Novel (Phenothiazinyl)vinyl‐pyridinium dyes and their potential applications as cellular staining agents

Article abstract of DOI:10.3390/ijms22062985

We report here the synthesis and structural characterization of novel cationic (phenothia-zinyl)vinyl‐pyridinium (PVP) dyes, together with optical (absorption/emission) properties and their potential applicability as fluorescent labels. Convective heating, ultrasound irradiation and mechano-chemical synthesis were considered as alternative synthetic methodologies proficient for overcoming drawbacks such as long reaction time, nonsatisfactory yields or solvent requirements in the synthesis of novel dye (E)‐1‐(3‐chloropropyl)‐4‐(2‐(10‐methyl‐10H‐phenothiazin‐3‐yl)vinyl)pyridin‐1‐ium bro-mide 3d and its N‐alkyl‐2‐methylpyridinium precursor 1c. The trans geometry of the newly synthe-sized (E)‐4‐(2‐(7‐bromo‐10‐ethyl‐10H‐phenothiazin‐3‐yl)vinyl)‐1‐methylpyridin‐1‐ium iodide 3b and (E)‐1‐methyl‐4‐(2‐(10‐methyl‐10H‐phenothiazin‐3‐yl)vinyl)pyridin‐1‐ium tetrafluoroborate 3a’ was confirmed by single crystal X‐ray diffraction. A negative solvatochromism of the dyes in polar solvents was highlighted by UV‐Vis spectroscopy and explanatory insights were supported by molecular modeling which suggested a better stabilization of the lowest unoccupied molecular orbitals (LUMO). The photostability of the dye 3b was investigated by irradiation at 365 nm in different solvents, while the steady‐state and time‐resolved fluorescence properties of dye 3b and 3a’ in solid state were evaluated under one‐photon excitation at 485 nm. The in vitro cytotoxicity of the new PVP dyes on B16‐F10 melanoma cells was evaluated by WST‐1 assay, while their intracellular localization was assessed by epi-fluorescence conventional microscopy imaging as well as one‐ and two‐photon excited confocal fluorescence lifetime imaging microscopy (FLIM). PVP dyes displayed low cytotoxicity, good internaliza-tion inside melanoma cells and intense fluorescence emission inside the B16‐F10 murine melanoma cells, making them suitable staining agents for imaging applications.

Full text of DOI:10.3390/ijms22062985

International Journal of
Molecular Sciences
Article
Novel (Phenothiazinyl)Vinyl-Pyridinium Dyes and Their
Potential Applications as Cellular Staining Agents
1
2
3
, Ana-Maria Craciun 3 , M aˇ d aˇ lina Nistor , Tamas Lovasz ,
2
1
Bianca Stoean , Dumitrita Rugina , Monica Focsan
4
1
1
, Castelia Cristea 1 , Luminita Silaghi-Dumitrescu ,
1
Alexandru Turza , Ioan-Dan Porumb , Emese Gál
Simion Astilean 3
,5
and Luiza Ioana Gaina *
1,
1
Research Center on Fundamental and Applied Heterochemistry, Faculty of Chemistry and
Chemical Engineering, Babe s¸ -Bolyai University, 11 Arany Janos Street, 400028 Cluj-Napoca, Romania;
sba2488@chem.ubbcluj.ro (B.S.); tlovasz@chem.ubbcluj.ro (T.L.); pdan@chem.ubbcluj.ro (I.-D.P.);
emese@chem.ubbcluj.ro (E.G.); castelia@chem.ubbcluj.ro (C.C.); lusi@chem.ubbcluj.ro (L.S.-D.)
Biochemistry Department, Faculty of Veterinary Medicine, University of Agricultural Science and
Veterinary Medicine, 3-5 Calea Manastur Street, 400327 Cluj-Napoca, Romania;
oliviapreda@gmail.com (D.R.); nistor.madalina@usamvcluj.ro (M.N.)
Nanobiophotonics and Laser Microspectroscopy Center, Interdisciplinary Research Institute in
Bio-Nano-Sciences, Babes-Bolyai University, 42 Treboniu Laurian Street, 400271 Cluj-Napoca, Romania;
monica.iosin@phys.ubbcluj.ro (M.F.); ana.gabudean@ubbcluj.ro (A.-M.C.);
2
3
simion.astilean@phys.ubbcluj.ro (S.A.)
Department of Mass Spectrometry, Chromatography and Applied Physics, National Institute for Research
and Development of Isotopic and Molecular Technologies, 67-103 Donat Street,
4
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
4
00293 Cluj-Napoca, Romania; alexandru.turza@itim-cj.ro
Biomolecular Physics Department, Faculty of Physics, Babe s¸ -Bolyai University, 1 M. Kogalniceanu Street,
00084 Cluj-Napoca, Romania
Correspondence: ioana.gaina@ubbcluj.ro; Tel.: +40-26-459-3833
5
Citation: Stoean, B.; Rugina, D.;
Focsan, M.; Craciun, A.-M.; Nistor,
M.; Lovasz, T.; Turza, A.; Porumb,
I.-D.; Gál, E.; Cristea, C.; et al. Novel
4
*
Abstract: We report here the synthesis and structural characterization of novel cationic (phenoth-
iazinyl) vinyl-pyridinium (PVP) dyes, together with optical (absorption/emission) properties and
their potential applicability as fluorescent labels. Convective heating, ultrasound irradiation and
mechanochemical synthesis were considered as alternative synthetic methodologies proficient
for overcoming drawbacks such as long reaction time, nonsatisfactory yields or solvent require-
ments in the synthesis of novel dye (E)-1-(3-chloropropyl)-4-(2-(10-methyl-10H-phenothiazin-3-
yl)vinyl)pyridin-1-ium bromide 3d and its N-alkyl-2-methylpyridinium precursor 1c. The trans geom-
etry of the newly synthesized (E)-4-(2-(7-bromo-10-ethyl-10H-phenothiazin-3-yl)vinyl)-1-methylpyri
din-1-ium iodide 3b and (E)-1-methyl-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium
(
Phenothiazinyl)Vinyl-Pyridinium
Dyes and Their Potential
Applications as Cellular Staining
Agents. Int. J. Mol. Sci. 2021, 22, 2985.
https://doi.org/10.3390/
ijms22062985
Academic Editor:
Konstantin Turoverov
0
tetrafluoroborate 3a was confirmed by single crystal X-ray diffraction. A negative solvatochromism
Received: 21 February 2021
Accepted: 11 March 2021
Published: 15 March 2021
of the dyes in polar solvents was highlighted by UV-Vis spectroscopy and explanatory insights were
supported by molecular modeling which suggested a better stabilization of the lowest unoccupied
molecular orbitals (LUMO). The photostability of the dye 3b was investigated by irradiation at 365 nm
in different solvents, while the steady-state and time-resolved fluorescence properties of dye 3b and
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
0
3a in solid state were evaluated under one-photon excitation at 485 nm. The in vitro cytotoxicity of
the new PVP dyes on B16-F10 melanoma cells was evaluated by WST-1 assay, while their intracellular
localization was assessed by epi-fluorescence conventional microscopy imaging as well as one- and
two-photon excited confocal fluorescence lifetime imaging microscopy (FLIM). PVP dyes displayed
low cytotoxicity, good internalization inside melanoma cells and intense fluorescence emission inside
the B16-F10 murine melanoma cells, making them suitable staining agents for imaging applications.
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/
Keywords: (phenothiazinyl)vinyl-pyridinium (PVP) dyes; sonochemistry; cellular staining; melanoma
cells; epi-fluorescence imaging; two-photon excited fluorescence lifetime imaging
4
.0/).
Int. J. Mol. Sci. 2021, 22, 2985. https://doi.org/10.3390/ijms22062985
https://www.mdpi.com/journal/ijms

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1. Introduction
In the last years, the methine dyes gained special attention in bioimaging due to
their high molar extinction coefficients, high fluorescence quantum yields, large range of
absorption and emission wavelengths reaching near infrared (NIR) domain and a good
ability to penetrate through the cell membranes. The applications of methine dyes in the
cell imaging are based on their selective accumulation in the target cellular organelles,
cytoskeletal components, DNA, RNA G-quadruplexes [
and many other biological macromolecules.
1], nucleotides, neurotransmitters,
Fluorescent probes, particularly emitting in NIR, based on polymethine dyes [
2],
polyfluorinated cyanine dyes [ ], and cyanine-benzothiazole hybrid system [ ] revealed
3
4
high selectivity towards mitochondria. The lysosomal compartment of different cell lines
such as HF-P4, BLM, U-2 OS and A-2058 were selectivity labeled by fluorescent Coumarin
Troger’s base derivatives with cyanine substituents [5]. The selective staining of the
cytoplasm was achieved by using different classes of organic dyes. For example, Schiff
bases-boron complexes were successfully used for the in vitro cytoplasmic staining in B16-
F10 murine melanoma cells [
employed in the selective staining of cytoplasm in live mouse embryonic fibroblasts [7].
Other methine derivatives such us triphenylmethane Brilliant Blue G [ ] and styryl
dye LDS 820 [ ], respectively, can be used as fluorescent NIR fluorescent labels for amyloid
6], while fluorophores containing thiophene moieties were
8
9
fibrils, which are associated with numerous neurodegenerative diseases. A large number
of commercially available or newly synthesized styryl dyes are currently in use or under
investigation for their ability to act as staining agents in cell and tissue imaging [10].
Continuing our previous investigations dedicated to the synthesis and characterization
of (phenothiazinyl)vinyl-pyridinium (PVP) dyes [11], in this work the series of cationic
0
methine dyes was extended with three new representatives (i.e., 3b
,
3a and 3d), exhibiting
structural adjustments designed to modulate their optical properties by introducing an
auxochromic bromo-substituent at the electron-donor phenothiazine unit 3b and to modify
the aggregation properties of the dye by attaching different alkyl chains and varying
0
the counter ion at the pyridinium electron withdrawing unit 3a
,
3d. In our pursuit for
finding greener synthetic alternatives to the traditional methods to carry out organic
synthesis, in our previous experiments the PVP dyes were conveniently synthesized by
microwaves assisted condensation of phenothiazine carbaldehyde with methylpyridinium
salts in dry media, while in this work we examine the benefits of ultrasound assisted
and mechanochemical synthetic procedures, as candidates for environmentally friendly
protocols proficient for overcoming drawbacks such as long reaction time, nonsatisfactory
yields or solvent requirements. Based on the documented potential of methine dyes for
practical applications as fluorescent markers the photophysical behavior of PVP dyes
in biological environment (i.e., B16-F10 murine melanoma cells) was investigated via
fluorescence imaging, as part of our search for novel cellular staining agents.
2. Results and Discussion
2.1. Synthesis of PVP
The synthetic path presented in Scheme 1 contains two reaction steps, the preparation
of the starting pyridinium salts 1a–c by the alkylation of 4-picoline with halogenated alkyl
derivatives, followed by the preparation of PVP dyes 3a–d by Knoevenagel condensation
of phenothiazine-carbaldehydes with the alkyl pyridinium salts.

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Scheme 1. The synthesis of (phenothiazinyl)vinyl-pyridinium (PVP) dyes and their precursors.
Different strategies have been addressed for the optimization of the reaction conditions.
Thus, the alkylation of picoline was achieved in homogeneous media using propanol
solution under stirring or ultrasound irradiation as well as under solvent free conditions,
to produce 1-(3-chloropropyl)-4-methylpyridin-1-ium bromide 1c (in 80% yield by heating
in homogeneous solution, 86% yield under ultrasound irradiation and 68% yield under
solvent free mechanical stirring). For the synthesis of PVP dye 3d convective heating (yield
7
0%), ultrasound irradiation (yield 80%) and solvent free mechanochemical procedures
(yield 50%) were also applied, the higher reaction yields being achieved by ultrasound
irradiation of the reaction mixture. A comparison between the outcomes of the use of
traditional convective heating with nonconventional alternative energy sources such as
ultrasound and mechanical mixing emphasizes the superiority of the methods using
homogeneous reaction media for the condensation reaction. The lowest yields obtained by
applying the mechanical stirring method are explicable taking into consideration that this
method cannot benefit from the improved heat and mass transfer due to high diffusion
rates in low viscosity medium ensured by the use of a solvent. Benefitting from a better
energy efficiency, the sonochemical method gave superior results in terms of product yield
and reaction rate as compared to convective heating procedure.
The structures of PVP dyes were assigned based on high resolution NMR spectra (ESI
1
0
Figures S1–S4). In the H NMR spectra of each PVP dyes 3a–d, 3a the signals of the vinyl
protons appeared highly deshielded and split in doublets with vicinal coupling constants
3
J 15.6 -16.5 Hz, pointing towards geometrical trans-isomers, in agreement with the stereo-
chemistry of the Knoevenagel condensation which produce the thermodynamically stable
E-alkenes. The same geometry was found in solid state by single crystal X-ray diffraction
0
(
XRD) for dyes 3a and 3b.
2
.2. X-ray Crystallographic Data
0
Crystal and molecular structure of dyes 3a and 3b were determined by single crystal
XRD. The compounds crystallize in the centrosymmetric P2 /c space group of monoclinic
1
crystal system.

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0
The molecular structure for the dyes 3a and 3b is illustrated in Figure 1 which shows
that the asymmetric unit consists of one phenothiazinyl vinyl-pyridinium cation and one
−
0
counter anion: tetrafluoroborate (BF4 ) in 3a or iodide in 3b, respectively.
0
Figure 1. Molecular structure plots for: (
a
) PVP 3a with tetrafluoroborate counter anion, (
b) PVP 3b with iodide
counter anion.
The E configuration of the double bond linking the phenothiazine and pyridinium
units which was assumed based on NMR data recorded in solution, was confirmed in the
0
solid state by XRD on crystals. The length of the vinyl double bond is 1.328 Å in 3a , and
1
.338 Å in 3b, respectively, both shorter than the reported value for dye 3c (1.343 Å [11]).
0
The folding angle between the aromatic rings of the phenothiazine unit displayed by 3a
was 179.45 and 170.13 for 3b, respectively, higher values as compared to 3c (146.9 [11]),
◦
◦
◦
suggesting that the phenothiazine conformation was shaped towards a planar geometry;
these structural features are supporting a better conjugation of the
PVP dyes 3a and 3b.
π
-electron system in
0
The nature of intermolecular interactions which assure the crystal structure stability
is by far driven by weak van der Waals C-H...C and ... interactions (Figure 2a). The
π
π
molecules are found to be aggregated in distinct stackings related by a two-fold screw axis,
◦
the angle between these stacking being 53.60 . The molecules in each stack are parallel to
each other, but there are two values recorded for the distances between the planes passing
through two adjacent molecules: one distance is 3.537 Å demonstrating
π
...
π
interactions
interactions
(
(
C4 ... C16 and C14 ... C2) and the other distance is 3.452 Å, presenting C-H ...
C9-H8A ... C16). For the dye 3b, C-H ... S interactions were shown to behave similarly to
π
conventional hydrogen bonds, although the sulphur is mildly electronegative compared to
oxygen [12].
The molecules of dye 3b are oriented in the oa-direction forming chain-like arrange-
ments, with iodide ions located in the spaces between these chains (ESI Figure S5).
−
+
Although the iodide anion (I ) is not found even near the pyridinium cation (N ), it
+
interacts with five other N nitrogen from different neighboring molecules with distances
in the range 3.938–5.664 Å (Figure 2b), these multiple electrostatic interactions contributing
to the stability of the compound.
0
The 3D supramolecular arrangements and structural stability of the compound 3a are
assured by a combination of C-H...F interactions between phenothiazine and pyridinium
cation to tetrafluoroborate anion and π... π interactions between the phenyl ring of phe-
nothizine to aromatic pyridinium fragment (Figure 2c). Donor to acceptor H...F contact
limits are characterized by moderate distances (2.459–2.610 Å), meanwhile the C-H...F
◦
◦
angles are situated between 126.65 and 170.56 .

Int. J. Mol. Sci. 2021, 22, 2985
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−
+
Figure 2.
(
a
) Molecular stacking in dye 3b; (
b
) the closest distances between I to N (nitrogen from pyridinium unit);
0
(
c) crystal packing of 3a seen along oc-axis.
2.3. Optical Properties
From Table 1 summarizing the structural characteristics and spectral data recorded
0
for PVP dyes 3a–d and 3a , it can be seen that the position of the absorption maxima
was not affected by neither counter ion (iodide, bromide, tetrafluoroborate), alkyl moiety
present in the pyridinium unit (methyl, butyl, 3-chloropropyl), nor N-functionalization
of the phenothiazine unit. Changes in the electronic structure, such as auxochrome para-
bromo-substitution of the phenothiazine heterocycle unit, did not play a major role in the
absorption spectra. An absorption band located around 458 nm with high molar absorption
5
−1
−1
coefficient values (
ε
~10 M
·
cm ) was observed for each PVP dye in agreement with
do not show fluorescence emission
possible * electronic transitions. PVP dyes 3a–d
π
-
π
in organic or aqueous solutions due to the presence of the halide counter ion susceptible
of photo-inducing the electron transfer to the dye’s LUMO, but show emission in solid
0
state (see discussion below and Figure 7). If the counter ion was changed in 3a with
trifluoroborate, this compound displayed emission both in solid state (720 nm) and solution
(
590 nm in water, 606 nm in MeOH, 605 nm in DMSO and acetone, respective 708 nm
0
in chloroform). A hypsochromic shift of the fluorescence emission for dye 3a in polar
protic solvents, in comparison to less polar aprotic (CHCl ) solvent was observed (see
3
0
ESI Figure S7), this behavior appearing also in the UV-vis spectra. The dye 3a displays a
relative low quantum yield in solution (e.g., in chloroform solution was 3.5% relative to
Rhodamine 101 standard).
0
The two-photon excited (TPE) fluorescence spectrum of dye 3a in DMSO solution
after excitation at 800 nm shows the emission maxima at 605 nm (see ESI Figure S8), value
corresponding to the one-photon excited fluorescence in DMSO.
The chemical stability and aggregation of the PVP dyes in solution was estimated by
monitoring the changes in the position of the visible absorption maximum, in different
solvents, upon continuous light irradiation (
λ
= 365 nm) and temperature increase up to
◦
40 C.

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0
Table 1. The structural characteristics, absorption and emission data recorded for PVP 3a
–
d
and 3a
.
Stokes
Shift
a
b
λ
λ
em
abs
Cpd.
0
−
−1
(cm )
R
H
H
Br
H
H
R
R”
X
(nm)
455
455
460
458
459
(nm)
749
Ref.
−
3
a
–CH3
–CH3
–CH3
–CH3
–CH3
I
8627
8089
6354
6886
6861
[11]
0
−
3
a
BF4
720 (605 *)
650
−
3
b
–C H
I
2
5
−
3
c
–CH3
–C H
I
669
[11]
4
9
−
3
d
–CH3
–(CH ) -Cl
Br
670
2 3
a
Conc. 10 M in DMSO solution; b λem in solid state for dyes 3a
–d and 3a ; * λem in DMSO solution (Conc.
0
0
−4
− 0
8
1
M) for 3a .
The PVP dyes appeared to be stable in water and TRIS-EDTA buffer solution, as ESI
Figure S9 shows unchanged pattern for the absorption spectrum displayed by dye 3b
in buffer solution after irradiation at 365 nm up to 90 min. It is known that in solution,
methine dyes can form aggregates imparting different photophysical behavior from those
observed for the monomeric species [13], but in the case of PVP in water solution there is no
evidence of aggregation, as it can be seen from the UV-Vis absorption spectra of dye 3b in
water recorded at different dye concentrations (Figure 3a). The absorption pattern typical
for a monomeric dye occurs in the linearity domain of Lambert Beer law, as presented in
Figure 3b.
Figure 3.
(a) UV-Vis absorption spectruma of dye 3b in water at different concentrations (5.28–169 µM); (b) plot of
absorbance maximum versus concentration.
Photostability is an important parameter in bioimaging applications and for this
reason the possible degradation of dye 3b under UV light irradiation was investigated by
UV-Vis spectroscopy in different solvents. Thus, solutions of 3b in water, DMSO, acetone,
chloroform, DCM and methanol, respectively, were irradiated with UV light at
λ = 365 nm
for 30 and 60 min, respectively, at room temperature and then the UV-Vis spectra were
recorded (Figure 4 and ESI Figures S10 and S11). No degradation of dye 3b was observed
neither in water nor in organic solvent. The spectra presented reveal that the exposure of
dye 3b at UV light do not induce hydrolytic effects and the optical properties were not
affected in water solution (Figure 4a). In the presence of less polar solvents (e.g., acetone,
chloroform) an hyperchromic effect proportional to the increase of irradiation time was
recorded, as shown in Figure 4c,d.

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Figure 4. Photostability of dye 3b under UV light irradiation (λex = 365 nm) for 30 and 60 min in solution: (a) water;
(
b) DMSO; (c) acetone; (d) chloroform.
The hyperchromic effect, more important in chloroform and methylene chloride
(
Figure 4d and ESI Figure S11) may possibly be due to an aggregation of the dye in the
◦
halogenated solvents. The hyperchromic effect appeared enhanced upon heating at 40 C
(
ESI Figure S12).
A hypsochromic shift of the visible absorption maxima was noticed for dye solutions
in polar protic (water, methanol) or aprotic (DMSO) solvents, in comparison to less polar
0
aprotic (CH Cl , CHCl ) solvents. The UV-Vis absorption maxima of dyes 3b
,
3d and 3a in
2
2
3
different solvents are summarized in supporting info ESI Table S1. For example, in Figure 5
0
is depicted the solvatochromism displayed by dyes 3b and 3a in solvents of different
polarities, emphasizing a red shift from 444 nm in water to 495 nm in DCM solution. The
−
0
modification in counter ion, iodide in 3b and tetrafluoroborate (BF4 ) in 3a , does not
induce changes in the UV-Vis absorption spectra.
In order to comprehend the negative solvatochromism displayed by the dyes 3a–d,
the experimental results were corroborated with the results of a computational approach
estimating the energy gap between the frontier molecular orbitals in vacuum and in the
presence of different solvents. The initial geometry optimization of the PVP dyes structure
obtained by molecular mechanics calculations in Spartan’06 [14] was used in quantum
calculations at semi-empirical molecular orbitals (PM3) and 6–31G(d) B3LYP DFT level of
theory [15
–17]. The generated lowest energy conformer of PVP dye was further used in the
calculus of quantum molecular parameters such as EHOMO, ELUMO and energy gap
∆
E (
∆E
HOMO–LUMO) in vacuum and methanol, DMSO, acetone, chloroform, and DCM solvents,
respectively.

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0
Figure 5. UV–Vis absorption spectra dye in different solvents: (a) 3b and (b) 3a .
The computed electron distribution in the molecular orbitals of PVP dyes 3a–d indi-
cated occupied frontier molecular orbitals (FMO) HOMO and HOMO-1 located predom-
inantly on the phenothiazine unit, whereas unoccupied molecular orbitals LUMO and
LUMO+1 were located predominantly on the pyridine core (plots of FMO for 3a–d ESI
Table S2). In Figure 6 are displayed the frontier molecular orbitals plots for dyes 3b and 3d
indicating the corresponding HOMO-LUMO energy gaps in two different solvents.
Figure 6. Plots of the frontier molecular orbitals and their energy in DMSO and DCM solvents for
dyes 3b and 3d computed at DFT level of theory.
Higher values for the computed FMO energy levels were found systematically in
the simulated presence of a solvent compared to vacuum conditions (ESI Table S3). The
noticeable increase of the LUMO energy levels in the presence of the polar solvent DMSO
is supporting the assumption of a blue shift of the absorption maximum generated by the
vertical mono-electron excitation process, in total agreement with the UV-vis absorption
experimental data.

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Going further, we investigated the steady-state and time-resolved fluorescence emis-
0
sion properties of dyes 3b and 3a in solid state, under one-photon excitation at 485 nm.
0
Upon excitation at 485 nm the novel 3b and 3a dyes displayed distinct fluorescence emis-
sions in the solid state (λem = 650 nm for 3b and λem = 720 nm for 3a ), likewise previously
0
reported PVP dyes 3a and 3c (λem = 749 nm for 3a and λem = 668 nm for 3c [11]).
0
Figure 7 presents the OPE-FLIM image of dyes 3b and 3a red powders deposited on
microscope cover glass along with the corresponding lifetime histograms of the images
and illustrative fluorescence spectra extracted from two regions of the samples.
0
Figure 7. OPE-FLIM image of dyes 3b
from ( ). (
image from (a,d). Excitation at 485 nm.
(a
) and 3a
(d
) in solid state. (
b,
e
) Lifetime histograms corresponding to FLIM image
a,
d
c,f) Representative one-photon excited fluorescence spectra extracted from the areas marked with 1–2 in FLIM
0
The recorded OPE-FLIM images demonstrates that dyes 3b and 3a display strong
fluorescence emission in solid state, under the used excitation conditions. Moreover, in
the case of dye 3b the lifetime histogram profile of FLIM image reveals the presence of
two lifetime components: one at 0.65 ns with the highest contribution and another one at
2
.3 ns with a lower contribution, which could be related to the degree of intermolecular
interactions occurring within the imaged aggregates which affect the excited state dynamic,
considering that the fluorescence lifetime decreases when the energy transfer increases in
0
molecular aggregates (Figure 7b). Similarly, the lifetime histogram profile of dye 3a reveals
two lifetime components, at around 1.25 and 3 ns, also with a lower contribution (Figure 7e).
Figure 7c,f presents two representative fluorescence spectra, for each of the dyes 3b and
0
3a
, extracted from the two different marked regions in the recorded OPE-FLIM images.
As seen in the spectra, the one-photon excited emission of dye 3b in solid state is localized
at around 655 nm with a small variation due to above mentioned aggregation effects, while
0
the maximum emission of dye 3a is localized around 720 nm.
Finally, to evaluate the potential of PVP dyes 3a–d for biological applications, we
assessed in vitro cytotoxicity by WST-1 assay on B16-F10 melanoma cells, while their cellu-
lar uptake and intracellular localization were analyzed by epi-fluorescence conventional
imaging as well as one- and two-photon excited confocal fluorescence microscopy.

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2.4. Staining Investigation on B16-F10 Melanoma Cells
2.4.1. Cell Viability
Dyes 3a
viability after 24 h of treatment (0–15
B16-F10 cells in the concentrations range used, which qualifies them as potential molecules
able to stain tumoral cells. For instance, the highest concentrations (13.7 and 14.7 M) of
dyes reduced viability with 24–30% (dye 3a), with maximum 34% (dye 3b), with 37% (dye
) compared to untreated control. No effect on the B16-F10 cell viability was observed
in dye 3d case. In this context, for further cellular imaging assays, the concentration of
3.7 µM was selected.
–
d
used herein to stain cells were tested on B16-F10 melanoma cells for their
µM). Figure 8 attests that no dye is cytotoxic on
µ
3d
1
Figure 8. Cell viability assessed by WST-1 assay. Dyes’ influence on B16-F10 melanoma cells treated viability after 24 h of
treatment, ( ) Dye 3a; ( ) Dye 3b; ( ) Dye 3c; ( ) Dye 3d. Data are expressed as mean SD (n = 3). Statistically significant
difference noted as *, ns—means statistically non-significant, for a value of p < 0.05, compared with untreated control.
a
b
c
d
±
2.4.2. Fluorescence Imaging
The cellular uptake of all synthesized PVP dyes 3a–d was the first step to evaluate their
potential for cellular staining. After incubating B16-F10 melanoma cells with dyes for 24 h,
the epi-fluorescence conventional images were acquired to evaluate their cellular uptake
capability. As shown in Figure 9a–d, PVP dyes 3a–d can be well-swallowed by B16-F10
melanoma cells, observing the green fluorescence in all cases, indicating consequently the
good cellular uptake capability.

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Figure 9. (a–d) Staining B16-F10 cells with dyes 3a–d appearing colored in green in conventional epi-fluorescence imaging.
Nuclei were stained with DAPI (blue). Excitation with blue light (450–490 nm). (e–h) Confocal OPE-FLIM images of B16-F10
melanoma cells incubated with dyes 3a–d. Excitation at 485 nm.
Specifically, Figure 9a shows that uptake of dye 3a was uniform in the cytoplasm
and nucleus by B16-F10 cells and stained both nuclei and cytoplasm of the cells as green
fluorescence can be clearly observed in both regions. Considering that nucleic acids are
mainly distributed in the nucleus, the collected fluorescence images led us to assume
that the dye 3a is able to simply diffuse through plasma membrane as well as nuclear
membrane, interacting with these two entities (i.e., DNA/RNA). However, when compared
with the following used dyes, dye 3a is less promising for efficient cells staining and—
implicitly—for the next fluorescence-based imaging applications. Dyes 3b (Figure 9b)
and 3d (Figure 9d) can be clearly seen in entire cytoplasm, but with high tendency to
accumulate in the perinuclear region. Even though experimentally determined octanol-
water partition coefficients for dye 3b comprising the hydrophobic 3-bromo-10-ethyl-
phenothiazine moiety (log p = 0.74) and dye 3c containing longer alkyl chain attached to
the pyridinium unit (log p = 0.71) indicate comparable overall hydrophobicity properties
(see ESI Figures S13 and S14), the existence of n-butyl chain seemed to favor the ability of
dye 3c to pass through the nuclear pores (Figure 9c).
It is important to note that no decrease of the fluorescence intensity was observed
during recording the fluorescence imaging, concluding that dyes do not present pho-
tobleaching in the cellular environment and, consequently, are well-suited as cellular
stain probes. The studied dyes appeared suitable for imagistic applications taking into
consideration their good ability of light absorption spotted by their strong absorption
maxima situated in the blue region of the Vis spectrum (455–460 nm with extinction
5
−1
−1
coefficients ε = 10 M cm ). Even though the dyes do not exhibit intense fluorescence
emission in solution or solid state, the very good separation of the excitation and emission
maxima (Stokes’s shifts 190–294 nm) as well as their obvious fluorescence in biological
environment, are overcoming this drawback.

Int. J. Mol. Sci. 2021, 22, 2985
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To better understand the localization of dyes 3a
–
d
inside the investigated cells we per-
formed confocal imaging assays on B16-F10 cells incubated with dyes 3a–d. Representative
OPE-FLIM images obtained under excitation at 485 nm are shown in Figure 9e–h along
with the intensity and lifetime scale bars. The results confirm that dye 3c has the highest
tendency to diffuse through the nuclear membrane and accumulate into nuclei, while dyes
3a, 3b and 3d are more suitable for the staining of the cytoplasmic region, considering that
only a small fluorescence signal can be detected from the nuclear region. Additionally, the
employed FLIM technique offers the advantage of discriminating different staining agents
based on their fluorescence lifetime. Here, the fluorescence of dyes 3a–d display different
lifetime inside cells, the shortest one being notably the emission of dye 3b.
Therefore, according to the fluorescence bioimaging studies it can be mentioned that
all synthesized compounds revealed their potential application as enhanced cytoplasmic
staining dyes with a low cytotoxicity. Moreover, confocal OPE-FLIM assays confirm that
dye 3c has a better nuclear staining capability.
Further, having in mind the possibility of using the synthesized PVP dyes as NIR
staining agents for TPE fluorescence imaging, a well-known noninvasive label-free and
high-resolution technique, we performed imaging assays on the same B16-F10 melanoma
cells incubated with the dyes 3a–d, under TPE at 800 nm. Nowadays, this in vitro non-
linear bioimaging technique represents a modern and high-performance tool for relevant
biological and living tissue research, a real alternative to traditional OPE, due to its unique
advantages, such as improved sensitivity and axial resolution, lack of background signal
and photodamage of living tissue, improved tissue penetration depth and cell viability,
as well as reduced photobleaching [18]. Moreover, confocal TPE fluorescence imaging
microscopy can be greatly improved when combined to FLIM approach, due to the im-
proved sensibility and contrast that can be achieved through the mapping the fluorescence
lifetime, as it is a parameter highly sensitive to environmental changes. Figure 10 presents
representative TPE-FLIM images of reference melanoma B16-F10 cells (Figure 10a) and
incubated with the different types of synthesized dyes 3a–d (Figure 10a–e).
Figure 10.
dye 3b collected inside cytoplasm. (
FLIM image from panel ( ). ( ) Lifetime histogram corresponding to TPE-FLIM image from (
inside cytoplasm compared to reference out of cell signal, from areas marked with x symbol in panel (
00 nm.
(
a
–
e
) TPE–FLIM images of control B16-F10 melanoma cells and incubated with dyes 3a
) Representative TPE fluorescence spectra extracted from the areas marked with 1-4 in
). ( ) Photostability of dye
). Excitation at
–d. (f) Lifetime decay of
g
c
h
c
i
3
8
b
c
As shown, under the employed NIR irradiation, the control unlabeled cells exhibit a
very weak TPE autofluorescence signal. In contrast, the TPE-FLIM images corresponding
to cells incubated with dyes 3a exhibit intense and traceable fluorescence signal which
–
d
proves the promising ability of the as-synthesized PVP dyes to perform as reliable NIR
staining agents.

Int. J. Mol. Sci. 2021, 22, 2985
13 of 21
After a closer examination of the images, we observed a different behavior of the
investigated dyes inside cells, under TPE excitation. Specifically, we observed that the
fluorescence of dye 3a can be detected mostly within the cellular cytoplasm with a small
amount inside the nuclei (Figure 10b). On the other hand, the signal of dye 3b is localized
mainly inside the cytoplasm (Figure 10c), being also the most intense from all samples,
whereas dyes 3c–d display a weak fluorescence signal within cytoplasm and much more
intense in the nuclei (Figure 10d–e), in good agreement with the observation from conven-
tional and OPE fluorescence imaging (see Figure 9). We believe that this behavior could be
related to a different degree of interaction that might occur, between the employed dyes
and different nuclear components. Additionally, similar to OPE-FLIM, the emission of
dye 3b displays a much shorter lifetime compared to the emission of dyes 3a, 3c and 3d,
which represent an additional advantage in imaging application as the interference with
cellular autofluorescence (few ns) becomes lower. The fluorescence lifetime histogram
extracted from the TPE-FLIM image corresponding to dye 3b (Figure 10c) and presented
in Figure 10h) reveals an average lifetime of TPE emission of 2 ns. When extracted from
an area from the cytoplasm region (marked with x in Figure 10c) the TPE fluorescence
lifetime decay displays a biexponential behavior. The fitting procedures reveal a lifetime of
2
.3 ns (35%) and a shorter one of 0.73 ns (65%), which can be assigned to different types
of molecular aggregates, in good agreement with the results from one-photon excited
fluorescence imaging of dye 3b in powder state, regarding the presence of two lifetime
components (see Figure 7b).
Furthermore, we extracted several TPE fluorescence spectra from different regions in
B16-F10 cells incubated with dye 3b, that proved to the most efficient and suitable for TPE
imaging. The spectra collected from the areas marked with 1–4 in TPE-FLIM image from
panel c (Figure 10c) are presented in Figure 10g. All spectra share similar features while the
maximum TPE fluorescence is located around 620 nm, at a lower wavelength compared
to the position of the band in solid phase, under one-photon excitation (see Figure 7c).
Finally, we investigated the stability of the TPE fluorescence signal of dye 3b inside cells,
under continuous irradiation. As shown in Figure 10i, the signal collected from the area
marked with x in panel (c) remains stable under continuous irradiation at 800 nm over
120 s tracking time, which further confirms the ability of dye 3b to perform as an efficient
and stable staining agent for NIR imaging.
3. Experimental Section
3.1. Materials and Methods
All the materials for synthesis, reagents, and solvents were obtained from commercial
suppliers and used without further purification unless otherwise noted.
Flash chromatography was performed on silica gel 60 (particle size 0.032–0.063 mm).
Thin layer chromatography was performed on Merck DCAlufolien, silica gel 60 F254
and components were visualized by UV VL-4LC. The melting points were determined in
capillaries with an Electrothermal 9100 instrument. Ultrasound irradiation was performed
on GT SONIC-P3 Ultrasonic Cleaner Washing Equipment. NMR spectra (1D, 2D-COSY, 2D-
HSQC and 2D-HMBC) were recorded at room temperature on Bruker Avance instruments
(
(
1H/13C: 400 MHz/100 MHz or 600 MHz/150 MHz) in solution (deuterated solvents
CDCl or DMSO). Elemental analyses were recorded on Thermo Scientific FlashEA
™
3
1
112. HRMS spectra recorded on Thermo Scientific LTQ Orbitrap XL. UV-Vis absorption
respective emission spectra in solvent were recorded with Perkin Elmer Lambda 35 and
Perkin Elmer LS55 spectrophotometers.
3
.2. Synthesis
The starting materials 1a
reported in our previous paper [11].
-(3-chloropropyl)-4-methylpyridin-1-ium bromide 1c
A. Classical procedure:
,
b and the dyes 3a, c were synthesized using the protocols
1

Int. J. Mol. Sci. 2021, 22, 2985
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In a round-bottom flask, 4-methylpyridine (0.051 mol, 5 mL) and 1-bromo-3-chloroprop
ane (0.05 mmol, 5.08 mL) in 15 mL of isopropyl alcohol were added. The mixture was
stirred at room temperature for 8 h and the reaction progress was monitored by TLC. The
formed solid was filtered and washed with petroleum ether leading to 10.2 g of white solid,
◦
yield 80%, m.p.120 C.
B. Ultrasound irradiation procedure:
In a round-bottom flask, 4-methylpyridine (0.051 mol, 5 mL) and 1-bromo-3-chloroprop
ane (0.05 mmol, 5.08 mL) in 5mL of isopropyl alcohol were added. The reaction mixture
was irradiated in an ultrasonic bath GT SONIC-P3/GT at 40kHz for 8 h and processed, as
indicated in the classical procedure, obtaining 11 g, yield 86%.
C. Solvent free reaction procedure:
In a round-bottom flask, 4-methylpyridine (0.051 mol, 5 mL) and 1-bromo-3-chloroprop
ane (0.05 mmol, 5.08 mL) were added, the mixture was stirred at room temperature for 8 h
and processed, as indicated in the classical procedure, obtaining 6.4 g, yield 68%.
1
2
H NMR (CDCl , 400 MHz)
δ
(ppm): 9.29 (d, 2H, J = 6.2 Hz, H Py), 7.79 (d, 2H,
3
J = 6.2 Hz, H3 ), 4.94 (t, 2H, J = 6.6 Hz, N -CH2,), 3.56 (t, 2H, J = 6.6 Hz, Cl-CH2),
.46-2.39 (m,2H, CH ), 2.46 (s, 3H, CH ), (see ESI S4).
(ppm): 159.3(qC ), 144.3(2C ), 128.7(2C ), 58.1 (N
+
Py
13
2
δ
C NMR (CDCl , 100 MHz)
2
3
3
4
2
3
+
CH2), 40.9(ClCH ), 33.8(CH ), 22.3
2 2
(
C
H ) (Figure 11). Anal. calcd. for: C H BrClN: C 43.14; H 5.23; N 5.59. Found: C 42.99;
3
9
13
H 5.25; N 5.56.
Figure 11. The structure of 1c and the numbering of the atoms in phenyl ring.
-bromo-10-ethyl-10H-phenothiazine-3-carbaldehyde 2a
7
In a 100 mL bottom flask, 0.01 mol (2.5 g) of 3-formyl-10-ethylphenothiazine was
dissolved in 25 mL of glacial acetic acid. To this mixture was added dropwise 0.0078 mol
(
1.25 g) of Br , previously dissolved in 10 mL of glacial acetic acid. A reddish-brown
2
precipitate was obtained that was stirred for 2 days, then water (100 mL) was added
and the resulting yellow precipitate was filtered off. The obtained solution was stirred
with 50 mL of toluene and the organic layer was separated, was dried over Na SO and
2
4
concentrated. The product was purified by column chromatography using silica gel. It
◦
was eluted with toluene to yield the product (1.77 g, 53%) as yellow solid, m.p. 95–97 C
◦
(
literature data 99–101 C [19].
1
H NMR (CDCl , 400 MHz)
δ (ppm): 9.78 (s, 1H, CHO), 7.62 (dd, 1H, J = 8.4 Hz,
J = 1.9 Hz, H ), 7.53 (d, 1H, J = 1.9 Hz, H ), 7.23 (dd, 1H, J = 8.7 Hz, J = 2.2 Hz, H ), 7.18
3
2
4
8
6
3
9
1
(
2
1
1
d, 1H, J = 2.2 Hz, H ), 6.88 (d, 1H, H , J = 8.4, H ), 6.72 (d, 1H, J = 8.7 Hz, H ), 3.92 (q,
1
13
9a
H, CH ), 1.42 (t, 3H, CH ). C-NMR (CDCl , 100 MHz,
δ
(ppm): 189.8 (
C
HO), 149,7 (C )
3
2
3
1
0a
3
6
4
8
2
7
5a
42.2 (C ), 131.1 (C ), 130.4 (C ), 130.2 (C ), 129.6 (C ), 128.1 (C ), 125.4 (qC ), 123.7(qC ),
9
4a
1
16.6(C ), 115.7(qC ), 114.5 (C ), 42.5(CH ), 12.7(CH ), (Figure 12). HR-MS, (ESI; m/z):
2
3
Figure 12. The structure of 2a and the numbering of phenothiazine ring.
M] + calcd. for C H BrNOS, 332.9817; Found 332.9807, (see ESI Figure S15).
[
15
12

Int. J. Mol. Sci. 2021, 22, 2985
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(
E)-4-(2-(7-bromo-10-ethyl-10H-phenothiazin-3-yl)vinyl)-1-methylpyridin-1-ium io-
dide 3b
In a round-bottom vessel, 7-bromo-10-ethyl-10H-phenothiazine-3-carbaldehyde
10 mmol, 3.3 g), 1,4-dimethylpyridin-1-ium iodide (11 mmol, 2.6 g) and catalytic
(
amount of piperidine were added in 50 mL of isopropyl alcohol. The reaction mixture was
stirred at room temperature for 3 h. The formed precipitate was filtered and successively
recrystallized from ethanol, affording desired product 3.0 g as a dark red acicular crystal
◦
with 56% yield, m.p. = 250 C.
1
(ppm): 8.81 (d, 2H, J = 9.2Hz, H2 Py), 8.13 (d, 2H,
H NMR (DMSO-d , 400 MHz)
J = 6.5 Hz, H ), 8.13 (d, 2H, J = 6.5 Hz, H3 ); 7.90 (d, 1H, J = 16 Hz, H vinyl); 7.54-7.55
δ
6
2
2
Py
Py
m, 2H, H2
,4
); 7.40 (d, 1H, J = 16 Hz, H
1
; 7.36-7.35 (m, 2H, H Ptz), 7.11 (d, 1H,
8,6
(
Ptz
vinyl)
J = 9.0 Hz, H9 ), 6.98(d, 1H, J = Hz, H
1
) 4.23 (s, 3H, N CH3); 3.94 (q, 2H, C
+
H
Py
2); 1.29 (t,
Ptz
Ptz
1
3
4
3
(
1
H, CH3), (see ESI S1, S2). C NMR (DMSO-d , 100 MHz)
δ
(ppm): 146.0(qC ), 145.5
6
9a
2
10a
3
6
4
8
2
qC ), 145.3(2C ), 143.0(qC ), 139.8(qC ), 130.8(C ), 130.2(C ), 129.4 (C ), 129.3 (C ),
Py
26.3 (C vinyl), 124.8(C vinyl), 123.5 (2C ), 122.8 (qC ), 121.8 (qC ), 117.7 (qC ), 116.1
Py
2
1
3
7
5a
4a
9
1
+
(C ), 114.7 (C ), 47.2 (N
CH ), 42.0 (NCH ), 12.7 (CH ) (Figure 13). Anal. calcd. for:
3 2 3
C H BrIN S: C 47.93; H 3.66; N 5.08; S 5.82. Found: C 48.05; H 3.70; N 5.10; S 5.80.
22
20
2
Figure 13. The structure of 3b and the numbering of the atoms in the molecule.
(
E)-1-(3-chloropropyl)-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium bro-
mide 3d
A. Classical procedure:
In a round-bottom vessel, 10-methyl-10H-phenothiazine-3-carbaldehyde (10 mmol,
.4 g), 1-(3-chloropropyl)-4-methylpyridin-1-ium bromide (11 mmol, 5.7 g) and catalytic
2
amount of piperidine were added in 50 mL of isopropyl alcohol. The reaction mixture
was stirred under reflux for 16h and the reaction progress was monitored by TLC. The
reaction mixture was cooled, the precipitate was filtered and successively recrystallized
from ethanol/acetonitrile (10:1, v/v) and isopropyl alcohol, affording desired product 3.1 g
◦
as a dark red solid with 70% yield, m.p. = 250 C.
B. Ultrasound irradiation procedure:
In a pear-shaped flask, 10-methyl-10H-phenothiazine-3-carbaldehyde (1 mmol, 0.24 g),
1
-(3-chloropropyl)-4-methylpyridin-1-ium bromide 7 (1.1 mmol, 0.57 g) and catalytic
amount of piperidine were added in 15 mL of ethylic alcohol. The reaction mixture
was irradiated in an ultrasonic bath GT SONIC-P3/GT at 40 kHz for 72 h. The reaction
mixture was filtered and processed, as in the classical procedure above, obtaining 0.36 g,
yield 80%.
C. Mechanochemical procedure:
In a mortar 10-methyl-10H-phenothiazine-3-carbaldehyde (1 mmol, 0.24 g), 1-(3-
chloropropyl)-4-methylpyridin-1-ium bromide 7 (1.1 mmol, 0.57 g) and catalytic amount
of piperidine were grinded with a pestle for 1h. The reaction mixture was successively
recrystallized from ethanol/acetonitrile (10:1, v/v) and isopropyl alcohol, affording desired
product 0.22 g (50% yield).
1
(ppm): 8.94 (d, 2H, J = 5.7Hz, H2 ); 7.96 (d, 2H,
H NMR (CDCl , 600 MHz)
δ
3
Py
3
); 7.59 (d, 1H, J = 16 Hz, H2
; 7.34 (d, 1H, J = 7.6 Hz, H2ptz); 7.19 (s, 1H,
J = 5.7 Hz,
H
Py
vinyl)
4
); 7.01(t, 1H, J = 7.6 Hz, H ptz); 6.93 (d, 1H, J = 16 Hz, H1vinyl); 6.89 (d, 1H, J = 7.1 Hz,
8
H
H
Ptz
Ptz
6
); 6.78(t, 1H, J = 7.1 Hz, H7 ); 6.63(d, 1H, J = 7.6 Hz, H2Ptz); 6.60 (d, 1H, J = 7.6 Hz,
Ptz

Int. J. Mol. Sci. 2021, 22, 2985
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H1 ); 4.89 (t, 2H, N CH2); 3.55(s, 3H, N-CH3); 3.50 (t, 2H, Cl-CH2), 2.37-2.42(m, 2H, CH2),
+
Ptz
13
4
9a
(
(
1
see ESI S3a,b). C NMR (CDCl , 150 MHz)
δ
(ppm): 153.7 (qC ), 147.6 (qC ), 143.9
3
Py
v1
2C ),140.9 (qC10a), 140.0 (qC ), 129.1 (C ), 128.9(C ), 127.7 (C ), 126.9 (C ), 126.2(C ),
2
3
2
v2
4
6
Py
23.9 (2C3 ), 123.4(C ), 123.1(qC ), 121.5 (qC ), 120.1 (C ), 114.6 (C ), 114.3 (C ), 57.6
8
4a
5a
7
9
1
Py
+
(
N
C
H ), 41.1 Cl-
2
C
H ), 33.7 (N
2
C
H ), 22.3 (
C
H ). Anal. calcd. for C H BrClN S; C 58.30;
3
2
23 22
2
H 4.68; N 5.91; S 6.77. Found: H 4.68; C 58.32; N 5.90; S 6.79.
(
E)-1-methyl-4-(2-(10-methyl-10H-phenothiazin-3-yl)vinyl)pyridin-1-ium tetrafluo-
0
roborate 3a
To a solution of dye 3a (9 mmol, 4.12 g) dissolved in 20 mL of MeCN, sodium tetrafluo-
roborate (10.9 mmol, 1.19 g) was added in a single portion. The reaction mixture was stirred
at room temperature for 5 h and a small amount of reddish-brown precipitate was observed.
The reaction mixture was concentrated by vacuum distillation, cooled and the solid residue
was filtered and recrystallized repeatedly from MeCN to give a tetrafluoroborate salt in
◦
8
5% yield (3.2 g) as a red-brown, microcrystalline solid m.p. 246 C with decomposition.
The single crystal was obtained by slow evaporation of a solution formed by dissolving
0
the compound 3a in an acetone:toluene mixture, 1:0.5 (v/v).
1
+
H NMR (400 MHz, Acetone-d , δ, ppm): 3.47 (s, 3H, N–CH ), 4.51 (s, 3H, N CH ),
.02–7.07 (m, 3H, H , H , H ), 7.18 (dd, 1H, J = 7.64 Hz, J = 1.5 Hz, H ), 7.26 (td, 1H,
J = 7.68 Hz, J = 1.5 Hz, H ), 7.47 (d, 1H, J = 16.3 Hz, H
dd, 1H, J = 8.5Hz, J = 1.8Hz, H ), 7.99 (d, 1H, J = 16.3 Hz, H vinyl), 8.27 (d, 2H, J = 6.7 Hz
H3Py), 8.92 (d, 2H, J = 6.7 Hz,
6
3 3
1
9
7
6
7
8
1
4
), 7.59 (d, 1H, J = 1.8Hz, H ), 7.63
vinyl
2
2
(
,
2
19
H
Py).
F
NMR (376 MHz, Acetone-d6,
δ
, ppm):
−
152.8.
Anal. calcd. for C H BF N S: C 60.30; H 4.58; N 6.70; S 7.67; Found: C 60.45; H 4.60; N
2
1
19
4
2
6.68; S 7.65.
3.3. X-ray Diffraction
Single crystal X-ray diffraction and refinement (SC)
A suitable single crystal of studied compound was placed on a fine nylon loop, was
coated in inert oil (PARATONE N) and mounted on the goniometer of a SuperNova
diffractometer. The diffractometer is equipped with dual micro-sources (Mo and Cu),
EoS CCD detector and the tube operates at 50 kV and 0.8 mA. The diffraction intensities
were collected at 120 K using CrysAlis PRO software [20]. The Lorentz, polarization and
absorption factors were corrected as well in CrysAlis PRO [20].
The crystal structure of dye 3b was solved by Direct Methods with SHELXS [21
]
solution program and refined by least squares minimization with SHELXL [22], which are
implemented in Olex2 software [23]. Nonhydrogenoid atoms were localized in the Fourier
difference map and were further refined anisotropically considering the displacement
isotropic parameter Uiso(H) = 1.2Ueq(C) for all CH, CH groups and 1.5Ueq(C) for all
2
CH groups. Hydrogen atoms were placed in idealized positions and treated as riding as
3
follows: ternary CH refined with riding coordinates (C-H = 0.98 Å), secondary CH refined
2
with riding coordinates (C-H = 0.97 Å), and idealized CH methyl groups refined as a
3
rotating group (C-H = 0.96 Å). Detailed crystallographic and structure refinement details
are given in Table 2.
All crystal packing and molecular drawings were generated using Mercury soft-
ware [24].
0
The CIF file of dyes 3a and 3b have been deposited with the Cambridge Crystallo-
graphic Data Centre, having the associated deposition numbers 2067603, respective 2024705.
A copy can be obtained free of charge on written application to CCDC, 12 Union Road, Cam-
bridge, CB2 1EZ, UK (Fax: +44-12-2333-6033); on request via e-mail to deposit@ccdc.cam.uk
or by access to http://www.ccdc.cam.ac.uk (accessed on 12 March 2021).

Int. J. Mol. Sci. 2021, 22, 2985
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Table 2. Crystal data and structure refinement for dye 3b.
Identification Code
Dye 3b
Empirical formula
Formula weight
Temperature/K
Crystal system
Space group
a/Å
C22H BrIN S
20
2
551.27
123.1
Monoclinic
P2 /c
1
17.6219(4)
7.8451(2)
15.0465(3)
90
101.306(2)
90
2039.75(8)
4
1.795
b/Å
c/Å
◦
α/
β/
γ/
◦
◦
3
Volume/Å
Z
g/cm
µ/mm
F (000)
3
ρ
calc
−
1
15.650
1080.0
0.1 × 0.15 × 0.17
CuKα (λ = 1.54184)
10.238 to 141.238
3
Crystal size/mm
Radiation
◦
2
Θ range for data collection/
Index ranges
Reflections collected
Independent reflections
−21 ≤ h ≤ 19, −9 ≤ k ≤ 5, −18 ≤ l ≤ 18
7087
3843 [Rint = 0.0299, Rsigma = 0.0447]
3843/0/246
Data/restraints/parameters
2
Goodness-of-fit on F
1.046
Final R indexes [I > = 2σ (I)]
R = 0.0372, wR = 0.0961
1 2
Final R indexes [all data]
R = 0.0453, wR = 0.1033
1
2
−
3
Largest diff. peak/hole/e Å
1.11/−1.06
3.4. Fluorescence Measurements
3.4.1. One-Photon Excited Fluorescence on Solid Samples
One-photon excited time-resolved fluorescence measurements (OPE-FLIM) on solid
samples were performed on a MicroTime200 time resolved confocal fluorescence micro-
scope system (PicoQuant) coupled with an SR-163 spectrograph and equipped with a New-
ton 970 EMCCD camera (Andor Technology) previously described [25]. We used for excita-
tion a laser diode, operating at 485 nm and the signal was filtered by a HQ519LP (Chroma,
Foothill Ranch, CA, USA) emission filter. Images were obtained with a 60 × /NA = 1.2
water immersion objective. For the fluorescence spectra extracted from different spots in
the solid sample deposited on microscope cover glass we used an integration time of 10 s,
with the diode working in continuous mode.
3
.4.2. Fluorescence Quantum Yield
0
The calculation of quantum yield for 3a dye in solution was performed using Rhodam
ine-101 as standard and the parameters employed are summarized in Table 3.
The experimental procedure was performed according to WW ϋrth et al. [26] with the
mention that the samples were diluted due to the large difference in quantum yield between
0
the standard used Rhodamine 101 [27] and 3a , and consequently the dilution factor was
introduced in the Equation (1).
2
Ast Fx n Dx
x
2
Φx = Φst·
·
·
·
(1)
Ax Fst n Dst
st

Int. J. Mol. Sci. 2021, 22, 2985
18 of 21
0
Table 3. Required parameters to calculate relative quantum yield for 3a .
Calculated/
Reported Value
Item
Samples
Expression
Rhodamine 101
(Ethanol + 0.01%
Φst
1 [26]
Quantum yield
HCl)
0
3
a (Chloroform sol.)
Φx
Ast
Ax
Fst
Fx
0.035
0.0265
0.07
Rhodamine 101
Absorbance at 525 nm
0
3a
Area of emission
spectrum
Rhodamine 101
19327.82
16255.18
1.85423
2.085
0
3a
Average refractive
index value of solvent
Rhodamine 101
nst
nx
0
3a
102
103
Rhodamine 101
Dst
Dx
Dilution ratio
0
a
3
3.5. Cell Culture and Staining Protocol
Reagents used for cell viability studies: Dulbecco’s modified Eagle’s medium (DMEM),
fetal bovine serum (FBS), glutamine, and antibiotics (penicillin/streptomycin mix) were
purchased from Gibco (Carlsbad, CA, USA). Cell proliferation reagent WST-1 was bought
from Roche Molecular Biochemicals (Indianapolis, IN, USA).
B16-F10 murine melanoma cell line was purchased from ATCC (American Tissue Cell
Culture) being maintained in standard cultured conditions: 95% humidity, 5% of CO and
2
◦
3
7 C. Cell culture media was supplemented with 10% FBS, glutamine (2%) and antibiotics
(penicillin/streptomycin mix, 1%). For viability evaluation, each dye in concentrations
ranging from 0 to 15 µM was administered to B16-F10 cells, for 24 h.
4
For fluorescence imaging, 8
bated with 13.7
×
10 cells/well, 2-well LabTek Chambered, were incu-
◦
µ
M PVP dye for 24 h, and kept at 37 C in humidified atmosphere with
5
% CO . The next day, the cells were washed three times with phosphate buffer (1
×
PBS,
pH 7.4) to remove the uninternalized dye, then were fixed by paraformaldehyde (4%) for
0 min at room temperature. A counterstaining with DAPI (0.5 g/mL) was performed to
see the localization of the dye comparing to the nucleus. Prior to and after this step the
cells were washed with PBS 1 . Finally, the fixed B16-F10 cells were investigated through
fluorescence microscopy as described in Section 3.7.
2
2
µ
×
3.6. Viability Assay
Viability assay was performed using WST-1 cell proliferation reagent. WST-1 reagent
(15 µL/well) was incubated for 30 min with treated B16-F10 cells, meanwhile being reduced
to its dye form. Its absorbance was measured at 420 nm by a HT BioTek Synergy microplate
reader (BioTek Instrument, Inc., Winooski, VT, USA). The results were expressed as survival
percent with respect to control. Cells considered to be control, with 100% of viability, were
incubated in DMEM only with WST-1.
3.7. Fluorescence Imaging
The conventional epi-fluorescence images of B16-F10 murine melanoma cells incu-
bated with the synthesized PVP dyes 3a–d were collected with an inverted Axio Observer
Z1 Carl Zeiss microscope), employing a Compact Light Source HXP 120 C metal halide
fluorescence light source. Concretely, the fluorescence was imaged with a G 365 excitation
filter (i.e., filter set 49 from Carl Zeiss) for visualization of the cell nuclei stained with
DAPI, and an excitation filter BP 470/40 (i.e., filter set 38 from Carl Zeiss) for localizing the
synthesized dyes inside the cells. The fluorescence images were captured using a 63
×
iris

Int. J. Mol. Sci. 2021, 22, 2985
19 of 21
oil immersion objective and a Zeiss AxioCam MRm monochrome microscope camera and
then processed using the ZEN software.
Confocal one-photon excited Fluorescence Lifetime Imaging Microscopy (OPE-FLIM)
assays on the B16-F10 murine melanoma cells post 24 h incubation with dyes 3a
performed using the MicroTime200 system as described in Section 3.4. The signal was
collected with a Plan N 40 objective with numerical aperture (NA) of 0.65 and filtered by
a 50 m pinhole. The laser power was set at 5 W. For the fluorescence spectra extracted
–d were
×
µ
µ
from the OPE-FLIM images, an integration time of 2 s was used.
Two-photon excited FLIM (TPE-FLIM) assays were performed using the previously
described MicroTime200 time-resolved confocal fluorescence microscope system (from
Pico-Quant) coupled to a Mira 900 Titanium: Sapphire tunable femtosecond laser (from
Coherent), operating at 76 MHz. The incubated B16-F10 cells were imaged under the
microscope under 800 nm laser beam excitation operating at 10 mW power, using the same
system configuration described previously [28]. The signal was subsequently collected
using a Plan N 40× objective with numerical aperture (NA) of 0.65 and spectrally filtered
by a FF01-750 SP emission filter (Semrock, USA). The TPE-FLIM images were acquired and
analyzed using the SymPhoTime 64 software provided by PicoQuant. TPE lifetime decay
curves and time traces were recorded from different regions of interest in the TPE—FLIM
images. OPE and TPE fluorescence spectra were extracted from FLIM images by using a
SR-163 spectrograph equipped with a Newton 970EMCCD camera from Andor Technology
coupled to an exit port of the main optical unit of MicroTime200 through a 50 µm optical
fiber. The integration time used for the TPE fluorescence spectra of dye 3b collected inside
0
cells was 60 s, while for the fluorescence spectra of dye 3a recorded in solution we used an
integration time of 2 s.
3.8. Lipophilicity Protocol
The lipophilicity protocol was adapted using previously reported methods [29,30].
(
1) Solvent preparation (water saturated with octanol and octanol saturated with water):
potassium phosphate aq buffer (0.1 M pH 7.0) and an equal volume of 1-octanol were
agitated to allow saturation, then left to separate overnight.
(
(
2) Preparation of dye stock solution: 100 µg / mL in DMSO.
3) A total of 150
absorbance of this initial aqueous solution was measured (A).
4) To the aqueous solution with the dye 1425 µL of 1-octanol (V ) was added.
w
µL of dye stock solution was added to 1425 µL of water (V ) and the
(
(
0
5) The sample was stirred for 2 h, then centrifuged (3500 rpm, 10 min) to separate the
phases.
(6) The absorbance of the separate aqueous solution (A’) was measured.
The lipophilicity of dyes 3b and 3c were calculated according to the Equation (2) and
the obtained values are presented in Table 4.
A − A0 Vw
logP = log (
·
)
(2)
A0
Vo
Table 4. Lipophilicity value for the dyes 3b–c.
0
Dye
A
A
Log P
3
3
b
c
0.535
0.453
0.088
0.080
0.748
0.711
4. Conclusions
To sum up, ultrasound irradiation is proposed as a convenient energy saving pro-
cedure for N-alkylation of pyridine derivatives as well as for Knoevenagel condensation
of methyl-pyridinium salts with phenothiazine carbaldehydes, generating high yields

Int. J. Mol. Sci. 2021, 22, 2985
20 of 21
(80–86%) of product in shorter reaction time as compared to convective heating or mechano
chemical procedures.
0
The optical properties of the PVP dyes 3a–d and 3a , described in this work are
disclosing absorption maxima situated in 440–450 nm range in water which appeared red
shifted to 495 nm when dissolved in low polarity organic solvents such as DCM and a good
photostability in solution upon irradiation at 365 nm. Additionally, the steady-state and
0
time-resolved emissive properties of dyes 3b and 3a in solid state were assessed under
one-photon excitation at 485 nm. Upon both conventional fluorescence microscopy (blue
light excitation (450–490 nm)) and confocal imaging (OPE (485 nm) and TPE (800 nm))
PVP 3a–d display fluorescence emission, making them suitable candidates for biological
cell staining, as emphasized by fluorescence microscopy images collected from stained
melanoma B16-F10 cells. All dyes were able to penetrate the plasma membrane and can
bind differently with DNA from nuclei. In this context, we assume that the synthesized
PVP dyes 3a–d described here can be successfully used as fluorescent staining agents for
in vitro imaging applications.
Supplementary Materials: The following are available online at https://www.mdpi.com/1422-006
7
/22/6/2985/s1.
Author Contributions: B.S., E.G., T.L., I.-D.P. Formal analysis, Investigation, Methodology, M.F.,
A.-M.C. Investigation, Methodology, Validation, Writing—original draft. A.T. Formal analysis
Writing—original draft; D.R., M.N. Validation, Investigation, Writing—original draft; L.I.G., C.C.
Conceptualization, Investigation, Methodology, Writing—original draft, Writing—review and editing;
S.A., L.S.-D. Supervision, project administration, funding acquisition. All authors have read and
agreed to the published version of the manuscript.
Funding: This work was supported by CNCS-UEFISCDI, Projects PN-III-P4-PCCF-2016-0142, within
PNCDI III.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data that support the findings of this study are available from the
corresponding author upon reasonable request.
Conflicts of Interest: The authors declare no conflict of interest.
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