Naphthalimide-based macrophage nucleus imaging probes

Article abstract of DOI:10.1016/j.ejmech.2020.112407

The photophysical properties of naphthalimide-based fluorophores can be easily tuned by chemical manipulation of the substituents on that privileged scaffold. Replacement of a OMe group at position 6 in 2-(hydroxyl)ethyl-naphthalimide derivatives by diverse amines, including 2-(hydroxyl)ethylamine, trans-(4-acetamido)cyclohexylamine and azetidine increases the solvatochromic (ICT) character, while this replacement in 2-(dimethylamino)ethyl-naphthalimide analogues (PET fluorophores) decrease their solvent polarity sensitivity or even reversed them to solvatochromic fluorophores. These fluorophores resulted macrophage nucleus imaging probes, which bind DNA as intercalants and showed low cytotoxicity in human cancer cells.

Full text of DOI:10.1016/j.ejmech.2020.112407

Journal Pre-proof
Naphthalimide-based macrophage nucleus imaging probes
Francisco Fueyo-González, Mar Fernández-Gutiérrez, Diego García-Puentes, Angel
Orte, Juan A. González-Vera, Rosario Herranz
PII:
S0223-5234(20)30378-0
DOI:
https://doi.org/10.1016/j.ejmech.2020.112407
EJMECH 112407
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 March 2020
Revised Date: 7 April 2020
Accepted Date: 27 April 2020
Please cite this article as: F. Fueyo-González, M. Fernández-Gutiérrez, D. García-Puentes, A. Orte,
J.A. González-Vera, R. Herranz, Naphthalimide-based macrophage nucleus imaging probes, European
Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112407.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Masson SAS.

Naphthalimide-based macrophage nucleus imaging probes
Francisco Fueyo-Gonzáleza, Mar Fernández-Gutiérrezc, Diego García-Puentesd, Angel Orteb,
Juan A. González-Veraa,b,*, Rosario Herranza,*

Naphthalimide-based macrophage nucleus imaging probes
Francisco Fueyo-Gonzáleza,e, Mar Fernández-Gutiérrezb, Diego García-Puentesc, Angel Orted, Juan A.
González-Veraa,d,*, Rosario Herranza,*
aInstituto de Química Médica (CSIC). Juan de la Cierva 3, 28006 Madrid, Spain.
bInstituto de Ciencia y Tecnología de Polímeros (CSIC), CIBER-BBN, Juan de la Cierva 3, 28006 Madrid, Spain.
cInstituto de Química Orgánica General (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain.
dDepartamento de Fisicoquímica, Unidad de Excelencia de Química Aplicada a Biomedicina y Medioambiente,
Facultad de Farmacia, Universidad de Granada, Campus Cartuja, 18071, Granada, Spain.
ABSTRACT
The photophysical properties of naphthalimide-based fluorophores can be easily tuned by chemical manipulation of the
substituents on that privileged scaffold. Replacement of a OMe group at position 6 in 2-(hydroxyl)ethyl-naphthalimide derivatives
by diverse amines, including 2-(hydroxyl)ethylamine, trans-(4-acetamido)cyclohexylamine and azetidine increases the
solvatochromic (ICT) character, while this replacement in 2-(dimethylamino)ethyl-naphthalimide analogues (PET fluorophores)
decrease their solvent polarity sensitivity or even reversed them to solvatochromic fluorophores. These fluorophores resulted
macrophage nucleus imaging probes, which bind DNA as intercalants and showed low cytotoxicity in human cancer cells.
Keywords: Fluorescence probes; Naphthalimide derivatives; Quinolimide derivatives; Macrophage imaging probes; DNA
intercalants
*Corresponding authors.
E-mail addresses: rosario@iqm.csic.es (R. Herranz); gonzalezvera@ugr.es (J. A. González-Vera)
e Current address: Department of Medicine, Translational Transplant Research Center, Immunology Institute, Icahn School of
Medicine at Mount Sinai, New York, USA.
1

1. Introduction
Hydrophobicity or hydrophilicity of local environment
which have been widely used as models for imaging cellular
activity [13-15]. We have found a striking capability of most of
the dyes to cross the nuclear membrane and bind DNA,
acting as efficient macrophage nuclei staining probes.
determines interaction activities of proteins and controls the
permeability of cell membrane compartments. Therefore,
polarity-sensitive fluorophores are powerful chemical tools for
studying biological systems. Donor-acceptor type dipolar
fluorophores have been widely used in this field as
intramolecular-charge-transfer (ICT) molecular probes [1-4].
Usually, these fluorophores exhibit red shifts in their emission
spectra and a decrease in their fluorescence intensities upon
increasing the environment polarity (“on-off” fluorophores). In
spite of the numerous applications of ICT fluorophores in
chemistry, biology and medical sciences, their low emission in
aqueous media has limited their application for monitoring
biological processes. On the other hand, the scarcity of
polarity sensing fluorophores of type “off-on“ when increasing
the environment polarity, which include acridine [5], pyrene-3-
carboxaldehyde [6], and 7-methoxy-4-methylcoumarin [7], has
also limited the monitoring of biological processes that imply
an increase in local hydrophilicity. Among the limited
examples of this type of sensors, recently, 2-(trans-(4-
hydroxy)-cyclohexylamino)-naphthalimide derivatives have
been described as biothiol sensors [8], 2-(2-aminoethyl)-
naphthalimide as in cellulo metal sensor [9], and some
BODIPY derivatives as sensors of local hydrophilicity in
dysfunctional lysosomes [10]. S. Singha et al. increased the
emission intensity of diverse ICT fluorophores in water by
replacing their dimethylamino group, usual donor group, by
several dialkylamines or bulky monoalkylamines, preferably 2-
(hydroxyl)ethylamine or trans-(4-acetamido)cyclohexyl-amine
[8]. The increase of fluorescence was explained due to
decreased non-radiative deactivation in H2O, by hindering the
interaction of the donor group with H2O through hydrogen
bonds. The increase in steric volume of the amino group also
reduced its rotational freedom, facilitating the charge transfer.
Fig. 1. New Naphthalimide- and quinolimide-based fluorophores 3-8
2. Results and discussion
2.1. Synthesis
As shown in Scheme 1, the 6-amino-naphthalimide
derivatives 3a, 5a, 6a and 8a were obtained from the
corresponding 6-bromo-naphthalimides 9a and 10a via
aromatic nucleophile substitution, by treatment with the
corresponding amine in EtOH at 120 ºC under MW irradiation.
In the case of the substitution with trans-4-amino-
cyclohexylamine in naphthalimides 11a and 12a, it was
necessary to carry out the reaction in sealed tube at 150-
180ºC in the presence of diisopropylethylamine (DIPEA). The
acetylation of the free amino group of 11a and 12a, by
treatment with acetic anhydride in CH2Cl2 yielded the
naphthalimides objective 4a and 7a. A similar methodology
was applied for the synthesis of the 9-[trans-(4-
We have recently described the switching from
naphthalimide and quinolimide-based (ICT) fluorophores 1
(Figure 1) to PET fluorophores by the simple substituent
manipulation of a hydroxyl to an N-dimethylamino group in
analogues 2 [11]. This switch transformed the “on-off” polarity
sensors N-(2-hydroxyethyl)-substituted derivatives 1a and 1b
into the “off-on” polarity sensors N-(2-(dimethyl)aminoethyl)-
substituted analogues 2a and 2b. In this way, we obtained
H2O and pH sensors and macrophage cytoplasm imaging
probes. Now, with the aim of increasing the fluorescence
emission of these fluorophores in H2O, we have studied the
replacement of the donor group OMe group of 1a,b and 2a,b
by better donor groups, such as several substituted amines,
concretely, 2-(hydroxy)ethylamine (Figure 1, 3 and 6), trans-
(4-acetamido)cyclohexylamine (Figure 1, 4 and 7) and the
good electrodonor group azetidine [12] (Figure 1, 5 and 8). In
general, this substituent manipulation has led to significant
enhancements in the quantum yields of these fluorophores in
water. We have also explored their application as in cellulo
imaging probes in macrophages, multi-functional immune
cells with key roles in host defense and tissue remodeling,
acetamido)cyclohexylamino-quinolimide
7b
from
the
corresponding 9-methoxy-quinolimide 2b.
2

Scheme 1. Synthesis of the naphtalimide derivatives 3a-8a and the quinolimide analogue 7b (a) 2-Hydroxyethylamina or azetidine, EtOH, MW
120ºC (20-85%); (b) trans-(4-amino)cyclohexylamine, DIPEA, EtOH, 150-180ºC (2-91%).
2.2. Photophysical properties
(F3C-CH2OH, MeOH and H2O) it would emit from the higher
energy LE state. The effect of the replacement of the OMe
group upon ε and ΦF of the naphthalimide derivatives 6a-8a
and the quinolimide 7b was diverse. In the azetidine
derivative 8a and the quinolimide 7b, the PET effect that we
We performed a thorough photophysical study of the
properties of the new naphthalimides 3a-8a and the
quinolimide 7b in solvents of diverse polarity (Table 1), in
order to rationalize the effect of new R2 substituents, when
compared with the OMe-substituted compounds 1a and 2a
(naphthalimides) and 2b (quinolimide) in comparison with
those of 1a, 2a and 2b, respectively.
had observed in 2a and 2b was absent, showing
a
solvatochromic behavior (typical of ICT fluorophores). In the
naphthalimides 6a and 7a the ε coefficients decreased, while
ΦF increased, mainly in non-polar solvents, eliminating the off-
on polarity effect previously observed in 2a. Therefore, in the
solvatochromic fluorophore 1a, the replacement of the MeO
group by hydroxyethylamine, azetidine or trans-(4-
acetamido)cyclohexylamine enhanced the solvatochromic
(ICT) character, while in the PET fluorophores 2a,b, that
replacement hinder the PET effect, leading either to
solvatochromic fluorophores in the case of the azetidine
derivative 8a and the quinolimide 7b or to low solvent polarity
sensitivity in the (hydroxyethyl)amine (6a) or the trans-(4-
acetamido)cyclohexylamine (7a) derivatives.
The replacement of the OMe group in the naphthalimide
carrying the 2-(hydroxyl)ethyl chain at position 2, 1a, by
amines led to increases in the solvatochromic behavior
characteristic of ICT–based fluorophores in 3a-5a. These
naphthalimides showed 65-100 nm bathochromic shifts in
abs
em
λmax
and λmax with respect to 1a. The increase in the
solvent polarity produced 10-40 nm bathochromic shifts in
both wavelength maxima, increased the extinction coefficients
and decreased the quantum yields.
In the naphthalimide carrying the 2-(dimethylamino)ethyl
chain, 2a, and in the quinolimide analogue, 2b, the
replacement of the OMe group by amines (compounds 6a-8a
and 7b) also produced bathochromic shifts of 65-100 nm in
abs
λmax
and λmaxem. Also, these wavelengths increased with
solvent polarity, except for the quinolimide derivative 7b,
em
where λmax
augmented from CH3Cl to acetone and
decreased from this solvent to H2O. This behavior could be
due to emission from two different excited states, the local
excited (LE) and the ICT state [16, 17]. In the less polar
solvents (CH3Cl, dioxane and acetone) 7b would emit from
the ICT state (higher λmaxem), while in more polar solvents
3

Table 1. Photophysical properties of the naphthalimides 1a-8a and
the quinolimides 2b and 7b.
2.3. Cytotoxicity in tumor cells
Several
2-[2-(dimethylamino)ethyl]-naphthalimide
b
Compda
Solvent
λmaxabs (nm)
ε (M-1cm-1) λmaxem (nm)
ΦF
derivatives have been described as antitumor agents [18],
such as mitonafide or amonafide [19, 20], which bind to DNA
as intercalants and inhibit topoisomerase II [21], forming and
CHCl3
360
359
357
373
366
375
366
358
359
371
367
376
428
422
430
438
438
445
433
426
435
450
447
451
443
428
437
449
449
460
426
422
433
437
438
445
430
422
436
452
441
446
441
430
437
453
449
460
377
367
370
380
376
381
470
466
462
483
471
471
14540
12080
13610
11950
12790
9650
8100
6700
9700
10100
8300
7200
11730
11370
11570
9970
12790
15780
10170
13120
8400
9230
8860
10630
13620
10820
11380
12720
14580
18660
6570
5810
6190
7530
4360
5220
6900
3600
6700
8300
7320
8170
9800
10380
8510
11830
7320
6540
3100
2800
4900
2900
3700
3200
3900
3470
4020
4225
3040
3800
428
426
434
449
442
458
428
425
433
450
444
460
502
501
516
523
525
527
497
491
510
524
522
527
510
501
521
527
528
533
501
499
511
530
527
531
499
489
511
529
525
534
506
502
518
534
527
533
457
457
467
495
493
505
554
562
572
539
526
533
0.57
Dioxane
Acetone
F3C-CH2OH
MeOH
H2O
CHCl3
Dioxane
Acetone
F3C-CH2OH
MeOH
H2O
CHCl3
Dioxane
Acetone
F3C-CH2OH
MeOH
H2O
CHCl3
Dioxane
Acetone
F3C-CH2OH
MeOH
H2O
CHCl3
Dioxane
Acetone
F3C-CH2OH
MeOH
H2O
CHCl3
Dioxane
Acetone
F3C-CH2OH
MeOH
H2O
CHCl3
Dioxane
Acetone
F3C-CH2OH
MeOH
H2O
CHCl3
0.60
0.43
0.32
0.57
0.30
0.20
0.22
0.14
0.63
0.50
0.63
0.86
0.97
0.82
0.55
0.26
0.17
0.98
0.84
0.77
0.51
0.56
0.23
0.98
0.68
0.72
0.44
0.40
0.16
0.61
0.69
0.99
0.56
0.94
0.57
0.51
0.35
0.77
0.40
0.66
0.71
0.81
0.78
0.87
0.20
0.32
0.18
0.12
0.17
0.04
0.63
0.23
0.26
0.16
0.12
0.03
0.02
0.05
0.02
stabilizing
the
ternary
complex
DNA-intercalant-
1a
topoisomerase II [22]. Besides, some quinolimide derivatives
have been also reported as antitumor compounds [23]. Taking
into account these reports, the naphthalimide and quinolimide
derivatives herein described were included in
a high
2a
3a
4a
5a
throughput screening (HTS) for cytotoxicity in non-small cell
lung cancer (A549), colon (HT29), breast (MDA-MD231), and
pancreas (PSN1) human cancer cell lines, using doxorubicin
as positive control and according to the National Cancer
Institute (NCI) protocols. None of the 2-(hydroxyl)ethyl-
naphthalimide derivatives 3a-5a showed citotoxicity at the
highest tested concentration (40 μM). In the 2-
(dimethylamino)-ethyl analogues, the naphthalimides 6a and
7a and the quinolimide 7b showed low cytotoxicities in the μM
range as the corresponding 6-methoxy-naphthalimide 2a and
the 9-methoxy-quinolimide derivative 2b (Table 2). These
values of cytotoxicity were of the same order than those
previously reported for mitonafide and amonafide [18, 23].
Table 2. Cytotoxicities (GI50)a of 2-(2-(dimethylamino)ethyl-
naphthalimides and 5-(2-(dimethylamino)ethyl-quinolimides
6a
7a
8a
2b
7b
Lung
A549
Colon
HT29
Breast
Pancreas
PSN1
Mouse
macrophages
Compd
MDA-MD-231
Doxorubicin 0.085
0.109
9.39
0.055
16.10
>33.4
10.10
5.44
0.052
16.10
>33.4
13.70
8.05
NDb
36.3
NDb
38.1
33.3
20.8
5.8
2a
2b
6a
7a
8a
7b
11.40
>33.4
11.90
7.55
>33.4
9.77
Dioxane
Acetone
F3C-CH2OH
MeOH
3.79
>30.9
>30.9
>30.9
>30.9
9.44
H2O[c]
CHCl3
7.79
5.67
8.26
Dioxane
Acetone
F3C-CH2OH
MeOH
H2O
CHCl3
Dioxane
Acetone
F3C-CH2OH
MeOH
aμM concentrations. bND: Non determined
2.4. Macrophage imaging probes
As already commented, although there are diversity of
fluorescent probes for imaging specific cellular organelles [24-
31], the number of polarity sensing fluorophores with “off-on”
features when increasing the environment polarity is very
limited and, in general, they have demonstrated limited
application due to quenching of their fluorescence by
protonation and to their low excitation and emission
wavelengths [32]. The scarcity of this type of fluorophores,
which emit in polar media, has limited the monitoring of
biological processes that imply an increase in local
H2O
aMeasured in duplicate at a 12 µM concentration. bQuantum yields calculated
with reference to cumarine 153 (in EtOH), except for 1a, 2a and 2b, where the
reference was quinine sulfate (in 0.1 M H2SO4).
4

hydrophilicity. In view of the photophysical behavior and low
cytotoxicity of the naphthalimides and quinolimides herein
described, we explored their application as in cellulo imaging
probes in macrophages. Therefore, mouse macrophages
were treated with a 5 μM concentration of each fluorophore
and, after one hour of incubation at 37 °C, the cells were
visualized by confocal microscopy. After laser excitation at
488 nm, the emission of the new naphthalimides 3a-8a was
observed with a 505-570 nm filter, while the emission of the
quinolimide 7b was observed with the GFP filter (525-615
nm). We also compare these results with the images of
previously published compounds 1a,b and 2a,b. As shown in
Figures 2 and 3, cells treated with the fluorophores carrying
the 2-(hydroxyl)ethyl chain at the imide ring 1a,b and 3a-5a
(Figure 2, A, C, and E, G, I) showed lower fluorescence than
low cytotoxicity is an objective of great interest for the
visualization of the cell nucleus in long-term studies.
cells
treated
with
fluorophores
carrying
the
2-
(dimethylamino)ethyl chain 2a,b, 6a-8a and 7b (Figure 2, B,
D, F, H, J and K), which displayed bright fluorescence. As
already described, cells treated with the 6-methoxy-
naphthalimide 2a or the 9-methoxy-quinolimide 2b displayed
bright fluorescence at their cytoplasm (Figure 2, B and D),
while cells treated with the 6-substituted amino-
naphthalimides 3a-8a or the quinolimide 7b displayed the
fluorescence at their nucleus (Figure 2, F, H, J, K), except for
the cells treated with the azetidine derivative 5a, which
displayed low fluorescence at the cytoplasm (G). According to
the ΦF of these fluorophores in H2O, cells treated with the
trans-(4-acetamido)-cyclohexylamine derivative 7a (Figure 2,
F) showed much brighter fluorescence than the corresponding
2-(hydroxyl)ethyl analogue 6a (Figure 2, J). Interestingly, cells
treated with the naphthalimide derivative 8a (Figure 2, H) or
with the quinolimide 7b (Figure 2, K) displayed bright
fluorescence, in spite of the low ΦF of these fluorophores in
H2O (78-86% lower than in CHCl3 or dioxane). This would
indicate their localization in a hydrophobic environment in the
nucleus.
The nucleus is the most prominent organelle of cells,
where ribosome synthesis and gene expression are regulated
[33], so its visualization is important to help answer questions
about these processes and others such as the genesis of
tumors or the action of some drugs. Among the limited
number of known nuclear fluorophores, 4',6-diamidino-2-
phenilindole (DAPI) and the Hoechst dyes, which bind to the
minor groove of DNA, and propidium iodide (PI), DNA
intercalant, have been the most used probes to stain cell
nuclei, due to its good cellular permeability and its specificity
for DNA [34]. However, these fluorophores are generally
mutagenic, have low water solubility and low photostability,
which limits their application in studies that require long
observation times. Other small fluorophores, used for the
visualization of the nucleus, such as, for example, various
styryl derivatives [35, 36], fluorescein [37], ruthenium (II) dyes
[38] and hemicianin [39], are used at very low concentrations,
due to problems of fluorescence quenching by aggregation. In
addition, these dyes usually suffer from notable
photobleaching after photoexcitation [40]. Therefore, obtaining
new nuclear probes, soluble in water, photostable and with
Fig. 2. Confocal microscopy images of mouse macrophages treated
with a 5 μM concentration of A) naphthalimide 1a; B) naphthalimide
2a; C) quinolimide 1b; D) quinolimide 2b; E) naphthalimide 4a; F)
naphthalimide 7a; G) naphthalimide 5a; H) naphthalimide 8a; I)
naphthalimide 3a; J) naphthalimide 6a; and K) quinolimide 7b. L)
Untreated control cells. Scale bars represent 50 µm.
5

Fig. 4. Cellular co-localization of the nuclear dye DAPI and the 2-(2-
(dimethylamino)ethyl)-naphthalimide derivative 7a in macrophages at
a 5 μM concentration. A) Macrophage cell nuclei stained with DAPI.
B) Macrophage cell nuclei stained with naphthalimide 7a. C) Overlay
of the two channels. Scale bars represent 50 µm.
2.5. Binding to DNA
Fig.3. Fluorescence relative intensity of the probes in macrophages.
Box-and-whisker plot represents the average fluorescence intensity in
pixels of confocal images (in Fig. 2) containing the studied
Having confirmed the nuclear localization of some of our
fluorophores, we studied the interaction of the naphthalimide
derivative 7a and the quinolimide analogue 7b with DNA, in
vitro, using salmon sperm DNA (FS-DNA) and the double
strand of the oligonucleotide R-13 (5’-GCGTACGCCAGCG-
3’) [42] as models of DNA. For this study, circular dichroism,
UV spectrometry and fluorescence excitation spectra of the
titration of constant concentrations of the fluorophores (7a: 16
μM and 7b: 20 μM) with increasing concentrations of FS-DNA
(8.1 μM till 2.0 mM base pairs) and oligonucleotide R-13
(0.165 μM till 4.4 mM) and the titration of a 4.3 μM
concentration of this oligonucleotide with increasing
concentrations of the fluorophores (0-430 μM) were recorded
[43, 44]. The behavior of both 7a and 7b was similar. Their
CD spectra showed a very small negative band at their
respective absorption maxima wavelengths (7a ≈ 450 nm and
7b ≈ 470 nm), indicative of their binding as intercalants. If the
binding would have been to the major or minor DNA grooves,
that band should have been stronger. Besides, the CD signal
increases slightly by increasing the concentration of the
fluorophore (shown in Figure 5 for 7a). These results are
characteristic of DNA intercalants [45]. Significant changes
were also observed in the UV and fluorescence excitation
spectra. The absorption intensity decreased, while
fluorescence intensity increased with increasing DNA
concentration. These results were also indicative of a binding
as intercalant [46]. In the case of the quinolimide derivative
7b, it was not possible to detect its fluorescence excitation,
probably due to its low ΦF in H2O. These results would explain
why although our fluorophores co-localizes with DAPI, there is
no competition between them, since, as mentioned, DAPI
binds to the minor DNA groove and is not intercalant.
fluorophores in mouse macrophages treated with
a
5
μM
concentration of probe. Boxes correspond to the 25 and 75%, with a
horizontal bar indicating the average value. Whiskers represent the
minimum and maximum values.
Taking into account the clear nuclear localization of the
naphthalimides carrying the 2-(dimethylamino)ethyl chain 6a-
8a, and the quinolimide 7b, in order to help to elucidate their
binding site, we explored whether these fluorophores co-
localized with DAPI. As shown in Figure 4 for 7a, the images
obtained with our fluorophores overlapped completely with
those obtained with DAPI, indicating coincidence in DNA
binding (Pearson colocalization coefficient of 0.929 ± 0.06,
Figure S5). In addition, two competition experiments were
performed between 7a and DAPI. In the first, a 1 μM
concentration of DAPI was added to the macrophages and,
after 1 hour of incubation at 37 °C and after checking that
DAPI was in the nucleus, the naphthalimide 7a was added in
increasing concentrations from 1 to 10 μM. In the second
experiment the order of addition of the fluorophores was
changed, adding compound 7a at 1 μM concentration and
increasing concentrations of DAPI from 1 μM to 10 μM. In all
cases, both fluorophores co-localized in the nucleus, without
observing displacement between them. These results
indicated that although both fluorophores bind to DNA, they
do at concentrations far from saturation or binding to non-
competing sites.
Taking into account the moderate cytotoxicity shown by
6a-8a and 7b in tumor cells, the cytotoxicity in mouse
macrophages RAW 264.7 (ECACC, Sigma P11) was also
determined by an Alamar blue assay [41]. After 24 h of
incubation at 37 °C, increasing concentrations up to 100 μM
of the studied fluorophores were added. All the compounds
showed low cytotoxicity (Table 2), with GI50 values greater
than 10 μM, except for the quinolimide 7b, with a GI50 of 5.8
μM.
6

good probes for macrophage imaging. While those with the
MeO group at the aromatic ring localized at the cytoplasm,
those where an amino moiety replaces the MeO group, such
as 6a-7a and 7b, are localized at the nucleus, where they
bind DNA as intercalants.
Fig. 5. CD spectra of R-13 with increasing concentrations of the
naphthalimide 7a.
Binding isotherms were obtained for 7a and 7b from the
representation of the absorbance against the DNA
concentration (Figures 6A and B). The affinity constants,
expressed as dissociation constants Kd with the DNA
concentration referred in terms of base pairs, were calculated
from these isotherms (see Supplementary materials for details
on the fittings), showing a good correlation between the
results of FS-DNA and R-13. The Kd values of 7a were 1.7 μM
and 1.3 μM with FS-DNA and R-13, respectively, whereas Kd
values of 8.9 μM and 3.5 μM were obtained for 7b with FS-
DNA and R-13, respectively. The stoichiometry of the
interaction represents the number of sites (base pairs)
occupied by the dyes upon binding. When interacting with FS-
DNA, we obtained the binding of 1 molecule of 7a for each 7
base pairs, and 1 molecule of 7b for each 9 base pairs. The
corresponding
binding
isotherms
to
the
shorter
oligonucleotide R-13 showed the binding of 1 molecule of 7a
for each 5 base pairs, and 1 molecule of 7b for each 6 base
pairs, indicating a higher level of saturation in the short
strands
Fig. 6. Binding isotherms obtained for (A) 7a and (B) 7b from the
representation of the absorbance against concentration of R-13.
3. Conclusions
The results herein described show how the photophysical
properties of naphthalimide-based fluorophores can be easily
tuned by chemical manipulation of the substituents on that
privileged scaffold. Thus, in the 2-(hydroxyl)ethyl-
naphthalimides, replacement of the OMe group at position 6
4. Experimental
4.1. Synthesis
4.1.1. General methods
em
by diverse amines leads to bathochromic shifts in their λmax
and to increases in their solvatochromic ICT character.
However, in the PET fluorophores, 2-(dimethylamino)ethyl-
naphthalimide derivatives, in general, that replacement leads
to increases in the fluorescence quantum yields in all
solvents, regardless of their polarity, loss of the PET
character, and in some cases to highly solvatochromic
fluorophores. Naphthalimides and quinolimides carrying the 2-
(dimethylamino)ethyl chain at the imide ring have resulted
All reagents were of commercial quality. Solvents were
dried and purified by standard methods. Analytical TLC was
performed on aluminum sheets coated with a 0.2 mm layer of
silica gel 60 F254. Silica gel 60 (230-400 mesh) was used for
flash chromatography. HPLC-MS was performed on a Sunfire
C18 (4.6×50 mm, 3.5 μm) column at 30°C, with a flow rate of 1
mL/min and gradient of 0.1% of formic acid in CH3CN (solvent
7

A) in 0.1% of formic acid in H2O (solvent B) was used as
mobile phase. Electrospray in positive mode was used for
ionization. NMR spectra were recorded using Varian Inova or
Mercury 400, and Varian Unity 500 spectrometers. The NMR
spectra assignments were based on COSY, HSQC, and
HMBC spectra. High resolution mass spectra (HRMS) were
recorded on an Agilent 6520 Q-TOF instrument with an ESI
source. MW experiments were carried out in sealed vessels in
a MW EmrysTM Synthesizer (Biotage AB), with transversal IR
sensor for reaction temperature monitoring. UV-visible
spectroscopy measurements were made at 25 Cº on a
Lambda 35, Perkin Elmer, UV-vis spectrophotometer; Starna
Cells (16.100-Q-10) 100 μL sub-micro cuvette, 1 cm path
length. Steady-state fluorescence emission spectra were
performed at 25 Cº either on a PerkinElmer LS 50B
57.9, 58.8, 103.7, 107.6, 120.0, 121.8, 124.1, 128.5, 129.3,
130.5, 134.0, 150.7, 162.9, 163.8. HRMS (ESI) m/z: Calc. for
C16H16N2O4 ([M+H]+): 301.1182, Found: 301.1176.
4.1.4. Synthesis of 6-((trans-4-aminocyclohexyl)amino)-2-
(2-hydroxyethyl)-1H-benzo[de]-isoquinoline-1,3(2H)-dione
(11a)
trans-1,4-Diaminocyclohexane (195 mg, 1.71 mmol) and
DIPEA (28 µL, 0.51 mmol) were added to a solution of the 6-
bromo-naphthalimide 9a (110 mg, 0.343 mmol) in EtOH (5
mL) and the mixture was heated at 120ºC in a closed tube for
5 days. After cooling at room temperature, the precipitated
solid was filtered off and the filtrate was evaporated to
dryness to give the desired naphthalimide 11a as a brown
solid (110 mg, 91 %). M.p. 280 ºC. HPLC-MS (gradient 5-
luminescence spectrometer or
a
JASCO FP-8300
1
95% of A en B, 10 min) tR = 4.61 min. H-NMR (DMSO-d6,
spectrofluorometer equipped with a 450 W xenon lamp for
excitation; Starna Cells (16.100F-Q-10) 100 μL sub-micro
cuvette, 1 cm path length.
400 MHz) δ: 1.25 and 1.49 (2m, 4H), 1.85 and 2.02 (2m, 4H),
2.62 (m, 1H), 3.57 (t, 2H, J = 6.5 Hz), 3.61 – 3.65 (m, 1H),
4.11 (t, 2H, J = 6.5 Hz), 6.82 (d, 1H, J = 9 Hz), 7.30 (d, 1H, J
= 8 Hz), 7.64 (t, 1H, J = 7 and 8.5 Hz), 8.23 (d, 1H, J = 9 Hz),
8.41 (dd, 1H, J = 1 and 7 Hz), 8.76 (dd, 1H, J = 1 and 8.5 Hz).
13C-NMR (DMSO-d6, 100 MHz) δ: 30.5, 34.8, 41.4, 49.9, 51.2,
58.0, 104.1, 107.4, 120.1, 121.9, 124.0, 128.8, 129.6, 130.6,
134.2, 149.8, 163.0, 163.9. HRMS (ESI) m/z: Calc. for
C20H23N3O3 ([M+H]+): 354.1812, Found: 354.1811.
4.1.2. Synthesis
of
6-bromo-2-(2-hydroxyethyl)-1H-
benzo[de]isoquinoline-1,3(2H)-dione (9a)[47]
2-Aminoethanol (48 μL, 0.79 mmol) was added to a
solution of 6-bromo-1H,3H-benzo[de]isochromene-1,3-dione
(200 mg, 0.72 mmol) in dry EtOH (3 mL) and the mixture was
heated at 68 ºC under MW irradiation for 10 min. After cooling
at room temperature, the precipitated solid was filtered and
dried under vacuum for 12 h to yield the corresponding 6-
bromo-naphthalimide 9a as a yellow solid (205 mg, 90 %).
M.p. 205 ºC. HPLC-MS (15-95 % gradient of A in B, 10 min)
tR = 8.07 min.1H-NMR (CDCl3, 400 MHz) δ: 2.19 (br s, 1H,),
3.92 (m, 2H), 4.39 (t, J = 5 Hz, 2H), 7.79 (dd, 1H, J = 7.5 and
8.5 Hz), 7.99 (d, J = 8 Hz, 1H), 8.36 (d, J = 8 Hz, 1H), 8.53
(dd, J = 1 and 8.5 Hz, 1H), 8.60 (dd, J = 1 and 7.5 Hz,
1H).13C-NMR (CD3OD, 100 MHz) δ: 43.0, 61.9, 122.1, 123.0,
128.3, 129.2, 130.8, 130.9, 131.4, 131.7, 132.5, 133.8, 164.7.
HRMS (ESI) m/z: Calc. for C14H11BrNO3 ([M+H]+): 319.9917,
Found: 319.9955 (0.09 %). Calc. for (M+H)+(-H2O): 303.9792,
Found: 303.9777 (100%).
4.1.5. Synthesis of N-(trans-4-((2-(2-hydroxyethyl)-1,3-
dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)amino)-
cyclohexyl)acetamide (4a)
Acetic anhydride (1mL) was added to a solution of the
naphthalimide 11a (40 mg, 0.11 mmol) in CH2Cl2 (5 mL) and
the solution was stirred at room temperature for 30 min. Then,
the reaction mixture was evaporated to dryness and the
residue was purified by flash chromatography, using 0-5%
gradient of MeOH in CH2Cl2 as eluent, to give the desired
acetylated derivative 4a as an orange solid (32 mg, 75%).
M.p. 116 ºC. HPLC-MS (gradient 5-95% of A in B, 10 min) tR =
7.29 min. 1H-NMR (DMSO-d6, 400 MHz) δ: 1.38 (m, 2H), 1.51
(m, 2H), 1.92 (s, 3H), 2.06 (m, 2H), 3.55 and 3.65 (2m, 2H),
4.28 (s, 4H), 6.89 (d, 1H, J = 8.5 Hz), 7.39 (d, 1H, J = 7.5 Hz),
7.68 (dd, 1H, J = 8.5 Hz), 7.82 (d, 1H, J = 7.5 Hz), 8.26 (d,
1H, J = 8.5 Hz), 8.44 (d, 1H, J = 8.5 Hz), 8.79 (d, 1H, J = 8.5
Hz). 13C-NMR (DMSO-d6, 100 MHz,) δ: 22.8, 30.5 and 31.0,
45.6, 47.3, 50.9, 61.1, 104.4, 107.2, 120.1, 121.6, 124.1,
129.1, 129.7, 130.8, 134.4, 150.0, 163.0, 164.0, 168.3. HRMS
(ESI) m/z: Calc. for C22H25N3O4 ([M+H]+): 396.1917, Found:
396.1918.
4.1.3. Synthesis
of
2,6-bis(2-hydroxyethyl)-1H-
benzo[de]isoquinoline-1,3(2H)-dione (3a)
2-Aminoethanol (275 µL, 4.6 mmol) was added to a
solution of the 6-bromo-naphthalimide 9a (150 mg, 0.46
mmol) in EtOH (5mL) and the mixture was heated at 120 ºC
under MW irradiation for 1.5 h. Afterwards, the solvent was
evaporated to dryness and the residue was purified by flash
chromatography, using 0-3% gradient of MeOH in CH2Cl2 as
eluent, to give the desired 2,6-bis(2-hydroxyethyl)-
naphthalimide 3a as an orange solid (50 mg, 36 %). M.p.
209 ºC. HPLC-MS (5-95% gradient of A in B, 10 min) tR = 5.62
min. 1H-NMR (DMSO-d6, 400 MHz) δ: 3.51 (c, 2H, J = 6 Hz,),
3.58 (t, 2H, J = 7 Hz), 3.71 (t, 4H, J = 6 Hz), 4.10 (t, 2H, J = 7
Hz), 6.75 (d, 1H, J = 8.5 Hz), 7.63 (t, 1H, J = 8 Hz), 7.70 (br s,
1H), 8.20 (d, 1H, J = 8.5 Hz), 8.38 (d, 1H, J = 7 Hz), 8.66 (d,
1H, J = 8.5 Hz). 13C-NMR (DMSO-d6, 100 MHz) δ: 41.3, 45.6,
4.1.6. Synthesis of 6-(azetidin-1-yl)-2-(2-hydroxyethyl)-1H-
benzo[de]isoquinoline-1,3(2H)-dione (5a)
Azetidine (178 µL, 2.65 mmol) was added to a solution of
the 6-bromo-naphthalimide 9a (170 mg, 0.53 mmol) in EtOH
(5mL) and the mixture was heated at 120 ºC under MW
irradiation for 1.5 h. After cooling at room temperature, the
precipitated solid was filtered, washed with EtOH (3×1mL),
8

and dried under vacuum for 12 h to yield the corresponding 6-
azetidin-1-yl-naphthalimide 5a as a red solid (50 mg, 36 %).
M.p. 222 ºC. HPLC-MS (gradient 5-95% of A in B, 10 min) tR
4.1.9. Synthesis of N-(trans-4-((2-(2-(dimethylamino)ethyl)-
1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)amino)-
cyclohexyl)acetamide (7a)
1
= 7.09 min. H-NMR (DMSO-d6, 400 MHz) δ: 2.47 (m, 2H),
Acetic anhydride (1mL) was added to a solution of the
naphthalimide 12a (14 mg, 0.028 mmol) in (9:1) CH2Cl2/dry
acetone mixture (1 mL) and the solution was stirred at room
3.59 (m, 2H), 4.09 (t, 2H, J = 7 Hz), 4.43 (t, 4H, J = 7.5 Hz),
4.79 (br s, 1H), 6.34 (d, 1H, J = 8.5 Hz), 7.52 (t, 1H, J = 8 Hz),
8.11 (d, 1H, J = 8.5 Hz), 8.24 (d, 1H, J = 8 Hz), 8.33 (d, 1H, J
= 7 Hz). 13C-NMR (DMSO-d6, 100 MHz) δ: 16.4, 41.4, 55.0,
57.9, 105.8, 108.2, 120.0, 121.6, 123.7, 129.8, 130.5, 132.6,
151.9, 162.8, 163.7. HRMS (ESI) m/z: Calc. for C17H16N2O3
([M+H]+): 297.1233, Found: 297.1222.
temperature for
6 h. Then, the reaction mixture was
evaporated to dryness and the residue was purified by flash
chromatography, using 1-10% gradient of MeOH in CH2Cl2 as
eluent, to give the desired acetylated derivative 7a as an
orange solid (12 mg, 80%). M.p. 241 ºC. HPLC-MS (30-95%
1
4.1.7. Synthesis
of
2-(2-(dimethylamino)ethyl)-6-((2-
gradient of A in B, 10 min) tR = 1.87 min. H-NMR (CD3OD,
hydroxyethyl)amino)-1H-benzo[de]isoquinoline-1,3(2H)-dione
(6a)
500 MHz) δ: 1.39 (m, 2H,), 1.51 (m, 2H), 1.85 (s, 3H), 1.94
(m, 2H), 2.11 (m, 2H), 2.76 (s, 6H), 3,19 (t, 2H, J = 6 Hz),
3.61 (m, 2H), 4.34 (t, 2H, J = 6 Hz), 6.74 (d, 1H, J = 9 Hz),
7.52 (dd, 1H, J = 7.5 and 8.5 Hz), 8.24 (d, 1H, J = 8.5 Hz),
8.39 (dd, 1H, J = 1 and 7.5 Hz), 8.49 (dd, 1H, J = 1 and 8.5
Hz). 13C-NMR (CD3OD, 125 MHz) δ: 22.7, 31.9, 32.3, 37.0,
44.6, 48.0, 52.6, 57.9, 105.5, 108.8, 121., 123.1, 125.4, 129.9,
131.6, 132.5, 136.3, 152.1, 165.7, 166.6, 172.6. HRMS (ESI)
m/z: Calc. for C24H30N4O3 ([M+H]+): 423.2390, Found:
423.2396.
Ethanolamine (52 µL, 0.86 mmol) was added to a
solution of the 6-bromo-naphthalimide 10a [48] (50 mg, 0.14
mmol) in EtOH (5 mL) and the mixture was heated at 120ºC
under MW irradiation for 3 h. Afterwards, the solvent was
evaporated to dryness and the residue was purified by
reverse C18 flash chromatography, using 0-30 % CH3CN in
H2O gradient as eluent, to give the desired naphthalimide 6a
as yellow syrup (11 mg, 20%). HPLC-MS (5-95% gradient of
1
A in B, 10 min) tR = 4.36 min. H-NMR (MeOD, 500 MHz) δ:
4.1.10. Synthesis of 6-(azetidin-1-yl)-2-(2-(dimethylamino)-
2.87 (s, 6H), 3.59 (t, 1H, J = 6 Hz), 3.88 (t, 1H, J = 6 Hz), 4.47
(t, 1H, J = 6 Hz), 6.86 (d, 1H , J = 8.5 Hz), 7.67 (dd, 1H, J =
7.5 and 8.5 Hz), 8.40 (d, 1H, J=8.5 Hz), 8.55 (m, 2H). 13C-
NMR (MeOD, 125 MHz) δ: 36.9, 44.6, 46.7, 58.0, 60.8,
105.3, 115.7, 122.0, 123.3, 125.6, 127.3, 129.7, 132.5, 136.3,
153.1, 165.9, 166.6. HRMS (ESI) m/z: Calc. for C18H21N3O3
([M+H]+): 328.1655, Found: 328.1661.
ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (8a)
Azetidine (75 µL, 1.08 mmol) was added to a solution of
the 6-bromo-naphthalimide 10a [48] (65 mg, 0.18 mmol) in
EtOH (5 mL) and the mixture was heated at 120ºC under MW
irradiation for 1.5 h. Afterwards, the solvent was evaporated to
dryness and the residue was purified by flash
chromatography, using 1-10% gradient of MeOH in CH2Cl2 as
eluent, to give the desired 6-(azetidin-1-yl)-naphthalimide 8a
as a yellow-orange solid (50 mg, 85 %). M.p. 152 ºC. HPLC-
MS (5-95% gradient of A in B, 10 min) tR = 5.25 min. 1H-NMR
(Cl3CD, 400 MHz) δ: 2.31 (s, 6H), 2.50 (p, 2H, J = 7.5 Hz),
2.61 (t, 2H, J = 7 Hz), 4.25 (t, 2H, J = 7 Hz), 4.41 (t, 4H, J =
7.5 Hz), 6.30 (d, 1H, J = 8.5 Hz), 7.42 (dd, 1H, J = 7 and 8.5
Hz), 8.14 (dd, 1H, J = 1 and 8.5 Hz), 8.29 (d, 1H, J = 8.5 Hz),
8.45 (dd, 1H, J = 1 and 7 Hz). 13C-NMR (CD3Cl, 100 MHz) δ:
17.3, 38.0, 45.9, 55.6, 57.2, 106.5, 110.4, 121.2, 122.9, 123.9,
130.3, 130.9, 131.4, 133.6, 152.8, 164.3, 165.0. HRMS (ESI)
m/z: Calc. for C18H20N4O2 ([M+H]+): 324.1580, Found:
324.1586.
4.1.8. Synthesis of 6-((trans-4-aminocyclohexyl)amino)-2-
(2-(dimethylamino)ethyl)-1H-benzo[de]-isoquinoline-1,3(2H)-
dione (12a)
trans-1,4-Diaminocyclohexane (80 mg, 1.90 mmol) and
DIPEA (26 µL, 0.46 mmol) were added to a solution of the 6-
bromo-naphthalimide 10a [48] (110 mg, 0.31 mmol) in EtOH
(5 mL) and the mixture was heated at 120ºC in a closed tube
for 2 days. Afterwards, the solvent was evaporated to dryness
and the residue was purified by reverse C18 flash
chromatography, using 0-15 % CH3CN in H2O gradient as
eluent, to give the desired naphthalimide 12a as dark yellow
solid (38 mg, 32%). M.p. 178 ºC HPLC-MS (30-95% gradient
of A in B, 10 min) tR = 4.5 min. 1H-NMR [(CD3)2CO, 400 MHz]
δ: 1.34, 1.52 and 2.15 (3m, 8H), 2.12 (s, 6H), 2.42 (t, 2H, J =
7 Hz), 3.26 (m, 1H), 3.66 (m, 1H), 4.09 (t, 2H, J = 7 Hz), 6.54
(d, 1H, J = 7.5 Hz), 6.82 (d, 1H, J = 8.5 Hz), 7.50 (t, 1H, J = 8
Hz), 8.23 (d, 1H, J = 8.5 Hz), 8.35 (d, 1H, J = 7.5 Hz), 8.47 (d,
1H, J = 8.5 Hz). 13C-NMR ((CD3)2CO, 100 MHz) δ: 30.0, 31.4
and 31.7, 36.9, 44.6, 51.0, 56.5, 58.0, 103.8, 108.5, 120.1,
122.3, 123.6, 127.1, 129.6, 130.0, 133.6, 149.0, 162.7, 163.5.
HRMS (ESI) m/z: Calc. for C22H28O2N4 ([M+Na]+): 403.2104,
Found: 403.2091.
4.1.11. Synthesis of 9-((trans-4-aminocyclohexyl)amino)-5-
(2-(dimethylamino)ethyl)-4H-benzo[de][2,6]-naphthyridine-
4,6(5H)-dione (12b)
trans-1,4-Diaminocyclohexane (25.3 mg, 33.5 μL, 0.6
mmol) and DIPEA (6 µL, 0.05 mmol) were added to a solution
of the 9-metoxi-quinolimide 2b (45 mg, 0.40 mmol) in EtOH (5
mL) and the mixture was heated at 150ºC in a closed tube for
7 days. Afterwards, the solvent was evaporated to dryness
and the residue was purified by flash chromatography, using
0-10 % MeOH in CH2Cl2 gradient as eluent, to give the
desired quinolimide 12b as red syrup (137 mg, 90%). HPLC-
9

MS (30-95% gradient of A in B, 10 min) tR = 4.5 min. 1H-NMR
[CD3OD, 400 MHz] δ: 1.44 (m, 4H)), 1.98 and 2.15 (2m, 4H),
2.25 (s, 6H), 2.54 (m, 2H), 2.87 (m, 1H), 3.58 (m, 1H), 4.15 (t,
2H, J = 7 Hz), 6.83 (d, 1H, J = 8.5 Hz), 8.12 (d, 1H, J = 4.5
Hz), 8.20 (d, 1H, J = 8.5 Hz), 8.83 (d, 1H, J = 4.5 Hz). 13C-
NMR (CD3OD, 100 MHz) δ: 31.6, 33.1, 45.8, 50.7, 51.8, 57.8,
106.2, 107.9, 123.9, 125.3, 130.5, 135.9, 137.7, 148.4, 133.6,
150.9, 164.5, 165.0.
where x and r denote the sample and standard, respectively,
A is the absorption at the excitation wavelength, I is the
integrated fluorescence intensity, and n is the refractive index
of the solvent.
4.3. HTS Cytotoxicity assays in tumor cells
A colorimetric assay, using the sulforhodamine B (SRB)
reaction, was adapted for a quantitative measurement of cell
growth and viability, following the technique described by
Skehan, P. A. et al. [52]. Cells (MDA-MB-231, A549, HT-29
and PSN1) were seeded in 96 well microtiter plates, at 5×103
cells per well in aliquots of 195 μL of RPMI medium, and they
were allowed to attach to the plate surface by growing in drug
free medium for 18 h. Afterwards, samples were added in
aliquots of 5 μL [dissolved in (3:7) DMSO/H2O]. After 48 h
exposure, cells were fixed by adding 50 μL of cold 50% (w/v)
trichloroacetic acid, and incubating at 4 ºC for 60 min. Then,
the plates were washed with deionized H2O and dried. 100 μL
of SRB solution (0.4% w/v in 1% acetic acid) was added to
each microtiter well and these were incubated at room
temperature for 10 min. Unbound SRB was removed by
washing with 1% acetic acid, the plates were air dried, and
the bound stain was solubilized with Tris buffer. Optical
densities were read on an automated spectrophotometer plate
reader at a single wavelength of 490 nm. Data analysis was
automatically generated by the high throughput screening
LIMS implemented at the laboratory. The three response
parameters GI50 (50% cell growth inhibition), LC50 (50% lethal
concentration), and TGI (total growth inhibition) were
extracted from concentration-response curves by linear
interpolation, according to the National Cancer Institute (NCI)
protocols [53].
4.1.12. Synthesis of N-(trans-4-((5-(2-(dimethylamino)ethyl)-
4,6-dioxo-5,6-dihydro-4H-benzo[de][2,6]-naphthyridin-9-
yl)amino)cyclohexyl)acetamide (7b)
Ac2O (1 ml) was added to a solution of the quinolimide
12b (7 mg, 0.018 mmol) in dry CH2Cl2 (1 mL), and the mixture
was stirred at room temperature for 1 h. Then, the solvent
was removed under vacuum and the residue was purified by
flash chromatography, using 0-5 % gradient of MeOH in
CH2Cl2 as eluent to give the acetylated derivative 7b as a red-
orange solid (5 mg, 65 %). M.p. 230 ºC. HPLC-MS (30-95%
gradient of A in B, 10 min) tR = 5.65 min. 1H-NMR (CD3OD,
500 MHz) δ: 1.38 and 1.50 (2m, 4H), 1.84 (s, 3H) 1.94 and
2.12 (2m, 4H), 2.48 (s, 6H), 2.86 (t, 2H, J = 6.5 Hz), 3.60 (m,
2H), 4.26 (t, 2H, J = 6.5 Hz), 6.86 (d, 1H, J = 8.5 Hz), 8.17 (d,
1H, J = 4.5 Hz), 8.25 (d, 1H, J = 8.5 Hz), 8.87 (d, 1H, J = 4.5
Hz). 13C- NMR (CD3OD, 125 MHz) δ 22.7, 30.7, 32.1, 37.8,
45.3, 52.0, 57.8, 64.3, 106.2, 107.6, 124.1, 125.5, 130.5,
136.2, 137.9, 148.4, 151.3, 164.9, 165.4, 172.6. HRMS (ESI)
m/z: Calc. for C23H29N5O3 ([M+H]+): 424.2343, Found:
424.2340.
4.2. Photophysical methods
Excitation and emission spectra of compounds were
determined for 12 μM solutions in solvents of diverse polarity.
The spectra were recorded between 300 and 690 nm (0.5 nm
increments and 0.1 s integration time) with excitation set at
the appropriate excitation wavelength. Slit widths were set to
15 nm for excitation and to 6 or 20 nm for emission,
depending on the observed emission intensity. All the spectra
were corrected for background fluorescence by subtracting a
blank scan of the solvent solution.
4.4. Macrophage culture and fluorescence visualization
Macrophages RAW 264.7 (ECACC, Sigma P11) were
used for testing our naphthalimide and quinolimide-based
fluorophores as imaging probes. The cells were cultured in
Dulbecco’s modified Eagle’s medium enriched with 100 mg/L
of sodium piruvate supplemented with 10% fetal bovine serum
(FBS), 200 mM L-glutamine, 100 u/mL penicillin and 100
μg/mL of streptomycin (complete medium). The cell culture
was maintained in humidified atmosphere at 37 ºC, with 5 %
of CO2 and 95 % of air. The morphology of the cells was
studied with an epifluorescence microscope Nikon TE-2000
and with the confocal Laser microscope (CLSM) Leica TCS
SP2 with 7 lines of laser (457, 477, 488, 496, 514, 543, 633
nm).
Fluorescence
quantum
yield
determination.
Fluorescence quantum yields (ΦF) were determined in
solvents of diverse polarity, and calculated with reference to a
standard, which was selected depending on the photophysical
properties of the corresponding fluorophore [49-51].
Coumarine 153 in EtOH (Φ = 0.53) was used as reference,
except for 1a and 2a,b that were calculated with reference to
quinine sulphate dihydrate in 0.1 M H2SO4 (Φ = 0.55). A 12
μM solution of the corresponding fluorophore was compared
to a 12 μM solution of the standard to assure that the
absorbance is less than 0.1 at identical excitation
wavelengths. The following equation was used to calculate
the quantum yield:
RAW 264.7 cells were grown to a density of 8 x 104
cells/mL in complete medium, in 24-well plates incubated for
24 hours. After this time, half of the growing wells were
treated with LPS (Polysaccharides of Escherichia coli, Sigma
Aldrich, Spain) at a concentration of 5 μg/mL for 2 h and,
then, treated with the fluorophores (5 μM). The other half of
the wells was treated directly with the flourophores at the
same concentration. The cells were left in the presence of the
Ix Ar n2 Φr
x
Φ =
AxIr n2
10
r

[6] K. Kalyanasundaram, J.K. Thomas, Solvent-dependent
fluorescence of pyrene-3-carboxaldehyde and its applications in
the estimation of polarity at micelle-water interfaces, J. Phys.
Chem., 81 (1977) 2176-2180.
fluorophores for 1 or 2 h. After this time, the cells were fixed
with glutaraldehyde (2.5% v/v in H2O) for 15 minutes. They
were washed with distilled water twice and mounted and
observed under the microscope. The images of cells treated
with our products were compared with images obtained with
the commercial nuclear fluorescent sensor DAPI (Invitrogen).
[7] J.S. Seixas de Melo, R.S. Becker, A.L. Macanita, Photophysical
behavior of coumarins as a function of substitution and solvent:
Experimental evidence for the existence of a lowest lying 1(n,π*)
state, J. Phys. Chem., 98 (1994) 6054-6058.
4.5. DNA Binding
[8] S. Singha, D. Kim, B. Roy, S. Sambasivan, H. Moon, A.S. Rao,
J.Y. Kim, T. Joo, J.W. Park, Y.M. Rhee, T. Wang, K.H. Kim, Y.H.
Shin, J. Jung, K.H. Ahn, A structural remedy toward bright
dipolar fluorophores in aqueous media, Chemical science, 6
(2015) 4335-4342.
The studies were carried out by means of titrations at a
constant concentration of fluorophore (16 μM for 7a and 20
μM for 7b), using UV-visible spectrometry and circular
dichroism (CD). The equipment used was the Jasco J-815
spectropolarimeter. Experimental conditions: 10 mM
phosphate buffer, pH = 7.10, T = 25 ° C, (200 mM NaCl only
in the titrations with the oligonucleotide). Inverse titrations, of
a 4.3 μM concentration of the oligonucleotide R-13 with
increasing concentrations of the fluorophores 7a and 7b (0-
430 μM) were also carried out.
[9] D. Singharoy, S. Chowdhury, S.S. Mati, S. Ghosh, K.
Chattopadhyay, S.C. Bhattacharya, Photoinduced electron
transfer switching mechanism of a naphthalimide derivative with
its solvatochromic behaviour: An experimental and theoretical
study with in cell investigations, Chem. - Eur. J., 23 (2017)
16516-16524.
[10] H. Zhu, J. Fan, H. Mu, T. Zhu, Z. Zhang, J. Du, X. Peng, d-PET-
controlled "off-on" polarity-sensitive probes for reporting local
hydrophilicity within lysosomes, Sci. Rep., 6 (2016) 35627.
[11] F. Fueyo-González, J.A. González-Vera, I. Alkorta, L. Infantes,
M.L. Jimeno, M. Fernández-Gutiérrez, M.C. González-García, A.
Orte, R. Herranz, Fluorescence mechanism switching from ICT
to PET by substituent chemical manipulation: Macrophage
cytoplasm imaging probes, Dyes and Pigments, 175 (2020)
108172.
Conflict of interest
The authors have no conflicts of interest to declare.
[12] J.B. Grimm, B.P. English, J. Chen, J.P. Slaughter, Z. Zhang, A.
Revyakin, R. Patel, J.J. Macklin, D. Normanno, R.H. Singer, T.
Lionnet, L.D. Lavis, A general method to improve fluorophores
for live-cell and single-molecule microscopy, Nat. Methods, 12
(2015) 244-250.
Acknowledgements
[13] A. Fernández, M. Vendrell, Smart fluorescent probes for imaging
macrophage activity, Chem. Soc. Rev., 45 (2016) 1182-1196.
The work was supported by grants SAF2012-32209,
FU2015-67284-R, CTQ2017-85658-R form the Spanish
Ministerio de Economía y Competividad, Agencia Estatal de
Investigación and the European Regional Development Fund;
and the CSIC grant 201580E073.
[14] Y. Li, T.-M. Liu, Discovering macrophage functions using In Vivo
optical imaging techniques, Front. Immunol., 9 (2018) 502/501-
502/520.
[15] J.S. Yoo, S.-C. Lee, Z.Y. Jow, P.Y.X. Koh, Y.-T. Chang, A
Macrophage-Specific Fluorescent Probe for Intraoperative
Lymph Node Staging, Cancer Res., 74 (2014) 44-55.
[16] K.A. Zachariasse, M. Grobys, T. von der Haar, A. Hebecker, Y.V.
Il'ichev, Y.B. Jiang, O. Morawski, W. Kühnle, Intramolecular
charge transfer in the excited state. Kinetics and configurational
changes, J.Photochem. Photobiol., A, 102 (1996) 59-70.
Supplementary Material
Supplementary material to this article can be found online
at
[17] C.J. Joedicke, H.P. Luethi, Time-dependent density functional
theory (TDDFT) study of the excited charge-transfer state
formation of a series of aromatic donor-acceptor systems, J. Am.
Chem. Soc., 125 (2003) 252-264.
References
[1] G.S. Loving, M. Sainlos, B. Imperiali, Monitoring protein
interactions and dynamics with solvatochromic fluorophores,
Trends Biotechnol., 28 (2010) 73-83.
[18] S. Banerjee, E.B. Veale, C.M. Phelan, S.A. Murphy, G.M. Tocci,
L.J. Gillespie, D.O. Frimannsson, J.M. Kelly, T. Gunnlaugsson,
Recent advances in the development of 1,8-naphthalimide based
DNA targeting binders, anticancer and fluorescent cellular
imaging agents, Chem. Soc. Rev., 42 (2013) 1601-1618.
[2] Z. Yang, J. Cao, Y. He, J.H. Yang, T. Kim, X. Peng, J.S. Kim,
Macro-/micro-environment-sensitive
biological imaging, Chem. Soc. Rev., 43 (2014) 4563-4601.
chemosensing
and
[19] M.F. Braña, J.M. Castellano, C.M. Roldan, A. Santos, D.
Vazquez, A. Jimenez, Synthesis and mode(s) of action of a new
series of imide derivatives of 3-nitro-1,8-naphthalic acid, Cancer
Chemother. Pharmacol., 4 (1980) 61-66.
[3] A.S. Klymchenko, Y. Mely, Fluorescent environment-sensitive
dyes as reporters of biomolecular interactions, Prog. Mol. Biol.
Transl. Sci., 113 (2013) 35-58.
[4] A.S. Klymchenko, Solvatochromic and Fluorogenic Dyes as
[20] M.F. Braña, A.M. Sanz, J.M. Castellano, C.M. Roldan, C. Roldan,
Synthesis and cytostatic activity of benz[de]isoquinoline-1,3-
diones. Structure-activity relationships, Eur. J. Med. Chem., 16
(1981) 207-212.
Environment-Sensitive
Probes:
Design
and
Biological
Applications, Acc. Chem. Res., 50 (2017) 366-375.
[5] A. Kellmann, Intersystem crossing and internal conversion
quantum yields of acridine in polar and nonpolar solvents, J.
Phys. Chem., 81 (1977) 1195-1198.
[21] M. Chau, J.L. Christensen, A.M. Ajami, R.L. Capizzi, Amonafide,
a topoisomerase II inhibitor, is unaffected by P-glycoprotein-
mediated efflux, Leuk. Res., 32 (2008) 465-473.
11

[22] Y.H. Hsiang, J.B. Jiang, L.F. Liu, Topoisomerase II-mediated
DNA cleavage by amonafide and its structural analogs, Mol.
Pharmacol., 36 (1989) 371-376.
imaging with organelle-specific emission, J. Mater. Chem. B, 4
(2016) 2614-2619.
[40] X. Ma, R. Sun, J. Cheng, J. Liu, F. Gou, H. Xiang, X. Zhou,
[23] M.F. Braña, A. Gradillas, Á. Gómez, N. Acero, F. Llinares, D.
Muñoz-Mingarro, C. Abradelo, F. Rey-Stolle, M. Yuste, J.
Campos, M.A. Gallo, A. Espinosa, Synthesis, biological activity,
and quantitative structure-activity relationship study of
azanaphthalimide and arylnaphthalimide derivatives, J. Med.
Chem., 47 (2004) 2236-2242.
Fluorescence
aggregation-caused
quenching
versus
aggregation-induced emission: A visual teaching technology for
undergraduate chemistry students, J. Chem. Educ., 93 (2016)
345-350.
[41] A. Schreer, C. Tinson, J.P. Sherry, K. Schirmer, Application of
Alamar blue/5-carboxyfluorescein diacetate acetoxymethyl ester
as a noninvasive cell viability assay in primary hepatocytes from
rainbow trout, Anal. Biochem., 344 (2005) 76-85.
[24] P. Gao, W. Pan, N. Li, B. Tang, Fluorescent probes for organelle-
targeted bioactive species imaging, Chem. Sci., 10 (2019) 6035-
6071.
[42] N.J. Buurma, I. Haq, Calorimetric and spectroscopic studies of
Hoechst 33258: Self-association and binding to non-cognate
DNA, J.Mol. Biol., 381 (2008) 607-621.
[25] H. Zhu, J. Fan, J. Du, X. Peng, Fluorescent Probes for Sensing
and Imaging within Specific Cellular Organelles, Acc. Chem.
Res., 49 (2016) 2115-2126.
[43] L. Hahn, N.J. Buurma, L.H. Gade,
A
Water-soluble
[26] W. Ma, S. Zhang, Z. Tian, Z. Xu, Y. Zhang, X. Xia, X. Chen, Z.
Liu, Potential anticancer agent for selective damage to
mitochondria or lysosomes: Naphthalimide-modified fluorescent
biomarker half-sandwich iridium (III) and ruthenium (II)
complexes, Eur. J. Med. Chem., 181 (2019) 111599.
tetraazaperopyrene dye as strong G-quadruplex DNA binder,
Chem. Eur. J., 22 (2016) 6314-6322.
[44] P. Thordarson, Determining association constants from titration
experiments in supramolecular chemistry, Chem. Soc. Rev., 40
(2011) 1305-1323.
[27] H. Yu, Y. Xiao, L. Jin, A Lysosome-Targetable and Two-Photon
Fluorescent Probe for Monitoring Endogenous and Exogenous
Nitric Oxide in Living Cells, J. Am. Chem. Soc., 134 (2012)
17486-17489.
[45] J.B. Chaires, Analysis and interpretation of ligand-DNA binding
isotherms, Methods in Enzymol., 340 (2001) 3-22.
[46] E.C. Long, J.K. Barton, On demonstrating DNA intercalation, Acc.
Chem. Res., 23 (1990) 271-273.
[28] L. He, Y. Li, C.-P. Tan, R.-R. Ye, M.-H. Chen, J.-J. Cao, L.-N. Ji,
Z.-W. Mao, Cyclometalated iridium(iii) complexes as lysosome-
targeted photodynamic anticancer and real-time tracking agents,
Chem. Sci., 6 (2015) 5409-5418.
[47] P.H. Grayshan, A.M. Kadhim, A.T. Peters, Heterocyclic
derivatives of naphthalene-1,8-dicarboxylic anhydride. III.
Benzo[k,l]thioxanthene-3,4-dicarboximides,
Chem., 11 (1974) 33-38.
J.
Heterocycl.
[29] W. Ma, L. Guo, Z. Tian, S. Zhang, X. He, J. Li, Y. Yang, Z. Liu,
Rhodamine-modified fluorescent half-sandwich iridium and
ruthenium complexes: potential application as bioimaging and
anticancer agents, Dalton Trans., 48 (2019) 4788-4793.
[48] A. Pardo, E. Martin, J.M.L. Poyato, J.J. Camacho, J.M. Guerra,
R. Weigand, M.F. Braña, J.M. Castellano, N-Substituted 1,8-
naphthalimide derivatives as high efficiency laser dyes, J.
Photochem. Photobiol., A, 48 (1989) 259-263.
[30] J. Li, Z. Tian, Z. Xu, S. Zhang, Y. Feng, L. Zhang, Z. Liu, Highly
potent half-sandwich iridium and ruthenium complexes as
lysosome-targeted imaging and anticancer agents, Dalton
Trans., 47 (2018) 15772-15782.
[49] C. Würth, M. Grabolle, J. Pauli, M. Spieles, U. Resch-Genger,
Relative and absolute determination of fluorescence quantum
yields of transparent samples, Nat. Protoc., 8 (2013) 1535-1550.
[31] W. Ma, X. Ge, L. Guo, S. Zhang, J. Li, X. He, Z. Liu,
Bichromophoric anticancer drug: Targeting lysosome with
rhodamine modified cyclometalated Iridium(III) complexes, Dyes
and Pigments, 162 (2019) 385-393.
[50] A.M. Brouwer, Standards for photoluminescence quantum yield
measurements in solution (IUPAC technical report), Pure Appl.
Chem., 83 (2011) 2213-2228.
[51] K. Rurack, M. Spieles, Fluorescence Quantum Yields of a Series
of Red and Near-Infrared Dyes Emitting at 600-1000 nm, Anal.
Chem., 83 (2011) 1232-1242.
[32] S. Uchiyama, K. Takehira, T. Yoshihara, S. Tobita, T. Ohwada,
Environment-sensitive
fluorophore
emitting
in
protic
environments, Org. Lett., 8 (2006) 5869-5872.
[52] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D.
Vistica, J.T. Warren, H. Bokesch, S. Kenney, M.R. Boyd, New
colorimetric cytotoxicity assay for anticancer-drug screening, J.
Natl. Cancer Inst., 82 (1990) 1107-1112.
[33] M.M. Yusupov, G.Z. Yusupova, A. Baucom, K. Lieberman, T.N.
Earnest, J.H.D. Cate, H.F. Noller, Crystal structure of the
ribosome at 5.5 Å resolution, Science, 292 (2001) 883-896.
[34] R.M. Martin, H. Leonhardt, M.C. Cardoso, DNA labeling in living
cells, Cytometry, Part A, 67A (2005) 45-52.
[53] R.H. Shoemaker, The NCI60 human tumour cell line anticancer
drug screen, Nat. Rev. Cancer, 6 (2006) 813-823.
[35] R.W. Dirks, H.J. Tanke, Styryl molecules light-up RNAs, Chem.
Biol., 13 (2006) 559-560.
[36] G. Song, Y. Sun, Y. Liu, X. Wang, M. Chen, F. Miao, W. Zhang,
X. Yu, J. Jin, Low molecular weight fluorescent probes with good
photostability for imaging RNA-rich nucleolus and RNA in
cytoplasm in living cells, Biomaterials, 35 (2014) 2103-2112.
[37] N. Stevens, N. O’Connor, H. Vishwasrao, D. Samaroo, E.R.
Kandel, D.L. Akins, C.M. Drain, N.J. Turro, Two color RNA
intercalating probe for cell imaging applications, J. Am. Chem.
Soc., 130 (2008) 7182-7183.
[38] N.A. O'Connor, N. Stevens, D. Samaroo, M.R. Solomon, A.A.
Marti, J. Dyer, H. Vishwasrao, D.L. Akins, E.R. Kandel, N.J.
Turro, A covalently linked phenanthridine-ruthenium(II) complex
as a RNA probe, Chem. Commun. , (2009) 2640-2642.
[39] C.Y.Y. Yu, W. Zhang, R.T.K. Kwok, C.W.T. Leung, J.W.Y. Lam,
B.Z. Tang, A photostable AIEgen for nucleolus and mitochondria
12

Highlights
•
•
New naphthalimide and quinolimide-based fluorophores are described
Properties of naphthalimide-based fluorophores have been tuned by substituent
manipulation
•
DNA intercalating properties provide macrophage nucleus fluorescence imaging

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: