Synthesis and antitumor activities of novel dipeptide derivatives derived from dehydroabietic acid

Article abstract of DOI:10.1016/j.bmcl.2014.02.001

A series of dipeptide derivatives from dehydroabietic acid were designed and synthesized as novel antitumor agents. The antitumor activities screening indicated that many compounds showed moderate to high levels of inhibition activities against NCI-H460, HepG2, SK-OV-3, BEL-7404, HeLa and HCT-116 cancer cell lines and that some displayed more potent inhibitory activities than commercial anticancer drug 5-fluorouracil. The mechanism of representative compound 7b was studied by AO/EB staining, Hoechst 33258 staining, JC-1 mitochondrial membrane potential staining, TUNEL assay, DNA ladder assay and flow cytometry, which exhibited that the compound could induce apoptosis in HeLa cells. Further investigation showed that compound 7b induced apoptosis of HeLa cells through a mitochondrial pathway.

Full text of DOI:10.1016/j.bmcl.2014.02.001

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1511–1518
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antitumor activities of novel dipeptide derivatives
derived from dehydroabietic acid
Xiao-Chao Huang a, , Meng Wang a, , Heng-Shan Wang a, Zhen-Feng Chen a, Ye Zhang a,b,
,
⇑
Ying-Ming Pan a,
⇑
a State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry & Pharmaceutical Science of Guangxi
Normal University, Yucai Road 15, Guilin 541004, Guangxi, PR China
b Department of Chemistry & Pharmaceutical Science, Guilin Normal College, Xinyi Road 15, Guangxi 541001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of dipeptide derivatives from dehydroabietic acid were designed and synthesized as novel anti-
tumor agents. The antitumor activities screening indicated that many compounds showed moderate to
high levels of inhibition activities against NCI-H460, HepG2, SK-OV-3, BEL-7404, HeLa and HCT-116 can-
cer cell lines and that some displayed more potent inhibitory activities than commercial anticancer drug
5-fluorouracil. The mechanism of representative compound 7b was studied by AO/EB staining, Hoechst
33258 staining, JC-1 mitochondrial membrane potential staining, TUNEL assay, DNA ladder assay and
flow cytometry, which exhibited that the compound could induce apoptosis in HeLa cells. Further inves-
tigation showed that compound 7b induced apoptosis of HeLa cells through a mitochondrial pathway.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 31 October 2013
Revised 20 January 2014
Accepted 1 February 2014
Available online 10 February 2014
Keywords:
Dehydroabietic acid
Dipeptide
Cytotoxicity
Apoptosis
Cancer is one of the primary causes of death globally, so search-
ing and developing effective anticancer drugs have greatly at-
tracted bioorganic chemists’ interest. After the success of
vinblastine, taxol and their related derivatives as antitumor drugs,
natural products have been seriously considered as a good tradi-
tional source of new antitumor drugs.1–6
of dipeptide derivatives derived from DHA has not been described.
Our present work in this paper is to design and synthesize a
series of DHA-dipeptide derivatives, and to evaluate their in vitro
antitumor activities. Furthermore, the mechanism of apoptotic
effects induced by the representative compound 7b is also
investigated.
As a natural occurring diterpenic resin acid, dehydroabietic acid
(DHA) and its derivatives has been found to have properties of
enhancing the inhibition activity of anticancer drug in various
cells, such as hepatocellular carcinoma cells, cervical carcinoma
cells, and breast cancer cells.7 Our previous study has also demon-
strated that DHA could be a useful tool in the synthesis of drugs
active against some tumour cells.8 Continuing our research pro-
gram on the synthesis of antitumor drugs effective, DHA skeleton
is chosen in the present work as active structural core and some
structural modifications are carried out to explore their antitumor
activities. Dipeptide, an important versatile bioactive moiety,9 is
able to improve the antitumor activity by constructing on a phar-
macy core.10–13 Many dipeptide derivatives have exhibited potent
inhibition activities against human tumors cells.9a,10–12 It is thus to
expect that introducing of dipeptide group on DHA skeleton may
contribute to potential antitumor activity. However, the study on
the synthesis, antitumor activities and apoptosis-inducing effects
DHA-dipeptide derivatives were synthesized as outlined in
Scheme 1. Compound 2 was synthesized by the treatment of phen-
ylalanine 1 with phthalic anhydride in the presence of acetic acid
according to the literature.13 Compound 3 was then obtained by
the condensation of compound 2 and oxalyl chloride, and it was
then treated with series of aromatic primary amines to offer com-
pounds 4. Compounds 5 were synthesized by the treatment of
compounds 4 with hydrazine hydrate in the presence of ethanol
at room temperature. DHA was treated with oxalyl chloride to offer
compound 6. Finally, compounds 7 were acquired by the conden-
sation of compound 6 and compounds 5 in the presence of trieth-
ylamine at room temperature. The structures of DHA-dipeptide
derivatives 7 were confirmed by 1H NMR, 13C NMR and high reso-
lution mass spectrum (HRMS).14
The in vitro antitumor activities of DHA-dipeptide derivatives
7a–7s were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT)9,15 assay against NCI-H460, HepG2,
SK-OV-3, BEL-7404, HeLa and HCT-116 tumor cell lines, with 5-flu-
orouracil (5-FU) as the positive control. The tested results were
shown in Table 1.
⇑
Corresponding authors. Tel./fax: +86 0773 2120958.
E-mail addresses: zhangye81@126.com (Y. Zhang), panym2005@sina.com (Y.-M. Pan).
Co-first author: These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2014.02.001
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

1512
X.-C. Huang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1511–1518
O
O
NH2
i
N
ii
iii
N
COCl
COOH
COOH
O
O
1
2
3
O
O
H2N
N
iv
NH R1
NH R1
O
O
R2
R2
5a-5s
4a-4s
R4
R3
vi
R4
R3
O
NH
NH R1
O
7
R2
v
R4
R3
7a: R1= H, R2 = H, R3 = H, R4 = H
7b: R1= H, R2 = H, R3 = OCH3, R4 = H
7c: R1= H, R2 = H, R3 = F, R4 = H
7d: R1= H, R2 = H, R3 =Cl, R4 = H
7e: R1= Cl, R2 = H, R3 =H, R4 = H
7f: R1= H, R2 = H, R3 =Br, R4 = H
7g: R1= H, R2 = H, R3 = CF3, R4 = H
7h: R1= H, R2 = OCH3, R3 =H, R4 = H
7i: R1= H, R2 = CH3, R3 =H, R4 = H
7j: R1= H, R2 = F, R3 =H, R4 = H
7k: R1= H, R2 = Cl, R3 =H, R4 = H
7l: R1= H, R2 = Br, R3 =H, R4 = H
7m: R1= H, R2 = NO2, R3 =H, R4 = H
7n: R1= H, R2 = CH3, R3 = CH3, R4 = H
7o: R1= H, R2 = Cl, R3 = CH3, R4 = H
7p: R1= H, R2 = Cl, R3 = Cl, R4 = H
7q: R1= H, R2 = Cl, R3 = F, R4 = H
7r: R1= OCH3 , R2 =H, R3 = H, R4 = OCH3
7s: R1= Cl, R2 = H, R3 = Hl, R4 = Cl
O
OH
DHA
O
Cl
6
Scheme 1. Synthetic route to target compounds 7a–7s. Reagents and conditions: (i) phthalic anhydride, CH3COOH, 50 °C; (ii) (COCl)2, CH2Cl2, rt; (iii) aromatic primary
amines, Et3N, CH2Cl2, rt; (iv) NH2NH2.H2O, CH3CH2OH; (v) (COCl)2, CH2Cl2, rt; (vi) Et3N, CH2Cl2, rt.
As shown in Table 1, most of DHA-dipeptide derivatives 7a–7s
displayed much higher inhibitory activity than DHA against NCI-
H460, HepG2, SK-OV-3, BEL-7404, HeLa and HCT-116 cell lines,
indicating the introduction of dipeptide group on DHA should
markedly improve the antitumor activity. Moreover, the substitu-
ent groups at 4-position of phenyl ring (R4) have important influ-
ence on the cytotoxic inhibition and the introduction of electron
donor substituents and halogen groups may result in the increase
of cytotoxic inhibition, while substituent groups at 3-position of
phenyl ring (R3) may lead to the decrease of cytotoxic inhibition.
Table 1 also revealed that, in NCI-H460 cell line assay, all the
compounds exhibited better inhibition than DHA (IC50
80.53 M), and most of compounds (such as 7a–7g, 7i–7m and
7o–7s) even displayed preferable cytotoxic activities than the com-
mercial anticancer drug 5-FU (IC50 = 36.04 M), with IC50 in the
range of 15.21–33.34 M, indicating good inhibition activities of
=
l
l
l
Table 1
Effect of compounds 7a–7s against cell viability of different cell lines
Compounds
IC50(lM)
NCI-H460
HepG2
SK-OV-3
BEL-7404
HeLa
HCT-116
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
28.20 1.42
17.22 1.04
22.83 2.45
15.82 1.43
15.21 1.86
28.68 1.25
28.85 2.36
40.42 3.45
32.35 2.48
26.08 3.21
26.34 2.41
33.34 3.45
32.83 2.65
36.98 3.25
26.10 2.23
26.98 2.46
26.77 2.54
27.44 2.48
15.78 1.75
80.53 2.11
36.04 2.54
27.71 1.64
12.40 0.88
27.10 3.21
26.30 2.43
25.26 2.54
34.64 1.98
21.61 1.43
29.50 2.46
26.90 3.21
23.86 1.87
24.65 1.68
22.89 1.46
25.26 2.23
42.78 4.21
36.90 2.32
20.42 1.88
32.91 2.46
38.07 2.75
23.43 2.66
76.76 3.68
26.98 2.37
27.36 1.57
23.75 1.76
20.07 1.22
26.34 2.34
13.52 1.33
28.68 2.46
36.34 3.54
30.52 2.13
29.84 2.12
33.92 3.14
30.62 2.76
38.80 4.21
20.07 1.23
51.79 4.56
37.82 2.31
33.15 1.27
30.54 2.85
53.75 3.51
21.58 2.18
75.00 2.21
24.43 0.41
16.70 0.56
15.34 0.78
21.01 1.27
33.59 2.52
15.80 2.43
16.90 2.36
36.10 2.38
38.53 3.33
34.56 3.21
27.45 2.87
23.79 2.34
19.95 2.61
27.05 2.15
48.16 3.11
41.16 3.45
44.69 2.46
26.06 2.12
56.34 3.31
30.98 2.88
34.70 2.23
26.80 2.23
18.89 1.22
4.94 0.88
30.33 3.26
15.67 0.76
27.43 1.39
37.79 2.78
25.48 2.65
35.33 2.31
27.38 2.56
35.77 2.45
32.52 3.21
34.76 2.12
45.85 3.15
44.10 3.65
27.43 2.31
32.14 2.41
32.30 3.42
40.85 3.67
39.27 2.55
36.84 2.56
24.10 1.29
35.24 3.26
24.43 0.40
10.84 1.13
12.22 1.56
10.29 0.64
15.20 1.46
10.47 0.96
25.79 1.23
18.19 1.32
13.91 0.58
13.06 1.24
25.13 0.92
12.82 1.02
14.34 1.43
10.11 0.75
28.79 1.46
11.12 0.86
19.26 2.13
14.94 1.35
29.35 2.32
80.48 2.31
7s
DHA
5-FU

X.-C. Huang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1511–1518
1513
these compounds on NCI-H460 cell line. The cytotoxic inhibition
screening results demonstrated that the introduction of dipeptide
moiety on DHA should clearly improve the antitumor activity
against NCI-H460 cell line. Among all the compounds, compound
as control for 24 h. The results were shown in Figure 1. As shown
in Figure 1, the HeLa cells treated with 7b from 12 to 24 h had
obviously changed. The nuclei markedly stained as yellow green
or orange. The phenomenon was associated with cell apoptosis.
Based on the above observation, the cells represented with an
apoptotic morphology. These findings indicated that compounds
7b could induce HeLa cells apoptosis.
Hoechst 33258, which stains the cell nucleus, is a membrane
permeable dye with blue fluorescence. Live cells with uniformly
light blue nuclei are obviously detected under fluorescence micro-
scope after treatment with Hoechst 33258, whereas apoptotic cells
have bright blue nuclei due to karyopyknosis and chromatin con-
densation. However, the nuclei of dead cells could not be stained.
7e exhibited the best cytotoxicity, with IC50 of 15.21
In HepG2 cell line assay, all compounds displayed better cyto-
toxicity than DHA (IC50 = 76.76 M), with IC50 in the range of
12.40–42.78 M, indicating that the introduction of dipeptide
lM.
l
l
group on DHA should obviously improve the antitumor activity
against HepG2 cell line. Moreover, many compounds (such as 7b,
7d, 7e, 7g, 7j–7m, 7p and 7s) even demonstrated better cytotoxic
inhibition than 5-FU, implying favorable inhibition activities of
these compounds. Among these compounds, compound 7b
showed the best inhibition, with IC50 of 12.40
In SK-OV-3 cell line assay, all the compounds displayed better
cytotoxicity than DHA (IC50 = 75.00 M), with IC50 in the range of
13.52–53.75 M, exhibiting that the introduction of dipeptide frag-
ment on DHA could enhanced the antitumor activity against SK-
OV-3 cell line. Moreover, compounds 7b, 7c, 7e, 7m and 7s even
demonstrated better cytotoxic inhibition than 5-FU. Compound
7e displayed the best inhibition on SK-OV-3 cell line among all
l
M.
HeLa cells treated with compound 7b at 10 lM from 12 to 24 h
were stained with Hoechst 33258. HeLa cells not treated with
the 7b were used as control at for 24 h. The results were given in
Figure 2. As shown in Figure 2, HeLa cells not treated with com-
pound 7b were normally blue (in the web version). It was worth
noting that, for 7b treatment, the cells displayed strong blue fluo-
rescence and indicated typical apoptotic morphology after 12 and
24 h. The observation revealed that compounds 7b induced apop-
tosis against HeLa cell line, consistent with the results for AO/EB
double staining.
l
l
the compounds, with IC50 of 13.52 lM.
In BEL-7404 cell line assay, except compounds 7g, 7h, 7n–7p
and 7r, other compounds displayed better cytotoxicity than DHA
(IC50 = 34.70 lM), with IC50 in the range of 15.34–34.56 lM, exhib-
iting that the introduction of dipeptide part on DHA should in-
crease the antitumor activity against BEL-7404 cell line.
Moreover, compounds 7a, 7b, 7c, 7e, 7f, 7k, 7l and 7q even demon-
strated better cytotoxic inhibition than 5-FU, with IC50 in the range
In order to further investigate the apoptosis-inducing effect of
target compound 7b, mitochondrial membrane potential changes
were designed and detected, using the fluorescent probe JC-1. JC-
1, which is a lipophilic cationic dye, can easily pass through the
plasma membrane into cells and accumulates in actively respiring
mitochondria. In the control cells, JC-1 could aggregate in mito-
chondria and present high red fluorescence. However, in cells
undergoing apoptosis, where the mitochondrial potential has col-
lapsed, JC-1 exists in the cytosol as a monomer which emits green
of 15.34–26.06
exhibited the best cytotoxic inhibition, with IC50 of 15.34
In HeLa cell line assay, all the compounds displayed better cyto-
toxicity than DHA (IC50 = 29.35 M), and even preferable than 5-
FU, with IC50 in the range of 4.94–28.79 M, exhibiting that the
l
M. Among these compounds, compound 7b
lM.
l
fluorescence. HeLa cells treated with compound 7b at 10 lM from
l
12 to 24 h were stained with JC-1 and not treated with the com-
pound 7b were used as control for 24 h. The results were shown
in Figure 3. The JC-1 monomer and J-aggregates were excited at
514 and 585 nm, respectively, and light emissions were collected
at 515–545 nm (green) and 570–600 nm (red). For fluorescence
microscopy, Figure 3 showed that cells not treated with the com-
pound 7b were normally red (in the web version), while for 7b
treatment, cells showed strong green fluorescence and indicated
typical apoptotic morphology after 12 and 24 h. Therefore, it could
be concluded that compound 7b induced apoptosis against HeLa
cell line. The results were identical with that of previous experi-
ment of AO/EB double staining and Hoechst 33258 staining.
Terminal-deoxynucleotidyl transferase meditated nick end
labeling (TUNEL) is a common method for identifying apoptotic
cells in situ by detecting DNA fragmentation. When the genomic
DNA is broken, the exposed 30-OH at the end of deoxynucleotide
transfer as the catalytic plus green fluorescent probes fluorescein
(FITC) labeled dUTP, which can be detected by fluorescence
microscopy or flow cytometry, as indicated by a green color. The
results were illustrated in Figure 4. For fluorescence microscopy,
Figure 4 revealed that HeLa cells treated with the compound 7b
at different time appeared in green (in the web version), indicating
that compound 7b significantly induced apoptosis against HeLa
cell line. The results were consistent well with the previous
experiments.
introduction of dipeptide section on DHA should evidently im-
prove the antitumor activity on HeLa cell line. It was important
to note that compound 7b showed the best antitumor activity in
this assay, with IC50 of 4.94 lM.
In HCT-116 cell line assay, except compounds 7d, 7f, 7h, 7k, 7l,
7p, 7q and 7r, other compounds displayed better cytotoxicity than
DHA (IC50 = 35.24 lM), with IC50 in the range of 15.67–34.76 lM,
exhibiting that the introduction of dipeptide segment on DHA
remarkably improve the antitumor activity against HCT-116 cell
line. Moreover, compounds 7b and 7s showed better inhibition
activity than 5-FU, with IC50 of 15.67 and 24.10 lM, respectively.
Apoptosis plays a central role in cancer, since its induction in
cancer cells is critical to a successful therapy.16 It is thus believed
that apoptosis assay may provide important information to preli-
minary investigation of the mode of action. Therefore, in the pres-
ent study, compound 7b, which exhibited good cytotoxic inhibition
in six cancer cell lines and could be used as a good representative
of compounds 7a–7s, was selected and its mechanism of growth
inhibition of HeLa cells was evaluated by acridine orange(AO)/ethi-
dium bromide(EB) staining,17 Hoechst 33258 staining,18 JC-1 mito-
chondrial membrane potential staining,19 TUNEL assay staining20
and flow cytometry.21
AO, which is a vital dye, can stain nuclear DNA across an intact
cell membrane, while EB can only stains cells that have lost their
membrane integrity. Therefore, after synchronous treating with
AO and EB, live cells will be evenly stained as green and early apop-
totic cells will be thickly stained as green yellow or show green
yellow fragments, while late apoptotic cells will be densely stained
as orange or display orange fragments and necrotic cells will be
stained as orange. The cytotoxicity of compound 7b at the concen-
Apoptosis is a fundamental cellular event during development
and is critical for the cytotoxicity induced by radiation or drugs
characterized by the cleavage of chromatin DNA into inter-nucleo-
somal fragments. Fragmentation of chromosomal DNA is the bio-
logical hallmark of apoptosis22 and can be detected by gel
electrophoresis.23 The DNA is cleaved to nucleosome-sized frag-
ments of approximately 180–200 base pairs, which results in the
formation of a ladder like pattern when the DNA is subjected to
tration of 10
lM against HeLa cells from 12 to 24 h was assayed by
AO/EB staining, and HeLa cells not treated with the 7b were used

1514
X.-C. Huang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1511–1518
(a)
(b)
(c)
Figure 1. AO/EB staining of compound 7b in HeLa cells. (a) Not treated with the 7b was used as control for 24 h and (b, c) treatment with compound 7b (10 lM) for 12 and
24 h, respectively.
(a)
(b)
(c)
Figure 2. Hoechst 33258 staining of compound 7b in HeLa cells. (a) Not treated with compound 7b was used as control for 24 h and (b, c) treatment with compound 7b
(10 M) for 12 and 24 h, respectively.
l
(a)
(b)
(c)
Figure 3. JC-1 mitochondrial membrane potential staining of compound 7b in HeLa cells. (a) Not treated with the 7b was used as control for 24 h and (b, c) treatment with
compound 7b (10
lM) for 12 and 24 h, respectively.
(a)
(b)
(c)
Figure 4. TUNEL assay of compound 7b in HeLa cells. (a) Not treated with the 7b were used as control for 24 h and (b, c) treatment with compound 7b (10 lM) for 12 and
24 h, respectively.
electrophoresis in an agarose gel.24 This ladder-like pattern is re-
garded as the biochemical hallmark of apoptosis.25 Herein, we used
agarose gel electrophoresis to detect the DNA fragment. The results
were shown in Figure 5. As shown in Figure 5, compared with the
control (lane 1), the addition of compounds 7b (lanes 2, 3 and 4)
lead to more obvious ladder-like pattern bands, indicating that
compounds 7b could induce apoptosis against HeLa cell lines.
The result was well consistent with that of fluorescence staining
and TUNEL assay.
The apoptosis ratios induced by compound 7b in HeLa tumor
cells were then quantitatively determined by flow cytometry
(Fig. 6). Four quadrant images were observed by flow cytometric
analysis: the Q1 area represented damaged cells appearing in the
process of cell collection, the Q2 region showed necrotic cells

X.-C. Huang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1511–1518
1515
contours. These results showed that compound 7b was able to in-
duce a loss of mitochondrial transmembrane potential and release
of mitochondrial cytochrome c into the cytosol. Therefore, it could
be concluded that the apoptosis induced by compound 7b in HeLa
cells may be through the intrinsic pathway.
Recently, oxidative damage to the mitochondrial membrane
due to increased generation of reactive oxygen species (ROS) has
been demonstrated to play a important role in apoptosis.28–30
Mitochondria has also been implicated as a source of ROS during
apoptosis. Reduced mitochondria membrane potential has recently
been shown to lead to increased generation of ROS and apoptosis.31
We herein studied the loss of mitochondrial transmembrane po-
tential resulting in the generation of ROS by assessing ROS gener-
ation caused by target compound 7b in vitro, using the fluorescent
probe 2,7-dichlorofluorescein diacetate (DCF-DA) determined by
fluorescence microscopy. Treated with compound 7b, cells exhib-
ited stronger fluorescence intensity in cytoplasm, while not treated
with compound 7b, cells under the same experimental procedures
were used as control and the fluorescence detected in these cells
were weak and spread all over the cells. For fluorescence micros-
copy, Figure 8 revealed that HeLa cells treated with the compound
7b appeared in stronger green fluorescence (in the web version),
indicating that compound 7b significantly induced apoptosis
against HeLa cell line. This phenomenon implied that the incre-
ment of ROS might play a role as an early mediator in compound
7b induced apoptosis. These findings pointed to an effect of com-
pound 7b on mitochondrial function and accumulation of ROS.
These features were cues for the induction of apoptosis.
Figure 5. DNA ladder assay. Compound 7b induced apoptosis-associated DNA
fragmentation in HeLa cells. The electrophoretic gel images of total cellular DNA
extracted from compound 7b treated HeLa cells and normal cultural HeLa cells.
HeLa cells were treated with compound 7b at 5, 10 and 15
The lane 1 was a normal cultural HeLa cells as control, and lane 2 was treated with
compound 7b at 5 M; lane 3 was treated with compound 7b at 10 M; lane 4 was
treated with compound 7b at 15 M, respectively.
lM for 24 h, respectively.
l
l
l
It is well-known that an increase of intracellular ROS can lead to
apoptosis. On the other hand, a decrease of ROS can also ruin the
stability of mitochondria, which is followed by a loss of mitochon-
drial membrane potential, release of cytochrome c into cytosol, and
cascade activation of caspases.27,28,30,32 The occurrence of the
apoptotic process should be confirmed by the presence of cas-
pase-3 and -9 (which played a crucial role and was a key element
in the execution phase of apoptosis). The enzyme activities of cas-
pase-3 and -9 were determined by spectrophotometry (Fig. 9).
After incubation with compound 7b for 24 h, the activities of cas-
pase-3 and -9 increased compared with the control. As shown in
Figure 9, the results clearly showed that an induction of cell apop-
tosis took place when the cells were treated with compound 7b,
and the stimulation of caspase-3 and -9 activities both increased
in a time dependent manner from 3 to 24 h.
In this study, a series of novel DHA-dipeptide derivatives were
designed and synthesized, and their cell growth inhibition activi-
ties against the NCI-H460, HepG2, SK-OV-3, BEL-7404, HeLa and
HCT-116 cell lines were evaluated using MTT assay. The in vitro
antitumor activities screening revealed that many compounds
exhibited better inhibition activities than the commercial antican-
cer drug 5-FU. The apoptosis-inducing activity of representative
compound 7b in HeLa cells were investigated by AO/EB staining,
and later period apoptotic cells, the Q3 area exhibited normal cells,
and the early apoptotic cells were located in the Q4 area. Figure 6
revealed that compound 7b could induce apoptosis in HeLa cells.
Apoptosis ratios (including the early and late apoptosis ratios) for
compound 7b were obtained after 12 h of treatment at the concen-
tration of 5 and 10 lM. The apoptosis of HeLa cells treated with
compound 7b increased gradually in a concentration manner.
The apoptosis ratios of compound 7b measured at different con-
centration points were found to 12.6%(5 lM) and 33.7%(10 lM),
respectively, while that of control was 2.3%. The results obviously
illustrated that compound 7b suppressed cell proliferation by
inducing apoptosis.
Mitochondria plays an important role in cell death by changing
its outer and inner membrane permeability and thus leading to
cytochrome c release and caspases activation.26,27 Herein, we dem-
onstrated that compound 7b could disrupt the function of mito-
chondria at the early stage of apoptosis and subsequently
coordinate caspase activation through the release of cytochrome
c. Treatment of HeLa cells was performed with different concentra-
tions of compound 7b for 12 h. As shown in Figure 7, nuclei with
karyopyknosis and conglomeration, a characteristic of apoptosis,
were observed under fluorescence microscopy in cells cultured
with compound 7b, while the control cells appeared with regular
(a)
(b)
(c)
Figure 6. Apoptosis ratio detection of compound 7b by Annexin V/PI assay. (a) Not treated with the 7b was used as control for 12 h and (b, c) treatment with compound 7b at
5 and 10 M for 12 h, respectively.
l

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X.-C. Huang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1511–1518
(a)
(b)
(c)
Figure 7. Induction of cytochrome c release assay of compound 7b in HeLa cells. (a) Not treated with the 7b were used as control for 12 h and (b, c) treatment with compound
7b (5, 10 M) for 12 h, respectively.
l
(a)
(b)
(c)
Figure 8. ROS generation assay of compound 7b in HeLa cells. (a) Not treated with the 7b were used as control for 12 h and (b, c) treatment with compound 7b (5, 10 lM) for
12 h, respectively.
(a)
(b)
Figure 9. Measurement of caspase-3 and -9 activities. Dose-dependent induction of caspase-3 (a) and -9 (b) in human HeLa cell line.
Hoechst 33258 staining, JC-1 mitochondrial membrane potential
staining, TUNEL assay, DNA ladder assay and flow cytometry. Fur-
thermore, compound 7b was found to induce apoptosis in HeLa
cells via the mitochondrial pathway, including a decrease of the
reactive oxygen species level, loss of mitochondrial membrane po-
tential, release of cytochrome c, activation of caspase-9 and cas-
pase-3. The above results demonstrate that the rational design of
DHA-dipeptide derivatives as novel antitumor leads is feasible.
The precise mechanism of action is currently under way.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.02.
001.
References and notes
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Z.-H.; Deng, G.; Ding, J.; Yang, Y.-X.; Feng, H.-J.; Li, Y.-Y. Bioorg. Med. Chem. Lett.
2010, 20, 6217–6221.
8. (a) Huang, X.-C.; Wang, M.; Pan, Y.-M.; Tian, X.-Y.; Wang, H.-S.; Zhang, Y.
Bioorg. Med. Chem. Lett. 2013, 23, 5283; (b) Huang, X.-C.; Wang, M.; Pan, Y.-M.;
Yao, G.-Y.; Wang, H.-S.; Tian, X.-Y.; Qin, J.-K.; Zhang, Y. Eur. J. Med. Chem. 2013,
69, 508.
Acknowledgments
This study was supported by 973 project (No. 2011CB512005,
2012CB723501), the National Natural Science Foundation of China
(No. 81260472 and 21362002), Guangxi Natural Science Founda-
tion of China (2011GXNSFD018010 and No. 2010GXNSFF013001),
the Guilin Scientific Research and Technological Development
Project (No. 20110106-2; 20120108-6), Bagui Scholar project and
the Foundation of Ministry of Education Innovation Team (No.
IRT1225).

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1517
9. (a) Nielsen, P. E. Pseudo-peptides in Drug Discovery; Wiley: New York, 2004; (b)
Leader, B.; Baca, Q. J.; Golan, D. E. Nat. Rev. Drug Disc. 2008, 7, 21; (c) Hancock, R.
E. W.; Scott, M. G. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8856.
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11. Ryppa, C.; Steinberg, H.; Biniossek, M.; Fainaro, R.; Kratz, F. Int. J. Pharm. 2009,
368, 89.
7.31 (s, 1H), 7.27 (d, J = 2.9 Hz, 2H), 7.24 (d, J = 2.1 Hz, 1H), 7.17 (d, J = 11.8 Hz,
2H), 7.14 (s, 1H), 7.04–7.00 (m, 3H), 6.91 (s, 1H), 6.83 (dd, J = 14.3, 3.7 Hz, 2H),
5.31 (s, 1H, NH), 5.04 (dd, J = 14.4, 8.4 Hz, 1H), 3.24–3.06 (m, 2H), 2.88–2.76 (m,
3H), 2.30 (d, J = 1.9 Hz, 1H), 2.10 (d, J = 3.9 Hz, 1H), 1.67–1.50 (m, 7H), 1.31 (s,
3H, CH3), 1.25 (d, J = 4.6 Hz, 6H, 2 ꢀ CH3), 1.23 (s, 3H, CH3). 13C NMR (100 MHz,
CDCl3):
d 179.87, 170.04, 146.74, 145.70, 136.72, 136.52, 134.71, 134.41,
12. Zitzmann, S.; Ehemann, V.; Schwab, M. Cancer Res. 2002, 62, 5139.
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5, 285.
131.74, 129.23, 128.71, 127.13, 126.92, 124.15, 124.04, 123.96, 123.91, 121.59,
55.46, 47.45, 47.35, 45.25, 44.68 , 37.87, 36.96, 33.48, 30.04, 25.15, 24.01,
20.68, 18.58, 16.32. HRMS (m/z) (ESI): calcd for
623.22491; found: 623.22068; Compound 7g: Yields 86.42%; 1H NMR
(400 MHz, CDCl3): 9.17 (s, 1H, NH), 7.33 (d, J = 2.1 Hz, 1H), 7.31 (d,
C
35H41BrN2O2 [M+Na+]:
14. (a) General procedure for the preparation of compounds 7: Compound 2 (1 mmol)
added to dry CH2Cl2 (15 mL) was stirred at 0 °C and oxalyl chloride (1.5 mmol)
was dripped into the mixture and stirred at room temperature for 6 h. After the
reaction, the solvent and excess oxalyl chloride was evaporated under reduced
pressure. Aromatic primary amines (1 mmol) and triethylamine (0.5 mmol)
were added to the mixture and stirred at room temperature for 0.5 h. After the
reaction, the solvent was evaporated under reduced pressure, and the crude
product was purified by chromatography on silica gel eluted with petroleum
ether/ethyl acetate (V:V = 6:1) to offer compound 4. Compound 4 (1 mmol) and
hydrazine hydrate (3 mmol) were added to ethanol (15 mL) and the mixture
was stirred at room temperature for 8 h. After the reaction was completed, the
solvent was evaporated under reduced pressure, and the crude product was
purified by chromatography on silica gel eluted with petroleum ether/ethyl
acetate (V:V = 3:1) to obtain compounds 5. DHA (1 mmol) added to dry CH2Cl2
(15 mL) was stirred at 0 °C and oxalyl chloride (1.5 mmol) was dripped into the
mixture and stirred at room temperature for 6 h. After the reaction, the solvent
and excess oxalyl chloride was evaporated under reduced pressure.
Compounds 5 (1 mmol) and triethylamine (0.5 mmol) were added to the
mixture and stirred at room temperature for 0.5 h. After the reaction, the
solvent was evaporated under reduced pressure, and the crude product was
purified by chromatography on silica gel eluted with petroleum ether/ethyl
acetate (V:V = 6:1) to offer compounds 7a–7s; (b) Experimental: NMR spectra
were recorded on a BRUKER AVANCE 500 NMR spectrometer in CDCl3. Mass
spectra were determined on a FTMS ESI spectrometer. Compound 7a: Yields
92%, 1H NMR (400 MHz, CDCl3): d 8.97 (s, 1H, NH), 7.40 (d, J = 7.5 Hz, 2H), 7.24
(d, J = 2.5 Hz, 3H), 7.21 (d, J = 8.2 Hz, 2H), 7.16 (d, J = 8.2 Hz, 1H), 7.07 (d,
J = 7.4 Hz, 1H), 7.04–6.97 (m, 2H), 6.84 (d, J = 1.4 Hz, 1H), 6.69 (d, J = 7.7 Hz,
1H), 5.06 (dd, J = 14.2, 8.1 Hz, 1H), 3.29–3.12 (m, 2H), 2.86–2.71 (m, 3H), 2.29
(d, J = 12.6 Hz, 1H), 2.13 (d, J = 12.5, 1H), 1.74–1.45 (m, 7H), 1.25 (s, 3H, CH3),
1.23 (d, J = 2.8 Hz, 6H, 2 ꢀ CH3), 1.19 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3): d
179.47, 169.87, 146.82, 145.68, 137.64, 136.83, 134.53, 129.33, 128.80, 128.66,
127.01, 126.89, 124.26, 124.00, 123.87, 120.12, 55.43, 47.40, 45.19, 38.17,
37.88, 37.03, 36.95, 33.48, 29.92, 25.17, 24.02, 20.82, 18.60, 16.39. HRMS (m/z)
d
J = 2.0 Hz, 1H), 7.25 (d, J = 2.3 Hz, 2H), 7.23 (d, J = 2.6 Hz, 2H), 7.15 (d,
J = 3.4 Hz, 2H), 7.08–6.99 (m, 2H), 6.85 (d, J = 1.4 Hz, 1H), 6.65 (d, J = 7.3 Hz,
1H), 5.04 (dd, J = 10.4, 7.4 Hz, 1H), 3.26–3.07 (m, 2H), 2.87–2.69 (m, 3H), 2.29
(d, J = 12.4 Hz, 1H), 2.11 (d, J = 11.7 Hz, 1H), 1.76–1.49 (m, 7H), 1.25 (J = 2.1 Hz,
6H, 2 ꢀ CH3), 1.23 (s, 3H,), 1.19 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3): d
179.61, 169.78, 146.74, 145.81, 136.55, 136.26, 134.38, 129.25, 129.18, 128.76,
128.68, 127.09, 126.88, 123.99, 121.23, 121.09, 55.40, 47.45, 45.21, 38.30,
37.86, 37.02, 33.46, 29.89, 25.14, 24.11, 24.01, 23.94, 20.82, 18.56, 16.35. HRMS
(m/z) (ESI): calcd for C36H41F3N2O2 [M+Na+]: 613.30178; found: 613.29834;
Compound 7h: Yields 85.23%; 1H NMR (400 MHz, CDCl3): d 8.89 (s, 1H, NH),
7.27 (d, J = 3.9 Hz, 2H), 7.16 (s, 1H), 7.14 (d, J = 3.7 Hz, 1H), 7.11 (d, J = 8.1 Hz,
1H), 7.00 (dd, J = 8.1, 1.7 Hz, 1H), 6.91–6.76 (m, 3H), 6.69 (d, J = 7.7 Hz, 1H),
6.63 (dd, J = 8.2, 1.9 Hz, 1H), 5.01 (dd, J = 14.4, 8.1 Hz, 1H), 3.76 (s, 3H, Ph-
OCH3), 3.27–3.09 (m, 2H), 2.85–2.69 (m, 3H), 2.28 (d, J = 12.7 Hz, 1H), 2.09 (d,
J = 12.5 Hz, 1H), 1.72–1.48 (m, 7H), 1.24 (s, 3H, CH3), 1.23 (d, J = 2.8 Hz, 6H,
2 ꢀ CH3), 1.18 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3): d 179.55, 169.87,
159.97, 146.88, 145.68, 138.82, 136.76, 134.54, 129.50, 129.28, 128.72, 127.03,
126.87, 124.06, 123.88, 112.30, 110.42, 105.54 , 55.51, 55.23, 47.41, 45.30,
37.89 (d, J = 9.5 Hz), 36.89, 33.47, 29.93, 25.18, 24.00, 20.76, 18.60, 16.34.
HRMS (m/z) (ESI): calcd for
C
36H44N2O3 [M+Na+]: 575.32496; found:
575.32074; Compound 7i: Yields 86.62%; 1H NMR (400 MHz, CDCl3): d 8.87
(s, 1H, NH), 7.27 (dd, J = 7.8, 2.6 Hz, 2H), 7.23 (d, J = 3.5 Hz, 2H), 7.21 (s, 1H),
7.15 (d, J = 8.2 Hz, 1H), 7.11 (dd, J = 7.2, 4.4 Hz, 2H), 7.00 (dd, J = 8.1, 1.8 Hz, 1H),
6.89 (d, J = 7.5 Hz, 1H), 6.84 (d, J = 1.5 Hz, 1H), 6.67 (d, J = 7.7 Hz, 1H), 5.05 (dd,
J = 14.2, 8.1 Hz, 1H), 3.27–3.12 (m, 2H), 2.85–2.71 (m, 3H), 2.28 (s, 3H, Ph-CH3),
2.20 (d, J = 12.5 Hz, 1H), 2.12 (dd, J = 12.5, 1.8 Hz, 1H), 1.78–1.51 (m, 7H), 1.25
(s, 3H, CH3), 1.24 (d, J = 7.0 Hz, 6H, 2 ꢀ CH3), 1.20 (s, 3H, CH3). 13C NMR
(100 MHz, CDCl3): d 179.45, 169.71, 146.81, 145.69, 138.66, 137.56, 136.81,
134.50, 129.34, 128.67, 128.63, 127.00, 126.86, 125.09, 124.02, 123.89, 120.75,
117.21, 55.41, 47.41, 45.31, 38.33, 37.89, 36.98, 33.45, 29.96, 25.20, 24.01,
21.39, 20.83, 18.62, 16.37. HRMS (m/z) (ESI): calcd for C36H44N2O2 [M+Na+]:
559.33005; found: 559.32629; Compound 7j: Yields 86.37%; 1H NMR
(ESI): calcd for
C
35H42N2O2 [M+Na+]: 545.31440; found: 545.31005;
Compound 7b: Yields 85.56%; 1H NMR (400 MHz, CDCl3): d 8.74 (s, 1H), 7.29
(d, J = 2.2 Hz, 1H), 7.27 (d, J = 2.3 Hz, 2H), 7.26 (d, J = 3.6 Hz, 3H), 7.15 (d,
J = 8.2 Hz, 1H), 7.00 (dd, J = 8.1, 1.7 Hz, 1H), 6.84 (d, J = 1.5 Hz, 1H), 6.75 (d,
J = 9.0 Hz, 2H), 6.66 (d, J = 7.5 Hz, 1H), 5.01 (dd, J = 14.3, 7.8 Hz, 1H), 3.77 (s, 3H,
Ph-OCH3), 3.26–3.12 (m, 2H), 2.86–2.71 (m, 3H), 2.27 (d, J = 12.4 Hz, 1H), 2.15–
2.08 (m, 1H), 1.75–1.44 (m, 7H), 1.24 (d, J = 1.9 Hz, 6H, 2 ꢀ CH3), 1.22 (s, 3H,
CH3), 1.19 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3): d 179.30, 169.49, 156.36,
146.84, 145.68, 136.83, 134.56, 130.71, 129.36, 128.66, 126.99, 126.88, 124.01,
123.87, 121.87, 113.96, 55.43, 55.27, 47.36, 45.20, 38.28, 37.86, 37.02, 36.99,
33.46, 29.96, 25.17, 24.00, 20.83, 18.59, 16.37. HRMS (m/z) (ESI): calcd for
(400 MHz, CDCl3): d 9.20 (s, 1H, NH), 7.37 (d, J = 10.9 Hz, 1H), 7.23 (d,
J = 5.4 Hz, 2H), 7.14 (t, J = 3.1 Hz, 2H), 7.10 (t, J = 5.7 Hz, 1H), 7.03–6.99 (m, 2H),
6.83 (t, J = 5.0 Hz, 1H), 6.80–6.72 (m, 2H), 6.67 (d, J = 7.8 Hz, 1H), 5.06 (dd,
J = 7.9, 2.6 Hz, 1H), 3.2–3.04 (m, 2H), 2.84–2.69 (m, 3H), 2.30 (d, J = 12.6 Hz,
1H), 2.10 (dd, J = 12.5, 1.8 Hz, 1H), 1.77–1.54 (m, 7H), 1.26 (s, 3H, CH3), 1.24 (d,
J = 6.9 Hz, 6H, 2 ꢀ CH3), 1.20 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3): d 179.61,
169.87, 146.73, 145.74, 139.22, 136.57, 134.42, 129.86, 129.75, 129.22, 128.66,
128.44, 127.07, 126.89, 123.99, 123.92, 115.29, 110.98, 55.43, 47.55, 47.48,
45.27, 38.29, 37.86, 37.08, 36.95, 33.45 , 29.86, 25.20, 23.98, 20.79, 18.57,
16.35. HRMS (m/z) (ESI): calcd for C35H41FN2O2 [M+Na+]: 563.30498; found:
563.30137; Compound 7k: Yields 85.54%; 1H NMR (400 MHz, CDCl3): d 9.24 (s,
1H, NH), 7.47 (d, J = 1.9 Hz, 1H), 7.22 (dd, J = 7.4, 2.3 Hz, 2H), 7.16 (d, J = 4.6 Hz,
2H), 7.11–7.07 (m, 1H), 7.03–7.00 (m, 2H), 6.87 (dd, J = 20.5, 5.8 Hz, 2H), 6.80–
6.67 (m, 2H), 5.08 (dd, J = 8.0, 3.1 Hz, 1H), 3.23–3.01 (m, 2H), 2.82 (dd, J = 14.2,
7.3 Hz, 3H), 2.32 (d, J = 7.3 Hz, 1H), 2.09 (dd, J = 12.5, 1.8 Hz, 1H), 1.77–1.59 (m,
7H), 1.27 (s, 3H, CH3), 1.25 (d, J = 2.1 Hz, 6H, 2 ꢀ CH3), 1.22 (s, 3H, CH3). 13C
NMR (100 MHz, CDCl3): d 179.54, 169.87, 146.72, 145.76, 145.74, 138.88,
136.58, 134.41, 134.34, 129.74, 129.22, 128.63, 128.44, 127.07, 126.90, 124.02,
120.11, 117.91, 55.43, 47.45, 46.88, 45.33, 38.55, 38.21, 36.97, 33.48, 29.84,
25.23, 24.01, 21.26, 20.82, 18.58, 16.38. HRMS (m/z) (ESI): calcd for
C
36H44N2O3 [M+Na+]: 575.32496; found: 575.32104; Compound 7c: Yields
83.28%; 1H NMR (400 MHz, CDCl3): d 9.07 (s, 1H, NH), 7.44–7.41 (m, 2H), 7.36–
7.32 (m, 3H), 7.24 (d, J = 8.2 Hz, 1H), 7.10 (dd, J = 8.2, 1.7 Hz, 2H), 6.98 (d,
J = 8.8 Hz, 2H), 6.93 (d, J = 1.5 Hz, 1H), 6.77 (d, J = 7.6 Hz, 1H), 5.10 (dd, J = 14.5,
8.0 Hz, 1H), 3.34–3.20 (m, 2H), 2.97–2.80 (m, 3H), 2.40 (d, J = 1.9 Hz, 1H), 2.20–
2.15 (m, 1H), 1.76–1.61 (dd, J = 32.7, 20.6 Hz, 7H), 1.33 (d, J = 2.1 Hz, 6H,
2 ꢀ CH3), 1.32 (s, 3H, CH3), 1.28 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3): d
179.65, 169.77, 146.76, 145.76, 136.64, 134.47, 133.62, 129.26, 128.73, 127.08,
126.86, 124.15, 124.02, 123.93, 121.81, 121.73, 115.53, 115.31, 55.38, 47.41,
45.20, 37.91, 36.98, 33.46, 29.91, 25.15, 23.99, 20.79, 18.58, 16.33. HRMS (m/z)
(ESI): calcd for
C
35H41FN2O2 [M+Na+]: 563.30498; found: 563.30241;
C
35H41ClN2O2 [M+Na+]: 579.27543; found: 579.27095; Compound 7l: Yields
Compound 7d: Yields 85.42%; 1H NMR (400 MHz, CDCl3): d 9.09 (s, 1H, NH),
7.34–7.31 (m, 2H), 7.23(dd, J = 7.6 Hz, 2H), 7.16 (dd, J = 7.3 Hz, 2H), 7.14 (dd,
J = 5.6 Hz, 2H), 7.04–6.93 (m, 2H), 6.85 (d, J = 1.3 Hz, 1H), 6.62 (d, J = 7.6 Hz,
1H), 5.03 (dd, J = 14.1, 8.1 Hz, 1H), 3.27–3.09 (m, 2H), 2.86–2.70 (m, 3H), 2.29
(d, J = 12.4 Hz, 1H), 2.10 (dd, J = 12.5, 1.8 Hz, 1H), 1.73–1.48 (m, 7H), 1.25 (d,
J = 2.1 Hz, 6H, 2 ꢀ CH3), 1.23 (s, 3H, CH3), 1.19 (s, 3H, CH3). 13C NMR (100 MHz,
84.55%; 1H NMR (400 MHz, CDCl3): d 9.25 (s, 1H, NH), 7.73 (d, J = 1.9 Hz, 1H),
7.33 (d, J = 3.3 Hz, 2H), 7.28 (d, J = 3.1 Hz, 2H), 7.18 (d, J = 8.0 Hz, 1H), 7.11–7.09
(m, 2H), 6.99 (d, J = 7.1 Hz, 2H), 6.94 (d, J = 1.5 Hz, 1H), 6.83 (d, J = 7.7 Hz, 1H),
5.12 (dd, J = 15.3, 7.6 Hz, 1H), 3.44–3.21 (m, 2H), 2.81 (d, J = 6.3 Hz, 3H), 2.40 (d,
J = 1.6 Hz, 1H), 2.15 (d, J = 1.8 Hz, 1H), 1.73–1.62 (m, 7H), 1.39 (s, 3H, CH3), 1.34
(s, 6H, 2 ꢀ CH3), 1.32 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3): d 179.74, 170.04,
146.84, 145.73, 139.01, 136.59, 134.69, 134.47, 130.06, 129.22, 128.69, 127.21,
127.01, 126.69, 124.11, 124.02, 122.95, 122.39, 118.44, 55.50, 49.02, 47.50,
45.23, 44.72, 37.81 , 37.00, 36.89, 33.45, 29.91, 25.11, 23.98, 21.78, 18.60,
16.33. HRMS (m/z) (ESI): calcd for C35H41BrN2O2 [M+Na+]: 623.22491; found:
623.22116; Compound 7m: Yields 89.37%; 1H NMR (400 MHz, CDCl3): d 9.87
(s, 1H, NH), 8.23 (t, J = 2.1 Hz, 1H), 7.44 (d, J = 8.2 Hz, 1H), 7.29 (d, J = 2.0 Hz,
3H), 7.20 (d, J = 2.9 Hz, 2H), 7.13–7.08 (m, 2H), 7.01 (t, J = 5.4 Hz, 1H), 6.94 (d,
J = 1.3 Hz, 1H), 6.86 (dd, J = 11.5, 4.6 Hz, 1H), 5.40 (s, 1H, NH), 5.28 (dd, J = 7.9,
4.5 Hz, 1H), 3.38–3.25 (m, 2H), 2.94–2.81 (m, 3H), 2.41 (d, J = 2.6, 1H), 2.27 (d,
J = 2.3 Hz, 1H), 1.87–1.72 (m, 7H), 1.40 (s, 3H), 1.34 (d, J = 2.3 Hz, 6H, 2 ꢀ CH3),
1.31 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3): d 179.89, 170.29, 148.33, 146.69,
145.85, 139.00, 136.41, 134.28, 129.48, 129.15, 128.64, 128.50, 127.12, 126.88,
125.41, 124.02, 118.61, 114.70, 55.50, 47.68, 47.60, 45.47, 37.31, 37.00, 33.39,
29.86, 25.28, 25.15, 24.00, 20.87, 18.58, 16.37. HRMS (m/z) (ESI): calcd for
CDCl3):
d 179.58, 169.70, 146.71, 145.81, 136.54, 136.24, 134.36, 129.24,
128.76, 128.68, 128.39, 127.08, 126.87, 123.98, 121.21, 121.06, 55.39, 47.45,
45.22, 38.24, 37.85, 37.61, 37.10, 36.92, 33.45, 29.88, 25.13, 23.98, 20.81, 18.55,
16.34. HRMS (m/z) (ESI): calcd for C35H41ClN2O2 [M+Na+]: 579.27543; found:
579.27190; Compound 7e: Yields 87.58%; 1H NMR (400 MHz, CDCl3): d 8.49 (s,
1H, NH), 8.33 (d, J = 8.3 Hz, 1H), 7.35–7.33 (m, 1H), 7.33–2.27(m, 3H), 7.25 (t,
J = 4.1 Hz, 2H), 7.15 (dd, J = 8.2, 4.6 Hz, 1H), 7.05 (dd, J = 7.7, 4.1, Hz, 1H), 7.02–
6.96 (m, 1H), 6.84 (s, 1H), 6.37 (d, J = 12.5 Hz, 1H), 4.95 (t, J = 7.3 Hz, 1H), 3.34–
3.12 (m, 2H), 2.88–2.65 (m, 3H), 2.28 (dd, J = 8.2, 4.9 Hz, 1H), 2.12–2.04 (m,
1H), 1.76–1.39 (m, 7H), 1.25 (s, 3H, CH3), 1.22 (d, J = 2.6 Hz, 6H, 2 ꢀ CH3), 1.20
(s, 3H, CH3). 13C NMR (100 MHz, CDCl3): d 179.25, 169.66, 146.77, 145.71,
136.54, 136.32, 134.57, 134.37, 129.21, 129.15, 128.91, 128.84, 127.53, 127.19,
126.88, 126.82, 124.91, 123.94, 123.83, 121.81, 55.38, 47.43, 45.34, 45.16,
37.82, 37.53, 36.95, 33.45, 29.68, 25.15, 23.97, 21.23, 20.70, 18.59, 16.32. HRMS
(m/z) (ESI): calcd for C35H41ClN2O2 [M+Na+]: 579.27543; found: 579.27102;
Compound 7f: Yields 84.21%; 1H NMR (400 MHz, CDCl3): d 9.20 (s, 1H, NH),
C
35H41N3O4 [M+Na+]: 590.29948; found: 590.29563; Compound 7n: Yields
85.49%; 1H NMR (400 MHz, CDCl3): d 8.83 (s, 1H, NH), 7.37ꢁ7.35 (m, 4H), 7.25

1518
X.-C. Huang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1511–1518
(d, J = 8.6 Hz, 3H), 7.08 (d, J = 9.2 Hz, 2H), 6.94 (d, J = 1.2 Hz, 1H), 6.77 (d,
16. Bunz, F. Curr. Opin. Pharmacol. 2001, 1, 337.
J = 7.7 Hz, 1H), 5.40 (s, 1H, NH), 5.12 (dd, J = 14.3, 8.0 Hz, 1H), 3.36ꢁ3.26 (m,
2H), 2.95–2.80 (m, 3H), 2.40 (d, J = 2.8 Hz, 1H), 2.30 (s, 3H, CH3-Ph), 2.27 (s, 3H,
CH3-Ph), 2.20 (d, J = 3.2 Hz, 1H), 1.89–1.58 (m, 7H), 1.34 (d, J = 2.1 Hz, 6H,
2 ꢀ CH3), 1.32 (s, 3H, CH3), 1.29 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3): d
179.28, 169.55, 146.83, 145.67, 136.98, 136.88, 135.33, 134.58, 132.57, 129.78,
129.37, 128.65, 126.97, 126.88, 124.03, 123.86, 121.47, 117.67, 55.36, 53.46,
47.39, 45.27, 38.29, 37.88, 36.99, 33.47, 29.99, 25.21, 24.01, 20.84, 19.77, 19.18,
18.62, 16.40. HRMS (m/z) (ESI): calcd for C37H46N2O2 [M+Na+]: 573.34570;
found: 573.34132; Compound 7o: Yields 85.52%; 1H NMR (400 MHz, CDCl3): d
9.20 (s, 1H, NH), 7.69 (d, J = 2.1 Hz, 1H), 7.29 (d, J = 5.6 Hz, 2H), 7.24 (d,
J = 6.7 Hz, 2H), 7.15 (d, J = 10.6 Hz, 1H), 7.13–7.07 (m, 2H), 7.00–6.95 (m, 1H),
6.90 (s, 1H), 6.80 (d, J = 7.8 Hz, 1H), 5.14 (dd, J = 14.1, 8.1 Hz, 1H), 3.29–3.08 (m,
2H), 2.96–2.74 (m, 4H), 2.38 (s, 3H, Ph-CH3), 2.23(d, J = 10.8 Hz, 1H), 1.81–1.52
17. General procedure for AO/EB staining: cells were seeded at a concentration of
5 ꢀ 104 cell/ mL in a volume of 2 mL on a sterile cover slip in six-well tissue
culture plates. Following incubation, the medium was removed and replaced
with fresh medium plus 10% fetal bovine serum and supplemented with
compound 7c (5
lM). After the treatment period, the cover slip with
monolayer cells was inverted on a glass slide with 20
lL of AO/EB stain
(100 mg/mL). Fluorescence was read on an Nikon ECLIPSETE2000-S
fluorescence microscope (OLYMPUS Co., Japan).
18. General procedure for Hoechst 33258 staining: cells grown on a sterile cover slip
in six-well tissue culture plates were treated with compounds for a certain
range of time. The culture medium containing compounds was removed, and
the cells were fixed in 4% paraformaldehyde for 10 min. After being washed
twice with PBS, the cells were stained with 0.5 mL of Hoechst 33258
(Beyotime) for 5 min and then again washed twice with PBS. The stained
nuclei were observed under an Nikon ECLIPSETE2000-S fluorescence
microscope using 350 nm excitation and 460 nm emission.
(m, 7H), 1.37 (s, 3H, CH3), 1.32 (d, J = 4.4 Hz, 6H, 2 ꢀ CH3), 1.30 (s, 3H, CH3). 13
C
NMR (100 MHz, CDCl3): d 179.45, 169.59, 146.75, 145.65, 136.52 , 134.37,
131.64, 130.67, 129.26, 129.17, 128.61, 128.55, 127.04, 126.89, 123.92, 120.69,
120.44, 118.14, 55.33, 54.96, 47.47, 38.39, 37.86, 36.99, 29.77, 25.17, 23.95,
21.19, 19.41, 18.59, 16.36. HRMS (m/z) (ESI): calcd for C36H43ClN2O2 [M+Na+]:
593.29108; found: 593.28748; Compound 7p: Yields 86.76%; 1H NMR
19. General procedure for mitochondrial membrane potential staining: JC-1 probe was
employed to measure mitochondrial depolarization in NCI-H460 cells. Briefly,
cells cultured in six-well plates after indicated treatments were incubated with
(400 MHz, CDCl3):
d
9.47 (s, 1H, NH), 7.67 (d, J = 2.1 Hz, 1H), 7.34 (d,
an equal volume of JC-1 staining solution (5 lg/ml) at 37 °C for 20 min and
J = 7.8 Hz, 2H), 7.29 (d, J = 5.6 Hz, 2H), 7.24 (s, 1H), 7.12–7.08 (m, 2H), 7.01–
6.93 (m, 2H), 6.72 (d, J = 7.6 Hz, 1H), 5.11 (dd, J = 6.7, 2.3 Hz, 1H), 3.36–3.12 (m,
2H), 2.97–2.80 (m, 3H), 2.81 (d, J = 6.6 Hz, 1H), 2.40 (d, J = 10.8 Hz, 1H), 1.82–
1.65 (m, 7H), 1.35 (s, 3H, CH3), 1.33 (d, J = 4.2 Hz, 6H, 2 ꢀ CH3), 1.29 (s, 3H,
CH3). 13C NMR (100 MHz, CDCl3): d 180.07, 169.91, 146.56, 145.72, 137.26,
136.42, 134.35, 132.49, 130.22, 129.13, 128.74, 127.28, 127.15, 127.03, 126.87,
123.99, 121.51, 119.01, 60.20, 55.55, 47.42, 45.39, 42.16, 37.98, 37.87, 37.12,
36.98, 33.44, 29.88, 25.13, 23.96, 20.82, 18.48, 16.34. HRMS (m/z) (ESI): calcd
for C35H40Cl2N2O2 [M+Na+]: 613.23645; found: 613.23269; Compound 7q:
Yields 85.58%; 1H NMR (400 MHz, CDCl3): d 9.33 (s, 1H, NH), 7.51 (dd, J = 6.6,
2.6 Hz, 1H), 7.25 (d, J = 7.3 Hz, 2H), 7.21–7.19 (m, 2H), 7.14 (s, 1H), 7.03–6.98
(m, 2H), 6.94 (d, J = 8.8 Hz, 1H), 6.85 (d, J = 1.4 Hz, 1H), 6.67 (d, J = 7.7 Hz, 1H),
5.05 (dd, J = 8.9, 5.7 Hz, 1H), 3.24–3.02 (m, 2H), 2.87–2.67 (m, 3H), 2.31 (d,
J = 11.4 Hz, 1H), 2.09 (dd, J = 12.7, 2.0 Hz, 1H), 1.73–1.52 (m, 7H), 1.27 (s, 3H,
CH3), 1.24 (d, J = 4.9 Hz, 6H, 2 ꢀ CH3), 1.20 (s, 3H, CH3). 13C NMR (100 MHz,
rinsed twice with PBS. Mitochondrial membrane potentials were monitored by
determining the relative amounts of dual emissions from mitochondrial JC-1
monomers or aggregates using an Nikon ECLIPSETE2000-S fluorescent
microscope. Mitochondrial depolarization is indicated by an increase in the
green/red fluorescence intensity ratio.
20. The TUNEL method was performed to label 30-end of fragmented DNA of the
apoptotic HeLa cells. The cells treated as indicated were fixed with 4% paraform
phosphate buffer saline, rinsed with PBS, then permeabilized by 0.1% Triton X-
100 for FITC end-labeling the fragmented DNA of the apoptotic HeLa cells using
TUNEL cell apoptosis detection kit. The FITC-labeled TUNEL-positive cells were
imaged under
a fluorescent microscopy by using 488 nm excitation and
530 nm emission.
21. (a) General procedure apoptosis ratios: Prepared NCI-H460 cells (1 ꢀ 106 cells/
mL) were washed twice with cold PBS and then resuspended gently in 500 lL
of binding buffer. Thereafter, cells were stained in 5
lL of Annexin V-FITC and
CDCl3):
d
179.78, 169.88, 146.67, 145.85, 136.49, 134.45, 134.35, 129.18,
shaken well. Finally, the cells were mixed with 5 L of PI, incubated for 20 min
l
128.67, 128.42, 127.10, 126.89, 124.01, 122.09, 119.53, 119.47, 116.38, 116.11,
55.46, 54.73, 47.50, 45.36, 42.24, 38.57 37.90, 37.17, 36.69, 33.46, 29.88, 25.14,
in the dark and subsequently analyzed using FACSCali-bur (Becton Dickinson).;
(b) General procedure cell cycle: NCI-H460 cell lines were maintained in
Dulbecco’s modified Eagle’s medium with 10% foetal calf serum in 5% CO2 at
37 °C. Cells were harvested by trypsinization and rinsed with PBS. After
centrifugation, the pellet (105–106 cells) was suspended in 1 mL of PBS and
kept on ice for 5 min. The cell suspensionwas then fixed by the dropwise
addition of 9 mL precooled (4 °C) 75% ethanol with violent shaking. Fixed
samples were kept at 4 °C until use. For staining, cells were centrifuged,
23.95, 20.85, 18.57, 16.38. HRMS (m/z) (ESI): calcd for
C35H40FCl2N2O2
[M+Na+]: 597.26600; found: 597.262089; Compound 7r: Yields 86.36%; 1H
NMR (400 MHz, CDCl3): d 8.24 (s, 1H, NH), 8.08 (d, J = 3.0 Hz, 1H), 7.31 (s, 1H),
7.27 (d, J = 2.6 Hz, 2H), 7.15 (d, J = 8.2 Hz, 2H), 6.76 (dd, J = 8.9, 3.0 Hz, 2H), 6.59
(dd, J = 8.9, 3.5 Hz, 2H), 6.42 (d, J = 7.4 Hz, 1H), 4.90 (d, J = 7.2 Hz, 1H), 3.80 (s,
3H, Ph-OCH3), 3.71 (s, 3H, Ph-OCH3), 3.22–3.17 (m, 2H), 2.84–2.73 (m, 3H),
2.29 (d, J = 12.4 Hz, 1H), 2.12–2.09 (m, 1H), 1.77–1.56 (m, 7H), 1.24 (s, 3H, CH3),
1.23 (d, J = 1.9 Hz, 6H, 2 ꢀ CH3), 1.20 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3): d
178.68, 169.22, 153.73, 146.81, 145.66, 142.31, 136.69, 134.66, 129.28, 128.77,
127.80, 127.02, 123.99, 123.82, 110.83, 108.82, 106.03, 56.17, 55.78, 55.39,
47.37, 45.15, 38.02, 37.87, 36.98, 33.46, 29.92, 25.16, 23.99, 20.75, 18.66, 16.36.
resuspended in PBS, digested with 500
lL of RNase A (250 mg/mL), and treated
with 25 L of propidium iodide (PI) (0.15 mM), then incubated for 30 min at
l
4 °C. PI-positive cells were counted with a FACScan Fluorescence-activated cell
sorter (FACS). The population of cells in each cell-cycle phase was determined
using Cell Modi FIT software (Becton Dickinson).; (c) Tong, G.; Poot, M.; Hu, D.;
Oda, D. Oral Oncol. 2000, 36, 236.
HRMS (m/z) (ESI): calcd for
C
37H46N2O4 [M+Na+]: 605.33553; found:
605.33052; Compound 7s: Yields 85.37%; 1H NMR (400 MHz, CDCl3): d 8.59
(s, 1H, NH), 8.46 (d, J = 1.9 Hz, 1H), 7.33 (d, J = 0.6 Hz, 1H), 7.28 (d, J = 1.9 Hz,
2H), 7.27 (d, J = 2.3 Hz, 2H), 7.14 (d, J = 3.2 Hz, 1H), 7.05–7.03 (m, 1H), 6.99 (d,
J = 6.1 Hz, 1H), 6.84 (d, J = 1.3 Hz, 2H), 6.34 (d, J = 7.3 Hz, 1H), 4.90 (dd, J = 7.0,
2.2 Hz, 1H), 3.14 (dd, J = 14.2, 8.3 Hz, 2H), 2.85–2.67 (m, 3H), 2.29 (d, J = 5.4 Hz,
1H), 2.08 (d, J = 2.2 Hz, 1H), 1.54–1.31 (m, 7H), 1.24 (s, 3H, CH3), 1.20 (d,
J = 3.0 Hz, 6H, 2 ꢀ CH3), 1.17 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3): d 179.41,
169.85, 146.70, 145.73, 136.36, 136.14, 135.28, 134.49, 133.30, 129.78, 129.17,
129.10, 128.91, 127.27, 126.88, 124.73, 123.96, 121.41, 55.39, 47.43, 45.17,
37.80, 37.18, 36.97, 33.46, 29.65, 25.17, 23.99, 21.25, 20.63, 18.60, 16.36. HRMS
(m/z) (ESI): calcd for C35H40Cl2N2O2 [M+Na+]: 613.23645; found: 613.23241.
15. General procedure for cytotoxic evaluation in vitro: NCI-H460, HepG2, SK-OV-3,
BEL-7404, HeLa and HCT-116 cells were seeded into 96-well microculture
plates and allowed to adhere for 24 h, respectively. After cells were exposed to
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aspirated and replenished with complete medium. IC50 was evaluated by MTT
tetrazolium dye assay. Each experiment was performed three times; (c)
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thrice, and all experiments yielded similar results. Measurements from all the
replicates were combined, and treatment effects were analyzed.
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