Synthesis and biological evaluation of novel indole-pyrazoline hybrid derivatives as potential topoisomerase 1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2019.126925

A series of novel indole-pyrazoline hybrid derivatives were designed, synthesized, and evaluated for topoisomerase 1 (Top1) inhibitory activity. Top1-mediated relaxation assays showed that our synthesized compounds had variable Top1 inhibitory activity. A

Full text of DOI:10.1016/j.bmcl.2019.126925

Journal Pre-proofs
Synthesis and Biological Evaluation of Novel Indole-Pyrazoline Hybrid Deriv-
atives as Potential Topoisomerase 1 Inhibitors
Bing Shu, Qian Yu, De-xuan Hu, Tong Che, Shang-shi Zhang, Ding Li
PII:
S0960-894X(19)30907-2
DOI:
https://doi.org/10.1016/j.bmcl.2019.126925
BMCL 126925
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
27 October 2019
15 December 2019
17 December 2019
Please cite this article as: Shu, B., Yu, Q., Hu, D-x., Che, T., Zhang, S-s., Li, D., Synthesis and Biological Evaluation
of Novel Indole-Pyrazoline Hybrid Derivatives as Potential Topoisomerase 1 Inhibitors, Bioorganic & Medicinal
Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.126925
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Synthesis and Biological Evaluation of Novel Indole-Pyrazoline
Hybrid Derivatives as Potential Topoisomerase 1 Inhibitors
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Bing Shua*, Qian Yua, b, De-xuan Hub, Tong Chec, Shang-shi Zhangc, and Ding
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Lib*
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aSchool of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006,
PR China.
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bSchool of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou University
City, 132 Waihuan East Road, Guangzhou 510006, P. R. China.
cCenter for Drug Research and Development, Guangdong Pharmaceutical University,
Guangzhou 510006, PR China.
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*Corresponding author E-mail address: shubing@gdpu.edu.cn (B. Shu);
liding@mail.sysu.edu.cn (D. Li).
Abstract:
A series of novel indole-pyrazoline hybrid derivatives were designed,
synthesized, and evaluated for topoisomerase 1 (Top1) inhibitory activity.
Top1-mediated relaxation assays showed that our synthesized compounds had
variable
Top1
inhibitory
activity.
Among
these
compounds,
3-(5-(naphthalen-1-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-1-(phenylsulfonyl)-1H
-indole (6n) was found to be a strong Top1 inhibitor with better inhibitory activity
than CPT and hit compounds. Our further experiments rationalized the mode of action
for this new type of inhibitors, which showed no significant binding to supercoiled
DNA.
Key words: topoisomerase 1, indole-pyrazoline hybrid derivatives, CPT, supercoiled
DNA

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DNA topoisomerase 1 (Top1) is overexpressed among various cancer cell lines
and widely considered as an essential nuclear enzyme to regulate the cellular
DNA-topological processes such as DNA replication, transcription, chromatin
assembly, recombination, and chromosome segregation, by relaxing supercoiled DNA
to alter DNA topology.[1-2] The active site tyrosine OH group (Tyr723 in human
Top1) could act as a nucleophile to attack DNA phosphodiester backbone at 3′-end of
the cutting strand to produce a single strand break, and subsequently attached to the
broken DNA, resulting in the formation of a binary enzyme-DNA covalent complex
(Top1 CC). Top1 inhibitors, such as Camptothecin (Fig. 1), bound to the interface of
Top1 CC, resulting in stable Top1 CC to prevent further religation of the broken DNA.
When replication and transcription machineries encounter the trapped Top1 CC, DNA
damage was generated, triggering cell death.[3-5] Hence the ability of inhibiting the
function of Top1, or trapping Top1 CC could be some effective strategies for inducing
DNA damage, which could eventually induce cancer cell apoptosis.[6]
On the base of mechanisms for molecular actions, compounds targeting Top1 or
Top1 CC could be classified into two categories: one is Top1 poison and the another
is Top1 catalytic inhibitor.[6] Top1 poisons are able to trap Top1 CC and prevent
further religation of the DNA single-strand breaks, resulting in DNA strand breaks
accumulation and DNA damage. Top1 catalytic inhibitors could inhibit catalytic DNA
cleavage reaction and prevent formation of Top1-DNA covalent complex, which are
different from Top1 poisons.[7] The first known Top1 poison is Camptothecin (CPT,
Fig. 1). To date, two structurally modified derivatives, topotecan and irinotecan have
been approved by FDA for cancer treatment, and other derivatives such as Belotecan
and 10-hydroxy Camptothecin, are in clinical trials (Fig. 1).[8-10] In addition, several
non-Camptothecin Top1 poisons have been reported and studied in preclinical or
clinical trials, including indolocarbazole (BMS-250749), dibenzonaphthyridinones
(ARC-111 and Genz-644282), indenoisoquinolines indotecan (LMP400), indimitecan
(LMP776) and MJ-III-65 LMP744, (Fig. 1).[11-12] Unfortunately, some camptothecins
suffered from disadvantages such as poor chemical stability, drug resistance,
gastrointestinal toxicity or significant side effects. Therefore, further discovery and

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development of novel Top1 inhibitors are required.
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Fig.1 Chemical structures of Camptothecin, Topotecan, Belotecan, Irinotecan,
10-hydroxy camptothecin, ARC-111, Genz-644282, LMP400, LMP776, LMP744,
BMS-250749, hit compound IP-1, IP-2, and designed compounds 6a-6t.
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In order to find small molecular ligands targeting Top1, we performed
Top1-mediated relaxation assay to screen our synthesized compounds, including
acridone derivatives, imidazole derivatives, ketone derivatives, quinoline derivatives,
and acridine derivatives. CPT, a well-known Top1 poison, was used as a positive
control. Top1-mediated relaxation assay has been previously characterized and
performed to block the formation of Top1-DNA covalent complex and research the
Top1 inhibitory activities of the targeted compounds.[5, 13-14] Briefly, supercoiled DNA
and Top1-DNA covalent complex could produce different migration bands in agarose
gel. Generally, DNA could be recognized by Top1 to attack the DNA phosphodiester
backbone, and covalently attached to the end of the broken DNA with formation of a
transient enzyme-DNA covalent complex (known as Top1 CC), resulting in a slower
migration band. On the contrary, supercoiled DNA alone could immigrate faster.[5,
13-14]
As shown in Fig. S1-2, we found that two indole-pyrazoline hybrids, IP-1 and
IP-2, (Fig. 1) could block the formation of Top1 CC to release supercoiled DNA at

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300 μM, indicating that indole-pyrazoline hybrids could be potential hits of Top1
inhibitors. However, other compounds such as acridone derivatives (B01-06),
imidazole derivatives (5a-5c), ketone derivatives (D01-03), quinoline derivatives
(Q01-04), and acridine derivatives (AD01-02) did not show obvious Top1 inhibition
ability at 300 μM concentration (Fig. S1-2). In order to investigate the effect for
introduction of various substituents and understand the structure-activity relationship
(SAR), a series of dual aryl substituted indole-pyrazoline hybrid derivatives (6a-6t,
Fig. 1) were designed and synthesized. Besides, their Top1 inhibition activity, binding
affinity to supercoiled DNA and molecular docking were evaluated and reported here.
The synthetic pathway for indole-pyrazoline hybrid derivatives 6a-6t was shown
in Scheme 1. 3-Acetylindole (1) was used as starting material to react with substituted
aldehydes (2a-h) through aldol condensation reaction catalyzed with NaOH in EtOH
solution to give the desired intermediates 2-en-1-ones (3a-h), which were used
directly for the next step reaction. Then, the obtained intermediates 3a-h were reacted
with phenylhydrazine hydrochloride under weak alkaline condition such as
triethylamine in EtOH solution to give key compounds 4a-h. Finally, the target
indole-pyrazoline hybrid derivatives 6a-6t were obtained through nucleophilic
substitution reaction between 4a-h and various arylsulfonyl chlorides (5a-c) in a
solution of benzyltriethylammonium chloride (TEBAC) and sodium hydroxide in
dichloromethane (Table I). The synthesized indole-pyrazoline hybrid derivatives
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were purified and their structures were determined by using H NMR, C NMR and
mass spectra. All compounds used for subsequent biological evaluations were
analyzed by using HPLC with their purity determined to be more than 95%.
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Scheme I. Reagents and conditions: (i) 5 °C, NaOH, EtOH; (ii) phenylhydrazine
hydrochloride, TEA, EtOH, 80 oC, yield 60-90%; (iii) TEBAC, NaOH,
dichloromethane, room temperature, yield 52-89%.
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Top1 inhibitory activity of the synthesized compounds was measured by using
Top1-mediated relaxation assay with CPT as
a
positive control, and
semiquantitatively expressed relative to CPT at 25 μM as follows: +++, more than
80% of the activity; ++, between 40% and 79% of the activity; +, less than 40% of the
activity, indicating no significant interaction between ligand and Top1. After the
DNA was incubated with Top1 in the absence or presence of final concentration of 25
μM compounds, the obtained mixture samples were electrophoresed, Gelred-stained
and results summarized as shown in Table 1 and Table S1. We found that hit
compounds IP-1 and IP-2 did not show inhibitory activity at 25 μM. Compared with
the hit compounds IP-1 and IP-2, some synthesized compounds displayed Top1
inhibitory activity. Among these compounds, compounds 6a, 6d-6e with
electron-donating OCH3 substitutive group at Ar1, and compounds 6q, 6s with
electron-donating isopropyl substitutive group had increased Top1 inhibition activity,
indicating that electron-donating group at Ar1 is helpful to improve Top1 inhibitory
activity. In comparison, compounds 6f-6g with weak electron-withdrawing p-Cl
substitutive group, compounds 6k-6m with weak electron-donating o-Br substitutive
group or compound 6t with electron-withdrawing m-NO2 substitutive group did not
show Top1 inhibition activity. Compound 6n with large aromatic group naphthalene
ring at Ar1, exhibited higher Top1 inhibition activity than CPT and hit compounds,
possibly because the π-π stacking interaction between 6n and Top1 blocked the
formation of Top1-DNA complex, which indicated that π-π stacking interaction could
be necessary for strong Top1 inhibitory activity. However, introduction of large
aromatic groups at Ar2 could produce unfavorable effect. For example, compounds
6c, 6g, 6j, 6m, 6p with naphthalene at Ar2 did not show any inhibition activity. As
mentioned before, compound 6n had the best inhibition activity to Top1, and
therefore, was selected for further investigation.

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Table 1
The structures and the in vitro Top1 inhibitory activities of the indole-pyrazoline
hybrid derivatives
Comp.
6a
Ar1
(p-OCH3)Ph-
(p-OCH3)Ph-
(p-OCH3)Ph-
(3,4,5-3OCH3)Ph-
(3,4,5-3OCH3)Ph-
(p-Cl)Ph-
Ar2
Ph-
Relaxation assaya
++
+
6b
6c
(p-CH3)Ph-
naphthoic-1-yl-
Ph-
+
6d
6e
++
++
+
(p-CH3)Ph-
Ph-
6f
6g
(p-Cl)Ph-
naphthoic-1-yl-
Ph-
+
6h
6i
Ph-
++
++
+
Ph-
(p-CH3)Ph-
naphthoic-1-yl-
Ph-
6j
Ph-
6k
6l
(o-Br)Ph-
+
(o-Br)Ph-
(p-CH3)Ph-
naphthoic-1-yl-
Ph-
+
6m
6n
6o
(o-Br)Ph-
+
naphthoic-1-yl-
naphthoic-1-yl-
naphthoic-1-yl-
(p-isopropyl)Ph-
(p-isopropyl)Ph-
(p-isopropyl)Ph-
(m-NO2)Ph-
-
+++
++
+
(p-CH3)Ph-
naphthoic-1-yl-
Ph-
6p
6q
6r
++
+
(p-CH3)Ph-
naphthoic-1-yl-
Ph-
6s
++
+
6t
IP-1
IP-2
-
+
-
-
+
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aTop1 inhibitory activity was semi-quantitatively expressed relative to CPT as
follows: +++, more than 80% of the activity; ++, between 40% and 79% of the
activity; +, less than 40% of the activity, indicating no significant interaction between
ligand and Top1. Every experiment was repeated at least twice independently.

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In order to further study the inhibition activity of preferred compound 6n to
Top1, a semi-quantitative Top1-mediated relaxation assay was performed.[7] As
shown in Fig. 2A-B, 6n had strong inhibitory activity in a dose-dependent manner.
The levels of supercoiled DNA were increased to 61% and 74%, in relation to whole
supercoiled DNA upon treatment with 25 and 125 μM compound 6n, respectively.
Besides, 6n showed stronger inhibitory activity than CPT, and under the same
condition only 41% and 48% supercoiled DNA remained for CPT. On the contrary,
CPT showed relatively high inhibitory activity at relatively low concentrations (1 and
5 μM).
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Fig.2 Semi-quantitative Top1-mediated relaxation assay. SC, supercoiled DNA; R,
relaxed DNA. Lane 1, supercoiled pBR322 DNA alone; Lane 2, DNA and Top1;
Lanes 3-7, DNA, Top1, and tested compound CPT (A) and 6n (B) at 0.2, 1, 5, 25,
125 μM.
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In order to know whether the inhibition effect was caused by affinity of the
compounds to Top1 or DNA, we studied the binding affinities of the synthesized
indole derivatives for supercoiled DNA. SPR assay was used as a highly efficient
method to evaluate the interaction between of the ligands and targeted DNAs.[15-17]
The binding constants KD values for the binding affinity of the indole-pyrazoline
hybrids to duplex DNA were determined as shown in Table S2 and Fig. S3A-B.
Ethidium bromide (EB), a well-known DNA intercalator, was used as a positive
control. We found that EB exhibited good binding affinity to duplex DNA with a KD
value of 1.2 μM (Fig. S3B). However, all synthesized compounds did not show
significant binding at 50 μM concentration, which might indicate no specific
interaction between the ligands and the DNA.

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In addition, the interaction between compound 6n and DNA was also studied by
using Top1-mediated unwinding assay as reported previously.[7] EB was used as a
positive control. As shown in Fig. S4, upon addition of increasing concentration of
EB, very clear unwinding effect appeared with supercoiled pBR322 DNA as
substrate. However, the representative compound 6n had no obvious unwinding
effect, which was different from the classical intercalation interaction.
Molecular docking was performed to provide a possible binding mode of 6n with
Top1-DNA complex. The ligand was docked to the ligand binding site of the
Top1−DNA−ligand ternary complex (PDB ID: 1SC7). The result of 6n with the
highest score was shown in Fig. 3. The aromatic framework stacked with the +1 and
-1 base pairs at the DNA cleavage site of Top1 CC. The sulfonyl group formed two
hydrogen bonds with Lys425 (3.0 Å) and Glu356 (3.6 Å), playing important role for
its Top1 inhibitory activity. In addition, Top1 has a moderate binding site with
Trp416 interacting with the aromatic group substituted with the sulfonyl group,
through a hydrophobic interaction. Importantly, the naphthalene ring stacked on the
scissile strand bases (G and T) through π-π stacking interaction, and the indole ring
stacked on the bases of non-cleaved strand (C and A). These results were consistent
with our experimental data for inhibition of Top1 by 6n.
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Fig. 3. Hypothetical binding mode of 6n in the ternary Top1-DNA-drug complex
(derived from PDB ID: 1SC7). The carbon atoms of 6n are shown in green, and the
base pairs are displayed in blue lines. All distances are measured from heavy atom to
heavy atom.
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In summary, twenty indole-pyrazoline hybrid derivatives were designed,
1
synthesized, and characterized by using H and 13C NMR and mass spectroscopic
analysis. Top1-mediated relaxation assays showed that compound 6n could act as
Top1 inhibitor with higher inhibitory activity than CPT and hit compounds. Besides,
our studies suggested that 6n stacked with the +1 and -1 base pairs at the DNA
cleavage site of Top1 CC without significant binding interaction with supercoiled
DNA. Further research about the biological activity and antitumor mechanism of
these potent compounds are currently going on.
Conflict of interest and Acknowledgements
The authors declare no competing financial interests. We thank the Guangdong
Medical Research Fund (Grant NO: B2019003), and Guangdong Provincial Education
Department Fund (Grant NO: 51340208) for financial support of this study.
Supplementary section
Details of experimental procedures, biological assays and analytical data for
novel compounds associated with this article can be found on pages S2-S17.
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Declaration of interests
☐ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

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☐ The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
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