Synthesis and biological evaluation of terminal functionalized thiourea-containing dipeptides as antitumor agents

Article abstract of DOI:10.1039/c6ra25590f

A series of antitumor agents based on terminal functionalized dipeptide derivatives containing the thiourea moiety were synthesized and evaluated for antiproliferative activity using a panel of cancer cell lines, and the effects and mechanism of apoptosis induction were determined. These compounds exhibited significant selectivity to different cancer cell lines with IC₅₀ values at micromolar concentrations. In particular, compound I-11 appeared to be the most potent compound, with an IC₅₀ = 4.85 ± 1.44 μM against the NCI-H460 cell line, at least partly, by the induction of apoptosis. Mechanistically, compound I-11 induced the activation of caspase-12 and CHOP, which triggered apoptotic signalling via the ROS-dependent endoplasmic reticulum pathway and arrested the cell cycle at the S phase. Thus, we concluded that dipeptide derivatives containing the thiourea moiety terminally functionalized by electron-withdrawing substituents may be potential antitumor agents for further investigation.

Full text of DOI:10.1039/c6ra25590f

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PAPER
Synthesis and biological evaluation of terminal
functionalized thiourea-containing dipeptides as
antitumor agents†
Cite this: RSC Adv., 2017, 7, 8866
Ri-Zhen Huang,‡ab Bin Zhang,‡a Xiao-Chao Huang,b Gui-Bin Liang,a Jian-Mei Qin,a
a
b
a
*
*
Ying-Ming Pan, Zhi-Xin Liao and Heng-Shan Wang
A series of antitumor agents based on terminal functionalized dipeptide derivatives containing the thiourea
moiety were synthesized and evaluated for antiproliferative activity using a panel of cancer cell lines, and the
effects and mechanism of apoptosis induction were determined. These compounds exhibited significant
selectivity to different cancer cell lines with IC50 values at micromolar concentrations. In particular,
compound I-11 appeared to be the most potent compound, with an IC50 ¼ 4.85 ꢀ 1.44 mM against the
NCI-H460 cell line, at least partly, by the induction of apoptosis. Mechanistically, compound I-11
induced the activation of caspase-12 and CHOP, which triggered apoptotic signalling via the ROS-
dependent endoplasmic reticulum pathway and arrested the cell cycle at the S phase. Thus, we
concluded that dipeptide derivatives containing the thiourea moiety terminally functionalized by
electron-withdrawing substituents may be potential antitumor agents for further investigation.
Received 21st October 2016
Accepted 11th January 2017
DOI: 10.1039/c6ra25590f
www.rsc.org/advances
potency since they exhibit cancer selective toxicity while avoid-
ing the shortcomings of the conventional chemotherapy.12
1 Introduction
Modern studies have also indicated that dipeptides as well as
their analogs exhibit anticancer activity in many human cancer
cells such as colon, gastric, cervical and breast cancer cells.13–16
However, most of the natural peptides consist of the L-form
a amino acids and due to the ubiquitous prevalence of pepti-
dases, they exhibit limited biostability, and consequently, low
bioavailability.17 The inability to cross epithelial layers,
including the blood–brain barrier, side effects and drug resis-
tance remain the major clinical challenges. Due to these limi-
tations, the development of pseudopeptides could lead to many
pharmacokinetic and pharmacodynamic advantages. This
strategy can be realized by introducing unnatural amino acids
instead of natural residues into the peptide framework, or by
the introduction of a non-peptidic scaffold into the peptidic
backbone, to lock a dened conformation of the peptide.18,19
These novel compounds provide new perspectives in drug
design by providing an entire range of highly specic and non-
toxic pharmaceuticals. Several non-peptidic skeletons have
been thus conjugated with peptides, and such analogs as
conjugates of paclitaxel, doxorubicin and daunorubicin with an
amino acid or peptides have demonstrated increased and more
selective anticancer activity than the drugs themselves.20–26
Among the anti-tumor drugs discovered in recent years,
thiourea derivatives27,28 possess potent anticancer properties.
Thiourea derivatives display a wide range of biological activity
including antibacterial, anti-fungal, antiviral, anthelmintic,
antituberculosis, and plant growth regulator properties.29–32 In
addition to increased stability, the incorporation of a thiourea
Peptides are among the most versatile bioactive molecules and
play crucial roles in the human body and other organisms.1
Because of their good solubility, permeability and bioavail-
ability, many peptide hormones and analogous short peptides
exert their action by binding to membrane receptors,2,3 or
possess signaling and regulatory functions.4 Accordingly, the
potential applications of these peptide-like structures are legion
and become immediately apparent. Recently, several peptide-
based derivatives have been in clinical trials, including borte-
zomib,5 carlzomib,6 DLS-10 (ref. 7) and ixazomib8 (Fig. 1).
Encouraged by these research results, our interest in investi-
gating peptide-based drugs for their potential therapeutic
effects has recently spurred us to examine the inuences of
dipeptide derivatives on antitumor properties.
Previous work has indicated that dipeptides and their
derivatives, in addition to enzyme inhibitors or drug carriers,
exhibit a wide spectrum of important bioactivities such as
antimicrobial, neuroprotective, antiviral and anticancer activi-
ties.9–11 In particular, anticancer peptides have shown relevant
aState Key Laboratory for the Chemistry and Molecular Engineering of Medicinal
Resources (Ministry of Education of China), School of Chemistry and
Pharmaceutical Sciences of Guangxi Normal University, Yucai Road 15, Guilin
541004, Guangxi, PR China. E-mail: whengshan@163.com
bPharmaceutical Research Center and School of Chemistry and Chemical Engineering,
Southeast University, Nanjing 211189, China. E-mail: zxliao@seu.edu.cn
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c6ra25590f
‡ These authors contributed equally to this work.
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Fig. 1 Chemical structures of peptide-based derivatives for clinical use.
moiety into the peptide sequence also provides access to addi- Hct-116 human colon tumor cells, and SKOV-3 human ovarian
tional binding interactions within the transition state confor- cancer cells. The commercial anticancer drugs 5-uorouracil
mation of the enzyme/substrate complex.33,34 Thiourea could (5-FU) and ubenimex (Ube) were positive controls. The assay
strongly enhance blocking, since sulfur is the weaker hydrogen results are shown in Table 1, where we see that most of the test
bond acceptor as compared to the amide carbonyl oxygen, and compounds exhibited moderate to high inhibitory activity
simultaneously enhance hydrogen-bonding due to the biden- against the tested tumor cell lines, indicating that the intro-
tate binding mode of the more acidic thiourea protons.35,36 duction of the thiourea moiety on the dipeptide could improve
Herein, we designed and synthesized a series of dipeptide anti-tumor activity. Compounds I-10, I-11 and I-32 showed good
thiourea derivatives as antitumor drugs. Their in vitro cytotox- inhibitory activity for all the tested carcinoma cell lines.
icities against ve selected tumor cell lines were evaluated. Compound I-9 showed strong inhibitory activity selectivity for
Moreover, the molecular mechanism of apoptosis in NCI-H460 MGC-803, as well as NCI-H460; I-31 showed selectivity for NCI-
cells by the representative target compound I-11 was also H40, and I-25 showed selectivity for MGC-803. The others also
investigated.
showed good inhibitory activity, except I-8, I-16, I-17, I-30, I-33,
I-34, for the tested carcinoma cell lines. In particular,
compound I-11 showed noteworthy anti-cancer activity in vitro,
with IC50 value of 4.85 mM against NCI-H460. The results
revealed that the analogs I-11, I-25, I-31 and I-32, obtained by
inserting the triuoromethyl or halogen moiety in positions R1
and R2 of the benzene ring, signicantly increased anti-
proliferative potencies. For example, compound I-25, with IC50
value of 9.13 mM, was more active than derivatives I-24 and I-26,
and undoubtedly emerged as one of the most active compounds
within this subset, thus suggesting that the insertion of
electron-withdrawing groups in positions R1 and R2 may have
resulted in certain steric electronic properties that enhanced
lipophilicity and the ability to penetrate the cellular membrane,
leading to an increased antiproliferative effect. On the other
hand, the presence of a weakly electron-withdrawing substit-
uent in positions R1 and R2 is harmful to the antiproliferative
effect: the derivatives I-33 and I-34 were not active against the
tested cell lines. A slight benecial effect was also observed with
the modications of the R3 and R4 positions: the derivative I-11
was more active than I-32. It is also important to note that the R1
and R2 substitutions seem more important for cytotoxicity than
R3 or R4 substitutions, since the variation of R3 or R4 slightly
changed the potency. These interesting results may indicate
2 Results and discussion
2.1 Chemistry
The general procedures for the synthesis of functionalized
dipeptide thioureas are shown in Scheme 1. First, Boc-amide
and aniline derivatives underwent acylation and deprotection,
and each amino acid was sequentially coupled to the peptide
chain from the C- to N-terminus in the presence of HOBT, EDCI
and Et3N. Aer obtaining the free amine dipeptide H by the
removal of the protective group, target compounds I were
synthesized by the condensation of phenylisothiocyanate,
according to a previously published report,37 with H in DCM at
room temperature. The structures of target compounds I were
then conrmed by 1H NMR, 13C NMR and high resolution mass
spectrometry (HRMS).
2.2 Biology activity
2.2.1 Cytotoxicity test. The in vitro cytotoxicity of the
synthesized compounds was evaluated by the methylthiazol
tetrazolium (MTT) assay in a panel of ve human cancer cell
lines, including MGC-803 human gastric cancer cells, NCI-H460
human lung cancer cells, HepG-2 human liver cancer cells,
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Scheme 1 General synthetic route for compound I.
that these compounds exert their antitumor activities by tar- concentrations of compound I-11. Cell cycle distribution was
geting different protein targets or signal pathways. The pres- investigated by ow cytometric analysis following staining of
ence of a bulky group in position R3 and R4 seems to be DNA with propidium iodide (PI). Aer treatment with
associated with a general increase in activity. In addition, the compound I-11 at different concentrations for 48 h, it was
inhibition activities of compounds I against HUVEC normal cell observed that G1 phase cells gradually decreased and G2 phase
lines were also estimated in Table 1. The results indicated that cells did not change signicantly, while S phase cells, compared
the cytotoxicity of most of the compounds against cancer cells with the control cells, gradually increased (Fig. 2 and 3). These
was much higher than the HUVEC normal cells, as compared to results suggest that target compound I-11 mainly arrested NCI-
5-FU and Ube, making them good candidates for anti-tumor H460 cells in the S phase.
drugs.
2.2.3 Compound I-11 induces apoptosis in NCI-H460 cells.
2.2.2 Cell cycle analysis. To determine the possible role of In order to conrm whether I-11-induced reduction in cell
cell cycle arrest in dipeptide thiourea derivative-induced growth viability was responsible for the induction of apoptosis, NCI-
inhibition, NCI-H460 cells were treated with different H460 cells were co-stained with PI and Annexin-V FITC, and
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Table 1 IC50 values (mM) of functionalized dipeptide thiourea derivatives I towards five selected tumor cell lines and normal cell lines, for 48 h
IC50a (mM)
Compd
MGC-803
NCI-H460
Hct-116
HepG-2
SKOV-3
HUVEC
I-1
I-2
I-3
I-4
I-5
I-6
I-7
I-8
25.82 ꢀ 1.12
24.51 ꢀ 0.83
40.01 ꢀ 0.86
20.85 ꢀ 1.06
30.43 ꢀ 1.00
38.95 ꢀ 0.71
26.38 ꢀ 1.21
>50
8.92 ꢀ 1.02
7.49 ꢀ 1.06
8.26 ꢀ 1.27
33.05 ꢀ 1.05
12.53 ꢀ 1.21
12.27 ꢀ 1.18
30.45 ꢀ 1.28
>50
23.11 ꢀ 1.22
24.32 ꢀ 1.29
38.37 ꢀ 0.93
21.39 ꢀ 0.99
31.20 ꢀ 1.04
39.48 ꢀ 1.25
25.41 ꢀ 1.33
>50
9.55 ꢀ 0.46
13.06 ꢀ 1.19
4.85 ꢀ 1.44
30.29 ꢀ 1.42
14.65 ꢀ 0.74
17.89 ꢀ 1.13
41.29 ꢀ 1.15
>50
29.34 ꢀ 1.07
26.17 ꢀ 1.01
39.25 ꢀ 1.14
21.06 ꢀ 0.98
27.77 ꢀ 0.94
38.62 ꢀ 0.68
30.17 ꢀ 0.90
>50
10.70 ꢀ 0.85
9.13 ꢀ 1.11
8.92 ꢀ 1.26
38.47 ꢀ 0.84
19.82 ꢀ 0.89
22.51 ꢀ 0.74
36.71 ꢀ 0.64
>50
27.16 ꢀ 1.16
29.05 ꢀ 0.96
41.27 ꢀ 1.02
26.79 ꢀ 1.10
34.57 ꢀ 0.85
42.07 ꢀ 1.18
30.95 ꢀ 1.23
>50
12.31 ꢀ 1.13
13.70 ꢀ 0.92
10.88 ꢀ 1.05
37.21 ꢀ 1.15
13.82 ꢀ 1.03
21.28 ꢀ 0.76
33.22 ꢀ 1.31
>50
24.58 ꢀ 0.91
29.96 ꢀ 0.87
44.32 ꢀ 1.11
28.28 ꢀ 0.89
30.99 ꢀ 1.35
39.24 ꢀ 0.49
31.84 ꢀ 0.75
>50
13.60 ꢀ 1.08
10.36 ꢀ 1.04
10.86 ꢀ 1.14
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
56.00 ꢀ 1.45
>50
I-9
I-10
I-11
I-12
I-13
I-14
I-15
I-16
I-17
I-18
I-19
I-20
I-21
I-22
I-23
I-24
I-25
I-26
I-27
I-28
I-29
I-30
I-31
I-32
I-33
I-34
I-35
I-36
5-FU
Ube
23.27 ꢀ 1.32
21.24 ꢀ 1.24
42.36 ꢀ 0.82
>50
>50
>50
>50
>50
>50
38.21 ꢀ 1.09
16.73 ꢀ 0.86
18.79 ꢀ 1.14
16.47 ꢀ 0.97
17.15 ꢀ 1.26
17.52 ꢀ 1.22
37.98 ꢀ 1.03
9.13 ꢀ 1.09
35.48 ꢀ 0.83
29.22 ꢀ 0.76
34.37 ꢀ 1.09
28.25 ꢀ 1.11
>50
41.89 ꢀ 0.97
14.95 ꢀ 0.59
18.51 ꢀ 0.88
15.63 ꢀ 0.58
17.31 ꢀ 1.31
17.66 ꢀ 1.23
40.82 ꢀ 1.79
10.62 ꢀ 1.14
31.67 ꢀ 1.49
28.80 ꢀ 1.37
32.46 ꢀ 1.06
29.58 ꢀ 1.28
>50
46.61 ꢀ 1.03
16.43 ꢀ 1.08
20.83 ꢀ 0.98
20.73 ꢀ 1.21
19.86 ꢀ 1.29
18.59 ꢀ 1.34
41.55 ꢀ 1.25
12.74 ꢀ 1.17
34.83 ꢀ 1.13
30.76 ꢀ 1.04
37.63 ꢀ 1.16
30.48 ꢀ 0.78
>50
38.95 ꢀ 1.19
20.13 ꢀ 1.16
24.45 ꢀ 1.27
19.44 ꢀ 0.95
21.86 ꢀ 1.18
21.05 ꢀ 1.28
40.79 ꢀ 0.71
14.32 ꢀ 1.20
36.24 ꢀ 1.21
31.81 ꢀ 1.26
38.24 ꢀ 0.91
31.65 ꢀ 0.88
>50
42.48 ꢀ 0.99
15.98 ꢀ 1.51
22.78 ꢀ 0.87
20.81 ꢀ 1.47
19.35 ꢀ 1.53
20.24 ꢀ 1.45
46.43 ꢀ 1.33
13.82 ꢀ 1.02
39.40 ꢀ 1.36
32.49 ꢀ 0.62
37.38 ꢀ 1.20
32.10 ꢀ 1.51
>50
13.75 ꢀ 1.23
8.79 ꢀ 0.77
>50
9.93 ꢀ 1.36
7.93 ꢀ 1.05
>50
11.59 ꢀ 0.94
13.78 ꢀ 1.15
>50
15.09 ꢀ 1.17
10.16 ꢀ 0.84
>50
10.26 ꢀ 0.96
11.62 ꢀ 1.08
>50
>50
>50
>50
>50
>50
41.65 ꢀ 1.02
42.93 ꢀ 1.36
46.92 ꢀ 2.03
>50
39.07 ꢀ 1.34
44.86 ꢀ 1.17
44.05 ꢀ 0.64
>50
42.69 ꢀ 0.36
41.78 ꢀ 1.04
40.57 ꢀ 0.61
>50
>50
>50
23.50 ꢀ 2.34
>50
30.98 ꢀ 0.73
>50
27.23 ꢀ 0.16
>50
a
IC50 values are presented as the mean ꢀ SD (standard error of the mean) from three separated experiments.
the number of apoptotic cells was estimated by ow cytometry were present in the control panel; in contrast, the percentage
(Fig. 4). Four quadrant images were observed by ow cytometric rose to 19.35% at the concentration of 5 mM aer treatment with
analysis: the Q1 area represented damaged cells that appeared I-11 for 24 h. At concentrations of 10 mM and 15 mM, there was
during the process of cell collection; the Q2 region showed a further increase to 42.3% and 43.4% aer treatment with I-11,
necrotic cells and later stage apoptotic cells; early apoptotic respectively. These results clearly conrm that compared with
cells were located in the Q3 area, and the Q4 area showed the control, compound I-11 effectively induced apoptosis in
normal cells. A dose-dependent increase in the percentage of NCI-H460 cells in a dose-dependent manner.
apoptotic cells was noted aer the cells were treated with
2.2.4 Morphological characterization of NCI-H460 cell
compound I-11 at the concentrations of 5 mM, 10 mM and 15 mM apoptosis by Hoechst 33258. In order to further validate cell
for 24 h. As shown in Fig. 4 and 5, few (4.79%) apoptotic cells apoptosis following treatment with compound I-11, NCI-H460
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Fig. 2 Investigation of cell cycle distribution of compound I-11 by flow cytometric analysis. (a) Untreated NCI-H460 cells as a control. (b, c, and
d) Cells were treated with increasing concentrations of compound I-11 (2 mM, 4 mM and 8 mM) for 48 h, respectively.
cells treated with compound I-11 at 10 mM from 6 to 24 h were I-11 increased the level of intracellular calcium within 6 h of
stained with Hoechst 33258. Control cells exhibited weak blue treatment, and the highest level of intracellular calcium was
uorescence (Fig. 6a); following treatment with compound I-11, observed aer treatment for 24 h. Therefore, these ndings
some cells emitted brilliant blue uorescence, and the nuclei of indicate that compound I-11 can signicantly increase the
NCI-H460 cells appeared hyper-condensed (brightly stained). intracellular level of calcium released.
The number of apoptotic nuclei containing condensed chro-
2.2.6 Compound I-11 disrupts mitochondrial membrane
matin increased signicantly when NCI-H460 cells were treated potential. Cytoplasmic calcium is rapidly taken up by the
with compound I-11 for 24 h, indicating that apoptosis of NCI- mitochondria resulting in mitochondrial swelling and loss of
H460 cells was induced by compound I-11 in a time-dependent mitochondrial membrane potential (MMP).39 The loss of MMP
manner.
is regarded as a limiting factor in the apoptotic pathway. In
2.2.5 Compound I-11 induces release of intracellular order to further investigate the antiproliferative effect of target
calcium. Conditions such as drug treatment, which interfere compound I-11, changes in MMP were detected using the
with ER function and induced endoplasmic reticulum (ER) uorescent probe JC-1, which exhibits potential dependent
stress and swelling, causing cavities or the accumulation of accumulation in mitochondria, indicated by a uorescence
unfolded proteins in the ER or misfolded proteins, play a crucial emission shi from red (ꢁ590 nm) to green (ꢁ545 nm). In the
role in the ER stress-related protein degradation pathway.38 The control cells, JC-1 aggregated in the mitochondria and showed
ER is the primary storage site for intracellular calcium, and strong red uorescence. However, in cells undergoing
pumps in the ER membrane maintain a calcium gradient apoptosis, where the mitochondrial potential had collapsed,
ꢁ1000-fold higher than that in the cytoplasm.39 To determine JC-1 was located in the cytosol as a monomer and emitted green
whether I-11 induced the release of intracellular calcium in NCI- uorescence. NCI-H460 cells treated with compound I-11 at 10
H460 cells, the calcium level was measured with and without mM from 6 to 24 h were stained with JC-1 for 24 h and cells not
(control) I-11 (10 mM) treatment for 6 to 24 h, using the uo- treated with compound I-11 were used as controls.
rescent probe uo-3-acetoxymethyl-ester (Fluo-3/AM) and ow
The results are shown in Fig. 8. The JC-1 monomer and
cytometry. In the control cells, the level of intracellular calcium J-aggregates were excited at 514 nm and 585 nm, respectively,
was lowest (Fig. 7). Treatment of NCI-H460 cells with compound and light emissions were collected at 515–545 nm (green) and
570–600 nm (red). Fluorescence microscopy (Fig. 8) showed that
cells not treated with compound I-11 were normally red (in the
web version), while cells treated with I-11 showed strong yellow-
green uorescence and typical apoptotic morphology aer 6 h,
12 h and 24 h, suggesting the occurrence of mitochondrial
depolarization by I-11.
2.2.7 Compound I-11 triggers ROS generation. Reactive
oxygen species (ROS) are highly harmful to cells as they initiate
oxidative stress and ultimately cause cellular damage. Excessive
ROS generation renders cells vulnerable to apoptosis.40 To
determine whether I-11 triggers ROS generation in NCI-H460
cells to induce apoptosis, the ROS level was measured with
and without (control) I-11 (10 mM) treatment for 6 to 24 h, using
the uorescent probe 2,7-dichlorouorescein diacetate (DCF-
DA) and ow cytometry. As shown in Fig. 9, compound I-11
induced an increase in ROS levels in NCI-H460 cells. Aer
exposure to 10 mM of compound I-11 for 24 h, the ROS level was
42.6%, which was more than three times that of the control.
Fig. 3 The percentages of NCI-H460 cells in different phases of cell
Taken together, these results show that I-11 caused oxidative
cycle are presented.
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Fig. 4 Apoptosis ratio detection of compound I-11 by Annexin V/PI assay. (a) NCI-H460 cells not treated with I-11. (b, c, and d) NCI-H460 cells
treated with compound I-11 at 5 mM, 10 mM and 15 mM for 24 h, respectively.
imbalance in NCI-H460 cells. This induction of oxidative burst we monitored the changes in apoptotic molecules related to the
is a key factor in the antiproliferative activity of compound I-11. ER stress signaling pathway. A number of key protein markers
2.2.8 Compound I-11 induces eIF2a phosphorylation. In involved in this signaling network were also examined by
general, ER stress removes proteins accumulated in the ER and western blot analysis. As shown in Fig. 10a, treatment of NCI-
promotes cell survival by inhibiting protein synthesis, while H460 cells with I-11 caused a signicant increase in the levels
overload or sustained ER stress induces cell death through of GRP78 and CHOP, in a dose-dependent manner. Further-
a series of signaling pathways, including the CHOP pathway, more, in comparison with the control cells, compound I-11
IRE1a-ASK1-JNK pathway and Caspase-12 pathway.38 The induced eIF2a and PERK phosphorylation.
pancreatic ER kinase (PKR)-like ER kinase (PERK), a trans-
2.2.9 Activation of caspases. Intracellular calcium plays
membrane protein kinase in the ER stress signaling network, a central role in the regulation of calpain activity.41 It is known
was previously shown to phosphorylate eIF2a in response to ER that calcium-activated calpain can cleave cellular proteins,
stress. The activation of PERK also leads to the induction of which results in cell swelling, membrane rupture, and cell
CHOP, which, as detailed later, is an important element in the death. Calpain and caspase are cysteine proteases and have
switch from pro-survival to pro-death signaling. To further important roles in the initiation, regulation and execution of
investigate the molecular mechanism of compound I-11 against cell death such as apoptosis and necrosis.42 As illustrated in
NCI-H460 cells to gain further insight into its mode of action, Fig. 10b, calcium overload was observed in NCI-H460 cells
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Fig.
5 Populations of apoptotic NCI-H460 cells treated with
Fig. 7 Effects of compound I-11 on the intracellular free Ca2+ levels in
NCI-H460 cells. After treatment with compound I-11 (10 mM) for 6 h,
12 h and 24 h, respectively, NCI-H460 cells stained with Fluo-3AM for
30 min were analyzed by flow cytometry.
increasing concentrations of compound I-11 (5 mM, 10 mM and 15 mM)
obtained by FACS analysis with PI and FITC-Annexin V staining.
following treatment with compound I-11; therefore, we
hypothesized that elevated caspase-12/caspase-4 and calpain
activity may be triggered by calcium overload. As shown in
Fig. 10b, target compound I-11 caused a signicant time-
dependent induction of caspase-12 and caspase-4 expression.
In addition, we also demonstrated that treatment with
compound I-11 signicantly activated the downstream death
receptor cascade, as evidenced by increased caspase-9 levels in
the cytoplasm, which may further amplify mitochondrial
membrane permeability.
3 Conclusion
A series of dipeptide thiourea derivatives were designed and
synthesized as potential antitumor agents with high selectivity.
These compounds exhibited signicant selectivity against
different cancer cell lines with IC50 values at micromolar
concentrations. It is noteworthy that further antitumor activity
screening revealed that some compounds exhibited better
Fig. 6 Hoechst 33258 staining of compound I-11 in NCI-H460 cells. (a) Cells not treated with compound I-11 were used as control. (b, c, and d)
Treatment with compound I-11 (10 mM) for 6 h, 12 h and 24 h, respectively.
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Fig. 8 JC-1 staining of compound I-11 in NCI-H460 cells. (a) Cells not treated with compound I-11 were used as control. (b, c, and d) Treatment
with compound I-11 (10 mM) for 6 h, 12 h and 24 h, respectively.
inhibitory activity than the commercial anticancer drug 5-Fu 1H-NMR and 13C-NMR spectra were recorded on a Bruker AMX-
and Ube. In particular, compound I-11 (IC50 ¼ 4.85 ꢀ 1.44 mM) 400 and AMX-500 spectrometer. High-resolution mass spectra
exhibited the best anticancer activity against the NCI-H460 cell (HRMS) were recorded using ESI and APCI ionization sources.
line and displayed more potent inhibitory activity than 5-Fu and
Ube. The apoptosis-inducing activity of representative
4.2 Experimental section
compound I-11 in NCI-H460 cells was investigated by Hoechst
33258 staining, JC-1 mitochondrial membrane potential stain-
ing, and ow cytometry. Simultaneous molecular mechanism
studies suggested that target compound I-11 induced apoptosis
in NCI-H460 cells through induction of ER stress-reactive
oxygen species. Furthermore, cell cycle analysis indicated that
compound I-11 arrested the NCI-H460 cell line in the S phase.
The possible mechanism involved in compound I-11 induced
apoptosis is shown in Fig. 11. Consequently, the rational design
of dipeptide thiourea derivatives offers signicant potential for
the discovery of a new class of antitumor agents. The precise
mechanism of this action requires further investigation.
4.2.1 General procedure for isothiocyanate compounds.
Toluene (20 mL) was added to the amine (10 mmol) and Et3N
(30.3 mmol). CS2 (30.3 mmol) was added while stirring, result-
ing in the precipitation of the dithiocarbamate. The reaction
mixture was stirred for 9 h at room temperature and then
ltered and dried. The resulting powder was suspended in the
ꢂ
CH2Cl2 (20 mL), and cooled to 0 C and then BTC (3.3 mmol)
was added. Aer completing the reaction at room temperature
for 1 h, reuxing was continued for 2 h. Insolubles were
removed by ltration, and the solvent was puried by chroma-
tography on silica gel eluted with petroleum ether to offer
isothiocyanate.
4.2.2 General procedure for compounds C and F. To
a stirred solution of N-t-butyloxycarbonyl-L-amino (5.5 mmol) in
DMF (10 mL), was added Et3N (12.5 mmol) and HOBT (7.5
4 Experimental
4.1 General information
The isothiocyanate was synthesized according to the litera- mmol). The mixture was allowed to stir for 1 h and then cooled
ture.34 Compound H was synthesized according to the litera- to 0 ꢂC and then EDCI (7.5 mmol) was added. Aer 1 h, a solu-
ture.43 All the chemical reagents and solvents used were of tion of aniline derivatives (5 mmol) in DMF (10 mL) was added
analytical grade. Aniline derivatives and all N-Boc protection to the above reaction mixture. The resulting mixture was stirred
amino acids during the synthesis were commercially available for 7 h at room temperature. Aer completion as monitored by
and purchased from Aladdin and Energy Chemical. Silica gel TLC, the reaction was quenched with water and extracted with
(300–400 mesh) used in column chromatography was provided EtOAc. The organic layer was washed with saturated brine
by Tsingtao Marine Chemistry Co. Ltd. Precoated silica gel solution, followed by drying over Na2SO4 and evaporating in
plates F-254 were used for thin-layer analytical chromatography. vacuo. The crude product was puried by column
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Fig. 9 ROS generation assay of compound I-11 in NCI-H460 cells. (a) Cells not treated with I-11 were used as the control for 24 h. (b, c, and d)
Treatment with compound I-11 (10 mM) for 6 h, 12 h and 24 h, respectively.
chromatography (EtOAc/petroleum ether) to give the pure
intermediates C and F.
1-(1-(1-(2,5-Dimethoxyphenylamino)-1-oxo-3-phenylpropan-2-
ylamino)-3-methyl-1-oxopentan-2-yl)-3-(3-nitrophenyl)thiourea (I-
1). Yield 77.4%. Mp 187.5–190.1 ꢂC. [a]D20 ¼ ꢃ29.2 (c 0.1, AcOEt).
1H NMR (400 MHz, DMSO-d6) d 10.15 (s, 1H), 9.06 (s, 1H), 8.84
(s, 1H), 8.61 (d, J ¼ 7.9 Hz, 1H), 7.97 (d, J ¼ 8.5 Hz, 1H), 7.92 (dd,
J ¼ 8.2, 1.5 Hz, 1H), 7.83 (dd, J ¼ 8.1, 1.2 Hz, 1H), 7.73 (d, J ¼
2.7 Hz, 1H), 7.58 (t, J ¼ 8.2 Hz, 1H), 7.33 (d, J ¼ 7.3 Hz, 2H), 7.23
(t, J ¼ 7.4 Hz, 2H), 7.16 (d, J ¼ 7.3 Hz, 1H), 6.94 (d, J ¼ 9.0 Hz,
1H), 6.62 (dd, J ¼ 8.9, 3.0 Hz, 1H), 5.00–4.90 (m, 1H), 4.82 (dd, J
¼ 13.0, 8.7 Hz, 1H), 3.76 (s, 3H), 3.68 (s 3H), 3.15 and 2.96 (dd, J
¼ 13.9, 4.7 Hz, 1H; dd, J ¼ 13.8, 9.7 Hz, 1H), 1.90–1.80 (m, 1H),
1.44 and 1.08 (ddd, J ¼ 13.1, 7.4, 3.1 Hz, 1H; td, J ¼ 13.7, 8.2 Hz,
1H), 0.91–0.73 (m, 6H, 2 ꢄ CH3). 13C NMR (100 MHz, DMSO-d6)
d 180.4, 170.9, 169.7, 152.9, 147.5, 143.1, 141.0, 137.6, 129.7,
129.3, 129.3, 128.0, 128.0, 127.8, 126.2, 117.9, 115.9, 111.8,
108.0, 107.5, 60.9, 56.2, 55.3, 55.3, 54.9, 37.5, 36.7, 24.3, 15.1,
4.2.3 General procedure for compounds D and H. To
a round ask was added C or F and a standard 50% TFA/CH2Cl2.
The sample was stirred at room temperature for 3 h. The solvent
was removed under reduced pressure and the residue was par-
titioned between NaHCO3 and EtOAc. The solvent of the organic
layer was removed under reduced pressure. The residue thus
obtained was puried by column chromatography (EtOAc/
petroleum ether) to give the deprotection of the Boc group to
unmask the free amine D and H.
4.2.4 General procedure for compounds I. Compounds I
were obtained by the condensation of isothiocyanates with F in
CH2Cl2 at room temperature. The structures were conrmed by
1H NMR, 13C NMR and HR-MS (see ESI†). The spectral data for
the representative compounds are shown in detail.
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Fig. 10 Western blotting analysis effect of compound I-11 on the expression of (b) caspase-9, caspase-3, caspase-12, calpain, caspase-4, (a)
CHOP, GRP78, p-PERK and p-eIF2a in NCI-H460 cells. NCI-H460 cells were treated with compound I-11 at 10 mM for 6 h, 12 h and 24 h,
respectively. Equal amounts of protein were loaded on SDS-PAGE gel for western blot analysis as described in the experimental section. b-Actin
was used as an internal control.
1H NMR (400 MHz, DMSO-d6) d 9.92 (s, 1H), 9.74 (s, 1H), 8.35 (d,
J ¼ 8.0 Hz, 1H), 7.96 (s, 1H), 7.83 (d, J ¼ 8.1 Hz, 1H), 7.46 (d, J ¼
8.9 Hz, 2H), 7.35 (dd, J ¼ 10.3, 8.1 Hz, 2H), 7.29 (d, J ¼ 7.0 Hz,
2H), 7.24 (t, J ¼ 7.4 Hz, 2H), 7.17 (d, J ¼ 7.1 Hz, 1H), 7.13 (d, J ¼
7.4 Hz, 1H), 6.87 (d, J ¼ 9.0 Hz, 2H), 4.85 (d, J ¼ 6.5 Hz, 1H), 4.70
(dd, J ¼ 13.9, 8.4 Hz, 1H), 3.72 (s, 3H), 3.09 and 2.95 (dd, J ¼
13.8, 5.2 Hz, 1H; dd, J ¼ 13.7, 9.3 Hz, 1H), 1.83 (d, J ¼ 6.1 Hz,
1H), 1.39 and 1.11–0.97 (dd, J ¼ 11.9, 5.6 Hz, 1H; m, 1H), 0.82 (d,
J ¼ 7.8 Hz, 6H, 2 ꢄ CH3). 13C NMR (100 MHz, DMSO-d6) d 180.3,
170.5, 169.1, 155.4, 141.2, 137.6, 132.5, 131.8, 130.0, 129.2,
129.2, 128.0, 128.0, 126.3, 123.4, 121.4, 121.4, 121.0, 120.4,
113.8, 113.8, 61.2, 55.1, 54.7, 37.5, 37.4, 24.4, 15.2, 11.4. ESI-
HRMS m/z calcd for C29H33ClN4O3S [M ꢃ H]ꢃ: 551.1889;
found: 551.1903.
N-(1-Oxo-3-phenyl-1-((3-(triuoromethyl)phenyl)amino)propan-
2-yl)-3-phenyl-2-(3-(3-(triuoromethyl)phenyl)thioureido)
propanamide (I-11). Yield 78.3%. Mp 143.8–150.6 ꢂC. [a]D20
¼
Fig. 11 The signaling pathways for apoptosis and cell cycle arrest
induced by compound I-11.
1
ꢃ26.0 (c 0.1, AcOEt). H NMR (500 MHz, DMSO-d6) d 10.45 (s,
1H), 10.15 (s, 1H), 8.80 (d, J ¼ 7.8 Hz, 1H), 8.10 (s, 2H), 7.85 (d, J
¼ 7.5 Hz, 1H), 7.80 (d, J ¼ 8.4 Hz, 1H), 7.59 (dd, J ¼ 17.8, 9.0 Hz,
2H), 7.50 (t, J ¼ 7.9 Hz, 1H), 7.42 (dd, J ¼ 15.7, 7.8 Hz, 2H), 7.33
(d, J ¼ 7.3 Hz, 2H), 7.28 (t, J ¼ 7.5 Hz, 2H), 7.22–7.16 (m, 3H),
7.15–7.10 (m, 3H), 5.18 (dd, J ¼ 12.3, 6.8 Hz, 1H), 4.74 (td, J ¼
8.4, 5.9 Hz, 1H), 3.27 (dd, J ¼ 13.8, 4.7 Hz, 1H), 3.13 (dd, J ¼ 13.9,
5.6 Hz, 1H), 3.00 (ddd, J ¼ 13.7, 7.7, 5.9 Hz, 2H). 13C NMR (125
11.3. ESI-HRMS m/z calcd for C30H35N5O6S [M + H]+: 594.2381;
found: 594.2355.
1-(3-Chlorophenyl)-3-(1-(1-(4-methoxyphenylamino)-1-oxo-3-
phenylpropan-2-ylamino)-3-methyl-1-oxopentan-2-yl)thiourea (I-
5). Yield 76.5%. Mp 169.9–173.5 ꢂC. [a]D20 ¼ ꢃ27.0 (c 0.1, AcOEt).
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MHz, DMSO-d6) d 179.8, 170.5, 170.4, 140.3, 139.5, 137.3, 136.9, 25.6, 19.5, 18.6, 15.4, 12.8, 11.4. ESI-HRMS m/z calcd for
130.1, 129.7, 129.6, 129.4, 129.0, 128.2, 127.9, 126.5, 126.3,
125.9, 125.2, 125.1, 123.1, 123.00, 122.9, 120.2, 119.9, 118.4,
C
27H37ClN4O2S [M + K]+: 555.1957; found: 555.2003.
1-(1-(1-(3,4-Dimethylphenylamino)-3-methyl-1-oxobutan-2-
115.5, 115.4, 57.5, 55.0, 37.5. ESI-HRMS m/z calcd for ylamino)-3-methyl-1-oxopentan-2-yl)-3-(3-nitrophenyl)thiourea (I-
C
33H28F6N4O2S [M + Na]+: 681.1729; found: 681.1722.
36). Yield 80.4%. Mp 194.5–195.4 ꢂC. [a]D20 ¼ ꢃ22.7 (c 0.1,
2-(3-(3-Chlorophenyl)thioureido)-N-(1-((3,5-dimethylphenyl) AcOEt). 1H NMR (500 MHz, DMSO-d6) d 10.17 (s, 1H), 9.83 (d, J ¼
amino)-1-oxo-3-phenylpropan-2-yl)-3-phenylpropanamide (I-16). 20.0 Hz, 1H), 8.85 (s, 1H), 8.14 (d, J ¼ 8.5 Hz, 1H), 8.09 (d, J ¼
Yield 83.1%. Mp 139.6–147.2 ꢂC. [a]D20 ¼ ꢃ37.0 (c 0.1, AcOEt). 1H 8.4 Hz, 1H), 7.90 (dd, J ¼ 8.2, 1.5 Hz, 1H), 7.83 (d, J ¼ 7.4 Hz,
NMR (500 MHz, CDCl3) d 9.16 (d, J ¼ 33.1 Hz, 2H), 8.69 (s, 1H), 1H), 7.57 (t, J ¼ 8.2 Hz, 1H), 7.35 (s, 1H), 7.29 (dd, J ¼ 8.1, 1.9 Hz,
7.63 (s, 1H), 7.43 (s, 1H), 7.24 (d, J ¼ 6.0 Hz, 2H), 7.15 (s, 3H), 1H), 7.03 (d, J ¼ 8.2 Hz, 1H), 4.95 (t, J ¼ 7.4 Hz, 1H), 4.27 (t, J ¼
7.07 (s, 2H), 6.96 (s, 3H), 6.85 (s, 1H), 6.78 (d, J ¼ 6.8 Hz, 5H), 8.0 Hz, 1H), 2.15 (d, J ¼ 10.6 Hz, 6H), 2.04 (dd, J ¼ 14.2, 7.1 Hz,
5.44 (s, 1H), 5.12 (s, 1H), 3.19–2.96 (m, 2H), 2.90 (d, J ¼ 23.0 Hz, 1H), 1.92–1.87 (m, 1H), 1.55–1.50 and 1.16–1.10 (m, 1H; m, 1H),
1H), 2.76 (s, 1H), 2.07 (s, 6H, 2 ꢄ CH3). 13C NMR (125 MHz, 0.95–0.87 (m, 9H, 3 ꢄ CH3), 0.85 (t, J ¼ 7.4 Hz, 3H, CH3). 13C
CDCl3) d 179.8, 171.0, 170.7, 139.7, 140.0, 139.0, 136.4, 136.2, NMR (125 MHz, DMSO-d6) d 180.4, 170.7, 169.55, 147.5, 141.1,
135.4, 134.3, 129.9, 129.6, 128.7, 128.3, 127.6, 127.2, 126.6, 136.5, 136.3, 131.1, 129.7, 129.6, 127.9, 120.6, 118.0, 116.9,
125.6, 124.6, 122.9, 119.4, 59.1, 56.1, 39.24, 38.1, 21.2. ESI- 115.9, 61.0, 59.0, 37.4, 30.5, 24.6, 19.6, 19.2, 18.8, 18.6, 15.3,
HRMS m/z calcd for C33H33ClN4O2S [M + Na]+: 607.1905; 11.3. ESI-HRMS m/z calcd for C26H35N5O4S [M ꢃ H]ꢃ: 512.2337;
found: 607.1900.
found: 512.2338.
N-(4-Bromophenyl)-2-(2-(3-(3-bromophenyl)thioureido)-3-
methylbutanamido)-3-methylpentanamide (I-20). Yield 88.2%. Mp 4.3 Cytotoxicity assay
126.7–131.1 ꢂC. [a]D20 ¼ ꢃ26.9 (c 0.1, AcOEt). 1H NMR (400 MHz,
The cell lines MGC-803, NCI-H460, Hct-116, HepG2, SKOV-3,
DMSO-d6) d 10.21 (s, 1H), 9.88 (s, 1H), 8.25 (d, J ¼ 7.9 Hz, 1H),
7.84 (d, J ¼ 8.0 Hz, 1H), 7.62–7.29 (m, 8H), 4.94 (s, 1H), 4.28 (t, J
¼ 8.0 Hz, 1H), 2.12 (dd, J ¼ 12.7, 6.4 Hz, 1H), 1.81 (d, J ¼ 5.4 Hz,
1H), 1.54 (s, 1H), 1.24–1.06 (m, 1H), 0.85 (dd, J ¼ 16.8, 6.6 Hz,
12H, 4 ꢄ CH3). 13C NMR (100 MHz, DMSO-d6) d 180.4, 170.7,
170.3, 139.1, 138.2, 131.6, 131.5, 131.2, 131.1, 124.2, 121.2,
115.6, 114.9, 61.2, 58.1, 36.2, 31.2, 24.6, 18.9, 18.2, 15.3, 10.9.
ESI-HRMS m/z calcd for C24H30Br2N4O2S [M + K]+: 635.0088;
found: 635.0080.
and HUVEC were obtained from the Shanghai Cell Bank in
the Chinese Academy of Sciences. MGC-803, NCI-H460, Hct-
116, HepG2, SKOV-3, and HUVEC cell lines were grown on 96-
well microtitre plates at a cell density of 10 ꢄ 105 cells per well
in DMEM medium with 10% FBS. DMEM and FBS were ob-
tained from Gibco-Thermo (BRL Co. Ltd., USA). The plates were
incubated at 37 ꢂC in a humidied atmosphere of 5% CO2/95%
air overnight. The cells were exposed to different concentrations
of compounds I and 5-Fu, and incubated for another 48 h. The
cells were stained with 10 mL of MTT in an incubator for about
4 h. The medium was thrown away and replaced by 100 mL
DMSO. The O.D. value was read at 570/630 nm using an enzyme
labeling instrument.
N-(3-Chloro-4-uorophenyl)-2-(2-(3-(3,4-dichlorophenyl)
thioureido)-3-methylbutanamido)-3-methylpentanamide
(I-25).
Yield 80.5%. Mp 129.5–133.2 ꢂC. [a]D20 ¼ ꢃ21.8 (c 0.1, AcOEt). 1H
NMR (400 MHz, DMSO-d6) d 10.31 (s, 1H), 10.01 (s, 1H), 8.28 (d, J
¼ 8.0 Hz, 1H), 8.16 (s, 1H), 7.98 (d, J ¼ 8.2 Hz, 1H), 7.92 (dd, J ¼
6.8, 2.3 Hz, 1H), 7.53 (d, J ¼ 8.7 Hz, 1H), 7.50–7.45 (m, 1H), 7.43
(dd, J ¼ 8.8, 2.3 Hz, 1H), 7.35 (t, J ¼ 9.1 Hz, 1H), 5.04–4.73 (m,
1H), 4.26 (t, J ¼ 8.1 Hz, 1H), 2.13 (dd, J ¼ 12.8, 6.5 Hz, 1H), 1.82
(d, J ¼ 6.2 Hz, 1H), 1.53 (d, J ¼ 7.2 Hz, 1H), 1.20–1.12 (m, 1H),
0.87 (dd, J ¼ 15.8, 8.4 Hz, 12H, 4 ꢄ CH3). 13C NMR (100 MHz,
DMSO-d6) d 180.7, 171.1, 170.8, 154.8, 152.4, 140.4, 136.4, 130.8,
130.6, 125.5, 123.5, 122.4, 120.9, 117.5, 117.3, 61.7, 58.5, 36.6,
4.4 Hoechst 333258 assay
NCI-H460 cells (2 ꢄ 106 cells) were seeded in six-well tissue
culture plates and exposed to compound I-11 (10 mM) for
different times. The cells were xed in 4% paraformaldehyde for
10 min, aer which the medium was discarded. The cells were
then washed twice with cold PBS and incubated with 0.5 mL of
Hoechst 33258 (Beyotime, China) in the dark for 5 min. Aer
5 min incubation, the cells were washed twice with cold PBS and
the results were analyzed using a Nikon ECLIPSETE2000-S
uorescence microscope using 350 nm excitation and 460 nm
emissions.
31.6, 25.0, 19.3, 18.6, 15.7, 11.2. ESI-HRMS m/z calcd for C24
-
H28Cl3FN4O2S [M + Na]+: 583.0875; found: 583.0870.
N-(1-((3-Chlorophenyl)amino)-3-methyl-1-oxobutan-2-yl)-3-
methyl-2-(3-(3,4,5-trimethylphenyl)thioureido)pentanamide (I-30).
Yield 83.6%. Mp 148.6–152.3 ꢂC. [a]D20 ¼ ꢃ11.7 (c 0.1, AcOEt). 1H
NMR (400 MHz, CDCl3) d 8.72 (s, 1H), 8.26 (s, 1H), 7.59 (t, J ¼
1.8 Hz, 1H), 7.45 (s, 1H), 7.34 (d, J ¼ 8.2 Hz, 1H), 7.13 (t, J ¼
4.5 Determination of mitochondrial membrane potential
8.1 Hz, 1H), 7.07–6.98 (m, 2H), 6.50 (s, 2H), 5.01 (t, J ¼ 7.7 Hz, The JC-1 probe (Beyotime, Haimen, China) was employed to
1H), 4.43 (t, J ¼ 8.0 Hz, 1H), 3.82 (s, 3H, CH3), 3.74 (s, 6H, 2 ꢄ measure mitochondrial depolarization in NCI-H460 cells.
CH3), 2.31–2.11 (m, 1H), 1.95–1.89 (m, 1H), 1.60–1.51 (m, 1H), Briey, NCI-H460 cells were seeded at the density of 2 ꢄ 106
1.17–1.06 (m, 1H), 0.99 (dd, J ¼ 6.7, 2.9 Hz, 6H, 2 ꢄ CH3), 0.91– cells mLꢃ1 of the DMEM medium with 10% FBS on 6-well plates
0.74 (m, 6H, 2 ꢄ CH3). 13C NMR (100 MHz, CDCl3) d 180.7, to the nal volume of 2 mL. The plates were incubated overnight
172.1, 170.1, 153.9, 153.2, 138.6, 134.7, 130.1, 124.8, 120.3, and then treated with compound I-11 (10 mM) for different
118.3, 103.6, 102.5, 63.1, 61.0, 60.2, 56.3, 56.2, 37.6, 30.5, times. The JC-1 probe was added 20 min aer replacing with
fresh medium. Cells were collected at 2000 rpm, rinsed twice
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with cold PBS and the mitochondrial membrane potential was concentrations were quantied by the Bradford method (BIO-
analysed in the FL-1 channel by ow cytometry (FACS Aria II; RAD) using Multimode varioscan instrument (Thermo Fisher
BD, USA).
Scientics). Equal amounts of protein per lane were applied in
12% SDS polyacrylamide gel for electrophoresis and transferred
to the polyvinylidine diuoride (PVDF) membrane (Amersham
Biosciences). Aer the membrane was blocked at room
temperature for 2 h in blocking solution, primary antibody was
added and incubated at 4 ꢂC overnight. Caspase-9, caspase-3
caspase-12, calpain, caspase-4, CHOP, GRP78, p-PERK and p-
eIF2a antibodies were purchased from Imgenex, USA. Aer
three TBST washes, the membrane was incubated with the
corresponding horseradish peroxidase-labeled secondary anti-
body (1 : 2000) (Santa Cruz) at room temperature for 1 h.
Membranes were washed with TBST three times for 15 min and
the protein blots were detected with chemiluminescence
reagent (Thermo Fischer Scientics Ltd.). The X-ray lms were
developed with developer and xed with xer solution.
4.6 Apoptosis analysis
NCI-H460 cells were seeded at the density of 2 ꢄ 106 cells mLꢃ1
of the DMEM medium with 10% FBS on 6-well plates to the nal
volume of 2 mL. The plates were incubated overnight and then
treated with different concentrations of compound I-11 for 24 h.
Briey, aer treatment with compound I-11 for 24 h, cells were
collected and washed twice with PBS, then resuspended in
Binding Buffer (0.1 M Hepes/NaOH (pH 7.4), 1.4 M NaCl, 25 mM
CaCl2) at a concentration of 1 ꢄ 106 cells mLꢃ1. The cells were
subjected to 5 mL of FITC Annexin V and 5 mL of propidium
iodide (PI) staining using the Annexin-V FITC apoptosis kit (BD,
Pharmingen), followed by the transfer of 100 mL of the solution
to a 5 mL culture tube and incubating for 30 min in the dark at
ꢂ
RT (25 C). The apoptosis ratio was quantied by system so-
5 Statistics
ware (CellQuest; BD Biosciences).
The data were processed by the Student's t-test, with the
signicance level P # 0.05, using SPSS.
4.7 Cell cycle analysis
The NCI-H460 cell line was treated with different concentra-
tions of compound I-11. Aer 48 h of incubation, cells were
washed twice with ice-cold PBS, xed and permeabilized with
ice-cold 70% ethanol at ꢃ20 ꢂC overnight. The cells were treated
Acknowledgements
This study was supported by the National Natural Science
Foundation of China (No. 81260472 21362002 and 21431001),
Special Research Found for the Doctoral Program of Higher
Education (No. 20134504110002), State Key Laboratory for
Chemistry and Molecular Engineering of Medicinal Resources,
Ministry of Science and Technology of China (CMEMR2016-
B06), Project Funded by the Priority Academic Program Devel-
opment of Jiangsu Higher Education Institutions (No.
1107047002).
with 100 mg mLꢃ1 RNase A at 37 C for 30 min aer washing
ꢂ
with ice-cold PBS, and nally stained with 1 mg mLꢃ1 of pro-
pidium iodide (PI) (BD, Pharmingen) in the dark at 4 ꢂC for
30 min. Analysis was performed with the system soware (Cell
Quest; BD Biosciences).
4.8 ROS assay
NCI-H460 cells were seeded into six-well plates and subjected to
various treatments. Cells were collected and washed with PBS
twice, then resuspended in 10 mM of DCFH-DA (Beyotime,
Haimen, China) dissolved in cell free medium at 37 ꢂC for
30 min in the dark, and then washed three times with PBS.
Cellular uorescence was quantied by ow cytometry at an
excitation of 485 nm and an emission of 538 nm.
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4.9 Calcium analysis
NCI-H460 cells were seeded into six-well plates and subjected to
various treatments. The cells were collected and washed with
PBS twice. Intracellular free Ca2+ levels in NCI-H460 cells were
detected using the Ca2+ specic uorescent probe, Fluo-3/AM
ꢂ
(Beyotime, Haimen, China), for 40 min at 37 C in PBS buffer.
Aer loading with the Fluo-3 dye, cells were washed with PBS
solution and cellular uorescence was quantied using ow
cytometry at wavelength of 515 nm.
4.10 Western blot
Total cell lysates from cultured NCI-H460 cells aer compound
I-11 treatments, as mentioned earlier, were obtained by lysing
the cells in ice-cold RIPA buffer with protease and phosphatase
inhibitor, and storing at ꢃ20 ꢂC for future use. The protein
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