Synthesis, DNA-binding abilities and anticancer activities of triazole-pyrrolo[2,1-c][1,4]benzodiazepines hybrid scaffolds

Article abstract of DOI:10.1016/j.bmcl.2013.10.004

We synthesized a new series of PBD-hybrid derivatives having tethered triazoles and investigated for their cytotoxicity. The studies indicated that cis-olefin compounds induce higher cytotoxicity with increase in the G1 cell cycle phase compared with the trans-compounds. Quantitative RT-PCR assay indicated that compounds (16a-d) induced G1 phase arrest through down-regulation of cyclin D1 and up-regulation of p21, p27, and p53 mRNA expressions. Compounds 16a-d induced A375 early apoptosis as detected by flow cytometry after double-staining with annexin V and propidium iodide. Moreover, the Western blot analysis showed that A375 treated by compounds (16a-d) resulted in decreased levels of Bcl-2 and Bcl-xL, increased levels of Bax and Bad, and caspase/PARP degradation to identify apoptotic cells.

Full text of DOI:10.1016/j.bmcl.2013.10.004

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6854–6859
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, DNA-binding abilities and anticancer activities of
triazole-pyrrolo[2,1-c][1,4]benzodiazepines hybrid scaffolds
c
Chung-Yu Chen b, Pei-Hua Lee a, Yong-Yong Lin c, Wen-Ting Yu a, Wan-Ping Hu c, , Chia-Chen Hsu ,
⇑
Ying-Ting Lin c, Long-Sen Chang d, Cheng-Tien Hsiao a, Jeh-Jeng Wang a, , Mei-Ing Chung b,
⇑
⇑
a Department of Medicinal and Applied Chemistry, College of Life Sciences, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
b School of Pharmacy, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
c Department of Biotechnology, College of Life Sciences, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
d Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung City, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized a new series of PBD-hybrid derivatives having tethered triazoles and investigated for
their cytotoxicity. The studies indicated that cis-olefin compounds induce higher cytotoxicity with
increase in the G1 cell cycle phase compared with the trans-compounds. Quantitative RT-PCR assay
indicated that compounds (16a–d) induced G1 phase arrest through down-regulation of cyclin D1 and
up-regulation of p21, p27, and p53 mRNA expressions. Compounds 16a–d induced A375 early apoptosis
as detected by flow cytometry after double-staining with annexin V and propidium iodide. Moreover, the
Western blot analysis showed that A375 treated by compounds (16a–d) resulted in decreased levels of
Bcl-2 and Bcl-xL, increased levels of Bax and Bad, and caspase/PARP degradation to identify apoptotic
cells.
Received 28 May 2013
Revised 22 September 2013
Accepted 2 October 2013
Available online 10 October 2013
Keywords:
Pyrrolo[2,1-c][1,4]benzodiazepine
Triazole
Apoptosis
Cytotoxicity
Ó 2013 Elsevier Ltd. All rights reserved.
Anticancer
Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) was isolated from
Streptomyces species and are known for their unique characteris-
tics to interact with DNA in minor groove.1 However, there are only
a few DNA-interactive agents that bind to DNA with high sequence
selectivity. Therefore, the development of small molecular DNA-
interactive agents with highly sequence-selective DNA-interactive
properties is considered to be one of the most important tasks. The
conjugate agents with active moieties of known antitumor and
antiviral agents are being designed and synthesized to provide
highly sequence-selective DNA-interactive properties and antitu-
mor activity.2 Based on above reason, a series of hybrid agents
PBD-indole was designed and synthesized to provide highly
sequence-selective DNA-interactive properties and antitumor
activity.3 Amongst them two compounds In-4C-PBD3a and In-6C-
PBD3b showed prominent selectivity against cancer cell lines
(Fig. 1A).
O
N
O
H
N
O
n
H
(A)
(B)
n
NH
N
O
N
O
n=0,3-6
O
O
n=1,3-6
n=4, In-4C-PBD
n=6, In-6C-PBD
Figure 1. PBD–indole and PBD–enediyne hybrid agent.
encouraged us to design and synthesize a diversity of PBD–
enediyne conjugate agents,6 which also shown good selectivity
activity against various cancer cell lines (Fig. 1B).
A vast number of pharmaceutical drugs and natural products
have been engineered to synthesize the triazole systems with sub-
stituent at the 1- and 4-positions.7 Most of them showed antimi-
crobial,8 anti-inflammatory,9 anticonvulsant,10 and anticancer
activities.11 In addition, there are numerous reports available on
the biological applications of such ring systems12 and descriptions
of their application in material chemistry.13 In the past few years,
the triazole moiety incorporated into natural and synthetic anti-
cancer agents such as 1,2,3-triazole-linked pyrrolobenzodiaze-
pine14a and 3-phenylpyrazolopyrimidine-1,2,3-triazole14b shows
Both of these two compounds have great significance in
developing potent anti-tumor agent that could be potentially
applied for both inhibiting melanoma cells and preventing mela-
noma metastasis.3a,b Moreover, the enediyne contains either DNA
intercalating groups4 or DNA minor groove binding functions5
⇑
Corresponding authors. Fax: +886 7 312 5339 (J.-J.W.).
E-mail addresses: wphu@kmu.edu.tw (W.-P. Hu), jjwang@kmu.edu.tw (J.-J.
Wang).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.10.004

C.-Y. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6854–6859
6855
N
N
N
MeO2C
N
O
N
N
O
H
3-5
N
R
MeO2C
H2N
N
N
O
N
N
N
N
N
N
O
MeO2C
CO2Me
R=OHCH2CH2CH2
3-phenylpyrazolopyrimidine-1,2,3-triazole
1,2,3-triazole-linked pyrrolobenzodiazepine
Figure 2. The antitumor agents of triazoles incorporated derivatives.
the corresponding E- and Z-PBD-triazole hybrid agents 16a–d
and 17a–d, respectively (Scheme 2).
After preparing the new series of PBD-triazole derivatives, we
evaluated the cytotoxicity of the PBD-triazoles analogues (16a–d
and 17a–d) in various human or mouse cells by MTT assay. As
shown in Table 1, cis-form agents (16a–d) were potently cytotoxic
in sensitive melanoma (A375, A2058, B16) and Ca9-22 oral cell
lines compared to that of trans-form agents (17a–d).
There was no significant cytotoxic effect in human dermal fibro-
blast cells. Because the agents 16a–d exhibited a higher inhibitory
activity on A375 cells compared to other cell lines at a concentra-
Figure 3. PBD–triazole derivatives.
potent cytotoxicity (Fig. 2). Herein, we report a new series of PBD-
triazole analogues via CuAAC reaction (Fig. 3), and verify whether
hybrid agents induced antiproliferation, leading to cell growth
cycle perturbation, induced G1 phase arrest through down-regula-
tion of cyclin D1 and up-regulation of p21, p27, and p53 mRNA
expressions and subsequent apoptotic cell death.
The synthesis of 2 (DC81) was carried out by our previously re-
ported method15 via 6 steps starting from 4-(benzyloxy)-5-meth-
oxy-2-nitrobenzoic acid (1, Scheme 1). The reaction of 2 with
commercially available reagents trans-1,4-dibromobutene and
cis-1,4-dichlorobutene in ethylmethylketone (MEK) and in the
presence of potassium carbonate formed compound 3 and 4,
respectively. Compounds 3 and 4 reacted with sodium azide and
catalytic amount of potassium iodide in methanol to give the cor-
responding PBD azide derivatives 5 and 6.
tion of 5
studies. To investigate the most effective time in this study, A375
cells were treated with 0, 1, 3, and 5 M of 16a–d and 17a–d for
lM, the A375 cells were selected as a model for further
l
0, 12, and 24 h. There was no significant cytotoxic effect against
A375 cells for 12 h (see Supplementary data). cis-Form agents in-
duced cytotoxic death of A375 cells in a dose- and time-dependent
manner. Compound 16a exhibited a higher inhibitory activity com-
pared to that of other agents on A375 cells. Based on the cytoxicity
data, IC50 were determined and shown in a subset table in Figure 4.
The triazoles moiety 21 was synthesized according to the re-
ported procedure (Scheme 3).17 In order to confirm whether conju-
gate agent is more effective as an antiproliferative agent than 2
(DC-81), 21 (triazoles moiety) or a combination of 2 and 21, we
used conjugate agent 16a for this issue. Our data indicate that
compound 16a exhibited a higher inhibitory activity compared to
that of other agents on A375 cells (Fig. 5).
The terminal alkyne derivatives 13–15 were synthesized from
the aryl iodides 7–9 as reported.16 Finally, the azide compounds
5 and 6 were individually subjected to CuAAC protocol to obtain
Meikrantz and Schlegel reported that the control of cell death is
linked to the cell cycle.18 Cells with a defective cell cycle are more
HO
O
NO2
OH
N
N
O
O
HO
O
i
H
H
Br
ii
N
N
O
1
O
O
2
3
N
O
H
N3
iii
N
O
O
5
HO
O
NO2
N
N
HO
O
O
H
H
i
ii
OH
N
N
Cl
O
O
O
O
1
2
4
N
O
O
H
iii
N
N3
O
6
Scheme 1. Syntheses of E/Z-PBD-azides. Reagents and conditions: (i) construction of PBD scaffold via 6 steps, Ref. 15; (ii) trans-1,4-dibromobutene or cis-1,4-dichlorobutene,
K2CO3, ethylmethylketone (EMK), reflux, 4 h, 94–95%; (iii) NaN3, KI, MeOH, rt, 12 h, 90–91%.

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C.-Y. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6854–6859
I
R
i
ii
TMS
R
R
10-12
7-9
R:2-OMe,3-OMe,4-OMe
13-15
R:2-OMe,3-OMe,4-OMe
R:2-OMe,3-OMe,4-OMe
N
O
O
R
H
N
O
H
N
N
iii
N
N
N
N3
O
13-15,20
R:H,2-OMe,3-OMe,4-OMe
O
O
6
R
R=H
16a, 62%
R=2-OMe 16b, 68%
R=3-OMe 16c, 68%
R=4-OMe 16d, 70%
N
O
O
N
H
N
O
O
N
H
N
N3
R
iii
N
N
O
5
13-15,20
R:H,2-OMe,3-OMe,4-OMe
O
R=H
17a, 65%
R
R=2-OMe 17b, 67%
R=3-OMe 17c, 70%
R=4-OMe 17d, 70%
Scheme 2. Synthesis of PBD-triazole and presynthesis for diversity of phenylalkynes. Reagents and conditions: (i) Pd(PPh3)4, CuI, Et3N, 90 °C, 6–12 h; (ii) K2CO3, THF, rt, 12 h;
(iii) 13–15 and 20, CuI, DMF:THF = 1:4, rt, 2 h, 65–70%.
Table 1
Cytotoxic activities of compounds in different human cells and mouse cell linea
Survival (%control)
Compound (5 lM)
DC81
16a
16b
58.4 0.2
16c
48.4 0.2
16d
17a
17b
58.4 0.2
17c
58.3 0.2
17d
69.2 0.3
A375 (melanoma)
RPMI7951 (melanoma)
A2058 (melanoma)
A549 (lung)
293T (kidney)
Ca9-22 (oral)
52.2 0.5
61.6 0.1
54.8 0.2
85.4 0.3
55.2 0.1
94.4 0.1
66.8 0.2
89.7 0.3
22.4 0.1
55.9 0.1
45.7 0.7
65.6 1.0
74.1 0.7
25.6 2.4
33.9 3.8
87.7 1.2
38.7 0.2
66.4 3.2
42.0 2.1
98.2 0.9
46.7 1.7
55.5 0.7
62.8 1.8
90.3 4.8
69.6 0.2
79.1 1.4
53.3 2.1
70.0 1.4
78.0 1.3
61.2 1.3
33.9 3.8
93.0 2.0
75.1 1.1
63.1 1.7
102.5 0.6
51.1 5.1
104.8 2.4
66.3 1.6
84.7 8.1
65.3 2.1
40.7 1.2
100.2 0.9
42.8 2.5
50.0 0.4
74.2 0.9
90.4 4.5
83 0.7
57.9 2.1
91.0 0.5
55.0 1.7
95.6 0.9
98.9 1.4
100.0 6.7
87.2 0.2
62.8 3.3
86.0 1.7
40.0 1.9
97.5 4.3
107.2 2.6
107.0 5.9
80.1 1.1
49.2 3.1
81.0 1.2
40.0 0.5
84.1 2.0
75.2 4.6
104.0 3.3
B16 (mouse melanoma)
Fibroblast
a
Cells were cultured with agents at a concentration of 5 lM for 24 h before growth and viability were assessed using the MTT assay.
vulnerable to some anticancer agents according to numerous pre-
clinical studies.19 Compounds manipulating the cell cycle may be
able to promote growth arrest, inhibit differentiation and induce
apoptosis. To verify whether cell damage might be attribute to
the cell cycle program or might have become arrested at any cell
cycle phase by PBD-triazole-induced apoptosis in A375 cells, the
DNA content of cell nuclei was measured by flow cytometric anal-
CDK activity, and p21 function as a sensor of cytostatic signals and
is mainly regulated by the p53 tumor suppressor gene.23 In our
study, we observed that PBD-triazoles caused cell cycle G1 arrest.
To further evidence the expression of possible G1 cell cycle-associ-
ated regulators, four genes (p53, p21, p27, and cyclin-D1) were de-
tected by quantitative RT-PCR (qPCR) experiments. A375 cells were
treated with agents at a concentration of 5 lM for 24 h. As shown
ysis. As shown in Table 2, cells-treated with 5
l
M PBD-triazole for
in Figure 7, we observed that 16a–d-induced G1 cell cycle arrest in
A375 cells might be through induction of p53, p21, and p27 fol-
lowed by a decrease in the expression of cyclin D1.
24 h significantly increased cell percentage at G1 phase and con-
comitantly, decreased cell populations at S and G2/M phase. In
addition, cis-form agents (16a–d) induced more G1 cell cycle arrest
in A375 cells than did trans-form agents (17a–d).
To investigate if the antiproliferative effect of 16a was a conse-
quence of DNA intercalation, we performed an in vitro DNA
unwinding assay. DNA intercalators enhance the unwinding of
negatively supercoiled DNA, which leads to a decreased mobility
in agarose gel during electrophoresis.20 As shown in Figure 6, it
Apoptosis is an important phenomenon in cytotoxicity induced
by anticancer drugs. The execution of apoptosis, or programmed
cell death24 is associated with characteristic morphological and
biochemical changes mediated by a series of gene regulation and
cell signaling pathways. Fluorescein isothiocyanate (FITC)-conju-
gated annexin V has been utilized to detect the externalization of
phosphatidylserine that occurs at an early stage of apoptosis. Pro-
pidium iodide (PI) is used as a marker of necrosis due to cell mem-
brane destruction.25 To elucidate whether compound-induced cell
death involved apoptosis or not, we performed a bi-parametric
cytofluorimetric analysis using annexin V and PI double-staining
showed that PBD-triazole compound 16a between
1000 M exhibited a dosage response. These data indicate that
16a may bind to DNA through intercalation.
0 and
l
Cyclin-dependent kinases (CDKs) play a crucial role in the inhi-
bition of cancer cell proliferation by chemotherapeutic agents.21
Overexpression of cyclin D1 has been reported in several types of
human cancer.22 p27 is considered the main regulator of G1 phase
as shown in Figure 8. Treatment of A375 cells with 5 lM agents
for 24 h induced apoptosis effects in 2.6% (Control), 23.1% (16a),
11.1% (16b), 16.4% (16c), and 12.1% (16d) of annexin V-FITC cells,

C.-Y. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6854–6859
6857
Table. In vitro cytotoxicity of tested compounds on human melanoma A375
Figure 5. Cell viability of 5
experimental conditions as in Table 1).
l
M compounds tested against A375 cells for 24 h (same
cells .a
Compound
IC50 (µM)
2.2
16a
16b
16c
16d
17a
17b
17c
17d
4.2
Table 2
3.6
Effect of PBD-triazoles on cell cycle distribution of A375 melanoma cellsa
2.7
4.5
Cell cycle
%sub-G1
%G1
%S
%G2/M
5.2
Control
16a
16b
16c
16d
17a
17b
17c
7.7 0.1
8.9 0.3
11.7 0.2
7.7 0.1
7.1 0.3
7.0 0.2
17.3 1.8
17.6 2.5
13.9 2.1
65.3 0.1
83.0 3.2
81.9 8.3
82.3 3.8
82.3 0.6
77.8 9.0
69.5 12.2
71.4 15.6
73.6 13.8
11.8 0.1
5.9 1.8
5.1 0.0
6.7 1.6
7.2 1.5
9.7 4.1
8.1 4.0
7.5 4.6
7.4 1.8
15.2 0.6
2.2 0.9
1.3 0.4
3.3 1.0
3.4 1.4
5.5 4.1
5.1 2.1
3.5 3.6
5.1 4.1
5.3
6.4
aIC50 (50% inhibitory concentration) represents the mean from dose-response
curves of three experiments.
Figure 4. Effect of PBD-triazoles on cell viability. A375 cells were seeded in a 96-
well plate at 10,000 cells per well and cultivated overnight until cell attachment.
PBD-triazoles at the indicated concentration was added into the culture media in
triplicate and incubated for 24 h before MTT reagent was added. The absorbance is
directly proportional to the number of living cells. ⁄P <0.05, ⁄⁄P <0.01, ⁄⁄⁄P <0.001
versus control group.
17d
a
Human A375 melanoma cells were treated with compounds, in triplicate at
M concentration for 24 h. Cells were harvested for cell cycle analysis by using
5
l
flow cytometer. Values are expressed as mean SD.
H
N
N
i
ii
Cl Si
N
N3 Si
18
20
19
21
Scheme 3. Synthesis of 5-phenyltriazole. Reagents and conditions: (i) NaN3,
PEG400, 60 °C, 2 h, 82%; (ii) CuI, DMF:MeOH = 1:1, 100 °C, 11 h, 80%, Ref. 17.
respectively. Our results showed that 16a–d agents induced more
apoptotic cells than that of the untreated control.
The anti-apoptotic proteins (Bcl-2, Bcl-xL) and the death-
promoting proteins (Bax, Bad) of the Bcl-2 family are considered
as the requisite gateway to the mitochondrial apoptotic pathway.26
Caspase-3 has been shown to be one of the most important cell
executioners for apoptosis.27 Poly (ADP-ribose) polymerase (PARP)
has been identified as a substrate for caspase-3. The expression of
apoptosis-related proteins was determined by Western blotting as-
say. Our results showed that 16a–d triggering apoptotic cell death
was associated with increased levels of Bax and Bad and decreased
levels of Bcl-2 and Bcl-xL. In addition, cell treatment with 16a in-
duced a concentration-dependent increase in Bax and Bad, cleaved
both caspase-3 and PARP whereas the levels of Bcl-2 and Bcl-xL
were reduced (Fig. 9A and B).
Figure 6. Compound 16a unwind plasmid DNA. Increasing concentration of
compound 16a were incubated with negatively supercoiled pBR322 (0.4 lg) for
12 h in TE buffer and then run for 2.5 h on a 1% gel.
In conclusion, the desired PBD-triazole hybrid analogues 16a–d
and 17a–d have been synthesized in good yields. Initially, we used
an MTT assay to evaluate the cytotoxicity of tested compounds in
seven human and one mouse cell lines. Our results indicated that
cis-form agents 16a–d were more effective as an anti-proliferative
agent than trans-form agents 17a–d. We hypothesized that the
anti-proliferative effects of 16a–d may be associated with cellular

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C.-Y. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6854–6859
Figure 7. Relative expression of cell cycle G1-related genes. A375 cells were treated
with 16a–d for 24 h. Gene expression was determined by qPCR and
normalized with b-actin expression in each sample. ⁄P <0.05 versus control group.
5 lM
Figure 9. Western blot analysis showed the effect of compounds on the expression
of apoptosis regulatory proteins. After exposure to 5 M 16a–d for 24 h (A) or
l
incubated with various concentrations of 16a (B), cell lysates were collected and
Western blotted with specific antibodies as indicated. For the internal control, the
same amounts of protein extract were also probed with antibody against actin.
Similar results were observed in three separate experiments.
cell entry into the S phase. Furthermore, compound 16a–d also in-
duced a marked increase of annexin V binding, decreased protein
expression of both Bcl-2 and Bcl-xL, increased levels of Bax and
Bad and caspase/PARP degradation to identify apoptotic cells.
Based on our results, compound 16a was found to be the most
effective of these PBD-triazole derivatives, which acts as a potential
antimelanoma agent.
Figure 8. PBD-triazoles induced externalization of PS. Dot plots for A375 cells
treated with 16a–d at a concentration of 5 M for 24 h and then stained with PI and
an annexin V-FITC conjugate specifically detected the exposure of PS residues at the
cell surface. Approximately 10,000 cells from each group were analyzed by flow
cytometry. Data shown are of a representative experiment repeated three times
with similar results.
l
Acknowledgement
We thank the National Science Council of the Republic of China
for financial support.
apoptosis. Because the cell cycle deregulation plays a critical role in
malignant
transformation
and
in
the
inefficacy
of
chemotherapy,23b the DNA content of cell nuclei was measured
by flow cytometric analysis. Our results showed that PBD-triazole
derivatives induce cell cycle G1 arrest on human melanoma A375
cells. Moreover, DNA unwinding assay indicated that PBD-triazole
anologue 16a may bind to DNA through intercalation. The cis-form
hybrid agents 16a–d induce p53 activation contributing to the
over-expression of p21 and p27, both being strong cyclin-depen-
dent kinase inhibitors, thereby inducing cell arrest G1 and blocking
Supplementary data
Supplementary data (preparation and characterization of com-
pounds 16a–d and 17a–d; copies of the 1H NMR and 13C NMR
for the new compounds. The purities of various resultant com-
pounds determined by HPLC) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl.2013.10.004.

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