Novel triazole and morpholine substituted bisnaphthalimide: Synthesis, photophysical and G-quadruplex binding properties

Article abstract of DOI:10.1016/j.molstruc.2019.02.073

Four bisnaphthalimides derivatives 3a, 3b, 5a and 5b with polyamine linkage have been synthesized and characterized. Different spectroscopic techniques, including UV–Vis spectroscopy, fluorescence spectroscopy, circular dichroism and fluorescence resonance energy transfer (FRET), have been used to explore the interaction of these compounds with G-quadruplex structures (Htelo, c-myc, c-kit). The compounds 3a, 3b and 5b show high affinity toward Htelo, c-myc and c-kit G-quadruplexes (K_a > 4.42 × 10⁶ M^?1), and exhibit more than 40-fold selectivity for the quadruplex versus CT DNA. Binding of 3a, 3b to telomeric and oncogenic G-quadruplexes induce strong enhancement of the fluorescence intensity (18–27-fold), whereas the duplex CT DNA induces much weaker fluorescence enhancement (around 2-fold). Being fluorescent, the cellular localization of 3a, 3b can be conveniently tracked by fluorescence imaging. The results indicate that 3a, 3b can readily be taken up by A549 lung cancer cells. Molecular docking studies indicate that 3a, 3b, 5a and 5b mainly bind in the groove and/or loop region of the G-quadruplexes, and hydrogen bonding plays an important role in the interaction of bisnaphthalimide derivatives with G-quadruplexes. CD spectroscopy and FRET melting assay indicate that 3a, 3b and 5b can promote the formation and the stabilization of human telomeric and c-myc G-quadruplex DNA. The compound 3b can inhibit the growth of A549 cells (IC₅₀ = 0.15 μM), with a much higher antitumor activity than amonafide.

Full text of DOI:10.1016/j.molstruc.2019.02.073

Accepted Manuscript
Novel triazole and morpholine substituted bisnaphthalimide: Synthesis, photophysical
and G-quadruplex binding properties
Zhize Ou, Zhiyuan Li, Yunyan Gao, Wenli Xing, Hongdan Jia, Huan Zhang, Na Yi
PII:
S0022-2860(19)30203-0
DOI:
https://doi.org/10.1016/j.molstruc.2019.02.073
MOLSTR 26230
Reference:
To appear in: Journal of Molecular Structure
Received Date: 11 December 2018
Revised Date: 5 February 2019
Accepted Date: 19 February 2019
Please cite this article as: Z. Ou, Z. Li, Y. Gao, W. Xing, H. Jia, H. Zhang, N. Yi, Novel triazole and
morpholine substituted bisnaphthalimide: Synthesis, photophysical and G-quadruplex binding properties,
Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/j.molstruc.2019.02.073.
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ACCEPTED MANUSCRIPT
Graphic abstract
The bisnaphthalimide, with high affinity toward G-quadruplex DNA, is able to inhibit
the growth of lung cancer A549 cells selectively over non-cancerous MRC-5 cells.

ACCEPTED MANUSCRIPT
Department of Applied Chemistry,
School of Science,
Northwestern Polytechnic University,
Xi’an 710072, P. R. China
E-mail: gaoyunyan@nwpu.edu.cn
Dear Editor:
Thank you for your e-mail and for the reviewers’ comments concerning our
manuscript entitled “Novel triazole and morpholine substituted bisnaphthalimide:
synthesis, photophysical and G-quadruplex binding properties” (Manuscript ID:
MOLSTRUC-D-18-03754). We have carried out some experiments and revised the
manuscript according to reviewers’ comments, with the revised parts marked in red.
We hope that could meet with their approval.
We would like to express our appreciation to you and to the reviewers for
suggesting how to improve our paper. We also responded point by point to the
referee’s comments as listed below, along with a clear indication of the location of the
revision in the manuscript.
Thank you.
Sincerely Yours,
Yunyan Gao
Professor of Chemistry
1

ACCEPTED MANUSCRIPT
Novel triazole and morpholine substituted bisnaphthalimide: synthesis, photophysical and
G-quadruplex binding properties
Zhize Ou, Zhiyuan Li, Yunyan Gao*, Wenli Xing, Hongdan Jia, Huan Zhang, Na Yi
The Key Laboratory of Space Applied Physics and Chemistry, Ministry of Education, Department
of Applied Chemistry, School of Science, Northwestern Polytechnical University, Xi'an 710072, P.
R. China.
Corresponding Author:
Yunyan Gao
Department of Applied Chemistry, School of Science
Northwestern Polytechnical University
Xi'an 710072
P. R. China
E-mail: gaoyunyan@nwpu.edu.cn
2

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Abstract
Four bisnaphthalimides derivatives 3a, 3b, 5a and 5b with polyamine linkage have been
synthesized and characterized. Different spectroscopic techniques, including UV-Vis spectroscopy,
fluorescence spectroscopy, circular dichroism and fluorescence resonance energy transfer (FRET),
have been used to explore the interaction of these compounds with G-quadruplex structures (Htelo,
c-myc, c-kit). The compounds 3a, 3b and 5b show high affinity toward Htelo, c-myc and c-kit
G-quadruplexes (Ka > 4.42×106 M-1), and exhibit more than 40-fold selectivity for the quadruplex
versus CT DNA. Binding of 3a, 3b to telomeric and oncogenic G-quadruplexes induce strong
enhancement of the fluorescence intensity (18 to 27-fold), whereas the duplex CT DNA induces
much weaker fluorescence enhancement (around 2-fold). Being fluorescent, the cellular
localization of 3a, 3b can be conveniently tracked by fluorescence imaging. The results indicate
that 3a, 3b can readily be taken up by A549 lung cancer cells. Molecular docking studies indicate
that 3a, 3b, 5a and 5b mainly bind in the groove and/or loop region of the G-quadruplexes, and
hydrogen bonding plays an important role in the interaction of bisnaphthalimide derivatives with
G-quadruplexes. CD spectroscopy and FRET melting assay indicate that 3a, 3b and 5b can
promote the formation and the stabilization of human telomeric and c-myc G-quadruplex DNA.
The compound 3b can inhibit the growth of A549 cells (IC50 = 0.15 ꢀM), with a much higher
antitumor activity than amonafide.
Keywords: G-quadruplex; bisnaphthalimide; anticancer drug; AutoDock; fluorescence probe
3

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1. Introduction
The Guanine-rich nucleic acid sequences are prevalent in human genes, especially in
telomere ends and promoters of some functionally important proto-oncogenes [1-3]. The guanine
tracts in these sequences can fold into G-quadruplexes (G4) via Hoogsten hydrogen bonds. There
are evidences that G4s are involved in a variety of biological processes [4,5]. Human telomeric
DNA consists of tandem repeats of the sequence 5’-d(TTAGGG)-3’, which protects chromosomes
from degradation and is essential for ensuring genomic stability. The telomerase is selectively
expressed in the majority of human cancer cells, and its activation is thought to be a critical step in
cellular immortalization and tumorigenesis. The telomeric G4 stabilizing ligands can inhibit
telomerase activity and induce growth arrest in cancer cells [6]. On the other hand, the
proto-oncogenes play critical roles in the regulation of programmed cell death or apoptosis. The
abnormal overexpress of the proto-oncogenes has recently been shown in tumors. For example,
the c-myc proto-oncogene, overexpressed in up to 80% of solid tumors, such as gastrointestinal,
ovarian and breast cancer tumors [7,8]. The proto-oncogenes can be transcriptionally repressed by
targeting a putative G4 with small molecules [9,10]. A large number of small molecules and metal
complexes, such as phorphyrin derivatives, carbazole derivatives, quindolines, peptide and
quarflxoxin can bind G4s and modulate the gene expression [11-16]. Thus, G4s are considered as
a promising new class of targets for the design of anticancer drugs [17-20].
Naphthalimide derivatives, as a class of DNA-binding agents, have been extensively explored
as antitumor agents [21]. However, naphthalimide-based anti-tumor drugs may cause serious side
effects, including central neurotoxicity and dose-limiting toxicity. In order to improve their
potency and reduce the side effects, naphthalimide derivatives, modified with fused aromatic rings,
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side chains and imide substituents have been synthesized [22-26]. Recently, the studies of the
sequence-specific binding of naphthalimides to DNA have been an important topic in the design of
novel drugs [27]. The trisubstituted naphthalimide derivatives can bind telomeric G-quadruplexes
with high affinity and show antiproliferation activity against various cell lines [28]. We have also
synthesized a series of naphthalimides modified with thiourea, imidazole and terpyridine
complexes, which possess high affinity (Ka > 106 M-1) and reasonable selectivity for telomeric
G-quadruplex DNA over duplex DNA [29-31].
It has been reported that the bisnaphthalimides bisnafide and elinafide present potent cellular
cytotoxicity and excellent in vivo antitumor activity, and have been selected for clinical trials [32].
The substituents at naphthalimide skeleton and linkers play crucial roles in determining the
sequence selectivity, photophysical properties and cytotoxic activity of bisnaphthalimides
derivatives [33-38]. However, to the best of our knowledge, there is no report about the interaction
between bisnaphthalimide and G-quadruplexes. In this study, the bisnaphthalimides with alkyl
amino linkages are synthesized. The flexible polyamine-bridge can facilitate the electrostatic
interactions with the loops and grooves of the phosphate backbone of G4 DNA [39]. The
morpholine and 1,2,3-triazole moieties are incorporated at the 4-position of the bisnaphthalene
ring to enhance their G4 binding ability and cytotoxic activity [40,41]. The association of
bisnaphthalimides with telomeric and oncogenic promoter G4s, including c-myc and c-kit
quadruplex, is investigated in detail by spectroscopic techniques and molecular docking studies.
Systematic investigation of the interaction between bisnaphthalimde and G4s can provide
structural insight into recognition of G-quadruplex by naphthalimides, and can be helpful for the
design of G-quadruplex targeting agents.
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2 Experimental
2.1 Materials
1-(4,5-Dimethyl-2-thiazolyl)-3,5-diphenylformazan
(MTT),
4,6-diamino-2-phenylindole
(DAPI), calf thymus DNA (CT DNA) and 4-bromo-1,8-naphthalic anhydride were purchased
from Sigma-Aldrich. The Htelo strand (5’-GGG-TTA-GGG-TTA-GGG-TTA-GGG-3’) was used
for
the
human
telomeric
studies.
The
oligomer
strand
and
(5’-TTG-AGG-GTG-GGT-AGG-GTG-GGT-AA-3’)
(5’-AGG-GAG-GGC-GCT-GGG-AGG-AGGG-3’) were used for c-myc and c-kit G4 studies,
respectively. All DNA oligomers were purchased from Sangon (Shanghai, China) and purified by
Waters 2695 Alliance HPLC (U.S.A). Other chemicals of analytical grade were obtained from
Beijing Chemical Plant. The organic solvents were distilled prior to use.
Unless otherwise stated, spectroscopic titration experiments were performed in HEPES
buffer (10 mM, pH 7.4). Stocking solutions of bisnaphthalimides (1 mM) were prepared in
dimethyl sulfoxide (DMSO).
2.2. Instrumentation
1H NMR spectra were measured on a Bruker Avance 400 spectrometer. 13C NMR
spectra were measured on a Bruker Avance 600 spectrometer. Chemical shifts were given
in parts per million (TMS as internal standard). Steady state absorption and fluorescence
spectra were recorded using a Hitachi UV-3010 UV-Vis spectrophotometer and a Hitachi
F-4600
spectrofluorimeter,
respectively.
For
quantum
yield
calibration,
9,10-diphenylanthralene in ethanol was measured under the same excitation and detection
conditions (fluorescence quantum yield of 9,10-diphenylanthralene in ethanol is 0.95) [42].
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2.3 Synthesis
2.3.1 4-morpholine-1-yl- 1,8-naphthalic anhydride (2)
The morpholine (0.94 mL, 10.8 mmol) was added to a suspension of 4-bromo-1,8-naphthalic
anhydride 1 (1.5 g, 5.4 mmol) in ethylene glycol (10 mL). The mixture was heated to reflux for 24
h under N2 atmosphere. The solvent was removed under vacuum. Then the residue was purified by
column chromatography on silica gel eluting with THF/CHCl3 (1/4, v/v) to give 1.33 g yellow
1
solid (yield, 86%). H NMR (CDCl3, 400 MHz) δ: 8.60 (d, 1H, J = 7.2 Hz), 8.54–8.48 (m, 2H),
7.78–7.75 (m, 1H), 7.29–7.26 (m, 1H), 4.07–4.05 (m, 4H), 3.36–3.33 (m, 4H).
2.3.2 N,N’-Bis[2-((4-morphine-1-yl)-naphthalimide)ethyl]propane-1,3-diamine (3a)
To a hot suspension of compound 2 (0.5 g, 1.75 mmol) and triethylamine (0.5 mL) in
1,4-dioxiane (5 mL), N,N-bis(2-aminoethyl)propane-1,3-diamine (140 mg, 0.875 mmol) was
added dropwise. The reaction mixture was refluxed for 48 h in dark. The solvent was removed
under vacuum. The residue was purified by column chromatography on silica gel eluting with
CH3OH/CHCl3 (1/9, v/v). The yellow solid was obtained of 0.445 g, yield of 63.3%. 1H NMR
(CDCl3, 400 MHz) δ: 8.57 (d, 2H, J = 7.2 Hz), 8.54 (d, 2H, J = 8.0 Hz), 8.42 (d, 2H, J = 8.4
Hz), 7.72 (m, 2H), 7.23 (d, 2H, J = 8.0 Hz), 4.28 (m, 4H), 4.02 (m, 8H), 3.25 (m, 8H), 2.960
(m, 4H), 2.757 (m, 4H), 1.86 (s, 2H), 1.69 (m, 2H). 13C NMR (CDCl3, 150 MHz) (ppm):
164.5, 164.0, 155.5, 132.5, 131.2, 130.0, 129.9, 126.1, 125.8, 123.2, 117.1, 114.9, 66.9,
53.4, 47.9, 47.7, 39.7, 30.0. MS (MAIDI-TOF): [M]+ 691.6, calcd for C39H42N6O6, 690.8.
2.3.3
N,N’-Bis[2-((4-morphine-1-yl)-naphthalimide)ethyl]-N,N’-dimethylpropane
-1,3-diamine (3b)
The compound 3a (100 mg, 0.145 mmol) was suspended in 10 mL of ice-cold methanol,
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followed by the addition of glacial acetic acid (0.126 g, 2.10 mmol), sodium
cyanoborohydride (62.9 mg, 1.0 mmol), and left stirring in the ice bath for 20 min. An
aqueous solution of 39% (v/v) formaldehyde (0.11 mL, 1.43 mmol) in 3 mL of methanol
was added dropwise to the reaction mixture over a period of 30 min. The ice bath was
removed and the reaction mixture was stirred at room temperature for 5 h. To adjust the pH,
25 mL of Na2CO3 solution (2%) was added, and the mixture was extracted with chloroform
three times (3×20 mL). The extract was washed with a saturated solution of NaCl, dried
over MgSO4, concentrated under vacuum. The solid obtained was purified by column
chromatography (CH3OH/CHCl3 =1/25, v/v). The yellow solid was obtained of 70 mg, yield of
1
42.6%. H NMR (CDCl3, 400 MHz) δ: 8.58 (d, 2H, J = 7.2 Hz), 8.52 (d, 2H, J = 8.0 Hz),
8.41 (d, 2H, J = 8.4 Hz), 7.69 (m, 2H), 7.23 (d, 2H, J = 8.0 Hz), 4.26 (m, 4H), 4.02 (m, 8H),
3.25 (m, 8H), 2.67 (m, 4H), 2.47 (m, 4H), 2.33 (s, 6H), 1.66 (s, 2H). 13C NMR (CDCl3,
150 MHz) (ppm): 164.3, 163.8, 155.5, 132.4, 131.1, 129.9, 129.8, 126.1, 125.8, 123.3,
117.2, 114.9, 66.9, 55.7, 54.6, 53.4, 42.3, 37.6, 29.7, 25.1. MS (MAIDI-TOF): [M]+ 719.7
calcd for C41H46N6O6, 718.8.
,
2.3.4 4-(4-Phenyl-[1,2,3]-triazol-1-yl)-1,8-naphthalic anhydride (4)
The compound 4 was prepared according to the literature with slight modification [43]. To a
suspension of 4-bromo-1,8-naphthalic anhydride 1 (1.0 g, 3.61 mmol) in 30 mL N-Methyl
pyrrolidone (NMP) was added a solution of sodium azide (0.70 g, 10.8 mmol) in water (3 mL).
The mixture was stirred vigorously for 5 h at 60 ℃, and the solution was poured into ice water.
The precipitated yellow solid was filtered to give 4-azido-1,8-naphthalic anhydride.
The crude product 4-azido-1,8-naphthalic anhydride and ethynylbenzene (0.37 g, 3.61 mmol)
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were suspended in DMF (10 mL). To this solution, copper(II) sulfate pentahydrate (21.0 mg, 0.08
mmol) was added, followed by sodium ascorbate (33 mg, 0.17 mmol). The mixture was heated at
60 °C for 14 h and poured into ice water. The product was collected by filtration. After washing
the precipitate with cold water and methanol, the desired product was obtained (0.98 g, 80 % yield)
as a yellow powder. 1H NMR (DMSO-d6, 400 MHz, ppm): 9.38 (s, 1H), 8.73–8.67 (m, 2H), 8.46–
8.44 (m, 1H), 8.24 (d, 1H, J = 8 Hz), 8.06–8.02 (m, 3H), 7.58–7.54 (m, 2H), 7.46–7.43 (m, 1H).
2.3.5 N,N’-Bis[2-(4-(4-Phenyl-[1,2,3]-triazol-1-yl)-naphthalimide)ethyl]propane- 1,3-diamine
(5a)
The procedure was similar to that of compound 3a, except that the compound 4 was used in
place of compound 2. Yield: 52.8%. 1H NMR (DMSO-d6, 400 MHz) (ppm) δ: 9.30 (s, 2H), 8.64–
8.59 (m, 4H), 8.34–8.29 (m, 2H), 8.15 (d, 2H, J = 8.0 Hz), 8.03–8.01 (d, 4H, J = 8.0 Hz), 7.97–
7.93 (m, 2H), 7.55–7.52 (m, 4H), 7.44–7.41 (m, 2H), 4.19–4.16 (m, 4H), 2.88–2.85 (m, 4H),
2.70–2.67 (m, 4H), 1.60–1.56 (m, 2H). 13C NMR (DMSO-d6, 600 MHz) (ppm) δ: 163.30, 162.77,
147.05, 137.53, 131.50, 130.38, 129.92, 129.04, 129.01, 128.81, 128.41, 128.35, 125.64, 125.50,
124.05, 124.02, 123.41, 122.61, 47.00, 46.39, 45.67, 28.42. MS (ESI-MS): calcd for C47H38N10O4,
806.87.
2.3.6
N,N’-Bis[2-(4-(4-Phenyl-[1,2,3]-triazol-1-yl)-naphthalimide)ethyl]-N,N’-
dimethylpropane-1,3-diamine (5b)
The procedure was similar to that of compound 3b, except that the compound 5a was used in
1
place of compound 3a. Yield: 53.0%. H NMR (DMSO-d6, 400 MHz) δ: 9.22 (s, 2H), 8.59–8.53
(m, 4H), 8.24 (d, 2H, J = 8.0 Hz), 8.09 (d, 2H, J = 8.0 Hz), 7.99 (d, 4H, J = 8.0 Hz), 7.89–7.85 (m,
2H), 7.53–7.50 (m, 4H), 7.43–7.39 (m, 2H), 4.07–4.03 (m, 4H), 2.48–2.45 (m, 4H), 2.35–2.26 (m,
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4H), 2.15 (s, 6H), 1.46 (s, 2H) . 13C NMR (DMSO-d6, 600 MHz) (ppm) δ: 163.02, 162.49, 146.99,
137.54, 131.53, 130.44, 129.90, 129.43, 128.99, 128.76, 128.40, 128.30, 125.58, 125.50, 123.96,
123.92, 123.21, 122.44, 54.85, 53.89, 42.13, 37.47, 18.53. MS (ESI-MS): 835.1M+, calcd for
C49H42N10O4, 834.92.
2.4 Determination of lipophilicity (logP)
To obtain octanol-saturated water (OSW) and water-saturated octanol (WSO), 100 mL of
water was stirred with 100 mL of octanol for 24 h, followed by centrifugation for 5 min. The
bisnaphthalimides were dissolved in 5.0 mL of OSW to a typical concentration of 0.05 mM and
then mixed with 5.0 mL WSO. The samples were mixed, stirred for 24 h at room temperature and
then centrifuged for 5 min. The layers were separated carefully, and the concentrations of
bisnaphthalimides in WSO and OSW were determined by UV-Vis spectroscopy. Partition
coefficients of bisnaphthalimides were calculated using the equation (eqn (1)):
logP = log ([compound]WSO/[compound]OSW
)
(1)
where [compound]WSO and [compound]OSW was the concentration of bisnaphthalimides in WSO
and OSW, respectively.
2.5 Fluorescence titration
To further investigate the interactions of 3a and 3b with DNA, fluorescence titration
experiments were carried out, in which the concentration of 3a and 3b is fixed at 10 ꢀM.
The changes in the emission were monitored by the titration of different concentration of
DNA.
2.6 UV-Vis absorption titration of DNA with bisnaphthalimide
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A typical titration of naphthalimide derivatives in HEPES buffer was performed by
using a fixed concentration of 20 ꢀM, to which the DNA stock solution was gradually
added up to saturation. After each addition, the solution was mixed and allowed to
re-equilibrate for 15 min before the absorption spectra were recorded. The binding constant
Ka was determined from a D/ꢁεap vs D plot according to the following equation (eqn (2))
[44]:
D/ꢁεap = D/ꢁε +1/[(ꢁε)Ka] (2)
where D is the concentration of DNA, ꢁεap = |εA – εF|, εA = Aobs/[compound], ꢁε = |εB –
εF|, and εB and εF correspond to the extinction coefficients of the DNA-compound adduct
and unbound compound, respectively.
2.7 Circular dichroism (CD) studies
The oligonucleotides were dissolved in water to yield a 100 ꢀM stock solution, and
then diluted using 10 mM Tris-HCl (pH 7.4) with or without KCl. Prior to use in the CD
assay, the DNA solution was annealed by heating to 95 °C for 5 min and slowly cooled to
room temperature, and stored at 4 °C overnight. After that, 400 ꢀL of 40 ꢀM
bisnaphthalimide was mixed with the annealed DNA. The final concentrations of
oligonucleotides and bisnaphthalimides were 10 and 20 ꢀM, respectively. The solution was
incubated at room temperature for 2 h before examination.
2.8 Fluorescence resonance energy transfer (FRET) melting assay
The FRET experiments were carried out in 10 mM lithium cacodylate buffer (pH 7.2)
containing 10 mM KCl and 90 mM LiCl. The fluorescently labeled oligonucleotide strand
FHtelo (5’-FAM-GGG-TTA-GGG-TTA-GGG-TTA-GGG-TAMRA-3’) and Fmyc
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(5’-FAM-TTG-AGG-GTG-GGT-AGG-GTG-GGT-AA-TAMRA-3’) were used as the
FRET probe, in which FAM and TAMRA was 6-carboxyfluorescein and
6-carboxytetramethylrhodamine, respectively. FHtelo and Fmyc were diluted from 20 ꢀM
stock solution to 0.2 ꢀM, and then annealed by heating to 90 °C for 5 min, followed by
slowly cooling to room temperature. After that, bisnaphthalimides were mixed with the
annealed fluorescently labeled oligonucleotide. The final concentrations of fluorescently
labeled oligonucleotide and bisnaphthalimides were 0.2 and 1.0 ꢀM, respectively.
Measurements were performed on a Hitachi F-4600 with excitation at 492 nm and
detection at 516 nm. The temperature of the solution was increased from 27 to 96 °C at
intervals of 0.5 °C/min, maintaining a constant temperature of 3 min before each
measurement. Final analysis of the data was carried out using Origin 9.0 (OriginLab
Corp.). The melting results were the average of two replicates.
2.9 Molecular docking studies
Docking studies were carried out using AutoDock (Version 4.2). The
bisnaphthalimides were optimized by Density Functional Theory (DFT/B3LYP) method
with 6-31G basis sets. The optimized structures were used to do the docking. The crystal
structures of the telomeric, c-myc and c-kit G-quadruplexes were obtained from the Protein
Data Bank (PDB). AutoDock Tools 1.5.6 was used to establish the Autogrid points and to
visualize the docked structure of G-quadruplex-ligand complex. In order to explore the
entire conformation of nucleic acids, the 3D grid box dimensions had been defined
including the entire DNA macromolecules.
2.10 Fluorescence imaging
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For the purpose of staining and imaging, A549 cells were seeded onto 35 mm cell
culture dishes and cultured for 24 h (cell confluence must be over 80%). The cells were
incubated with naphthalimides and/or DAPI for 2 h. The unbound molecules were washed
three times with PBS. Cells were imaged using an Olympus IX70 fluorescence microscope
(Olympus Corporation, Japan). The fluorescence of DAPI was excited by a 405 nm laser
and collected from 425 to 475 nm; the fluorescence of naphthalimides was excited by a
488 nm laser and collected from 500 to 600 nm. Florescent images were processed using
the Olympus FV10-ASW 1.6 viewer software.
2.11 Cytotoxicity assay
All tissue culture media and reagents were obtained from Hy-Clone, Thermo Scientific A549
and normal human lung fibroblast MRC-5 cells were maintained in bulbecco’s modified eagle
medium (DMEM) containing 10% fetal bovine serum. A549 and MRC-5 cells were plated at
2×105 per well in a Nunc 48 well plate and allowed to grow for 24 h. The cells were exposed to
increasing concentrations of bisnaphthalimides and incubated for 48 h. The loading medium was
then removed and the cells were fed with medium containing MTT. Cell viability was then
measured by reading the absorbance at 570 nm using a Thermo MK3 Multiscan microplate reader.
The signal was normalized to 100% viable (untreated) cells. Growth inhibition by
bisnaphthalimides was evaluated by IC50 (concentration of a drug causing 50% inhibition of cell
growth). Each growth inhibition experiment was repeated at least three times and the results were
expressed as mean ± standard deviation (SD).
3. Results and Discussion
3.1 Synthesis and photophysical properties of bisnaphthalimides
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The bisnaphthalimides have been known to possess strong DNA-binding affinity and
anticancer activity [45,46]. In the present study, four bisnaphthalimides 3a, 3b, 5a and 5b are
synthesized (Scheme 1). Reaction of 4-bromo-1,8-naphthalic anhydride with morpholine in the
presence of 2-ethoxyethanol affords compound 2. The bisnaphthalimides 3a is synthesized by the
reaction of the corresponding polyamine N,N-bis(2-aminoethyl)propane-1,3-diamine and 2 in a
1:2 ratio. The compound 3a can be converted to 3b by reductive amination with formaldehyde.
Reaction of 4-bromo-1,8-naphthalic anhydride with sodium azide results in
4-azido-1,8-naphthalic anhydride. Click reaction of ethynylbenzene with 4-azido-1,8-naphthalic
anhydride is performed in DMF at 60 °C for 14 h, affording 4. The bisnaphthalimides 5a and 5b
are synthesized according to a similar procedure as that of 3a and 3b. The lipophilicity (logP)
increases in the order of 3a (0.45) < 5a (0.60) < 3b (0.67) < 5b (0.89), demonstrating that
N-methylation of the linkage can enhance the lipophilicity of bisnaphthalimide [47]. All these
compounds are isolated and purified by flash chromatography on silica gel, and are structurally
characterized by 1H NMR, 13C NMR and mass spectrometry (Fig. S1-S12).
The spectroscopic properties of 3a, 3b, 5a and 5b in various solvents, including THF, ethanol
and HEPES buffer, are summarized in Table 1. In THF, the absorption spectra of 3a and 3b
display two bands centered at 258 and 387 nm, and the emission spectra show a peak around 512
nm (Fig.S13). The emission spectra of 3a and 3b are red shifted in polar solvents (Fig.S13b and
Table 1), which can be attributed to the intramolecular charge transfer (ICT) between the
morpholine group and the naphthalimide ring system [48]. For the triazole derivatives 5a and 5b,
the absorption spectra and the emission spectrum are slightly affected by the solvent polarity,
indicating relatively low charge transfer efficiency between triazole and naphthalimide moiety
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[49,50].
The quantum yields of 3a and 3b are moderate in THF (Table 1), and decrease dramatically in
polar solvents. On the other hand, 5a and 5b are weakly fluorescent in all solvents. These results
indicate that the substituent groups at the 4-position can affect the photophysical properties of
bisnaphthalimides.
3.2 UV-Vis absorption titration studies
It is well known that the naphthalimides are capable of binding to duplex DNA through the
major groove and/or via intercalation interaction. The interaction of 3a, 3b, 5a and 5b with
different DNA samples is investigated by absorption spectroscopy. In HEPES buffer solution
containing 100 mM KCl, the addition of CT DNA to the solution of 3a and 3b leads to
considerable hypochromicity (Fig. 1). By using a plot of D/ꢁεap versus D (Fig. 1a insets)
according to the Scatchard equation, the binding affinity (Ka) toward CT DNA can be determined.
The results are summarized in Table 2. The Ka values of 3a, 3b and 5b with CT DNA are in the
range of 5.72×104~1.08×105 M-1, similar to that of the 4-substituted bisnaphthalimide [51]. No
obvious spectral shift can be observed in such system, suggesting that these bisnaphthalimides
bind with CT DNA through groove interaction. As for 5a, addition of CT DNA leads to weak
decrease in the absorbance (within 5%), indicating weak interaction between 5a and CT DNA
(Fig.S14).
Upon incremental addition of the preannealed Htelo, c-myc and c-kit G4s, the absorption
spectra of 3a, 3b and 5b show 12.9~44.0% hypochromism without obvious red shift, indicating
that 3a, 3b and 5b interact with G4 through external binding mode (Fig. S15-S17). As shown in
Table 2, 3a, 3b and 5b show high affinity toward Htelo, c-myc and c-kit G4s (Ka > 4.42×106 M-1),
15

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comparable to those of reported good quadruplex binders, such as platinum complex, ruthenium
complex, tetraazaperopyrene, phthalocyanine and phenanthroline derivatives [52-55]. The
compounds 3a, 3b and 5b exhibit more than 40-fold selectivity for quadruplexes versus duplex in
the presence of potassium, similar to that of Pt(II) complexes [56]. As for 5a, it also shows good
affinity toward Htelo and c-kit quadruplexes, but low affinity toward c-myc (Table 2, Fig. S16c).
These results suggest that subtle structure modification, such as N-methylation of linkage, can
affect the binding affinity of bisnaphthalimides with G4s.
3.3 Fluorescence titration studies
We investigated the changes in fluorescence of 3a and 3b in the presence of G-quadruplexes,
as well as CT DNA. Fig. 2a presents the steady-state fluorescence spectra of 3a in the presence of
different amount of c-myc. In the absence of c-myc, 3a is weakly fluorescent in buffer solution.
Upon addition of c-myc, the fluorescence intensity increases gradually, with an increase of about
27.2-fold eventually. As for 3b, addition of c-myc also leads to a fluorescence enhancement of
about 20.2-fold at saturation (Fig. 2b). The emission peaks blue-shift obviously with the addition
of G4 DNA, suggesting that the naphthalimide moiety of 3a and 3b may penetrate into the
hydrophobic environment of G-quadruplex (Fig.S18-19) [57]. The titration curves with different
DNA reveal that the degree of fluorescence enhancement strongly depends on the types of DNA
(Fig. 2c and 2d). The telomeric and oncogenic G-quadruplexes induce strong enhancement of the
fluorescence intensity (18 to 27-fold, Table 3), whereas the duplex CT DNA induces much weaker
fluorescence enhancement (around 2-fold). In contrast to the linear structure of double-stranded
DNA, intramolecular G4s are globular structures derived from the sequence-dependent folding of
single-stranded DNA. The special structural characteristics make 3a and 3b interact with G4s with
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high selectivity [58,59]. These results indicate that 3a and 3b can serve as a light-up fluorescent
probe with good sensitivity and excellent selectivity toward G-quadruplex over duplex DNA (Fig.
S20). For 5a and 5b, the addition of G-quadruplex and duplex DNA results in a much smaller
enhancement, maybe due to the low fluorescence quantum yield of 5a and 5b in organic solvents
and buffer solution (Fig. S21).
3.4 Circular dichroism (CD) spectra studies
The CD spectroscopic studies are carried out to assess the structural changes of
G-quadruplexes upon interaction with 3a, 3b, 5a and 5b. In the presence of K+ ions, c-myc and
c-kit exhibit a parallel structure, with a positive band at 265 nm and a negative band at 240 nm
(Fig. 3a and Fig. S22a) [60]. The CD spectra of c-myc and c-kit are almost unaffected upon
addition of 3a, 3b, 5a and 5b, indicating that c-myc and c-kit maintain their conformations upon
interaction with 3a, 3b, 5a and 5b. In the case of Htelo, it shows a strong positive band at 295 nm,
with two weak positive peaks at 250 and 270 nm (Fig. 3b), characteristic of mixed
parallel/antiparallel-stranded G-quadruplex structure [61]. Compounds 3a, 5a, and 5b induce a
comparable stabilization of the mixed–hybrid structure as proved by the increase in the positive
peaks at 290 and 270 nm. However, addition of 3b leads to an increase in the positive band around
290 nm and an obvious decrease in the positive band at 250 nm, suggesting 3b can convert Htelo
into antiparallel G-quadruplex [62].
In the absence of K+ ions, c-myc, Htelo and c-kit exhibit two positive peaks at about 255 and
295 nm (Fig. 3c-d and Fig. S22b), indicating the coexistence of unfolded sequence and quadruplex
structure [63]. For c-kit, the peak position of the band remains unchanged upon addition of 3a, 3b,
5a and 5b, suggesting that these compounds could not induce the formation of c-kit quadruplex
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(Fig. S22b). As for c-myc, the positive peak at 265 nm is enhanced, and a negative peak at 240 nm
appears when interacting with 3a and 3b and 5b, suggesting that the parallel structure of c-myc
can be induced by 3a, 3b and 5b (Fig. 3c) [64]. Regarding Htelo, the intensity of the positive band
at 295 nm increases and a negative band at 265 nm appears upon addition of 3a, 3b and 5b,
suggesting that 3a, 3b and 5b can efficiently induce the formation of antiparallel quadruplex (Fig.
3d) [56]. On the other hand, the CD spectra of c-myc and Htelo show some changes after addition
of 5a. However, the resulting CD spectra are different from the characteristic CD spectra of
parallel c-myc quadruplex and antiparallel telomeric quadruplex. No induced CD spectra (ICD)
can be observed in the range of 300-400 nm, indicating that 3a, 3b, 5a and 5b may interact with
c-myc, Htelo and c-kit through external binding mode [65].
3.5 Molecular docking studies
To gain further insight into the binding mode of 3a, 3b, 5a and 5b with the G-quadruplex,
Molecular docking studies are carried out [66]. The X-ray crystal structures of the telomeric (PDB
ID: 1KF1), c-myc (PDB ID: 1XAV) and c-kit (PDB ID: 2O3M) are docked with the DFT
optimized molecular structures of 3a, 3b, 5a and 5b using AutoDock 4.2. The docking results of
3a, 3b, 5a and 5b in various binding site of G4s are showing in Fig. S23-25. These
bisnaphthalimides mainly bind in the groove and/or loop region of the G4s, in agreement with the
UV-Vis titration and CD experimental results. The representative docking results are shown in Fig.
4-5 and S26.
For the telomeric G-quadruplex (PDI ID: 1FK1), the best docked poses of 3a, 3b, 5a and 5b
have been shown in Fig. 4. The naphthalimide moieties of 3a and 3b are directed into the channel
formed between the TTA loop and G-tetrad. Hydrogen bond is formed between quadruplex and
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the polyamine linkage in 3a or the carbonyl groups in 3b (Fig. 4a-b). The naphthalimide moiety of
3b can enter the loop more deeply than that of 3a, which may arise from the larger lipophilicity of
3b than 3a [67]. Similarly, the naphthalimide moiety of 5b can penetrate into the loop of Htelo,
while that of 5a can’t (Fig. 4c-d). The triazole side-chains of 5a and 5b are properly directed into
the DNA grooves and filled the groove space, which may help lock the ligand onto the
G-quadruplex. The triazole moiety in 5a and 5b interacts with the groove through hydrogen bond
[68]. These results suggest that the increase in the lipophilicity by N-methylation of the linkages
can affect the binding model of bisnaphthalimides with G-quadruplex.
In the case of c-myc quadruplex (PDB ID: 1XAV), there are three loops bridging three
G-tetrad layers and connecting adjacent parallel strands [69]. The best docked poses of 3a, 3b and
5b have been shown in Fig. 5. Hydrogen bonds are formed between c-myc quadruplex and the
polyamine linkage in 3a, 3b and the triazole moiety in 5b, respectively. The docking results also
show that hydrogen bond exists between bisnaphthalimides and c-kit quadruplex (Fig. S24 and Fig.
S26).
3.6 FRET melting assay
The ability of bisnaphthalimides 3a, 3b, 5a and 5b to stabilize the G-quadruplex is further
evaluated by the FRET-melting assay. As shown in Fig. 6a, 3a and 3b appear as relatively strong
telomeric G-quadruplex stabilizers, with an increase in the melting temperature (ꢁTm) of around
10 ℃ (Table 4). In contrast, the ꢁTm induced by 5a and 5b is around 3 ℃. For c-myc, a
moderate increase in Tm values (ꢁTm ~4.7–7.4 ℃) is obtained after the addition of 3a, 3b and 5b
(Fig. 6b). Addition of 5a results in a small change in the Tm ( –0.3 ℃), suggesting that 5a weakly
interacts with c-myc G-quadruplex. The compounds 3a and 3b possess good interaction properties
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with G4 strands, with similar change in melting temperature of the G4s as that of polyaromatic
compounds [15] and previous reported mono-naphthalimides [28-31].
3.7 Cellular location studies
Being fluorescent, it is convenient to track the cellular localization of 3a and 3b by
fluorescence imaging. Fluorescence microscopy is used to study the localization of 3a and 3b in
A549 lung carcinoma cells. A convenient and reliable DNA-specific nuclear stain, DAPI is chosen
for costaining. As shown in Fig. 7, after incubation at 37 °C for 2 h, 3a, 3b and DAPI have entered
living cells. The green fluorescence of 3a, 3b and blue fluorescence of DAPI are observed. The
merged images show that 3a and 3b can readily be taken up by A549 lung cancer cells, but not
just localize in the nucleus.
3.8 Cytotoxicity of bisnaphthalimides
The cytotoxicity of 3a, 3b, 5a and 5b against non-small cell lung cancer cell line A549 and
noncancerous cells MRC-5 is assessed using the MTT assay (Fig. S27a). As shown in Table 5, 3a,
3b, 5a and 5b show good anticancer activities against the A549 cell lines, with the IC50 values in
the range of 0.15 to 1.04 µM. All the compounds are more effective in killing A549 cancer cells
than the standard drug amonafide. In particular, 3b is up to 25 times more effective in killing A549
cells than the reference drug. Importantly, 3a, 3b, 5a and 5b exhibit significantly less cytotoxicity
against a noncancerous human embryonic lung fibroblast cells MRC-5 cells (Fig. S27b), with the
IC50 values in the range of 0.95 µM to 2.22 µM. These results indicate that 3a, 3b, 5a and 5b are
promising anti-proliferative agents.
4. Conclusion
In conclusion, four bisnaphthalimides have been investigated for their potential G-quadruplex
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binding. The compounds 3a, 3b and 5b display high affinity for telomeric and oncogenic
G-quadruplexes. On the other hand, 5a only show high affinity for the c-kit and telomeric
quadruplex (Ka > 106 M-1), and interact with c-myc quadruplex and duplex CT DNA weakly.
Molecular docking analysis suggests that 3a, 3b, 5a and 5b interact with the groove and/or loop of
the G-qudruplex through hydrogen bonding. Fluorescence intensity of 3a and 3b is enhanced
dramatically in the presence of G-quadruplex. Fluorescence imaging further indicate that 3a and
3b can readily enter A549 cells. These compounds efficiently inhibit the growth of A549 cancer
cell lines with low cytotoxicity toward noncancerous cell MRC-5. These results indicate that the
bisnaphthalimide with G-quadruplex binding properties may possess tumor cell-selective
cytotoxic activity, which is helpful to design novel naphthalimide anticancer agent.
Acknowledgements
This research is supported by the Natural Science Foundation of Shaanxi Province
(2018JM2019, 2016JM2013), the National Natural Science Foundation of China (21073143), and
the NPU Foundation for Graduate Innovation (ZZ2017192).
Supporting Information Available
Supplementary absorption spectra, fluorescence spectra, CD spectra, Autodock analysis results,
and copies of NMR spectra and Mass spectra are included.
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