16),
28.82 (C-6), 25.60 (C-2), 21.42 (C-12), 21.42 (C-13), 18.81 (C-14),
16.31 (C-15). EI-MS: 299.16 [M + H+]. Calcd for C16H26O3S
(298.16): C, 64.39; H, 8.78; and O, 16.08. Found: C, 64.37; H,
8.79; and O, 16.09.
Fig. 2 Germacrone derivatives.
analyzed the results and explained the molecular mechanism of
eminent activities to c-Met kinase. Molecular dynamics simu-
lations were applied to carry out further evaluation of the
binding stabilities between compounds and their receptors.
Materials and methods
All chemicals were purchased from Aladdin or J&K Scientic
Ltd. China. Germacrone (99.99%) was obtained from Chengdu
Push Bio-technology Co., Ltd. Solvents were puried and dried
˚
based on standard procedures and stored over 3 A molecular
(3E,7E)-3,7-Dimethyl-10-(propan-2-ylidene)cyclodeca-3,7-
dien-1-yltriuoromethanesulfonate (3b). Yield: 64%, 1H NMR
(400 MHz, CDCl3) d 5.90–5.84 (m, 1H), 5.32 (dd, J ¼ 11.1, 9.6 Hz,
1H), 5.13 (ddd, J ¼ 11.7, 5.8, 4.6 Hz, 1H), 2.63 (dd, J ¼ 13.1,
11.3 Hz, 1H), 2.55 (dd, J ¼ 13.2, 9.4 Hz, 1H), 2.39 (dd, J ¼ 3.0,
2.0 Hz, 1H), 2.37 (s, 1H), 2.35 (dd, J ¼ 3.1, 2.0 Hz, 1H), 2.32 (d, J
¼ 1.3 Hz, 1H), 2.32–2.30 (m, 1H), 2.28 (d, J ¼ 3.7 Hz, 1H), 1.74 (s,
6H), 1.59 (s, 3H), 1.55 (s, 3H). 13C NMR (100 MHz, (D6) DMSO)
d 138.25 (C-11), 137.17 (C-4), 131.69 (C-10), 129.40 (C-1), 129.24
(C-7), 120.14 (C-5), 118.25 (C-16), 74.64 (C-8), 39.24 (C-3), 38.65
(C-9), 28.82 (C-6), 25.60 (C-2), 21.42 (C-12, C-13), 18.81 (C-14),
sieves. Reactions were tested by TLC using SILG/UV 254 silica-
gel plates. Flash chromatography (FC) was carried out by
silica gel column chromatography. 1H NMR and 13C NMR
spectra were obtained using a Bruker Digital NMR Spectrom-
eter, rep. d in ppm, J in Hz. EI-MS was obtained using a Waters
ZQ4000 instrument. Cells were obtained from the China Center
for Type Culture Collection of Wuhan University; c-Met kinase
was purchased from Millipore (Billerica, MA). RPMI-1640
culture medium and new-born calf serum were purchased
from Gibco (Grand Island, NY), and methyl thiazolyl tetrazo-
lium (MTT) was purchased from Amresco (Solon, OH).
16.31 (C-15). EI-MS: 353.41 [M
16H23F3O3S (352.13): C, 54.53; H, 6.58; F, 16.17; and O, 13.62.
+
H+]. Anal. calcd for
C
Synthesis of compounds
Found: C, 54.54; H, 6.59; F, 16.16; and O, 13.62.
Synthesis of 2. A suspension of LiAlH4 (3.8 mg, 0.1 mmol)
(3E,7E)-3,7-Dimethyl-10-(propan-2-ylidene)cyclodeca-3,7-
1
was added to a cold (ꢀ10 ꢁC) solution of 1 (21.8 mg, 0.1 mmol) dien-1-yl-ethanesulfonate (3c). Yield: 61%, H NMR (400 MHz,
in THF (15 mL) under argon and vigorous stirring. Aer 1 h CDCl3) d 5.86 (dd, J ¼ 5.1, 2.1 Hz, 1H), 5.30 (dd, J ¼ 11.0, 9.7 Hz,
(TLC control), the reaction was terminated by the addition of 1H), 5.15–5.08 (m, 1H), 3.48 (q, J ¼ 7.2 Hz, 2H), 2.58 (dd, J ¼
water (2 drops), 6 N NaOH solution (2 drops), and water (4 11.3, 9.2 Hz, 1H), 2.53 (dd, J ¼ 11.3, 7.5 Hz, 1H), 2.40–2.36 (m,
drops). The mixture was extracted with DCM (3 ꢂ 20 mL) to 1H), 2.34 (dt, J ¼ 4.3, 1.5 Hz, 1H), 2.31 (d, J ¼ 1.8 Hz, 1H), 2.30 (d,
obtain a crude product, which was puried by column chro- J ¼ 2.2 Hz, 1H), 2.28 (dd, J ¼ 4.5, 1.6 Hz, 1H), 2.27–2.20 (m, 1H),
matography (hexane/t-butyl methyl ether, 70/30) to obtain 2 1.72 (s, 6H), 1.56 (s, 3H), 1.54 (s, 3H), 1.36 (t, J ¼ 7.1 Hz, 3H). 13
C
(18.3 mg, 84%).
NMR (100 MHz, (D6) DMSO) d 138.25 (C-11), 137.17 (C-4), 131.69
Synthesis of 3a. A mixture of methanesulfonic acid (14.4 mg, (C-10), 139.40 (C-1), 129.24 (C-7), 120.14 (C-5), 74.64 (C-8), 44.10
0.15 mmol), SOCl2 (21.4 mg, 0.18 mmol), DMF (2 drops), and (C-16), 39.24 (C-3), 38.65 (C-9), 28.82 (C-6), 25.60 (C-2), 21.42 (C-
DCM (5 mL) was reuxed for 4 h to obtain methanesulfonyl 12), 21.42 (C-13), 18.81 (C-14), 16.31 (C-15), 8.09 (C-17). EI-MS:
chloride. Methanesulfonyl chloride (17.1 mg, 0.15 mmol) was
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313.47 [M + H+]. Anal. calcd for C17H28O3S (312.18): C, 65.35; H, with 200 mL in each well of a 96-well plate and treated with 0–
9.03; and O, 15.36. Found: C, 65.35; H, 9.01; and O, 15.36.
800 mmol Lꢀ1 of target products or 0.1% DMSO for 24 and 48 h.
(3E,7E)-3,7-Dimethyl-10-(propan-2-ylidene)cyclodeca-3,7- 20 mL of MTT was added and the mixture was incubated in the
dien-1-ylbenzenesulfonate (3d). Yield: 57%, 1H NMR (400 MHz, dark at 37 ꢁC for 4 h. Aer the removal of MTT, cells were treated
CDCl3) d 7.77 (dd, J ¼ 7.9, 1.4 Hz, 2H), 7.73–7.68 (m, 1H), 7.57 (t, with 150 mL of DMSO. The absorbances were determined at
J ¼ 7.8 Hz, 2H), 5.85 (dd, J ¼ 4.3, 3.0 Hz, 1H), 5.33 (dd, J ¼ 11.0, 590 nm using an automated microplate reader (SpectraMAX190,
10.0 Hz, 1H), 5.21–5.09 (m, 1H), 2.61 (dd, J ¼ 10.2, 8.1 Hz, 1H), Molecular Devices, USA).
2.56 (dd, J ¼ 10.2, 7.0 Hz, 1H), 2.43–2.40 (m, 1H), 2.40–2.36 (m,
1H), 2.35 (s, 1H), 2.35–2.33 (m, 1H), 2.33 (d, J ¼ 1.7 Hz, 1H), Inhibition of c-Met kinase
2.32–2.25 (m, 1H), 1.72 (s, 6H), 1.60 (s, 3H), 1.56 (s, 3H). 13C
NMR (100 MHz, (D6) DMSO) d 138.25 (C-11), 137.17 (C-4), 134.06
The inhibition of c-Met kinase was investigated as described in
a reported study.20 The IC50 values were determined using TR-
(C-16), 131.68 (C-10), 131.44 (C-19), 129.40 (C-1), 129.25 (C-18, C-
FRET. 6 His-tagged recombinant human c-Met residues (50 nM)
20), 129.23 (C-7), 127.83 (C-17, C-21), 120.14 (C-5), 74.64 (C-8),
974-end (Millipore) were cultured in a medium containing
39.24 (C-3), 38.65 (C-9), 28.82 (C-6), 25.60 (C-2), 21.42 (C-12, C-
2.5 mmol Lꢀ1 MnCl2, 10 mmol Lꢀ1 MgCl2, 20 mmol Lꢀ1 Tris,
13), 18.81 (C-14), 16.31 (C-15). EI-MS: 361.17 [M + H+]. Anal.
2 mmol Lꢀ1 DTT, and 0.01% Tween 20 with 5 mmol Lꢀ1 ATP and
calcd for C21H28O3S (360.18): C, 69.96; H, 7.83; and O, 13.31.
200 nmol Lꢀ1 5FAM-KKK-SPGEYVNIGFG-NH2 at room tempera-
Found: C, 69.95; H, 7.85; and O, 13.32.
ture for 60 min. Compounds were tested with increasing
concentrations. Reactions were terminated using IMAP stop
solution. Plates were incubated overnight and analyzed using
AiphaQuest.
(3E,7E)-3,7-Dimethyl-10-(propan-2-ylidene)cyclodeca-3,7-
dien-1-yl-4-methylbenzenesulfonate (3e). Yield: 62%, 1H NMR
(400 MHz, CDCl3) d 7.66 (d, J ¼ 8.1 Hz, 2H), 7.35 (d, J ¼ 7.8 Hz,
2H), 5.85 (dd, J ¼ 4.3, 3.0 Hz, 1H), 5.33 (dd, J ¼ 11.0, 10.0 Hz,
1H), 5.14 (ddd, J ¼ 6.9, 5.6, 2.9 Hz, 1H), 2.61 (dd, J ¼ 10.2, 8.1 Hz,
Molecular docking
1H), 2.56 (dd, J ¼ 10.2, 7.0 Hz, 1H), 2.42–2.37 (m, 2H), 2.37–2.35
Molecular docking was performed according to the related ref. 11.
The docking experiment was carried out using the CDocker
program, which was connected with the Accelrys Discovery Studio
2.5.5. The programs were adapted with an empirical scoring
function and a patented search engine.21,22 Briey, ligands were
(m, 2H), 2.34 (s, 3H), 2.34–2.32 (m, 1H), 2.32–2.28 (m, 1H), 1.72
(s, 6H), 1.60 (s, 3H), 1.55 (s, 3H). 13C NMR (100 MHz, (D6) DMSO)
d 144.27 (C-19), 138.25 (C-11), 137.17 (C-4), 133.84 (C-16), 131.69
(C-10), 129.93 (C-18, C-20), 129.40 (C-1), 129.24 (C-7), 128.01 (C-
17, C-21), 120.14 (C-5), 74.64 (C-8), 39.24 (C-3), 38.65 (C-9), 28.82
docked into the corresponding protein's binding site in compli-
(C-6), 25.60 (C-2), 21.42 (C-12), 21.42 (C-13), 21.26 (C-22), 18.81
ance with the protocol, which was generated by ligands from the
(C-14), 16.31 (C-15). EI-MS: 375.54 [M + H+]. Anal. calcd for
crystal structure of 3DKC with random hydrogen atoms and Gas-
C
22H30O3S (374.19): C, 70.55; H, 8.07; and O, 12.82. Found: C,
¨
teiger–Huckel charges, but not water. The ligands' other parame-
70.55; H, 8.05; and O, 12.84.
ters were default values except that the threshold was 1. The
structure of the receptor was minimized to 10 000 cycles using the
Powell method in DS 2.5.5. The geometries of all compounds were
optimized by the conjugate gradient method of TRIPOS. The
(3E,7E)-3,7-Dimethyl-10-(propan-2-ylidene)cyclodeca-3,7-
dien-1-yl-4-chlorobenzenesulfonate (3f). Yield: 52%, 1H NMR
(400 MHz, CDCl3) d 7.68 (d, J ¼ 7.8 Hz, 2H), 7.63 (d, J ¼ 8.1 Hz,
2H), 5.81 (dd, J ¼ 5.8, 1.4 Hz, 1H), 5.32 (dd, J ¼ 11.1, 9.8 Hz, 1H),
5.13 (ddd, J ¼ 6.9, 5.6, 2.9 Hz, 1H), 2.63–2.57 (m, 1H), 2.54 (dd, J
¼ 11.5, 8.1 Hz, 1H), 2.38 (dd, J ¼ 5.3, 1.8 Hz, 1H), 2.36–2.34 (m,
1H), 2.34–2.31 (m, 2H), 2.31–2.27 (m, 1H), 2.23 (dd, J ¼ 15.4,
convergence criterion was identied as 0.001 kcal molꢀ1
.
MD simulations
1.4 Hz, 1H), 1.72 (s, 6H), 1.59 (s, 3H), 1.54 (s, 3H). 13C NMR (100 The MD simulations were performed on the basis of molecular
MHz, (D6) DMSO) d 138.25 (C-11), 137.17 (C-4), 135.25 (C-19), docking using AMBER 10.0 for ligands and AMBER ff03 for
134.06 (C-16), 131.69 (C-10), 129.72 (C-17, C-21), 129.46 (C-18, proteins. The Gaussian 03 program was used to calculate partial
C-20), 129.41 (C-1), 129.24 (C-4), 120.14 (C-5), 74.64 (C-8), atomic charges at a neutral pH, with histidines 164 and 200
39.24 (C-3), 38.65 (C-9), 28.82 (C-6), 25.60 (C-2), 21.42 (C-12, C- protonated at the d position. The SHAKE algorithm was used to
13), 18.81 (C-14), 16.31 (C-15). EI-MS: 395.96 [M + H+]. Anal. restrict all the bonds, given the time step of 2 fs and cut-off
˚
calcd for C21H27ClO3S (394.14): C, 63.86; H, 6.89; Cl, 8.98; and O, distance of 8 A, with long-range electrostatic interactions
12.15. Found: C, 63.85; H, 6.88; Cl, 8.99; and O, 12.15.
treated with the particle mesh Ewald (PME) method.23,24 The
heating operation was carried out from 0 to 300 K in 50 ps using
Langevin dynamics at a constant volume and equilibrated for
100 ps at a constant pressure of 1 atm aer four steps of
Cell assay
Inhibition effects of the synthesized compounds on Bel-7402, minimizations, which included 2500 cycles of steepest descent
HepG2, A549 and HeLa cells were investigated according to minimization, followed by 2500 cycles of conjugated gradient
the related ref. 19. The cells were incubated in RPMI 1640 minimization. Heavy atoms of the receptor–ligand complex
2
complete culture medium consisting of 10% FBS, 100 U mLꢀ1 were restrained to 0, 10, 100, and 500 kcal (molꢀ1 A ) and were
˚
2
penicillin, and 100 mg mLꢀ1 streptomycin at 37 ꢁC in a humid- 10 kcal (molꢀ1 A ) during the heating and equilibration steps,
˚
ied atmosphere containing 5% CO2. Cell growth was evaluated whereas solvent molecules were not restricted. Finally, periodic
by MTT assay.10 Cells were inoculated at 1.0 ꢂ 104 cells per mL boundary conditions of 8 ns were employed for the whole
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system with a normal pressure of 1 atm and normal tempera- acetonitrile as a solvent, DMAP as a catalyst, and DCC as
ture of 300 K in the production step.
a dehydrator and can be widely applied.
Due to the distinct difference in polarity, compounds could
be easily separated. Structural modication of germacrone and
a systematic study showed that 8-hydroxyl group may require
further optimization studies and a range of substituents could
be suitable for this position.
MM-PBSA estimation of binding free energy DGpred
For each system, DGpred values were calculated using 100
snapshots from the last 1 ns trajectory with an interval of 10 ps
by the molecular mechanics Poisson–Boltzmann surface area
(MM-PBSA) method.25–28
Evaluation of biological activity
Biological activities were investigated by checking the effect of
germacrone and its derivatives on the growth of Bel-7402 cells,
HepG2 cells, A549, and HeLa cells. These cells were treated with
Results and discussion
Chemistry
The target compounds 2 and 3a–3f were synthesized, as germacrone and its derivatives at the concentrations of 12.5 mmol
described in Fig. 3. Different sulfonic acids reacted with (3E,7E)- Lꢀ1, 25 mmol Lꢀ1, 50 mmol Lꢀ1, 100 mmol Lꢀ1, 200 mmol Lꢀ1, 400
3,7-dimethyl-10-(propan-2-ylidene) cyclodeca-3,7-dienol (2) to mmol Lꢀ1, and 800 mmol Lꢀ1, respectively. An MTT assay was then
obtain (3E,7E)-3,7-dimethyl-10-(propan-2-ylidene) cyclodeca-3,7- applied at 24 h and 48 h. As shown in Table 1, the proliferations
dien-1-ylmethanesulfonate (3a), (3E,7E)-3,7-dimethyl-10-(propan- of HepG2, Bel7402, A549, and Hela cells were inhibited in a dose-
2-ylidene)cyclodeca-3,7-dien-1-yltriuoromethanesulfonate (3b), dependent manner, and the cytolytic activity was markedly
(3E,7E)-3,7-dimethyl-10-(propan-2-ylidene)cyclodeca-3,7-dien- inhibited at the same time. Moreover, the IC50 values of germa-
1-ylethanesulfonate (3c), (3E,7E)-3,7-dimethyl-10-(propan-2- crone against Bel-7402 cells, HepG2 cells, A549, and HeLa cells
ylidene)cyclodeca-3,7-dien-1-yl-benzenesulfonate (3d), and were about 173.54 mM, 169.52 mM, 179.97 mM, and 160.69 mM.
(3E,7E)-3,7-dimethyl-10-(propan-2-ylidene)cyclodeca-3,7-dien-1- The growth-inhibitory effect had no signicant differences aer
yl-4-chlorobenzene sulfonate (3f) via acyl chloride esterication incubation with germacrone and derivatives for 24 h and 48 h
reaction with yields of 67%, 61%, 64%, 57%, and 52%, respec- (Fig. 4); thus, there was no signicant inuence on the anti-
tively. The esterication reaction provided good yields using dry proliferation effect of compounds on Bel-7402 cells, HepG2
THF as a solvent and pyridine as an acid-binding agent. The cells, A549, and HeLa cells with an extended treatment time aer
residual target compounds (3E,7E)-3,7-dimethyl-10-(propan-2- 24 h. This might be caused by the saturation of intracellular drug
ylidene)cyclodeca-3,7-dien-1-yl-4-methylbenzenesulfonate (3e) concentration within 24 hours. Compounds 3e and 3f, in which R
were obtained via a DMAP/DCC esterication reaction with was substituted with benzene, showed high inhibition and 3e
a yield of 62%. This reaction was carried out using dry DCM or showed highest activity against HepG2 with an IC50 value of 56.22
Fig. 3 Synthesis of 3a–3f reagents and conditions: (a) LiAlH4, THF, r.t., 1 h; (b) SOCl2, DMF, Py, THF, r.t., 4 h; and (c) DMAP, DCC, r.t., 24 h.
Table 1 IC50 values of compounds for Bel-7402, HepG2, A549, and Hela cells
Bel-7402 IC50 (mM)
HepG2 IC50 (mM)
A549 IC50 (mM)
HeLa IC50 (mM)
Compound
24 h
48 h
24 h
48 h
24 h
48 h
24 h
48 h
1
2
173.54
167.08
122.23
88.87
105.05
92.00
177.21
166.32
125.68
92.44
110.14
91.02
169.52
155.20
119.09
79.98
98.54
84.67
56.22
64.12
174.56
161.54
121.08
77.80
95.65
87.74
57.18
62.99
179.97
177.21
127.68
97.74
111.28
101.35
84.47
177.21
185.24
131.45
95.52
115.69
97.74
160.69
157.31
125.96
100.25
108.97
102.80
88.64
167.78
163.15
129.44
97.28
105.88
108.36
89.26
3a
3b
3c
3d
3e
3f
71.78
77.04
74.68
79.45
81.00
95.65
91.25
95.24
99.11
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Fig. 4 Inhibition of germacrone derivatives on Bel-7402, HepG2, A549, and HeLa cells. (a) The effect of compounds on Bel-7402 cells. (b) The
effect of compounds on HepG2 cells. (c) The effect of compounds on A549 cells. (d) The effect of compounds on HeLa cells.
mM. The results of inhibition on c-Met showed that these deriv- derivatives owing to sesquiterpene structures of these
atives exhibited a stronger inhibitory effect than germacrone compounds; therefore, we chose this compound to perform the
(Fig. 5). Since the results suggested that the derivatives have good docking experiments. The 3DKC is the crystal structure of c-Met
activity, we could initially conrm that the designed compounds kinase in complex with ATP and its details can be obtained from
might be well worth investigation on the basis of molecular http://www.rcsb.org/pdb/explore/materialsAndMethods.do?
docking, molecular dynamics simulations, and preliminary structureId¼3DKC, which is widely used.29–31
activity tests. In conclusion, our strategy to design germacrone
Molecular docking suggested that these compounds were
derivatives was successful in obtaining the desired compounds, bound to the active site of the protein 3DKC (shown in Fig. 6).
which demonstrated a stronger inhibitory effect on cancer cells The binding energies of the complexes between each compound
and c-Met kinase. These compounds might be useful as lead (1–3f) and the active site of the receptor are shown in Table 2.
structures for the development of anti-cancer agents, and this The binding energies were higher when R was a benzene ring
study could supply important information for building a chem- and the compounds showed higher inhibition, of which
ical library and the basis for further research.
a substituted benzene ring had stronger binding energies and
inhibition. The interaction between the (E,E)-1,5-cyclo-
decadiene system and the protein crystal 3DKC seems to be
strong, and the binding forces were mainly hydrophobic forces,
whereas another clear interaction was the s–p interaction
Docking studies
In our previous study, we performed docking studies using
several PDB proteins including 3DKF, 2WGJ, 2RFS, and 3DKC,
in which only 3DKC interacted with germacrone and its
Fig. 5 Inhibition of compounds on c-Met Kinase.
3764 | RSC Adv., 2017, 7, 3760–3767
Fig. 6 Compact binding modes of all compounds (PDB code: 3DKC).
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Table 2 Binding energies of complexes between compounds and
3DKC
c-Met inhibition
rate (%)
–CDOCKER
Compounds
R
energy (kcal molꢀ1
)
1
2
3a
3b
3c
3d
—
–OH
Me
–CF3
–CH2CH3
Ph
27%
23%
24%
37%
31%
34%
29.4387
22.0488
22.7005
42.2213
32.4854
38.7086
3e
56%
65.5007
3f
44%
47.4545
Fig. 8 Plots of RMSD for all of the backbone atoms (A˚) vs. simulation
time (ns) for 3DKC in complex with 3e.
Table 3 The value of –CDOCKER energy
c-Met inhibition
rate (%)
DGpred energy
Compounds
R
(kcal molꢀ1
)
1
2
3a
3b
3c
3d
—
–OH
Me
–CF3
–CH2CH3
Ph
27%
23%
24%
37%
31%
34%
16.35
12.31
12.48
21.09
20.11
20.23
Fig. 7 Conformations of 3e.
between the benzene ring with LYS1110 and GLY1087.
Compound 3e showed the highest activity owing to the p–p
stacking interaction of the benzene ring and hydrophobic
interaction of methyl, as shown in Fig. 7.
3e
56%
28.36
3f
44%
25.41
experiment, DGpred values of less than ꢀ25 kcal molꢀ1 were
selected and used to evaluate 3DKC inhibitory activity. The
predicted DGpred value of 3e is ꢀ28.36 kcal molꢀ1, whereas the
predicted DGpred values of other compounds were more than
ꢀ25 kcal molꢀ1 (shown in Table 3). To save the computational
time and reduce the computational cost of the MD-based
screening studies, we did not consider the entropy contribu-
tion because of the extremely time-consuming nature of the
normal mode calculation of entropy for large systems. There-
fore, the calculated DGpred values in the present study were
inadequate owing to the omission of entropy contribution.
MD simulations and DGpred calculation
Building on the structural data from diverse experimental
sources, today's MD simulations permit the exploration of
biological processes in an unparalleled detail. Since molecular
docking studies reported a probably momentary binding mode,
which might be unreasonable and unstable, molecular
dynamics simulations were applied to carry out further research
of the binding stabilities between the compounds (1–3f) and
3DKC.
The crystal structures of the 3DKC complex with 1–3f were
used to investigate the reliability of molecular dynamics simu-
lations. The root mean square deviation (RMSD) curve and the
surface area curve of the 1 ns MD simulation indicated that the
structure of 3e was in a relatively stable state aer 1 ns and the
Conclusions
MD trajectories were quite smooth (shown in Fig. 8), whereas This study designed and synthesized novel bioactivity
other compounds were unstable with uneven MD trajectories compounds using the natural product germacrone as the lead
(as shown in ESI†). The MD parameters were suitable for the compound based on the design theory of alkylating agents. The
MD simulations.
results showed that the majority of compounds had moderate
Given identical MD conditions, the two 3DKC–ligand inhibition on cancer cells and c-Met kinase. These compounds
complexes were used to perform the molecular dynamics can be further studied as antitumor lead structures to lay the
simulations for eight nanoseconds. The RMSD curves of all foundation for developing anticancer compounds with clinical
compounds were stable during the MD simulations. In the value. The results of molecular docking and molecular
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dynamics simulations were very important in explaining the
molecular mechanism of eminent activities to c-Met kinase and
binding stabilities between the compounds and their receptors.
Moreover, these results can provide a theoretical basis for
further research on c-Met inhibitors.
stimulated murine macrophages, Int. Immunopharmacol.,
2009, 9(9), 1049–1057.
9 X. Xiao, Y. Zhao, H. Yuan, et al. Study on the effect of Rhizoma
curcuma Longa on gastrin receptor, J. Chin. Mater. Med.,
2002, 25(3), 184–185.
10 Y. Liu, W. Wang, B. Fang, et al. Anti-tumor effect of
germacrone on human hepatoma cell lines through
inducing G2/M cell cycle arrest and promoting apoptosis,
Eur. J. Pharmacol., 2013, 698(2), 95–102.
11 L. Ye, Y. Tian, Z. Li, et al. Design, synthesis and molecular
docking studies of some novel spiro[indoline-3,40-
piperidine]-2(1H)-ones as potential c-Met inhibitors, Eur. J.
Med. Chem., 2012, 50, 370–375.
12 P. Ma, T. Kijima, G. Maulik, et al. c-Met mutational analysis
in small cell lung cancer: novel juxtamembrane domain
mutations regulating cytoskeletal functions, Cancer Res.,
2003, 63(19), 6272–6281.
13 P. Ma, S. Jagadeesh, R. Jagadeeswaran, et al. c-Met
expression/activation, functions, and mutations in non-
small cell lung cancer, Proc. Am. Assoc. Canc. Res., 2004, 45,
1875.
Acknowledgements
This project was funded by the Special Fund of Applied
Research and Development of Guangdong Provincial Depart-
ment of Science and Technology (2015B020234009), Special
Fund for TCM supported by the State Administration of Tradi-
tional Chinese Medicine of China (201507004), Joint Natural
Sciences Fund of The Department of Science and Technology
and rst Affiliated Hospital of Guangdong Pharmaceutical
University (No. GYFYLH201317), Guangdong Natural Science
Foundation (2015A030313586), and Science and Technology
Planning Project of Guangdong Province (Guangdong Branch
Regulations [2015]110, 2016A020215159). The authors are
grateful to Mr Wei and Lanzhou University for molecular
docking experiments and MD Simulations.
14 D. Renzo, M. Olivero, T. Martone, et al. Somatic mutations of
the MET oncogene are selected during metastatic spread of
human HNSC carcinomas, Oncogene, 2000, 19(12), 1547–
1555.
References
1 W. Chen, Y. Liu, M. Gao, J. Wu, A. Wang and R. Shi, 15 J. Lee, S. Han, H. Cho, et al.
A novel germ line
Antiangiogenesis effect of essential oil from Curcuma
zedoaria in vitro and in vivo, J. Ethnopharmacol., 2011,
133(1), 220–226.
2 C. Chen, Y. Chen, Y. His, et al. Chemical Constituents and
Anticancer Activity of Curcuma zedoaria Roscoe Essential
juxtamembrane Met mutation in human gastric cancer,
Oncogene, 2000, 19(43), 4947–4953.
16 F. Yang, S. Praveena and C. Patrick, Met signaling: novel
targeted inhibition and its clinical development in lung
cancer, J. Thorac. Oncol., 2012, 7(2), 459–467.
Oil against Non-Small Cell Lung Carcinoma Cells in Vitro 17 M. Li, P. Yang, Y. Liu, et al. Low c-Met expression levels are
and in Vivo, J. Agric. Food Chem., 2013, 61(47), 11418–11427.
3 W. Seo, J. Hwang, S. Kang, et al. Suppressive effect of
Zedoariae rhizoma on pulmonary metastasis of B16
melanoma cells, J. Ethnopharmacol., 2005, 101(3), 249–257.
4 N. Dde, M. Souza, R. Neto, et al. Phytochemical analysis and
analgesic properties of Curcuma zedoaria grown in Brazil,
Phytomedicine, 2002, 9(5), 427–432.
5 B. Wilson, J. Abraham, V. Manju, et al. Antimicrobial activity
of Curcuma zedoaria and Curcuma malabarica tubers, J.
Ethnopharmacol., 2005, 99(1), 147–151.
prognostic for and predict the benets of temozolomide
chemotherapy in malignant gliomas, Sci. Rep., 2016, 6,
1–10.
18 T. L. Lemke, D. A. Williams and V. F. Roche, et al., Foye's
principles of medicinal chemistry, Wolters Kluwer:
Lippincott William and Wilkins, 7th edn, 2013.
19 W. Chen, Y. Liu, M. Li, et al. Anti-tumor effect of a-pinene on
human hepatoma cell lines through inducing G2/M cell
cycle arrest, J. Pharmacol. Sci., 2015, 3, 332–338.
20 J. Porter, S. Lumb, R. Franklin, et al. Discovery of 4-
azaindoles as novel inhibitors of c-Met kinase, Bioorg. Med.
Chem. Lett., 2009, 19(10), 2780–2784.
6 H. Matsuda, S. Tewtrakul, T. Morikawa, A. Nakamura and
M. Yoshikawa, Antiallergic principles from Thai zedoary:
structural requirements of curcuminoids for inhibition of 21 A. Jain, Surex: fully automatic exible molecular docking
degranulation and effect on the release of TNF-a and IL-4
in RBL-2H3 cells, Bioorg. Med. Chem., 2004, 12(22), 5891–
5898.
7 Y. Dang, X. Li, Q. Zhang and Y. Wang, Preparative isolation
and purication of six volatile compounds from essential
using a molecular similarity-based search engine, J. Med.
Chem., 2003, 46(4), 499–511.
22 A. Jain, Surex-Dock2.1: robust performance from ligand
energetic modeling, ring exibility, and knowledge-based
search, J. Comput.–Aided Mol. Des., 2007, 21(5), 281–306.
oil of Curcuma wenyujin using high-performance 23 Z. Li, Y. Cai, Y. Cheng, et al. Identication of Novel
centrifugal partition chromatography, J. Sep. Sci., 2010,
33(11), 1658–1664.
8 W. Cho, J. Nam, H. Kang, et al. Zedoarondiol isolated from
Phosphodiesterase-4D Inhibitors Prescreened by Molecular
Dynamics Augmented Modeling and Validated by Bioassay,
J. Chem. Inf. Model., 2013, 53(4), 972–981.
the rhizome of Curcuma heyneana is involved in the 24 M. Shuichi and P. Kollman, Settle: An analytical version of
inhibition of iNOS, COX-2 and pro-inammatory cytokines
via the downregulation of NF-kappa B pathway in LPS-
the SHAKE and RATTLE algorithm for rigid water models,
J. Comput. Chem., 1992, 13(8), 952–962.
3766 | RSC Adv., 2017, 7, 3760–3767
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
25 I. Massova and P. Kollman, Combined molecular
mechanical and continuum solvent approach (MM-PBSA/
GBSA) to predict ligand binding, Perspect. Drug Discovery
Des., 2000, 18(1), 113–135.
26 T. Hou, J. Wang, Y. Li and W. Wang, Assessing the
performance of the MM/PBSA and MM/GBSA methods. 1.
The accuracy of binding free energy calculations based on
analysis, molecular docking, and molecular dynamics
simulations, Bioorg. Med. Chem. Lett., 2010, 20(20), 6008–
6012.
29 S. G. Buchanan, J. Hendle, P. S. Lee, et al. SGX523 is an
exquisitely selective, ATP-competitive inhibitor of the MET
receptor tyrosine kinase with antitumor activity in vivo,
Mol. Cancer Ther., 2009, 8(12), 3181–3190.
molecular dynamics simulations, J. Chem. Inf. Model., 30 J. G. Christensen, R. J. Schreck, P. Kuruganti, et al. A selective
2011, 51(1), 69–82.
27 M. Liu, M. Yuan, M. Luo, et al. Binding of curcumin with
glyoxalase I: molecular docking, molecular dynamics
small molecule inhibitor of c-Met kinase inhibits c-Met-
dependent phenotypes in vitro and exhibits cytoreductive
antitumor activity in vivo, Cancer Res., 2003, 63(21), 7345.
simulations, and kinetics analysis, Biophys. Chem., 2010, 31 L. Wang, J. Ai, Y. Shen, et al. SOMCL-863, a novel, selective
147(1–2), 28–34.
28 L. He, F. He, H. Bi, et al. Isoform-selective inhibition of
chrysin towards human cytochrome P450 1A2. Kinetics
and orally bioavailable small-molecule c-Met inhibitor,
exhibits antitumor activity both in vitro, and in vivo, Cancer
Lett., 2014, 351(1), 143–150.
This journal is © The Royal Society of Chemistry 2017
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