Ferrocene appended naphthalimide derivatives: Synthesis, DNA binding, and in vitro cytotoxic activity

Article abstract of DOI:10.1016/j.jorganchem.2019.03.001

Three novel ferrocene appended naphthalimide derivatives (2a, 2b and 6) were synthesized and characterized by IR, ¹H NMR and mass spectroscopies. In electrochemical experiments, all of the ferrocene appended naphthalimide derivatives exhibited reversible one-electron oxidation in CV curves. And the DNA binding ability of the compounds was studied by ethidium bromide displacement experiments and UV–visible spectrophotometry. The ferrocene appended naphthalimide derivative 6 also exhibited the highest DNA binding ability in all the tested compounds. According to the viscosity results, all of the synthesized compounds displayed the partial intercalation binding mode to DNA duplex. Bis-naphthalimide compound 5 was used as reference compound to evaluate the synergistic effect of the ferrocene group in ferrocene appended naphthalimide derivative 6. The cytotoxicity of the synthesized compounds against 4 different human cancer cell lines (EC109, BGC823, SGC 7901 and HepG2) was studied by MTT assay. The ferrocene appended bis-naphthalimide derivative 6 showed the best cytotoxicity against tested human cancer cells in all the synthesized naphthalimide derivatives and control drug amonafide. The uptake of naphthalimide derivative 6 was proved by laser confocal images. In addition, the DNA damage induced by naphthalimide derivative 6 was studied by comet assay.

Full text of DOI:10.1016/j.jorganchem.2019.03.001

Accepted Manuscript
Ferrocene appended naphthalimide derivatives: Synthesis, DNA binding, and in vitro
cytotoxic activity
Deng-Guo Jia, Jia-An Zheng, Yan-Ru Fan, Jian-Qiang Yu, Xiu-Li Wu, Bo-Jin Wang,
Xin-Bin Yang, Yu Huang
PII:
S0022-328X(19)30079-8
DOI:
https://doi.org/10.1016/j.jorganchem.2019.03.001
JOM 20729
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 27 December 2018
Revised Date: 26 February 2019
Accepted Date: 1 March 2019
Please cite this article as: D.-G. Jia, J.-A. Zheng, Y.-R. Fan, J.-Q. Yu, X.-L. Wu, B.-J. Wang, X.-
B. Yang, Y. Huang, Ferrocene appended naphthalimide derivatives: Synthesis, DNA binding, and
in vitro cytotoxic activity, Journal of Organometallic Chemistry (2019), doi: https://doi.org/10.1016/
j.jorganchem.2019.03.001.
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ACCEPTED MANUSCRIPT
Ferrocene appended naphthalimide derivatives: Synthesis, DNA
binding, and in vitro cytotoxic activity
1,
†
1,
1,*
1
1
†
Deng-Guo Jia , Jia-An Zheng , Yan-Ru Fan , Jian-Qiang Yu , Xiu-Li Wu , Bo-Jin
Wang1, Xin-Bin Yang2, Yu Huang1,*
1
Key Laboratory of Hui Ethnic Medicine Modernization, Ministry of Education, Ningxia Engineering and
Technology Research Centre of Hui Medicine Modernization, College of Pharmacy, Ningxia Medical
University, 750004 Yinchuan, PR China
2 Rongchang Campus, Southwest University, 402460 Chongqing, PR China
*
Corresponding author: risefyr@163.com (Y. R. Fan); huangyunxmu@163.com (Y. Huang);
†The two authors contributed equally to this paper.
Abstract
Three novel ferrocene appended naphthalimide derivatives (2a, 2b and 6) were
synthesized and characterized by IR, 1H NMR and mass spectroscopies. In
electrochemical experiments, all of the ferrocene appended naphthalimide derivatives
exhibited reversible one-electron oxidation in CV curves. And the DNA binding ability
of the compounds was studied by ethidium bromide displacement experiments and UV-
visible spectrophotometry. The ferrocene appended naphthalimide derivative 6 also
exhibited the highest DNA binding ability in all the tested compounds. According to the
viscosity results, all of the synthesized compounds displayed the partial intercalation
binding mode to DNA duplex. Bis-naphthalimide compound 5 was used as reference
compound to evaluate the synergistic effect of the ferrocene group in ferrocene appended
naphthalimide derivative 6. The cytotoxicity of the synthesized compounds against 4
different human cancer cell lines (EC109, BGC823, SGC 7901 and HepG2) was studied

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by MTT assay. The ferrocene appended bis-naphthalimide derivative 6 showed the best
cytotoxicity against tested human cancer cells in all the synthesized naphthalimide
derivatives and control drug amonafide. The uptake of naphthalimide derivative 6 was
proved by laser confocal images. In addition, the DNA damage induced by naphthalimide
derivative 6 was studied by comet assay.
Keywords: naphthalimide derivatives, ferrocene, synthesis, cytotoxicity, DNA binding
1. Introduction
Cancer is the second cause of death and responsible for one-sixth of the deaths (estimated
9.6 million) all over the world in 2018 [1]. And the International Agency for Research on
Cancer (IARC) estimated that there would be 21.7 million new cancer cases and 13
million cancer deaths by 2030 [2]. The development of new cancer treatments and drugs
have been attracted great attention for decades. In 2017, FDA approved 12 oncology
products which were 26% of the approved products [3].
Organometallic compounds exhibited remarkable potential for the development of new
cancer drugs not only due to the direct cytotoxicity but also to the drug targeting and
active anticancer immune response ability [4]. Among the organometallic compounds,
ferrocene is one of the most well-known compounds in many areas of science, especially
in medicinal chemistry. It attracted great attention on account of the high stability,
lipophilicity, low toxicity, easy functionalization and mild reversible redox property [5].
Hence, ferrocenyl group conjugated nature products or drugs were synthesized and

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exhibited kinds of biological activities, including antitumor, antimalarial and antibacterial
activity [6]. In the development of ferrocene based drugs, the two most successful
examples were ferrocifen and ferroquine. The last one entered the clinical trials and
would be completed in 2019 [7]. The two compounds represent one type of ferrocene
derivatives, in which the ferrocenyl group replaced a portion of the lead compound [8].
And in another type, the ferrocenyl group directly attached to the lead compound, which
is a promising strategy to enhance the anticancer efficiency by attaching ferrocenyl group
to a DNA intercalator [5a]. Ferrocene appended intercalators were reported to exhibit far
more cytotoxicity or better selectivity towards cancerous cells than the single
intercalators [9].
1,8-naphthalimide, a well-known DNA intercalator, has been extensively investigated in
the development of antitumor agents [10]. Some of the naphthalimide derivatives, such as
amonafide, elinafide and bisnafide (Figure 1), have entered into phase II clinical trials
stage [10b, 11]. Among them, elinafide and bisnafide were typical bis-intercalator which
constructed by a polyamine linker and two naphthalimide groups. Such a strategy was
helpful to enhance the DNA binding ability and anticancer activity of the intercalator [12].
In our previous work, some bis-aryl compounds were designed and synthesized according
to the strategy [13]. And the DNA binding and cytotoxicity activity of the bis-aryl
compounds were systematically studied, which displayed significant advantage over the
mono-aryl ones.
Herein, ferrocene appended mono-naphthalimide and bis-naphthalimide derivatives were
synthesized to study the influence of linker and synergistic effect of the ferrocenyl group
to naphthalimide derivatives. The cytotoxicity of the synthesized compounds against

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human cancer cells was evaluated by MTT assay. And the DNA binding ability of the
compounds was studied by ethidium bromide (EB) displacement experiments, UV-visible
spectrophotometry and viscosity studies. According to the results of experiments, suitable
linker and the presence of the ferrocenyl group were helpful to enhance the cytotoxicity
and the DNA binding ability of the bis-naphthalimide derivative.
2. Materials and methods
2.1 General
All of the chemical reagents and solvents were obtained from commercial sources and
used directly without further purification, except THF. CT DNA was from Sigma-Aldrich.
N-Boc-bromoethylamine was synthesized according to the literature [14].
Electrochemical experiments were performed on a CH Instruments CHI-600
electrochemical workstation. Fluorescence spectra were recorded on a Hitachi F4600
spectrofluorometer at room temperature. UV-visible data were carried out on a Shimadzu
UV-2600spectrophotometer at room temperature. IR spectra were recorded on a
Shimadzu IRAffinity-1 spectrophotometer. 1H NMR and 13C NMR spectra were recorded
on a Bruker AVIII 400 spectrometer, with tetramethylsilane (TMS) as an internal
standard. Mass spectra were recorded on a Shimadzu LCMS-IT-TOF. Confocal
microscopy images were captured on an Olympus FV1000-IX81 Confocal Microscope.
Comet assay data were recorded with an Olympus BX53 fluorescence microscopy.
2.2 Synthesis
2.2.1 Synthesis of mono-naphthalimide derivative 1

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1,8-Naphthalimide (2 g, 10.14 mmol), hydroxyethyl amine and triphenylphosphine (5.3 g,
o
20.27 mmol) were dissolved in 50 mL anhydrous THF. After cooled to 0 C in an ice-
water bath, diisopropyl azodiformate (4.0 mL, 20.27 mmol) was added dropwise with
stirring in 30 minutes. Then the mixture was stirred under the protection of nitrogen at
room temperature for 5 hours. After removed the solvent by rotary evaporator, the crude
oil product was purified by column chromatography (ethyl acetate: methanol) to yield the
pure product as a white solid.
Naphthalimidoethylamine 1a
1
Yield: 51.64%. FTIR (KBr)
ν:
3348(NH2), 1698(C=O). H NMR (400 MHz, CDCl3) δ:
8.61 (dd, J = 7.2, 1.1 Hz, 2H, -ArH), 8.22 (dd, J = 8.2, 1.1 Hz, 2H, -ArH), 7.76 (dd, J =
8.2, 7.3 Hz, 2H, -ArH), 4.29 (t, J = 6.6 Hz, 2H, -CH2), 3.08 (t, J = 6.6 Hz, 2H, -CH2). 13C
NMR (100 MHz, CDCl3) δ: 164.62, 134.14, 131.72, 131.46, 128.35,127.07, 122.71,
43.24, 40.64. HR-MS, m/z: [M+H]+ 241.0939 (calcd for C14H12N2O2 : 241.0899).
+
2-(Naphthalimidoethylamino)ethylamine 1b
Yield: 58.33%. FTIR (KBr) ν
: 3433(NH2), 3243(NH), 1692(C=O). 1H NMR (400
MHz, CDCl3) δ: 8.56 (dd, J = 7.3, 1.2 Hz, 2H, -ArH), 8.18 (dd, J = 8.3, 1.2 Hz, 2H, -
ArH), 7.72 (dd, J = 8.2, 7.3 Hz, 2H, -ArH), 4.32 (t, J = 6.6 Hz, 2H, -CH2), 2.99 (t, J = 6.6
13
Hz, 2H, -CH2), 2.77 – 2.72 (m, J = 3.3 Hz, 4H, -CH2). C NMR (100 MHz, D2O) δ:
165.54, 135.67, 131.80, 130.98, 127.10, 126.99, 120.27, 46.76, 44.54, 36.64, 35.29. HR-
+
MS, m/z: [M+H]+ 284.1401 (calcd for C14H12N2O2 : 284.1392).
2.2.2 Synthesis of ferrocene appended mono-naphthalimide derivative 2

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Ferrocenecarboxaldehyde (0.53 g, 2.46 mmol) and compound 1 were dissolved in 50 mL
methanol. After refluxed for 3 hours, the mixture was cooled to room temperature. Then
sodium borohydride (0.2 g, 4.92 mmol) was added in batches with constant stirring at
room temperature. After the mixture was refluxed for 2 hours, the solvent was removed
by rotary evaporator to obtain the crude product. Then the crude product was dissolved in
50 mL dichloromethane, washed by 50 mL saturated sodium bicarbonate for two times,
50 mL water and 50 mL saturated brine in turn. The organic layer was dried by sodium
sulfate. After the organic solvent was removed by rotary evaporator, pure product was
obtained by column chromatography (ethyl acetate: methanol).
Ferrocene appended mono-naphthalimide derivative 2a
Yield: 56.48%. FTIR (KBr) ν
: 3316(NH), 1698(C=O), 491(Cp-Fe-Cp). 1H NMR
(400 MHz, DMSO-d6) δ: 8.49 (ddd, J = 16.6, 7.8, 1.1 Hz, 4H, -ArH), 7.88 (dd, J = 8.2,
7.3 Hz, 2H, -ArH), 4.25 – 4.17 (m, 4H, -C5H4), 4.11 (s, 5H, -C5H5), 4.07 (t, J = 1.9 Hz,
13
2H, -CH2), 3.57 (s, 2H, -CH2), 3.34 (s, 2H, -CH2), 2.91 (t, J = 6.4 Hz, 2H, -CH2). C
NMR (100 MHz, DMSO-d6) δ: 163.59, 134.24, 131.26, 130.67, 127.39, 127.19, 122.14,
68.17, 67.18, 47.60, 46.25. HR-MS, m/z: [M+H]+ 439.1082 (calcd for
+
C25H23FeN2O2 :439.1031).
Ferrocene appended mono-naphthalimide derivative 2b
1
Yield: 48.57%. FTIR (KBr)
ν
:
3369(NH), 1696(C=O), 495(Cp-Fe-Cp). H NMR (400
MHz, DMSO-d6) δ: 8.49 (ddd, J = 8.4, 5.1, 1.2 Hz, 4H, -ArH), 7.89 (dd, J = 8.3, 7.3 Hz,
2H, -ArH), 4.35 (t, J = 1.9 Hz, 2H, -CH2), 4.23 – 4.14 (m, 8H, -FcH), 3.79 (s, 2H, -CH2),
13
2.97 – 2.78 (m, 6H, -CH2). C NMR (100 MHz, DMSO-d6) δ: 163.54, 134.23, 131.29,

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130.67, 127.42, 127.18, 122.14, 68.15, 67.18, 47.84, 46.68, 46.37. HR-MS, m/z: [M+H]+
+
482.1529 (calcd for C27H28FeN3O2 :482.1453).
2.2.3 Synthesis of N-(tert-butyloxyoxycarbonyl)-bis(2-hydroxyethyl)aminoethylamine 3
To a solution of N-Boc-bromoethylamine (10 g ， 44.62 mmol) and N-
hydroxyethylenediamine (2.85 mL, 29.75 mmol) in 100 mL acetonitrile, anhydrous
potassium carbonate (20.56 g，150 mmol) was added. The mixture was refluxed for 40
hours. After filtrated to remove the solid, the filtrate was concentrated by
rotary evaporator to obtain the crude product. The pure oil product was purified by
column chromatography (ethyl acetate: methanol = 20:1, v/v). The yield of compound 3
was 20%. FTIR (KBr) ν:
3346(br, NH, OH), 1691(C=O). 1H NMR (400 MHz, CDCl3) δ:
3.90 – 3.74 (m, 4H, -CH2), 3.44 (q, J = 5.5 Hz, 2H, -CH2), 3.25 – 2.95 (m, 6H, -CH2),
13
1.44 (d, J = 4.1 Hz, 9H, -OC(CH3)3). C NMR (100 MHz, CDCl3) δ: 159.51, 79.95,
62.31, 60.64, 55.69, 38.17, 28.51. HR-MS, m/z: [M+H]+ 249.1802 (calcd for
+
C11H25N2O4 :249.1736).
2.2.4 Synthesis of bis-naphthalimide derivative 4
1,8-Naphthalimide (2.67 g ， 13.55 mmol), compound 3 (1.53 g, 6.16 mmol) and
triphenylphosphine (4.8 g，18.48 mmol) were dissolved in 50 mL anhydrous THF. After
cooled to 0 oC in an ice-water bath, diisopropyl azodiformate (3.7 mL，18.48 mmol) was
added dropwise with stirring in 30 minutes. Then the mixture was stirred under the
protection of nitrogen at room temperature for 5 hours. After removed the solvent by
rotary evaporator, the crude oil product was purified by column chromatography (ethyl
acetate: petroleum ether = 1:1, v/v) to yield the pure product. The yield of compound 4

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was 52.94%. FTIR (KBr) ν:
3349(NH), 1701(C=O). 1H NMR (400 MHz, CDCl3) δ: 8.51
(dd, J = 7.3, 1.1 Hz, 4H, -ArH), 8.24 (dd, J = 8.3, 1.1 Hz, 4H, -ArH), 7.73 (dd, J = 8.3,
7.3 Hz, 4H, -ArH), 4.74 (q, J = 7.6 Hz, 4H, -CH2), 3.77 (s, 6H, -CH2), 3.59 (s, 2H, -CH2),
13
1.32 (s, 9H, -OC(CH3)3). C NMR (100 MHz, DMSO-d6) δ: 164.08, 163.82, 134.32,
131.25, 130.70, 127.47, 127.12, 122.01, 78.45, 28.02. HR-MS, m/z: [M+H]+ 607.2551
+
(calcd for C35H35N4O6 : 607.2558).
2.2.5 Synthesis of reference compound 5
To a solution of Compound 4 (1.5 g，2.47 mmol) in 30 mL dichloromethane, 15 mL
trifluoroacetic acid (TFA) was added. After stirred for 2 hours, 80 mL saturated sodium
bicarbonate solution was slowly added. Then the water solution was extracted by 100 mL
dichloromethane for 3 times. The organic layer was combined and dried by sodium
sulfate. Dichloromethane was removed by rotary evaporator to obtain the pure product.
1
The yield of compound 5 was 89.6%. FTIR (KBr)
ν
:
3177(NH2), 1694(C=O). H NMR
(400 MHz, DMSO-d6) δ: 8.43 (dd, J = 8.3, 1.1 Hz, 4H, -ArH), 8.30 (dd, J = 7.3, 1.2 Hz,
4H, -ArH), 7.81 (t, J = 7.8 Hz, 4H, -ArH), 4.12 (t, J = 6.8 Hz, 4H, -CH2), 2.81 (t, J = 6.8
Hz, 4H, -CH2), 2.61 (t, J = 6.5 Hz, 2H, -CH2), 2.53 (d, J = 6.1 Hz, 2H, -CH2). 13C NMR
(100 MHz, DMSO-d6) δ: 163.42, 134.22, 131.22, 130.54, 127.32, 127.09, 121.97, 53.62,
+
51.59, 38.21, 37.64. HR-MS, m/z: [M+H]+ 507.2028 (calcd for C30H27N4O4 :507.1954).
2.2.6 Synthesis of ferrocene appended bis-naphthalimide derivative 6
Ferrocenecarboxaldehyde (0.42 g，1.96 mmol) and compound 5 (1.00 g，1.97 mmol)
were dissolved in 50 mL methanol. After refluxed for 3 hours, the mixture was cooled to
room temperature. Then sodium borohydride (0.15 g，4.0 mmol) was added in batches

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with constant stirring at room temperature. After the mixture was refluxed for 2 hours,
the solvent was removed by rotary evaporator to obtain the crude product. The crude
product was dissolved in 50 mL dichloromethane, washed by 50 mL saturated sodium
bicarbonate for two times, 50 mL water and 50 mL saturated brine in turn. The organic
layer was dried by sodium sulfate. After the organic solvent was removed by
rotary evaporator, pure product was obtained by column chromatography (ethyl acetate:
methanol = 3:1, v/v). The yield of compound 6 was 43.06%. FTIR (KBr) ν: 3349(NH),
1698(C=O), 485(Cp-Fe-Cp). 1H NMR (400 MHz, DMSO-d6) δ: 8.46 (d, J = 8.2 Hz, 4H,
-ArH), 8.33 (d, J = 7.2 Hz, 4H, -ArH), 7.84 (t, J = 7.8 Hz, 4H, -ArH), 4.17 – 4.14 (m, 4H,
-C5H4), 4.01 (s, 5H, -C5H5), 3.93 (t, J = 1.8 Hz, 2H, -CH2), 3.88 (t, J = 1.9 Hz, 2H, -CH2),
13
3.08 (s, 2H, -CH2), 2.83 (t, J = 6.8 Hz, 4H, -CH2), 2.67 (t, J = 6.2 Hz, 2H, -CH2). C
NMR (100 MHz, DMSO-d6) δ: 163.39, 134.17, 131.24, 130.54, 127.34, 127.08, 122.00,
68.31, 68.18, 67.32, 51.62, 37.61. HR-MS, m/z: [M+H]+ 705.2162 (calcd for
+
C41H37FeN4O4 :705.2086).
2.3 Water solubility
Excess amount (more than 10 mg for each compound) naphthalimide compounds were
added into 5 mL volumetric flask, respectively. After the addition of distilled water, the
mixture was ultrasonic treatment for 30 minutes until the compounds were dissolved
adequately in water to form a saturation solution. Then the saturation solution was stored
in dark for 24 hours. The supernatant was moved to centrifuge tubes and centrifuged for
15 minutes, then filtrated by microfiltration membrane to obtain the test solution. After
dilution, the absorbance of the test solution was measured by UV-Vis spectrophotometer.

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And the saturation solubility of the compounds was calculated according to the standard
curve of each compound.
2.4 Electrochemical studies
A three electrode system was used to study the electrochemical properties of the
ferrocene appended naphthalimide compounds. The working electrode, auxiliary
electrode and reference electrode were glassy carbon electrode, platinum electrode and
Ag/AgCl/KCl (sat.), respectively. The tested compounds were dissolved in 50% DMSO
water solution as storage solution (10 mmol/L). Then 1 mL compound solution was
diluted to 10 mL by 90% ethanol solution containing tetra-n-butylammonium perchlorate
(TBAP, 0.1 mol/L) as the supporting electrolyte. The scan rate was 100 mV/s in the
experiment.
2.5 Ethidium bromide displacement experiment
200 µL stock solution of EB (1 mg/mL) was added into a 50 mL volumetric flask. Then
diluted with PBS buffer solution (10 mmol/L, pH 7.4) to volume. To another 163 µL
stock solution of CT DNA (1 mg/mL) in 25 mL volumetric flask was diluted by the
prepared EB-PBS buffer solution to volume. The maximum fluorescence intensity of the
EB-DNA PBS buffer solution was measured on a Hitachi F4600 spectrofluorometer at
room temperature as F0. In 3 mL EB-DNA PBS buffer solution, increasing volume of the
compounds solution (5 mmol/L in DMSO, 5 - 15 µL) were added. And the maximum
fluorescence intensity of the mixture solution was measured as F. The quenching
constants were calculated according to the Stern-Volmer equation [15].
F0/F = 1 + Ksv[Compound]

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Where Ksv is the quenching constant.
2.6 UV-visible spectrophotometry
20 µL stock solution of naphthalimide compounds (5 mmol/L in DMSO) was diluted by
3 mL phosphate buffer (10 mmol/L, pH = 7.4). Increasing volume of CT DNA solution
(10-30 µL, 1 mg/mL) was added into the solution. The UV-Vis spectra of the compounds
were
recorded
by
a
Shimadzu
UV-2600
spectrophotometer.
And
the molar absorption coefficient of the compounds at 341 nm was calculated according to
the recorded data as εf. Then the solution was stirred and incubated at room temperature
for 10 min. After that, the UV-Vis spectra in the absence and presence of DNA were
recorded by a Shimadzu UV-2600 spectrophotometer. The binding constant Ka was
calculated from a D/∆εap vs D plot according to the following equation [16].
D/∆εap = D/∆ε + 1/[(∆ε)Ka]
Where D is the concentration of DNA, ∆εap = [εa-εf], εa = Aobs/[compound], ∆ε = [εb-εf],
εb and εf correspond to the extinction coefficients of the DNA-compound adduct and
unbound compound, respectively.
2.7 Viscosity studies
Naphthalimide compounds were dissolved in 30 % DMSO/water solution as storage
solution (5 mmol/L). 0.5 mL CT DNA (1 mg/mL) was diluted by 20 mL phosphate
buffer (10 mmol/L, pH 7.4) in an Ubblehode viscometer. After the mixture was
equilibrated in a water bath at 25 °C for 10 minutes, the flow time was measured by
stopwatch after varying the concentrations of compounds. The plot of (η /η0)1/3 vs r was

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obtained according to the flow time, where η and η0 were the flow time of presence and
absence of compounds, respectively, and r was equal to [compound]/[DNA].
2.8 Cytotoxicity assay
Human esophageal carcinoma cell (EC109), Human gastric carcinoma cell line
(BGC823), human gastric cancer (SGC7901) were cultured in RPMI 1640 medium, and
hepatoblastoma cell line (HepG2) were cultured in DMEM medium supplemented with
10% fetal bovine serum, 10 units/mL penicillin, and 10 units/mL penicillin streptomycin.
o
Cell culture was kept in 5% CO2 under humidified conditions at 37 C. The culture
solution was changed every other day, and the subcultures were performed with 0.25%
trypsin. Tested compounds were solubilized in RPMI 1640 or DMEM medium and
diluted to different concentration right after it will be used.
The thiazolyl blue tetrazolium bromide (MTT) assay was used to evaluate the
cytotoxicity of synthesized naphthalimide in vitro. Tumor cells were planted into 96-well
microtiter plates at a density of 5.0 × 103 cells/well. After cultured in 5 % CO2 under
o
humidified conditions at 37 C for 24 hours, various compounds medium solution were
added to obtain the final drug concentration of 0, 6.25, 12.5, 25, 50 and 100 µmol/L,
respectively. 50 µL MTT (5 mg/L) was added into each cell after 24 hours incubation.
For 4 hours later, the medium was removed and replaced by 150 µL DMSO to solubilize
the converted purple dye in culture plates. The absorbance of each cell was measured by
a Bio-rad 680 microplate reader at 550 nm. The IC50 which inhibits the growth of 50 % of
cells relative to nontreated control cells was calculated as the concentration of tested
compound by linear fitting.

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2.9 Cellular uptake and localization
After HepG2 cells were seeded into well plates 2 × 105 cells/well for 24 hours, compound
6 (6 µmol/L ) was added into the well plates. After incubation for 12 hours, the culture
supernatant was removed and the cells were washed by PBS buffer for 3 times. Then
paraformaldehyde solution was added and incubated with cells for 15 minutes to fix the
cells. After that, the cells were washed by PBS buffer again, then stained with DAPI. The
images were captured using an Olympus FV1000-IX81 Confocal Microscope.
2.10 Comet assay
HepG2 cells were planted into 6-well microtiter plates. After cultured in 5% CO2 under
o
humidified conditions at 37 C for 24 hours, compound 6 was added to obtain the final
drug concentration of 7, 11 and 15 µmol/L, respectively. 0.5 % DMSO was used as
control group. After incubation for 24 hours, cells were washed by PBS buffer for comet
test experiment.110 µL 0.8% normal melting point agarose was precoated on a
microscope slide. Then 100 µL 0.8% low melting point agarose with 104 cells was frosted
o
on the precoated microscope slide at 4 C. After that, the slide was immersed in a lysis
solution (2.5 mol/L NaCl, 100 mmol/L Na2EDTA, 10 mmol/L Tris, 1% Sodium
Sarcosinate, 1% TritonX-100, pH 10.0) for 3 hours. After the lysis immersion, the slide
was placed in a horizontal electrophoresis tank containing an electrophoretic solution (1
mmol/L NaEDTA, 300 mmol/L NaOH, pH 13.0) for 30 minutes. Then electrophoresis
was carried out at 22 V, 190 mA condition for 40 min. After electrophoresis, the slide
was washed by PBS buffer for 3 times and stained with EB. Comets were visualized with
an Olympus BX53 fluorescence microscopy. 100 cells were randomly selected for
recording the tail DNA% and tail moment.

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2.11 Statistical analysis
The significance of the differences between values obtained through comet assay was
evaluated using one-way ANOVA, followed by LSD t-test, statistically different from the
control: **p < 0.01.
3. Results and Discussion
3.1 Chemistry
The synthesis route of ferrocene appended naphthalimide derivatives was shown in
Scheme 1. In the synthesis of mono-naphthalimide derivatives, firstly, 1,8-naphthalimide
was reacted with the hydroxyl group of different amino alcohols via Mitsunobu Reaction.
And the amino terminus of the amino alcohols was condensed with
ferrocenecarboxaldehyde by Schiff's base reaction. Then the different Schiff bases were
reduced by sodium borohydride to yield the target compound 2. In the synthesis of bis-
naphthalimide derivative 6, due to the steric hindrance of the naphthalimide group, the
alkylation reaction of N-Boc-bromoethylamine with di(naphthalimidoethyl) amine 7
failed. Hence, a modified synthesis route of compound 6 was started from the
alkylation reaction of N-Boc-bromoethylamine with diethanolamine. Then the
intermediate 3 was condensed with 1,8-naphthalimide via Mitsunobu Reaction to obtain
compound 4. After compound 4 was deprotected by TFA to yield the reference
compound 5, the amino group of compound
5
was condensed with
ferrocenecarboxaldehyde and reduced by sodium borohydride to obtain the
1
target compounds 6. All of the synthesized compounds were characterized by IR, H
NMR, 13C NMR and mass spectroscopies.

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The IR spectral data of the ferrocene appended naphthalimide derivatives were shown in
table 1. The broad band at ～3350 cm-1 owed to the stretching of N-H and the sharp peak
at ～1698 cm-1 was due to the stretching of C=O. And the characteristic peak of the
1
ferrocene group was observed at ～490 cm-1 [17]. In the H NMR spectra of ferrocene
appended naphthalimide derivatives, the characteristic peaks of naphthalimide and
ferrocene group were displayed around 7.84-8.49 ppm and 4.01-4.25 ppm, respectively.
3.2 Electrochemical studies
The electrochemical property of the synthesized ferrocene appended naphthalimide
derivatives was studied by cyclic voltammetry (CV) in 90 % ethanol water solution. All
prepared ferrocene appended naphthalimide derivatives exhibited reversible one-electron
redox process (Figure 2), which was due to the formation of ferrocene/ferrocenium redox
couple [18]. The oxidation peaks of compound 2a, 2b and 6 were at 690 mV, 672 mV
and 689 mV, respectively. And the reduction peaks were at 604 mV, 580 mV and 599
mV, respectively. The ∆E values for the studied compounds were more than 59 mV,
which was attributed to the uncompensated solution resistance [19].
3.3 Ethidium bromide displacement experiment
EB is a typical DNA intercalator with strong fluorescence intensity when binding with
DNA duplexes. After the addition of other DNA intercalator into EB-DNA complex
solution, EB molecules which inserting into the DNA base pairs are replaced by the
intercalator and the fluorescence intensity of the EB-DNA complex solution is quenched
[20]. The fluorescence quenching constants are calculated by Stern-Volmer equation and
used to evaluate the DNA binding mode and binding ability of the intercalators [9c, 21].

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In our experiments, the fluorescence intensity of the EB-DNA complex solution was
decreased in the addition of synthesized naphthalimide derivatives (Figure 3). And the
fluorescence quenching constants were 1.53 × 105 L/mol, 1.70 × 105 L/mol, 2.04 × 105
L/mol and 3.37 × 105 L/mol for compounds 2a, 2b, 5 and 6, respectively. It was no doubt
that the fluorescence quenching constants of bis-naphthalimide compounds were larger
than the mono-naphthalimide compounds. To our surprise, the quenching constants of
bis-naphthalimide hybrid compound 6 were 1.65 times larger than that of the reference
compound 5. The enhanced quenching constants of ferrocene appended bis-
naphthalimide derivative 6 might be due to the groove binding of ferrocene group [22].
According to the calculated quenching constants, intercalation might be the binding mode
for all of the compounds.
3.4 UV-visible spectrophotometry
The EB displacement experiment is an indirect way to study the interaction of small
molecules with DNA. UV-Vis absorption study is another important technique to
evaluate the DNA binding ability of small molecules [9c, 23]. In an effort to further
understand the role of the ferrocene group in hybrid compound 6, UV-visible
spectrophotometry was carried out to evaluate the interaction of reference compound 5
and 6 with DNA. As shown in figure 4, the maximum absorption wavelength of the
compounds was at 341 nm, which was attributed to the absorption of the naphthalimide
group. The UV-Vis spectra of both compounds were showed a significant
hypochromic effect after the addition of DNA, which indicated the intercalation binding
mode between the compounds with DNA duplex [21]. According to the data, the binding
constant of compound 5 and 6 were calculated as 1.22 × 104 L/mol and 1.35 × 104 L/mol,

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respectively. Ferrocene appended bis-naphthalimide derivative 6 was exhibited higher
binding constant than the reference compound 5, which was in accordance with the
results of EB displacement experiments. That is to say, the conjugation of ferrocene in
the amino terminus is helpful to improve the DNA binding ability of naphthalimide
derivative.
3.5 Viscosity studies
Viscosity studies is a useful method to evaluate the binding mode of small molecules
with DNA. In typical intercalation binding mode, the aromatic ring intercalate between
the base pairs of DNA duplex and prolong the length of DNA, which increase the
viscosity of DNA solution. The viscosity results of the naphthalimide derivatives were
shown in figure 5. The classic DNA intercalator EB was used as reference compound.
Compared with EB, all the naphthalimide derivatives did not show the typical
intercalation binding mode with DNA. The viscosity of DNA solution was slightly
increased then dropped after the addition of mono-naphthalimide derivatives. The
appended ferrocene group disturbed the typical intercalation binding mode of
naphthalimide [24]. And the addition of bis-naphthalimide derivatives induced a dramatic
decline of the viscosity of DNA solution, which meant the partial intercalation of
naphthalimide groups [25].
3.6 In Vitro Cytotoxicity
The cytotoxicity of ferrocene appended naphthalimide derivatives and reference
compound 5 against 4 different cancer cell lines EC109, BGC823, SGC 7901 and HepG2
were evaluated by MTT colorimetric assay (Table 2). Although the tendency of the

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cytotoxicity was in accordance with that of the binding ability, the binding ability was not
the only factor which influenced the anticancer activity. According to the results of
mono-naphthalimide derivatives, the cytotoxicity of ferrocene appended naphthalimide
derivative 2a and 2b was lower than that of the control drug amonafide, which might be
due to the lack of protonated amino substituent group [26] and the low solubility in the
cell culture medium [27]. The water solubility of 2a and 2b was 1.60×10-4 and 1.53×10-3
mol/L, respectively (Table S1). The water solubility of 2b was 10 times than that of 2a.
Comparing with the ethylene amine linker of 2a, the extended diethylene amine linker of
2b improved the hydrophilicity. The amount of the amine motif rather than the length of
the linker improved the potency of naphthalimide [28]. For bis-naphthalimide derivatives,
although the dimeric naphthalimide derivatives attracted attention, the success of this
strategy depended on suitable linkers between two naphthalimide groups [29]. With the
help of flexible polyamine linker, the bis-naphthalimide compounds 5 and 6 displayed
higher cytotoxicity than the mono-naphthalimide ones. Importantly, the ferrocene
appended bis-naphthalimide derivative 6 was the most active compound in all of the
tested naphthalimide derivatives against cancer cell lines. The IC50 values of compound 6
were found to be 10.52 µmol/L, 7.76 µmol/L, 4.33 µmol/L and 6.79 µmol/L on EC109,
BGC823, SGC 7901 and HepG2 cells, respectively, which were much better than the
control drug amonafide and the reference compound 5. Considering the ability of
ferrocene in promoting the generation of free radicals [5b], the synergistic effect between
ferrocene group and bis-naphthalimide derivative significantly enhanced the cytotoxicity
[30].
3.7 Cellular uptake and localization

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Due to the strong green fluorescence and superior photo-stability, naphthalimide is
widely used in fluorescence chemosensors [31] and fluorescent cellular imaging agents
[10b]. Hence, the cellular uptake and localization of bis-naphthalimide derivative 6 were
able to study by confocal microscopy. DAPI, a nucleus-specific blue fluorescence dye
was used for co-localization study. As shown in figure 6, after 12 hours incubation of
HepG2 cells with compound 6, the cells were stained by compound 6 and green
fluorescence was observed (figure 6a). Blue fluorescence was observed for DAPI (figure
6b), which was located in the nucleus of the cells. The merged image (figure 6c)
confirmed the uptake of bis-naphthalimide derivative 6 which was located in the cytosol
and nucleus of the cancer cells.
3.8 Comet assay
Comet assay is a useful technique to evaluate the rate of DNA damages at the level of
individual cells [32]. As shown in figure 7, the undamaged cells exhibited comets with no
tails. After treated with bis-naphthalimide derivative 6, the cells showed comets with
detectable tails. Comparing with the control group, the tail DNA (%) and tail moment
ratio of treated cells increased with the increase of the concentration of bis-naphthalimide
derivative 6 (Table 3). The tail DNA (%) and tail moment ratio of the cells in the control
group were 2.07 ± 0.36 % and 0.89 ± 0.34, respectively. After treated by bis-
naphthalimide derivative 6, the tail DNA (%) increased from 6.54 ± 1.44 % to 14.96 ±
4.86 % and the tail moment ratio increased from 2.26 ± 0.72 to 126.14 ± 24.58 by
increasing the concentration from 7 µmol/L to 15 µmol/L. The DNA of HepG2 cells
showed significant damage (p < 0.01) induced by bis-naphthalimide derivative 6 at 15

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µmol/L. This results indicated that bis-naphthalimide derivative 6 was able to cause DNA
damage in cancer cells.
4. Conclusions
Herein, some ferrocene appended naphthalimide derivatives were synthesized and
characterized to evaluate the synergistic effect of the ferrocene group in anticancer
activity of napthalimide derivatives. And according to the results of EB display, UV-
visible spectrophotometry and viscosity studies, the ferrocene appended naphthalimide
derivatives exhibited partial-intercalation binding mode with DNA duplex. Ferrocenyl
group was helpful to enhance the DNA binding ability of bis-naphthalimide derivative.
Hybrid compound 6 was 6.45 to 17.62 times more toxicity than the reference compound
5 and control drug amonafide on tested cancer cell lines. The synergistic effect of
ferrocene group played an important role to enhance the cytotoxicity of bis-
naphthalimide derivative. And the cytotoxicity of compound 6 was relate to the DNA
damage in cancer cells.
Acknowledgments
This work was financially supported by the National Science Foundation of China (No
21362026, No 21867016), Young Talent Program of NingXia medical University and
Ningxia Medical University Key Project (XZ2018002).
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Figure 1. Representative naphthalimide derivatives in clinical trial.

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Scheme 1. Synthesis route of ferrocene appended naphthalimide derivatives. i) PPh3,
DIAD, ii) NaBH4, iii) K2CO3, iv) TFA

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Figure 2. The CV curves of ferrocene appended naphthalimide derivatives. (0.1 mmol/L
in 90% ethanol water solution, 0.1 mol/L TBAP, scan rate 100 mV/s)

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Figure 3. Stern-Volmer plot of the EB displacement experiments of naphthalimide
derivatives 2, 5 and 6. [DNA] = 20 µmol/L, [EB] = 10 µmol/L.

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Figure 4. UV-Vis spectra of naphthalimide derivatives 5 (a) and 6 (b) in phosphate buffer
(10 mmol/L, pH = 7.4). Inset: Plots of [DNA]/( εa-εf) vs. [DNA].

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o
Figure 5. DNA viscosity titration of naphthalimide derivatives at 25 C in phosphate
buffer (10.0 mM, pH 7.4)

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Figure 6. Confocal microscopic images of HepG2 cells treat with bis-naphthalimide
derivative 6 (a), DAPI (b) and merge (c).

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Figure 7. Photomicrographs of DNA damage evaluated by comet assay for HepG2
treated with different concentration of bis-naphthalimide derivative 6. (A) control group,
0 µmol/L (B) 7 µmol/L (C) 11 µmol/L (D) 15 µmol/L