Fluorescent mesoionic 1-(2-aryl-4H-thieno[3,4-d][1,2,3]triazol-2-ium-4-ylidene)ethan-1-olates: One-pot synthesis, photophysics, and biological behavior

Article abstract of DOI:10.1016/j.dyepig.2021.109777

This paper reports the one-pot synthesis of a rare type of mesoionic compounds, which are condensed mesoionics. A series of new bicyclic arylthienotriazoliumolates was synthesized by an improved synthetic method. This approach has a large scope of starting materials and was used to obtain diverse structures with outstanding photophysical properties. The pattern of substituents decorating the mesoionic ring was the important factor determining the electronic distributions of the synthesized compounds. Pull–pull (А–π–A), push–push (D–π–D), pull–push (A–π–D), and push–pull (D–π–A) systems were designed, synthesized and their absorption, emission, stability, and biological behavior were studied. Quantum chemical calculations were performed to deepen the knowledge about the absorption and emission properties of these systems and explain their unusual geometries and electronic structures. Confocal microscopy experiments showed that thienotriazoliumolates easily penetrated into living cells and selectively accumulated near the cell membrane, Golgi apparatus, and endoplasmic reticulum.

Full text of DOI:10.1016/j.dyepig.2021.109777

Journal Pre-proof
Fluorescent mesoionic 1-(2-aryl-4H-thieno[3,4-d][1,2,3]triazol-2-ium-4-
ylidene)ethan-1-olates: One-pot synthesis, photophysics, and biological behavior
Alexander K. Eltyshev, Nadezhda V. Chernysheva, Artem S. Minin, Varvara A.
Pozdina, Pavel A. Slepukhin, Enrico Benassi, Nataliya P. Belskaya
PII:
S0143-7208(21)00643-4
DOI:
https://doi.org/10.1016/j.dyepig.2021.109777
DYPI 109777
Reference:
To appear in: Dyes and Pigments
Received Date: 12 June 2021
Revised Date: 27 August 2021
Accepted Date: 1 September 2021
Please cite this article as: Eltyshev AK, Chernysheva NV, Minin AS, Pozdina VA, Slepukhin PA, Benassi
E, Belskaya NP, Fluorescent mesoionic 1-(2-aryl-4H-thieno[3,4-d][1,2,3]triazol-2-ium-4-ylidene)ethan-1-
olates: One-pot synthesis, photophysics, and biological behavior, Dyes and Pigments (2021), doi:
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Author contributions
Alexander Eltyshev: Synthetic and optical Investigation, preparing experimental data. Nadezhda
Chernysheva: Synthetic Investigation. Artem Minin: Biological investigations, preparing
biological experimental data. Varvara Pozdina: Biological investigations. Pavel Slepukhin: X-ray
experiment. Enrico Benassi: Analyze the results of density functional theory calculations, writing
the original draft. Nataliya P. Belskaya Conceptualization, analysis of the experimental data,
writing original draft.

Fluorescent mesoionic 1-(2-aryl-4H-thieno[3,4-d][1,2,3]triazol-2-ium-4-
ylidene)ethan-1-olates: One-pot synthesis, photophysics, and biological behavior
Alexander K. Eltyshev,a Nadezhda V. Chernysheva,a Artem S. Minin,ab Varvara A. Pozdina,c Pavel A.
Slepukhin,d Enrico Benassi,*e Nataliya P. Belskaya*ac
aUral Federal University, 19 Mira Str., Yekaterinburg 620002, Russia
bM. N. Mikheev Institute of Metal Physics, Ural Branch of Russian Academy of Science, 18 S.
Kovalevskaya Str., Yekaterinburg, 620108, Russia
cInstitute of Immunology and Physiology, Ural Branch of Russian Academy of Science, 20 S.
Kovalevskaya Str., Yekaterinburg, 620049, Russia
dPostovsky Institute of Organic Synthesis, Ural Branch of Russian Academy of Science, 22 S.
Kovalevskaya Str., Yekaterinburg, 620219, Russian Federation
eNovosibirsk State University, Pirogova Str. 2, 630090 Novosibirsk, Russia
Corresponding Authors: Prof. Belskaya Nataliya, Email: n.p.belskaya@urfu.ru;
Dr. Benassi Enrico, Email: ebenassi3@gmail.com.
Abstract: This paper reports the one-pot synthesis of a rare type of mesoionic compounds, which are
condensed mesoionics. A series of new bicyclic arylthienotriazoliumolates was synthesized by an
improved synthetic method. This approach has a large scope of starting materials and was used to obtain
diverse structures with outstanding photophysical properties. The pattern of substituents decorating the
mesoionic ring was the important factor determining the electronic distributions of the synthesized
compounds. Pull–pull (А–π–A), push–push (D–π–D), pull–push (A–π–D), and push–pull (D–π–A)
systems were designed, synthesized and their absorption, emission, stability, and biological behavior
were studied. Quantum chemical calculations were performed to deepen the knowledge about the
absorption and emission properties of these systems and explain their unusual geometries and electronic
structures. Confocal microscopy experiments showed that thienotriazoliumolates easily penetrated into
living cells and selectively accumulated near the cell membrane, Golgi apparatus, and endoplasmic
reticulum.
Keywords: Oxidative cyclization, thienotriazole, mesoionic heterocycles, fluorescence, DFT
calculations
1. Introduction
Mesoionic compounds are planar five- or six-membered heterocyclic dipolar compounds bearing at
least one side chain, and its α-atom takes part in a charge delocalization. A key difference between
mesoionic compounds and other heterocyclic compounds is their unique electronic structure. They could

be described rather as a combination of mesomeric and ionic structures than single covalent structure.
[1-10].
The synthesis and properties of mesoionic compounds have received considerable attention in the last
decade2,4,6-17 due to their wide spectrum of biological activities, including antitumor [11-14], anti-
inflammatory, antimicrobial [15,16], and antipyretic properties. Some condensed mesoionic 1,2,3-
triazolium-5-olates have shown immunosuppressant properties [17]. Mesoionic compounds have
fascinating chemistry and reactivity in 1,3-dipolar cycloadditions. Previous investigations revealed new
peculiarities in their reactivity, paving the way to use these compounds in click and bio-orthogonal
chemistry [2].
While mesoionic compounds are neutral structures, their high charge separation and mobile electrons
lead to a decreased energy gap between the highest occupied molecular orbital (HOMO) and lowest
unoccupied molecular orbital (LUMO). Therefore, mesoionics can be considered as promising
candidates for optoelectronic applications [18-24] and fluorophores for labelling and probe design [25-
28]. Stable mesoionic heterocycles are promising precursors for new nonlinear optical materials [29-
32].
Monocyclic mesoionic compounds are widely available. The most known mesoionics are sydnones
̈
and munchnones, which contain 1,2,3-oxadiazole units [2, 3, 8, 10, 11, 33] followed by azasydnone [34-
35] and other azoles such as 1,2,3-triazol-3-ium-5-olate and 4-aryl-1,3,2-oxathiazolyl-ium-5-olates [9,
29, 36-37]. Various approaches have been reported for the synthesis of these mesoionic compounds. In
contrast, condensed mesoionic heterocycles are less studied; methods for their synthesis are limited, and
their properties are not fully understood [38].
In a previous work, we proposed a method for the synthesis of new mesoionic compounds, resulting
in the first examples of arylthienotriazolium-olates (ATTs) [39]. We reported preliminary absorption
and emission properties based on a limited library of compounds [40]. In this article, we report an
improved method for preparing new mesoionics including this rare scaffold. The main aim of the present
study was to evaluate the optical properties of ATTs in detail using experimental (spectral) methods and
quantum mechanical calculations. Using the improved synthetic method, we obtained a set of new
compounds bearing different combinations of substituents at the aromatic rings (A and B in Scheme 1)
at opposite termini of the molecule (type I in Scheme 1). Moreover, we obtained new members of this
mesoionic class in which the anionic part of the ATT is non-aromatic (type II in Scheme 1).
2

Scheme 1 Retrosynthesis of targeted fused mesoionic 2-aryl-thieno-1,2,3-triazole-olates (ATTs).
Structures I and II cannot describe the exact electronic distribution in molecules of 1-(2-aryl-4H-
thieno[3,4-d][1,2,3]triazol-2-ium-4-ylidene)ethan-1-olates (see Scheme 3 and Quantum mechanical
calculations) and will be used throw the text of manuscript and Supporting Information only for the
simplification of presentation, as it usual have done in the most publications.
2 Experimental
2.1 Instruments
1Н NMR and 13C NMR spectra were recorded using a Bruker Avance II spectrometer (Karlsruhe,
Germany; 400 MHz for 1H and 100 MHz for 13C) and a Bruker Avance NEO spectrometer (Karlsruhe,
Germany; 600 MHz for 1H and 150 MHz for 13C). Chemical shifts are reported in parts per million (ppm)
relative to tetramethylsilane for 1H NMR and relative to the residual solvent signals for 13C NMR as an
external reference. Coupling constants (J) are given in hertz (Hz). Signal splitting patterns are described
as singlet (s), doublet (d), triplet (t), quartet (q), sextet (sext), quintet (quin), multiplet (m), broad (br),
doublet of doublets (dd), doublet of triplets (dt), or AA′XX′ (spin system of para-substituted benzene
with two different substituents). High-resolution mass spectra were obtained using an Agilent 1290
Infinity II high-performance liquid chromatography system equipped with a UV diode array detector
and tandem quadrupole time-of-flight accurate mass detector (Agilent 6545, Agilent Technologies Inc.,
Santa Clara, CA, USA).
Fourier transform infrared (FTIR) spectra were obtained using an attenuated total reflection (ZnSe)
FTIR spectrometer in the range of 4000–500 cm−1. Melting points were determined using a microscopic
melting point meter without correction. PXRD spectra were collected using an Xcalibur 3 diffractometer
based on the standard procedure (MoK irradiation, graphite monochromator, ω-scans with a 1° step at
295(2) K). The crystal structures were obtained by the slow evaporation of diluted solutions of
compounds 1p in a mixture of CHCl3/EtOH.
UV-Vis absorption spectra were recorded on a Shimadzu UV-1800 spectrometer. The fluorescence
of the sample solution was measured using a Hitachi F-7000 spectrophotometer (Tokyo, Japan). The
3

absorption and emission spectra were recorded in 1,4-dioxane, CHCl3, THF, EtOH, MeCN, DMF, and
DMSO using 10.00-mm quartz cells. The excitation wavelengths were the absorption maxima.
Atmospheric oxygen contained in the solutions was not removed. The concentration of the compound
in the test solution was 5.0 × 10−5 M for absorption and for fluorescence measurements. The absolute
quantum yields (QYs) were recorded from the solution, solid-state, and time-resolution studies were
recorded on a Horiba FluoroMax 4 spectrofluorometer (Kyoto, Japan) with a Quanta-φ integrating
sphere using FluorEssence 3.5 software.
Microscopic examination was performed using the equipment of the Shared Research Center of
Scientific Equipment SRC IIP UrB RAS. After staining and washing, living cells were examined using
a confocal laser scanning microscope (LSM-710, Carl Zeiss) with a multichannel QUASAR detector
(34 channels). The images were obtained using an immersion lens (40x/1.3 Oil). To obtain informative
fluorescent images, ZEN software was used in special lambda mode (λ mode), allowing for the
determination of the emission range with maximum contrast.
The reactions were monitored by analytical thin-layer chromatography (TLC) on aluminum-backed
silica-gel plates (Sorbfil UV–254). Visualization of the components was accomplished using short-
wavelength UV light (254 nm).
2.2 Reagents
2-Bromacetophenones
4a–d
and
2-chloracetone
4c
were
commercially
available.
Arylhydrazonothioacetamides 3a–i were synthesized as reported previously [41-44]. Commercially
available chemicals and solvents were used without further purification. Full experimental details for
the synthesis of compounds 1a–q, 5c, f, i, and o as well as their NMR spectra, photophysical
characteristics, and quantum mechanical calculation data are reported in the Supplementary Information
(SI). The products were purified by column chromatography using a silica-gel (0.035–0.070, 60 Ǻ) and
recrystallized from ethanol.
The molecular structures and synthetic procedure of the compounds 5c, f, i, o and 10a-p as well as
NMR spectra of new compounds; HRMS data; XRD data CCDC 2084371; details of photophysical
investigations, quantum-mechanical calculations, and the coordinates of compounds optimized at the
(TD-)DFT level were described in in the supporting information.
3 Results and discussion
3.1 Synthetic study
The pathway for the synthesis of 2-aryl-4H-thieno[3,4-d][1,2,3]triazol-2-ium-4-ylidenemethanolates
(ATTs) 1a–p from arylhydrazonothioacetamides 3a–i involved several stages (Scheme 2). The first step,
S-alkylation, was followed by heterocyclization to form arylazoaminothiophenes 5a–p. Oxidative
4

cyclization then led to the fusing of the 2-aryl-1,2,3-triazole ring to the thiophene moiety. Mechanistic
analysis showed that these reactions realized as tandem of the consequent transformations and could be
implemented in one step to obtain a large panel of ATTs without the isolation of the intermediate
thiophenes 5.
The preferable reaction conditions were chosen based on previously published data [39] and included
the initial heating of reagents with excess K2CO3 in EtOH for approximately 2.5–4.0 h. The solvent was
then evaporated under vacuum, and the next stage was carried out in pyridine with Cu(OAc)2 [41]. Base
is an important participant of the reaction; it takes part in (1) the activation of thioamides, (2) binding
the leaving halogen anion, (3) activating the CH2 group, and (4) facilitating the release of the ATT from
the metal complex after oxidation is complete. Thus, K2CO3 promotes every stage of the reaction and
was added in two-fold excess.
The one-pot approach was used to synthesize two sets of ATTs with different substituents on the
aromatic rings and different structures of the lateral anionic parts of the mesoionic molecules. The overall
yields ATTs ranged from 31% to 51% after the multistep transformation.
Scheme 2 The scope of the one-pot approach for the synthesis of ATTs 1a–p.
5

Table 1 Yields and conversion times for the synthesis of ATTs 1a–p by the one-pot procedure.
Entry Compds R1
NR2R3
R4
Yield,
5a–pa % 1a–p %
Yield,a
Time,b
min
2.5
3.0
3.0
5.0
1.5
2.5
3.0
2.5
2.5
3.0
3.0
3.0
3.5
2.0
3.0
2.5
1, 5
1
2
3
4
5
6
4-MeOC6H4 Morpholin-4-yl
4-MeOC6H4 Morpholin-4-yl
4-MeOC6H4 Morpholin-4-yl
4-MeOC6H4 Morpholin-4-yl
4-F3CC6H4 Morpholin-4-yl
4-F3CC6H4 Morpholin-4-yl
4-MeOC6H4
4-ClC6H4
4-CNC6H4
Pyridin-3-yl
4-MeOC6H4
4-NCC6H4
4-NCC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
2-ClC6H4
Me
-
40
35
33
55
31
30
31
47
50
40
40
35
36
51
31
33
a
b
c
d
e
f
-
53
-
-
55
-
7
8
9
4-NCC6H4
4-Ph
4-Ph
Morpholin-4-yl
Morpholin-4-yl
Pyrrolidin-1-yl
Pyrrolidin-1-yl
g
h
i
-
53
-
10
11
12
13
14
15
16
4-ClC6H4
j
k
l
m
n
o
p
4-MeOC6H4 Pyrrolidin-1-yl
4-MeOC6H4 Piperidin-1-yl
4-MeOC6H4 Azepan-1-yl
4-MeOC6H4 Morpholin-4-yl
4-MeOC6H4 Morpholin-4-yl
4-CF3C6H4 Morpholin-4-yl
-
-
-
-
40
-
Me
a Isolated yield achieved using the one-pot approach. bTime required for the one-pot method. Reaction
completion was confirmed by TLC.
The structures of the new compounds 1a–p, 5c, f, i and o were confirmed by NMR spectroscopy
(Figs. S1–S20, SI), high-resolution mass spectrometry, UV-Vis spectroscopy, fluorescence
spectroscopy, and comparison with the single-crystal X-ray crystallographic data of ATT 1o (Fig. 1;
Table S1 in SI).
The mass spectra of all bicyclic compounds 1a–p contain molecular ion peaks. The NMR spectra of
1
ATTs 1 have several peculiarities. Compared to the H NMR spectra of thiophenes 5, they contain no
signals from the NH2 group, and the signals of protons in the ortho positions of the aromatic rings are
downshifted by 0.5 ppm. Signals of protons of aromatic rings, especially ring A ortho-protons, are
13
broadened, the same as the signals of pyrrolidine and azepane fragments in compounds 1i, j, m. C
NMR spectrum of the ATT 1d was not registered owing to it weak solubility. ATT 1n spectra 1H and
13C exhibited double set of signals in ratio 6 : 1. It may be explained by the hindered in rotation of the
ring B due to the presence of chlorine atom at ortho-position and simultaneously increasing
intramolecular interactions of this atom. To confirm ATTs 1d and 1n purity and individuality, we have
included in SI HRMS qualitative analysis reports (Fig. S8 and S18, SI). In the FTIR spectra of the ATTs,
there are no absorption bands corresponding to the stretching vibrations of the N–H bonds of the amino
groups. The absorption bands corresponding to the stretching vibrations of the C=O bonds of the
carbonyl groups are observed in the range of 1580–1600 cm−1 (Experimental, SI).
6

b
a
c
Fig. 1 ORTEP view for ATTs 1o with thermal ellipsoids at the 50% probability level: (a) front view
with the weak intramolecular interactions; (b) side view and (c) molecular packing, respectively.
The structure of 1o was confirmed by the XRD analysis of a single crystal obtained by the slow
evaporation of solvents from a CHCl3-EtOH mixture. The compound 1o crystallized in a
centrosymmetric space group. The general view of the compound is shown in Fig. 1a, and the refinement
parameters are summarized in Table S1 (SI). Crystal structure analysis demonstrated that the
heterocyclic part of the molecule is planar; the aryl substituent is approximately located in the plane of
the heterocycle (Fig. 1b). C(12)-N(2) bond, binding heterocycle and aromatic ring is 1.431(3) Å. The
configuration of the N atom in the morpholine group indicates strong conjugation with the thiophene
ring. In particular, the N(8)–C(11) bond distance is 1.346(3) Å, and the N(8) atom is deviated from the
plane of the nearest C atom C(11)C(29)C(23) by a distance of 0.189 Å to form a planar trigonal pyramid.
The C(13)–C(14) bond distance in the acyl substituent is 1.406(4) Å, which is larger than typical C=C
bond. These bond distances clearly show the significantly bipolar structure with strongly conjugated
aromatic ring, morpholino- and carbonyl groups bound by the system of the π-bonds. Molecule 1o
exhibits various weak intramolecular interactions, which support its geometry and stability. The
molecular packing (Fig. 1c) shows a fairly ordered structure with distance between layers approximately
3.41 Å. An arrangement of molecules to each other provides the interaction of a positive site with a
negatively site and weak π–π rings interactions between the layers.
Classical approach to the organic molecules formation suggests for mesoionic compounds a set of
canonical structures with different combinations of separated charges. Unlike known mesoionic
compounds, the electronic distribution of ATTs molecule can be presented by a wide number of
variations of charge delocalization between the heteroatoms. Scheme 3 demonstrates them partially,
predominantly with the participation of atoms nitrogen and oxygen. The quite large set of the possible
7

resonance structures the remarkable distance between charge centers and localization of the negative
charge on the more electronegative atom (oxygen) provides stabilization and promotes the molecules
formation. According to this rule, forms B-C are more stable (see Quantum mechanical calculations data
and their discussion).
Scheme 3 Resonance of canonical structures of ATTs 1a–p.
3.2 Photophysical properties of the ATTs
The photochromic behavior of mesoionic compounds has been identified in solid since 1955.45
Sydnones are colorless or slightly yellow in the solid state. However, they rapidly develop a blue color
under UV irradiation with low QY [46, 47] Polycyclic sydnones show blue fluorescence with low QY
in DMSO/H2O mixture (50/50) [48]. Mesoionic oxazolones display bright-blue fluorescence under UV
irradiation [49].
The ATTs synthesized in this study are orange, red, and violet solids, and their solutions are red-
orange or burgundy in color under daylight and exhibit orange or red emission under irradiation (Figs.
2 and 5). The spectroscopic data of the ATTs are collected in Table 2. The corresponding absorption and
emission spectra are depicted in Fig. 2. The optical properties of ATTs 1b, h, i, and k were partially
described in our previous paper [40] and are presented here for comparison and to establish accurate
relationships between the structures and optical properties. The spectra of the ATTs show one or two
absorption bands in the UV-region and an additional band in the visible region that were sensitive to the
substitution patterns of the molecular termini (498–535 nm). The coefficient of molar extinction (ɛ) of
these compounds ranges from 6900 to 18800 M−1∙cm−1 (Table 2). The emission bands are located in the
red, orange, or yellow parts of the spectra (577–629 nm) and are the mirror images of the long-
wavelength laying absorption band.
The significant shift in the absorption band to the visible region versus monocyclic 2-aryl-1,2,3-
triazoles [50] or sydnones [45] or oxazolones [49] arises from the effective π-conjugative pathways
through the triazole and thiophene ring and the strong charge separation even in the GS. However, the
electronic nature and arrangement of the substituents causes significant shifts both in the absorption and
emission bands. It is obvious, the presence of electron-accepting substituents at aromatic ring A in the
ATTs led to a bathochromic shift and intramolecular charge transfer (ICT) for both the absorption and
8

emission bands. In contrast, the types, and positions of substituents at ring B had negligible effects (Figs.
2a–d; Table S3 Entries 5, 6, 7, and 17 in SI). The most significant bathochromic shifts were observed
for ATTs 1e and 1g (up to 626–629 nm). These results are in accordance with the resonance structures;
electron-accepting moieties strengthen the electron flow via the N2 atom, whereas substituents at ring B
are not in conjugation with the negatively charged atom oxygen and have only a slight influence on the
electronic system of the molecule. This observation is confirmed by the ATT 1p spectral data, which the
absorption and emission maxima are observed at 524 and 626 nm, respectively. Further confirmation is
provided by the optical characteristics of ATT 1n, which includes a substituent at the ortho-position of
ring B; both the absorption and emission maxima and QY were not significantly different from those of
1b (Table 2, Entries 2 and 15).
c
a
b
d
g
e
f
h
k
i
j
l
9

Fig. 2 (a,e,i) Absorption and (b,f,j) emission spectra (c = 5×10-5 M) of ATTs 1a-p in CHCl3 respectively
at room temperature). Photographs of the CHCl3 solutions of 1a-p under (c,g,k) daylight, and (d,h,l) with
UV radiation (λ = 365 nm), respectively.
Table 2 Photophysical propertiesa of ATTs 1a–p in CHCl3.
λem,
nm
Stokes shift,
nm/cm-1
85/2842
79/1569
74/2550
76/2639
98/2944
89/2781
94/2793
92/3182
80/2678
83/2725
73/2538
80/2688
76/2590
73/2510
91/3102
102/3110
Entry
Compd
λabs, nm
ɛ, M-1·cm-1
QY,b %
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
350, 506
352, 504
351, 505
35100, 15700
32200, 15700
27400, 18800
591
583
577
576
628
612
629
596
588
595
574
587
581
577
589
626
12.0
20.2
7.4
20.1
3.6
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
353, 500
23400, 11700
321, 366,530
307, 382, 523
322, 387, 535
364, 501
371, 508
329, 371, 514
349, 501
25400, 17600, 11000
22800, 11200, 13800
29000, 13600, 11700
29600, 7100
8600, 8100
21400, 13100, 10700
19500, 10300
6.8
2.9
17.2
17.6
10.4
25.9
15.2
23.2
12.2
12.4
3.8
352, 507
351, 505
350, 504
333, 498
23100, 11700
23900, 12500
17100, 8600
24600, 6900
322, 524
46400, 10800
a UV-Vis absorption and fluorescence wavelength at a concentration of 5×10-5 M. b Absolute quantum
yields.
The structure of the tert-amino group did not have a remarkable influence on the optical behaviors of
the ATTs because the shifts in the positions of the absorption or emission bands and QYs were similar
for ATTs 1b and k–m (Table 2, Entries 2 and 12–14).
The ATTs showed large Stokes shifts ranging from 2510 to 3182 cm-1, and this advantage expands
the application prospects of these compounds. The fluorescence QYs ranged from 2.9% to 25.9%, and
the best QY was achieved by ATT 1k. The moderate QY values can be attributed to the dissipation of
excitation energy during geometry relaxation or to the intersystem crossing and forbidden transition to
the closed triplet state. To verify this hypothesis, we determined the energy differences between the S1v
excited state and the closed (upper or lower) excited states (ΔEST) and plotted the dependence of QY on
the minimum ΔEST (upper or lower state) value for ATTs 1a–p (Table S10 and Fig. S25, SI). The lack
of an evident correlation suggests that intersystem crossing cannot be the main cause of such dissipation
of excitation energy. However, the previously mentioned possibility of a significant energy loss resulting
from the vibration of the B ring does not appear to be an appropriate explanation because the absence of
this ring in ATTs 1o and 1p does not lead to higher QYs. The results obtained for the set of substituents
and their combinations in the ATTs indicate the importance of the chlorine atom, which increased the
QY to 25.9% (Table 2, Entry 12). Along with these examples, it should be noted the high ATT 1d QY
10

(20.1), which structure includes heterocyclic ring B (Table 2, Entry 4). This hypothesis cannot be
confirmed by the data obtained and should be verified in future investigations.
3.3 Solvato(fluoro)chromic behaviors of ATTs
The effects of solvents with various polarities on the electronic absorption and fluorescent spectra of
ATTs were examined. In this study, the absorption bands of ATT 1e were shifted within 16–32 nm in
solvents with different polarities (Table 3 and Fig. 3). The strongest blue shift was observed in the polar
and protic solvent EtOH. In contrast DMSO (polar) and 1,4-dioxane (non-polar) had negligible effects
on the absorption and emission maxima. The ground state (GS) and excited state (ES) electron
distributions are, therefore, not significantly affected by dipole–dipole interactions with solvents. Dye
molecules that undergo small changes in permanent dipole moment upon excitation exhibit weak
solvatochromism. Thus, we can conclude that only the formation of hydrogen bonds with environment
(i.e., solvents or analytes) may interfere the electronic distributions of ATTs and change the positions of
their absorption and fluorescence maxima.
Table 3. Photophysical characteristics of ATT 1a and 1f in selected solvents (c = 5×10-5 M)
Entry
Compd
Solvent
λabs, nm
ɛ, M−1·cm−1
λem, nm QY, % Stokes shift,
nm/cm−1
1
2
3
4
5
6
7
8
9
1,4-Dioxane
THF
349, 513
349,514
350, 506
353, 497
348, 506
34000, 13400
30100, 11700
35100, 15700
28600, 12700
28600, 12700
603
601
591
580
597
601
626
612
610
622
629
7.0
7.6
12.0
9.0
5.2
6.2
1.5
6.8
1.3
0.8
0.5
90/2909
87/2816
85/2842
83/2879
91/3012
89/2892
93/2787
89/2781
101/3253
99/3043
99/2980
1a
CHCl3
EtOH
MeCN
DMSO
1,4-Dioxane
CHCl3
354, 512
32500, 14400
305, 383, 533
307, 382, 523
303, 382, 509
306, 385, 523
307, 394, 529
23900, 11600, 12100
22800, 11200, 13800
16200, 8200, 9900
25800, 11200, 13000
26200, 11100, 13200
1f
EtOH
MeCN
DMSO
10
11
The fluorescence QY was sensitive to solvent polarity, and the largest values of QY were obtained
in CHCl3 (approximately 2 to 12-fold higher than the QYs obtained in DMSO). This means that the
intermolecular interactions with CHCl3 molecules better stabilize the ATT excited states, than the
interactions
with
the
polar
DMSO
or
non-polar
1,4-dioxane
molecules.
11

c
a
b
d
g
h
e
f
Fig. 3 (a,e) Absorption spectra and (b,f) emission spectra of ATTs 1a (a,b) and 1f (e,f) in different
solvents. Photograph of solutions of ATTs 1a (c,d) and 1f (g,h) under (c,g) daylight, and under (d,h) UV
irradiation (λ = 365 nm), respectively, in different solvents: 1 - 1,4-Dioxane, 2-THF, 3 - CHCl3, 4 –
EtOH, 5 – MeCN, 6 - DMSO Absorption and emission (c = 5×10-5 M).
To obtain more information about the intramolecular interactions and their sensitivity to
various microenvironments, with the goal of expanding the application prospects of the ATTs,
we measured the absorption and emission spectra of ATTs 1j and 1o in the DMSO/H2O and 1,4-
dioxane/H2O mixtures with different solvent ratios (Fig. 4; Fig. S21 and Table S2, SI). As shown
by the obtained spectra (Fig. S21, SI), and images of the solutions (Fig. 4b,c), neither distortions
in spectral shape nor visible aggregates were observed in the studied mixtures. This indicates
that spectral methods are suitable for the investigation of different DMSO/H2O mixtures.
The analysis of the spectral data showed that the addition of water to the solutions of ATTs 1j
and 1o in DMSO led to blue shifts of 3–27 and 5–13 nm in the absorption and emission bands,
respectively (Table S3, SI). The absorption and fluorescent intensities decreased as the
proportion of water in the mixture increased (Fig. 4a). Strong interaction of ATTs with proton-
donating solvent or analyte molecules may be exploited to use the ATTs as sensors for these
probes.
12

b
c
a
Fig. 4 (a) Plot of emission intensity of ATTs 1j and 1o versus water fraction from 0% to 90% in the
mixture DMSO-H2O; Photographs of ATTs (b) 1j and (c) 1o solutions in DMSO-H2O mixture (c =
1×10-5 M).
3.4 Fluorescence lifetime of ATTs
The fluorescence lifetimes of ATTs 1a–o were determined in chloroform; a wider range of solvents,
viz. 1,4-dioxane, THF, EtOH, MeCN, and DMSO, was used for 1a (Table 4, Table S3). Upon least
square linear regression, the lifetime data of the ATTs accurately fit a double-exponential decay (Fig.
S22, SI), suggesting that two excited species existed in each compound [51]. The lifetimes obtained
ranged from 2.6 to 10.6 ns. Among the ATTs, the longest fluorescence lifetimes were obtained for 1b
and 1m. The radiative (kr) and non-radiative (knr) rate constants were also calculated (Table 4). The kr
values of the ATTs were obviously lower than the knr values. To understand the role of intersystem
crossing in this significant decrease in kr, we repeated the procedure applied for QY and plotted the
dependence of luminescence lifetime on ΔEST for ATTs 1a–o (Table S10 and Fig. S25a, SI), yet no
evident correlation was observed. However, the geometric relaxation cannot be considered as the main
reason for the stability of the ES structure. The fluorescence lifetimes of ATT 1o, which lack aromatic
B ring, are large but not as large as those of ATTs 1b and 1m bearing this aromatic ring B too. Thus, the
situation cannot not completely clarify by means of the interpretation of the sole collected experimental
data.
13

Table 4 Fluorescence lifetime (⟨τ⟩f), radiative (kr) and non-radiative (knr) decay parameters of ATTs 1a-
o.
Entry Compound
Solvent
1,4-Dioxane
THF
⟨τ⟩f (ns)
7.0
7.5
8.6
6.0
6.2
6.4
10.6
3.7
9.9
2.6
5.7
3.4
8.8
8.9
7.8
9.5
9.2
kr × 109 (s−1)
0.010
0.010
0.014
0.015
0.008
0.009
0.019
0.019
0.020
0.015
0.012
0.009
0.019
0.020
0.013
0.027
0.016
0.023
0.014
0.015
knr × 109 (s−1)
0.132
0.123
0.102
0.152
0.153
0.147
0.075
0.251
0.080
0.369
0.163
0.290
0.094
0.092
0.115
0.078
0.092
0.077
0.102
0.110
kr / knr
0.08
0.08
0.13
0.10
0.05
0.06
0.25
0.08
0.25
0.04
0.07
0.03
0.20
0.22
0.11
0.35
0.17
0.30
0.14
0.14
1
2
1a
3
4
5
6
CHCl3
EtOH
MeCN
DMSO
CHCl3
CHCl3
CHCl3
CHCl3
CHCl3
CHCl3
CHCl3
CHCl3
CHCl3
CHCl3
CHCl3
CHCl3
CHCl3
CHCl3
7
8
9
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
10
11
12
13
14
15
16
17
18
19
20
10.0
8.6
8.0
3.5 Solid-state emission of ATTs
Emission properties of the fluorophores were also investigated in solid state. All the compounds
exhibited broad emission bands in the region of 597–642 nm (Fig. 5 and Table 5). Most of the
compounds exhibited bathochromic shifts in comparison to their fluorescence maxima in CHCl3
solution. However, the emission QYs were small (0.2%–1.3%) and sometimes could not be detected
under the detection limit. This may be attributed to the effective excitation energy transition between
molecules owing to the effective π–π stacking (see above the discussion of X-ray data).
a
b
c
Fig. 5 (a,b) Normalized fluorescence spectra of ATTs 1a-e, g-p in the solid state and (c) photographs of
the solids of respective compounds in (left column) daylight and upon (right column) illumination at 365
nm.
14

Table 5 Spectral characteristics of ATTs 1a-e, g-o in a solid state.
Stokes shift,
Entry Compound λexca, nm λem, nm QY, %
nm/cm-1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
510
510
510
505
505
505
510
510
510
505
510
505
505
510
617
607
609
599
653
654
597
599
613
609
630
611
606
620
0.6
1.2
0.7
107/3400
97/3133
99/3187
94/3107
148/4488
149/4511
87/2857
89/2913
103/3295
104/3382
120/3735
106/3435
101/3300
110/3479
1a
1b
1c
1d
1e
1g
1h
1i
1j
1k
1l
1m
1n
1o
b
-
-
b
-
b
-
b
-
b
-
0.5
0.5
0.5
1.3
0.2
a Excitation wavelength; b very weak emission
3.6 Quantum mechanical calculations
The unusual electronic structures of ATTs and their outstanding optical properties encouraged us to
investigate these compounds using quantum mechanical calculations (see Supporting Information,
Computational Details). The molecular geometries of the ATTs optimized in chloroform solution are
depicted in Fig. S23 (SI), and the key geometric parameters are summarized in Tables S5–S7 (SI).
The ground-state geometries of the conformers of ATTs 1a–c, e, f, h, j, k, m, o, and p that could exist
due to the rotation of the side substituents were calculated. Since the differences in energy between
rotamers are small (Table S4, SI), we further calculated the excited states for the most stable conformers.
The results are shown in Tables S5–S7 (SI) and are discussed based on the atom labels shown in these
Tables and in Fig. 8. The predicted locations of the absorption bands are similar to the experimentally
observed positions with max deviations of 8 nm for absorption and 11 nm for emission (Tables 2 and 6).
In the GS and ES optimized geometry (Fig. S23, SI), the central heterocyclic core shows a planar
structure, and the aromatic ring A is coplanar to the heterocycle (θA = 1.04°–7.94° in GS and 0.02°–
1.54° in ES). The side chain on the C7 atom of the triazolothiophene core shows small deviation from
the heterocycle plane (within 0.04°–13.49°). The largest torsion was observed for the aromatic cycle B
and ranged from 31.08°–38.39°.
The lengths of the bonds differed from the standard lengths reported for double and single bonds in
GS [52]. Moreover, the bond lengths were noticeably affected upon ES relaxation. The most significant
changes were observed for the N(11)–N(4), C(7)–C(15), S(8)–C(9), C(1)–N(4), and N(4)–N(5) bonds.
The aromatic cycle A strongly participates in the conjugated system of the molecule; in contrast, the π
electrons of cycle B cannot effectively participate in charge transfer due to the large torsion angle.
15

However, this ring can influence the emission intensity due to its vibration and rotation during geometric
relaxation or due to the local electric field caused by these electrons on the neighboring
chromophoric/fluorophoric moiety. The presence of multiple weak intramolecular interactions is
confirmed by the computational results; they are responsible for an enhancement of the stability and
rigidity of the molecule.
The Electrostatic Potential (ESP) charge (Fig. 6; Tables S8 and S9, SI) computed for the GS and
vertical and relaxed ESs show that the overall picture is not as straightforward as depicted by means of
VB resonance structures (Scheme 3). The most significant negative charge is localized at O(16) and the
triazole N(5) and N(11); the amount of negative charge slightly decreases at O16 after excitation but
then recovers to the initial value after geometry relaxation. The positive charge is predominantly
associated with the N(2) atom of triazole and increases upon light absorption (the vertically excited,
Frank–Condon state) and relaxation to the S1r state. The N(12) atom of the tert-cycloalcylamine moiety
has centered less negative charge, which turned onto a positive charge in most compounds upon ES
geometry relaxation. Analogous alternative pictures are provided by the differences in electron densities
(Δ) between S1v → S0 and S1v → S1r states (Fig. S24, SI) and by the maps of the Molecular Electrostatic
Potential (MEP; vide postea). The changes in electron density upon excitation and relaxation are
common to all the considered compounds and involve all of the atoms of the heterocyclic core except
those belonging to the aromatic ring B, for which no changes were observed upon ES relaxation; this
represents the most important finding related to the electronic distribution for ATTs. We can therefore
conclude that charge redistribution occurs both upon excitation and relaxation.
S0
S1v
S1r
Fig. 6 ESP charges (in units of elementary charge) on the key heteroatoms of ATTs in GS and ESs.
Level of theory: (TD-)DFT ωB97-X[D] / 6-311++G** // IEF-PCM (UFF) (The ranges of the charge
are shown).
The frontier molecular orbitals (FMOs), the highest occupied molecular orbital (HOMO), the lowest
unoccupied molecular orbitals (LUMO), HOMO-LUMO energy, and HOMO-LUMO energy gap are
shown at Fig.7. Notably for these unusual compounds the difference between the HOMO-LUMO values
obtained (from 2.88 (1p) to 3.15 eV (1k)) is not equal to the excitation energy owing to the more
16

complicated electronic distribution and more configurations intervene in the description of the excited
state. The isosurfaces of the FMOs of the ATTs 1 are depicted in Fig. 7. The HOMOs of the ATTs are
mainly distributed over the central heterocyclic core. The aromatic cycles A is a nodal area for HOMOs,
whereas LUMO extends over it. Ring B is not involved either in HOMO or LUMO. The ES shows the
cognate pictures of HOMOs and LUMOs compared to the GS.
Fig. 7. Plot of HOMO and LUMO orbitals (along with their energies and energy gap) in the ground and
excited states for ATTs 1a-c, e, f, h, j, k, m, o, p (|Isovalue(MO)| = 0.02 a.u.). Level of theory: (TD-
)DFT ωB97-X[D] / 6-311++G** // IEF-PCM (UFF).
The studied systems undergo S1v ← S0 excitation that is primarily HOMO-LUMO in nature (Table 6,
cHL ~ 0.69). This transition is allowed and is characterized by moderate oscillator strength (f ~ 0.4-0.6);
among the investigated compounds, the highest value is observed for compound 1c bearing an electron-
donating substituent (MeO) on aromatic ring A and a strong electron-accepting group (CN) on ring B.
The electric dipole moments are large in modulus, as expected for mesoionic compounds (μ > 5 D) [54],
and decrease passing from S0 to S1v and S1r. Large changes in the dipole moment directions (up to 83.4°)
are observed upon relaxation.
17

Table 6 Computed absorption and emission propertiesa of ATTs 1a-c, e, f, h, j, k, m, o, p in solvated
phase (CHCl3). Level of theory: (TD-)DFT ωB97-X[D] / 6-311++G** // IEF-PCM (UFF).
Entry Compd
cH-L
λ01
f01
μ0
μ1v
θ0,1v
cL-H
λ10
f10
μ1r
θ0,1r
1
2
3
4
5
6
7
8
9
0.6866
0.6877
0.6857
0.6847
0.6869
0.6887
0.6876
0.6878
0.6878
0.6895
0.6876
508
504
502
525
517
509
508
499
506
497
524
0.5706
0.5892
0.6256
0.4802
0.5135
0.5444
0.4892
0.5519
0.5393
0.4628
0.3878
12.1
9.8
9.8
8.4
6.7
6.7
5.4
12.5 22.1
7.9 19.0
10.9 23.4
12.2 12.5
8.5
5.1
7.5
9.3
6.7
15.7
56.2
-0.6892
-0.6896
0.6882
0.6887
0.6901
0.6904
0.6904
0.6894
580
583
575
625
610
596
596
572
578
583
624
0.4153
0.4371
0.4797
0.3253
0.3573
0.3658
0.3658
0.4263
0.4146
0.3403
0.2728
7.3 42.4
6.1 70.1
9.5 55.3
7.0 87.8
13.4 58.2
7.6 69.8
11.1 66.2
11.5 29.4
7.8 62.1
4.8 80.8
8.9 83.4
1a
1b
1c
1e
1f
1h
1j
1k
1m
1o
1p
5.8
13.1
10.9
13.2
15.5
11.7
8.8
15.2 -0.6892
22.5 -0.6911
45.4 -0.6905
10
11
7.6
a
Absorption wavelength (λa, nm) and oscillator strength (f01); emission wavelength (λe, nm) and
oscillator strength (f10); moduli of the electric dipole moments of the ground state (μ0, D), of the vertical
FC excited state (μ1v, D), and of the relaxed excited state (μ1r, D); and angles formed by the dipole
moment vectors (θ0,1v,((deg) and θ0,1r (deg)).
The maps of the MEP provide information about possible interactions with electrophilic and
nucleophilic reagents and the formation of weak noncovalent interactions with analyte or solvent
molecules. The maps of MEP of 1a-c, e, f, h, j, k, m, o and p in GS and ES are presented in Fig. S26
(SI). Strongly electrophilic regions of the ATT molecules are associated with oxygen atom and the CN
groups in compounds 1c and 1f. Regions of negative electrostatic potential are generally localized over
hydrogen atoms, suggesting that hydrogen atoms are the favorable sites for interactions with
nucleophilic species. Regions of positive electrostatic potential are mainly located at tert-
cycloalkylamino groups and, to a lesser extent (as indicated by a light-blue color), around the hydrogen
atoms of ring A; these regions demonstrate the electrophilic reactivity of the ATT molecules. The
nucleophilic and electrophilic sites are not significantly different between the GS and ES. Moreover, the
activity of the sites depends on the substituents. Among the studied ATTs, 1f, 1j, and 1p show the most
active electrophilic centers; These ATTs indeed have electron-accepting substituents at rings A and B
(1f and 1j) or only in aromatic ring A (1p). The ATTs with the most active nucleophilic sites are clearly
compounds 1a and 1e, which bear the electron-donating MeO group at ring B.
3.7 Behavior in biological systems
To evaluate the application potential of the ATTs as new fluorophores in biological systems, a series
of confocal laser scanning microscopy experiments using the standard methodology [53] were carried
out. (Experimental details are presented in the SI). Solutions of 1a, 1b, 1h, 1j, 1k, and 1o in DMSO (c
= 10−6 mol/L) were diluted to 10 μL per 1 mL of nutrient medium. The ATTs were then incubated with
green monkey epithelial cells (Vero) or MIA PaCa-2 (human pancreatic cancer cell line) cells for 30
18

min at 37 °C in culture medium. The ATTs successfully stained both types of cells. While the
fluorescence intensity decreased during the observations, several images of average quality for the
relatively unstable 1b were obtained (Fig. S27, SI). From a series of lambda images taken during 30 s
of observation for 1a, 1h, 1j, 1k, and 1o, the decrease in integral fluorescence intensity was plotted over
time (Fig. 7; Figs. S30, S31, and S33, SI). Nevertheless, images of MIA PaCa-2 cells stained with the
relatively unstable 1b were obtained using a confocal microscope in lambda mode with laser excitation
at wavelengths of 405, 488, and 561 nm (Fig. S27, SI). The other samples were more photostable,
especially at λexc = 488 and 561 nm.
a
b
c
Fig. 8 (a) Graph of changes in the integral fluorescence intensity of a substance 1k during microscopic
observation. For visual clarity, the data is fitted by an exponential decay function (b) Images of MIA
PaCa-2 cells stained with the substance 1k, taken using a confocal microscope in lambda mode with
excitation with a laser with wavelengths of (b) 488 and (c) 561 nm.
Thus, high-quality images for 1a, 1b, 1h, 1j, 1k, and 1o were obtained, providing information about
the behavior of these compounds within the cells (Figs. 8-10, Figs S27-29,32 in SI). First, 1k
accumulated in the lipophilic parts of the cells. Morphological analysis indicated the accumulation of
ATTs in the cell membrane along with accumulation near the nucleus, likely in the Golgi apparatus,
which is also formed by lipophilic membranes (Figs. 8b and c). An unusual phenomenon was noted for
1h. Upon initial observation, 1h in the cells fluoresced in the orange-red range (600–650 nm); however,
the fluorescence in the red-orange part of the spectrum disappeared over time, while the fluorescence in
the green region, which was much weaker in intensity, persisted for much longer (Fig. 9, Fig. S28, SI).
The spectra obtained from a point inside the cell, which was fluorescent in the red range, and from the
periphery of the cell, which was fluorescent in the green range, are shown in Fig. 9c.
19

a
b
c
Fig. 9 Images of the Vero cells stained with the substance ATT 1h, obtained (a) at the very beginning
of the study and (b) after 30 s of observation, obtained in the lambda mode of a confocal microscope
with excitation by a laser with a wavelength of 488 nm; (c) Fluorescence spectrum of ATT 1h recorded
under excitation with a laser with a wavelength of 488 nm in two different parts of the cell.
The images obtained under different excitation wavelengths after staining with 1a were similar in
terms of the regions of fluorescence and the localization of the ATTs in the cells (Fig. S32, SI). It stained
a uniformly distributed reticular structure; based on the morphology, this structure is almost certainly
the endoplasmic reticulum. No noticeable accumulation of 1a was observed near the cell membrane.
ATT 1o was the most photostable among the investigated compounds. Over 30 s of observation, the
fluorescence intensity decreased by 30%–40% for all excitation wavelengths. The decrease in integral
intensity is plotted in Fig. S33 (SI). Fluorescence was observed in different regions. Red fluorescence
was evenly distributed throughout the cells (likely due to the staining of the endoplasmic reticulum),
while blue fluorescence was localized near the nucleus (staining of the Golgi apparatus is also possible).
Fig. 10 shows images of the cells before and after 30 s of observation.
20

a
b
c
d
e
f
g
h
Fig. 10 Images of the Vero cells stained with the substance ATT 1o, obtained (a,c,e,g) at the very
beginning of the study and (b,d,f,h) after thirty seconds of observation. Images were obtained in the
lambda mode of a confocal microscope with excitation by a laser with a wavelength of (a,b) 405, (c,d)
488, (e,f) 514 and (g,h) 561 nm.
The image obtained for cells which where staining with ATT 1o and excited with long-wavelength
(more than 405 nm) lasers, is similar to that for 1a. However, in this case, in addition to the reticular
structure (endoplasmic reticulum) inside the cell, an accumulation of matter at the periphery is also
visible, which makes it possible accurate conclude of staining the cell membrane. When excited with
lasers of 488 and 514 nm, it is seen that the membrane fluoresces is in the yellow range, and the inner
part of the cell is in the red, but this is a software processing artifact: the spectrum of fluorescence at the
periphery and inside the cell, in contrast to other substances described above, differ only with the
intensity of emission.
4. Conclusions and Outlook
We have synthesized and thoroughly characterized new family of mesoionic bicyclic condensed
heterocycles. The improved one-pot synthetic approach expands the library of these compounds and
provides a greater diversity of structures through the introduction of substituents with different
characteristics and their combinations, the introduction of heterocyclic fragments, and the variation of
the number of aromatic rings. Comprehensive spectral, theoretical, and biological investigations were
conducted and revealed some notable peculiarities of these compounds. The ATTs absorption and
emission characteristics demonstrate their outstanding and useful optical properties, including the
following: (1) absorption in visible region (498–535 nm); (2) yellow, orange, and red fluorescence (576–
21

629 nm) with strong contrast compared to the natural background; (3) large Stokes shift (2590–3110
cm−1); (4) tunable QY (up to 25.9%); (5) sensitivity to protic microenvironments; and (6) applicability
in water. These characteristics demonstrated the biocompatibility of the ATTs and their potential for
application in biological systems. (7) The ATTs exhibited double fluorescence; in addition to their
fluorescence in dilute solutions, they exhibited red emission in the solid state.
The combination of theoretical and experimental methods revealed several new features of the ATTs.
Charge localization, electronic distribution, HOMO–LUMO, and MEP map clarified the electronic
structure in GS and ES. This joint approach can be used to design new fluorophores with enhanced
photophysical properties and stability. The heuristic presentation of the ATT electronic system using the
typical pull−pull (А–π–A), push–push (D–π–D), pull–push (A–π–D) and push–pull (D–π–A) schemes
should be carefully evoked for design new ATTs fluorophores. The nature of the substituent at ring A is
the most important factor to consider to control the absorption and emission properties, whereas the
intensity and QY of the emission can be tuned by the specific substituent at ring B.
Finally, this study demonstrated the advantages of ATTs and, more importantly, the potential to
design new photoactive ATTs for possible application in biology and medicine. Moreover, some crucial
concepts and questions concerning the optical properties of mesoionic systems arose and need to be
addressed in future investigations.
Conflicts of interest
The authors declare no conflicts of interest.
Author contributions
Alexander Eltyshev: Synthetic and optical Investigation, preparing experimental data. Nadezhda
Chernysheva: Synthetic Investigation. Artem Minin: Biological investigations, preparing biological
experimental data. Varvara Pozdina: Biological investigations. Pavel Slepukhin: X-ray experiment.
Enrico Benassi: Analyze the results of density functional theory calculations, writing the original draft.
Nataliya P. Belskaya Conceptualization, analysis of the experimental data, writing original draft.
Acknowledgments
EA and BN thanks RFBR for financially supporting this work through the project No 19-33-
90184. The Siberian Branch of the Russian Academy of Sciences (SB RAS) Siberian
Supercomputer Centre is gratefully acknowledged for providing supercomputer facilities.
Electronic supporting information available. Experimental data; NMR spectra of new compounds;
XRD data CCDC 2084371; details of photophysical investigations, quantum-mechanical calculations,
and the coordinates of compounds optimized at the (TD-)DFT level.
22

References
[1] Baker W, Ollis WD. Meso-Ionic Compounds. Q. Rev Chem Soc 1957;11:15−29.
https://doi.org/10.1039/QR9571100015
[2] Porte K, Riomet M, Figliola C, Audisio D, Taran F. Click and Bio-Orthogonal Reactions with Mesoionic
Compounds. Chem Rev 2021;121:6718−6743. https://dx.doi.org/10.1021/acs.chemrev.0c00806
[3] Champagne PA, Houk KN. Influence of Endo- and Exocyclic Heteroatoms on Stabilities and 1,3-Dipolar
Cycloaddition Reactivities of Mesoionic Azomethine Ylides and Imines. J Org Chem 2017;82:10980−10988.
https://pubs.acs.org/doi/abs/10.1021/acs.joc.7b01928
[4] Ollis WD, Ramsden CA. Meso-ionic Compounds. Adv Heterocycl Chem 1976;19:1−122.
https://doi.org/10.1016/S0065-2725(08)60230-5
[5] Newton CG, Ramsden CA. Meso-Ionic Heterocycles (1976−1980). Tetrahedron 1982;38:2965−3011.
https://doi.org/10.1016/0040-4020(82)80186-5
[6] Gribble GW. Mesoionic Ring Systems. Synthetic Applications of 1,3-Dipolar Cycloaddition Chemistry
Toward Heterocycles and Natural Products. Weinheim: Wiley VCH; 2003. 753.
[7] Lopchuk JM. Mesoionics. In: Gribble GW, Editor. Metalation of Azoles and Related Five-Membered Ring
Heterocycles, Berlin: Springer; 2012; p. 381−413.
[8] Miao Q, Sun H. Recent Progresses in the Chemistry of Münchnones. Chin J Org Chem 2016;36:913-926.
https://doi.org/10.6023/cjoc201511047
[9] Nein YI, Morzherin YY. Synthesis of [1,2,3]triazolo[1,5-a]pyrazinium-3-olates. Chem Heterocycl Compd.
2014;50:1021−1026. https://doi.org/10.1007/s10593-014-1558-4
[10] Chandrasekhar RR, Nanjan MJ. Sydnones: a brief review. Mini-Rev Med Chem. 2012;12:1359−1365.
https://doi.org/10.2174/13895575112091359
[11] Dunkley CS, Thoman CJ. Synthesis and biological evaluation of a novel phenyl substituted sydnone series
as potential antitumor agents. Bioorg Med Chem Lett 2003;13:2899−2901. https://doi.org/10.1016/S0960-
894X(03)00487-6
[12] Abdulla ZM, Iyer RP, Akamanchi KG, Degani MS, Coutinho EC. J Heterocycl Chem 2011;48:38−49.
https://doi.org/10.1002/jhet.495
[13] Thatcher GRJ, Nicolescu AC, Bennett BM, Toader V. Nitrates and no release: contemporary aspects in
biological
and
medicinal
chemistry.
Free
Radical
Biol
Med
2004;37:1122−1143.
https://doi.org/10.1016/j.freeradbiomed.2004.06.013
[14] Huerta S, Chilka S, Bonavida B. Nitric oxide donors: Novel cancer therapeutics. Int J Oncol
2008;33:909−927. https://doi.org/10.3892/ijo_00000079
[15] Asundaria T, Patel NS, Patel KC. Novel 3-[4-(diethylamino)phenyl]-4-substituted-1-ylsulfonyl) sydnones:
Synthesis,
characterization
and
antimicrobial
studies.
Org
Commun
2010;3:2:30−38.
https://www.acgpubs.org/doc/20180802211253OC-0909-103.pdf
[16] Asundaria ST, Patel NS, Patel KC. Synthesis, characterization, and antimicrobial studies of novel 1,3,4-
thiadiazolium-5-thiolates. Med Chem Res 2012;21:1199−1206. https://doi.org/10.1007/s00044-011-9632-2
[17] Schulze B, Schubert US. Chem Soc Rev 2014;43:2522−2571. https://doi.org/10.1039/C3CS60386E
[18] Moura GL, Simas AM, Miller J. Mesoionic Rings as Efficient Asymmetric Bridges for the Design of
Compounds with Large Optical Nonlinearities. Chem Phys Lett 1996;257:639−646. https://doi.org/10.1016/0009-
2614(96)00601-X
23

[19] De Oliveira MB, Miller J, Pereira AB, Galembeck SE, De Moura GL, Simas AM. Mesoionic 2-N-
Cycloalkylamino-5-Alkyl1, 3-Dithiolium-4-Thiolates. Phosphorus, Sulfur Silicon Relat Elem 1996;108:75−84.
https://doi.org/10.1080/10426509608029640
[20] Lyra BF, De Morais SA, Rocha GB, Miller J, Moura GLC, Simas AM, Peppe C, De Athayde-Filho PF.
Thiazolium-5-Thiolates Mesoionic Compounds: Semiempirical Evaluation of Their First Static
Hyperpolarizabilities and Synthesis of New Examples. J Braz Chem So. 2010;21: 934−940.
https://doi.org/10.1590/S0103-50532010000500024
[21] Rakov N, De Araujo CB, Da Rocha GB, Simas AM, ́ Athayde-Filho PAF, Miller J. Two-Photon Absorption
in Mesoionic Compounds Pumped at the Visible and at the Infrared. Chem Phys Lett 2000;332:13−18.
https://doi.org/10.1016/S0009-2614(00)01261-6
[22] Rakov N, De Araujo CB, Rocha GB, Simas AM, ́ Athayde-Filho PA, Miller J. Reverse Saturable Absorption
and Anti-Stokes Fluorescence in Mesoionic Compounds Pumped at 532 nm. Appl Opt 2001;40:1389−1395.
https://doi.org/10.1364/AO.40.001389
[23] Bosco CA, Maciel GS, Rakov N, De Araujo CB, Acioli ́ LH, Simas AM, Athayde-Filho PF, Miller J. Probing
the Nuclear Susceptibility of Mesoionic Compounds Using Two-Beam Coupling with Chirp-Controlled Pulses.
Chem Phys Lett 2007; 449:101−106. https://doi.org/10.1016/j.cplett.2007.10.037
[24] Pilla V, De Araủjo CB, Lira BF, Simas MS, Miller J, Athayde-Filho PF. Nonlinear absorption of new
mesoionic
compounds.
Optics
Communications
2006;264:225−228
https://doi.org/10.1016/j.optcom.2006.02.012
[25] Mera A, Ito M, Nakayama A, Namba K. Nonlinear absorption of new mesoionic compounds. Chem Lett
2017;46: 539−542. https://doi.org/10.1016/j.optcom.2006.02.012
[26] Namba K, Mera A, Osawa A, Sakuda E, Kitamura N, Tanino K. One-Pot Synthesis of Highly Fluorescent
2,5-Disubstituted-1,3a,6a-triazapentalene. Org Lett 2012;14:5554−5557.
[27] Hayashi T, Osawa A, Watanabe T, Murata Y, Nakayama A, Namba K. Development of 1,3a,6a-
triazapentalene-labeled enterobactin as a fluorescence quenching sensor of iron ion. Tetrahedron Lett
2017;58:1961–1964. https://doi.org/10.1016/j.tetlet.2017.04.011
[28] J. Sawada, A. Osawa, T. Takeuchi, M. Kaneda, S. Oishi, N. Fujii, A. Asai, K. Tanino and K. Namba, Bioorg.
Med. Chem. Lett., 2016, 26, 5765–5769
[29] Cantillo D, Avalos M, Babiano R, Cintas P, Jiménez JL, Light ME, Palacios JC, Porro R. Stepwise Formation
́
of 1,3-Diazolium-4-thiolates by Münchnone Cycloadditions: Promising Candidates for Nonlinear Optics. J Org
Chem 2014;79:4201−4205. https://doi.org/10.1021/jo500349g
[30] Cai Z, Zhou M, Gao J. A New Type of Organic Donor-πAcceptor Compounds with Third-Order Nonlinear
Optical Properties. Opt Mater 2009;31:1128−1133. https://doi.org/10.1016/j.optmat.2008.12.003
[31] Srinivasan P, Kanagasekaran T, Gopalakrishnan RA. Highly Efficient Organic Nonlinear Optical Donor-
Acceptor
Single
Crystal:
L-Valinium
Picrate.
Cryst
Growth
Des
2008;8:2340−2345.
https://doi.org/10.1021/cg701143n
[32] Ouyang C, Liu J, Liu Q, Li Y, Yan D, Wang Q, Guo M, Cao A. Preparation of Main-Chain Polymers Based
on Novel Monomers with D-π-A Structure for Application in Organic SecondOrder Nonlinear Optical Materials
with
Good
Long-Term
Stability.
ACS
Appl
Mater
Interfaces
2017;9:10366−10370.
https://doi.org/10.1021/acsami.7b00742
[33] Bhattacharyya K, Ramaiah D, Das PK, George MV. Flash Photolysis Studies of Nitrile Imines and Related
Intermediates Photogenerated from Sydnones and Tetrazoles in fluid Solutions. J Photochem 1987;36:63−84.
https://doi.org/10.1016/0047-2670(87)87063-6
24

[34] Gettings ML, Thoenen MT, Byrd EFC, Sabatini JJ, Zeller M, Piercey DG. Tetrazole Azasydnone
(C2N7O2H) And Its Salts: High‐Performing Zwitterionic Energetic Materials Containing A Unique
Explosophore. Chem Eur J 2020;26:14530 – 14535. https://doi.org/10.1002/chem.202002664
[35] Bernard S, Audisio D, Riomet M, Bregant S, Sallustrau A, Plougastel L, Decuypere E, Gabillet S, Kumar
RA, Elyian J, Nguyet M, Koniev O, Wagner A, Kolodych S, Taran F. Bioorthogonal Click and Release Reaction
of
Iminosydnones
with
Cycloalkynes
Angew
Chem
Int
Ed
2017;56:15612−15616.
https://doi.org/10.1002/anie.201708790
[36] Eilertsen M, Allin SM, Pearson RJ. New 4-aryl-1,3,2-oxathiazolylium-5-olates: Chemical synthesis and
photochemical stability of a novel series of S-nitrosothiols. Bioorg Med Chem Lett 2018;28:1106–1110.
https://doi.org/10.1016/j.bmcl.2018.01.059
[37] Huang S, Wang Y, Hu C, Yan X. Computational study of 1,2,3-triazol-5-ylidenes with p-block element
substituents. New J Chem 2021;45:4802–4809. https://doi.org/10.1039/D1NJ00050K
[38] Abbott PA, Bonnert RV, Gaffrey MV, Cage PA, Cooke AJ. Fused mesoionic heterocycles: synthesis of
[1,2,3]triazolo[1,5-a]quinoline,
[1,2,3]triazolo[1,5-a]quinazoline,
[1,2,3]triazolo[1,5-a]quinoxaline
and
[1,2,3]triazolo[5,1-c]benzotriazine derivatives. Tetrahedron 2002;58:3185−3198. https://doi.org/10.1016/S0040-
4020(02)00269-7
[39] Belskaya NP, Koksharov AV, Eliseeva AI, Fan Z, Bakulev VA. Synthesis and oxidative cyclization of 3-
amino-2-arylazo-5-tert-cycloalkylaminothiophenes. Chem Heterocycl Compd. 2011;47:564− 570.
https://doi.org/10.1007/s10593-011-0799-8
[40] A.K. Eltyshev, N.V. Chernysheva, N.P. Belskaya, AIP Conf. Proc., 2021, in press.
[41] Gavlik KD, Lesogorova SG, Sukhorukova ES, Subbotina JO, Slepukhin PA, Benassi E, Belskaya NP.
Synthesis of 2‐Aryl‐1,2,3‐triazoles by Oxidative Cyclization of 2‐(Arylazo)ethene‐1,1‐diamines: A One‐Pot
Approach. Eur J Org Chem 2016;2016:2700–2710. https://doi.org/10.1002/ejoc.201600256
[42] Deryabina TG, Belskaia NP, Kodess MI, Dehaen W, Toppet S, Bakulev VA. [3+2]- Versus [4+2]-
cycloaddition reactions of 3-methylsulfanyl-2-arylazo-3-(pyrrolidin-1-yl)acrylonitriles with N-substituted
maleimides involving pyrrolidine-derived azomethine ylides. Tetrahedron Lett 2006;47:1853–1855.
https://doi.org/10.1016/j.tetlet.2005.12.130
[43] Belskaia NP, Deryabina TG, Koksharov AV, Kodess MI, Dehaen W, Lebedev AT, Bakulev VA. A novel
approach to fused 1,2,4-triazines by intramolecular cyclization of 1,2-diaza-1,3-butadienes bearing
allyl(propargyl)sulfanyl and cyclic tert-amino groups. Tetrahedron Lett 2007;48:9128–9131.
https://doi.org/10.1016/j.tetlet.2007.10.140
[44] Belskaya NP, Lesogorova SG, Subbotina JO, Koksharov AV, Slepukhin PA, Dehaen W, Bakulev VA. 1,3-
Dipolar cycloaddition of 3-alkylsulfanyl-2-arylazo-3-(tert-cycloalkylamino)acrylonitriles with N-methyl- and N-
phenylmaleimides. Tetrahedron 2015;71:1438–1447. https://doi.org/10.1016/j.tet.2014.12.087
[45] Nešpůrek S., Böhm S., Kuthan J. Photochromism of sydnones: Structural evidence for the blue
colored species from 3-(3-pyridyl)sydnone. Journal of Molecular Structure: THEOCHEM, 1986;136:
261–273. https://doi.org/10.1016/0166-1280(86)80141-5
[46] Nešpůrek S, Lukáš J, Böhm S, Bastl Z. Photochromism of 3-(3-pyridyl): an investigation by electron
spectroscopy for chemical analysis (ESCA) and molecular orbital calculations. J Photochem Photobiol Chem
1994;84:257–264. https://doi.org/10.1016/1010-6030(94)03870-8
[47] Veedu RN, Kvaskoff D, Wentrap C. Sydnone Photochemistry: Direct Observation of Earl’s Bicyclic Lactone
Valence Isomers (Oxadiazabicyclo[2.1.0]pentanones), Formation of Carbodiimides, Reaction Mechanism, and
Photochromism. Austr J Chem 2014;67:457–468. https://doi.org/10.1071/CH13536
25

[48] Plougastel L, Lamaa D, Yen-Pon E, Audisio D, Taran F. Tetrahedron 2020;76:131250.
https://doi.org/10.1016/j.tet.2020.131250
[49] Vasilescu M, Dumitraşcu F, Lemmetyinen H, Tkachenko N. Steady-State and Time-Resolved Spectroscopic
Characteristics
of
Mesoionic
Oxazolones
Solutions.
J
Fluoresc
2004;14:443–450.
https://doi.org/10.1023/B:JOFL.0000031825.21272.fc
[50] Gavlik KD, Sukhorukova ES, Shafran YM, Slepukhin PA, Benassi E., Belskaya NP Dyes and Pigments
2017;136: 229-242. 10.1016/j.dyepig.2016.08.015
[51] Valeur B, Berberan-Santos MN. Molecular Fluorescence. 2nd ed. Weinheim: Wiley VCH; 2013, 592.
[52] Cordero B, Gomez V, Platero-Prats AE, Reves M, Echeverґıa J, Cremades E, Barragan
F, Alvarez S Dalton Trans. 2008:2832-2838. https://doi.org/10.1039/B801115J
[53] Lugovik KI, Eltyshev AK, Suntsova PO, Slepukhin PA, Benassi E, Belskaya NP. Highlights on the
Road towards Highly Emitting Solid‐State Luminophores: Two Classes of Thiazole‐Based Organoboron
Fluorophores
with
the
AIEE/AIE
Effect.
Chem
Asias
J
2018;13:311–324.
https://doi.org/10.1002/asia.201701526
26

 New family of mesoionic bicyclic compounds was synthesized by the one-pot method
 Thienotriazolium-olates demonstrated outstanding optical properties
 The quantum chemical calculation revealed the peculiarities of the electronic structure of
thienotriazolium-olates
 Bioimaging experiments showed the easy penetration into the living cells