Synthesis of fluorogenic arylureas and amides and their interaction with amines: A competition between turn-on fluorescence and organic radicals on theway to a smart label for fish freshness

Article abstract of DOI:10.3390/molecules26051404

We describe the synthesis of fluorogenic arylureas and amides and their interaction with primary or secondary amines under air and light in organic-aqueous mixtures to give rise to a new class of persistent organic radicals, described on the basis of thei

Full text of DOI:10.3390/molecules26051404

molecules
Article
Synthesis of Fluorogenic Arylureas and Amides and Their
Interaction with Amines: A Competition between Turn-on
Fluorescence and Organic Radicals on the Way to a Smart Label
for Fish Freshness
Javier García-Tojal , José V. Cuevas , María-Josefa Rojo, Borja Díaz de Greñu, Carla Hernando-Muñoz,
José García-Calvo, Mateo M. Salgado and Tomás Torroba *
Department of Chemistry, Faculty of Science, University of Burgos, 09001 Burgos, Spain; qipgatoj@ubu.es (J.G.-T.);
jvcv@ubu.es (J.V.C.); mjrocam@ubu.es (M.-J.R.); bdpuertas@ubu.es (B.D.d.G.); chm1001@alu.ubu.es (C.H.-M.);
jgcalvo@ubu.es (J.G.-C.); mmsalgado@ubu.es (M.M.S.)
* Correspondence: ttorroba@ubu.es
Abstract: We describe the synthesis of fluorogenic arylureas and amides and their interaction with
primary or secondary amines under air and light in organic-aqueous mixtures to give rise to a new
class of persistent organic radicals, described on the basis of their electron paramagnetic resonance
(EPR), as well as UV–vis, fluorescence, NMR, and quantum mechanics calculations, and their
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
prospective use as multi-signal reporters in a smart label for fish freshness.
Citation: García-Tojal, J.; Cuevas, J.V.;
Rojo, M.-J.; Díaz de Greñu, B.;
Keywords: arylureas; turn-on fluorescence; organic radicals; smart label; volatile amines
Hernando-Muñoz, C.; García-Calvo,
J.; Salgado, M.M.; Torroba, T.
Synthesis of Fluorogenic Arylureas
and Amides and Their Interaction
1. Introduction
with Amines: A Competition
between Turn-on Fluorescence and
Organic Radicals on the Way to a
Smart Label for Fish Freshness.
Molecules 2021, 26, 1404. https://
doi.org/10.3390/molecules26051404
Persistent organic radicals are of immense importance for the preparation of new
hybrid materials with spin-probe and spin-label functions [ ], magnetic materials [ ], elec-
trical transport [ ], molecular spintronics [ ], and surface-confined electroactive molecules
for single-molecule switching devices [ ], and they remain integral in classical areas such as
catalysis for the oxidation of organic molecules [ ] and DNA damage [ ]. Persistent radical
cations [ ] and radical anions [ ], examples of organic mixed-valence compounds [10], are
1
2
3
4
5
6
7
8
9
Academic Editor: Narciso M. Garrido
also important, along with their neutral organic radical parents, for the discovery of new
organic materials and the development of improved devices. Amines and their derivatives
are extensively used as chemicals in abuse drugs, cosmetics, polymers, etc. [11]. They are
also important markers in food industries and medical diagnosis, as they are involved
in many biological processes, including as neurotransmitters [12]. The detection and dis-
crimination of amines are important issues in environmental protection and human health.
Common protocols for amine analyses are based on chromatographic methods [13], which
are quite time-consuming; therefore, the development of fluorescent sensors for amines
has become a desirable alternative given their simple instrumentation and fast response
Received: 9 February 2021
Accepted: 2 March 2021
Published: 5 March 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
time. Although fluorescent sensors lack selectivity in some applications [14–18], the use
of multiple-signal sensors can remediate this adverse selectivity. Herein, we present an
unusual family of new fluorogenic aminoaryl-indane-based probes that are also able to
form persistent radical species in the presence of primary or secondary amines, and we
describe their prospective use as multiple-signal fluorogenic reporters for the detection of
volatile amines.
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
2. Results and Discussion
The synthesis of arylureas used in this study was performed using an easy Suzuki re-
action between 5-bromoindanone and 4-aminobenzeneboronic acid pinacol ester, followed
by a Knoevenagel reaction with malononitrile and a reaction with selected mono- and
Molecules 2021, 26, 1404. https://doi.org/10.3390/molecules26051404
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1404
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bis-isocyanates (Scheme 1). To compare their fluorogenic properties, selected bis-amides
were also prepared in a straightforward reaction of the starting aminoindane derivative
and acid chlorides (Scheme 1).
Scheme 1. Synthesis of the chemical probes.
In this way, chemical probes 1–7 were prepared (Figure 1). Probe 1 was previously
reported as an excellent fluorogenic sensor for the determination of 3,4-methylenedioxym-
ethamphetamine (MDMA) from ecstasy tablets [19]. The interaction of primary and sec-
ondary amines with the fluorogenic probe was the key step in their selective discrimination
from tertiary amines, anilines, and heterocyclic amines. We observed that the presence
of primary or secondary biogenic amines initially promoted a sudden fluorescence in the
probe, which was followed by gradual emission extinction.
The kinetics of the process was specific for every reported amine, but the reason for
the emission extinction was unclear until we noticed that it was caused by the presence of
radical species generated after the probe/amine interaction. We selected a typical amine
derivative, pyrrolidine, and studied the interaction of pyrrolidine and
1 using titration
experiments monitored by electron paramagnetic resonance (EPR), as well as by NMR, UV–
vis and fluorescence spectroscopy, in order to understand the type of interaction involved
in the generation of the radical species. First, the effect of the addition of pyrrolidine to
an equimolecular solution of bis-diarylurea 1 in dimethylsulfoxide (DMSO) was studied
by X-band EPR measurements at room temperature under ambient light and air. Time-
dependent absorption was detected after faint discoloration of the dark orange solution
of 1. The signal was sensitive to the solvents used. The low solubility of 1 compelled
us to use DMSO, the only common solvent in which this urea is moderately soluble, but
a continuous decrease in the intensity of the EPR signal from minute 5 was observed if
DMSO was used as the only solvent. However, the radical life-time increased with the
addition of water; therefore, aqueous mixtures were used for the measurements. In some of
the experiments, acetone was added in an attempt to improve the resolution by decreasing
the viscosity of the solvent mixture, but no highly significant improvement was attained.
As a typical example, Figure 2 shows the evolution of the EPR spectra over time for a
mixture of
a mixture prepared from 100
of water, 100 L of acetone, and 100
by the addition of 100
L of a 10−2 M solution of pyrrolidine in DMSO. After shaking,
200 L of the non-deoxygenated mixture was pipetted into a flat cell. A spectrum with
1
and pyrrolidine at room temperature. The measurements were performed on
L of 10−2 M stock solution of
in DMSO diluted with 100
L of DMSO. The mixture was stirred for 1 h, followed
µ
1
µL
µ
µ
µ
µ
18 equally spaced lines was registered at room temperature, and its intensity increased for
the first 11–12 min and then decreased to disappearance within the next 40 h (Figure 2).

Molecules 2021, 26, 1404
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O
O
O
O
NH
NH
HN
HN
O
HN
NH
N
NH
HN
NH
HN
O
O
CN NC
CN
NC
CN NC
CN
NC
NC
CN
NC
CN
6
1
2
CN
CN
N
N
CN
NC
NC
NC
N
O
O
NH
HN
NH
NH
NH
NH
NH
NH
O
O
O
O
S
N
CN
NC
CN
NC
3
7
5
4
Figure 1. Chemical structures of the studied chemical probes.
dχ'' / dH
Intensity(5 min)
Intensity(11 min)
Intensity(17 min)
Intensity(24 min)
Intensity(30 min)
Intensity(36 min)
Intensity(43 min)
Intensity(16 h)
Intensity(40 h)
348
349
H (mT)
Figure 2. Electron paramagnetic resonance (EPR) spectral evolution with time for an equimolecular
solution of and pyrrolidine. Experimental details of the data collection: modulation amplitude
0.1 mT, time constant 40.96 ms, conversion time 327.68 ms, gain 6.32
104, power 20 mW, and
1
×
microwave frequencies 9.7410 (5 min), 9.7410 (11 min), 9.7410 (17 min), 9.7410 (24 min), 9.7460
(30 min), 9.7410 (36 min), 9.7410 (43 min), 9.7410 (16 h), and 9.7760 (40 h) GHz.
Once the experimental conditions were optimized, the best protocol consisted in
preparing an orange mixture containing 100
which was diluted by adding 100 L of water and 200
followed by the addition of 100
L of a 10−2 M solution of pyrrolidine in DMSO, at which
moment the color of the mixture slightly clarified. After shaking, 200 L of the final solution
µ
L of 10−2 M stock solution of
1
in DMSO,
µ
µL of DMSO and stirred for 2 h,
µ
µ

Molecules 2021, 26, 1404
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(2
×
10−3 M in DMSO/water 4:1 v/v) was pipetted into a flat cell, where the measurements
started 11 min after the addition of the amine. The obtained spectrum is shown in Figure 3
.
The best fit (correlation coefficient R = 0.9930) was reached when taking into account the
hyperfine coupling of the unpaired electron to two non-equivalent 14N nitrogen nuclei
1
(I = 1) and three sets of non-equivalent H hydrogen atoms (I = 1/2). The parameters
arising from the fit were g = 2.0035, AN1 = 0.180 mT (1N, 1.68 10−4 cm−1), AN2 = 0.060 mT
(1N, 0.56
×
10−4 cm−1), AH1 = 0.260 mT (1H, 2.−434
×
10−4 cm−1), AH2 = 0.120 mT (2H,
1.12 × 10−4 cm−1), AH3 = 0.060 mT (2H, 0.56
dχ'' / dH
×
10 cm−1), and line-width
∆
H = 0.055 mT.
Experimental
Fit
H (mT)
348
349
•
Figure 3. Experimental (blue line) and simulated (dashed red line) EPR spectra of radical [1]
in dimethylsulfoxide (DMSO)/water. Experimental details: modulation amplitude 0.02 mT, time
constant 40.96 ms, conversion time 327.68 ms, gain 6.32
frequency 9.7768 GHz.
×
104, power 20 mW, and microwave
The use of n-butylamine instead of pyrrolidine yielded a similar spectrum. A com-
parison between the EPR spectra of after the addition of pyrrolidine or n-butylamine is
1
given in Figure 4; the experimental conditions were the same as those reported for previous
pyrrolidine experiments, but the microwave frequency in the case of n-butylamine was
9.7750 GHz. An equimolecular solution of
to a very weak isotropic signal with a non-resolved hyperfine structure (g
0.500 mT (Figure S29 in Supporting Information). In the case of the interaction of
tetrabutylammonium cyanide, signals weaker than those obtained from and pyrrolidine
1
and DABCO in similar conditions gave rise
2.0035, H =
with
≈
∆
1
1
were registered, but they showed the same hyperfine pattern (Figure S30 in Supporting
Information). On the contrary, a very weak and unresolved initial signal was obtained
when aqueous NaOH (1 M) solution was added (Supporting Information). After one
day, this signal evolved to an intense isotropic Gaussian-type signal (g = 2.0029,
∆H =
0.580 mT), and the color of the solution became dark green (Figure S31 in Supporting
Information). The observed radical species are loosely related to stable radicals formed
from the decomposition of azobisisobutyronitrile (AIBN) [20,21], in which stabilization by
a capto-dative effect may be involved [22–25].
The EPR results proceeded in parallel to the reported fluorescent results for 1, which
was only sensitive to the presence of primary and secondary amines and generated an
intense fluorescent signal, but it was not sensitive to the presence of tertiary amines, with
the exception of a weak sensitivity to DABCO [19]. We then recorded the EPR spectra of
bis(aryl-benzyl)urea
, and diamides
2
(a fluorescent molecular ruler for
ω-amino acids) [26], bis-arylureas
3
–
5
6
and (the central diamide linker was used in fluorescent polyamide
7
chemical probes) [27] in the presence of 1 equiv. of pyrrolidine in the same conditions. In
spite of their low intensities and deficient resolutions, the measurements performed on
those bis- and mono-ureas suggested the attainment of the same EPR signal with different
intensities, with the most intense signals related to bis-urea systems in the order
1 >> 2 > 3

Molecules 2021, 26, 1404
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≈ 4 ≈ 5, indicating that bis-aryl-urea
than bis-benzylurea
However, no signals in the EPR spectra were obtained in the experiments with bis-amides
under the same experimental conditions; therefore, they were not further studied
1
produced a more intense and better resolved signal
2, and both produced more intense signals than mono-arylureas 3–5.
6–7
(Figure 5).
dχ'' / dH
pyrrolidine
butylamine
2.006
2.002
g
−3
10 M in DMSO/water
Figure 4. EPR spectra for the interaction of an equimolecular solution (2
4:1 v/v) of and pyrrolidine or n-butylamine. The measurements started at 20 min (pyrrolidine) and
6 min (n-butylamine) after the addition of the amine. Experimental details of the data collection:
modulation amplitude 0.01 mT, time constant 40.96 ms, conversion time 327.68 ms, gain 6.32
104,
×
1
×
and power 20 mW in all cases. Microwave frequencies were 9.7718 (pyrrolidine, three scans) and
9.7713 GHz (n-butylamine, four scans).
dχ'' / dH
1
2
3
4
5
6
7
g
2.006
2.002
Figure 5. A comparison of the EPR spectra of
solutions in DMSO/water 4:1 v/v). The measurements were carried out 11 min after the addition of
1
–
7
with pyrrolidine (2
×
10−3 M equimolecular
the amine. Experimental details of the data collection: modulation amplitude 0.02 mT, time constant
40.96 ms, conversion time 327.68 ms, gain 6.32
Microwave frequencies were 9.7732 (1), 9.7726 (2), 9.7724 (3), 9.7727 (4), 9.7716 (5), 9.7713 (6), and
×
104, power 20 mW, and three scans in all cases.
9.7715 GHz (7).
EPR intensity changes were accompanied by changes in the color of the solution,
from orange to pale yellow, after the addition of the amine to solutions. Therefore, we
performed UV–vis and fluorescence titrations with and pyrrolidine as a representative
1–5
1
case under the same conditions, generating changes in color and an intense fluorescent
signal in either mixed solvents, DMSO/water/acetone, or pure DMSO, which was followed
by kinetic measurements (see Figures S36–S41 in Supporting Information). The addition

Molecules 2021, 26, 1404
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of up to several equivalents of pyrrolidine in DMSO to a solution of
1
(5.0
×
10−5 M) in
DMSO at room temperature caused the appearance of a UV–vis absorption band centered
at 305 nm and the disappearance of the absorption band centered at 405 nm (Supporting
Information). In the interval between 0 and 1 equiv. of the amine, an isosbestic point at
336 nm was observed. During the titration experiment, we did not observe bands in the
650–900 nm region of the absorption spectrum, which are characteristics of the formation of
radical anion species [28,29], so their presence was excluded. UV–vis titration experiments
in a DMSO/water 4:1 (v/v) mixture produced similar results. We calculated the binding
constants from UV–vis and fluorescence titrations (Supporting Information), which were in
agreement with those previously reported for biogenic amines [19]. Therefore, the behavior
of 1 in the presence of pyrrolidine was similar to that observed for related amines, so we
assumed that the radical species was formed in the presence of all reported primary or
secondary amines.
The evolution of the radical species was followed by 1H and 13C NMR spectroscopy
of equimolecular solutions (2.5
1H NMR spectrum of
is shown in Figure 6 in blue. The purple spectrum in Figure 6 was
recorded immediately after the addition of pyrrolidine, which shows upfield displacements
in one of the NH peaks near 9.0, the closest aromatic proton signal at 8.25 (the aromatic
proton signals between 7.5 and 8.0 also underwent upfield changes), and downfield
displacement of one of the methylene groups at 3.2, indicated by the blue and purple
×
10−3 M) of
1 and pyrrolidine in deuterated DMSO. The
1
δ
δ
δ
δ
arrows, but the resolution of the spectrum did not change, indicating only hydrogen bond
interactions. The green spectrum of the same sample in Figure 6 was recorded after 8 h, in
which the NH protons produced a broad signal near 9 ppm, all aromatic protons between
δ
7.3 and 8.3 produced a large unresolved signal between
δ 8 and 7.6, and the methylene
signals appeared as large multiplets between 3 and 3.5, indicating a strong perturbation
δ
of the resolution (green arrows). The signals at
δ
7.5 and 7 did not undergo large changes.
8 h
O
HN
HN
CN
NC
NC
+
NH
O
1 min
CN
HN
O
HN
1
Figure 6. Evolution of the H NMR spectrum of equimolecular solutions (2.5
pyrrolidine in DMSO-d6.
×
10−3 M) of
1 +
The 13C NMR spectrum of
Figure 7 was recorded 8 h after the addition of pyrrolidine, which shows the disappearance
of the signals at 180 and 70 (corresponding to the exocyclic double bond), of the two peaks
at around 114 (corresponding to the two cyano groups), and of the methylene signal near
30. These changes are indicated by the blue and purple arrows. The spectrum also shows
the appearance of a new cyano group signal at 109, indicated by the red arrow. The signal
near 155 diminished, and the rest of the aromatic carbon signals between 150 and 123
experienced several changes, but the signals between 120 and 118 remained unchanged.
1 is shown in Figure 7 in blue. The purple spectrum in
δ
δ
δ
δ
δ
δ
δ
Apparently, the bis-aryl ether moiety was not affected by the presence of the radical species
1
in either H NMR or 13C NMR spectra, and all changes were in the arylindane moiety.

Molecules 2021, 26, 1404
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The most important changes affected the exocyclic dicyanovinyl group and the closest
methylene group, indicating the main positions where the radical species was localized.
The signals corresponding to the pyrrolidine group remained unchanged in all spectra.
We hypothesized that the addition of an electron to the cyanovinyl moiety produced
a weakening of the triple bond that thus accumulated negative charge on the nitrogen,
which acquired a more C=N character, a structure that can be stabilized by the capture of
hydrogen, or a C=C=N··H group in the presence of water. Both IR and NMR data pointed
to the weakening of the C≡N bond, in agreement with this hypothesis.
8 h
Figure 7. Evolution of the 13C NMR spectrum of equimolecular solutions (2.5
pyrrolidine in DMSO-d6.
×
10−3 M) of
1 +
A structure featuring all characteristics was subjected to quantum chemical calcula-
tions, as shown in Figure 8. The geometric structure of [1]• was optimized by using the
Becke three-parameter functional [30] and the Lee–Yang–Parr correlation functional [31
]
(B3LYP) at the 6-31G* basis set for all the elements, implemented in the Gaussian 03 (Revi-
sion C.02) program suite [32]. The unrestricted protocol was used for open-shell systems.
From the energy-optimized structures, the singly occupied molecular orbital (SOMO) is
represented in Figure 7. The SOMO is centered on the dicyanovinyl moiety and displays a
π
-antibonding character located over the C=N bond of the protonated nitrogen and on the
1-indan carbon atom and the aryl group, as well as -bonding over the indane aryl group.
The molecular orbital has slight contributions from -bonding interactions between the
π
σ
hydrogen atom and the ketenimine nitrogen atom, as well as the hydrogen atoms from the
alpha-methylene carbon atom of the five-membered ring. The large electronic delocaliza-
tion seems to be responsible for the high stability and long life of the radical. SOMO + 1 was
located on the arm of the molecule that was not affected by the formation of the radical. It
mainly has a
and a -antibonding character in the nitrile groups. Nevertheless, this orbital has a slight
π-bonding character in the C–C interactions of the nitrile groups and the 1-indane carbon
atom with the aryl fragment. SOMO + 1 was 2.03 eV (195.65 kJ mol−1, 46.76 kcal mol−1
higher in energy than SOMO. SOMO 1 is a -bonding molecular orbital delocalized over
the phenyl rings directly bonded to the ether oxygen atom and with slight -antibonding
interactions with a p orbital of this oxygen atom and with p orbitals of the urea nitrogen
atoms. SOMO
1 was 1.03 eV (99.24 kJ mol−1, 23.72 kcal mol−1) lower in energy than
π-antibonding character located over the aryl fragment near the nitrile groups
π
)
−
π
π
−
SOMO. The energy gap between SOMO and SOMO + 1 was within the visible light region,
1.55–3.26 eV (150–315 kJ mol−1, 35.85–75.29 kcal mol−1); therefore, light excitation and
proton transfer may delocalize the radical between the two arylindane moieties. This fact
explains why in 1H/13C NMR, both arylindane groups seem to be affected to the same
extent by the formation of the radical species. The interaction must be intramolecular.

Molecules 2021, 26, 1404
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Indeed, a mixture of
(the amine precursor of
an 8-hour 13C NMR recording. The fact that SOMO
arylether moiety explains the fact that this group appeared unchanged in all spectroscopic
experiments. Although the diarylether group seems to have little influence on the stability
of the radical species (there is no electronic interaction), this link seems to be optimal
for electron delocalization between the two indane moieties. An electronically isolated
1
, pyrrolidine, and 5-(4-aminophenyl)-1-dicyanomethyleneindane
1
) in deuterated DMSO showed vinyl signals at
δ 180 and 70 after
−
1 was localized exclusively at the di-
link such as the m-xylyl group in
2 produced a much weaker radical species in similar
conditions, and none of the conjugated bis-amide links, such as those in
radical species in the presence of pyrrolidines.
6–
7
, produced
SOMO − 1
SOMO + 1
SOMO
[1]•
•
Figure 8. Singly occupied molecular orbital (SOMO), SOMO + 1, and SOMO
−
1 for compound [
1
] .
From previous studies and the new experiments, we assumed that compound
1 was
a non-fluorescent compound, in contrast to fluorescent compound , because of a charge
2
transfer from the diarylether moiety to the fluorescent arylindane groups. The addition
of primary or secondary amines such as pyrrolidine or n-butylamine inhibited the charge
transfer by forming hydrogen bonds, thus giving rise to a fluorescent intermediate that
underwent electron transfer [33] from the amine group to the indane moiety, facilitated by
simultaneous proton transfer from water [34], giving rise to radical species characterized
using several spectroscopic techniques (Figure 9). A quick inspection of the evolution of
fluorescence (left) and paramagnetic signals (EPR, right) in Figure 9 clearly shows that
the extinction of fluorescence evolved in parallel to the increase in the EPR signal, so the
asymptotic minimum fluorescence was reached at roughly the same time as the maximum
EPR signal. We only observed the stable radical species [1
]•, and therefore, the radical
species that could form from the amine were reversed in the presence of water and air
compared to the neutral amine, which appeared unchanged in all NMR experiments.
These chemical probes are the first examples of multi-signal reporters for the discrimi-
nation between primary, secondary, and tertiary amines in which the usual UV–visible or
fluorescent signal is complemented by paramagnetic signals of the same samples at differ-
ent reporting times. The selectivity of the chemical probes for different types of amines
and the large types of signals offered by these probes, now including paramagnetic species,
will make them good starting points for the preparation of multi-signaling chemical probes
for the detection of biogenic amines such as histamine, putrescine, and cadaverine. These
amines were of particular interest due to their characteristics as markers for freshness in
fish samples. To compare the different increases in fluorescence, the quantum yield of 1 was
measured in DMSO in the presence of pyrrolidine, used as a reference, and then histamine,
putrescine, or cadaverine, immediately after the addition of the amine. The reference was
quinine sulfate in H2SO4 (0.05 M) (Figure 10). The results were very similar for all tested
amines, with pyrrolidine being a bit higher than the rest of the amines within the error of
the method (±0.02). In all cases, there was quite a noticeable fluorescence increase.

Molecules 2021, 26, 1404
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18,000
17,000
16,000
15,000
14,000
13,000
12,000
11,000
10,000
3500
3000
2500
2000
1500
1000
500
0
0
500
1000
1500
2000
2500
3000
0
500
1000
1500
2000
2500
3000
Time (s)
Time (s)
Figure 9. (Top) A summary of the formation and evolution of [
measured (black line) and fitting curve (red line) for the fluorescence intensity decay process of a 10:1
pyrrolidine/ solution, concentration C1= 5.0
10−5 M in 3:1:1 (v/v) DMSO/water/acetone. (Bottom
right) EPR spectral evolution with time for a solution of and pyrrolidine in DMSO/water/acetone
1
]•. (Bottom left) Fluorescence
1
×
1
under the conditions reported in Figure 2. Insets: Images of the solutions under white light (left) and
under UV light (366 nm) (right).
0.25
0.20
0.15
0.10
0.05
0.00
1
Pyrrolid. Histam. Putresc. Cadav.
Amine
Figure 10. Quantum yield of different combinations between 1 and amines in DMSO; comparison
between 1 without amine, pyrrolidine, histamine, putrescine, and cadaverine.
After testing in different conditions, the best conditions for the experiments were
determined to be as follows: three mackerel (Scomber scombrus) samples were bought in a
local market, the fish were cleaned, the spines were extracted, and the muscle was ground
(Philips HR 28308/B). Then, fish samples of 10
sealed with a septum. Half of the samples were incubated at 4 C, and the other half
±
0.2 g were placed in 20 mL vials and
◦

Molecules 2021, 26, 1404
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◦
1 C. Then, 3 mL of the gas phase of the vial was taken with a
were incubated at 25
±
syringe and bubbled into a probe solution at different times as the fish decayed over time.
The absorbance and fluorescence of the samples were measured at their maximum peaks,
and the differences between the obtained values and a reference solution were plotted
(Figure 11).
0.0
35
25 ºC
4 ºC
30
25 ºC
-5.0x10-2
4 ºC
25
20
-1.0x10-1
15
10
-1.5x10-1
5
0
-2.0x10-1
0
20 40 60 80 100 120 140 160 180
Time (hours)
0
20 40 60 80 100 120 140 160 180
Time (hours)
Figure 11. Evolution of the absorbance (left) and fluorescence (right) signals detected with solutions
of 1 after the decay of fish samples over several hours.
The process was repeated three times and the results were combined. The results
showed that the variation was quite low for refrigerated samples, but the emission of the
samples left at 25 ◦C clearly increased over time. The results indicate that the changes in
absorbance and fluorescence over time were closely related to the previous model amines.
While the change in fluorescence or absorbance was not very significant in refrigerated
samp◦les, the amine concentration in the gas phase clearly increased over time for samples
at 25 C. The increase in fluorescence was more visible by eyesight under 366 nm light than
by the naked eye (Figure 12).
−5
Figure 12. From left to right: (1) reference
1
(1.68
×
10 M in DMSO); (2) reference + air (bubbled);
◦
(3) reference + fish sample (bubbled) (3 days at 25 C); (4) reference + fish sample (bubbled) (1 day
at 25 ◦C); (5) reference + histamine (bubbled); (6) reference + putrescine (bubbled); (7) reference +
cadaverine (bubbled); (8) reference + pyrrolidine (bubbled); (9) reference + all amines (bubbled).
(Top) Under visible light; (Bottom) under UV light (366 nm).
Because it was easily visible under a UV light, the system could be used to create an
intelligent label for the detection of amines. To do so, the probe was dissolved in DMSO,
and a typical gelling agent (N-benzyloxycarbonyl-L-valyl-L-valine n-octadecylamide) [35
]
was added to the previous solution at 2 mg/0.5 mL DMSO, with 0.3% weight/volume

Molecules 2021, 26, 1404
11 of 17
being the minimum possible density to retain its gelation properties [36]. After that, a
sample of biogenic amine from rotten fish was pumped with a syringe onto the probe
(Figure S45 in Supporting Information), and a fading in the color and an increase in
bright fluorescence were immediately apparent and lasted for a long time (Figure 13). The
fluorescence decreased over time and disappeared after 12 h.
Figure 13. Response of a solution of
the addition of amines in the gas phase under UV light at 366 nm.
1
(5
×
10−5 M) in 0.25 mL of DMSO with 2 mg of gellant after
This process is potentially suitable for use as a smart label for the freshness of fish;
for example, it can be used with suspected rotten fish without opening the packaged fish
container (Figure 14).
Figure 14. Fluorescence increase in presence of biogenic amines in the gas phase generated from
rotten fish.
Using the combined techniques developed in this process, not only fluorescence but
also EPR could be used to characterize the freshness of fish while new applications of dual
fluorescence-spin-label probes [37] or sensor arrays [38] are developed for practical use.
3. Materials and Methods
The reactions performed with air-sensitive reagents were conducted under dry ni-
trogen. The solvents were previously distilled under nitrogen over calcium hydride or
sodium filaments. Melting points were determined in a Gallenkamp apparatus and are
not corrected. Infrared spectra were registered with potassium bromide tablets using a
Nicolet Impact 410 spectrometer (Madison, WI, USA). NMR spectra were recorded on
Varian Mercury-300 (Palo Alto, CA, USA) and Varian Unity Inova-400 (Palo Alto, CA,
USA) machines in DMSO-d6 or CDCl3. Chemical shifts are reported in ppm with respect to
residual solvent protons, and coupling constants (JX-X’) are reported in Hz. Mass spectra
were taken on a Micromass AutoSpec machine (Milford, MA, USA) by electronic impact
at 70 eV. Quantitative UV–visible measurements were performed with a Varian Cary 300
Bio UV spectrophotometer (Palo Alto, CA, USA) in 1 cm UV cells thermostated at 25 ◦C.
Fluorescence spectra were recorded on a Hitachi F-7000 FL spectrofluorometer (Minato-ku,
Tokyo, Japan) in the range 300–700 nm in 1 cm path length quartz cells thermostated at
25 ◦C. Solution X-band EPR spectra at room temperature were recorded by using flat cells
on a Bruker EMX spectrometer (Billerica, MA, USA) equipped with a Bruker ER 036TM
NMR-teslameter and an Agilent 53150A microwave frequency counter to fit the magnetic
field and the frequency inside the measuring cavity. Simulation of the EPR spectra was
performed by using the SimFonia program (Bruker, Billerica, MA, USA) and the public
domain WinSim program (WinSim Inc., Richmond, TX, USA), while spectra and graphics
were plotted with Kaleidagraph v3.5 software (Synergy Software, Reading, PA, USA).

Molecules 2021, 26, 1404
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Synthesis of 1-(4-methoxyphenyl-3-[4-(1-(dicyanomethyleneindan-5-yl)phenyl]urea
1
. 5-(4-Amino-
phenyl)indan-1-one: Pd(PPh3)4 (54 mg, 0.05 mmol) was added under nitrogen to a solution
of 5-bromoindanone (410 mg, 1.94 mmol) in a toluene/butanol/water 4:1:0.4 mixture
(50 mL), and the mixture was stirred for 30 min at room temperature. Then, solid 4-
aminobenzeneboronic pinacol ester (447 mg, 2.04 mmol) and Na2CO3 (1028 mg, 9.70 mmol)
were added, and the mixture was heated under reflux for 16 h. Then, the solvent was
evaporated under reduced pressure, and the residue was added to water (20 mL) and
extracted with CH2Cl2 (5
×
40 mL). The combined organic extracts were dried (Na2SO4)
and evaporated, and the residue was purified by flash chromatography (silica, 3 × 30 cm),
CH2Cl2, to obtain 5-(4-aminophenyl)indan-1-one (353 mg, 82 %) as a pale yellow solid,
mp 250–251 ◦C. IR (KBr, cm−1): 3429, 3335, 1678 (C=O), 1592, 1297, 1278, 811. H NMR
1
(DMSO-d6, 400 MHz)
6.68 (d, J = 8.6 Hz, 2H, ArH), 5.44 (s, br exch, 2H, NH2), 3.10 (m, 2H, CH2), 2.62 (m, 2H,
CH2). 13C NMR and DEPT (DMSO-d6, 400 MHz)
: 205.44 (C=O), 156.14 (C-NH2), 149.53,
δ: 7.71 (s, 1H, ArH), 7.60 (s, 2H, ArH), 7.48 (d, J = 8.6 Hz, 2H, ArH),
δ
146.91, 134.05, 127.90 (CHar), 125.84, 124.60 (CHar), 123.25 (CHar), 122.79 (CHar), 114.13
(CHar), 36.10 (CH2), 25.44 (CH2). MS (EI) m/z (%): 224 (M+ + 1, 17), 223 (M+, 100), 195
(18), 167 (8), 152 (9), 139 (4), 117 (7), 96 (3). HRMS (EI): calcd. for C15H13NO: 223.0997
(M+); found: 223.0998. 1-Dicyanomethylene-5-(4-aminophenyl)indane: Malononitrile
(200 mg, 3.03 mmol) and DABCO (120 mg, 1.07 mmol) were added under nitrogen to 5-(4-
aminophenyl)indan-1-one (200 mg, 0.90 mmol) dissolved in toluene (50 mL). The mixture
was heated under reflux for 8 h and then added to water (30 mL), stirred for 5 min, and
extracted with dichloromethane (5
(Na2SO4), and the solvent was evaporated under reduced pressure. The crude product
was purified by flash chromatography (silica, 3 30 cm), from CH2Cl2 to CH2Cl2:AcOEt
×
30 mL). The combined organic extracts were dried
×
(15:1), to obtain 1-dicyanomethylene-5-(4-aminophenyl)indene (59 mg, 95 %) as an orange
◦
solid, mp: >250 C (decomp.), IR (KBr, cm−1): 3491, 3382, 2221 (CN), 1627, 1600, 1561
1
(Car-Car). H NMR (DMSO-d6, 300 MHz)
δ
: 8.15 (d, J = 9.0 Hz, 1H, ArH), 7.75 (m, 2H,
ArH), 5.62 (s, br, exch,
2H, NH2), 3.23 (m, 2H, CH2), 3.11 (m, 2H, CH2). 13C NMR and DEPT (DMSO-d6, 75 MHz)
: 179.83 (C=C(CN)2), 156.43, 150.35, 147.42, 132.53, 128.20 (CHar), 125.34 (CHar), 124.90
2
×
ArH), 7.54 (d, J = 8.7 Hz, 2H, CH2), 6.66 (d, J = 8.7 Hz, 2H, 2
×
δ
(CHar), 124.72, 121.69 (CHar), 114.36 (CN), 114.15 (CHar), 113.95 (CN), 69.86 (C(CN)2),
34.65 (CH2), 29.41 (CH2). MS (EI) m/z (%): 272 (M+ + 1, 20), 271 (M+, 100), 243 (3), 214
(2), 206 (1), 204 (2), 189 (2), 135 (2). HRMS (EI): calcd. for C18H13N3: 271.1109 (M+);
found: 271.1108. 1-(4-methoxyphenyl-3-[4-(1-(dicyanomethyleneindan-5-yl)phenyl]urea
1: 1-Dicyanomethylene-5-(4-aminophenyl)indane (300 mg, 1.10 mmol) was added to 4,40-
diisocyanatodiphenylether (139 mg, 0.55 mmol) dissolved in CHCl3 (20 mL), and the
mixture was stirred at room temperature for 48 h. Cyclohexane (4 mL) was then added, and
the solid formed was filtered off, washed with CHCl3 (2
1-(4-methoxyphenyl-3-[4-(1-(dicyanomethyleneindan-5-yl)phenyl]urea
was obtained as a dark yellow solid, mp: >250 ◦C. IR (KBr, cm−1): 3316, 2221 (C
(C=O), 1598, 1557, 1510, 1239, 1184, 827. 1H NMR (DMSO-d6, 400 MHz)
: 8.90 (s, 2H,
ArH), 7.91 (s, 2H, 2 ArH),
ArH), 7.61 (d, J = 9.0 Hz, 4H,
×
4 mL), and dried. Compound
(337 mg, 77%)
N), 1654
1
≡
δ
2
×
NH), 8.73 (s, 2H, 2
×
NH), 8.25 (d, J = 9.0 Hz, 2H, 2
ArH), 7.77 (d, J = 9.0 Hz, 4H, 4
×
×
×
7.87 (d, J = 9.0 Hz, 2H, 2
×
4 × ArH), 7.46 (d, J = 9.0 Hz, 4H, 4 × ArH), 6.96 (d, J = 9.0 Hz, 4H, 4 × ArH), 3.30 (m, 4H,
2
×
CH2), 3.19 (m, 4H, 2
×
CH2). 13C NMR and DEPT (DMSO-d6, 100 MHz)
δ: 178.08
(C=C(CN)2), 156.27, 152.46, 151.95, 146.39, 140.92, 134.93, 133.73, 131.31, 127.81 (CHar),
125.95 (CHar), 125.40 (CHar), 123.26 (CHar), 120.11 (CHar), 118.82 (CHar), 118.41 (CHar),
114.09 (CN), 113.67 (CN), 71.23 (C(CN)2), 34.71 (CH2), 29.51 (CH2). MS (ESI) m/z (%): 795
(M+ + 1, 15). HRMS (ESI): calcd. for C50H34N8O3 + H+: 795.2827 (M+ + 1); found: 795.2832.
Synthesis of 1,3-bis[4-(1-dicyanomethyleneindan-5-yl)phenylureidomethyl)benzene
ethylene-5-(4-aminophenyl)indane (100 mg, 0.38 mmol) was added to m-xylylene diiso-
cyanate (15 L, 0.095 mmol) dissolved in CHCl3 (20 mL), and the mixture was stirred at
room temperature for 24 h. Then, a second portion of m-xylylene diisocyanate (15 L,
0.095 mmol) was added, and the mixture was stirred for an additional 24 h. The solid
2. 1-Dicyanom-
µ
µ

Molecules 2021, 26, 1404
13 of 17
formed after this time was filtered off, washed with CHCl3 (2
×
2 mL), and dried under
reduced pressure. Compound 1,3-bis[4-(1-dicyanomethyleneindan-5-yl)phenylureidom-
ethyl)benzene
cm−1): 3328, 2221 (C
400 MHz)
7.79 (d, J = 9.0 Hz, 2H, 2
ArH), 7.29 (m, 2H, 2
4.32 (d, J = 6.0 Hz, 4H, 2
DEPT (CDCl3, 100 MHz)
130.10, 128.22 (CHar), 127.58 (CHar), 125.72, 125.67 (CHar), 125.55 (CHar), 125.22 (CHar),
2
(104 mg, 75%) was obtained as a yellow solid, mp: 212–214 ◦C. IR (KBr,
1
≡
N), 1658 (C=O), 1604, 1557, 1320, 1219, 1184. H NMR (DMSO-d6,
δ
: 8.82 (s, 2H, NH), 8.17 (d, J = 9.0 Hz, 2H, 2
ArH), 7.65 (d, J = 8.6 Hz, 4H, 4
ArH), 7.20 (m, 2H, 2 ArH), 6.71 (t, J = 6.0 Hz, 2H, 2
CH2), 3.28 (m, 2H, CH2), 3.15 (m, 2H, CH2). 13C NMR and
: 179.60 (C=C(CN)2), 155.78, 154.63, 146.04, 141.21, 140.00, 133.07,
×
ArH), 7.82 (s, 2H, 2
ArH), 7.52 (d, J = 8.6 Hz, 4H,
NH),
×
ArH),
×
×
4
×
×
×
×
×
δ
122.60 (CHar), 117.54 (CHar), 113.66 (CN), 113.25 (CN), 70.61 (C(CN)2), 42.32 (2 CH2),
×
34.24 (CH2), 29.04 (CH2). MS (ESI) m/z (%): 753.27 (M+ + Na+, 94), 731.29 (M+ + 1, 100).
HRMS (ESI): calcd. for C46H34N8O2 + H+: 731.2877 (M+ + 1); found: 731.2864.
Synthesis of 1-(4-methoxyphenyl-3-[4-(1-(dicyanomethyleneindan-5-yl)phenyl]urea 3. 1-Dicyano-
methylene-5-(4-aminophenyl)indane (150 mg, 0.55 mmol) was added to p-methoxyphenyl
isocyanate (82 mg, 0.55 mmol) dissolved in CHCl3 (10 mL), and the mixture was stirred at
room temperature for 48 h. Cyclohexane (3 mL) was then added, and the solid formed was
filtered off, washed with CHCl3 (2
[4-(1-(dicyanomethyleneindan-5-yl)phenyl]urea
yellow solid, mp: 232–233 ◦C. IR (KBr, cm−1): 3320, 2221 (CN), 1658 (C=O), 1596, 1557,
1510, 1239, 1184, 1029, 823. 1H NMR (DMSO-d6, 300 MHz)
: 8.85 (s, 1H, NH), 8.56 (s,
×
2 mL), and dried. Compound 1-(4-methoxyphenyl-3-
3
(172 mg, 74%) was obtained as a dark
δ
1H, NH), 8.23 (d, J = 9.0 Hz, 1H, ArH), 7.90 (s, 1H, ArH), 7.86 (d, J = 9.0 Hz, 1H, ArH),
7.76 (d, J = 9.0 Hz, 2H, ArH), 7.58 (d, J = 9.0 Hz, 2H, ArH), 7.37 (d, J = 9.0 Hz, 2H, ArH),
6.87 (d, J = 9.0 Hz, 2H, ArH), 3.82 (s, 3H, CH3), 3.28 (m, 2H, CH2), 3.18 (m, 2H, CH2). 13C
NMR and DEPT (DMSO-d6, 75 MHz) δ: 180.04 (C=C(CN)2), 156.22, 154.52, 152.47, 146.33,
141.02, 133.63, 132.44, 131.05, 127.74 (CHar), 125.86 (CHar), 125.34 (CHar), 123.15 (CHar),
120.07 (CHar), 118.23 (CHar), 114.06 (CN), 113.94 (CHar), 113.63 (CN), 71.12 (C(CN)2),
55.13 (OCH3), 34.63(CH2), 29.45 (CH2). MS (EI) m/z (%): 421 (M+ + 1, 3), 420 (M+, 11),
297 (100), 271 (87), 149 (17), 123 (31). HRMS (EI): calcd. for C26H20N4O2: 420.1586 (M+);
found: 420.1582.
Synthesis of 1-[4-(N,N-dimethylamino)phenyl]-3-[4-(1-(dicyanomethyleneindan-5-yl)phenyl]urea
4. 1-Dicyanomethylene-5-(4-aminophenyl)indane (150 mg, 0.55 mmol) was added to p-
N,N-dimethylaminophenyl isocyanate (89 mg, 0.55 mmol) dissolved in CHCl3 (10 mL),
and the mixture was stirred at room temperature for 48 h. Cyclohexane (3 mL) was
then added, and the solid formed was filtered off, washed with CHCl3 (2
dried. Compound 1-[4-(N,N-dimethylamino)phenyl]-3-[4-(1-(dicyanomethylene-indan-
×
2 mL), and
5-yl)phenyl]urea
4
(160 mg, 67%) was obtained as a dark yellow solid, mp: 178–179 ◦C.
IR (KBr, cm−1): 3320, 2221 (C
≡
N), 1654 (C=O), 1596, 1565, 1518, 1320, 1242, 1185, 823.
: 8.81 (s, 1H, NH), 8.41 (s, 1H, NH), 8.25 (d, J = 9.0 Hz, 1H,
ArH),
ArH), 6.72 (d, J = 9.0 Hz, 2H,
CH3). 13C NMR and DEPT
: 180.06 (C=C(CN)2), 156.49, 152.63, 146.60, 141.28, 133.67, 130.96,
1H NMR (DMSO-d6, 300 MHz)
δ
ArH), 7.92 (s, 1H, ArH), 7.88 (d, J = 9.0 Hz, 1H, ArH), 7.76 (d, J = 9.0 Hz, 2H, 2
7.60 (d, J = 9.0 Hz, 2H, 2 ArH), 7.29 (d, J = 9.0 Hz, 2H, 2
ArH), 3.30 (m, 2H, CH2), 3.20 (m, 2H, CH2), 2.84 (s, 6H, 2
(DMSO-d6, 75 MHz)
×
×
×
2
×
×
δ
129.26, 127.80 (CHar), 125.89 (CHar), 125.42 (CHar), 123.16 (CHar), 120.32 (CHar), 118.22
(CHar), 117.54, 114.15 (CN), 113.73 (CN), 113.16 (CHar), 71.07 (C(CN)2), 40.64 (N(CH3)2),
34.61 (CH2), 29.43 (CH2). MS (EI) m/z (%): 433 (M+, 5), 297 (100), 271 (92), 162 (35), 136 (37).
HRMS (EI): calcd. for C27H23N5O: 433.1903 (M+); found: 433.1907.
Synthesis of 1-(4-methylthiophenyl)-3-[4-(1-(dicyanomethyleneindan-5-yl)phenyl]urea 5. 1-Dicya-
nomethylene-5-(4-aminophenyl)indane (150 mg, 0.55 mmol) was added to p-methylthiophe-
nyl isocyanate (91 mg, 0.55 mmol) dissolved in CHCl3 (10 mL), and the mixture was
stirred at room temperature for 48 h. Cyclohexane (3 mL) was then added, and the solid
formed was filtered off, washed with CHCl3 (2
methylthiophenyl)-3-[4-(1-(dicyanomethyleneindan-5-yl)-phenyl]urea
×
2 mL), and dried. Compound 1-(4-
(173 mg, 72%) was
5

Molecules 2021, 26, 1404
14 of 17
obtained as a dark yellow solid, mp: 209–210 ◦C. IR (KBr, cm−1): 3317, 2221 (CN), 1654
1
(C=O), 1596, 1546, 1495, 1324, 1281, 1192, 827. H NMR (DMSO-d6, 400 MHz)
δ: 8.91 (s,1H,
NH), 8.77 (s,1H, NH), 8.25 (d, J = 9.0 Hz, 1H, ArH), 7.91 (s, 1H, ArH), 7.87 (d, J = 9.0 Hz,
1H, ArH), 7.77 (d, J = 9.0 Hz, 2H, ArH), 7.60 (d, J = 9.0 Hz, 2H, ArH), 7.44 (d, J = 9.0 Hz,
2H, ArH), 7.23 (d, J = 9.0 Hz, 2H, ArH), 3.30 (m, 2H, CH2), 3.19 (m, 2H, CH2), 2.44 (s,
3H, CH3). 13C NMR and DEPT (DMSO-d6, 75 MHz)
δ: 180.05 (C=C(CN)2), 156.23, 152.28,
146.35, 140.80, 137.20, 133.72, 131.36, 130.17, 127.78 (CHar), 127.70 (CHar), 125.94 (CHar),
125.38 (CHar), 123.25 (CHar), 119.06 (CHar), 118.42 (CHar), 114.06 (CN), 113.64 (CN), 71.23
(C(CN)2), 34.69 (CH2), 29.50 (CH2), 15.94 (CH3). MS (EI) m/z (%): 436 (M+, 7), 297 (100),
271 (93), 165 (32). HRMS (EI): calcd. for C26H20N4OS: 436.1358 (M+); found: 436.1359.
Synthesis of N,N-bis-[4-(1-(dicyanomethyleneindan-5-yl)phenyl]pyridine-2,6-dicarboxamide 6. 2,6-
Pyridinedicarbonyl dichloride (75 mg, 0.37 mmol) dissolved in dichloromethane (10 mL)
was added dropwise to a chilled and stirred solution of 1-dicyanomethylene-5-(4-aminophe-
nyl)indane (200 mg, 0.74 mmol) and N,N-diisopropylethylamine (96 mg, 0.74 mmol)
in dichloromethane (50 mL). The mixture was left to warm to room temperature and
stirred for 24 h. The solid formed was filtered off, washed with CHCl3 (2
and dried. Compound N,N-bis-[4-(1-(dicyanomethyleneindan-5-yl)phenyl]pyridine-2,6-
×
3 mL),
◦
dicarboxamide
(KBr, cm−1): 3344, 2221 (C
400 MHz) (ppm): 10.79 (s, 2H, 2
J = 12.0 Hz, 2H, 2 ArH), 8.16 (d, J = 12.0 Hz, 2H, 2
ArH), 7.72 (m, 8H, 8 ArH), 3.30 (m, 4H, 2 CH2), 3.24 (m, 4H, 2
and DEPT (DMSO-d6, 75 MHz) : 179.95 (C=C(CN)2), 161.74, 156.13, 148.69, 146.00, 139.99
6
(197 mg, 79%) was obtained as a dark yellow solid, mp: >250 C. IR
≡
N), 1682 (C=O), 1565, 1526, 1340, 1188, 819. 1H NMR (CDCl3,
NH), 8.51 (d, J = 12.0 Hz, 2H, 2 ArH), 8.42 (d,
ArH), 8.10 (d, J = 9.0 Hz, 4H,
CH2). 13C NMR
δ
×
×
×
×
4
×
×
×
×
δ
(CHar), 139.00, 134.04, 133.80, 127.65 (CHar), 127.05, 126.20 (CHar), 125.35 (CHar), 123.61
(CHar), 121.09 (CHar), 113.93 (CN), 113.52 (CN), 71.50 (C(CN)2), 34.65 (CH2), 29.48 (CH2).
MS (ESI) m/z (%): 674 (M+ + 1, 12). HRMS (EI): calcd. for C43H27N7O2 + H+: 674.2299
(M+); found: 674.2295.
Synthesis of 5,50-(((6-cyclohexyl-[3,40-bipyridine]-20,60-dicarbonyl)bis(piperazine-4,1-diyl))bis
(pyridine-6,3-diyl))bis(indan-1-one) 7
. 6-(Piperidin-1-yl)-[3,40-bipyridine]-20,60-dicarboxylic
acid: Pd(PPh3)4 (58 mg, 0.05 mmol) was added under nitrogen to a solution of dimethyl
4-bromopyridine-2,6-dicarboxylate (822 mg, 3.0 mmol) in tetrahydrofuran (120 mL), and
the mixture was stirred for 30 min at room temperature. Then, solid 2-(piperidin-1-yl)-
5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (865 mg, 3.0 mmol) and cesium
carbonate (1.47 g, 4.5 mmol) dissolved in water (15 mL) were successively added, and the
mixture was heated under reflux for 24 h. Then, the solvent was evaporated under reduced
pressure, and the residue was suspended in water (10 mL). Then, NaOH solution (5% w/v
in H2O) was added dropwise until pH = 10, and the mixture was dissolved in tetrahy-
drofuran (60 mL) and heated under reflux for 1 h until complete hydrolysis of the diester
and monoester mixture. Then, the mixture was chilled in an ice bath, and HCl solution
(35% w/v in water) was added dropwise until pH = 1. Then, the solvent was evaporated
until reduced pressure was achieved, and the solid residue was dried under vacuum. The
solid residue was suspended in anhydrous dichloromethane (125 mL) under nitrogen,
oxalyl chloride (3.00 g, 24 mmol) and anhydrous dimethylformamide (0.2 mL) were added,
and the mixture was stirred overnight. Then, the solvent and the excess of oxalyl chlo-
ride were evaporated under reduced pressure, anhydrous methanol (5 mL, 124 mmol) in
dichloromethane (60 mL) was added under nitrogen, and the mixture was stirred overnight.
Then, the solvent was evaporated under reduced pressure, and the residue was purified
by flash chromatography (silica, 3
×
50 cm), from CH2Cl2 to CH2Cl2/MeOH 9:1 v/v, to
obtain dimethyl 6-(piperidin-1-yl)[3,40-bipyridine]-20,60-dicarboxylate as a light yellow
solid (522 mg, 49 %), mp: 146–148 ◦C, Rf (CH2Cl2/MeOH, 50:3): 0.83; 1H RMN (CDCl3,
300 MHz):
6.75–6.72 (d, J = 9.3 Hz, 1H, ArH); 4.04 (m, 6H, 3
9H, 3 = 165.4 (C=O), 159.6 (CAr), 148.7 (CHAr), 148.6
CH2); 13C RMN (CDCl3, 75 MHz):
δ
= 8.63 (s, 1H, ArH); 8.47 (s, 2H, ArH); 7.87–7.83 (dd, J = 9.3 Hz, 1H, ArH);
×
CH2); 3.66 (m, 4H, 3 CH2); 1.68 (m,
×
×
δ

Molecules 2021, 26, 1404
15 of 17
(CHAr), 147 (CHAr), 135.5 (CAr), 123.6 (CHAr), 119.1 (CAr), 106.5 (CHAr), 53.4 (CH3), 45.9
(CH2), 25.5 (CH2), 24.7 (CH2); MS (EI) m/z (%): 355 (M+, 100), 326 (55), 312 (30), 299 (20),
272 (32), 84 (28); HRMS (EI): calcd. for C19H21N3O4: 355.1532; found 355.1535. The diester
(512 mg, 1.44 mmol) was dissolved in a mixture of tetrahydrofuran (10 mL) and water
(1 mL), a solution of NaOH (5% w/v in H2O) was added dropwise until pH = 10, and the
mixture was heated under reflux for 1 h until complete hydrolysis of the diester. Then, the
mixture was chilled in an ice bath, and HCl solution (35% w/v in water) was added drop-
wise until pH = 1. Then, the solvent was evaporated until reduced pressure was achieved,
and the solid residue was extracted several times with dichloromethane/methanol 1:1
until complete extraction of the diacid. Evaporation of the solvent under reduced pressure
afforded pure 6-(piperidin-1-yl)-[3,40-bipyridine]-20,60-dicarboxylic acid as a light yellow
solid (448 mg, 95% from the diester, 47% from dimethyl 4-bromopyridine-2,6-dicarboxylate),
◦
1
mp: 244–246 C (decomp.); H RMN (CD3OD, 300MHz):
0.3H, ArH); 8.28 (s, 0.7H, ArH); 8.14–8.11 (d, J = 9.3 Hz, 1H, ArH); 7.10–7.07 (d, J = 9.3 Hz,
1H, ArH); 3.66 (m, 4H, 3 CH2); 1.69 (m, 6H, 3
CH2); 13C RMN (CD3OD, 75 MHz):
δ = 8.49 (s, 2H, ArH); 8.43 (s,
×
×
δ =
167.4 and 166.4 (C=O), 150.8 (CAr), 149.9 (CHAr), 140.2 (CHAr), 131.2 (CHAr), 128.8 (CAr),
124.7 (CHAr), 121.1 (CAr), 111.4 (CHAr), 26.6 (CH2), 25.2 (CH2); IR (KBr): 3609 (OH), 2786,
2167, 1648, 1340, 1108, 752 cm−1; MS (EI) m/z (%): 327 (M+, 75), 283 (92), 239 (45), 157 (41),
84 (100). HRMS (EI): calcd. for C17H17N3O4 + H+: 328.1292; found: 328.1291.
0
0
0
0
5,5 -(((6-Cyclohexyl[3,4 -bipyridine]-2 ,6 -dicarbonyl)bis(piperazine-4,1-diyl))bis(pyri-
dine-6,3-diyl))bis(indan-1-one)
7
: Oxalyl chloride (530.0
µ
L,0 ρ0= 1.478 g/mL, 6.17 mmol)
0
was added to a suspension of 6-cyclohexyl-[3,4 -bipyridine]-2 ,6 -dicarboxylic acid (294 mg,
0.90 mmol) in CH2Cl2 (100 mL) with three drops of DMF as a catalyst. The mixture
was stirred at room temperature for 24 h, and then the solvent and the excess of oxalyl
chloride were evaporated under reduced pressure. A solution of 1-dicyanomethylene-5-
(4-aminophenyl)indane (190 mg, 0.70 mmol) in DMF (55 mL) was added over the white
solid in an ice bath. Then, N,N-diisopropylethylamine (200 µL, ρ = 0.742 g/mL, 1.15 mmol)
and a spatula tip of 4-dimethylaminopyridine (DMAP) were added, and the mixture was
◦
stirred at 0 C for 15 min and then at room temperature for 24 h. A yellow precipitate
formed that was filtered off. The filtered solid was washed with CHCl3 (3
and CH2Cl2 (3 5 mL). Finally, the solid was collected, and traces of the solvent were
evaporated under reduced pressure to obtain 5,50-(((6-cyclohexyl-[3,40-bipyridine]-20,60-
×
10 mL)
×
dicarbonyl)bis(piperazine-4,1-diyl))bis(pyridine-6,3-diyl))bis(indan-1-one)
7 (466 mg, 62%)
as an orange solid, almost insoluble in all common solvents, mp: 212-213 ◦C (decomp.);
IR (KBr, cm−1): 3626 (NH), 3069, 2360, 2218, 1648 (C=O), 1558, 1527, 1337, 1188, 1122,
1005 cm−1; 1H NMR (DMSO-d6, 400 MHz)
δ: 11.21 (s, 2H, 2
×
NH), 8.72 (s, 1H, ArH), 8.61
(s, 2H, ArH), 8.31 (d, J = 8.4 Hz, 2H, ArH), 8.21 (m, 2H, ArH), 8.17 (d, J = 8.4 Hz, 4H, ArH),
8.00 (s, 2H, ArH), 7.95 (d, J = 8.8 Hz, 7H, ArH), 7.08 (d, J = 7.2 Hz, 1H, ArH), 3.70 (m, 4H,
2
×
CH2)), 3.35 (m, 4H, 2
×
CH2), 3.24 (m, 4H, 2
×
CH2), 1.68 (m, 2H, CH2), 1.61 (m, 4H,
2
×
CH2); MS (FAB) m/z (%): 833 (M+, 2), 834 (4), 835 (10), 836 (6), 837 (3). HRMS (EI):
calcd. for C53H39N9O2 + H+: 834.3299; found: 834.3298.
4. Conclusions
In conclusion, we describe a rare class of persistent radicals formed through the
interaction of fluorogenic arylurea derivatives and primary or secondary amines in the
presence of light, water, and air. The most persistent species of the series was studied using
spectroscopic techniques as well as quantum mechanics calculations. All results pointed to
a rare case of photoinduced proton-coupled electron transfer, although the slow kinetics of
formation and the evolution of the radical species from a formerly fluorescent transient
intermediate rendered the system more complicated than classic examples. The two best
examples of the formation of radicals having a well-resolved EPR hyperfine structure and
high intensity came from a well-known selective fluorogenic probe for biogenic amines or a
fluorescent probe for
ω-amino acids, therefore adding a new perspective to these and other
to-be-obtained fluorescent probes. These chemical probes are the first examples of multi-

Molecules 2021, 26, 1404
16 of 17
signal reporters for the discrimination between primary, secondary, and tertiary amines in
which the usual UV–visible or fluorescent signal is complemented by paramagnetic signals
of the same samples at different reporting times. The selectivity of the chemical probes
for different types of amines and the large types of signals offered by these probes, now
including paramagnetic species, make them good starting points for the preparation of
multi-signaling chemical probes for the detection of important organic metabolites. As an
immediate application, we developed a multi-signal smart label for the quick inspection of
freshness in fish.
Supplementary Materials: The following are available online, complete characterization of all
compounds, additional EPR measurements, UV–vis titrations, fluorescence measurements, detection
of biogenic amines, and quantum chemical calculations.
Author Contributions: Conceptualization, T.T.; methodology, EPR spectroscopy, J.G.-T.; investiga-
tion, UV–vis, and fluorescence spectroscopy, B.D.d.G.; investigation, synthesis, B.D.d.G., C.H.-M.,
M.M.S.; investigation, food technology, M.-J.R.; data curation, J.G.-C.; quantum chemical calculations,
J.V.C.; writing—original draft preparation, B.D.d.G., J.G.-C., T.T.; writing—review and editing, T.T.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the NATO Science for Peace and Security Programme (Grant
SPS G5536), the Junta de Castilla y León, Consejería de Educación y Cultura y Fondo Social Europeo
(Grant BU263P18), and the Ministerio de Ciencia e Innovación (Grant PID2019-111215RB-100).
Data Availability Statement: The data presented in this study are contained within the article and
also available in the supplementary material.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of compounds 1–7 are available from the authors.
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