Synthesis and characterization of benzotriazolyl acrylonitrile analogs-based donor-acceptor molecules: Optical properties, in vitro cytotoxicity, and cellular imaging

Article abstract of DOI:10.1016/j.dyepig.2021.109251

A series of eight new (E)-benzotriazolyl acrylonitrile derivatives were synthesized under reflux conditions via Knoevenagel condensation between acetonitrile analogs and a short series of aromatic aldehydes. X-ray diffraction analysis for one of the compounds was performed to determine the (E)-geometry of its double bond. The photoluminescent properties of all compounds in solution were also evaluated. These compounds exhibit strong blue, green and yellow emission under ultraviolet light excitation with fluorescence quantum yield in the 0.08–0.58% range. To determine the suitability of these compounds for use in cell-based analysis, cytotoxicity assays were performed on a representative molecule using a common mammalian cell line (HEK 293T cells) at different concentrations. The results showed limited toxicity (72 ± 16% viability) at the highest concentration tested (50 μM). Finally, confocal microscopy demonstrated that our compound was internalized into cells and localized to endosomes and/or lysosomes in a similar fashion to Dextran–Cascade Blue (DCB).

Full text of DOI:10.1016/j.dyepig.2021.109251

Dyes and Pigments 189 (2021) 109251
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Dyes and Pigments
journal homepage: http://www.elsevier.com/locate/dyepig
Synthesis and characterization of benzotriazolyl acrylonitrile analogs-based
donor-acceptor molecules: Optical properties, in vitro cytotoxicity, and
cellular imaging
Eugenio Hern ´a ndez-Fern ´a ndeza,*, Ana Sofia Ortega-Villarreal , Ma. Concepci o´ n García-L o´ pez ,
a
b
a
c
c
c
Rodrigo Chan-Navarro , Samuel Garrard , Gabriel A. Valdivia-Berroeta , Stacey J. Smith ,
c
c,**
Kenneth A. Christensen , David J. Michaelis
a
Universidad Aut o´ noma de Nuevo Le o´ n, Facultad de Ciencias Químicas, Pedro de Alba s/n, Ciudad Universitaria, 66455, San Nicol a´ s de Los Garza, Nuevo Le o´ n, Mexico
CONACYT-Universidad Aut ´o noma de Nuevo Le ´o n, Facultad de Ciencias Químicas, Pedro de Alba s/n, Ciudad Universitaria, 66455, San Nicol a´ s de Los Garza, Nuevo
b
Le ´o n, Mexico
c
Department of Chemistry and Biochemistry, Brigham Young University, 84602, Provo, UT, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of eight new (E)-benzotriazolyl acrylonitrile derivatives were synthesized under reflux conditions via
Knoevenagel condensation between acetonitrile analogs and a short series of aromatic aldehydes. X-ray
diffraction analysis for one of the compounds was performed to determine the (E)-geometry of its double bond.
The photoluminescent properties of all compounds in solution were also evaluated. These compounds exhibit
strong blue, green and yellow emission under ultraviolet light excitation with fluorescence quantum yield in the
Lysosome/endosome staining
Cellular imaging
Benzotriazole
Acrylonitrile analogs
Confocal microscopy
0
.08–0.58% range. To determine the suitability of these compounds for use in cell-based analysis, cytotoxicity
assays were performed on a representative molecule using a common mammalian cell line (HEK 293T cells) at
different concentrations. The results showed limited toxicity (72 ± 16% viability) at the highest concentration
tested (50 M). Finally, confocal microscopy demonstrated that our compound was internalized into cells and
μ
localized to endosomes and/or lysosomes in a similar fashion to Dextran–Cascade Blue (DCB).
1
. Introduction
The synthesis of acrylonitrile analogs has been pursued in recent
including anticancer [18], antituberculosis [19], antibacterial [20], and
antiproliferative [21], have been demonstrated in the literature.
The benzotriazole structural motif is also an important pharmaco-
phore in many biologically active molecules, and thus the synthesis of
compounds bearing this group has received significant interest [22].
Many bioactive heterocyclic compounds that contain a benzotriazole
fragment have exhibited outstanding properties in medicinal chemistry
including anticancer, antifungal, antibacterial, antiviral, antiparasitic,
and antioxidative activities [23]. Furthermore, the benzotriazole ring
system also undergoes a unique tautomerism phenomenon between the
1H- and 2H- forms, where the 1H-benzotriazole is the most stable in the
solid state and in solution [24,25].
years due to their unique chemical properties. They are a polyfunctional
class of molecules containing single, double and triple bonds, in
particular, vinyl and cyano groups are considered two of the most
important building blocks in organic synthesis [1]. Synthetic approaches
to this class of molecules involve Wittig reactions [2,3], McMurry
coupling reactions [4], Heck reactions [5], and more recently, conden-
sation reactions of the Knoevenagel type, using acid or base-catalyzed
condensation of active methylene moieties with carbonyl compounds
[
6–9]. In addition, the use of transition metal catalysts such as Pd, Cu, Ni
and Co, has also been described [10–12]. Acrylonitrile derivatives
bearing heterocyclic cores such as thiazoles [13], pyrazoles [14],
benzimidazoles [15], triazolopyridines [16], and benzotriazoles [17],
are considered valuable compounds and their biological activities,
The structural characteristics that allow benzotriazoles to form
hydrogen bonds and/or other type of molecular interactions, such as
π π
-
stacking and van der Waals forces, makes them capable of binding with
enzymes and receptors in biological systems [22]. For the benzotriazolyl
*
Corresponding author.
* Corresponding author.
E-mail addresses: eugenio.hernandezfr@uanl.edu.mx (E. Hern ´a ndez-Fern a´ ndez), dmichaelis@chem.byu.edu (D.J. Michaelis).
*
https://doi.org/10.1016/j.dyepig.2021.109251
Received 8 June 2020; Received in revised form 4 February 2021; Accepted 17 February 2021
Available online 23 February 2021
0
143-7208/© 2021 Elsevier Ltd. All rights reserved.

E. Hern a´ ndez-Fern a´ ndez et al.
Dyes and Pigments 189 (2021) 109251
acrylonitriles presented in this report, the combined presence of an
electron-rich donor (p-substituted benzaldehydes and benzotriazole
units) and an electron-deficient acceptor (cyano group) enables the se-
lective tuning of the photophysical and electronic properties of our
chromophores, resulting in a donor-acceptor (D-A) conjugated molec-
ular system [26,27]. In fact, D-A systems with several donor and
acceptor units have been reported for applications in chemistry, physics,
materials science, and biology, including their use in solar cells [28,29],
organic light-emitting diodes (OLEDs) [30,31], metal ions sensors
acrylonitrile derivatives 6a-d and 7a-d via Knoevenagel condensation.
Several p-substituted benzaldehydes with donor groups were screened in
the reaction, including 4-(4-morpholinyl)benzaldehyde, 4-(dimethyla-
mino)benzaldehyde, 4-(diethylamino)benzaldehyde and 4-(diphenyla-
mino)benzaldehyde 5a-d. Good yields of the desired products (42–78%)
were obtained after a short reflux (2 h) in ethanol in presence of
piperidine as base (Scheme 3). Compounds 6a-b and 7a-d were obtained
as yellow and orange solids and compounds 6c and 6d as yellow and
orange oils, respectively. Most of the products were obtained in pure
form and in good yields via filtration and washing of the resulting solids
from the reaction. The resulting benzotriazolyl acrylonitrile analogs
were soluble in common organic solvents such as acetonitrile, methanol,
ethanol, chloroform, ethyl acetate, dichloromethane, and dimethyl
sulfoxide but insoluble in hexane.
[
32–34], polymer staining applications [35], and as markers in security
documents [36,37]. Among them, the ability to dye cellular structures
for imaging purposes via fluorescence is of interest in the medical field
due to the valuable readout it can contribute to cell-based assays. Recent
advances of this technology include immunoassays that analyze
signaling pathways [38], screening for toxic chemicals [39], specific
organelle targeting and tracking [40], monitoring of cell states [41],
sensor and chemodosimeter studies of specific endogenous reactive
oxygen species (ROS) [42–44] and detecting metal ions in cancerous
cells [45].
The chemical structure of each compound was fully characterized by
1H NMR and 13C NMR and HRMS. 1H NMR data showed that vinylic
protons for compounds 6a-d appeared in a chemical shift within the
range from δ 7.62–7.78 ppm, while their corresponding isomers 7a-
d show those resonance signal with a slight displacement to higher
frequency regions (δ 8.35–8.39 ppm). It is noteworthy that compounds
6d and 7d, which incorporate the diphenylamino group in their struc-
ture, exert a greater deshielding effect on vinylic proton as can be seen in
Table 1.
In this sense, the synthesis of D-A molecules based on acrylonitrile
derivatives has received great attention in recent years due to their
chemical and biological properties; however, the synthesis of most of
these types of compounds involve various reaction steps, long reaction
times, and poor overall chemical yields. Therefore, the design and syn-
thesis of low-molecular weight compounds that present negligible
cytotoxicity and photophysical properties that enable them to be used as
cellular markers, continues to be an area of great interest today.
Taking the above precedence into account, our research groups
designed and synthesized a series of eight benzotriazolyl acrylonitrile
derivatives 6a-d and 7a-d via Knoevenagel condensation (Scheme 1).
2.2. Single-crystal X-ray diffraction analyses
The spectroscopic analysis of our benzotriazole products was not
sufficient at this stage to determine with confidence the geometry of the
double bond. Thus, in order to gain a better understanding of the
structure of our products, we grew X-ray-quality single crystals of
compound 7b, through slow evaporation of ethyl acetate/hexane solu-
tion at room temperature. The crystal structure of 7b confirmed the (E)-
geometry of the double bond and the highly planar nature of this class of
conjugated system (Fig. 1).
1
The compounds were fully characterized by NMR experiments ( H and
1
3
C) and by high resolution mass spectrometry (HRMS). The optical
properties of all the compounds were evaluated and the crystallographic
data was obtained for one compound (7b) to confirm the double bond
geometry of the product. Cytotoxicity was also determined, and confocal
microscopy characterization provide evidence for the utility of com-
pounds like 7b for bioimaging applications.
Compound 7b belongs to the triclinic space group P-1 and crystal-
lizes with two chemically similar but crystallographically different
molecules in the unit cell as shown in Fig. 2. The bond angle of
◦
◦
N2–C10–C11 (178.9 ) with torsion angle of N3–C10–C9–C6 (ꢀ 178.6 )
demonstrate that nitrile is a perfect linear group, which formed a
delocalization unit along the phenyl ring and double bond. In addition,
the dimethylamine group is also coplanar with the attached phenyl ring,
that is parallel to the benzotriazole unit.
2
. Results and discussion
2
.1. Synthesis of benzotriazolyl acrylonitriles 6a-d and 7a-d
The preparation of our desired benzotriazole derivatives 6a-d and
In the crystal, molecules are stacked through C2–H2B⋅⋅⋅N5 weak
interactions (d = 2.741 Å) within the 1D framework as shown in Fig. 3.
Detailed crystallographic parameters are included in Table 2.
7
a-d was conducted after obtaining their respective precursors 3 and 4
via bimolecular nucleophilic substitution (S 2) between benzotriazole 1
and bromoacetonitrile 2. Sonication in an ultrasonic bath led to much
faster reactions due to the presence of insoluble base (K CO ) in the
reaction, and product could be obtained in high yield in as little as 2 h
Scheme 2). The crude reaction mixture was purified by column chro-
N
2
3
2.3. Optical properties
(
The optical properties of 1H-benzotriazolyl acrylonitrile derivatives
6a-d and their corresponding 2H-tautomers 7a-d were next studied
(Table 3). Fig. 4a shows the electronic absorption spectra of 1H-ben-
zotriazolyl acrylonitrile derivatives 6a-d, and Fig. 4b shows those cor-
responding to 2H-tautomers 7a-d. In general, all the benzotriazolyl
acrylonitrile derivatives exhibit a main absorption band with maximum
wavelength ranging between 383 and 434 nm, which can be attributed
matography to give benzotriazole isomers 3 and 4 (75% vs 25%), which
result from the tautomeric effect of the benzotriazole unit. It’s important
to remark that 1H-isomer 3 is formed preferentially due to its higher
stability in the tautomeric form.
With the compounds 3 and 4 in hand, the next step was to use both
benzotriazolyl acetonitrile isomers for the preparation of the
Scheme 1. Designed and synthesized compounds in this work.
2

E. Hern a´ ndez-Fern a´ ndez et al.
Dyes and Pigments 189 (2021) 109251
Scheme 2. Synthesis of benzotriazolyl acetonitriles 3 and 4.
Scheme 3. Synthesis of benzotriazolyl acrylonitrile analogs 6a-d and 7a-d.
been previously reported for fluorescent salicylidenebenzoylhydrazones
47]. For benzotriazolyl acrylonitrile 6c a shoulder at 344 nm is also
evident and has been attributed to an n→ * transition of the nitrile
Table 1
[
1
H NMR spectroscopic data for vinylic protons in compounds 6a-d and 7a-d.
π
Compound
vinylic proton (δ, ppm)
Compound
vinylic proton (δ, ppm)
electron withdrawing group for similar molecules with donor-acceptor
design [48]. On varying the solvent polarity from aprotic to protic
from chloroform to acetonitrile and methanol, the shift of the longest
wavelength absorption band was so smaller than 7 nm (Table 3,
Table ESI-5, Fig. ESI-18, and Fig. ESI-19). This poor solvatochromic
response indicates the solvent do not affect the intramolecular interac-
tion between donor and acceptor groups in the ground state. Accord-
ingly, the optical band gap Egopt, which is in the semiconducting range
for all these molecules, decreases from 3.23 to 2.86 eV as the electron
donating character increases in each series.
6
6
6
6
a
b
c
7.72
7.66
7.62
7.78
7a
7b
7c
7d
8.37
8.36
8.35
8.39
d
to the
π
→
π
* (HOMO-LUMO) electronic transition. The position of the
maximum depends on the electronic delocalization through molecules
in both series. This delocalization is chemically modulated by varying
the electron donating groups in the para position of the terminal phenyl
ring (morpholinyl for 6a and 7a, dimethyl (diethyl) for 6b and 7b (6c
and 7c), or diphenyl for 6d and 7d) and by the nitrile electron with-
drawing group. In accordance with strong electron donating ability of
dimethyl (diethyl)amine compared to morpholine, the main absorption
bands of 6b and 7b (6c and 7c) exhibit a bathochromic shift compared
to 6a and 7a. Likewise, the lowest energy bands of the compounds with
diethylamine group (6c and 7c) appear at slightly higher energies in
relation to those reported for 6d and 7d, substituted with diphenylamine
groups. We found that 6d and 7d exhibit a bathochromic behavior with
The fluorescence spectra of 1H-benzotriazolyl acrylonitrile de-
rivatives 6a-d and their corresponding 2H-tautomers 7a-d are shown in
Fig. 5. Benzotriazolyl acrylonitriles 6a-c and their corresponding 2H-
tautomers 7a-c show broad blue and green emission bands in the
485–524 nm range, while derivatives 6d and 7d exhibit a broad yellow
peak, in agreement with the hypsochromic shift observed in the corre-
sponding absorption spectra. Moreover, the excitation and absorption
spectra are identical, which is consistent with the existence of just one
emitting state (benzotriazolyl acrylonitriles 6a-d as examples in Fig. ESI-
20).
respect to those of 6c and 7c due to their higher -delocalization, as has
π
been previously reported for trisheterocyclic systems with electron
donating amino groups [46]. The maximum absorption is also affected
by changing the position of the double bond on the benzotriazole from
the 1H or the 2H-tautomer. We found that the different electron
donating groups clearly affect the conjugated styryl segment, as has
An interesting fact is that the emission can be tuned from the blue
(6a, 6b and 7a) to the green (6c,d, and 7b,c) and yellow (7d) regions by
changing the electron-donating substituents in the para position on the
styryl ring. Stokes’ shift values are in the range of molecules that
dramatically change their geometry after excitation with non-radiative
3

E. Hern a´ ndez-Fern a´ ndez et al.
Dyes and Pigments 189 (2021) 109251
Fig. 1. Molecular structure of benzotriazolyl acrylonitrile 7b (CCDC 1864140) as determined by single-crystal X-ray diffraction analysis.
Fig. 2. Anisotropic displacement parameters depicted at the 50% probability level. Hydrogen atoms were omitted for clarity. Selected bond distances (Å) and angles
◦
(
): N3–C10 = 1.412(2); C10–C11 = 1.436(2); C6–C9 = 1.440(3); C9–C10 = 1.352(2); N2–C11 = 1.352(2); N3–C10–C9 = 122.5(1); C6–C9–C10 = 130.2(1);
N3–C10–C11 = 112.6(1).
Fig. 3. The 1D framework shown C2–H2B⋯N5 interaction within the crystal structure.
losses associated to fluorescence quenching [49]. On the other hand, the
low quantum yield measured under our experimental conditions [47,50]
stacking and coplanar arrangement exhibit high quantum yields [51].
Consequently, the elimination of either the benzotriazole (electro-
n-donating) or the nitrile (electron-withdrawing) groups, present in the
structure of this type of compounds, will allow us to get a better un-
derstanding of its photophysical properties (fluorescence quantum
yields), which are in progress and they will be reported in future studies.
Regarding fluorescence quantum yield, compounds showed values in
the 0.05–0.58% range, where a slightly higher φ value in solution was
found for most of the 2H-benzotriazolyl acrylonitrile derivatives (7a-c)
in comparison to their 1H-tautomers (6a-c), except for 7d, which
could be related due to charge transfer from the electron-donating –N
–
(
CH
2
CH
2
)
2
O, -Me
2
N (-Et
2
N) or -Ph
2
N to electron-acceptor (C
–
N)
groups, and by the donor character of benzotriazole group in the com-
pounds. Furthermore, the X-ray diffraction analyses for 7b, showed that
the relative face to face stacking distance of pairs of molecules was
7
.438 Å (Fig. ESI-17), which could also explain the low quantum yield,
as previously reported by Xue et al. for cyano-stilbene derivatives with
alkyl group, where the authors found that compounds without
π-π
4

E. Hern a´ ndez-Fern a´ ndez et al.
Dyes and Pigments 189 (2021) 109251
Table 2
2.4. In vitro cytotoxicity and cell imaging
Selected crystal data for compound 7b.
To determine the suitability of these compounds for use in cell-based
Compound
7b
analysis, we decided to perform a screening only for the compound 7b
Chemical formula
17 15 5
C H N
ꢀ 1
ꢀ 1
due to its good chemical yield (78%), high
ε (49 000 M cm ), λabs
M
289.34 g/mol
Triclinic
Crystal system
(400 nm), and large Stokes’ shift value (123 nm). Cytotoxicity assays
were performed by co-incubating various concentrations of this com-
pound with HEK 293T cells, a common mammalian cell line, and
determining cell viability after 24 h. The results of this assay showed
Space group
P-1
A
B
C
α
β
7.4380(6) Å
10.2915(6) Å
10.7640(7) Å
◦
◦
◦
limited toxicity of compound 7b for these cells up to 50
μ
M, the
65.377(4)
82.735(5)
71.907(4)
maximum tested concentration. Even at this concentration, the cells
maintained 72 ± 16% viability, indicating that the LC50 is similar to or
exceeds this value (Fig. 6). Many other “cell-safe” fluorophore con-
structs, such as Rhodamine 6G and PEI-DOCA20-Cy5.5, exhibit similar or
higher toxicities [56,57]. Therefore, compound 7b presents little risk of
toxicity in mammalian cell-based assays.
γ
V
3
712.00(9) Å
0.0414
0.1151
2
R
1
wR
2
Z
CCDC ID
1864140
Following cytotoxicity screening, we assessed how compound 7b
was internalized by cells and whether it localized to specific cellular
compartments, as occurred with similar compounds [58]. Therefore, we
co-incubated compound 7b in cell culture with dextran–Cascade Blue
(DCB), a 10 kD polysaccharide–fluorophore conjugate, and character-
ized localization of both 7b and DCB by confocal microscopy (Fig. 7).
DCB localizes to endosomes and lysosomes and is an effective marker of
these compartments within the cell [59,60]. We observed that com-
pound 7b co-localized broadly with DCB in HEK 293T cells, indicating
that 7b localizes to endosomes and/or lysosomes in a similar fashion to
DCB. A modified experiment (pulse-chase condition), in which DCB was
removed from cell media 4 h before imaging, sequesters DCB in lyso-
somes alone [61]. When this experiment was performed, DCB once again
co-localized with compound 7b, indicating that it is trafficked to lyso-
Table 3
Optical properties of benzotriazolyl acrylonitrile derivatives 6a-d and 7a-d in
CH CN.
3
4
Molecule
λ
abs
ε
M
cm
(10
Egopt
(eV)
λ
emi
Δ
ν
φ
(%)
SS
ꢀ
1
ꢀ 1
(cm )
(
nm)
(nm)
(nm)
ꢀ
1
)
6
6
6
6
7
7
7
7
a
b
c
383
394
401
406
395
400
407
434
6.07
6.49
6.67
7.39
5.72
4.90
7.40
3.18
3.23
3.14
3.09
3.05
3.14
3.10
3.04
2.86
485
490
492
555
521
523
524
577
5491
4973
4412
6612
6122
5880
5486
5710
0.08
0.05
0.09
0.58
0.15
0.12
0.11
0.55
102
96
91
d
a
b
c
149
126
123
117
143
d
somes, like DCB. These data indicate that 7b is
a
membrane-impermeable, fluid-phase marker like DCB.
The unique structure and excitation/emission profile of compound
showed a slightly lower value compared to 6d. However, according to
the desirable spectroscopic and photophysical criteria for bioimaging
applications, including absorption in the visible region (>400 nm), high
7
b, as compared to many other fluorophores, may provide special utility
as a cell-based fluorescent marker. Many fluorophores can be excited at
405 nm, but only a few emit near 550 nm as compound 7b does [62].
This large Stokes’ shift, together with its low cytotoxicity, may prove
useful for both fluorescence microscopy and flow cytometry for labeling
endosomes and lysosomes in cell-based assays. Due to its chemical
structure, it is anticipated that derivatization of compound 7b could be
performed to increase its utility in biological assays without introducing
substantial changes in the spectral character of the molecule.
ꢀ
1
ꢀ 1
absorption coefficient (>30 000 M
typically over 100 nm), our compounds successfully meet these re-
cm ), and large Stokes shift
(
quirements. The importance of this latter relies in the fact that a large
value helps to minimize cross-talk between the excitation source and the
fluorescent emission with high signal-to noise ratio. It also is useful to
avoid reabsorption of emitted photons leading to undesired background
interferences that might give false positive results and is critical to the
extremely high sensitivity of fluorescence imaging measurements
3
. Conclusions
[
52–55].
In this work, we present a straightforward and fast synthetic method
to obtain benzotriazolyl acrylonitrile derivatives with yields from 42%
CN at 25 C at concentrations from 1.90 to 2.80 × 10ꢀ 4 M.
◦
Fig. 4. Absorption spectra of a) 6a-d and b) 7a-d benzotriazolyl acrylonitrile derivatives in CH
3
5

E. Hern a´ ndez-Fern a´ ndez et al.
Dyes and Pigments 189 (2021) 109251
◦
ꢀ 4 ꢀ 6
3
Fig. 5. Emission spectra of a) 6a-d and b) 7a-d benzotriazolyl acrylonitrile derivatives in CH CN at 25 C at concentrations from 10 to 10 M.
uncorrected. Thin-layer chromatography was performed on pre-coated
sheets of silica gel 60 F254 (E. Merck). For column chromatography
2
30–400 mesh silica gel 60 (E. Merck) was used as stationary phase.
1
Proton nuclear magnetic resonance ( H NMR) data were acquired on an
Inova 300 MHz or a Bruker 400 MHz. Chemical shifts are reported in
delta (δ) units. Signals are reported as follows: s (singlet), d (doublet), t
(
triplet), q (quartet), m (multiplet). Coupling constants are reported in
1
3
hertz (Hz). Carbon-13 nuclear magnetic resonance ( C NMR) data were
acquired on an Inova at 75 MHz or Bruker 100 MHz spectrometer.
Chemical shifts are reported in ppm. Mass spectral data were obtained
using ESI techniques (Agilent, 6210 TOF).
4
.1.1. Procedure for the synthesis of benzotriazolyl acetonitriles 3 and 4
Benzotriazole 1 (1 equiv), K CO (2.2 equiv) and dry THF (30 mL)
2
3
were combined in a 250 mL flask. Then, bromoacetonitrile 2 (1.2 equiv)
was added dropwise and the reaction was left under an ultrasound bath
for 2 h. Reaction course was monitored by TLC analysis. After comple-
tion of the reaction, the solvent was removed, and the reaction mixture
was washed with water and extracted with ethyl acetate (3 × 30 mL).
Fig. 6. Cytotoxicity of compound 7b was assayed at concentrations from 0.6 to
5
0
μ
M. A decrease in cell viability compared to vehicle only control was only
2 4
The organic layer was dried over anhydrous Na SO and concentrated
noted in the 50 M treatment condition, which was not statistically significant
μ
under reduced pressure. Isolation of both isomer products was accom-
plished by column chromatography using a hexane/EtOAc (2:1) system.
(
p = 0.24). The positive control (EtOH killed cells) was significantly lower than
all other conditions. *, p < 0.05; NS, not significant.
4
.1.1.1. 2-(1H-benzo[d][1,2,3]triazol-1-yl)acetonitrile 3. (75%) Beige
to 78%. Our synthetic protocol benefits from the fact that most of our
products are easily purified by washing and filtration using ethanol. The
◦
1
solid, mp 86–87 C; H NMR (400 MHz, CD
7
1
3
2
OD): δ 5.96 (s, 2H, CH CN),
.44–7.48 (m, 1H, Harom), 7.59–7.63 (m, 1H, Harom), 7.82 (d, J = 8.4 Hz,
(
E)-geometry of the double-bond was confirmed for compound 7b by
13
H, Harom), 8.01 (d, J = 8.4 Hz, 1H, Harom); C NMR (100 MHz, CD
3
OD):
+
single-crystal X-ray diffraction. The optical properties showed that all
the compounds presented a low quantum yield of fluorescence from
δ 36.4, 110.7, 114.9, 120.5, 126.1, 129.8, 133.9, 146.8. HRMS (ESI )
calcd for C
+
8
H
7
N
4
[M+H] 159.06707, found 159.06712. Lit [63].
0
.05% to 0.58%; however, compounds 6a, 6d, and 7a-d presented a
large Stokes’ shift (greater than 100 nm) in fluorescence studies. Based
on this characteristic, compound 7b was selected to further determine
its toxicity to a common mammalian cell line (HEK 293T cells) and its
localization within these cells upon uptake. The results showed that 7b
has low toxicity and is internalized by cells and localized into endosomes
and/or lysosomes. Up to this point, these studies demonstrated that
compound 7b could be used as a cell-based fluorescent marker. Addi-
tionally, these types of derivatives could be further studied to confirm
their suitability to act as promising tools for fluorescent applications in
biological, medical, and other chemistry related fields.
4
.1.1.2. 2-(2H-benzo[d][1,2,3]triazol-2-yl)acetonitrile 4. (25%) Beige
solid, mp 78–80 ◦C; H NMR (500 MHz, CDCl
1
3
2
): δ 5.68 (s, 2H, CH ),
13
7
.44–7.46 (m, 2H, Harom), 7.87–7.89 (m, 2H, Harom); C NMR (125
+
MHz, CDCl
3
): 43.6, 112.3, 118.2, 127.7, 145.1. HRMS (ESI ) calcd for
[M+H]+ 159.06707, found 159.06716. Lit [19,21].
8 7 4
C H N
4
.1.2. General procedure for the synthesis of benzotriazolyl acrylonitriles
6
a-d, 7a-d
Precursor benzotriazolyl acetonitrile 3 or 4 (1 equiv) and piperidine
(
1.0 equiv) were dissolved in ethanol (30 mL) and stirred vigorously for
0 min at room temperature. Then, corresponding aldehyde 5a-d (1.0
1
4
. Experimental section
equiv) was added to the solution and reacted under reflux for 2 h. The
course of the reaction was monitored by TLC (7:3 hexane/EtOAc). After
solvent evaporation under vacuum, the crude product was filtered and
washed with ethanol. For derivatives 6c and 6d, the crude product was
purified on column chromatography using a hexane/EtOAc (9:1) eluent
system. Yields, melting points (mp) and spectroscopic data are reported
4
.1. Synthesis and characterization
All starting chemicals were obtained commercially as analytical re-
agent and used directly without any purification. Melting points were
determined on an Electrothermal Mel-Temp apparatus and are
6

E. Hern a´ ndez-Fern a´ ndez et al.
Dyes and Pigments 189 (2021) 109251
Fig. 7. a) Compound 7b (10
μ
M) was co-incubated in cell media with DCB (50
μ
M) and showed colocalization with DCB, suggesting that it also is present in
endosomes and lysosomes. b) In a modified assay (pulse-chase condition), DCB was removed from the cell medium 4 h before imaging, leaving DCB primarily in
lysosomes. Close analysis of the images reveals accumulation of both compound 7b and DCB in discrete puncta within the cell.
as follows:
.1.2.1. (E)-2-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(4-morpholinophenyl)
118.2, 120.6, 125.0, 127.8, 128.9, 131.9, 141.8, 146.3, 153.5. HRMS
+
O [M+H]+ 332.15114, found 332.14883.
(
ESI ) calcd for C19
H
18
N
5
4
◦
1
acrylonitrile 6a. (60%) Yellow solid, mp 207–208 C, H NMR (300
MHz, CDCl N), 3.90 (t, J = 4.6 Hz, 4H,
): δ 3.35 (t, J = 4.4 Hz, 4H, (CH
CH
O), 6.94 (d, J = 8.7 Hz, 2H, Harom), 7.46 (m, 2H, Harom), 7.72 (s,
H, Hvinylic), 7.85–7.89 (m, 3H, Harom), 8.12 (d, J = 8.3 Hz, 1H, Harom);
C NMR (75 MHz, CDCl ): δ 47.4, 66.6, 101.8, 110.3, 114.2, 114.9,
4.1.2.2. (E)-2-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(4-(dimethylamino)
)
2
phenyl)acrylonitrile 6b. (45%) Orange solid, mp 186–188 C, 1H NMR
◦
3
2
(
2
)
2
(300 MHz, CDCl
3
): δ 3.10 (s, 6H, (CH
3
)
2
), 6.74 (d, J = 9.0 Hz, 2H, Harom),
1
7.42–7.47 (m, 1H, Harom), 7.56–7.61 (m, 1H, Harom), 7.66 (s, 1H, Hvi-
nylic), 7.84 (d, J = 8.3 Hz, 1H, Harom), 7.86 (d, J = 9.0 Hz, 2H, Harom),
1
3
3
7

E. Hern a´ ndez-Fern a´ ndez et al.
Dyes and Pigments 189 (2021) 109251
8
1
1
.12 (d, J = 8.4 Hz, 1H, Harom); 13C NMR (75 MHz, CDCl
3
): δ 40.1, 99.5,
source coupled to a MACH3 kappa goniometer and Bruker Apex II CCD
detector. The SAINT [64] and SADABS [65] programs, as incorporated
in the APEX3 package, were employed to integrate, scale, and correct
the obtained data. The structure was solved using dual-space methods in
10.4, 111.8, 115.6, 117.7, 120.5, 124.8, 128.7, 132.1, 132.3, 143.0,
+
[M+H]+ 290.14057,
46.2, 152.8. HRMS (ESI ) calcd for C17
H
16
N
5
found 290.14083.
2
SHELXT [66] and refined against F using SHELXL [67].
4
.1.2.3. (E)-2-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(4-(diethylamino)
1
phenyl)acrylonitrile 6c. (42%) Yellow oil, H NMR (300 MHz, CDCl
CH
), 6.72 (d, J = 9.0 Hz, 2H, Harom), 7.42–7.47 (m, 1H, Harom),
.55–7.61 (m, 1H, Harom), 7.62 (s, 1H, Hvinylic), 7.83 (d, J = 8.4 Hz, 1H,
arom), 7.84 (d, J = 9.0 Hz, 2H, Harom), 8.11 (d, J = 8.4 Hz, 1H, Harom);
C NMR (175 MHz, CDCl ): δ 12.7, 44.8, 98.9, 110.4, 111.4, 115.8,
3
): δ
4
.3. Optical properties
1
.23 (t, J = 7.1 Hz, 6H, (CH
2
3 2
)
), 3.45 (q, J = 7.1 Hz, 4H,
(
CH CH
2
3 2
)
3
For the photophysical characterization, spectroscopic grade CH CN
7
from Aldrich was freshly distilled and the solutions were studied as
prepared, in order to avoid any solvolysis or photodegradation effect
H
1
3
3
[
68]. UV–Vis absorption and fluorescence spectra were measured on a
1
17.1, 120.5, 124.8, 128.7, 132.4, 132.5, 143.1, 146.2, 150.7. HRMS
Varian Cary 100 spectrophotometer and a PerkinElmer LS 50B spec-
trofluorometer, respectively. The optical band gap (Eg) was determined
from the intercept with the X axis of the tangent of the absorption
spectrum drawn at absorbance of 0.1. Emission spectra were recorded
with a PerkinElmer LS55 spectrofluorometer, by exciting 10 nm below
the longer wavelength absorption band. Fluorescence quantum yields
(φ) in solution were determined according to the procedure reported in
the literature [69], using quinine sulfate in H SO 0.1 M (φ (%) = 0.54 at
+
[M+H]+ 318.17187, found 318.17058.
(
ESI ) calcd for C19
H
20
N
5
4
.1.2.4. (E)-2-(1H-benzo[d][1,2,3]triazol-1-yl)-3-(4-(diphenylamino)
1
phenyl)acrylonitrile 6d. (44%) Orange oil, H NMR (300 MHz, CDCl ): δ
3
7
4
1
H
.10 (d, J = 8.8 Hz, 2H, Harom), 7.21–7.24 (m, 6H, Harom), 7.36–7.41 (m,
H, Harom), 7.47–7.53 (m, 1H, Harom), 7.62–7.67 (m, 1H, Harom), 7.78 (s,
H, Hvynilic), 7.82 (d, J = 8.8 Hz, 2H, Harom), 7.91 (d, J = 8.4 Hz, 1H,
2
4
1
3
arom), 8.16 (d, J = 8.4 Hz, 1H, Harom); C NMR (75 MHz, CDCl
3
): δ
310 nm) as the internal standard. Temperature was regulated at 25.0 ±
◦
1
1
02.4, 110.4, 114.8, 120.2, 120.6, 122.4, 125.0, 125.2, 126.3, 126.3,
0.5 C with a water circulating bath. Three solutions with absorbance at
+
28.9, 129.8, 129.9, 131.4, 141.4, 146.2, 151.5; HRMS (ESI ) calcd for
the excitation wavelength lower than 0.1 were analyzed for each sample
C
27
H
20
N
5
[M+H]+ 414.17187, found 414.17294.
and the φ was averaged. φ Measurements were determined by the
relative method and the quantum yield of the unknown, φ
x
, was calcu-
4
.1.2.5. (E)-2-(2H-benzo[d][1,2,3]triazol-2-yl)-3-(4-morpholinophenyl)
lated according to the following equation:
◦
1
acrylonitrile 7a. (67%) Yellow solid, mp 241–243 C, H NMR (300
MHz, CDCl N), 3.86 (t, J = 4.5 Hz, 4H,
): δ 3.33 (t, J = 4.6 Hz, 4H, (CH
CH
O), 6.92 (d, J = 9.0 Hz, 2H, Harom), 7.41–7.44 (m, 2H, Harom),
.88–7.94 (m, 4H, Harom), 8.37 (s, 1H, Hvynilic); C NMR (75 MHz,
CDCl ): δ 47.3, 66.6, 108.2, 114.2, 114.3, 118.2, 120.4, 127.8, 132.2,
2
A
R
E
x
.I
R
n
x
3
2
)
2
φ = φ .
x
R
.
.
(1)
2
x R x
A E I n
R
(
2 2
)
1
3
7
where φ
R
is the quantum yield of the standard, A is the absorbance of the
3
solution, E is the corrected emission intensity, I is the relative intensity
of the exciting light and n is the average refractive index of the solution.
Subscripts R and X refer to the reference and unknown analyte,
respectively.
+
+
1
37.4, 144.9, 153.4. HRMS (ESI ) calcd for C19
H
18
N
5
O [M+H]
3
32.15114, found 332.14907.
4
.1.2.6. (E)-2-(2H-benzo[d][1,2,3]triazol-2-yl)-3-(4-(dimethylamino)
phenyl)acrylonitrile 7b. (78%) Orange solid, mp 254–256 C, 1H NMR
◦
4
.4. In vitro cytotoxicity and cell imaging
(
300 MHz, CDCl
3
): δ 3.09 (s, 6H, (CH
3
)
2
), 6.73 (d, J = 9.1 Hz, 2H, Harom),
7
.40–7.43 (m, 2H, Harom), 7.88–7.93 (m, 2H, Harom), 8.36 (s, 1H, Hvy-
13
For cytotoxicity determination, HEK 293T cells were co-incubated
nilic); C NMR (75 MHz, CDCl
3
): δ 40.2, 106.4, 111.9, 114.9, 117.6,
+
with various concentrations of compound 7b and cell viability was
measured after 24 h via resorufin fluorescence. Cells were seeded at 20
1
18.1, 127.5, 132.5, 138.1, 144.8, 152.7. HRMS (ESI ) calcd for
[M+H]+ 290.14057, found 290.13784.
17 16 5
C H N
0
00 cells per well in a 96-well, tissue culture-treated optical plate
(
Nunc). Seeding and incubation were performed in full medium (Dul-
4
.1.2.7. (E)-2-(2H-benzo[d][1,2,3]triazol-2-yl)-3-(4-(diethylamino)
phenyl)acrylonitrile 7c. (34%) Orange solid, mp 190–191 C, 1H NMR
◦
becco’s Modified Eagle Medium supplemented with 10% fetal bovine
serum). After seeding, cells were left for 24 h to adhere to the wells, after
which growth media was removed and full medium added, containing
various concentrations of compound and up to 1% DMSO. For negative
(
300 MHz, CDCl
Hz, 4H, (CH CH
arom), 7.88–7.91 (m, 4H, Harom), 8.35 (s, 1H, Hvynilic); C NMR (75
MHz, CDCl ): δ 12.7, 44.8, 105.9, 111.5, 115.1, 116.9, 118.0, 127.4,
3
): δ 1.23 (t, J = 7.1 Hz, 6H, (CH
2
CH
3
)
2
), 3.45 (t, J = 7.1
2
3 2
)
), 6.71 (d, J = 9.1 Hz, 2H, Harom), 7.40–7.43 (m, 2H,
1
3
H
(
vehicle) control, cells were incubated with 1% DMSO in full medium
alone. Positive (dead) control included a 2 min wash in 85% ethanol/
5% H O before adding full medium. After a 20 h incubation with
compound or vehicle, resazurin in phosphate buffered saline was added
3
+
+
1
32.9, 138.1, 144.8, 150.6. HRMS (ESI ) calcd for C19
H
20
N
5
[M+H]
1
2
3
18.17187, found 318.16926.
ꢀ 1
at 20 g mL to each well. Cells were then allowed to incubate for an
μ
4
.1.2.8. (E)-2-(2H-benzo[d][1,2,3]triazol-2-yl)-3-(4-(diphenylamino)
additional 2 h, after which well fluorescence was measured using a
BioTek Synergy Hybrid plate reader at 590 nm emission and 560 nm
excitation. Normalized values were used to combine datasets; data
shown are averages of three independent replicates.
phenyl)acrylonitrile 7d. (43%) Orange solid, mp 180–181 C, 1H NMR
◦
(
300 MHz, CDCl
3
): δ 7.05 (d, J = 8.8 Hz, 2H, Harom), 7.15–7.21 (m, 6H,
H
H
arom), 7.33–7.45 (m, 6H, Harom), 7.82–7.92 (m, 6H, Harom), 8.39 (s, 1H,
1
3
vynilic); C NMR (75 MHz, CDCl ): δ 108.8, 114.2, 118.2, 120.2, 122.2,
3
Cell imaging was performed with a Leica DMi8 confocal microscope
1
25.2, 126.3, 127.9, 129.9, 131.8, 137.1, 145.0, 146.2, 151.4. HRMS
(
Leica Microsystems), exciting at 405 nm for both DCB and compound
b, and with emission filters of 420 ± 30 nm and 545 ± 30 nm for DCB
and compound 7b, respectively. HEK 293T cells were treated by co-
incubating with 10 M compound 7b and 50 M DCB for 16 h prior to
+
[M+H]+ 414.1787, found 414.16978.
(
ESI ) calcd for C27
H
20
N
5
7
4
.2. Single-crystal X-ray diffraction analyses
μ
μ
imaging, after which medium was removed. In pulse-chase assays, cells
were washed after 12 h with full media and incubated for an additional
Compound 7b was crystallized using slow evaporation in EtOAc/
hexanes to produce yellow single crystals for X-ray diffraction experi-
4 h in media containing 10 M compound 7b only before imaging. Cells
μ
◦
ments. High-resolution (0.84 A) data were collected at 100 K using Cu-
were washed once with full medium before imaging. Cells were imaged
◦
K
α
radiation produced by a Bruker-Nonius FR591 rotating anode X-ray
live in full medium at 37 C with 100% humidity and 5% CO
2
. Cell
8

E. Hern a´ ndez-Fern a´ ndez et al.
Dyes and Pigments 189 (2021) 109251
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Writing – original draft, Formal analysis; Ma. Concepci o´ n García-
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Acknowledgements
The authors thank CONACYT of M ´e xico for the financial support via
project CB-2015/256359. E.H.F. also thank CONACYT for sabbatical
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