Synthesis of carbazole derivatives containing chalcone analogs as non-intercalative topoisomerase II catalytic inhibitors and apoptosis inducers

Article abstract of DOI:10.1016/j.ejmech.2018.01.010

Novel topoisomerase II (Topo II) inhibitors have gained considerable interest for the development of anticancer agents. In this study, a series of carbazole derivatives containing chalcone analogs (CDCAs) were synthesized and investigated for their Topo II inhibition and cytotoxic activities. The results from Topo II mediated DNA relaxation assay showed that CDCAs could significantly inhibit the activity of Topo II, and the structure-activity relationship indicated the halogen substituent in phenyl ring play an important role in the activity. Further mechanism studies revealed that CDCAs function as non-intercalative Topo II catalytic inhibitors. Moreover, some CDCAs showed micromolar cytotoxic activities. The most potent compound 3h exhibited notable growth inhibition against four human cancer cell lines. Flow cytometric analysis revealed that compounds 3d and 3h arrested the HL-60 cells in sub G1 phase by induction of apoptosis. It was further confirmed by Annexin-V-FITC binding assay. Western blot analysis revealed that compound 3h induces apoptosis likely through the activation of caspase proteins.

Full text of DOI:10.1016/j.ejmech.2018.01.010

Accepted Manuscript
Synthesis of carbazole derivatives containing chalcone analogs as non-intercalative
topoisomerase II catalytic inhibitors and apoptosis inducers
Peng-Hui Li, Hong Jiang, Wen-Jin Zhang, Yong-Lian Li, Min-Cong Zhao, Wei Zhou,
Lan-Yue Zhang, Ya-Dong Tang, Chang-Zhi Dong, Zhi-Shu Huang, Hui-Xiong Chen,
Zhi-Yun Du
PII:
S0223-5234(18)30010-2
10.1016/j.ejmech.2018.01.010
EJMECH 10087
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 October 2017
Revised Date: 2 January 2018
Accepted Date: 4 January 2018
Please cite this article as: P.-H. Li, H. Jiang, W.-J. Zhang, Y.-L. Li, M.-C. Zhao, W. Zhou, L.-Y. Zhang,
Y.-D. Tang, C.-Z. Dong, Z.-S. Huang, H.-X. Chen, Z.-Y. Du, Synthesis of carbazole derivatives
containing chalcone analogs as non-intercalative topoisomerase II catalytic inhibitors and apoptosis
inducers, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.01.010.
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Graphical Abstract

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Title page
Synthesis of Carbazole Derivatives Containing Chalcone Analogs as
Non-intercalative Topoisomerase II Catalytic Inhibitors and Apoptosis Inducers
1
1
1
1
1
1
Peng-Hui Li , Hong Jiang , Wen-Jin Zhang , Yong-Lian Li , Min-Cong Zhao , Wei Zhou ,
1
1
1, 2
3
1, 4
Lan-Yue Zhang , Ya-Dong Tang , Chang-Zhi Dong , Zhi-Shu Huang , Hui-Xiong Chen ,
1
Zhi-Yun Du *
1
Institute of Natural Medicine & Green Chemistry, School of Chemical Engineering and Light
Industry, Guandong University of Technology, Guangzhou, 510006, China
2
Universite Paris Diderot, Sorbonne Paris Cite, ITODYS, UMR 7086 CNRS, 15 rue J-A de
Baif, 75270 Pairs Cedex 13, France
3
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China
4
CNRS, UMR8601, Laboratoire de Chimine et Biochimie Pharmacologiques et
Toxicologiques, CBNIT, Universite Paris Descartes PRES Sorbonne Paris Cite, UFR
Bio5medicale, 45 rue des Saints-Peres, 75270 Paris Cedex 06, France.
*
To whom correspondence should be addressed. Tel.:8620-39322235, Fax.: 8620-39322235,
e-mail: zhiyundu@gdut.edu.cn.
Abstract
Novel topoisomerase II (Topo II) inhibitors have gained considerable interest for the
development of anticancer agents. In this study, a series of carbazole derivatives containing
chalcone analogs (CDCAs) were synthesized and investigated for their Topo II inhibition and
cytotoxic activities. The results from Topo II mediated DNA relaxation assay showed that
CDCAs could significantly inhibit the activity of Topo II, and the structure-activity
relationship indicated the halogen substituent in phenyl ring play an important role in the
activity. Further mechanism studies revealed that CDCAs function as non-intercalative Topo
II catalytic inhibitors. Moreover, some CDCAs showed micromolar cytotoxic activities. The
most potent compound 3h exhibited notable growth inhibition against four human cancer cell
lines. Flow cytometric analysis revealed that compounds 3d and 3h arrested the HL-60 cells
in sub G1 phase by induction of apoptosis. It was further confirmed by Annexin-V-FITC
binding assay. Western blot analysis revealed that compound 3h induces apoptosis likely
through the activation of caspase proteins.
Keywork
Carbazole
Chalcone
Topoisomerase II
Cytotoxic
Apoptosis
1

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1
. Introduction
Cancer is characterized by uncontrolled, rapid and pathological proliferation of cancer cells
and is one of the most formidable afflictions in the world [1]. Thus, in the past decades,
researchers have been challenged by the task of finding effective approaches for the treatment
of cancer [2, 3]. Apart from surgery and radiotherapy, chemotherapy represents the most
important option for the treatment of cancer [4]. Lots of chemotherapy agents were proved to
be promising weapons against cancer. Etoposide, doxorubixin, and amonafide, for instance,
are anticancer drugs strongly inhibiting topoisomerase II (Topo II) [5]. Topo II is one of
promising targets for anticancer drugs able to interfere with the enzyme-DNA complex,
producing permanent DNA damages and triggering cell death [6]. Topoisomerase inhibitors
are widely used in the clinical treatment of cancer over 40 years. About 50% of
chemotherapeutic regimens use at least one drug that targets these enzymes [7]. However,
similar to other anticancer drugs, most Topo II inhibitors produce severe side effects,
including cardiotoxicity, specific luckemia and multidrug resistance [8–10]. Hence, search for
novel Topo II inhibitors with high efficacy and low toxicity is a highly active research field
[
11-16].
The carbazole scaffold is found in numerous bioactive natural products and synthetic
compounds with a wide range of biological activities, including antimalarial, antibacterial and
antitumor activity [17–19]. Moreover, investigations suggested that carbazole derivatives may
exert their antitumor effects by Topo II inhibition [20, 21]. In recent years,
modifications/substitutions of the carbazole ring have become one of the hot topics in the
exploitation of novel Topo II-targeting anticancer agents. For example (Figure 1), compound
2

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1
functions as a potential non-intercalative Topo II catalytic inhibitor [22]. Compound 2, as a
Topo II catalytic inhibitor, displayed a high cytotoxicity against HL-60 cell line [23].
Compound 3, as a Topo II poison, showed moderate cytotoxicity toward cancer cell lines [24].
These results demonstrated great interests in investigating carbazole derivatives as a novel
type of potential anticancer agents targeting Topo II.
Chalcone remained a fascination to researchers in the 21 Century due to its various
pharmacological effects, including anticancer, antioxidant, anti-inflammatory and
anti-infective activity [25-28]. Naturally occurring and synthetic chalcone analogs such as
compound 4, 5, and 6 (Figure 1) are of current interest as cytotoxic agents. These compounds
have been reported to inhibit cancer cells proliferation and induce apoptosis in various cancer
cell lines [29–31]. Several mechanisms of action have been reported for chalcone analogs
including: tubulin polymerization inhibition, apoptosis induction, and Topo II inhibition [32,
3
3].
In view of the above facts and to continue our efforts to develop Topo II inhibitors [34], we
report herein the design of a series of carbazole derivatives containing chalcone analogs
(
CDCAs) (Figure 1) on the basis of the scaffold-hopping approaches [35, 36]. The designed
compounds were prepared and their Topo II inhibition and cytotoxic activities were evaluated.
The most bioactive compounds were further examined for their mechanism of action. The
results indicated that CDCAs function as potential non-intercalative Topo II catalytic
inhibitors. Some of CDCAs exhibited potent cytotoxic activities with low micromolar IC50
against four human cancer cell lines. The leading compounds induced cancer cell apoptosis
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and this was demonstrated by cell cycle distribution, Annexin-V-FITC binding and Western
blot analysis.
2
. Chemistry
The synthetic pathway for CDCAs is shown in scheme 1. N-alkylation of carbazole was
carried out with appropriate benzyl bromides in the presence of KOH to form 1b–1i in 69–88%
yields. The N-subsititued-9H-3-carbaldehydes 2a-2i were synthesized from 1a–1i (1a was
commercially available) through Vilsmeier-Haack reaction in 62–81% yields. The next step
was the aldol condensation of the appropriate acetophenones with 2a–2i using NaOH as base.
Compounds 3a–3r with various substitutions on the phenyl ring were recovered in 63–88%
yields. Compounds 4a–4h were synthesized with different substituted benzyl groups on
N-position of the carbazole in 71–88% yields. The structure of the target compounds 3a-r and
4
a-h are shown in Table 1.
3
. Results and Discussion
3
.1. CDCAs as potent Topo II inhibitors
Topo II mediated DNA relaxation assay was performed to evaluate whether the synthesized
compounds were potent Topo II inhibitors. Etoposide served as control and pBR322 DNA
plasmid as substrate. In this assay, Topo II converts the negative supercoiled form of the
plasmid DNA into relaxed topoisomer which provides a less gel shift [37].
Compounds 3a–3r with different substitutions on the phenyl ring were first synthesized and
evaluated. As shown in Figure 2, compounds 3d, 3g, 3h, and 3j displayed strong Topo II
inhibitory activities at 50 µM. Among this series of compounds, 3d and 3g exhibited strong
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Topo II inhibition at 10 µM (Figure 2V). Specially, 3d displayed the strongest Topo II
inhibition at 5 µM. It can be observed that compounds 3d, 3g, and 3j, which contain a halogen
(
-F, -Cl, and –Br), respectively, on the p-position of the phenyl ring, exhibited better activities
than those with OMe (3a), morpholino (3c), CF (3e), I (3k) or with disubstitutions (3f and
3
3
o–3r). The inhibition potency decreased when the same substitutions (-F and –Cl) were
present on the ortho- or meta-positions of the phenyl ring (compounds 3l, 3m, and 3n). In
addition, compound with thiophene group (3i) in the place of the phenyl ring did not show
obvious Topo II inhibition at 50 µM. These results indicate that the phenyl ring, the
substituent in the phenyl ring as well as its position have significant impact on the inhibition
of Topo II activity, which should be maintained in the future for modifying structure.
In order to investigate the effect of the N-substitution on the inhibition potency, the ethyl
group was changed to benzyl groups, compounds 4a, 4d, 4e, and 4f showed strong Topo II
inhibitory activity at 50 µM (Figure 2III-V), other compounds 4b-c and 4g-h were much less
active at the same concentration. In addition, compounds 4e and 4f strongly inhibited Topo II
activity at 10 µM. It was found that compounds (4a, 4d, 4e, and 4f) with F as substituent on
the benzyl group showed much more potency to inhibit Topo II inhibition than the others with
H (4c), CH (4b), Cl (4g), and NO (4h). However, all of these N-substituted derivatives
3
2
showed a weaker Topo II inhibitory activity than 3d, indicating that the introduction of benzyl
group to N-position has an unfavorable effect on the inhibition of Topo II activity.
Topoisomerase I (Topo I)-mediated supercoiled pBR322 DNA relaxation assay was
carried out subsequently to study the effect of CDCAs on the catalytic activity of
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Topo I [38]. Results presented in Figure 3 showed that camptothecin (CPT), a
well-known Topo I inhibitor, strongly inhibited Topo I activity, while CDCAs had no
inhibitory effect on Topo I even at high concentration (50 µM). These results indicated
that CDCAs selectively inhibited the activity of Topo II.
As compounds 3d and 3g showed the best Topo II inhibition activity, these two leading
compounds were selected for further mechanism study.
3
.2. CDCAs as non-intercalative Topo II catalytic inhibitors
Topo II inhibitors act by two different ways: (1) one, is commonly referred as poisons, that
bind to and stabilize the Topo II-DNA cleavage complex, leading to the rapid DNA strand
break (e.g., etoposide and doxorubicin) and (2) the others, is commonly referred to as
catalytic inhibitors, that antagonize the ability of the enzyme to perform catalysis (e.g.,
merbarone and bisdioxopiperazines) [39].
To gain insight into the mode of action, a cleavage complex assay was performed to verify
whether compounds 3d and 3g function as Topo II posions or catalytic inhibitors. As shown in
Figure 4A, the well known Topo II poison etoposide produces discrete bands of linear DNA,
corresponding to its poison behavior. On the contrary, both representative compounds 3d and
3
g did not produce the same effects on DNA strand break as etoposide even at 100 µM. These
results indicated that these compounds may be Topo II catalytic inhibitors.
In order to determine the intercalating or non-intercalating ability of the leading compounds, a
DNA unwinding assay was carried out using supercoiled pBR322 DNA as substrate and
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ethidium bromide (EB) as control [40]. As shown in Figure 4B, EB (a classic DNA
intercalator) is able to transform the relaxed DNA into supercoiled DNA in the presence of
excess Topo I in a dose-dependent manner. However, neither compound 3d nor 3g could
intercalate into plasmids even at 100 µM, suggesting that these compounds are
non-intercalative Topo II catalytic inhibitors.
The ability of 3d to interact with DNA was further examined by fluorescence-based EB
displacement assay. The DNA bound form of EB has a stronger fluorescence emission than
free EB, so the displacement of EB from DNA could be monitored through the decrease of
fluorescence signal [41]. As shown in Figure 5A and 5B, addition of amsacrine (mAMSA, a
typical DNA intercalator) led to a significantly decrease in fluorescence intensity. In contrast
to mAMSA, compound 3d did not obviously alter the fluorescence intensity even at 100 µM,
indicating that 3d showed weak intercalating into DNA. In addition, UV-vis titration assay
was also employed to measure the binding affinity of 3d to calf thymus DNA (CT-DNA) [42].
Generally, interaction between a compound and the DNA would result in hypochromic and
bathochromic shifts in the absorption spectrum due to the strong stacking interactions
between the aromatic chromophore of the compounds and the DNA base pair, as well as the
electron transfer effect in the process. As shown in Figure 5C and 5D, addition of increasing
concentration of CT-DNA to 3d resulted in a weak hypochromic effect but without visible red
5
-1
shifts. The apparent binding constant (K ) of 3d to CT-DNA was 2.2× 10 M , indicating that
b
3
d exhibited weak interactions with CT-DNA. Taken together, these findings reveal that these
compounds function as non-intercalative Topo II catalytic inhibitors.
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3
.3. CDCAs as potent anticancer agents.
The cytotoxicity of CDCAs were evaluated against four human cancer cell lines, including
HeLa (human cervical cancer cell line), HL-60 (human acute leukemia cell line), A549
(
adenocarcinomic human alveolar basal epithelial cancer cell line), and PC-3 (human prostate
cancer cell line), by using the MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay as described by
Mosmann with modification [43]. Etoposide was tested as a reference compound.
(
As shown in Table 2, most of compounds displayed significant cytotoxicities toward the
tested cancer cell lines with low to moderate micromolar values of IC . Moreover, almost all
50
compounds showed a cytotoxic selectivity for the HL-60 cell line, and some of the tested
compounds such as 3h, 4a and 4d displayed a nanomolar level IC , which was consistent
5
0
with etoposide. However, there is no obvious correlation between Topo II inhibitory activity
and cytotoxicity for this series of compounds. Among them, 3h exhibited the best
anti-proliferation activity against cancer cells with an IC50 ranging between 0.29–2.16 µM.
However, this compound showed moderate Topo II inhibitory activity at 50 µM (Figure 2).
Compound 3d, 3g, and 3j the most potent Topo II inhibitors at 20 µM, displayed moderate to
weak cytotoxicity against all the tested cancer cell lines.
Compounds 4a-4h were synthesized on the basis of 3d. This series compounds also displayed
potent cytotoxic activity against the four cancer cell lines. Among them, 4d exhibited high
cytotoxicity with an IC ranging between 0.67–4.45 µM, which is more potent than that of 3d,
5
0
suggesting that a suitable N-benzylation could improve the cytotoxic effect.
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3
.4. CDCAs as potent apoptosis inducers
Cell cycle arrest is considered to be one of the crucial checkpoints of apoptotic cell death and
could be detected by the cell cycle arrest at sub G1 phase [45]. In order to study whether
CDCAs could inhibit cell proliferation by cell cycle-mediated apoptosis, the HL-60 cells were
treated with compounds 3d and 3h at 0.5-, 1-, and 2- fold of their IC values for 24 h and the
5
0
cell cycle distribution was analyzed using flow cytometry. As shown in Figure 6 and Table 3,
compounds 3d and 3h caused cell arrest at sub G1 phase from 13.14% to 74.73% and from
5
.88% to 67.29%, respectively, in a dose-dependent manner. This result demonstrated that
compounds 3d and 3h significantly induced cell cycle arrest at sub G1 phase and thereby
provoked the cell cycle-mediated apoptosis of HL-60 cells.
The cell cycle distribution studies suggested that compounds 3d and 3h could induce HL-60
cell apoptosis. This event was further examined in an FITC-label annexin V binding assay
[
46]. As shown in Figure 7A, after 24 h treatment with 3d and 3h, apoptotic cells increased
from 4.17% to 22.22% and from 3.38% to 52.88% respectively, in the early stage of apoptosis.
These results confirmed that 3d and 3h can trigger apoptotic cell death.
The expression of caspase-3, caspase-7, caspase-9, and poly ADP-ribose polymerase (PARP),
indicator proteins of apoptosis, in HL-60 cells were determined by using Western blot
analysis [47]. As shown in Figure 7B and 7C, it was found that cleaved caspase-3
(
C-caspase-3), caspase-7 (C-caspase-7), caspase-9 (C-caspase-9), and PARP (C-PARP)
significantly increased after treatment with 3h in a dose-dependent manner after 24 h. These
findings indicate that the leading compounds are apoptosis inducers.
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4
. Conclusion
In conclusion, a series of CDCAs were designed, synthesized and evaluated for their Topo II
inhibition and cytotoxic activity towards a panel of four human cancer cell lines. Topo II
inhibitory activity of these compounds was determined by Topo II-mediated pBR322 DNA
relaxation and cleavage complex assay. Compound 3d showed the most potent Topo II
inhibitory activity, and mechanism studies revealed that these compounds might function as
non-intercalative Topo II catalytic inhibitors. Most of CDCAs displayed potent cytotoxic
activity against four human cancer cell lines. However, no obvious correlation between Topo
II inhibition activity and cytotoxic effect of CDCAs was observed. Among the CDCAs,
compounds 3d, 3g, and 3j displayed strong Topo II inhibitory activity at 20 µM, especially
compound 3d showed potent Topo II inhibitory activity at 5 µM. However, they exhibited
moderate cytotoxic activity against all the tested cancer cell lines. On the contrary, compound
3
h, which displayed moderate Topo II inhibitory activity at 50 µM, exhibited high
cytotoxicity against the four cancer cell lines with an IC values ranging from 0.29–2.16 µM.
5
0
Subsequently, cell cycle distribution and annexin V-FITC/PI binding were employed to study
the action on cancer cells for CDCAs. The leading compounds 3d and 3h arrested the HL-60
cells at sub G1 phase of the cell cycle, leading to apoptosis in a dose-dependent manner. This
was further confirmed by annexin V-FITC/PI binding assay. Additionally, compound 3h
induced cancer cell apoptosis likely through activation of caspase proteins. Overall, the
carbazole derivatives containing chalcone analogs were demonstrated to be potent Topo
II-targeting anticancer agents with apoptosis-inducing activity. The leading compounds 3d
and 3h can be used as a new template to develop new anticancer agents.
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. Experimental section
5
5
.1. Synthesis and characterization
1
13
H and C NMR spectra were recorded using TMS as the internal standard in CDCl with a
3
Bruker BioSpin GmbH spectrometer at 400 MHz and 101 MHz, respectively. The chemical
1
shifts are reported in parts per million (ppm) relative to residual CHCl (δ = 7.26, H; δ = 77.0,
3
1
3
C) in the corresponding deuterium agent. The following abbreviations are used to designate
multiplicities: s, single; d, double; t, triplet; q, quartet; m, multiplet; dd, double-double; br s,
broad signal. Melting points (mp) were determined using an SRS-OptiMelt automated
melting point instrument without correction. High-resolution mass spectra (HR ESI-MS) were
recorded on Shimadzu LCMS-IT-TO. The purities of synthesized compounds were confirmed
by analytical HPLC performed with a dual pump Shimadzu LC-20AB system equipped with
an Ultimate XB-C18 column and eluted with methanol/water (70%) at a flow rate of 0.7
-1
mL·min , and the purities were proved to be higher than 95%. All of the reagents were
commercially available and purchased from Sigma-Aldrich Chemical Co., TCI Chemicals,
AlfaAesar, and Aladdin, which used without further purification.
5
.2. General method for the synthesized of 1b–1i
KOH (7 g, 125 mmol) was dissolved in acetone (15 mL) and the resulting solution was stirred
at room temperature for 30 min. Carbazole (3.3 g, 20 mmol) was then added and reacted for 4
h. The acetone (15 mL) containing appropriate benzyl bromide (30 mmol) was added into the
above solution and the resulting system was allowed to react overnight. When the reaction
mixture was poured into water (250 mL), the solid was filtrated. After recrystallization from
ethanol, a white solid was obtained in 69–88% yields.
1
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5
.2.1. Synthesis of 9-(4-fluorobenzyl)-9H-carbazole (1b)
Carbazole and 1-(bromomethyl)-4-fluorobenzene were used as reactants to give 1b. White
1
solid, yield: 83%. H NMR (400 MHz, CDCl ) δ 10.11 (s, 1H), 8.65 (d, J = 1.0 Hz, 1H), 8.19
3
(
d, J = 7.8 Hz, 1H), 7.83 – 7.95 (m, 1H), 7.53 – 7.48 (m, 1H), 7.45 – 7.39 (m, 2H), 7.37 –
7
.33 (m, 1H), 7.15 – 7.07 (m, 2H), 6.99 – 6.93 (m, 2H), 5.53 (s, 2H).
5
.2.2. Synthesis of 9-(4-methylbenzyl)-9H-carbazole (1c)
Carbazole and 1-(bromomethyl)-4-methylbenzene were used as reactants to give 1c. White
1
solid, yield: 76%. H NMR (400 MHz, CDCl ) δ 8.12 (d, J = 7.8 Hz, 2H), 7.44 – 7.40 (m, 2H),
3
7
.37 (d, J = 8.1 Hz, 2H), 7.28 – 7.20 (m, 2H), 7.07 – 7.02 (m, 4H), 5.48 (s, 2H), 2.28 (s, 3H).
5
.2.3. Synthesis of 9-benzyl-9H-carbazole (1d)
Carbazole and (bromomethyl)benzene were used as reactants to give 1d. White solid, yield:
1
8
6%. H NMR (400 MHz, CDCl ) δ 8.13 (d, J = 7.8 Hz, 2H), 7.45 – 7.41 (m, 2H), 7.37 (d, J
3
=
8.1 Hz, 2H), 7.27 – 7.23 (m, 5H), 7.16 – 7.13 (m, 2H), 5.52 (s, 2H).
5
.2.4. Synthesis of 9-(2-fluorobenzyl)-9H-carbazole (1e)
Carbazole and 1-(bromomethyl)-2-fluorobenzene were used as reactants to give 1e. White
1
solid, yield: 74%. H NMR (400 MHz, CDCl ) δ 8.13 (d, J = 7.8 Hz, 2H), 7.47 – 7.42 (m, 2H),
3
7
.39 (d, J = 8.1 Hz, 2H), 7.28 – 7.24 (m, 2H), 7.22 – 7.18 (m, 1H), 7.15 – 7.07 (m, 1H), 6.88
(
t, J = 7.5 Hz, 1H), 6.73 (t, J = 7.5 Hz, 1H), 5.57 (s, 2H).
5
.2.5. Synthesis of 9-(2,4-difluorobenzyl)-9H-carbazole (1f)
Carbazole and 1-(bromomethyl)-2,4-difluorobenzene were used as reactants to give 1f. White
1
solid, yield: 82%. H NMR (400 MHz, CDCl ) δ 8.13 (d, J = 7.8 Hz, 2H), 7.47 – 7.43 (m, 2H),
3
7
.37 (d, J = 8.2 Hz, 2H), 7.30 – 7.26 (m, 2H), 6.91 – 6.77 (m, 1H), 6.74 – 6.68 (m, 1H), 6.71
–
6.59 (m, 1H), 5.52 (s, 2H).
1
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5
.2.6. Synthesis of 9-(3-fluorobenzyl)-9H-carbazole (1g)
Carbazole and 1-(bromomethyl)-3-fluorobenzene were used as reactants to give 1g. White
1
solid, yield: 71%. H NMR (400 MHz, CDCl ) δ 8.14 (d, J = 7.7 Hz, 2H), 7.46 – 7.41 (m, 2H),
3
7
.33 (d, J = 8.2 Hz, 2H), 7.27 (d, J = 7.5 Hz, 2H), 7.24 – 7.19 (m, 1H), 6.95 –6.87 (m, 2H),
.82 (d, J = 9.5 Hz, 1H), 5.50 (s, 2H).
6
5
.2.7. Synthesis of 9-(4-chlorobenzyl)-9H-carbazole (1h)
Carbazole and 1-(bromomethyl)-4-chlorobenzene were used as reactants to give 1h. White
1
solid, yield: 69%. H NMR (400 MHz, CDCl ) δ 8.13 (d, J = 7.8 Hz, 2H), 7.47 – 7.39 (m, 2H),
3
7
.32 (d, J = 8.1 Hz, 2H), 7.25 (s, 2H), 7.22 (d, J = 8.4 Hz, 2H), 7.05 (d, J = 8.3 Hz, 2H), 5.47
(
s, 2H).
5
.2.8. Synthesis of 9-(4-nitrobenzyl)-9H-carbazole (1i)
Carbazole and 1-(bromomethyl)-4-nitrobenzene were used as reactants to give 1i. White solid,
1
Yield: 88%. H NMR (400 MHz, CDCl ) δ 8.17 – 8.13 (m, 2H), 8.11 (d, J = 8.7 Hz, 2H), 7.47
3
–
7.42 (m, 2H), 7.31 – 7.25 (m, 6H), 5.60 (s, 2H).
5
.3. General method for the synthesized of 2a–2i
DMF (0.4 ml) was added into a dried round-bottom flask and the system was cooled to 0 °C.
A solution of CHCl (3 ml) containing 1a–1i (5 mmol) was then added following the addition
3
of redistilled POCl (0.45 mL).Then, the solution mixture was heated to 90 °C for 8 h. After
3
most of the CHCl was removed the residue was poured into ice water and the pH was then
3
adjusted to 7-8 using NaHCO , the water layer was extracted with CH Cl and organic layer
3
2
2
was washed with water several times before being dried with Mg SO , after CH Cl was
2
4
2
2
removed, the crude product was purified by column chromatography using ethyl
1
3

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acetate/petroleum ether (1:10, V/V) as an eluent, and finally a primrose solid was obtained for
2
a–2i in 62–81% yields.
5
.3.1. Synthesis of 9-ethyl-9H-carbazole-3-carbaldehyde (2a)
1
Compound 1a was used as reactants to give 2a. White solid, yield: 79%. H NMR (400 MHz,
CDCl -d ) δ 10.08 (s, 1H), 8.62 (d, J = 1.1 Hz, 1H), 8.16 (d, J = 7.8 Hz, 1H), 8.02 (dd, J = 8.5,
3
6
1
.5 Hz, 1H), 7.59 – 7.51 (m, 1H), 7.48 (t, J = 7.6 Hz, 2H), 7.37 – 7.29 (m, 1H), 4.42 (q, J =
.2 Hz, 2H), 1.48 (t, J = 7.3 Hz, 3H).
7
5
.3.2. Synthesis of 9-(4-fluorobenzyl)-9H-carbazole-3-carbaldehyde (2b)
1
Compound 1b was used as reactants to give 2b. White solid, yield: 76%. H NMR (400 MHz,
CDCl ) δ 10.11 (s, 1H), 8.65 (d, J = 1.0 Hz, 1H), 8.19 (d, J = 7.8 Hz, 1H), 8.03 – 7.95 (m,
3
1
H), 7.53 – 7.48 (m, 1H), 7.46 – 7.38 (m, 2H), 7.36 – 7.30 (m, 1H), 7.17 – 7.05 (m, 2H), 7.01
6.93 (m, 2H), 5.53 (s, 2H).
–
5
.3.3. Synthesis of 9-(4-methylbenzyl)-9H-carbazole-3-carbaldehyde (2c)
1
Compound 1c was used as reactants to give 2c. White solid, yield: 62%. H NMR (400 MHz,
CDCl ) δ 10.10 (s, 1H), 8.65 (d, J = 1.2 Hz, 1H), 8.18 (d, J = 7.7 Hz, 1H), 8.01 – 7.94 (m,
3
1
2
H), 7.52 – 7.41 (m, 3H), 7.37 – 7.29 (m, 1H), 7.08 (d, J = 8.0 Hz, 2H), 7.03 (d, J = 8.1 Hz,
H), 5.52 (s, 2H), 2.29 (s, 3H).
5
.3.4. Synthesis of 9-benzyl-9H-carbazole-3-carbaldehyde (2d)
1
Compound 1d was used as reactants to give 2d. White solid, yield: 80%. H NMR (400
MHz, CDCl ) δ 10.10 (s, 1H), 8.65 (d, J = 1.1 Hz, 1H), 8.19 (d, J = 7.8 Hz, 1H), 8.02 – 7.94
3
(
m, 1H), 7.52 – 7.48 (m, 1H), 7.47 – 7.41 (m, 2H), 7.37 – 7.32 (m, 1H), 7.29 – 7.26 (m, 3H),
.15 – 7.12 (m, 2H), 5.56 (s, 2H).
7
1
4

ACCEPTED MANUSCRIPT
5
.3.5. Synthesis of 9-(2-fluorobenzyl)-9H-carbazole-3-carbaldehyde (2e)
1
Compound 1e was used as reactants to give 2e. White solid, yield: 77%. H NMR (400 MHz,
CDCl ) δ 10.11 (s, 1H), 8.65 (d, J = 1.0 Hz, 1H), 8.19 (d, J = 7.7 Hz, 1H), 8.03 – 7.95 (m,
3
1
H), 7.54 – 7.44 (m, 3H), 7.39 – 7.31 (m, 1H), 7.25 – 7.20 (m, 1H), 7.16 – 7.10 (m, 1H), 6.93
(
t, J = 7.3 Hz, 1H), 6.76 (t, J = 7.2 Hz, 1H), 5.61 (s, 2H).
5
.3.6. Synthesis of 9-(2,4-difluorobenzyl)-9H-carbazole-3-carbaldehyde (2f)
1
Compound 1f was used as reactants to give 2f. White solid, yield: 74%. H NMR (400 MHz,
CDCl ) δ 10.11 (s, 1H), 8.66 (d, J = 1.1 Hz, 1H), 8.19 (d, J = 7.8 Hz, 1H), 8.05 –7.97 (m, 1H),
3
7
6
.55 – 7.50 (m, 1H), 7.48 (d, J = 8.5 Hz, 1H), 7.43 (d, J = 8.2 Hz, 1H), 7.39 – 7.34 (m, 1H),
.93 – 6.87 (m, 1H), 6.78 – 6.72 (m, 1H), 6.71 – 6.63 (m, 1H), 5.57 (s, 2H).
5
.3.7. Synthesis of 9-(3-fluorobenzyl)-9H-carbazole-3-carbaldehyde (2g)
1
Compound 1g was used as reactants to give 2g. White solid, yield: 68%. H NMR (400 MHz,
CDCl ) δ 8.14 (d, J = 7.7 Hz, 2H), 7.46 – 7.41 (m, 2H), 7.33 (d, J = 8.2 Hz, 2H), 7.27 (d, J =
3
7
.5 Hz, 2H), 7.24 – 7.19 (m, 1H), 6.95 – 6.87 (m, 2H), 6.82 (d, J = 9.5 Hz, 1H), 5.50 (s, 2H).
5
.3.8. Synthesis of 9-(4-chlorobenzyl)-9H-carbazole-3-carbaldehyde (2h)
1
Compound 1h was used as reactants to give 2h. White solid, yield: 73%. H NMR (400 MHz,
CDCl ) δ 10.11 (s, 1H), 8.65 (d, J = 0.7 Hz, 1H), 8.19 (d, J = 7.7 Hz, 1H), 8.04 – 7.92 (m,
3
1
2
H), 7.52 – 7.48 (m, 1H), 7.44 – 7.33 (m, 3H), 7.24 (s, 2H), 7.06 (d, J = 8.3 Hz, 2H), 5.52 (s,
H).
5
.3.9. Synthesis of 9-(4-nitrobenzyl)-9H-carbazole-3-carbaldehyde (2i)
1
Compound 1i was used as reactants to give 2i. White solid, yield: 81%. H NMR (400 MHz,
CDCl ) δ 10.12 (s, 1H), 8.68 (d, J = 1.0 Hz, 1H), 8.22 (d, J = 7.8 Hz, 1H), 8.15 (d, J = 8.7 Hz,
3
1
5

ACCEPTED MANUSCRIPT
H), 8.04 – 7.96 (m, 1H), 7.54 – 7.50 (m, 1H), 7.40 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 8.2 Hz,
H), 7.29 (d, J = 8.9 Hz, 2H), 5.66 (s, 2H).
2
1
5
.4. General method for the synthesized of CDCAs
Appropriately substituted acetophenone (1 mmol) and 2a–2i (1 mmol) were dissolved in
EtOH (5 mL), 10% NaOH (0.5 mL) was added slowly. Then, the solution mixture was stirred
at room temperature for 2 h. The yellow solid was filtrated. After recrystallization from
ethanol, a yellow solid was obtained.
5
.4.1. Synthesis of (E)-3-(9-ethyl-9H-carbazol-3-yl)-1-(4-methoxyphenyl)prop-2-en-1-one
(3a). 1-(4-Methoxyphenyl)ethanone and 2a were used as reactants to give 3a. Yellow solid,
1
yield: 82%, mp: 176.1– 177.8 °C. H NMR (400 MHz, CDCl ): δ 8.40 (d, J = 1.4 Hz, 1H),
3
8
7
–
.17 (d, J = 7.7 Hz, 1H), 8.15 – 8.10 (m, 2H), 8.08 (d, J = 15.5 Hz, 1H), 7.86 – 7.78 (m, 1H),
.63 (d, J = 15.5 Hz, 1H), 7.56 – 7.50 (m, 1H), 7.49 – 7.41 (m, 2H), 7.35 – 7.29 (m, 1H), 7.07
1
3
6.99 (m, 2H), 4.40 (q, J = 7.2 Hz, 2H), 3.93 (s, 3H), 1.48 (t, J = 7.2 Hz, 3H). C NMR (101
MHz, CDCl ): δ 189.4, 163.2, 145.6, 141.4, 140.5, 131.7, 130.6, 126.3, 126.2, 123.5, 122.9,
3
+
1
3
21.5, 120.6, 119.7, 118.9, 113.8, 108.9, 108.8, 55.5, 37.8, 13.8. HR ESI-MS (M + H) m/z =
56.1640 (Cacld for C H NO : 356.1645) .
2
4
21
2
5
.4.2. Synthesis of (E)-3-(9-ethyl-9H-carbazol-3-yl)-1-phenylprop-2-en-1-one (3b).
Acetophenone and 2a were used as reactants to give 3b. Yellow solid, yield: 88%, mp:
1
1
7
62.4–164.1 °C. H NMR (400 MHz, CDCl ): δ 8.39 (s, 1H), 8.15 (d, J = 7.2 Hz, 1H), 8.11 –
3
.99 (m, 3H), 7.80 (d, J = 8.5 Hz, 1H), 7.65 – 7.47 (m, 5H), 7.47 – 7.39 (m, 2H), 7.33 – 7.27
1
3
(
m, 1H), 4.39 (q, J = 7.2 Hz, 2H), 1.47 (t, J = 7.2 Hz, 3H). C NMR (101 MHz, CDCl ): δ
3
1
6

ACCEPTED MANUSCRIPT
1
90.7, 146.5, 141.5, 140.5, 138.9, 132.4, 128.5, 128.0, 126.4, 126.0, 123.5, 122.9, 121.6,
+
1
20.6, 119.7, 119.2, 108.9, 37.8, 13.8. HR ESI-MS (M + H) m/z =326.1532 (Cacld for
C H NO: 362.1541)
23
19
5
.4.3. Synthesis of (E)-3-(9-ethyl-9H-carbazol-3-yl)-1-(4-morpholinophenyl)prop-2-en-1-one
(3c). 1-(4-Morpholinophenyl)ethanone and 2a were used as reactants to give 3c. Yellow solid,
1
yield: 81%, mp: 186.5–188.2 °C. H NMR (400 MHz, CDCl ): δ 8.38 (s, 1H), 8.15 (d, J = 7.7
3
Hz, 1H), 8.11 – 8.01 (m, 3H), 7.80 (d, J = 8.1 Hz, 1H), 7.61 (d, J = 15.7 Hz, 1H), 7.50 (d, J =
7
.1 Hz, 1H), 7.42 (d, J = 8.5 Hz, 2H), 7.32 – 7.27 (m, 1H), 7.01 (d, J = 8.0 Hz, 2H), 4.39 (q, J
13
=
7.1 Hz, 2H), 3.91 (t, J = 7.3 Hz, 4H), 3.35 (t, J = 8.1 Hz, 4H), 1.47 (t, J = 6.8 Hz, 3H). C
NMR (101 MHz, CDCl ): δ 188.3, 154.0, 145.0, 141.3, 140.5, 130.5, 129.5, 126.4, 126.3,
3
1
23.5, 121.3, 120.6, 119.6, 119.0, 113.6, 108.9, 108.8, 100.0, 66.6, 47.7, 37.8, 13.8. HR
+
ESI-MS (M + H) m/z =411.2057 (Cacld for C H N O : 411.2067).
2
7
26
2
2
5
1
7
7
1
1
1
.4.4. Synthesis of (E)-1-(4-chlorophenyl)-3-(9-ethyl-9H-carbazol-3-yl)prop-2-en-1-one (3d).
-(4-Chlorophenyl)ethanone and 2a were used as reactants to give 3d. Yellow solid, yield:
1
2%, mp: 192.4–193.9 °C. H NMR (400 MHz, CDCl ): δ 8.38 (s, 1H), 8.20 – 7.93 (m, 4H),
3
.79 (d, J = 8.4 Hz, 1H), 7.59 – 7.38 (m, 6H), 7.34 – 7.27 (m, 1H), 4.37 (q, J = 7.1 Hz, 2H),
1
3
.47 (t, J = 7.1 Hz, 3H). C NMR (101 MHz, CDCl ): δ 189.2, 147.0, 141.6, 140.5, 138.8,
3
37.2, 129.9, 128.8, 126.4, 125.8, 123.6, 122.9, 121.8, 120.7, 119.8, 118.5, 108.9, 108.9, 37.8,
+
3.8. HR ESI-MS (M + H) m/z =360.1136 (Cacld for C H ClNO: 360.1150).
2
3
18
5
.4.5.
Synthesis
of
(E)-3-(9-ethyl-9H-carbazol-3-yl)-1-(4-(trifluoromethyl)phenyl)prop-2-en-1-one
(3e).
1
7

ACCEPTED MANUSCRIPT
1
-(4-(Trifluoromethyl)phenyl)ethanone and 2a were used as reactants to give 3e. Yellow solid,
1
yield: 83%, mp: 159.6–161.3 °C. H NMR (400 MHz, CDCl ): δ 8.39 (s, 1H), 8.17 – 8.05 (m,
3
4
H), 7.78 (d, J = 7.7 Hz, 3H), 7.54 (d, J = 14.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.35 – 7.25
1
3
(
m, 1H), 4.40 (q, J = 7.4 Hz, 2H), 1.47 (t, J = 7.0 Hz, 3H). C NMR (101 MHz, CDCl ): δ
3
1
2
89.8, 147.9, 141.8, 141.7, 140.5, 133.9, 133.5, 128.7, 126.5, 126.4, 125.6, 124.8 (q, J =
72.3 Hz), 123.6, 122.8, 122.0, 120.7, 119.9, 118.6, 108.9, 37.8, 13.9. HR ESI-MS (M +
+
H) m/z =394.1412 (Cacld for C H NF O: 394.1413).
2
4
18
3
5
.4.6.
Synthesis
of
(E)-1-(benzo[d][1,3]dioxol-5-yl)-3-(9-ethyl-9H-carbazol-3-yl)prop-2-en-1-one
(3f).
1
-(Benzo[d][1,3]dioxol-5-yl)ethanone and 2a were used as reactants to give 3f. Yellow solid,
1
yield: 73%, mp: 177.1 – 179.4 °C. H NMR (400 MHz, CDCl ): δ 8.38 (s, 1H), 8.15 (d, J =
3
7
–
2
1
1
3
.7 Hz, 1H), 8.04 (d, J = 15.3 Hz, 1H), 7.79 (d, J = 8.3 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.62
7.46 (m, 4H), 7.47 – 7.38 (m, 2H), 7.33 – 7.27 (m, 1H), 6.92 (d, J = 8.2 Hz, 1H), 6.07 (s,
1
3
H), 4.39 (q, J = 7.3 Hz, 2H), 1.47 (t, J = 7.2 Hz, 4H). C NMR (101 MHz, CDCl ): δ 188.4,
3
51.4, 148.2, 145.9, 141.4, 140.5, 133.6, 126.3, 126.1, 124.4, 123.5, 122.9, 121.5, 120.7,
+
19.7, 118.7, 109.0, 108.9, 108.5, 107.9, 101.8, 37.8, 13.8. HR ESI-MS (M + H) m/z =
70.1427 (Cacld for C H NO : 370.1438).
2
4
19
3
5
1
7
8
.4.7. Synthesis of (E)-3-(9-ethyl-9H-carbazol-3-yl)-1-(4-fluorophenyl)prop-2-en-1-one (3g).
-(4-Fluorophenyl)ethanone and 2a were used as reactants to give 3g. Yellow solid, yield:
1
1
6%, mp: 166.8–168.5 °C. H NMR (400 MHz, CDCl ) H NMR (400 MHz, CDCl -d ) δ
3
3
6
.38 (d, J = 1.0 Hz, 1H), 8.19 – 8.01 (m, 4H), 7.83 – 7.75 (m, 1H), 7.57 – 7.49 (m, 2H), 7.45
1
8

ACCEPTED MANUSCRIPT
–
7.37 (m, 2H), 7.29 (t, J = 7.4 Hz, 1H), 7.23 – 7.15 m, 2H), 4.39 (q, J = 7.2 Hz, 2H), 1.46 (t,
1
3
J = 7.2 Hz, 3H). C NMR (101 MHz, CDCl ) δ 189.8, 147.9, 141.8, 141.7, 140.5, 133.9,
3
1
33.5 (d, J = 3.8 Hz), 128.7 (d, J =8.3 Hz), 126.5, 126.4, 125.6, 125.1, 123.6, 122.8, 122.0,
+
1
20.7, 119.9, 118.6 (d, J = 20.4 Hz), 108.9, 37.8, 13.9. HR ESI-MS (M + H) m/z =343.1439
(
Cacld for C H NOF: 343.1450).
23 18
5
1
1
1
.4.8. Synthesis of (E)-3-(9-ethyl-9H-carbazol-3-yl)-1-(p-tolyl)prop-2-en-1-one (3h).
-(P-tolyl)ethanone and 2a were used as reactants to give 3h. Yellow solid, yield: 63%, mp:
1
62.1–168.4 °C. H NMR (400 MHz, CDCl ) δ 8.38 (d, J = 1.1 Hz, 1H), 8.15 (d, J = 7.7 Hz,
3
H), 8.05 (d, J = 15.6 Hz, 1H), 7.99 (d, J = 8.1 Hz, 2H), 7.83 – 7.77 (m, 1H), 7.59 (d, J = 15.5
Hz, 1H), 7.53 – 7.47 (m, 1H), 7.46 – 7.40 (m, 2H), 7.35 – 7.26 (m, 3H), 4.39 (q, J = 7.2 Hz,
1
3
2
1
1
H), 2.45 (s, 3H), 1.46 (t, J = 7.2 Hz, 3H). C NMR (101 MHz, CDCl ) δ 190.2, 146.1,
3
43.2, 141.4, 140.5, 136.2, 129.3, 128.6, 126.3, 126.1, 123.5, 122.9, 121.6, 120.7, 119.7,
+
19.1, 109.0, 108.9, 37.8, 21.7, 13.8. HR ESI-MS (M + H) m/z =340.4362 (Cacld for
C H NO: 340.4371).
24
21
5
1
.4.9. Synthesis of (E)-3-(9-ethyl-9H-carbazol-3-yl)-1-(thiophen-2-yl)prop-2-en-1-one (3i).
-(Thiophen-2-yl)ethanone and 2a were used as reactants to give 3i. Yellow solid, yield: 67%,
1
mp: 157.5–159.8 °C. H NMR (400 MHz, CDCl ): δ 8.39 (d, J = 1.6 Hz, 1H), 8.21 – 8.09 (m,
3
2
–
7
H), 7.98 – 7.92 (m, 1H), 7.85 – 7.77 (m, 1H), 7.72 – 7.66 (m, 1H), 7.55 – 7.51 (m, 2H), 7.48
7.44 (m, 3H), 7.35 – 7.29 (m, 1H), 7.25 – 7.19 (m, 1H), 4.40 (q, J = 7.2 Hz, 2H), 1.48 (t, J =
1
3
.2 Hz, 3H). C NMR (101 MHz, CDCl ): δ 182.2, 146.1, 145.7, 1415, 140.5, 133.2, 131.3,
3
1
9

ACCEPTED MANUSCRIPT
1
28.1, 126.4, 126.3, 125.8, 123.5, 122.9, 121.7, 120.7, 119.7, 118.6, 109.0, 108.8, 37.8, 13.8.
+
HR ESI-MS (M + H) m/z =332.1022 (Cacld for C H NOS: 332.1023).
2
1
17
5
1
7
1
2
1
1
.4.10. Synthesis of (E)-1-(4-bromophenyl)-3-(9-ethyl-9H-carbazol-3-yl)prop-2-en-1-one (3j).
-(4-Bromophenyl)ethanone and 2a were used as reactants to give 3j. Yellow solid, yield:
1
9%, mp: 196.7–198.4 °C. H NMR (400 MHz, CDCl ): δ 8.37 (s, 1H), 8.14 (d, J = 7.7 Hz,
3
H), 8.05 (d, J = 15.5 Hz, 1H), 7.97 – 7.88 (m, 2H), 7.81–7.75 (m, 1H), 7.65 (d, J = 8.5 Hz,
H), 7.55 – 7.47 (m, 2H), 7.46 – 7.38 (m, 2H), 7.33 – 7.25 (m, 1H), 4.38 (q, J = 7.2 Hz, 2H),
1
3
.46 (t, J = 7.2 Hz, 3H). C NMR (101 MHz, CDCl ): δ 189.5, 147.2, 141.6, 140.5, 137.6,
3
31.8, 130.0, 127.4, 126.4, 125.8, 123.6, 122.9, 121.8, 120.7, 119.8, 118.5, 109.0, 37.8, 13.8.
+
HR ESI-MS (M + H) m/z =404.0625 (Cacld for C H NOBr: 404.0645).
2
3
18
5
.4.11. Synthesis of (E)-3-(9-ethyl-9H-carbazol-3-yl)-1-(4-iodophenyl)prop-2-en-1-one (3k).
1
-(4-Iodophenyl)ethanone and 2a were used as reactants to give 3k. Yellow solid, yield: 73%,
1
mp: 173.8–175.5 °C. H NMR (400 MHz, CDCl ) δ 8.39 (s, 1H), 8.15 – 8.07 (m, 4H), 7.82 –
3
7
.96 (m, 3H), 7.58 – 7.48 (m, 2H), 7.46 – 7.40 (m, 2H), 7.34 – 7.26 (m, 1H), 4.36 (q, J = 7.5
1
3
Hz, 2H) 1.47 (t, J = 7.2 Hz, 3H). C NMR (101 MHz, CDCl ) δ 189.8, 147.8, 141.7, 140.5,
3
1
1
28.7, 126.5, 126.4, 126.1, 125.7, 125.4, 123.5, 122.8, 121.9, 120.6, 119.8, 118.5, 109.0, 37.8,
+
3.8. HR ESI-MS (M + H) m/z =452.0514 (Cacld for C H INO: 452.0521).
2
3
18
5
1
8
1
.4.12. Synthesis of (E)-1-(3-chlorophenyl)-3-(9-ethyl-9H-carbazol-3-yl)prop-2-en-1-one (3l).
-(3-Chlorophenyl)ethanone and 2a were used as reactants to give 3l. Yellow solid, yield:
1
3%, mp: 164.7–166.6 °C. H NMR (400 MHz, CDCl ) δ 8.26 (s, 1H), 8.08 (d, J = 7.7 Hz,
3
H), 7.72 – 7.68 (m, 2H), 7.60 – 7.52 (m, 3H), 7.46 – 7.33 (m, 4H), 7.28 (d, J = 7.3 Hz, 1H),
2
0

ACCEPTED MANUSCRIPT
1
3
7
.16 (d, J = 16.0 Hz, 1H), 4.36 (q, J = 7.2 Hz, 2H), 1.44 (t, J = 7.2 Hz, 3H). C NMR (101
MHz, CDCl ) δ 194.2, 148.5, 141.7, 140.5, 139.7, 131.3, 131.0, 130.1, 129.3, 126.8, 126.4,
3
1
26.2, 125.4, 123.6, 123.4, 122.8, 122.2, 120.7, 119.8, 109.0, 37.8, 13.8. HR ESI-MS (M +
+
H) m/z =360.1140 (Cacld for C H ClNO: 360.1150).
2
3
18
5
.4.13. Synthesis of (E)-1-(2-chlorophenyl)-3-(9-ethyl-9H-carbazol-3-yl)prop-2-en-1-one
(3m). 1-(2-Chlorophenyl)ethanone and 2a were used as reactants to give 3m. Yellow solid,
1
yield: 88%, mp: 138.5–140.2 °C. H NMR (400 MHz, CDCl ) δ 8.38 (s, 1H), 8.17 (d, J = 7.7
3
Hz, 1H), 8.10 – 8.04 (m, 2H), 7.99 – 7.94 (m, 1H), 7.84 – 7.76 (m, 1H), 7.60 – 7.50 (m, 3H),
7
.48 – 7.40 (m, 3H), 7.36 – 7.29 (m, 1H), 4.38 (q, J = 7.2 Hz, 2H), 1.47 (t, J = 7.2 Hz, 3H).
1
3
C NMR (101 MHz, CDCl ) δ 189.1, 147.4, 141.6, 140.5, 140.4, 134.8, 132.3, 129.8, 128.5,
3
1
26.7, 126.5, 126.4, 125.6, 123.5, 122.8, 121.8, 120.6, 119.8, 118.3, 108.9, 37.8, 13.8. HR
+
ESI-MS (M + H) m/z =360.1140 (Cacld for C H ClNO: 360.1150).
2
3
18
5
1
8
1
7
.4.14. Synthesis of (E)-3-(9-ethyl-9H-carbazol-3-yl)-1-(2-fluorophenyl)prop-2-en-1-one (3n).
-(2-Fluorophenyl)ethanone and 2a were used as reactants to give 3n. Yellow solid, yield:
1
8%, mp: 154.6–157.1 °C. H NMR (400 MHz, CDCl ) δ 8.35 (s, 1H), 8.13 (d, J = 7.8 Hz,
3
H), 7.97 (d, J = 16.8 Hz, 1H), 7.86 – 7.75 (m, 2H), 7.56 – 7.47 (m, 2H), 7.45 – 7.38 (m, 3H),
.32 – 7.26 (m, 2H), 7.20 – 7.16 (M, 1H), 4.38 (q, J = 7.2 Hz, 2H), 1.46 (t, J = 7.2 Hz, 3H).
1
3
C NMR (101 MHz, CDCl ) δ 189.3, 147.6, 141.5, 141.2, 140.7, 134.3 (d, J = 3.8 Hz),
3
1
1
32.6, 129.5 (d, J = 8.2 Hz), 128.8, 126.9, 126.6, 126.2, 125.3 (d, J = 14.5 Hz), 123.7, 122.8,
21.6 (d, J = 20.4 Hz), 120.2, 119.4, 118.1 (d, J = 25.6 Hz), 108.7, 38.2, 14.6. HR ESI-MS
+
(
M + H) m/z =344.1439 (Cacld for C H FNO: 344.1444).
23
18
2
1

ACCEPTED MANUSCRIPT
5
.4.15.
Synthesis
of
(E)-1-(3,4-dimethoxyphenyl)-3-(9-ethyl-9H-carbazol-3-yl)prop-2-en-1-one
(3o).
1
-(3,4-Dimethoxyphenyl)ethanone and 2a were used as reactants to give 3o. Yellow solid,
1
yield: 72%, mp: 187.9–190.2 °C. H NMR (400 MHz, CDCl ): δ 8.39 (s, 1H), 8.15 (d, J = 7.6
3
Hz, 1H), 8.06 (d, J = 15.4 Hz, 1H), 7.82 – 7.74 (m, 2H), 7.70 – 7.58 (m, 2H), 7.55 – 7.47 (m,
1
2
1
1
H), 7.43– 7.35 (m, 2H), 7.32 – 7.27 (m, 1H), 6.96 (d, J = 8.4 Hz, 1H), 4.40 (q, J = 7.2 Hz,
1
3
H), 4.00 (s, 3H), 3.99 (s, 3H), 1.47 (t, J = 7.2 Hz, 3H). C NMR (101 MHz, CDCl ): δ
3
88.8, 153.0, 149.2, 145.6, 140.5, 131.9, 126.5, 126.2, 126.1, 123.5, 122.9, 122.8, 121.5,
+
20.6, 119.7, 118.7, 111.0, 108.9, 108.8, 56.1, 37.8, 13.8. HR ESI-MS (M + H) m/z
=
386.1751 (Cacld for C H NO : 386.1742).
25 23 3
5
.4.16. Synthesis of (E)-1-(3,4-dimethylphenyl)-3-(9-ethyl-9H-carbazol-3-yl)prop-2-en-1-one
(3p). 1-(3,4-Dimethylphenyl)ethanone and 2a were used as reactants to give 3p. Yellow solid,
1
yield: 71%, mp: 201.5–203.5 °C. H NMR (400 MHz, CDCl ): δ 8.41 (d, J = 1.4 Hz, 1H),
3
8
1
2
1
1
.18 (d, J = 7.7 Hz, 1H), 8.07 (d, J = 15.6 Hz, 1H), 7.96 – 7.80 (m, 3H), 7.62 (d, J = 15.6 Hz,
H), 7.58 – 7.50 (m, 1H), 7.47 – 7.41 (m, 2H), 7.38 – 7.26 (m, 2H), 4.42 (q, J = 7.2 Hz, 2H),
13
.40 (s, 3H), 2.36 (s, 3H), 1.48 (t, J = 7.2 Hz, 3H). C NMR (101 MHz, CDCl ): δ 190.5,
3
46.9, 141.6, 141.4, 140.5, 136.9, 136.6, 129.8, 129.6, 126.3, 126.2, 123.6, 122.9, 121.5,
+
20.7, 119.7, 119.3, 108.9, 37.8, 20.0, 19.8, 13.8. HR ESI-MS (M + H) m/z =354.1861 (Cacld
for C H NO: 354.1852).
25
23
5
.4.17.
Synthesis
of
(E)-3-(9-ethyl-9H-carbazol-3-yl)-1-(3-fluoro-4-methoxyphenyl)prop-2-en-1-one
(3q).
2
2

ACCEPTED MANUSCRIPT
1
-(3-Fluoro-4-methoxyphenyl)ethanone and 2a were used as reactants to give 3q. Yellow
1
solid, yield: 79%, mp: 173.2–175.4 °C. H NMR (400 MHz, CDCl ) δ 8.35 (s, 1H), 8.13 (d, J
3
=
7.7 Hz, 1H), 8.06 – 7.98 (m, 1H), 7.96 – 7.85 (m, 1H), 7.78 (d, J = 8.5 Hz, 1H), 7.54 – 7.47
(
m, 2H), 7.44 – 7.38 (m, 2H), 7.31 – 7.25 (m, 1H), 6.84 – 6.76 (m, 1H), 6.70 – 6.62 (m, 1H),
1
3
4
1
1
3
.38 (q, J = 7.2 Hz, 2H), 3.88 (s, 3H), 1.45 (t, J = 7.2 Hz, 3H). C NMR (101 MHz, CDCl ) δ
3
87.4, 145.7, 141.4, 140.4, 132.8, 132.4 (d, J = 3.3 Hz), 126.5, 126.2, 125.9, 123.4, 122.8,
22.7, 122.6, 121.8, 120.6, 119.6 (d, J = 8.1 Hz), 110.7, 110.6, 108.8, 101.9, 101.6, 55.8,
+
7.7, 13.8. HR ESI-MS (M + H) m/z =374.1562 (Cacld for C H NO F: 374.1551).
2
4
20
2
5
.4.18.
Synthesis
of
(E)-3-(9-ethyl-9H-carbazol-3-yl)-1-(2-fluoro-4-methylphenyl)prop-2-en-1-one
(3r).
1
-(2-Fluoro-4-methylphenyl)ethanone and 2a were used as reactants to give 3r. Yellow solid,
1
yield: 76%, mp: 181.4–183.6 °C. H NMR (400 MHz, CDCl ) δ 8.33 (s, 1H), 8.12 (d, J = 7.7
3
Hz, 1H), 7.99 – 7.93 (m, 1H), 7.77 (d, J = 8.6 Hz, 1H), 7.62 (d, J = 6.8 Hz, 1H), 7.53 – 7.47
(
m, 1H), 7.42 (m, 3H), 7.12 – 7.02 (m, 1H), 6.89 – 6.81 (m, 1H), 6.76 – 6.72 (m, 1H), 4.40 (q,
1
3
J = 7.2 Hz, 2H), 2.39 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H). C NMR (101 MHz, CDCl ) δ 189.4,
3
1
1
3
5
60.5, 158.0, 146.6, 141.5, 140.4, 134.0, 131.0 (d, J = 3.8 Hz), 126.4, 126.3, 125.7, 123.4,
23.0, 122.9, 122.8, 121.9, 120.6 (d, J = 21.2 Hz), 119.7 (d, J = 8.3 Hz), 116.2, 116.0, 108.9,
+
7.7, 20.5, 13.8. HR ESI-MS (M + H) m/z =358.1592 (Cacld for C H FNO: 358.1602).
2
4
20
.4.19.
Synthesis
of
(E)-1-(4-chlorophenyl)-3-(9-(4-fluorobenzyl)-9H-carbazol-3-yl)prop-2-en-1-one
(4a).
1
-(4-Chlorophenyl)ethanone and 2b were used as reactants to give 4a. Yellow solid, yield:
1
7
3%, mp: 231.6–233.7 °C. H NMR (400 MHz, CDCl ) δ 8.40 (s, 1H), 8.17 (d, J = 7.7 Hz,
3
2
3

ACCEPTED MANUSCRIPT
1
7
H), 8.09 – 7.99 (m, 3H), 7.82 – 7.70 (m, 1H), 7.58 – 7.45 (m, 4H), 7.37 (d, J = 8.5 Hz, 2H),
1
3
.34 – 7.30 (m, 1H), 7.14 – 7.10 (m, 2H), 7.70 – 7.92 (m, 2H), 5.50 (s, 2H). C NMR (101
MHz, CDCl ) δ 189.2, 162.3, 146.8, 142.1, 141.1, 138.9, 137.0, 132.2 (d, J = 3.8 Hz), 129.9,
3
1
1
28.9, 128.5 (d, J = 14.5 Hz), 126.7 (d, J =5.6 Hz), 126.4, 123.7, 123.0, 121.7, 120.7, 120.3,
+
18.8, 115.9, 109.4, 109.3, 46.1. HR ESI-MS (M + H) m/z =440.1123 (Cacld for
C H ClFNO: 440.1115).
28
19
5
.4.20.
Synthesis
of
(E)-1-(4-chlorophenyl)-3-(9-(4-methylbenzyl)-9H-carbazol-3-yl)prop-2-en-1-one
(4b).
1
8
1
–
1
1
-(4-Chlorophenyl)ethanone and 2c were used as reactants to give 4b. Yellow solid, yield:
1
2%, mp: 256.4–258.2 °C. H NMR (400 MHz, CDCl ) δ 8.38 (s, 1H), 8.15 (d, J = 7.7 Hz,
3
H), 8.06 – 7.98 (m, 3H), 7.77 – 7.69 (m, 1H), 7.54 – 7.44 (m, 4H), 7.40 – 7.34 (m, 2H), 7.32
1
3
7.28 (m, 1H), 7.13 – 6.97 (m, 4H), 5.47 (s, 2H), 2.28 (s, 3H). C NMR (101 MHz, CDCl ) δ
3
89.3, 147.0, 142.3, 141.2, 138.8, 137.5, 137.1, 133.5, 130.0, 129.6, 128.9, 126.7, 126.6,
26.4, 126.2, 123.7, 122.9, 121.7, 120.6, 120.2, 118.7, 109.5, 109.5, 46.6, 21.1. HR ESI-MS
+
(
M + H) m/z =436.1463 (Cacld for C H ClNO: 436.1458).
29
22
5
.4.21. Synthesis of (E)-3-(9-benzyl-9H-carbazol-3-yl)-1-(4-chlorophenyl)prop-2-en-1-one
(4c). 1-(4-Chlorophenyl)ethanone and 2d were used as reactants to give 4c. Yellow solid,
1
yield: 89%, mp: 239.6–241.2 °C. H NMR (400 MHz, CDCl ) δ 8.40 (s, 1H), 8.17 (d, J = 7.7
3
Hz, 1H), 8.09 – 7.97 (m, 3H), 7.81 – 7.69 (m, 1H), 7.56 – 7.44 (m, 4H), 7.41 – 7.37 (m, 2H),
1
3
7
1
.33 – 7.25 (m, 4H), 7.18 – 7.09 (m, 2H), 5.54 (s, 2H). C NMR (101 MHz, CDCl ) δ 189.2,
3
46.9, 142.3, 141.2, 138.8, 137.1, 136.5, 129.9, 128.9, 128.8, 127.7, 126.7, 126.6, 126.4,
2
4

ACCEPTED MANUSCRIPT
+
1
26.2, 123.7, 122.9, 121.7, 120.6, 120.2, 118.7, 109.5, 109.4, 46.8. HR ESI-MS (M + H) m/z
=
421.1233 (Cacld for C H ClNO: 421.1239).
2
8
20
5
.4.22.
Synthesis
of
(E)-1-(4-chlorophenyl)-3-(9-(2-fluorobenzyl)-9H-carbazol-3-yl)prop-2-en-1-one
(4d).
1
7
1
7
6
1
1
1
-(4-Chlorophenyl)ethanone and 2e were used as reactants to give 4d. Yellow solid, yield:
1
7%, mp: 271.3–272.8 °C. H NMR (400 MHz, CDCl ) δ 8.40 (s, 1H), 8.17 (d, J = 7.7 Hz,
3
H), 8.09 – 7.99 (m, 3H), 7.82 – 7.72 (m, 1H), 7.59 – 7.45 (m, 4H), 7.42 (d, J = 8.4 Hz, 2H),
.34 – 7.26 (m, 1H), 7.25 – 7.20 (m, 1H), 7.16 – 7.09 (m, 1H), 6.97 – 6.89 (m, 1H), 6.81 –
1
3
.73 (m, 1H), 5.59 (s, 2H). C NMR (101 MHz, CDCl ) δ 189.2, 160.4, 146.8, 142.2, 141.1,
3
38.8, 137.0, 129.9, 129.2 (d, J = 3.8 Hz), 128.9, 128.1 (d, J = 8.1 Hz), 126.7, 126.6, 126.4,
24.5 (d, J = 14.3 Hz), 123.7, 123.6, 123.0, 121.7, 120.7, 120.3, 118.8, 115.6 (d, J = 20.4 Hz),
+
09.4, 109.3 (d, J = 25.3 Hz), 40.5. HR ESI-MS (M + H) m/z =440.1123 (Cacld for
C H ClFNO: 440.1115).
28
19
5
.4.23.
Synthesis
of
(E)-1-(4-chlorophenyl)-3-(9-(2,4-difluorobenzyl)-9H-carbazol-3-yl)prop-2-en-1-one
(4e).
1
8
1
7
2
1
-(4-Chlorophenyl)ethanone and 2f were used as reactants to give 4e. Yellow solid, yield:
1
3%, mp: 264.2–265.7 °C. H NMR (400 MHz, CDCl ) δ 8.40 (s, 1H), 8.17 (d, J = 7.7 Hz,
3
H), 8.07 – 7.99 (m, 3H), 7.83 – 7.71 (m, 1H), 7.59 – 7.45 (m, 4H), 7.40 (d, J = 8.3 Hz, 2H),
.37 – 7.29 (m, 1H), 6.92 – 6.86 (m, 1H), 6.80 – 6.74 (m, 1H), 6.70 – 6.64 (m, 1H), 5.54 (s,
1
3
H). C NMR (101 MHz, CDCl ) δ 189.2, 162.5, 160.3, 146.7, 142.0, 140.9, 138.9, 137.0,
3
29.9, 129.0 (d, J = 3.8 Hz), 128.9, 126.8, 126.7, 126.5, 123.8, 123.0, 121.7, 120.7, 120.5,
2
5

ACCEPTED MANUSCRIPT
1
19.5 (d, J = 8.3 Hz), 118.9 (d, J = 14.6 Hz), 111.6 (d, J = 23.1 Hz), 109.2, 109.2, 104.3,
+
4
0.1. HR ESI-MS (M + H) m/z =457.1021 (Cacld for C H ClF NO: 457.1015).
2
8
19
2
5
.4.24.
Synthesis
of
(E)-1-(4-chlorophenyl)-3-(9-(3-fluorobenzyl)-9H-carbazol-3-yl)prop-2-en-1-one
(4f).
1
7
1
–
-(4-Chlorophenyl)ethanone and 2g were used as reactants to give 4f. Yellow solid, yield:
1
6%, mp: 271.8 – 273.4 °C. H NMR (400 MHz, CDCl ) δ 8.41 (s, 1H), 8.18 (d, J = 7.7 Hz,
3
H), 8.07 – 7.99 (m, 3H), 7.82 –7.70 (m, 1H), 7.58 – 7.45 (m, 4H), 7.38 – 7.30 (m, 3H), 7.26
1
3
7.21 (m, 1H), 6.98 – 6.89 (m, 2H), 6.82 (d, J = 9.5 Hz, 1H), 5.52 (s, 2H). C NMR (101
MHz, CDCl ) δ 189.2, 164.4, 162.0, 146.7, 142.1, 141.1, 139.2, 138.9, 137.0, 130.6 (d, J =
3
3
.8 Hz), 129.9, 128.9, 126.8, 126.7, 126.5, 123.7, 123.0, 121.9 (d, J = 8.3 Hz), 121.8, 120.7,
1
20.4, 118.9, 114.7, 113.5 (d, J = 15.3 Hz), 109.3 (d, J = 24.6 Hz), 46.3. HR ESI-MS (M +
+
H) m/z =440.1123 (Cacld for C H ClFNO: 440.1115).
2
8
19
5
.4.25.
Synthesis
of
(E)-3-(9-(4-chlorobenzyl)-9H-carbazol-3-yl)-1-(4-chlorophenyl)prop-2-en-1-one
(4g).
1
7
1
-(4-Chlorophenyl)ethanone and 2h were used as reactants to give 4g. Yellow solid, yield:
1
4%, mp: 283.2 – 285.0 °C. H NMR (400 MHz, CDCl ) δ 8.40 (s, 1H), 8.17 (d, J = 7.7 Hz,
3
H), 8.07 – 7.99 (m, 3H), 7.75 – 7.69 (m, 1H), 7.55 – 7.45 (m, 4H), 7.40 – 7.28 (m, 3H), 7.27
1
3
(
d, J = 8.7 Hz, 2H), 7.06 (d, J = 8.5 Hz, 2H), 5.50 (s, 2H). C NMR (101 MHz, CDCl ) δ
3
1
1
89.2, 146.7, 142.1, 141.3, 138.9, 137.1, 135.0, 133.6, 129.9, 129.1, 128.9, 127.7, 126.8,
26.7, 126.4, 123.7, 123.0, 121.7, 120.7, 120.4, 118.9, 109.4, 109.3, 46.2. HR ESI-MS (M +
+
H) m/z =456.0867 (Cacld for C H Cl NO: 456.0868).
2
8
19
2
2
6

ACCEPTED MANUSCRIPT
5
.4.26.
Synthesis
of
(E)-1-(4-chlorophenyl)-3-(9-(4-nitrobenzyl)-9H-carbazol-3-yl)prop-2-en-1-one
(4h).
1
7
1
7
1
1
-(4-Chlorophenyl)ethanone and 2i were used as reactants to give 4h. Yellow solid, yield:
1
1%, mp: 263.1 – 265.4 °C. H NMR (400 MHz, CDCl ) δ 8.43 (s, 1H), 8.20 (d, J = 7.7 Hz,
3
H), 8.14 (d, J = 8.7 Hz, 2H), 8.07– 7.97 (m, 3H), 7.81 – 7.71 (m, 1H), 7.58 – 7.46 (m, 4H),
1
3
.38 – 7.30 (m, 3H), 7.27 (d, J = 9.5 Hz, 2H), 5.63 (s, 2H). C NMR (101 MHz, CDCl ) δ
3
89.2, 147.6, 146.5, 143.9, 141.9, 140.8, 138.9, 137.0, 129.9, 128.9, 127.2, 127.0, 126.8,
24.2, 123.9, 123.1, 121.8, 120.9, 120.7, 119.2, 113.7, 109.1, 109.0, 46.2. HR ESI-MS (M +
+
H) m/z =466.1164 (Cacld for C H ClN O : 466.1159).
2
8
19
2
3
5
.5. Biological evaluation
5
.5.1. Topo II mediated DNA relaxation assay
Topo II assay kit from TopoGEN was used to determine the effects of CDCAs on DNA
relaxation catalyzed by Topo II. Relaxation assay were carried out according to the
manufacturer’s instructions with minor modifications. The reaction mixture of 200 ng of
pBR322 DNA (TaKaRa, Kyoto, Japan) plasmid and 1 U of Topo II was incubated without
and with CDCAs in a final volume of 20 µL in Topo II reaction buffer (50 mM Tris-HCl, pH
-
1
8
.0, 150 mM NaCl, 10 mM MgCl , 2 mM ATP, 0.5 mM DTT, and 30 µg·mL BSA). The
2
mixture incubated for 30 min at 37 °C and was terminated with 6 × stop buffer (5 µL per 20
µL reaction volume). Stop buffer contained 5% sarcosyl, 0.02% bromophenol blue, and 25%
glycerol. Reaction products were analyzed on a 1% agarose gel in TAE buffer (40 mM
Tris-acetate and 2 mM EDTA) and electrophoresis for 1.5 h at 75 V. Gels were stained for 30
2
7

ACCEPTED MANUSCRIPT
-
1
min in an aqueous solution of Ged Red (0.5 µg·mL ). DNA bands were visualized through
transillumination with UV light and then photographed with an Alpha Innotech digital
imaging system.
5
.5.2. Topo II mediated DNA cleavage assay
In brief, Topo II (6 U), 0.1 µg pBR322 DNA, and 100 µM 3d, 3g, or etoposide were employed
in a total of 20 µL of Topo II buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM MgCl2,
-1
2
mM ATP, 0.5 mM DTT, and 30 µg·mL BSA). After incubating for 6 min at 37 °C to reach
the cleavage relegation equilibrium, cleavage intermediates were trapped by adding 2 µL of 1%
SDS, followed by 2 µL of 250 mM NaEDTA at pH 8.0. Proteinase K was added (2 µL of 0.8
-
1
mg·mL ), and reactions were incubated for 30 min at 45 °C to digest the Topo II. Samples
were mixed with 2 µL of agarose gel loading buffer (30% sucrose, 0.5% bromophenol blue,
and 0.5% xylene cyanole FF in 10 mM Tris-HCl, pH 7.9), heated at 72 °C for 2 min, and
subjected to electrophoresis in a 1% agarose gel in TAE buffer for 1 h at 75 V. Gels were
-1
stained for 30 min in an aqueous solution of Ged Red (0.5 µg·mL ) and kept on
electrophoresis for 30 – 45 min at 75 V. Cleavage was monitored by the conversion of
negatively supercoiled plasmid to nicked DNA. DNA bands were visualized by UV light and
photographed with an Alpha Innotech digital imagingsystem.
5
.5.3. Topo I mediated DNA relaxation assay
In this relaxation assay, pBR322 DNA was used to determine the effects of CDCAs on DNA
relaxation catalytic by Topo I (TaKaRa, Kyoto, Japan). The reaction mixture was prepared
according to the manufacturer’s instructions with minor modifications, and incubated at 37 °C
2
8