Design, synthesis of 4,5-diazafluorene derivatives and their anticancer activity via targeting telomeric DNA G-quadruplex

Article abstract of DOI:10.1016/j.ejmech.2019.06.012

In our work, 19 novel 4,5-diazafluorene derivatives (11a-d, 12a-d, 13a-d, 14a-c, 15c, 16a-c) bearing a 1,3-disubstituted pyrazol/thioxothiazolidinone or thioxothiazolidinone-oxadiazole moieties were designed, synthesized, preliminarily explored for their

Full text of DOI:10.1016/j.ejmech.2019.06.012

Accepted Manuscript
Design, synthesis of 4,5-diazafluorene derivatives and their anticancer activity via
targeting telomeric DNA G-quadruplex
Kang Zhou, Jiachun Liu, Xuqiong Xiong, Mei Cheng, Xiaolin Hu, Suresh Narva,
Xiaoyin Zhao, Yanling Wu, Wen Zhang
PII:
S0223-5234(19)30529-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.012
EJMECH 11415
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 March 2019
Revised Date: 2 June 2019
Accepted Date: 3 June 2019
Please cite this article as: K. Zhou, J. Liu, X. Xiong, M. Cheng, X. Hu, S. Narva, X. Zhao, Y. Wu,
W. Zhang, Design, synthesis of 4,5-diazafluorene derivatives and their anticancer activity via
targeting telomeric DNA G-quadruplex, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.06.012.
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ACCEPTED MANUSCRIPT
Design, synthesis of 4,5-diazafluorene derivatives and their
anticancer activity via targeting telomeric G-quadruplex
Kang Zhoua,b,1, Jiachun Liua,b,1, Mei Chenga,b, Xiaolin Hua,b, Suresh Narvaa,b, Xiaoyin
Zhaoa,b, Xuqiong Xionga,b, Yanli Wanga,b, Yanling Wuc,*, Wen Zhanga,b,*
This kind of 4,5-diazafluorene derivatives with an extended planar aromatic system can interact with the
telomeric G-quadruplexes, inhibit the telomerase activity and consequently cancer cells proliferation.

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Design, synthesis of 4,5-diazafluorene derivatives and their anticancer activity
via targeting telomeric DNA G-quadruplex
Kang Zhoua,b,1, Jiachun Liua,b,1, Xuqiong Xionga,b, Mei Chenga,b, Xiaolin Hua,b, Suresh Narvaa,b,
Xiaoyin Zhaoa,b, Yanling Wuc,*, Wen Zhanga,b,*
a
b
Lab of Chemical Biology and Molecular Drug Design, College of Pharmaceutical Science and Institute of
Drug Development & Chemical Biology, Zhejiang University of Technology, Hangzhou, 310014, China.
cLab of Molecular Immunology, Virus Inspection Department, Zhejiang Provincial Center for Disease Control
and Prevention, Hangzhou, 310051, China
*Corresponding author. Lab of Chemical Biology and Molecular Drug Design, College of Pharmaceutical
Science, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou, 310014, PR China; Tel: +86-571-
88871507; Fax: +86-571-88871507; e-mail: wzhang63@zjut.edu.cn
1 These authors contributed equally to this work.
Abstract
In our work, 19 novel 4,5-diazafluorene derivatives (11a-d, 12a-d, 13a-d, 14a-c, 15c, 16a-
c) bearing a 1,3-disubstituted pyrazol/thioxothiazolidinone or thioxothiazolidinone-oxadiazole
moieties were designed, synthesized, preliminarily explored for their antitumor activities and in
vitro mechanism. All compounds showed different values of antiproliferative activity against
A549, AGS, HepG2 and MCF-7 cell lines through CCK-8. Especially, the compound 14c
exhibited the strongest activity and best selectivity against A549 cells with an IC50 1.13 µM and
an SI value of 7.01 relative to MRC-5 cells, which was better than cisplatin (SI = 1.80) as a
positive control. Experimental results at extracellular level demonstrated that compounds 14a-c
could strongly interact with the G-quadruplex(es) formed in a 26nt telomeric G-rich DNA, in
particular, the 14c exhibits quite strong binding affinity with an association equilibrium constant
(KA) of 7.04(±0.16) × 107 M-1 and more than 1000-fold specificity to G4-DNA over ds-DNA and
Mut-DNA at the compound/G4-DNA ratio of 1:1. Further trap assay ascertained that compounds
14a-c owned strong inhibitory ability of telomerase activity in A549 cells, suggesting that these
compounds have great possibility to target telomeric G-quadruplexes and consequently indirectly
inhibit the telomerase activity. In addition, it is worthy of note that the remarkable inhibitory
effects of 14a-c on the mobility of tested cancer cells were observed by wound healing assays.
Furthermore, molecular docking and UV-Vis spectral results unclose the rationale for the
interaction of compounds with such G-quadruplex(es). These results indicate that the growth and

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metastasis inhibition of cancer cells mediated by these 4,5-diazafluorene derivatives possibly
result from their interaction with telomeric G-quadruplexes, suggesting that 4,5-diazafluorene
derivatives, especially 14c, possess potential as anticancer drugs.
Keywords: 4,5-diazafluorene, telomeric G-quadruplex, telomerase activity, cancer cells,
antiproliferative activity
1. Introduction
Cancer morbidity and mortality are rapidly growing worldwide and cancer has become a
serious public health problem. The report of GLOBOCAN 2018 estimates on cancer indicates
that there will be 18.1 million new cases of cancer and 9.6 million deaths occurred from cancer in
2018 [1]. Especially, it is estimated that the cancer morbidity and mortality rates are high in
China, rank the top one in the world, accounting for over one-fifth of the new cancer cases and
cancer deaths occurred in the world in 2018. In both sexes combined, lung cancer is the most
commonly diagnosed cancer (11.6% of the total cases) and the leading cause of cancer death
(18.4% of the total cancer deaths). It is noteworthy that non-small cell lung carcinomas (NSCLC)
accounts for about 85% of all lung cancers. NSCLCs are relatively insensitive to chemotherapy,
compared to small cell carcinoma. The mortality rate of NSCLC patients remains high with
relapse, the five-year survival rate in stages I through IV significantly decreases due to the
advancement of the disease and is 47% for stage I, 30% for stage II, 10% for stage III, and 1% for
stage IV [2–5]. In addition, breast cancer is the most familiar malignancy among women. Over
the past decade, the number of breast cancer cases has been rising. According to statistics, there
are 2.08 million new breast cancer cases, approximately 11.6% of all newly diagnosed cancer
cases in 2018 [1]. Gastric cancer is still the third most common worldwide malignant tumor
disease; there exists a high regional difference in its morbidity. Especially in developing
countries, the morbidity of gastric cancer is high, estimated to account for about 8% of all new
cases of cancer, the number of new cases will rise to 1.7 million cases by 2030 [6,7].
Hepatocellular carcinoma (HCC), one of the most common malignancies in the world, is the third
leading cause of cancer death, the morbidity of HCC, which was highest in Asia and sub-Saharan
Africa, has increased in Western countries [8]. It was reported that 0.841 million new liver cancer
cases each year occurred in both men and women, with the mortality rate increased to 10.2% and

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5.6% respectively [1]. Collectively, various data indicate that cancer is a huge threat to human
health and a great challenge to medical research.
In recent years, a variety of clinical anticancer strategies (eg. chemotherapy, radiotherapy
and surgical resection) have been enduring to develop and progress. These treatments can
alleviate the symptoms of patients to a certain extent, but some cancers cannot be completely
cured by these therapies, the overall 5-year survival rate of patients is still very low. There are
also a number of disadvantages that cannot be ignored, such as patient compliance, side effects of
radiotherapy and chemotherapy, limitations of surgical resection, cell resistance, etc. These
problems should be responsible for the low survival rate of the patients. Therefore, the new target
therapy agents that highly effectively and preferentially induce apoptosis of cancer cells rather
than normal cells will bring hopes for the future.
On the other hand, telomerase shows dynamic activity in 85-90% cancer cells. Telomerase
can maintain the length of telomere as well as promote the process of cell cycle, so that the
cancer cells endow more vitality and avoid apoptosis in process of cell proliferation, while it is
also associated with the invasion and metastasis of cancer cells [9–13]. Hence, inhibition of the
telomerase activity may be a novel strategy for cancer chemotherapy. Special DNA sequences
containing (TTAGGG)n repeats in human telomeres [14–16] can fold into G-quadruplex (G4-
DNA) structures under approximate physiological conditions, which can effectively inhibit the
activity of telomerase and promote apoptosis [17–19], as depicted in Fig. 1. In addition, studies
have found that telomeric G4-DNA ligands can trigger telomere dysfunction and DNA damage
response, thereby inducing cellular senescence or apoptosis [20]. At the same time, with the study
of G-quadruplex probes, the dynamic folding and unfolding of G4-DNA can be traced and this
lays a foundation for further research [21-23]. Therefore, the telomeric G4-DNA ligands have
great potential to be developed as new target agents for the treatment of cancers.

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Fig. 1. The effects of telomerase on the proliferation, invasion and metastasis of cancer cells and development of
telomerase targeted drugs mediated by formation and stabilization of DNA G-quadruplex.
Scheme 1. Structure of the leading compounds and design strategy of the target compounds.
In our previous work [24], the phenanthroline derivative 1 (Scheme 1) exhibited potential
antiproliferative activity against A549 cell line with the half maximal inhibitory concentration
(IC50) of 1.48 µM and also showed significant inhibitory effect on the growth, migration of A549
cells by its possible interaction with telomeric DNA to stabilize G4-DNA structure to some
extent. However, the compound 1 had some defects that cannot be ignored, such as poor
solubility, weak stability to telomeric G4-DNA and inferior selectivity to cancer cells. This
implied that the compound 1, acting as a stabilizer of telomeric G-quadruplex, needs further
structural modification as a leading compound to develop cancer inhibitors. The another
compound 2 (Scheme 1), a kind of α-carboline derivatives, showed high levels of cytotoxicity
against HL-60, COLO 205, Hep3B, and H460 cells with IC50 values of 0.30, 0.49, 0.70, and 0.80
µM respectively [25]. Based on these previous findings, it is worthwhile to study whether the

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bioisostere structure (4,5-diazofluorene) of the carbazole and phenanthroline structures has the
better antitumor activity or not. Simultaneously considering too strong rigid conjugate plane of
1H-imidazo[4,5-f][1,10]phenanthroline skeleton bearing a pyrazole moiety designed in previous
work [24], possibly leading to their interaction instability and weak interaction with target DNA’s
and inferior cell selectivity, using a combination of bioisosterism principle using compounds 1
and 2 as the leading compounds, we designed, synthesized fifteen novel compounds (11a–d, 12a–
d, 13a–d, 14a–c) bearing a 1,3-disubstituted pyrazol moiety by substitution of the 1H-
imidazo[4,5-f][1,10]phenanthroline skeleton with a 4,5-diazofluorene skeleton (Scheme 1) and
evaluated their antitumor activity. On the other hand, rhodanine derivatives with a wide range of
biological activities, such as antibacterial [26], antiviral [27], antitumor [28] and lower blood
glucose [29], have already attracted plenty of attention. Compound 3 (Scheme 1) showed
significant antitumor activity against CEM cells with an IC50 value of less than 10 µM, indicating
that the rhodanine moiety of the compound played a key role in antitumor property [30]. In
addition, 1,3,4-oxadiazole is a privileged scaffold incorporated in many compounds presenting
potent antitumor activity. Compound 4 displayed highly effective inhibitory activity against Hela
cells with an IC50 value of 4.26 µM [31]. Likewise, based on combination principle, other four
novel compounds (15, 16a–c) (Scheme 1) bearing
a
thioxothiazolidinone or
thioxothiazolidinone-oxadiazole moieties were designed, synthesized and evaluated for their
antitumor activity.
2. Results
2.1 Chemistry
The synthetic route of the 4,5-diazafluorene derivatives 11–16 is depicted in Scheme 2.
Compounds 5, 6, 7a–d, 8a–d, 9a–d, 10a–c, 2-(4-oxo-2-thioxothiazolidin-3-yl)acetic acid and 4-
substituted benzohydrazides were reported in a previous paper [24,32–35]. Compounds 11a–d,
12a–d, 13a–d, and 14a–c were synthesized through the Knoevenagel reaction by heating a
mixture of compound 6 and 7a–d, 8a–d, 9a–d, 10a–c in the presence of ammonium acetate and
glacial acetic acid at 110 °C for 6 h. Compound 15 was synthesized through the Knoevenagel
reaction, then 15 was subjected to the condensation reaction with 4-substituted benzohydrazides
under phosphorus oxychloride condition (105 °C) for 5 h to obtain compounds 16a–c. The

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1
structures of the synthesized compounds were confirmed by H NMR, 13C NMR and Mass
spectral analysis.
Scheme 2. Synthesis of 4,5-diazafluorene target compounds 11–16.
2.2 In vitro anti-tumor activity evaluation by CCK-8 assay
To evaluate the antiproliferative abilities of novel synthesized compounds 11–16 against
A549 cells, AGS cells, HepG2 cells, MCF-7 cells and MRC-5 cells (normal human fetal lung
fibroblast cells as a reference) by Cell Counting Kit-8 technique [24], initially, four kinds of
cancer cells were exposed to 10 µM of compounds 11-16 for 36 h, respectively and the
progressive cell growth inhibition observed. As shown in Fig. 2A and Fig. S1, all the synthesized
derivatives presented different values of antiproliferative activity against all tested cancer cells. In
particular among all compounds, three compounds 14a-c exhibited the most potent activity at 10
µM, which was stronger than that of cisplatin as a positive control. Secondly, as shown in Fig.
2B, 2C, 2D, 2E and Table 1, after four kinds of cancer cells were treated with two-fold serial

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dilution of 14a–c and cisplatin for 36 h, it was found that 14a-c exhibited potent activity against
all tested cancer cells with IC50 values ranging from 1.13 µM to 11.77 µM. More notably, the 14c
possessed the best antiproliferative activity with IC50 values of 1.13 ± 0.02 µM, 1.81 ± 0.10 µM,
5.27 ± 0.07 µM against A549, AGS, MCF-7 cells respectively, and the strongest inhibitory effect
on A549 cells among all the tested four cancer cells. Furthermore, 14c displayed stronger
inhibitory activity than cisplatin with IC50 values of 11.90 ± 0.26 µM, 3.33 ± 0.01 µM, 6.89 ±
0.17 µM, respectively, and other similar structural tested compounds 14a, b at each of 1, 2 and 4
µM against A549 cells, AGS cells, MCF-7 cells but HepG2 cells.
Fig. 2. Viability of A549 cells treated with 10 µM of compounds 11–16 (A)，and A549 cells (B)，AGS cells (C)，
HepG2 cells (D)，MCF-7 cells (E)，and MRC-5 cells (F) treated with compounds 14a-c with 0, 1, 2, 4, 8 and 16
µM for 36 h，respectively. Cell viability was determined using Cell Counting Kit-8 assay. Cisplatin: positive
control; 1% DMSO is 1% DMSO solution in RPMI 1640 as a reference.

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Also, as shown in Fig. 2F and Table 1, after the treatment of MRC-5 cells with 14a–c and
cisplatin as a positive control, cytotoxicity results were obtained. The compounds 14a–c
presented moderate cytotoxicity against normal MRC-5 cells with IC50 values in the range of
5.93–9.81 µM, which are 1.72–7.01-fold higher than those of A549 cells. Among compounds
14a–c, 14c presented the strongest cytotoxicity against A549 cells over against normal MRC-5
cells with a biggest selectivity-index (SI) value of 7.01 and also showed better selectivity than
cisplatin with an SI value of 1.80.
Taken together, all these outcomes suggest that the derivatives 14a–c showed strong
cytotoxicity against four tested cancer cells, especially A549 cells, and 14c possessed strongest
inhibitory activity and best selectivity for A549 cells, implying that 14c may be explored as a
promising A549 cell inhibitor.
Table 1.
IC50 values (µM) for the A549, AGS, HepG2, MCF-7, and MRC-5 cell lines treated with the
desired compounds 14a-c and a positive control drug (cisplatin). The values are expressed as the
mean ± SD (triplicates).
Cancer cells
Cisplatin
14a
14b
14c
11.90±0.26 (1.80)b
3.33±0.01 (6.42)
3.26±0.07 (6.56)
6.89±0.17 (3.10)
21.39±0.29
3.44±0.10a(1.72)
2.93±0.12 (2.02)
3.32±0.04 (1.79)
6.70±0.27 (0.88)
5.93±0.09
4.15±0.14a (2.36)
5.52±0.20 (1.78)
6.43±0.13 (1.52)
11.77±0.29 (0.83)
9.81±0.34
1.13±0.02a (7.01)
1.81±0.10 (4.25)
3.48±0.08 (2.21)
5.27±0.07 (1.46)
7.92±0.15
A549
AGS
HepG2
MCF-7
MRC-5
a Statistically different to lung cancer cell line (A549 and MRC-5) (P < 0.05).
b Selectivity-Index
2.3 Interpretation of interaction mechanism of the novel compounds with the target(s) by
electrophoretic mobility shift assay (EMSA)
In order to explore whether the novel 4,5-diazafluorene derivatives with moderate
conjugated planar aromaticity can induce the formation and stabilization of telomeric G-
quadruplexes (G4-DNA’s) and consequently mediate the cancer cell apoptosis. We first carried
out electrophoretic mobility shift assay (EMSA) at the extracellular molecular level. The
sequences of newly designed 26nt telomeric DNA’s as target(s) in this experiment for the
examination of G4-DNA formation were listed in Table 2, containing a Tel26nt G-rich DNA (G-

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rich DNA) with four three-guanine repeats ((GGGTTA)4) which may form G4-DNA’s, a
complementary strand of Tel26nt C-rich DNA (C-rich DNA), a Tel26nt double-stranded DNA
(ds-DNA), and a Tel26 mutated single-stranded DNA (Mut-DNA) with a single G-to-C mutation
completely impossible to form any G4-DNA structures under our experimental conditions (a Tris
buffer containing 10 mM Tris–HCl, 10 mM KCl, 0.1 mM EDTA, pH = 7.4). To measure the
ability of 14a–c and 16c, which contains a rhodamine moiety and the same p-bromophenyl group
as 14c with strong inhibitory activity and possesses poor cytotoxicity to four examined cancer
cells, to induce the formation of and stabilize G4-DNA’s from the 26nt telomeric DNA sequences,
the properties of electrophoretic mobility shift for tested oligonucleotides treated with a serial
dilution of compounds were observed by using a Bio-Rad imaging detector (Fig. 3 and Fig. S2A).
Table 2.
The sequences of DNA oligomers used in this study.
DNA oligomers
DNA sequences
Tel26nt G-rich DNA (G-rich DNA)
Tel26nt C-rich DNA (C-rich DNA)
5'-TTAGGGTTAGGGTTAGGGTTAGGGTT-3'
5'-AATCCCAATCCCAATCCCAATCCCAA-3'
Tel26nt mutated single-stranded DNA (Mut-DNA)
Tel26nt double-stranded DNA (ds-DNA)
5'-TTAGCGTTAGCGTTAGCGTTAGCGTT-3'
5'-TTAGGGTTAGGGTTAGGGTTAGGGTT-3'
3'-AATCCCAATCCCAATCCCAATCCCAA-5'
5'-biotin–G-rich DNA
5'-biotin–Mut-DNA
5'-biotin–ds-DNA
5'-biotin–TTAGGGTTAGGGTTAGGGTTAGGGTT-3'
5'-biotin–TTAGCGTTAGCGTTAGCGTTAGCGTT-3'
5'-biotin–(TTAGGG)4TT-TATA-AA(CCCTAA)4-3'
As shown in Fig. 3, at least two kinds of G4-DNA conformations are formed in G-rich DNA
alone under our experimental conditions in gels, one for migrating faster and darker band is
attributable to an intramolecular G-quadruplex [36] and the other for migrating slower and very
faint band possibly ascribed to intermolecular G4-DNA’s, and(or) higher order structures relative
to a DNA Marker alone on Lane 1 in gel [36–43], but precise molecular structures should be
further interpreted by other high-resolution techniques such as nuclear magnetic resonance
(NMR) and X-ray crystallography. With increasing concentration of 14a–c and 16c, the faster-
moving species gradually decreased in a compound concentration-dependent manner and its
decreased amount and rate were different with addition of different compounds; among them, the
change upon addition of 14c was most noticeable and that upon addition of 16c a little. The

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slower-moving bands near 20bp, possibly being a mixture of intermolecular G4-DNA’s, higher
order structures and(or) compound/G4-DNA compounds, greatly changed in the presence of 14a–
c and 16c, relative to bands of G-rich DNA alone. However, as seen in Fig. 3, the species of both
slower and faster-moving bands gradually were converted to other some complicated products
with addition of more compounds 14a–c (≥3 µM), which possibly contain several species of
higher order structures and(or) compound/G4-DNA’s complexes [38,43], whereas no such change
for 16c was observed. In particular, 14c at the concentration of 12 µM could completely convert
the faster-migrating species to less lower-migrating ones near 20bp and more smearing species of
higher order structures and(or) compound/G4-DNA’s complexes. These data strongly suggest that
the examined compounds could differentially interact with 26nt telomeric G4-DNA’s to form
novel higher order structures and(or) compound/G4-DNA complexes and convert original DNA
structural species in the absence of compounds to the slower-migrating ones, with an order of
compounds exhibiting strongest such ability to induce the formation of and stabilize G4-DNA
structures: 14c>14a>14b>16c, which is in accord with the results at cellular level by CCK-8
assay.
Fig. 3. The ability of four 4,5-diazafluorene derivatives 14a–c and 16c to promote the formation of G4-DNA
structures by EMSA in a 26nt telomeric single-stranded DNA sequence (G-rich DNA). Lane 1: DNA Marker alone;
Lane 2: G-rich DNA alone in 1% DMSO containing buffer; Lane 3-6: G-rich DNA + compound 16c; Lane 7-10: G-
rich DNA + compound 14a; Lane 11-14: G-rich DNA + compound 14b; Lane 15-18: G-rich DNA + compound 14c.
Final concentration of DNA’s is 2 µM for single-stranded DNA (in single strand); the increasing concentration of
each compound is 1.5, 3, 6 and 12 µM from left to right.
In addition, as shown in Fig. 2S, the mobility properties of three kinds of DNA’s: C-rich
DNA, Mut-DNA, ds-DNA as negative oligonucleotides, and a 26nt telomeric G-rich RNA

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sequence, (UUAGGG)4UU, were also observed in the presence and absence of 14c with strongest
affinity to G4-DNA. Results indicated that 14c had no influence on bands for C-rich DNA, Mut-
DNA and G-rich RNA, only a bit influence on ds-DNA at a higher concentration of 24 µM which
is the two-fold concentration maximally used in the experiment in Fig. 3. Interestingly, based on
the moving position of bands for the C-rich DNA relative to the Marker, it was supposed that it
adopted a freedom coil form, not a completely linear form under our experimental conditions.
Collectively, these results demonstrate at extracellular molecular level that 4,5-diazafluorene
derivatives 14a–c and 16c, especially 14a–c, could specifically bind to the 26nt telomeric G4-
DNA and induce the conversion between species of and stabilize telomeric G4-DNA structures.
2.4 Tm values for telomeric G-quadruplexes in the presence/absence of 4,5-diazafluorene
derivatives
Melting curves are widely used to determine thermodynamic properties of folded nucleic
acid/protein structures including their stability and interaction with ligands [44,45]. The melting
temperature (Tm, °C), which is the mid-point of a melting curve at which the examined molecular
structures is 50% unfolded, is an important parameter to indicate the stability of examined nucleic
acid/protein structures in the presence/absence of ligands. Generally, the melting curve with a
standard forward “S” type for duplex nucleic acids and one with a standard inverted “S” type for
G4-DNA’s are measured due to hyperchromicity at 260 nm [46] and hypochromicity at 295 nm
[47], respectively. Thus, in order to assess binding affinity of 14a–c and 16c with G-rich DNA’s,
we measured melting curves in the presence/absence of 4,5-diazafluorene derivatives 14a–c and
16c by monitoring the UV absorbance of the G-rich DNA (Table 2) at 295 nm as a function of
temperature and obtained the Tm values. As shown in Table 3 and Fig. S3, the melting curves of
the G-rich DNA presented a standard inverted “S” type at 295 nm which is characteristic of a G-
quadruplex structure in the presence/absence of 14a–c and 16c, indicating that the G-rich DNA
could form a G4-DNA structure and the compounds may bind to such structure under experiment
conditions. Furthermore, the ∆Tm values of compounds 14a–c and 16c increased in a
concentration-dependent manner compared to the Tm (38.33 ± 0.07 °C) of the G-rich DNA alone.
It was noteworthy that the ∆Tm values for 14c are biggest relative to those for 14a, 14b and 16c at
each of used concentrations, of which the biggest ∆Tm value for 14c at 8 µM is 24.34 °C and the

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smallest one for 14b at 2 µM is 4.82 °C, implying that 14c could most strongly interact with and
stabilize the telomeric G4-DNA’s among all tested compounds.
Table 3.
Tm (°C) values for Tel-26nt G-rich DNA in the presence/absence of 4,5-diazafluorene derivatives
14a-c and 16c.
14a
14b
14c
Tm (°C) a
16c
Compound
concentration
0 µΜ
2 µΜ
4 µΜ
8 µΜ
38.33±0.07
43.97±0.11
45.57±0.09
48.56±0.06
38.33±0.07
43.15±0.04
43.96±0.06
45.71±0.09
38.33±0.07
53.11±0.08
56.08±0.05
62.67±0.11
38.33±0.07
43.44±0.09
44.91±0.12
46.98±0.13
aStandard deviation is given.
2.5 Specific interaction of 4,5-diazafluorene derivatives with telomeric G-quadruplexes
Circular dichroism (CD) spectroscopy is regard as an important method to explore the DNA
secondary structure, in particular G-quadruplex structure [48–50], but X-ray diffraction or NMR
spectroscopy. To clearly clarify the specific binding of 4,5-diazafluorene derivatives to target
DNA’s at extracellular molecular level, we examined the interaction properties of both
compounds 14c and 16c with 26nt telomeric DNA’s (G-rich DNA and ds-DNA) and Mut-DNA
(Table 2) by using the CD technique. The 14c and 16c are two representative compounds of 4,5-
diazafluorene derivatives, the former exhibiting strongest inhibitory activity and best selectivity
for A549 cells in CCK-8 assay (Fig. 2, Table 1) and strongest affinity to 26nt telomeric G-rich
DNA in EMSA (Fig. 3) and UV-melting experiments (Table 3); the latter with a
thioxothiazolidinone-oxadiazole moiety having weaker inhibitory activity against examined
cancer cells (Fig. 1) and weakest affinity to 26nt telomeric G-rich DNA in EMSA (Fig. 3).

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Fig. 4. The ability of 4,5-diazafluorene derivatives to specifically interact with G-quadruplex structures formed in a
26nt telomeric single stranded DNA sequence (G-rich DNA in Table 2) by CD (A and B) and SPR (C). Arrows at
240-245 nm (A) and 240-241 nm (B) denote the change of negative peak of telomeric G-quadruplex with the
increase of the 14C and 16C concentration, respectively; an arrow at 270 nm (A and B) denotes the should band of a
3+1 telomeric G-quadruplex structure; arrows at 290-292 nm (A) and 290-291.5 nm (B) denote the change of
positive peak of telomeric G4-DNA with the increase of the 14C and 16C concentration, respectively; arrows near
298.5 nm (A) and 300.5 nm (B) denote the isoelliptic points; arrows at 358 nm (A) and 377 nm (B) denote the
change of ICD positive band from 309-430 nm (A) and 311-435 nm (B) with the increase of the 14C and 16C
concentration, respectively. CD spectra for 26nt telomeric G-rich DNA sequences of human chromosome in 10 mM
K+ by titration of the compounds at the ratio of a compound to DNA (r), 0.0, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 at
concentrations of a compound ranging from 0.0 µM (red curve, highest positive peak for DNA alone) to 24 µM (cyan
curve, lowest negative peak). All experiments were performed in a buffer (pH 7.4) containing 10 mM Tris–HCl, 10
mM KCl, 0.1 mM EDTA, and a sample of 4 µM G-rich DNA (in single strand) dissolved in the buffer was incubated
for 24 h after annealing at 95 °C. Red solid curve: DNA alone; red dotted line: compound alone. (C) A typical SPR

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sensorgram for the interaction of the 14C with the G-quadruplex structure formed in a G-rich DNA (Table 2)
immobilized on the surface of a sensor chip SA. The binding response is changed with time. The data are globally
fitted to a 1:1 interaction model with a mass transfer effect. Experimental curves are shown in color and fitting
curves in black. The association phase was allowed to run for 300 sec to reach steady state and the dissociation phase
for 280 sec for all concentrations used. Concentrations of the 14C were used according to arrow direction: 6.25
(yellow), 12.5 (magenta), 25.0 (cyan), 50.0 (spring green), 100 (blue), 200 (gray), 400 (dark magenta), 600 (sky
blue), 800 (sea green), and 1000 nM (dark blue, highest curve), respectively. Each experiment was carried out in
triplicate.
Table 4.
Binding kinetic constants of 14c to DNA’s by SPRa.
DNA
ka [M-1s-1]
kd [s-1]
KA [M-1]
6.68(±0.12)b ×106
1.13(±0.08) ×103
9.49(±0.20) ×10-2
2.58(±0.10) ×10-2
7.04(±0.16) ×107
4.38(±0.13) ×104
G-rich DNA
ds-DNA
a ka, association rate constant with a unit of M-1s-1; kd, dissociation rate constant with a unit of s-1;
KA, association equilibrium constant with a unit of M-1; KA = ka/kd;
b Standard deviation is given in parentheses.
As shown in Fig. 4A, and B, CD spectrum of the G-rich DNA alone (red solid line)
presented a strong positive band at 290 nm, a shoulder band around 270 nm and a relatively
shallow negative band at 240 nm, which is characteristic of a hybrid type of G4-DNA structure,
also named a 3+1 G-quadruplex one [39,50,36], possibly including a small amount of other G-
quadruplex ones [50,51], which is in good agreement with EMSA results in Fig. 3; whereas two
compounds alone both are non-optically active, there existing not any CD bands (red dotted line).
No too much red shift with a typical positive band near 290 nm and a negative band near 240 nm
and the isoelliptic points at 358 nm and 377 nm were observed in the presence of 14c and 16c,
respectively, indicating the two-state nature of the structural transition between the quadruplexes
upon compound binding. More importantly, CD spectra of the G-rich DNA exhibited great
difference in peak intensity and the feature of induced CD (ICD) in the presence of 14c and 16c,
respectively. With gradual titration of 14c or 16c into the G-rich DNA at the ratios of
compound/DNA, 0.25:1, 0.5:1, 1.0:1, 2.0:1, 3.0:1, 4.0:1, 5.0:1 and 6.0:1, the 290 nm positive
peak intensity of the G4-DNA CD spectra progressively decreased up to saturation, and the 240
nm negative bands gradually became more negative up to a steady state, characteristic of a
mixture of G4-DNA structures with an external loop(s) [38,52], with induced CD (ICD) spectra

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ranging from 309 nm to 430 nm for 14c or from 311 nm to 435 nm for 16c; however, it was
found that 14c could cause bigger change in peak intensity and the feature of induced CD (ICD)
than 16c.
To investigate the binding selectivity of 4,5-diazafluorene derivatives to G4-DNA vs double-
stranded DNA, we have measured CD spectra of the 26nt telomeric double-stranded DNA (ds-
DNA, Table 2), in the presence and absence of representative 14c or 16c. As seen in Fig. S4A
and B, the CD spectrum of the ds-DNA alone was characteristic of double-stranded DNA
structure with strong positive and negative bands at 277 nm and at 241 nm, respectively [53].
With gradual titration of 14c or 16c into the ds-DNA at increasing concentrations of 4, 8, 16 µM,
only less change in positive/negative peak intensity of ds-DNA and no ICD were observed and
14c possessed slightly strong influence on ds-DNA than 16c, which is in line with the results of
Tm (°C) values (Table 3), suggesting that this kind of compounds own stronger selectively
binding to 26nt telomeric G4-DNA’s over ds-DNA. Also, CD spectra of Mut-DNA (Figs. S4C
and D) as a negative control was measured in the presence and absence of 14c or 16c under the
same experimental conditions as above used ones, which cannot form any G-quadruplex structure
due to the substitution of cytosine base for the middle guanine base of each of four three-
Guanine-runs in G-rich DNA (Table 2). These data showed that Mut-DNA alone was typical of a
freedom single-stranded DNA structure with a positive band at 281 nm and a negative band at
246.5 nm, and no influence was observed in the presence of compounds.
Collectively, this study suggests that the 14c could gradually convert the 3+1 G-quadruplex
structure formed in the G-rich DNA alone to a mixture of ones with an external loop(s),
consistent with gel electrophoresis results shown in Fig. 3, and specifically bind to 26nt telomeric
G4-DNA. Also, 16c can result in less change of the CD spectra of 26nt telomeric DNA’s.
To further evaluate the binding affinity, specificity and stoichiometry of 4,5-diazafluorene
derivatives to target DNA’s, we selected surface plasmon resonance (SPR) technique to have
examined the interaction natures of a representative compound 14c with G-rich DNA, Mut-DNA
and ds-DNA (Table 2). SPR is a valuable and extensively used method for the study on the
interaction between DNA’s and small molecules [54–56], especially in recent years on G-
quadruplexes [57,58] and telomerase inhibitors [59]. As shown in Figs. 4C, S5 and Table 4, the
quantitative SPR results provide strong evidence for a much greater binding affinity and

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specificity of the 14c to the 26nt human telomeric G4-DNA with an association equilibrium
constant (KA) of 7.04(±0.16) ×107 M-1 than to the double-stranded DNA sequence (ds-DNA,
Table 2) with a KA of 4.38(±0.13) ×104 M-1, indicating a more than 1000-fold increase in the
binding affinity to G4-DNA relative to ds-DNA. We employed a 1:1 global theoretical fitting
model with mass transport to experimental results. It was observed that experimental curves in
color and fitting curves in black shown in Fig. 4C are quite well fitting and binding of the
compound to the target DNA sequence at relatively higher concentrations of 800 to 1000 nM
reached to saturation to obtain better steady states, implying a quite possible 1:1 stoichiometry of
14c to the G4-DNA. In contrast, the 14c was found to bind weakly to ds-DNA relative to G4-
DNA (Fig. S5 and Table 4), which is in accordance with results by CD (Figs. 4A and S4A) and
Tm value analysis (Table 3). It is noticeable that almost no binding of the examined 14c to Mut-
DNA was observed at experimental concentrations, but there was large nonspecific binding at
higher concentrations and hence precise interaction parameters between the 14c and Mut-DNA
were not obtained. In short, our studies suggest that the 14c possesses quite strong binding and
well selective abilities to G4-DNA over ds-DNA and Mut-DNA at the compound/G4-DNA ratio
of 1:1. In addition, no 2:1 binding motif of 14c to G4-DNA was found despite high
concentrations being used.
2.6 In-vitro inhibition of telomerase activity by compounds 14a–c and 16c
Telomerase is highly expressed in a large proportion of human cancers relative to the very
low level in normal somatic cells and its major function is to maintain the length of telomeric
DNA by catalytically synthesizing telomeric DNA repeats. Because of such differential
expression between human cancer and normal somatic cells, telomerase has been regarded as an
anticancer target [60]. Thus, measurement of telomerase processivity/activity is very important to
assess the inhibitory properties of small molecules against telomerase and commonly performed
by the telomerase repeat amplification protocol (TRAP) assay [61]. As seen in Fig. 5, telomerase
activity in the presence/absence of compounds 14a–c in A549 cells was investigated by TRAP
assay and differential ladders of telomeric DNA were observed at the increasing compound
concentrations: 0, 2, 4, 8 µM. The result was found that the ability of 14a–c to inhibit telomerase
activity was in the order: 14c>14a>14b, with strongest telomerase inhibition activity for 14c
being 61.66 % at 8 µM relative to that without 14c (Fig. 5B). Furthermore, it was determined that

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the 14c can give rise to a good inhibitory effect on the telomerase activity of the positive control
cells in the kit, as shown in Fig. S6. This result suggests that the 4,5-diazafluorene derivatives
may directly or indirectly inhibit telomerase activity through their interaction with telomeric G4-
DNA, leading to cancer cell apoptosis.
Fig. 5. Ability of telomerase activity inhibition by compounds 14a, 14b and 14c. (A) Telomerase activity inhibition
mediated by compounds at the increasing concentrations, 0, 2, 4 and 8 µM in A549 cells. The arrow presents a 36 bp
internal control. (B) Telomerase activity was quantitated as the percent of the corresponding control. The mean of
three independent experiments with comparable results was shown. Error bars indicate ± SD.
2.7. Suppression of the examined cancer cell migration by compounds 14a–c
It was reported that the telomerase activity was generally closely related to the metastasis
and invasion of cancer cells [62,63]. In combination with the UV-melting assays and Trap assay,
the compounds 14a–c could strongly interact with and significantly stabilize telomeric G4-DNA
structures and might inhibit telomerase activity, thereby do the metastasis and invasion of cancer
cells. Therefore, we examined the effect of 14a–c on the migration of A549, AGS, HepG2 and
MCF-7 cells by in-vitro wound healing assay. As shown in Fig. 6, Fig. S7 and Table S1, all of
the compounds 14a–c can obviously inhibit the migration of the tested cancer cells in a
concentration-dependent manner and exhibited strongest migratory inhibitory effects on AGS
cells amongst four cancer cells at each of 0.1, 0.2, 0.4 µM. It was worth noting that 14c possessed
strongest inhibitory ability to four cancer cells amongst three compounds, with the order of cell
inhibitory ability being 14c, 14a and 14b, but for A549 at 0.4 µM and for HepG2 at 0.2 and 0.4
µM, which is partly supportive of the above-mentioned experimental results. These results
suggest that 4,5-diazafluorenes (14a–c) could effectively suppress the mobility of the four cancer
cell lines at the low level of drug concentration.

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Fig. 6. Effect of compounds 14a-c on the mobility of A549 (A), AGS (B), HepG2 (C) and MCF-7 (D) cell lines. The
mobility changes of A549 cells in the presence and absence of 14a–c at the concentration of 0, 0.1, 0.2, and 0.4 µM
for 48 h, respectively. Cell inhibitory rate (%) for suppression of the migration in cancer cells as a function of
concentrations of compounds 14a-c. The % cell inhibitory rate was calculated by the equation: cell inhibitory rate
(%) = (1-Ddrug/Dcontrol) × 100%, where Ddrug is mean distance of cell migration in drug group, Dcontrol is mean distance
of cell migration in control group; Values are the means ± S.D. (P< 0.05).
2.8. Docking and UV-Visible spectral studies on the interaction of 4,5-diazafluorenes with
telomeric G4-DNA
Such Structures of these small molecules with an extended planar aromatic system can
facilitate compounds easily stacking on the G-quartet through π-π interactions [39,64], which are
similar to a G-quartet in size and shape. Herein, LibDockin Discovery Studio 4.0 was utilized to
investigate the interactions of compounds 14a–c and 16c with the target, telomeric G-quadruplex
(PDB ID: 5MVB, a mode of a 3+1 typical telomeric G-quadruplex). As shown in Figs. 7, S8 and
Table 5, the results illustrated that the ligands 14a–c and 16c stack on the top capping of the 5’
tetrad, surrounded by the DNA bases A3, G4, G12, T13, T14, A15, G16 and G22. The ranking of
LibDock Score is consistent with the results obtained in the UV-melting, EMSA and CD studies.

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The 4,5-diazafluorene skeleton, pyrazol, rhodanine, 1,3,4-oxadiazole and phenyl ring all could
make intermolecular π-π interactions with DNA bases (magenta dash line). The
dimethylaminopropyl side chain at N1-position of pyrazol moiety can well extend to the loop
region at the 5’ end. Additionally, the intermolecular hydrogen bonds could be found between the
dimethylaminopropyl side chain and DNA bases A3, T13, T14 and A15 (green dash line in Fig.
S8). Surprisingly, some intramolecular H-Bond interactions were found between the
dimethylaminopropyl group and N14 in the pyrazol ring of compounds 14a-c. It gives rise to
more rigid conformations, which makes a good steric shape complementary between compounds
14a-c and G-quadruplex (i.e. 14c in Fig. 7). All these interactions and steric shape factor make
significant contribution to the potency. The ring number of compounds 14a-c is less than that of
16c, and the receptor-ligand complex of the former shows less π-π interactions. The better
potency of the former could be very likely due to the contribution of the N-substitutent group in
the pyrazol moiety. It might increase the potency if 1,3,4-oxadiazole group near the G-tetra center
is replaced with a hydrophobic aromatic ring or other multiple rings with substituents. Taken
together, the docking results indicate that 14a-c, especially 14c, can indeed quite fit to such G4-
DNA structure and strongly bind to the target at the 14a/G4-DNA ratio of 1:1 with a highest
LibDock score of 193.929 among the examined compounds, which supports the result of the
stoichiometry in SPR (Fig. 4C). These molecular docking data provide certain theoretical support
for experimental results and the next optimization.
Fig. 7. Highly occupied binding modes of compounds 14c and 16c with 5MVB. 5MVB is represented by its
soft atom charge surface in A, while its backbone model is shown in B. Compounds 14c and 16c extracted from
the complex are shown in C. 14c is shown in yellow CPK and stick models, and 16c is shown in blue CPK and
stick models. The compound 14c has the highest Molecular_3D_SASA (the total solvent accessible surface
area) of 695.86 Å2, while compound 16c has thirty less than that of the former.

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Table 5.
The LibDock scores and docking interactions of compounds (14a-c, 16c) with human telomeric
DNA G-quadruplex (PDB ID: 5MVB).
LibDock score
Compound
Interacting residues
Interacting atoms
(k.cal/mol)
A3, G4, G12
4,5-Diazafluorene - A3, G4, G12;
Pyrazol - A3, G12, G16;
14a
192.227
A15, G16, G22
Phenyl -G16, G22;
Intramolecular H-bond - 14a:H48 - 14a:N14
Intermolecular H-bond - 14a:H43 - A:DA3:N1;
14a:H49 - A:DA15:N3;
Hydrophobic - 14a:H51 - A:DA15
Halogen bond - A:DG16:N7 - 14a:F22.
A3, G4, G12
14b
191.007
4,5-Diazafluorene - A3, G4, G12;
Pyrazol - A3, G12, G16;
Phenyl - G16, G22;
T13, T14, G16, G22
Intramolecular H-bond
- 14b:H46 - 14b:N14;
14b:H51 - 14b:N14; 14b:H52 - 14b:N14;
Intermolecular H-bond - 14b:H42 - A:DA3:N1;
14b:H46 - A:DT14:OP2; 14b:H47 - A:DT14:OP2;
14b:H48 - A:DT13:O3'; 14b:H49 - A:DT14:OP2;
14b:H42 - A:DA3:N1; 14b:H46 - A:DT14:OP2;
Hydrophobic - A:DG16 - 14b:Cl22; A:DG22 -
14b:Cl22.
A3, G4, G12
G16
14c
193.929
4,5-Diazafluorene - A3, G4, G12;
Pyrazol - A3, G12, G16;
Phenyl - G16;
Intramolecular H-bond
- 14c:H47 - 14c:N14;
14c:H48 - 14c:N14;
Intermolecular H-bond - 14c:H43 - A:DA3:N1;
Hydrophobic - A:DG16 - 14c:Br22.
A3, G4, G12
4,5-Diazafluorene - A3, G4, G12;
Rhodanine - G4，G22;
16c
189.045
A15, G16, G22
1,3,4-oxadiazole - G16, G22;
Phenyl -A15, G16;
Intermolecular H-bond - A:DT14:H5'2 - 16c:Br28;
Hydrophobic - A:DA15 - 16c:Br28;
Pi-Sulfur -16c:S11 - A:DG4; 16c:S29 - A:DG22.
To gain insight into the interaction mode of 4,5-diazafluorenes with the telomeric G4-DNA,
we examined the UV-Vis spectral properties of a representative 4,5-diazafluorene compound, 14c,
gradually titrated with the 26nt telomeric G-rich DNA (G-rich DNA in Table 2). UV-Visible
spectroscopy is an important and extensively applied technique for interpreting the interaction

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mode of ligands with DNA. As seen in Fig. S9, the maximum absorption band of 14c is located
around 331.5 nm in the absence of G4-DNA. With the addition of G4-DNA, a striking spectral
change was observed that a flat and broad absorption band containing a dominant peak and a
shoulder one near 366.5 nm and 331.5 nm, respectively, occurred, the soret band exhibited more
hyperchromicity and bathochromically shifted from 331.5 nm to 366.5 nm at the increasing DNA
concentrations ranging from 0 to 40 µM, suggesting that the interaction of 14c with G4-DNA
takes places through external binding/stacking [65]. At the same time, another important fact is
that the isobestic point was not a sharp, tight single point, indicating that the interaction involves
multiple steps [65]. More importantly, when such interaction reached saturation, i.e. no change in
maximum absorption intensity and absorption wavelength with increasing compound
concentration, the 1:1 stoichiometry of the interaction of 14c with G4-DNA can be inferred (the
inset in Fig. S9). The UV-Visible data are in good agreement with results obtained by molecular
docking and SPR assays.
3. Discussion
The antiproliferative activities of all the synthesized 4,5-diazafluorene derivatives were
evaluated against A549, AGS, HepG2 and MCF-7 cells and MRC-5 cells as a control by CCK-8
assay and the results disclosed following some structure-activity relationships. SAR studies
revealed that various substitutions on the phenylpyrazole moiety had significant influence on
bioactivity. The R1 group of pyrazole ring plays a vital role in antiproliferative activity. Hydrogen
and aromatic substituents at the N-1 nitrogenation on the pyrazole ring like benzyl and 2,4-
dinitrophenyl groups did not influence on bioactivity. Compounds 11a–13d with such groups
exhibited moderate activity; while an aliphatic substituent, N,N1-dimethylaminopropyl group,
noticeably influences the bioactivity of examined compounds, leading to the findings that the
14a–c with such a group showed more excellent anti proliferative activity than the 11a–13d.
Another notable fact is that the effect of para substituents on the benzene ring on the bioactivity
of examined compounds was found in the following order: bromo, fluoro, chloro and methyl,
therefore, the 14c almost exhibited best activity and strongest affinity to the target in all finished
experiments amongst examined compounds. However, introduction of the thioxothiazolidinone
ring and the phenyloxadiazole moiety to 4,5-diazafluorene mother structure cannot obviously

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increase the bioactivity of compounds 15, 16a–c compared with 11a–13d, 14a–c. All in all, based
on the SAR study we conclude that modifications on 4,5-diazafluorene-pyrazole contribute to
the effort for finding new promising anticancer drugs.
In addition, in recent two decades, increasing evidence has shown that a large number of
small molecules possess the ability to inhibit telomerase and hence do antitumor activity. In
particular, small molecules that induce the telomeric guanine-rich single-stranded DNA to form
and stabilize G-quadruplex structures can block the function of telomerase, such as porphyrins,
acridines, and the natural product telomestatin [66–73]. Telomerase as a drug target is over
expressed in mostly cancer cells and it is closely associated the metastasis, invasion and apoptosis
of some cancer cells. Hence, we initially explored whether such resultant antitumor activity was
obtained by the interaction of the novel 4,5-diazafluorene derivatives with telomeric G4-DNA
which inhibits telomerase activity. Experimental results at extra/intracellular level are especially
inspiring that 14a–c derivatives had stabilizing ability towards human telomere G-quadruplex
DNA, in particular 14c, which are consistent with those by molecular docking studies, and
mediated the inhibition of telomerase activity. Furthermore, docking studies indicate that the 1-
N,N1-dimethylaminopropyl and 3-(4-bromophenyl) substituents on 14c have advantage over
others, when binding to G4-NDA. These data also imply that the introduction of 3-(3-(4-
bromophenyl)-1H-pyrazol-1-yl)-N,N-dimethylpropan-1-amine substituent on 4,5-diazafluorene
was successful.
4. Conclusion
In summary, 4,5-diazafluorene derivatives bearing phenylpyrazole and thioxo
thiazolidinone/thioxothiazolidinone-phenyloxadiazole moieties were designed, synthesized and
their antiproliferative activity evaluated against A549, AGS, HepG2 and MCF-7 cell lines, and
also their interaction mechanism with telomeric G4-DNA preliminarily explored. The compounds
14a–c with a phenylpyrazole group showed stronger inhibitory activities with IC50 values in the
range of 1.13–11.77 µM. In particular, the compound 14c is the most efficient antiproliferative
agent amongst the examined compounds including cisplatin as a positive control against A549
(IC50 = 1.13 µM), AGS (IC50 = 1.81 µM) and MCF-7 (IC50 = 5.27 µM) cells, but HepG2 cells
(IC50 = 3.4 µM). Likewise, the results by wound healing assay also are in agreement with the

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above results, showing that the inhibitory effects of 14a–c on the mobility of four cancer cells are
outstanding. 4,5-diazafluorenes bearing a phenylpyrazole group could effectively inhibit invasion
and metastasis of cancer cells and the 14c possesses strongest migration inhibitory ability, but for
HepG2 cells at 0.2 and 0.4 µM. Furthermore, based on EMSA, UV-melting, CD, SPR, UV-Vis
spectral assay, molecular docking study and TRAP assay results, 14a-c, especially 14c, can
strongly interact with 26nt telomeric G-quadruplexes and mediate telomerase activity decrease,
consequently causing the inhibition of cell proliferation. Our study demonstrate that 4,5-
diazafluorene derivatives with a phenylpyrazole group as a novel class of G4-DNA mediated
telomerase inhibitors may be developed as potential anticancer agents.
5. Experimental protocols
5.1. General
All solvents and chemicals were analytic pure, obtained from commercial suppliers and used
without further purification unless otherwise stated. A Millipore MilliQ water purification system
produced 18 MΩ water used to prepare buffer solutions. The melting points of analogues were
observed on the melting point detector (XT-5A). Reaction processes were monitored by TLC on
silica gel-precoated F254 Merck plates and the thin layer plates were examined under the UV
lamps (254 nm and 365 nm). 1H-NMR spectra were recorded in pure CDCl3/DMSO-d6 on Bruker
NMR spectrometers (AVANCE-III 500 MHz) using tetramethylsilane (TMS) as internal standard.
Chemical shifts were expressed in δ, ppm. Mass spectra were measured on an Agilent 6210 TOF
LC/MS (USA). Purity of compounds was analyzed by HPLC on a Shimadzu LC-20AT
instrument. DNA oligomers/primers were purchased from Sangon (Shanghai, China), and used
without further purification. These oligomers were purified by HPLC, identified by HPLC-CE
and TOF Mass, and exhibited single-band electrophoretic mobilities in denaturing
polyacrylamide gel electrophoresis with stated purities of ≥95%. The sequences of (biotin-
modified) DNA oligomers used are listed in Table 2. Solutions of the DNA oligomers were
prepared as follows: an oligonucleotide sample dissolved in a specified used buffer was heated at
95 °C for 5 min and allowed to slowly cool to room temperature at 1 °C/1 min over a period of
several hours and then incubated at 4 °C overnight. All cell lines were supplied by the Type
Culture Collection of the Chinese Academy of Sciences, Shanghai, China. Sensor chip SA were

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purchased from BIAcore AB (Sweden). All other commercially available reagents and solvents
were purchased and used without further purification unless otherwise stated. MilliQ water was
used to prepare buffer solutions.
5.1.1. General procedure for thesynthesis of target compounds 11–14.
A mixture of 4,5-diazafluorene 6 (1.0 mmol), ammonium acetate (4.0 mmol), and 1.1 mmol
7a–d, 8a–d, 9a–d, 10a–c respectively, in glacial acetic acid (20 mL) was heated at 110 °C with
stirring for 6 h. The reaction was monitored by TLC for completion. The resultant solution was
concentrated and neutralized with 20% aqueous ammonia. The white and yellow precipitate was
collected, washed with water and purified by silica gel chromatography
(dichloromethane/methanol, 15:1) to afford pure white products (11a–d, 12a–d, and 14a–c) and
yellow crystals (13a–d), respectively. The yield, melting point, purity and spectral data of each
compound are given as follows.
5.1.1.1.
5-((3-(4-Fluorophenyl)-1H-pyrazol-4-yl)methylene)-5H-cyclopenta[2,1-b:3,4-b']
dipyridine (11a)
1
Pale yellow solid; yield: 85%; m.p.:217–219 °C; purity by HPLC: 98.1%. H NMR (DMSO-d6,
500 MHz, ppm): δ 13.49 (br. s, 1H, N-H), 8.67 (dd, J = 5.0, 1.5 Hz, 1H, pyridine-H8), 8.63 (dd, J
= 5.0, 1.5 Hz, 1H, pyridine-H2), 8.48 (dd, J = 8.0, 1.5 Hz, 1H, pyrazol-H11), 8.22 (d, J = 7.5 Hz,
1H, pyridine-H6), 7.89 (br. s, 1H, C-H10), 7.72 (dd, J = 5.0, 1.5 Hz, 2H, benzene-H), 7.46 (dd, J =
5.0, 1.5 Hz, 1H, pyridine-H4), 7.34 (dd, J = 5.0, 1.5 Hz, 1H, benzene-H), 7.32-7.29 (m, 3H,
pyridine-H3,7 and benzene-H). MS (ESI) calcd for C21H13FN4: 340.11, found: 341.1 (M+H+).
5.1.1.2.
5-((3-(4-Chlorophenyl)-1H-pyrazol-4-yl)methylene)-5H-cyclopenta[2,1-b:3,4-b']
dipyridine (11b)
1
Pale yellow solid; yield: 83%; m.p.: 242–244 °C; purity by HPLC: 97.8%. H NMR (DMSO-d6,
500 MHz, ppm): δ 13.57 (br. s, 1H, N-H), 8.68 (d, J = 4.0 Hz, 1H, pyridine-H8), 8.64 (d, J = 4.0
Hz, 1H, pyridine-H2), 8.50 (d, J = 7.5 Hz, 1H, pyrazol-H11), 8.21 (d, J = 6.5 Hz, 1H, pyridine-
H6), 7.90 (br. s, 1H, C-H10), 7.71 (d, J = 7.5 Hz, 2H, benzene-H), 7.52 (br. s, 3H, pyridine-H4 and
benzene-H), 7.46 (dd, J = 7.5, 5.0 Hz, 1H, pyridine-H7), 7.34 (dd, J = 7.5, 5.0 Hz, 1H, pyridine-
H3). MS (ESI) calcd for C21H13ClN4: 356.08, found: 357.1 (M+H+).

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5.1.1.3.
5-((3-(4-Bromophenyl)-1H-pyrazol-4-yl)methylene)-5H-cyclopenta[2,1-b:3,4-b']
dipyridine (11c)
Pale yellow solid; yield: 88%; m.p.:251–253 °C; purity by HPLC: 98.5%. 1H NMR (DMSO-d6,
500 MHz, ppm): δ 13.55 (br. s, 1H, N-H), 8.67 (dd, J = 4.5, 1.0 Hz, 1H, pyridine-H8), 8.64 (dd, J
= 5.0, 1.5 Hz, 1H, pyridine-H2), 8.48 (dd, J = 7.5, 1.0 Hz, 1H, pyrazol-H11), 8.21 (d, J = 7.0 Hz,
1H, pyridine-H6), 7.89 (br. s, 1H, C-H10), 7.65-7.53 (m, 5H, pyridine-H4 and benzene-H), 7.46
(dd, J = 8.0, 5.0 Hz, 1H, pyridine-H7), 7.34 (dd, J = 8.0, 5.0 Hz, 1H, pyridine-H3). MS (ESI)
calcd for C21H13BrN4: 400.03, found: 401.0 (M+H+).
5.1.1.4. 5-((3-(P-tolyl)-1H-pyrazol-4-yl)methylene)-5H-cyclopenta[2,1-b:3,4-b']dipyridine (11d)
Yellow solid; yield: 81%; m.p.:178–180 °C; purity by HPLC: 98.3%. 1H NMR (DMSO-d6, 500
MHz, ppm): δ 13.46 (br. s, 1H, N-H), 8.67 (dd, J = 4.5, 1.0 Hz, 1H, pyridine-H8), 8.64 (dd, J =
4.5, 1.0 Hz, 1H, pyridine-H2), 8.45 (dd, J = 7.5, 1.5 Hz, 1H, pyrazol-H11), 8.28 (d, J = 7.0 Hz, 2H,
benzene-H), 7.86 (br. s, 1H, C-H10), 7.58 (d, J = 8.0 Hz, 2H, pyridine-H4,6), 7.45 (dd, J = 8.0, 5.0
Hz, 1H, benzene-H), 7.36 (dd, J = 8.0, 5.0 Hz, 1H, benzene-H), 7.28 (br. s, 2H, pyridine-H3,7),
2.33 (s, 3H, CH3). MS (ESI) calcd for C22H16N4: 336.14, found: 337.2 (M+H+).
5.1.1.5. 5-((1-Benzyl-3-(4-fluorophenyl)-1H-pyrazol-4-yl)methylene)-5H-cyclopenta[2,1-b:3,4-
b']dipyridine (12a)
White solid; yield: 74%; m.p.:232–234 °C; purity by HPLC: 98.6%. 1H NMR (CDCl3, 500 MHz,
ppm): δ 8.70 (dd, J = 4.5, 1.0 Hz, 1H, pyridine-H8), 8.63 (dd, J = 4.5, 1.0 Hz, 1H, pyridine-H2),
8.05-8.01 (m, 2H, benzene-H), 7.74 (s, 1H, pyrazol-H11), 7.72-7.69 (m, 2H, pyridine-H4,6), 7.45
(s, 1H, C-H10), 7.44-7.36 (m, 5H, benzene-H), 7.30 (dd, J = 7.5, 4.5 Hz, 1H, benzene-H), 7.12-
7.07 (m, 2H, pyridine-H3,7), 7.04 (dd, J = 8.0, 4.5 Hz, 1H, benzene-H), 5.44 (s, 2H, CH2). MS
(ESI) calcd for C28H19FN4: 430.16, found: 431.2 (M+H+).
5.1.1.6. 5-((1-Benzyl-3-(4-chlorophenyl)-1H-pyrazol-4-yl)methylene)-5H-cyclopenta[2,1-b:3,4-
b']dipyridine (12b)
White solid; yield: 77%; m.p.:260–262 °C; purity by HPLC: 98.7%. 1H NMR (CDCl3, 500 MHz,

26
ACCEPTED MANUSCRIPT
ppm): δ 8.70 (dd, J = 4.5, 1.0 Hz, 1H, pyridine-H8), 8.63 (dd, J = 5.0, 1.5 Hz, 1H, pyridine-H2),
8.03-8.01 (m, 2H, benzene-H), 7.73 (s, 1H, pyrazol-H11), 7.68-7.66 (m, 2H, pyridine-H4,6), 7.45
(s, 1H, C-H10), 7.44-7.41 (m, 2H, benzene-H), 7.40-7.36 (m, 5H, benzene-H), 7.31 (q, 1H,
pyridine-H7), 7.04 (q, 1H, pyridine-H3), 5.44 (s, 2H, CH2). MS (ESI) calcd for C28H19ClN4:
446.13, found: 447.1 (M+H+).
5.1.1.7. 5-((1-Benzyl-3-(4-bromophenyl)-1H-pyrazol-4-yl)methylene)-5H-cyclopenta[2,1-b:3,4-
b']dipyridine (12c)
White solid; yield: 78%; m.p.:263–265 °C; purity by HPLC: 98.3%. 1H NMR (CDCl3, 500 MHz,
ppm): δ 8.70 (dd, J = 5.0, 1.5 Hz, 1H, pyridine-H8), 8.62 (dd, J = 5.0, 1.5 Hz, 1H, pyridine-H2),
8.02-8.00 (m, 2H, benzene-H), 7.73 (s, 1H, pyrazol-H11), 7.62-7.59 (m, 2H, pyridine-H4,6), 7.54-
7.51 (m, 2H, benzene-H), 7.44 (s, 1H, C-H10), 7.43-7.36 (m, 5H, benzene-H), 7.30 (q, 1H,
pyridine-H7), 7.04 (q, 1H, pyridine-H3), 5.44 (s, 2H, CH2). MS (ESI) calcd for C28H19BrN4:
490.08, found: 491.1 (M+H+).
5.1.1.8.
5-((1-Benzyl-3-(p-tolyl)-1H-pyrazol-4-yl)methylene)-5H-cyclopenta[2,1-b:3,4-b']
dipyridine (12d)
White solid; yield: 75%; m.p.:231–233 °C; purity by HPLC: 97.8%. 1H NMR (CDCl3, 500 MHz,
ppm): δ 8.70 (dd, J = 4.0, 1.0 Hz, 1H, pyridine-H8), 8.63 (dd, J=4.5, 1.0 Hz, 1H, pyridine-H2),
8.10 (dd, J = 8.0, 1.5 Hz, 1H, benzene-H), 8.03 (dd, J = 8.0, 1.5 Hz, 1H, benzene-H), 7.74 (s, 1H,
pyrazol-H11), 7.62-7.61 (m, 2H, pyridine-H4,6), 7.51 (s, 1H, C-H10), 7.43-7.40 (m, 2H, benzene-
H), 7.39-7.36 (m, 3H, benzene-H), 7.31 (q, 1H, pyridine-H7), 7.23 (d, J = 7.5 Hz, 2H, benzene-
H), 7.05 (q, 1H, pyridine-H3), 5.45 (s, 2H, CH2), 2.38 (s, 3H, CH3). MS (ESI) calcd for C29H22N4:
426.18, found: 427.2 (M+H+).
5.1.1.9.
5-((1-(2,4-Dinitrophenyl)-3-(4-fluorophenyl)-1H-pyrazol-4-yl)methylene)-5H-
cyclopenta[2,1-b:3,4-b']dipyridine (13a)
1
Yellow solid; yield: 85%; m.p.:＞310°C; purity by HPLC: 98.5%. H NMR (DMSO-d6, 500
MHz, ppm): δ 9.11 (s, 1H, benzene-H17), 8.95 (d, J = 1.0 Hz, 1H, benzene-H), 8.71-8.70 (m, 2H,
pyridine-H2,8), 8.66 (d, J = 4.5 Hz, 1H, benzene-H), 8.56 (d, J = 7.5 Hz, 1H, pyrazol-H11), 8.31

27
ACCEPTED MANUSCRIPT
(d, J = 9.0 Hz, 1H, pyridine-H6), 8.25 (d, J = 8.0 Hz, 1H, pyridine-H4), 7.95 (s, 1H, C-H10), 7.74-
7.71 (m, 2H, benzene-H), 7.52-7.50 (m, 1H, benzene-H), 7.36-7.30 (m, 3H, benzene-H and
pyridine-H3,7). MS (ESI) calcd for C27H15FN6O4: 506.11, found: 507.1 (M+H+).
5.1.1.10.
5-((3-(4-Chlorophenyl)-1-(2,4-dinitrophenyl)-1H-pyrazol-4-yl)methylene)-5H-
cyclopenta[2,1-b:3,4-b']dipyridine (13b)
1
Yellow solid; yield: 85%; m.p.:＞310 °C; purity by HPLC: 98.4%. H NMR (DMSO-d6, 500
MHz, ppm): δ 9.12 (s, 1H, benzene-H), 8.96 (br. s, 1H, benzene-H16), 8.71 (d, J = 5.5 Hz, 2H,
pyridine-H2,8), 8.66 (d, J = 4.0 Hz, 1H, benzene-H), 8.56 (d, J = 7.5 Hz, 1H, pyrazol-H11), 8.31
(d, J = 9.0 Hz, 1H, pyridine-H6), 8.25 (d, J = 7.5 Hz, 1H, pyridine-H4), 7.95 (s, 1H, C-H10), 7.71
(d, J = 8.5 Hz, 2H, benzene-H), 7.56 (d, J = 8.0 Hz, 2H, benzene-H), 7.53-7.50 (m, 1H, pyridine-
H7) 7.36-7.34 (m, 1H, pyridine-H3). MS (ESI) calcd for C27H15ClN6O4: 522.08, found: 523.1
(M+H+).
5.1.1.11.
5-((3-(4-Bromophenyl)-1-(2,4-dinitrophenyl)-1H-pyrazol-4-yl)methylene)-5H-
cyclopenta[2,1-b:3,4-b']dipyridine (13c)
1
Yellow solid; yield: 83%; m.p.:＞310 °C; purity by HPLC: 98.6%. H NMR (DMSO-d6, 500
MHz, ppm): δ 9.12 (s, 1H, benzene-H17), 8.96 (d, J = 2.5 Hz, 1H, benzene-H), 8.72-8.69 (m, 2H,
pyridine-H2,8), 8.65 (dd, J = 6.0, 1.0 Hz, 1H, benzene-H), 8.54 (dd, J = 6.0, 1.0 Hz, 1H, pyrazol-
H11), 8.30 (d, J = 9.0 Hz, 1H, pyridine-H6), 8.24 (dd, J = 8.0, 1.0 Hz, 1H, pyridine-H4), 7.94 (s,
1H, C-H10), 7.68 (dd, J = 7.0, 2.0 Hz, 2H, benzene-H), 7.62 (dd, J = 7.0, 2.0 Hz, 2H, benzene-H),
7.50 (dd, J = 8.0, 5.0 Hz, 1H, pyridine-H7), 7.35 (dd, J = 8.0, 4.0 Hz, 1H, pyridine-H3). MS (ESI)
calcd for C27H15BrN6O4: 566.03, found: 567.0 (M+H+).
5.1.1.12. 5-((1-(2,4-Dinitrophenyl)-3-(p-tolyl)-1H-pyrazol-4-yl)methylene)-5H-cyclopenta[2,1-
b:3,4-b']dipyridine (13d)
1
Yellow solid; yield: 82%; m.p.:＞310 °C; purity by HPLC: 98.2%. H NMR (DMSO-d6, 500
MHz, ppm): δ 9.09 (s, 1H, benzene-H17), 8.94 (d, J = 2.5 Hz, 1H, benzene-H), 8.71-8.68 (m, 2H,
pyridine-H2,8), 8.66 (d, J = 4.5 Hz 1H, benzene-H), 8.54 (d, J = 7.5 Hz, 1H, pyrazol-H11), 8.30-
8.26 (m, 2H, pyridine-H4,6), 7.93 (s, 1H, C-H10), 7.58 (d, J = 8.0 Hz, 2H, benzene-H), 7.50 (dd, J

28
ACCEPTED MANUSCRIPT
= 8.0, 5.0 Hz, 1H, pyridine-H7), 7.35 (dd, J = 8.0, 5.0 Hz, 1H, pyridine-H3), 7.28 (d, J = 8.0 Hz,
2H, benzene-H), 2.32 (s, 3H, CH3). MS (ESI) calcd for C28H18N6O4: 502.14, found: 503.19
(M+H+).
5.1.1.13. 3-(4-((5H-cyclopenta[2,1-b:3,4-b']dipyridin-5-ylidene)methyl)-3-(4-fluorophenyl)-1H-
pyrazol-1-yl)-N,N-dimethylpropan-1-amine (14a)
o
1
Pale yellow solid; yield: 78%, m.p.:208–210 C; purity by HPLC: 98.7%. H NMR (DMSO-d6,
500 MHz, ppm): δ 8.67 (dd, J = 5.0, 1.0 Hz, 1H, pyridine-H8), 8.64 (dd, J = 5.0, 1.0 Hz, 1H,
pyridine-H2), 8.47 (dd, J = 8.0, 1.5 Hz, 1H, pyridine-H6), 8.34 (s, 1H, pyrazol-H11), 8.26 (dd, J =
8.0, 1.5 Hz, 1H, pyridine-H4), 7.86 (s, 1H, C-H10), 7.73-7.70 (m, 2H, benzene-H), 7.46 (dd, J =
8.0, 5.0 Hz, 1H, pyridine-H7), 7.32 (dd, J= 8.0, 5.0 Hz, 1H, pyridine-H3), 7.27-7.24 (m, 2H,
benzene-H), 4.29 (t, J = 6.75 Hz, 2H, CH2), 2.33 (t, J = 5.5 Hz, 2H, CH2), 2.20 (s, 6H, CH3),
2.08-2.03 (m, 2H, CH2). 13C NMR (CDCl3 + CD3OD-d4, 126 MHz, ppm): δ 163.95, 161.98,
157.45, 155.52, 151.05, 149.86, 134.16, 131.78, 131.51, 131.04, 129.81, 129.66, 128.84, 127.75,
123.17, 122.77, 122.26, 115.89, 114.02, 55.86, 50.15, 44.44, 27.12. MS (ESI) calcd for
C26H24FN5: 425.2016, found: 426.2083 (M+H+), 448.1902 (M+Na+).
5.1.1.14. 3-(4-((5H-cyclopenta[2,1-b:3,4-b']dipyridin-5-ylidene)methyl)-3-(4-chlorophenyl)-1H-
pyrazol-1-yl)-N,N-dimethylpropan-1-amine (14b)
1
Pale yellow solid; yield: 74%; m.p.:230–232 °C; purity by HPLC: 99.3%. H NMR (DMSO-d6,
500 MHz, ppm): δ 8.68 (d, J = 4.5 Hz, 1H, pyridine-H8), 8.65 (d, J = 4.5 Hz, 1H, pyridine-H2),
8.49 (d, J = 7.5 Hz, 1H, pyridine-H6), 8.35 (s, 1H, pyrazol-H11), 8.24 (d, J= 8.0 Hz, 1H, pyridine-
H4), 7.87 (s, 1H, C-H10), 7.70 (d, J = 8.5 Hz, 2H, benzene-H), 7.49 (d, J = 8.5 Hz, 2H, benzene-
H), 7.47-7.46 (m, 1H, pyridine-H7), 7.35-7.32 (m, 1H, pyridine-H3), 4.29 (t, J = 7.0 Hz, 2H,
CH2), 2.30 (t, J = 6.5 Hz, 2H, CH2), 2.18 (s, 6H, CH3), 2.08-2.02 (m, 2H, CH2). 13C NMR (CDCl3
+ CD3OD-d4, 126 MHz, ppm): δ 156.33, 154.37, 149.54, 148.76, 148.69, 133.34, 133.14, 130.72,
130.53, 130.20, 130.01, 128.61, 128.25, 127.93, 126.83, 122.21, 121.72, 121.39, 113.00, 55.24,
49.52, 44.21, 26.99. MS (ESI) calcd for C26H24ClN5: 441.1720, found: 442.1790 (M+H+),
464.1611 (M+Na+).