Synthesis and biological evaluation of novel carbazole-rhodanine conjugates as topoisomerase II inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.03.017

In this study, a series of carbazole-rhodanine conjugates was synthesized and evaluated for their Topoisomerase II inhibition potency as well as cytotoxicity against a panel of four human cancer cell lines. Among these thirteen compounds, 3a, 3b, 3g, and

Full text of DOI:10.1016/j.bmcl.2018.03.017

Accepted Manuscript
Synthesis and biological evaluation of novel carbazole-rhodanine conjugates as
topoisomerease II inhibitors
Hong Jiang, Wen-Jin Zhang, Peng-Hui Li, Jian Wang, Chang-Zhi Dong, Kun
Zhang, Hui-Xiong Chen, Zhi-Yun Du
PII:
S0960-894X(18)30196-3
https://doi.org/10.1016/j.bmcl.2018.03.017
BMCL 25671
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 January 2018
4 March 2018
5 March 2018
Please cite this article as: Jiang, H., Zhang, W-J., Li, P-H., Wang, J., Dong, C-Z., Zhang, K., Chen, H-X., Du, Z-
Y., Synthesis and biological evaluation of novel carbazole-rhodanine conjugates as topoisomerease II inhibitors,
Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.03.017
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Title Page
Synthesis and biological evaluation of novel carbazole-rhodanine conjugates as
topoisomerease II inhibitors
Hong Jianga, Wen-Jin Zhanga, Peng-Hui Lia,*, Jian Wangb, Chang-Zhi Donga, c, Kun Zhanga, d,
Hui-Xiong Chena, e, Zhi-Yun Dua*,
aInstitute of Natural Medicine & Green Chemistry, School of Chemical Engineering and Light
Industry, Guandong University of Technology, Guangzhou 510006, China.
bSchool of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, China.
cUniversite Paris Diderot, Sorbonne Paris Cite, ITODYS, UMR 7086 CNRS, 15 rue J-A de
Baif, 75270 Cedex 13 Pairs, France
dWuyi University, Jiangmen 529020, China.
eCNRS, 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
*Corresponding authors:
E-mail addresses: liphsiming@163.com (P. H. Li); zhiyundu@gdut.edu.cn (Z. Y. Du).
Abstract
In this study, a series of carbazole-rhodanine conjugates was synthesized and
evaluated for their Topoisomerase II inhibition potency as well as cytotoxicity against
a panel of four human cancer cell lines. Among these thirteen compounds, 3a, 3b, 3g,
and 3h possessed Topoisomerase II inhibition potency at 20 μM. Mechanism study
revealed that these compounds may function as Topo II catalytic inhibitors. It was
found that the electron-withdrawing groups on the phenyl ring of compounds played
an important role on enhancing both enzyme inhibition and cytotoxicity.
Keyword
Carbazole
Rhodanine
Topoisomerase II
1

Cytotoxic
Hybrid molecule
Human topoisomerase has been recognized as an important target in anticancer
drugs discovery. Two types of topoisomerase exist in humans, namely, type I
topoisomerase (Topo I) and type II topoisomerase (Topo II). Both isomers are nuclear
enzymes essential to resolve topological problems that occur during DNA
transcription, replication, and chromosome segregation1-3.Topo II is the target for
several commonly prescribed anticancer drugs, including etoposide, doxorubicin, and
mitoxantrone4. Since the identification of amsacrine as a Topo II-targeted anticancer
drug in 1984, about 50% of current treatment protocols still employ at least one drug
directed against topoisomerases5. However, drug resistance and the severe side effects
of Topo II-targeted drugs are an issue6-8. Novel and safer Topo II inhibitors for better
anticancer therapeutics are highly active research field9-14
.
Fig. 1. Hybrid molecule as novel Topo II inhibitors
A hybrid molecule comprising two pharmacophoric groups is often used for the
design of new drugs15. The hybrid may have increased potencies and/or modified
2

selectivity profiles compared to the corresponding single molecule. Accordingly,
several hybrid compounds have been designed, synthesized and identified as novel
Topo II inhibitors (Compounds 1, 2, 3, and 4; Fig. 1)16-19. Our previous studies
demonstrated that these conjugates were generally more potent activity than the
individual molecule alone (Compounds 5 and 6; Fig. 1)20,21. Continuing with our
interest in searching novel anticancer agents that targeting Topo II with high potency,
we designed and synthesized novel carbazole-rhodanine conjugates, which contain
biologically active carbazole and rhodanine moiety, as shown in Fig. 2.
Carbazole are of great interest due to their broad spectrum of biochemical effects
and pharmaceutical functions, including antimalarial, antibacterial and antitumor
activity22. In particular, there have been intense research efforts in recent years in the
design and development of carbazole derivatives as a new class of Topo II
inhibitors21,23,24. For example, compounds 7, 8, and 9 (Fig. 2) are function as potential
Topo II inhibitors and display high cytotoxicity against human cancer cell lines25-27
.
Another pharmacophoric group in the target compounds is rhodanine moiety,
which has been reported to present a variety of pharmacological activities, including
antibacterial, antiparasitic, anti-microbial, and anticancer28. In recent years, a lot of
efforts have focused on the anticancer activity of rhodanine compounds. For example
(Fig. 2), GSK1059615 (compound 10) is a reversible inhibitor of PI3Kα, which shows
potential anticancer activity29. Compound 11 displays significant anti-proliferation
activity against several cancer cell lines30.
3

Fig. 2. Design of carbazole-rhodanine conjugates as novel Topo II inhibitors
In the previous study, we reported a series novel Topo II inhibitors via a
pharmacophore hybridization strategy20,21,31,32. We found that carbazole derivatives
containing chalcone analogs displayed strong Topo II inhibition potency and
antiproliferation activity. The SAR study revealed that the benzyl group with different
substituent linked to carbazole play an important role on the activity21. To pursue our
mission to promote novel anticancer agents that target Topo II with high potency, we
introduced carbazole and rhodanine moiety into a new hybrid scaffold and linked the
benzyl groups with different substituent to carbazole[Fig. 2]. We found that these
newly synthesized compounds displayed potent Topo II inhibitory activity and
cytotoxic activity against four human cancer cell lines.
The synthetic route of carbazole-rhodanine conjugates is shown in scheme 1.
N-alkylation of carbazole was carried out with appropriate benzyl bromides in the
presence
of
KOH
to
form
1a–1m
in
53–84%
yields.
The
N-subsititued-9H-3-carbaldehydes (2a-2m) were synthesized from 1a–1m through
4

Vilsmeier-Haack reaction in 63–87% yields. The target compounds 3a–3m were
synthesized from 2a-2m through the Hornor-Wadsworth-Emmons reaction of
rhodanine moiety in 66–91% yield. The synthesized compounds were characterized
by 1H NMR, 13C NMR, IR, and HRMS (ESI), which was in full accordance with their
depicted structures and the structure of these compounds are shown in Table. 1.
Scheme 1. Synthesis route of carbazole-rhodanine conjugates. Reagents and conditions: (i) DMF,
appropriate benzyl bromide, K2CO3, rt, 24 h; (ii) POCl3, DMF, DCM, 0 – 90 °C, 8 h; (iii)
Rhodanine-3-acetic acid, NaOAc, acetic acid, 110 °C, 4 h.
The resulting synthetic 3a–3m were evaluated for their cytotoxic activities against
human lung adenocarcinoma A549, human cervical cancer Hela cells, human acute
leukemia HL-60 cells, and human chronic myeloid leukemia K562 cells using MTT
assay as described by Mosmann with modification31. Etoposide was taken as a
positive control. As shown in Table 1, most of compounds exhibited significant
cytotoxicities toward four cancer cell lines with low to moderate micromolar values of
IC50. Almost all the compounds showed a cytotoxic selectivity for the HL-60 and
K562 cell lines, and several compounds such as 3b, 3g, and 3h displayed a nanomolar
level IC50 toward HL-60, which was consistent with etoposide. It was found that the
substituent on the benzyl group had a significant effect on cytotoxic activity.
5

Compounds 3b, 3g, and 3h with a strong electron-withdrawing group (-NO2, -CN,
and –CF3, respectively) on the benzyl ring exhibited the most potent cytotoxic activity
against all the tested cancer cell lines with an IC50 ranging between 0.96–7.51 μM,
0.71–8.20 μM, and 0.43–5.06 μM, respectively. In the contrast, compounds 3e with
an electron-donating group (-CH3) on the benzyl ring exhibited poor cytotoxic
activity.
Table 1. Cytotoxic and Topo II inhibition activity of carbazole-rhoadnine conjugates.
IC50 (μM)
Topo II
Inhibitiona
Cpd.
R1
Hela
11.72
7.51
>50
A549
HL-60
3.51
K562
6.92
3a
2-F,4-Br
4-NO2
4-Br
9.44
3.94
++
++
-
3b
0.96
2.70
3c
>50
12.67
8.57
27.99
19.13
29.60
13.36
1.85
3d
4-F
>50
>50
+
3e
4-CH3
4-Cl
>50
>50
17.81
4.86
-
3f
28.30
5.06
8.20
15.14
>50
16.18
2.18
-
3g
4-CN
4-CF3
3-F,4-F
H
0.43
0.71
4.17
++
++
+
3h
3.40
2.16
3i
12.36
32.20
22.55
19.80
13.59
13.71
7.31
3j
3k
14.56
3.84
21.38
12.57
12.84
11.75
8.45
-
2-F,4-F
3-F
>50
-
3l
>50
8.29
1.82
-
3m
2-F
21.26
32.05
-
Etoposide
-
0.33
++
aThe relative potency Topo II inhibition of compounds are present as follows: -, no detectable
activity at 20 μM; +, weak activity at 20 μM; ++, strong activity at 20 μM.
6

Next, these compounds were tested in vitro against Topo II using etoposide as
positive control and pBR322 DNA as plasmid10-12. The results are present in Fig. 3
and Table 1 showed that most of tested compounds inhibited the Topo II-mediated
relaxation of pBR322 DNA at 50 μM (Fig. 3A). Among these compounds, 3a, 3b, 3g,
and 3h displayed strong Topo II inhibition potency at 20 μM (Fig. 3B). Specially,
compound 3b exhibited the most potent Topo II inhibition at 10 μM (Fig. 3C). It was
observed that compounds 3b, 3g, and 3h with a strong electron-withdrawing group
(-NO2, CF3, and –CN, respectively) on the benzyl ring, exhibited better activities than
those with a weak electron-withdrawing group (3c, 3d, 3f, 3i, 3k, 3l, and 3m). The
4-methyl substitution of the benzyl group (3e) did not improve the inhibition activity
at all. These results indicated that carbazole-rhodanine conjugates possessed
inhibitory activity on Topo II and the substitutions on the benzyl ring with an
electron-withdrawing group played an important role on the activity. Topo I-mediated
DNA relaxation assay was also carried out to test whether these compounds also
target Topo I11,12. Results presented in Fig. 3D showed that camptothecin, a
well-known Topo I inhibitor, strongly inhibited Topo I activity, while none of the
tested compounds possessed inhibitory activity on Topo I in 100 μM. The result
revealed that these compounds selectively inhibited the activity of Topo II.
7

Fig.3. (A, B, and C) Topo II inhibition potency of compounds with various concentrations. Lane
D, pBR322 DNA; lane T, pBR322 DNA + Topo II; lane E, pBR322 DNA+ Topo II + etoposide
(100 μM); other lanes, pBR322 DNA + Topo II + compounds. (D) Topo I inhibition potency of
compounds. Lane D: pBR322 DNA; lane T: pBR322 DNA + Topo I; lane C: pBR322 DNA +
Topo I + camptothecin (100 μM); other lanes: pBR322 DNA + Topo I + compounds (100 μM).
Topo II inhibitors are classified according to their ability to induce DNA
double-strand breaks with (Topo II poisons) or without (Topo II catalytic inhibitors)
the formation of a cleaved complex, reflecting different inhibition mechanisms1-3. To
gain insight into the mode of action, Topo II-mediated DNA cleavage assay was
carried out to verify whether these compounds function as Topo II poisons or Topo II
catalytic inhibitors. As shown in Fig. 4A, the well known Topo II poison etoposide
produces discrete bands of the linear DNA, corresponding to its poison behavior. In
8

contrast to etoposide, the linear form of the DNA is not visible in 3g and 3h even at
50 μM. These observations indicated that the tested compounds function as Topo II
catalytic inhibitor.
Topo II catalytic inhibitors have two types, namely, DNA intercalators and
nonintercalators. 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 ethidium bromide (EB; a classic DNA intercalator) as
control. As shown in Fig. 4B, EB is able to transform the relaxed DNA into
supercoiled DNA in the presence of excess Topo I. Compounds 3g and 3h displayed
the similar effect of EB in a dose-dependent manner, suggesting that these compounds
are intercalative Topo II catalytic inhibitors.
Fig. 4. (A) Topo II mediated cleavage of plasmid pBR322 DNA. Lane D: pBR322 DNA; lane T:
pBR322 DNA + Topo II; lane E: 100 μM etoposide + Topo II + pBR322 DNA; other lanes: 3g or
3h + Topo II + pBR322 DNA. The positions of supercoiled DNA (S), relaxed DNA (R), linear
DNA (L), and nicked DNA (N) are indicated. (B) The unwinding assay of 3g and 3h. Lane D,
pBR322 DNA; lane T, pBR322 DNA + Topo I; other lanes, pBR322 DNA + Topo I + 3g, 3h, or
EB with different concentrations.
9

In conclusion, we have synthesized a series of carbazoles-rhodanine conjugates
and evaluated their pharmacological activity. Of the compounds 3a, 3b, 3g, and 3h
could selectively impede Topo II function at 20 μM without affecting Topo I catalytic
activity. Mechanism studies revealed that these compounds might function as
intercalative Topo II catalytic inhibitors. These compounds displayed potential
cytotoxic activity against four human cancer cell lines. The electron-withdrawing
group on the benzyl ring enhancing both enzyme inhibition and cytotoxicity of these
compounds. This study may provide valuable information to researchers working on
the development of novel antitumor agents targeting Topo II.
Acknowledgements
The authors declare no competing financial interests. This research was supported by
National Science Foundation of China (grant number 21272043).
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Highlight
1. Synthesis of 13 novel carbazole-rhodanine conjugates.
2. These compounds as potent intercalative Topo II catalytic inhibitors.
3. These compounds displayed potential anti-proliferation activity.
13