Synthesis, electrochemistry, DNA binding and in vitro cytotoxic activity of tripodal ferrocenyl bis-naphthalimide derivatives

Article abstract of DOI:10.1016/j.jinorgbio.2021.111425

A series of tripodal ferrocenyl bis-naphthalimide derivatives were synthesized and characterized. All of the bis-naphthalimide derivatives exhibited good DNA binding ability which was confirmed by ethidium bromide (EB) displacement experiment and ultraviolet (UV)-visible absorption titration. And the binding mode of these compounds was proved to be a hybrid binding mode by experiments. The cytotoxicity of synthesized compounds against 4 different human cancer cell lines (EC109, BGC823, SGC7901 and HEPG2) was evaluated by thiazolyl blue tetrazolium bromide (MTT) assay. All of the bis-naphthalimide derivatives exhibited good anticancer activity than the positive control drug (amonafide), which was due to the promotion of reactive oxygen species (ROS) level in test cancer cells by the reversible one-electron redox process of ferrocenyl bis-naphthalimide derivatives. Although there was no obvious relationship between the binding constants and the chain length, the structure cytotoxicity relationship revealed that the linker of n = 3, m = 1 was the best choice for the tested tripodol bis-naphthalimide derivatives. Synopsis: A series of tripodal ferrocenyl bis-naphthalimide derivatives were synthesized to study the DNA binding ability and the cytotoxicity induced by reactive oxygen species. All of the compounds exhibited good DNA binding ability. And the structure cytotoxicity relationship revealed that the structure of 5h was the best choice.

Full text of DOI:10.1016/j.jinorgbio.2021.111425

Journal of Inorganic Biochemistry 219 (2021) 111425
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Synthesis, electrochemistry, DNA binding and in vitro cytotoxic activity of
tripodal ferrocenyl bis-naphthalimide derivatives
,
*
Yan-Ru Fana, Bo-Jin Wanga, Deng-Guo Jiaa, Xin-Bin Yangb, Yu Huanga
a Key Laboratory of Hui Ethnic Medicine Modernization, Ministry of Education, Ningxia Engineering and Technology Research Center of Characteristic Chinese Medicine
Modernization, College of Pharmacy, Ningxia Medical University, Yinchuan 750004, PR China
b Southwest University, Rongchang Campus, Chongqing 402460, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of tripodal ferrocenyl bis-naphthalimide derivatives were synthesized and characterized. All of the bis-
naphthalimide derivatives exhibited good DNA binding ability which was confirmed by ethidium bromide (EB)
displacement experiment and ultraviolet (UV)-visible absorption titration. And the binding mode of these
compounds was proved to be a hybrid binding mode by experiments. The cytotoxicity of synthesized compounds
against 4 different human cancer cell lines (EC109, BGC823, SGC7901 and HEPG2) was evaluated by thiazolyl
blue tetrazolium bromide (MTT) assay. All of the bis-naphthalimide derivatives exhibited good anticancer ac-
tivity than the positive control drug (amonafide), which was due to the promotion of reactive oxygen species
(ROS) level in test cancer cells by the reversible one-electron redox process of ferrocenyl bis-naphthalimide
derivatives. Although there was no obvious relationship between the binding constants and the chain length,
the structure cytotoxicity relationship revealed that the linker of n = 3, m = 1 was the best choice for the tested
tripodol bis-naphthalimide derivatives.
Bis-naphthalimide derivatives
Ferrocene
Synthesis
DNA binding
ROS
Synopsis: A series of tripodal ferrocenyl bis-naphthalimide derivatives were synthesized to study the DNA binding
ability and the cytotoxicity induced by reactive oxygen species. All of the compounds exhibited good DNA
binding ability. And the structure cytotoxicity relationship revealed that the structure of 5h was the best choice.
1. Introduction
the unique properties in the last decades. Due to the high stability, lip-
ophilicity, low toxicity, easy functionalization and mild revrsible redox
Cancer treatment is one of the enormous challenges in modern
medicine and basic medical science as cancer causes the huge numbers
of deaths and heavy economic costs [1]. Although many small organic
molecules and monoclonal antibodies have been approved to treat
cancer, platinum-containing drugs, including cisplatin, oxaliplatin and
carboplatin, still play an important role in cancer treatment [2]. How-
ever, the clinical application of these platinum-containing drugs was
restricted by their substantial side effects and drug resistance. Therefore,
a great number of new non‑platinum metallic compounds were designed
and synthesized to study their anticancer activities and mechanisms
[3–5].
property of ferrocene [6,7], ferrocenyl derivatives exhibited efficacy in
cancer, malaria, and many other diseases [8–13]. Ferrocifen, a novel
hybrid molecule of ferrocene and tamoxifen, is the most promising
molecule to cure breast cancer among the ferrocenyl derivatives. It was
reported that the crucial role of ferrocene group in hybrid ferrocenyl
derivatives to improve the cytotoxicity should be attributed to an
oxidation-related mechanism. This mechanism was based on the
reversible oxidation of ferrocene to ferrocenium, which generated
reactive oxygen species (ROS) resulting in oxidative DNA damage
[14–16]. Furthermore, the conjugation of ferrocene group into a com-
plex structure also promoted intercalation and/or non-covalent in-
teractions with DNA or proteins [17–20]. Hence, the cytotoxicity of
Ferrocene derivatives have become attractive to many scientists for
Abbreviations: 1H NMR, Proton nuclear magnetic resonance spectroscopy; CT DNA, Calf thymus DNA; CLSM, Confocal laser scanning microscopy; CV, Cyclic
voltammetry; DMSO, Dimethyl sulfoxide; DMEM, Dulbecco’s modified Eagle’s medium; DAPI, 4’,6-diamidino-2-phenylindole; DCFH-DA, 2,7-dichlorodi-hydro-
fluorescein diacetate; EB, Ethidium bromide; MS, Mass spectrometry; MTT, Methyl thiazolyl tetrazolium; TMS, Tetramethylsilane; TBAP, tetra-n-butylammonium
perchlorate; ROS, Reactive oxygen species; RPMI, Roswell Park Memorial Institute; UV, Ultraviolet.
* Corresponding author.
E-mail address: huangyunxmu@163.com (Y. Huang).
https://doi.org/10.1016/j.jinorgbio.2021.111425
Received 26 November 2020; Received in revised form 3 March 2021; Accepted 10 March 2021
Available online 19 March 2021
0162-0134/© 2021 Elsevier Inc. All rights reserved.

Y.-R. Fan et al.
Journal of Inorganic Biochemistry 219 (2021) 111425
some ferrocenyl derivatives which conjugated ferrocene group with
DNA intercalator was better than that of the intercalator alone [6,
21–23].
(m, 2H, -CH2). HRMS: [M + H]+ 189.1593 (calcd for C9H21N2O+2 :
189.1598).
N-tert-butoxycarbonyl-1, 5-pentanediamine 1c.
Naphthalimide derivatives, a kind of
π-deficient flat aromatic het-
Yield 96.76%. 1H NMR (400 MHz, Chloroform‑d) δ 3.13–3.10 (m,
2H, -CH2), 2.74 (s, 2H, -CH2), 1.53–1.41 (m, 4H, -CH2), 1.43 (s, 9H, -OC
(CH3)3), 1.39–1.29 (m, 2H, -CH2). HRMS: [M + H]+ 203.1755 (calcd for
erocycle compounds, have received enormous attention in the past de-
cades for the excellent fluorescent and biological properties [24–26].
Among the reported naphthalimide derivatives, ferrocenyl substituted
naphthalimide derivatives were synthesized and studied as electro-
chemical indicators or redox indicators [27,28]. The combination of
ferrocene and naphthalimide also exhibited other interesting properties.
In our previous work, the synthesized ferrocenyl naphthalimide de-
rivatives exhibited a synergistic effect between the ferrocene and
naphthalimide group in cytotoxicity against human cancer cells [29].
To further study the structure-activity relationship of the ferrocenyl
bis-naphthalimide derivatives, a series of ferrocenyl bis-naphthalimide
derivatives with different polyamine linkers were synthesized. All of
the compounds exhibited better cytotoxicity against tested human
cancer cells than that of the control drug. The cellular uptake of the
compounds and intracellular ROS generation abilities were proved by
using confocal microscope and flow cytometry, respectively. The DNA
binding abilities of these compounds were studied by spectroscopic
experiments including ethidium bromide (EB) displacement experi-
ments and UV-visible absorption titration. And the binding mode and
electrochemisty properties of the compounds were also investigated.
C
10H23N2O+2 : 203.1754).
N-tert-butoxycarbonyl-1, 6-hexanediamine 1d.
Yield 95.96%. 1H NMR (400 MHz, Chloroform‑d) δ 3.12–3.08 (m,
2H, -CH2), 2.86–2.83 (t, J = 7.3 Hz, 2H, -CH2), 1.63–1.60 (m, 2H, -CH2),
1.51–1.46 (m, 4H, -CH2), 1.43 (s, 9H, -OC(CH3)3), 1.36–1.33 (m, 2H,
-CH2). HRMS: [M + H]+ 217.1896 (calcd for C11H25N2O+2 : 217.1911).
2.2.2. General procedure of 2
1,8-naphthalimide (3.94 g, 20 mmol), dibromide alkane (60 mmol)
and anhydrous potassium carbonate (11.06 g, 80 mmol) were dissolved
in 50 mL CH3CN. Then the mixture was refluxed for 12 h. After filtration
to remove the insoluble substance, the filtrate was concentrated by a
rotary evaporator to obtain the crude product. The pure product was
purified by column chromatography (petroleum ether: ethyl acetate v/v,
8:1) to yield the pure product as a white solid.
N-(3-bromopropyl)-1, 8-naphthalimide 2a
Yield 54.55%. 1H NMR (400 MHz, DMSO‑d6) δ 8.50–8.44 (m, 4H,
-ArH), 7.87 (t, J = 7.2 Hz, 2H, -ArH), 4.17 (t, J = 7.0 Hz, 2H, -CH2), 3.61
(t, J = 6.7 Hz, 2H, -CH2), 2.23–2.17 (m, 2H, -CH2). HRMS: [M + Na]+
339.9944 (calcd for C15H12BrNO2Na+: 339.9944).
2. Experimental section
N-(4-bromobutyl)-1, 8-naphthalimide 2b
2.1. General
Yield 44.88%. 1H NMR (400 MHz, Chloroform‑d) δ 8.61 (d, J = 7.2
Hz, 2H，-ArH), 8.22 (d, J = 8.2 Hz, 2H, -ArH), 7.76 (t, J = 7.6 Hz, 2H,
-ArH), 4.23 (t, J = 7.1 Hz, 2H, -CH2), 3.48 (t, J = 6.5 Hz, 2H, -CH2),
2.04–1.96 (m, 2H, -CH2), 1.95–1.87 (m, 2H,-CH2). HRMS: [M + H]+
332.0285 (calcd for C16H15BrNO+2 : 332.0281).
All of the chemical reagents and solvents were obtained from com-
mercial sources and used directly without further purification. Calf
thymus (CT) DNA from Sigma-Aldrich was dissolved in water, and the
concentration of DNA solution was determined by UV-visible absorption
spectrometry using the molar absorption coefficient (6600 Mꢀ 1 cmꢀ 1) at
260 nm [30]. DNA binding ability was evaluated on a Hitachi F4600
spectrofluorometer and a Shimadzu UV-2600 spectrophotometer at
room temperature. Electrochemical properties of the compounds were
measured on a CH Instruments CHI-600 electrochemical workstation.
Proton nuclear magnetic resonance spectroscopy (1H NMR) spectra were
recorded on a Bruker AVIII 400 spectrometer, with tetramethylsilane
(TMS) as an internal standard. Mass spectra (MS) were recorded on a
Shimadzu LCMS-IT-TOF. Confocal microscopy images were captured on
an Olympus FV1000-IX81 Confocal Microscope. Cell ROS level was
measured via a BD Accuri C6 flow cytometry.
N-(5-bromopentyl)-1,8-naphthalimide 2c
Yield 56.52%. 1H NMR (400 MHz, DMSO‑d6) δ 8.50–8.43 (m, 4H,
-ArH), 7.86 (t, J = 8.3, 2H, -ArH), 4.05 (t, J = 7.6 Hz, 2H, -CH2), 3.54 (t,
J = 6.8 Hz, 2H, -CH2), 1.89–1.82 (m, 2H, -CH2), 1.70–1.62 (m, 2H,
-CH2), 1.49–1.41 (m, 2H, -CH2). HRMS: [M + Na]+ 368.0237 (calcd for
C17H16BrNO2Na+: 368.0257).
N-(6-bromohexyl)-1,8-naphthalimide 2d
Yield 40.56%. 1H NMR (400 MHz, DMSO‑d6) δ 8.50–8.45 (m, 4H,
-ArH), 7.87 (t, J = 7.6 Hz, 2H, -ArH), 4.04 (t, J = 7.2 Hz, 2H, -CH2), 3.52
(t, J = 7.2 Hz, 2H, -CH2), 1.83–1.77 (m, 2H, -CH2), 1.67–1.60 (m, 2H,
-CH2), 1.46–1.34 (m, 4H, -CH2). HRMS: [M + Na]+ 382.0399 (calcd for
C18H18BrNO2Na+: 382.0413).
2.2.3. General procedure of 3
2.2. Synthesis
A mixture of compound 1 (4.7 mmol), compound 2 (10 mmol) and
anhydrous potassium carbonate (6.5 g, 47.03 mmol) in acetonitrile (50
mL) were heated under reflux for 24 h. After the reaction mixture was
cooled to room temperature, the insoluble solid was removed by filtra-
tion, and the filtrate was concentrated in vacuo. The crude product was
purified by column chromatography (petroleum ether: ethyl acetate v/v,
1:1) on silica gel to afford compounds (3a–h) as a white solid.
3a
2.2.1. General procedure of 1
Mono-protection of diamine was synthesized following the general
method. Diamine (73.31 mmol) was dissolved in 25 mL dioxane and a
solution of di-tert-butyl pyrocarbonate (2 g, 9.16 mmol) in 10 mL 1,4-
dioxane was added dropwise at 0 ◦C. Then the mixture was stirred at
room temperature for 22 h. After removed the solvent by a rotary
evaporator, the residue was dissolved in water, and the insoluble residue
was removed by filtration. The water solution was extracted three times
with dichloromethane. Then the organic layer was combined and
washed two times with H2O. Finally, dichloromethane was removed by
a rotary evaporator to obtain the oil product.
Yield 65.38%. 1H NMR (400 MHz, DMSO‑d6) δ 8.41 (m, 8H, -ArH),
7.82 (t, J = 7.6 Hz, 4H, -ArH), 6.77 (t, J = 5.7 Hz, 1H, -NH), 4.07–4.01
(m, 4H, -CH2), 2.90–2.88 (m, 2H, -CH2), 2.49–2.48 (m, 4H, -CH2), 2.39
(s, 2H, -CH2), 1.78–1.71 (m, 4H, -CH2), 1.43–1.34 (m, 4H, -CH2), 1.33
(s, 9H, -OC(CH3)3). HRMS: [M + H]+ 663.3180 (calcd for C39H43N4O+6 :
663.3177).
N-tert-butoxycarbonyl-1, 3-propanediamine 1a
Yield 59.11%. 1H NMR (400 MHz, Chloroform‑d) δ 3.24–3.20 (m,
2H, -CH2), 2.77–2.8 (t, J = 6.6 Hz, 2H, -CH2), 1.67–1.61 (m, 2H, -CH2),
1.43 (s, 9H, -OC(CH3)3). HRMS: [M + H]+ 175.1445 (calcd for
C8H19N2O+2 : 175.1441).
3b
Yield 75%. 1H NMR (400 MHz, Chloroform‑d) δ 8.60–8.58 (m, 4H,
-ArH), 8.21–8.19 (m, 4H, -ArH), 7.75 (t, J = 7.2 Hz, 4H, -ArH), 4.21 (t, J
= 6.8 Hz, 4H, -CH2), 3.20–3.15 (m, 4H, -CH2), 3.05–3.03 (m, 2H, -CH2),
2.00–1.94 (m, 4H, -CH2), 1.86–1.83 (m, 4H, -CH2), 1.67 (s, 4H, -CH2),
1.60–1.56 (m, 2H, -CH2), 1.42 (s, 9H, -OC(CH3)3). HRMS: [M + H]+:
N-tert-butoxycarbonyl-1, 4-butanediamine 1b.
Yield 73.88%. 1H NMR (400 MHz, Chloroform‑d) δ 2.91–2.86 (m,
2H, -CH2), 2.51–2.48 (m, 4H, -CH2), 1.37 (s, 9H, -OC(CH3)3), 1.33–1.27
2

Y.-R. Fan et al.
Journal of Inorganic Biochemistry 219 (2021) 111425
691.3482 (calcd for C41H47N4O+6 : 691.3490).
-CH2), 3.13–3.03 (m, 6H, -CH2), 2.86–2.77 (m, 2H, -CH2), 1.81–1.68 (m,
10H, -CH2), 1.63–1.55 (m, 2H, -CH2). HRMS: [M + H]+ 647.3575 (calcd
for C40H47N4O+4 : 647.3592).
3c
Yield 73.56%. 1H NMR (400 MHz, Chloroform‑d) δ 8.56 (d, J = 7.6
Hz, 4H), 8.19 (d, J = 7.6 Hz, 4H, -ArH), 7.74 (t, J = 7.6 Hz, 4H, -ArH),
4.19 (t, J = 7.1 Hz, 4H, -CH2), 3.19–3.16 (m, 2H, -CH2), 3.01 (s, 6H,
-CH2), 1.92 (s, 4H, -CH2), 1.86–1.77 (m, 4H, -CH2), 1.66 (s, 2H, -CH2),
1.61–1.57 (m, 2H, -CH2), 1.48–1.41 (m, 4H, -CH2), 1.37 (s, 9H, -OC
(CH3)3). HRMS: [M + H]+ 719.3789 (calcd for C43H51N4O+6 : 719.3803).
3d
2.2.5. General procedure of 4c and 4e–h
Compound 3 (2.78 mmol) was dissolved in 20 mL hydrochloric acid
alcohol solution. Then the mixture was stirred overnight at room tem-
perature. After the solvent was removed, the crude product was washed
two times with 10 mL petrol ether. 50 mL saturated sodium bicarbonate
solution was added to the crude product, which adjusted the pH of the
mixture slightly basic. Then the water solution was extracted three times
by 50 mL dichloromethane. After the dichloromethane was dried by
sodium sulfate anhydrous, the pure products were obtained by removing
the dichloromethane on a rotary evaporator.
Yield 52.52%. 1H NMR (400 MHz, Chloroform‑d) δ 8.58–8.56 (m,
4H, -ArH), 8.20–8.18 (m, 4H, -ArH), 7.74 (t, J = 7.2 Hz, 4H, -ArH),
4.17–4.12 (m, 4H, -CH2), 3.17 (t, J = 6.0 Hz, 2H, -CH2), 3.01–2.97 (m,
6H, -CH2), 1.90–1.80 (m, 6H, -CH2), 1.78–1.71 (m, 4H, -CH2), 1.62–1.54
(m, 2H, -CH2), 1.50–1.41 (m, 8H, -CH2), 1.40 (s, 9H, -OC(CH3)3). HRMS:
[M + H]+ 747.4108 (calcd for C45H55N4O+6 : 747.4116).
3e
4c
Yield 87.20%. 1H NMR (400 MHz, DMSO‑d6) δ 8.40 (dd, J = 22.2,
7.8 Hz, 8H, -ArH), 7.80 (t, J = 7.6 Hz, 4H, -ArH), 3.98 (t, J = 7.4 Hz, 4H,
-CH2), 2.45 (t, J = 6.6 Hz, 2H, -CH2), 2.30–2.24 (m, 6H, -CH2),
1.63–1.56 (m, 4H, -CH2), 1.41–1.34 (m, 4H, -CH2), 1.32–1.21 (m, 8H,
-CH2). HRMS: [M + H]+ 619.3273 (calcd for C38H43N4O+4 : 619.3279).
4e
Yield 67.12%. 1H NMR (400 MHz, Chloroform‑d) δ 8.60–8.58 (m,
4H, -ArH), 8.21–8.19 (m, 4H, -ArH), 7.75 (t, J = 7.6 Hz, 4H, -ArH), 4.21
(t, J = 6.8 Hz, 4H, -CH2), 3.31–3.27 (m, 2H, -CH2), 3.20–3.16 (m, 4H,
-CH2), 3.10–3.06 (m, 2H, -CH2), 1.95–1.90 (m, 2H, -CH2), 1.89–1.83 (m,
4H, -CH2), 1.69–1.62 (m, 4H, -CH2), 1.41 (s, 9H, -OC(CH3)3). HRMS: [M
+ H]+ 677.3339 (calcd for C40H45N4O+6 : 677.3334).
3f
Yield 85.66%. 1H NMR (400 MHz, DMSO‑d6) δ 8.39–8.36 (m, 8H,-
ArH), 7.79 (t, J = 7.7 Hz, 4H, -ArH), 4.00 (t, J = 7.3 Hz, 4H, -CH2),
2.49–2.47 (m, 2H, -CH2), 2.37–2.32 (m, 6H, -CH2), 1.66–1.58 (m, 4H,
-CH2), 1.47–1.39 (m, 6H, -CH2). HRMS: [M + H]+ 577.2810 (calcd for
Yield 79.43%. 1H NMR (400 MHz, DMSO‑d6) δ 8.42–8.40 (m, 8H,
-ArH), 7.83 (t, J = 7.6 Hz, 4H, -ArH), 4.06 (t, J = 6.8 Hz, 4H, -CH2),
2.87–2.82 (m, 2H, -CH2), 2.35 (t, J = 5.6 Hz, 2H, -CH2), 1.75 (s, 4H,
-CH2), 1.65–1.62 (m, 2H, -CH2), 1.39–1.35 (m, 4H, -CH2), 1.33 (s, 9H,
-OC(CH3)3), 1.29–1.17 (m, 8H, -CH2). HRMS: [M + H]+ 705.3636 (calcd
for C42H49N4O+6 : 705.3647).
C
35H37N4O+4 : 577.2809).
4f
Yield 75.58%. 1H NMR (400 MHz, DMSO‑d6) δ 8.46 (dd, J = 18.8,
7.8 Hz, 8H, -ArH), 7.86 (t, J = 7.6 Hz, 4H, -ArH), 4.06 (t, J = 6.8 Hz, 4H,
-CH2), 3.13–3.08 (m, 4H, -CH2), 3.03–2.97 (m, 2H, -CH2), 2.79–2.74 (m,
2H, -CH2), 1.80–1.65 (m, 10H, -CH2), 1.63–1.55 (m, 2H, -CH2),
1.37–1.31 (m, 2H, -CH2). HRMS: [M + H]+ 605.3112 (calcd for
3g
Yield 67.07%. 1H NMR (400 MHz, Chloroform‑d) δ 8.60–8.58 (m,
4H, -ArH), 8.22–8.20 (m, 4H, -ArH), 7.76 (t, J = 7.6 Hz, 4H, -ArH), 4.22
(t, J = 6.9 Hz, 4H,-CH2), 3.17–3.15 (m, 4H, -CH2), 3.11–3.08 (m, 2H,
-CH2), 2.99–2.97 (m, 2H,-CH2), 1.98–1.95 (m, 4H,-CH2), 1.88–1.83 (m,
6H, -CH2), 1.49–1.47 (m, 2H, -CH2) 1.44 (s, 9H，-OC(CH3)3), 1.39–1.37
(m, 4H, -CH2). HRMS: [M + H]+ 719.3785 (calcd for C43H51N4O+6 :
719.3803).
C
37H41N4O+4 : 605.3122).
4g
Yield 82.38%. 1H NMR (400 MHz, DMSO‑d6) δ 8.39–8.34 (m, 8H,
-ArH), 7.78 (t, J = 7.2 Hz, 4H, -ArH), 3.99 (t, J = 7.3 Hz, 4H, -CH2), 2.42
(t, J = 6.8 Hz, 2H, -CH2), 2.34 (t, J = 6.9 Hz, 4H, -CH2), 2.26 (t, J = 7.2
Hz, 2H, -CH2), 1.65–1.58 (m, 4H, -CH2), 1.44–1.37 (m, 4H, -CH2),
1.30–1.21 (m, 4H, -CH2), 1.16–1.13 (m, 4H, -CH2). HRMS: [M + H]+
619.3274 (calcd for C38H43N4O+4 : 619.3279).
3h
Yield 48.72%. 1H NMR (400 MHz, DMSO‑d6) δ 8.42–8.31 (m, 8H,
-ArH), 7.78 (t, J = 8.0 Hz, 4H, -ArH), 3.99 (t, J = 7.2 Hz, 4H, -CH2), 2.84
(m, 2H, -CH2), 2.35 (t, J = 6.4 Hz, 4H, -CH2), 2.26 (t, J = 6.8 Hz, 2H,
-CH2), 1.65–1.57 (m, 4H, -CH2), 1.42–1.39 (m, 4H, -CH2), 1.33 (s, 9H,
-OC(CH3)3), 1.31–1.28 (m, 4H, -CH2). HRMS: [M + H]+ 677.3332(calcd
for C40H45N4O+6 : 677.3334).
4h
Yield 79.41%. 1H NMR (400 MHz, DMSO‑d6) δ 8.40–8.36 (m, 8H,-
ArH), 7.79 (t, J = 8.0 Hz, 4H, -ArH), 4.00 (t, J = 7.7 Hz, 4H, -CH2),
2.43 (t, J = 6.7 Hz, 2H, -CH2), 2.35 (t, J = 6.9 Hz, 4H, -CH2), 2.26 (t, J =
7.2 Hz, 2H, -CH2), 1.66–1.58 (m, 4H, -CH2), 1.45–1.38 (m, 4H, -CH2),
1.26–1.22 (m, 2H, -CH2). HRMS: [M + H]+ 577.2804 (calcd for
2.2.4. General procedure of 4a, 4b and 4d
Compound 3 (2.26 mmol) was dissolved in 20 mL hydrochloric acid
alcohol solution. Then the mixture was stirred overnight at room tem-
perature. The solvent was removed to obtain the pure product.
4a
C
35H37N4O+4 : 577.2809).
2.2.6. General procedure of 5
Ferrocene carboxaldehyde (0.42 g, 1.97 mmol) and compound 4
(2.36 mmol) were dissolved in 30 mL methanol (1 mL trimethylamine
was added for 4a, 4b and 4d), then the reaction mixture was refluxed for
3 h. After the mixture was cooled to room temperature, sodium boro-
hydride (0.15 g, 3.94 mmol) was slowly added. Then the mixture was
refluxed for another 2h. The solvent of the mixture was removed under
reduced pressure to obtain a yellow solid. The solid was solved in 100
mL dichloromethane and washed with 30 mL saturated sodium bicar-
bonate solution for three times, 30 mL water and 30 mL saturated brine
in turn. Then the organic layer was dried by sodium sulfate anhydrous
and concentrated on a rotary evaporator. The crude residue was purified
by column chromatography (MeOH:EA = 2:1–5:1) to obtain the pure
product as a yellow solid.
Yield 90.28%. 1H NMR (400 MHz, DMSO‑d6) δ 8.46–8.38 (m, 8H,-
ArH), 8.07 (s, 3H, -CH2), 7.84 (t, J = 7.6 Hz, 4H, -ArH), 4.09 (t, J =
6.8 Hz, 4H, -CH2), 3.21–3.17 (m, 4H, -CH2), 3.09–3.06 (m, 2H, -CH2),
2.80–2.75 (m, 2H, -CH2), 2.12–2.05 (m, 4H, -CH2), 1.79–1.71 (m, 2H,
-CH2), 1.62–1.55 (m, 2H, -CH2). HRMS: [M + H]+ 563.2650 (calcd for
C34H35N4O+4 : 563.2653).
4b
Yield 85.71%. 1H NMR (400 MHz, Chloroform‑d) δ 8.61 (dd, J = 7.3,
1.2 Hz, 4H, -ArH), 8.22 (dd, J = 8.3, 1.1 Hz, 4H, -ArH), 7.76 (t, J = 7.2
Hz, 4H -ArH), 4.22–4.13 (t, J = 7.6 Hz, 4H, -CH2), 3.41 (t, J = 6.8 Hz,
4H, -CH2), 1.91–1.85 (m, 4H, -CH2), 1.79–1.72 (m, 4H, -CH2), 1.55–1.40
(m, 8H, -CH2). HRMS: [M + H]+ 591.2963 (calcd for C36H39N4O+4 :
591.2966).
5a
4d
Yield 34.89%. 1H NMR (400 MHz, DMSO‑d6) δ 8.39 (dd, J = 8.3, 6.5
Hz, 8H,-ArH), 7.83 (t, J = 7.6 Hz, 4H, -ArH), 4.18 (t, J = 1.6 Hz, 2H,
-CH2), 4.08–4.05 (m, 9H,-FcH), 4.05–4.03 (t, J = 1.6 Hz, 2H, -CH2), 3.47
Yield 80.05%. 1H NMR (400 MHz, DMSO‑d6) δ 8.43 (m, 8H, -ArH),
7.86 (t, J = 8.0 Hz, 4H, -ArH), 4.07 (t, J = 6.8 Hz, 4H, -CH2), 3.35 (s, 8H,
3

Y.-R. Fan et al.
Journal of Inorganic Biochemistry 219 (2021) 111425
(s, 2H, -CH2), 2.55–2.48 (m, 6H, -CH2), 2.38 (t, J = 6.8 Hz, 2H, -CH2),
1.78–1.71 (m, 4H, -CH2), 1.45–1.37 (m, 4H, -CH2). HRMS: [M + H]+
761.2770 (calcd for C45H45FeN4O+4 : 761.2785).
2.4. Ethidium bromide (EB) displacement experiment
To study the competitive binding abilities of ferrocenyl bis-
naphthalimide derivatives with EB, EB displacement experiments were
carried out on a Hitachi F4600 spectrofluorometer at room temperature.
5b
Yield 39.28%. 1H NMR (400 MHz, DMSO‑d6) δ 8.39 (dd, J = 8.3, 4.8
Hz, 8H,-ArH), 7.80 (t, J = 7.6 Hz, 4H, -ArH), 4.09 (t, J = 1.8 Hz, 2H,
-CH2), 4.03–4.00 (m, 9H, -FcH), 3.98 (t, J = 1.8 Hz, 2H, -CH2), 3.30 (s,
2H, -CH2), 2.42 (t, J = 6.0 Hz, 2H, -CH2), 2.35 (t, J = 6.9 Hz, 4H, -CH2),
2.27 (s, 2H, -CH2), 1.66–1.59 (m, 4H, -CH2), 1.45–1.38 (m, 4H, -CH2),
1.33 (s, 4H, -CH2). HRMS: [M + H]+ 789.3095 (calcd for C47H49FeN4O+4 :
789.3098).
In a solution of EB-DNA mixture (EB 10 μmol/L, DNA 20 μmol/L), the
fluorescence spectra of the solution were recorded in the range of
530–750 nm with an excitation wavelength at λex = 515 nm. And the
maximum fluorescence intensity of the mixture solution was measured
as F0. An increasing volume of ferrocenyl naphthalimide derivatives
solution (5 mmol/L in DMSO) was added to the EB-DNA mixture solu-
tion. The fluorescence spectra of the mixture solution were recorded. As
a control experiment, an increasing volume of DMSO without any fer-
rocenyl naphthalimide derivatives was added into another EB-DNA
mixture solution. And the fluorescence spectra of the mixture solution
were recorded, too. The maximum fluorescence intensity of the mixture
solution was measured as F. The quenching constants were calculated
according to the Stern-Volmer equation [31].
5c
Yield 53.72%. 1H NMR (400 MHz, DMSO‑d6) δ 8.41 (dd, J = 21.8,
7.9 Hz, 8H, -ArH), 7.81 (t, J = 8.0 Hz, 4H, -ArH), 4.13 (t, J = 1.8 Hz, 2H,
-CH2), 4.06 (s, 5H, -FcH,), 4.00 (t, J = 1.6 Hz, 2H, -CH2), 3.98–3.96 (m,
4H, -FcH), 3.37 (s, 2H, -CH2), 2.45 (s, 2H, -CH2), 2.30–2.27 (m, 6H,
-CH2), 1.64–1.58 (m, 4H, -CH2), 1.41–1.29 (m, 12H, -CH2). HRMS: [M +
H]+ 817.3400 (calcd for C49H53FeN4O+4 : 817.3411).
5d
F0/F = 1 + Ksv[Com pound]
Yield 54.72%. 1H NMR (400 MHz, DMSO‑d6) δ 8.39 (dd, J = 17.2,
7.8 Hz, 8H, -ArH), 7.80 (t, J = 7.3 Hz, 4H, -ArH), 4.10 (t, J = 1.8 Hz, 2H,
-CH2), 4.03 (s, 5H, -FcH), 4.01–3.98 (m, 4H, -FcH), 3.96 (t, J = 1.8 Hz,
2H, -CH2), 3.34 (s, 2H, -CH2), 2.46 (t, J = 5.4 Hz, 2H, -CH2), 2.28–2.25
(dm, 6H, -CH2), 1.64–1.57 (m, 4H, -CH2), 1.34–1.31 (m, 16H, -CH2).
HRMS: [M + H]+ 845.3714 (calcd for C51H57FeN4O+4 : 845.3724).
5e
Where Ksv is the quenching constant.
2.5. UV-visible absorption titration
The intrinsic binding constants (Kb) were used to evaluate the DNA
binding ability of ferrocenyl naphthalimide derivatives. 5 μL stock so-
Yield 40.73%. 1H NMR (400 MHz, DMSO‑d6) δ 8.41–8.37 (m, 8H,-
ArH), 7.80 (t, J = 7.3 Hz, 4H, -ArH), 4.05 (t, J = 1.8 Hz, 2H, -CH2),
4.02–3.98 (m, 9H, -FcH), 3.93 (t, J = 1.8 Hz, 2H, -CH2), 3.31 (s, 2H,
-CH2), 2.48–2.45 (m, 2H, -CH2), 2.37–3.31 (m, 6H, -CH2), 1.66–1.58 (m,
4H, -CH2), 1.50–1.39 (m, 6H, -CH2). HRMS: [M + H]+ 775.2942 (calcd
for C46H47FeN4O+4 : 775.2941).
lution of ferrocenyl naphthalimide derivatives (5 mmol/L in DMSO) was
diluted by 3 mL phosphate buffer (10 mmol/L, pH = 7.4). The absorp-
tion intensities of the compound solution were recorded by UV-visible
spectrophotometry. Then an increasing volume of CT DNA solution
(5–25 μL, 3.38 mmol/L) was added into the solution and the absorption
intensities of compound-DNA mixture solution were recorded. The
intrinsic binding constants (Kb) of ferrocenyl bis-naphthalimide de-
rivatives with CT DNA were calculated from a D/Δεap vs D plot according
to the following equation [32].
5f
Yield 27.92%. 1H NMR (400 MHz, DMSO‑d6) δ 8.40–8.37 (m, 8H,
-ArH), 7.80 (t, J = 7.3 Hz, 4H, -ArH), 4.20 (t, J = 1.8 Hz, 2H, -CH2), 4.09
(s, 5H, -FcH), 4.06 (t, J = 1.8 Hz, 2H, -CH2), 4.01–3.98 (m, 4H, -FcH),
3.50 (s, 2H, -CH2), 2.54–2.50 (m, 2H, -CH2), 2.36 (t, J = 6.9 Hz, 4H,
-CH2), 2.28 (t, J = 7.1 Hz, 2H, -CH2), 1.66–1.59 (m, 4H, -CH2),
1.45–1.37 (m, 6H, -CH2), 1.35–1.28 (m, 2H, -CH2), 1.23–1.16 (m, 2H,
-CH2). HRMS: [M + H]+ 803.3248 (calcd for C48H51FeN4O+4 : 803.3254).
5g
/
/
/
D Δεap = D Δ
ε
+ 1 [(Δ
ε
)Kb ]
Where D is the concentration of DNA, Δεap = [εa
-
εf], εa = Aobs
/
[compound], Δε = [εb-εf], εb and εf correspond to the extinction co-
efficients of the DNA-compound complex and unbound compound,
respectively.
Yield 46.01%. 1H NMR (400 MHz, DMSO‑d6) δ 8.43–8.34 (m, 8H,
-ArH), 7.80 (t, J = 7.3 Hz, 4H, -ArH), 4.16 (t, J = 1.8 Hz, 2H, -CH2), 4.09
(s, 5H, -FcH), 4.06 (t, J = 1.8 Hz, 2H, -CH2), 4.01–3.98 (m, 4H, -FcH),
3.42 (s, 2H, -CH2), 2.46 (t, J = 7.2 Hz, 2H,-CH2), 2.35 (t, J = 6.9 Hz, 4H,
-CH2), 2.27 (t, J = 7.1 Hz, 2H, -CH2), 1.66–1.59 (m, 4H, -CH2),
1.45–1.38 (m, 4H, -CH2), 1.35–1.28 (m, 4H, -CH2), 1.17 (s, 4H, -CH2).
HRMS: [M + H]+ 817.3400 (calcd for C49H53FeN4O+4 : 817.3411).
5h
2.6. Viscosity studies
The binding mode of the ferrocenyl bis-naphthalimide derivatives
with CT DNA was assayed by monitoring the viscosity changes of DNA
solution after the addition of different derivatives in an Ubblehode
viscometer at 25 ◦C. 0.5 mL CT DNA (3.072 mmol/L) was added into 20
mL phosphate buffer (10 mmol/L, pH 7.4) in an Ubblehode viscometer.
After equilibrated at 25 ◦C for 10 min, the flow time of the DNA solution
was measured by a stopwatch as η0. Then an increasing volume of
compound solution (5 mmol/L) was added into the DNA solution, and
Yield 40.86%. 1H NMR (400 MHz, DMSO‑d6) δ 8.43–8.4 (m, 8H,
-ArH), 7.82 (t, J = 7.3 Hz, 4H, -ArH), 4.13 (m, 2H, -CH2), 4.07–4.05 (m,
9H, -FcH), 3.97 (t, J = 1.6 Hz, 2H, -CH2), 3.35 (s, 2H, -CH2), 2.46–2.41
(m, 6H, -CH2), 2.37 (t, J = 6.9 Hz, 2H, -CH2), 1.78–1.70 (m, 4H, -CH2),
1.37–1.30 (m, 4H, -CH2), 1.28–1.21 (m, 2H, -CH2). HRMS: [M + H]+
775.2948 (calcd for C46H47FeN4O+4 : 775.2941).
the flow time measured by a stopwatch as η, respectively. The plot of
(
η
/
η0
)
1/3 vs r was obtained according to the flow time, where r was equal
to [compound]/[DNA].
2.3. Electrochemical studies
2.7. Cytotoxicity assay
To measure the electrochemical properties of the ferrocenyl bis-
naphthalimide derivatives, a three-electrode system combined by
glassy carbon electrode, platinum electrode and Ag/AgCl/KCl (sat.) was
used. 1 mL compound solution (10 mmol/L) 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 experiments.
Cytotoxicity of the ferrocenyl naphthalimide derivatives was evalu-
ated by thiazolyl blue tetrazolium bromide (MTT) assay. The cells were
cultured in a humidified atmosphere at 37 ◦C in 5% CO2. Roswell Park
Memorial Institute (RPMI) 1640 medium with 10% fetal calf serum and
1% (v/v) penicillin/streptomycin was used as the culture medium of
Human Esophageal cancer cell EC109, Human gastric carcinoma cell
line BGC823 and human gastric cancer cell line SGC7901. Dulbecco’s
4

Y.-R. Fan et al.
Journal of Inorganic Biochemistry 219 (2021) 111425
modified Eagle’s medium (DMEM) supplemented with 10% fetal calf
serum, 1% (v/v) penicillin/streptomycin was used for cell culturing of
hepatoblastoma cell line HepG2. Briefly, 96-well plates were seeded
and washed with PBS. Then incubated with 2,7-dichlorodi-hydrofluores-
cein diacetate (DCFH-DA) for 20 min in dark at room temperature. After
DCFH-DA was removed, the cells were rinsed by PBS and collected in
1.5 mL centrifuge tubes by gentle centrifugation. Then the supernatants
were aspirated and cell pellets resuspended in 1 mL PBS to examine the
ROS level by flow cytometry.
with 100 μL of medium containing an appropriate number of cells (5000
for EC109, BGC823, SGC7901 and HepG2). The cells were allowed to
adhere for 24 h. Test compounds were dissolved in 20% DMSO and
diluted to different density with medium (0, 6.25, 12.5, 25, 50 and 100
μ
mol/L). After incubation for 24 h, each well was added 50
μ
L MTT (2
2.10. Statistical analysis
mg/mL, Sigma) and incubated for another 4 h. Then the medium of each
well was pipetted out and 150
μL of DMSO was added. The absorbance
The significance of the differences between values obtained through
cellular reactive oxygen species assay was evaluated using one-way
ANOVA or a two-tailed Student’s t-test, statistically different from the
control: P < 0.01.
of each well was read at 550 nm using a Bio-rad 680 microplate. The
cytotoxic effect was furnished as IC50, the concentration of extract that
reduces cell viability to 50% of the control.
3. Results and discussion
2.8. Cellular uptake and localization in vitro
3.1. Chemistry
The cellular uptake and localization of final compounds 5a, 5f, and
5h were visualized by confocal laser scanning microscopy (CLSM).
HepG2 cells (2 × 105 cells per well) were cultured in a confocal dish for
24 h. Then compounds 5a, 5f and 5h were added and incubated with
HepG2 cells for another 12 h, respectively. Before testing, HepG2 cells
The synthetic route for ferrocenyl bis-naphthalimide derivatives 5a-h
was described in Scheme 1. According to the synthetic route, firstly,
mono-Boc-protected diamines 1 were obtained by reacting excessive
diamines with di-tert-butyl pyrocarbonate. And 1,8-naphthalimide was
reacted with different dibromo-hydrocarbon via substitution reaction to
yield bromo-naphthalimide derivatives 2. Then the mono-Boc-protected
diamines 1 were reacted with bromo-naphthalimide derivatives 2 to
generate bis-naphthalimide derivatives 3. After the deprotection of
compounds 3 with acid, the amino deprotected intermediates 4 were
condensed with ferrocene carboxaldehyde by Schiff’s base reaction.
Then the mixture was reduced by sodium borohydride to yield the target
bis-naphthalimide derivatives 5. All of the synthesized compounds were
characterized by 1H NMR and mass spectroscopy.
were immobilized with 500
μL formaldehyde solution and the nucleus
was stained for 5 min using 4′,6-diamidino-2-phenylindole (DAPI) (5
μ
mol/L). Then the images were captured using an Olympus FV1000-
IX81 Confocal Microscope.
2.9. Cellular reactive oxygen species analysis
The concentration of ROS was detected with the Reactive Oxygen
Species Kit (Beyotime). According to the instruction, a total of 5 × 104
HepG2 cells were plated in each well and incubated with 3 mL culture
medium for 24 h. Subsequently, the culture medium was replaced with
another 3 mL culture medium containing tested compounds. After in-
cubation for another 24 h, the cells were detached by gentle pipetting
O
(Boc)2
Boc
H2N
n NH2
H2N
n N
H
i
n=1 Propylene diamine
n=2 Butane diamine
1a n=1
3a n=2 m=1
O
1b n=2
1c n=3
1d n=4
3b n=2 m=2
n=3 Pentamethylene diamine
n=4 Hexamethylene diamine
3c n=2 m=3
3d n=2 m=4
3e n=1 m=2
3f n=3 m=2
N
iii
O
m
O
O
m
H
N
N
N
3g n=4 m=2
NH
O
Boc
n
3h n=3 m=1
O
N
O
O
Br
Br
m
ii
m
Br
m=1 Dibromopropane
m=2 Dibromobutane
m=3 Dibromopentane
m=4 Dibromohexane
iv
2a m=1
2b m=2
2c m=3
2d m=4
4a n=2 m=1
4b n=2 m=2
4c n=2 m=3
4d n=2 m=4
4e n=1 m=2
4f n=3 m=2
O
5a n=2 m=1
5b n=2 m=2
5c n=2 m=3
5d n=2 m=4
5e n=1 m=2
5f n=3 m=2
H2N
O
CHO
N
O
Fe
O
n
O
m
m
H
N
N
N
N
N
m
m
N
v
n
4g n=4 m=2
5g n=4 m=2
O
Fe
O
4h n=3 m=1
5h n=3 m=1
O
Scheme 1. Synthesis route of ferrocenyl naphthalimide derivatives. i) dioxane, ii) K2CO3/CH3CN, iii) K2CO3/CH3CN, iv) HCl/CH3CH2OH, v) NaBH4/CH3OH.
5

Y.-R. Fan et al.
Journal of Inorganic Biochemistry 219 (2021) 111425
3.2. Electrochemical studies
Table 1
Electrochemical data by cyclic voltammetry of bis-naphthalimide derivatives.
We measured the electrochemical properties (Fig. 1 and Table 1) of
ferrocenyl bis-naphthalimide derivatives 5a-h by cyclic voltammetry
(CV) in TBAP solution (0.1 mol/L). The electrochemical properties of 5a-
h were similar to that of ferrocene under the same condition. Only a
couple of well-defined redox peaks were found in Fig. 1, which was
assigned to the ferrocenium/ferrocene couple of ferrocenyl in 5a-h. The
anode peak potentials of compounds 5a-h were 0.621 V–0.639 V and the
cathode peak potentials were 0.589 V–0.596 V, which was larger than
that of ferrocene under the same conditions. The oxidation potential of
compounds 5a-h has a significant positive shift compared with that of
ferrocene. It is well known that ferrocene undergoes a reversible one-
electron reversible oxidation to form ferrocenium ion. The ΔE of com-
pounds 5a-h were lower than that of ferrocene, and the current ratio of
anode peak current (Ipa) to cathode peak current (Ipc) for compounds 5a-
h is about 1. It was speculated that all of the ferrocene attached bis-
naphthalimide derivatives 5a-h exhibited reversible one-electron
redox process as the redox properties of ferrocene derivatives [33,34].
The redox properties of ferrocene derivatives often correlate with the
ROS generation ability [35,36]. And it was demonstrated that the
cytotoxic activity of ferrocenium salts was due to the generation of OH∙
in physiological conditions [37].
Compound
Epa
Epc
E1/2
ΔE
5a
0.621 V
0.634 V
0.637 V
0.639 V
0.636 V
0.632 V
0.628 V
0.628 V
0.496 V
0.556 V
0.550
0.589 V
0.592 V
0.596 V
0.595 V
0.595 V
0.593 V
0.592 V
0.589 V
0.45 V
0.065 V
0.084 V
0.083 V
0.089 V
0.082 V
0.078 V
0.072 V
0.078 V
0.092 V
5b
5c
0.554 V
0.550 V
0.554 V
0.554 V
0.556 V
0.550 V
0.404 V
5d
5e
5f
5g
5h
Ferrocene
3.3. Ethidium bromide (EB) displacement experiment
Ethidium bromide displacement experiment was a usually used
method to study the DNA binding ability of bioactive molecules. After
the classical intercalator EB binding with CT DNA, the fluorescence in-
tensity of EB-DNA complex was significantly increased. The competitive
binding of bioactive molecules with DNA displace EB from EB-DNA
complex, which decreased the fluorescence intensity of the EB-DNA
duplex [38–41]. After the addition of tested compounds solution into
EB/DNA mixture, the fluorescence intensity of the mixture solution was
decreased (Fig. S1). And the quenching constants were calculated by the
Stern-Volmer equation, respectively (Fig. 2). The quenching constants of
5a-h were listed in Table 2. In the control experiment, after the addition
of DMSO without any tested compounds, the fluorescence intensity of
EB-DNA complex was decreased only 8.8% (Fig. S2a). And if we
assumed the concentrations of DMSO were equal to the concentrations
of test compounds, the quenching constant of DMSO was calculated to
be 2.24 × 104 L/mol according to the Stern-Volmer equation (Fig. S2b).
The quenching constant of the control experiment was only half of the
Fig. 2. Stern-Volmer plot of the EB displacement experiments of bis-
naphthalimide derivatives. [DNA] = 20 mol/L, [EB] = 10 mol/L.
μ
μ
minimum quenching constant among the tested compounds. That was to
say, after excluding the quenching effect of DMSO, all of the tested
compounds showed strong competitive binding ability with DNA.
Bis-naphthalimide compound 5c exhibited the largest quenching con-
stant (1.53 × 105 L/mol) among the tested compounds. Although it was
reasonable to ascribe the strong fluorescence quenching to the interca-
lation of naphthalimide with DNA, we cannot ignore the electrostatic
interactions between the polyamine and DNA, which decreased the
fluorescence intensity by squeezing out the intercalated EB from DNA
[42–44]. Furthermore, despite some principles were postulated to pre-
dict the bis-intercalative binding of some bifunctional molecules
[45–47], such as Elinafide, it was not so easy to speculate on the DNA
binding mode of synthesized tripodal bis-naphthalimide compounds
with DNA for the conformational distortion [48]. Hence, the large dif-
ference of the quenching constants between the tripodal
bis-naphthalimide compounds might be due to the multiple binding of
different groups.
3.4. UV-visible absorption titration
To further verify the binding ability of bis-naphthalimide derivatives
5a-h with DNA, the UV-visible absorption titration experiments were
carried out. UV-visible absorption titration is a widely used method to
study the interaction between small molecules and DNA [49]. It re-
flected the interaction of intercalator and DNA. Generally, an inter-
calator binding with DNA results in the hypochromic effect and redshift
on the UV-visible spectra of the intercalator, which is due to the
π -π*
stacking between the intercalator and DNA base pairs [50,51]. In our
experiments, the absorption peaks of compounds 5a–h between the
wavelength of 300–400 nm decreased and slightly bathochromic shifted
Fig. 1. The CV curves of bis-naphthalimide derivatives. (1 mmol/L in 90%
ethanol water solution, 0.1 mol/L TBAP, scan rate 100 mV/s).
6

Y.-R. Fan et al.
Journal of Inorganic Biochemistry 219 (2021) 111425
Table 2
Quenching constants of bis-naphthalimide derivatives.
Compound
Ksv(×104 L/mol)
DMSO
2.24a
5a
5b
5c
5d
5e
5f
5g
5h
4.57
11.00
15.25
6.68
5.26
8.77
10.98
11.55
a
Assumption the concentrations of DMSO were equal to the concentrations of test compounds.
after the addition of CT DNA (Fig. S3). The obvious hypochromic effect
Table 3
(26%–39%) indicated that at least one naphthalimide group of
Binding constants of bis-naphthalimide derivatives.
bis-naphthalimide derivatives intercalated into the base pairs of DNA
[52,53]. However, it was still unknown whether the other naph-
thalimide group of bis-naphthalimide derivatives intercalated into the
base pairs of DNA. It was reported that the DNA binding constants of
bis-intercalation were quite large than that of mono-interalation [45,
46]. Hence, the intrinsic binding constants Kb were calculated according
Compound
Kb(×104 L/mol)
5a
5b
5c
5d
5e
5f
5g
5h
15.9
12.9
8.95
9.53
11.6
13.1
5.22
8.75
3.5. Viscosity studies
to the plot of [DNA]/(εa-εf) versus [DNA] to compare the DNA binding
Viscosity titration is a typical method to study the binding mode of
the selected compounds with DNA. Generally speaking, classical inter-
calation binding mode increases the length of DNA and the DNA vis-
cosity. However, partial intercalation binding mode bends or kinks the
DNA, which decreases the DNA viscosity [49,56,57]. Groove binding
and outside binding will not change the DNA viscosity [43]. When a
molecule bears two or more binding groups that binding with DNA at the
same time, the situation is complicated. Even a slight difference in
structure could induce a violent change in DNA viscosity. For porphyr-
in–anthraquinone hybrids, the extended flexible alkyl linkage of the
hybrids caused the trend of DNA viscosity to reverse from decrease to
increase in DNA viscosity experiments [58,59]. In our experiments, after
the addition of bis-naphthalimide derivatives 5a–h into CT DNA solu-
tion, the viscosities of the solutions were decreased (Fig. 4). Compared
with the viscosity curve of EB, a classic DNA intercalator, which
increased the viscosity of DNA solution, the decreased viscosity should
be attributed to the multiple binding of the naphthalimide, amino and
ferrocene groups with DNA, which condensed DNA into nanoparticles
[60].
mode of the final compounds 5a–h (Fig. 3). The calculated intrinsic
binding constants Kb were listed in Table 3. All of the tested compounds
exhibited strong binding ability with DNA. The binding constant of 5a
was 1.59 × 105 L/mol, which was the maximum binding constants
among the tested compounds. After taking into consideration the length
of the chain linking two naphthalimide groups and the steric effects of
the tripodal structure, it was speculated that the binding mode of
bis-naphthalimide compound 5a with DNA was mono-intercalation. And
the intrinsic binding constants decreased from 5a to 5d, which differed
in the linker length between two naphthalimide groups. Although the
linkers of 5b–d were long enough for bis-intercalation according to the
principles above [45,46], mono-intercalation might occur for these
compounds for the smaller intrinsic binding constants compared with
that of 5a. However, from 5e, 5b, 5f and 5g, which differed in the length
of ferrocenyl chain, the intrinsic binding constants increased slightly
from 5e–f and dropped to 5g. It seemed that excessively increasing the
ferrocenyl chain was not a good idea to enhance the binding ability. The
binding constant of 5a was only 3-fold higher than that of the minimum
value of 5g (5.22 × 104 L/mol), which meant the binding mode of other
bis-naphthalimide compounds was similar to 5a [45,46]. The difference
in the binding constants should be attributed to the steric effects and
other weak interactions with DNA, such as electrostatic interaction of
amino group or groove binding of the other naphthalimide group [54,
55].
3.6. In vitro cytotoxicity
The in vitro cytotoxicity of ferrocenyl bis-naphthalimide derivatives
5a–h was evaluated by MTT assays on four cell lines (EC109，BGC823，
SGC7901 and HepG2). Amonafide, a naphthalimide derivative with
potential therapeutic for solid tumors, was used as a positive control
under the same experimental conditions. The IC50 values of ferrocenyl
bis-naphthalimide derivatives 5a–h against the four cancer cell lines
were listed in Table 4. The IC50 values of final compounds 5a–h against
Fig. 3. UV-visible absorption titration plots ([DNA]/(εa
-εf) vs. [DNA]) of bis-
Fig. 4. DNA viscosity titration of bis-naphthalimide derivatives at 25 ◦C in
naphthalimide derivatives in phosphate buffer (10 mmol/L, pH = 7.4).
phosphate buffer (10.0 mM, pH 7.4).
7

Y.-R. Fan et al.
Journal of Inorganic Biochemistry 219 (2021) 111425
tested HepG2 cells under confocal microscopy. Compared with DAPI,
which was located in the nucleus and exhibited blue fluorescence, the
tested bis-naphthalimide derivatives were spread all over the cells
(Fig. 5). Compounds 5f and 5h were showed better selectivity toward the
cytosol than the nucleus of cancer cells. After the ferrocene group con-
jugated with bis-napthalimide linker, ferrocene derivatives were able to
penetrate into cells, which was one of the important factors to enhance
the cytotoxicity [61]. Although most of the bis-naphthalimide de-
rivatives were found in the cytosol, DNA damage was also a possible
mechanism to induce cell death [29,61].
Table 4
The cytotoxicity of bis-naphthalimide derivatives.
Compound
Cytotoxicity (IC50 μmol/L)
EC109
BGC823
SGC 7901
HEPG2
5a
4.76 ± 0.61
6.10 ± 0.46
5.45 ± 0.24
6.27 ± 0.72
9.66 ± 1.30
5.34 ± 0.37
6.40 ± 1.05
5.28 ± 0.32
129.00 ± 3.421
4.20 ± 0.77
7.20 ± 0.15
6.05 ± 0.54
6.71 ± 0.30
9.59 ± 1.61
5.41 ± 0.17
5.64 ± 1.37
3.40 ± 0.018
80.14 ± 0.26
6.37 ± 0.17
9.10 ± 0.92
5.64 ± 0.28
5.93 ± 0.72
10.18 ± 0.28
5.57 ± 0.79
7.45 ± 0.12
4.96 ± 0.38
52.17 ± 4.19
0.40 ± 0.010
6.12 ± 0.17
2.21 ± 0.18
2.71 ± 0.28
6.92 ± 1.10
2.60 ± 0.11
2.76 ± 0.20
3.24 ± 0.28
34.64 ± 1.38
5b
5c
5d
5e
5f
5g
5h
Amonafide
3.8. Cellular reactive oxygen species analysis
The cytotoxicity of ferrocene derivatives is related to the intracel-
lular ROS generation, which cause DNA oxidative damage [62]. To
study the effect of the bis-naphthalimide derivative on the cellular ROS
levels, DCFH-DA (a commonly used fluorescent probe for the detection
of ROS in the cells) was used. We found that incubation HepG2 cells with
the selected compound 5a resulted in the elevation of cellular ROS levels
in a dose-dependent manner (Fig. 6). Compared with the Control group,
the ROS level of experiment groups increased 14.3 and 17.7 times,
respectively. One-way ANOVA and LSD analysis showed that the two
groups of 5a were significantly different from the Control group. The
appended ferrocene group in the bis-naphthanimide derivatives signif-
icantly increased the cellular ROS level.
the four cell lines were much lower than that of the positive control
Amonafide, which should be attributed to the synergistic effect between
ferrocene group and bis-naphthalimide derivative as well as the suitable
partition coefficients of the hybrid molecules [29]. 5a exhibited the best
cytotoxic effects against three-quarters of tested cell lines among 5a–d
with the same ferrocene chain, except SGC 7901. And 5f was the most
cytotoxic compound among 5b and 5e–f against all of the tested cell
lines. Although the intrinsic binding constants Kb of 5a and 5f were the
maximum values in each group, it might be a coincidence. The cyto-
toxicity of other test compounds was not consistent with the trend of the
intrinsic binding constants in each group. Combining the naphthalimide
linker of 5a and the ferrocene chain of 5f, 5h with a linker of n = 3, m = 1
was synthesized to test the cytotoxicity. 5h exhibited better cytotoxicity
than that of 5a against BGC823 and SGC7901 cell lines, and better
cytotoxicity than 5f against three-quarters of tested cell lines, except
HEPG2. The linker of n = 3, m = 1 was the best choice of the tripodal
bis-naphthalimide derivatives.
4. Conclusion
Herein, 8 tripodal ferrocenyl bis-naphthalimide derivatives were
synthesized and characterized. All of the synthesized compounds
exhibited good cytotoxicity versus Amonafide. The optimized com-
pound 5h according to the structure-activity relationship exhibited
better cytotoxicity than the parent compounds 5a and 5f against most of
the tested cell lines, which indicated that the linker of n = 3, m = 1 was
the best choice of the tripodal bis-naphthalimide derivatives. Drug up-
take experiments exhibited that the combination of bis-naphthalimide
and ferrocene group was helpful to improve the cell penetrability of
3.7. Uptake and localization of selected compounds
The cell uptake experiment was carried out by incubating selected
bis-naphthalimide derivatives 5a, 5f and 5h with HepG2 cells. Bis-
naphthalimide derivatives exhibited strong green fluorescence in the
Fig. 5. Confocal microscopic images of HepG2 cells treat with bis-naphthalimide derivatives, DAPI and merge.
8

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Journal of Inorganic Biochemistry 219 (2021) 111425
Fig. 6. Intracellular ROS accumulation in HepG2 cells treated with compound 5a were analyzed. (A) Flow cytometry analyses of Hela cells incubated with Rosup and
0.05, 0.5 μmol/L 5a; (B) Mean fluorescence intensity (MFI) quantification by flow cytometry analyses. Rosup was set as positive control. Data is shown as mean ± SD
of three independent experiments. *P < 0.01.
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ferrocene. And cellular ROS analysis also showed that the cytotoxicity of
synthesized compounds was attributed to intracellular ROS generated
by ferrocene groups, which was attributed to the reversible one-electron
redox process of the ferrocene groups under physiological conditions.
The naphthalimide units in these compounds exhibited excellent DNA
binding ability, which was demonstrated by EB displacement and UV-
visible absorption titration experiments. However, there was no
obvious relationship between DNA binding ability and cytotoxicity. All
in all, the synergistic effect of ferrocene and intercalators provided an
interesting idea in anticancer drug development.
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Declaration of competing interest
There are no conflicts to declare.
Acknowledgments
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This work was financially supported by the National Science Foun-
dation of China (No 21362026, No 21867016), Ningxia Natural Science
Foundation (No. 2020AAC03170), the Key R&D Program of Ningxia
(No. 2018BFG02004), Ningxia High Education Scientific Research
Project (No. NGY2020032), Young Talent Program of NingXia medical
University and Ningxia Medical University Key Project (No. XZ2018002,
XZ2019002).
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Appendix A. Supplementary data
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Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jinorgbio.2021.111425.
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